iraqi j pharm sci, vol.22(1) 2013 flavonoid in the fruits of vitex agnus-castus l. 104 phytochemical study of flavonoid "casticin" present in the fruits of vitex agnus-castus l. cultivated in iraq aous a. shakir *1 and zainab j. awad ** * department pharmaceutics and clinical pharmacy, college of pharmacy, university of basrah, basrah, iraq. ** department of pharmacognosy, college of pharmacy, university of baghdad, baghdad, iraq. abstract this study detects the presence of an important flavonoid "casticin" in the fruits of vitex agnus-castus l. grown in iraq. the pharmaceutical importance of casticin arise from its consideration as anti-tumor substance and have cytotoxic effects, and the absence of any study concerning casticin content of this medicinal plant in iraq, gave this study its importance. this study concerned with the extraction, identification, isolation and purification of casticin from the fruits of vitex agnus-castus l. the extraction of this compound was carried out using two methods. identification of this compound was done by thin layer chromatography (tlc) in which three different solvent system has been tried. this identification was further augmented by using high performance liquid chromatography (hplc) and then this compound was isolated and purified. identification of the isolated casticin was carried out using melting point (m.p.), thin layer chromatography (tlc), and infrared spectroscopy (ir). this study confirms the presence of casticin in the fruits of vitex agnus-castus l. grown in iraq. key words : vitex agnus – castus , flavanoid , casticin انمسروع في انموجود في ثمار نباث شجرة ابراهيم( انكاستيسين)دراست كيموأحيائيت نهفالفينويذ انعراق أوش عبذانحسين شاكر * و زينب جهيم عواد ** * .، انبصزة ، انعزاقجبيعت انبصزة،كهيت انصيذنت ، انصيذالَببث ٔانصيذنت انسزيزيت فزع ** .، بغذاد ، انعزاقجبيعت بغذاد ،كهيت انصيذنت ،انعمبليز ٔانُببحبث انطبيت فزع انخالصت اٌ . ْذِ انذراست حخخض ببنكشف عٍ َٕع يٍ انفالفيُٕيذ ْٕٔ يبدة انكبسخيسيٍ في ثًبر َببث شجزة إبزاْيى انخي حًُٕ في انعزاق ست االًْيت انعالجيت نٓذِ انًبدة ٔانُبشئت يٍ كَٕٓب يبدة يضبدة نالٔراو انسزطبَيت ٔحًهكٓب حبثيزاث حيٕيت ضذ انسًٕو ٔنعذو ٔجٕد أي درا اسخخالص ٔكشف حُبٔنج ْذِ انذراست . راق حخُبٔل يحخٕٖ ْذا انُببث انطبي يٍ ْذِ انًبدة ْٕ انذي يعطي اًْيت نٓذِ انذراستسببمت في انع ببسخخذاو حمُيت ف فمذ حى شاالسخخالص ببسخخذاو طزيمخيٍ يخخهفخيٍ ، ايب انكحيث حى .يٍ ثًبر ْذا انُببث ٔفصم ٔحُميت ْذِ انًبدة ببسخخذاو يذيببث يخخهفت كٕسيظ َبلم ٔانكشف عُٓب ٔكذنك حمُيت كزٔيبحٕغزافيب األداء انعبني انسبئهت ٔبعذْب ،كزٔيبحٕغزافيب انطبمت انزليمت درجت : ي شًهج ٔكذنك اسخخذيج يجًٕعت يٍ انخمُيبث نهخحمك يٍ َٕعيت انًزكب انًفصٕل ٔ درجت َمبٔحّ ٔانج. حًج عًهيت انفصم ٔانخُميت ْذِ انذراست حؤكذ ٔجٕد يبدة انكبسخيسيٍ في ثًبر . االَصٓبر ٔ حمُيت كزٔيبحٕغزافيب انطبمت انزليمت ٔ كذنك يطيبف األشعت ححج انحًزاء .َببث شجزة إبزاْيى انخي حًُٕ في انعزاق .انكاستيسين،فالفينويذ ، النباث شجرة ابراهيم: انكهماث انمفتاحيت introduction vitex agnus-castus l. (family: verbanceae), figure (1), is an ornamental shrub or small tree small tree, approximately 1 to 6 m height, with aromatic odour, it is native to european, mediterranean and asian regions (1,2,3) . in iraq it is found in north areas (erbil, sulaimaniya, kirkuk, mosul and khanaqin), and it is also found in central areas as in baghdad (4) . the fruits as well as the leaves of vitex agnus-castus l. are known to contain the lipophilic flavonoids , mostly casticin , (synonym: vitexicarpin) the fruit is rich in casticin (5,6) and it is considered as finger print in hplc of vitex agnus-castus l (7) . the fruit extract of vitex agnus-castus l. has been shown to be effective for the treatment of premenstrual syndrome (pms), including premenstrual dysphoric disorder (pmdd) , that is a more severe form of (pms) (8,9) and it is used to normalize menstruation in women with shortened , lengthened or infrequent menstruation, particularly when low progesterone and luteal phase defects (10) . 1 corresponding author e-mail: ph_aous78@yahoo.com received:14/12/2011 accepted:3/4/2013 iraqi j pharm sci, vol.22(1) 2013 flavonoid in the fruits of vitex agnus-castus l. 105 it is also used to enhance fertility in women with progesterone deficiency (11) . the cytotxic activity of vitex agnus-castus l. fruit extract have been examined well and it was found that the extract inhibits the growth of breast, ovarian, cervical, gastric, colon and lung cancer cells (12) , casticin which found in the fruits of this plant showed inhibitory activity of both tlymphocyte and b-lymphocyte proliferation in-vitro and it may be a potential lead compound for treating inflammatory and immuno-related diseases or lymphomas in vivo (13) . also it has antifungal activity of against the plant pathogenic fungus and it has found that casticin has an inhibitory action against bacillus spp. and staphylococcus spp. (14) figure 1: vitex agnus-castus l. material and methods plant materials the plant materials (fruits) of vitex agnuscastus l. were collected from private garden, during the months of september and october (2010), they were cleaned and dried in oven at a temperature between (60-70)c for (4-5) hours, then these plant materials were coarsely powdered by mechanical grinder and weighted. a 100 g of dried powdered plant materials were extracted using two methods. extraction extraction method no. 1 (7) a quantity of 100 g of dried powdered fruit of vitex agnus-castus l. was macerated for 3 days at room temperature in aqueous ethanol (1 liter) 70% (v/v) for 3 days at room temperature and the resulting extract was filtered. the residue was extracted twice again as above. the obtained filtrates were combined and concentrated using rotary evaporator until we get dry extract, for the isolation of flavonoids, the extract was partitioned successively between an equal volumes of water and n-hexane, this step called (defatting step) then the residual water phase was extracted with an equal volume of ethyl acetate to draw all the flavonoids containing compounds in to the organic phase and the ethyl acetate phase was concentrated by means of rotary evaporator, then the dry extract was weighted and subjected for identification of flavonoids. figure (2) represents the general scheme for extraction of flavonoids from the fruits of vitex agnus-castus l. figure 2: general scheme for method (no. 1) for extraction of flavonoids from vitex agnuscastus l. extraction method no. 2(15) a quantity of 100 g of dried powdered fruit of vitex agnus-castus l. was macerated for 3 days at room temperature in 1 l of 90% methanol for 3 days at room temperature and the resulting extract was filtered. the residue was extracted twice again as mentioned above. the obtained filtrates were combined and concentrated using rotary evaporator until we get a dry extract, this step followed by step of successive partitioning of the dry extract between equal volumes of petroleum ether and water, this step called defatting step, then the water fraction was partitioned with equal volume with chloroform followed by partitioning with an equal volume with ethyl acetate to will draw all the falvonoids. the ethyl acetate phase was concentrated under reduced pressure till we get dry extract. the dry extract was weighted and subjected for identification and purifications of flavonoids. figure (3) represents the general iraqi j pharm sci, vol.22(1) 2013 flavonoid in the fruits of vitex agnus-castus l. 106 scheme for extraction of flavonoids from the fruits of vitex agnus-castus l. figure 3: general scheme for method (no. 2) for extraction of flavonoids from vitex agnuscastus l. identification of the flavonoid c .1: thin layer chromatography (tlc) using thin layer chromatography (tlc) technique for qualitative identification of casticin. in this identification:using a readymade aluminum plates of silica gel gf254. and a comparison was made with three different developing solvent systems that were (16) . solvent 1 (s1) : toluene: chloroform: acetone (8:5:7) solvent 2 (s2) : chloroform: acetone formic acid (75:16.5:8.5) solvent 3 (s3) : toluene: ethyl methyl ketone: formic acid (18:5:1) freshly prepared dimethyl formamide (dmf) solutions of the standard reference, casticin, and dmf solutions of the dried fruits of vitex agnus-castus l. extract were applied to tlc plates manually, and then developed by the ascending technique. the solvent migration limits was 10-12 cm from the base line. the above three developing systems were tried, and placed in a glass tank (22.5cm x 22cm x 7cm), and covered with a glass lid and allowed to stand for 45 minutes before use for saturation. after development, the plates were allowed to dry at room temperature and were detected using uvlight. two wave lengths were used 254 nm and 366 nm. c.2: high performance liquid chromatography ( hplc ) qualitative identification of casticin in extracts obtained from extraction methods above was authenticated by hplc, this identification was made by comparison of the retention time obtained at identical chromatographic conditions .the analyzed sample and the authentic standard conditions were: 1mobile phase: isocratic: acetonitrile / water (90% / 10%) + 0.1 n acetic acid 2column: c18 5 mm x 150 mm 3flow rate: 1 ml /min. 4detection: uv. detector at λ 258 nm. 5injection volume: 5 µl 6injection concentration: 1 mg /ml isolation and purification of the casticin after locating of casticin of the extract, preparative thin layer chromatography was done to isolate and purified casticin. the preparative tlc was performed by using a readymade glass plates 20x20 cm, which were coated with silica gel; layer thickness 1 mm; made by merck, germany, these plates were activated at 110c for one hour before use. the dried vitex agnus-castus l. fruits extract was dissolved in dmf, then applied as a concentrated solution in a raw of spots using capillary tubes, the mobile phase for casticin was s2=chloroform: acetone formic acid (75:16.5:8.5), the standard sample was applied in one side of the plate, the separated compound appeared as a band identified using uv-light detection method. the band was then assigned and the assigned silica gel was scrapped out and collected a beaker and mixed with hot dmf and then filtered. the silica gel on the filter paper was washed again with hot dmf. the dmf solutions were evaporated to dryness under reduced pressure to give the corresponding precipitates. these precipitates then recrystallized using aqueous dmf and maintained for tlc , hplc , ft-ir and measuring melting point. iraqi j pharm sci, vol.22(1) 2013 flavonoid in the fruits of vitex agnus-castus l. 107 identification and characterization of the isolated casticin for further identification of the isolated flavonoid, casticin, from the dried fruits of vitex agnus-castus l., the following methods were used: 1 .tlc : analytical tlc was performed by using a readymade plates of 20x20 cm, which are coated with silica gel (merck) layer of 0.25 mm thickness. the isolated flavonoid, casticin, obtained by preparative tlc was applied on silica gel plates as one spot by using a capillary tube along with the standard, the mobile phase was s2. 2. melting point : melting point was estimated by using electro-thermal melting point apparatus for the isolated compound and compared with that of the available standard of casticin. 3. ir : infrared spectra were recorded using kbr disk. 4. hplc analysis (conditions as in c.2) results and discussion extraction method results showed that extraction method no. 2 was better, because the percentage of yield of crude extract was higher than that of obtained from method no.1. in addition, quantitative estimation by hplc showed that flavonoids obtained in method no. 2 contain higher percentage of casticin than that obtained from method no. 1. as shown in table (1). table 1: percentages of crude extracts and casticin obtained from the fruits of vitex. agnus –castus. extraction method % yield of crude extract % yield of casticin method no. 1 12.1 6.8 method no. 2 13.7 7.2 identification of casticin by tlc the tlc of the extracts obtained from the fruits of vitex agnus-castus l by using the extraction method no1 and no2 , confirms the presence of casticin in these extracts in comparision with the standard by using different solvents . result showed the solvent no2 was the best solvent. as presented in table (2) and figure (4). table 2: rf values of flavonoid (casticin) as compared with the standard reference in different developing systems in tlc. s1 : toluene: chloroform: acetone (8:5:7) s2 : chloroform: acetone formic acid (75:16.5:8.5) s3 : toluene: ethyl methyl ketone: formic acid (18:5:1) uv 254 uv 366 f c f c figure 4: tlc of the fruit extract of vitex agnus-castus l. obtained by extraction no. 2 using silica gel gf 60 as adsorbent and (s2) as a mobile phase. (f= fruit extract, c= casticin standard) charactrization of isolated casticin tlc isolated compound ( casticin) appeared as a single spot having the rf value as that of reference standard. ir for further characterization of the casticin isolated from the dried fruit of vitex agnuscastus, ft-ir spectroscopy analysis was performed using casticin standard as a reference. the ir spectra of the isolated casticin compared with the authentic casticin standard gave us an identical results indicating that the isolated compound from vitex agnus-castus l. was casticin (17) as shown in figure (5). solvent system s1 s2 s3 rf of the standard casticin 0.4 0,45 0.77 rf of casticin of extract 0.39 0.44 0.75 iraqi j pharm sci, vol.22(1) 2013 flavonoid in the fruits of vitex agnus-castus l. 108 figure 5: ir spectra of the isolated casticin melting point the isolated compound was identified as casticin from its sharp melting point . since the compound showed a melting point of (184 – 186) c compared to casticin standard (185 –187)c. hplc analysis the hplc analysis of both of the authentic casticin and the isolated compound showed an identical retention time, which is considered as a conclusive evidence that the isolated compound was casticin.as shown in figures (6 and 7) figure 6: hplc analysis of casticin standard figure 7: hplc analysis of casticin isolated from vitex agnus-castus l. conclusion the phytochemical investigation of vitex agnus-castus l. fruits, grown in iraq revealed the presence of important medicinal natural product "casticin", which is a flavonoid. the extracted casticin was identified using tlc and hplc methods then it was purified and isolated from the dry crude extract by using a preparative tlc technique. the identification of the isolated compound, casticin, was made using tlc, hplc, melting point and ir spectroscopy. references 1. grieve, m.: a modern herbal (2 nd ed.)., dover publications, new york, 1982; vol. i : pp: 188. 2. the united states pharmacopeia. 29. rockville, md, united states pharmacopeia convention, 2005. 3. abel, g., goez, c., and wolf, h.: monographie vitex. in hagers handbuch der pharmazeutischen praxis, drogen p-z; r. hansel, keller, k., rimpler, ii., schneider. g., ed.; springer verlag: berlin, heidelberg, new york, london. paris, tokyo, hong kong. barcelona. budapest, 1994; pp: 11831196. 4. al-rawi a.: wild plants of iraq (3 rd ed.). ministry of agriculture and irrigation, repuplic of iraq, baghdad, 1988; pp: 6, 149. 5. abel, g.: experience with the analytical methods from raw extract to drug product. phytotherapy, 1999; vol. 20: pp: 147–148. 6. upton, r.: (chaste tree fruit) vitex agnuscastus: standards of analysis, quality control, and therapeutics. american herbal pharmacopoeia, santa cruz, california. iraqi j pharm sci, vol.22(1) 2013 flavonoid in the fruits of vitex agnus-castus l. 109 7. jarry, h. , spengler, b. , wuttke, w. and christoffel, v.: a in vitro assays for bioactivity-guided isolation of endocrine active compounds in vitex agnus-castus. s. maturitas, 2006; 55s: s26-s36. 8. pearlstein, t., steiner, m.: premenstrual dysphoric disorder. burden of illness and treatment update. journal of psychiastry and neuroscience, 2008; 33: 291–301. 9. prilepskaya, v.n., ledina, a.v., tagiyeva, a.v. and revazova, f.s.: vitex agnus castus: successful treatment of moderatr to sever premenstrual syndrome. maturitas, 2006; pp: 55-63. 10. mills, s. and bone, k.: principles and practice of phytotherapy. modern herbal medicine, london, churchill livingstone, 2000. 11. gerhard, i. , patek, a. , monga, b. , blank, a. , gorkow, c. and mastodynon, (r.): bei we iblicher sterilitt. forsch komplementarmed, 1998; vol. 5: pp: 272-278. 12. dixon-shanies, d. and shaikh, n.: growth inhibition of human breast cancer cells by herbs and phytoestrogens. oncol. rep., 1999; vol. 6: pp: 1383–1387. 13. you, k.m. , son, k.h. , chang, h.w. kang, s.s. and kim, h.p.: vitexicaipin, a flavonoid from the fruits of vitex rotundifolia, inhibits mouse lymphocyte proliferation and growth of cell lines in vitro. planta medica ,1998;. 64: 546-550. 14. wang, y. , hamburger, m. , gueho, j. and hostettmann, k.: antimicrobial flavonoids from psidia trinervia and their methylated and acctylated derivatives. phytochemistry, 1998; 28: 2323-2327. 15. d. e. webster, y. he, s. chen, g. f. pauli, n. r. farnsworth, z. j. wang, opioidergic mechanisms underlying the actions of vitex agnus-castus l.biochemical pharmacology 2011; 81 :170-177. 16. anderson, m. and markham, k.r.: flavonoids chemistry, biochemistry and applications, taylor and francis group, crc press, 2006; pp: 12,144-146. 17. silversteine, r.m. and webbster, f.x.: spectroscopic identification of organic compounds (6 th ed.). john wiley and sons inc., usa, 1998; pp: 57, 81-109. iraqi j pharm sci, vol.23(1) 2014 biochemical values in hemodialysis patients 14 measurement of some biochemical values in hemodialysis patients in baghdad hiba a.hassan *,1 * college of health and medical technology , baghdad, iraq. abstract one hundred of dialysis patients' mean age ( 51.18±8.28) years and one hundred healthy control group , where carried out from different hospitals of baghdad city , during the period between november /2012 until march/2013. blood samples were collected before dialyzing for estimation the concentration of urea, creatinine, uric acid, random blood sugar , calcium and cholesterol by enzymatic method detected spectrophotometerically. the aim of this study is to determine concentration of urea, creatinine, uric acid, rbs , calcium and cholesterol in hemodialysis patients in baghdad . the results showed that there were highly significant increases (p<0.01) in the mean of creatinine , urea , uric acid and rbs for patients (587.86µmol/l , 25.57 mmol/l, 414.36mmol/l and 6.47mmol/l) compared to the mean of healthy control (88.60 µmol/l , 5.07mmol/l ,270.76mmol/l and 4.65mmol/l ) was detected . also there was highly significant decrease(p< 0.01) in the mean of calcium and cholesterol for patients (1.93mmol/l and3.33mmol/l) compared to the mean of healthy control (2.34mmol/l and5.02mmol/l). it is concluded that in hemodialysis was associated with the higher levels of urea ,creatinine , uric acid and rbs with low level of cholesterol and calcium. key words: rbs (random blood sugar), sua (serum uric acid), cholesterol, urea, creatinine. فٌ بغداد انغسَم انكهوً ندى مرضي وحَوٍةانكَم بعض انقَمقَاس هبــــا عبد انحسَن حسن ،*1 * مييت اىتقْياث اىصحيت واىطبيت ، بغذاد ، اىعشاق . الخالصة ، اىنىىستشوه ،اىناىسيىً ، تشميض اىسنش اىعشىائي في ٍصو اىبىىيلىذساست تشميض جىهش اىبىه ، اىنشياتيْيِ ، حَط ٍجَىعت ٍشاقبت صحيت ، اىتجشبت سْت وٍائت ( 5,,5 ±81,15)اىعَش ٍائت ٍِ ٍشظً غسيو اىنيً ٍتىسط .ٍشظً غسيو اىنيً تٌ جَع عيْاث اىذً قبو ..112,حتً اراس / ,11,ّفزث في ٍستشفياث ٍختيفت ٍِ ٍذيْت بغذاد ، خاله اىفتشة بيِ تششيِ اىثاّي / اىتيذً ىتقذيش تشميض جىهش اىبىه و اىنشياتيْيِ وحَط اىبىىيل واىنىىستشوه واىناىسيىً و تشميضاىسنش اىعشىائي في اى اىذييضة في (p < 0.01 ) أظهشث اىْتائج أُ هْاك صيادة مبيشة ىيغايت اعتَذث بقياسها عيً االّضيَاث وتٌ قياسها بجهاص اىَطياف اىيىّي. ىيَشظً اىعشىائياىسنش تشميضواىنشياتيْيِ وجىهش اىبىه وحَط اىبىىيل ٍتىسط (587.86µmol/l , 25.57 mmol/l, 414.36mmol/l , 6.47mmol/l) ماُ هْاك أيعا ( µmol/l , 5.07mmol/l ,270.76mmol/l and 4.65mmol/l 88.60)ٍشاقبت صحيت ٍقاسّت ٍع ٍتىسط ٍقاسّت ٍع (3.33mmol/l and 1.93mmol/l)اىنىىستشوه و اىناىسيىً ىيَشظً في ٍتىسط (p < 0.01) ٍيحىظ ىيغايت اّخفاض . (5.02mmol/l and 2.34mmol/l)ٍشاقبت صحيت ٍتىسط تشميضاىسنش قذ استْتج ٍِ اىذساست اُ غسيو اىنيً ىت عالقت ٍع اىَستىياث اىعاىيت ىجىهش اىبىه واىنشياتيْيِ وحَط اىبىىيل وو .اىعشىائي وٍع اّخفاض ٍستىي اىنىىيستشوه واىناىسيىً انبونَك ، كونَستَرول ، انغسَم انكهوً . حامض انكهمات انمفتاحَة: introduction in medicine dialysis (from greek dialusis meaning dissolution, dia, meaning through, and lysis , meaning loosening or splitting) is a process for removing waste and excess water from the blood, and is used primarily to provide an artificial replacement for lost kidney function in people with renal failure (1). dialysis may be used for those with an acute disturbance in kidney function (acute kidney injury, previously acute renal failure), or progressive but chronically worsening kidney function–a state known as chronic kidney disease stage 5 (previously chronic renal failure or end-stage renal disease). the latter form may develop over months or years, but in contrast to acute kidney injury is not usually reversible, and dialysis is regarded as a "holding measure" until a renal transplant can be performed, or sometimes as the only supportive measure in those for whom a transplant would be inappropriate (2,3) . 1 corresponding author e-mail: hebaabdul@yahoo.com received:22 /4/2013 accepted: 5/1/2014 http://en.wikipedia.org/wiki/greek_(language) http://en.wikipedia.org/wiki/blood http://en.wikipedia.org/wiki/renal_replacement_therapy http://en.wikipedia.org/wiki/renal_function http://en.wikipedia.org/wiki/renal_failure http://en.wikipedia.org/wiki/renal_failure http://en.wikipedia.org/wiki/acute_kidney_injury http://en.wikipedia.org/wiki/acute_kidney_injury http://en.wikipedia.org/wiki/chronic_kidney_disease http://en.wikipedia.org/wiki/chronic_kidney_disease http://en.wikipedia.org/wiki/renal_transplant http://en.wikipedia.org/wiki/dialysis#cite_note-pendse-2 iraqi j pharm sci, vol.23(1) 2014 biochemical values in hemodialysis patients 15 in hemodialysis, the patient's blood is pumped through the blood compartment of a dialyzer, exposing it to a partially permeable membrane. the dialyzer is composed of thousands of tiny synthetic hollow fibers. the fiber wall acts as the semipermeable membrane. blood flows through the fibers, dialysis solution flows around the outside of the fibers, and water and wastes move between these two solutions. (4) the cleansed blood is then returned via the circuit back to the body. ultrafiltration occurs by increasing the hydrostatic pressure across the dialyzer membrane. this usually is done by applying a negative pressure to the dialysate compartment of the dialyzer. this pressure gradient causes water and dissolved solutes to move from blood to dialysate, and allows the removal of several litres of excess fluid during a typical 4-hour treatment. in the us, hemodialysis treatments are typically given in a dialysis center three times per week (due in the us to medicare reimbursement rules); however, as of 2007 over 2,500 people in the us are dialyzing at home more frequently for various treatment lengths (5) .studies have demonstrated the clinical benefits of dialyzing 5 to 7 times a week, for 6 to 8 hours. this type of hemodialysis is usually called "nocturnal daily hemodialysis", which a study has shown a significant improvement in both small and large molecular weight clearance and decrease the requirement of taking phosphate binders. (6) these frequent long treatments are often done at home while sleeping, but home dialysis is a flexible modality and schedules can be changed day to day, week to week. in general, studies have shown that both increased treatment length and frequency are clinically beneficial. (7) patients and methods patients this study included (100) dialysis patients (49 male and 51 female) from different hospitals of baghdad city( baghdad teaching hospital ,surgery specialist hospital and private nursing home hospital) patients' mean age for both sex ( 51.18±8.28) years and one hundred healthy control group(46 male and 64 female). selection of the patients depending on history of patients (included most of the patients have diabetes and excluded the patients which have other disease ) and the first time undergoing dialysis. sample collection from each patients' before dialyzing included in this study the blood was transferred into disposable plain tube and let stand for 30 minute to clot . serum was separated by centrifugation at 3000 rpm for 5 minute ,which was collected in plain tube and kept frozen unless analyzed immediately (8) . the serum was utilized for determination of blood urea, serum creatinine, serum uric acid, serum cholesterol, serum calcium and random blood sugar during the period between november /2012 until march/2013. commercial kits for biochemical analysis , the following kits were used: kits supplier kit for determination of serum glucose randox / united kindom kit for determination of serum urea biomaghreb/ ibn khaldountunisia kit for determination of serum creatinine biomaghreb/ ibn khaldountunisia kit for determination of serum cholesterol randox / united kindom kit for determination of serum calcium randox / united kindom statistical analysis suitable statistical methods were used in order to analyze and assess the results, includes the following (9) : 1descriptive statistics: a) statistical tables including observed frequencies with their percentages. b) summary statistic of the readings distribution (mean, sd, sem, minimum and maximum). 2 – inferential statistics: these were used to accept or reject the statistical hypotheses, they include repeated student test (t-test) by using spss program version-10. results and discussion a. distribution of patients and control according to mean age. the distribution of dialysis patients according to mean age is listed in table (1)shows the mean age have no significant difference( p>0.05) . but when comparing the mean age in present study with the mean age of other studies (4,10) shows decrease in the mean age .this result may be related to some environmental factors. http://en.wikipedia.org/wiki/dialysis#cite_note-7 http://en.wikipedia.org/wiki/dialysis#cite_note-8 http://en.wikipedia.org/wiki/dialysis#cite_note-9 http://en.wikipedia.org/wiki/dialysis#cite_note-10 iraqi j pharm sci, vol.23(1) 2014 biochemical values in hemodialysis patients 16 table (1) distribution of studied groups according to mean age / year. ns * : non significant at p>0.05 b.distribution of patients and control according to gender. in the table (2) showed a no significant differences( p>0.05) between gender for patients and control this results agree with other studies (11-13) which found no differences in the incidence and/or rate of progression of renal disease between men and women. table (2) distribution of studied groups according to gender groups c.s. p-value patients control gender male count 49 46 % within gender 49% 46% f.e.p.t. p=1.000 ns ⃰⃰ female count 51 64 % within gender 51% 64% total count 100 100 % within gender 100 % 100% ns * : non significant at p>0.05 c.distribution of patients and control according to the mean concentration of urea , creatinine , uric acid ,rbs ,calcium and cholesterol. table (3) shows a highly significant increase (p<0.01) in the mean concentration of urea , creatinine , uric acid and rbs in the studied groups (25.57 mmol/l , 587.86 µmol/ l , 414.36 mmol/l and 6.47mmol/l) respectively compared with control (5.07 mmol/l, 88.60 µmol/ l ,270.76 mmol/l and4.65mmol/l ) respectively. also there was highly significant decrease(p< 0.01) in the mean of calcium and cholesterol for patients (1.93mmol/l and 3.33mmol/l) compared to the mean of healthy control (2.34mmol/l and 5.02mmol/l). elevated creatinine and urea levels are likely evidence of decreased kidney function (3, 14,15) . while increase in uric acid may be caused secondarily by renal impairment which that the urate handling by the kidneys involves filtration at the glomerulus, reabsorption, secretion and, finally, post-secretory reabsorption at tubules which are handled by multiple organic anion transporters that have been recently identified such as the urate/anion exchanger, the human organic anion transporter and the urate transporter. consequently, elevated serum uric acid levels—as observed in our study—may result secondary to decreased glomerular filtration, decreased tubular secretion or enhanced tubular reabsorption. decreased urate filtration can contribute to the increased in uric acid of renal insufficiency (16,17,18) . but elevated in rbs due to most of the patients have diabetes which cause damage to the kidneys, and this condition can lead to kidney failure (19,20) . decreased calcium in present study which found when the kidneys fail, they are unable to process and filter the blood from waste products, decreasing its ability to reabsorb calcium and leading to loss of calcium in the urine (21,22) . serum cholesterol is often low in dialysis patients, probably because of malnutrition and chronic inflammation (23,24) . parameters groups no. mean std. dev. std. error 95% c. i. for mean min. max. lower bound upper bound age control 100 51.34 8.73 1.24 48.86 53.82 37 76 patients 100 51.18 8.28 1.17 48.83 53.53 37 71 ⃰⃰t-test for equality of means t d.f. sig. (2-tailed) c.s. 0.094 98 0.925 ns ⃰⃰ file://kidney-disease/vocabulary/kidney-failure/e/5496 iraqi j pharm sci, vol.23(1) 2014 biochemical values in hemodialysis patients 17 table (3) distribution of studied groups according to the mean concentration of urea , creatinine ,uric acid ,rbs, calcium and cholesterol. parameters groups no. mean std. dev. std. error 95% c. i. for mean min. max . lower bound upper bound urea (mmol/l) control 100 5.07 1.45 0.20 4.66 5.48 3.1 7.5 patients 100 25.57 7.39 1.05 23.47 27.67 14 41 creatinine (µmol/ l) control 100 88.60 20.91 2.96 82.66 94.54 62 124 patients 100 587.8 6 233.4 0 33.01 521.53 654.19 212 113 2 uric acid (mmol/l) control 100 270.7 6 85.12 12.04 246.57 294.95 180 410 patients 100 414.3 6 96.28 13.62 387.00 441.72 204 564 r.b.s (mmol/l) control 100 4.65 0.94 0.13 4.38 4.92 3.1 6.1 study 100 6.47 3.16 0.45 5.57 7.37 1.4 14.7 ca (mmol/l) control 100 2.34 0.19 0.03 2.29 2.40 2 2.7 patients 100 1.93 0.33 0.05 1.83 2.02 1.3 2.7 cholesterol (mmol/l) control 100 5.02 0.96 0.14 4.75 5.29 3.2 6.47 patients 100 3.33 0.55 0.08 3.17 3.48 2.17 4.26 * hs highly significance references 1. pendse s, singh a, zawada e.et al. initiation of dialysis. in: handbook of dialysis. 4 th ed. new york, ny, 2008:14– 21. 2. iseki k and fukiyama k. predictors of stroke in patients receiving chronic hemodialysis. kidney int 1996, 50: 1672– 1675 3. wolfe ra, hulbert-shearon te, ashby vb, et al. improvements in dialysis patient mortality are associated with improvements in urea reduction ratio and hematocrit, 1999 to 2002. am j kidney dis 2005,45:127-135. 4. locatelli1 and p pozzoni1. chronic kidney disease in the elderly: is it really a premise for overwhelming renal failure? kidney international 2006, 69, 2118– 2120. 5. eknoyan g, beck gj, cheung ak, et al. effect of dialysis dose and membrane flux in maintenance hemodialysis. n. engl. j. med. 2002, 347 (25): 2010–9. 6. locatelli f, martin-malo a, hannedouche t, et al. effect of membrane permeability on survival of hemodialysis patients". j am soc nephrol,2009,20 (3): 645–54. 7. ahmad s, misra m, hoenich n, daugirdas j. hemodialysis apparatus. in: handbook of dialysis. 4th ed. new york, ny, 2008:59-78. 8. tietz n.w.;fundamentals of clinical chemistry , philadelphia, w.b. saunders 1976, 729. 9. sorlie de. medical biostatistics & epidemiology : examination & board review. first ed. norwalk, connecticut, appleton and lange ,1995:47-88. 10. p. jungers, p. chauveau, and b. descamps-latscha et al. age and genderrelated incidence of chronic renal failure in a french urban area: a prospective parameters t-test for equality of means t d.f. sig. 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http://ndt.oxfordjournals.org.tiger.sempertool.dk/search?author1=peter+c.+thomson&sortspec=date&submit=submit http://ndt.oxfordjournals.org.tiger.sempertool.dk/search?author1=keith+simpson&sortspec=date&submit=submit iraqi j pharm sci, vol.22(2) 2013 steroidal sapogenin in the leaves of yucca aloifolia 1 phytochemical study of steroidal sapogenin “tigogenin” present in the leaves of yucca aloifolia cultivated in iraq nabaa m.ibrahem *, 1 and zainab j. awad * *department of pharmacognosy, college of pharmacy, university of baghdad, baghdad, iraq. abstract this study detects the presence of the most important steroidal sapogenin “tigogenin” in the leaves of yucca aloifolia widely cultivated in iraq. the absence of any study concerning the tigogenin content of this medicinal plant in iraq, and the industrial importance of tigogenin depending on its role as a precursor in the synthesis of some steroidal drugs, acquired this study its value. this study concerned with extraction, identification, isolation, and purification of tigogenin from the leaves of yucca aloifolia. extraction of this compound was carried out using two methods. identification of this compound was done using thin layer chromatography (tlc) where different solvent systems had been tried. libermann – burchard reagent was used for detection. this identification was further augmented by using high performance liquid chromatography (hplc) and then this steroidal saponin was isolated and purified. the identification of isolated tigogenin was carried out using melting point (m.p.), thin layer chromatography (tlc), infrared spectroscopy (ir) and high performance liquid chromatography (hplc) . this study confirms the presence of tigogenin in the leaves of yucca aloifolia cultivated in iraq. also the result of this study showed that the second extraction method was the best, because the amount of both extract and tigogenin were higher than one extraction method. key words : yucca aloifolia , steroidal saponin , "tigogenin" حيائيت للستيرويد الصابوني التيكوجينين الموجود في اوراق نباث اليوكاأدراست كيمو yucca aloifoliaالمسروع في العراق نبأ محمد ابراهيم ،*1 زينب جليل عوادو * * ، بغذاد ، انعزاق.جايعت بغذاد ،كهٛت انظٛذنت ،انعمالٛز ٔانُباحاث انطبٛت فزع الخالصة ْذِ انذراست حخخض بانكشف عٍ ٔجٕد اْى يادة سخٛزٔٚذٚت طابَٕٛت يٕجٕدة فٙ أراق َباث انٕٛكا انًزرٔع فٙ انعزاق ْٔٙ نهظُاعاث انذٔائٛت يادة انخٛكٕجٍُٛٛ . اٌ عذو ٔجٕد ا٘ دراست فٙ انعزاق حخُأل يحخٕٖ ْذا انُباث يٍ يادة انخٛكٕجٍُٛٛ انًًٓت بكَٕٓا انًادة االبخذائٛت نخظُٛع بعض االدٔٚت انسخٛزٔٚذٚت انًًٓت يثم انكٕرحزٌٔ ٔانٕٓريَٕاث انجُسٛت ٚبٍٛ لًٛت ْذِ انذراست . فٙ ًادة ْذِ انذراست حى اسخخالص ٔكشف ٔفظم ٔحُمٛت يادة انخٛكٕجٍُٛٛ انًٕجٕدة فٙ أراق َباث انٕٛكا . حٛث حى اسخخالص ْذِ ان باسخخذاو طزٚمٍٛ نهفظم ٔحى انكشف عُٓا بٕساطت طزٚمت كزٔياحٕكزافٛا انطبمت انزلٛمت , باسخخذاو يذٚباث يخخهفت كٕسٛظ َالم بٛزجارد . ٔكذنك بٕساطت حمُٛت كزٔياحٕكزافٛا االداء انعانٙ انسائم ٔبعذْا حًج عًهٛت –ٔانكشف عُٓا باسخخذاو كاشف نٛبزياٌ ذ اسخخذيج يجًٕعت يٍ انخمُٛاث نهخحمٛك يٍ َٕعٛت انًزكب انًفظٕل ٔدرجت َمأحّ ٔانخٙ شًهج : درجت انفظم ٔانخُمٛت . ٔل االَظٓار , كزٔياحٕكزافٛا انطبمت انزلٛمت , يطٛاف االشعت ححج انحًزاء ٔكذنك حمُٛت كزٔياحٕكزافٛا االداء انعانٙ انسائم .اثبخج ْذِ اق َباث انٕٛكا انًزرٔع فٙ انعزاق . كًا اظٓزث ْذِ انذراست باٌ انطزٚمت انثاَٛت انذراست ٔجٕد يادة انخٛكٕجٍُٛٛ فٙ أر سخخالص . نكٌٕ كًٛت انًسخخهض ٔيحخٕاِ يٍ ْذِ انًادة كاٌ اعهٗ يًااعطخّ انطزٚمت االٔنٗ نال ضمنالسخخالص ْٙ االف .التيكوجينينمادة ، صابونيتالموادالستيرويديتال،نباث اليوكاالكلماث المفتاحيت : introduction yucca aloifolia is a shrub or tree – like plant of the agavaceae family (1) , native to the deserts of the south – western united states and northern mexico (2) (figure 1). this plant contains several physiologically active phytochemicals. it is a rich source of steroidal sapogenins like sarsasapogenin and tigogenin and is used commercially as a steroidal saponin source (3) . these sapogenins are important starting materials for the semi synthesis of steroidal drugs especially the cortisone compound and steroidal hormones (4) . the saponins from yucca are the main medicinal agents in the plant that elevate the body’s production of cortisone possibly the herb’s ability to aid in arthritic pains (5) .saponins also provide anti – inflammatory relief as well as the ability to break up inorganic mineral obstructions and deposis (6) . 1 corresponding author e-mail:nabaamohammed14@yahoo.com received:12/5/2012 accepted:19/5/2013 iraqi j pharm sci, vol.22(2) 2013 steroidal sapogenin in the leaves of yucca aloifolia 2 yucca also has laxative properties and is also used to establish a flora balance in the gi tract. it is also speculated that yucca saponin block release of toxin from the intestines, which inhibit normal formation of cartilage (7) . both leaves and roots, function well as diuretic and emetics and has hair strengthening (8) . material and methods plant materials the plant materials (leaves) of yucca aloifolia l. were collected from private garden during the months of september and october (2010), they were cleaned and dried in oven at a temperature between (30 – 40) c for (4 – 5) hours then these plant materials were coarsely powdered by mechanical grinder and weighed. a 50 gm of dried powdered plant materials were extracted by using two methods. extraction extraction method no 1 (9) ; a 50 gm of dried powdered plant materials (leaves) were extracted in a soxhlet apparatus with 500ml dichloroethane for 20 – 24 hours. after that the plant material was dried , then reflexed with 500ml of 4n h2so4 for 3 hours. after cooling the mixture was filtered, the residue on the filter was washed with water, neutralized with 250ml 5% sodium bicarbonate solution to ph 7.5 and dried at 100 – 105c°. the dry hydrolysate ( the plant material ) was extracted with petroleum ether (b.p 60 – 80c°) in soxhlet apparatus for 10 hours. the extract was condensed to small volume under vacuum and then subjected to identification (figure 2). figure (1):yucca alifolia plant figure (2): general scheme for method no.1 for extraction of steroidal sapogenin tigogenin from the leaves of yucca aloifolia. iraqi j pharm sci, vol.22(2) 2013 steroidal sapogenin in the leaves of yucca aloifolia 3 extraction method no.2 (10) : a 50gm of dried plant material (leaves) was soaked in water for 24 hours and then extracted with 80% ethanol (500ml) in soxhlet extractor for 10 hours. then the residue was evaporated to dryness under vacuum. the dried residue was reflexed with 2n h2so4 in water containing 70% 2 – propanol (500ml) at 100c° for 4 hours. the mixture was cooled and evaporated under vacuum to remove the alcohol completely, and then the mixture was neutralized with 5%nh3 then partitioned with equal volume of petroleum ether (b.p 60 – 80°c) using sepratary funnel to give two layers, aqueous layer and the petroleum ether layer. the petroleum ether layer was taken and evaporated to dryness under vacuum and then subjected to identification (figure 3). figure (3): general scheme for method no.2 for extraction of steroidal “sapogenin tigogenin” from the leaves of yucca aloifolia. identification of the steroidal sapogenin “tigogenin”: c.1: thin layer chromatography (tlc): in this qualitative identification:using a ready-made aluminum plates of silica gel gf254, one detection method by using libermann – burchard reagen (11) in comparison with three different developing solvent systems that were (12-13) : solvent 1 (s1): chloroform:methanol (95:5) solvent 2 (s2): chloroform:petroleum ether: methanol (85:10:5) solvent 3 (s3): chloroform:acetone (80:20) c.2: high performance liquid chromatography (hplc): qualitative and quantitative estimations of tigogenin component in the crude extract obtained by extraction methods was carried out by using high performance liquid chromatography (hplc). the identifications were made by comparism of retention time of tigogenin component in the crud extracts with that of authentic standard at identical chromatographic conditions. hplc analysis was done by using the following conditions: 1) mobile phase: acetonitrile 100% 2) column: phenomenex ods 250mmx4.6mm, 5µm particle size. 3) column temperature: ambient 4) flow rate: 1ml/min 5) injection volume: 5µl 6) injection concentration: 1mg/ml detection: uv detector at λ 209nm. isolation and purification of tigogenin: isolation and purification of tigogenin was done by using the following steps: 1: fractionation by column chromatography: the final residue obtained from extraction method no2 (best method) was subjected to column chromatography using glass column (80cm x 5cm) packed with silica gel (0.063 – 0.200 mm) slurry in (250 ml) chcl3, in a ratio of 20gm of silica gel to each 1gm of the residue. a dry loading of the sample (residue) was used by dissolving it in small volume of iraqi j pharm sci, vol.22(2) 2013 steroidal sapogenin in the leaves of yucca aloifolia 4 chloroform and adsorbing it on small amount of silica gel of the same grade used for packing the column, then dried, grinded and applied to the top of column in order to prevent clogging. the column was eluted by gradient elution technique using chcl3: methanol with an increasing percentage of methanol from zero to 100% (the ratios of chcl3: methanol used were 100:0, 95: 5, 90:10, 85:15, 80:20, 70:30, 60:40, 50:50, 40:60, 25:75 and chcl3: methanol 0:100). the column developed by adding 50 ml of each eluent with collecting 5ml fractions, then monitored by tlc with solvent system (s1): chloroform: methanol (95:5). a total number of 100 fractions were obtained. those consecutive fractions, which have the same number of spots with the same rf values, were combined and concentrated to dryness to get major fractions. after that, the major fractions were subjected to thin layer chromatography with tigogenin reference standard and solvent system s1 chloroform : methanol (95:5). the results showed that the tigogenin compound was found in the major fraction no.5. 2: using preparative tlc plates: isolation of tigogenin compound is carried out using preparative tlc which was performed by using readymade plates of silica gel gf254 ( 20 x 20cm ) of 1mm thickness (merck). the major fraction (f 5) residue obtained from column chromatography applied as a concentrated solution in a row of spots using capillary tube four times on each plate (the spots should dry before the next application). the solvent system used was s1 (chloroform : methanol (95:5)) . the detection was done using libermann – burchard reagent in one side of the plate . the band corresponding to the tigogenin standared was scraped out and collected in a beaker, mixed with chloroform: methanol (95:5), stirred and left a side for one hour, then filtered. after evaporation of the solvent, the final filtrate gives yellowish precipitate . 3.purification of tigogenin by using charcoal material: the final solid product that has been isolated by preparative tlc was dissolved by heating insufficient quantity of methanol and a small amount of decolorizing charcoal was added, with stirring to the hot methanol solution until the supernatant liquid was almost colorless. then the hot solution was poured through filter paper into another flask. then the solvent was evaporated to give solid product (14) . identification and characterization of the isolated steroidal sapogenin tigogenin: 1. tlc: analytical tlc was performed by using ready made plate. the purified sapogenin tigogenin was applied on silica gel plate as one spot by using capillary tube along with its standard, using the solvent system (s1) chlorform : methanol (95;5) . the detection was done by using liebermann – burchared reagent . 2. melting point: the melting point of the purified sapogenin tigogenin was done and compared with that of the available standard tigogenin. 3. ftir:infrared spectra were carried out by using kbr disc for both purified sapogenin tigogenin and its standard. 4. hplc analysis: hplc analysis was made by comparism of retention times obtained at identical chromatographic conditions of analyzed purified sapogenin tigogenin and its standared. hplc conditions as mentioned previously results and discussion: extraction methods: two methods of extraction of steroidal sapogenin tigogenin were tried to select the best one. results showed that the method no.2 was better, because the yield of crude extract was higher than obtained from method no.1. in addition quantitative estimation by using hplc analysis showed that the amount of tigogenin obtained by method no.2 was much more compared with that obtained by method no.1 as showed in table (1). so, we select method no. 2 as an extraction procedure in our work. table (1): quantitative of crud extracts and tigogenin obtained from extraction methods. identification of tigogenin by tlc: tlc of the crud extracts obtained from the leaves of yucca aloifolia by using the extraction method no.1 and no.2, confirms the presence of tigogenin in these extracts in comparison with tigogenin standard . as presented in table (2) .and figures ( 4 ,5 and 6). extraction methods crud extract (mg) tigogenin % method no.1 2.9 3.86 method no. 2 3.5 5 iraqi j pharm sci, vol.22(2) 2013 steroidal sapogenin in the leaves of yucca aloifolia 5 table(2): rf values of tigogenin from yucca aloifolia leaves extract obtained by extraction methods no.1 and no.2 and its standard in different developing solvent systems in tlc solvent system s1 s2 s3 rf value of standard tigogenin 0.55 0.74 0.80 rf value of tigogenin in crud extract of method no1 0.54 0.75 0.80 rf value of tigogenin in crud extract of method no2 0.56 0.78 0.81 s1: chloroform:methanol (95:5) s2: chloroform:petroleum ether: methanol (85:10:5) s3: chloroform:acetone (80:20) t e1 e2 figure(4): tlc for yucca aloifolia leaves extract obtained by extraction methods no.1 and no.2 using silica gel gf 254 as adsorbent and (s1) as solvent system . visualization by liebermann-burcrhard spray reagent. t: tigogenin standard e1: extraction method no.1 e2: extraction method no. 2 t e1 e2 figure (5) : tlc for yucca aloifolia leaves extract obtained by extraction methods no.1 and no.2 using silica gel gf 254as adsorbent and (s2) as solvent system . visualization by liebermann-burcrhard spray reagent. t: tigogenin standard e1: extraction method no.1 e1: extraction method no. 2 t e1 e2 figure (6): tlc for yucca aloifolia leaves extract obtained by extraction method no.1 and no.2 using silica gel g 254 as adsorbent and (s3) as solvent system . visualization by liebermann-burcrhard spray reagent. t: tigogenin standard e1: extraction method no.1 e2: extraction method no.2 iraqi j pharm sci, vol.22(2) 2013 steroidal sapogenin in the leaves of yucca aloifolia 6 isolation and purification of tigogenin; one hundred fraction obtained from column chromatography were monitored by tlc. the consecutive fractions that have the same number of spots of the same rfvalues were combined to get 8 major fractions , which were concentrated to dryness and supjected to identification , as listed in table ( 3) . in the first 70 factions , fraction 1-10 gave one spot in tlc and were collected to give fraction one (f1) . fraction (11-20) gave one spot were collected to give fraction two (f2) , while fraction 21-30 gave 0ne spot and were collected to give faction three (f3), fraction 31-40 gave one spot and were collected to give the fraction four(f4) . fraction 41-52 gave two spots and were collected to give the major fraction five (f5) . fraction 5354 gave one spot and were collected to give the fraction six (f6 ) . fraction 55-57 gave two spots and were collected to give the fraction seven (f7). fraction 58-70 gave the faction eight (f8). in the last 30 factions no spots were appear in tlc examination. the results showed that the tigogenin is found in the fraction five (f5) therefore we selected this fraction to isolation thiscompound. table (3): major fractions obtained from column chromatography identification and characterization of the isolated tigogenin 1. analytical tlc isolated compound ( tigognin) appeared as a single spot having the rf value as that of reference standard . 2. measuring melting point the isolated tigogenin was identified from its sharp melting point of 200 202 ºc compared to standard tigogenin melting point 202-204 ºc . 3. ftir the ir spectra of isolated tigogenin was gave identical results with that of tigogenin standard, as showed in table (4) and figures(7 and 8 ) . table (4): the characteristic ir absorption bands (in cm-1) of the isolated tigogenin in comparison with that of tigogenin as reference standard (120) major fractions no. of collections 5 ml each no. of spots f1 1-10 1 f2 11-20 1 f3 21-30 1 f4 31-40 1 f5 41-52 2 f6 54-53 1 f7 55-57 2 f8 58-70 4 f9 70-100 negative functional group isolated tigogenin tigogenin standard assignment free o-h 3524 3522 free o-h stretching of alcohol o-h broad band (3390-3263) broad band (3389-3269) broad o-h stretching band indicate hydrogen bonding c–h 2929,2848 2941,2874 asymmetric and symmetric stretching of ch3 and ch2 groups c-h 1456,1373 1456,1373 c-h bending of ch2 and ch3 c-o 1242-1049 1242-1049 c-o stretching of aliphatic ether iraqi j pharm sci, vol.22(2) 2013 steroidal sapogenin in the leaves of yucca aloifolia 7 figure (7): ir spectrum of tigogenin standard figure (8): ir spectrum of isolated and purified tigogenin 4. hplc analysis the retention time for the isolated tigogenin was identical to the main peak of the standard reference as showed in figures ( 9 and 10 ) . figure (9):hplc analysis of isolated and purified tigogenin. iraqi j pharm sci, vol.22(2) 2013 steroidal sapogenin in the leaves of yucca aloifolia 8 figure (10) :hplc analysis of tigogenin standard. conclusions: phytochemical investigation of yucca aloifolia leaves, cultivated in iraq revealed the presence of important medicinal natural product “tigogenin” belong to steroidal saponin . tigogenin was extracted by using two extraction methods, and identified by using tlc and hplc method . isolation and purification tigogenin compound was made by using the following steps: fractionation by column chromatography, preparative tlc plates, and purification by using charcoal material. the identification of isolated tigogenin was carried out using melting point , thin layer chromatography , infrared spectroscopy and hplc. references 1. irish, gary . agaves ,yuccas .and related plants : agardener’s guide .timber press. 2000; .p.1 2. strecker’s giantskipper megathymus streckeri ( skinner , 1895).butterflies and moths of north america. 3. dewidar, am. ; elmunajjed , d. the steroid sapogenin constituents of agave americana , a. variegate and yucca aloifolia . planta med. 1970; 19 (1) : 87-91. 4. dewik , pm. : medicinal natural products , a biosynthetic approach (1 st ed) . chapter five , the mevalonate pathway . john wiley and sons ltd , chi chester , reprinted 1997 ; pp . 218-265. 5. bennett , cc. public information memo . new york : the arthritis founndation , feb 22, 1977. 6. cheek , pr. ; piacent , s. ; oleszek , w. antiinflmmatory and antiarthritic effects of yucca aloifolia : areview . j inflamm (lond) . 2006 ; 3: 6-13. 7. woese , c.; kandler , o. ; wheelis m. towards a natural system of organism : proposal for the domains archaea , bacteria and eucarya . proc natl acad sci usa . 1990; 87 (12) : 4576-4579. 8. woese, cr .; fox , ge.phylogenetic structure of the prokaryotic domain: the primary kingdoms . proc . natl. acad. sci. u.s.a. november 1977; 74 (11) : 5088 5090. 9. wang , j. ; chen , j.; yang, kd.and et al . study on the production condition of extraction in combination hydrolysis in situ for isolation diosgenin . zhongguo zhong yao zazhi . 2003; 28 (10) : 934-937. 10. kemertelidze , ep.; pkheidze, ta.: tigogenin from yucca glriosa apossible raw material for the synthesis of steroid hormonal preparation . phrmaceutical chemistry journal ,1972;6(12): 795 -797. 11. wagner, h.; bladt, s. and zgainski , em.: plant drug analysis, a thin layer chromatography atlas . springer – verlag , berlin , germany .1984; p.226. 12. harborene, jb.; phytochemical methods ( 1 st ed.). chapter two, phenolic compounds . chapman and hall , london , 1976; pp, 115 117pp. 13. waksmundzka – hajnos , m.; sherma, j.; kowalsk,t.: tlc of triterpenes ( including saponin). thin layer chromatography in phytochemistry (1 st ed) , crc press, taylor and francis group. usa . 2008; pp.528. 14. shriner, rl.; hermann, ckf.; morrill, tc.: preliminary examination , physical properties and elemental analysis , the systematic identificationof organic compounds (86 th ed). john wiley and sons inc., usa. 2004;pp:51-52. iraqi j pharm sci, vol.23(1) 2014 antimicrobial evaluation of new derivatives of coumarin 35 synthesis and antimicrobial evaluation of new 6 and7 substituted derivatives of coumarin rouaa a. muften * , kawkab y.saour ** and ammar a.razzak mahmood **,1 * ibn-zuhr hospital, ministry of health. baghdad, iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad baghdad,iraq. abstract a series of benzohydrazide derivatives attached to coumarin moiety at position 6 and 7 have been synthesized. the reaction of coumarin derivatives (coumarin i and ii) with p-nitrophenyl hydrazine yield schiff bases (compound1a and iia).these schiff bases were refluxed with benzoyl chloride to give benzohydrazide derivatives of coumarin substituted at its 6 or 7 nucleus position (ia1 and iia1).the reaction and the purity of the products were checked by thin layer chromatography (tlc). the structures of the final compounds and their intermediates were confirmed by their melting points, infra red spectroscopy, and elemental microanalysis(chn). compounds (ia1 and iia1) were evaluated for their preliminary antibacterial and antifungal activities. compound (iia1) has good antibacterial activity against staphylococcus aureus other than bacterial species, and was equivalent to ofloxacin as (standard drug). key word: coumarin, schiff base, benzohydrazide derivatives, antimicrobial activity. كمضاداخ 7و6تحضير والتقييم الثايولوجي لمشتقاخ الكومارين المعوضح في الموقعين للمايكروتاخ ،رؤى علي مفتن * كوكة يعقوب ساعور ** وعمارعثذ الرزاق محمود **،1 * . انعراق، بغذاد ،َزارة انصحت ،مسخشفى ابه زٌر ** . انعراق،بغذاد، جبمعت بغذاد ،كهيت انصيذنت، ميبء انصيذالويتيفرع انك الخالصح . ان حفبعم مشخمبث انكُمبريه )مركب 6َ7حم حخهيك سهسهت مه مركببث انبىسٌَبيذرازايذ انمخصهت بىُاة انكُمبريه في انمُلعيه ( (i ( َii( مع )p-nitrophenyl hydrazine اعطج لُاعذ شف, مركب )((1a(َ )iia نمذ اسخعمهج ٌذي انمُاعذ مع كهُريذ.)) (. نمذ جرث مرالبت جميع iia1 ,ia1) 6َ7انبىسَيم نيعطي مشخمبث انبىسٌَبيذرازايذ نهكُمبريه انمعُض عهى وُاحً في انمُلعيه ركببث انُسطيت َانمركببث انىٍبئيت انخفبعالث َانخبكذ مه ومبَة انمركببث بُاسطت كرَمُحغرافيب انطبمت انرليمت, كمب حم مخببعت سير انم َاثببث ٌُيخٍب مه خالل ليبش درجبث االوصٍبر َانخحهيم انطيفي نالشعت ححج انحمراء, َانخحهيم انذليك نهعىبصر.نمذ حم حمييم انفعبنيت staohylococcus( يمخهك فعبنيت جيذة ضذ (iia1( ضذ انجراثيم َانفطريبث,حيث َجذ ان انمركب iia1,ia1انمبذئيت نهمركبيه) aureus .دَن غيرٌب مه اصىبف انبكخريب َكبن مكبفئب نفعبيت االَفهُكسبسيه كذَاء ليبسي جراثيم .لل المضادجالفعاليح ، الكومارين، قواعذ شف ، مشتقاخ الثنسوهايذرازايذ الكلماخ المفتاحيح: introduction coumarins (2h-1benzopyran-2ones) are important oxygen containing fused heterocycles used in drugs and dyes. coumarins bound their class name to "coumarou" the vernacular name of tonaka bean, from which coumarin itself was isolated in 1820.coumarin and its derivatives represent one of the most active classes of compounds possessing a wide spectrum of biological activity (1-3) .novobiocin and chlorobiocin are established antimicrobials containing a coumarin skeleton (4) .many of these compounds have been proven to be active as,antibacterial (5-7) , antifungal (8) ,anti-inflamatory (9) ,anticoagulant (10) and anti tumor agents (11) . azomethine group (c=n) containing compounds typically known as schiff bases have been synthesized by condensation of primary amines with active carbonyl form. schiff bases from a significant class of compounds in medicinal and pharmaceutical chemistry with several biological applications that induce antibacterial and antifungal activities (12-16) . it's known that schiff bases react smoothly with acid chloride to give the corresponding addition products (17-21) . experimental section: 1. chemicals: the specific chemicals used in this work are supplied from himedia, fluka and sigma companies.all the solvents were of annular grade and used without further purification. 1 corresponding author e. mail: kubbaammar1963@gmail.com received:13 /4/2013 accepted:5 /2/2014 iraqi j pharm sci, vol.23(1) 2014 antimicrobial evaluation of new derivatives of coumarin 36 2. detection equipments: melting points were determined by capillary tube method on banested (uk). the ftir spectra were recorded on ftir spectrophotometer, shimadzu (japan) at the college of science(al-mustansaryia university).the chn microanalysis were carried out using euro a(elemental analyzer), (italy) performed in the college of science-almustansaryia university. thin layer chromatography (tlc) was run on silica gel gf254 (60), merck (germany) to check both purity of compounds and their reactions progress. the antimicrobial study of the synthesized final products was done in alkindy college of medicine-university of baghdad. synthesis: a. synthesis of 4-methyl -2 – oxo -2hchromene-6-carbaldehyde (compound i): methyl acetoacetate (0.28 mole, 30ml)was mixed with re-distilled trimethyl orthoformate (0.27mole,30 ml) in dry methanol (25)ml and then conc. hcl (0.3ml) was added. the mixture was distilled immediately to obtain methyl-β-methoxy crotonate. later polyphosphoric acid 100gm was added to a mixture of p-hydroxy benzaldehyde (0.05mole ,6.2 gm) and methylβmethoxy – crotonate (0.055 mole, 8 gm).the mixture was stirred for 90 minutes on steam bath at 80-85 °c and then poured into ice and water. the solid product was collected, washed and dried. re-crystallization was from ethanol (22) . percent yield, physical appearance, m.p., rf values are listed in table (1), while the ir characteristic absorption bands are listed in table (2). b. synthesis of 4methyl -6 ( 2 ( 4nitrophenyl) hydrazano) methyl) -2hchromene-2-one (compound ia) equimoles of compound (i) (0.01mole ,1.88gm) and p-nitrophenyl hydrazine (0.01mole,1.53gm) were dissolved in 25 ml of methanol, to the resulting mixture 4-5 drops of piperidine was added and the mixture was refluxed for 10 hours. after completing the reaction, the mixture was poured into crushed ice. the product obtained was filtered and purified from methanol (23) . percent yield, physical appearance, m.p., and rf values are listed in table (1) and the ir characteristic absorption bands are listed in table (2) and the elemental micro analysis data are listed in table (3). c. synthesis of n(chloro (4methyl -2 oxo-2hchromene 6 -yl) methyl)-n'-(4nitrophenyl) benzohydrazide (compound ia1). benzoyl chloride (0.01 mole, 1,2ml) was added drop wise to a solution of equimolar quantity of compound (ia), (0,01 mole,3.23gm),dissolved in 1,4 dioxane(15 ml) and the mixture was refluxed for 6-8 hrs. later the mixture was poured into ice with stirring. the product was filtered and purified from ethanol (24) , percent yield, physical appearance, m.p.,and rf values are listed in table (1),ir characteristic absorption bands are listed in table (2) and the elemental microanalysis data are listed in table (3). d. synthesis of 4-methyl-2-oxo-2hchromene-7-carbaldehyd (compound ii). a flask containing (45) ml conc. sulfuric acid was immersed in an ice bath and the temperature was kept below 10°c. a solution of (0,1 mole,12.2 gm) of 3-hydroxy benzaldehyde in (0.1 mole, 1.2 ml) of methyl acetoacetate was then added, and the temperature should be kept below 10°c during the time of addition. later the reaction mixture was kept at r.t for 48 hrs. after which, it was poured into a flask containing a mixture of ice and water, and the formed solid product was filtered off, washed with water, and recrystallized from ethanol (25) . percent yield, physical appearance, m.p., and rf values are listed in table(1), while the ir characteristic absorption bands are listed in table (2). e. synthesis of 4-methyl-7-((2-(4-nitrophenyl) hydrazono) methyl)-2h-chromen-2-one (compound iia). to a solution of compound (ii), (0.01 mole,1.88gm) in absolute ethanol (25 ml), an equimolecular amount of p-nitropheny hydrazine was added(0.01 mole,1.53 gm), to the above solution and a catalytic amount of piperidine (4-5) drops. the reaction mixture was heated under reflux for 10 hrs. it was then cooled at r.t, poured into crushed ice, filtered and purified from methanol (26) . percent yield, physical appearance, m.p., and rf values are listed in table (1), while the ir characteristic absorption bands are listed in table (2). f. synthesis of n-(chloro(4-methyl-2-oxo2h-chromene-7-yl)methyl)-n'-(4-nitro phenyl) benzohydrazide ( compound ii a1): compound (iia), (0.01 mole,3.23 gm) was dissolved in 1,4-dioxane (15 ml) and to which (0.01 mole,1.2 ml) of benzoyl chloride was added dropwise, and the mixture was refluxed for 6-8 hrs. afterwhich, the mixture was poured into ice with stirring. the product iraqi j pharm sci, vol.23(1) 2014 antimicrobial evaluation of new derivatives of coumarin 37 obtained was filtered and purified from ethanol (24) . percent yield, physical appearance ,m.p., and the rf values are listed in table (2).the elemental microanalysis data are listed in table (3). table (1) the physical appearance, percent yield, melting points and rf values of the synthesized compounds and their intermediates. solvent system rf value observed melting point °c yield % physical appearance compd. ethanol 8: benzene 2 0.8 185-188 63% dark brown crystal i petroleum ether(4060°c) 5: ethyl acetate 5 0.88 207-210 77% light brown crystal ia ethanol 8: benzene 2 0.84 110-112 89% light walnut powder ia1 petroleum ether(4060°c) 5: ethyl acetate 5 0.89 194-196 58% dark brown crystal ii ethanol 8: benzene 2 0.86 203-205 77% 93 light brown powde iia ethanol 8: benzene 2 0.83 115-118 light walnut powder iia1 table (2) the ir characteristic absorption bands of the synthesized compounds ir characteristic absorption bands v` (cm-1) compd. (1724) cm -1 for c=o of pyran ring and (1710)cm -1 for c=o stretching of aldehyde group i (1600) cm -1 for c=n stretching, (3400)cm -1 for n-h stretching and (1446) cm -1 and (1370) cm -1 for no2 stretching ia (1687) cm -1 c=o of amide group and (781) cm -1 for c-cl ia1 (1707)cm -1 for c=o of pyran ring and (1695)cm -1 for c=o stretching of aldehyde group ii (1579) cm -1 for c=n stretching,(3392)cm -1 for nh-stretching and (1516) cm -1 and (1385) cm -1 for no2 stretching iia 1687) cm -1 for c=o of amide group and (808) cm -1 for c-cl) iia1 table (3) the elemental microanalysis of the products ia1 and iiai antimicrobial activity a preliminary antibacterial and antifungal activity have been carried out according to well diffusion method. the prepared compounds have been studied for their antimicrobial activity in vitro against three tested bacteria ( staphylcoccus aureus, streptococcus spp., as gram positive bacteria and proteus spp., as gram negative bacteria, and fungi (candida spp.), were clinically activated and maintained on nutrient agar medium for testing antibacterial activity and sabouraud dextreose agar medium for antifungal activity. ofloxacin was used a standard drug for antifungal activity. the plates were incubated at 30°c for 72 hrs., for m.wt. n% h% c% value type compd. 463.5 9.07 9.11 3.89 3.84 62.1 59.3 calculated observed ia1 463.5 9.07 9.34 3.89 3.87 62.1 59.72 calculated observed iia1 iraqi j pharm sci, vol.23(1) 2014 antimicrobial evaluation of new derivatives of coumarin 38 the (fungal spp.), or 37°c for 24 hrs., for the bacterial spp.) (27) . the antimicrobial activity was evaluated by measuring the diameter of inhibition zone around the disk in (mm), as shown in table (4) and (5) respectively. table ( 4) the antibacterial activity of the tested compounds. zone of inhibition in mm. zone of inhibition in mm zone of inhibition in mm concentration µg/ml compd. proteus spp. streptococcus spp. staphylococcus aureus spp. no activity no activity no activity 2 4 8 1 5 9 3 µg/ml 25 µg/ml 60 µg/ml ia1 no activity no activity no activity 2 6 12 4 8 14 3 µg/ml 25 µg/ml 60 µg/ml iia1 6 11 17 7 13 18 8 10 16 3 µg/ml 26 µg/ml 60 µg/ml ofloxacin table (5) the antifungal activity of tested compounds zone of inhibition in mm candida spp. concentration µg/ml compd. 7 3 µg/ml ia1 9 25 µg/ml 16 60 µg/ml 6 3 µg/ml iia1 4 25 µg/ml 12 60 µg/ml 11 3 ketoconazole 27 25 30 60 results and discussion the present investigation involves the synthesis of new series of compounds involving the coumarin moiety as shown in scheme 1 and scheme 2; the pathway of synthesis of coumarin derivatives that have a characteristic aldehyde group substituted at the 6 or 7 positions of the coumarin nucleus as shown in compound (i) and (ii). schiff bases (1a,iia) were prepared by condensation of compound (i) and (ii) with pnitrophenyl hydrazine respectively using piperidine as catalyst; piperidine will increase the nucleophilicty of the amine group, and the reaction when followed by ir, showed the appearance of a characteristic absorption bands for (c=n) and (n-h), and disappearance of the band for (c=o) of aldehyde group as shown in table 2. the reactions between benzoyl chloride and schiff bases are type from addition to the (c=n) moiety, and the reactions when followed by ir; illustrated the disappearance of (c=n) band, and the appearance of (c=o) of amide band and the appearance of (c-cl) band as shown in table 2. the antimicrobial activities ( antibacterial and antifungal activities) have been carried out according to well diffusion methodology using ofloxacin and ketonazole as a standards respectively. all the results are fixed in table 4 and table 5. iraqi j pharm sci, vol.23(1) 2014 antimicrobial evaluation of new derivatives of coumarin 39 scheme 1: pathway of synthesis of compound (ia1) iraqi j pharm sci, vol.23(1) 2014 antimicrobial evaluation of new derivatives of coumarin 40 scheme 2: pathway of synthesis of compound (iia1) references 1. h.zuo, g.jose,z boli, b hynumoon, d soo shin, and m ghate, microwaveassisted synthesis of fluorinated coumarino sulfonamide, arkivok 2008,2,193-189 2. s lee; 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17:221233. 9. garazad, mm,muzychka,o.v,vovk, a.i, i.v., ogorodnichuk , a.s., modified coumarins 27. synthesis and antioxidant activity of 3substiuted 5,7dihydroxy-4methyl coumarins. ,chem. of nat. comp. ,2007;43,(1):19-23. 10. smitha, and r sanjeeva, zrcl4-catalyzed pechman reaction: synthesis of coumarins under solvent free conditions., synthetic comunications,2004;34(21): 3997-4003. 11. hamadi, c lidrissi, m saoud and ar nievas , h zarrouk, synthesis of some biologically coumarin derivatives, chem. hetero. comp. 2006; 42 (3): 320-325 12. abu-hussen, a. a. a., j.coord.chem., 2006;59, 157. 13. singh,k., barwa ,m.s .,tyagi, p., synthesis, charecterization and biological studies of co(ii),ni(ii),cu(ii) and zn(ii) complexes with bi-denntate schiff base derived by heterocyclic ketone , eur.j. med. chem., 2006;41(1),147-153 14. yang,w.,ruan, z., wang, y., van kirk, k., ma,z., arey, b.j., cooper,c.b., seehala,r., feyen, j.h.m and dickson, j.k., discovery and structure activity relationships of tri-substituted pyrimidines /pyridines as novel calcium channel sensing receptor antagonists, j.med .chem. 2009;52:1204. 15. mladenova, r.; 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(1999). 19. w.qin,sha long, mauro panunzio, schiff bases: a short survey on an evergreen chemistry tool, molecules,2013;18:1226412289 20. patai s.," the chemistry of the carbonnitrogen double bond", john wiely and sons,new york,1970,68. 21. scholz m., schumke a., and m.g. numchstolt, j.chem., 1962;2: 309 22. e.brand and m.sand berg, org.synth.,coll., 1943;2:49. 23. 23.za sizova; aa karasev; ll lukatskaya; mi rubtsov; ao dosohenko, theoretical and experimental chemistry, 2002; 38(3): 168-172. 24. hansa parekh; ashutosh joshi and ketan hirpani, indian. j. heterocycyclic. chem.,2004;13:221-224 25. 25.ahluwalia v.k., bhagat p., agarwal r., chandra r., intermediates for organic synthesis, i.k international, new delhi, 2005 p.276. 26. wadher s.j., karande n.a., borkar d,s, yeole p.g., synthesis and biological evaluation of schiff bases as antimicrobial agents, international j.chem.tech. research., 2009;1(4): 1297-1302. 27. kuete v.,tangmouo. j.g., penlap. v., mofo.f., lontsi.d: antimicrobial activity of tridesmostemon omphalocarpoides, j. ethnopharmacology.2006;104:5-11. iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 33 isolation and characterization of triterpenoid saponin hederacoside c. present in the leaves of hedera helix l. cultivated in iraq suhaib a. hussien *,1 and zainab j. awad ** *tallʼafar general hospital,nineva health directorate, ministry of health, tallʼafar, iraq. **department of pharmacognosy, college of pharmacy, university of baghdad, baghdad, iraq. abstract hedera helix l. plant belongs to the family araliaceae that provide a host of bioactive compounds (mainly saponins) of important biological activities, like spasmolytic, secretolytic, anti-inflammatory, and antibacterial activities. literature survey revealed that there was no previously study concerning h. helix l. which is cultivated in iraq, so we decided to carry out this study which include extraction, isolation, purification and identification of biologically important triterpenoid saponin hederacoside c from leaves of h. helix l. extraction of hederacoside c was carried out using two methods; in the first method maceration was done with methanol 99.8% and in the second method soxhlet extraction with ethanol 99.8%, was followed, then fractionation using column chromatography. preliminary identification of this saponin hederacoside c was done using thin layer chromatography (tlc) where different solvent systems had been tried. liebermann-burchard reagent where used for detection. the most suitable extraction method was fully described in this study. the characterization of the isolated hederacoside c was carried out using melting point (m.p.), thin layer chromatography (tlc), ft-ir, and high performance liquid chromatography (hplc). keywords: hedera helix l., hederacoside c, column chromatography. فصل وتىصيف الترايثنىيد الصاتىني )الهيدراكىسايد سي( المىجىد في اوراق نثات اللثالب .المستسرع في العراقالكثير صهية عسيس حسين *،1 و زينة جليل عىاد ** انؼشاق. ،ذهؼفش ،وصاسج انصحح ،دائشج صحح نُنىي ،*يسرشفً ذهؼفش انؼاو انؼشاق. ،تغذاد ،جايؼح تغذاد ،كهُح انصُذنح ، **فشع انؼمالُش واننثاذاخ انطثُح الخالصة: ( وهى َحرىٌ ػهً انكثُش ين انًشكثاخ انكًُُائُح و تشكم سئُسٍ انكالَكىسُذاخ araliaceaeَؼذ نثاخ انهثالب انكثُش ين ػائهح ) يضاد نالنرهاتاخ. و ( راخ انفؼانُح االحُائُح كًضاد نهرمهصاخtriterpenoid saponin glycosidesانصاتىنُح ثالثُح انرشاَثنىَذ ) شاق, نزا اصثح ين االهًُح دساسح هزا اننثاخ انًسرضسع فٍ انؼشاق. فٍ هزه ونظشاً نؼذو وجىد دساساخ حىل نثاخ انهثالب انكثُش فٍ انؼ ( ين اوساق نثاخ انهثالب انكثُش hederacoside cانذساسح ذى اسرخالص وكشف وفصم وذنمُح انكالَكىسُذ انصاتىنٍ انرشاَثنىَذ ) (h. helix l.حُث ذى االسرخالص تاسرخذاو ) ٍوانطشَمح انثانُح %99.8 طشَمرُن: انطشَمح االونً تاسرخذاو اننمُغ فٍ انكحىل انًثُه column) %,وين ثى انرجضئح تاسرخذاو كشوياذىكشافُا انؼًىد9...تاسرخذاو جهاص انسىكسهُد ويزَة انكحىل االثُهٍ chromatography.) ( ٍذى انكشف ػن انكالَكىسُذ انصاتىنhederacoside cتأس ) ,رخذاو ذمنُح كشوياذىغشالُا انطثمح انشلُمح (, وتؼذها ذًد ػًهُح انفصم و liebermann-burchardتاسرخذاو يزَثاخ يخرهفح كىسُظ نالم و انكشف ػنها تىاسطح كاشف ) اسرخذاو يجًىػح كزنك ذى .تانرفصُمانرنمُح. كًا ذى فٍ هزه انذساسح اخرُاس انطشَمح انًناسثح نالسرخالص وانفصم و انرنمُح و ششحها ( و دسجح نماوذها وانرٍ شًهد: )لُاط دسجح االنصهاس نهًشكة hederacoside cين انرمنُاخ نهرحمك ين انًشكة انًفصىل ) انًفصىل وكشوياذىغشافُا انطثمح انشلُمح ويطُاف االشؼح ذحد انحًشاء و كزنك ذمنُح كشوياذىغشافُا االداء انؼانٍ انسائهح( وانرٍ انمُاسٍ. hederacoside c)ها يغ نرائج )ذطاتمد نرائج الكلمات المفتاحية: نثات اللثالب الكثير, الهيدراكىسايد سي, كروماتىكرافيا العمىد. introduction hedera genus is one of the 55 genera in araliaceae family (1) . members of the genus are highly valued as ornamentals, and are commonly used in the landscape as well as indoors. recently examined species delimitations within hedera genus recognize 12 species, three subspecies, and one variety. hedera helix l (2) .(english ivy, common ivy) is an evergreen dioecious woody liana, one of the 15 species of the genus hedera. the whole plant leaves are coriaceous, 4-10 cm long and wide, cordate at the base. the lamina is palmately 3-5 lobed. the upper surface is dark green with a paler, radiate venation while the lower surface is more grenish-green and the venation is distinctly raised (3) . h. helix l. naturally grows in the western, central, and southern europe but has also been introduced to north america and asia (4) . saponins can be classified into groups based on the nature of the aglycone skeleton. the first group consists of the steroidal saponins, which are almost exclusively present in the monocotylednous angiosperms. 1 corresponding author e-mail: suhaib_aziz@yahoo.com received:20 /1/2014 accepted:25 /6/2014 iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 34 the second group consists of the triterpenoid saponins, which are most common and occur mainly in the dicotyledonous angiosperms (5) . the biologically active compounds responsible for the medicinal use of the plant are triterpenoid saponins (2.5-6%): the bidesmosidic glycosides of hederagenin and oleanolic acid (hederacoside c,b,d,e,f,g,h,i) and the monodesmoside αhederin. other groups of the identified compounds are represented by phenolics (flavonoids, anthocyanins, coumarins, and phenolic acids), amino acids, steroids, vitamins, volatile and fixed oils, β-lectins, and polyacetylenes. the pharmacological activities of hedera helix l. are: spasmolytic, secretolytic, anti-inflammatory, antimicrobial, antiviral, antifungal, protozoidal, hepatoprotective, antioxidant, hypoglycaemic, cytotoxic, antimutagenic, and antihyaluronidase activity (3) . figure 1: photo of h. helix l. (6) table(1): chemical structure of the major saponin glycosides of h. helix l. leaves (7,8) saponin glycoside r1 r2 r3 hederasaponin b h [α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl h hederasaponin c h [α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl oh hederasaponin d h α-l-arabinopyranosyl oh hederasaponin e oh α-l-arabinopyranosyl oh hederasaponin f h β-sulfate h hederasaponin g h [β-d-glucopyranosyl-(1→2)-α-l-arabinopyranosyl] oh hederasaponin i h β-d-glucuronopyranosl oh iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 35 approved therapeutic uses of h. helix l. extract: (9)  acute catarrh (inflammation) of the respiratory tract accompanied by coughing.  symptomatic treatment of chronic inflammatory bronchial diseases materials and methods plant materials the whole plant h. helix l. was collected during november 2012 from zayona district of baghdad, identified and authenticated by prof. dr. ali al-musawi, university of baghdad. the leaves were cut from the whole plant, cleaned gently, and dried in oven at 40 o c , then coarsely powdered by mechanical grinder and weighed. extraction methods of triterpenoid saponins from h. helix l. leaves: extraction method no.1 (10) thirty grams of the powdered leaves of h. helix l. were extracted by maceration in (450 ml) of methanol 99.8% for seven days at room temperature and then filtered. the filterate was evaporated at reduced pressure in the rotary evaporator to a thick residue. this residue was repeatedly washed with petroleum ether (b.p. 40-60 o c) to remove the chlorophyll and fatty materials.the process was continued till there were no traces of colouring matter in the petroleum ether. a thicky residue obtained which redissolved in (100 ml) of methanol 99.8%. to this methanolic solution diethyl ether was added. a white-yellowish precipitate of saponin was formed. the addition of diethyl ether was continued, until no further precipitate was formed. the precipitate was recovered by decantation ,then air dried at room temperature to yield the crude extract (4.3 g). scheme (1) shows the general procedure for this extraction method. extraction method no.2 (10) thirty grams of powdered leaves of h. helix l. were first extracted with (500 ml) of chloroform in a soxhlet apparatus for 18 hours. the residue left was next extracted with (500 ml) of ethanol 99.8% for 15 hours in soxhlet apparatus. the ethanolic extract was evaporated under reduced pressure to a residue which was next dissolved in (100 ml) of methanol 99.8%. to this methanolic solution diethyl ether was added. a white-yellowish precipitate of saponin was formed. the addition of diethyl ether was continued, until no further precipitate was formed. the precipitate was recovered by decantation, then air dried at room temprature to yield the dry crude extract (1.4 g ). scheme (2) shows the general procedure for this extraction method. scheme (1): the general schematic procedure for the extraction method no.1 iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 36 scheme (2): the general schematic procedure for the extraction method no.2 preliminary identification of the triterpenoid saponin glycosides identification of the triterpenoid saponin glycosides were carried out by thin layer chromatography (tlc) using ready made aluminum plates of silica gel gf254, and detection was done by liebermann-burchard reagent, using four different solvent systems. reference standard hederacoside c (chromadextm company-usa). different developing solvent systems were: s1= chloroform : methanol : water (55:37:7) (8) s2= n-butanol : glacial acetic acid : water (60:15:15) (11) s3= chloroform : glacial acetic acid : methanol : water (60:32:12:8) (12) s4= n-butanol : glacial acetic acid : water (40:10:50) (13) reagent used for detection liebermann-burchard reagent used for detection of triterpenoid saponin, and it is prepared by carefully adding 5 ml of acetic anhydride and 5 ml of concentrated sulfuric acid into 50 ml of absolute ethanol, while cooling in ice. spray the developed plate and heat it at 100 o c for 5–10 minutes. (14) isolation and purification of hederacoside c the dry crude extract obtained from extraction method no.1 of triterpenoid saponins was used for isolation and purification of hederacoside c, and performed as the following: fractionation by column chromatography two gram (2 g) of crude dry extract obtained from extraction method no.1 was subjected to column chromatography using glass column (50 cm x 2.5 cm) packed with a slurry of silica gel (0.063-0.200 mm) in chloroform (wet method), in a ratio of 20 g of silica gel to each 1 g of the extract. a dry loading of the sample (crude extract) was used by dissolving it in small volume of methanol and adsorbing it on small amount of silica gel of the same grade used for packing the column, then dried, grinded and applied to the column in order to prevent clogging, since the sample is insoluble in the initial solvent chloroform. the column was eluted by gradient elution technique using chloroform: methanol with an increasing percentage of methanol from zero to 45% (the ratios of chloroform: methanol used were 100:0, 90:10, 85:15, 80:20, 75:25, 70:30, 65:35, 60:40, and 55:45). the column developed by adding 100 ml of the eluent (chloroform : methanol), except for the ratios (60:40 and 55:45 of the eluent) the volumes of eluents were 200 ml and 100 ml, respectively then each 5 ml fractions were collected separately and monitored by tlc using developing solvent iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 37 system s1. a total number of 130 fractions were obtained. those consecutive fractions, which have the same number of spots with the same rf values, were combined and evaporated to dryness to get the major fractions, f1, f2, f3 and f4, as shown in table (2). table (2): major fractions obtained from column chromatography preparative tlc plates the major fraction (f3) obtained by column chromatography was applied as a concentrated solution in a row of spots using capillary tube four times on each plate (the spots should dry before the next application). the thickness of silica gel on the plate was 0.75 mm. the solvent system (s1) was placed in a glass tank (22.5 cm x 22 cm x 7 cm),and covered with a glass lid and allowed to stand for 45 minutes for saturation before use. the detection was done using liebermann-burchard spray reagent in one side of the plate and by uv at 254 nm wave length. the target band was recovered by scraping off the adsorbent at the appropriate places on the developed plates, and collected in a beaker, mixed with chloroform: methanol (60:40), stirred with gentle heating, then filtered. after evaporation of the solvent, the obtained residue was subjected to co-tlc with the reference standard of hederacoside c using different s1, s2, s3, and s4 as solvent systems for identification and checking the purity of isolated compound. qualitative estimation of hederacoside c using hplc technique qualitative estimation of hederacoside c was performed by using waters/ireland high performance liquid chromatography (hplc) in which identification was made by comparison of the retention time obtained at identical chromatographic conditions of the analyzed samples and authentic standard. the hplc condition for hederacoside c is listed in the following table (3): table (3): hplc condition for hederacoside c (15) results extraction of triterpenoid saponins from the dried leaves of h. helix l. were done by two extraction methods and the method no.1 showed higher yield of dry crude extract and hederacoside c than method no.2, as shown in table (4). so the method no.1 was chosen for isolation and purification. table (4): percentage of crude extracts obtained from extraction methods extraction method % yield of crude extract method no.1 14.4 % method no.2 4.65 % identification of triterpenoid saponin (hederacoside c) by tlc tlc of the major fraction (f3) obtained from fractionation by column chromatography of 2 g of crude extract from extraction method no.1, confirms the presence of hederacoside c in this major fraction in comparison with reference standard of hederacoside c, as represented in table (5) and figure (2). major fractions no. of collections 5 ml each no. of spots f1 35-50 2 f2 51-78 3 f3 79-115 4 f4 116-130 4 mobile phase gradient: solvent a=0.1% glacial aetic acid in water, solvent b=0.1% glacial acetic acid in acetonitrile, isocratic 5% b for 5 minutes, then increasing to 95% b over 20 minutes. column phenomenex c18 250 mm x 4.5mm, 5 µm particle size column temperature ambient flow rate 1 ml / min. injection volume 20 µl. injection concentration 2 mg /ml detection uv detector at λ 210 nm iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 38 table (5): the rf values of isolated hederacoside with its reference standard in different developing solvent systems using tlc technique: solvent system s1 s2 s3 s4 rf value of standard hederacoside c 0.72 0.44 0.35 0.5 rf value of isolated hederacoside c 0.72 0.43 0.34 0.5 figure 2: tlc of hederacoside c standard( st.hc ), the major fraction (f3), and isolated hederacoside c ( i.hc ), using four solvent systems (s1, s2, s3, s4) as developing solvent systems. detection by liebermann-burchard reagent. identification and characteriza-tion of the isolated hederacoside c tlc in analytical tlc using spiking technique in four different mobile phases, the isolated hederacoside c appears as a single spot having the same colour and rf value as the reference standard of hederacoside c, as shown in figure (2). measuring melting points the isolated compound was identified to be hederacoside c from its sharp melting point of (212-215 o c) compared to reference standard of hederacoside c melting point (214215 o c). ft-ir the ir spectrum of isolated triterpenoid saponin hederacoside c was recorded as kbr disc which gave identical results as compared with reference standard of hederacoside c, as shown in table (6) and figure (3): hplc (high performance liquid chromatography) qualitative and quantitative estimations of hederacoside c was done by using high performance liquid chromatography ( hplc) in which identifications were made by comparism of retention times obtained at identical chromatographic conditions of analyzed sample and authentic standard of hederacosid c, as shown in figure (4) and figure (5). table (6): the characteristic ir absorption bands in (cm -1 ) of the isolated hederacoside c: (16) functional group hederacoside c standard isolated cm2 compound assignment o-h 3495 3439 broad o-h stretching band indicating hydrogen bonding c-h 2962,2929,2875 2983,2937,2910 asymmetric and symmetric stretching of ch3 and ch2 c=c 1660 1647 stretching of c=c bond c=o 1700 1697 c=o stretching of carbonyl group c-h 1450,1380 1458,1365 asymmetric and symmetric stretching of ch3 and ch2 o-h 1410 1435 o-h bending of alcohol -c-o-c 1170 1122 stretching of ether s1 s2 s3 s4 iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 39 f ig u r e 3 : ir s p e c tr u m o f th e i so la te d h e d e r a c o si d e c iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 40 figure 4: hplc analysis of hederacoside c reference standard . figure 5: hplc analysis of isolated hederacoside c iraqi j pharm sci, vol.23(2) 2014 hederacoside c. in hedera helix l. 41 conclusions phytochemical investigation of h. helix l. leaves, which cultivated in iraq revealed the presence of important group of medicinal natural products belong to triterpenoid saponin glycosides. the crude extract fractionated by column chromatography, then hederacoside c isolated by preparative tlc. the isolated hederacoside c was by tlc, melting point, ft-ir, and hplc analysis. this needs to perform other pharmacological studies to highlight its actions as therapeutic agent. aknowledgement: i would like to express my deepest gratitude and appreciation to all teaching staff and students of college of pharmacy, university of baghdad. 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(2 nd ed.). springer– velag, berlin, germany;2009: 307. 13. j.b.harborne. phytochemical methods. john wiley & sons, inc. newyork, 1973. 14. waksmundzka-hajnos m., sherma j., kowalska t.: tlc of triterpenes (including saponins). thin layer chromatography in phytochemistry (1 st ed.), crc press, taylor & francis group, usa;2008:528. 15. hplc conditions of hederacoside c standard: chroma dex company, usa. (modified). 16. silversteine r. m., webster f. x.: spectrometric identification of organic compounds (6 th ed.). john wiley and sons inc.,usa,2005:81-100 iraqi j pharm sci, vol.31(1) 2022 determination of genistein by coupling reaction doi: https://doi.org/10.31351/vol31iss1pp278-284 278 flow injection spectrophotometric technique for determining of genistein in pure and supplements formulations through diazotization coupling reaction farqid faraj muhammed*, sadeem subhi abed*,1 *department of chemistry, college of science, university of baghdad, baghdad, iraq. abstract genistein (gen) is the major isoflavone found in soybeans, has a number of cardiovascular health benefits, postmenopausal syndrome and osteoporosis. a direct flow injection analysis method for estimation of gen in pure and supplements formulation was suggested. this system is based on a diazotization coupling reaction between procaine penicillin (pr) and gen in an alkaline medium. the formed orange dye has a maximum absorption at 416 nm. calibration curve was constructed over different gen concentrations with a linearity range of 10-100 µg/ml and a detection limit of 1.51 μg/ml. for the fia technique, all analytical factors were analyzed and optimized. the established method was successfully used to determine gen in the supplement formulations. keywords: genistein, flow injection analysis, spectrophotometry, procaine penicillin, diazotization coupling reaction. في المستحضرات النقية والمكمالت من خالل تفاعل تقنية طيف الحقن الجرياني لتقدير الجينيستين ازوتة واالزدواج 1*، و سديم صبحي عبد *فرقد فرج محمد قسم الكيمياء ، كلية العلوم ، جامعة بغداد ، بغداد ، العراق . * الخالصة موجود في فول الصويا بصورة رئيسية وله العديد من الفوائد الطبية لكثير من االمراض كأمراض الجينيستين هو مركب ايسوفاليفون الجينيستين القلب واألوعية الدموية وهشاشة العظام ومتالزمة ما بعد انقطاع الطمث . تم اقتراح طريقة تحليل حقن التدفق الجرياني المباشر لتقدير نية . تعتمد طريقة التقدير على تفاعل األقتران واألزوتة بين البروكائين بنسلين مادة دوائية تم استعمالها كاشف بصورته النقية وفي المكمالت الصيدال لتراكيز نانومتر . تم قياس األمتصاصية 416عند طول موجي صبغة ذات لون برتقالي وتقاس في التفاعل والجينيستين في وسط قاعدي . حيث تنتج ميكروغرام / مل . تم تقييم وتحسين جميع المتغيرات التحليلية 1.51ميكروغرام/ مل وكانت حدود الكشف 100-10مختلفة من الجينيستين المستخدمة في تطبيق الطريقة المحددة بنجاح. الكلمات المفتاحية: introduction flavonoids are a type of secondary metabolism molecule that occurs naturally in the world of plants. they are regarded as a quality indication for fruits and medicinal plants, and hence an important component to consider in the development of agricultural and manufactured products. many publications have been written throughout the years about the extraction and identification of flavonoids (1,2 ) .genistein (figure 1) is an isoflavone that acts as a phytoestrogen and an angiogenesis inhibitor. the chemical name comes from the fact that it was initially isolated in 1899 from the dyer's broom, genista tinctoria. the structure of the chemical was determined to be identical to that of prunetol in 1926. genistein was quantified by spectral methods (3-6), mass spectrometry (ms) with high performance liquid chromatography (hplc)(7), uv or electrochemical detection(8–16), gas chromatography-mass spectrometry (17), lc-ms method(18). figure 1. genistein 1corresponding author e-mail: sadeem.s@sc.uobaghdad.edu.iq received:7 /8/ 2021 accepted:15 /11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp278-284 iraqi j pharm sci, vol.31(1) 2022 determination of genistein by coupling reaction 279 the analysis method used in the present work is flow injection analysis. it's done by inserting a sample plug into a moving carrier stream (19,20). detection methods include spectrophotometry, fluorescence spectroscopy, atomic absorption spectroscopy, mass spectrometry, and other experimental methods. fia techniques have evolved into a wide range of applications. flow injection's application to real-world tests benefits from automated sample processing, good repeatability, adaptability to micro-miniaturization, chemical containment, waste reduction, and reagent economy in a system that runs at microliter levels (21,22). experimental work apparatus a digital spectrophotometer shimadzu mini uvvis 1240 was used for absorbance equipped with flow cell with 50 µl internal volume and 1 cm of bath length. it was with a peristaltic pump (shennchen, china) equipped with flexible vinyl tubes of an inner diameter of 0.5 to transfer the solutions. injection valve from knauer, germany was used to provide appropriate injection volumes of standard solutions in addition a reaction coil (rc) made of teflon with a 0.5 mm inner diameter was used. the manifold with two channels was used to determine gen spectrophotometric ally . the fia manifold used was depicted in figure.(2). figure 2. schematic of fia manifold for estimated gen . p: peristaltic pump, f.c: flow cell, r.c: reaction coil and w: waste. reagents and solutions standard gen 200 µg/ml : dissolving 0.02 (no. of mol) g of pure gen in 10 ml 0.5m of naoh in a small beaker, then transferring the solution to a 100 ml volumetric flask and completing the volume with distilled water to the mark . diazotized reagent solution: a solution of pr 0.003m was freshly prepared by dissolving 0.1712 g (no. of mol) of pr in 25 ml ethanol in beaker and cooling in ice bath. after that,3 ml,1m of hcl was added to it and stirring with cooling , then 0.0207(no. of mol) g of sodium nitrite (merck ) (0.003m) was added with constant stirring. then transferring the solution to a 100 ml volumetric flask and completing the volume by distilled water to the mark. sodium hydroxide (bdh, england) 1m : in a 250 ml volumetric flask, dissolve 10 g of naoh with distilled water and completed the volume to the mark by distilled water. hydrochloride acid (bdh , england) 1m : in a 500 ml volumetric flask, dilute 43.7 ml of 11.44 m concentrated hydrochloric acid with distilled water. preparation solutions of gen pharmaceutical forms: (200 µg/ml) an appropriate number of capsules (10 capsules) were emptied and weighted, and the average weight of one capsule's content was selected, and accurately weighted to equate to 0.02 g of gen and dissolved in 10 ml naoh 0.5m. then transferred to a 100 ml volumetric flask and completed to the mark with distilled water. serial dilutions were done to prepare the working solutions. general fia procedures working solutions for the process were made from gen stock solutions ranging from 10 to 100 g.ml-1. a 100µl portion of gen was injected into a stream of 0.003m procaine solution, then the solution is mixed with 0.1m naoh in 50cm reaction coil. at a total flow rate of 1.9 ml.min-1. at 416 nm, the orange dye absorption was measured. results and discussion preliminary studies were indicated that procain penicillin (1 ml of 0.003m) coupled with gen (1 ml of 200 µg .ml-1)in alkaline medium naoh (1 ml of 0.1m) and formed orange dye can be detected spectrophotometrically. the spectra of colored complex formed are shown in figure. 3. figure 3. a: absorption spectrum of azo dye formed when 20 µg.ml-1 of gen coupled with procaine penicillin in alkaline medium, b: blank. iraqi j pharm sci, vol.31(1) 2022 determination of genistein by coupling reaction 280 after chemical and physical variables have been optimized the calibration graph was performed to test the linearity of gen concentration (triplicate injections). two stages were necessary to complete the diazotization coupling reaction. pr was initially converted to a diazo compound by reacting with nitrous acid (nano2/hcl). the diazonium salt was then coupled with gen in para position of a poly phenolic molecule, producing orange dye in a basic medium in the second step, the phenolic nature of gen resulted in fast coupling with diazotized reagent, as shown in scheme 1. scheme 1. proposed mechanism for diazotization coupling reaction of gen with procaine optimization of chemical parameters the chemical and hydrodynamic parameters that could affect the reaction and the stability of the colored formed were studied by changing one variable at a time while keeping the others constant. the experiment was conducted using 20 µg.ml-1 of gen. the chemicals parameters, including the concentrations of reagent, medium, etc. were studied; the optimization of the concentration of (pr) was shown that when the concentration was increased up to 0.003 m, the absorbance increased. with increasing concentration, the absorbance was observed to decrease than it was in the previous concentration .therefore, 0.003 m concentration was selected for further used. the greater concentration leads to increase the blank signal as shown in figure 4. preliminary studied for type of alkaline medium reveals (naoh, koh and na2co3) that sodium hydroxide was the suitable base for this method. different concentrations of naoh were investigated in the range of (0.07 -0.9 m) and the concentration chosen was 0.1 m. the concentration greater than 0.1 m shown as an inhibitor for sensitivity of colored products, the results obtained are as shown in figure 5. iraqi j pharm sci, vol.31(1) 2022 determination of genistein by coupling reaction 281 figure 4. effect of the reagent concentration figure 5. effect of naoh concentration optimization the physical parameters effect of total flow rate total flow rate has a great role in fia system, since it is use to get the best reaction time and has a directed effect on sampling frequency. at higher flow rate the dispersion and reagent consumption were increased , therefore ,the absorbance and sensitivity decreased with increased flow rate. in this study, a flow rate range 0.45-4.95 ml.min-1 was used, and the optimum value was 1.9 ml.min-1.the current study indicated that the absorbance was decreased above 1.9 ml.min-1 flow rate because of dispersion and dilution . the result obtained from flow rate is shown in figure (6. a). the effect of the reaction coil length to enhance the sensitivity of the colored reaction and increase mixing of the reactants, the effect of reaction coil lengths was investigated .the dispersion of reactant zone probably increase with increasing the reaction coil length. different reaction coil lengths were studied in the range of 25 to 150 cm, and the coil length of 50 cm gave the maximum absorbance, therefore was selected and used in subsequent experiments(figure 6. b). the influence of the sample volume injected effect of injection volume was investigated with varying sample loops in the range of 75 to 200 μl. the highest absorbance was obtained at 150 µl, and this sample volume was used in subsequent experiments. the obtained result is shown in (figure 6.c). figure 6 . (a) the effect of total flow rate on reaction absorbance, (b) effect of reaction coil of the absorbance, (c) effect of the injection loop iraqi j pharm sci, vol.31(1) 2022 determination of genistein by coupling reaction 282 analytical characteristics the calibration curve was constructed under optimal conditions for gen estimation (table 1). table 2 shows the values for the calibration curve's intercept, slope, correlation coefficient, and molar absorptivity, as well as values for analytical statistical treatments. table 1. summary of the analytical parameters obtained from calibration graph, which indicated good linearity, highly reproducibility and low limit of detection accuracy and precision to determine the accuracy and precision, three different concentrations of standard solution of genistein were investigated in five replicates. for each concentration, the accuracy and precision were performed and the relative standard deviation rsd% was obtained with relative error e%. low values of the rsd% and e% indicated that method gave acceptable results. table 3 shows the precision and accuracy for the suggested method. table 2. analytical values of the techniques suggested for determinate gen parameter value regression equation y = 0.0153 x – 0.1223 correlation coefficient, r2 0.9989 linearity percentage ,r2 % 99.89 slope, b (µg ml-1) 0.0153 intercept, a 0.1223 linearity range (µg ml1) 10 100 standard deviation of the slope, sb 1.83 x 10-4 relative standard deviation rsd% 0.7 recovery range % 99.45 – 100.56 molar absorptivity , ɛ (l mol-1 cm-1) 4.135 x 103 loq, (µg ml-1) 2.67 lod, (µg ml-1) 0.88 standard deviation of the residuals, sy/x 1.66 x 10-2 sandell,s sensitivity (µg cm-2) 0.0654 table 3 . the precision and accuracy present found error sd% *rec.% *rsd% 20 19.89 -0.55 0.003 99.45 0.71 40 39.93 -0.17 0.002 99.83 0.27 50 50.28 0.56 0.002 100.56 0.22 *average of five determinations analytical applications (gns supplements) the current method was used to determine the amount of gen in capsules by analyzing three different concentrations (20,40, and 50 µl ml-1) under calibration graph conditions directly. according to the obtained results shown in table 4, the small values of calculated rsd% and e% refer to the repeatable and accurate of the suggested method. table 4. application of the proposed method for determining gen in pharmaceutical formulations by using direct method conc. gns alternative medicine solutions, inc.usa µg ml-1 found µg ml-1 error% rec.% rsd% 20 20.3 1.5 101.5 0.5 40 40.2 0.5 100.5 0.3 50 50.9 1.8 101.8 0.2 as shown in table 5, the calculated t and f-test values did not outperform the theoretical ones .when the suggested method and the ported uv method were evaluated, the findings revealed that there was no significant difference between them. value parameters 20 μg ml-1 conc. of gen 0.003 m conc. of pr 0.1 m conc. of naoh 1.9 ml.min-1 total flow rate 150 µl sample loop 50 cm reaction coil iraqi j pharm sci, vol.31(1) 2022 determination of genistein by coupling reaction 283 table 5. the tand f-tests were used to compare the proposed methods to the uv method preparation form proposed method classical uv method recovery % gen ( pure) 101.00 99.23 gen capsule 125 mg 99.27 101.34 t-calculate (t*=4.303) 0.13 f-calculate (f*=161.4) 1.44 the influence of foreign compounds was eliminated using standard addition method which applied under calibration curve conditions. good accuracy and precision was obtained .the results are shown in figure 7 and table 6. figure 7. standard addition method for determination of 20 µg.ml-1 of gen in supplements table 6. application of the proposed method for determining gen in pharmaceutical formulations by using standard addition method . pharmaceutical preparation proposed method classical uv method conc. of gen (µg.ml-1) *rec.% *rsd% conc. of gen (µg.ml-1) *rec.% *rsd% present found present found genistein capsule 125 mg 15 15.1 100.7 0.5 2 2.03 101.5 1 20 19.8 99.0 0.6 3 3.02 100.7 0.7 * average of five determinations conclusion according to the research study, no flow injection methods for estimating gen in medicinal preparations have been documented. the current study demonstrated that a new spectrophotometricflow injection approach for determining gen based on the diazotization coupling reaction using reagent pr at the microgram level was simple, fast, and robust. the suggested method has a high sensitivity, linearity, and cost effectiveness when compared to previous fia methods and other methods such as lc-ms, hplc, hptlc, gce, and hplc-uv. the recovery values were satisfactory, indicating the suggested method's excellent accuracy and reproducibility. the tand f-values proved that the proposed method and the classical method were in agreement. the method was sufficient for routine gen estimation in pure and pharmaceutical formulations. references 1. mayasa m.mohammed, sadeem subhi abed, simple spectrophotometric method for diosmin 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"flow injection tutorial". www.flowinjectiontutorial.com.retrieved 2016 :03 -28. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(1) 2012 phytochemical study of cynara scolymus l.cultivated in iraq 6 phytochemical study of cynara scolymus l. (artichoke) (asteraceae) cultivated in iraq, detection and identification of phenolic acid compounds cynarin and chlorogenic acid abdul mutalib a.g nasser* ,1 *department of pharmaceutical chemistry and pharmacognosy, baghdad college of pharmacy, baghdad, iraq. abstracts the leaves of globe artichoke, cynara scolymus family asteraceae/ compositea have long – used in traditional medicine and now included in british and european pharmacopeia, the british harbal pharmacopeia and complete german commission e monographs.the plant originally comes from mediterranean region and north africa and cultivated around the world. the flowers are used worldwide for nutrition purposes and the leaves for medical purposes including hepatic affections. the plant wildly distributed in iraq in the watery lines and boundary of the field.the plant contains many phytochemicals such as the bitter phenolic acids whose choleretic and hypocholestremic as these compounds are antioxidant. other materials to have other pharmacologic effect specially flavonoids. thus this work deals with phenolic acids cholrogenic acid and cynarin.this work includes cold extraction evaporation by freeze drying and separation inusing column chromatography, tlc and finally in high pressure liqued chromatography (hplc), the two important compound; chlorogenic acid and cynarin were separated and identified. key words: artichoke, cynara scolymus, poly phenolic acids, chlorogenic acid, cynarin. المستزرع في العراق. أيجاد وتشخيص cynara scolymusدراسة ميمياوية للنبات المسمى المرمبات الحلقية الفنولية السينارين وحامض النلوروجنل عبذ المطلب عبذ الغني ناصر* ،1 انعقاقٛز ٔانُباحاث انطبٛت ، كهٛت بغذاد نهصٛذنت ، بغذاد ، انعزاق .*فزع الخالصة يسخعًهت يٍ asteraceae/ compositaeيٍ انعائهت/ cynara scolymus كٙ انُباث انًسًٗأٔراق األرظٙ شٕ فٙ حٕض انبحز ًُٕٚٔيذكٕرة فٙ انذسخٕر انبزٚطاَٙ ٔاألٔربٙ كذنك فٙ انًجًٕعت األنًاَٛت. انُباث حقهٛذ٘ ٔقج غٕٚم كعالج َٕرحّ كغذاء ٔأٔراقّ نألغزاض انطبٛت ٔانخٙ حشًم أيزاض انًخٕسػ ٔشًال أفزٚقٛا ٔيُخشز فٙ جًٛع أَحاء انعانى حسخعًم األبٛط انُباث ٚحخٕ٘ عهٗ فٙ اغزاف انحقٕل انشراعٛت ٔخاصت قزب يصادر انًٛاِ ٔاألياكٍ انزغبت فٙ انعزاق.ًٕ ُٔٚخشز انُباث ُٚ . ذانكب انكٕنٛسخزٔل فٙ انذو ٔحًاٚت انكبذ كًٛٛأٚاث َباحٛت يُٓا يجًٕعت انحٕايط انفُٕٛنٛت انًزة ٔانخٙ نٓا خاصٛت عالج حخفٛط يسخٕٖ chlorogenicكذنك انًزكباث انفالفُٕدٚت ٔانخٙ نٓا خٕاص غبٛت نذا فأٌ ْذِ انٕرقت حبحث فٙ انًزكباث انحايعٛت انفُٕٛنٛت ٔخاصت acid ٔكذنكcynarinأشخًم انعًم عهٗ األسخخالص انكحٕنٙ انًائٙ انبارد ٔحى أسانت انسٕائم بٕاسطت انـ. freeze dryer ثى فصهج حى انخأكذ يٍ ْذٍٚ انًزكبٍٛ بٕاسطت أجٓشة انعغػ ٔ قٛقتانطبقت انز انًٕاد بٕاسطت كزياحٕغزافٛا انعًٕد انسائم ٔفٙ كزٔياحٕغزافٛا ( ٔحى حشخٛصٓا فٙ انجٓاس.hplcانعانٙ نهكزٔياحٕغزافٛا انسائهت ) مض الحلقية الفينولية : سينارين وحامض النلوروجنل . ، سينارا سنوليمص،الحوات المفتاحية : أرتيشوك)أرضي شومي(النلما introduction the importance of the plant cynara scolymus which is called artichoke or globe artichoke steamed from its used as edible material for nutrition and from its content of phenolic acid constituent in particular cynarin and chlorogenic acid (1, 7, 10) .the leaves of globe artichoke, cynara scolymus l.family asteraceae / compositae, have been long-used in traditional medicine and now included in british and european pharmacopeia (bp / ep) , the british harbal pharmacopeia ( bhp ) and the complete german commission e monographs (1) .the plant cynara scolymus l. originally comes from mediterranean region and north africa and also cultivated around the world (2,3) .the flowers are used worldwide with nutrition purposes and the leaves with medical purposes , broadly used in phytotherapy preparations with special indication in hepatic affections (3) .the plant is widely distributed in iraq and normally located and found in the outer lines of the fields, water lines and humid watery soil. the plant flourishes in winter and harvested in february and march. 1 corresponding author email : dr_abdulmutalibagm@yahoo.com received : 5/6/2011 accepted : 24/10/2011 iraqi j pharm sci, vol.21(1) 2012 phytochemical study of cynara scolymus l.cultivated in iraq 7 in north of iraq and kurdistan the people are used to eat the carpel of blooms (2) ; because of its nutritional value.the leaves of c. scolymus are characterized by the composition and high content of bitter phenolic acid compounds whose choleretic, hypocholestremic and heptatoprotective activity attributed (4) . at least to the antioxidant potential of artichoke extracts and of their phenolic compounds. constituents which are around 2% such as: caffeic acid, chlorgenic acid and cynarin, flavenoids (0.1 – 1 %) and essential oil (4) . pharmacological studies demonstrated that the extracts of c. scolymus and active principle cynarin (1,3 di-caffeoyl quinic acid ( c25h24o11 ) posses choleretic and hypocholesterolemic activity (5) .the extract of the plant also protect hepatocytes treated with carbontetrachloride (ccl4) from hepatic cellular necrosis. this activity related to the power antioxidant effect of phenolic acids (6) . recently pharmacological investigations and clinical reports published showed the efficacy and safety of artichoke extracts in treatment of hepatobiliary dysfunctions and abdominal pain (7) . cynara scolymus leaves cynara folium is the whole drug; consist of the dried or fresh basal leaves with coarsely toothed margin.the cut drug is composed of grayish green, tomintose and fleet like aggregate with pithy fragment of petioles and nervature as well as very long fine fibers. the lower leaf surface is densely pubescent, grayish and matted with pinnate venation. the upper leaf surface is glabrous and green (2) . the plant thistle – like cynara scolymus is perennial herb about 1.5 meter height; with pinnatified leaves 8 – 15 cm with wide flower head has obtuse – ovate fleshly involucres bracts (3) ; the pharmaceutical grad material consists exclusively of basal leaves, which up to 50 cm long and 25 cm wide; the deeply pinnotified leaf laminas is rarely entire margined but from flat, lance late segment with crossly serrate or finely toothed margin.the iraqi plant has thrown margin. no regular cultivation was adopted in iraq. the chemical constituents of leaves of cynara scolymus the chemicals which from important constituent of c. scolymus include three classes. (8,9,10) : 1. phenolic acids which include a combinations of caffeic acid and quinic acid. c. scolymus have the two important anti oxidant cynarin and chlorogenic acid, by the combination of 1, 3 – 0 – quinic acid with two molecules of caffeic acid to from 1, 3 di – 0 caffeoyl quinic acid (cynarin) and 5 – 0 – caffeoyl quinic acid (chrogenic acide). quinic acid caffeic acid chlorogenic acid cynarin 2. the flavenoids particularly glycoside of luteolin including luteoline-7glycoside ( cynaroside ) and luteoline-7rutinoside ( sculomoside). cynaroside – luteolin-7glylosyl scolomoside – luteolin-7rutinosyl iraqi j pharm sci, vol.21(1) 2012 phytochemical study of cynara scolymus l.cultivated in iraq 8 3. sesequiterpens; cynaropicrin and grosheimin lactons (13,14) . . cynaropicrin grosheimin other chemicals like sesequiterpin bselinene and caryphyllene also present (2) . the plant was registered by counsel of europe as a natural source of food flavoring (category n2). in usa the leaves used in beverage only with a maximum concentration (16 part per million).the german commission e recommended an average daily dose of 6 g. or equivalent dose of the extract for the treatment of dyspeptic problems.because the plant available in iraq as a weed and some has been cultivated and because the importance of such plant which extend worldwide; there for it was important to study such plant for its phenolic acid content and flavenoids as an important approach to use the plant medicinally in the health care and as a medicine, starting to study the phenolic acid content cynarin and chloragaic acid first. materials and methods 250 grams of dry basal leaves of cynara scolymus were collected from botanical garden of pharmacy college, university of baghdad and authenticated; were dried under shade and powdered; the dry powder was macerated with 1 liter of 80% methanol/ water for three days with occasional shaking. the methanolic extract was filtered and kept in the refrigerator the marc was macerated with another two 500 ml volumes of 80% methanol/ water each for two days, the filtrates were collected and mixed together with the first filtrate and evaporated under vacuum at 40⁰c in rotary evaporator (stuart rotatory evaporator; uk) to remove the methanol; the left collected water extract which was about 400 ml were separated into two parts, the first one (about 200 ml) shacked with three successive portion of 100 ml n butanol and the other shacked with three successive portion of 100 ml ethyl acetate the two portions were subjected to lyophilizati using virtis lyophilized usa 4.28 g. obtained from nbutanol portion and 2.74 g. obtained from ethylacetate portion dried and kept in refrigerator (9) . the same procedure was applied to 250 gm.of basal leaves of cynara scolymus which was obtained from controlled cultivated field in greaat district by ministry of agriculture.the quantities of the lyophilized n butanol and ethylacetate extracts were 7.78g. and 5.88 respectively. (10) . samples from two plant extracts were chromatographed and matched with standard cynarin obtained from china (changdu biopurify; china) using three mobile phases as fallow: s1= water: methanol: acetic acid (78.5: 20: 2.5 ml) s2= nhexan: aceton: chloroform: methanol (25: 25: 25: 25 ml) s3= ethyl acetate: formic acid: water (80: 10: 10 ml) the separated spots were detected on the chromatogram by using ultraviolet light at (254 – 366 nm); then sprayed by sulpheric acid and heating the plat to 110⁰c for five minutes.the rf values were calculated and the results were shown in table 1 and 2.to separate the phenolic acid in cynara scolymus column chromatography was used and gradient elution technique was applied, using two organic solvents ethyl acetate: methanol 100: 0; 80: 20; 60: 40; 40: 60; 20: 80; 0: 100 twelve fractions of 50ml were collected, using tlc to group the fraction; fraction 1; 2; 3; 4 and 5 show one compound, its rf value was 0.75 so they are collected together and called f1.fractions 6, 7, 8, and 9 showed nearly three spots having rf values 0.28, 0.29 and 0.3 respectively which were grouped together as f2 . fraction 10, 11 and 12 showed only two spots. high pressure liquid chromatography (hplc) was used under the fallowing conditions (11) : the instrument name: knaur (advance scientific instrument germany). the column used: hyperclone ods5 us; c18 v250 x4.6 mm. program used: chromoget. the loop capacity: 20 ml injection value. iraqi j pharm sci, vol.21(1) 2012 phytochemical study of cynara scolymus l.cultivated in iraq 9 detector: varian detector of variable ultraviolet light. the hplc chromatographic conditions applied. the flow rate of the mobile phase 1.3 ml/ minute. the mobile phase was: water: methanol: acetic acid (78.5: 20: 2.5 v/v). the detection: uv at 316nm injection volume: 20 µℓ of sample, filtered by millipore filter. the results obtained shown in table 3; 4 and 5: table -3shows the retention time of cynara scolymus standard. table -4shows the separated components of n – butanol layer fractions table -5shows comparison of retention times of standard phenolic acids and plant phenolic acid. using the same conditions the fraction of the nbutanol extract and the retention times of f2-6; f2-7; f2-8; f2-9 was recorded in table 4 and 5. results and discussion experimental studies in vitro and in vivo support some of the reputed use of artichoke. traditionally the choleretic and cholesterol lowering activity of globe artichoke have been attributed to cynarin and chlorogenic acid present in the leaves and carpel of the plant (2,7,10) .clinical trials investigating the use of globe artichoke powder and cynarin in treatment of hyperlipidaemia generally reports positive results; the benefits of such heptoprotective and heptoregenarating activity have been documented to cynarin in vitro and in animals. the flavonoid content of the plant have antioxidant activity and it was upregulate endothelial type nitricoxy synthese gene expressions in human endothelial cells. n. oxide (no) produced by endothelial nitric oxide synthese (enos) represent and antithrombotic and antiatheroseclorotic principle in vasculature which lead to provide protection against cardiovascular diseases (20) .the results obtained from the extract of the two organic solvent nbutanol and ethyl acetate support of the separation the two most important compounds the phenolic acid and the flavonoid the difference in polarity of the two compounds lead to phenolic acid prefer the nbutanol and the flavonoids prefer the ethyl acetate portion. another attempt to separate the more polar compound by shaking the two organic solvent extract with water but the results obtained (table 1 and 2) showed similar components together and worked as n butanol extract and ethyl acetate extract. it is obvious that the plant with controlled cultivation which include using fertilizers and herbicides will give more extractable materials and this need more investigation to evaluate the two important compounds phenolic acid and flavonoids with other components (13) .tlc and hplc result confirm the presence of the two phenolic acid compounds cynarin and chlorogenic acid (11,12, 15, 16, 17, 18, 19) ; table 1, 2, 3, 4 and 5 and figers 1, 2, 3 for the standard cynaria and chlorogenic acid and their combination, figure (4) for fraction (1) of butanol extract figure (5) for fraction (6) of butanol which shows clearly the two phenolic acid compound plus other compound including other chemicals. figs 6, 7, 8, show some phenolic acids and other compounds which needs more investigation in future.tables 3, 4, 5 shows the retention time of the standard two phenolic acids and the retention time of butanol extract table (4) and the standard retention time (21) .in table (4) many unidentified compounds are present which need further work to understand the complete photochemical found in the iraqi plant. the lack of some instruments and standard will hinder the investigation of other appeared component of the chromatogram. table 1: results of tlc (thin layer chromatography) for artichoke leaves from the (medicinal garden of the college of pharmacy/ university of baghdad) sample s1 s2 s3 ethyl acetate layer rf 1= 0.85 rf 2= 0.69 rf 3= 0.36 rf 1= 0.79 rf 2= 0.62 rf 1= 0.81 aqueous layer of ethyl acetate layer rf =0.87 rf = 0.79 rf = 0.53 aqueous layer of butanol layer rf = 0.91 rf = nil rf 1= 0.82 rf 2= 0.65 rf 3= 0.56 standard cynarin rf = 0.83 rf = 78 rf 1= 0.85 note: the results above calculated according to uv. & chemical identification using h2so4 in alcohol heating for 5 minutes in oven at 110⁰c. iraqi j pharm sci, vol.21(1) 2012 phytochemical study of cynara scolymus l.cultivated in iraq 10 table 2: results of tlc for artichoke leaves from (greaat controlled farm by agriculture college of baghdad university) sample s1 s2 s3 ethyl acetate layer rf 1= 0.86 rf 2= 0.71 rf 1= 0.8 rf 2= 0.78 rf 1= 0.86 rf 2= 0.68 rf 3= 0.55 aqueous layer of ethyl acetate rf 1= 0.84 rf 1= 0.81 rf 2= 0.79 rf 1=0.87 rf 2= 0.66 rf 3= 0.52 rf 4= 0.25 butanol layer rf 1= 0.86 rf 2= 0.70 rf 1= 0.82 rf 2= 0.77 rf 1=0.86 rf 2= 0.75 rf 3= 0.58 rf 4= 0.49 rf 5= 0.26 aqueous layer of butanol rf 1= 0.88 rf 2= 0.72 rf 3= 0.20 rf 1= 0.82 rf 2= 0.76 rf 1=0.86 rf 2= 0.75 rf 3= 0.68 rf 4= 0.60 rf 5= 0.50 rf 6= 0.25 standard cynarin rf = 0.83 rf = 0.78 rf = 0.85 table 3: standard of cynaria scolymus phenolic compounds after separation in hplc. name of the standard retention time (minutes) retention time (minutes) notes 1,3dicaffeoylquinic acid (cynarin) 4.783 ── ── caffeoyl quinic acid (chlorogenic acid) ── 5.467 ── mixture of 1, 3 dicaffeoyl quinic acid cynarin and caffoyol-quinic acid chlorogenic acid standard 4.683 second reading 4.467 6.583 second reading 6.067 at the same date table 4 : butanol layer fractions and their content from phenolic acids, after separation in hplc fraction peak number retention time (minutes) notes f2 (6) 1 2 3 4 5 6 1.650 2.483 3.983 4.383 5.050 5.817 unknown-need more investigation unknown unknown cynarin unknown chlorogenic acid f2 (7) 1 2 3 4 5 6 7 1.683 1.817 2.517 4.483 5.150 5.967 7.900 unknown unknown unknown cynarin unknown chlorogenic acid f2 (8) 1 2 3 4 5 6 1.650 2.550 2.867 4.717 6.550 7.583 unknown unknown unknown cynarin chlorogenic acid unknown f3 (9) 1 2.75 unknown f1: include fractions of the column 1, 2, 3, 4, 5. shows one peak at retention time 3:2 minutes which need further investigation in hplc. fig – 4 fractions of the column 10, 11, 12 show negative results in hplc. iraqi j pharm sci, vol.21(1) 2012 phytochemical study of cynara scolymus l.cultivated in iraq 11 table 5: comparison of retention times of standard phenolic acid with those of the plant nbutanol extracts after separation in hplc. name retention time name retention time standard ─── nbutanol extractfractions ─── 1,3dicaffeoylquinic acid (cynarin) 4.783 f2 (6) 4.383 5-0-caffeoylquinic acid (chlorogenic acid) 5.467 ─── 5.817 cynarin ─── f2 (7) 4.483 chlorogenic acid ─── ─── 5.967 cynarin ─── f2 (8) 4.717 cholrogenic acid ─── ─── 6.550 figure 1 : hplc analysis of cynarin standard. figure2 : hplc analysis of chlorogenic acid standard. figure 3: hplc analysis ofmixture of cynarin and chlorogenic acid standard. iraqi j pharm sci, vol.21(1) 2012 phytochemical study of cynara scolymus l.cultivated in iraq 12 figure 4: hplc analysis of f1 of butanol extract of cynaria scolyus of pharmacy college baghdad university. fraction of the column № 1,2,3,4,5. figure 5: hplc analysis of f2 of butanol extract of cynaria scolymus of pharmacy college baghdad university. fraction of the column № 6. figure 6: hplc analysis of f1: of butanol extract of cynaria scolymus of pharmacy college baghdad university. fraction of the column № 7. figure 7: hplc analysis of f2: of butanol extract of cynaria scolymus of pharmacy college baghdad university. fraction of the column № 8. iraqi j pharm sci, vol.21(1) 2012 phytochemical study of cynara scolymus l.cultivated in iraq 13 figure 8: hplc analysis of f3 of butanol extract of cynaria scolymus of pharmacy college baghdad university. fraction of the column № 9. references 1. trease and evans; phormacognosy; artichoke leaf 16 th ed. 2009 p. 184. 2. maria rosario alonso; maria del carmn garcia claudia garcia bonelli: validated hplc method for cynarin dctermination in biological sample: acta farm. bonaerense; 2006; 25(2):267 – 70. 3. p. perziosi: ii farmaco 1962; 17: 701 – 45.antioxident potential of artichoke axtract. 4. s. gorzaczany, r. filip, m. alonso 2001; choleretic and hypocholesterolemic activity of cynarin; j. ethenopharmacol 75: 291 – 4.through acta farm bonaerense 2006;25(2): 267-70 . 5. m. faure, e. lissi, r. torres and l. videla activity related to the power antioxidant effect of phenolic acids; phytochem pharmacol,1990; 29: 3773 – 5. 6. s. m. wittemer; m. ploch, t. winddeck, s. c. muller, 2005, phytomedicin 12 (1 – 2): 28 – 38. 7. herbal drugs and phytopharmaceutical; 2004. cynara folium (artichoke leaf) third ed. p.173 – 175. 8. neung tae jun, ki chang jang; j. appl. boil. chem.; 2007; 50(4) 244 -284 radical scavenging activity and content of cynarin. 9. kevin robards; j. of chromat graphy a. 2003; strategies for determination of bioactive phenols in plants, fruit and vegetable,2003; 657 – 691. 10. herbal medicine; artichoke, third ed. 2007; p.67 – 71. pharmaceutical press. 11. e. hvattum, rapid commun. 2002; 16; 655. 12. kevin robards; strateyies for determination of bioactive phenols in plants, fruit and vegetable; journal of chromatography a, 1000 ,2003; 657 – 691. 13. n brand z. constituents of cynara folium flavenoids; phytother ,1999; 20: 292 – 302. through herbal drugs and phytopharmacutical. 14. nbrand z. constituent of cynara folium bitter compound sesequitapine, phytother ,1990;11: 169 – 175. 15. bbv (bretange biotechnologie vegetable) fav health, quebee city dr. serge mabean, applied research center for plant breeding, biotechnology and quality. antioxidant activity of extracts of artichoke and by – products, 2005. 16. p. mattila, k. konko, m. eurola, concentration of phenols in the plant, j. agri food chem, 2001; 49: 2343. 17. w. k, li, hh. s fong, k. w. singletary; jf. fitzooff. j. lig. chromatogr. relat. technol ,2002;25: 397. gradient elution technique. 18. a. m. torres, t. manlastovicka, r. rezaaiyan, j. agric food chemi; 1987;35: 921. 19. x. q mu, q. shi, a. duan, t. t. x. dong; j. agric food chem, 2002;50: 4816. 20. huige li, ning xia, isolde brausch, ying yao and ulrich forstermann flavenoid from artichoke (cynara scolymus); up regulate endothelial type nitric – oxide synthase gen experssion in human endothelial cell. jpet. aspetjournals. org. ;2007. 21. jan frissche. christaon m. bindorff; eur. food res. technol, isolation, chroctevization and determination of minor artichoke (cynara scolymus l.) leap extract compounds. 2002;215: 149 – 157. iraqi j pharm sci, vol.31(1) 2022 valerian-carbamazepine interaction doi: https://doi.org/10.31351/vol31iss1pp220-224 220 effect of oral administration of valerian extract at different doses on pharmacokinetic parameters of carbamazepine in rabbits issam mohammed abushammala*,1 *department of pharmaceutics and industrial pharmacy, faculty of pharmacy, al-azhar university/gaza, , palestine. abstract carbamazepine (cbz) is a narrow therapeutic index drug used in the treatment of trigeminal neuralgia and psychiatric disorders. valerian (val) is a popular herbal product which should be prescribed to treat insomnia and anxiety. the study was designed to investigate the presence of significant pharmacokinetic (pk) interaction between valerian (val) at different concentrations on carbamazepine (cbz) pharmacokinetic parameters in healthy male rabbits. in an in vivo, parallel-randomized controlled trial, the rabbits in three groups "first (control), second and third" were given oral doses of cbz (50 mg/kg), for "second and third" groups (as test groups) rabbits were given (20 and 40 mg/kg/day) of the val respectively, as suspension in normal saline for eight consecutive days. on the eighth day, cbz was co-administered an hour after adding the last dose of val suspension. venous blood samples (1.0-1.5 ml) were obtained from rabbits' ears' marginal vein at predetermined different periods. the plasma of this blood separation was done using centrifugation and stored at -80°c, prior to analysis by using cbz chemiluminescent enzyme immunoassay detection kit. different pk parameters such as cmax, tmax, t½, ke, auc0-24 and auc0-∞ were determined for the three groups, applying statistical testing (anova). the results showed statistical insignificant differences for all pk parameters among the three groups with (p˃0.05). the findings showed that val at both concentrations is not likely to interfere with pk parameters related to cbz, further confirmation in humans shoud be done before these findings are applied to patient care. keywords: carbamazepine, cyp3a4, valerian, herb-drug interaction pharmacokinetic parameters. على عوامل بجرعات مختلفة حشيشة الهرثير التعاطي الفموي لمستخلص تأ حركية الكاربامازيبين في االرانب 1*،عصام محمد ابو شمالة . غزة، فلسطين –جامعة األزهر ، كلية الصيدلة م الصيدالنيات والصيدلة الصناعية،قس* الخالصة بتركيزات مختلفة على ( val) مستخلص حشيشة الهربين ( pk) فى عوامل حركية الدواءصممت الدراسة للتحقق من وجود تداخل معشاة في ذكور األرانب السليمة( cbz) للكاربامازبين عوامل حركية الدواء ، متوازية ، أعطيت مستخدمين ارانب سليمة صحيا ذات شواهد الثالث المجموعات في والثالثة)األرانب والثانية من ( األولى فموية للمجموعتين ( كلغم /ملغم cbz (50)جرعات بالنسبة والثالثة"؛ " الثانية ل ملحي عادي لمدة في محلو (val) مستخلص حشيشة الهرمن ( يوم / كلغم /ملغم 40و 20)أعطيت األرانب على التوالي ( كمجموعات اختبار) متتالية قاعدة على أيام إعطاء. ثمانية تمت ، الثامن اليوم من cbz في جرعة آخر إضافة من ساعة بعد مشترك الهربشكل حشيشة مستخلص val).) من الوريد الهامشي آلذان األرانب في فترات مختلفة ومن ثم تم إجراء فصل الدم ( مل 1.5-1.0)تم الحصول على عينات الدم الوريدي درجة مئوية قبل التحليل باستخدام مجموعة الكشف المناعي لإلنزيم الكيميائي 80-بالبالزما باستخدام الطرد المركزي وتم تخزينه في درجة حرارة للمجموعات الثالث ومن ثم تم تطبيق 0and auc 24-0, aucek½, , tmax, tmaxc-∞ ثل مختلفة م pkتم تحديد معلمات . الخاصة للكاربامازبين بين المجموعات عوامل حركية الدواءلجميع ( p˃0.05)أظهرت النتائج فروق ذات داللة إحصائية غير معنوية (. anova)االختبار اإلحصائي ، يجب إجراء مزيد cbzالمتعلقة بـ pkفي كال التركيزين من غير المحتمل أن يتداخل مع معلمات مستخلص حشيشة الهرتظهر النتائج أن . الثالث . من التجارب قبل تطبيق هذه النتائج في رعاية المرضى الحركية الدوائية. معامالت ، التفاعالت الدوائية ، مستخلص حشيشة الهر، الكاربامازيبين :المفتاحية الكلمات introduction herbal-drug interactions can be characterized as either pharmacodynamic or pharmacokinetic (pk) by nature. pharmacodynamic interactions may occur when constituents of herbal products have either synergistic or antagonist activity concerning a conventional drug meanwile, pk interactions result from alteration of absorption, distribution, metabolism, or elimination of a conventional drug by herbal product or other food supplements (1,2). 1corresponding author e-mail: issam.abushammala@uv.es received: 22/6/2021 accepted: 6/10 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp220-224 iraqi j pharm sci, vol.31(1) 2022 valerian-carbamazepine interaction 221 carbamazepine is a common drug used for treating partial and tonic-colonic seizers (3). cytochrome p450 (cyp450) monooxygenase is a superfamily of hemoproteins, which is responsible for the phase i metabolism of various xenobiotics and some endogenous substances as steroids. nearly 70–80% of all prescribed drugs are metabolized by the cyp system. cyp3a4 is involved in cbz metabolic sequences. carbamazepine has a narrow range of therapy and might show drug interaction with several drugs either by induction or inhibition of cyp3a4. despite its clinical popularity, cbz possesses several pk properties which make it a possible candidate to interact with substances upon coadministration, including synthetic drugs, pharmaceutical herbs and food. cbz is classified not only as an effective inducing agent of cyp450 system, but also as autoinduction agent (4-8). valerian (val) is the common name given to the crude drug consisting of underground organs of species of (valerianaceae). the major components of val include the monoterpenebornyl acetate and the sesquiterpene valerenic acid which, in addition to other types of sesquiterpene, are characteristic features of the species. the constituents of the volatile oil are variable due to population differences in genetics and environmental factors. val -as a traditional herbwas used to treat insomnia and anxiety. it is also considered as the largest attended nonprescribed sedative in european countries (9,10). herb/cbz interaction was catchy and crucial topic for many researchers all over the world (11,12). therefore, interaction potency should be taken into consideration upon using herbs as medication to avoid reduction in drug efficiency and/or increase the side effects of concomitantly administered drugs (13). possible herb-drug interaction of val-cbz is still underestimated. this research was conducted to study the effect of co-administration of val at different doses on pk parameters of cbz in rabbits. subjects and methods animals eighteen new zealand strains of adult male rabbits weighted (3.1-3.4 kg) and aged (7-9) months were divided into three groups (six subjects in each) and were used as animal model for the current drug – herb pk interaction study. all rabbits were kept under standard laboratory conditions in a 12-hour light/dark cycle at 25°c ± 2°c provided with pellet diet with free acces to water (ad libitum) and were fasted overnight before the experiments. study design and blood sampling the study design was parallel-randomized controlled trial. eighteen male rabbits were assigned into three groups. in the first group (control group), rabbits were given orally a volume equivalent to cbz (50 mg/kg) from a 2% oral suspension (tegretol, novartis). post dosing venous blood samples (1.0-1.5 ml) obtained from ear vein of the rabbits using special cannula (21g) at different time periods (0.50, 1.0, 1.50, 2.00, 2.50, 3.50, 4.0, 6.00, 12.00, 18.0 and 24.00 hr) (14). in the second and third groups (test groups), the rabbits were given a volume of cbz 2% oral suspension also in a dose of (50 mg/kg) at the same conditions as in the first group concurrently with volumes equivalent to (20 and 40 mg/kg/day, repectively) val suspension. this suspension was commercially prepared by pulverizing the film coated tablets of val relaxin, lab. pharma trenker) in normal saline. val suspension was given for eight continuous days. on the eighth day, cbz was administered one hour after administration of the last dose of val suspension, and blood was collected from rabbits from the second and the third groups at the same periods mentioned above. plasma was collected by centrifugation of samples and was stored at (-80°c) until analysis. the analysis was performed by cbz detection kits (cleia) and immulite 1000 immunoassay system. pk parameters determination the plasma level -time profile of cbz was constructed for the control and herb treated groups. the pk parameters of cbz in the control and test groups were calculated using model -independent approach (non-compartmental model (ncm)). winnonlin professional software (version 6.3, pharsight corporation, cary, nc) and (graphpad prism versión 4.00, san diego, ca, usa) were used in the calculation and statistical analysis of the following pk parameters: maximum plasma concentration (cmax), time to reach maximum plasma concentration (tmax), elimination rate constant (ke), elimination half-life (t1/2), area under plasma concentration -time curve up to 24 hours (auc0–24) and to the infinit time (auc0–∞) for the control and herb treated groups. statistical analysis data were presented as mean (plasma conc. of cbz) ± sd. differences in pk parameters of cbz upon administering without val (first group) and with val suspension at different concentrations (second and third groups) were assessed by analysis of variance (anova) using spss program (version 22.0) taking 95 % confidence interval and significant statistical differences as p value ˂0.05. iraqi j pharm sci, vol.31(1) 2022 valerian-carbamazepine interaction 222 results this study was carried out to determine the influence of val at two different concentrations (20 and 40 mg/kg/day) on pk parameters of cbz when given concurrently. the pk parameters of cbz in the control group (first group) were compared to those of the test groups treated with val 20 and 40 mg/kg/day (second and third groups respectively). plasma level -time profiles of cbz in the three groups are shown in figure 1. the calculated pk parameters: cmax, tmax, t1/2, ke, auc0-24 and auc0-∞ of the three groups were mentioned in table 1. in this study, cmax of cbz (first group) was 6.64±1.12 µg/ml and tmax was 1.95±0.22 h. the rabbits treated with val 20 mg/kg/day (second group) produced cmax and tmax of 6.82±1.25 µg/ml and 2.11±0.27 h, respectively. a slight, but statistically nonsignificant, increase of cmax and tmax of cbz were found when val was coadministered with cbz (second group). the systemic exposures according to auc0-∞ showed nonsignificant differences between the two groups (94.32±11.7 µg.hr/ml vs. 73.71±28.1 µg.hr/ml; p=0.063). other pk parameters including t1/2, ke and auc0-24 showed no significant differences between both groups (table 1). figure 1. concentration -time profile of cbz without val (first group) and with val (second and third groups) in rabbits. table 1. pharmacokinetic parmeters of cbz without val (first group) and with val (second and third groups) in rabbits. pk parameters groups mean ± sd p-value cmax (µg/ml) first group 6.64±1.12 0.770¥ 0.296§ second group 6.82±1.25 third group 6.41±1.13 tmax (hr) first group 1.95±0.22 0.054¥ 0.726§ second group 2.11±0.27 third group 2.10±0.20 t1/2 (hr) first group 13.70±4.40 0.055¥ 0.131§ second group 10.30±2.87 third group 8.91±2.47 ke (hr-1) first group 0.054±0.012 0.181¥ 0.052§ second group 0.053±0.025 third group 0.081±0.018 auc0-24 (µg*hr/ml) first group 76.49±13.85 0.360¥ 0.090§ second group 62.54±21.27 third group 55.53±20.50 auc0-∞ (µg*hr/ml) first group 94.32±11.7 0.063¥ 0.173§ second group 73.71±28.1 third group 64.30±27.1 n= 6 rabbits for each group. (¥) : p value of the differences between the first group and the second group. (§) : p value of the differences between the first group and the third group. t im e ( h ) p l a s m a c o n c e n t r a t i o n o f c b z (  g / m l ) 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4 0 2 4 6 8 f i r s t g r o u p ( c b z 5 0 m g / k g ) s e c o n d g r o u p ( v a l 2 0 m g / k g / d a y + c b z 5 0 m g / k g ) t h i r d g r o u p ( v a l 4 0 m g / k g /d a y + c b z 5 0 m g / k g ) iraqi j pharm sci, vol.31(1) 2022 valerian-carbamazepine interaction 223 the estimated model-independent pk parameters of cbz alone (first group) and coadministered with val 40 mg/kg/day (third group) were also listed in table 1. in this case, the differences in cmax and tmax of cbz between first and third groups were insignificant since, ( p > 0.05). furthermore, other pk parameters including t1/2 and ke of cbz in the first and third groups were also, statistically insignificant. besides, no significant differences were recorded between the first and third groups regarding the systemic exposures defined by auc0-24 and auc0-∞ (table 1). discussion since the antiepileptic cbz drug is given on a short-term basis, the opportunity of a significant clinical interaction between cbz and coadministered substances is common. the clinical sequence of interactions may be lack of efficiency, toxicity, unexpected effects, and vague side effects (15,16). regarding the lack of studies investigating val-cbz interactions, the present study was conducted. all rabbits enrolled in this study had finished the study period without any deviation. the isoform cyp3a4 of cyp450 is being a significant enzyme that is responsible for biotransformation of 70% of the drugs in use today. as antiepileptic regimens are normally given on a long-term basis, the opportunity of a clinically significant interaction between cbz and coadministered substances is considerably high. herbal medicines, dietary supplements, and food may interact with cbz pharmacokinetically and/or pharmacodynamically which leads to potential clinical consequences. one of the contributing factors towards increasing the incidence of herbdrug interaction is the increased popularity of herbal medicines (17). some herbal products such as cassia auriculata linn., piperine (an active compound in piper longum linn.), platycodonis radix, and polygonum cuspidatum were demonstrated to increase the plasma level/oral bioavailability of cbz through decreasing the metabolism of cbz or improving gastric solubility of cbz meanwhile, others like ginkgo biloba, hu-gan-ning pian, jiawei-xiao-yao-san, and xiao-yao-san decreased the plasma level/oral bioavailbaility of cbz through increasing the metabolism of cbz via cyp 3a4 induction (18-24). the results of the pk interaction study between cbz and val commercial dry extract at two different doses (20 and 40 mg/kg/day) in rabbits showed no statistical differences in pk parameters (p>0.05). similar results were found by jiang and his collaborators when studied the effect of berberine on pk of cyp3a4 and p-gp substrates (25), who found that a statistically insignificant differences after a 2-week pretreatment with bebeerine. the pk interaction study between saikoka-ryukotsu-borei-to and cbz in rats published by ohnishi and his collaborators showed a lack of pk interactions (26). similar findings were ontained by burstein et al. who demonstrated that treatment with st john’s wort for 14 days did not further induce the clearance of cbz (27). in other study abushammala et al. demonstrated that cbz had no significantly different pharmacokinetic (pk) parameters, namely, cmax, tmax, auc0-24, auc0-∞, t½, and ke, when it was given alone or concurrently with panax ginseng (28). conclusion herb-drug interaction is of great importance for patient safety, especially with the increased popularity of herbal medicines. pk interaction of cbz was recorded when it was coadministered with some herbs. in this study, val spspension that was prepared from valerian root extract showed minimal effect on pk profile of cbz. further research should also be performed by using higher val doses, a longer treatment duration and a larger sample size before the confirmation of the present results in humans. ethical statement the study was approved and performed under ethical principles laid down by the faculty of pharmacy, al-azhar university-gaza, palestine. conflict of interest no conflicts of interest relevant to this article. acknowledgments the authors wish to express deepest gratitude and thanks to all staff in the faculty of pharmacy at al-azhar university-gaza for their kind support, especially for dr. mai ramadan for her scientific support and consultancy. references 1. fugh-berman a. herb-drug interactions. lancet. 2000; 355(9198):134-138. 2. chavez ml, jordan ma, chavez pi. evidencebased drug-herbal interactions. life sci. 2006;78(18):2146-2157. 3. nolan sj, marson ag, weston j, tudur sc. carbamazepine versus phenobarbitone monotherapy for epilepsy: an 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al. interactions between traditional herbal and western medicines. lack of pharmacokinetic interactions between saiko-ka-ryukotsu-boreito and carbamazepine in rats. european journal of drug metabolism and pharmacokinetics. 2001;26(1, 2):129-135. 27. burstein a, horton r, dunn t, alfaro r, piscitelli s, theodore w. lack of effect of st john’s wort on carbamazepine pharmacokinetics in healthy volunteers. clin. pharmacol ther. 2000;68:605-612. 28. abushammala im, el-shaikh ali fk, abu shammaleh kf, taha mm, miqdad my. effect of panax ginseng on carbamazepine pharmacokinetics in rabbits. turk j pharm sci. 2021;25:18(1):17-20. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.25(1) 2016 gestational diabetes mellitus and hormonal alteration 37 gestational diabetes mellitus and hormonal alteration sura a.abdul sattar *, 1 ,amer h.abdulla * and aufaira sh. nsaif ** * department of chemistry, college of science, al-mustansiriya university , baghdad, iraq. ** national diabetes center, al-mustansiriya, baghdad, iraq. abstract gestational diabetes mellitus is known as carbohydrate intolerance first detected during pregnancy. pregnancy is periods of intense hormonal changes. the aim of the present study was to investigate a possible relation between the changes in serum hormones such as luteinizing hormone (lh) , follicle stimulating hormone(fsh), progesterone, and prolactin with gestational diabetes mellitus. thirty patients with gestational diabetes mellitus aged (22 -40) year attending the national center for treatment and research of diabetes/ al-mustansiriya university in baghdad and 29 controls aged (20-39) year were participated. hormonal tests including, fsh, lh, progesterone, and prolactin were detected by using enzyme linked fluorescent assay (elfa) kits. the demographic characteristics of gestational diabetes mellitus indicated that the most commonly affected age at (20-29) year (50%) ,60% of patients had body mass index(bmi) at ≥ 30kg/m 2+ , 76.6% of patients at first trimester of pregnancy, 23.3% had previous abortion,60% at the first pregnancy ,and 46.6% of patients had urine protein with one plus. a highly significant increase (p≤0.001) in fasting serum glucose(fsg), lh, fsh, progesterone, and prolactin were observed in sera of gestational diabetes mellitus patients in comparison to that of control pregnancy group. a non-significant correlation of fsg with age, bmi, lh, fsh, and progesterone were demonstrated. while a significant positive correlation of fbs with prolactin was found. it is conclude that higher prolactin level in pregnancy possibly played a role in gestational diabetes mellitus partly by impairing the functions of insulin, and result in hyperglycemia. keyword: gestational diabetes, fsh, lh, progesterone, prolactin. داء السكري الحملي والتغيراث الهرمىويت سري احمد عبد الستار ،*1 عبد هللا امر حسه،ع * وصيف ة شاكرريو عف ** * فزع انكًٛٛاء،كهٛت انعهٕو ، انضايعت انًسخُصزٚت ، بغذاد ، انعزاق . ** .انًزكش انٕطُٙ نبحٕد انسكز٘ ، انضايعت انًسخُصزٚت ، بغذاد ، انعزاق الخالصة دٔرة يٍ انحًم انذ٘ ًٚزم فخزة خالل يزة ألٔل اكخشف انكزبْٕٛذراحٙ ْٕ يعزٔف بعذو انخحًم ٔكًا داء انسكز٘ انحًهٙ اٌ انٓزيٌٕ يزم انذو فٙ انٓزيَّٕٛ انخغٛزاث بٍٛ يحخًهت عاللت عٍ ٔصٕد انحانٛت انٗ انكشف انذراست حٓذف .انًكزفت انٓزيَٕٛت انخغٛزاث ضاث يٍ انًزٚ 03ْذِ انذراست ضًج. انحًهٙ انسكز٘ يزض يع ٔانبزٔالكخٍٛ انبزٔصسخزٌٔ، انٓزيٌٕ انًحذ نهضزٚب،، انهٕحُٛٙ ًزكش انٕطُٙ نعالس ٔابحاد انسكز٘ فٙ انضايعت انًسخُصزٚت ببغذاد بًذٖ انٔحضزٔا انحًهٙ انسكز٘ داء يٍ ٚعاٍَٛ انهٕاحٙ حضًُج سُت . ( 02-23) بٍٛ يٍ انحٕايم غٛز انًصاباث بانسكز٘ بًذٖ عًز٘ حزأط 22ٔ سُت( 0322) بٍٛ عًز٘ حزأط ٔانبزٔالكخٍٛ باسخخذاو عذة االنفا انبزٔصسخزٌٔ، انٓزيٌٕ انًحذ نهضزٚب،، انهٕحُٛٙانٓزيٌٕ انفحٕصاث انٓزيَٕٛت (elfa)ٔكخهت ،٪(03) سُت( 22-23) سٍ حكٌٕ فٙ عادة حأرزا األكزز انحًهٙ أٌ نذاء نسكز٘ ًٕٚغزافٛت.اظٓزث َخائش انذراسّ انذ سابك، اصٓاض نذٚٓى٪ 20.0 انحًم، يٍ األٔنٗ انزالرت األشٓز ٍْ فٙ انًزٚضاث % ي50.00ٍٔ 03 ≥ انًزضٗ يٍ٪ 03 انضسى ل يعُٕٚت عانٛت ٔصٕد سٚادة بُٛج انُخائش . بمًٛت سائذ ٔاحذ انًزٚضاث يٍ٪ 00.0 ٔكاٌ بزٔحٍٛ انبٕل ل األٔل، ْٕ انحًم٪ 03 (p≤0.001) ٙفٙ ٔانبزٔالكخٍٛ انبزٔصسخزٌٔ، انٓزيٌٕ انًحذ نهضزٚب، انٓزيٌٕ انهٕحُٛٙ، يسخٕٖ كم يٍ سكز انذو انصٛايٙ ، ف حى ارباث َفسّ انٕلج فٙ. انحٕايم غٛز انًصاباث بذاء انسكز٘ نًضًٕعت حهك يع يمارَت انحٕايم انًصاباث بذاء انسكز٘ يصم دو حبٍٛ ٍحٛ فٙ انبزٔصسخزٌٔ ، انٓزيٌٕ انًحذ نهضزٚب،ٔ عذو ٔصٕد عاللت يعُّٕٚ نسكز انذو انصٛايٙ يع انعًز ، انٓزيٌٕ انهٕحُٛٙ، دٔر انٗ نّ انحًم فٙ انبزٔالكخٍٛ يسخٕٖ ارحفاع أٌ َسخُخش. انبزٔالكخٍٛ بٍٛ سكز انذو انصٛايٙ ْٔزيٌٕ عاللت يعُّٕٚ يٕصبت ٔصٕد .انذو فٙ انسكز ارحفاع إنٗ يًا ٚؤد٘ األَسٕنٍٛ، ٔظائف يٍ خالل انخعطٛم فٙ انحًهٙ انسكز٘ حذ يا فٙ االصابت بذاء سكر الحمل ، الهرمىن المحث للجريب ،الهرمىن اللىتيىي ، البروجستيرون ، البروالكتيه.الكلماث المفتاحيت: introduction gestational diabetes mellitus (gdm) is known as carbohydrate intolerance first revealed during pregnancy (1) . it is the most public metabolic disturbance of pregnant women, involves between 2% and 5% of pregnant women (2) . diabetes mellitus type1 and type 2 could be diagnosed newly or secondary to metabolic changes related to pregnancy (3) .gestational diabetes mellitus accounts for ninety percent of cases of diabetes 1 corresponding author e-mail: sura742003@yahoo.com. received: 10/1/2016 accepted: 12/4/2016 mailto:sura742003@yahoo.com iraqi j pharm sci, vol.25(1) 2016 gestational diabetes mellitus and hormonal alteration 38 mellitus in pregnancy, however eight percent of cases are preexisting type 2 (4) . gdm reveals usually between 24 and 28 weeks of gestation (5) .a large number of risk factors related to gdm increase have been fixed, including history of macrosomia, strong family history of diabetes, ethnicity, metabolic syndrome and obesity (2,6) .pregnancy is periods of intense hormonal changes (1) . women at risk for gestational diabetes have insulin resistance before conception. insulin resistance during pregnancy stems from a variety of factors, including alterations in growth hormone and cortisol secretion , human placental lactogen secretion , and insulinase excretion which is produced by the placenta and facilitates metabolism of insulin (7) . moreover, the steroid hormones such as progesterone and the estrogens have been shown to be involved in β-cell physiology and disturbance of the glucose insulin balance (8,9) . luteinizing hormone (lh) and folliclestimulating hormone (fsh) are omit gonadotropins are secreted from gonadotrophs cells in the anterior pituitary. most gonadotrophs secrete only lh or fsh, but some time both hormones (10) . in female, lh stimulates secretion of sex steroids from the gonads progesterone and estradiol (10) . progesterone is necessary for pregnancy reservation, and, in most mammals, lh is required for function and growth of corpora lutea (10) . lh levels will decrease if pregnancy occurs, and luteal function will instead be maintained by the action of human chorionic gonadotropin, which is similar to lh and secreted from the new placenta (11) . the prolactin(prl), is major stimuli for the adaptation of the endocrine pancreas during gestation (12) . prolactin is a polypeptide originally known as a pituitary hormone for its ability to promote lactation in response to the breast feeding (13) . besides its well-known lactogenic properties, prolactin is also a highly versatile hormone whose functions are related to reproduction, growth and development, metabolism, immune regulation, brain function, and behavior (14,15) . maternal prolactin increases synchronously to insulin during the second half pregnancy and stimulates β-cell proliferation, insulin production, and insulin secretion (16,17) . moreover, it is known that the glucose metabolic regulation impact of prolactin is not close to the period of pregnancy (15,18,19) . the aim of the present study was to investigate a possible correlation between the changes in serum hormones such as lh, fsh, progesterone, and prolactin with gestational diabetes mellitus. materials and methods case control study was used in the present where 30 women with gestational diabetes mellitus aged (22 -40) years as case and 29 age-matched of pregnant women aged (20-39) years as control attending the national center for treatment and research of diabetes/ almustansiriya university in baghdad. exclusion criteria were: age <20 years, diabetes diagnosed before pregnancy, physical disability, medications like oral corticosteroids and metformin, and multiple pregnancies. five milliliters of venous blood samples were collected from the gestational diabetes mellitus and the healthy pregnant as controls group then immediately transferred into plane tube and allowed to coagulate at room temperature then centrifuged at 3000 rpm for 5 min. the resulting serum was separated and stored at (-20°c) until use. bmi is being calculated directly as weight (in kilograms)/ height 2 (in meters). hormonal tests including, fsh, lh, progesterone, and prolactin were detected by using enzyme linked fluorescent assay (elfa) kits in mini vidas system and performed according to the manufacturer's instructions. statistical analysis the statistical software (spss v 15; chicago, il, usa) was used. the data were analyzed using unpaired t-test and person correlation coefficients. differences were considered significant when p< 0.05. results and discussion table 1 show the baseline characteristics of gestational diabetes mellitus where it was found that the most commonly affected age at (20-29) year(50%) ,60% of patients had bmi at ≥ 30kg/m 2+ , 76.6% of patients at first trimester of pregnancy, 23.3% had previous abortion,60% at the first pregnancy ,and 46.6% of patients had urine protein with one plus. https://en.wikipedia.org/wiki/pregnancy https://en.wikipedia.org/wiki/human_chorionic_gonadotropin https://en.wikipedia.org/wiki/human_chorionic_gonadotropin http://care.diabetesjournals.org/content/36/7/1974.long#ref-1 http://care.diabetesjournals.org/content/36/7/1974.long#ref-2 http://care.diabetesjournals.org/content/36/7/1974.long#ref-3 http://care.diabetesjournals.org/content/36/7/1974.long#ref-4 http://care.diabetesjournals.org/content/36/7/1974.long#ref-5 http://care.diabetesjournals.org/content/36/7/1974.long#ref-3 http://care.diabetesjournals.org/content/36/7/1974.long#ref-6 http://care.diabetesjournals.org/content/36/7/1974.long#ref-7 iraqi j pharm sci, vol.25(1) 2016 gestational diabetes mellitus and hormonal alteration 39 table (1): baseline characterization of gestational diabetes mellitus. category number (%) age in year 20-29 30-39 40-49 15(50%) 14(46.6%) 1(3.33%) bmi ≤19 20-24 25-29 ≥30 0(0%) 1(3.33%) 11(36.66%) 18(60%) trimester first second third 23(76.66%) 6(20%) 1(3.33%) pregnancy history previous abortion no abortion first pregnant 7(23.3%) 23(76.66%) 18(60%) urine protein trace + ++ +++ 5(16.66%) 14(46.66%) 9(30%) 2(6.66%) meanwhile a highly significant increase of bmi values was observed in gestational diabetes mellitus patients in comparison to that of control group (p=0.00) as shown in table 2. this result was in line with torloni mr et.al who reported that the gdm is positively associated with prepregnancy bmi where for every 1 kg/ m 2 increase in bmi, the prevalence of gdm increased by 0.92% (20) . the results represented in table 2 indicated highly significant increases (p=0.00) in fsg, lh, fsh, progesterone, and prolactin in sera of gestational diabetes mellitus patients in comparison to that of control pregnancy group.insulin secretion in women with gdm is defective and, therefore, is unable to rise adequately to compensate for the insulin resistance; the result is hyperglycemia (21) . lh and fsh play central roles in the hypothalamic-pituitary-gonadal axis, and, thus, conditions related to lh and fsh increases can be caused by pathology of either the hyperthalamus or pituitary (22) . lh increases almost always occurs in conjunction with follicle-stimulating hormone (fsh) increases because lh and fsh are secreted by the same pituitary gonadotrope cells. table (2): mean values of bmi, f.s.g, lh, fsh, progesterone, and prolactin levels in sera of patient and control groups. parameters group range (min-max) mean sd p value bmi (kg/m 2 ) control 19.00-28.40 23.5724 1.9063 0.00 patients 21.90-35.50 29.8967 3.0337 fsg (mg/dl) control 65.00-100.00 84.5172 32.6097 0.00 patients 130.40-260.00 180.0900 32.6097 lh (pg/ml) control 9.10-17.10 14.3966 1.8084 0.00 patients 9.70-30.10 19.6633 4.6668 fsh(pg/ml) control 8.80-18.70 15.8138 1.9913 0.00 patients 10.10-34.20 20.1533 5.3723 progesterone(pg/ml) control 10.20-104.00 44.7276 19.7237 0.00 patients 35.40-92.50 75.3533 12.7009 prolactin(pg/ml) control 13.70-42.10 27.2414 8.0499 0.00 patients 38.00-80.10 52.9900 12.2372 though a highly significant increase of progesterone level((p=0.00) was observed in the present study in sera of gestational diabetes mellitus in comparison to that level in control group (table 2), we demonstrated a nonsignificant correlation ((p˃0.05) of progesterone with fbs as shown in table 3. in previous studies of progesterone conflict data was reported where one of them has found association of progesterone with the development of gestational diabetes due to the enhancement of insulin resistance (23,24) , while another group did not find a correlation between progesterone level and an increasing risk of gestational diabetes mellitus (25) . in diabetes, the death of insulin-producing β-cells by apoptosis leads to insulin deficiency. the lower prevalence of diabetes in females suggests that female sex steroids protect from β-cell injury (26) . in addition straub et al. suggested that the progesterone, might contribute to the poor adaptation of insulin secretion and action to the increased requirements during pregnancy (27). http://www.ncbi.nlm.nih.gov/pubmed/?term=torloni%20mr%5bauthor%5d&cauthor=true&cauthor_uid=19055539 http://joe.endocrinology-journals.org/content/221/2/273.long#ref-40 iraqi j pharm sci, vol.25(1) 2016 gestational diabetes mellitus and hormonal alteration 40 table (3): correlations of fsg with age, bmi, and hormonal parameters. parameters r value p value age 0.302 0.105 bmi 0.252 0.179 lh -0.151 0.424 fsh -0.148 0.434 progesterone 0.173 0.360 prolactin 0.427 0.019 the present study indicated a highly significant increase in progesterone level in sera of gestational diabetes mellitus in compression to that of control group. this result was in agreement with guyton & hall who’s reported that no significant difference in plasma prolactin in normal or diabetic pregnant in fact it level might be lower in the pregnant with gdm (28) . while this result was in disagreement with the results of milasinovic l. et.al who reported that there were no significant differences in the level of plasma prolactin in normal or diabetic pregnancies; in fact its level might be lower in the pregnancies with gestational diabetes mellitus and, prolactin might have no effect on glucose intolerance during pregnancy (29) . arumugam r et al reported that prl up-regulates β-cell glucose uptake and employment, whereas glucose increases islet prl receptor expression and more effects of prl on cell cycle gene expression and dna synthesis (30) . the results presented in table 3 indicated a non-significant correlation of fsg with age, bmi, lh, fsh, and progesterone. on the other hand it was observed a significant positive correlation of fsg with prolactin. this result was in disagree with previous study which demonstrated that there is no correlation between the prolactin concentrations and the impairment of glucose tolerance and as a result the abnormal prolactin levels are not of pathophysiologic importance for the development of gdm (31) . conclusion in the light of the present results the findings confirmed that higher prolactin level in pregnancy possibly played a role in gestational diabetes mellitus partly by impairing the functions of insulin, and result in hyperglycemia. references 1. hawryluk j, grafka a, gęca t,and łopucki m.gestational diabetes in the light of current literature. pol merkur lekarski. 2015; 38(228):344-7. 2. ramos-román ma.prolactin and lactation as modifiers of diabetes risk in gestational diabetes horm metab res. 2011; 43(9):593-600. 3. mahmoud f, abul h, dashti a, al-jassar w, omu a. trace elements and cellmediated immunity in gestational and pregestational diabetes mellitus at third trimester of pregnancy. ta med acad. 2012; 41(2): 175-85. 4. thomas r moore, md; chief editor: george t griffing, m. diabetes mellitus and pregnancy: practice essentials. 2014. emedicine. medscape.com/article/127547overview. 5. hawryluk j, grafka a, gęca t, łopucki m . gestational diabetes in the light of current literature pol merkur lekarski. 2015; 38(228):344-7 6. wójcik m, chmielewska-kassassir m , grzywnowicz k, woźniak l, cypryk k .the relationship between adipose tissuederived hormones and gestational diabetes mellitus (gdm). endokrynol pol.2014; 65 (2) :134-42. 7. amanda “bird” hoffert gilmartin, serdar h ural, md, and john t repke, md. gestational diabetes mellitus. rev obstet gynecol. 2008 summer; 1(3): 129–134. 8. lenzen s &bailey cj.thyroid hormones, gonadal and adrenocortical steroids and the function of the islets of langerhans. endocrine reviews. 1984; 5:411–434. 9. straub sg, sharp gw, meglasson md & de souza cj. progesterone inhibits insulin secretion by a membrane delimited, nongenomic action. bioscience reproduction. 2001; 21 :653–666. 10. bowen r. gonadotropins: 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http://www.ncbi.nlm.nih.gov/pubmed/?term=lenzen%20s%5bauthor%5d&cauthor=true&cauthor_uid=24594616 http://www.ncbi.nlm.nih.gov/pubmed/?term=progesterone+induces+apoptosis+of+insulin-secreting+cells%3a+insights+into+the+molecular+mechanism http://www.ncbi.nlm.nih.gov/pubmed/?term=progesterone+induces+apoptosis+of+insulin-secreting+cells%3a+insights+into+the+molecular+mechanism http://www.ncbi.nlm.nih.gov/pubmed/?term=progesterone+induces+apoptosis+of+insulin-secreting+cells%3a+insights+into+the+molecular+mechanism http://jme.endocrinology-journals.org/search?author1=hisashi+masuyama&sortspec=date&submit=submit http://jme.endocrinology-journals.org/search?author1=yuji+hiramatsu&sortspec=date&submit=submit http://www.pnas.org/search?author1=cedric+le+may&sortspec=date&submit=submit http://www.pnas.org/search?author1=khoi+chu&sortspec=date&submit=submit http://www.pnas.org/content/103/24/9232.abstract?ijkey=e4e5b7fe1e45063cd61c011e65f18b3feafca745&keytype2=tf_ipsecsha#aff-2 http://www.pnas.org/search?author1=min+hu&sortspec=date&submit=submit http://www.pnas.org/search?author1=christina+s.+ortega&sortspec=date&submit=submit http://www.pnas.org/search?author1=evan+r.+simpson&sortspec=date&submit=submit http://www.pnas.org/content/103/24/9232.abstract?ijkey=e4e5b7fe1e45063cd61c011e65f18b3feafca745&keytype2=tf_ipsecsha#aff-3 http://www.pnas.org/search?author1=kenneth+s.+korach&sortspec=date&submit=submit http://www.pnas.org/content/103/24/9232.abstract?ijkey=e4e5b7fe1e45063cd61c011e65f18b3feafca745&keytype2=tf_ipsecsha#aff-4 http://www.pnas.org/search?author1=ming-jer+tsai&sortspec=date&submit=submit http://www.pnas.org/content/103/24/9232.abstract?ijkey=e4e5b7fe1e45063cd61c011e65f18b3feafca745&keytype2=tf_ipsecsha#aff-2 http://www.pnas.org/search?author1=franck+mauvais-jarvis&sortspec=date&submit=submit http://www.ncbi.nlm.nih.gov/pubmed/?term=skouby%20so%5bauthor%5d&cauthor=true&cauthor_uid=3510013 http://www.ncbi.nlm.nih.gov/pubmed/?term=k%c3%bchl%20c%5bauthor%5d&cauthor=true&cauthor_uid=3510013 http://www.ncbi.nlm.nih.gov/pubmed/?term=hornnes%20pj%5bauthor%5d&cauthor=true&cauthor_uid=3510013 http://www.ncbi.nlm.nih.gov/pubmed/?term=andersen%20an%5bauthor%5d&cauthor=true&cauthor_uid=3510013 http://www.ncbi.nlm.nih.gov/pubmed/3510013 iraqi j pharm sci, vol.31( suppl. ) 2022 combiflash and hplc doi: https://doi.org/10.31351/vol31isssuppl.pp54-61 54 phytosterol profile in iraqi lactuca serriola after purification and isolation by combiflash and hplc (conference paper )# murtadha saleh hussien*,1 and ayad a. al-hamashi** # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq **pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq abstract one of these plants utilized in traditional medicine is lactuca seriolla linn., which belongs to the asteraceae family. it goes by a variety of common names in the world, including prickly lettuce, wild lettuce, jagged lettuce, and kahu and khas. the work aimed to isolate and characterize some bioactive constituent(s) from the aerial part of lactuca serriola utilizing combiflash nextgen and high-performance liquid chromatography (hplc). lactuca serriola (aerial part) was extracted with 80% ethanol, then fractionated with hexane. then 250 mg of hexane extract was mixed with 4 g of silica gel and loaded in cartilage, then bounded to the gold column of combi flash using a solvent system comprised of ethyl acetate: n-hexane (10% ethyl acetate 90% hexane v/v) to elute the column. the fractions resulting were analyzed by thin layer chromatography and high-performance liquid chromatography (hplc). white needle-like crystalline substances were isolated via combi flash column chromatography. the isolated product was analyzed by thin layer chromatography and high-performance liquid chromatography (hplc). β-sitosterol (11 mg) and stigmasterol (7 mg) were obtained from 250 mg of hexane fraction. keywords: combiflash column chromatography, hplc, lactuca serriola, β-sitosterol, stigmasterol. العراقي بعد التنقية والعزل الكشف عن الستيرويدات النباتية في نبات الخس البري # ) بحث مؤتمر ( , الفالش كروماتوغرافي ( السائلة عالية األداء بواسطة)الفوتوغرافيا ** الهماشي علي عبداياد و 1*،حسين صالحمرتضى 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي العراق بغداد، بغداد، جامعة الصيدلة، كلية الطبية، فرع العقاقير والنباتات * كلية الصيدلة، جامعة بغداد، بغداد، العراق الكيمياء الصيدالنية، فرع ** الخالصة يُطلق عليه مجموعة العائلة النجمية . ، الذي ينتمي إلى .lactuca seriolla linn أحد هذه النباتات المستخدمة في الطب التقليدي هو في ، والخس ال الشائك، بما في ذلك الخس العالم،متنوعة من األسماء الشائعة البري ، ووالخس يهدف العمل إلى عزل .khas و kahu مسنن والكروماتوغرافيا combiflash nextgen باستخدام lactuca serriola منالهوئي وتوصيف بعض المكونات النشطة بيولوجيًا من الجزء ، ثم تجزئته مع الهكسان. soxhletبواسطة ٪ إيثانول 80)جزء هوائي( مع lactuca serriolaتم استخالص . (hplc) السائلة عالية األداء من فالش gold column ب ، ثم تم ربطها cartilage جم من هالم السيليكا وتم تحميلها في 4مجم من مستخلص الهكسان مع 250ثم تم خلط من . كومبي يتكون مذيب نظام اال ألزل (ethyl acetate 90% hexane v/v %10)باستخدام تحليل تم طريق جزاءالعمود. عن الناتجة األداء عالية السائلة والكروماتوجرافيا الرقيقة الطبقة طريق .(hplc) كروماتوجرافيا عن باإلبرة الشبيهة البيضاء البلورية المواد عزل تم كروماتوجراcombiflash nextgenكروماتوجرافيا بواسطة المعزول المنتج تحليل تم عالية . السائلة والكروماتوجرافيا الرقيقة الطبقة فيا الهكسان. مجم من جزء 250مجم( من 7مجم( وستيجماستيرول ) 11سيتوستيرول )-تم الحصول على بيتا .(hplc) األداء .ستيجماستيرول ، سيتوستيرول-بيتا، ((hplcالتحليل بواسطة ، combiflash nextgeالفصل بواسطة ، الخس البري فتاحية :الكلمات الم introduction plants have been utilized as a medicine source for thousands of years for most of the world's population (1). early civilizations in china, india, and the near east used medicinal plants as a source of sickness relief (2,3). per the world health organization (who), traditional medicines are used by more than 80% of the world's population for primary health care. the medical value of plants has related to the presence of chemical compounds that have various pharmacological effects on the human body. alkaloids, flavonoids, tannins, and phenolic compounds are examples of critical bioactive molecules (4). botany, chemistry, and pharmacology are all involved in researching medications derived from plants. botany deals with the classification and cultivation of plants. isolation, identification, and quantification of components in plant materials are all part of chemical characterization. pharmacology studies plant components' physiological effects on animal and human cells. 1corresponding author e-mail: mortadhasalih568@gmail.com received:28 /6 /2022 accepted:5 /9 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp54-61 iraqi j pharm sci, vol.31(suppl.) 2022 combiflash and hplc 55 taxol/paclitaxel, vinblastine, vincristine, etoposide, atropine, morphine, ergotamine, physostigmine, digoxin, and other critical medications utilized in modern medicine have all emerged from medicinal plant research. nowadays, linking folkloric uses of plants and expert knowledge of herbal medicine to modern research operations provides a noveltechnique that significantly increases the rate of drug discovery over random collection (5). asteraceae species involve many phytochemical components such as phytosterol, essential oils, lignans, saponins, polyphenolic compounds , phenolic acids, sterols, and polysaccharides that have medicinal effects (6). lactuca seriolla is one of these wild plants belonging to the asteraceae family (7). lactuca seriolla l in, a member of the asteraceae family, is an example of a traditional medicinal plant . prickly lettuce, wild lettuce , jagged lettuce , kahu, and kha are some of the common names used in iraq . lactuca serriola is a member of the asteraceae family that grows in arid conditions in the northern hemisphere and has a milky sap that oozes freely from wounds. when this comes into touch with air, it hardens and dries the sap contains lactucarium, which has antispasmodic, digestive, diuretic, hypnotic, tranquilizer , and sedative properties (8). phytosterols (also known as plant sterols) are essential components of plant cells. squalene, a part of the triterpene family, is the source of these chemicals. this set of molecules has 28 or 29 carbon atoms and one or two carbon-carbon double bonds, contributing to the wide range of natural structures. more than 250 compounds have been discovered so far. β-sitosterol (c-29), campesterol (c-28), and stigmasterol (c-29) are the most commonly encountered phytosterols, accounting for up to 98 percent of phytosterol dietary intake in humans (figure1) (9-11).’ figure 1. chemical structure of selected phytosterols. these substances are consumed by people and may have a substantial biological impact on maintaining and improving health (12). phytosterols compete with and block cholesterol absorption in the intestine because of their structural similarity to cholesterol, lowering ldl-c blood levels. agren et al. discovered that individuals with rheumatoid arthritis who consumed 732 mg of phytosterols daily had decreased ldl-c and total cholesterol levels (13). in addition to the physiological functions listed above, phytosterols perform a variety of essential roles in human health promotion. they can, for example, improve insulin resistance (14) and lipid metabolism (15), as well as reduce the risk of cancer (16), alzheimer's disease, and atherosclerosis-related cvds (17). in this work, we isolated and characterized several bioactive constituent(s) from the aerial part of lactuca serriola using flash chromatography and hplc. materials and methods collection of plant materials. the lactuca serriola plant materials (aerial part) were gathered at al-diwaniyah city, 180 km south of baghdad, between september and november 2021. the plant was identified, validated, and authenticated by prof. dr. sukaena abbas/ department of biology/ college of the science /university of baghdad. the plant was cleaned, allowed to dry in the shade at room temperature, ground up using an electrical miller, and weighed (18). extraction method (a hot method by soxhlet) a thimble was used for packaging 100 g of dried plant material (aerial portion). a round flask containing one liter of 85 percent ethanol was then filled, and the thimble was placed in a soxhlet extractor. approximately 22 hours at 70 c° were spent extracting the sample completely to exhaustion. using a rotary evaporator, the alcoholic extract was concentrated under reduced pressure, condensed to about 13 g, and then combined with 250 ml of distilled water to create a crude fraction (19). fractionation of extract a crude fraction of 13 g was partitioned with solvents (differ in polarity) like hexane, chloroform, ethyl acetate, and n-butanol (3x100 ml) for each fraction. the first three fractions dried over anhydrous sodium sulfate with except n-butanol fraction. the dried fractions were weighted and assigned for further analysis (20). chemical identification of phytosterol two types of chemical tests were used to recognize phytosterol compounds. 1liebermann-burchard test: a small amount of plant hexane extract was dissolved in 5 ml of chloroform, and the chloroform layer was then dried over anhydrous sodium sulfate. the mixture was then combined with ten drops of acetic anhydride iraqi j pharm sci, vol.31(suppl.) 2022 combiflash and hplc 56 and two drops of concentrated sulphuric acid. as oxidation proceeds in stages, the color is bluish green, indicating the existence of the steroidal nucleus. 2salkowski reaction: 2 ml of chloroform was used to dissolve a little amount of the hexane plant extract (f1), and then 3 ml of concentrated h2so4 was added slowly. due to the oxidation reaction, a reddish-brown hue was produced (21). preliminary identification of phytosterol (stigmasterol and β-sitosterol) by using tlc a small amount of the hexane extract was diluted with chloroform, and the resulting solution was spotted onto tlc plates that had been coated. the tlc plates were developed using particular solvent systems, examined individually with u.v. light at 236 nm, and then sprayed with the vanillinh2so4 reagent (22). isolation of proposed detected phytosterol by combi flash nextgen column chromatography from hexane fraction the purification process of flash chromatography is straightforward and requires little method development. with the teledyne isco combiflash nextgen flash chromatography system, you can automate processes with high productivity, create gradients with programmable parameters, separate peaks using u.v. light, and automatically identify columns and collecting tube racks (system dependent). its small size makes it an excellent personal system and makes it suitable for use in chemical hoods and other cramped indoor areas. in some settings, use on an open bench is permitted with the addition of an optional fraction collection enclosure. in comparison to conventional gravity-based column chromatography, separations that use a gas pressured solvent reservoir to speed up solvent flow can be completed more quickly. depending on spots that appear in tlc after the use proper solvent system (10% ethyl acetate and 90% n-hexane v/v), 250 mg of hexane extract mixed with 4 g silica gel, and this sample on silica is then poured into an empty cartridge, topped with a frit and loaded onto the system, combi flash using a solvent system comprised of ethyl acetate: n-hexane (10% ethyl acetate and 90% n-hexane v/v) to elute the column to obtain 28 fractions of 30 ml each. collected fractions were spotted on a tlc plate (figure 3)( 23). hplc analysis the hexane fraction components were separated utilizing the hplc system of knauer germany. the active components were compared to standard materials through u.v./v is absorption analysis. the retention time for the sample and standard were identified. the integrated peak area was used to assess the sample quantity, and the calibration curve content was derived by plotting the sample peak area against the concentration of the appropriate standard sample. the c18 hplc column (250  4.6) 5 m particles from the usa's water corporation were used to achieve the separation. the mobile phase used for phytosterol detection was meoh and acetonitrile of 30:70 meoh and acetonitrile, and the solvent system gradient was gradually increased to 0:100. the elution was sustained for 10 minutes, followed by 30:70 for 15 minutes in a flow rate of 1 ml/min. the wavelength of 210 nm was used for u.v. detection (24). component model company and origin 1 binary high-pressure gradient pump p6.1l knuaer, germany 2 diode array detector dad 2.1l knuaer, germany 3 sample loop (20µl) and injector d1357 knuaer, germany 4 analyses and system control software claritychrom, v 7.4.2.107 dataapex, czech republic result and discussion chemical tests were employed to identify phytosterol compounds in lactuca serriola, and the findings are shown in table 1. the libermannburchard reaction and salkowski reaction tests confirmed that steroids were present in the aerial part of lactuca serriola (25). table 1. result of the chemical tests for hexane fraction plant parts libermannburchard reaction salkowski reaction the aerial part of a plant + + stigmasterol and β-sitosterol in the aerial portion of lactuca serriola hexane extract were detected using tlc. the spot color and the rf value for stigmasterol and β-sitosterol were identical because the two compounds' structures are similar and only differ in the presence of a c22=c23 double bond in stigmasterol and a c22-c23 single bond in βsitosterol (figure 2) (26). iraqi j pharm sci, vol.31(suppl.) 2022 combiflash and hplc 57 figure 2. thin layer chromatogram for hexane fraction(h) with beta-sitosterol(b) and stigma sterol(s) std. using silica gel gf254nm as a stationary phase developed in hexane: ethyl acetate 9:1 as mobile phase. they were visualized by 5% h2so4 spray reagent followed by heating for 5-10 minutes at 110 c°. table 2. rf value of phytosterol obtained from hexane fraction of iraqi lactuca serriola compared to stigmasterol and b-sitosterol standard. standard solvent systems rf (retardation factor) value of standard rf (retardation factor) value of compound l2 stigmasterol hexane: ethyl acetate 9:1 0.26 0.25 b-sitosterol hexane: ethyl acetate 9:1 0.25 0.25 figure 3. thin layer chromatogram for hexane fraction (h)with fractions obtained by combi flash column chromatography after purification hexane fraction. using silica gel gf254nm as a stationary phase developed in (hexane: acetone 8:2 ) as mobile phase. visualized by 5% h2so4 spray reagent followed by heating for 5-10 minutes at 110 c°. the combi flash fractions were eluted through hplc for further separation. the compounds were compared with the respected standard material. hplc data indicated the presence of phytosterol. the content of the sample was computed using the calibration curve by plotting the peak area versus the concentration of the appropriate reference sample. the sample's quantity was determined by measuring the integrated peak area. the hplc analysis indicated that the combi flash fractions (12-20) contained phytosterol. on the other hand, combi flash fractions 19 and 20 contain stigmasterol only. and fractions 12,16 have a mixture of sitosterol and stigmasterol (figure 4). iraqi j pharm sci, vol.31(suppl.) 2022 combiflash and hplc 58 figure 4. hplc chromatogram analysis fractions contain stigmasterol [standard stigmasterol (-) sample (-)]. the retention time for isolated compound and stigmasterol standard is identical (16.9). figure 5. hplc chromatogram analysis fractions contain β‐sitosterol and stigmasterol(sample, standard). the retention time for isolated compound & β‐sitosterol standard is identical (24.1). iraqi j pharm sci, vol.31(suppl.) 2022 combiflash and hplc 59 figure 6. u.v. spectrum view using the diode array detector (dad) for β‐sitosterol peak. std, b-sitosterol standard absorbance(-). sample(-),β‐sitosterol peak absorbance at λ 210 nm. figure 7. u.v. spectrum view using the diode array detector (dad) for stigmasterol peak. std, stigmasterol standard absorbance(-). sample(-), stigmasterol peak absorbance at λ 210 nm. for quantitative analysis, the calibration curve was plotted using the area under the curve (auc) versus four concentration levels of stigmasterol and β-sitosterol standards from which the concentration of the proposed stigmasterol and β-sitosterol (l1 and l2) in the hexane fraction. the concentration was calculated applying a straight line equation, as illustrated in figures 6 and 7. the calculated concentration of proposed stigmasterol and β-sitosterol (l1 and l2) in the iraqi lactuca serriola is shown in table 2. iraqi j pharm sci, vol.31(suppl.) 2022 combiflash and hplc 60 figure 7. the calibration curve of stigmasterol figure 8. calibration curve of β-sitosterol table 2. the quantity of the plant's hexane fraction that contains the predicted stigmasterol and β-sitosterol (l1 and l2). samples peak area µg/ml of fraction µg/mg plant material stigmasterol 163.299 4.55566373 91.11327459 β-sitosterol 268.028 4.064778919 81.29557838 conclusion a rapid and effective method for isolating and purifying major plant sterol from lactuca serriola was performed. this method could provide reference substances for further phytochemical studies. to the best of our knowledge, this is the first report on separating phytosterol in medical plants using the flash column method in iraq. references 1. tripathi, leena, and jaindra nath tripathi. role of biotechnology in medicinal plants. tropical journal of pharmaceutical research. 2003;2 (2): 243-253. 2. salau, a. o., 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formulation and characterization of isradipine nanoparticle for dissolution enhancement rawaa m. hussien*,1 and mowafaq m. ghareeb** * department of pharmaceutics, college of pharmacy, al nahrain university, baghdad,iraq * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract isradipine belong to dihydropyridine (dhp) class of calcium channel blockers (ccbs). it is used in the treatment of hypertension, angina pectoris, in addition to parkinson disease. it belongs to the bcs class ii drug (low solubility-high permeability). the drug also suffers from extensive first pass metabolism, therefore it had low bioavailaility of approximately 15-24%. the aim of this study was to formulate and optimize a stable nanoparticles of this highly hydrophobic drug by solventantisolvent precipitation method to enhance in vitro dissolution rate and improve drug bioavailability ten formulas of isradipine nanoparticles were prepared by antisolvent precipitation method utilizing one of these polymers poloxamer 188, pva, and soluplus at different drugs: polymer ratios. the particle size, and polydispersity index (pdi) were investigated. among all the prepared nanoparticles formulas, formula (f9) which contain soluplus as a stabilizer at polymer: drug ratio of (1:0.75) and solvent: antisolvent ratio of (1:9) was considered as the optimum formula which showed increment in the solubility to about 10 times than that of the pure drug. the investigations of the drug–excipients compatibility studies by ftir and crystalline state by dsc, were done. moreover, the analysis by dsc of the nanoparticles of the selected formula (f9) indicated a reduction in the crystallinity and amorphization of the drug. in addition, ftir result indicates that hydrogen bond may be formed between soluplus and the drug, which it may have a contribution to increase affinity between them and resulted in enhanced solubility . the results of dissolution study showed a significant increase in dissolution rate through particle size reduction . so, it can be concluded that solventantisolvent precipitation method was an efficient method for preparation of isradipine nanoparticles for dissolution enhancement keywords: nanoparticles, isradipine, , dissolution rate enhancement يةجسيمات نانوية لتعزيز الذوبانك صياغة وتوصيف اإلسراديبين **موفق محمد غريب و 1*،رواء محمد حسين الصيدالنيات ،كلية الصيدلة ، جامعة النهرين ، بغداد ، العراق . فرع * . بغداد،العراق بغداد، جامعة الصيدلة، كلية ، الصيدالنيات فرع** الخالصة يستخدم في عالج ارتفاع ضغط الدم ، الذبحة الصدرية ، . اإلسراديبين ينتمي إلى فئة ديهيدروبيريدين من حاصرات قنوات الكالسيوم نفاذية -ذوبان منخفض )إنه ينتمي لالدوية المصنفة من الفئة الثانية حسب نظام تصنيف الصيدالنيات البيولوجي . باإلضافة إلى مرض باركنسون .٪24-15، وبالتالي فإن التوافر البيولوجي له منخفض بحوالي عملية ايض اولي واسعة النطاق في الكبد يعاني الدواء أيًضا من (. عالية بطريقة لهذا الدواء الغير محب للماءالهدف من هذه الدراسة هو صياغة جسيمات نانوية مستقرة من هذا الدواء شديد الكراهية للماء كان .لتعزيز معدل الذوبان في المختبر وتحسين التوافر الحيوي للدواء الترسيب بتغير المذيبات و poloxamer 188باستخدام أحد هذه البوليمرات الترسيب بتغيير المذيباتريقة تم تحضير عشر صيغ من جسيمات اإليزراديبين النانوية بط pva وsoluplus وقد تم فحص حجم الجسيمات ، ومؤشر التشتت المتعدد . نسب من دواء: بوليمر مختلفةفي(pdi.) : والمذيب( 0.75: 1) بنسبة دواء: بوليمر soluplusالتي تحتوي على ( f9)من بين جميع صيغ الجسيمات النانوية المحضرة ، اعتبرت الصيغة .مرات من الدواء النقي 10هي الصيغة المثلى التي تظهر الزيادة في الذوبان إلى حوالي ( 9: 1)نسبة مضاد المذيبات انوية للصيغة للجسيمات الن dscحيث يشير تحليل . dscوالحالة البلورية بواسطة ftirأجريت دراسات التوافق الدوائية بواسطة . إلى انخفاض في تبلور الدواء وعدم تشكيله( f9)المختارة والدواء ، والتي قد يكون لها مساهمة في soluplusإلى أن الرابطة الهيدروجينية قد تتشكل بين ftirباإلضافة إلى ذلك ، تشير نتيجة لذلك . الذوبان زيادة واضحه في معدل الذوبان من خالل تقليل حجم الجسيماتوأظهرت نتائج دراسة . زيادة التقارب بينهما وتؤدي إلى تعزيز الذوبان .، يمكن استنتاج أن طريقة ترسيب بتغير المذيبات كانت طريقة فعالة لتحضير الجسيمات النانوية لإليزراديبين لتعزيز الذوبان . زيادة الذوبانية، أسرادبين ، جسيمات نانوية الكلمات المفتاحية : 1corresponding author e-mail: rawaamohamed44@gmail.com received: 24/8/2020 accepted: 28/ 11/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp218-225 iraqi j pharm sci, vol.30(1) 2021 israd ipine nanoparticle 219 introduction frequent problems are result from a poor solubility of drug candidates in drug research and development. the aqueous solubility of the drug is a critical determinant of its dissolution rate, therefore limited dissolution rate arisie from low solubility which results in a low bioavailability of an orally administered drug(1). the solubility is a significant physicochemical property of a drug substance , mainly the aqueous solubility, so drug must be in solution to enter the systemic circulation and utilizes a therapeutic effect. however, 35-40% of new drugs discovered experience of poor aqueous solubility(2). numerous methods were improved for resolving the drug’s poor aqueous solubility(3). one of these approach is nanotechnology(4). nanoparticles (np)s are solid particles or particulate dispersions with size in the range of 10–1000 nm. within nanoparticle matrix the drug is dissolved, entrapped, absorbed, attached or encapsulated . according to the method of preparation, nanoparticles, nanospheres or nanocapsules can be obtained with different properties and release characteristics for an encapsulated therapeutic agent (5). the advantages of using nanoparticles for drug delivery result from their two main basic properties. first nanoparticles, due to their small size, can pass through smaller capillaries and uptake by cells, which permit an efficient drug accumulation at the target sites.second, the use of biodegradable materials for nanoparticle preparation allows sustained drug release within the target site over a period of days or even weeks(5). isradipine belongs to the dihydropyridine (dhp) class of calcium channel blockers (ccbs), isradipine binds to calcium channels with high affinity and specificity and inhibits calcium flux into cardiac and arterial smooth muscle cells(6). isradipine is a class ii drug according to bsc (7, 8). it is mainly absorbed from gastrointestinal tract after oral adminstration, undergoes extensive first-pass metabolism; as a result bio-availability is (15 to 24%) (8, 9). the aim of this study is to increase the solubility and enhance the dissolution rate of the poorly water-soluble (class ii) isradipine by the preparation of isradipine nanoparticles in size between 10-1000nm with studying preparation variables that affect the properties of prepared nanoparticles. materials and methods materials isradipine, polaxamer 188 (pxm 188), poly vinyl alcohol (pva) were purchased from hyper chem company,china, soluplus supplied by sigma-aldrich, germany. methanol supplied by avantor performance materials, norway. hydrochloric acid from grin land chemical comp, united kingdom. mannitol were purchased from jk . chemical. china . and filter syringe (0.45μm) supplied by chem lab, spain ,dialysis membrane 12000 da from schcuhardt, germany . na2hpo4, kh2po4 and deionized water were supplied by janeen for chemical and laboratory materials, baghdad, iraq. methods preparation of isradipine nanoparticles by solventantisolvent precipitation method : isradipine nanoparticles were prepared by using solvent/antisolvent precipitation technique (nanoprecipitation method). briefly, 10mg of isradipine was completely dissolved in water miscible organic solvent (methanol), then the obtained drug solution was added drop wise into the water containing one of the stabilizers (pva, pxm 188 , soluplus) at different ratios of drug to stabilizers which acts as an antisolvent as shown in table (1). precipitation of solid drug particles occurred immediately upon mixing. then, the precipitated nanoparticles stirred at agitation speed of 200 revolution per minute (rpm) on magnetic stirrer for 1 hour to allow the organic solvent to evaporate. and then lyophilized the selected formula using freeze drying system (labconco, usa) to obtain the nanoparticles powder (10). table 1.composition of isradipine nanoparticles formulas water (ml) methanol (ml) drug:polymer ratio polymer type formula no. 27 3 1:1 pva f1 27 3 1:0.5 pva f2 27 3 1:2 pva f3 27 3 1:1 pxm 188 f4 27 3 1:0.5 pxm 188 f5 27 3 1:2 pxm 188 f6 27 3 1:0.5 soluplus f7 27 3 1:0.75 soluplus f8 27 3 1:1.5 soluplus f9 27 3 1:2 soluplus f10 iraqi j pharm sci, vol.30(1) 2021 israd ipine nanoparticle 220 characterization of isradipine nanoparticles particle size and polydispersity index analysis particle size distribution analysis was done by using abt-9000 nano laser particle size analyzer which is a dynamic light scattering, works by measuring the intensity of light scattered by the molecules in the sample as a function of time, at a constant temperature of 25 ºc without dilution the samples. the average particle size was measured for all the prepared formulas. each sample was measured in triplicate. the polydispersity index (pdi), as measures for the width of the size distribution for each sample was also measured by the instrument (11). selection of optimized isradipine nanoparticle formula depending on the characterization study for the formulations of isradipine such as particle size, and pdi, the optimized formula was selected which should have reliable results in studying characterizations. the optimized formula was then subjected for further studies such as dsc, drug-excipient compatibility study. characterization of selected isradipine nanoparticles formula determination of saturation solubility of isradipine nanoparticles saturation solubility of the selected isradipine lyophilized powder was investigated in water, 0.1 n hcl and phosphate buffer ph 6.8 .in each case, the excess quantity of sample was added to 10 ml of solvent and agitated at 25°c in a water bath shaker for 72 hrs. after equilibration, the samples were centrifuged at 4000 rpm for 10 min, filtered using 0.45 μm millipore filters, suitably diluted and analyzed by measuring the absorbance using uv-visible spectrophotometer at 327nm, 328 nm, 329nm for 0.1n hcl, phosphate buffer ph 6.8, and water respectively, for the amount of drug dissolved (12-14). determination of isradipine content in nanoparticles the assay was performed using a lyophilized powder, equivalent to 2.5mg isradipine, that dissolved in 100 ml methanol. the samples were stirred and subsequently centrifuged at 6000 rpm for 10 min. the contents of isradipine in a sample were analyzed after suitable dilution using uv spectrophotometer at 326 nm. drug content was calculated by the equation. drug content = analyzed content theoretical content x 100 in-vitro dissolution profile of isradipine nanoparticle in-vitro dissolution test of isradipine nanoparticles was estimated using (paddle assembly) type ii dissolution test apparatus. accurately weighed lyophilized nanoparticles of the selected formula equivalent to 2.5 mg of isradipine was mixed with 5ml of the dissolution media and placed in a dialysis bag and rotated by a paddle at 50 rpm at 37 ± 0.5°c. in 500ml of dissolution medium, 0.1n hcl with 1% tween 20, to ensure sink condition . an aliquot of 5 ml samples was drawn from receiver compartment at predetermined time intervals and refilled the equal volume of fresh dissolution medium to conserve the constant volume. then, samples were filtered by 0.45μm filter syringe and assayed spectrophotometrically by uv–spectrophotometer at 327 nm (15).the same procedure was made for the pure drug . crystallinity study differential scanning calorimetry (dsc) analysis the dsc was used to assess the crystallainity properties of the various substances and formulations. pure isradipine powder and lyophilized nanoparticles (3-5mg) were sealed in the flat-bottomed aluminum pan of the differential scanning calorimeter (shimadzu dsc-60 plus, japan). data collection was achieved at a temperature range of 30–300°c and the heating rate was 10°c/min under nitrogen gas at a flow rate of 40 ml/min (16). compatibility study fourier transform infrared spectroscopy (ftir) the ftir spectra of pure isradipine, optimized polymer, and lyophilized nanoparticles were performed using ftir7600, (australia spectrophotometer). powders were mixed with potassium bromide (spectroscopic grade) and compressed into disks using hydraulic press before scanning from 4000 to 400 cm-1 (17). statistical analysis the experimental results were expressed as a mean triplicate sample ± standard deviation (sd) and were analyzed according to one-way analysis of variance (anova) using spss software at which the results would be significant when p < 0.05, and the results would be non-significant if p> 0.05. similarity factors (f2) was also calculated to compare dissolution profiles, where f2 more than 50 consider similar , while less than 50 consider dissimilar using dd solver program(17, 18). results and discussion characterization of isradipine nanoparticles particle size analysis and polydispersity index the particle size and pdi results of the prepared isradipine nanoparticles are shown in table (2). for all the prepared formulas, the average particle size and pdi were analyzed by particle size analyzer . the size of the particle was in the range of 77.34 – 1582.56 nm. the particle size determines the absorption and bioavailability of the drug; the smaller particle size of nanosize provides a larger iraqi j pharm sci, vol.30(1) 2021 israd ipine nanoparticle 221 surface area, which leads to faster drug release into aqueous medium (19). on the other hand, pdi which is a measure of the size distribution of the nanoparticles; were ranged from (0.005-0.395) depending on formulation variables, (0.005) indicating good uniformity,monodisperse system in the particle size distribution of nanoparticles while (0.395) indicated polydisperse system (20). table 2. the particle size and pdi of isradipine nanoparticles selection of optimized isradipine nanoparticle formula from the overall results of the particle size analysis, pdi, of nanoparticle formulations, formula f9 was selected as the best formula that characterized by a low particle size (77.34 nm), with pdi (0.005). therefore, the selected formula (f9) was subjected to further investigations of drugexcipient compatibility and crystalline state determination. characterization of selected isradipine nanoparticles: determination of saturation solubility of sradipine nanoparticles: the saturated solubility of the lyophilized isradipine powder of f9 was increased significantly (p <0.05), more than ten times in water, over the pure drug and about six times in hcl and more than ten times in phosphate buffer ph 6.8. this was due to the decrease in particle size which increased the surface area and due to the presence of surfactant within its composition that enhanced the solubility; as shown in figure (1) and table (3). figure 1. saturated solubility of pure isradipine and selected lyophilized formula f9 in different solvents table 3. saturated solubility of pure isradipine and selected lyophilized formula f9 in different solvents *sd standard deviation from the mean of a triplicate sample determination of isradipine content in nanoparticles drug content of the prepared isradipine nanoparticles was 96.89 ± 0.038%, which meet bp (british pharmacopeia) requirement and were within an acceptable range (95%-110%).(21) indicating that, there was no precipitation or lose of drug. in-vitro dissolution profile of lyophilized isradipine nanoparticle in-vitro drug release studies of isradipine nanoparticles of the selected formula (f9) and pure drug were conducted in 0.1n hcl to simulate invivo release in the stomach.(22) polydispersity index (pdi) particle size average(nm) drug:polymer ratio stabilizer formula no. 0.205 554.36± 0.012 1:1 pva f1 0.269 913.59±0.004 1:0.5 pva f2 0.023 769.1±0.143 1:2 pva f3 0.395 726.82±0.002 1:1 pxm 188 f4 0.332 1582.5±0.001 1:0.5 pxm 188 f5 0.246 1017.8±0.004 1:2 pxm 188 f6 0.279 120.9±0.120 1:1 soluplus f7 0.345 228.35±0.101 1:0.5 soluplus f8 0.005 77.34±0.003 1:0.75 soluplus f9 0.352 219.68±0.005 1:1.5 soluplus f10 solvent solubility of pure drug(mg/ml) mean ±sd* solubility of lyophilized isradipine nanoparticles (mg/ml) mean ±sd* hcl ph 1.2 0.040± 0.023 0.259± 0.012 phosphate buffer ph 6.8 0.010±0.187 0.106± 0.103 water 0.014±0.002 0.211± 0.015 iraqi j pharm sci, vol.30(1) 2021 israd ipine nanoparticle 222 comparing the dissolution profiles of the selected formula of isradipine nanoparticle, with the pure drug as a reference. it was observed that the f2 value equal to 23.53 indicating that, the dissolution of the selected formula of isradipine nanoparticles was dissimilar, faster than that of pure drug powder as shown in figure (2) . these results were expected according to noyes– whitney equation where the dissolution rate is directly proportionate to its surface area exposed to the dissolution medium and saturation solubility of the prepared drug dosage form (23). figure 2. dissolution profile for pure drug ,f9 (isradipine nanoparticles ) in 0.1n hcl at 37°c crystallinity study differential scanning calorimetry (dsc) analysis isradipine peak was clear in its dsc thermogram as a sharp endothermic peak around its melting point (170.3oc) such sharp endothermic peak indicates that isradipine used is in crystalline and pure state(24).the thermogramof pure isradipine is shown in figure (3). figure 3. differential scanning calorimetry of pure isradipine on the other hand, for lyophilized powder, the melting point of isradipine disappeared as in figure (4) giving a strong indication that the drug lost the crystallinity state and converted to an amorphous state(25). figure 4. differential scanning calorimetry of isradipine nanoparticle drugexcipients compatibility study fourier transform infrared spectroscopy (ftir) the ftir spectra of pure isradipine and lyophilized isradipine nanoparticle are shown in figures (5 , 6), respectively. pure isradipine exhibited characteristic peaks shown in table (4) at 3347cm–1 (n-h stretching), 2948cm–1 (c-h stretching), 1,702.84cm–1 (c=o stretching), and 1,490cm–1 (c=c vibration) (figure 5). all these peaks had appeared in the spectra of the selected formula (figure 6)(7). table 4.characteristic bands of pure isradipine no. type of bands pure drug 1 c-o vibration 1702.84 cm-1 2 c=c vibration 1490 cm-1 3 c-h stretching 2948 cm-1 4 n-h stretching of amide 3347 cm-1 from the ftir results, it was found there was no changes in the peaks of isradipine spectrum to that of the spectra of lyophilized isradipine nanoparticles f10, and all the functional groups in isradipine were maintained with low intensity due to dilution with the polymer .in addition hydrogen bond may be formed between soluplus and the drug shown as broad peak at 3363 , which it may have a contribution to increase affinity between them and resulted in enhanced solubility . iraqi j pharm sci, vol.30(1) 2021 isradipine nanoparticle 223 figure 5. ftir spectrum of pure isradipine figure 6.ftir of isradipine nanoparticle (f9) iraqi j pharm sci, vol.30(1) 2021 isradipine nanoparticle 224 conclusions based on the results obtained , the study concluded that nanoparticles is a promising approach to improve the solubility, dissolution rate and release of isradipine and solvent-antisolvent precipitation is an efficient method for preparation of isradipine nanoparticles. isradipine nanoparticles were successfully prepared using soluplus as stabilizer at ratios (1:0.75 drug:polymer) that gave a higher invitro release profiles compared to pure drug powder . dsc confirm that the crystalline structure of isradipine was lost and transformed to the amorphous state when the drug was converted into nanoparticles. drug–excipients compatibility studies revealed that hydrogen bond may be formed between soluplus and the drug, which it may have a contribution to increase affinity between them and resulted in enhanced solubility . acknowledgements the authors would like to thank department of pharmaceutics, college of pharmacy, university of baghdad for the completion of research. references. 1. pardeike j, strohmeier dm, schrödl n, voura c, gruber m, khinast jg, et al. nanosuspensions as advanced printing ink for accurate dosing of poorly soluble drugs in personalized medicines. international journal of pharmaceutics. 2011;420(1):93-100. 2. chaturvedi a, verma a. solubility enhancement of poorly water soluble drugs by solid dispersion. ijpsr. 2012;3(1):26. 3. goyal u, gupta a, rana ac, aggarwal g. self microemulsifying drug delivery system: a method for enhancement of bioavailability. ijpsr. 2012;3(1):66. 4. mallakpour s, behranvand v. polymeric 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iraqi j pharm sci, vol.30(1) 2021 isradipine nanoparticle 225 journal of pharmacy and pharmacology. 2012;64(7):931-43. 23. li w, yang y, tian y, xu x, chen y, mu l, et al. preparation and in vitro/in vivo evaluation of revaprazan hydrochloride nanosuspension. international journal of pharmaceutics. 2011;408(1-2):157-62. 24. park j-b, lee g-h, kang j-w, jeon i-s, kim jm, kim k-b, et al. improvement of photostability and dissolution profile of isradipine using inclusion complex. journal of pharmaceutical investigation. 2013;43(1):5561. 25. ramasamy t, khandasami us, ruttala h, shanmugam s. development of solid lipid nanoparticles enriched hydrogels for topical delivery of anti-fungal agent. macromolecular research. 2012;20(7):682-92. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.25(1) 2016 cardio-protective effects of ethanolic artichoke extract 1 the possible cardio-protective effects of ethanolic artichoke extract against 5fluorouracil induced cardiac toxicity in rats intesar t. numan * , maha n. hamad ** , ammar a. fadhil * and safa m. najim *,1  department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq.  department of pharmacognosy and medicinal plant, college of pharmacy, university of baghdad, baghdad, iraq. abstract cardiac toxicity can occur during the therapy with several cytotoxic drugs, including 5 fluorouracil (5fu). it is an antimetabolite that acts during the s phase of the cell cycle and is activated by thymidine phosphorylase into fluorodeoxyuridylate (5 fluoro 2'deoxyuridine 5'monophosphate, 5-fdump) that inhibits thymidylate synthase, thus preventing dna synthesis that leads to imbalanced cell growth and ultimately cell death. it is still a widely used anticancer drug, since 1957. the present study aimed to evaluate the possible cardio-protective effects of ethanolic artichoke extract (cynara scolymus l.) against 5-fluorouracil (5-fu) induced cardio-toxicity in rats by evaluating serum levels of alanine aminotransferase, aspartate aminotransferase and creatine kinase enzymes. methods: twenty -four female albino rats were randomly divided into 4 groups each group with 6 rats. group i: (negative control) received oral daily dose of dimethyl sulfoxide (dmso) (2 ml/kg /day) for 10 successive days. group ii: (positive control) received oral daily dose of dmso (2 ml/kg /day) for 10 successive days and subsequently administered single dose of 5-fu (150 mg/kg) by intraperitoneal injection on 8 th day in association with dmso. groups iii: received oral daily dose of ethanolic artichoke extract (200 mg/kg/day) for 10 successive days. groups iv: received oral daily dose of ethanolic artichoke extract (200 mg/kg/day) for 10 successive days with subsequently administered single intraperitoneal dose of 5-fu (150 mg/kg) on 8 th day in association with ethanolic extract. results: treatment of ethanolic artichoke extract prior 5-fu intoxication significantly attenuate the increase of serum alanine aminotransferase (alt), aspartate aminotransferase (ast) and creatine kinase (ck) enzymes activities caused by 5-fu-induced cardio-toxicity in rats. conclusions: results of the present finding suggest that the ethanolic artichoke extract may be an effective modulator in mitigating 5-fu induced cardiac toxicity in rats. keywords: ethanolic artichoke extract, 5-fluorouracil, cardio-protection, ast, alt and ck. نهمستخهص االٌثانىل ننبات االرض انشىكً ضد انسمٍه انقهبٍه انتاثٍر انىقائً انمحتمم فهىروٌىراسٍم فً انجرذان -5بعقار انمستخدمة انتصار طارق نعمان * ، مها نىري حمد ** ، عمار عامر فاضم * صفا مصطفى نجم و *،1 * .بغذاد،انعشاق خايعت بغذاد، فشع االدٌٔت ٔانسًٕو ،كهٍت انصٍذنت، ** .بغذاد،انعشاق بغذاد، خايعت انصٍذنت، كهٍت فشع انعمالٍش ٔانُباحاث انطبٍت ، الخالصه يعاد ْٕٔ. فهٕسٌٕٔساسٍم 5 رنك فً ٔبًا نهخالٌا انسايت انعمالٍش يٍ انعذٌذ يع انعالج اثُاء ححذد اٌ ًٌكٍ انمهبٍت انسًٍت 2 فهٕسٔ-5) فهٕسٔدٌٕكسٍٕسٌذٌالحً انى فٕسفٕسٔنٍض انثاًٌٍذٌٍ بٕاسطت ٌُٔشػ انخهٍت دٔسة يٍ( اط) يشحهت خالل ٌعًم اٌعً ْٔزا (اي اٌ انذي) انحايط انُٕٔي حشكٍب ًٌُع بذٔسِ ٔانزي ساَثٍض ثاًٌٍذنٍجعًم ًٌُع ٔانزي( يَٕٕفسفٌٕج' 5دٌٕكسٍٕسٌذًٌُ' نهسشغاٌ انًعادة االدٌٔت يٍ فهٕسٌٕٔساسٍم -5 ٌضال ال. انًطاف َٓاٌت فً انخهٍت يٕث ٔبانخانً نهخهٍت يخٕاصٌ انغٍش ًَٕ انى ٌؤدي االسض نُباث االٌثإَل نهًسخخهص انًحخًم انٕلائً انخاثٍش حمٍٍى إنى انذساست ْزِ حٓذف. 7551 عاو يُز ٔاسع َطاق عهى انًسخخذيت أٍٍَ نالَضًٌاث انًصم يسخٌٕاث بخمٍٍىٔرنك اندشراٌ فً فهٕسٌٕٔساسٍم -5 بعماس انًسخخذيت انمهبٍّ انسًٍّ ظذ انشٕكً – 751 ٔصَٓا ابٍط اَثى خشر ٔعششٌٔ اسبعت: انطشٌمت. كٍُاص انكشٌاحٍٍ ٔ أيٍُٕحشاَسفٍشاط جاسباسحاحٍ أيٍُٕحشاَسفٍشاط، ٔاحذة ٌٕيٍت خشعت جخشراٌ حهم سخت( انسهبٍت انًشالبت: )األٔنى انًدًٕعت: انخانً انُحٕ عهى يدًٕعاث اسبع انى حصٍُفٓا حى غى،211 جحهم خشراٌ سخت (االٌدابٍت انًشالبت: )انثاٍَت انًدًٕعت. يخخانٍت أٌاو 71 نًذة( ٌٕو/كغ/يم 2) سهفٕكسٍذ يٍثٍم ثُائً يٍ انفى غشٌك عٍ 5 عماس حهمج رنك ٔبعذ يخخانٍت أٌاو 71 نًذة( ٌٕو/كغ/يم 2) سهفٕكسٍذ يٍثٍم ثُائً يٍ انفى غشٌك عٍ ٔاحذة ٌٕيٍت خشعت :انثانثت انًدًٕعت. سهفٕكسٍذ يٍثٍم ثُائً يع بانخضايٍ انثايٍ انٍٕو فً انصفاق داخم انحمٍ غشٌك عٍ( كغى/يغى751) فهٕسٌٕٔساسٍم انخغزٌت بٕاسطت انفى غشٌك عٍ( كغى/يغى 211)انشٕكً االسض نُباث االٌثإَل يسخخهص يٍ ٔاحذة ٌٕيٍت خشعت حهمج خشراٌ سخت انشٕكً االسض نُباث االٌثإَل يسخخهص يٍ ٔاحذة ٌٕيٍت خشعت حهمج خشراٌ سخت: انشابعت انًدًٕعت.يخخانٍت اٌاو 71 نًذة االَبٕبٍت 751) فهٕسٌٕٔساسٍم -5ـ ببعذ رنك حمُٓا حى ثى يخخانٍت اٌاو 71 نًذة االَبٕبٍت انخغزٌت بٕاسطت انفى غشٌك عٍ( كغى/ يغى 211) ٔخٕد جحبٍُْزِ انذساست : انُخائح. انشٕكً االسض نُباث االٌثإَل يسخخهص يع بانخضايٍ انثايٍ انٍٕو فً انصفاق داخم( كغى/يغى انًعايهت اندشراٌ فً كاٌٍُض انكشٌاحٍٍ ٔ أيٍُٕحشاَسفٍشاط اسباسحاحٍج ، أيٍُٕحشاَسفٍشاط أٍٍَ يٍ نكم انًصم يسخٌٕاث فً اَخفاض 1 corresponding author e-mail: safamustafa_1985@yahoo.com received: 16 /9/2015 accepted: 30/12/2015 iraqi j pharm sci, vol.25(1) 2016 cardio-protective effects of ethanolic artichoke extract 2 : االسخُخاخاث.فهٕسٌٕٔساسٍم-5 بعماس انخسًى لبم يخخانٍت اٌاو 71 نًذة انشٕكً االسض نُباث االٌثإَل يسخخهص يٍ كغى/ يغى 211 يع فهٕسٌٕٔساسٍم 5عماس عٍ انُاحدت انمهبٍت انسًٍت حمهٍم فً فعاال كاٌ انشٕكً االسض ُباثاالٌخإَل ن يسخخهص اٌ إنى حشٍش انُخائح .نألكسذة ٔانًعاد نالنخٓاباث، كًعاد انًخعذدة انًفٍذة اإلظافٍت آثاسِ خالل يٍ اندشراٌ فً . ، اي اس تً ، اي ال تً و سً كًفهىروٌىراسٍم ، انحماٌة انقهبٍة 5االٌثانىنً ننبات االرض انشىكً ،انكهمات انمفتاحٍة: انمستخهص introduction cardiovascular disease isfescalating in recent years andgremains as a leading cause of mortality in developing countries (1) . it is can occur during the therapy with several cytotoxic drugs, including 5fluorouracil and may be the dose limiting factor in cancer treatment and hence tumor response (2) . cardiac toxicity includes a wide range of cardiac effects from small changes in blood pressure and arrhythmias to cardiomyopathy. in literature, different mechanisms of chemotherapy induced cardiac toxicity are postulated including cellular damage due to the formation of free oxygenjradicals and the induction ofnimmunogenic reactions with thedpresence of antigen presenting cells in the heart. moreover, the influence of the cytotoxic agents on certain phospholipids, especially cardiolipin, may also explain the development of cardiac toxicity (2) . 5fluorouracil (5-fu) is an antimetabolite thatfacts during the s phase of the cell cycle and is activated by thymidinefphosphorylase into fluorodeoxyuridylate (5 fluoro 2'deoxyuridine 5'monophosphate, 5-fdump) that inhibitsdthymidylate synthase, thus preventing dnagsynthesis that leads to imbalanced cellfgrowth and ultimately cell death (3-5 ) . it is a potentdantineoplastic agent commonly used for treatment of various malignanciesfincluding gastrointestinal, breast and head and neck cancer. in addition to bonevmarrow depression, gastrointestinal tract reaction, or even leucopenia anddthrombocytopenia (6) , it has diverse adverse effects such as cardiac toxicity, hepatotoxicity and nephrotoxicity which restrict its wide and extensive clinical usage, also it causes marked organ toxicity coupled with increased oxidative stress and apoptosis (7) . medicinal plants and theirdderivatives are widely used all over the world as medicinal, salutistic or functional food. some medicinalfplants are promising natural source (8) . artichoke (cynara scolymus l.) is one of the world’s oldest medicinal plants. it is anfimportant crop of ancient greece, grows in egypt, mediterranean area and other countries .it is belonging to the family (asteraceae) (9) . it has medical properties and used in traditional folk medicine mainly because of theirfcholeretic (increasing bile secreation), diuretic and hypocholesterolemic activities (10) . it is a good source of natural antioxidants such as vitamin c, hydroxycinnamic acids (9) and caffeoylquinic acid derivatives (cynarin and chlorogenic acid) (11) . artichoke plant is rich with flavonoids (luteolin, apigenin), which its potential protectiveveffect as antioxidant have been demonstrated for the extracts of this vegetable indreducing reactive oxygen species (ros) from stimulated human neutrophilfand in protection of hepatocyte from t-butyl hydrogen peroxide inducedfcytotoxicity (12) . the artichoke extracts were assessed for their protective role in the control of oxidative damagefto biological molecules (proteins, lipids and dna), caused by free radicals such as rcoo and/or oh, using the bcarotene/linoleate assay, the deoxyribose assay and the metmyoglobin assay. artichoke leaf extracts (ale) is known toshave potential antioxidant effect. several in vitro studies have showndthat the antioxidant potential effect of ale is dependent on radicaldscavenging and metal ion chelating effectfof its constituents such as cynarin, chlorogenic acid and flavonoids. pure constituents of ale have also been shown to produce less inhibitory activity on free radical production than the extract itself (13) . materials and methods chemicals and drugs 5-fluorouracil (5-fu) obtained from ebewe pharma, austria. dimethyl sulfoxide (dmso) and ethanol solvent obtained from warehouse chemicals of college of pharmacy/ university of baghdad. reagents standard assay rat’s kits for ast/got and alt/gpt were obtained from egyption company for biotechnology (s.a.e) and for ck obtained from biolabo sa, 02160 ,maizy, france. plant materials the plant was collected from the garden of medicinal plants at the department of pharmacognosy and medicinal plants / college of pharmacy / university of baghdad. the leaves of the plant were dried in shade at room temperature, then rendered into a fine powder by using electrical mill and weighed. extraction of the plant four grams of powdered leaves were extracted by maceration with 2500 ml of absolute ethanol for one week, then the extract was filtered and evaporated to dryness under reduced pressure by using rotary evaporator, iraqi j pharm sci, vol.25(1) 2016 cardio-protective effects of ethanolic artichoke extract 3 after that the collected amount was weighted (14) . preparation of the extract for injection a specific weights (4.5 gm.) from the dried ethanolic artichoke extract was dissolved in dimethyl sulfoxide (112.5 ml) to get a concentration of 40 mg/ml (as a stock solution) (13) . experimental animals twenty -four female albino rats of 1-2 month old (average body weight 150-200gm), were obtained from animal house of the college of pharmacy/ university of baghdad. the animals were acclimatized under standard laboratory conditions for 2 weeks prior to treatment .they had free access to standard diet and water. they were maintained under standard condition of temperature (30ºc), humidity and light / dark cycles. all the experimental studies were conducted inconformity with the guidance for care and standard experimental animals of our college ethical protocol. the animals were used in this study divided equally into four groups, each group with 6 rats they were treated as following: group i: (negative control) received oral daily dose of dmso (2 ml/kg /day) for 10 successive days. group ii: (positive control), received oral daily dose of dmso (2 ml/kg /day) for 10 successive days with administered single dose of 5-fu (150 mg/kg) intraperitoneally on 8 th day (7) in association with dmso. group iii: received 200 mg/kg/day (13) of ethanolic artichoke extract orally for 10 successive days. groups iv: received 200 mg/kg/day (13) of ethanolic artichoke extract orally 10 successive days with subsequently single intraperitoneal dose of 5-fu (150 mg/kg) on 8 th d (7) in association with the ethanolic artichoke extract (13) .after 24 h of the end of the experimental period (10 days), all the animals were anesthetized under light diethyl ether anesthesia and blood samples were collected in clean test tubes by intracardiac puncturing and allowed to clot at room temperature. biochemical assessment the serum was separated by centrifugation for 20 min at 3600 round per minute (r.p.m.) and stored into eppendorff tubes at – 20 °c to be used for determination of creatine kinase (ck), aspartate aminotransferase (ast) and alanine aminotransferase (alt) (13) . results and discussion data were subjected to statistical analysis, data were expressed as the mean values mean± standard deviation (sd) of samples. the statistical significance of the differences between various groups was determined by student unpaired ttest. differences were considered statically significant for p-value < 0.05. the effects of ethanolic artichoke extract on 5-fluorouracil (5-fu) induced cardiactoxicity in rats: 5-fu (group ιι) significantly (p<0.05) increased serum levels of ast, alt, and ck with respect to group ι. administration of ethanolic artichoke extract in association with 5-fu (group iѵ) significantly (p<0.05) decreases the serum levels of ast, alt and ck with respect to group ιι. groups ιιι show no significant differences (p<0.05) in alt and ck with respect to group ι, but there was significant difference in ast with respect to group i. while group iv showed significant elevation (p<0.05) in ast, alt and ck with respect to group ι, as shown in table (1). cardiac toxicity is one of the dangerous side effect of 5-fu , which often presents as myocardial ischemia, but to a lesser extent cardiac arrhythmia, hyper and hypotension, left ventricular dysfunction, cardiac arrest and sudden death (15-20) . the incidence of 5-fu induced cardiac toxicity varies between 0-35 % and this may depend on dose, cardiac comorbidity and schedule of chemotherapy (15, 16, 18) . the clinical handling of 5-fu-induced cardiac toxicity is difficult as the pathophysiological mechanisms underlying this cardiac toxicity remain undefined (15, 21) . however several mechanisms havebbeen proposed, including vascular endothelial damage followed by coagulation, ischemia secondary to coronary artery spasm, direct toxicity on the myocardium andfthrombogenicity due to altered rheological factors (21) . the pathogenesis of 5-fu induced cardiac toxicity may involve oxidative stress with increased levels of superoxide anion after 5fu treatment (22) .the activities of superoxide dismutase (sod) and glutathion peroxidase (gsh-px) were lowered in 5-fu treated guinea pigs (23) demonstrating a reduced antioxidant capacity. if not eliminated by cellular antioxidant systems, superoxide anions can generate the highly reactive and toxic hydroxyl radicals through the haber–weiss reaction, which is catalyzed by iron (24,25) . increased reactive oxygen species (ros) levels inside cells lead to oxidation of macromolecules, including lipids, nucleic acids, and proteins, thereby disturbing cellular functions (25) . in addition to ros, proinflammatory cytokines, such as tumourdnecrosis factorα (tnf-α) and interleukins (il)-1 and 6, also play an indirectdrole in production toxicity and organs iraqi j pharm sci, vol.25(1) 2016 cardio-protective effects of ethanolic artichoke extract 4 damage that generate by chemotherapy agents, as 5-fu (26) . the present study confirms the cardiac toxicity of 5-fu, as evidenced by the significantly (p<0.05) elevation in serum level of ast, alt and ck. detection of elevated concentrations of cardiac biomarkers in blood is a sign of cardiac injury which could be due to supply–demand imbalance, toxic effects, or haemodynamic stress (27) . creatine kinase (ck), ast, lactate dehydrogenase, myoglobin, and troponins are some of these markers (28) . the present study hasdshown a successful reduction in cardiac toxicity induced by 5-fu in albino rats after treatment with ethanolic artichoke extract, this was reflected by reduction in serum level of ast, alt and ck. this is give a strongly refers to the possible cardiac protective effects of artichoke extract against 5-fu induced cardiac toxicity in the rats. this protective effect of artichoke extract related to the power antioxidant effect of phenolic acids especially chlorogenic acid and cyanine (29) .when the biological activity of artichoke extracts is considered, the presence of luteolin-7glucoside and hydrolysable tannins, besides caffeoylquinic derivatives, in the phenolic fractionfof these extracts must be taken into account: all these phenolics possess a good antioxidantfactivityfagainst peroxyl and hydroxyl radicals by decreasing the release of ros, which is produced by effect of cytotoxic drugs, when assessed using the beta carotene/linoleate assay and the metmyoglobin assay (30) . table (1): effects of ethanolic artichoke extract (200 mg/kg) on the serum levels of ast, alt and ck in albino female rats with 5-fu induced cardiac toxicity, data are expressed as mean ± sd, n =6, p <0.05. standard deviation (sd), 5-fluorouracil (5-fu) and dimethyl sulfoxide (dsmo). treatment group n=6 type of treatment serum aspartate aminotransferase (s.ast) up to 40 iu/l (mean ± sd) serum alanine aminotransferase (s.alt) up to 40 iu/l (mean ± sd) serum creatine kinase (s.ck) up to 190 iu/l (mean ± sd) i dimethyl sulfoxide (dmso) only 27.33 ± 0.42 7.33 ± 0.81 51.33 ± 4.35 ii 5-fluorouracil (5-fu) 74.06 ± 11.52  32.53 ± 1.43  555.66 ± 67.77  iii 200 mg/kg of ethanolic artichoke extract 22.33 ± 2.60  7.2 ± 4.73 48.42 ± 9.40 iv 200 mg/kg of ethanolic artichoke extract + 5-fu 31 ± 1.93 s 16 ± 0.44  s 63.6 ± 5.05  s (): significant difference with respect to negative control group (p 0.05), (s): significant difference with respect to 5-fu treated group. conclusion the results of this study suggested that the ethanolic artichoke extract has protective effects against 5-fu-induced cardiac toxicity in albino female rats. however, before a conclusive statement can be made on the potential antioxidant activity of artichoke extract as an adjunct to 5-fu therapy, there is a need for further long-term chronic studies for different fractions of artichoke extracts. references 1. reddy ks, ansari nagar, new delhi. cardiovascular diseases in the developing countries: dimensions, determinants, dynamics and directions for public health action. public health nutr. 2002 feb; 5 (1a):231-7. 2. kirsten j m, schimmel dick j, and henkjan g, et al. chapter 6 cardiac toxicity of cytotoxic drugs cancer treatment reviews 2004; 30:181–191. 3. shanmugasundaram s, bharathithasan r and elangovan s, 5-fluorouracil-induced cardiac toxicity, indian heart j. 2002 janfeb; 54(1):86-7. 4. reza t. angina induced by 5-fluorouracil infusion in a patient with normal coronaries, am heart hosp j. 2010;8(2):111-12 5. costi mp., ferrari s. and venturelli a., et al. thymidylate synthase structure, function and implication in drug discovery. curr. med. chem. 2005; 12 (19): 2241–58. 6. yu b.t., sun x., and zhang z.r. enhanced liver targeting by synthesis of 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kristian t, johannes m and hugo k, et al. recommendations for the use of cardiac troponin measurement in acute cardiac care. european heart journal 2010; 31:2197–206. 28. sylvia archan and lee a. fleisher, from creatine kinase-mb to troponin. anesthesiology 2010; 112:1005–12. 29. wittemer sm, ploch m and windeck t, et al. bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids after oral administration of artichoke leaf extracts in humans. phytomedicin, 2005; 12 (1 – 2): 28 – 38. 30. lattanzio v, cardinali a and di venere d, et al. browning phenomena in stored artichoke (cynara scolymus l.) heads: enzymic or chemical reactions? food chemistry 1994; 50: 1–7. iraqi j pharm sci, vol.29(1) 2020 genotoxic evaluation for artificial food flavoring additive doi: https://doi.org/10.31351/vol29iss1pp55-61a 55 evaluation the incidence of genotoxic effects of artificial food favoring additives in bone marrow cells and spleen cells in mice nibras h. jasem* and ali f. hassan **,1 * ministry of health and environment ,technical affairs directorate, , baghdad, iraq. ** departments of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract genetic material is the most important component of cells because it contains the genetic information; hence any disruption to the structure chromosome of cells could lead to very bad results. genotoxicity use to evaluate the safety of any chemical compounds on genetic materials. artificial food flavoring additive are chemical substances to produce specific placebo effects added to foods but impart specific flavor to it. the present study evaluates the genotoxic effect of artificial food flavoring additive on structure of chromosomes at three different concentrations (50%, 100%and 150%) on both bone marrow cells and spleen cells in mice for fourteen successive days. it was found that artificial food flavoring additive at concentration (50% and 100%) show not significant increase in total chromosomal aberration in both bone marrow cells and spleen cells when compare to negative control (p>0.05) meanwhile at concentration 150% it causes a significant increase when compare to negative control (p<0.05) .the results have been showed that artificial food flavoring additive had a genotoxic effect at (50%, 100% and 150%). keywords: artificial food flavoring additive, bone marrow cells, spleen cells, genotoxicity, chromosome بسبب استخدام االضافات المنكهة الصناعية في خاليا تقييم احتمالية حدوث تشوهات كروموسومية نخاع العظم وخاليا الطحال في الفئران 1**,علي فارس حسنو *نبراس حسب هللا جاسم العراق ، دائرة الشؤون التكنولوجية بغدادوزارة الصحة والبيئة، * كلية الصيدلة، جامعة بغداد، العراق، السمومو االدويةفرع ** الخالصة تعتبر المادة الوراثية من اهم مكونات الخلية وذلك الحتوائها على المعلومات الوراثية للخلية ولذا اي تعرض سئ لهذة المادة يكون لة عواقب وخيمة على الخلية . فحص السمية الجينية واحد من اهم الفحوصات التي تستعمل لمعرفة تاثير المواد الكيمائية على الشكل العام تعتبر المنكهات الصناعية واحدة من اكثر المركبات استعماال بالوقت الحالي حيث تضيف نكهة مميزة للطعام بدون التاثير على مات.للكروموسو تم تحضير ثالثة حيث للكروموسوم.خواصة العامة . الهدف من البحث هو معرفة التاثيرات السمية الجينية للمنكهات الصناعية على الشكل العام ( وتم اعطاؤها لمدة اربعة عشر يوما للفئران وفي اليوم الخامس عشر تم %150و %100. %50ختلفة من هذة المنكهات الصناعية )تراكيز م ( %100و%50بتراكيز ) ان االضافات المنكهة وقد أظهرت االنتائج. الجينية.استخالص خاليا نخاع العظم وخاليا الطحال لفحص التاثيرات السمية وان (p>.05)تسبب زيادة غير معنوية للمجموع التكسرات الكوموسومية لخاليا نخاع العظم وخاليا الطحال عند مقارنتها بمحموعة السيطرة السالبة حموعة السيطرة السالبة للمجموع التكسرات الكوموسومية لخاليا نخاع العظم وخاليا الطحال عند مقارنتها بم بسبب زيادة معنوية %150تركيز (p<0.05) .المنكهة الصناعية لديها تاثير سام على المادة الوراثية وان هذي التاثيرات السامة تزداد مع زيادة االضافاتان ان نستنتج وبذلك يمكن التركيز . .لجينية , الكوموسومات الكلمات المفتاحية:االضافات المكنهة الصناعية , خاليا نخاع العظم, خاليا الطحال , السمية ا introduction artificial food flavoring additive is chemical substances to produce specific flavor when added to foods (1). in the past, different techniques have been used to preserve foods and make them more desirable by addition different natural compounds. in the present days, there is huge orientation for use different types of food additive to the foods (2). food additives and their metabolites are subjected to different types of toxicological analysis before marketing (3). a previous study shows that about 75% of the western diet is made up of different types of processed foods; it has been show that the individual consume 8-10 pounds of food additives per year (4). unfortunately, the children considered as the higher consumer for the food with these food additive (5). children are higher consumer for calories compared to adults because their activity is higher as compare to adult (6). again, the blood-brain barrier, being poorly developed early in life, which in turn affect the blood flow as well as permitting a toxic substance to passively cross into the central nervous system (7). there are very little studies for the evaluation of this compound to the human compared to the animal studies (8-9). genotoxicity is destruction in the genetic materials within the cell which may lead to bad consequences like mutagenesis or cancer development (10). the dna in a human cell undergoes several thousand to a million damaging events in every day which most of them are detected by repair system (11). 1corresponding author e-mail: ali_1371982@yahoo.com received: 3 / 7 / 2019 accepted:16 / 9 / 2020 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp55-61a mailto:ali_1371982@yahoo.com iraqi j pharm sci, vol.29(1) 2020 genotoxic evaluation for artificial food flavoring additive 56 any dna mutations inherit if the dna damage is not repaired by repair system before mitosis (12]. cell division is a process in all living organisms. during cell division, eukaryotic cell is divided; dna replication and cell growth take place in a coordinated way to ensure correct division and formation of progeny cells containing intact genomes (13, 14]. at the end of eukaryotic cells division cycle the cells that generate either another copy of themselves or to generate gametes (sex cells) (15]. materials and methods artificial food flavoring additive has been bought from the iraqi market. preparation of solutions for different artificial flavoring additive concentration. three different concentrations of artificial flavoring additive (16] 1. 50% artificial flavoring additive solution. prepared by dissolving 50 grams of artificial flavoring additive in sufficient amount of distilled water to complete the volume to 100 ml of .then, the solution is mixed by a vortex. 2. 100% artificial flavoring additive solution. prepared by dissolving 100 grams of artificial flavoring additive in sufficient amount of distilled water to complete the volume to 100 ml of .then, the solution is mixed by a vortex. 3. 150% artificial flavoring additive solution. prepared by dissolving 150 grams of artificial flavoring additive in sufficient amount of distilled water to complete the volume to 100 ml of .then the solution is mixed by a vortex. experimental model sixty albino swiss mice (mus musculus) were used for two experiments. they were supplied by animal house of tikrit university. their weights were 23-27 gram. they were divided into five groups; each was kept in a separate plastic cage. the animals were maintained at a temperature of 23 – 25°c, and they had free excess to food (standard pellets) and ad libitum of water. the animals were divided into five groups (six mice of each) as follow: group1: mice were treated with distilled water. this group was served as negative control the dose was given orally for fourteen successive day days. group2: mice were treated with a single dose (20mg/kg) of methotrexate giving intraperitoneally. this group was served as a positive control. group3: mice were orally treated with (50%) of artificial food flavoring additive for fourteen successive days. group4: mice were orally treated with (100%) of artificial food flavoring additive for fourteen successive days. group5: mice were orally treated with (150%) of artificial food flavoring additive for fourteen successive days. the different concentration of artificial food additive was given orally instead of drinking water. mice were sacrificed by (spinal dislocation). samples of bone marrow cells and spleen cells were taken and genotoxic analyses were carried out as described later (17]. evaluation of genotoxicity after fourteen days of treatment, all animals were injected intraperitoneally with 1mg/kg colchicine, and then two hours later they are sacrificed by cervical dislocation. bone marrow samples were aspirated from the femur bone and processed using aseptic technique and spleen cells samples for evaluation of mitotic index, but only bone marrow cells have been used for evaluation of micronucleus appearance (18]. statistical analysis all the results were expressed as mean± standard deviation (m±std). the data were analyzed by utilizing a computerized statistical package for the social sciences (microsoft office excel 2010) program. unpaired student t-test was performed for each group pair includes a comparison between negative control and tests groups after fourteen days of treatment. p-values< 0.05 were considered to be statistically significant. two factors with no replication anova test have been performed for each group pair includes a comparison between different test groups after fourteen days of treatment. p-values< 0.05 were considered to be statistically significant. results in table (1),in bone marrow cells , artificial food flavoring additive at concentration 50% show not significant increase in total chromosomal aberration , chromosomal break, chromosomal gap and ring chromosome when compared to negative control (p>0.05) the rest individual chromosomal aberration show a significant increase at same concentration when compare to negative control (p<0.05). at concentration 100% artificial food flavoring additive show non-significant increase in total chromosomal aberration, chromosomal break and ring chromosome when compared to negative control(p>0.05) , the rest individual chromosomal aberration show a significant increase at same concentration when compare to negative control (p<0.05).at concentration 150% artificial food flavoring additive show significant increase in all individual and total chromosomal aberration except ring chromosome when compare to negative control (p>0.05). iraqi j pharm sci, vol.29(1) 2020 genotoxic evaluation for artificial food flavoring additive 57 artificial food flavoring additive at concentrations (50% and 100%) show a significant decrease in , chromosomal break, chromosomal gap , chromatid break, chromatid gap , acentric chromosome , dicentric chromosome, when compare to positive control (p<0.05) meanwhile artificial food flavoring additive at concentration (150%) show non-significant decrease in, chromosomal break, chromosomal gap, chromatid break, chromatid gap , acentric chromosome , dicentric chromosome, when compare to positive control (p>0.05). artificial food flavoring additive show non-significant decrease in total chromosomal aberration at concentrations (50%, 100% and 150%) when compared to positive control (p>0.05). artificial food flavoring additive show significant differences in total chromosomal aberration and individual chromosomal aberrations at concentrations (50%, 100% and 150%) when compare between each other (p>0.05). in table (2),in spleen cells , artificial food flavoring additive at concentration 50% show non-significant increase in total chromosomal aberration , acentric chromosome, dicentric chromosomal and ring chromosome when compared to negative control (p>0.05) the rest individual chromosomal aberration show a significant increase at same concentration when compare to negative control (p<0.05). at concentration 100% artificial food flavoring additive show not significant increase in total chromosomal aberration and ring chromosome when compare to negative control (p>0.05) , the rest individual chromosomal aberration show a significant increase at same concentration when compared to negative control (p<0.05).at concentration 150% artificial food flavoring additive show significant increase in all individual and total chromosomal aberration except ring chromosome when compare to negative control (p<0.05). artificial food flavoring additive at concentrations (50% and 100%) show significant decrease in chromosomal gap , chromatid break, chromatid gap , acentric chromosome and dicentric chromosome, when compare to positive control (p<0.05) meanwhile artificial food flavoring additive at concentration (150%) show nonsignificant decrease in individual and total chromosomal aberrations when compare to positive control (p>0.05). artificial food flavoring additive at three different concentrations show significant differences in all individual and total chromosomal aberration except ring chromosome, chromosomal break and chromosomal gaps when compare among each other’s (p<0.05). iraqi j pharm sci, vol.29(1) 2020 genotoxic evaluation for artificial food flavoring additive 58 table 1. individual and total chromosomal aberrations in bone marrow cells after exposure to different concentrations of artificial foo d flavoring additive in mice  data are expressed as mean±s.d; n=6 animals in each group;  *significantly different compared to distilled water (negative control) (p<0.05);  values with non-identical small letters superscripts (a,b,c) consider significant different when compared with methotrexate (positive control) (p<0.05).  values with non-identical capital letters superscripts (a,b,c) significant different when compared between tests groups (p<0.05). bone marrow cells chromatid break chromatid gap deletion dicentric chromosome acentric chromosome ring chromosome chromosome breaks chromosome gap total chromosomal aberration distilled water (negative control) 0.062± 0.008 0.064± 0.005 0.238 ± 0.027 0.190± 0.012 0.210± 0.007 0.036± 0.011 0.078 ± 0.008 0.056± 0.011 0.934± 0.081 methotrexate(mtx) (positive control) 20mg/kg 0.196± 0.021*a 0.218± 0.013*a 0.356 ± 0.015 *a 0.612± 0.019*a 0.866± 0.035*a 0.056± 0.017 a 0.130 ± 0.012 *a 0.142± 0.036*a 2.518± 0.273*a artificial flavoring additive at concentration 50% 0.094± 0.015*ab 0.094± 0.021*ab 0.280± 0.022*ab 0.228± 0.015*ab 0.244± 0.025*ab 0.030± 0.020 ab 0.084 ± 0.015 ab 0.070± 0.019 ab 1.14± 0.087 aa artificial flavoring additive at concentration 100% 0.106± 0.021*bc 0.108± 0.016*bc 0.314± 0.018*bc 0.296± 0.046*bc 0.278± 0.026*bc 0.041± 0.015 ba 0.090 ± 0.043 ba 0.100± 0.025*ba 1.454± 0.100 ba artificial flavoring additive at concentration 150% 150mg/kg 0.210± 0.016*ca 0.236± 0.021*ca 0.376± 0.017*ca 0.450± 0.142*ca 0.798± 0.076*ca 0.084± 0.043 ca 0.126 ± 0.025 *ca 0.126± 0.021*ca 2.15± 0.088*ca iraqi j pharm sci, vol.29(1) 2020 genotoxic evaluation for artificial food flavoring additive 59 table 2. individual and total chromosomal aberrations in spleen cells after exposure to different concentrations of artificial food flavoring additive in mice . spleen cells chromatid break chromatid gap deletion dicentric chromosome acentric chromosome ring chromosome chromosome breaks chromosome gap total chromosomal aberration distilled water (negative control) 0.046± 0.009 0.062± 0.001 0.210 ± 0.016 0.182± 0.008 0.200± 0.016 0.024± 0.006 0.060 ± 0.012 0.044± 0.011 0.826± 0.077 methotrexate(mtx) (positive control) 20mg/kg 0.200± 0.016*a 0.198± 0.026*a 0.294 ± 0.027 *a 0.490± 0.016*a 0.752± 0.059*a 0.050± 0.007*a 0.118 ± 0.022 *a 0.134± 0.023*a 2.259± 0.180*a artificial flavoring additive at concentration 50% 0.072± 0.018*ab 0.072± 0.008*ab 0.252± 0.030*ab 0.192± 0.008 ab 0.218± 0.008 ab 0.025± 0.017 ab 0.078 ± 0.008 *ab 0.064± 0.013*ab 0.964± 0.086 ab artificial flavoring additive at concentration 100% 0.086± 0.011*bc 0.094± 0.009*bc 0.288± 0.013*ba 0.282± 0.053*bc 0.270± 0.026*bc 0.033± 0.016 aa 0.086 ± 0.052 *aa 0.088± 0.045 *aa 1.35± 0.097 ba artificial flavoring additive at concentration 150% 150mg/kg 0.202± 0.016*ca 0.218± 0.013*ca 0.316± 0.015*ca 0.430± 0.055*ca 0.768± 0.072*ca 0.044± 0.024 aa 0.104 ± 0.089 *aa 0.114± 0.021*aa 1.994± 0.090*ca  data are expressed as mean±sd; n=6 animals in each group;  *significantly different compared to distilled water (negative control) (p<0.05);  values with non-identical small letters superscripts (a,b,c) consider significant different when compared with methotrexate (positive control) (p<0.05).  values with non-identical capital letters superscripts (a, b, c) significant different when compared between tests groups (p<0.05). iraqi j pharm sci, vol.29(1) 2020 genotoxic evaluation for artificial food flavoring additive 60 discussion the artificial food additive contains different chemicals substances considered as the major active chemical that responsible for the imparting specific flavor that given, the major active substances are (monosodium glutamate, disodium inosinate and disodium guanylate). monosodium glutamate (msg) is used in the food industry as a flavor enhancer which gives umami taste (19]. a previous studies have examined the toxicity of monosodium glutamate, it have been found that the use of this chemical associated with the induction of oxidative stress in different experimental animals after giving of chronic doses of monosodium glutamate (20]. glutamic acid has been proposed as one of the amino acids using by the body during gluconeogenesis (21]. increased influx of substances into the cells has been associated with increase the incidence of oxidative stress (22]. this has been corroborated in more recent reports in which hyperglycemiainduced mitochondrial dysfunction and endoplasmic reticulum stress, promote reactive oxygen species (ros) accumulation that, in turn, promote cellular damage and contribute to many complications can be development and progression. ros can directly damage lipids, proteins or dna and modulate intracellular signaling pathways, such as mitogen-activated protein kinases and redox-sensitive transcription factors causing changes in protein expression and, therefore, irreversible oxidative modifications(23-24]. hyperglycemia , induced by monosodium glutamate , is also known to increase the incidence of glucose autooxidation and labile glycation or intracellular activation of the polyol pathway which in turn it induce an oxidative degradation of the glycated protein which lead to increase of reactive oxygen species formations (25]. oxidative stress is an imbalance between the production of reactive oxygen species (ros) and the antioxidant capacity of the cell. ros have been classified as harmful by-products of the normal aerobic metabolism process of the mitochondria and the increase of its production associated with large variety of diseases besides these harmful effects, if the production of ros is under control, it plays physiological roles especially in cell signaling and regulating cell redox homeostasis (26]. one of the most dangerous deleterious effect of ros is the disruption of cell division by interfering with many cellular components especially the genetic materials, when the body was unable to regulate high levels of ros leading to many diseases characterized by both neurodegeneration and bone marrow failure as well as cancer (27], other previous finding showed that the overproduction of reactive oxygen species lead to chromosomal instability, which in turn increase the incidence of cancer and aging (28). disodium inosinate and disodium guanylate, it is typically sold in a 50:50 mixture of the two ribosides. this combination with glutamates which imparts the umami (29].these two chemical substances have been estimated for the ability of induction of chromosomal aberration, the test was achieved in vitro using a chinese hamster fibroblast cell line they found that both of these compounds cause a chromosomal aberration in these cell lines (30]. conclusions the results showed that artificial food flavoring additive had a genotoxic effect at (50%, 100% and 150%). references 1. ismael a, rahiemaa. effect of saccharin on albino rats’ blood indices and the therapeutic action of vitamins c and e. hum exp toxicol,2011; 30 (2):129-137. 2. karen l, graham mw, dominic pw, vyvyan hc. synergistic interactions between commonly used food additives in a developmental neurotoxicity test, toxicolsci 2006;90(1):178–187 3. mccann d, barrett a, cooper a, crumpler d, dalen l, grimshaw k, et al. food additives and hyperactive behavior in 3-year-old and 8/9-year old children in the community: a randomized, double-blinded, placebo-controlled trial. lancet, 2007;370 (9598):1560–7. 4. 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chemtoxicol 1984; 8:623-636. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://www.ncbi.nlm.nih.gov/pubmed/?term=collado%20m%5bauthor%5d&cauthor=true&cauthor_uid=16079833 http://www.ncbi.nlm.nih.gov/pubmed/?term=gil%20j%5bauthor%5d&cauthor=true&cauthor_uid=16079833 http://www.ncbi.nlm.nih.gov/pubmed/?term=efeyan%20a%5bauthor%5d&cauthor=true&cauthor_uid=16079833 http://www.ncbi.nlm.nih.gov/pubmed/?term=guerra%20c%5bauthor%5d&cauthor=true&cauthor_uid=16079833 http://www.ncbi.nlm.nih.gov/pubmed/?term=schuhmacher%20aj%5bauthor%5d&cauthor=true&cauthor_uid=16079833 https://www.ncbi.nlm.nih.gov/pubmed/?term=barradas%20m%5bauthor%5d&cauthor=true&cauthor_uid=16079833 http://www.ncbi.nlm.nih.gov/pubmed/16079833 https://www.ncbi.nlm.nih.gov/pubmed/?term=chen%20z%5bauthor%5d&cauthor=true&cauthor_uid=16079851 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http://www.ncbi.nlm.nih.gov/pubmed/16950796 https://www.ncbi.nlm.nih.gov/pubmed/?term=ayed-boussema%20i%5bauthor%5d&cauthor=true&cauthor_uid=21994239 https://www.ncbi.nlm.nih.gov/pubmed/?term=rjiba%20k%5bauthor%5d&cauthor=true&cauthor_uid=21994239 https://www.ncbi.nlm.nih.gov/pubmed/?term=mnasri%20n%5bauthor%5d&cauthor=true&cauthor_uid=21994239 https://www.ncbi.nlm.nih.gov/pubmed/?term=moussa%20a%5bauthor%5d&cauthor=true&cauthor_uid=21994239 https://www.ncbi.nlm.nih.gov/pubmed/?term=bacha%20h%5bauthor%5d&cauthor=true&cauthor_uid=21994239 https://www.ncbi.nlm.nih.gov/pubmed/21994239 https://www.researchgate.net/scientific-contributions/2133550754_joshi_b https://www.ncbi.nlm.nih.gov/pubmed/?term=farombi%20eo%5bauthor%5d&cauthor=true&cauthor_uid=16955747 https://www.ncbi.nlm.nih.gov/pubmed/?term=emerole%20go%5bauthor%5d&cauthor=true&cauthor_uid=16955747 https://www.ncbi.nlm.nih.gov/pubmed/?term=ukoha%20ai%5bauthor%5d&cauthor=true&cauthor_uid=16955747 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https://www.ncbi.nlm.nih.gov/pubmed/?term=kitada%20m%5bauthor%5d&cauthor=true&cauthor_uid=12874441 https://www.ncbi.nlm.nih.gov/pubmed/?term=kashiwagi%20a%5bauthor%5d&cauthor=true&cauthor_uid=12874441 https://www.ncbi.nlm.nih.gov/pubmed/?term=kashiwagi%20a%5bauthor%5d&cauthor=true&cauthor_uid=12874441 https://www.ncbi.nlm.nih.gov/pubmed/?term=kikkawa%20r%5bauthor%5d&cauthor=true&cauthor_uid=12874441 https://www.ncbi.nlm.nih.gov/pubmed/?term=haneda%20m%5bauthor%5d&cauthor=true&cauthor_uid=12874441 https://www.ncbi.nlm.nih.gov/pubmed/?term=fiorentino%20tv%5bauthor%5d&cauthor=true&cauthor_uid=23448484 https://www.ncbi.nlm.nih.gov/pubmed/?term=prioletta%20a%5bauthor%5d&cauthor=true&cauthor_uid=23448484 https://www.ncbi.nlm.nih.gov/pubmed/?term=zuo%20p%5bauthor%5d&cauthor=true&cauthor_uid=23448484 https://www.ncbi.nlm.nih.gov/pubmed/?term=folli%20f%5bauthor%5d&cauthor=true&cauthor_uid=23448484 https://www.ncbi.nlm.nih.gov/pubmed/23448484 https://www.ncbi.nlm.nih.gov/pubmed/?term=lyons%20tj%5bauthor%5d&cauthor=true&cauthor_uid=26366051 https://www.ncbi.nlm.nih.gov/pubmed/?term=jenkins%20aj%5bauthor%5d&cauthor=true&cauthor_uid=26366051 https://www.ncbi.nlm.nih.gov/pubmed/?term=samper%20e%5bauthor%5d&cauthor=true&cauthor_uid=14570235 https://www.ncbi.nlm.nih.gov/pubmed/?term=nicholls%20dg%5bauthor%5d&cauthor=true&cauthor_uid=14570235 https://www.ncbi.nlm.nih.gov/pubmed/?term=melov%20s%5bauthor%5d&cauthor=true&cauthor_uid=14570235 https://www.ncbi.nlm.nih.gov/pubmed/14570235 https://www.ncbi.nlm.nih.gov/pubmed/?term=campagnol%20pc%5bauthor%5d&cauthor=true&cauthor_uid=22391056 https://www.ncbi.nlm.nih.gov/pubmed/?term=dos%20santos%20ba%5bauthor%5d&cauthor=true&cauthor_uid=22391056 https://www.ncbi.nlm.nih.gov/pubmed/?term=terra%20nn%5bauthor%5d&cauthor=true&cauthor_uid=22391056 https://www.ncbi.nlm.nih.gov/pubmed/?term=terra%20nn%5bauthor%5d&cauthor=true&cauthor_uid=22391056 https://www.sciencedirect.com/science/article/pii/s0309174012000484#! https://www.ncbi.nlm.nih.gov/pubmed/22391056 https://www.ncbi.nlm.nih.gov/pubmed/?term=hayashi%20m%5bauthor%5d&cauthor=true&cauthor_uid=6381265 https://www.ncbi.nlm.nih.gov/pubmed/?term=nohmi%20t%5bauthor%5d&cauthor=true&cauthor_uid=6381265 https://www.ncbi.nlm.nih.gov/pubmed/?term=sawada%20m%5bauthor%5d&cauthor=true&cauthor_uid=6381265 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.24(2) 2015 poisoning cases in the poisoning consultation center 22 evaluation of poisoning cases in the poisoning consultation center and forensic medicine institute within baghdad area faris h.maedie * , hasan alhaddad * and saad a. hussain *, 1 * department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract social factors may affect the available sources of toxic substances and causes of poisoning, and these factors may change over time. additionally, understanding the characteristics of poisoning cases is important for treating such patients. therefore, the present study investigated the characteristics of poisoning cases in baghdad poisoning consultation center (pcc) and forensic medicine institute (fmi). data on all poisoning cases reported in pcc and fmi during 2013 were retrospectively obtained from medical records. total of 1131 reports of poisoning cases (1082 from pcc and 49 from fmi) were analyzed according to age, sex, geographical distribution and causes of poisoning according to the type and class of poisoning agent. the results showed that most of the poisoning case are from urban area, and the incidence in male is greater than that in females. in both centers, the higher percent of poisoning occurred within the age range 11-20 years. regarding the type of poisons, zinc sulphide and carbon monoxide represent the cause of poisoning reported in mfi, while metals (mostly copper) and drugs (mostly cns depressants) represent the major causes of toxicity reported in pcc. in conclusion, among the elements that are common between the two centers are the age distribution of the cases. rodenticides and metals represent the major causes of poisoning cases reported in baghdad during 2013. the study results suggest that it is necessary to continuously collect data of patients admitted to emergency departments with toxic poisoning at multiple centers. keywords: poisoning, consultation center, forensic medicine. وهعهد الطب العدلي في هدينة بغداد تقيين حاالت التسون في هركز استعالهات السوىم فارس حويد هعيدي * ، حسن الحداد * 1،*سعد عبدالرحون حسين و * . فشع األدٌٔخ ٔانغًٕو، كهٍخ انصٍذنخ، خبيؼخ ثغذاد، ثغذاد، انؼشاق الخالصة ٔأعجبة انزغًى. ْٔزِ انؼٕايم لذ رزغٍش يغ يشٔس انٕلذ. انغبيخ انًصبدس انًزبحخ يٍ انًٕاد ػهى لذ رؤثش انؼٕايم االخزًبػٍخ فً ٓؤالء انًشضى. ٔنزنك، حممذ ْزِ انذساعخ ننؼالج كَٕٓب يٍ يزطهجبد افٓى خصبئص حبالد انزغًى يٍ انًٓى ثبإلضبفخ إنى رنك، . ٔلذ رى انحصٕل ػهى ثغذاد( فً يذٌُخ fmi( ٔيؼٓذ انطت انؼذنً )pccيشكض اعزؼاليبد انغًٕو )خصبئص حبالد انزغًى فً ثأثش سخؼً يٍ انغدالد انطجٍخ. ٔلذ رى رحهٍم يب 3102خالل pcc ٔfmiثٍبَبد ػٍ خًٍغ حبالد انزغًى انزً أػهٍ ػُٓب فً ( ٔفمب نهغٍ ٔاندُظ ٔانزٕصٌغ اندغشافً ٔأعجبة fmiيٍ pcc ٔ94يٍ 0103رمبسٌش ػٍ حبالد انزغًى )يٍ 0020يدًٕػّ ، ٔاإلصبثخ فً فً ثغذاد هزغًى. ٔأظٓشد انُزبئح أٌ يؼظى انحبالد ْى يٍ انًُطمخ انحضشٌخن انؼٕايم انًغججخئخ فمب نُٕع ٔفٔانزغًى ٔ ػبيب. ٔفًٍب ٌزؼهك 31-00يٍ انزغًى داخم انفئخ انؼًشٌخ َغجخ يئٌٕخ أػهى حصهذانزكٕس أكجش يٍ رنك فً اإلَبس. فً كم انًشاكض، انًغزٓذفخ ًؤعغبد انانزغًى انزً أػهٍ ػُٓب فً حبالد يؼظى ك ٔأٔل أكغٍذ انكشثٌٕ رًثم عجتكجشٌزٍذ انضَفأٌ كم يٍ ُٕع انغًٕو، ث كزئبة( رًثم األعجبة انشئٍغٍخ يضبداد اال)ٔيؼظًّ يٍ انًؤثشاد انؼمهٍخانُحبط( ٔثبألخص انًؼبدٌ )ٔرجٍٍ اٌ ، فً حٍٍ ثبنذساعخ اٌ ُبصش انزً ًْ يشزشكخ ثٍٍ انًشكضٌٍ ًْ انزٕصٌغ انؼًشي نهحبالد. . فً انخزبو، يٍ ثٍٍ انؼpccفً زغًى انزً رى رغدٍهٓبنه . َزبئح انذساعخ رشٍش إنى 3102يجٍذاد انمٕاسض ٔانًؼبدٌ رًثم األعجبة انشئٍغٍخ نحبالد انزغًى انزً أػهٍ ػُٓب فً ثغذاد خالل ػبو إنى ألغبو انطٕاسئ فً يشاكض يزؼذدح.انًصبثٍٍ ثحبالد انًحبنٍٍ انًشضى يٍأٌ يٍ انضشٔسي خًغ انجٍبَبد ثشكم يغزًش الكلوات الوفتاحية : التسون، استعالهات السوىم، الطب العدلي . introduction poisoning is a serious health problem in many developed countries, but it is still poorlydefined in developing countries (1-4) . more than 9 million natural and synthetic chemicals are used worldwide, and the no. keeps increasing definitely (5) . in iraq, the problem is getting worse with time, as newer drugs and chemicals are developed in vast numbers, and there are no stringent rules and regulations for their dispensing and use. pesticides are the most common cause of poisoning. according to world health organization (who) estimates, approximately 3 million pesticide poisonings occur worldwide each years, leading to more than 220 000 deaths. developing countries like sri lanka and india report high rates of toxicity and death (6,7) . however, in iraq there is limited local information on the toxicity of pesticides or other toxicants due to insufficient cases documentation. in 1956, an outbreak of human poisoning by mercury occurred in northern iraq, which caused more than 100 hospitalized patients and at least a 12 fatalities (8) . furthermore, in 1971-72 a massive outbreak of alkylmercury poisoning, caused by the ingestion of treated seed occurred in iraq. 1 corresponding author e-mail: saad_alzaidi@yahoo.com received: 26/4/2015 accepted: 29/9/2015 iraqi j pharm sci, vol.24(2) 2015 poisoning cases in the poisoning consultation center 23 this case has discussed during november 1974 in an international conference that organized in baghdad by the world health organization in conjunction with the iraqi ministry of health and the swedish international development authority (9) . there are increasing number of poisoning cases reported in iraq, the reason could be attributed to the increasing number of toxic chemicals and their large-scale use without proper regulations and direction for use and storage. banned products also continued to flow into the market like household agents , cleaning products, products that contain dimethylfumarate (dmf), or medicines that have further widened the spectrum of toxic products to which people may be exposed. the scale of the problem is enormous due to the increased incidence of morbidity and mortality (2) . improvement in the preventive and management approaches can be enhanced by identification of high risk population, susceptible groups within the population, identification of chemicals and commercial products implicated in poisoning cases in the community (10-12) . in iraq, clinical and toxicological diagnostic and treatment facilities are usually not satisfying due to the lack of well-trained personnel and lack of appropriate treatment. moreover, the weak communication between doctors, medical personnel, and the poisoning control center (pcc) make it difficult for good information flow between the medical team. according to that, we conducted this study to determine the poisoning cases reported in 2013 in baghdad pcc and the forensic medicine institute (fmi). however, we discussed the main problem facing pcc in the treatment of poisoning cases. material and methods poisoning cases received by the medical referral system (written request, direct contact, samples and calls) to the baghdad poisoning consultation center (pcc) or the forensic medicine institute in baghdad (fmi), are analyzed during a period of one years (january-december 2013); the total number of cases reported in baghdad pcc was 1082 (58.4 males and 41.6% females), and in the fmi was 49 (53.1 males and 46.9 females), during the year 2013. the reports form obtained from the pcc included the time, date, mode of inquiry, enquirer’s name and address, patient’s or victim’s age, sex, chief complaint, symptoms and treatments given. the report form obtained from the fmi include victim’s sex, age, address, and the cause of death. the age, sex, geographical distribution and causes of poisoning according to the type and class of poisoning agent are analyzed. results a total of 1131 reports of poisoning cases reported in baghdad pcc and fmi during a period of one year (januarydecember 2013), were retrieved and evaluated. there were 1082 cases of a positive result documented in the medical records of baghdad pcc, and 49 cases in the fmi. geographical distribution of poisoning cases reported in baghdad pcc indicated that poisoning cases from urban area were 731 (67.5%), and those reported in suburban area were 351 (32.5%) as shown in figure 1. figure (1): geographical distribution of poisoning cases reported in baghdad pcc during 2013. meanwhile, the geographical distribution of poisoning cases reported in the fmi showed that cases from urban region represent 65.3% (32 cases), while those reported from suburban regions represent 34.7% (17 cases), as shown in figure 2. figure (2): geographical distribution of poisoning cases reported in the forensic medicine institute during 2013. iraqi j pharm sci, vol.24(2) 2015 poisoning cases in the poisoning consultation center 24 when the reported cases of poisoning were ranked according to gender bases, table 1 showed that the poisoning cases were higher in males than females (632, 58.4% vs. 450, 41.6%), and the maximum number of cases (during 2013) are reported during the following months: september (126) > july (117) > may (105) > august (101). the percent of poisoning in males reported in baghdad pcc is greater than that reported in the fmi (58.4% vs. 53.1%), while the percentage of poisoning within females reported in the fmi is greater than that reported in baghdad pcc (46.9% vs. 41.6%), within the same period of time (2013) (figure 3). table (1): distribution of poisoning cases according to gender and month of the year in baghdad pcc during 2013. month male no. (%) female no. (%) total january 40 (54.8) 33 (45.2) 73 february 65 (65.7) 34 (34.3) 99 march 51 (54.3) 43 (45.7) 94 april 48 (52.7) 43 (47.3) 91 may 54 (51.4) 51 (48.6) 105 june 45 (57.7) 33 (42.3) 78 july 80 (68.4) 37 (31.6) 117 august 58 (57.4) 43 (42.6) 101 september 67 (53.2) 59 (46.8) 126 october 39 (59.0) 27 (41.0) 66 november 38 (62.3) 23 (37.7) 61 december 47 (66.2) 24 (33.8) 71 total 632 (58.4) 450 (41.6) 1082 figure (3): gender distribution of poisoning cases reported in baghdad pcc and forensic medicine institute during 2013. the results of the present study showed that maximum number of poisoning cases were reported within the age group 11-20 years (32%), followed by the other age groups as follow: 1-10 years (23%)> 21-30 years (17%), 31-40 years (11%)> 51 years and above (10%) (figure 4). figure (4): distribution of poisoning cases reported in baghdad pcc during 2013 according to the age ranges. moreover, distribution of poisoning cases reported in the fmi according to the age range revealed that maximum number of cases are reported within the age group 11-20 years (32%), similar to that reported in baghdad pcc; the remaining cases are distributed as follow: 1-10 years (20%)>31-40 years (18%)> 21-30 and 1-10 years (12% each)> 51 years and above (6%) (figure 5). figure (5): distribution of poisoning cases reported in the forensic medicine institute during 2013 according to the age ranges. regarding the type of agents that predispose to the poisoning cases reported in the fmi, figure 6 showed that zinc phosphide and carbon monoxide are the major causative agents (32.6% and 30,4%, respectively), while hydrogen sulphide, kerosene and ketamine predispose to equal percent of poisoning cases iraqi j pharm sci, vol.24(2) 2015 poisoning cases in the poisoning consultation center 25 (8.1% each), followed by cefotaxime (4.1%); while thallium, carbamazepine, phenine diamine, and multi-drugs formulations are responsible for the least amount of poisoning (2.1% each). figure (6): distribution of poisoning cases reported in the forensic medicine institute during 2013 according to the poison type. in baghdad pcc, the highest percent of reported poisoning cases were attributed to metals (82.2%), while unspecified types of drugs predispose to 7.1% cases, followed by pesticides and amanita phalloids, which represent 4.2% and 0.5% of the reported cases at that center, respectively (figure 7). figure (7): distribution of poisoning cases reported in baghdad pcc during 2013 according to the class of the poisoning agent. copper was found to be the causative agent behind 89.1% of poisoning cases reported in baghdad pcc during 2013, while the other types of metals are ranked as follow: magnesium (4.1%)> lead (3.5%)> zinc (2%)> thallium (1.2%)> lead+copper (0.1%) (figure 8). figure (8): distribution of metal-induced poisoning cases reported in baghdad pcc during 2013 according to the metal type. regarding the type of pesticides that predispose to the reported cases of poisoning in baghdad pcc during 2013, zinc phosphide seems to be the causative agent behind 42% of cases, followed by the organophosphates and their mixtures with organochlorines, which predispose to 29% and 11.2% of the cases, respectively (figure 9). meanwhile, warfarin and organochlorine compounds predispose to equal percent of poisoning cases (4.5% each), followed by the other agents that predispose to equal percent of poisoning cases (2.2%each) during 2013 (figure 9). figure (9): distribution of pesticidesinduced poisoning cases reported in baghdad pcc during 2013 according to the specific type of pesticides. table 2 showed that multi-drugs formulations were responsible for 20.8% of poisoning cases reported in baghdad pcc during 2013, followed by carbamazepine (14.3%). meanwhile, diazepam and paracetamol predispose to equal percent of iraqi j pharm sci, vol.24(2) 2015 poisoning cases in the poisoning consultation center 26 cases (7.8% each), while chlorpheniramine, alprazolam, and tramadol predispose to equal percent of poisoning cases (6.5 each). table 2 indicated that 5.1% of reported cases were due to ingestion of diphenhydramine, while amitriptyline, imipramine, benzhexol, and codeine were found to be the causative agents in 2.6% of poisoning cases for each one of them. the other types of drugs were found to be responsible for equal percent of poisoning cases (1.3% each). table (2): incidence of drugs-induced poisoning reported in baghdad pcc during 2013 distributed according to drug type. name of drug no. of cases percent carbamazepine 11 14.3 diazepam 6 7.8 amitriptyline 2 2.6 niflumic acid 1 1.3 chlorpheniramine 5 6.5 diphenhydramine 4 5.1 paracetamol 6 7.8 atenolol 1 1.3 alprazolam 5 6.5 metronidazole 1 1.3 imipramine 2 2.6 tca 1 1.3 clonazepam 1 1.3 cinnarizine 1 1.3 oxazepam 1 1.3 benzhexol 2 2.6 tramadol 5 6.5 codeine 2 2.6 rifampicin 1 1.3 methamphetamine 1 1.3 glibenclamide 1 1.3 lithium 1 1.3 multi-drugs 16 20.8 total 77 100 discussion in emergency settings, the proportion of patients with poisoning differs depending on which agents are defined as toxic and classification of these toxic agents. toxic poisoning may be broadly or improperly defined, and the definition may differ in different countries and societies, and over time (13) . additionally, collection and analysis of nationwide data on poisoning plays a key role in informing policies for toxic substances, including the production and sales management of toxic substances as well as, establishment of poison control centers and stores of the required antidotes (14,15) . therefore, relying on treatment data from other countries is problematic and cannot be easily interpreted according to the local findings. in usa for example, nationwide toxic poisoning centers participate in the toxic poisoning surveillance system (tess), and data are stored in a database operated by the american association of poison control centers (aapcc) (16) . however, there are no standardized nationwide guidelines for such classifications in other countries including iraq (17) . the present study shed a light on some of the available data in two centers that concern with reporting poisoning cases in baghdad. comparative evaluation was not possible due to inconsistency of reported details in the two centers (baghdad pcc and fmi), and this was considered as a sounded limiting factor of the present study. moreover, since the study followed the retrospective approach, the data are presented as appeared in the patient's files. the present study focused on providing a comprehensive view about some important data related to the characteristics of iraqi patients with toxic poisoning that were reported in baghdad pcc and fmi during 2013. however, thorough comparison of data between these two institutions are not amenable due to inconsistence of registration approaches and inadequacy of certain information. with respect to the age groups of patients, the results indicate some degree of similarity, where the age group 11-20 years demonstrates the higher rate of poisoning; the geographical distribution of the cases also indicate similar incidence pattern in both centers. in 1992, song et al. reported that patients in their 20s were the highest proportion (35.7%) of patients with toxic poisoning (18) . additionally, lee et al. (1996) also found that the most common age group with poisoning patients were in their 20s (46.8%) (19) . meanwhile, kang et al. (1999) also reported that 23.7% of patients with toxic poisoning were in their 20s (20) . in other countries, burillo-putze et al. (2003) reported that the average age of patients with toxic poisoning in spain was 33 years (21) , while xiang et al. (2007) reported that most patients iraqi j pharm sci, vol.24(2) 2015 poisoning cases in the poisoning consultation center 27 with toxic poisoning admitted to emergency departments in usa were aged 35-44 years (22) . in baghdad, and according to the patient's data abstracted from the two centers, highest incidence of poisoning were found in teenage and young people, and then declined in older ages. comparison with the data presented by other investigators may be not exactly accurate, because the data presented in the current study not represent the national situation in iraq. in the present study, poisoning was more common in males than females according to reports of baghdad pcc, while the opposite situation was predicted in the fmi during 2013. the wide variation between sample sizes from both institutions could be the reason behind this result, and this finding seems to be in tune with that reported by other investigators, while not with others. lee et al. reported a male-to-female ratio of 1:173 in poisoned patients (19) . similarly, the proportion of females among poisoning patients was reported to be 52.1%, according to so et al. (14) , 53.3% according to kang et al. (20) , and 68.4% according to song et al. (18) . given that female patients are exposed to toxic substances at a higher rate than male patients, and the main cause of poisoning being consumption of toxic material while committing suicide, this finding is most likely related to the high rate of suicidal attempts among women (23) . according to xiang et al., 56.7% of all suicide attempts are made by women (22) . the previously mentioned data are in tune with the fmi reports. in contrast to the iraqi fmi data, burillo-putze et al. reported that 56% of poisoning are reported in men in spain (21) , which was in tune with the data reported in baghdad pcc during 2013. although the present study do not clarify whether exposure to toxic substances accidental or for committing suicide, most cases of the reported cases of poisoning in other countries are due to consumption of toxic substances for committing suicide, while accidental poisoning are the second most common cause. burillo-putze et al. reported that 77.7% of patients admitted to emergency departments with acute toxic poisoning had attempted suicide, which are similar to our study findings: 74.9% and 72.0% of admissions in 2003 and 2011, respectively, were due to suicide attempts (21) . similar rates were reported by other investigators (18-20) . regarding medication-related poisoning cases, the proportion of toxic poisoning cases due to prescribed drugs tended to increase over time, pharmacies were the most common source of toxic materials followed by other types of stores (24) . the finding of the present study was not in tune with the previous one, where drugs related cases are not the primary cause, as reported in baghdad pcc. the present study showed that during 2013, the most poisoning cases reported in baghdad pcc were admitted to emergency departments in september, july, may and august, respectively. this finding may not be consistent with other reports due to many social and geographical variations (25) . the most important factor to consider in the treatment of acute toxic poisoning is the causative substance. decontamination and treatment methods as well as antidotes used are different according to toxic substances. therefore, access to data on substances commonly associated with toxic poisoning is very important for medical staff dealing with patients in emergency departments. the substances most commonly causing poisoning vary depending on the patient’s society, for example, the ease of purchase and acquisition of toxic substances. in the present study, exposure to the rodenticide zinc phosphide and carbon monoxide were the most common causative agents in reported in fmi during 2013, while in baghdad pcc exposure to toxic metals represents the major cause within the same period. such discrepancy could be attributed to the difference in sample size, or that the fmi reports include only the death cases referred for forensic evaluation. several studies have reported that pesticides were the most commonly used agents for poisoning in many regions worldwide. however, the present study findings showed that pesticides contributed only for 7.1% of the poisoning cases within baghdad area; the social class of the referred cases to baghdad pcc were mostly from urban regions, and might explain the small contribution of pesticides in poisoning cases. this finding was not consistent with that reported by others (26,27) . in addition to pesticide poisoning, the other common types of expected poisonous agents were medicines like benzodiazepines, analgesics, anti-depressants and others. the present study showed that multi-drugs formulations represent the major causative agent of drugs-related poisoning, followed by other drugs especially cns depressants and anticonvulsants. medicines are used as poisoning agents for intentional poisoning in developed countries (28) and urban areas. in certain places, street drugs and otc medicines like paracetamol, antihistamines are also used for intentional poisoning (29, 30) . iraqi j pharm sci, vol.24(2) 2015 poisoning cases in the poisoning consultation center 28 conclusions the present study evaluates the characteristics of poisoning cases reported in baghdad pcc and fmi. among the elements that are common between the two centers are the age distribution of the cases. rodenticides and metals represent the major causes of poisoning cases reported in baghdad during 2013. the study results suggest that it is necessary to continuously collect data of patients admitted to emergency departments with toxic poisoning at multiple centers. acknowledgment the authors thank the college of pharmacy, university of baghdad for support, and the staff of baghdad pcc and fmi for technical support. the data were presented by faris h. maedie as bsc graduation project references 1. seneviratne b, thambipillai s. pattern of poisoning in developing agricultural country. br j prev soc med 1974; 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(asteraceae) is one of echinops genus species that spread in syria, lebanon, and palestine. phytochemicals found in this species roots have been extracted with gradient polarity solvents, and primary screening of the secondary metabolites was established. then, the phenolic compounds content was determined with folin-ciocalteu reagent and flavonoids content with aluminum chloride reagent. the free radicals scavenging activity was evaluated for all extracts with dpph• in a 96-well microplate. the selectivity study indicates that ascorbic acid and reducing sugars didn't exist in the extracts. the identification tests showed the presence of polyphenols like flavonoids and coumarins. the methanolic extract of the e.polyceras roots was the most effective scavengers of free radicals (84% in 30 min) with phenolic compounds content 575.5 mg gae/g de and flavonoids content 130 mg qe/ g de, and the chloroform extract was the least effective as free radical scavenging (45% in 30 min) as the phenolic compounds content was 222.5 mg gae/g de and flavonoids content 57.5mg qe/ g de. in conclusion, the phenolic compounds and flavonoids from echinops polyceras boiss. are effective in free radicals scavenging and protecting from diseases caused by oxidative stress. keywords: antioxidants, polyphenols, flavonoids, free radicals, and echinops polyceras boiss دراسةُ الفعاليّة الُمضادة لألكسدة لعديدات الفينول والفالفونوئيدات الُمستخلَصة من جذور نبات echinops polyceras النامي في سورية *ميس خازم و 1،عيسى العّساف* كليّة الصيدلة، جامعة دمشق، دمشق، سورية في قسم العقاقير * الخالصة فرط تُعدّ الجذور الُحّرة نواتج طبيعّية لعمليات االستقالب، وتتميّز أنها غير ثابتٍة وذات عمٍر قصير وُمتفاعلة بشدّة، ويُمكن أن يُسبب echinopsإنتاجها أمراضاً تنكسيّة مثل السرطان. تتميّز ُمضادات األكسدة مثل عديدات الفينول بدورها الوقائّي ضد الجذور الُحّرة. يُعدّ نوع polyceras boiss. أحد أنواع جنسechinops .استُخلصت الُمكّونات الفعّالة الموجودة في جذور هذا الذي ينتشر في سورية ولبنان وفلسطين -لفينولّي )باستخدام كاشف فولينالنوع باستخدام ُمحاّلٍت ُمتدّرجة القطبّية، وقد تّم بعد ذلك إجراء تحّرٍ أولّي عن الُمستقلبات الثانوّية. تّم تحديد الُمحتوى ا استخدام كاشف سيكالتو( والفالفونوئيدّي )باستخدام كاشف كلوريد األلمنيوم(، ومن ثّم تقييم الفعاليّة الكاسحة للجذور الُحّرة للخالصات المدروسة ب dpph• ن كّلٍ من حمض األسكوربيك والسكريات حفرة. أظهرت نتائج دراسة االنتقائّية خلو الخالصات م 96وذلك باستخدام طبٍق للزرع ذو الكحولّي الُمختزلة، كما أظهرت تفاعالت نتائج الكشوفات األوليّة وجود عديدات الفينول مثل الفالفونوئيدات والكومارينات. وقد أبدى الُمستخلص 130والفالفونوئيدّي mg gae/g de 575.5دقيقة( حيث بلغ الُمحتوى الفينولّي 30خالل %84أفضل نشاط في كسح الجذور الُحّرة وبتقدير ) mg qe/ g de( حيث بلغ الُمحتوى الفينولّي 30خالل %45، وكانت أدنى فعالية ُمضادة لألكسدة لُمستخلص الكلوروفورم )222.5دقيقة mg gae/g de 57.5والفالفونوئيدّيmg qe/ g deفالفونوئيدات تمتلك فعّالّية في كسح . يُمكن تلخيص ما سبق بأن الُمركبات الفينوليّة وخاصةً ال .الجذور الُحّرة وبالتالي الوقاية من األمراض الناتجة عن الشدة التأكسديّة boiss echinops polycerasعديدات الفينول، الفالفونوئيدات، الجذور الُحّرة وُمضادات األكسدة، الكلمات المفتاحيّة: introduction free radicals are products of normal cellular metabolism, they are atoms or molecules which contain one or more unpaired electrons in a valency shell. the odd number of electron(s) of a free radical makes it unstable, short-lived and highly reactive (1). the excessive production of free radicals is considered to be an important cause of oxidative damage in biomolecules, such as proteins, lipids, and dna, this damage leads to numerous degenerative diseases (2), such as cancer, atherosclerosis, gastric ulcer, and other conditions (3). antioxidants are molecules that can prevent or delay the oxidation of substrate, these compounds have a high affinity for free radicals and scavenge them to protect our health (4). polyphenols are strong antioxidants that have a protective role against oxidative stress caused by excess free radicals (5). the mechanism of the protective action of phenolic compounds in plants relies on the antioxidant activity that scavenges free radicals, protection of lipid peroxidation, and the chelation of toxic metals (6). 1corresponding author e-mail: issa.alassaf.92@gmail.com received: 18/ 5/2021 accepted:11 / 7 /2021 published online first: 2021-12-12 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp261-268 iraqi j pharm sci, vol.30(2) 2021 antioxidant activity of echinops polyceras 262 flavonoids are a large subgroup of the family of phenolic compounds, and because of the presence of multiple hydroxyl groups in their structure flavonoids have reducing properties (7). echinops (asteraceae) is a genus that includes 120 species of perennials, annuals, and biennials plants. these species are found in eastern and southern europe, tropical and north africa, and asia (8). echinops polyceras boiss. is a perennial herb, 40-60 cm, sometimes with very fine and short whitish glandular hairs in the lower part. basal leaves congested, oblong-lanceolate, pinnatipartite into short lobes armed with short yellow spines. heads generally abundantly, with about 4.5-5 cm in diameter (not including cornigerous bracts). partial involucre of non-cornigerous headlets about 2 cm, pale green. corolla is white to pale bluish, anthers are greyish-violet, and the flowering time is june – july (9). this species spread in syria, lebanon, and palestine (10). the aim of this study was to evaluate the phytochemicals and the free radicals scavenging activity of echinops polyceras boiss root since no previous studies have distinguished the chemical constituents and the biological effects of this plant. materials and methods chemicals gallic acid (was purchased from avonchem), quercetin (from sigma-aldrich), 2,2-diphenyl-1-picrylhydrazyl dpph (from tci), distilled water, absolute ethanol (from merck), absolute methanol (from sigma-aldrich), ethyl acetate (from sham lab), chloroform (from merck), glacial acetic acid (from bdh), hydrochloric acid (from himedia), sulfuric acid (from himedia), folin ciocalteu (from sigmaaldrich), sodium carbonate (from scharlau), aluminum chloride (from scharlau), potassium acetate (from merck), ascorbic acid (from panreac quimica slu), magnesium metal turnings (from chem-lab), ferric chloride (from panreac), potassium iodide (from eurolab), bismuth nitrate (from himedia) and mercuric chloride (from himedia), picric acid (from panreac eu), iodine crystals (from honeywell), 3,5-dinitrobenzoic acid (from titan biotech), gelatin. apparatus 96-well microplate reader (biotek), rotary evaporator. plant material the whole plant of flowering echinops polyceras boiss. was collected from ma'aret sednaya (rif-dimashq, syria) in july 2019, and authenticated by dr. imad alkadi (department of plant biology, damascus university, syria). the roots were separated from the rest of the plant parts, then dried in shade and powdered. extraction of the plant roots: dried and powdered roots of echinops polyceras boiss. (20 g) were extracted with 200 ml of each of gradient polarity solvents: distilled water, ethanol 50%, absolute methanol, methanol + ethyl acetate (1:1), and chloroform, at room temperature with shaking for seven days. the five extracts were evaporated separately by a rotary evaporator, and the extraction yield was calculated by the equation: yield% = (weight of evaporated extract/ weight of roots powder) ×100 phytochemical identification echinops polyceras roots extract was assessed for the existence of flavonoids, coumarins, tannins, anthraquinones, alkaloids, saponins and cardiac glycosides. test for flavonoids lavonoids were identified by uv (366 nm) fluorescence after the addition of 1 ml of 5% aluminum chloride in ethanol (11).  magnesium metal (0.5 g) was added to 5 ml of ethanolic extract then 1 ml of concentrated hcl was added. a pink or red coloration that disappears on standing for 3 minutes indicates the presence of flavons (shinoda test). test for coumarins ethanolic extract (5 ml) was evaporated, and the residue was dissolved in 2 ml of hot distilled water, then few drops of this solution were put on a filter paper and the fluorescence under uv light was examined. an intense blue fluorescence indicates the presence of coumarins. test for tannins  ferric chloride test: ethanolic extract (1 ml) was added into a test tube then 2 to 3 drops of 10% of ferric chloride (fecl3) solution were added, and observed for a dark green (hydrolysable tannins) or dark blue (condensed tannins) coloration (12).  gelatin test: tested extract (1 ml) was placed in a test tube, then 2 drops of 1 % gelatin solution with 10% sodium chloride were added. a white precipitate formation indicates the presence of tannins (13). test for anthraquinones:  borntrager test: e. polyceras roots powder (1 g) was extracted with 10 ml of chloroform for 10 minutes and filtered, then 2 ml of ammonia were added. the formation of red color in the aqueous layer indicates the presence of free anthraquinones (12).  modified borntrager test boil 1g of the plant material with 2ml of dilute sulphuric acid, 2ml of 5% aqueous ferric chloride solution for 5 minutes and continue the reaction as borntrager test. the formation of red iraqi j pharm sci, vol.30(2) 2021 antioxidant activity of echinops polyceras 263 color in the aqueous layer indicates the presence of anthraquinone glycosides (13). test for alkaloids ethanolic extract (20 ml) was evaporated, and the dry residue dissolved in 5 ml of hcl (2n) and filtered. then, few drops of mayer, dragendroff, wagner and hager reagents were added. the formation of white, orange, reddish-brown and yellow precipitates respectively indicate the presence of alkaloids (12, 13). test for saponins to 0.5 g of the aqueous extract, 20 ml of hot water were added into a test tube, the tube was shaken vigorously. the formation of a stable foam indicates the presence of saponins (12). test for cardiac glycosides  keller killiani test: to 2 ml of the ethanolic extract, 1 ml of glacial acetic acid was added with one drop of 5% fecl3 and 1 ml concentrated h2so4. the formation of reddish-brown color at the junction of the two liquid layers, and the bluish-green color at the upper layer indicate the presence of cardiac glycosides (14).  kedde’s test: evaporate the chloroform extract of the roots, then add one drop of 90% alcohol and 2 drops of the reagent (2% 3,5-dinitro benzoic acid in 90% alcohol), an alkaline solution (20% sodium hydroxide solution) was added. purple color is produced in the case of the presence of β unsaturated-o lactones (13). determination of total phenolic content (tpc) total phenolic content was determined by a micro colorimetric method described by ainsworth & gillespie (15): 200 mg of each extract were dissolved with 2 ml of methanol 95% (vol/vol). 100 µl of each sample were transferred to 2 ml microtubes and were mixed with 200 µl of 10% (vol/vol) folin–ciocalteu reagent, the mixture was vortexed thoroughly. then 800 µl of na2co3 (700 mm) was added into each tube, and the assay tubes were incubated at room temperature for two hours. 200 µl of samples, standard (gallic acid), and blank (200 µl of 10% (vol/vol) folin–ciocalteu reagent with 800 µl of 700 mm na2co3) were transferred to a clear 96-well microplate, and the absorbance of each well was read at 765 nm in triplicate. gallic acid calibration curve the calibration curve was established with nine dilutions of gallic acid standard at concentrations of (12, 24, 36, 48, 60, 84, 96, 108, 120) mg/l. then the absorbance was read at 765 nm using the microplate reader. tpc of the extracts: total phenolic content was calculated as gallic acid equivalents (gae) in 1 g of dried extract (de) using the regression equation between gallic acid standard concentrations and absorption at 765 nm. specificity the specificity of folin–ciocalteu method was checked by detection of the presence of some reducing compounds like reducing sugars and ascorbic acid.  detection of reducing sugars using fehling's test: 1 ml of the ethanol extract was diluted with 1ml of water in a test tube, then 20 drops of boiling fehling’s solution (a and b) was added. the formation of a precipitate red-brick in the bottom of the tube indicates the presence of reducing sugars (12).  detection of ascorbic acid using a spectrophotometric method: determination of λmax of ascorbic acid (16) ascorbic acid (0.1 g) was dissolved with distilled water in a volumetric flask (100 ml), then 1 ml of this solution was transferred into another 100 ml volumetric flask with the addition of 10 ml of 0.1 n of hydrochloric acid, distilled water was used to complete the rest volume to 100 ml. the λmax was determined by a spectrophotometric scan between 200-300 nm. scanning of extract solution aqueous extract (1 g) was also dissolved with distilled water in a volumetric flask (100 ml), then 1 ml of the solution was transferred into another 100 ml volumetric flask with 10 ml of 0.1 n of hydrochloric acid, then distilled water was used to complete the rest volume to 100 ml. the absorbance was detected at the λmax of ascorbic acid. determination of total flavonoids content (tfc) flavonoids content was determined according to chang et al. protocol (17): 200 mg of each extract were mixed with 1.5 ml of 95% ethanol, 100 µl of 10% alcl3 (w/v) solution, and 100 µl of 1 mol/l potassium acetate solution were added, and the assay tubes were incubated at room temperature for 30 min. 200 µl of samples, standard (quercetin) and blank (100 µl of 10% alcl3 (w/v) with 100 µl of 1 mol/l potassium acetate) were transferred to a clear 96-well microplate and read the absorbance of each well at 420 nm in triplicate. quercetin calibration curve the calibration curve was established with seven dilutions of quercetin standard at concentrations of (6, 12, 24, 30, 36, 48, 60) mg/l. then the absorbance was read at 420 nm using the microplate reader. tfc of the extracts total flavonoids content was calculated as quercetin equivalents (qe) in 1 g of dried extract (de) using the regression equation between quercetin standard concentrations and absorption at 420 nm. evaluation of free radicals scavenging activity (rsa) iraqi j pharm sci, vol.30(2) 2021 antioxidant activity of echinops polyceras 264 the dpph• radical scavenging activity was evaluated according to cheung et al. method (18) with some modification by choi et al. briefly, 160 µl of 0.2 mm dpph• in methanol were mixed with 40 µl of the extracts or standards (ascorbic acid, gallic acid, and quercetin) in a 96-well microplate. the mixtures were left to stand at room temperature, and the absorbance at 520 nm was measured against methanol as a blank after 10 and 30 min. free radicals scavenging activity (rsa) was determined by the equation: rsa% = 100 × 𝐀𝟎−𝐀𝐬 𝐀𝟎 where: a0: absorption of dpph• solution as: absorption of dpph• solution after 10 and 30 min of the sample addition. results extraction yield the extraction yield% of the extracts is shown in table (1). aqueous extract showed the highest yield (10.5%) followed by hydroethanolic 50%, methanol, methanol: ethyl acetate (1:1), and chloroform extracts with yields of 7, 4, 2.75, and 1%, respectively. table 1． extraction yield extracts yield% dh2o 10.5 etoh 50 7 meoh 4 meoh+etoac 2.75 chcl3 1 phytochemical identification the results of the identification tests are shown in table (2). table 2 ． phytochemical identification of e. polyceras roots flavonoids aluminum chloride + shinoda test + coumarins fluorescence + tannins ferric chloride test + gelatin test anthraquinones borntrager test modified borntrager alkaloids mayer dragendroff wagner hager saponins foam test + cardiac glycosides keller killiani test kedde’s test gallic acid calibration curve gallic acid concentrations and their absorbances are shown in table (3). also, the linearity and the regression equation are shown in figure (1). table 3. gallic acid concentrations and their absorbances figure 1. gallic acid calibration curve tpc of the extracts total phenolics content in the extracts was presented in table (4). the methanolic extract showed the highest content of phenolic compounds (575.5 mg gae/g de), followed by meoh: etoac (1:1), etoh 50%, distilled h2o, and chcl3 extracts, respectively. the tpc results are shown in figure (2). concentration (mg/l) ā 765 0 0 12 0.174 24 0.317 26 0.428 48 0.595 60 0.721 84 1.011 96 1.13 108 1.223 120 1.378 iraqi j pharm sci, vol.30(2) 2021 antioxidant activity of echinops polyceras 265 table 4.tpc, tfc and rsa% of the standards and the extracts. samples tpc±sd (gae/ g de) tfc±sd (qe/ g de) rsa% time for rsa≥ 90% 10 min 30 min ascorbic acid 91.21 93.85 < 10 min gallic acid 83.52 87.54 > 30 min quercetin 85.71 89.59 > 30 min dh2o 403.5 ±0.335 103 ±0.541 76.61 82.65 > 30 min etoh 50 486 ±0.335 119 ±0.207 77.39 81.55 > 30 min meoh 575.5 ±0.358 130 ±0.435 78.96 84.07 > 30 min meoh+etoac 521.5 ±0.503 123 ±0.281 73.47 83.28 > 30 min chcl3 222.5 ±0.276 57.5 ±0.405 35.16 45.27 > 30 min figure 2. tpc in 1 g of the dried extracts all results of tpc are presented as the mean of three replicates ± sd (p < 0.05) specificity  detection of reducing sugars: the result of fehling's test indicates the absence of reducing sugars because the red-brick precipitate does not exist.  detection of ascorbic acid using a spectrophotometric method: determination of λmax of ascorbic acid: ascorbic acid solution showed the maximum absorbance at 240 nm, according to figure (3). figure 3. the scanning of ascorbic acid solution scanning of extract solution the scanning of the extract solution is shown in figure (4), it indicates that the extract of e. polyceras roots is free of ascorbic acid. figure 4. the scanning of roots extract solution. quercetin calibration curve the concentrations of quercetin and their absorbances are shown in table (5). also, the linearity and the regression equation are shown in figure (5). iraqi j pharm sci, vol.30(2) 2021 antioxidant activity of echinops polyceras 266 table 5. quercetin concentrations and their absorbances figure 5. quercetin calibration curve tfc of the extracts flavonoids content in the extracts is also presented in table (4). as the methanolic extract showed the highest phenolic amount, it contained the highest flavonoid content (38.9 mg qe/ g de), followed by meoh: etoac (1:1), etoh 50%, distilled h2o and chcl3 extracts, respectively. the tfc results are shown in figure (6). figure 6. tfc in 1 g of the dried extracts all results of tfc are presented as the mean of three replicates ± sd (p < 0.05) rsa of the extracts dpph• assay revealed that the methanolic extract was the most effective in free radicals scavenging after 30 min (≈ 84.1 %), compared with the other extracts: meoh: etoac (1:1), dh2o, etoh 50%, and chcl3, they scavenge 83.28, 82.65, 81.55, and 45.27 % of dpph• radical, respectively. the scavenging activities after 10 and 30 min for the standards: [ascorbic acid (aa), gallic acid (ga) and quercetin (q)] and the studied extracts are shown in figure (7). the dpph• scavenging activities after 10 and 30 min, total phenolic, and flavonoid contents of the extracts is shown in table (4). figure 7.rsa% of standards and studied extracts after 10 and 30 min statistical analysis all experiments were accomplished in triplicate. the results were expressed as the mean± standard deviation (sd). one-way analysis of variance (anova) was carried out to identify significant differences between experimental groups, using microsoft excel 2019. differences were considered significant (p < 0.05). correlation ratio was calculated between: total phenolic content and flavonoids content of the roots extracts, it was ≈ 98%. free radical scavenging activity and total phenolic content, it was ≈ 82% free radical scavenging activity and flavonoids content, it was ≈ 89% discussion neither the chemical composition nor the biological effects of echinops polyceras boiss. have been studied previously. in this study, a primary chemical screening has been established, the polyphenols and flavonoids contents have been determined and the free radicals scavenging activity has been evaluated for five different extracts of this plant. the results of extraction yield of e. polyceras boiss. roots (h2o> etoh 50%> meoh 100% > meoh+ etoac (1:1) > chcl3) -which descended according to the polarity of the solvent may refer to the polarity of the extracted compunds, or the presence of the secondary metabolites as glycosides more than free aglycons because the solvents have a crucial role in the type of the secondary metabolites found in the extracts (19). the phytochemical screening revealed the existence of polyphenols as an important component in the concentration (mg/l) ā 420 0 0 6 0.058 12 0.109 24 0.196 30 0.235 36 0.285 48 0.363 60 0.456 iraqi j pharm sci, vol.30(2) 2021 antioxidant activity of echinops polyceras 267 extract, especially flavonoids and coumarins, because of that the content of total phenols and flavonoids were determined. ascorbic acid and reducing sugars are reducing compounds that may interfere with the antioxidant activity of the phenolic compounds in the extracts (20), so, they were identified in the specificity tests, which indicates the absence of ascorbic acid and the reducing sugars. the methanolic extract of e. polyceras roots showed the highest content of total phenols (575.5 gae/ g de) and flavonoids (194.5 qe/ g de), while the chloroform extract showed the lowest contents 222.5 gae/ g de and 94 qe/ g de respectively. based on these results, the examined extracts have significant antioxidant and free radicals scavenging effects, the most effective extract among them was the methanolic extract (84% in 30 minutes), while the chloroform extract was the less effective (45% in 30 minutes), this may be explained by the content of phenols and flavonoids in the extracts. according to the correlation ratio, the free radicals scavenging activity is correlated to the phenolic content and flavonoids content with correlation ratio 82% and 89%, respectively, these results indicate that the phenolic compounds especially flavonoids are effectively contributed to the scavenging activity. also, we noticed that there is a high correlation between phenolic compounds and flavonoids (98%), this may indicate that the majority of phenols in the studied extracts are flavonoids and these compounds were responsible for most of the activity. the phenolic content may contribute directly to the antioxidant activity (21). studied extracts scavenging activities of free radicals were close to the scavenging activities of gallic acid and quercetin standards, this may be explained by the obvious content of phenolic compounds especially flavonoids. depending on the positive result of shinoda test, the extract of roots contains flavone type of flavonoids, the structure-activity relationship study of these compounds indicates that the hydroxyl groups, the 3,4-catechol structure in the b-ring, the 2-3 double bond, and 4-oxo function are key factors for the antioxidant activity of the flavonoids (22). the aqueous extract which can use as an infusion in traditional medicine (23) showed a good effect in scavenging free radicals (≈83 in 30 minutes). all the results of this study showed that e. polyceras roots can be a valuable source of polyphenols such as flavonoids, which have a crucial role as antioxidants and free radicals scavengers, this can predict the ability to use this species in the treatment of oxidative stress illnesses. this plant needs more studies about the safety, the toxicological effects and the determination of the therapeutic dose. conclusion the study was concluded that our study is the first report about echinops polyceras bois. roots, where primary identification tests of secondary metabolites were established, and the phenolic compounds and flavonoids contents were determined in different extracts, using micro methods in a 96-well microplate. also, the scavenging activity of free radicals was evaluated using the dpph• radical. our in vitro results were good enough to make this species a good source of effective antioxidants which can be used to prevent oxidative stress illnesses. the in vivo and the safety studies should be fulfilled. acknowledgments the authors would like to thank the staff of the department of pharmacognosy in the faculty of pharmacy damascus university and the staff of the leishmania center of epidemiological and biological studies, damascus university, especially, prof. chadi soukkarieh and dr. hassan alkhouri for providing research facilities. references 1. phaniendra a, jestadi db, periyasamy l. free radicals: properties, sources, targets, and their implication in various diseases. indian j clin biochem. 2015 jan;30 (1):11-26. 2. supasuteekul c, nonthitipong w, tadtong s, likhitwitayawuid k, tengamnuay p, sritularak b. antioxidant, dna damage protective, neuroprotective, and α-glucosidase inhibitory activities of a flavonoid glycoside from leaves of garcinia gracilis. rev. bras. farmacogn. 2016 jun;26(3):312-320. 3. shon my, kim th, sung nj. antioxidants and free radical scavenging activity of phellinus baumii (phellinus of hymenochaetaceae) extracts. food chem. 2003 sep;82(4):593-7. 4. ramos‐tovar e, muriel p. free radicals, antioxidants, nuclear factor‐e2‐related factor‐2 and liver damage. j appl toxicol. 2020 jan;40(1):151-68. 5. tsao r. chemistry and biochemistry of dietary polyphenols. nutrients. 2010 dec;2(12):123146. 6. kalinowska m, gryko k, wróblewska am, jabłońska-trypuć a, karpowicz d. phenolic content, chemical composition and anti-/prooxidant activity of gold milenium and papierowka apple peel extracts. sci rep. 2020 sep;10(1):1-5. 7. vicente o, boscaiu m. flavonoids: antioxidant compounds for plant defence... and for a healthy human diet. not bot horti agrobo. 2018 jan;46(1):14-21. 8. khadim ej, abdulrasool aa, awad zj. phytochemical investigation of alkaloids in the iraqi echinops heterophyllus (compositae). iraqi j pharm sci. 2014;23:26-34. iraqi j pharm sci, vol.30(2) 2021 antioxidant activity of echinops polyceras 268 9. feinbrun-dothan n. flora palaestina/3. ericaceae to compositae/by naomi feinbrundothan text text. israel academy of sciences and human.; 1978. 10. mouterde p. nouvelle flore du liban et de la syrie. 11. sabatier s, amiot mj, tacchini m, aubert s. identification of flavonoids in sunflower honey. j. food sci. 1992 may;57(3):773-4. 12. zohra sf, meriem b, samira s, muneer ma. phytochemical screening and identification of some compounds from mallow. j nat prod plant resour. 2012;2(4):512-6. 13. de s, dey yn, ghosh ak. phytochemical investigation and chromatographic evaluation of the different extracts of tuber of amorphaphallus paeoniifolius (araceae). int j pharm biol res. 2010;1(5):150-7. 14. bhatt s, dhyani s. preliminary phytochemical screening of ailanthus excelsa roxb. int j curr pharm res. 2012;4(1):87-9. 15. ainsworth ea, gillespie km. estimation of total phenolic content and other oxidation substrates in plant tissues using folin–ciocalteu reagent. nat protoc. 2007 apr;2(4):875-7. 16. davey mw, montagu mv, inze d, sanmartin m, kanellis a, smirnoff n, benzie ij, strain jj, favell d, fletcher j. plant l‐ascorbic acid: chemistry, function, metabolism, bioavailability and effects of processing. j sci food agric. 2000 may;80(7):825-60. 17. mammen d, daniel m. a critical evaluation on the reliability of two aluminum chloride chelation methods for quantification of flavonoids. food chem. 2012 dec;135(3):1365-8. 18. choi y, jeong hs, lee j. antioxidant activity of methanolic extracts from some grains consumed in korea. food chem. 2007 jan;103(1):130-8. 19. azwanida nn. a review on the extraction methods use in medicinal plants, principle, strength and limitation. med aromat plants. 2015; 4: 196. 20. sánchez-rangel jc, benavides j, heredia jb, cisneros-zevallos l, jacobo-velázquez da. the folin–ciocalteu assay revisited: improvement of its specificity for total phenolic content determination. anal. methods. 2013;5(21):5990-9. 21. rammohan a, bhaskar bv, camilo jr a, gunasekar d, gu w, zyryanov gv. in silico, in vitro antioxidant and density functional theory based structure activity relationship studies of plant polyphenolics as prominent natural antioxidants. arab j chem. 2020 feb;13(2):3690-701. 22. heim ke, tagliaferro ar, bobilya dj. flavonoid antioxidants: chemistry, metabolism and structure-activity relationships. j nutr biochem. 2002 oct;13(10):572-84. 23. alachkar a, jaddouh a, elsheikh ms, bilia ar, vincieri ff. traditional medicine in syria: folk medicine in aleppo governorate. nat. prod. commun. 2011 jan;6(1). baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 the resveratrol adverse effect in mice doi: https://doi.org/10.31351/vol31iss1pp167-175 167 estimation the safety of parenteral resveratrol in mice rehab am. jawad *,1, hayder b sahib** *ministry of health and environment, baghdad, iraq ** department of pharmacology and toxicology, college of pharmacy, al-nahrain university, baghdad, iraq abstract resveratrol is polyphenolic compound has many biochemical and biological effects on several organs. therefore, resveratrol can be used to treat many diseases. the aim was to evaluate resveratrol safety when used in a parenteral single bolus dose. this study was conducted on 60 mice (30 males and 30 females) both sexes weighing 25-35g were divided into 6 groups (5animals per group) for each sex. all mice groups given 1% dmso and five different doses of resveratrol (5, 2.5, 1.25, 0.625, 0.312) g/kg intra-peritoneally given to five groups respectively. the mice were continuously monitored during 14 days. the number of deaths, changes in general behavior, changes in physiological activity, and signs of toxicity were reported. on day 15 blood was collected using a jugular vein puncture to obtain blood samples for hematological and biochemical analysis. all mice were euthanized under anesthesia. the heart, lung, liver, kidney, and gonads were dissected and sent for histopathological study. the result showed that at dose 0.312gm/kg neither signs of toxicity nor death were detected. the ld50 dose was 1.18 g/kg for female and 1.07 g/kg for male mice. the body weight change, biochemical and hematological assay, revealed that at doses (1.25,0.625,0.312) g/kg for both sexes no significant changes had reported in comparison with the control group (p˃0.05). histopathological examination revealed that at doses 1.25 g/kg for both sexes no significant tissue changes had reported in comparison with the control group (p˃0.05). in conclusion resveratrol at lower doses showed non-observed adverse effect while at high doses, showed dose dependent toxicity when used as single bolus dose intraperitoneally keywords: acute toxicity, intraperitoneally, histopathology, resveratrol, biochemical assay ل بالحقن في الفئرانتقدير سالمة الريسفيراترو ** و حيدر بهاء صاحب 1*،رحاب عبد المطلب محمد جواد العراق بغداد، والبيئة، وزارة الصحة * العراق بغداد، النهرين، جامعة ، صيدلة الكلية والسموم، فرع االدوية ** الخالصة له العديد من التأثيرات البيوكيميائية والبيولوجية على العديد من األعضاء. لذلك ، يمكن استخدام الريسفيراترول هو مركب بوليفينوليك . ريسفيراترول لعالج العديد من األمراض. كان الهدف هو تقييم سالمة ريسفيراترول عند استخدامه في جرعة بلعة مفردة بالحقن داخل الصفاق مجموعات )خمسة فئران لكل مجموعة(. 6إناث(. تم تقسيم كل فئران من الذكور واإلناث إلى 30كور و ذ 30فأر ) 60أجريت هذه الدراسة على 0.625، 1.25، 2.5، 5%(و خمس جرعات مختلفة من ريسفيراترول ) 1أعطيت جميع مجموعات الفئران ماده) الدي ام اس اوبتركيز اقل من يوًما. تم اإلبالغ عن عدد 14جموعات على التوالي. تمت مراقبة الفئران بشكل مستمر خالل ( جم / كجم داخل الصفاق تعطى لخمس م0.312، ثقب الوريد الوفيات والتغيرات في السلوك العام والتغيرات في النشاط الفسيولوجي وعالمات السمية. في اليوم الخامس عشر ، تم جمع الدم باستخدام ل الدم والكيمياء الحيوية. تم قتل جميع الفئران تحت التخدير. تم تشريح القلب والرئة والكبد والكلى والغدد الوداجي للحصول على عينات الدم لتحلي جم/كجم لم يتم الكشف عن العالمات السميه والموت .كانت 0.312التناسلية وإرسالها لدراسة التشريح المرضي أظهرت النتائج أنه عند الجرعه جم/كجم. أظهر تغير وزن الجسم ، المقايسة 1.07جم/كجم بينما للذكور تساوي 1.18الحيوانات المختبريه لالناث هي من %50الجرعه التي تقتل جم/كجم لم تسجل أي تغيرات معنوية مقارنه بمجموعه التحكم)قيمه بي اكبر من 0.625,0.312, 1.25البيوكيميائية والدمية ، أنه عند الجرعات (. في 0.05جم/كجم لكال الجنسين لم تسجل تغيرات معنوية في األنسجة مقارنة بمجموعة التحكم)قيمه بي اكبر من 1.25(. عند الجرعه 0.05 عند الختام ، أظهر ريسفيراترول عند الجرعات المنخفضة تأثيًرا ضاًرا غير ملحوظ بينما عند الجرعات العالية ، أظهر سمية تعتمد على الجرعة داخل الصفاق. استخدامه كجرعة مفردة الكلمات المفتاحية : السمية الحادة ، داخل الصفاق ، التشريح المرضي ، ريسفيراترول، المقايسة البيوكيميائية. introduction resveratrol is a polyphenolic compound found in at least 70 plant species. its phytoalexin has activity against viruses, bacteria, and fungi. obtained by biotechnological synthesis from yeasts or by chemical methods. it is found in a discrete amount in several human foods such as grapes, pomegranate, mulberries, peanuts, apple, tomato, and dark chocolate(1,2,3). resveratrol has many biochemical and biological effects on several organs. for this reason, resveratrol can be used to 1corresponding author e-mail: rehab.ph@yahoo.com received: 18/7/2021 accepted:20 / 9/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp167-175 iraqi j pharm sci, vol.31(1) 2022 the resveratrol adverse effect in mice 168 treat many diseases. the curative effect of resveratrol is derived from its antimicrobial, antiinflammatory, anti-viral anti-cancer, anti-oxidant, anti-hyper-lipidemic, anti-hypertensive, antidiabetic. in addition to cardioprotective, neuroprotective, and androgen lowering effect on theca-interstitial cells of the ovary (1,4,5). also, it acts as phytoestrogen due to its similarity in structure to diethylstilbestrol. other uses are calories restriction (weight loss), and anti-aging (2,6,7). moreover, it has a therapeutic effect on the liver in iron overload (8). many clinical studies demonstrated the above activity of resveratrol (3). acute toxicity is the capability of any material to cause severe biological injury or death soon following a single dose exposure; the goal of this study is aimed for acute toxicity testing and lethal dose required to kill 50% of tested animals (ld50) was estimated. materials and methods materials resveratrol as a dry powder have been purchased from hangzhou hyper chem. limited/china. dimethyl sulfoxide (dmso) is a solvent obtained from chem-lab nv, belgium. 4% formaldehyde in phosphate buffer saline has been purchased from edutek/india. hematoxylin and eosin stain purchased from bdh/england. all other kits used in biochemical and hematological have been obtained from roche/germany and cusabio/usa. samples preparation resveratrol cas 501-36-0/99% freshly prepared as stock solution equivalents to 5.0, 2.5, 1.25, 0.625, 0.312 gm/kg by ( by dissolving each concentration in separated volumetric flask in dmso and then diluted gradually with distilled water to give the required strength solution with concentration of dmso 1% (9). experimental animals sixty swiss albino mice weighing between 25-35 g had been purchased from the center for drug control and research in baghdad/ iraq. all handling and procedure process to the animal conducted with direction in the guide for the use and care of experimental animals of the animal ethics committee al-nahrain university/ college of pharmacy “. animals were left over seven days in the animal care facility of al-nahrain university/ college of pharmacy in a light/ dark cycle with regular feeding with rodent chow and ad libitum. the environment of the place was well ventilated with fresh air and the temperature was set to standard levels (23 ± 2 °c). method the study of acute toxicity was performed following the organization of economic cooperation and development (oecd) guideline for chemical testing(10). thirty male and thirty female swiss albino mice weighing (25-35) g each were randomly distributed into control group and five treated groups, containing five animals per group. all animals were freely reach their water & food and were permitted to familiarize with the laboratory conditions for seven days before the test. all mice groups given 1% dmso and five different doses of resveratrol (5,2.5,1.25,0.625,0.312) g/kg respectively. the acute toxicity testing was performed according to previous studies (11,12). in which mice were continuously monitored for the first 4 h and then every hour for the next 24 h and at 6 hourly intervals for the next 48 h after administration of resveratrol. then the number of death, changes in general behavior and other physiological activity, signs of toxicity such as changes in weight, skin, hair, eyes, mucous membranes, secretions and excretions, autonomic activity, and other cns signs of toxicity such as ( drowsiness, loss of gait, convulsion, tremor)were reported the observation period is 14 days. all mice were weighed and data collected at day zero (before any treatment had been received), at day 7 from the first dose, and on day 14(13). then on day 15 blood was collected using a jugular vein puncture(14)(approximately (1 ml) for hematologic analysis put in ethylenediaminetetraacetic acid (edta) tubes and for clinical biochemistry assay, the blood for the hematological assay (hemoglobin (hgb) concentration and wbc count) was immediately analyzed using diagon d-cell60. the blood for the clinical biochemistry assay (ast, alt, alp, bilirubin, creatinine, and urea) was centrifuged for 10 min at 3000 rpm to isolate plasma and deposited at -20 °c until reviewing for clinical biochemistry using cobas/roch apparatus. (15,11,16). then all animals were euthanized by cervical dislocation under light chloroform anesthesia. on the 15th day after administration of the treatment, the heart, lungs, livers, kidneys, and sex organs were dissected and fixed in a 10% neutral buffer formalin and processed effectively to study histopathological changes. histopathologists using a zeiss imager m2 microscope fitted with an axiocamhrc camera (carl zeiss microscope) to observe histopathological changes statistical analysis all data were collected, tabulated and statistically analyzed using social sciences software statistical package (ssps) software version 20. the result was presented as means ±sd one-way analysis of variance (anova) followed by a t-test (2-tail) was used to compare between groups. the level of significance was set at the p values <0.05. iraqi j pharm sci, vol.31(1) 2022 the resveratrol adverse effect in mice 169 results and discussion table 1 and table 2 show signs of acute toxicity of resveratrol in observation period and the number of dead for female and male mice respectively. at dose 5 g/kg and 2.5 g/kg of resveratrol all female and male mice died after the sign of toxicity (loss of gait, muscular fasciculation, convulsion, diarrhea, lacrimation, salivation finally muscle weakness, paralysis, dyspnea, and death). at 1.250 g/kg dose the symptoms of toxicity were less intense and the number of mortalities was decreased. this may be due to resveratrol has an oh group that binds to acetylcholine esterase enzyme (ache) and suppresses its activity in a concentration-dependent manner, so excessive accumulation of acetylcholine at the neuromuscular junction and synapses causes symptoms of both muscarinic and nicotinic toxicity. besides at dose 1.250 gm/kg, there is a symptom of dehydration (piloerection and sunken) (17), which may be due to loss of fluid through diarrhea. while (pale footpad and ear) may refer to shock or anemia that reversible in some mice which indicates the ability of detoxification(12), that resveratrol is extensively metabolized by phase ii detoxification enzyme in the liver, its metabolism, and its metabolite are correlated with the presence of two genes ( sulfotransferase and udp-glucuronosyltransferase (18). at the dose of 0.625 g/kg no signs of toxicity only in the first hours' diarrhea may be due to stress or side effect of resveratrol. this finding is in agreement with another study that reported the side effect of resveratrol is diarrhea regardless the route of administration (2), one mouse died from each group female and male were detected during 14 days of the acute toxicity trial span. at dose 0.312g/kg neither signs of toxicity nor death detected during 14 days of the acute toxicity. this result in agreement with previous studies that reported resveratrol has a dose-dependent inhibitory effect on both acetylcholine esterase and butyrylcholinesterase activity (20,21). from these data concluded the dose of 0.312 g/kg consider the non-observed adverse effect level (noael) and this result was confirmed by a histopathological study that showed no morphological changes in the examination organs. table 1 .signs of acute toxicity of resveratrol in observation period and number of dead female mice. t/d: number of mice treated/number of total deaths. the duration of observation =14 days. (+), (++), (+++) means slightly, moderately, and intensively increased respectively. dose g /kg t/d observance period sign of toxicity no. of dead mice 5 5 /5 5 min-15min loss of gait, muscular fasciculation, convulsion, dyspnea, lacrimation, and death (+++). 3 15min-4h hypoactivity, diarrhea atypical locomotion (back limbs falling abdominal contract, dyspnea, death (++). 1 4 h-6h atypical locomotion, piloerection, dyspnea, and death (+). 1 2.5 5 / 5 5 min-15min loss of gait, muscular fasciculation, convulsion, dyspnea, lacrimation, and death (+++). 2 15min-4h hypoactivity, diarrhea atypical locomotion (back limbs falling, dyspnea, and death (++). 1 4 h-6h atypical locomotion, piloerection, dyspnea, and death (+). 1 6-24 h atypical locomotion, piloerection, dyspnea, and death (+). 1 1.25 5 /3 10-15 min loss of gait, muscular twitching and death (+). 0 15 min-4h atypical locomotion (back limbs falling) hypoactivity, hyperventilation, and death (++). 0 6-24 h hypoactivity, piloerection, atypical locomotion (back limbs falling) pale foot pads and ear finally death. 1 24-48 h hypoactivity. 1 48 h-14 d no sign of. toxicity 0 0.625 5 / 1 1 -6 h hypoactivity. 0 6-24 h diarrhea (steaky stool in the anus), hypoactivity 0 24h-48 h hypoactivity, sunken, piloerection, and death. 1 48 h-14 d no sign of. toxicity 0 0.312 5 / 0 1h-6 h hypoactivity 0 24 h-14 d no sign of toxicity 0 iraqi j pharm sci, vol.31(1) 2022 the resveratrol adverse effect in mice 170 table 2. signs of acute toxicity of resveratrol in observation period and number of dead male mice t/d: number of mice treated/number of total deaths. the duration of observation =14 days. (+), (++), (+++) means slightly, moderately, and intensively increased respectively. figure 1 shows the dose-response curve of resveratrol for female mice groups.and calculate the ld50 dose through the equation y=40.375 ln(x)+43.003 and it was 1.18 g/kg. figure 2 shows the dose-response curve of resveratrol for male mice groups shows the lethal dose that kills fifty percent of male mice (ld50) of resveratrol calculated through the equation y=40.377 ln(x)+47.003 and it was 1.07 g/kg. the data concluded from both figures shows that resveratrol has dose dependent toxicity. the more toxic substance has lower ld50. figure 1& 2 also showed the present of mortality was 100 percent in the first two doses while the percent of mortality was decreased in slight variation between male and female which may be referred to gender effect. figure 1 .dose-response curve of resveratrol for female mice groups. dose g /kg t/d observance period sign of toxicity no. of dead mice 5 5 /5 5 min-15min loss of gait, muscular fasciculation, convulsion, dyspnea, lacrimation, and death (+++). 3 15min-4h hypoactivity, diarrhea atypical locomotion (back limbs falling abdominal contract, dyspnea, death (++). 1 4 h-6h atypical locomotion, piloerection, dyspnea, and death (+). 1 2.5 5 / 5 5 min-15min loss of gait, muscular fasciculation, convulsion, dyspnea, lacrimation, and death (+++). 2 15min-4h hypoactivity, diarrhea atypical locomotion (back limbs falling, dyspnea, and death (++). 1 4 h-6h atypical locomotion, piloerection, dyspnea, and death (+). 1 6-24 h atypical locomotion, piloerection, dyspnea, and death (+). 1 1.25 5 /3 10-15 min loss of gait, muscular twitching and death (+). 1 15 min-4h atypical locomotion (back limbs falling) hypoactivity, hyperventilation, and death (++). 1 6-24 h hypoactivity, piloerection, atypical locomotion (back limbs falling) pale foot pads and ear finally death. 1 24-48 h hypoactivity. 0 48 h-14 d no sign of. toxicity 0 0.625 5 / 1 1 -6 h hypoactivity. 0 6-24 h diarrhea (steaky stool in the anus), hypoactivity 0 24h-48 h hypoactivity, sunken, piloerection, and death. 1 48 h-14 d no sign of. toxicity 0 0.312 5 / 0 1h-6 h hypoactivity 0 24 h-14 d no sign of toxicity 0 iraqi j pharm sci, vol.31(1) 2022 the resveratrol adverse effect in mice 171 figure 2. dose-response curve of resveratrol for male mice groups. bodyweight changes were tabulated at day zero (before any dose given), day seven, and day 14, statistically analyzed in mean ±sd and summarize in table 3 and three figures. figure 3 represents bodyweight changes between male and female mice at dose 1.250gm/kg and shows no significant changes in body weight compared to the control group (p > 0.05). figure 4 represents body weight changes between male and female mice at dose 0.625gm/kg and shows no significant changes in body weight compared to the control group (p > 0.05). figure 5 represents body weight changes between male and female mice at dose 0.312gm/kg and shows no significant changes in body weight compared to the control group (p > 0.05). the change in animal body weight has been used as a reliable predictor of the drug or chemical's side effects on the animal. (22),and the loss in body weight from the control would reflect the toxicity of the material(11,23) also the change in animal body weight may indicate drug change the metabolic events and growth rate of the tested treated mice groups(24).from this results concluded resveratrol has no toxic effect on the metabolic events and growth rate at these single doses because there are no significant changes in body weight of treated mice at (1.250 g/kg,0.625 gm/kg, and 0.312gm/kg). table 3. body weight changes weakly. group sex mean± sd weight at day 0 (g) weight at day 7(g) weight at day 14(g) control m 28.6 ± 2.70 31.66 ± 3.3 32.98 ± 3.3 f 29. 2± 1.9 31.96 ± 1.8 34.48 ± 2.1 group i m 30.38 ± 5.3 37 ± 0.1 39.55 ± 1.7 f 30.9±3.8 32.7±0.8 33.98±0.95 group ii m 27.32±4.1 27.72±2.3 30.5±1.50 f 28.6±5.5 30.5±3.3 31. 87±2.9 group iii m 33.14±4.8 32.1±6.0 34.12±5.4 f 28.6±4.8 29.3±4.9 31±4.7 m refer to male, f refer to female. group 1: 1.25 g/kg resveratrol, group ii: 0.625g/kg resveratrol, group iii: 0.312g/kg resveratrol figure 3 weight changes between male and female mice have received 1.25gm/kg resveratrol and their controls at day zero, seven, and on day fourteen. where mmd0, cmmd0, fmd0, cfmd0, mmd7, cmmd7, fmd7, cfmd7, mmd14, cmmd14, fmd14, and cfmd14 represent, male mice day zero, its control, female mice day zero, its control, male mice day seven, its control, female mice day seven, its control, male mice day fourteen, its control, female mice day fourteen and its control, respectively. iraqi j pharm sci, vol.31(1) 2022 the resveratrol adverse effect in mice 172 figure 4 weight changes between male and female mice have received 0.625 gm/kg resveratrol and their controls at day zero, seven and on day fourteen. where mmd0, cmmd0, fmd0, cfmd0, mmd7, cmmd7, fmd7, cfmd7, mmd14, cmmd14, fmd14, and cfmd14 represent, male mice day zero, its control, female mice day zero, its control, male mice day seven, its control, female mice day seven, its control, male mice day fourteen, its control, female mice day fourteen and its control, respectively. figure 5 weight changes between male and female mice have received 0.312 gm/kg resveratrol and their controls at day zero, seven, and on day fourteen. where mmd0, cmmd0, fmd0, cfmd0, mmd7, cmmd7, fmd7, cfmd7, mmd14, cmmd14, fmd14, and cfmd14 represent, male mice day zero, its control, female mice day zero, its control, male mice day seven, its control, female mice day seven, its control, male mice day fourteen, its control, female mice day fourteen and its control, respectively. according to the table 4 that shows there are no significant changes between male and female mice concerning hematological and biochemical changes compared to their control (p > 0.05 )) hematological and biochemical changes are of essential importance for the detection of pathophysiological changes in animals. moreover, deviations in hematological parameters are capable of signifying toxicity-induced hemolysis (11). also, hb level can signify renal failure (impairment erythropoietin synthesis) and may indicate toxicity that induces hemorrhage or hemolysis. enzymatic and non-enzymatic biochemical parameters (e.g., alanine transaminase (alt), alkaline phosphatase (alp), aspartate transaminase (ast), and bilirubin) which are often used to indicate liver damage. the enzyme alanine transaminase (alt) and aspartate transaminase (ast) are the mitochondrial enzyme mostly found in the liver, skeletal muscles, and kidneys. so, elevate ast level indicates either liver damage or cardiac infarction and also, may indicate muscle injury (25) bilirubin and albumin are a good marker for liver function while urea and creatinine parameters are used to indicate kidney damage. (26) so, the results of this study indicate the resveratrol is safe to the liver and kidney which are the main organs for xenobiotic detoxification. iraqi j pharm sci, vol.31(1) 2022 the resveratrol adverse effect in mice 173 table 4 the serum profile and hematological assay after 14 days for group i: 1.25 g/kg resveratrol, group ii: 0.625g/kg resveratrol, group iii: 0.312g/kg resveratrol for both sex of mice after administration as a single intra-peritoneal route. serum and blood profile control male control female group i male: 1.25 g/kg resveratrol male group male ii: 0.625g/kg resveratrol group iii male: 0.312g/kg resveratrol group i female: 1.25 g/kg resveratrol male group female ii: 0.625g/kg resveratrol group iii female: 0.312g/kg resveratrol ast u/l 264±8.20 199.4±2.1 267±2.82 266±9.89 264.6± 1.27 223.9± 1.55 202.05± 1.34 200± 2.54 alt u/l 44.65±6.1 46.5±9.89 47.2±9.47 45.8±9.75 45± 1.41 49.75± 4.59 48.25± 3.88 47.25± 7.00 alp u/l 64 ±2.54 47±4.24 67.7 ±0.28 66.1±0.98 65.15± 0.21 50.75± 2.47 48.5± 9.19 47.75± 3.18 bilirubi n t mg/dl 0.15±0.07 0.1±0.14 0.2±0.14 0.15±0.07 0.05± 0.07 0.2± 0.14 0.15± 0.07 0.1± 0.14 albumi n g/dl 2.9±0.42 2.85±0.35 3±0.28 2.85±0.49 2.75± 0.35 3.05± 0.07 2.95± 0.07 2.7± 0.42 creatin ine mg/l 0.3±0.14 0.35±0.07 0.45±0.35 0.4±0.28 0.35± 0.21 0.45± 0.35 0.4± 0.14 0.35± 0.21 urea mg/dl 31.4±2.26 32±2.12 32.4±0.56 31.55±3.0 31.5± 2.12 33.5± 0.28 32.85± 0.35 32.3± 0.98 wbc ×10 ⁹/l 5.9±0.14 4.95±0.77 6.95±0.49 6.1±0.98 6± 0.14 5.95± 0.49 5.35± 0.77 5.2± 0.14 hgb g/dl 14.95±0.6 14.85±0.2 14±1.27 14.3±0.56 14.7± 0.56 14.15± 1.34 14.55± 0.77 14.65± 0.63 figure 6 .shows selected images with total magnification 400 h&e stain figure 6 shows selected images with total magnification 400 h&e stain were (a) represent liver tissue for mice received resveratrol shows no hepatic tissue changes such as hepatocyte iraqi j pharm sci, vol.31(1) 2022 the resveratrol adverse effect in mice 174 degeneration, no fatty changes or necrosis.no interstitial inflammatory cell infiltration or fibrosis. when compare to control liver tissue (b). (c) represent renal tissue exposed to resveratrol shows neither interstitial inflammatory cell infiltrate nor fibrosis, and normal glomeruli when compared with control renal tissue (d). (e) represent heart tissue for mice exposed to resveratrol shows there was no inflammatory cell infiltrate in the interstitial or perivascular spaces, and no myocyte damage or necrosis compare to control heart tissue. (f). (g) represent the lung tissue of mice exposed to the resveratrol displaying there was no capillary obstruction, no alveolar epithelial cell necrosis, no interstitial or intra-alveolar edema or hemorrhage, no inflammatory cell penetration in the interstitial space, and no hyaline membranes lining the alveolar ducts compare to control lung tissue. (h) . (i) represent testis tissue from mice given resveratrol and found normal seminiferous tubule thickness in the slice, normal spermatogenesis, no tubular wasting, no leydig cell hyperplasia, and no thickening of the seminiferous tubules' basement membrane compare to appearance and structure of the control testis tissue (j). 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2014 cefixime nanocrystals 1 preparation and evaluation of cefixime nanocrystals ahmed a. hussein * and hasanain sh. mahmood 1,** * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmaceutics, college of pharmacy, university of karbala, karbala, iraq. abstract drug nanocrystals are nanoscopic crystals of the parent compound with dimensions less than 1 µm. a decrease in particle size will lead to an increase in effective surface area in the diffusion layer, which, in turn, increases the drug dissolution rate. drug nanocrystals are one of the most important strategies to enhance the oral bioavailability of hydrophobic drugs. cefixime is the first member of what is generally termed the third generation orally active cephalosporins. these third generation cephalosporins are distinct from the older β-lactam antibiotics in their intensive antibacterial activity against a wide range of gram-negative bacteria. the aim of this study is to prepare nanocrystals of cefixime as a capsules dosage form in order to increase its oral dissolution rate and bioavailability. the cefixime nanocrystals were prepared by solvent/antisolvent precipitation method. certain amount of drug was dissolved in water miscible solvent (methanol used in this study), then this solution was injected (at certain speed) into water containing stabilizer. upon injection, precipitation of cefixime nanocrystal will occur immediately; this precipitate is sonicated at 37 ˚c for 30 min. then lyophilized. powder of nanocrystal was obtained and filled into capsules. the physicochemical interaction between drug and addatives was studied using ftir, dsc, results show that the best formula of cefixime nanocrystals prepared by dissolving 20mg/ml of cefixime in methanol, then 5ml was injected at 60ml/hr rate to a 50ml pvp solution as stabilizer in concentration 0.05%, then lyophilized to obtain the cefixime nanocrystal powder. the resulted mean particle size was 9-11 nm and the dissolution rate was significantly higher than that of the raw cefixime powder (p>0.05). keywords: cefixime trihydrate, nanocrystals, anti-solvent precipitation, pvp, hpmc, poloxamer 188. تحضير وتقييم البلورات النانوية للسفكسيم أحمد عباس حسين * حسنين شاكز محمودو **،1 * . انؼشاق ،بغذاد ،صبيؼت بغذاد ،كهٍت انصٍذنت ،فشع انصٍذالٍَبث ** .انؼشاق ،كشبالء ،صبيؼت كشبالء ،كهٍت انصٍذنت ،فشع انصٍذالٍَبث الخالصة اٌ االَخفبض فً حضى .يبٌكشٔيٍخش 1يٍ انًشكب األصهً يغ أبؼبد ألم يٍ انحضىانبهٕساث انُبٌَٕت ًْ بهٕساث َبٌَٕت انبهٕساث انُبٌَٕت ًْ .انضسًٍبث ٌؤدي إنى صٌبدة فً انًسبحت انفؼبنت فً طبمت االَخشبس، ٔانخً بذٔسْب حضٌذ يٍ يؼذل اَحالل انذٔاء . ٔاحذة يٍ أْى االسخشاحٍضٍبث نضٌبدة انخٕافش انحٍٕي نألدٌٔت شحٍحت انزٔببٌ فً انًبء ػٍ طشٌك انفى ْزا انضٍم يٍ .ػضٕ فً يب ٌسًى ػًٕيب ببنضٍم انزبنذ نهسٍفبنٕسبٕسٌٍ انفؼبنت ػٍ طشٌك انفىانسٍفٍكسٍى ْٕ أٔل grameٌخخهف ػٍ االصٍبل انسببمت نهًضبداث انحٌٍٕت بضٌبدة فؼبنٍخّ انًضبدة نهبكخٍشٌب انسبنبت انضشاو ( انضٍم انزبنذ)انسٍفبنٕسبٕسٌٍ negative).) نبهٕساث انُبٌَٕت نذٔاء انسٍفٍكسٍى ػهى شكم كبسٕل يٍ أصم صٌبدة يؼذل انزٔببٌ ٔ انخٕافش حٓذف يٍ ْزِ انذساست انى ححضٍش ا . انحٍٕي ػٍ طشٌك انفى حٍذ حى ارابت كًٍت يؼٍُت يٍ . انًزٌب انًضبد / حضشث انبهٕساث انُبٌَٕت نهسٍفٍكسٍى ببسخخذاو طشٌمت انخشسٍب بٕاسطت انًزٌب .، رى حى حمٍ ْزا انًحهٕل فً انًٍبء انًحخٕي ػهى يزبج( اسخخذو انًٍزبَٕل فً ْزِ انذساست ) نًبء انذٔاء فً يحهٕل لببم نأليخضاس يغ ا ( sonicator)ػُذ انحمٍ، ٌحذد حشسب نهبهٕساث انُبٌَٕت نهسفكسٍى ػهى انفٕس ؛ ٌؤخز انشاسب ٌٕٔضغ فً صٓبص انًٕصبث انصٕحٍت . أخز انًسحٕق ٔػبًء ػهى شكم كبسٕل . صًٍذرى ٌضفف ببنج . دلٍمت 30يئٌٕت نًذة 37 بذسصت حشاسة يم يٍ انسٍفٍكسٍى فً انًٍزبَٕل، حى /يهغ20حظٓش انُخبئش أٌ أفضم صٍغت نهبهٕساث انُبٌَٕت نهسٍفٍكسٍى َخضج ػٍ طشٌك إرابت ٪ ، رى صفف ببنخضًٍذ نهحصٕل ػهى يسحٕق 0.05كًزبج بخشكٍض pvp يم يحهٕل ٌحخٕي ػهى 50يم يٍ ْزا انًحهٕل فً 5حمٍ َبَٕيخش، ٔ يؼذل االَحالل أػهى بكزٍش يٍ يسحٕق انسٍفٍكسٍى 11-9 حضى انضسًٍبث انُبحضت ْٕيخٕسظ . بهٕساث انسٍفٍكسٍى انُبٌَٕت . (p<0.05)انخبو . 188، بولوكساميز pvp ،hpmc، المذيب المضاد / بواسطة المذيبالتزسيب السيفيكسيم ، البلورات النانوية ، : الكلمات المفتاحية 1 corresponding author e-mail: hasanain@live.com received:11 /1/2014 accepted:4 /3/2014 iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 2 introduction over the last years, the number of poorly soluble drugs has steadily increased. estimates state that 40% of the drugs in the pipelines have solubility problems (1, 2, and 3) . progress in high throughput screening methods leads to an even greater amount of newly discovered drugs that have poor water solubility. other literature states that about 60% of all drugs coming directly from synthesis are nowadays poorly soluble (4) . poor solubility in water correlates with poor bioavailability. if there is no way to improve drug solubility it will not be able to be absorbed from the gastrointestinal tract into the bloodstream and reach the site of action. there are many ways to solubilize certain poorly soluble drugs. but these methods are limited to drugs with certain properties in regard to their chemistry (eg, solubility in certain organic media) or for example to their molecular size or conformation (eg, molecules to be incorporated into the cyclodextrin (cd) ring structure) (5) . apart from that, the usage of surfactants or cosolvents is also possible, but sometimes leads to increased side effects and other disadvantages (eg, organic solvent residues). the micronization of drug powders to sizes between 1 and 10 μm in order to increase the surface area, and thus the dissolution velocity, is not sufficient to overcome bioavailability problems of many very poorly soluble drugs of the biopharmaceutical specification class ii. a consequent step was to move from micronization to nanonization. (6) . the size reduction leads to an increased surface area and thus according to the noyes-whitney equation (7) to an increased dissolution velocity. therefore nanonizationis a suitable way to successfully enhance the bioavailability of drugs where the dissolution velocity is the rate limiting step. moreover, below a critical size of 1–2 μm, the saturation solubility is also a function of the particle size. it increases with decreasing particle size below 1000 nm. therefore, drug nanocrystals possess increased saturation solubility. cefixime is the first member of what is generally termed the third generation orally active cephalosporins. these third generation cephalosporins are distinct from the older βlactam antibiotics in their intensive antibacterial activity against a wide range of gram-negative bacteria. (8) cefixime is freely soluble in methanol but has low aqueous which affects its rate of absorption, resulting in a low and variable oral bioavailability of about solubility and poor dissolution in gastric fluid 40-50 % (9) . it has been classified as biopharmaceutical classification system class ii drug (10) .therefore we aimed to prepare cefixime nanocrystals as a capsule dosage form in order to increase its oral dissolution rate and bioavailability. material and method materials cefixime was supplied by "samaraa drug industry, iraq".poloxamer 188 was purchused from “himedia laboratories, india”, polyvinylpyrrolidone (pvp) from “riedel de haen ag seelze, honnover, germany”, hpmc from “himedia laboratories, india”, methanol from “gcc analytical reagent, uk”, magensium stearate from “barlocher,gmbh, germany”, hydrochloric acid and potassium dihydrogen phosphate from “bdh chemical ltd, england” and sodium hydroxide from “carlo erba reagents,(spain)”. method preparation of cefixime nanocrystals cefixime nanocrystals were prepared by the precipitation technique which is also called antisolvent precepitation method . cefixime was dissolved in a methanol (5 ml) at room temperature, this was poured into 50 ml of water containing different types of surfactants and subsequently stirred at agitation speed of 250 round per minute (rpm) on magnetic stirrer for 1 hour to allow the volatile solvent to evaporate. addition of organic solvents at certain rate by means of a syringe positioned with the needle directly into surfactant containing water. variable drug to surfactant ratio was used. (‎11) twenty four formulas (f1-f24) were prepared by this technique demonstrated in table (1) with their composition. characterization of nanoparticles particle size analysis particle size determination of the prepared formulas (f1-f24) was done by using abt9000 nano laser particle size analyzer at scattering angle 90º. the average particle size reflects the size of those particles which constitute the bulk of the sample volume was measured after performing the experiment in triplicates . the polydispersity index (pdi) of each formula was also determined as a measurment for the width of the size distribution , it is a parameter to define the particle size distribution of nanoparticles obtained from a particle analyzer. pdi is an index of width or spread or variation within the particle size distribution. the analyzer also determines the specific surface area for each sample. iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 3 table 1: components of various formulas.  control freeze drying of the prepared nanocrystals after the evaluation of the prepared formulas (f1-f24), the formula with lowest particle size was selected and lyophilized using vacuum freeze dryer at a controlled temperature of −44 ◦ c and the pump operating at a pressure of 2.5 × 10 pascal over a period of 48–72 hour. the yielded powder was used for further studies and also it is used to prepare the capsules. determination of present yield and entrapment efficiency: nanocrystals after being dried were weighed and the yield was calculated as a percentage of the total weights of starting material (polymer and drug) introduced into the system (this represents the theoretical weight of nanocrystals), and the actual weight of nanocrystals obtained. the percent yield was calculated using equation 1 (12) : %yield = actual weight of nanocrystals gained theoretical weight of nanocrystals × 100 eq.(1) the entrapment efficiency of nanocrystals was determined from the theoretical and actual drug contents. the percent entrapment efficiency was calculated using equation 2 (12) . % entrapment efficiency (ee) = actual drug content theoretical drug × 100 eq.(2) preformulation studies of the prepared nanocrystals powder the flowability of a powder is of critical importance in the production of pharmaceutical dosage forms in order to get a uniform feed as well as reproducible filling of capsules, otherwise high dose variations will occur. the powder flowability of prepared cefixime nanocrystals were characterized by angle of repose (13) . formula cefixime conc. mg/ml volume injected ml poloxamer 188 conc% pvp conc % hpmc conc % solution vol ml injection speed ml/hr c * 20 5 50 60 1 20 5 0.025 50 60 2 20 5 0.05 50 60 3 20 5 0.1 50 60 4 20 5 0.2 50 60 5 20 5 0.025 50 60 6 20 5 0.05 50 60 7 20 5 0.1 50 60 8 20 5 0.2 50 60 9 20 5 0.025 50 60 10 20 5 0.05 50 60 11 20 5 0.1 50 60 12 20 5 0.2 50 60 13 20 2.5 0.025 50 60 14 20 2.5 0.025 50 60 15 20 2.5 0.025 50 60 16 20 10 0.1 50 60 17 20 10 0.1 50 60 18 20 10 0.1 50 60 19 10 5 0.025 50 60 20 10 5 0.025 50 60 21 10 5 0.025 50 60 22 5 5 0.0125 50 23 5 5 0.0125 50 60 24 5 5 0.0125 50 60 iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 4 in vitro dissolution profile of cefixime nanocrystals capsules dissolution studies of capsules containing cefixime nanocrystal were performed in dissolution apparatus using the paddle method according to usp 30. capsules containing cefixime nanocrystal (equivalent to 200 mg cefixime) were dispersed on 900 ml of dissolution media (0.1 n hcl ph of 1.2 and phosphate buffer ph 7.2) at 37˚c ± 0.5 and rotated at 100 rpm. five milliliter samples were withdrawn for analysis periodically and replaced by the same volume of fresh media maintained at 37 ○ c ± 0.5. the withdrawn samples were filtered. the amount of cefixime released in the medium was diluted and determined spectrophotometrically at λmax of 285 nm. the concentration of cefixime released at different time intervals was determined using the equation obtained from the calibration curve. each in vitro release study was performed in triplicate. after each time interval , the average value of absorbance was computed and finally the percent of mean drug release was plotted against time to obtain dissolution profiles (11, 14) . fourier transform infrared spectroscopy (ft-ir) the fourier transform infrared spectroscopy (ft-ir) spectrum were studied to detect any sign of interaction or complexation may occur between cefixime and stabilizers used in the preparation of the nanocrystals and with the excipients used in the preparation of capsules. the spectrum was obtained using ft-ir shimadzu 8300 japan . samples which studied were pure drug , pvp and lyophilized powder of selected formula (f6). all these samples were grounded and mixed thoroughly with potassium bromide, at 1:5 (sample : potassium bromide) weight ratio. the spectrum obtained was in between the wave number of 4000-400 cm -1(15, 16) . differential scanning calorimetry (dsc) study dsc can be used to determine the compatibility between the drug and excipients and also used to evaluate the crystalline state of drug especially when converted to nanocrystals. thermal characteristics of the same materials that examined in ftir study were determined by an automatic thermal analyzer system (shimadzu, dsc– 60, japan). accurately weighed samples (5mg) were placed in nonhermetically aluminium pans and heated at the rate of 20 ºc/minute against an empty aluminium pan as a reference covering a temperature range of 50 ºc to 300 ºc (17, 18) . powder x-ray diffraction analysis (pxrd) x-ray diffraction is used to study the atomic and molecular structure of crystalline substances such as drugs and excipients. xrays diffraction patterns (diffractograms) can be used to confirm the crystalline nature of a sample. therefore, this information is used to verify whether the substances are crystalline or amorphous .pxrd diffractograms of the pure drug and lyophilized powder of f6 were recorded using shimadzu diffractometer 6000 (shimadzu , japan) with input voltage at 220v/50hz . surface morphology studies scanning electron microscopy (sem) the morphology of raw drug and cefixime nanocrystals were examined by scanning electron microscope (vega3 tescan czech republic) operated with a secondary detector at an acceleration voltage of 10 kv and at 100x magnification for raw drug and 15 kv and 10 kx for f13 . the morphology of raw drug was done by direct deposition of powder on double-sided carbon tape and coated with gold. while for liquid nanocrystals sample was prepared by the droplet evaporation technique. a droplet of liquid was deposited on doublesided carbon tape and dried at room temperature for the evaporation of water and then coated with gold (19) . atomic force microscopy (afm) afm is capable of scanning the surfaces in controlled environmental conditions and is complementary to sem imaging and also can measure the particle size of the nanoparticles accurately . droplets of selected formulas were deposited on freshly cleaved mica and dried 15 minutes in oven. particle size,histogram of particle size distribution and 3d surface morphology of each formulawere obtained (19) . results and discussion particle size results of the prepared cefixime nanocrystal most of the prepared cefixime nanocrystal formulas showed a particle size result within nano range except formula 16 wide variations from 9nm to 651nm. only one formula occur out of nanorange (f16) which gave a particle size range of 3539 4456nm. as shown in table (2). it has been shown that formula 6 score the lowest average particle size, so this formula selected for further evaluation. also it has been seen that pvp provided the lowest particle size when used as stabilizer. this could be due to pvp high affinity for both hydrophilic and hydrophobic surfaces (20) . and this means that pvp has a higher affinity to adsorb cefixime than poloxamer 188 and hpmc. iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 5 table (2): particle size, specific surface area and polydispersity index results of various formulas. formula average particle size (nm) specific surface area (ssa) (m 2 /g) polydispersity index (pdi) c 250-315 7.64 0.263 1 397-629 4.94 0.018 2 28-31 71.45 0.337 3 140-223 13.44 0.018 4 140-199 13.91 0.012 5 199-281 9.13 0.009 6 9-11 201.18 0.585 7 88-125 21.28 0.015 8 88-125 22.09 0.011 9 70-88 26.20 0.429 10 39-44 50.38 0.439 11 281-500 6.43 0.021 12 315-500 5.86 0.028 13 397-500 4.98 0.013 14 199-281 9.68 0.012 15 281-500 6.80 0.031 16 3539-4456 0.52 0.000 17 177-250 10.58 0.013 18 445-561 4.47 0.009 19 353-500 5.36 0.011 20 39-50 48.48 0.292 21 111-140 17.88 0.120 22 353-561 29.65 0.173 23 62-79 29.65 0.173 24 315-445 6.15 0.011 percent yield and entrapment efficiency of cefixime nanocrystals: the percentage yield of cefixime nanocrystals of the selected formula (f6) was 87.6% and entrapment efficiency was 88%. flowability study of the prepared nanocrystal powder (angle of repose) angle of repose was measured for the selected nanocrystal formula (f6), to observe the flow properties of powder. results show that the formula had poor flowability (angle of repose 50.19˚). so magnesium stearate (mg.st.) 0.5% was added to improve the flow properties of the formula to insure efficient filling into capsules during the manufacturing process (21) . in vitro release study in vitro drug release studies of the capsules containing selected formula, raw drug and marketed cefixime capsules (cefix ® ) were carried out in 0.1n hcl containing 0.1% methanol and phosphate buffer ph (7.2) to simulate in vivo release in both stomach and intestine. methanol was added to the 0.1n hcl solution to increase cefixime solubility and hence reach to sink condition. results show significant increase (p<0.05) in dissolution rate of all the selected formula than the raw drug and the marketed one (figure 1). these results expected according to noyes– whitney equation where the solid dissolution rate is directly proportional to its surface area exposed to the dissolution medium. also these results agreed to that of mansouri et al. where they prepared ibuprofen nanoparticles by using solvent/antisolvent precipitation technique and found that the prepared ibuprofen nanoparticles had a dissolution rate 2.33 times more than that of raw drug (22) . iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 6 figure 1: the dissolution profile of the selected formula (f6) at 37ºc ± 0.5 ºc and 50 rpm. a: in 0.1n hcl +0.1% methanol media.b: in phosphate buffer ph(7.2) fourier transform infrared spectroscopy (ftir) the infrared spectra of raw cefixime, pvp, magnesium stearate and cefixime nanocrystals selected formulas (f6) were studied by ftir spectroscopy using kbr disc (fig. 2-5). the ft-ir spectra of pure cefixime (figure 2) powder display a characteristic – nh2 absorption peak at 3296 cm -1 , which is a normal range of absorption of primary amines. it exhibits a strong band for c=o stretching of the nonconjugated carboxylic acid at 1770 cm -1 whereas the second band which is expected to shift to lower frequency (owing to conjugation) appears as an overlapping band. the carbonyl of cyclic as well as acyclic amide appears at 1670 cm -1 . these peaks are agreed with the reported one which indicates the purity of the drug (23) . the results indicate that there was no chemical incompatibility between drug and polymer as all the characteristic ir peaks related to pure drug were also appear in the ir spectrum of the formulas. figure 2: ft-ir spectrum of pure cefixime . iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 7 figure 3: ftir spectra of pvp. figure 4: ftir spectra of cefixime nanocrystals using pvp as stabilizer + mg.st. (f6). figure 5: ftir spectrum of mg. st. iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 8 differential scanning calorimetry (dsc) dsc thermogram (figuer 6) of cefixime trihydrate shows broad endothermic peak at 217 0 c, which is its melting point as cefixime melt with decomposition over the range 218225ºc. before melting point, dsc shows board endothermic peak at 125 0 c which represent the removal of hydrates from cefixime trihydrate (24) . the dsc thermogram of the selected formulas (f6) (figure 9) shows absence of the melting point peak, this result can be attributed to the change in the crystilanity state of the prepared formulas to the amorphous state (which also confirmed by xrd) and not to chemical interaction as ftir study showed that there was no chemical interaction between the drug and the excipients. more over the board endothermic peak suggests that these formulas are product of physical mixture of the drug and the polymer used to prepare them, if it is a reaction product which might have formed during the formulation, it has given rise to short range of melting process with 2 to 3 0 c, which has not happened in this case, it confirms the drug used in the formulation is in the free state rather than in the chemically reacted form. drug is freely available to the system whenever administered (25) . figure 6: dsc curve of cefixime trihydrate. 50.00 100.00 150.00 200.00 250.00 temp [c] -10.00 -8.00 -6.00 -4.00 -2.00 0.00 mw dsc 3.78x100mi n 120.14x100c figure 7: dsc curve of pvp. 100.00 200.00 300.00 temp [c] -30.00 -20.00 -10.00 0.00 mw dsc 4.04x100mi n 123.32x100c figure 8: dsc curve of mg.st. 100.00 200.00 300.00 temp [c] -0.00 10.00 mw dsc 88.50x100c 222.53x100c figure 9: dsc curve of cefixime nanocrystals formula using pvp as stabilizer + mg.st. (f6) . 50.00 100.00 150.00 200.00 250.00 temp [c] -10.00 0.00 10.00 mw dsc 8.67x100min 217.00x100c figure 10: dsc curve cefixime and pvp (1:1) physical mixture. powder x-ray diffraction (xrd) the results obtained from dsc reasonably agreed with the results obtained by pxrd. the change in the crystalline state of cefixime nanocrystals was further confirmed by x-ray diffraction. the x-ray patterns of the pure cefixime(figures 11)displayed the presence of numerous narrow and symmetrical characteristic diffractionpeaks with high intensity this indicated the crystalline structure of the drug , while xrd of prepared formula (figure 12) showed no sharp peak and less intensity of the diffraction peak when compared to that of raw drug indicating that the crystalline structure of cefixime was lost and converted into amourphous form. this results agreed with previous researches where junghanns et al. stated that depending on the production technology, processing of drug 50.00 100.00 150.00 200.00 250.00 temp [c] -20.00 -10.00 0.00 mw dsc 4.07x100min 125.12x100c 8.67x100min 217.43x100c iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 9 microcrystals to drug nanoparticles can lead to an either crystalline or to an amorphous product, especially when applying precipitation. in the strictest sense, such an amorphous drug nanoparticle should not be called nanocrystal. however, often one refers to “nanocrystals in the amorphous state” (6) . figure 11: xrd diffractogram of cefixime trihydrate. figure 12: xrd diffractogram of formula 6. surface morphology studies scanning electron microscope (sem) the shape of nanocrystals obtained from the selected formula (f6) visualized by scanning electron microscope. formula 6 magnified at 5 kx, 10 kx and 50 kx. (figure 13). it has been noticed from sem images that the formula gave uniform submicron sized particles and this results will confirmed by afm. figure 13: sem images of (f6). iraqi j pharm sci, vol.23(2) 2014 cefixime nanocrystals 10 atomic force microscope (afm) the atomic force microscope (afm) is one kind of scanning probe microscopes (spm) which are instruments that measure the properties of surfaces. afm is capable of scanning the surfaces in controlled environmental conditions and is complementary to sem imaging. with the high precision of the afm, in principle it is possible to determine the dimensions of nanoparticles with high accuracy. afm allows the visualization of samples with resolution in three dimensions x-, yand z-directions in atmospheric or submerged conditions (26, 27) . the morphological analysis and particle size of formula (f6) performed by afm (figure 14). results show spherical shaped nanoparticles with a size of 50-150 nm as it approved by the histogram of particle size distribution (figure 15). the formulation was found to be stable and no aggregation of particles could be observed. the particle size obtained by afm was larger than that measured by abt-9000 nano laser particle size analyzer, this variation because particle size analyzer is only capable of giving information about the volumetric mean diameter of a great number of particles, but results on the real size distribution are rather difficult to obtain. for a precise determination of single particle dimensions, size and distribution microscopic techniques are required (26) . figure 14: afm image (f6). figure 15: afm average size histogram of (f6). references 1. lipinski ca. poor aqueous solubility an industry wide problem in drug discovery. am pharm rev. 2002; 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46 (1) : 803-14. iraqi j pharm sci, vol.24(1) 2015 novel nitrogenous heterocyclic compounds with biological activity 49 synthesis of some novel nitrogenous heterocyclic compounds with expected biological activity as antimicrobial and cytotoxic agents. mohammed s. farhan *,1 and kawkab y. saour * * department of pharmaceutical chemistry, college of pharmacy, university of baghdad,baghdad,iraq. abstract this study includes synthesis of some nitrogenous heterocyclic compounds linked to amino acid esters or heterocyclic amines that may have a potential activity as antimicrobial and/or cytotoxic. quinolines are an important group of organic compounds that possess useful biological activity as antibacterial, antifungal and antitumor .8-hydroxyquinoline (8-hq) and numerous of its derivatives exhibit potent activities against fungi and bacteria which make them good candidates for the treatment of many parasitic and microbial infection diseases. these pharmacological properties of quinolones aroused our interest in synthesizing several new compounds featuring heterocyclic rings of the quinoline derivatives linked to amino acid ester or heterocyclic amine with the aim of obtaining a pharmacologically active compounds.o-alkylation has been done on( 8-hydroxyquinoline ) to get (o-alkylated ester) derivatives which are deesterfied to get acetic acid derivatives, then coupled with amino acid that have protected carboxyl group (amino acid esters) or heterocyclic amine by using conventional solution method for peptide synthesis as a coupling method. the proposed analogues were successfully synthesized and the processing of the reactions confirmed by tlc ,the synthesized analogues with the proposed structures as they were characterized and proved by melting point, infrared spectroscopy (ir) and elemental microanalysis. the tested analogues showed cytotoxic activity on the hep-2 cell line (tumor of larynx) with inhibitory concentration percent of (ic %) range (32.43 % 49.55%) and showed that the tested compounds had variable antimicrobial activities against selected bacteria and yeast when compared with selected standard drugs. keywords: quinolones, 8-hydroxyquinoline , n-heterocycles biological activity. مع تىقع الحاويح على الىتروجيهومتجاوسح ر غيال الجذيذج تخليق تعض المركثاخ الحلقيح يحوسرطاو ميكروتيحكمضاداخ الفعاليح الحيىيح لها محمذ سىجار فرهان *،1 كىكة يعقىب ساعىر و * * . انعشاق ،بغذاد،خبيعت بغذاد،كهيت انصيذنت ،فشع انكيًيبء انصيذالَيت الخالصة غيش يشكببث حهميتأ حخعًٍ ْزِ انذساست حخهيك بعط انًشكببث انحهميت غيش انًخدبَست انًمخشَت بأسخشاث األحًبض االييُيت أٔ يعبداث نهسشغبٌ /انخي لذ حًخهك فعبنيت َشيطت كًعبداث نهدشاثيى ٔٔ يخدبَست حبٔيت عهى يدًٕعت اييُيت .ٔسشغبَيت ًعبداث بكخيشيت ٔفطشيتكحًخهك انكٕيُٕنيُبث اًْيت يًيزة كأحذ انًشكببث انععٕيت ٔانًسخحعشاث انطبيت ٔانخي حسخخذو يششحت خيذة نعالج انعذيذ يٍ ب ٔانعذيذ يٍ يشخمبحّ أَشطت فعبنت ظذ انفطشيبث ٔ انبكخيشيب انخي خعهٓ) ْيذسٔكسيكٕيُٕنيٍ-8)اظٓش ال ْٕ األكثش إثبسة نالْخًبو ، ٔرنك (ْيذسٔكسيكٕيُٕنيٍ-8)يٍ خًيع يشخمبث انٓيذسٔكسيكٕيُٕنيٍ .األيشاض انطفيهيت ٔ اندشثٕييت نذٔائيت أثبسث اْخًبيُب بخحعيش عذة يشكببث خذيذة حخًيز بحهمبث انغيش يخدبَست بسبب خصبئصّ انًخعذدة انٕظبئف ، ْزِ انخصبئص ا بٓذف انحصٕل غيش يخدبَست حبٔيت عهى يدًٕعت اييُيت يشكببث حهميتأ نًشخمبث انكٕيُٕنيٍ يشحبطت بإسخشاث األحًبض األييُيت .عهى انًشكببث انصيذالَيت انفعبنت ٔ يٍ ثى ححهم االسخش (o-alkylated ester)نهحصٕل عهى يشخمبث )ْيذسٔكسيكٕيُٕنيٍ -8(عهى o-alkylationحى إخشاء غيش يخدبَست حبٔيت يشكببث حهميتأ نهحصٕل عهى يشخمبث حبيط أنخهيك ٔيٍ ثى سبطٓب يع يخخهف اسخشاث األحًبض االييُيت ث كطشيمت ناللخشاٌ ٔحكٕيٍ أصشة اييذيت نهحصٕل عهى ببسخخذاو غشيمت انًحهٕل انخمهيذيت نصُبعت انببخيذا عهى يدًٕعت اييُيت ٔلذ حًج يشالبت سيش انخفبعالث .اٌ عًهيت حخهيك انًشكببث انًطهٕبت لذ حًج ببحببع غشيمت انخفبعم يخعذد انخطٕاث.انًشكببث انُٓبئيت نُٓبئيت ٔانخبكذ يُٓب يٍ خالل ليبس حى حشخيص انخشكيب انكيًيبئي نهًشكببث انٕسطيت ٔا .ببسخخذاو كشٔيبحٕغشافيب انطبمت انشليمت .دسخبث االَصٓبس ٔانخحهيم انطيفي نألشعت ححج انحًشاء ٔانخحهيم انذليك نهعُبصش بُسبت حشكيز يئٕيت (سو انحُدشةت )ٔأظٓشث انُخبئح انخي اخشيج عهى انًشكببث انًصُعت َشبغ سًي عهى َٕع يٍ انخاليب انسشغبَي بث ييكشٔبيت أحًخهك فعبني الحًخهكانًصُعت ٔكزنك اظٓشث انُخبئح اٌ انًشكببث % ( 5443 %6654) يثبطت حخشأذ يب بيٍ ال ب ٔانفطشيبث انًخخبسة ببنًمبسَت يع االدٔيت انميبسيت انًخخبسة ايعب في ْزِ انذساستبكخشيانظذ يخخهفت .الحلقيح الغير متجاوسح والحاويح على الىتروجيهالفعاليح الحيىيح للمركثاخ ، هيذروكسيكىيىىليه -8 ،كىيىىلىنالكلماخ المفتاحيح : 1 corresponding author e-mail: mohmadsinjar@gmail.com received:10 /1/2015 accepted: 28/4/2015 mailto:mohmadsinjar@gmail.com iraqi j pharm sci, vol.24(1) 2015 novel nitrogenous heterocyclic compounds with biological activity 50 introduction heterocyclic compounds are acquiring more importance in recent years which display biological activities. (1) heterocyclic compounds particularly five and six member heterocyclic have attracted the attention of pharmaceutical community over the years due to their therapeutic values. (2) poly functionalized heterocyclic compounds containing nitrogen, sulphur, oxygen as heteroatom play important roles in the drug discovery process. (3) analysis of drugs in late development stages or in the market shows that 68% of them are heterocycles. (4) n-heterocycles have found wide-spread applications as key components of a large number of biologically-active natural products. examples include antibiotics such as penicillin and cephalosporin, alkaloids such as vinblastine and morphine and fungal natural product like cyclosporine a , all exhibiting interesting biological properties. however, synthetic n-heterocyclics have also found widespread use in pharmaceuticals as anticancer agents, analeptics, analgesics, hypnotics and antioxidants (5) .quinoline is one of the most popular n-heteroaromatic compounds incorporated into the structures of many pharmaceuticals. many quinolinecontaining compounds exhibit a wide spectrum of pharmacological activities, such as antiplasmodial (6), , cytotoxic (7) , antibacterial (8) , antiproliferative (9), ,antimalarial (10) , and anticancer activity (11) . these pharmacological properties of quinolines and their derivatives had attracted worldwide attention in the last few decades because of their wide occurrence in natural products and drugs (12,13) . quinoline derivatives also have been shown to exhibit awide variety of pharmacological activities including effects on cancer and nowadays it is reported that the incorporation of quinolone nucleus could alter the course of reaction as well as the biological properties of the synthesized compounds (14, 15). . materials and methods materials 8-hydroxyquinoline was purchased from avonchem (u.k). absolute ethanol, absolute methanol, acetone, and chloroform were purchased from gcc (germany). absolute isopropyl alcohol, ethylbromoacetate , diethyl ether, ethyl acetate, hydrazine hydrate, hydrochlorica cid, n-methyl morpholine (nmm), petroleum spirit and thionyl chloride were purchased from bdh (u.k).coumarin was purchased from himedia (india). d-alanine, 1-hydroxy benzotriazole (hobt), and n,ndimethyl formamide (dmf), were purchased from fluka ag(switzerland). all other reagents were of analytical grade. methods of identification general methods were used for purification and identification of the synthesized analogues including: • thin layer chromatography: • melting points: • infrared spectra: • elemental microanalysis synthesis esterification of amino acids synthesis of dalanine methyl ester hcl compound (a.1) (16) a suspension of d-alanine (5 mmol, 0.445g) in (100ml) of absolute methanol, was cooled down to –15 ºc then thionyl chloride (5 mmol, 0.37 ml) was added drop by drop(the temperature should be kept below –10 0 c) , the reaction mixture was left at 40 0 c for 3hr, then reflux started for other 3hr and left at room temperature overnight, the solvent was evaporated to dryness under vacuum, redissolved in methanol and evaporated, this process was repeated several times and recrystallize from methanol–ethyl acetate (3:1) synthesis of 1-aminoquinolin-2(1h)-one (b.1) (17) a solution of (2.96g, 20 mmol) coumarin and excess hydrazine hydrate (99%), in 25 ml of absolute ethanol was refluxed for 24 h, left at room temperature(r.t.) for one week , then the solvent was evaporated to dryness and the solid product was washed with cold ethanole, and recrystallized from chloroform to give yellow crystals. tlc was done indicate the disappearance or lightening of coumarin spot and new spot was resulted. synthesis of ethyl-quinoline-8-yl oxyacetate. compound 1 (18) a mixture of 8hydroxyquinoline(12.5 mmol), ethyl bromoacetate (12.5 mmol) and k2co3 (17.9 mmol) in 50ml dry acetone was refluxed for about 20 hr at 70 0 c. after cooling, the mixture was evaporated to dryness and the residue was partitioned between chloroform (50 ml) and water (50 ml). the organic phase was dried with anhydrous sodium sulphate (na2so4), filtered and evaporated to dryness. the residue was recrystallized from ethanol. synthesis of 2(quinoline-8-yloxy) acetic acid (qaa) compound 2 (19) a solution of compound 1 (1.5g, 7.38mmol) in methanol (25 ml) and 5% naoh (6 ml) was heated under reflux for 4 h. after cooling, the solution was evaporated to dryness and the residue was dissolved in 10 ml water and acidified with 1n hcl( 6 ml). the iraqi j pharm sci, vol.24(1) 2015 novel nitrogenous heterocyclic compounds with biological activity 51 white precipitate was filtered, dried and crystallized from methanol. coupling method and reagents conventional solution method for peptide synthesis was used as a coupling method between the carboxy protected amino acids or heterocyclic amine and carboxy derivatives of quinolone. dicyclohexylcarbodiimide (dcc) was used in the peptide bond formation as the coupling reagent, while 1-hydroxybenzotriazole(hobt) was used to decrease racemization and to increase the yields (20) . synthesis of methyl 2-(2-(quinolin-8yloxy)acetamido)propanoate ( comp.3 ) to a stirred solution of d-ala. methyl ester hcl (comp. a.1) (2mmol, 0.28g) in (25ml) of dimethylformamide (dmf), (2mmol, 0.22ml) of n-methylmorpholine (nmm) was added with stirring for 10 min., then (2mmol,0.406 g) of (comp. 2) was also added, and the mixture was cooled down to (-10 0 c) then (4.0mmol, 0.540g) of hobt and (2.0mmol,0.412 g ) of dcc were added with stirring, which was continued for 2 days at 0 0 c and then at room temperature for 5days. the reaction mixture evaporated to exclude dmf and redissolved in chloroform from which the n,n–dicyclohexyl urea (dcu) was filtered off. the clear filterate washed three times with 5% sodium bicarbonate solution, 0.1n hcl, once with distilled water, and with saturated sodium chloride solution. the chloroform layer was dried with anhydrous magnesium sulphate and evaporated under vacuum; the resulted product was collected, recrystallized from (methanol: chloroform) (5:1). synthesis of n-(2-oxoquinolin-1(2h)-yl)-2(quinolin-8-yloxy)acetamide ( comp. 4) to a stirred solution of compound 2 (0.406g, 2.0 mmol) in (20ml) of dmf, (0.32 g, 2.0 mmol ) of compound b1 was added, the mixture was cooled down to (-10 0 c) then (0.56g, 0.4 mmol) of hobt and (0.412g ,2 mmol) of dcc were added with stirring, which was continued for 2days at 0 0 c and then at room temperature for 5 days. the reaction mixture evaporated to exclude dmf and re dissolved in chloroform from which the n,n–dicyclohexyl urea (dcu) was filtered off. the clear filterate washed three times with 5% sodium bicarbonate solution, 0.1n hcl, once with distilled water, and with saturated sodium chloride solution. the chloroform layer was dried with anhydrous magnesium sulphate and evaporated under vacuum; the resulted product was collected, recrystallized from (methanol: acetone) (2.5:1) . biological activity (21,22) antimicrobial activity the antimicrobial of the synthesized final products was done in ibn sena research center / research and development authority/ ministry of industry and minerals_iraq. a preliminary antibacterial and antifungal activity has been carried out according to agar well diffusion method the prepared compounds had been studied for their antimicrobial activity in vitro against three tested bacteria (staphylococcus aureus) as gram positive bacteria and( e. coli. ,pseudomonas aeruginosa) as gram negative bacteria and the yeast (candida albicans) were clinical isolated and maintained on nutrient agar medium for testing antibacterial activity and sabaroud agar medium for antifungal activity. compounds were dissolved in dmso.ciprofloxacin, gentamycin and cephalexin were used as a standard antibiotic for antibacterial activity and clotrimazole was used as a standard drug for antifungal activity. sensitivity assay agar well diffusion assay was carried out by using bacterial suspension of about (1.5×10 8 cfu/ml) obtained from mcfarland turbidity standard (number 0.5).this was used to inoculate by swabbing the surface of mueller hinton agar (mha) plates. excess was air-dried under a sterile hood, and in each agar plate of tested bacteria five wells were made and (30µl) of each concentration was added in it. the plates were incubated at 28 °c for 48 hours (fungi spp.) or 37 °c for 24 hours (bacteria) and the antimicrobial activity was evaluated by measuring the diameter of the inhibition zone (iz) around the disc in mm. the assessment of antibacterial activity was based on measurement of the diameter of inhibition zone formed around the well, and show that the zone of inhibition varied with the increasing of concentration of the tested compounds, as show in table (4) . cytotoxic activity study ( 23) a preliminary in vitro cytotoxicity assay (cell viability assay on cancer cell line) for some of the final compounds (3 and 4) has been carried out in the biotechnology research center / university of al-nahrain. actually, the in vitro cytotoxicity assays with cultured cells are widely used to evaluate chemicals including cancer chemotherapeutics, pharmaceuticals, biomaterials , natural toxins, antimicrobial agents and industrial chemicals because they are rapid and economical. iraqi j pharm sci, vol.24(1) 2015 novel nitrogenous heterocyclic compounds with biological activity 52 cytotoxicity assay has been done by using neutral red uptake assay method. this method has been used for cytotoxicity assay of compounds (3 and 4). a set of two fold in four concentrations (3, 1.5, 0.75, 0.375 μg/ml) was made for each product and the exposure time of the assay was 24hrs. result and discussion synthetic part a. the reaction pathways the aim of our research is to synthesize quinoline derivatives coupled to different amino acid ester or heterocyclic amine (figure 1). the overall synthesis strategy based one four major lines: 1. amino acid derivatives the amino acids were activated by thionyl chloride to get acyl chloride that attacks either ethanol or methanol to get ethyl or methyl esters of the selected amino acids. 2. alkylation of 8-hydroxyquinoline by ethyl bromoacetate in presence of anhydrous potassium carbonate base to get (o-alkylated ester) derivatives which are deesterfied by using naoh solution to get acetic acid derivatives. 3. peptide bond formation conventional solution method for peptide synthesis used as a coupling method between the carboxy-protected amino acids or 1-amino quinolone derivatives with acetic acid side chain of quinolone. the dcc/ hobt coupling reagents used for peptide bond formation. the overall reaction pathway is shown in the following figure 1 . figure (1): scheme of overall pathway of synthesis of n-heterocyclic compounds. iraqi j pharm sci, vol.24(1) 2015 novel nitrogenous heterocyclic compounds with biological activity 53 b. strategy of synthesis the strategy of synthesis started from the esterification of amino acids then proceeded to the synthesis of quinolone-8-yloxy acetic acid from 8-hydroxyquinoline and finally coupling between the acid and the amino group of the amino acid esters, and as follows: 1) esterification of amino acids the esterification of carboxyl group of amino acids is normally used as an amino acid protecting group. esterification of carboxyl group enhances the nucleophilic character of amine group and allows its subsequent acylation (24) . 2) 8-hydroxyquinoline can be alkylated at hydroxyl group by alpha-haloester of ethylbromoacetate which is widely used as alkylating agent to o-, nand sgroups. triethylamine or potassium carbonate act as a base which make a nucleophilic attack to the hydroxl group (-oh) of the above heterocyclic and deprotonate it and convert it to negative charge group which will then make a nucleophilic attack to the ethylbromoacetate which have bromide group that is a good leaving group to get ethyl acetate ester derivative of the above heterocyclic (compound 1). (25) the mechanism of o-alkylation by alkyl halide is a nucleophilic substitution as shown in figure (2) (26) . figure (2): scheme of the o-alkylation of 8-hydroxyquinoline 3) peptide coupling method: there is many ways to form a peptide bond (30) , however in this work; the direct coupling with dcc/hobt method was used. this method is characterized as being simple, efficient, no racemization, rapid, and leading to a good yield at r. t. (31) . the overall reaction resembles the dehydration process to form the amide bond (figure 1) .the properties of synthesized new compounds are shown in table (1) while the data obtained from elemental microanalysis are shown in table (2).on the other hand the characteristic ir bands are shown in table (3). 4) removal of ethoxy group from compounds 1 were done by alkaline catalyzed deesterification (saponification) to get the corresponding conjugated bases, that acidified by mineral acid (hcl) to get the corresponding carboxylic acid (27,28) (compound 2). the mechanism of saponification was proposed by ingold as shown in figure (3) 29 ). figure (3): scheme of saponification of compound 1. iraqi j pharm sci, vol.24(1) 2015 novel nitrogenous heterocyclic compounds with biological activity 54 table (1): the identification parameters of the synthesized compounds symp. compound name yield % physical appearance m.p. o c rf value a1 d-alanine methyl ester. hcl 78 off-white crystals 98-102 0.73a 0.69b b1 1-amino-quinoline-2-(1h)-one 73 yellow crystals 128130 0.90a 0.63c 1 ethyl 2-(quinolin-8-yloxy)acetate 77 yellow crystals 52-54 52-53 0.74c 0.79d 2 2-(quinolin-8-yloxy)acetic acid 68 palle-yellow crystals 210214 0.30c 0.25d 3 methyl 2-(2-(quinolin-8yloxy)acetamido)propanoate 60 white crystals 208210 0.88b 0.36c 4 n-(2-oxoquinolin-1(2h)-yl)-2-(quinolin8-yloxy)acetamide 50 faint-yellow powder 217220 0.90d table( 2): the elemental microanalysis of the synthesized compounds table (3): the characteristic ir bands of the synthesized compounds; (measured in cm-1) cpd no. m. wt. chemical formula calculated ∕ found c% h% n% o% 3 288.30 c15h16n2o4 62.49 61.94 5.59 6.10 9.72 10.58 22.20 21.38 4 345.35 c20h15n3o3 69.56 70.44 4.38 4.25 12.17 12.26 13.90 13.05 a1 3325, 3277 asym. and sym. str. of nh2 ; 2928, 2850 asym. and sym. ch3, ch2 str.;1743 c=o str. ester; 1570nh bend.; 1215 c-o str. ester; 1435 ch2 bend.; 1375 ch3 bend. . b1 3306, 3186 asym. and sym. str. of nh2; 3057 c-h ar. str of benzene; 1662 c=o str. of amide; 1514nhbend.; 1118 c-n str.;1597,1548,1458 c=car. str. . 1 (3053 c-har str.), (2989assym , 2875 sym. c-halif. str.), (1743.7 c=o str. ester), (1504 , 1446 c=car str.), (1219 c-o str. ester), (1118 ar-o-c str.),1653 c=n str. . iraqi j pharm sci, vol.24(1) 2015 novel nitrogenous heterocyclic compounds with biological activity 55 table (3): continued the characteristic ir bands of the synthesized compounds; (measured in cm -1 ) asym=asymmetric , sym.=symmetric , str.=stretching , bend.=bending , ar.=aromatic, alif=alifatic table (4): the antibacterial activity of the tested compounds.(3 and 4 ) 2 (3462 oh str. of cooh), (3053 c-har. str.), (2937assym,2833sym c-halif. str.), (1716 c=o str. of cooh), (1600 , 1562,1454 c=car str.), (1118 ar-o-c str.). 3 3323 nh str.), (3036 ch ar. str.), (2928assym,2850symchalif.str.), (1743.7 c=o str. ester), (1678c=o str. amid) ,(1624 c=n str.), (1535-n-h bend.) ,(1572,1564,1502 c=car str.), (1242 ar-oc str.). 4 (3323 nh str.), (3032 ch ar str. ), (2928 assym. , 2850 sym chalif. str.), (1683 c=o str. amide), (1537 nh bend. amide ii), (15701435 c=c str.), (1242 ar-o-c str.). compound no. zone of inhibition in mm e. coli atcc10536 staphy. aureus atcc6538 pseud. aurogenosa atcc15442 3 100µg/ml no activity no activity 13 200µg/ml 13 no activity 14.5 4 100µg/ml no activity no activity 15 200µg/ml 13.5 no activity 15 ciprofloxacin 100µg/ml 20.5 22 30 200µg/ml 26 32 35 gentamycin 200µg/ml 20 20 14 cephalexin 600µg/ml 24 36 no activity iraqi j pharm sci, vol.24(1) 2015 novel nitrogenous heterocyclic compounds with biological activity 56 table (5): the antifungal activity of the tested compounds.( 3 and 4) according to the following equation, the cytotoxic effect for tested compounds expressed as ir % at transmittance wave length 492 nm as showed in table 6 inhibition rate% = x100 table (6): the initial cytotoxic effect (cell viability assay ) on hep 2 cell line of compounds ( 3 and 4) by neutral red assay method. cpd. no. concentration time 24 hrs. 3μg/ml 1.5μg/ml 0.75μg/ml 0.375μg/ml control/ 0μg/ml 3 0.1396 88.49 11.51 0.1353 85.768 14.23 0.1066 67.575 32.43 0.1093 69.286 30.71 0.15775 100 0 abs. at 492nm cell viability% i.r.% 4 0.1026 65.05 34.97 0.09 57.05 42.95 0.0796 50.45 49.55 0.0836 52.99 47.01 0.15775 100 0 abs. at 492nm cell viability% i.r.% abs.= absorbance i.r.%= inhibition rate percent summary of the results 1)from the result in table (4), all tested compounds had low or no activity against e.coli and no activity against staphylococcus aureus when compared with ciprofloxacin, gentamycin and cephalexin. while all tested compounds showed high activity against pseudomonas aeruginosa when compared to cephalexin, and comparable activity against same bacteria when compared to gentamycin and finally ,the tested compounds showed low activity against pseudomonas aeruginosa when compared to ciprofloxacin. from the results in table (5), the tested compounds showed a moderate to good activity against candida albicans. when compared to clotrimazole. 2)the cytotoxic study was done on hep-2 cell line passage (75), exposure time =24hrs. staining is neutral red stain. when the cancer cell line (hep-2) was treated with these products the result showed significant cytotoxic effect in tested samples in comparison with the control. the toxic effect varied from one sample to another, all samples showed a significant toxicity (p < 0.05) started from 3μg/ml to the 0.375μg/ml. the inhibitory concentration percent (ic %) was estimated, and the result was varied among samples as shown in table (6). conclusions from the antimicrobial and cytotoxic activity studies, compound 4 showed the best activity. according to pharmacokinetics properties and cationic nature of comp.4, comp.4 may cause dna intercalation through dna topoisomerase/gerase chelation or other binding sites. references 1. padmavathi v., subbaiah drcv, mahesh k, lakshmi tr.; synthesis of and bioassay of 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intraocular pressure doi: https://doi.org/10.31351/vol30isssuppl.pp1-8 1 the effect of anabasis articulata stems extract on lowering intraocular pressure in the glaucoma rat model(conference paper )# waleed k. abdulsahib* # 9th scientific conference conference sponsored by college of pharmacy , university of baghdad 25-26 august 2021 *department of pharmacology and toxicology, college of pharmacy, alfarahidi university, baghdad, iraq. abstract high intraocular pressure (iop) is a recognized risk factor for glaucoma and optic nerve injury, and it is one of the primary causes of vision loss globally. anabasis articulata (aa) is a desert plant found in iraq. the extract of aa is used for the treatment of diabetes, fever, eczema, and kidney infections. the study aims to evaluate the antioxidant effect of methanol extract of aa on intraocular pressure in the glaucoma rat model. fortytwo rats were allocated into seven groups, each with six animals: group 1 (normal), group 2 (control), in which animals were induced to have elevated iop by betamethasone suspension injection, groups 3,4 and 5 for evaluating the effect of 50,100 and 150 mg/kg/day of the tested extract, respectively, and the remaining two groups (group 6 and 7) for evaluating oral acetazolamide and topical timolol 0.5% respectively. betamethasone was used for the induction. measure the iop every 2 days for 2 weeks. the daily dose of aa extract (50 mg/kg/day) for 6 days significantly reduces intraocular pressure (p ˂ 0.05), from (34.23± 0.58) to (32.83± 1.38) mmhg when compared with the control group. in group 4, iop decreased significantly from (35.5±1.37) to (31.35±0.40) mmhg (p ˂ 0.05) after 1 week of treatment. in group 5, the significant (p˂ 0.001) iop reduction from (35.66±0.39) to (31.88±0.74) mmhg started on day 6 and continued until the end of the experiment, reaching (24.53±0.53) mmhg (p˂ 0.001). the antioxidant and anti-angiogenic properties of aa make it a promising adjuvant treatment for glaucoma. keywords: anabasis articulata, glaucoma, antioxidant, extract, betamethasone, intraocular pressure. في خفض ضغط العين لداء الزرق المستحدث في anabasis articulataتأثير مستخلص سيقان ال #) بحث مؤتمر ( الجرذان 1*،وليد خليل عبد الصاحب 2021اب 26 – 25جامعة بغداد ، # المؤتمر العلمي التاسع لكلية الصيدلة *فرع االدوية والسموم، كلية الصيدلة، جامعة الفراهيدي،بغداد، العراق الخالصة أحد يعتبر ارتفاع ضغط العين لإلصابة بداء الَزَرق وكذلك إصابة العصب البصري ، و يؤديهو عامل خطير ارتفاع ضغط العين .األسباب الرئيسية لفقدان البصر على مستوى العالم .هو نبات صحراوي موجود في العراق ويستخدم لعالج مرض السكري والحمى واألكزيما وكذلك التهابات الكلى (anabasis articulata)العجرم على ضغط العين المستحدث في الجرذان. تم تقسيم اثنين جرملنبتة الع تقييم التأثير المضاد لألكسدة لمستخلص الميثانولمن الدراسة هو الهدف )مجموعة التحكم( ، حيث تم استحداث 2)طبيعة( ، المجموعة 1إلى سبع مجموعات ، كل مجموعة تحوي ستة حيوانات: المجموعة جرذاوأربعين 100و 50الثالثة والرابعة والخامسة استخدمت لتقييم تأثيرعن طريق حقن معلّق البيتاميثازون ، المجموعة في هذه المجموعة المرتفع ضغط العين ( لتقييم تأثير األسيتازوالميد الفموي والتيمولول 7و 6ملغم / كغم / يوميا من المستخلص على التوالي ، والمجموعتان المتبقيتان )المجموعة 150و مجم / كغم / 50اظهرت نتائج الجرعة اليومية من المستخلص ) .ينكل يومين ولمدة أسبوع ٪ على التوالي. تم قياس ضغط العين 0.5الموضعي ( مم 1.38± 32.83( إلى )0.58± 34.23، حيث انخفض الضغط من ) (p˂ 0.05) أيام انها تقلل بشكل ملحوظ من ضغط العين 6يوم( ولمدة ( 0.40± 31.35( إلى )1.37± 35.5ل ملحوظ من )الضغط في المجموعة الرابعة بشك زئبق بالمقارنة مع المجموعة الضابطة. وكذلك انخفض 31.88( إلى )0.39± 35.66من ) للضغط(p˂ 0.001) ، بدأ االنخفاض المعنوي 5بعد أسبوع من العالج. وفي المجموعة (p ˂0.05) مم زئبق . استنتج من الدراسة ان ( (p˂ 0.001) ( مم زئبق0.53± 24.53( مم زئبق في اليوم السادس واستمر حتى نهاية التجربة ، ليصل إلى )±0.74 تجعله عالًجا مساعدًا واعدًا لداء الزرق. الخصائص المضادة لألكسدة والمضادة لتولد األوعية لنبتة العجرم .داء الزرق، البيتاميثازون ،ضغط العين،مضاد االكسدة ،الكلمات المفتاحية: العجرم introduction high intraocular pressure (iop) is a recognized risk factor for glaucoma and optic nerve injury, (1), and it is one of the most common causes of blindness in the world (2). glaucoma affected approximately 64.3 million people worldwide in 2013, putting them at risk of blindness. this number is expected to rise to 76.0 million in 2020 and 111.8 million in 2040 (3). the buildup of aqueous humor (ah) in the anterior chamber, which is mostly caused by the eye's failure to discharge aqueous fluid effectively, results in a high iop(4). blood flowing through the ciliary body's arteries is the main cause of aqueous humor. in the posterior chamber, the ciliary body between the iris and lens secretes aqueous humor , the fluid of this region 1corresponding author e-mail: waleed.abdelsahib@alfarahidiuc.edu.iq received: 18/8/2021 accepted: 14/9 /2021 published online first: 2022-1-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30isssuppl.pp1-8 iraqi j pharm sci, vol.30(suppl.) 2021 anabasis articulata and lowering intraocular pressure 2 moves from the posterior to the anterior chamber between the cornea and the iris before being emptied from the eye at the iridocorneal junction(5). increased iop is thought to be a chief risk factor for the gradual loss of retinal ganglion cells (rgcs). according to previous research, the longer the iop rise, the more severe the optic nerve consequences (6). medication, surgery, and laser therapy are all common glaucoma treatments. ocular hypotensive medications either reduce or improve trabecular meshwork, schlemm's canal, and uveoscleral outflow(7). on the other hand, most clinical medications can produce adverse effects, and natural plant extracts may provide an alternate medication source(8). herbal medicines have grown in importance as a subject of study for health care around the world (9). anabasis articulata (aa), also known as ajrem, eshnan, or berry bearing glasswort, is a desert plant found in iraq, algeria, syria, and egypt. in folk medicine, aa is used to treat diabetes, fever, eczema, and kidney infections(10). many studies after the phytochemical screening on aa discovered the availability of coumarins, anthraquinones, unsaturated sterols or triterpenoids, alkaloids, saponin, phenolics, flavonoids, iridoids, and tannins as active components that may have many pharmacological effects (11). additionally, recent studies have shown that the main ingredients identified by gas chromatography-mass spectrometry (gc-ms) of methanol extracts are tannins, saponins, flavonoids, phenolics, and alkaloid compounds (12). aa has recently become the focus of research due to the diversity of its composition and effectiveness. however, the scientific literature on the beneficial effects of aa on glaucoma patients is limited. thus, the goal of this study is to evaluate the effect of aa on intraocular pressure in a glaucoma rat model. materials and methods preparation of plant extract anabasis articulata (forssk.) moq. (amaranthaceae) stems were obtained from a baghdad-based herbal apothecary and authenticated by assistant professor dr. ibrahim salih abbas (ph.d. medicinal plants, pharmacognosy department, college of pharmacy, almustansiriyah university, iraq). the plant's specimen (99334) was deposited in the herbarium of al farahidi universityfaculty of pharmacy. the maceration process (cold process) was carried out by soaking plant stems (powdered) in a container stopped with 70% v/v ethanol and allowing for 72 hours of frequent turmoil at room temperature in the bath, after which the crude material was extracted using a vacuum-concentrated rotary evaporator and kept in a dry bottle and firmly screened. experimental animals fortytwo sprague dawley rats of both sexes, aged about 8 months were used in the experiment. animals maintain a 12-hour light/dark cycle and are kept at 25 ± 3 cᵒ. rats were free to access clean water ad libitum and rodent diet. the institutional animal care and use committee of al farahidi university, pharmacy college approved the research protocol, and the conduction of the work was performed according to the association of research in vision and ophthalmology (arvo). group 1: animals were injected 0.2 ml distilled water subconjunctivally (sq) and this group is considered a normal group. group 2: animals were induced to have elevated iop by 0.2 ml betamethasone suspension sq injection. groups 3,4, and 5: these groups were used to evaluate the effect of 50,100, and 150 mg/kg/day of the herb, respectively. the remaining two groups (groups 6 and 7) were used to evaluate oral acetazolamide (at a dose of 78 mg/kg/day) and topical timolol 0.5 percent (twice daily), respectively, and these groups were considered positive. asteroid suspension of 0.03 ml betamethasone containing a mixture of (betamethasone dipropionate 2 mg and betamethasone sodium phosphate 5mg) / ml was used for induction of elevated iop. (diprofos*, msd)(13). syringe gauge 30 was used (pic, uk) for subconjunctival steroids. after injection, one drop of antibiotic ofloxacin (pioneer, iraq) was used to prevent future infections of the eye (14). in all animal groups, the right eye was used to induce chronic glaucoma models by weekly sq betamethasone for 4 weeks(15), and the left eye was used to evaluate whether oral test extracts had any adverse effects on the eyes. after 7 days of induction, rats with an iop increase of more than 32 mmhg were included in the test group. accupen handheld tonometer (accutome, usa) is used to measure intraocular pressure every 2 days. all readings were taken in the morning (10 am) to exclude diurnal changes in iop. three doses of methanol extract of aa stems (50, 100, and 150 mg/dose/day) were orally administered by gastric tube gavage for 14 days to study the effect of the extract in controlling elevated iop. bodyweight was measured every week to study the adverse effect of steroids and the potential beneficial effect of aa. a blood sample was withdrawn before and after treatment with aa to measure the serum level of lactate dehydrogenase (ldh), malondialdehyde (mda), glutathione peroxidase (gp), catalase (cat), and superoxide dismutase (sod).in previous studies, the safety of the extract was studied, and the ld 50 was even calculated. the doses used have no toxic effects. statistical analysis all data are represented in this study as mean± standard deviation( sd) ( 6 animals for each group). bodyweight changes, serum level of the biochemical parameters, and iop were investigated iraqi j pharm sci, vol.30(suppl.) 2021 anabasis articulata and lowering intraocular pressure 3 using one-way analysis of variance (anova) using version 23 of ibm spss and then multiple comparison tests of type ttukey. statistical significance is defined as a p-value ˂ 0.05. results bodyweight effect in a steroid-induced model of glaucoma as displayed in a table (1) and figure 1, a significant decrease in mean body weight (p ˂ 0.05) was detected in the second group (control group), but not in the first group 1 (normal group). after 7 days of oral intake of 100 mg of the tested extract, a significant increase (p ˂ 0.05) in average body weight was observed in group 4 compared to group 2, as shown in table (1 ). group 5 shows a significant (p ˂ 0.05) increase in body weight after 14 days of treatment compared to the control group. the weight of animals in the fourth group after 2 weeks increased significantly (p ˂ 0.05) when compared to the third group. although the administration of 150 mg (group 5) resulted in a greater increase in body weight, there was no significant difference when compared to group 4 (p ˂ 0.05). acetazolamide did not affect body weight when compared to the control group (p=0.88). table 1. effect of anabasis articulata extracts on the body weight in the betamethasone-induced chronic glaucoma model. groups bodyweight (gram) before induction after 7 days of induction after 14 days of induction normal 216.33± 2.25 227.83± 5.03 252.83± 4.62 control 212.83± 1.94 196.66± 2.80 * 207.33± 1.75 * aa treatment 50 mg 213.66± 3.38 199.83± 4.79 208.16± 2.13 aa treatment 100 mg 217.83 ± 5.41 210.50± 6.74 ** 218.83± 6.64 ** aa treatment 150 mg 219.83± 3.71 206.00± 7.21 215.00± 4.47 ** acetazolamide 217.16± 4.53 202.66±4.08 208.83± 3.76 results are denoted as mean± standard deviation (n=6). * p ˂ 0.05 means there is a significant difference compared with the normal group, ** p ˂ 0.05 means there is a significant difference compared with the control group. aa: anabasis articulate figure 1. the effect of induction agent (betamethasone suspension), anabasis articulata (50, 100, and 150 mg/ kg/ day) and (50 mg/kg/day) for acetazolamide on body weight (n= 6). * p less than 0.05 denote a significant difference when compared to the normal group, **p less than 0.05 denote a significant difference compared to the control group. data represented as mean± sd. error bar represents the standard deviation of the mean. effect of anabasis articulata on iop subconjunctival injection of steroids in the second group of rats increased the intraocular pressure from (18.75±0.44) to (34.58±0.97) mmhg. compared with the normal group, these changes are significant (p ˂ 0.05), as shown in figure 2. one week later, the increase in intraocular pressure in the control group was still significant (p˂ 0.001) (34.76±0.9 mmhg). in addition, the intraocular pressure at the end of the study was still high (35.01±0.69 mmhg) (p˂0.001) compared to the normal group (18.66±0.45). figure 2 shows the daily dose of aa extract (50 mg/kg/day) for 6 days significantly reduces intraocular pressure (p ˂ 0.05), from (34.23± 0.58) to (32.83± 1.38) mmhg when compared with the control group. the maximum drop in intraocular pressure (28.51±0.52 mmhg) (p˂0.05) was reached on day 14. in group 4, iop decreased significantly from (35.5±1.37) to (31.35±0.40) mmhg (p ˂ 0.05) after 1 week of treatment. on day 14, there was the greatest pressure reduction (24.76±0.69), which was significant iraqi j pharm sci, vol.30(suppl.) 2021 anabasis articulata and lowering intraocular pressure 4 compared with the control group (p˂ 0.001), as shown in figure 3. on days 6 and 12, group 4 produces a significant iop reduction when compared to group 3 (p ˂ 0.05). in group 5, the significant (p˂ 0.001) iop reduction from (35.66±0.39) to (31.88±0.74) mmhg started on day 6 and continued until the end of the experiment, reaching (24.53±0.53) mmhg (p˂ 0.001), as shown in figure4. no superiority of 150 mg over 100 mg. positive control groups: group 6 (acetazolamide) and group 7 (timolol) significantly reduced intraocular pressure (p˂ 0.001) from (35.31±0.77) to (30.58± 0.49); from (36.03±0.49) to (28.11±0.64) mmhg respectively,as presented in figure 5 and 6. figure 7 shows that all doses of aa reduce the iop significantly after 2 weeks of treatment. finally, acetazolamide and timolol were more effective in reducing iop during the study period (p ˂ 0.05) compared to all doses of the extract. figure 2. effect of anabasis articulata stems extract (50 mg/kg/day) on mean iop in a chronic glaucoma model in rats. *p ˂ 0.05 means there is a significant difference compared with the control group. figure 3. effect of anabasis articulata stems extract (100 mg/kg/day) on mean iop in a chronic glaucoma model in rats. *p ˂ 0.05 means there is a significant difference compared with the control group. figure 4. effect of anabasis articulata stems extract (150 mg/kg/day) on mean iop in a chronic glaucoma model in rats. *p ˂ 0.05 means there is a significant difference compared with the control group. figure 5. comparison between different doses of anabasis articulata stems extract and topical timolol (0.5%) on mean iop in a chronic glaucoma model in rats. *p ˂ 0.05 means there is a significant difference compared with the control group, **p ˂ 0.05 means there is a significant difference compared with groups 3,4, and 5. figure 6. the comparison effect between different doses of anabasis articulata stems extract and topical timolol (0.5%) on mean iop in a chronic glaucoma model in rats. *p ˂ 0.05 means there is a significant difference compared with the control group **p ˂ 0.05 means there is a significant difference compared with groups 3,4, and 5. iraqi j pharm sci, vol.30(suppl.) 2021 anabasis articulata and lowering intraocular pressure 5 figure 7. the comparison effect between different doses of anabasis articulata stems extract, acetazolamide, and topical timolol (0.5%) on mean intraocular pressure after 2 weeks of treatment in a chronic model of glaucoma in rats. *p ˂ 0.05 means there is a significant difference compared with the normal group, **p ˂ 0.05 means there is a significant difference compared with the control group. effects of anabasis articulata extract on serum levels of some oxidative stress parameters. table (2) shows the effect of 3 doses of the extract on the serum levels of five biological parameters in steroid-induced glaucoma after 2 weeks of treatment. in group 2 (control) the serum levels of ldh & mda increased significantly (p˂0.05), whereas those of gp, cat, and sod decreased significantly (p˂0.05) when compared to the normal group. after 2 weeks of oral administration of extract in all treatment groups (3,4 and 5), led to a trend of significantly decreased in the serum levels of ldh and mda and a significant increase in the serum level of gp, cat, and sod (p˂0.05) compared to control group. compared with the third group, the second group showed the preferred effect in reducing the levels of ldh and mda and increasing the levels of gp, cat, and sod (p˂0.05). in addition, the antioxidant activity of the fourth group was more significant than that of the fifth group (p˂0.05), resulting in the effect of the fourth group being better than that of the fifth group. table 2. the effect of anabasis articulata extract on serum levels of biological parameters in a chronic model of glaucoma in rats after 2 weeks of treatment. groups ldh (u/l) sod (u/mg) mda (mmol/ mg) cat (u/ mg) gp (u/ mg) normal 1769.94± 12.70 116.35± 1.77 1.17± 0.130 65.73± 0.41 1.15± 0.022 control 2319.42± 6.94* 71.14± 0.84* 1.43± 0.031* 25.47± 0.15* 0.56± 0.017* treatment 50 mg 1320.26± 12.53 a 86.32± 0.53 a 1.23± 0.037 a 30.77± 0.28 a 0.64± 0.036 a treatment 100 mg 1486.91±7.15 ac 101.48± 1.32 ac 1.33± 0.037 35.49± 0.33 ac 0.95± 0.017 ac treatment 150 mg 1416.41±3.66 ad 95.99± 1.38 ad 1.14± 0.034 ad 31.44± 0.22 ad 0.83± 0.020 ad results are denoted as mean± standard deviation (n=6). * p ˂ 0.05 denote a significant difference when compared to the normal group, a p ˂ 0.05 denote a significant difference when compared with the control group, c p˂0.05 denote a significant difference when compared with the 50 mg treatment group. d p˂0.05 denote a significant difference when compared with the 100 mg treatment group. ldh: lactic dehydrogenase; sod: superoxide dismutase; mda: malondialdehyde; cat, catalase; gp, glutathione peroxidase. discussion glaucoma is the world's second-largest reason of blindness, in which the optic nerve and rgcs gradually degenerate (16). high iop is still the most well-recognized risk factor for glaucomatous optic nerve injury, even though other variables may play a role in glaucoma. (17). the effect of aa extract on iop in a rat model of steroid-induced glaucoma was investigated in this work. steroid-induced glaucoma resulted in a noteworthy elevation in iop and a drop in mean rat weight, according to the findings. steroid-induced iop elevation is said to be dose-dependent and associated with known systemic adverse consequences such as weight loss. (13). although weight gain is a common side effect of glucocorticoids in humans (18), irritation of the stomach is moreover prevalent, which could lead to appetite and weight loss in rats(18). however, after 14 days of aa administration, bodyweight loss slightly recovered when compared to the control group. iop increased above 32 mmhg in the steroid groups, iraqi j pharm sci, vol.30(suppl.) 2021 anabasis articulata and lowering intraocular pressure 6 indicating that the glaucoma model was efficiently produced. the experiment results displayed that all doses of aa reduce iop noticeably after 2 weeks, with 100 mg/kg/day being the most effective. the control group had considerably higher ldh and mda levels and marked lowering effects on sod, gp, and cat levels, indicating that the glaucoma rats were under a lot of oxidative stress. after fourteen days of treatment, 50 mg of aa administered daily caused an apparent decrease in ldh and mda levels and a momentous rise in sod, gp, and cat levels in the experimental groups. groups 4 and 5 demonstrated a similar trend, but with greater antioxidant effects than group 3. the experimental results show that aa extract increased the glaucoma rats' intrinsic antioxidant capabilities. in addition to laser treatment and surgical procedures, the most frequent treatment method for glaucoma is lowering iop with medical therapy. to reduce iop, medical therapy employs two mechanisms: boosting outflow drainage and inhibiting aqueous humor formation. (19). according to reports, antioxidant pretreatment markedly reduces the influence of oxidative stress on the trabecular meshwork (tm) (20). it has been reported that aa has a high clearance rate of free radicals of 2',2'-diphenyl-1-picrahydrazyl (dpph)(21,22). the high content of tannins, saponins, flavonoids, phenolics, and alkaloid compounds in aa is attributed to its antioxidant activity (21). there is growing evidence that reactive oxygen species be a factor in the pathogenesis of primary open-angle glaucoma. (23). the association between antioxidant component content, in vivo / in vitro antioxidant abilities, and iop-reducing effect suggests that oxidative stress is important in the progress of iop elevation and glaucoma-related lesions (20). tnfα is a pro-inflammatory cytokine with antiinflammatory and neuroprotective properties. furthermore, this cytokine has two different receptors: tnf-r1 and tnf-r2, and its activity varies depending on which of these two receptors is activated. stimulation of the first receptor results in the recruitment of immune cells, which causes inflammation, as well as the activation of enzymes that cause oxidative stress (18). the second, on the other hand, support tissue homeostasis and promotes tissue regeneration, and so plays an active role in neuroprotection (20). this result requires stimulation of the nfkb pathway by tnf-r2(24), which translocates into the nucleus of cortical neurons and likely protects against excitotoxicity. tnf-, on the other hand, has been proven in vitro to trigger rgc cell death by activating caspase-3 and -8 or by oxidative stress produced by mitochondrial malfunction. (25). the increment in ros production can also result in the death of the neuronal cell. the ros that activates the nfkb pathway in glial cells induces inflammation, which then stimulates nadph oxidase, which produces more ros, creating a vicious cycle. as a result of the herbs' antioxidant activity, this vicious cycle will be broken. based on our findings, the antioxidant effect of aa could be linked to reduce iop and decrease rgcs death. oxidative stress is caused by an elevation in ros that exceeds the tissue's antioxidant capacity, which contributes to the aging process by generating and accelerating cellular senescence. the defective mitochondrial function in glaucoma patients' tm cells makes these cells abnormally vulnerable to ca++ stress, resulting in iop control failure. in glaucoma patients, several inflammatory molecules are upregulated. vascular endothelial growth factor (vegf), interleukins, and tnf are a few examples (23). the optic axon is affected by vegf activators such as hypoxia and no, though the mechanism is unknown(26). elevated vegf levels in the anterior segment may be the primary cause of the remodeling process in tm tissues (16). most of these mediators can induce extracellular matrix remodeling and change functions of cytoskeletal in the tm during glaucoma(27). specifically, il6, which increases in response to oxidative stress, il8 can modify the permeability of schlemm's canal endothelial cells and has also been connected to the induction of senescence and the modification of barrier functions of the tm endothelium in pig eyes (28). previous research has shown that aa has an important anti-angiogenesis effect by inhibiting vegf, and this action will increase the extract's efficacy in lowering iop and preventing the progression of glaucoma disease(22,29). furthermore, male and female rats given aa for acute and chronic toxicity in previous studies revealed no observable adverse effects at doses greater than 5000 mg/kg, indicating that aa is safe for use as a therapeutic supplement (12). iraqi j pharm sci, vol.30(suppl.) 2021 anabasis articulata and lowering intraocular pressure 7 figure 8. supposed mechanism of action of oral administration of anabasis articulata for treatment of glaucoma. oxidative stress can cause alteration in the conventional outflow pathway, leading to iop elevation and induce apoptosis that leads to rgc death (30). tnf-α: tumor necrosis factorα; nf-kb: nuclear factor-kappa; mmp: mitochondrial membrane potential; iop: intraocular pressure; vegf: vascular endothelial growth factor. conclusion the antioxidant and anti-angiogenic properties of aa make it a promising adjuvant treatment for glaucoma patients. more studies with larger sample sizes are needed to evaluate the longer-lasting effects of aa treatment. acknowledgments the author wishes to express gratitude to al farahidi universitypharmacy college for your assistance in providing experimental rats. conflict of interests there are no 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http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 treatment modalities among patients on maintenance doi: https://doi.org/10.31351/vol31iss1pp95-101 95 adherence to different treatment modalities among patients on maintenance hemodialysis maha a. abdul-jabbar*1 and dheyaa j. kadhim** * al nuaman general hospital, ministry of health and environment, baghdad, iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract end stage renal disease is a well-known global public health problem. maintenance hemodialysis is considered a life-saving treatment for patients with such disease. this treatment method that requires patients to be adherent to hemodialysis attendance, dietary and fluid recommendations as well as adherence to prescribed medications to ensure success. the aim of the current study was to assess adherence, perception, and counseling among hemodialysis patients to different modalities of treatment (fluid restriction, dietary recommendations, medications, and hemodialysis schedules). a cross-sectional study carried out on hemodialysis patients who attended to the dialysis centers at alkarama teaching hospital and madinat alimamain alkadhimain teaching hospital. the arabic version of the “ end stage renal disease-adherence questionnaire ” was used in assessing adherence, perception, and counseling. the number of recruited patients was 200 adult patients (113 men and 87 women). the average of the total adherence score lies within the moderate adherence (984.9 ±174.2). patients adherence to the hemodialysis session was good as representing by high scores of adherence for (hemodialysisattendance, episode of hemodialysis-shortening and duration of hemodialysis-shortening) while the lowest adherence score (126.0 out of 200) was for following the fluid restriction. in terms of adherence categories, the majority (61%) of the patients had good adherence, 33.0% had moderate adherence with 6% had poor adherence. age had a significant positive association with the total adherence score. all patients perceived their hemodialysis management as highly/very important. on the other hand, some patients perceived their adherence to the recommended diet as moderately (7%) or less important (7%). regarding the frequency of counseling received by patients for different treatment modalities. the negative answers represented 58.5% of answers about the how important is to follow a proper diet, and 25.0% of answers about the importance of taking prescribed medications as ordered. accordingly, the overall adherence of hemodialysis patients to different treatment modalities was less than optimum with fluid and diet adherence representing most challenging tasks in the health care of hemodialysis patients. keywords: adherence, hemodialysis, chronic kidney disease, esrd-adherence questionnaire, iraq الدمويالكلى المستمرين على غسيل المرضىلدى المختلفة العالج بطرقااللتزام **جبار كاظم ضياء و 1 ، * الجبار عبدعامر مها .العراق بغداد، والبيئة، الصحة وزارة العام، النعمان مستشفى * .العراق بغداد، بغداد، جامعة الصيدلة، كلية فرع الصيدلة السريرية، ** الخالصة ه المزمن الكلى غسككي . العالم مسككت على المعروفة المتزايدة العامة الصككحية المشككك من النهائية المرحلة في الكلى مرض يعد غسكي وع ج الم صك فة االلتزام باألدوية هذه الع ج طريقة تتطلب. النهائية المرحلة في الكلى مرض من يعان ن الذين المرضكى حياة إلنقاذ إجراء بين المشكك رة وتقديم ، واإلدراك ، االلتزام تقييم ه الحالية الدراسككة من الهدف كان. نجاحها لضككمان والتقييدا المتعلقة باألغذية والسكك ائ الكلى أجريت هذه الدراسكة المقطعية (. الكلى غسكي وجداول ، واألدوية ، الغذائية والت صكيا ، السك ائ تقييد) المختلفة الع ج لطرق الكلى غسكي مرضكى تم. التعليمي الكاظمين اإلمامين مدينة ومسكتشكفى التعليمي الكرامة مسكتشكفى في الكلى غسكي مراكز إلى حضكروا الذين الكلى غسكي مرضكى على المشم لين المرضى عدد بلغ. واالستشارة ، واإلدراك ، االلتزام لتقييم النهائية المرحلة في الكلى بمرض االلتزام استبيان من العربية النسخة استخدام كان(. 174.2± 984.9) المعتدل االلتزام معدل ضككمن االلتزام نقاط مجم ع مت سكك يقع(. امرأة 87 و رج ا 113) بالغ مريض 200بالدراسككة كانت بينما( الكلى غسكي تقصكير ومدة حاال تقصكير ، الحضك ر) لككككك االلتزام من عالية درجا يمث ألنه جيداا الكلى غسكي بجلسكة المرضكى التزام ٪ 33.0 و ، المرضى التزام جيد من٪( 61) الغالبية لد كان ، االلتزام فئا حيث من. الس ائ ل لتزام بتقييد( 200 من 126.0) التزام درجة أق غسككي جلسككا أن المرضككى جميع اعتبر. االلتزام نقاط مجم ع مع كبير إيجابي ارتباط للعمر كان. ضككعي التزام لديهم٪ 6 و معتدل التزام لديهم معتدل االهمية به الم صككى الغذائي بالنظام التزامهم أن المرضككى بعض اعتبر ، أخر ناحية من. األهمية شككديدة/ للغاية مهمة بهم الخاصككة الكلى من٪ 58.5 السكككلبية اإلجابا مثلت. المختلفة الع جية للطرق المرضكككى يتلقاها التي االسكككتشكككارة بتكرار يتعلق فيما٪(. 7) االهمية قلي أو٪( 7) لمرضى العام االلتزام كان ، لذلك وفقاا. كما وصفت األدوية تناول أهمية ح ل اإلجابا من٪ 25.0 و ، سليم غذائي نظام اتباع أهمية ح ل اإلجابا الصكككحية الرعاية في صكككع بة األكثر المهام الغذائي والنظام بالسككك ائ ويمث االلتزام األمث ال ضكككع دون المختلفة الع ج بطرائق الكلى غسكككي الكلى. غسي لمرضى العراق النهائية، المرحلة في الكلى بأمراض االلتزام استبيان المزمنة، الكلى أمراض الدموي، الكلى غسيل االلتزام ،: المفتاحية لكلمات 1corresponding author e-mail: maha.2020.clinical@gmail.com received: 21/5/2021 accepted: 22/8 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp95-101 iraqi j pharm sci, vol.31(1) 2022 treatment modalities among patients on maintenance 96 introduction chronic kidney disease (ckd) is a progressively decrease in renal function, which may ultimately lead to renal failure requiring transplant or dialysis for survival (1). worldwide, in 2017, about 1.2 million people died due to ckd. the worldwide all-age rate of mortality from ckd increased by about 41.0% between 1990 and 2017. in 2017, about 697 million cases of all-stage ckd were reported, for a worldwide prevalence of 9.1%. the worldwide all-age prevalence of ckd increased by about 29.0% since 1990, however; the agestandardized prevalence remained stable (2). a patient registry system is not available in most of developing countries. as a result, the accurate number ckd and end-stage renal disease (esrd) patients are difficult to be obtained. the recorded prevalence of esrd in middle east area varies, between 818 per million populations (pmp) in lebanon and 52 pmp in iraq. the mean prevalence of esrd in the whole middle east is 430 pmp (3). adherence is defined as “ the extent to which a person's behavior corresponds with the agreed recommendations of a healthcare provider in terms of taking medications, following a recommended diet and/or executing lifestyle changes” (4). generally, the world health organization (who) reported that long‐term adherence to therapy among patients having chronic diseases in developed countries was about 50% and that the consequences of such non‐adherence are poorer health-outcome and increased in the overall healthcare costs (5). with respect to ckd patients, adherence to therapy is essential to prevent unnecessary progression and serious complications of this disease (6). for patients with esrd, chronic hemodialysis (hd) is a life-saving treatment (7), however; hd replaces about 10% of the normal kidney function. the average hd patient takes about 6–10 drugs a day in combination with many dietary restrictions. these complex therapeutic regimens place a significant burden on the patients and usually make them dependent on health-care providers for many aspects of their treatment (8). the successful hd treatment depends highly on long-term adherence to different aspects of therapy such as regular dialysis attendance, fluid restriction, as well as adherence to medications and dietary advices. despite that, adherence to such recommendations which involve lifestyle changes are frequently associated with significant difficulties for hd patients and the non-adherence rate to medications and lifestyle changes is highly recognized as a big problem in dialysis. the recorded prevalence of non-adherence among hd patients varies widely, ranging from 22%-74% (9, 10). non-adherence to hd results in many metabolic and cardiovascular disorders. additionally, the risk of hospitalization was found to be increased due to non-adherence to diet, fluid, and medications treatment (4). as a result, non-adherence is associated with increased mortality and healthcare-resource utilization and decreased in quality of life (qol) of patients undergoing hd (9). in contrast, enhanced treatment adherence has been linked with better outcomes (11). in iraq, patients with esrd are mostly treated by chronic hd in governmental hospitals. assessment of adherence rate for hd patients will give the healthcare providers the opportunity to implement interventional actions to minimize economic and health consequences related to nonadherence. the aim of the current study was to assess adherence, perception, and counseling among hd patients regarding different treatment modalities (fluid restriction, dietary recommendations, medications, and hd schedules). patients and methods patients the current study was a cross-sectional study carried out on already diagnosed esrd patients on hd, who attended to the dialysis centers at alkarama teaching hospital and madinat al imamain alkadhimain teaching hospital, from november 2020 to january 2021. inclusion criteria the inclusion criteria of the study were: 1-patients with esrd who were aged 18 years and above of both genders and willing to participate in the study. 2-patients should be on regular hd for at least 6 months. exclusion criteria patients who provide incomplete information were excluded from the current study. data collection demographic characteristics were collected by using a data sheet that includes age, gender, social status, educational level, residency, occupation, body mass index (kg/m2), monthly income, smoking and alcoholic status, medications used, and the number and type of comorbidities. in addition, the arabic version of the esrdadherence questionnaire (esrd-aq) had been used to assess adherence. the questionnaire items distributed into five sections: the first section contained general and disease related information while the remaining four sections measures adherence to hd sessions, adherence to dietary recommendations, adherence to medications, and adherence to fluid restriction. these questions were scored and response of patients to these questions was summed to calculate the adherence behavior subscale. according to esrd-aq, higher scores means higher adherence to the measured behavior. the esrd-aq total score was categorized into iraqi j pharm sci, vol.31(1) 2022 treatment modalities among patients on maintenance 97 three levels: poor (< 700), moderate (700-999) or good adherence (1000-1200) (12). ethical approval the research proposal which explains the objectives behind the current study and the intended methods for collecting data was administered to the college of pharmacy, university of baghdad and the approval was obtained from scientific and ethical committee. in addition, approval was obtained from the iraqi ministry of health. while approval from the patients to participate in the study was obtained verbally. administration of the questionnaires when the patients arrived to the dialysis center for hd sessions, they were interviewed and asked if they agree to participate in the current study, if they agreed, then a clarification of the questionnaire’s questions were given while the patients were filling the research questionnaire till it had been filled completely. statistical analysis descriptive statistics (means, standard deviation, frequencies and percentages) were conducted for all study items. data was analyzed using statistical package for the social sciences (spss) software version 25. non-parametric tests were used due to not normal distribution of the included variables. the multiple regression analysis was conducted to measure the relationships between the independent variables (personal characteristics and duration of analysis) and the outcome variable (total adherence score). p-value of less than 0.05 was considered statistically significant. results the number of recruited patients was 200 adult hd patients (113 men and 87 women). the average age of the patients was 50.4 ±15.0 years. most (85.5%) of the participants had secondary school or lower education. more than two-thirds were unemployed and had low-income (˂0.5 million iraqi dinars monthly). more than three-quarters (79.0%) lived in urban areas (table 1). the average of the total score lies within the moderate adherence (984.9 ±174.2). the findings show that patients adherence to the hemodialysis session was good as representing by high scores of adherence for the first three items (hd–attendance, episode of hd-shortening and duration of hd-shortening if shortened). in contrast, the lowest reported adherence score (126.0 out of 200) was for following the fluid restriction recommendations (table 2). in terms of adherence categories, the majority (61%) of the patients had good adherence to their management regimens. however, 33.0 had moderate adherence and 6% had poor adherence (table 3). table 1. socio-demographic and clinical characteristics of patients mean ± sd or number of patients (%) age (mean ± sd) 50.41± 15.01 gender male 113 (56.5) female 87 (43.5) social status single 32 (16.0) married 133 (66.5) divorced 10 (5.0) widowed 25 (12.5) education level illiterate 28 (14.0) primary school 70 (35.0) secondary school 73 (36.5) university 29 (14.5) living place urban 158 (79.0) rural 42 (21.0) profession employed 57 (28.5) unemployed 143 (71.5) income (million iraqi dinars) ˂0.5 133 (66.5) 0.5-1.0 50 (25.0) ˃1.0 17 (8.5) duration of dialysis (mean ± sd) years 3.42 ± 2.58 total medications (mean ± sd) 8.09 ± 2.69 total comorbidities (mean ± sd) 1.88 ± 1.08 iraqi j pharm sci, vol.31(1) 2022 treatment modalities among patients on maintenance 98 table 2. mean (± sd) of adherence score for various treatment modalities. adherence item range of score mean (sd) hd– attendance “ during the last month, how many dialysis treatments did you miss completely? ” 0-300 260.00 (79.89) episode of shortening hd “ during the last month, how many times have you shortened your dialysis time? ” 0-200 185.00 (44.27) duration of shortening hd if shortened “during the last month, when your dialysis treatment was shortened, what was the average number of minutes ? ” 0-100 90.13 (26.51) adherence to medication “ during the past week, how often have you missed your prescribed medicines? ” 0-200 181.50 (35.55) adherence to fluid restriction “ during the past week, how often have you followed the fluid restriction recommendations? ” 0-200 126.00 (71.23) adherence to dietary restriction “ during the past week, how many times have you followed the diet recommendations? ” 0-200 142.25 (65.18) total adherence score 0-1200 984.88 (174.20) total score=summation of the 6 questions.. table 3. the adherence categories according to the esrd-adherence questionnaire adherence categories frequency percent good 122 61.0 moderate 66 33.0 poor 12 6.0 total 200 100.0 poor adherence=< 700; moderate adherence =700-999; good adherence =1000-1200. all patients perceived their hemodialysis management as highly/very important. similarly, almost all (96%) of patients perceived their adherence to medicines as high/very important. on the other hand, some patients perceived their adherence to the recommended diet as moderately (7%) or less important (7%). similarly, fluid intake restriction was less popular compared to the adherence to hemodialysis and medicines since 13% perceived limiting fluids as moderate important and 2% perceived it as with little or no importance. the highest score (4.82± 0.39) of perceive importance was for the adherence to hemodialysis while the lowest score was for limiting fluids intake (table 4). table 4.the importance perceptions of following the recommended dialysis, medicines, fluid and food. items highly/very important n (%) moderately important n (%) little/not important n (%) mean of score (sd) “ how important do you think it is to follow your dialysis schedule? ” 200 (100%) 0.0 (0.0%) 0.0 (0.0%) 4.82 (0.39) “ how important do you think it is to take your medicines as scheduled? ” 192 (96%) 8 (4%) 0.0 (0.0%) 4.74 (0.53) “ how important do you think it is to limit your fluid intake? ” 170 (85%) 26 (13%) 4 (2%) 4.38 (0.82) “ how important do you think it is important for you to watch your diet daily? ” 172 (86%) 14 (7%) 14 (7%) 4.51 (1.04) total perception of therapy importance score 18.44 ± (1.71) highly important=5; very important =4; moderately important =3; little important=2; not important =1. age out of eight characteristics (independent variables) had significant (p-value< 0.05) positive association with the total score of management adherence (outcome variable) according to the multiple linear regression. that means when the patient age increase, the total management adherence score increases as well (table 5). iraqi j pharm sci, vol.31(1) 2022 treatment modalities among patients on maintenance 99 table 5. multiple linear regression of factors influencing the total score of management adherence predictor (independent variable) standardized coefficients pvalue beta age (years) 0.208 0.016* gender -0.075 0.321 social status -0.032 0.688 education level -0.113 0.161 living place -0.057 0.435 profession 0.030 0.715 total medications 0.037 0.616 total comorbidities -0.124 0.104 dependent variable: total adherence score. r square=0.164. *significant at the 0.05 level. four items in the esrd-aq were about frequency of counseling received by patients for different treatment modalities (table 6). the negative answers were “never, rarely, irregularly or only when there are an abnormal test results”. collectively, these negative answers represented 58.5% of answers concerning how importance is to follow a proper diet, 54.5% about the importance of dialysis treatment, 41.0 % about the importance of fluid restriction and 25.0% about the importance of taking their medications as prescribed. table 6. frequency of counseling received by patients for different treatment modalities items every dialysis treatment n (%) every week n (%) every month n (%) every 2 to 3 months n (%) every 4 to 6 months n (%) when i have abnormal blood or other test results n (%) rarely n (%) irregularly n (%) never n (%) other (specify) n (%) a 8 (4) 14 (7) 56 (28) 9 (4.5) 3 (1.5) 40 (20) 33 (16.5) 12 (6) 24 (12) 1 (0.5) b 7 (3.5) 16 (8) 108 (54) 17 (8.5) 2 (1) 24 (12) 12 (6) 10 (5) 4 (2) c 18 (9) 16 (8) 68 (34) 14 (7) 1 (0.5) 39 (19.5) 16 (8) 12 (6) 15 (7.5) 1 (0.5) d 3 (1.5) 7 (3.5) 60 (30) 11 (5.5) 2 (1) 69 (34.5) 27 (13.5) 9 (4.5) 12 (6) item a: “ how often does a medical professional (your doctor, nurse, dietician, or other medical staff) talk to you about the importance of staying for the entire dialysis time during your dialysis treatment? ” item b: “ how often does a medical professional (your doctor, nurse, dietician or other medical staff) talk to you about the importance of taking medicines as ordered? ” item c: “ how often does a medical professional (your doctor, nurse, dietician or other medical staff) talk to you about the importance of fluid restriction? ” item d: “ how often does a medical professional (your doctor, nurse, dietician or other medical staff) talk to you about the importance of following a proper diet? ” discussion to manage the esrd successfully, hd patients should be adherent for many all aspects of their treatment including complete attendance to hd, adherence to the prescribed medications, fluid restrictions, and dietary advices (13). in this study, adherence behaviors of esrd patients on chronic hd were measured and analyzed. based on demographic results of the current study (table 1), the majority of hd patients had low socioeconomic status, as demonstrated by the high unemployment level (71.5%), low monthly income (66.5% had less than 0.5 million iraqi dinars), and low educational levels. comparable findings were found in a study conducted in saudi arabia in which high levels of unemployment (66.20%), low monthly incomes (72.85%), and low educational levels were found among hd patients at governmental kidney centers (14). the esrd-aq which was used in the current study is the first tool to measure all components of adherence behaviors of esrd patients and it is reliable, valid and easy to administer (15). relatively high scores of adherence for (hd–attendance, episode of hd-shortening and duration of hdshortening if shortened). in contrast, the lowest reported adherence score (126.0 out of 200) and (142.25out of 200) were for following the fluid restriction and dietary recommendations, respectively (table 2). generally, adherence to different hd treatment modalities was less than optimum with 39.0% of hd patients had an overall score corresponding to moderate/or poor adherence (table 3). this finding is comparable to a study conducted in palestine and found about 45.0% of hd patients had these two suboptimum adherence levels (12). adherence to dietary advices and fluid restriction is very important for success of treatment (13). nonadherence may increases complication rates, healthcare costs, and decreased survival (16-18). the iraqi j pharm sci, vol.31(1) 2022 treatment modalities among patients on maintenance 100 findings that hd patients are more adherent to dialysis than dietary or fluid restrictions are similar to previous studies (19-21). this may be related to the need for higher motivations and more appropriate skills and knowledge to follow dietary and fluid advice. in addition, the lowest reported adherence score for following the fluid restriction and dietary recommendations is supported and may be partially explained by the finding that perception of importance of following the fluid and dietary recommendations were the lower compared to perceptions toward adherence to medications and hd sessions (table 4). age out of eight characteristics (independent variables) had significant (p-value< 0.05) positive association with the total score of management adherence (outcome variable) according to the multiple linear regressions. that means when the patient age increase, the total management adherence score increases as well (table 5). other previous studies have also showed that older age was associated with more adherence to fluid restriction and prescribed medications (19, 22, 23). furthermore, it has also been reported that dietary adherence of dialysis patients improves with older age,19,20 and the odds of missing at least one dialysis session in a month were higher in patients aged <55 years (24). possible explanations may be that the younger patients may perceive themselves as less liable for the negative health consequences while, on the other hand; older patients may possess more organized lifestyle that accommodates different treatment regimen demands (25), confirming the occurrence of an ‘‘intentional non-adherence’’ (26). the finding that young patients are more likely to be non-adherent to treatment may be associated with poorer qol in the future and higher mortality rates among these hd patients. in the current study, marital status and level of education were not found to be a detrimental factors for adherence. in contrast, other studies have found a positive role for marital status (8, 27) and education (7) on level of adherence. regarding the frequency of counseling received by patients for different treatment modalities. the negative answers were less than optimum for all treatment modalities with negative answers ranging from 25.058.5% (table 6). it seems that patients’ counseling are important in building patients’ general perception for various treatment modalities which may significantly affect their adherence. the current study had a few limitations. the first limitation was incorporating hd patients from only two centers in baghdad city, so the data did not fully represent all iraqi hd patients. second limitation was the self-reported design of the questionnaire used in the current study. studies showed that there may be a disagreement between the actual adherence and the self-reported adherence (28,29). the third limitation was its cross-sectional nature. a longitudinal study design might be better suited to establish causal relationships and would help to investigate changes of over time. conclusions the current study showed that the overall adherence of hd patients to different treatment modalities was less than optimum with fluid and diet adherence representing the most challenging aspects in the health care of hemodialysis patients. finally, older patients had higher odds of being more adherent. references 1. genevra 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14(5): 324–327. 26. hussey lc, gilliland k. compliance, low literacy, and locus of control. nursing clinics of north america. 1989; 24(3): 605–611. 27. sabi k., noto-kadou-kaza b., amekoudi y., tsevi m., sylla f., et al. medication adherence of 65 patients in hemodialysis in togo. me´decine et sante´ tropicales. 2014 ; 24 : 172176. 28. katherine k., maria v., benjamin a., susan e., anne w., vicki h., et al. the agreement of patient-reported versus observed medication adherence in type 2 diabetes mellitus (t2dm). bmj open diabetes research and care. 2016;4(1): 1-5. 29. harsha t., nalyn s., rachel c., cristian p., james p., john e., et al. differences between self-reported and electronically monitored adherence among patients receiving antiretroviral therapy in a resource limited setting. aids. 2012;26(18):2399–403. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate doi: https://doi.org/10.31351/vol28iss1pp113-123 113 synthesis and preliminary anticancer evaluation of 6-mercaptopurine – methotrexate conjugate as possible mutual prodrug asmaa s. al-darraji*,1 and mohamed h.mohamed** *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq abstract small molecules drug conjugate mutual prodrug design (smdc) composed of folate and lethal agent conjugate, rigidly bonded via hydrophilic bridge and self immolative disulfide bond ; represent new interesting approaches for cancer treatment , the component of smdc intended for targeting folate receptor , along with greater conservation of component until reaching the target tumor tissue . the designing and synthesis of compound vi and viii derived from 6-mercaptopurine (6-mp) and methotrexate ( mtx) conjugate altogether as mutual prodrugs were processed forward successfully by multistep reaction procedures , and by thin layer chromatography (tlc) for preliminary detection of products and their intermediates, along with their purity. the structures of two final compounds and their intermediates were proclaimed by melting point measurement, infrared spectrometry and ¹hnmr analysis given results greatly correspond with theoretical proposed chemical structure of synthesized compounds. furthermore, cytotoxic activity evaluation on cell line level had been done for two final compounds against human breast tumor cell (mcf-7) and human ovarian tumor cell (sko-3) types of cancer cell line and the results were confirmed which show greater cytotoxic tumor activity of two final compounds, while compound vi possess optimal activity proportional with increased number of 6 -mp molecules. keywords: smdc, 6mp, methotrexate, cytotoxic activity. ميركابتوبيورين والميثوتركسسيت كمقدمات -6من تصنيع وتقييم مضاد سرطاني اولي لمدمج دوائي دوائيه محتمله **محمد و محمد حسن 1*،اسماء صفوان الدراجي . فرع الكيمياء الصيدالنية، كلية الصيدلة، جامعة بغداد، بغداد، العراق* الخالصه طان بقوه الفوليت والماده القاتله ويرتب اقتران من يتكون ثنائية فعالية ذو ان تصميم المقترن الدوائي ذو الجزيئات الصغيره كمقدم دوائي الستهداف مهياة( smdcلعالج السرطان. مكونات )لالهتمام ومثيرة جديدة مناهجتمثل عن طريق جسر محب للماء ،وثنائي السلفايد الذاتيه التحلل ؛ السرطانيه المقصوده .مع الحفاظ وبقوه لمكوناتها الى ان تصل الى االنسجه الفوليت مستقبالت نجاحبميركابتوبيورين والميثوتركسيت معا كمقدمات دوائيه ذات فعاليه ثنائيه قد تم -6المشتقين من اقتران viii و vi تصميم وتصنيع المركبين تركيب , الرقيقة الطبقة كروماتوغرافيا بواسطة المركبات نقاوة من والتاكد التفاعالت جميع مراقبة وتم, الخطوات متعدد التفاعل طرق باتباع المغناطيسي النووي الرنين وتحليل, الحمراء تحت لالشعة الطيفي التحليل, االنصهار درجة بقياس اثباته تم الوسطية مركباتهم مع النهائيين المركبين للبروتون النهائيين للمركبين ويالخل السمي التاثير تقييم تم ات المصنعه ،واعطت نتائج تنطبق بصوره كبيره مع التركيب الكيميائي المفترض نظريا للمركب لنهائيين اضد خاليا سرطان الثدي وخاليا المبيض السرطانيه وتم تثبيت النتائج التي اظهرت درجه عاليه من السميه ضد الخاليا السرطانيه للمركبين ميركابتوبيورين . -6الفعاليه المثاليه التي تتناسب مع زياده عدد جزيئات vi،بينما يمتلك المركب الفعاليه القاتله للخاليا السرطانيه .، الميثوتركسييت ،ميركابتوبيورين smdc ،6) ) الكلمات المفتاحيه : introduction 6-mercaptopurine, is thiopurine related structurally and belong to antimetabolite group of medication(1) ,it has been discovered in 1948 , published and approved as suitable for human medical use in 1953(2), followed by announcement that it is the safest and relevant effective medication for public health(3). 6-mp possess limited spectrum against tumor cell, for instance , the use of this medication limited for the treatment of lymphocytic leukemia during acute phase , chronic myeloid leukemia(4), other use apart from cancer treatment when use concomitantly with other medication for treatment auto-immune disease such as crohn's disease (5)and other inflammatory complex disease such as ulcerative colitis (6),specifically 6-mercaptopurine and methotrexate use together during maintains phase of child nonhodgkin leukemia as the most successful combination for treatment with high curative rates(7) . 1corresponding author e-mail: asmaasafwan2016@gmail.com received:14 /11/2018 accepted:18 /2/2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp113-123 mailto:asmaasafwan2016@gmail.com iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate 114 probably 6-mp possesses many disadvantages that make its use highly confused; first pharmacokinetic consideration , include weak water solubility , poor oral absorption ,also subsequent lower bioavailability , shorthalf -life less than 1 hour , perhaps slower onset of action ; effect not seen furthest than 3-6 months after initiation of therapy , also in liver convert to inactive metabolite 6thiouric acid which may revised the balance between the active and inactive metabolite(8) , 6-mp associated with serious life-threatening myelosuppression, if untreated, may lead to chronic bone marrow failure exacerbate to secondary cancer(9) , also cancer cell shows characteristic rapid resistance shortly after initiation of therapy.(10) prodrug design is chemical modification of parent drug, designed specifically to enhance pharmacokinetic profile and/or to achieve sitespecific drug delivery(11), the pharmacological activities of parent drug residue are sufficiently retained and recovered only either by enzymatic/non-enzymatic biotransformation.(12,13)6mp find to have greater application in practice, once for improving its water solubility combined with characteristic stability and rapid releasing capability by combining this medication with hyaluronic acid via carbonylvinyl sulfide bridges (14) and other achieving targeting action by concentrate drug in one body compartment much greater than the other such as cis-3-(9h-purin-6-ylthio)acrylic acid (pta) prodrug targeting tumors with up-regulated gsh levels, and b, s-(9h-purin-6-yl)-l-cysteine are ( prodrug selectively kidney delivered , concentrate 6-mp probably approximate 2.3 to 90 fold higher than plasma and liver ) (15) prodrugs of the 6mercaptopurine (6-mp) antitumor agent both need glutathione mediated metabolic activation (16) , also another two 6mp derivative 6-[5-pyridine -4-yl1, 2, 3, 4-oxadiazole -2-(yl) dithiol]-9h-purine and 9h-purine -6-yl-benzyl dithio carbamate, both possess cytotoxicity level much higher than 6mp, potentiates high inhibitory cytotoxic capability against renal cell and ovarian cell cancer(17). folate receptor (cancer cell biomarker) (18) , frα and frβ antagonist show invaluable mean in cancer treatment since these receptor show efficient up regulation in tumor tissues compared with normal tissues ,thus potentiate tumor metastasis , also play a role in cancer development ,and progression(19) methotrexate, belong to the antimetabolite class of medication of antifolate type and effective chemotherapeutic agent for diverse cancer treatment, closely resemble folic acid structurally property rendering mtx as effective competitive inhibitor for folate as enzymatic co-factor for cellular vitality (20, 21). the work focused on the synthesis of two mutual prodrugs as (smdc) with molecular weight not exceed 1000dalton, both designed by conjugation of 6mp and mtx through viable enzymatically hydrolyzed amide and significant disulfide bonds. materials and methods most of chemicals for research need were supplied by (hyperchem company/ china); these chemicals include 6-mpand mtx, dicyclo hexyl carbodiinmide (dcc), and 1-hydroxy benzotriazole (hobt),while cysteine amino acid was supplied by (sinopharm/china), and 4-nitrophenylchloroformate from (tci /china), purity determination and reaction progress following up realized by thin-layer chromatography (tlc), it was prepared by running diverse mobile phase on stationary phase prepared from coating silica gel f-254(type60) on thin film of aluminum(chmlab group/ spain ).the final products and their intermediacy were revealed by attendant iodine vapor or simple irradiation with uv lamp(254nm), featuring melting points of final products and their intermediates were measured by capillary tube method, results accession on electric melting point instrument (stuart value / england ) , and they are uncorrected. infrared spectra were accomplished by (biotech / england) utilize kbr disc. ¹hnmr analysis performed on (nanalysis company / canada). synthesis of methyl cysteinate hydrochloride (compound i) (22) a solution of l-cysteine (41.26 mmol ,5g) in absolute methanol(50 ml), was prepared and cooled to -15 °c, then thionyl chloride (49.2 mmol,3.567 ml) was added drop wise over 10 minute with persistent stirring (the temperature was kept at 15°c) , this mixture was allowed to cool at room temperature before being transferred and refluxed for 3 hrs. excess solvent was removed under vacuum, then 60ml of diethyl ether was added, and allowed to stand in refrigerator overnight, the bulky product filtered to produce compound i,% yield= 98%, m.p(142°c), ir (kbr disc),(ʋ cmˉ¹): 33982511 (nh3+ str),2954(c-h) str. of ch3,2555(s-h) stretching,1743(c=o) carbonyl ester stretching ,1577 and 1516-1496(nh3+) salt a sym. and sym. bending, 1441 and1404a sym. and sym. (c -h)of ch3 bending, 1246and1205 (c=o-o)a sym. and sym. stretching , 1068 (c-n) stretching of aliphatic ester.¹hnmr (60mhz, dmsod6 ,δ=ppm): 1.4(1h,s,sh), 3.02(3h,s,ch3) ,2.83,2.67 (2h,q,ch2), 4.13,4.28(1h,d,ch), 8.75(2h,d,nh2). synthesis of 7h-purine-6-thiolate potassium salt (compound ii) (23) solution of 6 –mercaptopurine (32.5 mmol ,5g) in 300ml ethanol , and ethanolic solution of koh ( 32.5 mmol , 1.82g ) was added and stirred for half of an hour with heating followed by further half of an hour at room temperature , the result precipitate was filtered to give compound ii, iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate 115 %yield=92%,m.p=340°c, ir (kbr disc),(ʋ cmˉ¹):3431cmˉ¹(n-h) stretching of purine ,2931and 2900 cmˉ¹(c-h) a sym.and sym.stretching of imidazole, 1587-1331 cmˉ¹(c=c,c=n) ring stretching ,1331-1254 cmˉ¹(c-n) stretching of imidazole ,904-806 cmˉ¹(c-h) out of plane bending ,706-658 cmˉ¹(c=c) out of plane bending. synthesis of1-((7h-purin-6-yl)thio)pyrrolidine2,5-dione (compound iii) to cold solution of compound ii (10.51mmol ,2g) in 100 ml anhydrous dmf , a cold solution of n-bromo succinimide nbs (10.51mmol, 1.87 g) in 20 ml acetone was added with continuous stirring and cooling, the color change from green to orange with complete conversion into clear solution had observed , solution left in ice bath for 3 hours , followed by overnight stirring at room temperature ,kbr filtered out , cold water was added to filtrate ,the solution kept in refrigerator for 2 days ,precipitate filtered to give compound iii, % yield =65%, m.p=195°c, ir (kbr disc),(ʋ cmˉ¹):3464 cmˉ¹(n-h) stretching of purine, 3089and3053 cmˉ¹(c-h) a sym.andsym. stretching of imidazole ,2976 and 2821 cmˉ¹ (c-h) stretching of methylene of succinimide ,1649 cmˉ¹(c=o) stretching of imide, 1236 cmˉ¹(s-n) stretching ,924 cmˉ¹succinimde ring vibration .¹hnmr (60mhz, dmsod6 ,δ=ppm): 2.68,2.83(4h,d,ch2), 8.54 (1h,s,ch) pyrimidine, 8.61 (1h,s,ch) imidazole ,13.75(1h,s,nh). synthesis of methyl s-((7h-purin-6-yl) thio) cysteinate hydrochloride salt (compound iv)(24) mixture of compound i (4.04 mmol,0.68 g) and compound iii (4.04mmol,1.007gm) in 20 ml dimethylformamide ( dmf) was refluxed for 3 hours , the mixture then was stirred overnight at room temperature ,then 20ml cold water was added and the resulted precipitate was filtered to give compound iv, %yield=50%,m.p =270°c, ir (kbr disc),(ʋ cmˉ¹):3431 cmˉ¹(n-h) stretching of purine ,3093and3024 cmˉ¹ a sym. and sym. (c-h) stretching of imidazole ,2779 cmˉ¹ sym. stretching of cysteine methylene group ,1739 cmˉ¹(c=o) stretching of ester, 1664-1466 cmˉ¹(c=c,c=n) stretching pyrimidine ,1568 cmˉ¹(n-h) bending of cysteine methyl ester ,1377 cmˉ¹(c-h) bending of methyl of cysteine methyl ester, 1221 cmˉ¹(c=oo)stretching ,509 cmˉ¹(s-s) stretching .¹hnmr (60mhz, dmsod6 ,δ=ppm): 2.67(2h,ch2) of ester ,2.83(1h,ch) of ester,3.62(3h,ch3) of cysteine methyl ester ,8.14(1h,ch) pyrimidine ,8.31(1h,ch) ,13.69(1h,nh) imidazole . synthesis of 2-((4-(((4-(l2-azaneyl)-2-(((4nitrophenoxy) carbonyl) amino) pteridin-6-yl) methyl) (methyl) amino) phenyl) carbamoyl)pentanedioic acid (compound v) (25) a solution of para –nitrophenylchloroformate (2.2mmol,0.4434g) was added to cooled solution of methotrexate (1.1 mmol, 0.5g) and triethylamine ( tea) (2.2 mmol,0.306m) 10ml anhydrous dmf, the mixture was stirred at room temperature for1 hour, then 2equimolar 0.02m hcl added ,and iced-water added andthe resulted precipitate filtered to give compound v .% yield =73%, m.p=109°c, ir (kbr disc),(ʋ cmˉ¹): 3618-2400cmˉ¹(o-h) stretching of carboxyl , ,3116 and 3030 cmˉ¹(ch)stretching of phenyl rings of 4-npc and methotrexate respectively , 2966and2941 cmˉ¹(c-h) a sym. stretching of methyl andmethylene respectively ,1784 and 1662 cmˉ¹(c=o) overlapping of carbamate stretching and carboxylic acid ,1641,1601,1446 cmˉ¹(amide iandii,c=c,c=n)stretching,1520and1350(no2) asym. and sym. stretching respectively,1306(o-h) out of plane also(c-o) stretching appear at same region ,1227and1190 cmˉ¹( c=o-oando-cc)stretching .¹hnmr (60mhz, dmsod6 ,δ=ppm):1.13(2h,ch2 ) methylene β to amide, 2.68(2h,ch2)methylene δ to amide ,2.84 (1h,ch) methine α to amide ,3.17(3h,ch3)n-ch3 ,4.77(2h,ch2)methylene next to nch3,7.34,7.75,7.9,8.15 (8h,ch) methine of phenyl of 4-npc ,7.08 ,6.7 ,6.97 ,6.84(4h,ch) methine of benzene mtx, 8.27 (1h,nh) of amide ,8.58(1h,nh) of carbamate ,8.0(1h,ch)of petridine. synthesis of 5-methyl 1-(triethyl-l5-azaneyl) (4-( ( ( 2 (3-(3-((7h-purin-6-yl)disulfaneyl)-1-methoxy 1-oxopropan-2-yl)ureido)-4-(3-(3-((9h-purin-6-y l )disulfaneyl)-1-methoxy-1-oxopropan-2-yl) ureido ) pteridin – 6 yl ) methyl ) (methyl ) amino ) benzoyl)glutamate (compound vi)(26 ) a solution of compound iv( 1.25mmol,0.3847g) in 5ml anhydrous dmf was added to solution of compound v (0.625mmol,0.5g) and tea (1.875mmol.,0.2609ml) in 5ml dmf at room temperature, mixture was stirred at room temperature overnight, then 10 ml of distilled water was added, the resulted precipitate was filtered and dried to give compound vi ,%yield =50%, m.p=not measured compound as gel , ir (kbr disc),(ʋ cmˉ¹): 3400 cmˉ¹(n-h) stretching of purine and 2°-amide ,3677-2400 cmˉ¹(n-h)of triethylamine salt,2976and2939 cmˉ¹a sym. stretching of methyland methylene respectively ,1724 cmˉ¹ (c=o)stretching of ester,1641cmˉ¹ (c=o)stretching of amide and urea,1604 ,1543,1439 cmˉ¹(c=c)also (n-h)bending appear in same region , (c=n) appear in same region ,1336 cmˉ¹(ch2)twisting of cysteine ,1290 cmˉ¹(c-nh) stretching of purine iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate 116 ,1205,1173,1022 cmˉ¹(c=o-o,o-c-c) stretching of ester ,945-700 cmˉ¹(c-h)out of plane bending ,700509 cmˉ¹(n-h) out of plane . ¹hnmr (60mhz, dmsod6 ,δ=ppm): 1.14(9h,ch3)of triethylaminesalt,1.26 (6h,ch2),1.79 (2h,ch2 ) methylene βto mtx amide ,2.66 (2h, ch2 )δ to mtx amide , 2.77 1h,ch)αto mtx amide, 3.0 (3h,ch3 )of n-ch3 ,3.58(ch3,3h),3.82(ch3,3h) of o-ch3 ,4.47 (2h,ch2 ) methylene next to nch3,6.87(1h,ch)of benzene ring ,7.02 (1h,ch) of benzene ring ,7.97,7.91(1h,ch) of benzene ring ,8.12(1h,ch)of pteridine ,8.70 (2h,ch) of pyrimidine, 9.03(2h,ch) of imidazole,9.3 (1h,nh) of amide of mtx ,10.66 (1h,nh) of urea . synthesis of (s)-5-((1h-benzo[d][1,2,3]triazol-1yl)oxy)-2-(4-(((2,4-diaminopteridin-6yl)methyl)(methyl)amino)benzamido)-5oxopentanoic acid(compound vii)(27) solution of mtx (1.1mmole ,0.5g ) was prepared by dissolving of the defined weight of former in 10ml dmf ,solution then reserved at 0°cwith constant stirring ,tea (1.1mmole ,0.15ml) was dropped wise on previous mixture ,followed by dcc(1.1mmol,0.226g), 1-hobt(1.1mmol,0.148g) addition respectively .solution then allowed to stand overnight at0°c, followed by addition equimolar of 0.02m hcl then mixture of 30% acetone / diethyl ether was then added to precipitate dcu( dicyclo urea ) , filtrate volume diminished under vacuum to give compound vii. % yield =63%,m.p=78-82°c, ir (kbr disc),(ʋ cmˉ¹):3406-3207 cmˉ¹ (n-h) stretching and(c-h)stretching of phenyl ring ,30002500 cmˉ¹(o-h) free hydroxyl stretching,2929 cmˉ¹(c-h) a sym. stretching of methylene ,1720 cmˉ¹,(c=o) stretching of ester ,1657 cmˉ¹(c=o) of carboxylic acid , 1633 cmˉ¹ (c=o) of amide stretching ,1603,1556,1508 cmˉ¹ (c=n) overlap with (c=c)stretching , 1385 cmˉ¹(c-h)(o-h) bending ,1290-1252 cmˉ¹(c-n) stretching and(c-h) in plane bending . synthesis of triethyl-l4-azaneyl n2-(4-(((6,8diaminonaphthalen-2yl)methyl)(methyl)amino)benzoyl)-n5-(1methoxy-1-oxo-3-((4,5,6,7-tetrahydro-9h-purin-6yl)disulfaneyl)propan-2-yl)glutaminate (compoundviii)(28) compound vii (0.87mmol ,0.5g ) was dissolved in 10ml anhydrous dmf , then tea (2.61mmol ,0.36ml) was added carefully drop by drop to mixture ,then compound iv(0.87mmol,0.273g) was added to previous mixture , the latter had then transferred and reserved at 0°c for 3 days ,followed 2 days at 15°c,(*reaction followed up by tlc),initial precipitation of byproduct did by diethyl ether, then product precipitation by adding 120ml acetone and the resulted precipitate was filtered to give compoundviii,% yield =65%,m.p=104-110°c, ir (kbr disc),(ʋ cmˉ¹):3600-2150 cmˉ¹(n-h)stretching of ammonium salt ,3357and 3180 cmˉ¹(n-h) a sym.andsym. of amine ,3030 cmˉ¹(c-h)sym. stretching of benzene , 1664(c=o) stretching of secondary amide ,1549 cmˉ¹(c=o)stretching of carboxylate anion ,1643and1603(amide iandii)also (nh2)bending appear in same region ,1549 cmˉ¹ ,1508,1475,1446 cmˉ¹(c=c)stretching of benzene ,1398 cmˉ¹(c-h) methylene scissoring overlap with methyl scissoring also,(c=n) appear in same region ,1334 cmˉ¹(c-h) methylene twisting ,1254,1201(c=o-o)and(c-o-c)of ester stretching ,1078 cmˉ¹(c-n)stretching of aromatic amine ,1034and764 cmˉ¹(c-h) in plane and out of plane bending ,852and833 cmˉ¹(n-h) wagging ,704-606 cmˉ¹(n-h)amide out of plane bending .¹hnmr (60mhz, dmsod6 ,δ=ppm):1.14 (9h,ch3 )methyl of triethylamine mtx, 1.26 (6h,ch2) of tea ,2.45 (2h,ch2 ), 2.68(2h,ch2)methylene σ to amide , 2.8 (1h,ch) α to amide, 3.05 and 3.17(6h,ch3) nch3 and o-ch3,3.39 (2h,ch2)methylene next to n-ch3 ,6.83(1h,nh) of amide of mtx,7.63(1h,ch)of benzene ,7.77(1h,ch) of benzene ,8.12(1h,nh) of pyrimdine ,8.31(1h,ch)of imidazole ,8.54(1h,ch)of pteridine. cytotoxicity assay of compound vi, viii (29) the cytotoxicity were determined for both targeting mutual prodrug (vi,viii) by utilization mtt assay method on sko-3andmcf-7malignant cell ,and this study was performed in iraq biotech company .while the results was compared with 6mp as standard for our work ,the antitumor potency was dedicated on measurement of cell viability (mtt)assay, cell line were labeled on 1x104cells/well and by using 96 wells plates andafter 24hrs , compounds with their consecutive standard tested at different conc. , 72hrs time need for close contact between cell and samples before viability measurement ,then their medium replace by 28µl of 2 mg/ml solution of mtt and incubation for furthest 2.5hrs at 37°c, then mtt solution removed ,while the rest crystals collected and incubated after solubilization in dmso for quarter of an hr at 37°c with stirring. absorbency was determined on micro plate's reader (492nm), and percent of cytotoxicity calculated as in following equation cytotoxicity% = (a-b/a)*100 where a, b are the optical density of control and the optical density of test respectively. (30) half maximal inhibitory concentration (ic50) the quantitive values represent in molar concentration of drug need to inhibit intended biological process by half.(31) the ic50 of standard and compound vi and viii against mcf-7 and sko-3 all mentioned in tables (1) and (2). iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate 117 results and discussion chemistry the synthesis of compound i is a process of protection of cysteine amino acid, and this is done by esterification on carboxyl group ,activation initially by thionyl chloride (socl2) , these in the presence of anhydrous methanol lead to the substitution of methyl moiety on carboxyl and (compound i) produce. next step is to enhance of nucleophilicity of thiol group of 6-mp and these criteria had been achieved by converting of thiol group primarily into its corresponding potassium salt ,followed by thiolate substitution on electrophile n-bromosuccinimde ,and (compound iii) liberate .then compound i react with compound iii at neutral ph and in slow rate reaction process to produce compound iv. in other hand, mtx is protected on its two 1°amine group by carbamate formation by reaction of mtx in anhydrous dmf with 2equimolar of 4-nitrophenylchloroformate to produce compound v, same dry condition in same solvent required for compound vi synthesis by reaction of compounds v and iv with stirring at 25°c for 24 hrs. on other hand, mtx protected on its γ carboxyl and again in anhydrous dmf by reacting it with 1-hydroxbenzotriazole, process by carbodiimide coupling and compound vii produce. finally, to produce compound viii reaction of compounds ivand vii take place in condition in which reaction retained at lower temperature and in dry medium. scheme (1 ) synthesis of compounds (i-iv) iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate 118 scheme ( 2 ) synthesis of mutual prodrug vi. iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate 119 scheme (3) synthesis of mutual prodrug viii cytotoxic activity cytotoxic assay of the tested compound against mcf-7 type of tumor cell herein , % of cytotoxicity,ic50% results of standard and the two synthesized vi,viii were shown in figure 1, while statistical data of them shown in table1. figure (1) cytotoxic activity of 6mp, compound vi, viii against mcf-7 cell line. iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate 120 table ( 1) cytotoxic activity of 6-mp, compound viii and compound vi against mcf-7 cancer cell (*mean ±sem is mean and standard error of mean, p value is probability value, ns: non-significant value, **** significant value) cytotoxic assay of the tested compounds against sko-3 type of tumor cell % of cytotoxicity, ic50% results of standard and the two synthesized vi, viii were shown in figure 2, while statistical data of them shown in table 2. figure (2) anti-proliferative activity of 6mp, compound vi, viii against sko-3 cell line compound number ic50% inhibitory concentration µg/ml cytotoxicity mean ± sem p value 6-mp 29.72 6.25 (µg/ml) 25.67 ±2.404 ns 12.5(µg/ml) 40.00±1.732 <0.0001(****) 25(µg/ml) 46.67±2.333 <0.0001(****) 50(µg/ml) 52.33±2.848 <0.0001(****) 100(µg/ml) 58.67±1.85 <0.0001(****) compound viii 13.51 6.25 (µg/ml) 34.67±1.202 <0.0001(****) 12.5(µg/ml) 49.67±2.404 <0.0001(****) 25(µg/ml) 62.67±2.028 <0.0001(****) 50(µg/ml) 69.33±2.333 <0.0001(****) 100(µg/ml) 75.67±2.333 <0.0001(****) compound vi 10.44 6.25 (µg/ml) 41.00±1.528 <0.0001(****) 12.5(µg/ml) 55.67±2.333 <0.0001(****) 25(µg/ml) 70.00±1.732 <0.0001(****) 50(µg/ml) 77.67±1.202 <0.0001(****) 100(µg/ml) 87.00±1.155 <0.0001(****) iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate conjugate 121 table (2) statistical results of cytotoxic activity of 6-mp, compound viii and compound vi against sko3 cancer cell (mean ±sem is mean and standard error of mean, p value is probability value, ns: nonsignificant value, **** significant value) the results of cell line study against mcf7andsko-3 malignant cell show that compound vi possess the optimal inhibitory activity compared to compound viii and this activity is at optimal value versus mcf-7 compared to sko-3types of cancer cell, while compound viii have moderate potency against same cancer cell mentioned earlier, and both synthesized compounds have anticancer potency much higher than standard 6-mp. conclusion two new mutual prodrugs were synthesized, both by conjugation of 6-mp and mtx through viable amide bond and reversible disulfide bond on relatively different positions relative to mtx molecules. these compounds were also evaluated on cell line level and results compared with standard and revealed that these prodrugs possess cytotoxic concentration against selected cancer cell much higher compared to standard. ir, ¹hnmr analysis results gave certainty that required compounds were obtained, while in vitro preliminary cytotoxic activity evaluation by mtt assay performance leads to conclusion that synthesized compounds have maximal potency on cancer cell compared to standard at much lower dose. lead in conclusion that lower doses of two prodrugs may give same or evenly higher response compared to standard drug. compound number ic50% inhibitory concentration µg/ml cytotoxicity mean ± sem p value 6-mp 41.14 6.25 (µg/ml) 21.07 ±1.453 ns 12.5(µg/ml) 38.33± 0.8819 <0.0001(****) 25(µg/ml) 42.33 ±1.453 <0.0001(****) 50(µg/ml) 49.33± 3.180 <0.0001(****) 100(µg/ml) 57.00± 3.51 <0.0001(****) compound viii 16.23 6.25 (µg/ml) 32.00± 2.082 <0.0001(****) 12.5(µg/ml) 47.00± 1.732 <0.0001(****) 25(µg/ml) 60.33 ±2.603 <0.0001(****) 50(µg/ml) 64.33± 3.283 <0.0001(****) 100(µg/ml) 73.67 ±2.028 <0.0001(****) compound vi 11.17 6.25 (µg/ml) 37.33± 1.453 <0.0001(****) 12.5(µg/ml) 54.33 ±2.603 <0.0001(****) 25(µg/ml) 67.00± 1.528 <0.0001(****) 50(µg/ml) 72.33 ±1.764 <0.0001(****) 100(µg/ml) 81.00 ±2.082 <0.0001(****) iraqi j pharm sci, vol.28(1) 2019 6-mercaptopurine –methotrexate 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release (sr) strips of sodium montelukast smlt , which is selective leukotriene antagonist , used for patients suffered from mid-night asthma , were prepared successfully ,using different polymers, like guar gum , carrageenan , and xanthan gum , by solvent casting method . the results obtained by this study revealed ,that best fast dissolving film of smlt was loaded in carrageenan polymer 57% w\w (30mg.) , with acceptable physical properties, like film thickness , elastic endurance and surface ph . besides to that , the disintegration time , and cumulative 80% drug release were estimated 22 seconds and 3.7 minutes , respectively .on the other hand , the sustained release film ( fsr6-7 ) as a second layer , appear to be the most promised layer of smlt loaded in 50 % w\w (15mg.) pvp k17 , with respect to its film thickness, elastic endurance and , surface ph . moreover the , the disintegration time of the second layer film was 21seconds , and the time for 50% drug release was 15 minutes extended for 4hours of 100% drug release . meanwhile , the investigation of possible interference between the drug , and, polymers used revealed no evidence of this effect ,using ftir , and sem technique . in an attempt to evaluate the extended release effect of the selected formula , singulair® plain tablet as a marketed product was used , the result gave more than 4 hours of selected formula , compared with 30 minutes to marketed product singulair® . the overall results candidates the selected fsr6-7 formula as a promised bi layer buccal fast , as compliance and extended therapy for asthmatic midnight patients. keywords: montelukast sodium, dual buccal strips, guar gum, xanthan gum, carrageenan. جحضيروجشخيص المووحيلوكبسث صوديوم كشرائح فموية مزدوجة بطيئة الححرر خليل يحيى اسمبعيل ،*1 * .نعشاق،ابغذاد،خبيعت بغذاد ،كهٛت أنصٛذنت ،فشع أنصٛذالَٛبث الخالصة نهٛكٕحشاٍُٚٛ, ٔانًغخعًم نًشضٗ انششائر انفًٕٚت انًضدٔخت بطٛئت انخسشس نهًَٕخٛهٕكبعج ٔانز٘ ٚعخبش انًضبد ٔانًخخص كصًغت انكٕاس,ٔانكبسخُٛبٌ ٔانضاَثٍٛ. بأعخعًبل طشٚقت انقشظ انًزٚب . انشبٕ انهٛهٙ قذ زضش بُدبذ بأعخعًبل بٕنًٛشاث يخخهفت أٌ انُخبئح انًغخخهصت فٙ ْزِ انذساعت أظٓشث بشكم افضم ششٚست عشٚعت انزٔببٌ نهًَٕخٛهٕكبعج يسًهت فٙ بٕنًٛش م ٔاالط انٓٛذسٔخُٛٙ انغطسٙ .يقبٕنت,يثم عًك انششٚست , يطبطٛت انخسً يهغى. بصفبث فٛضٚبٔٚت03ٔصٌ (\% ) ٔص75ٌانكبسخُٛبٌ .دقٛقت عهٗ انخٕانٙ 5.5ثبَٛت ,22% يٍ انعقبس قذ بهغج 03أضبفت انٗ رانك فأٌ ٔقج أَسالل ٔحسشس ظٓشث االكثش ٔاعذة نهًَٕخٛهٕكبعج بخسًٛهٓب ،كطبقت ثبَٛت 5-6ٔيٍ انُبزٛت االخشٖ فأٌ انششٚست بطٛئت انخسشس ف ط س َغبت انٗ عًك انششٚست , يطبطٛت انخسًم ٔاألط انٓٛذسٔخُٛٙ بٕنًٛش انبٕنٙ فُٛم ببٚشٔنٛذٌٔ يهغى. ف57ٙٔصٌ ( \% ) ٔص73ٌ انغطسٙ . دقٛقت ٔنفخشة 57يُّ فٙ % 73ثبَٛت, ٔٔقج حسشس انعقبس ل 25عالٔة عهٗ رانك فأٌ ٔقج اَسالل انطبقت انثبَٛت قذ بهغ أيكبَٛت انخذاخم بٍٛ انبٕنًٛش انًغخخذو ٔانعقبس , نى ٚظٓش ا٘ أشبسة انٗ ْزا % .كًب أ533ٌعبعبث نُغبت حسشس 4صيُٛت أيخذ ث انٗ ٔفٙ يسبٔنت نخقٛٛى حأثٛش انخسشس انطٕٚم انخأثٛش بأعخعًبل حكُهٕخٛب يطٛبف األشعت حسج انسًشاء , ٔحكُهٕخٛب انًغر االنكخشَٔٙ . عبعبث بفخشة 4َت, زٛث أظٓشث انُخبئح حسشس انعقبس عهٗ أيخذاد نهخشكٛبت انًخخبسة, فأٌ يغخسضش انغُكٕنٛشاندُٛظ قذ أعخخذو نهًقبس نًغخسضش انغُكٕنٛش اندُٛظ . دقٛقت 03 كخشكٛبت ٔاعذة نششائر فًٕٚت يضدٔخت ٔيالئًت نعالج يشضٗ انشبٕ انهٛهٙ . 5-6أٌ انُخبئح انكهٛت حظٓش انخشكٛبت ف ط س .الكبرجيىبن ، صمغة الزاوثيه، صمغة الكوار ، ، فموية مزدوجةشرائح ، المووحيلوكبسث صوديوم الكلمبت المفحبحية : 1 corresponding author e-mail: ybmmaz@yahoo.com received:17 /10/ 2015 accepted:17/11/2015 iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 62 introduction now a days the synchronized of asthmatic attach at night , became one of the worsening condition of respiratory system (1) , most of the patient suffering from this disease occurred during the mid-night hours and dusty bad weather , mainly for children and elderly (2) . so according to these findings , the drug therapy should be prescribed in a manner to result therapeutic steady state concentration at specific period (3) . consequently , the administration of the therapy prepared in two types of drug in a loading dose , and maintenance dose at bed time with a programmed drug release in early a hours of next day morning give a best effective therapy than the ordinary controlled delivery system (4) . montelukast sodium smlt is selective leukotriene antagonist used for the management and treatment of acute asthma and alveoli constriction in preparations formulated as a tablets, oral dispersible tablets in equivalent weight of 10 mg. per dose montelucast mlt as equivalent base of smlt salt derivative (5) . fast disintegrating buccal film is the most developed recent product of oro-buccal dosage form due to acceptability by the formulators. they can improve the activity of the medicines by dissolving within seconds in oral cavity by means of saliva presence without chewing and no need of water for administration. so it gives fast absorption and higher bioavailability of drugs due to local high blood flow in the buccal cavity materials and methods materials montelucast sodium ( gift from stada drug industry , vietnam ), guar gum, xanthan gum, carrageenan and are obtained by provizer pharma co. india, dimethyl phthalate , glycerin, ribose sugar obtained by sigma-aldrich co. usa, fructose , lactic acid ,polyvinypyrrolidone k17 , eudragit rs are given as a gift by al-sharq al-owset ,and orange flavor is from ( merck labs. ,germany ) ,the other remaining reagents and materials were of analytical grade , obtained by bdh chemicals ltd poole, england, gcc analytical reagents, uk, and fluka chemi ag, switzerland . methods preparation of rapidly dissolving layer table (1), illustrates that eighteen formulas as fast release layer were prepared using a modified solvent casting method (6) ; with each circular film surface area approximately 7.07 cm 2 is loaded with equivalent weight 5mg mlt.the amounts of polymers were weighed and dissolved in a beaker containing 10ml of distilled water maintained at 40°c overnight to ensure a uniform dispersion of different (w/v)% solutions. meanwhile the smlt and other excipients were dissolved in 10ml of distilled water in another beaker. the drug solution was added to the polymer solution and mixed using magnetic stirrer for two hour. the resulting solution was left for 30 minutes to remove all air bubbles entrapped, the resultant solution was cast onto 12 cm – diameter petri dish and dried in the oven at 40 o c for 24 hours. the film was slowly and carefully removed from the petri dish, and checked for any imperfections, cut into 3cm diameter circular films to gain the equivalent dose per strip (5mg.). preparation of sustained release layer six formulas as sustained release strips ( fsr1-fsr2 ) were prepared alone using spray technique , as a second layer on the surface of the optimized fast dissolving layer (f7), it was prepared by dissolving the smlt and polymers with other excipients in 10ml of ethanol 95 v\v % , then , and sprayed the solution via nozzle on to other side of the dried optimized formula of the fast dissolving layer in petri dish in a uniform distribution to permit fast drying without deterioration of the fast dissolving layer , then allowed to dry at a room temperature for 24 hours to ensure complete evaporation of all solvent traces (7) , the dried bi-layer films ( fsr-f7 ) for each fsr were carefully removed from the petri dish and cut into 3 cm in diameter circular films. samples were packed in an amber glass container until further analysis. each sample strip contain smlt 10.38mg , equivalent to 5 mg. mlt in each layer, and total weight 46 mg. ( 22mg. f7 and 24mg. fsr ) . physical cracterization avisual inspection properties such as homogeneity, color, transparency and surface of films were evaluated for all prepared oral films. (8) bweight variation the 7.07cm 2 strip of smlt was divided into equal four pieces in the cast film. and each film was taken and then differences in weight was observed. (10) cthickness measurements the thickness of each film was determined at eight different locations (two from each four corners) using vernier caliper micrometer. iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 63 table (1): composition of the sodium montelukast fast and sustained dissolving layers mechanical characterization to indicate a good elasticity , film strength of the cast was examined by measuring many parameters like modulus, strain, percent of elongation and the folding endurance .the films used for investigating the tensile properties were cut around a standard template (dumbbell) according to american society for testing and materials international test method for thin plastic sheeting. (9) the tensile properties of the films were evaluated by stretching the dumbbell-shaped sections to break using a universal testing machine. the breaking load in newton [n] and elongation percent were measured (11) . atensile strength (ts) evaluation of this type of oral strip films ,tensile strength is used, which is defined as a the maximum stress applied to a point at which the strip specimen breaks. it is calculated by the applied load at rupture divided by the cross-sectional area of the strip as given in equation below and was expressed in force per unit area: mega pascal (mpa) (12) . belongation at break (e) elongation was estimated by dividing the extension at the moment of rapture of the specimen by the initial gage length of specimen which represents hundred percent according to equation (1) below: %e = [(ls – l0) / l0] x 100 … equation 1 celastic modulus (em) this parameter is the measure of stiffness of the strip it was calculated as the slope of the linear portion of the stress – strain curve. the result was expressed in force per unit area (mpa) (12) . f/a = em [(ls – l0) / l0] … equation 2 where: f =breaking load (n) a = cross-sectional area of the sample em = is the modulus of elasticity. l0 = is the initial gage length of the specimen ls = is the length of the film after elongation. d strain strain has been used as an indicator of the overall mechanical quality of the film (12) ... equation 3 efolding endurance the endurance of each fold was measured manually for the prepared films. a strip (7.07cm 2 ) area of a film was cut and repeatedly folded at the same place till it broke; the number of times of the film could be folded at the same place without breaking gives the value of folding endurance. folding endurance more than 300 indicating that the formulation good tough and flexible (13). drug content uniformity four circular strips (3cm in diameter ) are placed in 100ml phosphate buffer ph 6.8 solution and kept on magnetic stirrer for 1hr iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 64 for first fast dissolving layer and 24hrs for the strips of two layers (fast dissolving layer with sustained release layer). solution was suitability diluted then the uv absorbance of the solution was measured at smlt λmax, and the drug content was determined (14) . measurement of surface ph the surface ph of the films was determined in order to investigate the possible irritation of buccal mucosa, the strip to be tested was moistened with 0.5ml of distilled water and kept for 1hr. the ph probe electrode must be in contact with the surface of formulation and allowing equilibrating for 1 minute and the average of triplicate measurements for each was indicted and reported (15) . disintegration test (16) in-vitro disintegration time 1drop method in this method one drop of distilled water was dropped by a pipette onto the oral films. therefore the films were placed on a glass slide and placed planar on a petri dish. the time until the film dissolved and caused a hole within film was measured. 2petri dish method two milliliters of distilled water was placed in petridish and one film was added on the surface of the water and the time required until the oral film was dissolved completely was measured. in-vitro drug release the profile of the smlt dissolution was performed according to determine the dissolution release profile of the prepared smlt oral strips . dissolution medium was 900ml of phosphate buffer ph 6.8 and( 0.1 n) hcl at 37 ± 0.5 o c with a rotation speed of (50) r.p.m. beside to that , the release profile of the singulair ® . tablet (as references) was also determined using 900ml of the same test environments. (10 ml ) of samples were taken at time intervals (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 and 30 minutes) for dissolution of fast dissolving layer while continue in sampling every 30 minutes reach to 4 hours for the strips contain the two layers (fast dissolving layer and sustained release layer) at 50 rpm with 900 ml volume of fresh 0.1 n hcl. both( 0.1n) hcl and phosphate buffer ph 6.8 were replenished after each sampling. samples were filtered through 0.5 µm membrane filter and the concentration of the dissolved smlt was determined using spectrophotometric technique at smlt λmax ( 340nm ) , the percent release of smlt from film was measured. the obtained results represent the mean of three findings . meanwhile the drug released period (t80%) and percentage drug dissolve in two minutes (d2min) were used for the first fast dissolving layer of the strip while the sustained and uniform releasing for the second sustained release layer. (17) compatibility studies a spectroscopy of the formula (ftir) samples are grinded and mixed with potassium bromide. the spectrum was obtained ( for all formulas ) between the wave number 4000-400 cm -1 . b-scanning electron microscope scanning electron microscope of the selected formula for both fast dissolving layer and sustained layer were confirmed by direct deposition of the film on double-side carbon tape and coated with gold, the sample visualized using scanning electron microscope operated with a secondary detector at different acceleration voltage and at different magnification. (17) . statistical analysis the anova statistical analysis were used, as (probability >0.05) insignificant results, and (probability < 0.05) ,as a significant results . results and discussions all the prepared fast dissolving films appeared homogenous, transparent, with faint light off white color , and smooth surface properties which they contain (guar gum, xanthan gum, and carrgeenan polymers ). the bi-layered formulas are smooth, show homogeneous, cloudy off white with formulas prepared by eudraget rs® and white with formulas prepared by pvp. (18) effect of different types of polymers formulas (f1-f9) in ( table 1) were used to study the effect of polymer type ( xanthan gum, guar gum and carrgeenan) and their weights (30 , 35 , and 40mg.) as the physical and mechanical properties of the prepared smlt fast dissolving layer. results showed in table (2) that the lowest disintegration time of the film forming polymer is gave by carrgeenan polymer f7 which have disintegration time (22sec.) and with good mechanical properties (folding endurance more than 300) and surface ph with in normal physiological mouth ph (6.6) range of oral cavity . films of guar gum (f4) and (f5) showed in-vitro disintegration time 29 seconds and 31 seconds respectively but these polymers give brittle films with weak folding endurance ( 1 fold ) , which is in a consistent iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 65 with results obtained by harsha et.al for tramadol (19) . while films of xanthan gum (f1f3) have good mechanical properties with good peeling off and good appearance , but with a long disintegration time (100127 seconds) , which may be attributed to the interaction of drug with dimethyl phthalate molecules which is enriched with multi hydroxyl groups that formed hard ester bonds, and the long disintegration time of this polymer due to the swelling property of this polymer which make a gel like layer on the surface of the film upon contact with aqueous media lead to prevent penetration of water to the film, this swelling property increased with increasing of the polymer concentration., which is similar to the dicyclomine drug (20) . also , the results in table (2) revealed different thickness of flake films depending, on the type and concentration of the,, film forming polymer used where the thickness of the film increased as the polymer concentration increased. at the same time for each polymer type where the thickness of film increased lead to delay the time required for, disintegration. a very low standard deviation value indicates the uniform thickness of the films. surface ph was found to be in the range (6.6 – 7.1) which close to salivary ph, which indicates that the films have less potential to irritate the oral mucosa, so they are comfortable films. table (2): physical and mechanical properties of the prepared smlt oral fast dissolving layer formula f1-f9 / values are presented as mean ± sd (n=3). effect of different concentrations of carrageenan polymer according to the physical and mechanical properties, carrageenan gum was selected as an optimized suitable polymer for prepare fast dissolving smlt layer with respect to its disintegration time , ph and mechanical strength .on the other hand , formulas (f8, and f9) of different carrageenan concentrations were further evaluated to get an optimum suitable polymer concentration by study their dissolution profile parameters.table (3) shows that the formula (f7) has the highest d2 min percent (39.24%) and the lowest t80% (3.7min) compared with formulas f8 and f9 , indicating that carrgeenan at this concentration gave lowest interaction between with smlt , allowing higher solubility to the drug , and the higher dissolution rate appear (21) as shown in figure (1). table (3): effect of carrageenan concentration on the in-vitro dissolution parameters of fast dissolving smlt layers in phosphate buffer ph 6.8. at 37 °c. formula no. t80% (min) d2 min (%) f7 3.7 39.24 f8 8.1 20.66 f9 9.7 13.76 drug content (%) surface ph folding endurance in-vitro dt (sec) film * thickness (mm) formulation code 99.4±0.55 6.9±0.001 300 100±0.032 0.077±0.0001 f1 x a n th a n g u m 94.6±0.54 6.8±0.002 300 110±0.043 0.092±0.0001 f2 94.4±0.53 6.8±0.002 300 127±0.041 0.99±0.0001 f3 99.1±0.52 7.1±0.001 1 29±0.035 0.035±0.0001 f4 g u a r g u m 94.8±0.51 6.8±0.003 1 31±0.039 0.043±0.0001 f5 98.8±0.54 6.5±0.001 1 55±0.058 0.048±0.0001 f6 98.7±0.54 6.6±0.002 300 22±0.031 0.039±0.0001 f7 c a r r g e e n a n g u m 93.5±0.52 6.8±0.001 300 43±0.034 0.042±0.0001 f8 91.1±0.52 6.9±0.002 300 69±0.030 0.046±0.0001 f9 iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 66 figure (1): effect of carrageenan polymer on the dissolution profile of the prepared smlt fast dissolving layer in ph 6.8 phosphate buffer at 37 o c. (results are expressed as mean, n=3). effect of combined plasticizers ratios table (4) shows the effect of changing the concentration of a dual combined dimethylpthalate – glycerin ( 1:1) ratio . results revealed that there is no significant difference (p<0.05) in the disintegration time of smlt fast dissolving layer upon changing the concentration of dimethylpthalate – glycerin ( 1:1) ratio . mean while figure (2) revealed also that thee is no significant difference in the release profile of smlt , which may be attributed to to the effect of both plasticizers alone, the same result was obtained when losartan fast dissolving oral strip film formulated with the combined glycerinated resins as plasticizers , which they show little enhance in dissolution rate ( 18 ) . table (4): effect of glycerinated dimethylphthalate concentration on the in-vitro dissolution parameters in phosphate buffer ph 6.8. at 37°c figure (2): the. effect of glycerinated dimethylphthalate concentration ,on the release profile of fast dissolving layer of smlt in ph 6.8 phosphate buffer at 37 o c (results are expressed as mean, n=3). effect of lactic acid as a saliva stimulant the formulas f13 and f 14 were prepared using different concentrations of lactic acid as a stimulant , table 5 , revealed that no effect of film thickness differences occurred . meanwhile the surface ph decrease significantly ( p< 0.05 ) for both f13 and f14,this may be attributed to the acidic nature of lactic acid , while the disintegration time decrease to 15 and 11 seconds for both f13 and f14 compared with 22 second for f7.this result also reported by shweta kalyanand et al, where get an enhancement in the dissolution of atenolol fast dissolving film by using weak acid stimulants. (22) on the other hand adding lactic acid to the prepared smlt fast dissolving layer showed an enhancement in the dissolution release profile compared with (f7) as discussed in table (5) which represented by figure (3). this result also reported by deep ak et al. were they got an increase in disintegration and enhance in dissolution release profile of cinnarizine fast dissolving films by increasing the amount of citric acid from 2% to 4% w/w (23) . formula no. t80% (min) d2 min (%) f10 3.9 38.7 f11 3.1 39.5 f12 3.4 40.2 f10 6mg. f11 9mg. f12 12mg. iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 67 table (5): physical properties of the prepared smlt fast dissolving layer f13 and f14 / values are presented as mean±sd (n=3), (n*=5). figure (3): the effect of lactic acid stimulant on the dissolution profile of the prepared smlt fast dissolving layer in ph 6.8 phosphate buffer at 37 o c. (results are expressed as mean, n=3). sustained release layer formulas (fsr1-7 to-fsr6-7) , were utilized to study the effect of polymer type (eudraget® rs100, and pvp k17), with different concentrations on the physical properties of the prepared smlt bilayer strip. the results showed in table (6) for formulas prepared by using eudraget® rs100 and pvp k17 in different ratios (fsr1-7 tofsr3-7) show decease in disintegration time as the eudraget® rs100 concentration increased ; this is due to the swelling property of the hydrophilic polymer (eudraget® rs100) which form gel like layer cause reduction of water permeation and disintegration this swelling property increased with increasing hydrophilic polymer concentration, this result is in a consistent with the result obtained by shalini mishra et al., (24) . while for formulas prepared by pvp k17 in different concentrations (fsr4-7 tofsr6-7) shows also a reduction in the disintegration time with increase addition of pvp k17, which may be attributed to the higher solubility of pvp k17 (24) . the dose of smlt used is (10mg) divided as (5mg) for each layer (fast release layer and sustained release layer), so the dissolution profile evaluated in a manner that 50% of the drug (5mg) must release fast with in short duration of time (as its fast release layer) while the rest 50% must be released in a sustained manner in a comparison with fast dissolving layer . on the other hand , the other formulas , do not show any significant variations, concerned with film thickness , surface ph , and drug content percentages. according to the these results, the formula (fsr6-7) which is subjected into bi phasic sustained layer composed from pvp k17 only selected as an optimized formula with low disintegration time (21 seconds), besides to first 50% of drug released with (15 minutes) and the remaining drug released in a sustained stable manner reach (4 hours) , as shown in table (7) . drug content (%) surface ph in-vitro dt (sec) film * thickness (mm) formulation code 97.92±0.56 6.1±0.001 15±0.034 0.070±0.001 f13 l a c ti c a c id 4 m g . 98.61±0.51 5.5±0.001 11±0.015 0.068±0.001 f14 .l a c ti c a c id 6 m g . iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 68 table (6): physical properties of the prepared smlt sustained release layer values are presented as mean ±sd (n=3) table (7): in-vitro dissolution parameters of different formulas in 0.1n hcl. and 37°c temp. compatibility of smlt and polymers ftir of the prepared fast dissolving film f7 and sustained release film frs6-7 were utilized to investigate the interaction of the drug and polymers used , it was seen that there was no significant shift with ftir spectra by comparing with the spectra of smlt powder , and polymer used as a physical mixture of smlt with polymer as shown in figures 4, 5 and 6 that indicated lack of the possibility of interaction between smlt and polymers used in the preparation of bi layer films. on the other hand ,figure (7) shows the morphology of both layers , the fast dissolving layer (7a) and sustained release layer (7b) of the prepared smlt bi layer oral strip. the fast dissolving layer (carrageenan layer) show clear, smooth surface without pores. while the sustained release layer (pvp k17 layer) showed clear porous surface , which may be referred to the rapid diffusion and evaporation of solvent which easily occurred with low polymer concentration, this result , also reported by ym jagtap et al (24) which revealed that porous structure is useful to enhance the disintegration of the sustained release layer. in a comparison of the dissolution release profile of the selected fast release layer formula (f7) with the release profile of the marketed smlt oral tablet (singulair ® ), the result indicated no significant difference (p>0.05) in cumulative percent of smlt release from prepared formula (f7) and the marketed oral plain tablet (singulair ® ) as shown in figure (7). drug content (%) surface ph in-vitro dt (sec) film * thickness (mm) formulation code 99.97±0.56 6.1±0.001 95±0.011 0.108±0.001 fsr1-7 e u d r a g e t ® r s 1 0 0 99.53±0.51 5.8±0.002 90±0.014 0.144±0.001 fsr2-7 98.36±0.55 6.4±0.001 82±0.013 0.131±0.001 fsr3-7 98.95±0.50 6.2±0.001 46±0.018 0.124±0.001 fsr4-7 p v p k 1 7 99.54±0.50 6.0±0.002 42±0.032 0.104±0.001 fsr5-7 99.67±0.53 5.9±0.001 21±0.014 0.086±0.001 fsr6-7 formula no. t50% of drug released (min.) t100% of drug released (min.) fsr1-7 22 225 fsr2-7 27 240 fsr3-7 25 230 fsr4-7 26 240 fsr5-7 20 270 fsr6-7 15 240 iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 69 figure (4): ftir spectrum of smlt pure powder figure (5): ftir spectrum of formula 7 smlt fast dissolving film figure (6): ftir spectrum of fsr7-6 smlt sustained release layer iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 70 (a) fast dissolving smlt layer (b) sustained release smlt layer figure (7) sem of fsr6-7 bi layer smlt buccal strip figure (5): a comparison of cumulative dissolution profile of the prepared smlt fast dissolving film and marketed smlt oral plain tablet (singulair ® ) in ph 6.8 phosphate buffer at 37 o c. acknowledgement the author is grateful to the , awamedica pharmaceutical industry ,for their help and collaboration in supply chemicals . references 1. skloot g. nocturnal asthma : mechanism and management ,the mount sinai journal of med. , 2002; 69 : 140-147 . 2. martin r. j. , schlegel s. b , chronobiology of asthma , am , j. , respiratory criteria care medical , 1998 ;158 : 1002-1007. 3. brahamankar d. m. , biopharmaceutics and pharmacokinetic, m . k . jain for vallabh prakshan , ap-53a , itampura ,delhi-110088 ,2009;, chapter 15 , p.336 . m m iraqi j pharm sci, vol.24(2) 2015 montelukast sodium as a dual sustained release buccal strips 71 4. mihir p., gandhi nirvi, et al.; formulation, development and evaluation of dissolving oral 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delivery: international journal of drug development and research. 2012;4(2): 408-417. 21. ym jagtap, rk bhujbal, an rande, ns ranpise, effect of various polymers concentrations on physicochemical properties of floating microspheres. indian journal of pharmaceutical sciences. 2012;.74( 6): 512-520. 22. shweta kalyanand mayank bansal, recent trends in development of oral dissolving film. pharmtech. 2012;4 (2): 725-733. 23. siriporn okonogi and satit puttipipatkhachorn, dissolution impropvement of high drug-loaded solid dispersion. aaps pharm sci tech. 2006; 7(2): article 52. 24. shalini mishra, g. kumar, p. kothiyal, formulation and evaluation of buccal patches of simvastatin by using different polymers. the pharma innovation. 2012;1(7): 87-92. http://www.ncbi.nlm.nih.gov/pubmed?term=%22salama%20r%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22chan%20hk%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22young%20pm%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed/19534707 http://www.ncbi.nlm.nih.gov/pubmed/19534707 iraqi j pharm sci, vol.30(2) 202 omega-3 diazepam as antiepileptic drug against yohimbine induced clonic seizure in mice doi: https://doi.org/10.31351/vol30iss2pp241-248 241 possible protective effects of omega 3, diazepam and their combination against yohimbine-induced clonic seizure in mice: comparative study manal a. ibrahim *,1, alaa r. khudhair ** and nada n. al-shawi ** department of pharmacology and toxicology, college of pharmacy, university of basrah, basra, iraq.  department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract yohimbine is actually confirmed in the united states to be utilized for erectile dysfunction; and recently such drug has become commonly used in body-building communities for its presumed lipolytic and sympathomimetic effects. but ingestion of such drug can bring about epileptic effects. many antiepileptic drugs can be utilized to counteract myoclonic seizure; furthermore, diazepam can be used to oppose such type of seizure; in addition, surrogate therapeutic options such as omega 3 may also be utilized. in this study, twenty-four (24) mice of both sexes weighing 20-25g were randomly-allocated into 4 groups (6 animals each group) as follows: group iyohimbine-induced clonic seizure [mice orally-administered dmso (10%), and after 30 min, animals sc. injected with 45mg/kg yohimbine). group iidiazepam-treated as standard drug: it is sc. injected at a dose of 2mg/kg, and after 20 minutes, yohimbine at a dose of 45mg/kg is sc. injected. group iiiomega 3 (40 mg/kg) is orally-administered, and after 30 min 45mg/kg yohimbine is sc. injected. group ivcombination of omega 3 (40mg/kg) is orally-administered then and after ten minutes, diazepam was ip injected 2mg/kg then after 20 minute, 45mg/kg yohimbine is sc. injected. the result of this study showed that omega 3 has non-optimal antiepileptic effect; where, it is un-able to reduce the onset and frequency of epilepsy tone in mice during several time periods (30-120min); and data weren’t significantly different when compared to diazepam. but omega 3 reduced onset of epilepsy in combination with diazepam when compared with diazepam alone. as well as omega 3 caused small percent changes frequency of epilepsy tone through 120 min when compared with other groups. results of the current research suggested omega 3 fatty acid when is combined with diazepam produced significant role for reducing onset of epilepsy against yohimbine-induced seizure model. keyword: omega, diazepam, yohimbine, epilepsy. ا ضد النوبات االرتجاجية التي يسببها موالديازيبام ومزيجه 3اآلثار الوقائية المحتملة ألوميغا دراسة مقارنة: الفئراناليوهمبين في **ندى ناجي الشاوي و **االء راضي خضير ،1 ، *منال عبد الخالق ابراهيم بصره ،العراق. ،البصرةجامعة فرع االدوية والسموم ،كلية الصيدلة،* .بغداد ،العراق جامعة بغداد، كلية الصيدلة، فرع االدوية والسموم ،** الخالصة في االستخدام عالج ضعف االنتصاب ؛ ومؤخًرا ، أصبح هذا الدواء شائعلتم تأكيد استخدام اليوهمبين بالفعل في الواليات المتحدة صرع.اثار الالودي. لكن تناول هذا الدواء يمكن أن يؤدي إلى للجهاز محلل للدهون ومحاكي كآلثاره المفترضة االجساممجتمعات بناء ديد من األدوية المضادة للصرع لمواجهة نوبة الرمع العضلي. عالوة على ذلك ، يمكن استخدام الديازيبام لمقاومة هذا النوع من يمكن استخدام الع .3النوبات ؛ باإلضافة إلى ذلك ، يمكن أيًضا استخدام خيارات عالجية بديلة مثل أوميغا حيوانات في كل مجموعة( 6مجموعات ) 4جراًما إلى 25-20نسين بوزن فأًرا بشكل عشوائي من كال الج 24 تخصيصفي هذه الدراسة ، تم دقيقة ، 30٪( ، وبعد dmso 10) التي يسببها اليوهمبين ]التي تم تناولها عن طريق الفم النوبة االرتجاجية -على النحو التالي: المجموعة األولى مجم / كجم 2(. المجموعة الثانية: ديازيبام تعامل كدواء قياسي: يحقن بجرعة تحت الجلد مجم / كجم من اليوهمبين 45بـ )الحيوانات يتم حقنها مجم / كجم( يتم تناوله عن 40) 3أوميغا -. المجموعة الثالثة تحت الجلدمجم / كجم 45يوهمبين بجرعة يتم حقن ال، دقيقة 20، وبعد تحت الجلد مجم / كجم( يتم تناوله 40) 3مزيج أوميغا -. المجموعة الرابعة تحت الجلدمجم / كجم 45يوهمبين بجرعة يتم حقن الدقيقة 30طريق الفم ، وبعد مجم / كجم 45يوهمبين بجرعة يتم حقن الدقيقة ، 20داخل الصفاق ثم بعد مجم / كجم الديازيبام 2عن طريق الفم ثم بعد عشر دقائق ، تم حقن بـ .تحت الجلد ة الصرع في الفئران وبتأثيرمضاد للصرع غير مثالي. حيث يكون غير قادر على تقليل ظهور وتكرار ن له 3هذه الدراسة أن أوميغا ائجأظهرت نت قللت من ظهور الصرع مع 3دقيقة( ؛ ولم تختلف البيانات اختالفًا كبيًرا عند مقارنتها بالديازيبام. لكن أوميغا 120-30خالل عدة فترات زمنية ) دقيقة بالمقارنة مع 120ة الصرع خالل وبتسبب في تغيرات طفيفة في نسبة تكرار ن 3زيبام بالمقارنة مع الديازيبام وحده. وكذلك أوميغا الديا دوًرا مهًما في تقليل ظهور الصرعيؤدي الدهني عندما يقترن بالديازيبام 3حمض أوميغا انلبحث الحالي لنتائج المقترحة الالمجموعات األخرى. ضد نموذج النوبات التي يسببها اليوهمبين. .الكلمة الرئيسية: أوميغا ، ديازيبام ، يوهمبين ، الصرع 1corresponding author e-mail: manal.ibrahim@uobasrah.edu.iq received: 17/3/2021 accepted:11 /7 /2021 published online first: 2021-12-12 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp241-248 iraqi j pharm sci, vol.30(2) 202 omega-3 diazepam as antiepileptic drug against yohimbine induced clonic seizure in mice 242 introduction yohimbine is a sympatholytic drug used to treat impotence in male patients (1). yohimbine possessed physiological and behavioral effects after systemic administration that includes cns actions which arise from its highly lipophilic and crosses the blood–brain barrier (bbb) (2). moreover, it also has peripheral effect by increasing the release of norepinephrine (ne). the dual effect of yohimbine can be produced by its antagonistic effect on the central alpha-2-receptors and can act as sympathomimetic agent (3). the sympathomimetic, aphrodisiac, and hallucinogenic properties of yohimbine has succumb resurgence as a street drug; where, it is also used by the body builder community as a nutritional supplement for its presumed lipolytic and sympathomimetic effects; but, such drug was reported to produce a sever acute neurotoxicity in a case of a body builder (4, 5). epilepsy is one popular cns disorder that can cause considerable morbidity and mortality (6); and it is a disabling popular chronic illness of neurological disorder (7). furthermore, it has variety of etiologic backgrounds and it is diverse group of disorders that accompanying with seizure appearance (8) which include rapid head and eye movement that extend or transient depletion of consciousness to spasms and uncontrolled muscle contractions (9). many investigators demonstrated that less than 70% of patients stricken with epilepsy achieve seizure control with the available antiepileptic drugs. however, for the remaining 30%, who suffer from intractable epilepsy, treatment options are limited to different multi-drug therapies or surgery if applicable (10). thus, alternative treatments for patients with refractory epilepsy are therefore needed. moreover, various types of antiepileptic drugs have several complications and crucial undesirable effects such as agranulocytosis and hepatotoxicity demand new drugs with more advisable margins of safety and more acceptable (11). omega 3 fatty acids are n-3 polyunsaturated fatty acids (n-3 pufa) that are available in marine oils and sea diet, which are fundamental for neurological function and progression (12). the n-3 pufa are principle structural components of neuronal membranes, and are concerned in cell signaling and modification neurotransmission (13). research in animals and human has explained a beneficial effect of high n-3 pufa intake in several neurological disorders, including attention deficit disorder and alzheimer’s disease (ad) (14). but, there were conflicting results concerning the antiepileptic effect of omega 3; where, researchers reported that supplementation with omega 3 fatty acid can suppress the epileptic seizures while others mentioned that such fatty acids can elevate the seizure (15). thus, this study is designed to evaluate the effect of omega 3 against yohimbine-induced clonic seizure in comparison with 2mg/kg diazepam as a reference drug. materials and method the yohimbine powder was dissolved in dimethylsulfoxide (dmso) to produce a stock solution of 0.2mg/ml; and the dose of yohimbine (45mg/kg). diazepam (bristol, uk) as solution for injection was diluted with 10% dmso to produce a stock solution of 0.1mg/ml; and the dose of diazepam was prepared according to the body weight of the animal to produce (2mg/kg). omega 3 (tad, germany) was diluted in dmso to produce a stock solution of 0.4mg/ml; where, the dose of omega 3 is (40mg/kg). twenty-four mice weighing 20-25g were obtained from the college of pharmacy, university of baghdad. nimals were kept in the animal house of the department of pharmacology and toxicology, college of pharmacy, university of baghdad, at 25 ± 2 °c and light: dark cycle of 12:12 h for 1week before starting experiments. animals were provided with standard rodent pellet diet and the food was withdrawn 12h before the experiment, though water was allowed ad libitum. all experiments were performed according to the guidelines of laboratory animals' care and the ethical guidelines for the investigations on experimental animals. groups of animal twenty-four mice of both sexes weighing 20-25g were randomly allocated into 4 groups (6 animals each group) as follows: gr iyohimbine-induced clonic seizure: mice were orally-administered 10% (dmso), then after 30 minutes, animals were sc. injected with 45mg/kg yohimbine (16). gr iidiazepam as reference drug: mice were ip injected with a dose of 2mg/kg diazepam (17), then after 20 min, animals were sc. injected with 45 mg/kg yohimbine gr iiiomega 3-administered animals: mice were orally-administered omega 3 at a dose 40mg/kg by gavage tube (18); then after 30 min, yohimbine at a dose of 45mg/kg was sc. injected gr ivcombination therapy: mice were orally-administered omega 3 at a dose 40mg/kg by gavage tube then after ten min, diazepam was ip injected at a dose of 2mg/kg. then after 20 min from diazepam injection, yohimbine at a dose of 45mg/kg was injected sc. (16-18). iraqi j pharm sci, vol.30(2) 202 omega-3 diazepam as antiepileptic drug against yohimbine induced clonic seizure in mice 243 measured parameters in adult mice or rats, the clonic seizure starts as forelimbs rhythmic movements, often accompanied with facial clonus that can be either unilateral or a synchronized bilateral clonus with or without rearing and the tail erection (straub tail) (19). mice were observed for in a range of 30-120 min (20) for: 1the onset clonic seizures. 2the number of clonic seizures attack at 30-60min, 60-90min, and 90-120min after yohimbine injection. 3measurement of the percent (%) changes in number of clonic seizures attack induced by yohimbine hcl at 120min according to the following equation: % change= (mean of control group-mean of treated group)/control × 100 statistical analysis the results were expressed as mean ± s.e.m. the significance of differences between the mean values has been calculated using unpaired student t-test. comparison among multiple groups was made by using analysis of variance (anova). p-values less than 0.05 (p<0.05) were considered significant for data of this work. result observation of the onset of epilepsy seizure within 30 min mice of group i (10% dmso + yohimbine) exhibit onset of epilepsy at 5±0.258 min; while in group ii (diazepam + yohimbine) mice, the onset of epilepsy is 24.2 ±0.478 min; and there was a significant different (p<0.05) in the onset of epilepsy in minutes by comparing such time in group ii to that of group i as shown in table 1 and figure 1. in group iii (omega 3 fatty acids + yohimbine) mice, the onset of epileptic seizure is 5.8±0.307min; and there were non-significant differences (p>0.05) in the onset of epilepsy in group iii mice compared to that time in group i mice (10% dmso + yohimbine). (5±0.258 min). table 1 and figure 1. in group iv [combination therapy (omega 3 fatty acids + diazepam + yohimbine)] mice, the onset of epilepsy is 29.3±0.66min; and there were significant different (p<0.05) in the onset of epilepsy between group iv and group i (10% dmso + yohimbine); (5±0.258 min); similarly, there were significant differences (p<0.05) in the onset of epilepsy between group iv and group iii (5.8±0.307min) as shown in table 1 and figure 1. furthermore, by comparing the onset of epilepsy among group ii (diazepam + yohimbine), with groups iii (omega 3 fatty acids +yohimbine hcl) there were significant differences (p<0.05) in the onset of epilepsy. table 1 and figure 2. also, by comparing the onset of epilepsy among group ii (diazepam + yohimbine), with and group iv (omega 3 fatty acids +diazepam + yohimbine hcl), there were also significant differences (p<0.05) in the onset of epilepsy. table 1 and figure 2. observation of the number of clonic seizures attack at 30-60min in the studied groups in table 1 and figure 2, there were significant different (p<0.05) in the number of clonic seizure in group i (yohimbine-treated) (16.5±0.846) compared to groups ii (diazepam +yohimbine hcl) (1.33±0.494) and group iv (omega 3fatty acids +diazepam + yohimbine hcl) (0.833±0.477). furthermore, there were non-significant (p>0.05) difference in the number of clonic seizure in group i (16.5±0.846) compared to group iii (15.16±0.477) as shown in table 1 and figure 2. moreover, table 1 and figure 2 showed that there were significant differences (p<0.05) in the number of clonic seizure in group ii (diazepam + yohimbine) (1.33±0.494) compared to that number in group iii (omega 3 fatty acids + yohimbine hcl) (15.16±0.477); but, there were non-significant differences (p>0.05) in the number of clonic seizure in group ii (1.33±0.494) compared to group iv mice (0.833±0.477). in addition, there were significant different (p<0.05) in the number of clonic seizure in group iii (omega 3 fatty acids + yohimbine) (15.16±0.477) compared to that in group iv (omega 3 fatty acids + diazepam + yohimbine hcl) mice (0.833±0.477). observation of the number of clonic seizures at 6090min in the studied groups table 1 and figure 3 showed that, there were significant different (p<0.05) in the number of clonic seizures at 60-90min observed in group i (yohimbine-treated) (12.3±0.557) compared to that in groups ii (diazepam +yohimbine hcl) (0±0) and group iv (omega 3 fatty acids + diazepam+ yohimbine hcl) (0±0); but there were nonsignificant difference (p>0.05) in the number of clonic seizures at 60-90min observed in group i (12.3±0.557) compared to that in group iii mice (11.66±0.614). also table 1 and figure 3 showed that there were significant difference (p<0.05) in the number of clonic seizures at 60-90min in group ii (diazepam + yohimbine hcl) (0±0) compared to that in group iii (omega 3 fatty acids + yohimbine hcl) (11.66±0.614); but there were non-significant different (p>0.05) in the number of clonic seizures at 60-90min in group ii (0±0) compared to that in group iv mice (0±0). additionally, there were significant different in the number of clonic seizures at 60-90min (p<0.05) in group iii (omega 3 fatty acids +yohimbine hcl) (11.66±0.614) compared that in group iv (omega 3fatty acids +diazepam +yohimbine hcl) mice (0±0). table 1 and figure 3. iraqi j pharm sci, vol.30(2) 202 omega-3 diazepam as antiepileptic drug against yohimbine induced clonic seizure in mice 244 observation the number of clonic seizures at 90-120min in the studied groups table 1 and figure 4 showed that there were significant different (p<0.05) in the number of clonic seizure at 90-120min in group i (yohimbinetreated) mice (6.5±0.341) compared to that in group ii (diazepam +yohimbine hcl) (0±0) and group iv (omega 3 fatty acids +diazepam + yohimbine hcl) mice (0±0); but there were nonsignificant (p>0.05) in the number of clonic seizure at 90-120min in group i (6.5±0.341) compared to that in group iii mice (5.66±0.494). moreover, there were significant difference (p<0.05) in the number of clonic seizure at 90120min in group ii (diazepam + yohimbine hcl) (0±0) compared to that in group iii (omega 3 fatty acids +yohimbine hcl) mice (5.66±0.494); but there were non-significant difference (p>0.05) in the number of clonic seizure at 90-120min in group ii (0±0) compared to that in group iv mice (0±0). table 1 and figure 4. in addition, table 1 and figure 4 showed that there were significant difference (p<0.05) in the number of clonic seizure at 90-120min in group iii (omega 3 fatty acids +yohimbine hcl) (5.66±0.494) compared to that in group iv mice (omega 3 fatty acids +diazepam +yohimbine hcl) (0±0). percent (%) changes in number of clonic seizures in studied groups within 120min in table 1 and figure 5 showed that there were significant different (p<0.05) in percent changes in number of clonic seizures attack at 120min between group i (yohimbine-treated) (0%) compared to that in group iii mice (omega 3 fatty acids + yohimbine hcl). (13.846%). in contrast, there were non-significant (p>0.05) different in percent changes in number of clonic seizures attack at 120min in group ii (diazepam + yohimbine hcl) (100%); but there were significant different (p<0.05) in percent changes in number of clonic seizures attack at 120min in group iii (omega 3 fatty acid + yohimbine hcl) each compared to that in group iv mice (omega 3fatty acids +diazepam + yohimbine hcl) (100%) as that shown in table 1 and figure 5. table 1. onset of clonic seizures during 30min,number of clonic seizure during different period from 30120min that induced by yohimbine and other treatment groups and percent changes in number of clonic seizures within 120 min symbol *refers to difference between group i and other groups. symbol; # refers to difference between group ii and other groups; symbol ¥ refers to difference between group iii and other groups; and symbol € refers to difference between group iv and other groups. treatment group n=6 type of treatment onset of clonic seizures (30min) (mean ± s.e.m) number of clonic seizures at 30-60min (mean ± s.e.m) number of clonic seizures at 60-90min (mean ± s.e.m) number of clonic seizures at 90-120min (mean ± s.e.m) percent changes in number of clonic seizures within 120min i yohimbine hcl 5±0.258# € 16.5± 0.846#€ 12.3± 0.557#€ 6.5± 0.341#€ 0% ii diazepam+ yohimbine hcl 24.2 ±0.478*¥ € 1.33± 0.494*¥ 0 ± 0*¥ 0± 0*¥ 100% iii omega 3 fatty acids + yohimbine hcl 5.8±0.307#€ 15.16± 0.477#€ 11.66± 0.614#€ 5.66± 0.494#€ 13.846% iv omega 3 fatty acids + diazepam + yohimbine hcl 29.3±0.66* #¥ 0.833± 0.477*¥ 0 ± 0*¥ 0± 0*¥ 100% iraqi j pharm sci, vol.30(2) 202 omega-3 diazepam as antiepileptic drug against yohimbine induced clonic seizure in mice 245 figure 1. mean±se of the onset of clonic seizures(min) that induced by yohimbine hcl (group i) and other treated groups, diazepam+ yohimbine hcl (group ii), omega 3 fatty acids +yohimbine hcl (group iii) and omega3fatty acids +diazepam +yohimbine hcl (group iv). symbol *refers to difference between group i and other groups. symbol; # refers to difference between group ii and other groups; symbol ¥ refers to difference between group iii and other groups; and symbol € refers to difference between group iv and other groups. figure 2. mean±se of the number of clonic seizures at 30-60min that induced by yohimbine hcl (group i) and other treated groups (diazepam+ yohimbine hcl (group ii), omega 3 fatty acids +yohimbine hcl (group iii) and omega 3 fatty acids +diazepam +yohimbine hcl (group iv). symbol *refers to difference between group i and other groups. symbol; # refers to difference between group ii and other groups; symbol ¥ refers to difference between group iii and other groups; and symbol € refers to difference between group iv and other groups. figure 3. mean±se of the number of clonic seizures at 60-90min that induced by yohimbine hcl (group i) and other treated groups (diazepam+ yohimbine hcl(group ii), omega 3 fatty acids +yohimbine hcl (group iii) and omega 3 fatty acids +diazepam + yohimbine hcl (group iv). symbol *refers to difference between group i and other groups. symbol; # refers to difference between group ii and other groups; symbol ¥ refers to difference between group iii and other groups; and symbol € refers to difference between group iv and other groups. figure 4. mean±se of the number of clonic seizures at 90-120min that induced by yohimbine hcl (group i) and other treated groups (diazepam+ yohimbine hcl(group ii), omega 3 fatty acids +yohimbine hcl (group iii) and omega 3 fatty acids +diazepam +yohimbine hcl (group iv). symbol *refers to difference between group i and other groups. symbol; # refers to difference between group ii and other groups; symbol ¥ refers to difference between group iii and other groups; and symbol € refers to difference between group iv and other groups. iraqi j pharm sci, vol.30(2) 202 omega-3 diazepam as antiepileptic drug against yohimbine induced clonic seizure in mice 246 figure 5. the percent of changes in the number of clonic seizures attack induced by yohimbine hcl (group i) and other treated groups (diazepam+ yohimbine hcl (group ii), omega 3 fatty acids +yohimbine hcl (group iii) and omega 3 fatty acids +diazepam +yohimbine hcl (group iv) at 120min. discussion the α2-antagonist yohimbine has a diversity of effects on the noradrenergic, dopaminergic, serotonergic and gamma aminobutyric acid (gabaergic) neurotransmission. yohimbine-inducted seizures, not only intermediated through impairment of gabaergic transmissions but furthermore, by an endogenous enhancement of the excitatory amino acid transmission (21); and it was and still vastly used in diverse experimental studies in vitro (22, 23, 24) and in vivo in aware animals (25, 26, 27). in the present study, yohimbine-induced seizures in all groups of the experiment i, ii, iii, and iv in various time sequences from 30 min after seizure induction by yohimbine to 120 min. table 1 and figures (1-4). this is parallel with other studies also showed that yohimbine can excite seizure assay (28, 29). pharmacological studies revealed that prostaglandin e2 and other pro-inflammatory mediators were upraised in the animal design of epilepsy (30). it has been reported that both of the enzymes, phospholipase a2 (pla2) and oxidoreductase cyclooxygenase-2 (cox2) can be invigorated by epilepsy; where, the pla2 enzyme can liberate arachidonic acid (aa) from membrane phospholipids, and the cox2 can transfer aa to pro-inflammatory mediators (31). polyunsaturated fatty acids (pufas) docosahexaeno¬ic acid (dha, 22: 6n-3) are major constituents of membrane phospholipids in brain tissue (32). diets insufficient in omega 3 pufa lead to decreased dha in the brain and enhanced turnover of arachidonic acid to eicosanoids, an effect which is defeat by restoring the omega 3 to diet (33). researchers reported that the proinflammatory mediators have been decreased by omega-3 fatty acid eicosapentaenoic acid (epa) through inhibiting pla2 and cox2 enzymes (34). others reported that n-3 pufa possessed beneficial therapeutic effect in patients suffering from a neurological disorder such as epilepsy, in spontaneous, and in recurrent seizure (35). that chronic treatment by omega-3 encourages neuroprotection and positive plastic exchanges in the brain of rats with epilepsy (34), with a reduced in neuronal death in ca1 and ca3 subfields of the hippocampus. this may be assigned to n-3 pufas ion channel intonation, and antiinflammatory activity (36). the current study showed that omega-3 have partial antiepileptic effect but non-significant ,it was unable to decrease the number of clonic seizure-induced by yohimbine (group iii) from the onset of induction to 120 min compared to that in yohimbine-treated (group i) mice; in other word, its effect was not-significant (p>0.05); also, the onset of seizure induction times in group iii mice were shorter than that observed in group ii mice; but, the positive antiepileptic effect of omeg3 that include the onset clonic seizures in group iv mice (combination of omega 3 with diazepam) was significant compared to that in diazepam alone (group ii) mice; but, the effect of omeg3 that include the onset time for induction of epilepsy in group iv mice was significantly prolonged compared to that in group iii (omega 3 fatty acid against yohimbine-treated); moreover the beneficial effect of omega3 in group iv rats, there were reduction in the number of epilepsy when omega 3 combined with diazepam during various time intervals 30-120 minutes compared to that in diazepam-treated (group ii) mice but, was nonsignificantly different (p>0.05). as well as in figure 5, the positive effect of omega 3 (group iii) was shown; where, there were clear difference in the percent of epileptic frequency compared to that of yohimbine hcl (group i). the reason for the non-optimal significant activity of omega 3 against yohimbine-induced clonic seizure may be related to short duration of treatment with such fatty acid as researcher mentioned that, prolonged oral administration of alpha-linolenic acid (an omega-3 fatty acid) and linoleic acid in a 1:4 mixture protected rats from having seizures in four different epilepsy models (34). moreover, investigators reported that 200 mg/kg/day of omega 3 intraperitoneally (i.p) injected for 21 days possessed antiepileptic effect (37). conclusion omega 3 fatty acid produced nonsignificant effect; 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antiepileptic drugs. int. res j pharm. app sci. 2013; 3(3): 18-23. 36. vera c. terra1, ricardo m. arida, guilherme m. et al. the utility of omega3 fatty acids in epilepsy more than just a farmed tilapia!. arq neuropsiquiatr 2011; 69(1):118-121. 37. taha ay, filo e, ma dw, mcintyre bw. dose-dependent anticonvulsant effects of linoleic and alpha-linolenic polyunsaturated fatty acids on pentylenetetrazol induced seizures in rats. epilepsia .2009; 50: 72–82. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 effects of zinc sulfate on mitoxantrone-induced cardiotoxicity doi: https://doi.org/10.31351/vol29iss1pp115-122 115 effects of two different doses of zinc sulfate on serum troponin i 3 enzyme level and cardiac malondialdehyde contents in mitoxantroneinduced cardiotoxicity in rats taif m. maryoosh *,1, nada n. al-shawi ** and eman s. salih*** * ministry of health and environment ,wassit health department,iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. *** department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad, iraq. abstract mitoxantrone antineoplastic drugs used in the treatment cancers. the mitoxantrone' may cause cardiotoxicity which is irreversible and dose-dependent. zinc is regarded as a necessary element for cell division and the production of dna and protein; furthermore, it has a main role in alleviating cardiovascular diseases; and may have protective outcomes in coronary artery disease. the current research is designed to investigate the effects of two different doses of zinc sulfate on mitoxantrone-induced cardiotoxicity in rats. forty-eight (48) adult albino rats of both sexes were utilized in this study; the animals were randomly classified into six groups of 8 animals each. group i: rats were received distilled water (negative control). group ii: orally-administered zinc sulfate (15mg/kg/day) group iii: orally-administered zinc sulfate (30mg/kg/day) group iv: rats intraperitoneally injected with a mitoxantrone at a dose of (2.5 mg/kg) to reach total cumulative dose of 7.5 mg/kg on day 20 (positive control). group v: orally-administered zinc sulfate at a dose (15mg/kg/day) and an intraperitoneal injection of mitoxantrone at a dose (2.5 mg/kg) was administered to reach the total cumulative dose of 7.5 mg/kg on day 20. group vi: orally-administered zinc sulfate at a dose (30 mg/kg/day), and an intraperitoneal injection of mitoxantrone at a dose (2.5 mg/kg) to reach the total cumulative dose of 7.5 mg/kg on day 20. forty-eight (48) hours after the end of treatment duration (i.e. at day 22 nd), each animal was euthanized by diethyl ether and ketamine. then, after cervical dislocation, blood was obtained by cardiac puncture and then serum was prepared to estimate troponin i 3 enzyme levels, and the heart of each animal was excised for homogenate preparation to estimate of malondialdehyde contents. it was found that oral administration of zinc sulfate [(15mg/kg/day with total cumulative dose (7.5 mg/kg) of mtxn] (group v), resulted in a non-significant (p>0.05) differences in both serum level of troponin i 3 enzyme, and malondialdehyde (mda) contents in heart tissue homogenate compared to the corresponding level and content in group of rats ip injected with total cumulative dose of 7.5 mg/kg of mtxn (group iv). moreover, there were significant reductions (p<0.05) in serum level of troponin i 3 of rats orally-administered zinc sulfate [(30 mg/kg/day) with total cumulative dose (7.5mg/kg) of mtxn] (group vi) compared to the corresponding enzyme levels in serum of -group iv rats that ip injected with total cumulative dose of 7.5mg/kg of mtxn and -group v rats that orally-administered zinc sulfate [(15mg/kg/day with total cumulative dose (7.5 mg/kg) of mtxn]. furthermore, there were significant reductions (p<0.05) in mda contents in heart tissue homogenate of group vi rats compared to the corresponding contents in heart tissue homogenate of group iv rats. according to above results it can be concluded that zinc sulfate at a dose of (30 mg/kg/day) (in group vi rats) diminished the cardiotoxicity induced by mitoxantrone as indicated by the reduction of serum troponin i 3 enzyme level but non-significant difference in lipid peroxidation marker (malondialdehyde) compared to (15 mg/kg/day) (in group v rats). keywords: mitoxantrone, cardiotoxicity, zinc sulfate, troponin i 3 enzyme, malondialdehyde contents ومحتوى في مصل الدم troponin i 3 ال معلى أنزي جرعتين مختلفتين من كبريتات الزنك تأثير عقار ميتوزانترون في الجرذانبواسطة ة سمية القلب المستحث فيالمالونداي الديهايد ***صالح ايمان سعديو **ندى ناجي الشاوي، 1*،طيف محمد مريوش العراق ،فرع صحة واسط وزارة الصحة والبيئة، * ، كلية الصيدلة، جامعة بغداد، العراقالسموم و االدوية فرع** كلية الصيدلة، جامعة بغداد، العراق ،السريرية المختبرية العلوم فرع ** الخالصة ويعتمد يعد ال عكوس، المايتوزانترون عن الناتج القلب ان تسمم. انواع السرطانات عالج في يستخدم لألورام عالج مضاد هوالمايتوزانترون .العالج من سنوات بعد يحدث وقد ، الجرعة على 1corresponding author e-mail: tayfmaruish@yahoo.com received: 29/ 7 /2019 accepted: 12/ 10/2019 iraqi journal of pharmaceutical scienc https://doi.org/10.31351/vol29iss1pp115-122 iraqi j pharm sci, vol.29(1) 2020 effects of zinc sulfate on mitoxantrone-induced cardiotoxicity 116 يعد الزنك معدنًا أساسيًا النقسام الخاليا وتوليف الحمض النووي والبروتين ؛ عالوة على ذلك ، فإن هذا المعدن له دور مهم حاالت أمراض القلب واألوعية الدموية و يبدو أن له آثار وقائية في مرض الشريان التاجي واعتالل عضلة القلب.تخفيف في جرعتين مختلفتين من كبريتات الزنك على سمية القلب المستحثة بواسطة الميتوزانترون في اعطاء رصممت هذه الدراسة الستقصاء آثا الجرذان. حيوانات لكل 8( جرذا بالغا من كال الجنسين. تم تقسيم الحيوانات بشكل عشوائي في ست مجموعات من 48تم استخدام ثمانية وأربعون ) المجموعة ملغ / كغم/ يوم كبريتات الزنك فمويا. 15: المجموعة الثانيةمجموعة السيطرة(. : الماء المقطر )المجموعة األولىمنها. ملغم/ كغم( 2.5بجرعة ) لمايتوزانترونحقنة داخل الصفاق با . المجموعة الرابعة:ملغ /كغم/ يوم كبريتات الزنك فمويا30: الثالثة ملغ/كغم/ يوم كبريتات الزنك 15: المجموعة الخامسة. 20/ كغم في اليوم ملغم 7.5للوصول إلى الجرعة التراكمية اإلجمالية البالغة 7.5ملغم/كغم( للوصول إلى إجمالي جرعة تراكمية قدرها 2.5الصفاق بجرعة ) داخل الحقن بطريقة مايتوزانترونفمويا مع إعطاء داخل الحقن بطريقة مايتوزانترونملغ/كغم/ يوم كبريتات الزنك فمويا مع إعطاء 30: المجموعة السادسة. 20ملغم / كغم في اليوم 20ملغم/كغم في اليوم 7.5ملغم/ كغم( للوصول إلى الجرعة التراكمية اإلجمالية البالغة 2.5الصفاق بجرعة ) ثقب داخل القلب وبعد ذلك تم إعداد المصل تم الحصول على الدم عن طريقبعد خلع العنق، وفي اليوم الثاني والعشرين من الدراسة التروبونين؛ كما وتم استئصال قلب كل حيوان من أجل تحضير التجانس لتقدير محتوى المالونداي 3لتقدير مستويات نشاط انزيم القلب الديهايد. ملغ/ 7.5ملغ/كلغ/ يوم( مع الجرعة التراكمية اإلجمالية )15كبريتات الزنك التي اعطيت فمويا للجرذان بجرعة )وقد أظهرت النتائج ان ( في مستوى نشاط المصل من انزيم التروبونين p>0.05كلغ( من ميتوزانترون داخل الصفاق )المجموعة الخامسة( لم تنتج فرق معنوي ) i 3 ومحتوى المالونداي الديهايدانس األنسجة القلبية مقارنة بمستوى نشاط المصل من االنزيم المقابل في تج ومحتوى المالونداي الديهايد ملغم/كغم من الميتوزانترون )المجموعة الرابعة( 7.5في مجموعة من الجرذان التي تم حقنها داخل الصفاق جرعة تراكمية إجمالية قدرها ومحتوى المالونداي و i3( في مستوى نشاط المصل لمحتويات إنزيم التروبونين p<0.05؛على العكس، كان هناك انخفاض معنوي ) ملغم/ كغم/ يوم( مع الجرعة التراكمية 30في أنسجة القلب المتجانسة من الجرذان التي اعطيت جرعة فموية من كبريتات الزنك ) الديهايد ومحتوى المالونداي الديهايدة السادسة( مقارنة بمستويات نشاط اإلنزيم داخل الصفاق )المجموع مايتوزانترونملغم/كغم( من ال 7.5الكلية ) )المجموعة المايتوزانترونملغم/كغم من 7.5في مجموعة من الجرذان التي تم حقنها داخل الصفاق بجرعة تراكمية إجمالية قدرها الرابعة(. ملغ/ كغم/ يوم( )جرذان المجموعة السادسة( قللت من تسمم القلب المستحث 30الزنك بجرعة ) كبريتات وبذلك يمكن االستنتاج ان مايتوزانترون من خالل خفض مستوى التروبونين في مصل الدم مع اختالف غير معنوي في محتوى بروكسيد الدهون بواسطة ال جرذان المجموعة الخامسة(.ملغ/كغم/يوم )15)المالوندايالديهايد( مقارنة مع كبريتات الزنك بجرعة .ومحتوى المالونداي الديهايد ، سمية القلب ، كبريتات الزنك ، تروبونين القلب الميتوزانترون : المفتاحية الكلمات introduction mitoxantrone (mtxn) is one of the most generally used antineoplastic drug used in treatment of acute leukemia ,breast and prostate cancer, lymphoma, and also in the treatment of multiple sclerosis (ms) because of its immunosuppressive features(1,2). the mtxn prompted cardiotoxicity is irreversible, and depend on dose. the risk of mtxn-induced cardiac adverse effects can be seriously increased with cumulative dose > 140 mg/m2 (2). clinically, the cardiotoxicity produced by mtxn can cause a reduction in congestive heart failure (chf) and left ventricular stroke volume (3); furthermore, authors stated that the cardio-protective properties of several pharmacologic drugs and a plant have been confirmed by researchers; where, pyridoxine, statins and artichoke have been revealed to be potentially protective against various cardiotoxic agents(4,5,6). zinc (zn), is one of the main trace elements in the body. it is involved in homeostasis role in oxidative stress (os). it is required for the metabolic activity of the body's enzymes that involved carbohydrates, fats, proteins the metabolism; and it has an important role in states of cardiovascular diseases (cvds); and it may have cardio-protective effects in heart disease (7).the aim of the study is to investigate the effects of two doses of zinc sulphate on mtxn –induced cardiotoxicity. materials and methods preparation of drugs solution mitoxantrone (mtxn) vial (20mg/10ml) was diluted with 10ml d.w to obtain 1mg /ml to be ip injected at a dose 2.5 mg/kg. each capsule of zinc sulfate (220mg) was reconstituted in 22ml of d.w to obtain 10mg/ml to be administered by oral gavage at doses 15mg/kg, and 30mg/kg (8, 9 ). animals forty-eight (48) healthy adult wistar albino rats of both sexes (24 male and 24 female), three months old, weighing range 150240 gm were utilized in this research; they were gotten from in the animal house of college of science, university of dhi qar. rats were maintained in the college of science, wasit university under normal conditions of temperature, humidity and under a 12 h light/dark cycle .rats were supplied commercial pellets and tap water throughout the experiment period. the animals had no manifestation of any illness upon examination. they were left for two weeks without interference for acclimatization. the study was accepted by the graduate studies and the scientific committees of the college of pharmacy, university of baghdad. iraqi j pharm sci, vol.29(1) 2020 effects of zinc sulfate on mitoxantrone-induced cardiotoxicity 117 experimental protocol healthy rats were randomly allocated into six groups, each containing 8 rats' (4 males and 4 females) as follows: group i: rats received 0.5 ml/day of distilled water (dw) intraperitoneally (ip) for 20 days. this group served as a negative control. group ii: rats orally-administered zinc sulfate at a dose of (15mg/kg/day) alone for 20 days. group iii: rats orally-administered zinc sulfate at a dose of (30mg/kg/day) alone for 20 days (8) group iv: rats intraperitoneally (ip) injected with a mitoxantrone (mtxn) at a dose (2.5 mg/kg) on day 0, 10, 20 to reach total cumulative dose of 7.5 mg/kg on day 20 ( 9 ). this group served as positive control. group vrats orally-administered zinc sulfate at a dose (15mg/kg/day) for 20 days, with an ip injection of mitoxantrone (mtxn) at a dose (2.5 mg/kg) was administered at the day 0, 10, 20 to reach total cumulative dose of 7.5 mg/kg on day 20. group virats orally-administered zinc sulfate at a dose (30 mg/kg/day) for 20 days, and an ip injection of mitoxantrone (mtxn) at a dose (2.5 mg/kg) was administered at the day 0, 10, 20 to reach total cumulative dose of 7.5 mg/kg on day 20. forty-eight (48) hour after the end of the treatment duration (i.e. at day 22 nd), each animal was euthanized by diethyl ether and ketamine. then, after cervical dislocation, two and a half (2.5) ml blood was obtained from each rat by cardiac puncture and then was transferred into a gel tube and left to clot for 30 minutes before centrifugation for 15 minutes at 3000 round per minute (rpm) to obtain serum, which was collected using rubber micropipette, divided into small aliquots in labeled eppendorf tubes, and kept frozen at -50 ºc until analyzed. the serum was utilized for the estimation of troponin i 3; where, the estimation is based on sandwich-enzyme-linked immunosorbent assay (elisa) (10). serum level of troponin i 3 was expressed as pg/ml. furthermore, cardiac tissues were obtained to prepare heart tissue homogenates for the determination of mda contents with the use of phosphate buffer saline (pbs ph 7.4) which also based on eliza (11). the contents of mda in heart tissue homogenate were expressed as ng/ml. statistical analysis statistical analysis was done by statistical package for social sciences (spss) version 24. results were calculated as mean ± standard error of means (sem). comparison among groups was done by using a one-way analysis of variance (anova). the statistically significant differences were considered when p<0.05. results table 1 and figure 1showed that there were non-significant differences (p>0.05) in serum level of troponin i 3 in group of rats orally-administered zinc sulfate (15mg/kg/day alone for 20 days) (group ii) compared to the corresponding enzyme level in serum of negative control rats (group i). mean±sem of serum of troponin i 3 levels were 155.09±7.56 vs. 152.47±3.90, respectively. similarly, table 1 and figure 1 showed that there were nonsignificant differences (p>0.05) in serum level of troponin i 3 in group of rats orallyadministered zinc sulfate (30mg/kg/day alone for 20 days) (group iii) compared to the corresponding level in serum of negative control rats (group i). mean±sem of serum of troponin i 3 enzyme levels were 150.80±2.966 vs. 152.47±3.90, respectively. furthermore, rats ip injected with a total cumulative dose of 7.5mg/kg of mtxn (group iv) (positive control) produced significant elevation (p<0.05) in serum level of troponin i 3 compared to the corresponding level in serum of negative control rats (group i). mean±sem of serum ctn-i 3 levels were respectively, 203.71±4.76and 152.47±3.90. meanwhile, table 1 and figure 1 also showed that oral administration of zinc sulfate [(15mg/kg/day with total cumulative dose (7.5 mg/kg) of mtxn] (group v), resulted in a non-significant (p>0.05) difference in serum level of troponin i 3 compared to the corresponding serum level in group of rats ip injected with total cumulative dose of 7.5 mg/kg of mtxn (group iv). moreover, there was a significant reduction (p<0.05) in serum level of troponin i 3 in rats orally-administered zinc sulfate [(30 mg/kg/day) with total cumulative dose (7.5mg/kg) of mtxn] (group vi) compared to the corresponding levels in serum of rats ip injected with total cumulative dose of 7.5mg/kg of mtxn (group iv). mean±sem of serum level of troponin i 3 enzymes were 174.1±2.8 vs. 203.71±4.76 pg/ml, respectively. table 1and figure 1. in addition there were significant reduction (p<0.05) in serum levels of troponin i 3 in rats orally-administered zinc sulfate [(30mg/kg/day) with total cumulative dose (7.5mg/kg) of mtxn] (group vi) compared to the corresponding levels in serum of rats orally-administered zinc sulfate [(15mg/kg/day with total cumulative dose (7.5mg/kg) of mitoxantrone (mtxn)] (group v). mean±sem of serum troponin i 3 enzymes iraqi j pharm sci, vol.29(1) 2020 effects of zinc sulfate on mitoxantrone-induced cardiotoxicity 118 were respectively, 174.1±2.8 vs. 195.25±6.7 pg/ml. table 1 and figure 1. table 1. effects of various treatments on serum cardiac troponin i 3 (ctn-i 3) levels in studied rats' groups group/treatment serum cardiac troponin i 3 (ctn-i 3) levels (pg/ml) mean ± sem group i/ negative control [distilled water (dw)] 152.47±3.90 group ii/zinc sulfate (15 mg/kg/day) 155.09±7.56 group iii/zinc sulfate (30 mg/kg/day) 150.80±2.966 group iv/mitoxantrone (mtxn) (7.5 mg/kg) (total cumulative dose) 203.71±4.76* group v/zinc sulfate (15 mg/kg/day) with mitoxantrone (mtxn) (7.5 mg/kg) 195.25±6.7 group vi/zinc sulfate (30 mg/kg/day) with mitoxantrone (mtxn) (7.5 mg/kg) 174.1±2.8 # data expressed as mean ± standard error of means (sem). *: p<0.05: significant difference compared to negative control rats (group i). # p<0.05: significant difference compared to group iv, and group v. number of rats in each group=8. figure 1. bar chart showing serum cardiac troponin i 3 (ctn-i 3) level (pg/ml) in various experimental rats' groups. group i: negative control [distilled water (dw)]; group ii: zinc sulfate (15mg/kg/day); group iii: zinc sulfate (30mg/kg/day); group iv: mitoxantrone (mtxn) (7.5mg/kg); group v: zinc sulfate (15mg/kg/day) with mitoxantrone (mtxn) (7.5 mg/kg); group vi: zinc sulfate (30 mg/kg/day) with mitoxantrone (mtxn) (7.5mg/kg). *: significant difference (p<0.05) with respect to the negative control group. (group i) # p<0.05: significant difference compared to group iv, and group v. table 2 and figure 2 showed that there were non-significant differences (p>0.05) in mda contents in heart tissue homogenate in group of rats orally-administered zinc sulfate (15 mg/kg/day alone for 20 days) (group ii) compared to corresponding contents in negative control rats (group i). mean±sem of in mda contents in heart tissue homogenate were 44.48±1.88 and 42.40±.92, respectively. similarly, there were non-significant differences (p>0.05) in mda contents in heart tissue homogenate in the group of rats orallyadministered zinc sulfate (30 mg/kg/day alone for 20 days) (group iii) compared to the corresponding contents in negative control rats (group i). mean±sem in mda contents in heart tissue homogenate levels were 43.91±1.61 and 42.40±.92, respectively. furthermore, rats ip injected with a total cumulative dose of 7.5 mg/kg of mtxn (group iv) produced significant elevation (p<0.05) in mda contents in heart tissue homogenate compared to the corresponding contents in heart tissue homogenate in negative control rats (group i). mean±sem of mda contents in heart tissue homogenate contents were 59.41±4.1and 42.40±.92, respectively. table 2 and figure 2. moreover, table 2 and figure 2 also showed that administration of zinc sulfate [(15mg/kg/day with total cumulative dose (7.5 mg/kg) of mtxn] (group v), resulted in a nonsignificant (p>0.05) difference in mda contents in heart tissue homogenate compared to the corresponding contents in rats ip injected with total cumulative dose of 7.5 mg/kg of mtxn (group iv). mean±sem of mda contents in heart tissue homogenate levels were 54.97±1.67 and 59.41±4.1, respectively. furthermore, there were significant reduction (p<0.05) in mda contents in heart tissue homogenate of rats orally-administered zinc sulfate [(30 mg/kg/day) with total cumulative dose (7.5mg/kg) of mtxn] (group vi) compared to the corresponding contents in heart tissue homogenate of rats ip injected with total cumulative dose of 7.5mg/kg of mtxn (group iv). mean±sem of mda contents were 49.91±2.08 and 59.41±4.1 (ng/ml), respectively. iraqi j pharm sci, vol.29(1) 2020 effects of zinc sulfate on mitoxantrone-induced cardiotoxicity 119 in addition, table 2 and figure 2 showed that there were non-significant differences (p>0.05) in mda contents in heart tissue homogenate of rats orally-administered zinc sulfate [(30mg/kg/day) with total cumulative dose (7.5mg/kg) of mtxn] (group vi) compared to mda contents in heart tissue homogenate of rats orally-administered zinc sulfate [(15mg/kg/day with total cumulative dose (7.5mg/kg) of mtxn] (group v). mean±sem of mda contents in heart tissue homogenate levels were 49.91±2.08 and 54.97±1.6 (ng/ml), respectively. table 2.effects of various treatments on malondialdehyde (mda) contents in heart tissue homogenate in studied rats' groups group heart homogenate mda (ng/ml) mean ± sem group i/ negative control [distilled water (dw)] 42.40±.92 group ii/zinc sulfate (15 mg/kg/day) 44.48±1.88 group iii/zinc sulfate (30 mg/kg/day) 43.91±1.61 group iv/mitoxantrone (mtxn) (7.5 mg/kg) (total cumulative dose) 59.41±4.1* group v/zinc sulfate (15 mg/kg/day) with mitoxantrone (mtxn) (7.5 mg/kg) 54.97±1.6 group vi/zinc sulfate (30 mg/kg/day) with mitoxantrone (mtxn) (7.5 mg/kg) 49.91±2.08 # data expressed as mean ± standard error of means (sem). *: p<0.05: significant difference compared to negative control rats (group i). # p<0.05: significant difference compared to group iv. number of rats in each group=8 figure 2. bar chart showing mda contents in heart tissue homogenate in various experimental rats' groups. group i: negative control [distilled water (dw)]; group ii: zinc sulfate (15mg/kg/day); group iii: zinc sulfate (30mg/kg/day); group iv: mitoxantrone (mtxn) (7.5mg/kg); group v: zinc sulfate (15mg/kg/day) with mitoxantrone (mtxn) (7.5 mg/kg); group vi: zinc sulfate (30 mg/kg/day) with mitoxantrone (mtxn) (7.5mg/kg). *: significantly different (p<0.05) with respect to the negative control group. (group i) # p<0.05: significant difference compared to group iv. discussion mitoxantrone (mtxn) is an antineoplastic agent used to treat several types of cancers and on multiple sclerosis (ms); such drug showed a high incidence of cardiotoxicity, which was reported to depend on several factors; where, age and cumulative dose being two important factors (12). in the current study, ip injection of mtxn can cause destruction of the myocardial cell membrane, which becomes more permeable; thus, serum troponin i 3 level were significantly elevated (p<0.05) compared to the corresponding levels in negative control rats' group (table 1 and figure 1). this comes in tune with a study of others (13). biomarkers are molecules which occur naturally and can be used as an indicator of a particular disease state or some other physiological state of an organism. troponins were medium-sized proteins which have an important role in regulating the cardiac muscle contractile elements (myosin and actin) (14). furthermore, troponins are well-established biomarkers for the discovery of heart damage in both human and animals; where, levels may increase after 2-3 hours; and serum levels of ctni and ctnt components of the troponin complex of muscle cells, are of importance in the diagnosis of myocardial damage in several conditions, including acute myocarditis (15,16, 17). iraqi j pharm sci, vol.29(1) 2020 effects of zinc sulfate on mitoxantrone-induced cardiotoxicity 120 the serum levels of troponin i 3 to detect myocardial destruction induced by mtxn was not previously estimated; but the study performed by herman eh, et al at 2001 (18) and by the determination of troponin t levels showed that there were high levels of the biomarker in rats with chronic doxorubicinand mtxn-induced cardiotoxicity . thus, to our knowledge the current research is the first to estimate the serum level of troponin i 3 enzyme in mtxn-induced cardiotoxicity; thus, we did not have a chance to compare results obtained from this study with others concerning this respect. the results of the present study also showed that oral administration of zinc sulfate [(15mg/kg/day with total cumulative dose (7.5 mg/kg) of mtxn] (group v), resulted in a non-significant (p>0.05) difference in serum level of troponin i 3 compared to the corresponding serum level in group of rats ip injected with total cumulative dose of 7.5 mg/kg of mtxn (group iv). moreover, there were significant reduction (p<0.05) in serum level of troponin i 3 of rats orally-administered zinc sulfate [(30 mg/kg/day) with total cumulative dose (7.5mg/kg) of mtxn] (group vi) compared to the corresponding levels in serum of rats ip injected with total cumulative dose of 7.5mg/kg of mtxn (group iv) (table 1 and figure 1). zinc is a vital element in preserving the normal structure and physiology of cells. it appears to have protective effects in heart diseases (19). furthermore, authors reported ischemia and infarction may lead to release of zinc from proteins and cause myocardial damage. in such states, replacing with zinc has been shown to recover cardiac function and prevent further destruction (20). moreover, authors reported that rodents administered zinc salts during reperfusion has been stated to decrease arrhythmias and improve myocardial function (19). additionally, to assess if mtxn is able to initiate or enhance lipid peroxidation, the most known secondary product of lipid peroxidation, mda, was determined; where, it is one of the best indicators of os (21). results of this study exhibited that the cardiac tissue mda content was significantly elevated (p<0.05) in mtxn-treated rats (group iv, positive control) compared to the corresponding cardiac contents in negative control (group i) rats (table 2 and figure 2); these results are in line with the work of others. the increment in the product of lipid peroxidation (mda) may be considered as a causative factor in mtxninduced acute cardiotoxicity (22, 23) furthermore, results of this study showed that that oral administration of zinc sulfate [(15mg/kg/day with total cumulative dose (7.5 mg/kg) of mtxn] (group v), resulted in a non-significant (p>0.05) difference in mda contents in heart tissue homogenate compared to the corresponding contents in heart tissue in group of rats ip injected with total cumulative dose of 7.5 mg/kg of mtxn (group iv). moreover, there were significant reduction (p<0.05) in mda contents in heart tissue homogenate of rats orallyadministered zinc sulfate [(30 mg/kg/day) with total cumulative dose (7.5mg/kg) of mtxn] (group vi) compared to the corresponding mda contents in heart tissue homogenate of rats ip injected with total cumulative dose of 7.5mg/kg of mtxn (group iv). authors revealed that zn, a necessary metal that is integral to the activity of many metalloenzymes required for cellular functions; and it has critical, anti-atherogenic, antiinflammatory and antioxidant actions to protect against various oxidative stresses moreover, the authors describe that there is a strong basis to consider the strategy of zn supplementation to cardioprotect the heart against mi (24). furthermore, asri-rezaei siamak et al (2017) observed that, zinc sulfate administration could protect against lipid peroxidation, due to anti-oxidative effect that showed by a reduction in mda contents in rats. furthermore, others reported that zinc-induced a very efficient antioxidant in scavenging various free radicals [metallothionein (mt)] synthesis in the rat heart which in turn may alleviate diabetic cardiotoxicity (8). in addition, at 2015 hala attia et al reported that addition of zn to glibenclamide can significantly reduce mda in cardiac tissues compared to glibenclamide alone; this can support the powerful antioxidant effect of zn and potential cardio-protection from oxidative damage (25). conclusion zinc sulfate at a dose of (30 mg/kg/day) (in group vi rats) diminished the cardiotoxicity induced by mitoxantrone as indicated by the reduction of serum troponin i 3 enzyme level but non-significant difference in lipid peroxidation marker ( malondialdehyde) compared to (15 mg/kg/day) (in group v rats). iraqi j pharm sci, vol.29(1) 2020 effects of zinc sulfate on mitoxantrone-induced cardiotoxicity 121 acknowledgments this article was abstracted from a ph.d. thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. authors deeply indebted to dr. saleema s. salman, the university of baghdad, college of pharmacy for her valuable assistance in statistical analysis. references 1. seiter, k. toxicity of the topoisomerase ii inhibitors. expert opinion on drug safety. 2005; 4: 219–234. 2. kingwell e, koch m, leung b,et al. cardiotoxicity and other adverse events associated with mitoxantrone treatment for ms. neurology 2010; 74:1822–1826. 3. avasarala, j r, cross a h, clifford d b, et al. siegel, b. a., & abbey, e. e. rapid onset 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https://www.ncbi.nlm.nih.gov/pubmed/?term=bodor%20gs%5bauthor%5d&cauthor=true&cauthor_uid=8319322 https://www.ncbi.nlm.nih.gov/pubmed/?term=d%c3%a1vila-rom%c3%a1n%20vg%5bauthor%5d&cauthor=true&cauthor_uid=8319322 https://www.ncbi.nlm.nih.gov/pubmed/?term=d%c3%a1vila-rom%c3%a1n%20vg%5bauthor%5d&cauthor=true&cauthor_uid=8319322 https://www.ncbi.nlm.nih.gov/pubmed/?term=delmez%20ja%5bauthor%5d&cauthor=true&cauthor_uid=8319322 https://www.ncbi.nlm.nih.gov/pubmed/?term=apple%20fs%5bauthor%5d&cauthor=true&cauthor_uid=8319322 https://www.ncbi.nlm.nih.gov/pubmed/8319322 https://www.ncbi.nlm.nih.gov/pubmed/?term=dores-sousa%20jl%5bauthor%5d&cauthor=true&cauthor_uid=25582955 https://www.ncbi.nlm.nih.gov/pubmed/?term=duarte%20ja%5bauthor%5d&cauthor=true&cauthor_uid=25582955 https://www.ncbi.nlm.nih.gov/pubmed/?term=seabra%20v%5bauthor%5d&cauthor=true&cauthor_uid=25582955 https://www.ncbi.nlm.nih.gov/pubmed/25582955 https://www.ncbi.nlm.nih.gov/pubmed/?term=van%20dalen%20ec%5bauthor%5d&cauthor=true&cauthor_uid=15010064 https://www.ncbi.nlm.nih.gov/pubmed/?term=van%20der%20pal%20hj%5bauthor%5d&cauthor=true&cauthor_uid=15010064 https://www.ncbi.nlm.nih.gov/pubmed/?term=bakker%20pj%5bauthor%5d&cauthor=true&cauthor_uid=15010064 https://www.ncbi.nlm.nih.gov/pubmed/?term=bakker%20pj%5bauthor%5d&cauthor=true&cauthor_uid=15010064 https://www.ncbi.nlm.nih.gov/pubmed/15010064 https://www.ncbi.nlm.nih.gov/pubmed/16600075 iraqi j pharm sci, vol.29(1) 2020 effects of zinc sulfate on mitoxantrone-induced cardiotoxicity 122 22. vefki g k, i̇brahim m, süleyman d, hülya a, mete k,et al. the protection of the myocardium by amifostine against mitoxantrone-induced acute cardiotoxicity in rats.turk j hematol. 2010; 27: 62. 23. gurhan k, ibrahim m, hulya a, mete k, s uleyman d, et al. is amifostine effective on the protection of mitoxantrone-induced acute cardiotoxicity? a biochemical and histopathological experimental study. blood 2006; 108: 4364 24. nouf m a, hala a a, raeesa a m, nawal m a, maha a. preventive effects of selenium yeast, chromium picolinate, zinc sulfate and their combination on oxidative stress, inflammation, impaired angiogenesis and atherogenesis in myocardial infarction in rats. j pharm pharm sci 2013; 16(5): 848-867. 25. hala a, nouf a, nawal a, laila f. the combination of zinc and glibenclamide limits cardiovascular complications in diabetic rats via multiple mechanisms. pak j pharm sci. 2015; 28(2):499-508. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://www.ncbi.nlm.nih.gov/pubmed/25730804 https://www.ncbi.nlm.nih.gov/pubmed/25730804 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 liver protective effect of thymoquinone doi: : https://doi.org/10.31351/vol30iss2pp50-57 50 thymoquinone attenuates immune mediated liver injury induced by concanavalin a in mice halla a. ahmad*,1 and sarmed h. kathem** * ministry of health and environment, medical city health directorate, baghdad, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract autoimmune hepatitis is an inflammatory disease and its incidence has been increasing. the features of hepatitis are the release of inflammatory cytokines, the elevation of ast and alt, and hepatocyte apoptosis and necrosis. concanavalin a considered as essential model represents the acute immune-mediated liver damage in rodents. thymoquinone is well known herbal compound that exert hepatoprotective, anti-inflammatory, and antioxidant activity. in this study, we focus on the immunoregulatory and liver protective effect of thymoquinone in a mouse model of concanavalin a-induced liver injury. twenty-four male mice were randomly divided into four groups each containing six animals: negative control group, concanavalin a model group, thymoquinone 15mg/kg group, and thymoquinone 30mg/kg group. blood was collected to detect the activities of alanine transaminase (alt) and aspartate transaminase (ast) as well as liver tissue for the detection of tumor necrosis factor α (tnf α), nf-kb, and interferon γ (ifn γ) levels, after 8 hours of concanavalin a injection. injecting mice with concanavalin resulted in a dramatic increase in serum level of both alt and ast which indicate severe liver tissue damage with a significant increase in inflammatory cytokines tnf α, and inf γ levels, with a notable increase in nf-kb gene expression in mice liver tissue homogenate. thymoquinone pretreatment revealed a dose-dependent increase in liver tissue protection. conclusion: thymoquinone has hepatoprotective and immune modulatory effects in concanavalin a induced immune mediated liver damage. thymoquinone exerted its effect through attenuating nf-κb and its downstream effectors tnf α and inf γ in a dose dependent manner. keywords: concanavalin a, liver injury, thymoquinone, alt, ast, tnf-α, ifn-γ, nf-кb. نالثيموكينون يخفف من إصابة الكبد المناعية المستحثه بالكونكانافالين أ لدى الفئرا ** مكاظ سرمد هاشم و 1*،الكريم احمد بدهالة ع وزارة الصحة والبيئة، دائرة مدينة الطب، بغداد، العراق * اق**فرع االدوية والسموم، كلية الصيدلة، جامعة بغداد، العر الخالصة التهاب الكبد المناعي الذاتي هو مرض التهابي .تتمثل سمات التهاب الكبد المناعي في إطالق السيتوكينات ، االلتهابية ، وأرتفاع ذجآ اساسيا مستوى انزيمات الكبد )االنين امينو ترانسفيراس واسبارتات امينوترانسفيراس( وموت الخاليا المبرمج والنخر. كونكانافالين أ يعتبر نمو ات ومضادًا ليمثل تلف الكبد المناعي في القوارض. الثيموكينون هو مركب عشبي معروف جيدًا له فوائد كثيرة كحماية الكبدً و مضادًا لاللتهاب سببها حقن لألكسدة, وفي دراستنا نركز على التأثير المناعي والوقائي لمادة اللثيموكينون في نموذج فأر مصاب بمرض التهاب الكبد المناعي والتي ي .مادة الكونكانافالين أ ستة حيوانات ، مجموعة السيطرة السلبي، تم تقسيم أربعة وعشرين فأر من الذكور بشكل عشوائي إلى أربع مجموعات تحتوي كل مجموعة على مجم / 30/ كجم ، مجموعة ثيموكينون مجم 15مجموعة النموذج لمرض التهاب الكبد المناعي حقنت بمادة الكونكانافالين أ ، مجموعة ثيموكينون tnfأنسجة الكبد للكشف عن عامل نخر الورم)(, وكذلك astوأسبارتات أميناز) (altكجم. تم جمع الدم للكشف عن أنشطة ترانس أميناز أالنين) α) و , nf-κb و interferon-(ifn γ) ساعات من حقن كونكانافالين أ 8بعد. مما يشير إلى تلف شديد في أنسجة الكبد مع ast و alt يؤدي حقن الفئران بمادة الكونكانافلين أ إلى زيادة كبيرة في مستوى المصل لكل من في أنسجة كبد الفئران, كشفت المعالجة nf-κb ، مع زيادة ملحوظة في التعبير الجيني inf و tnf α توكينات االلتهابيةزيادة ملحوظة في السي .المسبقة بالثيموكينون عن زيادة في حماية أنسجة الكبد وينو ن الثايموكوينون له تأثيرات مناعية و وقائية للكبد في حالة تلف الكبد المناعي الذي يسببه الكونكانافالين أ . يمارس الثايموك: األستنتاج يرون كاما بطريقة تأثيره من خالل تقليل التعبير الجيني للنيوكليرفاكتر كابا بي وبالتالي التقليل من مستويات التيومر نكروسز فاكتر الفا واالنتيرف تعتمد على الجرعة. alt ،ast ،tnf α ،ifn γ ،nf-bالكلمات المفتاحية: كونكانافالين أ ، إصابة الكبد ، ثيموكينون ، 1corresponding author e-mail: taifhalla@gmail.com received: 7/9/2020 accepted: 13/ 2/2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp50-57 iraqi j pharm sci, vol.30(2) 2021 liver protective effect of thymoquinone 51 introduction the liver in the human body considered as the largest hard organ, approximately making 2% of body weight in adult, the liver is responsible for many important tasks that play a critical role in all physiological systems and supporting the function of many organs. the exclusive vasculature of the liver allows it to perform the removal of exogenous antigens and pathogens from the systemic circulation, and the degradation of waste products and toxins (1). several essential functions of the liver are local and systemic host defense including inflammatory reaction and both innate immunity and the more specific adaptive immunity (2). hepatitis is defined as inflammation of the liver. hepatic inflammation can be caused by alcohol, viruses, medication use, or exposure to toxins, and autoimmune diseases (3). in immunemediated hepatitis, the distinct distribution of hepatic chemokine receptors and its secretion that regulate the accumulation of immune response cells, and the obvious anatomical parts of the liver with its unique liver sinusoids and its paired portal and arterial blood supply permit the mobilization of distinct populations of circulating cells that present large surface for the interaction of lymphocyte and local cell, all these features in enable liver to carry an efficient immune response by recruitment of the standard lymphocytes (4). the innate immune system always activated before the adaptive immune system (5). the liver is highly perfused with innate immune cells, like natural killer (nk) cells, nkt cells, kupffer cells, and dendritic cells (dc), which result in exclusive immune responses against microbial pathogens (6)(7). macrophages (kupfer cells) in innate immune response display outstanding plasticity and can differentiate into macrophage 1 (m1 that linked with type 1 cytokines such as il-6, tnf α, and inf γ) and macrophage 2 (m2 subsets show the potent pro-inflammatory response) (8). adaptive immunity which is the cellular immunity plays an essential role in the pathogenesis of hbv related acute liver failure. the clearance of the virus depends principally on two ways: the straight killing of infected hepatocytes by cytotoxic t lymphocytes (ctls), or by the release of ifn γ(9). t helper cell responses and ctl responses are of significant importance in immune-mediated liver damage and viral control (10). the concanavalin a model is a distinctive and well-established model for inspecting t-cell and macrophage dependent liver damage in mice, which closely resemble the pathological changes and pathogenesis mechanisms of autoimmune hepatitis patients, and is considered as the best experimental model for immune-mediated liver damage study up to now (11). the predominant and extremely special activation of t cells of concanavalin a provides a suitable model for straight exploratory mechanisms of t cell-mediated hepatitis, which holds similarities to human autoimmune or acute viral hepatitis along with immune-mediated drug hepatotoxicity and may therefore help to explore new treatment options (12). in this study, evaluation of the liver protective effect of thymoquinone. thymoquinone is a principal active constituent of black seed oil nigella sativa. the chemical formula is 2-isopropyl5-methyl1,4-benzoquinones. thymoquinone is richly present in volatile oil in addition to fixed oil in n. sativa seeds (13). the important medical effect of thymoquinone can be linked to suppression of the nuclear factor-kappa b (nf-κb) signaling. most studies showed that thymoquinone suppresses tnf α and nf-κb activation with dose-dependent manner (14). material and method reagents concanavalin a, and thymoquinone were purchased from (hangzhou hyper chemicals) to induce immune mediated liver injury. tnf α and inf γ elisa kites were purchased from beijing solarbio science/china, and used to measure these mediators in liver tissue. nf-κb mrna expression detected using real time pcr, the primer was purchased from macrogen, korea, and the primer sequence used showed in table (1). alt and ast measured using randox kits using colorimetric method. table 1. reverse and forward primer sequence primer sequence nfkb1-f 5`-aagacaaggagcaggacatg-3` nfkb1-r 5`-agcaacatcttcacatcccc-3` ywhaz-f 5`-gatgaagccattgctgaacttg-3` ywhaz-r 5`-gtctccttgggtatccgatgtc-3` animals twenty-four adults' albino male mice weighing (20-30) gram were purchased from the animal house of college of pharmacy/university of baghdad and maintained under normal conditions of temperature, humidity, and light/dark cycle. the animals were fed commercial pellets and tap water throughout the experimental period. the study was approved by the scientific and ethical-committees of the college of the pharmacy/university of baghdad. experimental protocol twenty-four mice were divided into 4 groups, each group with 6 mice as follows: negative control group: animals in this group received 0.1 ml distilled water orally for 4 days starting day 1, with a single dose of 0.1 ml normal iraqi j pharm sci, vol.30(2) 2021 liver protective effect of thymoquinone 52 saline on day 4 by retro-orbital iv route and sacrificed after 8 hours. this group served as a negative control group. model control group: animals in this group received 0.1 ml distilled water orally for 4 days starting day 1, with a single dose of 0.1 ml concanavalin a 20mg/kg on day 4 by retro-orbital iv route and sacrificed after 8 hours. this group served as an immune mediated liver damage model group. thymoquinone 15mg/kg group: received 0.1ml thymoquinone (15 mg/kg dissolved in corn oil) by oral gavage for 4 successive days starting day 1, with a single dose of concanavalin a 20mg/kg on day 4 and sacrificed after 8 hours. thymoquinone 30mg/kg group: received 0.1ml thymoquinone (30 mg/kg) by oral gavage for 4 successive days starting day 1, with a single dose of concanavalin a 20mg/kg on day 4 and sacrificed after 8 hours. samples collection blood and serum samples collection on day 5 and exactly 8 hrs. after concanavalin a injection, about 1.5 ml of blood was drawn from the retro-orbital area of mice. blood were let to stand in room temperature for 30 mins for serum collection. blood centrifuged for 15 min. at 5000 rpm in cold centrifuge at 4 ˚c. the serum was collected and frozen for alt and ast determination. preparation of liver tissue homogenate after euthanization, and dissection of each mouse, liver was quickly excised, rinsed in ice-cold normal saline. after that, 100 mg of liver tissue were placed in eppendorf tube containing 1ml of trizol™ reagent and gently mixed by vortex and kept frozen for later use to measure mrna nf-κb level. take 150 mg from the remaining liver tissues were placed in eppendorf tube containing 1.5 ml of phosphate buffer saline. the liver tissues were homogenized by electrical homogenizer. then the homogenate centrifuged with a cold centrifuge for 15 minutes at 5000 rpm and the supernatant fluid collected and kept frozen for later use to measure tnf α and inf γ. statistical analysis the numeric data presented in the study were expressed as mean ± standard error of the mean (se). all statistical analyses were carried out using the statistical package of social science (spss) software version 25. intergroup comparisons were made using (student's t test). differences between the means were considered significant at p<0.05. results effect of thymoquinone pretreatment on serum alt and ast level data obtained from this study showed that concanavalin a administration in a dose 20mg /kg causes a significant and dramatic elevation of serum alt (122.14 ± 3.33 iu/l) and ast(183.65 ± 5.11 iu/l) levels (p ˂ 0.05), indicating severe liver injury in comparison to the negative control group alt (3.895 ± 1.05 iu/l) and ast (56.5 ± 4.56 iu/l) levels as shown in table (2) and figures (1) and (2). when mice pretreated with thymoquinone 15mg /kg resulted in significant decrease in alt (37.09±10.74 iu/l) and ast(113.84 ± 13.86 iu/l) levels (p ˂ 0.05) in comparison to the model control group received concanavalin a, group treated with thymoquinone dose 30mg /kg resulted in significant and massive reduction of alt (31.12 ± 3.82 iu/l) and ast (80.1 ± 11.27 iu/l) levels (p ˂0.05) in comparison to the model control group, as shown in the table (1) and figures (1) and (2). effect of thymoquinone pretreatment on tnf α level. analysis of data of present study shows that the administration of concanavalin a 8 hours before animal sacrificing results in significant (p˂ 0.05) and dramatic elevation of tnf α level (44.59 ± 2.05 pg/mg) in comparison to the negative control group (12.79 ± 0.53 pg/mg). this result in 3fold duplication of tissue tnf α level, as shown in figure (3) and table (2). pretreatment of animals with thymoquinone 15 mg/kg 4 days before injection with concanavalin a resulted in significant (p˂0.05) lowering in tnf α (24.27 ± 2.1pg/mg) in comparison to the model group (44.59 ± 2.05 pg/mg), doubling up the dose of thymoquinone to 30 mg /kg resulted in significant (p˂0.05) attenuation of tnf α level (17.67 ± 1.58 pg/mg) in comparing to the lower dose, these results indicate the dose dependent reduction effect of thymoquinone on tissue tnf α level as shown in table (1) and figure (3). effect of thymoquinone pretreatment on inf γ level. mice administered concanavalin a 20mg /kg by the retro-orbital rout in this study showed significant (p˂0.05) and notable elevation of the immunological marker inf γ by 4 folds (2.23 ± 0.052 pg/mg) in comparison to the negative control group which administered normal saline only (0.61 ± 0.046 pg/mg), as shown in table (2) and figure (4). the groups which were treated with thymoquinone showed dose-dependent attenuation of inf γ tissue level, 15mg /kg dose of thymoquinone was given to the mice showed a significant (p˂0.05) decrease in inf γ level (1.39 ± 0.02 pg/mg) in comparison to the model group (2.23 ± 0.052 pg/mg), doubling the dose of thymoquinone to 30mg /kg result in even more significant (p˂0.05) depletion of inf γ level (0.81 ± 0.018 pg/mg) as shown in table (1) and figure (4). effect of thymoquinone pretreatment on nf-κb gene expression as demonstrated in table (2) and figure (5), concanavalin a administration resulted in significant (p˂0.05) increase in hepatic gene iraqi j pharm sci, vol.30(2) 2021 liver protective effect of thymoquinone 53 expression of nf-κb by 3 folds (3.56±0.55 fold) compared to the non-treated negative control group (1.11±0.34 fold). on the other hand, treatment with thymoquinone (15mg/kg) showed a significant (p˂0.05) attenuation of nf-κb mrna expression (2.03±0.29 fold) compared to experimental model group (3.56±0.55 fold). further escalation in thymoquinone dose to (30mg/kg) resulted in more significant (p˂0.05) drop in nf-κb mrna level (1.6±0.16 fold) compared to experimental model group (3.56±0.55 fold), in this case the gene expression of nf-κb reduced by (30%). these data clearly revealed a dose-dependent attenuation of thymoquinone against nf-κb surge due to con a. table 2. effect of thymoquinone pretreatment on serum alt and ast levels, and levels of inf γ, tnf α, and nf-κb in liver tissue homogenate of mice: group type of treatment tissue inf γ (pg /mg) mean ± sem tissue tnf α (pg/mg) mean ± sem serum alt (iu/l) mean ±sem serum ast (iu/l) mean ± sem mrna nfκb level (fold) mean ±sem negative control group normal saline 0.61 ± 0.05 12.79 ± 0.53 3.895 ± 1.05 56.5 ± 4.56 1.11± 0.34 con a group con a 2.23 ± 0.05 # 44.59 ± 2.05 # 122.14±3.33 # 183.65±5.11 # 3.58 ± 0.54 # tq 15mg/kg group con a +tq 15mg/kg 1.39 ± 0.02 * 24.27 ± 2.1 * 37.09±10.74 * 113.84 ± 13.86 * 2.02 ±0.29* tq 30mg/kg group con a +tq 30 mg/kg 0.81 ± 0.018 * 17.67 ± 1.58 * 31.12 ± 3.82 * 80.1 ± 11.27 * 1.59 ±0.16* data are expressed as mean ± standard error of means (sem). (#) significant difference (p<0.05) compared to the negative control group, (*) significant difference (p<0.05) compared to the model control group, con a: concanavalin a, tq: thymoquinone, tnf α: tumor necrosis factor-alpha, inf γ: interferon-gamma, nf-κb: nuclear factor kappa b. figure 1.effect of thymoquinone pretreatment on serum alt level. (#) significant difference (p<0.05) compared to the experimental negative control group, (*): significant difference (p<0.05) compared to the model control group, con a: concanavalin a, tq: thymoquinone, n: negative control group figure 2.effect of thymoquinone pretreatment on serum ast level. (*): significant difference (p<0.05) compared to the model group, (#): significant difference (p<0.05) compared to the negative control group, con a: concanavalin a, tq: thymoquinone, n: negative control group figure 3.effect of thymoquinone pretreatment on tnf α level. (*): significant difference (p<0.05) compared to the model control group, (#): significant difference (p<0.05) compared to the negative control group, con a: concanavalin a, tq: thymoquinone, n: negative control group . iraqi j pharm sci, vol.30(2) 2021 liver protective effect of thymoquinone 54 figure 4. effect of thymoquinone pretreatment on inf γ level. (*): significant difference (p<0.05) compared to its corresponding model control group, (#): significant difference (p<0.05) compared to the negative control group, n: negative control group, con a: concanavalin a, tq: thymoquinone, inf γ: interferon gamma. figure 5.effect of thymoquinone pretreatment on nf-κb level. (*): significant difference (p<0.05) compared to the model control group, (#) significant difference (p<0.05) compared to the negative control group, n: negative control group, con a: concanavalin a, tq: thymoquinone, nf-κb: nuclear factor kappa b discussion hepatitis is defined as inflammation of the liver. hepatic inflammation can be caused by alcohol, viruses, medication use, exposure to toxins, or autoimmune diseases (15). both innate and adaptive immunity contribute to the pathogenesis of immune-mediated liver injury (9). concanavalin a is a plant agglutinin extracted from brazilian rubber beans that were used to study liver injury. concanavalin a model is a representative and easily built model of autoimmune hepatitis, it was shown to bind to mannosyl sugar residues of the insulin receptor in the liver, in addition the binding of concanavalin a to hepatocytes, endothelial cells, and kupffer cells is also reported (16). concanavalin a can modify the major histocompatibility complex (mhc) structure to produce inflammatory reactions, by activating macrophages and cd4+ t cells, which release tnf α, il-1β, il-6, and other inflammatory factors that damage hepatic cells (17). thus, concanavalin a treatment is a good simulation of the clinical onset of autoimmune hepatitis (aih) and viral hepatitis. the model also has the advantages of utilizing a simple extraction and causing liverspecific damage (injury to other organs is not obvious), thus providing a reliable animal model for clinical research in basic immunology (18). many research efforts recently oriented to explore the potential therapeutic activity of natural products develop more effective therapeutic strategies. in the present study, the work dedicated to investigate the immunomodulatory and liver protective effect of herbal active constituent thymoquinone using concanavalin a as a model of immune induced hepatitis. thymoquinone is one of the major bioactive components of the volatile oil of n. sativa seeds. studies showed that thymoquinone has immunomodulatory and anti-inflammatory effects, that prevent the biosynthesis of important mediators in inflammatory processes and reduce proinflammatory cytokines such as interleukins (ils) and tnf α. besides, thymoquinone showed the immunomodulatory role in cellular and humoral immunity (19)(20). analysis of data obtained from the current study revealed that concanavalin a administration by retro-orbital route result in a remarkable elevation in both ast and alt serum activities in comparison to the negative control group which received normal saline through retroorbital rout. injection of concanavalin a result in liver damage which can alter the permeability of the membrane, and cause the release of some hepatic specific enzymes. therefore, abnormally high levels of serum alt and ast indicate hepatic damage. these observations are in agreement with the studies of other researchers. previous study has shown that even doses as low as 1.5mg/kg are already capable of inducing mild immune-mediated hepatitis with slightly elevated transaminase levels but no further manifestation of liver disease (12). in another study, even a dose of 10mg/kg gives a significant result and rising the serum activity of alt and ast with obvious manifestations of immune-mediated liver damage (21). concerning thymoquinone treatment, results obtained from the present study revealed a thymoquinone hepatoprotective effect. this hepatoprotective effect concluded from the remarkable attenuation in alt and ast serum activities that were elevated by concanavalin a administration. this result is the first to demonstrate a hepatoprotective effect of thymoquinone in immune mediated liver damage, that means an immune modulatory activity has been proposed in the present study. however, other studies also showed hepatoprotective activity of thymoquinone using other experimental models, these models were induced liver damage in way that did not include a direct immune induction activity. these models include lipopolysaccharide (lps)-, sepsis-, methotrexate-, metal-, induced liver damage among other models (22)(23)(24)(25). each model induces liver damage in a unique mechanism but not through iraqi j pharm sci, vol.30(2) 2021 liver protective effect of thymoquinone 55 direct immune activation as con a does. the importance of immune mediated liver damage is to simulate human autoimmune and viral hepatitis, which considered an upgrowing health issue globally (26). for example, thymoquinone exerts a hepatoprotective effect against ccl4-induced liver damage. in this model the suggested mechanism of protection is antioxidant activity of thymoquinone as ccl4 induced the damage through oxidative stress mechanism (27). in another model, acetaminophen-induced liver damage is also reversed by administration of thymoquinone. similarly, the suggested mechanism of protection is the antioxidant activity of thymoquinone (28). furthermore, liver damage is also induced by nafld (non-alcoholic fatty liver disease) where thymoquinone also exerted a beneficial effect through metabolic modulation and antioxidant activity (29). antioxidant activity of thymoquinone is a well-known effect observed from many studies (30). the dose dependent hepatoprotective effect of thymoquinone were also observed in metal-induced liver damage. thymoquinone showed an attenuating effect on alt and ast in pb-induced hepatotoxicity that might be clinically useful in pb intoxication (24). however, pb-induced liver damage is through direct effect on enzymes and oxidative stress. however, the present study is the first to provide a clue that thymoquinone have immune mediated hepatoprotective activity as the damage induced by concanavalin a is via direct immune induction (11). in the present study effect from taking concanavalin a on tnf α 8 hours before animal sacrificing results in dramatic elevation tnf α value. this suggests that the pro-inflammatory cytokine tnf α play important role in the progression of concanavalin a-induced hepatitis. concanavalin a attachment to the endothelial lining of hepatocytes that mainly rich in kupffer cell ( liver macrophage ) and liver sinusoidal endothelial cells (lsec), therefor t cells become activated to cd4+ and secreting different immunogenic cytokines mostly tnf alpha (31). similar observations have been made by other study that revealed that serum level of tnf α dramatically increased after 1 hour from concanavalin a injection in dose 15mg/kg (32). results obtained from the analysis of data of the present study about thymoquinone groups improve its dose-dependent effect on immunomodulatory cytokine tnf α, that 15mg/kg dose of thymoquinone give significant reduction, 30mg/kg dose of thymoquinone dose resulted in a more remarkable decrease in tnf α in comparison to the model control group. other researcher approved that thymoquinone may be a potential small-molecule inhibitor to modulate tnf α induced signaling in rheumatoid arthritis synovial fibroblast (ra-fls), the molecular mechanism through which thymoquinone modulates tnf α response in ra-fls, is through thymoquinone effect on tnf α-induced mapk pathways, which are integral signaling mediators of tnf α-induced downstream inflammatory proteins in ra pathogenesis, that thymoquinone selectively inhibits tnf α-induced phosphorylation of p38 and jnk in a dose-dependent manner (33). mice administered concanavalin a in this study showed notable elevation of the immunological marker inf γ in comparison to the negative control group which administered normal saline only. results of this study were consistent with those of other authors who improved that concanavalin a administration results in a significant and remarkable increase in stimulation of nf-κb followed by liberation of inflammatory cytokine ifn γ which have a great role in inducing and maintaining inflammatory induced liver injury (34) (11). the animal groups which were treated with thymoquinone showed dosedependent attenuation of inf γ tissue value, doubling the dose of thymoquinone result in even more significant depletion of inf γ in comparison to the concanavalin a model group. results of the current study were consistent with another study, where they utilized guinea pig instead of mice as an animal model of asthma, and they provide that single dose of thymoquinone (3 mg/kg injected intraperitoneally) results in a significant decrease of inf γ level (35). concanavalin a-induced hepatic injury is associated with the release of proinflammatory cytokines such as tnf α and ifn γ. production of these and other inflammatory mediators mainly depends upon activation of nfκb, a ubiquitous transcription factor that regulates several genes involved in inflammation. a recent report has also shown that the expressions of the nfkb gene significantly decreased by thymoquinone administration in both dose and time-dependent manner (14). conclusion according to 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university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 73 guggulusome a novel vesicular carriers for enhanced transdermal delivery vivek dave *,1 , sachdev yadav * , swapnil sharma * and nitesh panwar ** * department of pharmacy, banasthali university, banasthali-304022, rajasthan, india. ** goenka college of pharmacy, lachhmangarh road, sikar-332313, rajasthan, india. abstract the present work describes guggul as a novel carrier for some anti-inflammatory drugs. guggulusomes containing different concentration of guggul with aceclofenac were prepared by sonication method and characterized for vesicle shape, size, size-distribution, ph, viscosity, spread ability, homogeneity, and accelerated stability in-vitro drug permeation through mouse skin. the vesicles exhibited an entrapment efficiency of 93.2 ± 12%, vesicle size of 0.769 ± 3μm and a zeta potential of 6.21mv. in vitro drug release was analyzed using franz’s diffusion cells. the cumulative release of the guggulusomes gel (g2) was 75.8% in 18 hrs, which is greater than that all the gel formulation. the stability profile of prepared system assessed for 45 days. the vesicular suspension was kept in sealed vials (10ml) at 4 ± 2ºc and at room temperature for 45 days no change was showned in entrapment efficiency. the optimized guggulusomes formulation showed transdermal flux 216.1μg/cm²/hr. the result advocates the potential of guggulusomes formulation to treat diseases where facilitated penetration of the drug into muscle and synovial fluid is desirable. in the end of the tests guggulusomes gel g2 with carbopol 934k was the most stable. the paw edema and percentage inhibition of carrageenan-induced paw edema in rats treated with aceclofenac guggulusomes gels and commercial formulation of aceclofenac gel marketed gel. the formulation g2 were devoid of any irritation potential and no edema formation was observed in any case. irritation score for aceclofenac guggulusomes gels was zero, which indicated its acceptability for topical administration. keywords: aceclofenac, gel, carbopol, guggulu carrier. introduction new drug delivery system that offers numerous advantages compared to conventional dosage forms. such systems often use macromolecules as carriers for the drugs (1) . guggul is used as an antiinflammatory, anti-hyperlipidemic, antiobesity, anti-cancer and is also used in cosmetics as anti-wrinkle, anti-acne in dermatitis etc. if aceclofenac drug is incorporated in guggul, it may give the synergistic effect (2) . by this concept of guggulusomes we can reduce the dose amount of these drugs due its synergistic effect and minimize the dose dependent side effects of these drugs. guggul is an ayurvedic extract sourced from the resin of the commiphora mukul tree of india. guggul contains guggulsterones, compounds that primarily promote healthy lipid metabolism. guggul also shows initial promise for supporting joint function and comfort. guggul moderated lipid peroxide levels, revealing important antioxidant action for lipid support. additional proposed mechanisms for the lipid modulating potential of guggul includes supporting liver enzyme and cellular membrane function, promoting receptor function, enhancing bile acid excretion, and promoting thyroid function. in addition, guggulsterones have been shown to support platelet function and fibrinolytic activity, helping to maintain blood vessel health and cardiovascular support (3,4) . guggul shows anti-inflammatory action as well as promises to deliver the drugs. the carrier that results is yet to be described named as ‘guggulusomes’ it has mixed properties of liposome, solid lipid particles and multiple emulsion etc. the purpose of this research was to find that guggul which has been proven medicinally could also function as a drug carrier. aceclofenac, an nsaid, has been recommended orally for the treatment of rheumatoid arthritis and osteoarthritis. it also has anti-inflammatory, antipyretic, and analgesic activities. the short biological halflife (about 4 h) and a higher dosing frequency make aceclofenac an ideal candidate for sustained release. the oral administration of aceclofenac causes gastrointestinal ulcers and gastrointestinal bleeding with chronic use. because of gastrointestinal bleeding, it also causes anemia. using the transdermal route eliminates these side effects, increases patient compliance, avoids first-pass metabolism, and maintains the plasma drug level for a longer period of time. 1 corresponding author e-mail: vivekdave1984@googlemail.com received: 21/2/2014 accepted: 24/4/2014 iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 74 guggul is one of the very ancient ayurvedic drugs, having been first described in atharva veda (2000 b.c). according to sushrut samhita, guggul is, when taken orally, curative of obesity, liver dysfunction, internal tumors, malignant sores and ulcers, urinary complaints, intestinal worms, leucoderma, sinus, edema and sudden paralytic seizures. it is also considered a cardiac tonic (5) . when drug materials, dyes or marker etc. are triturated with guggul in presence of water, the globules, particles or the vesicles formed in the dispersion, take them up. this uptake of is not limited to only lipophilic materials, the hydrophilic materials are taken-up as well. this behavior of guggul is analogous to the liposomes forming behavior of phospholipids. the entrapped moieties show much slower dialysis diffusion as compared to their true solution counterparts. the skin acts as a major target as well as a principle barrier for topical/transdermal (tt) drug delivery. the stratum corneum plays a crucial role in barrier function for tt drug delivery. despite major research and development efforts in tt systems and the advantages of these routes, low stratum corneum permeability limits the usefulness of topical drug delivery. to overcome this, methods have been assessed to increase permeation. one controversial method is the use of vesicular systems, such as liposomes, ethosomes and niosomes, whose effectiveness depends on their physicochemical properties (6) . hence the aim of present study was to develop sustained topical guggulusomes gel of aceclofenac and to evaluate with respect to various in vitro evaluation tests. presently no scientific reports are available on the formulation of topical drug delivery system of guggulusomes gel of aceclofenac. materials and methods purification of guggul guggul, which is a oleo-gum-resins, obtained from plant sources i.e. commiphora wightii or commiphara mukul (mukul myrrh tree) is one of such material, with main source species of family burceacea. visible impurities like sand, glass, stone impurities were removed from guggul manually. guggul was broken in to small pieces, put it in to muslin cloth bag and heat in a beaker with distilled water. the muslin cloth bag is hanged in a beaker so that the material remains immersed in liquid. the heating was continued until all guggul is converted in to liquid and coming out of muslin cloth bag. the content of muslin cloth is thrown; liquid is filtered and heated until soft mass is formed. (7) the soft mass is thus obtained was then dried in sunlight and triturated with mortar with addition of small amount of ghee. aceclofenac was received as a gift sample from lupin research park, pune, guggul (resin) was extracted from commiphora mukul tree which was authenticated by taxonomist, ethanol, propylene glycol, chloroform, from qualigens, mumbai, carbopol 934k and hpmc 15cps from sd fine-chemical limited, mumbai, all chemicals used were of analytical grade. preparation of guggulusomes aceclofenac guggulusomes were prepared using different concentrations of guggul lipid, ethanol, propylene glycol and aceclofenac as given in the (table 1). guggulusomes and drug were dissolved in ethanol and propylene glycol. the mixture was heated to 30ºc in water bath. in this solution distilled water was added slowly in a fine stream with a constant mixing (mechanical stirrer, remi equipment, mumbai) at 700rpm in a closed vessel. the temperature was maintained at 30ºc during the experiment. the mixing was continued for 5 minutes. the preparation was stored at 4ºc. guggulusomes prepared by the above procedure were subjected to sonication at 4ºc using probe sonicator in 3 cycles of 5 minutes with 5 minutes rest between the cycles. (8) incorporation into gel carbopol 934k 1%w/v and hpmc 15cps 1 % was soaked in minimum amount of water for an hour. guggulusomes suspensions 20 ml containing aceclofenac (200mg) was added to the swollen polymer under stirring. petroleum jelly was melted and liquid paraffin was mixed and the mixture was added to above prepared solution and mixed well. stirring was contained were then incorporated in gel with continuous stirring at 700 rpm in a closed vessel and maintained at temperature 30ºc until homogeneous guggulusomes gels were achieved. the ph was then adjusted to neutral using triethanol amine (tem) and stirred slowly till a gel was obtained. ph measurement of the formulations using ph meter by dipping the glass electrode completely into the semisolid formulation so as to cover the electrode (8-10) . fourier transform infrared spectroscopy infrared spectroscopy was conducted using a shimadzu ftir 8300 spectrophotometer and the spectrum was recorded in the region of 4000 to 400cm-1. the procedure consisted of dispersing a sample (drug and drug-excipient mixture, 1:1 ratio) in kbr (200-400mg) and compressing into discs by applying a pressure of 5 tons for 5min in a hydraulic press. the pellet was placed in the light path and the spectrum was iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 75 obtained. all spectra were collected as an average of three scans at a resolution of 2cm -1 . differential scanning calorimetry differential scanning calorimetry was performed by using dsc-60. the instrument comprised of calorimeter (dsc 60), flow controller (fcl 60), thermal analyzer (ta 60) and operating software ta 60 from (shimadzu corporation, japan.) the samples were placed in aluminium pans and were crimped, followed by heating under nitrogen flow (30ml/min) at a scanning rate of 5 o c/min from 25 o c to 200 o c. aluminium pan containing same quantity of indium was used as reference. the heat flow as a function of temperature was measured for both the drug and drug-excipient mixture. duplicate determinations were carried out for each sample. optical microscope observation and vesicular shape and surface morphology the guggulusomes dispersion was spread on the glass slide using a glass rod. formation of multilamellar vesicles was confirmed by examining the guggulusomes suspension under an optical microscope with the magnification power of 40x and 100x. (olympus cx41, philippines) photographs of vesicles were taken using olympus camera. scanning electronic microscopy (sem) was also conducted to characterize the surface morphology of the guggulusomes vesicles were analyzed by scanning electron microscopy (sem). prior to analysis, the guggulusomes were mounted onto doublesided tape that has previously been secured on copper stubs and coated with platinum, then analyzed at different magnifications. vesicle size and size distribution the vesicle size, size distribution and zeta potential of optimized guggulusomes formulation were determine by the dynamic light scattering (dls) using a computerized inspection system (malvern zetamaster (zem 500962, malvern, uk). the electric potential of guggulusomes, including its stern layer (zeta potential) was determine by injecting the diluted system into a zeta potential measurement cell. determination of viscosity a brookfield viscometer was used to measure the viscosities of all the formulations of gel prepared. a spindle (lv1) was rotated at 50rpm. samples of gel formulations were left to settle over 30 minutes at the temperature 32 ± 1°c before measurements were taken. viscosity was determined by using the following formula (11) . viscosity = dial reading × factor determination of spreadability one gram of gel is placed between two plates of 20×20cm. the mass of the upper plate was 125gm. after one minute the spreading diameter was measured. the spreadability was calculated by using the following formula (12,13) . s = mt/l where s = spreadability, m = weight of upper slide, l = length of the glass slide, t = time. determination of ph ph is an important parameter to be considered in gels as irritation on skin, compatibility and drug release may be altered. the ph for skin should ranges in between 6.4 to 6.8 (14) . determination of homogeneity it is visually tested by vision test and also by elegance effect. the +ve sign indicates the confirmation of good clarity and good elegance effect while –ve sign indicates the non homogeneity and non elegance effect (1416) . determination of drug content ten gram of the gel was taken and dissolve in 100ml buffer (ph 6.8) and shaken continuously until dissolved. the solution was ultrasonicated for 15 minutes. after filtration, the drug was suitably diluted and analyzed at uv spectrophotometer at 276nm (15) . entrapment efficiency the entrapment capacity of aceclofenac guggulusomes was measured by the ultracentrifuge method vesicular preparations containing 1% aceclofenac were kept overnight at 4˚c and centrifuged in a ultracentrifuge (remi) equipped with tla-45 rotor at 4˚c, at 30,000rpm for 2h. aceclofenac was assayed both in the sediment and in the supernatant. the entrapment capacity of aceclofenac was calculated from the relationship [(t-c)/t] 100, where t is the total amount of aceclofenac that is detected both in the supernatant and sediment, and c is the amount of aceclofenac detected only in the supernatant (16,17) . in-vitro permeation studies the in-vitro skin permeation of aceclofenac from guggulusomes formulation was studied using franz’s diffusion cell. the in-vitro diffusion of the drug through mouse skin was performed. the mouse skin soaked in a buffer for 6-8 hours. the two cell compartments will be held together with a clamp. the receptor compartment has a volume of 11 ml and will be filled with ph 6.4 buffer. it will be kept at 37°c by circulating water through an external water jacket. after 30min of equilibration of the membrane with the receptor solution, 200μl of the drug iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 76 solution will be applied in the donor compartment by means of a pipet. the donor compartment will be then covered with parafilm to prevent evaporation of the solvent. the receptor solution will be continuously stirred by means of a spinning magnetic bar, at 500rpm. receptor solution samples, 2.0ml aliquots, was withdrawn through the sampling port of the receptor compartment at various time intervals. the cells will be refilled with receptor solution to keep the volume of receptor solution constant during the experiment. the sample was withdrawn and replaced by 2.0ml with ph 6.4 phosphate buffer saline. the drug concentrations in the aliquot were determined at 276 nm against appropriate blank. this experiment was done in triplicate and average value was reported. (5,6) permeation enhancers these are compound, which promote skin permeability by altering the skin as a barrier to the flux of a desired penetrant. the flux, j, of drug across the skin can be written as: j = d dc/ dx where d is the diffusion coefficient and is a function of the size, shape and flexibility of the diffusing molecule as well as the membrane resistance, c is the concentration of the diffusing species, x is the spatial coordinate. the in vitro skin permeation of acelecfenac from guggulusomes formulation was studied using diffusion cell specially designed in our laboratory according to the literates. the effective permeation area of the diffusion cell is 2.013cm². the temperature was maintained at 37 ± 0.5ºc. (5,6) in-vivo efficacy study the anti-inflammatory activity of aceclofenac guggulusomes gels formulations was evaluated by the carrageenan-induced rat hind paw edema method (18,19) . the experimental protocol was designed and approval of institutional animal ethics committee (iaec) (reg. no. 0436) was obtained. healthy albino wistar rats of either sex weighing between 150-200 g were obtained from the disease free small animal house of ccshau, hisar. the animals were housed in institutional animal house under standard conditions with free access to food and water. anti-inflammatory activity of the aceclofenac guggulusomes gels was compared to the marketed gel of aceclofenac (ac rub). six albino wistar rats were divided into three groups of five animals each as follows: group 1 (control group): animals were treated with plain guggulusomes gels. group 2 (standard group): animals were treated with aceclofenac (ac rub). group 3 (carbopol 934k): animals were treated with guggul lipid loaded guggulusomes gels formulation. group 3 (hpmc): animals were treated with guggul lipid loaded guggulusomes gels formulation. inflammation was induced by sub-plantar carrageenan injection and after 1 hour, formulations were applied topically on the inflamed paw of rats by gently rubbing with index finger and the volume of the paw was measured. the thickness of paw was measured at 1h time intervals till 5h after carrageenan injection. a digital vernier caliper (aerospace, china) was used for measuring paw thickness of rats (20,21) . the percentage inhibition of inflammation was calculated by the following formula: percentage inhibition = [(c-t)/c] x 100] where c = control paw edema, t = test paw edema. draize skin irritation test lastly, each formulation was assessed for irritancy by conducting modified draize skin irritation tests on male white new zealand rabbits (3-4 kg) obtained from. approval for the use of animals in the study was obtained from the banasthali university. animal ethics committee (banasthali university. rajasthan, india, ref. no. bu/bt/184/11-12). for this purpose, a dorsal area on each restrained animal was shaved and then tape stripped three times to detach several upper layers of the stratum corneum. a 0.5 ml aliquot of each test guggulusomes gels was used in these areas which were then covered with a surface. after 4h, the gels were removed and the rabbits were observed over 14 days for signs of erythema, edema and ulceration. on days 1, 3, 7 and 14, visually-apparent cutaneous changes were assigned scores ranging between 0 and 4 with higher numbers signifying greater skin damage. the mean erythema scores were recorded depending on the degree of erythema: no erythema = 0, slight erythema (barely perceptible-light pink) = 1, moderate erythema (dark pink) = 2, moderate to severe erythema (light red) = 3, and severe erythema (extreme redness) = 4. stability study all regulatory bodies accept only real time data for any drug or pharmaceutical for all purpose of assessing the shelf life and only accelerated stability studies my serve as a tool for formulation screening and stability issues related to shipping or storage at room temperature. the accelerated stability studies were carried out in accordance with the ich guidelines. the ability of vesicles to retain the drug was assessed by keeping the iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 77 guggulusomes suspension at different temperature. optimized guggulusomes formulation was selected for stability studies of vesicles. the vesicular suspension was kept in sealed vials (10ml) at 4 ± 2ºc and at room temperature for 45 days. percent entrapment was determined at different time intervals. it was observed that the guggulusomes system was more stable at 4 ± 2°c. results and discussion fourier transform infrared spectroscopy the possible interaction between the drug and the excipients were studied by ir spectroscopy. ir spectra of pure aceclofenac, its physical mixture with guggul lipids and the prepared formulation are shown in figure 1. pure aceclofenac showed major peaks at 3317.3, 2970.2, 2935.5, 1716.5, 1589.2, 1506.3, 1479.3, 1344.3, 1280.6, 1255.6, and 665.4 cm -1 the result revealed no considerable change in the ir peaks of aceclofenac in the physical mixture or in the prepared crystals when compared to pure drug there by indicating the absence of any interaction. figure (1) ftir spectra of a) aceclofenac b) guggle lipid c) carbopol 934k d) hpmc e) formulation g2 f) formulation g6 . differential scanning calorimetry the results of dsc studies are given in figure 2. pure aceclofenac showed a sharp endotherm at 157.2°c corresponding to its melting point/transition temperature. there was no appreciable change in the melting endotherms of the physical mixture (aceclofenac + polymer) compared to pure drug. this observation further supports the ir spectroscopy results , when our optimised formulation g2 showed a sharp endothern on 154.7°c which indicated the absence of any interactions between drug and additives used in the preparation. however there was slight decrease in the melting point of the drug. it was also observed that there was a noticeable reduction in the enthalpy of the formulation with compare to aceclofenac formulation g2 showed -3.6j/mg. iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 78 . figure 2: dsc spectra of a) aceclofenac b) guggle lipid c) carbopol 934k d) formulation g2 optical microscope observation and vesicular shape and surface morphology the aceclofenac guggulusomes dispersion was spread on the glass slide using a glass rod. formation of multi-lamellar vesicles was confirmed by examining the aceclofenac guggulusomes suspension under an optical microscope with the magnification power of 40x and 100 x. (olympus cx41, philippines) as showed in figure 3. photographs of vesicles were taken using olympus camera. surface morphology and three-dimensional nature of guggulusomes were further confirmed by sem, justifying the vesicular characteristics possessed by this novel carrier figure 4. figure 3: optical microscope observation of guggulusome a) 40x b) 100x figure 4: sem of guggulusome formulation g2 vesicle size and size distribution vesicular sizes are the basic parameter on the basis of which the formulations were optimized. the effect of guggul lipid and ethanol concentration on the size distribution of guggulusomes vesicles was investigated using dynamic light scattering (dls) method. formulation g 2 has concentration of guggul lipid 3% with carbopol 934k and g 6 have concentration of guggul lipid 3% with hpmc but ethanol concentration is same. the data shows narrow particle size distribution with an average vesicles size of 0.769μm of formulation g2 and formulation g6 particle size was shown 1.240µm. the size of the vesicles was increase with increasing guggul lipid concentration. determination of viscosity viscosity was measured by brookfield viscometer for all the formulations of gel. a spindle (lv1) was rotated at 50rpm. samples of gel formulations from g1 to g8 at the temperature 32°c±1° were taken as showed in (table 1). viscosity was determined from 7485±0.563 to 8760 ±0.316 as showed in (table 1). determination of spreadability spreadability is expressed in terms of time in seconds taken by two slides to slip off from gel and placed in between the slides under the direction of certain load. lesser the time taken for separation of two slides, better the spreadability. (12-15) the aceclofenac guggulusomes formulation g1 to g8 having optimum viscosity and spreadability as showed in (table 1). the value is lie between 15.6±0.445 to 20.84±0.239. determination of ph and homogeneity accurately 2.5 gm of each gel formulation was weighed and dispersed in 25 ml of purified water. the ph of the dispersion was measured by using digital type of ph meter. (5,6) the guggulusomal gel formulation g1 to g8 ph lie between 6.2 to 6.7 and from the results it is concluding that all the gel formulation showed good appearance and homogeneity as showed in (table 1). iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 79 table (1) composition and characteristics of aceclofenac guggulusomes gel entrapment efficiency the entrapment efficiency of guggulusomes was determined for all formulation from g1 to g8. effect of ethanol concentration was observed on percent entrapment of guggulusomes. the entrapment efficiency was maximum for formulation g2 (93.26%) and minimum for formulation g5 (79.15%). solubility of aceclofenac also increased when ethanol was used in concentration. therefore, the drug also entrapped in the core of the vesicles. (5,16,17) the entrapment efficiency increased with an increase in concentration of guggul lipid but, above 3% of guggul lipid concentration there was no significant increase in percent entrapment. in formulations g1 to g4, percentage guggul lipid concentration was varied 2% to 5% and the concentration of ethanol is same that is 5%. similarly in formulations g5 to g8, percentage guggul lipid concentration was varied 2% to 5% and the concentration of ethanol is same that is 4%. this indicates that guggul lipid play an important role in percent entrapment. it is shown that increase in the guggul lipid concentration increases entrapment but, above 3% guggul lipid concentration there was no significant increase in the entrapment efficiency. the results show that as the concentration of ethanol increased from the entrapment efficiency increased as showed in (table 1) and (figure 5). figure 5: percentage entrapment efficiency of guggulusome composition in % (w/w) g1 g2 g3 g4 g5 g6 g7 g8 drug 1 1 1 1 1 1 1 1 guggul lipid 2 3 4 5 2 3 4 5 ethanol (ml) 5 5 5 5 4 4 4 4 propylene glycol 1 1 1 1 1 1 1 1 liquid paraffin 1 1 1 1 1 1 1 1 white petroleum jelly 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 carbopol 934k 1 1 1 1 hpmc 15cps 1 1 1 1 water q.s q,s q.s q.s q.s q.s q.s q.s characterization % entrapment efficiency 83.0± 3 93.2± 6 91.0± 8 89.5± 1 79.15±5 93.12±1 89.78±7 82.25±4 vesicle size (µm) 0.769 1.240 viscosity (cps) 7485± 0.563 8020 ± 0.746 8274± 0.131 8465 ± 0.487 8015± 0.431 8453± 0.869 8584± 0.351 8760 ± 0.316 spreadibility 15.68± 0.949 17.36± 0.429 15.6± 0.445 16.16± 0.127 19.12± 0.854 20.84± 0.239 18.45± 0.45 19.15± 0.276 homogeneity gel strength (n/g) ++ ++ ++ ++ ++ ++ ++ ++ ph 6.7 6.6 6.5 6.7 6.8 6.7 6.2 6.5 j (flux) (µg/cm²/hr) 196.8 216.1 199.6 197.54 201.8 209.4 195.5 192.0 r(regression coefficient) 0.9886 0.9464 0.991 iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 80 in-vitro permeation study release study of different formulations was undertaken in a modified diffusion apparatus containing mouse skin. all the formulations had propylene glycol as a skin penetration enhancer. the in vitro drug releases of various formulations are shown in figure 6. figure 6: in-vitro drug release profile from guggulusomes formulation the cumulative release of the guggulusomes gel (g2) was 75.8% in 18hrs, which is greater than that all the gel formulation. the formulation was having carbopol 935 as polymer from g1 to g4 and formulation g5 to g8 having hpmc as polymer. ethanol provides the vesicles with soft flexible characteristics, which allow them to more easily penetrate into deeper layers of the skin. when guggul lipid concentration was increased, there was increase in drug permeation. use of propylene glycol also influenced the amount of drug permeation. propylene glycol also acts as permeation enhancer, which increases permeability of vesicle through biological membrane due to synergistic effect with ethanol on bilayer of the vesicles. formulation (g1) containing 2% guggul lipid showed 69.7% drug release within 18h. formulation (g2) containing 3% guggul lipid showed 75.81 % drug release within 18h. where as formulation (g3 and g4) containing 4% and 5% guggul lipid showed 61.1 % and 52.8% drug release within 18h. in formulation g1 to g4 carbopol 934 used as base. similarly formulation (g5) containing 2% guggul lipid result showed 63% drug release within 18h. and formulation (g6) containing 3% guggul lipid result showed 71.8% drug release within 18h. where as formulation (g7 and g8) containing 4% and 5% guggul lipid showed 56.4 % and 58% drug release within 18h. in-vivo efficacy study (table 2) shows the results of paw edema and percentage inhibition of carrageenaninduced paw edema in rats treated with aceclofenac guggulusomes gels and commercial formulation of aceclofenac gel (ac rub). the control group consisted of rats treated with plain aceclofenac gel base. the results revealed a significantly higher inhibition of carrageenan-induced paw edema in rats treated with guggul lipid-loaded guggulusomes gels as compared with the control animals. however, an highly significant inhibition of carrageenan induced paw edema was observed in animals treated with commercial reference product (ac rub) gel in comparison with the control during the entire 5h duration of the study. further, the results of percent inhibition of paw edema produced by reference products were statistically higher as compared to the guggul lipid loaded guggulusomes gels. the study shows that the guggul lipid loaded guggulusomes gels possesses fair antiinflammatory activity but it is not as good antiinflammatory as the commercial product of aceclofenac. thus, aceclofenac guggulusomes gels is a potential anti-inflammatory formulation. aceclofenac guggulusomes gels were devoid of any irritation potential and no edema formation was observed in any case. irritation score (18,20) (primary skin irritation index) for aceclofenac guggulusomes gels was zero, which indicated its safety and acceptability for topical administration. stability study the ability of vesicles to retain the drug was assessed by keeping the guggulusomes suspension at different temperature. optimized guggulusomes formulation g2 was selected for stability studies of vesicles. the vesicular suspension was kept in sealed vials (10ml) at 4 ± 2ºc and at room temperature for 45 days. percent entrapment was determined at different time intervals. it was observed that the ethosomal system was more stable at 4 ± 2°c showed in (table 3). iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 81 table( 2) paw thickness and percentage inhibition (paw edema) in different groups at various time intervals. values are the mean ± sem (n = 5) table( 3)stability study of optimized formulation (g2) aceclofenac guggulusomes time ( days) percent entrapment (4 ± 2°c) percent entrapment ( rt) 1 92.1±0.2 91.1±0.8 15 91.0±0.8 89.8±0.6 30 90.4±0.6 85.2±0.4 45 87.9±0.7 82.9±0.3 mean ± sd, n = 3, rt = room temperature conclusion the result advocates the potential of guggulusomes gel formulation to treat rheumatic disease where facilitated penetration of the drug into muscle and synovial fluid is desirable. in light of the data obtained from experimental work we can expect the guggulusomes gel formulation to be safe and very efficient as a drug carrier for systemic as well as topical delivery of drug, holding future in effective transdermal delivery. it is envisaged that this particular formulation should be the basis of further studies in the clinically relevant environments. acknowledgement authors would like to thank to prof. aditya shastri, honorable v.c., banasthali university, rajasthan, india. references 1. rolland a. pharmaceutical particulate carrier, therapeutic application, marcel dekker., usa 2002; 61: 1-10. 2. altern. anti-inflammatory avtivity of guggul, the health med., 2003; 9: 74-79. 3. indian herbal pharmacoepia. revised new edition. mumbai, indian drug manufactures association., 2002, 134-143. 4. jain a., gupta v. b., chemistry and pharmacological profile of guggula review, indian journal of traditional knowledge., 2006; (5): 478-483. 5. dave v., kumar d., lewis s., paliwal s., ethosome for enhanced transdermal drug delivery of aceclofenac, international journal of drug delivery., 2010; (2): 81-92. 6. vyas s. p., dixit v. k., advanced in liposomal therapeutics, 1st ed. new delhi, cbs publishers and distributors., 2001; 231-239. 7. scott a., gum guggul and some of its steroidal constituents review of toxicological literature, integrated laboratory systems. inc. research triangle park, north carolina. 2005; 1-39. 8. touitou e., dayan n., bergelson l., godin b., eliaz m., ethosomes-novel vesicular carriers for enhanced delivery: characterization and skin penetration properties, j. control. release., 2000; (65): 399-403. 9. touitou e., composition of applying active substance to or through the skin, us patent. 1996; 5,716,638. 10. touitou e., composition of applying active substance to or through the skin, us patent. 1998; 5,540,934. 11. nokhodchi a., james l., philip h., rubinstein m. h., the effects of group treatment paw volume (mm)* (percentage inhibition) 1 h 2 h 3 h 4 h 5 h i control 5.37 ± 0.13 5.26 ± 0.19 5.42 ± 0.22 5.56 ± 0.12 5.43 ± 0.07 ii standard 4.24 ± 0.37 (21.02%) 4.12 ± 0.43 (21.6%) 4.36 ± 0.32 (19.5%) 4.29 ± 0.17 (22.8%) 4.13 ± 0.28 (23.9) iii g2 5.14 ± 0.14 (4.2%) 5.08 ± 0.13 (3.4%) 5.00 ± 0.24 (7.7%) 4.97± 0.15 (1%) 5.03 ± 0.11 (7.3) iv g6 4.68 ± 0.19 (1.2%) 4.72 ± 0.16 (1%) 4.88 ± 0.18 (9.9%) 4.80 ± 0.19 (1.3%) 4.73 ± 0.20 (1.2%) iraqi j pharm sci, vol.23(1) 2014 guggulusome a novel vesicular carriers 82 compression rate and force on the compaction properties of different viscosity grades of hydroxypropylmethylcellulose2208, international journal of pharmaceutic., 1996;129: 2131. 12. jones d. s., woolfson a. d., brown a. f., texture, viscoelastic, and mucoadhesive properties of pharmaceutical gels composed of cellulose polymers, int. j. pharm., 1997; (151): 223-233. 13. duggin g., softening skin with emollient ingredients, manufacturing chemist., 1996, (6): 27-31, 14. kotwal s. m., shankar v., immobilized invertase biotechnology advances., 2009; (27): 311-322. 15. ubaidulla u., reddy v. s., ruckmani k., ahmad f. j., khar r. k., transdermal therapeutic system of carvedilol: effect of hydrophilic and hydrophobic matrix on in vitro and in vivo characteristics, aaps pharmscitech., 2007; (8): 13-20. 16. touitou e., godin b., dayan n., weiss c., piliponsky a., levi-schaffer f., intracellular delivery mediated by an ethosomal carrier, biomaterials., 2001; 22: 3053 -3059. 17. dayan n., touitou e., carriers for skin delivery of trihexyphenidyl hci: ethosomes vs. liposomes, biomaterials., 2000; (21):1879 1885. 18. bamgbose s. o. a., noamesi b. k., studies on cryptolepine. ii: inhibition of carrageenan induced oedema by cryptolepine, planta med., 1981; (4):3926. 19. sewell r. d. e., spencer p. s. j., antinociceptive activity of narcotic agonist and partial agonist analgesics and other agents in the tail immersion test in mice and rats, neuropharmacology., 1976; (11):683-8. 20. rice d. p., ketterer d. j., restrainer and cell for dermal dosing of small laboratory animals, lab anim sci., 1977; (1):72-5. 21. ngo m. a., maibach h. i., dermatotoxicology: historical perspective and advances, toxicol appl pharmacol., 2010; (2):225-38. iraqi j pharm sci, vol.31( 2 ) 2022 progression and prediction factors for covid-19 doi: https://doi.org/10.31351/vol31iss2pp39-49 39 descriptive, prospective observational studystudying possible prediction factors for disease severity and progression among a sample of covd 19 patients in iraq duaa m .habib*, 1 and zinah m. anwer** * ministry of health and environment, baghdad, iraq **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq abstract coronavirus has affected many people around the world and caused an increase in the number of hospitalized patients and deaths. the prediction factors may assist the clinician in identifying patients who are at high risk of complications and require further medical attention. we aimed to study the possible relationship between c reactive protein level and the severity of symptoms and its effect on the prognosis of the disease. and determine patients who require closer respiratory monitoring and more aggressive supportive therapies to avoid poor prognosis. the data was gathered using medical record data, the patient's medical history, and the onset of symptoms, as well as a blood sample to test the c-reactive protein level. the patients were divided into three groups based on the severity of the disease. a descriptive, prospective observational study of 246 patients over the age of 18 years discovered that c-reactive protein levels were significantly higher in more severe cases than in mild cases, and that older patients with high levels of ast, tsb, urea, creatinine, and crp were associated with the need for a high flow of oxygen, an intensive care unit, a longer length of hospitalization, and have a high mortality rate. the study concluded several predictor factors for the disease (covid-19) severity, duration of hospitalization, icu admission and need for oxygen therapy. key wordscovid-19, prediction factors, severity of disease, length of hospitalization, intensive care unit, death. دراسه عوامل التنبؤ المحتملة لشدة المرض وتطوره في عينة من -دراسة وصفيه مستقبليه المحتمله في العراق 19مرضى كوفيد ** زينه مظفر انور و 1 ،*دعاء محمد حبيب ، العراق بغدادصحة والبيئة، دائرةوزاره الصحة * ، بغداد ، العراق بغداد الصيدلة جامعة السريرية، كليه ةالصيدلفرع ** الخالصة اصاب فايروس كورونا العديد من االشخاص حول العالم وتسبب في زياده اعداد المرضى في المستشفيات وزياده الوفيات. من الممكن عوامل التنبؤ الطبيب لتصنيف المرضى الذين يحتاجون عنايه طبيه اكثر لتقليل الوفيات وتدهور حاله المريض. الهدف من الدراسه هو ان تساعد ى معرفة العالقه المحتمله بين مستوى البروتين التفاعلي سي وشده االعراض وتاثيره على تدهور االعراض ، وتحديد المرضى الذين يحتاجون ال عالجات داعمه اكثر للتقليل من تدهور االعراض. تم جمع البيانات باالعتماد على السجالت الطبيه ،التاريخ المرضي وبدايه االعراضوتم اوكسجين و 246اخدعينه دم الجراء فحص البروتين التفاعلي سي قسم المرضى حسب شده المرض الى ثالثه مجاميع. دراسه وصفيه مستقبليه المحتمله ل عاما وجدت ان مستوى البروتين التفاعلي سي يكون اعلى بشكل كبير في الحاالت الشديده منه في الخفيفه،كما وجدت 18هم اكثر من مريض اعمار تين التفاعلي ان كبار السن والذين لديهم مستوى عالي من انزيم ناقلة األمين أسبارتات ،تحليل البليروبين الكلي، اليوريا، الكريتنين، ومستوى البرو .استنتجت ي كان لها عالقه كبيرة باالحتياج لمستوى عالي من االوكسجين ، وحده العنايه المركزه، مده بقاء اكثر في المستشفى ، ومعدل وفيات اعلىس كوفيد مرض لشده التنبؤيه العوامل من العديد وجود وال 19الدراسه المركزه العنايه وحده الى الدخول المستشفى، في البقاء مده الى ، حاجه .االوكسجين .عوامل التنبؤ ، شده المرض ، مده البقاء في المستشفى ، وحده العنايه المركزه ، والوفاة 19كوفيد -الكلمات المفتاحيه introduction since december 2019, the coronavirus disease, sever acute respiratory syndrome coronavirus-2 (sars-cov-2) has had a very negative effect on humans (1). the pathogen that triggers covid-19 is a novel coronavirus ( ncov) called sever acute respiratory syndrome coronavirus-2 (sars-cov-2) sars-cov-2 also known as novel corona virus disease-2019 [covid-19-ncov] that was first identified in late january 2020 (2).coronaviruses belong to the subfamily coronavirinae, one of the two members of the family coronaviridae (3). the world health organization (who) announced covid-19 a worldwide pandemic on11 march 2020 (4). 1corresponding author e-mailduaamohammed8888@gmail.com received: 26 / 8 /2021 accepted: 15 / 11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp39-49 iraqi j pharm sci, vol.31(2) 2022 progression and prediction factors for covid-9 40 coronavirus first appeared in iraq on 24 february 2020, in al-najaf city, south of baghdad. two weeks later, the iraqi ministry of health (moh) revealed that 101 instances of covid-19 had been tested positive in 14 provinces, with 9 deaths. baghdad city accounted for nearly 40% of those cases (5). infected individuals are thought to be the most important source of sars-cov-2 infection. droplets and close contact with covid19 patients appear to be the most prevalent method of transmission, sars-cov-2 is mostly transferred from symptomatic patients by coughing or sneezing (6).globally, as of 3 june 2021, there have been 171,292,827 confirmed cases of covid-19, including 3,687,589 deaths, reported to who , as of 2 june 2021, a total of 1,581,509,628 vaccine doses have been administered. the reported cases in iraq, from 3 january 2020 to, 3 june 2021, was 1,210,105 with 16,436 deaths, as of 25 may 2021, a total of 442,234 vaccine doses have been administered (7). the incubation period ranges from 0 to 24 days, with an average of 5–7 days. individuals of any age are susceptible to infection, including neonates and pregnant women. the majority of patients present with mild to moderate symptoms (8). acute respiratory distress syndrome, septic shock, metabolic acidosis, coagulation malfunction, and multiple organ dysfunction syndromes can all occur in severe instances and the death rate for patients with severe covid-19 is rather significant (9). patients with concomitant conditions may be more likely to develop severe covid-19 (10). liver damage has been known to occur during the course of the disease in severe condition (11). acute kidney damage (aki) has been documented in severely ill sars-cov-2 patients, particularly those with underlying medical conditions (12). c-reactive protein (crp) is a widely used diagnostic marker for determining the severity of chronic inflammation. it is an acute phase response protein that appears in the blood 6–10 hours after tissue injury, has a plasma half-life of 19 hours, and doesn't require a memory response to be produced(13). c-reactive protein is produced predominantly by hepatocytes in the liver, but also by smooth muscle cells, macrophages, endothelial cells, lymphocytes, and adipocytes in the body (14). the study aimed to find the possible relationship between c reactive protein (crp) level and the severity of symptoms and its effect on the prognosis of the disease among a sample of covid19 patients in iraq and to identify patients who require closer respiratory monitoring and more aggressive supportive therapies to avoid poor prognosis. during the study, we searched for additional predictors, such as another laboratory parameter, chronic disease, and schedule medication use, to see whether they might be utilized as predictors. methods study design and patients this is a descriptive, prospective observational study which was conducted between november-13th2020 and march-21st -2021. the data was collected from 246 iraqi patients aged 18 years or older who were admitted to al shefaa center and al-hayat hospital in baghdad and were confirmed to have covid-19 infection by using real-time polymerase chain reaction (pcr) and chest computed tomography (ct). inclusion criteria • inpatient positive for covid-19. • agree to participate in the study. • aged ≥ 18 years exclusion criteria • the patient who voluntarily left the hospital before his health condition improved. • patient admitted after the onset of symptoms for more than two weeks. • immunocompromised patient. • patients who died within a few days after being discharged from the hospital.( discharge or death was the end point of patient follow-up; some patients were needed to return to the hospital, and a few died just days after being discharged. • cancer patient. data collection within 24 hours of the patient being admitted to the ward, the data was based on the medical records, the patient’s medical history and the onset of symptoms recorded by the patients, and a blood sample was taken to test the c-reactive protein level by using the genrui and beckman device in the laboratory department. follow up the progression of symptom within three weeks of hospitalization, the patients were placed into groups based on the severity of the disease, the recovery group were discharged patients and there was no need to be admitted to an intensive care unit, the worsening group were patients who need an intensive care unit (icu) and a high flow of oxygen supply, and the mortality group include died patients. clinical classification clinical classification was carried out according to the ministry of health guidelines, with following standards 1. mild typemild clinical symptoms without pneumonia in chest imaging performance. 2. moderate typefever, respiratory tract and other symptoms with pneumonia in imaging performance. 3. severe typeany one condition of the followings respiratory distress, respiratory rate ≥ 30 times/min; fingertip oxygen saturation ≤ 93% in resting state; arterial oxygen partial pressure/fraction of inspired oxygen ≤300 mmhg. iraqi j pharm sci, vol.31(2) 2022 progression and prediction factors for covid-9 41 4. critical typeany one condition of the followings respiratory failure need mechanical ventilation; shock; other organs failure need intensive care unit (icu) monitoring and treatment (15). statistical analyses descriptive statistics (means, standard deviation, frequencies and percentages) were conducted for all study items. data was analyzed using the statistical package for the social sciences (spss) software, version 25. for association of continuous variables, we used correlation while for categorical variable, we used chi-square. additionally, kruskual wallis test and mann whitney test are applied to investigate differences among means for continuous ( not normally distributed variables)the kruskal wallis test was used to determine if there are statistically significant differences between the severity of the disease and many biomedical parameters, use this test instead of anova as that when we checked the normality test we found that the data was not normally distributed, anova was incorrect to use because the parametric test need to assume normality. depending on the assumptions we made that data's are not normally distributed, the non-parametric test mann-whitney test used instead of independent ttest. k wallis & m whitney tests are non-parametric test and applied to investigate differences among means .p-value of less than 0.05 was considered statistically significant. results the study included 246 patients with covid-19. their average age was 58.28 ± 14.824. the majority were men (54.5%). patients were admitted with severe condition (38.6 %) and 70.7% recovered. approximately half (49.6%) of the patients were taking one or more chronic medication(s) before admission as illustrated in table-1. table-1. descriptive characteristics of the patients. character subcategory n % gender female 112 45.5 male 134 54.5 smoker former smoker 89 36.5% disease severity mildmoderate 71 28.9 severe 95 38.6 critical 80 32.5 total 246 100.0 patient outcome recovered 174 70.7 died 72 29.3 number of chronic diseases 1.00 62 39.0 2.00 48 30.2 3.00 28 17.6 4.00 16 10.1 5.00 5 3.1 total 159 100.0 number of chronic medications 1.00 36 29.5 2.00 33 27.0 3.00 23 18.9 4.00 15 12.3 ≥5.00 15 12.3 total 122 100.0 minimum maximum mean std. deviation bmi 17.3 58.6 31.0324 6.88222 age 18 93 58.28 14.824 bmi=body mass index (n=237) age (n=244) there were no differences in the severity of disease among the patients related to smoking and gender as shown in table-2. iraqi j pharm sci, vol.31(2) 2022 progression and prediction factors for covid-9 42 table 2. the difference in the disease severity according to the gender and smoking. covid-19 severity critical mildmoderate sever p-value smoking no count 48 46 61 .825 % within 31.0% 29.7% 39.4% yes count 31 25 33 % within 34.8% 28.1% 37.1% gender female count 37 36 39 .460 % within 33.0% 32.1% 34.8% male count 43 35 56 % within 32.1% 26.1% 41.8% pearson chi-square with the p-value of (0.001) age was related to disease severity, need of intensive care unit admission and the use of high flow of oxygen supply. it is the mean that as the age increase disease severity, the need of intensive care unit admission and the use of high flow of oxygen supply was also increase as illustrated in table-3. table 3. the differences in need for oxygen (cpap and nrm), icu admission and disease severity according to patient age. n mean rank for age p-value cpap& no need to use cpap 182 111.53 .001* need to use cpap 62 154.70 nrm& no need to use nrm 56 90.91 .001* need to use nrm 188 131.91 icu admission& no need icu admission 181 110.43 .001* need icu admission 63 157.17 severity⁑ mild-moderate 71 77.92 .001* severe 94 122.94 critical 79 162.04 &mann-whitney test and ⁑kruskal wallis test. * the mean difference is significant at the 0.05 level. cpap= continuous positive airway pressure. nrm=non rebreather mask. the severity of disease was related to high level of c-reactive protein as demonstrated in table-4. table 4. the difference in c-reactive protein according to the disease severity. n mean rank for crp mg\l p-value severity mild-moderate 58 30.59 .000* sever 80 117.27 critical 62 144.27 total 200 * significant (p-value <0.05) according to kruskal wallis test; crp = c-reactive protein. from 246 patients, crp was tested for 200 patients only. iraqi j pharm sci, vol.31(2) 2022 progression and prediction factors for covid-9 43 the differences among the three groups of severity were significant according to post-hoc (pairwise analysis) mild-moderate vs severe, mild-moderate vs critical and severe vs critical as shown in figure -1 figure 1. pairwise comparisons of severity. each node shows the sample average rank of severity – numeric there is asymptomatic significant between mild-moderate vs severe, mild-moderate vs critical and severe vs critical as shown in table-5 table 5. the sample average rank of severity. sample 1sample2 sig. adj. sig 1.000-2.000 .000 0.000 1.000-3.000 .000 0.000 2.000-3.000 .006 0.017 1-mild-moderate ,2-sever,3-critical. each row tests the null hypothesis that the sample1 and sample2 distributions are the same. asymptomatic significances (2-sided tests) are displayed. the significance level is .05. with a p-value of (0.001), 62 patients with chronic illness (39.0%) have severe symptoms, and around 67 (42.1%) have critical symptoms. and schedule medication usage is evident in 51 (41.8%) and 49 (40.2%) of those with severe and critical symptoms, respectively as illustrated in table-6. table 6. the association between patient severity of covid-19 and having chronic disease/medication parameter severity mildmoderate severe critical p-value chronic disease no count % 41 33 13 .001* 47.1% 37.9% 14.9% yes count % 30 62 67 chronic medication 18.9% 39.0% 42.1% 0.001* no count % 49 44 31 39.5% 35.5% 25.0% yes count % 22 51 49 each row tests the null hypothesis that the sample 1 and sample 2 distribution are the same * significant (p-value <0.05) according to the pearson chi-square. three antidiabetic medications had significant association with the covid-19 severity included metformin, insulin, dipeptidyl peptidase-4 inhibitor (dpp4), and angiotensinogen ii receptor blocker (arbs) were also significantly associated, while others were not as shown in table-7. table 7. the association between the individual chronic medication and the severity of covid-19 disease. disease severity p-value 1.00 2.00 3.00 metformin no count 65 73 69 .030* % within 31.4% 35.3% 33.3% yes count 6 22 11 % within 15.4% 56.4% 28.2% insulin no count 70 86 69 .023* % within 31.1% 38.2% 30.7% yes count 1 9 11 % within 4.8% 42.9% 52.4% sulfonylurea no count 61 75 71 .186 % within 29.5% 36.2% 34.3% yes count 10 20 9 % within 25.6% 51.3% 23.1% dpp4 no count 68 80 74 .032* iraqi j pharm sci, vol.31(2) 2022 progression and prediction factors for covid-9 44 % within 30.6% 36.0% 33.3% yes count 3 15 6 % within 12.5% 62.5% 25.0% sgle2 § no count 71 93 80 .335 % within 29.1% 38.1% 32.8% yes count 0 2 0 % within 0.0% 100.0% 0.0% acei § no count 68 89 74 .709 % within 29.4% 38.5% 32.0% yes count 3 6 6 % within 20.0% 40.0% 40.0% b-blocker no count 64 78 66 .303 % within 30.8% 37.5% 31.7% yes count 7 17 14 % within 18.4% 44.7% 36.8% statin no count 69 85 72 .150 % within 30.5% 37.6% 31.9% yes count 2 10 8 % within 10.0% 50.0% 40.0% antiplatelet no count 63 80 68 .691 % within 29.9% 37.9% 32.2% yes count 8 15 12 % within 22.9% 42.9% 34.3% disease severity p-value 1.00 2.00 3.00 arbs no count 62 73 56 .038* % within 32.5% 38.2% 29.3% yes count 9 22 24 % within 16.4% 40.0% 43.6% ccbs no count 68 83 69 .116 % within 30.9% 37.7% 31.4% yes count 3 12 11 % within 11.5% 46.2% 42.3% *significant (p-value < 0.05) according to pearson chi-square. § fisher's exact test. arb=angiotensinogen ii receptor blocker; dpp4=dipeptidyl peptidase-4 inhibitor (anti hyperglycemic); sgle2=sodium–glucose co-transporter-2; acei=angiotensin converting enzyme inhibitor. ccbs=calcium channel blockers. severity1=mild-moderate; 2=severe; 3=critical iraqi j pharm sci, vol.31(2) 2022 progression and prediction factors for covid-9 45 age with the (p-value0.001), ast (p-value0.002), tsb (p-value0.002), urea (p-value0. .001), creatinine (p-value0. .001), crp (p-value0. .001) were related to higher mortality rate as shown in figure-2. figure 2.the significant (p-value < 0.05) associations between the levels of 11 biomedical parameters and the patient death probability. ldh=lactate dehydrogenase; ast=aspartate aminotransferase; tsb=total serum bilirubin; crp=c reactive protein. the numbers in the y-axis (mean rank) mean as these parameter increase the death outcome increase and it’s not limited value. these results may help clinicians to identify on admission which patients with covid19 are at high risk of deterioration. discussion the study reported numerous risk factors that may predict poor outcomes. the current study found that elderly patients experienced more severe covid-19 symptoms and a higher intensive care unit (icu) admission rate and needed more oxygen therapy and stayed longer in hospital compared to younger patients. shengmei niu and his colleagues (2020) in china studied clinical characteristics of old patients infected with covid-19 and reported that covid-19 infection in elderly individuals is associated with a high number of severe infections and a high mortality rate (16). another study of aline mendes, (2020) studied predictors of inhospital mortality in older patients with covid-19 and reported hospitalization for covid-19 older individuals was an independent risk factor for death (17). this may be related to his immune system that considered to weaken with age. overproduction of cytokines furthermore, the activity of t-cells and bcells decreases with age this might speed up virus replication and prolong pro-inflammatory reactions, causing serious health problems (18,19). the current study resulted that gender had no influence on the severity of the disease, requirement for an icu, or the length of hospitalization. in addition to that, there is no substantial difference in the need for oxygen treatment based on gender. hongdou li 1 (2020) in china studied on 5319 covid-19 patients and reported that there isn't much of a distinction between males and females only in the age categories of 15 to 50 years old is there a variation in the incidence risk for different genders, where males have a slightly greater incidence risk than females after the age of 15, although the difference is insignificant in those over 50 (20). the study does not support a previous one of jonathan kopel (2020) which studied rasial and genderbased difference in covid-19 and reported that through an increase in the innate and humoral response, increased estrogen levels in female covid-19 patients may iraqi j pharm sci, vol.31(2) 2022 progression and prediction factors for covid-9 46 lower the severity and fatality of covid-19 fatalities (21). this difference may be related to 84.8% of females in the study aged over 45 years were defined as post-menopausal women and with last period menopause. differences in susceptibility to viral infections are most likely attributable to intrinsic differences in male and female immune systems, as a result of more powerful humoral and cellular immune responses, females mount a greater immunological response to viral infections than males (22) .most cells of the innate and adaptive immune systems, including t cells, b cells, neutrophils, macrophages, dendritic cells (dc), and natural killer (nk) cells, express estrogen receptors, during the menstrual cycle, when estrogen levels are high, more regulatory t cells are present, it is widely recognized that the aging process has an impact on sexual dimorphism in terms of immunocompetence and disproportionality (23). in terms of smoking, the study found that smoking has no significant effect on the severity of the disease and the need for an icu, and the length of hospitalization, and has no significant effect on the need for oxygen therapy. this may be related to the immune system of a current smoker is more tolerant and less reactive, compared to patients who have never smoked, whose immune system may be more suitable for triggering a cytokine release syndrome, that could be associated with covid-19related high mortality(24). meta-analysis of roengrudee patanavanich md, llm, (2020) in usa studied the association of smoking with covid-19 progression, and found that smokers have 1.91 times the odds of progression in covid19 severity than never smokers (25). the study found that c-reactive protein (crp) has significant correlation with disease severity and mortality. severe covid19 individuals with high crp levels may be related to the overproduction of inflammatory cytokines. although cytokines combat germs, when the immune system becomes overactive, it can cause lung tissue damage. in individuals with covid19, inflammatory cytokines and tissue damage cause crp production (26). this result agrees with a study done by weifeng shang1and his colleague in china (2020) who studied the value of clinical parameters in predicting the severity of covid‐19 and reported that crp in the severe group was significantly higher than that in the non-severe group, and crp was an independent risk factor for severe covid‐ 19(27). and with ashaqali1 (2021) studied myoglobin and c reactive protein are efficient and reliable early predictors of covid 19 associated mortality and reported that crp was specific risk factors related to mortality and highly correlated to organ failure in covid-19 disease (28). patients with chronic disease/medication experienced more severe symptoms with a percent of 42.1, 40.2 critical cases respectively reported in the study. nancy chow, (2020), studied preliminary estimates of the prevalence of selected underlying health conditions among patients with coronavirus disease 2019 — united states, february 12–march 28, 2020 and reported that people who have underlying health issues tend to have a higher risk of severe covid-19–related disease than those who don't (29). the current study found that metformin had significant association with the covid-19 severity, this explained by metformin might theoretically enhance the availability of ace2 in the respiratory tract via the ampk–phospho-ace2 axis, promoting sarscov-2 infection and worsening covid-19 illness (30). xu cheng, and his colleague (2020)in china agree with this by studied metformin is associated with higher incidence of acidosis, but not mortality, in individuals with covid-19 and pre-existing type 2 diabetes of 1,213 and reported that metformin usage was linked to an increased risk of acidosis, particularly in patients with severe covid19(31). for the use of insulin the study was found that insulin had a significant association with the covid-19 severity. insulin therapy was linked to an increase in systemic inflammation and a worsening of important organ damage (32). shayan riahi (2020) in usa that studied insulin use, diabetes control, and outcomes in patients with covid-19 showed home insulin treatment was linked to a higher risk of death (33). this study found that, the peptidase-4 inhibitor (dpp4) was also have a significant association with covid-19 severity. dpp-4 regulates the immune system by activating t cells via the nuclear factor-b pathway, and therefore its suppression may have an unfavorable effect on the immunological response to viral infection, especially as lower numbers of t cells have been linked to covid-19 severity (34). dr. rinkoo dalan, and his colleague (2020) in singapore supported this by studying the association of hypertension and diabetes with covid-19 severity and immune signatures found that patients using dpp4 inhibitors were more likely needs to be admitted to the icu (35). the study found another drug that has a significant association with the disease severity, which is angiotensin receptor blockers (arbs). one theory is that sars-cov-2 utilizes ace2 receptors for cellular entrance, and that blocking the receptor with acei/arbs prevents viral attachment, entry, and multiplication. continuous use of these medicines, on the other hand, may result in overexpression of ace2 receptors through a negative feedback mechanism, increasing coronavirus binding to target cells (36). this agree with a study carried out by murat selçuk in turkey (2020) that studied is the use of ace inb/arbs associated with higher in-hospital mortality in covid-19 pneumonia patients? and found that the iraqi j pharm sci, vol.31(2) 2022 progression and prediction factors for covid-9 47 use of arbs therapy might be associated with an increased in-hospital mortality in patients who were diagnosed with covid-19 pneumonia (37). the current study's mortality results are linked to high levels of urea and creatinine. the etiology of this might be complex, with direct effects of the coronavirus on renal parenchyma, such as ace2 expression in urinary organs, being one possibility. secondary, the kidney may be damaged by the deposition of immune complexes of viral antigen or virus-induced particular immunological effector mechanisms (specific t-cell lymphocyte or antibody). third, virus-induced cytokines or mediators may have an effect on the immune system (38). this was supported by, lei chen (2020) in london who studied risk factors for death in 1859 subjects with covid-19 and confirmed that scr (serum creatinine) on admission was shown to be more useful in predicting in-hospital death in patients with covid-19 and aki who had an increased baseline scr (39). another study by yemao liu (2021) in china who studied kidney function indicators to predict adverse outcomes of covid-19 and found that blood urea nitrogen and scr levels were shown to be related with a higher risk of death (40). this study found that, aspartate aminotransferase (ast) and total serum bilirubin (tsb) were also linked to high mortality. though the specific mechanism of covid19-induced liver injury is unknown, several ideas have been presented, including direct viral cytotoxicity via ace-2, drug-induced liver injury, immunemediated damage, and passive congestion (41). zeyang dingand (2020) in china studied the association of liver abnormalities with in-hospital mortality in patient with covid19 and reported that early start of increase ast and direct bilirubin patient treated with covid19 was found to be an independent predictor of in-hospital mortality (42). and a retrospective cohort study of zeming liu (2020) in china studied bilirubin levels as potential indicators of disease severity in coronavirus disease patients and reported those with high stb levels showed a greater mortality risk (43). limitations of the study includesome information was obtained from relatives due to the patient's tiredness, which may not guarantee the accuracy of the information and not all laboratory parameters are performed on admission but rather according to the doctor's discretion. conclusion the study found several predictors for the disease (covid-19) severity, duration of hospitalization, icu admission and needed for oxygen therapy including crp, liver and renal function tests. old age, having chronic disease and taking scheduled medications are also predictors for oxygen need, icu admission or having more severe covid-19 symptoms. additionally, several biomedical parameters including age, crp, ast, tsb, urea and creatinine had significant association with higher probability of patient death due to the disease (covid-19). references 1. sipahi, oguz resat. 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( طفل مصاب بالتهاب المعد349( من مجموع )40 بينما أظهر النمط الجيني g2. أظهر األطفال الذين تم تلقيحهم نسبة أعلى بكثير من g2و g9و g1تم العثور على توزيع الطرز الجينية ليكون g1 ية في توزيع طول اإلسهال بين الطرز الجينية المختلفة. يغير التطعيم بشكل ملحوظ في األطفال غير الملقحين. ال توجد فروق ذات داللة إحصائ توزيع النمط الجيني وقد يخلق هذا الوضع تحديات لفعالية لقاحات الفيروسة العجلية والتخطيط لسياسات مستقبلية. ، النمط الجيني لفيروس الروتا.®( rotarixالكلمات المفتاحية: فيروس الروتا ، لقاح ) introduction rotavirus infection is the most significant cause of acute gastroenteritis in children worldwide (1). annually, about 150 million episodes of diarrhea in children which need hospital care, andnearly 500,000 deaths globally for children beneath 5 years were attributed to rotavirus infection (2). in iraq, rotavirus is a major cause of nosocomial infectious diarrheaof nearly (18.5%), occurring primarily among children younger than 5 years of age (3, 4). 1corresponding author e-mail: pharm.mrdha@uomustansiriyah.edu.iq received: 15/1/2021 accepted: 23/5 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp167-176 iraqi j pharm sci, vol.30(2) 2021 rotavirus genotypes after rotarex vaccine 168 rotavirus can infect children numerous times during their lives with first infection, after three months of age, is most probably cause severe diarrhea and dehydration5.young children with primary rotavirus infection do not have immunity against re-infection, nevertheless such an infection protects against the progress of clinically severe disease process during rotavirus new infection6. the primary rotavirus infection induces an antibody response which is predominantly a serotype-specific neutralizing type, while subsequent infections, yet with the same serotype of rotavirus, commonly elicit a cross-reactive broader serotype response (7). vp4 and vp7, the two outer capsid proteins, contribute to the development of immunity by stimulating the production of neutralizing antibody (8). nevertheless, after rotavirus infection, the antibody response is more generalized and involves antibodies against different other rotavirus proteins like vp2, vp6, nsp2, and nsp4 9.the diversity in the strains of rotavirus, beside the genetic drift and genetic reassortment mechanism by which wild-type rotavirus strains can evolve, represent a huge challenge to modern health care3. globally, a great diversity in circulating wild-type strains have been established with main strains causing severe disease alter from year to year and from region to region 10. the findings of phase iii clinical studies in several countries revealed that rotarix® offers sustained good protection against severe cases of rotavirus gastroenteritis during the first two years of child life, with broad protection produced against each of the five main rotavirus strains that circulate globally (g1, g2, g3, g4, and g9) (7). improving living standards and personal hygiene is sufficient to reduce risk of having diarrhea in children particularly in developing countries. consequently, progress of an efficient and secure vaccine turns into a priority reduce poor clinical outcome represented by recurrent primarycare attendance, hospitalization or probably death(11,12). in previous reports, existing licensed rotavirus vaccines have been shown to be effective and well-tolerated (13,14). studies performed in infants in high income settings like europe and north america demonstrated vaccine efficacy exceeding 90% (15). in middle income settings of latin america, south africa and far east asia, vaccine efficacy ranged from 72% to 83% (16), while in low income settings in asia and africa, vaccine efficacy ranged from 39% to 49% (17,18). evaluation of the outcomes of vaccination in early introduced countries is a big globaldifficulty for policy makers, and it is necessary to review if benefits balance the costs, and maintain wider spread of these vaccines. in the developing countries, rotavirus vaccines continued to be frequently assessed due to easy approach to reach target populations with much more strain diversity and immunogenicity of vaccines thatcould decrease immunization program performance (19). the present study is designed to evaluate the genetic characterization of rotavirus genotyping among children less than five years old diagnosed with acute gastroenteritis in babylon provincewho have received two doses of rotarixselected from the population registry. materials and methods patients thisstudy was done as a cross sectional one enroll a total of (349) sample population composed of children attended three primary health centers and babylon hospital for maternal and pediatrics in babylon province were screened for positive rotavirus infection.samples were collected during the period from october 2016 to august 2017. the study inclusions include; children older than 6 months and less than five years of age, selected from primary health centers presented with gastroenteritisinduced diarrhea (the duration of diarrhea defined by the duration since onset of diarrhea until admission or examination of the children). the children are defined as vaccinated against rotavirus according to the following categories: •vaccinated: 44children with positive rotavirus who have received two doses of rotarix® at age 2months and 4 months (minimum 4 weeks in between), of which the last dose was preceding 14 days before onset of symptoms. •non vaccinated: 125children with positive rotavirus with absence of written records for rotarix® vaccination in the vaccination registry or medical record20. only 40 children sample with positive rotavirus gastroenteritis were used to examine the rotavirus genotyping distribution. meanwhile thestudy exclusion includesthe followings: 1. infant ˂ 6 months of age and children more than 5 years of age. 3. history of children hypersensitivity to the vaccine. 4. history of gastrointestinal tract congenital malformation. 5. childrenwith history of intussusception. 6. history of severe combined immunodeficiency disease. sample size was calculated using raosoft website sample size calculator. assuming the margin of error of 5% and the confidence level is 95%, the total number of children registered at the above center were 3500 children then the sample size will be of minimally 347 children which is the minimum sample size for stratified analysis (21). iraqi j pharm sci, vol.30(2) 2021 rotavirus genotypes after rotarex vaccine 169 genotyping and molecular identification for rotavirus by rtpcr: all rotavirus-positive samples were confirmed by rt-pcr. the rna extraction was carried out according to world health organization(2009) protocol and faiza, l.et.al (2008) (10,11). 1. rna extraction 1. a) stool sample preparation 1five hundred mg of stool was suspended in 5 ml of phosphate buffer saline (ph 7) 2the suspension was homogenized by vortexing for 30 sec 3the homogenized suspension then centrifuged at 3000 rpm for 4 min. 4the clear supernatant was transferred for sterilized nucleases free tube. 1. b) rna extraction 1from the supernatant of the clarified 10% stool suspension 250 µl was pipetted into sterile 1.5-ml eppendorf tube and mixed with 750 µl of trizol by vortixing for 30 sec and incubated for 5 min at room temperature. 2two hundreds µl of chloroform was added to each tube and mixed by vortixing for 30 sec, then the tubes were incubated at room temperature for 3 min 3tubes then centrifuged at 12,000 rpm for 5 min at 4°c. 4the clear, upper aqueous phase was carefully transferred into a new tube. 5two volumes of cold isopropanol were added and mixed gently by inverting the tube several times. 6the tubes then incubated for 20 min at 20°c. 7genomic dsrna precipitated by centrifugation at 12,000 rpm at 4°c for 15 min. 8the supernatant was pipetted carefully and the tubes were left for drying at room temperature. 9the pellet then suspended in 20 μl of nucleases free water. 10immediately the extracted dsrna was used for downstream processes. 1. c) rotavirus genomic dsrna electrophoresis non-denaturing polyacrylamide gel electrophoresis was carried briefly as the following: 1the electrophoreses device glasses were washed and dried by alcohol, then assembled in the device according to the manufacture directions. 2resolving gel (10%) preparation: five ml of dh2o, 3.1 ml of 30% acryl stock and 1.2 ml resolving buffer, ph 8.8 were mixed and degased by vacuum for 5 min. then5 μl temed and 140 μl of 10% aps were added and briefly mixed by swirling. 3immediately the mixture was poured in to the device by disposable pasteur pipette until 1 cm beneath the comb teeth ends, covered by 3ml of water, and incubated in 37°c for 45 min to polymerize. 4spacer gel 4% was prepared by mixing 2.5 ml of dh2o, 0.6 ml of 30% acryl stock and 0.5 ml of spacer buffer, ph 6.8, the mixture was degased under vacuum for 5 min. then 2 μl temed and 60 μl of 10% aps were added and mixed briefly by swirling. 5immediately the mixture was poured in to the device by disposable pasteur pipette and the wells forming comb was inserted gently to its location. 6the gel was left for 45 min to polymerize at room temperature. 7when the gel is fullypolymerized, the gel was submerged in 0.5x running buffer, and the comb was removed, then the wells were carefully washed by the buffer. 8fifteen μl of the sample were mixed with 5 μl of loading buffer and loaded by mechanical pipet. 9the electrophoresis run was carried out under a constant voltage (150 v) fore about 2h. 10the gel then was dissembled and stained by silver stain as described below. 1. d) silver staining 1the gel was fixed by 50 ml of fixing solution 1 on an orbital shaker and stay rotated at room temperature for 30 min. 2the fixing solution 1 was replaced with50 ml of fixing solution 2 and the gel was rotated for 30 min at room temperature on the orbital shaker. 3the agno3 solution (50ml) was prepared just before use and added to the gel after the aspiration of fixing solution 2. the gel was rotated for 30 min in the dark. 4the silver nitrate staining solution was aspirated and the gel was washed twice with water for 2 min each time. 5preparing the developing solution by adding the naoh to the previously prepared formaldehyde and water solution. 6the developing solution was added to the gel, and agitated by hand for 30 sec to remove any black precipitate. 7the developing solution was aspirated and a new developing solution was added and rotated until rna bands were visible. 8the developing solution, was aspirated and the stopping solution was added and the gel was rotated at room temperature for 5-10 min. 9the gel was then rinsed in distilled water and imaged by digital camera . iraqi j pharm sci, vol.30(2) 2021 rotavirus genotypes after rotarex vaccine 170 1. e) cdna synthesis 1ten μl of extracted nucleic acid was transferred to a pcr tube and denatured at 97oc for 5 min. then chilled immediately on ice for 2 min. 2five μl of the denatured dsrna and 1 μl of each primer vp7-f and vp7-r (20 pmoles/μl) were added to rtmastermix (bioneer), the total volume was completed to 20 μl by nucleases free water. 3the tubes were incubated at 37oc for 1h. and then the reaction was stopped by incubation at 95oc for 5 min. 4the tubes then were chilled on ice for 2 min. 5the synthesized cdna can be used or stored at 20oc until use. 1. f) first-round pcr the first round pcr reaction mixture compositions are listed in table (1); table1.first round pcr reaction mixture. no component volume 1 cdna 5 μl master mix (promega) 10μl vp7-f primer 1μl vp7-r primer 1μl nucleases free water 3 μl total 20 μl the pcr mixture incubated in thermocycler according to the protocol listed in table (2); table 2.the pcr mixture thermocycling 1. g) genotyping pcr rotavirus genotyping was carried out according to world health organization (2009) protocol by nested multiplex pcr.the pcr reaction mixture components are listed in table (3). the pcr reaction was carried out by employing thermocycling protocol listed in table (4). table 3. reaction mixture components for rotavirus genotyping. table 4. thermocycling conditions for rotavirus genotyping . 1. h) agarose gel electrophoresis agarose gel electrophoresis was done according to the protocol described below in brief: 1the gel (2.5%) was prepared by dissolving 1.25 g of agarose in 50 ml of 0.5x tbe buffer and heated by microwave oven for 2 min. no stage temperature incubation time cycle number initial denaturation 94 c° 2min 1 denaturation 94 c° 1 min 35 annealing 52 c° 1min polymerization 72 c° 1min final polymerization 72 c° 5min 1 no component volume 1 first round pcr product 1 μl master mix (promega) 12.5 vp7-r primer 0.5μl g1 primer 0.5μl g2 primer 0.5μl g3 primer 0.5μl g4 primer 0.5μl g8 primer 0.5μl g9 primer 0.5μl g10 primer 0.5μl g12 primer 0.5μl nucleases free water 7 μl total 25 μl no stage temperature incubation time cycle number initial denaturation 94°c 4min 1 denaturation 94°c 1 min 30 annealing 42°c 2min polymerization 72°c 1min final polymerization 72°c 5min 1 iraqi j pharm sci, vol.30(2) 2021 rotavirus genotypes after rotarex vaccine 171 2the homogenized agarose then cooled to 55°c by water bath. 3a 50 μl of ethidium bromide stock (1 mg/ml) solution was added to the gel and mixed by swirling. 4then the gel poured in to the gel tray and let to polymerize for 30 min. 5after full polymerizationthe gel then is transferred to the electrophoresis devise and submerged with 0.5% tbe running buffer. 6pcr product (5 μl) was loaded carefully by mechanical pipet to the gel wells. 7the electrophoresis was carried out by setting the device on 100 volts and 40ma for about 60 min. 8the gel was then imaged and the image analyzed to determine the genotype. 1. i) gel image analyzingto determine rotavirus genotypes according to world health organization (2009) protocol each genotype will produce a specific pcr product.the product for each genotype are listed in table (5). table 5.the intended pcr product for each rotavirus genotype. no genotype intended pcr product (bp) 1 first round copy of gene 9 881 genotype g8 754 genotype g3 682 genotype g1 618 genotype g2 521 genotype g4 452 genotype g10 387 genotype g12 266 genotype g9 177 table 6.the pcr primer sequence . no primer name primer sequence 1 primer vp7-f atg tat ggt att gaa tat acc ac 2 primer vp7-r aac ttg cca cca ttt ttt cc 3 primer g1 caa gta ctc aaa tca atg atg g 4 primer g2 caa tga tat taa cac att ttc tgt g 5 primer g3 acg aac tca aca cga gag g 6 primer g4 cgt ttc tgg tga gga gtt g 7 primer g8 gtc aca cca ttt gta aat tcg 8 primer g9 ctt gat gtg act aca aat ac 9 primer g10 atg tca gac tac aga tac tgg 10 primer g12 ccg atg gacgtaacgttgta statistical analysis the spss 20.0.0, minitab 17.1.0, graphpad prism 7.0 software package used to make the statistical analysis. two samples t test used to analyze the differences in means between two groups. discrete variables presented using their number and percentage, chi square test used to analyze the discrete variable.p value considered when appropriate to be significant if less than 0.05. results genotyping of rotavirus results of rotavirus genomic dsrna stain, agarose gel electrophoresis, and nested multiplex pcr were shown in figures (1, 2 and 3). figure 1. non denatured polyacrylamide gel electrophoresis of rotavirus genomic dsrna stained with silver stain. (lanes 1,2,3,and 4: represent rotavirus genomic dsrna segments, lane l: dsdna 100bp step ladder ) iraqi j pharm sci, vol.30(2) 2021 rotavirus genotypes after rotarex vaccine 172 figure 2. agarose gel electrophoresis of first round pcr. ( lanes 1-9 : 881 bp of vp7 amplified fragment , lane l : 100 bp step dna ladder). figure 3.rotavirus genotyping by nested multiplex pcr. ( lanes 1,7,10 and 12: g9 genotype , lanes 4 and 8: g1 genotype , lanes 2 and 9: g2 genotype , lane 3: mixed infection by g1 and g2 genotypes , lane 5: mixed infection by g1 and g9 genotypes , lane 11: mixed infection by g2 and g9 genotypes , lane 6: empty lane). distribution of rotavirus genotypes the rotavirus genotypes were found to be g1, g2, and g9, table (7). the g1 represents the majority of positive infected children (35.0%), then g9 (32.5%), and g2 (27.5%).mixed genotypes (g1 + g9 or g 2+ g9)represent(5.0%). significant difference was noticed among all rotavirus genotyping distribution (p ˂ 0.05) regardless of vaccination status of infected children. table 7. distribution of rotavirus genotype(n=40). genotype number(n=40) percentage (%) g1 14 35.0 g2 11 27.5 g(1 or 2) + g9 2 5.0 g9 13 32.5 p value 0.029٭ number of patients (n) and percentage (%) chi-square test was used p value < 0.05 ( significant) ٭ iraqi j pharm sci, vol.30(2) 2021 rotavirus genotypes after rotarex vaccine 173 duration of diarrhea and association with genotypedistribution in table (8a),vaccinated children with positive rotavirus infection were significantly associated with longer duration of diarrhea compared to non-vaccinated children(p< 0.01). in table (8b),no significant difference was seen between the duration of diarrhea in positive infected children according to genotype distribution. table 8a. duration of diarrhea of study population duration of diarrhea(days) not vaccinated (n=125) vaccinated (n=44) or 95% ci of or p value ٭٭0.002 2.065 – 1.181 1.561 1.1 ± 4.4 1.3 ± 3.6 data presented as mean ± sd, binary logistic regression or: oddratio ci: confidence interval, ٭٭ p value < 0.01( highly significant) table 8b. association between duration of diarrhea and rotavirus genotype g1 g2 g9 p value 3.4 ± 1.4 3.9 ± 1.4 4.1 ± 1.4 0.470ns data presented as mean ±sd of diarreaha duration (days) , ns is considered non-significant association between genotype of vaccination status vaccinated children exhibited significantly higher percentage of g2 (58.3%) while g1 and g9 were presented more considerably in non vaccinated children (42.3%) as in table (9).the mixed genotypes were as follows; (non vaccinated g1 + g9 or non vaccinated g 2+ g9) one samples for each mixed type were excluded from the association. table 9. association between rotavirus genotyping and vaccination status number of patients (n) and percentage (%) chi-square test was used p value < 0.05 ( significant)٭ discussion acute diarrhea in children caused by rotavirus infection is considered as the major public health concern in the developing countries (22), including iraq. about 70% episodes of acute infectious diarrhea causedby viruses in the pediatric age23.especially during the first few years of life; rotavirus infection is endemic worldwide and frequentlylinkedto morbidity and mortality in high rates particularly in developing countries due to poor nutrition and reduced health care (24,25). the two currently licensed rotavirus vaccines, rotarix and rotateq, both are very effective in reducing severe diarrhea in vaccinated and unvaccinated children although the incidence and significance of this vaccine-acquired diarrhea remain to be determined 26,also both vaccines have been associated with a low risk of intussusceptions among vaccinated infants27 in our previously published data (28,29) ,the percentage of positive rotavirus infection was (48%) among all vaccinated and non vaccinated children28, and after the assessment of the effectiveness of rotarix®vaccine, the results revealed that non vaccinated children had a higher percentages of positive rotavirus infection (64.8%) in comparison to vaccinated children (29). large percentage of samples will need to be analyzed by rt-pcr to determine the genotypes of the strains not typeable with serotyping monoclonal antibodies (10), the results of the current showed that there were considerable differences in genotypes among both vaccinated and not vaccinated children. vaccinated children exhibited the following genotype pattern g1 (25%), g2 (58.3%), g9 (16.7%) while unvaccinated children exhibited the following genotyping g1 (42.3%), g2 (15.4%), g9 (42.3%), with reference to the pcr primer sequence (10). in pre-vaccination era, abood et al, (2013) genotyping pattern was presented as following genotype distribution of g1 (48.57%), g2 (22.14%) and g9 (11.42%)30, which was close to the predominance pattern of the unvaccinated children22. in an observational, prospective, multicentre, hospital-based case–control study in belgium reported similar findings to the current study, where g2p[4] strains were more prevalent in vaccinated than in unvaccinated cases and that hospitalized rotavirus gastroenteritis caused by heterotypic g2p[4] rotavirus strains, meanwhile in the unvaccinated group, g1p[8] and g2p[4] genotypes were almost equally (31). there was a significant association (p <0.001) between rotavirus vaccination status and genotype (29). the immunological responses providingboth homotypic and heterotypic protectionnormally resulted after rotavirus infection or oral vaccination,which reflecthigher effectiveness of monovalent vaccine against circulating homotypic non vaccinated vaccinated p value number 26 12 g1 11 (42.3%) 3 (25.0%) 0.024٭ g2 4 (15.4%) 7 (58.3%) g9 11 (42.3%) 2 (16.7%) iraqi j pharm sci, vol.30(2) 2021 rotavirus genotypes after rotarex vaccine 174 g1p[8]rotavirus strains, compared with the circulating heterotypicg2p[4] rotavirus strains (31). brazil introduced the rotarix g1p[8] vaccine in 2006, surveillance study after using the monovalent rotavirus vaccine,the predominance of the g2p[4] genotype is temporal and that other unusual genotypes may appear with time (32,33). in a previous hospital-based korean study between 2011 and 2014, no significant difference in the genotypedistribution between vaccinated children andthose unvaccinated, both of majority g2p [4], this observation could be due to small number of vaccinatedchildren (n = 19) (34). there was shifting in the abundance of rotavirus genotype towards g2 with greater reduction in the percentages of g1 and g9, though doesn’t affect the duration of diarrhea caused by rotavirus gastroenteritis.the proposed explanation is that changes in the seasonal pattern of rotavirus disease was also observed after vaccine introduction, including delays in the start of the rotavirus season, a shorter duration of seasons and blunting of seasonal peaks (35), thus, whether the differences in rotavirus genotypes due to natural seasonal variation or vaccine-induced selection pressure is unclear. currently, differences in genotype-specific vaccine effectiveness and the resulting influence on distribution of co-circulating rotavirus strains, probably help to explain the increase in the proportion of g2p[4] strains. recent data from longitudinal study in africa among hospitalized children aged <5 years, revealed predominance of g2 genotype [33.0%]followingrv1 introduction36.this finding based on previous studies that showed a lower point of vaccine effectiveness against rotavirus gastroenteritis caused by the heterotypic g2p[4] strains than against rotavirus gastroenteritis caused by fully homotypic g1p [8], subsequently this translates to a higher prevalence of g2p[4] strains in vaccinated cases compared with unvaccinated cases (37,38). in line with such findings, adlhoch et al. reported that g2p[4] genotypes were more frequently found in breakthrough cases vaccinated with the monovalent vaccine (39,40). according to all previous findings,strain diversity and genotype variation are likely driven by natural mechanisms rather than vaccine pressure, hence, monitoring genotypes, whole genomic characterization of circulating rotavirus strains before and after vaccine introduction will help assess whether vaccines are affecting the evolution of the rotavirus genome. moreover,the changing in the model of genotype distribution of rotavirus following vaccine introduction is very crucial in deciding the future policy of immunization and assessing vaccine effectiveness. it may also explain why children are still susceptible to infection although they were fully vaccinated and why the prevalence of rotavirus gastroenteritis remains high after five years of introducing rotavirus vaccine in the iraqi national immunization program. conclusion rotavirus gastroenteritis remains higher after five years of introducing rotavirus vaccine in the iraqi national immunization program.vaccinated children exhibited significantly higher percentage of g2rotavirus genotype.no significant difference in the distribution of the length of diarrhea among different genotypes.monitoring genotypes, whole genomic characterization of circulating rotavirus strains before and after vaccine introduction will help assess whether vaccines are affecting the evolution of the rotavirus genome. acknowledgements 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gurgel. effectiveness of rotavirus vaccines againstrotavirus infection and hospitalization inlatin america: systematic review and metaanalysisinfectious diseases of poverty (2016) 5:83 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 felodipine lphns doi: https://doi.org/10.31351/vol31iss1pp119-129 119 investigation of lipid polymer hybrid nanocarriers for oral felodipine delivery: formulation, method, in-vitro and ex-vivo evaluation hayder kadhim drais1,*, ahmed abbas hussein** *ministry of health and environment, babil health directorate, babil, iraq. **baghdad college of medical sciences, baghdad, iraq. abstract the antihypertensive felodipine is a calcium-channel blocking agent. it is practically insoluble in water and shows low oral bioavailability (15%-20%). this investigation aims to formulate and characterize felodipine lipid polymer hybrid nanocarriers (lphns) to be given orally by two nanovesicles formulating methods and make comparative analysis through characterization process and in vitro and ex vivo intestinal permeation evaluation. the felodipine lphns formulations (hf1-hf3) were prepared by the new microwave-based method and that felodipine lphns formulations (hf4-hf6) were prepared by a single emulsification solvent evaporation technique (seset). all formulations (hf1-hf6) enter the characterization process. the felodipine lphns formulations (hf1-hf6) were prepared successfully and undergo different characterization processes to make a comparative study between formulations prepared by different methods. it was found that formulas prepare by a microwavebased method are most superior to the seset. the felodipine lphns formulations hf1-hf3 has lower particle size, lower pdi and higher zeta potential, significantly higher (p< 0.05) dissolution rate, and significantly higher (p< 0.05) intestinal permeation study than the felodipine lphns formulations hf4-hf6. the microwave-based method is a very successful technique in preparing felodipine lphns formulations (hf1-hf3) and prepotent to the seset. all the felodipine lphns formulations (hf1-hf6) show extended drug release nanosystem. keywords: lipid-polymer hybrid nanocarriers, felodipine , microwave based method, seset. التحقيق في تحضيرناقالت نانوية عالجية هجينة من الدهن والبوليمر لتسليم الفلودبين عن طريق خارج الجسم الحي وخالل األنسجة الحية الفم:وذلك بمقارنة الطريقة مختبريا ** عباس حسين احمد و 1*،حيدر كاظم دريس . العراق بابل، بابل، دائرة صحة والبيئة، وزارة الصحة * .العراق بغداد، ، الطبيةكلية بغداد للعلوم ** الخالصة إنه غير قابل للذوبان عمليا في الماء ويظهر التوافر الحيوي عن طريق الفم . فيلوديبين الخافض للضغط هو عامل يحجب قنوات الكالسيوم التي يتم إعطاؤها عن طريق الفم ( lphns)الهدف من هذا البحث هو صياغة وتوصيف ناقالت النانو الهجينة الفلوديبين الدهنية (. 20٪-٪ 15) . النانوية وإجراء تحليل مقارن من خالل عملية التوصيف وتقييم نفاذية األمعاء في المختبر وخارج الجسم الحي النواقلمن خالل طريقتين لصياغة بتقنية تبخير المذيبات األحادية lphns hf4-hf6بطريقة جديدة تعتمد على الميكروويف وتم تحضير تركيبات فلوديبين hf1-hf3تم تحضير بنجاح ( hf1-hf6)الفلوديبين lphnsتم تحضير تركيبات . في عملية التوصيف( hf1-hf6)كيبات تدخل جميع التر . seset االستحالب وجد أن الصيغ المحضرة بطريقة تعتمد على . وخضعت لعمليات توصيف مختلفة إلجراء دراسة مقارنة بين الصيغ التي تم تحضيرها بطرق مختلفة على حجم جزيئات أقل ، lphns hf1-hf3تحتوي تركيبات فيلوديبين . ألحادية االستحالبالميكروويف هي األكثر تفوقًا على تبخير المذيبات ا ملحوظ pdiوأقل بشكل أعلى ذوبان ومعدل ، أعلى زيتا بكثير ( p <0.05)وإمكانات أعلى معوية نفاذية تركيبات ( p <0.05)ودراسة من lphns الفلوديبينhf4-hf6 .على الميك في تحضير تركيبات فلوديبين تعتبر الطريقة المعتمدة ناجحة للغاية تقنية -lphns hf1روويف hf3 ومتفوقة على seset . تُظهر جميع تركيبات فلوديبينlphns hf1-hf6 نظام نانوي ممتد إلطالق العقاقير. ، طريقة تعتمد على الميكروويف ، تبخير المذيبات األحادية االستحالب بوليمر ، فيلوديبينال من الدهن وناقالت نانوية هجينة : الكلمات المفتاحية introduction it became clear that nanotechnology has entered into the development of most areas of life, including the development and manufacture of medicines (1). despite the great discoveries of drugs, they remain ineffective until a safe way is found to deliver them to the circulatory system or site targeting. felodipine is the calcium-channel antagonist. its use in the treatment of hypertension and angina pectoris. it is practically insoluble in water and undergoes a hepatic first-pass effect that leads to low oral bioavailability (15%-20%) (2). the oral route of drug administration is most prevalent where and has many merits such as its safe, easy to administer therapeutic agent, not associated with pain as in the intravascular route. but the oral route has some demerits such as intestinal and hepatic first-pass effect and is associated with low bioavailability deera2020@gmail.com :mail-corresponding author e1 received: 12/6/2021 accepted: 5/ 9/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp119-129 iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 120 for drugs that have low solubilities such as felodipine and low permeability(2). in addition to oral route obstacles, the conventional oral delivery system has many demerits such as poor patient amenability, increased opportunity of dose missing of a drug with a short half-life for which frequent taking is required and presence of typical peak and valley in the blood concentration/time curve lead to fluctuations in therapeutic agent level this will create adverse effects particularly in therapeutic agent with low therapeutic index (3). nanoparticles played a major role in carrying therapeutic agents and delivering them to the place of effectiveness. the presence of nanoparticles led to an increase in solubility, an increase in the surface area for drug release, and thus increased absorption and bioavailability (4). there are many nanoparticles used in drug delivery, but they are associated with some obstacles that interfere with drug efficacy. the most important obstacles are low drug loading, drug expulsion, and drug instability after administration and in long-term storage. the lipid polymer hybrid nanocarriers (lphns) system is a nanoparticulate system for drug delivery. it protects the encapsulated drug from obstacles associated with another nanoparticle system(5-7). the lphns consist of lipid content and polymeric ingredient (8). the lipid content enhances the solubility of hydrophobic drugs, improves membrane permeability leads to increase bioavailability. the presence of polymer within the structure of lphns provide more control to release of loaded drug. the hybridization between lipid and polymeric ingredients creates a nanoparticulate system which is lphns that characterized mainly by toughness, robust nanoparticles, provide extended-release drug delivery, higher drug payload, and high stability in the human circulatory system and during formulation storage(9). there are many methods of lphns preparation with some limitations such as: the presence of impurities in final products, costly, require high time that delays research field, and low stability. the newly microwave-based method is employed with great success in the formulation of a highly advanced nano system which is lphns and characterized by economical, absence of impurities associated with other nanoparticle preparation methods, inexpensive, high stable of final formulation (10-12). the single emulsification solvent evaporation technique (seset) is widely used in the preparation of nanocarriers. the seset has been successfully used to loading a variety of hydrophobic therapeutic agents with good reproducibility, high yield, and ease of scaling up (13). this study aims to prepare and characterize oral felodipine lipid polymer hybrid nanocarriers (lphns) by two nanoparticle preparing methods to make a comparative study through the characterization process and in vitro and ex vivo intestinal permeation evaluation. material and methods materials the labrasol, peg laurate, and peg oleate were purchased from beijing yibai biotechnology co., ltd. china. the methanol, ethanol, kcl, hcl, kh2po4, and na2hpo4 grin land chemical comp. the u.k. the felodipine, lauric acid, polysorbate 80, polysorbate 20, span 80, propylene glycol, and naoh were purchased from nanjing duly biotech co., ltd. china. the aniseed oil, argan oil, and cardamom oil were purchased from hemani international kepz, karachi, pakistan. the olibanum oil was purchased from aiemad for plant oil products. iraq. the fenugreek oil was purchased from bar-sur-loup grasse (a.m). methods the microwave-based method the hydrophobic blend was prepared under a magnetic stirrer device at 1000 rpm for 5 minutes. it contains felodipine, lauric acid and chitosan that was dissolved in cardamom oil : peg-laurate. the hydrophilic blend contains distilled water, polysorbate 80 and propylene glycol related to optimized amounts. by the application of microwave device for less than 15 seconds to the mixture of the two blends and under magnetic stirrer device at 1000 rpm for adequate time (seconds to minutes according to a final volume of dosage form), a colloidal dispersion system of felodipine lphns will be prepared. the optimized felodipine lphns formulations (hf1-hf3) as shown in table (1), are immediately used for the study or lyophilized to be filled in hard gelatin capsules. in the lyophilization process (freeze-drying), the samples were first frozen at -20 °c for 2 h, then transferred at -80 oc for 22 h., and then lyophilized at 0.001 mbar at -104 °c for 24 h using lactose 10%(w/w) as a cryoprotectant (10,14). iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 121 single emulsification solvent evaporation technique (seset) in this method, the felodipine and the chitosan are dissolved in organic solvent and mix with the lipophilic phase, then it was added into an aqueous phase containing peg laurate: polysorbate 80: propylene glycol. under magnetic stirrer device and probe or ultra-sonication process result in the formation oil in water (o/w) emulsion. the organic solvent is removed by a rotary evaporator, yielding the felodipine lipid polymer hybrid nanocarriers. the optimized felodipine lphns formulations (hf4-hf6) as shown in table (1), are immediately used for investigation or pass through freeze drying to fill in hard gelatin capsules. in freeze-drying, the samples were first frozen at -20 °c for 2 h, then transferred at -80 oc for 22 h., and then lyophilized at 0.001 mbar at -104 °c for 24 h using lactose 10% as a cryoprotectant (15,16). table 1. the selected felodipine lphns formulations (hf1-hf6) for characterization and optimization. formulation code method of preparation felodipine % (w/w) cardamon oil % (w/w) lauric acid % (w/w) chitosan % (w/w) peg-(400) laurate :polysorbate 80: propylene glycol % (w/w) distilled water % (w/w) up to hf1 microwave based method 1 8 2 0.2 17.5:8.75:8.75 100 hf2 microwave based method 1 8 2 0.25 20:10:10 100 hf3 microwave based method 1 8 2 0.35 22.5:11.25:11.25 100 hf4 seset 1 8 2 0.2 17.5:8.75:8.75 100 hf5 seset 1 8 2 0.25 20:10:10 100 hf6 seset 1 8 2 0.35 22.5:11.25:11.25 100 characterization of the felodipine lphns formulations(hf1-hf6) globule size determination the globule size is determined by the nanoparticle analyzer model sz-100 nanopartica series instruments from horiba scientific company. the photo correlation spectroscopy (pcs), is a technique that has been used to determine the particle size of felodipine lphns formulation. the experiments were performed in triplicate (17). polydispersity index (pdi) determination the uniformity of the nanosystem of colloidal attributes is determined by the polydispersity index (pdi). it is measured by pcs technique. as pdi value increases, the uniformity of nanocarriers of the felodipine lphns formulations will decrease. the experiments were performed in triplicate (17). zeta potential (zp) measurement the zp is an index to determine the stability of the colloidal dispersion system that is affected by dlvo forces. it is measured by pcs technique. the zp determines the surface charge which can develop around nanocarriers in a dispersion medium. the experiments were achieved in triplicate (17). entrapment efficiency (ee) and drug loading (dl) determination the entrapment efficiency of felodipine lphns formulation is determined by the indirect method through, 0.1 ml of freshly prepared felodipine lphns formulation was taken and add to it 9.9 ml ethanol for dilution. the obtained colloidal dispersion was centrifuged for 15 minutes at 10000 rpm. the supernatant layer was removed and filtered through a 0.45 μm filter. the filtrated liquid was diluted in a sufficient amount of ethanol and analyzed by an ultraviolet (uv) spectrophotometer at 361.5 nm. this study was performed in three trials (18). the encapsulation efficiency (ee) can be indirectly determined by the following equation (1): ee (%)= [(weight of felodipine in lphns)/𝑇𝑜𝑡𝑎𝑙 felodipine amount)] ×100 ………..… (1) the drug loading (dl) expressed in percentage (%) is the quantity of active pharmaceutical agents https://www.horiba.com/uk/scientific/products/particle-characterization/particle-size-analysis/details/sz-100-7245/ https://www.horiba.com/uk/scientific/products/particle-characterization/particle-size-analysis/details/sz-100-7245/ iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 122 present in the nanocarriers divided by the total quantity of lipid present in the nanosystem. it is measured by the equation(2): dl (%)=[(weight of felodipine in lphns)/weight of lphns] × 100 ………..… (2) the ph determination it has a great effect on the solubility of the therapeutic agents, the formulation attributes, tolerability, formulation stability, and the therapeutic agent's activity. the ph determination is an important factor in felodipine lphns formulations due to its relation to the stability and activity of pharmaceutical nanocarriers. the alteration of ph may be related to chemical interactions that can affect product quality. the digital ph meter employs to measure the ph of the felodipine lphns formulations. the study was achieved in triplicate (19). percent of light transmittance measurement it is an important parameter that ascertains colloidal attributes of felodipine lphns formulations. the percent of light transmittance was determined by a uv-visible spectrophotometer to preserve double distilled water as blank at 600 nm. results were being taken in triplicate (20). in vitro felodipine release experiment the experiment was achieved for felodipine lphns (hf1-hf3) prepared by microwave-based method and felodipine lphns (hf4-hf6) that is prepared by single emulsification solvent evaporation technique and compare it with drug dissolution from pure drug suspension using the combination method of (usp dissolving type i apparatus dialysis bag technique).two dissolution media have been used which are hcl buffer ph 1.2 + 0.3 % polysorbate 80 solution and phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solution. the dissolution medium volume is 900 ml for each experiment at 37 ± 0.5 o c with constant stirring at 50 rpm. the drug amount in each of felodipine lphns (hf1-hf6) and pure drug suspension was 5mg of felodipine. samples were withdrawn at predetermined intervals of time (5, 10, 15, 30, 60 minutes, and 2,4,8,12,24,36 hours) and filtered by microfilter paper of 0.45 µm pore size and compensate by equal withdrawn volume. at 361.5 nm, the felodipine concentration determines by spectrophotometrically(21,22). the study was performed in triplicate and the results were analyzed using anova statistical test at level p< 0.05. the drug liberation kinetics study performed by fit the obtained kinetic data into the various kinetic equations. the value of regression coefficients (r2) will explain the resultant model. the mechanism of felodipine release was obtained from the slope of the korsmeyer peppas equation (23,24). ex-vivo intestinal permeation study the study of ex-vivo intestinal permeation was performed using the non-everted sac technique (25,26). the fasted male sheep weighing about 16 kg was slain and anatomized under license university of baghdad/ college of pharmacy. the small intestine is a tested region that isolated and mesentery residue was removed and washed with normal saline solution. several pieces of 5 cm in length and 2 cm diameter of the small intestine was produced after the cutting process. insert in each piece that ligates from one end one capsule of (felodipine lphns hf1-hf6 ) and (felodipine drug suspension) where all capsules contain 5mg of felodipine and add 4.5 g of phosphate buffer ph 6.8 solution and tied the other end. insert the tested segments in 900 ml of liquid which is phosphate buffer ph 7.4 solution+0.3% polysorbate 80mg using apparatus 1 rotating basket (biobase meihua trading co., ltd.). at predetermined time intervals (5,10,15,30,60,90,120,150,180,210, 240 minutes) samples (5 ml) were taken and filtered by microfilter paper(0.45 µm) then find the felodipine concentration by uv spectrophotometer where the wavelength is 361.5 nm. the replenishment with an equal volume of withdrawn sample by diffusion liquid at once. the study was achieved in three trials and the outcome was analyzed statistically by anova test at p< 0.05. the effective membrane permeability was achieved by equation (3). m = peff s cd tres ………..… (3) where, m = therapeutic agent amount that absorbed peff = permeability coefficient (effective membrane permeability). cd = drug concentration at donor compartment tres = residence time of drug in gi lumen. s = surface area available for absorption statistical analysis the research experimental data documented as mean ± sd (n=3). a statistical study was performed by analysis of variance (anova) where a p-value less than 0.05 indicates a significant outcome(36). results and discussion preparation of felodipine lipid polymer hybrid nanocarriers the felodipine lphns formulations (hf1hf3) were prepared by the new microwave-based method according to specified concentrations of components as shown in table (1). the microwave's role in the formulation process was according to the following mechanism: when microwave passes through the ingredient of felodipine lphns formulation, the hydrogen bond, vander walls bonds, electrostatic interactions, and hydrophobic forces between the molecules undergo temporary breakage. the microwave causes dipolar rotation and ionic conduction that cause molecular oscillation and molecular collisions that raise the thermal energy of the system lead to weak bonds disruption. the increased thermal energy for the iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 123 system create tamed molecules that is more favorable and faster to get self-assembly by the magnetic stirrer agitation to produce felodipine lphns formulation(10,14). the felodipine lphns formulations (hf4-hf6) were prepared by single emulsification solvent evaporation technique (seset) according to specified concentrations of components as shown in table (1). the mechanism of seset in felodipine lphns formulation depends on the formation of oil/water emulsion that finally produces hybrid nanocarriers through a single formulation step(15,16). characterization of the prepared felodipine lphns formulations the following tests were utilized to characterize the prepared felodipine lphns systems: globule size determination the results were hf1 (146.2 nm); hf2 (94.2 nm); hf3 (75.1 nm); hf4 (743.2 nm); hf5 (169.7) and hf6 (179.2 nm). the outcomes indicate that all felodipine lphns formulations( hf1-hf6) have nanoscale particle size and colloidal dispersion attributes. the analysis of variance indicates that there is a significant (p<0.05) decrease in particle size as the increase the concentration of peg laurate: polysorbate 80: propylene glycol blend at constant lipid content. polydispersity index (pdi) determination pdi is a homogeneity parameter that determines the size distribution of nanocarriers within a colloidal dispersion system. its value ranges from 0 to 1. the smaller values near zero indicate a more homogenous globule size distribution while large values that approach 1 indicate a wider globule distribution (27). the results of felodipine lphns formulations (hf1-hf6) are hf1 (0.547); hf2 (0.54); hf3 (0.335); hf4 (0.888); hf5 (0.551) and hf6 (0.411). the outcome explains that there is a significant (p<0.05) decrease in pdi as the increase the concentration of peg laurate: polysorbate 80: propylene glycol blend at constant lipid concentration. zeta potential (zp) measurement the zeta potential is a parameter employ to measure the surface charge of felodipine lphns. it explains the physical stability of some colloidal dispersion systems. the results of felodipine lphns formulations (hf1-hf6) are hf1 (2.3 mv); hf2 (10.3 mv); hf3 (10.2 mv); hf4 (0.6mv); hf5 (-2.8mv) and hf6 (1.3mv). the felodipine lphns formulations(hf1-hf6), depend on non dlvo forces which are steric forces and hydration forces to stabilize the nanovesicles. therefore the low value of zeta potential of lphns formulations(hf1hf6) do not affect the physical stability and remain to withstand the process of nanocarrier aggregation because the stabilization process performed by non dlvo forces (28) the anova explain there is a nonsignificant (p < 0.05) correlation between independent variables and zeta potential factor. entrapment efficiency (ee) and drug loading (dl) determination the entrapment efficiency and felodipine loading is an important factor that employs for the evaluation and characterization of felodipine lphns formulations. the ee outcome of felodipine lphns formulations (hf1-hf6) are hf1 (85.443 % w/w ); hf2 (84.81w/w); hf3 (84.177% w/w); hf4 (84.2% w/w); hf5 (83.4% w/w ) and hf6 (82.8% w/w). it was found that the preparations (hf1-hf3) are more ee than preparations (hf4hf6). the anova indicates there is a significant (p > 0.05) correlation between independent variables and ee parameter.the dl outcome of felodipine lphns formulations (hf1-hf6) are hf1 (8.544% w/w ); hf2 (8.481% w/w); hf3 (8.418% w/w); hf4 (7.7% w/w); hf5 (7.5% w/w ) and hf6 (7.4% w/w). it was found that the preparations (hf1-hf3) are more dl than preparations (hf4-hf6) due to lower particle size and lower lipid content. the anova indicates there is a significant (p > 0.05) correlation between independent variables and the dl factor. the ph determination the ph outcome of felodipine lphns formulations (hf1-hf6) are hf1 (4.2); hf2 (4.1); hf3 (4.3); hf4 (4.1); hf5 (4.1 ) and hf6 (4.2). the result shows that the felodipine lphns formulations (hf1-hf6) had a suitable acidic ph value in the range of (4.1 – 4.3) that is better for oral administration(29). also that the preparations (hf1hf3) are nearly similar ph values of preparations (hf4-hf6). the analysis of variance shows a significant relationship (p<0.05) between independent variables and ph parameter. percent of light transmittance measurement the percent (%) of light transmittance is an attractive parameter to explain physically the colloidal properties of felodipine lphns formulations (hf1-hf6).the results of light transmittance percentage of felodipine lphns formulations (hf1-hf6) are hf1 (94.3%); hf2 (95.2%); hf3 (92.3%); hf4 (89.2%); hf5 (90.1%) and hf6 (90.8%).the outcomes show that the preparations (hf1-hf3) are more transparent than preparations (hf4-hf6) and all felodipine lphns formulations (hf1-hf6) gave features of colloidal dispersion (30). the analysis of variance indicated a significant relationship between independent variables and light transmittance percentage at a level (p<0.05). iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 124 in vitro felodipine release experiment the experiment was done using the combinational method which is (usp type i (basket) dialysis bag technique in two dissolution media which are hcl buffer ph 1.2 + 0.3 % polysorbate 80 solution and phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solutions. the outcome ascertains there is no burst release of the therapeutic agent from all felodipine lphns formulations (hf1-hf6) and there was an extended-release process over 36 hours from all felodipine lphns formulations (hf1-hf6) (46). in dissolution medium of hcl buffer ph 1.2 + 0.3 % polysorbate 80 solutions as shown in figures (1,2,3), the felodipine release profile was significantly higher (p-value <0.05) in dissolution rate for hf3 and was significantly lower (p-value < 0.05) in dissolution rate of the pure drug. the comparability profile of felodipine release for formulas have similar concentration with different preparation method was explained as following: hf1> hf4, hf2> hf5 and hf3> hf6 while the comparability profile of the felodipine release from lphns formulation (hf1-hf6) and the pure drug suspension explains in the following descending order: hf3 > hf 6 > hf 2 > hf 5 > hf 1 > hf 4 > pure drug suspension. the result indicates that formulas were prepared by microwave-based method to provide a higher dissolution rate in comparison to formulas were prepared by seset. this is due to the microwave-based method was prepared formulas (hf1-hf3) has lower particle size than formulas (hf4-hf6) which prepared by seset, which provide higher surface area for exposure to the dissolution medium lead to increase the rate of felodipine dissolution. in dissolution medium of phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solution as shown in figures (4,5,6), the felodipine release profile was significantly higher (p-value <0.05) in dissolution rate for hf2 and was significantly lower (p-value < 0.05) in dissolution rate of pure drug suspension. the comparability profile of felodipine release for formulas have similar concentration with a different preparation method was explained as following: hf1> hf4, hf2> hf5 and hf3> hf6 while the comparability profile of the felodipine release from felodipine lphns formulations (hf1-hf6) and the pure drug suspension explains in the following descending order: hf2 > hf 3 > hf 1 > hf 6 > hf 5 > hf 4 > pure drug suspension. the result indicates that formulas were prepared by a microwave-based method to provide a higher dissolution rate in comparison to formulas were prepared by seset. this is due to the microwavebased method was prepared formulas (hf1-hf3) has lower particle size than formulas (hf4-hf6) which prepared by seset, which provide higher surface area for exposure to the dissolution medium lead to an increased rate of felodipine dissolution. in the dissolution rate of felodipine profile, it was observed that the pure drug suspension gives a lower release rate of the therapeutic agent in comparison to all optimized lphns formulations (hf1-hf6) because that lphns formulations (hf1-hf6) have a large surface area that exposes to the dissolution medium and this will allow a higher interaction with the dissolution medium that increases dissolution rate (47,48). the analysis of variance indicated a significant (p<0.05) relationship between independent variables and felodipine release. figure 1. in vitro felodipine release profile from lphns formulations hf1,hf4 and the pure drug at hcl buffer ph 1.2 + 0.3 % polysorbate 80 solutions, the values of mean ±sd (n=3). figure 2. in vitro felodipine release profile from lphns formulations hf2,hf5 and the pure drug at hcl buffer ph 1.2 + 0.3 % polysorbate 80 solutions, the values of mean ±sd (n=3). iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 125 figure 3. in vitro felodipine release profile from lphns formulations hf3,hf6 and the pure drug at hcl buffer ph 1.2 + 0.3 % polysorbate 80 solutions, the values of mean ±sd (n=3). figure 4. in vitro felodipine release profile from lphns formulations hf1,hf4 and the pure drug suspension at phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solutions the values of mean ±sd (n=3). figure 5. in vitro felodipine release profile from lphns formulations hf2,hf5 and the pure drug suspension at phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solutions. figure 6. in vitro felodipine release profile from lphns formulations hf3,hf6 and the pure drug suspension at phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solutions. kinetic analysis of release drug the kinetic data were summarized in table (3) and table (4), were obtained from in vitro release of felodipine lphns formulations (hf1-hf6) and the pure drug suspension at hcl buffer ph 1.2 + 0.3 % polysorbate 80 solution and phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solutions. it was fitted to different models that determine the mechanism of therapeutic agent release. the kinetic models that employ in the investigation were zero-order, firstorder kinetic, higuchi model, and korsmeyerpeppas model. the outcome indicates higuchi's model was obtained due to the higher regression coefficient (r2) is obtained for it. this indicates felodipine was release from the monolithic system. the felodipine liberation from lphns formulations (hf1-hf6) following non fickian/anomalous dissolution (diffusion and erosion) due to the drug liberation exponent were significantly higher (p<0.05) than 0.43(23). iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 126 table (2). summary of characterization results of felodipine lphns formulations (hf1-hf6). code globule size (nm)* pdi* zeta potential (mv)* entrapmen t efficiency % (w/w)* drug loading % (w/w)* ph* percent of light transmittance* release percent of felodipine in hcl buffer ph 1.2 + 0.3 % polysorbate 80 solutions at 24 hours* release percent of felodipine in phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solutions at 24 hours* hf1 146.2±10.095 0.547± 0.0502 2.3± 0.264 85.443±5.0 58 8.544±0.478 4.2±0.435 94.3± 0.8 74.21± 0.7 89.882± 1.268 hf2 94.21± 1.587 0.54± 0.078 10.3±0.721 84.81± 4.013 8.481±0.354 4.1±0.264 95.2± 0.964 77.682± 0.532 90.575± 0.238 hf3 75.1±2.816 0.335± 0.005 10.2±0.754 84.177±2.5 61 8.418±0.256 4.3± 0.36 92.3± 0.888 81.154± 0.872 90.2± 0.435 hf4 743.2±8.861 0.888± 0.007 0.6± 0.264 84.2± 4.529 7.7± 0.608 4.1±0.132 89.2± 0.869 69.21± 0.642 80.6± 1.587 hf5 169.7± 9.553 0.551± 0.005 -2.8±1.081 83.4± 3.984 7.5± 0.529 4.1±0.264 90.1± 1.058 74.98± 1.048 85.3± 0.264 hf6 179.2± 8.827 0.411± 0.011 1.3± 0.2 82.8± 3.862 7.4± 0.435 4.2±0.264 90.8± 0.964 78.1± 1.228 87.1± 0.754 *values are expressed as mean ± sd (n=3). iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 127 table 3. the correlation coefficient (r2) and release exponent (n) of different kinetic models of felodipine lphns (hf1-hf6) and the pure drug released in hcl buffer ph 1.2 + 0.3 % polysorbate 80 solutions. table 4. the correlation coefficient (r2) and release exponent (n) of different kinetic models of felodipine lphns (hf1-hf6) and the pure drug released in phosphate buffer ph 6.8 + 0.3 % polysorbate 80 solutions. ex-vivo intestinal permeation study the permeability coefficient (cm/min) was calculated after obtaining felodipine flux (μg/ml) as shown in table (5). the comparative study was performed on the felodipine lphns formulations (hf1-hf3) that prepare by microwave-based method and felodipine lphns formulations (hf4hf6) that prepare by seset. it was found that the permeability coefficient (cm/min) has the following descending order hf1 > hf4, hf2 > hf5, hf3> hf6. this indicates that formulas (hf1-hf3) were prepared by microwave-based method provide a higher permeation rate across the intestinal membrane in comparison to formulas (hf4-hf6) were prepared by seset. the outcomes of the ex vivo intestinal permeation experiment indicate that the permeability coefficient (cm/min) of felodipine was significantly higher (pvalue <0.05) for f3 and was significantly lower ( pvalue < 0.05) for pure drug suspension. the comparability profile of the felodipine release from lphns formulation (hf1-hf6) and the pure drug suspension explains in the following descending order: hf3 > hf 6 > hf 2 > hf 5 > hf 1 > hf 4 > pure drug suspension as shown in figures (7,8,9). it was noted that the pure drug suspension gives a lower intestinal flux in comparison to all felodipine lphns formulations(hf1-hf6) due to increase solubility and intestinal permeation with felodipine lphns as lipid-based technology. in addition, the nanosize of these lipid-based carriers provide a large surface area for drug release and rapid absorption to the systemic circulation (25,26). the analysis of variance indicated a significant(p-value <0.05) relationship between independent variables and ex vivo intestinal permeation factor. table 5. the slope and permeation coefficient for felodipine from lphns formulations (f1-f9) and pure drug suspension through non-everted sheep intestine. formulation code flux (μg/ml) permeability coefficient (cm/min) hf1 0.0211 0.000559 hf2 0.0213 0.000564 hf3 0.0226 0.000599 hf4 0.0199 0.000527 hf5 0.0211 0.000559 hf6 0.022 0.000583 pure drug suspension 0.006 0.000159 korsemeyer-peppas model higuchi model first order model zero order model formulation code n r2 r2 r2 r2 0.5064 0.948 0.982 0.866 0.971 hf1 0.496 0.934 0.979 0.8821 0.968 hf2 0.4914 0.919 0.984 0.906 0.958 hf3 0.516 0.960 0.983 0.756 0.969 hf4 0.504 0.942 0.992 0.870 0.972 hf5 0.494 0.929 0.984 0.885 0.968 hf6 0.4133 0.908 0.965 0.89 0.843 pure drug suspension korsemeyer-peppas model higuchi model first order model zero order model formulation code n r2 r2 r2 r2 0.7304 0.924 0.989 0.965 0.895 hf1 0.6774 0.964 0.988 0.968 0.891 hf2 0.6356 0.9794 0.9884 0.9563 0.917 hf3 0.649 0.968 0.996 0.909 0.936 hf4 0.645 0.98 0.993 0.937 0.911 hf5 0.622 0.983 0.992 0.944 0.905 hf6 0.4795 0.9318 0.9755 0.958 0.939 pure drug suspension iraqi j pharm sci, vol.31(1) 2022 felodipine lphns 128 figure 7. permeation of felodipine from lphns formulations hf1, hf4 and pure drug suspension through non-everted sheep intestine, the values of mean ±sd (n=3). figure 8. permeation of felodipine from lphns formulations hf2, hf5 and pure drug suspension through non-everted sheep intestine, the values of mean ±sd (n=3). figure 9. permeation of felodipine from lphns formulations hf3, hf6 and pure drug suspension through non-everted sheep intestine, the values of mean ±sd (n=3). conclusion the microwave-based method is a very effective and reproducible technique in preparing felodipine lphns formulations (hf1-hf3) and prepotent to the seset that prepare felodipine lphns formulations (hf4-hf6). all the felodipine lphns formulations (hf1-hf6) show colloidal dispersion properties but the (hf1-hf3) 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26(1):765-72. doi: 10. 1080 /10717544 .2019 .1642420 this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 20 microencapsulation of green coffee beans (coffea canephora) extract using whey protein concentrate muhammad ali husni***, akhmad kharis nugroho*, teuku nanda saifullah sulaiman*and nanang fakhrudin**1 *department of pharmaceutics, faculty of pharmacy, universitas gadjah mada, ,indonesia. **department of pharmaceutical biology, faculty of pharmacy, universitas gadjah mada, indonesia. ***department of pharmacy, faculty of mathematics and natural sciences, universitas syiah kuala, , indonesia. ***doctorate program, faculty of pharmacy, universitas gadjah mada, indonesia. abstract coffee bean contains bioactive compounds including caffeine and chlorogenic acid (cga) that have a stimulant effect and are used for combating fatigue and drowsiness, and enhancing alertness. however, when the coffee bean was processed in the form of green coffee bean (gcb) extract, it has an unpleasant flavour and limitations instability, activity, and bioavailability. this study aimed to produce microcapsules of the gcb (coffea canephora) ethanolic extract containing considerable amounts of the bioactive compounds for nutraceutical supplements. the gcb ethanolic extract was microencapsulated by spray drying using a whey protein concentrate (wpc) biopolimer. the particle size (psa), morphology (sem), and physicochemical characteristics (uv and lc-ms/ms), as well as radical scavenging activity (dpph) of the microcapsule were determined. we found that the microencapsulation yield was 95.85% of the extract, with the particle mean of volume diameter was 1.312 µm (span value: 1.285 µm). the morphology of the microcapsule particles was microspheres with wrinkle and shrivel surface. the microcapsule demonstrated the caffeine content of 15.25%, the cga content of 8.52%, the total phenolic content of 1.8 % (1794.7 mg/100g of the gallic acid equivalent (gae)), and the radical scavenging activity of 179.23 µg/ml. the wpc can be used to encapsulate the gcb extract by using spray drying microencapsulation to produce a high yield microcapsule with a smaller and narrower particle diameter. this microencapsulation was able to engulf and package unpleasant flavor and aroma, and to preserve considerable amounts of the bioactive compounds. keywords: spray drying, coffee, microcapsule; caffeine, chlorogenic acid. introduction coffee beans are the second-largest most traded commodity(1,2) and one of the most popular beverages in the world(3,4). the top five coffeeproducing countries are brazil, vietnam, indonesia, columbia, and ethiopia, which account for almost 65% of the total global coffee production(5). the coffee plant (genus-coffea; family-rubiaceae) consists of more than 70 species. among them, coffea arabica (arabica) and coffea canephora (robusta) commonly consumed by human beings as beverages and they account for almost 75% and 25% of the total coffee beans produced in the world, respectively(5). coffee contains various bioactive compounds, such as cellulose, soluble carbohydrates, insoluble polysaccharides, nonvolatile and volatile compounds, aliphatic acids, nitrogenous compounds, polyphenols, proteins, lipids, amino acids, vitamins, and minerals. among those bioactive natural compounds, chlorogenic acid (cga) and caffeine are the two most important compounds from a nutraceutical perspective(5). by drinking coffee, people expect to gain stimulating effect, get better taste and aroma sensation(6), reduce fatigue and drowsiness(7), and enhance alertness(8). these effects are associated with the presence of its main phytochemical constituent, caffeine, and cga. the effects of coffee consumption on human metabolism and psychological aspect have been reviewed by de melo pereira et al(9). coffee consumption is benefiting human health. it reduces the risk of various disorders such as type 2 diabetes, cardiovascular disease, neurological diseases (parkinson’s, alzheimer’s, cognitive impairment, and dementia), suicidal behaviour, cancer, hepatic injury and cirrhosis, human gut microbiota symbiosis, and gastrointestinal problems (9). coffee is lauded for its aroma and flavor, which are imparted by certain constituents such as alkaloids, trigonelline, proteins, amino acids, phenolics, and carbohydrates. on the other hand, the composition of these constituents is also responsible for the unpleasant odour and taste such as bit, sour, pungent, rancid, and repulsive that can cause nausea and vomiting(5). 1corresponding author e-mail: nanangf@ugm.ac.id received:28 /4/2021 accepted: 4/7 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 21 therefore, before serving for consumption, the gcb is processed through fermentation, soaking, drying, defatting, decaffeinating, and blending or roasting to reduce or eliminate unpleasant odour and taste. however, the processing of gcb causes changes in chemical and physical parameters(10) which alter the concentration, composition, structure, and activity of phyto-constituents and lead to the formation of new compounds. likewise, caffeine and cga have been limited by their stability against oxidative degradation during process and storage, and bio-accessibility from the matrix during gastrointestinal digestion(11). the alternative methods that can be done are extracting, fractionating, or isolating the phytoconstituents, but the extracts and fractions still leaving an unpleasant odour and taste of solvents, while the isolates are derived in a complicated way. though, each process has its advantages and disadvantages related to stability, time, storage, and cost. a promising approach to overcome those problems that are related to the bioactive constituents in gcb is by spray drying microencapsulation using whey protein concentrate (wpc) biopolymer. wpc is one of the polymeric shell materials that has been applied in gelation technology, thermal stabilization, and emulsification. whey is the liquid remaining after the precipitation and curd removal during cheese production(12). the important components of whey are soluble proteins, such as β-lactoglobulin (β-lg), α-lactalbumin (α-la), immunoglobulin (ig), bovine serum albumin (bsa), lactoferrin and lactoperoxidase enzymes, and caseinomacropeptide; lactose; lipids; and mineral salts. wpc provides a positive effect for the body against many diseases including cardiovascular diseases, nerve disorders, digestive problems, cancer, diabetes, osteoporosis, stress, and obesity. it can also enhance immune systems and demonstrates antimicrobial properties benefiting human health(13). whey proteins might increase the overall nutritional value of gcb and preserve the quality and property of the bioactive components in gcb. besides, it also serves as a proper vehicle for bioactive compounds. the gelation property of the globular proteins (β-lg) in wpc allows the development of microand nano structures-based formulations such as microgels, nanohydrogels, nanofibrils, and nanotubes. several studies have reported the use of wpc as wall material to microencapsulate various plant-based products, such as peanut(14), grape(15), vanillin(16), drumstick(17), apple jus(18), and phenolic extract(19). the use of wpc as wall material to microencapsulate the extract of gcb (as a core material) using spray drying has not been reported. the present study aimed to prepare the formula and to produce microencapsulation of the extract of gcb using wpc. the product yield, particle size distribution, particle morphology, radical scavenging activity, total phenolic content, caffeine content, and cga content were investigated. materials and methods chemicals and plant material the analytical grade of n-hexane, ethyl acetate, and ethanol was purchased from merck (usa). the instant wpc (iwpc 80) was purchased from milkspecialties (usa). the chromatography grade of caffeine and cga were purchased from sigma-aldrich (st. louis, usa). the coffee bean of the coffea canephora was collected from the local plantation in suka makmur timur, bener meriah district, gayo highland, aceh, indonesia. the red cherry of the coffee bean was harvested from 10 years old plant. coffee beans preparation the coffee bean (5 kg) was pulped and dried under indirect sunlight for 7 days. the parchment of the coffee bean was pulped and the bean was dried again for additional 7 days. the silver skin of the coffee bean was pulped and the beans were dried for 3 days to obtain dried gcb. the dried gcb was ground by blender and sifted (sieve number 40). the coarse gcb was collected in the vessel and stored in a dark and dry place before the extraction process (figure 1). extractions procedure the extraction procedure was performed by using maceration according to khoddami et al(20) procedure with some modifications (figure 1). 200 g of coarse gcb was macerated with 1 l of nhexane at room temperature for 24 hours and then filtered by a vacuum-assisted filter. the filtrate was collected and then n-hexane residue was dried and then re-macerated with 1 l of ethyl acetate at room temperature for 24 hours and then filtered. the filtrate was collected and then the ethyl acetate residue was dried and then macerated with 1 l of ethanol (70%) at room temperature for 24 hours and filtered. taken together, three filtrates were collected from this maceration process and all filtrates were evaporated by using a rotary evaporator (buchi). the concentrated filtrates were collected in the dark bottles, weighed, and stored in a refrigerator. the maceration process was done in triplicate. microencapsulation process the microencapsulation was processed by using a spray dryer (gea niro) according to the abrahão et al(21) procedure (figure 1). the extract (100 mg) was dispersed in wpc solution (0.1%) and diluted with water to obtain 200 µg/ml of feed solution. the feed solution was homogenized by using an ultra-turrax (ika t18) at 40 °c, 1,000 rpm for 45 minutes, and then stored in a refrigerator overnight to allow the complete formation of https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 22 monomer dispersion. the spray drying was performed at an inlet temperature of 180 °c, outlet air temperature of 70 °c, and feed flow rate of 5 ml/min. the microcapsule was collected in an airtight plastic bag, weighed, and stored at a desiccator for analysis. the spray drying microencapsulation was performed in triplicate. figure 1. scheme of gcb preparation, extraction, and microencapsulation process. the extraction was macerated gradually using n-hexane (defatted), ethyl acetate (decaffeinated), and ethanol solvents (analytical grade). microencapsulation performed by processing the feed solution (200 µg/ml) containing extract (100 mg) and wpc (0.1%) using a spray dryer (180 °c inlet temperature, 70 °c outlet temperature, and 5 ml/min feed flow rate). etoac: ethyl acetate; etoh: ethanol; wpc: whey protein concentrate. extracts and microparticles yield the extracts yield was calculated according to the fikry et al(22). extracts yield (%) was expressed as a ratio of the weight of the extracted solids and the initial sample weight. the physical properties of the extract were determined visually and by tactile. the microcapsule yield was calculated according to the gonçalves et al(23). microcapsule yield (%) was expressed as the ratio of the mass of microcapsule obtained in the spray dryer output and the solid content of the initial feed solution. the physical microcapsule was determined visually and by tactile. particle size distribution and morphology the particle size was analyzed by a particle size analyzer (psa) (beckman coulter ls 13 320) according to the kusmayadi et al(24) procedure. the sample was dripped on the instrument, and the particle size of the sample was recorded by using a zeta potential analyzer (beckman coulter delsa tm). then, the span was calculated using an equation: span = [(d90 d10)/d50] where d90: the portion of particles with diameters below this value is 90%; d50: the portions of particles with diameters smaller and larger than this https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 23 value are 50%; d10: the portion of particles with diameters smaller than this value is 10%. particle morphology was examined by using a scanning electron microscope (sem) (leo 40 xvp) equipped with ray-x microanalysis systems (quantax eds and espirit software) according to kuck & noreña(25) procedure. the sample was adhered to a double-sided adhesive tape, nailed to a stub, and then coated with vacuum gold. the sem conditions were operated at 15 kv using 1.0 to 7.0k magnification. caffeine content the caffeine content was determined using lc-ms/ms according to the previous methods described by tine et al(26) and vinson et al(27). the lc instrument was a flexar lc perkin-elmer with flexar lc pe200 column oven (luna 3u c18 column). the ms/ms conditions were carried on an ab sciex 3200 qtrap fitted with an apci ion source. the apci was operated in the positive mode. the 5 mg of sample and caffeine standard were dissolved in acetonitrile/h2o (1:1 v/v) to obtain the final concentration solution of 50 µg/ml and filtered using polytetrafluoroethylene (0.2 µm). caffeine standard solutions were diluted with acetonitrile/h2o (1:1 v/v) to obtain calibration curves with 5 points concentration range of 2-10. the caffeine (2 µg/ml) standard solutions were directly infused at the flow rate of 10 µl/min in the ms/ms apparatus. multiple epi mass spectra of caffeine were recorded of m/z 195 at 138 da. the software for data acquisition and data analysis was analyst 1.5.2. each experiment was done in triplicate. chlorogenic acids content the cga content was determined according to tine et al(26) and vinson et al(27) procedures by using lc-ms/ms with some modifications. the lc instrument was a flexar lc perkin-elmer with flexar lc pe200 column oven (luna 3u c18 column). the ms/ms conditions were carried on an ab sciex 3200 qtrap fitted with an apci ion source. the apci was operated in the negative mode. the 5 mg of sample and standard were dissolved in acetonitrile/h2o (1:1 v/v) to obtain the final concentration solution of 50 µg/ml and filtered using polytetrafluoroethylene (0.2 µm). the cga standard solutions were diluted in acetonitrile/h2o (1:1 v/v) to obtain calibration curves with 5 points concentration range of 10-50 µg/ml. the cga (20 µg/ml) standard solutions were directly infused at the flow rate of 10 µl/min in the ms/ms apparatus. multiple epi mass spectra of cga were recorded of m/z 359 at 191 da. the software for data acquisition and data analysis was analyst 1.5.2. each experiment was done in triplicate. total phenolic assay a total phenolic content (tpc) was determined according to the abrahão et al(21) procedure by using a spectrophotometer (shimadzu uv-1601). shortly, 0.07 g of the samples were diluted in 25 ml of distilled water at 25 °c and stirred by a vortex. 200 μl of the sample solution was transferred to tubes containing 200 μl of folinciocalteu's reagent. then, 200 μl of saturated calcium carbonate solution (10% w/v) was added together with 2 ml of distilled water and incubated in the dark at room temperature for 60 min. the absorbance value was measured using a spectrophotometer at 765 nm. the tpc was calculated using linear regression obtained from the standard curve of gallic acid (0–80 μg/ml). the tpc value was expressed in milligram of gallic acid equivalent (gae) per gram of dry matter. radical scavenging activity assay radical scavenging activity was determined according to the abrahão et al(21) procedure by using the 2,2-diphenyl-1picrylhydrazyl (dpph) method in a spectrophotometer (shimadzu uv-1601). shortly, 0.07 g of the samples were diluted in 25 ml of distilled water at 25 °c and stirred by a vortex. 2.5 ml of the sample solution was pipetted into tubes containing 1 ml of dpph solution (0.3 mm) reagent followed by stirring. after 30 min, the absorbance was recorded in the spectrophotometer at 518 nm. ethanol (1.0 ml) containing a sample solution (2.5 ml) was used as a blank. dpph solution (1.0 ml; 0.3 mm) in ethanol (2.5 ml) was used as a negative control. the absorbance values were converted to radical scavenging activity percentage using the following equation: %rsa=100 {[(abs s abs b)] x 100/abs c} where %rsa: radical scavenging activity percentage; abs s: sample absorbance; abs b; blank absorbance; abs c; control absorbance. results and discussion extracts and microparticles yield prior to extraction using ethanol, the gcb was extracted with n-hexane and ethyl acetate to remove the nonpolar components such as lipids, fats, and oils. we found that the extraction yield was 2.38%. this extraction yield was lower that the previous studies(21,28–30) because we performed a preextraction process using relatively nonpolar solvents to obtain an enriched extract with fewer nonpolar constituents. additionally, the difference in the type of coffee bean and the extraction method (solvent, duration, temperature, solvent-sample ratios) might also affected the extraction yield. https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 24 table 1. the yield and physical characteristics of the gcb extract obtained from the extraction, and the microcapsule product. samples yields (%) physical characteristics (visual and tactile) gcb extract 2.38 ± 1.15 semi-solid formed, yellowish-green color, smooth sandy texture bitter flavor, coffee distinctive odor, and stung ethanol aroma microcapsule 95.85 ± 3.60 fine powder formed, smooth, greenish-yellow color, bitter flavorless, savory flavor, roasted coffee, and milk distinctive odor, and fragrant aroma data are expresed as mean ± sd (n = 3). gcb: green coffee bean the extract was microencapsulated using a spray drying method and yielded a 95.85% microcapsule. the yield and characteristics of the extract and microcapsule product were showed in table 1. interestingly, the yield of microcapsule of the gcb extract in this study was an abundant. the other plant-based products reported previously, the microencapsulation yield of vanilla extract, peanut sprout extract, red grape juice were reported 94.4, 89.01, and 39.00%, respectively(14–16). also, the utilization of wpc in gcb extract microencapsulation to the other material using maltodextrin (40-84%)(28–31) and arabic gum (60%)(32). this indicated that the type of wall material affected microencapsulation yield. other factors that might affect microencapsulation efficiency are the inlet temperature(33–35) and cyclone efficiency(23,33,36) in the spray drying process. microcapsule size in this study, it was found that the mean volume diameter and span of the microcapsule were 1.312 and 1.285 µm, respectively (figure 2). these data demonstrated that the microcapsule particle has a homogeneous and uniform relatively, as well as narrower of size. the reported publications previously, the mean value of volume diameter was variated. calva-estrada et al(16) reported the feret diameter of 16.25 µm for the microcapsule of natural vanilla extract using wpc; oliveira et al(15) reported the de brouckere mean diameter was ranging from 4.77 to 13.56 µm for microcapsule of red grape juice using wpc; abrahão et al(21) reported the volume diameter of 15.78 µm for microcapsule of spent espresso coffee grounds using whey protein isolate (wpi); whereas desai et al(28) reported the particle size of 2.3 µm for microcapsule of green coffee extract using maltodextrin. a study by desai et al(31) has reported that a particle size of 0.08 µm for the extract of green coffee using maltodextrin with a nanospray dryer. these indicated that our data showed a typical microcapsule particle size. the particle size is determined by several processes including spray mesh size, solid particle concentration (viscosity), inlet temperature, spray rate, pump circulation rate, type of surfactant, and solvent(33). microcapsule morphology the microcapsule morphology was observed with scanning electron microscopy (sem). the result showed that the particle was a spherical dense, smooth, wrinkle, shrivel, homogenize, compact, and porous. we found that the particles of microcapsule were dispersed throughout the particles or molecules matrix (figure 3). the particle morphology was similar to the other studies(16,19,21,28,31). indeed, the particle morphology in this study was in line with arpagaus et al(33) showing that microcapsule particle has the morphology of dense, hollow, porous, and encapsulated structures with spherical, wrinkled, shriveled, or doughnut-like shapes(33). clearly, the microcapsule morphology of the microcapsule particle obtained in this study was microspherical. the spherical geometry of the microcapsule is determined by the wall material(23), feed property (material type, solid concentration, solvent, and surfactant), and drying temperature(33). generally, the slow-drying process results in more compact particles, while the fast-drying process yields hollow particles(33). https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 25 figure 2. the particle diameter of the microcapsule measured with psa (ls 13 320; optical model: fraunhofer.rf780d; fluid: h2o). data are expresed as mean ± sd (n = 3). d90: the portion of particles with diameters below this value is 90%; d50: the portions of particles with diameters smaller and larger than this value are 50%; d10: the portion of particles with diameters smaller than this value is 10%. span = (d90 – d10)/d50 figure 3. the sem analysis results of microcapsule (condition=vacc=15.0kv). (1) 1.0k magnifications, 100 µm; (2) 2.0k magnifications, 50 µm; (3) 4.0k magnifications, 20 µm; and (4) 7.0k magnifications, 10 µm. https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 26 caffeine content in the lc-ms/ms analysis, the retention time of caffeine appeared at 4.22 min (figure 4a) with the molecular ion m/z 195.081 in the positive ion mode (figure 5). this was in line with perrone et al(37) and davies & wasan(38) reported that the precursor ion (m/z) of caffeine was 195 with the retention time 4-5 min. the linear regression curve of the caffeine (2-10 µg/ml) was y = 1536.1x + 315.3 (r2 = 0.998). by using this linear regression curve, the caffeine content of the microcapsule and extract was determined. we found that the caffeine content of the extract and the microcapsule was 18.23 and 15.25%, respectively (table 2). the caffeine content of the microcapsule was lower than the extract (0.2 times) with an encapsulation efficiency was 80%. the result was in accordance with the previous studies showing the decrease of caffeine content after encapsulation. this occurred not only in the microencapsulation process with wpc as wall material but also with the use of other wall materials. in addition, the difference in the type of coffee extract also contributed to the variability of caffein content. desai et al(28) reported the caffeine content of gcb and its microcapsule (using maltodextrin) were 12.78 and 11.98 mg/g, respectively; whereas abrahão et al(21) showed that the caffeine content of spent espresso coffee ground extract and its microcapsule (using wpc) was 6.12 and 2.54 mg/g, respectively. in line with these data, our result also demonstrated that part of the caffeine was lost during the microencapsulation process. caffeine is a thermo-stabile alkaloid. caffeine loss might be due to the water-soluble properties of caffeine whereby partially carried by the water vapor eliminated(5). figure 4. the chromatogram of caffeine standard (a), etoh extract (b), and microcapsule (c). positive ion mode of time-of-flight mass spectrometry electrospray ionization (tof ms esi+). retention time 4.22 min figure 5. the spectrum of caffeine standard. positive ion mode of time-of-flight mass spectrometry electrospray ionization (tof ms esi+). ion fragmentation m/z 195.081 https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 27 chlorogenic acids content based on the chromatogram in the figure 6a, the retention time of cga was at 4.79 min with the molecular ion m/z 353.14 in the negative ion mode (figure 7). this finding is in line with previous publications showing the retention time of cga was 4-5 min with m/z of the molecular ion were 352.7(39) and 353(27). figure 6. the chromatogram of the cga standard (a), etoac extract (b), and microcapsule (c). negative ion mode of time-of-flight mass spectrometry electrospray ionization (tof ms esi-). retention time 4.79 min figure 7.the spectrum of cga standard. negative ion mode of time-of-flight mass spectrometry electrospray ionization (tof ms esi-). ion fragmentation m/z 353.14 the regression curve of cga standard (10-50 µg/ml) was obtained y = 1426.8x + 7848.8 (r2 = 0.999). this linear regression curve was used for the determination of cga content in the microcapsule and extract. we found that the cga content of the extract and microcapsule was 10.88 and 8.52 %, respectively (table 2). cga content of the microcapsule was lower than the extract (0.28 times). our data were in agreement with the other studies reporting the decrease of cga content after microencapsulation. similar with the decrease in caffeine content, the cga content was also affected by the difference in wall material and type of coffee extract. a previous study demonstrated that the https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 28 cga content for the green coffee extract and microcapsule (using maltodextrin) were 10.23 and 9.9 mg/g, respectively(28). abrahão et al(21) showed the cga content for the spent espresso coffee ground extract and microcapsule (using wpc) were 3.71 and 1.57 mg/g, respectively. cga is a thermolabile phenolic compound that is susceptible to degradation. the loss of cga content due to the encapsulation process in this study is 21.69% or 0.28 times lower than the extract. the encapsulation process might trigger isomerization, epimerization, lactonization, and other degradation that alter the chemical structure cga(21,40). total phenolic contents the total phenolic content of the extract and the microcapsule were 219 and 1794.7 mg gae/100 g, respectively (table 2). the total phenolic content of the microcapsule was higher than the extract (7.19 times). the result was in line with other studies that reported the increase of tpc is associated with the use of wpc as a wall material. díaz-bandera et al(41) reported the utilization of wpc in the microencapsulation of roselle extract that increased the tpc from 72.06 to 89.09 mg gae/100 g (0.24 times). abrahão et al(21) also reported the same showing that the microencapsulation of spent espresso coffee grounds using wpc increased the tpc from 757 to 1284 mg gae/100 g (0.7 times). interestingly, the enhancement of tpc in the microencapsulated extract in this study was an abundant relatively. on contrary, other studies have reported a decrease in tpc after microencapsulation of gcb using maltodextrin. calva-estrada et al(16) reported the utilization of wpc for the encapsulation of vanilla extract significantly reduced the tpc from 0.89 and 0.19 g gae/100 g (3.68 times); whereas desai et al(28) reported the decrease in tpc from 350.28 to 174.07 mg gae/100 ml (1.01 times) in green coffee extract after microencapsulation. the changes in the tpc of the microcapsule extract could be due to the formation of polymerization of phenolic compounds as a result of the higher inlet temperature (180 °c) during the encapsulation process. furthermore, the changes might be due to the physicochemical transformation of the phenolic compounds upon interaction with whey proteins as a wall material that lead to the formation distict chemical structures(21,41,42). table 2. parameter values of the extract and the microcapsule parameters gcb extract microcapsule radical scavenger (ic50 (µg/ml) 109.84 ± 0.17 179.23 ± 4.83 total phenolic content (mg gae/100 g) 219 ± 0.02 1794.7 ± 77 chlorogenic acid content (%) 10.88 ± 0.11 8.52 ± 0.05 caffeine content (%) 18.23 ± 0.08 15.25 ± 0.06 data are expresed as mean ± sd (n = 3). gcb: green coffee bean; ic50: half maximal inhibition concentration; gae: gallic acid equivalent radical scavenging activities to determine the antioxidant activity of the extract, we performed a radical scavenging activity using dpph as a reagent. we found that the ic50 of the extract and microcapsule were 109.84 and 179.23 µg/ml, respectively. the ic50 of the microcapsule was higher than the extract (table 2), indicated that the microcapsule has a lower antioxidant activity compared to the extract (0.62 times). the decrease in the radical scavenging activity was might be due to the effect of the encapsulation process and the use of wpc. the decrease in antioxidant activity after microencapsulation using wpc was also observed in the vanilla extract(16), but not in the microencapsulation using maltodextrin(31) and glucomannan(35). this result suggested that utilization of wpc as a wall material in gcb extract microencapsulation was not as effective as maltodextrin or glucomannan in terms of its ability as a radical scavenger. the use of maltodextrin as a wall material in gcb extract microencapsulation increased the antioxidant capacity from 414.8 to 425.9 μmol trolox/g(31); whereas the use of glucomannan as a wall material in the microencapsulation of roasted coffee extract increased dpph scavenging activity from 619.21 to 813.3 µm trolox/g(35). the radical scavenging activity of the microcapsule is determined by many factors, including the temperature during the process. as the process of microencapsulation in this study used the inlet and outlet temperature of 180 and 70 °c, respectively, the thermolabile compounds, including those that have radical scavenging activity might be degraded or altered. the high temperature during the encapsulation process could lead to pre-oxidative reactions and other reactions which could change the native compounds or generate artefacts(16,35). moreover, different types of wall material for the encapsulation could also affect the antioxidant potential due to its interaction with the phytochemical constituents(40,41). although the total phenolic content of the microcapsule was higher than the extract, the radical scavenging activity is lower than the extract. this indicated that the compounds generated by the microencapsulation process and interaction with wpc were not able to scavenge the dpph radicals. other antioxidant assays with a https://doi.org/10.31351/vol31iss1pp20-31 iraqi j pharm sci, vol.31(1) 2022 microencapsulation of green coffee extract doi: https://doi.org/10.31351/vol31iss1pp20-31 29 distinct mechanism of action were required to assess the antioxidant potential of the microcapsule. conclusion in summary, we found that wpc was effective as a wall material in gcb extract microencapsulation using spray drying. the microcapsule showed smaller and narrower span particles with microspheres geometry, compact morphology, and wringkled surface of particle structure. the microcapsule was able to carry and protect considerable amounts of bioactive compounds. it preserved gcb bioactive compounds including caffeine and chlorogenic acid. it also engulfed and packaged the unpleasant flavor and aroma in the gcb extract. this method can be applied to formulate innovative products based on coffee beans for healthy pharmaceutical ingredients, food, and beverages. conflicts of interest the authors declared no conflict of interest. acknowledgments the authors thank direktorat jenderal pendidikan tinggi (ditjen dikti) of indonesia for financial support wthin beasiswa program pascasarjana-dalam negeri (bpp-dn) no: 1405.29/e4.4/2015. references 1. bae j, hong kh. optimized dyeing process for enhancing the functionalities of spent coffee dyed wool fabrics using a facile extraction process. polymers. 2019 mar 28;11(4):574. 2. ijanu em, kamaruddin ma, norashiddin fa. coffee processing wastewater treatment: a critical review on current treatment technologies with a proposed alternative. appl water sci. 2019 nov 18;10(1):11. 3. gaascht f, dicato m, diederich m. coffee provides a natural multitarget pharmacopeia against the hallmarks of 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secnidazole dental in situ gel doi: https://doi.org/10.31351/vol31iss2pp50-61 50 preparation and in-vitro evaluation of secnidazole as periodontal in-situ gel for treatment of periodontal disease dheyaa a. raheema *,1 and hanan j. kassab ** * pharmacy department of alawyaa children hospital, baghdad, iraq **college of pharmacy, university of baghdad, baghda ,iraq abstract this study aims to develop a thermosensitive mucoadhesive periodontal in situ gel of secnidazole for local release of drug for treatment of periodontitis, in order to increase the drug residence time and to increase patient compliance while lowering the side effects of the drug. cold method was used to prepare 30 formulas of secnidazole periodontal in situ gel, using different concentrations of thermosensitive polymers (poloxamer407 alone or in combination with poloxamer 188) and methyl cellulose (mc ) or hydroxypropyl methylcellulose (hpmc k4m )in different concentrations used as mucoadhesive polymer and the resultant formulations were subjected to several tests such as gelation temperature gt, appearance and ph value. the formulas with the most appropriate gt were subjected to in-vitro drug release. three formulas were chosen with appropriate release, f6 (15% p407, 1% mc), f29 (18%p407,3% p188, 0.8% hpmc) and f30 (18%p407,3% p188, 1% hpmc). these formulas were subjected to mucoadhesive force, viscosity, drug content, spreadability, gelation time and fourier transform infrared (ftir) compatibility studies. the results indicates that formula f29 and f30 have best gelation temperatures (33°c, 32°c) gel strength (1.5h,2h) mucoadhesive force of (17.1, 23.4 dyne/cm2 ) and in-vitro drug release (98.2%, 100%) respectively during 3.5h and gelation, time about 10 seconds for both formulas and ftir spectrum study show absence of important interaction between secnidazole and the polymers used. keywords; in-situ gel, methylcellulose, hpmc, poloxamer, secnidazole تحضير والتقييم المختبري لــــ سكنيدازول هالم بالموقع لعالج التهاب اللثةال **حنان جالل كساب و 1، *ضياء عبد الحسن رحيمة قسم الصيدلة في مستشفى أطفال العلوية، بغداد، العراق * ، العراق بغداد بغداد، جامعة الصيدلة، كلية الصيدالنيات، **فرع الخالصة الذي يتصلب في الموقع وتحسينه لزيادة وقت تواجد الدواء وتسليم secnidazoleالهدف الرئيسي من هذه الدراسة هو تحضير هالم تركيبة للهالم )الذي يتصلب 30قد تم تحضيرالدواء المحلي مما يزيد من امتثال المريض ويقلل من اآلثار الجانبية لألدوية ويقدم معدل شفاء أسرع ال للحرارةفي متحسسة بوليمرات باستخدام الباردة بالطريقة على poloxamer 407 and poloxamer 188) موقع( تساعد بوليمرات مع ) واعتماد درجة الحرارة methylcellulose( mc) and hydroxypropyl methyl cellulose (hpmc)))االلتصاق على الغشاء المخاطي التي لها اختيار التراكيبالالزمة للتحول من السائل الى الهالم القريبة من درجة حرارة الجسم الطبيعية، كأساس في اختيار التركيبة المناسبة، تم الى تحرر الدواء خارج الجسم و كان احسن التراكيب التي هذه التراكيبخضعت وقد الجسم.هالم قريبة من درجة حرارة تحول الىدرجة حرارة (الذين يحويان االتي f30و f29و التركيبتين ) (p407, 1% mc %15) التي تحوي f6التركيبة ثالث تراكيب، أظهرت تحرر مناسب هي f29 (18%p407,3% p188, 0.8% hpmc) f30 (18%p407,3% p188, 1% hpmc)قوة االلتصاق للغشاء اختبارهم من حيث . وتم ( لبيان أي تعارض كيميائي ftirالطيف تحت الحمراء ) صودراسة امتصا التحول الى الهالم ووقت واالنتشارالدوائي والمحتوى واللزوجةالهالمي بين المواد. )بأفضل درجات f30و f29تتمتع الصيغتان ) ، (درجة مئوية 32و 33حرارة تحول الى الهالم ، قوة الصق ( ساعة 2و 1.5 و قوة الهالم 10حوالي الهالم ووقت تكون ، ساعة 3.5على التوالي خالل ( ٪100٪ و98.2) في المختبر العقار تحرر ( 2داين/سم 23.4 و 17.1 )مخاطي .ليمرات المستخدمةسيكنيدازول والبوالعقار بين كيميائيشير إلى عدم وجود تفاعل ت ftirدراسة طيف هما. ثواٍن لكل من متحسس للحرارة، سيكنيدازول بالموقع،هالم الكلمات المفتاحية : introduction periodontitis is an inflammatory disease of supporting tissues of teeth caused by specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with periodontal pocket formation, gingival recession or both (1) . gingivitis, is the mildest form among periodontal disease(2). periodontitis affects nearly 60% of the world’s elderly population and 50% of the adult population (2). male’s susceptibility is more than females for chronic periodontitis(3) . dheiaalzobaidy@yahoo.com :mail-corresponding author e1 received: 27/8 /2021 accepted:15 /11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp50-61 iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 51 heavy smokers have a high risk of occurrence of chronic periodontitis (4). in addition, it can be associated with other serious health conditions such as diabetes, cardiovascular disease and stroke (5). current concepts of the etiology of periodontitis implicate a bacterial infection as the primary cause of the disease (6) . the main cause of periodontitis is that bacterial flora gradually shifts to anaerobic status, in addition to periodontal pockets, which provide a favorable environment for the growth and proliferation of some anaerobic bacterial species(7). porphyromonas gingivalis, tannerella forsythia, filifactor alocis, (a gram-positive anaerobe) (8), and treponema denticola have shown to be the most common cause of periodontal disease (9,10) . the non-surgical treatments are enough for patients with early or moderate disease which include scaling and root planning (srp) (bacteria’s mechanical removal). supplemental use of local antibiotics, local antiseptic drugs, systemic antibiotics have been shown to provide some additional benefit compared with debridement alone (11). nitroimidazoles (such as metronidazole) have excellent activity against anaerobic microorganisms due to their bactericidal activity, broad spectrum of activity and rapid onset of action (12). while secnidazole, has a longer terminal elimination halflife than commonly used drugs in this class. therefore, the treatment interval will be shorter and significantly more effective than the treatment using other imidazole drugs (13) . secnidazole is a second-generation of 5nitroimidazole antimicrobials and has selective activity against many anaerobic gram-positive and gram-negative bacteria and protozoa (14) , it has molecular weight of 185.18g/mol, melting point is 76°c, a water solubility is nearly 34 mg/ml at 27°c, partition coefficient is 0.27 and half-life of 17 hours (15), and is approved for dental infections(16) . local application into periodontal pocket could be very advantageous both in prolonged drug delivery, preventing systemic side effects (17), ease of application; especially in situ gelling system, as they are applied as liquids that gel upon contact with the oral cavity, selectively targeting a limited number of diseased sites that were unresponsive to conventional therapy and possibly enhanced treatment results at due mucoadhesion and drug retention (18). secnidazole vaginal in situ gel formulations were prepared by karthick et al., using 0.45% of carbopol 940 with 0.35% hpmc k4m and the 0.35% of carbopol 940 with 0.35% hydroxy propyl cellulose hpc these formulations also released less than 50% of drug in simulated vaginal fluid at the end of 8 hours (19). while, narayana et al., formulated in situ vaginal gels of secnidazole, based on ion activated systems using gellan gum (0.10.75% w/v) and sodium carboxy methylcellulose to prolong the release of secnidazole (1% w/v) (20). for periodontal delivery, a previous study of secnidazole and of serratiopeptidase was performed by priyanka et al. using sodium alginate as an insitu gel polymer (1%), and hpmc e50lv (18%w/w) to modulate the gel strength and the bioadhesive force, (21) also, dento-oral gels of secnidazole were prepared by gad et al. by using 3% w/w mc and 5% w/w hydroxy ethyl cellulose hec with hpmc or carbopol 934 or carbopol 971 (1 or 3% w/w) (22). also secnidazole in situ implant to modify the release of secnidazole over 24hours using pla(poly lactic acid) and plga(copoly lactic glycolic acid) polymers(23). gelation temperature gt is the crucial criteria for the selection of the appropriate thermo-sensitive formula of poloxamers. poloxamers are synthetic triblock copolymers of poly (ethylene oxide)-poly (propylene oxide)-poly (ethylene oxide) (peo-ppopeo), poloxamer 407 (p407) has 70% peo while poloxamer 188 (p188) has 30% peo(24,25) . poloxamer 407 (p407) as a thermosensitive polymer is mostly used in range 15-20 % (w/w) to achieve the desired sol-gel thermal transition near body temperatures (33-35ºc) (26). due to their amphiphilic nature, at critical micelle temperature cmt, p407 molecules assemble to form spherical micelles at the critical micelle concentration cmc, with a dehydrated hydrophobic ppo core surrounded by hydrated swollen peo chains, packing and entanglements of micelles will increase with temperature results in a 3d lattice structures (27,28) . poloxamers form gel at the critical micelle concentration cmc and critical micelle temperature cmt, the higher the peo % in the poloxamer, the higher is the cmc and cmt, thus p188 has a higher cmc and cmt than p407 and the crucial determining factor of gt is the type and concentration of the poloxamer (28). although the mucoadhesive polymer may alter the gt but this is secondary to the effect of poloxamer (29). the aim of this study is the preparation of a thermosensitive in-situ periodontal gel for the local intrapocket administration of secnidazole, that will gel near physiological temperature, using the thermo responsive polymers (poloxamer 407 and poloxamer 188) with mucoadhesive polymers (methyl cellulose or hydroxypropyl methylcellulose). iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 52 materials and method secnidazole, poloxamer 407 p407, poloxamer 188 p188 and methyl cellulose mc (63000 da) were purchased from baoji guo kang bio-technology co., ltd. china. hydroxypropyl methyl cellulose hpmc (k4m) from hangzhou hyper chemicals limited, zhejiang, china. all other chemicals and solvent were of analytical reagent grade. method of in situ gel preparation in situ gel of secnidazole sc was prepared by cold method (30). a predetermined amount of poloxamer p407 (15-18% w/v) alone or in combination with p188 (2-4% w/v) was gradually added to cold water at 4 °c in a beaker with continuous agitation at a speed of 500 rpm for 2 hours using a magnetic stirrer, then the poloxamer solution was left overnight for complete hydration (at 4 °c) to ensure the formation of a clear and viscous solution of poloxamer. secnidazole at 1% w/v (16,20)was then added to poloxamer solution in the next day, using magnetic stirrer. the viscosity enhancement polymer dispersions were prepared beforehand, hpmc was added with continuous mixing to hot water (70°c), while mc was added slowly and gradually to water at room temperature with continuous mixing and their dispersion was left overnight for complete hydration. preservative (methylparaben mp and glycerin gl) and the viscosity enhancement polymers hpmc and mc were added to the poloxamerdrug mixture with continuous stirring. the final dispersion was kept in refrigerator for another night at 4°c. finally, the volume was completed with purified water (31). secnidazole periodontal in-situ gel components are shown in table 1 table 1. composition of sc periodontal in-situ gel h2o to (ml) sc g gl (ml) mp (g) hpmc (g) mc. (g) p188 (g) p 407 (g) formul a no. 100 1 2 0.01 0.2 15 1. 100 1 2 0.01 0.4 15 2. 100 1 2 0.01 0.6 15 3. 100 1 2 0.01 0.25 15 4. 100 1 2 0.01 0.5 15 5. 100 1 2 0.01 1.0 15 6. 100 1 2 0.01 1.5 15 7. 100 1 2 0.01 0.25 4 15 8. 100 1 2 0.01 0.5 4 14 9. 100 1 2 0.01 0.2 17 10. 100 1 2 0.01 0.4 17 11. 100 1 2 0.01 0.6 17 12. 100 1 2 0.8 17 13. 100 1 2 0.01 0.25 17 14. 100 1 2 0.01 0.5 17 15. 100 1 2 0.01 0.25 4 17 16. 100 1 2 0.01 0.5 4 17 17. 100 1 2 0.01 0.2 4 17 18. 100 1 2 0.01 0.4 4 17 19. 100 1 2 0.01 0.2 3 17 20. 100 1 2 0.01 0.4 3 17 21. 100 1 2 0.01 0.1 2 17 22. 100 1 2 0.01 0.2 2 17 23. 100 1 2 0.01 1 18 24. 100 1 2 0.01 0.1 2 18 25. 100 1 2 0.01 0.2 2 18 26. 100 1 2 0.01 0.2 3 18 27. 100 1 2 0.01 0.4 3 18 28. 100 1 2 0.01 0.8 3 18 29. 100 1 2 0.01 1 3 18 30. iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 53 characterization of prepared sc periodontal insitu gel determination of sol -gel transition temperature to determine the gelation temperature test tube tilting method was employed, two ml of the formula was placed in a test tube with 1 cm diameter and sealed with parafilm, to be immersed in cold water (at 4 °c) in a water bath. (medical sources co., ltd., china), the temperature was increased 3°c at the beginning, then when the temperature approached the desired gelation temperature (around 30°c) it was increased by 1°c and kept constant in each temperature degree for ten minutes. when the test tube was tilted 90° and there is no flow of the formula, this was recorded as the gelation temperature (32). appearance and ph determination all formulations were checked visually for their clarity, color and if there are any suspended particles and this is done against black and white background (33). in addition, the ph of all the formulas was measured using a digital ph meter (hanna instruments). the ph meter probe was immersed in each formula and this is achieved in triplicate and takes the average as the ph of in-situ gel formula(34) . in vitro release studies secnidazole release from the selected sc periodontal in situ gel formulations were done by placing 5ml of the prepared formulation (f6, f7f10 f11, f29, f30) into the dissolution jar filled with 500 ml simulated saliva fluid ssf ph 6.8(35) using usp dissolution apparatus type ii (paddle type) for a period of 3.5 h with rotation rate 50 rpm at 37°c. volume of (5ml) were taken at different time intervals and checked spectrophotometrically at 320 nm(22,23). the formulas that showed acceptable release were chosen for further evaluation. kinetic modeling of secnidazole release the mechanism of release of secnidazole from the selected formulas that showed acceptable release were analyzed by fitting the release data into zero, first, higuchi, and korsmeyer peppas equations. using a ddsolver excel microsoft addin program and k and r2, were obtained for each equation, and n value for korsmeyer peppas equation at 60% of release (36). drug content accurately, 1 ml of the formulation (equivalent to 10mg/ml sc) from the selected sc periodontal in situ gel formulas were diluted to 10 ml with ssf (37), and 1 ml of this solution was diluted again to 10 ml with ssf. finally, the absorbance was measured at the maximum absorption wavelength using uv spectrophotometer. determination of gelling capacity the gelling capacity depend on the formulation properties like gelling time and time required for the formed gel to dissolve in a specific environment. gelling capacity was measured by placing a drop of the formula in a vial containing two ml of freshly prepared simulated saliva fluid equilibrated at 37 °c and assessing visually the gel formation and record the time for gel to be formed and the time required for the formed gel to dissolve. gelation time was classified in three groups depending on the gel stiffness, gelation time and duration (38). viscosity determination the selected formulas were maintained at physiological temperature (at 34-37 °c) with the aid of the water bath (medical sources co., ltd., china) to form a gel. (20,34)the viscosity was measured using fungilab smart viscometer fitted with spindle r2, the spindle was rotated from 6 to 100 rpm, and the rotation speed was increased gradually and allowed to rotate for two minutes before viscosity measurement were recorded in pascal per second (pa. s -1). determination of mucoadhesive force mucoadhesive force is the adherence power of the formulations to the epithelial mucosa. the modified balance method was used with a plastic beaker at one facet and on the other facet of the balance, then a vial was geared up with the oral mucosa of the sheep (with a thickness of 0.6 mm obtained from slaughtered sheep) taken immediately after sacrifice. the oral mucosa was outfitted on the backside of the vial. the vials were maintained at temperature (32–34 °c) for 10 minutes, then 1 ml of the sc periodontal in-situ formula was placed in a watch glass beneath the vial fitted with the oral tissue, then the vial was pressed down onto the gel for one minute as preliminary contact time. water was added to the beaker on the other side of the balance gradually until complete separation of the mucosa from the formula as shown in figure (1). the mucoadhesive force was measured by the weight of water needed to separate the dental in-situ formula from the oral mucosa by equation (1): 𝐷𝑒𝑡𝑎𝑐ℎ𝑚𝑒𝑛𝑡 𝑓𝑜𝑟𝑐𝑒 ( 𝑑𝑦𝑛𝑒 𝑐𝑚2 ) = 𝑚×𝑔 𝐴 ……….. eq (1) where, m is the required weight (in g), g is the acceleration (980 cm/s2) due to the gravity, a is the exposed oral tissue area which is 3.14 cm² in all preparations (39). iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 54 figure 1. mucoadhesive force modified balance method. determination of gel strength a sample of 3 g of each selected sc periodontal in-situ gel formula was placed in a 5 ml cylinder, after complete sol to gel transition and kept in water bath at (33°c). a mass of 5g was placed on the upper surface of the gel. the gel strength was recorded as the time required by the mass to move down 0.5 cm into the formed gel (33). spreadability test the spreadability was checked for the selected formulas after gelation was completed, by dropping 0.1 g of gel on the center of glass slide (20 x20 cm in size), and another glass slide with the same dimensions, covered the initial slide. the initial gel diameter was measured (in cm.) and after placing a 100 g weight on upper slide glass for 5 minutes, the final diameter of spread gel after weight was removed, the difference between the two diameters is the measure of spreadability (40). statistical analysis the results of the statistical analysis showed that the value of f calculated for the tests conducted is smaller than the level of significance (p<0.05), which leads to the rejection of the null hypothesis and thus there are significant or substantial differences between the mean of the treatments. results and discussion determination of sol-gel transition temperature as seen in table 2a, increasing the concentration of hpmc caused a reduction in gt when comparing formulas containing p407 15% f1, f2 and f3 (hpmc 0.2, 0.4, and 0.6%), with formulas containing p407 17% f10, f11, f12, and f13, (hpmc 0.2,0.4, 0.6 and 0.8%), formulas containing p407 17%, p188.3% , f18, f19 (hpmc 0.2,0.4%), formulas containing p407 17%, p188.4% f20, f21 (hpmc 0.2,0.4%), formulas containing p407 18%, p188.3% f27, f28, f29 and f30 (hpmc 0.2, 0.4, 0.6, 0.8 and 1%). the gelation temperature lowering effect due to hpmc k4m could be attributed to the capability of hpmc to join to the chain of poly-oxyethylene in the poloxamer moieties (41). that will lead to increase the dehydration of poloxamer, resulting in an increase in the complexity of neighboring molecules as well as intermolecular hydrogen bonding drastically increasing which produce gelation at much lower degree of temperature (28,37,41). table 2. a gelation temperature gt, appearance, ph of the formulated sc periodontal in-situ gel with hpmc as mucoadhesive polymer appearance at refrigerator temperature (4°c) ph gt hpmc p188 p407 no. clear liquid 6.42±0.15 38±0.70 0.2 15 f1 clear liquid 6.62±0.10 36±1.00 0.4 15 f2 clear liquid 6.68±0.10 26±0.97 0.6 15 f3 clear liquid 6.52±0.12 34±0.50 0.2 17 f10 clear liquid 6.65±0.05 32±0.59 0.4 17 f11 clear liquid 6.81±0.05 24±0.90 0.6 17 f12 clear liquid 7.07±0.12 21±0.86 0.8 17 f13 clear liquid 6.60±0.08 41± 1.25 0.2 4 17 f18 clear liquid 6.78±0.08 39± 1.00 0.4 4 17 f19 clear liquid 6.03±0.15 35± 1.00 0.2 3 17 f20 clear liquid 7.02±0.15 33± 1.32 0.4 3 17 f21 clear liquid 6.43±0.10 37± 1.3 0.2 3 18 27 clear liquid 6.76±0.15 35± 1.15 0.4 3 18 28 clear liquid 6.94 ±0.05 33± 1.00 0.8 3 18 29 clear liquid 7.13 ±0.10 32± 1.04 1 3 18 30 iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 55 while the effect of mc on gt is concentration and molecular weight dependent, as seen in table 2-b. this may be due to mc hydrophobic interaction with poloxamer molecules will enhance cmc formation and mdecrease gt and enhance gel formation(42).opposite to expectation, increasing mc concentration increased the gt of the formulation, (42), due to the high molecular weight of mc (63000 da) as seen in table 2b, according to these results different polymers used alter gt significantly, since the gelation of methylcellulose is an entropy-driven process (43). at first while increasing the concentration of mc it will undergo intermolecular hydrogen bonding through its unmodified hydroxyl groups with hydrophilic part of poloxamer 188 and water molecules forming a network of macromolecular structure that interfere with cmc formation and increasing gt which is responsible for the formation of a stiff gel and cause drastic increment in gelation temperatures in formulation containing poloxamer 188 and mc combinations. mc being a thermosensitive polymer with a gt between 50-70°c (depending on the molecular weight of mc). it is impossible to have sol.-gel transition at temperatures lower than 51 ºc because the hydrophobic association between the methyl groups of mc are not formed at this temperature range, this is not obvious at lower concentrations of mc(43). table 2 .b gelation temperature gt, appearance, ph of the formulated sc periodontal in-situ gel with mc as mucoadhesive polymer appearance all prepared formulas were translucent, and clear. the turbidity observed during preparation was found to disappear and regain clarity after overnight standing at refrigerator (44). all the hpmc containing formulas were clear at low temperature and formed a translucent gel at higher temperature. while some of the mc containing formulas (f6, f7, f8 and f9) separated into two phases when the temperature was raised. in formula f8 (15 % p407 4% p188 0.25% mc) and f9 (15 % p407 4% p188 0.5% mc) poloxamer 407 concentrations was (15%) which is too low for appropriate gelation (45), poloxamer 188(4%) further delay gelation temperature of this mixture(45), for this reason high temperature is required for poloxamer solution to reach gelation temperature and this high temperature concomitantly with low concentration of mc used (0.25-0.5) the hydrogen bonding between mc and water will be weaker, leads to decrease water retention ability of mc and instead mc chain to chain aggregate through its hydrophobic parts, simultaneously stronger network of hydrogen bonds between poloxamer and water is formed, diluting the poloxamer, and poloxamer fails to reach cmc (43) so when the water returns to the poloxamer solution and at this stage we can see two separated layers one with clear appearance (phase separation), which represent the poloxamer solution and more thick and turbid layer represent the mc semi gel solution as seen in table (2). figure 2 shows the phase separation of poloxamer solution with mc, the same concept applies for f6 formula which almost converted to gel at 32°c but when the temperature is increased to more than 65°c the gel collapse and phase separation occur. appearance at high temperature (> 50°c) appearance at 4°c ph gt mc. p188 p407 no. clear liquid 7.10±0.05 26±0.76 0.25 15 f4 clear liquid 7.00±0.15 28±1.10 0.5 15 f5 thick (2 layers) clear liquid 6.93 ±0.15 32±1.00 1.0 15 f6 thick (2 layers) clear liquid 5.92±0.06 above 60 1.5 15 f7 thick (2 layers) clear liquid 6.43±0.065 above 50 0.25 4 15 f8 thick (2 layers) clear liquid 6.12±0.05 above 50 5 4 5 f9 clear liquid 6.48±0.12 20±1.00 0.25 17 f14 clear liquid 6.44±0.02 22±0.76 0.5 17 f15 clear liquid 6.33±0.10 41±0.76 0.25 4 17 f16 clear liquid 6.11±0.06 51± 1.25 0.5 4 17 f17 clear liquid 6.92±0.05 32± 1.65 0.1 2 17 f22 clear liquid 6.64±0.05 45± 1.24 0.2 2 17 f23 clear liquid 6.33±0.10 25± 1.08 1 18 f24 clear liquid 7.07±0.05 32± 1.08 0.1 2 18 f25 clear liquid 6.71±0.05 33± 1.3 0.2 2 18 f26 iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 56 figure 2.phase separation of f6 when the temperature was raised to 65° c ph measurement the ph value is important in oral in-situ gel formulations, for good patient acceptance and compliance (46).the ph of selected oral in-situ gel formulas (f6, f29and f30) ranged between (6.93) and (7.13) as shown in table (2). the ph range is acceptable with oral ph. the ph of all formulations was within the range that would not cause any irritation upon administration; and close to oral ph (47) . selection of the appropriate formula according to the results of the sol-gel transition temperature 7 formulas were chosen for further evaluation with the appropriate sol-gel transition temperature near body temperature with mc (f6, f26) and with hpmc (f10, f20, f21, f29 and f30) in vitro release studies the release of sc from the selected formulas in ssf is seen in figure 3. mucoadhesive polymer used at low concentration f10, f20 (0.2% hpmc), f21 (0.4% hpmc) and f26 (0.2 % mc) showed rapid drug release time. increasing the concentration of the mucoadhesive polymer, as in formulas f6 (1%mc) , f29 (0.8%hpmc) and f30 (1% hpmc) figure (4), the release of sc was slower due the higher concentration of the cellulosic mucoadhesive polymer in these formulation which may be explained to their ability to increase the formulations viscosity as well as their ability to distort or squeeze the extra-micellar aqueous channels of poloxamer micelles through which the drug diffuses thereby delaying the release process as seen in previous studies (48). for this reason, at low hpmc or mc concentration the gel layer surrounding poloxamer micelles are thinner and easily penetrated by the dissolution medium, while at high concentration this gel layer is more condensed and need time for erosion and dissolution, that’s to say, polymer concentration has a significant effect on secnidazole release (49) . another factor is hpmc is more hydrophilic, than mc resulting in a faster rate of polymer swelling and hydration and a large increase in drug release (49). figure 3. the percent release of sc with time in sff (ph 6.8) at 37°c. iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 57 figure 4.the percent release of sc with time in sff (ph 6.8) at 37°c several studies in which mucoadhesive polymers such as mc or hpmc were used and increasing the concentration of these polymers leads to extension of time of drug release and enhances gel strength and each of these characterizations differ widely in each study depending on concentration and type of polymer responsible of gelation process used beside the effect of mucoadhesive polymer (30,48,50,51). kinetic modeling of secnidazole release according to the results obtained from the release data seen in figure 3, the kinetic data modeling was performed for f6, f29 and f30, is shown in table (3). the release of secnidazole obeys korsmeyer peppas non fickian diffusion according to the n value. drug release from hydrophilic polymers like (mc and hpmc) will be based upon all factors that influence the swelling and erosion of the gel such as the composition of the formulation, the characteristics of the drug itself, as well as the properties of the polymers in the product (52).the progressive swelling of polymers leads to changes the porosity of the polymers that affect the diffusional release of a drug (53). table 3.mathematical release kinetics of secnidazole from oral in-situ gel . f6 f29 f30 first order k1 0.029 0.018 0.019 r2 0.9594 0.9480 0.9509 zero order k0 0.618 0.589 0.597 r2 -0.1712 0.4110 0.4447 higuchi kh 7.952 7.377 7.471 r2 0.7046 0.9412 0.9303 korsmeyerpeppas to 60% release k kp 4.083 5.643 2.482 n 0.769 0.632 0.865 r2 0.9967 0.9999 1.0000 drug content the drug content of the selected sc oral insitu gel formulas (f6, f29 and f30) was in the range of (88.2%–101%) which is acceptable according to the usp (54), indicating high content, uniformity of the in-situ gel formulas and suitability of the preparation method. the results are presented in table (4). table 4.the drug content in the selected sc periodontal in situ gel formulas drug content f6 88.2% f29 98.2% f30 100% gelation time and gel capacity gelling time was measured to test the time the formula will remain in gel form before been dissipated by the ssf, and the gelation time was inspected by visual examination. the grading and gelling capacity are shown in table 5. all the formulas showed good gelation time and capacity due to the higher concentration of both the poloxamer and the hpmc. the selected sc oral in situ formula f29 and f30 containing hpmc (k4m) (from 0.8 to 1%) showed excellent gelation time and gel capacity, due to the high concentration of poloxamer 407 (18%) and high concentration of hpmc which possess high molecular weight. in addition, f6 has good gelation capacity this is, due to high concentration of mc (1%) used which enhance viscosity and improve gelation properties but take more time to form in-situ gel this may be due to the lower poloxamer 407 concentrations, 15% used in this formula, so both gelation time and iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 58 gelation temperature are affected significantly by types and concentration of polymer used. table 5. the gelation time and gelling capacity of sc periodontal in situ formulas formula code gelation time (sec.) gelling capacity** f6 10 ++ f29 10 +++ f30 10 +++ (+)gelation occurs after few minutes, remains for few minutes, and dispersed rapidly. (++)gelation occurs at once and remains for 8 hours (+++)gelation occurs at once and remains for more than 8 hours. viscosity determination the viscosity is important since lower viscosity formulas will drain from the oral cavity. the viscosity measurements for selected formulations f29, f30, and f6 at different rpm are shown in figure 5. the formulations showed pseudoplastic rheology, shear thinning and decrease in viscosity with increase velocity, the viscosity was significantly dependent on the polymeric content and effected by their concentration, 0.8 % hpmc (f29), 1% hpmc (f30) or 1% mc (f6) with relatively high concentration of poloxamer 407,which assist formulation to be fluids before and during administration at room temperature, they can be easily injected by means of a periodontal syringe, allowing the formulation to get access to the entire pocket (28). figure 5.viscosity (pa/s) of f6, f29 and f30 at different rpm at 37°c determination of gel strength formulations with higher concentration hpmc (f30) relative to poloxamer shows higher gel strength than those with lower concentration of hpmc (f29) relative to poloxamer polymers (55,56), while, mc (f6) showed more gel strength in comparison with hpmc k4m polymer (f29, f30), this might be related to the formation of more interlocking forces (hydrogen bond, van der waals) between the poloxamer and mc polymer more than with poloxamer and hpmc (56), which means the polymer type and concentration have significant effects on gel strength. table 6. gel strength, mucoadhesive force and spreadability of sc oral in-situ gel selected formulas (mean ±sd) (n=3) formula code gel strength (h) mucoadhesive force (dyne/ cm2 ) spreadability (cm) f6 24±0.03 8991.04 ± 862.54 3.7 ±0.3 f29 1.5±0.06 5338.34± 359.54 3.3 ±0.3 f30 2±0.06 7305.22± 511.54 2.8 ±0.2 determination of mucoadhesive force table 6 shows the mucoadhesive force of the selected formulas. formula f30 has a much higher mucoadhesive force than f29 due to higher concentration of hpmc in f30. the mucoadhesive force is the result of hydrogen bonding between the polymers and oligosaccharide chain of the mucus, lining the mucus membrane. the mucoadhesive force increases significantly due to an increase in the number of penetrating hydrophilic chain to mucus glycoprotein as the concentrations of polymer increase. mucoadhesive forces depends on the nature and the concentration of viscosity enhancement polymers, as the polymer concentration increases (57–59). the high molecular weight of mc and the high concentration of mc in f6 may allow extensive adhesion to the oral mucosa, since high concentration of mc enhances the rheological synergy between mc and mucin(60). when the temperature increases, over and around 30 °c, nonnewtonian behavior occurs corresponding to chain– chain interaction due to the hydrophobic character of methylcellulose. this behavior is in good agreement with the hypothesis of kobayashi et al (61). a high molecular weight of mc have the more effect on muco-adhesion(62). the mucoadhesive force of the oral in-situ gel formulas should be enough to resist saliva fluid flow in oral cavity to maintain good contact with oral mucosal iraqi j pharm sci, vol.31(2) 2022 secnidazole dental in situ gel 59 membranes, especially when placed in the dental pocket and is useful for treatment of periodontitis. spreadability test from the results shown in table 6, all the selected formulas have spreadability from 2.3to 3.7 cm, in f29 and f30 at high concentration of the poloxamer and viscosity enhancement polymer hpmc, the viscosity of the sc periodontal in-situ formula increase and the spreadability decreased significantly (63), while in f6 showed higher spreadability, which may be due to low poloxamer 407 concentration 15% . conclusion intra pocket drug delivery system is an attractive and promising way to deliver antimicrobial agent into periodontal pocket to achieve local enhanced gingival crevicular drug concentration 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hydrogel based on methylcellulose/xyloglucan for periodontitis. j sol-gel sci technol. 2019;89(2):531–42. 61. kobayashi k, huang c, lodge tp. thermoreversible gelation of aqueous methylcellulose solutions. macromolecules. 1999;32(21):7070–7. 62. shaikh r, raj singh t, garland m, woolfson a, donnelly r. mucoadhesive drug delivery systems. j pharm bioallied sci. 2011;3(1):89– 100. 63. harish nm, prabhu p, charyulu rn, gulzar ma, subrahmanyam evs. formulation and evaluation of in situ gels containing clotrimazole for oral candidiasis. indian j pharm sci. 2009;71(4):421–7. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 75 synthesis, characterization, and antimicrobial evaluation of new ceftriaxone derivatives kasim s. hmood * and amer n. elias *, 1 * department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract the present study was designed to synthesize a number of new ceftriaxone derivatives by its involvement with a series of different amines, through the chemical derivatization of its 2aminothiazolylgroup into an amide with chloroacetyl chloride, which on further conjugation with these selected amines will produce compounds with pharmacological effects that may extend the antimicrobial activity of the parent compound depending on the nature of these moieties. ceftriaxone was first equipped with a spacer arm (linker) by the action of chloroacetyl chloride in aqueous medium and then further reacted with seven different aliphatic and aromatic amines which resulted in the production of the aimed final target products. the syntheses have been carried out following simple methodology in excellent isolated yields. the structure of the synthesized derivatives has been characterized by elemental microanalysis (chn), ftir spectroscopy, and other physicochemical properties. all the final synthesized compounds were screened for their antimicrobial activity against selected microorganisms and showed excellent antibacterial and antifungal activities in comparison to ceftriaxone, cephalexin and fluconazole. the new ceftriaxone derivatives have broadened the antimicrobial spectrum of the parent compound in having an extra antifungal activity in addition to its original antibacterial activity. keywords: ceftriaxone, 2-aminothiazole, antimicrobial activity, antifungal activity, ceftriaxone derivatives. اكسونييم الفعالية المضادة للميكروبات لمشتقات جديدة للسيفترايوتوصيف وتق عينتص قاسم سلمان حمود * و عامر ناظم الياس *،1 .فشع اٌىٍّاء اٌظٍذالٍٔت ، وٍٍت اٌظٍذٌت ،جاِعت بغذاد ، بغذاد ، اٌعشاق * الخالصة طّّج اٌذساست اٌحاٌٍت ٌخظٍٕع عذد ِٓ ِشخماث اٌسٍفخشٌاوسْٛ اٌجذٌذة ٚ رٌه بّشاسوخٗ ِع سٍسٍت ِٓ االٍِٕاث اٌّخخٍفت ِٓ ٌمذ اٌخاطت بٗ بٛاسطت واشف وٍٛسٌذ اٌىٍٛسٚأسٍخًٍ ٚاٌزي عٕذ الخشأٗ بعذ رٌه -إٍِٛثاٌضًٌٍٚ-2خالي ححضٍش االِاٌذ ِٓ ِجّٛعت اي ًُي ٔخج ِٛادا ٌٙا حاثٍشاث فاسِاوٌٛٛجٍت لذ ٌٕجُ عٕٙا حٛسع فً ٔشاط اٌّشوب االَ ضذ اٌٍّىشٚباث اعخّادا عٍى بخٍه االٍِٕاث اٌّخخاسة س .طبٍعت ٘زٖ اٌّجاٍِع بفعً ِادة وٍٛسٌذ اٌىٍٛسٚأسٍخًٍ فً ِحٍظ ِائً ٚ ِٓ ثُ حّج ِفاعٍخٗ بعذ٘ا ( سابظ)ٚ ٌٙزا فمذ جٙض اٌسٍفخشٌاوسْٛ اٚال بزساع فاطً ٌمذ اجشي اٌخظٍٕع بٛاسطت اسخخذاَ اٌطشق إٌّٙجٍت . اٌٍفاحٍت ٚ اسِٚاحٍت ِخخٍفت اٌخً أخجج اٌّٛاد إٌٙائٍت اٌّبخغاةبسبعت إٍِاث .اٌبسٍطت ٚ بعٛائذ فظً ِّخاصة ُِيٍَضث اٌخشاوٍب اٌىٍٍّاٌٚت ٌٙزٖ اٌّشوباث باسخخذاَ اٌخحًٍٍ اٌذلٍك ٌٍعٕاطش ٚ غٍش٘ا ِٓ , اطٍاف االشعت ححج اٌحّشاء, (chn)ٌمذ حُ فحض فعاٌٍت وافت إٌٛاحج إٌٙائٍت اٌّضادة ٌٍٍّىشٚباث ضذ عذد ِخخاس ِٓ ٘زٖ االحٍاء اٌذلٍمت ٚ اٌخً . اٌخٛاص اٌفٍضٌٛوٍٍّاٌٚت . وٛٔاصٚياٌسٍفاٌٍىسٍٓ ٚ اٌفٍٛ, اظٙشث فعاٌٍت حسٕت جذا ضذ اٌبىخٍشٌا ٚ اٌفطشٌاث باٌّماسٔت ِع اٌسٍفخشٌاوسْٛ أظٙشث اٌّشخماث اٌجذٌذة ٌعماس اٌسٍفخشٌاوسْٛ حٛسعاً فً طٍف اٌّشوب االَ فً فعاٌٍخٗ ضذ اٌٍّىشٚباث ِٓ خالي اِخالن فعاٌٍت جذٌذة .ضذ اٌفطشٌاث اضافتً اٌى ٔشاطٗ االطًٍ ضذ اٌبىخٍشٌا ة للفطريات ، مشتقات دة للميكروبات ، الفعالية المضادا،الفعالية المضالفعالية امينوثايزول ،-2، السيفترياكسون :مفتاحية لالكلمات ا .السيفتراياكسون introduction the irresponsible usage of drugs and among which the antimicrobial agents has affected the general population and specially the immunocompromised patients since it resulted in the corresponding increase of diseases caused by bacteria, fungi and viral species. frankly speaking the widespread use of antibacterial and antifungal drugs has resulted in the increased resistance of the bacterial and fungal infections towards these drugs and has led to serious health hazards. the resistance that faced the wide spectrum antibacterial agents which increased dramatically in recent years has prompted the discovery and modification towards new antifungal and antibacterial drugs (1, 2) , since the infections caused by these tough mutant microorganisms pose a serious challenge to the medical community and highlighted the importance and urgent need for new, more potent and selective non-traditional antimicrobial agents (3) . 1 corresponding author e-mail: amerelias40@yahoo.com received: 4/5/2014 accepted:22 /10/2014 mailto:amerelias40@yahoo.com iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 76 the rational planning of new synthetic prototypes in drug design and development has been based on the combination of pharmacophoric moieties of different bioactive substances (4) and using a series of methods of structural modification that aim, a priori, at the generation of new compounds presenting optimized pharmacodynamic and pharmacokinetic properties, exploring bioactive substances‟ fragments (fragmentbased drug design) (5) , active metabolites of drugs (6) , bioisosterism (7, 8) , selective optimization of side effects of drugs (9) and drug latentiation (10) . the drug latentiation strategy, in principle, uses a functional group in a drug as merely a handle for the introduction of a moiety that confers on the new entity some desirable characteristic to improve its pharmaceutical utility; more frequently, the group is intimately connected with the pharmaceutical deficiency and its masking directly addresses the deficiency. of the commonly occurring drug functional groups, perhaps greatest effort has been directed at temporarily masking the amino group. the most easily identified liability of candidate amino drugs is their tendency to ionize under physiological conditions, leading to poor membrane penetration by passive diffusion. the impact of this is amplified for the large number of amino drugs that are required to penetrate the blood brain barrier in order to reach their pharmacological targets. a second issue that can affect the development of amino drugs is instability. an example of this is the tendency of primary amines to undergo first-pass metabolism due to n-acetylation and oxidation by monoaminooxidase (mao) (11) . low water solubility, poor stability and low permeability through biological membranes often hinder the clinical development of biologically active amino compounds (12, 13) . the major advantage in designing derivatives of amino drugs is the general robustness of amine derivatives particularly those, such as amides, in which the capacity to ionize has been obviated (14, 15) . on the other hand, the very robustness of amino derivatives means that subtle drug targeting effects can be achieved if an appropriate local vector can be identified and accommodated in the design process (16) . derivatives of amino compounds such as piperazine, 2-aminothiazole and others were reported earlier in the literature in that they have an extended and diverse biological activity when compared to their parent compounds (17-28) . sometimes, they may even attain a complete change in their biological responses as illustrated for the n-alkyl and naryl derivatives of piperazine in that they possess antibacterial and antifungal activities which are even not present in their parent compound, the well-known anthelmintic piperazine (17, 18, 20, 21, 26, and 27) . thiazole derivatives have been found to possess many pharmacological properties and are widely implicated in biochemical processes. the 2-aminothiazole can be used as a thyroid inhibitor in the treatment of hyperthyroidism or for its antibacterial activity (29, 30) . recent studies using prion-infected neuroblastoma cell lines have suggested that the 2-aminothiazole may be used as a therapeutic drug for prion diseases (31) . members of this class of thiazoles are known to possess antibacterial activity against both gram-positive and gram-negative bacteria, for e.g. ceftriaxone, a semi-synthetic third generation cephalosporin that even shows excellent activity for the treatment of infection due to methicillin-susceptible staphylococcus aureus (32) . ceftriaxone is marketed for parenteral use with a relatively long half-life and it is stable to the β-lactamases particularly those produced by gram-negative organisms (33, 34) , which is due to the fact it is a competitive, irreversible beta-lactamase inhibitor and has good inhibitory activities against the clinically important plasmid mediated β-lactamase that is most frequently responsible for the transferred drug resistance (33, 34) . it has excellent anti gram-negative activity. it contains a highly acidic heterocyclic system on the 3-thiomethyl group; where this unusual ring system is believed to confer the unique pharmacokinetic properties to this agent. it kills bacteria by interfering in the synthesis of the cell wall. ceftriaxone has been effective in treating infections due to other „difficult‟ organisms such as the multidrug-resistant enterobacteriaceae (35) . so far, the modifications of the thiazole ring have proven to be highly effective with improved potency and lesser toxicity (30) . in continuation of the increased interest in the chemistry of functionalized chloroacetamide derivatives in drug discovery, because of the high mobility of chlorine atom and the reactive n-h group, therefore, compounds containing chloroacetamide moiety are known to be useful synthetic scaffolds for the design of many heterocyclic systems (36, 37) . in this context patten and coworkers have reviewed the synthesis of a variety of the 2aminothiazoles and their substituted derivatives (by first introducing the αchloroamides of this amine and later by conjugating it with other amino compounds iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 77 such as piperidine, morpholine and others), and evaluating the antibacterial and antifungal activities of these synthesized products (38) . it was observed that the substituted thiazoles have very good antibacterial and antifungal activities against all the tested samples whereas the chemotherapeutic agents used were active against some specific samples (38) . therefore, the present study was designed to synthesize a number of new ceftriaxone derivatives; by first converting its 2aminothiazolylmoiety into the α-chloroamide derivative which on further conjugation with selected amines will result in the production of the final compounds; that may possibly broaden the antimicrobial spectrum of their parent ceftriaxone. materials and methods materials and equipments 2-aminothiazole and potassium carbonate were purchased from himedia (india), chloroacetyl chloride and imidazole were purchased from fluka ag (switzerland), morpholine was purchased from lobachemie (india), piperidine was purchased from bdh (england), hydrazine hydrate (80% w/v) was purchased from qualikems (india), and pyrrolidine and n-methylpiprazine were purchased from sigma (germany ). ceftriaxone sodium was donated thankfully by the arab company for antibiotics industries (acai) (baghdad, iraq). the quality of all these chemicals together with the other ones used throughout the study and obtained from standard commercial sources were of analar grade and used without further purification. the melting points were determined by the open capillary method using thomas hoover melting point apparatus (england) and were used uncorrected. cooling of reactions when needed was done using a julabo chiller vc (f30) (gmbh, germany). infra-red spectra were recorded in kbr disc on shimadzu ftir 8400 spectrometer (japan), at the college of pharmacy, university of baghdad and the college of science, university of al-mustansiriyah. elemental microanalysis was performed at the jordanian university using chn elemental analyzer (euro-vector ea3000a, italy) and at the department of chemistry, college of science, al-mustansiriyah university, using chns elemental analyzer (euro-vector ea, italy). the progress of the reaction was monitored by ascending thin layer chromatography which was run on kieslgel gf254 (60) aluminum plates (e. merck, germany), which was used as well to check the purity of the product. the synthesized compound was revealed either by derivatization or reactivity toward iodine vapor or by irradiation with uv254 light. chromatograms were eluted using methanol: acetone: water (2:2:1) solvent system. chemical tests such as the sodium fusion and the carboxylic acid tests were run to check the presence or absence of chlorine (39) and the free carboxylic acid group (40) in all the synthesized compounds. the antimicrobial evaluation was performed at the department of biology, college of science, university of baghdad. experimental section a. chemical synthesis 1. synthesis of (6r,7r)-7-((z)-2-(2-(2chloroacetamido)thiazol-4-yl)-2(methoxyimino)acetamido)-3-(((2-methyl-5,6dioxo-1,2,5,6-tetrahydro-1,2,4-triazin-3yl)thio)methyl)-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (intermediate compound a) intermediate (a) was synthesized according to the synthetic pathway depicted in scheme 1 and in accordance to the previously reported procedure in reference 41 for the synthesis of α-chloroamides in water. an accurately weighed amount of ceftriaxone disodium (10 mmol, 6.62 gm) dissolved in 20 ml of distilled water was placed in a 100ml round bottom flask. triethylamine (et3n or tea) (10 mmol, 1.4 ml) was added to this solution and the mixture was cooled in an ice salt bath at (-10 o c). chloroacetyl chloride (40 mmol, 3.2 ml) was added drop wise to the above mentioned mixture with constant stirring over a period of one hour, while keeping the temperature at (10 o c). the mixture was left to stir overnight and the product was isolated as a faint yellowish white precipitate. the ph of the supernatant liquid was measured and it was found to be strongly acidic around 3.5-4.0. the precipitated material was washed three times with 50 ml of distilled water, and then left to dry. this derivative was recrystallized from acetone: methanol (1:1). the percent yield, physical appearance, melting point, and rf value of the synthesized intermediate compound are listed in table 1, while its elemental microanalysis and the ftir spectral data assignments of bands are shown in tables 2 and 3 respectively. iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 78 scheme (1): the synthetic scheme of the intermediate compound (a) 2. the chemical synthesis of the final ceftriaxone derivatives (target compounds b1-b7) (42) . to a stirred mixture of the intermediate compound (a) (5 mmol, 1.57 gm) and k2co3 (10 mmol, 0.98 gm) in 50 ml of acetone, was added one of the following amines (5 mmol): morpholine (0.48 ml), pyrrolidine (0.42 ml), piperidine (0.44 ml), hydrazine hydrate (0.5 ml), n-methylpiprazine (0.54 ml), imidazole (0.335 gm), and 2-aminothiazole (0.5 gm) and then the resultant mixture was refluxed for 1618 hours at 60-70 o c. later the solvent was evaporated under reduced pressure, and the precipitate obtained was collected and washed several times (3x) with 5 ml portions of acetone. the solid obtained after washing showed only one tlc spot which corresponds originally to the intended product appeared during the reaction followup. later the obtained product was recrystallized from aqueous ethanol. the synthetic scheme of the final ceftriaxone derivatives (target compounds b1-b7) is shown in scheme 2. the percent yield, physical appearance, melting points, and rf values of the synthesized compounds are listed in table 1, while their elemental microanalysis and the ftir spectral data assignments of bands are shown in tables 2 and 3 respectively. scheme( 2): the synthetic scheme of the final ceftriaxone derivatives (target compounds b1-b7) iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 79 table( 1): the percent yield, physical appearance, uncorrected melting points, and rf values of the synthesized intermediate (a) and target compounds (b1-b7) compound chemical name chemical formula physical appearance % yield melting point o c rf a (6r,7r)-7-((z)-2-(2-(2chloroacetamido)thiazol-4-yl)-2(methoxyimino)-acetamido)-3-(((2methyl-5,6-dioxo-1,2,5,6-tetrahydro1,2,4-triazin-3-yl)thio)methyl)-8-oxo5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid c20h19cln8o8s3 white powder 65 172-174 0.26 b1 (6r,7r)-7-((z)-2-(methoxyimino)-2(2-(2-morpholinoacetamido)-thiazol4-yl)acetamido)-3-(((2-methyl-5,6dioxo-1,2,5,6-tetrahydro-1,2,4-triazin3-yl)thio)methyl)-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2carboxylic acid c24h27n9o9s3 yellow powder 88 245 0.5 b2 (6r,7r)-7-((z)-2-(methoxyimino)-2(2-(2-(pyrrolidin-1-yl)acetamido)thiazol-4-yl)acetamido)-3(((2-methyl-5,6-dioxo-1,2,5,6tetrahydro-1,2,4-triazin-3yl)thio)methyl)-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2carboxylic acid c24h27n9o8s3 brown powder 93 230 0.55 b3 (6r,7r)-7-((z)-2-(methoxyimino)-2(2-(2-(piperidin-1-yl)acetamido)thiazol-4-yl)acetamido)-3(((2-methyl-5,6-dioxo-1,2,5,6tetrahydro-1,2,4-triazin-3yl)thio)methyl)-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2carboxylic acid c25h29n9o8s3 pale yellow powder 88 220 0.64 b4 (6r,7r)-7-((z)-2-(2-(2hydrazinylacetamido)-thiazol-4-yl)-2(methoxyimino)-acetamido)-3-(((2methyl-5,6-dioxo-1,2,5,6-tetrahydro1,2,4-triazin-3-yl)thio)methyl)-8-oxo5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid c20h22n10o8s3 dark yellow powder 87 220 0.66 b5 (6r,7r)-7-((z)-2-(methoxyimino)-2(2-(2-(4-methylpiperazin-1yl)acetamido)thiazol-4-yl)acetamido)3-(((2-methyl-5,6-dioxo-1,2,5,6tetrahydro-1,2,4-triazin-3yl)thio)methyl)-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2carboxylic acid c25h30n10o8s3 pale brown powder 86 247-249 0.68 iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 80 table (2): the elemental microanalysis (chns) of the synthesized intermediate (a) and target compounds (b1-b7) compound chemical formula mol. wt. value type c h n s a c20h19cln8o8s3 630.02 calculated observed 38.07 38.319 3.03 3.042 17.76 17.791 15.24 15.312 b1 c24h27n9o9s3 681.72 calculated observed 42.28 41.958 3.99 3.898 18.49 18.267 14.11 14.551 b2 c24h27n9o8s3 665.72 calculated observed 43.3 44.147 4.09 4.12 18.94 19.22 14.45 14.5 b3 c25h29n9o8s3 679.75 calculated observed 44.17 44.674 4.3 4.360 18.55 18.393 * b4 c20h22n10o8s3 626.65 calculated observed 38.33 38.198 3.54 3.444 22.35 22.103 * b5 c25h30n10o8s3 694.76 calculated observed 43.22 43.352 4.35 4.411 20.16 19.985 * b6 c23h22n10o8s3 662.68 calculated observed 41.69 40.923 3.35 3.282 21.14 21.938 14.52 14.274 b7 c23h22n10o8s4 694.74 calculated observed 39.76 40.145 3.19 3.22 20.16 20.704 18.46 18.314 * the analysis was done at the jordanian university which lacks the detection of sulfur. b6 (6r,7r)-7-((z)-2-(2-(2-(1h-imidazol1-yl)acetamido)thiazol-4-yl)-2(methoxyimino)-acetamido)-3-(((2methyl-5,6-dioxo-1,2,5,6-tetrahydro1,2,4-triazin-3-yl)thio)methyl)-8-oxo5-thia-1-azabicyclo[4.2.0]oct-2-ene-2carboxylic acid c23h22n10o8s3 light brown powder 90 231-233 0.56 b7 (6r,7r)-7-((z)-2-(methoxyimino)-2(2-(2-(thiazol-2-ylamino)acetamido)thiazol-4-yl)acetamido)-3(((2-methyl-5,6-dioxo-1,2,5,6tetrahydro-1,2,4-triazin-3yl)thio)methyl)-8-oxo-5-thia-1azabicyclo[4.2.0]oct-2-ene-2carboxylic acid c23h22n10o8s4 yellow powder 89 184-186 0.58 iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 81 table (3): the characteristic ir bands of the synthesized intermediate (a) and target compounds (b1-b7). sym. chemical structure ir band [kbr] v cm -1 interpretation a 3379 (n-h) stretching vibration of secondary amide 3103 heteroaromatic (c-h) stretching vibration 2940 aliphatic (c-h) asymmetric stretching vibration 2840 aliphatic (c-h) symmetric stretching vibration 1774 (c=o) stretching vibration of β-lactam 1713 (c=o) stretching vibration of –cooh group 1658 (c=o) stretching vibration of secondary amide 1631 oxime c=n stretching mode 1586 stretching vibration of (c=c) in the thiazole ring 1448 (c-h) bending vibration of –ch2 group 1413 (c-h) bending vibration of –ch3 group 1311 (c-o) stretching of –cooh group 1246, 1222 (c-h) wag in terminal -ch2cl 1184 (c-o) stretching vibration 1097, 1045 (c-o) stretching in -och3 and (n-o) in oxime 852 (c-cl) stretching vibration b1 3439 (n-h) stretching vibration of secondary amide 2980 aliphatic (c-h) asymmetric stretching vibration 2890 aliphatic (c-h) symmetric stretching vibration 1761 (c=o) stretching vibration of β-lactam 1712 (c=o) stretching vibration of –cooh group 1643 (c=o) stretching vibration of secondary amide 1608 oxime c=n stretching mode 1595 stretching vibration of (c=c) in the thiazole ring 1460 (c-h) bending vibration of –ch2 group 1375 (c-h) in-plane bending vibration of -ch2 group 1300 (c-o) stretching of –cooh group 1215 (c-n) stretching of amino group 1184 (c-o) stretching vibration 1107, 1039 (c-o) and (n-o) stretching in -och3 and oxime 808 heteroaromatic (c-h) out of plane bending b2 3375 (n-h) stretching vibration of secondary amide 2941 aliphatic (c-h) asymmetric stretching vibration 2854 aliphatic (c-h) symmetric stretching vibration 1769 (c=o) stretching vibration of β-lactam 1716 (c=o) stretching vibration of –cooh group 1670 (c=o) stretching vibration of secondary amide 1637 oxime c=n stretching mode 1586 stretching vibration of (c=c) in the thiazole ring 1460 (c-h) bending vibration of –ch2 group 1369 (c-h) bending vibration of –ch3 group 1261 (c-o) stretching of –cooh group 1220 (c-n) stretching of amino group 1070, 1041 (c-o) and (n-o) stretching in -och3 and oxime iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 82 833 heteroaromatic (c-h) out-of-plane bending b3 3400 (n-h) stretching vibration of secondary amide 2941 aliphatic (c-h) asymmetric stretching vibration 2840 aliphatic (c-h) symmetric stretching vibration 1769 (c=o) stretching vibration of β-lactam 1712 (c=o) stretching vibration of –cooh group (shoulder) 1654 (c=o) stretching vibration of secondary amide 1604 oxime c=n stretching mode 1537 stretching vibration of (c=c) in the thiazole ring 1460 (c-h) bending vibration of –ch2 group 1367 (c-h) in-plane bending vibration of –ch3 group 1292 (c-o) stretching of –cooh group 1215 (c-n) stretching of amino group 1101, 1039 (c-o) and (n-o) stretching in -och3 and oxime 823 heteroaromatic (c-h) out-of-plane bending b4 3462-3311 (n-h) stretching vibrations of primary –nh2 group around 3462 and 3400 cm -1 coupled with the band between 3350-3310 cm -1 for the secondary amine of the reacted hydrazine 3425 (n-h) stretching vibration of secondary amide 2945 aliphatic (c-h) asymmetric stretching vibration 2825 aliphatic (c-h) symmetric stretching vibration 1759 (c=o) stretching vibration of β-lactam 1710 (c=o) stretching vibration of –cooh group (shoulder) 1635 (c=o) stretching vibration of secondary amide 1604 oxime c=n stretching mode (shoulder) 1550 stretching vibration of (c=c) in the thiazole ring 1525 (n-h) bending vibration of secondary amide 1456 (c-h) bending vibration of –ch2 group 1375 (c-h) bending vibration of –ch3 group 1272 (c-o) stretching of –cooh group 1207 (c-n) stretching of amino group 1134, 1041 (c-o) and (n-o) stretching in -och3 and oxime 806 heteroaromatic (c-h) out-of-plane bending b5 3419 (n-h) stretching vibration of secondary amide 2945 aliphatic (c-h) asymmetric stretching vibration 2823 aliphatic (c-h) symmetric stretching vibration 1761 (c=o) stretching vibration of β-lactam 1710 (c=o) stretching vibration of –cooh group (shoulder) 1651 (c=o) stretching vibration of secondary amide 1604 oxime c=n stretching mode (shoulder) 1558 stretching vibration of (c=c) in the thiazole ring 1440 (c-h) bending vibration of –ch2 group 1371 (c-h) in-plane bending vibration of –ch3 group 1290 (c-o) stretching of –cooh group 1215 (c-n) stretching of amino group 1107, 1037 (c-o) and (n-o) stretching in -och3 and oxime 833 heteroaromatic (c-h) out-of-plane bending 3394 (n-h) stretching vibration of secondary amide 2943 aliphatic (c-h) asymmetric stretching vibration iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 83 b6 2872 aliphatic (c-h) symmetric stretching vibration 1751 (c=o) stretching vibration of β-lactam 1715 (c=o) stretching vibration of –cooh group 1664 (c=o) stretching vibration of secondary amide 1650 (c=o) stretching vibration of amide 1606 oxime c=n stretching mode 1535 stretching vibration of (c=c) in the thiazole ring 1440 (c-h) bending vibration of –ch2 group (shoulder) 1375 (c-h) in-plane bending vibration of –ch3 group 1292 (c-o) stretching of –cooh group 1215 (c-n) stretching of amino group 1109, 1041 (c-o) and (n-o) stretching in -och3 and oxime 833 heteroaromatic (c-h) out-of-plane bending b7 3454 (n-h) stretching vibration of secondary amide 3342 (n-h) stretching vibration of secondary amine 2943 aliphatic (c-h) asymmetric stretching vibration 2823 aliphatic (c-h) symmetric stretching vibration 1751 (c=o) stretching vibration of β-lactam 1720 (c=o) stretching vibration of –cooh group 1653 (c=o) stretching vibration of secondary amide 1616 oxime c=n stretching mode 1550 stretching vibration of (c=c) in the thiazole ring 1464 (c-h) bending vibration of –ch2 group 1377 (c-h) in-plane bending vibration of –ch3 group 1276 (c-o) stretching of –cooh group 1207 (c-n) stretching of amino group 1103, 1041 (c-o) and (n-o) stretching in -och3 and oxime 856 heteroaromatic (c-h) out-of-plane bending notes: 1. it was noticed that the (c=o) stretching vibration of the newly formed amide in the intermediate (a) and the final compounds (b1-b7) and that of the other amide already present in ceftriaxone were overlapping each other. 2. it is clear that the (c-cl) stretching vibration appeared in the intermediate (a) at 852 cm -1 has disappeared in the final target compounds (b1-b7). b. the antimicrobial evaluation of the newly synthesized ceftriaxone derivative the antimicrobial activity was determined for the newly synthesized ceftriaxone derivatives by the well diffusion method for screening the in vitro antibacterial activity against the selected gram positive (staphylococcus aureus) and gram negative bacteria (e. coli) and screening the antifungal activity against the selected fungus (candida albicans) (43) . ceftriaxone, cephalexin were used as references for testing the antibacterial activity while fluconazole was used as a reference for testing the antifungal activity. the synthesized compounds and references were dissolved in dmso (also was used as a control), to prepare a 500 μg/ml stock solution. two dilutions of the synthesized compounds and reference compounds were prepared to have 250 and 125 μg/ml respectively (44) . wells were made in mueller hinton agar for bacteria and sabouraud dextrose agar for c. albicans. plates were seeded with 0.1 ml of 10 8 cfu / ml of bacteria, and with 10 6 cfu / ml of c. albicans. triplicates of each concentration for each microorganism species were prepared. the inoculated plates were incubated at 37° c for 24 hr. the diameter of the inhibition zones were measured for each plate (45) . the inhibitory zones for each of the synthesized and reference compounds against each of the tested microorganisms are listed in tables 4 and 5. iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 84 table (4): the antibacterial activity data of the synthesized target compounds compound zone of inhibition in mm staphylococcus aureus e. coli 125 μg/ml 250 μg/ml 125 μg/ml 250 μg/ml b1 26 32 22 29 b2 18 18 15 17 b3 31 35 27 30 b4 23 27 22 25 b5 27 30 29 30 b6 26 27 22 29 b7 26 27 27 28 dmso fluconazole cephalexin 30 38 33 37 ceftriaxone 32 34 32 33 table (5): the antifungal activity data of the synthesized target compounds compound zone of inhibition in mm c. albicans 125 μg/ml 250 μg/ml b1 26 27 b2 16 16 b3 30 32 b4 25 28 b5 25 31 b6 25 26 b7 22 27 dmso fluconazole 33 37 cephalexin ceftriaxone notes: 1. dmso was used as a control for both the antibacterial and antifungal evaluations and it did not show any inhibitory activity against both the bacteria and fungus used in these tests. 2. running ceftriaxone and cephalexin in the antimicrobial tests showed that these two chemotherapeutic agents didn't have any antifungal activity originally, while fluconazole on the other hand showed that it lacks any antibacterial activity. results and discussion the scope of the research includes first of all equipping ceftriaxone with the desired spacer moiety by acylating its 2-aminothiazolylgroup with chloroacetyl chloride which subsequently forming its α-chloroamide derivative (intermediate a). secondly the latter was further conjugated with several amines in order to produce the final target compounds (b1-b7). the strategy that lies behind the synthesis of this system is in the fact that the chemical synthesis of amides are conducted in organic solutions or in a mixture of organic and aqueous solutions (schotten–baumann conditions) where the organic reagents are generally dissolved in the organic solution and treated with aqueous base (46) . the development of alternative methods for achieving amide synthesis in high yield and/or in a stereospecific manner is of great current interest. it was reported earlier that the classical reaction between the corresponding amine and chloroacetyl chloride (2 equiv.) in dry tetrahydrofuran (or dichloromethane) at 10 o c in the presence of freshly distilled triethylamine, gave very low yields of the desired α-chloroamides. furthermore, changing to solvents such as iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 85 dimethylformamide, dichloromethane, chloroform or diethyl ether did not increase significantly the yield of the products, and the reaction only proceeded in ethyl acetate, but in low yields (47) . hence, the focus was directed towards alternative synthetic methods and it was found that the amide coupling could be conducted in water in the presence of a medium of bases such as et3n, khco3, k2co3 or naoh, where the desired products were formed as solids in acceptable yields, under ambient conditions (41) . varying the reaction conditions such as solvent and base did not yield any improvement. the problem seemed to stem from the insolubility of the amine, which was only fully soluble in alcohols and water (48) . however, due to the lack of solubility of the amine in the organic solution; it was decided to carry out a modification of this method using only water as the solvent. moreover, instead of using an excess of the amine, four equiv. of chloroacetyl chloride were used, which were added dropwise over 1 h to the aqueous amine solution. the solution was left to stir overnight and the desired product was isolated as a precipitate in yields of 65% with no need for further purification. hence, the two-phase procedure mentioned earlier was not necessary and in fact the modification which was carried out here enabled the successful isolation of the desired product with minimal effort. accordingly, the free 2-aminothiazolyl group of ceftriaxone sodium was found to react in water with the spacer compound (chloroacetyl chloride) in the presence of a suitable base such as et3n and this will result in the formation of the intended corresponding α-chloroamide (49) . the mechanism of this reaction is an acyl nucleophilic substitution which occurs selectively at the acyl carbon atom in chloroacetyl chloride because of the greater reactivity of nucleophiles toward acid chlorides compared to alkyl chlorides. the reasons for this selectivity are attributed to the differences in the electrophilicity of the two carbon atoms in chloroacetyl chloride. besides that the electronic, and steric factors also play a role in this selectivity, since it is easier for the nucleophile to attack the carbon of the planar carbonyl group in the acid chloride than to attack the tetrahedral carbon in the –ch2cl group. the reaction is carried out with triethylamine, which acts as a base to neutralize the hydrogen chloride (hcl) formed (50, 51) . later the selected amines are coupled with the synthesized intermediate compound (a) for the synthesis of the target compounds (b1-b7), since the amine n functions as a nucleophile and attacks the electrophilic c of the alkyl chloride displacing the chloride and creating the new c-n bond via an alkyl nucleophilic substitution reaction (sn2), to yield the corresponding amine alkylated product. the sn2 mechanism is concerted and proceeds through a single rate-determining transition step and it begins when the reactant is attacked by a nucleophile from the side opposite the leaving group, with bond making occurring simultaneously with bond breaking between the carbon atom and the leaving group. the transition state has trigonal bipyramidal geometry shape with a pentacoordinated carbon. with the loss of the leaving group, the carbon atom again assumes a pyramidal shape; however, its configuration is inverted and this inversion is often called the walden inversion as shown in scheme 3 (52-55) . nucleophile reactant transition state product leaving group (note: the product presents the walden inversion in being stereochemically inverted) scheme( 3): the mechanism of the sn2 reaction the structures of the synthesized compounds were confirmed by using ftir, elemental microanalysis (chns), and other physicochemical parameters (tables 1, 2, and 3). the ftir spectrum of ceftriaxone sodium obtained in kbr pellets was in excellent agreement with that reported earlier in the literature (56) . the synthesized compounds (a and b1-b7) showed several characteristic sharp bands in the ir region, where the appearance of the band near 3500-3400 cm -1 that represent the free nh stretching vibration of the formed secondary amide was accompanied with the disappearance of the two peaks near 3500 and 3400 cm -1 of the n-h stretching modes of primary amine of the 2aminothiazolylmoiety, and this was accompanied together with the appearance of the signal near 1640 cm -1 that represent the c=o stretching frequency of the carbonyl group in simple, open chain, secondary amides. it is also noticed that the (c-cl) stretching vibration appeared in the intermediate (a) at 852 cm -1 has disappeared in all the final target compounds (b1-b7). the appearance of the signal in the region of 17201706 cm -1 represents the carbonyl group http://www.chem.ucalgary.ca/courses/350/carey5th/ch08/ch8-11.html http://www.chem.ucalgary.ca/courses/350/carey5th/ch08/ch8-4.html iraqi j pharm sci, vol.23(2) 2014 n ew ceftriaxone derivatives 86 stretching vibration of bonded aliphatic acids which is accompanied also with the appearance of very broad, intense o-h stretching absorption in the region of 33002500 cm -1 that corresponds also to carboxylic acid dimers. the two stretching vibrations that represent the carboxylate anion present previously in ceftriaxone sodium at 1602 and 1398 cm -1 have disappeared from the spectrums of all synthesized compounds. it is worthwhile to mention that the chemical tests performed on all the synthesized compounds showed the appearance of chlorine in the intermediate compound (a) and its disappearance in the corresponding final compounds, while the carboxylic acid test showed the presence of the free carboxylic acid group in the synthesized intermediate and final derivatives. the elemental microanalysis (table 2) revealed good agreement with the calculated percentages. the newly synthesized ceftriaxone derivatives showed moderate to good antibacterial and antifungal activities. both compounds b3 and b5 showed good activity compared to both cephalexin and ceftriaxone but compound b3 was more potent than ceftriaxone in its antibacterial activity against staphylococcus aureus. in addition all compounds showed moderate to good antifungal activity but compounds b3 and b5 were more potent than others. the antimicrobial data presented earlier showed that the synthesized target compounds with the structural changes done with the parent ceftriaxone molecule have gained an expansion in their antimicrobial activity to include antifungal properties in addition to the already retained antibacterial ones. the data showed also that ceftriaxone itself lacks any activity against fungi in its antimicrobial spectrum. conclusion the results obtained in the present study showed that it is possible to synthesize new derivatives of ceftriaxone by linking its molecule through a spacer to appropriately chosen amines, since these amines may give by their presence additional properties to the ceftriaxone parent compound. this was evidenced since the synthesized ceftriaxone derivatives showed marked antibacterial and antifungal activities when compared with ceftriaxone itself, cephalexin and with fluconazole. therefore, these results illustrate that these ceftriaxone derivatives have gained an expansion in their antimicrobial spectrum to include an added antifungal activity to it, while retaining the original 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microbiological bioassay", anal. methods, 2012, 4: 2490-2498. http://www.sciencedirect.com/science/article/pii/s0040403909007898 http://www.sciencedirect.com/science/article/pii/s0040403909007898 http://www.sciencedirect.com/science/article/pii/s0040403909007898 http://www.sciencedirect.com/science/article/pii/s0040403909007898 http://www.sciencedirect.com/science/article/pii/s0040403909007898 http://www.sciencedirect.com/science/article/pii/s0040403909007898 http://www.sciencedirect.com/science/article/pii/s0040403909007898 http://www.sciencedirect.com/science/journal/00404039 http://pubs.rsc.org/en/results?searchtext=author%3af.%20juliusburger http://pubs.rsc.org/en/results?searchtext=author%3as.%20masterman http://pubs.rsc.org/en/results?searchtext=author%3ab.%20topley http://pubs.rsc.org/en/results?searchtext=author%3aj.%20weiss iraqi j pharm sci, vol.22(1) 2013 serum levels of total ige and interleutines, in children with asthma. 110 serum levels of total ige, il-12, il-13 and il-18 in children patients with asthma. ghadah m. saleh al-quraishi *,1 * department of biology, college of science, university baghdad, baghdad,iraq. abstract t-cell activation and alteration of cytokine levels are involved in the pathogenesis of asthma. however, the profile of circulating t-lymphocyte subsets and related cytokines during asthmatic attacks is still unclear. we compared the serum concentrations of proinflammatory cytokines interleukine-18( il-18) and interleukine-12(il-12), t-helper 2 (th2) cytokine interleukine-13(il-13 ) and immunoglobuline-e ( ige) in 27 asthmatic children and 21 sex and age matched healthy control subjects. serum cytokines and ige concentrations were measured by enzyme-linked immunosorbent assay. serum il-13 , il-18 and ige concentrations were significantly higher in asthmatic patients than normal control subjects ( il-13: median 9.73 versus 4.43 pg/ml, p<0.05 ; il-18: 76.81 versus 35.41 pg/ml, p<0.05 ; ige: 225.44 versus 37.94 iu/ml, p<0.05 . asthmatic patients showed a decreased serum il-12 concentration 93.57 versus 122.83 pg/ml, p<0.05 . in conclusion, it was suggested that il-13 and il-18 might have a potential role in controling lymphocyte responses in asthmatic patients specially in children through an ige-mediated pathway. key words: asthma, interleukine-12, -13, -18, immunoglobuline-e. ندى االطفال il-12,il-13,il-18انكهي وانحركيات انمناعية igeانمستوى انمصهي نكم من انمصابين بانربو غادة محمد صانح *،1 * .قسٌ عيً٘ اىحٞاج ، ميٞح اىعيً٘ ، جاٍعح تغذاد ، تغذاد ، اىعزاق الخالصة ، عٍَ٘ا. ىٖا دٗر فٜ إٍزاضٞح اىزت٘( اىساٝر٘مْٞاخ)ٗذغاٝز ٍسر٘ٝاخ اىحزمٞاخ اىَْاعٞح ( t-cells)إُ ذحفٞش اىخالٝا اىيَفاٗٝح اىَ٘ج٘دج فٜ اىذً ٗاىساٝر٘مْٞاخ اىرٜ ذْرجٖا خاله ٕجَاخ اىزت٘ ٍا ساىد غٞز ( t-lymphocyte subsets)اىخالٝا اىيَفاٗٝح إُ اّ٘اع ٗمذىل ( il-12) 12-ٗاالّرزى٘مِٞ( il-18) 18-االّرزى٘مِٞ: فٜ ٕذٓ اىذراسح ذٌ ٍقارّح اىرزامٞش اىَصيٞح ىيساٝر٘مْٞاخ اىراىٞح. ٗاضحح e-تاالضافح اىٚ قٞاص اىرزمٞش اىَصيٜ ىيني٘تٞ٘ىِٞ اىَْاعٜ( t-helper 2)اىذٛ ْٝرج ٍِ اىخالٝا اىيَفاٗٝح ( il-13) 13-االّرزى٘مِٞ (ige ) ٙذٌ قٞاص ذزامٞش االّرزى٘مْٞاخ ٗاالٍّٞ٘٘مي٘تٞ٘ىِٞ اىَْاعٜ. طفو صحٜ مَجَ٘عح سٞطزج 21طفو ٍصاب تاىزت٘ ٗ 27ىذ-e إسدٝادا ٍعْ٘ٝا ىذٙ il-13 ٗil-18 ٗigeأظٖز ذزمٞش مو ٍِ (. elisa)ط تاالّشٌٝ ت٘ساطح اخرثار االٍرصاص اىَْاعٜ اىَزذة 76.81اىَر٘سط : pg/ml ; il-18 4.43ٍقاتو 9.73pg/mlاىَر٘سط : il-13) االطفاه اىَصاتِٞ تاىزت٘ ٍقارّح تَجَ٘عح اىسٞطزج pg/ml 35.41ٍقاتو pg/ml ; ige : 226.44اىَر٘سط iu/ml 37.94ٍقاتو iu/ml ( )0.05>p) فٜ حِٞ أظٖز ،il-12 إّخفاظا 122.83ٍقاتو pg/ml 93.57اىَر٘سط : il-12) غٞز ٍعْ٘ٛ فٜ ذزمٞشٓ ىذٙ االطفاه اىَصاتِٞ تاىزت٘ ٍقارّح تَجَ٘عح اىسٞطزج pg/ml ) ُىذىل فإّٔ ٝعرقذ ا ،il-18 ٗil-13 ىَٖا دٗر فعاه فٜ اىسٞطزج عيٚ اسرجاتح اىخالٝا اىيَفاٗٝح ىذٙ ٍزضٚ اىزت٘ ٗخاصح . igeاالطفاه ٌٍْٖ ٗتطزٝقح ٝشرزك خالىٖا . e-، واالميونوكهوبيونين انمناعي 18 -، 13 –، 12 –انربو ، االنترنوكين :انكهمات انمفتاحية introduction asthma is a common respiratory disorder worldwide, it is a heterogenous disease, in which genetic plus environmental factors may contribute to its initiation and continuance (1) . allergic asthmatic patients generally develop the disease early in life , usually in infancy or childhood. the attack usually occur upon exposure to allergens, the total serum immunoglobuline-e(ige) concentration is frequently elevated but sometimes remain normal (2) . allergen-induced ige synthesis can trigger eosinophils, basophils and mast cells to release cytokines for the differentiation of thelper 2 (th2) cells to secrete interleukine-4 (il-4) 1 corresponding author e-mail: ghadahalquraishi@yahoo.com received:19/5/2012 accepted:6/4/2013 iraqi j pharm sci, vol.22(1) 2013 serum levels of total ige and interleutines, in children with asthma. 111 , interleukine-5 (il-5), interleukine-10 (il-10) and interleukine-13 (il-13) as well as the release of proinflammatory, vasoactive and fibrogenic factors ( histamine, peptide leukotriens, platelet activating factor, tryptase, ect.) that are responsible for symptoms of asthma (3) . allergic airway diseases are associated with skewed th2 cytokine production although the underlying cause of this aberrant immune response is not well understood (4) . interleukine12 (il-12) is a critical determinant of t-helper1 (th1) mediated immune responses, as it has been shown that deficiency in this cytokine can lead to th2-polarized immune responses (5) .in peripheral lymphocytes of the th1 type, il-12 induces the synthesis of interferon-γ (ifn-γ), interleukine-2 (il-2) and tumer necrotic factor (tnf), the production of il-12, tnf and ifn-γ is inhibited by il-10 (6) . il-12 is also involved in the selection of immunoglobulin isotypes, it markedly inhibits the synthesis of ige by peripheral blood mononuclear cells stimulated with il-4 (7) . interleukine-13 is an immunoregulatory cytokine generated predominantly by activated th2 cells and it shows functional properties with il-4 (8) . il-13 shares a receptor component signaling pathway and many biological activities with il-4. in fact, il-13 is also an antiinflammatory cytokine that plays a unique role in the optimal induction and maintenance of ige production and ige-mediated allergic responses when il-4 production is low or absent (9,10) . il13 has been recognized as a key cytokine mediating allergic airway inflammation and airway remodeling in asthma which is characterized by mucous hypersecretion , airway hyper-responsiveness and sub-epithelial fibrosis (11) . reports demonstrated that in asthmatics il4+ and il-13+ cells present within the airway smooth muscle were predominantly expressed by mast cells , suggesting that il-4 and il-13 may play an important role in mast cell-airway smooth muscle interactions (12) . interleukine-18 formerly called interferon gamma inducing factor, is a novel proinflammatory cytokine related to the il-1 family (13) , it is produced by a wide range of cells and involved in the pathogenesis of several inflammatory diseases such as asthma (14) . it was recently demonstrated that il-18 acts on t-cells to induce airway inflammation and airway hyper-responsiveness , as well as , stimulating th1 cells to produce cytokines and chemokines responsible for the airway infiltration and inflammatory responsiveness , also activating mast cells and basophils (15) . however , there are few studies investigating the relationship between il-12, il-13, il-18 and total ige in asthmatic patients, especially in children . in this study, we studied the levels of these markers in children patients with asthma . materials and methods study population the population of this study consisted of 27 patients with asthma (15:males and 12:female) , ages ranged from 3-12 years (mean ± se: 7.0 ± 0.89) . allergic asthmatic patients who have been attending the zahraa allergic center of al-karkh hospital with clinical diagnosis of asthma, i.e. : history of recurrent wheeze, cough and dyspnea in the previous 12 months. 21 healthy children matched for age (12:males and 9:females ) were recruited as control individuals, with the following exclusion criteria : history of childhood asthma, family history of asthma, a febrile illness or chest infection within the previous four weeks, or episodes of cough and wheezing in the past 12 months, and subjects with a serum total ige value of > 100 iu/ml . measurement of serum total ige, il-12, il-13 and il-18 levels a forearm venous blood sample (5 ml) was drawn from all subjects for complete blood count, cytokine and ige estimation upon recruitment. blood was allowed to clot and was then centrifuged and sera was collected and divided into a number of eppendorf tubes, stored at -20˚c and thawed immediately before analysis. serum concentration of total ige was measured by elisa method (human, germany). serum levels of il-12, il-13 and il-18 were also measured by elisa method ( biosource, belgium ), ( immunotech, france ), ( mbl, germany ) respectively. the minimal detection levels of cytokines using this method were 2.0 pg/ml for il-12, 1.5 pg/ml for il-13 and 12.5 pg/ml for il-18, cytokine levels below the detection limits were considered as zero. iraqi j pharm sci, vol.22(1) 2013 serum levels of total ige and interleutines, in children with asthma. 112 statistical analysis data on circulating il-12, il-13, il-18, and ige levels are presented as mean ± sem. a paired t-test was used to compare the serum level of the studied parameters between asthma patients and controls, also using a special elisa software (biorad lab, inc.)to draw the standard curves of il-12, il-13, il-18 and ige. statistical analysis was assumed for p values lower than 0.05. results serum concentrations of ige, il-13, and il18 were significantly higher in asthmatic patients compared to control subjects. ige :226.44 ± 52.33 iu/ml versus 37.94 ± 12.42 iu/ml,p<0.05;il-13: 9.73 ± 1.72 pg/ml versus 4.43 ± 0.59 pg/ml , p<0.05; il-18: 76.81 ± 22.44 pg/ml versus 35.41±3.17 pg/ml, p>0.05 fig 1.a,b,c . whereas, serum concentration of il-12 were lower in asthmatic patients compared to control subjects il-12: 93.57 ± 12.17 pg/ml versus 122.83 ± 19.16 pg/ml showed no significant differences in there results, as shown in fig 1.d . figure 1 : serum concentrations of a. ige, b. il-13, c. il-18,and d. il-12 in both asthma patients and healthy controls. discussion asthma is a chronic inflammatory disease of the airways characterized by mucus gland hyperplasia, basement membrane thickening and eosinophil infiltration (16). serum levels of ige was shown to be higher in asthmatics compared to control subjects as a result of type 1 hypersensitivity response (fig.1.a), the results of this study is supported by data that have been published by (sandstrom, 2009) (17) . on the other hand, bronchial hyper responsiveness and airway inflammation can be elicited through ige independent mechanisms as documented in an experimental model of asthma (18) . furthermore, a genetic study in humans involving il-13 gene polymorphism has clearly shown a relationship between susceptibility to asthma which was independent on serum ige levels (19) . serum ige values decline with age in the general population , as the immune system undergoes characteristic changes with aging. most of t-cell function are depressed in elderly individuals and the accumulation of cd45ro+memory cells in elderly individuals result in a reduced ability to respond to new antigens, and a retained ability to respond to recall antigens as long as the memory cells remained present and functional (20) . interlukine-12 was shown to be involved in inhibiting ige levels,enhancing ifn-γ production which also inhibit ige levels (7). serum levels of il-12 in asthmatics showed no elevation compared to control subjects in this study, (fig 1.d) . although high ige levels was detected in asthmatics, and as we know asthma is a th2 disease, in which the th1 cytokine (il-12) does not play a vital role in the pathogenesis of the disease. patients with both th1 and th2 mediated disease like type 1 diabetes mellitus and asthma display different pattern of il-12 and il-18 expression with much higher levels of both il-12 and il-18 compared to their levels in patients with one disease only and controls (29) . serum levels of il-13 was higher in asthmatics compared to control subjects as results reveled in this study,( fig 1.b), thus pointing to an inflammatory mediator in asthma patients which was also presented in other studies (21,22) . therefore, elevation in serum levels of both il-13 and il-5 in asthmatics may point towards an ongoing systemic th2 inflammatory response (23) . the source of elevated serum il-13 levels in asthmatics is not clarified. however, there is evidence to suggest that the circulating pbmcs may be a major source of circulating il13 in asthmatics (24) . in addition, the pbcms producing il-13 are higher in proportion after exposure to allergens (25) . increased expression of il-13 has been documented in the bronchial iraqi j pharm sci, vol.22(1) 2013 serum levels of total ige and interleutines, in children with asthma. 113 mucosa of asthmatics, therefore spillover of il13 from inflamed airways to peripheral blood cannot be discounted as a possibility for the raised serum il-13 levels observed in asthmatics (26). il-13 blocking antibody has been tested in animal models of asthma with some success (27), also, gene therapy directed against il-4 receptors may represent another new approach in controlling airway inflammation in asthma (28). higher serum il-18 levels were found in asthmatics as compared to control subjects as presented in (fig.1.c). il-18 serum levels may partly reflect disease activity in asthmatic patients, il-18 is mainly secreted by activated monocytes/macrophages and kupffer cells as well as other cells (30). il-18 and il-12 have both been shown to be strong cofactors for th1 cell activation and stimulate th1 cytokines. however, it was thought that il-18 might act as a coinducer of both th1 and th2 cytokines, it was suggested that elevated il-18 levels in asthma patients did not affect ifn-γ production and might enhance t-lymphocyte-mediated inflammation (15). it is speculated that il-18 might partly reflect asthma exacerbation through activation of t-lymphocyte, monocyte, and granulocyte-triggered inflammation with the ifn-γ independent pathway, which may explain il-18 contribution to th2 cytokine dominant disease in a clinical setting. furthermore, histamine does dependently stimulate the production of il-18 in human pbmcs, most of the histamine is stored in mast cells in asthmatic airway and is released quickly to bronchial tissue during ige-dependent asthmatic response (31). increased serum il-18 production in asthmatic patients may partly stimulated by histamine release. on the other hand, many viral and bacterial infections also may stimulate il-18 generation (32). some studies reveled detectable ifn-γ in some asthmatic patients although asthma is a th2 cytokine dominant disease, in which th1 cytokine production is suppressed (33). in conclusion, it was suggested that il-18 and il-13 might have a potential role in controlling lymphocyte responses in asthmatic patients and such elevation of these cytokines might induce different immunologic responses in a th2 cytokine dominant disease. references 1. akinbami,l.j. and schoendorf,k.c. trends in childhood asthma: prevalence, health care utilization and mortality. pediatrics., 2002; 110(2) : 315-322. 2. mediaty,a. and neuber,k.. total and specific ige decreases with age in patients with allergic rhinitis, asthma and insect allergy but not in patients with atopic dermatitis. immunity and aging., 2005;may;2:9. 3. marone,g asthma: recent advances. immunol.today;1998; 19:5-9. 4. ngoc,p.l.; gold,d.r.; tzianabos,a.o; et al. cytokines, allergy, and asthma. curr.opin.allergy clin.immunol., 2005;apr; 5(2): 161-166. 5. wills-karp,m. il-12/il-13 axis in allergic asthma. j.allergy clin.immunol., 2001; jan;107(1):9-18. 6. hamza,t.; 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12(7): 596-603. 30. okam,o.h.; tsutsui,h.; kashiwamura,s.; et al. interleukine-18: a novel cytokine that augments both innate and acquired immunity. adv.immunol., 1998;70: 281-312. 31. kohks,k.h.; nishibori,m.; iwagaki,h.; et al. histamine is a potent inducer ofil-18 and ifnγ in human peripheral blood mononuclear cells. j.immunol., 2000; 164: 6640-6646. 32. lauw,f.n.; simpson,a.j.; prins,j.m.; et al. elevated plasma concentrations of ifnγ and the ifnγ-inducing cytokines interleukine(il)-18,il-12, and il-15 in severe melioidosis. j.infect.dis., 1999; 180: 1878-1885. 33. vande van,p.k.; boeige,l.c.; de groot,e.r.; et al. reduced production of il-12 and il-12dependent ifnγ release in patients with allergic asthma. j.immunol., 1997;158: 55605565. iraqi j pharm sci, vol.25(1) 2016 spectrophotometric determination of olanzapine 42 the spectrophotometric determination of olanzapine via coupling with diazotized p-nitroaniline sahar r. fadhel *,1 , najwa i. abdulla ** and intidhar d. sulaiman ** * department of chemistry, college of science, university of diyala, diyala, iraq. ** department of chemistry, college of education for pure sciences (ibn al-haitham), university of baghdad, baghdad, iraq. abstract a new spectrophotometric method has been developed for the assay of olanzapine (oln.) in pure and dosage forms. the method is based on the diazocoupling of (oln.) with diazotized p-nitroaniline in alkaline medium to form a stable brown colored water-soluble azo dye with a maximum absorption at 405 nm. the variables that affect the completion of reaction have been carefully optimized. beer’s law is obeyed over the concentration range of (0.5-45.0 μg.ml -1 ) with a molar absorptivity of 1.5777×10 4 l.mol -1 .cm -1 . the limit of detection was 0.3148 μg.ml -1 and sandell’s sensitivity value was 0.0198 μg.cm -2 . the proposed method has been applied successfully to the determination of olanzapine in tablet pharmaceutical preparations. keywords: spectrophotometry, olanzapine, diazotization, p-nitroaniline, tablet dosage form. ويترواويليه-ت باراووالوسابيه باقتراوه مع العامل المؤزالتقدير الطيفي لال سحر ريحان فاضل *،1 وجوى اسحق عبد هللا ، ** اوتظار داّود سليمان، ** * انعراق. ،دٍاني ،جايعت دٍاني ،لسى انكًََاء, كهَت انعهوو ** انعراق،بغداد ،جايعت بغداد ،انھَثى( ابٍ) أنصرفت نهعهوو انخربَت كهَت،انكًََاء لسى الخالصة ٌ الخرا عهي انطرٍمت حعخًد. انصَدالََت انًسخحضراث فٌ و انُمَت بصورحه طَفَت جدٍدة نخمدٍر االوالَسابٍَ طرٍمت حطوٍر ىح فٌ ذائبه بٌُ نوٌ ذاث يسخمرة صبغه نخكوٍٍ لاعدً فٌ وسظ َاٍخرواَهٍَ انداٍازوحاٍسً-انباراانداٍازو نعمار االوالَسابٍَ يع كاشف بدلت انخفاعم اكخًال فٌ حؤثر انخٌ انعوايم يٍ درسج انعدٍد. َاَويخر 405 انًوجٌ انطول عُد ايخصاص اعظى اعطج انًاء انخٌ يم.ياٍكروغراو ( 45,0 – 0,5)انخراكَس يا بٍَ يٍ يدى ضًٍ بَر نمد اَطبك لاَوٌ .انفضهي نهخفاعم انظروف عهي نهحصول -1 . 10يوالرٍت حمدر حَث اعطج ايخصاصَه 4 يول.نخر 1.5111× -1 سى. -1 يم.ياٍكروغراو 0.3140انكشف يساوٍا نـ نمد كاٌ حد . -1 ايا سى.ياٍكروغراو 0.01.0 حساوً ساَدل فمد كاَج حساسَت -2 فٌ حمدٍر االوالَسابٍَ فٌ بُجاح خرحتمانً انطرٍمت حطبَك حى . يسخحضراث انحبوب انصَدالََت. ويترواويليه, مستحضرات الحبوب الصيدالوية.-, اوالوسابيه, االزوته, باراالطيفي التقديرالكلمات المفتاحية: introduction olanzapine (oln.) )c17h20n4s( is known chemically as 2-methyl-4-(4-methyl 1piperazinyl) 10h-thieno [2,3-b] [1,5] benzodiazepine (scheme 1), has molar mass of 312.439 g.mol -1 and it is a yellow crystalline solid substance with a melting point of 195ºc. (1,2) it is an atypical antipsychotic drug used in the treatment of schizophrenia and other psychotic syndromes (3) . scheme (1):the structural formula of olanzapine. (1) the mode of action of olanzapines’ of antipsychotic activity having efficacy in schizophrenia is unknown, (4) it has been proposed that drug achieve this seemingly highly selective approach to treatment is the antagonism of a specific serotonin receptor, 5ht2a .this receptor can be found on the axon terminal of neurons that produce dopamine. when serotonin activates those receptors, the release of dopamine decreases. by building into the typical antipsychotics a mechanism to block the 5-ht2a receptors from serotonin, the dopamine release is increased. (3,5) since its introduction in 1996 in over 84 countries (6) (oln.) was determined by using several methods that have been reported for the analysis in pure form, dosage forms or in combination with other drugs. these methods include spectrophotometry, (7-10) hplc, (11,12) flow injection analysis, (13) electro-analytical method, (14,15) and capillary electrophoresis (16) . 1 corresponding author e-mail: saharchemst2011@yahoo.com received: 19 /1/2016 accepted: 12/4/2016 iraqi j pharm sci, vol.25(1) 2016 spectrophotometric determination of olanzapine 43 the aim of the present study is to develop an accurate, simple and sensitive spectrophotometric procedure for the determination of olanzapine in pure and tablet dosage forms. the method is based on the coupling of olanzapine with diazotized p-nitroaniline in basic medium to form a colored complex. in addition, the reaction conditions were studied univariatly one-factor-a time to provide an optimized analytical response. experimental apparatus a pg instrument, uvvisible spectrophotometer model t80 (u.k) with 1 cm matched quartz cells was used for the absorbance measurements. sartorius bl 210s electronic balance was used for weighing the samples. hot plate with magnetic stirrer, ijlassco, india. materials and methods all chemicals used were of analytical reagent grade and were obtained from bdh and panreac. olanzapine standard powder was kindly provided by the state company for drug industries and medical appliances (sdi), samara-iraq. the pharmaceutical preparation; zyprexa  tablet (5 mg) lilly (spain), zyprexa  tablet (10 mg) lilly (spain) and olan  tablet (5 mg) micro (india) were purchased from local markets. olanzapine stock solution [1000 µg.ml -1 ] the stock solution of (oln.) was prepared by dissolving an accurately weighed 0.1000 g of pure drug in 5 ml of 0.1 m hcl and the volume was made up to the mark in a 100 ml volumetric flask with distilled water. the stock solution was protected from light and stored at 5 ºc. olanzapine working solution [100 μg.ml -1 ]: prepared by diluting 10 ml of the stock solution to 100 ml in a volumetric flask with distilled water. diazotized p-nitroaniline reagent solution, (dpna), [0.01 m] p-nitroaniline (pna) (0.2762) g was dissolved in 50 ml distilled water and 6.7 ml of concentrated hcl was then added to this solution with stirring. the mixture was heated to obtain a clear solution, transferred to 200 ml volumetric flask, and cooled to (0-5) c in an ice-bath. nano2 (0.1380) g was then added and the mixture was stirred vigorously. five minutes later, the solution was made up to the mark with cold distilled water. the solution is kept in a brown bottle into refrigerator and used after three hours of preparation. it is stable for at least 72 hours. sodium hydroxide solution (~2 m): prepared by dissolving (8.000) g of naoh in a suitable volume of distilled water and the volume was made up to the mark in 100 ml volumetric flask. olanzapine tablets solution [1000 µg.ml -1 ] seven tablets of both strengths of zyprexa and ten tablets of oln-5 were accurately weighed and separately, grinded into fine powder and mixed well then the average weight was calculated for each brand. an amount of the powder equivalent to (0.5928) g, (0.2972) g and (0.5848) g (containing 0.0200 g of the drug olanzapine) of zyprexa-5 mg, zyprexa 10 mg and oln-5 mg respectively was accurately weighed, dissolved in 5 ml of 0.1 m hcl and stirred for 10 min to ensure complete dissolution of the drug, then transferred into 20 ml volumetric flask and diluted to the mark with distilled water to get 1000 µg.ml -1 of (oln.). the solution was filtered by using whatman filter paper no.41 to avoid any suspended or un-dissolved material before use. working solution 100 µg.ml -1 was freshly prepared and analyzed by the recommended procedure. general recommended procedure for calibration in a series of 10 ml volumetric flasks, 1 ml of 0.01 m of the diazotized pnitroaniline solution, aliquots of working drug solution (100 µg.ml -1 ) in the range (0.05, 0.1, 0.3, 0.5……4.5) ml were added to each flask followed by 1 ml of 2 m sodium hydroxide with shaking and allowed to stand for 5 min. the contents were diluted to the mark with distilled water and mixed well. after 5 min, the absorbance of the brown colored azo-dye was measured at 405 nm against the reagent blank prepared in the same manner without the analyte. results and discussion absorption spectra for primary test the primary test for the present method involved diazotization of p-nitroaniline followed by coupling with olanzapine (oln.). the test was done by adding 1 ml of 0.005 m diazotized p-nitroaniline in 10 ml volumetric flask, followed by the addition of 1 ml of 100 µg.ml -1 (oln.) with shaking. 1 ml of 1 m naoh was then added to the above mixture. the contents were diluted to the mark with distilled water. the absorbance and λmax of the colored azo-dye was measured against the reagent blank prepared in the same manner without the analyte. (figure 1) shows that the maximum absorption was obtained at a wavelength of 405 nm. iraqi j pharm sci, vol.25(1) 2016 spectrophotometric determination of olanzapine 44 figure (1): absorption spectra of: (a) the complex of 10 µg.ml -1 (oln.) with diazotized p-nitroaniline against reagent blank, (b) blank solution against distilled water under the primary test conditions. optimization of reaction variables the various parameters related to the colored product formation have been studied by varying the parameters one at a time and controlling all others fixed and the optimum conditions have been selected 1. effect of the diazotized p-nitroaniline concentration the optimum diazotized p-nitroaniline concentration was estimated by adding 1ml of various concentrations [0.0050.030] m of diazotized p-nitroaniline reagent solution, the results showed that 0.01 m of the reagent solution is sufficient for production of maximum and reproducible color intensity (figure 2). therefore, the recommended concentration of diazotized p-nitroaniline was chosen to be 1 ml of 0.01 m and used for all subsequent measurements. figure (2): effect of diazotized pnitroaniline concentration on the color development in the determination of 10 µg.ml -1 (oln). 2. effect of type of the base the effect of different alkaline solutions with concentration of 1 m on the absorption intensity of the colored azo-dye formed was investigated. it was found that sodium hydroxide gave the maximum absorption intensity of the colored product which is used for the subsequent work, (table 1). table (1): effect of different bases. alkaline medium [1m] absorbance naoh 0.216 koh 0.188 na2co3 0.104 nh4oh 0.025 2. effect of sodium hydroxide concentration the stability of the formed azo dye product depends upon the nature of reaction medium. (17) the formed azo dye was found to have a reasonable stability when the reaction medium was rendered alkaline via the addition of 1ml of 2 m sodium hydroxide solution which was optimum and recommended for the subsequent work, (figure 3). figure (3): effect of sodium hydroxide concentration on the color development in the determination of 10 µg.ml -1 (oln.). 4. effect of coupling reaction time the optimum time of coupling reaction was determined by choosing different time periods (0-20) min for development the color of azo-dye at room temperature, it was found that 5 minutes period was required for full color development as shown in (table 2). table (2): effect of coupling reaction time. time (min) absorbance 0 0.305 5 0.392 10 0.362 15 0.314 20 0.288 5. effect of order of mixin the effect of different orders of component addition on chromogen formation was studied by changing the order for three times as iraqi j pharm sci, vol.25(1) 2016 spectrophotometric determination of olanzapine 45 shown in (table 3). results shows that mixing order number one was recommended and thus was followed in the subsequent experiments, since it resulted in obtaining maximum absorbance. table (3): variation of absorbance with reactants addition order on the determination of 10 µg.ml -1 (oln.). no sequence absorbance 1 diazotized reagent + drug + base 0.392 2 diazotized reagent + base+ drug 0.330 3 drug + base + diazotized reagent 0.327 6. stability under the aforementioned optimum condition, the effect of time on the formation of the azo-dye product was investigated by allowing the reaction to proceed by varying periods. it was found that the absorbance reach to a maximum constant value after 5 min, and the color of the azo product was nearly stable for at least 60 min as shown in (figure 4). figure (4): the stability of colored reaction product with time. final absorption spectra when 10 µg. ml -1 of (oln.) is treated with diazotized pnitroaniline reagent, under the aforementioned optimum conditions, an absorption peak is obtained showing intense brown azo dye absorption at 405 nm, the reagent blank showed almost nil absorption at this maximum wavelength as shown in (figure 5). figure (5): absorption spectra of: (a) the complex of 10 µg.ml -1 (oln.) with iazotized p-nitroaniline against reagent blank, (b) blank solution against distilled water under the optimum conditions. calibration curve and analytical data according to the optimum experimental conditions, linear calibration graph for (oln.) was obtained (figure 6), which shows that beerʼs law was obeyed in the concentration range of (0.545.0) µg.ml -1 . the regression equation, correlation coefficient, molar absorptivity, sandell's sensitivity, limit of detection (lod) and limit of quantification (loq) were given in (table 4). table (4): optical characteristics and statistical data for the determination of (oln.). parameter value λmax (nm) 405 color brown regression equation y=0.0505[(oln.) µg.ml -1 ]-0.0119 linearity range (µg.ml -1 ) 0.5 45 calibration sensitivity (ml.µg -1 ) 0.0505 correlation coefficient (r) 0.9994 correlation of linearity (r 2 ) 0.9989 molar absorptivity (l.mol -1 .cm -1 ) 1.5777x10 4 sandellʼs sensitivity (µg.cm -2 ) 0.0198 l.o.d. (µg.ml -1 ) 0.3148 l.o.q. (µg.ml -1 ) 1.0495 iraqi j pharm sci, vol.25(1) 2016 spectrophotometric determination of olanzapine 46 figure(6): calibration curve for the determination of (oln.) under optimum conditions. nature of the dye product job's method (18) and mole ratio method (19) have been used in the determination of the reaction ratio of (oln.) with p-nitroaniline reagent. the obtained results in (figures 7 and 8) showed that 1:1 (oln.) to diazotized p nitroaniline reagent ratio is obtained. hence the azo-dye may have the proposed mec hanism illustrated in (scheme 2). figure (7): continuous variation method for reaction (oln.) with diazotized pnitroaniline figure (8): mole ratio method for (oln.) with diazotized p-nitroaniline. scheme (2):the suggested reaction mechanism between dpna and (oln.). iraqi j pharm sci, vol.25(1) 2016 spectrophotometric determination of olanzapine 47 comparison of the methods (table 5), shows the comparison between some analytical variables of the present method with another spectrophotometric methods in literature. precision and accuracy the precision and accuracy for the determination of (oln.) via the proposed method were studied by calculating the values of coefficient of variation (c.v%) and percentage of relative error (er %), for three replicates at three different concentration levels of (oln.) drug. the results in (table 6) show acceptable values for accuracy and precision. interference study the effect of various excipients, which may be present in pharmaceutical products and affecting the reaction between (oln.) and diazotized pnitroaniline, was studied. optimum experimental conditions, were employed to determine 10μg.ml -1 concentration of (oln.). (table 7) shows that the studied excipients did not interfere in the present method. application in pharmaceutical preparation the application of the method for the assay of olanzapine in drugs has been applied successfully, and the results obtained were listed in (table 8) for each sample in three replicates table (5): analytical parameters for the analysis of olanzapine by the proposed ethodcomparing to the methods. methods linear range μg.ml -1 (ε) l.mol -1 .cm -1 correlation coefficient ( r) c.v% range ref. proposed method 0.5-45.0 1.5777x10 4 0.9994 0.033-0.080 … spectrophotometric 4.0-20.0 1.74×104 0.9998 0.355-0.822 6 spectrophotometric 5.0-160.0 0.60 x 10 3 0.9999 0.820–0.910 7 spectrophotometric 5.0 -40.0 ---0.9980 0.120-0.590 9 spectrophotometric 0.4 -8.0 2.08 × 10 4 0.9994 0.669-2.278 20 spectrophotometric 40.0 -0.4 4.375 × 10 3 0.9999 0.270-0.700 21 rp-uplc 25.0-150 ---0.9990 0.210-0.330 10 hplc 2.5-20.0 ---0.9999 0.150-0.460 11 table (6): evaluation of accuracy and precision for the determination of (oln.) by proposed method. conc. of (oln.) μg.ml -1 er% c.v% taken found* 10.000 9.951 -0.490 0.458 18.000 18.013 0.072 0.013 32.000 32.046 0.143 0.033 *average of three measurements. table (7): recovery values for 10 µg.ml -1 of (oln.) in the presence of different excipients. excipients olanzapine conc. recovery (%) name conc. (µg.ml 1 ) taken (µg.ml -1 ) found (µg.ml -1 ) lactose 500 10.000 9.981 99.810 glucose 500 9.839 98.390 sucrose 500 9.990 99.900 starch 1000 9.971 99.710 magnesium stearate 1000 9.952 99.520 iraqi j pharm sci, vol.25(1) 2016 spectrophotometric determination of olanzapine 48 table (8): application to the olanzapine concentration measurement in tablets. sample weight found* (mg) concentration (µg.ml -1 ) recovery % c.v % taken found* zyprexa 5 mg 5.026 10.000 10.052 100.523 0.469 5.027 30.000 30.166 100.553 0.285 zyprexa 10 mg 10.145 10.000 10.145 101.450 0.514 10.031 30.000 30.093 100.310 0.107 olan 5 mg 4.882 10.000 9.947 99.473 0.280 4.986 30.000 29.917 99.725 0.185 *average of three measurements. conclusion the proposed method permits rapid, precise, and accurate determination of olanzapine. it makes use of simple reagents, which an ordinary analytical laboratory can afford. the method was found to be free from interference by the excipients. the wide applicability of the new procedure for routine quality control was well established by the assay of olanzapine in pure form and in pharmaceutical preparations. references 1. british harmacopoeia on , er ajesty s stationary office, london, 2013. 2. andrew, w.; pharmaceutical manufacturing encyclopedia, 3 rd edition, elsevier saunders, 2013; 5-6. 3. kassahun, k.; mattiuz, e. nyhart, e.; obermeyer, b.; gillespie, t.; murphy, a.; goodwin, r. m.; tupper, d.; callaghan, j. t. and lemberger, l.; disposition and biotransformation of the antipsychotic agent olanzapine in humans. drug metab. dispos, 1979; 25(1): 81. 4. eris, f. p.; psychiatric advanced practice nursing. a biopsy-chosocial foundation for practice, f.a. davis company , 2012; (170). 5. richard, a. l. pharmacology for nursing care, 8 th , elsevier saunders ; 2013; 343. 6. ring, b. j.; catlow, j. and et al; identification of the human cytochromes p450 responsible for the in vitro formation of the major oxidative metabolites of the antipsychotic agent 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r. and rathore, r.p. analytical method development and validation of olanzapine in formulated product, world journal of pharmaceutical research, 2015; 4(4): 1690-1699. 12. basavaiah, k.; rajendraprasad, n.; and vinay, n.k.; isocratic high-performance liquid chromatographic assay of olanzapine: method development and validation, hindawi publishing corporation, isrn analytical chemistry, 2014; 1-6. 13. zhao, f.; fan, q. and cai, h.; flowinjection chemiluminescence determination of olanzapine using nchlorosuccinimidecalcein reaction sensitized by zinc (ii), luminescence, 2014; 29(3): 219-224. 14. manal, a. e.; electrochemical studies for the determination of quetiapine fumarate and olanzapine antipsychotic drugs, advanced pharmaceutical bulletin, 2013; 3(2): 339-344. 15. anwar, a. w. and shabaan, a.; new type of pvc-membrane ion-selective iraqi j pharm sci, vol.25(1) 2016 spectrophotometric determination of olanzapine 49 electrodes and their applicability to determine some antidepressant drugs, sci. pap. univ. pardubice ser., 2010; a16: 33–55. 16. hillaert, s.; snoeck, l. and vanden, b.w.; optimization and validation of a capillary zone electrophoretic method for the simultaneous analysis of four atypical antipsychotics, journal of chromatography a. 2004; 1033(2): 357– 362. 17. mallikarjuna, h.; lokesh, k. s.; shivaprasad, k. h. and venugopala, k. r.; novel spectrophotometric methods for the assay of an antiepileptic – oxcarbazepine, world journal of pharmacy and pharmaceutical sciences, 2014; 3(7): 815-831. 18. job, p.; spectrochemical methods of analysis, wiley, new york, 1971; 346. 19. christian, g. d.; analytical chemistry, fifth ed., wiley, new york, 1994; 385– 386. 20. revanasiddappa, h. d. and deepakumari, h. n.; highly sensitive spectrophotometric method for the quantitative determination of olanzapine in its pure and in pharmaceutical dosage forms, journal of scientific & industrial research, 2014; 73: 41-45. 21. nagabhushana, k.; giri, a.; saritha, b. and sreenivasulu, r. t. assay of olanzapine in pharmaceutical formulations by visible spectrophotometry using cadmium (ii), international journal for pharmaceutical research scholars, 2014; 3(i-2): 418-425. iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 9 neutrophil / lymphocyte ratio is not correlated with disease activity in rheumatoid arthritis patients ehab m. mikhael *,1 and turathn.ibrahim ** * department of clinical pharmacy college of pharmacy university of baghdad, baghdad ,iraq. ** department of clinical pharmacy college of pharmacy al-mustansria university, baghdad, iraq. abstract rheumatoid arthritis is a chronic systemic inflammatory disease. inflammation leads to joint damage and increases the risk of cardiovascular diseases. neutrophil lymphocyte ratio (nlr) is a measure of inflammation in many diseases. therefore, we aimed to evaluate the usefulness of nlr to detect inflammation in ra, and its correlation to ra disease activity indices and some hematological parameters. a cross-sectional study involving 24 patients with active rheumatoid arthritis (ra) who are using mtx participated in this study. all patients were clinically evaluated using disease activity score of 28 joints (das28) and simplified disease activity index (sdai), whereas functional disability was assessed by health assessment questionnaire disability index (haqdi); moreover, blood specimen of each patient was used for measuring erythrocyte sedimentation rate (esr), c – reactive protein (crp), rheumatoid factor (rf), hemoglobin (hb), white blood cells (wbc) count, platelets and red blood cells (rbcs) count, and nlr ratio.nlr was positively correlated with esr and inversely correlated with hb, but it didn’t show any correlation with other clinical and laboratory parameters. in conclusion nlr is less correlated with inflammation and not suitable to monitor disease activity in ra patients using mtx. keywords: rheumatoid arthritis, inflammation, neutrophil lymphocyte ratio. نسبة كريات الدم البيط العدلة إلى كريات الدم البيط اللوفاوية الترتبط هع فاعلية الورض عند الوصابين بالتهاب الوفاصل الروهاتىيدي ايهاب هضر هيخائيل ،*1 وتراث نبيل ابراهين ** * انؼشاق.تغذاد،فشع انصٛذنح انسشٚشٚح ،كهٛح انصٛذنح ،جايؼح تغذاد ، ** انؼشاق .تغذاد،، انجايؼح انًسرُصشٚحفشع انصٛذنح انسشٚشٚح ، كهٛح انصٛذنح ، الخالصة انرٓاب انًفاصم انشٔياذٕٚذ٘ يشض انرٓاتٙ يزيٍ ْٔزا االنرٓاب ٚؤد٘ إنٗ ذهف انًفصم ٔكًا ٚزٚذ يٍ خطٕسج األيشاض انمهثٛح نك انٕػائٛح. إٌ َسثح كشٚاخ انذو انثٛط انًؼرذنح إنٗ كشٚاخ انذو انثٛط انهًفأٚح ٚؼرثش لٛاسا نُسثح االنرٓاب فٙ كثٛش يٍ األيشاض. ٔنز فائذج ْزِ انُسثح نمٛاس يسرٕٖ االنرٓاب ػُذ يشظٗ انرٓاب انًفاصم انشٔياذٕٚذ٘ ٔػاللح ْزِ انُسثح تفاػهٛح يشض كاٌ ْذفُا ذمٛٛى يصاتا تانرٓاب انًفاصم انشٔياذٕٚذ٘ ٔانزٍٚ ٚسرؼًهٌٕ 42انرٓاب انًفاصم انشٔياذزيٙ ٔتؼط انًؼاٚٛش انذيٕٚح. شًهد انذساسح ٔيهحك فاػهٛح انًشض (das28)يفصم 42شٚشٚا تٕاسطح يؼٛاس فاػهٛح انًشض ل انًٛثٕذشكسٛد. ذى ذمٛٛى جًٛغ انًشظٗ س يهحك انؼجز. إظافح نزنك ػُٛاخ انذو سحثد نمٛاس –, أيا يمذاس اإلػالح فرى ذمًّٛٛ تٕاسطح اسرثٛاٌ ذمٛٛى انصحح (sdai)انًثسط َسثح ذشسة كشٚاخ انذو انحًش, تشٔذٍٛ سٙ انفؼال, يمٛاس انشٔياذٕٚذ, انًٕٓٛغهٕتٍٛ, ػذد كشٚاخ انذو انحًش ٔانثٛط ٔانصفٛحاخ ط انؼذنح إنٗ كشٚاخ انذو انثٛط انذيٕٚح ٔ َسثح كشٚاخ انذو انثٛط انؼذنح إنٗ كشٚاخ انذو انثٛط انهًفأٚح. إٌ َسثح كشٚاخ انذو انثٛ انهًفأٚح كاٌ نٓا ػاللح اٚجاتٛح يغ َسثح ذشسٛة كشٚاخ انذو انحًش ٔػاللح ػكسٛح يغ انًٕٓٛغهٕتٍٛ ٔنكُٓا نى ذظٓش أ٘ ػاللح يغ تالٙ أٚح ذشذثط تؼاللح ظؼٛفح انًؼاٚٛش انسشٚشٚح ٔانًخرثشٚح. ٚسرُرج يٍ رنك إٌ َسثح كشٚاخ انذو انثٛط انؼذنح إنٗ كشٚاخ انذو انثٛط انهًف يغ االنرٓاب ٔغٛش يالئًح نًراتؼح فاػهٛح انرٓاب انًفاصم انشٔياذٕٚذ٘ نهًشظٗ انزٍٚ ٚسرؼًهٌٕ يٛثٕذشكسٛد. . كريات الدم البيط العدلة الى كريات الدم البيط اللوفاويةالكلوات الوفتاحية: التهاب الوفاصل الروهاتىيدي, التهاب, نسبة introduction rheumatoid arthritis (ra) is a chronic systemic inflammatory disease of unknown etiology that characterized by both articular and extra articular features (1) . local inflammation of the joints is correlated to joint damage (2) whereas systemic inflammation increases the risk of atherogenesis and coronary heart disease in ra patients (3) . several studies have explored the relationship between systemic inflammation and cardiovascular mortality (4, 5) .systemic inflammation can be measured by using a variety of biochemical and hematological markers (6) crp is a strong predictor of future 1 corresponding author e-mail:ehab_pharma84@yahoo.com received:3/11/2012 accepted:19/5/2013 iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 10 cardiovascular events in individuals both with and without overt cardiovascular disease (cvd) (7) . additionally, crp correlates directly with the presence of atherosclerosis in patients with ra (8) whereas; esr is significantly associated with the risk of cvd in ra (9). neutrophil lymphocyte ratio (nlr) is an important measure of systemic inflammation as it is readily available and could be calculated easily (10) . many studies found that nlr is a useful measure to detect inflammation and predict long term outcomes in patients with renal failure, cancer and heart diseases (11 -13) . aim of the study to evaluate the usefulness of nlr to detect inflammation in ra, and its correlation to various ra disease activity indices and some hematological parameters. patients and methods a cross-sectional study was conducted in baghdad teaching hospital, rheumatology unit from december 2011 to may 2012. a total of 24 patients (7 males and 17 females) with active ra were involved in this study. patients were diagnosed to have ra by the rheumatologist according to american college of rheumatology (acr) classification criteria for ra (14) . all patients included in the study signed an informed consent form according to the declaration of helsinki. ethical approval was obtained from the ethics committee of baghdad university, college of medicine, department of medicine. patients with diseases other than rheumatoid arthritis were excluded from the study. demographic data of patients were reported regarding their age, duration of the disease, and medication history (table 1). laboratory investigations blood specimens were taken from all patients. hematological investigations that include complete blood count (wbc, rbc and platelet), differential wbc count and hb were measured using hematology auto analyzer (ruby – cell – dyn 08h56 – 02) from abbott company usa. esr was measured by westergren method (15) . crp was measured semi quantitatively according to method of singer et al using serial dilutions of serum; each dilution was mixed with a latex reagent and observed for the presence of agglutination (16) using a ready made kit (agapee, switzerland) whereas rf was measured qualitatively (16) by a ready made kit (spectrum, egypt). clinical evaluation the 28 joints included bilateral knees, shoulders, elbows,wrists, metacarpophalangeal and proximal interphalangeal joints, were palpated to count the number of tender and swelling joints. the patients were asked to mark on the vas of 0 – 100mm according to their global assessment of their general health and pain. the physician marked on the vas of 0-10 cm according to the physician global assessment of the disease activity. disease activity was measured by both das28 and sdai. das28 was calculated from the tjc, sjc and esr according to the following formula [17]: das28 = {(0.56 • √[tjc28]) + (0.28 • √[sjc28]) + (0.70 • in[esr])} • 1.08 whereas sdai was calculated by the following formula [18]: sdai = crp + tjc + sjc + vas (0-10) + ega (0-10)functional status of the patients was measured using health assessment questionnaire disability index [19]. additionally morning stiffness of each patient was calculated according to patient approximate. statistical analysis spss version 12 was used for data input and analysis. shapiro wilk test (web version) used to check if data is normally or abnormally distributed. spearman correlation coefficient was used to assess the correlation between abnormally distributed continuous variables. all p values used were asymptotic and two sided. values with p < 0.05 were considered significant. results table (1) showed general demographic data for the patients that participated in this study. table (2) showed the values (mean ± sd) for the different studied variables, when these valuescorrelated, with neutrophil/lymphocyte ratio there was a significant positive correlation with esr (r = 0.495, p = 0.014) and a significant negative correlation with hb (r = -0.426 , p = 0.034), crp has a weak positive correlation that didn’t achieve statistical significance , whereas other parameters didn’t show a significant correlation with nlr (table 3). iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 11 table (1): general demographic data of the patients parameter patients with moderate disease activity patients with high disease activity all participated patients age, years mean  sd 5112.11 45.2411.08 46.9211.44 female percent 14.85 88.24 66.67% disease duration, years mean  sd 6.714.57 5.885.16 6.134.91 drug used (mtx/hcq) (5/2) 17/0 (22/2) rf positive n (%) 5 (71.4) 9 (52.94) 14 (58.33) smoking percent 42.86 0 12.5% mtx = methotrexate; hcq = hydroxychloroquine; sd = standard deviation; rf = rheumatoid factor. table (2): clinical and laboratory parameters in ra patients participated in table (3): correlation of different parameters with nlr the study jc = tender joint count; sjc = swelling joint count; vas = visual analogue scale; ega = evaluator global assessment; das28 = disease activity score 28 joints; sdai = simplified disease activity score; crp = c – reactive protein; esr = erythrocyte sedimentation rate; rf = rheumatoid factor; hb = hemoglobin; wbc =white blood cells; rbc = red blood cell haqdi = health assessment questionnaire disability index. jc = tender joint count; sjc = swelling joint count; vas = visual analogue scale; ega = evaluator global assessment; das28 = disease activity score 28 joints; sdai = simplified disease activity score; crp = c – reactive protein; esr = erythrocyte sedimentation rate; rf = rheumatoid factor; hb = hemoglobin; wbc =white blood cells; rbc = red blood cell haqdi = health assessment questionnaire disability index. parameter correlation coefficient p value tjc -0.001 0.997 sjc 0.051 0.813 vas 0.179 0.404 ega 0.232 0.276 morning stiffness -0.139 0.516 das28 0.351 0.093 sdai 0.150 0.484 crp (mg/dl) 0.368 0.077 esr (mm/hr) 0.495 0.014 hb ( gm/dl) -0.435 0.034 wbc (cell/ nano liter) 0.306 0.146 rbc (cell/ nano liter) -0.084 0.698 platelet (cell/ nano liter) 0.339 0.105 haqdi 0.146 0.496 parameter value (mean  sd) tjc 10.544.53 sjc 5.133.03 vas 54.1725.70 ega 5.132.35 morning stiffness 26.238.1 das28 5.431.46 sdai 27.9213.62 crp ( mg/dl) 1.652.34 esr ( mm/hr) 3822.14 rf 0.580.50 hb ( g/dl) 12.281.62 wbc (cell/ nano liter) 9.753.27 rbc (cell / nano liter) 4.670.76 platelet (cell/ nano liter) 28887.82 haqdi 1.360.74 iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 12 additionally table (4) showed that nlr not differ significantly between highly active and moderately active ra (p = 0.07), while esr, crp and das28 values varied significantly (p<0.05) between moderately and highly active ra patients. table (5) showed a highly positive correlation between vas and ega. table (4): comparison of some parameters between patients with highly active ra and those with moderately active ra parameter patients with moderate ra disease activity (7) patients with high ra activity ( 17) p value nlr 2.110.46 3.171.68 0.070 das28 3.851.49 6.080.85 0.000 crp 0.430.90 2.152.57 0.047 esr 18.8614.96 45.8819.86 0.004 nlr= neutrophil lymphocyte ratio; das28 = disease activity score of 28 joints; crp = c – reactive protein; esr = erythrocyte sedimentation rate table (5): correlation between visual analogue sale (vas) and evaluator global assessment (ega) disease activity number of cases vas ega spearman correlation coefficient p value moderate 7 31.4320.35 27.1418.90 0.924 0.003 high 17 63.8921.18 60.5616.97 0.795 0.000 moderate and high 24 54.825.35 51.222.97 0.867 0.000 discussion this study showed that nlr as a measure of inflammation in ra patients was positively correlated with esr, similar finding was observed when nlr was evaluated as a measure of inflammation in ulcerative colitis patients (20) . moreover, nlr was inversely correlated with hb, however, there is no any study in this respect and it is the 1 st time to get such result. this finding is acceptable since hb in ra patients is inversely correlated with inflammation and ra disease activity (21) . regarding crp, there is a modest but a non significant correlation with nlr, there is an agreement of this finding in a trial that followed up patients with cancer (22) . according to the above, and since nlr is well correlated with esr and not well correlated with crp, so nlr may be not sufficient laboratory test to predict cvd risk in ra patients and further studies areneeded to evaluate the association of nlr with the severity and extent ofcoronary atherosclerosis. results from the current study also showed that nlr didn’t correlate with ra clinical parameters like tender joint count, swelling joint count and haqdi, this result may be rationale since it has been found that many of clinical parameters that were used to diagnose ra like tjc, sjc, morning stiffness and haqdi were less correlated with inflammation (23-25). this study showed a direct positive correlation between vas and ega and also showed that there is no correlation between vas and ega with nlr. this finding can be explained according to the finding of naredoetal (24) at which vas was not correlated with inflammation ( especially with esr) and since this study showed that nlr was directly correlated with esr, so it can be concluded that there is no correlation between vas or ega with nlr . additionally hematological parameters like wbc, rbc and platelets count were not correlated with nlr. there was very complex hematological parameters in active ra patients that use mtx who participated in this study, since active ra disease is associated with anemia, leucocytosis and thrombocytosis (26,27) , whereas mtx may causes neutropenia and thrombocytopenia (28) . consequently absence of correlation between nlr and hematological parameters may result from the unsuitable patient sample selection and further studies are needed to correlate between nlr and hematological parameters in ra patients using dmards other than mtx. iraqi j pharm sci, vol.22(2) 2013 neutrophil/ lymphocyte ratio in rheumatoid arthritis 13 finally, results from the current study showed that nlr is not correlated with ra disease activity as measured by either das28 or sdai; similarly fauziaimtiazet al also found that there was a non significant relationship between nlr and rheumatoid arthritis (29). this finding was strengthened by absence of statistical difference in nlr between patients with moderately and those with highly active ra, and only crp and esr showed a significant difference between the 2 groups of patients similar to that of das28; consequently esr and crp is better than nlr to detect ra disease activity. conclusion nlr is less correlated with inflammation and not suitable to monitor disease activity in ra patients using mtx. reference 1. manole cojocaru, inimioara mihaela cojocaru, isabela silosi , et al. extraarticular manifestations in rheumatoid arthritis. maedica (buchar) 2010; 5(4): 286–91. 2. conaghan pg, o'connor p, mcgonagle d, et al. elucidation of the relationship between synovitis and bone damage: a randomized magnetic resonance imaging study of individual joints in patients with early rheumatoid arthritis. arthritis rheum. 2003;48(1):64-71. 3. naveedsattar, david w. mccarey, hilary capell, et al. explaining how “highgrade” 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http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20al-ghamdi%2ba%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20attar%2bsm%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20attar%2bsm%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed?term=syed%20km%5bauthor%5d&cauthor=true&cauthor_uid=19078065 http://www.ncbi.nlm.nih.gov/pubmed?term=pinals%20rs%5bauthor%5d&cauthor=true&cauthor_uid=19078065 http://www.ncbi.nlm.nih.gov/pubmed/19078065## http://www.ncbi.nlm.nih.gov/pubmed?term=gutierrez-ure%c3%b1a%20s%5bauthor%5d&cauthor=true&cauthor_uid=8849378 http://www.ncbi.nlm.nih.gov/pubmed?term=molina%20jf%5bauthor%5d&cauthor=true&cauthor_uid=8849378 http://www.ncbi.nlm.nih.gov/pubmed?term=garc%c3%ada%20co%5bauthor%5d&cauthor=true&cauthor_uid=8849378 http://www.ncbi.nlm.nih.gov/pubmed?term=garc%c3%ada%20co%5bauthor%5d&cauthor=true&cauthor_uid=8849378 http://www.ncbi.nlm.nih.gov/pubmed/8849378 http://www.ncbi.nlm.nih.gov/pubmed?term=imtiaz%20f%5bauthor%5d&cauthor=true&cauthor_uid=22281066 http://www.ncbi.nlm.nih.gov/pubmed?term=shafique%20k%5bauthor%5d&cauthor=true&cauthor_uid=22281066 http://www.ncbi.nlm.nih.gov/pubmed?term=mirza%20ss%5bauthor%5d&cauthor=true&cauthor_uid=22281066 http://www.ncbi.nlm.nih.gov/pubmed/22281066 http://www.ncbi.nlm.nih.gov/pubmed/22281066 iraqi j pharm sci, vol.31(1) 2022 anti-angiogenic of moringa oleifera leaves extract doi: https://doi.org/10.31351/vol31iss1pp225-232 225 anti-angiogenic screening of moringa oleifera leaves extract using chorioallantonic membrane assay wong wing kei* and nisha shri chengama raju *,1 *department of pharmacology, faculty of pharmacy, segi university kota damansara, petaling jaya, malaysia abstract angiogenesis is defined as the physiological process resulting in formation of new blood vessels from pre-existing vessels. however, angiogenesis in cancer will lead to tumor growth and metastasis. therefore, antiangiogenesis is one of the ways to slow down growth and spreading of tumor. moringa oleifera is also known as a “miracle tree” which has high nutritive value and various therapeutics effects in different parts of the plant. this study aims to determine the anti-angiogenic property of moringa oleifera leaves extract by using chick chorioallantoic membrane (cam) assay. the extracts were prepared by decoction method using methanol (99.8%) and water. the qualitative phytochemical screening was carried out for both methanol and aqueous extracts. the fertilised chicken eggs were divided into six groups which include negative control group (phosphate-buffer saline with ph 7.4), positive control group (sunitinib), 50% and 100% methanol extract, 50% and 100% aqueous extract. the anti-angiogenic effect of moringa oleifera leaves extract was determined by calculating the number and percentage decrease in blood vessels in post-24 and post-48 hours of treatment. statistical analysis by one-way anova has shown significant (p<0.05) percentage reduction in the blood vessels between each treatment group after 48 hours of treatment. among all the extracts, 100% aqueous extract of moringa oleifera was found to have highest anti-angiogenic effect with the greater percentage decrease in blood vessels (81.33%) in post-48 hours of treatment. furthermore, the anti-angiogenic effect of moringa oleifera leaves was found to be increased when the concentration of the moringa oleifera extract was increased. moringa oleifera leaves with various phytochemicals was found to possess anti-angiogenic potential. y.leaves, chick chorioallantoic membrane (cam) assa moringa oleiferaenic screening, angiog-keywords: anti introduction angiogenesis is defined as the formation of new blood vessels to allow nutrient supply, gaseous exchange and removal of waste products to a localized tissue. it plays an important role in the process of wound healing, tissue repair, tissue growth, foetal development and cancer (1). angiogenesis is regulated by various mediators, where the most common type of mediators is growth factors and cytokines. the examples of growth factors include vascular endothelial growth factor (vegf), tumor necrosis factor-alpha (tnf-α), fibroblast growth factor (fgf), platelet-derived growth factor (pdgf) and angiopoietin (2). however, the mediators can be divided into stimulator or activator and inhibitor, hence the balance between stimulatory and inhibitory mediators are extremely important in the process of angiogenesis. normally, when the formation of new blood vessels is required, an “angiogenic switch” will be turned on by increasing the activator growth factor (3). in recent years, there are many studies regarding angiogenesis inhibition. the mechanism of inhibition of angiogenesis is by blocking the formation of new blood vessels which further slows down the cancer progression and reduces release of metastatic cells into the circulation (4). hence, angiogenesis inhibition is one of the mechanisms in anticancer therapy. moreover, angiogenesis inhibition is not restricted to any specific types of tumor cell because all of the solid tumors are based on angiogenesis (5). therefore, inhibition of angiogenesis has become a new target for anticancer action. angiogenesis inhibitor consists of different benefits such as fewer side effects, less resistance compare to anti-cancer medication and accessibility of endothelial cell in blood vessels are better than tumor cell (6). plant extracts are rich in bioactive compounds that can decrease the growth rate of cancer cell and induce cancer cell apoptosis by inhibiting angiogenesis. this can lead to suppression or eradication of cancer. phytochemicals play an important role in preventing development and growth of tumour. however, further studies are still required to explore and identify more therapeutic plants with antiangiogenic effects. furthermore, the side effect and toxicity of plant-based anti-cancer drug was found to be fewer compared to conventional chemotherapeutic agents (7). 1corresponding author e-mail: nishsri@gmail.com received: 8/8/2021 accepted: 12/10 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp225-232 iraqi j pharm sci, vol.31(1) 2022 anti-angiogenic of moringa oleifera leaves extract 226 moringa oleifera is rich in different types of phytochemicals which makes it as a good source of anti-cancer agents. furthermore, moringa oleifera leaves are proved to possess various medicinal properties, such as analgesic activity, antimicrobial activity, antidiabetic activity, antiinflammatory activity and antihypertensive activity (8–13) . the anti-cancer effect of moringa oleifera leaves was well-studied and established by certain researchers but studies regarding antiangiogenic effect of moringa oleifera are least (14,15). hence, this research was conducted to study the antiangiogenic effect of moringa oleifera leaves extract by in-ovo chick chorioallantoic membrane (cam) assay. materials and methods sample preparation and extraction moringa oleifera leaves powder with product name lohas® was purchased from a shop named kait lifestyle store located at desa parkcity, kuala lumpur. approximately 25 g of moringa oleifera leaves powder was dispersed in methanol and distilled water separately. the mixtures were boiled by placing over a water bath for two hours setting the temperature of the water bath closer to the solvent boiling point. aqueous extraction was done by placing the water bath at 80℃ and methanol extraction was done at 50℃. the mixtures were then filtered and the filtrate was concentrated by placing in an oven at 60℃ till complete solvent evaporation and dried extracts are obtained. the percentage yield was calculated and the dry extracts were stored separately with appropriate label in the refrigerator at 4 °c. preliminary qualitative analysis of phytochemicals preliminary phytochemical screening was done for both extracts by the following procedure (16,17) 1. alkaloid (dragendroff’s and wagner’s tests) approximately 1 ml of plant extract was added to 0.5 ml of 2% hydrochloric acid (hcl) then heated in a water bath for two minutes. the mixture was filtered and treated with three to four drops of dragendroff’s reagent. formation of orange-brown precipitate indicates presence of nitrogen containing compounds. about 1 ml of plant extract was treated with 2 ml of wagner’s reagent. reddish-brown precipitate was obtained indicating the presence of alkaloids. 2. flavonoids (alkaline reagent test) approximately 1 ml of plant extract was mixed with 0.5 ml of distilled water. the mixture was treated with 0.5 ml of 10% sodium hydroxide (naoh) solution. formation of yellow colour solution indicates presence of flavonoid. 3. saponins (foam test) approximately 1 ml of plant extract was added with 0.5 ml of distilled water in a test tube then closed with test tube stopper. the mixture was shaken vigorously for 5 minutes. formation of foam indicates presence of saponin. 4. tannins (ferric chloride test) approximately 1 ml of plant extract was heated on a water bath for two minutes then cooled. three drops of 10% ferric chloride (fecl3) solution were added into the extract. formation of dark green colour indicates presence of tannin. 5. steroids (salkowski test) approximately 1 ml of plant extract was added with 0.5 ml of chloroform. then 0.5 ml of concentrated sulfuric acid (h2so4) was added drop by drop on the sides of the test tube containing the mixture. formation of red or brown colour ring at the lower part of the chloroform layer indicates presence of steroid. preparation of treatment solutions the study was conducted among six treatment groups which included positive control (sunitinib), negative control (phosphate buffer saline), aqueous moringa oleifera extract (50% and 100%) and methanolic moringa oleifera extract (50% and 100%). phosphate buffer saline (pbs) was prepared according to the formula from the united states food and drug administration (fda) (18). the ph of pbs solution was measured using eutech instruments cyberscan ph 510 and adjusted to ph 7.4 by adding a few drops of naoh or hcl. the pbs solution was sterilised by autoclave at 121 °c for 15 minutes. sunitinib 0.01 μg/ml solution was prepared by serial dilution from the stock solution (100 mg/ml) using pbs. the different concentrations of extracts namely, 50% methanol extract, 100% methanol extract, 50% aqueous extract and 100% aqueous extract were prepared by dissolving concentrated extract in pbs. the required amount of concentrated extract was weighed using an analytical balance and the concentrated extract was dissolved in pbs. the mixture was stirred until the concentrated extract has fully dissolved and stored in the refrigerator at 4 °c. preparation of cam membrane for antiangiogenic screening fertilised chicken eggs were used in cam assay. the fertilised eggs were purchased from a local hatchery farm at klang. the eggs were cleaned by using wet sponge and tissues papers to remove any dirt, stain and marks on the surface of the shell. the incubator was disinfected with 70% alcohol to remove if any microbes present. incubator was pretreated for 3 days to ensure the temperature maintained inside falls within the range of 37.0 to 37.5 °c and humidity within the range of 55-60 %. the temperature and humidity of incubator were set at 37.5 °c and 55-60 % respectively (19). eggs were turned manually in horizontal direction three to four times daily for five days throughout the incubation period. this was done to iraqi j pharm sci, vol.31(1) 2022 anti-angiogenic of moringa oleifera leaves extract 227 increase the viability rate of the embryo and prevent the sticking of cam with the shell (20). egg candling (figure 1) was performed on the fourth day (day-4) of incubation by using a torchlight in a dark room to provide a clear visualization to identify the development of embryo. figure 1. egg candling air sac displacement and window grafting was done on the fifth day (day-5) of incubation (figure 2). to displace air sac, one small hole was made on the air sac area which is located at the blunt end of the egg and another small hole was made on the avascular area using a needle. then, air in the air sac area was sucked out through the small hole of blunt end by using a rubber pipette pump. figure 2. regions identified during embryo development. after successful air sac displacement, 1 cm x 1 cm square was drawn on the eggshell and cut with a penknife. the outer membrane located under the eggshell was gently ripped off with a forceps to expose cam layer (figure 3). then, the window was sealed with paraffin tape and returned to the incubator (21,22). figure 3. window for accessibility to cam. cam assay the eggs were divided into six groups and appropriate treatment solutions were applied on sixth day (day-6) of incubation. it was carried out in biosafety cabinet and all the apparatus required in the process were disinfected with 70% alcohol to minimize contamination. the treatment solutions were taken out from the refrigerator to reach room temperature before applying on the cam. the filter paper discs of 5 mm diameter were made using whatman filter paper. the discs were soaked into the treatment solutions and airdried to remove the excess solution. soaked and dried discs were applied onto the cam membrane labelled appropriately (figure 4). . figure 4. application of sample treated filter paper disc on cam. after applying sample discs, the cam was observed under a stereomicroscope and images were captured (figure 5). the windows were sealed with paraffin tape and returned to incubator for further incubation and changes of blood vessels in cam. the cam layer was further observed under stereomicroscope after 24-hours and 48-hours of sample application which was noted as post-24 hours and post-48 hours changes respectively. images of cam after 24 and 48 hours were captured for further analysis. iraqi j pharm sci, vol.31(1) 2022 anti-angiogenic of moringa oleifera leaves extract 228 figure 5. observation of cam under stereomicroscope. the percentage increase/ decrease in blood vessels for post-24 and post-48 hours of treatment were calculated using the formula(23). percentage increase/decrease in blood vessels = difference in number of blood vessels after treatment number of blood vessels before treatment x 100% one-way anova and post-hoc test analysis of data was done using statistical software spss version 26. results pharmacognostical parameters table 1. percentage yield, physical characteristics and phytochemical screening of moringa oleifera leaves extracts. aqueous extract methanol extract extractive yield (g%) 11.27 1.68 colour straw yellow dark green consistency of dried extracts fine powder coarse granules odour characteristic characteristic alkaloids (dragendroff’s test) present present alkaloids (wagner’s test) present present flavonoids (alkaline test) present present saponins (foam test) present present tannins (ferric chloride test) present present steroids (salkowski test) present present cam assay table 2. changes in chorioallantoic membrane after sample treatment. treatment group duration of treatment (hours) 0 24 48 negative control positive control iraqi j pharm sci, vol.31(1) 2022 anti-angiogenic of moringa oleifera leaves extract 229 counited table 2. changes in chorioallantoic membrane after sample treatment. figure 6. percentage reduction in blood vessels post-24 hours of treatment figure 7. percentage reduction in blood vessels post-48 hours of treatment discussion qualitative phytochemical testing revealed presence of various types of phytochemical components in both the extracts investigated (table 1). angiogenesis inhibition was assessed by counting the number of blood vessels. decrease in the number of blood vessels in the area of interest reveals the inhibition angiogenesis. the negative control group showed no reduction in blood vessels, as an alternative the number of blood vessels has increased in post-24 and post-48 hours of treatment contributing to percentage increase in blood vessels of about 22.59% and 36.83% respectively. therefore, pbs which was used as solvent for dilution of extracts does not show any effect on angiogenesis and did not interfere with the normal angiogenesis process in the embryo. hence, it can be concluded as pbs does not cause any damage to the cell and will not affect the cell growth (24). sunitinib is an angiogenesis inhibitor, approved by u.s food and drug administration. it is a multitargeted receptor tyrosine kinase (rtk) inhibitor which mainly inhibits the pdgf receptor and vegf receptor which is important in angiogenesis progression (25). ic50 of sunitinib for angiogenesis suppression was found to be 0.01μm treatment group duration of treatment (hours) 0 24 48 aqueous extract50% aqueous extract100% methanol extract50% methanol extract100% iraqi j pharm sci, vol.31(1) 2022 anti-angiogenic of moringa oleifera leaves extract 230 (26) and the same concentration of sunitinib was selected to compare the antiangiogenic potential of moringa oleifera extracts. hence, sunitinib served as positive control sample in this study. different concentrations of moringa oleifera leaves extract (50% and 100%) showed reduction in the number of blood vessel on the sample treated region on cam after 24 and 48 hours of treatment (table 2). it was observed that aqueous extract with 100% concentration has the highest percentage reduction in blood vessels after 24 hours and 48 hours of treatment compared to other treatment groups. the treatment group with the weakest anti-angiogenesis effect was 50% methanol extract which has the lowest percentage reduction of blood vessels in post-24 hours and post-48 hours of treatment (figure 6 and 7). the one-way anova test result and posthoc test result for post-24 hours of treatment showed an insignificant difference in post-24 hours of treatment. but for the one-way anova test result of post-48 hours there was statistically significant difference (p<0.05). the anti-angiogenesis activity was found to increase with the increase of concentration of both aqueous and methanol extracts. this showed that moringa oleifera leaves extracts possess concentration-dependent anti-angiogenesis effect. this research findings has encountered an agreement with the study of pachava et al. (23) and dharani et al. (27) that proposes the anti-angiogenesis properties of moringa oleifera leaves were dosedependent manner. the main flavonoids found in moringa oleifera leaves were myricetin, kaempferol and quercetin (28). kaempferol has been reported to possess anticancer activity against pancreatic cancer cells by suppressing cell proliferation and inducing cancer cell apoptosis (29). in addition to the above mentioned mechanism, kaempferol also suppresses angiogenesis (30). luo et al. have reported that kaempferol causes angiogenesis inhibition in cam. further, he also concluded that kaempferol also responsible for inhibition of vegf expression in ovarian cell lines (31). moreover another study has also reported anti-angiogenesis effect of kaempferol through regulated vegf and fgf signalling pathway (32). furthermore, another flavonoid, quercetin has been reported to show anticancer activity in various types of cancer (33). the mechanism of quercetin includes inhibition of cell proliferation, inhibition of angiogenesis, promote apoptosis in cancer cell and altering the cell cycle progression (34). one of the study reported quercetin to have anti-angiogenesis effect by targeting vegf2 receptor thereby resulting in downregulation of akt/mtor/p70s6k signaling pathway that contributes to restrain of tumour growth (35). in summary, both flavonoids were found to suppress vegf receptor that leads to anti-angiogenesis activity. qualitative analysis of the extracts in the present study also revealed presence of flavonoids which could have contributed for the antiangiogenic property of moringa oleifera leaves. conclusion this study reports anti-angiogenesis effect of methanolic and aqueous extract of moringa oleifera leaves screened by in-ovo cam assay. different phytochemicals including flavonoids, alkaloids, saponins, tannins and steroids are present in both the extracts. in present study, 100% aqueous moringa oleifera leaves extract has shown significant (p<0.05) reduction in the 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2020;121:109604. 35. pratheeshkumar p, budhraja a, son yo, wang x, zhang z, ding s, et al. quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting vegfr2 regulated akt/mtor/p70s6k signaling pathways. plos one. 2012 oct;7(10):e47516. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 mucoadhesive clotrimazole vaginal hydrogel 19 preparation and in-vitro evaluation of mucoadhesive clotrimazole vaginal hydrogel basma i. abdul –aziz * and nawal a. rajab **,1 ⃰ national center for drug quality control and research, ministry of health, baghdad, iraq. ⃰⃰ ⃰ department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract clotrimazole (clo) is an antimycotic imidazole derivative applied locally for the treatment of vaginal yeast infections. in this study, clo was formulated as vaginal mucoadhesive hydrogel, using different types of mucoadhesive polymers to ensure prolonged contact between active ingredient and vaginal mucosa. physicochemical properties of the prepared formulas were evaluated as a visual inspection, ph, swelling index, spreadability, and mucoadhesive characteristics, in addition to an in-vitro drug release. the influence of type and concentration of polymers as cmc-na (1.5, 2.5, and 3.5%w/w), carbopol 940( 0.25, 0.5, and 1 %w/w) and poloxamer 407 (15, 25, 30%w/w) on clo release from the prepared gels were also investigated. all the different concentrations of each polymer gave a percent of drug release profile inversely proportional with the polymer concentration. the lowest concentration showed faster release within the first half an hour of 99± 2% of drug release , while cmc-na at 3.5% (f2) demonstrated the least release of 40 % after three hours. on the other hand, poloxamer 407 of 25% gel (f7) produced the highest release of 98%, while carbopol 1% (f5) and cmc-na (f1) produced 85% and 80% ,respectively. all the prepared formulas were following higuchi kinetic model. based on overall result, clo can be formulated as mucoadhesive vaginal hydrogel using 25%poloxamer as the best prepared formula. key words: hydrogel, clotrimazole, mucoadhesive. كهىتريًازول بشكم هالو يائي باستخذاو قىاعذ نًادة تحضير و تقييى خارج انجسى انحي هبهيًيخاطيت االنتصاق نالستخذاو ان 1،** نىال عياش رجبو *عًاد عبذ انعسيسبسًت * .انًشكض انٕطُي نهشقابت ٔانبحٕد انذٔائيت ,انعشاق , بغذاد ** . قسى انصيذالَياث, كهيت انصيذنت , صايعت بغذاد, انعشاق , بغذاد انخالصت فطشياث انًٓبم . في ْزِ انذساست حى يعخبش عقاس انكهٕحشيًاصٔل ٔاحذا يٍ يشخقاث االيًيذاصٔل يسخخذو يٕضعيا في عالس ححضيش انكهٕحشيًاصٔل بصيغت ْالو يائي نّ انقابهيتعهٗ االنخصاق بانضذساٌ انًخاطيت . االَخفاخ,قابهيت االَخشاس َسبت ،انكيًيائيت يزم)انٕصف انخاسصي ,االط انٓيذسٔصيُي-حى حقيى انصيغ يٍ حيذ انخٕاص انفيضيأيت االنخصاق(باالضافت انٗ حقيى ححشس انذٔاء خاسس انضسى .حًج دساست انعٕايم انًؤرشة في ححشسانذٔاء كُٕع ٔحشكيض انقٕاعذ ٔقابهيت 25-15) ٔ 040 (% يٍ انكاسبابٕل 1 25,0 -5,0(% يٍ انكاسبٕكسي يزيم سيهيهٕصصٕديٕو ٔ)5,3 5,2 -5,1انضالحيُيت ) .404 (% يٍ انبٕنٕكساييش30 في صًيع انصيغ انًحضشة باخخالف يع صيادة حشكيض انبٕنيًشعكسيا حخُاسب بصٕسة عايتكهٕحشيًاصٔل ت ححشس كًيٌ ا ثٔقذ ظٓش % خالل َصف انساعت االٔنٗ نهضيغ رٔاث انخشاكيض انقهيهت نكافت انبٕنيًشاث 2±00حشاكيضْا, حيذ اٌ ححشس انذٔاء كاٌ بًعذل % خالل رالد ساعاث . يٍ انُاحيت االخشٖ , اٌ 40بًقذاس %5,3م سهيهٕص صٕديٕو ,بيًُا ابطئ ححشس في حشكيبت كاسبٕكسي يزي 040انكاسبابٕل ى%( بعذ يشٔس ساعخيٍ ٔيٍ ر09اظٓشث اعهٗ ححشسنهذٔاء) %(25) 404انبٕنٕكساييش انخشكيبت انحأيت عهٗ %(.اٌ ححشسانذٔاءيٍ انٓالو انًائي نكافت انصيغ انًحضشة حخضع نقإٌَ 90صٕديٕو) يزيم سيهيهٕص انكاسبٕكسي ٔاخيشا %(95) انعانى ْيضٕشي . % انبٕنٕكساييش25بُاءا عهٗ انُخائش انًسخحصهت اربج باٌ دٔاء انكهٕحشيًاصٔل يًكٍ حشكيهّ بصيغت ْالو يائي يٓبهي باسخخذاو 404. هالو يائي ، كهىتريًازول ، االنتصاق انًخاطي :انكهًاث انًفتاحيت introduction vaginal delivery as a route of drug administration is currently of a great interest to scientists and pharmaceutical industry (1) . many different approaches have been tested to develop vaginal drug delivery system (dds) that can meet clinical and patient requirements (2) . considerable attention has been focused on the development of vagina l (dds) that providing a gradual release of drug over time. one interesting group of auxiliary agent is the mucoadhesive polymers, which are the basis of newly designed system. the most widely used mucoadhesive vaginal (dds) is (hydrogel) (3, 4) .hydrogel is one of the 1 corresponding author e-mail:nawalayash@yahoo.com received:24 /11/2013 accepted:4 /3/2014 iraqi j pharm sci, vol.23(1) 2014 mucoadhesive clotrimazole vaginal hydrogel 20 upcoming classes of polymer-based controlled release (dds) due to several factors among them are their hydrophilicity, biocompatibility mucoadhesive characteristics (5) . also gels are easy to manufacture, comfortable, and have ability to spread onto the surface of mucosa to achieve an intimate contact with vaginal mucosa. moreover, because of their high water content and their rheological properties, they provide hydration and lubrication action (6) . clotrimazole is an imidazole derivative with a broad spectrum of antimycotic activity, the drug is a weak base described as a white to pale yellow crystalline powder, practically insoluble in water, freely soluble in chloroform and methanol (7, 8) .the objective of this study is to formulate 1% clotrimazole vaginal mucoadhesive hydrogel using three different types of polymers with different concentration of each.the physicochemical characteristics of gel and mucoadhesive property of selected formulas were also evaluated. materials and method materials clotrimazole and methyl paraben were supplied by (samara drug industry), sodium carboxy-methylcellulose (bdh chemicals ltd, pool, england), cabopol 940(hi media lab.,ltd, mumdai, india), poloxamer 407. (sigma, germany ), monobasic potassium phosphate (fluka , switzerland), tween 80 (merk-schuncherdt, germany). method preparation of clotrimazole vaginal gels: nine formulas of clo hydrogel were prepared by cold mechanical method (9) as shown in (table 1). pre weighed amount of polymers was dispersed in sufficient quantity of distilled water (containing 1% tween 80), the dispersion was homogenized using magnetic stirrer for 1 hour and then left to for 24 hours for complete swelling and equilibration of polymer. weighted amount of clo and methyl paraben were added with continuous stirring. in case of carbopol 940 containing gel, [ specific amount of triethanolamine (tea) drop by drop was added with continuous mixing] and the final weight was completed to 100g with the aqueous solution. the formulas were placed in refrigerator to complete the formation of hydrogel (10) . table (1) composition of clotrimazole vaginal hydrogels formulas (%w/w). * sodium carboxymethyl cellulose ** triethanol-amine evaluation of the prepared hydrogel formulas visual examination the examination considered a series of visual characteristics (consistency, homogeneity) (11) . ph determination the ph of the prepared gel was measured using ph – meter by putting the tip of the electrode into the gel and after 2 minutes the result was recorded (12). swelling study one gram sample from each formula was soaked into 5ml of phosphate buffer ph 4.0 and left for a specific time, and then the excess buffer was removed and reweights the samples again. this test done for time intervals after one and three hours. the following formula will be used to calculate the swelling ratio. swelling ratio= ws-w0 / w0 × 100 where ws is the weight of the swollen hydrogel at time t and wº is the initial weight (13) . distilled water methyl paraben tween 80 tea* * poloxamer 407 carbopol 940 cmc-na* clotrimazol e formula no. up to 100 0.4 1 ------3.5 1 f1 up to 100 0.4 1 ------2.5 1 f2 up to 100 0.4 1 ------1.5 1 f3 up to 100 0.4 1 2.0 --1.0 --1 f4 up to 100 0.4 1 1.0 --0.5 --1 f5 up to 100 0.4 1 0.5 --0.25 --1 f6 up to 100 0.4 ----30 ----1 f7 up to 100 0.4 ----25 --1 f8 up to 100 0.4 ----15 ---1 f9 iraqi j pharm sci, vol.23(1) 2014 mucoadhesive clotrimazole vaginal hydrogel 21 spreadability measurement spreadability was measured on the basis of“slip” and “drag” characteristics of gels. 1.0gm of gel was placed on the center of ground slid plate and spread over an area of 1 cm diameter. the gel was then sandwiched between two slides by the application of 200 gm weight. the spread diameter was recorded after 5 minutes, and measured in cm. this measured area was taken as comparative values for spreadability, (diameter of the spread circle – initial diameter) (12) . estimation of drug content by hplc analysis the content of clo in prepared formulas was determined using hplc analysis method stated in the united state pharmacopeia (usp) for clo assay (8) . hplc from knauer ,germany was utilized. a specified quantity of the gel equivalent to 50 mg was stirred using ultrasound with about 30 ml of the diluents [a mixture of 25% phosphate buffer (4.35mg/ml of k2hpo4) and 75% methanol] and then the volume was made up to 50 ml with same diluents. the above solution was further diluted to obtain solution having concentration of 0.2mg/ml. the chromatographic system was carried out using the following specifications: -column: 4.6-mm x25 –cm; 5µm packing l1. -mobile phase: acetonitrile and buffer (3: 1), filtered and degassed. (buffer: 4.35mg/ml of dibasic potassium phosphate) -detector: uv 254nm. -flow rate: 1.5 ml/min. -injection size: 20 µl. mucoadhesive strength measurement the mucoadhesive strength of selected formula was determined by measuring the weight required to detach the gel from the sheep vaginal mucosal tissue by using a modified chemical balance. a section of vaginal mucosa was cut from the sheep’s vaginal cavity and instantly fixed with mucosal side out onto each glass vial using a rubber band. the vials with vaginal mucosa were stored at 37°c for five minutes; the vial was attached to the balance instead of one of the pans by a height-adjustable hook. next, glass plate was placed under the mucosal tissue .the gel was placed on the glass plate; then, the height of the vial was adjusted so that the gel could touch the mucosal tissue which was allowed to adhere for three minutes to the gel (preload time) (14) . on the other side of the balance, a plastic cup was placed to collect water, and balanced with the other side. water was added drop by drop to the plastic cup until the weight of water in the cup detached the glass slide from the mucosal tissue. the detachment stress (dyne/cm 2 ), was determined from the minimal weights that detached two surfaces from each other, was calculated using the following equation f= 980 m/π r 2 where f is the detachment force (dyne/cm 2 ) per unit area of mucosa (cm 2 ) (π r 2 in which r is the radius of the vial), (r =1.2 cm) by the balance weight m (g) and 980 is acceleration due to gravity (cm/sec 2 ). in-vitro dissolution test the in vitro release of clo from all hydrogel formulas was performed by using dissolution apparatus type 2 (paddle type).a weighing quantity of a gel ( 10 gm that contain 100 mg clo ) was uniformly spread on petri dish 4.5cm in diameter, and this was immersed in dissolution jar filled with 900ml dissolution media ( phosphate buffer ph 4.0 b.p. containing 2% tween 80 ) at 37˚c (15) . the paddle was placed about 2cm above the petri dish and rotated at speed of 50 rpm. samples of 5ml were withdrawn at intervals of 30, 60, 120, and 180 minutes and were replaced with equal volume of the fresh buffer solution each time to maintain constant volume. samples were analyzed for clo by hplc (16). variable affecting release profile effect of different polymer concentration on the release profile of clotrimazole the effect of different concentrations of the used polymer on the release profile of the drug was studied; all formulas were subjected to this study. effect of different types of polymers on the release profile of clotrimazole one formula was selected from each polymer cmc-na, carbapol 940 and poloxamer 407 ( the highest release profile ). comparion between these formulas were achieved to show the effect of the polymer on the release. release kinetics to analyze the mechanism and rate of drug release from prepared gel, the dissolution data obtained were fitted to kinetic equations including zero order, first order, higuchi and korsmeyerpeppas. r 2 value was used to show the best fit model to be selected (17). iraqi j pharm sci, vol.23(1) 2014 mucoadhesive clotrimazole vaginal hydrogel 22 results and discussion macroscopic feature: visual inspection of prepared gel indicated the homogeneity of all formulas, no phase separation, with semi-rigid texture and white to off white smooth gel. this result is similar to that defined in references (7, 18). ph the ph of all formulas were almost neutral as show in table (2 ). table (2) ph of prepared formulas swelling index swelling of the polymer depends on the concentration of the polymer, degree of cross linking, ionic strength and presence of water (18). increasing in crosslinker concentration shows a direct negative effect due to tighter structure (19) . tablet (3) shows that f1 and f2 started swelling rapidly and continued for three hours due to high amount of hydroxyl group in each cellulose unit which contributing in presences of hydrogen bound (20) . f4 shows less swelling index at the beginning but polymer when fully hydrated swelled in phosphate buffer and surface disintegration of the gel occurred at the end of three hours (21). the swelling ratios of ionic hydrogels were often an order of magnitude higher than those of neutral gels because of the presence of intermolecular interactions. (f7-f9) being contain neutral gel have no swelling appearance may be due to full hydration of the polymer (20, 22). table (3) swelling ratio of prepared formulas swelling ratio w/w after 3 hours ± s.d. swelling ratio w/w after 1 hour formula no. 80% ± 1.8% 50% ± 1% f1 150% ± 0. 23% 45% ± 1. 2% f2 50% ± 0.5% 0% f3 30% ± 0.65% 8% ± 0.3% f4 --f5 --f6 --f7 --f8 --f9 - : swelling ratio is negligible because the polymer used is water soluble (20) . spreadability measurement all prepared gels using different concentrations of different polymers were spreadable on the tissue surface as show in table (4). spreadibilty of prepared gels was decreased as the polymer concentration increased as in formula (f1,f5,f7). it should be mentioned that the addition of polysorbate 80 improved the physical characteristics including spreadability, consistency and skin feel duo to an increase in the diameter of the spread circle of the gel (23) . table (4) spreadability values of prepared gels. formula no. f1 f2 f3 f4 f5 f6 f7 f8 f9 spread-ability cm ± s.d 1.5 ±0.1 2 ±0.08 3 ±0.14 1.5 ±0.08 2.2 ±0.1 2.8 ±0.12 0.4 ±0.02 0.6 ± 0.04 3 ±0.15 mucoadhesive strength measurement the mucoadhesive strength of the prepared gels was affected by polymer type and it was found in the following order: poloxamer 407 >carbopol 934 > cmc na (f8, f4, and f2) respectively as illustrated in table (5). this is due to the fact that mucoadhesive strength depends on the polymer structure, molecular weight, and other physicochemical properties which are varied according to the polymer type (24, 25) . table (5) result of force required for detachment formula no. f2 f4 f8 detachment stress (dyne/cm 2 ) ±s.d. 4009.6 ± 2.7% 5310.1 ± 3.5% 6783.8 ± 4.3% ph formula no. 6.4 ± 0.10 f1 6.4 ± 0.11 f2 6.2 ± 0.08 f3 7.3 ± 0.06 f4 7.4 ± 0.05 f5 7.1 ± 0.08 f6 7.3 ± 0.07 f7 7.0 ± 0.10 f8 7.1 ± 0.085 f9 iraqi j pharm sci, vol.23(1) 2014 mucoadhesive clotrimazole vaginal hydrogel 23 content uniformity a validated hplc method was used for determination of clo content in the gels. table 6 indicated that the results obtained were within the limit of usp monograph (90-110) % of the theoretical content of the prepared gel (8) . table (6) drug content uniformity formula no. f1 f2 f3 f4 f5 f6 f7 f8 f9 % of original amount od drug ± s.d. 103 ± 0.20 104 ± 0.30 99 ± 0.80 95.2 ± 0.23 97.5 ± 1.00 101 ± 1.20 98.8 ± 0.43 97.6 ± 0.66 99 ± 0.50 effect of polymer concentration on the in vitro release it was found in formulas f1, f2 and f3 that the amount of drug release from cmc-na hydrogel was decreased with increasing polymer concentration as in figure (1). this result could be attributed to an increase in the density which leads to increase in diffusional path way of drug from more sticking cellulose matrix (26). figure (2) illustrates the difference in percent of drug release which was attributed to the mesh size of the network. the mesh size depends on the grade of crosslinking and the chemical structure of the monomers, so the diffusivity of the incorporated drug was effected (27). the hydrogel with low degree of crosslinking (f6) was fast released. on the other hand, gradual release of drug occurred in f4 due to the tighter network (28) . figure 1: effect of cmc-na concentration on the release of clotrimazole from three different formulas. figure 2: effect of different carbopol 940 concentration on the release of clotrimazole from three different formulas. figure (3) showed that the increase in polyxamer 407 concentration led to decrease the release rate (f7f9). it is hypothesized that the drug is released by diffusion through the extra micellar water channels of the hydrogels matrix, and higher concentration of polyxamer 407 causes smaller size of water channels (29). figure 3: effect of different poloxamer 407 concentration on the release of clotrimazole from three different formulas. effect of polymer types on in vitro release three formulas of different types of polymers which show best release profile were chosen for this study and illustrated in figure (4). poloxamer 407 (f8) performed higher release with control rate when compared with other formulas. this due to the mechanism of drug delivery via poloxamer which occurred by diffusion and dissolution of the gel at administered site. poloxamer presented as complete dissolution in three hours, and this feature can be used to prepare pharmaceutical formulation. the amount of clo released, was found in the following order: poloxamer ˃carbopol and cmc, this may be due to higher viscosity which was responsible for hindering drug release from gel matrix (30) . figure 4: effect of different polymers on the clotrimazole release from different formulas. iraqi j pharm sci, vol.23(1) 2014 mucoadhesive clotrimazole vaginal hydrogel 24 figure 4: effect of different polymers on the clotrimazole release from different formulas. mathematical modelling of drug release profile according to the results obtained and shown in table (7), the release of clo from polymer network forming hydrogel for formulas f1-f3, f4, f6 and f9 obeys higuchi release kinetic as there (r 2 )values gave high results, while f4, f7, and f8 follow zero-order kinetics. the mechanism of release depending on the composition of hydrogel, geometry, preparation technique and environmental conditions during drug release (17). table (7) mathematical finding of clotrimazole release from repared gels. formula no. r 2 krossmeyr-peppas n zero order first order higuchi f1 0.979 0.861 0.997 0.22 f2 0.982 0.845 0.990 0.221 f3 0.730 0.733 0.961 f4 0.999 0.861 0.973 0.1 f5 0.831 0.754 0.992 f6 0.730 0.733 0.962 f7 0.997 0.891 0.986 0.216 f8 0.999 0.876 0.974 0.167 f9 0.734 0.734 0.970 conclusion the present study demonstrates that the clo confirm a promising vaginal mucoadhesive hydrogel since it provided highest release profile with maximum adhesion property especially for poloxamer 407 of 25% (f8), in addition to the acceptable physiochemical parameter such as 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"development of miconazole nitrate thermosensitive bioadhesive vaignal gel for vaginal candidiasis". american journal of advanced drug delivery 2013; 1(3): 358368. iraqi j pharm sci, vol.21(1) 2012 treatment of idiopathic male infertility 14 effect of l-carnitine, multivitamins and their combination in the treatment of idiopathic male infertility # jehan j. aram * , kassim j. al-shamma **,1 and ansam n. al-hassani * * college of pharmacy, hawler medical university, erbil, iraq. ** college of pharmacy, university of baghdad, baghdad, iraq. abstract the aim of the present study is to investigate and compare the efficacy of l-carnitine, multivitamins and their combination therapies on semen characteristics in idiopathic male infertility. idiophathic infertile patients were randomly divided into three groups who had received three different treatment regimens for three months: group a (45 patients) has received 2 grams daily of l-carnitine alone; group b (55 patients) had received the combination of l-carnitine (2 grams daily) plus one tablet daily of multivitamins (stresstabs ® ); and group c (29 patients) had received one tablet daily of multivitamins alone. the study was started on 1/11/2009 and completed on 31/3/2010 and performed at rizgari teaching hospital in hawler city/erbil governorate. thirty fertile male volunteers were used as a control group as well. seminal fluid analysis has been done before treatment and then monthly after treatment for three month. the results of the present study clearly demonstrated that the combination therapy of l – carnitine and multivitamins was more efficient than each drug therapy alone and this evidenced by improvement and significant increase in the semen parameters; sperm concentration (72%), sperm count (79%), actively motile sperm (29%), and progressive motile sperm count (125%) when compared to pretreatment values than either l – carnitine or multivitamins therapy alone (53%, 60%, 19% and 84%), (21%, 30%, non significant, 34%) respectively.the results demonstrated that the combination therapy of l-carnitine and multivitamins was more efficient and produced more significant improvement in semen characteristic than either therapy alone, and that lcarnitine therapy was more efficient than multivitamins.improvement in these semen parameters can aid in the treatment of idiopathic male infertility. key words : l-carnitine, multivitamins, infertility. كارَتيٍ وانفيتاييُاث انًتعددة وكالهًا سىيت نعالج يزض عقى انذكىر يجهىل انسبب -تقييى نـ جيهاٌ جالل آراو * ، قاسى جهيم انشًاع **،1 اَساو َاجي انحسُي و * .* كهٛح انصٛذنح ، خايؼح ْٕنٛش انطثٛح ، استٛم ، انؼشاق كهٛح انصٛذنح ، خايؼح تغذاد انطثٛح ، تغذاد ، انؼشاق** الخالصة نًرؼذدج أٔكالًْا يؼا ػهٗ كاسَرٍٛ أٔانفٛرايُٛاخ ا -ْذف ْزِ انذساسح ْٕ انرحمك ػٍ ٔ يماسَح كفاءج ػالخاخ نـ إٌ خصائص انسائم انًُٕ٘ )حدى انسائم انًُٕ٘، ذشكٛض انحٛايٍ، ػذد انحٛايٍ، شكم انحٛايٍ، حشكح انحٛايٍ، ػذد انحٛايٍ راخ انحشكح أشٓشح ذحسٍٛ ْزِ انخصائص خالل ثالث إنٗانًرمذيح، ٔػذد انخالٚا انًذٔسج( .فٙ ػمى انزكٕس يدٕٓل انسثة فٙ يحافظح استٛم، ْادفح فٙ ْزِ يٍ انؼالج ٔالكرشاف انطشٚمح انصحٛحح نرمذٚى انثٛاَاخ انًخرثشٚح نرحهٛم انسائم انًُٕ٘ ٔانرٙ ٚرى ػًهٓا فٙ يحافظح استٛم. نًذج األخشثالثح يدايٛغ كم يُٓا اخز ػالج ٚخرهف ػٍ إنٗيشٚض ) ٔانؼمى يدٕٓل انسثة ( تطشٚمح ػشٕائٛح 921انذساسح ذى ذمسٛى يسرًشج: شأشٓثالثح كاسَرٍٛ ٕٚيٛا، -غشاو يٍ نـ 2يشٚض ( ذُأنٕا 54) –أ –انًدًٕػح كاسَرٍٛ يغ حثح ٔاحذج يٍ انفٛرايُٛاخ انًرؼذدج ) سرشٚسراتض -غشاو يٍ نـ 2يشٚض ( ذُأنٕا 44) –ب –انًدًٕػح ® .) سرشٚسراتض يشٚض ( ذُأنٕا حثح ٔاحذج يٍ انفٛرايُٛاخ انًرؼذدج ) 21) –ج –انًدًٕػح ® .) يرطٕع ركش خصة. نمذ ذًد يراتؼح انًشضٗ 03) انكَٕرشٔل ( فٙ ْزِ انذساسح ٔانرٙ ذضًُد أخشٖٔلذ ذى اخز ٔذسدٛم يدًٕػح ( رٔ انًُٕرج t. ْزا ٔلذ ذى اسرخذاو فحص )أشٓشٔذى ذحهٛم انسائم انًُٕ٘ لثم انثذء تأخز انؼالخاخ ٔشٓشٚا خالل اخز انؼالج نًذج ثالثح يٍ انؼالج، ٔلذ اػرثشخ لًٛح احرًانٛح انصذفح أشٓشخٙ نهًماسَح تٍٛ انمٛى لثم انؼالج ٔانمٛى انًماتهح نٓا تؼذ شٓش ٔشٓشٍٚ ٔثالثح انضٔ (p راخ )كاسَرٍٛ انًسرخذو -نـ إٌ. نمذ أظٓشخ َرائح ْزا انثحث تصٕسج ٔاضحح 3034إحصائٛح ػُذيا ذكٌٕ لًٛرٓا الم يٍ أًْٛح ٚحسٍ ذشكٛض انحٛايٍ، ػذد انحٛايٍ، َسثح انحٛايٍ انًرحشكح تصٕسج فؼانح، أٌيسرًشج يًكٍ أشٓشٛا نًذج ثالثح غشاو ٕٚي 2تدشػح ٔػذد انحٛايٍ راخ انحشكح انًرمذيح ػُذ انشخال انزٍٚ ٚؼإٌَ يٍ انؼمى ) لهح ذشكٛض انحٛايٍ أ لهح سشػرٓا أ كالًْا( يدٕٓل انسثة، كاسَرٍٛ ٔ انفٛرايُٛاخ انًرؼذدج نًذج انؼالج َفسٓا ْٕ اكثش كفاءج ) حصٕل صٚادج يًٓح احصاءٚاً فٙ -ٔاٌ انؼالج انز٘ ٚدًغ تٍٛ نـ % تانرؼالة يماسَح يغ انمٛى انًماتهح نٓا لثم انؼالج ( يٍ 924%، 21%، 21%، 22تًمذاس أػالِخصائص انسائم انًُٕ٘ انًزكٕسج %، غٛش يٓى 03%، 29ٚاً فٙ خصائص انسائم انًُٕ٘ انًزكٕسج اػالِ تًمذاس كاسَرٍٛ نٕحذِ ) حصٕل صٚادج يًٓح احصاء -ػالج نـ ٚساػذ فٙ أٌ% تانرؼالة يماسَح يغ انمٛى انًماتهح نٓا لثم انؼالج ( نرحسٍٛ خصائص انسائم انًُٕ٘ ْزِ ٔيٍ انًًكٍ 05احصاءٚاً، يٍ ػالج يدًٕػح انفٛرايُٛاخ أفضمٚكٌٕ أٌٍ كاسَرٍٛ يٍ انًًك -نزنك، ػالج نـ تاإلضافحػالج ػمى انشخال يدٕٓل انسثة. َظاو انؼالج انز٘ ذى ذطثٛمّ فٙ ْزِ انذساسح فٙ ذحسٍٛ خصائص انسائم انًُٕ٘ ػالج ػمى انشخال يدٕٓل انسثة. تاسرخذاوانًرؼذدج كارَتيٍ ، قيتاييُاث ، عقى . -انكهًاث انًفتاحيت : نـ # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1corresponding author email : drkassim_alshamaa@yahoo.com received : 22/3/2011 accepted : 15/11/2011 iraqi j pharm sci, vol.21(1) 2012 treatment of idiopathic male infertility 15 introduction male infertility means the male is unable to impregnate the female because of male factors.it represents 25-40% of the total causes of infertility (1) .although the etiology of male infertility is not clear in most cases, different treatment options have been suggested to increase sperm count and motility (2) . these treatment options include general, medical and surgical management. medical treatment includes gonadotrophins, androgens, corticosteroids, antibiotics, alpha1sympathomimetics, anticholinergics, antiestrogens, aromatase inhibitors, and alternative therapy which is considered the safest among the treatment options and include antioxidants (multivitamins) and l-carnitine (3) . carnitine is a zwitterionic amino acid (3hydroxy-4-trimethylamino-butyic acid). it is found in different food items and derived endogenously from lysine and methionine (2) . l-carnitine is an essential cofactor that could accelerate lipid metabolism and has a pivotal role in mitochondrial β-oxidation of long-chain fatty acids for cellular energy production (4,5) . l-carnitine and l-acetylcarnitine are highly concentrated in the epididymis and play a crucial role in sperm metabolism and maturation (6) .the redox system in the spermatozoa regulates the processes that are crucial for fertilization (7) , but increased reactive oxygen species (ros) observed in semen of infertile men might cause cellular damage and this have brought about the widespread use of antioxidants (8) . many vitamins such as vitamin c, vitamin e, vitamin b12, and many other antioxidants were used to improve sperm quality for the treatment of idiopathic oligoasthenozoospermia (9) . in addition, sperm concentration was increased in a number of studies on subfertile men after treatment with zinc and folic acid (10, 11) . the aim of the present study is to investigate and compare the efficacy of l-carnitine, multivitamins and their combination therapies on semen characteristics (seminal fluid volume, sperm concentration, sperm count, sperm morphology, sperm motility, progressive motile sperm count and round cells count) in idiopathic male infertility. patients and methods settings: this randomized interventional study has been accomplished cooperatively with the laboratory of the department of pharmacology in the college of pharmacy/ hawler medical university(hawler medical university is besiderizgari teaching hospital) , the laboratory of rizgari teaching hospital, dr. hunar abdul qadir's clinic, and shai and luay laboratories in hawler city / erbil governorate/ iraqi kurdistan region. the practical work of this research has been started on 1/11/2009 and completed on 31/3/2010. the research was authorized and approved by the board and ethical committee of hawler medical university, erbil, iraq. subjects and patients control group:included 30 fertile male volunteers diagnosed by the urologist, their ages ranged between 21 to 38 years; the results of their seminal fluid analyses were within the normal values and this test was done and recorded for them one time only. patients' inclusion criteria : were infertile or subfertile young males diagnosed by the urologist with age range between 20 to 42 years, absence of leukocytospermia in the semen samples taken before the start of the treatment period, and absence of endocrinopathies or known etiology or female infertility factors. protocol: all 129 patients had received their treatments for 3 months. group a (45 patients) had received 2 grams per day of l-carnitine (nature’s bounty, inc./usa) in 4 divided doses. group b (55 patients) had received 2 grams per day of l-carnitine in 4 divided doses plus 1 tablet daily of multivitamins with zinc (stresstabs ® weyth healthcare/ canada ). group c (29 patients) had received 1 tablet of stresstabs ® daily.seminal fluid analysis was done and recorded for each patient before treatment and monthly during the treatment. collection of semeninal fluid: samples were collected following a period of sexual abstinence from 2 to 3 days to no longer than 7 days. most of the patients had collected their semen samples in the laboratory and analysis was performed within 30 minutes by the same examiner. average readings were taken. questionnaire paper contained the following questions were prepared and answered by the patients and the controls. 1. the name of the patient. 2. the age of the patient and his wife. 3. mobile number. 4. years of marriage. 5. years of infertility. 6. number of children. 7. smoker or alcoholic. 8. past and recent medical history. 9. past and recent surgical history. 10. past and present illnesses. iraqi j pharm sci, vol.21(1) 2012 treatment of idiopathic male infertility 16 11. weight of the patient (if obese then look for the cause). 12. the type of the diet. 13. the occupation of the patient. 14. exposure to toxins like lead, mercury, pesticides, or radiation. 15. presence of female infertility factors. 16. the date of starting the interventional treatment. 17. the assigned dates of the sperm analysis during the treatment. 18. the assigned date of ending the interventional treatment. laboratory procedures: seminal fluid analysis done (according to strasinger and dilorenzo, 2008 (12) , who 2010 (13) ) had included the following: recording the semen color, odor, viscosity, liquefaction, and ph of seminal specimen; measurement of seminal fluid volume, sperm count and concentration, sperm morphology, sperm motility (percentage of active, sluggish, immotile sperms, and progressive motile sperm count, (sperm count x percent active motile count), and round cell count. statistical analysis: all the seminal fluid and sperm parameters in all of the groups were represented as the mean ± the standard error (se). paired sample t-test was applied to compare between the values before treatment and their corresponding values after 1, 2, and 3 months of treatment. p values of < 0.05 were considered statistically significant. *p<0.05 represents significant difference between before treatment and after treatment. different letters represent significant difference between controls and the patients before treatment. results twenty idiopathic infertile patients were dropped out during the treatment before we had reached to the above final numbers of participated patients. group a patients table 1: effects of l-carnitine treatment on the mean ± se of seminal fluid volume, sperm concentration, and sperm count in idiopathic infertile patients(n=45) compared to controls (n=30). patients/controls seminal fluid volume (ml) sperm concentration (million/ml) sperm count (million/ ejaculate) controls 2.63 ± 0.13 63.26 ± 10.08 a 168.96 ± 31.82 a patients before treatment 2.63 ± 0.20 20.08 ± 1.59 b 51.42 ± 5.41 b 1 month after treatment 2.67 ± 0.15 24.82 ± 2.42 * 67.23 ± 7.21 * 2 month after treatment 2.66 ± 0.14 28.82 ± 3.23 * 74.16 ± 8.37 * 3 month after treatment 2.74 ± 0.17 30.88 ± 2.63 * 82.41 ± 8.78 * *p<0.05 represents significant difference between before treatment and after treatment. different letters represent significant difference between controls and the patients before treatment. table 2: effects of l-carnitine treatment on the mean ± se of sperm motility (represented as percent of total) in idiopathic infertile patients(n=45) compared to controls (n=30). patients/controls active motile sperm (%) sluggish motile sperm (%) immotile sperm (%) progressive motile count (million/ejaculate) controls 51.50 ± 1.59 a 31.16 ± 0.94 17.33 ± 1.21 a 91.10 ± 20.97 a patients before treatment 30.11 ± 2.52 b 33.44 ± 1.85 36.44 ± 3.51 b 18.02 ± 2.40 b 1 month after treatment 30.66 ± 2.58 35.00 ± 1.74 34.33 ± 3.24 23.88 ± 3.82 2 month after treatment 33.55 ± 2.42 * 34.44 ± 1.48 32.00 ± 2.94 * 27.42 ± 4.09 * 3 month after treatment 35.77 ± 2.70 * 34.77 ± 1.55 29.44 ± 3.30 * 33.22 ± 4.77 * *p<0.05 represents significant difference between before treatment and after treatment. different letters represent significant difference between controls and the patients before treatment. iraqi j pharm sci, vol.21(1) 2012 treatment of idiopathic male infertility 17 group b patients table 3: effects of l-carnitine and multivitamins treatment on the mean ± se of seminal fluid volume, sperm concentration, and sperm count in idiopathic infertile patients(n=55) compared to controls (n=30). patients/controls seminal fluid volume (ml) sperm concentration (million/ml) sperm count (million /ejaculate) controls 2.63 ± 0.13 63.26 ± 10.08 a 168.96 ± 31.82 a patients before treatment 2.48 ± 0.14 12.96 ± 1.13 b 30.58 ± 2.86 b 1 month after treatment 2.52 ± 0.12 18.41 ± 1.65 * 44.98 ± 4.43 * 2 month after treatment 2.48 ± 0.10 20.07 ± 1.50 * 51.05 ± 4.58 * 3 month after treatment 2.45 ± 0.08 22.43 ± 1.57 * 54.99 ± 4.30 * *p<0.05 represents significant difference between before treatment and after treatment. different letters represent significant difference between controls and the patients before treatment table 4: effects of l-carnitine and multivitamins treatment on the mean ± se of sperm motility (represented as percent of total) in idiopathic infertile patients(n=55) compared to controls (n=30). patients/controls active motile sperm(%) sluggish motile sperm(%) immotile sperm (%) progressive motile count (million /ejaculate) controls 51.50 ± 1.59 a 31.16 ± 0.94 17.33 ± 1.21 a 91.10 ± 20.97 a patients before treatment 22.81 ± 2.22 b 32.58 ± 1.77 44.60 ± 3.18 b 8.02 ± 1.05 b 1 month after treatment 25.63 ± 2.20 34.36 ± 1.39 40.18 ± 2.84 13.40 ± 2.03 * 2 month after treatment 26.63 ± 2.02 * 36.00 ± 1.17 * 37.34 ± 2.42 * 15.15 ± 1.96 * 3 month after treatment 29.36 ± 2.27 * 35.81 ± 1.46 34.90 ± 3.15 * 18.03 ± 2.19 * *p<0.05 represents significant difference between before treatment and after treatment. different letters represent significant difference between controls and the patients before treatment table 5: effects of multivitamins treatment on the mean ± se of seminal fluid volume, sperm concentration, and sperm count in idiopathic infertile patients(n=29) compared to controls (n=30). patients/controls seminal fluid volume (ml) sperm concentration (million/ml) sperm count (million/ ejaculate) controls 2.63 ± 0.13 63.26 ± 10.08 a 168.96 ± 31.82 a patients before treatment 2.43 ± 0.17 20.37 ± 1.11 b 47.68 ± 3.61 b 1 month after treatment 2.39 ± 0.14 22.37 ± 1.31 * 52.72 ± 4.44 2 month after treatment 2.46 ± 0.14 23.89 ± 1.78 * 57.68 ± 4.97 * 3 month after treatment 2.50 ± 0.11 24.72 ± 1.70 * 62.17 ± 5.29 * *p<0.05 represents significant difference between before treatment and after treatment. different letters represent significant difference between controls and the patients before treatment table 6: effects of multivitamins treatment on the mean ± se of sperm motility (represented as percent of total) in idiopathic infertile patients(n=29) compared to controls (n=30). patients/ controls active motile sperm (%) sluggish motile sperm (%) immotile sperm (%) progressive motile count (million/ ejaculate) controls 51.50 ± 1.59 31.16 ± 0.94 17.33 ± 1.21 91.10 ± 20.97 a patients before treatment 46.06 ± 1.47 34.24 ± 1.39 19.68 ± 1.65 21.81 ± 1.66 b 1 month after treatment 45.68 ± 1.12 33.96 ± 1.39 20.34 ± 1.40 24.29 ± 2.28 2 months after treatment 46.03 ± 1.43 34.13 ± 1.26 19.82 ± 1.45 26.52 ± 2.58 * 3 months after treatment 46.89 ± 1.72 33.44 ± 1.21 19.65 ± 1.71 29.29 ± 3.19 * *p<0.05 represents significant difference between before treatment and after treatment. different letters represent significant difference between controls and the patients before treatment iraqi j pharm sci, vol.21(1) 2012 treatment of idiopathic male infertility 18 table 7: percentages of increase in sperm concentration, sperm count, actively motile sperm, and progressive motile sperm count measured after 3 months of treatment when compared with their corresponding values before treatment in group a, b, and c patients. parameter measured percentage of increase in the semen parameters measured after 3 months when compared with values before treatment group a patients group b patients group c patients sperm concentration 54% 73% 21% sperm count 60% 80% 30% active sperm motility 19% 29% non significant (ns) progressive motile sperm count 84% 125% 34% no significant differences were found between the values of round cell count in the patients of different groups before and after treatment and those of the control subjects. the above results had demonstrated that l-carnitine at a daily dose of 2 gm/day for a treatment period of 3 months can improve sperm concentration, sperm count, percentage of actively motile sperm, and progressive motile sperm count among men with idiopathic oligoand/or asthenozoospermia, and the combination of l-carnitine and multivitamins for the same period of treatment is more efficient than either l-carnitine or multivitamins alone(in the form of stresstabs®) for the improvement in these semen parameters and can aid in the treatment of idiopathic male infertility. in addition, lcarnitine may be better than the use of oral multivitamins in the dosage regimens applied in this study in improving semen parameters and treatment of idiopathic male infertility. moreover, the use of the progressive motile sperm count in the seminal fluid analysis is more indicative for the improvement in semen parameters than the sperm count and percentage of actively motile sperms separately, since it represents the product of multiplying the sperm count by the percentage of actively motile sperm. discussion other similar studies have suggested an improvement in semen parameters of men with low sperm motility following treatment with oral carnitine (14, 15, 2) . these observations suggest a role of carnitine or acetylcarnitine as empiric therapy for idiopathic asthenospermia (16) .the significant effect of l-carnitineon increasing sperm concentration, sperm count, and percentage of actively motile sperm in this study could be due to or explained by of which is that l-carnitine is an essential cofactor that could accelerate lipid metabolism and has a pivotal role in mitochondrial β-oxidation of long-chain fatty acids for cellular energy production (4,5) . there is also an unknown effect of l-carnitine in sertoli cell– spermatogenic line interaction, an action on the postmeiotic phases of spermatogenesis (for example, on the chromatin stability or mitochondrial function of spermatocytes or spermatids), or an improvement in the quality of the epididymal microenvironment, reducing gamete phagocytosis at this level while increasing ejaculated spermatozoa (17) . in addition, carnitine administration increases prostaglandin e2 concentration (18) , which affects sperm count (19) . moreover, carnitine protects cell membrane and dna against damage induced by free oxygen radicals. it also prevents protein oxidation and lactate oxidative damage (20, 9) . hence, it acts as an “anti-aging” substance, protecting against damage induced by free oxygen radicals (17) .in the same manner, there was a significant increase in the progressive motile sperm count, since it is a product of sperm count and the percentage of active motile sperm. a number of studies have suggested a beneficial role of antioxidants or anti-ros drugs (vitamin e, vitamin c and gsh), including improved sperm quality and increasing fertilizing capacity (3) .vitamin e is one of the major membrane protectants against ros and lipid peroxidation (21) . because vitamin e is a chainbreaking antioxidant and not a scavenging antioxidant, it would be expected to offer protection to membrane components without influencing ros generation (22) . vitamin e, on the other hand, acted as an effective antioxidant and significantly enhanced the capacity of human spermatozoa for spermoocyte fusion. vitamin b12 and folic acid are important in cellular replication, especially for the synthesis of rna and dna, and deficiency states have been associated with decreased sperm count and motility (23) . vitamins b1, b2, b3, b5, and b6 are important in many metabolic pathways in cells in general (and sperm in particular), and any deficiency in these vitamins can lead to defects in sperm metabolism and normal iraqi j pharm sci, vol.21(1) 2012 treatment of idiopathic male infertility 19 functions (24) .although pregnancy was not a principal end point in many studies (15, 14) , including this study, as it is difficult to avoid the many confounding variables acting on naturally induced fertilization and subsequent pregnancy, one pregnancy was recorded in group b patients after 2 months of treatment. in conclusion the present study had demonstrated that l-carnitine at a daily dose of 2 gm/day for a treatment period of 3 months can improve sperm concentration, sperm count, percentage of actively motile sperm, and progressive motile sperm count among men with idiopathic oligoand/or asthenozoospermia, and the combination of lcarnitine and multivitamins for the same period of treatment is more efficient than either l-carnitine or multivitamins alone references 1. yao m.w.m., and schust d.j. infertility. berek j.s, editor. novak's gynecology. 13 th edition. usa: lippincott williams and wilkins.2002; pp: 973-1048. 2. peivandi s., karimpour a., and moslemizadeh n. effects of l-carnitine on infertile men's spermogram: a randomized clinical trial. j reprodfertil;2010; 10(4): 41-44. 3. schiff j.d., ramirez m.l., and bar-chama n. medical and surgical management of male infertility. endocrinolmetabclin n am; 2007; 36: 313-331. 4. jeulinc., and lewin l.m. role of free lcarnitine and acetyl-l-carnitine in postgonadal maturation of mammalian spermatozoa. hum reprod update; 1996; 2: 87-102. 5. kerner j., and hoppel c. genetic disorders of carnitine metabolism and their nutritional management. annu rev nutr;1998; 18: 179-206. 6. agrawal a., and said t.m. carnitines and male infertility. j urol;2005; 174(4 pt 1): 1369. 7. leclerc e., de lamirande e., and gagnon c. regulation of protein-tyrosine phosphorylation and human sperm capacitation by reactive oxygen derivatives. free radicbiol med;1997; 22: 643-656. 8. aitken r.j.the human spermatozoon-a cell in crisis.j reprodfertil;1999;115: 1-7. 9. zhou x., liu f., and zhai s. effect of lcarnitine and/or l-acetylcarnitine in nutrition treatment for male infertility: a systematic review. asia pac j clinnutr; 2007;16(suppl 1): 383-390. 10. wong w.y., merkus h.m., thomas c.n., menkveld r., zielhuis g.a., and steegerstheunissenr.p.. effects of folic acid and zinc sulphate on male factor subfertility: a double blind, randomized, placebocontrolled trial. fertilsteril; 2002; 77: 491498. 11. ebisch i.m., pierik f.h., de jong f.h., thomas c.m.g., and theunissen s.r.p.m.. does folic acid and zinc sulphate intervention affect endocrine parameters and sperm characteristics in men? int j androl; 2006;29(2): 339-345. 12. strasinger s.k. and dilorenzom.s.. semen. urinalysis and body fluid 5 th edition . usa: f.a. davis company. 2008; pp: 199 – 208 13. world health organization (who). who. laboratory manual for the examination and processing of human semen. 2010;5 th edition switzerland: who press. 14. arnaud a., schved j. f., gris j. c., costa p., navratil h., and humeauc.. tissue-type plasminogen activator level is decreased in human seminal plasma with abnormal liquefaction.fertilsteril; 1994;61: 741-745. 15. vitali g., parente r., and melottic..carnitine supplementation in human idiopathic asthenospermia: clinical results. drugs expclin res; 1995; 21: 157159. 16. sigman m., glass s.r.n., campagnone j.m.s., and pryor j.l..carnitine for the treatment of idiopathic asthenospermia: a randomized double-blind, placebocontrolled trial. fertilsteril; 2006; 85: 1409-1414. 17. lenzia., lombardo f., sgro p., salacone p., caponecchia l., dondero f., et al.. use of carnitine therapy in selected cases of male factor infertility: a double-blind crossover trial. fertilsteril;2003; 79(2): 292-300. 18. vicari e., and calogeroa.. effects of treatment with carnitines in infertile patients with prostate-vesiculoepididymitis. hum reprod; 2001;16: 23382342. 19. cavallini g., ferraretti a.p., gianaroli l., biagiotti g., and vitali g. cinnoxicam and l-carnitine/acetyl-l-carnitine treatment for idiopathic and varicocele-associated oligoasthenospermia. j androl; 2004;25: 761-770. 20. arduinia..carnitine and its acyl esters as secondary antioxidants? am heart j; 1992; 123: 1726-1727. 21. halliwell b., and gutteridgej.m.c.. free radicals in biology and medicine. 2 nd iraqi j pharm sci, vol.21(1) 2012 treatment of idiopathic male infertility 20 edition. uk: clarendon press;1989; 253259. 22. sharma r.k., pasqualotto f.f., nelson d.r., thomas a.j., and agrawala.. relationship between seminal white blood cell counts and oxidative stress in men treated at an infertility clinic. j androl ; 2001;22: 575-583. 23. sinclair s. male infertility: nutritional and environmental considerations. altern med rev; 2000; 5(1): 28-38. 24. champe p.c., harvey r.a., and ferrier d.r. lippincott's illustrated reviews: biochemistry. 4 th edition. china: lippincott williams and wilkins. 2008;pp: 181-200, 373-394. iraqi j pharm sci, vol.23(2) 2014 new β-adrenoceptor blockers 42 synthesis of new β-adrenoceptor blocking agent including 1,3,4 thiadiazole with expected adrenoceptor blocking activity salah h. zain al-abdeen *,1 and ahlam j. qasir ** * tall'afar general hospital, nineva health directorate, ministry of health, tall'afar , iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract β-adrenergic blocking agents, mostly comprising of β-amino alcohols, are of pharmaceutical significance and have received major attention due to their utility in the management of cardiovascular disorders including hypertension, angina pectoris, cardiac arrhythmias and other disorders related to the sympathetic nervous system. most compounds available for clinical use belong to the aryloxypropanolamine series, which is considered the second generation of β-blocking agents. the present study includes the synthesis of compounds with an n-substituted oxypropanolamine moiety attached to the 1, 3, 4-thiadiazole derivatives. according to this information, eight compounds were synthesized and characterized by ir spectra and elemental microanalysis that confirmed the structural formula of these compounds. keywords: β-adrenoceptor blockers, 1,3,4-thiadiazole, schiff base ثايادايازول -4,3,1مركباث جديدة كمثبطاث نمستهماث بيتا األدرينانيت متضمنا حهقت تحضير درينانيتكمثبطاث ا ذاث فعانيت متوقعت صالح حسن زين انعابدين *،1 رو احالو جميم قصي ** * . انؼشاق ، حهؼفش،وصاسة انصحت ،صحت َُُىي دائشة ،يسخشفً حهؼفش انؼاو ** . انؼشاقبغذاد ، ،جايؼت بغذاد ،كهُت انصُذنت ،فشع انكًُُاء انصُذالَُت . الخالصة راث أهًُت دوائُت ػانُت ولذ حظُج باهخًاو ,انكحىالث األيُُُتوانخٍ حخأنف يؼظًها يٍ ,يزبطاث يسخهًاث بُخا األدسَُانُت حؼخبش ػذو اَخظاو , انزبحت انصذسَت, كبُش َظشا نفائذحها فٍ انخذبُش انؼالجٍ الضطشاباث انمهب واألوػُت انذيىَت بًا فٍ رنك اسحفاع ضغط انذو وفشة نهؼالس انسشَشٌ حُخًٍ انً اٌ أكزش انًشكباث انًج. اوٌدضشباث انمهب وغُشها يٍ االضطشاباث انًخؼهمت بانُظاو انؼصبٍ انسًب اٌ انبحذ انًمذو َشخًم ػهً حخهُك .صُف اسَم اوكسٍ بشوباَىل ايٍُ انخٍ حؼخبش يٍ انجُم انخاٍَ نًزبطاث يسخهًاث بُخا األدسَُانُت فمذ , نًؼهىياثاػخًادا ػهً هزِ ا .راَاداَاصول -4,3,1 بًشخماث حهمتيشكباث فُها يجًىػت اوكسٍ بشوباَىل يؼىضت االيٍُ يشحبطت حى حصُُغ رًاٌ يشكباث وحشخُصها بىاسطت االشؼت ححج انحًشاء وانخحهُم انذلُك نهؼُاصش وانخٍ اربخج صحت حشاكُب انًشكباث .انًحضشة .ثايا دايازول ، قواعد شيف – 4, 3,، ا ةمثبطاث مستهماث بيتا االدريناني :انكهماث انمفتاحيت introduction β-adrenergic receptors (β-ars) are g protein-coupled receptors (gpcrs) that mediate physiological responses to adrenaline and noradrenaline. these receptors are the molecular targets for some of the most commonly prescribed drugs in the history of medicine. there are three receptor subtypes in this family: β1-ar is found at its highest levels in the heart and brain (1) , β2-ar is more widely expressed in bronchial and vascular smooth muscle cells (2) , and β3-ar is found at its highest levels in adipose tissue (3) . all three receptors couple primarily to gαs to stimulate adenylyl cyclase, but can also couple to gαi in some cells under certain conditions (4) . there is an additional βadrenoceptor subtype which has been identified in cardiac tissue and is a putative, atypical subtype classified as the β4 adrenoceptor ( 5) . likewise, β-adrenoceptor antagonists have also been classified as β1 and β2-blockers. most of the developed β-blockers belongs either to arylethanolamine or aryloxypropanolamine classes, where the aryloxypropanolamines are more potent β blockers than the corresponding arylethanolamines, and most of the β-blockers currently used clinically are aryloxypropanolamines (6) . in the current study it was aimed to replace the aryl nucleus of β adrenoceptor blocker by heterocyclic derivatives (5-amino-1, 3, 4-thiadiazole-2-thiol derivative) as an isostere in an attempt to improve β1 affinity by synthesizing the following compounds which are: 1 corresponding author e-mail:salah_hart@yahoo.com received: 20/1/2014 accepted: 6/7/2014 iraqi j pharm sci, vol.23(2) 2014 new β-adrenoceptor blockers 43 1-(4-methylpiperazin-1-yl)-3-(4-((5-(4nitrobenzylthio)-1,3,4-thiadiazol-2ylimino)methyl) phenoxy)propan-2-ol. compound 13 1-(2,6-dimethylpiperidin-1-yl)-3-(4-((5-(4nitrobenzylthio)-1,3,4-thiadiazol-2ylimino)methyl) phenoxy)propan-2-ol. compound 14 1-(4-((5-(4-nitrobenzylthio)-1,3,4-thiadiazol-2ylimino)methyl)phenoxy)-3-(piperidin-1-yl) propan-2-ol. compound 15 1-(4-((5-(4-nitrobenzylthio)-1,3,4-thiadiazol2-ylimino)methyl)phenoxy)-3-(pyrrolidin-1yl) propan-2-ol. compound 16 1-(4-((5-(4-chlorobenzylthio)-1,3,4-thiadiazol2-ylimino)methyl)phenoxy)-3-(4methylpiperazin -1-yl)propan-2-ol. compound 17 1-(4-((5-(4-chlorobenzylthio)-1,3,4-thiadiazol2-ylimino)methyl)phenoxy)-3-(2,6-dimethyl piperidin-1-yl)propan-2-ol. compound 18 1-(4-((5-(4-chlorobenzylthio)-1,3,4-thiadiazol2-ylimino)methyl)phenoxy)-3-(piperidin-1-yl) propan-2-ol. compound 19 1-(4-((5-(4-chlorobenzylthio)-1,3,4-thiadiazol2-ylimino)methyl)phenoxy)-3-(pyrrolidin-1yl) propan-2-ol. compound 20 thiadiazole ring: thiadiazole is a heterocyclic compound containing two nitrogen atom and one sulfur atom as part of the aromatic five-membered ring. they occur in nature in four isomeric forms as 1,2,3-thiadiazole (i); 1,2,4-thiadiazole (ii); 1,2,5-thiadiazole (iii) and 1,3,4thiadiazole (iv). 1, 3, 4-thiadiazole are important because of their versatile biological actions which exhibits a wide variety of biological activities such as cns depressants (7) , hypoglycemic (8) ,antinflammatory (9) , antimicrobial (10) ,antihypertensive (11) , antifungal (12) ,anticancer (13) and antioxidant activity (14) . among them 2,5-disubstituted 1,3,4thiadiazoles are associated with diverse biological activity probably virtue of –n=c-s grouping. thiadiazole moiety acts as a “hydrogen binding domain” and “two-electron donor system”. thiadiazoles carrying mercapto, amino substituents can exists in many tautomeric forms (15) and considered as useful intermediates in organic synthesis. moreover, these groups can react with electrophiles, for instance some alkylation or schiff base reactions that take place at sor n atom (16) . schiff bases have gained great importance because of physiological and pharmacological activities associated with them. there has been considerable attention to the chemistry of schiff bases because of their wide range of applications in many fields including biological, organic, inorganic and analytical chemistry. they are used as pigments, dyes, catalysts, intermediates in organic and inorganic synthesis and polymer stabilizers (17) . schiff bases containing heterocyclic rings are known to show cytotoxic, anticonvulsant, antimicrobial, anticancer, antifungal, antimalarial, antiviral, antidepressant and enzyme inhibitor activities (18) . furthermore, some schiff bases are used in ion sensors and electrochemical sensors to empower detection with enhanced selectivity and sensitivity (19) . experimental work chemicals and equipments acetic anhydride, carbon disulfide, pchlorobenzyl chloride, p-nitrobenzyl chloride, n-methyl piprazine, piperidine, pyrrolidine, phydroxy benzaldehyde, thiosemicarbazide, 2,6 dimethylpiperidine. all the solvents and materials used were of analar type and used without further purification. infrared spectral determination was performed for all compounds in kbr disk, using ftir at the college of pharmacy, university of baghdad. elemental analysis has been done using carlo erba elemental analyzer euro vector, and was done at al-al bayt university in al-mafraq (jordan). chemical synthesis synthesis of heterocyclic ring 5-amino-1,3,4thiadiazole-2-thiol (compound 1) (20) to thiosemicarbazide (0.1 mole, 10gm) suspended in anhydrous ethanol (40 ml), anhydrous sodium carbonate (0.054 mole, 5.82 gm) was added together with carbon disulfide (0.12 mole, 10.1 gm). the reaction mixture was heated with stirring under reflux for (5 hr.). the completion of the reaction was indicated by tlc. the solvent was largely removed under reduced pressure by using rotary evaporator. the residue was dissolved in water (44 ml), and acidified with concentrated hydrochloric acid 37% (8.8 ml). the product was recrystallized from ethanol/water to give the pure compound. the physical appearances, percentage yield, melting point, and rf values are listed in table (1). synthesis of series a compounds synthesis of n-(5-mercapto-1,3,4-thiadiazol2-yl)acetamide (21) .(compound 2) a mixture of (0.022 mole, 3g ) of compound 1 and (0.11 mole, 10ml) of acetic iraqi j pharm sci, vol.23(2) 2014 new β-adrenoceptor blockers 44 anhydride containing (0.5ml) of concentrated sulphuric acid was heated in a steam-bath for 1hr. later the mixture was poured into (60 ml) of cold water and the mixture was boiled to decompose the excess of acetic anhydride. when cold, filter the residual insoluble acetyl derivative and wash with little of cold water and recrystalize from distilled water or aqueous ethanol. the physical appearances, percentage yield, melting point and rf value were listed in table (1). table (1): the physical appearances, percentage of yield, melting points, rf values of the intermediate and final compounds . a) chloroform: ethylacetate: methanol (2:2:1), b) toluene: ethylacetate:formic acid(5:4:1) c) n-hexane: isopropanol: methanol (9: 0.9:0.1), d) n-hexane: isopropanol: methanol (8.5: 1.2:0.3) synthesis of n-[5-(4-nitrobenzylthio)-1,3,4thiadiazol-2-yl]acetamide (22) .(compound 3) to a mixture of compound (2) (1mmole ,0.175g) and p-nitrobenzyl chloride (1mmole, 0.1715g) in ethanol (15ml), koh(1.1mmole, 66mg in 5 ml of h2o) was added drop wise while the mixture was stirred at room temperature overnight. then water (20 ml) was added and the separated solid was filtered off, washed with h2o, and crystallized from ethanol: water (90:10).the physical appearances, percentage yield, melting point and rf value were listed in table (1). synthesis of n-[5-{(4-chlorobenzyl)thio}1,3,4-thiadiazolyl] acetamide.(compound 4) the same procedure was carried out as mentioned previously for compound (3), but starting with p-chlorobenzyl chloride (1mmole, 0.161g) which was coupled with compound (2)(1mmole, 0.175g ).the physical appearances, percentage yield, melting point and rf value were listed in table (1). synthesis of 5-(4-nitrobenzylthio)-1,3,4thiadiazol-2-amine (23) . (compound 5) a mixture of (0.01mole, 3.1g) compound (3), 6ml of concentrated hcl and 40ml ethanol was refluxed in oil bath for 3hrs. after a part of ethanol had evaporated, the obtained suspension was allowed to cool slowly. the solid mass was filtered and crystallized from water. the physical appearances, percentage yield, melting point and rf value were listed in table (1). synthesis of 5-[(4-chlorobenzyl)thio]-1,3,4thiadiazol-2-amine. (compound 6 ) the same procedure was carried out as mentioned previously for compound (5) but starting with compound (4) (0.01mole, 3g). the physical appearances, percentage yield, melting point and rf value were listed in table (1). synthesis of (e)-4-[{(5-(4-nitrobenzyl)thio)1,3,4-thiadiazol-2-ylimino]methyl phenol.(compound 7) the mixture of compound (5) (0,002mole, 0.53g) and p-hydroxybenzaldehyde (0.0023mole, 0.29g) in 25ml ethanol with 2 drops of concentrated h2so4 was refluxed for 6 hr. then, the solution was concentrated and kept overnight bellow 20˚c. the solid mass separated was filtered and washed with hot water: ethanol (1:1, 20ml). recystallization of the product from 95% ethanol was carried out. the physical appearances, percentage yield, compound no. physical appearance %yield melting point (˚c) rf values observed reported a b c d 1 faint yellow crystal 69% 232-234 230-232 0.8 0.67 ---- 2 pale yellow powder 93% 295-296 ------- 0.76 0.41 ---- 3 off-white powder 58.9% 252-254 ------- 0.74 0.64 ---- 4 pale yellow powder 60% 209-211 ------- 0.88 0.71 ---- 5 pale yellow powder 73.9% 168.5-170 ------ 0.67 0.4 ---- 6 white powder 76% 161-163 162-164 0.93 0.49 ---- 7 yellow powder 40% 193-196 ------ 0.42 0.62 ---- 8 bright yellow powder 41% 202-204 ------ 0.53 0.71 ---- 13 orange powder 28% 143-146 ---------- 0.23 0.26 14 dark yellow powder 28.3% 99-102 ---------- 0.20 0.24 15 grey yellow powder 26% 109-112 --------- 0.21 0.23 16 yellow powder 33.5% 110-113 --------- 0.18 0.2 17 dark yellow powder 38.7% 146-148 --------- 0.24 0.34 18 yellow powder 42.6% 148-151 --------- 0.21 0.3 19 grey yellow powder 43.8% 150-152 --------- 0.23 0.31 20 dark yellow powder 39.6% 136-138 --------- 0.19 0.28 iraqi j pharm sci, vol.23(2) 2014 new β-adrenoceptor blockers 45 melting point and rf value were listed in table (1). synthesis of (e)-4-((5-(4-chlorobenzylthio)1,3,4-thiadiazol-2-ylimino)methyl phenol.(compound 8) the same procedure was carried out as mentioned previously for compound (7), but starting with compound (6) (0.002mole, 0.51g) which was coupled with phydroxybenzaldehyde (0.0023mole, 0.29g). the physical appearances, percentage yield, melting point and rf value were listed in table (1). the overall reaction steps are shown in scheme (i) as shown bellow synthesis of series b compounds (epichlorohydrin – substituted amines ) this series include the following compounds:  1-chloro-3-(piperidin-1-yl)propan-2-ol (compound 9)  1-chloro-3-(2,6-dimethylpiperidin-1yl)propan-2-ol (compound10)  1-chloro-3-(pyrrolidin-1-yl)propan-2-ol (compound11)  1-chloro-3-(4-methylpiprazin-1-yl)propan-2ol (compound 12) these compounds (9-12) were synthesized by mixing equimolar quantities of epichlorohydrin and amines including (piperidine, 2,6dimethylpiperidine, pyrrolidine, nmethylpiprazine ) in methanol at room temperature (not exceeding 25 ˚c) for (48 hrs.). the resulting chloropropanolamines table (2) (9-12) were stored in refrigerator and were used without further purifications (24) . iraqi j pharm sci, vol.23(2) 2014 new β-adrenoceptor blockers 46 table (2): synthesis of series b compounds (9-12) synthesis of final products (compounds (1320)) the synthesis was accomplished by the oalkylation of the final compound of series a (compound 7 & 8) with those of series b compounds including (9-12). as in the following procedure: to a stirred solution of one of the series a (7, 8) compounds (0.01 mol.) in methanol containing potassium hydroxide (0.01mol.), a solution of one of series b compounds (9-12) (0.012 mol.) was added drop wise using a dropping funnel. the reaction mixture was refluxed on steam bath for (5-9 hrs.). the mixture was cooled to room temperature and the volume was then reduced under vacuum. the crude product was precipitated by gradual addition of water, collected and washed several times with water. crystallization of all solid products was achieved using absolute ethanol (25) .the physical appearances, percentage yield, melting point and rf value were listed in table (1), the elemental analysis results are presented in table (3), while the ir data are shown in table (4). results and discussion the 2amino – 5 – mercapto -1 ,3 , 4 thiadiazole was synthesized through steps of reactions starting from thiosemicarbazide with carbon disulphide in a basic medium. in the acetylation of compound (1), where this step includes the synthesis of amide; it was done by treatment of an amine with acetic anhydride in the presence of a few drops of sulphuric acid as catalyst. conversion of the amino group into the acetamido group by acetylation modifies the interaction of the nitrogen lone pair with the π-electron system of the aromatic ring so that the ring is less powerfully activated toward electrophilic attack. alkylation of compound (2) which includes the synthesis of thioether or alkyl sulphide , it was done by treatment of a thiol with a base, such as koh, giving the corresponding thiolate ion (rsˉ).which undergoes reaction with a primary or secondary alkyl halide to give a sulfide. the reaction occurs by an sn2 mechanism, analogous to the williamson synthesis of ethers. thiolate anions are among the best nucleophiles known, and product yields are usually high in these sn2 reactions. then the amide group of compound (3&4) is hydrolyzed (amide hydrolysis) by heating in either aqueous acid or aqueous base mediums. acid hydrolysis reaction occurs by nucleophilic addition of water to the protonated amide, followed by transfer of a proton from oxygen to nitrogen to make the nitrogen a better leaving group and for subsequent elimination. the steps are reversible with the equilibrium shifted towards the product by the protonation of the nh2 in the final step. reaction of compounds (5&6) with aromatic aldehyde under acidic conditions afforded the corresponding schiff bases. the mechanism of schiff base formation is a reversible, acid catalyzed process, begins with nucleophilic addition of the primary amine to a carbonyl group (aldehyde or ketone) followed by a transfer of a proton from nitrogen to oxygen to yield a neutral amino alcohol or carbinolamine. protonation of the carbinolamine oxygen by an acid catalyst then converting the (-oh) group into a better leaving group (–oh2 + ), and e1like loss of water produces an iminium ion which after the loss of a proton from nitrogen gives the schiff base and regenerate the acid catalyst to afford series a compounds. the compounds from 2-7 & 8 were reported earlier in the literature but they were synthesized without protecting the amino or thiol groups present in compound 2 since both -sh &-nh2 are susceptible for the reagent cl-ch2-c6h4-x, in addition the compound amine used chemical structure of series b 9 piperidine 10 2,6-dimethylpiperidine 11 pyrrolidine 12 n-methylpiprazine iraqi j pharm sci, vol.23(2) 2014 new β-adrenoceptor blockers 47 synthesized products from 2-7&8 have different melting point and without recording their elemental analysis. this study has taken care of the susceptibility of the amino group and it was therefore protected. furthermore, all synthesized compounds were with different melting points, in addition, the final compounds synthesized have elemental analysis which comply with the expected theoretical data. while the preparation of series b compounds was accomplished by mixing equimolar amounts of epichlorhydrin with primary or secondary amines in methanol at room temperature (not exceeding 25 ˚c) for 48 hrs. in the reaction of amines and epichlorhydrin, a considerable variety of products may be obtained by varying temperature, molar ratio of reactants, reaction media, and basicity of the amine. in the synthesis of the final compounds actually includes the synthesis of an ether. it was done by heating an alkyl halide with the salt of an alcohol (e.g. potassium alkoxide).the procedure is recognized as an adaptation of the williamson method for the synthesis of ethers. the reactions are therefore often run as a twostep process with pre-formation of the alkoxide salt and subsequent addition of the alkyl halide (sn2 reaction). table (3): elemental microanalysis of the final compounds (13-20) compound molecular weight chemical formula % elemental microanalysis element calculated observed 13 528 c24h28o4n6s2 c 54.545 54.475 h 5.30 5.556 n 15.90 16.368 o 12.121 11.551 s 12.121 12.05 14 541 c26h31o4n5s2 c 57.67 58.21 h 5.73 5.84 n 12.939 12.5 o 11.829 11.66 s 11.829 11.79 15 513 c24h27o4n5s2 c 56.14 56.560 h 5.263 5.361 n 13.645 13.184 o 12.475 12.417 s 12.475 12.478 16 499 c23h25o4n5s2 c 55.31 55.81 h 5.01 5.11 n 14.02 13.61 o 12.825 12.6 s 12.825 12.87 17 517.5 c24h28o2n5cls2 c 55.652 57.142 h 5.41 4.865 n 13.526 13.50 s 12.367 12.808 18 530.5 c26h31o2n4cls2 c 58.812 59.999 h 5.843 5.628 n 10.556 9.819 s 12.064 12.015 19 502.5 c24h27o2n4cls2 c 57.313 58.361 h 5.373 4.70 n 11.144 11.418 s 12.736 12.670 20 488.5 c23h25o2n4cls2 c 56.499 56.61 h 5.117 5.335 n 11.463 11.39 s 13.1 13.135 iraqi j pharm sci, vol.23(2) 2014 new β-adrenoceptor blockers 48 table (4): characteristic ir absorption of the final compounds compound band(cm -1 ) interpretation 13 3296.35 3107.32 2966.52,2947.23,2839.22 1681.93 1633.71-1598.99 1575.84 1508.33, 1342.46 1240.23, 1049.28 1178.51, 1014.56 894.97, 858.32 oh stretching of oxypropanolamine side chain. ch stretching of aromatic ring. ch asymmetrical and symmetrical stretching vibration of ch3 &ch2. c=n stretching of side chain (schiff base). c=n stretching of heterocyclic ring aromatic c=c stretching asymmetrical & symmetrical n-o stretching respectively. asymmetrical & symmetrical c-o stretching of ether. aromatic ch in-plane bending. aromatic ch out of plane bending. 14 3286.70 3076.46 2931.80, 2868.15,2854.65 1687.71 1598.99 1573.91 1517.98, 1344.38 1247.94, 1053.13 1192.01 858.32, 800.46 oh stretching of oxypropanolamine side chain. ch stretching of aromatic ring. ch asymmetrical and symmetrical stretching vibration of ch3 &ch2. c=n stretching of side chain (schiff base). c=n stretching of heterocyclic ring aromatic c=c stretching asymmetrical & symmetrical n-o stretching respectively. asymmetrical & symmetrical c-o stretching of ether. aromatic ch in-plane bending. aromatic ch out of plane bending. 15 3302.13 3109.25 2929.87, 2854.65 1685.79 1597.06 1571.99 1508.33, 1344.38 1236.37, 1055.06 1188.15, 1014.56 858.32, 800.46 oh stretching of oxypropanolamine side chain. ch stretching of aromatic ring. ch asymmetrical and symmetrical stretching vibration of ch2. c=n stretching of side chain (schiff base) c=n stretching of heterocyclic ring. aromatic c=c stretching asymmetrical & symmetrical n-o stretching respectively. asymmetrical & symmetrical c-o stretching of ether aromatic ch in-plane bending. aromatic ch out of plane bending. 16 3284.77 3076.46 2962.66 1676.14 1598.99 1571.99 1514.12, 1344.38 1244.09, 1051.20 1190.08, 1014.56 893.04, 858.32 oh stretching of oxypropanolamine side chain. ch stretching of aromatic ring. ch asymmetrical stretching vibration of ch2. c=n stretching of side chain (schiff base). c=n stretching of heterocyclic ring. aromatic c=c stretching asymmetrical & symmetrical n-o stretching respectively. asymmetrical & symmetrical c-o stretching of ether aromatic ch in-plane bending. aromatic ch out of plane bending 17 3311.78 3101.54 2937.59, 2827.64 1681.93 1600.92 1577.77-1500.62 1238.30, 1060.85 1296.16, 1014.56 1091.71 893.04, 879.54 oh stretching of oxypropanolamine side chain. ch stretching of aromatic ring. ch asymmetrical and symmetrical stretching vibration of ch3 &ch2. c=n stretching of side chain (schiff base). c=n stretching of heterocyclic ring. aromatic c=c stretching asymmetrical & symmetrical c-o stretching of ether. aromatic ch in-plane bending. aromatic c-cl stretching. aromatic ch out of plane bending. iraqi j pharm sci, vol.23(2) 2014 new β-adrenoceptor blockers 49 table (4): characteristic ir absorption of the final compounds conclusion the synthesis of the proposed compounds was successfully achieved by applying the reported procedures and their chemical structures were characterized and confirmed by spectral and elemental analyses. the following points were attempted to improve the β1 selectivity and may improve the pharmacokinetic properties of these βadrenoceptor blockers. 1. replacement of the aryl nucleus of βadrenoceptor blocker by a heterocyclic ring (5-amino-1, 3, 4 thiadiazole-2-thiol) as an isostere in an attempt to improve β1 affinity. 2. introduction of imine group in the proposed β-adrenoceptor antagonist in an attempt to increase affinity to β1-adrenoceptor. 3. using different derivatives of amines at oxypropanolamine side chain to improve cardiac β1 selectivity and affinity. 4. incorporating alkyl or arylalkyl group in the series a compounds may improve the pharmacokinetic properties of these β adrenoceptor blockers. acknowledgment the authors gratefully thank the college of pharmacy, university of baghdad, for supporting this research. references 1. frielle t, collins s, daniel kw, caron mg, et al., "cloning of the cdna for the human β1-adrenergic receptor", proc natl acad sci usa. 1987;84:7920–7924. 2. dixon ra, kobilka bk, strader dj, benovic jl, et al.," cloning of the gene and cdna for mammalian β-adrenergic receptor and homology with rhodopsin", nature, 1986;321:75–79. 3. emorine lj, marullo s, briend-sutren mm, patey g, et al., "molecular characterization of the human beta 3adrenergic receptor", science, 1989;245:1118–1121. 4. xiang y, devic e, kobilka b, "the pdz binding motif of the β1 adrenergic receptor modulates receptor trafficking and signaling in cardiac myocytes", j. biol. chem., 2002;277:33783–90. 18 3317.56 3091.89 2968.45, 2935.66 1681.93 1600.92 1575.84-1500.62 1238.30, 1062.78 1091.71 1296.16, 1016.49 839.03, 736.81 oh stretching of oxypropanolamine side chain. ch stretching of aromatic ring. ch asymmetrical stretching vibration of ch3 & ch2. c=n stretching of side chain (schiff base). c=n stretching of heterocyclic ring. aromatic c=c stretching. asymmetrical & symmetrical c-o-c stretching of ether. aromatic c-cl stretching. aromatic ch in-plane bending. aromatic ch out of plane bending. 19 3311.78 3095.75 2968.45, 2850.79 1681.93 1602.85 1575.84-1500.62 1238.30, 1064.71 1296.16, 1016.49 1091.71 879.54,738.74 oh stretching of oxypropanolamine side chain. ch stretching of aromatic ring. ch asymmetrical and symmetrical stretching vibration of ch2. c=n stretching of side chain (schiff base). c=n stretching of heterocyclic ring. aromatic c=c stretching. asymmetrical & symmetrical c-o stretching of ether. aromatic ch in-plane bending. aromatic c-cl stretching. aromatic ch out of plane bending 20 3315.63 3093.82 2968.45, 2850.79 1681.93 1602.85 1575.84-1500.62 1240.23, 1064.71 1296.16, 1016.49 1091.71 879.54, 839.03, oh stretching of oxypropanolamine side chain. ch stretching of aromatic ring. ch asymmetrical and 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46 : 1125-29. 16. karsovskii k, bulgakov ak, andrushko ap, et al., "antimicrobial and tuberculostatic activity of 5-aryl(hetaryl)1,3,4-oxadiazole-2-thiones and their derivatives", pharm. chem. j., 2000;34:115-117. 17. spînu c, pleniceanu m, tigae c, "biologically active transition metal chelates with a 2thiophenecarboxaldehyde derived schiff base: synthesis, characterization and antibacterial properties", turk. j. chem., 2008;32:487. 18. jarrahpour a, khalili d, de clercq e, salmi c, et al.,"synthesis, antibacterial, antifungal, antiviral activity evaluation of some new bis-schiff base of isatin and their derivatives", molecules,2007;12:1720-1730. 19. parra m, hernandez s, alderete j, "metallomesogens derived from chelating schiff-bases containing 1,3,4-oxadiazole and 1,3,4-thiadiazole: synthesis, characterization and study of mesomorphic properties", j. chil. chem. soc., 2003;48:57-60. 20. petrow v, stephenson o, thomas aj, wild am, j. chem. soc., 1952;2:1509. 21. gyogyszer c, vegeyeszeti e, termekek rt, gyara, (ep 789583) (1958). 22. mohammadhosseini n, azadipour a, letafat b, vosooghi m, et al.," synthesis and in vitro anti helicobacter pylori activity of 2-(substituted benzylthio)-5-(5nitro-2-furyl)-1,3,4-thiadiazole derevatives", turk j. chem, 2009;33:471478. 23. oktay a, irfan kö, barbaros n, "synthesis and investigation of inhibition effects of new carbonic anhydrase inhibitors", bioorg. & med. chem., 1997;5(3):515-518. 24. gaetner vr , "alkyl 2, 3 –epoxypro pylamines ", tetrahydron, 1967;23:2123. 25. a.j, "synthesis of 2-propanolamine derivatives with expected β-adrenoceptor blocking activity", ph.d. thesis, baghdad university, baghdad, 1997, pp.46. iraqi j pharm sci, vol.28(2) 2019 lactobacillus against antibiotics associated diarrhea 179-https://doi.org/10.31351/vol28iss2pp174 doi: 174 the protective effect of lactobacillus against ciprofloxacin and levofloxacin associated diarrhea in sample of iraqi patients suha n. muhsin*,1 and ali f. hassan** *departments of pharmacognosy, college of pharmacy, university of thiqar, thiqar, iraq. * departments of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract fluoroquinolones drugs are important classes of wide spectrum antibacterial agents that are active against a wide range of gram-negative and gram-positive pathogens; they are divided into four generations. specific types of antibiotics have been associated with side effect like diarrhea, this called (collateral damage), which may occur due to drug-resistant organisms and the unwanted development of colonization or infection with multidrugresistant organisms. this damage is mostly related to levofloxacin and ciprofloxacin. the aim of the current study was to compare the incidence of collateral damage between two quinolone antibiotic derivatives (ciprofloxacin and levofloxacin) and to evaluate the activity of lactobacillus to reduce the collateral damage. this study was carried out on 100 patients. administration of ciprofloxacin, levofloxacin each alone or in combination with lactobacillus; the character of diarrhea and the grade of diarrhea was studied before and after 10 days of administration each dosing protocol. the results for this study, there are a significant increase in the incidence of diarrhea for all groups when comparison between before and after treatment diarrhea was made; a number of patients with diarrhea in group 1 after finish the treatment was not significantly higher when compared with group 2 (p>0.05); meanwhile, number of patients with diarrhea in group 4 after finish the treatment was significantly lower when compared with group 3 (p<0.05). it can concluded that , the use of ciprofloxacin and levofloxacin associated with incidence of collateral damage represented as diarrhea and levofloxacin is the least risk of this damage, and using of lactobacillus with levofloxacin was better results than the other three groups. key words: collateral damage, ciprofloxacin, levofloxacin, lactobacillus. السبروفلوكساسين و االسهال المصاحب الستعمال تقييم التاثير الوقائي للالكتوباسيالس ضد المرضى العراقيين عينة من الليفوفلوكساسين في **و علي فارس حسن 1*،محسنناظم ا هس *فرع العقاقير ، كلية الصيدلة ، جامعة ذي قار ، ذي قار ، العراق . .العراق ،بغداد،جامعة بغداد،كلية الصيدلة، فرع االدوية** الخالصة البكتريا ة الغرام والموجب البكترياة ضد يفعال طيف والتي تمتلك من مضادات البكتيريا الواسعة ال من األنواع المهمةأدوية الفلوروكوينولون تعد أرتبطت بتأثير جانبي مثل االسهال, هذا مايعرف بالضرر الجانبي الذيمن المضادات الحيوية أنواع محددةتقسم الى اربعة أجيال. و السالبة الغرام مقاومه لعدة أدوية. هذا الضرر هو معظمه يتعلق عضياتء وتطور غير مرغوب به لالستيطان أوعدوى مع مقاومة للدوا نتيجة لعضياتحصل ي بالسبروفلوكساسين والليفوفلوكساسين. ين سين مشتقين من مجموعة الكوينولون )سبروفلوكسايلمقارنة امكانية ظهوراالضرار الجانبية بين مضادين حيو من الدراسة الحالية هوالهدف ان أدوية اعطاءم . تهذه الدراسة تم تنفيذها على مائة مريض. و ليفوفلوكساسين( وتقييم فعالية الالكتوباسلس في تقليل ظهوراالضرار الجانبية يام من أ, تم دراسة حالة االسهال وتدرجات األسهال درست قبل وبعد عشرة الالكتوباسلسمع وبشكل مفرد أو الليفوفلوكساسين االسبروفلوكساسين هناك زيادة معنوية في حدوث األسهال لكل المجاميع عند المقارنة فيما بين قبل وبعد معالجة األسهال, اظهرت النتائج ان أعطاء الجرعة المتفق عليها. وان ,(p<0.05)وعة الثانية ان عدد المرضى الذين يعانون من األسهال في المجموعة األولى بعد نهاية المعالجة هو أعلى معنويا بالمقارنة مع المجم استخدام ان. (p<0.05)عدد المرضى الذين يعانون من االسهال في المجموعة الرابعة هو أقل معنويا عند المقارنة بالمجموعة الثالثة ن ن حيث الضرر, واطرا محدوث االضرار الجانبية المتمثلة باالسهال و الليفوفلوكساسين هو االقل خب ارتبطالسبروفلوكساسين والليفوفلوكساسين المجاميع الثالث االخرى. بخالفافضل النتائج أعطىستخدام الالكتوباسلس مع الليفوفلوكساسين أ : االضرار الجانبية, سبروفلوكساسين, ليفوفلوكساسين, الكتوباسلس.ةالكلمات المفتاحي introduction fluoroquinolones are synthetic drugs having broad spectrum of antibacterial activity and they are broadly used to treat or prevent infectious diseases (1). they are greatly active against both gram positive and gram – negative microorganisms and have metabolic properties that are promising for treating extensive types of infections; they are active against streptococcus pneumonia, mycoplasma pneumoniae, chlamydophila pneumonia and pseudomonas aeruginosa (2). ali_1371982@yahoo.commail:-or ecorresponding auth1 received: 3/7 /2019 accepted: 6/9 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp174-179 iraqi j pharm sci, vol.28(2) 2019 lactobacillus against antibiotics associated diarrhea 175 furthermore, fluoroquinolones inhibited either dna gyrase, which is an adenosine triphosphatehydrolyzing topoisomerase ii enzyme to keep safe the state of supercoiling in replicating and nonreplicating chromosomes of bacteria or -action of topoisomerase iv (3). moreover, fluoroquinolones indicated for the treatment of different types of infections like urinary tract infections, chronic bacterial prostatitis, acute uncomplicated cystitis in females (4), skin and skinstructure infections, bone and joint infections (5) and -gastrointestinal infections (6). ciprofloxacin is a second-generation fluoroquinolone, is carboxylic acid derivatives of fluoroquinolones, and the most beneficial and it is widely used among fluoroquinolone drugs (7). levofloxacin is a third generation fluoroquinolone which is the optical s-(-) isomer of ofloxacin. levofloxacin have an extended antibacterial range and new different uses when compared with older generations of fluoroquinolone (8, 9). antibiotic associated diarrhea is a most important type of collateral damage that associated with the uses of specific type of antibiotics. the major causes of this adverse effect are the presence of drug-resistant organisms and the inadequate development of colonization or occurrence of multidrug-resistant pathogens (10, 11). lactobacilli are live bacterial agents without any adverse effects (12). lactobacillus considered a probiotics which use as a dietary supplements. lactobacillus containing different types of bacteria including (lactobacillus species, bifidobacterium species, and others). the clinical use of lactobacillus was supports health by improving the normal flora of the gastrointestinal tract (git) (13). moreover, lactobacillus can also compete with microorganisms for food and adhesion places on the gastrointestinal mucosa (14). besides probiotics can regulate local and systemic immune responses by enhancing the secretion of immunoglobulin iga (15). the aim of the current study was to compare the incidence of diarrhea associated with the use of two quinolone antibiotic derivatives (ciprofloxacin and levofloxacin) and evaluate the protective activity of lactobacillus against diarrhea associated with these two antibiotics. patients and methods patients the current prospective study was carried out on 100 patients who diagnosed by gynecologist or urologist or internist having one type of infections which is either typhoid fever , urinary tract infections or gynecological diseases during august 2018 to march 2019. the age of patients range from (18-60) years. study design a prospective study of cohort of 100 patients, divided as: * group 1include 25 patients, taking ciprofloxacin (ciprodar) (r) 500mg tablet twice daily for 10 day (16). * group 2includes 25 patients, taking ciprofloxacin (ciprodar) (r) 500 mg tablet twice daily (16) plus lactobacillus (vitalactic b) (r) 50 mg capsule twice a day for 10 days (17). * group 3include 25 patients, taking levofloxacin (tavanic) (r) 250 mg tablet twice daily for 10 days (16). * group 4include 25 patients, taking levofloxacin (tavanic) (r) at dose 250 mg tablet twice daily (16) plus lactobacillus (vitalactic b) (r) 50 mg capsule twice a day for 10 days (17) inclusion criteria the inclusion criteria for this study were: 1patients did not use antibiotic for last three months 2patients did not have diarrhea 3patients in both sexes at age range from (18-65) years exclusion criteria the exclusion criteria for this study were: 1patients with bowel diseases like (irritable bowel syndrome). 2patients in at age less than 18. 3patient with allergic reaction against fluoroquinolone. diarrhea grade diarrhea grade was divided as follow (grade 1 less than 4 times /day over baseline, grade 2 between 4-6 times /day over baseline, grade 3 more than 7 times /day over baseline, grade 4 lifethreatening diarrhea (18). statistical analysis all results were expressed as frequencies for each category with calculation of percent for each frequency. data were analyzed by utilizing computerized statistical package for the social sciences (spss) program version 24. chi square test was performed for test groups to evaluate dependency, if p values < 0.05 were considered to be significantly dependent. ethical consideration all patients included in this study were informed about the aim of the study and they agree to participate in this study and their consent was obtained. results characteristics of diarrhea table 1 and figure 1 showed that, in group 1 there are a significant differences in the presence of diarrhea when comparison was made between patients before starting treatment protocols (4%) and after finishing the treatment protocol (68%) (p<0.05). iraqi j pharm sci, vol.28(2) 2019 lactobacillus against antibiotics associated diarrhea 176 moreover, in group 2, there are significant differences in the presence of diarrhea when comparison was made between patients before stating treatment protocols (4%) and after finishing the treatment protocol (44%) (p<0.05).table 1 and figure 1. furthermore, table 1 and figure 1 showed that in group 3, there are significant differences (p<0.05) in the presence of diarrhea when comparison was made between patients before stating treatment protocols (0%) and after finishing the treatment protocol (44%). moreover, in group 4, there are significant differences in the presence of diarrhea when comparison was made between patients before stating treatment protocols (4%) and after finishing the treatment protocol (24%) (p<0.05). table 1. the presence of diarrhea in patients before and after a specific medication protocol. patients with diarrhea patients without diarrhea pvalue group 1 before 1(4%) 24(96%) 5.31e05 after 17(68%) 8(32%) group 2 before 1(4%) 24(96%) 0.0132 after 11(44%) 14(56%) group 3 before 0(0%) 25(100%) 0.0117 after 11(44%) 14(56%) group 4 before 1(4%) 24(96%) 0.0415 after 6(24%) 19(76%) number of patients for each group =25, chi square test use to evaluate the dependency in the presence of diarrhea before treatments and after the treatment in the study groups * group 1 taking ciprofloxacin 500mg tablet twice daily for 10 day. * group 2 taking ciprofloxacin 500 mg tablet twice daily plus lactobacillus 50 mg capsule twice a day for 10 days * group 3 taking levofloxacin 250 mg tablet twice daily for 10 days * group 4 taking levofloxacin at dose 250 mg tablet twice daily plus lactobacillus 50 mg capsule twice a day for 10 days the value between two brackets represents a percent of patients with or without diarrhea. figure 1. incidence of diarrhea before and after a specific medication protocol . group 1 patients taking ciprofloxacin 500mg tablet twice daily for 10 day. group 2 patients taking ciprofloxacin 500 mg tablet twice daily plus lactobacillus 50 mg capsule twice a day for 10 days group 3 patients taking levofloxacin 250 mg tablet twice daily for 10 days group 4 patients taking levofloxacin at dose 250 mg tablet twice daily plus lactobacillus 50 mg capsule twice a day for 10 days. table 2 showed that in group 1, the number of patients having diarrhea after the end of treatment was (17 patients) which is higher when compared to group 2 patients (11patients). furthermore, there are a significant differences in the presence and absence of diarrhea in group 1 as compare to patients in group 2 (p<0.05). moreover, in group 3, a number of patients having diarrhea was (11 patients) after the end of treatment which is higher when compared with group 4 in which the number of patient having diarrhea was (6 patients). additionally, there are significant differences (p<0.05) in the presence and absence of diarrhea in group 3 as compare to patients in group 4. iraqi j pharm sci, vol.28(2) 2019 lactobacillus against antibiotics associated diarrhea 177 table 2. comparison in the incidence of diarrhea for patients after taking either ciprofloxacin or levofloxacin with patients taking ciprofloxacin and lactobacillus or levofloxacin and lactobacillus. patients with diarrhea patients without diarrhea p-value group 1 17 8 0.0873752 group 2 11 14 group 3 11 14 0.0005764 group 4 6 19 -data express as total number of patients, n=25, chi square was use to evaluate the dependent between two study group in which (p<0.05) considered as significant dependence * group 1 taking ciprofloxacin 500mg tablet twice daily for 10 day. * group 2 taking ciprofloxacin 500 mg tablet twice daily plus lactobacillus 50 mg capsule twice a day for 10 days. * group 3 taking levofloxacin 250 mg tablet twice daily for 10 days * group 4 taking levofloxacin at dose 250 mg tablet twice daily plus lactobacillus 50 mg capsule twice a day for 10 days the value between two brackets represents a percent of patients with or without diarrhea. table 3showed that in group 1, a number of patients having diarrhea was (17) after the end of treatment; meanwhile, in group 3 (11 patients) having diarrhea; and, there are no significant differences (p>0.05) in the presence and absence of diarrhea in group 1 as compared to that in group 3 patients. additionally, table 3 showed that, in group 2, number of patients having diarrhea was (11) after the end of treatment; meanwhile, in group 4 (6 patients) having diarrhea; and, there are no significant differences in the presence and absence of diarrhea in group 2 as compared to that in group 4 patients (p>0.05). table 3. comparison in the incidence of diarrhea for patients after taking ciprofloxacin with levofloxacin and patients taking ciprofloxacin plus lactobacillus with patient taking levofloxacin plus lactobacillus. patients with diarrhea patients without diarrhea p-value group 1 17 8 0.087375 group 3 11 14 group 2 11 14 0.135515 group 4 6 19 -data express as total number of patients, n=25, chi square was use to evaluate the dependent between two study group in which (p<0.05) considered as significant dependence * group 1 taking ciprofloxacin 500mg tablet twice daily for 10 day. * group 2 taking ciprofloxacin 500 mg tablet twice daily plus lactobacillus 50 mg capsule twice a day for 10 days. * group 3 taking levofloxacin 250 mg tablet twice daily for 10 days * group 4 taking levofloxacin at dose 250 mg tablet twice daily plus lactobacillus 50 mg capsule twice a day for 10 days the value between two brackets represents a percent of patients with or without diarrhea. discussion the fluoroquinolone class of antimicrobial agents which is active against an extensive range of multi-resistant microorganisms due to its mode of action against different molecular targets than other antibacterial classes (19). antibiotic associated diarrhea or collateral damage considered a serious adverse effect associated with the use of fluoroquinolones antibiotic therapy; such type of adverse effect had especial concern when initiating empirical antibiotic therapy, especially in patients who are seriously immune-compromised or ill (20). in tables 1, 2, and 3, all the groups of patients have a diarrhea after finishing the treatments protocol and this induction was significant differences when compare with same group before the treatment protocol started, besides the addition of lactobacillus to the ciprofloxacin show a notsignificant improvement in the diarrhea but the addition of lactobacillus to the levofloxacin show a significant improvement in the induction of diarrhea when compared to levofloxacin alone. at the same time in (table 4), there are significant differences in the grade of diarrhea in groups1, 2, 3 and 4 when iraqi j pharm sci, vol.28(2) 2019 lactobacillus against antibiotics associated diarrhea 178 compare between before start treatment and after finish the treatment protocol. margaret em, et al at 2003 showed that drugs belong to fluoroquinolone group may have the ability to prompt diarrhea (21). probiotics (live microorganisms) can give a health advantages on the host when taken in a sufficient amounts (22). authors mentioned that the purpose behind taking probiotics in gastrointestinal disorders is based on the theory that probiotics may help in normalization of an unbalanced flora in git. moreover, probiotics may promote intestinal health by the induction of immunity, competition for nutritional components of foods, or the inhibition of epithelial invasion (23). the clinical study for lactobacillus showed that it had the ability to prevent many clinical conditions, such as the treatment of lactose intolerance and the prevention and treatment of nosocomial diarrhea (24). guandalini s, et al (2000) reported that when lactobacillus has been used in adults and children having an acute diarrhea and treated with lactobacillus, the recurrence of diarrheal symptoms were reduced (25). results of the current study are matched with those of previous one; where, the use of ciprofloxacin and levofloxacin alone associated with increasing the incidence of diarrhea and at the same time, ciprofloxacin has the ability to induce diarrhea higher than that produced by levofloxacin as seen in table 2; furthermore, the addition of lactobacillus to the treatment protocol caused reduction in the incidence of diarrhea in each treatment (groups 2 and 4), and the reduction in the incidence was significantly higher in group 4 when compared to the incidence of diarrhea in group 2. the same findings have been seen concerning the grade of diarrhea. conclusions the use of ciprofloxacin and levofloxacin associated with increasing the incidence of collateral damage represented as diarrhea; and levofloxacin produced the least risk of this damage; and the use of lactobacillus with levofloxacin produced better results. references 1. robicsek a, strahilevitz j, jacoby g a, et al. fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. nature medicine 2006; 12(1): 83-88. 2. john sb, mary aj. the use of systemic and topical fluoroquinolones. the american academy of pediatrics 2011; 128(4):1034-1045. 3. hooper dc. mode of action of fluoroquinolones. drugs 1999;58 (2):6-7 4. andriole vt. overview of quinolone development, the quinolones: past, present, and future. cid 2005; 41: s113–9. 5. prabodh cs, ankit j, sandeep j, et al. ciprofloxacin: review on developments in synthetic, analytical, and medicinal aspects. journal of enzyme inhibition and medicinal chemistry 2010; 25(4): 577-589 6. schaad ub. fluoroquinolone antibiotics in infants and children. infect dis clin north am. 2005; 19(3):617-28. 7. fatima r abdul, nehad a taher, ashraf s hassan, enaam h batah. the effect of coumarin derivatives (compounds) on the vibrio cholerae isolates from different clinical iraqi sources. iraqi j pharm sci 2017; 26 (1):32-39 8. une t, fujimoto t, sato k, et al. in vitro activity of dr-3355, an optically active ofloxacin. antimicrob agents chemother. 1988; 32(9):1336-1340. 9. fu kp, lafredo sc, foleno b, et al. in vitro and in vivo antibacterial activities of levofloxacin (1ofloxacin), an optically active ofloxacin. antimicrob agents chemother. 1992; 36(4):860-866. 10. paterson dl. collateral damage from cephalosporin or quinolone antibiotic. cid. 2004; 38(4):341-345. 11. borgmann s. ciprofloxacin: one drug – numerous collateral damages. j bacteriol parasitol. 2012; 3(6):1000e107 12. gregor r. the importance of guidelines in the development and application of probiotics. current pharmaceutical design 2005; 11(1): 1116 13. beverly cm, clarence ec. constipation, diarrhea, and irritable bowel syndrome. in: chisholm-burns m. a., schwinghammer t.l. (eds). pharmacotherapy principles & practice. 4th ed. mc graw hill education 2016; 21: 333348 14. lee yk, puong ky, ouwehand ac, salminen s. displacement of bacterial pathogens from mucus and caco-2 cell surface by lactobacilli. j. med. microbiol. 2003; 52: 925–30. 15. ali mm, emad mm, nawal mj, zeinab r. a comparative biochemical study of proteins profile in iraqi children and adolescent with? thalassemia. iraqi j pharm sci 2010; 9(2):19-23 16. cheol k, jieun k, dae wp, baek nk, u-syn h, seung l, et al. clinical practice guidelines for the antibiotic treatment of community-acquired urinary tract infections. journal of infection and chemotherapy 2018; 50(1):67-100 17. hyun js, jin y k, sung aj, seong ek, hye sp, yoolwon j, et al. effect of probiotic lactobacillus ( lacidofil ® cap ) for the prevention of antibiotic-associated diarrhea : a prospective , randomized, double-blind, multicenter study. j korean med sci. 2010; 25 (12): 1784-1791 18. zijun hs, huang ys, hubin h, et al. a systematic review and meta-analysis of the risk https://www.ncbi.nlm.nih.gov/pubmed/?term=schaad%20ub%5bauthor%5d&cauthor=true&cauthor_uid=16102652 https://www.ncbi.nlm.nih.gov/pubmed/16102652 http://bijps.uobaghdad.edu.iq/index.php/bijps/issue/view/45 iraqi j pharm sci, vol.28(2) 2019 lactobacillus against antibiotics associated diarrhea 179 of diarrhea associated with vandetanib treatment in carcinoma patients. onco. targets and therapy 2016; 9:3621–3631 19. hooper dc. new uses for new and old quinolones and the challenge of resistance. clin infect dis. 2000; 30:243–54. 20. paterson dl. collateral damage from cephalosporin or quinolone antibiotic therapy. clin infect dis. 2004; 38 (4):s341-5 21. margaret em, anthony dh, eli p, et al. fluoroquinolone use and clostridium difficile– associated diarrhea. emerg infect dis. 2003; 9(6): 730–733 22. goldenberg jz, lytvyn l, steurich j, et al. probiotics for the prevention of pediatric antibiotic-associated diarrhea. cochrane database syst. rev. 2015: cd004827. 23. 23-rolfe rd. the role of probiotic cultures in the control of gastrointestinal health. j. nutr. 2000; 130: 396s–402s 24. shaukat a, levitt md, taylor bc, et al. systematic review: effective management strategies for lactose intolerance. ann intern med. 2010; 152: 797-803. 25. guandalini s, pensabene l, zikri ma, dias ja, et al. lactobacillus gg administered in oral rehydration solution to children with acute diarrhea: a multicenter european trial. j pediatr gastroenterol nutr. 2000; 30: 54-65 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa doi: https://doi.org/10.31351/vol30iss1pp179-188 179 investigation of some phytochemical compounds found in anchusa strigosa l. grown naturally in iraq suroor a. ghalib*,1 and enass j. kadhim* *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad,baghdad,iraq abstract anchusa strigosa l.: perennial herb, with hairs especially on the leaves., flowers generally regular. commonly named (lisan althour) in iraq, from boraginaceae family. the plant contain phenolic acids, flavonoids, alkaloids,sterols and terpenoids .whole plant part deffated n-hexane for 24 hours. the deffated plant material extracted using absolute methanol by soxhlet apparatus until complete exhaustion, the extract fractionated by solvents of different polarity: petroleum etherchloroform ethylacetateand n-butanol respectively. the n-butanol fraction hydrolyzed with 5% hcl for 5 hours by reflex to break down the glycosidic linkage. rosmarinic acid, caffeic acid ,genistein and silybin were isolated from ethyl acetate fraction by preparative thin layer chromatography which identified by high performance liquid chromatography hplc, fourier transforms infrared (ftir) spectra, thin layer chromatographytlc and melting point. since the plant contain alkaloids so acid-base extraction was performed for crude extract resulting from maceration of the plant parts in methanol (cold method) to obtain the alkaloid that isolated by preparative thin layer chromatography and then identified by fourier transforms infrared (ftir) spectra and thin layer chromatography(tlc). the aim of this research was to carry out a phytochemical study of this plant since no previous phytochemical investigation work had been done on this species in iraq keywords: anchusa strigosa ,rosmarinic acid, sterol ,acid –base extraction. في الذي ينموطبيعيانبات لسان الثورالبومي في الموجودة النباتية الكيميائية المركبات بعض دراسة العراق *و ايناس جواد كاظم 1*،سرور عبد االمير غالب .، بغداد، العراق فرع العقاقير والنباتات الطبية ، كلية الصيدلة ، جامعة بغداد* لخالصةا , اوراقها ذوات شعيرات ,ازهارها منتظمة الشكل.االسم الشائع لها في العراق ) لسان الثور( وهي فصلي او معمر لسان الثورهوعشب تم ازالة الدهون من اجزاء من عائلة الحمحميات .يحتوي النبات على الفالفينويدات,االحماض الفينولية ,االلكالويدات ,الستيروالت والتيربينويدات. باستخدام لحد النفاذ ساعة في الهكسان. تمت عملية االستخالص لالجزاء المنزوعة الدهون في محلول الميثانول 24واسطة تنقيعها لمدة النبات الكاملة ب خالت االثيل و -كلوروفورم -البتروليوم ايثر جهاز السكسوليت ثم تمت تجزئة المستخلص بعدة مذيبات عضوية ذات قطبية مختلفة شملت بالتتابع : ساعات .تم فصل حامض الروزمارينيك ,حامض 5لمدة %5بيوتانول االولي . تم كسر االصرة الكاليكوسيدية باستخدام حامض الهيدروكلوريك ال مطياف الكشف تحت الحمراء ,الفصل الملون من جزء خالت االثيل بتقنية طبقة السليكا التحضيرية وشخصت بواسطة الكافئين,جنيستين وسيليبين طة الكفاءة ,اختبار درجة االنصهار وتقنية الفصل الملون بطبقة السليكا الرقيقة.نظرا الحتواء النبات على االلكالويدات تم استخالص بواسعالي لويد لكاالحامض والقاعدة للحصول على االلكالويدات من مستخلص الخام الناتج من تنقيع النبات في الميثانول )الطريقة الباردة(للحصول على اال مطياف الكشف تحت الحمراء.يهدف الذي تم فصله بواسطة تقنية طبقة السليكا التحضيرية وشخص بواسطة تقنية الفصل الملون بطبقة السليكا الرقيقة, الحث الحالي الجراء دراسة كيمو نباتية للنبات لعدم وجود دراسة سابقة لهذا النوع في العراق. . والقاعدةستخالص بواسطة الحامض الا، الستيروالت، حامض الروزمارينيك،لسان الثور الكلمات المفتاحية : introduction hardy annual biennial or perennial herb (1), usually with hairs on the leaves, the flowers generally regular (2). commonly named (lisan althour) in iraq, from boraginaceae family (3) that distributed in the temperate, usually in mediterranean and tropical regions; in iraq is common on the road sided in middle and southern regions (1). the plants of boraginaceae family contain naphthaquinones, flavonoids, terpenoids and phenols however, these plants also have hepatotoxic pyrrolizidine alkaloids. (4) plants of the boraginaceae family are traditionally used in the treatment of fever, asthma, kidney stones, wound healing, treatment of arthritis, sprains or dislocation of the joints and bone fractures (5) flowers of anchusa italica retz. and anchusa strigosal. used as tea: tonic to invalids and children; lower pulsation. it is a substitute of anchusa offisinalis used as a diaphortic and diuretic (6). in turkey some anchusa species used traditionally as wound healing and diuretic agent and others as demulcent, expectorant, analgesic, sedative (7) in jordon and palestine the root decoction is used as diuretic, for abdominal pain and for treatment of gastric ulcer, while the leaves juice is applied externally for skin disease, arithritis and wounds (8,9). 1corresponding author e-mail: suroor.amir@yahoo.com received: 29/ 8/2020 accepted: 21/ 11/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp179-188 iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa 180 different species of anchusa extract have many pharmacological activities ; methanolic extract of anchusa officinalis l .has great antioxidant activity as free radical scavenging due to phenolic and flavonoids content,(10)while this activity is great for aqueous extract of anchusa strigosa (11) antihyperglycemic activity of a. strigosa in diabetic rats showed a significant decrease in the fasting blood glucose and an increase in the serum insulin levels after administration that possibly was either by increasing the pancreatic secretion of insulin from existing β-cells or by release from the bound form (12) the uses of anchusa strigosa extract has neuroprotective activity against amyloid toxicity ; reducing levels of amyloid secretion from cells and reduced γ-secretase activity (13). the volatile oil of anchusa strigosa l. has strong antibacterial activity against both gram positive and gram negative bacteria in a high concentration (14), the antioxidant activity due to phenolic and flavonoids content of anchusa italica may be beneficial in ischemic patients (15) on the other hand a. strigosa aqueous and methanol extracts has anti arithritis activity (16) also finding that the petroleum ether fraction of anchusa strigosa has effective anti-ulcer profile (17). anchusa strigosa contain many secondary metabolites including pyrolizidine alkaloids with high concentration in the leaves followed by the flowers and finally by the roots such as retrorsine, trachelanthamidine, supinidine , platynecine and heliotridine (9,18) ,flavonoids and phenolic acid content of this plant including catechins, quercitin,rosmarinic acid caffeic acid and others (19,20) ,this plant also contains steroids and terpenoids as tormentic acid. (5) this study aim is to carry out a phytochemical study of iraqi anchusa strigosa for isolation of some phenolic acid,flavonoids and alkaloid. experimental section plant material the whole plant of anchusa strigosa l. of family (boraginaceae) was collected from khalow bazian near kirkuk during april at the flowering stage and the plant was authenticated by dr. abdul hussein alkhiat, specialist in plant taxonomy in college of sciences/ university of erbil. extraction anchusa strigosa whole plant parts were cleaned, dried at room temperature for 7 days, pulverized by mechanical milled and then weighed. about (500gm) of powdered plant defatted by maceration in pure n-hexane for 24 hours, filtered through a wattman paper, then filled in the thimble and extracted with sufficient amount of absolute methanol by a soxhlet extractor until complete exhaustion. this extract was concentrated using rotary evaporator. after complete evaporation of the solvent, dry extract was weighted and dissolved in 350 ml water, partitioned with 350 ml (3times) petroleum ether, chloroform, ethylacetate and butanol. each fraction evaporated by rotary evaporator, dry, weighted and revealed for preliminary test. the n-butanol fraction was hydrolyzed by reflex with 10% hcl, and then the hydrolyzed fraction was taken with ethyl acetate then dried for further investigation. preliminary phytochemical examination of crude methanolic extract: to identify the phytochemicals in the methanolic extract, general phytochemical screening was performed (21) alkaloids test by wagner’s reagent to 2-3 ml filtrate few drops of wagner’s reagent were added to test for the presence of alkaloids; if positive it gives brown yellow precipitate. test for polyphenol (ferric chloride test) three ml of methanolic extract was mixed with (5 ml) of distilled water. to this solution 4-5 drops of 5% ferric chloride solution was added. development of bluish black color indicates the presence of polyphenol. test for sterols h2so4 test: one ml of methanolic extract was mixed with (1 ml) acetic acid, to which equal volume of concentrated sulfuric acid was added from the side. the development of blue, green ring at the interface indicates the presence of sterol. test for saponins a bout 2 gm of the powdered extract was boiled in 20 ml of distilled water in a water bath and filtered, 10 ml of the filtrate mixed with 5 ml of distilled water in test tube and shaken vigorously. the presence of saponins indicated by a characteristic persistent froth at least 1cm in height. detection of beta-sitosterole and tormentic acid by thin layer chromatography few milligrams from the petroleum ether fraction were suspended in about one ml of absolute methanol, applied to a readymade analytical tlc plate precoated with silica gel gf254, and developed in two mobile phases: chloroform: methanol (100:10) and toluene: ethylacetate: chloroform (5:1:4) after development, the plates were allowed to dry at room temperature and the separated spots were detected by liebermann burchard reagent used for identification of steroidal compounds (22). isolation of flavonoids and phenolic acid compounds by preparative layer chromatography plc from the ethyl acetate fraction flavonoids and phenolic compounds were isolated by preparative layer chromatography plc from the ethyl acetate fraction of a. strigosa preparative silica gel gf254 plate of 20×20 cm dimension with a layer thickness of 1cm. reactivated iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa 181 by heating at 1200c for 15–20 min, then cooled for the application of the sample. one mobile phase: chloroform: methanol: formic acid (75:20:5) for ethyl acetate fraction was used, placed in jar, the jar was lined with a filter paper closed tightly, and left for saturation. sample application was done by dissolving 1g of the sample in absolute methanol and applied to the baseline of preparative plc plate using capillary tubes, the marked bands were scrapped out of the preparative plate on separated papers using a fine spatula. each band’s powder was introduced in an individual clean and dry conical flask, a sufficient quantity of absolute methanol was added, and the flasks were shaken on a warm water bath, filtered through microporous filter (seaterd) then with filter paper. the solvent was evaporated under reduced pressure using rotary evaporator. the isolated flavonoid and phenolic compounds from ethyl acetate fraction were identified by hplc, tlc, ftir and melting point. detection of isolated compounds by thin layer chromatography (tlc) analytical tlc was performed for each separated compound by using: chloroform: methanol: acetic acid (87.5:10:2.5) solvent system for isolated caffeic acid &: chloroform: methanol: formic acid (75:20:5) solvent system for isolated rosmarinic acid to measure the rf value (retardation factor) for that compound and comparing it with standard material. detection of isolated compounds by hplc analysis the samples and standard that detected by hplc include: ethyl acetate, n-butanol fraction before hydrolysis, four isolated bands, rosmarinic acid std., caffeic acid std., apigenine std., kaempferol std., genistein std., silybin std. each sample was dissolved in 200 microliter methanol prior to inject in hplc system. the separation was achieved on c18 column (knuaer, germany) (250 -4.6 mm i.d., 5 µm particle size, 80 å pore size ( . the mobile phase contains 1% aq. acetic acid solution (solvent a) and acetonitrile (solvent b), the flow rate was adjusted to 1 ml/min, the column was thermostatically controlled at 280c and the injection volume was kept at 20 μl . a gradient elution was performed by varying the proportion of solvent b to solvent a as shown below. time (min) mobile a (%) mobile b (%) flowrate ml/min 0 90 10 1 ml/min 28 60 40 1 ml/min 39 40 60 1 ml/min 60 10 90 1 ml/min hplc chromatograms were detected using a photo diode array uv detector at three different wavelengths (272, 280 and 310 nm). (23) detection of isolated compounds by ftir analysis the isolated compounds subjected to ftir analysis to identify the functional groups. the following condition and apparatus were used shimadzu 3800 ft-ir/ japan in the pharmaceutical chemistry department at al mustansiriyah university / college of science. detection of isolated compounds by melting point using melting point apparatus stuart melting point /smp30. acid-base extraction of alkaloids from crude extract part of the crude extract from hot method &all extract obtained from cold method (maceration of whole plant part in absolute methanol for 24 hours) were first defatted with n-hexane and partitioned with water to remove pigment and fatty materials(150 ml *3) then the aqueous part is treated with nh4oh to ph10 to liberate free alkaloid then equal volume of chloroform is added to separator funnel partitioned and the lower organic layer was collected & dried then acidified with 5% h2so4 to ph 2., to this layer add nh4oh to ph10 and partitioned with chloroform, the chloroform layer now contains free tertiary alkaloids (24) . this fraction symbolized as fk isolation and purification of alkaloid from fk fraction by preparative layer chromatography the alkaloid isolated by preparative layer chromatography plc from fk fraction of a. strigosa preparative silica gel gf254 plate of 20 ×20 cm dimension with a layer thickness of 1cm. reactivated by heating at 120oc for 15–20 min, then cooled for the application of the sample. mobile phase: methanol: water: formic acid (25:2:73) was used, placed in jar, the jar was lined with a filter paper closed tightly, and left for saturation. sample application was done by dissolving 1g of the sample in absolute methanol and applied to the baseline of preparative plc plate using capillary tubes, the marked bands were scrapped out of the preparative plate on separated papers using a fine spatula. each band’s powder was introduced in an individual clean and dry conical flask, a sufficient quantity of absolute methanol was added, and the flasks were shaken on a warm water bath, filtered through iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa 182 microporous filter (seaterd) then with filter paper. the solvent was evaporated under reduced pressure using rotary evaporator. the isolated alkaloid then identified by tlc, ftir. detection of isolated compounds by thin layer chromatography (tlc) analytical tlc was performed for separated compound by using methanol: water: formic acid (25:2:73) and methanol: water: formic acid (50:50:2) as solvent system for isolated compound then sprayed with dragendorff reagent for identification of alkaloid. detection of isolated compounds by ftir analysis the isolated compounds subjected to ftir analysis to identify the functional groups. the following condition and apparatus were used shimadzu 3800 ft-ir/ japan in the pharmaceutical chemistry department at mustansiriya university / college of science. results phytochemical investigation for methanolic extract of aanchusa strigosa table 1 showed the major active constituents present in the crude methanolic extract of a. strigose. the present study for the anchusa strigosa showed the presence of medicinally active qualitatively analyzed and the results are presented in table 1.the positive results, based on the presence or absence of color change, polyphenols, alkaloid, sterols and saponins gave positive (+) results. table1. phytochemical analysis of anchusa strigosa extract phytochemical components result alkaloid + polyphenol + sterol + saponin + detection of beta-sitosterole and tormentic acid by thin layer chromatography: depending on the color of standards spots that appear after heating and spraying tlc plate with liebermannburchard reagent; beta sitosterol appeared as pink to red color while tormentic acid as purple color. isolation of flavonoids and phenolic acid compounds by preparative layer chromatography plc from the ethyl acetate fraction: preparative plc was used to isolate and purify flavonoids & phenolic compounds; using chloroform: methanol: formic acid (75:20:5) solvent system for developing the isolation. as shown in figure (1), four bands (a,b,c,d) representing the four isolated compounds respectively (r,ca, g,s) . figure 1. preparative layer chromatography for ethyl acetate fraction developed in chloroform: methanol: formic acid (75:20:5) & detect under uv light at 254 nm (a: r, b:ca, c: g, d: s) iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa 183 hplc chromatogram for the ethyl acetate fraction hplc analysis were performed for the ethyl acetate fraction (figure 2, 3) and each isolated compound as shown in (figures 4,5,6,7). figure 2. hplc chromatogram of ethyl acetate fraction. figure 3. hplc chromatogram of nbutanol fraction before hydrolysis figure 4. hplc chromatogram of the isolated r compound & standard rosmarinic acid iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa 184 figure 5. hplc chromayogram of the isolated ca compound &standard caffeic acid figure 6. hplc chromatogram of the isolated g compound &standard genistein figure 7. hplc chromatogram of the isolated s compound &standard silybin tlc for the isolated compounds this was carried out to insure the purity of the isolated compound which was isolated by scraping the isolate bands of the preparative plc by comparing the retardation factor of isolated compound with standard. retardation factor for isolated r compound compared with that of standard rosmarinic acid after developing the tlc plate in chloroform: methanol: formic acid (75:20:5); they have same rf value (0.468), retardation factor for isolated ca compound compared with that of standard caffeic acid after developing the tlc plate in chloroform: methanol:acetic acid (87.5:10:2.5); they have approximate rf value (0.178 for standard & 0.142 for isolated compound), retardation factor for isolated g compound was (0.837) after developing the tlc plate in chloroform: methanol: formic acid (75:20:5) , retardation factor for isolated s compound; they have approximate rf value (0.863 for standard & 0.818 for isolated s ) after developing the tlc plate in chloroform: methanol: acetic acid (87.5:10:2.5 ) and all the plates detected at 254 nm uv light. iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa 185 ftir analysis for the isolated compounds figure 8. ftir spectrum of isolated r compound figure 9. ftir spectrum of isolated ca compound iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa 186 figure 10.ftir spectrum of isolated g compound figure 11. ftir spectrum of isolated s compound melting point  isolated r compound melt at 171 to 175 °c which match standard rosmarinic acid.  isolated ca compound melt at 221-224 °c which match standard caffeic acid.  isolated g compound melt at 298300 °c which match standard genistein.  isolated s compound melt at162–163°c which match standard silybin. isolation and purification of alkaloid from fk fraction by preparative layer chromatography preparative plc was used to isolate and purify the compound that detected as alkaloid by spraying the developing plate by dragendorff reagent; methanol: water: formic acid (25:2:73) was used as solvent system for developing the isolation. detection of isolated compound by thin layer chromatography (tlc) this was performed to insure the purity of the isolated compound which identified by spraying the plate with dragendorff reagent and measuring the retardation factor (rf) after developing the tlc plate ; founding the rf value was (0.193) by using methanol: water: formic acid(25:2:73) and was (0.654) by using methanol: water: formic acid (50:2:50) as solvent system. iraqi j pharm sci, vol.30(1) 2021 anchusa strigosa 187 ftir analysis for the isolated compound figure 16. ftir spectrum of isolated ak compound discussion hplc, ir analysis and melting point carried out in this study to identify and confirm the presence of rosmarinic acid, caffeic acid, genistein and silybin, and pyrolizidine alkaloid in the iraqi anchusa strigosa. conclusion the results of this study exhibited the presence of phenols i.e., rosmarinic acid, caffeic acid and flavonoids, i.e., genistein, silybin in ethyl acetate fraction, pyrolizidine alkaloid after acid – base extraction of crude plant material. acknowledgments financial provision and deeply grateful to the college of pharmacy, university of baghdad, for giving us the opportunity and facilities to accomplished this work. references 1. chakravarty hl. plant wealth of iraq, vol. 1. ministry of agriculture and agrarian reforms. baghdad. 1976; 506.page 29. 2. al-zubaidy am, tobakari sr. a comparative systematic study of the genus symphytum l.(boraginaceae) with new first record of the species symphytum tuberosuml. from iraq. plant archives. 2018;18(2):2068-76 3. al-douri na. a survey of medicinal plants and their traditional uses in iraq. pharmaceutical biology. 2000 jan 1;38(1):74-9 4. dresler s, szymczak g, wójcik m. comparison of some secondary metabolite content in the seventeen species of the boraginaceae family. pharmaceutical biology. 2017 jan 1;55(1):6915 5. boskovic i, đukić da, maskovic p, mandić l, perovic s. phytochemical composition and antimicrobial, antioxidant and cytotoxic activities of anchusa officinalis l. extracts. biologia. 2018 nov 1;73(11):1035-41. 6. ali al-rawi and h.l.chakravarty:medical plants of iraq.volume15 ,1964, page 13 7. taban k, eruygur n, üstün o. biological activity studies on the aqueous methanol extract of anchusa talic e l. subsp. talic (ten.) coutinho. marmara pharmaceutical journal. 2018 sep 1;22(3). 8. said o, khalil k,fuldes s,azeizeh h.ethnopharmacological survey of medicinal plants in israel,the golan heights and the west bank region. j ethnopharmacol 2002; 83:25165 9. al-khalil s.asurvey of plants used in jurdinian traditional medicine.int j pharmacogen1995;33:317-23 10. jakovljević d, vasić s, stanković m, topuzović m, čomić l. the content 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alkaloids and glycosides from anchusa strigosa. planta medica. 2003 sep;69(09):835-41 21. evans wc. trease and evans’ pharmacognosy. 16th ed. edinburgh london new york philadelphia st louis sydney toronto 2009: saunders ltd; 2009. 616 p 22. waksmundzka-hajnos m., sherma j., kowalska t.:thin layer chromatography in phytochemistry (1sted.). crc press. usa: taylor& francis group 2008 23. seal, t. quantitative hplc analysis of phenolic acids, flavonoids and ascorbic acid in four different solvent extracts of two wild edible leaves, sonchus arvensis and oenanthe linearis of north-eastern region in india. journal of applied pharmaceutical science, . 2016. 6(2), 157-166 24. ezghayer ma, kadhim ej. uplc-esi-ms/ms and various chromatographic technique for identification of phytochemicals in populus euphratica oliv. leaves extract. iraqi journal of pharmaceutical sciences. 2020 jun 24; 29 (1) :94 -114. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.24(2) 2015 trace elements in pediatric patients with otitis media effusion 72 measurement of serum trace elements (zinc, copper, magnesium and iron) concentrations in pediatric patients with otitis media with effusion in iraq najwan k. fakree *,1 * department of clinical laboratory science, collage of pharmacy, university of baghdad. baghdad, iraq. abstract otitis media with effusion (ome) is a common disease especially among young children (before school age) and it is one of the common causes of acquired hearing loss in childhood. pediatric patients with ome are usually undernourished. the purpose of this study is to determine whether the serum levels of trace elements (zinc, copper, magnesium, iron) have a role in the development of ome in children. this study carried out on 55 children and subdivided them into two groups. group 1 (patient group) consist of 30 children suffering from ome and group 2 (control group) included 25 apparently healthy children. serum levels of zinc, copper, magnesium and iron were measured for both groups. comparison the results between the two groups showed that group 1(patients) had significantly lower serum zinc, copper, and iron levels than group2 (p <0.05). meanwhile the differences between the two groups in terms of serum values of magnesium were not statistically significant (p>0.05). this study postulated that serum levels of zinc, copper, and iron may have a role in the development of ome in children. while, serum magnesium may not have effect on the development of ome in children. keyword: pediatrics, otitis media, trace elements. قٍاس تراكٍز انعىاصر انىادرج فً مصم انذو ) انزوك، انىحاس ، انمغىسٍىو وانحذٌذ ( عىذ انمرضى تانتهاب االران انىسطى االوصثاتً فً انعراق االطفال انمصاتٍه فخريوجىان قٍصر ،*1 فشع انعهىو انًخرثشيح انسشيشيح، كهيح انصيذنح ، جايعح تغذاد ، تغذاد ، انعشاق .* الخالصة انرهاب االرٌ انىسطً االَصثاتي هى احذ االيشاض انشائعح خاصح عُذ االطفال ) قثم سٍ انًذسسح( وهى احذ االسثاب انًؤديح انً انطفىنح. وحيس اٌ االطفال انًصاتيٍ تانرهاب االرٌ انىسطً االَصثاتي هى عادج يعاَىٌ يٍ سىء انرغزيح . نهزا ضعف انسًع انًكرسة في يشحهح ذوز اصثح انغشض يٍ هزِ انذساسح هى نرحذيذ فيًا ارا كاٌ يسرىياخ انعُاصش انُادسج )صَك , َحاط , يغُسيىو ,حذيذ( في يصم انذو نها دوسفي ح طفم وذى ذقسيًهى انً يجًىعريٍ . انًجًىعح االونً )يجًىعح 55صثاتي نذي االطفال . هزِ انذساسح اجشيد عهً انرهاب االرٌ انىسطً االَ يٍ االطفال االصحاء . ذى 55طفم يعاَىٌ يٍ انرهاب االرٌ انىسطً االَصثاتي و انًجًىعح انثاَيح )انكىَرشول( وذشًم 03انًشضً( وذشًم انًغُسيىو وانحذيذ في يصم انذو نالطفال في كهرا انًجًىعريٍ. تعذ يقاسَح انُرائج تيٍ انًجًىعريٍ ذثيٍ اٌ في قياط يسرىياخ انضَك , انُحاط , . في حيٍ نى يكٍ انًجًىعح االونً كاَد يسرىياخ انضَك, انُحاط وانحذيذ في يصم انذو اقم تصىسج يهحىظح يقاسَح يع انًجًىعح انثاَيح غُسيىو في يصم انذو عُذ انًجًىعريٍ. هزِ انذساسح افرشضد اٌ يسرىياخ انضَك, انُحاط وانحذيذ في يصم االخرالف يهحىظ في يسرىياخ انً يهعة انذو يًكٍ اٌ يكىٌ نها دوس في طثيعح حذوز انرهاب االرٌ انىسطً االَصثاتي عُذ االطفال. تيًُا يسرىي انًغُسيىو في يصم انذو ستًا ال .لدوس في حذوز هزا انًشض عُذ االطفا . االطفالطة انعىاصر انىادرج ، انتهاب االرن انىسطى ، انكهماخ انمفتاحٍح : introduction otitis media with effusion (ome) is the most common ear disease in children; it is characterized by the accumulation of fluid in the middle ear space behind an intact tympanic membrane without symptoms of acute inflammation. it may lead to complications such as hearing loss, speech and language delay or poor balance (1, 2) . approximately 90% of children experience ome at some time before school age, with peak incidence in 6 months to 4 years (3) . pathogenesis of ome is not well understood, but a low-grade infection, especially with species similar to acute otitis media, eustachian tube dysfunction, inflammatory response following acute otitis media and complex interactions of biochemical, immunologic and inflammatory mediators in middle ear and adenoid hypertrophy have all been implicated (1, 4, 5) . the oxidative stress and the deficiency of antioxidants may be one of the factors leading to the pathogenesis of ome (6, 7) . 1 corresponding author e-mail:najwankaisar@yahoo.com received: 4/10/2015 accepted: 15 /12/2015 iraqi j pharm sci, vol.24(2) 2015 trace elements in pediatric patients with otitis media effusion 73 in ome (which is considering as inflammatory disease in the middle ear) oxygen radicals are blamed in the pathogenesis of inflammation. infections are one of the reasons of increased reactive oxygen species production (8) . excessive production of reactive oxygen species which is known as oxidative stress may lead to increase the duration of middle ear inflammation by disturbing the immune function of leucocytes through damaging to the membrane lipids. also dna damage by free radicals decreases production of certain critical factors by leucocytes and lowered their proliferation capacity (6) . moreover, oxidative stress and free radicals causes damage to the cellular dna and proteins to the cilia in the middle ear (change the structure of cilia) leading to slow down the cilia activity and inhibit cellular regeneration (9) . under normal physiological conditions enzymatic and non enzymatic antioxidant defenses protect aerobic organisms against the action of free radicals (7) .one of the most important enzymatic antioxidants is superoxide dismutase (sod) (10) . the cytoplasmic copper/zinc-sod, one of three forms of sod, contains copper and zinc as cofactors and is believed to have a predominant role in the first step of antioxidant defense (7) . ceruloplasmin, the copper-containing protein of extracellular fluids, has been shown to have important antioxidant properties towards peroxidising lipids (8) . this suggests that the risk of middle ear infection may potentially affect by factors (dietary or otherwise) which impair copper or zinc status (2) . magnesium has an indirect antioxidant effect. it act as a cofactor on enzymatic reactions which have a role on energy production, thus activity of glutathione reductase (antioxidants) may decrease due to dietary deficiency of magnesium leading to free-radicals induced protein oxidation and lesion to tissues (11, 12) . bacterial infections are considered as one of the etiological factors in ome (6) . iron is an integral component of enzyme myeloperoxidase (mpo) activity which is used by neutrophil for killing process of bacteria by production of highly toxic reactive oxygen species intermediates (hydroxyl radicals) (13) . poor iron status are associated with decreased neutrophil function and impaired their bactericidal activity, thymic atrophy with reduction of t-lymphocytes numbers, defective proliferation of t helper (th)-1 and th-2 lymphocytes, reduction in the activity of natural killer cell , reduction in the interleukin-2 levels produced by lymphocytes and decreased in the macrophage migration inhibition factors production (13) . in developing countries, studies on middleear diseases and their influence with micronutrient status may provide valuable information on the knowledge of the pathogenesis of middle-ear infection, prevention and treatment. our hypothesis is that low serum values of trace elements (zinc, copper, magnesium and iron) may be a contributory factor in the development of ome in children. patients and methods this study was carried out at ear-nose – throat (ent) department in al yarmook teaching hospital. the study consists of 55 children 29 male and 26 female with age range between 3 -6 years (with a mean age of 4.6± 1.06). they were divided into two groups: 1-group 1(patients group): consisted of 30 patients diagnosed to have ome. 2-group 2(control group): included 25 healthy children without any complaint in the ear-nosethroat region. no infection or any other systemic diseases. patients group were diagnosed to have ome by using otoscope, otomicroscopic and tympnometric evaluation by specialized physician. venous blood samples of all children were taken. serum zinc, copper, magnesium and iron measurements were obtained calorimetrically using commercial diagnostic kits (1417) . the reference ranges for the trace elements measured are listed in table 1. table (1): the reference range for the trace elements measured. trace element reference ranges zinc (umol/l) 10.7-17.6 copper (umol/l) 4.72-23.6 magnesium (mmol/l) 0.8-1.0 iron (umol/l) 6.6-26 results the serum trace elements values of both patient group (group 1) and control groups (group 2) are shown in table 2. comparison between two groups showed that group 1(patients) had significantly lower serum zinc levels than group 2 (control) p < 0.05. (serum levels of zinc in group 1 and group 2 were 8.7303 ±1.5503 and 16.8039± 2.4528(umol/l) respectively) also patients group had significantly lower serum copper values as comparing with control group (p <0.05) (serum copper levels were 14.2097± 4.3439 in group 1 and 18.1874± 3.01628 (umol/l) in group 2). as shown in table 2 and figure 1. according to the results in table 2 although the serum magnesium levels in group 1 was lower than in group 2 (0.8349± 0.08903 and 0.8645± 0.09598 (mmol/l)) respectively there was no significant difference between the group’s p > 0.05 (figure 1). the serum iron levels of patients and control groups were 16.9850± 3.0323 and 19.4356± 3.1020 (umol/l) respectively. the patient http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3889347/#cr6 http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3889347/#cr7 http://www.ncbi.nlm.nih.gov/pmc/articles/pmc3889347/#cr6 iraqi j pharm sci, vol.24(2) 2015 trace elements in pediatric patients with otitis media effusion 74 group had significantly lower serum iron as comparing with control group (p <0.05) (table 2 and figure1). table ( 2): serum levels of zinc, copper, magnesium and iron in groups group 1 (patients group). group 2 (control). data were expressed as mean± standard deviation (sd). n= number of children. the statistical significant differences in the concentrations of trace elements determined in group 1 and group 2 were investigated using student t-test) * p ≤ 0.05 figure (1): serum concentrations (mean serum concentrations) of trace elements in control group and patients group of children. discussion ear disease consider as one of the major health problem particularly in developing countries (2) . a close interaction among nutrition, infection, and health has been recognized since many decades. adequate intake of micronutrients is essential for the proper function of the immune system (18) . nutritional factors may play a contributory role in the middle ear diseases (2, 8) . zinc is an essential trace element. it plays structural, regulatory, and catalytic roles in the body. dietary zinc deficiency impairs immune function and resistance to infection by suppressing lymphocytes and neutrophils activity, decreased phagocytosis by macrophages, decreased functions of natural killer cell , and decreased antibody responses among other (19) . some studies have found that children with vitamin a, zinc and iron deficiency were more susceptible to upper respiratory and ear infections (20, 21, 22) . in a previous study in bangladeshi children, zinc supplementation significantly reduced the incidence of suppurative otitis media in those children (2) . our results showed that patients in group 1 (ome) had significantly lower serum zinc values than those of group 2 (healthy control). copper has a specific regulatory role in the development and expression of the immune reactions. it has been demonstrated that copper deficiency reduced numbers and functions of neutrophil, decrease function and proliferation of tlymphocytes and reduced macrophages activity (18) . also copper attends to important catalytic functions in a number of enzymes such as cu,zn superoxide dismutase (sod), cytochrome oxidase , and ceruloplasmine. thus impairing copper or zinc status (due to dietary or otherwise factors) markedly reduced tissue cu, zn-sod (which consider as enzymatic antioxidant defenses important in ear disease) and cause peroxidative damage and mitochondrial dysfunction (12) . in a comprehensive review of literatures, no human research specifically examining the relation between copper status and middle ear diseases was identified. in our results, we found that group 1 children had significantly lower serum copper levels than those of group 2. magnesium has a relationship with the immune system both in innate and acquired immune response (23) . in human, antibody production and susceptibility to infections have been associated with alack of magnesium (23) . also magnesium has indirect antioxidant effect, and a dietary magnesium deficiency may reduce glutathion reductase activity (antioxidant defense) leading to radical-induced protein oxidation (11, 12) . previous study showed that the levels of magnesium and iron were significantly depressed in children suffering from the exudative otitis media comparing with the control group (24) . in our study there were no differences in the level of mg between patients and healthy children. iron deficiency is the most widespread nutritional deficiency worldwide, especially in children living in poor resource countries (13) . iron is essential for normal development of immune system and studies indicated that individuals with iron deficiency showed impairment of cell mediated immunity, in addition phagocyte microbicidal function, natural killer cell activity and mucosal immunity are reduced leading to increase susceptibility to infections (13, 25) . in a previous study by margareta b.et al children with frequent middle ear infections trace element group 1 n= 30 group 2 n= 25 p value zinc (umol/l) 8.7303 ± 1.5503 16.8039± 2.4528 0.000 copper (umol/l) 14.2097± 4.3439 18.1874± 3.0162 0.000 magnesium (mmol/l) 0.8349± 0.08903 0.8645± 0.09598 0.241 iron (umol/l) 16.9850± 3.0323 19.4356± 3.1020 0.005 iraqi j pharm sci, vol.24(2) 2015 trace elements in pediatric patients with otitis media effusion 75 seemed to account for most of the differences in the serum levels of iron and zinc between the patients and controls (26) . other study by golz a et al concluded that the children with iron-deficiency anemia have higher prevalence of episodes of acut otitis media in comparison to healthy (27) . these findings are in accordance with our results, we found that serum levels of iron in patients group were significantly lower than those in control group children. the antioxidant and micronutrient status of the patients group may be a significant factor in predisposition to ome. we found that serum levels of zinc, copper, and iron in group 1 (ome) were significantly lower than group 2(control). however, there were not statistically significant differences between both groups regarding serum magnesium levels. we speculated that deficiency of serum zinc, copper, and iron have a role in pathogenesis of ome in children. conclusion this study postulated that deficiency of serum zinc, copper, and iron levels may have a role in pathogenesis of ome in children. further clinical studies needed to elucidate the effect of other micronutrient and vitamins status in pathogenesis of ome in children. references 1. poopak izadi, mohammad-ebrahim, nahid kalid, et al. effect of zinc on treatment of otitis media with effusion . medical journal of islamic republic of iran. 2010; 24(3): 126132. 2. elemraid ma, makenzie ij, fraser wd. nutritional factors in the pathogenesis of ear disease in children: a systemic review. ann. trop. paediatr. 2009; 29: 8599. 3. tos m. epidemiology and natural history of secretary otitis. am j. 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vol.23(1) 2014 nasal carriage of mrsa 83 determine nasal carriage of methicillin resistant staphylococcus aureus mrsa in young adult college student ebtihal n. saeed *,1 ,hanan i.omer al-deen * , maysoon a. merdaw * * department of clinical laboratory sciences , college of pharmacy , university of baghdad , baghdad, iraq abstract present study was carried out to find prevalence of mrsa in healthy individual of second stage students, college of pharmacy/baghdad university. a total of 74 student selected between age 18-23 years old were included in this study, nasal swabs collected and subjected to many diagnostic standard bacteriological identification methods. culture, colonial morphology, gram stain, mannitol fermentation, coagulase ,gelatinasetest, dnaase, mr/vp and antimicrobial susceptibility test was performed on tryptic soy agar by modified kirby-bauer muller hinton disc diffusion method and the result show that out of 74 nasal swabs,67(90.5%) were mrsa positive isolates, 21(31.4%) of them were mannitol ferment and 46(68.6%) non mannitol fermenter, among these isolates 33(44.6%)male and 41(55.4%) female, there was no significant sex difference in the prevalence of staph. aureus, while show decrease in prevalence with age group,(54%),(27%),(9.5%) alternatively, mrsa positive isolates indicated relatively high rate of resistance to antibiotics, so to vancomycin 3(4.5%),ciprofloxacin 4(6%) , tetracyclin 9(13.5%) ,gentamycin 6(9%), erythromycin 15(22.5%) and keflex 20(30%). this study show a high prevalence of mrsa carriage in young adult college student (healthy people) that indicating the spread of mrsa in the community which consider high risk of spreading infections also we isolate non mannitol fermenter (mrsa) staph. aureus that need further moleculer analysis to prove it. keywords: staphylococcus aureus , mrsa, modified kirby-baur methods. ليه في سيت المقاومت للمضاد الحيىي مثفتحديد الحامليه لبكتريا المكىراث العىقىديت االو البالغيه االصحاء مه طالب الجامعت وىري سعيد ابتهال ،*1 عمر الديه ،حىان ابراهيم * مرداو ، ميسىن عبد الزهرة * * العلْم الوختبشٗة الغشٗشٗة ، كل٘ة الص٘ذلة ، جبهعة بغذاد . فشع الخالصة (mrsa)اجشٗت ُزٍ الذساعة لوعشفة هعذل اًتشابس بتتشٗاب الوتاْسال العٌيْدٗاة الويبّهاة للولابد الل٘إْ الو غال٘ي ّ الوغاوب 47فٖ الوجتوع ب٘ي الٌبط االصلبء ّ رلك ببخذ عٌ٘بل عشْائ٘ة لطالة الوشحلة الذساع٘ة ال بًَ٘ فاٖ كل٘اة الصا٘ذلَ / جبهعاة بغاذاد بعاذد ( ّ قااذ اان عااضل البتتشٗااب ّ شخ٘صااِب باابلطشا البتت٘شٗااة الو بل٘ااة هااي دساعااة الوااضسّع البتت٘ااشٕ ّ اات 32-81هغاالة باا٘ي االعواابس %( لْٕ علٔ بتتشٗاب الوتاْسال 9..5 74عضلة , 47التشام ّ الفعبل٘بل الببْٗك٘و٘بّٗة , ّقذ ّ جذ اًَ هي ب٘ي الوغتعوشال ّ صبغة %( ُاٖ ي٘اش هخوااش 71.7 77هٌِاب ُاٖ هخواش لغاتش الوابًتْل بٌ٘واب %(28.7 38( ّببًاَ (mrsaالعٌيْدٗاة الويبّهاة للو غال٘ي ( عاٌة لإْ اعلأ ًغابة هاي 85-81يبسًة بوجتوعبل اخشٓ , كوب ّجذ ببى االعوبس ب٘ي لغتش الوبًتْل ّ عتبش ُزٍ الٌغبة عبل٘ة ببلو %( هواب 5.9عاٌة بٌغابة 38%( ّهي ثن االعوبس التٖ ُٖ ضوي الفئة العوشٗة االكبش هاي 34( عٌة 38-.3%( ّ ب٘ي 97العضالل جٌظ ال ٗؤثش فاٖ الٌغابة ح٘اي الْٗجاذ فاشا با٘ي الازكْس ّ االًاب الاى ٗذل علٔ ٌبقص هعذل التْاجذ للبتتشٗب هع التيذم ببلعوش كوب ب٘ي %( , 7.9كوااااب ا ِااااشل الذساعااااة اى البتتشٗااااب الوعضّلااااة اِااااش بعاااار الويبّهااااة لولاااابدال ح٘ب ٘ااااة اخااااشٓ ه اااا الفبًتْهبٗغاااا٘ي %( . .2التفلتااظ %( ّ اخ٘ااشا 33.9%( ّاالسثشّهبٗغاا٘ي 82.9%( , التتشاعاابٗتل٘ي 5%( الجٌتبهبٗغاا٘ي 7البغبشّفلْكغبعاا٘ي باااا٘ي الذساعااااة خطااااْس اصدٗاااابد ًغاااابة اًتشاااابس البتتشٗااااب فااااٖ الوجتوااااع ّ التااااٖ بااااذّسُب ضٗااااذ هااااي ًغاااابة االصاااابببل الوتتغاااابة فااااٖ .ّ كوااب باا٘ي الذساعااة اًتشاابس ًااْع هااي ُاازٍ البتتشٗااب ي٘ااش الوخوااش لغااتش الواابًتْل hospital acquired infectionالوغتشااف٘بل .molecular analysisالتٖ عتبش عتش جذٗذ ّ لتبج بذّسُب الٔ دساعة جضٗئ٘ة لت ب٘تِب ّالويبّهة للو غل٘ي ّ . عدلتي بىر المب، طريقت كير المقاومت للمضاذ الحيىي مثسليه المكىراث العىقىديتالكلماث المفتاحيت: introduction staphylococcus aureus is a major pathogen responsible for nosocomial and community acquired infections (1, 2) . methicillin resistant staph. aureus (mrsa) has emerged as a nosocomial pathogen of a major worldwide importance and is an increasingly frequent cause significant morbidity and mortality (3) , colonized person can serve as a reservoir for the nosocomial spread of (mrsa) . active surveillance and timely identification of (mrsa) colonization of patients is an important infection control activity that help to prevent nosocomial spread and is cost effective (4,5) . acquired infection with staph. aureus have until very recently corresponding author e. mail: ibtihalnoori@gmail.com received:6/10/2013 accepted:4/5/2014 iraqi j pharm sci, vol.23(1) 2014 nasal carriage of mrsa 84 been reliably treated with b-lactam antibiotics, b-lactam antibiotics normally bind to penicillin binding proteins (pbps) in the cell wall, resulting in the disruption of synthesis of the peptidoglycan layer and death of bacterium. since a lactam cannot bind to low affinity pbps; synthesis of the peptidoglycan layer and cell wall synthesis are able to continue cited by (2) mrsa infection often require systematic antibiotic therapy and are an important health care burden since they increase treatment costs and patient morbidity ,mrsa carriage in many communities is largely unknown and it varies in different geographical regions ,so to control the spread of disease continuing study is needed to asses geographical distribution and epidemiology of infection and develop strategies to that ( 2 ,6) . the spread of mrsa can also be potentially minimized by prevention of risk factors such as previous antibiotic use, day care attendance , contact with a health care workers or nursing home resident, residence in a long term care facility, diabetes mellitus ,hospitalization, admission to intensive care unit, intravenous drug use, invasive indwelling devices, hemodialysis or peritoneal dialysis, naso gastric tube, gastrostomy ,and external feeding ,mechanical ventilation, endotracheal tube, tracheostomy tube ,surgical procedures ,immune suppression ,chronic illness (2,7) . the anterior nares are the primary reservoir of staph. aureus in both adult and children (8) nasal carriage of staph . aureus is important because most of staph .aureus infection occur in person who are colonized with this organisms, this staph .aureus carriage has been assumed to be one of the risk factors for subsequent infection (8) ,there for recognition of persons colonized orinfected with staph .aureus is recommended for preventing the spread of the organisms within hospitalsor communities (4,8) . staphylococcus aureus —methicillin resistant mannitol fermenter negative strain were first isolated and reported as subtype of "antario epidemic" strain (mrsa-1) known in canada lab.2003 by using unenriched media such as brain heart infusion (bhi) and incubate for 24-48 hours then sub cultured on blood agar, improve isolation and detection of these un usual strain which first reported by antario researchers need further molecular analysis (9, 10) . so the aim of this study is to estimate the frequency of mrsa staph .aureus in community and control the spread of disease which need continuing study to asses geographical distribution and epidemiology of infection and develop strategies to that (3) . materials and methods sample collection: nasal swabs of 74 student within age limit 21-23 years were collected for purpose of the study during a period from (october--feb.2010), the specimens were collected with sterile cotton swabs available commercially the swabs was introduced 2-3 centimeter in the nasal cavity and rotated 4-5 times both clock wise and anticlock wise before with draw. each sample was labeled with code number and various other information including age, sex, location, etc. were recorded. the sample was transported to laboratory of microbiology in sidethe college of pharmacy and immediately inoculate on the special media (bhi, msa, blood agar media) for diagnosis (2, 11, 12). culture and sensitivity to be sure that none of the staph. aureus were lost, nasal swabs were inoculated into brain heart infusion broth, non selective media containing 0.5% salt,75 mg/loztreonam, 5mg/l ceftizoxime (9,10) overnight incubation for 24hrs,37c°, then subcultured on blood agar and mannitol salt agar to indicate the type of hemolysin and fermentation of mannitol. both hemolytic mannitol fermenter yellowish colonies and non hemolytic, non mannitol fermentor pink colonies, are subsequently identified by the gram stain, catalase test, slide and tube coagulase test, gelatinase test, mr/vp test (1,2,12,13) . also, the colonies subcultured on tryptic soy agar (tsa) media for further examination. antibiotic susceptibility test: all the identified staph. aureus isolates from nasal swabs subjected to in vitro susceptibility test modified kirby-bauer disc diffusion methods (2,14) antibiotics used in the study were methicillin (10mcg), tetracycline (30 mcg), ciprofloxacin (30mcg), erythromycin (15mcg), vancomycin (30mcg), gentamycin (10mcg) and keflex (30 mcg), all the disc were obtained from (oxoid) . quality control for the test: in this study the accuracy of the overall testingprocedure was monitored by using stap. aureus atcc 25923 as reference strain. statistical analysis the computer programmer ssps (statistical package for social sciences) version 17.0 was employed to manipulate the statistical analysis of present study results. the results were presented as percentage frequencies. significant differences were assessed by student t-test in which p<0.05 was considered significant. result table 1 and 2 show that among 74 student under study, 33(44.5%) male and 41 (55.5%) iraqi j pharm sci, vol.23(1) 2014 nasal carriage of mrsa 85 female, staph. aureus could be isolated from 67 (90.5%) nasal swabs sample, among these isolates 29(43.3%) from male, while 38 (56.7%)from female. there was no significant sex difference in colonization of staph .aureus between male and female, while the study showed that highest colonization of staph .aureus in age group 18-19 years old (54%) follow by 20-21, age group (27%) and over 21 years old (9%), and only 7 which equalto (9.5%) show negative result means no staph. aureus isolates, 3 (4.1%) 0f them male and 4 (5.4%) female . table( 1) number of nasal swabs in correlation with age group and sex age group yrs. number of swabs no % male no % female no % 18-19 43(58) 22(29.7) 21(28.3) 20-21 24(32) 6(8.1) 18(24.3) 22> 7(10) 5(6.8) 2 (2.7) total no. 74(100) 33(44.7) 41(55.3) table (2) frequency of staphylococcus aureus(mrsa) isolates , nasal carriage by age and sex group age group yrs. number of mrsaisolated no % male no % female no % 18-19 40(54.0) 20(27.0) 20(27.0) 20-21 20(27.0) 5(6.75) 15(20.2) 22> 7(9.5) 5(6.75) 2(2.70) total no. 67(90.5) 30(40.5) 37(50.0) total no. of sample 74 , no. of negative isolates 7(9.54) , male 3(4.1%) and female 4(5.4%) table 3-show the identification methods for staph. aureus, and we can see all the mannitol fermenter staph. aureus isolates show strong slide and tube coagulase test dnaase test, gelatinase test, mr/vp test as mention in reference lab. while mannitol fermenter negative 46 (68.6%) give weakly positive tube coagulase test andmostly negative for other test, which means that n.m.f. staph aureus are different in some properties (new strain reported in 2003) (10) . table 4show the sensitivity pattern according to number of staph aureus isolates, 67 (90.5%)were methicillin resistant mrsa which divided in to21(31%) mannitol fermenter and 46 (68.6%) non mannitol fermenter, indicated relatively some resistant to other antibiotics such as for keflex (kf) 30%, erylhromycin (e) 22.5%,tetracyclin (te) 13.5%,vancomycine (v) 4.5% and for ciprofloxacin (cp) 6%. table (3) identification methods used to diagnose staphylococcus aureus test used type and no. result tube coagulase 21 (+) 46(w+) * slide coagulase 67 (+) mannitolferment 21 (+) 46(-) gelatinase 21 (+) 46(-) dna ase 21 (+) 46(-) catalase 67 (+) mr/vp 67 +/ *w=weakly positive non mannitol ferment 46 table( 4 ) sensitivity pattern according to no. of isolated staphylococcus aureus (mrsa) antibiotics used mannitolfermention total no. 21(31.4%) non mannitolfermention 46(68.6%) sensitivity no % resistance no. % sensitivity no % resistance no. % vancomycin 19 28.4 2 2.9 45 67.2 1 1.49 tetracyclin 19 28.4 2 2.9 39 58.2 7 10.4 erythromycin 16 23.9 5 7.5 36 53.7 10 14.9 keflex 14 20.9 7 10.4 33 49.2 13 19.4 gentanycin 20 29.9 1 1.5 41 61.2 5 7.5 cifrofloxacin 20 29.9 1 1.5 43 64.2 3 4.4 iraqi j pharm sci, vol.23(1) 2014 nasal carriage of mrsa 86 discussion study on staphylococcus aureus nasal carriage rates in various populations have been investigated in the developed countries with temperate climate (10) , but nosuch study among healthy population had been reported, in india so far researchers reported that nasal carriage of staph. aureus varied in different communities, (10) manyinvestigators have reported an increasing incidence of ca-mrsa infections in community settings (8,15) such increase without the usual risk factors related to mrsa infection or colonization have given the surveillance for staph. aureus greater significance. so due to association between the carriages of staph.aureus and subsequent infection (8) evolution of colonization prevalence may be useful for the estimation of potential staph.aureus causing disease (8) . the result of present study showed that nasal carriage of staphylococci was as high as (90.5%) among 74 nasal swabs which are under study table (1, 2), this result is slightly higher than what reported in previous studies on college student in brazil (40.8%) (l6) ,and in more recent studies in india reported that the rate of approximately (88.2 %)found to be positive staphylococcus and (52.2%) of them mrsa were isolated (15) , other found different ratio such as in two studies with pre-clinical medical student showed that (35. 2%) and (29%) were staph. aureus nasal carriers (11,12) . the prevalence of mrsa in the apparently healthy community of eask sikkim was estimated to be (11.1%)a total of 129 (46.1%) among 280 healthy individual screened were nasal carriers of staph. aureus ,similar findings were reported by anwar et al, cited by majuandar d et al (23) . in their study in lahore, pakistan who screened 1024 and 636 apparently healthy persons from urban and rural area respectively for nasal carriage of staph. aureus and mrsa and reported thaturban area prevalence of nasal carriers of staph. aureus was estimated to be(16.99%) but rural areas was (11.32%) and the prevalence of mrsa in urban areas was found to be(22.98%) against ( 11.11%) in rural areas (8) . in a study by lamikanra and colleagues (15) it was observed that (56.4%) of healthy nigerian students were nasal carriers of staph. aureus, tanakaetal. while studying staph. aureus in healthy individuals in japan reported (24.3%) of them to be nasal carriers (11) , another study conducted at university of taxas, reported that 99 (58%) of 170 isolates are staphylococcus aureus (22) . also we found that there was no statistically significant difference in the prevalence of staph. aureus between male and female subjected in the present study, this finding was contrary to that observed in the study done in nigreian population where females harbored staph. aureus more often than males (15) , but most studies agree with our result that no significant sex differences (15, 19, 20, 21) and all of them agree with that increasing of colonization on healthy population in older adult between 18-40 years old which affected by many factors some of them depend on sampling, quality of culture media, using enriched media, population under study, geographical area,civilization of the country little knowledge about factors that make one person to be chronic carriers or transient carriers (2) , so it need further investigation and epidemiological studies including genotyping to understand the dynamics of spread of mrsa in the community in more details. table (3) shows that from the 67 (90.5%) mrsa isolates 21, (31.4%) were mannitol fermenter and 45 (68.6%) were non mannitol fermenter staph. aureus, and this strain was first reported as subtype of epidemic strain c mrsa-1 in canada research lab. (10) , this strain of n.m.f. staph. aureus which is community strain found to be different in there biochemical activity from m.f. mrsa staph. aureus such as weakly positive tube coagulase test and gelatinase negativeand mr/vp variable this strain need further molecular analysis because it show high prevalence in community so further study is need. in table (4) the sensitivity pattern of mrsa isolates 67 (90.5%) to antibiotics show rate of resistance toward some antibiotics, keflex(30%), erythromycin(22.5%), tetracycline (13.5%), gentamycin (9%), ciprofloxacin (6%) and vancomycin (4.5%). pantandrai (17) reported rate of resistant for staph. aureus, some antibiotics, ampicillin (38.1), erythromycin (33.3%1, cloxacilin ( 14.3%), gentamycin (9.5%)and methicillin (9.5%) respectively. in comparison to these result we can see that our value nearly equal to some values, while other study like in nepal medical study show high rate of resistant for mrsa isolates it show resistance toward cloxacilin (68.8%) , follow by ofloxacin (40.7%), tetracycline (15.6%), erythromycin (9.4%),ciprofloxacin (6.3%) , and vancomycin (3.1%). the different in antibiotic resistant pattern may be due to different in '' community strain, personal factors such as antibiotic therapy. iraqi j pharm sci, vol.23(1) 2014 nasal carriage of mrsa 87 references 1. skov r, smyth r, larsen ar, bolmostrom a, karlsson a. phenotypic detection of methicillin resistance in staphylococcus aureus by disk diffusion testing and e test on muller-hinton agar. j. clin. microbiol. 2006; 44: 43959 . 2. rijal kr, phari n, shrestha bk, nepal ak, paudel b, and skalko n, prevalence of methicillin resistant staphylococcus aureus in school children of pokara-nepal. med.coll. j. 2003; 10(3): 192-195. 3. rubin rj, harrington ca, poon a, dietrich k, green ja, and moiduddin a. (1999).the economic impact of staphylococcus aureus infection in new york city hospital . emerg. lnfect. dis. 5, 9-17. 4. muto ca, jernigan ja, ostrowsky be, richet hm, jarvis wr, boyce jm, and farr bm. (2003). shea guideline for preventing nosocomial transmission. epidemiol. 24, 362-386 .(cross ref.) . 5. wernitz mh, keck s, swidsinki s, schulz s, &vei sk. (2005). cost analysis of hospital wide selective screening program for methicillin-resistant staphylococcus aureus (mrsa) carriers in the context of diagnosis related (drg) payment clin. microbiol.infect.11,466-471 .(cross ref.) (medline). 6. susuan v , leonie c , auton y petal . carriage of methicillin resistant staphylococcus aureusin a queens land indigenous community med. 1 agust. 2006; 184:556-9 . 7. cohen pr. community -aquired methicillin-resistant staphylococcus aureus skin infections; areview of epidemiology, clinical features management, and prevention. lntel.j.dermatol.2007 ;46:1-11 . 8. sooko k, lee jy, baek jy, peck kr, rhee jy, kwon kt, heo st, ahn km and song jh. characterization of staphylococcus aureus.nasal carriage from children attending an outpatient clinic .in seoul,korea.microbial . drug .resistance volume14, number 1,2008. 9. martel c, ramotark, ouellet c, etal.2001 mannitol-negative methicillin-resistant staphylococcus aureus(mn mrsa):are you missing them abstracts j3.69th.annual meeting of the canadian association of clinical microbiology and lnfectious diseases. victoria ,b . c. 10. cmpt m032-3 nasal swab: mrsa positive-un graded educational challenge (mannitol-negative strain of mrsa,subtype of ( mrsa-1) .clinical microbiology proficiency testing,august 2003. 11. brown dfj, hawkey pm, edwards dl etal, guidelines for the laboratory diagnosis and susceptibility testing of methicillin resistant staphylococcus aureus(mrsa).j. antimicrobial chemotherapy 2005;56:1000-18. 12. collee jg, marmion bp, fraseragetal. mackie and mccartney practical medical microbiology, ed.l4th.churchill livingstone, united kigdom,1996 . 13. forbes ba, sahm df ,weissfeld as. bailey &scotts diagnostic microbiology ,10th edition ,mos by lnc.usa 1998. 14. world health organization guidelines for antimicrobial susceptibility testing who coilaborating center for surveillance of antimicrobial resistance.egypt,1996. 15. lamikanar a, paul bd , akinwole ob , paul mo . nassal carriage of staphylococcus aureus in a population of healthy nigerian students .j. med. microbial .1995 19 ,211-216 . 16. kaplan sl, hultenkg ,gonzalez be etal. three years surveillance of communityaquired staphylococcus aureus infection in children. clin.lnfect.dis. 2005;40:1785-91. 17. pant j ,raisk occurrence of staphylococcus aureus in hospital environment and staffs in teaching hospital in katmandu, nepalassos. medi. lab .sci2007 ;8:7 2_7 3 . 18. soysal a, sahim ,yagci a, barlant i,bakir m. the low rate of methicillin – resistant staphylococcus aureus in turkish chidren .japanse j.infect dis.2006;59:195 – 6 . 19. 1r a 20-uemura e, kakinohana s, higa n, toma c, nakason n, comparative characterization of staphylococcus aureus isolates from throat and nose of healthy volunteers. japanse j .lnfect. dis.2004;57:2t-24 . 20. lo. wt. linwj , tseng mh etal . nasal carriage of singl clone of community acquired methicillin– resistant staphylococcus aureus among kindergarten attendees northern taiwan ,bimed central infect . dis 2007 , 15-4 . iraqi j pharm sci, vol.23(1) 2014 nasal carriage of mrsa 88 21. al faro c ,mascherdenen m, fergie j . etal prevalence of methicillinresistant staphylococcus aureus nasal carriage in patients admitted to driscoll children’s hospital , pediatr infect . dis. j.2006;25:459-61 . 22. partes ka , tories am ; gracia lb . etal nasal carriage of methicillinresistant staphylococcus aureus in university student . braz j. infect. dis. vol. 14 no. 3 salvador may / june 2010. 23. majuandar d. barua a, paul b. prevalence of nasal carriage of methicillinresistant staphylococcus aureus in healthy population of gangtokeast sikkim jimas octobers – december 2008.vol.21,no.4 . iraqi j pharm sci, vol.30(2) 202 pharmacists' attitudes & practice towards minor ailments doi: https://doi.org/10.31351/vol30iss2pp225-230 225 community pharmacists' attitudes and practice in the management of minor ailments ehab mudher mikhael*,1 * department of clinical pharmacy , college of pharmacy , university of baghdad , baghdad, iraq abstract this study aimed to assess the attitudes and practice of pharmacists regarding the management of minor ailments in iraqi community pharmacies. a cross-sectional study for 320 community pharmacists was conducted during february 2020 using a newly developed and validated questionnaire. only 4.4% of pharmacists preferred not to deal with minor ailment cases. minority (15.6%) of participated pharmacists referred more than half of minor ailment cases they face to the physician. regarding the assessment of minor ailments using wwham technique, “what are the symptoms” was the most commonly asked question by pharmacists. only 49.1% mentioned that they asked all wwham questions. on the other hand, most pharmacists (90%) mentioned that they educated their patients about the dosing regimen. meanwhile, less than 10% of pharmacists mentioned that they provided their patients with all possible information about their medications. all demographic factors had no effect on the pharmacists' usage of wwham technique and in pharmacist's role in patient counseling or education. in conclusion minor ailment services that provided by the iraqi community pharmacists' who participated in this study was poor at which most pharmacists do not use wwham technique appropriately and also fail to provide their patients with the required medication counseling and education. keywords: minor ailments, community pharmacists, attitudes and practice. ممارسات وانطباعات صيادلة المجتمع في عالج االمراض البسيطة 1*،ايهاب مضر ميخائيل .الصيدلة، جامعة بغداد، بغداد، العراقفرع الصيدلة السريرية ،كلية * الخالصة هدف هذه الدراسة هو لتقييم انطباعات وممارسات الصيادلة لعالج االمراض البسطية في صيدليات العراق. تم اجراء دراسة مقطعية من الصيادلة المشاركين فضلوا %4.4باستخدام استبيان تم أنشاؤه والتحقق من صحته حديثا. فقط 2020صيدلي مجتمعي خالل شباط 320شملت ( يقومون باحالة اكثر من نصف الحاالت المرضية %15.6عدم التعامل مع الحاالت المرضية البسيطة. نسبة قليلة من الصيادلة المشاركين بالدراسة ) فأن السؤال عن االعراض كان أكثر البسيطة التي يواجهونها للطبيب. فيما يخص تشخيص الحاالت المرضية البسيطة باستخدام طريقة )ووهام(, من المشاركين %49.1سؤال استخداما من قبل الصيادلة, ولكن السؤال عن االجراء المتبع من قبل المريض لعالج حالته كان االقل استخداما. فقط اهم نظام جرعات العالج. ولكن اقل ( يعلمون مرض%90ذكروا انهم يقومون باستخدام جميع اسئلة )ووهام(. ومن جانب اخر, فأن معظم الصيادلة ) من الصيادلة يرفدون المرضى بجميع المعلومات المتوفرة عن االدوية المصروفة للعالج. جميع العوامل الديموغرافية لم يكن لها تأثير %10من الحاالت المرضية البسيطة المجهزة خدمةعلى استخدام الصيادلة لنظام )ووهام( وال على دور الصيدلي بنصح وتثقيف المرضى. نستنج من ذلك ان ل من قبل معظم صيادلة المجتمع العراقيون المشاركون في هذه الدراسة ضعيفة حيث ان معظم الصيادلة اليستخدمون نظام )ووهام( بالشكل االمث وكذلك فان معظم الصيادلة اليقومون بالتثقيف والنصح الدوائي لمرضاهم. .االنطباعات والممارسات ،صيادلة المجتمع ، البسيطة اض االمرالكلمات المفتاحية : introduction the pharmacist's role in the community pharmacy has changed nowadays from just a dispenser of medication to provider of professional services such as reviewing the appropriateness of prescribed medications, and more commonly management of minor ailments (1). minor ailments can be defined as ‘common, uncomplicated and/or self-limited conditions which can be managed with over the counter (otc) medications and without complicated medical interventions (2). seeking pharmacist's advice about minor ailment is more common than physician consultation since this advice is faster and free of charge (3). meanwhile, providing minor ailments services by a community pharmacist can reduce the workload pressure on physicians in their clinics and in hospitals (4). besides that offering nonprescription medication to treat minor ailments enhances individual's confidence to manage their own health and wellbeing (5). despite all these benefits for management of minor ailments by community pharmacists, this job may be dangerous if inappropriately performed. in this regard, one study showed that approximately 20% of hospital admissions were due to otc medications-related problems (6). thus to provide minor ailment services appropriately by community pharmacists, the pharmacist must assess each case carefully and refer cases with alarming feature to a physician while manage minor cases only (7). 1corresponding author e-mail: ehab_pharma84@yahoo.com received: 27/3/2021 accepted: 20/6 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp225-230 mailto:ehab_pharma84@yahoo.com iraqi j pharm sci, vol.30(2) 202 pharmacists' attitudes & practice towards minor ailments 226 in this regard, wwham mnemonic (w stand for: who is the patient; w: what are the symptoms; h: for how long such symptoms being present; a: what action does the patient take to treat the problem; and m: account for what other medications is the patient is already using), is the most commonly and widely used acronym for assessment of minor ailment in community pharmacies (8). on the other hand, the decision to manage the minor ailment case usually involves dispensing some medications to the patient, followed by providing the patient with suitable education and counseling about the dispensed medication (9). these actions (assessment of the case and patient counseling) are highly appreciated because inappropriate supply or use of otc medications can harm patients (10,11). only few small pilot studies were conducted to assess the role of iraqi pharmacists in the management of few minor ailments such as common cold and diarrhea (12,13). therefore, the current study was aimed to assess the attitudes and practice of a large sample of pharmacists towards the management of minor ailments in iraqi community pharmacies. methods study design this study was a community based crosssectional study conducted during february 2020. the study was ethically approved by the ethical committee at college of pharmacy/university of baghdad. recruitment of pharmacists to participate in this study was performed through sharing the study name and aim with a link to the questionnaire in an official facebook page for iraqi pharmacists (almultaqa al-saidalani). all pharmacists who accepted to participants in the study were asked to fill in the electronic version of the newly developed and validated questionnaire. the participation in the questionnaire was open for 10 days. development and validation of the questionnaire a questionnaire was developed by the author of this study based on information from symptoms in the pharmacy book (14). the questionnaire consisted from 2 main parts: the first part included 3 questions to assess pharmacists' demographic data, while the second one involved 4 questions to assess pharmacists' attitudes and practice in regard to management of minor ailments, as shown in table 1. for content validity, the questionnaire was sent by email to a panel of 5 experts in clinical pharmacy, social pharmacy, and pharmacy practice. the experts were asked to rate each item in the questionnaire using a 3 – point scale: essential; useful but not essential and not necessary. all experts agreed on essentiality of all questionnaire items. table 1. community pharmacist's attitude and practice regarding the management of minor ailments (cpapp) questionnaire part questions the answer 1.demographic data 1. your age 20-30 years , 31-40 years 41-50 years , more than 50 years 2. gender male and female 3. how many years are you working in a community pharmacy? less than 3 years , 3-10 years , 11-20 years , more than 20 years 2. the pharmacist's attitude and practice in management of minor ailment cases. 4. when a patient asks you for management of his/her complain. in such a case what will be your main focus? treatment of the case assessment of the case to know patient treatment preference don't try to deal with the case 5.what is the approximate referral rate to physician for patients who ask you to treat them less than 10% , 10-30% , 31-50% more than 50% 6. when you communicate with the customer (patient) who asks you for treating his/her condition, what do you ask? note: you can choose more than one answer who is the patient? what are the symptoms? how long do you have such symptoms? do you take any action to treat these symptoms (this case)? do you use any other medications? 7. regarding treatment with otc medications, on what you always focus while recommending a treatment to the patient? note: you can choose more than 1 answer dosing regimen (e.g., once, twice daily, etc…) time to take the medication (e.g., morning or night, etc…) taking the medication in regard to meal medication storage possible medication side effects foods or drugs to avoid while taking the medication treatment time scale iraqi j pharm sci, vol.30(2) 202 pharmacists' attitudes & practice towards minor ailments 227 statistical analysis data input was done using microsoft excel 2010. categorical variables were presented as numbers and percentages. chi square test was used to assess the difference among different demographic variables affecting on the pharmacist's role in diagnosis of minor ailments and in patient counseling. chi square testing was performed using the following online calculator "http://www.quantpsy.org/chisq/chisq.htm". p values less than 0.05 were considered significant. results at the end of the study, 320 pharmacists were completed filling the questionnaire. majority of this study participants were females with less than 3 years of working experience. on the other hand, more than half (59.7%) of participants were within the age range of 20-30 years, while only 1.5% of participants were older than 50 years old. further details about demographic data are given in table 1. table 1.demographic data of participants parameter number (percent) age 20-30 31-40 41-50 more than 50 years 191 (59.7%) 100 (31.3%) 24 (7.5%) 5 (1.5%) gender male female 151 (47.2%) 169 (52.8%) working experience less than 3 years 3-10 years 11-20 years more than 20 years 155 (48.4%) 113 (35.3%) 42 (13.1%) 10 (3.1%) regarding the attitudes of iraqi community pharmacists toward management of minor ailment cases, only 4.4% of pharmacists mentioned that they preferred not to deal with minor ailment cases, while others (56.6%) mentioned that they dealt with such cases and mainly focused on assessment of the case before managing it. the greatest percentage of participated pharmacists (40%) referred 10-30% of minor ailment cases they face to the physician. meanwhile, few (15.6%) of the participants mentioned that they referred more than half of minor ailment cases they face to the physician. further details are given in table 2. table 2. the attitudes of community pharmacists towards the management of minor ailments. parameter values number (%) the main focus of the pharmacist while dealing with cases of minor ailments assessment 181 (56.6%) treatment 98 (30.6%) knowing patient preference 27 (8.4%) don't try to deal with such cases 14 (4.4%) referral to physicians less than 10% 50 (15.6%) 10-30% 128 (40%) 31-50% 92 (28.8%) more than 50% 50 (15.6%) regarding the assessment of minor ailments using wwham technique, “what are the symptoms” was the most commonly asked question by pharmacists (95.6%), while questioning about the action that was taken by the patient was the least (68.1%). only 49.1% mentioned that they asked all wwham questions. further details are given in table 3. table 3. the role of community pharmacists in diagnosis of minor ailments question usually asked by the pharmacist who is the patient? 254 (79.4%) what are the symptoms? 306 (95.6%) how long? 287 (89.7%) action taken 218 (68.1%) medications 250 (78.1%) all wwham questions 157 (49.1%) on the other hand, most pharmacists (90%) mentioned that they educated their patients about the dosing regimen, whereas only 21.6% of pharmacists mentioned that they reminded their patients about the method of medication storage. about 2/3 of participated pharmacists counseled their patient about the best way to take their medications. meanwhile, less than 10% of pharmacists mentioned that they provided their patients with all possible information about their medications. further details are given in table 4. http://www.quantpsy.org/chisq/chisq.htm iraqi j pharm sci, vol.30(2) 202 pharmacists' attitudes & practice towards minor ailments 228 table 4. the role of community pharmacists in patient education about medications for management of minor ailments education parameter usually performed by the pharmacist dosing regimen (e.g., once, twice daily, etc…) 288 (90%) time to take the medication (e.g., morning or night, etc…) 226 (70.6%) taking the medication in regard to meal 217 (67.8%) medication storage 69 (21.6%) possible medication side effects 141 (44.1%) foods or drugs to avoid while taking the medication 162 (50.6%) treatment time scale 176 (55%) educating the patient with all possible information 30 (9.4%) all studied factors including pharmacists' gender, age, working experience, and their main focus while dealing with cases of minor ailments had no significant effect on the pharmacists' usage of wwham technique in assessment of minor ailments. details are given in table 5. all demographic factors including pharmacists' gender, age, working experience had no significant effect on the pharmacists' role in patient education and counseling about medications (table 6). table 5. factors affecting pharmacist's usage of the wwham technique parameter asking all wwham questions asking some of wwham questions p value gender male 78 (49.7%) 73 (44.8%) 0.3805* female 79 (50.3%) 90 (55.2%) age 20-30 92 (58.6%) 99 (60.7%) 0.531* 31-40 53 (33.8%) 47 (28.8%) 41-50 9 (5.7%) 15 (9.2%) more than 50 years 3 (1.9%) 2 (1.2%) working experience less than 3 years 69 (43.9%) 86 (52.8%) 0.150* 3-10 years 60 (38.2%) 53 (32.5%) 11-20 years 25 (15.9%) 17 (10.4%) more than 20 years 3 (1.9%) 7 (4.3%) the main focus of the pharmacist while dealing with cases of minor ailments assessment 96 (61.1%) 85 (52.1%) 0.249* treatment 45 (28.7%) 53 (32.5%) knowing patient preference 12 (7.6%) 15 (9.2%) don't try to deal with such cases 4 (2.5%) 10 (6.1%) *= non significant effect (p value>0.05) table 6. factors affecting pharmacist's counseling role parameter providing the patient with full medication education providing the patient with partial information about the medication p value gender male 16 (53.3%) 135 (46.6%) 0.479* female 14 (46.7%) 155 (53.4%) age 20-30 16 (53.3%) 175 (60.3%) 0.663* 31-40 12 (40%) 88 (30.3%) 41-50 2 (6.7%) 22 (7.6%) more than 50 years 0 (0%) 5 (1.7%) working experience less than 3 years 14 (46.7%) 141 (48.6%) 0.340* 3-10 years 8 (26.7%) 105 (36.2%) 11-20 years 7 (23.3%) 35 (12.1%) more than 20 years 1 (3.3%) 9 (3.1%) *= non significant effect (p value>0.05) iraqi j pharm sci, vol.30(2) 202 pharmacists' attitudes & practice towards minor ailments 229 discussion participants of this study were mainly young females with less than 3 years of working experience. this finding highly reflects the reality in iraq, since the number of pharmacy colleges had been tripled in the last decade, besides that most of pharmacy college students in iraq were females (15). this study showed that only minority (4.4%) of community pharmacists preferred not to deal with minor ailment cases, while the majority of them managed such cases. lack of competence and training may be the expected reason for those who did not prefer to deal with minor ailment cases (16). this study demonstrated that 40% of participated pharmacists mentioned that the referral rate of minor ailment cases ranged between 10-30%. referral rate in other studies that assessed the role of community pharmacists in management of diarrhea and ocular allergy was very close to that in the current study and ranged between 14-31% (17,18). regarding the assessment of minor ailments using wwham technique, asking about symptoms was mentioned by 90% of pharmacists, while questioning about the action taken by the patient was the least asked question by pharmacists (68.1%). two other studies, one conducted in iraq and the other in slovakia, that assessed the role of the community pharmacists in the management of common cold also found that asking for symptoms was commonly asked question by 40% and 49.3% of pharmacists, respectively (12,19). additionally, the current study found that only 49.1% of pharmacists mentioned that they ask all wwham questions; whereas al hassan ws and colleagues found that only 11% of community pharmacists in duhok, an iraqi governorate, use wwham technique appropriately (20). despite the general similarity of the aforementioned studies with the current study, the results obtained in these studies were lower than that reported in the current study (12,19,20). this may be due to the use of a different method to assess the pharmacist's role in diagnosing minor ailments in these studies (a direct method through simulated patient technique) from that used in the current study (indirect method through filling in a self-reported questionnaire). in this regard, results obtained from self-reported questionnaires are usually biased and exaggerated (21). this study also found that most pharmacists (90%) educated their patients about the dosing regimen of the dispensed medication(s) for management of minor ailment cases. similarly, information about medication dose and dosing regimen was the most common information that was given to patients by the ethiopian community pharmacists (22,23). meanwhile, less than 10% of pharmacists provided their patients with all possible information about their medications. similarly, only 15% of community pharmacists in duhok, an iraqi governorate, provided their patients with appropriate counseling and education about the dispensed medication for treatment of minor ailments (20). additionally, two other pilot studies that were conducted in baghdad (the capital of iraq) found that pharmacists provided their patients with counseling on dosing frequency mainly while neglecting other information about medication usage (13). this study demonstrated that all demographic factors including pharmacists' gender, age and working experience had no effect on the pharmacist role in assessing minor ailment case and in educating and counseling patients about the dispensed medications. similarly, a malaysian study found that gender and educational experience had no effect on perceptions and attitudes of the pharmacist toward management of minor ailments (16). on the other hand, the lack of educational and training programs to the graduated pharmacists in iraq, in addition to the lack of sufficient incentives toward providing minor ailment services may be the main reasons behind the poor practice of managing minor ailments by all iraqi community pharmacists, without regard to their gender, age or working experience. despite the fact that this study involved a large sample size, but lack of information about the governorate at which each pharmacist work may be a limitation for this study. conclusion majority of participated pharmacists preferred dealing with minor ailment cases. however, minor ailment services that provided by the iraqi community pharmacists' who participated in this study was poor at which most pharmacists did not use wwham technique appropriately and also failed to provide their patients with the required medication counseling and education. so, it is highly recommended for iraqi pharmacists to join continuous medical educational programs and training workshops regarding the management of minor ailments. reference 1. melton bl, lai z. review of community pharmacy services: what is being performed, and where are the opportunities for improvement?. integr pharm res pract. 2017;6:79–89. 2. fielding s, porteous t, ferguson j, et al. estimating the burden of minor ailment consultations in general practices and emergency departments through retrospective review of routine data in north east scotland. fam pract . 2015;32(2):165-72. 3. porteous t, ryan m, bond cm, hannaford p. preferences for self-care or professional advice for minor illness: a discrete choice experiment. br j gen pract. 2006;56:911–917. iraqi j pharm sci, vol.30(2) 202 pharmacists' attitudes & practice towards minor ailments 230 4. hall g, cork t, white s, berry h, smith l. evaluation of a new patient consultation initiative in community pharmacy for ear, nose and throat and eye conditions. bmc health serv res. 2019;19(1):285. 5. bourbeau j, collet j, schwartzman k, ducruet t, nault d, bradley c. economic benefits of self-management education in copd. chest j. 2006;130:1704–1711. 6. caranasos g j, steward rb, cluft le. druginduced illness leading to hospitalization. jama,1974; 228, 713-717. 7. curley le, moody j, gobarani r, et al. is there potential for the future provision of triage services in community pharmacy? j pharm policy pract. 2016;9:29. 8. sarriff a, nordin n, hassali ma. starzdrp: a step-by-step approach for pharmacy triage services. malaysian j phar. 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wiley & sons, 2014. 15. al-lela oq, al tukmagi hf, salih mrm, et al. pharmacy education in iraq: history and developments 1936-2012. am j pharm pharmacol. 2014; 1(4): 51-55. 16. selvaraj, a., redzuan, a.m. & hatah, e. community pharmacists’ perceptions, attitudes and barriers towards pharmacist-led minor ailment services in malaysia. int j clin pharm. 2020; 42(2): 777–785. 17. bilkhu p, wolffsohn js, taylor d, gibson e, hirani b, naroo sa. the management of ocular allergy in community pharmacies in the united kingdom. int j clin pharm. 2013;35(2):190–4. 18. driesen a, vandenplas y. how do pharmacists manage acute diarrhoea in an 8-month-old baby? a simulated client study. int j pharm pract. 2009;17(4):215–20. 19. mináriková d, fazekaš t, minárik p. jurišová e.assessment of patient counselling on the common cold treatment at slovak community pharmacies using mystery shopping. saudi pharm j. 2019; 27(4):574-583. 20. al hassan ws, al habeeb qs. self-medication needs and practice of community pharmacies in duhok. edorium j public health 2019;6:100023p16wh2019. 21. rosenman r, tennekeoon v, hill lg. measuring bias in self-reported data. int j behav healthc res. 2011; 2(4): 320–332. 22. ayele aa, mekuria ab, tegegn hg, gebresillassie bm, mekonnen ab, erku da. management of minor ailments in a community pharmacy setting: findings from simulated visits and qualitative study in gondar town, ethiopia. plos one. 2018; 13(1): e0190583. 23. surur as, getachew e, teressa e, hailemeskel b, getawns, erku da. self-reported and actual involvement of community pharmacists in patient counseling: a cross-sectional and simulated patient study in gondar, ethiopia. pharm pract (granada). 2017;15(1):890. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://www.ncbi.nlm.nih.gov/pubmed/31053122 https://www.ncbi.nlm.nih.gov/pubmed/31053122 https://www.ncbi.nlm.nih.gov/pubmed/29444752 https://www.ncbi.nlm.nih.gov/pubmed/29444752 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5754123/ http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 42 preparation, characterization, and in vivo evaluation of daptomycin poly(d,l-lactide-co-glycolide) microspheres laith h. samein *,1 *department of pharmaceutics, college of pharmacy, university of baghdad , baghdad, iraq abstract polymeric microsphere devices occupy a wide range in the field of controlled drug delivery. subcutaneous injectable preparations of poly(lactide-co-glycolide) (plga) microsphere of daptomycine were prepared by solvent extraction/evaporation technique using different copolymers ratio and molecular weights. four formulations were prepared (f1-f4) and characterized in term of particle size, surface morphology, bulk density and porosity in addition to the drug content. the effects of the above parameters on the in-vitro release study were evaluated. these formulas were evaluated also for their in-vivo release profile using rat (as an animal model) and their serum daptomycin concentration were evaluated accordingly. a trapezoidal method was used to estimate the cumulative auc parameter and the effect of copolymers ratio and molecular weights on the shape of auc for four formulas were evaluated. simulation study was performed (for 7 or 10 days dosing of f1 and f2 and 15 day dosing for f3 and f4) as a predictor of the steady state concentration and other pharmacokinetics parameters. it was inferred from this study that by using tailored formulation approach, long and short acting preparations of injectable daptomycin microsphere could be developed helping to provide the clinicians more flexibility to select the suitable preparation according to the patient need. key words: daptomycin, trapezoidal,microsphers ,simulation . تىهايسين باستخذامبلذواء داتحضير وتىصيف وتقيين داخل الجسن poly(d,l-lactide-co-glycolide) ككراث هجهريت ليث حوزة سوين ،*1 . انؼشاق،بغذاد ،خبيؼت بغذاد،كهيت انصيذنت ،فشع انصيذالَيبث * الخالصة ححضيش حقٍ ححج اندهذ نذٔاء . حى راث انخحشس انًسيطش ػهيّفي يدبل األدٔيت حيض كبيشاألخٓضة انًكشٔيت انبٕنيًشيت ححخم ببسخخذاو َسبت بٕنيًشاث يخخهفت ٔ انًزيب ش يبٕاسطت حقُيت اسخخشاج / حبخ ( plga )انذابخٕيبيسيٍ بشكم يكٕساث يدٓشيت يغ حدى اندسيًبث ، يٕسفٕنٕخيب انسطح، انكثبفت َبحيت يٍ ى حقييًٓبٔ ح ( f1f4 ) األٔصاٌ اندضيئيت . ٔحى إػذاد أسبؼت حشكيببث حى حقييى ْزِ كزنك . ححشس انذٔاء يخخبشيبأػالِ ػهٗ دساست ؼٕايم. حى حقييى حأثيش انذٔاءان ٖانظبْشيت ٔانًسبييت ببإلضبفت إنٗ يحخٕ . ٔقذ اسخخذيج طشيقت شبّ يُحشف انذٔاء في انذوحشكيضحؼييٍ انصيغ في اندسى انحي ببسخخذاو انفئشاٌ )كًُٕرج حيٕاَي ( ٔ خشٖ سبؼت صيغ. الن انًسبحت ححج انًُحُي اندضيئيت ػهٗ شكم ٓببٕنيًشاث ٔ أصاَانانخشاكًي ٔحأثيش َسبت ححج انًُحُيانًسبحت نخقذيش حبنت انخشكيض نببػخببسِ يؤششا f3 ٔ f4 يٕو اندشػبث ل f1 ٔ f2 ٔ01 ايبو نهصيغ 01أٔ 7أخشيج دساست انًحبكبة ) نًذة اٌ حقٍ دابخٕيبيسيٍ يكشٔيت ْزِ انذساست ببسخخذاو َٓح صيبغت يصًًت ، يٍسخذالل ًسخقشة ٔانًؼهًبث انذٔائيت األخشٖ. حى االان ٔفقب نحبخت انًشيض الخخيبسانًسخحضش انًُبسبيًب يسبػذ ػهٗ حٕفيش انًضيذ يٍ انًشَٔت نألطببء قصيشة ٔطٕيهت االيذ ايكٍ حطٕيشِ تىهايسين ، شبه هفرق ، كراث هجهريت .بداالكلواث الوفتاحيت: introduction controlled release delivery systems were being developed to address many difficulties associated with traditional methods of administration. one of the most important advantages of controlled drug delivery is reduced frequency of administration, a thing that significantly improves patient compliance and convenience with a consequent improvement in the efficacy of the treatment (1) . due to their attractive properties, microspheres occupy a unique position in controlled delivery technology, and have shown to control release profiles of drugs having a wide range of molecular weights, applying different types of polymers. biodegradable polymers are the most interesting type since it hold several advantages including their biocompatibility and biodegradability where it would degrade into nontoxic, absorbable subunits which 1 corresponding author e-mail: dr_laith_2006@ yahoo.com received: 19/11/2013 accepted: 18/2/2014 iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 43 would be subsequently metabolized, in addition to that these polymers exhibit a predictable erosion so it do not exhibit dose dumping and would retain its characteristics even after depletion of the drug. the release of the active agent occurs by either gradual bioerosion of the drug containing polymer matrix, or by cleavage of unstable bonds by which the drug is coupled to the polymer matrix so the release rate is governed by the biodegradation process (2) . biodegradable polymer includes synthetic and natural type. the linear polyesters including poly lactic acid-co-glycolic acid (plga) are the most widely investigated type (3,4) . the exact kinetics of drug release could be determined and fine-tuned by careful design of the microsphere composition including the type of polymer used, the polymer molecular weight, the copolymer composition, the nature of any excipients added to the microsphere formulation and the microsphere size, all of these factors have a strong impact on the delivery rates (5) . daptomycin is novel cyclic lipopeptide antibiotic with molecular weight 1620.67 g/mol. it's a tridecapeptide comprising several non-proteinogenic amino acids with an nterminal decanoyl fatty acid side chain and a decapeptide lactone core that resulting from the cyclization of the thr-4 hydroxyl group onto the c-terminal carboxylate. daptomycin is produced from the fermentation culture of streptomyces roseosporus and it shares a similar structure, and possibly a related mode of action, to other acidic lipopeptide antibiotics, these include the calciumdependent antibiotics (6-8) . because of its unique structure which consists of a 13member amino acid, a noval mechanism of bactericidal action has been achieved which involves insertion of the lipophilic daptomycin tail into the bacterial cell membrane, causing rapid membrane depolarization and a potassium ion efflux. this is followed by arrest of dna, rna and protein synthesis (9-11) . daptomycin is highly water soluble, this is due to its predominantly acidic nature and the negative charge at neutral ph while the presence of lipid tails and some hydrophobic amino acids offer amphipathic properties to its structure. the pka values for individual daptomycin residues at neutral media are 2.9, 3.5, 4.3, 4.7, and 10.5 while its melting point is 215°c (12,13) . on 2003, the food and drug administration (fda) approved daptomycin for the treatment of complicated skin and skin structure infections in adults. in 2006 an indication was added for staphylococcus aureus right-sided infective endocarditis. daptomycin provides a useful alternative to standard therapies for both methicillin susceptible (mssa) and methicillin-resistant (mrsa) staphylococcus aureus, and in some cases, vancomycinresistant enterococcus (vre) (14) . adult dosing 4-6mg/kg/day and it available in 500mg of lyophilized powder per10 ml vial for iv injection over a two minute period or by infusion over a thirty minute period (13) . in accordance to above, the delivery of daptomycin using polymeric carriers, dosed subcutaneously or intramuscularly, is an effective strategy in mitigating patient compliance concerns and related issues as it ensures adherence to therapy leading to improved patient outcomes. this fact is further corroborated by several publications that have emphasized the development and clinical use of long acting dosage forms used in the treatment of systemic and life-threatening infections caused by gram-positive organisms (15) . thus, the plga polymer is an ideal delivery matrix for daptomycin that could provide initial and sustained levels based on the choice of the polymer used. therefore, the goal of this study was to develop and subsequently investigate the suitability of using plga polymers having varying properties like copolymer composition and molecular weight to provide tailored invivo release of such novel antibiotic via the subcutaneous route. materials and methods materials daptomycin (molecular weight 1619.7086, soluble in water; sparingly soluble in acetonitrile, and soluble in dichloromethane) was purchased from cipla ltd., bombay, india. plga having varying molecular weights (15 and 131kda of 75:25 plga, 30 kda of 50 : 50 plga, 82 kda of 65 : 35 plga) was purchased from boehringer ingelheim (ingelheim, germany) and alkermes (cambridge, ma, usa). all other chemicals were obtained commercially as analytical grade reagents. method preparation of microspheres the four plga copolymer ratios and molecular weights evaluated were:(a)15 kda plga, 75 : 25 lactide : glycolide (formula f1),(b)30 kda plga, 50 : 50 lactide : glycolide (formula f2) , (c)82 kda plga, 65 : 35 lactide : glycolide (formula f3),(d)131 kda plga, 75 : 25 lactide : glycolide (formula f4). the microspheres were prepared by a solvent extraction/evaporation method and recovered by filtration (16) . briefly, a solution of drug and iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 44 polymer (10–20% polymer concentration) in dichloromethane was injected into an aqueous continuous phase at a ratio between 250 and 350 parts of polymer phase: aqueous phase, under stirring with a silverson l4r mixer (silverson machines, ma, usa) at 5000 rpm. subsequently, the solvents were removed by stirring after which the microspheres were recovered by filtration, suspended in a suitable vehicle, filled into vials, and freeze dried. characterization the microspheres were characterized for mean particle size, surface morphology, bulk density, drug content, and in-vivo performance. particle size particle size distribution of the microspheres prior to vialing was determined using a laser diffraction technique (malvern 2600c particle sizer, malvern, uk). the particles were suspended in 0.05% tween 80 and counted using a laser sensor. the average particle size was expressed as volume mean diameter in microns (μm) (17) . surface morphology the surface morphology was examined by scanning electron microscopy (sem) (hitachi s800, japan) at an appropriate magnification, after palladium/gold coating of the microsphere sample on an aluminum stub (18) . bulk density bulk density was determined by transferring known quantity of micro spheres to graduated cylinder and tapping 100 times from a vertical distance of approximately 0.5 inches at 2 sec interval. the tapping process was repeated until the volume occupied by particles remained unchanged. the final volume was recorded as tapped volume, 𝑉𝑏, and the tapped bulk density (g/cc) was calculated as 𝑀/𝑉𝑏, where “𝑀” was the weight of microspheres employed (19) . drug content daptomycin content in the microspheres was analyzed by a reverse phase hplc method using the stationary phase, hypersil 5 ods in a stainless steel column, 100 x 4.6 mm (thermo-sep electron corp., waltham, ma, usa) with a guard column of the same material. the mobile phase was 0.2 m phosphate buffer (ph 5.5) and acetonitrile (70:30). the pump flow rate was 1.5 ml/min. detection was by uv absorbance at 223 nm (model uvd170u, dionex corp., sunnyvale, ca, usa). serum (100 μl) was mixed with acetonitrile (100 μl), allowed to stand for 5 min and centrifuged at 13000g for 5 min. an aliquot of 10 μl of the supernatant was injected (20) .measurements were performed in triplicate. drug content (%) was expressed as the weight of drug in microspheres/weight of microspheres × 100. encapsulation efficiency (%) was also calculated for the four formulations. in-vivo study four groups of male sprague-dawley rats (per group) weighing approximately 300 g were used to evaluate in-vivo performance of daptpmycin microspheres. the microspheres were injected subcutaneously at the back of the neck (15–30 mg/kg daptomycin/rat) after reconstitution with 0.9% of sodium chloride for injection, usp. blood samples were collected from the tail vein. the samples were centrifuged in microtainer tubes (becton dickinson, franklin lakes, nj) and serum was collected. serum samples were frozen and stored at −20°c until analysis. serum samples were analyzed using a validated method (21) . simulation studies multiple dosing pharmacokinetic (mdp) studies are commonly used to help the selection of an appropriate dosing regimen for a given formulation (22) . since daptomycin follows linear pharmacokinetics after multiple intravenous dosing, the plasma concentrations observed after multiple dosing of daptomycin can be linearly related to the dose and can be predicted from the auc after administration of a single dose (23) . therefore, this linearity allows simulations of multiple dose pharmacokinetics after continual dosing to be performed using the superposition principle. in the current study, simulations of serum invivo levels were obtained after subcutaneous administration of formulations f1 and f2 ( single dose at 15 mg/kg), and formulations f3 and f4 (single dose at 30 mg/kg) were performed using the superposition principle. a 7and 10-day dosing regimen was used with formulations f1 and f2 while a 15-day dosing was used with formulations f3 and f4. a total of 4 doses were selected for the simulation study as this would be an interpreter of steady state concentrations for this molecule. results and discussions characterization of daptomycin microspheres particle shape, size, and morphology the scanning electron microscope (sem) images of formulations f1-f4 are provided in figure 1. the scanning showed that the microspheres having a spherical shape with a smooth nonporous surface and homogeneous particle size distribution. particle size analysis revealed that formulas f1,f2,f3 and f4 had a mean volume diameter of 20.0, 19.8, 25.3, and 23.6 μm, respectively (table1). the mean volume diameter was found to be similar in iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 45 formulas f1and f2, both prepared from lower molecular weight plga, while the same was true for formulas f3 and f4, manufactured using higher molecular weight plga. table (1) properties of daptomycin plga microspheres formulation f1 f2 f3 f4 plga type 75 : 25 50 : 50 65 : 35 75 : 25 polymer mw 15 kda 30 kda 82 kda 131 kda drug content, % 30 34 44 44 bulk density, g/ml 0.99 1.1 1.0 1.36 mean particle size (μm) 20.0 19.8 25.3 23.6 dose of daptomycin 15 mg/kg 15 mg/kg 30 mg/kg 30 mg/kg f1 f2 f3 f4 figure 1: scanning electron micrographs of daptomycin plga microspheres of different formulations according to the published literatures, the particle size was found to have an important effect on the drug release in the field of drug delivery. for instance, a reduction in particle size is a common strategy to enhance dissolution rate of soluble drugs (24) . particle size remains one of the key parameters that affect the degradation rate of the plga polymer matrix and thereby drug release rates (25) . a reduction in particle size generally depicts an increase in surface area to volume ratio, resulting in a large surface area available for the dissolution media penetration into the particles and also for a rapid escape of any polymeric degradation products. additionally, with plga microsphere dosage forms, the initial burst release phenomenon depends on particle size. yagnesh bhatt et al, reported that the processing conditions employed during preparation of microparticles determine the properties of the microparticles, such as particle size and the reduction in particle size was found to have dramatic effect on the initial rapid release from bovine serum albumin (bsa) loaded plga microparticles (26) . thus, the initial burst effect depends on two parameters: (a) amount of drug loosely associated with the surface and (b) drug entrapped in the easily accessible porous network. for smaller sized particles, the amount of surface associated drug is expected to be large, and hence, initial burst is not unexpected (27) . based on the small particle size of the daptomycin microspheres, it was inferred that an initial burst would be exhibited by all the formulations evaluated. however, a shorter duration of release was expected for formulations a and b, due to lower molecular weight. this suggests that the in vivo behavior of daptomycin from plga microspheres could be manipulated to provide varying duration of action. bulk density the results of bulk density studies are summarized in table 1. bulk density values for the formulations were varied greatly and were determined to be 0.99, 1.1, 1.00, and 1.36 g/cc for formulations f1,f2,f3 and f4, respectively. these data suggest that all four formulations showed intermediate to high bulk density values. between the formulations, a comparison of the data revealed the lowest values for formulas f1 and f3, while formula http://www.hindawi.com/journals/jph/2013/831381/tab1/ iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 46 f4 exhibited the highest bulk density value, with an intermediate bulk density value for formulation f2. the evaluation of bulk density would provide an important information about the porous network in the drug loaded microspheres. thus, any variation in the density or porosity influences the other parameter and hence, impacts drug release behavior. low bulk density values are a qualitative indicator of the porous network inside the microspheres. additionally, low bulk density values are also observed with irregular or nonspherical microspheres that display nonoptimal packing. further, these values can also be correlated with specific surface area and onset of mass loss. microspheres with high bulk density typically exhibit low values of specific surface area. conversely, microspheres with a highly porous network will have a low bulk density and thus a faster drug release rate (28, 29) . it was concluded from the bulk density data, that the specific surface area was the lowest for f4, but the highest for formulations f1 and f3. generally, low bulk density (high porosity) values in microspheres translate to faster drug release; same finding has been seen by byrne r et al who studied the influence of the porous microstructure on the drug release from aluminosilicate pellets (30) . hence, certain predictions have been made with bulk density and particle size data: (a) particle size values for the four formulations were similar implying that the impact of this parameter on drug release would be comparable across formulations f1–f4, and (b) due to slightly lower bulk density values for formulations f1 and f3, they were expected to show a higher initial burst than formulations f2 and f4. drug content results of drug content for daptomycin plga microspheres, as determined by hplc, are presented in table 1, f1 had the lowest drug content (30%) while f2 had 34% and formulations f3 and f4 had the highest drug loading (44%). a noteworthy observation was that the encapsulation efficiency was 100% for all the microsphere formulations. these results suggest that the solvent extraction/evaporation method is suitable method for the preparation of daptomycin microspheres. in-vivo studies serum levels of daptomycin for formulations f1-f4 serum levels of daptomycin after administration of formulas f1 and f2, of 15 mg/kg dose, and formulas f3, and f4 of 30 mg/kg dose, are shown in figure 2. in general, formulations f1-f4 exhibited similar release profiles in that they show an initial burst release followed by a brief decline leading to a secondary peak and a final slow decay phase for the four formulations. figure2: in-vivo release of daptomycin plga microspheres ( formulations f1 and f2 ( 15 mg/ kg dose) ,and formulations f3 and f4 ( 30 mg/kg dose). as expected, f1 exhibited the highest initial burst (81 ng/ml) followed by a sharp drop that characterized the trough (21 ng/ml, day 1) leading to a second peak of around 39 ng/ml at day 4, after which levels exhibited a slow decline to day 15 (figure 2). in comparison, f2 exhibited an intermediate initial burst (45 ng/ml) followed by very slight dip in levels (43 ng/ml, day 1) and a secondary peak where values were comparable to the initial burst and trough (39 ng/ml, day 4), with a slow drop in levels till the last time point (day 15). with formulations f1 and f2 administered of 15 mg/kg dose, the short duration of action (15 days) was expected and attributed to a http://informahealthcare.com/action/dosearch?action=runsearch&type=advanced&result=true&prevsearch=%2bauthorsfield%3a%28byrne%2c+r.+s.%29 iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 47 combination of the properties of plga polymer (copolymer ratio and molecular weight) and microspheres (bulk density and drug content). the high initial burst for formulation f1 was attributed to a combination of small particle size and low bulk density that allowed for easily accessible drug residing on the surface or in the pores of the microspheres to be released rapidly in-vivo, while the intermediate burst for formulation f2 was recognized for its high bulk density (low porosity). for formulations f3 and f4, administration of 30 mg/kg dose, the duration of action was significantly longer than formulations f1 and f2 (figure 2). with formulation f3, initial levels were low (29 ng/ml, 6 hours), dropping even lower to reach a trough value of 18 ng/ml at day 1, then serum daptomycin values rose sharply to reach 61 ng/ml by day 4. the true secondary peak level for formulation f3 was achieved by day 8 (78 ng/ml) after which levels dropped equally sharply to reach about 3 ng/ml by day 30. unlike formulation f3 where initial burst was lowest, intermediate burst levels were observed with formulation f4 (48 ng/ml) that dropped to a stark trough value of 5 ng/ml (day 1). after the trough, serum daptomycin values began a steady ascent to reach 51 ng/ml (day 8) after which levels once again dropped to reach a final minimum of 3 ng/ml by day 30 (figure 2). serum daptomycin profiles obtained for formulations f3 and f4 can be explained on the basis of the in-vitro characterization results. as stated in section 3.1.3, a low to intermediate burst was expected for formulations f3 and f4. since the bulk density and drug content values were high, a low to intermediate burst implied that the drug remaining in the microspheres would be released in a more sustained fashion. considering the factor of in the higher lactide content and high polymer molecular weight, the extended duration in-vivo release was expected for these two formulations. between formulations f3 and f4, the former was manufactured from a 65: 35 plga polymer and hence, faster release of drug to reach a secondary peak was predicted; the invivo results are in agreement with predicted behavior of these polymeric formulations. cumulative auc for formulations f1,f2,f3 and f4 the cumulative area under the curve (auc), a key pharmacokinetic parameter, for the four formulations, as calculated by the commonly used trapezoidal method (equation 1), is shown in table 2. table (2) cumulative auc for daptomycin plga microspheres. where (t) is time in hours and (c) represents serum concentration of daptomycin in ng/ml. results from auc calculations indicate that formula f1 exhibited the lowest cumulative auc through 15 days (480 ng/ml.day), with a slight increase in the value for formula f2 (549 ng/ml.day). the lower cumulative auc values for formulations f1 and f2 were attributed to the low polymer molecular weights and low drug content for both formulations. a closer examination of the data revealed that despite the high burst with f1 that contributed about 3% to the cumulative auc, the net contributions of the time points after the secondary peak were similar to that of f2. this was attributed to the lack of the characteristic peak and trough release profile observed with f2 (figure 2) where burst release contributed a meager 1% to the total cumulative auc. for this reason, the total cumulative auc value for f2 was slightly higher than f1. formulations f3 and f4, administered at a higher dose (30 mg/kg), demonstrated cumulative auc values of 1300 and 1232 ng/ml.day through 30 days, respectively, which were higher than those observed with formulations f1 and f2 that were administered at 15 mg/kg dose (table 2). these formulations exhibited low to intermediate initial burst; therefore, the percent of cumulative auc contributed by this phenomenon was less than 0.4% for formulations f3 and f4. a lower amount of initial burst also suggested that the extended duration of plga release was due to iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 48 daptomycin entrapped in the polymer that was released slowly upon hydrolytic degradation of the 65:35 or 75:25 lactide : glycolide copolymer. in general, analysis of cumulative f1-f4 revealed the following significant points:(a) the contribution of initial burst towards the total auc for all formulations was minor (equal to or less than 3%).(b) daptomycin was well entrapped in the plga polymer matrix and was responsible for over 97% of the cumulative auc invivo.(c)the cumulative auc obtained with f3 and f4 was nearly 2 to 3 times greater than that observed with f1 and f2, suggesting that by selection of an appropriate polymer molecular weight and proportions, the release of daptomycin from plga microsphere would be customized. simulation of multiple dosing figure 3 shows serum levels for formulations f1 and f2, after 4 doses, when administered weekly or once every 10 days. once weekly and 10-day dosing regimen were selected for formulations f1 and f2, where the duration of action was short. once weekly simulation for f1 revealed that pulsatile behavior was to be expected in-vivo, similar to what was observed with administration of a single dose. simulations for doses 2-4 show that levels between 40 and 110 ng/ml are easily achieved with weekly dosing with a slightly lower range for the 10day dosing. with formulation b, weekly dosing provides serum levels ranging between 50 and 80 ng/ml while 10-day dosing affords slightly lower levels, in a manner similar to that observed with f1. the difference between the maximum and minimum serum levels for f2 was the smallest among all the formulations evaluated. irrespective of the dosing regimen, figure 3 indicates that steady state levels are attained between doses 2 and 4 for f1 and f2. figure 3: simulation of multiple dosing regimens after 15 mg/kg dose of formulas f1and f2 ,a: every 7 days ,b:every 10 days. a 15-day dosing regimen was performed on formulations f3 and f4, where the duration of action was considerably longer (figure 4). the 15-day simulation for formulations f3 and f4, shows that drug release from the latter formulation was pulsatile. however, serum levels ranged between 30 and 100 ng/ml for both batches through 4 doses. this infers that formulations f3 and f4, tailored to release drug for an extended duration, and it would be excellent candidates for 15-day administration. such type of therapy has the added benefit of reducing the number of injections required to initiate and maintain adherence to therapy. overall, simulations for the four formulations suggest that the daptomycin plga microspheres provide a suitable initial burst and maintain release over a period of time invivo. 0 20 40 60 80 100 120 140 0 5 10 15 20 25 30 s e ru m d a p to m y ci n e (n g /m l) time (day) f1 :plga(50:50) 30 kda f2 :plga(75:25)15 kda a: 7 days dosing 0 20 40 60 80 100 120 0 10 20 30 40 50 s e ru m d a p to m y ci n (n g /m l) time (hr) f1:plga(50:50)30 kda f2:plga(75:25)15 kda b: 10 days dosing iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 49 figure 4: simulation of multiple dosing regimens(30mg/kg every 15 days, total=4 doses) for daptomycin plga microsphere (f3 and f4) steady state levels a comparison of the average steady state concentration for formulations f1-f4 is shown in figures 5 and 6. the average steady state concentrations were computed for the four formulations and was found in range of 54 and 64 ng/ml for weekly dosing of formulations f1 and f2, with slightly lower levels (39 and 46 ng/ml, respectively) for 10-day dosing( figure 5). a similar calculation for formulations f3 and f4 (15-day dosing) revealed steady state levels of 67 and 63 ng/ml, respectively ( figure 6). figure 5: average steady state concentration for formulation f1 and f2 , a :7day dosing b:10 day dosing 0 20 40 60 80 100 120 0 10 20 30 40 50 60 70s e ru m d a p to m y ci n ( n g /m l) time(hr) f3:plga(65:35)82kda f4:plga(75:25)131kda 0 50 100 150 0 5 10 15 20 25 30s e ri u m d a p to m y ci n (n g /m l) time(hr) f1:plga(50:50)30kda f2:plga(75:25)15kda a 0 20 40 60 80 100 120 0 5 10 15 20 25 30 35 40 45 s e ru m d a p to m y ci n (n g /m l) time(hr) f1:plga(50:50)30 kda f2:plga(75:25)15 kda steady state f1 steady state f2 b iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 50 figure 6: average steady state concentration of formulation f3 and f4 steady state values from the simulation studies provide information on the in-vivo behavior of the four formulations. for f1, dosed weekly, a high burst is expected after which levels drop nearly 35 ng/ml to reach 60 ng/ml and release drug in a sustained fashion through the 4-week dosing interval. slightly higher and constant steady state levels are expected when f2 is dosed weekly. as expected, steady state levels for a 10-day dosing regimen are lower for f1 and f2 (figure 5). for the higher molecular weight longer acting plga formulations, higher levels could be achieved with 15-day dosing. in fact, the steady state levels achieved are higher than the initial burst and can be attributed to drug entrapped in the polymeric matrix. these results bear strong clinical significance in that drug levels in vivo can be tailored to suit patient needs using a systematic scientific approach. indeed, steady state levels for weekly, 10-day, or 15-day dosing range between 45 and 65 ng/ml, allowing the clinician to utilize a variety of dosage forms for a shorter or longer duration of therapy that is patient specific. such an approach is highly effective in the treatment of patient populations with skin , lungs , and blood infections . conclusion preparation of injectable depot of daptomycin antibiotic encapsulated within plga microspheres is an excellent delivery mechanism that offers the possibility of sustained drug release over a long duration of time. in this study, 4 long-acting formulations of varying molecular weight and copolymer compositions were developed with the purpose of illustrating that such formulation modification can provide medical professionals suitable choices in designing therapeutic strategies to treat patients with varying clinical needs. in-vivo experiments done in rats, revealed that daptomycin formulations would be suitable for weekly, 10day or, 15-day dosing and would achieve steady state levels by the second dose. the results showed the value of the tailored formulation approach in developing longacting daptomycin injectable depot preparations. thus, proper selection of polymer composition and molecular weight will enable customizing drug release from plga formulations and reduction in the frequency of dosing. refrences 1. gaurav t, ruchi t, birendra s, bhati l, pandey s, pandey p, etal. drug delivery systems: an 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e, barriere s.l. telavancin pharmacokinetics and pharmacodynamics in patients with complicated skin and skin structure infections and various degrees of renal function. antimicrobial agents and chemotherapy. 2012; 56(4):2062-2066. 23. barry h, david b, michael f, robert d. daptomycin pharmacokinetics and safety following administration of escalating doses once daily to healthy subjects. antimicrobial agents and chemotherapy. 2003; 47(4): 1318-1323. 24. steffy b, jomon n, en bijin, icey c, pramod k, valsalakumari j. microcrystalliation of glipizide: effect of type of stabilizer on particle size, solubility and dissolution. research journal of pharmaceutical, biological and chemical sciences.2013;4(2):405-409 25. siepmann j , faisant n , akiki j ,richard j , benoit j.p . effect of the size of biodegradable microparticles on drug release: experiment and theory. journal of controlled release. 2004; 96(1):123–134. 26. yagnesh b, dushyant s. influence of additives on fabrication and release from protein loaded plga microparticles. journal of chemical and biological researches. 2012; 4(3):1708-1715. 27. ruma m, somasree r, biswarup d, amit k n. ethyl cellulose microparticles containing metformin hcl by emulsification-solvent evaporation technique: effect of formulation variables. isrn polymer science.2012:17 28. meral y, kandemir c. indomethacinloaded microspheres: preparation ,characterization and in-vitro evaluation regarding ethylcellulose matrix material. turkish journal of pharmaceutical sciences. 2008;5(3): 129-142. http://www.cubicin.com/pdf/prescribinginformation.pdf http://www.cubicin.com/pdf/prescribinginformation.pdf http://www.sciencedirect.com/science/article/pii/s0168365904000379 http://www.sciencedirect.com/science/article/pii/s0168365904000379 http://www.sciencedirect.com/science/article/pii/s0168365904000379 http://www.sciencedirect.com/science/article/pii/s0168365904000379 http://www.sciencedirect.com/science/article/pii/s0168365904000379 http://www.sciencedirect.com/science/journal/01683659 http://www.sciencedirect.com/science/journal/01683659 http://www.sciencedirect.com/science/journal/01683659/96/1 iraqi j pharm sci, vol.23(1) 2014 daptomycin poly(d,l-lactide-co-glycolide) microspheres 52 29. microspheres and particles handling guide. polymersciences, inc. chemistry beyond the ordinary. polysciences europe gmbh, u.s. corporate headquarters.1-16. avalible at www.polysciences.com. 30. bryne r, deasy p. use of porous aluminosilicate pellets for drug delivery. journal of microencapsulation. 2005; 22 (4):423-437. http://www.polysciences.com/ iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 6 design, synthesis and kinetic study of coumarin-based mutual prodrug of 5-fluorouracil and dichloroacetic acid yasser f. mustafa * and nohad a. al-omari *,1 * department of pharmaceutical chemistry, college of pharmacy,university of mosul, mosul, iraq. abstract on the basis of known coumarin-based prodrug system, a novel coumarin-based mutual prodrug of 5-fluorouracil and dichloroacetic acid was designed, synthesized and evaluated as a promising oral chemotherapeutic agent basing on in vitro stability study in hcl buffer (ph 1.2) and in phosphate buffer (ph 7.4), as well as in vitro release study in human serum. the chemical structure of prodrug was confirmed by analyzing its ftir, 1 h nmr, 13 c nmr and ms-esi spectra. the results of in vitro kinetic study indicated that the prodrug was significantly stable in hcl and in phosphate buffers, and was hydrolyzed in human serum followed pseudo first order kinetics. keywords: coumarin-based prodrug, 5-fluorouracil, dichloroacetate, kinetics. تصمٍم، تصنٍع ودراسة حركٍة بادئ الذواء التبادلً المرتكز على الكىمارٌن الخلٍك حامض الكلىر وثنائً فلىروٌراسٍل -٥لـ ٌاسر فخري مصطفى * نهاد عبذ الىهاب العمريو ،*1 * ، انؼراق . جايؼة انًىصم،كهٍة انصٍذنةفرع انكًٍٍاء انصٍذالٍَة ، الخالصة -٥ اػحًادا ػهى َظاو بادئ انذواء انًرجكز ػهى انكىيارٌٍ، جى جصًٍى وجصٍُغ بادئ دواء جبادنً غٍر يأنىف يكىٌ يٍ فهىروٌراسٍم وثُائً انكهىر حايض انخهٍك، وجقًٍه كؼالج كًٍٍائً فًىي واػذ اسحُادا انى دراسة االسحقرارٌة خارج جسى انكائٍ انحً (، باالضافة انى ٧,٤( ويحهىل انفىسفٍث انبفري )أس هٍذوجًٍُ ١,٢نبفري )أس هٍذوجًٍُ فً يحهىل حايض انهاٌذروكهىرٌك ا دراسة انححرر فً يصم انذو انبشري خارج جسى انكائٍ انحً. انحأكذ يٍ انشكم انكًٍٍائً نبادئ انذواء يٍ خالل جحهٍم اطٍافه نألشؼة جحث انحًراء، انرٍٍَ انُىوي انًغُاطٍسً نههٍذروجٍٍ جى كربىٌ باالضافة نطٍف انكحهة. نقذ أشارت َحائج انذراسة انحركٍة خارج جسى انكائٍ انحً انى اسحقرارٌة بادئ انذواء انحبادنً بشكم ونه كبٍر فً يحهىل حايض انهاٌذروكهىرٌك انبفري ويحهىل انفىسفٍث انبفري وجحههه فً يصم انذو انبشري يحبؼا حركٍات انرجبة انزائفة األونى. .، ثنائً الكلىر حامض الخلٍك ، الحركٍة فلىروٌراسٍل --٥الذواء المرتكز على الكىمارٌن ، المفتاحٍة : لكلماتا introduction the classical antimetabolite, 5fluorouracil (5-fu), exerts its antitumor effect by inhibiting thymidylate synthase (1) and by misincorporating into dna or rna to inhibit its normal function (2) . it is widely used since its discovery in 1957 as a single agent in treatment of colorectal cancer and as a part of various regimens in treatment of breast, head, neck and upper gastrointestinal tumors (3) . however, 5-fu suffers from many drawbacks; first, it can cause many toxic effects as gastrointestinal disorders, myelosuppression, oral mucositis and cardiotoxicity (4) .secondly, its efficiency is limited by the short plasma half-life after intravenous administration due to the activity of dihydropyrimidine dehydrogenase (dpd), and irregular absorption with unpredictable plasma level after oral administration (5) . thirdly, the resistance of some tumors to chemotherapeutic effect of 5-fu; such resistance is a result of high expression of thymidine phosphorylase or low reserves of reduced folates (6) . to tackle these problems, many approaches have been developed and evaluated; the most important one is the use of prodrug strategy to yield 5-fu prodrugs that can avoid certain routes of degradation [7] or can target the tumor cells (8) . 1 corresponding author e-mail: nohad.alomari@gmail.com received: 3/11/2015 accepted: 2/1/2016 iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 7 there is no doubt that prodrug strategies play a critical role in the development of drug delivery and drug discovery; one of these strategies is a coumarin-based prodrug system that is important for preparing esterasesensitive prodrugs of alcohols, amines and peptides (9) . this system (scheme 1) has several advantages such as the facile lactonization when an acyl group (r) is hydrolyzed by esterase and the safety profile of the final product, coumarin (10) . to date, this system is successfully used to prepare several prodrugs of opioid peptide (11) , non-peptide analgesic (12) and peptidomimetic (13) . o n r''r' o r o esterase rcooh o n r''r' oh spontaneous hnr'r'' o o scheme( 1): the illustration of a coumarin-based prodrug system for amine. metabolic abnormity is a phenotypic trait of cancer cells. it is observed that cancer cells generally utilize glycolysis for energy production rather than oxidative phosphorylation. thus, pyruvate is converted to lactate through anaerobic metabolism rather than its conversion to acetyl-coa by action of pyruvate dehydrogenase (pdh) in aerobic glucose metabolism, this is called warburg effect. pyruvate dehydrogenase kinase (pdk) can inactivate pdh in many glycolytic phenotypes including cancer and switch the metabolism from anaerobic glycolysis to aerobic oxidation which is proved to be detrimental to tumor growth (14-16) . dichloroacetate (dca) has been known for many years as an experimental drug for treating certain metabolic disorders (e.g. inborn errors of metabolism) and as a promising agent in cancer therapy (17) . dca is a well-known inhibitor of mitochondrial pdk; thus it can reverse warburg effect, restore mitochondrial function, induce apoptosis and diminish the growth advantage of highly glycolytic tumors (18) . it is approved that the co-administration of dca with 5-fu potentiates the antitumor activity of 5-fu in treatment of different types of cancer (19) . in addition, there are many reports indicated the beneficial effect of dca in the treatment of glioblastoma, metastatic carcinomas, ovarian cancer, colorectal cancer, endometrial cancer, lung cancer and breast cancer (2022) . the aim of this work was to synthesize a novel coumarin based mutual prodrug of 5fu and dca starting from coumarin and to evaluate it as a promising oral chemotherapeutic agent by monitoring its in vitro stability in hcl buffer (ph 1.2) and in phosphate buffer (ph 7.4), as well as its in vitro release in human serum. experimental materials all chemicals and solvents used in this work were purchased from commercial sources and used without further purification. coumarin was purchased from sigma-aldrich, dca and lialh4 from tokyo chemical industry (tci) and the others from fluka. instruments the instruments used for structure elucidation of the prodrug were: bruker avance drx-400 mhz (germany) to scan nmr spectra that were expressed in part per million upfield to tms as an internal standard, shimadzu lcms-2020 single quadrupole liquid chromatograph mass spectrometer (japan) with electrospray ionization source to measure the mass spectrum. the lcms and nmr spectra were performed in japan via japan food research laboratories (jfrl). bruker-alpha atr-ftir spectrophotometer (germany) to record the ir spectrum that performed in college of pharmacy/university of mosul. melting points were determined, using open capillary method, on an electrochemical cia 9300 melting point apparatus (uk) and were uncorrected. the instrument used to identify uv spectra and to follow kinetic study was carrywinn uv varian uv/visible spectrophotometer. the purity of compounds and the completion of reactions were checked by thin layer http://www.google.iq/url?url=http://www.tcichemicals.com/en/gb/&rct=j&frm=1&q=&esrc=s&sa=u&ved=0ccuqjbawbmovchmizyoagls8xwivzd4uch26iweu&usg=afqjcnf-kv_p74jd43otznj1q-nrf4cvqa http://www.google.iq/url?url=http://www.tcichemicals.com/en/gb/&rct=j&frm=1&q=&esrc=s&sa=u&ved=0ccuqjbawbmovchmizyoagls8xwivzd4uch26iweu&usg=afqjcnf-kv_p74jd43otznj1q-nrf4cvqa iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 8 chromatography (tlc) using precoated silica gel plates (60g f254, merck) and the spots on chromatograms were localized via uv light (at 366 nm). structures, the calculated molecular formula and molecular weights were carried out by using chemdraw ultra 2010. synthesis dichloroacetyl chloride (23). freshly distilled thionyl chloride (10 ml) was added dropwise to a cold solution of dichloroacetic acid (2.1 ml, 25 mmol) in 10 ml dry ether with continuous stirring under anhydrous condition. the mixture was heated at 40°c for 30 minutes and then refluxed for two hours. the solvent and the excess of thionyl chloride were removed under reduced pressure. the product was obtained by distillation at 108°c as slightly yellowish liquid with 82% of yield and λmax (ethanol) = 244 nm. o o (1) oh oh (2) lialh4, 0°c 15 min oh o si (3) tbdm s-cl, dm ap 0°c, 14 h o o si o (4) clcl dichloroacetyl chloride k2co3, ch2cl2, 4 h thf: h2o: hoac (1:1:3) 50 o c, 1 h o cl cl o (5) oh o cl cl o o (6) m no2, chcl3 reflux, 20 h o cl cl o (7) 10 o c, 155 min o oh naclo2, h2o2 5-fu, dcc, dmap (prodrug) tea, rt, overnight o o n nh o o f cl cl o scheme (2): synthetic pathway of coumarin-based mutual prodrug. (z)-2-(3-hydroxypropenyl)phenol (2). a solution of 1 (3.65 g, 25 mmol) in 50 ml dry ether was placed in an ice bath and treated with a solution of 1.0 m of pure lialh4 in dry ether (1.9 g of lialh4 dissolved in 50 ml, 50 mmol). after stirring for 15 min, 5 % hcl (25 ml) was added to the reaction at 0°c. then the solution was adjusted to ph 5 with 1m hcl and extracted with ether (3×50 ml). the ether layer was dried over na2so4, filtered and evaporated. the residue was dissolved in iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 9 ethanol, filtrated and evaporated to afford the desired product. (z)-2-(3-(tert-butyldimethylsilyloxy)propenyl) phenol (3). a solution of compound 2 (3.43 g, 22.8 mmol) in 40 ml dry thf was placed in an ice bath. to this solution, tert-butyldimethylsilyl (tbdms) chloride (3.79 g, 25 mmol) dissolved in 35 ml dry thf was added at 0°c. then n,n-dimethylaminopyridin (dmap) (4.18 g, 34 mmol) in 40 ml dry thf was added in a dropwise manner. after stirring for 14 hours at 0°c, the solution was filtered and evaporated to remove the thf. the residue was re-dissolved in ethyl acetate (50 ml) and washed with 1 m hcl (2 × 27 ml), 5 % nahco3 (22 ml) and h2o (22 ml). the ethyl acetate layer was dried over na2so4, filtered and evaporated. the residue was then crystallized from chcl3. (z)-2-(3-(tert-butyldimethylsilyloxy)propenyl) phenyl dichloroacetate (4) to a solution of compound 3 (2.65 g, 10 mmol) in 25 ml dry ch2cl2 treated with k2co3 (600mg), solution of dichloroacetyl chloride (1 ml, 10 mmol) in 10 ml dry ch2cl2 was added dropwise for 30 minutes. the reaction mixture was refluxed for 4 hours. the progress of reaction was monitored by tlc using ether: ethyl acetate (1:1) mixture as a mobile phase. the reaction mixture was washed with water (2×25 ml); the organic layer was dried over anhydrous na2so4 and concentrated in rotary evaporator to dryness. the residue was re-dissolved in ethyl acetate, filtrated and evaporated to afford the desired product. ( z ) -2 ( 3 hydroxypropenyl )phenyl dichloroacetate (5) to a solution of compound 4 (3 g, 8 mmol) in thf (20 ml), water (20 ml) was added. this was followed by dropwise addition of acetic acid (60 ml). the mixture was stirred at 50ᴼc for one hour and then evaporated to remove thf, water and acetic acid under reduced pressure. ethyl acetate (50 ml) was added to the residue, which was washed with 5 % nahco3 (2 × 25 ml) and water (2 × 25 ml). the ethyl acetate layer was dried over na2so4, filtered and evaporated. the desired product was then crystallized from ethanol. (z)-2( 3-oxopropenyl)phenyl dichloroacetate (6) a suspension of 5 (2.09 g, 8 mmol) and mno2 (3.48 g, 40 mmol) in chcl3 (50 ml) was heated at reflux for 20 hours. the hot mixture was filtered, washed with warm chcl3 (2×25 ml) and the solvent was evaporated under reduced pressure. the residue was re-dissolved in 30 ml acetone, filtered and the filtrate was evaporated to afford the desired product. (z)3[2(dichloroacetyloxy) phenyl ] acrylic acid (7) a solution of sodium chlorite (660 mg, 7.33 mmol) in water (6 ml) was added dropwise very slowly to a stirred mixture of compound 6 (1.04 g, 4 mmol) in acn (15 ml), sodium dihydrogen phosphate (102 mg, 0.85 mmol) in water (1.65 ml), and 30 % hydrogen peroxide (0.5 ml, 4.17 mmol). during the addition, the reaction temperature was kept at 10ᴼc in a cold water bath and oxygen evolution from the solution was observed visually until the end of the reaction. when oxygen evolution was ended (about 155 minutes from the first addition of sodium chlorite), a small amount of sodium sulfite (0.05 g) was added to destroy the unreacted hocl and h2o2. the solution was acidified with 1 m hcl to ph 2. the mixture was then extracted with ethyl acetate (2×50 ml). the combined ethyl acetate layer was washed with saturated sodium chloride solution (2 × 25 ml), dried over anhydrous na2so4, filtered and evaporated. the residue was re-dissolved in acn, treated with aqueous solution of 10% nahco3 to ph 6.5 and filtered. the filtrate was acidified with 1m hcl to ph 2.5 and the desired product obtained by filtration. (z)2 [ 3( 5 – fluorouricyl )-3-oxopropenyl] phenyl dichloroacetate (prodrug) to a cold solution of 7 (1.1 g, 4 mmol) in 50 ml freshly distilled dmso, 5-fu (0.52 g, 4 mmol), dcc (1 g, 4.8 mmol), dmap (40 mg, 0.33 mmol) and triethylamine (0.6 ml , 4 mmol) were sequentially added to the reaction mixture. the solution was stirred overnight at room temperature. after addition of methanol (10 ml) and acetic acid (0.75 ml), the mixture was stirred for one hour and then neutralized with 10% aqueous nahco3 solution. the resulting solid was filtered and the filtrate was evaporated by rotary evaporator. the resulted residue was washed with (20 ml×3) distilled water and then dried. the desired product was crystallized from a mixture of chloroform and petroleum ether. kinetic study the synthesized prodrug was subjected to chemical hydrolysis in buffers of physiological ph values, and enzymatic hydrolysis in human serum. these hydrolytic reactions were monitored by double beam uv/visible spectrophotometer for decreasing in the concentration of prodrug with the time by applying a beer’s law equation, that is: iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 10 absorbance = ԑ × l × c where ԑ is the molar extinction coefficient or called absorbance coefficient. l is the path length of cell holder (2cm). c is the concentration. stability study in acidic buffer (ph 1.2) and in basic buffer (ph 7.4). the in vitro chemical hydrolysis of the prodrug was studied in (0.1 m) hydrochloric acid buffer (ph 1.2) and in (0.1 m) phosphate buffered saline (ph 7.4). a sample (5μmol) of prodrug was dissolved in 2 ml anhydrous dmso in a 100ml beaker. to this solution, 48 ml preheated buffer solution was added with gentle stirring to achieve a final concentration of 100 μm. at the end of addition, the time was detected and the resulted solution was kept at a constant temperature (37 ± 1°c) using a water bath. then, the solution was divided into a set of ten test tubes; each one would contain 5 ml. at selected time intervals of 30, 60, 90, 120, 150, 180, 210 and 240 minutes, a test tube was removed from a water bath and its content extracted with 2 ml ch2cl2. aliquot (2 ml) was withdrawn from aqueous layer and estimated at defined λmax on uv/visible spectrophotometer to determine the remaining concentration of prodrug. release study in serum. the in vitro enzymatic hydrolysis of the prodrug was studied in serum; a sample (2.5μmol) of prodrug was dissolved in 2 ml phosphate buffered saline in a 50ml beaker. to this solution, 23 ml preheated serum was added with gentle stirring to achieve a final concentration of 100 μm. at the end of addition, the time was detected and the resulted solution was kept at a constant temperature (37 ± 1°c) using a water bath. then, the solution was divided into a set of ten test tubes; each one would contain 2.5 ml. at selected time intervals of 30, 60, 90, 120, 150, 180, 210 and 240 minutes, a test tube was removed from a water bath and its content extracted with 2 ml ch2cl2. aliquot (2 ml) was withdrawn from aqueous layer and estimated at defined λmax on uv/visible spectrophotometer to determine the remaining concentration of prodrug. results and discussion the oral use of 5-fu was forsaken during the past decades because of its irregular absorption (24) and unpredictable plasma level with high intraand inter-individual variations due to the fickle activity of dpd in the gastrointestinal mucosa [25] . although the oral use of dca is well tolerated, but it may cause many gastrointestinal (e.g. nausea, vomiting and heartburn) [26] and central nervous system (e.g. neuropathy, confusion and twitching) related side effects that are highly dose dependent [27] . in an attempt to optimize the drug-like properties of 5-fu and dca, a novel coumarin-based esterase-sensitive mutual prodrug was designed, synthesized and evaluated depending on in vitro stability study in hcl buffer (ph 1.2) and in phosphate buffer (ph 7.4), as well as on in vitro release study in human serum. synthetic part the prodrug was synthesized through a sequence of 7 linear steps starting from coumarin; this synthetic pathway (scheme 2) can be considered as a modification to that described by wang et al [28] . the first step involved reduction of coumarin to an open ring diol with lithium aluminum hydride (lialh4). higher temperature than 0°c and/or longer reaction time may lead to reduce the exocyclic double bond whereas the use of commercially available lialh4 may lead to lower the yield to high extend. the following steps involved selective protection of allylic hydroxyl group resulted from the previous step with tbdms-chloride, and acylation of free phenolic hydroxyl group with dichloroacetyl chloride. when aromatic ester is formed, the tbdms ether was cleaved under acidic condition to afford allylic hydroxyl group that converted to carboxylic acid group in two steps. coupling of the free carboxylic acid with 5-fu was carried out by using dicyclohexyl carbodiimide (dcc) as an activating agent. iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 11 table( 1): the physicochemical properties of compounds (1-8). compound number physical appearance yield % m.p. (ᴼc) rf chloroform: acetone (4:1) λmax (nm)e 1 white needle like crystals ------68-70 0.696 315 2 white crystals 41 148-151 0.337 286 3 white crystals 76 122-124 0.556 281 4 white needle like crystals 79 86-88 0.601 307 5 white crystals 92 102-105 0.478 304 6 white powder 68 119-122 0.576 316 7 white crystals 89 154-156 0.489 311 prodrug white needle like crystals 82 136-138 0.589 326 table (2): the calculated molecular formula, molecular weight and elemental analysis of compounds (1-8). comp. no. molecular formula m. wt. elemental analysis %c %h %cl %f %n %o %si 1 c9h6o2 146.14 73.96 4.14 ------------21.90 ---- 2 c9h10o2 150.17 71.98 6.71 ------------21.31 ---- 3 c15h24o2si 264.44 68.13 9.15 ------------12.10 10.62 4 c17h24cl2o3si 375.36 54.40 6.44 18.89 --------12.79 7.48 5 c11h10cl2o3 216.10 50.60 3.86 27.16 --------18.38 ---- 6 c11h8cl2o3 259.08 50.99 3.11 27.37 --------18.53 ---- 7 c11h8cl2o4 275.08 48.03 2.93 25.78 --------23.26 ---- prodrug c15h9cl2fn2o5 387.15 46.54 2.34 18.31 4.91 7.24 20.66 ---- the structure of compounds (2-7) was characterized by monitoring the presence and/or absence of specific functional groups using the ftir spectrophotometer, whereas the prodrug structure was established by analyzing its ftir, 1 h nmr, 13 c nmr and mass spectra. 1 h-nmr (dmso-d6) spectrum of the prodrug (figure 1) showed the chemical shift of the following protons: δ 11.95 ppm (s, 1h, nh), δ 7.97 ppm (d, 1h, -fc=ch-n), δ 7.72 ppm (d, 1h, ar-ch), δ 7.30-7.45 ppm (m, 4h, aromatic), δ 6.45 ppm (d, 1h, ch=ch-co), and δ 6.25 ppm (s, 1h, chcl2). 13 c-nmr (dmso-d6) spectrum of the prodrug (figure 2) reported the chemical shift of the following carbons: δ 164.75 ppm (o-co-), δ 160.90 ppm (=ch-co-n), δ 158.08 ppm (cf-co-), δ 154.03 ppm (-c-o), δ 150.05 ppm (n-conh), δ 143.55 ppm (ar-ch), δ 141.39 ppm (cf), δ 131.88, 127.91, 124.48, 116.93, 116.68 ppm (aromatic), δ 126.09 ppm (fc=ch), δ 118.87 ppm (-ch-co-n) and δ 73.53 ppm (chcl2). iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 12 figure (1): 1 h-nmr (dmso-d6) spectrum of the prodrug. figure (2): 13 c-nmr (dmso-d6) spectrum of the prodrug. iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 13 ftir spectrum of the prodrug revealed the characteristic absorption band for the following functional groups: ν 797 cm -1 (ccl), ν 1167 cm -1 (c-f), ν 1648 cm -1 (c=o, 3ᴼ amide), ν 1668 cm -1 (c=o, 2ᴼ amide), ν 1732 cm -1 (c=o, ester), ν 2822 cm -1 (-ch), ν 3037 cm -1 (=ch) and multiple bands for n-h at ν 3114.33, 3153, 3186 cm -1 . ms-esi spectrum (m/z) of the prodrug operated in a positive mode ( figure 3) characterized the mass of the following products: 388 [m+h] + , 402 [m+nebulizer gas,ch3], 410 [m+na] + , 276 [mo=c=cl2], 248 [m-(o=c=cl2 & co)]. figure (3): ms-esi spectrum (m/z) of the prodrug operated in a positive mode. kinetic part under experimental conditions, the stability study in hcl buffer (ph 1.2) and in phosphate buffer (ph 7.4) showed a significant stability of prodrug with half-lives of about 33hr and 18hr respectively. in human serum, the prodrug was liberated 5-fu and dca followed pseudo first order kinetics (figure 4) with half-life of about 7hr. data obtained from the kinetic study (average of three trials) were listed in table 3, while the kinetic parameters listed in table 4. iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 14 table (3): data obtained from kinetic study. absorbance medium time (min.) x (m×10 6 ) a-x (m×10 6 ) ln a/a-x 0.0568 ph 1.2 0 0 100.000 0 0.0596 ph 7.4 0 100.000 0 0.0531 serum 0 100.000 0 0.0562 ph 1.2 30 1.0391 98.9609 0.0104 0.0584 ph 7.4 1.8940 98.0160 0.0200 0.0505 serum 4.8206 95.1794 0.0494 0.0556 ph 1.2 60 2.0722 97.9278 0.0209 0.0574 ph 7.4 3.7521 96.2479 0.0382 0.0484 serum 8.8512 91.1488 0.0927 0.0551 ph 1.2 90 3.0282 96.9718 0.0308 0.0561 ph 7.4 5.8725 94.1275 0.0605 0.0458 serum 13.7848 86.2152 0.1483 0.0544 ph 1.2 120 4.2077 95.7923 0.0430 0.0553 ph 7.4 7.2148 92.7852 0.0749 0.0436 serum 17.9397 82.0603 0.1977 0.0539 ph 1.2 150 5.1002 94.8998 0.0523 0.0542 ph 7.4 9.1189 90.8811 0.0956 0.0419 serum 21.0923 78.9077 0.2369 0.0533 ph 1.2 180 6.1620 93.8380 0.0636 0.0533 ph 7.4 10.5705 89.4295 0.1117 0.0391 serum 26.3653 73.6347 0.3061 0.0528 ph 1.2 210 7.0246 92.9754 0.0728 0.0519 ph 7.4 12.9195 87.0805 0.1383 0.0376 serum 29.2399 70.7601 0.3459 0.0522 ph 1.2 240 8.0371 91.9629 0.0838 0.0511 ph 7.4 14.1847 85.8153 0.1529 0.0355 serum 33.1450 66.8550 0.4026 a = conc. of prodrug at zero time and (a-x) = conc. of prodrug remaining for any time. table (4): parameters obtained from kinetic study. ph1.2 ph 7.4 serum ԑ = 284 ԑ = 298 ԑ = 265.5 λmax = 313 nm λmax = 338 nm λmax = 332 nm t1/2 = 1991.38 min t1/2 = 1087.91 min t1/2 = 420.80 min kobs = 0.000348 min -1 kobs = 0.000637 min -1 kobs = 0.001647 min -1 ε = absorbance coefficient and kobs = observed rate constants of hydrolysis. iraqi j pharm sci, vol.25(1) 2016 coumarin based mutual prodrug of 5-fluorouracil 15 figure (4): pseudo first order slope of the in vitro hydrolysis of prodrug in human serum. conclusion this work reported the first attempt to utilize a coumarin-based prodrug system to deliver two active moieties which are 5-fu and dca. thus, it is believed that the synthesized compound is a first agent belongs to a new prodrug strategy, the coumarin-based mutual prodrug system, and is a promising oral chemotherapeutic agent. further studies are recommended to establish the result obtained from this work. references 1. longley db, harkin dp and johnston pg. 5-fluorouracil: mechanisms of action and clinical strategies. nat rev cancer 2003; 3: 330-338. 2. phua lc, mal m, koh pk, cheah py, chan ec and ho hk. investigating the role of nucleoside transporters in the resistance of colorectal cancer to 5fluorouracil therapy. cancer chemother pharmacol 2013; 71: 817-823. 3. yoshihiro m, victoria r, tracy aw, neil b, hiroshi and alan et. synergistic 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jw. quantitative evaluation of dichloroacetic acid kinetics in human: a physiologically based pharmacokinetic modeling investigation. toxicology 2008; 245:35-48. 27. kaufmann p, engelstad k, wei y and et al. dichloroacetate causes toxic neuropathy in melas: a randomized, controlled clinical trial. neurology 2006; 66: 324-330. 28. wang b, zhang h and wang w. chemical feasibility studies of a potential coumarin-based prodrug system. bioorg med chem lett 1996; 6: 945-950. iraqi j pharm sci, vol.28(1) 2019 substituted 1, 3, 4-thiadiazole as antibacterial agents doi: https://doi.org/10.31351/vol28iss1pp124-130 124 synthesis of acetylenic derivatives of a substituted 1, 3, 4-thiadiazole as antibacterial agents anwar a. tamer*,1 and ahlam j. qassir* *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq abstract thiadiazole is a heterocyclic compound that exhibits a wide variety of pharmacological activities such as anticancer, antibacterial, antifungal, antimicrobial, anti‐inflammatory, analgesic and anticonvulsant. 2, 5 disubstituted 1, 3, 4-thiadiazole constitutes an important class of compounds for new drug development because it acts as "hydrogen binding domain" and "two-electron donor system." it also serves as a constrained pharmacophore. this research highlights the recently synthesized schiff base and mannich base derivatives and investigation of their chemical and biological behavior. depending on this information’s new derivatives of 1, 3, 4-thiadiazole were synthesized and in the hope of having some activities as antibacterial and antifungal. these are: 1. n-(5-((4-(piperidin-1-yl) but-2-yn-1-yl) thio)-1, 3, 4-thiadiazol-2-yl) acetamide compound (4). 2. 1-(4-chlorophenyl)-n-(5-((4-(piperidin-1-yl) but-2-yn-1-yl) thio)-1, 3, 4 thiadiazol-2-yl) methanimine compound (6). 3. 1-(4-chlorophenyl)-n-(5-(prop-2-yn-1-ylthio)-1, 3,4thiadiazol-2-yl)methanimine compound(7). the characterization of mentioned compounds was performed by ftir spectroscopy, 1hnmr, measurements of their physical properties, and studying of biological activity of the synthesized compounds by well diffusion method. keywords: 1, 3, 4 thiadiazole, schiff base, mannich base كعناصر مضادة للبكتريامعوضة ثياديازول4,3,1تحضير مشتقات أستيلينية لحلقة *أحالم جميل قصير و 1*,أنوار عدنان تمر العراق. ،بغداد، جامعة بغداد ،كلية الصيدلة،فرع الكيمياء الصيدالنية* الخالصة ثياديازول هو مركب غير متجانس ويظهر انواع عديدة من الفعاليات الصيدالنية مثل مضاد للسرطان،مضاد للبكتريا، مضاد للفطريات، ثياديازول ثنائي التعويض يمثل صنف مهم من المركبات لتطوير ادوية 2،5مضاد للميكروبات،مضاد لاللتهابات،مسكن لاللم ومضاد للتشنجات. وذلك بسبب انه يعمل ك"مجال رابط للهيدروجين" و"نظام واهب أللكترونين" هو ايضا يسلك كخاصية دوائية مقيدة.هذا البحث يسلط الضوء جديدة ات قعلى مشتقات كقواعد شف وقواعد مانخ المصنعة حديثا مع البحث في خواصها الكيميائية والحيوية. اعتمادا على هذه المعلومات تم تحضير مشت ثياديازول على امل ان يكون لها فعالية مضادة للبكتريا والفطريات وهذه المركبات هي: 4، 3، 1دة ل جدي ( 4آيل( اسيتامايد المركب)-2-ثياديازول4،3،1 -آيل( ثايو(-1-آين -2-آيل(بيوت -1-)بايبيريدين -4))-5)-ن 1 (.6يل(ميثان إمين المركب) -2-ثياديازول 4،3،1-آيل( ثايو( -1-آين -2-آيل( بيوت -1-)بايبيريدين -4))-5)-ن -كلوروفينيل( -4)-1 2 (.7آيل(ميثان إمين المركب)-2-ثياديازول 4،3،1-آيل ثايو(-1-آين-2-)بروب 5) –ن –كلوروفينيل( -4)-1 3 ات لنووي المغناطيسي للبروتون والخصائص الفيزيائية للمركبتم تشخيص المركبات المذكورة بأستخدام:مطياف االشعة تحت الحمراء والرنين ا المحضرة وكذلك دراسة الفعالية الحيوية للمركبات المصنعة بواسطة طريقة االنتشار. ثياديازول , قاعدة شف , قاعدة مانخ . 4, 3, 1 الكلمات المفتاحية : introduction heterocyclic compounds are the cyclic organic compounds which contain at least one heteroatom, the most common heteroatoms are the nitrogen, oxygen, and sulfur but heterocyclic rings containing other heteroatoms are also widely known (1). heterocyclic compounds are considered as one of the principal classes of organic compounds, which are used in many biological fields, due to their activities. biological molecules such as dna and rna, chlorophyll, hemoglobin, vitamins and many more contain the heterocyclic ring in the significant skeleton (2). heterocycles are used in the development of several pharmaceutically essential compounds in a wide manner. the nitrogen and sulfur heterocyclic systems are important because of their physicochemical properties like lipophilicity with relevance to the design of new drugs (3). 1corresponding author e-mail: noor.adnan85@yahoo.com received: 12/12/2018 accepted: 27 /2/ 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp124-130 iraqi j pharm sci, vol.28(1) 2019 substituted 1, 3, 4-thiadiazole as antibacterial agents 125 thiadiazole nuclei have antimicrobial, antiinflammatory, anticancer, anticonvulsant, antidepressant, antioxidant, radio protective, antileishmanial activities , antimicrobial, antitubercular, antifungal, analgesic, oxidative inhibitors, anti hpylori, herbicides, dyes, lubricants and analytical reagents (4)..(6). the mannich reaction is a crucial c–c bond forming reaction that is widely used in the synthesis of many biologically active natural compounds (7). mannich reactions are three component condensation reactions involving carbonyl compounds, which exist as enol–keto tautomeric forms, formaldehyde and a primary or secondary amine (8). mannich bases are known to have potent activities like anti-inflammatory, anticancer, antibacterial, antifungal, anticonvulsant, antitubercular, analgesic, antiviral, antihistamine activities and in agrochemicals such as plant growth regulators(9)...(11). schiff bases are an essential class of compounds due to their flexibility, structural similarities with natural biological substances and the presence of imine (-n=ch-) which is involved in the mechanism of transformation and racemization reaction in the biological system. these novel compounds could also act as valuable ligands whose biological activity has been shown to increase on complexation (12). also, they have a wide range of biological activities especially anti-bacterial (13), anti-inflammatory (14), anti-fungal (15), anti-tumor (16) anti-oxidant (17), antimicrobial, anthelmintic, anti-inflammatory, analgesic .antipyretic, antitubercular, diuretic, hypoglycemic, anticonvulsant, anti-hiv, cytotoxic (18) recently, the severe infectious diseases caused by gram positive and gram negative pathogenic bacteria have inflated to threat level around the world. this increases, as well as the emergence of bacteria immune to ordinarily used antibiotics, has resulted in the need to develop new categories of antibacterial agents to conflict infections (19). material and methods chemicals used during the synthesis were supplied by hyper-chem (china). completion of reactions and the purity of compounds were monitored by thin-layer chromatography (tlc), using silica gel gf254 (type 60) pre-coated aluminum sheets, merck (germany) exposed to uv254 nm light. two solvent systems were used ethyl acetate: hexane (3:7) and methanol: hexane (8:2). melting points were detected by using stuart smp3 melting point apparatus in open capillary tubes, and are uncorrected. the infrared spectra were performed in kbr disc, (υ, cm-1), using ftir spectrophotometer (shimadzu, wqf-520, japan). 1hnmr spectra were obtained on (nmrready-60 spectrophotometer, 60mhz nanalysis corp, canada) using deuterated acetone and dmsod6 as solvents and tms as an internal standard. chemical synthesis the target compound were synthesized by multistep reaction as shown in scheme 1 . synthesis of 2-amino-5-mercapto 1, 3, 4 thiadiazole (1) (20): thiosemicarbazide (0.043 mole, 4g) was suspended in absolute ethanol (30ml) in a round flask (250ml), anhydrous sodium carbonate (0.021 mole,2.23g) and carbon disulfide (0.125 mole,9.5g) were then added respectively with continues stirring. reflux to the reaction mixture was done for five hours; then allowed to cool to room temperature and filtered. the filtrate was subjected to evaporation under vacuum then cold distilled water (90 ml) was added, followed by acidification with concentrated hcl drop by drop, a white-yellowish precipitate was formed, the precipitate was obtained by filtration, and washed with distilled water, re-crystallized using hot distilled water. product 2-amino-5-mercapto 1, 3, 4 thiadiazole (1) was yellow powder, yield 70%, m.p: 230-232°c, reported (230-232°c), ir: (3325 and 3244) nh2 stretching, (1604) nh2 bending, (1550) c=n stretching, (640) c-s stretching. synthesis of 5-(2-propynylsulfanyl)-1, 3, 4thiadiazol-2-ylamine (2)(21) to a sterried solution of 2-amino-5-mercapto 1, 3, 4 thiadiazole compound (1) (0.1mole, 13.3g) in absolute ethanol (200ml), a solution of potassium hydroxide (0.1mole, 5.6g) in 100 ml absolute ethanol was added. then to the reaction mixture propargyl bromide (0.11 mole 13.3 g) was added drop wise. reflux to the reaction mixture was done for one hour then the reaction mixture was cooled to room temperature, filtered. the filtrate was poured into cold d.w (150 ml), yellow precipitate separated out. the product (2) was yellow crystals, yield 68%, m.p:126-129°c, ir: (3294) for ≡c-h, (3294 and 3263) nh2 stretching, (2978 and2785) ch2 stretching, (2360) c≡c stretching, (1608) nh2 bending, (1492) ch2 bending. synthesis of n-(5-(prop-2-yn-1-ylthio)1,3,4thiadiazol-2-yl)acetamide(3) (22): a mixture of compound (2) (0.022 mole, 3.762g) and acetic anhydride containing 0.5 ml of concentrated h2so4 (0.11 mole, 10 ml), was heated in a steam bath for 1 hour. then the mixture was cooled then poured into 60 ml of cold water. after that, the mixture was boiled to decompose the excess of acetic anhydride. the mixture was left to cool then filtered, and the product was washed with cold water and recrystallized from d.w. iraqi j pharm sci, vol.28(1) 2019 substituted 1, 3, 4-thiadiazole as antibacterial agents 126 compound (3) was off-white powder, yield 70%, m.p:200-203°c, ir: (3255) stretching of (c-h) of triple bond, (3155) nh amide stretching, (2866 and2785) stretching of ch2 and ch3, (2360) stretching of c≡c, (1689) (c=o) of amide stretching, (1558) nh amide bending, (1446-1337) bending of ch2 and (ch3). synthesis of mannich base: n-(5-((4-(piperidin-1yl) but – 2 – yn – 1 – yl ) thio ) – 1 ,3 ,4 thiadiazol2-yl)acetamide(4)(21) to the solution of compound (3) (0.003mole, 0.639g) in free peroxide dioxane (10 ml), paraformaldehyde (0.003 mole, 0.09 g) was added. then piperidine (0.003 mole, 0.3g) and cuprous chloride (catalytic amount) were added. heat the reaction mixture was done by using a water bath at 70-80°c for 3 hours. finally, the reaction mixture was cooled down to room temperature, filtered; the filtrate was poured into ice water mixture (25 ml). the product compound (4) was brown powder, yield 56%, m.p: 138-140ºc, ir :( 3140) stretching of nh amide, (2897 and 2762) stretching of c-h (ch2) and (ch3), (2360) stretching of c≡c, (1701) stretching of(c=o) of amide, (1593) bending of nh amide, (1435) and (1369) bending of c-h(ch2) and(ch3). the 1hnmr spectrum of the compound (4 ) displayed a peak at(δ=1.30ppm) as multiplate for 6 protons of piperidine ring, multiplate peak at (δ=2.13ppm) for 4h of piperidine, singlet peak at(δ=2.22ppm) 3h for ch3 of amide, singlet peak at(δ=3.15ppm) 2h for ch2 beside n, singlet peak at(δ=4.04ppm) 2h for ch2 beside s, and singlet peak at(δ=12.75ppm) 1h for amide n-h. synthesis of deprotected compound 5-((4(piperidin-1-yl)but-2-yn-1-yl)thio)-1,3,4-thiadiazol – 2 amine(5) (23) a mixture of protected compound (4) (0.01 mole, 3g), concentrated hcl (6ml), and ethanol (40 ml) was refluxed for 3 hours in oil bath. after that, the reaction mixture was subjected for evaporation to get rid of a part of ethanol. then filtered, the precipitate obtained was recrystallized from d.w. the product (5) was off-white powder, yield 64.5%, m.p:118-120 ºc, i.r: , (3379 and 3307) nh2 stretching, (2947 and 2881) stretching of c-h of (ch2), (2364) c≡c stretching, (1666 and 1589) nh2 bending, (1462) bending of c-h(ch2). synthesis of 1-(4-chlorophenyl)-n-(5-((4(piperidin-1-yl)but-2-yn-1-yl)thio)-1,3,4 thiadiazol – 2 – yl ) methanimine compound(6)(24) compound (5) (0.002 mole, 0.536g) was suspended in 25 ml of absolute ethanol. pchlorobenzaldehyde (0.002 mole, 0.28g) in 25 ml of absolute ethanol solution was added with few drops of glacial acetic acid. the mixture was then refluxed for 8 hours and later left it overnight at room temperature. the solvent was evaporated in vacuum to get 1-(4-chlorophenyl)-n-(5-((4-(piperidin-1-yl) but-2-yn-1-yl) thio)-1, 3, 4 thiadiazol-2-yl) methanimine compound (6). compound (6) was recrystallized from methanol. the compound (6) was gray powder, yield 48%, m.p: 247-250 ºc, i.r: (3070) stretching of ch of aromatic ring,(2947 and 2881) stretching of ch (ch2), (2360) c≡c stretching, (1700) c-h stretching of n=c(imine), (1593 and 1442) c=c stretching of aromatic ring, (1500) bending ch(ch2),(1037) in plane bending of c-h of aromatic ring, (860) out of plane bending of c-h of aromatic ring, (686) c=c bending of aromatic ring. 1hnmr for compound (6) recorded the following important signals, (δ=1.29 ppm) as a multiplate peak attributed to 6h of piperidine ring. a triplet peak at δ= (2.04 ppm) 4h of piperidine ring beside n, singlet peak at (δ=3.02 ppm) 2h for ch2 beside s and 2h beside n overlapped, c-h proton of c=n appear as singlet peak at (δ=10.05 ppm). (δ= 7.48 and 7.61ppm) as singlet peak for aromatic h ortho to cl, (δ= 7.98 and 8.11ppm) as singlet peak for aromatic h meta to cl. synthesis of schiff base derivatives (1-(4chlorophenyl)-n-(5-(prop-2-yn-1-ylthio)-1, 3, 4 thiadiazol – 2 – yl ) methanimine compound (7) (24) compound (2) (0.002 mole, 0.342g) was suspended in 25 ml of absolute ethanol. pchlorobenzaldehyde (0.002 mole, 0.28g) in 25 ml of absolute ethanol solution was added with few drops of glacial acetic acid. the mixture was refluxed for 8 hours left overnight. the solvent was evaporated in vacuum and the residue was recrystallized from methanol. the product (7) was off-white powder, yield 56%, m.p:106-110ºc, ir (3271)≡c-h stretching, (3093) c-h stretching of aromatic ring, (2947 and 2835) stretching of c-h(ch2), (2360) c≡c stretching, (1697) c-h of n=c stretching (imine), (1585 and 1423) c=c of aromatic ring stretching, (1489) bending of c-h (ch2), (1014 in plane bending of c-h of aromatic ring, (850)out of plane bending of c-h of aromatic ring, (700) c=c of aromatic ring bending. 1hnmr for compound (7) recorded the following important signals, (δ=3. 84 ppm) for 2h of ch2 beside s and c-h binded to triple bond, singlet peak at (δ=7.33 ppm) 4h of aromatic protons, and peak at (δ=8.96 ppm) for 1h of h-c=n as a singlet. antimicrobial activity the antimicrobial activity of the final compounds was evaluated in the university of baghdad / college of education for pure sciencesibn al-haitham by the advisory office of the central service laboratory.a preliminary antibacterial have been carried using the well diffusion method. the synthesized compounds were evaluated for their antimicrobial activity in vitro iraqi j pharm sci, vol.28(1) 2019 substituted 1, 3, 4-thiadiazole as antibacterial agents 127 against three types of tested microorganisms (staph. aureus., and bacillus subtilis as a gram-positive bacteria) and (klebsiella pneomonae, and e. coli) as a gram-negative bacteria) were clinically activated and maintained on nutrient agar for examining antibacterial activity. ampicillin was used as a standard drug for antibacterial activity. ir of compound (7) result and discussion synthesis of compound (1) the 2-amino-5-mercapto-1, 3, 4thiadiazole was synthesized by steps of reactions starting from thiosemicarbazide with carbon disulfide in basic medium (25). synthesis of compound (2) compound (2) was prepared by alkylating the potassium salt of compound (1) with propargyl bromide. it is logical to assume that the alkylation step followed an sn2 mechanism. the reaction is started by nucleophilic attack of the sulfide anion on the propargyl bromide affording the desired alkylated thiadiazole derivative. no allylic rearrangement was observed (26). synthesis of compound (3) in the acetylation of compound (2), where this step includes the synthesis of amide; it was done by treatment of an amine with acetic anhydride in the presence of a few drops of sulphuric acid as catalyst. switching of the amino group into the acetamido group by acetylation modifies the interaction of the nitrogen lone pair with the π-electron system of the aromatic ring so that the ring is less powerfully activated toward electrophilic attack (27). synthesis of compound (4) mannich reaction is a nucleophilic addition reaction that involves the condensation of a compound with active hydrogen(s), with an amine (primary or secondary) and formaldehyde (any aldehyde) (9). synthesis of compound (5) acid hydrolysis reaction occurs by nucleophilic addition of water to the protonated amide, followed by transfer of a proton from oxygen to nitrogen to make the nitrogen a better leaving group and for subsequent elimination. the steps are reversible with the equilibrium shifted towards the product by the protonation of the nh2 in the final step (28). synthesis of compound (6) and (7) the mechanism of schiff base formation is a reversible, acid catalyzed process, begins with nucleophilic addition of the primary amine to a carbonyl group (aldehyde or ketone) followed by a transfer of a proton from nitrogen to oxygen to yield neutral amino alcohol or carbinolamine. protonation of the carbinolamine oxygen by an acid catalyst then converting the (-oh) group into a better leaving group (–oh2), and e1like loss of water produces an iminium ion which after the loss of a proton from nitrogen gives the schiff base and regenerate the acid catalyst to afford compounds (6) and (7)(29)(30). iraqi j pharm sci, vol.28(1) 2019 substituted 1, 3, 4-thiadiazole as antibacterial agents 128 scheme (1) synthesis of compounds (1) to (7) iraqi j pharm sci, vol.28(1) 2019 substituted 1, 3, 4-thiadiazole as antibacterial agents 129 antibacterial activity table (1) the antibacterial activities of synthesized compounds. compound conc. mg/ml klebsiella pneumonia g-ve e.coli g-ve bacillus subtilis g+ve staphylococcus aureus g+ve comp.(4) 0.01 13 13 comp.(6) 0.01 13 15 11 comp.(7) 0.01 13 15 13 24 ampicillin std. 0.01 25 comp.(4) 0.02 16 16 16 comp.(6) 0.02 17 20 16 comp.(7) 0.02 19 17 17 25 ampicillin std. 0.02 25 (-)= no activity, (+) = slightly active (inhibition zone in between 5-10 mm), (++) = moderately active (inhibition zone in between 10-15 mm), (+++) =highly active (inhibition zone more than 15 mm). the recorded data in table (1) lead to the following conclusions: all the synthesized compounds showed antimicrobial activity against g (+ve) and g (-ve) bacteria, but some of them showed no activity against (staphylococcus aureas) (compound 4 and compound 6) at concentration (0.01) mg/ml and even in concentration (0.02 mg/ml). compound (7) showed activity against previously mentioned g (+ve) (staphylococcus aureas) but at higher conc. showed higher antibacterial activity and it is schiff base derivative, its benzene ring containing an ewithdrawing group (cl) at the para position for compounds (6) which are a combination of mannich base and schiff base derivative showed moderate to higher antibacterial activity at both concentrations against tested bacteria. conclusions new derivatives of 2-amino 5-mercapto1, 3, 4 thiadiazole were successfully synthesized using the conventional method. the synthesis of these proposed compounds was successfully performed by the stated procedures as previously described. the results obtained from this investigation were achieved according to the data shown by the physical and chemical analysis including (tlc, melting point, ftir and 1hnmr analysis). compound 4, 6 & 7 exhibit good antimicrobial activity comparable with marketable compounds. the antimicrobial evaluation indicated that the newly synthesized compounds showed moderate to high antimicrobial activity in comparison to ampicillin for gram-positive bacteria and also have highest anti-microbial activity for gram negative bacteria. the compound (7) showed an excellent antimicrobial activity, and highest activity against staphylococcus areus compared to ampicillin. acknowledgments the authors gratefully thank the college of pharmacy, university of baghdad, for supporting this research. references 1. mishra g, singh ak, jyoti k. review article on 1, 3, 4-thiadiazole derivatives and its pharmacological activities. int j chem tech res. 2011; 3:1380-93. 2. al-mulla a. a review: biological importance of heterocyclic compounds. der pharma chemica. 2017; 9(13):141-7. 3. balaji k, bhatt pr, mallika d, jha an. design, synthesis and antimicrobial evaluation of some mannich base derivative of 2 (2-substituted)-5amino-thiadiazoles. international journal of pharmacy and pharmaceutical sciences. 2015;7:145-9. 4. kokila. p, sarju. p, rinku. p, rekha. p. a simple and efficient procedure for synthesis of biologically active 1,2,4-triazolo-[3,4-b]1,3,4-thiadiazole -2-aryl-thiazolidine-4-one derivatives. res. j. chem. sci.2011; vol. 1, 1821. 5. bhuva h, sahu d, shah b, modi dc, patel mb. biological profile of thiadiazole. pharmacol online. 2011;1:528-45. 6. singh. a.k, mishra. g , jyoti.k, review on biological activities of 1,3,4thiadiazolederivatives journal of applied pharmaceutical science; 2011: vol.5, 44-49. 7. barta p, fülöp f, szatmári i. mannich baseconnected syntheses mediated by ortho-quinone methides. beilstein journal of organic chemistry. 2018 mar 6;14(1):560-75. 8. demirbas a, sahin d, demirbas n, karaoglu sa. synthesis of some new 1, 3, 4-thiadiazol-2ylmethyl-1, 2, 4-triazole derivatives and iraqi j pharm sci, vol.28(1) 2019 substituted 1, 3, 4-thiadiazole as antibacterial agents 130 investigation of their antimicrobial activities. european journal of medicinal chemistry. 2009 jul 1;44(7):2896-903. 9. bala s, sharma n, kajal a, kamboj s, saini v. mannich bases: an important pharmacophore in the present scenario. international journal of medicinal chemistry. 2014;2014. 10. manjula ps, sarojini bk, narayana b, raj cd. an exploration on the synthesis and bioapplications of derivatives of heterocyclic mannich bases. journal of fundamental and applied sciences. 2016;8(1):115-75. 11. a. balakrishnan, dr. a. sankar. study on the synthesis, characterization and antimicrobial activity of the new mannich base benzimidazolyl phenyl methyl acetamide and its metal complexes.ijppr human journal. 2017; 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(eds:): amines react with carbonyl compounds. in: organic chemistry. oxford university press inc., new york, 2001, pp 349-352. iraqi j pharm sci, vol.24(1) 2015 antibacterial evolution of bis heterocyclic derivatives 59 synthesis and antibacterial activity of bis heterocyclic derivatives of 1,3,4-thiadiazole ahlam a. al-jubouri ٭ ,1 and ahlam j. qasir٭٭ .department of registration , ministry of health, baghdad, iraq ٭ .department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq ٭٭ abstract the chemistry of heterocyclic sulphur and nitrogen containing compounds have a great role in the field of scientific studies, the 2-amino 5-mercapto-1,3,4-thiadiazole ring for instance, has gained more importance in recent years because they are considered as potent biologically active nucleus. in this study disulfide derivative can be obtained by oxidation with hydrogen peroxide of thiol group of the heterocyclic 2-amino 5-mercapto-1,3,4-thiadiazole ring to obtain compound (3) with expected antibacterial activity. in order to use it as a diazo component to prepare some new bis azo compounds as possible antibacterial agents, the reaction of two primary amino groups on both sides of disulfide dimer with sodium nitrite was carried out to prepare diazounium salt which was coupled with different coupling compounds to form the azo linkage. the reaction steps and the purity of the products were confirmed by thin layer chromatography (tlc) and melting points measurement .the chemical structure of the final compounds were characterized and confirmed by measuring their ft-ir spectroscopy and elemental microanalysis(c, h, n &s).these compounds were screened for their antibacterial activity which was done on four different strains of bacteria by well diffusion method. these compounds show moderate to good activity against tested gram positive bacteria and low or no activity against gram negative bacteria compared to the standard compounds. key words: disulfide dimer, diazo compound, antibacterial activity. ٤, ١,٣كمشتقاث مركباث ثنائيت حهقيت غير متجانست ن بكتريانه ةفعانيت مضادو خهيقت زول ياثياد احالو عذنان انجبوري ،*٣ جميم قصيرو احالو ٭٭ ٭ انؼشاق. ،بغذاد ،ٔصاسة انصحت انخسجٍم،لسى ٭٭ . قانؼشا ،دبغذا ،جايؼت بغذاد ،كهٍت انصٍذنت ،فشع انكًٍٍاء انصٍذالٍَت الخالصة ٕي انكبشٌج ٔانُخشٔجٍٍ حًخهك دٔس ػظٍى فً يجال انذساساث انؼهًٍت , فؼهى خاٌ كًٍٍاء انًشكباث انحهمٍت غٍش انًخجاَست انخً حح اكخسبج اًٍْت اكبش فً انٕلج انشاٍْ الَٓا حؼخبش َٕاة فؼانت صٔل ٌاثٍاد ٤, ١,٣يٍشكبخٕ – ٥ايٍُٕ -۲سبٍم انًثال انحهمت صٔل باكسذة يجًٕػت ٌاثٍاد ٤, ١,٣يٍشكبخٕ – ٥ايٍُٕ -۲ فً ْزِ انذساست حى ححعٍش يشخك ثُائً انكبشٌج نهحهمت .˝بإٌنٕجٍا كب انُٓائً االٔل رٔ فؼانٍت يخٕلؼت كًعاد انثإٌل فً ْزِ انحهمت غٍش انًخجاَست بٕاسطت بٍشٔكسٍذ انٍٓذسٔجٍٍ نهحصٕل ػهى انًش اصٔ نخحعٍش بؼط يشكباث ثُائً االصٔ انجذٌذة راث فؼانٍت يعادة ثُائً ْزا انًشكب كًكٌٕ نًشكباث ؼًال.نغشض اسخبكخشٌانه نذاٌا صٍَٕٔو انزي حى ػهى جٓخً ثُائً انكبشٌج يغ َخشٌج انصٕدٌٕو نخحعٍش يهح ا االٔنٍتار حى يفاػهت يجًٕػخً االيٍٍ بكخشٌانه حى انخاكذ يٍ خطٕاث انخفاػم َٔمأة انُٕاحج بٕاسطت كشٔياحٕغشافٍا انطبمت انشلٍمت) .سبطّ بًشكباث يخخهفت نخكٌٍٕ اصشة االصٔ tlc) نٓا .انخشاكٍب انكًٍٍائٍت نهًشكباث انُٓائٍت لذ حى حشخٍصٓا ٔانخاكذ يُٓا بٕاسطت لٍاط اغٍاف االشؼت سٔ لٍاط دسجاث االَصٓا اسبؼت ؼًال( .كزنك حى انخؼشف ػهى فؼانٍخٓا كًعاداث نهًٍكشٔباث باسخc,h,n&sش)ححج انحًشاء ٔانخحهٍم انكًً انذلٍك نهؼُاص لهٍهت أ نًشكباث فؼانٍت يخٕسطت انى جٍذة ظذ انبكخشٌا يٕجبت انغشاو ٔفؼانٍتزِ اْ اظٓشثانمشص .نمذػضالث يخخهفت بطشٌمت اَخشاس فً ْزِ انذساست بانًماسَت يغ انًشكباث انمٍاسٍت.ؼًهت يؼذٔيت حجاِ انبكخشٌا سانبت انغشاو انًسخ ثنائي داي سهفايذ ، مركباث انذايازو، انفعانيت انمضادة نهبكتريا .انكهماث انمفتاحيت : introduction the rapidly expanding population of immunocompromised patient results in a corresponding increase of diseases caused by bacteria, fungi and other yeast. infections caused by these microorganisms pose a serious challenge to the medical community and highlight the importance and urgent need for new, more potent and selective antimicrobial agents. the incidence of bacterial infections has increased dramatically in recent years (1) . drug resistance by microorganisms is of increasing importance as the phenomenon has considerable impact on human and animal health. the prevalence of clinical drug resistance has unfortunately increased significantly in recent decades due to the use 1 corresponding author e-mail: ahlamaljubouri@yahoo.com received:14 /1/2015 accepted: 18/5/2015 iraqi j pharm sci, vol.24(1) 2015 antibacterial evolution of bis heterocyclic derivatives 60 and misuse of antimicrobial drugs and many infectious diseases can no longer be treated effectively with common anti-infective drugs. antimicrobial resistance is not limited to bacterial species and in the 1990s resistance emerged to one of the most potent antifungal agents, fluconazole. antifungal resistance is particularly problematic as diagnosis is often delayed and there are relatively few antifungal treatments approved for use by the fda (2) .heterocyclic play an important role in biochemical processes because the side groups of the most typical and essential constituents of living cells are based on aromatic heterocycles. between them, sulfur and nitrogen-containing heterocyclic compounds have maintained the interest of researchers through the development of organic synthesis. some of them are tetrazoles, fused thiazoles, thiadiazoles, oxadiazoles, triazoles, which are structural subunits of several biologically active compounds (3) . thiadiazole derivatives possess biological activity probably conferred to them by the strong aromaticity of this ring system, which leads to great in vivo stability and generally, a lack of toxicity for higher vertebrates, including humans. thiadiazole can act as the bio-isosteric replacement of the thiazole moiety. so it acts like third and fourth generation cephalosporin (4) . 1, 3, 4thiadiazole ring containing compounds represent an important class of heterocyclic nitrogen compounds and their derivatives are characterized with a broad spectrum of biological activity in both agrochemical and pharmaceutical fields (5,6) . many 1,3,4thiadiazoles derivatives have been used as “privileged” scaffolds to produce substances of interest in numerous therapeutic areas (7) such as antituberculosis (8,9) , anti-inflammatory (10) ,analgesic (11) ,anticonvulsants (12) , antihypertensive (13) , antioxidant (14) , anticancer (15) ,antiviral (16) ,antidepressant (17) , anti-hiv, antiproliferative activities (18) . compounds possessing 1, 3,4-thiadiazole ring system show antifungal, bacteriostatic as well as anthelmintic effects (19,20) . furthermore, five membered heterocyclic compounds show various types of biological activity among them 2,5-disubstituted 1,3,4-thiadiazoles are associated with diverse biological activities probably, due to –n=c-sgrouping (21) . hence the current work is the need for the development of novel antimicrobial agents to combat the bacterial infections. materials and methods aceticanhydride, 4-aminophenol, carbon disulfide, 1-naphthol, 2-naphthol, thiosemicarbazide. all the solvents and materials used were of analar type and used without further purification. the ascending tlc was run on silica gel f-254 (type 60) precoated aluminum sheets, for checking the purity of the products as well as monitoring the progress of the reaction. the final products and their intermediates were detected by reacting with iodine vapor or by irradiation with uv light. infrared spectral determination was performed for all compounds in kbr disk, using ftir at the college of pharmacy, university of baghdad and at the college of science al-mustansiriya university. elemental microanalysis has been done using elemental analyzer euro vector, and was done at the college of science, al-mustansiriya university. chemical synthesis synthesis of p-hydroxy acetanilide (compound 1) (22) . in a 100 ml conical flask containing paminophenol (25 mmole, 2.725g) and water (7.5ml). acetic anhydride (31.7 mmole, 3ml) was added with constant shaking. the mixture was stirred vigorously and warmed on a water bath for about 15 minutes until the solid dissolves completely. upon cooling, phydroxy acetanilide was precipitated which was collected by suction filtration , then washed with a little cold water, drained well and recrystallized from hot water (about 18.5ml) and dried.the physical appearances, percentage yield, melting point,and rf values are listed in table 1. synthesis of 5-amino-1, 3, 4-thiadiazole-2 thiol (compound 2) ( 23) . to thiosemicarbazide (100 mmole, 10g) suspended in absolute ethanol (40 ml), anhydrous sodium carbonate (54 mmole, 5.82 g) were added and carbon disulfide (120 mmole, 10.1 g). the reaction mixture was heated with stirring under reflux for five hours .the completion of the reaction was indicated by tlc. the solvent was largely removed by rotatory evaporator and the residue was dissolved in water (44 ml), then acidified with concentrated hydrochloric acid (8.8 ml). the product was recrystallized from ethanol/water to give a pure compound. the physical appearances, percentage yield, melting point, and rf values are listed in table 1, the elemental analysis results are presented in table 2 while the ir data are shown in table 3. synthesis of 5, 5’dithiobis (2-amine -1, 3, 4thiadiazole) (compound 3) ( 24) . hydrogen peroxide (3.1ml, 30%) was added drop wise to a suspension of compound (2) (10 mmole, 1.33g) in ethanol (30ml) with continuous stirring for 1 hour at room temperature, a yellow precipitate was formed, this precipitate was collected by filtration, iraqi j pharm sci, vol.24(1) 2015 antibacterial evolution of bis heterocyclic derivatives 61 washed with hot ethanol to afford the pure product and dried in oven at 60 o c to provide compound (3).the physical appearances, percentage yield, melting point and rf value are listed in table 1, the elemental analysis results are presented in table 2 while the ir data are shown in table 3. synthesis of 5, 5’ dithiobis ( 2amino -1, 3, 4-thiadiazole) diazonium salt (compound 4) ( 25 ) . compound 3 (1.32 g, 5mmole) was dissolved in 6 ml of concentrated hydrochloric acid and 6 ml of water in a suitable beaker; the resulting solution was stirred and cooled by immersing in a bath of crushed ice; throughout the reaction the temperature was kept below 5 o c. a cold solution of (0.75g, 11mmole) sodium nitrite in (5 ml) water was placed in a dropping funnel which was cooled using crushed ice, then it was added dropwise into the first solution in the ice bath with continues stirring ,the temperature should not be allowed to rise above 10°c.the last quantity of the sodium nitrite solution was added more slowly and after stirring for 3-4 minutes, a drop of the solution diluted with 4 drops of water was tested with potassium iodide-starch paper; if no immediate blue color was obtained at the point of contact with the paper, a further amount of sodium nitrite solution was added. the testing was continued every 5 minutes until an immediate blue color was obtained. a solution of sulfamic acid (1.5ml) of 2% w/v was added and stirring was continued for 20 minutes. the diazonium salt formed was used immediately in the following synthesis of compounds (4a4c). synthesis of 1,1'-(1z,1'z)disulfanediylbis(1,3,4thiadiazole-5,2-diyl)bis(diazene-2,1diyl)dinaphthalen-2-ol(compound 4a) (26) . 2-naphthol (1.44 g, 10 mmole) was dissolved in (8 ml) of (10 %) naoh in a suitable beaker immersed in an ice bath. the solution was stirred vigorously and the temperature was kept below 5°c by the addition of crushed ice. the cold diazonium salt solution from the previous step (compound 4) was placed in a dropping funnel, then it was added drop by drop to the cooled, stirred 2naphthol solution; a deep red color was developed and a red crystals soon separated. at the end of the addition the mixture was stirred for 3hours in the ice bath. then the solution was filtered through buchner funnel, washed well with water, and recrystallized from glacial acetic acid (3ml); washed with a little absolute ethanol to eliminate acetic acid and dried. the physical appearances, percentage yield, melting point and rf value are listed in table 1, the elemental microanalysis results are presented in table 2 while the ir data are shown in table 3. synthesis of 4,4'-(1z,1'z)-5,5'-disulfanediylbis(1,3,4-thiadiazole-5,2-diyl)bis(diazene-2,1diyl)dinaphthalen-1-ol (compound 4b) ( 26) . 1-naphthol (1.44 g, 10 mmole) was dissolved in (8 ml) of (10 %) naoh in a suitable beaker immersed in an ice bath. the solution was stirred vigorously and the temperature was kept below 5°c by the addition of crushed ice. the same procedure was carried out as in the synthesis of compound (4a). the deep bluish violet color was developed and a deep blue crystals soon separated. at the end of the addition the mixture was stirred for 3 hours in the ice bath. then the solution was filtered through buchner funnel, washed well with water, and recrystallized from glacial acetic acid (3ml); washed with a little ethanol to eliminate acetic acid and dry upon filter paper. the physical appearances, percentage yield, melting point and rf value were listed in table1, the elemental microanalysis results are presented in table 2 while the ir data are shown in table3. synthesis of n , n '( 3 , 3 '( 1 z , 1 'z)-5,5'disulfanediylbis(1,3,4-thiadiazole5,2diyl)bis(diazene-2,1-diyl)bis(4-hydroxy3,1-phenylene)) diacetamide (compound 4c) (26) . p-hydroxyacetanilide (compound 1) (1.51 g; 10 mmole) was dissolved in (8 ml) of (10 %) naoh in a suitable beaker immersed in an ice bath. the solution was stirred vigorously and the temperature was kept below 5°c by the addition of crushed ice. the same procedure was carried out as in the synthesis of compound (4a and4b) .the deep brown color was developed and a brown crystals soon separated. at the end of the addition the mixture was stirred for 1h in the ice bath. then the solution was filtered through buchner funnel, washed well with water, and recrystallized from glacial acetic acid (3ml); washed with a little absolute ethanol to eliminate acetic acid and dried. the physical appearances, percentage yield, melting point and rf value are listed in table 1, the elemental microanalysis results are presented in table 2 while the ir data are shown in table 3. iraqi j pharm sci, vol.24(1) 2015 antibacterial evolution of bis heterocyclic derivatives 62 table (1): physical appearance, percentage of yield, melting points, rf values of intermediates and final compounds. compound no. physical appearance %yield observed melting point (˚c) reported melting point (c) rf values solvent system 1 grey crystal 83.6% 168-169 169 0.72 a 2 pale yellow powder 73.4% 241-243 240-242 0.87 b 3 yellow powder 58.7% 233-235 233-235 0.92 c 4a deep red powder 81.8% 81 (decom.) --------0.92 d 4b deep blue powder 66.1% 170 (decom.) -------- 0.71 d 4c brown crystal 85.3% 201-203 -------0.88 a a) toluene: methanol (9:1), b) chloroform: ethylacetate: methanol (2:2:1) c) n-hexane: isopropanol: methanol (9: 0.9:0.1), d) chloroform: ethanol (8:2) table (2): elemental microanalysis of the final compounds table (3): characteristic ir absorption bands of intermediates and final compounds elemental microanalysis % empirical formula molecular weight compound observed calculated element 18.201 18.181 c c4h4n6s4 264 3 1.465 1.515 h 31.943 31.818 n 48.391 48.48 s 50.167 50.174 c c24h14o2n8s4 574 4a 2.434 2.439 h 19.521 19.512 n 22.288 22.299 s 50.079 50.174 c c24h14o2n8s4 574 4b 2.441 2.439 h 19.57 19.512 n 22.331 22.299 s 40.798 40.816 c c20h16o4n10s4 588 4c 2.712 2.721 h 23.912 23.809 n 21.698 21.768 s compound band(cm -1 ) interpretation 1 3321 3109 2991, 2881 1651 1608 ,1560 1506 1437 1437 ,1369 1255 , 1224 1170 1107 ,1014 853 ,802 o-h stretching of phenol and n-h stretching of amide. c-h stretching of aromatic ring. c-h asymmetrical and symmetrical stretching of ch3. c=o stretching of amide and c=n stretching of heterocyclic ring aromatic c=c stretching n-h bending of amide. o-h in plane bending c-h bending of ch3. aromatic c-h in-plane bending. c-o stretching of phenol aromatic c-h out of plane bending. iraqi j pharm sci, vol.24(1) 2015 antibacterial evolution of bis heterocyclic derivatives 63 table (3): continued characteristic ir absorption bands of intermediates and final compounds antibacterial activity assessment (27) . the antibacterial activity of the synthesized compounds was investigated in comparison with cefotaxime (30 μg/ disc) and amoxicillin (25 μg/disc) which were used as a reference antibacterial activity against grampositive bacteria (staphylococcus aureus, staphylococcus epidermidis) and gramnegative bacteria (escherichia coli, klebsiella pneumonia ) antibacterial activities of each compound were evaluated by well diffusion method using mueller–hinton agar as culture media (27) . the synthesized compounds were dissolved in dimethylsulfoxide to prepare the stock solution (20mg/ml) and the solution was diluted with dimethylsulfoxide: distilled water (1:5) to obtain the required concentrations of 0.4, 0.8and 1.0 mg/ml. the petri dishes were inoculated with (30 μl) separately of each concentration of the synthesized compounds for each well and incubated at 37 °c for 24 h. to ensure that the solvent had no effect on the bacterial growth, a control was performed with the test medium supplemented with dmso at the same dilutions as used with the tested compounds. at the end of the period the inhibition zones formed on media were measured with a zone reader in millimeters. the inhibition zone values are summarized in table 4. compound band(cm -1 ) interpretation 2 3329 ,3246 2642 1606 1548 1172 n-h stretching vibration of primary amines. s-h stretching of thiol. c=n stretching of heterocyclic ring. n-h bending of primary amine. c-n stretching of heterocyclic ring 3 3255 1635 1134 n-h stretching vibration of primary amines. c=n stretching of thiadiazole ring moiety. c-n stretching of thiadiazole ring moiety. 4a 3286 3053 1627 1508 ,1600 1464 1274 ,1242 1213 844 , 813 o-h stretching vibration of 2-naphthol. c-h stretching of aromatic ring. c=n stretching of aromatic ring. c=c stretching of heteroaromatic ring. n=n stretching. aromatic c-h in-plane bending. c-n stretching. aromatic c-h out of plane bending. 4b 3248 3061 1620 1595,1541 1498 1253 ,1230 1207 871 ,839 ,748 682 o-h stretching of 1-naphthol. c-h stretching of aromatic ring. c=n stretching of aromatic ring. c=c stretching of heteroaromatic ring. n=n stretching. aromatic c-h in-plane bending. c-n stretching. aromatic c-h out of plane bending. c=c bending of aromatic ring. 4c 3284 ,3306 3091 2933 1637 1608 1560 1535 1500 1386 ,1367 1257 , 1224 1170 871 ,839 ,748 677 o-h stretching and n-h stretching of amide. c-h stretching of aromatic ring. c-h stretching of ch3. c=o stretching of amide. c=n stretching of heteroaromatic ring. c=c stretching of aromatic ring. n-h bending of amide. n=n stretching. c-h bending of ch3. aromatic c-h in-plane bending. c-n stretching. aromatic c-h out of plane bending. c=c bending of aromatic ring. iraqi j pharm sci, vol.24(1) 2015 antibacterial evolution of bis heterocyclic derivatives 64 table (4): the antibacterial screening data for final compounds. zone of inhibition in mm compound no. klebsiella pneumonia escherichia coli staphylococcus epidermidis staphylococcus aureus -ve -ve 19 19 12 μg/ well 3 -ve -ve 22 22 24 μg/ well -ve -ve 22 23 30 μg/ well -ve -ve -ve 20 12 μg/ well 4a -ve -ve -ve 20 24 μg/ well 8 8 -ve 22 30 μg/ well -ve -ve -ve 7 12 μg/ well 4b -ve -ve -ve 8 24 μg/ well -ve -ve -ve 16 30 μg/ well -ve -ve 25 25 12 μg/ well 4c -ve -ve 29 26 24 μg/ well -ve -ve 30 29 30 μg/ well -ve -ve -ve -ve ------dmso 26 25 18 -ve 30 μg/ disc cefotaxime 15 -ve 13 15 25 μg/ disc amoxicillin results and discussion the synthesis of the designed compounds was successfully achieved by following the stated procedures as shown in (scheme 1). the fourier transform infrared (ft-ir) spectra of the final synthesized compounds and their intermediates showed the characteristic bands of absorption by which they were identified.ir data help not only to identify the final compounds, but also they are advantageous to follow up the reactions depending on the appearance or disappearance of specific group frequencies. the values of the interesting bands of these spectra are presented in table (3). for compound (3),we noticed the absence of the s-h stretching band at 2624 cm -1 and the compound was insoluble in 5% koh solution but soluble in 5% hcl solution due to presence of basic group (nh2) on both sides of compound and absence of acidic group ( -sh) ,for compound (4a), we noticed the appearance of n=n stretching band at 1464 cm -1, ,the disappearance of n-h primary amine stretching bands of dimer at 3255 cm -1 and appearance of o-h of 2naphthol stretching bands at 3286 cm -1 ; and the color of the compound was changed from yellow to deep red and it was soluble in 5% koh solution but for compound (4b),we noticed the appearance of n=n stretching band at 1498 cm -1 ,the disappearance of n-h primary amine stretching bands of dimer at 3255cm -1 and appearance of o-h of 1-naphthol stretching bands at 3248 cm -1 ; the color of the compound was changed to deep blue and it was soluble in 5% koh solution, and for compound (4c),we noticed the appearance of n=n stretching band at 1500 cm -1 ,the disappearance of n-h primary amine stretching bands of dimer at 3255 cm -1 and the appearance of o-h of phenol stretching bands and n-h secondary amide stretching band at 3306 -3284cm -1 ;the color of the compound was changed to brown and it was soluble in 5% koh solution. the newly synthesized compounds were screened for their antibacterial activity. from the result in table (4),compound 4c showed highly antibacterial activity against gram positive bacteria ( iraqi j pharm sci, vol.24(1) 2015 antibacterial evolution of bis heterocyclic derivatives 65 staphylococcus aureus, staphylococcus epidermidis ) tested in all three concentrations used compared with the standard (amoxicillin) and no activity against gram negative bacteria (klebsiella pneumonia., escherichia coli ) at tested concentrations compared with the standard (cefotaxime).final compounds except 4b showed high activity at all concentrations against gram positive bacteria (staphylococcus aureus). the tested compounds except compound 4c (at concentration 30μg/ml) showed no activity against gram negative bacteria (klebsiella pneumonia, escherichia coli) in all three concentrations. . scheme (1): synthesis of intermediates and final compounds. conclusion the synthesis of the designed compounds was successfully achieved by following the stated procedures as previously described. characterization and structural formulas of the synthesized compounds were characterized and confirmed by determination their melting points, decomposition points, rf values, infrared spectroscopy (ir) and elemental microanalysis. most of these compounds showed good antibacterial activity comparable with the standard compounds. the zone of inhibition of the final compounds shows the disulfide dimer of 2-amino 5 mercapto 1, 3, 4-thiadiazole ring and its derivatives exhibit potent bioactivities against gram positive bacteria. this is may be attributed to the different groups substituted on the amino groups of dimer through diazotization reaction. however, for compounds 4a and 4b, the position of hydroxyl group in naphthol ring showed wide variation in potency against bacteria. this may be reflected by differences in physiochemical properties or may have different affinity for bacterial cell wall that favor compound 4a compared to compound 4b. iraqi j pharm sci, vol.24(1) 2015 antibacterial evolution of bis heterocyclic derivatives 66 acknowledgment the authors gratefully 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27. ramezan a. a., ali m., hosseini s., khadijeh m., zadegan m., a method for antibiotic susceptibility testing: applicable and accurate. jundishapur j. microbiol. 2012;5(1):341-345. . iraqi j pharm sci, vol.28(2) 2019 co-administration of omega-3 with lornoxicam doi: https://doi.org/10.31351/vol28iss2pp159-164 159 effects of omega-3 co-administered with therapeutic dose of lornoxicam on male rats' liver israa a. majeed*1, nada n. al-shawi** * ministry of health and environment , dyala health directorate, dyala, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad-iraq. abstract lornoxicam drug is an enolic acid derivative (oxicam) of the non-steroidal anti-inflammatory drugs class. it is used to relieve pain and inflammation in rheumatic disease and osteoarthritis and other inflammation. furthermore, such drug has side effects similar to other nsaids, most commonly gastrointestinal and headache. severe but seldom adverse effects include bleeding, bronchospasms and the extremely rare stevens–johnson syndrome. adverse effects on liver were not previously-investigated. omega-3 fatty acids are poly-unsaturated fatty-acids (including α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid) have roles in human physiology. they possess anti-inflammatory effect, reduce blood pressure; in addition to other health effects. the aim of this study is to investigate whether lornoxicam, omega 3 fatty acid, or co-administration of omega 3 with lornoxicam have adverse effects on liver of healthy male rats. twenty-eight adults male rats weighing 180-200g were used in this study and the animals were randomly divided into four groups of seven rats each. group i: negative control/rats intraperitoneally injected with normal saline in a dose 5ml/kg/day; group ii: rats intraperitoneally injected with lornoxicam at dose 0.7 mg/kg/day; group iii: rats orally-administered omega-3 only at a dose 185mg/kg/day; group iv: rats co-administered omega-3 (185mg/kg/day) orally and intra-peritoneal injection of lornoxicam (0.7 mg/kg/day). duration of treatment was 14-day; and at day 15 of the study, the liver of each rat was excised for the preparation of tissue-homogenate to be utilized for the estimation of alt, ast, tnf-alpha and il-10. omega-3 can reduce signs of inflammation through the reductionof tnf-alpha level and elevation of il-10 with a significant reduction in alt enzyme activity level in rats' liver tissue homogenate. in conclusion, omega-3 poly-unsaturated fatty-acids may have a protective effect against hepatocytes inflammation when co-administered with lornoxicam. keywords: lornoxicam, omega 3, liver enzymes, tnf-alpha, il-10. الجرذانذكور على كبد اللورنوكسيكاممع عقار 3 ألوميكاتأثير األعطاء المتزامن ل **د.ندى ناجي الشاوي و 1*،أسراء احمد مجيد ، العراق. ديالى، ديالىوزارة الصحة والبيئة، دائرة صحة * الصيدلة،جامعة بغداد،بغداد،العراق.،كلية االدوية والسمومفرع ** الخالصة يعد عقار اللورنوكسيكام من مشتقات حامض األينوليك )األوكسيكام( من صنف األدوية المضادة لاللتهاب الغير ستيرويدية يستخدم لعالج ض الى ذلك، فان عقار اللورنوكسيكام لديه اعرا حاالت االلتهاب كالتهاب المفاصل الرثياني أو الداء المفصلي التنكسي والتهابات اخرى. باألضافة لكن نادرا و جانبية مشابهة لتلك التي تسببها االدوية المضدة لاللتهابات الغير ستيرويدية )االكثر شيوعا( على الجهاز الهضمي، أعراض جانبية شديدة تم بحثها سابقا.ماتحدث كمتالزمة ستيفنس جونسون. األعراض الجانبية لهذا العقار على الكبد لم ي الفا، حامض االيكوسابنتانويك و حامض الدوكوسا -من الدهون غير المشبعة المتعددة تتضمن )حامض اللينوليك 3 تعد حوامض األوميكا هيزانويك( لديها أدوار لفسلجة االنسان كامتالكها خاصية مضادة لاللتهاب، تقلل ضد الدمن باالضافة الى تأثيرات صحية اخرى. (82ثمانية وعشرون )الجرذان. تم استخدام ذكورعلى كبد اللورنوكسيكاممع عقار 3 ألوميكالالهدف من هذه الدراسة هو تقييم االعطاء المتزامن لصفاقحقنت داخل ا السيطرةمجموعات وفي كل مجموعة سبعة حيوانات. المجموعة االولى: مجموعة أربعبالغا قُسمت عشوائيا الى ذكرا جرذا 521جرعة فموية: اعطيت الثالثة, المجموعة اللورنوكسيكامغ/كغم من عقار لم 0.7 حقنت داخل الصفاقمحلول ملحي عادي, المجموعة الثانية: ب . نوكسيكاماللورغ/كغم لم 0.7حقنة داخل الصفاقمع 3 األوميكاغ/كغم من لم 521 الرابعة: اعطيت جرعة فموية, المجموعة 3 -االوميكا غ/كغم منلم في (tnf-alpha, il-10)و السايتوكينات (alt, ast)ى االنزيمات من اجراء الدراسة تم استئصال الكبد لغرض تقييم مستو51في اليوم وخفض بصورة معنوية il-10و رفع نسبة tnf-alphaيقلل من عالمات االلتهاب بواسطة تقليل نسبة 3 األوميكانسيج الكبد. وجدت النتائج أن يمكن أن يكون له تأثير وقائي ضد ألتهاب خاليا الكبد في حال أعطي بالتزامن 3 األوميكافي نسيج الكبد. ويمكن األستنتاج بأن altمستوى انزيم .اللورنوكسيكاممع عقار il-10( ، tnf – alpha،انزيم الكبد ،عامل التنخر الفأ ) 3 -األوميكا اللورنوكسيكام ، الكلمات المفتاحية : 1corresponding author: israa.ahmed10@yahoo.com received: 16/6 /2019 accepted: 21/8 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp159-164 mailto:israa.ahmed10@yahoo.com iraqi j pharm sci, vol.28(2) 2019 co-administration of omega-3 with lornoxicam 160 introduction the liver is the fundamental and major site of drugs-metabolism; and non-steroidal antiinflammatory drugs (nsaids) are the most widely utilized medications for pain-control and inflammation in several countries (1). the main cause of the limitation for the use of such drugs class is the gastrointestinal (gi) adverse effects such as the bleeding ulcers and hepatic adverse effects (2). lornoxicam is a potent nsaid with analgesic, antipyretic and anti-inflammatory effects that used to relieve the pain of postoperative in orthopaedic surgery (3) (4). moreover, lornoxicam caused adverse effects during its hepatic metabolism (5). omega-3 poly-unsaturated fatty-acid (pufas) including docosahexaenoic acid (dha) and eicosapentaenoic acid (epa) is an essential oil for the development of fetal and health aging; it plays a role in anti-inflammatory process and cell membrane viscosity (6,7). furthermore, it has been reported that the intake of omega-3 pufa may provide a protective impact against the most common adverse effects of nsaids such as cardiovascular and gastrointestinal (8). the drugliver interaction and drug-drug interaction must also be considered. authors reported that drug–drug interactions (ddis) have been noticed when nsaids are co-administered with some common medications (9); thus, the safety profile of nsaids alone, and in combination with other medications and supplements needs to be assessed in healthy status. the aim of this study is to investigate whether lornoxicam, or omega 3 fatty acid, or coadministration of omega 3 with lornoxicam have adverse effects on liver of healthy male rats. materials and methods animals twenty eight (28) healthy adult albino male rats, weighing 180-200gm were used in this study; they were obtained and maintained in the animal house of the college of pharmacy, university of baghdad under conditions of controlled humidity and temperature and light-dark cycle. animals were fed standard laboratory pellets and tap water ad libitum during the experiment duration. this study was approved by the scientific committee of the department of pharmacology and toxicology, and by the scientific committee of the college of pharmacy/ university of baghdad. lornoxicam and omega 3 fatty acids lornoxicam vial of 8mg/2ml was purchased from ms pharma (jordan), and omega-3 capsules of 1000mg pure fish-oil from (ams) america medic and science (usa). experimental protocol rats were randomly divided into four groups, seven animals each as follows: group i: rats were intraperitoneally (ip) injected normal saline of 5ml/kg/day dose for 14 days. this group served as a negative control. group ii: rats were ip injected with lornoxicam 0.7mg/kg/day for 14 days (10). group iii: rats were orally-administered omega-3 at a dose of 185mg/kg/day by gavage tube for 14 days (11). group iv: rats were administered a combination of oral omega-3 (185mg/kg/day) with ip lornoxicam (0.7mg/kg/day) for 14 days. twenty-four hour after the end of treatment duration i.e. at day 15 the animals were euthanized by diethyl ether and then after cervical dislocation, liver of each animal was quickly excised and washed with ice-cold phosphate buffer saline (pbs) (ph 77.2) to descend excessive blood, then the liver tissue was weighed, and then cut down to a small pieces and 1gm of liver tissue was placed in tube containing 9ml of ice-cold pbs to prepare 10ml of tissue homogenate; the liver tissue then homogenized by the homogenizer after putting the tube containing tissue in beaker containing ice. the homogenate then centrifuged on 10000 rpm for 15 minutes in a cold centrifuge and the supernatant then used to estimate alt and ast enzymes levels; in addition to cytokines [tumor necrosis factor-alpha (tnf-α), and interleukine-10 (il-10)]. determination of alanine aminotransferase (alt) activity level the principle of this assay uses enzymelinked immune-sorbent assay (elisa) based on biotin double antibody sandwich technology. standards and samples are pipetted into the wells that pre-coated with alt monoclonal antibody and then incubated at 37⁰c for 60 minutes. after incubation, anti-alt antibodies that labeled with biotin were added to unite with streptavidin-hrp to form an immune-complex. un-bound enzymes then removed after incubation and washing; then substrate a and b were added. the solution turned blue and changed to yellow by the effect of acid. the shades of solution and the concentration of rat alt were positively correlated (12). the assay range: 1u/l-200 u/l and the sensitivity: 0.52 u/l. determination of aspartate aminotransferase (ast) activity level the principle of this assay depends on enzyme-linked immune sorbent assay (elisa) based on biotin double antibody sandwich technology. standards and samples are pipetted into the wells that are pre-coated with ast-monoclonal antibody and then incubated at 37⁰c for 60 minutes. after incubation, ast-antibodies that labeled with biotin were added to unite with streptavidin-hrp, which forms the immune complex. un-bound enzymes were removed after incubation and washed with washing buffer; then substrate a and b were iraqi j pharm sci, vol.28(2) 2019 co-administration of omega-3 with lornoxicam 161 added. the solution were turned to blue and changed to yellow with effect of acid. the shades of solution and the concentration of ast are positively correlated (13). the assay range: 1u/l-200u/l; and the sensitivity: 0.51u/l. determination of tumor necrosis factor-alpha (tnf-alpha) level the principle of this assay depends on enzyme-linked immune sorbent assay (elisa) based on biotin double antibody sandwich technology. standards and samples are pipetted into the wells that are pre-coated with tnf-α monoclonal antibody and then incubated at 37⁰c for 60 minutes. after incubation, tnf-α -antibodies that labeled with biotin were added to unite with streptavidin-hrp, which forms the immune complex. un-bound enzymes were removed after incubation and washed with washing buffer; then substrate a and b were added. the solution were turned to blue and changed to yellow with effect of acid. the shades of solution and the concentration of rat's tnf-α is positively correlated (14). assay range: 5ng/l-1000ng/l. sensitivity: 2.51ng/l determination of interleukin-10 (il-10) levels the principle of this assay depends on enzyme-linked immune sorbent assay (elisa) based on biotin double antibody sandwich technology. standards and samples are pipetted into the wells that are pre-coated with il-10-monoclonal antibody and then incubated at 37⁰c for 60 minutes. after incubation, il-10-antibodies that labeled with biotin were added to unite with streptavidin-hrp, which forms the immune complex. un-bound enzymes were removed after incubation and washed with washing buffer; then substrate a and b were added. the solution were turned to blue and changed to yellow with effect of acid. the shades of solution and the concentration of il-10 are positively correlated (14). assay range: 3pg/ml-900pg/ml. sensitivity: 1.51pg/ml. statistical analysis unpaired student t-test was utilized to estimate the difference between two groups. the significance of differences among various groups was determined by one-way analysis of variance (anova). differences were statistically considered significant for p-value less than 0.05 and data were expressed as means ± standard error of the mean (sem). results table 1 showed that in group of rats intraperitoneally injected with therapeutic dose of lornoxicam (0.7mg/kg) for 14 days (group ii) there were non-significant (p>0.05) differences in both enzymes activity levels (alt and ast) in liver tissue homogenate compared to the corresponding levels in negative control rats (group i). table 1 showed that in group of rats intraperitoneally injected with therapeutic dose of lornoxicam (0.7mg/kg) for 14 days (group ii) there were significant reduction (p<0.05) in tnf-α level in liver tissue homogenate compared to negative control rats (group i). in contrast, there was nonsignificant difference (p>0.05) in il-10 level in group ii rats' liver tissue homogenate treated with therapeutic dose of lornoxicam (0.7mg/kg) compared to negative control rats (group i). table1. effect of therapeutic dose of lornoxicam on liver tissue enzymes (alt and ast) and cytokines (tnf-α, il-10) levels in male rats (n=7). group mean±sem of alt (iu/l) mean±sem of ast (iu/l) mean±sem of tnf-α ng/l mean±sem of il10 pg/ml group i 20.568±1.94 11.761±0.59 281.17 ± 18.3 129.33 ± 3.9 group ii 22.8991±2.242 11.326±1.104 237.5 ± 17.6 * 122.28 ± 17.4 -data presented as means ± standard error of the mean (sem). *: p<0.05: significant difference compared to negative control group. -group i: negative control (ns); group ii: lornoxicam-treated rats (0.7 mg/kg). alt: alanine aminotransferase; ast: aspartate aminotransferase; tnf-α: tumour necrosis factor-alpha; il10: interleukin-10; n: number of animals table 2 showed that in group of rats orallyadministered omega 3 (185mg/kg) alone for 14 days (group iii) there were non-significant difference (p>0.05) in alt, ast enzymes activity , and tnfα level in liver tissue homogenate compared to negative control rats (group i). in contrast, a significant elevation (p<0.05) in il-10 level in group iii rats' liver tissue homogenate orallyadministered omega 3 (185mg/kg) was shown compared to negative control rats (group i). iraqi j pharm sci, vol.28(2) 2019 co-administration of omega-3 with lornoxicam 162 table 2. effects of omega-three (ω-3) fatty acids alone on the liver tissue liver tissue enzymes (alt and ast) and cytokines (tnf-α, il-10) levels in male rats. (n=7). group mean±sem of alt (iu/l) mean±sem of ast (iu/l) mean±sem of tnf-α in liver tissue (ng/l) mean±sem of il-10 in liver tissue (pg/ml) group i 20.568±1.94 11.761±0.59 281.17 ± 18.3 129.33 ± 3.9 group iii 22.313±1.696 12.927±1.763 285.003 ± 28.32 148.2 ±16.77* -data presented as means ± standard error of the mean (sem). *: p<0.05: significant difference compared to negative control group. -group i: negative control (ns); group iii: omega 3-treated rats (185mg/kg). alt: alanine aminotransferase; ast: aspartate aminotransferase; tnf-α: tumour necrosis factor-alpha; il10: interleukin-10; n: number of animals table 3 showed that male rats coadministered therapeutic dose of lornoxicam (0.7mg/kg) intraperitoneally plus orallyadministered omega 3 (185mg/kg) once daily for 14 days (group iv) showed a significant elevation (p<0.05) in both enzyme activity levels (alt, and ast) in liver tissue homogenate each compared to the corresponding levels in male rats ip injected with therapeutic dose of lornoxicam (0.7mg/kg) (group ii); similarly, there were significant elevation (p<0.05) in both enzymes levels (alt, and ast) in liver tissue homogenate in male rats coadministered therapeutic dose of lornoxicam (0.7mg/kg) ip plus orally-administered omega 3 (185mg/kg) once daily for 14 days (group iv) compared to the corresponding levels in male rats orally-administered omega 3 alone (group iii). table 3 showed that male rats coadministered therapeutic dose of lornoxicam (0.7mg/kg) ip plus orally-administered omega 3 (185mg/kg) once daily for 14 days (group iv) showed a non-significant difference (p>0.05) in the level of tnf-α in liver tissue homogenate compared to the corresponding levels in male rats ip injected with therapeutic dose of lornoxicam (0.7mg/kg) (group ii); but, there was a significant reduction (p<0.05) in the level of tnf-α in liver tissue homogenate in male rats coadministered therapeutic dose of lornoxicam (0.7mg/kg) ip plus orally-administered omega 3 (185mg/kg) once daily for 14 days (group iv) compared to the corresponding levels in male rats orally-administered omega 3 alone (group iii). additionally, table 3 showed that male rats coadministered therapeutic dose of lornoxicam (0.7mg/kg) ip plus orally-administered omega 3 (185mg/kg) once daily for 14 days (group iv) showed a significant elevation (p<0.05) in the level of il-10 in liver tissue homogenate compared to the corresponding levels in male rats ip injected with therapeutic dose of lornoxicam (0.7mg/kg) (group ii); in contrast, there was non-significant difference (p>0.05) in the level of il-10 in liver tissue homogenate in group iv (rats co-administered therapeutic dose of lornoxicam (0.7mg/kg) ip plus orally-administered omega 3 (185mg/kg) once daily for 14 days compared to the corresponding levels in male rats orally-administered omega 3 alone (group iii). table 3. effect of co-administration of therapeutic dose of lornoxicam and omega 3 on the liver tissue enzymes (alt and ast) and cytokines (tnf-α, il-10) levels in male rats compared to omega 3 and lornoxicam-treated group. (n=7). group mean±sem of ast (iu/l) mean±sem of ast (iu/l) mean±sem of tnf-α ng/l mean±sem of il-10 pg/ml group ii 22.8991±2.242a 11.326±1.104a 237.5 ± 17.6 a 122.28 ± 17.4 a group iii 22.313±1.696a 12.927±1.763a 285.003 ± 28.32 b 148.2 ±16.77 b group iv 19.913±1.487b 13.908±1.531a 241.8 ± 15.2 a 140.12 ± 17.1 b -data presented as means ± standard error of the mean (sem). values with non –identical capital letters (a, and b) are significantly different (p<0.05). -group ii: lornoxicam-treated (0.114mg/kg); group iii: omega 3-treated rats (185 mg/kg); group iv: omega 3 (185mg/kg) plus lornoxicam-treated (0.7mg/kg). alt: alanine aminotransferase; ast: aspartate aminotransferase; tnf-α: tumour necrosis factor-alpha; il10: interleukin-10; n: number of animals. iraqi j pharm sci, vol.28(2) 2019 co-administration of omega-3 with lornoxicam 163 discussion results obtained from this study showed that therapeutic dose of lornoxicam (0.7mg/kg) intraperitoneally injected for 14 days (group ii) produced non-significant (p>0.05) differences in both enzymes activity levels (alt and ast) in liver tissue homogenate compared to the corresponding levels in negative control rats (group i) as shown in table 1. this could be attributed to the antiinflammatory effects of such drug. results obtained from the current study are inconsistent with those obtained by others (15); moreover, in the present study, tnf-α levels in liver tissue homogenate were significantly reduced (p< 0.05) and non-significant (p>0.05) in il-10 level in liver tissue homogenate of group ii (rats treated with therapeutic dose of lornoxicam (0.7mg/kg) compared to negative control rats (group i); this may indicate that lornoxicam used alone, may have the ability to reduce the inflammatory response during the study period. authors illustrated the adverse effects of other drugs and chemicals on rats liver (16) (17); but, no previous studies were reported in order to compare results of this study with others concerning the effect of lornoxicam used at therapeutic dose alone on rats' liver. it has been reported that tnf-α is a proinflammatory cytokine involved in various biological processes including regulation of cell proliferation, differentiation, apoptosis and immune response; and as a cell signaling protein involved in systemic inflammation. it is principally produced by -activated macrophages-, many other cell types. the deregulatory production of tnf has been implicated in a variety of diseases and drug-use (11) (18). moreover, results showed in table 3 revealed that levels of enzymes alt and ast in liver tissue homogenate were respectively, significantlyreduced (p<0.05) (alt) and non-significantly changed (p>0.05) (ast) when omega-3 was coadministered with lornoxicam for 14 days compared to lornoxicam-treated rats. furthermore, table 3 also showed that tnf-α levels in liver tissue homogenate were not significantly changed (p>0.05) when omega-3 was co-administered with lornoxicam for 14 days compared to lornoxicam-treated rats, respectively; but, there were significant reduction (p<0.05) in the level of tnf-α in liver tissue homogenate of group iv rats (combination) compared to the corresponding levels in male rats orally-administered omega 3 alone (group iii) (table 3). this may be due to synergistic effects of combination treatment. additionally, table 3 showed that male rats coadministered therapeutic dose of lornoxicam (0.7mg/kg) intraperitoneally plus orallyadministered omega 3 (185mg/kg) once daily for 14 days (group vi) showed a significant elevation (p<0.05) in the level of il-10 in liver tissue homogenate compared to the corresponding levels in male rats injected with therapeutic dose of lornoxicam (0.7mg/kg) (group iii); this elevation of il-10 may be due to a compensatory manner to regulate inflammatory mediators. authors reported that il-10 is an immunomodulatory cytokine that may regulate the immune response by inhibiting the proliferation of certain immune cells and promoting the proliferation of others; this in turn may reduce the production of inflammatory cytokines; and may promote the secretion of antibodies, which bind to specific foreign molecules, thereby inactivating those molecules and marking them for destruction by other immune cells (19). furthermore, authors mentioned omega 3 had minimal side effects, and even at high doses administration, it was elevating levels of hepatic enzymes and not exhibit liver injury (20). in conclusion, the current study is the first that highlight the effect of utilizing alone, lornoxicam, or omega 3 on rats' liver; and again, it is the first that investigates the effect of combination of omega 3 co-administered with lornoxicam on rat's liver; thus, we did not have a chance to compare results of this study with others. the present study proved that, omega-3 can reduce signs of inflammation through the reductionof tnf-alpha level and elevation of il-10 with a significant reduction in alt enzyme activity level in rats' liver tissue homogenate. references 1. shah s, mehta v. controversies and advances in non-steroidal anti-inflammatory drug (nsaid) analgesia in chronic pain management. postgrad med j. 2012; 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"is there a link between tnf gene expression and cognitive deficits in depression?". acta biochim pol. 2017; 64 (1): 65-73. 19. manuela g. neuman, ph.d. cytokines— central factors in alcoholic liver disease. alcohol research and health. 2003; 27 (4):309. 20. harris ws, ginsberg hn, arunakul n, shachter ns, windsor sl, et al. safety and efficacy of omacor in severe hypertriglyceridemia. j cardiovasc risk 1997; 4(5-6): 385-91. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice doi: https://doi.org/10.31351/vol31iss1pp154-166 154 evaluation the effectiveness of phenolic compound of salvia frigida on induced atopic dermatitis in experimental mice zahraa y. hassan*,1, tuka y. hassan** and ahmed r. aburaghif* * department of pharmacology and therapeutics, college of medicine, al‑nahrain university, baghdad-iraq. ** ministry of health and environment, baghdad-iraq. abstract to evaluate the effectiveness of phenolic compound of salvia frigida on induced atopic dermatitis (ad) of mice. forty mice were included in the study, divided in to four groups (10 mice/group): apparently healthy, induced ad without treatment, induced ad treated with tacrolimus 0.1% ointment, and induced ad treated with phenolic compound of salvia frigida cream 5%. examination of histopathology was done and skin homogenates levels also measured. levels of wbc, eosinophil, skin tissue homogenate of il-13 and il-4, serum ige, and histopathological scores were significantly increased among induced non treated ad group in comparison with the control group. comparisons of non-treated induced ad group with salvia frigida or tacrolimus treated groups; shows a significant reduction in the levels of all studied parameters’ (wbc, eosinophil, skin tissue homogenate of il4and il-13, serum ige, observational severity score, and histopathological scores) after the application of tacrolimus 0.1% ointment. while after the application of phenolic compound cream 5%, it shows a significant reduction in the levels of all parameters except those of (eosinophil, ige, and il-13). the comparison between the effect of topical application of tacrolimus and phenolic compound on the studied variables shows that the levels of epidermal thickness was significantly lower after application of phenolic compound among studied groups, while the levels of wbc and inflammatory cell were significantly lower after application of tacrolimus among studied groups. in conclusion, the use of these therapeutic agents that target ige, il-4 and il-13 could be promising in the treatment of ad. keywords: phenolic compound, salvia frigida , atopic dermatitis, tacrolimus, interleukin-4, interleukin-13 التحسسي الجلد التهاب على تاكروليموس مع بالمقارنة فريجيدا لسالفيا الفينولي تقييم فعالية المركب الفئران المختبرية في المستحث *رغيف أحمد أبوو *حسن * تقى يونس ،1*، حسن يونس زهراء * .العراق ، بغداد ، النهرين جامعة ، الطب كلية ، والمداواة الصيدلة قسم .العراق ، بغداد ، والبيئة الصحة وزارة ، الرصافة صحة مديرية ، العامة الصحة دائرة ** الخالصة مقسمة ، الدراسة في فأًرا أربعين تضمين تم. المختبرية الفئران في التأتبي الجلد التهاب على فريجيدا لسالفيا الفينولي المركب فعالية لتقييم بتاكروليموس ُمعالج التأتبي الجلد بالتهاب ُمحفَّزة ، عالج التأتبي دون الجلد بالتهاب ُمحفَّزة ، صحية(: مجموعة/ فئران 10) مجموعات أربع إلى مستويات وقياس المرضي التشريح فحص إجراء تم٪ . 5 فريجيدا سالفيا كريم من الفينول معالج بمركب التأتبي الجلد بالتهاب ُمحفَّزة و ، مرهم٪ 0.1 الدم في واالجسام المضادة أي 4و االنترلوكين 13في االنترلوكين والحمضية الخاليا والبيضاء كريات الدم في مستويات وجد زيادة. الجلد تجانس الُمحفَّزة المجموعة بين مقارنات. الضابطة المجموعة مع بالمقارنة المعالجة غير المستحثة المجموعة بين ملحوظ بشكل المرضية األنسجة ونتائج المعلمات جميع مستويات في كبيًرا انخفاًضا يُظهر ؛ تاكروليموس أو فريجيدا بسالفيا المعالجة المجموعات مع المعالجة غير التأتبي الجلد بالتهاب العوامل جميع مستويات في ذات داللة احصائية انخفاًضا الفينولي المركب الكريم تطبيق بعد يظهر بينما تاكروليماس. مرهم وضع المدروسة بعد الفينولي والمركب للتاكروليموس الموضعي التطبيق تأثير بين المقارنة أظهرت .أي( المضادة واالجسام 4االنترلوكين ، الحمضيةالخاليا ) تلك باستثناء كانت بينما المدروسة، المجموعات بين الفينول المركب تطبيق بعد ملحوظ بشكل أقل كانت البشرة سماكة مستويات أن المدروسة المتغيرات على يكون أن المحتمل من. المدروسة المجموعات تاكروليموس بين عقار تطبيق بعد ملحوظ بشكل أقل االلتهابية والخليةالبيضاء كريات الدم مستويات .التأتبي الجلد التهاب عالج في أي مفيدًا المضادة واالجسام 4االنترلوكين و 13 النترلوكين تستهدفا التي العالجية العوامل هذه استخدام 13 إنترلوكين ، 4 إنترلوكين ، تاكروليموس ، التأتبي الجلد التهاب ، فريجيدا سالفيا ، الفينول مركب: األساسية الكلمات introduction atopic dermatitis (ad) also known as atopic eczema, it is a common familial chronic inflammatory skin disease, determined by xerosis (increased water loss through the skin), itching, scaly and erythematous skin lesions, and high serum levels of ige. between 10 to 20% of children and 1 to 3% of adults worldwide affected by it and has negative medical and social effect on patients and their families. about 85% of affected children develop the disease before the age of 5 years (60% before the age of 1). patients may get off of this condition (improvement during puberty is a common phenomenon). it may persist or appear for the first time into adulthood (1). 1corresponding author e-mail: zahraahassan793@gmail.com received: 26/6/2021 accepted: 14/ 9/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp154-166 iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 155 individuals with ad have frequent and sometimes severe bacterial and viral infections skin infections. herpetic eczema (caused by the herpes simplex virus) it is known to occur mainly in ad patients. nearly 80-100% of patients have ad disease colonization of staphylococcus aureus bacteria on their skin (which often forms from a heterogeneous mixture), compared to only 5-30% of the normal population (2). ad treatment should be geared towards restoring the skin barrier which includes moisturizing and repairing the skin, reducing itching and reducing inflammation when necessary. therefore, the successful treatment of ad requires a polymorphic approach that involves patient and caregiver education, optimal skin care practices, anti-inflammatory treatment with topical corticosteroids and/or topical calcineurin inhibitors, and skin infections treatment (3) tacrolimus is the generic name for the macrolide immunosuppressant formerly known by its experimental name fk506. tacrolimus was the first discovered while examining for activity of antibacterial of a multitude compound. this macrolide is produced by streptomyces tsukubaensis, a bacterium found in the soil near tsukuba, japan. (4) it shows a good penetration through the skin due to its small size (molecular weight 822) and can be used to improve the severity of ad through its immune regulation and improve control of acute attacks and prevention of new ones due its mechanism of action as immune regulation. (5, 6). tacrolimus has side effects, such as skin burning and itching (7). accordingly, effective therapy with fewer side effects is required for treatment of ad. the world health organization encourages, promotes and facilitates effective herbal health programs (8). salvia plant is the largest genus of the lamiaceae family. it has around 1000 species distributed over the world (9). salvia frigida is one of the most medicinal plants that is used frequently in turkey (10) . the acetone extract of the aerial parts of salvia frigida has been tested previously. two oleanane type (erythrodiol, olean-12-ene-3β-ol) and two cycloartane type triterpenoids (24methylenecycloartanol, cycloartanol) with the compounds α –amyrin, and β –sitoserol were isolated and identified (11) . pharmacological activity of salvia frigida extract are: xanthine oxidase inhibition, antioxidant activities (12), anticholinesterase effect (13), and anticancer activities (14). plant phenolic compounds (pcs) are biologically generated, secondary metabolites. it is found universally in the plant kingdom (15). the antioxidant properties of pcs and flavonoids are thought to be mediated by scavenging free radicals such as ros and rns, and to suppress formation of ros and rns by inhibition specific enzymes or chelating trace minerals needed for their production, and finally by up regulating or protecting the antioxidant defense system (16, 17). although the currently used medications in the treatment of ad are effective in managing the disease; adverse reactions may decrease their usefulness (7). accordingly, the present study was designed to evaluate the effectiveness of phenolic compound of salvia frigida on induced atopic dermatitis mice model through their effect on wbc, eosinophil, serum ige, tissue homogenate of il4 and il13, observational severity score, and histopathological score. the study also aimed to compare the anti-inflammatory effect of phenolic compound of salvia frigida with tacrolimus on induced atopic dermatitis mice model. materials and methods a randomized prospective, controlled animal study was carried out. this study was conducted from 1st of november 2020 to 30th of april 2021, in the department of pharmacologycollege of medicine-al nahrain university. the protocols for the animal experiment used were carefully reviewed for ethical and scientific care procedures and approved by alnahrain university – college of medicine review council (approval number 857 in 28/9/2020). experimental design and animal groups a total of 40 healthy adult male albino mice (25-30g) collected from the animal house. the mice were housed in animal house in a good ventilated isolated place; with a room temperature of 20-24°c. the animals were left for seven days to acclimatize to the animal room conditions and allowed free access to water and ad libitum feeding. the animals were housed in animal house, at college of veterinary medicine in a good ventilated isolated place; with a room temperature of 20-24°c, and kept light for 12 hours. the practical part of the study was directed at college of veterinary medicine, university of baghdad, baghdadiraq. ten mice were chosen randomly and considered as a healthy control group and compared with other induced groups. thirty mice treated with 1-chloro-2, 4-dinitrobenzene (dncb) induced ad and randomly divided into three groups 10 mice/group: (induced ad mice non treated, induced ad mice treated with tacrolimus 0.1% ointment, and induced ad mice treated with phenolic compound of salvia frigida cream 5% topically). topical treatment was applied once daily at 9:00 am for 21 days. induction mouse model of dncb-induced atopic dermatitis mice described ad skin through shaving hair from dorsal of skin then 150 μl of 1% dncb in 3:1 (v/v) acetone/olive oil solution was topically applied once to the exposed skin. five days after dorsal hair removal, 0.2% dncb dissolved in an iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 156 acetone: olive oil mixture (3:1 vol/vol) was applied to challenge the dorsal skin (150 μl) three times a week for 3 weeks. after the visual confirmation of skin sensitization, mice were treated with test samples. (18) figure 1. figure 1. normal skin lesion without induction (a), induced atopic dermatitis skin lesion (b) (c). plant material the aerial parts of salvia frigida were extracted and authenticated in november, 2020 by department of pharmacognocy and medicinal plants / college of pharmacy/ al-mustasryiah university (iraq). salvia frigida extraction: 150 gm of shadedried pulverized leaves was defatted by maceration with hexane for 24 h then allowed to dry at room temperature. the defatted plant materials were extracted using soxhlet apparatus in which the powder packed in the thimbles and extracted with 1.75 l of aqueous methanol 85% as a solvent extraction for 24 hours. the extract was filtered and the solvent was evaporated under reduced pressure using a rotary evaporator to get 12 gm dry extract. 4 gm from the residual was suspended in 100ml water; about 3-4 ml of 5% sodium hydroxide was added to obtain a basic solution having ph 10 and partitioned with ethyl acetate (3*100 ml) (19, 20). the aqueous layer collected and evaporated to dryness which represents the phenolic compounds rich fraction that was used. preparation of phenolic compound 5% cream 5 gm of phenolic compound extracted from salvia frigida was weighted and dissolved in 3 ml of alcohol and shaking it for 4 minutes until it dissolved completely and became clear, after that we complete the weight to 100 gram with aquasoft cream (ajanta company) and shake the combination for 5 minutes by spatula.(21) high performance liquid chromatography (hplc) for quantitative and qualitative detection plant extractions the identification was made by (hplc) by comparing the retention times obtained at identical chromatographic conditions between extract fraction and standards. the concentration for the extraction of phenolic compound was quantitative determined by comparing the peak area of the standard with of the sample (22) . treatment protocols the topical applications of treatments (tacrolimus 0.1% ointment (23) and phenolic compound of salvia frigida 5% cream)(24) were applied to atopic dermatitis area of animal for 21 days once daily at 9 am starting from the fifth day of induction. parameters of study the following parameters are used to compare the results between experimental groups after day 21 of treatment: wbc and eosinophil count, serum ige, il-4 and il-13 were measured in skin tissue homogenate for mice with atopic dermatitis skin lesion, histopathological evaluation of atopic dermatitis skin lesion and compared with those of controls, and assessment of observational severity score. animal sacrificing, dissection, histological analysis, skin tissue homogenate preparation, and assessment of observational severity score at the 21th day of the treatment, we took the whole number of mice from each study groups and anesthetized through a piece of cotton socked with ether put with the mouse inside a closed jar for few minutes to ensure be anesthetized by inhalation, iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 157 blood sample collected (1ml) in edta tube for cbc and serum ige, then sacrificed by cervical dislocation and atopic dermatitis skin area was cut by sharp blade; this skin wound was dissection into two equal pieces one for the histological analysis and the second for the preparation of skin homogenate. the remaining mice from each group were subjected to the same procedure at the 21th day of the treatment. histological section preparation dorsal skin samples were collected from each animal in study groups and fixed in 10% formaldehyde paraffin embedded and cut into 6 μm sections. deparaffinized sections were stained with ordinary hematoxylin and eosin (h&e) to determine inflammatory degree and histological changes associated with atopic dermatitis (25) . assessment of histopathological changes of skin sections histopathological follow-up procedures were used for the skin samples taken from each group on the 21 days of treatment. histopathological changes of skin of each specimen were evaluated and scored by semi quantitative scoring systems for the evaluation of mouse model histopathology include epidermal hypertrophy, hyperkeratosis, parakeratosis, erosion, inflammatory cell infiltration, and extracellular edema, each scored from 0 to 3 (0 no abnormality, 1+ slight, 2+ mild, and 3+ moderate)(26), has been examined by pathologist and carried out in histopathology department /ibn sina university of medical and pharmaceutical sciences to observe the changes in tissues. skin tissue homogenate preparation the second piece of skin obtained were washed with normal saline, and rinsed with chilled phosphate buffer saline (1x pbs), put with filter paper and weighed. each 100 mg of skin wound tissue was homogenized with 1 ml of (1x pbs) with the aid of tissue homogenizer (27) for 1 minute at 4 °c, and must be stored overnight at 20°c. two freeze-thaw cycles must be performed to break the cell membranes; the homogenates were centrifuged for ten minutes at 2000 rpm at 2-8 °c. the supernatant was obtained and stored at –20°c to the assay of il-4 and il-13 levels in the tissue. the quantitative measurement of ige, il-4 and il13 (principle of the assay) serum ige: (the enzyme-linked immunosorbent assay). elisa kit for the estimation of ige was obtained from cusabio\china kit. specific different antibodies can be measured quantitatively by the enzyme-linked immunosorbent assay (elisa). after incubating the tested serum in an antigen-coated polystyrene plat or tube, enzyme specifically labeled anti-immunoglobulin is then added and the remaining in the plate after washing will give a measure to the quantity of specifically related antibody in the serum. the procedure depends on the insolubilization of specific antigens by passive adsorption to a solid phase (plate), example polystyrene phase (28). the procedure is done according to the manufacturer's instructions. skin tissue homogenate of il-4 and il-13 elisa kit for the estimation of il-4 and il-13 was obtained from cusabio\china kits was established on the base of sandwich enzyme-linked immunosorbent assay technology. antiil-4 and antiil-13 antibodies were precoated onto 48-well plates. and as detection antibodies, the biotin conjugated antiil-4 and antiil-13 antibodies were used. we added; the standards, test samples and biotin conjugated detection antibodies to the wells subsequently, and washed with wash buffer. hrp-streptavidin was added and unbound conjugates were washed away with wash buffer. to visualize hrp (horseradish peroxidase) enzymatic reaction tmb (3, 3′, 5,5′tetramethylbenzidine) substrates were used. tmb (3, 3′,5,5′-tetramethylbenzidine) was catalyzed by hrp to produce a blue color product which changed into yellow in accordance to adding acidic stop solution. the density of yellow color is proportional to amount of il-4 and il-13 of the sample captured in plate. we read the optical density absorbance at 450nm in a microplate reader, and then the concentration of il-4 and il-13 was calculated by comparing the optical density of the samples to that of standard in the corresponding microtiter plate. the concentration of il-4 and il-13 in each sample was expressed in pg/ml for comparison of the results with those of controls concentration (29) . assessment of observational severity score the severity of ad on the dorsal area was evaluated for each group on the 21th days of treatment. the evaluation of erythema, dryness, erosion and edema scored as 0 (none), 1 (mild), 2 (moderate), and 3 (severe). clinical skin score was defined as the summation of each individual scores, range from 0 to 12 (30) . statistical analysis data of the study were collected, analyzed, and presented using microsoft office excel 2010 and statistical package for the social sciences spss software version 23. numeric variables were expressed as mean ± sd and all statistical comparisons were made by means of independent ttest and anova test. when p ≤0.05 was considered statistically significant, and highly significant when p ≤ 0.01. the correlation was done between observational severity score, il-4, and il-13 using pearson correlation test. iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 158 results comparison between control and non-treated atopic dermatitis induced group regarding wbc, eosinophil, serum ige, skin tissue homogenate of il-13 and il-4, histopathological scores, and observational severity score: inflammatory signs have been seen from the first day in all induced non treated group. the levels of wbc, eosinophil, skin tissue homogenate of il-13 and il-4, and serum ige, were significantly increased among induced non treated atopic dermatitis group in comparison with control group (p=0.01, p=0.001, p=0.004, p<0.001 and p<0.001 respectively). table 1, figure 2 . table 1. comparison between controls and non-treated atopic dermatitis induced group regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-13 and il-4: variables mean ±𝐒𝐃 non-treated group mean ±sd control group p* value wbc (x103 /µl) 10±2.1 3.3 ± 2 0.01 eosinophil (x103 /µl) 2.5±0.02 0.0±0.0 0.001 ige(ng/ml) 26.62±5.15 15.59±8.65 0.004 il13 (pg/ml) 57.8±105.29 22.303±68.76 <0.001 il4 (pg/ml) 22.11±6.21 6.68±3.01 <0.001 *independent sample t test where p significant at ≤ 0.05 and high significant at <0.001 figure 2. comparison between means of controls and non-treated atopic dermatitis induced group regarding wbc, eosinophil, serum ige, skin tissue homogenate of il-13 and il-4.; ad: atopic dermatitis. wbc: white blood cells, ige: immunoglobulin e, il-13: interleukin 13, il-4: interleukin 4. results are expressed as mean ± sd, p is significant at ≤ 0.05 observational severity score and histopathological changes showed a significant high elevation among induced non treated group than among controls, p<0.01. table 2, figure 3. figure 4 shows the histopathological changes in topically induced ad group in comparison with controls. iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 159 table 2. comparison between controls and non-treated atopic dermatitis induced group regarding histopathological scores, and observational severity score. variables mean ±𝐒𝐃 non-treated group mean ±sd control group p* value epidermal thickness 3.50±0.52 0.0±0.0 <0.001 hyperkeratosis 3.00±0.81 0.0±0.0 <0.001 parakeratosis 3.40±0.69 0.0±0.0 <0.001 erosion 1.50±0.52 0.0±0.0 <0.001 inflammatory cell 2.60±0.51 0.0±0.0 <0.001 extracellular edema 2.50±0.52 0.0±0.0 <0.001 observational severity score 10.00±.81 0.0±0.0 <0.001 *independent sample t test where p significant at ≤ 0.05 and high significant at <0.001 figure 3. comparison between means of controls and non-treated atopic dermatitis induced group regarding histopathological scores and observational severity score; ad: atopic dermatitis; results are expressed as mean ± sd, p is significant at ≤ 0.05 iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 160 figure 4. histopathological changes in topically induced ad group (b) in comparison with controls (a) (10x): ordinary hematoxylin and eosin stain. comparisons of non-treated atopic dermatitis induced group with each of (salvia frigida treated group and tacrolimus treated group); regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13, observational severity score, and histopathological score: according to the comparison between nontreated atopic dermatitis induced group and phenolic compound of salvia frigida treated group; skin tissue homogenate of il4 and wbc were affected by the treatment with phenolic compound of salvia frigida 5% cream topically which appear clearly in the result tabulated in table (3) that variables were significantly decreased from those of non-treated induced ad group (p=0.002, and p=0.042 respectively) table 3. a significant improvement in the histopathological parameters and in the observational severity score after treatment with topical 5% phenolic compound of salvia frigida was observed compared with atopic dermatitis induced group. table 3, figure. 5. table 3. comparisons of non-treated atopic dermatitis induced group with phenolic compound of salvia frigida treated group regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il13, observational severity score, and histopathological score. variables mean±sd non treated mean±sd salvia p* wbc (x103 /µl) 10±2.1 7.6± 3.03 0.042 eosinophil (x103 /µl) 0.05± 0.06 0.03±0.03 0.166 ige(ng/ml) 26.62±5.150 20.36±5.92 0.32 il13 (pg/ml) 57.776±10.529 37.244±18.002 0.06 il4 (pg/ml) 22.11±6.21 11.59±2.23 0.002 epidermal thickness 3.50±0.52 1.00±0.66 0.001 hyperkeratosis 3.00±0.81 1.60±0.51 <0.001 parakeratosis 3.40±0.69 1.20±0.78 <0.001 erosion 1.50±0.52 0.40±0.51 <0.001 inflammatory cell 2.60±0.51 1.80±0.78 0.015 extracellular edema 2.50±0.52 1.10±0.56 <0.001 observational severity score 10.00±0.81 3.70±1.33 <0.001 *independent sample t test where p significant at ≤ 0.05 ad: atopic dermatitis iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 161 figure 5. histopathological changes in topically ad induced group (b) in comparison with phenolic compound of salvia frigida treated group (a) (10x): ordinary hematoxylin and eosin stain. comparisons of non-treated atopic dermatitis induced group with tacrolimus treated group; regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13, observational severity score, and histopathological score: comparison between topically tacrolimus treated group and non-treated induced atopic dermatitis group postulated in table 4 which elucidate clearly that the levels of wbc, eosinophil, serum ige, il-13 and il-4 in mice received tacrolimus 0.1% ointment topically were significantly lower than the corresponding levels in ad induced non-treated group, (p=0.02, p=0.013, p=0.022, p=0.025 and p<0.001 respectively). table 4. observational severity score and histopathological changes (epidermal thickness, hyperkeratosis, parakeratosis, erosion, inflammatory cell infiltrate, and extracellular edema) were significantly reduced in tacrolimus treated group in comparison with those non-treated ad induced group, p<0.001. table 4, fig. 6. table 4. comparisons of non-treated atopic dermatitis induced group with tacrolimus treated group regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13, observational severity score, and histopathological score. variables mean±sd non treated mean±sd tacrolimus p* wbc (x103 /µl) 10±2.1 6.03± 2.02 0.02 eosinophil (x103 /µl) 0.05± 0.07 0.020± 2.02 0.013 ige(ng/ml) 26.62±5.150 16.0±6.08 0.022 il13 (pg/ml) 57.776±10.529 31.82±21.3 0.025 il4 (pg/ml) 22.11±6.21 9.05±4.03 <0.001 epidermal thickness 3.50±0.52 1.20±1.22 <0.001 hyperkeratosis 3.00±0.81 1.60±0.51 <0.001 parakeratosis 3.40±0.69 1.20±0.78 <0.001 erosion 1.50±0.52 0.20±0.42 <0.001 inflammatory cell 2.60±0.51 1.70±0.42 0.001 extracellular edema 2.50±0.52 1.20±0.51 <0.001 observational severity score 10.00±0.81 4.50±1.08 <0.001 *independent sample t test where p significant at ≤ 0.05 ad: atopic dermatitis iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 162 figure 6. histopathological changes in topically ad induced group (b) in comparison with tacrolimus treated group (a) (10x): ordinary hematoxylin and eosin stain. the comparison between three groups (phenolic compound of salvia frigida , tacrolimus treated groups, and non-treated atopic dermatitis induced group regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13, histopathological changes and score in comparison between the effect of topical tacrolimus and phenolic compound on the studied variables, the level of epidermal thickness was significantly lower after phenolic compound of salvia frigida treatment among studied groups (p=0.025). the level of wbc and inflammatory cell were significantly lower after tacrolimus treatment among studied groups (p=0.04 and p=0.046 respectively). reduction of erosion was more significant among tacrolimus treated groups, p<0.001. table 5, figure (7, 8, 9) table 5. comparison between non treated atopic dermatitis induced group with each of salvia frigida and tacrolimus treated groups (by one way anova test) regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13. variables mean±sd salvia group mean±sd tacrolimus group mean±sd nontreated p wbc (x103 /µl) 7.6± 3.03 6.03± 2.02 10±2.1 0.04 eosinophil (x103 /µl) 0.03±0.03 0.020± 2.02 1± 0.07 <0.001 ige(ng/ml) 20.36±5.92 16.0±6.08 26.62±5.150 0.029 il13 (pg/ml) 37.24±18.0 31.82±21.3 57.8±10.529 0.022 il4 (pg/ml) 11.59±2.23 9.05±4.03 22.11±6.21 <0.001 epidermal thickness 1.00±0.66 1.20±1.22 3.50±0.52 0.025 hyperkeratosis 1.60±0.51 1.60±0.51 3.00±0.81 <0.001 parakeratosis 1.20±0.78 1.20±0.78 3.40±0.69 <0.001 erosion 0.40±0.51 0.20±0.42 1.50±0.52 <0.001 inflammatory cell 1.80±0.78 1.70±0.42 2.60±0.51 0.046 extracellular edema 1.10±0.56 1.20±0.51 2.50±0.52 <0.001 observational severity score 3.70±1.33 4.50±1.08 10.00±0.81 <0.001 iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 163 figure 7. comparison between non treated atopic dermatitis induced group with each of salvia frigida and tacrolimus treated groups (by one way anova test) regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13. wbc: white blood cells, ige: immunoglobulin e, il-13: interleukin 13, il-4: interleukin 4. results are expressed as mean ± sd, p is significant at ≤ 0.05. figure 8. comparison between phenolic compound of salvia frigida and tacrolimus treated groups (by one way anova test) regarding histopathological changes and observational severity score. results are expressed as mean ± sd, p is significant at ≤ 0.05. iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 164 figure 9. comparison between phenolic compound of salvia frigida treated group (a) and tacrolimus treated group (b) (10x): ordinary hematoxylin and eosin stain. correlations between observational severity score and ige, il-4 and il-13 in all studied groups table 6 revealed that the levels of ige, il4, and il-13 were correlated positively and significantly with observational score of mice subjected to the present study. p<0.05 table 6. correlations between observational severity score, ige, il-4 and il-13 in all studied groups. discussion according to the above results, comparison between apparently healthy group and ad induced non-treated group shows significant inflammation signs, in addition to significant increase in thickness and in the level of observational severity score among ad induced non-treated group. similar to this result, a study showed an increase in all types of wbc in ad induced non-treated group (31) signs of inflammation, histopathological changes, and observational severity score after application of 5% phenolic compound or 0.1% tacrolimus ointment topically, were significantly declined when compared with non-treated induced ad group. this indicates that the anti-atopic effect of phenolic compound of salvia frigida is similar to the effect of tacrolimus. in consistent with that, some studies found that salvia plant has anti-inflammatory effect among ad treated group with phenolic compound (32, 33) many other studies confirm these results, concluded that the properties of salvia plant include antiinflammatory, anticancer, anticholinesterase, antimicrobial, antimalarial and antioxidant (34, 35). it has been reported that there was a significant improvement in overall quality of life had been obtained and maintained throughout a 4week tacrolimus treatment study period, as well as the improvements in erythema, pruritus and sleeplessness (36). when compare between salvia frigida and tacrolimus treated groups in the present study, salvia treated group shows a significant reduction in epidermal thickness after 3 weeks of treatment when compare with tacrolimus treated groups while tacrolimus treated group shows more significant reduction in wbc count and inflammatory cells in comparison to others. the reduction of erosion was more pronounced among tacrolimus treated groups. similarly, a study revealed that the application of different topical treatment apart on the atopic dermatitis like skin lesions reduced the inflammatory response on damaged skin barrier, which is caused by foreign allergic substances such as dncb, and suppress the elevation of blood concentrations of histamine (37, 38) the levels of ige, il-4 and il-13 were correlated positively and significantly with observational severity score of mice model in this study. this finding appeared to be consistent with il13 il4 observational severity score ige r .532 .476 .393 p value .002** .008** .032* il13 r .440 .278 p value .015* .013* il4 r .680 p value .000** **. correlation is significant at the 0.01 level (2-tailed). *. correlation is significant at the 0.05 level (2-tailed). iraqi j pharm sci, vol.31(1) 2022 salvia frigida effect on atopic dermatitis in mice 165 another one that reported a positive correlation among same parameters (39). these results indicate that using of these therapeutic agents that targeting ige, il4, and il13 will probably useful in treatment of atopic dermatitis. conclusion topical application of tacrolimus ointment or phenolic compound of salvia frigida seems to be effective in the treatment of atopic dermatitis through their abilities to decrease wbc, eosinophil, serum ige, skin tissue homogenate of il4, and il13; as well as improving histopathological picture and reducing observational severity score, with more effectiveness of tacrolimus than of salvia frigida in the treatment of atopic dermatitis. a significant positive correlation was observed between serum ige, skin tissue homogenate il4, il13 and observational score for atopic dermatitis mice model. the use of phenolic compound of salvia frigida that target ige, il4, and il13 could be promising l in the treatment of atopic dermatitis. acknowledgment the authors are grateful to the department of pharmacology college of medicine, al‑nahrain university, for giving the opportunity and facilities to achieve this work. conflicts of interest the authors declare no conflicts of interest. funding support the authors declare that they have no funding support for this study. reference 1. fuxench zcc. atopic dermatitis: disease background and risk factors. in management of atopic dermatitis. springer, cham, 2017; vol. 1027: 11-19. 10.1007/978-3-319-64804-0_2 2. nutten s. atopic dermatitis: global epidemiology and risk factors. ann nutr meta, 2015; 66(suppl 1):8-16. doi: 10.1159/000370220 3. tanei, r. atopic dermatitis in 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indused hyperuricemia in mice. asian journal of pharmaceutical and clinical research, 2019; vol 12, issue 4:211-217. 10.22159/ajpcr.2019.v12i4.32096. 39. yousif ad, abu-raghif ar. the effect of topical dapsone in comparison with tacrolimus on dncb induced atopic dermatitis in mice, int. j. res. pharm. sci, 2020; volume 11, issue 4: 2050-2062. doi:10.26452/ijrps.v11ispl4.4419 this work is licensed under a creative commons attribution 4.0 international license. https://doi.org/10.1093/glycob/cwf068 https://www.sciencedirect.com/science/journal/03785173/276/1 http://dx.doi.org/10.26452/ijrps.v11ispl4.4419 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(suppl.) 2021 community pharmacists and modified release doi: https://doi.org/10.31351/vol30isssuppl.pp40-47 40 evaluation of community pharmacists knowledge, attitude and practice towards modified release dosage forms (conference paper )# noor yousif fareed*, amenah m. mohammed ** , suhair murtada* # 9th scientific conference conference sponsored by college of pharmacy , university of baghdad 25-26 august 2021 *department of pharmaceutics, college of pharmacy, university of basra, basra, iraq. **department of pharmaceutics, college of pharmacy, university of tikrit, tikrit, iraq. abstract to evaluate knowledge, practice and attitude of community pharmacists in basra regarding modified release dosage forms which are widely used for many therapeutic purposes in pharmacy practice. the current study was conducted among certified pharmacists in basra governoratesouth of iraq. data collection was carried out by a self-developed questionnaire in a cross-sectional study. a total number of 175 community pharmacists responded to the questionnaire. the majority worked in over the counter drugs based dispensing pharmacies located in the center of the city. only 38% of respondents correctly answered the first question in the knowledge section. there was a major positive agreement (75 %) towards medical representatives' rule in promoting the prescribing of modified release products by physicians. avoiding crushing and breaking of solid oral modified release drugs were identified by the majority (70%) of participants. correlation analysis showed a 22.8 correlation coefficient between knowledge and practice which was statistically significant. there was a statistically significant higher knowledge and practice scores in males than females. the result of this study demonstrates the lack of knowledge in many aspects regarding modified release dosage forms with several negative attitudes towards and practicing errors regarding modified release dosage forms. the conduction of a brief educational program would be very beneficial in bringing basic theoretical knowledge with practicing points of interest and promote a more positive attitude toward this unique class of novel drug delivery system. keywords: modified release products, community pharmacists, knowledge, attitude, practice, basra. تقييم معرفة الصيادلة المجتمعيين، الموقف والممارسة تجاه نماذج الجرعات ر مؤتمر () بحث ُمعدَّلة التحرُّ # *عاشور سهير مرتضىو **، آمنة مصطفى محمد1،*نور يوسف فريد 2021اب 26 – 25جامعة بغداد ، # المؤتمر العلمي التاسع لكلية الصيدلة *فرع الصيدالنيات، كلية الصيدلة، جامعة البصرة، بصرة، العراق. العراق.**فرع الصيدالنيات، كلية الصيدلة، جامعة تكريت، صالح الدين، الخالصة ر المستخدمة بصورة واسعة للعديد تقييم معرفة، ممارسة وموقف صيادلة المجتمع في البصرة فيما يتعلق بأشكال الجرعات ُمعدَّلة التحرُّ جمع البيانات من األغراض العالجية في ممارسة الصيدلة.أجريت الدراسة الحالية على الصيادلة المعتمدين في محافظة البصرة جنوب العراق. تم صيدلياً من المجتمع المحلي. غالبية المستجيبين يعمل في صيدليات تقع في مركز المدينة وتعتمد نظام 175من خالل استبيان.استجاب لالستبيان المعرفة. كان ٪ من المستجيبين عن االجابة بصورة صحيحة عن السؤال األول في قسم ٣٨صرف األدوية التي ال تحتاج لوصفة طبية . تمكن فقط ٪( في تعزيز وصف المنتجات المعدلة من قبل الطبيب. تم تحديد تجنب سحق وكسر 75هناك اتفاق إيجابي كبير تجاه قاعدة المسؤولين الطبيين ) المعرفة والموقف كان بين 22.٨. أظهر تحليل االرتباط أن معامل ارتباط (٪ 7٠األدوية الصلبة المعدلة عن طريق الفم من قبل غالبية المشاركين ) ذا داللة إحصائية. أظهر الذكور درجات معرفة وممارسة ذات داللة إحصائية أعلى من اإلناث. ر مع العديد من المواقف السلبي ة تظهر نتيجة هذه الدراسة نقص المعرفة في العديد من الجوانب المتعلقة بأشكال الجرعات ُمعدَّلة التحرُّ ق بأشكال جرعة اإلطالق المعدلة.إن إجراء برنامج تعليمي موجز سيكون مفيدًا جدًا في جلب المعرفة النظرية األساسية وممارسة األخطاء فيما يتعل مع ممارسة نقاط االهتمام وتعزيز موقف أكثر إيجابية تجاه هذه الفئة الفريدة من نظام توصيل األدوية الجديد. ر، صيادلة المجتمع، المعرفة، الموقف، الممارسة، البصرة .الكلمات المفتاحية: الجرعات ُمعدَّلة التحرُّ introduction modified release dosage forms can be defined as drug products that alter the timing and/or the release pattern of the active drug substance (1). they come into different classes including extended release, delayed release, repeated action and targeted release dosage forms (2). extended release can be further classified into controlled and sustained depending on the rate of release whether it is predetermined or not respectively (3). 1corresponding author e-mail: noor.fareed@uobasrah.edu.iq received: 27/8/2021 accepted: 15/11 /2021 published online first: 2022-1-12 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30isssuppl.pp40-47 iraqi j pharm sci, vol.30(suppl.) 2021 community pharmacists and modified release 41 modified-release release dosage forms can provide several benefits. firstly, the reduction in dosing frequency and fluctuation in circulating drug concentration (4). secondly, increasing patient compliance and decreasing dosing frequency (5). finally, enhancing overall drug bioavailability and pharmacokinetic properties (6). however, it also has disadvantages summarized mainly in dose dumping and low potential for dosage adjustment and higher cost compared to conventional dosage forms (7). the knowledge, attitude and practice (kap) survey ca collect quantitative and qualitative information depending on specific questions formatted in standardized questionnaires. it is mostly used as a tool to address misconceptions or misunderstandings that might be an obstacle towards the implementation of a certain behavior change (8). over the years, several drugs were marked as modified products and proved their success in therapy. nevertheless, kap insufficiency among healthcare professionals was noticed. as the core pharmaceutical services represented by promoting the correct and safe use of medications (9). therefore, pharmacists were selected as a target for this research. once the level of participants is determined, it woul n be defined as a quantitative method that is used to d be easier to detect what elements are mostly deficient and suitable interventions will be directed towards the defect. it is expected that educational intervention will provide this goal as it was helpful in similar areas (10). previous study in benghazi was conducted to evaluate the knowledge and perception of pharmacy practitioners toward sustained release dosage forms. the study showed that most patients and physicians lack knowledge about the advantages and disadvantages of these dosage forms which can discourage use. also, it is the pharmacy practitioner duty to enhance their understanding and promote the full benefit of using these dosage forms (11). another study at khartoum locality revealed that community pharmacists have a moderate knowledge with a general negative attitude and an acceptable practice regarding these drugs with different delivery patterns (12). the aim of the present investigation is to address the knowledge, attitude and practice of community pharmacists in basra regarding the modified release dosage forms. the required information is obtained by a pre-validated questionnaire and the data collected were evaluated by different descriptive and inferential statistical tools. methods questionnaire the knowledge, practice and attitude of community pharmacists were assessed by a self-developed questionnaire in a cross-sectional study. the questionnaire was approved by the scientific committee in the clinical pharmacy, college of pharmacy, basra university. the test-retest method was used to evaluate the reliability of the questionnaire. this is done by carrying out a pilot study on thirty participants. ten days later, the same participants were asked to re-fill the questionnaire. the first section, knowledge, consisted of 6 questions. each of the second and third sections consisted of four questions related to attitude and practice respectively. each correct answer in the knowledge and practice section was marked with 1 while wrong or don’t know answers carried 0 marks. this gave a total score in the range of 0 – 6 for the knowledge section and from 0 to 4 in the practice section. in the attitude section, positive attitude with agreement was marked with 3, neutral carried 2 marks while negative attitude with disagreement were given 1 according to likert scale (13, 14). this gave a score range from (13) for the attitude section. the reliability of the questionnaire data was evaluated using cronbach alpha coefficient (15). study design this study was an observational crosssectional, pharmacists in basra governorate were provided with questionnaires by a researcher. data collection was carried out through the period from 12th of january to 15th march in 2020. the purpose of the study was illustrated briefly to the pharmacists, then they were requested to fill in the questionnaire. the questionnaires were either collected by the researcher or returned by the participants themselves after half an hour. statistical analysis data analysis was carried out by the spss program, version 17.0. the demographic data were coded and the answers were marked according to each section's requirements. the demographic data of the participants and their responses were described by descriptive statistics in terms of frequency and percentages. for determining the appropriate inferential statistical test, the responses in the three domains of the questionnaire were tested for normality using kolmogorov-smirnov test. as the normality condition was not achieved, nonparametric tests, mann-whitney for two u tests and kruskal wallis h test for more than 2, were used for analyzing the significance of each demographic characteristic on each domain in the questionnaire. for each test, a p-value of less than 0.05 was considered statistically significant. results the reliability of the questionnaire was confirmed since there was no significant difference between the pilot studies earlier performed. there were 175 responses from participants to the questionnaire. the demographic data of the questionnaire participants are shown in table (1). the cronbach alpha coefficient was used where its iraqi j pharm sci, vol.30(suppl.) 2021 community pharmacists and modified release 42 value was (0.67). this ensures the ability of the study tool to measure the dimensions and prove their validity and reliability. it was found that the majority of participants around 72.57% belonged to the age (2435) years and only 4% had age 46 years and over. the number of females that participated in the questionnaire was higher than males which were 57.71% and 42.28% respectively. regarding the qualification of participants, about 77.14% had b.sc. in pharmacy and just 2.85% had a specialty in pharmaceutical science. approximately half of the participants had 1-5 years of practice in community pharmacy. the percentage of pharmacists that had (1-5) years after graduation was approximately equal to the percentage that had more than 10 years from graduation which was 33.14% and 34.28% respectively. concerning the location of pharmacies, high percent of pharmacies around 78.28% located in the center of basra city and the majority of the pharmacies (66.85%) were dependent on over the counter (otc) mode of dispensing, i.e., the drugs were dispensed by the pharmacists. knowledge about modified release dosage forms pharmacists’ knowledge about modified release dosage forms was evaluated by using six questions relating the basic theoretical background and practical evidence from marketed dosage forms as shown in table 2. less than half of participants answered the first and the third question in the knowledge section correctly. the majority of participants were unable to distinguish the different categories of modified release products by symbols printed on their packaging. also, the risk of dose dumping is higher with modified products than immediate as they mostly contain more drugs so dosage failure is more serious. on the other hand, more than threequarters (78% and 94 %) of participants realized the benefit obtained from these dosage forms by increasing drug bioavailability and decreasing adverse drug reactions and increasing patient compliance, respectively. in addition, a high percentage of participants (68%) were aware that not all drugs could be formulated as modified release formulations. moreover, a large proportion of participants (71%) considered modified release formulations to be more sophisticated dosage forms and require more complicated preparation steps if compared with immediate release formulations. table 1. the demographic data for all participants demographic characteristic frequency n (%) age 24-35 years 127 (72.57) 36-45 years 41 (23.42) 46 years and over 7 (4) gender female 101(57.71) male 74(42.28) qualification bsc pharmacy 135 (77.14 ) pharmaceutics specialist 5 (2.85 ) msc in pharmacy 24 (13.71 ) phd in pharmacy 11 (6.28 ) years in community pharmacy practice less than 1 year 31 (17.71 ) 1-5 years 78 (44.57 ) 6-10 years 31 (17.71) more than 10 years 35 (20 ) years from graduation less than 1 year 16 ( 9.142) 1-5 years 58 (33.142) 6-10 years 41 (23.42) more than 10 years 60 (34.28) pharmacy location center of the city 137(78.28) countryside 38(21.71 ) mode of dispensing in the pharmacy otc by the pharmacist 117 (66.85) by prescription 58 (33.142) data presented as number and percentage, n = 175 iraqi j pharm sci, vol.30(suppl.) 2021 community pharmacists and modified release 43 table 2. frequency of responses to questions in knowledge section question yes (n %) no (n %) i do not know (n %) k1 the terms, controlled release –extended release – delayed release and zero order kinetic are interchangeable 94 (54) 66 (38*) 15 (9) k2 modified release formulations increase drug bioavailability and decrease adverse drug reactions 136 (78 *) 28 (16) 11 (6) k3 modified release formulations cause more side effects than immediate release formulation if inappropriately formulated. 60(34*) 83 (47) 32 (18) k4 reduction of the dosing frequency results in enhancement in patient compliance and therapeutic outcomes when using modified release formulations. 164(94 *) 5(3) 6 (3) k5 all drugs can be formulated as modified release formulations, they are considered good candidate with good therapeutic outcomes 32(18 ) 119 (68*) 24 (14) k6 modified release formulations are more sophisticated dosage forms in comparison with immediate release formulation and involve more complicated preparation steps. 125(71*) 11 (6 ) 39 (22) data presented as number and percentage, n = 175 *correct answer attitudes towards modified release dosage forms to investigate the attitudes of pharmacists towards controlled release dosage forms, four questions were designed. their responses were described in table (3). approximately half of participants had a positive attitude about a1 and a3, about 53% agreed that the development of controlled release medication could reduce the financial expenditure with improvement of physicochemical and pharmacokinetic characteristics of the drug. similarly, around 49% of participants agreed that switching the medication from immediate release to controlled release could significantly affect the therapeutic outcomes. in addition, high percent of participants (75%) were in agreement with the remarkable effect of medical representatives in prescribing controlled release medication by physicians and only (2%) of participants had disagreement with this question. moreover, the majority of participants (60%) had a positive attitude towards a4, they agreed that the controlled release is a worthy formulation and its therapeutic outcomes rationalized its higher price compared to immediate release. table 3. frequency of responses to questions in attitude section question agree (n %) neutral (n %) disagree (n %) a1 development of modified release formulations decrease the financial expenditure on developing new medicine with enhanced pharmacokinetic and physicochemical properties 92(53) 56(32) 27(15) a2 medical representative activities play a major role in doctors prescribing modified release formulation 132(75 ) 39(22 ) 4(2) a3 changing the patient medication from immediate release formulation to modified release formulation significantly effects on the therapeutic outcomes of the disease 86(49) 55(31) 34(19) a4 modified release formulations are considered cost effective as the additive price compared to immediate release provide enhanced therapeutic outcomes. 105(60 ) 43(25) 27(15) data presented as number and percentage, n = 175 practice variables concerning the practice of community pharmacists toward control release formulation, six questions were directed to the participants. approximately three quarters (70%) of participants answered p1 incorrectly. changing from modified dosage form to immediate release causes dramatic change in patient response especially in critical illness. however, the majority of participants could answer p2, and p3 correctly, as crushing and breaking the modified release dosage forms destroys their unique release characteristics. furthermore, in p4, there were very limited correct answers. the side effect profile and drug interactions should be iraqi j pharm sci, vol.30(suppl.) 2021 community pharmacists and modified release 44 monitored as they are part of the pharmacodynamics response to the medication. table 4. frequency of various responses to questions in practice section question yes n (%) no n (%) i do not know n (%) p1 it is not necessary to consult the prescriber before changing the medication of a patient from modified to immediate in case of patient request or unavailability. 38(22) 123 (70) 14 (8) p2 modified release preparation can be cut to provide the required lower doses than the labeled amount 9 (5) 146 (83) 20 (11) p3 crushing modified release medication to use them in ng tube or with liquids is possible 21(12) 123(70) 31 (18) p4 it is not necessary to check for drug -drug interaction or monitor side effects with modified release formulations. 151(87) 6(3) 18 (10) data presented as number and percentage, n = 175 practice multiple choice questions. the responses to this section showed that a great number of participants (87%) stated that the majority of modified release formulations are available in the market as oral dosage forms, followed by transdermal then subcutaneous and very few topical and other nonmentioned are available. responses to this section are illustrated in table 5. table 5. practice multiple choice questions. data presented as number and percentage spearman correlation coefficient this test was used to evaluate the relationship between the three domains of the kap questionnaire. the results are shown in table 6 below. table 6. correlation between kap responses . variable spearman correlation coefficient p-value knowledge , attitude 0.073 0.335 knowledge, practice 0.228 0.002* practice, attitude 0.048 0.531 *statistically significant at p < 0.05 mann-whitney u test and kruskal wallis h test these tests were used to evaluate the significance of each demographic characteristic on each domain in the questionnaire. the results of these two tests were summarized in table 7 below. table 7. mean score with respect to demographics variable k-score pvalue a-score pvalue p-score p-value 0.638+0.202 2.461+0.374 0.654+0.245 age 24-35 years 88.12 0.382 89.13 0.037* 87.65 0.301 36-45 years 83.65 92.44 84.68 46 years and over 111.36 41.5 113.86 gender female 81.19 0.031* 88.01 0.998 79.14 0.003* male 97.29 87.99 100.09 majority of modified release formulations available in the market are oral 153 87% transdermal 12 7% subcutaneous 6 3% others 3 2% topical 1 1% the therapeutic category for which the majority of modified medications are prescribed cardiovascular 87 50% endocrine 47 27% anticancer 17 10% antimicrobial 13 7% cns drugs 11 6% iraqi j pharm sci, vol.30(suppl.) 2021 community pharmacists and modified release 45 continued table 7. qualification bsc pharmacy 87.39 0.426 87.39 0.966 89.34 0.045* pharmaceutics specialist 82.6 98.5 91.6 msc in pharmacy 79.88 89.46 68.1 phd in pharmacy 109.14 87.55 113.36 years in community pharmacy practice less than 1 year 92.89 84.6 78.5 1-5 years 85.75 0.734 89.38 0.956 79.32 0.012* 6-10 years 82.65 85.81 105.69 more than 10 years 93.43 89.87 100.09 years from graduation less than 1 year 102.63 0.502 86.44 0.806 77.63 0.181 1-5 years 90.04 93.04 79.59 6-10 years 81.27 86.91 96.8 more than 10 years 86.73 84.28 92.88 pharmacy location center of the city 86.87 0.561 88.74 0.706 89.3 0.48 countryside 92.08 85.33 83.3 mode of dispensing in the pharmacy otc by the pharmacist 85.48 0.332 89.78 0.499 88.86 0.726 by prescription 39.09 84.41 86.26 *statistically significant at p < 0.05 discussion as far as we know, this is the first study in iraq to assess kap regarding modified release dosage forms. regarding the knowledge section, the inability of community pharmacists to distinguish between different types of dosage forms and hazard of dose dumping were probably due to the lack of educational programs or the proper implementation of the theoretical knowledge into practice (16). similar findings were reported in another study performed in sri lanka in 2019. the study samples were academics working at medical faculties of the state universities and having an mbbs degree (17). for the attitude section, there was a predominant agreement of 60% of participants about the cost effectiveness of the modified release product. this could be explained by better disease control and decreasing the effort of nursing staff and probably their number in each shift in case of hospitalized patients. a study performed by zaid an in palestinian during 2009 to evaluate the knowledge and practice of pharmacist and doctors regarding sustained release product showed that the majority of the both groups agreed with the cost saving potential of the modified release products (18). on the other hand, the role of medical representatives and their promotional activities generally plays a major role in increasing the prescribing of certain medication or dosage forms (19). the case is similar for modified release products as reported in our results with a major agreement. an important finding in the practice section is that 83 % and 70 % of participants were aware that modified release products should not be splitted or crushed, respectively. this was also reported in a study conducted by gafar ma (2017) in sudan in which kap regarding tablet splitting was estimated. iraqi j pharm sci, vol.30(suppl.) 2021 community pharmacists and modified release 46 the hazards of modified release tablet deformation prior to swallowing were identified by 64.4% of the respondents (20). similarly, another study in benghazi medical centre, libya during 2019 to evaluate the knowledge about tablet crushing among pharmacists, doctors and nurses. pharmacists showed extraordinary knowledge represented by 96.2% of them giving correct answers as compared to doctor and nurse where correct responses were 75 and 20 % respectively (21). the correlation between the three domains of the questionnaire was estimated by spearman correlation coefficient (22, 23). the results are given by table 6. it can be noticed that there is a positive correlation that is statistically significant between knowledge and practice (p value = 0.002, p < 0.05). this means that as the knowledge increases, the practice errors are reduced and enhanced practice outcomes. the results of our study indicate that males showed better knowledge and practice scores than females as shown in table 7. there was a statistically significant difference between knowledge and practices of males and females (k, p value = 0.031, p, p value = 0.003). this result was not documented in a research before but could be explained by the fact that male pharmacists are involved more than females in the conferences and drug companies’ promotional activities, so they would get more information from this close contact with these authorities. also, there was a statistical difference between pharmacists with different qualifications in concern with the practice section. phd holders displaying significantly better (p, p value = 0.045, < 0.05) practice score than other participants with bsc., msc. and diploma. knowledge of the ph.d. qualified pharmacist was also the highest compared with others but the difference is statistically insignificant. this is perhaps due to longer years of academic study better focused on the particular specification of several advanced drug delivery systems including the modified release product. it is expected that the attitude of people having a specialty in pharmaceutics was more positive than other categories as a result of their knowledge with the specific techniques employed to enhance the performance in these dosage forms. it was also noticed that there is a significant difference between the participants with respect to their actual years of practice in the community pharmacy. as the years of experience increased, the mean rank of the specific category increased accordingly in all of the three domains. however, the difference is statistically significant in the practice section only (p, p value = 0.012). another important finding of this study was that the difference in kap domains is statistically insignificant between the different levels in the duration after graduation, pharmacy location, dispensing modes. similar findings could not be found in literature but this can be indicative that the same information was reachable to pharmacists in the governorate regardless of their pharmacy location or dispensing mode. limitations of the study the number of participants is the major limiting factor in this study. conclusions the result of this study demonstrates lack of knowledge in many aspects regarding modified release dosage forms. this leads to a variety of negative attitudes towards these products. also, several practice errors were the result of this knowledge gap and misunderstanding among community pharmacists in basra city. the result of this study demonstrates the need for educational programs for community pharmacists that implement the basic theoretical background into practical examples and real cases in dealing with this important class of dosage forms. such programs would provide a great benefit for career development and fill the gap in case of misconception or lack of knowledge. acknowledgement the authors wish to acknowledge all the participants in the study. also, the academic experts for their opinions in the validation of the questionnaire. references 1. murugesan s, gowramma b, lakshmanan k, reddy karri vvs, radhakrishnan a. oral modified drug release solid dosage form with special reference to design; an overview. curr drug res rev. 2020;12(1):16-25. 2. qiu y, zhou d. understanding design and development of modified release solid oral dosage forms. journal of validation technology. 2011 apr 1;17(2):2332. 3. gujral g, kapoor d, jaimini m. an updated review on modified release tablets. journal of drug delivery and therapeutics. 2018 jul 14;8(4):5-9. 4. niraj, v.k., srivastava, n., singh, t. and gupta, u. sustained and controlled drug delivery systemas a part of modified release dosage form. international journal of research in pharmaceutical and nano sciences. 2015;4(5):347-364. 5. dutta s, sengupta m. modified release dosage form and drug delivery. j pharm res. 2009 nov;2(11):1728-1739. 6. treacy d, flanner h, guttendorf r, burnside b, inventors; advancis pharmaceutical corp, assignee. enhanced absorption of modified release dosage forms. united states patent application us 11/021,309. 2005 jun 30. iraqi j pharm sci, vol.30(suppl.) 2021 community pharmacists and modified release 47 7. mamidala rk, ramana v, sandeep g, lingam m, gannu r, yamsani mr. factors influencing the design and performance of oral sustained/controlled release dosage forms. int. j. pharm. sci. and nanotech. 2009 nov 30;2(3):583-594. 8. pillay m. knowledge, attitude and practices (kap) survey. 9. kopciuch, 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and the “laws” of statistics. adv in health sci educ . 2020;625–632 . 15. santos jr. cronbach’s alpha: a tool for assessing the reliability of scales. journal of extension. 1999 apr;37(2):1-5. 16. silva bb, fegadolli c. implementation of pharmaceutical care for older adults in the brazilian public health system: a case study and realistic evaluation. bmc health serv res. 2020:20-37. 17. fernando mg, priyadarshi ki, shanika lg, samaranayake nr. knowledge, perceptions and practices on modified release tablets among prescribers: a preliminary study. journal of health sciences and innovative research. 2020 dec 25;5-14. 18. zaid an. attitude and perception of patients and health care practitioners toward oral sustained release dosage forms in palestine. saudi pharmaceutical journal. 2010 oct 1;18(4):251256. 19. shraim ny, al taha ta, qawasmeh rf, jarrar hn, shtaya ma, shayeb la, sweileh wm. knowledge, attitudes and practices of community pharmacists on generic medicines in palestine: a cross-sectional study. bmc health services research. 2017 dec 1;17(1) :1-9. 20. gafar ma, yousef ba, ibrahim a, osman za. knowledge, attitude, and practice of community pharmacists toward tablet splitting and crushing at omdurman locality: a cross-sectional study. current medical issues. 2021 apr 1;19(2):94102. 21. abdrabba f, salama r, fallah g, almahd a, salem s. knowledge, attitudes and practices of health care practitioners regarding crushing or splitting oral solid dosage forms in benghazi medical centre, libya. lebda medical journal. 2021 jan 29;7(1):252-258. 22. mukaka mm. a guide to appropriate use of correlation coefficient in medical research. malawi medical journal. 2012;24(3):69-71. 23. sedgwick p. spearman’s rank correlation coefficient. bmj. 2014 nov 28;349: g7327. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 effects of kappa carrageenan on cell lines and human dna doi: https://doi.org/10.31351/vol30iss1pp189-195 189 the pharmacological effects of kappa carrageenan on different human cell lines and genomic dna: an in vitro study aseel j. ali*,1 , hajer i. abdulla** and marwan s. al-nimer*** * ministry of health and environment, al ma'amon dental care centre, baghdad, iraq. ** department of dentistry, alesraa university college, baghdad, iraq. *** department of pharmacology, imam ja'afar al-sadiq university, baghdad, iraq. abstract carrageenan extract is a compound of sulfated polyglycan that is taken out from red seaweeds. being hydrocolloid in nature, carrageenan has gelling, emulsifying and thickening properties allowing it to be commonly used in the oral healthcare products and cosmetics. due to its bioactive compounds, carrageenan has been shown to have antimicrobial, antiviral, and antitumor properties. the purpose of this work is to study the probable use of carrageenan on the diseases that are related to oral cavity and on the genomic dna in in vitro experimental model. in this study, the effects of κ-carrageenan on four different cell lines related to the cancer and normal cells which cultured on selective media were done. moreover, the effect of κ-carrageenan on the dna molecule using an in vitro model was investigated in order to explain the antiproliferative effect of carrageenan. kappa-carrageenan inhibited the cancer cell growth and fibroblast cell lines growth (in vitro) experimental model. in addition, κcarrageenan solution completely and significantly damaged the dna molecule by the evidence that the mean ± sd absorbance of the mixture of κ-carrageenan and dna solution is 0.0 ± 0.0. this study shows that the κcarrageenan pharmaceutical preparations exert biological activities as anticancer in vitro studies. keywords: carrageenan, cell line, dna, antitumor, in vitro. التأثيرات الدوائية لمادة كابا كراجينان على خطوط الخاليا البشرية و د.ن.أ الجينومي : دراسة داخل األنبوب ***مروان صالح النمرو **،هاجر ابراهيم عبد هللا 1*،أسـيل جاسـم علي التخصصي لطب االسنان، بغداد، العراق.وزارة الصحة والبيئة ، مركز المأمون * ** قسم طب الفم، ،كلية االسراء الجامعة ، بغداد،العراق *** قسم علم االدوية،جامعة االمام جعفر الصادق، بغداد،العراق . الخالصة الكراجين بطبيعته الغروية المائية كاراجينان هومركب يحتوي على البولي گاليكان الكبريتي المتعدد وهو مستخلص من الطحلب االحمر. اظهر يمتلك خواص هالمية،مثخنة ومستحلبة تسمح باستخدامه بكثرة في مستحضرات الصحة الفموية والكماليات.وبسبب مركباته الحياتية الفعالة فقد للكراجبينان في خطوط الخاليا السرطانية لقد هدف هذا البحث الى دراسة التطبيق الممكن .الكراجينان امتالكه خواص مضادةللخاليا السرطانية تم دراسة تأثير كراجينان في أربعة والخاليا الليفية وتاثيره في الحمض النووي الجيني البشري في نموذج مختبري خارج الزجاج )في األنبوب( جينان في اثباط نمو الخاليا المزروعة ، كما وامتدت انواع من الخاليا السرطانية والطبيعية والتي تم زرعها في وسط انتقائي لمعرفة مدى تأثير كرا وقد بينت هذه الدراسة ان كابا كراجينان يمنع خطوط الخاليا الدراسة لتشمل تأأثير كاراجينان في الحمض النووي في أنموذج تجريبي خارج الزجاج. جزيئة الحمض النووي الجيني البشري. يستدل من ذلك ان الكراجينان السرطانية والخاليا الليفية في التجارب االختبارية باالضافة الى أذى تام في . الدراسات المختبريةي الذي استعمل في هذه الدراسة على شكل محلول لديه نشاط مضاد للسرطان ف .في الزجاج ، ضد االورام، كراجينان،خطوط الخاليا البشرية المختلفة،الدنا الكلمات المفتاحية: introduction the carrageenan extract is a (sulfated – polyglycan) that is taken out from red seaweeds from the genera (gigartina ,chondrus, eucheuma and iridaea (1,2). carrageenans are mainly utilized as a diet improver because of its emulsifying, thickening and gelling activities making it a vegetarian substitute for the gelatin (2). in addition to their use in food, carrageenans are generally used as excipients in a personal lubricant, toothpaste, many cosmetics besides many of the pharmaceutical products (3). commercially, three forms are there of carrageenan that are presented (kappa, lambda and iota) that are differing in degree and composition of sulfation in the polymeric structures. (figure 1). these actions of carrageenan have been considered, particularly on the animal model, where it had been reported that carrageenan extract has anti-microbial (4,5) and anti-tumor criteria (6,7). the tissue of the oral cavity is mainly formed by oral-keratinocytes, which are stratified squamous epithelium that form a main barrier to physical, chemical and microbial against agents that can cause localized cellular injury (8). 1corresponding author e-mail: dentist_aseel@yahoo.com received: 11/ 9 /2020 accepted:21 / 11 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp189-195 iraqi j pharm sci, vol.30(1) 2021 effects of kappa carrageenan on human cell lines and genomic dna 190 these cells are also actively involved in the proinflammatory route through the making of some cytokines(9,10). some of the toothpastes and oral gels contained carrageenan, and if exposure to an extended period will cause contact with oral cells besides the oral microorganisms. as stated before, carrageenan possessed anti-microbial activity (11). in earlier work, κ-carrageenan oligo-saccharides from kapaphycus stratum; in vitro and in vivo were proved to have immuno-modulation action on s180 bearing mice (12,13). chemical adjustment of carrageenan oligosaccharides can increase their antioxidant activities (14). the aim of the study is to evaluate the effect of κ-carrageenan on the human cancer and fibroblast cell lines and on the human genomic dna in in vitro experimental study. figure 1 chemical structure of κ-carrageenan materials and method materials κ-carrageenan purchased from sigma-aldrich (usa)(15) cell line: • ahmed majeed 2003(am3) transplantable mammary adenocarcinoma line. • hela established by (george gey) at the johns hopkins medical school. •primary rat embryo fibroblast (ref) provided and established from the iraqi centre for cancer & medical genetics research, baghdad, iraq (iccmgr). • rhabdomyosarcoma (rd) cell line (got from the pelvic rhabdomyosarcoma of a seven-years-old caucasian girl) (16,17). methods effect of κ-carrageenan on cancer cell line and fibroblast cells line in this study, four types of the cell lines are used. these are hela cell, rhabdomyosarcoma, mammary cell carcinoma, and fibroblast cells. media and solutions preparing for the (in vitro cell culture) experiment: 1. rosswell-park-memorial/ institute rpmi/1640 media the procedure of the media is made as following: a. the (rpmi/1640) powder with the (hepes.) buffer, with l-glutamine (8.2g) was dissolved in a (400) ml of the distilled water, and afterwards other constituents were added. b. the sodium-bicarbonate powder is equal to 1.1 gm streptomycin (0.25 ml) of (1gm) vial that is dissolved within a (5 ml) of distilled water. c. ampicillin (0.5)ml of a (500) mg vial that is dissolved in (5 ml )of distilled water. d. fetal calf serum/fcs (100 ml) afterward this volume is accomplished for up to (500) ml with distilled water and this media was sterilized with nalgen filter by a 0.2µm filtering unit (16,17). 2. phosphate buffer saline/pbs solution. this solution is made up using the following procedure: a. firstly dissolving an amount of 5 gm (pbs) powder in a (400) ml of distilled water, and then the other constituents were added. b. streptomycin (0.25) ml of (1gm vial was dissolved in a 5 ml of distilled water). c. ampicillin 0.5 ml of (500 mg) vial was dissolved in (5 ml) of distilled water). afterwards the volume is completed up to 500 ml by adding distilled water and this media was sterilized by nalgen filter with a (0.2µm) filter unit. 3. trypsine/versen s olutions the solution is made as the following: a. dissolving amount of (5.05gm)trypsine/versene powder in a (400 ml) of distilled water, and after that adding other constituents. b. adding sodium-bicarbonate powder amount of 1 gm. c. streptomycin (0.25) ml of (1gm) vial is dissolved in a 5 ml of distilled water. d. ampicillin amount of (0.5 ml) of a 500 mg vial which is dissolved in a 5 ml of distilled water).then the volume is finished to (500ml) with distilled water and then media is sterilized by nalgen filter with (0.2)µm filtering units(16,17). 4. fetal calf serum(fcs) this serum is thermally deactivated, set for uninterrupted use for the tissue culture media and it was sterilized(16,17). 5. preserving serum free media / sfm. the media was made by the same process that is described for rpmi./ media except that the fcs was not added. iraqi j pharm sci, vol.30(1) 2021 effects of kappa carrageenan on human cell lines and genomic dna 191 solution of methyl-thiazolyl tetrazolium /mtt. 3-dimethylthiazol-2-yl-2,5-diphenyltetrazoliumbromide (0.2 gm), which was dissolved in a 100 ml of the pbs in order to get (2 mg /ml) concentration of the dye. this solution was filtered by using a 0.2µm millipore filter to get rid of any blue color formazan material, and then kept in sterile, dark, glass container at a (4˚c). the solution must be used within 2weeks of its preparation (16,17). types of the cell lines used in this study 1. hela cell: this cervical cancer cell line of human which was firstly recognized by “george gey” at the medicine school of johns hopkins in the year 1951 obtained from a mother 31-year-old that had four children, her name is “henrietta lacks”. the hela cell were different from another cervical cancers explant in which they raised horribly in culture, could be too violently. passage/13-14that was used in the study, and the rpmi-1640 media with a (5%) fcs was used in preserving these cells. 2. rhabdomyosarcoma (r.d. cell line): this human cell line (rd) derived from a biopsy sample got from the pelvic rhabdomyosarcoma of a (7 years old) caucasian girl. the passage of (20-21) used in this study, and the rpmi-1640 media with 5% /fcs was used in preserving these cells. 3. ahmed/mohammaed/nahi-2003(amn-3) cell line: this is a murine mammary-a type of adenocarcinomacell line is a derivative of the initially (in-vivo) passage for the spontaneous mammary adenocarcinomas of a female balb-c mouse. passage/182-183 was utilized in this work, and the rpmi/1640 media with 5% fcs utilized in preserving these cells. 4. the primary rats embryo fibroblasts/r.e.f the r.e.f cell line is recognized and delivered by the help of dr. ahmed m. alshammery from the (iccmgr). these cells of this standard rats cell lines were a combination of epithelial and fibroblastic cell with ordinary chromosomal image. tumorigenicity trial of the cell lines has shown no tumor or growth development in the rats that are injected during 3 months of observing. passage of (77/78) was utilized in this study, and the rpmi/1640 media with 5%/ fcs was used in preserving these cells. four cell lines are subjected to carrageenan. those cells are (hela, fibroblasts mammary-amn3, and rhabdomyosarcoma. these cells are stored in a deep freezing as stock cells at the national centre of cancer. such cells are rebooted before the testing. many trials of the cell revival are made so that to get a single layer cell in a precise falcon volume of 25 ml which can be accustomed under the microscope to search for the presence of monolayer cell. cell cultivation was carried on according to the instructions of the national center of cancer in baghdad, using the following procedures: 1st step: during the first day, when the development of a single layer cells that have been formed in the cell falcons which is a precise falcon of 25 ml of volume, the last growth media got rid of and washed with the phosphate buffer saline (pbs) buffer only once, then add amount of 0.5-1.0 ml trypsin-versin solution, shaking the mixture and allowed to set down for 1 min. then, at that moment, a gentle shake by hand is followed for a minute, after that add 10 ml of sterile growth media in amount of 5% of fetal calf serum to the cells in the falcons, then start to pipette in order to diffuse the cells within the newly made media. then the isolated cells moved into a germ-free microplate. a whole volume of (200) µl cells suspension or cell suspension-culture media (which is considered as control) is moved to a number of wells of a microplate. the microtitre plate is protected with a (plate-cover) to avoid the opportunity of impurity and then incubated at a temperature (37ºc) for 24 -hours so that to maintain a single layer cells progress. 2ndstep: during the second day meaning after 24hour of cell culturing sin microplates, when the monolayer cell growth is formed in the microtitre plate, culture media was thrown away and the subsequent measurements of culture media and then carrageenan was added in a number of wells  200 µl of cell cultures media  200µl of the cultured media were added into the wells with the monolayer cells growing  175 µl of the culture media having no cells in addition to 25µl of 0.5%w/v freshly made κcarrageenan solution.  the prepared κ-carrageenan solution poured into the wells with monolayer cells growth. the solutions of carrageenan were sterilized by filtering using a 0.2µm millipore/filter before the adding step. the microtitre plate was covered by a plate cover to inhibit the possibility of impurity and then was incubated at a 37ºc for 24 hours so that to get a mono-layer cell growth. 3rd step: after 24 hours, an amount of 30µl m.t.t dye was added into all of the wells in dark room (to prevent oxidation of the dye), covering the microtiter plate by a foil and is kept for incubation for about two hours at a 37.5ºc in the incubator. then the wells containing solution discarded from the plate and, 100µl of dimethyl/sulfoxide added to each well and then the plates were shaken for 15 minutes using horizontal shaker. then the absorbance of all wells were recorded at 540nm by the elisa-reader effect of κ-carrageenan on the genomic dna of human the genomic dna of human has been generously gotten from prof. dr. adil al huseiny, at the medicine department, college of medicine/ diyala university. briefly genomic dna of human is extracted from the anti-coagulated blood by edta by using the (proteinase k) solutions and iraqi j pharm sci, vol.30(1) 2021 effects of kappa carrageenan on human cell lines and genomic dna 192 genomic purification kit/ geneaid, taiwan, and this process of extraction is followed like the instruction of the manufacturer company of the kit. absorbance of the extracted dna, which is dissolved in the phosphate buffer solutions, recorded wavelength of 260nm and 280nm using a uv-visible spectrophotometer. a ratio of absorbance at λ260nm to λ280nm approximately equal to 1.8-2.0, indicating the genomic dna contaminated with rna. a total number of six genomic dna obtained from six participants were tested with 0.5% (w/v) κ-carrageenan; where, 5µl κ-carrageenan was added to 5µl genomic dna in a quartz cuvette then completed the volume to the 4ml by distilled water. the absorbance of genomic dna was recorded at 260nm wavelength using a uv-visible spectrophotometer. a decrease in the absorbance of genomic dna compared with the absorbance before adding κ-carrageenan indicated dna damage (a term known as hypochromasia). while an increase of the genomic dna absorbance after adding κcarrageenan, indicating separation of the dna strands (a term known as hyperchromasia). statistical analysis: descriptive and inferential statistics were done by the use of the microsoft excel /2007 program. all results were stated as numbers (%), and every time possible as mean and standard deviation (sd). data were analyzed by the utilization of unpaired two/tailed student's t-test by taking (p ≤ 0.05) as the lowest limit of the significance. results effect of κ-carrageenan on the cancer cell line κ-carrageenan inhibits the development of cancer cell and fibroblasts cell lines in-vitro experimental model. it significantly (p≤0.05) stops the growth of hela cell/line by 81.2% (the mean absorbance value at λ 540 nm is reduced from 0.706 to 0.133 (figure2). figure 2 the effect of κ-carrageenan on hela cell growth in vitro experimental model. the results expressed as mean ± sd (n=8). κ-carrageenan completely and significantly suppressed the growth of mammary cell carcinoma i.e. the inhibitory percent is 100% (the mean absorbance value at λ 540 nm is reduced from 0.108 to 0.0 (figure 3). κ-carrageenan failed to suppress the growth of rhabdomyosarcoma, in fact it improves the growth of these cells by the evidence that the mean absorbance value is increased from 0.463 to 0.524, i.e. 13% increment (figure 4). figure 5 shows that κ-carrageenan completely and significantly suppressed the growth of fibroblast cells i.e. the inhibitory percent is 100% (the mean absorbance value at λ 540 nm is reduced from 0.196 to 0.0. figure 3 the effect of κ-carrageenan on mammary cell growth in vitro experimental model. the results expressed as mean ± sd (n=8). figure 4 effect of κ-carrageenan on the rhabdomyosarcom growth in vitro experimental model. results expressed as mean ±sd(n=8). iraqi j pharm sci, vol.30(1) 2021 effects of kappa carrageenan on human cell lines and genomic dna 193 figure 5 effect of κ-carrageenan on the fibroblast cell growth in vitro experimental model. results expressed as mean ±sd(n=8) effect of κ-carrageenan on the human genomic dna the mean ± sd concentration of human genomic dna that isolated from 6 subjects was 1.783±0.397 µg/ml which corresponding to the absorbance of 0.0178±0.0039 at wavelength of 260 nm. kappa(κ) carrageenan solution completely and significantly (p<0.001) damaged the dna molecule by the evidence that the mean ± sd absorbance of the mixture of κ-carrageenan and dna solution is 0.0 ± 0.0 (figure 6) figure 6 effect of κ-carrageenan solution (0.5% w/v) on the human genomic dna in vitro experimental model.results are expressed as mean±sd of six subjects. discussion the results of this study showed that κcarrageenan significantly prevent growth of fibroblasts, hela cell, and mammary cells; while, its effect against rhabdomyosarcoma cell is negligible, indicating its anti-growth effect is specific. these observations agreed with a previous study that showed the specificity of carrageenan as anticancer; furthermore, it is imperative to declare that the anti-cancer effect of carrageenan is strictly associated with the molecular weight, carbohydrates structure and the contents and the linking site of sulfur group (18). in previous results of the in vivo and in vitro experimental study, it has been found that degraded iota (ι)-carrageenan inhibits the osteosarcoma growth in established xenograft tumor models in mice and enhanced survival rate of animals (in-vivo study) as well as it inhibits the growth human osteosarcoma cell line. the authors suggested that carrageenan induced apoptosis (not necrosis), and arrest the cell cycle at g1 phase (19). in another study, the small molecular-weight, highly-sulfated lambda λ-carrageenan oligosaccharide inhibits the angiogenesis (a process that is involved in initiation and also promotion of cancer), as well as inhibits the cellular invasion, migration and proliferation (20). mi et al 2008 linked those effects to the upregulation of apoptotic genes like tnf-alpha, p-53, caspase 8, caspase 3 and increase level of active caspase 3 (21). tobacman and walters (2001) used transmission electron microscopy to illustrate the interaction between mammary myoepithelial cells of human and lambda-carrageenan and found that carrageenan entered the cells by membrane-associated endocytic vesicles and accumulate in endosomes and lysosomes (22) . as a result of the release of proteolytic enzymes from the distorted lysosome, the mammary myoepithelial cells are destroyed. on the other hand, tobacman et al (2001) verified that increasing intake of numerous gums with carrageenan associates positively with high frequency of breast carcinoma (23). authors found that carrageenan oligosaccharides can inhibit the growth of hela cell as well as the endothelial cell via a mechanism related to inhibition of the heparanase enzyme activity; moreover, this finding pointed out that carrageenan inhibited the cell growth at the molecular level (20). the effects of κ-carrageenans on fibroblast indicate that this substance is not free from harmful effect on the normal human cells. this observation is of great importance because carrageenan is pharmaceutically formulated as salt cast film in dressings for drug delivery to wound and to allow active adherence to and protection for the wound (24). according to results obtained from the current study, it is expected that healing of the wound is either delayed or even not occurred. the results of this study demonstrated that κcarrageenan induced dna (human genomic) damage in in vitro model. these results are in agreement with other studies that used carrageenan as inflammatory inducing agents. cuzzocrea et al (2001) demonstrated that intra-pleural injection of carrageenan into rats caused dna damage in lung tissue that accompanied with generation of reactive nitrogen species (rns) (25); moreover, this important finding should be considered in the oral medicinepractice because, the soluble carrageenan is used as a pharmaceutical formula of the bio-adhesive buccal mucosa in which the carried medication is released and dissolved in saliva within 40 minutes (26). therefore, using of such formula may induce dna iraqi j pharm sci, vol.30(1) 2021 effects of kappa carrageenan on human cell lines and genomic dna 194 damage or oral mucosa. bhattacharyya et al (2014) demonstrated an increase in m-rna expression in a mouse colonic epithelium and the colonic epithelial cells of human (27). there is no evidence in human that dietary carrageenan induced expression of mrna in the cells of large intestine. also bhattacharyya et al (2008) found that carrageenan induced cell necrosis rather than apoptosis in the colonic epithelial cell lines of human (ncm460) and in the primary colonic epithelial cells of humans; moreover, there is evidence that carrageenan can induce dna damage and arrest the cycle of the cell (28). on the other hand, weiner (2014) concluded that, using dietary carrageenan is safe and there is no evidence that it has undesirable effects on the normal cell growth or on the reproductive system (29). conclusions the results showed that the effects of carrageenan against cancer cell line prohibit the claim that the dietary carrageenan plays a role in carcinogenesis; whereas, its effect against fibroblast pointed to limit its uses in tissue injuries as it may limit the healing process. the damaging effect of carrageenan on the human genomic dna in in vitro experimental study should be supported by further studies including in vivo experimental animal models to demonstrate the safety of using carrageenan. acknowledgment the authors express their thanks to the prof. dr. adil h. alhusseiny for generously providing us the genomic 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additive carrageenan stimulates wnt/ β-catenin signaling in colonic epithelium by inhibition of nucleoredoxin reduction. nutr cancer. 2014; 66(1):117-27. 27. bhattacharyya s, borthakur a, dudeja pk, tobacman jk. carrageenan induces cell cycle arrest in human intestinal epithelial cells in vitro. j nutr. 2008 mar; 138(3):469-75. 28. weiner ml. food additive carrageenan: part ii: a critical review of carrageenan in vivo safety studies. crit rev toxicol. 2014; 44(3):244-69. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 algae biocompounds possible role during covid-19 doi : https://doi.org/10.31351/vol30iss2pp16-22 16 algae bioactive constituents and possible role during covid-19 pandemic (a review) sayed rashad*,1 and ghadir a. el-chaghaby** *regional center for food and feed, agricultural research center, giza, egypt. abstract nowadays, the use of natural bio-products in pharmaceuticals is gaining popularity as safe alternatives to chemicals and synthetic drugs. algal products are offering a pure, healthy and sustainable choice for pharmaceutical applications. algae are photosynthetic microorganisms that can survive in different environmental conditions. algae have many outstanding properties that make them excellent candidate for use in therapeutics. algae grow in fresh and marine waters and produce in their cells a wide range of biologically active chemical compounds. these bioactive compounds are offering a great source of highly economic bio-products. the present review discusses the phytochemical and bioactive compounds present in algae biomass and their potent biological activities. the review focuses on the use of alga in therapy and their pharmaceutical applications with special reference to the possible preventive and therapeutic role of algae against covid-19. keywords: algae, bioactivity, therapeutic role, bio-products, covid-19, pharmaceutical covid-19المكونات النشطة بيولوجياً في الطحالب و دورها المحتمل اثناء جائحة )مراجعة( سيد رشاد احمد*,1 و غدير علي الشغبي ** .مصر،الجيزه ،مركز البحوث الزراعية ،المركز االقليمي لالغذية واالعالف* ةالخالص في الوقت الحاضر ، يكتسب استخدام المنتجات الحيوية الطبيعية في المستحضرات الصيدالنية شعبية كبيرة كبدائل آمنة للمواد الكيميائية ا للتطبيمات الصننيدالنية. الطحالب عبارة ع االدوية الُمصنننع و كائنات . وفي هذا السننياف ف م منتجات الطحالب دمدم ايارط يبيعيا ويننحيطا ومسننتدامط ا دقيمة دموم بعملية التمثيل الضننوئي يمكن ا البماف في وروف بيةية مختل.ة. دتمتا الطحالب بالعديد م الخصننائم المميزه التي دجعل ا مرشنن ا ممتازط حط ميائية النشننطة بيولوًيطا. لالسننتخدام كعال . فنجد ام الطحالب دنمو في المياه العذبة و البحرية و دنتف في االياها مجموعة واسننعة م المركبات الكي ا رائعطا للمنتجات الحيوية االقتصننادية. و يناقا هذا البح المركبات الكيميائية النبادية والنشننطة مراًعةدمدم هذه المركبات النشننطة بيولوًيطا مصنندرط الطحالب في العال ودطبيماد ا الصنننيدالنية ما بيولوًيطا الموًودة في الكتلة الحيوية للطحالب وأنشنننطت ا البيولوًية. و دركز الدراسنننةعلد اسنننتخدام .covid-19إشارة ااية إلد الدور الوقائي والعالًي المحتمل للطحالب ضد الكلمات المفتاحية : الطحالب ؛ النشاط الحيوي؛ الدور العالجي ؛ المنتجات الحيويه؛ فيروس كورونا؛ المنتجات الصيدالنيه introduction natural algae products have, for a long time, been commonly explored for human use in food and in medical care (1). algae are very simple organisms containing chlorophyll, they consist of one single cell or colonies of grouped cells that are essentially not very connected to each other, which makes them of a polyphyletic nature (2). algae are grouped into two main types namely, macroalgae and microalgae. microalgae are photosynthetic unicellular microorganisms that are commonly 1 to 400 μm long and are unnoticed for the naked human eye (3,4). whereas, macroalgae, also known as seaweed are large and multicellular aquatic photosynthetic plant-like organisms(5). compared to plants, both types of algae have higher growth and biomass productivity and fresh water may not be needed for the cultivation of algae(6). algae have the ability to be cultivated in all types of water e.g. fresh water, brackish water, seawater and even wastewater(7). it has been extensively proven that both macroalgae and microalgae contain a broad variety of high-value bioactive compounds that can be used as medicinal compounds, nutritious foods and natural pigments (3). algae are good source of nutrients and bioactive compounds such as antioxidants, fiber, vitamins, minerals, pigments, lectins, steroids, halogenated compounds, proteins, polyunsaturated fatty acids, polysaccharides and other lipids (2,8–12). algae also contain other bioproducts such as carotenoids and amino acids(13) . all these bio-products are now being sold for human food and for health use. in the pharmaceutical industry algae are considered as a possible secondary highly bioactive metabolites source that may be useful for developing new pharmaceuticals (14). 1corresponding author e-mail: sayed_rashad79@hotmail.com received:23 /1/2021 accepted:1 /3 /2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp16-22 iraqi j pharm sci, vol.30(2) 2021 algae biocompounds possible role during covid-19 17 several experimental studies were carried out on the bioactive chemical compounds derived for human gain and welfare from algal biomass. in the present review, a summarized overview of the phytochemical and bioactive compounds present in algae biomass and their potent biological activities is presented. also, the review points out the pharmaceutical, economic and future prospects for algae role during the present covid-19 pandemic. algae algae represent a diverse group of largely photosynthetic aquatic organisms(9,15). algae can be unicellular, colonial or filamentous, leading to a wide diversity in overall cell morphology (16). they are present both in aquatic and in fresh water and have a short duplication period, making them one of the most quickly developing species(17). algae have diverse methods of assessing and effectively using ambient carbon dioxide and nutrients for biomass production (8,18). the cell walls of algae are thin, rigid but variable in their conformation and they surround the plasma membranes (16). in a water body, algal development is a dynamic process characterized by a wide variety of chemical, physical and biological progressions in the interior and it is influenced by a range of environmental factors in the outside. the key factors affecting algal growth have been established as total nitrogen, phosphorus, water temperature, and light strength, and their synergistic effects contribute to algae growth(19). algae constitute a group of photosynthetic eukaryotic species(20) that are either autotrophic which use water, light, carbon dioxide and other nutrients to produce their own food or polyphyletic which are large and diverse. algae on the basis of their morphological characteristics comprise microalgae made up of only one cell or macroalgae (seaweed) that use many different cells to function (21) and they can be identified in freshwater, marine habitats and also on wet rocks (22). microalgae microalgae are part of the subgroup of algae comprising thousands of types and are in essence photosynthetic. they are categorized as cyanobacteria, rhodophytes, chlorophytes and chromophytes(23). microalgae comprises photosynthetic microorganisms that are prokaryotic or eukaryotic, they are single or multicellular in structure (24). in freshwater and marine environments, microalgae constitute one of the most diverse classes of microorganisms(25). in taxonomy, microalgae denote eukaryotic microorganisms. however, microalgae include in their widest description prokaryotic cyanobacteria, green algae, diatoms, etc.(26). the green algae, which belong to phylum chlorophyta and contain common generations such as chlorella, dunaliella, and haematococcus are a particularly significant group of microalgae. another large group is the phylum heterokontophyta diatoms and includes common genera such as phaeodactylum (22,27-29). microalgae represent a source of immense bioactive compounds such as antiviral, antibacterial, antifungal and anticancer(30) that can be used for commercial applications. various microalgae pharmaceutical products have high potential, but their commercialization is still in its initial stages and can be perceived as a portal to the industry worth some billion dollars in the near future (31). the genetic potential of microalgae for different bioactive agents is amazing. these microorganisms, as in the pharmaceutical industry, have been shown to be able to manufacture certain compounds in the biotechnological spotlights for use and marketing(32,33). macroalgae macroalgae, as a fresh and renewable compound source, are currently being researched for both pharmaceutical and nutraceutical applications(25). they can be categorized according to the presence of distinctive pigments into green algae (chlorophyceae), brown algae (phaeophyceae) and red algae (rhodophyceaeae) (34,35). according to (36) there are 11017 species of macroalgae on earth and they are distributed as 1901 , 7083 and 2033 for green ,red and brown algae, respectively. marine macroalgae have been a significant source of biological and natural compounds including antidiabetic, vitamins, antibacterial and antiviral agents. macroalgae are renowned for their excellent health, ecological health with a high content of minerals and nutritious fibres (37). bioactive constituents of algae algae are rich resources for many compounds that are bioactive, including primary and secondary metabolites and that could be used in the pharmaceutical sector as possible candidates of interest (28). algae have been proved to be vitamins and vitamin precursors source including ascorbic acid, riboflavin and tocopherol (38). algae include a wide variety of medicinal drugs, vaccines, nutritive elements, proteins, which are not otherwise available or which are very expensive to obtain from plant and animal sources(33,39). antioxidants inflammations and oxidative stress especially in the presence of chronic diseases linked to antioxidant system fragility, are key factors that increase the severity of covid‐19. these data endorse the advice for the supplementation of antioxidants as effective covid‐19 strategies (40). antioxidants perform an essential role in the regeneration of damaged cells (41). in order to avoid these diseases and health issues, antioxidants iraqi j pharm sci, vol.30(2) 2021 algae biocompounds possible role during covid-19 18 terminate the attacks of reactive oxygen species and dramatically controlled oxidative reactions and thus antioxidants are of utmost importance (42). antioxidants have a critical role in the prohibition of many disease such as cancer, autoimmune disorders, neurological degeneration and coronary heart. nowadays, many experts are interested in finding healthy and satisfactory natural antioxidants. algae were shown to resist oxidative damages and persist despite exposure to reactive oxygen species and this indicates that algae cells have protective antioxidant defense systems (43). natural antioxidants present in many algae are essential bioactive compounds, which play a key role in protecting cells against oxidative damage in various diseases and aging processes. (43). sevral algae species showed potential antioxidant activity. zouaoui and co-authors(44) categorized the algal antioxidants into two groups; (1) water-soluble and (2) fatsoluble. some of the studies reporting the antioxidant properties of algae species are summarized below. anti-oxidant ability has been reported to be correlated with filamentous, green marine algae of chaetomorf species including c. aerea, c. crassa, c. linum and c. brachygona. in contrast with other plant species c. linum displayed the highest antioxidant potential with a relatively low minimum inhibitory concentration ic50 (1.484 ± 0,168 mg ml 1), a highest flavonoid content (18.177 ± 2.238 mg /g as rutin equivalent) and a relatively low phenolic content (2,895 ± 0,415 mg/g as gallic acid equivalent) (45). the six brown algae, sargassum linearifolium, sargassum vestitum, hormosira banksia, phyllospora comosa, padina sp., and sargassum podocanthum were reported to have antioxidant activity that was correlated to their phenolic compounds content and the six algae were found to contain the potent antioxidant compound “fucoxanthin”(46). in the four edaphic algae vaucheria geminate, pleurochloris pyrenoidosa, botrydiopsis eriensis, and scenedesmus obliquus, seven phenol compounds were identified (resorcinol, gallic acid, chlorogenic acid, syringic acid, caffeic acid, coumaric acid and ferulic acid). the four species showed high total antioxidant capacity that ranged from 6.66 to 36.33 ( mg of ascorbic acid/g) and also they showed up to 97.37% inhibition for dimethyl phenyl hydrazyl (dpph) free radical (28). the main algal species used to manufacture astaxanthin belongs to the genus haematococcus, but it is also produced by some chlorella species, such as chlamydomonas nivalis. astaxanthin is highly important due to its high antioxidant ability and associated health benefits(47). for its antioxidant qualities, ethanolic and aqueous extracts from seaweed polysiphonia and laurencia have been compared. the content of flavonoids, phenol, and tannins was higher in polysiphonia. also, polysiphonia displayed higher levels of antioxidant activity relative to laurencia for both ethanol and water extracts (48). methanol extracts of the red seaweed, chondrococcus hornemannii and spyridia fusiformis were reported to have high antioxidant activity owing to their phytochemical composition including flavonoids, tannin, alkaloids, saponin and steroids (49). the methanol extract of blue green algae, anabaena sp. exhibited high antioxidant activity with phenolic content equal to 57.06 gallic acid equivalent (mg/gm) and dpph radical scavenging activity 48.62±0.29% at 100 mg/ml concentration with an ic50 value of 101.81 mg/ml(50). antivirals viral infections are a big public health concern causing multiple illnesses that endanger health. many investigations seek to detect novel antivirals in order to monitor and disseminate these contagious conditions(51). synthetic antiviral drugs were developed to diminish the virus infections ‘complications. on the other hand, serious side effects and development of some resistant mutants of the virus were documented, particularly for longterm antiviral medication(52). in many studies, researches were driven towards investigating natural sources of antiviral drugs. the antiviral properties of macroalgae have a defensive function against many species of viruses(53,54). the antiviral activity against certain retroviruses have been found to be compatible with bioactive compounds isolated from microalgae. steroids and algae-extracted glycolipids have hiv bioactivity(55). algae bioactive constituents especially algae derived-polysaccharides have been demonstrated to have potential antiviral activity that could be effective in the treatment of viral infections. in the present review, we are summarizing some of the studies that highlight algae role as antiviral agent(56). methanol extract of spirulina platensis isolated from the nile river in egypt recorded a pronounced antiviral activity with 50% reduction of viral titer. the observed antiviral activity of spirulina was attributed to its content of sulphoquinovosyl diacylglycerol(57). sulphoquinovosyl diacylglycerol is a natural sulpholipid extensive biological activities such as inhibitory effects on dna polymerase and hivreverse transcriptase, p-selectin receptors, the aids virus, telomerase, and inflammation/proliferation (58). the red microalga porphyridium cell-wall sulphated polysaccharide showed remarkable antiherpes simplex virus type (hsv 1, 2), and herpes zoster virus type 1 and 2 (hsv 1, 2) variicella sp (vzv) (59). iraqi j pharm sci, vol.30(2) 2021 algae biocompounds possible role during covid-19 19 sulfolipids with remarkable antiviral activity against herps simplex virus type1 (hsv-1) were isolated from different marine algal species (laurencia popillose, galaxoura cylindriea, ulva fasciata, dilophys fasciola and taonia atomaria) (60). the algal extracts of spirogyra showed anti-hsv activity in different phases of the multiplication processes of herpes simplex virus type 1 (hsv 1) and type 2 virus (hsv-2). alkaloids, essential oils and terpenoids were the major active compounds against hsv found in spirogyra spp ethanolic extract (61). aqueous extract from the red macroalga laurencia obtusa inhibited the replication of the viruses: influenza b, a (h3n2) and a (h1n1) (62). marine species of seaweed osmundaria obtusiloba extract revealed high levels chikungunya virus (chikv) infection (63). the anti-hiv-1 activity of the bioactive compound fucoidan extracted from two different marine macroalgae dictyota bartayesiana and turbinaria decurrens was proved (63). murine leuchemia virus (mulv), and cell transformation by murine-sarcoma virus (musv124) in cell culture have been substantially inhibited by polysaccharides isolated from the red microalgae porphyridium sp. (64). lectins were extracted and purified from canavalia brasiliensis , c.maritima , dioclea. lasiocarpa, d. sclerocarpa , amansia multifida , bryothamniom seaforthii , hypnea musciformis, meristiella echinocarpa and solieria filiformis. the isolated lectins were efficient for inhibiting 18 different viruses, including hiv and influenza viruses (65). antiviral activity against the type 1 herpes simplex virus, hsv-1 virus and the rift valley fever virus (rvfv) were investigated and proved for the carragene sulfated polysaccharides isolated from red alga acanthophora and brown alga clathratus hydroclathrus(66). covid-19 disease progression the emergence of coronavirus (covid19) began last november in wuhan, china, exhibiting pneumonia-like symptoms in patients. by the end of january 2020, the world health organization (who) declared covid-19 as a pandemic (67). coronaviruses are enveloped viruses that have a single-stranded, positive-sense rna genome on their surface carrying spike protein. sars-cov2 has high transmissibility and has contributed to a public health epidemic worldwide. symptoms of covid-19 range from mild flu-like illness to a potentially fatal syndrome of acute respiratory failure or fulminant pneumonia. since 2012, more than 800,000 individuals have died as a result of sars cov-1 and mers cov. in march 2020, the pandemic was declared a pandemic(68). covid-19 has an unceasingly rising mortality rate of 0.5-1 percent. an easy way to resist viral infection and reduce fatalities has been to improve immunity. there are currently no drugs and/or vaccines that can help mitigate this viral disease, but spread can be limited by the use of masks and social distancing(69). the healthcare systems of the entire world are struggling to solve this pandemic. millions of lives have been disrupted because of mandatory isolation or quarantines(70). algae prospective during covid-19 pandemic during the current covid-19 pandemic, scientist all over the world are seeking for exploring new drugs to prevent the infection and to treat the illness but this may take time to verify. in this respect, algae bioactive compounds having antioxidant and antiviral activities could be explored as possible supportive therapy. some of the recently tested algal compounds have been promising indications that they are both pharmacologically and commercially efficient antiviral agents and can be used worldwide on a commercial basis to eliminate the infectious pathogen responsible for the covid-19 pandemic worldwide (71). according to pereira and critchley (72) marine algae species possess large amounts of sulphate polysaccharides of complex structures, which have been shown to inhibit the replication of viruses. the probable antiviral therapeutic antiviral agents against sars-cov-2 include molecules such as lectin, carrageen, sulphated polysaccharides, ulvans and fucoidans. the capacity of algae-based nutraceuticals, in particular spirulina, to improve immunity to viral diseases has already been clinically documented. spirulina is considered as a potential bioresource of inhibitory therapeutic-value peptides that may be investigated in the treatment and inflammation of extreme symptoms associated with betacoronavirus, like covid-19 (69) . conclusion presently, most of the researchers are seeking for the development of successful prevention and treatment therapies to prevent infection and cure of covid-19. natural bioactive compounds could offer an excellent solution in getting a handle on this pandemic. in this review, we summarized some important studies that discussed the antioxidant, and antiviral properties of algaebased nutraceuticals and bioactive compounds. in those studies, algae bioactive ingredients were proven to serve as immune-boosting and therapeutic agents. the present review shed the light about the use and pharmaceutical applications of algae in therapy, with particular reference to the possible iraqi j pharm sci, vol.30(2) 2021 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j appl phycol. 2020;2009–11. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 30 potentiometric transducers for the selective recognition of risperidone based on molecularly imprinted polymer najwa i. abdulla *,1 and hamsa m.yaseen * * department of chemistry, college of education for pure sciences (ibn-al-haitham), university of baghdad ,baghdad, iraq. abstract graphite coated electrodes (gce) based on molecularly imprinted polymers were fabricated for the selective potentiometric determination of risperidone (ris). the molecularly imprinted (mip) and nonimprinted (nip) polymers were synthesized by bulk polymerization using (ris.) as a template, acrylic acid (aa) and acrylamide (aam) as monomers, ethylene glycol dimethacrylate (egdma) as a cross-linker and benzoyl peroxide (bpo) as an initiator. the imprinted membranes and the nonimprinted membranes were prepared using dioctyl phthalate (dop) and dibutylphthalate (dbp) as plasticizers in pvc matrix. the membranes were coated on graphite electrodes. the mip electrodes using (aa) and (aam) showed a near nernstian and nernstian response with slopes of 55.2±0.1 and 59.0±0.2 mv/decade, correlation coefficient (r 2 ) 0.9997 and 0.9999, a linear response for a concentration range of (1.0×10 -6 1.0×10 -2 ) m and (5.0x 10 -7 to 1.0 x 10 -2 ) m respectively. the response time of the prepared electrodes was less than 30 seconds. the electrode responses were stable in a ph range (4-8). the electrodes exhibited good selectivity over a wide range of interference. the most effective electrode (ris-mip gce) was used to determine the concentration of (ris.) in some pharmaceutical formulations. the electrodes could be successfully used within (7and13) weeks respectively without any drift. keywords: ion-selective electrodes, molecularly imprinted polymer, pvc membrane coated graphite electrode, risperidone. متحسسبت جهدية للتعييه اإلوتقبئي للرسبيريدون المعتمد علً بىليمرات الطبعة الجزيئية وجىي إسحق عبد هللا ،*1 همسة مىعم يبسيهو * * د، ثغداد ، انعزاق. ثغدا لسى انكيًيبء ، كهيخ انززثيخ نهعهىو انصزفخ اثٍ انهيضى، جبيعخ الخالصة رى رحضيز ألطبة كزافيذ نجىنيًزاد طجعذ ثعمبر )انزسجيزيدوٌ( نهزعييٍ األَزمبئي نهذا انعمبر.حيش حضزد ثىنيًزاد زيهيك انطجعخ انجشيئيخ وانجىنيًزاد غيز انًطجىعخ ثطزيمخ ثهًزح انًجًم ثإسزعًبل )انزسجيزيدوٌ( كمبنت )طجعخ(، حبيض االك واألكزيم أيبيد كًىَيًزاد فعبنخ ،أصيهيٍ كاليكىل صُبئي يضيم أكزيهذ كزاثظ رمبطعي وثيزوكسيد انجُشويم كجبدئ. حضزد أغشيخ انطجعخ انجشيئيخ واألغشيخ غيز انًطجىعخ ثجعضزح دلبئك ثىنيًز انطجعخ انجشيئيخ وانجىنيًز غيز انًطجىع في فضبالد صُبئي األوكزيم عمبر نزمديز انكزافيذ الطبة ثهب طهيذ انًحضزح األغشيخ انجيىريم كًذيجبد يهدَخ في يزعدد كهىريد انفبيُيم. هذِ وفضبالد صُبئي )انزسجيزيدوٌ(. أظهزد ألطبة انطجعخ انجشيئيخ نهزسجيزيدوٌ انًعزًدح عم حبيض األكزيهيك و واألكزيم أيبيد كًىَيًزاد فعبنخ إَحداراد خطيخ يدي رزاكيش ( يع0,9999و 0,9999( ثًعبيالد اررجبط ) 59.0 – 55.2)mv/decadeلزة َيزَسزيخ وَيزَسزيخ يمدارهب m ( (1.0×10ثيٍ رززاوح -6 –1.0×10 -2 m( (5.0×10و -7 –1.0×10 -2 m( 3.3×10 يمدارهب وحدود رحسس -7 10×2.0و -7 ( عهً صبَيخ.وأظهزد األلطبة أسزجبثخ يسزمزح في يدي دانخ حبيضيخ رزاوحذ 00انزىاني. كًب كبٌ سيٍ اسزجبثخ األلطبة انًحضزح ألم يٍ ( في رعييٍ رزكيش ris-mip gceوإَزمبئيخ جيدح ثىجىد انعديد يٍ األصُبف انًزداخهخ. إسزعًم انمطت األكضز رأصيزا ) (8 – 4) ( أسجىع عهً انزىاني دوٌ حدوس إَحزاف في 30و9انزسجيزيدوٌ في ثعض انًسزحضزاد اندوائيخ.أيكٍ إسزعًبل األلطبة نفززح ) انجهد. ية آيىوية ،بىليمرات الطبعة الجزيئية ، أقطبة كرافيث مطلية بأغشية متعدد كلىريد الفبيىيل ، الرسبيريدون.الكلمبت المفتبحية : أقطبة إوتقبئ introduction risperidone (ris.) belongs as a chemical structure to an important antipsychotic medication chemical group called "benzioxazoles" (1,2) that is mainly used to treat schizophrenia , schizoaffective disorder, the mixed and manic states of bipolar disorder, and irritability in people with autism. (ris.) undergoes hepatic metabolism and renal excretion. (2) it is a second-generation atypical antipsychotic agents (sgas) which are broadspectrum antagonists of dopamine, alpha noradrenergic, serotonin and histamine receptors. the structural formula of (ris.) is shown in scheme (1). scheme (1): the structural formula of risperidone. 1 corresponding author e-mail: hamssa28_munam@yahoo.com received: 30/5/ 2015 accepted: 3/11/2015 http://en.wikipedia.org/wiki/schizophrenia http://en.wikipedia.org/wiki/schizoaffective_disorder http://en.wikipedia.org/wiki/schizoaffective_disorder http://en.wikipedia.org/wiki/bipolar_disorder http://en.wikipedia.org/wiki/bipolar_disorder http://en.wikipedia.org/wiki/atypical_antipsychotic http://en.wikipedia.org/wiki/atypical_antipsychotic http://www.crazymeds.us/pmwiki/pmwiki.php/sources/glossary#antagonists http://www.crazymeds.us/pmwiki/pmwiki.php/sources/glossary#dopamine http://www.crazymeds.us/pmwiki/pmwiki.php/sources/glossary#serotonin http://www.crazymeds.us/pmwiki/pmwiki.php/sources/glossary#receptors iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 31 the mechanism by which atypical drugs achieve this highly selective approach to treatment is the antagonism of a specific serotonin receptor, 5-ht2a .this receptor can be found on the axon terminal of neurons that produce dopamine.when serotonin activates those receptors, the release of dopamine decreases. by building into the typical antipsychotics a mechanism to block the 5ht2a receptors from serotonin, dopamine release is increased (3, 4) . (ris.) was determined by using other techniques; spectrophotometric (5) , high performance liquid chromatography(hplc) (6) and solid phase extraction (spe) (7) . molecular imprinting is a process in which functional and cross-linking monomers are copolymerized in the presence of the target analyte (the imprint molecule), which acts as a molecular template. (8, 9) the functional monomer initially interacts with the imprint molecule to form a multi-action spot (complex). (10) after polymerization, their functional groups are held in position by the highly cross-linked polymeric structure (8, 9) . additionally, the steric arrangement of these interactions around a given substrate (molecular template) is a crucial aspect necessary for the creation of binding pockets providing complementary size, shape, and functionality for preferentially facilitating selective recognition along with a high affinity toward the target. thus, the recognition process in mips may be described in analogy with mechanisms established for enzyme – substrate-complexes such as the lock-and-key (11) . the synthesis of mips usually involves a parallel process involving synthesis of a control non-imprinted polymer (nip) under conditions identical to those of the mip except that the template is absent. in principle, the nip is entirely analogous to the mip except that any binding sites within its porous structure are non-selective. the nip can therefore be used as a benchmark for assessing the selectivity of the mip (12) . experimental apparatus the ftir spectra of mips and nips were obtained using ftir-8400s spectrometer (shimadzu, japan). a shimadzu 1800 uvvisible, double beam spectrophotometer (kyoto–japan) with 1 cm path length quartz cell. all potentiometric measurements were made at room temperature with a ph/mv meter philips pw9421, england. the potentiometric measurements were recorded using the fabricated ris-mip membrane coated on a homemade graphite electrode in conjugation with a reference saturated calomel electrode (sce). the ph values were recorded using wtw gmbh , inolab ph 7110 germany ph meter. reagents the entire chemical used were reagent grade with highest purity and used as received without further purification. acrylic acid (aa) (99.0% himedia ltd), acrylamide (aam) (99.0%, fluka), benzoyl peroxide (bpo) (75%,acros organics), ethylene glycol dimethacrylate (egdma) (95%, bdh), dioctyl phthalate (dop) (99%, bdh), tetrahydrofuran (thf) (99.0%, riedel-dehaen). highmolecular-weight polyvinyl chloride (pvc) (99.5%, bdh). for ph control, sodium hydroxide (0.1 m) and hydrochloric acid (0.1 m) were used. pharmaceutical grade risperidone powder received in pure form (99.99 %) were provided from (arab pharmaceutical manufacturing company, jordan) as a gift from the university of science and technology amman, jordan. pharmaceutical grade domperidone and cephalexin powders received in pure form (99.99 %) were provided as a gift from state company for drug industries and medical appliances samara-iraq (sdi). respal caplets (2mg), joswe medical (jordan) and rispond tablets (4mg), india were purchased from local markets. synthesis of ris-mips mips for (ris.) were prepared using a bulk polymerization procedure. 3.6542 mmol of the template (ris) was dissolved in 5 ml of chloroform in a thick walled glass tube. 14.6168 mmol of functional monomer acidic (aa) or basic (aam), 73.084 mmol of crosslinker (egdma), and 0.32 mmol of initiator (bpo) were then added to the above solution respectively. the mixture was degassed by purging nitrogen for 10 minutes and shacked in ultrasonic water bath. the reaction flask was then removed, sealed and heated at 70°c in a water bath for 15 min. to allow initiation of the reaction. orange colored polymers with a rigid structure were formed. schemes (2) and (3) illustrate the synthesis of mips for (ris.) based on (aa) and (aam) as acidic and basic monomers. iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 32 scheme (2): ris-mip synthesis, using acrylic acid (aa) as an acidic functional monomer. scheme (3): ris-mip synthesis, using acrylamide (aam) as a basic functional monomer. non-reacted species (excessive reagents or template) were removed from the polymers by consecutive washout of the particles with chloroform then diethyl ether and were dried at room temperature overnight. the polymers were then crushed grounded using mortar and pestle and sieved to particles size 150μm (using 100 mesh sieve). the template (ris.) was removed by repeated washing the mips successively with 100 ml portions of 30% (v/v) acetic acid / water solution. the elimination of (ris.) from the mips was confirmed by measuring the absorbance of the washout solution at 274 nm. after the polymer was completely dried at ambient temperature, it was used as an active material in the selective sensor. a control non-imprinted polymer (nip) was prepared according to the same procedure, but excluding the target molecule, (ris.) (13, 14) . construction of the mip membrane graphite electrode the sensing pvc membrane was prepared by mixing 0.17 g of high molecular weight pvc, 0.4 g of plasticizer either dop or dbp and 0.02 g of the mip or nip particles. after homogenization, 2-3 ml of thf was added and stirred. the graphite coated electrode (gce) was constructed by dipping a graphite rod (3 mm diameter and 10 cm long) in the above solution and allowing it to dry in air. the procedure was repeated until the coating thickness is about 1.0 (±0.1) mm. (13, 15) emf measurements the performance of the electrode was investigated by measuring the e.m.f. using the following cell: hg(l) | hg2cl2(s), kcl(sat'd) || test solution | mip membrane | ge preparation of 1.0×10 -2 m (4.105 mg/ml) ris.ʼs stock solution a standard stock solution of (ris.) was prepared by dissolving accurately weighed 0.4105 g of pure drug in 10 ml of 0.1 m hcl and the volume was completed up to the mark in 100 ml volumetric flask with distilled water. working solutions were freshly prepared by subsequent dilutions. potentiometric measurements of the proposed sensors all potentiometric measurements were carried out at room temperature (25±2˚c) in stirred solutions. aliquots from standard drug solution 1.0×10 −2 m were diluted to obtain a series of solutions with concentrations ranged (5.0×10‐ 9 ‐1.0×10‐ 2 ) m in 50ml volumetric flasks. 30ml of each solution was transferred to a beaker and the e.m.f. values were recorded using (ris.) electrode in conjunction with a reference sce after stabilization to ±0.2 mv and the calibration curve was plotted. the calibration plot was used for measuring unknown concentrations under the same conditions. (16) binding experiments binding experiments were carried out by placing 0.02 g of ris-mip in contact with 10.0ml (ris.) solutions ranged (0.005–0.32) mm .the mixtures were shacked for 2h at (25˚c±2) and the solid phase was separated by centrifugation 6000) rpm) for 40 minutes. the concentrations of free drugs in the supernatant were detected by uv-vis spectrophotometry at 238 nm. the amount of drugs bound to the polymer was calculated by subtracting the concentration of free drug from the initial drug concentration. the data obtained were used for a scatchard analysis. (16, 14) determination of (ris.) in pharmaceutical formulations mip electrode for (ris.) based on (aam) as basic monomer was applied for the direct and standard addition determinations of (ris.) in two pharmaceutical formulations; respal® (2mg) caplets and rispond (4mg) tablets. ten tablets were finely ground, homogenized, then 0.2546 gm of respal®, iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 33 0.3572gm of respond were weighed accurately and separately, dissolved in 12ml of 0.1nhcl. the mixture was sonicated for 20min to aid dissolution and left to stand for (1 h). the solution was filtered by using whatman filter paper no.41 to avoid any suspended or un-dissolved material before use. the resulting filtrate was transferred into a 25 ml volumetric flask and the volume was completed to the mark with distilled water to get 1.0×10 -3 m. working solutions were freshly prepared by subsequent dilutions with distilled water, and analyzed by the recommended procedure. results and discussion ftir of acidic and basic mips of (ris.) the ftir spectra of (ris.), control nonimprinted and molecularly imprinted polymer based on (aa) as a functional monomer prepared using radical bulk polymerization are shown in figure (1). both polymers have similar ir spectra indicating the similarity in the backbone structure. in the ir spectra, the absorptions due to sp 2 -ch str.(3228)cm -1 , sp 3 ch str. 2peaks(2985,2956) cm -1 , c=o str.(1732) cm -1 , c=c str.residual(1668) cm -1 , sp 3 -ch scissoring (1456) cm -1 , sp 3 -ch rocking (1390) cm -1 , o–c str.( 1257) cm -1 , c–o acryl str.(1159) cm -1 , c–o alkoxy str.( 1047) cm 1 ,sp 2 -ch ben. out of plane (960) cm -1 were observed. a) (a) (b) (c) figure (1): ftir of (a): (ris.) (b): ris-mip(aa) after the removal of (ris.) (c) : ris-nip(aa). iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 34 in addition to backbone similarity concluding, those absorbances attributed to the previous groups for the molecularly imprinted polymer are relatively stronger than for non-imprinted polymer. from this comparison it was found that presence of imprint molecule (ris.) causes incorporation of ethylene glycol dimethacrylate in the preparation of polymer to be increased. (17) on the other hand the ftir spectra of (ris.), control non-imprinted and molecularly imprinted polymer based on (aam) as a functional monomer prepared using radical bulk polymerization are shown in figure (2). both polymers have similar ir spectra indicating the similarity in the backbone structure. in the ir spectra, the absorptions due to -nh2 str. 2peaks (3435, 3370) ) cm -1 , sp 2 -ch str.( 3200) ) cm -1 , sp 3 -ch str.( 2957) ) cm -1 , ester c=o str.( 1726) ) cm -1 , amide c=o str.( 1690) ) cm -1 , c=c str. residual(1645) ) cm -1 , nh ben.( 1535) ) cm -1 , sp 3 -ch scissoring(1456) ) cm -1 , sp 3 -ch rocking(1382)cm -1 , o–c str.( 1295) ) cm -1 , cn str.( 1261) ) cm -1 , c–o acryl str.( 1159) ) cm -1 , c–o alkoxy str.( 1051) ) cm -1 , sp 2 -ch ben.out-of-plane(948) ) cm -1 were observed. in addition to backbone similarity concluding, those absorbances attributed to the previous groups for the molecularly imprinted polymer are relatively stronger than for non-imprinted polymer. from this comparison it was found that presence of imprint molecule (ris.) causes incorporation of ethylene glycol dimethacrylate in the preparation of polymer to be increased (17) . . (a) (b) figure (2): ftir of (a): ris -mip(aam) after the removal of (ris.), (b) : ris -nip(aam). electrodes performance characterization of the primary analytical features [according to iupac recommendations] of graphite electrodes based on (ris.) mip or nip particles incorporated in pvc membrane and plasticized with dop was investigated (18) .the results are shown in table (1). ris – mip sensors using aa and aam as monomers displayed slopes of 55.2  0.1 and 59.00.2 m v decade -1 respectively . the sensors showed linear responses in the range of (1.0×10 -6  1.0 ×10 -2 ) m and (5.0×10 -7  1.0× -2 ) m of (ris) , with detection limits of 3.3×10 -7 m ,respectively as shown in figures (3,4) . iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 35 the life time of electrodes were estimated by their periodical recalibration and calculations of slope and limit of detection. a drift in the performance of electrodes i and iii was noticed after 7 and 13 weeks respectively which indicate the end of electrodes lives. two membrane compositions were investigated by using two types of plasticizers (dop and dbp) as shown in figure (5).the results shown in table (1) for electrodes (iii and v) indicate that membrane plasticized with (dop) displayed better performance than that plasticized with (dbp). the (dop) appeared to be more compatible with the ris-mip as it produces a homogenous and clear membrane having better slope and longer life time than (dbp). table (1): the characteristics of the ( ris.) gce based on mips and nips using different functional monomers and plasticizers. figure (3): calibration curve of ris-mip (i) and ris-nip (ii) gces based on (aa) as a functional monomers and dop as plasticizer. figure (4): calibration curve of ris-mip (iii) and ris-nip (iv) gces based on (aam) as a functional monomers and dop as plasticizer. type of plasticizer (dop) as plasticizer (dbp) as plasticizer electrode no. i ii iii iv v type of electrode ris-mip (aa) ris-nip (aa) ris-mip (aam) ris-nip (aam) ris-mip (aam) slope 55.2±0.1 36.855 59.0±0.2 26.8 47.9±0.9 correlation coefficient (r 2 ) 0.9997 0.9975 0.9999 0.9796 0.9988 linear range(m) (1.0×10 -6 1.0×10 -2 ) (5.0×10 -5 1.0×10 -2 ) (5.0×10 -7 1.0×10 -2 ) (5.0×10 -7 1.0×10 -2 ) (5.0×10 -7 1.0×10 -2 ) lod(m) 3.3×10 -7 0.0×10 -6 2.0×10 -7 4.0×10 -7 3×10 -7 response time (s) 15-20 15-20 15-20 15-20 30 life time(week) 7 13 3 rsd% 0.1329 0.2989 1.9951 re% -0.0767 -0.1717 -2.2513 iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 36 figure (5): the effect of using dop (iii) and dbp (v) plasticizers on the characteristics of (ris.) gces based on mip using (aam) monomer. effect of ph the effect of ph on the potential readings of the electrodes (i and iii) was studied by immersing a combined glass electrode, (ris.) gce and a saturated calomel reference electrode in 50 ml beakers containing 25 ml aliquots of 1.0×10-3 and 1.0×10-4 m of (ris.) aqueous solutions. the ph of each solution was adjusted by 0.1n sodium hydroxide or hydrochloric acid solutions. the potential reading at each ph value ranging 1-10 was recorded. a plot of ph values versus the potential of the electrode is illustrated in figure (6). the electrode potential did not affect by the ph change in the range (4 – 8) which considered as the working ph range of the proposed sensor.at ph lower than 4 the electrode potential decreased due to the formation of di-protonated form of (ris.) where nitrogen atom of the pyrimidine ring is the preferred site for the second protonation (19). at ph value higher than 8, the decreased electrode potential might attribute to the deprotonation form of (ris.) which lead to the precipitation of the drug in the test solution. figure (6): ph effect on potential response of ris-mip(aam) electrode. the selectivity study the influence of the interfering ions on the response behavior of ion selective membrane electrode is usually described in terms of selectivity coefficients. the selectivity coefficients for (ris.) over a wide range of interfering molecules, monovalente and di-valent ions were determined by the separate solutions method (ssm) using electrode (iii) and listed in table (2).the selectivity coefficients calculated for (ris.) over each interference at aa= ab in a series of concentrations ranged (1.0×10 -7 -1.0×10 -2 ) m for each of the two separate solutions ,i.e. the (ris.) solutions and the interference solutions, were below the unity which indicates that these species do not interfere in the determination of (ris.) . the values of the selectivity coefficients were calculated according to the following equation (20) : ( ) ( ⁄ ) plots of selectivity of electrode (iii) to interfering molecules and ions are shown in figure (7) figure (7): selectivity of the electrode (iii) to interfering species by separate solution methods (ssm). iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 37 table (2): selectivity coefficient values of some interfering species calculated by separate solution method using electrodes (iii). validation of the proposed method the proposed sensor no.(iii) was successfully applied for the determination of (ris.) in pure solutions. four solutions of (ris.) with different concentrations were prepared and analyzed with triplicate measurements. the validation of the method was achieved by the accuracy and precision test of the proposed sensor (iii) expressed in terms of percent relative error and percent relative standard deviation for inter-day (repeated over 3 days) and intra-day (repeated independently three times a day) measurements of the prepared solutions. the results are shown in table (3). the recoveries of (ris.) for each concentration were also calculated. the results have shown good accuracy and precision. table (3): validation of the proposed method for the determination of (ris.) in pure form *average of three determinations interfering species at aa= ab 1.0×10 -7 m 1.0×10 -6 m 1.0×10 -5 m 1.0×10 -4 m 1.0×10 -3 m 1.0×10 -2 m k + 2.8×10 -3 1.5×10 -3 1.6×10 -4 1.8×10 -5 1.9×10 -6 2.9×10 -7 mg +2 2.9×10 -6 3.6×10 -6 1.4×10 -6 4.6×10 -7 2.5×10 -7 5.4×10 -8 tartaric acid 7.6×10 -3 2.9×10 -3 2.6×10 -4 5.6×10 -5 1.8×10 -5 1.7×10 -6 glycine 2.8×10 -3 1.5×10 -3 1.6×10 -4 1.8×10 -5 1.9×10 -6 2.9×10 -7 valine 2.1×10 -1 8.2×10 -2 1.0×10 -2 7.6×10 -4 7.3×10 -5 7.6×10 -6 glucose 4.2×10 -1 1.5×10 -1 1.2×10 -2 1.2×10 -3 1.2×10 -4 1.0×10 -5 sucrose 3.6×10 -3 1.1×10 -3 9.2×10 -5 9.3×10 -6 8.9×10 -7 5.2×10 -8 lactose 1.4×10 -1 6.8×10 -2 6.8×10 -3 8.6×10 -4 1.5×10 -4 1.2×10 -5 starch 1.4×10 -1 8.2×10 -2 8.2×10 -3 8.6×10 -4 1.9×10 -4 1.4×10 -5 cephalexin 2.1×10 -1 8.2×10 -2 1.0×10 -2 1.1×10 -3 1.9×10 -4 1.8×10 -5 domperidone 2.7×10 -1 11×10 -1 1.1×10 -2 1.2×10 -3 1.7×10 -4 1.1×10 -5 intra-day taken [ris.] mole /l *found [ris.] mole /l re % rsd % recovery % 1.0000×10 -6 1.0200×10 -6 2.0000 0.6792 102.0000 1.0000×10 -5 1.0400×10 -5 4.0000 1.9208 104.0000 1.0000×10 -4 9.9800×10 -5 -0.2000 2.2672 99.8000 1.0000×10 -3 9.9700×10 -4 -0.3000 2.2682 99.7000 inter-day taken [ris.] mole /l found [ris.] mole /l re % rsd % recovery % 1.0×10 -6 1.0500×10 -6 -1.0000 2.2300 105.0000 1.0×10 -5 1.0400×10 -5 2.0000 2.3046 104.0000 1.0×10 -4 9.7200×10 -5 -2.8000 2.2404 97.2000 1.0×10 -3 1.0100×10 -3 1.0000 2.2389 101.0000 iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 38 determination of (ris.) in pharmaceutical formulations the direct, the standard addition (s.a) and multiple standard addition (m.s.a) methods were applied for the determination of (ris.) in two types of pharmaceutical formulations; rispal @ 2mg caplets and rispond 4mg tablets. the results obtained using graphite coated electrode no.(iii) are listed in table (4). table (4): analysis of (ris.) in some pharmaceutical formulations by the direct and the standard addition methods using electrode (iii). *average of three determinations multiple standard addition (m.s.a) method has exhibited better results over the direct method. the method showed rsd% ranged between (0.0653-0.4965) and rec% ranged between (99.0400 101.1500). the reported percent recoveries of the drugs in table 4 were calculated using the values reported by the manufacturer which was obtained using the recommended assay by the british pharmacopoeia. binding characteristics to understand the way how small molecules interact with adsorbent surface, the mode of binding and site distributions in the interaction, adsorption isotherms are an important tool obtained by plotting of the equilibrium concentrations of bound mip versus free drug. in liquid-phase applications of mips, a molecule in solution interacts with binding sites in a solid adsorbent, and after equilibrium the free drug concentration becomes constant and is easily quantified to plot the corresponding adsorption isotherm. (21) this was determined by plotting the binding capacity (q) against the free (ris.). q was calculated according to the following equation: (22) ( ) where q is the binding capacity of mips (µmol/g), ci is the initial (ris.) concentration (mm), cfree is the final (ris) concentration (mm), vs is the volume of test solution (l) massmip is the mass of the dried mip (g). the adsorption isotherms obtained after shaking varying concentrations of (ris.) with the synthesized particles for 2 hours in a thermostatic water bath shaker at 25°c were plotted in figs. (8). sample pot. methods taken [ris.] m found [ris.] m weight found mg/ dosage *rsd% rec % respal 2mg direct 1.0000×10 -4 0.9400×10 -4 1.8800 2.2451 94.0000 1.0000×10 -5 0.9400×10 -5 1.8800 5.2477 94.0000 1.0000×10 -6 0.9880×10 -6 1.9760 0.1168 98.8000 sam 1.0000×10 -4 1.0409×10 -4 2.0818 0.7433 104.0900 1.0000×10 -5 1.0137×10 -5 2.0274 1.1628 101.3700 1.0000×10 -6 1.0904×10 -6 2.1808 0.4336 109.0400 msa 1.0000×10 -4 1.0010×10 -4 2.0020 0.3539 100.1000 1.0000×10 -5 1.0009×10 -5 2.0018 0.4965 100.0900 1.0000×10 -6 0.9917×10 -6 1.9834 0.3920 99.1700 rispond 4mg direct 1.0000×10 -4 0.9120×10 -4 3.6480 0.1064 91.2000 1.0000×10 -5 9.9900×10 -5 3.996 1.8148 99.9000 1.0000×10 -6 0.9910×10 -6 3.9640 0.8159 99.1000 sam 1.0000×10 -4 1.0129×10 -4 4.0516 1.2912 101.2900 1.0000×10 -5 0.9889×10 -5 3.9556 1.1724 98.8900 1.0000×10 -6 0.9678×10 -6 3.8712 0.4419 96.7800 msa 1.0000×10 -4 0.9905×10 -4 3.9620 0.1426 99.0500 1.0000×10 -5 0.9904×10 -5 3.9616 0.0761 99.0400 1.0000×10 -6 1.0115×10 -6 4.0460 0.0653 101.1500 iraqi j pharm sci, vol.24(2) 2015 potentiometric transducers for the selective recognition of risperidone 39 a1 a2 b1 b2 figure (8): a1,a2,b1,b2 are binding isotherm and scatchard plots of rismip(aa) and ris-mip(aam) respectively. in general, adsorption data showed that the binding capacity of ris-mips increased with increasing initial concentrations of (ris.), leading to saturation at higher concentrations. the binding parameters of the binding rismips were calculated by scatchard analysis, with the following equation: where cfree is the free analytical concentration at equilibrium (mm), qmax is the maximum apparent binding capacity. kd is the dissociation constant at binding site. the equilibrium dissociation constant was calculated from the slopes and the apparent maximum number of binding sites from the y-intercepts in the linear plot of q/ci vs. q as shown in fig. (8).the scatchard plot for mip based on (aa) as a monomer was not linear in all (ris.) concentration ranges, suggesting that the binding sites in the mip were not uniform. the plot shows two distinct sections that can be regarded as straight lines, revealing two types of distributing binding sites in the mip. the equilibrium dissociation constant kd1(aa) and the apparent maximum amount qmax1(aa) for the higher affinity binding sites were calculated to be 34.6021 µm and 10.8547 µmol/g respectively for the dry polymer. using the same treatment, kd2(aa) and qmax2(aa) for the lower affinity binding sites were calculated to be 6.9638 µm and 10.7716 µmol/g respectively. in contrast, when (aam) was used as a monomer, the scatchard plot was linear in all concentration ranges, suggesting that the binding sites were homogeneous and of one type. the equilibrium dissociation constant kd(aam) and the apparent maximum amount qmax(aam) for the higher affinity binding sites were calculated to be 84.0336 µm and 31.1765 µmol/g respectively for the dry polymer (12) . according to the equilibrium dissociation constants (kd) of the two acidic and basic ris -mips, it was noticed that basic ris-mip has homogenous binding site distributions which lead to better results in its application than acidic ris-mip which exhibited high (kd) value as well as heterogeneous binding sites distributions. (11, 22) conclusions the molecular imprinting technique was employed to produce (ris.) host – tailored sensors incorporated in pvc matrix and coated on a graphite electrode for potentiometric transduction. the electrodes were successfully used for the determination of (ris.) in their pure form and pharmaceutical formulations. the mip based electrodes using (aam) showed a higher affinity for the templates (ris.) than (aa) based electrodes in the sense of their nernsation slopes. the electrodes exhibit a short response time good stability, sensitivity, selectivity and life time more than two months. the rebinding procedure confirms the performance of (ris.) mip gce based on (aam) as basic monomer. references 1. halter m. jordan .varcarolis , foundations of psychiatric mental health nursing a clinical approach, saunders, an imprint of elsevier inc.; 2014; 58-62. 2. schatzberg a. f. and nemeroff c. b. .textbook of psychopharmacology4 th ed., the american psychiatric publishing inc., 2009; 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81(1): 631–639. 20. umezawa y. ; bühlmann p.; umezawa k. ; tohda k. and amemiya s. potentiometric selectivity coefficients of ion-selective electrodes. part i. inorganic cations, pure appl. chem.,).2000; 72(10): 1851–2082. 21. j. r. l., guerreiro m., sales g. f., moreira f. t. c. and rebelo t. s. r. selective recognition in potentiometric transduction of amoxicillin by molecularly imprinted materials. eur food res technol. 2011; 232: 39–50. 22. öpik a. , menaker a., reut j., and syritski v. molecularly imprinted polymers: a new approach to the preparation of functional materials. proceedings of the estonian academy of sciences, materials science. 2009; 58(1): 3–11. http://www.jourlib.org/search?kw=torres%20v&searchfield=authors http://www.jourlib.org/search?kw=pablo;%20sep%c3%balveda%20c&searchfield=authors http://www.jourlib.org/search?kw=m.%20jacqueline;%20von%20plessing%20r&searchfield=authors http://www.jourlib.org/search?kw=carlos;&searchfield=authors http://www.scielo.cl/scielo.php?script=sci_serial&pid=0717-9707&lng=es&nrm=iso http://iupac.org/publications/analytical_compendium/ http://iupac.org/publications/analytical_compendium/ http://www.ncbi.nlm.nih.gov/pubmed/?term=alparone%20a%5bauthor%5d&cauthor=true&cauthor_uid=21764365 iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel doi: https://doi.org/10.31351/vol31isssuppl.pp32-44 32 formulation variables effect on gelation temperature of nefopam hydrochloride intranasal in situ gel (conference paper) # ammar a. alabdly*,1 and hanan j. kassab ** # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 * department of pharmaceutics, college of pharmacy, anbar university, anbar, iraq ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract nefopam (nf) hcl is a non-narcotic centrally-acting, non-opioid benzoxazocine analgesic used to relieve acute and chronic pain. it exhibits low bioavailability (about 36%) due to its first-pass degradation in the liver. intranasal administration has been proposed as a new route for targeting active brain sites and enhancing the bioavailability of nf hcl by bypassing hepatic metabolism. in situ gel of nf hcl was prepared by the cold method using different concentrations of poloxamer 407 (polox 407), poloxamer 188 (polox 188), hydroxypropylmethyl cellulose (hpmc k4m), carbapol 934 (car934), methylcellulose (mc) and hyaluronic acid(h.a) polymers. the results showed that identification tests including differential scanning calorimetry (dsc), fourier transform infra-red spectroscopy (ftir), and spectrophotometric λ max determination are superimposed with references, solubility study shows that nf hcl is suitable to be administered intranasally; compatibility studies reveal incompatibility of n.f hcl with hpmc k4m and car934; meanwhile, no interaction with mc and h.a. in conclusion, the obtained results revealed the feasibility of the produced nf hcl intranasal in situ gel. keywords: nefopam hydrochloride, thermosensitive in situ gel, poloxamer 407, poloxamer 188. موقعي داخل االنف لعالج النيفوبام تأثير بعض متغيرات الصياغة على درجة حرارة تكون الهالم ال # مؤتمر( )بحث هايدروكلورايد ** جالل كساب وحنان 1*،العبدلي دعبد المجيعمار 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي العراق. االنبار، االنبار، جامعة الصيدلة، كلية الصيدالنيات، فرع * العراق. بغداد، بغداد، جامعة الصيدلة، كلية الصيدالنيات، فرع ** الخالصة األمد، فئتها الكيميائية البنزوكسازوسين، والطويلينتمي عالج النيفوبام الى فئة االدوية غير المخدرة من مسكنات االآلم لالستخدام القصير .تجاوز هذه المشاكلل نف لالللتحضير كهالم موقعي لذلك يرشح .الكبدتكسره في ل %( 36)يبلغ ف يواجه مشكلة في توافره الحيوي و و هيدروكسي بروبيل مثيل 188و بولوكزامر 407تم تحضيير الصيغة باستخدام الطريقة الباردة عن طريق مزج تراكيز مختلفة من بولوكزامر و أظهرت دراسة الذوبانية المصادر، أظهرت النتائج تطابق المواد المستخدمة مع ، و مثيل سليلوز. 934 سليلوز ، حمض الهايلورونك ، كاربوبول اما التوافقية فأظهرت عدم توافق المزيج الذي يحتوي هايدروكسي بروبيل مثيل سليلوز و الكاربوبول بينما الهيئة،قابلية استخدام النيفوبام على هذه يستنتج من ذلك ان النتائج المستحصلة كشفت عن القدرة لتصييغ النيفوبام كهالم موقعي الهايلورونك و المثيل سليلوز. أظهرت توافق استخدام حمض داخل االنف لتحسين توافره الحيوي. . 188، بولوكزامر 407الكلمات المفتاحية : نيفوبام هايدروكلورايد ، هالم موقعي متحسس للحرارة ، بولوكزامر introduction a direct nose to brain delivery system is an excellent way to increase absorption to the central nervous system via the olfactory zone positioned at the top of the nasal cavities crossing the blood-brain barrier to reach the brain. nose-to-brain delivery offers many advantages including the absence of stomach and pancreatic enzymes, mucus's ph neutrality in the nasal cavity, less dilution by the gastrointestinal tract contents, and the mucosa are more permeable to drugs than the gastrointestinal system. additionally, it diminishes the therapeutic dosage and associated adverse effects(1). this mode of administration may be advantageous for treating serious brain diseases like tumors or deteriorating neural illnesses such as parkinson and alzheimer (2) or as migraine therapy(3). n.f hcl is a non-narcotic centrally-acting, non-opioid benzoxazocine analgesic used to relieve acute and chronic pain (4) and chemical name of 5methyl -1 -phenyl -1,3,4,6tetrahydro2,5-benzoxazocine; hydrochloride as seen in figure 1 and molecular weight of 289.8 log p ~ 3.4(5). 1corresponding author e-mail: amar.abdulmajeed1200a@copharm.uobaghdad.edu.iq received: 28/ 6/ 2022 accepted: 5/9 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp32-44 iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 33 figure 1. chemical structure of nefopam hcl orally administration is associated with low bioavailability (about 36%) due to its first-pass degradation in the (6) and shows several common side effects related to peripheral action of nefopam hydrochloride, such anticholinergic side effects include urinary retention, dry mouth, and nausea (7). n.f hcl has a pka of 8.98, s0877t65dc vo it will be mainly in an ionized form in nasal mucosa ph (4.5-6.5) (8) mnhuitrewerjkgoi9 /and a molecular weight lower than 300,allow a better penetration via passive diffusion (9). also, the log p is higher than 35 therefore, penetration is predicted to be over 80% (10). in situ gel is a solution that undergoes sol-gel transformation by a specific physiological trigger such as body temperature in the case of thermosensitive in situ gel. 3 dalia m.n. abouhussein et al. develop rivastigmine noseto-brain targeted poloxamer based thermosinsitive mucoadhesive in situ gel(11). accordingly, the current study aims to explore the factors affecting on formulation and gelation temperature of polxamer 407 based intranasal in situ gel of nefopam. materials and methods materials nefopam (nf) hcl, hyaluronic acid (h.a), and hpmc k4m were acquired from (baoji guokang,ltd–china),car934,polox 407, and polox 188 were purchased from ( eastman chemical company – usa), benzalkonium chloride (bnz) was obtained as a gift from ( the state company for drug industry and medical appliances, sdi – iraq), every other substance, including reagents, was of analytical grade. methods characterization of nf hcl identification tests to confirm the active pharmaceutical ingredients (api) and excipients' identities and purities. determination of the melting point and differential scanning calorimetry (dsc) of nf hcl the melting point was determined using the capillary tube approach (12), and the thermal behavior was evaluated using a dsc60 (shimadzu 60 plus japan). a small quantity of nf hcl. was sealed in ordinary aluminum pans and then heated at rate of 10°c every min. over a range of temperatures 50°c to 300°c. fourier transform infra-red spectroscopy (ftir) ftir is effective method for identifying pure compounds and compatibility studies between the active pharmaceutical ingredient (api) and the excipient(s) (13,14). ftir was performed using lambda 7600 australia instrument for nf hcl, hpmc k4m, car934, h.a, mc, and physical mixture. the ftir spectrum of n.f hcl intranasal gel of the selected formulas and the excipients used were recorded using ftir spectrometer between spectral bands 4000 400 cm-1. the sample was mixed in a mortar with potassium bromide (kbr), then compressed into a tiny disc using a hydraulic press. standard calibration curves of nf hcl the standard calibration curves of nf hcl in double distilled water (d.d.w), simulated nasal fluid (snf, cacl2.2 h2o 0.32 g/l , nacl 7.45 g/l, and kcl 1.29 g/l.)(15), and aqueous phosphate buffer saline (pbs) at ph 7.4, were made by controlled dilutions of the stock solution (1mg/ml) at various concentrations (50–350 g/ml). each solution's absorbance was measured at the n.f hcl λmax. plotting was done between the measured absorbance and the corresponding concentration. solubility study of nf hcl the test tube method was used to measure the solubility of drugs in an aqueous buffer, simulated nasal fluid, and deionized water by adding an excess amount of nf hcl in 10 ml of solvents mentioned above in a glass tube, then stored at 34 oc takes 72 hours to establish an equilibrium state in a shaker water bath. after that, a solution centrifuged for 15 minutes at 3000 rpm ; then, the solution is filtered using 0.45 μm filter paper and a final concentration of nf hcl measured after a suitable dilution to be read by uv-vis (16). this test is checked in triplicate manner. iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 34 preparation of nf hcl intranasal in situ gel the cold approach was applied to formulate different compositions of mucoadhesive thermosensitive in situ gel as detailed in table 1. different concentrations of poloxamer 407 (polox 407) (17-20%) were blended with or without poloxamer 188 (polox 188) (3-4%) in cold double distilled water (d.d.w) immersed in ice with continuous stirring for 4-6 hours. the obtained cloudy mixture was stored in the refrigerator at 4 oc for 24 hours to get a clear solution. the gelation temperature was determined in triplicate for each formula; only compositions with a gelation temperature in the range of 30-36 oc (17) were selected for further addition to the final formula. table 1. compositions of intranasal in situ gel polymer mixture (p). p no. polox 407 (%w/v) polox 188 (%w/v) d.d.w q.s ml 1 17 10 2 18 10 3 19 10 4 20 10 5 17 4 10 6 18 4 10 7 19 4 10 8 20 4 10 9 17 3 10 10 18 3 10 11 19 3 10 12 20 3 10 after that, different concentrations of different mucoadhesive polymers (hpmc k4m, car934,h.a, and m.c) as shown in table 2. were added slowly to a selected polymer mixture with continuous stirring for 30 min at 50 rpm. the slow stirring speed was applied to prevent foam formation that can affect the mucoadhesive polymer solubility in poloxamer solution. then n.f hcl and bnz at a concentration (20 mg/ml) and (0.01 % w/v), respectively, were added slowly with continuous slow stirring for the same reason mentioned above, and complete the volume with double distilled water up to 10 ml. finally, the mixture was stored for 24 hours at 4 oc to eliminate a formed foam. table 2. compositions of the formulated intranasal in situ gel with mucoadhesive polymers (p.i.). p.i. no. nf mg polox 407(%) polox 188(%) hpmc k4m (%) h.a (%) car934 (%) m.c (%) 1 200 17 1 2 200 19 4 1 3 200 18 3 1 4 200 19 3 1 5 200 17 0.75 6 200 19 4 0.75 7 200 18 3 0.75 8 200 19 3 0.75 9 200 17 0.5 10 200 19 4 0.5 11 200 18 3 0.5 12 200 19 3 0.5 iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 35 continued table 2. p.i. no. nf mg polox 407(%) polox 188(%) hpmc k4m (%) h.a (%) car934 (%) m.c (%) 13 200 17 0.75 14 200 19 4 0.75 15 200 18 3 0.75 16 200 19 3 0.75 17 200 17 0.5 18 200 19 4 0.5 19 200 18 3 0.5 20 200 19 3 0.5 21 200 17 0.25 22 200 19 4 0.25 23 200 18 3 0.25 24 200 19 3 0.25 25 200 17 0.3 26 200 19 4 0.3 27 200 18 3 0.3 28 200 19 3 0.3 29 200 17 0.2 30 200 19 4 0.2 31 200 18 3 0.2 32 200 19 3 0.2 33 200 17 0.1 34 200 19 4 0.1 35 200 18 3 0.1 36 200 19 3 0.1 37 200 17 0.3 38 200 19 4 0.3 39 200 18 3 0.3 40 200 19 3 0.3 41 200 17 0.2 42 200 19 4 0.2 43 200 18 3 0.2 44 200 19 3 0.2 45 200 17 0.1 46 200 19 4 0.1 47 200 18 3 0.1 48 200 19 3 0.1 gelation temperature the gelation temperature is determined in triplicate by visual inspection of the glass tube when it is tilted at 90 degrees. gelation temperature recorded when no movement of solution occurred as a result of conversion into a gel. each glass tube that contained 2 ml of the formulation was kept in the water bath at 25 oc; then, the temperature raised gradually at a rate of 1 oc / min, and at the end of the minute, a glass tube titled at 90 0 in a horizontal position to show if the solution is converted into a gel (18). appearance visual inspection of selected formulas against white and black paper in triplicate were conducted to examine the occurrence of any incompatibility due to an interaction between api with excipients or among excipients themselves. a change in color or precipitation was considered as a sign of incompatibility (19). compatibility study ftir was carried out on several separated formulas observed by appearance test to compare its ftir spectra with the original spectra of drug and polymers. iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 36 ftir was performed with the same method mentioned previously in ftir section. gelling capacity by adding 1 ml of the system to a vial containing 2 ml of freshly prepared and equilibrated at 34° c simulated nasal fluid, and visually observing the gel formation and noting the time for gelation every 5 sec. and the time taken for the gel formed to dissolve, noting desolvation every 15 min., the gelling capacity was determined in triplicate(20). ph of nf hcl intranasal in situ gel the ph of the selected p.i formulas was measured by using hi98107 ph meter, hanna, italy, in triplicate at room temperature using ph meter. ph 4 and ph 7 standard buffer solutions were utilized to calibrate the equipment (21). the osmolarity of nf hcl intranasal in situ gel osmotic pressure was measured by using a osmomat 030 cryoscopic osmometer. the osmometer was zeroed by purified water and then 300 mosmol nacl (1%nacl) standard solution for calibration. then, 50 µl of the p.i formulas were used in a clean, dry microtube. after checking for bubbles of air , the measurement performed. (22), and the results were recorded in triplicate. mucoadhesive strength the weight needed to separate a preparation from nasal mucosa of sheep using a modified pan balance for p.i formulae yielded the mucoadhesive detachment force. within 30 minutes of the sheep's sacrifice, we were able to retrieve a sheep nose from the slaughterhouse. the sheep's nasal mucosa was preserved in 0.9% nacl. it was prepared for usage after blood and bony cartilage were taken out of the mucosal membrane. a glass vial was used to hold the mucosal side. before they were connected to the equipment, these vials were held at 34 °c for 5 minutes. one side included a modified syringe vial that was inverted and held 1 ml of formula, and the other side contained a small plastic beaker that had previously been weighted. the syringe held specific amounts of each formulation (1 ml). by permitting two minutes of contact time, it was possible to confirm that tissue glued to a petri dish underneath the syringe was in intimate contact with the formula. the weight increased in gradually in the plastic pan on the other side until the formula separated from the tissue. the bioadhesive force, which represents the minimal weight necessary for tissues to detach from the surface for any formulation, is determined using the equation; mucoadhesive force (dynes/cm2 ) = mg / a where m is the smallest weight in grams needed to separate two vials, g is the acceleration gravity which is (980 cm/s2), and a is the exposed area of the tissue. for each measurement, the newly formed nasal mucosa was employed (21). results and discussion melting point determination the temperature at which nf hcl melted by the method mentioned above was found to be 260 °c, which coincides with the reported melting point (23). differential scanning calorimetry (dsc) the dsc of nf hcl, shown in figure 2 , agrees with the reference that it exhibits an endothermic peak at 259.8 oc (24) both melting point and dsc give good evidence for the purity of crystalline drug powder. figure 2. dsc thermogram of n.f hcl iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 37 fourier transform infra-red spectroscopy (ftir) ftir was carried out on drug and polymers powder, as shown in the figures 3 and 4, r figure 3. ftir of nf hcl spectra is compatible with the characteristics absorption peaks of n.f hcl ftir spectra, at 3440 cm-1 ,3049 cm-1 , 2947 cm-1 , 2440 cm-1 , 1464 cm-1 , 1437 cm-1 , 1357 cm-1, 1107 cm-1 , 1028 cm-1 , and 760 cm-1 ,correlates to c-o-c of ring, substituted aromatic ring, c-h of alkane(s), resonance of amine salt nh+ , c=c of substituted aromatic ring, c3-n, c-o stretch of cyclic ether, o-disubstituted aromatic ring, and mono-substituted and o-disubstituted aromatic rings, respectively. these findings are superimposed with reference (25). figure 4. ftir for hpmc k4m (a), h.a (b), mc (c), car934 (d), polox 407 (e), and polox 188 (f). spectra show characteristics peaks of hpmc at 3650-2800 cm-1 broad peak, 1653 cm-1, 1450 cm-1, and 1026 cm-1, that are corresponded to a broad range of hydroxyl hbond that overlap and cover sp3 c-h stretching of alkane, cellulose nitrate ester group that converted from cellulose hydroxyl groups, spectra of poly-(vinyl alcohol) o-h deformation spectra of a secondary alcohol, and c-o-c spectra, respectively. spectra coincide with reference (26). h.a peaks at 3500-3000 cm-1,2891 cm-1, 1603 cm-1, and 1030 cm-1 are indicated for broad spectra of h-bond of hydroxyl of acidic, alcoholic, and amine groups of hyaluronate, ch stretching of ring moieties, cooh group of an acidic group, and c-o-c ether group. that agrees with reference (27). mc peaks at 3600-3050 cm-1,2926 cm-1, 1640 cm-1, 1456 cm-1, and 1046 cm-1; these are spectra for broad h-bond spectra of acidic and alcoholic -o.h. group that overlap weak c-h stretching peak at 3000-3100 cm-1, c-h stretching of cyclic alkane, cellulose nitrate ester group that converted from cellulose hydroxyl groups, poly(vinyl alcohol), and co-c peak, respectively. these peaks are compatible with the reference peaks(28). iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 38 car934 peaks at 3500-2500 cm-1, 1711 cm-1, and 1165 cm-1, correlate to carboxylic dimer h-bond cooh that hide sp3 c-h stretching peak, c=o, and c-o stretching mode, respectively. that is match the reference(29). polox 407 , and polox 188 has two characteristic peaks appear at 2868 cm-1 and 1100 cm-1, representing the reading of r-ch3 and c-o-c peaks, respectively. these are agreed with the reference(30,31). standard calibration curves nf hcl shows a maximum absorption peak at 266 nm. that is compatible with the results of references (32,33). accordingly, standard calibration (std) curves figure 5 were used to calculate saturated solubility of nf in different media. figure 5. standard calibration curves of nf hcl in different solvents solubility the solubility of nf hcl in aqueous buffer saline at ph 7.4, simulated nasal fluid ph 6.4, and deionized water ph 7, shown in table 3. table 3. saturated solubility of nf hcl in different solvents. solvent saturated solubility (mg/ml) (mean±sd, n=3) aqueous buffer 21.957 ± 0.05 snf 13.2 ± 0.11 deionized water 21.679 ± 0.07 the fact that only (100 – 250 µl ) can be instilled in a human nose (34) makes a potent drug with high solubility essential for intranasal formulations. drugs with poor water solubility at high strengths are challenging to synthesize without employing significant amounts of solubilizing agents, which might possibly harmful excipients. (35). gelation temperature initially sol-gel temperature was measured for polymer mixtures that composed of only poloxamer 407 and poloxamer 188 as shown in table 4. table 4. temperature of gelation for intranasal in situ gel polymer mixture (p). p no. polox 407 (%w/v) polox 188 (%w/v) d.d.w q.s ml sol-gel temp. (mean±sd, n=3) 1 17 10 32.1 ± 0.653 2 18 10 29.5 ± 0.430 3 19 10 27.3 ± 0.120 4 20 10 23.8 ± 0.115 5 17 4 10 42.4 ± 0.346 6 18 4 10 38.5 ± 0.152 7 19 4 10 35.1 ± 0.247 8 20 4 10 28.4 ± 0.432 9 17 3 10 40.6 ± 0.461 10 18 3 10 36.0 ± 0.154 11 19 3 10 32.3 ± 0.280 12 20 3 10 27.8 ± 0.316 only p formula with gelation temperature 30-36 oc 15 were selected for further addition of mucoadhesive polymers and n.f hcl. iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 39 formulations inclusion criteria and exclusion criteria gelation occurs at room temperature if the temperature is lower than 32oc, which makes it difficult to manufacture, handle, and administer. the formulation will remain liquid at body temperature if the gelation temperature is higher than 34oc, resulting in nasal drainage. drug and additives may increase or decrease gelation temperature. as a result, the formulation's gelation temperature must fall between the following range: 30-36ºc for p formula to allow study the effect of drug and additives on gelation temperature. and the p.i formulations of table 5 must have gelation temperature 32-34 oc. for the same reasons mentioned above, any formula above 36 oc or below 30 oc for p formula , and any p.i formula above 34 oc or below 32 oc are excluded from further studies. results from table 4 indicated that polox 407 or polox 188 alone could not provide a suitable gelation temperature. only formula ( p no. 1 ) with (17% polox 407) show gelation temperature at suitable range. in cases of mixtures of polox 407 and polox188, several formulations ( p no. 7,10,and 11) are gelled at the included temperature.table 5 show that (p.i no. 1, 4, 5, 8, 9, 12 ,17 ,20, 21, 24, 28, 29, 32,33,36,41,44,and 45) are gelled within included temperature. table 4 and 5 show that as the concentration of polox 407 increase, the gelation temperature decreases due to aggregation and formation of critical gel concentration (cgc),which become more easily as the concentration increases, and as the concentration of polox188 , hpmc k4m, h.a, m.c, and car934 increases, the sol-gel temperature increase because these polymers will build up inside a gel texture of polox407 and inhibit gel formation so that the critical gel temperature will be higher(36). table 5. gelation temperature(s-g) of the p.i formula. p.i 1 2 3 4 5 6 7 8 s-g temp (mean±sd, n=3) 33.9 ± 0.113 40.1 ± 0.425 38.2 ± 0.236 34 ± 0.364 33.2 ± 0.168 39.4 ± 0.536 40.2 ± 0.325 33.1 ± 0.364 p.i 9 10 11 12 13 14 15 16 s-g temp (mean±sd, n=3) 33.2 ± 0.424 40.2 ± 0.244 41.3 ± 0.461 32.2 ± 0.145 34.1 ± 0.295 41.2 ± 0.196 42.1 ± 0.325 34.2 ± 0.212 p.i 17 18 19 20 21 22 23 24 s-g temp (mean±sd, n=3) 33.2 ± 0.315 40.4 ± 0.213 41.5 ± 0.116 34 ± 0.187 34 ± 0.285 41.9 ± 0.314 39.8 ± 0.412 33.9 ± 0.440 p.i 25 26 27 28 29 30 31 32 s-g temp (mean±sd, n=3) 34.8 ± 0.173 41.7 ± 0.238 42 ± 0.315 34 ± 0.164 34 ± 0.253 42.1 ± 0.426 40.2 ± 0.154 33.1 ± 0.332 p.i 33 34 35 36 37 38 39 40 s-g temp (mean±sd, n=3) 33.9 ± 0.227 37.8 ± 0.463 38.9 ± 0.352 33.1 ± 0.109 35.9 ± 0.267 39 ± 0.315 40.7 ± 0.573 38.8 ± 0.012 p.i 41 42 43 44 45 46 47 48 s-g temp (mean±sd, n=3) 34 ± 0.173 38.5 ± 0.532 38.9 ± 0.425 34 ± 0.316 33.1 ± 0.305 35.7 ± 0.311 36.9 ± 0.130 35.8 ± 0.189 iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 40 appearance appearance and clarity test reveal formation of cloudy and precipitate at the bottom of container for formulas that contain hpmc k4m and car 934 in all concentrations. table 6. shows that p.i formula no. (1-12) and (25-36) are cloudy with precipitate, and one selected precipitated cloudy formula for hpmc k4m (p.i no.4) and car934 (p.i no.26) are subjected to ftir scan to check its compatibility and reason of separation. table 6. appearance (p.a) results (cloudy -, very cloudy --, clear +, glassy ++, precipitate (ppt.)) p.i 1 2 3 4 5 6 7 8 p.a ppt. ppt. ppt. - ppt. ppt. ppt. ppt. ppt. p.i 9 10 11 12 13 14 15 16 p.a ppt. ppt. ppt. ppt. ++ + + + p.i 17 18 19 20 21 22 23 24 p.a ++ + ++ + ++ ++ ++ ++ p.i 25 26 27 28 29 30 31 32 p.a ppt. - ppt. - ppt. - ppt. ppt. - ppt. ppt. ppt. p.i 33 34 35 36 37 38 39 40 p.a ppt. - ppt. ppt. ppt. + + + + p.i 41 42 43 44 45 46 47 48 p.a + + + + ++ + + + compatibility study physicochemical compatibility between nf hcl and different excipients was studied using ftir to examine any interactions between the nf hcl and the excipients used in the p.i formulation. the spectrums were compared to the spectrum of nf hcl alone. ftir spectra of p.i no. 4 and 26. are shown in figure 6. figure 6. ftir of p.i no. 4 (a) and p.i no. 26 (b). in comparing with ftir of pure drug figure 3., we find that a peak has lower intensity with the absence of peaks at 2800 cm-1 – 1460 cm-1 region and absence of a characteristic peak at 1000 cm-1 – 1100 cm-1 of cyclic c-o-c; these might be resulted from the interaction of n.f hcl with hpmc k4m and car934. despite the critical need to verify apiexcipient compatibility, there is currently no globally acknowledged standard for evaluating such interactions; ftir and appearance are straightforward methods often used in analytical labs (13). iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 41 incompatibility is characterized as a change that occurs and an unfavorable product is produced, which may influence the pharmaceutical product's safety, effectiveness, stability, and appearance (19). gelling capacity gelation time is an important feature as gelation temperature, because the formula should transform into gel upon instillation but also remain as gel for the period of time sufficient for the drug release. only clear and un precipitated formulas are checked. gelation time is described in table 7. table 7. gelation time and gelling capacity of pi formulas gelling capacity (hr:min) (mean±sd, n=3) gelation time (sec.) (mean±sd, n=3) p.i formula no. 6:15 ± 0.00 56.66 ± 2.886 13 6.1 ± 0.086 60 ± 0.00 16 5.2 ± 0.173 46.66 ± 2.886 17 4:15 ± 0.00 48.33 ± 2.886 20 5.1 ± 0.086 46.66 ± 2.886 21 3.63 ± 0.317 40 ± 0.00 24 5.1 ± 0.086 50 ± 0.00 41 3.816 ± 0.317 46.66 ± 2.886 44 5.1 ± 0.173 50 ± 0.00 45 the gelation study was conducted in snf (ph 6.5). all the formulations on contact with the gelation medium had undergone sol-to-gel transition due to the presence of gel-forming polymers such as poloxamer. gelation characteristics of the formulations showed that increasing the concentration of poloxamer 407 increased the gelation time, while the combination with poloxamer 188 showed less gelling capacity as seen in table 7. also the rise in the concentration of ha and m.c increased the time of gelation for the formulas (3) , meanwhile different concentrations of different polymers show non-significant effect on gelation time. ph of selected nf hcl intranasal in situ gel the degree of ionization of a drug molecule intended to be administered intranasally depends on two factors, the dissociation constant of the drug and the ph value (37) of nasal fluid (4.5-6.5) (38). the ph value of the formula plays a significant role in the ionization process of the molecule so that it will affect its solubility and penetration capacity; the intranasal formulation should have a controlled ph range (4.5-6.5) (38) to be tolerable. increased ph increases the likelihood of nasal cavity infections by inhibiting the lysozymes responsible for eliminating them, while a decrease in its value will irritate the tissue (2). the ph range of selected formulas as shown in table 8 were 5.7-6.1 , which are accepted to be used intranasally and compatible with the nasal ph range 4.5 – 6.5 (38) at this ph range, the drug nf hcl will be mainly in ionized form to be completely soluble in the formula. table 8. the ph value of selected ip formulas. p.i 13 16 17 20 21 24 41 44 45 ph (mean±sd, n=3) 5.7 ± 0.243 5.8 ± 0.152 5.9 ± 0.216 5.9 ± 0.022 6.1 ± 0.171 6.1 ± 0.142 6 ± 0.415 6 ± 0.104 6.1 ± 0.028 osmolarity of nf hcl intranasal in situ gel osmotic pressure is required for all biological activities that involve solute diffusion or fluid transfer across membranes. while hypotonic formulation induces epithelial cells to expand and promote water intake and drug absorption, hypertonic solutions increase cell shrinking and decrease the likelihood of release. the formulation should, however, range from 200 to 600 mosm/l. to protect the nasal mucosa's since these changes might have a harmful impact during chronic treatment. finally, hypertonic formulations stimulate mucociliary clearance, iraqi j pharm sci, vol.31(suppl.) 2022 nefopam hcl intranasal in situ gel 42 increasing medication excretion (2). table 9 shows the osmolarity range 590 – 311 was within an acceptable range; osmolarity is directly proportional to the concentration of thermosensitive and mucoadhesive polymers, with h.a it shows the significant impact on osmolarity. table 9. osmolarity value of selected p.i formula p.i 13 16 17 20 21 24 41 44 45 osmolarity (mean±sd, n=3) 543 ± 0.081 590 ± 0.102 484 ± 0.119 552 ± 0.258 403 ± 0.317 461 ± 0.163 358 ± 0.401 396 ± 0.259 311 ± 0.066 mucoadhesive strength the residence time of the p.i formulas in the nose can be prolonged by the mucoadhesive force of the gel. the mucoadhesive force of the p.i formulas should be enough to supply good opposition to the gel mucocilliary clearance. the oligosaccharide sequence of the mucin glycoprotein in the mucus membrane forms a hydrogen bond with the polymer in the formula, producing the mucoadhesive force. evaluation of the mucoadhesive strength in terms of detachment stress demonstrated (table 10) that formulations with higher concentrations had improved mucoadhesive qualities. table 10. shows the mucoadhesive force of the selected p.i formulations mucoadhesive force (dyne/cm2) detachment weight (g) (mean, n=3). p.i formula no. 8776.31 7.03 ± 0.055 13 9388.03 7.52 ± 0.170 16 8089.68 6.48 ± 0.271 17 9125.86 7.31 ± 0.320 20 7502.93 6.01 ± 0.076 21 8401.78 6.73 ± 0.176 24 3458.09 2.77 ± 0.116 41 4431.85 3.55 ± 0.273 44 3283.31 2.63 ± 0.079 45 mucoadhesive strength for formulations containing h.a (p.i no. 13,16,17,20,21,and 24) show two three folds higher mucoadhesive detachment force than that of formulations that containing m.c (p.i no. 41,44,and 45) , due to the ha capacity to include hydrogen bonding , and large molecular weight (39). also, poloxamers have shown mucoadhesive effect. the faster fluid uptake from the mucus layer that enables the polymer chain to penetrate the mucin network and establish adhesive contacts is thought to be the cause of the higher mucoadhesive strength of the delivery system, which may lead to enhance retention and improved absorption. strong mucoadhesive force of the p.i can avoid the drainage of the drug from the nose, causing greater absorption through mucosal tissues and extended retention. the nasal mucosal membrane, however, can be harmed by excessive mucoadhesive force (i.e., more than 10,000 dyne/cm2 gel) (40).all formulations does not topped the highest limit and hence appeared as formulations with optimal mucoadhesion properties. conclusion from these research findings, it was determined that n.f hcl intranasal in situ gel that is prepared by the cold method could be formulated by poloxamer 407 as a gel-forming polymer with poloxamer 188 to modify its gelation temperature, along with hyaluronic acid and methylcellulose as mucoadhesive polymers, these studies highlighted the impacts of polymer type and concentrations on gelation temperature. it was concluded that n.f hcl p.i formulas (13,16,17,20,21,24,41,44 and 45) could be used to formulate nf hcl intranasal mucoadhesive in situ gel. further studies on the compatibility, drug release, and permeation are recommended. iraqi j pharm sci, vol.31(2) 2022 nefopam hcl intranasal in situ gel 43 references 1. moinuddin s, 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attribution 4.0 international license http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 anemia and qol of breast cancer patients doi: https://doi.org/10.31351/vol31iss2pp62-70 62 impact and association of anaemia severity and its treatment with quality of life of breast cancer patients in malaysia fares mohammed saeed muthanna*, bassam abdul rasool hassan**,1, ali haider mohammed***, mahmathi karuppannan****and abdulrasool m. wayyes** *department of pharmaceutical care, school of pharmacy, walailak university, thailand **department of pharmacy, al rafidain university college, baghdad, iraq ***school of pharmacy, monash university malaysia, selangor, malaysia **** department of pharmacy practice, universiti teknologi mara,selangor, malaysia abstract anaemia is a crucial issue among cancer patients and need to be treated properly. high incidence of anaemia in patients with cancer have been associated with several physiological manifestations, leading to decreased quality of life (qol). the current study aimed to assess the severity of anaemia, evaluate the current treatment guideline of anaemia, and to determine the association between the level of anaemia and its treatment on quality of life of breast cancer patients in malaysia. this prospective study conducted among breast cancer patients in multicancer centers in malaysia including three follow ups after receiving their chemotherapy. clinical data were collected from their medical records and at each follow up, they asked to fill up a functional assessment chronic therapy (fact-an) questionnaire. descriptive and inferential statistical analysis were done using spss. the mean age of participants was 52 ± 11 years old, and out of 120 participants, 32% received antianaemic treatments including 87% of them were prescribed with iron supplementation and only 13% received combination of blood transfusion and iron therapy. surprisingly, none of the participants received erythropoietin stimulating agents (esas). statistical tests also indicated a significant association between anti-anaemic treatments with haemoglobin level and qol scores. however, this association was insufficient to significantly improve qol or palliate anaemia severity among participants. this study showed a great evidence that, the current practice of anaemia treatment (iron therapy) among breast cancer patients in malaysia’s healthcare setting, was not sufficient to palliate anaemia severity or to improve patients’ qol. there is still a lot of gaps to improve in the management of anaemia among breast cancer patients to show a significant improvement in haemoglobin level. therefore, respective organisations and oncologists are required to raise awareness about the optimal treatment of anaemia among breast cancer patients, as a result, improve their general wellbeing. keywords anaemia, quality of life, breast cancer تأثير وترابط شدة فقر الدم وعالجه مع جودة حياة مرضى سرطان الثدي في ماليزيا فارس محمد سعيد مثنى* ، بسام عبد الرسول حسن ** ، علي حيدر محمد*** ، مهماتي كاروبانان **** و عبد الرسول محمود ويس** قسم الرعاية الصيدالنية ، كلية الصيدلة ، جامعة واليالك ، تايالند * قسم الصيدلة ، كلية الرافدين الجامعة ، بغداد ، العراق ** كلية الصيدلة ، جامعة موناش ماليزيا ، سيالنجور ، ماليزيا *** ، كلية الصيدلة ، جامعة مارا التكنولوجية ، سيالنجور ، ماليزيا الصيدلة السريريةقسم *** الخالصة طان يعد فقر الدم من القضايا الحساسة بين مرضى السرطان ويحتاج إلى العالج المناسب. ارتبط ارتفاع معدل اإلصابة بفقر الدم لدى مرضى السر لعالجية الحالية ا بالعديد من المظاهر الفسيولوجية ، مما أدى إلى انخفاض جودة الحياة. تهدف الدراسة الحالية إلى تقييم شدة فقر الدم ، وتقييم الدالئل ين مرضى لفقر الدم ، وتحديد العالقة بين مستوى فقر الدم وعالجه على جودة حياة مرضى سرطان الثدي في ماليزيا. أجريت هذه الدراسة التحليلية ب البيانات السريرية من سرطان الثدي في مراكز متعددة ألمراض السرطان في ماليزيا و شملت ثالث متابعات بعد تلقي العالج الكيميائي. تم جمع الوصف اإلحصائي التحليل إجراء تم المزمن. للعالج الوظيفي التقييم استبيان بملء منهم الطلب تم ، متابعة كل وفي الطبية المرضى ي سجالت ٪ عالجات 32، تلقى مشارًكا 120عاًما ، ومن بين 11± 52عمر المشاركين واالستنتاجي باستخدام برنامج التحليل األحصائي. كان متوسط ٪ فقط مزيًجا من نقل الدم والعالج بالحديد. والمثير للدهشة أن أيا من 13٪ منهم تم وصفهم بمكمالت الحديد وتلقى 87مضادة لفقر الدم بما في ذلك العال بين كبير ارتباط وجود إلى أيًضا اإلحصائية االختبارات أشارت إرثروبويتين. عالج يتلق لم ومستوى المشاركين الدم لفقر المضادة جات ، وأظهرت الهيموجلوبين ونوعية الحياة. ومع ذلك ، لم يكن هذا االرتباط كافياً لتحسين نوعية الحياة بشكل ملحوظ لتخفيف فقر الدم بين المشاركين طان الثدي في بيئة الرعاية الصحية في هذه الدراسة دليالً قوياً على أن الممارسة الحالية لعالج فقر الدم )العالج بالحديد( بين مرضى سر 1corresponding author e-mail: bassamsunny@yahoo.com received: 29/8 /2021 accepted: 15/11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp62-70 iraqi j pharm sci, vol.31(2) 2022 anemia and qol of breast cancer patients 63 رضى لم تكن كافية لتقليل شدة فقر الدم أو لتحسين نوعية حياة المرضى. ال يزال هناك الكثير من الثغرات لتحسين التعامل مع فقر الدم بين م ماليزيا ، هذا األمر بسرطان الثدي ليتم من خالله مالحظة تحسن كبير في مستوى الهيموجلوبين. لذلك ، يتعين على المنظمات وأطباء األورام الخبيثة المعنية برفع مستوى الوعي حول العالج األمثل لفقر الدم بين مرضى سرطان الثدي ، ونتيجة لذلك ، سوف تتحسن صحتهم العامة. لكلمات المفتاحية فقر الدم ، جودة الحياة ، سرطان الثديا introduction breast cancer is the most prevalent form of cancer among females globally (1). it is the second most common cancer among malaysian women, accounting for approximately 31% of total cancer cases (2). one of the major side effects is anaemia (3). anaemia is common in cancer, its incidence ranging from 63% (4) to 83% (5) amongst solid cancer patients. incidence of anaemia in cancer patients was reported to be 41% before receiving chemotherapy but increased to 43.1% upon antineoplastic therapy (6). increased incidence of anaemia among cancer patients will negatively impact both patients qol (7), worsening the progression of disease and affect anti-cancer treatments (8). this can significantly reduce their survival rate, specifically among those treated with chemotherapy (9-13). health related quality of life (hrqol) is defined as a multi-dimensional aspect that could be used as a prognostic indicator for treatment of breast cancer patients and for survival (14, 15) . it has been found that hrqol of cancer patients is affected negatively and significantly from the moment of being diagnosed with cancer. i.e. negative impact will set in once the person hears the word “cancer” (16). during the last decade, hrqol has become an important factor in the management of breast cancer. evaluation of qol in breast cancer patients is becoming increasingly important in health care and in assessing treatment outcomes (17). improving of qol primarily would help in the development of health care medicine and treatment of breast cancer patients (18). a previous literature study suggested that information and data provided by cancer patients via qol assessments, is very helpful for clinical decision-making and better patient treatment (19). therefore, evaluating anaemia treatment and its impact on cancer patients qol found to be an essential matter. this study used the functional assessment cancer therapy of anaemia (fact-an) scales to evaluate the effect of anaemia on qol among malaysian breast cancer patients. the purpose for using fact-an is that it covered all aspects of qol domains (physical well-being (pwb), social well-being (swb), emotional wellbeing (ewb), and functional well-being (fwb) which was necessary to reflect the real quality of care among cancer patients comparing to other scales. in addition, fact-an is a specific scale for anaemia condition compared to other scales that are in general. both tools are valid, reliable, easy to understand, and fast to be filled and completed by participants (5-10 min). regarding qol and breast cancer patient, a study has been conducted in china in 2016 to detect the effects of social support, financial situation and clinical factors on breast cancer patients, to evaluate qol. a total number of 1,160 breast cancer patients have been included; the quality of life is evaluated using the functional assessment of cancer therapy-breast cancer that comprise of five domains. the results reveal that financial and stoical support has affected qol significantly (11). another trial that has been conducted in korea in 2013 included 534 breast cancer patients in the study. results showed that qol among patients with a history of receiving chemotherapy were affected negatively as compared to those without chemotherapy treatment (12). in addition to that, a study conducted in morocco had 1,463 breast cancer patients who enrolled in the study. results indicated that financial support and fear of death were the two main factors that affect qol negatively (13). in malaysia, there is a lack of information regarding qol among breast cancer patients. up to date only limited trials have been conducted in malaysia to detect the factors affecting qol among breast cancer patients. results from a study have demonstrated that as the age increases, the qol for breast cancer patients also improve (3). moreover, several studies revealed that the management of anaemia needs to be evaluated regularly in order to ensure that it is up to date and help to achieve better health outcome (20 , 21). this is due to the fact that evaluation and treatment of anaemia will significantly increase the response to antineoplastic therapy, improve qol, increase survival and improve clinical outcomes (22). a large number of studies demonstrated and recommended the use of erythropoiesis-stimulating agents (esas) in the treatment of anaemia among cancer patients (23). taking into consideration the potential risk of esas which in some cases cause thrombosis, esas suggested to be the best medication that significantly increase hemoglobin levels, reduce the need of blood transfusion requirements, and improve quality of life, (22 , 23). however, to the best of our knowledge, internationally, there are limited studies focused on detecting the effects of anemia on breast cancer patients’ qol. besides, up to date, there is no study in malaysia that focuses on detecting the negative effects of anemia on the breast cancer patients qol. hence, the current study aimed to detect and evaluate treatment given to breast cancer patient suffering from anaemia and to determine its impact on cancer patients qol. iraqi j pharm sci, vol.31(2) 2022 anemia and qol of breast cancer patients 64 method study design and setting this is a prospective, observational, and longitudinal design among breast cancer patients in multi-cancer centres in malaysia which are hospital kuala lumpur (hkl), university malaya medical centre (ummc), and institute kanser negara (ikn), putrajaya, malaysia. the present study conducted for 8 months starting from july 2019 to march 2020. sample size was calculated using raosoft online sample size calculator. the calculation was based on 50 % response distribution, 5 % margin of error and 95 % confidence interval. the online software foundation is based on widely utilized descriptive studies sample size estimation formula. setting the response distribution to 50% is the most conservative assumption (raosoft inc). the assumption that the response rate is 50 % was based on the idea that both responses and response rates were completely unknown since only limited studies conducted in malaysia which were similar to the current study and the response rate was more than 50% (24) . based on the incidence of anaemia (10-40%) (6) of breast cancer patients, with a confidence interval of 95% and a margin of error at 5%,.the sample size fell between 95-135 patients. these numbers were derived based on an estimated total population of 125 -207 breast cancer patients attending hospitals regularly .the total number of patients who met the inclusion criteria in this study were 120 female anaemic breast cancer patients. anaemic breast cancer patients identified based on blood analysis results i.e., haemoglobin level in their medical files. inclusion criteria stipulated were patients aged 18 and above, hb ≤ 12 g/dl, diagnosed with breast cancer regardless of stage, currently receiving chemotherapy and able to understand as well as sign the informed consent. while the exclusion criteria stipulated were patients with haematological, inherited disease, pancreatitis or dementia. besides that, those receiving different management procedure such as radiotherapy, biological therapy or endocrine therapy were also excluded from the study. research instrument and data collection procedure upon identification, breast cancer patients were approached to participate in this study and data was collected using convenient sampling method. anaemic cancer patients were identified based on blood analysis results in their medical files. the specific patient was approached to participate in the study. the patient was then briefed regarding the purpose of the study. upon agreeing, a consent form was provided to be signed. subsequently, patients were provided with the functional assessment of cancer therapyanaemia (fact-an) questionnaire which has been validated using pre-test and pilot study and results of cronbach’s alpha was 0.891 indicated that questionnaires yields reliable. this questionnaire was available in two languages (english and bahasa melayu) from the original developer so that it was provided according to patient’s language preference (i.e. bahasa melayu or english version) to measure qol (25). permission to use the research instruments (both english and bahasa melayu version) was obtained from the developers and validated accordingly. the questionnaire took 10-15 minutes to complete, and patients were followed-up for the next two cycles of chemotherapy (total three cycles). besides that, hb levels were measured prior to each cycle by a well-trained staff and hb readings were extracted from the patients’ medical records. severity of anaemia corresponds to 10.0–10.9 g/dl as mild, 7.0–9.9 g/dl as moderate, and less than 7.0 g/dl as severe (26). in addition, demographic, clinical, and anaemia treatments data, were collected from patients’ medical records. data analysis data was analysed via spss version 25. estimated mean scores and 95% confidence intervals were calculated for qol score and hb levels, evaluated based on all three follow ups. difference in mean scores for all three follow ups was observed and findings were expressed as mean and standard deviation (sd). computation of results was done in accordance with fact-g group guidelines. a repeated measured anova was used to detect the association between hb level and qol scores with time across the three follow-ups, and one-way repeated measured manova was performed to identify the relationship between antianaemic medications and both dependent variables (qol and hb levels) along the three subsequent follow ups. to overcome problems related to data mining, post hoc analysis was conducted using bonferroni adjustments to identify differences between each cycle. significance level was set at p < 0.05 for all analyses. ethical approval all the aspects and protocols of this study were reviewed and approved by clinical research centre (crc) of uitm ( rec/392/19), hkl (hcrc.iir-2019-07-163), ikn (ikn/500-5/1/25 jid 4 (18), ummc & medical research ethical centre (mrec) (nmrr -18-3902-45218). researcher adhered to the principles of the declaration of helsinki and the malaysian good clinical practice guidelines. iraqi j pharm sci, vol.31(2) 2022 anemia and qol of breast cancer patients 65 results from the 120 respondents, the majority were elderly (n=89; 74.2%) with mean age of 52.63 years (± sd 11.27), malay (n = 77; 64.2%), married (n=108; 90%), and postmenopausal (n=87; 72.5%) (6). only 39 (32.5%) patients received anti-anaemic management. out of the 39 patients who received anaemia treatment, about 87% of the patients were treated with only iron supplements plus multivitamins while 13% received a combination of iron products with multivitamins and underwent blood transfusion and none of the patients received erythropoiesis-stimulating agents (esas). furthermore, more than two third of the anaemic patients (n = 81, 67.5%) were not treated for anaemia. additional clinical and demographic data are shown in table 1. table 1 demographic data in breast cancer patients undergoing chemotherapy (n= 120) variable n (%) mean age 52.63 (sd 11.27) age (years ) ≥ 60 89 (74.2%) < 60 31 (25.8%) race malay 77 (64.2%) indian 14 (11.7%) chinese 27 (22.5) others 2 (1.7%) marital status married 108 (90%) single 8 (6.7%) divorced 4 (3.3%) stage of breast cancer stage i 5 (4.2%) stage ii 29 (24.2 %) stage iii 62 (51.7 %) stage iv 24 (20 %) anti-anaemic medications treated 33 (27.5%) un treated 81 (67.5%) un detected 6 (5%) type of anti-anaemic treatment iron therapy & multi-vitamins 34 (87.2%) blood transfusion 2 (5.1%) mixed 3 (7.7%) esas 0 (0%) treated = all patients received anti-anaemic treatment , un-treated = patients who did receive any type of antianaemic medication ,un detected = patients who received anti-anaemic medication either at 1st or 2nd or 3rd interviews . esas = erythropoietin stimulating agents . mixed = iron therapy & multi-vitamins+ blood transfusion haemoglobin levels and qol across three consecutive follow ups the total average mean (i.e., sum of 3 follow-up means) and standard deviation for overall hb levels was 10.34 g/dl ± 0.73 (mean ± sd). specifically, the hb mean for patients for the first follow-up was 10.64 ± 0.85, 10.26± 0.85 for the second follow-up, and 10.13 ± 0.83 g/dl for the third follow-up. results revealed a decline in hb levels across the three follow ups (i.e., increased anaemia severity across the three follow ups). similarly, the total average mean and sd for overall qol was 96.38 ±16. 15. the mean of qol at first follow-up (108.96±20.94) was higher than the mean of qol at second follow-up (95.11 ±17.58). the mean of qol at third follow-up was the lowest (85.06 ± 25.88) signifying a decline in the qol among anaemic cancer patients along the three follow ups as shown in table 2. iraqi j pharm sci, vol.31(2) 2022 anemia and qol of breast cancer patients 66 table 2.mean of hb level (severity of anaemia) and qol in breast cancer patients (n= 120) hb g/dl (mean ±sd) g/dl total average hb 10.34 ± 0.73 hb g / dl mild (10-12) g/dl 78 (65%) moderate (8-10) g/dl 41 (34.2%) severe (6-8) g/dl 1 (0.83.3%) qol total (0-188) 96.38 ±16.15 qol fact-an 1st follow up (0-188) 108.96 ± 20.94 2nd follow up (0-188) 95.11±17.58 3rd follow up (0-188) 85.06 ± 25.88 hb – hemoglobin association between the level of hb , qol , and anti-anaemic treatments one-way repeated measured manova was used to determine if there was any significant interaction between (anti-anaemia treatments) as independent variable and (hb and qol) as dependent variables at three follow ups. results indicated a statistically significant difference in the mean of hb levels and qol scores based on antianaemic medications (table 3). univariate analysis between anti-anaemic treatment and both hb levels and qol scores across the three follow ups indicated a significant association between antianaemic treatment with hb levels across three follow ups and qol scores at the three follow-ups. however, this association was not enough to improve the hb level and qol of participants. table 3. association between anti-anaemic medications and hb level + qol at 3 follow ups variables f(df) f sig η2 anti-anemic treatment hb 1st follow up 2.117 6.8 0.002 0.10 hb 2nd follow up 12.4 <0.001 0.17 hb 3rd follow up 3.9 0.023 0.06 qol 1st follow up 3.4 0.036 0.05 qol 2nd follow up 6.9 <0.001 0.10 qol 3rd follow up 1.6 0.020 0.02 η2= partial eta square discussion in malaysia, to our best knowledge, the current study is the first of its kinds to evaluate the association between anaemia severity and its treatment with quality of life among breast cancer patients. it is proved that cancer-related anaemia (cra) adversely affects quality of life (7,27) and is associated with reduced overall survival (28). correction of anaemia in cancer patients has the potential to improve treatment efficacy and as a result increase survival (29). pourali et al, mentioned that prevalence and severity of anaemia among breast cancer patients in their advanced stages have been well documented, but the same cannot be said amongst those in the earlier stages. hence signifying the value of this study as it focuses on detection and evaluation of anti-anaemic treatment among breast patients across various stages (6). imran et al., 2019, who conducted a study amongst female breast cancer patients in saudi arabia to assess their qol mentioned that there was a direct relationship between choice of treatments used for breast cancer patients, side effects, medical issues, and qol (18). the current obtained data indicate that, the mean for haemoglobin level declined across the three follow ups. meaning the number of patients who were suffering from moderate anaemia was increasing. this indicates that treatments used for treating and/or palliating anaemia within this study may not be enough to improve hb levels. in line with these findings, a study denotes that cra treatment is only considered effective when haemoglobin level improves significantly, specifically among those with mild and moderate anaemia (22). moreover, another study highlights that the main objectives for cra treatment is to palliate and/or treat anaemia besides improving qol among cancer patients with anaemia (30). however, results from the current study shows otherwise. concerning anaemia treatment, our data showed that only 32.5% of our respondents received general anti-anaemic treatment (i.e., non-specific antianaemic treatment). a large majority did not receive any kind of anti-anaemic medications. among those who received treatment, almost all received iron supplements and vit b-complex or multivitamins, iraqi j pharm sci, vol.31(2) 2022 anemia and qol of breast cancer patients 67 while only two respondents were treated with blood transfusion. none were treated with erythropoiesisstimulating agents (esas). as per our data, the use of iron supplements did not improve haemoglobin level across the three follow ups. this finding is consistent with another study conducted in ethiopia, where only 32% out of its total respondent were treated with supportive treatments which include blood transfusion and iron supplements (31). based on the suggestion and recommendation by hassan ba et al., (21), the future studies should evaluate and improve the guideline used for treating anaemia among cancer patients (21). this suggestion stemmed from results of their prospective study, which was conducted in a cancer center in penang, malaysia. their results found that treatment of anaemia among solid cancer patients was neither effective nor specific. their efforts in evaluating treatment were one that is novel and crucial. in addition, a previous study concluded that clinicians and oncologists need to evaluate the negative effects of anaemia towards cancer patients qol and treat it accordingly (32). moreover, another researchers mentioned that it is important to determine incidence of anaemia among cancer patients and its impact on cancer patients qol as it helps identify correct method of treatment (i.e. the right treatment method will significantly enhance qol among cancer patients) (22). besides, evaluating and detecting proper treatment methods and guidelines for treating mild and/or moderate anaemia among cancer patients should be the foremost objective (22). this was precisely the focus of the current study. bohlius et al. (20) , also mentioned in their review article that the treatment guideline used for cra, especially among cancerpatients suffering from non-curative and advanced cancers like breast cancer, need to be evaluated and updated. besides, there is a need for evaluating the use of erythropoiesis-stimulating agents (esas) among cancer patients to highlight benefits and risks of using esas (20). the asco guideline recommends using esas for treatment of cra in early or advanced stages of breast cancer (20). statistically, our results showed a significant negative association between anaemia severity with type and pattern of anaemia treatment. this indicates that neither the type nor the pattern of anaemia treatment used was appropriate enough to palliate or solve the main problem i.e., anaemia. moreover, results across the three follow ups further confirmed that anaemia treatment type and pattern did not improve both anaemia severity and qol. therefore, emphasising a need for rectification. busti et al. (22) explains that iron therapy is only useful in enhancing efficacy of esas and reducing the need of blood transfusion. iron therapy is not to be used as main protocol for treatment of anaemia, as it is in this setting. busti et al. also recommends that iron therapy should be replaced with esas for the treatment of anaemia in cancer patients (22). effectiveness of esas in treating anaemia in comparison with oral iron has been substantiated by busti et al., 2018, which showed that the use of esas is far more superior than the use of oral and intravenous iron in treating anaemia among cancer patients (22,33). a reliable indicator of effectiveness of anaemia treatment is the improvement haemoglobin (hb) level. specifically, 2g/dl within 4-8 weeks from treatment initiation. otherwise, it is considered ineffective and must be modified accordingly. hence strongly endorses findings from the current study. moreover, hassen et al. highlights in his study the major and essential need to determine the proper treatment, so that it can significantly improve qol among breast cancer patients receiving chemotherapy (34). besides, evaluating the impact of anaemia among breast cancer patients on their qol will significantly help the clinicians to determine several positive outcomes (34). direct interviews assessing qol were found to be highly advantageous in guiding healthcare professionals to attain a clear idea about the patient’s qol, to evaluate patient’s medical situation and to tailor treatment accordingly so as to avoid medical problem that may negatively affect patient’s qol (35,36). barnadas et al., (37), denotes the necessity to understand impact of cancer disease-associated complications such as anaemia among others, as well as palliative care treatments towards breast cancer patients qol. moreover, chen et al. (38) mentions that studies focused on detecting qol among women suffering from breast cancer is the cornerstone for evaluating clinical situation, clinical decision making as well as health policy or reimbursement decisions. though these may be currently obscure, it strongly advocates the importance and novelty of this study. overall, there are two key points based on our findings. the first is anaemia treatments did not significantly improve hb levels and qol scores. even if there were some significant association between anaemia treatments with anaemia severity and/or qol, there were no improvements of the dependent variables (hb and qol) across the three follow ups. secondly, anaemia treatment (i.e., the entire treatment plan) must be replaced with a proper treatment guideline designed specifically to treat anaemia among cancer patients in malaysia. the reason being though significant associations were found among anti-anaemic treatment used with anaemia severity and qol, the effect of the treatment remains weak. meaning it neither improved anaemia severity nor patients qol, as indicated by low value of partial eta squared in accordance to guideline by cohen, miles and shevlin (39). iraqi j pharm sci, vol.31(2) 2022 anemia and qol of breast cancer patients 68 limitations although this study answers the research questions set, several limitations need to be acknowledged. this study was conducted in a few cancer-centres and only breast cancer patients who received chemotherapy and gave a consent, were interviewed. therefore, further studies are required to be conducted among different type of cancers at various number of oncology centers. conclusion findings from this study conclude that anaemia treatments used among anaemic breast cancer patients were ineffective enough to palliate anaemia severity or improve qol quality of life. therefore, there is an urgent need to improve anaemia treatment guideline for effective management of anaemia as well as to improve qol among breast cancer patients. there is a need to use strategies that will unfetter the qol of cancer patients so that they will have a better sense of control over their illness and treatment. acknowledgment the author would like to thank all the staffs at respective hospitals for their assistance and generosity in providing access to the medical records at the clinic. funding the authors declare that there was no funding for this work. conflicts of interest the authors have declared no conflicts of interest for this article. references 1. ferlay j, soerjomataram i, dikshit r, et al. cancer incidence and mortality worldwide: sources, methods and major patterns in globocan 2012. international journal of cancer. 2015:136(5):e359-86. 2. lim gcc, rampal s, yahaya h. 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j., miles, j., & shevlin, m. applying regression and correlation: a guide for students and researchers .2001: (p. 272). this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 fimasartan and methotrexate-induced nephrotoxicit doi: https://doi.org/10.31351/vol31iss1pp87-94 87 the ameliorative effect of fimasartan against methotrexate-induced nephrotoxicity in rats maryam rasheed abd*,1 and ali faris hassan* *department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract drug-induced acute kidney injury is a serious disorder. oxidative stress has a key role in its initiation and progression. in this study, the possible ameliorative effect of fimasartan against methotrexate-induced nephrotoxicity was investigated in comparison with α-tocopherol in rats. wistar rats were allocated into six groups and treated as follows: group ӏ received water on a daily basis for 8 successive days; group ӏӏ received methotrexate (20 mg/kg) on day 1, followed by water for 7 successive days; group ӏӏӏ received fimasartan (3 mg/kg/day) for 7 successive days; group iv received α-tocopherol (1 g/kg/day) for 7 successive days; group v received methotrexate (20 mg/kg) on day 1, followed by fimasartan (3 mg/kg/day) for 7 successive days; a nd group vi received methotrexate (20 mg/kg) on day 1, followed by α-tocopherol (1 g/kg/day) for 7 successive days. finally, after euthanization of each animal by diethyl ether, the samples were collected for analysis. administration of fimasartan and α-tocopherol resulted in a significant decline in serum creatinine and urea, a significant reduction of renal malondialdehyde, and a significant elevation of renal superoxide dismutase-1 compared to the methotrexate-treated rats. in conclusion, fimasartan has ameliorative effects, comparable to those of α-tocopherol, on methotrexate-induced nephrotoxicity in rats. keywords: nephrotoxicity, methotrexate, oxidative stress, fimasartan, α-tocopherol. المستحثة بواسطة الميثوتريكسيت في الجرذانللفيماسارتان ضد السمية الكلوية رالمحّسنالتأثي * علي فارس حسن و 1*، مريم رشيد عبد .العراق ، ، بغدادبغداد جامعة ، الصيدلة كلية والسموم، االدوية فرع * الخالصة لإلجهاد التأكستدي دو ييستط فط ودو و وتقاهموف فط و و االعتالل الكلوي الحاد الناتج عن استتددام اددوية وو اطتاراص يتحط راير فط لقا توكوفيرولالد استتة، تف فحا التأ ير المحستتن المحتمف للقيماستتا تال طتتد الستتمية الكلوية المستتتحمة بواستتاة الميموتريكستتي م ا ة باد ايام متتالية، المجموعة 8مجموعة االولى تل الماء يوميا لمدة الجرذالف تف ت سيف جرذال ويستا الى س مجاميع وتف اعااؤوا على النحو اآلتط: ال ايتام متتتاليتة، المجموعتة المتالمتة تل ت 7كغف( فط اليوم ادول تالوتا تل ط المتاء يوميتا لمتدة / ملغف 20المتا يتة تل ت جرعتة واوتدة من الميموتريكستتتتيت ايام متتالية، المجموعة 7اليوم( لمدة / كغف/ غف 1 لقا توكوفيرولمجموعة الرابعة تل ادايام متتالية، ال 7اليوم( لمدة / كغف/ ملغف 3القيماسا تال ايام متتالية، 7اليوم( لمدة / كغف/ ملغف 3كغف( فط اليوم ادول تالوا القيماستتتا تال / ملغف 20الدامستتتة تل جرعة واودة من الميموتريكستتتي اليوم( لمدة / كغف/ غف 1 لقا توكوفيرولكغف( فط اليوم ادول تالوا تل ط اد/ ملغف 20واودة من الميموتريكستي والمجموعة الستادستة تل جرعة إيميف اإليمر وجمع العينات منها لقحصتتتهاف تج عن إعااء القيماستتتا تال ة نايط االرويف للحيوا ات بواستتت تف اجراء ال تف ايام متتاليةف فط الدتام، 7 وا تقتاع كييرفط ا ييف التديهتايتد داي متالولفط المصتتتتف وا دقتار كيير فط ال اليو يتا والكريتاتينين يتاتفط مستتتتتوتنتاها كيير يروللقتا توكوفواد لقا ، م ا بة لتأ يرات ادتأ يرات محستنةالقيماستا تال فط ستيج الكلى م ا ة بالجرذال المعاملة بالميموتريكستي ف كتستتنتال، لد 1-ديستموتا القاي التسمف الكلوي الناتج عن الميموتريكسي فط الجرذالف ، طدوفيرولتوك .ألفا توكوفيرولفيماسارتان، ، اإلجهاد التأكسدي الكلوي، ميثوتريكسيت، التسمم المفتاحية: الكلمات introduction acute kidney injury (aki) is a serious medical problem characterized by a rapid and reversible decline in renal function (1). it is associated with a high risk of irreversible renal injury, poor prognosis, and high healthcare costs (2). many important medications have been reported to cause aki, which limits their clinical usefulness (3). among these agents, methotrexate (mtx), which is a widely used antimetabolite, has been reported to cause aki in about 12% of patients receiving high dose-mtx (hdmtx) (4). hdmtx therapy, defined as the administration of mtx in doses exceeding (500 mg/m2), is generally used in chemotherapy against various malignancies (5). mtx-induced aki is a complex process that arises from tubular obstruction via precipitation of mtx and its metabolites within the renal tubules (5,6), as well as direct tubular toxicity linked to inflammation, mitochondrial dysfunction, and increased production of reactive oxygen species (ros) in renal tissue (4-8). . 1corresponding author e-mail: maryamrasheed.a.r@gmail.com received:12 /6/2021 accepted: 14/8 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp87-94 iraqi j pharm sci, vol.31(1) 2022 fimasartan and methotrexate-induced nephrotoxicit 88 given that oxidative stress (os) has a key role in the development and progression of mtx-induced renal injury, many studies have been directed to identify interventions that promote the antioxidant defences of the cells in order to circumvent mtx-induced aki development or its complications, and the results are encouraging (7, 9-11). previous researches have proved that α-tocopherol (α-toc), which is the predominant and most biologically active form of vitamin e, can mitigate renal injury and inhibit renal fibrosis due to its potent antioxidant and antiinflammatory properties (12-14). furthermore, high doses of α-tocopherol were shown to protect the renal and hepatic tissues against oxidative damage induced by various drugs (14). fimasartan (fms) is an efficacious and potent angiotensin ii receptor blocker (arb) that was recently developed and approved in korea as an antihypertensive medication (15). it is metabolically stable and chiefly excreted via the bile, and its use exhibited a good safety profile (15-17). experimental data proposed that fimasartan exert organ-protecting effects beyond its hypotensive action and a previous study revealed that it has a protective role against renal inflammation and fibrosis through the induction of the antioxidant pathway (15,18,19). besides, cho et al. (2018) found that fimasartan preserves kidney structure and function in a murine model of ischemia-reperfusion injury through its anti-inflammatory and antiapoptotic properties (20). all these factors make fimasartan an attractive candidate to be examined as a renoprotective adjuvant to the standard mtx chemotherapy. in view of the above considerations, this study was conducted to examine the possible ameliorative activity of fimasartan, in comparison to α-tocopherol, against mtx-induced renal injury in rats, pointing to its ability to suppress os. materials and methods chemicals, drugs, and kits (+)-α-tocopherol was obtained from santa cruz biotechnology inc. (texas, usa). fimasartan potassium trihydrate was purchased from novachemistry (loughborough, uk). methotrexate (50mg/2ml) solution for injection was supplied from mylan s.a.s. (saint-priest, france). diethyl ether (romil ltd, cambridge, uk) and phosphatebuffered saline (euroclone, s.p.a., milan, italy) were also used in the study. all enzyme-linked immunosorbent assay (elisa) kits utilized in the study were obtained from mybiosource, inc. (california, usa) and include: rat blood urea nitrogen (bun) elisa kit, rat creatinine (cr) elisa kit, rat malondialdehyde (mda) elisa kit, and rat superoxide dismutase [cu-zn] (sod-1) elisa kit. animal selection thirty-six adult wistar rats (8 weeks old) of both sexes, weighing 150-240 g, were used in the present study. they were obtained from and maintained in the animal house at the college of pharmacy/university of baghdad under conditions of controlled temperature, humidity and light periodicity (12-hour light/dark cycle). they were fed commercial pellets and tap water ad libitum throughout the experimental period. to get adapted, these rats were routinely handled and acclimatized for 7 days in the above-stated conditions before drug administration. experimental protocol this study was approved by the scientific and ethical committees of the college of pharmacy/university of baghdad. the rats employed in this study were randomly divided into six groups of six rats each, as follows: group ⅰ (negative control group): rats received sterile water for injection in a volume of (6 ml/kg) intraperitoneally (21) for 8 days starting from day 1. group ⅱ (mtx group): rats received a single dose of mtx (20 mg/kg) intraperitoneally on day 1, followed by daily intraperitoneal (ip) administration of sterile water for injection (6 ml/kg) for 7 days starting from day 2 (22). group ⅲ (fms group): rats received a daily ip injection of fimasartan (3 mg/kg/day) for 7 successive days. a solution of (0.05% w/v) fms was prepared by dissolving fimasartan potassium trihydrate in water on the day of administration (18,23,24). group ⅳ (α-toc group): rats received αtocopherol (1 g/kg/day) orally for 7 successive days (14). group ⅴ (mtx plus fms group): rats received a single dose of mtx (20 mg/kg) intraperitoneally on day 1, followed by daily ip injection of fimasartan (3 mg/kg/day) for 7 successive days starting from day 2. group ⅵ (mtx plus α-toc group): rats received a single dose of mtx (20 mg/kg) intraperitoneally on day 1, followed by daily administration of αtocopherol (1 g/kg/day) orally for 7 successive days starting from day 2. samples collection and preparation of kidney tissue homogenate after twenty-four hours from the final drug administration, blood samples were withdrawn from the carotid artery (at the neck) and collected in gel activated tubes and allowed to stand for 30 minutes to clot. then, it was centrifuged at 3000 rpm for 15 minutes using a centrifuge (eba 20, andreas hettich gmbh & co. kg, germany) to obtain serum (25). the obtained sera were utilized for the estimation of urea and creatinine levels. next, all rats were sacrificed by cervical dislocation under diethyl ether anaesthesia and kidney tissues were isolated and processed for analysis (25). briefly, the kidneys were rapidly excised, cleaned from fatty tissues, and washed with a pre-cooled pbs (ph=7.4, 4˚c) to rinse away any residual blood. then, each kidney was blotted on https://www.google.com/search?q=dallas&stick=h4siaaaaaaaaaopge-luz9u3sdpotupt4gazjyoktbsyk63084vse_myqxjlmvpzudhwgamjkywliuulquxfi1jzxbjzchkld7ayaga6bbtqtwaaaa&sa=x&ved=2ahukewip6qaot9hvahvxwoskhtwuax4qmxmoatafegqihbad iraqi j pharm sci, vol.31(1) 2022 fimasartan and methotrexate-induced nephrotoxicit 89 filter paper, weighed, and chopped into fine pieces. for each rat, the left kidney was used to prepare the kidney tissue homogenate by adding 0.4 g of the minced tissue and 3.6 ml of pbs (ph=7.4, 4˚c) into a tube (26). homogenization was then accomplished using a tissue homogenizer (dyna-passion® wt130, success technic industries, selangor, malaysia) at set 3 for 1 minute at 4˚c. samples were kept on ice throughout all the above-mentioned steps. the resultant suspension was then subjected to a freezethaw cycle and centrifuged in a refrigerated centrifuge (hermle labortechnik gmbh, germany) at 10,000 rpm for 10 minutes at 4˚c. the resultant supernatant was immediately collected and stored at −20˚c until the day of analysis when it was used for the estimation of mda and sod-1 levels (26,27). biochemical analysis serum levels of kidney function biomarkers, blood urea nitrogen (bun) and creatinine (scr), were measured using elisa kits according to the manufacturers’ instructions. moreover, to assess the oxidant/antioxidant status in the tissue, the concentrations of mda and sod-1 were quantified in the renal tissue homogenate by sandwich elisa method according to the kit manufacturers’ instructions (11, 22). measurements of the relative kidney weight (kidney index) on the morning of sacrifice day, the bodyweight of each rat was measured. then, the rats were euthanized and the weights of right and left kidneys were measured immediately after harvesting the kidneys from the rat carcasses. then, the kidney-to-body weight ratio, also called the relative kidney weight or kidney index (ki), had been calculated for each rat by dividing the total right and left kidney weights by the total body weight of the rat, then multiplying the result by 100 (9). statistical analysis data were expressed as mean ± standard deviation (sd). analysis was carried out using statistical package for social sciences (spss, version 25) software. the differences between the groups were evaluated by a two-way analysis of variance (anova). the differences among the groups were considered statistically significant at a p value of less than 0.05 (p<0.05). results effects on serum markers of kidney function (table 1) and (figures 1 and 2) revealed that administration of mtx in group ii resulted in a significant increase of bun and scr levels compared to the negative control group (group i) (p<0.05). at the same time, there were no significant differences in the fms and α-toc groups when compared to group i (p>0.05). besides, comparing the fms group and α-toc group with the mtx group revealed significant differences between them (p<0.05). interestingly, rats in the mtx plus fms group showed a significant decrease in bun and scr levels compared to the mtx group (p<0.05). similarly, rats in mtx plus α-toc group showed significantly lower levels compared to the mtx group (p<0.05), as shown in (table 1) and (figures 1 and 2). by comparing the levels of bun and scr in rats treated with mtx alone (group ii), fms alone (group iii), and mtx followed by fms (group v), the obtained results showed that there were significant differences between the three groups (p<0.05). likewise, when we compare bun and scr levels among the mtx group, α-toc group, and mtx plus α-toc group, statistically significant differences between them can be noted (p<0.05), as shown in (table 1) and (figures 1 and 2). the same table also showed a significant difference in bun levels when we compare the mtx plus fms group to the mtx plus α-toc group (p<0.05), which has significantly lower bun levels compared to the mtx plus fms group. however, there is a nonsignificant difference in scr levels when we compare the mtx plus fms group to the mtx plus α-toc group (p>0.05). table 1. effects of fimasartan on the serum levels of bun and creatinine. groups serum bun (mmol/l) serum cr (mmol/l) i. negative control group 1.79±0.46 1.03±0.48 ii. mtx group 2.86 ± 0.79*aa 2.73±0.61*aa iii. fms group 1.78±0.62ѱb 1.201±0.69ѱb iv. α-toc group 1.74±1.27ѱb 1.42±0.32ѱb v. mtx + fms group 2.05±0.34δc 1.79±0.92δc vi. mtx + α-toc group 1.69±0.24βc♣ 1.83±0.48βc • the data are expressed as mean ± sd, number of rats in each group = 6 • superscript (*) indicates significant differences when groups ii, iii and iv are compared to the negative control group (p<0.05) • superscript (ѱ) indicates significant differences when group iii and iv are compared to the mtx group (p<0.05) • superscript (δ) indicates a significant difference when group v is compared to group ii (p<0.05) • superscript (β) indicate a significant difference when group vi is compared to group ii (p<0.05) iraqi j pharm sci, vol.31(1) 2022 fimasartan and methotrexate-induced nephrotoxicit 90 • small letter superscripts (a, b, c) indicate significant differences among the groups (ii, iii, v) (p<0.05) • capital letter superscripts (a, b, c) indicate significant differences among the groups (ii, iv, vi) (p<0.05) • superscript (♣) indicates a significant difference when group v is compared to group vi (p<0.05) figure 1.effects of fimasartan on the serum levels of bun. figure 2. effects of fimasartan on the serum levels of creatinine. effects on renal lipid peroxidation and antioxidant parameters table 2 and figures 3 and 4 revealed that administration of mtx in group ii resulted in a significant increase in renal mda levels, coupled with a significant decrease in renal sod-1 contents, as compared to the negative control group (group i) (p<0.05). at the same time, there were no significant differences in the levels of mda and sod-1 in the fms and α-toc groups as compared to group i (p>0.05). besides, comparing the fms group and α-toc group with the mtx group revealed significant differences between them (p<0.05). notably, the rats in mtx plus fms group showed a significant decrease in mda along with a significant increase in sod-1 levels as compared to the mtx group (p<0.05). similarly, rats in mtx plus α-toc group showed significantly decreased mda and increased sod-1 levels as compared to the mtx group (p<0.05), as shown in (table 2) and (figures 3 and 4). by comparing the levels of mda and sod-1 in the renal tissue homogenate of rats in the mtx group, fms group, and mtx plus fms group, the obtained results showed that there were significant differences between the three groups (p<0.05). likewise, when we compare the renal mda and sod-1 levels among the mtx group, α-toc group, and mtx plus α-toc group, statistically significant differences between them can be noted (p<0.05), as shown in (table 2) and (figures 3 and 4). the same table also showed a nonsignificant difference in renal mda levels when we compare the mtx plus fms group to the mtx plus α-toc group (p=0.05). however, mtx plus fms group has significantly higher renal sod-1 levels compared to mtx plus α-toc group (p<0.05). table 2.effects of fimasartan on the renal mda and sod-1 levels. groups renal mda (nmol/ml) renal sod-1 (ng/ml) i. negative control group 8.44±1.22 149.11±11.35 ii. mtx group 16.58±1.03*aa 109.66±19.21*aa iii. fms group 9.07±0.94ѱb 151.87±23.87ѱb iv. α-toc group 8.32±1.265ѱb 148.51±18.82ѱb v. mtx + fms group 11.69±0.99δc 158.36±16.86δc♣ vi. mtx + α-toc group 10.82±0.59βc 135.23±20.83βc • the data are expressed as mean ± sd, number of rats in each group = 6 • superscript (*) indicates significant differences when groups ii, iii and iv are compared to the negative control group (p<0.05) • superscript (ѱ) indicates significant differences when group iii and iv are compared to the mtx group (p<0.05) • superscript (δ) indicates a significant difference when group v is compared to group ii (p<0.05) • superscript (β) indicate a significant difference when group vi is compared to group ii (p<0.05) iraqi j pharm sci, vol.31(1) 2022 fimasartan and methotrexate-induced nephrotoxicit 91 • small letter superscripts (a, b, c) indicate significant differences among the groups (ii, iii, v) (p<0.05) • capital letter superscripts (a, b, c) indicate significant differences among the groups (ii, iv, vi) (p<0.05) • superscript (♣) indicates a significant difference when group v is compared to group vi (p<0.05) figure 3.effects of fimasartan on the renal mda levels. figure 4. effects of fimasartan on the renal sod1 levels. effects on the relative kidney weight (kidney index) as shown in (table 3) and (figure 5), mtx administration in group ii resulted in a significant increase in ki as compared to the untreated rats (group i) (p<0.05). at the same time, there were no significant differences in the fms and α-toc groups rats as compared to the untreated rats in group i (p>0.05). besides, comparing the fms group and αtoc group with the mtx group revealed significant differences between them (p<0.05). remarkably, the rats in the mtx plus fms group showed a significant decrease in ki as compared to the mtx group (p<0.05). likewise, rats in mtx plus α-toc group showed a significantly decreased ki as compared to the mtx group (p<0.05), as shown in (table 3) and (figure 5). by comparing the ki of rats in the mtx group, fms group, and mtx plus fms group, the obtained results showed that there were significant differences between the three groups (p<0.05). likewise, when we compare among mtx group, α-toc group, and mtx plus α-toc group, statistically significant differences between them can be noticed (p<0.05), as shown in (table 3) and (figure 5). the same table also showed a nonsignificant difference in ki when we compare the mtx plus fms group to the mtx plus α-toc group (p>0.05). table 3.effects of fimasartan on the kidney index (ki). groups ki i. negative control group 0.69±0.074 ii. mtx group 1.12±0.29*aa iii. fms group 0.696±0.04ѱb iv. α-toc group 0.74±0.03ѱb v. mtx + fms group 0.71±0.12δc vi. mtx + α-toc group 0.725±0.11βc • the data are expressed as mean ± sd, number of rats in each group = 6 • superscript (*) indicates significant differences when groups ii, iii and iv are compared to the negative control group (p<0.05) • superscript (ѱ) indicates significant differences when group iii and iv are compared to the mtx group (p<0.05) • superscript (δ) indicates a significant difference when group v is compared to group ii (p<0.05) • superscript (β) indicate a significant difference when group vi is compared to group ii (p<0.05) • small letter superscripts (a, b, c) indicate significant differences among the groups (ii, iii, v) (p<0.05) • capital letter superscripts (a, b, c) indicate significant differences among the groups (ii, iv, vi) (p<0.05) • superscript (♣) indicates a significant difference when group v is compared to group vi (p<0.05) iraqi j pharm sci, vol.31(1) 2022 fimasartan and methotrexate-induced nephrotoxicit 92 figure 5.effects of fimasartan on the kidney index (ki). discussion aki induced by hdmtx represents a serious challenge, especially among hospitalized patients, with a high risk of progression to irreversible renal impairment. moreover, the nephrotoxicity induced by mtx is of special importance because mtx is eliminated primarily by the kidneys (4,7). hence, if mtx-induced aki developed, excess amounts of the drug and its metabolites will accumulate in the body, leading to enhancement of other mtx toxicities including hepatotoxicity, myelosuppression, and neurotoxicity (5,28). oxidative stress has been wellestablished as a key player in the pathogenesis of mtx-induced renal injury (9,29). previous investigations reported that mtx can promote ros production by inducing mitochondrial dysfunction and activating nadph oxidases, which generate excessive amounts of ros, especially superoxide anion, as their main products (22). the resultant oxidative burst, coupled with impairment of the antioxidant defences of the body; can result in oxidative damage of important cellular components including lipids, proteins, and nucleic acids; contributing to lipid peroxidation and cell death (22,30). in the current study, the administration of mtx to the rats resulted in renal dysfunction evidenced by the significant increase in serum creatinine and bun levels; which have been widely utilized to assess renal impairement in numerous preclinical studies (31-33); combined with a significant increase in renal mda levels (a lipid peroxidation marker), and a significant decrease in renal contents of the antioxidant enzyme, sod-1. these results were similar to previous studies, indicating the important role of oxidative stress in the development of peroxidative damage and renal injury upon exposure to mtx (10,12,22). mtx-induced nephrotoxicity was further confirmed by a significant increase in ki in the mtx group compared to the untreated rats, confirming the harmful effects of mtx on the kidneys, since any change in ki from normal is an indicator of renal toxicity (9,34,35). importantly, the present study showed that fimasartan ameliorated the renal injury induced by mtx, evidenced by the significant decrease in the serum levels of renal function parameters and ki, coupled with a significant decrease in renal mda and a significant increase in renal sod-1 contents, reflecting a restoration of the cellular redox balance in the kidney. these results are consistent with previous researches that reported the renoprotective effects of fimasartan and other arbs in various drug-induced renal injury models (18,20,35,36). a study by kim et al. (2015) in a mice model of unilateral ureteral obstruction showed that the ameliorating effect of fimasartan against renal oxidative stress, inflammation, and fibrosis was mediated through upregulation of the antioxidant enzymes (including sod-1) along with counteracting the effects of angiotensin ii (ang ii), which is a major component of the renin-angiotensin-aldosterone-system (18). they found that the locally expressed ang ii in the kidneys contributed to the oxidative stress by enhancing nadph oxidases, and blockade of ang ii/angii type 1 receptor signalling by fimasartan reduced the oxidative stress and inflammation induced by ang ii (18). similarly, in another preclinical study, fimasartan was found to preserve renal structure and function in an ischemia/reperfusion injury model by preventing apoptosis induced by the inflammatory pathway (20). notably, the mechanisms offered by fimasartan and other renoprotective arbs are the same, where it was reported that irbesartan displayed a protective role in gentamicin-induced nephrotoxicity via its antioxidant effect (36). similarly, losartan showed a renoprotective effect through counteracting oxidative stress in an ischemic renal injury model (37). consequently, the beneficial effects of fimasartan against renal injury can be attributed to its antioxidant activity mediated by the enhancement of the endogenous antioxidants, with the resultant attenuation of lipid peroxidation in renal tissue. on the other hand, α-tocopherol appears to have antioxidant effects which contribute to its renoprotective effects. in agreement, many investigators verified that α-tocopherol can mitigate the nephrotoxicity induced by several agents, and the protective effect was credited to its antioxidant and anti-inflammatory properties (12,38,39). however, fimasartan appears to be a more powerful antioxidant than α-tocopherol, since it resulted in a more significant increase in the renal sod-1 levels. conclusion in conclusion, the present study revealed that treatment of rats with fimasartan have ameliorative effects, that are comparable to those of α-tocopherol, against mtx-induced nephrotoxicity through boosting the antioxidant defences in the kidneys; with fimasartan being more effective than α-tocopherol since it increased the renal sod-1 levels to a greater extent. iraqi j pharm sci, vol.31(1) 2022 fimasartan and methotrexate-induced nephrotoxicit 93 acknowledgement the data of this article were abstracted from the m.sc. thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the authors are extremely grateful to the college of pharmacy/university of baghdad for supporting this work. references: 1. soares s, c r souza l, t cronin m, m waagagasser a, f grossi m, r franco g, et al. biomarkers and in vitro strategies for nephrotoxicity and renal disease assessment. nephrol ren dis. 2020;5(1):1–14. 2. darmon m, joannidis m, schetz m. focus on critical care nephrology. intensive care med. 2019;45(9):1288–91. 3. soares s, c r souza l, t cronin m, m waagagasser a, f grossi m, r franco g, et al. biomarkers and in vitro strategies for nephrotoxicity and renal disease assessment. nephrol ren dis. 2020;5(1):1–14. 4. howard sc, mccormick j, pui c, buddington rk, harvey rd. preventing and managing toxicities of high‐dose methotrexate. oncologist. 2016;21(12):1471–82. 5. ramsey lb, balis fm, o’brien mm, schmiegelow k, pauley jl, bleyer a, et al. consensus guideline for use of glucarpidase in patients with high‐dose methotrexate induced acute kidney injury and delayed methotrexate clearance. oncologist. 2018;23(1):52–61. 6. ahmed w, zaki a, nabil t. prevention of methotrexate-induced nephrotoxicity by concomitant administration of garlic aqueous extract in rat. turkish j med sci. 2015;45(3):507–16. 7. aladaileh sh, hussein oe, abukhalil mh, saghir sam, bin-jumah m, alfwuaires ma, et al. formononetin upregulates nrf2/ho-1 signaling and prevents oxidative stress, inflammation, and kidney injury in methotrexate-induced rats. antioxidants. 2019;8(10):1–18. 8. gai z, gui t, kullak-ublick ga, li y, visentin m. the role of mitochondria in drug-induced kidney injury. front physiol. 2020;11:1–13. 9. hassanein ehm, shalkami ags, khalaf mm, mohamed wr, hemeida ram. the impact of keap1/nrf2, p 38 mapk/nf-κb and bax/bcl2/caspase-3 signaling pathways in the protective effects of berberine against methotrexate-induced nephrotoxicity. biomed pharmacother. 2019;109(october 2018):47–56. 10. abdel-daim mm, khalifa ha, abushouk ai, dkhil ma, al-quraishy sa. diosmin attenuates methotrexate-induced hepatic, renal, and cardiac injury: a biochemical and histopathological study in mice. oxid med cell longev. 2017;2017:3281670. 11. aldossary sa. protective effect of hesperidin against methotrexate-induced nephrotoxicity in rats. life science journal. 2019;16(2):18–22. 12. abdel-daim mm, aleya l, el-bialy be, abushouk ai, alkahtani s, alarifi s, et al. the ameliorative effects of ceftriaxone and vitamin e against cisplatin-induced nephrotoxicity. environ sci pollut res. 2019;15248–54. 13. wu tk, pan yr, wang hf, wei cw, yu yl. vitamin e (α-tocopherol) ameliorates aristolochic acid-induced renal tubular epithelial cell death by attenuating oxidative stress and caspase-3 activation. mol med rep. 2018;17(1):31–6. 14. jilanchi s, nematbakhsh m, bahadorani m, talebi a, eshraghi-jazi f, mansouri a, et al. vitamin e is a nephroprotectant agent in male but not in female in a model of cisplatininduced nephrotoxicity. isrn nephrol. 2013;2013:1–6. 15. angeli f, verdecchia p, trapasso m, pane m, signorotti s, reboldi g. pk/pd evaluation of fimasartan for the treatment of hypertension current evidences and future perspectives. expert opin drug metab toxicol. 2018;14(5):533–41. 16. lee hy, oh bh. fimasartan: a new angiotensin receptor blocker. drugs. 2016;76(10):1015–22. 17. han se, jeong sh, kang hj, hong ms, paek e, cho h, et al. safety and efficacy of fimasartan with essential hypertension patients in real world clinical practice: data from a post marketing surveillance in korea. transl clin pharmacol. 2018;26(3):118–27. 18. kim s, kim sj, yoon he, chung s, choi bs, park cw, et al. fimasartan, a novel angiotensin-receptor blocker, protects against renal inflammation and fibrosis in mice with unilateral ureteral obstruction: the possible role of nrf2. int j med sci. 2015;12(11):891–904. 19. yang x, sun j, kim tj, kim yj, ko sb, kim ck, et al. pretreatment with low-dose fimasartan ameliorates nlrp3 inflammasomemediated neuroinflammation and brain injury after intracerebral hemorrhage. exp neurol. 2018;310(august):22–32. 20. cho jh, choi sy, ryu hm, oh ej, yook jm, ahn js, et al. fimasartan attenuates renal ischemia-reperfusion injury by modulating inflammation-related apoptosis. korean j physiol pharmacol. 2018;22(6):661–70. 21. machholz, e., mulder, g., ruiz, c., corning, b.f., pritchett-corning, k.r. manual restraint and common compound administration routes in mice and rats. j. vis. exp. 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2020 nsaids for appendectomy in children doi: https://doi.org/10.31351/vol29iss1pp123-133 123 comparing the efficacy of paracetamol, diclofenac, and ketorolac on post-appendectomy outcomes in children and adolescents majeed n. abdul majeed *,1 and zinah m. anwer ** *clinical pharmacy department in college of pharmacy,university of al-kafeel, karbala, iraq. ** clinical pharmacy department in college of pharmacy, university of baghdad, baghdad, iraq abstract acute appendicitis is one of the most frequent abdominal conditions that face children population and needs urgent surgical intervention and appendectomy until now represent standard treatment for uncomplicated cases of appendicitis. nausea, vomiting and pain after surgery are the most frequent issues facing patients and affecting patient quality of life and responsible for many cases of readmission after surgery. ketorolac and diclofenac represent the most commonly prescribed non-steroidal anti-inflammatory used in postoperative setting and they cause many side effects as gastrointestinal, kidney and cardiac adverse effect in addition to increased risk of bleeding. paracetamol is currently among the most frequently prescribed medication worldwide and it can be used safely for all age groups. this study aimed to compare the analgesic efficacy and safety of paracetamol, diclofenac, and ketorolac when used after appendectomy and to assess their efficacy regarding nausea and vomiting in children and adolescents. a randomized, single-blinded, comparative, observational prospective clinical study was carried out on patients diagnosed with acute appendicitis and assigned for emergent appendectomy between october 2018 to may 2019 in alzahraa teaching hospital in al-najaf province, iraq.120 patients were randomly distributed into three groups who received diclofenac sodium suppositories (2mg/kg), iv paracetamol )15mg/ kg every 6 hr.(, and iv ketorolac )0.5mg/ kg( immediately after surgery. all patients were observed for pain, nausea and vomiting and bleeding. patients received ketorolac had a high percentage of the decrease in pain score between 30 and 60 min. after surgery, while paracetamol was the next and diclofenac sodium was the last. regarding nausea and vomiting after surgery, ketorolac had higher percentage of the decrease in nausea, vomiting score during first day after surgery followed by diclofenac and paracetamol respectively. the present study also showed that there is no significant difference between groups regarding bleeding after surgery. it is concluded that ketorolac has higher analgesic efficacy compared with paracetamol and diclofenac with no risk of nausea and vomiting and bleeding when used after appendectomy. keywords: postoperative pain, ketorolac, diclofenac, paracetamol, appendectomy. دراسة مستقبلية عشوائية سريرية لمقارنة تأثير براسيتامول ودايكلوفيناك وكيتوروالك على نتائج ما بعد عملية استئصال الزائدة الدودية عند األطفال واليافعين. مجيد نبيل عبد المجيد *،1 و زينة مظفر أنور** ، كربالء ، العراق . فرع الصيدلة السريرية ، كلية الصيدلة ، جامعة الكفيل* بغداد ، بغداد ، العراق .فرع الصيدلة السريرية ، كلية الصيدلة ، جامعة * الخالصة التهاب الزائدة الدودية واحد من أكثر االمراض التي تصيب االطفال والتي تحتاج الى تدخل جراحي سريع وطارئ وتعتبر عملية استئصال ٪ من ٣الزائدة الدودية العالج الرئيسي للحاالت غير المعقدة من المرض. حصول المضاعفات بعد عملية استئصال الزائدة شيء نادر ويحدث بنسبه حياتهم ل في الحاالت غير المعقدة. ويعتبر الغثيان والتقيؤ وااللم بعد العملية من أكثر المشاكل شيوعا والتي تواجه المرضى وتؤثر على طبيعة االطفا ر الستيروئدية ومسؤولة عن الكثير من حاالت الدخول الى المستشفى بعد العملية. الدايكلوفيناك والكيتورالوك من أكثر االدوية المضادة لاللتهابات غي ما صرفا وخصوصا بعد العملية وتملك العديد من االثار الجانبية كالتآثيرات الجانبية على الجهاز الهضمي والكلى والقلب ومخاطر النزيف بين مقارنة فعالية وآمان البراسيتامول الذي يعتبر من االكثر االدوية صرفا حول العالم والذي يوصف بامان لكل الفئات العمرية. الهدف من الدراسة هو الدودية في االطفال البراسيتامول والدايكلوفيناك والكيتوروالك على تسكين االلم وتقييم فعاليتها على تقليل الغثيان والتقيؤ بعد عملية استئصال الزائدة واليافعين. المرضى المشخصين بالتهاب الزائدة الدودية والمرشحين الجراء عملية استئصال الزائدة دراسة سريرية مستقبلية عشوائية اجريت على ال مريضا ١٢٠في مستشفى الزهراء التعليمي في محافظة النجف في العراق وتضمنت الدراسة ٢٠١٩وايار ٢٠١٨الدودية للفترة بين تشرين االول ملغ/كغم( ٢ساعات( والثانيةاخذت تحاميل الدايكلوفيناك ) ٦ملغ/كغم كل ١٥وريدي )براسيتامول ئيا على ثالث مجاميع االولى اخذتوزعوا عشوا ملغ/كغم( بعد العملية مباشرة. تمت متابعة المرضى في الدراسة من حيث االلم والغثيان والتقيؤ وحدوث ٠،٥والثالثة اخذت خذوا كيتوروالك وريدي ) النزف بعد العملية. 1corresponding author e-mail:ph.majeednabeel@gmail.com received: 4/8/2019 accepted: 22/ 10/2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp123-133 iraqi j pharm sci, vol.29(1) 2020 nsaids for appendectomy in children 124 دقيقة من العملية يليه البراسيتامول ٦٠و ٣٠يملك النسبة االكبر من المرضى الذين لديهم اقل درجه الم بعد اثبتت الدراسة بان الكيتوروالك الول والدايكلوفيناك تتابعا وكذلك اثبتت ان الكيتوروالك يملك النسبة االكبر من المرضي الذين لديهم اقل درجة للغثيان والتقيؤ في اليوم ا ناك ثم البراسيتامول اخيرا. كما اثبتت الدراسة الحالية انه ال يوجد اختالف واضح بين مجموعات الدراسة فيما من العملية يليه الدايكوفي الكيتوروالك يملك اعلى فعالية كمسكن الم مقارنة بالبراسيتامول والدايكلوفيناك وبدون خطر نستنتج انيتعلق بحدوث النزف بعد العملية. لنزف بعد عملية استئصال الزائدة الدودية.االصابة بالغثيان والتقيؤ وا ، كيتوروالك، دايكلوفيناك، براسيتامول، عملية استئصال الزائدة الدوديةالم بعد العملية الكلمات المفتاحية: introduction acute appendicitis is considered one of the most frequent abdominal condition that faces children population and needs an urgent surgical procedure (1). appendectomy till now represents standard treatment for uncomplicated cases of appendicitis (2) . appendectomy can perform either by open surgery or laparoscopic procedure. it accounts for more than half of the operations that are done in the emergency room and constitutes about 10-30% of pediatric emergency operations(3,4). surgical treatment of acute appendicitis by open surgery approach is considered a clean-contaminated surgery and still has good outcomes with acceptable complication rate (11.1%) and mortality rate of less than 0.5% which is related to the operation itself (5-8). the complications after appendectomy are rare and include surgical site infection (1.2-12%), intra-abdominal abscess (1.8-8%), small intestine obstruction (0-1.9%) and lesser percent present with stump leakage and stump appendicitis(9,10). nausea and vomiting (n,v) and pain after surgery are the most frequent issue facing patients(11,12). previous studies stated that about 30% of patients suffer from mild pain, 30% present with moderate pain and the rest percent 40% suffer from severe pain after surgery (13). children usually face periods of healing after discharge from hospital and back home, during this period, children have significant postoperative pain which significantly affects the patient’s quality of life(14). many medications are used to treat postoperative pain include opioids, non-steroidal antiinflammatory drugs (nsaids), and paracetamol. however, these medications are not free from side effects, which can produce respiratory depression and gastrointestinal problems (15). the aim of treating postsurgical pain is to achieve good pain control with minimum side effects (16). post-operative nausea and vomiting (ponv) is one of the common issues in practice of anesthesia, since it causes many complications as surgical wound opening, unplanned admission, delay return to normal activity and dehydration(17). ponv is influenced by many factors, which include personal factors and surgical factors. pain represents common post-surgical risk factor of ponv(18). after surgery patients favor to have pain rather than (n,v)(19). after hundreds of years of advances, the mainstay of pain therapy is still the opioids, which are considered a very potent pain killer but associated with many unwanted side effects like: respiratory depression, sedation, hypotension, bradycardia, nausea and vomiting (n,v) pruritus and inhibition of bowel function(16). nonsteroidal anti-inflammatory drugs are a group of medications used in modern medicine to relieve pain and inhibit inflammation(20). the main mechanism by which nsaids produce their efficacy is inhibition of prostaglandin synthesis by inhibiting the first enzyme in prostaglandin synthesis which is called cyclooxygenase (cox)(21). nsaids are widely used for both children and adults but not without side effects, where they cause gastrointestinal, kidney and cardiac adverse effects in addition to increased risk of bleeding(22). ketorolac and diclofenac represent the most commonly prescribed nsaids used in the postoperative setting. ketorolac has potent analgesic activity but a moderate antiinflammatory effect(23). ketorolac intravenously is used to manage moderate to severe pain after surgery(24). it is considered a reasonable option to avoid the use of opioids in children in addition to decrease the cost of treatment with comparable efficacy to morphine where 0.5-1.5 mg\kg of iv ketorolac has comparable analgesia of 0.1 mg \kg of iv morphine with lowest adverse effect(25). on the other hand, diclofenac has potent analgesic, anti‐inflammatory, and antipyretic properties. diclofenac has a potency greater than other nsaids since it is like celecoxib in cox2 selectivity and low risk for bleeding(22). it is used safely in children and is frequently given rectally for acute pain after surgery(24). paracetamol is currently among the most frequently prescribed medication worldwide. it can be used safely for all age groups and it constitutes step no.1 of the world health organization ladder of analgesia(26,27). paracetamol acts as a good pain reliever and fever reduction but it has no anti-inflammatory action. it differs from opiates since the lack of respiratory depression effect and does not cause addiction also it differs from nsaids since the iraqi j pharm sci, vol.29(1) 2020 nsaids for appendectomy in children 125 lack of unwanted gastrointestinal, renal or cardiac side effect(28). due to the faster onset of action and good tolerability profile, paracetamol as the i.v route is used currently to relieve pain immediately after surgery(28,29). paracetamol works by a mechanism not clearly understood until now even after many years of discovery and clinical use(28) . the aim of this study was to compare the analgesic efficacy and safety of paracetamol, diclofenac, and ketorolac when used after appendectomy and to assess their efficacy regarding postoperative nausea and vomiting in children and adolescents. patients and methods this study was conducted after approval by graduate studies committee of the college of pharmacy\ university of baghdad and scientific and ethical committee of researchers of al-najaf health directorate and after taking the permission of patient’s parents. a prospective, randomized, singleblinded, comparative, observational clinical trial was carried out on 120 patients diagnosed as acute appendicitis and assigned for emergent appendectomy surgery (mean age was7.52.74 years) at pediatric surgery department, alzahraa teaching hospital, al-najaf province, iraq. collecting data and following up was done from october 2018 to may 2019. the number of female patients was 85 (70.8%), while the number of male patients was 35(29.2%). the follow-up period was one month for each patient and it is done either by phone calls or interviewing with patients when they visited the consultant unit at pediatric surgery department, al-zahraa teaching hospital postoperatively or at private clinics of a surgeon. the inclusion criteria for enrolling patients in the study were: 1 patients diagnosed with acute appendicitis, 2 aged between 4-14 years with no history of allergy or contraindication to ketorolac, diclofenac or paracetamol. 3 taking the same analgesia for 3 consecutive days without any rescue analgesic and taking the same antibiotic regime mentioned later in this study. exclusion criteria involved 1 patient who disagree to participate in the study, 2 patients with known hypersensitivity or contraindication to ketorolac, diclofenac, and paracetamol, 3concomitant therapy with warfarin or heparin or high-dose aspirin (>1000 mg/day), those with the established cardiovascular disorder or uncontrolled hypertension and who had received antibiotics within 72 hours of admission. 4 those with complicated or perforated appendicitis. 5 patient with less than one month of follow up. patients fulfilling the eligibility criteria were blindly and randomly allocated into one of the three groups, each group contained 40 patients. the randomization was done by the preparation of a list on the computer by the researcher that was coded to contain only the number and the name of the drug without knowledge of the patients and surgeons. after the surgery was done, the patient takes the same drug that presents in the list according to the number present in the previously prepared list. group (a): patients received paracetamol 15 mg /kg every 6 hrs (intravenously), group (b): patients received diclofenac 2 mg/kg per rectal route twice daily (in order to determine the amount of medication needed per dose in patient taking diclofenac suppositories, the suppository was scraped-off by using sharp surgical blade, sectioned longitudinally then weighted by using radwang electronic balance), and group (c): patients received ketorolac 0.5 mg /kg (intravenously)once daily . the patients were transferred to the recovery room and received analgesia immediately after surgery and continue to use it for three days. the patients receive the same antibiotics regime (the antibiotic regimen was continued for five days after surgery and it consisted of iv ceftriaxone with a daily dose of 50 mg/ kg and iv metronidazole with a dose of 7.5 mg/ kg every 8 hr.). the data collection sheet designed by the researcher to obtain information related to the patient directly from him or from his relatives. postoperative pain, and (n,v) were assessed by using a validated scale or asking patients and the data were obtained by direct interview with the patients on the first day (day of surgery at the hospital). postoperative pain was assessed in each group by using the faces pain scale-revised (fps-r)(30,31), which is considered as a selfreport measure of pain intensity that was developed to be used in children with age range of 4-16 years. this scale contains six face expression with different emotion scores (0, 2, 4, 6, 8 and 10), the child should be referred to face express that coordinate with his/her pain feeling. the patients assessed their pain intensity by using this scale at 30 minutes and 60 minutes after surgery and the score was recorded. postoperative nausea and vomiting were assessed using baxter animated retching iraqi j pharm sci, vol.29(1) 2020 nsaids for appendectomy in children 126 faces (barf)(32) which is characterized by six characters with different degrees of vomiting (0, 2, 4, 6, 8 and 10) that represent the severity of (n,v) in a manner to make this scale easy to be administered by a child. the patients should be referred to expressions that matched their feelings. the assessment of (n,v) was done at the end of the first 24 hr. postoperatively. also, taken into consideration if the antiemetic was prescribed, the type and name and the dose should be taken. the postoperative wound bleeding was also assessed for over one month after surgery. statically analysis data were summarized, analyzed and presented using statistical package for the social sciences (spss) version 23 software for windows. numeric data were expressed as mean± standard deviation and range, whereas, categorical data like frequency were expressed as number and percentages. fisher’s exact test (chi square test could not be applied) was used to assess the statistical significance in distribution between different discrete variables (p-values< 0.05) were considered to be statistically significant. the calculation of percentage of change was achieved according to following equation: % change = original number new number ÷ original number × 100 if answer is a negative number, then this is a percentage increase while a positive number indicates percentage decrease. results demographic characteristic of the study showed for the total of 120 patients, 85 (70.83%) of patients were male and 35 (29.17%) were female patients (in table 1). regarding pain score at 30 min. after surgery, there1is1a significant 1differencebetween, groups of study (p-value-<0.05) as showed in (table 2) and (fig.1), the highest frequency of patients in the ketorolac group (77.5%) was found at score 2, the highest frequency of patients in the diclofenac group (42.5%) at score 2whereas the highest frequency of patients in paracetamol group (37.5%) was seen at score 4. regarding pain score at 60 min. after surgery, there1is a1 significant1 difference 1between1 groups of study (p-value1>0.05) as showed in (table 3) and (fig.2), the highest frequency of patients in ketorolac group (72.5%) was found at score 0, regarding diclofenac, the highest frequency of patients (62.5%) presented at score 2 while the highest frequency of patients in paracetamol group (37.5%) was seen at score 6. table 1. demographic characteristics of the study groups. diclofenac sodium (n=40) paracetamol (n=40) ketorolac (n=40) total age(years) mean ± sd range (min-max) 6.75±2.9 (4-14) 6.73±2.48 (4-13) 8.76±2.33 (5-13) 7.41±2.73 (4-14) weight (kg) mean ± sd 22.85±7 22.85±6.09 26.29±5.76 24±6.46 body mass index (bmi)(kg/m2) average ± sd 17.8±2.53 17.06±2.67 17.17±2.09 17.34 ±2.46 gender male no.(%) 32(80%) 27(67.5%) 26(65%) 85 (70.83%) female no.(%) 8(20%) 13(32.5%) 14(35%) 35 (29.17%) residency urban no.(%) 23(57.5%) 24(60%) 22(55%) 69 (57.5%) rural no.(%) 17(42.5%) 16(40%) 18(45%) 51(42.5%) family history positive no.(%) 11(27.5%) 10(25%) 7(17.5%) 28 (23.33%) negative no.(%) 29(72.5%) 30(75%) 33(82.5%) 92 (76.67%) work no work no.(%) 23(57.5%) 22(55%) 10(25%) 45(37.5%) part time student no.(%) 17(42.5%) 18(45%) 30(75%) 75(62.5%) iraqi j pharm sci, vol.29(1) 2020 nsaids for appendectomy in children 127 table 2. pain score after 30 minutes of surgery for the study groups. diclofenac sodium no. (%) paracetamol no. (%) ketorolac no. (%) p-value number2of1patients at1score 0 0(0%) 2(5%) 5(12.5%) 5.4823e-8a number1of1patients at1score 2 17(42.5%) 8(20%) 31(77.5%) number of patients at score 4 15(37.5%) 15(37.5%) 4(10%) number of patients at score 6 5(12.5%) 10(25%) 0(0%) number of patients at score 8 3(7.5%) 5(12.5%) 0(0%) number of patients at score 10 0(0%) 0(0%) 0(0%) a : fisher’s exact test figure 1. percent of the frequency of pain score for the study groups after 30 minutes of surgery. note: there are (0%) at score 10 of all three groups, table 3. pain score after 60 minutes of surgery for the study groups. diclofenac sodium no. (%) paracetamol no. (%) ketorolac no. (%) p-value number of patients at score 0 2(5%) 7(17.5%) 29(72.5%) 1.1719e-13a number of patients at score 2 25(62.5%) 15(37.5%) 11(27.5%) number of patients at score 4 11(27.5%) 6(15%) 0(0%) number of patients at score 6 2(5%) 9(22.5%) 0(0%) number of patients at score 8 0(0%) 3(7.5%) 0(0%) number of patients at score 10 0 (0%) 0(0%) 0(0%) a : fisher’s exact test iraqi j pharm sci, vol.29(1) 2020 nsaids for appendectomy in children 128 figure 2.percent of the frequency of pain score for the study groups after 60 minutes of surgery. note: there are (0%) at score 10 of all three groups according to the percent of differences in pain score, (table 4) and (fig.3) shows that a high percentage of the decrease in pain score between 30 and 60 min. after surgery was seen in the ketorolac group while paracetamol was next and diclofenac sodium was the least. table 4. percent of changes in1pain1score between1301minutes and1601minutes1for the study group. figure 3. percent of changes in pain score between 30 minutes and 60 minutes of surgery for the study groups. diclofenac sodium paracetamol ketorolac percent of changes in patients at score 0 0% 250% 480% percent of changes in patients at score 2 47% 87.5% 64% percent of changes in patients at score 4 27% 60% 100% percent of changes in patients at score 6 60% 10% 0% percent of changes in patients at score 8 100% 40% 0% percent of changes in patients at score 10 0% 0% 0% % change = original number new number ÷ original number × 100 iraqi j pharm sci, vol.29(1) 2020 nsaids for appendectomy in children 129 regarding (n,v) score after surgery, there is a significant difference between the groups of study (p-value <0.05) as shows in (table 5) and (fig.4), highest frequency of patients in ketorolac group (82.5%) found at score 2, regarding diclofenac sodium highest frequency of patients (37.5%) presented at score 4 while highest frequency of patients in paracetamol group (27.5%) seen at score 4. table 5. nausea and vomiting score for the study group. diclofenac sodium no. (%) paracetamol no. (%) ketorolac no. (%) p-value number of patients at score 0 0(0%) 8(20%) 5(12.5%) 1.3951e-8a number of patients at score 2 19(47.5%) 9(22.5%) 33(82.5%) number of patients at score 4 15(37.5%) 11(27.5%) 2(5%) number of patients at score 6 3(7.5%) 8(20%) 0(0%) number of patients at score 8 1(2.5%) 3(7.5%) 0(0%) number of patients at score 10 2(5%) 1(2.5%) 0(0%) a : fisher’s exact test figure 4. percent of frequency of nausea and vomiting score for the study groups. regarding bleeding after surgery, there is no significant difference between the number of patients with bleeding when compared to the number of patients without bleeding (p<0.05) as shown in (table 6). table 6.bleeding after surgery. diclofenac sodium no. (%) paracetamol no. (%) ketorolac no. (%) p-value patients with bleeding 1(3%) 0(0%) 1(3%) 1a patients without bleeding 39(97%) 40(100%) 39(97%) a : fisher’s exact test iraqi j pharm sci, vol.29(1) 2020 nsaids for appendectomy in children 130 discussion acute appendicitis is a common health problem in children and after appendectomy patients need medications to relieve their pain and nausea and vomiting. as shown in the demographic data, male gender was greater than female. this finding is in concordance with many studies which stated that appendicitis is more common among males(33-35). the mean age of patients enrolled in this study was 7.41±2.73 years, this finding ties well with many studies that showed a high frequency of appendicitis between 7-19 years(36-38). this may be explained by the effect of life stress in this group of patients as a type of psychological trauma(39) while, it is difficult to explain that in children under the age of 7 years , who have a lower incidence. in the present study, the majority of patients had a negative family history, this does not agree with most of the studies showing family history as an important parameter in predicting acute appendicitis(40-43). this finding may be due to the methodology characteristics of this study. in the present study, ketorolac has a higher analgesic efficacy when compared to paracetamol and diclofenac used postappendectomy. this clearly appeared in three previous studies done in different countries showing that ketorolac had a higher analgesic effect than diclofenac and paracetamol after surgery(44-46), and also agrees with another retrospective study in 2016 showing that ketorolac after appendectomy significantly decreases the pain score during the first day of surgery in pediatric patients(47). this conclusion was also presented with different surgical procedures both in adults(48-50) and children(47,50). another study in korea showed that iv paracetamol (1g) has the same level of pain relief after surgery as iv ketorolac (30 mg) in patients who underwent thyroidectomy, this made paracetamol an effective alternative to ketorolac in case of mild to moderate pain after surgery(51). ketorolac and diclofenac have similar analgesic efficacy in another study(52). this result may explain the high analgesic potency of ketorolac where it differs from other nsaids that it is used for management of moderate to severe pain after surgery and the role of intravenous dosage form where ketorolac has a rapid onset of an action (30 minutes)(53). in the present study, ketorolac less with nausea and vomiting than diclofenac and paracetamol. this result is in concordance with another study in 2012 that showed ketorolac’s effect to decrease (n,v) after surgery(54) while, another study stated that the use of 30 mg ketorolac produces a superior analgesia and antiemetic effect after mixed ambulatory surgeries when compare with 4 mg of dexamethasone and 12 mg of betamethasone(55). the ability of ketorolac to decrease the rate of (n,v) may be related to its potent analgesic activity to reduce (n,v) which is associated with pain and decrease the need for antiemetic agents after surgery. the present study showed that there is no significant difference between patients who had bleeding and those without in all groups of the study. these finding is supported by a previous cohort study involve 35 hospitals with participants take 10,272 injectable ketorolac courses(56). the present study showed that there is a little association between use of ketorolac and overall bleeding risk and this risk increases with increasing the dose of ketorolac(57). this finding also presented in another three systemic studies that state there is no significant association between the use of nsaids rather than aspirin and bleeding risk(58-60). the other two studies stated that ketorolac is considered a good choice for patients after surgery since it is not associated with risk of bleeding after surgery(61,62). this study had several laminations 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systematic review of the risk of postoperative bleeding with perioperative non-steroidal antiinflammatory drugs (nsaids) in plastic surgery . european journal of plastic surgery 2018; 41(5):505–510 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://link.springer.com/journal/238 https://link.springer.com/journal/238 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.22(1) 2013 arabidopsis thaliana extract and blood glucose level 115 study the effect of arabidopsis thaliana extract on reducing blood glucose level in diabetic white albino mice khaleel i. rashid *,1 , abedaljasim m. aljibouri ** ,abdulameer j. zayer ** , luma b. khalid ** and ahmed abdul-munaem ** * college of health and medical, baghdad, iraq. ** biotechnology center technologies-baghdad, alnahrain university, baghdad ,iraq. abstract this study was designed to evaluate the effect of aqueous extract of arabidopsis thaliana seeds on reducing glucose level for white albino mice. twenty adults mice were used, divided randomly into four groups (five mice per each group). the first group (normal mice) was administrated with 0.1 ml of distilled water as a control, the second group (normal mice) was administrated with 0.1 ml of the plant extract, whereas the third and fourth groups (diabetic mice) were administrated with single dose of alloxan (150 mg/kg of the body weight) to induce diabetes, and the fourth group was administrated with 0.1 ml of the plant extract for 10 days, then blood glucose level was measured for all of the experimental animals (diabetic and non diabetic). results showed clear increasing in glucose levels in the diabetic mice, while significant reduction was recorded in glucose levels of the normal mice that was treated with the plant extract as compared with the control group. these results indicate that arabidopsis thaliana seeds aqueous extract possesses a hypoglycemic effect. key words: arabidopsis thaliana, glucose level, albino mice زفٌ خفض هستوى الكلوكو arabidopsis thalianaًببت اراى الفبر دراسة تأثَر هستخلص فٌ دم الفئراى البَضبء الوصببة ببلسكر خلَل ابراهَن رشَذ *،1 ، عبذالجبسن هحَسي الجبورً ** ، عبذاالهَر جواد زاٍر ** ، لوي برهبى خبلذ ** احوذ عبذ الوٌعن و ** * . ، العشاقبغذاد ، كلُت الخقٌُبث الصحُت والطبُت ** .، بغذاد ، العشاق خبهعت الٌهشَي ،هشكض بحىد الخقٌُبث االحُبئُت الخالصة علً هسخىي السكش فٍ دم arabidopsis thalianaصووج هزٍ الذساست لخقُُن حأثُش الوسخخلص الوبئٍ لبزوس ًببث اراى الفبس خشعج (. فئشاى لكل هدوىعت 5)قسوج عشىائُآ الً اسبعت هدبهُع , فأسة فٍ هشحلت الٌعىج 20اسخعولج فٍ هزٍ الخدشبت . الفئشاى البُعبء كغن هي /مهلغ 200حشكُض) هللخش هي هسخخلص آراى الفأس 0,1الودوىعت الثبًُت خشعج , هللخش هي الوبء الوقطش 0,1الودوىعت االولً ألسخحذاد هشض السكشٌ (هي وصى الفأس .كغن / هلغن 150) والودوىعت الثبلثت والشابعت حقٌج بدشعت هفشدة هي االلىكسبى, (وصى الفأس بعذهب حن قُبط هسخىي الكلىكىص بذم الحُىاًبث الوخخبشة. اَبم 10هللخش هي هسخخلص الوبئٍ ولوذة 0,1وقذ خشعج الودوىعت الشابعت وًقصبى واظح فٍ هدوىعت , (الوصببت)الوعبهلت ىبٌُج الٌخبئح صَبدة واظحت فٍ هسخىي الكلىكىص فٍ هدوىعت الفئشا(. صبَت والسلُوتالن) وكزلك ًقصبى فٍ هسخىي الكلىكىص للفئشاى السلُوت الودشعت ببلوسخخلص , الفئشاى الوصببت والخٍ خشعج ببلوسخخلص الوبئٍ للٌببث . حشُش هزٍ الٌخبئح الً اى الوسخخلص الوبئٍ لبزوس آراى الفبس حوخلك حبثُش خبفط للسكش(. السُطشة)الطبُعُت ببلوقبسًت هع الودوىعت .أراى الفبر ، هستوى السكر، الفئراى االلبٌَو :الكلوبت الوفتبحَة introduction approximately 0.7% of the world's population suffers from insulin-dependent diabetes mellitus (1) . it has been estimated that the incidence of diabetes will double to approximately 300 million in the next 25 years. to date, insulin therapy is the only effective treatment for type 1 diabetes and is also generally required for the treatment of type 2 diabetes as the disease progresses. therapyrequires the regular monitoring of blood glucose levels , combined with frequent injection of insulin , in order to avoid the severe debilitating secondary complications associated with chronic hypoglycemia. meeting this demand will necessitate the development of more cost-effective, higher capacity production in the near future (2) . presently, there is growing interest in herbal remedies due to the side effects associated with the oral hypoglycemic agents (therapeutic agent) for the treatment of diabetes mellitus. corresponding author e-mail: dr.khaleel2011@yahoo.com received:12 /11/2012 accepted: 4/5/2013 iraqi j pharm sci, vol.22(1) 2013 arabidopsis thaliana extract and blood glucose level 116 so the traditional herbal medicines are mainly used which are obtained from plants, it plays important role in the management of diabetes mellitus (3) . in recent years, herbal medicines have started to gain importance as a source of hypoglycemic agents. marles and farnsworth estimated that more than 1000 plant species are being used as folk medicine for diabetes (4) . biological actions of the plant products used as alternative medicines to treat diabetes are related to their chemical composition. herbal products or plant products are rich in phenolic compounds, flavonoids, terpenoids, coumarins, and other constituents which show reduction in blood glucose levels .(5, 6, 7) . several species of herbal drugs have been described in the scientific and popular literature as having antidiabetic activity (8) . due to their perceived effectiveness, fewer side effects in clinical experience and relatively low costs, herbal drugs are prescribed (9) . best reported for the presence of insulin-like substances in plant materials like green tops of onions, lettuce leaves, green bean leaves, barley roots, beet roots and others (10) . the discovery of this hormone in tissues of the higher plants as well as in yeast opened up a new field of research in plant metabolism and afforded another remarkable example of parallelism between certain physiological processes in the plant kingdom with the animal kingdom. pancreatic insulin’s influence on glycogen formation provoked a theoretical concept on the existence of insulin or insulin like protein hormones in organisms rich in glycogen. collip’s efforts on extracts of yeast and onion were successful in altering glucose metabolism. the term glucokinin was proposed by him in order to differentiate insulin of plant origin from that of animals. following collip’s discovery charles best reported on insulin like material in germinating potatoes, rice and even in beetroot (11, 12) .arabidopsis thaliana, a small, annual flowering, dicotyledonous plant, was discovered by johannes thal (hence, thaliana) in the harz mountains in the sixteenth century. arabidopsis is a member of the brassicaceae family, which includes important crops. it has no agronomic significance, but offers important advantages for basic research in genetics and molecular biology (13) . the aims of this research are to determine the active compounds in arabidopsis plant seeds, for anti-diabetic treatments of patients with high glucose level in their blood, and to achieve commercial and economic source of insulin. materials and methods plant material seeds of arabidopsis thaliana were obtained from dr. enas muhgin in ebn-albetar centerbaghdad. the plant was cultivated in north of iraq and used in genetic engineering experiments in research centers. it was authenticated by biology department-college of sciencebaghdad university. preparation of the extract the powdered material of seeds (50 gram) added to 250 ml of distilled water, left over night on stirrer, the extract then dried under reduced pressure and was subjected to various chemical tests to detect the presence of different active phytoconstituents like alkaloids, tannins, flavonoids, saponins, terpens and steroids (14) . detection of some active compoundscolorimetric test this detection was carried out using chemical reagents and depends on appearance of the color to determine the presence of the compound only. detection of tannins (10 gram) of plant powder was mixed with 50 ml distilled water in a magnetic stirrer. the mixture was boiled in a boiling water bath for few minutes, then filtered and the filtrate was treated with few drops of 1% lead acetate solution. the development of greenish-blue precipitate is an indicator for the presence of tannins (15). detection of saponins saponins were detected by two methods: the first method, aqueous extract of a. thaliana seeds powder was shaken vigorously with distilled water in a test tube. the formation of foam standing for a time indicates a positive result. the second method, five milliliters of aqueous extract of the plant was added to 1-3 drops of 3% ferric chloride solution, a white precipitate was developed which indicates a positive result (16) . detection of terpenes and steroids (1 ml) of ethanolic extract was participated in a few drops of chloroform, then a drop of acetate anhydride and drop of concentrated sulfuric acid were added, brown precipitate appeared which representing the presence of terpene, and the appearance of dark blue color after few minutes would represent the present of steroids. the color is due to the hydroxyl group (-oh) of the steroids reacting with the reagents and increasing the conjugation of the unsaturation in the adjacent fused ring. since this iraqi j pharm sci, vol.22(1) 2013 arabidopsis thaliana extract and blood glucose level 117 test uses acetic anhydride and sulfuric acid as reagents, caution must be exercised so as not to receive severe burns (17). detection of flavonoids flavonoids were extracted by well established method of harborne 1984, and the procedure has also been followed by several others authors. the extraction protocol which has been carried out in this investigation is only for detection of flavonoids in seed extract of a. thaliana. ethanolic extract was partitioned with petroleum ether (1:1v/v), the aqueous layer was mixed with the aluminum solution. the appearance of dark color is an evidence for the presence of flavonoids. flavonoids react with the reagent and give colour reactions. spraying reagents 5% fehling solution and 1% alcl3 solution are exclusively used to detect flavonoids (17) . detection of alkaloids (10 gram) of the extract was boiled with 50 ml of distilled water and 4% of hydrochloric acid was added, then the solution was filtered and cooled. 0.5 ml of the supernatant was tested with mayer solution, appearance of white precipitate indicates the presence of alkaloids (17) . experimental animals healthy 20 adult albino male mice of swiss albino strain were obtained from the animal house of biotechnology research center, alnahrain university. the age of the mice was 8 weeks, and the weight was 25 gram. the animals were housed in plastic cages, which were cleaned and sterilized weekly with 70% ethanol. five mice kept in each cage with natural 14 hours light , 10 hours dark , and a controlled temperature at (24-28) c ͦ . the animals were fed chow and water ( the protocol was proved by institutional animal ethical committee jkkmmrf/cp/phd/ 2008) . induction of diabetes the animals were fasted for 24 hours, then diabetes was induced by a single intraperitoneal (ip) injection of alloxan monohydrated dissolved in distilled water at a dose of 150 mg/kg of mice body weight in volume of 0.1 ml. the diabetic state was confirmed 48 hours after alloxan injection. blood glucose value was reached 260 mg/dl which indicate hyperglycemia (140 mg/dl as standard before treatment), and there was 5% mortality in animals treated with alloxan. surviving mice with fasting blood glucose level 250 mg/dl or higher were included in this study (18). experimental groups the animals were divided into four groups (five mice per each group), and the groups were treated as following: first group, control, normal mice administrated with 0.1 ml distilled water. second group, normal mice administrated with 0.1 ml of arabidopsis seed extract. third group, diabetic mice administrated with 0.1 ml of distilled water. fourth group, diabetic mice administrated with 0.1 ml of arabidopsis seed extract. blood sample collection for 10 days after the experiment, blood samples were collected every two days (2, 4, 6, 8, 10) days, from the tail vein of the mice under the experiment, and glucose was assayed immediately using glucometer apparatus. results results chemical detection the chemical test of the active compounds in arabidopsis thaliana seed extract showed in table (1) indicated that the aqueous extract of this medicinal plant contains tannins, favonoids, alkaloids, saponins, terpenes and steroids. table (1): chemical detection of some active compounds in arabidopsis thaliana seed extract anti-hyperglycemic activity the effect of treatment with aqueous extract of arabidopsis thaliana on blood glucose levels in normal and diabetic mice are reported in table 2. blood glucose levels of the treated mice were significantly higher than normal mice (control). injection of 0.1 ml alloxan induced diabetes in the experimental animals as data showed, higher glucose level reached 249.2 mg/dl (as the average of five investigation) for the diabetic mice, and while it was recorded 166 mg/dl when it was injected with 0.1 ml of arabidopsis extract no. compound result 1 tannins + 2 flavonoids + 3 alkaloids + 4 saponins + 5 terpenes and steroids + 6 glycoside (+) means positive detection , (-) means negative detection iraqi j pharm sci, vol.22(1) 2013 arabidopsis thaliana extract and blood glucose level 118 (200 mg/kg of b. wt). it was reached 163 mg/dl with diabetic mice which were treated with swine insulin. from these results, significant decrease in blood glucose levels was obtained as compared with normal mice (control treatment) which recorded 164.4 mg/dl. table 2: anti-hyperglycemic effects of arabidopsis thaliana seed extract (200 mg/kg of body weight) on induced diabetic mice group/treatment dose (0.1 ml) after 2 days (mg/dl) after 4 days (mg/dl) after 6 days (mg/dl) after 8 days (mg/dl) after 10 days (mg/dl) avg (mg/dl) normal mice (control) 142 168 162 169 181 164.4 diabetic mice treated with alloxan only 260 250 242 248 248 249.2 diabetic mice treatment with insulin 168 164 162 163 164 163 diabetic mice treated with extract 173 171 161 166 159 166 discussion the plant extract studied could be an answer to the people seeking for therapeutic agents from natural sources which is believed to be more efficient with a little or no side effects when compared to the synthetic chemotherapeutic agents. the present experiment indicated that aqueous extract of arabidopsis seeds exhibited a potent blood glucose lowering properties in diabetic white albino mice. recombinant human insulin in the model plant species of a. thaliana seeds was produced by cory et al. (2) . weili et al., reported that an important therapeutic protein human insulin-like growth factor 1(higf-1) called somatomedin c, was expressed in arabidopsis thaliana seeds via oleosin fusion technology. the biological activity of the higf-1 as an oleosin-higf-1 fusion protein in vitro was demonstrated by using human neuroblastoma cells (19) . the hypoglycemic activity shown in this study may be related to the presence of flavonoids compounds which had very pronounces effect in the seed extract of this plant. flavonoids may preserve β-cell function by reducing oxidative stress-induced tissue damage and therefore protect against the progression of insulin resistance to type 2 diabetes. a prospective study in finland showed that the intakes of some specific types of flavonoids including quercetin and myricetin were inversely associated with risk of incident type 2 diabetes. in addition, emerging evidence shows that oxidative stress may be involved in the pathogenesis of chronic inflammation underlying insulin resistance, diabetes and cardiovascular disease (20, 21) . many literature reports suggest the existence of proteins with functions similar to proteins that are members of insulin pathways characteristic of vertebrates. localization of insulin-like protein in plant tissues and it has functions in connection with carbohydrate metabolism (22) . results of this study are in harmony with the results demonstrated by sheng et al. who found a hypoglycemic activity in momordica charantia and canavalia ensiflormis seed extracts. the results are also in agreement with panahi et al who found that transgenic plants such as, arabidopsis thaliana producing recombinant human insulin-like growth factor-1 (higf-1), the plant-derived higf-1 caused differentiation of human neuroblastoma cell line sh-sy5y, indicating its biological activity (23, 24, 25) . it was concluded that it was plant insulin, the presence of insulin-like molecule was recently demonstrated in the seed extract of arabidopsis thaliana , this protein may be responsible for the lowering of blood glucose concentrations when it was injected in diabetic mice. the hypoglycemic activity of the insulin-like protein from seed extract of this plant was similar to that of commercial swine insulin used as control. references 1. winter, j., neubauer, p., glockshuber, r. and rudolph, r. “increased production of human proinsulin in the periplasmic space of escherichia coli by fusion to dsba”. j. biotechnol. 84: pp175–185, 2001. iraqi j pharm sci, vol.22(1) 2013 arabidopsis thaliana extract and blood glucose level 119 2. cory, l. n.; joseph, g. b.; elizabeth, w. m.; richard, g. k.; joseph, g.; nancy, a. m. and maurice, m. m. ” transgenic expression and recovery of biologically active recombinant human insulin from arabidopsis thaliana seeds “ plant biotechnology journal, vol.4 issue 1, pp:77–85, 2006. 3. patel, k. and srinivasan, k. “ plant foods in the management of diabetes mellitus: vegetables as 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in alloxaninduced diabetic rats. african journal of microbiology research, 3(5): pp 287-291, 2009. 19. weili, l.; linguo, li.; kunlon, li.; juan, lin.; xiaofen, sun. and kexuan, t.“ expression of biologically active human insulin-like growth factor-1 in arabidopsis thaliana seeds via oleosin fusion technology. int. union of biochemistry and molecular biology. inc. vol. 58: 139146,2011. 20. knekt, p.; kumpulainen, j.; jarvinen, r.; rissanen.,h.; heliovaara, m.; reunanen, a.; hakulinen, t. and aromaa, a.” flavonoid intake and risk of chronic diseases”. am j clin nutr76 :560– 568,2002. 21. esposito k, nappo f, marfella r, giugliano g, giugliano f, ciotola m, quagliaro l, ceriello a, giugliano d: inflammatory cytokine concentrations are acutely increased by hyperglycemia in humans: role of oxidative stress. circulation106 :2067– 2072,2002. 22. xavier-filho, j.; oliveira, aea.; silva, l. b.; azevedo, cr. and venancio, tm.” plant insulin or glycokinin: a conflicting issue. bras. j. plant physiol., 15: 67-78, 2003. 23. sheng, q.; yao, h.; xu, h.; ling, x. and he, t.” isolation of plant protein from momordica charantia seeds by gel filtration and rp-hplc.zhang. yao cal, 27: 414416, 2004. 24. panahi, m., alli, z., cheng, x., belbaraka, l., belgoudi, j., sardana, r., phipps, j., and altosaar, i. insulin like growth factor (in weili, et al. , 2011) trans . genic res. 13 , 245–259, 2004. 25. menand, b., desnos, t., nussaume, l., berger, f., bouchez, d. and meyer, c. expression and disruption of the arabidopsis tor (target of rapamycin) gene. proc natl. acad. sci. ; 99:6422-6427, 2002. iraqi j pharm sci, vol.23(2) 2014 protective effect of terminalia arjuna bark on induced oxidative nephrotoxicity 89 evaluation of protective effect of different doses of terminalia arjuna bark ethanolic extract on cisplatin induced oxidative nephrotoxicity in rats venkateshwarlu eggadi *,1 , sharath chandra korupozu * , bharath kumar korupoju ** , sharavanabhava bandaru sheshagiri, shiva kumar r *** and venkateshwar rao jupally **** * department of pharmacology, vaagdevi college of pharmacy, hanamkonda, warangal, telangana, india. ** department of pharmacology, kakatiya institute of pharmaceutical sciences, pembarthi, warangal, telangana, india. *** singhania university, pacheri, jhunjhun, rajastan,india . **** department of pharmaceutical chemistry, talla padmavathi college of pharmacy, urus, kareembad, warangal, telangana, india. abstract cisplatin (cp), a platinum compound, is one of the most active cytotoxic drugs used for cancer treatment. nephrotoxicity is severe dose limiting side effect of this drug. abnormal production of reactive oxygen species (ross) leading to oxidative stress has been implicated in kidney toxicity by cisplatin. here the study was aimed to evaluate nephroprotective effect of ethanolic extract of terminalia arjuna bark (eetab) at the doses (200 & 400 mg/kg, body weight) against cisplatin (7.5 mg/kg, i.p) induced nephrotoxicity in rats. the evaluation was done by measuring % change in body weight, renal function tests such as blood urea nitrogen (bun), serum creatinine (cr), serum total protein (tp) and also kidney sod (superoxide dismutase),cat (catalase), gsh (reduced glutathione) and mda (malondialdehyde) levels altered by cisplatin administration. rats treated with eetab2 (400mg/kg) significantly (p<0.001) reduced the elevated levels of bun, cr, tp, mda and significantly (p<0.001) increased the levels of sod, gsh, and cat by restoring kidney architecture. in conclusion eetab2 (400mg/kg) attractively showed the protection against cisplatin nephrotoxicity. keywords: cisplatin, nephrotoxicity, terminalaia arjuna, reactive oxygen species, renal function tests. introduction cisplatin [cis-diamminedichloroplatinum (ii)] is a platinum containing drug used in various cancers (1) . nephrotoxicity is one of the serious dose limiting side effect of the drug (2) , leading to acute kidney failure which is a major clinical problem seen in 20% of patients despite the use of hydration and diuretics (3, 4) . the concentration of cisplatin in proximal tubule cells of s3 segment is 5 times more when compared to serum (5, 6) . the mechanism by which cisplatin targets the cancer cells is different from its action on proximal tubule cells (7) . cisplatin is conjugated to glutathione in the proximal tubule cells and gets bio transformed to a reactive thiol, which is a potent nephrotoxin. the pathway is γ-glutamyl transpeptidase and cysteine-s-conjugate β lyase dependant (8) . free radical generation in the renal tubule cells and its subsequent lipid peroxidation have been suggested to be a main cause for cisplatin induced nephrotoxicity (4, 9) . terminalia arjuna (family: combretaceae) is an ancient indian medicine used for different ailments (10) . so, far the activities reported are anti-atherogenic (11) , analgesic (12) , wound healing (13) , antioxidant (14) , hepatoprotective (15) , hypolipidemic (16), antiulcer (17) , anti-inflammatory, immunomodulatory and antinociceptive activities (18) .the phytoconstituents of the terminalia arjuna bark include triterpinoids (arjunic acid, arjunolic acid and terminic acid), glycosoides (terminoside a and arjunetin), βsitosterol, flavonoids (arjunolone, arjunone, bicalein, luteolin, gallic acid, quercetin and kampferol), tannins (punicallin, punicalagin, and casuarin) (10) . the flavonoid content in 100 g of terminalia arjuna bark is 5698 ± 531 mg (19) . ethanol was selected as solvent for extraction since the extraction efficacy of components showing antioxidative properties are in the following order: ethanol > methanol > acetone (20) . utilizing the antioxidant, free radical scavenging and anti-inflammatory activity of terminalia arjuna bark, the present study was aimed to evaluate its protective effect on cisplatin induced oxidative nephrotoxicity in rats. 1 corresponding author e-mail: eggadivenkey@gmail.com. received:27 / 1 /2014 accepted: 19/ 8 /2014 iraqi j pharm sci, vol.23(2) 2014 protective effect of terminalia arjuna bark on induced oxidative nephrotoxicity 90 materials and methods animals adult male wistar albino rats weighing 170 to 200g were obtained from the animal facility of albino laboratories, hyderabad. they were housed in polypropylene cages and maintained controlled temperature (22 ± 3 o c) with a 12 hr light/dark cycle. during the experimental period they were to free access to food and water ad libitum. the experimental protocol was approved by the institutional animal ethics committee (iaec) of vaagdevi college of pharmacy, warangal, india (1047/ac/07/cpcsea). drug and chemicals cisplatin (50mg/50ml) was a marketed product obtained from neon laboratories (code 66618), urea/bun kit of reckon diagnostics pvt. ltd. both creatinine and total protein test kit of cpc diagnostics pvt. ltd and all other chemicals used were of analytical grade obtained commercially. plant collection terminalia arjuna stem bark was obtained from the nearby areas of warangal forests. the obtained bark was authenticated by dr. vatsavaya s. raju, plant botanist, kakatiya university, warangal and a voucher specimen (no.1873) has been deposited at the herbarium of department of botany. plant extraction the terminalia arjuna bark was shade dried and powdered using grinder. about 1000g of bark powder was subjected to maceration using 1000 ml 80% v/v ethanol for about 10 days. after the 10 th day supernatant should be decanted and filtered through whatman no.1 filter paper. filtered extract was concentrated under vacuum pressure at 45 o c using rotating vacuum evaporator (heidolph manufacturers). the percentage yield of the extract was about 7.96%. phytochemical screening phytochemical screening of the ethanolic extract of terminalia arjuna bark revealed the presence of triterpinoids, flavonoids, tannins, glycosides (21) . acute toxicity sudies acute oral toxicity studies were performed as per organization for economic cooperation and development guidelines (oecd 423). thirty male swiss albino mice (20‑25 gm) were selected for acute toxicity study. the animals were fasted overnight and the fractions were administered orally at doses of 100, 400, 800, 1500 and 3200 mg/kg body weight. the animals were closely observed for the first 24 h for any toxic symptoms and for 72 h for any mortality (22) . experimental design twenty four wistar albino rats were selected and they were divided into four groups each containing six rats. cisplatin was injected intraperitoneally (i.p.) at the dose of 7.5 mg/kg body weight for induction of nephrotoxicity in rats (2) . all the animals were sacrificed on 6 th day of cisplatin administration. the experimental design was given below group i single intraperitoneal (i.p) injection of 0.5 ml isotonic saline was given on 5 th day and 0.5% sodium carboxy methyl cellulose (sod cmc) suspension was administered orally for 10 days (normal control). group ii single intraperitoneal (i.p) injection of cisplatin (7.5 mg/kg) was given on 5 th day (disease control). group iii ethanolic extract terminalia arjuna bark i (eetab i: 200 mg/kg, p.o) + cisplatin (7.5 mg/kg). rats were administered (200 mg/kg) orally for 10 consecutive days in addition to cisplatin which was administered as a single intraperitoneal dose on the 5 th day of the experiment 1 h prior to eetab i dose. group iv ethanolic extract terminalia arjuna bark ii (eetab ii: 400 mg/kg, p.) + cisplatin (7.5 mg/kg). rats were administered with ethanolic extract terminalia arjuna bark (400 mg/kg) orally for 10 consecutive days in addition to cisplatin which was administered as a single intraperitoneal dose on the 5 th day of the experiment 1 h prior to eetab ii dose. at the end of the experiment (i.e. six days after cisplatin administration), body weights of group ii, group iii and group iv rats were weighed. sample preparations serum sample blood sampling was withdrawn by retro orbital puncturing after anaesthesia. blood was collected in eppendorf and left at room temperature for 10 minutes to clot, and centrifuged at 3000 rpm at -4 o c. separated serum was used for accessing renal function tests. tissue sample all the animals were sacrificed by cervical dislocation under slight anaesthesia, kidneys were dissected out; left kidney separated and was immediately minced in ice cold phosphate buffer saline (pbs) (0.05m, ph 7) to obtain 1:9 (%w/v) whole homogenate. homogenate obtained was centrifuged at 3000 rpm and separated supernatant was stored at 80 o c for the estimation of antioxidants. the iraqi j pharm sci, vol.23(2) 2014 protective effect of terminalia arjuna bark on induced oxidative nephrotoxicity 91 right kidney fixed in 10% formalin and used for histopathological examinations. assessment of renal functions estimation of blood urea nitrogen it was estimated using enzopak urea/bun kit of reckon diagnostics pvt. ltd. estimation of serum creatinine and serum total protein levels creatinine and total protein in serum were estimated using identi -creatinine and -total protein test kit of jeev diagnostics pvt. ltd, using auto analyser (turbochem, india). assessment of kidney oxidative stress estimation of renal lipid peroxidation (mda) the concentration of mda levels in kidney homogenate was determined based up on the reaction with thiobarbituric acid (23) taking lipid peroxidation as an index. mda levels were measured spectrophotometrically at 532nm, using an extinction coefficient of 1.56 x 10 5 m -1 cm -1 and expressed as nanomoles of mda per g of tissue. estimation of reduced glutathione (gsh) gsh concentration in the kidney homogenate was measured by the method of ellman (24) and was expressed as μg/mg tissue. estimation of superoxide dismutase (sod) sod in the kidney homogenate was estimated by the method of misra and fridovich (25) and was expressed as u/mg of tissue. one unit of sod is defined as the amount of enzyme required to produce 50% inhibition of epinephrine auto oxidation. estimation of catalase (cat) cat was estimated by using the method of aebi (26) . catalase activity was expressed as μmoles of h2o2 utilized min -1 .mg -1 of protein. estimation of creatinine in urine creatinine in urine was determined using identi creatinine test kit of cpc diagnostics pvt. ltd, using auto analyser (turbochem, india). histopathological studies 10% formalin fixed kidney was sectioned at 5μm thickness and embedded in paraffin. later they were stained with hematoxylin and eosin (h&e). the stained sections were examined under microscope for determination of tissue pathological changes. a maximum of 10 fields of each slide were examined and score for the severity of changes in architecture was given. the scoring was done as none (-), mild (+), moderate (++), severe (+++) changes. statistical analysis results were expressed as mean ± sd of six rats in each group. statistical significance of any difference in each parameter among the groups was evaluated by one-way anova, followed by dunnett’s multiple comparison tests using graph pad prism software (version 5.01). p value <0.05 was considered as statistically significant. results acute oral toxicity study the ethanolic extract of terminalia arjuna bark did not show any mortality and toxic manifestations up to the dose of 3200 mg/kg. according to oecd guidelines, for acute toxicity, an ld50 dose of 2000 mg/kg and above is categorized as unclassified, and hence, the extract is found to be safe. based on the acute toxicity studies, the doses 200 mg/kg and 400 mg/kg of the terminalia arjuna bark ethanolic extract have been selected as therapeutic doses. the effect of ethanolic extracts of terminalia arjuna bark on body weight significant body weight loss (p<0.01) were seen in rats intoxicated with cisplatin compared to normal control. moreover, ethanolic extracts of terminalia arjuna bark (eetab i & ii) were significantly decreased the loss in body weight caused by cisplatin (p<0.05), but still significantly different from those of control rats as shown in figure 1. the effect of ethanolic extracts of terminalia arjuna bark on renal functional tests a-the effect of ethanolic extract of terminalia arjuna bark on bun significant (p<0.01) rise in serum bun were seen in rats intoxicated with cisplatin compared to normal control. furthermore, eetab ii produced highly significant (p<0.001) reduction in the levels of bun, which was elevated by cisplatin compared to eetab i. (figure2). b-the effect of ethanolic extract of terminalia arjuna bark on serum creatinine levels significant (p<0.01) rise in serum creatinine levels were seen in rats intoxicated with cisplatin compared to normal control. additionally, eetab ii produced very high significant reduction (p<0.001) in the levels of serum creatinine, which was elevated by cisplatin compared to eetab i (figure 3). c-the effect of ethanolic extract of terminalia arjuna bark on serum total protein levels figure 4 showed that there were very high significant (p<.001) rise in serum total protein levels in rats intoxicated with cisplatin compared to normal control. eetab ii significantly (p<0.01) reduced the increased levels of serum creatinine by cisplatin compared to eetab i as shown in figure 4. iraqi j pharm sci, vol.23(2) 2014 protective effect of terminalia arjuna bark on induced oxidative nephrotoxicity 92 the effect of ethanolic extract of terminalia arjuna bark on kidney mda levels figure 5 showed that very high significant (p<0.001) rise in kidney mda levels were seen in rats intoxicated with cisplatin compared to normal control. moreover, eetab ii was highly significantly (p<0.001) reduced the increased levels of kidney mda levels compared to eetab i. the effect of ethanolic extract of terminalia arjuna bark on kidney antioxidant levels a-the effect of ethanolic extract of terminalia arjuna bark on kidney gsh levels very high significant (p<0.001) reduction in kidney gsh levels were seen in rats intoxicated with cisplatin compared to normal control (figure 6). the intended figure also showed that eetab ii significantly (p<0.001) increased kidney levels of gsh, which was reduced by cisplatin compared to eetab i (p<0.05). b-the effect of ethanolic extract of terminalia arjuna bark on kidney sod levels figure 7 showed that very high significant (p<0.001) reduction in kidney sod levels in rats intoxicated with cisplatin compared to normal control (figure 7). furthermore, the intended figure demonstrated that eetab ii significantly increased kidney levels of sod, which was reduced by cisplatin compared to eetab i (p<0.01). cthe effect of ethanolic extract of terminalia arjuna bark on kidney cat levels very high significant (p<0.001) reduction in kidney cat levels were seen in rats intoxicated with cisplatin compared to normal control (figure 8). eetab ii produced very high significant (p<0.001) increase in kidney levels of cat, which was reduced by cisplatin compared to eetab i (p<0.01) (figure 8). dthe effect of ethanolic extract of terminalia arjuna bark on serum creatinine levels significant (p<0.001) rise in serum creatinine levels were seen in rats intoxicated with cisplatin when compared with normal control (figure 9). eetab ii produced very high significant (p<0.001) reduction of the increased levels of serum creatinine by cisplatin when compared with eetab1 (figure 9). -30 -20 -10 0 10 normal control ci spl ati n control eetab1 (200 mg/kg) + ci spl ati n eetab2 (400 mg/kg) + ci spl ati n *****### treatment groups % c h a n g e i n b o d y w t figure (1): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on % change in body weight. n=6, ### p < 0.001 vs normal control, ** p<0.01 vs cisplatin control, *** p < 0.001 vs cisplatin control. 0 20 40 60 80 normal control ci spl ati n control eetab1 (200 mg/kg) + ci spl ati n eetab2 (400 mg/kg) + ci spl ati n ** *** ### treatment groups b u n ( m g /d l) figure (2): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on blood urea nitrogen. n=6, ### p < 0.001 vs normal control, ** p<0.01 vs cisplatin control, *** p < 0.001 vs cisplatin control. iraqi j pharm sci, vol.23(2) 2014 protective effect of terminalia arjuna bark on induced oxidative nephrotoxicity 93 0 2 4 6 normal control ci spl ati n control eetab1 (200 mg/kg) + ci spl ati n eetab2 (400 mg/kg) + ci spl ati n ** *** ### treatment groups se ru m c re a ti n in e ( m g /d l) figure (3): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on serum creatinine levels. n=6, ### p < 0.001 vs normal control, ** p<0.01 vs cisplatin control, *** p< 0.001 vs cisplatin control. 0 2 4 6 8 10 normal control ci spl ati n control eetab2 (400 mg/kg) + ci spl ati n eetab1 (200 mg/kg) + ci spl ati n ** *** ### treatment groups se ru m t o ta l p ro te in ( g /d l) figure (4): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on serum total protein levels. n=6, ### p < 0.001 vs normal control, ** p<0.01 vs cisplatin control, *** p < 0.001 vs cisplatin control. 0 10 20 30 40 normal control ci spl ati n control eetab1 (200mg/kg) + ci spl ati n eetab2 (400mg/kg) + ci spl ati n ** *** ### treatment groups m d a ( n m o l/ g m ) figure( 5): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on kidney mda levels. n=6, ### p< 0.001 vs normal control, ** p<0.01 vs cisplatin control, *** p< 0.001 vs cisplatin control. iraqi j pharm sci, vol.23(2) 2014 protective effect of terminalia arjuna bark on induced oxidative nephrotoxicity 94 0 5 10 15 20 normal control ci spl ati n control eetab1 (200 mg/kg) + ci spl ati n eetab2 (400 mg/kg) + ci spl ati n *** ### * treatment groupsg s h ( m ic ro g m /m g o f w et t is su e) figure ( 6): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on kidney gsh levels. n=6, ### p< 0.001 vs normal control, * p<0.05 vs cisplatin control, *** p< 0.001 vs cisplatin control. 0 2 4 6 8 10 normal control ci spl ati n control eetab1 (200 mg/kg) + ci spl ati n eetab2 (400 mg/kg) + ci spl ati n ** *** ### treatment groups s o d ( u /m g ) figure (7): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on kidney sod levels. n=6, ### p< 0.001 vs normal control, ** p<0.01 vs cisplatin control, *** p< 0.001 vs cisplatin control. 0 50 100 150 200 normal control ci spl ati n control eetab1 (200 mg/kg) + ci spl ati n eetab2 (400 mg/kg) + ci spl ati n ** *** ### treatment groups c a t (m ic r o m o le s / m in / m g ) figure ( 8): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on kidney catalase levels. n=6, ### p< 0.001 vs normal control, ** p<0.01 vs cisplatin control, *** p< 0.001 vs cisplatin control. iraqi j pharm sci, vol.23(2) 2014 protective effect of terminalia arjuna bark on induced oxidative nephrotoxicity 95 0 1 2 3 4 5 normal control ci spl ati n control eetab1 (200 mg/kg) + ci spl ati n eetab2 (400 mg/kg) + ci spl ati n ### ** *** treatment groups s e r u m c r e a ti n in e (m g /d l) figure (9): effect of different doses of ethanolic extracts of terminalia arjuna bark (eetab i and ii) on serum creatinine levels. n=6, ### p < 0.001 vs normal control, ** p<0.01 vs cisplatin control, *** p< 0.001 vs cisplatin control. histopathological studies in normal control hematoxylin and eosin stained kidney sections revealed normal glomerulus and tubules with regular anatomy in (figure 10a). rats treated with cisplatin showed degenerating glomeruli and tubules (figure 10b). in eetab i (200 mg/kg, body weight) treated rats large number degenerating tubules were observed (figure 10c). however rats treated with eetab ii (400mg/kg, body weight) revealed predominant morphology which is nearer to normal with occasional degenerating tubules (figure 10d). figure( 10): photo micrograph of rat kidney tissues h and e stained (45x): (a) normal control group. (b) cisplatin control group. (c) eetab1 (200 mg/kg, body weight) treated plus cisplatin. (d) eetab1 (400 mg/kg, body weight) treated plus cisplatin. here in photos thick and thin arrow represents glomeruli and tubules, respectively. a b c d iraqi j pharm sci, vol.23(2) 2014 protective effect of terminalia arjuna bark on induced oxidative nephrotoxicity 96 table (1): histopathological examination and scoring of rat kidney injury. groups tubular cell swelling tubular casts tubular dilation epithelial necrosis glomerular hyper cellularity normal control cisplatin control +++ +++ +++ +++ +++ eetab1 ++ ++ ++ ++ ++ eetab2 + + + + + severity of the histopathological changes in kidney were scored using scale of none (-), mild (+), moderate (++), and severe (+++) damage. discussion cisplatin is an effective agent used against various solid tumours; nephrotoxicity is the severe dose limiting side effect of the drug (27) . in the present study, rats were intoxicated with cisplatin (7.5 mg/kg). noticeable increase in the levels of blood urea nitrogen, serum creatinine, serum total protein and urine creatinine levels were observed. administration of eetab2 significantly (p<0.001) attenuated the levels of the blood urea nitrogen, serum creatinine, serum total protein and urine creatinine levels to nearer normal values (figure 2, 3, 4 and 9). increased content of serum creatinine in rats intoxicated with cisplatin may be due to the up regulation of guadino acetate methyl transferase (gamt) enzyme (28) . free radical generation and lipid peroxidation by cisplatin is responsible for its renal toxicity. cytotoxic effect exerted by free radicals is the cause for peroxidation of membrane phospholipids finally leading to change in permeability and loss of membrane integrity (29) . in the present study, intraperitoneal administration of cisplatin resulted in increased levels of mda in kidney. eetab2 treated rats showed significant (p<0.001) reduction in kidney mda levels (figure 5). reduction in mda levels may be due to inhibition of lipid peroxidation by βsitosterol present in bark (30) . reduction of gsh levels in cisplatin intoxicated rats may be due to its direct conjugation with the drug (31) . activities of antioxidant enzymes such as cat, sod were reduced significantly in gsh depletion condition (32) . in the present study rats intoxicated with cisplatin showed decline in kidney cellular antioxidants such as gsh, sod and cat. administration of eetab ii significantly (p<0.001) restored the enzyme levels (figure 6, 7 and 8). it has been reported that the bark of terminalia arjuna contains compounds that may have beneficial effects, for example flavonoids have antioxidant-; and sitosterol has anti-inflammatory-properties (33) . the significant reduction in the body weights were observed in rats intoxicated with cisplatin which may be due to gastric toxicity caused by cisplatin (34, 35) . administration of eetab i or ii to rats intoxicated with cisplatin resulted in increment in body weight compared to cisplatin-treated animals, this may be due to the symptomatic relief provoked by the intended extract against oedema-induced by cisplatin. conclusion intoxication of rats with cisplatin increased oxidative stress and kidney damage, which is illustrated by substantial increase in pathological parameters with concurrent decrease in antioxidant parameters. here the present data reveals nephroprotective activity of ethanolic extract of terminalia arjuna bark (400 mg/kg, bw) on cisplatin induced oxidative nephrotoxicity by normalising pathological and antioxidant parameters. further investigation need to be carried out for the individual phytoconstituents which are responsible for nephroprotection. conflict of interests no conflict of interests. references 1. hanigan, m. h. and devarajan, p. “cisplatin nephrotoxicity: molecular mechanisms”. cancer theraphy. 2003; 1.47–61 2. sahu, b. d k; rentam, k. k. r.; putcha, u. k.; kuncha, m vegi, g. m and sistla, r.“carnosic acid attenuates renal injury in an experimental model of rat 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(adrs). this assumption was based on many possible factors such as hormonal or behavior differences, and it was not clearly identified since the female gender was not preferred to be enrolled in many clinical trials. the primary aim of this study was to assess the extent of possibly relevant gender differences in drug–adrs regarding causality, severity, preventability, seriousness, expectedness and outcome. while the secondary aim was to assess for which group of drugs and for which adrs gender differences are identified most often. the study was a retrospective one that depends on processing a specially selected group of data obtained from the iraqi pharmacovigilance center database. the data included consisted of 3833 individual case safety reports sent during the period from 1st january 2017 to 31st december 2019. it was found that the reported adverse drug reactions for females (60.84 %) were much more than males (39.16 %). in addition, significant differences in age group distribution of adverse drug reactions were found in which females in their reproductive age had more adverse drug reactions while the older adult males were more likely to suffer adverse drug reactions if compared with the same age groups from the opposite gender. the highest type of adverse drug reactions for both genders were those that fall in the skin and subcutaneous tissue disorders (26.4 % in females) and (22.6 % in males) with statically significant difference between the two genders. while the highest group to cause adverse drug reactions was the systemic anti-infective agents with a greater chance ‘statistically significant’ in females to suffer a side effect from this group of medications (40.8 %) compared to male gender (35.5 %). the frequency of serious adverse drug reactions was significantly more prevalent in females (45.4 %) than for males (41.3 %) while the fatal outcome was significantly more observed in males (0.8 %) as compared with females (0.2 %). the expectedness analysis gave the finding that for each gender, the chances to get an expected adr were nearly equal. keywords: pharmacovigilance, adverse drug reactions, iraqi pharmacovigilance center, gender differences, iraq. العراقي للمركز عنها االبالغ تم والتي اختالفات الجنسين في االعراض الجانبية لألدوية لدى البالغين الدوائية لليقظة ** و منال محمد يونس *جبار كاظم ضياء، 1*، أثير عبد الرزاقأريج .فرع الصيدلة السريرية ،كلية الصيدلة، جامعة بغداد، بغداد، العراق* .مدير المركز العراقي لليقظة الدوائية، دائرة االمور الفنية، وزارة الصحة، العراق** الخالصة االعراض الجانبية بين الجنسين محل جدل لدى الدارسين لسنوات عديدة. هذه الفرضية قد تولدت لطالما كانت إحتمالية وجود إختالفات في ة بسبب وجود اختالفات متعددة كالهورمونية او السلوكية و غيرها. و لم يتم الكشف عن هذا التباين بشكل واضح بسبب عدم إشراك اإلناث بصور دوية. إن الغاية الرئيسية من هذه الدراسة هو بيان إمكانية وجود إختالفات محتملة بين الجنسين خاصة كافية أثناء التجارب السريرية السابقة على اال لهدف الثانوي باألعراض الجانبية لألدوية متمثلة في إختالفات في قياس السببية والشدة و إمكانية الوقاية و الجدية و توقع الحصول و النتيجة، أما ا لدوائية و ألي األعراض الجانبية يعود اإلختالف بين كال الجنسين. هذه الدراسة تمت بأثر رجعي على بيانات منتقاة بصورة فهو تقييم ألي المجاميع ا سنوات للفترة الزمنية من األول من كانون 3خاصة من قاعدة بيانات مركز اليقظة الدوائي العراقي. و قد شملت التقارير المرسلة الى المركز خالل . خالل دراسة العينة البحثية تمت مالحظة أن التقارير المرسلة لحاالت من 2019ولغاية الحادي والثالثين من كانون األول لعام 2017م الثاني لعا % فقط. باإلضافة الى ذلك، وجدت إختالفات كبيرة في 39.16% من المجموع الكلي للعينة بينما َشَكلت تقارير الرجال 60.84اإلناث كانت تشكل ل زيع الفئات العمرية وكانت نسبة النساء البالغات في سن الخصوبة إحصائيا هي األكثر عرضة لإلصابة باألعراض الجانبية، بينما كان الرجاتو جنس اآلخر. ن الالبالغين من األعمار األكبرسناً هم األكثر عرضة للمعاناة من األعراض الجانبية فيما إذا ما قورنت النسبة بالفئة العمرية المماثلة م % في 22.6% في اإلناث و 26.4 هذا وقد كانت األعراض الجانبية الواقعه ضمن فئة الجلد و إضطرابات أنسجة تحت الجلد هي األعلى و بنسبة الحيوية الذكور مع وجود إختالف ذو داللة إحصائية بين هذه النسب. في حين أن أعلى مجموعة دوائية تسبيباً لألعراض الجانبية هي المضادات مقارنة %40.8المخصصة لالستخدام الجهازي العام مع وجود فرصة أكبر "ذات داللة إحصائية" لدى اإلناث لحصول األعراض من هذه األدوية حين % في 41.4% مقارنتا بالذكور 45.4%. و كان تواتر األعراض الجانبية الجدية او الخطيرة أكثر إنتشارا بين االناث 35.5بجنس الذكور %. وقد بين تحليل التوقعات أنها كانت متساويا تقريبا بالنسبة لكل 0.2% بالمقارنة مع اإلناث 0.8أن النتيجة المميتة كانت أكثر إنتشارا لدى الذكور من الجنسين. ختالفات الجنسين ، العراق.إ ، العراقي الدوائية اليقظة مركز ، الضارة الدوائية التفاعالت ، الدوائية اليقظةالكلمات المفتاحية: 1corresponding author e-mail: pharmacologicaleffect@gmail.com received: 4/5/2021 accepted: 11/7 /2021 published online first: 2021-12-12 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp249-260 iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 250 introduction from the very early beginning of the human history, human noticed that there were undesirable effects accompanied the benefits while using medications. many events such as the tragic disaster of thalidomide urged for the development of a well-defined and organized system to monitor drug safety and detect any possible harm to ensure that similar events will never be repeated in the future(1), therefore pharmacovigilance science was developed. pharmacovigilance (pv) is “the science and activities relating to the detection, assessment, understanding and prevention of adverse effects or any other drug-related problem” as defined by the world health organization (who)(1). adverse drug reaction (adr) is defined by the later as “any noxious or unintended response to a drug, which occurs at doses normally used in man for prophylaxis, diagnosis or therapy of disease, or for modification of physiological function”(2). the spontaneous reporting system which depends on the physician, pharmacist, any health care provider or any other person is the greatest source of newly defined adrs in recent years. the reasons behind its importance are; its immediate availability after the drug is released to the market(2) and its great ability of detecting rare adrs that may be missed during the clinical trials(3). this system has its limitations such as the under reporting by health care providers and the high percentage of false positive reports in addition to the low quality of some information reported(2). other sources of information includes clinical studies, observational studies and the randomized controlled trials(2). vigiflow which represents the who international database of the adrs reported to the uppsala monitoring center by the means of spontaneous reports or so called the individual case safety reports (icsr) is considered a rich mine of raw information that could be processed in countless ways to obtain various important information(4). in iraq, there are very small number of studies that make use of the vigiflow database belonging to the iraqi pharmacovigilance center (iphvc) that could enrich the iraqi individuals in addition to the world knowledge about adrs. many studies have found that female gender suffers more adrs than male gender without any distinct clear reasons(5–8). these variances may be related to hormonal, pharmacodynamics and pharmacokinetic reasons(7,9–11) or may be simply because female gender is consuming more drugs and different medication groups than those used by males (7,9,12–14) or even due to behavioral causes(6,15). the pharmacovigilance parameters and demographic patient’s characteristics inspection may give a better idea about adrs differences between male and female. the primary aim of this study was to assess the extent of possibly relevant sex differences in drug–adrs reported to the iraqi pharmacovigilance center regarding causality, severity, preventability, seriousness, outcome and expectedness. while the secondary aim was to assess for which drugs and for which adrs sex differences are identified most often. subjects and method this is a pharmacovigilance retrospective study that deals with the data obtained from the online world health organization-uppsala monitoring center (who-umc) database known as (vigiflow) which contain individual case safety reports (icsrs). only icsrs that belong to the iraqi pharmacovigilance center (iphvc) were accessed after taking the required legal permissions. reports in the period of three years (from the 1st of january 2017 to the 31st of december 2019) concerning adult patients (≥ 18 years) -which were found to be (7080) reportswere collected and analyzed. general exclusion criteria that applied were: reports that not specify the patient’s gender (217 reports), reports in which the reporter didn’t describe if the adr was serious or not (311 reports) and reports that didn’t mention the action taken to deal with the reported adr (2487 reports). additional specific exclusions were done manually while processing each icsr separately which were: duplicated reports (32 reports), gender specific adrs that cannot be compared between both sexes such as vaginal bleeding in female or male impotence (19 reports). gender specific drugs that prescribed for a specific condition related to one gender only such as oral contraceptives (54 reports), reports including vaccines as a suspect of causing the adr (9 reports), reports that did not include the name of the suspect drug (13 reports), reports that didn’t contain the details of the adr, but only mentioned that there was an adr that took place (16 reports), reports about blood and auxiliary products such as plasma and packed rbcs (40 reports), reports about the local reactions that appears after doing the allergy tests (44 reports), reports about the intentional over dose or suicidal attempts (2 reports), 3 reports that were excluded because it contained counterfeit medicine and product quality issues. in addition to that, in icsrs that contain multiple drug suspects or multiple adrs, some of their drug-adr combinations (26 combination) were omitted for missing action taken to deal with a drug suspect or for being a gender specific adr. the resulted study sample was consisted of (3833) reports that have (3972) drugs as suspects, (6153) adrs and (6407) drug-adr combinations to be processed and statistically analyzed. the differences in the total number of icsrs, the total number of suspect drugs and the total number of adrs are due to the fact that in a single icsr, iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 251 there may be more than one suspect or more than one adr. the number of drug-adr combinations is expected to be the largest number among them because in icsrs containing several suspects or adrs, each combination was recorded and analyzed seperatedly. for each icsr that included in this study, patient’s gender was recorded as it is the most important parameter in this study since the study is depending on finding the differences between the two sexes. for the age of patients, it was recorded and grouped into 3 intervals so that it would be easier to compare and study. these age groups were 18-45 years, >4565 years and above 65 years’ intervals representing female reproductive age, post-menopausal age and elderly age groups respectively. adverse drug reactions classification the adrs were classified using the highest level of hierarchy in the medical dictionary for regulatory activities (med dra) which is called the system organ classes (soc) that contain 27 major classes(16). suspected drugs classification the drugs mentioned in the extracted data were classified according to the anatomical therapeutic chemical (atc) classification system, which classify all the medical compounds depending on its different characteristics, it consists of 5 levels. this study concentrates on the 1st level which has fourteen anatomical /pharmacological groups (17). for the drug combinations that were suspected to cause an adr, it was recorded and dealt with as a single suspect and it was left without any distinct atc code, but referred to as ‘combination’ while doing the drug suspect calculations. causality assessment using the who-umc algorithm (18), a causality assessment was done to predict the certainty of association between the administration of a suspected drug in a specific report and the adr that took place. (table 1) illustrate the descriptions of each level in the used causality assessment. for this criteria, drug-adr combinations were compared between both genders. table 1. the who-umc criteria for causality assessment (18) causality term assessment criteria certain event or laboratory test abnormality with plausible time relation to exposure _ cannot be explained by diseases or other drugs _ response to withdrawal plausible (pharmacologically, pathologically) _ event definitive pharmacologically or phenomenological. _ challenge causes definite recurrence. probable/likely _ event or laboratory test abnormality with reasonable time relation to exposure _ unlikely to be explained by diseases or other drugs _ response to withdrawal clinically reasonable _ rechallenge not required or possible. possible _ event or laboratory test abnormality with reasonable time relation to exposure _ could also be explained by diseases or other drugs _ information on withdrawal may be lacking or unclear. unlikely _ event or laboratory test abnormality with time relation to exposure that makes an association improbable (but not impossible) _ diseases or other drugs provide plausible explanations. conditional/ unclassified _ event or laboratory test abnormality _ more data for proper assessment needed, or _ additional data being examined. unassessable/ unclassifiable _ report suggesting an adverse reaction _ cannot be judged because information is insufficient or contradictory _ data cannot be supplemented or verified. severity the modified hartwig and seigel criteria were used to assess the severity of the drug-adr combinations by categorizing them into 7 ascending levels starting from (level 1) which is mild and requires no interpretations to (level 7) that describes a lethal adr (19). these levels are explained clearly in (table 2). iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 252 table 2，hartwig’s severity assessment scale(19) level of severity the criteria level 1 an adr occurred but required no change in treatment with the suspected drug level 2 the adr required that treatment with the suspected drug be held, discontinued, or otherwise changed. no antidote or other treatment requirement was required. no increase in length of stay (los) level 3 the adr required that treatment with the suspected drug be held, discontinued, or otherwise changed. and/ or an antidote or other treatment was required. no increase in los level 4 any level 3 adr which increases length of stay by at least 1 day. or the adr was the reason for the admission level 5 any level 4 adr which requires intensive medical care level 6 the adverse reaction caused permanent harm to the patient level 7 the adverse reaction either directly or indirectly led to the death of the patient 2 expectedness it was evaluated by reviewing the summery of product characteristics (smpc or spc). drug-adr combinations were categorized into two groups: either ‘expected’ if the adr is mentioned previously or ‘unexpected’ if it is not recorded previously in the smpc(20). preventability: for evaluation of preventability, the modified schumock and thornton criteria were applied to each drug-adr combination, the criteria depend on answering 7 questions that discuss several points about the drug safety and conditions of administration with a simple answer of yes or no(21). if any of these questions was answered with ‘yes’, the adr would be considered to be preventable. while if all the 7 answers were ‘no’ it will be recorded as a non-preventable adr. in case of having any questions with unclear answer while assessing an icsr, this will be labeled as a possibly preventable adr (table 2-3). table 3．schumock and thornton preventability assessment criteria(21) question yes no 1 was there a history of allergy or previous reaction to the drug? 2 was the drug involved inappropriate for patient's clinical condition? 3 was the dose, route, or frequency of administration inappropriate for the patient's age, weight or disease? 4 was there any required therapeutic drug monitoring, or other laboratory tests not performed? 5 was a drug interaction involved in the adr? 6 was poor compliance involved in the adr? 7 was a toxic serum concentration or a laboratory? monitoring test documented? seriousness the seriousness was covered depending on the icsr seriousness assessment method, which is adopted by the iraqi pharmacovigilance center (figure 1)(3). relying on the judgment of the person that made the spontaneous report, the adr that took place was categorized as serious or not. iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 253 iii. management of adverse drug reaction: drug (s) discontinued [ ] yes [ ] no improvement on discontinuation [ ] yes [ ] no hospitalization (following the adr) [ ] yes [ ] no [ ] already hospitalized do you consider the reaction to be serious? [ ] yes [ ] no if yes, please tick (  ) to indicate why the reaction is considered to be serious: [ ] patient died due to the reaction [ ] involved or prolonged in patient hospitalization [ ] life threatening [ ] involved persistent or significant disability of incapacity [ ] congenital anomaly [ ] medically significant, please give details:-------------------------------------------------------- treatment given: [ ] no [ ] yes, please specify ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- figure 1.seriousness assessment in the iraqi icsr (3) action taken information regarding the action taken found in the original iraqi icsr (figure 1) was very important in this study for the identification of the severity level that previously discussed in this paper. outcome: it can be found on the original iraqi icsr on (section ii) as shown in (figure 2) this section will tell the reader if the adr happened recovered or not, and if the outcome was unknown or fatal. the comparison of this property can give an idea of which gender has better odds in its adr experience. ethical approval this study has been approved by the scientific committee of the university of baghdad/ college of pharmacy and the iraqi ministry of health (moh)/ department of research and development before it was started. ii. details of advese drug reaction (adr) onset date: ----/----/--- outcome: [ ] recovered (date): --------- [ ] not recovered yet dd/mm/yy [ ] fatal (date of death ) ------[ ] unknown end date: ----/----/---- dd/mm/yy duration: ----------------------min, hour, day, week, month, year figure 2. the outcome recording in the iraqi icsr (3) statistical analysis all the data were tabulated and organized using microsoft excel 2016. ibm statistical package for the social sciences (spss) program version 26 was used in the statistical calculations. descriptive statistics such as counting the percentages and frequencies were done. chi-square test was applied to compare between both genders and search for any significance. yate’s chi square test was adopted in case of having small values that is below 5 to compare. p values which is less than 0.05 was considered to be statistically significant. results from the total of 3833 icsrs, females had 2332 reports (60.84%) while males had 1501 (39.16%). age group distribution demonstrated in (table 1) shows that for younger adults that having an age in the array of (18-45 years), the possibility of having an adr in the female (63.6 %) is more than that of the male gender (55.5 %) of the total specific gender population, and this was found to be statistically significant with a p value of (0.0000005). while for the older age group of (>45 65 years) the chances of males to have an adr is more (30.8 %) compared to (25.9 %) in females, the same was found in the next age group that contains adults of (> 65 years) which shows greater chance in male (13.7 %) than female (10.5 %) with a p value of (0.001 and 0.002) respectively. iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 254 table 1. age group distribution of adrs age group (years) number and percentage of icsr freq. and percentages in female freq. and percentages in male gender difference (p value) 18-45 2316 (60.4 %) 1483 (63.6 %) 833 (55.5 %) 0.0000005* >45-65 1068 (27.9 %) 605 (25.9 %) 463 (30.8 %) 0.001* > 65 449 (11.7 %) 244 (10.5 %) 205 (13.7 %) 0.002* total 3833 (100.0 %) 2332 (100.0 %) 1501 (100.0 %) *significant (p-value <0.05) according to chi square test. adrs: adverse drug reactions, icsr: individual case safety report comparing the adr classes’ distribution between both genders depending on the soc system classification shown in (table 2) gave the picture that the highest type of adrs for both genders were those that fall in the skin and subcutaneous tissue disorders (26.4 % in females) and (22.6 % in males) with a statically significant difference between the two genders. the second one was found to be the gastrointestinal disorders but without any statistical significant difference. respiratory, thoracic and mediastinal disorders came in the third place with (11.9 %) in females and (10.1 %) in males which make it a statistically significant difference (p value 0.037). the group of disorders that came in the fourth stage is those that fall under the general disorders with the following percentages for female and male (10.5 % and 11.2%) respectively, with no significant differences. the nervous system adrs were having statistically significant differences (p value 0.02), with higher values for the male gender (10.3 %) while females were (8.6 %). other classes had less percentages and no statistically significant differences between both genders. a statistically significant gender differences were found (table 3), while testing which medication group -by using the atc classificationis suspected to be responsible for making the highest number of adrs in both genders. by taking a deeper look, it was found that the highest group to cause adrs was the systemic anti-infective agents with a greater chance ‘statistically significant’ in females to suffer a side effect from this group of medications (40.8 %) compared to male gender (35.5 %), the cardiovascular group was in the second place with a statistically significant difference toward the male gender (13.9 %) more than females (8.5 %). the third was the alimentary tract group with approximately equal chances for both male (9.4 %) and female (9.2 %). the nervous system came in the next place with males having (9.3 %) and females (8.8 %) so relatively equal chances is present. the fifth group that responsible for making high numbers of adrs was the antineoplastic and immune-modulating agents, this group was found to cause adrs in females (9.7 %) more than males (5.8 %) with statistically significant differences (p value 0.00001). another important finding that was recorded in this study is that, males are more prone to have an adr due to a drug-drug interaction with a statistically significant difference (p value 0.03). other groups shown in (table 3) are participating in less extent in the overall percentage of causing adrs and has no statistically significant differences, except for a group that has low participation but a statistically significant difference which was the systemic hormonal agents that was clearly causing more adrs in males (4.1 %) than females (2.9 %) (p value 0.047). iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 255 table 2. distribution of adr according to the soc system in both genders. soc system freq. and percentage of total reports freq. and percentage in female freq. and percentage in male gender differences (p value) blood and lymphatic system disorders 39 (0.6 %) 25 (0.7 %) 14 (0.6 %) 0.769 cardiac disorders 253 (4.1 %) 150 (3.9 %) 103 (4.4 %) 0.396 ear and labyrinth disorders 38 (0.6 %) 20 (0.5 %) 18 (0.8 %) 0.241 eye disorders 118 (1.9 %) 65 (1.7 %) 53 (2.3 %) 0.128 gastrointestinal disorders and hepatobiliary disorders 1251 (20.3 %) 755 (19.8 %) 496 (21.1 %) 0.229 general disorders and administration site conditions 663 (10.8 %) 400 (10.5 %) 263 (11.2 %) 0.402 immune system disorders 246 (4.0 %) 155 (4.1 %) 91 (3.9 %) 0.696 infections and infestations 55 (0.9 %) 34 (0.9 %) 21 (0.9 %) 1.000 injury, poisoning and procedural complications 34 (0.6 %) 17 (0.4 %) 17 (0.7 %) 0.154 investigations 89 (1.4 %) 53 (1.4 %) 36 (1.5 %) 0.656 metabolism and nutrition disorders and endocrine disorders 116 (1.8 %) 72 (1.9 %) 44 (1.9 %) 0.956 musculoskeletal and connective tissue disorders 103 (1.7 %) 62 (1.6 %) 41 (1.7 %) 0.731 neoplasms benign, malignant and unspecified and congenital, familial and genetic disorders 10 (0.2 %) 5 (0.2 %) 5 (0.2 %) 0.441 nervous system disorders 568 (9.2 %) 326 (8.6 %) 242 (10.3 %) 0.022* psychiatric disorders and social circumstances 92 (1.5 %) 61 (1.6 %) 32 (1.3 %) 0.372 renal and urinary disorders and reproductive system and breast disorders 49 (0.8 %) 29 (0.8 %) 20 (0.8 %) 0.702 respiratory, thoracic and mediastinal disorders 689 (11.2 %) 451 (11.9 %) 238 (10.1 %) 0.037* skin and subcutaneous tissue disorders 1537 (25.0 %) 1005 (26.4 %) 532 (22.6 %) 0.00089* vascular disorders 203 (3.3 %) 119 (3.1 %) 84 (3.6 %) 0.339 total 6153 (100.0 %) 3804 (100.0 %) 2349 (100.0 %) adrs: adverse drug reactions, soc: system organ classes. *significant (p-value <0.05) according to chi square test. iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 256 table 3. drug atc group in both gender atc group frequencies and percentage freq. and percentage in female freq. and percentage in male gender difference (p value) a 368 (9.3 %) 222 (9.2 %) 146 (9.4 %) 0.853 b 292 (7.4 %) 173 (7.2 5%) 119 (7.6 %) 0.578 c 423 (10.6 %) 206 (8.5 %) 217 (13.9 %) 0.00000007* comb. † 33 (0.8 %) 14 (0.6 %) 19 (1.2 %) 0.030* d 35 (0.9 %) 24 (1.0 %) 11 (0.7 %) 0.342 g 24 (0.6 %) 15 (0.6 %) 9 (0.6 %) 0.862 h 135 (3.4 %) 71 (2.9 %) 64 (4.1 %) 0.047* j 1537 (38.7 %) 984 (40.8 %) 553 (35.5 %) 0.00087* l 324 (8.2 %) 234 (9.7 %) 90 (5.8 %) 0.00001* m 264 (6.6 %) 158 (6.5 %) 106 (6.8 %) 0.749 n 358 (9.0 %) 213 (8.8 %) 145 (9.3 %) 0.603 p 5 (0.1 %) 1 (0.0 %) 4 (0.3 %) 0.153‡ r 137 (3.4 %) 79 (3.3 %) 58 (3.7 %) 0.447 s 23 (0.6 %) 12 (0.5 %) 11 (0.7 %) 0.396 v 14 (0.4 %) 8 (0.3 %) 6 (0.4 %) 0.780 total 3972 (100.0 %) 2414 (100.0 %) 1558 (100.0 %) atc; anatomical therapeutic chemical. a; alimentary tract and metabolism. b; blood and blood forming organ. c; cardiovascular system. d; dermatological agents. g; genitourinary system and sex hormones. h; systemic hormonal preparations. j; anti-infective for systemic use. l; antineoplastic and immune modulating agents. m; musclo-skeletal system. n; nervous system. p; antiparasitic agents, insecticides and repellants. r; respiratory system. s; sensory organs. v; various. * significant (p-value <0.05) according to chi square test. † comb. refers to the adrs caused by drug combinations (drug-drug interactions) ‡ yates’ chi square test was adopted because of the small values (below 5). when comparing the seriousness of adrs between both genders (table 4), a statistically significant difference (p value 0.001) was found which indicate that the reported adrs were considered to be more serious in female (45.4 %) than for males (41.3 %) according to the initial reporter judgment. table 4. seriousness of adr in both gender seriousness freq. and percentage in total icsr female frequency and percentage male frequency and percentage gender difference (p value) no 3456 (56.2 %) 2076 (54.6 %) 1380 ( 58.7 %) 0.001* yes 2697 (43.8 %) 1728 (45.4 %) 969 (41.3 %) total 6153 (100.0 %) 3804 (100.0 %) 2349 (100.0 %) * significant (p-value <0.05) according to chi square test. studying the outcome of adrs in both genders shown in (table 5), demonstrates that there were statistically significant differences. the fatal outcome was more observed in male gender (0.8 %) as compared with females (0.2 %). the recovery was more detected in females (77.1 %) than males (74.1 %) while the unknown outcome was recorded more frequently in males (13.2 %) than female (10.4 %). other outcome subgroups that include the recovery with sequelae, adrs that not recovered till the time of reporting and the adrs that still under the recovery phase were without any statistically significance differences. iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 257 table 5. outcome of the adr for each gender. outcome freq. and percentage in total freq. and percentage in female freq. and percentage in male gender difference (p value) fatal 24 (0.4 %) 6 (0.2 %) 18 (0.8 %) 0.00019* not recovered 524 (8.5 %) 324 (8.5 %) 200 (8.5 %) 1.000 recovered 4673 (75.9 %) 2933 (77.1 %) 1740 (74.1 %) 0.006* recovered with sequelae 20 (0.3 %) 14 (0.4 %) 6 (0.3 %) 0.451 recovering 208 (3.4 %) 132 (3.5 %) 76 (3.2 %) 0.620 unknown 704 (11.4 %) 395 (10.4 %) 309 (13.2 %) 0.0009* total 6153 (100 %) 3804 (100 %) 2349 (100 %) * significant (p-value <0.05) according to chi square test.. the causality assessment (table 6) showed that the major category found in the study sample was the probable which counted for (66.1 %) of the total number of drug-adr combinations followed by the possible category (28 %). there were significant differences in the probable and the possible categories only. females adrs, according to these findings, are more obvious and related easier to the suspect drug because the probable category has higher percentage in the female gender (68.5 %) compared with male gender (62.2 %), while the possible category has higher male findings (31.4 %) compared with the females (25.8 %). for other categories, values were closely the same without any statistically significant differences. table 6. causality assessment of adr. causality by who method freq. and percentage in total reports female frequency and percentage male frequency and percentage gender difference (p value) certain 15 (0.2 %) 9 (0.2 %) 6 (0.2 %) 0.882 conditional 265 (4.1 %) 161 (4.1 %) 104 (4.3 %) 0.710 possible 1791 (28.0 %) 1023 (25.8 %) 768 (31.4 %) 0.0000012* probable 4234 (66.1 %) 2714 (68.5 %) 1520 (62.2 %) 0.0000002* unlikely 102 (1.6 %) 55 (1.4 %) 47 (1.9 %) 0.097 total 6407 (100 %) 3962 (100 %) 2445 (100 %) *significant (p-value <0.05) according to chi square test. the evaluation of severity criteria shows that most of the reported adrs were considered to be from level 2 of the hartwig’s severity assessment scale which represents adrs that needs only to discontinue administrating of the suspect agent as a management to the harm ensued. there were statistically significant differences between both genders in two levels of severity only. these were the third level which refers to the adrs that need to stop giving suspect drugs in addition to the use of a drug or an antidote for treating the resulted harm, this level was significantly higher in female gender (9.2 %) compared to male (7.2 %). the other level that also showed a statistically significant difference was the seventh level, adrs that cause death of the patient directly or indirectly, which was found to be higher in male gender (0.8 %) than female (0.2 %) (table 7). iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 258 table 7. severity of adr in both gender severity level freq. and percentage in all icsr female freq. and percentage male freq. and percentage gender difference (p value) level 1 791 (12.3 %) 474 (12.0 %) 317 (13.0 %) 0.236 level 2 4211 (65.7 %) 2600 (65.6 %) 1611 (65.9 %) 0.826 level 3 540 (8.4 %) 364 (9.2 %) 176 (7.2 %) 0.0053* level 4 785 (12.3 %) 481 (12.1 %) 304 (12.4 %) 0.727 level 5 40 (0.6 %) 26 (0.7 %) 14 (0.6 %) 0.680 level 6 12 (0.2 %) 9 (0.2 %) 3 (0.1 %) 0.520 ‡ level 7 28 (0.4 %) 8 (0.2 %) 20 (0.8 %) 0.0002* total 6407 (100 %) 3962 (100 %) 2445 (100 %) *significant (p-value <0.05) according to chi square test. ‡ yates’ chi square test was adopted because of the small values (below 5). females and males had no statistically significant differences in the preventability patterns of adrs in both possibly preventable and preventable adrs, while the non-preventable subgroup were totally not found in the study population because some of the important information needed to mark the cases as a non-preventable adr were not recorded in the icsrs. the resulted findings are recorded in (table 8). table 8. preventability of adr in both sex. preventability of adr total report freq. and percentage female freq. and percentage male freq. and percentage gender difference (p value) possibly preventable 6170 (96.3 %) 3823 (96.5 %) 2347 (96.0 %) 0.303 preventable 237 (3.7 %) 139 (3.5 %) 98 (4.0 %) total 6407 (100 %) 3962 (100 %) 2445 (100 %) the expectedness analysis showed in (table 9) gave the finding that for each gender the expectedness of adrs were nearly equal. table 9: expectedness of adr for each gender expected adr freq. and percentage in total freq. and percentage in female freq. and percentage in male gender difference (p value) no 2070 (32.3 %) 1277 (32.2 %) 793 (32.4 %) 0.867 yes 4337 (67.7 %) 2685 (67.8 %) 1652 (67.6 %) total 6407 (100 %) 3962 (100 %) 2445 (100 %) discussion this study revealed the characteristics of the reported adrs to the iphvc. some of the obtained results were nearly similar to previous readings from different countries and the other findings were unique in the studied population. the predominance of female gender in the total number of icsrs is an expected result that resembles many previous studies(6,15,22,23). this is a multifactorial fact with no clear explanation, that may be due to pharmacological, biological, social and behavioral differences between both genders(6–8). during their reproductive age, females are more susceptible to have adrs than males from the same age group. this finding cannot be explained by female use of extra -gender specificdrugs such as oral contraceptives since cases containing contraceptive suspects were excluded from the study sample, neither could be related to the total number of population of each gender since it is approximately equal in our country according to official publications(24). this finding is similar to many previous studies such as; the sex related vigibase global data analysis of the last 50 years(23) and the tran et al work which analyzed 10 years data collected from “glaxo wellcome-sunnybrook drug safety clinic” records in canada searching for gender differences(6). this age group variation could be due to the fact that females during their reproductive age seek more medical attention and had twice more doctor visits than males and are more likely to report any medical concerns and look for medical advice (11,23). while for older age groups (>45-65 years) and (> 65 years), the male gender was recorded to have more adrs than females. this could be because males were found to have greater hospital stays in the ages above 44 years which might mean more medications and complications (11). the biggest group of drugs that found to cause iraqi j pharm sci, vol.30(2) 2021 gender differences in adverse drug reactions 259 adrs were antibiotics for systemic use and it was higher in the females. a possible explanation of this finding is the increasing number of cesarean section operations in iraq in the last years(25) which has led to the increasing use of antibiotics for prophylactic reasons. while the cardiovascular system agents were higher in the male gender. this could be reasonable if taking in consideration that; males are diagnosed with cardiovascular diseases more often than females(26), and the prevalence of both stroke and heart disease is higher in men worldwide(27). this will result in more male gender related consumption of cardiovascular agents(28) and as a result, more adrs will appear and be reported for those agents. the observed differences in the soc systems of adrs distribution in both genders could be explained by the presence of variances in the used medications by each gender (28) which has led to the appearance of different sorts of adrs. in the current study, the frequency of serious adrs was significantly more prevalent in females than for males (table 4), these results contradict the global analysis of the vigiflow database which found that males had more serious adrs than females (23). also, it was found that females were taking treatments for the adrs more than males and males had a higher mortality due to adrs than females. these findings were similar to the global review of vigiflow database(23). the higher mortality in male gender could be explained by three reasons identified here which are: males not tend to seek medical help early during their adr experience as women do (23), they tend to use different classes of medications (28) in addition to that, they suffer from adrs in older age groups (table 1). the males had less clear adrs outcome since more females are reported to be recovered from their adrs and more males had unknown outcome (table 5), also the causality assessment for the males are less linked to the use of medications than the female gender (table 6). the causality assessment may explain, for some extent, the outcome results of the current study where the adrs are more clearly linked to medications used by females so it is easier to manage these reactions either by giving treatment or by stop giving the causative drugs while in male gender the causality is less obvious. for the expectedness and the preventability, there were no differences between both genders. although the current study has a large sample size over a relatively extended period of time, it has several limitations such as its dependence on the spontaneous reports only which means a lot of important adrs that occur in practice and passed without reporting were not identified here. the study is of a retrospective type that restricted by the available information only which caused the exclusion of numerous reports missing essential data and that may affect the study statistical results. also, the available reported data did not have the same quality as they are disclosed by different sources and qualifications which may lead to bias. conclusions and recommendations in conclusion, the current study showed possibly relevant differences between males and females in the adrs spontaneously reported to the iraqi pharmacovigilance center indicating that gender may be a risk factor for (development of adrs for some class of drugs, some types of adrs, seriousness of adrs, as well as the outcome of these adrs). further work is required to elucidate the mechanisms explaining the differences observed between male and female patients. in addition, more efforts 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[cited 2021 jun 6]. available from: https://population.un.org/wpp/download/stand ard/population/ 25. shabila np. rates and trends in cesarean sections between 2008 and 2012 in iraq. bmc pregnancy childbirth [internet]. 2017;17(1):4– 9. available from: http://dx.doi.org/10.1186/s12884-016-1211-6 26. pilote l, dasgupta k, guru v, humphries kh, mcgrath j, norris c, et al. a comprehensive view of sex-specific issues related to cardiovascular disease. cmaj. 2007;176(6). 27. bots sh, groepenhoff f, eikendal alm, tannenbaum c, rochon pa, regitz-zagrosek v, et al. adverse drug reactions to guidelinerecommended heart failure drugs in women: a systematic review of the literature. jacc hear fail. 2019;7(3):258–66. 28. putignano d, bruzzese d, orlando v, fiorentino d, tettamanti a, menditto e. differences in drug use between men and women: an italian cross sectional study. bmc womens health. 2017;17(1):1–7. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 13 evaluation of the clinical use of metformin or pioglitazone in combination with meloxicam in patients with knee osteoarthritis; using knee injury and osteoarthritis outcome score mohammed m. mohammed *,1 , kassim j. al-shamma ** and nizar a. jassim *** * department of clinical pharmacy, college of pharmacy, university of al-mustansiryah, baghdad, iraq. ** department of therapeutics and clinical pharmaceutics, baghdad college of pharmacy, baghdad, iraq. ***department of rheumatology, college of medicine, university of baghdad, baghdad, iraq. abstract osteoarthritis is the most prevalent arthritic disease and a leading cause of disability. the pathogenesis of osteoarthritis involves multiple etiologies, including variable degree of synovial inflammation. metformin and pioglitazone could potentially reduce the levels and activity of inflammatory mediators. this may consider as a new therapeutic approach added to the current used drugs in an attempt to decrease the pain, inflammation, and improve daily activity and quality of life in patients with knee osteoarthritis. this study designed to evaluate the clinical utility of using metformin or pioglitazone as antiinflammatory agents in combination with non-steroidal anti-inflammatory drugs (nsaid) of selective type of cyclooxygenase-2 (cox-2) inhibitor, meloxicam, in the treatment of knee osteoarthritis (oa). randomized, double blinded clinical study was performed on 98 patients who have symptomatic and radiologic evidence of painful oa of the knee (57 patients only completed the study). patients were allocated into three groups, group (a); 20 patients treated with meloxicam (15mg/day) alone, group (b); 20 patients treated with metformin (1000mg/day) + meloxicam (15mg/day) and group (c); 17 patients treated with pioglitazone (15mg/day) + meloxicam (15mg/day). the treatment was followed for 12 weeks through measurement of the clinical effects of drugs each 7 days, using the knee injury and osteoarthritis outcome score (koos) system. the results showed that metformin or pioglitazone, when used in combination with nsaid resulted in significant improvement in the components of koos, higher than that produced by meloxicam when used alone. in conclusion, administration of metformin or pioglitazone as adjuvant therapy to nsaid, meloxicam, in oa patients produced very well characterized analgesic and antiinflammatory activities, and improves the therapeutic profile of meloxicam. keywords: metformin, pioglitazone, osteoarthritis, knee injury, koos. ة للوتفىرهٍي او الباٌىكلٍتازوى كعالج هساًذ للوٍلىكسٍكام فً الورضى تقٍٍن الفعالٍت السرٌري الوصابٍي بالتهاب الركبت غٍر الرثىي، باستخذام ًظام كىز لتقٍٍن حالت الورٌض هحوذ هحوىد هحوذ *،1 ، قاسن جلٍل الشواع ** و ًسار عبذ اللطٍف جاسن *** * .اىؼشاق،تغذاد اىَسرْصشٝح،ظاٍؼح ،ميٞح اىصٞذىح اىصٞذىح اىسشٝشٝح ،فشع ** .اىؼشاق ،تغذاد ، صٞذىحتغذاد ىوميٞح ، اىؼالظاخ ٗاىصٞذىح اىسشٝشٝحفشع *** .ميٞح اىطة ، ظاٍؼح تغذاد ، تغذاد ،اىؼشاق الخالصة ٍشض اىرٖاب اىَفاصو غٞش اىشش٘ٛ ٕ٘ اىَشض االمصش اّرشاسا تِٞ اٍشاض اىَفاصو ٗاىسثة اىشئٞسٜ فٜ ظؼو اىَشٝط غٞش الٝ٘ظذ حٞس, ٍرفاٗذحاه ٓذٖاب اىضىٞيٜ فٜ اىَفاصو تذسظاخهاال ٍِ ظَْٖاأسثاب اىَشض ٍرؼذدج , قادس ػيٚ اىحشمح تص٘سج غثٞؼٞح فٜ اىَقاً االٗه اىٚ اىحذ ٍِ ذٖذف االسرشاذٞعٞاخ اىؼالظٞح اىحاىٞح ىزىل ّعذ اُ, ٍشظٚ اىرٖاب اىَفاصو غٞش اىشش٘ٛه شافٜػالض ػقاس مال ٍِ فاػيٞحاىؼذٝذ ٍِ اىذساساخ اىَصََح ػيٚ اىَْارض اىحٞ٘اّٞح اٗ ػيٚ االّساُ اىٚ أشاسخ .األىٌ ٗذحسِٞ ٗظٞفح اىَفصو ا ّٖعا ػالظٞا ظذٝذا ٝعاف اىٚ ٍعاداخ ًٍَا ٝعؼئ, ٗسطاء االىرٖاب فٜ اىحذ ٍِ ٍسر٘ٝاخ ّٗشاغ صُٗاىَرف٘سٍِٞ ٗػقاس اىثاٝ٘ميٞرا االىرٖاب غٞش اىسرٞشٗٝذٝح اىَسرخذٍح حاىٞا فٜ ٍحاٗىح ىيرقيٞو ٍِ االىٌ ٗاالىرٖاب ٗذحسِٞ اىفؼاىٞح اىٍٞ٘ٞح ّٗ٘ػٞح حٞاج اىَشظٚ اسرخذاً ػقاس ػِ ظَحاىْا ٕزٓ اىذساسح ىرقٌٞٞ اىفائذج اىسشٝشٝح خصٌَ ٙ رىلاء ػوٗتِ. اىَصاتِٞ تاىرٖاب ٍفصو اىشمثح غٞش اىشش٘ٛ فٜ ػالض , اىَٞي٘مسٞناًك -2-اىَصثطح الّضٌٝ االمسذج اىحيقٜ اىسرٞشٗٝذٝح ٍعاداخ االىرٖاب غٞشاحذ ٍغاىثاٝ٘ميٞراصُٗ اىَرف٘سٍِٞ اٗ . اىرٖاب ٍفصو اىشمثح غٞش اىشش٘ٛ أمَو )ٍشٝعا̿ ٍَِ ىذٌٖٝ أػشاض ٗأدىح سشٝشٝح ػيٚ األصاتح تاىرٖاب ٍفصو اىشمثح اىَسثة ىألىٌ 98أظشٝد ٕزٓ اىذساسح ػيٚ ذٌ ػالظٌٖ تؼقاس اىَٞي٘مسٞناً (ٍشٝط 20) -أ-اىَعَ٘ػح : ٗذٌ ذقسٌٞ ٕؤالء اىَشظٚ اىٚ شالز ٍعاٍٞغ(. ٍشٝعا̿ فقػ 57اىذساسح 500)ٳظافح اىٚ ػقاس اىَرف٘سٍِٞ ( ٍيغٌ ٍٝ٘ٞا 15)قاس اىَٞي٘مسٞناً ذٌ ػالظٌٖ تغ( ٍشٝط 20) -ب–اىَعَ٘ػح , (ٍيغٌ ٍٝ٘ٞا 15) ٳظافح اىٚ ػقاس اىثاٝ٘ميٞراصُٗ ( ٍيغٌ ٍٝ٘ٞا 15)ذٌ ػالظٌٖ تؼقاس اىَٞي٘مسٞناً ( ٍشٝط 17) -ض–اىَعَ٘ػح , (ساػح 12ٍيغٌ مو (. ٍيغٌ ٍٝ٘ٞا 15) 1 corresponding author e-mail: phd_pharm@yahoo.com received: 11/5/2014 accepted: 10/6/2014 iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 14 .koosٍشاقثح اىَشظٚ ٗذقٌٞٞ فؼاىٞح االدٗٝح اىَسرخذٍح ٍِ خاله فحص االسرعاتح اىسشٝشٝح ىيؼالض اسث٘ػٞا̿ تاسرخذاً ّظاً ٗذٌ فٜ ٍؼْ٘ٛ ػقاس اىَٞي٘مسٞناً ٝؤدٛ اىٚ ذحسِ ٍغأظٖشخ ّرائط اىذساسح تأُ اسرخذاً ػقاس اىَرف٘سٍِٞ اٗ اىثاٝ٘ميٞراصُٗ . ذيل اىرٜ ٝسثثٖا ػقاس اىَٞي٘مسٞناً ى٘حذٓب ٍقاسّح koosٍسر٘ٙ اىَؼاٝٞش اىخاصح تْظاً ػقاس اىَرف٘سٍِٞ أٗ اىثاٝ٘ميٞراصُٗ ىؼقاس اىَٞي٘مسٞناً ٝحذز ذأشٞشا̿ ٗاظحا̿ فٜ اىرقيٞو ٍِ ٍسر٘ٙ ظافَٔٝنِ االسرْراض تأُ ا مَا . ض اىَصاحثح ىٔ مَا ٝحسِ اىفؼاىٞح اىٍٞ٘ٞح ّٗ٘ػٞح حٞاج اىَشٝط ٍِ خاله اىرأشٞش اىَعاد ىالىرٖاب ٗاىَسنِ ىألىٌاالىٌ ٗاالػشا .اىؼالظٞحٝسٌٖ فٜ ذحسِٞ فؼاىٞرٖا اُ اسرخذأٍ ٍغ ٍعاداخ االىرٖاتاخ غٞش اىسرٞشٗٝذٝح .الرثىي، اصابت الركبت، ًظام كىز لتقٍٍن حالت الورٌض اسبىعٍاهتفىرهٍي، باٌىكلٍتازوى، التهاب الركبت غٍر :الكلواث الوفتاحٍت introduction osteoarthritis (oa) is a progressive musculoskeletal condition that involves the deterioration of articular cartilage and subsequent subchondral bone erosion (1) . although the exact pathophysiology of the condition has not been uncovered yet, it is generally considered to be caused by a combination of cumulative mechanical stresses from aging, destructive biochemical changes taking place in the synovial membrane, and apoptosis of chondrocytes (2) . clinically, there are several classes of treatments for oa, including nonpharmacological , pharmacological , and surgical treatment modalities. however, these treatments provide largely symptom relief, and do not halt the progression of the disease (3) . non-steroidal anti-inflammatory drugs (nsaids) are the most commonly used medications to treat oa. however, these drugs may elicit adverse effects particularly gastrointestinal ulcerations (4) . moreover, some of these agents have been reported to disrupt extracellular matrix metabolism, particularly proteoglycans synthesis (5) . prolonged consumption of these drugs can result in severe adverse effects. consequently, there is an urgent need for new strategies in oa therapy which can improve symptoms and are safe for clinical use over long periods of time (6) . the ability of metformin and pioglitazone to reduce the intensity of pain and inflammation that contributed to the pathophysiology of oa, with no serious adverse effects, has been reported in many animal model studies (7,8) . this may considered a new therapeutic approach added to the currently used nsaids to improve pain, inflammation, and quality of life in patients with knee oa. it has been shown that metformin can serve as potential drug to treat inflammation-related disorders (8) . the specific anti-inflammatory mechanism of metformin is not clearly understood. however , several studies demonstrated that the pharmacological action of metformin goes beyond mere glycemic control , decreasing markers of inflammation and contributing to the reduction of oxidative stress (9,10) . metformin dose-dependently reduced the production of nitric oxide (no) and prostaglandin e2 (pge2) and suppressed the mrna and protein levels of inducible nitric oxide synthase (inos) and cox-2 in lipopolysaccharidesactivated macrophages (11) . pioglitazone is potent and highly selective agonist for the nuclear receptor peroxisome proliferatoractivated receptors gamma (ppar-γ) and to a lesser extent ppar-α (12) . through ppar-γmediated effects, pioglitazone improve insulin sensitivity and also have pleiotropic effects on insulin secretion, lipid and adipose tissue metabolism, body fat distribution, and vascular endothelial function (13) .the anti-inflammatory effects of ppars are mainly mediated by either inhibiting the induction of proinflammatory cytokines or stimulating the production of anti-inflammatory molecules (14) . many in vitro studies have been shown that ppar-γ is expressed and functionally active in chondrocytes, and those ppar-γ activators modulate the expression of several genes considered essential in the pathogenesis of oa. ppar-γ activation inhibits the il-1induced expression of inos, metalloproteinase-13 (mmp-13), cox-2, and pge2 in chondrocytes (1517) . this study designed to evaluate the clinical utility of using metformin or pioglitazone as anti-inflammatory agents in combination with nsaid of selective type of cox-2 inhibitor, meloxicam, in the treatment of knee osteoarthritis (oa). patients and methods a double blind clinical study was carried out on (98) randomly selected patients (29 males and 69 females) with painful osteoarthritis (oa) of the knee, at the outpatients clinic in baghdad teaching hospital with age range 36-71 years (59.2  7.3). all selected patients have symptomatic and radiological evidence of oa in one or both knee joints. they were informed about the nature and the aim of the study. during selection of patients, certain exclusion criteria iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 15 were followed to exclude unsuitable patients including; 1. patient with hypertension, ischemic heart diseases or diabetes mellitus. 2. patient with hepatic or renal impairment and those who are on treatment with drugs, which interfere with the tested drugs. 3. patients who have active peptic ulcer or damage. 4. patients with end-stage radiological events of joint destruction. 5. patients with positive history of bladder cancer. 6. patients with positive history of allergic reactions to any one of the known tested drugs. 7. patient who misses one time of blood sampling or treatment assessment indicated in this study and/or his medication for any reason. 8. pregnant or lactating female patients. the selected patients were randomly allocated into three groups as follow: group a, includes 32 (11 males and 21 females) patients with negative git risk factors, treated with meloxicam tablets (15mg/day) taken at night for 12 weeks (20 patients only completed the study). group b, includes 36 (9 males and 27 females) patients with negative git risk factors, treated with meloxicam tablets (15mg/day) taken at night and metformin (500mg/12 hours) for 12 weeks (20 patients only completed the study). group c, includes 30 (9 males and 21 females) patients with negative git risk factors, treated with meloxicam tablets (15mg/day) taken at night and pioglitazone (15mg/day) for 12 weeks (17 patients only completed the study). effects of drug treatment were assessed each seven days by clinical evaluation and direct interview with patients through a questionnaire method known as knee injury and osteoarthritis outcome score (koos) (18) . the results were expressed as mean ± sem; paired t-test and anova were used to examine the degree of significance; p values less than 0.05 were considered significantly different. results effect on pain score before enrolment in the study (zero time), oa patients demonstrated poor pain control with their previous therapy, manifested by low pain score which indicate severe or extreme symptoms of pain in most of patients (table 1). treatment with meloxicam alone resulted in significant increase in pain score from the first week (22.11%) compared to pre-treatment value, reaching maximum level at week twelve (88.81%) at the end of the study. however, combination of meloxicam with metformin resulted in significantly higher levels of improvement in the pain score started from the first week of treatment (51%) and remain elevated to the end of the study (170.95%). while addition of pioglitazone to meloxicam resulted in significant increase in pain score started from the first week (30.07%) reaching maximum level at week nine (146.62%), and remain around this level until the last week of the study (twelve weeks) (table 1). the obtained data showed that maximum level of improvement in pain score was gained in patients treated with combination of meloxicam and metformin which was significantly higher than that observed in patients treated with combination of meloxicam and pioglitazone at corresponding durations. however; these two groups demonstrated significant improvement in pain score compared to patients treated with meloxicam alone at corresponding duration. effects on symptom score at zero time (before starting treatment), all selected oa patients showed poor management of oa symptoms, manifested by low score of symptoms according to the outcome of koos (table 2). treatment with meloxicam alone resulted in significant elevation in symptom score compared to pretreatment value from the first week (15.57%) with maximum elevation at the last three weeks of the study (54.52%, 56.01%, and 55.03%). treatment with combination of meloxicam and metformin resulted in significantly higher levels of improvement in the symptom score started from the first week of treatment (21.99%) and maximum score achieved at week eight (113.64%) and remain elevated to the end of the study. however, the combination of pioglitazone and meloxicam resulted in time dependent significant increase in symptom score, reaching maximum level at the last two weeks of the study (87.08%, and 86.52%) relative to pre-treatment value, respectively. parallel improvement in symptoms score were obtained by using combinations of meloxicam with either metformin or pioglitazone through first seven weeks of treatment. from week eight to the end of study; combination of meloxicam with metformin showed significant improvement compared to that of meloxicam and pioglitazone combination at corresponding duration. both of these combination groups showed significantly different improvement in symptom score along the study period compared to patients group of meloxicam alone-treatment (table 2). iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 16 table(1): effects of treatment with meloxicam alone, combination of meloxicam + metformin, and meloxicam + pioglitazone on pain score in osteoarthritic patients. duration (weeks) pain score of koos meloxicam (15 mg/day) no. of patient=20 meloxicam(15mg/day)+ metformin(1000mg/day) no. of patient=20 meloxicam (15mg/day)+ pioglitazone(15mg/day) no. of patient=17 0 32.07  1.08 29.98  1.08 31.53  1.45 1 39.16  1.14 * a 45.27  1.16 * b 41.01  1.13 * a 2 44.16  1.05 * a 52.08  0.94 * b 49.67  0.89 * c 3 47.24  0.92 * a 56.66  1.02 * b 54.08  1.04 * c 4 49.3  0.85 * a 61.38  1.01 * b 58.17  1.27 * c 5 52.08  0.85 * a 66.11  0.87 * b 61.92  0.91 * c 6 52.91  0.84 * a 71.58  0.83 * b 66.99  0.95 * c 7 55.41  0.79 * a 76.65 0.89 * b 74.01  1.04 * c 8 55.83  0.8 * a 79.42  0.76 * b 75.8  0.85 * c 9 57.49  0.83 * a 80.98  0.72 * b 77.76  1.09 * c 10 59.16  0.73 * a 80.81  0.64 * b 77.76  1.01 * c 11 60.41  0.78 * a 81.09  0.77 * b 77.76  0.82 * c 12 60.55  0.85 * a 81.23  0.8 * b 76.78  0.75 * c data are expressed as mean  sem. * p>0.05 significant difference compared to pre-treatment value within the same group. values with non-identical superscripts (a, b & c) among different groups are significantly different (p>0.05) at corresponding duration. iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 17 table (2): effects of treatment with meloxicam alone, combination of meloxicam + metformin, and meloxicam + pioglitazone on symptoms score in osteoarthritic patients. duration (weeks) symptoms score of koos meloxicam (15 mg/day) no. of patient=20 meloxicam(15mg/day)+ metformin(1000mg/day) no. of patient=20 meloxicam (15mg/day)+ pioglitazone(15mg/day) no. of patient=17 0 35.71  1.37 34.1  1.69 37.39  1.37 1 41.27  1.02 * a 41.6  1.01 * a 43.48  0.76 * b 2 44.28  0.91 * a 48.22  0.92 * b 48.1  1.07 * b 3 48.03  0.99 * a 52.32  1.08 * b 52.73  1.13 * b 4 51.07  0.86 * a 56.07  0.74 * b 55.04  0.87 * b 5 51.97  0.71 * a 62.85  0.79 * b 62.6  1.06 * b 6 53.22  0.93 * a 66.23  0.65 * b 66.8  0.96 * b 7 52.5  0.69 * a 69.28  0.75 * b 69.32  0.69 * b 8 52.85  0.88 * a 72.85  0.95 * b 68.69  0.78 * c 9 54.1  0.94 * a 72.14  0.85 * b 68.2  0.92 * c 10 55.18  0.75 * a 70.89  0.83 * b 69.3  0.76 * c 11 55.71  0.79 * a 72.67  0.87 * b 69.95  0.75 * c 12 55.36  0.84 * a 72.49  0.78 * b 69.74  0.54 * c data are expressed as mean  sem. * p>0.05 significant difference compared to pre-treatment value within the same group. values with non-identical superscripts (a, b & c) among different groups are significantly different (p>0.05) at corresponding duration. effects on daily living activity (adl) score in table 3, adl score was found relatively low before starting treatment in all patients (zero time) enrolled in study. in patients group treated with meloxicam, adl score showed significant increase, started after one week of treatment and reaching maximum score at the end of the study (13.98% and 80.82%) compared to pre-treatment value, respectively, with no significant differences among the mean values of last three weeks of the study. these values are significantly lower than those produced by combination treatment of meloxicam with metformin and meloxicam with pioglitazone at the same period of time. addition of metformin to meloxicam resulted in significant improvement of adl score after one week treatment (32.73%) compared to pre-treatment value and reach maximum improvement at the last four weeks of the study. the levels of improvement in adl score produced by treatment with meloxicam and pioglitazonee combination remained significantly higher than baseline from week one (33.78%) to the end of study (106.53%), with maximum level of improvement recorded at week eight (118.58%) as shown in (table 3). treatment with combination of meloxicam and metformin showed higher improvement of adl scores which are significantly different compared to that resulted by treatment with meloxicam alone, and with combination of meloxicam and pioglitazone, particularly at the last five weeks of study. iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 18 table (3): effects of treatment with meloxicam alone, combination of meloxicam + metformin, and meloxicam + pioglitazone on daily living activity (adl) score in osteoarthritic patients. duration (weeks) adl score of koos meloxicam (15 mg/day) no. of patient=20 meloxicam(15mg/day)+ metformin(1000mg/day) no. of patient=20 meloxicam (15mg/day)+ pioglitazone(15mg/day) no. of patient=17 0 34.77  1.72 38.38  1.37 37.36  1.06 1 39.63  0.99 * a 50.94  0.89 * b 49.98  1.04 * b 2 42.35  1.1 * a 57.35  0.93 * b 56.13  1.33 * b 3 45.44  1.12 * a 62.94  0.88 * b 66.17  0.91 * c 4 51.46  0.87 * a 65.88  0.8 * b 70.67  0.86 * c 5 53.22  0.87 * a 75.36  0.99 * b 77.85  0.64 * c 6 54.4  0.93 * a 79.88  0.83 * b 80.45  0.64 * b 7 56.83  0.84 * a 82.19  0.78 * b 81.66  0.78 * b 8 51.82  0.98 * a 84.38  0.73 * b 81.66  0.96 * c 9 60.95  0.66 * a 85.99  0.8 * b 81.35  0.8 * c 10 62.65  0.81 * a 87.46  0.79 * b 78.98  0.84 * c 11 62.57  0.7 * a 86.94  0.72 * b 77.68  0.79 * c 12 62.87  0.8 * a 86.87  0.84 * b 77.16  0.99 * c data are expressed as mean  sem. * p>0.05 significant difference compared to pre-treatment value within the same group. values with non-identical superscripts (a, b and c) among different groups are significantly different (p>0.05) at corresponding duration. effects on sport/recreation score: table 4; revealed low sport/recreation score at zero time levels before starting drug treatment with treated drugs. treatment with meloxicam alone resulted in significant increase in sport/recreation score started after first week (16.19%) compared to pre-treatment value, reaching maximum level (59.05%) at the end of the study (12 weeks). combination of meloxicam with metformin resulted in significantly higher level of improvement in sport/recreation score after the first week of treatment, which found to be comparable to those produced by combination of meloxicam with pioglitazone (28.7%, 17.04%) compared to pre-treatment values, respectively. at the end of the study; significant difference was achieved between the two combination groups, with preference to meloxicam and metformin group over meloxicam and pioglitazone group in improving sport/recreation score (117.39%, 75% compared to pre-treatment value, respectively) (table 3). both of combination groups revealed a significant elevation in sport/recreation score compared to patients group treated with meloxicam alone at the same period of study. iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 19 table (4): effects of treatment with meloxicam alone, combination of meloxicam + metformin and, meloxicam + pioglitazone on sport/recreation score in osteoarthritic patients. duration (weeks) sport / recreation score of koos meloxicam (15 mg/day) no. of patient=20 meloxicam(15mg/day)+ metformin(1000mg/day) no. of patient=20 meloxicam (15mg/day)+ pioglitazone(15mg/day) no. of patient=17 0 26.25  0.8 28.75  1.35 25.88  1.23 1 30.5  0.8 * a 37  1.11 * b 30.29  1.17 * a 2 32  0.76 * a 44  1.12 * b 35.59  1.04 * c 3 33.75  0.8 * a 46.75  0.98 * b 38.24  1.05 * c 4 34.75  0.68 * a 48.5  0.73 * b 38.82  1.01 * c 5 36.75  0.98 * a 49.75  0.92 * b 40  0.96 * c 6 37  1.05 * a 54.5  1.02 * b 40.88  1.15 * c 7 37.5  0.85 * a 59  0.93 * b 41.76  1.05 * c 8 39.25  0.75 * a 59.75  0.85 * b 43.24  0.73 * c 9 39.5  0.88 * a 62.25  0.68 * b 44.12  0.77 * c 10 39.25  0.83 * a 62.75  0.77 * b 45.59  0.84 * c 11 41.25  0.95 * a 62.5  0.85 * b 45.59  0.95 * c 12 41.75  0.91 * a 62.5  0.93 * b 45.29  0.8 * c data are expressed as mean  sem. * p>0.05 significant difference compared to pre-treatment value within the same group. values with non-identical superscripts (a, b & c) among different groups are significantly different (p>0.05) at corresponding duration . effects on quality of life score (qol) at zero time (before treatment), all patients showed relatively low qol score. there is a clear evidence for good response to treatment with all used medications concerning qol score, indicating that drug therapy improves not only the disease state and clinical features, but also the patient's mood. during the first week of treatment , all groups demonstrated time-dependent improvement in qol score compared to baseline value of each group, with the priority to patients group treated with combination of meloxicam and metformin (28.87%). the presented data also showed that treatment with meloxicam alone resulted in a slower improvement pattern of qol score compared to that produced by treatment of meloxicam and metformin combination at the end of the study (50%, 89.98%) compared to pre-treatment values, respectively. addition of pioglitazone to meloxicam resulted in significant improvement in qol score compared to that of meloxicam alone at corresponding duration, particularly within the last few weeks of study. the presented data, clearly demonstrated the advantage of adding metformin to meloxicam concerning improvement of qol score in comparison to that produced by meloxicam alone and combination of meloxicam with pioglitazone (table 5). iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 20 table (5): effects of treatment with meloxicam alone, combination of meloxicam + metformin, and meloxicam + pioglitazone on quality of life (qol) score in osteoarthritic patients. duration (weeks) qol score of koos meloxicam (15 mg/day) no. of patient=20 meloxicam(15mg/day)+ metformin(1000mg/day) no. of patient=20 meloxicam (15mg/day)+ pioglitazone(15mg/day) no. of patient=17 0 25  1.36 28.13  1.4 25.37  1.56 1 27.81  1.24 * a 36.25  0.86 * b 27.57  1.52 * a 2 32.19  0.94 * a 40.63  0.96 * b 28.31  1.21 * c 3 33.13  0.92 * a 42.78  1.04 * b 30.15  1.23 * c 4 33.75  0.84 * a 44.69  0.82 * b 33.09  0.71 * a 5 34.69  0.85 * a 45.63  0.8 * b 35.29  0.92 * a 6 35.31  0.94 * a 46.88  0.85 * b 35.66  0.89 * a 7 35.94  0.89 * a 47.5  0.95 * b 36.38  0.96 * a 8 35. 63  0.92* a 50.63  1.01 * b 38.97  1.01 * c 9 36.25  0.86 * a 51.88  1.02 * b 39.34  1.04 * c 10 37.49  0.9 * a 53.13  0.85 * b 39.71  0.92 * c 11 37.5 0.78 * a 53.76  0.84 * b 40.44  0.95 * c 12 37.5 0.78 * a 53.44  0.96 * b 41.18  0.94 * c data are expressed as mean  sem. * p>0.05 significant difference compared to pre-treatment value within the same group. values with non-identical superscripts (a, b & c) among different groups are significantly different (p>0.05) at corresponding duration. discussion osteoarthritic pain is generally described as a sharp ache or a burning sensation in the associated muscles and tendons. oa can cause a crackling noise (called "crepitus") when the affected joint is moved or touched and patients may experience muscle spasms and contractions in the tendons. occasionally, some patients reported increased pain with movement, cold weather, high humidity, and/or a drop in barometric pressure (19) . the pain of oa includes both nociceptive and nonnociceptive components and is associated with abnormally excitable pain pathways in the peripheral and central nervous systems (20, 21) . furthermore; unrelieved pain leads to serious negative consequences, like those observed with poor pain score belong to oa patients before treatment (table 1), with many other physiological effects associated with increased catabolic demands (22) . there is evidence that nsaids are superior to paracetamol for pain relief in patients with osteoarthritis (23) , but they are also associated with more adverse effects. the major concern is serious gastrointestinal, renal and cardiovascular complications, with risk increasing with age, concurrent use of other medications and duration of therapy. nsaids help relieve pain, swelling and stiffness but they do not alter the progression of osteoarthritis (24) . since cartilage tissues of osteoarthritic patients contain no pain receptors. so, sensation of pain likely results from inflammatory mediators. pro-inflammatory agents interleukin-1 (il-1), tumor necrosis factor alpha (tnf-α), as well as the growth iraqi j pharm sci, vol.23(2) 2014 metformin or pioglitazone in treatment of knee osteoarthritis 21 factors have all been shown to induce cox-2 expression which produces measurable quantities of prostaglandins. on the other hand, the anti-inflammatory cytokines il-4 and il-13, as well as the immunosuppressive glucocorticoids, were shown to decrease cox2 levels (25) . in this respect, the beneficial effects of metformin and pioglitazone in reducing pain and symptoms in patients with oa can be explained according to the nature of the biological activity which can be attributed to many factors including the anti-inflammatory and antioxidant effects. the mechanism by which metformin or pioglitazone regulate the inflammatory response is poorly understood, but many studies on animal models demonstrated their inhibitory effect on the expression of the pro-inflammatory mediators and oxidative stress markers. metformin, the well-known adenosine monophosphateactivated kinase (ampk) activator, can suppress cox-2 and inos mrna and protein expression dose dependently (26) . metformin ability to reduce the intensity of pain, mainly associated with its effects on the profile of inflammatory cytokines (i.e., tnf a, il-1β, il-6, and il-10) and adipokines (27) , it significantly prevented the increased levels of pro-inflammatory mediators like tnf-α, il1β, il-6, and il-18 in many inflammatory disorders, moreover; metformin prevented the expression of cox-2, inos, and decreased the levels of no and pge2 in cell culture media (28) , an evidence which support the observed effect of reducing the consequence of pain in oa patients. pioglitazone is potent and highly selective agonist for the nuclear receptor, ppar-γ. activation of ppar-γ has been shown to exhibit anti-inflammatory and anti-catabolic properties and to be protective in animal models of oa (29) . study by (mrgenweck, 2013) reported that ppar-γ could be emerged as a new pharmacotherapeutic target for chronic pain; ppar-γ activation blocks the development of, and reduces established neuropathic pain (30) , the possible mechanism of neuroprotection by ppar-γ agonist, pioglitazone, may involve modulation of inflammatory reaction and oxidative stress (31) . pioglitazone, via ppar-γ activation, reduce the effects of il-1β-induced cox-2 expression by interfering with oxidative stress and role of ros (32) . ppar-γ agonist, pioglitazone, has been reported to reduce the severity of experimental oa. this effect was associated with a reduction in the levels of mmp-13 and il-1β, which are known to play an important role in the pathophysiology of oa lesions (33) . the lack of satisfaction of patients and doctors with nsaids treatment reflected by that fewer than 20% of patients with hip or knee oa, in whom nsaids treatment initiated, are still taking the same drug 12 months later (18) . the typical patient with oa is middle aged or elderly, and complains of knee, hip, hand, or spine pain. in most cases, the patient experience pain and stiffness in and around the affected joint, causing a decrease in function and activity. the onset of these symptoms is mostly insidious, and pain typically worsens with the use of the affected joint, but usually is alleviated with rest, while morning stiffness is lasting less than 30 minutes in common (34) . osteoarthritis is a common debilitating joint disorder, affecting large sections of the population, which results in high morbidity; significant disability and impaired quality of life (35) . accordingly, the primary goals of oa treatment are to relief pain, minimizing disability and limit the progression of the disease. because most patients with oa are elderly people who have co-morbidities and are more susceptible to side effects of chronically used medications, care must be taken to individualize therapy on the bases of a patient's need and to minimize potential drug toxicity (36) . in this study, the high level of improvement in pain score observed with the use of metformin compared to meloxicam alone or in combination with pioglitazone (table 3 and 5) may correspond to the reported improvement in the daily activities and quality of life score, where metformin, and to less extent pioglitazone, improve all parameters in koos system when added to meloxicam, compared to using meloxicam alone. according to the results of this study, the addition of either metformin or pioglitazone to nsaids could constitute a new promising way to reversing or retarding the progression of degenerative processes that predispose to pain and other consequent symptoms in oa. conclusion co-administration of metformin or pioglitazone with meloxicam, in oa patients produced very well characterized analgesic and anti-inflammatory activities, and improves the therapeutic profile of meloxicam. references 1. goldring sr, goldring mb. clinical aspects, 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https://doi.org/10.31351/vol31iss1pp251-269 251 in silico, in vitro studies of anti-oxidant and anthelminthic abilities of phytoconstituents from rhynchosia cana (wild.) dc. praveena yempada*,1, arya lakshmi marrisetti*, ganga rao battu , girija sastry vedula* *pharmacognosy and phytochemistry research division, university college of pharmaceutical sciences, andhra university, visakhapatnam, andhra pradesh, india abstract helminthiasis is a significant economic burden on grazing cattle. increased resistance to currently available synthetic anthelmintics used to treat helminthiasis, and anthelmintic residues in meat and dairy products pose a significant worldwide health threat. these obstacles require the development of new anthelmintics capable of combating drug resistance while also exhibiting improved safety profiles. rhynchosia cana (fabaceae) is a herb that has historically been used as a worm expeller. to evaluate the phytochemical profile and explore the anti-oxidant and anthelmintic effects of different extracts of rhynchosia cana (r. cana) by in silico and in vitro methods. using standardised chemical tests as defined in the literature, phytochemical research was carried out. using 2,2-diphenyl-1-picrylhydrazyl (dpph) and hydrogen peroxide (h2o2) radicals scavenging assay, in vitro free radical scavenging behaviour of different extracts was quantitatively estimated, whereas in-vitro anthelmintic activity was measured against pheretima posthuma (p. posthuma) (annelida). the molecular docking analysis was then carried out to establish compounds with good efficiency for anti-oxidant activity against the catalase, superoxide dismutase, glutathione-s-transferase, glutathione reductase, glutathione peroxidase and tubulincolchicine enzyme for anthelmintic activity. furthermore, adme/t profiles have been tested by admet sar. the various extracts of r cana potentially inhibited the reactive oxygen species (ros) and possessed anti-oxidant activity. in anti-oxidant assays, the ic50 values ranged from 62.08 to 440.08 μg/ml for perc, earc, and merc. all the extracts demonstrated anthelmintic behaviour on p. posthuma that was dose-dependent and statistically relevant. on the other side, molecular docking analysis reveals that gallocatechin has the best fitness score of -7.1 kcal/mol with tubulin-colchicine enzyme; rhynchosin, luteolin-3',4'-dimethyl ether, isoorientin and orientin has the best fitness scores with different targets related to the oxidation process. in addition, all compounds were in the array of expected properties to fulfil the lipinski law of five to be accepted as drug -like potential. the observation indicates that the r. cana possesses anti-oxidant and anthelmintic activity in vitro and in silico assays. however, further research was needed to elucidate their primary molecular mechanism of action, safety, toxicity, and bioavailability. keywords: anthelmintic, antioxidant, rhynchosia cana (wild.) dc., in silico, in vitro studies introduction throughout the globe, the usage of medicinal plants predates the advent of antibiotics and other modern medicines. it has been demonstrated that higher plant origin products are efficient sources of chemotherapeutic agents without underscoring side effects. (1, 2). in treating chronic illnesses, including alzheimer's disease, many culinary herbs and spices have been assessed for their biological activities (3-5). reactive oxygen species (ros)/free radicals have been implicated in developing over 100 diseases (6-10). evidence has been given in laboratory, clinical, and epidemiological trials to support ros's cancer aetiology function (11). there are anti-oxidant protection mechanisms for all aerobic organisms, including humans, which protect against oxidative destruction. nevertheless, the natural anti-oxidant protection mechanisms may be ineffective, and thus dietary consumption of anti-oxidant components is necessary and suggested. interest in seeking natural anti-oxidants for use in foods or pharmaceutical goods to supplement synthetic anti-oxidants, which are limited due to their undesirable reactions, such as carcinogenicity, has recently risen dramatically. therefore, plants represent the principal reservoir of natural anti-oxidant molecules capable of removing or neutralising the deleterious ros. helminth infections were prevalent in marginalised, lowincome, and resource-constrained regions of the world, with over 1 billion people in developing areas of sub-saharan africa, asia, and the americas infected with one or more helminth species (12). humans utilised plants to cure a variety of illnesses, but the practice was eventually abandoned. researchers have grown increasingly interested in alternative medicines derived from plants due to their structural variety, low toxicity, ease of accessibility, and varied mode of action. (13-15). 1corresponding author e-mail: navya.praveena.26@gmail.com received: 25/5/2021 accepted:20 /11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp251-269 iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 252 the use of medicinal plants can significantly decrease medical expenditures in developing nations, which is sadly plagued by hunger and poverty. in developing nations, these diseases have a significant effect, provided that there is no regular supply of a new drug or that the parasitic strain has established a tolerance to the available medicine. besides, multidrug-resistant and extremely drug-tolerance helminths are observed in patients in developed countries. on the other side, pain is a massive global phenomenon that decreases living standards and significantly affects well-being and the economy (16, 17). it will also minimise all the risks involved with the drugs that are currently accessible by utilising a herbal plant with significant biological activity against pathogens and acute or chronic pain. rhynchosia cana (r. cana) belongs to the family fabaceae, which has high medicinal value. there are approximately 300 rhynchosia species distributed all over the world. this plant finds use in traditional medicine viz., the bark decoction for dysentery (18), leaves used for wounds, cuts, boils, and rheumatic pains. the phytochemicals of r. cana explored were vitexin, vicenin-2, orientin, isoorientin (19). the flowers of r. cana demonstrate anti-inflammatory and antipyretic activity (20). recent studies also confirmed the genus' pharmacological and biological activities, such as anti-oxidant, antimicrobial, anti-inflammatory, antiangiogenic, and antityrosinase antiproliferative and allelopathic activities (21). ethical concerns about the use of animals in science have increased significantly in recent years, necessitating exploring alternative techniques like in silico approach that do not include experimental animals, thus reducing animal use. methods were developed in silico to identify and model toxic effects in humans and the environment. (22, 23) in silico methods help integrate more intelligently diverse up-to-date computational and experimental techniques than a set of exploratory laboratory analyses (24). in medicinal chemistry, methods such as virtual screening are used in silico to evaluate plant compound pharmacological behaviour and receptor associations, and these strategies are cost-effective and straightforward (25). various online programming systems have been developed for in-silico research by different laboratories. detailed pharmacological profiles of phytoconstituents and novel biological activities of these phytoconstituents are expected using these programmes. pass is based on a comprehensive analysis of structure operation relationships and allows more accurate predictions for phytoconstituents belonging to a new chemical class (26, 27). the purpose of this analysis was to examine computationally the possible biological effects of the primary compounds found in the extract of rhynchosia cana, with an emphasis on anti-oxidants, a property implicated in several pathologies, and anthelmintic properties, also to quantify potential risks of toxicity. materials and methods collection of plant material rhynchosia cana whole plant (leaves, root and stem) was collected from tirumala hills, district chittoor, andhra pradesh, india, in november 2016. it was authenticated by dr madhava chetty, department of botany, sri venkateswara university, tirupati. the plant parts gathered were washed with water and dried at room temperature in the shade. the dry components were ground coarsely. for the duration of the analysis, the powdered plant was stored in an airtight, lightresistant package. extraction procedure powdered plant material (500 g) was taken in separate beakers and immersed in 1000 ml of petroleum ether, ethyl acetate, and methanol; along with constant shaking, the beaker with the contents was kept for 14 days. preliminary phytochemical analysis all the extracts were qualitatively tested using different chemical methods for different classes of phytoconstituents. carbohydrates were detected using the molisch's test; proteins were detected using either biuret test or millon's test and amino acids using ninhydrin test. steroids were detected by salkowski, libermann-burchards and libermann's tests, alkaloids were identified with freshly prepared dragendroff's, mayer's, hager's and wagner's reagents by the presence of turbidity or precipitation. the flavonoids were detected using four tests, shinoda, sulphuric acid, aluminium chloride, lead acetate and sodium hydroxide. tannins were detected by gelatin, lead acetate, potassium dichromate, and ferric chloride. the froth, emulsion, and lead acetate tests were applied for the detection of saponins. steroids were detected using a combination of acetic anhydride with sulphuric acid or acetic chloride with sulphuric acid. sample extracted with chloroform was treated with sulphuric acid to test for the presence of terpenoids. ammonia solution and ferric chloride solutions were used to detect the presence of anthraquinones. (28-30). in-vitro anti-oxidant activity dpph radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (dpph), radical scavenging assay was performed according to blois (31) and desmarchelier et al. (32), using ascorbic acid as a reference standard?) hydroxyl radical scavenging activity hydroxyl radical scavenging behaviour was calculated by the halliwell et al. technique (33). iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 253 anthelmintic activity collection and authentication of worm the indian earthworms pheretima posthuma (annelida) employed in the present study were collected from moist and muddy soil of the paderu region, visakhapatnam, andhra pradesh, india, the average size earthworm being 6-8 cm. worms were washed with normal saline to remove all the faecal matter. it was authenticated by the department of zoology, andhra university. visakhapatnam. in-vitro anthelmintic activity the anthelmintic activity was performed according to mehta et al. on the adult indian earthworm pheritima posthuma. a total amount of 51 earthworms had collected and were divided up into groups, each made up of three worms. various concentrations (5 mg/ ml, 10 mg/ml, 15 mg/ml, and 20 mg/ml) of the extracts, i.e., r. cana petroleum ether extract (perc), r. cana ethyl acetate extract (earc), and r. cana methanol extract (merc), as well as standard (mebendazole), had been prepared in distilled water of 10 ml. the control group was treated with only distilled water. the earthworms have been formerly washed in regular water before these were launched into 10 ml of the corresponding petri dish. promptly after releasing the earthworms in the concerned petri dishes, the time of launching was stated, and also consequently, the motility of the earthworms was observed. time of paralysis was seen if the earthworms revealed no activities apart from when worms have trembled very and also the time of death was seen after finding that earthworms neither relocated as soon as shiver extremely neither when showered with hot water (40–50°c) (34-36). gc-ms analysis using agilent technology gc systems with the gc-7890a/ms-5975c model, the gc-ms study of bioactive compounds of methanolic extracts of r. cana was carried out. drug-likeliness the phytochemical components of the compounds were determined by using drulito software (37, 38). in silico docking studies the docking studies of the compounds vitexin, isovitexin, vicenin-2, orientin, and isoorientin were carried out using the interaction between ligand and target protein tools from pyrx and discovery studio biovia 2020. for the present study, five enzymes of the cellular anti-oxidant mechanism, viz. catalase (pdb id: 1dgh), superoxide dismutase (pdb id: 5ytu), glutathione-s-transferase (pdb id: 5h5q), glutathione reductase (pdb id: 1xan), and glutathione peroxidase (pdb id: 13gs), were selected and to determine the anthelmintic activity, β-tubulin (pdb id: 1oj0) was used and to estimate the inhibitory potential of the constituents and metabolites of r. cana on these (figure 1). figure 1. 2d representation of various ligands from rhynchosia cana iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 254 admet analysis admet of ligands refers to their pharmacokinetic properties, which must be investigated to determine their role inside the system. the admet inheritance of ligands was investigated using admetsar. (39, 40). results phytochemical screening preliminary phytochemical screening of various extracts of r. cana extracts revealed the presence of several phytoconstituents listed in table 1. table 1. preliminary phytochemical analysis of various extracts of rhynchosia cana " +" present "-" absent perc: petroleum ether extract of rhynchosia cana; earc: ethyl acetate extract of rhynchosia cana; merc: methanol extract of rhynchosia cana in-vitro anti-oxidant activity dpph radical scavenging activity the dpph scavenging function was used to monitor the capacity of the different rhynchosia cana extracts to mop up free radicals. the ic50 values of perc (360.88), earc (162.38), merc (62.08), and ascorbic acid (38.97) were obtained using the linear regression equation. figure 2 and table 2 show the percentage inhibition of various extracts of r. cana and reference standard over a range of concentrations. ic50 = (0.5-b)/a hydrogen peroxide radical scavenging activity the hydrogen peroxide radical scavenging potential of various extracts of r. cana was evaluated using the hydrogen peroxide (h2o2) scavenging method. the results are shown in figure 2 and table 2. figure 2. anti-oxidant activity of various extracts of r. cana (a) dpph scavenging activity (b) hydrogen peroxide scavenging activity. values are expressed as mean±sem (n=3). one-way anova followed by multiple tukey’s comparison test. *p < 0.05, as compared to ascorbic acid. test for specific phytoconstituent perc earc merc alkaloids + flavonoids + + tannins + + steroids + + volatile oils + + saponins + fats and fixed oils + + proteins + carbohydrates + acidic compounds + iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 255 table 2. dose-dependent dpph free radical scavenging and h2o2 radical scavenging activity of different extracts of r. cana values are expressed as mean±sem (n=3). one-way anova followed by multiple tukey’s comparison test. *p < 0.05, as compared to ascorbic acid. in-vitro anthelmintic activity on pheretima posthuma worms, the antihelmintic function of different extracts was calculated. the results indicate that the degree of anthelmintic activity is directly proportional to the concentration of the extract, which ranges from 5 to 20 mg/ml. the paralysis time and death time was enlisted in table 3 and represented in figure 3. figure 3. the results are presented as a mean±standard deviation, (n=3). one-way anova was used to evaluate the results, followed by dunnett's examination.(a) paralysis time for various extracts of rhynchosia cana with standard mebendazole. (b) death time for various extracts of rhynchosia cana with standard mebendazole.*, p<0.05, %, p<0.01 and $, p<0.001 versus standard. method treatment % inhibition of radical scavenging activity ic50 (µg/ml) 12.5 µg/ml 25 µg/ml 50 µg/ml 100 µg/ml 200 µg/ml dpph free radical scavenging (% inhibition) perc 20.15±1.84 26.51±0.55 38.12±1.48* 46.15±2.61 61.25±2.43* 360.88 earc 24.52±0.63 36.15±1.24* 45.22±1.54 58.15±3.51 69.12±1.86* 162.38 merc 28.22±2.11 38.15±1.66 51.22±0.69 64.15±1.26 76.12±1.52* 62.08 ascorbic acid 28.21±1.52 40.51±2.84 58.22±3.51 72.15±2.71 80.12±1.36 38.97 h2o2 scavenging activity (% inhibition) perc 20.21±2.62 24.51±0.84* 31.22±1.82 36.15±2.51 46.12±0.55 440.08 earc 26.33±1.85 38.61±2.51 46.42±1.61 56.31±1.44 68.62±2.51 276.03 merc 30.22±1.63 38.51±1.52 52.33±2.51 60.14±1.56* 72.52±1.44 75.77 ascorbic acid 36.23±1.36 48.51±2.44 61.84±0.89 75.61±2.66 82.32±2.81 37.45 iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 256 table 3.anthelmintic activity of various extracts of r. cana against pheretima posthuma treatment / dose paralysis time death time control (water) mebendazole (5 mg/ml) 9.21±0.32 15.62±0.11 mebendazole (10 mg/ml) 6.81±0.64 10.32±0.62 mebendazole (15 mg/ml) 4.65±0.21 6.55±0.22 mebendazole (20 mg/ml) 2.62±0.15 3.02±0.18 perc (5 mg/ml) 22.54±2.12* 38.12±2.51* perc (10 mg/ml) 15.45±0.51 27.22±0.36 perc (15 mg/ml) 13.26±2.44% 24.15±3.15% perc (20 mg/ml) 12.51±1.51$ 18.25±1.19$ earc (5 mg/kg) 14.12±1.52% 25.81±1.98% earc (10 mg/kg) 10.63±2.15* 18.32±0.64* earc (15 mg/kg) 7.81±1.23 11.66±0.32 earc (20 mg/kg) 4.74±1.12$ 8.63±1.51$ merc (5 mg/kg) 10.22±0.61% 18.63±1.44% merc (10 mg/kg) 8.45±2.11 13.68±0.66 merc (15 mg/kg) 5.11±0.23* 8.51±2.95* merc (20 mg/kg) 2.98±0.49$ 5.51±0.66$ values are expressed as mean±sem (n=3). dunnett's test after a one-way anova was used for evaluating the paralysis and death time, *, p<0.05, %, p<0.01 and $, p<0.001 versus standard. perc= petroleum ether extract of r. cana; earc= ethyl acetate extract of r. cana; merc: methanol extract of r. cana gc-ms analysis eleven peaks were shown in the gc-ms chromatogram analysis of the methanolic extract of r. cana (figure 4), indicating 11 phytochemical constituents. eleven phytocompounds were characterised and defined about the constituents' mass spectra and the nist library (table 4). figure 4.gc-ms spectral analysis of methanol extract of rhynchosia cana (merc) iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 257 table 4. biologically active compounds derived from r. cana s. no retention time compound name canonical smiles area (%) molecular formula molecular weight 1. 6.252 orientin c1=cc(=c(c=c1c2=cc(=o)c3=c(o2)c(=c(c=c3o)o) c4c(c(c(c(o4)co)o)o)o)o)o 8.68 c21h20o11 448.4 2. 6.647 luteolin 3',4'-dimethyl ether coc1=c(c=c(c=c1)c2=cc(=o)c3=c(c=c(c=c3o2)o c4c(c(c(c(o4)c(=o)o)o)o)o)o)oc 1.89 c23h22o12 490.4 3. 8.292 genistein c1=cc(=cc=c1c2=coc3=cc(=cc(=c3c2=o)o)o)o 10.98 c15h10o5 270.24 4. 8.917 alpha-terpineol cc1=ccc(cc1)c(c)(c)o 5.53 c10h18o 154.25 5. 11.334 vitexin c1=cc(=cc=c1c2=cc(=o)c3=c(o2)c(=c(c=c3o)o)c 4c(c(c(c(o4)co)o)o)o)o 48.23 c21h20o10 432.4 6. 12.149 isoorientin c1=cc(=c(c=c1c2=cc(=o)c3=c(o2)c=c(c(=c3o)c4 c(c(c(c(o4)co)o)o)o)o)o)o 2.85 c21h20o11 448.4 7. 13.324 rhynchosin c1=cc(=c(c=c1c2=c(c(=o)c3=cc(=c(c=c3o2)o)o) o)o)o 10.14 c15h10o7 302.23 8. 15.698 isovitexin c1=cc(=cc=c1c2=cc(=o)c3=c(o2)c=c(c(=c3o)c4 c(c(c(c(o4)co)o)o)o)o)o 2.37 c21h20o10 432.4 9. 16.595 cubenol cc1ccc(c2c1(ccc(=c2)c)o)c(c)c 10.32 c15h26o 222.37 10. 17.940 vicenin-2 c1=cc(=cc=c1c2=cc(=o)c3=c(c(=c(c(=c3o2)c4c( c(c(c(o4)co)o)o)o)o)c5c(c(c(c(o5)co)o)o)o)o)o 6.02 c27h30o15 594.5 11. 19.241 gallocatechin c1c(c(oc2=cc(=cc(=c21)o)o)c3=cc(=c(c(=c3)o)o )o)o 2.32 c15h14o7 306.27 iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 258 drug likeliness the physicochemical properties of 11 active compounds, i.e., orientin, luteolin 3',4'-dimethyl ether, genistein, alpha-terpineol, vitexin, isoorientin, rhynchosin, isovitexin, cubenol, vicenin-2 and gallocatechin were investigated using the drulito program. except for four compounds, the remaining compounds followed lipinski's law. (table 5). table 5. physicochemical properties of active compounds and accordance with the rule of drug-likeliness ligand mw logp alogp hba hbd tpsa amr nrb natom nacidic rc nrigidb naromring nhb no. of violations orientin 448.4 -0.36 -3.22 11 0 35.53 114.1 3 32 0 4 32 2 11 2 luteolin 3',4'dimethyl ether 490.4 0.55 -2.65 12 0 80.29 124.4 6 35 0 4 32 2 12 2 genistein 270.24 1.043 -0.39 5 0 26.3 78.92 1 20 0 3 21 2 5 0 alphaterpineol 154.25 2.369 1.122 1 0 0 47.92 1 11 0 1 10 0 1 0 vitexin 432.4 -0.71 -2.66 10 0 35.53 112.5 3 31 0 4 31 2 10 0 isoorientin 448.4 -0.36 -3.22 11 0 35.53 114.1 3 32 0 4 32 2 11 0 rhynchosin 302.23 2.263 -1.24 7 0 26.3 83.44 1 22 0 3 23 2 7 1 isovitexin 432.4 -0.71 -2.66 10 0 35.53 112.5 3 31 0 4 31 2 10 0 cubenol 222.37 4.054 1.364 1 0 0 66.87 1 16 0 2 16 0 1 0 vicenin-2 594.5 -2.55 -5.09 15 0 44.76 144.9 5 42 0 5 41 2 15 2 gallocatechin 306.27 1.2 -1.5 7 0 9.23 82.67 1 22 0 3 23 2 7 0 iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 259 molecular docking studies molecular docking was used to identify potential anti-oxidants and anthelmintic candidates against various targets using phytoconstituents obtained from r. cana. these 11 compounds were docked to the target enzymes, and their docking results were assessed. the top three scored compounds with multiple targets were an excellent representation of anti-oxidant and anthelmintic activity. refer to table 6 for detailed molecular interactions of the ligand with the targeted proteins. studies on molecular interactions biovia discovery studio was used to predict the rigid docking effects. the best binding sites for protein-ligand interaction were identified in tables 7-12 and figures 5-9. figure 5. molecular docking (molecular surface view) and 2d representation of interactions between various ligands with the human erythrocyte catalase (pdb id: 1dgh). (a) orientin, (b) luteolin-3',4'dimethyl ether, (c) rhynchosin, and (d) ascorbic acid iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 260 figure 6. molecular docking (molecular surface view) and 2d representation of interactions between various ligands with the human sod1 complexed with isoproterenol. (a) isoorientin, (b) isovitexin, (c) rhynchosin, and (d) ascorbic acid figure 7. molecular docking (molecular surface view) and 2d representation of interactions between various ligands with the human gpx4 complex with gxpep-1 (pdb id: 5h5q). (a) isoorientin, (b) orientin, (c) rhynchosin, and (d) ascorbic acid iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 261 figure 8. molecular docking (molecular surface view) and 2d representation of interactions between various ligands with the human glutathione reductase in complex with a xanthene inhibitor (pdb id: 1xan). (a) luteolin-3',4'-dimethyl ether, (b) orientin, (c) genistein, and (d) ascorbic acid. figure 9. molecular docking (molecular surface view) and 2d representation of interactions between various ligands with glutathione s-transferase complexed with sulfasalazine (pdb id: 13gs). (a) rhynchosin, (b) luteolin-3',4'-dimethyl ether, (c) vicenin-2, and (d) ascorbic acid iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 262 table 6. molecular docking of selected compounds from r. cana with various targets associated with oxidation and helminthiasis. ligands binding affinities (kcal/mol) 1dgh 5ytu 5h5q 1xan 13gs 1oj0 gallocatechin -8.8 -6 -6.1 -7.4 -7.2 -7.1 rhynchosin -9.8 -6.8 -6.1 -8.1 -8.5 -6.2 genistein -9.6 -5.9 -5.5 -8.2 -7.1 -5.8 α-terpineol -6.6 -4.2 -4.9 -5.2 -4.8 -5.4 cubenol -8.6 -5 -5 -6.4 -5.8 -5.2 vitexin -8.1 -6.2 -6.1 -7.6 -6.7 -4.5 orientin -11.3 -6.3 -6.2 -8.7 -7.1 -4 isovitexin -7.8 -7 -5.6 -8.1 -7.3 8.7 isoorientin -8.1 -7.4 -7.5 -8 -7.3 9.6 vicenin-2 -7.8 -6.3 -2.4 -7.9 -7.5 22.8 luteolin-3',4'-dimethylether -10.5 -6.8 -5.6 -9 -8.4 -4 ascorbic acid -6.2 -4.6 -5.7 -6.5 -5.8 mebendazole -5.4 table 7. interactions of catalase (pdb id: 1dgh amino acid residues with ligands at receptor sites. ligands binding affinity, δg (kcal/mol) amino acids involved and distance (a°) hydrogen binding interactions hydrophobic interactions electrostatic interactions orientin -11.3 his a:362 (4.56), ala a:133 (3.59) his a:75 (4.96, 5.55), val a:146 (4.82), ala a:133 (4.64), asn a:148 (5.41) arg a:354 (6.57) luteolin-3',4'dimethylether -10.5 arg a:72 (4.92), arg a:112 (3.37), ser a:114 (4.49) phe a:161 (4.41, 5.01), met a:350 (3.59), pro a:158 (4.08), pro a:162 (4.74), arg a:354 (5.14, 5.39), ala a:357 (5.03, 5.09), val a:74 (4.89, 5.90), arg a:72 (7.13) rhynchosin -9.8 tyr a:358 (5.97), val a:73 (3.45), thr a:361 (3.76), arg a:365 (2.17) ala a:133 (6.33), val a:146 (5.61), his a:75 (4.50), tyr a:358 (5.90), arg a:72 (4.10, 5.51), thr a:361 (3.76), gly a:131 (4.07) ascorbic acid -6.2 arg a:72 (4.85), arg a:112 (3.77), his a:362 (5.63) iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 263 table 8. interactions of superoxide dismutase (pdb id: 5ytu) amino acid residues with ligands at receptor sites. ligands binding affinity, δg (kcal/mol) amino acids involved and distance (a°) hydrogen binding interactions hydrophobic interactions electrostatic interactions isoorientin -7.4 ser a:102 (3.59), asp a:101 (3.62), leu a:84 (6.22), thr a:88 (3.73) lys a:75 (5.05, 5.32), ile a:99 (6.10), pro a:74 (5.06, 6.10), asn a:86 (5.45) isovitexin -7 thr a:88 (4.00), asp a:101 (4.58), ser a:102 (3.50) ile a:99 (4.72, 5.06), pro a:74 (4.78, 6.07), lys a:75 (5.79) rhynchosin -6.8 pro a:74 (4.70), asn a:86 (3.85), glu a:100 (3.92) ile a:99 (4.02, 4.29, 5.62), pro a:74 (4.90, 5.11, 6.35) ascorbic acid -4.6 glu a:100 (2.68), asp a:101 (4.40), ser a:102 (3.43, 3.72) lys a:75 (4.37) table 9. interactions of glutathione-s-transferase (pdb id: 5h5q) amino acid residues with ligands at receptor sites. ligands binding affinity, δg (kcal/mol) amino acids involved and distance (a°) hydrogen binding interactions hydrophobic interactions electrostatic interactions isoorientin -7.5 phe a:127 (5.47), asp a:50 (4.83), met a:129 (4.60), lys a:58 (5.52) val a:54 (4.27, 4.57, 6.19), lys a:58 (5.00) asp a:48 (6.55, 5.63), asp a:128 (6.81), lys a:58 (5.52) orientin -6.2 asp a:128 (2.93, 4.09), met a:129 (4.69), met a:53 (5.07) val a:54 (4.45), lys a:58 (6.26) asp a:48 (6.29, 6.31)) rhynchosin -6.1 lys a:58 (5.56), met a:129 (5.28, 5.37) val a:54 (4.34, 4.70, 5.06), lys a:58 (5.06) asp a:48 (6.54), asp a:128 (6.99) ascorbic acid -5.7 ile a:156 (3.78), glu a:184 (4.75, 5.12), arg a:179 (5.13) gly a:181 (3.74), gly a:155 (3.86) iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 264 table 10. interactions of glutathione reductase (pdb id: 1xan) amino acid residues with ligands at receptor sites. ligands binding affinity, δg (kcal/mol) amino acids involved and distance (a°) hydrogen binding interactions hydrophobic interactions electrostatic interactions luteolin-3',4'dimethylether -9 val a:370 (3.68) ala a:342 (5.23, 6.75), cys a:58 (4.50), gly a:62 (4.01) asp a:331 (5.76), cys a:63 (4.68, 6.15) orientin -8.7 ser a:30 (4.34), leu a:337 (3.59) leu a:338 (6.10), lys a:66 (5.44), cys a:63 (4.17), ile a:198 (4.99) cys a:63 (4.13) genistein -8.2 cys a:63 (4.05), asp a:331 (4.18) cys a:58 (5.20), lys a:66 (6.71), ile a:198 (4.84, 6.81), ser a:177 (4.20), asp a:331 (6.93) ascorbic acid -6.5 ser a:30 (3.74), cys a:58 (4.51), thr a:57 (4.29), asp a:331 (4.19) thr a:57 (3.87), thr a:339 (4.24) table 11. interactions of glutathione peroxidase (pdb id: 13gs) amino acid residues with ligands at receptor sites. ligands binding affinity, δg (kcal/mol) amino acids involved and distance (a°) hydrogen binding interactions hydrophobic interactions electrostatic interactions rhynchosin -8.5 asn a:66 (4.70), arg a:70 (6.02), cys a:101 (5.93), asn b:66 (3.76), asp a:94 (4.93) asp b:98 (5.74), glu a:97 (5.29), arg a:13 (6.27) luteolin-3',4'dimethylether -8.4 cys b:101 (5.86), ser a:165 (2.61, 3.58), asp b:94 (4.60) cys b:101 (5.86), arg a:13 (4.24) arg a:13 (7.48), asp a:98 (5.33), glu a:97 (5.38), glu a:97 (4.81) vicenin-2 -7.5 arg b:70 (5.74), asn b:66 (3.57), ser b:65 (3.25), asp a:98 (4.96), cys a:101 (4.56) l;ys a:102 (4.90), cys b:101 (5.71, 7.30), cys b:101 (5.63, 7.13) glu b:97 (4.87) ascorbic acid -5.8 asn a:66 (4.43), arg b:70 (5.69), asp a:94 (4.09), asp a:98 (3.99) glu a:97 (5.52) iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 265 table 12. interactions of βtubulin (pdb id: 1oj0) amino acid residues with ligands at receptor sites. ligands binding affinity, δg (kcal/mol) amino acids involved and distance (a°) hydrogen binding interactions hydrophobic interactions electrostatic interactions gallocatechin -7.1 ser a:138 (3.73), gly a:140 (2.75), leu a:139 (4.89), val a:169 (3.57), asn a:204 (5.12), asn a:226 (4.67) cys a:12 (5.76), val a:169 (6.00), glu a:181 (4.23, 7.60) rhynchosin -6.2 gly a:140 (4.64), asn a:204 (5.45) cys a:12 (4.86), val a:169 (5.42, 5.56), ile a:16 (5.97) genistein -5.8 asn a:204 (4.85), asn a:226 (3.61) cys a:12 (4.73, 5.18), val a:169 (5.35), ile a:16 (6.18) met a:233 a (5.21, 5.50, 5.58) mebendazole -5.4 his a:6 (5.65), gln a:134 (4.72), ser a:165 (3.66) phe a:167 (3.99), val a:229 (6.54) met a:233 (5.21, 5.50, 5.58) adme/t analysis by admetsar the admet properties of ligands were determined using admetsar (table 13). both substances demonstrated adequate human intestinal absorption (hia) and blood-brain barrier penetration (bbb). none of them has been determined to be carcinogenic. none of the compounds was found to be ames-negative. the following table 13 summarises the hia, bbb, and ld50 tests performed on compounds. iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 266 table 13.adme/t analysis of various phytoconstituents from r. cana ligand absorption distribution metabolism toxicity bbb hia caco2 p-glyco protein substrate renal organic cation transporter subcellular localization cyp450 2c9 substrate cyp450 2d6 substrate cyp450 3a4 substrate cyp450 1a2 inhibitor cyp450 2c9 inhibitor cyp450 2d6 inhibitor cyp450 2c19 inhibitor cyp450 3a4 inhibitor ames toxicity carci nogens fish toxicity (mg/l) rat acute toxicity (mol/kg) orientin 0.6742 0.9442 0.9163 0.8777 0.8909 0.5728 0.8041 0.8767 0.6032 0.8355 0.9071 0.9476 0.9240 0.8310 0.7232 0.9553 0.9771 2.3664 luteolin 3',4'dimethyl ether 0.9068 0.6737 0.6036 0.6546 0.9436 0.6622 0.8072 0.9161 0.5871 0.8108 0.8959 0.9307 0.8909 0.7521 0.8536 0.9469 0.7843 2.7471 genistein 0.6785 0.9877 0.7002 0.5000 0.9075 0.7606 0.7672 0.9105 0.6821 0.9254 0.8949 0.9232 0.8881 0.7960 0.9638 0.9276 0.4316 0.8689 α-terpineol 0.9568 0.9941 0.7505 0.5466 0.8024 0.4268 0.8255 0.8650 0.6082 0.8390 0.6034 0.9322 0.7049 0.8411 0.9133 0.7414 0.6781 1.5603 vitexin 0.6472 0.9442 0.9163 0.5836 0.8909 0.5728 0.8041 0.8767 0.6032 0.8355 0.9071 0.9476 0.9240 0.8310 0.7232 0.9553 0.9771 2.3664 isoorientin 0.6472 0.9442 0.9163 0.5836 0.8909 0.5728 0.8041 0.8767 0.6032 0.8355 0.9071 0.9476 0.9240 0.8310 0.7232 0.9553 0.9771 2.3664 rhynchosin 0.5116 0.9833 0.8367 0.5510 0.9242 0.7742 0.8088 0.9110 0.6630 0.9249 0.8949 0.9230 0.6945 0.7054 0.5905 0.9390 0.2432 3.1831 isovitexin 0.6472 0.9442 0.9163 0.5836 0.8909 0.5728 0.8041 0.8767 0.6032 0.8355 0.9071 0.9476 0.9240 0.8310 0.7232 0.9553 0.9771 2.3664 cubenol 0.9404 1.000 0.8378 0.7280 0.7956 0.4847 0.8351 0.7109 0.9032 0.9160 0.9784 0.9555 0.8607 0.8508 0.9032 0.9160 0.2472 2.4872 vicenin-2 0.6871 0.9156 0.9096 0.6277 0.8732 0.5454 0.8119 0.8714 0.6173 0.8801 0.9182 0.9445 0.9102 0.8760 0.6401 0.9478 1.1546 2.1951 gallocatechin 0.5331 0.9654 0.8956 0.5972 0.9458 0.4440 0.8227 0.8771 0.6345 0.9046 0.9071 0.9231 0.9041 0.8309 0.7658 0.9539 0.9422 1.8700 iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 267 discussion as a possible source of new medicinal agents, plant-derived natural products have gained interest. because of their potent pharmacological functions, low toxicity, and cost-effectiveness, the therapeutic properties of plants have been examined. in addition, several of the currently available treatments come from natural ingredients, reflecting the significance in the drug development phase of medications having biological origins. therefore, it is necessary to research medicinal plants to determine the active ingredient in natural products for curing diseases and then synthesise the defined active compounds in the laboratory (41-43). with this insight, the herb, r. cana, in vitro approaches have been studied to assess anti-oxidants; anthelmintic function utilising p. posthuma accompanied by an in silico molecular docking and adme/t analysis research. free radicals, also known as reactive oxygen species (ros), are produced in the body during biological metabolism. ros contributes to multiple human diseases, including diabetes and cancer. anti-oxidant molecules have the potential to regulate certain free radicals or neutralise them. the most important route is to scavenge free radicals under which phenolic compounds interrupt free radical chain reactions. r. cana has been shown to have a more significant potential for neutralising ros. with the rise in plant concentration suggesting dose-dependent function, anti-oxidant activities have been improved. extracts could be able to limit their ability as a measure of possible anti-oxidants. by breaking the free radical chain by contributing hydrogen atoms, the diminishing ability of the extracts can serve as an indicator of potential antioxidant activities. when a ligand binds to a binding site in any conformation, molecular docking scores represent the energy of interactions in the binding area. a ligand's conformation is a three-dimensional structure or arrangement of its atoms or pharmacophoric groups that can be used to determine the interaction energy. the following table 6 summarises the top phytochemicals that function as unique protein ligands. the highest docking value of -11.3 kcal/mol with orientin was found for the catalase among the gc-ms eluted phytoconstituents. the other compounds, luteolin-3',4'-dimethylether, rhynchosin and ascorbic acid, have interacted well with the catalase target with binding energies of 10.5, -9.8 and -6.2 kcal/mol, respectively (table 7 fig. 5). isoorientin was found to be effective against superoxide dismutase and glutathione-s-transferase enzymes with binding scores of -7.4 kcal/mol, whereas ascorbic acid was found to be -4.6 kcal/mol. the ligand luteolin-3',4'-dimethyl ether was effective with a docking score of -9.0 kcal/mol regarding the glutathione reductase enzyme, whereas ascorbic acid was found to be -6.5 kcal/mol. rhynchosin displayed a docking score of -8.5 kcal/mol with the target glutathione peroxidase, whereas ascorbic acid was found to be -5.8 kcal/mol. by observing the in silico analysis, the phytoconstituents of r. cana were effective against free radicals and could be a potent anti-oxidant source compared to ascorbic acid. additionally, we evaluated the molecular docking of several compounds to demonstrate the molecular interaction between the compounds and the protein, which enables us to depict the behaviour of the molecules in the coupling site of the targeted proteins and to illustrate the biochemical process underlying the anthelminthic activity. as a consequence of the results (as shown in table 12), it is determined that gallocatechin (-7.1 kcal/mol), rhynchosin (-6.2 kcal/mol), and genistein (-5.8 kcal/mol) all had substantial docking scores similar to the reference medication mebendazole (-5.4 kcal/mol). all three compounds had a higher docking score than the conventional medication mebendazole. docking analysis indicates that these compounds, particularly gallocatechin, may be a promising candidate for a novel anthelmintic drug. gc-ms analysis revealed important phytocompounds in r. cana (figure 5 and table 4), including the major gallocatechin, rhynchosin, genistein, α-terpineol, cubenol, vitexin, orientin, isovitexin, isoorientin, vicenin-2 and luteolin-3',4'dimethyl ether, which have been detected in previous studies with r. cana (44). it is understood that these compounds have crucial anti-oxidant efficacy. (44). as oxidative stress is implicated in the pathophysiology of several human diseases, antioxidant substances are pretty significant. (45). the effective dose for anthelmintic activity was calculated to be 20 mg/ml merc for our research, with 2.98 and 5.51 min for the time of paralysis and death, respectively. it took more time to paralyse and death for the rest of the concentration. the extracted power was inversely proportional to the time required to paralyse the worms. studies demonstrate pretty clearly that the methanol extract has a significant anthelmintic effect. as indicated in folk medicine, the above observations support the usage of an anthelmintic. to demonstrate the molecular relationship between compounds and proteins, we also examined the molecular docking of selected compounds, which enables us to represent the behaviour of these molecules at the coupling site of targeted proteins and clarify the biochemical mechanism of anthelminthic action. it is inferred from the findings (as seen in table 12) that gallocatechin (-7.1 kcal/mol) and rhynchosin (-6.2 kcal/mol) displayed substantial docking scores similar to those of mebendazole (-5.4 kcal/mol), the reference drug. a relatively better docking score than the standard medication, mebendazole, is gallocatechin and iraqi j pharm sci, vol.31(1) 2022 in silico, in vitro studies on rhynchosia cana 268 rhynchosin. it is clear from the outcome of the docking study that these compounds, particularly gallocatechin, maybe a good candidate for a new anthelmintic agent. the adme and toxicity analyses revealed that all compounds met lipinski's rule of five for being recognised as having drug-like potential in terms of improved pharmacokinetic properties with fewer adverse effects. conclusion we can assume from the above discussion that this plant can play a prominent role in the antioxidant and anthelmintic activity. our molecular docking analysis showed that rhynchosin, luteolin3',4'-dimethyl ether, isoorientin, orientin, and gallocatechin could be valuable sources for developing novel anti-oxidants and anthelmintic agents. however, further 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radicals and other reactive species in disease. e ls. 2015:1-9. doi: 10.1002/9780470015902.a0002269.pub3. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension doi: https://doi.org/10.31351/vol31iss1pp43-56 43 improvement of the solubility and dissolution characteristics of risperidone via nanosuspension formulations. hayder emad jabar *,1 and shaimaa nazar abd-alhammid ** *department of pharmaceutics, college of pharmacy, university of al-ameed, karbala, iraq *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract risperidone is an atypical antipsychotic drug that is used for treating schizophrenia, bipolar mania, and autism. risperidone rebalances dopamine and serotonin to improve thinking, mood, and behavior by working on dopamine and serotonin α2receptor antagonism. risperidone has poor solubility and high permeability through the intestine, so it belongs to biopharmaceutical classification system (bcs) class ii exhibits poor oral biopharmaceutical properties. the aim of the present work was to improve solubility and dissolution of risperidone by preparing nanosuspension using different stabilizers and different solvents in a method known as solvent-antisolvent precipitation method. twenty-eight formulas were prepared and evaluated particle size, pdi, (ee), zeta potential, and in-vitro dissolution studies. the results showed that particle size of nanosuspension was nanosized for all formulations. the best formula (f13) has particle size (40.9) nm containing a (soluplus) as a stabilizer in ratio 1:1 with drug by using acetone as a solvent in ratio 1:5 with water which was act as anti-solvent with stirring speed 1000 rpm and e.e % was 98%. for self-dispersible dry nanosuspension, the selected formula (f13) shown fast dispersibility of less than 1 min. and complete in-vitro dissolution to about 30 min. in 0.1n hcl. xrd and dsc indicate the transformation of a crystalline form of risperidone into an amorphous form. the stability studies of the best formula (f13) suggest that estimated shelf life was about 4 years. in conclusion; the formulation of poorly water-soluble risperidone as nanosuspension significantly improved the dissolution rate of drug and enhanced its solubility. keywords: risperidone, nanosuspension, solubility, particle size. نانوية معلقة للريسبيريدون عن طريق تركيبات تحلل خارج الجسمتحسين خصائص الذوبان و ** شيماء نزار عبد الحميدو 1, *حيدر عماد جبار .قالعرا كربالء، جامعة العميد، ، فرع الصيدالنيات، كلية الصيدلة* . قبغداد، العرا جامعة بغداد، فرع الصيدالنيات، كلية الصيدلة، * الخالصة والتوحد. القطب ثنائي واالضطراب الشخصية انفصام لعالج يستخدم الذهان لمرض نمطي غير دواء هو الريسبيريدون دواء وتونين الريسبيريدون يعيد توازن الدوبامين والسيروتونين ليدعم وظائف التفكير والمزاج والتصرف من خالل عمله على مستقبالت الدوبامين والسير المعاكسة تصنيف ومستقبالت لنظام الثاني بالتصنيف يصنف ولذلك األمعاء خالل من عالية نفاذية وله الماء في الذوبان قليل الريسبيريدون الفا. الصيدالنيات البيولوجية كدواء له خصائص ضئيلة باالستخدام الفموي. الهدف من البحث هو دعم ذوبانية وتحلل الريسبيريدون من خالل تحضير نانوي معلق النانوي. كصيغة المعلق لتحضير المذيب ومضاد بالمذيب ترسيب مثبتات بأ بطريقة ,soluplus, poloxamer 188ستخدام poloxamer 407, pvp k30)) .لحجم ثمانية وعشرون تركيبة قيم لها اومذيبات مختلفة)ميثانول, اسيتون أو االيثانول( بطريقة ترسيب المذيب أظهرت أن الحجم الحبيبي لمعلق الريسبيريدون كان ضمن الحجم جهد الزيتاودراسة تحلل المادة خارج الجسم.. النتائجالدواء والحبيبي ونسبة انحباس مع الدواء الخام بإستخدام االسيتون 1:1كمثبت بنسبه soluplusوالتي تحوي nm (40.9) ولها حجم حبيبي (f13)النانوي. وأفضل تركيبة كانت .للمعلق النانوي الجاف ذاتي % 98نحباس دوائي تقدر ب إدورة بالدقيقة وبنسبة 1000مع الماء مضاد للمذيب بسرعة دوران 1:5كمذيب بنسبة أشارا dscو 0.1n hcl. xrdالحامضي دقيقة في وسط 30الذوبان تظهر سرعة ذوبانية أقل من دقيقة وتحلل كامل خارج الجسم بحوالي )للريسب تحول الشكل البلوري أن العمر االفتراضي للتركيبة ( ما يقارب األربع f13يريدون الى غير متبلور. دراسة استقراريه الدواء تشير إلى سنوات. نستنتج ان تحضير الريسبيريدون قليل الذوبان في الماء كمعلق نانوي أدى الى تحسن كبير في حل الدواء وعزز قابليته في الذوبان. .ذوبانية يدون، معلق نانوي، حجم الجسيم،ريسبيرالكلمات المفتاحية: introduction biopharmaceutical classification is such a system that provides a profile for each drug substance based on its permeability and solubility at several media. accordingly, drugs are classified into four groups and become a benchmark in the regulation of bioequivalence of oral drug products. these classes are the following (1): class i (highly soluble, highly permeable), class ii (poorly soluble, highly permeable), class iii (highly soluble, poorly permeable), and class iv (poorly soluble, poorly permeable). decrease particle size leads to increase solubility and enhance dissolution. many techniques were used to decrease particle size. nanotechnology-based approaches used to enhancement of drug solubility rely on reducing drug size to nanoparticles (2). nanosuspension is one of nanotechnology approaches, which is a biphasic system consisting of pure drug particles dispersed in an aqueous vehicle in which the diameter of the 1corresponding author e-mail: hyderimad@gmail.com received: 19/2/2021 accepted: 25/7 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp43-56 iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 44 suspended particle is less than 1µm in size (3). an essential role of stabilizers is to compensate for the additional non-bound energy of newly revealed surfaces. rigorously wetting drug particulates, hindering ostwald ripening, and agglomeration of nanosuspension particulates are the most important advantages of adding pharmaceutical stabilizers (4). the widely used techniques of stabilization are steric and/or electrostatic stabilization, both ionic surfactants and charged polymers act as electrostatic stabilizers and non-ionic surfactants act as steric stabilizers (5). maintenance of stability of nanocrystalline structure is of great significance results from polymer steric stabilization most widely applied pharmaceutical excipients as polymeric stabilizers are (soluplus, poloxamer 188, poloxamer 407 and pvp k30) k30). risperidone is a benzisoxazole atypical antipsychotic, reported being an antagonist at dopamine d2, serotonin (5-ht2), adrenergic (α1 and α2), and histamine (h1) receptors. it is given orally for the treatment of schizophrenia and other psychoses and in short-term treatment of acute manic or mixed episodes associated with bipolar disorder. risperidone is effective for treating positive and negative symptoms of schizophrenia owing to its affinity for its loose binding affinity for dopamine d2 receptors and additional 5-ht antagonism compared to firstgeneration antipsychotics, which are strong, non-specific dopamine d2 receptor antagonists(6). figure 1. chemical structure of risperidone (6) this study aimed to formulate risperidone nanosuspension by the use of solventantisolvent technique. an attempt to enhance risperidone solubility and improve dissolution rate. materials and method materials risperidone, soluplus purchased from basf/ switzerland and all other solvents, disodium hydrogen phosphate, potassium dihydrogen phosphate, and other stabilizers purchased from china. preparation of risperidone nanosuspension by liquid solventanti-solvent precipitation 2 mg of risperidone was dissolved in 1 ml methanol (acetone or ethanol) which is an organic solvent and sonicated in an ultrasonic bath for 15 min. on the other hand, 5ml of deionized water containing, stabilizers (soluplus, poloxamer 188, poloxamer 407, and pvp k30) in ratio 1:1 that act as the antisolvent system. the following step is the addition of organic solvent slowly into aqueous solution at rate of (0.5 ml per min) of using a syringe pump, under mechanical agitation at different speeds (300, 1000, or 2000 rpm) with aid of a hotplate magnetic stirrer at room temperature (7). then, evaporation of the organic solvent was done after stirring the sample at low stirring speeds for a half-hour at 25oc temperature to get coveted nanosuspension (8). change in solvent and anti-solvent volume and stirring speed was done to formulate different risperidone nanosuspension preparation formulas shown in table (1). determination of saturation solubility solubility of saturation is determined by putting an excessive amount of pure risperidone powder determined in 10 ml in various media where 0.1n hcl with 2% w/v brij 35, water with 2% w/v brij 35, phosphate buffer ph 6.8 with 2% w/v brij 35, and methanol were used. excess amount of drug was added to all of prepared media and kept in an (incubator shaker) at 25 °c, and after three days, prepared solution has been put in a centrifuge at 4000 rpm for duration of 10 min. then, filtration by filter syringe of 0.45μm and dilution with particular solution of supernatants was done afterward. absorbance was calculated according to calibration curves and it is determined at a wavelength for each media using uvspectrophotometer(carry win uv, varian, australia) and solubility (9). measurement of particle size and polydispersity index size of particle and index of polydispersity were determined by using the nano brook 90plus particle size analyzer which is a dynamic light scattering, act by measuring the intensity of light scattered by the molecules in the sample as a function of time, at a scattering angle of 90º, and a constant temperature of 25 ºc. dilute suspensions prepared, a short ultrasonication is sometimes auxiliary to separate loosely-held agglomerates. 2 or 3 ml of nanosuspension is required to make the measurements. disposable, acrylic square cells used for aqueous and alcohol suspensions (10). iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 45 table 1 .composition of risperidone nanosuspension using different parameters stirring speed(rpm) ratio solvent ratio stabilizer stirring speed(rpm) ratio solvent ratio stabilizer 1000 1: 5 acetone 1: 1 pvp k-30 f15 1000 1: 5 methanol 1: 1 soluplus f1 1000 1: 5 ethanol 1: 1 pvp k-30 f16 1000 1: 5 methanol 1: 1 pvp k-30 f2 1000 1: 5 acetone 1: 1 poloxamr188 f17 1000 1: 5 methanol 1: 1 poloxamer188 f3 1000 1: 5 ethanol 1: 1 poloxamr188 f18 1000 1: 5 methanol 1: 1 poloxamer407 f4 1000 1: 5 acetone 1: 1 poloxamr407 f19 1000 1: 10 methanol 1: 1 soluplus f5 1000 1: 5 ethanol 1: 1 poloxamr407 f20 1000 1: 15 methanol 1: 1 soluplus f6 300 1: 5 methanl 1: 1 soluplus f21 1000 1: 10 methanol 1: 1 pvp k-30 f7 2000 1: 5 methanl 1: 1 soluplus f22 1000 1: 15 methanol 1: 1 pvp k-30 f8 300 1: 5 methanl 1: 1 pvp k-30 f23 1000 1: 10 methanol 1: 1 poloxamer188 f9 2000 1: 5 methanol 1: 1 pvp k-30 f24 1000 1: 15 methanol 1: 1 poloxamer188 f10 300 1: 5 methanol 1: 1 poloxamer188 f25 1000 1: 10 methanol 1: 1 poloxamer407 f11 2000 1: 5 methanol 1: 1 poloxamer188 f26 1000 1: 15 methanol 1: 1 poloxamer407 f12 300 1: 5 methanol 1: 1 poloxamer407 f27 1000 1: 5 acetone 1: 1 soluplus f13 2000 1: 5 methanol 1: 1 poloxamer407 f28 1000 1: 5 ethanol 1: 1 soluplus f14 iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 46 determination of entrapment efficiency of a drug (ee) in nanosuspension nanosuspensions of different ratios were centrifuged at about 5000rpm for 20 min. at 4°c using a cooling ultracentrifuge. the amount of free drug was detected by calculating the absorbance of an appropriately diluted sample of supernatant at 280 nm using a uv spectrophotometer (carry win uv, varian, australia). for each formula, the experiment was repeated three times and the average was calculated. (ee) could be calculated by the following equation: e.e%= (the total drug in a formula – free drug) *100 / total drug in formula .... (1) in vitro dissolution of risperidone nanosuspensions dissolution study of prepared nanosuspension was done under sink condition using paddle-type ii according to usp pharmacopeia. briefly, freshly an accurately weighed plain drug (2mg), and prepared nanosuspensions of (f1, f2, f5, f6, f13, and f21) formulas were placed in a pretreated dialysis bag (dialysis membrane); soaked in dissolution media overnight before use; and fitted with a paddle then dispersed in 900ml of 0.1n hcl with 2% w/v brij35 solution as dissolution medium. the paddle has been built up to be rotated at 100 rpm maintained at 37 ± 0.5°c. an aliquot of (5ml) samples was withdrawn from the receiver compartment at predetermined time intervals (5, 10, 15, 20, 30, 45, 60,120) min and replenished with the equivalent volume of fresh dissolution medium to preserve the constant volume. and then, samples were analyzed for risperidone content spectrophotometrically after proper dilution using uv-spectrophotometer scanning at 200400nm. the experiment repeated in triplicate for each formulation (11). zeta potential evaluation of nanosuspension zeta-potential evaluated by the use of zetasizer (zetasizer nano zs, malvern instrument, worcestershire, uk) (12). the characteristics of surface charge were studied to assess the stability of the prepared nanosuspension and lyophilized nanoparticle formulas. the minimum limit needed for electrostatic stabilization of nanosuspension is ± 30 mv and for steric stabilization of ± 20 mv (13) freeze drying of nanosuspension freeze drying was used to convert the optimum formula to dry powder, later for further evaluation. mannitol used as a cryoprotectant at 3% w/v. about 400ml of optimized formula was prepared and freezedried to yield a dry powder for evaluation. four flasks froze in a deep freezer at -20°c for 24 hr. the frozen flasks were attached to the vacuum port of the device, then four flasks each containing 100ml of nanosuspension, instrument operated till dry powder yielded. sublimation of solvent from frozen samples took 48 -72hr (14). effect of stirring speed on the particle size of risperidone nanosuspension three different speeds were used to show their effect on the formulation of risperidone nanosuspension. speeds of 300 and 2000 rpm used in the formulas (f21-f28) were used to study this effect effect of solvent/antisolvent ratio change on the particle size of risperidone nanosuspension study the effect of change in solvent/anti-solvent ratio in nanosuspension where the change in volume of deionized water (anti-solvent) from 50 ml to 100 ml and 150 ml. formulas (f5-f12) were used (15). effect of polymer type when using different solvents on the particle size of risperidone nanosuspension by changing the solvent (methanol)in formulas (f1-f5) to acetone and ethanol. formulas (f13-f20) were used (16). characterization of lyophilized powder saturation solubility of lyophilized powder three dissolving solvents water, 0.1 n hcl with ph 1.2, and phosphate buffer ph 6.8 were used to investigate the saturation solubility of lyophilized formula (f13). an excess amount of drug was added to each test tube containing the above-mentioned solvents then were shaken for at least 48hr in a water bath shaker at 25 °c. then filter with a conventional filter paper before reading for each sample. sample test tubes were centrifuged at 6000 rpm for 15 min. the absorbance of the supernatant was recorded and a calibration curve was used to determine the amount of drug dissolved in the specific volume of each dissolving media (17). the in-vitro dissolution profile of lyophilized risperidone nanoparticle in-vitro dissolution test of risperidone lyophilized powder was estimated using (paddle assembly) type ii dissolution test apparatus. accurately weighed lyophilized powder equivalent to 2 mg of risperidone was placed on a pretreated dialysis bag and fitted to the paddle which rotates at 100 rpm at 37 °c± 0.5 in 900ml of dissolution medium 0.1n hcl. an aliquot of 5-milliliter samples was withdrawn from the receiver compartment at predetermined time intervals and refilled the iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 47 equal volume of fresh dissolution medium to conserve the constant volume. the same procedure was applied on lyophilized powder with the same conditions at 100 rpm rotation of paddle containing phosphate buffer (ph 6.8). then samples were filtered by 0.45μm filter syringe and assayed spectrophotometrically by uv–spectrophotometer. results obtained were compared using similarity factors and the experiment was repeated in triplicate (18). differential scanning calorimetry (dsc) dsc can be used to evaluate the crystalline state of drug especially when converted to a lyophilized powder. 8-10 mg sample of the pure risperidone and lyophilized powder of the selected formulations heated at a scanning rate of 10°c/min. this done under dry nitrogen flow (100 ml/min) between 30 and 300° against an empty aluminum pan as a reference using dscshimadzu 60 with tda trend line software (19). x-ray powder diffraction (xrpd) analysis patterns for pure risperidone and nanoparticles were analyzed using an xrd6000, shimadzu-japan. the continuous scan range of 5-80 degree. 40 (kv) was for the operating voltage, 30ma for the current, scan step size of 0.050° (2θ), and scan step time of 60 sec (20). scanning electron microscopy (sem) study the morphology of pure drug and lyophilized formula were examined by (vega3tuscan) by direct deposition of powder on double-sided carbon tape and coated with gold at 1k, 2k, 5k, and 500x magnification then coated with gold (21). stability study in specific capsules, the selected formula powder was filled and stored at three various 40°c, 50°c, and 60°c temperatures degrees for 90 days, at an interval of 14 days, samples were dissolved in 100 ml methanol. and then, the drug concentration was measured spectrophotometrically at λ max considering this solvent as blank. k at 25°c was predicted from the arrhenius plot and the date of expiration was determined (22). statistical analysis the results experiments were expressed as a mean triplicate sample ± standard deviation (sd) and the analysis was done with the help of one-way analysis of variance (anova) using spss software at which the results would be significant when p < 0.05, and the results would be non-significant if p> 0.05 (23). difference factor (f1), similarity factors (f2) were also calculated to compare dissolution profiles, using dd solver program. the difference factor (f1) measures the percent error between two curves over all time points. the similarity factor (f 2) was introduced by moore and flanner (24). results and discussion determination of risperidone saturation solubility the solubility study was performed in four different media, 0.1n hcl, phosphate buffer ph 6.8 methanol, and distilled water. risperidone is a weak base with pka value of 8.76 and expected to be more soluble in an acidic medium of about 1.3 mg/ml. in contrast, risperidone solubility in phosphate buffer was very low with a solubility value of 0.61mg/ml. risperidone was soluble in organic solvent(methanol) in 6.1mg/ml. on the other hand, the water solubility of risperidone was the lowest at about 0.019 mg /ml which considered practically insoluble. characterization of the prepared risperidone nanosuspension determination of the particle size and polydispersity index the estimated average particle size was in the range of (40.9 nm – 1286 nm) and were illustrated in table (2). on the contrary, the pdi which measures the size distribution of the nanoparticles; were ranged from (0.1-0.62) depending on formulation variables, (0.1) indicating good uniformity, a monodisperse system in the particle size distribution of nanoparticles while (0.62) indicated polydisperse system. effect of stirring speed of the anti-solvent system when the stirring was 300 rpm the process failed to nanosizing the stabilizer around the drug due to the stuffiest energy required to achieve the mixing. besides that, high stirring speed (2000 rpm) not always favored due to high agitation with the formation of foams that separate drug particles from the vehicle medium which act as an obstacle in the preparation process, thus an optimum speed (1000 rpm)is required to have optimum drug particles within the acceptable range (25). effect of solvent/antisolvent ratio the results illustrated in figure (2). it can be seen that when the ratio increases between the solvent and water increase particle size due to decrease the quality of mixing of media because of the decrease in specific stirrer speed to the solution that need to nucleation causing growth formation and increase flocculation (26). increase in water volume decrease stabilizer in water leading to decrease stabilization and increase particle size. formulas from f5to f12 were significant differences (p<0.05) in particle size and pdi of these formulas. iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 48 table 2. particle size, polydispersity index, and e.e. of formulas from 28 formula data are expressed as mean ± sd, p <0.05. standard deviation (sd) (mean±sd) n=3. zeta potential e.e% pdi particle size(nm) zeta potential e.e, pdi particle size(nm) -20 96.26±0.2 0.35 335 f15 -14.59 97.2±0.2 0.173 51.2 f1 -19.2 95±0.1 0.34 160.3 f16 -16.24 92± 0.2 0.264 77.8 f2 -18.5 92±0.14 0.3 260 f17 -12.76 90±0.21 0.353 233.5 f3 -16.7 89±0.2 0.36 450 f18 -11.39 87± 0.2 0.465 245.5 f4 -18.9 88±0.3 0.3 502 f19 -10.2 88±0.05 0.26 134.3 f5 -11.55 84±0.15 0.327 492 f20 -11.55 92±0.05 0.19 92.8 f6 -16 90.1±0.2 0.4 140 f21 -12 85±0.05 0.518 162 f7 -11.4 87±0.1 0.38 210 f22 -10.3 90±0.05 0.37 216.9 f8 -10 92±0.19 1.8 371 f23 -9.1 88± 0.2 0.48 602 f9 -9.2 88±0.05 0.36 449 f24 -8.9 94±0.2 0.31 867 f10 -6.9 93±0.2 0.3 308.5 f25 -15.1 84±0.2 0.6 271 f11 -15.7 82±0.2 0.14 811 f26 -16 88±0.2 0.6 333 f12 -16 94±0.3 0.3 815 f27 -18.8 98±0.2 0.1 40.9 f13 -17.8 84±0.3 0.4 1286 f28 -17 94.2±0.2 0.178 92.15 f14 figure 2. the effect of solvent/antisolvent on the particle size features on the prepared risperidone nanosuspension effect of polymer type on particle size when using different solvents acetone and ethanol were used in the preparation of nanosuspension in the formulas (f13f20) and compared to the formulas prepared with methanol (f1, f2, f3, and f4). for acetone, as shown in figure (3), the ratio of the drug to the stabilizer was fixed to a 1:1 ratio to study the effect of the type of the solvent. formula f13 in which acetone was used as a solvent and soluplus as a stabilizer showed the smallest particle size among all formulations with other stabilizers within an optimum range of 40 nm and a pdi was 0.101 which indicate a good uniform particle size distribution. for formulas, f15 and f17 showed almost similar results of particle size 335 nm and 260 nm and a pdi of 0.34 and 0.32 which indicate almost homogenous particle size distribution. while f19 formula showed a bigger particle size of 502 nm and pdi of 0.308 which is similar to other formulas. this due to the strength of solvents polarity, where the acetone considered less polar and best miscible with water than methanol or ethanol, so risperidone highly solubility in acetone (27). compared with other solvents (methanol, ethanol) that gave a good miscibility degree to a solvent carry the drug and facilitate diffusion when mixed by dripping with antisolvent media that contain a stabilizer leading to the formation of highly stabilizing nanosuspension by reducing the surface energy of the fine particles and effective energy barrier to prevent agglomeration and crystal growth. besides, there is a fact that the soluplus is soluble up to (50%, 45%, and 25%) than water in acetone methanol and iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 49 ethanol consecutively, where the process of precipitation method and before the solvent was evaporated the solvent could be contributed by made complete dissolved miscible component whether it was the drug or the stabilizer (28) figure 3. effect of polymer type when using acetone on particle size for f13, f15, f17, and f19 for methanol as a solvent result in figure (4) showed that formulas f1 and f2 showed particle sizes of 51 nm and 77.8 nm respectively but the f2 formula showed more uniform particle size distribution with pdi of 0.17. formulas f3 and f4 presented a similar pattern of particle size and their distribution form of 233.5 nm and 245.5 nm respectively, and a pdi was 0.353 and 0.465 respectively. these results indicate similar superiority in using both stabilizers soluplus and pvp k30. figure 4. effect of polymer type when using methanol on the particle size of f1, f2, f3, and f4 for ethanol as a solvent, results are shown in figure (5). f14 formula where soluplus used showed the smallest significant particle size of 92 nm (p<0.05) and provided a uniform particle size distribution within the formulation with a pdi of 0.101. while f16 formula where pvp k30 was used presented particle size 160.3 nm with almost a uniform homogenous size distribution with pdi of 0.34. formulas f18 and f20 showed about the same results when used poloxamer 188 and poloxamer 407 with a particle size of 450 nm with pdi of 0.36 and 490 nm with pdi of 0.32 respectively. iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 50 figure 5. effect of polymer type when using ethanol on particle size for the formulations f14, f16, f18, and f20 in – vitro drug release study the dissolution profile of the prepared risperidone nanosuspension formulas is illustrated in figure (6). the results showed a significant increase in the dissolution rate of the risperidone nanosuspension formulas which reach 100% release within 45 min when compared to the pure drug that reached 56 % release within the same time frame. also, the f13 formula presented the fastest complete release within 35 min, while pure drug showed the slowest incomplete release profile within the specified time. figure 6. the release profile of risperidone nanosuspension in 0.1n hcl to validate these findings, the fit factors have been used and expressed by two factors; f1 the difference factor, and f2 the similarity factor. for two dissolution profiles to be considered similar and bioequivalent, f1 should be between 0 to 15 whereas, f2 should be in the range of 50 to 100. the similarity between f13 of the best release profile with the pure drug release profile showed the similarity value (f2) was 23.7 which is less than 50 which indicates a significant improvement in the release behavior. also, the difference factor value was 84.2 which confirmed the previous similarity test results and conclude the greater and faster release pattern of the best formula f13. f13 formula was selected to investigate the best release profile in two different mediums 0.1n hcl and 6.8 phosphates and the comparison was shown in figure (7). it can be seen that the f13 formula showed a faster release profile in 0.1n hcl than phosphate buffer ph 6.8. in terms of validation, the similarity and difference factors were calculated and provided that, the similarity factor obtained was equal to 26.7 and the difference factor was equal to 84.4. according to these data results, 0.1n hcl is considered the best medium for drug release of the selected f13 formula. this comparison in similarity and difference factor were significantly improved. figure 7. the release profile of f13 nanoparticle in phosphate buffer ph 6.8 and 0.1n hcl selection of the best formula according to the previous results of all required tests of particle size determination, polydispersity index, entrapment efficiency and invitro drug release it can be concluded that the f13 nanosuspension formula was the best formula with the smallest particle size of 40.9 nm and pdi value of 0.101 and zeta potential -18 showed highest drug entrapment efficiency of about 98.7 which will be subjected for further characterization after lyophilization and converted to nanoparticle by determining zeta potential, powder x-ray properties, drug excipient compatibility, in vitro drug release after lyophilization by freeze-drying process solidification of risperidone nanosuspension by freeze-drying the selected optimized formula f13 was freeze-dried to obtain the powder form of the prepared nanosuspension, enhanced amorphization, and subjected for further studies of solubility and invitro drug release. characterization of the lyophilized selected formula self-dispersibility of freeze-dried powder in aqueous medium the selected f13 lyophilized powder was tested for self-dispersibility in aqueous media. the iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 51 powder was completely dispersed within seconds in the aqueous media. the dispersed formula was tested for the particle size and polydispersity index and the results showed that the particle size was 60.5 nm which is higher than the particle size of the same formula when it was nanosuspension and the pdi of the lyophilized powder was 0.115 which is also higher which was 0.101 the zeta potential of the lyophilized f13 formula showed -18.96 mv which is similar to the value obtained before lyophilization which indicates the robust formulation by the action of the selected polymer with risperidone drug particles and last durable after freeze-drying. these results indicate that the lyophilized f13 formula provided an accepted effective property of the nanosuspension formula which makes it potential for further study. solubility study of the selected lyophilized formula solubility study done at three different media; water, 0.1n hcl, and phosphate buffer ph 6.8(all with 2% w/v brij 35). the results illustrated in figure (8) and showed risperidone water solubility has significantly increased by about 105 times when compared with the one before lyophilization. risperidone showed higher solubility in 0.1nhcl that results in higher and faster drug release when compared to the pure drug about 13.5 folds these were responsible for the significantly enhanced (p<0.05) saturation solubility. figure 8. solubility of lyophilized f13 in distilled water, phosphate buffer ph 6.8 and 0.1n 0.1n hcl in – vitro drug release study of the lyophilized selected formula the dissolution study of the selected lyophilized f13 formula was performed in 0.1n hcl with 2% w/v brij35 and compared with the pure drug and the results presented in figure (9). it can be seen that lyophilized f13 formula nanosuspension showed a very characteristic enhancement in drug release when compared to the pure drug with faster and complete drug release within 30 min when compared to the pure drug. this advancement in drug release was confirmed by using the similarity factor and showed 27.4 (f2<50) and the different factor was 83.6 (f1>50) which is explained due to better dissolution characteristics which in turn theoretically will boost the dissolution and absorption rate of risperidone nanosuspension and eventually its bioavailability. figure 9. drug release percentage of the lyophilized risperidone differential scanning calorimetry (dsc) lyophilized f13 was selected to investigate the crystalline purity after lyophilization and the result is presented in figure (10). the figure shows a sharp peak at 170°c, and it is similar as reported and indicates the purity of the drug (29). on the other hand, an endothermic broad peak at 162°c is shown for the lyophilized risperidone. broad endothermic peak is including lower in melting points, broadening, shifting, and reducing in intensities supported the suggestion of a reduction in the crystalline state of risperidone optimized formula with a significant increment in the amorphous state that formed in lyophilization (30). iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 52 figure 10. dsc of pure risperidone and the lyophilized f13 x-ray powder diffraction (xrpd) the diffractograms of pure drug and lyophilized f13 were shown in figures (11) and (12), respectively. it can be seen that there is a significant change in the x-ray pattern after the preparation of the nanosuspension formulation. the pure drug showed high intense peaks at 14°, 15°, 19°, 21°, 22°, 23°, 24°, 33°, and 34° that indicates a high degree of crystallinity of the pure risperidone. although, after the preparation of the lyophilized powder, leaser intensity peaks are observed which indicates a significant change in the crystallinity nature of the drug to be more amorphous. it has been noted that the emergence of new peaks at 31°, 39°, and 43° are related to the mannitol that has been added to the lyophilized powder (31). these changes indicate higher stability of the prepared lyophilized powder with the enhancement of water wettability and dissolution profile (32). figure 11. xrd diffraction of pure risperidone iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 53 figure 12. xrd diffraction of the lyophilized f13 scanning electron microscopy (sem) the sem pictures are shown in figures (13) and (14). the sem of the pure powder showed coarse agglomerates of risperidone crystals that packed together forming larger particles. lyophilized f13 showed a homogenous dispersion of small flake irregular particles within a nanosized range contributed to cryoprotectants employed in nanoparticles where the freeze-drying process could influence the morphology. the surface area of nanoparticles affects dissolution. the larger surface area could increase the dissolution rate of nanoparticles (33). figure 13. sem of risperidone pure powder iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 54 figure 14. sem of lyophilized selected f13 stability study of the selected f13 lyophilized powder the prepared risperidone lyophilized powder was investigated for stability study at three accelerated temperatures 40°c, 50°c, and 60°c for 16 consecutive weeks. the f13 lyophilized powder was subjected to the study and the results are illustrated in figure (15). table 3.the degradation constant of the selected formula f13 lyophilized powder the temperature in ºc k(week-1) 40 ºc 1.84 × 10-3 50 ºc 3.454× 10-3 60 ºc 7.83× 10-3 expiration date for risperidone in f13 lyophilized powder determined through the application of the following equation: t90% remaining = 0.105 / k25 figure 15. accelerated degradation of the selected f13 lyophilized powder at 40 ⸰c, 50 ⸰c, and 60 ⸰c the degradation constant was calculated through constructing arrhenius plot as shown in figure (16). t90% was determined according to the previously mentioned equation and found to be 205.09 weeks that equal to 3.9 years. figure 16. arrhenius plot of the prepared risperidone in the selected f13 lyophilized powder for estimation of the expiration date iraqi j pharm sci, vol.31(1) 2022 risperidone nanosuspension 55 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department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract high frequencies of multidrug resistant organisms were observed worldwide in intensive care units which is a warning as to use the only few effective antimicrobials wisely to reduce selective pressure on sensitive strains. the aim of the current study is to asses the compliance of the currently followed antibiotic prescribing pattern in the intensive care unit in an iraqi hospital with the international guidelines.a cross-sectional study was done in the intensive care unit (icu) of the surgical specialties hospital, medical city in bagdad from the 30 th of november 2011 to the 5 th of may 2012.patients were followed until they were discharged or died to see any change in condition, response to drugs, devices used, and medications. during the period of the study, there were 46 patients admitted to the icu of whom 23 (50%) were males and 23 (50%) were females. the age range of patients was between 16 and 85 years. the mean of age of patients was 44.52 (sd±18.45) years. all patients underwent culture and sensitivity test as soon as they were admitted to the icu, but out of 46 patients only 16 (34.78%) of them have culture and sensitivity tests results retrieved.the number of patients, in whom the antibiotics were prescribed or changed according to culture and sensitivity tests, was six patients only (13.04%). ceftriaxone was the most commonly used antibiotic as an empiric treatment followed by ampicillin-cloxacillin combination and meropenem, while clarithromycin and ciprofloxacin were among the least used. the bacterial culture and sensitivity tests of different samples obtained from the patients showed that the most effective antibiotic was tobramycin (90%) followed by ciprofloxacin and levofloxacin (69.2%) for each and the least effective (bacterial resistance) was ceftriaxone (33.3%) and cefotaxime (28.5) among others. in conclusion, there is a critical need for reviewing the trend of antibiotic use in the icus depending more on lab. tests to identify the most effective drugs and to minimize the emergence of resistant infection. keywords: icu, antibiotic resistance, culture and sensitivity tests. طريقت استخذام المضاداث الحيويت في وحذة العنايت المركسة في مذينت الطب في بغذاد زينت مظفر النعمت ،*1 الصُدلة،جبمؼة بغداد، بغداد، الؼساق.فسع الصُدلة السسَسَة، كلُة الخالصة نظرسا لثررسو وجررىد الثب ربم الة بومررة للؼدَرد مر ا دوَررة فرٍ و رردام الؼ بَرة الةسكرجو ال بررد مر الدىجرر رد دا الة رربدام الىصرابم البيُرة للة ربدام الهرد مر الدزا رة الحبلُرة ترى لد ُرُا امدررب الحُىَة بحثةة للد لُل م ال غط ػلرً السرت م الحسب رة. لدزا رة الحبلُرة م بؼُرة اجسَر فرٍ ا الحُىَة الةديؼة بلُب فٍ و دو الؼ بَة الةسكجو فٍ مسدشاً ػساقرٍ مرغ االىصرابم الةديؼرة دولُرب. .1321 اَبز 5إلً 1322تشسَ الربنٍ 03و دو الؼ بَة الةسكجو فٍ مسدشاً الجسا بم الد صصُة فٍ مدَ ة البب فٍ بغداد م تا مدببؼة الةسضً الداخلُ للى دو دً خسوجها أو مىتها لةؼسفة أٌ تغُُس فٍ الحبلة وا دجببة لألدوَة و كررل اججهرجو الةسرد دمة . وا دوَة الةسد دمة ا نرب 7( م 53) 710( م الركىز و 53) 10مسَ ب ادخل الً و دو الؼ بَة الةسكجو م ها 64خت فدسو الدزا ة، كبن ت بك ( ػررب ال خ ررغ جةُررغ 25.65±)ا نحررسا الةؼُرربزٌ 51ال66 ػررب وكرربن مدى ررط ػةررس الةسضررً 55الررً 24ذوٌ الائررة الؼةسَررة مرر 24الةسضً ػ د الدخى لسدتة الؼ بَة الةسكجو خديبزام الحسب ُة للة بدام الحُىَةال ولثر ترا الحصرى ػلرً ندرب ت ا خديربزام ض الوكبن ػدد الةسضً الرَ تا وصف او تغُُرس الة ربدام الحُىَرة اػدةربدا ػلرً ندرب ت اخديربزام مسَ 64( مسَض ف ط م 45ال06) 7( ف ط. واظهسم الدزا ة ان دواء ادساَبكسىن ا كرس شُىػب فٍ ا د دا كؼتج اولٍ ثا َلُ الردواء الةسكرب 36ال20) 4الحسب ُة بنررر ا دوَرررة الدبلُرررة كتزَرسومبَسرررُ و ُيسوفلىكسب رررُ ا قرررل ا رررد دامب. مررر ا ميسرررُلُ والثلىكسب رررلُ ثرررا دواء ا َةُيُ رررُا. وك واوضح ندرب ت اخديربزام الحسب رُة الدرٍ ترا الحصرى ػلُهرب فرٍ الدزا رة الحبلُرة ان الة ربد الحُرىٌ ا كررس فؼبلُرة ترى تىبسامبَسرُ لثرررل م هةرررب وا قرررل فؼبلُرررة فرررٍ م بومرررة اليثدسَرررب ترررى 7( .1ال46 7( وَلُررر ببلاؼبلُرررة دواء ُيسوفلىكسب رررُ و اللُاىفلىكسب رررُ )63) 7(.5ال715( والسُاىتبكسُا )1ال00 ادساَبكسىن ) فٍ ال دب بد م الد ىَ الً ان ت بك بجة مب ة ػبدو ال ظس فٍ طسَ ة ا د دا الة بدام الحُىَة فٍ و دام الؼ بَة الةسكرجو ء ا كرس فبػلُة للد لُل م الؼدوي الة بومة لتدوَة.وا ػدةبد ػلً الدحلُتم الة ديسَة لدحدَد الدوا . اختباراث الحساسيت ،مقاومت المضاداث الحيويت ،لكلماث المفتاحيت: وحذة العنايت المركسةا 1 corresponding author e-mail: zena_alnema@yahoo.com. received: 21/2/2016 accepted: 17/5/2016 mailto:zena_alnema@yahoo.com iraqi j pharm sci, vol.25(1) 2016 trend of antibiotic use in the icu 51 introduction intensive care units (icu) are epicenters for the emergence of antibiotic-resistant gramnegative bacteria (1,2) , and multi-resistant grampositive infections, largely due to the inappropriate use of antimicrobials (3) . in addition to that, rapid patient turnover, immunological susceptibility of acutely ill patients, and frequent contact between healthcare workers and patients, facilitate cross-transmission. antibiotic stewardship programs are considered important to reduce antibiotic resistance, but the effectiveness of strategies such as, for instance, antibiotic rotation, have not been determined rigorously (1) . the patterns of such resistance clearly vary among hospitals. inadequate empirical antimicrobial treatment has also resulted in greater patient morbidity, higher mortality rates, and increased healthcare costs (4) . high frequencies of multidrug resistant organisms were observed in intensive care units which is a warning as to use the only few effective antimicrobials wisely to reduce selective pressure on sensitive strains (5) . hospital-acquired pneumonia often occurs as ventilator-associated pneumonia is the most frequent hospital infection in icus. early adequate antimicrobial therapy is an essential determinant of clinical outcome, its diagnosis and management are complex. consequently, its prevention becomes a cornerstone in daily clinical practice (6,7) . inadequately treated patients had a higher complicated pathogen risk score (cprs) compared to those who received adequate therapy. this shows that therapy based on local experiences may be sufficient for patients with low cprs but inadequate for those with high cprs (7) . therefore, guideline-adherent initial intravenous antibiotic therapy is clinically superior, saves lives and is less expensive than non-guideline adherent therapy. using a cprs can be a useful tool to determine the right choice of initial intravenous antibiotic therapy (7) . the aim of the current study is to assess the compliance of the currently followed antibiotic prescribing pattern in the intensive care unit in iraqi hospitals with the international guidelines. methods a cross-sectional study was done in the intensive care unit (icu) of the surgical specialties hospital, medical city in bagdad from the 30 th of november 2011 to the 5 th of may 2012. the icu of the specialty hospital had 16 beds with 2 isolated rooms each containing 1 bed, there is also a side lab for simple and emergency investigations. the icu receives either postoperative patients mainly neurosurgery or severely injured patients and sometimes medical patients with neurological diseases. admission is usually the responsibility of the senior physician of the icu. the staff of the icu consists of seniors specialist in anesthesia and intensive care and senior residents with trained nursing staff. clinical pharmacist is also a part of the medical team. since most of the patients were unable to talk normally, because of being critically ill or intubated (by endotracheal tube or tracheostomy), data were taken mainly from case sheets of patients and from medical staff (doctors, nurses, pharmacists, and lab technicians) samples taken for culture and sensitivity tests included urine, tracheostomy tube, blood, sputum, cerebrospinal fluid, and ventriculo-peritoneal shunt. culture and sensitivity tests were done using kirby bauer disk diffusion method (8) . patients were followed until they were discharged or died to see any change in condition, response to drugs, devices used, medications (duration of treatment, route of administration, dosage, side effects, and frequency). children were excluded because the study was designed for adults only. the followings were reported for each patient: systemic disease, diagnosis, devices used, causes of each admission, culture results, antibiotic(s) used, and indication for each and the outcome of the patient (discharged or died). chi square was used to calculate the differences between groups using p value below 0.05 as significant. results during the period of the study, there were 46 patients admitted to the icu of whom 23 (50%) were males and 23 (50%) were females. the age range of patients was between 16 and 85 years. the mean of age of patients was 44.52 (sd ±18.45) years. the duration of admission to icu ranged from one to 142 days. the mean of duration of admission was 27.45 (sd ±32.2) days. the fate of the patients who were admitted to icu during the period of study was as follows: 23 patients retuned back to their original wards to complete their therapy, 22 patients died (mortality rate 47.8%) and one patient was transferred outside iraq to complete his treatment. iraqi j pharm sci, vol.25(1) 2016 trend of antibiotic use in the icu 52 all patients were receiving medical treatment according to their condition including empirical antibiotics depending on the clinical state of the patient and the experience of the physician. all patients underwent culture and sensitivity test as soon as they were admitted to the icu, but out of 46 patients only 16 (34.78%) of them have culture and sensitivity tests results retrieved and the total number of tests performed for these 16 patients was 25 tests and as follows: urine: five culture and sensitivity tests, blood: seven culture and sensitivity tests, sputum: three culture and sensitivity tests, tracheostomy swab: eight culture and sensitivity tests, cerebrospinal fluid: one and ventriculoperitoneal shunt swab: one test. the mean time needed for culture and sensitivity test results to return back to the icu was 9.58 (sd± 4.66) days, range (1-27 days). the number of patients, in whom the antibiotics were prescribed or changed according to culture and sensitivity tests, was six (13.04%) patients only. the mean duration for empiric treatment was 5.32 (sd ±5.37) days, the empirical antibiotics were used in different combinations according to the condition of the patient and the experience of the physician, ceftriaxone was the most commonly used antibiotic as an empiric treatment (35.5%) followed by ampicillin–cloxacillin combination (6.6%) and meropenem (4.4%), while clarithromycin, and ciprofloxacin were among the least used (2.2%) for each (table 1). table (1): antibiotics used for empiric treatment. antibiotic (s) number of patients percentage of patients (%) 1 ceftriaxone 16 35.5 2 ceftriaxone + metronidazole. 3 6.6 3 ceftriaxone + ampiclox*. 3 6.6 4 ceftriaxone + ampiclox + metronidazole. 3 6.6 5 meropenem. 2 4.4 6 meropenem + ceftriaxone 2 4.4 7 meropenem + metronidazole 1 2.2 8 meropenem + azithromycin 1 2.2 9 meropenem + vancomycin 1 2.2 10 meropenem + clarithromycin + ciprofloxacin 1 2.2 11 ceftriaxone + vancomycin 1 2.2 12 ceftriaxone + ciprofloxacin 1 2.2 13 ceftriaxone + amikacin 1 2.2 14 ceftriaxone + amikacin + ampiclox 1 2.2 15 ceftriaxone + azithromycin 1 2.2 16 ceftazidime 1 2.2 17 ceftazidime + ampiclox 1 2.2 18 ceftazidime + metronidazole 1 2.2 19 ceftazidime + teicoplanin 1 2.2 20 azithromycin 1 2.2 21 vancomycin + metronidazole + cefotaxime 1 2.2 22 cefotaxime + metronidazole +ampiclox +ceftriaxone 1 2.2 *ampiclox: ampicillin-cloxacillin combination. postoperative patients represented the majority of the patients admitted to the icu, they were 21 out of 46 patients, followed by patients with motor neuropathy 7 out of 46 patients, (table 2). types of bacteria isolated from the patients were as follows: out of 16 who have culture test, results of 8 cultures show no growth of bacteria, 7 cultures with one species of bacteria, and 6 cultures with two species of bacteria, (table 3). samples taken for culture and sensitivity tests included urine, tracheostomy tube, blood, sputum, cerebrospinal fluid, and ventriculoperitoneal shunt swab, and many bacteria were isolated, and the sensitive or resistant antibiotics were displayed in details in table 4. iraqi j pharm sci, vol.25(1) 2016 trend of antibiotic use in the icu 53 table (2): diseases of patients admitted to icu. disease no. of patients 1 postoperative neurological surgery 14 2 postoperative nonneurological surgery 7 3 motor neuropathy 7 4 cva 5 5 unknown diagnosis 1 6 rta 2 7 miscellaneous pulmonary fibrosis acute pancreatitis lung cancer metastasis kyphosis head trauma burn acute respiratory distress severe chest infection hypovolemic shock 10 1 1 1 1 1 1 2 1 1 total 46 table (3): types and frequency of bacteria isolated from patients of the icu. *six patients have two species of bacteria. table (4): causative bacteria isolated from patients and their sensitivity to antibiotics in each sample in the icu sample causative organism sensitive antibiotic(s) resistant antibiotic(s) 1 urine klebsiella spp. amikacin, nitrofurantoin. ceftazidime, cefalothin, cefotaxime, ceftriaxone, co-trimoxazole, cefotetan, cefoxitin, ciprofloxacin, levofloxacin, gentamicin, tobramycin, tetracycline, chloramphenicol, piperacillin. klebsiella spp. cotrimoxazole. amikacin, aztreonam, ceftazidime, cefalothin, cefotetan, cefoxitin, ciprofloxacin, levofloxacin, gentamicin, tobramycin, tetracycline, chloramphenicol, nitrofurantoin. acinetobacter spp. tetracycline, gentamicin, tobramycin. amikacin, ampicillin, aztreonam, amoxiclav*, ceftazidime, cefuroxime, cefalothin, co-trimoxazole, nitrofurantoin, ceftriaxone, cefixime. 2 tracheost omy swab acinetobacter spp. tetracycline, doxycycline, minocycline. amikacin, ceftazidime, cefotaxime, ceftriaxone, gentamicin. baurkholderia spp. cotrimoxazole. ceftazidime. pseudomonas spp. amikacin, ceftazidime, ciprofloxacin, levofloxacin, gentamicin, tobramycin. piperacillin. acinetobacter spp. amikacin, aztreonam, ceftazidime, cefotaxime, ceftriaxone, ciprofloxacin, levofloxacin, gentamicin, tobramycin, tetracycline. piperacillin. pseudomonas spp. amikacin, ciprofloxacin, levofloxacin, gentamicin, tobramycin. piperacillin. type of bacteria isolated from patients frequency of bacteria isolated from patients* percentage of bacteria isolated from patients klebsiella spp. 7 53.8 acinetobacter spp. 5 38.46 pseudomonas spp. 3 23.07 baurkholderia spp. 2 15.3 proteus spp. 1 7.6 coagulase –ve staphylococci 1 7.6 iraqi j pharm sci, vol.25(1) 2016 trend of antibiotic use in the icu 54 table (4): (continued) causative bacteria isolated from patients and their sensitivity to antibiotics in each sample in the icu sample causative organism sensitive antibiotic(s) resistant antibiotic(s) pseudomonas spp. amikacin, ceftazidime, ciprofloxacin, levofloxacin, gentamicin, tobramycin. piperacillin. proteus spp. amikacin, cefotaxime, ceftriaxone, cefotetan, cefoxitin, ciprofloxacin, levofloxacin, tobramycin. ceftazidime, piperacillin, tetracycline, chloramphenicol. acinetobacter spp. nil 3 sputum baurkholderia spp. cotrimoxazole. ceftazidime. klebsiella spp. cefotetan, cefoxitin, ciprofloxacin, levofloxacin, gentamicin, tobramycin, tetracycline, chloramphenoicol. piperacillin. acinetobacter spp. cefotetan, cefoxitin, ciprofloxacin, levofloxacin, gentamicin, tobramycin, chloramphenicol. tetracycline. 4 blood klebsiella spp. tetracycline. amikacin, aztreonam, ceftazidime, cefotaxime, ceftriaxone, cefotatan, cefoxitin, co-trimoxazole, ciprofloxacin, levofloxacin, gentamicin. coagulase –ve staphylococci tetracycline, doxycycline, vancomycin. co-trimoxazole, clarithromycin, erythromycin, aztreonam, ciprofloxacin, levofloxacin, gentamicin, chloramphenicol, penicillin, clindamycin, methicillin. klebsiella spp. ciprofloxacin, levofloxacin piperacillin. 5 csf** klebsiella spp. amikacin, cefotetan, cefoxitin, ciprofloxacin, levofloxacin, gentamicin, tobramycin, tetracycline, chloramphenoicol. ceftazidime, cefotaxime, ceftriaxone, piperacillin. 6 vp*** shunt swab klebsiella spp. amikacin, cefotetan, cefoxitin, ciprofloxacin, levofloxacin, gentamicin, tobramycin, tetracycline, chloramphenoicol. aztreonam, ceftazidime, cefotaxime, ceftriaxone, piperacillin. *amoxiclav: amoxicillin clavulanic acid combination. **csf: cerebrospinal fluid. ***vp shunt: ventriculoperitoneal shunt. the bacterial culture and sensitivity tests of different samples obtained from the patients showed that tobramycin has the greatest sensitivity (90%) while the resistance was low (10%) and this difference is significant (p value=0.0003), whereas ceftriaxone sensitivity was low (33.3%) and the resistance was high (66.7%) and this is a non significant difference (p value= 0.248). ciprofloxacin and levofloxacin were the second effective antibiotics with high sensitivity (69.2%), (table 5). iraqi j pharm sci, vol.25(1) 2016 trend of antibiotic use in the icu 55 table (5): results of culture and sensitivity tests* antibiotic number of sensitive results (%) number of resistant results (%) total sensitivity tests p value 1 doxycycline 2(100)** 0(0) 2 0.04(s) 2 minocycline 1(100)** 0(0) 1 0.157(ns) 3 vancomycin 1(100)** 0(0) 1 0.157(ns) 4 tobramycin 9(90) 1(10) 10 0.0003(s) 5 ciprofloxacin 9 (69.2) 4 (30.8) 13 0.049(s) 6 levofloxacin 9 (69.2) 4 (30.8) 13 0.049(s) 7 amikacin 8(66.6) 4(33.4) 12 0.102(ns) 8 tetracycline 7(63.6) 4(36.4) 11 0.200(ns) 9 gentamicin 8(61.5) 5(38.5) 13 0.239(ns) 10 cefoxitin 4(57.1) 3(42.9) 7 0.592(ns) 11 cefotetan 3(50) 3(50) 6 1(ns) 12 co-trimoxazole 3(42.8) 4(57.2) 7 0.592(ns) 13 nitrofurantoin 1(33.3) 2(66.7) 3 0.414(ns) 14 ceftriaxone 2(33.3) 4(66.7) 6 0.248(ns) 15 cefotaxime 2(28.5) 5(71.5) 7 0.108(ns) 16 ceftazidime 3(23) 10(77) 13 0.006(s) 17 aztreonam 1(20) 4(80) 5 0.057(ns) *regardless of the sample taken **these results may not reflect the true ratio because of low number of test performed. (s) = significant difference. (ns)= non-significant discussion in the present settings, the icu receives patients referred from either postoperative or critically ill due to trauma, and usually they are attached to many machines and instruments example endotracheal tube, foley catheter, central line …etc. which are potential sources of cross infection between patients. in the current study, every patient received antibiotic treatment prescribed by the senior physician in charge and usually it is prescribed based on clinical basis and experience to start with, and this is what was applied in five tertiary care hospitals in germany as stated by wilke m et al who showed that therapy based on local experiences may be sufficient for patients with low complicated pathogen risk score (cprs) but inadequate for those with high cprs (7) . in the present study samples for culture and sensitivity were taken from all patients on admission, but only 16 of 46 patients have their culture and sensitivity results back. this indicates a major breakdown on the care system and implies a comprehensive review of the weak points that delay or corrupt the management line of the patients regarding treating their infections. also, in the present study, the mean time for culture and sensitivity results to come back to rcu was 9.58 days (range 1-27 days), this is a long period. this problem is also a challenge in the medical practice even in the in european countries, as there is a wide difference between many countries in retrieving the results of the culture test, for example in uk, 2 hr. are needed from collection to incubation while in germany 20 hr. are needed for incubating the bacteria due to remote laboratories (9) , still these periods are much shorter than that in our laboratories. this is not acceptable delay and this for sure will impede or derange the course of treatment and delay the recovery of patients and may encourage resistance emergence of fatal bacterial infections. there are many studies abroad looking for new methods to shorten the period of detecting the causative organisms in the patients so as to improve the selection of the antibiotics as early as possible and by this improve the outcome, such as a study which used new spectrometry method which provides rapid pathogen identification in critically ill patients with the ability to rule out infection within 6 hours. this has potential clinical and economic benefits (10) . a new computer-assisted infection (cai) monitoring software program has been developed for use in an intensive-care unit (icu) (11) . iraqi j pharm sci, vol.25(1) 2016 trend of antibiotic use in the icu 56 the commonest bacteria isolated from patients in this study was klebsiella (53.8%), and the least was coagulase-negative staphylococci (7.6%). this result differs from that of a study done in spain which found that the microorganisms most frequently isolated in patients with inadequate empirical antimicrobial treatment were: coagulasenegative staphylococci (29.5%), acinetobacter baumannii (27.3%), enterococcus faecalis, pseudomonas aeruginosa, enterobacter cloacae, proteus mirabilis, escherichia coli, and candida species (4.5% each) (4) . whereas another study, done in turkey found that pseudomonas spp. were the most frequently isolated gram-negative species (26.8%), followed by klebsiella spp. (26.2%). escherichia coli, acinetobacter spp. and enterobacter spp. were the other commonly isolated organisms (2) . in the present study ceftriaxone was the main antibiotic used as an empiric treatment, this may be due to an experience with this drug, its availability, its opened dispense with no limitation and its broad spectrum of action. now, if we compare the results of culture and sensitivity test in the present study, we can notice clearly that 9 (90%) tests out of 10 tests done shows that tobramycin was the most effective against bacteria but it was not used clinically because it was unavailable during the period of the study. the 2 nd most effective antibiotic against bacteria was ciprofloxacin and levofloxacin 9 (69.2%) sensitive tests out of 13 tests for each. while the most frequently used antibiotic in empiric treatment (ceftriaxone) was one of the least effective antibiotic on isolated bacteria 2 (33.3%) sensitive tests as revealed by culture test. this infrequent use of ciprofloxacin may explain its potential activity against isolated bacteria as revealed by culture studies, and this in turn should alert the clinicians and the clinical pharmacists to review their selections of the empirical therapy drugs and to decrease their use of ceftriaxone as first choice antibiotic in the empirical treatment. ceftriaxone resistance was noted by madani n et al who studied the resistance of bacteria in icu against several antibiotics and revealed that 75.0% of klebsiella, 31.9% of e. coli and 68.4% of enterobacter spp. were resistant to ceftriaxone and 10% of enterobacter spp. to imipenem and 13.5% pseudomonas spp. were resistant to imipenem. they concluded that bacterial resistance was high in morocco (12) . in contrary to the present study, the results of a study done from 2005 to 2010, showed that ciprofloxacin-resistant e. coli strains (37.1%) was increased, as well as imipenemresistant (36.4%) and ciprofloxacin-resistant (37.1%) strains of p. aeruginosa (13) . in another study, increasing ciprofloxacin-resistance was evident for k. pneumoniae (14) . levin pd (2007) determined risk factors associated with ciprofloxacin resistance in clinical bacterial isolates from icu patients which includes prior use of fluoroquinolones and duration of hospitalization prior to icu admission (15) . metronidazole is widely used antibiotic in our clinical practice and unfortunately the culture and sensitivity tests for anaerobic bacteria are unavailable in most of our centers including our settings in the present study, so we can’t evaluate its efficacy against bacteria, but in the recent years, metronidazole has appeared to lose some of its effectiveness in clostridium difficile infection (cdi) management and vancomycin is now recognized as the first-line treatment of severe cases (16) , although, many studies proved that the rate of cdi in icu patients is low, but the infection affects severely ill patients, and is associated with high mortality (17,18,19) . in the current study the mortality rate was 47.8%, and this is very high when compared with a study done in usa in 2009 which revealed that the mortality rate range from 9.3% to 13.3% (20) . the high mortality rate in the present study may partly be caused by sepsis and inappropriate use of antibiotics as shown in the results. also another study supports claims that the availability of medical and nursing staff is associated with the survival of critically ill patients (21) . in conclusion, there is a critical need for reviewing the trend of antibiotic use in the icus depending more on laboratory tests to identify 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parameters in children from al-fallujah city in iraq asmaa a. shukur ,*,1 , dawser k.ismail * and kawther m. ibrahim * * department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract environmental exposures to lead remain a serious problem in the developing and industrializing countries. children are the highest risk aged-group for lead poisoning. this study was designed to assess lead exposure in al-fallujah city by analyzing blood lead levels in children and adults and to explain the relationship between blood lead levels, hematological parameters and ferritin levels in the children. the study was performed on-(90) subjects, (65children and 25 adults).venous blood samples were taken for estimation of hematological parameters, serum ferritin levels and blood lead levels. the children group was subdivided into four groups as: group (a) (low ferritin, low hb), group (b) (low ferritin, normal hb), group (c) (normal ferritin, low hb) and healthy control group (d) (normal ferritin, normal hb).the results of this study demonstrating that all children groups: group (a) (n=14), group (b) (n=7), group(c) (n=17) and group (d) (n=27) had blood lead levels above the acceptable level 10µg/dl. there was significant increase in blood lead levels (10%) in the control group of children compared to adult group (p<0.05). in addition showed a significant increase in blood lead levels(52%)in group (a) and (29%) in group(c) compared with control group(p<0.001,p<0.01) respectively, while no significant increase in blood lead levels was shown in group (b). thus the current study showed that elevated mean blood lead level above the acceptable limit of (10µg/dl) in all children groups, suggesting that iron deficiency anemia may amplify the effect of lead contamination in the environment. key words: blood lead levels, hematological parameters, ferritin, iron deficiency anemia. جركيز انرصاص وانحغيرات انحاصهة بدالئم اندو الطفال انعراق في يديُة انفهىجة انعالقة بيٍ اسًاء عبد شكر ،*1 و كىثر يحًد ابراهيى اسًاعيم ، دوسر خهيم * ، مييخ اىظيذىخ ، جبٍعخ ثغذاد ، ثغذاد ، اىعزاق .اىسَىً االدويخ وفزع الخالصة اىزعزع اىجيئي ىيزطبص ٍبساه يَثو ٍشنيخ جذيخ في اىجيذاُ اىْبٍيخ و اىجيذاُ اىظْبعيخ و أُ األؽفبه هٌ اىفئخ اىعَزيخ األمثزرعزػبً ىيزسٌَ ثبىزطبص.اىهذف ٍِ هذٓ اىذراسخ هىرقييٌ اىزعزع ىيزطبص في األؽفبه و اىجبىغيِ في ٍذيْخ اىفيىجخ ٍِ خاله ٍعبىٌ ٍنىّبد اىذً و ٍسزىيبد اىفزيزيِ ،ذً .ومذىل رىػيخ اىعالقخ ثيِ ٍسزىيبد اىزطبص في اىذً رذييو ٍسزىيبد اىزطبص في اى رٌ أخذ عيْبد دً ٍِ األشخبص اىذيِ شَيزهٌ اىذراسخ .ثبىغ( 56ؽفو و 56( شخض ثيْهٌ)09 (في األؽفبه.وقذ أجزيذ اىذراسخ عيً ذً, و ٍسزىيبد اىزطبص في اىذً. رٌ رقسيٌ ٍجَىعخ األؽفبه اىزي خؼعذ ىزقذيز ٍعيَبد رنىيِ اىذً, وٍسزىيبد اىفزيزيِ في ٍظو اى ( )اىفيزيزيِ ٍْذفغ,خؼبة اىذً bٍجَىعخ) ،()اىفيزيزيِ ٍْخفغ,خؼبة اىذً ٍْخفغ(aىيذراسخ اىً ارثع ٍجبٍيع : ٍجَىعخ ) فيزيزيِ وخؼبة اىذً ( )اىcontrol( )اىفيزيزيِ ؽجيعي,خؼبة اىذً ٍْخفغ(, و ٍجَىعخ اىسيطزح)cؽجيعي(, ٍجَىعخ ) ، (7()اىعذد=b(,ٍجَىعخ)41( )اىعذد=aٍجَىعخ ) :ؽجيعي(.ٍسزىيبد اىزطبص في مو ٍجبٍيع األؽفبه اىذيِ شَيزهٌ اىذراسخ (.وأُ هْبك سيبدح 10µg/dl( ثيغذ امثز ٍِ اىذذ اىَقجىه )57( )اىعذد=control( وٍجَىعخ اىسيطزح)47( )اىعذد=cٍجَىعخ) ٍسزىي اىزطبص ىذي األؽفبه في ٍجَىعخ اىسيطزح ثبىَقبرّخ ٍع ٍجَىعخ اىجبىغيِ اىزي خؼعذ في ٍعذه )%499ٍعْىيخ ) ( c%( في ٍجَىعخ )50( و)a%( في ٍجَىعخ )65أُ ٍسزىيبد اىزطبص في اىذً اسدادد ثظىرح ٍعْىيخ ) .( (p<0.05ىيذراسخ (.ّزبئج هذٓ bرشهذ اىذراسخ سيبدح ٍعْىيخ في ٍجَىعخ ) ( عيً اىزىاىي في ديِ ىp<0.01،p<0.001ٌثبىَقبرّخ ٍع ٍجَىعخ اىسيطزح) (في أؽفبه ٍذيْخ اىفيىجخ. واىْزبئج اىزي 10µg/dlاىذراسخ رشيز اىً اررفبع ٍعذه اىزطبص اىً ٍسزىي اعيً ٍِ اىذذ اىَقجىه ) رىطيْب اىيهب رقززح أُ فقز اىذً ثسجت ّقض اىذذيذ ينجَزرأثيزاىزيىس اىجيئي ثبىزطبص. غهيسيريم بهيُيث ، اندهىٌ انصهبة انًجهرية ، جسيًات انثيىفهييٍ انُاَىية . انًفحاحية:انكهًات introduction lead is a nonessential metal, its presence in the body at any level could be considered as contamination (1) . it is a divalent cation (pb +2 ), exists in both organic and inorganic forms (2) .it occurs naturally in the earth's crust . it is ubiquitous in the environment of industrialized cities because it comes mainly from human activity (3) .the elimination half– time of lead in blood is approximately 30-35 days (4) ; therefore blood lead concentrations relatively reflect the exposure history of the previous few months (5) . 1 corresponding author e-mail:asmaa-9071@yahoo.com received:23/2/2013 accepted:27/5/2013 iraqi j pharm sci, vol.22(2) 2013 blood lead levels in children from al-fallujah city 16 lead is stored in bone, with elimination half – time estimates of 20-30 years (6) .the mechanisms of lead toxicity include the ability of lead to mimic or compete with calcium, and to interact with proteins by binding with every available functional group (7) .the most critical effects are those on; heme biosynthesis, the nervous system and kidney (8) .lead interferes with heme biosynthesis by altering the activity of several enzymes ,one of the most sensitive hematological effects is inhibition of the enzyme delta-amino levulinic acid dehydratase (alad) resulting in accumulation of 6-amino levulinic acid (ala) in blood, urine, and soft tissues, including brain (9) . in addition, ala undergoes auto oxidation, generating free radicals that may contribute to toxicity, and promotes oxyhemoglobin oxidation (10) . the mitochondrial enzyme ferrochelatase is the second enzyme in the heme biosynthetic pathway inhibited by lead. ferrochelatase catalyzes the transfer of iron from ferritin into protoporphyrin to form heme. inhibition of this enzyme results in increase of the substrates erythrocyte porphyrin (ep), and zinc protoporphyrin (zpp) (11) . at relatively high levels of lead exposure, anemia may occur due to the interference with heme synthesis and also to red cell destruction .leadinduced anemia occur in children at blood lead levels of 40µg/dl (12) . lead impair the activity of erythrocyte pyrimidine -5'-nucleotidase ,resulting in increase in pyrimidine nucleotides in red blood cells ;which lead to a deficiency in maturing erythroid elements, if sufficiently severe, result in induction of basophilic stippling and premature erythrocyte hemolysis; thus decreased red blood cells counts and eventually anemia (13) . anemia is a common condition worldwide, although the burden is highest in the developing countries where nutrient deficiencies and chronic infections are revalent (14) . iron deficiency anemia is the main cause of anemia in children and pregnancy (15) .the most accurate initial diagnostic test for iron deficiency anemia is the serum ferritin measurement (16) . the aim of this study was to assess the magnitude of lead exposure in children living in al-fallujah city and explain the relationship between blood lead levels, hematological parameters and serum ferritin levels in children. materials and methods this study was carried out on a total of 90 subjects living in al-fallujah city grouped as follows: children group (n=65), age range (411years) (7.26±2.38, mean±sd) and adults group (n=25), age range (20-45years) (30.08±7.43, mean±sd). the children group was subdivided into four groups as shown in (table1). a venous blood sample (8ml) was taken from each child (after obtaining their parents’ consent) and (from each adult a venous blood sample of (5ml) was taken for estimation of blood lead levels). blood samples from each child divided into three tubes. table (1): children groups. the first tube (2ml in plain tube) for separation of serum to estimate serum ferritin by immunoradiometric assay method using ready – made kit for this purpose and automated gama counter. the second tube (1 ml in edta tube) used for estimation of hematological parameters using automated device. these parameters included hemoglobin (hb), packed cell volume (pcv), and mean corpuscular volume (mcv). the third tube (5ml in edta tube) used for estimation of lead by using flame atomic absorption spectrophotometer (aas). the cutoff values of the studied parameters are listed in (table2).the results were expressed as the mean standard deviation (sd).the ttest was performed to assess the significance of the differences between the mean blood lead levels in adults and children group and the variables (hb, mcv, pcv, and ferritin levels) investigated in children groups. p values of less than 0.05 were considered significant. group (a) n=14) low ferritin, low hb. group (b) (n=7) low ferritin, normal hb. group (c) (n=17) normal ferritin, low hb. controlgroup (d) (n=27) normal ferritin, normal hb. iraqi j pharm sci, vol.22(2) 2013 blood lead levels in children from al-fallujah city 17 table(2): the cutoff values of the studied parameters. results the data presented in (table 3) showed a significant increase in the mean blood lead level (10%)in control children group (17±3.17µg/dl;mean±sd) compared with adult group (15.36 ± 3.29 µg /dl ; mean ± sd), (p<0.05).(table 4), and (figure 1) showed a significant increase in blood lead levels (52%) in group (a) (25.85±1.75µg/dl), and (29%) in group(c) (22±5.67µg/dl) compared with control group, (p<0.001, p<0.01) respectively, while no significant differences was shown in group (b) compared to control group (p>0.05). table (3): comparison of blood lead levels between adults and children groups. data are presented as (mean±sd). n=number of subjects. * show significance between adults and control children group (significance were evaluated by t-test), *p<0.05. control children (normal ferritin, normal hb) and pb (lead). table (4): comparison of blood lead levels among children groups. control d a (n=14) b (n=7) c (n=17) pb (µg/dl) 17±3.17 25.85±1.75 19. 58±5.94 22±5.67 data are presented as (mean±sd). n=number of subjects. * show significance between control and other groups (significance between groups were evaluated by t-test), **p<0.01, *** p<0.001and # nonsignificant. group (a) (low ferritin, low hb), group (b) (low ferritin, normal hb), group(c) (normal ferritin, low hb), control group (normal ferritin, normal hb) and pb (lead). figure (1): the blood lead levels in children groups (table 5) showed a significant decrease in the hb 10%), mcv (16%) , pcv (14%) and ferritin levels (87%) in group(a) (10.49±0.62g/dl,68.69 ±4.2µm³, 32.21±1.79%, 6.86±3.33ng/ml) respectively compared with control group (12.4±0.75g/dl, 81.77±5.05µm³, 37.47±2.03%, 52.83±19ng/ml), (p<0.001),while there was only statistically significant decrease in ferritin levels(85%) in group (b) (8.11±2.98ng/ml) compared to control group, (p<0.001).in group (c) ( table 5) showed a significant decrease in the levels of hb (13%), mcv(16%), and pcv(9%) (10.83±0.66g/dl, 68.98±.30µm³, 34.0±1.89%) respectively compared with control group, (p<0.001, p<0.001, and p<0.01). studied parameters cutoff values blood lead levels ≥10µg/dl (18) ferritin levels ≤ 12ng/ml (19) pcv <3334% (19),(20) mcv <73 µm³ (20) hb < 5years 5-11years < 11g/dl < 11.5g/dl (19) adult n=25 control children n=27 15.36±3.29* 17±3.17 pb (µg/dl) *** iraqi j pharm sci, vol.22(2) 2013 blood lead levels in children from al-fallujah city 18 table(5): comparison of hematological parameters and serum ferritin level among children groups. c (n=17) b (n=7) a (n=14) control d (n=27) 10.83±0.66*** 11.98±0.48 # 10.49±0.62*** 12.4±0.75 hb (g/dl) 68.98±4.30*** 79.25±8.12 # 68.69±4.2*** 81.77±5.05 mcv(µ³m) 34.0±1.89** 37.1±2.06 # 32.21±1.79*** 37.47±2.03 pcv% 55.21±14.69 # 8.11±2.98*** 6.86±3.33*** 52.83±19 ferritin (ng/ml) data are presented as mean ±sd. n=number of subjects. * shows significance between control and other groups (significance between group were evaluated by t-test), *p<0.05, **p<0.01, *** p<0.001, and # non-significant. group(a)(low ferritin, low hb), group(b)(low ferritin, normal hb) , group(c)(normal ferritin, low hb), control(normal ferritin, normal hb), hb(hemoglobin), mcv(mean corpuscular volume), pcv(packed cell volume). discussion there are considerable variations in the sources and pathways of lead exposure (18)(20) .in al-fallujah city it is attributable to miscellaneous sources; which may be more significant than universal exposure .there is a number of cement, fire brick, and ceramic plants at about 5 km to the east of fallujah. ; the high traffic density (19) ; presence of large numbers of gasoline electricity generators and also this city undergoes two major military operations and there is no doubt that war can leave a terrible legacy of pollution behind; bombs, missiles, shells, and bullets flood the environment with lead, nitrates, nitrites, hydrocarbons, phosphorous, radioactive debris, corrosive and toxic heavy metals (21),(22) . this study showed that blood lead level in the control children group was significantly (10%) more than adult group living in the same area (table3). the results of this study indicated that children living in al-fallujah city exposed to high lead levels in their environment; where their mean blood lead level twice as high as the cutoff value of 10µg/dl (17) . in addition recent studies indicated that there was no safe threshold for the neurological adverse effects of lead in children, like impairment of cognitive functioning and academic achievement could occur at blood lead levels below 5µg/dl (23) . however the basis of the special concern for infants and children relates to certain structural, functional and behavioral differences between them and adults (3) . children are at increased risk for lead exposure, as well as for adverse effects, because: children have behavioral characteristics (outdoor activity, less concern for hygienic conditions, hand –to –mouth activities or pica), which increase the risk of lead exposure; children eat and drink more per unit of body weight than adults, so that their relative lead intake is increased (24) ; lead absorption in the g.i.t. is substantially higher in children, about 50% ,compared with about 10% in adults (25) ; there is a greater prevalence of nutritional deficiencies (e.g. iron and vitamin d) among children , which enhance absorption of lead from the g.i.t. (26) ;the bloodbrain barrier is not yet fully developed in young children and neurological effects of lead occur at lower thresholds than adults (27) .the results of this study showed significant decrease in hb, mcv, pcv and ferritin levels in group(a) (10%, 16%, 14% and 87%) respectively.(table5), (p<0.001), indicated that children in this group suffered from iron deficiency anemia (16) ; while there was a significant increase in mean blood lead level (52%) [(table4), (figure1)], (p<0.001) compared to control group. this result is in agreement with previous study in which iron deficiency anemia strongly associated with observed toxicities of lead (28) ,other studies showed a strong correlation between proportions of children with elevated blood lead levels and low iron levels (29),(30) ,while other studies showed that iron deficiency present even at low lead levels (31),(32) . the hematological findings showed that only mean ferritin level in group (b) was significantly decrease (85%) compared to control group (p<0.001) (table5). this is due to the depletion of stored iron only (19) ; while there was no significant difference in the mean blood lead levels in this group compared to control group [(table4),(figure1)].the results are in agreement with a previous study in which ferritin depletion alone has no influence on the blood lead levels in children (33) . the results of this study showed significant decrease in hb, mcv and pcv in iraqi j pharm sci, vol.22(2) 2013 blood lead levels in children from al-fallujah city 19 group (c) (13%, 16%, and 9%), respectively (table5), (p<0.001, p< 0.001, p< 0.01), with a significant increase in mean blood lead level (29%) [(table 4), (figure 1)], (p<0.01) compared to control group. there are other studies showed significant association between anemia and blood lead levels (34),(35) and this is consistent with the results of our study, while others showed poor correlation (36) , or no correlation (37) . our results could be explained by referring to iron homeostasis, which is regulated at the level of iron absorption, since increase iron absorption would be consequently increase lead absorption, because ferrous ion transported across the apical membrane by divalent metal transporter (dmt1) which is of broad specifity, including manganese, copper, zinc, cadmium, and lead cations (38) . absorption of intestinal iron is regulated by three mechanisms .the first is influenced by recent dietary iron intake" dietary regulator". the second, iron absorption can be modulated in response to body iron stores "store regulator" it is capable of changing the amount of iron absorbed to a limited extent. the third, a signal communicates the state of bone marrow erythropoiesis (developing erythrocytes in the bone marrow) to the intestine "erythropoietic regulator" (39) .when red-cell production in the bone marrow is accelerated, absorption of intestinal iron is increased due to stimulation of the "erythropoietic regulator" (40) , which has the dominant function in the control of iron absorption, and it could produce an increase in intestinal iron absorption much greater than that which the" store regulator" could produce (41) .this may explain our findings that only in group(a); there is stimulation of the "erythropoietic regulator”; which resulted in significant increase in blood lead levels, while in group(b) only the "store regulator" is stimulated and there was no significant increase in blood lead levels. this is not consistent with our results in group (c), because according to our hematological findings in this group anemia may be due to infection or inflammation, even when the illness was mild and brief (42) , in which the usual flow of iron from reticulo-endothelial and parenchymal cells as well as iron absorption is decreased (39) , there may be misclassification of iron status due to the coexistence of anemia of infection or inflammation and iron deficiency anemia ; when inflammatory disease and iron deficiency coexist, serum ferritin values may be within the normal range; because ferritin as an acute phase protein may be elevated by infection or inflammation (43) . school children (the age range of our studied group) carry the heaviest burden of intestinal parasitic infestation (44) , and there is a strong association of hookworm with increased prevalence of iron deficiency anemia (45) , also mild inflammatory conditions such as upperrespiratory infections and otitis media, which remain common in children can be a major source of error in diagnosing iron deficiency (42) , and under estimation of iron deficiency by using serum ferritin levels (15) . this may be one of the limitations in this study and the coexisting iron deficiency anemia in group(c) may be the predisposing factor in the significant increase in blood lead levels in this group. therefore it is important for further study to include the measurement of serum transferrin receptor, to differentiate between iron deficiency anemia (ida) and anemia of inflammation or infection, because it remains normal in the anemia of infection or inflammation, but elevated with ida (46) . the results of this study showed that blood lead levels in children were below the levels that cause anemia (figure 1), since it is uncommon for lead poisoning to produce anemia and microcytosis unless it is severe (47) . therefore the hematologic findings in our study were the result of iron deficiency anemia and anemia of infection or inflammation. conclusion the results of this study showed that children living in alfallujah city are suffer from a high risk of environmental lead exposure indicated by the elevated mean blood lead level above the acceptable limit of (10µg/dl), while the adults from the same city had significantly lower blood lead levels than children. the results of this study suggest that iron deficiency anemia may amplify the effects of lead contamination in the environment by increasing the intestinal absorption of both iron and lead; 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122:1240-1248. 44. thi le h., brouwer i.d., verhoef h., nguyen k.c., and kok f.j. anemia and intestinal parasite infection in school children in rural vietnam. asia pac.journal of clinical nutrition.2007;16:716-723. 45. mupfasonid.,karbushib.koukounari a.,ruberanziza e., kaberuka t., kramer m.h., mukabayira o., et al. polyparasite helminthic infections and their association to anemia and under nutrition in northernrwanda.plos.neglectedtropical diseases.2009; 3:1-10. 46. goyal r., das r., bambery p., garewal g. serum transferrin receptor-ferritin index shows concomitant iron deficiency anemia and anemia of chronic disease is common in patients with rheumatoid arthritis in north india. indian journal of pathology &microbiology.2008; 51:102-104. 47. cohen a.r., trotzky m.s., pincus. reassessment of the microcytic anemia of lead poisoning. medical journal of the american academy of pediatrics.1981; 67:904-906. iraqi j pharm sci, vol.23(2) 2014 lipid profile , fasting blood sugar and colelithiasis 51 lipid profile and fasting blood sugar analysis in patients with cholelithiasis rajaa n.al-atrakchey * , mohammed a.taher **,1 and ibtehal n.saeed ** * imam al – hussein medical city ,karbala ,iraq. ** department of clinical laboratory sciences, college of pharmacy, university of baghdad,baghdad,iraq. abstract cholelithiasis is one of the commonest surgical problems and one of the most common gastrointestinal diseases throughout the world but its pathogenesis remains unclear. many theories have been proposed forward to explain the mechanism of stone formation. it is not fully clear if symptomatic gallstone disease is associated with a specific pattern of some biochemical abnormalities, as lipid profile and fasting blood sugar in serum of patients. this study was designed to estimate lipid profile and fasting blood sugar in the sera of patients with cholelithiasis in comparison with normal individuals (control). in this study, 104(male=16, female=88) were symptomatic gallstone patients (aged 42.79± 12.18 years), and 38(male=6 and female=32) were apparently healthy controls (aged 40.03± 7.47 years). blood samples were collected from symptomatic gallstones patients before their cholecystectomy operation. over night fasting, blood samples were collected from all subjects to evaluate serum lipid profile: total cholesterol (tc), triglyceride (tg), high density lipoprotein-cholesterol (hdl-c), low density lipoprotein-cholesterol (ldl-c), very low density lipoprotein-cholesterol (vldl-c) and fasting serum glucose (fsg). there was a significant increase (p<0.05) in serum: tc, tg, ldl-c, vldl-c and fsg of patients with cholelithiasis compared to the apparently healthy controls. the study also showed that there was a significant decrease (p<0.05) in serum hdl-c in gallstone patients compared to control. in conclusion, cholelithiasis was associated with lipid profile and fasting serum glucose abnormality that be the cause or the effect of gallstone formation. these findings should be taken into consideration while treating gallstone patients. key words: gallstones, cholesterol, cholecystectomy. صيغت اندهىٌ ويستىي سكر اندو انصبئى عُد انًرضً وانًصببيٍ بحصً انًرارةتحهيم رجبء َعًت اييٍ االطرقجٍ * ، يحًد عببس طبهر **،1 و ابتهبل َىرٌ سعيد ** * .،مشبالء،اىؼشاقاىحسٍِ اىطبٍت االٍبً ٍذٌْت ** . فشع اىؼيً٘ اىَخخبشٌت اىسشٌشٌت ، ميٍت اىصٍذىت ، خبٍؼت بغذاد ،بغذاد ،اىؼشاق الخالصة ٌؼذ ٍشض حصى اىصفشاء أحذ اىَشبمو اىدشاحٍت اىشبئؼت ٗٗاحذا ٍِ اٍشاض اىدٖبص اىٖعًَ االمثش شٍ٘ػب ح٘ه اىؼبىٌ ىنِ بشنو ٗاظح أّ ٍِ غٍش اىَؼشٗف . ٗقذ ٗظؼج اىنثٍش ٍِ اىْظشٌبث ىخ٘ظٍح آىٍت حشنو اىحصبة. اٍشاظٍخٔ ٌنخْفٖب بؼط اىغَ٘ض فً ٗ سنش اىذً فً حبىت اىصٍبً ٍسخٌ٘بث شحً٘ اىذً ٍثو اىخغٍشاث اىنٍٍَبئٍت اىحٌٍ٘ت اسحببغ ٍشض حصى اىصفشاء بَْػ ٍؼٍِ ىبؼط فً ٍصو اىَشظى اىزٌِ ٌؼبُّ٘ ٗ سنش اىذً فً حبىت اىصٍبً ٍسخٌ٘بث اىشحًٕ٘زٓ اىذساست ىَؼشفت ٗقذ حٌ حصٌٍَ .ٍصو دً اىَشظى (. ٍدَ٘ػت اىسٍطشة)ٍغ االشخبص االصحبء ٍِ حصى اىصفشاء ٍقبسّت سْت ، 12.18± 42.79 اسٌٌَٕثيُ٘ ٍدَ٘ػت اىَشظى ٍؼذه اػٌ( رم٘س 16اّثى ٗ 88)ٍئت ٗاسبؼت اشخشك فً ٕزٓ اىذساست . سْت 7.47± 40.03 ٌَثيُ٘ ٍدَ٘ػت اىسٍطشة ٍؼذه اػَبسٌٕ( رم٘س 6اّثى ٗ 32)ٗثَبٍّت ٗثالثُ٘ . ػٍْبث اىذً حٌ خَؼٖب ٍِ ٍشظى حصى اىصفشاء قبو ػَيٍت اسخئصبه اىَشاسة ، اىذُٕ٘ ( tc)اىن٘ىٍسخشٗه اىنيً :شحً٘ اىَصو ش٘اموػٍْبث اىذً حٌ سحبٖب ٍِ اىَشظى ٗاالصحبء بؼذ اىصً٘ ىٍال ىقٍبس اىَشحبػ اىبشٗحٍِ اىشحًَ رٗ اىنثبفت اى٘اغئت, ( hdl-c)اىَشحبػ ببىن٘ىسخشٗه اىبشٗحٍِ اىشحًَ رٗ اىنثبفت اىؼبىٍت، ( tg)اىثالثٍت س اىذً فً حبىت ، ٗسل ( vldl-c)خذا اىَشحبػ ببىن٘ىسخشٗه اىبشٗحٍِ اىشحًَ رٗ اىنثبفت اى٘اغئت , ( ldl-c)ببىن٘ىسخشٗه (. (fsg اىصٍبً ٗم٘ىسخشٗه اىبشٗحٍِ اىشحًَ رٗ اىنثبفت اى٘اغئت اىن٘ىٍسخشٗه اىنيً ٗاىذُٕ٘ اىثالثٍت: فً ٍصو اىذً (p <0.05)ٗخذث صٌبدة ٍؼٌْ٘ت اىَشاسة ٍقبسّت فً اىَشظى اىزٌِ ٌؼبُّ٘ ٍِ حصى ٗ سنش اىذً فً حبىت اىصٍبًخذا ٗم٘ىسخشٗه اىبشٗحٍِ اىشحًَ رٗ اىنثبفت اى٘اغئت اىذً ىن٘ىسخشٗه اىبشٗحٍِ اىشحًَ رٗ اىنثبفت اىؼبىٍت فً ٍصو (p<0.05)مَب اظٖشث اىذساست ٗخ٘د اّخفبض ٍؼْ٘ي .ببألصحبء حبىت اىصٍبً ٗ سنش اىذً فًحصى اىَشاسة ٍشحبػ بخغٍش ٍسخٌ٘بث شحً٘ اىذً ّسخْخح اُ ٍشض . ّفس اىَشظى ٍقبسّت ببألصحبء فً . ٌْبغً أُ حؤخز ٕزٓ اىْخبئح بؼٍِ االػخببس أثْبء ػالج ٍشظى حصى اىَشاسة ىزا. اىحصبةىخنٌِ٘ اىخً قذ حنُ٘ ّبحدت اٗ ٍسببت .استئصبل انًرارة, كىنسترول , حصً انًرارة : انكهًبث انًفتبحيت 1 corresponding author e-mail:sad5554@yahoo.com received:1 /3/2014 accepted:28 /9/2014 iraqi j pharm sci, vol.23(2) 2014 lipid profile , fasting blood sugar and colelithiasis 52 introduction the presence of stones in the gallbladder is referred to as cholelithiasis (from the greek: chol, "bile" + lith-, "stone" + iasis-, "process") (1) . cholelithiasis or gallstone disease (gd), is one of the most prevalent gastrointestinal diseases, with a substantial burden to health care systems (2) . gallstones (gs) are abnormal masses of a solid mixture of cholesterol crystals, mucin, calcium bilirubinate, and proteins that have affected people for centuries (3) . gallstone formation is multifactorial, and known risk factors are advancing age, female gender, genetics ethnicity, obesity, rapid weight loss, diet, drugs, and activity (4) . the association of gallbladder (gb) stone disease with metabolic abnormalities such as diabetes, dyslipidemia, obesity, and hyperinsulinemia has supported the hypothesis that gb stone formation is a type of metabolic syndrome (5,6) . gallstones are classified as cholesterol, pigmented or mixed stones based on their chemical composition (7,8) . the role of serum lipid in the etiology of cholelithiasis is very important and particularly in cholesterol gallstones in which serum lipid profile are altered which is suggestive of metabolic syndrome. research suggests that metabolic syndrome is a risk factor for gallstones (9) . low levels of high-density lipoprotein cholesterol (hdl-c) and high triglycerides are associated with gallstone disease (10) . cholecystectomy is the standard and definitive treatment for symptomatic gallbladder stones and can be performed regardless of the type, number, and size of the stones (11,12) . it is effective and safe, with low rates of complications (14%) and mortality (0.17%). for cholesterol gallstones, current medical treatment includes: litholytic therapy (stone dissolution) by oral bile acid litholysis with chenodeoxycholic acid and/or ursodeoxycholic acid (udca) (13,14) , and lithotripsy (stone shattering) (15) . the aim of our study is to evaluate the biochemical changes in some serum parameters (lipid profile and fasting serum glucose) in gallstone patients compared to controls. subjects and methods the study was carried out on patients with clinical and imaging features confirming symptomatic cholelithiasis admitted to imam al-hussein medical city, safeer al-hussein surgical center and maitham al-tammar surgical hospital in karbala city from february to october 2013. patients with symptomatic gallstones had a history of (pain located in the epigastrium and/or right upper quadrant; recurrent symptoms occurring at different intervals; and episodes lasting 30 minutes or more. the pain may present with one or more of the following: associated nausea and vomiting; radiating to the back and/or right infrasubscapular region; and causing one to awaken from sleep in the middle of the night (16) , an abdominal ultrasonography is the standard diagnostic test for gallstone detection (17) ). a total number of 104 patients were included in this study, among which 88 females and 16 males in age group ranged between 18-75 years with a mean ±sd (42.79 ± 12.18). these were compared with 38 (age and sex matched) healthy controls (32 females, 6 males), with mean age ±sd (40.03 ± 7.47). the patients were selected not have liver cirrhosis, viral hepatitis, renal failure, thyroid disease, asthma and diabetes mellitus nor pancreatitis and gallbladder cancer nor those taking antihyperlipidemic drugs that may interfere with the data obtained. samples cllection after an overnight fasting, 5 milliliters venous blood samples were collected from patients before laparoscopic cholecystectomy and from healthy volunteers in plain tubes. after allowing the blood to clot at room temperature for about 30 minutes, blood samples were centrifuged at 5000 rpm for about 5 minutes to obtain serum. the serum samples were stored at -20 0 c until analysis was performed. biochemical assay methods biochemical assay was done at imam alhussein medical city, laboratory department using architect plus, abbott4000, automated auto-analyzer. the assay was include: serum total cholesterol (18) , serum triglyceride (19) , serum high density lipoprotein-cholesterol (20) , serum low density lipoprotein-cholesterol and very low density lipoprotein-cholesterol were calculated using friedewald formula (21) , and serum glucose level (22) . statistical analysis the following statistical data analysis approaches were done through the statistical package for social sciences (spss 17) and excel application were used in order to analyze and assess the results of the study (23) :  statistical tables.  mean value and standard deviation (sd).  bivariate comparisons were examined for parameters using pearson rank correlation coefficients (r). iraqi j pharm sci, vol.23(2) 2014 lipid profile , fasting blood sugar and colelithiasis 53  graphical presentation.  student's t-test was used to examine the degree of significance. p values less than 0.05 was considered significant. results table (1) and figures (1), (2) and (3) showed that the levels of total cholesterol, ldl, vldl, tg, and fasting serum glucose were significantly higher (p<0.05), while the levels of hdl were significantly lower (p<0.05) in the sera of gallstone patients in comparison with healthy control group. table (1): serum lipid profile and fasting serum glucose in gallstone patients and controls. data are expressed as mean ±sd (standard deviation), n=number of patients or controls. tg: triglyceride, hdl: high density lipoprotein, ldl: low density lipoprotein, vldl: very low density lipoprotein and fbs: fasting serum glucose. p <0.05 : significant. figure 1: mean serum total cholesterol (cholesterol), triglyceride (tg) and low density lipoprotein (ldl) in patients with cholelithiasis and healthy controls (milligram per deciliter). figure 2: mean serum high density lipoprotein (hdl) and very low density lipoprotein (vldl) in patients with cholelithiasis and healthy controls (milligram per deciliter). figure 3: mean of serum fasting blood sugar (fbs) in patients with cholelithiasis and healthy controls (milligram per deciliter). parameters/ (mg/dl) mean ± sd patients n=104 controls n=38 pvalue total cholesterol 197.64 ± 49.68 178.78 ± 37.60 <0.05 tg 180.006 ± 50.00 159.71 ± 25.12 hdl 46.78 ± 11.062 52.59 ± 8.53 ldl 115.76 ± 37.694 97.24 ± 21.015 vldl 36.83 ± 17.82 30.20 ± 7.82 fsg 112.73 ± 25.53 101.88 ± 28.50 iraqi j pharm sci, vol.23(2) 2014 lipid profile , fasting blood sugar and colelithiasis 54 table (2): pearson rank correlations between parameters. *significant at p <0.05 and ** high significant at p <0.01 table (2) showed that there were positive correlations between:  serum total cholesterol and tg (r= 0.323, p< 0.05), serum total cholesterol and ldl (r= 0.76, p< 0.05), and serum total cholesterol and vldl (r= 0.42, p< 0.05).  serum tg and vldl (r= 0.53, p< 0.05).  serum ldl and vldl (r= 0.24, p< 0.05). discussion the present study observe low serum hdl levels and high serum triglyceride, total cholesterol and ldl levels in patients with cholelithiasis which is in agreement with other studies (24,25) , virupaksha hs et al (10) , channa na et al, found that lipids elevation in cholelithiasis, seems to play a major contributing role in the pathogenesis of gallstones in females of up to 45 years age (25) . the elevation of serum total cholesterol and tg levels in patients may be due to: gallstone patients have abnormal secretory mechanism for bile acids and phospholipids, decrease bile acids and phospholipids (which solubilize cholesterol in the bile) will increase cholesterol precipitation (26) ,and some of gallstone patients may present with metabolic syndrome which is a cluster of symptoms such as glucose intolerance, high total cholesterol, hyperinsulinemia, increased vldl and/or total cholesterol, decrease hdl and hypertension who indicate that the metabolic syndrome is one of the risk factors for gallstone disease (27) . previous study described a decrease in hdl in gallstone patients, and there will be a return to the normal condition after gallstone removal (28) . the high ldl was seen in gallstone patients in this study either due to abnormal secretary function and/or prolonged high fatty diet and agree with zhao et al (26) , and down regulation of ldl-apob receptors by inhibition of ldl-apob receptor gene expression (29) . hyperlipidemia are strong risk factors in cholelithiasis as estimated previously (30) . life style and dietary modification are effective measures for the prevention of cholelithiasis (31) . the previous observation indicate that medications used to treat dyslipidemia may be of value in the prevention and treatment of cholelithiasis (32) . there was significant positive correlation (r= 0.53, p<0.05) between serum vldl and tg in patients with cholelithiasis in the present study. cholelithiasis is associated with hypertriglyceridemia (10,33) . hypertriglyceridemia is associated with decreased hdl-cholesterol (hdl-c) and increased small dense ldl (34) . ldl particles are formed as vldl lipoproteins lose triglyceride through the action of lipoprotein lipase and they become smaller and denser (fewer fat molecules with same protein transport shell), containing a higher proportion of cholesterol esters (35,36) . a positive correlation (r = 0.32, p< 0.05) between serum parameters (mg/dl) t . c h o le st e r o l t g h d l l d l v l d l f b s t. cholesterol 1 .323 ** .127 .760 ** .427 ** -.030 .000 .140 .000 .000 .725 tg .323 ** 1 .060 .123 .532 ** .123 .000 .489 .155 .000 .153 hdl .127 .060 1 -.012-.006-.044 .489 .894 .944 .610 ldl .760 ** .123 -.0121 .243 ** -.023 .000 .155 .894 .004 .786 vldl .427 ** .532 ** -.006.243 ** 1 .067 .000 .000 .944 .004 .440 fbs -.030.123 -.044-.023.067 1 .725 .153 .610 .786 .440 http://en.wikipedia.org/wiki/vldl http://en.wikipedia.org/wiki/lipoprotein_lipase http://en.wikipedia.org/wiki/lipoprotein_lipase http://www.rpi.edu/dept/bcbp/molbiochem/mbweb/mb2/part1/lipoprot.htm iraqi j pharm sci, vol.23(2) 2014 lipid profile , fasting blood sugar and colelithiasis 55 triglyceride and serum total cholesterol in patients with cholelithiasis was observed in the present study, which is in agreement with the other study demonstrated that there was a significant positive correlation between total cholesterol and tg levels (37) . in present study, fasting serum glucose (fsg) was significantly higher (p<0.05) in gallstone patients compared to healthy controls, in agreement with other studies performed by simona tîrziu, et al. (38) and jindal n, et al. (39) "they reported that fasting glucose levels were significantly increased in gallstone patients as compared to controls". gallstone disease appeared strongly associated with fasting glycemia (40) . there was a positive correlation between prevalence of gallstone with higher fasting glucose. the possible mechanisms for this association may be as follows: hyperglycemia inhibits bile secretion from the liver and disturbs gallbladder contraction (41) . gallstone disease appears to be strongly associated with metabolic syndrome, and the more the components of metabolic syndrome, the higher the prevalence of gallstone disease, and elevated fasting blood glucose is one of component of metabolic syndrome that associated with gallstone disease (42) . conclusions gallstone disease is associated with some biochemical abnormalities (elevation of total cholesterol, triglyceride, ldl, vldl, fasting serum glucose level and decrease in hdl level compared to control) that may be the cause or the 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during cholesterol gallstone formation in rabbit model. world j. gastroenterol. 1998; 4(4): 337-9. 27. guraya sy. reappraisal of the management of cholelithiasis in diabetics. saudi med j. 2005; 26:1691-1694. 28. tang wh : serum and bile lipid level in patients with and without gallstone. j. gastroenterol. 1996; 31(6): 823-7. 29. smith af, beckett gj, walker sw, et al: clinical biochemistry, blackwell science, usa. 1998; pp 165-74. 30. hung s, liao k, lai s, et al. risk factors associated with symptomatic cholelithiasis in taiwan: a population-based study. bmc gastroenterology. 2011; 11:111-1. 31. tsai cj, leitzmann mf, willett wc, et al: dietary carbohydrates and glycaemic load and the incidence of symptomatic gall stone disease in men. gut 2005, 54(6):823-828. 32. zak a, zeman m, hrubant k, et al: effect of hypolipidemic treatment on the composition of bile and the risk or cholesterol gallstone disease. cas lek cesk 2007, 146(1):24-34. 33. sun h, tang h, jiang s, et al: gender and 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ali i, et al. effect of cholelithiasis and cholecystectomy on serum lipids and blood glucose parameters. archives of international surgery. 2013; 3(2): 97-101. 40. cojocaru c, pandele gi: metabolic profile of patients with cholesterol gallstone disease. rev med chir soc. med nat iasi. 2010; 114:677-682. 41. chen cy, lu cl, huang ys, et al: age is one of the risk factors in developing gallstone disease in taiwan. age ageing. 1998; 27:437-441. 42. chen ly, qiao qh, zhang sc, et al: metabolic syndrome and gallstone disease. world j gastroenterol. 2012; 18(31): 4215-4220. http://en.wikipedia.org/wiki/special:booksources/1568273908 iraqi j pharm sci, vol.24(1) 2015 iraqi tribulus 68 phytochemical investigation and antioxidant activity of iraqi tribulus terrestris nabaa m. ibrahim * and enas j. kadhim *,1 * department of pharmacognosy and medicinal plant , college of pharmacy,university of baghdad,baghdad,iraq. abstract the aim of the present study was to characterize the iraqi tribulus terrestris for the presence of biologically active phyto-chemicals using methanolic extracts of the plant (aerial parts) by gas chromatography –mass spectrometry (gc/ms), while the mass spectra of the compounds found in the extract was matched with the national institute of standards and technology (nist) library , in addition to study the antioxidant activity of plant extract , results confirmed the presence of therapeutically potent compounds in the iraqi tribulus terrestris extract predominantly alkaloids, flavonoids, saponins, tannins and terpenoids. antioxidant potential of iraqi tribulus terrestris herbal preparations was evaluated by determination of blood glutathione, serum ascorbic acid and serum superoxide dismutase in rats. the obtained results demonstrated that t. terrestris preparations possess a significant antioxidant activity. keywords: iraqi tribulus terrestris, phytochemical investigation, anti-oxidant activity. دراست المكوناث الكيمائيت والفعاليت المضادة لالكسدة لنباث ذقن الشيخ العراقي نبأ محمد ابراهيم * و ايناس جواد كاظم ،*1 * فزع العمالٍز َالىباحاث الطبٍت ،كلٍت الصٍذلت ، جامعت بغذاد ، بغذاد ، العزاق . الخالصة المُاد الكٍمٍائٍت الفعالت المُجُدة فً االجشاء الٍُائٍت للمسخخلص الكحُلً لىباث عزالً شخٍصالٍذف مه ٌذي الذراست ٌُ ح َمماروت الىخائج مع الىخائج gc/ms مطٍاف الكخلت-الفصل الكزَماحُجزافً للغاساث ٌسمى )دله الشٍخ( اَ شمشك باسخخذام طزٌمت ( باالضافت الى دراست الفعالٍت المضادة لالكسذة لمسخخلص الىباث . nistالمثبخت فً المعٍذ الُطىً للمعاٌٍز َالخكىُلُجٍا) شُفاث كٍمٍائٍت محذدة لذ حمج على المسخخلص المٍثاوُلً ان عملٍت الكشف الىُعً الخمٍٍذي لالٌضاث الثاوٌُت المخخلفت مه لبل ك .لالجشاء الٍُائٍت مه الىباث َاشارث الىخائج ان ٌذي االجشاء ححخُي على الملٌُذاث, فالفٍىٌُذاث، صابُوٍاث , مُاد دباغٍت َ حزبٍىاث الىخائج ان المسخخلص الكحُلً لذًٌ فعالٍت حضمىج ٌذي الذراست اٌضا الكشف عه الفعالٍت المضادة لالكسذة لىباث الشمشك َاظٍزث .ةجٍذة ضذ االكسذ نباث ذقن الشيخ العراقي ، الدراست الكيميائيت ،الفعاليت المضادة لألكسدة .:الكلماث المفتاحيت introduction tribulus terrestris (family: zygophyllaceae ) is a perennial creeping herb widely distributed in iraq. it is regarded as an aphrodisiac in addition to its beneficial claims on various ailments such as urinary tract infections, inflammations, oedema and ascites (1) . in iraq t. terrestris(figure-1) is used in folk medicine as tonic, aphrodisiac, analgesic, astringent, stomachic, antihypertensive, diuretic, lithontriptic and urinary antiinfective (2) .the aphrodisiac property of this plant extract was examined in rats (3) . administration of tribulus extract to humans and animals improves libido and spermatogenesis (4) .clinical studies showed that this plant improved reproductive function, including increased concentration of hormones such as estradiol, with testosterone being very slightlyinfluenced, thereby improving reproductive function, libido and ovulation (5) . free radicals and reactive oxygen species are generated in living cells as a result of different biochemical and physiological processes, they are the causative agents for many chronic diseases, such as cancer, diabetes, aging and other degenerative diseases in humans due to oxidative damage of proteins, lipids and dna (6) . plants are the valuable sources of natural products for maintaining human health, more than 80%of population across the world use traditional medicine including compounds derived from medicinal plants. therefore, such plant should be investigated to better understand their properties, safety and efficiency (7) . the aim of this work was to investigate the chemical constituents and antioxidant activity of methanolic extract of aerial parts of a newly studied plant widely and wildly distributed in our country iraq. 1 corresponding author e-mail: dr.enasjawad@yahoo.com received: 8/3/2015 accepted: 18 /5/2015 iraqi j pharm sci, vol.24(1) 2015 iraqi tribulus 69 plant material and methods the aerial part of tribulus terrestris(family: zygophyllaceae) was collected from kirkuk, a city in the north of iraq, 236 kilometers (147 mi) north of baghdad. the plant was authenticated by the national herbarium at abu-graib, the plant leaves were dried in the shade for several days at room temperature and then grinded as powder and weighed. figure(1):iraqi tribulus terrestris the experimental work is divided into • the experimental preliminary phytochemical screening of various secondary metabolites like alkaloids, flavonoids, steroids, tannins, saponins, anthraquinioin and terpenoids in the plant. • extraction of different active constituents. • gc-ms analysis of methanolic extract of the plant. • investigation of the antioxidant activity of methanolic extract of aerial parts of plant preliminary qualitative phytochemical analysis chemical tests were carried out using the methanolic extracts from plant using standard procedures to identify the active constituents (810) : test for alkaloids alcoholic extract (10 ml) was stirred with 5 ml of 1% hcl on a steam bath. mayer’s (1.35gm mercuric chloride in 60ml water + 5gmpotassium iodide in 10ml water )and wagner’s reagents (1.27g of iodine and 2g of potassium iodide in 100ml of water) were added, white and reddish brown color precipitate respectively, were taken as evidence for the presence of alkaloids. test for flavonoids lead acetate test: lead acetate 10% (1 ml) solution was added to 5ml of alcoholic extract, the formation of a yellowishwhite precipitate was taken as a positive test for flavonoids. tests for steroids liebermann-burchard test: extract (3ml) was treated with chloroform, acetic anhydride and drops of sulphuric acid was added. the formation of dark pink or red color indicates the presence of steroids. test for tannins plant material (10mg) in 10ml distilled water was filtered, and then the filtrate (3ml) + 3ml of fecl3 solution (5%w/v) were mixed. the formation of a dark green or blue black precipitate was considered an indication for the presence of tannins. tests for anthraquinones borntrager’s test: alcoholic extract of 3ml was shaken with 3 ml of benzene, filtered and 5 ml of 10% ammonia solution was added to the filtrate. the mixture was shaken and the development of a pink, red or violet color in the ammonical (lower) phase indicates the presence of free anthraquinoin. test for terpenoids alcoholic extract (2ml) was dissolved in chloroform (2ml) and evaporated to dryness. concentrated sulphuric acid (2ml) was then added and heated for about 2 min. a grayish color was considered an indication for the presence of terpenoids. preparation of extract shade-dried coarsely powdered aerial parts of tribulus plant was defatted with hexane for 24 hours then allowed to dry at room temperature. the defatted plant material was extracted with 85% methanol in soxhlet apparatus until complete exhaustion. the alcoholic extract was evaporated under reduced pressure at a temperature not exceeding 40c • to give a dark-brown residue designated as a crude extract. animals healthy adult 30 male mice weighing 120150gm were used in this study. the animals had free access to a standard commercial diet and water; they were kept in rooms maintained at 25-27°c. the animals were divided randomly into three groups; each group consisted of ten male mice: group 1: received 100 mg/kg body wt. of 85 % methanolic extract of the plant. group 2: received 50 mg/kg body wt. of85 % methanolic extract of the plant. group 3: served as control group and received only 2% gum acacia (0.2ml). iraqi j pharm sci, vol.24(1) 2015 iraqi tribulus 70 the extracts were suspended in distilled water using tween 20, and the dose was orally administered once daily for 4 weeks. at the end of treatment, blood samples were collected centrifuged and serum was separated for the determination of the following: 1blood glutathione content according to the method described by beutler (11) . 2serum superoxide dismutase activity, the method was carried out according to the pyrogallol method of marklund (12) . 3serum ascorbic acid was estimated by the method of jagota (13) . gc-ms analysis instruments and chromatographic conditions gc-ms analysis was carried out on gcms-qp2010 shimadzu system comprising a gas chromatographinterfaced to a mass spectrometer instrument employing the following conditions : column vf-5ms fussed silica capillary column (30.0m x 0.25mm x 0.25μm, composed of 5% phenyl/95% dimethylpolysiloxane), operating in electron impact mode at 70ev; helium (99.999%) was used as carrier gas at a constant flow of 1. ml/min and an injection volume of 0.5μl was employed (split ratio of 10:1) injector temperature 240 0c ion-source temperature 200 c ₒ . the oven temperature was programmed from 100 c ₒ (isothermal for 3 min), with an increase of 10c ₒ /min, to 240 c ₒ , ending with a 9min isothermal at 270 c ₒ . mass spectra were taken at 70ev; a scan interval of0.5 seconds and fragments from 40 to 440da. total gc running time is 30min. results and discussion the results of preliminary qualitative phytochemical of iraqi tribulus terrestris are given in table-1.the results of preliminary phytochemical screening of plant extracts showed the presence of alkaloids, flavonoids, tannins, saponins and terpenoids and the absence of steroids and anthraquinoin. many researchers reported that the concentration of secondary metabolites are varying from plant to plant belong to the same genus and even in the different parts of the same plant (14) , this is due to many factors like environmental heterogeneity, since the effect of environmental heterogeneity is highly scaledependent. it may create high niche diversity and hence allow species to coexist at a large spatial scale (15) , also the high complexity and heterogeneity of soil, like(soil structure, texture and depth, moisture retention characteristics, aeration) create a big variation in the chemical constituents even in the same country (16) . identification of components by gc-ms: interpretation on mass spectrum of gcms was done using the database of national institute of standard and technology (nist) having more than 62,000 patterns. the mass spectrum of the unknown component was compared with the spectrum of the known components stored in the nist library . the results of gc-ms analysis led to the identification of number of compounds from the methanol extract of iraqi tribulus plant. gc-ms chromatogram showed 46peaks, indicating the presence of 46 compounds (figure-2) and (table2).many of these components reported in this plant for the first time like monoterpene [example terpineol] sesquiterpenes: [2,3,8,8-tetramethyltricyclo2ene], [1 ,4 – dimethyl – 8 isopropylidenetricyclodecane, alkaloids like (3-methoxy-2-pyrazinyl)-2-methyl-1-propanol and thiazole, saturated fatty acid[example myristic acid]coumaran and many phenolic compounds, coumaric acid, linoleic acid, arachidic acid and oleic acid. table(1): phytochemical screening of tribulus extract terpenoids anthraquinoin saponins tannins steroids flavonoids alkaloids + + + + + +, represent presence and absence of phytoconstituents respectively. iraqi j pharm sci, vol.24(1) 2015 iraqi tribulus 71 figure(2): gc-ms chromatogram of methanolic extract of iraqi tribulus terrestris table(2): phytocomponents identified in the methanolic extracts oftribulus terrestris peak# r.time area% name 1 4.168 0.56 3-penten-2-one, 4-methyl 2 4.257 0.39 1-butanol 3 4.534 0.24 hexanal dimethyl acetal 4 4.981 0.68 glycerin 5 5.592 0.97 trimethylsilylmethanol 6 6.092 1.54 4-hexenoic acid, 2-(phenylsulfonyl)-, methyl ester, (e) 7 6.626 1.50 coumaric acid 8 7.081 12.75 2-hexanol, 2-methyl 9 8.300 2.29 archidic acid 10 8.552 0.30 2-pentanone, 4-hydroxy 11 8.800 0.19 nonanal dimethyl acetal 12 9.049 0.48 1-hexanol, 2-ethyl 13 9.242 0.77 linoleic acid 14 9.341 0.74 silane, [2-(2-methoxyethoxy)ethoxy]trimethyl 15 9.729 0.32 propanoic acid 16 9.821 0.81 myristic acid 17 10.923 0.27 (r)-(-)-2,2-dimethyl-1,3-dioxolane-4-methanol 18 10.982 0.26 1,4-dimethyl-8-isopropylidenetricyclodecane 19 12.323 0.35 cyclohexanol, 1-methyl-4-(1-methylethylidene) 20 13.767 38.72 stearic acid 21 14.219 0.27 (2-benzyloxy-2-oxiran-2-ylethoxy)-t-butyldimethylsilane 22 14.585 0.30 cyclononasiloxane, octadecamethyl iraqi j pharm sci, vol.24(1) 2015 iraqi tribulus 72 table(2): contained phytocomponents identified in the methanolic extracts of tribulus terrestris the groups treated with alcoholic extracts of aerial part of iraqi tribulus showed a significant increase in blood glutathione level, serum superoxide dismutase activity and serum ascorbic acid level comparing with control group table( 3): mean blood glutathione content, serum ascorbic acid and serum superoxide dismutase among group of rats treated alcoholic extracts of t. terrestris. group (2) group (1) control group tested parameter 4.15 ± 0.21* 8.53 ± 0.317* 1.92 ± 0.052 blood glutathione (mg/ gm hb) 17.5 ± 1.03 * 30.2 ± 2.06* 10.23 ± 0.6 superoxide dismutase (μg/ml ) 6.54 ± 1.03* 12.09 ± 0.46* 3.23 ± 0.43 ascorbic acid (μg/ml ) group (1): treated with100 mg/kg body wt. of 85 % methanolic extract of the plant group (2): treated with 50 mg/kg body wt. of 85 % methanolic extract of the plant. * significantly different from control value. discussion in much of the developing countries, 70– 95% of the populationrely on traditional medicines for primary care, and between70% and 90% of populations in industrialized world use traditional medicines under the titles “complementary”, “alternative”,or “nonconventional” (17) . plants have formed the basis for traditional medicinal systems for thousands of years, with the first records dating from about 2600 bc in mesopotamia. traditional knowledge of medicinal plants has always guided the search for new cures. in spite of the advent of modern high throughput drug discovery and screening techniques, traditional knowledge systems have given clues to the discovery of valuable drugs. in the present study, methanolic extract of the iraqi tribulus terrestriswas analyzed for the first time for the presence of different secondary metabolites which could be of medicinal & economic value and study antioxidant activity of crude extract of this iraqi plant. the comparison of the mass spectrum with the nist database library gave more than 90% match as well as a confirmatory compound structure match. this work will help to identify the compounds, which may be used in body products, drugs, pharmaceutical and therapeutic value since peak# r.time area% name 23 14.641 0.07 n-cbz-glycylglycine p-nitrophenyl ester 24 15.099 0.62 phenylethyl alcohol 25 15.465 1.59 3,7,11,15-tetramethyl-2-hexadecen-1-ol 26 15.816 0.58 dodecanal 27 16.134 0.68 d-mannotridec-6-ene-1,2,3,4,5-pentaol 28 16.361 0.42 heptacosanoic acid, methyl ester 29 16.544 0.23 heptanoic acid, 3-buten-1-yl ester 30 16.874 0.55 thiazole, 4-ethyl-2,5-dimethyl 31 17.409 0.33 methyl(methyl 3,4-di-o-methyl-.alpha.-dmannopyranoside)uronate 32 17.409 2.24 2-pentadecanone, 6,10,14-trimethyl 33 18.322 0.15 ethanone, 1-phenyl-2-(phenylsulfonyl) 34 18.891 0.48 z-25-tetratriaconten-2-one 35 19.136 12.43 hexadecanoic acid, methyl ester 36 19.303 0.46 2,3,8,8-tetramethyltricyclo-2ene 37 19.734 0.18 phenol, 3,5-bis(1,1-dimethylethyl) 38 20.515 0.99 methyl 10-methyl-hexadecanoate 39 20.932 0.22 1,4-dimethyl-8-isopropylidenetricyclodecane 40 21.089 0.03 hexanedioic acid, bis(2-ethylhexyl) ester 41 21.709 0.2 diethyl phthalate 42 21.961 1.9 benzofuran, 2,3-dihydro 43 23.241 3.59 octadecanoic acid, methyl ester 44 23.762 1.73 9-octadecenoic acid, methyl ester 45 24.231 0.77 3-methoxy-2-pyrazinyl)-2-methyl-1-propanol 46 25.211 0.43 oleic acid iraqi j pharm sci, vol.24(1) 2015 iraqi tribulus 73 many components isolated from this plant reported for the first time, also the present study results were confirmed the traditional uses of this plant as an antioxidant, antiinflammatory, antispasmodic agent due to different secondary metabolites constituents like flavonoids, essential oil, alkaloids , saponins and others . the characteristic antioxidant properties of t.terrestris may cause significant increase in blood glutathione level, serum superoxide dismutase activity and serum ascorbic acid level. based on the results obtained in this study, it could be said that t.terrestris plant powder contains chemical constituents of pharmacological and nutritional significance. however, it is recommended that further work be carried out to isolate and purify the bioactive constituents in this plant powder using various extraction solvents with a view to characterizing their molecular structure, formula, weight as well as evaluating their safety or otherwise (toxicity) for human and other animal use. acknowledgment many thanks to college of pharmacy in university of baghdad for their support and our deepest gratefulto al-mustansiriya university .college of science, department of chemistry for their help in running gc-ms and many thanks to college of science in baghdad university for their help in estimating antioxidant activity of our plant. references 1. gauthaman k, ganesan a. the hormonal effects of tribulus terrestris and its role in the management of male erectile dysfunction – an evaluation using primates, rabbit and rat. phytomedicine 2008; 15:44. 2. s. h. majeed, and m. j. mahmood. herbs and medicinal plants in iraq between traditional medicine and scientific research. 1st ed. baghdad:dar althaowra for publishing., 1988, p. 40. 3. anand r, patnaik gk, kulshreshtha dk, dhawan bn. activity of certain fractions of tribulus terrestris fruits against experimentally induced urolithiasis in rats. ind j expbiol 1994;32:548–552. 4. koumanov f, bozadjieva e, andreeva m, platonva e, ankov v, clinical trial of tribestan. experiment med 1982; 1:2–4. 5. gauthaman k, adaikan pg, prasad rn. aphrodisiac properties of tribulus terrestris extract (protodioscin) in normal ancastrated rats. life sci 2002; 71: 13851396. 6. harman d., aging: phenomena andtheories. ann ny acadsci, 1998;854:1-7. 7. ogbole o. o., gbolade a. a, ajaiyeobae. o. , ethno-botanical survey of plants used in treatment of inflammatory diseases in ogun state of nigeria. european journal of scientific research, , 2010;43 (2): 183191 8. kokate c. k., gokhale s. b., purohit a. p. a text book of pharma-cognosy. 29th ed., nirali prakashan, 2009, p. 635. 9. harborne j.b. phytochemical methods, a guide to modern techniques of plant analysis.1st ed. london: chapman and hall; new york, 1973, p.278. 10. sarker s. d., latif z., gray a. i. natural products isolation. 2 nd ed. humana press, totowa, new jersey, 2005, p .515. 11. beutler e, duron o and kellely b (1963). improved methods for determination of blood glutathione.journal of laboratory and clinical medicine, 61: 882-888. 12. marklund s and marklund g. involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase.eur. j. biochem., 1974; 47: 469-474. 13. .jagota s and dani h. a new colorimetric technique for estimation of vitamin c. biochemistry, 1982;127: 178-182. 14. abdul k. k., palwasha a., ayeesha m., safdar ali k. ,rasool b.t.: response of plant parts and age on the distribution of secondary metabolites on plants found in quetta. pak j bot 2009; 41(5): 2129-35. 15. pausas j. g.1 , austin m.: patterns of plant species richness in relation to different environments: an appraisal. journal of vegetation science 2001; 12: 153-166. 16. karlovsky p.: secondary metabolites in soil ecology. volume 14,1st ed., springer-verlag berlin, heidelberg ,2008, 293p. 17. robinson, m.m., zhang, x. the world medicines situation traditional medicines: global situation, issues and challenges. who press, world health organization. 2011. iraqi j pharm sci, vol.28(2) 2019 guggulsterone and cyclophosphamide-induced renal toxicity doi: https://doi.org/10.31351/vol28iss2pp180-185 180 inhibition of nf-κb pathway by guggulsterone in the protective effects of cyclophosphamide-induced renal toxicity ali m. al-joda* and munaf h. zalzala**,1 * ministry of health and environment ,babil , babylon, iraq ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, iraq. abstract cyclophosphamide which acts as cytotoxic alkylating agent can induce a renal damage through the toxic metabolites which result from metabolic activation of cyclophosphamide by cytochrome p-450 inside hepatocyte and develop renal toxicity by direct binding with cellular organelles in the urinary tract cells. guggulsterone is a sterol derived from plant has ability to bind to farsenoid x receptor, mineral corticosteroid receptor, androgen receptor, glucocorticoid receptor and estrogen receptor. it efficiently decreases the proinflammatory response by downregulate some of genes that implicate in production and regulation of interleukins (il-2, il-4, il-6 and tnf-α). in this study, the albino rats were divided to four groups: control group treated with vehicle, cyclophosphamide group, cyclophosphamide with guggulsterone group and guggulsterone alone group. guggulsterone doses were administered orally (25 mg/kg/day) for 8 consecutive days while the cyclophosphamide (150 mg/kg) was administered intraperitoneal at day 5 of experiment. at the end of experiment, the kidney index and the serum level of tumor necrosis factor (tnf-), urea and creatinine were determined, also the nf-κb p65 and catalase tissue activities were measured with histopathological assessment. the daily dose of guggulsterone produced a significant reduction in the serum level of tnf-a, urea, creatinine, and tissue activities of nf-κb, and catalase enzyme. the histological feature was also improved. these data represent the protective effect of guggulsterone against the cyclophosphamide renal toxicity. key words: guggulsterone, nf-κb, cyclophosphamide, renal toxicity. سيكلوفوسفاميدالكوكيلستيرون والتاثير الوقائي للسمية الكلوية للبواسطة bk-nfتثبيط الـ 1،**لةمناف هاشم زلز و *علي محمود الجودة العراق . ، بابل ، بابل، دائرة وزارة الصحة والبيئة * .العراق،بغداد ،جامعة بغداد ،كلية الصيدلة والسموم ، فرع االدوية** الخالصة سيكلوفوسفاميد ، عامل ألكيل نشط سامة للخاليا ، له سمية كلوية بسبب األيضات السامة الناتجة عن تنشيط السيكلوفوسفاميد بواسطة ن طريق الربط المباشر لعامل ألكيل مثل األكورولين في المسالك في الكبد ومن ثم إفراز الكلى لهذه األيضات السامة ع p-450السيتوكروم ميكروسوم ومستقبالت الكورتيكوستيرويد المعدنية ومستقبالت xالبولية. غوغولستيرون هو ستيرول نباتي لديه القدرة على الربط بمستقبالت الفارسويد عة كل فعال من االستجابة المؤدية لاللتهابات عن طريق الحد من مجمواألندروجين ومستقبالت الجلوكوكورتيكويد ومستقبالت اإلستروجين. انه قلل بش في هذه الدراسة تم تقسيم الفئران أن (. tnf-αو il-2, il-4 ،il-6) االنترلوكينات مثل متنوعة من الجينات التي تنطوي على تنظيم إنتاج ومجموعة سيكلوفوسفاميد ، وسيكلوفوسفاميد مع مجموعة غوغلسترون البيضاء إلى أربع مجموعات: المجموعة الضابطة التي عولجت بمركبة ، 021أيام متتالية بينما كان سيكلوفوسفاميد ) 8مغ / كغ / يوم( لمدة 52ومجموعة غوغلسترون وحدها. سلعت جرعات غوغلسترون عن طريق الفم ) ، ( tnf-αتحديد مؤشر الكلى ومستوى المصل لعامل نخر الورم ) من التجربة. في نهاية التجربة ، تم 2مغ / كغ( يزرق داخل الصفاق في اليوم والكاتالز في النسيج الكلوي مع تقييم التشريح المرضي. تنتج الجرعة اليومية من nf-κb p65واليوريا والكرياتينين ، كما تم قياس أنشطة ، وأنزيم الكاتالز في النسيج الكلوي. كذلك nf-κb، وأنشطة ال ، واليوريا ، والكرياتينين tnf-αغوغلستيرون انخفاًضا كبيًرا في مستوى مصل تم تحسين الميزة النسيجية أيًضا. تمثل هذه البيانات التأثير الوقائي للغلغلسترون ضد سمية السيكلوفوسفاميد في الكلى. . سمية كلوية ، سيكلوفوسفاميدلل، bk-nf، الكوكيلستيرون الكلمات المفتاحية: introduction cyclophosphamide, a strong cytotoxic alkylating agent, is unique member of nitrogen mustards and broadly used to treat a variety of malignant and nonmalignant conditions. it is somewhat inactive in vitro and transformed to the active form in vivo[1]. the drawback of medical usages of cyclophosphamide owing to non-specific cytotoxicity which produce several adverse effects in multi-organs primarily in bone marrow, liver, testes, heart, kidney, and urinary bladder (1). the main mechanism of cyp induce renal injury is the formation of reactive toxic metabolites subsequent to metabolism of parent drug by hepatic cytochrome p-450 which followed by the renal excretion of these toxic metabolites that act as an alkylating agent (e.g. acrolein) with bind to several cellular organelles of the urinary tract. the acrolein has high reactivity to react with numerous target sites in cells of kidney and liberation of free radical which result in diminution of cellular thiols or stimulation of nfκb(2). 1corresponding author: munafzalzala@copharm.uobaghdad.edu.iq received: 6 /7 /2019 accepted: 6/9 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp180-185 mailto:munafzalzala@copharm.uobaghdad.edu.iq iraqi j pharm sci, vol.28(2) 2019 guggulsterone and cyclophosphamide-induced renal toxicity 181 cyclophosphamide administration can prompt tubular fibrosis, tubular necrosis, glomerular nephritis, interstitial edema, cortical tubular vacuolization, mild hemorrhagic cortex and then renal dysfunction. similarly, it has been demonstrated that administration of cyp result in an acute inflammation in the urinary bladder (3). it has also been identified as a product and also an initiator of lipid peroxidation(2). guggul resin take out from guggul tree named (commifora mukul) of family burseraceae. it has been used in ayurvedic remedy for many years(4). the key active constituent of guggul is guggulsterone (gs) which is a plant sterol with two isomers, eand z-guggulsterone, that accountable for hypolipidemic action(5). the two isomers of plant sterol has capability to bind and activate several nuclear receptors such as fxr, androgen receptor, mineral corticosteroid receptor, estrogen receptor, and glucocorticoid receptor .gs efficiently reduce the pro-inflammatory response by downregulation of variety of genes that involve in regulation and production of il-2, il-4, il-6 and tnf-alpha (6–8). also, the suppression of nf-κb and nf-κb regulated gene may define the effects of gs against atherosclerosis, diabetes, osteoarthritis, psoriasis and other inflammatory diseases (9, 10). this study was designed to evaluate the protective effect of gs against renal toxicity of cyclophosphamide. material and method drug and chemical cyclophosphamide was purchased from baxter oncology (frankfurt, germany). guggulsterone was purchased from xi’an geekee biotech, china. rat nf-kb/p65 elisa kit and tnfalpha (elisa) kit were purchased from elabscience biotechnology co. ltd. creatinine kit and urea kit for rats were purchased from biolabo sas, les hautes rives, maizy, france. measurement of catalase performed by manual method affording by m. h. hadwan and h.n. abed. (2016)(11) animal in a randomized, controlled animal study plan, 32 wistar albino adult male rats weighing (150-250) were obtained from the animal house of department of pharmacology and toxicology /collage of pharmacy of baghdad university. all animals were reserved in a area controlled for temperature at 25 ± 2, humidity 50-70 % and 12h lighting cycle followed a possible hygienic condition and permitted to eat and drink freely throughout the experiments. after 2 weeks of acclimatization, animals were weighed and their initial weights were recorded. the rats were divided equally to four groups, group i (control), group ii (cyclophosphamide-treated group cyp) where the rats treated with an oral dose of the blank suspension for 8 consecutive days, at day 5, a single dose of 150 mg/kg of cyclophosphamide injected intraperitoneal (i.p) and group iii (cyclophosphamide with guggulsterone cyp+ gs group) the rats were treated with an oral dose of 25 mg/kg/day of guggulsterone suspension for 8 consecutive days, at day 5, a single dose of 150 mg/kg of cyclophosphamide injected intraperitoneal (i.p), and group iv (guggulsterone gs) where the animal were treated with an oral dose of 25 mg/kg/day of guggulsterone suspension for 8 consecutive days. all the animals were euthanized at day nine after record the body weight. determination of organ index in the start of experiment, altogether wister albino rats have been weighed and when rats have been euthanized by cervical decapitation. kidney were collected and kidney index being measured by dividing kidney weight by entire body weight (12). biochemical analysis at the end of experiment, the animals were anesthetized with ethyl ether and the blood samples were collected from the heart puncture and allow all blood samples to clot for 1 hour at room temperature and the serum were separated by centrifugation for 15 minute on 10000 g speediness in a cold centrifuge. all samples are kept at -20°c for subsequent measuring the serum level of tumor necrosis factor (tnf-), urea and creatinine. measuring the level of nf-κb p65 and catalase activity in tissue homogenate: a 10% of kidney tissue homogenate was prepared and utilized to determine the concentration of nf-κb p65 by elisa kits by following to sandwich-elisa principle. the concentration of rat nf-κb in the samples were calculated by comparing the od of the samples to the standard curve[13]. catalase activity was measured by incubating the enzyme sample in 1.0 ml substrate (65 mmol/ml hydrogen peroxide in 60 mmol/l sodium–potassium phosphate buffer, ph 7.4) at 37 °c for three minutes. the reaction was terminated with ammonium molybdate. absorbance of the yellow complex of molybdate and hydrogen peroxide is measured at 374 nm against the blank. histological assessment tissues sample from the kidney was collected and fixed in 10% buffer formalin for histological examination by staining with hematoxylin and eosin (h&e) and investigated under light microscope (using 40x magnification). statistical analysis all results are presented as mean ± sd. the p-value < 0.05 are considered a significant for all data. the unpaired student's t-test was obtained to determine significance of differences between the mean values of each group (14). results as presented in table 1, the nephrotoxic effect of cyclophosphamide represent a significant elevation in renal level of nf-κb in comparison to control group. also, this effect produced a significant reduction in antioxidant activity of iraqi j pharm sci, vol.28(2) 2019 guggulsterone and cyclophosphamide-induced renal toxicity 182 catalase enzyme in the kidney tissue. these toxic effects of cyclophosphamide were reduced significantly by the oral doses of guggulsterone by decreasing the renal level of nf-κb and increases in the action of catalase in the renal tissue. as well as, the serum level of urea and creatinine were significantly elevated in the cyp group compared to control group. however, the administration of the oral guggulsterone with cyp (cyp+gs) significantly reduce the urea and creatinine levels (p<0.05) compared to cyp treatment group. table 1. effect of guggulsterone on the kidney parameters in the cyclophosphamide-treated rats. parameter group i (control) group ii (cyp) group iii (cyp+gs) group iv (gs) kidney nfκb level (ng/gm) 6.76  0.65 1.36  0.53 * 8.66  1.73# 6.98  0.52# kidney catalase activity (u/g) 2.51  0.12 2.06  0.24 * 2.305  0.22 # 2.54  0.06 # serum tnf-α (pg/ml) 93.25  40.66 722.7  186.6 * 462.38  197.91 # 101.85  45.97 # serum urea (mg/dl) 23.60  7.15 33.24  7.74 * 24.40  7.54 # 21.88  5.79# serum creatinine (mg/dl) 0.80  0.27 1.797  0.50 * 0.93  0.69 # 0.66  0.53 # kidney index 6.19  0.44 8.06  1.04* 7.05  0.74# 6.35  0.64# * is significantly different compared with control group (p<0.05). # is significantly different compared with cyp group (p<0.05). table 1 shows that the kidney index in the cyp treated animals was significantly increase compared to the control group (p<0.05). the administration of gs with cyp cause a significant reduction in the kidney index (p<0.05) compared to cyp treated group. all parameters were not significantly altered in the gs treated rats (group iv) compared to control group. figure 1. influence of guggulsterone (gs) administration on patho-histological structure of kidney in cypinduced toxicity in rats. photo-micrographs of hematoxylin and eosin-dyed kidney sections (400x) viewing: (a) control, (b) cyclophosphamide group, (c) cyclophosphamide + guggulsterone administration group and (d) guggulsterone (gs) only groups. in figure 1a, the control group displays a normal kidney tissue with normal glomeruli and tubules. however, cyclophosphamide treatment group as in figure 1b the image was revealed a widespread degenerative damage (red arrow) and cell death of epithelium lining of renal tubules. figure 1c, the cyclophosphamide + guggulsterone treatment showed narrow degenerative area and iraqi j pharm sci, vol.28(2) 2019 guggulsterone and cyclophosphamide-induced renal toxicity 183 little area of cellular death in the proximal tubules cells. whereas the gs only group (figure 1d), showed an extensive matching view to the normal tissue of normal glomeruli and normal renal tubules but there is infrequent of degenerative region of renal tubules. discussion the effect of cyclophosphamide induced significant increase in the nf-κb amount in renal tissue as compared to the control group. cyp prompted nephrotoxicity particularly at the proximal renal tubules in the rats due to oxidative stress and production of pro inflammatory cytokines which are controlled by stimulation of transcription factor nfκb which is accountable for the kidney damage(15,16). the renal tubular cells also have uptake and efflux transporters that implicated in the magnification the concentrations of cyp and /or its metabolites in the cells(17). the administration of guggulsterone with a cyp causes significant decreases in the activity of the transcription factors nf-κb compared to cyp only treated group owing to gs down regulate the expression of constitutive nf-kb activity and blocked ikb𝛼 phosphorylation besides it enhanced separation of inhibitory retained protein by inhibition of ikb kinase activation, therefore reducing p65 phosphorylation and don’t permit the nuclear translocation. the outcomes of this study come to an agreement with other studies[18-20]. therefore, gs decreased inflammation by suppression nf-κb signaling and has a brilliant impact on protection against cyp induced renal toxicity . the administration of cyp induce an oxidative damage and lipid peroxidation that result in a decrease in the catalase enzyme level. at high dose of cytotoxic cyp, it generates toxic metabolites with additional amount of reactive oxygen specie s(ros) for example super oxide, hydroxyl radical and hydrogen peroxide[21]. intracellular oxidant/antioxidant equilibrium system was interrupted due to high level of ros and decline of anti-oxidant enzyme activity. oxidative stress (os) might also cause catalase expenditure which consider antioxidant enzymes[22]. table 1 shows that cyp can produce a significant decrease in catalase activity compared to control group and significantly increase when gs added with cyp. in this study, the role of gs improves the structure and function of kidney, as a minimum in part by anti-oxidant effect that is mediated by increase renal tissue catalase activity. the suppression of nf-κb signaling and decrease release of pro-inflammatory cytokine which induce the inflammation, reduce generation of ros and its effects. also, previous studies showed that guggulsterone has an anti-oxidant activity(23,24). similarly, cyp treatment could also result in cellular injury by interfering with mitochondrial electron transport system which may interrupted oxidant/antioxidant balance through buildup of ros and induce the release of tnf-α [2,25,26]. numerous studies supposed that the formation of reactive oxygen species and os were the key intermediaries for initiation of inflammatory pathways (27,28). in the present study, a significant surge in tnf-α level in serum after treatment with cyp compared to control group and significant reduction in the serum level of tnf-α when gs is administered (table 1). the induction of tnf-α level was decresed in gs treatment due to their protective consequence against cyppromoted inflammation. this is consistent with numerous previous studies reported the anti-oxidant and antiinflammatory action of gs (7,8,29). the serum creatinine and urea is the typical serum markers being used to diagnose acute kidney injury[30]. treatment with cyp prompted acute kidney injury that was proven by the rise in renal biomarkers, serum creatinine and urea and the reasonable histopathological injuries in kidney [30]. in our study, the treatment of cyclophosphamide to rats prompted renal injuries and cause a significant increase of urea and creatinine serum level as compare to the control group (table 1). the intraperitoneal administration of guggulsterone (25 mg/kg) to rats was prevent the elevation of urea and creatinine levels which represent the biomarkers for the renal function. these outcomes might approve the protecting consequence of gs against cyp induced kidney damage and consistent in with the previous studies that reported the protective effect of guggulsterone from toxicant in kidney[32,33]. the protection mechanism of guggulsterone demonstrated by anti-oxidant properties as potent scavenger of free radical and inhibition of nf-κb pathway. the histo-pathological testing also brought further endorsement for the protective action of guggulsterone against cyclophosphamide induced renal toxicity. renal tissue (fig. 1) of cyp treated rats display extensive region of degenerative and apoptosis of epithelium lining with dilatation of renal tubules. this consistent with previous researches which stated cyclophosphamide induced histological and pathological changes, os and lipid peroxidation (31,34,35). the oral guggulsterone at dose of 25 mg/kg to rat for 8 days with intra-peritoneal injection of cyclophosphamide had revealed less histological alterations (figure1c), over narrow region of degenerative area and little zone of apoptosis in the cells of proximal renal tubules. this effect approve the protective consequence of guggulsterone treatment against the cyclophosphamide prompted renal injury. histological interpretation is in agreement with preceding reports that stated the protective action of iraqi j pharm sci, vol.28(2) 2019 guggulsterone and cyclophosphamide-induced renal toxicity 184 guggulsterone against other kidney toxicants (32,33). references 1. motawi tmk, sadik nah, refaat a. cytoprotective effects of dl-alpha-lipoic acid or squalene on cyclophosphamide-induced oxidative injury: an experimental study on rat myocardium, 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nephrotoxicity. bioorg med chem lett. 2017;27(14):3156–61. 33. ramesh b, sainath sb, karuna r, reddy ss, manjunatha b, sudhakara g, et al. effect of commiphora mukul gum resin on hepatic and renal marker enzymes, lipid peroxidation and antioxidants status in pancreas and heart in fructose fed insulin resistant rats. beni-suef univ j basic appl sci. 2015;4(4):269–78. 34. manda kl, bhatia al. prophylactic action of melatonin against cyclophosphamide-induced oxidative stress in mice. cell biol toxicol. 2003;19(6):367–72. 35. gunes s, sahinturk v, uslu s, ayhanci a, kacar s, uyar r. protective effects of selenium on cyclophosphamide-induced oxidative stress and kidney injury. biol trace elem res. 2018;185(1):116–23. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 flurbiprofen oral film 53 formulation and evaluation of flurbiprofen oral film shaimaa n. abd-alhammid * , 1 and haider h.saleeh ** * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. ** ministry of health, pediatric hospital, baghdad, iraq. abstract fast dissolving film can be defined as a dosage form, which when placed in the oral cavity. it will rapidly disintegrate and dissolves to release the medication for oral mucosal absorption or allow for the gastrointestinal absorption to be achieved when swallowed. flurbiprofen is non-steroidal anti-inflammatory agent with antipyretic and analgesic properties and can be used in low doses 8.75 mg as analgesic and anti inflammatory agent in sore throat infection. this study aims to formulate flurbiprofen as oral dissolving films, to improve the effective relief of pain with severe sore throats with little or no adverse effect. nine formulas were prepared using solvent-casting method, and the effect of different formulation variables on the physical and mechanical properties of the prepared films, besides to the drug release behavior was evaluated. it was found that, the prepared oral film of flurbiprofen that contains hydroxypropyl methylcellulose alone showed the fastest invivo disintegration time (30 sec.) among other investigated polymers. the drug release rates was also observed the prepared formula f1 which contains hpmc in concentration of (54% w/w), peg 400 (16% w/w) showed the fastest disintegration time 30seconds, drug release was 77.5% within 2minutes with satisfactory mechanical properties. the overall results suggested that the prepared formula of flurbiprofen can be conveniently administered orally in the form of an oral film for sore throat infection. keyword:oral strip, fluriprofen, hpmc polymer. تصييغ وتقييم الفلوربابروفين كشرائط فموية شيماء نزار عبدالحميد * ،1 صالح حيدر هيثم و ** .انعشاق، بغذاد ، كهٛت انظٛذنت جايعت بغذاد * .انعشاق ، بغذاد،ٔصاسة انظحت يستشفٗ انطفم انًشكض٘ ** الخالصة تفكك تفأَٓا ٚعطٗ فًٕٚا أ عٍ طشٚك انفى ٔانتٙ يا إٌ ٚتى ٔضعٓاشكم دٔائٙ حذٚث بأَٓاانششائح سشٚعت انزٔباٌ ًٚكٍ تعشٚف زٔب نتحشٚش انذٔاء نٛتى ايتظاطٓا عٍ طشٚك انغشاء انًخاطٙ انفًٕ٘ أٔ ٚسًح نٓا باأليتظاص عٍ طشٚك انجٓاص انٓضًٙ ٔانز٘ تٔ بهعّ.ٚتحمك عُذ ّٚ يع خظائض خافضّ نهحشاسِ ٔيسكُّ نالالو ,ًٔٚكٍ أستخذايّ فٙ جشعاث الستٛشٔدانضاد نالنتٓاباث يٚبشٔفٍٛ ,ْٕ افهٕسب أٌ األعشاع ٔكًضاد نالنتٓاب فٙ عذٖٔ انتٓاب انحهك ٔانبهعٕو .ٔيٍ انًعشٔف اٜالوفٙ يعانجت يههٙ غشاو 8,,5يُخفضت بانفى رائبتٚبشٔفٍٛ بشكم ششائح اانفهٕبإعطاء أٌ ٚبشٔفٍٛ ًٚكٍ أٌ تكٌٕ يشتبطّ بانجشعّ انذٔائٛت ٔبانتانٙ يٍ انًتٕلعاهفهٕسبن انجاَبٛت .أدَٗ أ بذٌٔ أعشاع جاَبٛتحذ ب انحهكانتٓاب تعًم عهٗ تمهٛم االالو فٙ تى تحضٛش تسعت طٛغ دٔائٛت بطشٚمت انمشظ كًا تى دساست انًتغٛشاث انًختهفت ٔ انخٕاص انفٛضٚأٚت ٔ انًٛكاَٛكٛت نهششائظ انًحضشة ٔحذِ أظٓشث أسشع ْٛذسٔكسٙ بشٔبٛم يثٛم سهٛهٕص شس انذٔاء. نمذ ٔجذ اٌ انششائظ انًحضشة انًحتٕٚت عهٗ إضافت إنٗ دساست تح ثاَٛت كزنك ضًٍ يجًٕعت يٍ انبٕنًٛشاث تى دساست تحشس انذٔاء اٚضا. 03صيٍ تفكك ( w/w% )85يثٛم سهٛهٕص بتشكٛض( انتٙ تحتٕ٘ عهٗ ْٛذسٔكسٙ بشٔبٛم f1) 1األٔنٗ ف انًحضشة انظٛغتنمذ أظٓشث انُتائج أٌ ثاَّٛ َٔسبّ تحشس 03( ْٙ االفضم بٍٛ انظٛغ االخشٖ يٍ َاحٛت صيٍ انتفكك فٙ انجسى w/w% )11بتشكٛض ٚكٕل,ٔبشٔبٛم اثٛم كال ٚبشٔفٍٛ اسبأيكاَٛت استخذاو انفهٕ إنٗأجًانٙ انُتائج تشٛش % ٔكزنك أظٓشث خٕاص يٛكاَٛكّٛ يمبٕنت .,,انذٔاء خالل دلٛمتاٌ ٚكٌٕ . انفىالنتٓاباث ٔانتٙ تعطٗ فًٕٚا كششائح سشٚعت انزٔباٌ بوليمر . hpmc ، موية ،فلوربايبروفينفالشرائط الالكلمات المفتاحية: introduction oral drug delivery has been known for decades as the most widely utilized route of administrated among all other routes that have been employed for systemic delivery of a drug via various pharmaceutical products of different dosage forms (1) . drug delivery through the oral cavity offers many advantages, the oral mucosa is conveniently and easily accessible and therefore allows uncomplicated application of dosage form, furthermore the oral mucosa is hurt against local stress or any damage and show fast cellular recovery after such incident (2) . 1 corresponding author e-mail:shaimaa-alsamariai@yahoo.com. received: 14/12/2013 accepted:12 /4/2014 iraqi j pharm sci, vol.23(1) 2014 flurbiprofen oral film 54 fast dissolving films (fdf) is a type of oral drug delivery system for the oral delivery was developed based on the technology of the monolithic transdermal patches. this delivery system consists of a thin film which is simply placed on the patients tongue or mucosal tissue of cheek (3) . a film or strip can be defined as a dosage form that employs a water-dissolving polymer (generally a hydrocolloid, which may be a bioadhesive polymer), which allows the dosage form to quickly hydrate adheres, and dissolves when placed on the tongue or in the oral cavity. these oral thin films or strips which are flexible one similar in size, shape and thickness to a postage stamp (2x3 cm) and can be packaged in multi dose containers or individually pouched (4) . flurbiprofen is a non-steroidal antiinflammatory drug, the structural formula are shown in figure 1 (5) . flurbiprofen is a drug that exhibits antiinflammatory, analgesic, and antipyretic activities in animal models. the mechanism of action of flurbiprofen, like that of other nonsteroidal anti-inflammatory drugs, is not completely understood but may be related to prostaglandin synthetase inhibition (5) . the present study is undertaken to prepare flurbiprofen oral dissolving films of (8.75 mg), to improve patient compliance, reduce the frequency of administration and to obtain greater therapeutic efficacy. figure 1: the chemical structure of flurbiprofen experimental work materials flubiprofen fdc limited , india, citric acid panreac , barcelona, espana, gelatin medichem enterprise(shanghais) co. limited, china, glycerin searle company, england, hydroxy propyl methylcellulose (hpmc e-15) sodium carboxymethyl cellulose ( cmc na) sigma-aldrich, usa, mannitol riedel-dehaen , germany, polyethylene glycol 400 (peg400) j.t baker,china, poly oxyethylene sorbitan monooleate (tween 80) sinopharm chemical reagent co.,ltd, potassium dihydrogen ortho phosphate, kh 2po4 sd fine-chem. limited,mumbai. methods characterization of flubiprofen determination of melting point the melting point of flurbiprofen was determined according to the method stated by usp (6) a compact column of flurbiprofen powder was prepared by inserting a small quantity of the powder into a one side sealed capillary glass tube. the tube was moderately tapped on a solid surface to form column of powder in the bottom of the tube, which is then positioned in electrical melting point apparatus and monitored until complete melting of the powder where the temperature reading was recorded. determination of λ max accurately weighed 10mg of pure flurbiprofen was transferred to 100ml volumetric flask. the drug then dissolved and diluted with phosphate buffer ph 6.8 to get concentration of 100µg/ ml of stock solution (7) . from stock solution aliquot was prepared to get concentration of 10 µg /ml and scanned over the wavelength range 200-400 nm against phosphate buffer 6.8 using spectrophotometer .the spectrum of absorbance versus wavelength was recorded using uvspectrophotometer and analyzed for the absorbance maximum (of λ max)-the wavelength at which the highest absorbance was observed. construction of calibration curves from stock solution 0.1 mg/ml aliquots were prepared 2, 4, 6, 8, 10, 14, 16 ml and transferred to 100ml volumetric flasks and diluted to get concentration of 2, 4, 6, 8, 10, 14 ,16 µg /ml, respectively . the absorbance of solution was measured at 247 nm using uvvisible spectrophotometer against phosphate buffer ph6.8 as a blank. the plot of absorbance versus concentration µg /ml is plotted& data was subjected to linear regression analysis. preparation of flurbiprofen oral films method of preparation of rapidly dissolving films nine formulas were prepared (f1-f9)with their composition shown in table (8), using solvent casting method, each film with surface area approximately 4 cm 2 is loaded with 8.75 mg flurbiprofen which is equivalent to about as a base. the area and number of films prepared for each batch can be calculated as follow (8) : total area of petri dish was 154 cm 2 each film area = 2×2 = 4 cm 2 number of films in batch =154/4 =38.5 iraqi j pharm sci, vol.23(1) 2014 flurbiprofen oral film 55 total drug load = 8.75×38.5 = 341.25 mg (flurbiprofen) an aqueous dispersion( solution 1) of film forming polymer was prepared by dissolving 1.053 gm hpmc in 50 ml distilled water ,then add on it 39mg citric acid , 39mg mannitol, and 312mg of peg. then the dispersion allowed to stir for 3 hours &kept at room temperature for 20 hour to remove all air bubble entrapped and then solution 2 is prepared by dissolving 341.25mg flurbiprofen in 50 ml ethanol and 78 mg of tween 80 (surfactant ) were added with a a constant stirring for 45 min. both solution (1 & 2) stirred for 1hour , and were used after at least 24 hours in the refrigerator to rest and remove all the air bubbles entrapped, then was cast onto 14 cm –diameter petri dish and was dried in the oven at 40 ºc for 24 hours. the films were carefully removed from the petri dish, checked for any imperfections and cut into the required size (2 x 2 cm 2 ) to deliver the equivalent dose per strip. the samples were stored in glass container until further analysis. film samples with air bubbles, cuts or imperfections were excluded from the study. table(1) composition of the prepared flurbiprfen oral films formulas. characterization of flurbiprofen oral films drug content uniformity five films unit of each formulation were taken in separate 100ml of volumetric flasks, 100m of ph 6.8 phosphate buffer was added and continuously stirred for 24 hr using water bath shaker. the solutions were filtered, diluted suitably and analyzed at 247 nm in a uv-spectrophotometer. the average of flurbiprofen was calculated. visual inspection properties such as homogeneity, color, transparency and surface of the oral films were evaluated for all the prepared formulas visually (9) . weight variation the weight variation of the flurbiprofen oral film was done by weighting twenty films individually and the average weight was calculated. for the film to be accepted, the weight of not more than two films deviate from the average weight by no more than 7.5% and no film deviates by more than 15% (10) . thickness measurements the thickness of each film was measured at five different locations (centre and four corners) using vernier caliper micrometer .the data are represented as a mean±sd of three replicate determinations. (11) folding endurance the folding endurance of randomly selected films was determined by repeatedly folding one film at the same place till it break or folded maximum 250 times (12) . the data are represented as a mean of three replicate determinations. surface ph measurement the surface ph of oral film was determined in order to investigate the possibility of any side effects in vivo. as an acidic or alkaline ph, may cause irritation to the oral mucosa. oral film was slightly wet with the help of water. then the ph was measured by ph paper (13) . the data are represented as a mean of three replicate determinations. mannitol tween 80 flurbiprofen glycerin citric acid peg 400 gelatin hpmc +cmc 50: 50 cmc hpmc formula no. 3.25 mg 2mg 8.75mg 1 mg 8mg 27 mg 1 3.25mg 2 mg 8.75 mg mg 1 8 mg mg27 2 3.25 mg 2 mg 8.75 mg 1 mg 8 mg 21 mg 3 3.25 mg 2mg 8.75 mg 1mg 8 mg 21mg 4 3.25mg 2mg 8.75mg 1mg 8mg 27 mg 5 3.25mg 2mg 8.75mg 1mg 8mg 21 mg 6 3.25mg 2mg 8.75mg 1mg 8mg 21mg 7 3.25 mg 2mg 8.75mg 8mg 1mg 21mg 8 3.25mg 2mg 8.75mg 10mg 1mg 21mg 9 iraqi j pharm sci, vol.23(1) 2014 flurbiprofen oral film 56 in-vivo disintegration study the time required for complete disintegration in the oral cavity was collected from three healthy volunteers. the volunteers were told about the purpose of the test. before the test, the mouth cavity was rinsed with a cup of water (48) . the film was placed on the tongue and subsequently the tongue was gently moved. the time required for disintegration in mouth was measured with a stopwatch and recorded as a disintegration time (14) . the data are represented as a mean of three replicate determinations. in-vitro dissolution study the in vitro dissolution test was carried out for all formulas in a usp basket dissolution apparatus type 1 (15) .a 4-cm 2 sample of film was exactly weighed. the dissolution medium was 900 ml of phosphate buffer ph 6.8 (51) . the rotation speed was 100 rpm at 37±0.5°c.10 ml aliquot of the dissolution medium was withdrawn at specific time intervals, and replaced with 10 ml of the phosphate buffer. the drug release was analyzed spectrophotometrically at λ max 247 nm. one film was placed into each vessel. the data are represented as a mean of three replicate determinations. result and discussion characterization of flurbiprofen determination of melting point the melting point of flurbiprofen measured was 115 ⁰c; this result is the same as reported (16) which indicates the purity of the drug powder. determination of λ max scanning the diluted solutions of flurbiprofen in phosphate buffers (ph 6.8) by uv spectrophotometer at 200400 nm gave the λ max found was 247 nm as reported (16) . construction of calibration curve calibration curve of flurbiprofen in phosphate buffers (ph 6.8) are represented in figure2. figure 2: calibration curve of flurbiprofen in phosphate buffer (ph 6.8) and 37 °c. evaluation of flurbiprofen oral films drug content uniformity all the prepared films were found to contain a uniform quantity of the drug. the preparations met the criteria of british pharmacopeia content uniformity (85115) % of the label claim. on this basis, it was found that the drug was dispersed uniformly throughout the film (17) . visual inspection hpmc films were transparent, colorless, thin and soft, and those prepared from sodium carboxy methyl cellulose (na cmc) were opaque, white and combination of hpmc and na cmc have semi-transparent appearance as shown in figure 3, while gelatin films were excluded from visual inspection because of rough surface , aggregation appearance ,and very poor film forming capacity. figure 3: formula f1 containing hpmc (hydroxy propyl methyl cellulose) polymer weight variation the results reveal that: the average weights for all the prepared formulas were uniform and comply with the referred values as showed in the table 2. thickness measurements table 2 gives the average thickness value of 3 films for each formula. a very low standard deviation value is indicating that the method used for the formulation of films is reproducible and give films of uniform thickness and hence dosage accuracy in each film can be ensured (18) . surface ph study the surface ph of all films was found between (6.2 -7.4) which is within the range of salivary ph.6.2 -7.4. no significant difference was found in surface ph of different films, and no mucosal irritation (19) . folding endurance the results were reported in table 2 were the folding endurance was found to be higher in f1(>300 ) due to the flexibility iraqi j pharm sci, vol.23(1) 2014 flurbiprofen oral film 57 nature and the concentration of the polymer (hpmc), while in formula f8(298) was, formula f9(180) was due to change of plasticizer from peg400 to glycerol and increase the concentration of plasticizer (20) . disintegration time in vivo the results table 2 showed that both changing the plasticizer types and concentration had non-significant difference on the mouth disintegration time of oral films. this because the two plasticizers are water soluble and will diffuse out of polymeric films in aqueous media generating void spaces in the film through which diffusion of fluid occurs, facilitating film disintegration (21) . table( 2)the physicochemical parameters of the prepared flurbiprofen oral films standard deviation from mean n=3 in vitro dissolution in vitro release was carried out in usp basket type dissolution apparatus .f1 formulation showed that the drug was rapidly released 77.5% with in 2 minute and formulation f3 release 34.04% with in 2 minute as shown in figure 4 and this is because increasing the concentration of hpmc e-15 may increased the release due to the leakage of the soluble part of the film during dissolution which left pores for drug release (22) . on other hand f2 formula has low release which is 26.6% when compared with formula f4 which is 36% which is drug release within 2 minute so increase cmc concentration increases the viscosity of the gel surrounding the film upon hydration and leads to the formation of a gel layer with longer diffusional path therefore decrease drug release (23) . figure4: effect of polymer type and concentration on the release profile of flurbiprofen in f1, f2, f3, and f4 phosphate buffer ph 6.8 and 37 °c. 0 20 40 60 80 100 120 0 5 10 15 20 25 30 35 40 % o f th e d ru g r e le a se d time(min.) f1 f2 f3 f4 hpmc 27 mg cmc 27 mg hpmc 21 mg cmc 21mg formula code thickness (mm) surface ph invivo dt ( sec) weight variation (mg) % drug relase within 2min. folding endurance f1 0.1 6.91 30.0 47 2 77.5 >300 f2 6.73 92.32 4 2 26.3 1 f3 6.913 32 44 4 34.04 70 f4 6.86 90 4 2 36 4 f5 ------------ f6 ------------ f7 4 7 6.66 50 36 32 2 f8 2 6.78 65 4 22.16 298 f9 4 2 6.75 60 4 52 180 iraqi j pharm sci, vol.23(1) 2014 flurbiprofen oral film 58 the combination of hpmc and na cmc show more retardation in the release from formula f8, which is 22.6% within two minutes as shown in figure 5. figure 5: effect of plasticizer type and concentration on the release profile of flurbiprofen in f7, f8and f9 phosphate buffer ph 6.8 and 37 °c. conclusion on the basis of the results obtained; hydroxy propyl methyl cellulose showed the fastest in vivo disintegration time. in addition, an acceptable mechanical properties and dissolution behavior were achieved. also glycerin was the best plasticizer as it showed an improvement in mechanical and physical characteristics of the flurbiprofen oral film. references 1. gavaskar b, kumar sv, sharan g. overview on fast dissolving films. int. j. pharmacy and pharma. sci. 2010; 2(3):2933. 2. gauri s, kumar g. fast dissolving drug delivery and its technologies. the pharma innovation. 2012; 1(2):34-39. 3. dixit r p, puthli s p.oral strip technology: overview and future potential, journal of controlled release. 2009; 139: 94-107. 4. ghosh t k, chatterjee d j, pfister w r. quick dissolving oral dosage forms: scientific and regulatory considerations from a clinical pharmacology and biopharmaceutical perspective. in: ghosh t k and pfister w r( eds). drug delivery to the oral cavity: molecules to market. n y, usa: crc press. 2005; 337-356. 5. maryadele j o’neil (ed ) the merck index. an encyclopedia of chemicals, drugs and biological. merck research laboratories division of merck co., inc., whitehouse station, nj, 2006, 14 th ed. 6. the united states pharmacopoeia (usp) 30, nf 25, 2006, usa: the united states pharmacopeial convention inc. 7. philip a k, pathak k. osmotic flow through asymmetric membrane: a means for controlled delivery of drugs with varying solubility. aaps pharm. sci. tech. 2006; 7(3): 1-11. 8. wei-qin t. practical aspects of solubility determination in pharmaceutical preformulation, in: augustijns p, brewster me, (eds) solvent systems and their selection in pharmaceutics and biopharmaceutics, new york: springer. 2007: 138-140. 9. mishra r, amin a.formulation development of taste masked rapidly dissolving films of cetirizine hydrochloride. pharm. technol. 2009; 33(2): 48-56. 10. semalty m, semalty a, kumar g. formulation and characterization of mucoadhesive buccal films of glipizide. indj. pharm sci. 2008;70:43-48. 11. sapkal n p, kilor v a, daud a s, bonde m n. development of fast dissolving oral thin films of ambroxol hydrochloride: effect of formulation variables, journal of advanced pharmaceutical research.2011; 2(2): 102-109. f7: peg 400 f8: glycerin 8mg f9: glycerin 8 mg time (min.) % o f d r u g r e le a se d iraqi j pharm sci, vol.23(1) 2014 flurbiprofen oral film 59 12. jadhav s d, kalambe r n, jadhav c m, tekade b w, patil v r. formulation and evaluation of fast dissolving oral film of levocetirizine dihydrochloride. int j pharm pharm sci. 2012; 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(2008), 21(3): 241-248. 16. vemala et al. colon specific controlled release matrix tablets of flurbiprofen: development and charetaraziation. asian j pharmaclin res.2012; 5(4): 9296. 17. cilurzo f, cupone i e, minghetti p, buratti s, gennari c, montanari l, diclofenac fast-dissolving film: suppression of bitterness by a taste-sensing system, drug dev. ind. pharm. 2011; 37(3): 252–259. 18. garsuch v, breitkreutz j. novel analytical methods for the characterization of oral wafer. eur. j. pharm. and biopharm. 2009; 73:195–201. 19. cilurzo f, cupone i e, minghetti p, selmin f, montanari l. fastdissolving films made of maltodextrins. eur j pharm biopharm 2008; 895–900. 20. chen, m., tirol, g., schmitt, r., chien, cand dualeh, a., film forming polymers in fast dissolve oral films. aaps annual meetings posters and papers, t3200, 2006. 21. vijayasri k et al. montelukast sodium oral thin films: formulation and evaluation .asian j pharm res, 2012; 5(4): 266-270. 22. garsuch v, breitkreutz j. novel analytical methods for the characterization of oral wafer, eur. j. pharm. and biopharm. 2009; 73:195–201. 23. jyothi s a, mounika p. formulation development and evaluation of oral thin filmsdiphenhydramine hcl. ijsr, 2013; 4(9): 3484-3488. iraqi j pharm sci, vol.24(1) 2015 spectrophotometric determination of clonazepam 25 spectrophotometric determination of clonazepam in pure and dosage forms using charge transfer reaction hind hadi *, 1 * chemistry department, college of science, university of baghdad, baghdad, iraq. abstract a rapid, sensitive and without extraction spectrophotometric method for determination of clonazepam (clo) in pure and pharmaceutical dosage forms has been described. the proposed method was simply depended on charge transfer reaction between reduced clo (n-donor) and metol (nmethyl-p-aminophenol sulfate) as a chromogenic reagent (πacceptor). the reduced drug, with zinc and concentrated hydrochloric acid, produced a purple colored soluble charge-transfer complex with metol in the presence of sodium metaperiodate in neutral medium, which has been measured at λmax 532 nm. all the variables which affected the developed and the stability of the colored product such as concentration of reagent and oxidant, temperature and time of reaction were investigated and optimized. the linearity of the method was observed within a concentration range 5-40 μg ml -1 clo and with a correlation coefficient not less than 0.998, while the molar absorbitivity and sandell sensitivity were 3.473×10 3 l.mole -1 .cm -1 and 0.0909 μg cm -2 respectively. the present work includes also the usage of the benesi–hildebrand equation for the evaluation of the association constant and molar absorptivity of the colored complex .finally the proposed method was successfully applied for the determination of clo in tablets. keywords: spectrophotometric ; clonazepam ; metol ; charge-transfer reaction. انتقذير انطيفي نهكهونازيباو في االشكال اننقية وانصيذالنية باستخذاو تفاعم انتقال انشحنة هنذ هادي عبذ هللا ،*1 * لسى ،خايعح تغذاد، تغذاد ، انعراق. انعهىو،انكًُُاء ،كهُح الخالصة نرمذَر عمار انكهىَازَثاو فٍ انشكم انُمٍ وانًسرسضراخ سرَعح وتذوٌ اسرخالص َرضًٍ انثسث طرَمح طُفُح تسُطح انصُذالَُح تاسرخذاو انًطُاف انضىئٍ .ذعرًذ انطرَمح انًمررزح تثساطح عهً ذفاعم اَرمال انشسُح نهكهىَازَثاو )يٍ َىع ياَر nكثرَراخ(انًخرسل تىاسطح انخارصٍُ وزايض انهُذروكهىرَك انًركس يع كاشف انًُرىل( n-اتار-يثُم-)يسرمثم يٍ أيُُى فُُىل( ( تىخىد يُراتُراَىداخ انصىدَىو كعايم يؤكسذ وفٍ وسظ يرعادل زُث َركىٌ َاذح ارخىاٍَ يسرمر ورائة فٍ انًاء أعطً πَىع ثرج عهً اسرمرار وذطىر انُاذح انًهىٌ ذى ذثثُرها واَدادها. ؤانً َاَىيُرر. خًُع انًرغُراخ 535أعهً ايرصاص عُذ طىل يىخٍ ارذثاط ياال خسء تانًهُىٌ وتًعايم 40 -5ر انرسى انثُاٍَ انخطٍ ناليرصاص يماتم انرركُس تأٌ انخطُح كاَد ضًٍ يذي انرركُس َشُ × 4733 إنً يساوَح انًىالرَح االيرصاصُح لًُح وكاَد 9980 عٍ َمم 3 نرر.يىل 00 -0 .سى -0 ساَذل زساسُح ولًُح 0909 0ياَكروغراو .سى -5 اَضا اسرخذاو يعادنح تُُُسٍ هُهذتراَذ السرخراج ثاتد ذدًُع انًعمذ وايرصاصه . َرضًٍ انثسث انًىالرٌ. أخُرا ذى ذطثُك انطرَمح تُداذ عهً انسثىب انساوَح عهً انكهىَازَثاو. تفاعم انتقال انشحنة.، انميتول، انكهونازيباو، انكهمات انمفتاحية: انتقذير انطيفي introduction clonazepam (clo) which is chemically known as 5-(o-chlorophenyl)-1, 3-dihydro-7nitro-2h-1, 4-benzodiazepin-2-one (figure1) is a very important drug. it is and medically considered as an anticonvulsant drug which is broadly used in the controlling of epilepsy. the most effective action of clo is treatment of absence seizures, myoclonic seizures, and infantile spasms (1, 2) . for the importance of clo compound, the literature review contains several researches that deal with its estimation. different methods for clo identification have been reported in all dependant pharmacopeia (36) such as ir spectroscopy method, whereas for the assay purposes a potentiometric and hplc methods have been reported. figure (1) clonazepam 1 corresponding author e-mail: hindhadi13@yahoo.com received: 21 /12/2014 accepted: 25 /3/2015 o2n n n h o cl iraqi j pharm sci, vol.24(1) 2015 spectrophotometric determination of clonazepam 26 many researches involved different methods for the determination of clonazepam in different matrices ( biological fluids and pharmaceutical forms) have been reported. these methods include chromatography (high performance liquid chromatography) (7, 8) , voltametry using carbon nanotubes modified electrode (9) , electrochemiluminescence sensors (10, 11) , derivative spectrometry (12,14) , gas and liquid chromatography-mass spectroscopy (15, 16) . from the literature search it was very clear that there are only a few spectrophotometric works for the determination of clo. therefore the present work describes the development of a cheap, simple and fast spectrophotometric method based on charge transfer (c.t) reaction between clo as donor and metol as acceptor in the presence of sodium metaperiodate. the purple charge transfer complex was measured at a maximum wave length of 532 nm. all the factors affected the reaction or the product were studied and optimized, and the method was satisfactorily applied for determination of clo in tablets. experimental apparatus the spectral and absorbance measurements in this work were performed using a digital double beam recording spectrophotometer (shimadzu uv-visible 260) using 1-cm quartz cells. materials and reagents all chemical reagents were supplied in a pure form and utilized for the current study. highly pure clonazepam (clo) was obtained from the state company for drug industries and medical appliance (sdi/iraq), while tablets that contain clo were obtained from market sources under the brand names (rivotril 0.5 and 2mg clonazepam/roche farma-spain).  reduction solution of clonazepam (500 μg ml -1 ) about 0.0500 g of pure clo was dissolved in a small volume of ethanol and then transferred into 50 ml volumetric flask and complete the volume to the mark with the same solvent. into a beaker of 150 ml, the previous solution was transferred. then a 20 ml of distilled water with 20 ml of concentrated hydrochloric acid (37%, 11.64 n), and 3 g of zinc powder were added and the mixture was mixed then allowed to stand for 15 min at ambient temperature (25 ºc).finally the reduction mixture was filtered and transferred into 100 ml volumetric flask, and diluted to the mark with distilled water to obtain 500 μg ml -1 of clo reduced solution (17) . the reduction solution is stable more than one week if kept in refrigerator. an appropriate dilution of stock solution using distilled water was performed to obtain a working solution.  blank solution this solution was prepared exactly as the previous reduction solution prepared but without presence of clo. this solution was added to the all blanks prepared in all experiments to minimize the error.  metol (n-methyl-p-aminophenol sulfate) reagent solution(bdh) (3×10 -2 m) this aqueous solution was freshly prepared by dissolving about 1.0331 g of reagent (m.wt=344.38 g/mol) in 100 ml distilled water, and stored in dark bottle.  sodium metaperiodate (spi) solution (0.1m) the oxidant solution in this concentration was obtained by dissolving 2.1360 g of spi and diluting to 100 ml with distilled water in volumetric flask.  solutions of pharmaceutical tablets the preparation of a 500 μg ml -1 stock solutions of clo from the tablets was done by taking twenty tablets of the commercial drug after weighting and pulverization, an amount of the powder corresponding to 50 mg of clo was dissolved in 30 ml of ethanol, shaked well and filtered into a 50 ml volumetric flask. the residue was washed with ethanol and finally the volume was made up to 50 ml with ethanol. then the reduction procedure which was described earlier was accomplished by transferring this solution into 125 ml beaker and followed the previous steps. further appropriate diluted solutions of pharmaceutical tablets were made using distilled water.  solution of blank pharmaceutical tablets similar procedure was followed for drug free tablets, to exclude the interference that may occur with excipient of tablets. recommended procedure and calibration curve into a sequence of 25 ml standard flasks (7 flasks), increasing volumes (0.25-2 ml) from reduced standard clo stock solution were transferred to cover the calibrated range (5-40 μg ml -1 of clo). to each flask a volume of 1.0 ml of 3×10 -2 m metol solution, and 0.5 ml of 0.1m sodium metaperiodate solution were added and the contents of the flasks were diluted to the mark with distilled water and mixed well. the absorbances of resulting solutions were measured after 10 min at 532 nm at ambient temperature (25°c) against reagent blank containing all materials except clo. alternatively the corresponding calibration curve and regression equation were iraqi j pharm sci, vol.24(1) 2015 spectrophotometric determination of clonazepam 27 constructed. for the optimization of conditions and in all later experiments, a solution of 500 μg of clo was used in a final volume of 25 ml (i.e. 20 ppm). stoichiometry of charge transfer reaction using job’s method and mole ratio method the stoichiometry of charge transfer reaction was calculated using equimolar of reduced clo and metol (1.584×10 -3 m) at constant oxidant concentration adopting job’s method of continuous variation (18) , and mole ratio method. in job’s method a series of solutions was prepared in which the total volume of drug and reagent was constant (10 ml). while in mole ratio method an increasing volume of metol (0.25, 0.5, 1, 1.5, 2 ml) was added to 1 ml of reduced clo at constant oxidant concentration. in various proportions of both drug and reagent, the solutions were mixed and diluted with distilled water in 25 ml volumetric flask, then the absorbance was measured at optimum wavelength and under optimal time and temperature against a reagent blank. the results obtained from figure 2 and 3 show that a 1:1 (drug to reagent) chargetransfer complex formed between clo and metol. figure (2): job’s method figure(3): mole ratio method estimation of association constant into a series of 25-ml volumetric flasks (7 flasks) an increasing volume of 0.25–1.5 ml of 1.584×10 -3 m standard solution of clo were transferred followed by 1.0 ml of 30 mm metol solution, and 0.5ml of 0.1m sodium metaperiodate solution. the detailed procedure under section 3 was then followed for this method. alternatively the association constant and standard free energy were calculated dependant on benesi–hildebrand equation. results and discussion absorption spectra among different reactions used for spectrophotometric determination of drugs, charge transfer reactions considered as a very sensitive and adequate method of analysis. based on formation a charge-transfer complex between reduced clo as electron-donor with selected π-acceptor (metol) in aqueous medium, a simple and rapid spectrophotometric method for clo determination was adopted in the present work. electron donor and acceptor compounds produce a new band of absorption at a specific maximum wavelength belong to the new complex. the reaction of reduced clo with metol results in the formation of a charge transfer complex of the n–π* type which absorbs at 532 nm (figure 4). figure(4): absorption spectra of (1) 40 μg/ml of clo treated as described under procedure and measured against blank (2) the reagent blank measured against distilled water and (3)40 μg/ml of solution of reduced clo only. reaction mechanism depending on the result obtained from job’s method about the stoichiometry of the reaction which showed that 1:1 (drug to reagent) charge-transfer complex has been formed between reduced clo and metol reagent at 532 nm, therefore, the formation of product possibly occurs as illustrated in scheme 1 (19) . optimization of charge transfer reaction conditions the experimental factors (reagents concentrations, order of addition, temperature and stability time) that affecting on development, stability and sensitivity of charge transfer complex were carefully considered. as we mentioned previously all experiments were done using 500 µg of clo in final volume 25 ml (i.e. 20 µg ml -1 ) and the absorbance measurements were carried out after 10 min of mixing of the reagents at laboratory ambient temperature (25 ± 2°c). iraqi j pharm sci, vol.24(1) 2015 spectrophotometric determination of clonazepam 28 sheme1: proposed mechanism of the reaction effect of reagent volume to examine the effect of volume of reagent, various concentrations of metol were added (0.5-2.0 ml of 30mm) to a fixed amount of reduced clo solution. a 1 ml of 30 mm solution was found enough to develop the color to its full intensity and gave a maximum absorbance of product (figure5), and was considered to be optimum for the next experiments. figure (5): effect of volume of metol effect of sodium metaperiodate concentration the effect of sodium metaperiodate (naio4) concentration were studied using different volumes over range (0.5-2.5 ml) from 0.1m of naio4 solution which were added to a reduced clo solution in the presence of 1 ml of 3×10 -2 m of metol, it was found that 0.5 ml of this oxidant was enough to give a maximum absorbance and full intensity (figure 6). the absorbance was decreased with increase of the volume of oxidant that added to the mixture of reaction, this may be due to the increase in the absorbance of the blank with each addition of sodium metaperiodate. figure (6): effect of volume of oxidant effect of reaction medium and order of addition the charge transfer product was only formed in acidic medium, while a precipitate had been formed in a basic medium. it is found that the acidic media used for reduction of clo was enough for reaction to proceed. usually a blank for clo solution containing all the contents of reduction material except clo, was used as a blank of reaction. different orders of addition of reagents were experimented (figure 7) and it was found that the order of addition of reagents cited under general procedure (clo+metol+naio4) was used in all following experiments, and was efficient in producing the aimed results and reaction progression. figure (7): effect of order of addition (d: drug, o:oxidant, r:reagent) effect of temperature and reaction time different temperatures were examined to study the effect of temperature on the color intensity of the charge transfer product. the experiment showed that the charge transfer complex formed between reduced clo and metol is formed immediately at an optimum ambient temperature of (25°c±2), furthermore the absorbance decreased with decreasing temperature (0°c), while at high temperature (60°c) the complex had been damaged. during following the progress of color intensity of the charge transfer complex with the time, the experimental results revealed that the color intensity reach a maximum after the reduced clo solution had been reacted with no2 n n h o nh2 n n h o zn/hcl clo reduced clo naio4 purple charge transfer complex cl cl n n n h o cl nch3 o h2 : + metol metol iraqi j pharm sci, vol.24(1) 2015 spectrophotometric determination of clonazepam 29 metol and naio4 for 10 min, therefore, a 10 min development time was suggested as the optimum reaction time and remain stable for 90 min (figure 8). figure (8): stability of charge transfer complex with the time analytical performance calibration curve and linearity after employing the optimum conditions described earlier under general procedure, linear absorbance-concentration plot (fig.9) over the concentration range of 125-1000 μg/25 ml or 5-40 μg ml -1 of clo with a correlation coefficient of 0.9984 were obtained. the visual characteristics such as molar absorpativity, limit of detection (lod), limit of quantification (loq) and sandell’s sensitivity (20) were summarized in table 1. figure(9): calibration curve table (1): analytical data obtained from proposed method parameter value regression equation y= 0.0110x 0.0267 linear range (μg ml -1 ) 5-40 correlation coefficient(r 2 ) 0.9984 lod (μg ml -1 ) 3.2427 loq (μg ml -1 ) 10.8089 reproducibility (%) 0.7-2.6 average of recovery(%) 99.1-104.1 molar absorbativity (l.mole -1 .cm -1 ) (ε=b×m×1000; m= m.wt (g mol -1 )) 3.473×10 3 sandell’s sensitivity (μg.cm -2 ), s (s = m/ ε) 0.0909 accuracy and precision in order to establish the accuracy and precision of the method, three different concentrations of standard solutions of clo were analyzed in four replicates. the results presented in table (2) indicate that the proposed method gave a reasonable precision and accuracy. table (2): accuracy and precision of the proposed method conc. of clo, μg ml -1 error %* rec .%* rsd %* present found 20.00 20.82 4.11 104.11 4.32 30.00 29.73 -0.89 99.10 1.17 40.00 40.54 1.35 101.35 1.07 *average of four determinations. method specificity (study of pharmaceutical additives) the proposed charge transfer reaction is carried out only between donor compound (reduced clo) and other acceptor, therefore tablets excipients such as talc, starch, lactose, polyvinyl pyrrolidone and magnesium stearate did not interfere with the assay due to absence of acceptor characteristic. experimental obtainable recoveries of spiked pure drug with previous excipients were ranged between (99.6-101.2%) and that means no interferences were observed in present work. analytical applications in order to examine if the proposed method is adequate for clo quality control, the proposed method was applied to the quantitative determination of clo in its dosage forms. two doses of tablets containing 0.5 and 2mg of clo have been analyzed and the results in table 3 (good precision <2.6 and high recoveries best than 98%) showed obviously compatibility of the proposed method for drug analysis. the new method was compared effectively with the british pharmacopeia's standard method (ultra violet method) (4) , which depends on tand f-tests respectively (20) . the obtained results tabulated in table 4 (calculated values <<tabulated values) showed that there was no significant differences in accuracy or precision between two methods at 95% confidence level. iraqi j pharm sci, vol.24(1) 2015 spectrophotometric determination of clonazepam 30 table (3): application of the proposed methods for determination of clo in pharmaceutical tablet forms drug form conc. of clo, μg ml -1 error%* rec.%* rsd%* present found rivotril® (0.5mg, roche farma, spain) 20.00 19.88 -0.62 99.38 1.86 30.00 30.12 0.41 100.41 0.71 rivotril® (2.0 mg, roche farma, spain) 20.00 19.83 -0.86 99.15 2.60 30.00 29.42 -1.92 98.08 2.57 *average of four determinations. table (4): the comparison of the proposed method with standard method using tand fstatistical tests drug form proposed method standard method[4] rec.% ( ̅ rec.% ( ̅ clo pure 101.52 2.274 100.50 0.322 rivotril/0.5mg 99.90 0.013 99.80 0.018 rivotril/2.0mg 98.62 1.952 99.50 0.188 s** 1.091( =2.119) =0.263) t (2.776)* 0.088 (n1 + n2 – 2) = 4 f (19.000)* 8.047 n1= 3 , n2=3 * indicates table value. **s = pooled standard deviation √ , t = | ̅ ̅ | √( ) ̅ ̅ in addition a paired t-test (20) was conducted between the samples from three sources by either method of analysis i.e. charge transfer method with standard method as shown in table 5. a t-value for n-1 degree of freedom = 4.303. calculated t-value=0.124 for n-1 at α 0.05 (95%), two tailed indicate that since 0.142<<4.303, therefore it can be regarded that there is no difference in using the two methods. table ( 5): paried t-test for proposed method with classical method sample amount found xd ̅d ̅√ / at 95% at 95% proposed method classical method clo pure 101.52 100.50 1.020 0.078 0.953 0.142<<4.303 rivotril/0.5mg 99.90 99.80 0.100 rivotril/2.0mg 98.62 99.50 -0.885 xd: difference between two methods, ̅ difference mean, difference sd, , n=no of sample. association constant (benesi–hildebrand equation) the most important factors that associated with the stability of the complex and should be calculated during the study of the charge transfer reactions are the association constant and standard free energy change of product complex. these can be obtained by applying the benesi–hildebrand equation (21, 22) which depends on reacting two species one of them is in large excess concentration, in order that its concentration will be unchanged on the construction of the complex. iraqi j pharm sci, vol.24(1) 2015 spectrophotometric determination of clonazepam 31 where [aº] is the total concentrations of acceptor, [dº] is the total concentrations of donor, a ad is the absorbance of the charge transfer complex, ad and are the molar absorptivity and association constant of the of the complex respectively. in order to obtain the association constant, molar absorpativity and standard free energy of the charge transfer complex product, a simple plotting of [a0]/a ad versus 1/[d0] (fig. 10) was adopted. the intercept and slop of a straight line obtained were used to calculate the corresponding wanted factors (table 6). the value of the standard free energy change (δgº) of the product complex is determined by the following equation: δgº= -2.303rt log where r is the universal gas constant (8.314 j mol -1 deg -1 ) and t is the absolute temperature in kelvin and δgº were calculated to be 18.1418 kj/mol as presented in table 6. the negative charge of δgº value refers to the spontaneously of the reaction. figure (10): benesi–hildebrand plot for clo and metol complex table(6): association constant, molar absorptivity values and calculated free energy from benesi–hildebrand plots for the complexes formed intercept slope (l.mol -1 .cm -1 ) log δgº, j/mol (δgº=-2.303rt log ) δgº, kj/mol 0.000501 3.311e-07 1997.681 1511.87 3.1795 -18141.8 -18.1418 conclusions charge transfer reaction is considered as one of the most important reaction used for drug analysis. the present work describes a simple and cheap method for determination of clo in its dosage forms depend on this 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university of baghdad, baghdad,iraq. abstract the presence of the most important steroidal sapogenin “tigogenin” in the leaves of agave americana cultivated in iraq was detected. the absence of any study concerning the tigogenin content of this medicinal plant in iraq, and the industrial importance of tigogenin depending on its role as a precursor in the synthesis of some steroidal drugs, acquired this study its value. this search include extraction, isolation, purification and dentification of tigogenin from the leaves of agave americana. extraction of this compound was carried out using two methods. identification of this compound is carried out by using thin layer chromatography (tlc) where three different mobile phase have been used. detection is done by using libermann – burchard reagent .high performance liquid chromatography (hplc) is also used for further identification of tigogenin and then this steroidal saponin was isolated and purified. isolated tigogenin is identified by using thin layer chromatography (tlc), melting point (m.p.), infrared spectroscopy (ir) and high performance liquid chromatography (hplc) . this study confirms the presence of tigogenin in the leaves of agave americana cultivated in iraq. also the result of this study showed that the second extraction method is better, because the amount of both tigogenin and extract obtained from method no.2 are more than one extraction method. key words : agave americana , steroidal saponin , tigogenin. اغا األ نباث اوراق في الموجود التيكوجينين الصابوني للستيرويد كيموأحيائيت دراست العراق في المسروع رؤى محمد ابراهيم ،*1 و زينب جليل عواد * ** .، انعزاقجايعت بغذاد،كهٛت انظٛذنت، انعمالٛز انطبٛت فزع الخالصة انعزاق فٙ انًزرٔع غافاأل َباث أراق فٙ يٕجٕدة طابَٕٛت سخٛزٔٚذٚت يادة اْى ٔجٕد عٍ بانكشف حخخض انذراست ْذِ انذٔائٛت نهظُاعاث انًًٓت انخٛكٕجٍُٛٛ يادة يٍ انُباث ْذا يحخٕٖ حخُأل انعزاق فٙ دراست ا٘ ٔجٕد عذو اٌ . انخٛكٕجٍُٛٛ يادة ْٔٙ فٙ . انذراست ْذِ لًٛت ٚبٍٛ انجُسٛت ٔانٕٓريَٕاث انكٕرحزٌٔ يثم انًًٓت انسخٛزٔٚذٚت االدٔٚت بعض نخظُٛع االبخذائٛت انًادة بكَٕٓا ْذِ اسخخالص حى حٛث غاف.األ َباث أراق فٙ انًٕجٕدة انخٛكٕجٍُٛٛ يادة ٔحُمٛت ٔفظم ٔكشف اسخخالص حى انذراست ْذِ َالم كٕسٛظ يخخهفت يذٚباث باسخخذاو , انزلٛمت انطبمت كزٔياحٕكزافٛا طزٚمت بٕساطت عُٓا انكشف ٔحى نهفظم طزٚمٍٛ انًادةباسخخذاو عًهٛت انفظم حًج ٔبعذْا انسائم انعانٙ االداء كزٔياحٕكزافٛا حمُٛت بٕساطت ٔكذنك . بٛزجارد نٛبزياٌ كاشف باسخخذاو عُٓا ٔانكشف , االَظٓار درجت : شًهج ٔانخٙ َمأحّ ٔدرجت انًفظٕل انًزكب َٕعٛت يٍ نهخحمٛك انخمُٛاث يٍ يجًٕعت اسخخذيج ٔلذ . ٔانخُمٛت ٔجٕد انذراست ْذِ اثبخج. انسائم انعانٙ االداء كزٔياحٕكزافٛا حمُٛت ٔكذنك انحًزاء ححج االشعت يطٛاف , انزلٛمت انطبمت كزٔياحٕكزافٛا االفضم ْٙ نالسخخالص انثاَٛت انطزٚمت باٌ انذراست ْذِ اظٓزث كًا . انعزاق فٙ انًزرٔع األغاف َباث أراق فٙ انخٛكٕجٍُٛٛ يادة . نالسخخالص االٔنٗ انطزٚمت يًااعطخّ اعهٗ كاٌ انًادة ْذِ يٍ ٔيحخٕاِ انًسخخهض كًٛت نكٌٕ .التيكوجينين مادة الموادالستيرويديتالصابونيت،،اغا األ نباث: المفتاحيت الكلماث introduction agave americana is succulent plants that are present in abundance in swaziland especially in the middleveld and lowveld . agave plant is a monocotyledon that refer to the agavaceae family. it is a native of mexico and other parts of tropical america. it is described by rigid, fleshy and hard-surfaced leaf grow directly out from the central stock to form a dense rosette. common names include american aloe , maguey, or century plant (1) . a.americana crude extract have been report to possess insecticidal and molluscicidal properties (2) and also have antisporulant activity versus sclerospora graminicola (3) . the leaves of this plant possess angiotensin converting enzymes that are represent a powerful medicine for hypertension treatement. in addition, the leaves is a rich source of steroidal sapogenins like sarsasapogenin ,hecogenin and tigogenin 1 corresponding author e-mail: received: 4/8/ 2015 accepted: 7/11/2015 iraqi j pharm sci, vol.24(2) 2015 steroidal sapogenin “tigogenin” in the leaves of agave americana 42 which can be ustilized in the production of semisynthetic corticosteroids. active constituent of a. americana such as tigogenin is used as starting material for the synthesis of other steroids (2) . antibacterial activities of a. americana leaf extract is attributed to the presence of saponins (4) . saponins have immune-stimulating activity , hemolytic, antiinflammatory and expectorative. in addition, saponins displayed antimicrobial activity particularly against bacteria, fungi and protozoa. the antifungal activity of steroidal sapogenin has been demonstrated particularly versus agricultural pathogens (1) and other activities for this type of compounds include hypoglycemic, antitumor, immunoregulatory (5) . material and methods plant materials the leaves of agave americana l. were collected from garden of college of pharmacy of baghdad university during november (2014). they were cleaned and dried in oven at a temperature between (30 – 40) for (16) hours then these plant materials were coarsely powdered by mechanical grinder and weighed. 50 gm of dried powdered plant materials were extracted by using two methods. extraction extraction method no.1 (6) : 50 gm of powdered leaves of a. americana l. were macerated with 300 ml of methanol 99.8% at 25 ₒ c for one week. after evaporation of the methanol under vacuum, the extract is dissolved in 50 ml of h2o and then extracted with ethyl acetate by separatory funnel (3 × 50 ml , 30 min each), the organic phase is evaporated to dryness under vacuum, and then subjected to identification as shown in figuer (2). figure (1):agave americana plant cultivated in iraq powdered leaves (50 gm) maceration in 300 ml of methanol at 25  c for one week filteration filterate drying residue dissolved in water extracted with ethylacetate by separatory funnel (3 × 50 ml , 30 min each). ethylacetate layer aqueous layer evaporated to dryness (0.84gm) figuer (2): general scheme for method no.1 for extraction of steroidal sapogenins from the leaves of agave americana l. iraqi j pharm sci, vol.24(2) 2015 steroidal sapogenin “tigogenin” in the leaves of agave americana 43 extraction method no.2 (7) 50 gm of dried powdered leaves was refluxed in water (500 ml) for 3 hrs.the aqueous extract is filtered then concentrated in oven at 50 ₒ c then resuspended in an ethanol :water (80:20) solution and maintained at 25 ₒ c for 18 hrs to separated polysaccharides.then the precipitated polysaccharides were separated by filteration and the hydroalcoholic extract was concentrated. the hydroalcoholic extracts was subjected to a hydrolysis reaction under reflux for 3 hrs in a hydroethanol solution 80% ( 30 ml ) containing 5 ml of conc. hcl.the reaction product was neutralized with 10% naoh and then extracted with ethylacetate to yield the sapogenins, after removal of the solvent using a rotary evaporator . the extract was evaporated to dryness under vacuum, and then subjected to identification , shown in figure (3). powdered leaves (50 g) extracted with 500 ml distilled water for 3 hrs filtered and concentrated in oven at 50 ₒ c resuspended in an ethanol : water (80:20) and maintained at 25 ₒ c for 18 hrs prepcipitated hydroalcoholic extract (polysaccharide ) acid hydrolysis by reflux for 3 hr neutralization by 10%naoh extracted with ethylacetate by separatory funnel ethylacetate layer evaporated to dryness(1.2 gm) figure(3):general scheme for method no.2 for extraction of steroidal sapogenin from the leaves of agave americana l. identification of the steroidal sapogenin “tigogenin”: the preliminary identification of steroidal sapogenin (tigogenin)was performed by using: 1thin layer chromatography (tlc): in this qualitative identification:used a ready-made aluminum plates of silica gel gf254 and detection is done by libermann – burchard reagent (8) in comparison with three different developing solvent systems that were (9 ,10) : solvent 1 (s1): hexan :ethylacetate (1:1) solvent 2 (s2): chloroform:methanol (95:5) solvent 3 (s3): benzene:acetone (90:10). 2preperative high performance liquid chromatography (phplc): qualitative estimations of tigogenin component in crude extract obtained by extraction methods was carried out by using preperative high performance liquid chromatography (phplc). the qualitative estimations are performed by comparing retention time of tigogenin component in the crude extracts with that of authentic standard at identical chromatographic conditions. hplc analysis was done by using the following conditions (11) . iraqi j pharm sci, vol.24(2) 2015 steroidal sapogenin “tigogenin” in the leaves of agave americana 44 1. mobile phase : methanol 100% 2. column:phenomenex c18 250 mm x 4.6 mm, 5 µm particle size. 3. column temperature: ambient 4. flow rate : 10 ml / min 5. injection volume : 2 ml 6. injection concentration: 1 mg /ml 7. detection: uvdetector at λ 254 nm. isolation and purification of tigogenin isolation and purification of tigogenin was done by using the following steps: 1.using preparative tlc plates isolation of tigogenin compound is carried out using preparative tlc . the crude extracts obtained by extraction method no.2 was dissolved in ethylacetate then apply by using capillary tube in a row of spots as a concentrated solution five times on each plate (the spots must dry before other application) . the thickness of silica gel on the plate was 0.75 mm. the solvent system used was s3 (benzene : acetone (90:10)) . the detection was done using libermann – burchard reagent in one side of the plate . the band is scraped out that corresponding to the tigogenin standard and put in a beaker, added chloroform: methanol (95:5) with stirring then left a side for hour, and filtered. after evaporation of the solvent, the final filtrate gives yellowish precipitate . 2.purification of tigogenin by recrystalization the final solid product that has been isolated by preparative tlc was dissolved by heating insufficient quantity of methanol and decolorizing charcoal( small amount) is added , with stirring to the hot methanol solution until supernatant liquid is almost colorless. then the hot solution is filtered and then the methanol is evaporate to obtain powder (12) . results and discussion extraction methods two methods of extraction of steroidal sapogenin tigogenin were tried to select the best one. results showed that the method no.2 was better, because the yield of crude extract was higher than obtained from method no.1. in addition quantitative estimation by using hplc analysis showed that the amount of tigogenin obtained by method no.2 was much more compared with that obtained by method no.1 as showed in table (1). so, we select method no. 2 as an extraction procedure in our work. table (1): quantitative of crud extracts and tigogenin obtained from extraction methods. identification of tigogenin by tlc: tlc for the crude extracts from agave americana leaves obtained by using the extraction method no.1 and no.2, confirms the presence of tigogenin in these extracts in comparison with tigogenin standard . as presented in table (2) and figures ( 4 ,5 and 6). table(2): rf values of tigogenin from agave americana l. leaves extract obtained by extraction methods no.1 and no.2 and its standard in three different mobile phases in tlc. solvent system e1 e2 s s1 0.671 0.685 0.671 s2 0.746 0.733 0.746 s3 0.446 0.43 0.43 s1: hexan : ethylacetate (1:1) s2: chloroform : methanol (95:5) s3: benzene : acetone (90:10) e1 :crude. extra ct of method no.1 e2: crude extract of method no.2 s: tigogenin standard extraction method yield of crude extract (g) % yield of crude extract % yield of tigogenin method no.1 0.84gm 1.68% 0.23% method no.2 1.2gm 2.4% 0.43% iraqi j pharm sci, vol.24(2) 2015 steroidal sapogenin “tigogenin” in the leaves of agave americana 45 e2 t e1 figure(4): tlc for agave americana leaves extract obtained by extraction methods no.1 and no.2 using silica gel gf254 as adsorbent and (s1) as solvent system . visualization by liebermann-burcrhard spray reagent. t: tigogenin standard e1: extraction method no.1 e2: extraction method no.2 e2 t e1 figure (5) : tlc for agave americana leaves extract obtained by extraction methods no.1 and no.2 using silica gel gf254 as adsorbent and (s2) as solvent system . visualization by liebermann-burcrhard spray reagent. t: tigogenin standard e1: extraction method no.1 e2: extraction method no.2 e2 t e1 figure (6): tlc for agave americana leaves extract obtained by extraction method no.1 and no.2 using silica gel gf254 as adsorbent and (s3) as solvent system . visualization by liebermann-burcrhard spray reagent. t: tigogenin standard e1: extraction method no.1 e2: extraction method no.2 identification and characterization of the isolated tigogenin 1. analytical tlc isolated compound ( tigognin) appeared as a single spot having the same color and rf value to that of standard . 2. melting point the purified tigogenin is characterized from its sharp melting point of 200 202 ºc compared to standard tigogenin melting point 202-204 ºc 3. ftir the ir spectra of isolated tigogenin was gave identical results with that of tigogenin standard, as showed in table (4) and figure (7). 4. hplc analysis the retention time for the isolated tigogenin was identical to the main peak of the standard reference as showed in figures ( 8 and 9 ) . iraqi j pharm sci, vol.24(2) 2015 steroidal sapogenin “tigogenin” in the leaves of agave americana 46 table (4): ): the most significant group frequencies from ft-ir spectrum of isolated tigogenin . functional group group frequency wave number ( cm -1 ) assignment free o-h 3533 free o-h stretching of alcohol o-h broad band (3431-3238) broad o-h stretching band indicate hydrogen bonding c–h 2926,2848 2962,2875 asymmetric and symmetric stretching of ch2 and ch3 groups c-h 1456,1367 c-h bending of ch2 and ch3 c-o 1242-1041 c-o stretching of aliphatic ether figure (7): ir spectrum of isolated tigogenin figure (8):hplc analysis of isolated and purified tigogenin. figure (9) :hplc analysis of tigogenin standard. iraqi j pharm sci, vol.24(2) 2015 steroidal sapogenin “tigogenin” in the leaves of agave americana 47 conclusions phytochemical investigation of agave americana leaves, cultivated in iraq confirm the presence of “tigogenin” steroidal sapogenin as potent medicinal natural product . tigogenin was extracted by using two extraction methods, and identified by using tlc and hplc method . isolation and purification tigogenin compound was made by using the following steps: preparative tlc plates, and purification by using charcoal material. the isolated tigogenin is identified by using thin layer chromatography , melting point , infrared spectroscopy and hplc. acknowledgements i would like to acknowledge prof. dr. ali al-musawi, in college of sciences, university of baghdad. for identification of agave americana plant. my deepest gratitude and appreciation to all teaching staff and students of college of pharmacy, 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biological activities and distribution of plant saponins. journal of ethnopharmacology. 2004;94:.219-243. 6. chen p-y, chen c-h, kuo c-c; et al : cytotoxic steroidal saponins from agave sisalan. planta med .2011; 77: 929–933. 7. santos& branco :'' gc-ms characterization of sapogenins from sisal waste and a method to isolate pure hecogenin ''.bioresources. 2014; 9 (1) : 1325-1333. 8. wagner, h.; bladt, s. and zgainski , em.: plant drug analysis, a thin layer chromatography atlas . springer – verlag , berlin , germany .1984; p.226 . 9. harborene, jb.; phytochemical methods ( 1 st ed.). chapter two, phenolic compounds . chapman and hall , london , 1976; pp, 115117. 10. stahl e.: thin layer chromatography hand book, 1999; pp. 694. 11. peana at, moretti md, manconi v, et al: anti-inflammatory activity of aqueous extracts and steroidal sapogenins of agave americana. planta med .1997;63(3): 199202. 12. shriner, rl.; hermann, ckf.; morrill, tc.: preliminary examination , physical properties and elemental analysis , the systematic identificationof organic compounds (86th ed). john wiley and sons inc., usa. 2004; pp.51-52. iraqi j pharm sci, vol.25(1) 2016 oxidative stress and some trace elements in type 2 diabetes mellitus 17 estimation of oxidative stress and some trace elements in iraqi men patients with type 2 diabetes mellitus abdulrahman r. mahmood *,1 * department of chemistry, college of education for pure sciences (ibn-alhaitham), university of baghdad, baghdad, iraq. abstract type 2 diabetes mellitus (t2dm) is a chronic disorder that is associated with the imbalance of trace elements which are involved in many functions especially enzyme activities. changes in the levels of serum elements probably can create some complications in type 2 diabetes mellitus. previous experimental and clinical studies report that oxidative stress plays a major role in the pathogenesis and development of (t2dm). however, the exact mechanism of oxidative stress could contribute to and accelerate the development of (t2dm). the aim of this study contained the following sections: firstly, to determine some biochemical parameters in subjects with type 2 diabetes mellitus (t2dm) like lipid peroxidation marker, malondialdehyde (mda), total antioxidant capacity (tac), and glycated haemoglobin (hba1c). secondly, to determine serum and urine levels of zinc as trace element and serum level of iron, then to compare those parameters with that for age matched healthy individuals. the study performed on 45 iraqi men, who attended baghdad teaching hospital. they were grouped into to 2 groups based on fasting serum glucose (fsg), and (hba1c) value first group was included 20 healthful persons with a1c <6.4% as control non diabetic group, second group was included 25 patients with a1c>6.4% as t2dm. outcomes of this study demonstrated a clear increase in t2dm in fasting serum glucose, ( hba1c), mda values, urine concentration of zinc , and serum level of iron compare to control.t2dm shows a significant reduction in both tac and serum level of zinc ,if compared with control group . keywords: type 2 diabetes mellitus, trace elements, lipid peroxidation. تقذٌر جهذ التبكسذ وبعط العىبصر الىزرة لمرظى عراقٍٍه مصببٍه بذاء السكري الىىع الثبوً عبذ الرحمه رشٍذ محمىد ،*1 * خاِعح تغذاد ، تغذاد ، اٌعشاق . ، (ا١ٌٙثُ اتٓ )لسُ اٌى١ّ١اء ، و١ٍح اٌرشت١ح ٌٍعٍَٛ اٌظشفح الخالصت ٘ٛ اػطشاب ِزِٓ ٠رشافك ِع اخرالي ِسر٠ٛاخ اٌعٕاطش إٌزسج اٌرٟ ذشاسن فٟ اٌعذ٠ذ ِٓ اٌٛظائف ِشع اٌسىش إٌٛع اٌثأٟ االٔز١ّ٠ح.اْ اٌرغ١شاخ فٟ ِسرٜٛ ِظً ٘زٖ اٌعٕاطش ستّا ذٌٛذ تعغ اٌرعم١ذاخ فٟ ِشع اٌسىش إٌٛع اٌثأٟ خاطح اٌفعا١ٌاخ .اٌذساساخ اٌردش٠ث١ح ٚاٌسش٠ش٠ح اٌساتمح اظٙشخ تاْ خٙذ اٌراوسذ ٠ّىٓ اْ ٠ٍعة دٚسا سئ١س١ا فٟ ِشػ١ح ٚذطٛس ِشع اٌسىش إٌٛع اوسذ ٠ّىٓ اْ ذساُ٘ فٟ ذسش٠ع ٚذط٠ٛش ٘زا اٌّشع.اٌثأٟ .ِٚع رٌه فاْ اال١ٌح اٌذل١مح ٌدٙذ اٌر اٌثأٟ فٟ اٌّشػٝ اٌز٠ٓ ٠عأْٛ ِٓ ِشع اٌسىشٞ إٌٛع اٌّعا٠ش اٌى١ّٛح٠ٛ١ح ذمذ٠ش، أٚال اٌٙذف ِٓ ٘زٖ اٌذساسح ذؼّٓ (t2dm) , ِاٌْٛ ثٕائٟ األٌذ٠ٙا٠ذِثً ,ِؤشش االوسذج اٌفٛل١ح (mda) ,ٌّؼاداخ األوسذج حاٌى١ٍ حاٌسع (tac) ٓٚا١ٌّٙٛغٍٛت١ .ثا١ٔا ذمذ٠ش ِسرٜٛ اٌزٔه فٟ ِظً اٌذَ ٚفٟ االدساس ٚوزٌه (fsg)سىش اٌظائُِظً اػافح ٌفحض ِسرٜٛ (hba1c)اٌّسىش ذمذ٠ش ِسرٜٛ اٌحذ٠ذ فٟ ِظً اٌذَ ٌذٜ ٘ؤالء اٌّشػٝ , ِٚماسٔح إٌرائح ِع ِدّٛعح اشخاص اطحاء ِطاتمح تاٌعّش ٌّدّٛعح ٌرٍمٟ اٌعالج.ذُ ذمس١ُّٙ اٌٝ ِسرشفٝ تغذاد اٌرع١ٍّٟاٌز٠ٓ أدخٍٛا اٌٝ ، ِٓ عشالٟ سخً( 54.اخش٠د اٌذساسح عٍٝ )اٌّشػٝ .(hba1c)ِدّٛعر١ٓ عٍٝ اساس ِسرٜٛ سىش اٌذَ اٌظائُ ٚا١ٌّٙٛغٍٛت١ٓ اٌّسىش ِش٠ؼا (04وّدّٛعح ػاتطح ِٓ االطحاء,اٌّدّٛعح اٌثا١ٔح ذاٌفد ِٓ)a1c<6.4%( فشد ل١اس02اٌّدّٛعح االٌٚٝ ذاٌفد ِٓ ) تاعرثاسُ٘ ِشػٝ اٌسىشٞ إٌٛع اٌثأٟ.ac1>6.4%ل١اس ِشع اٌسىشٞ إٌٛع اٌثأٟ ِماسٔح ِع اٌّدّٛعح اٌؼاتطح فٟ وً ِٓ ِسرٜٛ ِظً فٟأظٙشخ ٔرائح ٘زٖ اٌذساسح اسذفاعا ِع٠ٕٛا االدساس,ِٚسرٜٛ اٌحذ٠ذ فٟ ,ِسرٜٛ اٌزٔه فٟ (mda),ِؤشش االوسذج اٌفٛل١ح(hba1c),ا١ٌّٙٛغٍٛت١ٓ اٌّسىش (fsg)سىش اٌظائُ ِظً اٌذَ. فٟ ح١ٓ اظٙشخ اٌذساسح أخفاػا ِع٠ٕٛا ٌذٜ ِدّٛعح اٌّشػٝ ِماسٔح تاٌّدّٛعح اٌؼاتطح فٟ اٌسعح اٌى١ٍح ٌّؼاداخ ِٚسرٜٛ اٌزٔه فٟ ِظً اٌذَ. (tac)االوسذج الوسذج ,لذ ذٍعة دٚسا ٘اِا فٟ ِشع اْ اٌشذ اٌراوسذٞ اٌزٞ ذسثثٗ اٌدزٚس اٌحشج ِع أخفاع ل١ّح ِؼاداخ ا االسرٕراج٠ّىٓ فٟ ِظً اٌذَ (tac)ِٚسرٜٛ اٌحذ٠ذ فٟ ِظً اٌذَ ٚذٕالض ِسر٠ٛاخ اٌزٔه ٚ (mda)اٌسىشٞ إٌٛع اٌثأٟ تسثة ذزا٠ذ ِسر٠ٛاخ االكسذة الفىقٍت للذهىن.، العىبصر العئٍلت ،مرض السكري الىىع الثبوً الكلمبث المفتبحٍت: introduction diabetes mellitus is a long term chronic disease marked by derangements in sugar, lipid, and protein metabolism caused by abnormalities in insulin release, 1 corresponding author e-mail:abdo_aljumaily@yahoo.com received:3 /11/2015 accepted: 19/1/2016 iraqi j pharm sci, vol.25(1) 2016 oxidative stress and some trace elements in type 2 diabetes mellitus 18 insulin action, or both. in 1979, the national diabetes data group developed a categorizing and identifying plan for diabetes (1) . the level of hba1c relies on the level of glucose in the blood stream and the timeperiod of hyperglycemia. the changes in the level of hba1c in diabetic patients can be used to follow the efficiency of treatment for the diabetes.t2dm, is associated with increased metabolic processes and oxidative stress .the trace elements are important co-factors in these events. impaired metabolism of these minerals play a role in the progression of t2dm and later development of complications. trace element participate in production of reactive oxygen species (ros), which contribute to oxidative stress. oxidative stress contributes to the pathogenesis of many diseases including t2dm affect a cascade of reaction series resulting in cellular injury and illness (2) . malondialdehyde is an indicator of fat peroxidation and rises in oxidative stress status. free mda, the chemically active form, helps as a marker of last injury (3-6) . antioxidants are unique organic compounds that are active in the prevention of very rapid harmful chemical chain reactions with oxygen or nitric oxide, that is, oxidation reactions. antioxidants interact with the free radicals before they are successfully interacting with other molecules, which can provide defense against oxidation reaction (7) . trace elements form a part of daily eating are known to play major role in the maintenance of health (8) . literature survey shows that some trace elements like zinc plays an important role in synthesis ,storage and secretion of insulin ,also increase the insulin ability to bind to its receptor (9) , also serving as cofactor or components for enzyme systems included glucose metabolism (10) . on the other hand, serum iron effects on glucose metabolism even in non-existence of increased iron overload or even in a state of iron deficiency. iron is a transition of metal and a possible catalyst in cellular reactions through fenton reaction: these free radicals can be quite harmful, particularly for cellular components such as lipids, proteins and nucleic acids. free radicals, when oxygen present, reacts with membranes lipids double bonds generating lipid peroxides that can spread the damage throughout the membrane. in erythrocytes, ros can induce hemolysis. free radicals can oxidize lateral amino acidic groups, impairing protein function by promoting cross-linkage formation such as disulfuric bond, causing mutation in protein structure or folding .also dna structure can be damage or modified by free radicals, through induction mutations in azotate bases (this show how ros can be responsible of cellular aging and cancer induction), resulting in a state of oxidative stress and finally cell damage death (11) . experimental part patient selection and blood sampling in the present study, 45 iraqi men, who attended baghdad teaching hospital, classified into a couple of groups. first group includes 20 apparently healthy subjects {by clinical examination and with no history of diabetes (a1c <6.4%)}. their age ranged (45± 10) years. second group included 25 patients with type 2 diabetes (a1c>6.4%), their age ranged (52±12) years diagnosed by specialized physician. it was adopted hba1c in the diagnosis of t2dm according to a report published in 2009 by an international expert committee on the role of hba1c in the diagnosis of diabetes recommended that hba1c can be used to diagnose diabetes and that the diagnosis can be made if the hba1c level is ≥6.5% (12). blood was collected by vein puncture into plain tubes with no edta. serum was separated from the cells by centrifuging at 3500 rpm, and then divided into small portions and kept frozen until analysis. determination of serum glucose levels glucose was determined after enzymatic oxidation in the presence of glucose oxidase (13) . the formed hydrogen peroxide composed reacts within the catalysis of peroxidase, with phenol and 4aminophenazone to form a red-violet quinone imine dye as indicator. determination of glycated hemoglobin (hba1c) nycocard hba1c is a boronate affinity assay traceable to the international federation of clinical chemistry and laboratory medicine (ifcc) reference method for measurement of hba1c. the reagent contains agents called lyse erythrocytes and precipitates hemoglobin specifically, as well as a blue boronic acid conjugate that binds cis-diols of glycated hemoglobin (14) . determination of malondialdehyde (mda) the concentration of mda in serum was determined according to buege and iraqi j pharm sci, vol.25(1) 2016 oxidative stress and some trace elements in type 2 diabetes mellitus 19 aust method of enzymology (15) . mda formed from breakdown of poly unsaturated fatty acid (pufa). mda reacts with thiobarbituric acid (tba) to give pink color that is read at λ max535nm. mda was calculated according to the molar extinction factor of 1.56×105 (16) . determination of total antioxidant capacity (tac) abts [2, 2-azino-bis-(3ethylbenzthiazoline sulphonate)] is incubated with a peroxidase (metmyoglobin) and h2o2 to produce the radical cation abts · + . this has a relatively stable blue-green color, which is measured at λ = 600nm. antioxidants in the added sample cause repression of this color production to a degree which is proportional to their level (17) . trace metals determination levels of the selected metals were estimated by flam atomic absorption spectrophotometer (aa-7000, shimadzu, japan). the amount of radiant energy absorbed is proportional to its concentration. the serum diluted to 10 ml with hydrochloric acid in 10 ml centrifuge test tube. the diluted samples displayed directly to the flame. zinc was measured at λ = 213.86 nm, whereas iron was measured at λ = 248.33nm. statistical analysis student’s t-test was applied to compare the significance of the difference in the mean values of between studied groups, and (p < 0.05) was considered statistically significant, while p<0.001 was considered highly significant. (microsoft office 2013–excel). results and discussions table (1) shows the serum levels of some biochemical measures in two studied groups. the fasting serum glucose levels show a high significant increase (p<0.001) for patients (190 ± 18.60) mg/dl compared to control (80±7.5) mg/dl. hyperglycemia plays a critical role in the growth and progression of diabetic nephropathy, but the mechanism is not clear. the possible sources of raised oxidative stress might involve increased production of free radicals or weakened antioxidant protection system, enhances free radicals formation in diabetes. our results are in agreement with the recent study that reports an increase in oxidative stress as depicted by increase in mda with the concomitant decrease in tac as compared to controls. mda concentration increases with the severity of t2dm. tac summarizes the overall activity of antioxidants and antioxidant enzymes. the cooperation between different antioxidant pathways provides greater protection against attack by ros, compared to any single compound. thus the overall antioxidant capacity may give more relevant biological information compared to that obtained by the measurement of individual biomarkers (18) . table (1): levels of fsg, hba1c, mda and tca in sera of patients with t2dm and healthy subjects. p-value: probability, sd: standard deviation. fsg: fasting serum glucose. hba1c: glycated haemoglobin. +9 mda: malondialdehyde. tac: total anti-oxidant. results of glycated hemoglobin display a significant increase (p<0.001) in sera of patients (8.10±1.30) opposed to control (5.40±0.11) values. the classification of the groups in the current study was depending on the levels of hba1c to assess a diabetic from a non-diabetic individual. it has been reported that the prevalence and interference between intermediate hyperglycemia was defined by hba1c (5.7-6.4) %. this range was proposed as an indicator of type2 diabetes mellitus, since the advantage of using hba1c is less day to day variability compared with glucose tolerance test and fasting glucose levels (19) . according to world health organization (who), the american diabetes association (ada) has suggested 5.7 – 6.4% as the high risk range, therefore we can use hba1c levels in diagnosis of t2dm (20). results of mda showed highly significant increase (p<0.001) in patients (5.60±0.82) µmol/l compared to control (1.75±0.20) µmol/l. parameters control mean ±sd patients mean ±sd pvalue fsg(mg/dl) 80± 7.5 190± 18.60 p <0.001 hba1c (% of total hb) 5.40± 0.11 8.10± 1.30 p <0.001 mda (µmol/l) 1.75± 0.20 5.60± 0.82 p <0.001 tac (mmol/l) 0.80± 0.26 0.29± 0.12 p <0.001 iraqi j pharm sci, vol.25(1) 2016 oxidative stress and some trace elements in type 2 diabetes mellitus 20 the present results are compatible with reported data that strongly confirmed the evidence that diabetic patients were susceptible to oxidative stress and higher blood glucose level had an association with free radical-mediated lipid peroxidation. there appears to be mismatch between oxidant and antioxidant defence systems in type 2 diabetic individuals (21) . results of the present study consist with others who found that oxidative stress is increased in diabetes and the over generation of reactive oxygen species (ros) is a direct consequence of hyperglycaemia. diabetes patients have more severe oxidative stress than normal persons (22) . total antioxidant capacity values showed highly significant decrease (p<0.001) for patients (0.29±0.12) mmol/l as compared to the controls, (0.80±0.26) mmol/l. our results are in good agreement with the recent study that reports an increase in oxidative stress as depicted by increase in mda with the concomitant decline in tac as compared to controls (23) . total antioxidant capacity summarizes the total activity of antioxidants and antioxidant enzymes. the collaborating between diverse antioxidant pathways gives greater defense against assault by reactive oxygen species, compared to any solo compounds. therefore, the general antioxidant capacity may provide further relevant biological data compared to that achieved by measuring of individual biomarkers (24) . results of the present study were in agreement with a study indicated that the oxidative stress was boosted and there was an imbalance between oxidant – antioxidant defense according to the seriousness of the diabetic nephropathy. in patients at the acute phase of the disease decreased total antioxidant capacity may produce to abnormal lipid peroxidation, resulting in a high rate of glomerular injury. on the other side, continued lipid oxidation may produce to diminished antioxidant activity (25) . as shown in table (2) serum of zinc level is significantly decrease (p <0.05) for patients (0.58±0.11)mg/dl as compared with control (0.90±0.52) mg/dl, whereas urine level of zinc increases significantly (p <0.05) for patients (0.71±0.31) mg/dl compared with control (0.43±0.12) mg/dl. serum zinc levels of diabetics were obviously lower than those of non-diabetics; also, urine zinc levels were higher in diabetics than non-diabetics studied. the cause of decreased level of serum zinc in diabetes may be due to increased urinary excretion due to reduction in renal function associated with disease, gastrointestinal malabsorption genetic factors or signs of function during which zinc acts as a defence mechanism (26) . zinc and insulin levels in the pancreas modify in the same orientation in a diversity of conditions in humans (27) , it is helpful in creation, store, and secretion of insulin (28) . the current results demonstrate that the level of zinc is reduced in the serum of diabetic patients (table-2). the loss of these metals might be assigned to weakened absorption or to the excess excretion of these minerals in urine in these individuals, which may indicate a deficiency of these metals in serum of diabetic subjects and that is compatible with the literature (29) . results of serum fe in table (2) showed a significantly increase (p<0.05) in patients (0.83±0.01) mg/dl compared to control (0.69±0.05) mg/dl, which including the radical forms due to oxidation of ferrous form. recent studies shows an increase in iron could expect the danger of evolving type 2 diabetes, while the reduction in iron level is protective (30) . tissue iron excess will rise the generation of free radicals which in turn magnifies the steps implicated in inflammatory damage (31) . iron is closely related to oxidative stress. highly toxic free radicals, such as hydroxide and the super oxide anion, which leads to lipid peroxidation, which generated by iron through the fenton reaction (32) . table (2): levels of serum zn and fe concentration, urine zn concentration in patients with t2dm and healthy subjects. conclusion oxidative stress, which caused by free radicals production with impairment of antioxidant, play an importance role in t2dm, because of the increasing levels of mda and serum level of iron and 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magnesium levels in type 2 diabetes. int. j. diabetes devel. countries,2006 ;26:122-3. 29. kazi tg, afridi hi. copper, chromium, manganese, iron, nickel, and zinc levels in biological samples of diabetes mellitus patients. biol trace elem res,2008 ;122:1-18. 30. nan hee kim et al, jung heon oh, kyung mook choi, young hyen kim and sei hyu baik. serum ferritin in healthy subjects and type 2 diabetes patients. younsei medical journal, 2000; 41: 387 – 392. 31. jonathan e shaw and donald j chisholm. epidemiology and prevention of type 2 diabetes and the metabolic syndrome. journal of clinical endocrinology and metabolism, 2003; 179(7): 379 -383. 32. josé manuel fernández-real, abel lópez-bermejo, and wifredo ricart. cross-talk between iron metabolism and diabetes. diabetes, 2002; 51: 2348 – 2354. iraqi j pharm sci, vol.22(1) 2013 lipase activity of acinetobacter baumannii 82 physicochemical factors affected the partial purified lipase activity of acinetobacter baumannii “local isolates” huda j. mohammed *,1 * department of clinical laboratories sciences, college of pharmacy, university of baghdad, baghdad, iraq. abstract microbial lipases today occupy a place of prominence among biocatalysts owing to their ability to catalyze awide variety of reactions in aqueous and nonaqueous media, a.baumannii were isolated from different clinical specimens from hospitalized patients from baghdad hospitals and were detected by biochemical tests and api20e system. the percentage of isolation was (16.6%), a. baumannii is an increasingly multidrug – resistant (mdr), it showed high level of resistant to ceftriaxon, colistin, piperacillin, co-trimoxazol, tertracycline, carbenicillin, amoxicillin, penicillin g, gentamicin and ceftazidim , wherease the isolates were highly sensitive to imipenem, ciprofloxacin, meropenem, amikacin, and cefotaxime. the isolated strians of a.baumannii were screened for the production of lipase by using rhodamine b agar media. one of the isolated strians exhibited a greater clear zone than the others, indicated higher lipase activity. the lipase in present study was partially purified by ammonium sulphate at the concenterations of 30% and 80%, the 30% ammonium sulphate showed higher activiy of the enzyme than 80% in which the enzyme activity was slightly decreased indicating that the better purification was at the 30% concenteration of ammonium sulphate.various physicochemical parameters such as ph, temperature and metal ions were studied in order to determine the optimum conditions for lipase production. the production of lipase by a. baumannii was optimum at 35c, ph 7.0 and was enhanced by the ca ++ and na + wherease inhibited by zn ++ ions. key words : acinetobacter baumannii, lipase enzyme, partial purification. المنقي جزئيا من جرثومت يبيزالعوامل الفزيوكيمائيت المؤثرة علي فعاليت انزيم الال acinetobacter baumannii هدى جاسم محمد *،1 الخالصة حى عزل ,ث حياحيت في االوساط انسائهت وغيش انسائهت الاَزيًاث اناليبيزانًايكشوبيت ححخم انيىو يكاَت يهًت نذوسها انكبيش كًحم يسخشفياث يخخهفت في بغذاد وحى حشخيصها باالعخًاد عهى االخخباساث انكيًىحياحيت يشضى ساقذيٍ فيانبكخشيا يٍ ًَارج سشيشيت يخخهفت يٍ -:يُها وكاَج أٌ هزِ انبكخشيا واسعت انًقاويت نهًضاداث انحياحيت ,% 16,6وكاَج َسبت انعزل ( api20e)وَظاو (ceftriaxon, colistin, piperacillin, co-trimoxazol, tertracycline, carbenicillin, amoxicillin, penicillin g, gentamicin and ceftazidim) . (.imipenem, ciprofloxacin, meropenem, amikacin, and cefotaxime)نهًضاداث في حيٍ كاَج حساست وكاَج عزنت واحذة فقظ يُخجت ( rhodamine b agar)حى اخخباس قابهيت انبكخشيا انًعزونت عهى اَخاج أَزيى اناليبيز باسخخذاو وسظ وأظهش انخشكيز ,%80و% 30و طشيقت انخشسيب بكبشيخاث األيىَيىو بخشكيز حًج انخُقيت انجزئيت ألَزيى اناليبيز بأسخخذا, نألَزيى بشكم فعال وهزا يشيش انى اٌ عًهيت انخُقيت انجزئيت حكىٌ افضم عُذ % 80يٍ كبشيخاث األيىَيىو فعانيت أَزيًيت عانيت اعهى يٍ حهك عُذ انخشكيز % 30 . يٍ كبشيخاث األيىَيىو% 30انخشكيز ودسجت 7يًيائيت انخي حؤثش عهى فعانيت األَزيى وحبيٍ اٌ انشقى انهذسوجيُي األيثم ألَزيى اناليبيز هى حى دساست انظشوف انفزيائيت وانك سيهيزيت وحبيٍ ايضا اٌ فعانيت األَزيى حخحفز بىجىد ايىَاث انكانيسىو وايىَاث انصىديىو وحخثبظ انفعانيت بىجىد 35انحشاسة انًثهى هي . .ايىَاث انخاسصيٍ .، التنقيت الجزئيتانزيم الاليبيز، acinetobacter baumannii :المفتاحيت الكلماث introduction lipases are the special kind of esterase belonging to subclass 1 of hydrolytic enzyme class 3 and have been assigned sub-sub class 3.1.1 due to their specificity for carboxylic acid ester bonds, these enzymes are widely distributed in nature and have been found in many corresponding author e-mail:h.mastermaster@yahoo.com received: 14/10/2012 accepted: 16/3/2013 iraqi j pharm sci, vol.22(1) 2013 lipase activity of acinetobacter baumannii 83 species of animals, plants, bacteria, yeast and fungi . apart from their wide distribution , their presence in microorganisms is most interesting because of their potential application in various industries ranging from the use in laundry detergent to stereo-specific biocatalysts table 1 (1) . table 1: list of industrial applications of lipases the extracellular bacterial lipases are of considerable commercial importance, as their bulk production is much easy (2,3) . bacterial lipases are greatly influenced by nutritional and physicochemical factors, such as temperature, ph, nitrogen and carbon sources, presence of lipids, inorganic salts, agitation and dissolved oxygen concentration (3) . each application requires unique properties with respect to specificity, stability, temperature and ph dependence or ability to catalyze (4) . some stains of a. baumannii have the ability to produce different extracellular enzymes including lipases enzymes (5) , which are amongst the most widely used biocatalysts due to their ability to catalyze diverse reactions, they reduce fats to fatty acids and glycerol (6) . the genus acinetobacter is classified under the family moraxellaceae and comprises strictly aerobic, gram-negative bacilli, sometimes difficult to decolorize, thus appearing gram positive. it often resemble gram negative cocci in direct smears from positive blood cultures performed in liquid media , while the coccobacillary forms predominate on solid culture media and bacillary forms predominate in liquid media, it is non-motile, lactose non fermenting, oxidase negative, catalase positive (7). acinetobacter spp. are widely distributed in soil, water, sewage, food and can occasionally be cultured from skin, mucous membranes, secretions and from hospital environment, they are part of indigenous microflora of oral and upper respiratory ,genitourinary and lower gastrointestinal tracts as well as of skin (8,9) . acinetobacter spp. are opportunistic pathogens that readily colonize in patients with compromised host defenses, thereby penetrating deep into the host tissue which serves as a prerequisite for an infection (10). acinetobacter spp. can cause various types of infections, including pneumonia, septicemia, peritonitis, endocarditis, meningitis, arthritis, corneal infections, urinary tract infections, skin and wound infections and bacteremia (11) . these infections are mainly caused by acinetobacter baumannii , and the mortality rate in patients with a. baumannii in intensive care unit fluctuates widely, depending on patient characteristics, such as age and immune status (12) . the risk factors for a. baumannii infections include hospital size > 500 beds, include : previous antimicrobial therapy, long stay in an intensive care unit (icu), catheterization, mechanical ventilation and surgery (13) . industry action product of application dairy food hydrolysis of milk, fat, cheese ripening,modification of butter fat development of flavoring agent in milk cheese and butter bakery food flavor improvement shelf life propagation beverages improved aroma alcoholic beverages e.g, sake wine food dressings quality improvement maysoin dressing and whippings health food transesterification health food meat and fish flavor development meat and fish product fat removal fats and oils transesterification and hydrolysis cocoa butter, margarine fatty acids, glycerol mono and diglycerides laundry reducing biodegradable strains cleaning cloths cosmetics esterification skin and sun-tan creams, bath oil etc industry action product of application agrochemicals esterification herbicides such as phenoxypropionate pharmaceuticals hydrolysis of expoyester alcohols produce various intermediates used in manufacture of medicine. fuel industries transesterification biodiesel production pollution control hydrolysis and transesterification of oils and grease to remove hard stains, and hydrolyze oil and greases. iraqi j pharm sci, vol.22(1) 2013 lipase activity of acinetobacter baumannii 84 acinetobacter strains are often resistant to antimicrobial agents , and therapy of infection can be difficult, susceptibility test should be done to help select the best antimicrobial drugs for therapy, resistance is more common in a. baumannii than in strains of other acinetobacter. spp. (9) . during the last three decades, clinicians are faced with increasing infections of resistant a. baumannii strains to almost all clinically applicable antibiotics (14,15) , by the late 1990s, carbapenems and polymyxins were only remaining useful agents that could combat severe infections with these organisms in certain hospitals. to date, however, carbapeneme resistant strains are accumulating worldwide (16) , a worrying development gives the paucity of alternative treatment agent (17) . a. baumannii respond most commonly to gentamicin, amikacin and to newer penicillins or cephalosporins (9,18) . the specific aim of the current study is: estimating the optimal ph, temperature, and some metal ions for the better activity of the partial purified lipase from a.baumannii to find out its usage in appropriate industry. materials and methods isolation and identification specimens of blood (n=20), urine (n=20), and pus from skin lesions (n=20) where collected from hospitalized patients from various hospitals in baghdad city from the period of march 2012 to july 2012. all specimens were characterized by standard biochemical tests and by api 20 e system. antimicrobial susceptibility testing in vitro activity of the routinely used antibiotics against all isolated strains of a. baumannii (n=10) was tested on mueller hinton agar by disk diffusion (kirby – bauer method) (19) . the antibiotics included imipenem (10 mcg), ciprofloxacin (5 mcg), meropenem (10 mcg), penicillin g (10 mcg), ceftriaxone (30 mcg), colistin (10 mcg), cephotaxime (30 mcg), piperacillin (100 mcg), gentamycin (10 mcg), tetracyclin (30 mcg), amikacin (30 mcg), amoxicillin (25mcg), ceftazidim (30 mcg), cotrimoxazol (25 mcg), and carbencillin (100 mcg). the selection of antibiotics was in accordance with clinical and laboratory standards institute (clsi) guidelines. lipase production from a. baumannii the bacterial strains of a. baumannii were grown on nutrient agar containing olive oil (1% w/v) and rhodamine b 0.001% to assay the production of lipase on rhodamine b agar plates (20) . partial purification of lipase enzyme a. baumannii was grown in nutrient broth for 20 hrs at 37°c.the cell free supernatant, prepared by centrifugation (8000 rpm, 20 min), was passed through a 0.45μm pore size membrane and then ammonium sulphate was added to achieve 30% saturation. the suspension was further centrifuge (8000 rpm, 20 min, 4°c) and the ammonium sulphate was added to the supernatant to reach 80% saturation. the precipitates (0-30%) and (0-80%), collected by centrifugation, were separately dissolved in minimal volume of 20 mm tris buffer(ph7.0) at 4 ° c and was dialyzed against the same buffer to remove residual ammonium sulphate (2) . characterization of lipase lipase assay the hydrolytic activity was tested by trimetric method, 20 mm phosphate buffer / arabic gum (1%w/v) and the substrate (olive oil) 1/1 (v/v) were made up to a total volume of 10 ml. the reaction cocktail was thoroughly mixed and equilibrated at 20°c and 1 ml of crude enzyme was added after being previously incubated at the same temperature. the reaction was left at room temperature for exactly 30 min and stopped by adding 3 ml of 95% ethanol. the released fatty acids from oil substrate during enzymatic hydrolysis were titrated to neutralization with 50 mm naoh in presence of thymolphtalein as indicator. a blank was prepared for each sample where the enzyme was inactivated by heating at 95°c for 15 min. one unite of lipase activity was expressed a micro equivalents of fatty acid released from a triglyceride in 30 min at ph 7.0 at 20°c (21) . effect of temperature on lipase activity lipolytic activity of the partial purified enzyme was determined after 1 hr. of incubation at temperatures 25, 35, 45, 55, 65, 75, 85 c , the activity was measured by using tween 20 as a substrate and the reaction mixture was incubated at the previous temperatures (22) . effect of ph on lipase activity the effect of ph on the activity of partial purified lipase was studied at various ph in the range of (3-10) using tween 20 as a substrate. buffer solutions (20 mm) of different ph values were used which includes acetate buffer (3,4,5,6), phosphate buffer (7,8,9,10). the partial purified enzyme was incubated for 1 h. in buffers with varying ph values at 30  c and the lipolytic activity was measured under standard conditions (22) . iraqi j pharm sci, vol.22(1) 2013 lipase activity of acinetobacter baumannii 85 effect of some metal ions on lipase activity for determining the effect of metal ions on lipase activity, the enzyme were per-incubated with 1 mm of metal ions for 1 h. at 30  c.the chloride salts of metal ions tested were ca ++, na + and zn ++ (21) . results and disscussion atotal number of a.baumannii that isolated during the study period was 10(16.6%), out of 60 collected different clinical specimens (4) were isolated from the blood samples, (3) were isolated from pus samples and (3) were isolated from urine samples table (2). the isolation percentage of a.baumannii in recent study nearly to that obtained by shareek et al. (23) , and to that obtained by andreza et al. (13) . table 2: number of a. baumannii isolates from different clinical samples colonies of pure culture of the above isolates were chacterized by using morphological and biochemical charcteristics table( 3). table 3:the morphological and biochemical charcteristics of a. baumannii the ten isolates were coccobacilli, nonmotile, catalase positive, oxidase negative, produce acid from (glucose, xylose, galactose, mannose, rhamnose, lactose, maltose), developing colorless to slightly pink colonies on macconkey agar, non hemolytic on blood agar, negative for nitrate reduction test, in addition, the identification was confirmed by api 20 e system, the isolates of a.baumannii acquired the number (0204042) according to the results obtained from the api 20 e system stripfigure 1. figure1 :the result of api20e system for a. baumannii the current results of morphological, biochemical test and api 20e similar to that recorded by xu etal. (24) , and by paul etal (19) . in the past decade, acinetobacter baumannii has emerged as amajor nosocomial pathogen in many parts of the world, resulting in devastating outcomes in terms of mortality and mobidity (18) . table (4) showed the results of antibiotics susceptibility test of a.baumannii , all isolates of a.baumannii were highly resistant to ceftriaxon, colistin, piperacillin, co-trimoxazol, tertracycline, carbenicillin, amoxicillin, penicillin g, gentamicin, ceftazidim, wherease all of them, were sensitive to imipenem, ciprofloxacin, meropenem, amikacin, and cefotaxime figure 2. figure 2:the sensitivity of a.baumannii to and imipenem (10 mcg) ipm and meropenem (10mcg) mem clinical samples number of isolates blood n=20 4 pus n=20 3 urine n=20 3 total number =60 10(16.6%) test result morphology coccobacilli motility non-motile growth on macconkey agar slightly pink colonies nitrate reduction positive hemolysis non-hemolytic catalase positive oxidase negative acid from : glucose positive xylose positive galactose positive mannose positive rhamnose positive lactose positive maltose positive iraqi j pharm sci, vol.22(1) 2013 lipase activity of acinetobacter baumannii 86 table 4: the antibiotics suscebtibility patterns of ten isolates of a. baumannii there are recent reports of increasing resistance to these important drugs, documented all over the world; prakasam et al., reported high level of resistance to piperacillin, ceftazidim, gentamicin ,and co-trimoxazol (18) . shareek etal., reported that a. baumannii were sensitive to meropenem, imipenem, cefotaxime, amikacin and ciprofloxacin (23) , other reports indicate that the resistance rate for meropenem, imipenem, amikacin and ciprofloxacin were relatively low, however they were almost more than 30 % (24) . awide spectrum of antimicrobial resistance mechanisms is exhibited by a. baumannii, a part from its intrinsic resistance mainly due to low permeability of the outer membrane to certain antibiotics,production of β lactamases , mutations in antibiotic targets as well as constitutive expression of certain efflux pumps (25). avast number of a. baumannii lipases have a wide range of potential applications in the hydrolysis, esterification and transesterification of triglycerides (2). lipase production by a. baumannii was confirmed on rhodamine b plates containing olive oil, the colonies showed orange fluorescence colour when exposed to u.v. light, this is due to the formation of complex between cationic rhodamine b and the uranyl fatty acid ion (21) , the results obtianed from rhodamine b agar indicated that only one isolate which obtained from pus was strong producer of lipase enzyme. the above results were also recorded by many researchers like saeed etal. (1) , iftkhar etal. (20) , and jagtap etal. (26) . the enzyme was partialy purified using ammonium sulphate precepitation method, the maximum total activity, specific activity, yielded (14.3 u/ml, 18.8 u/mg , and 88.2%) were detected at 30% ammonium sulphate respectively ,wherease by increasing the ammonium sulphate concenteration to 80% a slight decrease in total activity was obtianed table( 5). table 5: partial purification of lipase from a. baumannii by using ammonium sulphate some authors recorded that lipase activity increased at 30 % ammonium sulphate than in 80%, this may be due to the fact that ammonium sulphate at high concenterations could result in drop in ph and consequently a loss in enzyme activiy (26,27) . the optimum production temperature for lipase production by a. baumannii was at 35c, no production was detected at 65c, 75 c and 85 c as shown in figure 3. figure 3: effect of various temperature on lipase activity antibiotics symbol resistance% sensitivity% imipenem (10 mcg) ipm 10(100%) ciprofloxacin (5mcg) cip 10(100%) meropenem (10mcg) mem 10(100%) penicillin g(10mcg) p 2(20%) 8(80%) ceftriaxone (30mcg) cro 10(100%) colistin (10 mcg) cl 10(100%) cephotaxime (30mcg) ctx 3(30%) 7(70%) piperacillin (100mcg) prl 10(100%) gentamycin (10mcg) cn 6(60%) 4(40%) tetracyclin (30 mcg) te 10(100%) amikacin (30 mcg) an 10(100%) amoxicillin (25mcg) amx 10(100%) ceftazidim (30 mcg) caz 8(80%) 2(20%) co-trimoxazol (25mcg) sxt 10(100%) carbencillin (100mcg) py 10(100%) stage total activity (u/ml) specific activity (u/mg) yield % purifica tion factor culture filtrate 17.2 4.2 100.0 1.0 30% 14.3 18.8 88.2 4.5 80% 13.8 18.1 84.7 4.3 iraqi j pharm sci, vol.22(1) 2013 lipase activity of acinetobacter baumannii 87 hasan etal., recorded that the optimum temperature for lipase production by acinetobacter spp. was 40 c (28), while gupta etal., recorded that the optimum temperature for lipas production by acinetobacter spp. was 25c , the bacterial lipases generally have optimal temperature range of 3060c, however reports exist on bacterial lipases with optimal temperature in both lower and higher ranges (2) . the ph range of a. baumannii lipase was detectable over a wide range between (3-10) wih an optimum ph value at 7 and no lipase production was detected at acidic ph (3.0, 4.0 ,5.0, 6.0) and exreme alkaline conditions (ph 10.0) figure 4. figure 4: effect of various ph on lipase activity the initial ph of the growth medium is important for lipase production, largely, bacteria prefer ph around 7.0 for best growth and lipase production (3). the ph results are in accordance with huang etal. (29) , and salah etal. (30) , the enzyme was stable at ph range (7-8) and also retained 90% of its activity as reported by kasana etal., (31) . the effect of some metal ions on the activities of a. baumannii lipase was also investigated, it was found that nacl, cacl2 enhance the lipolytic activity of partial purified lipase and inhibited by zn ++ (table 6). the observation that lipase activity was significantly enhanced in the presence of these metal ions probably reflects the abilityof these salts to react with free fatty acids. sharma etal., reported that lipase activity was enhanced by the presence of na + and ca ++ (32). similar kind of work has also reported by kumer etal. (33) , and by patil etal. 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activity of acinetobacter baumannii 89 of its alkaline lipase. j. basic microbiol.,2008, 48(3):207-12. 32. sharma r, chisti y, banerjee u, production, purification, characterization, and applications of lipases, j.biotechnol., 2001, 19 (2) : 627662. 33. kumerh., kim sh, lee ys, lee sc, zhou y, kim cm, et al. gene cloning, purification, and characterization of a cold-adapted lipase produced by acinetobacter baumannii. j microbiol.biotechnol 2009;19(2):128–35. 34. park i., kim s., lee y., lee s., zhou y., kim c., ahns.,. gene cloning, purification and characterization of a cold adapted lipase produced by acinetobacter baumannii bd5. j.microbiol. biotechnol.2009, 19: 128-135. iraqi j pharm sci, vol.30(1) 2021 patient safety culture in iraqi community pharmacies doi: https://doi.org/10.31351/vol30iss1pp66-75 154 evaluating patient safety culture in iraqi community pharmacies basma sh. jassim*,1 and mohammed y. jamal** * ministry of health and environment, aldiwaniyah health department, al diwaniyah, iraq ⁎⁎department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq abstract patient safety is the main issue in health care organization, the agency for healthcare research and quality defines it as, “freedom from accidental or preventable injuries produced by medical care. thus, practices or interventions that improve patient safety are those that reduce the occurrence of preventable adverse events”. the purpose of this study was to evaluate iraqi pharmacist perception about the culture of patient safety. as well as estimate whether safety is a principal issue in their pharmaceutical practice. this study was carried out on 435 pharmacists who are working in community pharmacies in various iraqi provinces. a survey was distributed via the internet during the period from may to june 2020. a community pharmacy questionnaire was used to evaluate the awareness of pharmacists regarding the culture of patient safety. a result of this study showed that the patient counseling field was the most positive one among the studied domains with score 68.8% of positive awareness and 70.4% of the pharmacists indicated that they inform patients with needed information about their new prescriptions. in contrast, staffing and work pressure scored the lowest positive response (36.55%). although 66.7% of the participants stated they have the appropriate number of staff in their pharmacies to deal with the workload. keywords: iraqi pharmacist, patient safety culture, community pharmacy. المريض في صيدليات المجتمع العراقي تقييم ثقافة سالمة **و محمد ياوز جمال 1*،جاسم شاكر سمة ب وزارة الصحة والبيئة ، فرع صحة الديوانية ، الديوانية ، العراق.* فرع الصيدلة السريرية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق .** الخالصة منظمة الرعاية الصحية ، وتعرفها وكالة أبحاث الرعاية الصحية والجودة بأنها "التحرر من تعتبر سالمة المرضى قضية رئيسية في ن سالمة اإلصابات العرضية أو التي يمكن الوقاية منها الناتجة عن الرعاية الطبية. وبالتالي ، فإن الممارسات أو التدخالت التي تعمل على تحسي السلبية التي يمكن الوقاية منها. الهدف من هذه الدراسة هو تقييم تصور الصيدلي العراقي عن المرضى هي تلك التي تقلل من حدوث األحداث 435ثقافة سالمة المرضى. باإلضافة إلى تقدير ما إذا كانت السالمة هي القضية الرئيسية في ممارستهم الصيدالنية ، أجريت هذه الدراسة على حزيران إلى ايار عبر اإلنترنت خالل الفترة من الستبيان ف المحافظات العراقية. تم توزيع اصيدليًا يعملون في صيدليات المجتمع في مختل لتقييم الوعي بثقافة سالمة المرضى بين الصيادلة. تظهر نتيجة هذه الدراسة أن مجال إرشاد المرضى صيدلية المجتمع . تم استخدام استبيان2020 ٪ من الصيادلة أشاروا إلى أنهم يطلعون المرضى 70.4٪ من الوعي اإليجابي و 68.8كان األكثر إيجابية بين المجاالت المدروسة بنسبة ٪(. على الرغم من أن 36.55لمقابل ، سجل التوظيف وضغط العمل أدنى استجابة إيجابية )بالمعلومات الالزمة عن الوصفات الجديدة. في ا ٪ من المشاركين ذكروا أن لديهم العدد المناسب من الموظفين في صيدلياتهم للتعامل مع عبء العمل.66.7 . دلي عراقي ، ثقافة سالمة المريض ، استبيان صيدلية المجتمع ، المحافظات العراقيةالكلمات المفتاحية: صي introduction indeed, iraqi society considers pharmacist as a trusted source for medical advice besides the traditional role of community pharmacy in medication supply(1). community pharmacies recognized internationally as the most easily accessible and cost-effective sites for introducing healthcare services(2). pharmacists working in the community pharmacies can considerably add to national work aimed for the enhancement of patients care and quality of life(3). since pharmacists are usually responsible for optimizing drug therapy and preventing medication errors, an inspection of pharmacists' safety culture in various settings can provide insight into their understanding and help identify weakness domains in order to improve them(4). a public concern is globally raising around patient safety issue in various health care settings(5). institute of medicine (iom) has publishing its first statement about patient safety and patient hurt "to err is human: building a safer health system", which has highlight the topic of safety as well as preventable medical errors cost at the forefront of healthcare practice and the national conversation (67). the culture of patient safety is explained as “the beliefs, values, and norms regarding how members of an organization should behave and includes policies, procedures, and processes to improve quality and safety”(8). 1corresponding author e-mail: ph.bassmashakir@gmail.com received: 15/8/2020 accepted: 27/ 10/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp66-75 iraqi j pharm sci, vol.30(1) 2021 patient safety culture in iraqi community pharmacies 155 it is very important to establish and maintain a solid base of safety culture which identified as an essential constituent of using medication safely(8). despite all efforts to minimize the occurrence of patient hurt, actually every 20 patients there was one patient subjected to a preventable injury(9). worldwide, medication errors are considered a problem which requires serious efforts (10). it is explained as “any preventable event that may cause or lead to inappropriate medication use or patient harm while the medication is in the control of the health care professional, patient, or consumer” (11). the occurrence of such errors were documented as a pricey issue for health care organization in addition to its negative influence on patients and health care staff(12). drug dispensing is considered as the main task in pharmaceutical practice, which in turn make it an important source of such errors, thus it can form a persistent risk to the safety of the patient (13). one of the notable reasons for errors in pharmacy practice are distractions and interruptions(14). in its recent issue of questionnaires about the culture of safety the american agency for healthcare research and quality (ahrq) published the pharmacy survey on patient safety culture, which aimed to rising consciousness regarding safety culture assessment in community pharmacy (15). accordingly, the purpose of this study was doing to evaluate iraqi pharmacist's perceptions who are working in community pharmacies about the culture of patient safety. as well as, it estimates whether safety is a primary issue in their practice. subject and method participants this study was a cross-sectional one that involved 435 pharmacists working in community pharmacies in many iraqi provinces, from may to june 2020. the number of males participants was 195 (44.8%) while the number of females was 240 (55.2%). inclusion criteria pharmacists who are working in community pharmacies exclusion criteria other workers in community pharmacy such as pharmacist assistant method the questionnaires assessing the culture of patient safety using precise comprehensive questionnaires designed for community pharmacy workers is known as the pharmacy survey on patient safety culture(16-17). the survey used with some modifications to suite iraqi community pharmacy system (for example there is no documentations for mistakes in iraqi pharmacies). the survey form contains 36 items assembled into three-part which is working in this pharmacy, communication and work pace, the last one patient safety and response to mistakes. these three part assessed 11 important fields of patient safety culture(18). also, contains an overall rating question section and background questions about participant additional items were added to this section. it is distributed in the english language. the survey items were previously validated (pilot study). five-point scale was designated for agreement or frequency type of answers was applied for responses measuring. administration of the questionnaires it was an electronic survey using google form. the survey was distributed via the internet in professional groups of iraqi pharmacists and as a mixed form (hardware and software) in alqādisiyyah provinces to the pharmacists working in community pharmacies. the participation was optional, anonymous and no incentive was offered to the participants. statistical analysis analysis was done using statistical package for social science (spss, version 22, ibm, new york, usa). descriptive statistics (means, standards deviations, frequencies, and percentages) of the participants were calculated. agreement responses which include “strongly agree”, “agree”, “most of the time”, and “always” were combined to “positive” responses, while disagreement covers the following responses “strongly disagree” and “disagree” “never” and “rarely” was compiled into “negative” response. “don’t know” were excepted from answers. measurement of dimensions through calculations of positive responses means, for negatively worded items the negative answers combined to confirm dimensions positivity. participants categorized according to their duration of working in the pharmacy into three groups: the first group has experience up to one year, the second group with 1-3 years of working, and the last group with longer duration of working more than three years of experience in the pharmacy. kruskal-wallis test (nonparametric test used for skewed distribution data) was used to measure the difference among participants regarding measured items according to working years results the study recruited 435 pharmacists with an average age of 30.11 years. from 16 iraqi provinces, baghdad (31%) and al-qādisiyyah (30.3%) the most common participating provinces where showed in (table1). iraqi j pharm sci, vol.30(1) 2021 patient safety culture in iraqi community pharmacies 156 table1. provinces of the participants province frequency percent baghdad 135 31.0 alqādisiyyah 132 30.3 dhi-qar 39 9.0 najaf 27 6.2 babylon 26 6.0 karbala 24 5.5 salahuddin 12 2.8 wasit 9 2.1 basra 7 1.6 diyala 7 1.6 muthanna 5 1.1 anbar 4 .9 maysan 4 .9 kirkuk 2 .5 erbil 1 .2 dohuk 1 .2 total 435 100.0 more than three-quarters (77.0%) of the participating pharmacists had a bachelor’s grade as demonstrated in (table2) table 2. the participating pharmacist credentials degree frequency percen t bachelor 335 77.0 master 52 12.0 high diploma 24 5.5 phd 11 2.5 board of pharmacy 13 3.0 total 435 100.0 (figure 1) demonstrated that the largest portion of the participants those working three or more years figure1. the participants’ experience years in community pharmacies safety culture assessment table 3 demonstrates the percentage of each type of response which appears as positive, negative, and neutral responses for each item in the questionnaire form. patient counseling domain record 68.8 % (mean of positive responses for patient counseling item 66.4, 69.7, 70.4) considered the highest positive domain, the second-highest is teamwork with 68.5% (mean of positive responses for teamwork item 74.3, 61.8, 69.4), and the third dimension was organizational learning and continuous improvement with 68.3% (mean of positive response for organizational domain item 71.5, 69.7, 63.7), while the lowest field with 36.55% (mean of positive response of first and third item 39.8, 66.7 and negative response for the second and fourth item 24.3, 15.4) was staffing, work pressure, and pace. positive response to other domains ranged from 47.4% to 61.6%. the overall positive perception of safety between pharmacists was 61.6%. the statement “the staff treat each other with respect” scored high agreement response with an average of 3.82 (± 1.15), in other word 74.2% of the pharmacists reported positive response about the statement. table 3. illustrations response type and percentage to survey item positive% neutral % negative% 1. physical space and environment 66.9 16.1 14.9 “this pharmacy is well organized”. 45.5 19.8 32.9 “ this pharmacy is free of clutter/untidiness” 58.2 18.4 18.4 “the physical layout of this pharmacy supports good workflow” positive% neutral % negative% 2. teamwork 74.3 10.6 14.3 “the staff treat each other with respect” 61.8 14.5 22.3 “ staff in this pharmacy clearly understand their roles and responsibilities” 69.4 14.5 14.3 “staff work together as an effective team”. positive% neutral % negative% 3. staff training and skills 55.4 16.60 25.1 “pharmacy assistants/helpers in this pharmacy receive the training they need to do their jobs” iraqi j pharm sci, vol.30(1) 2021 patient safety culture in iraqi community pharmacies 157 continued 3. illustrations response type and percentage to survey item. 64.1 15.4 19.1 “staff in this pharmacy have the skills they need to do their jobs well” 61.6 17.0 20.0 “staff who are new to this pharmacy receive an adequate orientation” 60.9 16.6 21.1 “staff get enough training from this pharmacy” 4. communication openness 47.1 33.8 17.5 “ staff ideas and suggestions are valued in this pharmacy” 68.1 13.8 17.0 “staff feel comfortable asking questions when they are unsure about something” 59.3 16.8 20.2 “ it is easy for staff to speak up to their pharmacy manager (chief pharmacist) or pharmacy owner about patient safety concerns in this pharmacy” . positive% neutral % negative% 5. patient counseling 66.4 16.1 16.1 “ pharmacists in this pharmacy encourage patients to talk to pharmacists about their medications”. 69.7 13.6 14.7 “ our pharmacists spend enough time talking to patients about how to use their medications” 70.4 15.2 12.7 “ our pharmacists tell patients important information about their new prescriptions” positive% neutral % negative% 6. staffing, work pressure, and pace 39.8 29.2 27.4 “staff take adequate breaks during their shifts” 34.0 33.1 24.3 “we feel rushed when processing prescriptions” 66.7 15.2 7.8 “ we have enough staff to handle the workload”. 33.8 25.5 15.4 “interruptions/distractions in this pharmacy (from phone calls, faxes, customers, etc.) make it difficult for staff to work accurately”. positive% neutral % negative% 7. communication about prescriptions across shifts 40.9 32.0 22.3 “we have clear expectations about exchanging important prescription information across shifts” 49.7 20.9 22.7 “ we have standard procedures for communicating prescription information across shifts” 51.7 23.4 12.6 “the status of problematic prescriptions is well communicated across shifts” positive% neutral % negative% 8. communication about mistakes 56.6 20.0 22.3 “ staff in this pharmacy discuss mistakes”. 55.6 23.0 6.9 “ when patient safety issues occur in this pharmacy, staff discuss them. 61.8 18.2 6.4 “in this pharmacy, we talk about ways to prevent mistakes from happening again” positive% neutral % negative% 9. response to mistakes 54.7 23.7 17.7 “staff are treated fairly when they make mistakes” 70.3 16.1 10.6 “this pharmacy helps staff learn from their mistakes rather than punishing them” 65.7 19.8 10.3 “we look at staff actions and the way we do things to understand why mistakes happen in this pharmacy” 37.2 26.7 27.4 “staff feel like their mistakes are held against them”. positive% neutral % negative% 10. organizational learning—continuous improvement 71.5 14.0 10.8 “when a mistake happens, we try to figure out what problems in the work process led to the mistake” 69.7 16.8 9.4 “when the same mistake keeps happening, we change the way we do things” 63.7 18.4 14.0 “ mistakes have led to positive changes in this pharmacy” positive% neutral % negative% 11. overall perceptions of patient safety 27.1 20.0 48.3 “this pharmacy places more emphasis on sales than on patient safety” 66.9 19.3 9.9 “this pharmacy is good at preventing mistakes”. 69.7 16.6 11.5 “the way we do things in this pharmacy reflects a strong focus on patient safety” assessment of pharmacist according to the experience years in pharmacy regarding work in this pharmacy, participants assessed according to experience years in the pharmacy, significant finding (p-value < 0.5) reported in this section. in other word, pharmacists with experience longer than three years, state pharmacy members were more aware of their task than other groups of participants as showed in (table 4). another part the communication and work pace. pharmacists who worked in pharmacy longer than three years demonstrated significant difference (p-value < 0.5) regarding interruptions/distractions in their pharmacies as compared to those who worked less than three years as showed in (table 5). iraqi j pharm sci, vol.30(1) 2021 patient safety culture in iraqi community pharmacies 158 table 4. difference in responses to working in this pharmacy items according to experience years. item years of working categories n mean rank p-value this pharmacy is well organized <1 100 199.63 .438 13 139 214.60 ≥3 184 216.76 total 423 staff treat each other with respect <1 102 204.05 .371 13 143 211.95 ≥3 184 223.44 total 429 pharmacy assistants/helpers in this pharmacy receive the training they need to do their jobs <1 96 219.78 .361 13 138 199.50 ≥3 186 213.87 total 420 staff in this pharmacy clearly understand their roles and responsibilities <1 100 205.80 .029* 1-3 141 197.55 ≥3 186 230.88 total 427 this pharmacy is free of clutter/untidiness <1 100 202.96 .490 13 141 210.85 ≥3 184 220.11 total 425 staff in this pharmacy have the skills they need to do their jobs well <1 101 222.57 .681 13 141 210.00 ≥3 185 212.37 total 427 the physical layout of this pharmacy supports good workflow <1 94 202.06 .516 13 135 199.39 ≥3 182 212.93 total 411 staff who are new to this pharmacy receive adequate orientation <1 99 201.26 .388 13 143 213.48 ≥3 185 221.22 total 427 staff work together as an effective team <1 100 203.79 .394 13 141 209.37 ≥3 185 221.90 total 426 staff get enough training from this pharmacy <1 99 204.49 .569 13 142 212.90 ≥3 186 219.90 total 427 iraqi j pharm sci, vol.30(1) 2021 patient safety culture in iraqi community pharmacies 159 table 5. the difference in communication and work pace items according to years of working categories item years of working categories n mean rank p-value it is easy to speak up with pharmacist <1 101 211.31 .498 13 137 200.17 ≥3 180 215.59 total 418 staff ideas and suggestions are valued in this pharmacy <1 101 204.99 .705 13 144 215.24 ≥3 181 216.87 total 426 pharmacists in this pharmacy encourage patients to talk about their medications <1 100 199.48 .373 1-3 143 218.01 ≥3 184 218.78 total 427 staff take adequate breaks during their shifts <1 101 203.50 .391 1-3 138 203.72 ≥3 181 219.57 total 420 we have clear expectations about exchanging important prescription information across shifts <1 95 192.73 .112 13 140 198.86 ≥3 177 219.94 total 412 staff feel comfortable asking questions when they are unsure about something <1 101 210.53 .243 1-3 143 203.64 ≥3 184 225.12 total 428 we have standard procedures for communicating prescription information across shifts <1 95 191.31 .105 13 130 192.15 ≥3 179 215.96 total 404 our pharmacists spend enough time talking to patients about how to use their medications <1 101 204.73 .416 1-3 141 207.15 ≥3 182 220.96 total 424 staff in this pharmacy discuss mistakes <1 102 204.20 .281 1-3 144 208.42 ≥3 182 225.09 total 428 we feel rushed when processing prescriptions <1 91 202.16 .836. 13 132 200.88 ≥3 173 194.76 total 396 our pharmacists tell patients important information about their new prescriptions <1 102 218.32 .790 13 142 214.48 ≥3 181 208.84 total 425 we have enough staff to handle the workload <1 101 222.48 .037⁎ 13 141 191.78 ≥3 182 223.01 total 424 iraqi j pharm sci, vol.30(1) 2021 patient safety culture in iraqi community pharmacies 160 continued table 5. when patient safety issues occur in this pharmacy, staff discuss them <1 100 203.57 .838 13 139 212.22 ≥3 179 210.70 total 418 the status of problematic prescriptions is well communicated across shifts <1 97 195.60 .125 13 136 195.26 ≥3 177 218.79 total 410 in this pharmacy, we talk about ways to prevent mistakes from happening again <1 102 203.67 .749 13 141 211.82 ≥3 178 214.55 total 421 interruptions/distractions in this pharmacy make it difficult for staff to work accurately <1 96 186.72 .027* 13 138 200.14 ≥3 180 224.22 total 414 overall rating of pharmacists to their pharmacy regarding patient safety the participating pharmacists rated the patient safety standards of their pharmacies as good with average 3.38 (±0.9). more specifically, 82.3% of pharmacists rated their pharmacy patient safety standards between good and excellent as shown in the figure below. figure 2. overall rating of pharmacists to their pharmacies regarding patient safety issues. discussion culture of safety has grown to have a significant issue in research in the subject of patient safety and there is a developing consideration to maintain efficient interventions to enhance safety (19) measurement of safety culture is essential because the organizational culture as well as team attitudes have been shown to affect patient outcomes and may work as tools to observe progress (20).result from this study showed the highest agreement and positivity in the patient counseling field which is similar to the result of study conducted in wisconson in the united states of america (aboneh et al 2020) (16). community pharmacists have a principal role in effectively use of medication ,enhancing patient outcomes, as well as avoiding misuse of medication (21). during the medication course, it was verified that self-administration was complicated and susceptible to many errors (22). the efficient communication among patients and pharmacists is an essential factor in affecting the quality of patient counseling, and has an impact on healthcare outcomes, mutual trust, and patient satisfaction,(23). teamwork scored second place in this study which is a similar findings to the result of study conducted in wisconson in the united states of america (aboneh et al 2020) (16). regarding pharmacy practice, teamwork is essential for improving patient care. (24). favorable interaction between pharmacy members, can efficiently improve work processes and deal with unaccepted events that may occur during the daily work(25). constantly pharmacy team can communicate with patients and realize the causes that influence patients’ ability to control their chronic disorders(26). the field of organizational learning-continuous improvements recorded in third place in word of agreement response . many researches show that medication errors attributed to different causes can be categorized into personal and organizational(27). persistent quality improvement focused on organization work process to figure out the source of errors and optimize patient safety(28). in the health system ,culture of blame is an essential cause of unaccepted medical errors(29). trust in the organization is a critical factor for establishing the culture of safety (30). regarding the result of study staffing work pressure domain recorded lowest positive response. this is similar to study conducted in community pharmacies in abu dhabi in the united arab emirates (alslubi et al 2019)(31). it has also shown similarity to a study conducted in malaysia( sivanandy et al’s 2016) (32).and the same results were found in the previous wisconson study (aboneh et al study 2020)(16) .in this study, 33.8% (around one-third) of the participant report their pharmacies to have interruptions which can influence workflow. despite that 66.7% of the participants have stated iraqi j pharm sci, vol.30(1) 2021 patient safety culture in iraqi community pharmacies 161 they have the appropriate number of staff in their pharmacies to deal with the workload, workrelated pressure has an adverse influence on quality associated event, that reach to patient or those stopped before dispensing(33). an additional suitable solution is training technicians who might able to reduce pharmacists dispensing workload,(34).there was a significant difference between pharmacist according to the experience year in pharmacy and distraction and interruptions. in other words, with a longer period of work in the pharmacy, interruptions of healthcare provider was a more considerable workload and interfere with workflow (35). most of the pharmacists (n=211) rate their pharmacies good about the standers of safety in their practice, which is similar to the result of study previously mentioned wisconson study (aboneh et al study 2020) were (92 %) of participant rate their pharmacies standers as good (16). conclusion iraqi community pharmacists have shown patient safety priority orientation, and there was a notable collaboration and communication between pharmacy workers. also, they show an interest in the pharmacy environment and workflow. references 1. ibrahim ir, al tukmagi hf, wayyes a. attitudes of iraqi society towards the role of community pharmacists. innovation in pharmacy. 2013;4(2):1–5. 2. hillier-brown f, bambra c, thomson k, balaj m, walton n, todd a. the effects of community pharmacy 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pharmacy practice. 2012;20(6):417–421. 35. reddy a, abebe e, rivera aj, stone ja, chui ma. interruptions in community pharmacies: frequency, sources, and mitigation strategies. research in social and administrative pharmacy. 2019;15(10):1243–1250. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.28(1) 2019 synthesis and anti-inflammatory study of new ibuprofen derivatives doi: https://doi.org/10.31351/vol28iss1pp131-137 131 synthesis, characterization and preliminary study of the anti-inflammatory activity of new pyrazoline containing ibuprofen derivatives mayada r. al-nakeeb*,1 and tagreed n-a. omar* *department of pharmaceutical chemistry, college of pharmacy, baghdad university, baghdad, iraq. abstract new ibuprofen derivatives containing variously substituted pyrazoline have been synthesized, characterized by ft-ir and 1h-nmr spectroscopy and evaluated preliminarily for their anti-inflammatory activity. in vivo anti-inflammatory activity of the final products (5a‐f) was studied in rats using egg‐white induced edema method of inflammation. all of them showed anti-inflammatory activity compared to the control group (propylene glycol) except compound 5f that showed more anti-inflammatory activity than ibuprofen. keywords: ibuprofen, pyrazoline, anti-inflammatory على ةالمضادة لاللتهاب لمشتقات جديدة لاليبوبروفين الحاوي ةللفعالي ةاولي ةتخليق و تشخيص ودراس البيرازولين * عمر تغريد نظام الدينو 1*،رياض النقيب ميادة *فرع الكيمياء الصيدالنية، كلية الصيدلة، جامعة بغداد، بغداد،العراق. الخالصة تحت الشعةا بمطياف تشخيصها تم قد و مختلفة، بمجاميع معوض بيرازولين تحتوي لالبوبروفين جديدة مشتقات تخليق تم لاللتهاب لمضادةا الفعالية دراسة تم. لاللتهاب المضادة لفعاليتها اولي تقييم و للبروتون المغناطيسي النووي الرنين مطياف و الحمراء عاليةف اظهرت المركبات كل. المختبرية الجرذان عند البيض بزالل المستحدثة الوذمة طريقة باساستخدام( و – أ5) النهائية للمركبات .االبوبروفين من اكثر لاللتهاب مضادة فعالية اظهر الذي( و5) المركب ماعدا‘كاليكول البروبيلين بمجموعة مقارنة لاللتهاب مضادة .الكلمات المفتاحية: ايبوبروفين، بيرازولين، مضاد التهاب introduction one of the most useful medication used in primary health care are nsaid, because of their analgesic, antipyretic and anti-inflammatory activities. inflammation in rheumatoid arthritis and osteoarthritis is known to be reduced by the use of nsaid, also they enhance the recovery and mobility(1). nsaid act by the same mechanism of action, through inhibiting cyclooxygenase enzymes cox that are responsible for the production of prostaglandins. those enzymes are different in their regulation, distribution and biological functions(2). there are two isoforms of cox enzymes: cox-1 is constitutively normally expressed in most tissues, and prostaglandins controlled by cox-1 mediate cytoprotection of gastric mucosa and platelet aggregations in addition to some other physiological processes(3).cox–2 is not detected in normal tissues and selectively induced in response to pro-inflammatory stimuli such as, cytokines and growth factors(4). classical nsaids, such as ibuprofen, inhibit both isoforms of the enzymes(5). cox-3 is a splice variant/isoenzyme of cox-1 and, may have been named cox-1b. it was initially reported to be expressed in canine cerebral cortex. in humans cox-3 is found in highest concentrations in the brain and heart. the advantage of cox-3 is that it could explain the pharmacological actions of drugs such as acetaminophen and other antipyretic analgesic which are weak inhibitors of cox-1 and cox-2, but penetrate easily into the central nervous system(6). the major limitation to long term use of nsaids in therapy is the increased risk of gastrointestinal( gi) ulceration (7). a well-accepted fact that the gi side effect of acidic nsaids is a result of direct irritation of gastric mucosa, because of the carboxylic group, and indirectly because of cox-1 inhibition that reduce the levels of protective prostaglandins. inhibition of prostaglandin synthesis in the gi tract lead to increase gastric acid secretion and decrease trophic effects on epithelial mucosa(8) . 1corresponding author e-mail: mralnakeeb@yahoo.com received: 24/12/2018 accepted: 20/3/2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp131-137 https://doi.org/10.31351/vol28iss1pp131-137 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.28(1) 2019 synthesis and anti-inflammatory study of new ibuprofen derivatives 132 in order to increase the analgesic and antiinflammatory activity and reduce the side effects, recent researches focus on designing new compounds by derivitization of the carboxylic group of nsaid with heterocyclic systems, pyrazoline(9,10). pyrazolines constitute an interesting class of heterocycles due to their synthetic flexibility and effective biological activities. they have been shown to possess a broad range of physiological activities such as antimicrobial, analgesic, antiamoebic, anticancer, antidepressant, and antiinflammatory activities (11–15) . the direction of the current work is to synthesize potent non‐steroidal anti‐inflammatory agents that are derivatives of ibuprofen by joining a group of pyrazoline ring to the carboxylate group of ibuprofen, using spacer arm glycine ester(16,17), and then evaluating them as anti‐inflammatory agents. materials and methods chemicals and solvents used during synthesis were of analar type and were purchased from (fluka, england, india and germany). ibuprofen (general company for pharmaceuticals industries and medical appliances, samarra, iraq). the progress of the reaction and checking the purity of the products were determined by thin-layer chromatography (tlc), using silica gel gf254 (type 60) pre-coated aluminium sheets, merck (germany) exposed to uv-254nm light. chromatograms were eluted by using three solvent systems: a /ethyl acetate: petroleum ether (1:1). b / ethanol: toluene (4:6). c /chloroform: methanol (85:15). melting points were measured by using stuart smp3 melting point apparatus in open capillary tubes, and are uncorrected. the infrared spectra were performed using ft-ir (iraffinity-1) spectrophotometer, shimadzu, japan. 1hnmr spectra were recorded on nmready-60 spectrometer model with tetra methylsilane as an internal standard, chemical shift were expressed as (δ= ppm) and coupling constant in (hz). chemical synthesis general method for synthesis of chalcones derivatives (1a-f) in a 96% ethanol (22ml) acetophenone (1.18 ml, 10 mmole) and derivatives of aromatic aldehydes (a-f) (10 mmole) were dissolved (table 1). sodium hydroxide (40%, 10 ml) solution was added gradually. the solution was stirred in an ice bath until solidified, then kept in cold condition overnight. then the solid was diluted with cold water and acidified with 2n hcl, filtered then recrystallized by ethanol(18). table (1 )aromatic aldehydes and weights used aldehydes’ name product r weight (gm) benzaldehyde ia h 1.06 4-chlorobenzaldehyde ib cl 1.4 4-nitrobenzaldehyde ic no2 1.51 4-hydroxybenzaldehyde id oh 1.22 4methoxybenzaldehyde ie och3 1.36 4-dimethylaminobenzaldehyde if n(ch3)2 1.49 1,3-diphenylpropenone (c15h12o) (compound 1a): colour: pale yellow crystals. yield: 90%. m.p.:5658 ºc. rf: a=0.6. ft-ir: 3055 (ch asymmetric stretching aromatic), 3028 (ch symmetric aromatic stretching), 1662 (c=o), 1604, 1573 (c=c aromatic). 3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (c15h11ocl) (compound 1b): colour: off white powder. yield: 80%. m.p.: 112-114 ºc. rf: a=0.5. ft-ir: 3064 (ch asymmetric aromatic stretching), 3016 (ch symmetric aromatic stretching), 1658 (c=o), 1597, 1589 (c=c aromatic), 821 (c-cl). 3-(4-nitrophenyl)-1-phenylprop-2-en-1-one (c15h11no3) (compound 1c): colour: deep yellow crystals. yield: 75%. m.p.: 156-158 ºc. rf: a=0.54. ft-ir: 3070 (asymmetric c-h stretching aromatic), 1654 (c=o), 1589, 1577 (c=c aromatic), 1516 (no2 asymmetric stretching), 1334 (no2 symmetric stretching). 3-(4-hydroxyphenyl)-1-phenylprop-2-en-1-one (c15h12o2) (compound 1d): colour: light yellow crystals. yield: 60%. m.p.: 182-184 ºc. rf: a=0.7. ft-ir: 3213 (oh stretching), 3070 (asymmetric ch stretching aromatic), 3032 (symmetric ch stretching aromatic), 1647 (c=o), 1597, 1554 (c=c aromatic), 1346 (oh bending), 1215 (c-o). 3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one c16h14o2) (compound 1e): colour: yellow crystals. yield: 80%.m.p.: 73-75ºc. rf: a=0.6. ft-ir: 3059(asymmetric ch stretching aromatic), 3017 (symmetric ch stretching aromatic), 2954(asymmetric ch stretching), 2839 (symmetric c-h stretching), 1654 (c=o), 1689, 1573(c=c aromatic), 1257(c-och3), 1257 (c-o). iraqi j pharm sci, vol.28(1) 2019 synthesis and anti-inflammatory study of new ibuprofen derivatives 133 3-(4(dimethyl amino) phenyl)-1-phenylprop-2-en1-one (c17h17no) (compound 1f): colour: bright orange red needle like crystal. yield: 85%. m.p.: 110-112ºc. rf: a=0.7. ft-ir: 2912 (asymmetric ch stretching), 2846 (symmetric ch stretching), 1647 (c=o), 1597, 1558 (c=c aromatic), 1157 (c-n). synthesis of ethyl amino acetate hydrochloride (c4h10no2cl) (compound 2): glycine (2 g, 26.6 mmol) to be esterified was suspended in absolute ethanol (50 ml) and cooled to 5ºc. thionyl chloride (2.3 ml, 32 mmol) was added slowly over a period of 15 minutes, and the reaction mixture was refluxed for 3 hours. the solvent was then evaporated to dryness under reduced pressure. finally the residue was purified by recrystallization from ethanol: diethyl ether .colour: white crystals. yield: 92%. m.p.: (144-145) ºc, (145-146) ºc lit.. rf c=0.5. ft-ir: 1743 (c=o) , 1246(c-o-c), 1176 (c-n) (17). ethyl 2-(2-(4-isobutylphenyl) propanamido) acetate chemical synthesis (c17h25no3) (compound 3): synthesis of ibuprofen acid chloride: ibuprofen (1g, 4.8 mmol) was suspended in 1 ml dry chloroform and excess of thionyl chloride (1.2 ml, 16.9 mmol) was added drop wise over a period of 3-5 minutes, then refluxed for 3 hours. evaporation under reduced pressure to remove excess gases was done. ibuprofen acid chloride was obtained as a faint yellow oily residue(19). the reaction of ibuprofen acid chloride with glycine ethyl ester: triethylamine (tea) (1.35 ml, 9.6mmol) was added to a suspension of glycine ethyl ester hydrochloride (0.676g, 4.8 mmol) in dry dichloromethane (dcm) (30 ml) at 25°c. the reaction mixture was stirred for 3hours. to this mixture, ibuprofen acid chloride (prepared above) dissolved in 1 ml dry chloroform was added drop wise with continuous stirring, keeping the temperature about 5ºc and left stirring overnight. after evaporating the solvent under vacuum, ethyl acetate 30ml was added to the residue, filtration was done to remove the precipitate. the ethyl acetate layer was washed with 1n hcl, followed with distilled water, and then it was washed with 5% nahco3 solution and distilled water. the ethyl acetate layer was dried over anhydrous magnesium sulphate, filtered, and the filtrate was evaporated under vacuum to an oily residue, that represents the compound (3). colour: yellow oil. yield: 80%. rf: b=0.6. ft-ir: 3309 (nh amide) , 1739 (c=o ester) , 1654 (c=o amide) (20). n( 2 – hydrazinyl – 2 – oxoethyl ) -2(4isobutylphenly) propanamide chemical synthesis (c15h23n3o2) (compound 4): compound (3) (0.8 g, 3 mmol ) was dissolved in 10 ml methanol, then hydrazine hydrate ( 1.5 ml ,30 mmol) was added, the reaction mixture was refluxed for 9 hours, then left over night with continuous stirring, then the solvent was evaporated under reduced pressure. white precipitate appeared upon the addition of iced water, filtered washed with cold water, dried and washed with ether for further purification. colour: white powder. yield: 80%. m.p.: 100-112ºc. rf: b=0.5. ft-ir: 3305 (nh amide) , 3228 and 3190 (nhnh2) , 1678 (c=o 2º amide) (21). n-(2( 3 ,5 – diphenyl – 4 ,5-dihydro-1h-pyrazol1-yl)-2-oxoethyl ) – 2 ( 4 – isobutylphenyl ) propanamide (compounds 5 ( a – f ) ) chemical synthesis: compound (4) (0.4gm, 1.44 mmol) was dissolved in absolute ethanol, together with chalcones (1a-f) of (1.44 mmol) , then few drops of glacial acetic acid (gaa) was added. the reaction mixture was refluxed for 24 hours and followed by using tlc, till single spot is obtained. the solvent was evaporated to dryness under reduced pressure; the residue was triturated with petroleum ether. then ether was added, white precipitate appeared, filtered and collect the filtrate, evaporate to dryness(22). n-(2-(3,5-diphenyl-4,5-dihydro-1h-pyrazol-1-yl)2-oxoethyl)-2-(4-isobutylphenyl) propanamide (c30h33n3o2) compound 5a: colour: off white powder. yield: 60%. m.p.: 66-88ºc. rf a= 0.62. ft-ir: 3278 (nh amide), 1647 (c=o), 1593 (c=n). 1hnmr (60 mhz, dmso-d6, ppm): 0.75-0.85 (6h, d, two ch3 ibuprofen), 1.22-1.33 (3h, d, ch3 ibuprofen), 1.76-1.86 (m, ch ibuprofen), 2.3 (2h, d, ch2 ibuprofen), 3.63 (3h, m, ch ibuprofen and ch2 pyrazoline), 4.21 (3h, m, ch2 glycine and ch of pyrazoline ring), 6.94-7.44 (14h, m, ar-h), 8.10 (1h, br. s, nh amide). n-(2-(5-(4-chlorophenyl)-3-phenyl-4,5-dihydro1h-pyrazol-1-yl)-2-oxoethyl)-2-(4isobutylphenyl)propanamide (c30h32cln3o2) compound 5b: colour: off white powder. yield: 62%. m.p.: 62-65ºc. rf a=0.65. ft-ir: 3294 (nh amide), 1647 (c=o), 1593 (c=n), 813 (c-cl). 1hnmr (60 mhz, dmso-d6, ppm): 0.75-0.85 (6h, d, two ch3 ibuprofen), 1.21-1.33 (3h, d, ch3 ibuprofen), 1.76-1.86 (1h, m, ch ibuprofen), 2.3 (2h, d, ch2 ibuprofen), 3.63 (3h, ch ibuprofen and ch2 pyrazoline), 4.25 (3h, m, ch2 glycine and ch of pyrazoline ring), 6.94-7.15 (13h, m, ar-h), 8.12 (1h, br. s, nh amide). 2( 4 – isobutylphenyl ) – n ( 2 -(5-(4nitrophenyl)-3-phenyl – 4 , 5 – dihydro 1h – pyrazol – 1 – yl )-2-oxoethyl) propanamide (c30h32n4o4) compound 5c: colour: yellow powder. yield: 70%. m.p.: 76-78ºc. rf a=0.55. ftir: 3305 (nh amide), 1651 (c=o), 1620 (c=n), 1514 (no2 asymmetric stretching), 1334 (no2 symmetric stretching). 1hnmr (60 mhz, dmsod6, ppm): 0.74-0.84 (6h, d, two ch3 ibuprofen), 1.19-1.32 (3h, d, ch3 ibuprofen), 1.75-1.85 (1h, m, ch ibuprofen), 2.29 (2h, d, ch2 ibuprofen), iraqi j pharm sci, vol.28(1) 2019 synthesis and anti-inflammatory study of new ibuprofen derivatives 134 3.63 (3h, m, ch ibuprofen and ch2 pyrazoline) ,4.36 (3h, m, ch2 glycine and ch of pyrazoline ring) , 6.93-7.82 (13h, m, ar-h) , 8.18 (1h, br. s, nh amide). n-(2-(5-(4-hydroxyphenyl)-3-phenyl-4,5 -dihydro1h-pyrazol-1-yl)-2-oxoethyl)-2-(4-isobutylphenyl ) propanamide ( c30h33n3o3 ) compound 5d: colour: light brown powder. yield: 70%. m.p.: 82-84ºc. rf a=0.7. ft-ir: 3232 (nh amide), 3217 (phenolic oh), 1647 (c=o), 1604 (c=n). 1hnmr (60 mhz, dmso-d6, ppm): 0.75-0.85 (6h, d, two ch3 ibuprofen) , 1.22-1.32 (3h, d, ch3 ibuprofen), 1.8 (1h, m, ch ibuprofen), 2.31 (2h, d, ch2 ibuprofen), 3.66 (3h, ch ibuprofen and ch2 pyrazoline ring), 4.10 (3h, ch2 glycine and ch pyrazoline ring) , 6.81-7.43 (13h, m, ar-h) , 8.03 (1h, br. s, nh amide), 9.82 (1h, br. s, oh). 2( 4isobutylphenyl ) – n ( 2 ( 5 ( 4methoxyphenyl)-3phenyl – 4 , 5 – dihydro 1h – pyrazol – 1 -yl)-2-oxoethyl) propanamide (c31h35n3o3) compound 5e: colour: off white powder. yield: 60%. m.p.: 60%. rf a=0.6. ft-ir: 3294 (nh amide), 1647 (c=o), 1604 (c=n). 1hnmr (60 mhz, dmso-d6, ppm): 0.75-0.85 (6h, d, two ch3 ibuprofen), 1.22-1.33 (3h, d, ch3 ibuprofen), 1.78-1.86 (1h, m, ch ibuprofen) , 2.32.4 (2h, d, ch2 ibuprofen), 3.73 (6h, m, ch ibuprofen and -och3 overlapped with ch2 pyrazoline), 4.21 (3h, ch2 glycine and h of pyrazoline ring), 6.94-7.44 (13h, m, ar-h) , 8.05 (1h, br. s, nh amide),. n-(2-(5-(4-(dimethyl amino) phenyl)-3-phenyl-4,5 dihydro-1h-pyrazol-1-yl)-2-oxoethyl)-2-(4isobutylphenyl) propanamide (c32h38n4o2) compound 5f: colour: red powder. yield: 65%. m.p.: 60-62ºc. rf a=0.55. ft-ir: 3298 (nh amide), 1647 (c=o), 1600 (c=n), 1168 (n-ch3). 1hnmr (60 mhz, dmso-d6, ppm): 0.74-0.85 (6h, d, two ch3 ibuprofen), 1.2-1.3 (3h, d, ch3 ibuprofen), 1.78-1.86 (1h, m, ch ibuprofen) , 2.4 (2h, d, ch2 ibuprofen), 2.9 (6h, s, n(ch3)2), 3.56 (3h, ch ibuprofen and ch2 pyrazoline), 4.2 (3h, ch2 glycine and ch pyrazoline), 6.73-7.43 (m, 13h arh), 8.11 (1h, br. s, nh amide). pharmacology anti-inflammatory assessment study in vivo, anti‐inflammatory effects of the chemically synthesized products (5a‐f) were assessed using egg‐white induced paw oedema. measuring the decrease of paw thickness is the basis for the evaluation of the ant‐inflammatory activity of the desired compounds(23). albino rats of either sex weighing (170±10 g) were supplied by iraqi centre for cancer and medical genetic research and were housed in university of technology under standardized conditions for 10 days for acclimatization. animals were fed commercial chow and had free access to water. animals were taken to the laboratory, one hour before the experiment, and were separated into eight groups (each group consist of 6 rats) as follows(24): group a: six rats considered as control and injected with the vehicle intra peritoneally (i.p.) (propylene glycol 50% v/v). group b: six rats treated with ibuprofen as reference substance in a dose of 50mg/kg suspended in propylene glycol. group c‐h: six rats for each group injected with the tested compounds (5a‐f), also suspended in propylene glycol(25), in doses that are illustrated in table 2. the paw width was measured by vernier calliper at seven time periods (0, 30, 60, 120, 180, 240, and 300 min.) after drug delivery. potent inflammation can be created by injecting of 0.05ml of undiluted egg-white subcutaneously into the planter side of the hind paw of the rats 1/2 hr. after i.p. delivery of the target compounds including standard and control . the data was stated as the mean ± standard error of mean (sem) and results were investigated for statistical significance using student t‐test (two sample assuming equal variances) for comparison between mean values. however, comparisons between different groups were made using anova: two factors without replication. probability (p) value of less than 0.05 was considered significant. ibuprofen which is given in a dose of 50mg/kg, so; the doses of synthesized compounds are calculated as in table 2: m. wt. of ibuprofen = 206.29 (50mg / kg) / m. wt. of reference drug = dose /m. wt. of the tested compound (25). table (2) compounds with their molecular weight and dose compound molecular wt. weight dose (mg/ kg) ibuprofen 206.29 50.00 5a 467.60 113.335 5b 502.05 121.685 5c 512.60 124.242 5d 483.60 117.21 5e 497.60 120.61 5f 510.67 123.77 results and discussion chemistry chalcones (1a-f) were synthesized based on aldol condensation by reacting aromatic aldehydes with acetophenone in ethanol with naoh 40% solution. chalcones were characterized by ftir that shows appearance of c=o stretching at (16621647). glycine ethyl ester hcl (2) was prepared by refluxing glycine with thionyl chloride in absolute iraqi j pharm sci, vol.28(1) 2019 synthesis and anti-inflammatory study of new ibuprofen derivatives 135 ethanol. ftir shows characteristic band of ester c=o stretching at (1743). compound (3) was synthesized from reacting ibuprofen acyl chloride with compound (2), and was characterized by ftir to show nh stretching at (3305), c=o stretching of ester at (1739) and c=o stretching of amide at (1654). compound (4) was prepared by refluxing compound (3) with hydrazine hydrate in methanol. ftir shows characteristic band of hydrazide of nhnh2 stretching at 3224 and 3190. the final product (5) was synthesized by refluxing chalcones with compound (4) in ethanol using glacial acetic acid as a catalyst. the appearance of bands c=n stretching at (1620-1593), were characteristic of pyrazoline derivatives. all the steps involved in synthesis of targets compound were shown in (scheme 1). iraqi j pharm sci, vol.28(1) 2019 synthesis and anti-inflammatory study of new ibuprofen derivatives 136 the anti‐inflammatory activity evaluation of the tested compounds the anti‐inflammatory activity of the tested compounds has been evaluated in comparison with their vehicle (control group) and ibuprofen. table 3 explains the effect of tested compounds 5a‐f in comparison to control and ibuprofen. discussion all tested compounds employed imperative reduction of paw oedema in comparison to the effect of propylene glycol 50%v/v (control group). the effect of ibuprofen and all tested compounds started at time 120 min except 5f which has started at 60 min. indicating rapid onset of action. the effect of target compounds and ibuprofen continued till the end of experiment. compound 5d and 5e showed comparable effect to ibuprofen at all experimental time while compounds 5a, 5b and 5c produced significantly lower inhibitory effect than ibuprofen at time 120‐240 minutes. remarkably, compound 5f exerted significantly higher paw edema reduction than ibuprofen at 60‐240 min. all tested compounds have showed comparable effect to that of ibuprofen at time 300 minutes. table( 3 )the anti‐inflammatory effect of control, ibuprofen and compounds 5a‐f on egg‐white induced paw edema in rat # #non‐identical superscripts (a, b and c) among different tested compounds are regarded significantly different (p < 0.05);*significantly different compared to control (p < 0.05). data are expressed in mm paw thickness as mean ± sem. n= number of rats. time (0) is the time of i.p. injection of ibuprofen, tested compounds and propylene glycol. time (30) is the time of injection of egg white to induce edema. percent of inhibition in paw oedema thickness the percent of inhibition of paw oedema thickness at each time interval was calculated from the mean effect in control and tested animals according to the equation(26): % inhibition= [vc ‐vt /vc] ×100 where vc and vt are the mean paw thickness of the control group and tested group (at t‐time zero) respectively. the comparison among the ibuprofen, compounds 5a‐f was presented in table 4. table (4 )percent of inhibition of inflammation for ibuprofen and compounds 5a‐f on egg‐white induced paw oedema in rats. time (min.) ibuprofen 5a 5b 5c 5d 5e 5f 60 4.6% 2.9% 4% 5% 2.3% 3.5% 45% 120 56% 32% 34% 32% 50% 50% 72% 180 69% 57% 53% 53% 66% 70% 88% 240 81% 70% 62% 65% 79% 81% 97% 300 83% 68% 72% 64% 51% 62% 92% conclusion new pyrazoline containing ibuprofen derivatives have been synthesized and characterized successfully. all of the synthesized compounds demonstrated anti-inflammatory activity in the control group of rats. out of them, compound 5f showed better anti-inflammatory activity than that of ibuprofen, since it has electron donating group n(ch3)2. references 1. al-shidhani a, al-rawahi n, al-rawahi a, murthi p s. non-steroidal anti-inflammatory drugs (nsaids) use in primary health care centers in a’seeb, muscat: a clinical audit. oman med j. 2015;30(5):366–371. 2. croff lj. use of nsaids in treating patients with arthritis. arthritis res ther. 2013;15(3):1– 10. 3. badrey mg, abdel-aziz hm, gomha sm, p a w t h ic k n e ss ( m m ) / n = 6 compoun d time (min) 0 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assessment of new ibuprofen analogues containing imidazole-4 one derivatives. j glob pharma technol. 2018;10(03):134–141. 26. verma s, jain a, gupta vb, stract ab. synergistic and sustained anti-inflammatory activity of guguul with the ibuprofen : a preliminary study. int j pharma bio sci. 2010;1(2):1–7. iraqi j pharm sci, vol.30(2) 2021 carvone and intestinal mucositis doi: https://doi.org/10.31351/vol30iss2pp58-63 58 carvone attenuates irinotecan-induced intestinal mucositis and diarrhea in mice ban w. abbas *,1 and sarmed h. kathem ** * ministry of health and environment, medical city health directorate, baghdad, iraq. ** college of pharmacy, university of baghdad, pharmacology and toxicology department abstract intestinal mucositis is referring to inflammatory or ulcerative lesions of the oral or gastrointestinal tract; one of the main reasons is treatment with cancer chemotherapy. the prodrug irinotecan is converted by carboxylesterase to the active metabolite sn-38, conjugated by ugt enzyme to sn-38g and then deconjugated by β-glucoronidase produced by intestinal bacterial flora to produce sn-38. irinotecan induces intestinal mucositis and diarrhea due to increased concentration of its active metabolite (sn-38).to evaluate the protective effect of carvone, i.p injection of (75mg/kg/day) of irinotecan for 4 days to induce intestinal mucositis, carvone administered to mice orally for 6 days starting from day 1. results showed that carvone (50mg/kg and 100mg/kg) significantly and by dose-dependent manner attenuated body weight loss (-9.39±1.56 vs. -23.21±1.65 %), diarrhea scores (0.50±0.244 vs. 2.67±0.211) and serum tnf-α level (1361.44±55.075 vs. 3402.12±321.56 ng/ml) compared to experimental model group. in conclusion, carvone exerted a dose dependent anti-inflammatory and protective effect by attenuation irinotecan-induced intestinal mucositis. keywords: intestinal mucositis,iirinotecan, carvone, body weight variation, diarrhea score, tnfα level. الكارفون يثبط االلتهاب المعوي واالسهال المستحث بواسطة االيرينوتيكان في الفئران **سرمد هاشم كاظم و 1*،عباسوليد بان . العراقبغداد ، ، دائرة مدينة الطبوزارة الصحة والبيئة ،* .، بغداد ، العراق ، كلية الصيدلة، جامعة بغداداالدوية والسموم فرع ** الخالصة يشير التهاب الغشاء المخاطي المعوي إلى اآلفات االلتهابية أو التقرحية في الفم أو الجهاز الهضمي ؛ أحد األسباب الرئيسية هو جرعة ، مترافق بواسطة إنزيم carboxylesterase بواسطة sn-38 يتم تحويله إلى مستقلب نشطهو دواء أولي irinotecan .عالية من العالج الكيميائي ugt إلى sn-38g ثم يتم تفكيكه بواسطة β-glucoronidase إلنتاجالبكتريا الموجودة بصورة طبيعية باالمعاء الذي تنتجه sn-38 يتسبب irinotecan زيادة تركيز في التهاب الغشاء المخاطي المعوي واإلسهال بسبب sn-38 المعوي ويؤدي إلى زيادة اإلجهاد التأكسدي. لدراسة التأثير مجم / كجم / يوم( لمدة 75) ip يسبب التهاب الغشاء المخاطي ، التهاب الغشاء المخاطي المعوي الناجم عن حقن -irinotecan الوقائي للكارفون في أيام بدًءا من اليوم األول ، أدى إلى انخفاض كبير في فقدان وزن 6عن طريق الفم للفئران لمدة أيام. أظهرت النتائج أن الكارفون الذي تم تناوله 4 في البالزما tnfα ( ومستوى0.211± 2.67مقابل 0.244± 0.50( ، ودرجة اإلسهال )1.65± 23.21-مقابل 1.56± 9.39-الجسم ) جموعة التحكم في النموذج. في الختام ، كارفون له تأثير وقائي في التهاب الغشاء ( مقارنة بم321.56± 3402.12مقابل ±55.075 1361.44) .المخاطي واإلسهال الناجم عن اإلرينوتيكان الكلمات المفتاحية:التهاب الغشاء المعوي, ايرينوتيكان, كارفون, تغير وزن الجسم, درجة االسهال,عامل نخر الورم الفا introduction mucositis is an inflammatory or ulcerative lesions of the oral or gastrointestinal tract. infectious disease, immune deficiency and medications as with high-dose cancer therapy could be considered as one of main causative factors for mucositis (1). the condition was developed by breakdown of the mucosal barrier leading to: significant ulceration of the oral cavity and git leading to passing of bacteria into the systemic circulation, increasing the risk of infections, an imbalance between secretion and absorption, demonstrated as diarrhea(2). chemotherapy induced diarrhea (cid) being a principal reason for pain and reduced quality of life during cancer treatment because it is one of the most debilitating adverse effects of chemotherapy (3). the progression of mucositis can be divided into five phases: initialization, activation, signal amplification, ulceration and healing phase(4). severe diarrhea leading to dehydration, renal insufficiency, malnutrition, fever, neutropenia, infectious complications, or serious electrolyte imbalances can result in hospitalization (5). the presence of cid can impact on providers to modulate chemotherapeutic agents, decrease treatment doses, delay therapy, or even cease therapy, resulting in potentially worsened clinical outcomes(5). irinotecan hydrochloride is a prodrug that is metabolically activated to 7-ethyl10hydroxycamptothecin (sn-38) by carboxylesterase 2 in the blood and liver, and thereafter sn-38 is conjugated in the liver by uridine diphosphate glucuronosyl transferase 1a1 to produce a βglucuronide conjugate, sn-38g(6). 1corresponding author e-mail: ban.walid.alsaffar@gmail.com received: 12/9/2020 accepted: 13/2 /2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp58-63 iraqi j pharm sci, vol.30(2) 2021 59 irinotecan and sn-38 are a potent inhibitor of dna topoisomerase i, a fundamental enzyme that controls dna structural alterations by relaxing dna supercoil as well as relegating cleaved dna strands throughout dna transcription and replication (6). two types of diarrhea induced by irinotecan can be distinguished: early and late-onset diarrhea. early-onset diarrhea begin during, or immediately after, drug infusion and is occur due to increased cholinergic activity, which triggers intestinal contractility and diminish the absorptive capacity of the mucosa(7) late-onset diarrhea appear approximately 8–10 days after irinotecan infusion and is recognized by a more severe course, which is probably occur due to damage of the intestinal mucosa as a result of increased oxidative stress by biliary-secreted or intestinally deconjugated sn38(8). the treatment of cid involves non pharmacologic and pharmacologic interventions to inhibit diarrhea and careful serial estimation to abolish significant volume depletion or comorbidities that would need particular intervention or hospitalization(9). anti-diarrheal therapies used are: opioid, octreotide, budesonide, intestinal alkalization, alteration of intestinal micro flora [prebiotic and probiotic, antibiotics], d-saccharic acid 1,4-lactone (sal)] and glutamine. different plants extract found to have a protective effect against irinotecan-induced intestinal mucositis and diarrhea, as kampo kampo (japanese \chinese herbal) and mentha spicata l. (spearmint) the plant that we will study its assumed protective effect against cid . the main components of mentha spicata oil were carvone (40.8% ± 1.23%) and limonene (20.8% ± 1.12%), followed by 1,8-cineole (17.0% ± 0.60%), β-pinene (2.2% ± 0.25%), cis-dihydrocarvone (1.9% ± 0.49%), and dihydrocarveol (1.7% ± 0.31%)(10), in term of biological uses, mentha spicata used as antispasmodics and anti-platelets and insecticides(11). carvone (p-mentha-6, 8-dien2-one) is a chiral monoterpene ketone that can be achieved by distillation. it occurs naturally in two enantiomer forms: 1. s (+)carvone (the major component in the essential oil of caraway [carum carvi]. 2. r(-) carvone (which is existing in the essential oil of spearmint [ mentha spicata] (12) (r)-(−)-carvone gives ant nociceptive activity (13), both (r)-(−)carvone and (s)-(+)-carvone decreased peripheral nerve activity, likely through the blockade of voltagegated sodium channels(12). carvone has an obvious role as it has been reported to exert anticancer effect (14), it was manifests various possible biological activities namely antioxidant, antimicrobial, nematicidal and antidiabetic effect (15). materials and methods chemicals and drugs irinotecan vial (100mg/5ml) was purchased from kocak pharma, turkey. r-(-) carvone was purchased from sigma, usa. tnf-α elisa kit purchased from shanghai, china. preparation of stock solution of carvone a stock solution of carvone was prepared by dissolving 0.2 ml of carvone in 10 ml of corn oil then the solution is mixed by vortex to obtain a final concentration of (1.5 mg/0.1 ml) according to the carvone dose for mice (50mg/kg) (16). another stock solution of carvone was prepared by dissolving 0.4 ml of carvone in 10 ml of corn oil then the solution is mixed by vortex to obtain a final concentration of (3 mg/0.1 ml) according to the carvone dose for mice (100mg/kg) (16). experimental protocol twenty -four albino female mice weighing (25-50 g) were brought from animal house of college of pharmacy, baghdad university. animals were maintained on standard conditions of temperature, humidity, and light /dark cycle. they had free access to standard diet and water. mice were divided into 4 groups, each group with 6 mice as following and illustrated in figure 1: group i: six animals received a single oral daily dose of 0.1ml of normal saline for 7 successive days starting day 1. this group served as a normal control group. group ii: six animals received a single dose of intraperitoneal irinotecan (75mg/kg) for 4 successive days starting day 1. this group served as a model control group. group iii: six animals received a single dose of intraperitoneal irinotecan (75mg/kg) for 4 successive days starting day 1 with 0.1ml of carvone of dose 50mg/kg by oral gavage for 6 successive days starting day 1. group iv: six animals received a single dose of intraperitoneal irinotecan (75mg/kg) for 4 successive days starting day 1 with 0.1ml of carvone of dose 100mg/kg by oral gavage for 6 successive days starting day 1. administration of carvone done by oral gavage at 8am and 8pm daily from day 1 through day 6. euthinazation has done on day 7 by diethyl ether followed by cervical dislocation. iraqi j pharm sci, vol.30(2) 2021 60 figure1. schematic representation of the experimental model design. serum collection: on day 7, about 0.5ml of blood was drawn from retro-orbital area and collected in eppendorf tube and let to completely coagulated, then centrifuged for 15 min. at 5000 rpm in a cold centrifuge at 4 °c. the serum was then collected and froze for later use to measure tnf-α. body weight measurement mice body weights were recorded at 8am daily. weight values were expressed as a percentage of weight variation in relation to the baseline weight(17). weight variation (%) = {(w2-w1)/w1}* 100 w1= baseline mouse weight w2 = weight of mouse during the study period. diarrhea score measurement the diarrhea scores were estimated daily starting 24 hr. after the last irinotecan dose between day 5 and 7. the intensity of delayed-onset diarrhea was recorded at 8:00 am daily after the last irinotecan dose till euthanization (figure 1). diarrhea was estimated using standard reference scale (18), as shown in table1. table 1. diarrhea score reference scale. (18) extent of diarrhea manifestations score normal normal stool or absent 0 slight slightly wet and soft stool 1 moderate wet and unformed stool with moderate perianal staining of the coat 2 severe watery stool with severe perianal staining of the coat 3 determination of serum tnf-α level the principle of tnfα determination in serum is based on enzyme-linked immunosorbent assay (elisa) that was use the sandwichelisa technique. the micro elisa plate has been precoated with antibody specific to mouse tnfα(19). statistical analysis values expressed as mean± standard error mean (sem). mice body weight values presented as weight variation percent ±sem. all statistical analyses were carried out using the statistical package of social science (spss) software version 25. differences were considered significant at p<0.05. intergroup comparisons were made by student's t-test. results effect of carvone on serum tnf-α level the data presented in table 2 and figure 2 showed that administration of irinotecan in the model control group (group ii) resulted in a significant increase in inflammation (p<0.05) compared to the normal control group (group i) resulted in tnfα serum levels elevation from 3402.12 ± 321.56pg/dl to 858.7±47.79pg/dl respectively. administration of carvone 50mg/kg (group iii), and carvone 100mg/kg (group iv), produce a significant (p<0.05) reduction in serum tnfα levels (2210.48± 188.29pg/dl, 1361.45 ± 55.07pg/dl) respectively compared to model group (group ii, 3402.12 ± 321.56pg/dl) as shown in table 2 and figure 2. this interesting result indicates a dosedependent anti-inflammatory effect of carvone and a counteracting role in irinotecan-induced mucositis. table 2. effects of carvone on serum tnfα level in irinotecan-induce intestinal mucositis in mice data are expressed as mean ± standard error of means (sem). (*) significantly different (p<0.05). groups type of treatment tnf α (pg/dl) i normal saline 858.70±47.79 ii irinotecan 3402.12 ±321.56 * iii carvone 50mg/kg +irinotecan 2210.48± 188.29 * iv carvone 100mg/kg +irinotecan 1361.44±55.075 * iraqi j pharm sci, vol.30(2) 2021 61 figure 2. effect of carvone on serum tnf-α level in irinotecan-induces intestinal mucositis. (*) denotes significant difference (p<0.05) compared with experimental model group (group ii). effect of carvone on mice body weight analysis of mice weight variation percent revealed that significant (p<0.05) weight loss started from day 2 in all groups except non-treated control group (figure 3). mice received irinotecan only (group ii) showed significant (p<0.05) weight loss from day 2 till euthanization. on day 7, a maximum weight loss variation percent recorded of (23.21±1.65%). on the other hand, treatment with carvone (50mg/kg) significant (p<0.05) attenuated weight loss compared to model group on day 7, where the weight variation percent calculated was (13.78±1.62%). furthermore by increasing the dose, treatment with carvone (100mg/kg) significantly (p<0.05) hampered weight loss on days 5, 6 and 7 compared to experimental model. interestingly, on day 7 the weight variation percent calculated was (9.39±1.56%). analysis of the data of weight variation percent clearly revealed that carvone exerted a dose-dependent attenuation of weight loss induced by irinotecan (figure 3). figure 3. effect of carvone treatment on mice body weight variation in irinotecan-induced intestinal mucositis and diarrhea. (*) denotes significant difference (p<0.05) compared with experimental model group (group ii). effect of carvone on diarrhea score data obtained from the present study showed that diarrhea scores began to rise on day 5 in the model control group (group ii) and other treatment groups 24 hours after the last irinotecan injection as shown in table 4.on day 7, mice in model control group (group ii) suffered from severe diarrhea as manifested from the significant (p<0.05) high diarrhea score (2.67±0.211) compared to the normal control group (0). however, treatment with carvone (50mg/kg) resulted in significant (p<0.05) attenuation of the diarrhea scores (1.00±0.258) compared to model group. increasing the dose of carvone to (100mg/kg) resulted in significant (p<0.05) and more reduction in diarrhea scores (0.05±0.244) compared to model group. iraqi j pharm sci, vol.30(2) 2021 62 table 4. effect of carvone treatment on diarrhea score in irinotecan-induced intestinal mucositis. group treatment groups diarrhea score (mean ± sem) d4 d5 d6 d7 i normal saline 0 0 0 0 ii irinotecan 0 0.17±0.167 1.00±0 2.67±0.211 iii carvone 50mg/kg±irinotecan 0 0 .83±0.167 1.00±0.258 * iv carvone 100mg/kg±irinotecan 0 0 0 0.50±0.244 * (*): significant difference (p< 0.05). (d): days of treatment. discussion mucositis is referring to inflammatory or ulcerative lesions of the oral or gastrointestinal tract(1). not only it is associated with an adverse symptoms which include pain, vomiting and diarrhea but it may limit patients’ ability to tolerate treatment if not adequately prevented and managed(20). chemotherapy-induced mucositis being a principal reason for pain and reduced quality of life during cancer treatment (3). irinotecan induce intestinal mucositis and late onset diarrhea, the pathogenesis of this toxic side effect is that irinotecan and its active metabolite,sn-38, can cause direct damage to the intestinal mucosal cells, leading to dna damage and generation of reactive oxygen species due to oxidative stress (4) furthermore irinotecan can stimulate several signaling pathways, such as the (nf)-kb, increase the level of the inflammatory markers such as tumor necrosis factor (tnfα) and interleukin (il)-1β and elevate the expression of inflammation related factor such as prostaglandin e₂ (pge2)(21). in the present study, results showed a significant increase in tnfα serum level in mice confirmed irinotecan-induced intestinal mucositis mediated by inflammation. a previous study by (odília antunes fernandes, simone. 2018) confirmed that tnfα plays a crucial role in mucositis(22), moreover, this study showed significant decrease in mice body weight with increase in diarrhea score. this result confirmed by other study and explained by lowering in intestinal absorptive capacity lead to diarrheal episodes, fluid loss and body weight loss(23). tnfα serum level results showed significant decrease during the use of carvone at dose 100mg/kg rather than the other dose of carvone (50mg/kg), this effect may be due to increasing the dose, carvone has antioxidant and anti-inflammatory effect (24) leading to scavenging of ros, decrease oxidative stress, inhibition of pro-inflammatory cytokines and decrease inflammation. the successful reduction in serum tnfα in mice treated with carvone confirmed by significant reduction in mice body weight loss and diarrhea score, weight loss occur due to diarrhea and decreased food intake, carvone has antioxidant and ant-inflammatory effect leading to reduction in inflammation, mucositis and diarrhea. in addition to its antispasmodic effect (25) which leads to improvement of food intake and decrease diarrhea. conclusion the present study results conclude that carvone exerted a dose dependent anti-inflammatory and protective effect by attenuation irinotecaninduced intestinal mucositis. through reduction of inflammatory marker tnf-α and other observed parameters including weight loss and reduced diarrhea scores. references 1. peterson de, bensadoun rj, roila f. management of oral and gastrointestinal mucositis: esmo clinical practice guidelines. ann oncol. 2011;22(suppl. 6):78–84. 2. van sebille yza, stansborough r, wardill hr, bateman e, gibson rj, keefe dm. management of mucositis during chemotherapy: from pathophysiology to pragmatic therapeutics. curr oncol rep. 2015;17(11). 3. skinner r. nephrotoxicity of cancer treatment in children. ped health. 2010;4(5):519–38. 4. araújo rs, de barros alb. intestinal mucositis induced by chemotherapy: an overview. j mol pharm org process res. 2015;03(03):18–9. 5. benson ab, ajani ja, catalano rb, engelking c, kornblau sm, martenson ja, et al. recommended guidelines for the treatment of cancer treatment-induced diarrhea. j clin oncol. 2004;22(14):2918–26. 6. bao x, wu j, kim s, lorusso p, li j. pharmacometabolomics reveals irinotecan mechanism of action in cancer patients. j clin pharmacol. 2019;59(1):20–34. 7. silber jh, fridman m, smith m, simon r, therapy c, program e. cpt1-induced cholinergic effects in cancer patients iraqi j pharm sci, vol.30(2) 2021 63 ondansetron compatible with sodium acetate. 2019;3–4. 8. brandi g, dabard j, raibaud p, di battista m, bridonneau c, pisi am, et al. intestinal microflora and digestive toxicity of irinotecan in mice. clin cancer res. 2006;12(4):1299– 307. 9. stein a, voigt w, jordan k. review: chemotherapy-induced diarrhea: pathophysiology, frequency and guidelinebased management. ther adv med oncol. 2010;2(1):51–63. 10. snoussi m, noumi e, trabelsi n, flamini g, papetti a, de feo v. mentha spicata essential oil: chemical composition, antioxidant and antibacterial activities against planktonic and biofilm cultures of vibrio spp. strains. molecules. 2015;20(8):14402–24. 11. abdul jabbar aas, kathem sh. the protective effect of mentha spicata ethanolic extract on irinotecan-induced mucositis in mice. iraqi j pharm sci. 2019;28(1):37–43. 12. gonçalves jcr, alves a de mh, de araújo ae v., cruz js, araújo dam. distinct effects of carvone analogues on the isolated nerve of rats. eur j pharmacol. 2010;645(1–3):108–12. 13. gonçalves jcr, oliveira fds, benedito rb, de sousa dp, de almeida rn, de araújo dam. antinociceptive activity of (-)-carvone: evidence of association with decreased peripheral nerve excitability. biol pharm bull. 2008;31(5):1017–20. 14. ding x, chen h. anticancer effects of carvone in myeloma cells is mediated through the inhibition of p38 mapk signalling pathway, apoptosis induction and inhibition of cell invasion. j buon. 2018;23(3):747–51. 15. muruganathan u, srinivasan s. beneficial effect of carvone, a dietary monoterpene ameliorates hyperglycemia by regulating the key enzymes activities of carbohydrate metabolism in streptozotocin-induced diabetic rats. biomed pharmacother. 2016;84:1558–67. 16. muruganathan u, srinivasan s, indumathi d. antihyperglycemic effect of carvone: effect on the levels of glycoprotein components in streptozotocin-induced diabetic rats. j acute dis. 2013;2(4):310–5. 17. bastos ccc, ávila phm de, filho ex dos s, ávila ri de, batista ac, fonseca sg, et al. use of bidens pilosa l. (asteraceae) and curcuma longa l. (zingiberaceae) to treat intestinal mucositis in mice: toxico-pharmacological evaluations. toxicol reports. 2016;3:279–87. 18. sakai h, sagara a, matsumoto k, hasegawa s, sato k, nishizaki m, et al. 5-fluorouracil induces diarrhea with changes in the expression of inflammatory cytokines and aquaporins in mouse intestines. plos one. 2013;8(1):1–10. 19. zhang s, garcia-d’angeli a, brennan jp, huo q. predicting detection limits of enzyme-linked immunosorbent assay (elisa) and bioanalytical techniques in general. analyst. 2013;139(2):439–45. 20. sonis s, elting l, keefe d, nguyen h, grunberg s, randolph-jackson p, et al. unanticipated frequency and consequences of regimen-related diarrhea in patients being treated with radiation or chemoradiation regimens for cancers of the head and neck or lung. support care cancer. 2015;23(2):433–9. 21. ouyang m, luo z, zhang w, zhu d, lu y, wu j, et al. protective effect of curcumin against irinotecan-induced intestinal mucosal injury via attenuation of nf-κb activation, oxidative stress and endoplasmic reticulum stress. int j oncol. 2019;54(4):1376–86. 22. odília antunes fernandes s. repurposing the thalidomide to the treatment of irinotecaninduced intestinal mucositis: an old drug to a new use. glob j pharm pharm sci. 2018;6(2). 23. van vliet mj, harmsen hjm, de bont esjm, tissing wje. the role of intestinal microbiota in the development and severity of chemotherapy-induced mucositis. plos pathog. 2010;6(5):1–7. 24. nogoceke fp, barcaro imr, de sousa dp, andreatini r. antimanic-like effects of (r)-(-)carvone and (s)-(+)-carvone in mice. neurosci lett. 2016;619:43–8. 25. souza fvm, da rocha mb, de souza dp, marçal rm. carvone: antispasmodic effect and mode of action. fitoterapia. 2013;85(1):20–4. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(2) 2014 quality of life of diabetes 99 quality of life of patients with type ii diabetes mellitus in alhilla city-iraq haydar f. al tukmagi 1,* and miamin a. moussa ** * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** babil health directorate, ministry of health, babil, iraq. abstract diabetes mellitus is a global problem nowadays due to increase the disease cases all over the world, in both the developed and developing countries which may affect the quality of life (qol ) of diabetic patients. this study was conducted to assess the quality of life of patients with type 2 diabetes mellitus (dm) and to determine some selected clinical and sociodemographic factors that affect the quality of life of these patients in al hila city-iraq. this was a cross sectional study in which 100 patients with type 2 diabetes mellitus attending diabetic outpatient clinics of merjan teaching hospitalal hila. to assess the quality of life of those diabetic patients, the world health organizations quality of life assessment (whoqol) was applied, but in a short version questionnaire and abbreviated form called (whoqol-bref). concerning the results of this study, it was found that the patients responded fairly well on the questionnaire used, 39% had good score, 47% had fair score and 14% had poor score. for physical health domain 17% had poor score, for social domain 22% had poor score, for psychological domain 19% and environmental domain 18% had poor score. it was concluded from the results that although it supports previous reports in which qol of patients with dm were fairly good but this disease is significantly affects physical health, social relations and environment. furthermore, there is association with marked impairment in aspects of quality of life relating to mental health and psycho-social functioning and at least some aspects of physical health. given this, and given its high prevalence, greater attention is needed to watch dm as a public health problem. the fact that dm is “normative” should not be taken to infer that it is benign. keywords: diabetes mellitus, quality of life. العراق –النوع الثانً فً مذٌنت الحلت نمط حٍاة مرضى السكر التكمجًحٍذر فخري *،1 و مٍامن عبذ الرضا موسى ** .ثغذاد،ثغذاد،انؼشاق انصٍذنخ،جبيؼخ كهٍخ ،نخ انسشٌشٌخانصٍذ فشع* ** . ، انؼشاقثبثم ،ٔصاسح انصحخ ثبثم،دائشح صحخ الخالصة انزأثٍش ٌٔكًٍ ،يٍ انًًكٍ اػزجبس رضاٌذ حبالد االصبثّ ثًشض انسكشي ْزِ االٌبو كًشكهّ ػبنًٍّ ٌؼبًَ يُٓب انؼبنى اجًغ، انًجبل ٔخبصخ انًجبالد جًٍغ فً انحٍبح َٕػٍخ ٔجٕدح أسهٕة فً انسكشي يشض ٌحذثٓب انزً انزغٍشاد فً انًشض نٓزا األسبسً .ٔانؼبغفً االجزًبػً انجذًَ، ػهى رؤثش انزً ٔانسكبٍَخ ٔاالجزًبػٍخ انسشٌشٌخ انؼٕايم ٔرحذٌذ 2 انًُػ يٍ انسكشي نًشظى انحٍبح َٕػٍخ نزقٍٍى صًًذ ْزِ انذساسّ انؼٍبدح انخبسجٍخ نهسكشي فً يسزشفى يشجبٌ انزؼهًًٍ فً يشاجؼً يٍ يشٌط100 انذساسخ فقذ شًهذ .انًشظى نٓؤالء انحٍبح َٕػٍخ .انؼبنًٍخ انصحخ نًُظًخ اسزجٍبٌ اسزًبسح ثبسزخذاو انحٍبح َٕػٍخ ٔقًٍّذ يذٌُخ انحهّ نهُشبغ ثبنُسجخ .سدٌئخ %14 ٔ يزٕسطخ %47 ٔ جٍذح حٍبح َٕػٍخ ًٌهكٌٕ انًشظى يٍ %39 ػبيخ ثصٕسح أَّ انذساسخ خهصذ َزبئج نهٕظغ ثبنُسجخ أيب ،يٍ انًشظى % 22 سدٌئّ ػُذ كبَذ االجزًبػٍخ انؼالقبد ٔاٌ ،يٍ انًشظى% 17ػُذ سدٌئخ كبَذ انجذًَ .يٍ انًشظى % 18 سدٌئّ ثُسجخ ػُذْى انحٍبح َٕػٍخ ٔانجٍئً فكبَذ ,يُٓى %19سدٌئّ ػُذ كبَذفانُفسً رؼزجش ثهذَب فً انسكشي يشظىل انحٍبح َٕػٍخ يٍ خالل انُزبئج انزً رٕصهذ انٍٓب انذساسّ،، يٍ انًًكٍ اػزجبس ثبَّ ثبنشغى يٍ اٌ انجذٍَخ ٔاالجزًبػٍّ ٔانُفسٍخ ٔ انجٍئٍخ فقذ رأثشد ثشكم يهحٕظ فعال ػٍ رنك اسرجبغ انسكشي ثبنعشس انجبنغ فً أٔجّ انُبحٍخ نكٍ. جٍذِ َٔزٍجخ نزنك ٔالَزشبس . َٕػٍخ انحٍبح انًزؼهقخ ثبنصحخ انؼقهٍخ ٔانٕظبئف األجزًبػٍخ ٔانُفسٍخ ٔثذسجخ أقم األٔجّ انًزؼهقخ ثبنصحخ انجذٍَخ . ٌٕجت أٌ ٌؼطى اْزًبو أكجش نهسكشي ثأػزجبسِ يشكهخ صحخ ػبيخ ، انكجٍش فً انؼبنىي انسكشيشض .داء السكري ، نمط الحٍاة :الكلماث المفتاحٍت 1 corresponding author e-mail: h.tukmagi@hotmail.com received:24 /9/2014 accepted: 23/11/2014 iraqi j pharm sci, vol.23(2) 2014 quality of life of diabetes 100 introduction it is very clear nowadays that diabetes mellitus (dm) is one of the chronic noncommunicable diseases that can affects all world population whether developed or nondeveloped countries (1) many reports from world health organization (who) stated that the life style and quality of life of diabetic patients can strongly affected by the disease. diabetes mellitus is common both in the developed and the developing world. in iraq, according to the national chronic noncommunicable diseases risk factor survey done in 2006, the prevalence of dm is 10.4%. women seem to be at a greater risk for type ii dm; this may be due to enhanced sensitivity to a western lifestyle in certain ethnic groups. traditionally considered a disease of adults, type ii dm is increasingly diagnosed in children in parallel with rising obesity rates. type ii dm is now diagnosed as frequently as type i dm in teenagers in the united states (1-3) . in spite of lacking of diabetic patients’ assessment incorporating their perception of health status, psycho-social status and other aspects of life are affected by the disease. quality of life (qol) is defined by the who as “individuals' perceptions of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns” (4-7) . the significance of qol in diabetes can be addressed in the following perspectives. first is to supplement objective clinical or biological measures of disease. the subjective factors are especially important for people with dm because there is a significant difference in how clinicians, diabetes and the general public perceive the effect diabetes has upon qol (8) . second is to assess the need for health care. the researches have indicated that qol is a useful surrogate maker for a multitude of factors and can provides useful information of how well a patient is coping with diabetes overall (9) . third is to evaluate the effectiveness of interventions. because the overall goal for the treatment of all diabetes is to prevent acute and chronic complication, while preserving a good quality of life for the patient the true impact of a successful medical intervention can be understood in the way that the treatment has a positive influence on patients’ immediate and/or future well-being (10) . fourth is to predict the health o utcome. that is, in individuals with a variety of chronic diseases qol is known to be diminished and has been shown to predict long-term outcomes, disease progression, and response to therapy (11) . the diabetes-related changes may cause the disability in physiological, psychological and social function. at first, the mobility impairment and decline of activity of daily life (adl) related to diabetes complications and common co-morbidities have a significant impact on patients’ qol (1113) . it is already known that people with diabetes are much more likely to have a physical limitation than those without diabetes after controlling possible confounding factors and was one of the strongest direct predictors of recovery of mobility difficulty (14). it is recommended that use of both general and disease-specific measures of qol facilitates comparisons of findings across studies and disease treatment as well as providing insight into the mechanisms specific to diabetes self-management (11) . specifically, generic measures are necessary to compare outcomes across different populations and interventions. on the other hand, diseasespecific measures are more sensitive for the detection and quantification of small changes that are important to clinicians or patients (15) . patients and method this study was carried out at diabetic outpatient clinic of merjan teaching hospital . 100 patients enrolled in this cross sectional study which conducted to determine the quality of life of diabetic patients and to find the association between that quality and certain variables including (age, sex, occupation, residence, educational level, marital status, family history of diabetes and duration of diabetes). the study duration continued from june the 1st to september the 1st 2013. all patients visited the diabetic outpatient clinics of merjan teaching hospital during period of data collection .a sample of 100 hundred diabetic patients were collected during that period. during period of data collection, patients were interviewed by questionnaire from those visited the diabetic outpatient clinics of merjan teaching hospital. in addition to age ,sex ,occupation, residence, educational level ,marital status, family history of diabetes and duration of diabetes , the questionnaire consist of four domains including (physical health, psychological wellbeing , social relationship, environmental status) from them score was obtained to determine the quality of life . for each question there is a score and the median score is calculated in each domain. a score of mean ± sd on each domain is considered fair, iraqi j pharm sci, vol.23(2) 2014 quality of life of diabetes 101 a score of ˂ mean-1 sd is poor and a score of ˃ +1sd is good. the dependent variable for this study was the quality of life of diabetic patients while the independent variables of this study were including (age, sex, occupation, residence, educational level, marital status, family history of diabetes and duration of diabetes). statistical analysis was carried out using spss version 18. categorical variables were presented as frequencies and percentages. continuous variables were presented as (means ± sd). pearson’s chi square (x2) test was used to find the association between the categorical variables. analysis of variance ( anova) was used to compare means between two groups. a pvalue of ≤ 0.05 was considered as significant. results hundred consecutive attendees of the diabetes clinic met the inclusion criteria during the course of the study; 52 were females and 48 were males. for overall qol, 39 (39%) had good score,47 (47%) had a fair score and 14(14%) had poor score, table (1). table (1): relationship between qualities of life outcomes and sociodemographic variables. variables overall quality of life good % fair % poor % total % all patients 39 39% 47 47% 14 14% 100 100% sex female male 14 25 26.9 52 29 18 55.7 37.5 9 5 17.3 10.6 52 48 52% 48% marital status single married widowed divorced 1 32 4 2 33.3 45 18.1 50 1 32 12 2 33.3 45 54.5 50 1 7 6 0 33.3 0.098 27.2 0 3 71 22 4 3% 71% 22% 4% the qol score is high in all the domains except in environmental domain at which the majority of patients had fair to poor score and only (13) patients had good score, in the four domains the qol score is higher in males than in females, for example, in physical health domain more than half (27) patients from (42) patients who had good score were males and the remaining were females, table (2). table (2): relationship between quality of life outcomes and sociodemographic variables according to domains. variables domain 1 physical health domain 2 psychological well being domain 3 social relation ship domain 4 environmental good fair poor good fair poor good fair poor good fair poor sex female male 42 15 27 28 15 13 30 22 8 43 16 27 34 19 15 23 17 6 53 24 29 33 19 14 14 9 5 13 2 11 63 40 23 24 10 14 marital status single married widowed divorced 42 1 36 3 2 28 1 19 7 1 30 1 16 12 1 43 1 34 5 3 34 1 26 6 1 23 1 11 11 0 53 0 43 10 0 33 2 20 8 3 14 1 8 4 1 13 2 9 1 1 63 0 49 13 1 24 1 13 8 2 discussion the present study found that the overall perception of (hrqol) of patients with dm type 2 was affected by the disease. the result of this study showed that (43%) patients with dm experienced a fair qol. however this study has been agreed with the study that has been done in mosul (16) . this study it was seen that there is an increase in hrqol with increasing mean age of the patients; this is different to the results of mosul study (16) as well as another study in saudi arabia .this study showed that there is no effect of gender on qol, however this result was in agreement with other studies which iraqi j pharm sci, vol.23(2) 2014 quality of life of diabetes 102 showed that female highly associated with poor qol due to high prevalence of obesity in female patients (17-19) . duration of dm in this study has significant association with qol, good qol is related to duration of dm of less than 6 years due to short time for complication of dm to appear. however this finding was in agreement with mosul study but disagreement with a study in saudi arabia showed that the duration of illness had no significant effect on qol (17) . findings from another study from sweden showed that there is a strong relationship between the qol and the duration of disease in which patients suffering from the disease for more than 5 years had better qol (20) . although, those findings are related to good life style and the good treatment with best control. issa et al, showed in his study in 2006 that environmental domain is the only one that affected by the duration of the illness (18) . in the current study, it has been shown that means of physical health, social relation and environmental domains have been strongly differ by qol items; meanwhile there was no significant mean different of psychological wellbeing domain by qol items. the social domain which assesses personal relationships showed a high mean score in (hrqol) of our patients; this probably is due to a very large extent to high degree of satisfaction to the items of this domain. this finding however was consistent with mosul study as well as other study by awadalla et al who reported that there is no difference in the score of patients with diabetes in relation to general population on the social relationships domain (19) . it is worthy of note that patients with a good level of social support and had strong family care giver support system. it was found from previous studies and researches about the relation between qol and dm, that good psychological adjustment of the patients is depending on the family members supports (19) . our study showed that environment domain has been demonstrated that the majority of patients got fair score. however, this was in agreement with previous study , meanwhile differs from the finding of other studies (21,22) , which revealed that: the score of the environment domain was much lower than the other three domains and this could be due to bad environmental condition in these localities. finally, this was a cross-sectional study. conceivably, poor quality of life could be conducive to dm. although the direction of the observed associations cannot be determined on the basis of the present study, it may be noted that findings from prospective epidemiological studies strongly support the role of (whoqolbreef) in iraq, for predicting adverse health outcomes in diabetic patients. notable strengths of the current study were the recruitment of a large, general population sample of diabetic patient, widelyused measures of quality of life and the assessment of dm symptoms. conclusion the study is in agreement with other studies and reports that found in spite of affected physical health of diabetic patients, but still those diabetic patients are with fairly good qol. for further supporting results concerning the effect of dm type 2 on qol, it is highly recommended to do future studies on high number of iraqi diabetic patients. acknowledgement the author thanks university of baghdad for support and the al-hila directorate of health for technical aid. references 1. valentine u, odili lu, oparah a, et al. quality of life of people with diabetes in benin city as measured with whoqolbreef, the internet journal of law, healthcare and ethics, 2010; 6 (2): 1-11. 2. king h. global burden of diabetes 1995 2025: prevalence, numerical estimates and 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tolerance. zhonghnaya fang yi xue za zhi, 2001; 35(1): 21-29. 21. eljedi a, mikolajczyk rt, kraemer a, laaser u. health-related quality of life in diabetic patients and controls without diabetes in refugee camps in the gaza strip: a cross-sectional study bmc public health 2006; 6:268. 22. aghamollaei t, eftekhar h, shojaeizadeh d, et al. behavior, metabolic control and health-related quality of life in diabetic patients at bandar abbas diabetic clinic.iranian j publ health, 2003; 32: 54–59. iraqi j pharm sci, vol.30(2) 2021 factors impacting hematological markers during pregnancy doi: https://doi.org/10.31351/vol30iss2pp153-157 153 association of age, parity and body mass index with hemoglobin and serum ferritin levels in pregnant women in baghdad city mayada m. moustafa *, ali a. kasim*,1, rawaa dawood al-janabi** *department of clinical laboratory sciences, college of pharmacy, university of baghdad, iraq **baghdad teaching hospital, baghdad medical city, baghdad, iraq abstract hemogloin (hb) and serum ferritin levels are used to assess anemia in pregnancy. some studies referred to the influence of maternal age, body mass index (bmi) and parity on hb and serum ferritin levels. the study aimed to examine the possible association of maternal hb and serum ferritin with maternal age, parity, and bmi in a sample of pregnant women in baghdad. ninety healthy pregnant women, grouped in three equal groups according to the pregnancy trimester, and thirty apparently healthy non-pregnant women from baghdad were enrolled in this observational study. blood and serum samples were obtained for the estimation of hb and serum ferritin levels. the pooled data of participants showed a negative correlation between parity and each of blood hb concentrations (r= -0.147, p=0.046) and plasma ferritin levels (r= -0.186, p= 0.038). the negative correlation of parity with blood hb concentration was reported in participants in the third trimester of pregnancy (r= -0.270, p=0.048); and between parity and plasma ferritin levels in the second (r= -0.088, p= 0.046) and third (r= -0.398, p=0.029) trimester pregnant. the study did not report a significant correlation between age and bmi with blood hb concentrations or serum ferritin levels in pregnant women at any trimester of pregnancy. there is a negative correlation between parity and each of blood hb concentration and serum ferritin levels in pregnant women in baghdad. while, there is no such correlation with maternal age and bmi at any trimester of pregnancy. keywords: anemia, hemoglobin, ferritin, parity, pregnancy كتلة الجسم مع مستويات الهيموغلوبين والفيريتين في العمر وعدد مرات االنجاب ومؤشر ارتباط النساء الحوامل في مدينة بغدادالمصل لدى **داود الجنابي اءور و 1 *،علي عبد الحسين قاسم ،*مصطفى فق ومميادة فرع العلوم المختبرية السريرية ، كلية الصيدلة ،جامعة بغداد ،بغداد ، العراق * العراق . بغداد، الطب،مدينة التعليمي،مستشفى بغداد ** الخالصة أشارت بعض الدراسات إلى تأثير عمر األم ومؤشر كتلة . ومستويات الفيريتين في الدم لتقييم فقر الدم أثناء الحمل يستخدم الهيموغلوين هيموجلوبينال مستويات هدفت الدراسة إلى فحص االرتباط المحتمل بين. على مستويات الهيموغلوبين والفيريتين في الدم عدد مرات االنجابو الجسم .في مدينة بغداد ومؤشر كتلة الجسم في عينة من النساء الحوامل عدد مرات االنجابو العمرمع لحاملا للمرأةوالفيريتين الى ؛ اضافةثالث مجموعات متساوية وفقًا لثلث الحمل الى قسيمهنتم توتم تسجيل تسعين امرأة حامل يتمتعن بصحة جيدة، : والطرق المشاركين .نسب الهيموجلوبين والفيريتين حديدتم الحصول على عينات الدم والمصل لت. ه الدراسة القائمة على المالحظةثالثين امرأة غير حامل من بغداد في هذ ,r= -0.147)تراكيز الهيموغلوبين في الدم عدد مرات االنجاب و كل من بين عكسيةأظهرت البيانات المجمعة للمشاركين وجود عالقة p=0.046) مصلومستويات الفيريتين في ال(r= -0.186, p= 0.038). =r) في الثلث الثالث من الحمل حوامل اللواتي كنلدى التم مالحظته تركيز الهيموغلوبين في الدم و العكسي بين عدد مرات االنجاباالرتباط 0.270, p=0.048) ; الثلث الثاني الحوامل اللواتي كن في في المصلوفيريتين عدد مرات االنجابوبين(r= -0.088, p= 0.046) والثالث(r= -0.398, p=0.029) من الحمل. من ثلثالهيموغلوبين أو الفيريتين لدى النساء الحوامل في أي تركيز مؤشر كتلة الجسم مع واالدراسة وجود عالقة معنوية بين العمر سجللم ت .الحمل بينما ، . الفيريتين لدى النساء الحوامل في بغداد والهيموغلوبين مستوياتكل من و عدد مرات االنجاببين عكسيةتوجد عالقة ارتباطية من الحملثلث ال يوجد مثل هذا االرتباط مع عمر األم ومؤشر كتلة الجسم في أي .الحمل ،عدد مرات االنجاب فقر الدم ، الهيموغلوبين ، الفيريتين ، المفتاحية :الكلمات introduction globally, anemia during pregnancy represents one of the most prevalent public health problems, causing negative maternal and fetal health effects (1) .the world health organization (who) defines anemia in pregnancy as the condition where hemoglobin (hb) concentration is less than 11g/dl, at any trimester of pregnancy (2) . 1corresponding author e-mail: ali.qasem@copharm.uobaghdad.edu.iq received: 26/2/2021 accepted: 2/5 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp153-157 iraqi j pharm sci, vol.30(2) 2021 factors impacting hematological markers during pregnancy 154 while, according to the centers for disease control and prevention guidance, anemia in pregnancy is when hb concentration less than 11 g/dl at the first trimester and less than 10.5 g/dl in the second or third trimesters (3) . it is estimated that 56% of pregnant women in low and middle-income countries and more than 10% in high-income countries have anemia. (1) iron deficiency is the primary cause of anemia in about 75% of all anemia cases during pregnancy.(4) other causes that are prevalent in developing countries involve micronutrient insufficiencies such as folic acid, vitamin a and vitamin b12; some parasitic infections including malaria and hookworm; and chronic infections like tuberculosis and acquired immunodeficiency syndrome (5-7) . in pregnancy, without supplementary iron, hb concentration declines due to physiological hemodilution. serum ferritin is sensitive indicator of body iron stores, and it is considered as a reliable indicator of iron status in pregnant women. (8) at the beginning of pregnancy, serum ferritin concentrations vary more widely than corresponding hb values (9). some studies referred to the influence of maternal age, body mass index (bmi) and parity on hb and ferritin levels (10-12). the aim of the present work is to study the possible association of maternal hb and serum ferritin with maternal age, parity, and bmi in a sample of iraqi pregnant women. subjects and methods a cross-sectional study conducted at the antenatal clinic in baghdad teaching hospital in baghdad city, the capital of iraq, during the period extending from may to july 2019. ninety pregnant women, aged 18-45 years, were divided into three equal groups according to the trimester of pregnancy; and 30 apparently healthy non-pregnant married women to serve as control group, were enrolled in the study. participants with history of diseases or treatments that interfere with blood hb concentrations or serum ferritin levels were excluded from the study. data regarding participants’ age, bmi (calculated as weight in kilograms (kg) divided by height in meters squared (m2)) and parity, are collected and recorded using a data collection sheet designed for the purpose of the study. five ml blood samples were collected, 2 ml in edta tube for estimation of hb and 3 ml in plain tube for serum separation. the collected anticoagulated blood samples were used for hb concentrations measurement using an automated hematology analyzer. while, serum samples were stored at -20°c until the time of estimation of ferritin levels by enzyme-linked immuno-sorbent assay (elisa) test. ethical considerations the ethics committee of the college of pharmacy, university of baghdad approved the study. informed consent was obtained from each participant before participation in the study. statistical analysis statistical analysis was performed using spss® software version 22 for windows. data are presented mainly as mean values and standard deviation (sd) or frequencies and percentages. shapiro wilk test was used to check the distribution normality for the dependent variables, blood hb concentrations and serum ferritin levels. mannwhitney u test was conducted to examine differences between means of two groups, while kruskal-wallis h test was used to compare means among three or more groups. the pearson’s correlation coefficient (r) was used to evaluate the association between variables. a p value < 0.05 was considered statistically significant. results the present study showed a significant differences between the means of blood hb concentration and plasma ferritin levels of the pregnant and non-pregnant participants (p= 0.001 and p= 0.033; respectively). meanwhile there is no significant difference between the means of age, bmi and parity of the pregnant and non-pregnant participants (p≥0.05) (table 1). table 1. participants’ characteristics variable non-pregnant participants n=30 pregnant participants n=90 p-value age (year) 24.53 ± 6.16 26.06 ± 6.08 0.122 bmi (kg/m2) 23.02 ± 1.94 24.29 ± 1.97 0.050 parity 2.10 ± 1.27 2.32 ± 1.61 0.113 0 3 (10) 13 (14.4) 1 6 (20) 13 (14.4) 2 12 (40) 29 (32.2) 3 4 (13.3) 14 (15.6) ≥4 5 (16.7) 21 (23.4) hb (g/dl) 12.74 ± 1.64 10.84 ± 1.78 0.001 <11 4 (13.3) 47 (52.2) >11 26 (86.7) 43 (47.8) ferritin (ng/ml) 54.81 ± 19.23 36.86 ± 14.76 0.033 iraqi j pharm sci, vol.30(2) 2021 factors impacting hematological markers during pregnancy 155 comparison of participants’ characteristics by the trimester of pregnancy showed that there is significant differences among means of bmi (p< 0.001), blood hb concentrations (p< 0.001) and serum ferritin levels (p=0.029) of the studied groups (table 2). pairwise comparisons revealed that bmi mean of the participants in the third trimester of pregnancy is significantly higher than that of the non-pregnant or the first trimester pregnant participants. while, the mean of blood hb concentrations of the participants in the third trimester of pregnancy is significantly lower than that of the non-pregnant or the first trimester pregnant participants. moreover the mean of blood hb concentrations of the participants in the second trimester of pregnancy is significantly lower than that of the non-pregnant participants. finally, mean serum ferritin levels of the participants in the third trimester of pregnancy is significantly lower than that of the non-pregnant participants. table 2.participants’ characteristics by the trimester of pregnancy variable study groups p-value non-pregnant participants 1st trimester 2nd trimester 3rd trimester age (year) 24.53 ± 6.16 23.72 ± 5.42 26.91 ± 4.92 26.18 ± 6.14 0.480 bmi (kg/m2) 23.02 ± 1.94 23.75 ± 1.88 24.06 ± 1.93 25.07 ± 1.90a,b <0.001 parity 2.10 ± 1.27 2.70 ± 1.66 2.3 ± 1.47 1.93 ± 1.64 0.287 hb (g/dl) 12.74 ± 1.64 11.60 ± 1.78 10.87 ± 1.74a 10.06 ± 1.52a,b <0.001 ferritin (ng/ml) 54.81 ± 19.76 47.88 ± 14.41 34.99 ± 16.86 27.71 ± 10.84a 0.029 a significant difference from the non-pregnant participants b significant difference from participants in the first trimester of pregnancy correlation tests of the pooled data of pregnant participants of age, parity and bmi with blood hb concentrations and serum ferritin levels, showed that there is a negative correlation between parity and each of blood hb concentrations (r= 0.147, p=0.046) and plasma ferritin levels (r= 0.186, p= 0.038). other correlation pairs testing did not show a significant correlation (p>0.05) (table 3). correlation tests of age, parity and bmi with blood hb concentrations and serum ferritin levels of the study groups showed that there is a negative correlation between parity and blood hb concentrations in the third trimester pregnant (r= 0.270, p=0.048); and between parity and plasma ferritin levels in the second (r= -0.088, p= 0.046) and third (r= -0.398, p=0.029) trimester pregnant. other correlation pairs testing among the study groups did not show a significant correlation (p>0.05) (table 4). table 3. correlation tests of age, parity and bmi with blood hb concentrations and serum ferritin levels of the pregnant participants . correlated variables r p-value age vs. hb 0.195 0.062 age vs. ferritin 0.079 0.459 bmi vs. hb 0.134 0.198 bmi vs. ferritin 0.175 0.100 parity vs. hb -0.147 0.046 parity vs. ferritin -0.186 0.038 table 4. correlation tests of age, parity and bmi with blood hb concentrations and serum ferritin levels of the study groups. correlated variables control 1st trimester 2nd trimester 3rd trimester r p-value r p-value r p-value r p-value age vs. hb 0.065 0.735 0.045 0.815 0.382 0.104 0.310 0.095 age vs. ferritin 0.006 0.974 0.034 0.860 0.015 0.938 0.219 0.245 bmi vs. hb 0.286 0.125 0.275 0.141 0.077 0.685 0.267 0.153 bmi vs. ferritin 0.100 0.599 0.164 0.386 0.120 0.528 0.104 0.586 parity vs. hb -0.271 0.074 -0.039 0.083 -0.051 0.079 -0.270 0.048 parity vs. ferritin -0.039 0.084 -0.045 0.089 -0.088 0.046 -0.398 0.029 discussion the study showed that 52.2% (47 of 90) of the pregnant participants are anemic with mean hb concentration less than 11 gm/dl. the prevalence of anemia in pregnant women in the present study is higher than that reported by a recent study conducted in baghdad (33.8%) which was conducted on larger cohort (400 participants).(13) yet, the prevalence of anemia in pregnant women in low and middle-income countries was reported to be iraqi j pharm sci, vol.30(2) 2021 factors impacting hematological markers during pregnancy 156 56%. (1) the present study also showed that serum ferritin levels in pregnant women showed a significantly lower value than that of the nonpregnant. physiologic anemia occurring during a healthy pregnancy is attributed mainly to increase in the plasma volume without sufficient increase in red cells count. (14) in young adult women, the stored iron is roughly about one tenth of the iron incorporated in hemoglobin. (15, 16) during pregnancy, the red cell volume expands to about 24– 31% of the non-pregnant levels; (17, 18) this expansion of red cell volume results in depletion of the iron stores in most of pregnant women and hence serum ferritin levels. (19) on the other hand, the present study showed that 13.3% (4 of 30) of the nonpregnant participants have mean hb concentration less than 11 gm/dl. using the cut-off point of hb for diagnosis of anemia in non-pregnant women is 11.9 will escalate the percent of anemic non-pregnant participants to 23.3% (7 of 30). the prevalence of anemia in the non-pregnant group is high, but it is still slightly lower than that reported in iraq as of 2016 (29.1%). (20) hypervolemia and the increase in fetal weight may represent the main causes of the increase in bmi with the advancement in gestational age. the results showed a negative correlation between parity and each of blood hb concentration and serum ferritin levels, which suggests a reduction of iron store with increasing parity. the results of this study in this regard occur in accordance with many reports, (21, 22) and disagree with others that reported no association between high parity and anemia or low iron stores. (23-25) moreover, some studies reported a reduction in risk of anemia with high parity. (26, 27) increased risk of bleeding during pregnancy as well as during and after delivery may represent the link between anemia and low iron store and increasing parity. several mechanisms were proposed to explain the increased risk of bleeding with increased parity; yet, none of these mechanisms have been proven.(28) the study did not show any significant correlation between age and bmi with blood hb concentrations or serum ferritin levels in pregnant women at any trimester of pregnancy. this result do not agree with the results of other studies. for example, mocking et al. has reported a positive correlation between bmi in first trimester and blood hb concentrations in indonesian and ghanaian women;(29) while, ramussen et al. has reported a significant association of blood hb concentrations during pregnancy with maternal age, and with body mass index; with no correlation with serum ferritin levels (11) . conclusion anemia is very prevalent among pregnant women in baghdad. there is a negative correlation between parity and each of blood hb concentration and serum ferritin levels. while, there is no such correlation with maternal age and bmi at any trimester of pregnancy. the present study has some limitations. first, the causality between the correlated variables cannot be determined because of the nature of cross-sectional study. second, being a single center study on relatively small sample size, prevent the finding to be generalized on pregnant women in baghdad. multi-center and larger cohort study is recommended. references 1. black re, victora cg, walker sp, bhutta za, christian p, de onis m, et al. maternal and child undernutrition and overweight in low-income and middle-income countries. lancet. 2013;382(9890):427-51. 2. world health organization. iron deficiency anaemia: assessment, prevention and control: a guide for programme managers. 2001. 3. cdc. recommendations to prevent and control iron deficiency in the united states. mmwr recomm rep. 1998;47:1–29. 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in hematology. 2015;52:339–47. 20. world health organization. global health observatory data repository/world health statistics: http://apps.who.int/gho/data/node.main.1?lang =en (last accessed on jan, 2020) 21. rizk d, khalfan m, ezimokhai m. obstetric outcome in grand multipara in the united arab emirates. a case control study. archives of gynecology and obstetrics. 2001;264:194-8. 22. kumari as, badrinath p. extreme grandmultiparity: is it an obstetric risk factor? european journal of obstetrics & gynecology and reproductive biology. 2002;101(1):22-5. 23. toohey js, keegan ka, jr., morgan ma, francis j, task s, deveciana m. the "dangerous multipara" : fact or fiction? american journal of obstetrics & gynecology. 1995;172(2):683-6. 24. eugene m, abedinego o. grandmultiparity: is it really an independent predictor of adverse pregnancy outcomes? saudi journal for health sciences. 2017;6(2):77-82. 25. humphrey m. is grand multiparity an independent predictor of pregnancy risk? a retrospective observational study. the medical journal of australia. 2003;179:294-6. 26. king pa, duthie sj, ma hk. grand multiparity: a reappraisal of the risks. international journal of gynecology & obstetrics. 1991;36(1):13-6. 27. silva ljp. grand grand multiparity. journal of obstetrics and gynaecology. 1992;12(5):301-3. 28. aliyu m, jolly p, ehiri j. high parity and adverse birth outcomes: exploring the maze. birth (berkeley, calif). 2005;32:45-59. 29. mocking m, savitri ai, uiterwaal cspm, amelia d, antwi e, baharuddin m, et al. does body mass index early in pregnancy influence the risk of maternal anaemia? an observational study in indonesian and ghanaian women. bmc public health. 2018;18(1):873. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://apps.who.int/gho/data/node.main.1?lang=en http://apps.who.int/gho/data/node.main.1?lang=en http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 seroprevalence of cmv in women with bad obstetric history in babil/iraq doi: https://doi.org/10.31351/vol30iss2pp106-112 106 seroprevalence of cmv in women with bad obstetric history in babil/iraq maha diekan abbas*,1 and solomon egbe** * department of biotechnology, college of biotechnology, al-qasim green university, al-qasim, iraq. ** epidemiology department, edo state health management board, edo state, nigeria. abstract placental dysfunction and fetal central nervous system infestation caused by human cytomegalovirus (hcmv) is the leading cause of congenital non-genetic neuro-developmental problems of the newborn worldwide. although the highest rates of congenital infection and cmv seroprevalence occurs in developing countries like iraq, there remains a paucity of data from that part of the world. this descriptive case control study was undertaken in babylon/ iraq to determine the local seroprevalence of cmv in women of child bearing age and to identify the socio-demographic factors associated with it. this study found a seropositivity peak amongst the 26-35 yr olds which declined in the 36 – 45 yr olds. however, the evidence of current infection was stable at 25% among the 26-35 yr olds and the 36 – 45 yr old women. overall, seropositivity was at 77.32%, a susceptibility rate was at 22.68%, and seropositivity for igg was highest among the educated, those living in overcrowded settings, and those with poor obstetric histories. our study concludes that cmv screening of women in the al hamza district in babylon/iraq and the availability of advice on how to prevent the infection can be beneficial for health outcomes. keywords: cmv, cytomegalovirus, women, eliza, poor obstetric history, iraq, babil االنتشار المصلي للفيروس المضخم للخاليا لدى النساء ذوات تاريخ الوالدة السيئ في بابل / العراق **سليمان إغبي و 1* ، مها ديكان عباس .العراق ، القاسمقسم التقنيات االحيائيه، كلية التقنيات االحيائيه ، جامعة القاسم الخضراء ، * .قسم الوبائيات ، مجلس إدارة الصحة بوالية إيدو ، والية إيدو ، نيجيريا** الخالصة السبب الرئيسي (hcmv) المضخم للخاليا البشرييعد الخلل الوظيفي في المشيمة و / أو إصابة الجهاز العصبي المركزي للجنين بسبب الفيروس المصلي لمشاكل النمو العصبية الخلقية غير الوراثية للمواليد في جميع أنحاء العالم على الرغم من أن أعلى معدالت العدوى الخلقية واالنتشار البيانات من هذا الجزء من العالم. أجريت هذه الدراسة للفيروس المضخم للخاليا تحدث في البلدان النامية مثل العراق ، ال يزال هناك ندرة في في النساء في سن اإلنجاب ، وللتعرف على العوامل االجتماعية cmv الوصفية للشواهد في بابل / العراق لتحديد االنتشار المصلي المحلي لفيروس عاًما والتي انخفضت 35و 26شخاص الذين تتراوح أعمارهم بين والديموغرافية المرتبطة به. وجدت هذه الدراسة ذروة اإليجابية المصلية بين األ سنة. 45 36سنة والنساء بين 35-26٪ بين 25عاًما. ومع ذلك ، فإن األدلة على اإلصابة الحالية كانت مستقرة عند 45-36في الفئة العمرية كانت األعلى بين المتعلمين ، والذين igg ، واإليجابية المصلية لـ٪ 22.68٪ ، ومعدل الحساسية 77.32كانت اإليجابية المصلية اإلجمالية عند يعيشون في ظروف مزدحمة ، وأولئك الذين لديهم تاريخ توليدي ضعيف. خلصت دراستنا إلى أن فحص الفيروس المضخم للخاليا للنساء في منطقة .مكن أن يكون مفيدًا للنتائج الصحيةالحمزة في بابل / العراق وتوافر النصائح حول كيفية الوقاية من العدوى ي الكلمات المفتاحية: الفيروس المضخم للخاليا ، النساء ، اليزا ، تاريخ الوالدة الضعيف ، العراق ، بابل introduction worldwide, 0.1-0.2% of all pregnancies result in permanent disabilities of varying degree because of active hcmv infection. disabilities caused by hcmv in the newborn include blindness, deafness, microcephaly, cerebral palsy, intellectual disability, developmental delay, and in rare cases death (1). such disabilities may be lifelong and / or severe. hcmv is the leading cause of infectious (congenital) neurologic handicap in the newborn (2). globally, 0.5 – 1.1% of all pregnancies occur in women with active hcmv infection. some of these infections are a reawakening of dormant historic episodes. 90% of these pregnancies result in the birth of asymptomatic babies, however, 5-10% of babies who are asymptomatic at birth eventually die of hcmv related defects (3). 1corresponding author e-mail: mehaalzubaidy@yahoo.com received: 23/1/2021 accepted: 15/3 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp106-112 iraqi j pharm sci, vol.30(2) 2021 seroprevalence of cmv in women with bad obstetric history in babil/iraq 107 epidemiology globally, there is epidemiologic variation in hcmv infection in women of childbearing age; varying from 30.4% (in ireland) to 98.9% (in turkey) (4, 5). the seroprevalence rate in women with a bad obstetric history (boh) varies from 14.2% (iran) to 91.05% (india) (6, 7). amongst arab countries, seroprevalence in pregnant women varies from 77.8% (iraq) to 88% (jordan) (8, 9). for arab women with boh, it varies from 4.8% (iraq) to 95% (jordan) (10). study aims and objectives although there is an accumulation of information on hcmv seroprevalence worldwide, there is however little data informing the seroprevalence of hcmv local levels in iraq. the objective of the current study is to investigate the seroprevalence of cmv in women of child bearing age with a boh in the al hamza district in babylon/iraq and the socio-demographic variables which might affect seropositivity. materials and methods study design this is a descriptive case—control study. the total study population involves (n=115) women of childbearing age (15-45 years old). to ensure a good spread of the subjects, women with bad obstetric history were identified and recruited from the private labs and the primary healthcare centers situated in urban and rural areas in the al hamza district in babylon governorates. twent-five of the women were excluded as they did not meet the study criteria and had missing data. of the remaining (n=90) members of the study population, 54 (60%) had boh and considered as study group subjects while the 36 (40%) had average to good obstetric history and considered as control group subjects. collection of data members of the project team visited primary health care centers and the private labs in the region on a regular basis. possible candidates were identified, approached, interviewed and were suitable, included in the study population once informed consent and permission had been sought and obtained. four groups of patients were recruited. group 1 – pregnant women with average to good obstetric history. group 2pregnant women with bad obstetric history. group 3 – non-pregnant women with average to good obstetric history. group 4non-pregnant women with bad obstetric history. subjects were interviewed to exclude those whose boh was due to other causes e.g. uterine or cervical abnormalities (e.g. cervical incompetence), chromosomal abnormalities in either spouse, poorly controlled hyperthyroidism, diabetes mellitus, hypertension, hypothyroidism, renal disorders, a history of foetal chromosomal or genetic anomalies. collection of blood samples: blood samples were collected from january 2018 until december 2019. around 5—10 ml of blood was collected from vein for each case and placed in a sterile container with strict aseptic conditions. the serum was then separated and stored in aliquots at −20 ◦c until further processing. elisa essay serum samples collected from the study and control groups subjects were diagnosed for cmv igm and igg antibodies using cyto megalo virus elisa kits which are commercially available. the kit was purchased from calbiotech, cm028m and cm028g for igm and igg respectively. elisa test was performed according to the manufacturer’s instructions (calbiotech). the results were processed using a microwell elisa reader and read at an optical density 450 nm. data analysis using microsoft excel spreadsheet, the collected data were compiled, assembled and the odds ratios computed for suitable analysis. using chi squared test via spss (v.24), the association between categorical data was examined. accuracy, especially in instances of smaller sample sizes, was ensured using power analysis. association between variables was determined using bivariate regressionline analysis to compute odds ratio. cmv infection determinants were ascertained through regression line analysis and odds ratio, while standardising for confounding factors such as age, residential or educational status. results a total of (n=115) women were identified and pooled for this study. incomplete data were recorded for 25 of the women in the study sample. the women in the sample were categorized into further subgroups using different factors such as education level, pregnant or not pregnant, residential status (overcrowding or not overcrowded), bad obstetric history or normal obstetric history and whether or not they aborted (table 1). iraqi j pharm sci, vol.30(2) 2021 seroprevalence of cmv in women with bad obstetric history in babil/iraq 108 table 1. sample population with recorded versus missing igg and igm results data summary valid missing total n percent n percent n percen t sample population with recorded v missing cmv_igm result. 90 78.3% 25 21.7% 115 100% sample population with recorded v missing cmv_igg result. 90 78.3% 25 21.7% 115 100% of the 90 women whose data was complete, 34 were in the 15-25 yr old age group, 48 in the 26-35 yr old age group, and 8 in the 36 – 45 yr age group. 23 of the 34 (67%) of the 15-25 yr age group were positive for igg, 40 of the 48 (83%) of the 26-35 yr age group were positive for igg, and 6 of the 8 (75%) of the 36-45 yr age group were positive for igg (table 2). table 2. prevalence of cmv igg among different age groups for igm, 7 of the 34 (20.59%) of the 15-25 yr age group were positive for igm, 12 of the 48 (25%) of the 26-35 yr age group were positive for igm, and 2 of the 8 (25%) of the 36-45 yr age group were positive for igm (table 3). table 3. prevalence of cmv igm among different age group there were age disparities in the distribution of igg and igm seroprevalence among the study population. starting at 33.33% in the 15-25 yr old, igg seroprevalence peaked at 57.97% in the 26 35 yr olds before declining to 8.7% in the 36-45 yr old. a similar pattern is seen for igm starting at 33.33% amongst the 15-25 yr old, peaks at 57.14% in the 26-35 yr old and declining to 9.52% in the 36-45 yr old. age range cmv_igg total negative positive 15-25 yr old women 11.00 23.00 34.00 32.35% 67.65% 100.00% 26-35 yr old women 8.00 40.00 48.00 16.67% 83.33% 100.00% 36-45 yr old women 2.00 6.00 8.00 25.00% 75.00% 100.00% total 21.00 69.00 90.00 23.33% 76.67% 100.00% age range cmv_igm total negative positive 15-25 yr old women 27.00 7.00 34.00 79.41% 20.59% 100.00% 26-35 yr old women 36.00 12.00 48.00 75.00% 25.00% 100.00% 36-45 yr old women 6.00 2.00 8.00 75.00% 25.00% 100.00% total 69.00 21.00 90.00 76.67% 23.33% 100.00% iraqi j pharm sci, vol.30(2) 2021 seroprevalence of cmv in women with bad obstetric history in babil/iraq 109 table 4. seroprevalence of cmv igg and igm in regards to pregnancy, abortion, education, boh and residential status age groups boh pregnancy status residential status education status aborted 15-25 yr. old women 26-35 yr. old women 36-45 yr. old women normal pregnanc y boh not pregnant pregnant no overcrowding over crowding uneducated educated not aborted aborted cmv_igm negative 27.00 36.00 6.00 34.00 39.00 27.00 44.00 8.00 63.00 11.00 52.00 35.00 39.00 39.13% 52.17% 8.70% 46.58% 53.42% 38.03% 61.97% 11.27% 88.73% 17.46% 82.54% 47.3% 52.7% cmv_igm positive 7.00 12.00 2.00 5.00 18.00 13.00 9.00 1.00 20.00 2.00 19.00 5.00 18.00 33.33% 57.14% 9.52% 21.74% 78.26% 59.09% 40.91% 4.76% 95.24% 9.52% 90.48% 21.74% 78.26% pearson x2 value 0.23 4.47 3.04 0.78 0.76 0.65 p value >0.05 0.034 >0.05 >0.05 >0.05 >0.05 cmv_igg negative 11.00 8.00 2.00 15.00 6.00 11.00 10.00 2.00 20.00 4.00 13.00 16.00 6.00 52.38% 38.10% 9.52% 71.43% 28.57% 52.38% 47.62% 9.09% 90.91% 23.53% 76.47% 72.73% 27.27% cmv_igg positive 23.00 40.00 6.00 24.00 51.00 29.00 43.00 7.00 63.00 9.00 58.00 24.00 51.00 33.33% 57.97% 8.70% 32.00% 68.00% 40.28% 59.72% 10.00% 90.00% 13.43% 86.57% 32.00% 68.00% pearson x2 value 2.75 10.57 0.97 0.02 1.06 11.64 p value >0.05 0.001 >0.05 >0.05 >0.05 0.001 bold values indicated a significant difference iraqi j pharm sci, vol.30(2) 2021 seroprevalence of cmv in women with bad obstetric history in babil/iraq 110 overall, the seroprevalence of cmv within the study sample was 77.32%. igg levels were highest among those living in overcrowded situations (90%), those who had abortions (68%), and the educated (86.57%). interestingly, igg levels were highest (68%) among those with a poor obstetric history (study group), compared to 32% for those with moderate to good obstetric history (control group). evidence of current infection, i.e. positive igm levels was highest amongst those in overcrowded situations (95.24%), those with a poor obstetric history (78.26%), those who had abortions (78.26%), and the educated (90.48%). contrary to other studies the seroprevalence of igm amongst pregnant women (40.91%) was lower than in the women who were not pregnant (59.09%). the converse was true of igg levels being 59.72% (pregnant) and 40.28% (not pregnant) (table 4). the chi squared values and p-values indicate that x2 figures for women with a bad obstetric history, both for igg and igm was significant at a p value of below 0.05 at 95% confidence interval (ci). this was also true of those women with aborted fetus. this association was also confirmed by the odds ratio, the x2 boh * cmv_igm was 4.47 (p value = 0.034) while the x2 of boh * cmv_igg was 10.57 (p value=0.001) and the x2 of cmv_igg * abortion was 11.64 with (p value=0.001). there is however no significant correlation (p value>0.05) found between cmv_igg/ igm and residency, educational level, age group and pregnancy status. discussion according to the who (11), the seroprevalence of hcmv in women of child bearing age in the european region is 63-76%, this is lower than that in the east mediterranean region (88-95%), the region of the americas (69-87%), the west pacific region (86-94%), the african region (8294%), and the south east asian regions (82-94%) (12). other studies undertaken in other parts of iraq have found seroprevalence rates in the region of 95.7% in kirkuk (13). the overall seroprevalence of cmv within this study sample was 77.32%, this study found hcmv seroprevalence highest amongst 26-35 yr old women but declining with age. this study found a susceptibility rate of 22.68%, which is higher than the average for the country. the study found an association of current infection with abortion and boh (control group). other findings of this study include the higher rates of seroprevalence amongst the educated and among those living in overcrowded households. the higher seroprevalence amongst 26-35 yr old is aligned with previous studies which have attributed this to the exposure of these women to school age children, especially as their own children begin to attend school (14). the finding of higher rates amongst the educated and amongst those living in overcrowded households is also in alignment with previous studies (15). the overall prevalence of 77.32% and the susceptibility rate of 22.68% highlights the need for routine screening in antenatal clinics. this study shows that while the average rates of cmv seroprevalence in iraq may be over 95% (12), there are regional variations which require local adjustment of national policies on cmv infection prevention and management. scholars have argued against routine screening because of high levels of seroprevalence, cost, extensive viral strain diversification, and the inconsistent and ineffective treatment options available for hcmv (16). however, the arguments against routine screening, awareness promotions, prevention campaigns, and treatment may be predicated on a general underestimation or a lack of understanding of the enormity of the challenge posed by hcmv infection as a disease burden. hcmv is highly adapted to its human host, it is a member of the hsv group. primary infection with the virus results in an initial period of viral replication and shedding in body fluids including saliva, breast milk, urine, and in genital secretions. this is followed by a viraemia and, in some people, an infectious mononucleosis phase. finally, the host develops a generalized immune response involving the entire immune system. thereafter the infection enters a latent phase when viral levels are either low or even absent to detection but present in cd14+ and also in the cd33+ & 34+ cells of the mononuclear cells of the peripheral blood and bone marrow respectively. these sites then serve as reservoirs for future reactivation at opportune moments (17). this cycle perpetuated by the virus accounts for the delayed onset of symptoms in congenitally infected babies, the initial asymptomatic infection in many, and therefore, the presumption in many quarters that babies born to mothers with prior hcmv antibodies have normal outcomes. yet nothing could be further from the truth. the diversification of the virus creates a potential new threat to those who are already seropositive. the fact that the source of such diversity has been found to be endogenous makes those who are seropositive or living in a high seroprevalence environment particularly at risk (18). it is not surprising therefore, that the greatest burden of hcmv burden is found in developing countries where there is also a high level of seropositivity. high viral diversification continues to present challenges to the development of effective vaccines. a challenge which is amplified by the fact that large proportions of congenitally infected babies are the product of mothers with demonstrable hcmv antibodies (19). the consistent finding of an association between poorer socioeconomic conditions such as overcrowding, breast milk transmission and high hcmv seroprevalence offers an opportunity to iraqi j pharm sci, vol.30(2) 2021 seroprevalence of cmv in women with bad obstetric history in babil/iraq 111 address this problem while an effective vaccine is awaited (15). contrary to previous conception that higher seroprevalence confers immunity, the high viral diversity of hcmv predisposes the already seronegative women to reinfection with a different strain from the community or from endogenous viral reactivation. thus, higher seroprevalence may actually predispose to higher likelihood of infection. therefore, the higher seroprevalence of hcmv in the rural and/or urban areas of babylon should not occasion complacency to prevention and treatment but rather is the more reason for intervention. educating women and children about hcmv preventive measures is known to be effective (20). basic preventive measures like: teaching people to wash their hands regularly, catch their sneezes and body fluids and dispose of this in the appropriate way, avoiding the sharing of cutleries, plates and cups, educating mothers not to put babies’ pacifiers in their mouths if it has been in a child’s mouth, or advising mothers to avoid kissing children on the lips, have been shown to be feasible practices observable by mothers when adopted and effective in limiting the spread of infection. other measures include the introduction and reinforcement of these messages in antenatal clinics, children’s clinics, and hospitals using relevant staff holding classes and demonstrations with women and their husbands. there is also an opportunity to use billboards or posters in the hospital to adorn the walls of the waiting rooms or consulting rooms and wards where women are likely to see them. all such posters, classes, advice, and discussions should take place and employ cultural and language sensitive media, observing the complexities and subtleties of local customs and practices to drive home the message. conclusion this is the first study into the seroprevalence of hcmv amongst women of childbearing age in al-hamza district in babylon/iraq. the study found that a significant number of women were susceptible to hcmv infection, this finding contrasts the average of over 95% seroprevalence rate for the country. this study highlights the need, as the search for an effective vaccine and the debate over the cost benefit of routine screening continues, for seroprevalence data of hcmv in local populations to be researched and documented. this is because there is a need for age stratified, population based hcmv seroprevalence data in the planning of demographically appropriate modelling and strategic intervention programs for local hcmv infection in the al hamza district in babylon/ iraq. bridging 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infected infants. plos pathog. 2011; 7:e1001344. doi:10.1371/journal .ppat.1001344. 20. adler, s.p., and nigro, g. prevention of maternal-fetal transmission of cytomegalovirus. cmv transmission, cid. 2013; 2013:57 (suppl 4) • s189. . baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals doi: https://doi.org/10.31351/vol31iss1pp202-219 202 development of novel paracetamol/naproxen co-crystals for improvement in naproxen solubility amal f.al-dulaimi, *,1 myasar al-kotaji, ** and faris t. abachi * *department of pharmaceutical chemistry, college of pharmacy, university of mosul, mosul, iraq. ** department of pharmaceutics, college of pharmacy, ninevah university, mosul, iraq. abstract co-crystals are new solid forms of drugs that could resolve more than one problem associated with a drug’s formulation like solubility, stability, bioavailability, mechanical and tableting properties. this work aims to prepare multi-drug co-crystals consisting of paracetamol and naproxen to improve the solubility performance. a preliminary theoretical study for estimating the possible bonding between the co-crystal components (paracetamol and naproxen) was performed using the chemoffice program. the solvent evaporation method was used to prepare paracetamol/naproxen co-crystal in three different molar ratios. the characterization of the prepared co-crystals was performed by fourier transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, powder x-ray diffraction, and field emission scanning electron microscopy. in addition, a solubility study was conducted to compare the water solubility of pure paracetamol and naproxen with co-crystals solubility. the result of the theoretical bonding study revealed a high possibility for bonding between paracetamol and naproxen. the solvent evaporation technique was a successful method for the production of paracetamol/naproxen co-crystals in the three explored molar ratios 1:1, 2:1, and 1:2, which was proved by the different characterizing techniques. the solubility study exhibited an enhancement in naproxen solubility by more than two times in (1:1) and (1:2) paracetamol/naproxen co-crystals in addition to a little increase in paracetamol solubility. in conclusion, this work succeeded in the formation of new paracetamol/naproxen co-crystals, which can be considered as a new promising technique for the formulation of these two drugs with an obvious enhancement in crystallinity and naproxen solubility. this could be exploited in the preparation of tablets with possible improvement in dissolution and bioavailability. however, further work is needed to prove this assumption. keywords: co-crystal, powder x-ray diffraction, naproxen, solubility. ذوبانية النابروكسين نيلتحسجديدة من الباراسيتامول/نابروكسين مشتركة تطوير بلورات * ذنون العباجي فارس و **ميسر القوطجي 1، *أمل فخرالدين الدليمي موصل، العراق. جامعة الموصل، الصيدلة، الصيدالنية، كليةفرع الكيمياء * . العراق ، موصل نينوى، جامعة الصيدلة، كلية الصيدالنيات، فرع ** الخالصة من جديدة صلبة أشكال هي المشتركة الذوبان، البلورات مثل الدواء بتركيبة مرتبطة مشكلة من أكثر تحل أن يمكن التي األدوية . على الصياغة بشكل أقراصالقابلية واالستقرار، والتوافر البيولوجي، والخصائص الميكانيكية و تم إجراء دراسة .ذوبانيةن اليهدف هذا العمل إلى تحضير بلورات مشتركة متعددة األدوية تتكون من الباراسيتامول والنابروكسين لتحسي )الباراسيتامول والنابروكسين( باستخدام برنامج بين مكونات البلورة المشتركة لتقدير الترابط المحتمل . تم استخدام chemofficeنظرية أولية لبلورات المشتركة المحضرة عن ل شخيصتم إجراء ت مختلفة. والريةطريقة تبخير المذيبات لتحضير بلورات الباراسيتامول / نابروكسين بنسب م لمسحوق، والمجهر لطريق التحليل الطيفي باألشعة تحت الحمراء، والتحليل الحراري الوزني، والمسعرات التفاضلية، وانحراف األشعة السينية في الماء ينالنقي الباراسيتامول والنابروكسيناإللكتروني لمسح االنبعاث الميداني. باإلضافة إلى ذلك ، أجريت دراسة قابلية الذوبان لمقارنة ذوبان مع ذوبان البلورات المشتركة. 1corresponding author e-mail: amal.php1@student.uomosul.edu.iq received:9 /8/2021 accepted: 6/10/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp202-219 iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 203 والنابروكسين. كانت تقنية تبخير المذيبات طريقة ناجحة إلنتاج كشفت نتيجة دراسة الترابط النظري عن إمكانية عالية لالرتباط بين الباراسيتامول الطرق التشخيصية من خالل ثباتهاوالتي تمت ا ، 2: 1 و ، 1: 2، 1: 1 دروسةية المربلورات الباراسيتامول / نابروكسين في النسب الثالث الموال : 1)المحضرة بالنسب بلورات الباراسيتامول / نابروكسين ن مرتين فيزيادة في ذوبان النابروكسين بأكثر م يةأظهرت دراسة الذوبان .المتنوعة .باإلضافة إلى زيادة طفيفة في ذوبانية الباراسيتامول ( 2: 1( و )1 دة جديدة نجح في تكوين بلورات مشتركة جديدة من الباراسيتامول / نابروكسين ، والتي يمكن اعتبارها تقنية واع وبذلك يمكن االستنتاج أن هذا العمل مع تعزيز واضح في التبلور وذوبانية النابروكسين. يمكن استغالل ذلك في تحضير األقراص مع التحسن المحتمل في الذوبان نالدوائييلصياغة هذين إلثبات هذا االفتراض. بحاثوالتوافر البيولوجي. ومع ذلك ، هناك حاجة إلى مزيد من اال ية.نابروكسين، الذوبان كة، حيود األشعة السينية للمسحوق،الكلمات المفتاحية: البلورة المشتر introduction the discovery of new active pharmaceutical agents is the cornerstone in the drug industry. however, how to formulate these active pharmaceutical ingredients (apis) into effective dosage forms is the critical step. the selection of the proper formula that ensures a good drug delivery to the site of action inside the body is controlled by different factors, most of which are related to the drug physical and chemical properties (1). the most important physicochemical property of api is solubility, other properties could be related to lipophilicity, permeation, and pka (2). solubility is a physical property of a compound, it is defined as the amount of solute dissolved in a solvent at a certain temperature (3). about 40% of marketed drugs and about 90% of apis exhibit poor solubility, which ends up with low oral absorption and therefore a very low bioavailability; consequently a reduction in therapeutic effect (4). naproxen is a non-steroidal antiinflammatory drug (nsaid) that is classified as a class ii drug according to the biopharmaceutics classification system (bcs) because of its high membrane permeability and poor aqueous solubility. naproxen exhibits a variable bioavailability after oral administration due to its low solubility and/or poor dissolution in aqueous media. the key to improve the bioavailability of naproxen and produce a therapeutically effective dosage form is to design it in a more water-soluble form (5). different techniques were used to increase naproxen solubility including the use of a salt form of naproxen (naproxen sodium) (6), liquid-pellet formulation, (7) or incorporation of microcrystals naproxen into pulsincap device (5). some of these techniques improve solubility at a certain ph values only. however, the formulation of naproxen with a suitable hydrophilic co-former in the form of co-crystal could be an alternative technique to ensure that naproxen will have enhanced solubility across all ph ranges (8). paracetamol (acetaminophen) is one of the most commonly used drugs around the world as an analgesic and antipyretic. one of the problems with paracetamol tablet production is its poor compressibility. however, the change in paracetamol crystal structure by co-crystallization showed an enhancement in its mechanical properties. a new compressible form of paracetamol tablet with a good hardness was formulated by karki and his colleagues using the co-crystal engineering technique of paracetamol with four different co-formers (oxalic acid, naphthalene, theophylline, and phenazine) (9). paracetamol combination therapy with an nsaid as a single tablet had exhibited a superior pharmacological activity than nsaids alone (10). for instance, the formulation of low-doses of naproxen with paracetamol as a combination therapy to relief arthrosis pain demonstrated a better analgesic effect with a decrease in the incidence of toxicity and side effects which are accompanied with the treatment by high-doses of naproxen alone (11). co-crystal is a different solid phase of the compound. it contains two or more materials joined together by non-covalent bonds. the pharmaceutical co-crystal consists of api that binds non-covalently with either active pharmaceutical former and this is known as single-drug co-crystal, or the api is joined with another api and it is called multiple-drug cocrystal (12,13). the advantages of multi-drug co-crystal over the conventional method of combinational drugs production are the improvement in solubility, dissolution, stability, bioavailability, mechanical characteristics and tableting properties of both or at least one drug component in multi-drug co-crystal. in addition, co-crystal production exhibited other advantages including the simplicity, and low cost of production (14). the creation of co-crystal was facilitated by using some co-crystal design techniques like studying the possibility of hydrogen bond formation which is the most common non-covalent bond involved in co-crystal production (15). different methods are used in co-crystal manufacturing. solvent evaporation is the most common method for co-crystals production (16). it depends on the formation of a clear solution of cocrystal components in a certain solvent, and upon evaporation of the solvent, supersaturation will occur leading to an increase in the co-crystal formed in the mixture. this technique gives a good quality cocrystal suitable for single x-rays diffraction (17). this study aims to formulate a multi-drug co-crystal between paracetamol and naproxen, using solvent evaporation technique, to enhance the solubility of paracetamol and naproxen. iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 204 materials and methods materials paracetamol and naproxen are gifted from pioneer drug company/sulaymaniyah/iraq. absolute ethanol and methanol were purchased from scharlau, spain. methods co-crystal design a computational method for the prediction of non-covalent bond formation (hydrogen bond and van der waals forces) could help in co-crystal design. this computational method is performed by chemoffice program 2016 in order to predict the possibility of weak bonds formation between paracetamol as acceptor or donor and the selected nsaids (naproxen) as donor or acceptor (15). preparation of co-crystals and physical mixtures: the physical mixture was prepared as a control to the co-crystal by simple mixing of the two drug powders in molar ratios (1:1, 2:1, and 1:2) similar to that used in co-crystals preparation (18). the solvent evaporation method was used to prepare the multi-drugs co-crystals. the required amount of each drug was placed in a beaker to which 5 ml of ethanol was added, and the solution was stirred at 200-300 rpm using a hot plate magnetic stirrer (accuplatetm, labnet international, pc4200, mexico) until a clear solution was obtained. the solution was further heated (45-50°c) for 10 minutes, after which the resultant solution was poured into a petri dish and let it to dry at room temperature (19). the weight of all physical mixtures and prepared co-crystals with their molar ratios is illustrated in table (1). table 1. all prepared co-crystals and physical mixtures with their different molar ratios. preparation methods molar ratios number of moles of paracetamol (mol) the quantity of paracetamol (mg) number of moles of naproxen (mol) the quantity of naproxen (mg) symbol physical mixtures 1:1 0.001 151.2 0.001 230.3 c1 2:1 0.002 302.4 0.001 230.3 c2 1:2 0.001 151.2 0.002 460.6 c3 solvent evaporation mixtures 1:1 0.0005 75.6 0.0005 115.15 n1 2:1 0.001 151.2 0.0005 115.15 n2 1:2 0.0005 75.6 0.001 230.3 n3 co-crystal characterization methods a. fourier transform infrared spectroscopy (ftir) ftir spectra were obtained using the bruker-ftir apparatus (germany) over a range of 4000–500 cmˉ1. a small amount of the sample was put onto a platinum disk and 10.000–15.000 psi pressure was applied (20). b. thermo gravimetric analysis (tga) tga detects the changes that occur in the mass as a function of temperature. the instrument used for tga measurement was (mettler toledo tga/dsc1, switzerland). sample weighing (7-12) mg was placed in an aluminum pan and heated over a temperature range of 30-500°c. the rate of heating was increased by 10 ºc/min under a dry nitrogen atmosphere (with a flow rate of 50 ml/min). the analysis of tga/dsc data was achieved by using the stare software system (21). c. differential scanning calorimetry (dsc) differential scanning calorimetry (dsc) curves were obtained using shimadzu dsc plus 60 (japan) apparatus. a sample of 2 to 3 mg was sealed in a flat bottomed aluminum pan and heated at 100 ml/min nitrogen flow rate. the thermal behavior of the sample was evaluated at 10˚c /min scanning rate and temperature increased up to 500˚c (22). d. powder x-ray diffraction (pxrd) pxrd is measured by diffractometer (dx2700bh, haoyuan instrument co., ltd, china) with cu kα radiation source at 40 kv and 30 ma and the 2θ value ranging from (0°-80°). the scanning rate was 4°/min (23). the obtained results were analyzed by using the computer program originpro 2021b (learning edition, origin lab, usa). e. field emission scanning electron microscopy (fesem): the morphology of the prepared co-crystal and its physical mixture was observed by using field emission scanning electron microscopy (fesem) from zeiss sigma, germany 300hv. the scanned sample was loaded on the aluminum stamp and fixed by using carbon adhesive tape. after that, it was covered with a thin layer of gold and observed at various magnification powers by the a high resolution field-emission scanning electron microscope (24) saturation solubility study solubility study was performed by using a shaking water bath (stuart scientific, sbs30, uk). excess amounts of pure paracetamol, naproxen, and the prepared co-crystals were placed in conical flasks and 10 ml distilled water was added to each flask to iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 205 obtain a supersaturated solution. these solutions were allowed to stand at 37 ± 1˚c for 24 hours in a water bath shaker to achieve a complete saturation (25). after that, each solution was filtered by a 0.45 μm membrane filter, diluted by suitable diluent (15 methanol: 85 water) for paracetamol and absolute methanol for naproxen and analyzed by shimadzu uv-spectrophotometer.the determination of the content is carried out by simultaneous technique by ashour et al 2015 at 242 nm and 331 nm wavelengths for paracetamol and naproxen, respectively (26,27). statistical analysis the values of the solubility study were expressed as a mean ± standard deviation (sd). statistical analysis was performed by applying a oneway anova test followed by the tukey test. the difference is considered significant when the p value is ≤ 0.05. results and discussion co-crystal design the co-crystal design was dependent on molecular mechanics to theoretically calculate the strength and energy of bond formation between paracetamol and the selected nsaid (naproxen). molecular mechanics was used to assuming the total bonding energy between two molecules which came from the summation of different energies required for stretching, bending, stretch-bending, torsional, electrostatic, van der waals, and non-van der waals attractions (28). in general, the lower total energy required for bonding indicates a more stable and easier bond formation between the two molecules. theoretical calculation of the total energy of bond formation between (paracetamol-naproxen) was 26.5571 kcal/mole which could be considered as low total energy for bond formation, as demonstrated in figure (1a). all possible hydrogen bonds between paracetamol and naproxen are illustrated in figure (1 b). in addition to intermolecular hydrogen bonds, other weak interactions like van der waals forces may be involved in (paracetamol-naproxen) cocrystal formation. figure 1. molecular mechanics study of (paracetamol-naproxen) bonding. a)theorotical calculation of (paracetamol-naproxen) bonding energy. b) 3-d structure of possible bonding between paracetamol and naproxen. where the gray balls are carbon atoms, the white balls are hydrogen atoms, the red balls are oxygen atoms, and the blue ball is nitrogen atom. the dotted lines represented the hydrogen bonds between the two drugs. the results of the molecular mechanics theory indicated a higher possibility for bonding and cocrystal formation between paracetamol and naproxen drugs. praparation of co-crystals and physical mixtures the solvent evaporation technique was used for co-crystals production. ethanol was used as a solvent since it is non-toxic in low doses, suitable for oral preparations, and evaporates easily. moreover, both paracetamol and naproxen have a good solubility in it (29). the products of the solvent evaporation method were white, needle-like structures. co-crystal characterization methods a. fourier transform infrared spectroscopy (ftir) ftir is a useful tool in confirming the presence of new solid forms like co-crystal. the hydrogen bonding causes a change in stretching frequencies and vibrational bending, leading to the shift of the band to different wavenumbers and increase the width of the band (30). the important iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 206 functional groups of paracetamol that could participate in intermolecular bonding in the ir spectrum includes phenolic o–h stretching at (3315.60) cm-1, n–h stretching at (3152.62) cm-1, and amide n–c=o stretching at (1647.24) cm-1, as represented in figure (2). while for naproxen, the ir spectrum includes carboxylic o–h stretching at (3108.57) cm-1, carboxylic acid ho–c=o stretching at (1717.44) cm-1, and methoxy ch3-o-c stretching at (1167.10) cm-1, as illustrated in figure (3). the c–h stretching whether aromatic or aliphatic in paracetamol was between (2700 and 2800) cm-1, in comparison with (2965.94) cm-1 in naproxen. figure 2. ftir spectrum of paracetamol. figure 3. ftir spectrum of naproxen. regarding the prepared 1:1 co-crystal mixture (n1), there were significant changes in the ir spectrum from its parent drugs and physical mixture (c1). the appearance of o-h stretching at wavenumber (between 3350 and 3400) cm-1 in the ir spectrum of n1 (figure 4b) in comparison with (3315.60) cm-1 in pure paracetamol could indicate its participation in hydrogen bond formation. in the physical mixture c1 (figure 4 a), the o-h stretching did not appear in the ir spectrum, this may be due to the overlapping with the n-h bands. also, the shifting in n-h stretching region to (3157.83) cm-1 and the peak became sharper with the shifting of methoxy group stretching from (2965.09) cm -1 wavenumber to (2941.51) cm -1 may indicate the presence of intermolecular bonding. the formation of new band at (1555.20) cm-1 could be related to (n-c=o) bending. all of these changes with the increase of the tone between (19002400) cm-1 could indicate the formation of a hydrogen bond between paracetamol and naproxen in n1(31). iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 207 figure 4. ftir spectrum of 1:1 paracetamol/ naproxen mixtures. a) c1 physical mixture. b) n1 prepared co-crystal mixture. the 2:1 prepared co-crystal mixture (n2) had changes in ir spectrum in comparison with its physical mixture (c2), as demonstrated in figure5 (a and b), there was the shifting of o-h stretching to (3313.06) cm-1 and n-h stretching from (3148.34) to (3153.79) cm-1. also, there was the formation of new bands at (3103.49, 2600.99, 1796.36, and 1323.87) cm-1 in n2. all of these changes could indicate the presence of intermolecular bonding in n2. a b iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 208 figure 5. ftir spectrum of 2:1 paracetamol/ naproxen mixtures.a)c2 physical mixture. b) n2 prepared co-crystal mixture. regarding n3, which represents the (1:2) ratio of paracetamol-naproxen prepared co-crystal mixture, there was a shifting in n-h stretching from 3145.43 cm-1 to 3151.49 cm-1 and the band became broad and all of the bands had moved to higher absorbing transmittance. the n3 was also different from its physical mixture (c3) by the shifting of the (n-c=o) band from (1675.92) to (1594.78) cm-1 and the band became wider. in addition, there was the shifting of (c-o) stretching related to the amide and carboxylic acid in (1499.02) and (1219.99) cm1 wavenumbers to (1458.86) and (1170.29) cm1, respectively, as indicated in figure 6 (a and b). this could indicate the participation of the carboxylic acid of naproxen with the amidic (nc=o) in paracetamol to form a hydrogen bond. . b a iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 209 figure 6. ftir spectrum of 1:2 paracetamol/ naproxen mixtures. a) c3 physical mixture. b) n3 prepared co-crystal mixture. the interpretation of ir results indicated the presence of bonding between paracetamol and naproxen in all three ratios of the co-crystal mixtures prepared by solvent evaporation method. however, further diagnostic techniques are needed to confirm co-crystals formation. b. thermo gravimetric analysis (tga) tga measurements of the pure drugs and the prepared co-crystal mixtures approved that there was no entrapped water inside the materials (no solvate or hydrate form) because there is no mass loss occurred before 200°c. for paracetamol, the onset of mass loss began at 300°c. the paracetamol loses 50% of its mass by reaching ≈ 354°c and only 10% of the paracetamol mass will remain intact at about 375°c, as shown in figure (7 a). while in pure naproxen, the initial decomposition temperature (idt) was ≈ 237°c and the temperature at which 50 % of the mass was lost or decomposed (t 50%) was at 300°c, as demonstrated in figure (7 b). a b iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 210 figure 7. tga of pure paracetamol (a) and tga of pure naproxen (b). for the prepared paracetamol-naproxen co-crystals, there were changes in their tga measurements in comparison with their physical mixtures and parent drugs, like n3 (1:2 paracetamol-naproxen co-crystal mixture) when compared with the physical mixture (c3). in n3, the idt was 250°c, as in figure (8 a), in comparison with 240°c for c3. the mass loss in n3 required a higher temperature (310°c) to lose 50% of its mass while in c3, 50% of its mass was lost when the temperature reached 290°c, as demonstrated in figure (8 b). differences between the prepared co-crystals and their parent drugs were observed in n1 and n2. the idt of n1 and n2 were 250°c and 257°c, respectively. while the temperature at which the co-crystal lost 50% of its mass was 365°c for n1 and about 330°c for n2, as demonstrated in figure 9 (a and b). the tga of the prepared co-crystals gave an indication about their differences from their parent drugs paracetamol and naproxen, as their decomposition temperature was higher than naproxen and lower than paracetamol. while the physical mixture c3 had, approximately, similar tga profile to the naproxen alone (figure 7 b). tga measurements could exclude the formation of hydrate or solvate solid phase in the prepared mixtures, so this confirms that the products of the solvent evaporation method are co-crystals. a b iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 211 figure 8.tga of n3 co-crystal (a) and tga of (c3) physical mixture (b). figure 9. tga of n1 co-crystal (a) and tga of n2 co-crystal (b). b a iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 212 c. differential scanning calorimetry (dsc) the dsc measurements of paracetamolnaproxen prepared co-crystals were compared with the dsc analysis of pure drugs and their physical mixtures. the pure drug exhibited a single sharp peak around its melting point and a second broad peak appeared at a higher temperature which represented the decomposition of the melted drug, and it matches with the tga recorded idt. for paracetamol, the sharp melting peak value was 175.10°c (figure 10 a), while in naproxen the peak value equals 160.46°c, as illustrated in figure (10 b). the idt of paracetamol in the dsc was around 300°c, and for naproxen, the decomposition process was completed before the sample reaching 300°c. in both cases, the dsc results gave a similar decomposition profile to the tga measurements of paracetamol and naproxen, as in figure (7 a) and (7 b), respectively. figure 10. dsc of paracetamol (a) and naproxen (b). the n1, n2, and n3 paracetamol-naproxen cocrystals exhibited a single sharp endothermic peak in the dsc measurement for each mixture. the n1 cocrystal exhibited a peak at 147.41°c (figure 11 a) while in the n2 and n3 co-crystals the peak was formed at 145.45°c and 151.70°c, respectively as illustrated in figures (11 b) and (11 c). the dsc analysis of these three co-crystals showed distinctly different thermal profiles from the individual parent drugs and their physical mixture. this could indicate the formation of co-crystals with the three different ratios. figure 11. dsc of n1 (a), n2 (b), iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 213 figure 11. dsc of n3 (c) co-crystals and physical mixture c1 (d). the physical mixture (c1) showed the dsc profile of the eutectic mixture. the eutectic mixture is produced when the components of the mixture start to melt at a lower temperature than the melting points of their starting material (32). c1 physical mixture demonstrates a single sharp peak on dsc thermal analysis at 144.77°c, as indicated in figure (11 d), which might be attributed to the formation of the eutectic mixture. however, this peak appears at a different temperature from all three prepared cocrystal mixtures (n1, n2, and n3), and this confirms that these three products are co-crystals rather than simple physical mixtures. d.powder x-ray diffraction (pxrd): pxrd is used to confirm the formation of a new solid phase when there is a variation in the xrd patterns from the parent materials. pxrd was measured by diffractometer and the obtained results were analyzed using the computer program originpro 2021b. the characteristic peaks of pure paracetamol at 2 theta° of the pxrd pattern (figure 12) are 12.3°, 15.7°, 18.2°, 20.5°, 23.6°, 24.5°, 26.7°, 32.8°, and 37.0°. on the other hand, naproxen characteristic peaks at 2 theta° (figure 12 ) are: 6.8°, 12.8°, 13.6°, 17.0°, 19.2°, 20.2°, 22.8°, 24.0°, 27.5°, 28.0°, 28.7°, and 30.1°. figure 12.x-ray powder diffraction pattern of pure paracetamol and pure naproxen. the (1:1) paracetamol-naproxen co-crystal (n1) exhibited a completely different xrd pattern from their pure drug components. the characteristic peaks at 2 theta° values are 6.6°, 12.4°, 13.5°, 16.6°, 18.8°, 19.7°, 22.1°, 23.2°, 23.7°, 24.1°, 28.2°, 32.2°, 33.4°, 37.3°, 40.4°, and 45.3°, as shown in figure 13. iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 214 figure 13.x-ray powder diffraction pattern of n1. the characteristic peaks of the (2:1) paracetamol-naproxen co-crystal (n2) were at the following 2 theta° values: 6.4°, 13.6°, 15.4°, 16.6°, 18.8°, 19.7°, 20.2°, 22.0°, 23.3°, 23.7°, 24.1°, 26.4°, 26.2°, 28.2°, 31.1°, 33.4°,40.4°, and 45.3°, as illustrated in figure 14. figure 14. x-ray powder diffraction pattern of n2. the (1:2) paracetamol-naproxen co-crystal (n3) exhibited the following characteristic peaks: 6.6°, 12.5°, 13.6°, 16.7°, 17.9°, 18.8°, 19.7°, 22.3°, 23.8°, 26.4°, 27.2°, 27.7°, 28.4°, 31.1°, 33.5°, 40.5°, and 45.5°, as demonstrated in figure 15. iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 215 figure 15. x-ray powder diffraction pattern of n3. besides the different pxrd characterization peaks of n1, n2, and n3, there were differences in the 2 theta° value that gave the higher intensity peak in comparison with paracetamol and naproxen. in n1 the higher intensity was equal to 3423, which is correspondent with the 2 theta° value of 12.4°, while in n2 and n3 the highest intensities were 3362 (2 theta°= 22°) and 5710 (2 theta° =12.5°), respectively. these different xrd patterns indicate the presence of different solid phases in n1, n2, and n3 in comparison with their pure drugs. this is in accordance with the work of abbas et al (33), who applied a similar pxrd explanation in confirming the formation of naproxen/ nicotinamide co-crystal. the pxrd results confirmed the previous conclusions obtained from ftir, tga, and dsc data, which indicated the formation of paracetamolnaproxen co-crystals when prepared by using the solvent evaporation method in the three ratios 1:1 (n1), 2:1 (n2), and 1:2 (n3). e. field emission scanning electron microscopy (fesem) the fesem was used to indicate the differences in the morphology and surface shape between the co-crystal and its physical mixture. the n1 co-crystal fesem image had been displayed as an example to confirm the difference between the prepared co-crystal (figure 16, upper row) and its physical mixture c1 (figure 16, lower row). both fesem images at 1 micrometer and 2 micrometers scale manifested a high crystallinity of n1 cocrystal with the improvement that occurred in the shape of co-crystal when compared with c1. these morphological changes may lead to the enhancement of the flowability and improvement in poor tableting properties (24). iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 216 images of n1 co-crystals figure 16. fesem images of n1 co-crystals (upper row) and fesem images of c1 physical mixture (lower row). saturation solubility study saturated solubility study was conducted to compare the solubility of n1 (1:1), n2 (2:1) and n3 (1:2) paracetamol-naproxen co-crystals with the saturated solubility of their parent drugs (paracetamol and naproxen). paracetamol water solubility as a pure powder was found to be (13.104 ± 0.952) mg/ml at 37 ± 1˚c and this result was close to the paracetamol solubility recorded in literature which is around 17 mg/ml (34). the three co-crystals (n1, n2, and n3) showed a higher saturated solubility of paracetamol when compared with the solubility of pure paracetamol, as demonstrated in figure 17. however, all those increases in saturated solubility of the paracetamol component of the three co-crystals are considered statistically non-significant (p-value is more than 0.05). figure 17. solubility of pure paracetamol and its co-crystals n1, n2 and n3; all values are expressed as mean ± sd. in general, naproxen solubility in water is very low. pure naproxen saturated solubility was found to be (189.129 ±12.548) µg/ml at 37 ± 1˚c and this result is comparable to the recorded solubility in the scientific literature (35). all three co-crystals n1, n2, and n3 exhibited a significant enhancement in naproxen water solubility in comparison with water solubility of pure naproxen, as demonstrated in figure 18. iraqi j pharm sci, vol.31(1) 2022 paracetamol/naproxen co-crystals 217 figure 18. solubility of pure naproxen and its co-crystals n1, n2 and n3; all values are expressed as mean ± sd. the highest solubility was observed in n3 (1:2) paracetamol-naproxen co-crystal with (450.398±1.205) µg/ml. n1 (1:1) paracetamolnaproxen co-crystal demonstrated the secondhighest solubility (421.989±1.206) µg/ml, while n2 (2:1) paracetamol-naproxen co-crystal had the lowest solubility (293.295±16.877) µg/ml. the increase in saturated solubility in all the three cocrystals (n1, n2, and n3) is considered statistically significant (p-value ≤ 0.05). the increase in paracetamol and naproxen solubility, when they were formulated in co-crystals form, might be due to the higher co-crystallization lattice energy which leads to an increase in solubility. in addition, the decrease that occurs in melting points (thermal stability) of the co-crystals in comparison with both parent materials (paracetamol and naproxen) as confirmed by the dsc analysis may contribute to the enhancement of solubility in co-crystals. the increase in solubility was more obvious in the naproxen case due to it is very low water-saturated solubility. naproxen solubility was increased by 2.2, 1.6, and 2.38 times in the case of n1, n2, and n3 co-crystals, respectively, when compared with pure naproxen solubility. this may be attributed to the co-crystal high energy that made co-crystal behave in a similar manner to amorphous form when dissolved in water by maintaining a supersaturated state which leads to giving a higher saturated solubility in comparison with their starting materials (36,37). the improvement in naproxen solubility by co-crystal formation was reported in some previous researches, such as the naproxennicotinamide co-crystal (33). however, to our best knowledge, no work on naproxen multi-drug cocrystals was conducted before. conclusion multi-drug co-crystals formation is a simple technology to produce a new physical form of drugs with improved drug properties such as solubility without deteriorating the chemical properties or the pharmacological action. this work concluded that the production of paracetamol: naproxen by solvent evaporation method was feasible and demonstrated enhanced naproxen solubility with possible improved mechanical properties which enabled these co-crystals to be formulated as tablet dosage forms in the future. acknowledgment the researchers would like to express their thanks to the \ college of 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evaluate the toxicity of a substance. rutin is one of the flavonoid compounds with a variety of pharmacological effects. the aim of the study is to calculate the lethal dose that affect fifty percent of the mice used in the experiment (ld50). thirty swiss albino male and 30 non-pregnant female mice have been divided equally and randomly into 5 treated groups and one control group (n=5) rutin has been administered with concentrations 5, 2.5.1.25,0.625 and 0.312 g/kg administered as a single dose intraperitoneally (ip) while the control group received 1% dmso (ip). animals were observed for any morbidity and mortality for 14 days. after 14 days the animal blood collected for biochemical and hematological analysis then all animals are euthanized for histopathological evaluation. the results showed the ld50 was 1.51 g/kg for male mice while for female mice was1.49 g/kg. no significant changes were observed at dose of 1.25glkg (female) and 0.625, 0.312 glkg (both sexes) in body weight measurements and in biochemical or hematological assays. moreover no significant histopathological changes were reported compared to control.. it can be concluded that rutin is practically a non-toxic substance. keywords: acute toxicity, rutin, ld50%, intraperitoneally, histopathology, dmso, biochemical and hematological assay. السمية الحادة للروتين في الفئراندراسة 1*،حيدر بهاء صاحب و * زينة محمد حامد . وزارة الصحة والبيئة، دائرة مدينة الطب، بغداد، العراق* اقفرع االدوية والسموم، كلية الصيدلة، جامعة بغداد، العر** الخالصة أحد مركبات الفالفونويد ذات التأثيرات الدوائية المتنوعة ، وتهدف الدراسة تعد السمية الحادة خطوة لتقييم سمية مادة ما ، ويعتبر الروتين اناث غير حوامل بالتساوي 30ذكر و 30٪ وتقييم سمية الروتين في الفئران. تم تقسيم الفئران البيضاء السويسرية 50إلى حساب الجرعة المميتة جم / كجم جرعه واحده ، 0.312و 5،2.5.1.25،0.625( بتركيز 5ة )ن = مجموعات ُمعالجة ومجموعة سيطرة واحد 5وبشكل عشوائي إلى يوم يتم جمع الدم الجراء التحليالت الباوكيمائيه والدمويه ومن ثم يتم 1٤يوم. بعد ال 1٤داخل الصفاق ثم التغيرات السريريه والوفيات تسجل لمده ld50% 1.51 امراض انسجه. كانت النتيجة لذكور الفئران البيضاء السويسرية قتل جميع الحيوانات لجمع االعظاء الحيويه وارسالها الختصاص جم / كجم ووزن الجسم والمقايسة البيوكيميائية والدمية ، أظهر الفحص التشريحي المرضي أنه عند ld50%1.49 جم / كجم بينما كانت لالناث مع مجموعة التحكم. لذلك ، يعتبر (p˃0.05) الجنسين ال يوجد فرق معنويجم / كجم لكال 0.312، 0.625جم / كجم )لإلناث( و 1.25الجرعة .نجم / كجم لكال الجنسي 0.312الروتين مادة غير سامة عمليًا مع أقل تأثير ضار ملحوظ بجرعة . dmsoفحص الكيمياء الحيوية والدم ، داخل الصقاق ، التشريح المرضي ، ld50%السمية الحادة ، روتين الكلمات المفتاحية : introduction in last years, compounds that are derived from herbal or natural origin widely take the world interest as a complementary supplement in many diseases (1) rutin, is one of the flavonol compounds its chemical structure consists of aglycon part (quercetin) that is linked to glycoside moiety (rhamnose or called rutinose sugar part) (2) a study conducted in 1936 on citrus fruits and listed as one of vitamins ( vitamin p) (3) rutin is present in many vegetables, fruits, plant such as ruta graveolens, eucalyptus spp. , sophora japonica and buckwheat (4,5). rutin has many biological and pharmacological effects such as anti-inflammatory, antioxidant, antiangiogenic (6,7) moreover it has been found effective in many chronic diseases such as diabetes mellitus, hypertension, inflammatory bowel syndrome, hyperlipidemia, and alzheimer's disease(8-11). the aim of this study is to investigate the acute toxicity of rutin that can kill 50%of treated male and female swiss albino mice intraperitoneally at different concentrations over 14 days. materials and methods acute toxicity study was done according to the guidelines of the world health organization (who) (12) .thirty male and 30 non-pregnant female swiss albino mice with an average weight between 25-30 g (purchased from the national center for drug control and research) that is randomly and equally divided into five treated groups and one control group (n=5) for each male and female mice groups. 1corresponding author e-mail: haider_bahaa@yahoo.com received: 6/4/2021 accepted: 20/6 /2021 published online first: 2021-12-12 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp231-240 iraqi j pharm sci, vol.30(2) 2021 the study of toxicological activity of rutine 232 all mice have been kept in cages for 1 week in the pharmacology laboratory at al-nahrain university/collage of pharmacy; where the study is done for environmental accommodation and all conditions such as temperature, food, and water have been adjusted. rutin (cas number 153-18-4 /usp has been purchased from sigma-aldrich) prepared freshly of 5, 2.5, 1.25, 0.625 and 0.312 g/kg. (13,14) different doses of rutin have been injected intraperitoneally (ip) as a single dose according to the bodyweight while control group has been received 1%dmso as a negative control. the animals were observed for any sign of mortality or morbidity for 15, 30, 60. 120 and 240min for the first 24hrs after the administration of rutin; and the observation last for 14 days (13,15). weighting all mice at zero (before injection), seven and 14 days after administration of rutin. (16) after 14 days, blood was collect from the jugular vein (17,18) the collected blood was centrifuged at 4000 rpm for 10 min for biochemical analysis such as alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp), bilirubin, creatinine, urea, and albumin serum level. while for hematological analysis blood collected in a tube containing anticoagulant such as ethylenediaminetetraacetic acid (edta) for measuring white blood cell (wbc) count and hemoglobin(hgb) level (19)(20). after blood collection, all animals have been euthanized by cervical dislocation to collect vital organs such as ( liver, kidney, heart, lung and sex organs)to make histopathological examinations after preserving vital organs in 4% formalin (21,22). statistical analysis the results were introduced as (as meansˍ+sd standard deviation) one-way analysis of variance (anova) and ssps statistical software version (20) has been used in statistical analysis of data and the level of significance was set as p>0.05a significant, highly significant (23). results after administration of rutin at different concentration ip, the percentage of mortality of both male and female mice at dose 5 and 2.5g/kg were 100% and male at dose 1.25 also 100% while for female at the same dose were 60% other next doses there is variation in the percentage of mortality between male and female mice where at dose 0.625 g/kg the percentage of mortality for both sex were 60% while at dose 0.312 g/kg the percentage were 0% and 20% for male and female respectively. however, the sign observed can be summarized in table (1) and table (2). where (+), (++) and (+++) represent a degree of acute toxicity as slight, moderate and severe respectively. table 1.sign observed after rutin administration to male mice dose gm/kg t/m the period at sign observed sign of toxicity no.of mice dead at this period 5 5/5 5 mint_15 mint clonic convulsion, loss of gait, miosis, muscular fasciculation , tachycardia, dyspnea, lacrimation, death(+++) 2 15mint_4hrs hypoactivity, asthenia, abdominal contract, diarrhea, atypical locomotion, back limbs failling, bardycardia, dyspnea, death(++) 2 4hrs_6hrs bradycardia, atypical locomation, piloerection, asthenia ,dyspnea and death(+) 1 2.5 5/5 5 mint_15 mint clonic convulsion, loss of gait, miosis, muscular fasculation, tachycardia, dyspnea ,lacrimation, death(+++) 2 15mint_4hrs hypoactivity, asthenia ,abdominal contract, diarrhea, atypical locomotion ,back limbs failling , bardycardia, dyspnea, death(++) 1 4hrs_6hrs bradycardia,atypical locomation,piloerection,asthenia,dyspnea and death(+) 1 24-48 hrs. hypoactivity 1 1.25 5/5 5min_15 min clonic convulsion, loss of gait,miosis,muscular fasculation,tachycardia,dyspnea,lacrimation,death(++) 0 15mint_4hrs hypoactivity, asthenia, abdominal contract ,diarrhea, atypical locomotion, back limbs failling, bardycardia, dyspnea, death(++) 0 4hrs_6hrs bradycardia, atypical locomotion, piloerection, asthenia, dyspnea, and death(+) 1 24-48 hrs. hypoactivity, sunken eye 4 iraqi j pharm sci, vol.30(2) 2021 the study of toxicological activity of rutine 233 continued table 1. dose gm/kg t/m the period at sign observed sign of toxicity no.of mice dead at this period 0.625 5/3 10_15 mint loss of gait, muscular fasciculation, miosis, tachycardia (+) 0 15mint_4hrs asthenia, atypical locomotion(back limbsfalling, abdominal control, hypoactivity, hyperventilation ++ 0 6 _24 hrs asthenia, hypoactivity, piloerection, atypical locomotion (back limbs failing) and pale food pads, and pale ear 2 24-48 hr hypoactivity, sunken eye 1 0.312 5/0 1_6 hr tachycardia, hypoactivity, stool in the anus 0 24 hrs_14 days no sign of toxicity, no death 0 table 2. sign observed after rutin administration to female mice: dose gm/kg t/m the period at sign observed sign of toxicity no.of mice dead at this period 5 5/5 5min-15 min clonic convulsion, loss of gait, miosis ,muscular fasculation, tachycardia, dyspnea, lacrimation, death(+++) 2 15min-4 hrs hypoactivity, asthenia, abdominal contract, diarrhea, atypical locomotion, back limbs failing, bradycardia, dyspnea, death(++) 1 4hrs-6hrs bradycardia, atypical locomotion, piloerection, asthenia, dyspnea, and death(+) 1 2.5 5/5 5 mint_15 mint clonic convulsion, loss of gait, miosis, muscular fasciculation, tachycardia, dyspnea, lacrimation, death(+++) 2 15mint_4hrs hypoactivity, asthenia, abdominal contract, diarrhea, atypical locomotion, back limbs failling,bardycardia, dyspnea, death(++) 1 4hrs_6hrs bradycardia, atypical locomotion, piloerection, asthenia, dyspnea, and death(+) 1 24-48 hrs hypoactivity 1 1.25 5/3 5min_15 min clonic convulsion, loss of gait, miosis, muscular fasciculation, tachycardia, dyspnea, lacrimation , death(+++) 0 15mint_4hrs hypoactivity ,asthenia ,abdominal contract, diarrhea, atypical locomotion , back limbs failing , bardycardia , dyspnea, death(++) 1 4hrs_6hrs bradycardia,atypical locomation, piloerection, asthenia, dyspnea and death(+) 1 6-24hrs hypoactivity, bluish mucous membrane of the mouth, and tail 1 0.625 5/3 10_15 mint loss of gait, muscular fasculation,miosis,tachycardia (+) 0 15mint_4hrs asthenia, atypical locomotion(back limbs falling, abdominal control, hypoactivity, hyperventilation ++ 0 6 _24 hrs asthenia, hypoactivity, piloerection, atypical locomotion (back limbs failing) and pale food pads, and pale ear 2 24-48 hrs hypoactivity 1 0.312 5/1 1_6 hr tachycardia, hypoactivity, stool in the anus 0 24 hrs_48hr hypoactivity, bluish mucous membrane of the mouth, and tail 1 48hr-14 days no sign of toxicity 0 iraqi j pharm sci, vol.30(2) 2021 the study of toxicological activity of rutine 234 from the calculation of both percentage of mortality and dose giving for both male and female mice, a dose-response curve has been drawn to calculate ld50% as shown in figure (1) and (2) for male and female respectively where lethal dose was 1.51 g/kg for male mice while female mice were 1.49 g/kg. figure 1. dose-response curve of rutin in male mice. figure 2.dose-response curve of rutin in female mice. by measuring the body weight at day 0, 7 and 14 to all treated and control group the bodyweight tend to increase gradually with time to all treated and control groups of male and female mice, however, there is no significant difference (p˃0.05) in body weight changes by comparing treated male and female mice groups that have been received 1.25, 0.625 and 0.312g/kg of rutin with the control group at day 0, 7 and 14 as shown in figure (3) and (4) of treated and control male and female mice respectively. figure 3. body weight changes of treated groups of male mice receiving 0.625 g/kg and 0.312 g/kg of rutin at day 0, 7 and 14 where cmd0, rmbd0, rmcd0, cmd7, rmbd7, rmcd7, cmd14, rmbd14, and mcd14 represent control group, group of mice receive 0.625 g/kg 0.312 g/kg at day 0, control group, group of mice receive 0.625 g/kg, 0.312 g/kg at day 7, control group, group of mice receive 0.625 g/kg, 0.312 g/kg at day 14 respectively. figure 4.body weight changes between control and treated groups of female mice receiving 1.25, 0.625 and 0.312 g/kg of rutin at day 0, 7 and 14 where cfd0, rfad0, rfbd0 , rfcd0, cfd7, rfad7, rfbd7,rfcd7, cfd14, dfa14, rfbd14, rfcd14 represent control group, group of mice receive 1.25 g/kg, 0.625g/kg 0.312 g/kg at day 0, control group, group of mice receive 1.25 g/kg, 0.625g/kg 0.312 g/kg at day 7, control group, group of mice receive 1.25 g/kg, 0.625g/kg 0.312 g/kg at day 14 respectively. biochemical and hematological analysis of treated mice that have been received 1.25, 0.625 and 0.312g/kg for both sex revealed that there is no significant difference (p˃ 0.05) when comparing with control groups. as shown in table (3). iraqi j pharm sci, vol.30(2) 2021 the study of toxicological activity of rutine 235 table 3. serum and blood profile of treated male mice receiving 0.652, 0.312 g/kg rutin and female mice receiving 1.25, 0.652, 0.312 g/kg and control mice groups. serum and blood profile cm (mean± sd) cf (mean± sd) male 0.625 (mean± sd) male 0.312 (mean± sd) female 1.25 (mean± sd) female 0.625(mean± sd) female 0.312(mean ± sd) ast u/l 263.7±8.34 216.25±6.57 266.55±9.12 264.6±1.27 219.5±10.60 217.5±9.19 217±4.24 alt u/l 53.7±3.81 60±2.82 57±4.24 55±4.24 64.5±4.94 62.5±0.70 61.5±3.53 alp u/l 74±8.48 68±2.82 77±4.24 76.5±10.60 71±4.24 69±5.65 68.8±1.83 bilirubin t mg/dl 0.1±0.14 0.1±0.14 0.15±0.07 0.05±0.07 0.2±0.14 0.15±0.07 0.1±0.14 albumin g/dl 3.05±0.21 3.2±0.14 3.1±0.14 3± 0.28 3.15±0.21 3.05±0.35 2.9±0.28 creatinin e mg/dl 0.4±0.14 0.4±0.28 0.5±0.14 0.45±0.35 0.55±0.35 0.5±0.14 0.45±0.07 urea mg/dl 30.5±2.12 31.45±2.75 31.5±3.53 31±1.41 32.6±1.27 31.6±2.68 31.5±2.12 wbc ×10 ⁹/l 6±0.28 4.95±0.35 6.45±0.77 6.3±0.28 5.95±0.07 5.2±0.28 5.05±0.35 hgb g/dl 14.25±0.77 13.7±0.84 13.9±0.14 14±1.41 13.35±0.49 13.4±0.56 13.5±0.70 the results of histopathological examination of vital organs after staining with hematoxylin and eosin (h and e) stain revealed there are no significant histopathological changes in tissue (p˃0.05) of rutin treated mice at dose 1.25, 0.625 and 0.312 g/kg for male and female mice when compare with the tissue of control group as shown in the following figures: figure 5. represent section of liver x400 that stain with hematoxyline and eosin stain (h&e) where section of liver look like normal histological structure apperance in which central vein and the hepatocyte cell arranged as thread around it. there are no fibrosis or hepatocyte inflammation or necrosis where a,b,c,d,e,f represent groups of female that have been 1.25 , 0.625, 0.312 g/kg of rutin ,control group, groups male mice reciving 0.625,0.312 g/kg rutin respectively.no significant difference when compare all male and female mice treated at all dose with control dose. iraqi j pharm sci, vol.30(2) 2021 the study of toxicological activity of rutine 236 figure 6. represent section of kidney x400 that stain with hematoxyline and eosin stain (h&e) that show there is no infilteration of inflammatory cell in interstitial space , normal tubular vascular and glomerular a,b,c,d,e,f represent groups of female that have been receiving 1.25 , 0.625, 0.312 g/kg of rutin ,control group, groups male mice reciving 0.625,0.312 g/kg rutin respectively. no significant difference when compare all male and female mice treated at all dose with control dose. figure7. represent section of heart x 400 that stain with hematoxyline and eosin stain (h&e) that show normal structure of heart with no infilteration of inflammatory cell in perivascular or interstitial regions , no injury or necrosis, where a,b,c,d,e,f represent groups of female that have been receiving 1.25 , 0.625, 0.312 g/kg of rutin ,control group, groups male mice reciving 0.625,0.312 g/kg rutin respectively. no significant difference when compare all male and female mice treated at all dose with control dose. iraqi j pharm sci, vol.30(2) 2021 the study of toxicological activity of rutine 237 figure 8.represent section of lung x10 that stain with hematoxyline and eosin stain (h&e) that show normal structure of lung that consist of normal alveoli, no damage in the alveolar septa or alveolar cell necrosis , there is no inflammatory cell or edema in interstitial space or intra-alveolar region, there is no congestion or hemorrhage in capillary or alveolar duct where a,b,c,d,e,f represent groups of female that have been receiving 1.25 , 0.625, 0.312 g/kg of rutin ,control group, groups male mice receiving 0.625,0.312 g/kg rutin respectively. .no significant difference when compare all male and female mice treated at all dose with control dose. figure 9. represent section of testis and ovary where testis show normal structure of seminiferous tubules that inside it consist of numerous sperm and normal spermatogenesis, no atrophy in leyding cells or tubules or injury of basement membrane of tubules. while ovary consist of normal primary follicle and graafian follicle with no change and normal ovarian stroma a,b,c,d,e,f,g represent groups of female that have been receiving 1.25 , 0.625, 0.312 g/kg of rutin ,control group female,control group male, groups male mice iraqi j pharm sci, vol.30(2) 2021 the study of toxicological activity of rutine 238 receiving 0.625,0.312 g/kg rutin respectively. no significant difference when compare all male and female mice treated at all dose with control dose. discussion the capability of a substance to make harmful, toxic effect or even death after single short exposure can be named acute toxicity study (11). in the present study, male and female swiss albino injected as a single dose intraperitoneally at a different concentration, (5 and 2.5 g/kg), of rutin showed high mortality rate (100%). this may be due to that rutin have the ability to inhibit acetylcholinesterase (ache) in dose dependent manner (24) this inhibition done due to oh group of rutin can bind to the anionic site in the ache and inhibit its activity (25). this inhibitory activity of cholinesterase (acetylcholinesterase and buterylesterase) had been studied in vitro and in vivo (26,27). the increase acetylcholine activity at its receptors (nicotinic and muscarinic) may lead to cholinergic crisis that is show in tables 1 and 2 such as clonic convulsion, loss of gait, miosis, muscular fasculation, tachycardia, dyspnea, lacrimation, urination that finally lead to respiratory failure , cyanosis and death(28,29) subsequent dose show significant difference in the mortality rate and survival rate between male and female mice this may be due to sex effect where cytochrome p450activity in female higher than in male (11) or may be due to il-6 level , after its stimulated synthesis by any stimulus , its level will sustained in circulation in male mice more than female mice (30). the sign of toxicity in lower dose is less extensive and resolve step by step with time (31). the ld50 of rutin in male 1.51 g/kg while to female 1.49 g/kg . body weight of animals have been used as a good indicator of the side effects of the drugs or chemical on the animal (32). reduction in body weight of the mice when administered certain drugs or material over a certain time may be indicated of the harmful effect of that drugs (33)while there is no significant difference in body weight gain from normal diet compare with the control group with good water and food consumption may indicate drug did not change the metabolic events of the tested treated mice groups and not affect growth rate (34). it is concluded that mice treated with rutin do not show a significant difference in body weight compared with the control group. ast, alt, alp use as an indicator of liver dysfunction or damage due to various causes such as a toxin, drugs, viral that increase in the serum level(35). alt is an enzyme specific to the liver while ast is an enzyme present in other tissue such as heart, muscle (36). increase level of alp may be due to bile duct obstruction, cholestasis. hepatitis (37). albumin and bilirubin is another marker for liver function(38). decrease level of albumin can be account to low diet intake, toxicity inside the body, increase catabolism and kidney problem (39) while increase level of it can be cleared mostly as high protein diet (40). creatinine and urea consider as a marker of kidney function (32). hematological analysis was done to evaluate the risk of hematological changes due to it considers important and sensitive parameter where toxic material or drugs targeting on it that lead to detect the toxicity of drugs and physiopathological alteration in a biological system(34,10) alteration in hematological analysis such as hemoglobin (hgb) level may indicate toxicity that induces hemorrhage and hemolysis(41) or dysfunction of renal system while white blood cell(wbc) increase level of it may indicate there is an activation of the immune system due to either injury of liver, kidney or other tissue or infection(42) rutin treated groups in male and female in doses (1.25, 0.625, and 0.312 g/ kg) show no significant difference when compare with the control group and all results of the biochemical analysis revealed it lie in the normal range that is confirmed by histopathological examination for (liver, kidney, heart, lung and sex organs) that show no significant changes of treated tissue when compare with control tissue groups(43). other studies show that rutin when administer orally for 13 weeks to rat was safe till 2000 mg/kg (44) while other study show when administered orally to rats as a single(acute toxicity study) and repeated dose(subacute toxicity study) is safe till 5000 mg/kg (45). in the present study, the lowest observed adverse effect loae was seen at dose 0.312 g/kg and according to” loomis and hayes classification” is classified as a practically non-toxic substance (43) . conclusion acute toxicity study of rutin revealed that it is a practically non-toxic substance 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vol.22(1) 2013 esters and amides derivatives of naproxen 120 synthesis and preliminary pharmacological evaluation of esters and amides derivatives of naproxen as potential anti-inflammatory agents tagreed n – a . omar *, 1 * department of pharmaceutical chemistry, college of pharmacy, baghdad university, baghdad, iraq. abstract 4-chloro and 4nitro substituted phenol and aniline incorporated to a carboxylic group of naproxen a well-known non-steroidal anti-inflammatory drug (nsaid) to increase bulkiness were synthesized for evaluation as a potential anti-inflammatory agents with expected cox-2 selectivity. in vivo acute antiinflammatory activity of these compounds (i-iv) was evaluated in rats using egg-white induced edema model of inflammation in a dose equivalent to (2.5 mg/kg) of naproxen. all tested compounds produced a significant reduction in paw edema with respect to the effect of propylene glycol 50% v/v (control group). moreover, compounds i and iv might show higher effect comparable to that of naproxen and to that of compounds ii & iii which may attribute to the higher effect of nitro group than chloride group. the results of this study indicate that esterification and amidation of naproxen with selected pharmacophoric groups enhance or maintain its anti-inflammatory activity. keywords: anti-inflammatory; naproxen derivatives. تخليق وتقيين دوائي اولي لوشتقات استرية واهايذية للنابروكسين كوضادات التهاب هحتولة *عور تغريذ نظام الذين ،1 * .فشع انكًٛٛاء انصٛذالَٛت ، كهٛت انصٛذنت ،جايعت بغذاد ، بغذاد ، انعشاق الخالصة َاٚخشٔ يعٕضاث انفُٕٛل ٔاالَٛهٍٛ حى ادخانٓى كم عهٗ حذٖ عهٗ يجًٕعت انكاسبٕكغٛم نهُابشٔكغٍٛ ْٕٔ احذ ادٔٚت -4كهٕسٔ ٔ –4 انًعشٔفت نضٚادة ضخايخٓا قذ صُعج نخقًٛٛٓا كعُصش يضاداث االنخٓاباث غٛش انغخٛشٔٚذٚت نًعشفت صٚادة فعانٛخٓا ٔاَخقائٓا ألَضٚى كٕكظ . 2-كٕكظ ٚت يخٕقعت ألَضٚىقٕ٘ يضاد نالنخٓاب يع اَخقائ داخم جغى انجشر بطشٚقت اعخحذاد ٔريت بأعخخذاو صالل انبٛض ( 4 –1) قًٛج انفعانٛت انًضادة نالنخٓاب انحاد نهًشكباث نقذ انبشٔبهٍٛ جًٛع انًشكباث انخٙ حى فحصٓا اعطج َخائج اٚجابٛت بانًقاسَت يع حأثٛش . يٍ انُابشٔكغٍٛ( كهغى / يهغى 2.5 )ٔبجشعت حكافئ 2اثشث بقٕة عهٗ فعانٛت انُابشٔكغٍٛ اعهٗ يٍ انًشكبٍٛ 4ٔ 1باألضافت نزنك انًشكبٍٛ (. كًجًٕعت قٛاط)حجى / حجى % 50كالٚكٕل َخائج ْزِ انذساعت حشٛش انٗ اٌ اعخشاث ٔاياٚذاث انُابشٔكغٍٛ يع.ٔٚعٕد رنك سبًا نقٕة حأثٛش يجًٕعت انُاٚخشٔ يقاسَت يع انكهٕساٚذ 3ٔ . يُخقاث حضٚذ أ ححفظ فعانٛخّ كًضاد نالنخٓاباث حشكٛبٛت يجًٕعت عالجٛت هضادات التهاب ، نابروكسين :الكلوات الوفتاحية introduction non-steroidal anti-inflammatory drugs represent one of the most widely used classes of drugs, and are used primarily for treatment of osteoarthritis , rheumatoid arthritis and other inflammatory disorders; however, the use of nsaids is significantly limited by their ability to induce the formation of erosions and ulcers in the gastrointestinal (gi) tract (1) . the mechanism of action principally responsible for most of the nsaids seems to act by inhibition of prostaglandin (pg) synthesis causing almost complete blockade of the activity of the precursor enzymes , cyclooxygenase (2) . cyclooxygenase is a rate limiting enzyme for prostaglandin synthesis (3) . the three isoenzymes of cox (cox-1, cox-2 and cox3) have been identified (4, 5) though cox-3 activity in human has not been confirmed (6) .cox-1 is constitutively expressed, widely distributed and has "housekeeping" function. it is of particular importance in maintaining gastric mucosal integrity, renal function and homeostasis (7) . cox-2 is highly induced in settings of inflammation by cytokines and inflammatory mediators or physiological stress (8, 9) . however, cox-2 also is constitutively expressed in certain areas of kidney, brain, reproductive tract (10) , the vascular system (11) , in wound healing, lung and bone (12) . 1 corresponding author e-mail: tagomar77@yahoo.com received: 18/2/2013 accepted: 4 /5/2013 however, since the identification of cyclooxygenase-2 (cox-2), the field of inflammation and particularly the search for effective nsaids with fewer adverse effects has iraqi j pharm sci, vol.22(1) 2013 esters and amides derivatives of naproxen 121 greatly intensified. increasing number of experimental and clinical data support the role of selective cox-2 inhibitor in anti-inflammatory processes and the involvement of cox-1 inhibition in the side effects associated with using nsaids (13) . selective cox-2 inhibitors differ from traditional nsaids in two major ways; coxibs are less likely to result in nsaid-induced gastropathy, and they do not inhibit platelet function (14) . as a result , selective cox-2 inhibitors elicit less clinically significant gi damage and bleeding than conventional nsaids (15) . naproxen (1) is one of the most used nsaids for the treatment of arthritic pain. it can induce gi side effects ranging from stomach irritation to ulceration and bleeding. these gi complications are believed to be determined from the mixed effect of irritation caused by blockage of pg biosynthesis in the gi tract and direct action of free carboxylic groups in nsaids. (16,17) studies have shown that derivitization of the carboxylate moieties in nsaids such as indomethacin (2) and meclofenamic acid(3) result in the generation of potent and selective cox-2 inhibitors. (18) also amidation of diclofenac (4) with 4 (methyl sulfonyl) aniline pharmacophore (19) and amidation of ibuprofen (5) with 4 amino benzene sulphonamide (20) results in the generation of potent cox-2 inhibitors. in the view of this background, the present study was conducted to synthesis, and preliminary evaluate ester and amide derivatives of naproxen as new non-steroidal antiinflammatory agent with expected selectivity toward cox-2 enzyme using 4-chloro and 4 nitro substituted phenol and aniline. figure 1: chemical structure of some nsaids experimental section iraqi j pharm sci, vol.22(1) 2013 esters and amides derivatives of naproxen 122 chemistry the synthetic pathways for the designed target compounds (i-iv) are illustrated in schemes 1 & 2. h3co ch3 r-cooh + ho r1 dcc dmap rcoo r = compound i r1 = no2 compound ii r1 = cl naproxen r1 scheme1: synthesis of target compounds (i and ii) scheme2. synthesis of target compounds (iii and iv). pharmacology albino rats of either sex weighing (150 ± 10 g) were supplied by the animal house of the college of pharmacy, university of baghdad, and were housed in the same location under standardized conditions. animals were fed commercial chaw and had free access to water ad libitum. animals were divided into six groups as follow: group a: six rats served as control; and treated with the vehicle (propylene glycol 50% v/v). group b: six rats treated with naproxen in a dose of 2.5mg/ kg (21) .suspended in propylene glycol 50%. group c-f: six rats/group treated with the tested compounds (i-iv) respectively in doses that determined below. (suspended in propylene glycol 50%). anti-inflammatory activity: the anti-inflammatory activity of the tested compounds was studied using the egg-white induced hind paw edema model (22) . acute inflammation was produced by a subcutaneous injection of undiluted egg-white (0.05 ml) into iraqi j pharm sci, vol.22(1) 2013 esters and amides derivatives of naproxen 123 the plantar side of the left hind paw of the rats; 30 min after i.p. administration of the drugs or their vehicle. the paw thickness was measured by vernea at seven time intervals (0, 30, 60, 120, 180, 240, and 300 min) after drug administration. the data was expressed as the mean ± sem (standard error of the mean) and results were analyzed for statistical significance using student t-test (two sample assuming equal variances) for comparison between mean values. while comparisons between different groups were made using anova (analysis of variance): two factors without replication. probability (p) value of less than 0.05 was considered significant. general all reagents and anhydrous solvents were used as received from the commercial supplier (merck –germany, sigma – aldrich – germany , bdh – england and fluka –usa). naproxen was supplied from sdi company, iraq. melting points were determined by capillary method on electric melting point apparatus – england. thin layer chromatography (tlc) was run on silica gel (60) f254, merck –germany to check the purity of the products as well as monitoring the progress of reactions. the identification of compounds was done using u.v. detection and chromatograms were eluted by chloroform: methanol (85:15). ft-ir spectra were recorded by using shimadzu –japan spectrophotometer and the determination of spectrum was performed by using kbr disk. chn microanalysis was done by using euro ea3000 elemental analyzer –italy. hplc analysis was done using knauer analyzer – germany. general procedure for the esterification of naproxen: synthesis of compounds (i & ii). a reaction mixture containing naproxen (0.84 mmol) in 6ml of anhydrous ch2cl2 was treated with dcc (0.92 mmol), dmap (0.084 mmol) and 4-chlorophenol (or 4-nitrophenol) (0.92 mmol). after stirring at room temperature for 5 h, the reaction mixture was filtered and the filtrate was concentrated in a vacuum (in vacuo). the residue was diluted with water (30ml) and extracted with ethyl acetate (2 × 30 ml). the combined organic solution was washed with 5% acetic acid (acoh) (2 × 30 ml), 1 n sodium hydroxide (naoh) (2 × 30) ml), and water (100 ml) dried magnesium sulfate (mgso4), and filtered, and the solvent was removed in vacuo to give the final compounds i & ii. (23) . the final products were obtained as solids and the recrystallization was carried out by dissolving the compound in ethyl acetate and addition of petroleum ether (80–100) °c to the filtrate until turbidity occurred and then keeping in a cold place overnight. the mixtures were filtered while cold and the precipitate was collected to give the final compounds i & ii. 4-nitrophenyl 2( 6 – methoxynaphthalen – 2 – yl )propanoate (compound i): white crystal (62% yield); m.p. 208–209 °c; rf = 0.64; ir (cm −1 ): 2,945, 2,843 (c-h) , 1,742 (c=o) of ester, 1,597 and 1,518 (c=c of aromatic), 1,141(c-o); chno calculated (c20h17no5): c, 68.37; h, 4.88; n, 3.99; o, 22.77 found: c, 68.75; h, 4.73; n, 3.81; o, 23.44. 4-chlorophenyl2( 6methoxynaphthalen-2-yl ) propanoate (compound ii): white powder (55% yield); m.p. 185-186 °c; rf = 0.73; ir (cm− 1 ): 2,941, 2,852 (c-h), 1,753 (c=o) of ester, 1,602 and 1,523 (c=c of aromatic), 1,146(c-o); chno calculated (c20h17clo3): c, 70.49; h, 5.03; o, 14.08 found: c, 71.82; h, 5.15; o, 14.75. synthesis of acid anhydride derivatives of naproxen . 2(6 – methoxynaphthalene -2-yl) propanoic anhydride the anhydrides intermediate was obtained when two mmols of naproxen (0.46 g) was dissolved in tetrahydrofuran (thf) (30 ml), and then one mmol of dicyclohexyl carbodiimide (dcc) (0.206 g) was added. the reaction mixture was continuously stirred at room temperature for 4 hours, whereby a white precipitate of dicyclohexylurea (dcu) was formed, which then removed by filtration. the solvent was evaporated under vacuum to yield anhydride (24) as a white powder (75% yield); m.p. 128–130 °c; rf = 0.45. ir (cm−1): 1,801 and 1,737 of anhydride (symmetric and asymmetric), 1,606 and 1,471 (aromatic), 1,313, 1,224, 1,159 c-(c=o)-o-(c=o)-c of anhydride. general procedure for the amidation of naproxen: synthesis of compounds (iii & iv). a mixture of anhydride derivatives of naproxen (2.65 g, 6 mmol), 4-chloro aniline (1.53 g, 12 mmol) or 4-nitro aniline (1.65g, 12 mmol), zinc dust (0.011 g, 0.168 mmol), glacial acetic acid (1.1 ml, 19.2 mmol) and dioxane (35 ml) were placed in a flask equipped with reflux condenser, and boiling stones were added. the reaction mixture was refluxed gently for 90 min, the solvent was evaporated under vacuum, the residue was dissolved in ethyl acetate, washed with sodium bicarbonate (nahco3) (10%, 3×), iraqi j pharm sci, vol.22(1) 2013 esters and amides derivatives of naproxen 124 hydrochloric acid (hcl) (1 n, 3×), and distilled water (3×), and filtered over anhydrous magnesium sulfate (mgso4). the filtrate was evaporated under vacuum to give the final compounds i-iv. the final products were obtained as solids and the recrystallization was carried out by dissolving the compound in ethyl acetate and addition of petroleum ether (80–100°c) to the filtrate until turbidity occurred and then keeping in a cold place overnight. the mixtures were filtered while cold and the precipitate was collected to give the final compounds (iiii & iv) (24) . 2( 6methoxynaphthalen – 2 – yl ) – n ( 4 chlorophenyl)propanamide (compound iii) white crystal m.p. 218–220 °c; rf = 0.68; ir (cm −1 ): 3,298 (n-h) of secondary amide, 1,658 (c=o) of secondary amide, 1,595 and 1,516 (c=c of aromatic); chns calculated (c20h18clno2): c, 70.69; h, 5.34; n, 4.12; o, 9.42; found: c, 71.23; h, 5.29; n, 3.89; o, 9. 82. 2( 6 – methoxynaphthalen – 2 yl ) – n ( 4 nitrophenyl)propanamide (compound iv) white crystal m.p. 175-176 °c; rf = 0.68; ir (cm −1 ): 3,286 (n-h) of secondary amide, 1,667 (c=o) of secondary amide, 1,597 and 1,523 (c=c of aromatic); chns calculated (c20h18clno2): c, 68.56; h, 5.18; n, 8.00; o, 18.27; found: c, 67.94; h, 5.27; n, 7.89; o, 18.81. results and discussion the most widely used primary test to screen new anti-inflammatory agents is based on the ability of a compound to reduce local edema induced in the rat paw following injection of an irritant agent (25) . when egg-white is injected into the paw of rats, a substantial induction of cox-2 is observed at 2 hours coinciding with enhanced prostaglandins (pgs) and local edema (26) . table (1) shows the effect of naproxen (reference) and propylene glycol (control) on egg-white induced paw edema in rats. the differences in paw thickness readings among control and naproxen groups indicates that the method used in this study (paw edema) is a valid method and can effectively be used for the assessment of the anti-inflammatory effect of the newly synthesized compounds as shown in figure 2. table 1: effect of naproxen (reference) and propylene glycol (control) on egg white induced paw edema in rats. figure 2. effect of naproxen (reference), and propylene glycol (control) on egg-white induced paw edema in rats. time (30) is the time of egg-white injection. table ( 2 ) shows the effect of the tested compounds (i-iv) with respect to control and reference group (naproxen). all tested compounds effectively limited the increase in paw edema, the effect of all tested compounds started at 60 minutes (significantly different compared to control). however, the effect of all tested compounds continued till the end of the experiments with statistically significant (p > 0.05) reduction in paw edema, as shown in figure 3. table 2: effect of control, naproxen and compounds (i-iv) on egg-white induced paw edema rats. naproxen ( n = 6 ) control ( n = 6 ) time ( min ) 4.43 ± 0.06 4.48 ± 0.08 0 6.48 ± 0.13 6.55 ± 0.14 30 6.80 ± 0.07  7.63 ± 0.04 60 6.64 ± 0.03  7.05 ± 0.16 120 6.16 ± 0.11  6.75 ± 0.11 180 5.86 ± 0.05  6.50 ± 0.09 240 5.70 ± 0.07  6.12 ± 0.08 300 iraqi j pharm sci, vol.22(1) 2013 esters and amides derivatives of naproxen 125 non-identical superscripts (a & b) among different tested compounds are considered significantly different (p < 0.05); * significantly different compared to naproxen (p < 0.05). figure 3: effect of naproxen, propylene glycol, compounds i, ii, iii and iv on eggwhite induced paw edema in rats. results are expressed as mean ± sem (n = 6 for each group). time (30) is the time of egg-white injection. the comparison between the tested compounds and naproxen shows that at time 0– 60 minutes there are no differences with naproxen between groups; however at the interval time 120–300 minute, compounds i and iv show significantly higher effects than naproxen while compound ii expressed a comparable effect to that of naproxen at the interval time 60–240 minute, and compound iii showed a comparable effect to that of naproxen for all the experimental times. conclusions an in vivo anti-inflammatory study showed that esterification and amidation of naproxen maintained or increased its anti-inflammatory activity. these structural pre-requisites for cox2 selectivity can mainly attributed to the occupancy of the side pocket (val.509) in cox-2 enzyme. compounds ii and iii showed a comparable effect to that of naproxen, while compounds i and iv might show higher effects comparable to that of naproxen and to that of compounds ii & iii which may attributed to the higher effect of nitro group than chloride group. references 1. john l wallace ; linda vong. nsaidinduced gastrointestinal damage and the design of gi-sparing nsaids. current opinion in investigational drugs 2008; 9(11):1151-1156. 2. non-steroidal anti-inflammatory drugs (nsaids), medicinal chemistry, ashutosh kar, (4 th ed), new age international publishers, new delhi, 2007; 522-535. 3. laurance, d.r.; bennett, p.n.; brown, m.j, clinical pharmacology (9th ed.), churchill livingstone, london, 2003, pp. 280. compound iv ( n =6 ) compound iii ( n =6 ) compound ii ( n =6 ) compound i ( n =6 ) naproxen ( n = 6 ) control ( n = 6 ) time ( min ) 4.46 ± 0.13 4.41 ± 0.11 4.45 ± 0.16 4.40 ± 0.07 4.43 ± 0.06 4.48 ± 0.08 0 6.50 ± 0.11 6.56 ± 0.07 6.46 ± 0.11 6.43 ± 0.05 6.48 ± 0.13 6.55 ± 0.14 30 6.71 ± 0.07  6.88± 0..13 6.86 ± 0.03  6.70 ± 0.11 6.80 ± 0.07  7.63 ± 0.04 60 6.30 ± 0.12  b 6.70 ± 0.13  a 6.56 ± 0.03  a 6.26 ± 0.13  b 6.64 ± 0.03  a 7.05 ± 0.16 120 5.77 ± 0.11  c 6.26 ± 0.11  a 6.10 ± 0.15  a 5.49 ± 0.09  b 6.16 ± 0.11  a 6.75 ± 0.11 180 5.38 ± 0.05  b 5.98 ± 0.05  a 5.80 ± 0.05  a 5.23 ± 0.04 b 5.86 ± 0.05  a 6.50 ± 0.09 240 5.09 ± 0.07  b 5.67 ± 0.07  a 5.40 ± 0.07  c 4.95 ± 0.07 b 5.70 ± 0.07  a 6.12 ± 0.08 300 iraqi j pharm sci, vol.22(1) 2013 esters and amides derivatives of naproxen 126 4. marnett, l.j.; 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al., ester and amide derivatives of the nonsteroidal anti-inflammatory drug, indomethacin, as selective cyclooxygenase-2 inhibitors, j. med. chem, 2000, 43, 28602870. 24. monther, f. mahdi.; abdul-rassoul, w.; samira, f, synthesis and preliminary pharmacological evaluation of amino benzene sulfonamide derivatives of http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20h%26%23x000e9%3btu%20po%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20riendeau%20d%5bauth%5d iraqi j pharm sci, vol.22(1) 2013 esters and amides derivatives of naproxen 127 diflunisal as anti-inflammation agents. iraqi j. pharm. 2008, 7–8, 42–49. 25. winter, c.a; risley, e.a.; nuss, g.w,: carrageenan-induced edema in hind paws of the rat as an assay for anti-inflammatory drugs. proc. soc. exp. bio. med, 1962, 111: 544-547. 26. seibert, k.; zhang, y.; leahy, k.; masferrer, j.; et al, pharmacological and biochemical demonstration of the role of cyclooxygenase-2 in inflammation and pain, proc. natl. acad. sci. 1994, 91: 12013. iraqi j pharm sci, vol.31(suppl,) 2022 drug information resources in iraq doi: https://doi.org/10.31351/vol31isssuppl.pp25-31 25 drug information resources in iraqi community pharmacies (conference paper) # mohanad yasir al-radeef *,1 and khalid saud saleh** # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of clinical pharmacy, college of pharmacy, university of tikrit, tikrit, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of tikrit, iraq abstract drug information resources are the information that is used in medications discovery, utilization, and management. little information about different types of resources used by iraqi community pharmacists is known. therefore, the objectives were to determine drug information resources' type do the pharmacists used and the common drug information questions they faced during their work in community pharmacy. a cross-sectional descriptive study was conducted in different iraqi provinces and online self-reported survey was introduced through google form software to an appropriate sample of graduated pharmacists who were working in a private community pharmacy and having at least one year of experience between february 27 and may 15, 2021. the researchers received 402 usable surveys. british national formulary was used by (47%) of the surveyed pharmacists to find specific information, followed by "pharmacotherapy(s) and applied therapeutics" (16.9% for both). on the other hand, internet was used by (93%) of the surveyed pharmacists and google search engine (65%) and medscape application (62%) were frequently surfed to find specific drug information and (81%) of pharmacists trusted in this information and passed them to consumers. safety of drugs during pregnancy and lactation periods was the most frequently question received from the patients (60.7%). in conclusion pharmacists prefer to surf specific internet websites to collect specific information about medicines and they referred to pharmaceutical textbooks if available at their pharmacies to get such information. the pharmacist is the person who is more often accessed by patients and the patients follow pharmacist's instruction for specific drug related questions. keywords: information, community pharmacy, resource, iraq, internet. # ) بحث مؤتمر ( مصادر المعلومات الدوائية في صيدليات المجتمع في العراق **الحص ودو خالد سع 1*،مهند ياسر الرديف 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي ع الصيدلة السريرية، كلية الصيدلة، جامعة تكريت، تكريت، العراق فر * فرع االدوية والسموم، كلية الصيدلة، جامعة تكريت، تكريت، العراق ** الخالصة على الرغم مصادر المعلومات الدوائية هي المصادر المختصة بتوفير معلومات موضوعية وحديثة عن األدوية واستخدامها. هدفت هذه الدراسة من أهمية هذه المصادر المتنوعة وتوافرها اال ان القليل منها معروف ويشيع استخدامه من قبل الصيادلة في العراق. صيدليات لألسئلة الدوائية الشائعة التي واجهوها أثناء عملهم في باإلضافةوائية التي يستخدمها الصيادلة لتحديد مصادر المعلومات الد . 2021ايار 15الى 2021شباط 27أجريت هذه الدراسة المجتمعية المستعرضة في عدة محافظات عراقية خالل الفترة من .المجتمع ارة استبيان تم اعدادها من قبل المؤلفين اعتمادا على االدبيات المنشورة في هذ استمتم جمع المعلومات عبر اإلنترنت عن طريق وشملت عينات مالئمة من الصيادلة المتخرجين الذين يعملون في صيدلية خاصة ولديهم لج ونماذج ج برنامج المجال وتم توزيعها عبر من الصيدليات (%47)استطالعاً تم استالمها. تواجد كتاب الوصفات الوطنية البريطانية في اثنينو ةمئ اربع خبرة ال تقل عن عام واحد. لكليهما(. من ناحية أخرى، تم استخدام اإلنترنت من قبل %16.9التطبيقية ) يليه كتاب العالج الدوائي والعالجات الدراسة،التي شملتها شملهم (93%) الذين الصيادلة للعثور على (%62و% 65) ،االستطالعمن مدسكايب البحث كوكل وتطبيق محرك منهم اعتمدوا هذه المواقع. كانت سالمة استعمال األدوية أثناء معلومات دوائية محددة ونسبة عالية من الصيادلة كانوا يثقون بالمعلومات المتوفرة في يستنتج من هذه الدراسة تفضيل الصيادلة لتصفح مواقع إنترنت محددة الحمل والرضاعة من بين األسئلة األكثر تلقيا من المرضى. صيدلياته في متوفرة كانت إذا الصيدالنية الكتب إلى باالضافة األدوية عن موثوقة معلومات على هذه للحصول على للحصول م المعلومات كون العديد من المراجعين يثقون بالمعلومات المقدمة من قبل الصيدالني ويعملون على تطبيقها. ، االنترنيتالعراق ،المصادر ،صيدلية المجتمع ،: المعلوماتمفتاحيةالكلمات ال introduction drug information (di) resources, also called medication information, drug information, or sometimes drug informatics, are basically included discovery, utilization, and management of information in the medications usage. they should include the identification, pharmacokinetics, cost, and dose in addition to adverse effect profile of medicines(1). 1corresponding author e-mail: mohanadyasir@tu.edu.iq received: 1/7 /2022 accepted:17 /8 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp25-31 iraqi j pharm sci, vol.31(suppl.) 2022 drug information resources in iraq 26 drug information resources were defined by other authors as “a resource dedicated to provide objective, independent and current information on drugs and their use, and communicate to the different categories of users for better understanding and benefit of patients”(2). three categories of di resources are available depending on their originality: primary, secondary or tertiary(3). primary di sources are the original resources on which the study is based. these include scientific articles and journals showing the results of the experimental research, meetings proceedings, symposia and conferences, patents, dissertations, and technical reports(3). note that not all journal articles are regarded as primary literature, such as review articles that summarize the literature; these are categorized as tertiary resources(4). upon publishing, the information of primary resources serves as the basis for secondary di sources. they belong to indexing and abstracting systems that are organized and provided easy method of retrieving primary resources. these involve indexing and abstracting systems, medline database search, works of criticism and interpretations, or otherwise ‘add value’ to the newly reported primary literature information(5). tertiary di sources involve summary of data that was retrieved from the primary literature. they are represented as reference textbooks, pharmacopoeias, treatment guidelines, list of essential drugs, drug bulletins, drug formularies, and drug compendia(6). the best di sources are those that provide highly accurate and relevant information which is simple to be applied(7). appropriate di is vital for pharmacists in order to correct the use of drugs and to improve patient outcome. common situations in community pharmacy like adverse reactions of drugs, the use and safety of drugs during pregnancy and lactation periods, and drug-drug interactions need the access to di sources(8). the international pharmaceutical federation (fip) states that '' it is the responsibility of the pharmacist to ensure that the patient receives the required information for the quality use of medications ''. in contrast to this, failure to provide high quality drug information may lead to several detrimental results(9). an ethiopian study reported that lack of appropriate di that is belonged to the adverse effects among the populations led to high level of self-medication practice(10). another ethiopian study reported that (76%) of 50 prescribers enrolled reported to have an access to upto-date di, while from 30 pharmacists, (43.3%) did not get any access to such di(11). also, from a total of 1875 pharmacists enrolled in a jordanian study, only one-fifth of them stated that they depend on di lists for specific information(9). as the pharmacists were became the primary source of information on medicines and had the respect as well as appreciation of the healthcare team providers everywhere in the world; they should maintain their frontline position as information experts and providers, they must provide accurate, precise, current, and unbiased information and must be extremely effective, skilled, and competent when looking for and providing such information(12). it has been indicated that di provided by pharmacists in the settings of community pharmacy are usually associated with better patient satisfaction with pharmaceutical care services(13). in addition to that, it has been reported that patients do usually have a wide variety of questions about their medicines; even though the internet is becoming a significant source of information, the pharmacist can still be extremely helpful in giving patients di about their medications(14). little is known about the types of di sources frequently used by pharmacists in iraq, despite the significance and accessibility of a variety of di sources for healthcare provider members; therefore the objectives of the current study were to find out the type of di resources do the community pharmacists in iraq were used to find specific drug information in addition to determine the most common drug information questions they faced during their work in community pharmacy. subjects and methods this cross-sectional descriptive study was done in different iraqi provinces by using a webbased self-reported electronic survey that was introduced through google form software. the survey link was electronically distributed through different social media groups (facebook, whatsapp, telegram) of iraqi pharmacists only between february 27 and may 15, 2021. inclusion criteria involved graduated pharmacists who are working in a private (community) pharmacy and having worked at the same pharmacy for at least a year. approximately 20,000 pharmacists were registered in syndicate of iraqi pharmacists at the time of the study. based on this and using a confidence interval of 95%, a standard deviation of 0.5, and a margin of error of 5%, the minimum sample size required was 290 pharmacists. no validated questionnaires in this context were available. consequently, a standardized questionnaire was developed by the authors based on available literatures. the questionnaire was first developed to obtain general data (gender, age, experience duration, location and type of the pharmacy, and graduation degree), accessible di resources, how often you access them, the availability of tertiary textbooks in the pharmacy, the use internet for di purposes and the most common websites used for them, and the kinds of questions that the patients asked the pharmacists who were surveyed. the pretest (pilot study) for the survey was conducted in february 2021 to assess the clarity of the data that would be gathered. there were no iraqi j pharm sci, vol.31(suppl.) 2022 drug information resources in iraq 27 incentives provided. the survey taken was both voluntary and anonymous. this study was approved by the research ethics committee at the college of pharmacy at tikrit university, sallahaddin, iraq (no. 2021 – 10). the data were reviewed, organized, tabulated and analyzed by using spss version 23. continuous variable are expressed by using their mean ± standard deviation (sd), while discrete variables were presented using their number and percentages. results only 402 of the total of 472 surveys that the researchers received were usable (because some of the participants were not graduated pharmacists and some had less than one year of experience at their pharmacy). pharmacists from seventeen governorates in iraq were participated in this survey and pharmacists from baghdad had the highest percentage of participants (40.3%) followed by pharmacists from sallahaddin (17.4%), diyala (7%), kirkuk (6.5%) and babil (5.5%). the remaining participants from other governorates are represented in table 1. the mean ages for these participants were (29.76 ± 4.652) years. approximately three-quarters (64.4%) of the participants were less 35 years, of them, 218 (54.2%) were females. the vast majority of the participants (92%) were gained a bachelor degree. two hundred and ninety two pharmacists were worked in pharmacies presented in urban area while the remaining 110 pharmacists were worked in those presented in rural area. more than half of these pharmacies (62.2%) were offered both prescriptions only and over the counter medications while the remaining (29.6% and 8%) were offered over the counter medications and prescriptions only medications respectively. these data are expressed in table 2. table 1. number and percentage of pharmacists who participated in this study with their governorate (n = 402) governorate no. % baghdad 162 40.3 sallahaddin 70 17.4 diyala 28 7.0 kirkuk 26 6.5 babil 22 5.5 karbala 16 4. thi-qar 14 3.5 basra 12 3.0 najaf 10 2.5 ninevah 8 2.0 wasit 8 2.0 maysan 8 2.0 other* 24 4.5 *: participants from al-anbar, dohuk, sulaymanyya, erbil and muthana. table 2. general information of pharmacists and their pharmacies (n = 402). parameter no. % age (years) 25 35 260 64.4 35-45 126 31.4 more than 45 16 4.0 gender male 184 45.8 female 218 54.2 academic degree bachelor 370 92.0 master of science 22 5.5 doctor of philosophy 10 2.5 pharmacy location urban area 292 72.6 rural area 110 17.4 pharmacy type prescription medication only 32 8.0 over the counter medication only 120 29.6 both types 250 62.2 the available tertiary textbooks resources in the pharmacies reported in this study were the “british national formulary” (bnf) (47.3%) followed by "pharmacotherapy(s) and applied therapeutics" (16.9% for both). the "drug information handbook and iraqi drugs guide" were available in the (15.9% and 15.4% respectively) of the included pharmacies. figure 1 shows other resources. iraqi j pharm sci, vol.31(suppl.) 2022 drug information resources in iraq 28 figure 1. tertiary textbooks available in the pharmacy where the participants currently in (a hard copy or soft copy). in order to find specific drug information, (93%) of the enrolled pharmacists used the internet during work time; on the other hand (7%) never used it for di purposes as shown in figure 2. for the majority of the time, pharmacist enrolled searched for di using general search engines like google (65.2%),while medscape was relied on by (62.7%), wikipedia by (25.9%), drugs.com by (24.9%), webmd by (18.4%), mayo clinic by (13.9%) and food and drug administration (fda) by (10.4%) as shown in figure 3. figure 2. percentage of participants using the internet during working hours in the pharmacy to find a specific information for certain drug. figure 3. web sites the participants most commonly surfed to find specific drug information during working in the pharmacy. iraqi j pharm sci, vol.31(suppl.) 2022 drug information resources in iraq 29 when asking the pharmacists '' do you trust the drug information gained from the internet and forward it to the patients? (81.1%) of them answered with ''yes'' while the remaining (18.9%) answered with ''no'' as shown in figure 4. figure 4. percentage of participants trusting the drug information gained from the internet and forward them to the patients. the most common type of questions received and forced the enrolled pharmacists to seek different di resources to get an exact answer were those related to drug use and safety during pregnancy and lactation periods (60.7%) followed by the adverse effect of certain drug (51.2%), dose of certain drug (50.2%), drug interactions (46.3%), and pediatric safety (38.3%). other requests are shown in figure 5. figure 5. questions the participants were received and looked for appropriate answer in different resources during working in the pharmacy. discussion the results of advancements in healthcare lead to an increasing demand for pharmacists as primary di providers to healthcare professionals and to the population. as professional support for pharmaceutical care, pharmacists should become the primary source of drug knowledge(15). patient care, medication error hazard, and medication compliance are all positively affected by pharmacist interventions. according to a recent cochrane database review, pharmacist intervention can improve patient behavior and compliance and improve physician prescribing in addition to the expanding role of pharmacists on improving patient outcomes, health care utilization, and cost(16). the results showed that most of pharmacists participated in this survey were from baghdad and this is related to the huge population in this city. therefore, a large number of pharmacists were required to provide the appropriate pharmaceutical care for people. in addition, (54.2%) of the participants were females, their mean age was (29.76 ± 4.652) years and (64.4%) of these participants were less 35 years. the vast majority of the participants (92%) were gained a bachelor degree. in line with a study of mohammed and al-razaq that was conducted in baghdad city only, in which 150 pharmacists accept to participate in it, their age was ranging from 23 to 65 years and (42%) of these participants were less than 33 years. the participants' females had the highest percentages (56.7%) that were qualified as bachelor pharmacists (63.3%)(17). while in qadus et al study more than (80%) of the participants were less than 35 year-old, (68.5%) were females and (80%) were gained bachelor degree(9). the study showed that bnf is the most available usable tertiary textbooks resources in the pharmacies (47.3%). in according with this result, iraqi j pharm sci, vol.31(suppl.) 2022 drug information resources in iraq 30 al-tabakha et al reported that all the pharmacists enrolled in their study were had and use the bnf for further information regarding medicines while working in community pharmacies (18). in contrast, bnf was only (15%) available and ranked fourth among the surveyed community pharmacies in the study conducted in kuwait(19). the bnf is released twice annually (march and september). it provides concise, clear, and authoritative information on the selection and clinical use of medications. it also includes details about the medications offered in the united kingdom, such as their pharmacology, dosages, side effects, and contraindications. it also includes information about their legal classification, proprietary names, and generic names, as well as the cost of the medications(18). the bnf's accessibility can be partially attributed to the fact that most pharmacists are familiar with it and that it is less expensive than hardware and software di resources. the other important resources like applied therapeutics and pharmacotherapy hand book. possibly because clinical pharmacy bachelor courses of iraqi pharmacy programs depend on these textbooks that the pharmacists were became familiar with them therefore (16.9%) of the pharmacies had these textbooks(20). in response to the question: ''do you use the internet during work in the community pharmacy to get a specific di?'' (93%) were replied ''yes''. the same result was reported by al-tabakha et al (18) while jaradat and sweileh reported that (29%) of the community pharmacies surveyed were had an internet access for di(14). although there may be a huge of di on the internet that isn't necessarily covered in textbooks, their quality is frequently in doubt(21). some pharmacists indicated that they did not use it for di purpose due to the load during work hours or they didn't have to because they didn't have much faith in the internet-based information. a sizable percentage of participants (65.2%) said they get their internet di from the google search engine, followed by medscape (62.7%). next to e-mail and product research, search engines used to find health information on the web are listed as the third most popular internet usage. google is the one of these search engines that is used the most frequently(1). on the other hand, medscape is particularly useful for pharmacists since pharmacy-specific topics are addressed in a section of this application(1). in a study evaluated online di databases when answering infectious diseasespecific queries, polen et al reported that medscape on top databases for completeness (95%) and answered significantly more questions compared to other databases(22). in an open question ''do you trust the drug information gained from the internet and forward it to the patients?'', the answer by relatively large proportion was ''yes''. because websites offered by the government, universities, and non-profit organizations may offer good quality of di, this demonstrates the widespread belief that internet websites contain high quality di. however, the provided information ought to be current and backed up by pertinent references(23). the most common type of information requested by the clients and provided by the pharmacists in this study were those related to drug use and safety in pregnancy and lactation periods (60.7%). this finding was also reported by altabakha et al (18) in contrast to this, a palestinian study reported that the most common question reported was that related to drug price (30%) and only (6%) requested pregnancy and lactation drug safety information(14). this could mean that patients in these various settings have a variety of concerns. evaluating the safety of medicines in pregnancy or lactation is a challenge that most physicians will face at some time in their careers. almost all women will want some type of drug therapy while they are pregnant or breastfeeding. such questions may be passed to the pharmacists who care for women in their reproductive years on a daily basis. in fact, a pharmacist may be the first health care professional to meet a patient when she knows she is pregnant, or a physician may ask the pharmacist to recommend safer treatment options for pregnant and lactating females(24). this study has limitations. it's possible that some of the targeted population was not included in the online survey, which would have limited the findings' ability to be generalized and the 402 surveyed pharmacists might not demonstrate the entire pharmacists working in iraq at the time of the study. also the response rate for this study could not be estimated, which may lead to nonresponse bias. therefore, a careful interpretation of the findings is required. in conclusion the study results reported that most of the pharmacists prefer to surf specific internet websites to collect specific information about medicines. at the same time they referred to pharmaceutical textbooks if available at their pharmacies to get such information. in addition to these findings, this study also reported that the consumers are more often accessed the community pharmacists and followed pharmacists for specific drug related questions. the pharmacist needs to be educated about the various di resources and how to evaluate information obtained, particularly via the internet. references 1. shields km, park sk. drug information resources. in: malone pm, malone mj, park sk. eds. drug information: a guide for pharmacists 6e. new york, ny: mcgraw-hill; 2018. 2. nova-manosalva m.a., lópez-gutiérrez j.j., cañas m. drug information centers: an iraqi j pharm sci, vol.31(suppl.) 2022 drug information resources in iraq 31 overview to the concept. rev. colomb. cienc. quím. farm. 2016; 45(2), 243-255. 3. alamgir, a.n.m. origin, definition, scope and area, subject matter, importance, and history of development of pharmacognosy. in: therapeutic use of medicinal plants and their extracts: volume 1. progress in drug research, 2017;(73). springer, cham. 2017. 4. kier kl and goldwire m. drug information resources and literature retrieval. in: accp updates in therapeutics 2018: pharmacotherapy preparatory review and recertification course 7th edition. american college of clinical pharmacy, 2018. 5. anandabaskar, n. drug information. in: raj, g., raveendran, r. 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psychotropic medications doi: https://doi.org/10.31351/vol29iss1pp166-173 166 the prevalence of potentially inappropriate prescribing in geriatric patients with psychiatric disorders in iraq mustafa k. mahmood*,1 and zinah m. anwer*  department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract potentially inappropriate prescribing is the prescribing of a medication that may cause more harm than benefit. the elderly population aged 65 years or older is more prone to potentially inappropriate prescribing because of the alterations in their physiology, pharmacokinetics, and pharmacodynamics as well as polypharmacy and comorbidities. beers list is a screening tool that helps doctors to detect potentially inappropriate prescribing in geriatric patients and is designed to solve this problem. the aim of this study was to measure the prevalence of potentially inappropriate prescribing among psychiatric geriatric patients using the beers criteria as an assessment tool and to find the relationship between pip and the duration of hospitalization, comorbidities, and polypharmacy in elderly. this cross-sectional study was carried out using electronic medical records in ibn rushd psychiatry and addiction hospital in baghdad and 369 patients were included. the mean age of the patients was (68.59 ± 3.75 years) and 177 (48%) of them had comorbidities, 100 (27.1%) of them had polypharmacy and 17 (4.6%) stayed in the hospital for more than 3 weeks, the most used drug classes were antipsychotics in (39.9%) of patients and benzodiazepines in (17.6%) of patients. the prevalence of potentially inappropriate prescribing according to beers criteria was found to be 74.3% among study patients, the most prevalent inappropriately used drug class was benzodiazepines, and there was a significant association between the prescribing of a potentially inappropriate medication with gender (p=0.018), with comorbidities (p=0.022), and a very significant association with polypharmacy (p<0.001) keywords: potentially inappropriate prescribing, potentially inappropriate medication, beers criteria, geriatric patients. المصابين بامراض نفسية في العراقكبار السن الغير مالئم لمرضىانتشار الوصف الدوائي *مظفر انور ةزينو 1*,محمود مصطفى كاظم بغداد،العراق. جامعة بغداد، فرع الصيدلة السريرية ،كلية الصيدلة،* ةالخالص سنة 65ان شريحة كبار السن لالعمار .مالئمته هو وصف اي دواء من الممكن تسببه بضرر يفوق نفعهالوصف الدوائي المحتمل عدم االفراط الدوائي و باالضافة الىاو اكثر هم اكثر عرضة للوصف الدوائي الغير مالئم بسبب تغيرات في الفارماكوكاينتك و الفارماكوداينامك و من هذه الدراسة الهدف .فحص تساعد االطباء لكشف الوصف الدوائي المحتمل عدم مالئمته لكبار السنالئحة بيرز هي اداة . االمراض المصاحبة استخدام هي قياس انتشار الوصف الدوائي الغير مالئم لكبار السن المصابين بامراض نفسيه باستخدام الئحة بيرز كاداة تقييم و ايجاد العالقة بين ود بالمستشفى, االمراض المصاحبة و االفراط الدوائي.االدوية الغير مالئمة و فترة الرق مراض النفسية و االدمان في بغداد و شمل في مستشفى ابن رشد لال اجريت هذه الدراسة المستعرضة باالعتماد على سجالت المرضى االلكترونية يعانون من امراض مصاحبة, اكانو( منهم %48) 177و سنة( 68,59 ± 3,75كان ) اعمارهممتوسط ,مريض 369الدراسة هذه في دوية استخدم ااكثر صنف . اسابيع 3من الكثرفي المستشفى ارقدو ( منهم%4.6) 17يعانون من الفرط الدوائي و ا كانو ( منهم27.1%)100 ( من المرضى.%17.6( من المرضى و البينزوديازبينات في )%39.9كان مضادات الذهان في ) الدراسة, المرضى المشاركين في هذهبين %74.3دوائي المحتمل عدم مالئمته لكبار السن استنادا الى معيار بيرز هو وجد ان االنتشار للوصف ال ذات اهمية بين وصف دواء من المحتمل عدم مالئمته احصائية اكثر صنف دوائي غير مالئم استخدم كان البينزوديازبينات, و كان هناك عالقة مع الفرط الدوائي عالية احصائية (, وعالقة ذات اهميةp=0.022(, مع االمراض المصاحبة )p=0.018) جنس المريضلكبار السن مع (p<0.001.) .مرضى كبار السن ،االدوية المحتمل عدم مالئمتها،الئحة بيرز،الوصف الدوائي المحتمل عدم مالئمتهالكلمات المفتاحية : 1corresponding author e-mail: mustafa.kazum@gmail.com received: 26/ 8/2019 accepted:7 /12 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp166-173 mailto:mustafa.kazum@gmail.com iraqi j pharm sci, vol.29(1) 2020 pip psychotropic medications 167 introduction potentially inappropriate prescribing (pip) can be defined as the prescribing of a medication that the risk of adverse drug reaction may outweigh the benefit of the medication especially if there is a safer alternative available(1). the elderly population which is aged 65 years or older has been increasing rapidly, since the last century are very susceptible to the problem of pip because of the alterations in their pharmacodynamics and pharmacokinetics as well as other drug-related problems such as polypharmacy and having multiple co-morbid diseases (2,3). there are many consequences of pip, including increased adverse drug events such as cognitive impairment, falls and fractures which can lead to increase emergency room visits and prolonged hospitalization and increased health care use and cost(4–6). it is challenging to prescribe for geriatric patients with mental disorders because there are many factors influencing prescribing options that can cause alterations in pharmacokinetics such as changes in renal clearance, liver metabolic activity, brain volume, lean body mass and albumin binding this can lead to increased sensitivity to drugs effect especially in the central nervous system (7). in addition, geriatrics with psychiatric disorders usually take several medications for the treatment of their mental disorders as well as other diseases, which increases the risk of drug-drug and drugdisease interactions.(8) patients with mental disorders may be less able to express their discomfort or drugrelated problems such as side effects especially in patients with dementia, therefore they may communicate their discomfort as aggression or agitation(9). this miscommunication may be perceived as worsening of their mental disease rather than a drug-related adverse effect. lastly, the geriatric population are often excluded from most clinical trials, typically clinical trials conducted in adult population include patients between the ages of 18 and 64 years which causes a limited evidence base for medication use and safety in this population(10,11). this fact leads to the need of a screening tool that helps doctors to measure the appropriateness of the medications to be prescribed to the elderly and the first list for potentially inappropriate medications (pims) made was called the beers criteria and was created in 1991 making it the longest-running criteria for detecting pims in the elderly, the criteria were revised and updated many times over the years and adopted by the american geriatric society, the criteria consist of five categories including drugs to be avoided regardless of condition and drugs to be avoided due to a certain disease/syndrome and drug interaction and drugs to be avoided due to drugdrug interaction(12). the aim of this study was to measure the prevalence of pip among psychiatric geriatric patients using the beers criteria as an assessment tool and to find the relationship between pip and the duration of hospitalization, comorbidities, and polypharmacy in such patients. patient and methods study design this cross-sectional observational study was carried out using the medical records of patients admitted to ibn rushd psychiatry and addiction hospital which is a large psychiatric teaching hospital in baghdad, iraq. the researcher retrospectively reviewed inpatient medical records over several years (from july 2011 to september 2018) and recorded any inappropriately prescribed cases. inclusion criteria for this study, the inclusion criteria were: 1patient aged 65 years or older 2patient admitted for more than 24 hours 3patients received pharmacological therapy 4patients information was included in the hospital electronic medical record. data collection the research reviewed medical records using a data collection sheet that was specifically designed by the research team to match study goals and it included the following information: 1the age at the time of admission 2gender 3diagnosis 4administered medications 5length of stay 6comorbidities screening tool to assess the appropriateness of prescribed medications the 2015 beers list was used, the following table 1 shows a summary of the criteria used in this study for a medication to be considered inappropriate and was directly derived from the 2015 beers criteria, these criteria were selected to measure the inappropriateness of psychotropic medications only. iraqi j pharm sci, vol.29(1) 2020 pip psychotropic medications 168 table 1 .summary of the criteria used in this study(12) administrative arrangement and ethical approval the study proposal was approved by the ethical committee at the university of baghdad college of pharmacy and permission from ibn-rushd hospital was obtained before conducting this study. the college ethical committee decided that informed consent is not required from patients in case of de-identification patients’ names, addresses and date of birth in addition to lacking direct researcher-patient contact. statistical analysis data were subjected to statistical analysis; data were expressed as mean± standard deviation (sd) of samples. the statistical significance of the differences between various groups was determined by chisquare test using spss software version 22. differences were considered statically significant for p-value < 0.05. results a total number of 6129 medical records were reviewed for the period from july 2011 to september 2018 and 369 patients met the criteria for this study. demographic data mean ± sd of age was 68.59 ± 3.75 years ranging from 65 to 95 years with a male to female ratio of 1.13:1 and 77% of the study patients were aged ≤ 70 years. they have been prescribed a total of 1376 medications with a median of 4 drugs per patient and polypharmacy defined as receiving five or more medications were found in 100 (27.1%) of patients the most frequent diseases that were diagnosed among patients were schizophrenia (33.06%), psychosis with depression (15.44%), then by bipolar disorder (11.32%), psychotic episode due to alcohol consumption (8.94%), and alzheimer disease (4.06%). iraqi j pharm sci, vol.29(1) 2020 pip psychotropic medications 169 the most prescribed drugs were quetiapine (13.44% of all prescribed drugs), procyclidine (9.3%) and haloperidol (7.92%). table 2. patients characteristics and administered medication age: mean (sd) years 68.59(3.75) females: n (%) 160 (43.4%) benzodiazepines: n (%) 239 (17.6%) antipsychotics: n (%) 550 (39.9%) antidepressant: n (%) 213 (14.79%) antiepileptics as mood stabilizer: n (%) 138 (13.28%) anticholinergics for ep*: n (%) 128 (9.3%) other medications: n (%) 63 (4.56%) drugs prescribed per patient: median, )iqr( 4, )2( patients with comorbidities 177 (48%) patients prescribed with 5 drugs or more: n (%) 100 (27.1%) the duration of hospitalization in 216 patients (58.5%) was less than one week, 136 (36.9%) from one week to three weeks and 17 (4.6%) stayed for more than three weeks. among the 369 patients, 177 patients (48%) were complaining from comorbidities the most prevalent diseases were hypertension (63.8%) and diabetes mellitus (35.6%). prevalence of pip pims regardless of patient condition in this study, the overall number of pims that were prescribed inappropriately regardless of patient condition was 323. the most frequent inappropriately prescribed medications were: diazepam (18.27%), quetiapine (16.72%) and alprazolam (10.84%). the distribution of non-conditional pims is shown in table . *extrapyramidal side effect. table 3 . potentially inappropriate medication regardless of patient condition potentially inappropriate medication no. percentage (%) antidepressant 49 15.17% amitriptyline 26 8.05% clomipramine 19 5.88% imipramine 2 0.62% paroxetine 2 0.62% second generation antipsychotics 86 26.63% quetiapine 54 16.72% olanzapine 21 6.50% risperidone 11 3.41% first generation antipsychotics 27 8.36% haloperidol 16 4.95% flupenthixol 6 1.86% chlorpromazine 3 0.93% trifluoperazine 1 0.31% fluphenazine 1 0.31% benzodiazepine 161 49.85% diazepam 59 18.27% alprazolam 35 10.84% clonazepam 30 9.29% chlordiazepoxide 24 7.43% lorazepam 13 4.02% non-benzodiazepine 2 0.62% zolpidem 2 0.62% 323 100.00% iraqi j pharm sci, vol.29(1) 2020 pip psychotropic medications 170 pips due to drug-condition interaction there was 71 pims prescribed to the study patients as follows: benzodiazepines in patients with delirium (30.99%) and benzodiazepines in patients with dementia (19.72%), and antipsychotics in patients with dementia (19.72%). the most frequent class of medications present was benzodiazepines as recorded in 54.94%. table 4 .potentially inappropriate medication regarding patient condition potentially inappropriate medication no. percentage (%) syncope 6 8.45% olanzapine 6 8.45% delirium 28 39.44% benzodiazepine 22 30.99% antipsychotics 5 7.04% chlorpromazine 1 1.41% dementia 28 39.44% benzodiazepine 14 19.72% antipsychotics 14 19.72% falls 7 9.86% benzodiazepine 3 4.23% antipsychotics 3 4.23% anticonvulsant 1 1.41% parkinson 2 2.82% antipsychotics 2 2.82% 71 100.00% the pims due to drug-drug interaction 355 pims were found and this occurred most frequently by inappropriate prescriptions of benzodiazepines (42.5%), followed by antidepressants and antipsychotics (29.6%, and 26.7%) table 5 .potentially inappropriate medication due to drug-drug interaction potentially inappropriate medication no. percentage (%) benzodiazepines 151 42.54% antidepressants 105 29.58% antipsychotics 93 26.20% anticholinergics 6 1.69% 355 100.00% iraqi j pharm sci, vol.29(1) 2020 pip psychotropic medications 171 the total prevalence of pip the prevalence inappropriate psychotropic medications was 74.3%, while the remaining 25.7% received appropriately prescribing medications. figure 1 . prevalence of potentially inappropriate prescribing in this study. the association of pips with certain demographic data, comorbidities, and duration of hospitalization the study revealed that 78.9% of male patients in this study received psychotropic medications inappropriately, with statistically significant association (p=0.018) between gender of patients and prescription of inappropriate psychotropic medications, this study also found a statistically significant association (p=0.022) between the presence of comorbidities and pims prescription, and there was a highly significant association between polypharmacy and being prescribed a potentially inappropriate medication (p<0.001). table 6. association of pips with patient factors *significant difference according to the chi-square test discussion the results of the study shows that the antipsychotics were the most widely used medication by (39.9%) of the total number of geriatric patient enrolled in this study but it wasn’t the most frequent inappropriately prescribed medication due to the exception made by the criteria to geriatric patients diagnosed with schizophrenia, bipolar disorder or acute psychotic episodes that threaten to cause harm to self or others(12). there are several factor that lead to the high prevalence of pips in 74.3% of this study patients such as: most patients admitted due to acute psychological conditions or agitation which in turn required aggressive therapeutic plans to improve patient’s condition, combined with the lack of safer alternative interventions which lead to the increased use of anxiolytics, hypnotics and antipsychotic injections; other factors that may have caused a false increase in pips is due to a flaw in the criteria itself for not providing a rule regarding a duration of medication use to be considered inappropriate which lead to many medications used only once variable pips total (%) n= 369 pvalue yes (%) n= 274 no (%) n= 95 age (years) ≤ 70 213 (74.5) 73 (25.5) 286 (77.5) 0.857 > 70 61 (73.5) 22 (26.5) 83 (22.5) gender male 165 (78.9) 44 (21.1) 209 (56.6) 0.018* female 109 (68.1) 51 (31.9) 160 (43.4) comorbidities yes 141 (79.7) 36 (20.3) 177 (48.0) 0.022* no 133 (69.3) 59 (30.7) 192 (84.3) duration of hospitalization (weeks) < one 155 (71.8) 61 (28.2) 216 (58.5) 0.331 one three 107 (78.8) 29 (21.3) 136 (36.9) > three 12 (70.6) 5 (29.4) 17 (4.6) polypharmacy ≥5 medications 88 (88) 12 (12) 100 (27.1) 0.0002* <5 medications 186 (69.1) 83 (30.9) 269 (72.9) iraqi j pharm sci, vol.29(1) 2020 pip psychotropic medications 172 by the patients to be counted as potentially inappropriate(13–16). however, the result of the present study is similar to (m gutiérrez-valencia et al., 2017)(14) which measured a pip prevalence of 71.5% in hospitalized geriatric patients in acute setting in spain, and also similar to (g. fond et al.,2016)(17) which measured prevalence of pip of psychotropic medications in hospital setting after discharge in france and found it to be 76.1%, another study measuring pips in psychiatric hospital in the netherlands (s. rongen et al.,2016)(16) found a much lower prevalence of 47%. the second major result of this study is that benzodiazepines was the most prevalent pim used, this is also similar to other studies such as (m gutiérrez-valencia et al., 2017)(14) in spain which measured the impact of hospitalization on pip and found benzodiazepines as the most inappropriately prescribed medication, other studies concerning elderly patients treated with psychotropic medications in spain such as (x. vidal et al.,2016)(18) found benzodiazepines as the most prescribed medications as well as the most inappropriately prescribed medication, as mentioned before this result is most likely due to the acute nature of most admissions. it was interesting to notice the differences in prevalence of polypharmacy in geriatric patients among different studies concerning with the subject of potentially inappropriate prescribing ranging from as high as 95% in the study by (mf. najjar et al,.2018)(13) in saudi arabia, 86.5% in the study by (m gutiérrez-valencia et al., 2017)(14) in spain, 79% in the study by (s. rongen et al.,2016)(16)in the netherlands to 29% in a study by (h. cho et al,.2018)(15) in south korea despite being defined in all these studies as patients receiving 5 or more medications, it was also found to be a significant factor for the prescribing of an inappropriate medication in all of these studies similarly to the result of this study even though we found polypharmacy only in 27% of the study participants which can be explained by the methodology of this study which included only listing the medications in the electronic medical chart which doesn’t include medications used for other diseases such as hypertension or diabetes. gender and age were considered to be a significant factor in the prescribing of pim is some studies (g. fond et al.,2016)(17) and (h. cho et al,.2018)(15), and considered to be insignificant in others (x. vidal et al.,2016)(18) , this can be explained by differences in study settings, prescribed medications, and discrepancies in other factors. conclusions there is a high prevalence of potentially inappropriate prescribing among geriatric patients with psychiatric disorders in iraq in addition the factors affecting potentially inappropriate prescribing were gender (male), having a comorbid disease and taking more than 5 medications and found to be strongly associated with being prescribed a potentially inappropriate medication. references 1. o’mahony d, o’sullivan d, byrne s, o’connor mn, ryan c, gallagher p. stopp/start criteria for potentially inappropriate prescribing in older people: version 2. age ageing. 2015;44(2):213218. 2. sera lc, mcpherson ml. pharmacokinetics and pharmacodynamic changes associated with aging and implications for drug therapy. clin geriatr med. 2012;28(2):273-286. 3. nobili a, garattini s, mannucci pm. multiple diseases and polypharmacy in the elderly: challenges for the internist of the third millennium. j comorbidity. 2011;1:28-44. 4. montastruc f, duguet c, rousseau v, bagheri h, montastruc j-l. potentially inappropriate medications and adverse drug reactions in the elderly: a study in a pharmacovigilance database. eur j clin pharmacol. 2014;70(9):1123-1127. 5. wong j, marr p, kwan d, meiyappan s, adcock l. identification of inappropriate medication use in elderly patients with frequent emergency department visits. can pharm j / rev des pharm du canada. 2014;147(4):248-256. 6. cahir c, fahey t, teeling m, teljeur c, feely j, bennett k. potentially inappropriate prescribing and cost outcomes for older people: a national population study. br j clin pharmacol. 2010;69(5):543-552. 7. corsonello a, pedone c, incalzi ra. agerelated pharmacokinetic and pharmacodynamic changes and related risk of adverse drug reactions. curr med chem. 2010;17(6):571-584. 8. dolder cr, mckinsey j. antipsychotic polypharmacy among patients admitted to a geriatric psychiatry unit. j psychiatr iraqi j pharm sci, vol.29(1) 2020 pip psychotropic medications 173 pract. 2011;17(5):368-374. 9. ahmed aia, van den elsen gah, van der marck ma, olde rikkert mgm. cannabinoids for pain in dementia: the good, the bad, and the ugly. j am geriatr soc. 2014;62(5):1001-1002. 10. herrera ap, snipes sa, king dw, torresvigil i, goldberg ds, weinberg ad. disparate inclusion of older adults in clinical trials: priorities and opportunities for policy and practice change. am j public health. 2010;100(suppl 1):s105. 11. shenoy p, harugeri a. elderly patients’ participation in clinical trials. perspect clin res. 2015;6(4):184. 12. samuel mj. american geriatrics society 2015 updated beers criteria for potentially inappropriate medication use in older adults. j am geriatr soc. 2015;63(11):2227-2246. 13. najjar mf, sulaiman sas, al jeraisy m, balubaid h. the impact of a combined intervention program: an educational and clinical pharmacist’s intervention to improve prescribing pattern in hospitalized geriatric patients at king abdulaziz medical city in riyadh, saudi arabia. ther clin risk manag. 2018;14:557-564. 14. gutiérrez-valencia m, izquierdo m, malafarina v, et al. impact of hospitalization in an acute geriatric unit on polypharmacy and potentially inappropriate prescriptions: a retrospective study. geriatr gerontol int. 2017;17(12):2354-2360. 15. cho h, choi j, kim ys, et al. prevalence and predictors of potentially inappropriate prescribing of central nervous system and psychotropic drugs among elderly patients: a national population study in korea. arch gerontol geriatr. 2018;74(september 2016):1-8. 16. rongen s, kramers c, o’mahony d, feuth tb, olde rikkert mgm, ahmed aia. potentially inappropriate prescribing in older patients admitted to psychiatric hospital. int j geriatr psychiatry. 2016;31(2):137-145. 17. fond g, fajula c, dassa d, brunel l, lançon c, boyer l. potentially inappropriate psychotropic prescription at discharge is associated with lower functioning in the elderly psychiatric inpatients. a cross-sectional study. psychopharmacology (berl). 2016;233(13):2549-2558. 18. vidal x, agustí a, vallano a, et al. elderly patients treated with psychotropic medicines admitted to hospital: associated characteristics and inappropriate use. eur j clin pharmacol. 2016;72(6):755-764. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 campsis grandiflora doi: https://doi.org/10.31351/vol31iss1pp176-183 176 isolation of beta-sitosterol and evaluation of antioxidant activity of iraqi campsis grandiflora flowers sarah saad hasson *,1, ibrahim saleh abbas ** and bahir abdul razzaq mshimesh*** *department of pharmacognosy and medicinal plants, college of pharmacy, mustansiriyah university, baghdad, iraq. **department of pharmacognosy and medicinal plants, college of pharmacy, mustansiriyah university, baghdad, iraq. ***department of pharmacology and toxicology, college of pharmacy, mustansiriyah university, baghdad, iraq. abstract campsis grandiflora (bignoniaceae) is a fast growing deciduous climber, the dried flowers have been used as a carminative, blood tonic, and febrifuge in chinese traditional medicine. this plant has an antiinflammatory, anti-oxidant, anti-depressant, and anti-bacterial effect; with a beneficial role in stagnant blood and endometriosis conditions. in this study, the detection of beta-sitosterol in the hexane extract of iraqi c.grandiflora flowers was performed using thin layer chromatography (tlc) and high performance liquid chromatography(hplc); while the isolation done by preparative layer chromatography then structure elucidation of isolated compound was done by ftir and 1hnmr. furthermore, assessment of the anti-oxidant activity of the ethyl acetate extract of iraqi c.grandiflora flowers using three different methods and total flavonoid content, then measuring the pearson’s correlation coefficient between these methods. the results showed that the hexane extract of iraqi c.grandiflora flowers contain beta-sitosterol compound and the ethyl acetate extract of this plant possesses an excellent anti-oxidant effect using the single-electron transfer (set) pathway in scavenging the free radicals, and this activity attributed to the potent antioxidant i.e. polyphenols. keywords: anti-oxidant activity, betasitosterol, campsis grandiflora, total flavonoids content. ألزهار نبات البوق الزاحف العراقي لألكسدة ةالمضاد مركب البيتا سيتوستيرول وتقييم الفعاليةعزل *** مشيمش باهر عبد الرزاق و ** ابراهيم صالح عباس ،1*’ سارة سعد حسون العراق ، المستنصرية، بغداد الجامعة، الصيدلة كلية الطبية، فرع العقاقير والنباتات * العراق ، بغداد ، الجامعة المستنصرية ، كلية الصيدلة الطبية، فرع العقاقير والنباتات ** العراق ، بغداد ، المستنصريةالجامعة ، الصيدلةكلية والسموم، فرع االدوية *** الخالصة تم ايلتددام الزهو المجضضة لذاا النبات كماد اا د للري ، منطل هو متسلل نضيلس يلريل النمو ، )عائلة البنونية( نبات البوق الزاحف ا مل دو للدم ، وميلاد للمم فس الط التقليد الصلينسه هاا النبات لت تيرير ميلاد لهلتذابات ض ميلاد لةكسلد ض ميلاد لهكتضاد وميلاد للب تيري د ايلة تم ال طلف عم مرك البيتا يليتويلتيرو فس مسلتدلك الذ سلار نبها نبات مضيد فس حاالت كود الدم واالنتباذ البطانس الرحمسه فس هاه ال بينما تم العز بوايللطة ( hplcوال روماتوارافيا السللائلة عالية اندا ) (tlc)البوق الزاحف العراقس بايللتددام كروماتوارافيا الطبقة الرقيقة عهو عل ذلك ، (hnmrو ftirكجر ال يميائس للمرك المعزو بوايلطة) رم تم توضلي السلتر ((plcكروماتوارافيا الطبقة التميليرية وقياس ممتوى بييلللتددام رهر ارق مدتلضة تم تقييم النطلللاا الميلللاد لةكسلللد لمسلللتدلك ليللليتات اتيايب نبها نبات البوق الزاحف العراقس لظذرت النتائج لر مسلللتدلك الذ سلللار مم لبها نبات البوق الزاحف الضهفونويد ال لسض رم حسلللاد معامب اال تباا )بيريلللور( بيم هاه الطرقه ا ميلادما لةكسلد بايلتددام ا ممتابم مسلا نقب العراقس يمتو عل مرك البيتا يليتويلتيرو ولر مسلتدلك ليليتات اتيايب لذاا النبات يمتلك تيريرم إل ميادات انكسد القوية ماب البوليضينو هالجاو المر ، و يُعزى هاا النطاا كس فس (set) اتل ترور الضرد . محتوى الفالفونويد الكلي ، نبات البوق الزاحف، بيتاسيتوستيرول ، الكلمات المفتاحية: الفعالية المضادة لالكسدة introduction campsis grandiflora (bignoniaceae) is a fast growing deciduous climber, native to central and southern china; its flowers are hermaphrodite, curled, and bright orange-red in color. this plant blooms for about eight months, between the last week of march and the second week of october(1).flowering of c.grandiflora plant belongs to the cornucopia pattern, in which a large number of plant flowers are produced in each inflorescence for about several months(2). the dried flowers of this plant, also known as ''aborticide'' in chinese folk, have been used as a carminative, blood tonic, febrifuge, and depurative diuretic in chinese traditional medicine. rheumatoid pains and menstrual problems exacerbated by blood stagnation, swelling breast after child birth, rubella, bleeding rectum, and diabetes have been treated with a decoction of this flowers(3,4). c.grandiflora flowers have several pharmacologic activities e.g. cellular protection and anti -oxidant effect(3), anti 1corresponding author e-mail: sara.saad.hassoon@gmail.com received: 27/8/2021 accepted: 22/9 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp176-183 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 177 -depressant effect(3), with a beneficial role in stagnant blood, and endometriosis conditions(6).the objective of this study was detection and isolation of the beta-sitosterol present in the iraqi c.grandiflora flowers with the evaluation of the anti-oxidant effect and total flavonoid content of the ethyl acetate extract of iraqi c.grandiflora flowers and measuring the correlation between these methods. materials and methods plant material collection flowers of campsis grandiflora were collected from baghdad – hay aljamea`a in july 2020. the plant material was authenticated at the university of baghdad / college of sciences by a professional taxonomist. flowers were separated from the rest of the plant and washed thoroughly. it was then dried under shade conditions at room temperature for seven days. after that, the dried flowers were ground in a mechanical grinder and finally stored in a glass container at 4◦c. chemicals and reagents beta-sitosterol and quercetin standards were purchased from changdu biopurify; hexane, ethanol, acetone, dpph, tptz, abts, sodium nitrite were obtained from sigma aldirch. ethyl acetate, methanol, fecl3 hexahydrate, potassium persulfate, chloroform and toluene were supplied from alpha chemika, ascorbic acid was purchased from merk, sulphuric acid and aluminum chloride were obtained from bdh. preparation of extract for the detection and isolation of compound 1 powdered plant (100g) was extracted by soxhlet apparatus with n-hexane (1200ml) till exhaustion. the extract was dried using a rotary evaporator, weighted and labelled as hexane extract (he)(7). tlc analysis an aliquot of hexane extract dissolved in about 2ml of hexane then applied to analytical tlc plate against standard beta-sitosterol, and developed in the following mobile phases(mp) (8–11): • mp1: aceton:hexane (1:3) • mp2: hexane :ethyl acetate (7:2) • mp3: toluene: chloroform :ethyl acetate (5:4:1) • mp4: chloroform :acetone (9:1) the plates were sprayed with anisaldehyde – sulphuric acid reagent followed by heating for the detection of distinct spots. hplc analysis one milligram from hexane extract and standard beta-sitosterol was dissolved separately in hplc grade methanol (1ml) using a mobile phase consisting of acetonitrile:methanol(70: 30, v/v). the flow rate was 1 ml/min, and the detector was monitored at 210 nm(12). isolation of compound 1 hexane extract (1.5 gram) was dissolved in hexane and conducted on preparative layer chromatography plates (plc) against standard betasitosterol(13) and developed in mp2 mobile phase(10). the detection was done by spraying the plates' side by anisaldehyde-sulphuric acid reagent followed by heating. the bands at rf = 0.32 were scrapped off and the scrapped silica then eluted with warmed hexane, the purity of the c1 compound was confirmed by analytical tlc using the mp4 mobile phase. spectral analysis of the isolated compound 1 the isolated compound was subjected to different spectral analysis e.g. ftir and 1hnmr. preparation of extract for the anti-oxidant assay twenty-five gram of powdered plant material was defatted with 250 ml n-hexane by soxhlet apparatus. the marc was further extracted with ethanol 80% (250 ml) till exhaustion also by using the soxhlet apparatus. firstly, the ethanolic extract was dried by rotary evaporator and weighted. secondly, the dried extract was dissolved in 20 ml distilled water and partitioned with ethyl acetate (3×50 ml). the upper layer was collected, dried by rotary evaporator, weighted, labelled as ethyl acetate extract (eae) and stored at 4◦c while the lower aqueous layer was discarded(14). the dpph [ 1,1–diphenyl – 2 picryl–hydrazyl] assay the dpph assay was performed using a colourimetric method described by anna floegel and coworkers(15) in which a methanolic dilution of dpph was used. (2.95) ml of dpph solution (1 mm dpph in 80% methanol ) was mixed with (0.05) ml of eae or ascorbic acid (concentrations ranging from 12.5 to 200 mcg per 1 ml methanol). the assay was performed in triplicate, and the developing mixture was incubated in the dark for 30 minutes with aluminium foil over it. at 517 nm, a decrease in the absorbance was observed. the control was prepared by replacing the amount of eae or standard by 80% methanol. the findings were reported as a percentage of radical scavenging and estimated using the formula below(16): % 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = (𝐴0 − 𝐴1) 𝐴0 × 100 𝐴0 is the absorbance of the control, 𝐴1 is the absorbance of plant extract (eae) or standard ascorbic acid. the abts [2,2'–azino – bis (3– ethylbenzothiazoline–6–sulphonic acid) diammonium salt ] assay a colourimetric method(17) has been used to measure the antioxidant activity of eae against abts•+ radical in which aqueous solutions (1:1) of abts (7 mm) and potassium persulphate (140 mm) were mixed. then, this mixture was incubated in a iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 178 dark place for 12 hours at room temperature enabling the radical abts•+ to formed. after that, (1.96) ml from the previous solution was mixed with (0.04) ml of eae or ascorbic acid (concentrations ranging from 12.5 to 200 mcg per 1 ml ethanol) and let to sit at room temperature. the assay was performed in triplicate, and the decrease in the absorbance was measured at 734 nm. the control was prepared by replacing the amount of eae or standard by ethanol, and the findings were reported as a percentage of radical scavenging using the previously mentioned formula. the frap (ferric reducing antioxidant power) assay the frap assay was carried out using a colourimetric method(18) in which 3.8 ml of frap reagent was mixed with 0.2 ml of eae or ascorbic acid (concentrations ranging from 12.5 to 200 mcg per 1 ml methanol). the frap reagent (10:1:1) was made by incorporating 10 parts of sodium acetate buffer solution (300 mm, ph=3.6) with 1 part from each of ferric chloride hexahydrate (20 mm) and tptz (10 mm). the assay was performed in triplicate, and the mixture was kept at 37◦c for 30 minutes. the absorbance was determined at 593 nm, and the control was made by replacing the diluted sample with the same volume of methanol. the findings were expressed in microgram of ascorbic acid equivalents (aaes) per milligram of dried eae(19) and calculated using the formula from the ascorbic acid standard curve: 𝑦 = 0.0025𝑥 + 0.1802 𝑦 is the sample's absorbance at 593nm, 𝑥 is the concentration of ascorbic acid equivalent measured in mcg/ml. the total flavonoids content (tfc) the total flavonoids content of iraqi c.grandiflora was estimated by using the aluminium chloride colourimetric method(20) in which 1ml of plant eae extract (100 mcg/ml) or standard quercetin solution (5,10,20,40,60,80 and 100 mcg/ml ) was mixed with 4ml of distilled water and then adding 0.3 ml of sodium nitrite solution (5%) to all test tubes. after five minutes, 0.3 ml of aluminium chloride solution (10%) was applied, followed subsequently by 2 ml of sodium hydroxide solution (1%). the assay was performed in triplicate, and the mixture was mixed well, then, the absorbance was measured at 510 nm against blank. total flavonoid content (tfc) has been determined using an equation derived from the quercetin standard curve(20) 𝑦 = 0.0009𝑥 + 0.0283 y is the sample's absorbance at 510 nm, x is the concentration of the flavonoids in the sample measured in mcg/ml. statistical analysis the pearson’s correlation coefficient (r) was computed using the spss-16.0 (statistical packages for social sciencesversion 16), the correlation is significant at (p≤0.05). results and discussion tlc results phytosterol, such as beta-sitosterol, was reported in c.grandiflora flower extract(21). the results in (table1) showed a matching in the rf values of both beta-sitosterol and compound 1(c1) spots in four mobile phases, and this finding suggested the presence of beta-sitosterol in hexane extract of iraqi c.grandiflora flower, as shown figure.1. table 1. the rf values of compound 1(c1) as compared with the rf values of standard betasitosterol. figure1.analytical tlc for the hexane extract and beta-sitosterol using mp1 solvent system:(a) c1 spot, (b) standard beta-sitosterol spot. hplc results the peak at (6.973) minutes in the hplc analysis of hexane extract of iraqi c.grandiflora flowers was matched the peak of standard beta sitosterol with a retention time (6.913) minutes, as shown in figure 2. mobile phase the rf value of c1 the rf value of standard betasitosterol mp1 0.51 0.50 mp2 0.32 0.33 mp3 0.42 0.43 mp4 0.69 0.67 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 179 figure2.the hplc analysis: (i) hexane extract of iraqi c.grandiflora flowers, (ii) standard beta-sitosterol. isolation of compound1 hexane extract of c.grandiflora flowers was applied to plc plates to give 52 mg (3.46 % w/w) of c1, and then the purity was confirmed using analytical tlc plate, as shown in figure 3. fourier transform infrared spectrometry (ftir) of compound1 the wavenumbers of the isolated compound (c1) showed a characteristic broad peak at 3242-3439 cm-1 that confirmed a hydroxyl group with peaks at 3007 and 1641 cm-1 that established a double bond. these results were confirmed by comparing the wavenumbers with the reported literature(22),as in figure 4 figure 3. isolation and purity confirmation of c1 from hexane extract: (i) plc plate for isolation using mp2, (ii) tlc plate for purity confirmation using mp4, (a)c1 spot, (a') standard beta-sitosterol spot. figure 4. the ir spectrum of the isolated compound 40060080010001200140016001800200024002800320036004000 1/cm 45 52.5 60 67.5 75 82.5 90 97.5 %t 34 39 .1 9 33 77 .4 7 33 50 .4 6 30 07 .1 2 29 26 .1 1 28 54 .7 4 17 45 .6 4 16 41 .4 8 14 54 .3 8 13 73 .3 6 12 42 .2 0 11 51 .5 4 1 08 0. 17 10 28 .0 9 93 9. 36 86 4. 14 75 8. 05 71 3. 69 57 8. 66 53 0. 44 48 4. 15 1 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 180 nuclear magnatic resonance (nmr) of compound1 the isolated compound 1 (c1) exhibited four distinct chemical shifts in the 1hnmr spectrum (figure5), which reflected the active functional groups. the chemical shifts began at 0.65 parts per million (ppm), which correspond to the hydrogens of saturated hydrocarbons, followed by the allylic protons, which have a chemical shift of 2.1 ppm. at 4.62 ppm, a proton next to a carbon adjacent to heteroatom can be observed. finally, at 5.32 ppm, the vinylic proton can be seen. figure 5. the 1hnmr spectrum of c1 the results of the different chromatographic techniques and various spectral analysis proved that the compound 1 was beta-sitosterol. the dpph assay results the dpph radical is a popular substrate for quickly determining the antioxidant activity of biological samples because of the simplicity of the assay and the stability of radical. as the dpph radical is quenched by the antioxidant using the single electron transfer (set) and hydrogen atom transfer (hat) reaction mechanisms, the color of the dpph solution changes from purple to yellow and the absorbance was read at 517 nm(23). the results of dpph assay were illustrated in table (2) and more visually expressed in figure. 6. table 2.the dpph-radical scavenging activity of c.grandiflora eae dpph :1,1-diphenyl-2-picryl-hydrazyl, eae: ethyl acetate extract of iraqi c.grandiflora. figure 6.the antioxidant activity of eae measured by dpph method and compared with ascorbic acid standard: (eae) ethyl acetate extract of iraqi c.grandiflora,(dpphassay)1,1diphenyl-2-picryl-hydrazyl assay. the abts assay results the abts can react with potassium persulfate in an oxidation reaction in which the colored-abts radical is formed. the degree of decolorization indicates that the antioxidants had the ability to transfer electrons or hydrogen atoms to inactivate this radical(24). the results of abts assay were illustrated in table (3) and more visually expressed in figure 7. dpph assay conc. of vit c or extract (mcg/ml) % scavenging activity of vit c % scavenging activity of eae extract 12.5 22.90 17.63 25 39.00 40.43 50 57.60 48.68 100 74.06 52.50 200 86.03 71.69 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 181 table 3.the abts-radical scavenging activity of c.grandiflora eae. abts: [2,2'–azino – bis (3– ethylbenzothiazoline – 6– sulphonic acid) diammonium salt], eae: ethyl acetate extract of iraqi c.grandiflora. the frap assay results in frap assay, the antioxidant activity was assessed by measuring the capacity of the antioxidants in the eae to convert ferric (fe+3) to ferrous (fe+2) in a redox-linked colorimetric assay using single electron transfer (set) as a reaction mechanism(25). the results were expressed as ascorbic acid equivalents (aaes), measured in microgram ascorbic acid per milligram dried eae, depending on the equation from ascorbic acid standard curve (table 4). figure 7. the antioxidant activity of eae measured by abts method and compared with ascorbic acid standard: (eae) ethyl acetate extract of iraqi c.grandiflora, (abts assay ) 2,2'azino–bis(3–ethylbenzothiazoline–6– sulphonic acid) diammonium salt. table 4. the antioxidant activity measured by the frap method. each value represents the mean±sem of 3 samples; frap: ferric reducing antioxidant power, eae:ethyl acetate extract of iraqi c.grandiflora, aaes: ascorbic acid equivalents. the total flavonoids content result the main antioxidant compounds in plants are phenols, which have an aromatic ring that enables the unpaired electrons in their arrangement to be stabilized and relocated, allowing electrons and hydrogen atoms to be donated from their hydroxyl groups(18). since phenolic compounds are one of the main classes of compounds known to serve as primary antioxidants, and the litreture have shown a good correlation between the total flavonoids content (tfc) and the antioxidant activity(23), it's essential to calculate the amount of these compounds that present in the eae. the results of the total flavonoids content illustrated in table (5). table 5. the total flavonoid content of eae of iraqi c.grandiflora. each value represents the mean±sem of 3 samples; eae: ethyl acetate extract of iraqi c.grandiflora. the pearson's correlation coefficient results the data of pearson's correlation coefficient between the three different antioxidant methods and between these methods and tfc were summerized in table (6). abts assay conc. of vit c or extract (mcg/ml) % scavenging activity of vit c % scavenging activity of eae extract 12.5 23.43 15.97 25 37.93 15.35 50 52.43 28.89 100 64.7 37.92 200 71.96 45.10 eae concentration (mcg/ml) eae absorbance (nm) mcg ascorbic acid equivalent (mcg/ml) aaes (mcg/mg dried extract) 12.5 0.190±0.005 3.92±2.309 313.6±184.7 25 0.216±0.017 14.58±7.055 583.46±282.2 50 0.265±0.011 34.18±4.42 683.73±88.4 100 0.362±0.025 72.72±10.283 727.2±102.8 200 0.590±0.005 163.92±2.309 819.6±11.5 eae concentration (mcg/ml) eae absorbance (nm) mcg quercetin equivalent (mcg/ml) mcg quercetin equivalent /mg eae 100 0.0903±0.0008 68.925±0.979 689.25±9.799 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 182 table 6.the pearson's correlation coefficient. dpph assay :1,1-diphenyl-2-picryl-hydrazyl assay ; abts assay : 2,2'–azino – bis (3– ethylbenzothiazoline – 6– sulphonic acid) diammonium salt assay ; frap assay : ferric reducing antioxidant power assay ; tfc : total flavonoid content ; * : means correlation is significant at (p≤0.05). the results demonstrated that iraqi c.grandiflora ethyl acetate extract possesses strong antioxidant capacity measured with dpph, abts, and frap assays compared with ascorbic acid. these findings were consistent with another study in which c.grandiflora ethyl acetate extract exhibit the highest radical scavenging potency against dpph and abts radicals with ic50=1.625mcg/ml; this result was excellent when compared to ic50 of ascorbic acid (8.6 mcg/ml), also the ic50 of other fractions such as ethanol, petroleum, butanol, and water were (8.75, 32.25, 10, 7.6 mcg/ml), respectively(3). there was a positive correlation between the dpph, abts, and frap assays, and this may be explained by the fact that these assays were suitable and accurate for determining the plant extracts' overall antioxidant capacities; these results were agreed with another research(26) in which a high correlation between these methods have been showed. a significantly high correlation coefficient was obtained from the dpph and fraps assays (0.976) with a p-value ≤0.05. these findings suggested that the antioxidants in eae of iraqi c.grandiflora were used the single-electron transfer (set) pathway in scavenging the free radicals; since the dpph and frap assays were based on the set pathway as a reaction mechanism. there was a negative correlation between the antioxidant assays and the tfc, and these findings pointed that, although there are a relatively high amount of flavonoids in the extract but there are various factors affecting the anti-oxidant activity of a substance and results in negative correlation, these include substance solubility, ph of the medium, oxidation state, etc(23). these results suggested that flavonoids not only the major contributor to the antioxidant effect of this plant , but there are other phytochemicals i.e. the non-flavonoidal compounds such as acetoside, caffeic acid, gallic acid, etc. that reported in c.grandiflora flower extract may contribute to this excellent activity since these compounds also exhibited a pronounced antioxidant effects(27,28). conclusion beta-sitosterol was isolated form the hexane extract of iraqi c.grandiflora flowers, and this compound was confirmed with different chromatographic and spectroscopic techniques. moreover, 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testing methods on antimicrobial and antioxidant activities of selected medicinal essential oils. int food res j. 2017;24(6):2616– 24. 27. gülçin i. antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). toxicology. 2006;217(2–3):213–20. 28. cardinali a, pati s, minervini f, d’antuono i, linsalata v, lattanzio v. verbascoside, isoverbascoside, and their derivatives recovered from olive mill wastewater as possible food antioxidants. j agric food chem. 2012;60(7):1822–9. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 89 preparation and evaluation of liquid and solid self-microemulsifying drug delivery system of mebendazole ahmed a. hussein *,1 * department of pharmaceutics, college of pharmacy, university of baghdad,baghdad, iraq abstract the aim of present study was to develop solid and liquid self-microemulsifying drug delivery system of poorly water soluble drug mebendazole using aerosil 200 as solid carrier. microemulsions are clear, stable, isotropic liquid mixtures of oil, water and surfactant, frequently in combination with a co-surfactant having droplet size range usually in the range of 20-250 nm. oleic acid, tween 80 and polypropylene glycol were selected as oil, surfactant and co-surfactant respectively and for preparation of stable smedds, micro emulsion region was identified by constructing pseudo ternary phase diagram containing different proportion of surfactant: cosurfactant (1:1, 2:1 and 3:1), oil and water. in brief s/ cos mix means surfactant to co-surfactant and oil were mixed at ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 manner. to the resultant mixtures, water was added drop wise till the first sign of turbidity in order to identify the end point and after equilibrium; if the system became clear then the water addition was continued. prepared optimised formula of microemulsion was evaluated for sem, particle size analysis, polydispersity index, phase separation, viscosity determination, zeta potential, invitro dissolution study and in vivo studies. the optimized microemulsion was converted into solid form by spray drying technique by using aerosil 200 as solid carrier. prepared smedds was characterized for same parameters as that of microemulsion. solid smedds of mebendazole prepared using aerosil 200 by spray drying technique showed good drug content uniformity. after reconstitution it formed microemulsion with micrometric range. in-vitro drug release and in-vivo plasma drug concentration of microemulsion and smedds was much higher than that of marketed praparation. hence lipid based drug delivery system may efficiently formulate microemulsion and it can be solidified easily by spray drying technique which enhances dissolution rate and thus concomitantly bioavailability. in conclusion ,self micro emulsifying drug delivery system has become promising tool to overcome shortcomings associated with conventional delivery. kew words: self-microemulsifying drug delivery system, microemulsion, mebendazole. تحضٍر وتقٍٍى نظاو الٌصال اندواء عهى شكم سائم وصهة ذاتً االستحالب انى انًبٍندازول حسٍن احًد عباس ،*1 .انؼشاق،ثغذاد،خبيؼخ ثغذاد،كهٛخ انصٛذنخ ،فشع انصٛذالَٛبد * الخالصة انزٔثبَٛخ انًجُٛذاصٔل ثأسزخذاو انٓذف يٍ انذساسّ انًقذيخ ْٕ رطٕٚش َظبو صهت ٔسبئم رارٙ االسزسالة نهؼقبس انقهٛم االٚشٔصٚم كُبقم. انًسزسهت انًبٚكشٔ٘ شفبف ٔيسزقش ٔيضٚح يٕزذ يٍ انضٚذ ٔانًبء ٔانسٛشفكزبَذ ٔغبنجب يبٚسزخذو يغ ٔانجشٔثهٍٛ كالٚكٕل ٚسزخذيٌٕ كضٚذ 02َبَٕيٛزش. االٔنٛك اسذ ,انزٍٕٚ 052-02كٕسٛشفكزبَذ نّ زدى قطشِ ٚزشأذ يٍ ذ ٔكٕسٛشفكزبَذ ثبنزؼبقت ٔرى رؼٍٛٛ انًسزسهت انًبٚكشٔ٘ ثبسزخذاو يخطط ثالثٙ انسبنخ ٚسزٕ٘ َست يخزهفّ يٍ ٔسٛشفكزبَ انسٛشفكزبَذ ٔانكٕسٛشفكزبَذ. نهًضٚح انُبرح ٚضبف انًبء رذسٚدٛب نسٍٛ أل يُطقّ يٍ انزؼكش نكٙ رؼٍٛٛ َقطخ انُٓبٚخ ثؼذ انزٕاصٌ ارا اصجر انُظبو ٔاضر يؤشش اضبفخ انًبء. انصٛغّ االيثم انًسضشح يٍ انًسزسهت انًبٚكشٔ٘ قٛٛى نًسر انًدٓش االنكزشَٔٙ, زدى اندضٚئّ,ٚسزًش ثؼذ رنك انزشزذ انًزؼذد , زبنخ االَفصبل، ٔقًٛخ انهضٔخخ، صٚزب انًسزًهخ، فٙ دساسخ زم انزدبسة انًخزجشٚخ ٔانذساسبد انًدشاح. انًسزسهت كُبقم صهت. انًسزسهت انًبٚكشٔ٘ 022هت ثٕاسطخ ثخبش رقُٛخ انزدفٛف ثبسزخذاو االٚشٔسٛم انًبٚكشٔ٘ االيثم رى رسٕٚهّ انٗ ص 022انصهت ٚقٛٛى نُفس انؼٕايم نهًسزسهت انًبٚكشٔ٘ انسبئم. انًسزسهت انًبٚكشٔ٘ انصهت نهًجُٛبدٚضٔل انًسضش ثبسزخذاو اٚشٔسٛم ٚظٓش يسزٕٖ خٛذ نهًسزٕٖ. بٚكشٔ٘ ركٌٕ ثبنًذٖ انًبٚكشٔ٘. يؼذل رسشٚش انؼقبس داخم ٔخبسج اندسى نهًسزسهت انًبٚكشٔ٘ اػهٗ ثؼذ اػبدح انزٔثبَّٛ انًسسهت انً يٍ انًسضش ردبسٚب. ثبنزبنٙ ٔخذ اٌ َظبو انؼقبس انًسزُذ نهذٌْٕ ًٚكٍ اٌ ٚكٌٕ يؤثش كًسزسهت يبٚكشٔ٘ ًٔٚكٍ اٌ ٚزصهت ثسٕٓنخ نؼقبس ٔنزنك رضداد انزٕافش انجبٕٚنٕخٙ. انُظبو انزارٙ انًسزسهت نهؼقبس اصجر ثٕاسطخ رقُٛخ ردفٛف انشرار ٔانز٘ ٚضٚذ يٍ يؼذل رسشس ا كزقُّٛ ٔاػذِ نزدبٔص االَظًّ انزقهٛذّٚ. . يبٍندازولنظاو دوائً ذاتً االستحالب ، يستحهة ياٌكروي ،نكهًات انًفتاحٍة :ا 1 corresponding author e-mail: ahmed_abbas201383@yahoo.com received: 5/4/2014 accepted:4/5/2014 iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 90 introduction microemulsions are clear, stable, isotropic liquid mixtures of oil, water and surfactant, frequently in combination with a co-surfactant having droplet size range usually in the range of 20-250 nm. its aqueous phase may contain salt and/or other ingredients, and the "oil" may actually be a complex mixture of different hydrocarbons and olefins. if we compare it with ordinary emulsions , microemulsions form upon simple mixing of the components and do not require the high shear conditions generally used in the formation of ordinary emulsions (1) . there are two basic types of microemulsions one is direct (oil dispersed in water, o/w) and other is reversed (water dispersed in oil, w/o). in ternary systems such as microemulsions, where two immiscible phases (water and oil) are present with a surfactant, the surfactant molecules may form a monolayer at the interface between the oil and water, with the hydrophobic tails of the surfactant molecules dissolved in the oil phase and the hydrophilic head groups in the aqueous phase (2) . as in the binary systems (water and surfactant or oil and surfactant), selfassembled structures of different types can be formed. a self microemulsifying drug delivery system (smedds) typically comprises a mixture of surfactant, oil and drug which when introduced in the body to form droplet of approximately the same size range as those observed in the microemulsion system (3) . the release of the drug compound from smedds takes place upon its partitioning into the intestinal fluids during droplet transport and disintegration along the gastrointestinal tract. it was proposed that two main factors, small particle size and polarity of the resulting oil droplets determine the efficient release of the drug compound from smedds. in o/w microemulsions, however, the impact of the polarity of the oil droplets is not very significant because the drug compound reaches the capillaries incorporated within the oil droplets (3) . many studies carried out in animals for the assessment of the oral bioavailability of hydrophobic drugs formulated in o/w emulsions indicated better absorption profiles but, the use of these systems is limited due to their poor physical stability and the large volumes needed. thus, smedds may be a promising alternative to orally administered emulsions because of their relatively high physical stability and ability to be delivered in standard soft gelatine capsule (4) . mechanism of self emulsification self-emulsification takes place when the entropy change favoring dispersion is greater than the energy required to increase the surface area of the dispersion. the free energy of a conventional emulsion formulation is a direct function of the energy required to create a new surface between the oil and water phases. the two phases of the emulsion tend to separate with time to reduce the interfacial area and thus the free energy of the systems (5) . the conventional emulsifying agents stabilize emulsions resulting from aqueous dilution by forming a monolayer around the emulsion droplets, reducing the interfacial energy and forming a barrier to coalescence. on the other hand, emulsification occurs spontaneously with sedds because the free energy required to form the emulsion is either low and positive or negative. it is necessary for the interfacial structure to show no resistance against surface shearing in order for emulsification to take place (4) . the ease of emulsification was suggested to be related to the ease of water penetration into the various liquid crystal (lc) or gel phases formed on the surface of the droplet. the interface between the oil and aqueous continuous phases is formed upon addition of a binary mixture (oil/non-ionic surfactant) to water. this is followed by the solubilisation of water within the oil phase as a result of aqueous penetration through the interface. this will occur until the solubilisation limit is reached close to the interphase. further aqueous penetration will lead to the formation of the dispersed lc phase (3) . in the end, everything that is in close proximity with the interface will be lc, the actual amount of which depends on the surfactant concentration in the binary mixture. thus, following gentle agitation of the selfemulsifying system, water will rapidly penetrate into the aqueous cores and lead to interface disruption and droplet formation (3) . according to reiss, the energy required to increase the surface area of the dispersion for self emulsification process bear less importance when compared to the entropy change that favours dispersion. self microemulsification is related to the free energy. that is free energy of conventional emulsion is direct function of the energy essential to create a new surface between the oil and water phases and can be described by equation (4) , dg = s n p r 2s where, dg is the free energy related to the process, n is the number of droplets of radius, r and s represents the interfacial energy. the iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 91 emulsifying agent forms a monolayer of emulsion droplets , and hence reduces the interfacial energy, and providing the barrier to avoiding coalescence. in case of self emulsification , the free energy required to form the microemulsion is either very low or positive or negative. emulsification requires very low energy involves destabilisation through contraction of local interfacial regions (1) . the aim of present study was to develop smedds of poorly water soluble drug mebendazole using aerosil 200 as solid carrier. microemulsion was prepared using oleic acid, tween 80 and propylene glycol as oil, surfactant and co-surfactant respectively and was converted to solid smedds by adsorbing it on aerosil 200. prepared optimised formula of microemulsion was evaluated for sem, particle size analysis, polydispersity index, phase separation, viscosity determination, zeta potential, in vitro dissolution study and in vivo studies. materials and methods materials the following materials were used : mebendazole ( ciron pharma ltd. mumbai). oleic acid, tween-80 ,propylene glycol, dimethyl sulfoxide, methanol (research lab fine chem industry, mumbai). dialysis membrane (12000 da) (hi media), sodium hydroxide and acetonitril (sd fine chem). methods solubility study the solubility of mebendazole in various oils, surfactant, and cosurfactant was determined by: an excess amount of mebendazole powder was added to 2 ml of vehicle (oil, surfactant, cosurfactant), shaken in a mechanical shaker at 37 0 c for 48 hrs, and centrifuged at 5,000 rpm for 15 min. supernatant was diluted with methanol for the quantification of mebendazole and analysed by uv spectrometer (2) . preparation of microemulsion construction of pseudo-ternary phase diagram oleic acid, tween 80 and polypropylene glycol were selected as oil, surfactant and cosurfactant respectively and for preparation of stable smedds, microemulsion region was identified by constructing pseudo ternary phase diagram containing different proportion of surfactant: co-surfactant (1:1, 2:1 and 3:1), oil and water. in brief s/ cos mix means surfactant to co-surfactant and oil were mixed at ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1 manner. to the resultant mixtures, water was added drop wise till the first sign of turbidity in order to identify the end point and after equilibrium; if the system became clear then the water addition was continued (6-8) . selection of optimised ratio all ratio of s/co gives region in pseudo ternary phase diagram and compared among them to select optimized ratio. preparation of mebendazole microemulsion mebendazole was added to the mixtures of oil, surfactant, and co-surfactant with varying percentage as described in table 1 and then an appropriate amount of water was added to the mixture drop by drop with constant stirring on magnetic stirrer. microemulsions containing mebendazole was obtained spontaneously on stirring the mixtures. all the formulations were stored at appropriate temperature. table( 1)composition of microemulsion formulation at 2:1 (oil to s/cos) evaluation of microemulsion the optimised formulations were evaluated for the following characteristics: optical transparency optical transparency of the formulas was determined by inspecting the sample in clear and transparent container under the presence of good light against reflection into the eyes, and viewed against black and white illuminated background. phase separation microemulsion system were subjected to centrifugation at 15,000 rpm for a period of 15 batch code mebendazole(mg) oleic acid (mg) tween-80 (mg) propylene glycol (mg) water (ml) b1 5.18 1.5 20 10 68.5 b2 5.18 2.5 20 10 67.5 b3 5.18 3 20 10 67 b4 5.18 4 20 10 66 b5 5.18 5.5 20 10 64.5 b6 5.18 6.5 20 10 63.5 b7 5.18 8.5 20 10 61.5 b8 5.18 10.5 20 10 59.5 b9 5.18 12.5 20 10 57.5 iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 92 minute and examined for any change in phase separation (9,10) . emulsification test self emulsification ability of surfactant was assessed to select the best surfactant from a large pool of surfactant. selected oil and surfactant were mixed in 1:3 ratio heated at 40-50 0 c and vortexed to form a homogenous mixture at room temp (25 ± 1 0 c). 500 mg of oilsurfactant mixture was dispersed in 500ml double distilled water in a glass beaker, was prepared under gentle stirring. visual test was used to assess self emulsification of surfactant in terms of dispersibility, ease of emulsification and appearance using a grading system (11) . measurement of globule size the average globule size was measured using a nanophox (nx0088), cross corrélation. the measurement was performed at 25°c (12). polydispersity index polydispersibility which determines size range of particles in the system, it is expressed in terms of polydispersibility index (pdi). an ideal smedds should be widely distributed with particles less than 100 nm and so pdi should be less than 0.3 or in other words particles having size more than 100 nm should be maximum up to 23% (13) . (d0.9 d0.1) / d0.5 i. e. (x90 x10) / x50 viscosity measurement the viscosity of microemulsion was measured using a brookfield viscometer equipped with the spindle no. 64. the measurement was performed at ambient temperature of a single formula or for all formulas and it reflects interdroplet interaction (14) . zeta potential the magnitude of the zeta potential gives an indication of the potential stability of the colloidal system. if all the particles have a large negative or positive zeta potential they will repel each other and there is dispersion stability. if the particles have low zeta potential values then there is no force to prevent the particles coming together and there is dispersion instability. zeta potential is determined by using zetasizer (13) . sem analysis the morphology of mebendazole loaded smedds (globules) prepared under the optimum condition was observed under scanning electron microscope. invitro study dialysis membrane was used to carry out mebendazole release from its suspension. mebendazole suspension containing 10 mg of drug was placed into dialysis bag (himedia, molecular weight cut off 12000 da). invitro drug release of all formulas was carried out using usptype ii dissolution apparatus (paddle type). the dissolution medium, 900 ml 0.1 n hcl and 1% sls was placed into the dissolution flask maintaining the temperature at 37 ± 0.5 0 c and speed of 100 rpm. dissolution studies were carried out for 2 hr. 5 ml of aliquot was withdrawn at an interval of 5, 10, 20, 40, 60, 80, 100, 120 min. after collecting the sample, the dissolution medium was replenished with the same volume of fresh medium, and the sample was filtered.the samples were then analyzed at 300nm by uv visible spectrophotometer (15,16) . release kinetic modelling to analyze the in-vitro release data, various kinetic models were used to describe the release kinetics. zero order rate equation (equation-1) describes drug release rate is independent of its concentration in the systems. the first order equation (equation-2) describes the release from system where release rate is concentration dependent. higuchi (1963) described the release of drugs from insoluble matrix as a square root of time dependent process based on fickian diffusion (equation-3). the hixson-crowell cube root law (eqution-4) describes the drug release from systems where there is a change in surface area and diameter of particles or tablets (hixson and crowell, 1931). korsmeyer et al (1983) derived a simple relationship which described drug release from a polymeric system (eqution-5). c = k0 t (1) where, k0 is zero-order rate constant expressed in units of concentration/time and t is the time. log c= log c0 kt / 2.303 (2) where, c0 is the initial concentration of drug and k is first order constant. q = k t 1/2 (3) where, k is the constant reflecting the design variables of the system. q0 1/3 – qt 1/3 = khc t (4) where, qt is the amount of drug released in time t, q0 is the initial amount of the drug in tablet and khc t is the rate constant for hixsoncrowell rate equation. mt / m∞ = kt n (5) where mt / m∞ is fraction of drug released at time t, k is the rate constant and n is the release exponent (7) . plots were made cumulative % drug release vs. time for zero order kinetic model, log cumulative of % drug remaining vs. time for first order kinetic model, cumulative % iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 93 drug release vs. square root of time for higuchi model, log cumulative % drug release vs. log time for korsmeyer model and cube root of cumulative % drug release vs. time hixson-crowell cube root. in vivo study hplc specification an analysis was carried on jasco hplc instrument equipped with quaternary gradient pump pu-2089 plus, photo diode array detectormd-2018 plus and hi-q-sil c18 column (250 mm x 4.6mm, 5μ, particle size (17) . prepartion of stock solution 25 mg of pure mebendazole was transferred in 25 ml of volumetric flask and dissolve in 15 ml of dmf, sonicated for 15 min. and then finally volume made upto mark by dmf to form stock soln of 1000μ g/ml. the further dilutions made by the developed mobile phase i.e (acetonitril : phosphate buffer 55:45 at ph 6.5 adjusted with ortho-phosphoric acid) to get concentration range of 10-60 μ/ml by taking appropriate aliquots from the above stock solution. in vivo bioavailability measurement the in-vivo studies of optimised formulations of mebendazole, an microemulsion (test) marketed formulation (standard), normal saline (control) was performed in rats. all animals care and procedures were conducted according to the guiding principles in the use of animals in toxicology. albino wistar rats weighing 200 250 ± 20 g were fasted for 10–12 hours prior to the experiments but were allowed free access to water (18) . each groups comprises of 6 rats. the rats in each group were administered orally with 2.5 mg of mebendazole preparations (liquid smedds and marketed preparation) as test and standard respectively. then, 0.25 ml of blood was collected from the right or left retro-orbital cavity using 1-ml needle at predetermined time intervals and 0.1 ml of plasma was separated by centrifugation of blood samples at 10,000 rpm for 15 min . plasma samples were stored at -20 0 c until further analysis. the plasma was then deproteinized with 0.9 ml of acetonitrile and was vortexmixed for 5 min and then centrifuged at 8000g for 2 min. the residue was reconstituted with 100 μl of acetonitrile and 50 μl of the resulting solution was analyzed by hplc as mentioned above (19) . stability studies of microemulsion optimised formula of liquid microemulsion were subjected to accelerated stability study at 25 ° c ± 2 ° c and 60% rh, 30 ° c ± 2 ° c and 65% rh, 40 ° c ± 2 ° c and 75% rh respectively in stability chamber (biotech india) for 1, 2 and 3 months. after completion of time, samples were drawn and analyzed for physical parameters. preparation of solid smedds by spray drying aerosil 200 (500 mg) was suspended in 200 ml methanol by magnetic stirring. the liquid smedds (12 ml) was then added with constant stirring, and the solution was kept stirring at room temperature for 15 min to obtain a good suspension of aerosil 200 (20) . the suspension was spray dried by using a spray dryer (labultima model no.lu-222advanced) and following condition were maintained. evaluation of solid smedds ir spectroscopy the ir spectra of smedds containing solid carrier (aerosil 200) were recorded using fourier transform infra-red spectrophotometer (shimadzu 8400-s) with diffuse reflectance principle. sample preparation involved, drying of potassium bromide (kbr), drug and excipients in the oven to get rid of any moisture content then mixing the sample with kbr by triturating in glass mortar. finally preparing of pellet and placing in the sample holder. the spectrum was scanned over a frequency range 4000 – 400 cm -1 (21) . dilution study by visual observation dilution study was done to observe the effect of dilution on solid microemulsion, because dilution may better mimic the condition of stomach after oral administration. in this method, solid smedds (100 mg) was introduced into 100 ml double distilled water in a glass beaker that was maintained at 37 0 c and the contents mixed gently using a magnetic stirrer. the emulsification ability of smedds judged qualitatively “good” when clear microemulsion formed and “bad” when there was turbid or milky white emulsion formed after stopping of stirring (22) droplet size determination optimised formulation of solid microemulsion is dispersed in 100 ml of water and kept it on magnetic stirrer for 1 min. sample were allowed to stand for 10 min so that colloidal silicon dioxide particle could sediment. the supernatant was filtered through coarse filter (whatman paper) and the filtrate was used for globule size analysis. the average globule size was measured using a nanophox (nx0088), cross corrélation. the measurement was performed at 25°c (23) . polydispersity index polydispersibility which determines size range of particles in the system, it is expressed in terms of polydispersibility index (pdi). an iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 94 ideal smedds should be widely distributed with particles less than 100 nm and so pdi should be less than 0.3 or in other words particles having size more than 100 nm should be maximum up to 23%. (d0.9 d0.1) / d0.5 i.e (x90 x10) / x50 drug content drug content was estimated by extracting mebendazole from solid microemulsion. in brief solid microemulsion was dissolved in sufficient quantity of methanol. solution was bath sonicated for 10-15 min for extraction of the mebendazole in methanol and filtered. the absorbance of filtrate was read at 300 nm on uvvisible spectrophotometer (24) invitro drug release studies dialysis membrane was used to carry out release of mebendazole suspension. mebendazole smedds containing 10 mg of drug was placed into dialysis bag (himedia, molecular weight cut off 12000 da). invitro drug release of all formulas was carried out using usptype ii dissolution apparatus (paddle type). the dissolution medium, 900 ml 0.1 n hcl and 1% sls was placed into the dissolution flask maintaining the temperature at 37 ± 0.5 0 c and speed of 100 rpm. dissolution studies were carried out for 2 hours. 5 ml of aliquot is withdrawn at an interval of 5, 10, 20, 40, 60, 80, 100, 120 min. after collecting the sample, the dissolution medium was replenished with the same volume of fresh medium, and the sample was filtered. the samples were then analyzed at 300nm by uvvisible spectrophotometer. in vivo study hplc specification procedure same as that in evaluation of microemulsion. in vivo bioavailability measurement procedure same as that in evaluation of microemulsion. result and dicussion solubility studies one important consideration when formulating a self emulsifying formulations is avoiding precipitation of drug on dilution in the gut lumen in-vivo. therefore the component used in the system should have high solubilisation capacity for the drug, ensuring the solubilisation of the drug in the resultant d9-ispersion. oleic acid shows the highest solubilisation capacity than other oil for mebendazole (5.1816 mg/ml ) , followed by tween 80 (9.7579 mg/ml ) and propylene glycol (1.9250 mg/ml ) . thus for further study oleic acid as oil and tween 80 and propylene glycol as surfactant and co-surfactant respectively was selected. construction of phase diagram the pseudo ternary phase diagrams for different surfactant: co-surfactant ratios are as shown in figure 1. all diagrams showed the maximum amount of surfactant, cosurfactant required to form a microemulsion. highlighted part in each phase diagram shows the region in which two immiscible phases exist. whereas all plotted points indicate the instantaneous formation of microemulsions for respective oil to water ratios with specific amount of surfactant and cosurfactant. from diagrams it can be concluded that with at ratio of surfactant: cosurfactant 2:1 the large microemulsion region was observed compared to 1:1 and 3:1. therefore 2:1 ratio of surfactant and co-surfactant was selected for preparation of microemulsion. (a) (b) (c) figure 1: pseudoternary phase diagram (a) ratio 1:1 (b) ratio 2:1 (c) 3:1. water surfactant oil iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 95 evaluation of microemulsion optical transparency all formulas of liquid smedds were transparent . phase separation study none of the microemulsion formulation showed signs of phase separation on centrifugation at 15,000 rpm for 15 minutes. this result provided a rapid and full proof identification of the system as micro emulsion, and which is sign of stability of microemulsion (25,26). emulsification test a visual test was carried out to assess self emulsification of liquid smedds in 100 mi of distilled water at 37 0 c under gentle agitation. all solid smedds formulations showed spontaneous microemulsification (< 1 min) and there was no sign of phase separation of microemulsification. determination of globule size an increase in ratio of oil phase resulted in the proportional increase in particle size. formula b1 shows the least globule size (figure2) as compare to the all six microemulsion formulas. it is well known that the addition of surfactants to the microemulsion system causes the interfacial film to stabilize and condense, while the addition of cosurfactant causes the film to expand thus the relative proportion of surfactant has varied effect on the droplet size (27) . figure 2: droplet size distribution of microemulsion. (batch no b1) polydispersity index table 2 showed polydispersity index of microemulsion. table (2) polydispersity index of microemulsion batch pdi b1 0.360 b2 0.280 b3 0.291 b4 0.311 b5 0.320 b6 0.281 viscosity measurement it was observed that there is increase in viscosity as increasing oil concentration. formula b9 which contains higher amounts of oil therefore it shows highest viscosity(155 cps) as compared to all formulas. formula b1 shows least viscosity (78 cps) so it contain less oil in formula (28) . zeta potential determination a dividing line between stable and unstable aqueous dispersions is generally taken at either +30 or -30 mv. particles with zeta potentials more positive than +30 mv are normally considered stable. particles with zeta potentials more negative than -30 mv are normally considered stable (13) . figure 3 given that zeta potential was 31.5 mean result microemulsion normally stable. figure 3: zeta potential of microemulsion sem photomicrograph of microemulsion figure 4 showed sem of microemulsion (batch b1). figure 4. sem of microemulsion (batch b1) figure 4. sem of microemulsion (batch b1) in vitro dissolution study in-vitro drug release profile of mebendazole was carried out for developed formulation (microemulsion) and marketed formulation. after 2 hr, the amount of drug released from marketed formulation was 66.95% ± 0.04562 and for microemulsion was 101.1 % ± 0.213 as showen in figure 5. 0 10 20 30 40 50 60 70 80 90 100 c u m u la ti v e d is tr ib u ti o n q ( x ) / % 0 1 2 3 4 5 6 7 d e n s it y d is tr ib u ti o n q * ( x ) 0.5 1.0 5 10 50 100 500 1000 5000 10000 p article size / nm iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 96 the best fit model was found to be koresmayer peppas kinetic model equation plot (r2 = 0.9985 microemulsion) indicating the dissolution rate limited drug release from a smedd formulation. the n value (0.1818) indicate the mechanism of drug release was super case ii transport. figure 5: percentage drug release of microemulsion and marketed formulation in vivo studies figure 6 shows the plasma concentration profiles of mebendazole after oral administration of various formulations to rats. the auc of mebendazole in optimised formula increased 2 fold compared with that of mebendazole in the orally administrated marketed preparation (10.1464 vs. 5.7758 μg/ml * h ). the cmax was higher 3 fold in microemulsion with that of the orally administrated suspension (3.6954 vs 0.2445). thus, the oral bioavailability of mebendazole increases 2 fold in microemulsion as compared to marketed preparation. the improved oral bioavailability of mebendazole in the microemulsions could be explained by the combination of the following effects: (1) significantly improved solubility of mebendazole by microemulsions which could keep the drug as the soluble form during the gastrointestinal dilution and permeation process; (2) the synergistic effect of oil and surfactants as absorption enhancers; (3) the inhibition of p-gp efflux (29) . figure 6: (a) plasma drug concentration of marketed preparation (b) plasma drug concentration of microemulsion stability studies there are no phase separation found after optimised formulations of microemulsion were subjected to accelerated stability study at 25 ° c ± 2 ° c and 60% rh, 30 ° c ± 2 ° c and 65% rh, 40 ° c ± 2 ° c and 75% rh respectively in stability chamber (biotech india) for 1, 2 and 3 months. evaluation of solid smedds ir spectrum the ftir absorption spectrum of mebendazole and its solid smedds is shown in figure 7. ftir spectrum of mebendazole showed all the peaks corresponding to the functional groups present in the structure of mebendazole. the band at 1720 cm -1 is due to c = o amide stretching, 1650 cm -1 is due to c=o stretch, 2795 cm -1 is due to c-h stretch, 3415 cm -1 is due to n-h stretching. 0 10 20 30 40 50 60 70 80 90 0 50 100 150 p e r c e n t d r u g r e le a se d time(min.) mktd. formulation smedds iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 97 (b) figure 7: ir spectrum of (a) mebendazole (b) solid smedds dilution study dilution study was done to observe the effect of dilution on smedds, because dilution may better mimic the condition of stomach after oral administration. in this method, smedds (100 mg) was introduced into 100 ml double distilled water in a glass beaker that was maintained at 37 0 c and the contents mixed gently using a magnetic stirrer. the emulsification ability of smedds judged qualitatively “ good ” when clear microemulsion formed and “bad” when there was turbid or milky white emulsion formed after stopping of stirring (30, 31) . droplet size measurement the droplet size (figure 8) of selected formula of solid smedds (batch b1) was 401 nm. figure 8. droplet size distribution of smedds formulation polydispersity index polydispersity index of selected formula of solid smedds (batch b1) was 0.281. drug content percentage of drug content in selected formula of solid smedds (batch b1) was within usp limit (97.22 %). in vitro dissolution studies in-vitro drug release profile of mebendazole was carried out for developed formulation (smedds). after 2 hr, the amount of drug released from the smedds was 85.47% ± 0.1614 as showen in figure 9.the best fit model was found to be koresmayer peppas kinetic model equation (r 2 = 0.9985) indicating the dissolution rate limited drug release from a solid smedd formulation. the n value (n = 0.1496) indicate the mechanism of drug release was super case ii transport. 0 10 20 30 40 50 60 70 80 90 100 c u m u la ti v e d is tr ib u ti o n q ( x ) / % 0 1 2 3 4 5 6 7 8 d e n s it y d is tr ib u ti o n q * ( x ) 0.5 1.0 5 10 50 100 500 1000 5000 10000 p article size / nm b a iraqi j pharm sci, vol.23(1) 2014 self-microemulsifying drug delivery system of mebendazole 98 figure 9: percentage drug release of smedds and marketed formulation in vivo study figure 10 shows the plasma concentration profiles of mebendazole after oral administration of various formulations to rats. the auc of mebendazole in optimised formulation increased 1.8 fold compared with that of mebendazole in the orally administrated marketed preparation (8.3145 vs. 5.7758 μg/ml * h). the cmax was 6 fold higher in smedds as compared with that of the orally administrated suspension (6.2015 vs 0.2445). thus, the oral bioavailability of mebendazole was 1.8 fold higher in (smedds) as compared to marketed preparation. the improved oral bioavailability of mebendazole in the microemulsions could be explained by the combination of the following effects: (1) significantly improved solubility of mebendazole by microemulsions which could keep the drug as the soluble form during the gastrointestinal dilution and permeation process; (2) the synergistic effect of oil and surfactants as absorption enhancers; (3) the inhibition of p-gp efflux (29) . conclusion self micro 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-vitro and in vivo evaluation. yong-mei y. , fude c. , chao-feng m. et.al., journal of controlled release 140 (2009) 86–94. 30. soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm jeffrey bethony. et. al., seminar vol 367 may 6, 2006. 31. study of cosurfactant effect on nanoemulsifying area and development of lercanidipine loaded self nano emulsifying drug delivery system. nitin parmar. et. al., colloids and surface b : biointerfaces 86(2011) 327-338. iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students doi: https://doi.org/10.31351/vol31iss1pp102-108 102 nasal carriage of vancomycinand methicillin-resistant staphylococcus aureus among intermediate students of urban and rural schools of muthanna province in iraq saad m. hantoosh*,1 *al-muthanna education directorate, ministry of education, al-muthanna, iraq abstract staphylococcus aureus is one of the common causative agents of infections, from asymptomatic carriers to healthy individuals. it can colonize anterior nares of carriers with a high capability to resist different antibiotics. students are susceptible to bacterial infection due to some factors, including poor health habits and surrounding school conditions. this study screened the rate of vancomycinand methicillinresistant staphylococcus aureus nose carriers among secondary students in rural and urban schools and its association with some sociodemographic factors. the study sample included 300 male/female students aged 15-20 years from 12 schools of rural and urban areas during the period from november 2020 till may 2021. it was found that males are 2.3 times more of mrsa nose carriage and the rate of infections was higher in rural schools than urban whether among males or females. the prevalence of mrsa was 72/300 (24%) among students with 15/72 (21%) mdr-mrsa isolates with high resistance to clindamycin and erythromycin at rate 46% and 42% respectively, and a resistance ranging between (20-26) % for gentamycin, levofloxacin, trimethoprim/sulfamethoxazole, rifampin, and nitrofurantoin with high sensitivity to vancomycin at 4% of resistance. there was no significant association between mrsa incidence with both medication and chronic diseases despite the 19% of students were self-medicating. most schools were suffering from a shortage of potable water, disinfectants, and first aid materials. students lack health awareness about transmissible diseases with unhealthy habits spread among students, as specialized health teams did not visit most schools. keywords: methicillin, vancomycin, mrsa nose carriage, muthanna province معدل الحمل االنفي بالمكورات العنقودية الذهبية المقاومة للفانكومايسين والمثيسيلين لطالب المتوسطة في المدارس الحضرية والريفية لمحافظة المثنى في العراق سعد سالم حنتوش *،1 وزارة التربية ، المثنى ، العراق مديرية تربية المثنى ، * الخالصة المكورات العنقودية المكورات العنقودية الذهبية هي أحد العوامل الشائعة المسببة للعدوى من حامليها بدون أعراض إلى األفراد األصحاء. بعض ممكن ان تستعمر الفتحات االنفية األمامية مع قدرة عالية على مقاومة المضادات الحيوية المختلفة. الطالب عرضة للعدوى البكتيرية بسبب كورات العنقودية الذهبية العوامل، بما في ذلك العادات الصحية الخاطئة والظروف المدرسية المحيطة. فحصت هذه الدراسة معدل الحمل األنفي للم جتماعية المقاومة للفانكومايسين والميثيسيلين بين طالب المرحلة الثانوية في المدارس الريفية والحضرية ودراسة مدى ارتباطها ببعض العوامل اال لمحافظة لريفية والحضرية من المناطق ا مدرسة ۱۲سنة من ۲۰و ۱٥طالب وطالبة تراوحت أعمارهم بين ۳۰۰والديموغرافية. شملت الدراسة مرة وكان معدل اإلصابة أعلى في المدارس الريفية منه في المناطق الحضرية ۲.۳المثنى. وجد أن الذكور هم أكثر عرضة لإلصابة من االناث بمقدار ۲٧/۳۰۰لين )المرسا( بين الطالب هي سواء بين الذكور أو اإلناث. كانت نسبة وجود بكتيريا المكورات العنقودية المقاومة للمضاد الحيوي المثيسي متعددة المقاومة للمضادات الحياتية. كانت مقاومة العزالت البكتيرية عالية للكليندامايسين واإلريثروميسين بمعدل ( ٪۲۱) ٥۱/۲٧كان منها ٪(٤۲) )٤۲٪ و٤6 بين تراوحت المقاومة على التوالي، بينما سلفاميثوكسازول، ريفامبين، ( ٪ لجنتامايسين، ليفوفلوكساسين، و۲6 -٪۲۰ / تريميثوبريم ٪ فقط. لم يكن هناك ارتباط معنوي بين االصابة بالمرسا مع كل من نسبة التداوي الذاتي ٤ونتروفورانتوين مع حساسية عالية للفانكومايسين بمقاومة من نقص بمياه الشرب، المطهرات، ومواد االسعافات ٪. معظم المدارس كانت تعاني ۱۹واألمراض المزمنة بالرغم ان نسبة التداوي الذاتي كانت الصحي الفرق ان كما الصحية الغير العادات بعض الى باإلضافة االنتقالية االمراض حول الصحي الوعي الى يفتقرون كانوا والطلبة ة االولية المتخصصة لم تزور معظم المدارس. ل االنفي بالمرسا، محافظة المثنىكلمات مفتاحية: الميثيسيلين، الفانكومايسين، معدل الحم introduction staphylococcus aureus is a leading source of infections ranging from superficial skin infections (ssti) to invasive infections and death (1). different types of infections caused by s. aureus including skin infections, bacteremia, bone infections, endocarditis, food poisoning, pneumonia, and toxic shock syndrome (2). one species of the staph germ, called methicillin-resistant staphylococcus aureus (mrsa), is not easy to cure because mrsa is not eliminated by certain antibiotics used to treat other staph germs. 1corresponding author e-mail: saadmuslim85@gmail.com received: 13/6/2021 accepted:22 /8 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp102-108 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 103 for an extended period, methicillin-resistant s. aureus has been recognized as a pathogen associated with healthcare settings, but in the 1990s, community-associated infections of mrsa had emerged. mrsa infections cost millions of dollars and tens of thousands of patients around the world, with severe infections causing deaths between different ages (3). some healthy persons usually have staph germs on their body parts like skin and noses, and these persons are known as carriers, but they contagious to other people (4). carriers of staph germs are at risk to be colonized by staph that makes them sick. the most common transmission methods of staphs are skin-to-skin contact and personal items such as towel and clothing then enters damaged skins through scratches and cuts. the infections can diffuse deeper and affect the blood and other organs such as the heart, brain, lungs causing lifethreatening infections (5). many community members are at a high level of risk of infection with mrsa, including athletes, prisoners, hospitalized patients, students, and military personnel (6). students are considered among the community groups at risk of mrsa infection, as most of the risk factors are existent in schools, especially in iraq, such as overcrowding of students in classrooms and poor sanitation, as indicated by many previous iraqi studies (7, 8). in addition, the level of health education is low among students in iraq, which is a direct cause of the spread of many transmissible diseases among them (9). therefore, there is an urgent need to focus more scientific research to discover some factors that may increase the spread of infection among students. subjects and methods population and study design: a cross-sectional survey study was conducted during november 2020 till may 2021 and worked on collecting nasal swabs with relevant medical information from high school students in rural and urban schools for both males and females aged 15-20 years. the 300 student that equally distributed as 150 participants with 75 males and 75 females for both of urban and rural schools, participants who abstained from giving the nasal swabs (n = 18) and who did not send the results of the questionnaire (n = 15) were excluded from the study. the questionnaire included a set of health information collected to investigate whether they are risk factors for infection with mrsa included medical counsel, chronic diseases, and medication in addition to investigating some unhealthy habits of students such as the rate of washing hands and exchanging personal items. most of the answers were simplified in order not to include long and complex choices that may confuse the student's reliability and accuracy in the answer. detection of methicillin-resistance s. aureus and antibiotics susceptibility tests: both or one of the anterior nares were sampled with normal saline moistened sterile rayon-tipped swab and then placed in amies transport medium (oxoid, uk) and all specimens were cultured within a maximum of two days. each swab was cultured on blood agar (himedia, india) and incubated for 24 hours at 37°c. colonies with β-hemolysis were picked up and inoculated on both mannitol-salt agar (oxoid, uk) and mrsa chromagar (himedia, india) with incubation for 24 hours at 37°c. mannitol fermenter with positive green-bluish colonies on mrsa chtomagar were confirmed by slide/tube coagulase test (10). the confirmed s. aureus isolates were stored for long preservation in 15% glycerol brain heart infusion broth at minus 30°c. the antibiotic susceptibility profile of methicillin-resistant s.aureus isolates was determined according to clsi, (2017) (11) using kirby-bauer disc-diffusion method against eight antibiotics of different classes as following (gentamycin 10 µg, levofloxacin 5 µg, erythromycin 15 µg, clindamycin 2 µg, nitrofurantoin 300 µg, trimethoprimsulfamethoxazole 1.25/23.75 µg and rifampin 5 µg) with etest for vancomycin 0.016-256 µg. statistical analysis: the electronic questionnaire data that included the students' answers were received, sorted individually, then entered into excel and then into the spss version 23. the chi-square test of independence and binomial logistic regression test were performed to find out the degree of correlation between the incidence of mrsa nose carriage and other factors. the significant differences between male/female and rural/urban were analyzed using mann–whitney u test. a pvalue that ≤ 0.05 is statistically significant. results the sociodemographic information showed that the proportion of participants that received medication was 33% with 14% of medical consultation which indicating about 19% of students underwent treatment without medical examination (table 1). about 10% of the students were found to have arranged of chronic diseases including diabetes, asthma, physical urticarial, and psoriasis. iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 104 table 1.sociodemographic characteristics of 300 elementary school participants in al-muthanna province it was obvious that the health teams affiliated with the ministry of health or education did not visit most schools, as 78% of students indicated that they were not visited or examined by any health teams. in addition, schools do not consistently have the simplest health requirements, such as water and soap, as all students who were asked about the availability of water and soap answered that they are not available at all times, which indicates that schools suffer from a shortage of water and detergents most of times. the lack of basic requirements for hygiene was not the only problem, as the questionnaire showed that schools lack first aid materials such as gauzes and wound disinfectants, as the students who were exposed to wounds and scratches (n = 93) during the school times only 27% of them received primary treatment for wounds, while the rest did not receive any type of treatment where their scratches and wounds remain susceptible to contamination. the sources of bacterial infection were not limited to poor school health facilities, as there was either deliberate negligence or ignorance on the part of the students. the results indicated that 61% of students borrow or exchange clothes among themselves, such as sports clothes. the biochemical and cultural properties of nose swabs revealed that 127/300 samples were βhemolytic and only 78/127 samples were mannitol fermenter while 72/78 samples were coagulasepositive and grew on mrsa chromagar (figure 1). figure 1. positive growth of mrsa/vrsa isolates on chromagar with greenish colonies due to the cleavage of chromogenic substrate the factors of resident, medication, chronic diseases, and the medical consultation had no correlation with the mrsa nose carriage except the factor of gender, which has been proven to be associated with and contribute to increasing the prevalence of mrsa infections where males have a 2.3 probability higher for mrsa carriers than females (table 2). characteristics categories frequency n=300 percentage residence urban 150 50 rural 150 50 gender male 150 50 female 150 50 chronic disease yes 31 10 no 269 90 medication consultation yes 42 14 no 258 86 medication yes 100 33 no 200 67 mrsa carriage yes 72 24 no 228 76 characteristic urban (n= 150) rural (n=150) total (n=300) chronic disease 15(10%) 16(11%) 31(10%) medical consultation 19(13%) 23(15%) 42(14%) medication 51(34%) 49(33%) 100(33%) mrsa carrier 30(20%) 42(28%) 72(24%) iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 105 table 2. chi-square test of association between mrsa nose carriage and sociodemographic background of students. characteristics mrsa carrier yes no correlation residence urban 30 120 0.105 rural 42 108 gender male 47 103 0.003 female 25 125 medical consultation yes 10 32 0.975 no 62 196 medication yes 22 78 0.57 no 50 150 chronic disease yes 7 24 0.845 no 65 204 no significant difference between male and female regarding treatment intake, medical consultation, and chronic diseases rate, wherefore these factors were excluded as possible reasons for high mrsa colonization among male (47/72, 65%) in comparison of female (25/72, 35%). although the results of the statistical analysis did not indicate the existence of statistically significant differences between male and female infections in the rural and urban schools, it is still higher in rural than urban schools, if the infections increased by seven in the rural, the statistical difference would be 0.025 (figure 2). figure 2. the prevalence of mrsa nasal carriage among male and female in urban and rural schools the antibiotic susceptibility profile of 72 mrsa isolates was interpreted according to the guideline of clsi, (2017) (11), the isolates showed a varying resistance to the eighth antibiotics, where the highest resistance was recorded against clindamycin and erythromycin at rate above 40% in comparison to the resistance of other antibiotics of gentamycin, levofloxacin, rifampin, trimethoprim-sulfamethoxazole, and nitrofurantoin which was ranging between 20% to 25% with high sensitivity to vancomycin at rate of 4% of resistance as shown in figure (3). 15/72 mdrmrsa isolates were detected at rate 21% (12 mrsa and 3 vrsa) figure 3.antibiotic susceptibility profile of 72 mrsa isolates against eight antibiotics agents of different classes discussion the results of questionnaire indicated that about 19% of students take treatments or what is known as self-medication without medical consultations which were more among the urban than the rural population as noted in table (1). taking treatments without medical advice may not contribute to treating the disease, but rather causes an exacerbation of the health problem and the development of some side effects such as the development of drug allergy in the patient and an increase in antibiotic resistance and may even cause iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 106 death (12). the cultural level has a role in increasing self-medication, as the higher the educational level of the family, the more self-medication will be resorted to, and this explains its rise in urban areas (13, 14). about 10% of students suffer from chronic diseases mainly diabetes, asthma, and physical urticarial with no difference between urban and rural students. while some students suffer from respiratory and health problems, it was found that most of the schools in iraq are destroyed and lack the most basic elements of a healthy environment, the most important of which are good ventilation and clean classrooms (15). the present study also revealed that all students indicated that they had never been visited or checked up by any of specialist health teams. there are many ways of transmitting bacterial infection among the members of society, including exchanging or borrowing personal items for infected persons, including clothes (16). in the sense of the results, more than half of the students showed a lot of behavior, which is one of the main causes of bacterial transmission such as the exchange and borrowing of sports clothes between them because iraqi schools lack educational health programs that teach students some healthy behaviors (17). the failure was also evident by the health teams affiliated to the ministry of health and ministry of education, which either visited schools nominally or did not visit schools at all (18). it is not surprising that schools suffer from a shortage of water; students do not find water for washing most of the time, in addition to the lack of safe drinking water (6, 8). among the most important causes of bacterial infection is the poor or neglectful treatment of wounds. even superficial wounds need direct cleaning and sterilization, as open wounds are susceptible to bacterial infection (19). about 73% of the students who were exposed to wounds during school time did not receive the initial treatment for wounds such as sterilization or putting on gauze, and this is completely consistent with a previous study that indicated the same problem where some students had to return to their homes to seek treatment which means that their wounds remain for several hours, exposed to bacterial contamination (20). the issue goes beyond poor sanitation and school environment, as some schools lack first aid materials (21) and even if they are available, it has been found that educational personnel are not trained or lack primary medical information and have not been engaged in training courses on how to do first aid (22). the results of the cultural properties showed that about a quarter of the students 24% of the students were mrsa carriers. the percentage of mrsa nose carriers is considered high, but it is expected in iraq whereas in a similar study in kurdistan, iraq, the percentage was very close to the current study, as it was about 20% in general, whether in rural or urban students (23) but in another similar study, hussein et al., (2019) (24) reported that the percentage of students carrying mrsa was about half. mrsa isolates found to have 100 percent of resistance to cefoxitin and oxacillin with about 40% of resistance to clindamycin and erythromycin especially among mdr-mrsa isolates that agree to a large extend with the results of kistler et al., (2018) (25), montravers and eckmann (2020) (26), and ullah et al., (2020) (27). over the past 10 years, there has been an increased resistance to clindamycin where the resistance ratio is doubled 8 times (26). kistler et al., (2018) (25) reported that the resistance to clindamycin and levofloxacin increased consistently several times over the past years. although vancomycin was the most effective agent with the lowest resistance reaching 4% in comparison to other antibiotics, there was a worrying number of increasing resistance to vancomycin reaching 14% by mrsa isolates recovered from clinical samples (28). the multi-drug resistance isolates can be defined as that resist to one or more agents of three or more antimicrobial classes (29). about 21% of mrsa isolates were multi-drug resistant isolates that were higher than results obtained by relatively similar study pathak et al., (2010) (30) and close to arali et al., (2016) (31) while no similar studies were found about iraq. the degree of correlation or influence of some factors that may contribute to an increase in mrsa nose carriage was studied. these factors did not prove their role in raising the infection, such as the self-medication and chronic diseases, but the factor of gender was very influential as the results showed that males are more susceptible to infection than females. the gender was found to significantly affect the increase in mrsa infections among males for reasons that have not been determined (32). it was also indicated that the residence factor was not statistically significant to increase mrsa infections, but at the same time, it had a role in increasing the number of infections between males and females in rural students more than in urban, as shown in figure 2. males were more affected by mrsa that may be due to multiple reasons, including levels of some vitamins such as vitamin d, as well as elevated smoking habits among males (33). among the other reasons that may increase the incidence of mrsa among males is the difference in some habits among them, for example, the level of sterilization and hand hygiene, in general, males are less sterilized than females that may be due to mingling between males or the practice of some sports (34). also, among the possible reasons that may increase the infection rate of mrsa among males are some habits such as going to public places such as football and sports halls and barber shops that may lead to sharing shaving tools and sports clothing (35). iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 107 it was noted that housing in rural areas contributes to an increase in the incidence of mrsa nose carriage. there are three main strains of mrsa which are livestock acquired mrsa la-mrsa, community acquired ca-mrsa, and hospital acquired ha-mrsa (36). the reason for the increase in mrsa nose carriage among students in rural areas may be due to the transmission of mrsa from animals to humans, either through direct contact with animals or indirect contact with animals' products such as milk, meat, and wool (37). the coexistence between infected animals and humans, or vice versa leads to the transmission of infection between them. where it was 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http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 anadrol overdose on liver function in male rats doi: https://doi.org/10.31351/vol31iss1pp65-71 65 study the effects of anadrol overdose on liver function in male rats zahra sami*,1 and ahmed obaid* * department of biotechnology, faculty of biotechnology, al-qasim green university, babylon, iraq abstract anadrol (oxymetholone) is an active androgenic anabolic steroid that has been clinically studied in numerous diseases since the 1960s. it is used in the treatment of anemia and the replacement of male sex steroids. unfortunately, in attempts to improve physical performance, anadrol could be misused by athletes, that can lead to poisoning contributes to hepatotoxicity. the aim of this study was to investigate the impact of anadrol on the liver function in rat model, via assessment of liver enzymes and histopathological study. a forty male rats, weights about (200-300 gm), aged 8-12 weeks, after acclimatization, the rats were randomly divided into four groups (10 rats in each group) as follow: control group (in which all rats were administered normal saline (ns) via oral gavage), anadrol 10 mg/kg (iran-tehran company) group (in which all rats were administered anadrol 10mg/kg via oral gavage), anadrol 20 mg/kg group (in which all rats were administered anadrol 20mg/kg via oral gavage), and anadrol 30 mg/kg group (in which all rats were administered anadrol 30mg/kg via oral gavage), the oral administration had continued for 8 weeks in single daily dose regimen. at the end of study liver function enzymes such as alanine aminotransferase & aspartate aminotransferase were measured via chemical analysis. then histopathological study was done on the liver tissue in the four experimental groups. male rats that treated with anadrol displayed high level of liver enzymes, including as alanine aminotransferase & aspartate aminotransferase, as compared with control group. on the other hand, histopathological study exhibited significant injurious changes in the hepatic tissue in anadrol groups comparing with control. when anadrol given in high doses results in hepatic injury, that can be cleared via elevated levels of hepatic enzymes and liver histopathological changes. keywords: anadrol, hepatic injury, alt, ast, anabolic androgenic steroid دراسة تأثير االنادرول على وظائف الكبد في نموذج الجرذان *عبيد حسين احمد و 1*، زهراء سامي محمد التقانات االحيائية، كلية التقانات االحيائية، جامعة القاسم الخضراء، بابل، العراق *فرع الخالصة يتم الستينيات. منذ األمراض من العديد في سريريًا دراسته تمت قد , البنائية االندروجينية للستيرويدات الفعال هوالشكل االنادرول, االنادرول جنسية الذكرية. لسوء الحظ، في محاولة لتحسين األداء البدني، يمكن أن يساء استخداماستخدامه في عالج فقر الدم تعويض الهرمونات ال من قبل الرياضيين، مما قد يؤدي إلى التسمم المؤدي الى تسمم الكبد. على وظائف الكبد في نموذج الجرذان، من خالل تقييم أنزيمات الكبد والدراسة االنادرول الهدف من هذه الدراسة هو التحقق من تأثير .( أسبوًعا ، بعد التأقلم 12-8جم( ، و أعمارهم ) 300-200النسيجية للكبد.تم استخدام أربعين جرذ مختبري من الذكور، تتراوح أوزانهم ) ) تم إعطاء جميع مجموعة السيطرةكل مجموعة( على النحو التالي: فئران في 10تم تقسيم الجرذان عشوائياً إلى أربع مجموعات ) ) ايران( -شركة طهران( مجم / كجم 10 االنادرول مجموعةعن طريق الجرعة الفموية و لمدة شهرين( (ns) الجرذان المحلول الملحي طبيعي ) تم مجم / كجم 20 االنادرول مجموعةية و لمدة شهرين( عن طريق الجرعة الفمو ملغم/ كغم 10تم إعطاء جميع الجرذان االنادرول بجرعة ) تم إعطاء مجم / كجم 30 االنادرول مجموعة عن طريق الجرعة الفموية و لمدة شهرين( ملغم/ كغم 20إعطاء جميع الجرذان االنادرول بجرعة لمدة شهرين(.عن طريق الجرعة الفموية و ملغم/ كغم 30جميع الجرذان االنادرول بجرعة عن طريق التحليل الكيميائي. ثم أجريت الدراسة النسيجية astو alt في نهاية الدراسة تم قياس إنزيمات وظائف الكبد بما في ذلك ا في ذلكأظهرت الفئران التي عولجت باألنادرول مستوى مرتفعًا من إنزيمات الكبد، بم.المرضية على أنسجة الكبد في المجموعات التجريبية األربع alt وastمقارنة بمجموعة السيطرة. من ناحية أخرى ، . مجموعات في الكبدية األنسجة في كبيرة ضارة تغيرات المرضية النسيجية الدراسة مجموعة االنادرول أظهرت مع مقارنة خالل المستويات المرتفعة لإلنزيمات الكبدية والتغيرات بجرعات عالية تؤدي إلى إصابة الكبد، والتي يمكن اثباتها من عندما تعطى االنادرول.السيطرة .النسيجية الكبد ِّطات البنائي ينيَّة الكلمات المفتاحية: االنادرول، اإلصابة الكبدية، االنزيم ناقل امين االالنين، االنزيم ناقل أمين األسبارتات ، الُمنش ة اآلْندُروج 1corresponding author e-mail: zahraasami2020@gmail.com received: 25/5/2021 accepted:1 /8/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp65-71 iraqi j pharm sci, vol.31(1) 2022 anadrol overdose on liver function in male rats 66 introduction anadrol is an active androgenic anabolic steroid that has been clinically studied in numerous diseases since the 1960s. it is used in the treatment of anemia and the replacement of male sex steroids as a stimulator of bone marrow cells also it is used in some illnesses to improve general weakness. unfortunately, in attempts to improve physical performance, anadrol could be misused by athletes and is therefore classified as 'controlled substance schedule iii.' anadrol poisoning contributes to hepatotoxicity, prostatic hypertrophy, azoospermia, and impotency (1). as a testosterone 17-α derivative, anadrol demonstrate its anabolic effects via one of two mechanisms, either by direct activation of androgen receptors or indirectly by activation of specific estrogen receptors after its conversion to estradiol. the next step is that transportation of free testosterone into the cytosol of target cells and tissues, then either make binding with androgen receptors or undergo reduction, through the activity of 5α-reductase (cytoplasmic enzyme), into 5αdihydrotestosterone (dht). the latter mediator, 5αdihydrotestosterone (dht), will make stronger binding with androgen receptor (2.5 times) as compared with testosterone. after binding the drugreceptor complex will undergo conformational and structural changes, that result in entry of the drug molecules into the nucleus, followed by direct binding with hormone response elements (hres), which include specific sequences of dna nucleotides, then lead to gene expression and finally end with the required androgenic effects (2). anadrol, which had been approved as anabolic steroid by food and drug administration (fda), considered the potent one in body building as comparing to other anabolic steroids, in such condition body builder can get about 14.5 pounds/100 pounds of their weight (3). furthermore, it is also cheaper, have higher activity, but mandatory monitoring of liver function should be done routinely (4, 5). supraphysiologic-dose anabolic-androgenic steroid (aas) use is associated with physiologic, cognitive, and brain abnormalities similar to those found in people at risk for developing alzheimer’s disease (6) . androgen's treatment may result in liver dysfunction, adenomas, and adenocarcinomas, and patients should have liver function tests performed every 3 to 6 months and hepatic lesions assessed by ultrasonography every 6 months (5). long-term supraphysiologic-dose aas exposures are associated with abnormalities in liver and kidney (7). liver toxicity associated with the use of anabolic steroids can be arranged from mild to lifethreatening condition including liver transaminases level elevation, fatty liver, chronic vascular injury, and lipid profile changes hepatic cell carcinoma. some of these injuries can be reversed by discontinuation of steroids, but other will be irreversible even drug cessation (8). it is well known that the illegal abuse of such anabolic steroids consider a growing factor to result in documented drug induced liver injury (dili) that consequently result in severe liver dysfunction (9). anadrol can result in significant increase in the level of liver transaminases like ast & alt (10). when used, in double blind study in the treatment of hiv-wasting syndrome, anadrol result in elevate hepatic enzymes including ast & alt into five folds (11, 12). materials and methods animal grouping forty adult male rats weighted about (200300 gm), aged 8-12 weeks, and were brought from the college of science, university of babylon. animals were harbored in the animal house with a temperature controlled 20-25ºc and 60-65% humidity with a fitted 12 hours light and 12 hours dark cycle for 14 days before the start of the experiment. also, the rats were free to access food and water. in this study, the rats were divided randomly into 4 equal groups, 10 rats in each group, and as the following: 1. control group: rats in this group administered equivalent volume of normal saline (ns) via oral gavage route daily for 8 weeks (13). 2. anadrol 10mg group: rats in this group administered anadrol in a dose of 10 mg/kg via oral gavage route daily for 8 weeks (14, 15). 3. anadrol 20mg group: rats in this group administered anadrol in a dose of 20 mg/kg via oral gavage route daily for 8 weeks (16). 4. anadrol 30mg group: rats in this group administered anadrol in a dose of 30 mg/kg via oral gavage route daily for 8 weeks (17). at the end of study animals were sacrificed via anesthesia, then blood and hepatic tissue samples collection had been done as below. preparation of drug anadrol 50 mg tablet (iran-tehran company) was obtained and dissolved in normal saline as a vehicle to get anadrol solution, then given via oral gavage according to animal’s body weight (13). sample collection at the end of study, animals were anesthetized with ketamine (50mg/kg) and xylazine (10 mg/kg) (5). then blood sampling was done via direct cardiac puncture, furthermore, animals were sacrificed and hepatic tissues were obtained. blood sampling withdrawn blood was let to clot in gel tube then centrifuged at 4000 × g for 10 min to get serum, that directly sent for chemical analysis. iraqi j pharm sci, vol.31(1) 2022 anadrol overdose on liver function in male rats 67 tissue sampling after animal scarification with anesthesia hepatic tissues were obtained and preserved in 10% formalin until histopathological study was done. liver function analysis to get liver function parameters that include serum ast and serum alt, chemical analysis was done measured with a fully automatic biochemical analyser (fuji dri-chem nx500). briefly 10 μl of serum is deposited on a fuji drichem slide tp-piii. after depositing, the specimen spreads uniformly on the special spreading layer then reacts with reactive reagent that released from reagent layer to form color. the slide is incubated at 37 °c for a fixed time in the fuji dri-chem analyzer and the optical reflection density is measured at 540 nm. the optical reflection density is then converted into the total protein concentration using a calibration curve preinstalled in the analyzer (18). histopathological analysis histological specimens from the liver were prepared at the cancer research unit, faculty of medicine, university of kufa. liver samples were fixed in 10% buffered formalin for at least 24 h before processing, as described previously (19). briefly the fixed tissues were embedded into the paraffin wax followed by the dehydration process with a series of increasing concentrations of ethanol to remove the free or bound water. the embedded tissues were sliced using a microtome into the tiny section 5 μm. for histological assessment, the liver sections were mounted on plain glass slides and routinely stained with hematoxylin and eosin (he) staining. he-stained sections were observed for any abnormalities of histopathological features under a light microscope at 100×, 200×, and 400×. hepatic histopathology scoring the degree of liver injury was scored based on the grading system done by the previous study (20), in which hepatocyte necrosis determined by the percentage of cell swelling, increase cytoplasmic eosinophilia, and nuclear changes including pyknosis (shrinkage), karyorrhexis (fragmentation), and karyolysis (nuclear loss). in addition to mildmoderate inflammatory changes. as shown via table 1 below: table 1. induced liver injury scoring system score description 0 (−) normal–no hepatocytes necrosis 1 (+) minimal–mild focal, limited to centrilobular region less than 25% of affected lobules are necrotic 2 (++) mild-moderate focal and multifocal central to midzonal lobular region 50% affected lobules are necrotic 3 (+++) moderate to severe multifocal (centrilobular-portal region) 75%>x>50% affected lobules are necrotic 4 (++++) severe multifocal x>75% affected lobules are necrotic statistical analysis statistical analysis was performed using spss 26 (spss, inc., chicago, il, usa). analysis of variance (anova) with lsd post-hoc test was used to investigate differences between groups. while histological differences were confirmed using kruskal-wallis with mann-whitney u-test. statistically, the present data significance was defined as p ≤0.05 (21). results the effect of anadrol on liver function to investigate the effects of anadrol on liver function, liver function parameters including serum alt and serum ast were carried out in experimental groups via chemical analysis. the effect of anadrol on the levels of alt anadrol 10 mg, 20 mg, and 30 mg groups demonstrated a significant (p < 0.05) higher levels of alt as compared with that of control group. furthermore, anadrol 20mg, 30mg groups showed a significant (p < 0.05) higher levels of alt as compared with anadrol 10mg group. on the other hand, the study showed there is no significant elevated in alt level in anadrol 30mg group when compared with anadrol 20mg. these findings as shown in figure 1: iraqi j pharm sci, vol.31(1) 2022 anadrol overdose on liver function in male rats 68 figure 1. the mean serum alt level (u/l) in the four experimental groups: data are expressed as mean ± sd; *p <0.05 versus corresponding control; # p <0.05 versus anadrol 10 mg. the effect of anadrol on the levels of ast anadrol 10 mg, 20 mg, and 30 mg groups showed a significant (p < 0.05) higher levels of ast as compared with ast level of control group. additionally, anadrol 20 mg, 30 mg groups exhibited a significant (p < 0.05) higher levels of ast when compared with anadrol 10mg group. also, the current study showed there is no significant elevated in ast level in anadrol 30mg group when compared with anadrol 20 mg group. these results were summarized in figure 2: figure 2. the mean serum ast level (u/l) in the four experimental groups: data are expressed as mean ± sd; *p <0.05 versus corresponding control; # p <0.05 versus anadrol 10 mg. the histopathological effects of anadrol on hepatic tissue according to used scoring system the histopathological results of hepatic tissue of rats of the four experimental groups are summarized by the following table 2 and figure 3. table 2. hepatic histopathological damage percentage and score of the four experimental groups. h is to p a th o lo g ic a l s c o ri n g groups damage % score control 0 0 anadrol 10mg/kg 10 1 anadrol 20mg/kg 19 1 anadrol 30mg/kg 46 2 figure 3.mean rank of liver damage in the four experimental groups control group control hepatic tissue had normal architecture without hepatocytes necrosis with clear cell boundaries. according to the used scoring system, the severity of injury showed a zero degree of damaging (score mean = 0 and represent 0%of damage) all rats in this group show normal histopathological findings 100% as shown in figure 4: iraqi j pharm sci, vol.31(1) 2022 anadrol overdose on liver function in male rats 69 figure 4.photomicrograph of rat liver section of control group shows liver normal histology, h&e stain 40 x. anadrol 10 mg group anadrol 10 mg group hepatic tissue had focal, limited to centrilobular region necrosis with mild changes in cell boundaries. in the term of histopathological grading from normal hepatic tissue, rats in this group showed up to 25% of affected lobules are necrotic as shown in figure 5. figure 5. photomicrograph of rat liver section of anadrol 10mg/kg group shows mild centrilobular inflammation (blue arrow) surrounded by normal hepatocyte (yellow arow), h&e stain 40 x. anadrol 20 mg group anadrol 20 mg group hepatic tissue focal and multifocal central to midzonal lobular region. in the term of histopathological grading from normal hepatic tissue, rats in this group showed up to 50% of affected lobules are necrotic as shown in figure 6. figure 6. photomicrograph of rat liver section of anadrol 20mg / kg group shows moderate centrilobular inflammation (blue arrow) with increased cytoplasmic eosinophilia (yellow arrow) of surrounding hepatocyte, h&e stain 40 x. anadrol 30 mg group anadrol 30 mg group hepatic tissue moderate to severe multifocal (centrilobularmidzonal-portal region) are necrotic. in the term of histopathological grading from normal hepatic tissue, rats in this group showed up to 75% of affected lobules are necrotic as shown showed in figure 7. figure 7. photomicrograph of rat liver section of anadrol 30mg / kg group shows severe portal inflammation (blue arrow), multifocal hepatocyte damage (green arrow), with increased cytoplasmic eosinophilia (yellow arrow) of surrounding hepatocyte, h&e stain 40 x. discussion the effects of anadrol on liver present study showed significant changes in liver function among the four experimental groups, that included its effects on the liver markers such as alt & ast enzymes, as clarify through the following sections. iraqi j pharm sci, vol.31(1) 2022 anadrol overdose on liver function in male rats 70 the effect of anadrol on the level of alt the current study demonstrated a significant elevated alt level in the three anadrol pretreated groups compared with the control group. these finding are consistent with previous studies (13, 22) such pathological changes, that indicated liver injury, can be attributed to major or minor changes in cell membrane integrity lead to significant changes in liver enzyme activities (23, 24). the effect of anadrol on the level of ast additionally, present study showed a significant elevated ast level in the three anadrol pretreated groups compared with control group. these finding are similar with other studies (11, 12, 25), that showed elevated level of ast during androgenic anabolic steroids usage. these findings, that indicated liver injury, can be explained by increased metabolic rate of such xenobiotic by liver, in addition to increasing hepatocyte permeability and change in cellular integrity (23, 24, 26). more interestingly the present study, depending on the microscopic examination of the liver of rats from four experimental groups and revealed variable histopathological findings showed that these anadrol pretreated groups significantly had hepatic tissue injury as compared with control group. the histopathological damage score in ranged from normal in control group, mild, moderate, and severe in anadrol pretreated groups histopathological findings in the theses anadrol pretreated groups were associated with cellular swelling, increased cytoplasmic eosinophilia, rbcs extravasation, and nuclear changes (pyknosis, karyorrhexis, and karyolysis). in addition to inflammatory changes. similar results also experienced by previous study (24), that found such injury occur due to the fact that the biotransformation of xenobiotic compounds is accumulated in the liver. conclusion this work found that high doses of anadrol lead to liver injury. further, it was found that this organ injury confirmed by the elevated level of hepatic specific injury markers, including alt and ast, in addition to the histopathological changes that revealed the hepatic tissue injury. references 1. abdollahi m, pakzad m. oxymetholone. in: wexler p, editor. encyclopedia of toxicology (third edition). oxford: academic press; 2014. p. 744-6. 2. pavlatos am, fultz o, monberg mj, vootkur a, pharmd. review of oxymetholone: a 17alpha-alkylated 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doi : https://doi.org/10.31351/vol29iss1pp76-87 76 enhancement of aqueous solubility and dissolution rate of etoricoxib by solid dispersion technique mustapha m. abdul-rahman*,1 and fatima j. jawad** * ministry of health and environment ,kirkuk general hospital, kirkuk health directorate. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract etoricoxib (exb) is a highly selective cox-2 inhibitor which belongs to the non-steroidal antiinflammatory drug (nsaid). exb is a class ii drug according to the biopharmaceutical classification system (bcs), which possess a very low aqueous solubility in water. in the present study, many trials were made to improve the aqueous solubility and dissolution rate of exb by solid dispersion technique. eighteenth exb formulas were formulated as a solid dispersion using a variety of hydrophilic polymers (as carriers) including poloxamer 407 (pxm 407), poloxamer 188 (pxm 188) and polyethylene glycol 4000 (peg 4000) at different drug: polymer ratios (1:1, 1:3 and 1:5). these formulas were prepared by two methods; solvent evaporation and fusion method. the prepared formulas were evaluated for practical yield percent (py %), drug content, saturated solubility and release rate, fourier transforms infrared spectroscopy (ftir), differential scanning calorimetry (dsc) and powder x-ray diffraction (pxrd). it was found that the solubility was affected by the polymer type and the method of preparation. the polymers (as carriers) used to prepare exbsolid dispersion showed improvement in the solubility in the following descending order; pxm 407>pxm 188> peg 4000. the optimum formula (sd15) composed of the drug: pxm 407 at a ratio of 1:5 was prepared by solvent evaporation showed 7.76 folds (676.40%) solubility improvement as compared to pure exb. the optimum formula showed a release rate of 99.8% through the first 15 min. the advance characterization of the selected formula indicated the possible transformation of the drug to the amorphous state. keywords: exb, solid dispersion, pxm 407, pxm 188, peg 4000. تعزيز قابلية الذوبان ومعدل االنحالل لالتوريكوكسيب بتقنية المنتشر الصلب مصطفى مظفر عبد الرحمن*،1 و فاطمة جالل جواد ** العراق.، دائرة صحة كركوك العام،مستشفى كركوك والبيئة،ة حوزارة الص * العراق. بغداد،، جامعة بغداد الصيدلة،كلية ، فرع الصيدالنيات * * الخالصة إيتوريكوكسيب . 2 -بصورة انتقائية عالية كمثبط لإلنزيم كوكس الستيروئيدية ويعملمن ادوية مضادات االلتهاب غير إيتوريكوكسيب هو الثانية وفقًا لنظام تصنيف المستحضرات الصيدالنية الحيوية، والذي يمتلك قابلية ذوبان مائي منخفضة للغاية في الماء. من الدرجةيصنف هو دواء .من خالل تقنية المنتشر الصلب ومعدل الذوبانفي هذه الدراسة تم إجراء عدة محاوالت لتحسين الذوبانية لعقار االتوريكوكسيب كصلب منتشر باستخدام مجموعة متنوعة من البوليمرات المحبة للماء )كحامالت( و عشر صيغةتم تصييغ ثماني : 1,1: 1بنسب دواء: بوليمر مختلفة ) peg 4000) اثلين كاليكول )والبولي (pxm 188)188،بولكزامر (pxm 407) 407بولوكزامرتشمل طريقة تبخير المذيبات واالنصهار. تم تقييم الصيغ المحضرة بتقييم النسبة المئوية للعائد العملي، محتوى بطريقتين؛(. حضرت هذه الصيغ 5: 1, 3 و حيود األشعة السينية dsc)) الكالوريفرق المسح ،((ftirت الحمراء المشبع ومعدل التحرر. تم تطبيق مطياف االشعة التح الدواء، الذوبان pxrd).في هذه الدراسة ) اظهرت البوليمرات المستخدمة تحسنًا في قابلية الذوبان التحضير. فقد المستخدم وبطريقةلقد اظهرت النتائج بأن ذوبانية الدواء تتأثر بنوع البوليمر التالي؛في الدواء بالترتيب التنازلي peg4000< pxm 188< pxm 407( أظهرت الصيغة المثلى .sd15 :المكونة من الدواء )pxm 407 دواء:بولمر( المحضرة 5: 1بنسبة( ٪( تحسن في الذوبانية مقارنة مع اإليتوريكوكسيب النقي. وأظهرت الصيغة المثلى معدل 676.40أضعاف ) 7.76بواسطة تبخير المذيبات زيادة للصيغة المثلى إلى احتمال تحول الدواء إلى الحالة الغير متبلورة. يشير التحليل المتقدمدقيقة. 15٪ خالل أول 99.8 تحرر .بولي اثلين كاليكول. بواوكزامر , الصلب المنتشر , إيتوريكوكسيب :لكلمات المفتاحية 1corresponding author e-mail: :mustaphaalkhalidi@gmail.com received: 16 / 6 /2019 accepted:21 / 9/2019 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp76-87 iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 77 introduction etoricoxib (exb) is a cyclooxygenase-2 (cox-2) specific inhibitor which belongs to non-steroidal antiinflammatory drugs (nsaids). cyclooxygenase-2 physiological role is the synthesis of prostanoid mediators of pain, inflammation and fever. exb selectively inhibits the cyclooxygenase-2(cox-2) isoenzyme action while sparing cyclooxygenase-1 (cox-1), which is presented in the gastrointestinal tract, kidneys and platelets (1). therefore, the potential gastrointestinal adverse effects and impacts on platelet aggregation are less than other non-selective cox inhibitors (2). etoricoxib is used to relieve the symptoms of rheumatoid arthritis, osteoarthritis, acute gout, chronic musculoskeletal pain (including chronic low back pain), and primary dysmenorrhoea (3). etoricoxib has nomenclature is 5-chloro-6'methyl-3[4(methylsulfonyl) phenyl] -2, 3’bipyridine and a molecular weight of 358.84 g/mol (figure 1). it has a molecular formula of c18h15cln2o2s, a partition coefficient (logp) of 3.9, and it presented as a whitish to creamish coloured powder (4). figure 1.structural formula of exb (5) etoricoxib is classified as a class ii based on the biopharmaceutical classification system, due to its low aqueous solubility and thus reduced dissolution rate, which may induce challenges to the formulator (6). poor aqueous solubility is a significant problem that can delay the action of the drug and impart difficulties to the drug formulation. the enhancement of drug solubility has a significant impact on improving dissolution behaviour, which later on improving drug pharmacokinetics and therapeutic efficacy (7). to enhance the low aqueous solubility of the drug and improve its dissolution, several strategies can be employed such as particle size reduction, use of surfactants, ph adjustment, and complexation with cyclodextrin and solid dispersion (8). solid dispersion was first introduced in 1961 by sekiguchi and obi as a method for enhancing aqueous solubility and dissolution rate of poorly soluble drugs. the proposed solid dispersion is an eutectic mixture of a hydrophobic drug dispersed in an inert hydrophilic carrier or matrix (9). the purpose of the present study was to enhance the solubility and dissolution rate of exb by solid dispersion using different carriers in different ratios and different preparation methods. the study was done to compare and investigate the best carrier type, ratio and method to enhance exb solubility and dissolution rate. materials and methods materials etoricoxib (virdev, india), poloxamer 407 (pxm 407) and poloxamer 188(pxm 188) from sigma, usa), polyethylene glycol 4000 (peg 4000) from (ineos chemicals, belgium), methanol (sigma-aldrich co., germany). all other reagents were of analytical grade. methods preparation of exb-solid dispersion by the solvent evaporation method etoricoxib solid dispersion was prepared by solvent evaporation method using different polymers (as carriers) including pxm 407, peg 4000, and pxm 188 at three different ratios (1:1, 1:3, 1:5) (drug: carrier ratio w:w) as shown in table (1). the carriers and drug were accurately weighed and dissolved in methanol separately by utilizing a magnetic stirrer. then followed by mixing of the two solutions by a magnetic stirrer as well. the solvent was evaporated by leaving it in an oven at 40°c for 24 hr. the resultant dried mass was pulverised and sieved through sieve no.60 then stored in a desiccator for further investigations (10). preparation of exb-solid dispersion by the fusion method the fusion method involved liquefying specific amounts of carriers and drug at ratios (1:1, 1:3, and 1:5) (drug: carrier ratio w: w) as shown in table (1). the carrier was first melted in porcelain petri-dish slightly above its melting point. the drug was added to the molten carrier followed by constant stirring for five minutes then cooled down using an ice bath. the dried mass was then pulverised and sieved through sieve no.60 and stored in a desiccator for further investigations (11). iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 78 table 1. composition of exb solid dispersion formula number carrier drug: carrier ratio (w:w) preparation method sd1 pxm 188 1:1 fusion sd2 pxm 188 1:3 fusion sd3 pxm 188 1:5 fusion sd4 pxm 407 1:1 fusion sd5 pxm 407 1:3 fusion sd6 pxm 407 1:5 fusion sd7 peg 4000 1:1 fusion sd8 peg 4000 1:3 fusion sd9 peg 4000 1:5 fusion sd10 pxm 188 1:1 solvent evaporation sd11 pxm 188 1:3 solvent evaporation sd12 pxm 188 1:5 solvent evaporation sd13 pxm 407 1:1 solvent evaporation sd14 pxm 407 1:3 solvent evaporation sd15 pxm 407 1:5 solvent evaporation sd16 peg 4000 1:1 solvent evaporation sd17 peg 4000 1:3 solvent evaporation sd18 peg 4000 1:5 solvent evaporation characterization of exb-solid dispersion determination of exb saturated solubility an excess amount of exb pure powder was added to 10 ml of distilled water, 0.1n hcl and phosphate buffer ph 6.8. the mixtures were placed in a shaking water bath at a constant temperature of 25°c for 72 hr. span. then the mixture in suitable tubes was placed in the centrifuge for 15 min at 3000 rpm. the solutions were filtered utilizing a 0.45μm filter syringe, and concentration of the filtrate was measured by uv –spectrophotometer at λmax 234 nm to determine the solubility of exb (12) determination of practical yield per cent (py %) of the prepared exb solid dispersion the practical yield per cent (py %) of the prepared exbsolid dispersion was conducted to know the efficiency of each preparation method. the (py %) was determined by calculating the ratio of the actual weight of the obtained solid dispersion on the theoretical mass of drug and carrier (equation 1) (13). py(%) = practical mass(sd) theoretical mass(drug + carrier) × 100 eq. (1) determination of exb content in the prepared solid dispersion the exb solid dispersion equivalents to 50mg of exb was dissolved in 40 ml methanol using 50 ml volumetric flask and followed by sonication for 15 min. the volume was made up to 50 ml using methanol. after that, the resultant drug solution was diluted with methanol; the drug solution was assayed for drug content using uv-spectrophotometer at λmax 234 nm (14). the percentage of drug content in the obtained solid dispersion was determined by using the following (equation-2) (15). drug content% = actual weight of exb theoretical weight of exb × 100 eq. (2) determination of saturated solubility of exb in solid dispersion the solubility of the prepared exbsolid dispersion was carried out in aqueous media. the solubility study was performed by adding excess amounts of exb-solid dispersion into screw-capped vials containing 10 ml of water. the tightly sealed vials were kept in a water bath shaker at 25°c for 48hr. the saturated solutions were then analysed using uv-spectrophotometer at λmax 234 nm (16). in-vitro dissolution studies of exb-solid dispersion in-vitro dissolution of exb-solid dispersion was determined and compared with pure exb drug by using usp xxii rotating paddle apparatus i. etoricoxib-solid dispersion equivalents to 60 mg of the pure drug was dispersed in dissolution medium surface. the dissolution medium employed for drug release study was 900 ml of phosphate buffer (ph 6.8) containing 1% w/v sodium lauryl sulphate (sls) to maintain sink conditions. the temperature was set at 37±0.5°c using a iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 79 thermostatic water bath with shaking by the paddle rotating at 100 rpm (4). five millilitres samples were withdrawn after 5, 10, 15, 20, 30, and 45 min. sink condition was kept by replacing every withdrawn sample with an equal volume of fresh phosphate buffer (ph 6.8 with 10%w/v sls). the samples were filtered via a 0.45μm filter membrane and assayed using uv-spectrophotometer at λmax 234 nm (17). factors affecting dissolution behaviour of exb from solid dispersion effect of polymer type the impact of different polymer types (pxm 188, pxm 407 and peg 4000) on the drug release of prepared solid dispersion was studied by the formulas sd12, sd15 and sd18, respectively. these formulas had the same ratio of drug: polymer (1:5) and prepared by solvent evaporation method. the results were compared to the release of pure drug. the ratio of drug: polymer (1:5) was chosen to study this effect since the highest solubility was obtained by this ratio for all the used polymers compared to other ratios. effect of the drug: carrier ratio the impact of the different drug: polymer ratio on the drug release of prepared solid dispersion was studied with formulas sd13 (1:1), sd14 (1:3) and sd15 (1:5). since all of them were prepared by solvent evaporation method using pxm 407 as a carrier. the results were compared to the release of pure drug. effect of preparation methods the effect of preparation method on the drug release of prepared solid dispersion was studied with formulas sd6 which was prepared by fusion method and sd15 which was prepared by solvent evaporation method. these formulas were chosen since both of them were prepared using the same polymer (pxm 407) and with the same drug: polymer ratio (1:5) but by different methods. selection of the optimum formula the selection of the optimum formula was depended on the solubility study and the dissolution profile of exb solid dispersion. evaluations of the selected formula fourier transforms infrared spectroscopy (ftir) the ftir analysis of pure exb, pxm 407 and the selected formula was performed to investigate drug-polymer intermolecular interaction. the samples were compressed with potassium bromide (kbr) as a disc, and analyzed by ftir apparatus (shimadzu8000, japan); the scanning range was 4000400 cm-1 (18). differential scanning calorimetry (dsc) thermal characteristics of the selected formula, pure exb and the purely related polymer were analyzed by an automatic thermal analyzer system (shimadzu, dsc-60, and japan). each sample (5 mg) was placed in none hermetically aluminium pan and heated at rate 10°c/ min over temperature 5°c to 300°c. the analysis was carried out under the atmosphere flow conditions (19). powder x-ray diffraction (pxrd) powder x-ray diffraction was utilized to analyze the crystallinity of the pure exb and selected formula. the pxrd measurement was performed under the following conditions: the target metals cu, filter kα, 45kv voltage, 30ma current. samples scanned over a 2θ range of 30-80° at a step size of 0.02° (20). statistical analysis the results of the experiments were given as a mean for triplicate samples ±standard deviation (std) and were analyzed according to a one-way analysis of variance (anova). the test level of (p < 0.05) was considered to be statistically significant, using microsoft excel 2010. results and discussion characterization of exb-solid dispersion determination of exb saturated solubility the solubility of exb in different media (water, 0.1n hcl and phosphate buffer ph 6.8) at 25 °c was determined. table 2 showed that the equilibrium saturated solubility for exb was highly ph-dependent. since, the solubility decreased from 24.23 mg/ml (sparingly soluble) at ph 1.2 of 0.1 n hcl to 0.076 mg/ml (practically insoluble) at ph 6.8 phosphate buffer. this result was in agreement with previous study (21). iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 80 table 2.the saturated solubility of exb in different media at 25°c. solvent solubility (mg/ml) mean±std (n=3) solubility definition distilled water 0.089 ± 0.1 practically insoluble 0.1 n hcl 24.23 ± 0.4 sparingly soluble phosphate buffer 6.8 0.076 ± 0.2 practically insoluble determination of practical yield percent (py %) and content of the prepared exbsolid dispersion the (py %) of the prepared exb-solid dispersion was determined to investigate the best method for the preparation of solid dispersion granules. both solvent evaporation and fusion methods gave acceptable py % ranged from 94-99.3%, as shown in table 3. the results elucidated that both fusion and solvent evaporation methods are comparable. the results are associated with the best entrapment of exb particles by carrier utilized in both preparation methods. moreover, the content of exb loading in solid dispersion was found in a range of 94.13% 103.9% in all prepared formulations, which was consistent with the same range of u.s. pharmacopoeia requirements (90-110%). the above results suggested a uniform distribution of exb particles within polymers (as a surfactant) used in all prepared formulas (22). table 3. the practical yield percent (py %) and drug content of exb solid dispersion using different methods formula number preparation method practical yield percent (py %) drug content mean±std (n=3) sd1 fusion 97.42% 98.62 ±0.01 sd2 fusion 96.6% 98.23 ±0.03 sd3 fusion 98.8% 99.05 ±0.01 sd4 fusion 97.88% 103.9 ±0.05 sd5 fusion 99.43% 97.9 ±0.02 sd6 fusion 98% 98.11 ±0.01 sd7 fusion 98.17% 98.61 ±0.14 sd8 fusion 98% 94.13 ±0.06 sd9 fusion 96% 95.11 ±0.01 sd10 evaporation 98% 97.56 ±0.01 sd11 evaporation 95.6% 99.08 ±0.02 sd12 evaporation 99.3% 99.70 ±0.12 sd13 evaporation 94% 97.03 ±0.01 sd14 evaporation 94.35% 95.40 ±0.04 sd15 evaporation 95.4% 97.7 ±0.06 sd16 evaporation 98.8% 99.03 ±0.01 sd17 evaporation 96.6.% 98.8 ±0.03 sd18 evaporation 96.06.% 99.8 ±0.12 determination of saturated solubility of exb in solid dispersion it was found that there was no significant difference among pxm 188, pxm 407 and peg 4000 at 1:1 (drug: polymer) ratio in enhancing solubility using solvent evaporation method as shown in figure 2. while, figure 3 shows that a significant (p< 0.05) enhancement in solubility was obtained by using pxm 407 at 1:3 (drug: polymer) ratio using solvent evaporation method. the best result (0.691 mg/ml) was found when pxm 407 was utilized as a carrier at a ratio of 1:5 w: w (drug: polymer) the solid dispersion was prepared by solvent evaporation method as shown in figure 4. the enhancing in the aqueous solubility of 7.76 folds (676.40%) with pxm 407 as compared with pure exb was due to the hydrophilic nature of the carriers and the hydrogen bonding formation between exb and these carriers (23). the high hydrophilic nature of pxm 407 in comparison with other carriers is contributed to increasing the wettability and high solubility of the exb particles (24). iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 81 figure 2. effect of carrier type of exb solid dispersion at ratio (1:1) on the solubility of exb in distilled water at 25°c . figure 3. effect of carrier type of exb solid dispersion at a ratio (1:3) on the solubility of exb in distilled water at 25°c . figure 4. effect of carrier type of exb solid dispersion at a ratio (1:5) on the solubility of exb in distilled water at 25°c in-vitro release of exb solid dispersion effect of polymer type at a constant ratio of drug: polymer (1:5), pxm 407(sd15) showed exb release of 99.8% within the first 15 min in comparison with other the types of carriers and pure exb. the release percentage after 15 min were 82.21%, 77.76% and 21.19% for pxm 188(sd12), peg 4000(sd18) and pure exb, respectively as shown in figure 5. this significant result (p˂0.05) was attributed to the enhanced solubility of exb solid dispersion. these results were due to the increase of the wettability and possible decrease in the crystallinity of exb. the outcome was a consequence of the interaction of exb with many propylene oxide hydrophobic segments of pxm 407 (25). the use of surface-active agents (e.g. poloxamer) may overcome precipitation and the recrystallization of the drug from the amorphous state during the cooling or solvent removal step of the preparation process or storage which was considered as one of the disadvantages of solid dispersion (26). figure 5. effect of polymer type of exb solid dispersion prepared by solvent evaporation method with the drug: polymer ratio 1:5 on the release profile of exb in phosphate buffer ph 6.8 dissolution medium at 37°c effect of the drug: carrier ratio the effect of the different drug: carrier ratio on the dissolution behaviour of exb solid dispersion is shown in figure 6. the figure represents the release profile of exb drug from solid dispersion prepared by the solvent evaporation method using pxm 407 as a carrier as well as pure exb. it was observed that as the amount of pxm 407 increased in formulas (sd13, sd14, sd15), the drug release from solid dispersion was increased significantly (p<0.05). formula sd15 exhibited the highest the percent of release by (99.8%) within the first 15 min as compared with sd13 (61.3%), sd14 (74.09%) pure exb (21.19%). the enhanced in-vitro dissolution rate of exb solid dispersions that were formulated with higher ratios of the water-soluble carriers was attributed to the improved drug wettability, high probability of a reduction in particle size and possible conversion to the amorphous forms which was confirmed later by dsc and pxrd (27). iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 82 figure 6. effect of exb: pxm 407 ratio on the per cent drug release profile from exb solid dispersion formulated by the solvent evaporation method in phosphate buffer ph 6.8 dissolution medium at 37°c. effect of preparation methods the dissolution profile of exb binary solid dispersion was affected by the preparation techniques. figure 7 revealed the amount of exb released from the formulas contain pxm 407 at a ratio 1:5 that formulated by solvent evaporation (sd15) and fusion (sd6) methods. the amount of exb released after 15 min was found to be 99.8% for (sd15) and 81.5% for (sd6). therefore the binary system of exb and polymer (as a carrier) tend to release exb as fine particles when it touched the dissolution medium as compared to the fusion technique. the higher release privilege was attributed to the uniform distribution of the drug and the surface activity of the pxm 407 in the molecular dispersion (28). figure 7.effect of preparation methods on the release of exb from pxm 407 solid dispersion of ratio (1: 5) in phosphate buffer at ph 6.8 dissolution medium maintained at 37°c. selection of the optimum formula the optimum formula was (sd15) which composed of exb-pxm 407 solid dispersion at a ratio of 1:5 that was formulated by the solvent evaporation method. it was characterised by enhanced solubility, and the highest amount of exb released within a short time. therefore it was subjected to further invitro evaluation studies. evaluation of optimum formula fourier transforms infrared spectroscopy (ftir) the ftir spectrum of the pure exb (figure 8) showed characteristic peaks of c–h stretching vibration at 2964 cm−1 and c = n stretching vibration at 1597cm−1, 1564 cm−1 and 1493 cm−1. prominent peaks at 1431 cm−1, 1298 cm−1, 1144 cm−1 and 1086 cm−1 indicate s=o stretching vibrations. c–cl stretching vibration was denoted by the presence of peaks at 841 cm–1, 775 cm–1 and 729 cm–1, these results were in agreement with previous studies (29). the ftir spectra of optimum formula (sd15), as shown in figure 10, was examined and compared with ftir of exb pure (figure 8) powder and pxm 407 (figure 9) spectrum separately. in sd15 spectrum all peaks of exb were almost diffused except peaks at 777, 841 and 1599 cm−1, while the other peaks could no longer be noticeable. the characteristic peaks of pxm 407 were 1111 cm−1 (c-o intense) and 2887 cm–1(c–h stretching) presented at the same position and became more intensified in the solid dispersion. however, no additional peak was observed in the solid dispersions declared the absence of any chemical interaction between exb and carrier (30). iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 83 figure 8. ftir spectrum of exb figure 9. ftir spectrum of pxm 407. iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 84 figure 10.ftir spectrum of the best formula (sd15) differential scanning calorimetry (dsc) the dsc thermograms of exb and pxm 407 were represented in figures 11 and 12 show a sharp endothermic peak at 138.00°c and 58.2°c, respectively. they represented the melting point of exb and pxm 407, which, revealed the crystalline nature of the drug and the polymer as previously reported (31,32). figure 13 represented the dsc thermogram of sd15. there was a single endothermic peak at 55.9°c which was around the pxm 407 melting point while, the absence of endothermic peak of exb (at 138.0°c) might be attributed to the existence of exb in an amorphous state rather than its original crystalline state (33). figure 11.the dsc thermogram of exb. iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 85 figure 12. the dsc thermogram of pxm 407 figure 13.the dsc thermogram of the sd15 powder x-ray diffraction (pxrd) powder x-ray diffraction was utilized to analyze the crystallinity of the substance such as pure exb and sd15. the degree of crystallinity was resolved (34). the pxrd analysis of exb and optimum formula (sd15) were given in figures 14 and 15, respectively. the drug characteristic peaks were observed at 16.59°, 15.52° and 18.14° at a 2θ value with intensity 4750, 2480 and 2060, respectively. while the pxrd pattern of optimum formula (sd15) showed characteristic peaks at 19.15°, 23.28° and 22.75° with an intensity of 2320, 2120 and 700 respectively. the intensity of the characteristic peaks of the pure drug(16.57°, 15.49° and 18.13°) were markedly reduced to (600, 310 and 300) indicating the drug crystalline was converted to amorphous upon molecular dispersion (35). figure14. x-ray diffraction pattern of pure exb iraqi j pharm sci, vol.29(1) 2020 solid dispersion of etoricoxib with hydrophilic carriers 86 figure 15. x-ray diffraction pattern of the sd15 conclusions based on the results obtained from the present study,it can be concluded that the solubility of exb (class iι drug) was successfully enhanced by solid dispersion technique, and pxm 407 surface active agent was the best carrier at a ratio of 1:5 drug: polymer. moreover, the solvent evaporation method was the preferred method for the preparation of exb solid dispersion in contrast to the fusion 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pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghda. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 natural products: promising therapy for sars cov-2 doi : https://doi.org/10.31351/vol30iss1pp29-40 29 natural products as a promising therapy for sars cov-2; an overview noor s. jaafar*,1 and iman s jaafar ** *department of pharmacognosy and medicinal plants ,college of pharmacy , university of baghdad, baghdad, iraq ** department of pharmaceutics ,college of pharmacy , mustansiryah university, baghdad, iraq abstract recently emerging pandemic sars cov-2 conquered our world since december 2019. continuous efforts have been done to find out effective immunization and precise treatment stetratigies. a way from therapeutic options that were tried in sars cov-2, an increased attention is directed to predict natural products and mainly phytochemicals as collaborative measures for this crisis. in this review, most of the mentioned compounds specially flavonoids (biacalin, hesperidin, quercetin, luteolin,, and phenolic (resveratrol, curcumin, and theaflavin) exert their effect through interfering with the action of one or more of this proteins (spike protein, papain-like protease, 3-chymotrypsin-like cysteine protease, and rna‐dependent rna polymerase) that are involved in viral life cycle beside the anti-inflammatory effect of these compounds. the triterpenoids (celastrol, escin and glycyrrhizin) and the alkaloids (lycorine and cepharanthine) mediated their effect mainly through anti-inflammatory activity. glycyrrhetinic acid (glycyrrhizin metabolite) dawn regulates ace-2, and reduces protease expression, thus reduce viral entry. this review may be representing an initial step in long path for designing the successful and effective treatment or vaccine for this pandemic. keywords: sars cov-2, remedsiver, antiviral, phytochemical, interleukin. :نظرة عامة cov-2المنتجات الطبيعية كعالج واعد لسارس **صباح جعفر و ايمان 1،*نور صباح جعفر بغداد،العراق جامعة بغداد، والنباتات الطبية ،كلية الصيدلة،فرع العقاقير بغداد،العراق فرع الصيدالنيات ،كلية الصيدلة، الجامعة المستنصرية ، الخالصة . وقد بذلت جهود مستمرة الكتشاف التحصين الفعال وإالجراءات 2019الناشئة مؤخًرا عالمنا منذ ديسمبر cov-2 جائحة السارس زتغ .محددةالعالجية ال ، يتم توجيه اهتمام متزايد للتنبؤ بالمنتجات الطبيعية والمواد الكيميائية النباتية بشكل cov-2بعيدا عن الخيارات العالجية التي تم استعمالها لسارس فونويد )بايكالين ، هيسبيريدين ، أساسي كعالجات مساندة في هذه األزمة . في هذه المراجعة، معظم المركبات المذكورة وخاصة مركبات الفال تأثيرها من خالل التداخل مع عمل واحد أو أكثر من هذه البروتينات تظهرفينوالت )ريسفيراترول ، كركمين ، وثيافالفين( الكويرسيتين ، لوتيولين ، و na‐dependent rna rlike cysteine protease, and -chymotrypsin-3like protease, -spike protein, papain ( polymerase) ، التي تشارك في دورة الحياة الفيروسية بجانب التأثير المضاد لاللتهابات لهذه المركبات. الترايتيربينويدات )سيليسترول ، إيسين حمض الجليسيررتينيك )مستقلب جليسيررهيزين( والقلويدات )ليكورين وسيفارانثين( تعطي تأثيرها بشكل رئيس من خالل النشاط المضاد لاللتهابات. ، ويقلل من تعبير البروتياز ، وبالتالي يقلل من دخول الفيروس.قد تمثل هذه المراجعة خطوة 2الجليسيررهيزين( يثبط اإلنزيم المحول لألنجيوتنسين .أولية في مسار طويل لتصميم ناجح وعالج أو لقاح فعال لهذا الوباء , ريميدسفير,مضاد فايروسي,المركبات النباتية الكيميائية,انترلوكينز.cov-2السارس الكلمات المفتاحية: introduction one of the main pathogens that mainly goals the respiratory system in human is coronavirus (cov), which is be allied to the coronaviridae family. under the electron microscope cov signifies spike like projections on its outer surface granting it a crown-like appearance; therefore the designation coronavirus (1). covs are miniature in size as well as consist of a single stranded rna .the cov family subgroups include alpha (α), beta (β), gamma (γ) in addition to delta (δ) coronavirus (2) . 1corresponding author e-mail: nooraldahan201@gmail.com received: 31/8/2020 accepted: 21/11 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp29-40 iraqi j pharm sci, vol.30(1) 2021 natural products: promising therapy for sars cov-2 30 earlier outbreaks of cov consist of middle east respiratory syndrome (mers)-cov as well as the severe acute respiratory syndrome (sars)-cov which have been previously categorized as agents that form abundant public health hazard (3). recently coronavirus disease 2019 (covid-19), a new developed respiratory disease resulted from severe acute respiratory syndrome coronavirus 2 (sarscov-2), has turn out to be pandemic (4). sars-cov-2 is contemplating a novel human infecting β cov. phylogenetic studies of the sars-cov-2 genome show that the virus is closely correlated (by 88% identity) to two bat-derived sars like covs and genetically distinct from sars-cov (by around 79% similarity) as well as mers-cov (5).these studies point out that bats may aid as the virus natural host , signifying that mammals are the almost certainly link between sars-cov-2 and humans (6). transition numerous reports have proposed that human to human transmission is a possible route for sars-cov-2 infection dispersal (7). all age groups are prone to infection which is transmitted via large droplets generated by both symptomatic and asymptomatic people. the infection can be transmitted even before onset of symptoms during patient coughing and sneezing. even on clinical recovery patients could be infectious provided that the symptoms last (8). it has been stated that the virus can endure from 2 hours to few days in the droplets on the surfaces or else ground and may infect individuals at a distance of about 1.8 meters radius (9). infection is acquired either by inhalation of the droplets or touching the mouth, nose, as well as eyes after contact with contaminated objects or surfaces. both stool and contaminated water may also contain virus. as a result consequent transmission through aerosolization and/or fecal oral route is strongly hypothesized (10). pathogenesis afterwards human body entrance , sarscov-2 primary enters the host cells prior to replication (11). the upper respiratory tract mucosal lining epithelium is the main site for viral replication. additional multiplication occurs in lower respiratory tract and alimentary mucosa (12). angiotensin converting enzyme 2 ( ace2) which is broadly expressed in heart , lung and kidney has been recognized as the functional receptor by which sars-cov-2 enters the body mucosa (13). the binding of the virus spike (s) protein (separated to the s1 and s2 part) to ace2 activate sars-cov-2 infection. the attachment of the virus to the target host cells surface is expedites via binding of s1 part of the virus s protein to the ace2 cellular receptor of the host. viral s protein priming then requires s protein cleavage of s1 from s2 (and at another s2’ site) by the host cell serine protease tmprss2. the fusion of the cell membranes of both virus and host is driven by the s2 subunit of the virus (14). moreover, variance response through t helper cells (type 1 and type 2) activates cytokine storm and both respiratory dysfunction besides hypoxemia induced by sars-cov-2 can cause myocardial cells impairment (15). this progress is related to inflammatory cytokines elevation including il2, il7, il10, gcsf, ip10, mcp1, mip1a in addition to tnfα (16). symptoms the clinical manifestations of sars-cov2 varying from asymptomatic infection to acute respiratory distress syndrome (ards) and multiple organ impairment. the average incubation period assessed to be 5.2 days (17). commonly observed symptoms include fever, sore throat, cough, myalgia or fatigue, dyspnoea, headache, diarrhea as well as conjunctivitis (16, 18). in some severe cases serious respiratory syndromes, pneumonia, kidney impairment and even death may occur. severe clinical patterns are witnessed in individuals with coexisting health conditions like cardiovascular disease, diabetes, lung disease and cancer (15, 19). adverse outcomes are also more common in aged as well as frail patients (20). diagnosis the initial identification of sars-cov-2 patients is based on the clinical manifestation incidence related to early disease stage development. different diagnostic methods are used as confirmatory test for patients with sars-cov-2. real-time polymerase chain reaction (rt-pcr) considered as a standard diagnostic test which is based on the recognition of the sars-cov-2 sequence (21). since the virus rna has been isolated from both upper as well as lower respiratory tract samples, the collection of nasopharyngeal besides oropharyngeal swabs is the main method for diagnosis (22). detection of sars-cov-2 rna in stool and blood specimens has also been proved by numerous studies (23, 24). distinctive range of laboratory anomalies can be considered as an additional nonspecific laboratory characteristics for patients with sarscov-2 (15, 25). an elevated level of serum c-reactive protein, alanine aminotransferase, lactate dehydrogenase and aspartate aminotransferase can be observed. high percentage of patients showed reduced albumin level (26). hematological abnormality include lymphopenia, mild thrombocytopenia, prothrombin time prolongation and elevated d-dimer values (25, 27, 28). both d-dimer and lymphopenia to a minor degree seem like to have the main prognostic associations (28). besides to the previously designated methods, antigen-antibody reaction based kits offer rapid diagnostic technique. this test exploits immune reaction of combined human antibodies i.e., immunoglobulin m (igm) in addition to igg through spike proteins of sars-cov-2 from the blood sample of patient (29). iraqi j pharm sci, vol.30(1) 2021 natural products: promising therapy for sars cov-2 31 additional specific and more sensitive diagnostic tool for sars-cov-2 is computerized topography or ct. related features of ct images are shown in the majority of patients including ground glass opacity and bilateral patchy infiltrates (30). conventional chest x ray is less sensitive tool comparing to ct. in a very limited cases, ultrasound has been used as a diagnostic tool (31). treatment presently, there are no definite drugs or vaccine for potential treatment of sars-cov-2 till now. treatment strategies are basically symptomatic and supportive. nevertheless, to manage this disease some perspectives have been used (32). the single choice available is the practice of broad-spectrum antiviral agents such as nucleoside analogues as well as hiv-protease inhibitors that could weaken virus infection till availability of precise antiviral drug. in preclinical trials for both sars-cov and mers-cov infections, remdesivir as per a nucleotide analogue has been stated to be effective agent through acting on the coronaviruses polymerase. remdesivir acts by impeding into the rna chain of budding viral and then results in its premature termination (33). the combination of lopinavir/ritonavir, a protease inhibitor demonstrated a potent efficacy against sars-cov-2. a reduction in the viral load in sars-cov-2 patients was also observed with this combination (34). another antiviral agent which is suggested as probable therapy is nelfinavir, a selective hiv protease inhibitor that revealed strong sars-cov-2 inhibition (35). other therapeutic options suggested for treatment are arbidol (antiviral agent with a broadspectrum activity)(36), interferons (antiviral cytokines)(37), monoclonal antibody(38), convalescent plasma (plasma acquired from individuals recovered from sars-cov-2)(39) and intravenous immunoglobulin (40). conceivable natural products against sars-cov-2 resveratrol it is a (trans-3,4′,5-trihydroxystilbene) (40) (figure 1). this polyphenol was at first detected in 1940 in white hellebore roots and synthesized by not less than 72 plant species, grapes, cranberries, and huzhung are included (41, 42). resveratrol is a phytoalexin exhibits antiviral effect against several viruses as pseudorabies virus, african swine fever virus, herpes simplex virus, influenza a virus, human immune deficiency virus, and respiratory syncytial virus (43, 44). lin et al (2017) proved the antiviral action of resveratrol against mers-cov. the antiviral effect was the product of several mechanisms which ultimately result in opposing viral-induced apoptosis and promotion or stimulation of cell survival (42). marenella perspective (2020) was that resveratrol might be effective supportive management for sars cov-2. ace2 (angiotensinconverting enzyme -2) receptors are the target of sars-cov. receptors–virus interaction influences viral entrance, clearance, and it has a crucial role in determining arsd (acute respiratory distress syndrome) symptom severity. following viral infection, the expression of the receptors is downregulated which may affect pathogenesis and disease prognosis (45). horne jr and a college perspective' (2020) was based on previous studies in the animals and in vitro study on human aortic smooth muscles, these studies demonstrate that resveratrol alone or dietary fat lessening in combination with resveratrol causes ace2 upregulation. they suggest a probable mechanism whereby dietary fat reduction and/or rise resveratrol intake may modify responses to sarscov 2 (46).wahedi hm et al (2020) in a molecular docking studies showed that stilbene-based natural constituents and especially resveratrol might be an auspicious sars-cov 2 drug candidate which disrupted sars-cov-2 spike s glycoprotein and human ace2 receptor complex (47). figure 1. chemical structure of resveratrol (48) curcumin it is a polyphenolic compound [1,7-bis(4hydroxy-3-methoxyphenyl)-1,6-hep tadiene 3,5dione] (figure 2), and the main curcuminoid obtained from turmeric ( curcuma longa).various interesting biological activities have been displayed by curcumin, such as antimicrobial, antioxidant, anticancer, antiviral, and others (49, 50). zahedipour et al (2020) mentioned a suitable justification based on modern and previous studies (curcumin impeding viral cell entry, inhibiting virus encapsulation and viral protease enzyme as well as modifying diverse cellular signaling pathways) that encourage the planning of modern studies and clinical trials using curcumin as a therapeutic agent for sars-cov-2 (51). chen et al (2020) evaluate the effect of a combination of phytochemicals (vitamin c, curcumin, and glycyrrhizic acid abbreviated as vcg plus) against the coronavirus utilizing systems biology. the study revealed that this combination can have an action on 88 hub targets that are closely coupled and linked with immune and inflammatory responses. vcg plus can modulate or regulate the innate immune response by influence nod-like and iraqi j pharm sci, vol.30(1) 2021 natural products: promising therapy for sars cov-2 32 toll-like signaling pathways to stimulate interferon generation, activation and balancing t-cells, and for regulating the inflammatory response by inhibiting of certain signaling pathways (pi3k/akt, nf-κb, and mapk). the inhibition of inflammatory response, in turn, prohibit cytokine storm which is observed in certain sars-cov-2 (52). in addition to the proved antiviral effect of curcumin against other viruses. rosha et al (2020) postulated that this phytochemical probably beneficial in sars-cov 2 patients through various actions as; antithrombotic activity since an elevated number of thrombotic events occur in those patients, anti-cytokines and antifibrotic effects could assist in lung involvement. the main pathway for sars-cov-2 cell entry might be interposed by curcumin (53). figure 2. chemical structure of curcumin (54) biacalin baicalin is (7-glucuronic acid, 5,6dihydroxyflavone, or 7-o-glycoside 0f biacalein) (figure 3). it is the main bioactive compound from the root of scutellaria baicalensis, also it is present in the thymus vulgaris l. leaves (55, 56). chu et al (2015) proved through in vitro and in vivo (in mice) the antiviral activity of biacalin against influenza virus a (h1n1) as a powerful inducer of interferongamma (ifn-𝛾) in major ifn-𝛾 producing cells (57). jelic d et al (2016) showed in two assays that biacalin (the glycoside) and biacalein (the aglycone) are significant inhibitors for src tyrosine kinase, inhibition of this receptor is associated with the decreased generation of inflammatory cytokines, one of which il-6 in lps-stimulated thp-1 cells. the inhibitory effect (anti-inflammatory effect) of biacalein is more than that of biacalin. in mts cytotoxicity assay in thp-1 cells both of these flavonoids demonstrated no cytotoxicity (56). haixia su et al (2020) investigated the inhibitory effect of biacalin and biacalein vs sarscov-2 3clpro. (58). 3clpro is a 3-chymotrypsinlike cysteine protease this enzyme rules replication of coronavirus and is crucial for its life cycle (59). the antiviral effect of these compounds was also evaluated against a clinical isolate of sars-cov-2 in vero e6 cells. biacalin and biaclein have a distinctive binding mode with sars-cov-2 3clpro which was determined by x-ray protein crystallography. a core of substrate-binding pocket formed by the interaction of two catalytic residues, the first is decisive s1/s2 subsites and the second is an oxyanion loop. to this pocket, the biacalin is ensconced acting or behaving as a shield opposite the catalytic dyad to avoid the peptide substrate from getting near the active site. so haixia su et al. stated that biacalin because of its simple chemical structure, ubiquitous mode of action and powerful antiviral effect along with safety profile could be tried as antiviral agent (58). figure 3. chemical structure of (a) biacalin and (b) biacalein (56) hesperidin it is a flavonoid glycoside, its aglycone is hesperetin (figure 4). hesperidin was initially isolated from the citrus peel (60) and it is present in large amounts in sweet oranges and lemons (61). hesperidin is used in diosmin manufacturing (62, 63). hesperidin has anti-inflammatory, antiviral, antioxidant, and other pharmacological activities (61). haggag et al (2020) visualized the probability of using hesperidin as a prophylactic and adjuvant curative candidate for sars-cov-2. haggag declared this vision according to previously reported outcomes that confirm the effectiveness and safety of this phytochemical. hesperidin bans sars-cov-2 ingress to the lung cell through the disruption of ace2 and sars-cov-2 receptor binding domain interaction by targeting the binding interface of sars-cov-2 spike and ace2 human receptors. hesperidin ameliorates the host cellular immunity, minify inflammatory mediators’ release. simultaneous administration of a mixture of hesperidin and diosmin along with heparin is essential for protection against thromboembolism (62, 63). meneguzzo et al (2020) mentioned that hesperidin could be an effective antiviral agent against sars cov-2 and its further mutations. hesperidin is outperforming the other natural molecules as well as antiviral drugs that were advised in sars-cov-2 clinical trials as the binding tendency of it for all the major viral and cellular targets is robust. hesperidin can interrupt the viral infection at different stages starting from virus entrance to the steward human cell, to the viral genome transcription and replication. it was iraqi j pharm sci, vol.30(1) 2021 natural products: promising therapy for sars cov-2 33 suggested that hesperidin might be effective prophylaxis since it prohibits virus spreading to other cells, it has a high safety index, short life span in the human body, and at high doses, hesperidin showed no sign of cytotoxicity. hesperitin also displayed a good binding affinity to one or more targets (64). figure 4. chemical structure of :a. hesperidin b. hesperitin (60) quercetin is flavonol aglycone; (3,3′,4′,5,7 pentahydroxy-flavone) (figure 5), found in various vegetables and fruits. quercetin demonstrated numerous effects such as antioxidant, antiproliferative, antibacterial, antiviral, and others (65, 66). previous studies reported that quercetin could reduce mortality resulted from severe pandemic influenza a complications (66). in a previous study, quercetin was shown to inhibit sars-cov entry to host cell (67). biancatelli et al (2020) pointed for the use of a combination of vitamin c and quercetin for prophylaxis and early management of sara cov2 respiratory tract infections. quercetin affects various enzymes and targets that are involved in the virus life cycle as reverse transcriptase enzymes, sars 3cl protease in sars-cov. hydroxyl groups in quercetin and its derivatives inhibit sars 3cl protease through binding to gln189 site on this enzyme. because sars cov-2 protease 3cl has the same binding site (gln189) as sars-cov so quercetin assumed to be effective for this pandemic. vitamin c display immunomodulatory effect, boosting interferon formation, restrictive cytokinemediated organ damage, enhance survival rate in lethal infection. regarding this combination, a synergistic effect is gained, vitamin c is an antioxidant compound, has the ability for the generation of quercetin from oxidized quercetin derivatives. at the same time, both of these nutraceuticals exert antiviral and immunomodulatory effects (68). glinsky (2020) showed the feasibility of using putative combinations one composed of quercetin and vitamin d and the other quercetin, vitamin d, and estradiol for sars-cov-2. these combinations were more effective as treatment agents showed statistically more powerful effects on expression of sars-cov-2 target genes as compared to monotherapies these agent separately or in combination alter the expression of genes encoding sars-cov-2 targets to varying degree which ultimately modulate the functions of viral proteins (69). figure 5. chemical structure of quercetin (69) luteolin this flavone aglycone displayed antiviral effect (figure 6). theoharides (2020) discussed the effective role of luteoline in sars cov-2. luteolin suppress viral entrance to host cell through binding to the sars-cov-2 spike s glycoprotein. serine proteases, like sars‐cov 3cl protease and other proteases were inhibited by luteolin. this flavone has anti-inflammatory effect. tetramethoxyluteolin (luteolin derivative) decrease the secretion of the tnf and il‐1β (pro‐inflammatory cytokines) from the mast cells. chemokines ccl2 and ccl were also inhibited (70). figure 6. chemical structure of luteolin (71). theaflavin theaflavin is a polyphenolic compound that has a benzotropolone skeleton (figure 7), it results from catechin oxidative condensation. this reddish-orange pigment (theaflavin) mainly founded in fermented tea, and anyhow very small quantities might be present in non-fermented tea (green tea) (72, 73). theaflavin has antiviral and anti-inflammatory properties (74). chen et al (2005) in a study using fluorogenic substrate assay demonstrated that theaflavin-3,3′-digallate inhibit sars-cov 3clpro more than theaflavin which might be explained by iraqi j pharm sci, vol.30(1) 2021 natural products: promising therapy for sars cov-2 34 the absence of gallate group in theaflavin (75). lung et al (2020) based on molecular docking studies demonstrated the probability of using theaflavin as a lead compound for evolving an inhibitor for sars cov-2. according to the result of lung's study theaflavin has the ability to docking in or targeting the catalytic pocket nearer the active site of rdrp (rna‐dependent rna polymerase) in mers‐cov, sars‐cov, and sars‐cov-2. rdrp is crucial for catalyzing the rna replication from rna templates. in the used molecular docking studies, theaflavin possess the lowest idock score and lower binding energy in the catalytic pocket of sars‐ cov‐2 rdrp which may be explained by the fact that further hydrogen bonds along π‐cation interaction were formed between theaflavin and the catalytic pocket of rdrp in sars‐cov‐2 (76). figure 7. chemical structure of theaflavin (72) glycyrrhizin is a licorice bioactive compound that belongs to an acidic type of saponin glycosides (figure 8). this glycoside exhibits various clinical effects, one of which is the antiviral effect (77). luo p et al (2020) perspective debates the possible use of glycyrrhizin as a sars-cov-2 drug candidate based on its various pharmacological actions. the proposed mechanisms for using glycyrrhizin to conflict sars-cov2 may be via binding to ace2, thus prevent the formation of sars-cov2 and ace2 complex, inhibition of proinflammatory cytokines, thrombin, and cumulating iros (intracellular reactive oxygen species). airway exudates overproduction is inhibited while stimulating endogenous interferon (78). harald murck (2020) discussed the protective effect of glycyrrhizin in sars cov-2 patients and its role in mitigating disease severity. his point of view was based on two main effects related to glycyrrhizin and its metabolite glycyrrhetinic acid (ga). ga firstly restrain an enzyme named 11-beta-hydroxysteroid dehydrogenase type 2, through the inhibition of this enzyme cortisol activate mineralocorticoid receptors which in turn dawn regulate ace2 expression and thus reduce entry points of sars cov-2. type 2 transmembrane serine protease serves as a cofactor for virus uptake by ace2. ga reduces protease expression and offers a supplementary mechanism that limits virus entrance (79). ga exerts antiinflammatory effect through antagonism of toll-like receptor 4, and thus decreases inflammation in many tissues including lungs. the production of inflammatory cytokines as tnfα, il6, and il1ß are reduced. as a result, ga neutralizes the mitigation of the ace2 protective anti-inflammatory effect gained from reduced ace2 expression due to ga used. ace2 possesses an anti-inflammatory effect by the formation of angiotensin 1-7 and angiotensin 1-9 (79). figure 8. chemical structure of a. glycyrrhizin and b. glycyrrhetinic acid (80) escin escin is a natural mixture of triterpenic saponins rather than a pure compound (figure 9), consisting of a, b, c and d escin, it is extracted from the horse chestnut seeds (81). previous studies reported the potent escin anti-inflammatory and anti-edematous actions (82). michelini et al (2018) proved the antiviral effect of β-escin against hsv-1 infection (herpes simplex virus type 1). by the meaning of screening assay escin showed inhibitory effect vs sars-cov (83) . gallelli et al (2020) demonstrated the possibility of using escin in combination with another antiviral agent as remdesivir as an effective therapeutic option for sars cov-2 patients suffering from acute lung injury. gallelli and colleagues based in their outcome on the powerful anti-inflammatory effect of escin that is analogous to dexamethasone and methylprednisolone. in the late stage of sars cov-2 infection, the cytokine storm produces acute lung injury, escin might reduce the secretion of inflammatory mediators in addition to attenuate the acute lung injury. several on ongoing clinical trials using escin oral and intravenous dosage form in sara cov-2 patients (84). iraqi j pharm sci, vol.30(1) 2021 natural products: promising therapy for sars cov-2 35 figure 9. chemical structure of escin (81) celastrol is a pentacyclic triterpenoid of quinone methide type (figure 10), extracted from the root bark of tripterygium wilfordii. it is used for the treatment of many conditions; tumors and rheumatoid arthritis in particular (85). celastrol targets nfkb, anti-inflammatory, antioxidant mediators, and certain cell signaling molecules, exhibiting a potent anti-inflammatory effect (86). in a previous study celastrol along with other quinone methide triterpeniods proved as a potent inhibitor for sars-cov 3clpro (87). law et al (2020) concluded that celastrol is an effective therapeutic option for sars cov-2 based on its powerful anti-inflammatory effect. celastrol perhaps acts through inhibition of lipopolysaccharide and subsequent protein and mrna expression that is encoding for proinflammatory cytokines like il-6, il8, and mcp-1 (monocyte chemoattractant protein 1). the reduction in pro-inflammatory cytokines suppresses the phosphorylation step of the nf-κb, ikkα/β and iκbα. by suppression of this step, p56 is inactivated and the cascade of the inflammatory process is inhibited (88). figure 10. chemical structure of celastrol (88) lycorine is a pyrrolo[de]phenanthridine ring-type alkaloid (89) which was isolated from bulbs of narcissus pseudonarcissus (90). lycorine proved in a study to have an antiviral effect against sars-cov (severe acute respiratory syndrome associated coronavirus) (91). shen et al (2020) showed the antiviral effect of 36 compounds against alpha and beta coronavirus that infect human among these virus hcov-oc43 which cause encephalitis in mice. lycorine demonstrated powerful antiviral action against multiple genetically distinct coronavirus in vitro and protected mice against lethal hcov-oc43 infection in vivo (92). zank et al (2020) examined the antiviral effect of lycorine along with gemcitabine, and oxysophoridine vs sars-cov-2 in cell culture. these compounds demonstrated a dose-dependent antiviral effect that is not related to compound mediated cytotoxicity. lycorine demonstrates various mechanisms as an antiviral agent for viruses other than sars-cov-2, but for the sars-cov-2 antiviral mechanism probably related to hosting factors modulation instead of targeting viral factors directly (93). figure 11. chemical structure of lycorine (94) cepharanthine this is benzylisoquinoline alkaloid (figure 11), isolated from stephania cephalantha. it has anti-inflammatory, antioxidant properties used for the treatment of leukemia, alopecia, and other diseases (95). rogosnitzky et al (2020) et al (2020) in a review showed that cepharanthine might be a drug of choice and therapeutic option for sars cov-2. cepharanthine interfered with certain enzymes and mediators generation that are involved in the viral entrance, replication, and in a cytokine storm. among these deactivations of the nuclear factorkappa b (nf-κb), cyclooxygenase expression, lipid peroxidation, nitric oxide, and cytokine formation (96). jeon et al (2020) evaluated the antiviral effect of 48 fdaapproved drugs as a candidate for sars cov-2 using chloroquine, lopinavir, and remdesivir as standards or reference drugs. among the 48 tested drugs only 24 demonstrated the antiviral effect for sars cov-2, cepharanthine is included (97). iraqi j pharm sci, vol.30(1) 2021 natural products: promising therapy for sars cov-2 36 figure 12. chemical structure of cepharanthine (98) other natural products narkhede et al (2020) reported in a study using molecular docking studies the potential antiviral effect of several phytochemicals including berberine, tryptanthrine, indirubin, indican rhein, βcaryophyllene, β-sitosterol, these compound act via inhibition of viral protease (99). conclusion different phytochemicals and natural products were tried as possible treatment for sars cov-2. alkaloids, phenolic, terpenes, and others were examples of these compounds. flavonoids and phenolics were the compounds that are discussed in this review followed by triterpenes and lastly alkaloids. the pharmacological activities of these candidates were mediated through the direct effect on virus life cycle and / or via the anti-inflammatory effect which are beneficial in cytokine storm. almost all the acquired data or information based on molecular docking studies (theoretical studies) and or previous studies related to mers and sars cov. further, in vitro and in vivo studies are required to find out the effective treatment for this 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international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 doi: https://doi.org/10.31351/vol30isssuppl.pp48-59 48 a pharmacoeconomics study for anticoagulants used for hospitalized covid-19 patients in al-najaf al-ashraf city –iraq(conference paper )# fatimah baqer al-qubbanchi*,1 and fadya yaqoob al-hamadani* # 9th scientific conference conference sponsored by college of pharmacy , university of baghdad 25-26 august 2021 ∗department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract hematologic complications are one of the major consequences in patients with covid-19 infection. anticoagulants were used to mitigate covid-19 related coagulopathy such as enoxaparin and unfractionated heparin.to investigate the differences between enoxaparin and unfractionated heparin using a cost-effectiveness analysis that compares the clinical outcome and the costs of two anticoagulants. . a retrospective review of medical records of hospitalized, severe to critical covid-19 infected patients was conducted at al-amal hospital at al-najaf city-iraq from august 2020 to june 2021. d-dimer level, length of stay (los), and survival rate were used to assess the effectiveness, and the cost of both medications was also evaluated for comparison. . one hundred and forty-four covid-19 infected patients were enrolled and divided into heparin group n=72, and enoxaparin group n=72. covid-19 infected patients had a higher level of d-dimer than the reference range (2534.675 ng/dl). no significant differences in average d-dimer between both genders. there was a significant difference between patients' ages ≥60 years and patients <60. higher d-dimer levels were associated with a higher mortality rate. heparin was more effective in decreasing d-dimer levels than enoxaparin which inversely increased the d-dimer levels. additionally, heparin was associated with higher survival rate compared to enoxaparin. it was associated with a longer duration of stay in hospital than enoxaparin however, no significant difference was observed. heparin cost/per patient/per day was less than enoxaparin. . heparin was a more cost-effective anticoagulant therapy compared to enoxaparin, it was associated with a lower cost and better effect. keywords: pharmacoeconomic cost-effectiveness, covid-19, anticoagulants, heparin, enoxaparin. مدینة النجف الراقدین في ۱۹-كوفید التخثرالمستخدمة لمرضى راسة االقتصادیات الدوائیة لمضاداتد #( بحث مؤتمر ) االشرف/العراق *فادیة یعقوب الحمدانيو ۱*،فاطمة باقر القبانجي ۲۰۲۱اب ۲٦ – ۲٥جامعة بغداد ، # المؤتمر العلمي التاسع لكلیة الصیدلة بغداد، بغداد/العراقفرع الصیدلة السریریة، كلیة الصیدلة، جامعة * الخالصة ، حیث یتم استخدام مضادات التخثر للتخفیف ۱۹-المضاعفات المرتبطة بالدم ھي احد التبعات الرئیسیة للمرضى المصابین بفایروس كوفید تخدام تحلیل الفعالیة االقتصادیة اكتشاف الفرق بین من مضادات التخثر (الھیبارین و االینوكزابارین) باسز من التخثر، كاالینوكزابارین والھیبارین اصابة شدیدة الى حرجة الدراسة كانت مراجعة باثر رجعي لسجالت المرضى المصابین الذي یقارن النتائج والكلفة الثنان من مضادات التخثر. تم استخدام معدل دي دایمر، ، حیث۲۰۲۱الى یونیو ۲۰۲۰في مستشفى االمل في محافظة النجف في العراق، من آب بفایروس كورونا والراقدین مریض في الدراسة مقسمة ۱٤٤تضمین تم مدة الرقود في المشفى، ومعدل النجاة لقیاس الفعالیة الدوائیة و تم احتساب كلفة الدواء فقط في التحلیل. رضى المصابین بفایروس كورونا كان وقد اظھرت النتائج بأن الم .مریض تلقوا عالج االینوكزابارین ۷۲مریض تلقوا عالج الھیبارین و ۷۲الى نغ/دل). ولم یكن ھناك فرق ملحوظ بین الجنسین في المعدل االساسي للدي ۲٥۳٤٫٦(من الحد الطبیعي المحدد في المصادرلدیھم معدل دي دایمر . كما یبدو ان المعدالت عاما ٦۰من صغرعاما وا ٦۰كان ھنالك فرق ملحوظ في المعدل االساسي للدي دایمر بین االعمار اكبر ویساوي .دایمر دایمر. االعلى للدي دایمر كانت مرتبطة بشكل ملحوظ بمعدل وفیات اعلى. الھیبارین كان اكثر فعالیة من االینوكزابارین في تقلیل مستویات الدي كان یرتبط بمدة بقاء اطول في المستشفى بالمقارنة ھكما انبالمقارنة مع االینوكزابارین . نجاة اعلىمعدل یرتبط بباالضافة الى ذلك فان الھیبارین كان .بما یخص الكلفة فان كلفة الھیبارین كانت اقل من كلفة االیناكزابارین / للمریض الواحد/ لیوم واحد) مع االینوكزابارین لكن الفرق كان غیر ملحوظا. .حیث انھ یرتبط بكلفة اقل وفعالیة افضلالھیبارین كان ذو فعالیة اقتصادیة اعلى بالمقارنة مع االینوكزابارین، ، مضادات التخثر، ھیبارین، اینوكزابارین.۱۹-الكلمات المفتاحیة: االقتصادیات الدوائیة، الفعالیة االقتصادیة، كوفید introduction coronavirus is a newly discovered virus(1), it is deemed a pandemic by who in january/2020 (2), where the first case of covid-19 was reported in december 2019 in wuhan, china, from that date, 180 million cases were reported globally until june 25.2021, with 3.9 million deaths. in iraq, 1.3 million cases were reported, with 17,000 deaths (3). symptoms of covid-19 infection cause respiratory syndrome, which overlaps with other viral syndromes. it includes fever, headache, fatigue, shortness of breath, diarrhea, cough, and myalgias(4). furthermore, abnormalities could be seen in chest xray and computed tomography (ct)(5). 1corresponding author e-mail: fatima.alhussainy1994@gmail.com received: 28/8/2021 accepted: 15/11 /2021 published online first: 2022-1-12 https://doi.org/10.31351/vol30isssuppl.pp48-59 iraqi j pharm sci, vol.30(suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 doi: https://doi.org/10.31351/vol30isssuppl.pp48-59 49 iraqi journal of pharmaceutical science laboratory findings include leukopenia and lymphopenia (6), elevated levels of aminotransferase, c-reactive protein (crp), ddimer, s.ferritin, and lactate dehydrogenase (ldh). it also leads to cardiac(7), hematologic(8), renal(9), and other complications. thromboembolic events occur in patients with covid-19, with the highest risk occurring in critically ill patients(10) where the incidence of thromboembolism among hospitalized patients with covid-19 ranged from 25 to 53% (11). it has been found that infection with covid-19 is related to an increase in padua prediction score >4 in 40% of patients, (padua score was developed to estimate risk of venous thromboembolism (vte) in hospitalized medical patients (12) where a score >4 indicates a higher risk of venous thromboembolism (13). covid-19 infection potentiates all 3 components of virchow’s triad (endothelial dysfunction, hypercoagulable state, and stasis). it increases the risk of thrombosis, endothelial dysfunction which is triggered by angiotensin-converting enzyme 2 (ace2), and result in an increase in d-dimer, fibrin/fibrinogen. in addition, thrombin time is affected and becomes shorter(14,15)while prothrombin time, and activated partial thromboplastin time appears to be longer (16). currently, the world health organization (who)(17), recommend prophylaxis dose of anticoagulants, low molecular weight heparin (enoxaparin) 40 mg by subcutaneous injection every 24h: if bmi > 40 kg/m2 or weight > 120 kg: enoxaparin 40 mg by subcutaneous injection every 12h. or unfractionated heparin (ufh) 5000 units by subcutaneous injection every 8 or 12h: if bmi > 40 kg/m2 or weight > 120 kg: 7500 units q12h or 5000 units every 8h. enoxaparin and unfractionated heparin are both on the who model list of essential medicines; enoxaparin has the advantage of daily dosing and the suggested duration of standard thromboprophylaxis is until hospital discharge. if therapeutic dosing is prescribed, clinicians should be aware of the increased risk of bleeding, including major bleeding requiring transfusion (e.g. gastrointestinal) or clinically significant bleeding even if transfusion is not required (e.g. intracranial). factors influencing the choice of agent include: the availability of laboratory monitoring (needed for unfractionated heparin); requirement for rapid reversibility (favors unfractionated heparin); presence of severe renal dysfunction (favors unfractionated heparin); interaction with other drugs used to treat covid-19 (especially direct oral anticoagulants);, convenience (least with unfractionated heparin, most with direct oral anticoagulants); and suspicion of heparininduced thrombocytopenia (favors fondaparinux or direct oral anticoagulants). for therapeutic or intermediate intensity anticoagulation, patients should have baseline creatinine, platelet count, prothrombin time or international normalized ratio, and partial thromboplastin time. patients on therapeutic dosing of unfractionated heparin require monitoring of partial thromboplastin time or anti-factor xa levels and ideally platelet count (18). as the treatments of covid 19 infection continues to evolve, the health service provider needs to understand the effect of potential treatments on the primary outcomes (e.g. mortality, mechanical ventilation, duration of hospital stay); understand their effects with regards to different parameters such as; age, respiratory support requirement, disease severity, and race/ethnicity together with the immensity of clinical benefit, as this will be necessary to make the best decision. (19). of note, the covid-19 pandemic did not affect the health only but its effects have been extended to the social and economic aspect therefore, many studies focused on estimating the cost of covid-19 disease and its treatments to understand the impact on economic aspects (20,21). other researches were studied the clinical effects of covid-19 treatments, however, there is a paucity in studies conducted to understand and explain both cost and effectiveness of covid-19 treatments. (see discussion section). objective the study aimed to conduct a costeffectiveness analysis comparing the clinical outcome and the costs of two anticoagulant injections (unfractionated heparin and low molecular weight heparin (enoxaparin)) used to treat hospitalized, severe-critical covid-19 infected patients. methodology study design and patients the study was a retrospective review of medical records for hospitalized patients diagnosed with severe critical covid-19 infection. inclusion criteria hospitalized patients with covid-19 infection, age > 18 years, non-pregnant, and received one of the injectable anticoagulants, for 3 days and more, with at least two measurements for the d-dimer (the first one before receiving https://doi.org/10.31351/vol30isssuppl.pp48-59 iraqi j pharm sci, vol.30 (suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 50 the treatments and the second one at the last day of receiving the treatments. patients who were not fit these criteria were excluded from the study. sample size searching in patient's medical records continue until the two arms became equal, heparin arm n=72, enoxaparin arm n=72, with allocation ratio 1:1, 1100 patient's records were reviewed until equality had been achieved. the equation for finite population (22) was used to estimate the sample size where the confidence level is 95%, the estimated sample size is 139, which means the strength of the sample size is more than 100%, also the equality of the two arms gives the sample size a statistical strength. study setting and ethical approval the study was conducted at al-amal hospital, at al-najaf city from (augast 2020 to june 2021) after obtaining the approval from the scientific committee of the university of baghdad/ college of pharmacy and the iraqi ministry of health/ al-najaf department of health/ department of research and development. data sourcing all medical data were taken from patient's medical records, and the cost of treatments was taken from the drugs store which supplied al-amal hospital. cost-effectiveness analysis of two injectable anticoagulants (unfractionated heparin and low molecular weight heparin (enoxaparin)) was conducted. outcome measures the clinical outcomes of original injectable anticoagulants (low molecular weight heparin (enoxaparin) and unfractionated heparin) were assessed using d-dimer levels, duration of hospitalization, and survival rate. where d-dimer level aids in the diagnosis of venous thromboembolism (vte) (23,24). in the beginning, some demographic assessments were done to know if the baseline readings of d-dimer were affected by gender and age to avoid bias. gender distribution was demonstrated as male and female, the age distribution was categorized into groups (<60 years and ≥ 60 years). there was no distribution according to race or ethnicity in this study. then analysis was conducted to ensure the normality of distribution of the sample, the age and comorbidities of the two arms were compared. then the average baseline of ddimer in patients with covid-19 infection was assessed to understand the effect of covid-19 on d-dimer levels. the effects of original injectable anticoagulants (heparin and enoxaparin) on ddimer level, length of stay, and survival rate were then assessed. average of differences between 1st and 2nd reading of d-dimer /per day/per patient was considered as a primary clinical outcome of anticoagulants therapies). it was calculated by dividing the difference between the 1st and 2nd ddimer reading of the patient by the number of days the patient received the treatment, then the summation of the differences per day divided by the number of patients who received the treatment. secondary outcomes: length of stay was calculated as an average for each treatment group. the survival rates during hospitalization days of the patients were calculated using kaplan-meier to show the survival rate over time of hospitalization. also, it was calculated in the descriptive method, where it is equal to number of patients who survived at the end of hospitalization time divided by the total number of patients who received the treatment. both the kaplan-meier method and the descriptive method gave the same survival rate. economic outcome athe costs of medications were taken from the drugs store which supplied al-amal hospital during the same period of data collection, so there was no need to count the inflation rate or the discount factor. bthe cost of hospitalization: the average cost of one day of hospitalization was 53.6 $us, which includes the cost of health service providers, cost of medical instruments, cost of medication, and non-medical costs. this cost was taken from the iraqi ministry of health/department of financial planning in 2017. the inflation factors in iraq were then multiplied with the average cost for the period from 2017 to 2021(25). cwillingness to pay: the average maximum willingness to pay of iraqi people was obtained from the responses of 375 people who were participated in a web-based survey, hypothetical scenarios were used to ask people about the maximum amount they are willing to pay for special benefits using multiple-choice questions. the survey was conducted by the author at the same period of conducting the study. so there was no need for inflation rate or the discount factor, and there was no need for sensitivity analysis because there were 375 responses. cost-effectiveness analysis cost vs effect was represented by: cost consequence analysis method, cost-effectiveness ratio cer method while incremental costeffectiveness ratio was conducted when needed. iraqi j pharm sci, vol.30 (suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 51 to conduct a cost-effectiveness analysis, two methods were used: 1st by using the cost-effectiveness plane, and the 2nd method is called incremental net benefit analysis (inb) which considers maximum willingness to pay as an effector on cost-effectiveness analysis. statistical analysis all data were collected, assembled, analyzed using a microsoft excel spreadsheet, the effect of the treatments on the indicator was calculated as an average change in number per patient per day and compared using independent student t.test. additional statistical analysis to assess the impact of treatments upon the survival outcome variable. kaplan-meier survival curves were plotted to measure the survival rates using statistical package for the social sciences software (spss) version 24. the normality of continuous variables was measured using the shapiro-wilk test. not normally distributed variables were tested using non-parametric tests. results d-dimer value had been used to evaluate the effectiveness of anticoagulants (heparin and enoxaparin). before conducting the comparison, the d-dimer baseline average was calculated for covid-19 infected patients before receiving any treatment. see (table 1) . table 1. d-dimer baseline average for hospitalized, covid-19 infected patients before receiving any treatment. indicator average normal average p-value n d-dimer baseline average for hospitalized patients with covid-19 infection 2534.675 ng/dl ± sd=2923 <500 ng/dl 0.0005 *103 144 one sample student t.test, p-value < 0.05 is significant. the effect of demographic characteristics on ddimer level the average baseline d-dimer value was calculated regarding the demographic variations to understand if the d-dimer was influenced by gender and age, and to understand if the baseline measurement had an effect on the final status of the patients, died or survived. d-dimer baseline average before receiving the treatment was higher in males than females, but the difference was non-significant (p-value >0.05). d-dimer baseline average was higher in patients age ≥60 years than patients age <60 years. the difference in average according to age was significant. (p-value <0.05). higher d-dimer levels seem to be associated with a higher mortality rate, the ddimer baseline average was significantly higher in patients who died than patients who survived (p-value <0.05). see (table 1-2)(figure1-1). table 2. d-dimer baseline average according to gender (male, female), age, and final status. demography average d-dimer ng/dl sdv ng/dl percentage% n p. value total 2534.675 ± 2923 100 144 heparin group 3349.8 ±3081.5 50 0.00005 enoxaparin group 1637.76 ±1634.03 50 gender male 2649.95 ± 3365.729 58.2 84 0.7 female 2374.10 ± 2213.854 41.8 60 age (years) ≥60 years 3177.33 ± 3514.531 54.4 78 0.04 <60 years 1763.06 ± 1699.574 45.6 66 final status died` 3166.263 ± 3422.71 55.9 80 0.04 survived 1729.94 ± 1829.12 44.1 64 this table measured the difference in means of d-dimer (continous variable) according to binary (categorized variable) using independent t-test. , p-value < 0.05 is significant. iraqi j pharm sci, vol.30 (suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 52 figure 1. d-dimer baseline average according to gender (male, female), age, and final status. comparison of age and comorbidities of heparin and enoxaparin groups a comparison was performed between the age and comorbidities of the group who received unfractionated heparin, and the group who received low molecular weight heparin (enoxaparin). the analysis showed that there were no significant differences with regard to the age and comorbidities of both groups of treatment. (p-value >0.05). see (table 1-3). table 3. average age of hospitalized patients who received anticoagulants (heparin and enoxaparin), and the type and percentage of comorbidities for each group. indicator heparin enoxaparin p-value average age (years) 61.9 ±15.9 58.5 ± 12.9 0.159 type and percentage of comorbidities (co-existed diseases) 0.86 hypertension 73.6 70.8 0.84 diabetes mellitus 47.2 45.8 0.9 ischemic heart disease 29.2 18 0.17 asthma 6.9 4.2 0.47 renal disease 2.77 0 0.16 liver disease 0 4.2 0.08 type and percentage of comorbidities from patients vital signs 0.87 severe infection 68.05 68.05 1 critical infection 31.9 31.9 1 o2 supplementation 100 100 1 s. creatinine ≥1.5 mg/dl 12.5 0 0.0027 s. urea > 20 mg/dl 100 100 1 s. urea>100 mg/dl 8.3 12.5 0.43 esr > 40 mm/hr 68.05 65.3 0.74 blood pressure >130/80 mm hg 19.4 20.8 0.85 blood pressure < 90/60 mm hg 2.77 5.55 0.41 heart rate >100 bpm 26.38 22.22 0.61 heart rate <60 bpm 5.55 2.77 0.41 low grade fever 37.5-38.5 c̊ 18 5.55 0.029 high grade fever >38.5 c 0 0 1 independent student t.test used to compare all comorbidities of the two groups (bold), chi-square test used to compare each comorbidity (categorical) with the other group, p-value < 0.05 is significant clinical outcome the primary outcome for assessing the effectiveness of heparin and enoxaparin was the effect of those two medications on reducing ddimer levels, heparin was significantly more effective on reducing d-dimer levels than enoxaparin, enoxaparin had a negative outcome in reducing d-dimer levels (p-value <0.05). see (table 1-4) and (figure 1-2). iraqi j pharm sci, vol.30 (suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 53 table 4. the effect of anticoagulant therapies on the level of d-dimer of hospitalized patients with covid-19. independent student t.test, p-value < 0.05 is significant . figure 2. the effect of anticoagulant therapies on the level of d-dimer of hospitalized, severe –critical covid-19 infected patients. the secondary outcome for assessing the effectiveness of anticoagulants (heparin and enoxaparin) was an average length of stay (los) in hospital, the group who received enoxaparin had a shorter average for length of stay (los) than the group who was treated with heparin, but the difference on (los) was nonsignificant (p-value >0.05). see (table 1-5) and (figure 1-3). table 5. average length of stay in hospital (days) for patients with covid-19 infection who received anticoagulants therapies (heparin and enoxaparin). indicator heparin enoxaparin p-value average length of stay (days) 13.7 ± 8.1 12.3 ± 9.9 0.37 independent student t.test, p-value < 0.05 is significant. figure 3. average length of stay in hospital (days) for patients with severe-critical covid-19 infection who received anticoagulants therapies (heparin and enoxaparin). the last outcome used to assess the effectiveness of heparin and enoxaparin was the survival rate during hospitalization time, it is calculated by dividing the number of patients who survived at the end of the period of treatment by the number of total patients who received the treatment n=72 then multiplied by 100%. the group of patients who were treated with heparin showed a higher survival rate (lower mortality rate) during hospitalization days than the group of patients who were treated with enoxaparin during hospitalization days, the difference between the survival rate of the two groups was significant (p-value<0.05). see (table 1-6), (table 1-7) and (figure 1-4). table 6. survival rate and test of equality of survival distributions for hospitalized patients with covid-19 infection who received injectable anticoagulants (heparin, enoxaparin) indicator heparin enoxaparin survival rate 55% 35% test chisquare f sig. indicator heparin enoxaparin p-value average of 1st d-dimer reading nd/dl 3349.806 1637.769 0.00005 average of 2nd d-dimer reading ng/dl 3012.108 2479.214 0.29 difference between average d-dimer before treatment and after treatment (delta) decreased 337.698 increased 841.445 0.01 average difference of d-dimer per day per patient decreased 24.4/ng/dl/day ± 226.614 increased 154.701ng/dl/day ± 504.6 0.01 iraqi j pharm sci, vol.30 (suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 54 log rank (mantelcox) 5.332 1 .021 chi-square test, p-value < 0.05 is significant. table7.case processing summary, 1=heparin, 2=enoxaparin, event =survival. figure 4. kaplan meier survival curve during hospitalization (days) for original heparin=1, and original enoxaparin =2, for hospitalized, severe –critical covid-19 infected patients. status 1= survival, test = log rank (mantel-cox). costs of anticoagulants costs of anticoagulants converted from iraqi dinars into u.s dollars, and then the average cost of treatments only was calculated for the patient per one day. the total cost of heparin for 72 patients who were treated with doses of 986 days was calculated, and the cost of enoxaparin for 72 patients who were treated with doses of 889 days was also calculated. the cost of hospitalization was calculated by multiplying the average cost of one day of hospitalization by the average duration of hospitalization. (table 8) table 8. costs of treatment with anticoagulants (heparin and enoxaparin) of hospitalized, severe –critical covid-19 infected patients. treatment/cost heparin enoxaparin cost us$/dosage form 2.28 /5ml 2.95/4000uni t average cost us$ /day/patient 2.08±0.5 /day/patient 9.44±1.9/day /patient total cost for 72 patients in us$ during hospitalization days. 1,885.7/986 days/72 patients 8,279.91/889 days/72 patients the average cost (us$) of hospitalization for each patient 809.122 726.44 treatment total n n of events censored n percent 1 72 32 40 55.6% 2 72 47 25 34.7% overall 144 79 65 45.1% iraqi j pharm sci, vol.30 (suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 55 presentation of cost vs effectiveness table 9. presentation of cost vs effectiveness of anticoagulants (heparin and enoxaparin) for hospitalized, severe –critical covid-19 infected patients. cost-effectiveness analysis cost-effectiveness plane heparin had a lower cost and higher effect compared to enoxaparin so it is located at the negative side of y-axes= -7.36, and the positive side of x-axes= +179.1, at quadrant (ii) which means heparin is cost-effective (dominant). see (figure 5) figure 5 .cost –effectiveness plane ,graphical presentation of cost – effectiveness of anticoagulants used for hospitalized severecritical covid-19 infected patients. incremental net benefit analysis inb= (lambda * (effect of heparin – effect of enoxaparin)) –(cost of heparin – cost of enoxaparin) equation 1. incremental net benefit equation. average willingness to pay to decrease the level of d-dimer from the abnormal average of covid-19 infected patients to the normal range was= 45.9$. the d-dimer level should be reduced by 2034.67 ng/dl to be within the normal range. so wtp to reduce one unit of d-dimer was = 45.9$/2034.67 ng/dl= 0.022$ / 1 ng/dl of d-dimer inb = (0.022$ * (24.4-(-154.701))-(2.08$9.44$)= +11.3 the positive result means that heparin is more cost effective than enoxaparin. 1. cost consequence analysis treatment heparin enoxaparin total cost in $ us for 72 patients 1,885.7/986 days/72 ptients 8,279.91/889 days/72 patients average cost in $ us per day per patient 2.08 ± 0.5$/day/patient 9.44 ± 1.9$ /day/patient outcome decreased -24.4 ± 26.614ng/dl/day increased +154.70 ± 504.6 ng/dl/day average d-dimer difference per day survival rate 55% 35% the average length of stay (days) 13.7 ± 8.1 12.3 ± 9.9 2. average cost-effectiveness ratio (cer): average cost in $ us per unit (1ng/dl) of d-dimer changed per day per patient 2.08/24.4= 0.085 $ u.s per 1 ng/dl of d-dimer decreased 9.44/-154.7= 0.061 $ u.s per 1 ng/dl of d-dimer increased cost in $ u.s per one percent increase in survival rate 1,885.7 $/55= 34.28$ per one percent of survival rate 8,279.91$ /35= 236.56 $ per one percent of survival rate the average cost in $ u.s of decreasing hospitalization duration for one day. 809.122 $/13.7day= 59.06 $ per day 726.44$/12.3 day= 59.06 $ per day 3. incremental cost-effectiveness ratio: heparin compared to enoxaparin outcome incremental cost-effectiveness ratio (icer) the average change in d-dimer level per day (2.08$-9.44$)/(-24.4(ng/dl)-154.7(ng/dl))= 0.041$ saved per extra unit of ddimer decreased. survival rate (1,885.7 $8,279.91 $)/55%-35%= 319.71$ saved per one percent increase in survival rate. iraqi j pharm sci, vol.30 (suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 56 discussion the current study has shown that levels of d-dimer of covid-19 infected patients were higher than normal range (p.value <0.05). there are many reasons which might contribute to this rise in ddimer values in covid-19 patients such as: i) infection which can cause the release of proinflammatory cytokines, thus causing an inflammatory storm(26). ii) some patients with covid-19 have different degrees of hypoxia and inflammation which could lead to thrombosis or increased oxygen consumption(27). iii)severe infection or acute inflammation caused by sepsis could also affect blood coagulation(28) therefore, ddimer tests are extremely useful for the diagnosis of thrombotic diseases hence, patients with covid-19 were reported to have a hypercoagulable state(29). d-dimer levels are associated with the severity of covid-19 infection, where higher ddimer levels were associated with a high mortality rate. number of studies have shown that the severity of patients with covid-19 was significantly related to d-dimer concentrations. meanwhile, the severe covid-19 patients tend to have a higher concentration of d-dimer when compared with nonsevere patients. this suggests that d-dimer could be used to evaluate the severity of infection(30-31) the d-dimer levels in patients who died from the infection were significantly higher than those of surviving cases (32) where 71% of patients who died from covid-19 were found to have met the disseminated intravascular coagulation (dic) standard(16). critical d-dimer values are associated with advanced age, male gender, dyspnea, hypertension, coronary heart disease, diabetes, and cerebrovascular disease (p < 0.05). (33). results of this study found that there is no effect of gender on d-dimer levels while, abnormal d-dimer values were identified in patients over 60 years old age (p < 0.001). other studies suggest that higher d-dimer levels are associated with the male gender(33), and others showed it is associated with the female gender where women were at a higher risk of developing thrombotic disorders in covid19 infection(34). overall, it seems that, there is an association between age and d-dimer levels (33,34). of note, the results of the present study have shown that treatment with heparin was more effective in decreasing d-dimer levels and mortality rates than enoxaparin, but it was associated with a longer duration of stay. these results are in contrast with other studies which found that enoxaparin was more effective than heparin as anticoagulant therapy for covid-19 infected patients. patients who administered enoxaparin had a lower mortality rate, lower icu admission rates, and shorter hospital / icu stays than those who received unfractionated heparin(35). previous studies have suggested that enoxaparin may be more effective than unfractionated heparin in certain cases of treatment and prophylaxis of coagulopathies., for example in the prevention of venous thromboembolism (vte)(36,37). furthermore, some studies reported that enoxaparin treatment in covid-19 might be effective not only as anticoagulants but also has an anti-inflammatory effect. therefore, starting enoxaparin treatment in the earlier stage will decrease the risk of micro-thrombosis in vital organs(38). this controversy in results leaves several questions and possibilities. it might be due to the different effects of anticoagulants in different ddimer values. patients with d-dimer levels < 1 µg/ml did not appear to benefit from anticoagulation while patients with d-dimer levels > 10 µg/ml derived the most benefit(39). in addition, different fda indications were reported for enoxaparin(40) and heparin(41). the label of enoxaparin includes the prophylaxis and treatment of deep vein thrombosis (dvt) with or without pulmonary embolism (pe) in various settings, while the label of heparin includes similar prophylactic indications as well as the treatment of a broader spectrum of acute embolic events including peripheral arterial embolism and embolism in the setting of atrial fibrillation. it is worth mentioning that, the results of this study may be influenced by unmeasured variables which are not recorded in this dataset, such as; prothrombin time (pt), partial thromboplastin time (ptt), the international normalized ratio (inr), erythrocyte sedimentation rate (esr), respiratory rate (rr), inflammatory cytokines (cytokine storm). other contributory factors which might have a role in the findings are: the circumstances of storage of the biological anticoagulants; the correct doses and methods of administrations for anticoagulants; different circumstances of carrying out d-dimer tests because the proficiency of testing are highly variable from one mthod to another. notebly, changing the type or magnitude of units from that recommended by the manufacturer are associated with as much as a 20-fold increase in the failure of proficiency testing of d-dimer. (24) with regards to the cost, heparin has a lower cost than enoxaparin, (this includes only the cost of medication), taking into account the difficulties in estimating other costs such as indirect costs, non-medical costs, and additional costs that result from side effects of the treatment. iraqi j pharm sci, vol.30 (suppl) 2021 pharmacoeconomic study for anticoagulants for covid-19 57 on the other hand, other studies revealed that enoxaparin is associated with a significant costsaving impact when used for therapy for patients with venous thromboembolism compared to iv heparin (42,43). in contrast, other studies reported that enoxaparin is associated with a non-significant reduction in total hospital costs compared with the appropriate use of ufh prophylaxis(44).overall, the results of the present study motivate further studies to investigate reasons for differences in the outcomes and future trials that could enable the development of a more efficacious standard of practice in regards to the administration of anticoagulants in covid-19 patients. prospective analysis comparing the efficacy of enoxaparin and unfractionated heparin is warranted (35). conclusion originator heparin was a more costeffective anticoagulant therapy compared to originator enoxaparin, it had a better effect in decreasing d-dimer level and higher survival rate, where the differences in the effect on those two outcomes were significant. in addition, heparin was associated with a lower cost, treatment with heparin has resulted in positive inb= 11.3, where a positive result means that heparin is more cost-effective than enoxaparin. the two methods of pharmacoeconomic analysis have revealed that heparin was more cost-effective than enoxaparin in treating covid-19 infected patients. references 1. coronavirus [internet]. who.int. 2021 available from: https:// www. who.int/healthtopics /coronavirus#tab=tab_1 2. timeline: who's covid-19 response [internet]. who.int. 2021 available from: 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ambrose j, youssef i, griffiths e et al. cost reduction associated with heparin-induced thrombocytopenia panel ordering for enoxaparin versus heparin for prophylactic and therapeutic use: a retrospective analysis in a community hospital setting. avicenna journal of medicine. 2018;8(04):133-138. 42. weinberg, richard & lichtig, leo & caprini, joseph & merli, geno & vogenberg, f randy & rph, cost implications of using unfractionated heparin or enoxaparin in medical patients at risk for venous thromboembolic events. (2006).p and t. 31, 43. amin a, lin j, lenhart g, schulman k. economic outcomes in patients at risk for venous thromboembolism receiving appropriate enoxaparin or unfractionated heparin prophylaxis. blood. 2008; 112 (11) :1285 -1285 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ study design and patients the study was a retrospective review of medical records for hospitalized patients diagnosed with severe critical covid-19 infection. all medical data were taken from patient's medical records, and the cost of treatments was taken from the drugs store which supplied al-amal hospital. cost-effectiveness analysis of two injectable anticoagulants (unfractionated heparin and low molecular weight heparin (enoxaparin)) was conducted. outcome measures the clinical outcomes of original injectable anticoagulants (low molecular weight heparin (enoxaparin) and unfractionated heparin) were assessed using d-dimer levels, duration of hospitalization, and survival rate. where d-dimer level aids in the diag... in the beginning, some demographic assessments were done to know if the baseline readings of d-dimer were affected by gender and age to avoid bias. gender distribution was demonstrated as male and female, the age distribution was categorized into gro... then analysis was conducted to ensure the normality of distribution of the sample, the age and comorbidities of the two arms were compared. then the average baseline of d-dimer in patients with covid-19 infection was assessed to understand the effect... the effects of original injectable anticoagulants (heparin and enoxaparin) on d-dimer level, length of stay, and survival rate were then assessed. average of differences between 1st and 2nd reading of d-dimer /per day/per patient was considered as a primary clinical outcome of anticoagulants therapies). it was calculated by dividing the difference between the 1st and 2nd d-dimer reading of the ... secondary outcomes: length of stay was calculated as an average for each treatment group. the survival rates during hospitalization days of the patients were calculated using kaplan-meier to show the survival rate over time of hospitalization. also, it was calculated in the descriptive method, where it is equal to number of patients who su... economic outcome athe costs of medications were taken from the drugs store which supplied al-amal hospital during the same period of data collection, so there was no need to count the inflation rate or the discount factor. bthe cost of hospitalization: the average cost of one day of hospitalization was 53.6 $us, which includes the cost of health service providers, cost of medical instruments, cost of medication, and non-medical costs. this cost was taken from the ira... cwillingness to pay: the average maximum willingness to pay of iraqi people was obtained from the responses of 375 people who were participated in a web-based survey, hypothetical scenarios were used to ask people about the maximum amount they are w... cost-effectiveness analysis cost vs effect was represented by: cost consequence analysis method, cost-effectiveness ratio cer method while incremental cost-effectiveness ratio was conducted when needed. to conduct a cost-effectiveness analysis, two methods were used: 1st by using the cost-effectiveness plane, and the 2nd method is called incremental net benefit analysis (inb) which considers maximum willingness to pay as an effector on cost-effective... statistical analysis all data were collected, assembled, analyzed using a microsoft excel spreadsheet, the effect of the treatments on the indicator was calculated as an average change in number per patient per day and compared using independent student t.test. additional statistical analysis to assess the impact of treatments upon the survival outcome variable. kaplan-meier survival curves were plotted to measure the survival rates using statistical package for the social sciences software (spss) version 24. the normality of continuous variables was measured using the shapiro-wilk test. not normally distributed variables were tested using non-parametric tests. iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 1 formulation and evaluation of nystatin microparticles as a sustained release system sara t.ismail *,1 , myasar m. al-kotaji * and ahlam a. khayrallah * * department of pharmaceutics, college of pharmacy, mosul university, mosul, iraq. abstract nystatin is the drug of choice for treatment of cutaneous fungal infections with main disadvantage that is the need for multiple applications to achieve complete eradication which may reduce patient compliance. microparticles offer a solution for such issue as they are one of sustained release preparations that achieve slow release of drug over an extended period of time. the objectives of this study were to fabricate nystatin-loaded chitosan microparticles with the ultimate goal of prolonging drug release and to analyze the influence of polymer concentration on various properties of microparticles. microparticles were prepared by chemical cross-linking method using glutaraldehyde as cross-linking agent. five formulas, namely n1c1, n1c2, n1c3, n1c4 and n1c5, were prepared and the effect of drug to polymer ratio was studied with respect to drug loading, encapsulation efficiency, particle size and morphology. furthermore the prepared microparticles were subjected to various physico -chemical studies, such as drugpolymer compatibility by fourier transform infrared spectroscopy (ftir) and invitro drug release characteristics. microparticles obtained from n1c1, n1c2 and n1c3 formulas were regular in shape with mean particle size ranging between 1µm and 10µm. n1c5 formula was resulted in particles with irregular shape while n1c4 showed a blend of microparticles and deformed particles. the effect of chitosan concentration on drug loading and entrapment efficiency was studied. the results showed increment in these parameters that was directly proportional to the increment in polymer concentration. percentage yield showed a significant increment which was related to the increment in the ratio of chitosan used during the study. ftir results showed no interactions between nystatin and chitosan. dsc studies proved the crystalline nature of nystatin and chitosan. on other hand, the thermogram of loaded microparticles showed the absence of endothermic peak corresponding to nystatin which may indicate the loss of the crystalline nature of the drug presented inside the microparticles. in vitro release studies resulted in 95.6% release of nystatin for n1c1 after 15 hours. n1c1 appeared to be promising in formulating microparticles that provide nearly complete release of the drug within15 hours. this formula can be selected in future work to be formulated as topical gel that prolongs the release of nystatin. keywords: nystatin, chitosan, glutaraldehyde, chemical cross-linking. التصييغ و التقيين الوختبري للجزيئبث الوبيكرويت للٌستبتيي كٌظبم تحرير دوائي طىيل األهذ سبرة طه اسوبعيل *،1 هيسر هحوذ القىطجي ، * أحالم أحوذ خيرهللا و * * ق.انعشا ،انًٕصم ،خبيعخ انًٕصم ،كهٛخ انصٛذنخ ،لسى انصٛذالَٛبد لخالصةا ٚعزجش انُسزبرٍٛ انعالج انًثبنٙ إلصبثبد اندهذ انفطشٚخ يع رعًُّ عٛت سئٛسٙ ْٕٔ انحبخخ إنٗ االسزعًبل انًزعذد نغشض نهًشبكم انًشبثٓخ ٔرنك نكَٕٓب إحذٖ انٕصٕل إنٗ انشفبء انزبو يًب ٚؤد٘ إنٗ رمهٛم انزضاو انًشٚط. رمذو اندضٚئبد انًبٚكشٔٚخ حال رحعٛشاد انزحشٚش غٕٚهخ األيذ ٔانزٙ رحمك رحشٚشا ثطٛئب نهذٔاء ٚسزًش نفزشاد يًزذح يٍ انضيٍ .انغشض األٔل يٍ ْزِ انذساسخ ْٕ فٕٓ دساسخ رأثٛش رصُٛع خضٚئبد يبٚكشٔٚخ يٍ انكبٚزٕسبٌ يحًهخ ثبنُسزبرٍٛ ْذفٓب انُٓبئٙ إغبنخ رحشٚش انذٔاء, أيب انغشض انثبَٙ حعشد اندضٚئبد انًبٚكشٔٚخ ثطشٚمخ انشثػ انكًٛٛبئٙ ٔثبسزخذاو رشكٛض انكبٚزٕسبٌ عهٗ يخزهف خصبئص اندضٚئبد انًبٚكشٔٚخ. نذساسخ رأثٛش َسجخ انجٕنًٛش (n1c1,n1c2, n1c3, n1c4 and n1c5) كهٕرشانذٚٓبٚذ كعبيم سثػ.رى رحعٛش خًس صٛغ انزغهٛف ٔ انخصبئص انشكهٛخ ٔحدى اندسًٛبد. خععذ اندضٚئبد انًبٚكشٔٚخ انًحعشح نذساسبد عهٗ رحًٛم انذٔاء, كفبءح ٔ كزنك دساسخ خصبئص رحشٚش انذٔاء. كبَذ ftirٔ انجٕنًٛش ثٕاسطخ خٓبص ءفٛضٕٚكًٛبئٛخ يزعذدح يثم دساسخ انزٕافك ثٍٛ انذٔا . يكى 11يكى ٔ 1ُزظًخ انشكم يع يزٕسػ حدى ٚزشأذ ثٍٛ ي n1c1 , n1c2 ٔ n1c3اندضٚئبد انًبٚكشٔٚخ انُبردخ يٍ انصٛغ خهٛػ يٍ اندضٚئبد انًبٚكشٔٚخ انزمهٛذٚخ ٔ خضٚئبد n1c4خسًٛبد غٛش يُزظًخ انشكم ثًُٛب أظٓشد انصٛغخ n1c5أَزدذ انصٛغخ دح فٙ ْزٍٚ انعبيهٍٛ ٔكبَذ ْزِ يشْٕخ. رًذ دساسخ رأثٛش رشكٛض انكبٚزٕسبٌ عهٗ رحًٛم انذٔاء ٔ كفبءح انزغهٛف. أظٓشد انُزبئح صٚب انضٚبدح راد عاللخ غشدٚخ ثبنضٚبدح فٙ رشكٛض انجٕنًٛش. أظٓشد انُسجخ انًئٕٚخ نهًحصٕل صٚبدح يعزجشح ٔانزٙ عضٚذ إنٗ انضٚبدح فٙ dscذ دساسخ عذو ٔخٕد أ٘ رفبعم ثٍٛ انُسزبرٍٛ ٔ انكبٚزٕسبٌ. أثجز ftirَسجخ انكبٚزٕسبٌ انًسزخذو خالل انذساسخ. أظٓشد َزبئح انطجٛعخ انجهٕسٚخ نهُسزبرٍٛ ٔ انكبٚزٕسبٌ. يٍ خٓخ أخشٖ, أظٓش انشسى انحشاس٘ نهدضٚئبد انًبٚكشٔٚخ انًحًهخ ثبنذٔاء غٛبة لًخ 1 corresponding author e-mail: gardeenea84@ yahoo.com received: 28/2/2015 accepted: 31/5/2015 iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 2 ايزصبصٛخ يمبثهخ نُمطخ اَصٓبس انُسزبرٍٛ يًب ًٚكٍ أٌ ٚذل عهٗ فمذاٌ انشكم انجهٕس٘ نهذٔاء انًٕخٕد داخم اندضٚئبد. أسفشد دساسخ َزبئح n1c1سبعخ. أظٓشد انصٛغخ ,1ٔ رنك ثعذ يشٔس n1c1% ثبنُسجخ نهصٛغخ 9,,6انزحشٚش انذٔائٙ فٙ انًخزجش عٍ رحشٚش سبعخ. ْزِ انصٛغخ ًٚكٍ أٌ رخزبس فٙ عًم ,1نًبٚكشٔٚخ ٔرنك ثزٕفٛشْب رحشٚش شجّ كبيم نهذٔاء ثعذ ٔاعذح فٙ رصٛٛغ اندضٚئبد ا يسزمجهٙ نزصٛٛغ شكم دٔائٙ يٕظعٙ ْاليٙ غٕٚم األيذ يٍ انُسزبرٍٛ. : ًستبتيي, كبيتىسبى, كلىترالذيهبيذ, ربط كيوبئي.الوفتبحيت الكلوبث introduction the development of new delivery systems for the controlled release of drugs is one of the most interesting fields of research in pharmaceutical sciences. microparticles are one of sustained release preparations that achieve slow release of drug over an extended period of time. microparticles are micrometric matrix systems essentially spherical in shape having size ranging between 1μm 1000μm (1) . microparticles can be used for the controlled release of drugs, vaccines, antibiotics and hormones. microparticles have many advantages such as providing a larger surface area and possessing an easier estimation of diffusion and mass transfer behavior. besides these, the encapsulated small molecules could diffuse out of the barrier with precise kinetics modeling and control-release of drugs to the body fluid (2) . candida albicans infection is a problem of growing clinical importance worldwide. literature data point out that about 96% of all opportunistic mycoses are caused by candida species (3) . nystatin is a polyene antifungal antibiotic characterized by a potent broad-spectrum antifungal action against a variety of fungal pathogens including candida, aspergillus, histoplasma, and coccidioides (4) . successful eradication of cutaneous fungal infections requires 2-4 applications of all available topical dosage forms of nystatin (5) . consequently, the aim of this study was to fabricate nystatin-loaded, sustained-release formulation that would prolong the release of the drug at the site of application, reduce the frequency of application, reduce the amount of drug administered and improve patient compliance and acceptance. chitosan was selected as a release-retardant polymer and the chemical crosslinking was selected as the technique. to find the best formula (the one with the highest drug content, best encapsulation efficiency, optimum particle size and morphology), different formulas were prepared using different ratios of chitosan. materials and methods materials nystatin was obtained as a gift from ninavah drug industry (ndi). chitosan was obtained from johnson cao, us. span 80 is purchased from bdh ® (uk). liquid paraffin, methanol and glutaraldehyde were obtained from tedia ® (usa). glacial acetic acid was obtained from thomas baker (uk) while hexane obtained from poch ® (poland). preparation of microparticles glutaraldehyde crosslinking method was employed for preparation of microparticles (6) . accurately weighed amounts of polymer and drug (see table 1 for the different amounts of drug and polymer) were added step by step into 2% acetic acid solution. the resultant nystatinchitosan dispersion was added drop wise by syringe with 23g needle into a continuous phase consisting of 125 ml of light liquid paraffin containing 0.5% of span 80 as an emulsifying agent. the mixture was stirred for 30 minutes at 1500 rpm using a 4-blade mechanical stirrer to form a w/o emulsion. a drop-by-drop solution of a measured quantity of aqueous glutaraldehyde (25% v/v) was added to the prepared emulsion at 15, 30, 45, and 60 minutes. stirring was continued for 2.5 hours to obtain microparticles which were washed first with hexane and then with distilled water to remove the adhered liquid paraffin and glutaraldehyde. finally the resultant microparticles were filtered, dried at room temperature and stored in a desiccator for further evaluation. physicochemical characterization morphological characteristics and particle size analysis: a microscopical image analysis technique was applied for determination of particle size. the morphology and particle sizes were determined with motic ® digital microscope equipped with imaging accessory. the particle diameters of 200 microparticles were measured randomly by supplied software and the average particle size was determined (7) . in order to be able to define a size distribution or compare the characteristics of particles with many different diameters, the size distribution can be broken down into different size ranges, which can be presented in the form of a histogram. determination of drug loading and entrapment efficiency a known quantity of microparticles (10 mg) was triturated by mortar and pestle and extracted with 10 ml methanol (containing 5% acetic acid). the resultant solution was centrifuged and the obtained supernatant was diluted appropriately with respective solvent iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 3 system (8) . absorbance was measured at 305 nm using labomed ® uv-vis spectrophotometer and concentration of nystatin in microparticles was calculated according to the standard regression. each determination was made in triplicate. the drug loading of prepared microsphere was determined by applying the following equation (9) : drug loading (%) = entrapment efficiency represents the proportion of the initial amount of drug incorporated/associated into/with the microparticles (10) . this parameter was determined by applying the following equation (11) : entrapment efficiency (%) = determination of percentage yield the relative yield was calculated based on the amount of microparticles of each formulation obtained relative to the amount of the total weight of the polymer and drug used in the dispersed phase. the percentage yield was calculated according to the following equation (12) . yield (%) = fourier transforms infrared spectroscopy (ftir) drug-polymer interactions were studied by ftir spectroscopy. the spectrum was recorded for chitosan, pure nystatin, blank chitosan microparticles, physical mixture of blank chitosan microparticles and nystatin and for nystatin loaded chitosan microparticles using bruker ® (germany) infrared spectrophotometer (13) . differential scanning calorimetry (dsc) thermal analysis was performed using dsc (shimadzu dsc plus, japan). approximately 10 mg of each sample (chitosan, pure nystatin, blank chitosan microparticles, physical mixture of blank chitosan microparticles and nystatin in the ratio of (1:1) and nystatin loaded chitosan microparticles) were used. each sample was placed in a sealed aluminum pan and heated at a scanning rate of 10ºc/min from 30ºc to 250ºc (14) . invitro drug release studies nystatinloaded chitosan microparticles equivalent to (10) mg of nystatin were subjected to in-vitro drug release studies to ascertain the drug release pattern of nystatin from the prepared microparticles formulations. microparticles were introduced in a beaker containing (300) ml of phosphate buffer solution (ph 5.5), used as the release medium. the beaker was rotated at 100 rpm and maintained at 32±0.5°cin a thermostat shaking water bath (15, 16) . an aliquot of (5) ml was withdrawn periodically at 1, 2, 3, 4, 5, 6, 9, 12 and 15 hours and replaced with a same volume of fresh medium each time. the withdrawn samples were filtered, then diluted appropriately and analyzed with labomed ® uv–vis spectrophotometer at 305 nm to determine the amount of drug released. drug release kinetics to examine the drug release kinetics, the cumulative release data were fitted to models representing zero-order (q v/s t), first-order (log (q0-q) v/s t) and higuchi‟s square root of time (q v/s t 1/2 ), respectively, where q is the cumulative percentage of drug released at time t and (q0-q) is the cumulative percentage of drug remaining after time t (17) . to determine the mode of drug release, the initial 60% drug release values were fitted to the korsmeyer–peppas model: mt/m∞ = kt n , where mt/m∞ is the fraction( ≤ 60%) of drug released at time t, k is the drug release rate constant, and n is the release exponent. the n value is employed for the characterization of the mechanism of solute release from formulation (18) . statistical analysis all experiments were repeated at least three times. results are expressed as means ± standard deviation (sd). statistical analysis was carried out employing one-way anova test. a p-value ≤ 0.05 was considered statistically significant. results and discussion preparation of microparticles different formulas of nystatin microparticles were fabricated successfully using chemical cross-linking method (table 1). the formula n1c5 has shown very high viscous dispersion that was difficult to be extruded from a 23g needle, so it was applied by a disposable syringe without needle. chitosan has 1 primary amino and 2 free hydroxyl groups for each c6 building unit. due to the availability of free amino groups in chitosan, it carries a positive charge and thus it reacts with many negatively charged compounds (6) . chitosan could be covalently cross-linked with glutaraldehyde through its amino groups. the aldehyde groups of the glutaraldehyde formed covalent imine bonds with the amino groups of chitosan, due to the resonance established with the adjacent double ethylenic bonds via a schiff reaction (19) . iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 4 table (1): composition of nystatinloaded chitosan microparticles formulas particle size analysis and morphological characteristics optical microscope images show that n1c1, n1c2 and n1c3 formulas have uniform spherical shaped microparticles while n1c5 microparticles were irregularly shaped and highly aggregated in nature and practically difficult to be distinguished as individual microparticles. in other hand, n1c4 showed a blend of microparticles and deformed particles (figure. 1). it was noted that as the concentration of chitosan increased, the color of microparticles changed from light yellow to dark brown. the size distribution profiles of the microparticles are shown in figure 2. the particle size of all formulas was ranged from 1µm to 10 µm. the results showed that particle size was affected significantly (p<0.05) by the polymer concentration, as the chitosan concentration increased a larger mean size of microparticles was obtained (table. 2). this effect may due to the increase in the viscosity of the droplets (due to the increase in concentration of polymer solution) (11) . the increase in solution viscosity led to a decrease in stirring efficiency hence result in difficulty in dispersion and subdivision of droplets and thus increased the resultant microsphere size (20) . figure (1): optical microscope images of nystatin loaded microparticles formula code drug: polymer ratio (w/w) nystatin (mg) chitosan (mg) aq. glutar -aldehyde solution (ml) aqueous acetic acid solution (ml) n1c1 1:1 100 100 15 10 n1c2 1:2 100 200 15 10 n1c3 1:3 100 300 15 10 n1c4 1:4 100 400 15 10 n1c5 1:5 100 500 15 10 iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 5 figure(2): histogram showing the effect of increasing chitosan concentration on frequency distribution analysis of nystatin loaded microparticles formulas determination of drug loading and entrapment efficiency the concentration of chitosan had significant (p<0.05) effect on the drug loading and entrapment efficiency of nystatinloaded microparticles which is reflected by the variations in the percentage of drug loading and entrapment efficiency that observed in all formulations as shown in table 2. better entrapment efficiency were achieved by increasing the ratio of chitosan which complies with patel et al. who obtained the same results with tramadol-loaded chitosan microspheres (21) . senthil et al. revealed that entrapment efficiency was also increased with the higher polymer concentration (22) . another study conducted by palanisamy et al. also achieved highest drug entrapment with drug: polymer ratio of 1:3 indicating that polymer concentration has a great impact on this parameter (23) . increase in the concentration of chitosan increases the yield of the prepared microparticles and thereby resulting in higher drug entrapment levels (24) . it was further observed that the drug entrapment was proportional to the size of the microparticles (21) . the contribution of a high polymer concentration to drug loading can be interpreted in two ways. first, the high viscosity of the polymer solution would be expected to decrease the diffusion of the drug into the external phase which would result in high entrapment efficiency (8, 25) . second, the higher polymer concentration produce large size microparticles in which the loss of drug from surface during washing process is lesser in comparing to small microparticles. thus size of microparticles is also affecting the drug loading (26, 27) . determination of percentage yield it was observed that as the polymer ratio increased, the product yield percentage also significantly (p<0.05) increased (table 2). the low percentage yield in n1c1 in relative to other formulas may be due to microparticles loss during washing process. table ( 2): drug loading (%), entrapment efficiency (%), yield (%) and particle diameter of nystatinloaded microparticles formulas. fourier transforms infrared spectroscopy (ftir) owing to that each specific chemical bond often has unique energy absorption; ftir spectroscopy has been extensively applied to identify the presence of certain functional groups or chemical bonds in a molecule. spectral data could confirm the stability of the drug during microparticles preparation and the absence of any drugpolymer interaction. ftir spectra of pure nystatin, chitosan, blank chitosan microparticles, physical mixture of blank chitosan microparticles and nystatin and the spectrum of nystatin loaded chitosan microparticles are shown in figure 3. nystatin displays characteristic absorption bands at about 1702 cm −1 due to the stretching vibration of carbonyl group (28) . on the other hand, the characteristic absorption of the chitosan formula code drug: polymer ratio w/w drug loading* (%) entrapment efficiency* (%) percentage yield* (%) particle diameter (µm)* n1c1 1:1 43.9±1.6 88.9±0.4 82.15±3 4.19±1.6 n1c2 1:2 28.6±0.8 91.2±0.4 86.67±4 4.65±1.7 n1c3 1:3 21.6±0.5 93.5±0.6 93.57±2.2 4.81±1.6 n1c4 1:4 17.8±0.4 96.2±0.5 95.33±2.1 7.91±2.9 n1c5 1:5 15.9±0.1 97.2±0.6 97.12±1.1 nd *: mean± standard deviation, nd: not determined file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_21 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_22 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_23 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_24 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_21 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_8 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_25 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_26 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_27 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_28 iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 6 situated at 1647 cm -1 due to c=o stretching in amide group ( amide i vibration) and at 1576 cm -1 due to n–h bending in amide group (amide ii vibration) (29) . some changes can be observed after chitosan cross-linking with glutaraldehyde. the characteristic peak at 1642 cm -1 is due to schiff base (c=n) formed by a cross linking reaction between the amino group and the aldehydic group of glutaraldehyde. moreover, no absorption is observed at ~1715 cm -1 , related to the free aldehyde group which further confirms the cross-linking reaction (18) . ftir spectra of nystatin loaded microparticles and blank polymer microparticles showed the same characteristic absorption peaks with the exception of characteristic band value at ~1700 cm -1 for loaded microparticles which can be assigned to the c=o group of the drug. this result clearly indicated the stability of the drug during the microparticles preparation and revealed the absence of any drugpolymer interaction. figure( 3): ftir spectra of pure nystatin (a), chitosan (b), blank chitosan microparticles (c), physical mixture of nystatin and blank microparticles (d) and nystatin loaded microparticles (e) d b a c e file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_29 file:///d:/new%20journal%2012/sara/النسخة%20النهائية%20مع%20التصحيحات%20%20الكاملة.docx%23_enref_18 iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 7 differential scanning calorimetry (dsc) the thermograms of nystatin (a), chitosan (b), physical mixture of chitosan and nystatin (c), plain microparticles (d) and loaded microparticles (e) are shown in figure 4. nystatin presented a single, well defined sharp endothermic peak at 165.5 ◦ c which is corresponded to the melting point of nystatin. chitosan showed initial endothermic peak at 75.15 ◦ c which can be correlated with loss of water associated to hydrophilic groups of polymer (30, 31) . physical mixture thermogram showed an endothermic peak at 164 ◦ c due to the presence of nystatin, which reveals the absence of interactions between the nystatin and chitosan. thermogram of drug-loaded chitosan microparticles showed the same peaks observed in blank microparticles, but there was no peak corresponding to nystatin. this might be an indication of loss of crystallinity of nystatin presented within the prepared micoparticles (21, 24, 29) . 100.00 200.00 temp [c] -2.00 0.00 2.00 4.00 mw dsc 165.51x100c 50.00 100.00 150.00 200.00 temp [c] 2.00 3.00 4.00 5.00 6.00 7.00 mw dsc 75.15x100c 50.00 100.00 150.00 200.00 250.00 temp [c] 2.00 4.00 6.00 8.00 mw dsc 72.03x100c 164.40x100c 50.00 100.00 150.00 temp [c] 4.00 6.00 8.00 10.00 12.00 mw dsc 96.51x100c 50.00 100.00 150.00 200.00 temp [c] 0.00 5.00 mw dsc 86.95x100c figure 4: dsc thermograms of pure nystatin (a), chitosan (b), physical mixture of chitosan and nystatin (c), plain microparticles (d) and loaded microparticles(e). a b c d e iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 8 invitro drug release studies the cumulative percentage of nystatin release from chitosan microparticles versus time was drawn and represented graphically (figure 5). figure (5): cumulative % of nystatin released versus time. the invitro drug release plot exhibited a biphasic mode with an initial „burst‟ release for the different plots. thereafter, a slow release phase was started. the initial rapid release of drug might due to fast dissolution of drug molecules attached to the surface of the microparticles while subsequent slow but steady release was related to diffusion of drug molecules present in the core of the microparticles (20) . this is in agreement with kumar et al. who found that increasing the concentration of chitosan leads to decrease the release of indomethacin from chitosan microparticles (29) . khalandar et al. who prepared nasal microspheres of sumatriptan using different concentrations of chitosan also concluded that the drug release is retarded by increasing polymer ratio (32) .the results show that an increase in polymer ratio reduced the first phase (burst phase) significantly (p< 0.05) as is evident from t50% value (time required to release 50% of the loaded dose) as seen in table 3. nystatin was released from microparticles in a controlled manner for about 15 hours. the particle size would be expected to influence the rate of drug release (29) . this can explain the higher initial burst release of drug from n1c1 which has a smaller particle size. the overall release of nystatin from chitosan microparticles (from n1c1 to n1c4) decreased as chitosan concentration increased, suggesting that drug release could be controlled by varying chitosan concentration. n1c1 released the highest cumulative percentage of nystatin as 95.6% was released after 15hr. n1c2 did not show much variation. the remaining formulas (n1c3 and n1c4) exhibited less drug release. at the end of dissolution test , the release of nystatin was found to be incomplete in all formulas; this could be due to the relatively slow erosion of the polymeric substance under dissolution test conditions, with a consequential slow release of entrapped drug from the matrices (33) . drug release kinetics the invitro release data were plotted according to different models, and these curves were used to draw some conclusions regarding the mode of drug release from the microparticles. the curve fitting and plotting was performed in excel (microsoft software inc., usa). correlation coefficient (r 2 ) values were calculated for the curves obtained by the regression analysis of the plots. the model with the highest correlation coefficient was considered to be the best fitted model. as table 3 shows, the kinetic model fits more appropriately with the higuchi matrix model (linear nature of curve). this was confirmed by high values of regression coefficients obtained in all formulas. all other models produced curvilinear plots with lower values of regression coefficient. to find out the mechanism of drug release, the obtained drug release data were fitted in korsmeyer-peppas model. this equation was used to calculate the value of release point (n) which gave an additional evidence for the diffusion controlled mechanism. the values of (n) for all formulas suggested a non-fickian (anomalous) diffusion mode of nystatin release from chitosan microparticles, for spheres, when n= 0.85 it indicates case-ii (swelling – controlled drug release). case-ii is the relaxational release drug transport mechanism that is associated with stresses and state-transition in hydrophilic glassy polymers which swell in water or biological fluids. this term also includes polymer disentanglement and erosion (33) . when n value is between 0.43 and 0.85 (0.43 < n < 0.85) it corresponds to non-fickian (anomalous) model while if n≤ 0.43, it indicates fickian diffusion mechanism (34) . non-fickian refers to a combination of both diffusion and erosion to obtain controlled dug release (10) . typically, the diffusion process consists of an initial “burst” release of drug at or near the surface of the microsphere followed by the additional release of drug from the pores of the microsphere. erosion occurs by hydrolysis of the polymer matrix that generate pores which expose interior pockets of nystatin to the bathing liquid. for continuous release, the diffusion and erosion process must balance each other to allow the drug to diffuse out of the microsphere at a constant rate (35) . iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 9 table (3): release mechanism and kinetic model of nystatin release from chitosan microparticles in pbs (ph 7.4) (n=3). (r 2 = correlation coefficient, n = diffusion exponent from eq. mt/m∞= kt n ). conclusion in an attempt to formulate nystatin as sustained release microparticles by chemical cross-linking method, five different formulas were prepared and evaluated. chitosan concentration had a substantial effect on the main parameters of microparticles (particle size, drug loading, entrapment efficiency and yield) as any increase in polymer ratio was accompanied by a similar increase in the aforementioned parameters. in vitro release studies of microparticles showed that n1c1 formula has resulted in higher release of nystatin from the loaded microparticles after 15 hours of study (95.6% release). the kinetic study demonstrated that nystatin release from the prepared microparticles is likely to follow higuchi matrix model. from the aforementioned results, chitosan appears to be a good choice as a polymer for producing a modified release microparticles. the produced microparticles have been found to have a good potential for prolonged nystatin release and therefore can be beneficial for use in treatment of fungal infections. in the future, the optimized nystatin microspheres formula will be incorporated in a topical gel that will be evaluated for its possible utilization in the prolongation of nystatin release. references 1. patel hk, patel pr, brahmbhatt tj, s s, suthar bmh, patel a. sustained release microparticles: a review. american journal of pharmtech research. 2011;1(4):108-126. 2. kumar ks, reddy pj, sekhar kc. a review on microsphere for novel drug delivery system. journal of pharmacy research 2012;5(1):420-424. 3. bondaryk m, kurzątkowski w, staniszewska m. antifungal agents commonly used in the superficial and mucosal candidiasis treatment: mode of action and resistance development. postep derm alergol 2013;xxx(5):293–301. 4. spulber m, fifere a, anamaria d-a, fifere n. nystatin–polyethylene oxide conjugates with enhanced solubility in water. j incl phenom macrocycl chem 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2012;4(1):125-132 11. prabu sl, shirwaikar a, shirwaikar a, kumar a. formulation and evaluation of sustained release microspheres of rosin containing aceclofenac ars pharm. 2009;50(2): 51-62. 12. umakanthareddy ma, sreeramulu j, punna s. formulation development of losartan potassium microspheres using natural polysaccharides and their in-vitro evaluation. rjpbcs. 2012; 3(2):725-734. formula code n release mechanism r 2 release kinetic t50% (hour) firstorder higuchi model zeroorder korsmeyer– peppas n1c1 0.45 anomalous transport 0.96 0.98 0.85 0.99 higuchi 4.5 n1c2 0.52 anomalous transport 0.93 0.96 0.79 0.99 higuchi 5.54 n1c3 0.58 anomalous transport 0.90 0.95 0.79 0.99 higuchi 6.75 n1c4 0.61 anomalous transport 0.89 0.95 0.80 0.92 higuchi 10.18 iraqi j pharm sci, vol.24(2) 2015 nystatin microparticles 10 13. kaushik d, sardana s, mishra d. in-vitro characterization and cytotoxicity analysis of 5-fluorouracil loaded chitosan microspheres for targeting colon cancer. indian jpharm educ 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lin m-f. a kinetic study of thermal degradations of chitosan/polycaprolactam blends. macromolecular research. 2004;12(5):466-473. 31. sarmento b, ferreira d, veiga f, ribeiro an. characterization of insulin-loaded alginate nanoparticles produced by ionotropic pre-gelation through dsc and ftir studies. carbohydrate polymers 2006;66:1–7. 32. khalandar kd, sudhakar y, jayaveera k. chitosan based nasal microspheres of sumatriptan: formulation and in-vitro evaluation. rjpbcs. 2011; 2(3):489-498. 33. singhvi g, singh m. review: in-vitro drug release characterization models. ijpsr. 2011;ii( i):77-84. 34. siepmann j, peppas na. modeling of drug release from delivery systems based on hydroxypropyl methylcellulose ( hpmc). advanced drug delivery reviews 2001;48:139–157. 35. machado ha, abercrombie jj, you t, deluca pp, leung kp. release of a wound-healing agent from plga microspheres in a thermosensitive gel. biomed research international. 2013:111. iraqi j pharm sci, vol.23(2) 2014 synthesis of new cephalosporins of improved activity 24 synthesis of new cephalosporins of expected improved activity and resistance against -lactamases shakir m. alwan *,1 and ameer h. kadhim * * department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract the development of new cephalosporins with improved activity against resistant microbes, such as, mrsa (methicillin resistant staph. aureus), p. aeruginosa, is of high potential. chemical synthesis of two new series of thiadiazole linked to cysteine (series 1) and cephalosporins containing thiadiazole linked to cysteine through disulfide bond (series 2) were achieved. the chemical structures of the synthesized compounds were confirmed using spectral (ft-ir, 1 h-nmr) and elemental microanalysis. the incorporation of privileged chemical moieties, such as, thiadiazole, schiff base, cysteine and sulfonamide, has been found to have great contribution to the antimicrobial activities. compounds of series 1 (1b-d), containing a schiff base or a sulfonamido moiety, showed reasonable activity and were less potent than cephalexin with respect to e. coli and staph. aureus. the new cephalosporins (series 2) showed remarkable activities on e. coli (62.5-15.6µg/ml) and staph. aureus (31.2-62.5µg/ml) when compared with cephalexin (250 and 125 µg/ml) respectively. moreover, compounds 1 and 3 showed very promising activity against mrsa (250 and 500µg/ml) respectively. the incorporation of a sulfonamido moiety to the cephalosporin molecule was successfully achieved. this is a very interesting finding which may open a new approach in the synthesis of newer cephalosporins. keywords: cephalosporins, cysteine, schiff base, sulfonamides, thiadiazole. يازولاثايا د -1,3,4تشخيص وتقييم اولي للفعالية المضادة للبكتريا لمشتقات جديدة لمجموعة وتخليق السيفالوسبوريناتالمرتبطة مع شاكر محمود علوان 1،* و امير حسيه كاظم * * .فشع انكٍمٍبء انصٍذالوٍت ،كهٍت انصٍذنت ،جبمعت بغذاد ، بغذاد ، انعشاق الخالصة .ان حطٌُش سٍفبنُسبُسٌىبث جذٌذة راث فعبنٍت ضذ انمبٌكشَببث انممبَمت مثم انمكُساث انعىمُدٌت انزٌبٍت انممبَمت نهمثٍسٍهٍه (mrsa) انضائفت انضوجبسٌت َ (p. aeruginosa) نمذ حم انخخهٍك انكٍمٍبئً الثىٍه مه انسالسم انجذٌذة نهثٍبداٌضَل .رَ اٌمٍت عبنٍت َ سٍفبنُسبُسٌىبث انحبٌَت عهى انثٍبدٌبصَل انمخصم ببنسٍسخبٌه بُاسطت اصشة ( 1سهسهت )انمخصم ببنحبمض االمٍىً انسٍسخبٌه ححٌُم فُسًٌٍ مطٍبف األشعت )ل انخحبنٍم انطٍفٍت حم حشخٍص انخشاكٍب انكٍمٍبئٍت نهمشكببث انمحضشة ببسخعمب( . 2سهسهت )انذاٌسهفبٌذ ان ادخبل مجبمٍع (.انٍبٌذسَجٍه َانىبٌخشَجٍه, انكبسبُن)َلٍبط طٍف انكخهت ( ححج انحمشاء َانشوٍه انىَُي انمغىبطٍسً نهبشَحُن انٍت فً انفعبنٍبث انمضبدي َجذ نٍب مسبٌمت ع, سٍسخبٌه َمجمُعت انسهفُن امٍذَ, لبعذة شف, كٍمٍبئٍت مخمٍضة مثم انثٍبدٌبصَل اظٍشث فعبنٍت ممبُنت َكبوج , انحبٌَت عهى مجبمٍع لُاعذ شف اَ انسهفُن امٍذَ شف ( a-d) 1مشكببث انسهسهت االَنى . نهمبٌكشَببث انسٍفبنُسبُسٌىبث . اسخثىبئٍت ضذ انضائفت انضوجبسٌتفعبنٍبث َ(e. coli) الم لُة مه انسٍفبنٍكسٍه ضذ انبكخشٌب االششٌكٍت انمُنُوٍت ارا مب لُسوج مع ( مم/مبٌكشَغم 31.2-62.5)َ انمكُساث انعىمُدٌت انزٌبٍت ( مم/مبٌكشَغم 15.6-62.5( )2سهسهت )انجذٌذة جذا ضذ انمكُساث اظٍشث فعبنٍت َاعذي 3َ 1ببالضبفت انى انمشكببث . عهى انخُانً( مم/مبٌكشَغم 125َ 250)انسٍفبنٍكسٍه نمذ حم بىجبح اضبفت مجمُعت انسهفُن امٍذَ انى . عهى انخُانً( مم/مبٌكشَغم 500َ 250)انعىمُدٌت انزٌبٍت انممبَمت نهمثٍسٍهٍه ان انخُصم انى ٌزي انىخٍجت انمشجعت جذا ٌمكه ان ٌفخخ افبق جذٌذة فً حخهٍك . نجضٌئت انسٍفبنُسبُسٌه c7 انسهسهت انجبوبٍت عهى . فبنُسبُسٌىبث جذٌذة ححُي ٌزي انصٍغت انكٍمٍبٌَتسً . الثياديازول،السلفوواميد ،قاعدة شف ، السيستايه ، سيفالوسبوريىاثال :الكلماث المفتاحيت introduction the development of new antibiotics has been a very important task in providing the proper means for treating resistant strains of organisms that previously had been susceptible to older antibiotic (1) . the elucidation of biochemical mechanisms of microbial resistance to antibiotics, such as the inactivation of penicillins and cephalosporins by β-lactamases, has stimulated the research in the development of semisynthetic analogs that resist microbial biotransformation (2) . the evolution of hospital-acquired strains of staphylococci resistant to penicillin and of g () bacilli, such as, pseudomonas and klebsiella spp., e. coli, and others, often resistant to several antibiotics has become a serious medical problem (3) . 1 corresponding author e-mail: shakmawales@yahoo.co.uk. received: 27/1/2014 accepted:11 /6/2014 mailto:shakmawales@yahoo.co.uk iraqi j pharm sci, vol.23(2) 2014 synthesis of new cephalosporins of improved activity 25 cephalosporins are derived from cephalosporanic acid substituted at c7 acyl side chain and at c3 positions of the cephem. these derivatives may have different antibacterial spectra, β-lactamase sensitivity/resistance and pharmacokinetic properties (4) . the newer generations of cephalosporins have generally focused on two parameters: broadening the spectrum to include activity towards resistant pathogens and have anti-pseudomonal activity, as well as improving the pharmacokinetic properties (3, 5) . cephalosporins linked to privileged chemical moieties, such as amino acids act as false substrate to the enzymes involved in bacterial cell wall synthesis (6) . prodrugs of ceftizoxime containing an amino acid on the aminothiazol moiety was synthesized and found with potential activity and improved physicochemical properties and oral absorption (7) . cephalosporins containing acyl derivatives of certain amino acids at the c3 side chain showed improved aqueous solubility at physiological ph (8) . thiadiazole ring was previously incorporated at c3 and c7 positions of the cephem, as privileged heterocyclic moiety. cephalosporins containing thiadiazole showed good antibacterial activity against both g (+) and g (-) bacteria, such as, cefuzonam, cephazoline, ceftobiprole and ceftaroline (9, 10) . cefuzonam exhibited excellent activity against p. aeruginosa (11) . cefazopran derivatives had anti-methicillin resistant staph. aureus (mrsa) activity (12, 13). ceftobiprole and ceftaroline fosamil are fifth generation cephalosporins containing thiadiazole moiety at c7 position with activity against mrsa, penicillin-resistant strep. pneumonia, p. aeruginosa and enterococci (14,15) . cephalosporins containing a 1,3,4-thiadiazole moiety linked through a sulfide or a disulfide bond in the acyl side chain were found to be equipotent to cephalexin (10) . this finding was supported when compared with antibiotics containing disulfide bonds and showed marked activities (16) . schiff bases have been reported for their wide range of biological activities, such as, antitumor (17) , anti-tuberculosis (18) , antimicrobial (19, 20) . sulfonamido moiety as privileged chemical entity has been reported to possess antimicrobial (21, 22) , antiviral (23) , anticancer (24) and antimalarial (25) activities. the development of new cephalosporins with improved activity against resistant microbes, such as, mrsa, p. aeruginosa, is of great interest. the privileged chemical moieties, such as, thiadiazole, schiff base, cysteine and sulfonamide, have been considered to be incorporated into the c7 acyl side chain of the cephalosporin molecule to achieve one or more of the desired goals. to the best of the authors' knowledge, sulfonamido moiety has not been incorporated within cephalosporins molecules, so far. experimental section general methods melting points were determined (uncorrected) using electrical melting point apparatus, electro-thermal 9300, usa. the infrared spectra were performed in kbr disc by ftir spectrophotometer/ shimadzu. elemental micro-analyses (chn) were performed by euro-vector ea 3000a, italy. 1 h-nmr spectra was recorded using nmr bruker 500 mhz−avance iii and chemical shifts were recorded in parts per million (ppm). 1 h-nmr and chn analyses are kindly performed by faculty of science/university of jordon. tetramethylsilane was used as reference. chemical synthesis the synthesis of the target compounds of series 1 and 2 (1a-d and 1-3) were achieved, as illustrated in schemes (1-3). chemical synthesis of compounds of series1 5,5'-disulfanediylbis(1,3,4-thiadiazol-2-mine) 1a this compound was synthesized by oxidation of 2-amino-1,3,4-thiadiazole-5-thiol using hydrogen peroxide (10) , as illustrated on scheme 1. hydrogen peroxide (7.5mmol, 2.67 ml) was added drop wise to a suspension of 5amino-1,3,4-thiadiazole-2-thiol (7.5mmol, 1g) in absolute ethanol (10 ml) with continuous stirring for 1hr at room temperature. a yellow precipitate was formed, collected by filtration, washed excessively with distilled water and crystallized from hot ethanol and the product was dried in an oven at 70 ºc. the product was collected as yellow powder, yield 92%, m.p. 221-223 o c, the ir spectrum (ʋ, cm -1 ); 3265, 3088 (nh2 stretching), 1637 (c=n stretching), 1610 (c=n stretching), 1552, 1505 (nh2 bending) and 1138 (c-s stretching). 2-amino-3-((5-amino-1,3,4-thiadiazol-2-yl)-di sulfanyl) propanoic acid 1b this compound 1b is prepared by a sulfhydryl-disulfide exchange reaction performed to produce cysteine linked to 5amino-1,3,4-thiadiazole-2-thiol (10, 26) . this reaction involves a nucleophilic attack (sn2) of the thiolate anion of cysteine to the disulfide (1a) as the electrophile, as shown in scheme 1. an aqueous solution (20 ml) of cysteine (3.7 mmol, 0.45g) was added to a suspension of 1a (3.7 mmol, 1g) in potassium chloride solution (2 m, 10ml) and the mixture was iraqi j pharm sci, vol.23(2) 2014 synthesis of new cephalosporins of improved activity 26 adjusted to ph 7.5 by potassium hydroxide (5%). the mixture was vigorously stirred for 24 hrs at room temperature. the reaction mixture was filtered to remove the unreacted compound 1a and the filtrate was neutralized with acetic acid (5%) and placed in a refrigerator. the precipitated product was formed, collected and washed with distilled water, recrystallized from ethanol and dried in an oven at 70 °c. the product was collected as white powder, yield 64%, m.p. 252 o c (decomposed).the ir spectra (ʋ, cm -1 ) showed the characteristic bands; 3250-2950 (broad band of oh and nh2 stretching), 2918 (asymmetric c-h stretching of methylene), 2858 (symmetric c-h stretching of methylene), 1654 (c=o stretching of carboxyl), 1622 (c=n thiadiazole), 1585 (nh2 bending). the 1 h-nmr spectra (δ, ppm); 2.93 (dd, 1h, -ch2-), 3.12 (dd, 1h, -ch2-), 3.5 (t, 1h, ch). the elemental analysis for c5h8n4o2s3; calculated: found; c, 23.8:23.11, h, 3.2:3.36; n, 22.2:22.95. 2-(benzylideneamino)-3-((5-(benzylideneamino) -1,3,4-thiadiazol-2-yl) disulfanyl)propanoic acid 1c formation of schiff bases of 1,3,4thiadiazole and cysteine 1b was performed using benzaldehyde in the presence of a catalytic amount (3-4 drops) of concentrated h2so4 (27) , as illustrated on scheme (1). compound 1b (3.9mmol, 1g) was dissolved in dmf (30ml) containing 3-4 drops of concentrated h2so4 and refluxed with benzaldehyde (7.9mmol, 0.8ml) for 6 h. the solvent was evaporated under vacuum and the residue was washed thoroughly with distilled water, dried in an oven at 70 °c and triturated with petroleum ether (2x10ml) and crystallized from ethanol. this afforded compound 1c, which was dried in an oven at 70°c. the product was collected as pale brown powder, yield 65%, m.p. 195-197 o c. ir spectra (ʋ, cm 1 ); displayed the following; 3400-3100 (oh stretching of carboxyl), 1700-1600 (broad band of c=o carboxyl, imines of thiadiazole and imines of schiff bases. 1 h-nmr spectra (δ, ppm); 2.8 (dd, 1h, methylene), 3.09 (dd, 1h, methylene), 3.5 (t, 1h, ch), 7.3-7.78 (t, 1h, c-h and t, 1h, c-h overlapped with d, 1h, c-h and d, 1h, c-h and s, 1h, hc=n). the elemental analysis was calculated for c19h16n4o2s3; calculated: found; c, 53.25:54.55, h, 3.76:3.68, n, 13.07:13.31 2-(4-chlorophenylsulfonamido)-3-((5-(4-chloro phenylsulfonamido)-1,3,4-thiadiazol-2-yl)-di sulfanyl) propanoic acid 1d this is a sulfonamido-thiadiazole compound 1d, which is synthesized by reacting 1b with 4-chloro-benzenesulfonyl chloride. this is a nucleophilic attack of the amine groups on the sulfonyl moiety with the liberation of hcl which is neutralized by na2co3 in the media, as illustrated in scheme (1). an aqueous solution (30ml) of compound 1b (3.9mmol, 1g) containing na2co3 (7.8mmol, 0.82g) was cooled to 0ºc using an ice-bath. 4-chloro-benzenesulfonyl chloride, (7.8mmol, 1.65g) dissolved in dry tetrahydrofuran (10ml) was added drop wise over a period of 30min and the reaction mixture was continuously stirred for 2hrs at 0 c (28) . the mixture was further stirred for 4 hrs at room temperature and the volume was reduced to 10ml and then hcl solution (5%) was added to acidify the mixture to ph 5. the mixture was stored in a refrigerator overnight. the product 1d was collected as a white precipitate, washed with acetone (2x15ml), recrystallized from ethanol and dried in an oven at 70°c. the product was collected as white powder, yield 77%, m.p. 257-259°c. the ir spectra (ʋ, cm -1 ); recorded the following bands; 3200-2900 (o-h stretching of carboxyl, n-h stretching of sulfonamide, ch stretching of aromatic ring), broad band at 1680-1610 (represents c=o stretching of carboxyl, c=n stretching of imine and c=c aromatic), 1510 (n-h bending of sulfonamide), 1276, 1161 (s=o stretching of sulfonamide). 1 h-nmr spectra (δ, ppm), 3.2 (d, 1h, methylene), 3.3 (d, 1h, methylene), 4.2 (t, 1h, ch), 7.57 (s, 2h, chlorophenyl, adjacent to cl at c2 and s, 2h, chlorophenyl, adjacent to cl at thiadiazole), 7.79 (s, 2h, chlorophenyl, adjacent to so2 at c2 and s, 2h, chlorophenyl, adjacent to so2 at thiadiazole). the elemental analysis was calculated for c17h14cl2n4o6s5; calculated: found; c, 33.94:35.19, h, 2.35:2.27, n, 9.31:9.92. iraqi j pharm sci, vol.23(2) 2014 synthesis of new cephalosporins of improved activity 27 nn s hs nh2 nn s sh2n nn s nh2s ethanol/h2o2 1a stirring 1 hr rt hs h2 c h c cooh nh2 1koh solution ph 7.5/24 hrs rt nn s sn s ch2 h c n cooh ref lux 6 hrs chhc 1b dm f/h 2 so 4 1c nn s sh2n s h2 c ch nh2 cooh cl s clo o 0 c 2h rs /rt 4 hr s nn s sn h s ch2 h c h n cooh ss 1d o o o o cl cl th f c oh 2acetic acid 5% scheme (1) chemical synthesis of compounds series 1 chemical synthesis of compounds of series 2 7-(2-(benzylideneamino)-3-((5-(benzylidene amino)-1,3,4-thiadiazol-2-yl)disulfanyl)propan amido)-3-methyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid 1 the synthesis of this compound was achieved by reaction of compound 1c with 7aminodesacetoxy cephalosporanic acid (7adacd) by the mixed anhydride method with ethylchloroformate (ecf) (29) and as shown on scheme 2. compound 1c (2.33mmol, 1g) was dissolved in a mixture (30 ml) of dry acetone and dmf (1:2) containing tea (2.33mmol, 0.32ml) and the mixture was placed in an ice bath at (-5 to -10°c). a solution of ecf (2.33 mmol, 0.22 ml) was added to the above mixture over a period of 10 min with continuous stirring, which was continued for further 30 min. 7-adaca (2.33mmol, 0.5g) was dissolved in 10ml of na2co3 (2.33mmol, 0.24g) solution in distilled water previously cooled to 0°c and was added at once to the above mixture with stirring for 4 hrs. the solvent was then evaporated and the resultant precipitate was washed with hcl solution (3%) and recrystallized from ethanol. the product was collected as brown powder, yield 58%, m.p. 261-263°c. the ir spectra (ʋ, cm -1) ; showed the following bands: 3271 (n-h stretching of amide), 3250-2950 (o-h stretching of carboxyl), 3047 (c-h stretching of aromatic ring), 1759 (c=o stretching of βlactam), 1691 (c=o stretching of carboxyl), 1620-1500 (broad band of c=n of schiff base, c=n of thiadiazole, c=c of cephem, c=c aromatic ring and n-h bending). 1 h-nmr spectra (δ, ppm); 1.83 (s, 3h, ch3), 3.04 (dd,1h, methylene), 3.07 (dd, 1h, c2 methylene), 3.16 (dd,1h, c2, methylene), 3.28 (dd, 1h, methylene), 3.87 (t, 1h, α-ch), 4.96 (s, 1h, c6-h), 5.3 (s, 1h, c7-h), 7.3-7.5 (t, 1h, c-h and t, 1h, c-h overlapped with d, 1h, c-h and d, 1h, c-h of aromatic) 7.79 (s, 1h, hc=n). the elemental analysis was calculated for c27h24n6o4s4; calculated: found, c; 51.9:53.76, h; 3.87:3.59, n; 13.45:13.02. 7-(2-amino-3-((5-amino-1,3,4-thiadiazol-2-yl) disulfanyl)propanamido)-3-methyl-8-oxo-5thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid 2 this compound was prepared by reaction of compound 1with hcl solution adjusted at ph 2 (30) and as shown on scheme 2. compound 1 (1.6mmol, 1g) was suspended in distilled water (20 ml) and cold hcl solution (1n) was added drop wise and the ph was adjusted to 2 and the mixture was placed in an ice-bath and stirred for 2 hrs. the reaction mixture was neutralized by nahco3 solution (5%). the resultant precipitate was excessively washed with distilled water and dried in an oven at 70°c. the product was triturated with petroleum ether (2x10ml) to afford compound 2. the product was obtained as pale brown powder, yield 50%, m.p. 274276 °c the ir spectra (ʋ, cm -1 ); showed the following bands; 3475-2974 (broad band of o h carboxyl, nh2 (aliphatic and aromatic ) iraqi j pharm sci, vol.23(2) 2014 synthesis of new cephalosporins of improved activity 28 and n-h amide), 1739 (c=o stretching of βlactam), 1718 (c=o stretching of carboxyl), 1695 (c=o stretching of amide), 1666 (c=n of imine thiadiazole), 1580-1592, 1510 (broad band of n-h bending of n-h, nh2). 1 h-nmr spectra (δ, ppm), 1.85 (s, 3h, ch3), 2.86 (dd, 1h, methylene), 3.03 (dd, 1h, c2 methylene), 3.13 (dd, 1h, c2 methylene), 3.16 (dd, 1h, methylene), 3.5 (t, 1h, α-ch), 4.96 (s, 1h, c6h), 5.3 (s, 1h, c7-h). the elemental analysis was calculated for c13h16n6o4s4; calculated: found, c; 34.8:35.93, h; 3.6:3.38, n; 18.74:19.57. nn s sn s ch2 h c n c o h n s n ch3 ho o o ch hc 1 1c 1. ecf/tea -5 to -10 oc 1h 2. 7-adaca/na 2 co 3 4hs nn s sh2n s ch2 h c nh2 c o h n s n ch3 ho o o 1.h c l 1n /ph 2 0 oc 2 hrs 2.n a2 c o 3 /ph 7 2 3. hcl 5% scheme (2) chemical synthesis of compounds 1 and 2 7-(2-(4-chlorophenylsulfonamido)-3-((5-(4chlorophenylsulfonamido)-1,3,4-thiadiazol-2yl)disulfanyl)propanamido)-3-methyl-8-oxo-5thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 3 the synthesis of this compound was achieved by reaction of compound 1d by the mixed anhydride method with ecf (29) and as previously described. the chemical synthesis is illustrated on scheme 3 compound 1d (1.25mmol, 1g) was suspended in a mixture (30 ml) of dry acetone and dmf (1:2) containing tea (1.25mmol, 0.17ml) was placed in an ice bath at (-5 to 10c°). a solution of ecf (1.25mmol, 0.12ml) was added and the mixture was treated as previously described for compound 1. the product was obtained as white powder, yield 56%, m.p. 291-293 o c. the ir spectra (ʋ, cm 1 ); 3483 (n-h stretching of amide), 3300-3100 (broad band of o-h and n-h stretching of carboxyl and sulfonamide respectively), 1788 (c=o stretching of β-lactam), 1735 (c=o stretching of carboxyl), 1681 (c=o of amide), 1597 (c=n of aromatic), 1246, 1155 (s=o stretching of sulfonamide). 1 h-nmr spectra (δ, ppm); 1.85 (s, 3h, ch3), 2.89(d, 1h, methylene), 3.16 (d, 1h, c2 methylene), 3.19 (d, 1h, c2 methylene), 3.51 (d, 1h, methylene), 3.55 (t, 1h, a-c-h), 4.99 (s, 1h, c6-h), 5.33 (s, 1h, c7-h), 7.49 (s, 2h, chlorophenyl, adjacent to cl at c2 and s, 2h, chlorophenyl, adjacent to cl at thiadiazole), 7.73 (s, 2h, chlorophenyl, adjacent to so2 at c2 and s, 2h, chlorophenyl, adjacent to so2 at thiadiazole). the elemental analysis was calculated for c25h22cl2n6o8s6; calculated: found; c, 37.64:36.48, h, 2.78:2.93, n, 10.53:10.99. 2. 7-adaca na 2 co 3 4hs 3. hcl 5% 1d nn s sn h s ch2 h c nh c o h n s n ch3 ho o o s 3 scl cl o o o o 1. ecf/tea -5-10 oc 1h scheme (3) chemical synthesis of compound 3 iraqi j pharm sci, vol.23(2) 2014 synthesis of new cephalosporins of improved activity 29 antimicrobial evaluation (determination of mic values) the determination of mic values of the synthesized compounds of series 1 and 2 was achieved using the broth microdilution method (31) . a panel of certain microorganisms, such as, staph. aureus, e. coli, p. aeruginosa and mrsa were used and their stock suspensions measured as 1 × 10 6 cfu/ml was inoculated. cephalexin sodium was used as reference standard. the full experimental details are described below: broth microdilution method mic values of compounds (1b-d and 1-3) were determined against the tested microorganisms using the microdilution method in 96-well plates. an aliquot (150 μl) of the mueller hinton broth was used to fill the first experimental well, while the other wells were filled with (100 μl). a aliquot (50 μl) of the tested compound as sodium salt (2mg/ml) was added to the first well to make a total volume of 200µl. double-fold serial dilution was then carried out across the wells of the plate. the overnight batch culture of the microorganisms (10 μl) was used to inoculate each well to achieve an inoculum size of approximately 1 × 10 6 cfu/ml. the plates were incubated for 24 h at 37°c. the mic values were calculated visually according to the degree of turbidity against mcfarland standard (31) . negative controls (well without sample or reference standard) and positive controls (with cephalexin) were used. each mic value was determined in duplicate and the average was calculated and the results are listed on table (1). results and discussion the ft-ir characteristic bands shown in the spectra of the synthesized compounds were used to identify and confirm their chemical structures depending on the appearance and disappearance of certain chemical groups and consequently their bands. the ir spectrum of compound 1a (5,5'-disulfanediylbis(1,3,4thiadiazol-2-amine)), showed sharp peak of the imine at 1637cm -1 and very distinguished two absorption bands for the primary amines at 3265cm -1 and 3088cm -1 . the physical and chemical properties of this compound 1a confirmed its chemical structure, as it is only soluble in dmf and aqueous acidic solutions and it is insoluble in water, alcohols, chloroform or acetone. the ir spectrum of compound 1b displayed the characteristic bands of the carboxyl group of cysteine appeared in the range of 3250-2950 cm -1 . this broad band overlapped the two amino groups of thiadiazole and the cysteine. the appearance of bands at 2918 and 2858cm -1 for the asymmetric and symmetric vibration of the methylene and the carbonyl of carboxyl at 1654cm -1 of cysteine were clearly recorded. 1b is insoluble in water and hot ethanol, soluble in dmf and alkaline or acidic aqueous solutions. the ir spectrum of compound 1c (schiff bases of 1b) displayed a broad band at 17001600cm -1 which refers to the four imines (two imines of thiadiazole and the two schiff bases) and the c=o of carboxyl group. compound 1c is soluble in ethanol and acetone on contrary of compound 1b. the ir spectra of compound 1d showed broad band at 3200-2900cm -1 representing the carboxyl group and the two nh of the sulfonamide group. appearance of a broad band at 1680-1610cm -1 represents the c=o stretching of carboxyl, the c=n stretching of imine and c=c of aromatic. the spectrum also displayed clear bands at 1510cm -1 and 1467cm -1 for nh bending. the sulfon groups (o=s=o) recorded characteristic bands at 1276 and 1161cm -1 , which were not displayed by compound 1b. compound 1d has noticeable water solubility compared with its precursor (1b) and this is may be due to the 4chlorophenylsulfonyl moiety. the ir spectrum of compound 1 displayed a characteristic band at 3271cm -1 representing the nh stretching of amide. a broad band at 3250-2950cm -1 may refer to the presence of carboxylic group and characteristic bands at 1759cm -1 and 1691cm -1 indicate the c=o of βlactam and c=o of carboxyl respectively. a broad band at 1620-1500cm -1 represents c=n of schiff bases, c=n of thiadiazole, c=c of cephem and nh bending. the physicochemical properties of compound 1 were clearly differentiated from its precursors (7-adaca and 1c). the ir spectra of compound 2, showed two amino groups (aliphatic and aromatic) and a broad band at 3475-2974 cm -1 for the oh of carboxyl, amines and nh of amide. c=o stretching of β-lactam, carboxyl and amide appeared at 1739, 1718 and 1695 cm -1 respectively. the liberated free amine groups of compound 2 have enabled the compound to be converted to the hydrochloride salt and separated from the aqueous solution. the ir spectra of compound 3 displayed the characteristic band at 3483cm -1 of nh stretching of amide, a broad band at 34003100cm -1 represents the oh of carboxyl and nh of sulfonamide. characteristic bands of the β-lactam, c=o of carboxyl and c=o of amide are displayed at 1788, 1735 and 1681cm -1 respectively. c=n of thiadiazole appeared at 1597cm -1 and the sulfon groups (o=s=o) recorded characteristic bands at 1246 and 1155cm -1 . iraqi j pharm sci, vol.23(2) 2014 synthesis of new cephalosporins of improved activity 30 1 h-nmr spectra (δ, ppm) of compounds of series 1 and 2 showed the characteristic peaks for the protons, as expected. all the synthesized compounds were prepared as sodium salt and dissolved in d2o, therefore, protons on oand ncould not be detected. the 1 h-nmr spectra of compounds 1b-d, 1 and 2 displayed the phenomenon of complex spin-spin splitting (32) with regard to the methylene protons in the cysteine moiety. this phenomenon has led to the appearance of doublet of doublet, which was clearly shown on spectra of these compounds. in the 1 hnmr spectra the chemical shifts of different protons have been distinct and the spin-spin splitting patterns have been straightforward. however, different kinds of protons in a molecule have overlapping signals, as previously reported (33) . therefore, the five aromatic ring protons gave a complex (overlapping pattern), even though they are not all equivalent. this observation was noticed in compounds 1c and 1 but was not detected in compounds 1d and 3, since these compounds contain 4-chlorophenyl moiety and the positions of the protons are clearly affected by the inductive effect of chlorine atom. 1 hnmr spectra of compounds 1-3 displayed characteristic peaks of the protons of the cephem nucleus and the characteristic peaks appeared for cysteine moiety, as previously shown for compounds 1b-d. compounds 1 and 3 displayed the characteristic peaks for the aromatic substitution, while, compound 2 did not display the aromatic protons, simply because the aromatic substitution was removed. antimicrobial evaluation (determination of mic values) the mic values of the synthesized compounds (series 1 and 2) were determined in comparison with cephalexin. compound 1b showed no activity against the microorganisms used in a concentration of < 500µg/ml (table 1). compound 1c was less potent than cephalexin against staph. aureus, while it has no activity against the other bacteria used. compound 1d showed activity only against e. coli and was less potent than cephalexin. notably, 1,3,4-thiadiazole derivatives showed significant antibacterial activity against staph. aureus and e. coli (34, 35) . the new cephalosporins 1 and 3 showed very interesting results (mic values of 500 and 250 µg/ml, respectively) against p. aeruginosa in comparison with cephalexin, which showed no activity at <500 µg/ml. compounds 1-3 have much higher activity against e. coli (15.6-250µg/ml), when compared with cephalexin (250µg/ml). compound 3 was the most potent (15.6µg/ml) among this series of new cephalosporins against e. coli. the new cephalosporins 1-3 showed very interesting activity against staph. aureus and the mic values ranged between (31.2-62.5µg/ml) in comparison with cephalexin (125µg/ml). moreover, compound 1 was the most potent of the series with mic value of 31.2µg/ml. cephalosporins 1 and 3 showed very promising and satisfactory results (250 and 500 µg/ml, respectively) against mrsa in comparison with cephalexin, which has no activity at all (31) at concentration of < 500µg/ml. moreover, compound 1 was more potent than compound 3 with a margin of one fold. the detailed antimicrobial results are stated on (table 1). the interesting activities of the new cephalosporins 1 and 3 against p. aeruginosa and mrsa may indicate their resistance against the β-lactamases produced by these microorganisms. this is an expected observation, due to the presence of 2 benzylideneamino or 2-(4chlorophenylsulfonamido) moieties in the acyl side chain at the α-carbon adjacent to the βlactam ring. these two moieties may provide steric effect and consequently may be considered as isosteric replacement for the alkoxyimino moiety, which is essential for the protection against β-lactamases. it is worth mentioning that compound 3, which is a new cephalosporin containing 1,3,4thiadiazole linked to a sulfonamido moiety comprising a new approach of incorporation of sulfonamide within the cephalosporin molecule. iraqi j pharm sci, vol.23(2) 2014 synthesis of new cephalosporins of improved activity 31 table( 1 ): mic values (µg/ml) of the synthesized compounds compound pseudomonas aeruginosa atcc 9027 escherichia coli atcc 8739 staphylococcus aureus atcc 29213 mrsa atcc 43300 cephalexin ------250 125 ------ 1b ------------------------ 1c ------------500 ------ 1d ------500 ------------ 1 500 62.5 31.2 250 2 ------250 62.5 ------ 3 250 15.6 62.5 500 keynote: ---= no growth acknowledgement the authors are very grateful for the university of baghdad for supporting this research work. the great efforts of dr. amal g. al-bakri / pharmaceutics and microbiology department / faculty of pharmacy / university of jordan, for performing the antimicrobial evaluation are deeply acknowledged. references 1. abdulrasool m. m., jawad a. h. and shneine j. k., synthesis, characterization and evaluation of biological activity of new heterocyclic compounds containing 1,2,4triazole and 1,3,4-thiadiazole rings. international journal of applied science and technology, 2012, 2:10, 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antimicrobial activities of 1, 2, 4-triazole and 1,3, 4-thiadiazole derivatives of 5-amino-2hydroxybenzoic acid, e-j. chem., 2008; 5(4), 963-968. iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs doi: https://doi.org/10.31351/vol31iss1pp32-42 32 safety profile of biological drugs in clinical practice: a retrospective pharmacovigilance study elaaf f. hassaan*,1, dheyaa j. kadhim* and manal m. younus** *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** iraqi pharmacovigilance center, directorate of technical affairs, ministry of health and environment, baghdad, iraq. abstract biological drugs have an active substance that is made by a living organism or derived from a living organism. they are one of the important therapy options used in a wide range of diseases especially lifethreatening diseases. biological therapy opens new opportunities for treating different diseases for which drug therapy is minimal, but they have considerable differences in the safety consequences in comparison with nonbiological drugs. the aim of the current study was to assess the post-marketing safety profile of biological drugs used in iraqi hospitals by the analysis of the reported adverse drug reactions regarding their severity, seriousness, preventability, expectedness, and outcome. it is a retrospective study of the individual case safety reports from the iraqi pharmacovigilance center/ministry of health. there were 446 individual case safety reports in the research, involving 899 adverse drug reactions. rituximab was found to be the drug with the highest number of adverse drug reactions with 241 adverse drug reactions (26.81% out of total adverse drug reactions). most of the adverse drug reactions were related to general disorders and administration site conditions (22.25%). regarding severity of adverse drug reactions, the majority of adverse drug reactions were observed in moderate levels [level 4 (26%), and level 3 (18%)]. the severe adverse drug reactions in patients below 18 years age group were significantly higher compared to adults and elderly. seriousness assessment showed that the majority of adverse drug reactions were serious (52%). rituximab was the drug for which the highest number of serious adverse drug reactions was reported (41.28% of total serious adverse drug reactions), most of the adverse drug reactions (66%) were probably preventable. fatality outcome was reported for 3% of adverse drug reactions while 43% of adverse drug reactions were recovered/resolved. keywords: safety profile, adverse drug reactions, iraqi pharmacovigilance center, biological drugs. جية في الممارسة السريرية: دراسة استرجاعية لليقظة الدوائية يولواسالمة االدوية الب ايالف فاضل حسان *،1، ضياء جبار كاظم * ومنال محمد يونس** العراق.فرع الصيدلة السريرية، كلية الصيدلة، جامعة بغداد، بغداد، * المركز العراقي لليقظة الدوائية، دائرة االمور الفنية، وزارة الصحة والبيئة، بغداد، العراق ** ةالخالص واحدة من خيارات العالج الهامة وهي مادتها الفعالة من كائن حي أو مشتقة من كائن حي تتكون التيالعقاقير البايولوجية هي األدوية ة المستخدمة في مجموعة واسعة من األمراض وخاصة األمراض التي تهدد الحياة. يفتح العالج البايولوجي فرًصا جديدة لعالج األمراض المختلف ختالفات كبيرة في عواقب السالمة مقارنة باألدوية غير البايولوجية. الهدف من الدراسة الحالية التي يكون العالج الدوائي فيها ضئياًل ، لكن لها ا ة المبلغ عنها هو تقييم برنامج األمان بعد التسويق لألدوية البيولوجية المستخدمة في المستشفيات العراقية من خالل تحليل التفاعالت الدوائية الضار رجعي لتقارير سالمة الحاالت الفردية من مركز ذات أثردراسة الا ، وإمكانية الوقاية منها ، والتوقع ، والنتيجة. ، وخطورته بشدتها فيما يتعلق تفاعل دوائي ضار. وجد أن ٨٩٩في البحث ، بما في ذلك لحالة السالمة الفردية تقرير ٤٤٦التيقظ الدوائي العراقي / وزارة الصحة. كان هناك ٪ من إجمالي التفاعالت ٢٦٫٨١تفاعل دوائي ضار )٢٤١اء الذي يحتوي على أكبر عدد من التفاعالت الدوائية الضارة معريتوكسيماب هو الدو ٪(. فيما يتعلق بشدة ٢٢٫٢٥) و اضطراب موضع الحقن الدوائية الضارة(. كانت معظم التفاعالت الدوائية الضارة مرتبطة باضطرابات عامة كانت (٪١٨) ٣٪( ، والمستوى ٢٦) ٤، لوحظت غالبية التفاعالت الدوائية الضارة في مستويات معتدلة ]المستوى الضارة التفاعالت الدوائية عاًما أعلى بشكل ملحوظ مقارنة بالبالغين وكبار السن. أظهر تقييم ١٨الشديدة في المرضى الذين تقل أعمارهم عن الضارة التفاعالت الدوائية ٪(. كان ريتوكسيماب هو الدواء الذي تم اإلبالغ عن أكبر عدد من التفاعالت ٥٢كانت خطيرة )الضارة ت الدوائية االخطورة أن غالبية التفاعال الخطيرة( ، وربما كان من الممكن الوقاية من معظم التفاعالت الدوائية الضارة ٪ من إجمالي التفاعالت الدوائية ٤١٫٢٨الدوائية الضارة الخطيرة ) .معالجتهاتم الضارة ٪ من التفاعالت الدوائية ٤٣٪ من التفاعالت الدوائية الضارة بينما ٣م اإلبالغ عن نتيجة الوفيات لـ ٪(. ت٦٦) الضارة .يولوجيةاالعراقي ، األدوية الب ةالدوائي اليقظة، التفاعالت الدوائية الضارة ، مركز سالمة االدويةالكلمات المفتاحية : 1corresponding author e-mail: elaf.hassan1206@copharma.uobaghdad.edu.iq received:5 /5/2021 accepted:11 /7 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp32-42 iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs 33 introduction pharmacovigilance is defined by the world health organization (who) as “the science and activities related to the detection, assessment, understanding, and prevention of adverse effects and other drug related safety problems”. pharmacovigilance is aimed to avoid the adverse reactions of drugs that may occur in humans during the life cycle of these drugs. furthermore, pharmacovigilance includes identifying prescription errors, a lack of effectiveness data, off-label use, acute and chronic toxicity, drug-related mortality evaluation, drug abuse and misuse, and adverse drug interactions with chemicals and other medications (1). a pharmacovigilance research may be clinical, epidemiological, experimental (such as detecting an adverse effect in animals and establishing the mechanism required for human protection), or diagnostic (based on imputable methods). as a result, pharmacovigilance is a tool for specifically describing and optimizing a drug's benefit/risk ratio throughout its life cycle (2). the international conference of harmonization (ich) guidelines for good clinical practice (gcp) had defined adverse drug reactions as "any response to any medicinal product which was noxious and unintended and occurs at doses normally used in humans for prophylaxis, diagnosis or therapy of disease or the modification of physiological function which includes the terms out of marketing authorization such as off-label uses, overdose, misuse, abuse, and medication errors" (3). biological drugs are an effective treatment choice for a variety of diseases, especially those that are life-threatening. biological medicinal products have an active ingredient that was produced or derived from a biological source. these include medicinal substances derived from living-cells or organisms. “biologics include a wide variety of molecules, e.g., hormones, growth factors, interleukins, monoclonal antibodies, which differ in size and structural complexity (e.g., their molecular mass ranges from 5 kda for insulin to more than 150 kda for monoclonal antibodies)” (4). in most countries, the production and use of biological drugs is booming, as these drugs provide new therapeutic options for diseases for which drug therapy is restricted. they are a clinical innovation, but they also reflect an unknown environment with negative side effects and events that endanger patient safety (5). adverse events associated with these agents are typically attributable to an increase in documented pharmacologic activities, such as the risk of infections and malignancies, or to immunologic and infusion reactions, such as the production of antidrug antibodies as a result of the protein nature of these agents (6). many biologics, such as monoclonal antibodies, have a longer half-life and duration of action than small molecules. they are usually injectable medications that can cause mild cutaneous or hypersensitivity reactions. since biologics may trigger immune reactions including mild hypersensitivity, infusion reactions, and crossreactions with endogenous molecules, immunogenicity is a major safety concern. this may result in a loss of efficacy or deficiency syndromes (e.g., thrombocytopenia as a result of neutralizing antibodies blocking endogenous thrombopoietin after treatment with recombinant thrombopoietin or neutralizing antibodies with human growth hormone) (7). biological drugs can cause certain immune responses by producing “anti-drug” antibodies. specific adverse effects are related to several biological drugs due to their mechanism of action. this motivated the researchers to further evaluate the benefit/risk ratio of these drugs (8). biological drugs have specific characteristics that include a complicated manufacturing procedure, restricted evaluation of the preclinical to clinical data, and increased possibility of immunogenicity. for biological drugs, as well as for all drugs, pharmacovigilance is necessary to discover, detect, and characterize the adverse drug effects in the postmarketing safety profile due to the inherent limitations of clinical trials (9). the most prevalent suspected adverse drug reactions (adrs) for biologics, according to a vigibase study, were "infections and infestations," "surgical and medical procedures," and "neoplasms benign, malignant, and unspecified." (10). in the italian spontaneous reporting system, administration-site conditions, infections, and neoplasms were more likely to be diagnosed with biologics than with non-biologics8. because of the inclusion criteria and limited sample size in premarketing clinical trials, rare and unexpected adverse effects are difficult to identify. furthermore, biological drugs may be associated with adverse effects (aes) that are unrelated to their mechanism of action, such as the production of anti-drug antibodies (11). minor variability (microheterogeneity) is possible but must be kept within acceptable limits to ensure positive benefit-risk profiles in biologics generated with recombinant dna technology. even within or between batches of the same biologic, microheterogeneity can be observed, particularly when the manufacturing process is modified, as it may be during the drug's commercial lifespan. “natural variability is intrinsic to all biologics, and strict controls are often in place during processing to ensure that it does not impact the way the drug functions or its protection” according to the european medicines agency (ema)(4). the aim of the current study was to determine the safety profile of biological drugs used in iraqi public hospitals by evaluating the adrs that occur with these drugs (regarding their severity, seriousness, iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs 34 preventability, expectedness, and outcome) using the iraqi pharmacovigilance center (ipvc) database. materials and methods individual case safety reports (icsrs) submitted to the iraqi pharmacovigilance center / ministry of health are the subject of this review, which is a retrospective analysis (sent from 2009 to the end of 2020). before beginning the report, the iraqi ministry of health/department of research and development and the college of pharmacy/university of baghdad's scientific committees gave their approval. the data source is vigiflow – iraq. vigiflow is a database that belongs to uppsala monitoring center (umc) that is a who collaborating center for adrs from many national centers around the world (12). the inclusion criteria were to choose all the icsrs for biological drugs available in the vigiflow – iraq database. exclusion criteria were duplicated reports and non-relevant reports (medication error, and not mentioning the adrs). the icsrs included in the study were analyzed for demographic distribution, adrs classification, severity, expectedness, preventability, seriousness, and outcomes. age distribution was: neonate (4 weeks ), child (1-12 years), adolescent (13-18 years), adult (over 18 years), and elderly (over 65 years) (13). the adrs were listed using the system organ classification (soc), which categorizes adverse reactions according to the affected systemorgan (14). the severity was assessed using the modified hartwig and seigel severity scale (table 1) (15). it classified adrs into seven severity categories. levels 1 and 2 are considered mild, levels 3 and 4 are considered moderate, and levels 5, 6, and 7 are considered severe(16). the smpc for medicinal drugs, which is a primary reference guide that advises healthcare professionals on how to use the medicinal product safely and effectively, is used to assess the expectedness of adrs (17). as a result, adrs were categorized as “expected” if they were mentioned in the smpc and “unexpected” if they were not (18). the schumock and thornton criteria (table 2) were used to determine whether adrs could be prevented. this criterion has three parts in its updated form: preventable, probably preventable, and non-preventable. section a comprises five questions while section b has four questions. all the answers are categorized as “yes” or “no”. adrs were “definitely preventable” if the answer was “yes” to one or more questions in section a. if answers were all negative, then we proceeded to section b. adrs were “probably preventable” if the answer was “yes” to one or more questions in section b. if answers were all negative, then we proceeded to section c. in section c the adrs were non-preventable (19). the seriousness was determined using guidelines established by the national pharmacovigilance center or regional centers located in health directorates in iraq. these criteria are available in the icsr, which is a paper reporting format for all adrs encountered in iraqi hospitals. if the included seriousness in the icsr was correct, the seriousness was selected; otherwise, the seriousness was evaluated by the researcher (figure 1) (20). according to the who, the outcome of each icsr was registered and classified into one of the following categories: (1) recovered / resolved, (2) recovering /resolving, (3) not recovered/not resolved / ongoing, (4) recovered / recovered with sequelae, (5) fatal, and (6) unknown in the case of missing details (12). table 1. the modified hartwig and seigel severity scale (15). level of severity the criteria level 1 an adr occurred but required no change in treatment with the suspected drug level 2 the adr required that treatment with the suspected drug be held, discontinued, or otherwise changed. no antidote or other treatment requirement was required. no increase in los. level 3 the adr required that treatment with the suspected drug be held, discontinued, or otherwise changed. and/ or an antidote or other treatment was required. no increase in los level 4 any level 3 adr which increases length of stay by at least 1 day. or the adr was the reason for the admission. level 5 any level 4 adr which requires intensive medical care. level 6 the adverse reaction caused permanent harm to the patient. level 7 the adverse reaction either directly or indirectly led to the death of the patient. adr: adverse drug reaction; los: length of stay iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs 35 table 2. schumock and thornton preventability assessment criteria (19). no yes question section a: definitely preventable adr was there a history of allergy or previous reaction to the drug? 1 was the drug involved inappropriate for patient's clinical condition? 2 was the dose, route, or frequency of administration inappropriate for the patient's age, weight or disease state? 3 was a toxic serum concentration or a laboratory monitoring test documented? 4 was there a known treatment for adr? 5 section b: probably preventable adr was there any required therapeutic drug monitoring, or other laboratory tests not performed? 6 was a drug interaction involved in the adr? 7 was poor compliance involved in the adr? 8 were preventative measures not prescribed or administered to the patient? 9 section c: non-preventable adr if all the above criteria not fulfilled 10 adr: adverse drug reaction figure 1. seriousness assessment in the individual case safety report (20). statistical analysis the extracted data from the icsr reports was arranged in excel spreadsheets, then the parameters' criteria were applied, and the findings were displayed in bar charts, before being statistically analyzed with the “statistical package for the social sciences (spss) program” version 26. descriptive statistics were used to quantify the frequency and the percentage of each adr reported and the chi-square test ”to demonstrate the significance of the relations between gender and age group with the severity, seriousness and outcome of the adrs. the p-value for the data sets were calculated and the value of less than 0.05 was considered a statistically significant relation. results during the study period, a total of 899 adrs (from 446 icsr reports) were reported corresponding to 15 biological drugs. the icsrs analysis showed that reports for the female gender were more than the reports for the male gender (60.09% females’ reports versus 31.61% males’ reports, and 8.30% of the reports had no information for the gender). regarding age group, the icsrs were assessed to the following categories: adult 291(65.25%), unknown 110 (24.66%), elderly 28 (6.28%), child 9 (2.02 %), adolescent 7 (1.57 %) and neonate 1(0.22%), respectively. pharmacists were responsible for the majority of the reports, with 215 (48.21% of total reports) (table 3 ). regarding the frequencies of adrs and icsrs for each of the 15 biological drugs, the highest number adrs was for rituximab with 241 adrs (26.81%) while the highest number of icsrs was for interferon beta-1a with 112 reports (25.11%) (table 4). according to adrs classification based on the soc system. “it was found that most of the adrs were related to general disorders and administration site conditions with 200 adrs (22.25%), followed by skin and subcutaneous tissue disorders with 126 adrs (14.02%), respiratory, thoracic and mediastinal disorders with 114 (12.68%), and gastrointestinal disorders with 98 adrs (10.90%) as shown in (table 5).” do you consider the reaction to be serious? yes no if yes, please tick (✓) to indicate why the reaction is considered to be serious: the patient died due to the reaction involved or prolonged inpatient hospitalization life threatening involved persistent or significant disability or incapacity congenital anomaly medically significant, please give details: treatment given: no yes (please specify):----------------- iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs 36 table 3. age group, gender, and reporter qualification distribution of icsrs.” gender number of icsrs (%) female 268 (60.09 %) male 141(31.61%) n/a 37 (8.3%) age group number of icsrs (%) adult 291(65.25%) unknown 110 (24.66% elderly 28 (6.28%) child 9 (2.02 %) adolescent 7 (1.57 %) neonate 1( 0.22%) reporter qualification number of icsrs (%) pharmacist 215 ( 48.21%) consumer or other nonhealth professional 104 (23.32%) physician 68 (15.25%) other health professional 59 (13.23%) n/a : not available , icsr: individual case safety report table 4. icsrs and adrs for each of the biological drugs.” drug name adrs no. & (%) icsrs no. & (%) rituximab 241 (26.81%) 91 (20.4%) interferon beta-1a 200 (22.25%) 112 (25.11%) infliximab 113 (12.57%) 70 (15.7%) trastuzumab 101 (11.23%) 51 (11.43%) etanercept 70 (7.79%) 47 (10.54%) filgrastim 45 (5.01%) 25 (5.61%) bevacizumab 35 (3.89%) 14 (3.14%) bortezomib 29 (3.23%) 12 (2.69%) epoetin alfa 22 (2.45%) 12 (2.69%) interferon alfa 2b 20 (2.22%) 1 (0.22%) interferon beta1b 9 (1%) 5 (1.12%) natalizumab 6 (0.67%) 2 (0.45%) adalimumab 5 (0.56%) 2 (0.45%) peginterferon alfa 2a 2 (0.22%) 1 (0.22%) aflibercept 1 (0.11%) 1 (0.22%) total 899 (100%) 446 (100%) table 5. adrs classification based on the soc system. adrs in system organ classification number & (% ) of adr general disorders and administration site conditions 200 ( 22.25%) skin and subcutaneous tissue disorders 126 (14.02%) respiratory, thoracic and mediastinal disorders 114 (12.68%) gastrointestinal disorders 98 (10.90%) nervous system disorders 86 (9.57%) musculoskeletal and connective tissue disorders 48 (5.34%) immune system disorders 38 (4.23%) investigations (e.g., ejection fraction decreased) 30 (3.34%) infections and infestations 25 ( 2.78%) cardiac disorders 23(2.55%) eye disorders 20 (2.22 %) surgical and medical procedures 17 (1.89%) injury, poisoning and procedural complications 15 (1.67%) psychiatric disorders 13 (1.45%) renal and urinary disorders 12 (1.33%) neoplasms benign, malignant and unspecified (including cysts and polyps) 9 (1.00%) blood and lymphatic system disorders 8 (0.88 %) metabolism and nutrition disorders 8 (0.89%) hepatobiliary disorders 3 ( 0.33%) product issues (suspected counterfeit product) 2 (0.22%) social circumstances 2 (0.22%) ear and labyrinth disorders 1(0.11%) reproductive system and breast disorders 1 (0.11%) congenital , familial and genetic disorders 0 (0%) endocrine disorders 0 (0%) pregnancy, puerperium and perinatal conditions 0 (0%) regarding severity of adrs, the majority of adrs were observed in level 4 (26%), followed by level 3 (18%) and level 2 (13%) . with respect to expectedness of the adrs, the expected adrs represented 68% while the unexpected adrs counted for 32% of the total adrs. concerning preventability assessment of adrs, more than half (66%) of the adrs were probably preventable, 4% were preventable, and 30% were non-preventable. seriousness assessment showed that serious adrs account for the majority of the adrs with 52% of the adrs, while non-serious adrs were 46% for iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs 37 the encountered drugs. only 2% of the adrs were missing some data to assess their seriousness and are presented in the n/a (not available) category. outcome analysis of the adrs showed that in 44% of the cases the outcome was unknown, and most of the adrs were in the recovered category with 43% as illustrated in (table 6). the relation between the severity of the reported adrs to the gender of the patients were tested. it was found that there is a significant relation between the severity level of the adrs and the gender of the patients as shown in (table 7). the mild adrs were significantly higher in male patients, while moderate adrs were significantly higher in females. there is no significant difference in severe adrs between male and female. the relation between the severity of the adrs and the age of the patients were also tested. the severe adrs in patients below-18 age group was significantly higher and the mild adrs was significantly lower compared to adults and elderly (table 8). “rituximab was the drug for which the highest number of serious adrs was reported with 194 serious adr (41.28% of total serious adrs), followed by trastuzumab with 76 serious adr (16.17% of total serious adrs) and infliximab with 59 serious adr (12.55% of total serious adrs) as shown in (table 9).” table 6. severity levels, expectedness, preventability and outcome of adrs reported for biological drugs.” severity assessment number of adrs ( % ) level 1 80 (9%) level 2 117 (13%) level 3 164 (18%) level 4 236 (26%) level 5 11 (1%) level 6 10 (1%) level 7 37(4%) under assessment 244 (27%) expectedness number of adrs ( % ) expected 610 (68 %) unexpected 289 (32 %) preventability number of adrs ( % ) probably preventable 593 (66%) non-preventable 296 (30%) preventable 37 (4%) outcomes number of adrs ( % ) unknown 397(44%) recovered 383(43%) recovering 45(5%) not recovered / ongoing 39(4%) fatal 29(3%) recovered with sequelae 6(1%) table 7. severity levels distribution among gender.” severity / gender male female p-value mild count & % within gender 93 (41.52%) 92 (24%) 0.00000621* moderate count & % within gender 106 (47.32%) 263 (68.67%) 0.0000002* severe count & % within gender 25 (11.16%) 28 (7.31%) 0.10492778 *significant (p-value <0.05) according to chi square test. table 8. severity levels distribution among age groups. severity / age group below 18 adult elderly p-value mild count & % within age group 7 (12.5%) 160 (33.4%) 22 (33.33%) 0.006* moderate count & % within age group 36 (64.285%) 284 (59.29%) 40 (60.6%) 0.765 severe count & % within age group 13 (23.214%) 35 (7.3%) 4 (6.06%) 0.0002* *significant (p-value <0.05) according to chi square test. iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs 38 table 9. seriousness of the adrs. n/a no. & (%) no no. & (%) yes no. & (%) drug name 8 (53.33%) 39 (9.42%) 194 (41.28%) rituximab 3 (20.00%) 22 (5.31%) 76 (16.17%) trastuzumab 0 (0.00%) 54 (13.04%) 59 (12.55%) infliximab 0 (0.00%) 32 (7.73%) 38 (8.09%) etanercept 2 (13.33%) 5 (1.21%) 22 (4.68%) bortezomib 0 (0.00%) 16 (3.86%) 19 (4.04%) bevacizumab 0 (0.00%) 26 (6.28%) 19 (4.04%) filgrastim 1 (6.67%) 185 (44.69%) 14 (2.98%) interferon beta-1a 0 (0.00%) 9 (2.17%) 13 (2.77%) epoetin alfa 0 (0.00%) 14 (3.38%) 6 (1.28%) interferon alfa 2b 1 (6.67%) 1 (0.24%) 4 (0.85%) natalizumab 0 (0.00%) 6 (1.45%) 3 (0.64%) interferon beta1b 0 (0.00%) 0 (0.00%) 2 (0.43%) peginterferon alfa 2a 0 (0.00%) 4 (0.97%) 1 (0.21%) adalimumab 0 (0.00%) 1 (0.24%) 0 (0.00%) aflibercept 15 (1.67% of total adrs)) 414 (46.05% of total adrs) 470 (52.28 % of total adrs) total n/a : not available discussion awareness about clinical information of side effects secondary to biologic agents will improve the use of biologic agents and improve outcomes of patients. biological drugs require special pharmacovigilance” considerations, and more regular monitoring to ensure their efficacy and safety (21). biological drugs are more commonly associated with adverse events than synthetic drugs (approximately 20% of existing drugs are biological drugs), some of which are serious and even lethal (22, 23). according to the findings of this research, adrs associated with biological drugs were more prevalent among female patients. a similar trend in results was recorded in previous studies such as a study conducted in spain and found that 82.9% of adrs were reported in females (24). another research in the united states discovered that females recorded 75.5 % of adrs (25), and in italy, studies found that 54.3% (26) and 71.3% (8) of adrs were associated with females. females are at an elevated risk for a variety of causes, including gender-related variations in pharmacokinetics, immunological, and hormonal factors, as well as differences in drug usage by females versus males (27). analyzing the adrs of biological drugs with the consideration of patients' age showed that the main age group in the reported adrs was in adults followed by the elderly. this may be due that the use of biologics in the pediatric population is still limited because of unknown long‑term safety profile and absence of large‑scale studies (28). the results also revealed that the reporters of the adrs were primarily pharmacists which specifies that the “pharmacovigilance” responsibility in the healthcare facilities is more carried by pharmacists. among the biological drugs related to the adrs encountered in the present study, rituximab had 241 adrs (26.81% of total adrs) which probably because rituximab is one of the important drugs that is used in combination with chemotherapy and it is more effective as a firstor second-line therapy than chemotherapy alone in providing tumor remission and patient survival in the treatment of nonhodgkin’s lymphoma (nhl), chronic lymphocytic leukemia (cll). also, it is frequently used for treating resistant and special cases of moderately to severely active rheumatoid arthritis (ra) which is prevalent in about 1% of the iraqi population (29). “it is worth mentioning that the most reported adrs in the present study were related to general disorders and administration site conditions, followed by skin and subcutaneous tissue disorders, respiratory, thoracic, and mediastinal disorders, and gastrointestinal disorders.” while most adrs recorded in other different studies were related to infections (25, 26, 30, 31), general disorders and administration site conditions(32-34), and the skin or iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs 39 subcutaneous tissues(8, 32-35). injection site reactions (isrs) are a local phenomenon defined as a constellation of symptoms, including swelling, erythema, pruritus, and pain around the site of injection. isrs are major complications of all fdaapproved injectable biological agents, both in adults and children, with studies showing an incidence rate of 0.5-40% (36). inappropriate injection techniques, injection close to blood vessels, the chemical and physical properties of the injected drug and a reaction to the vehicle component are probable causes for the irritative reactions (37) which represent general disorders and administration site conditions. regarding the severity of adrs reported in the current study, the majority of the adrs were classified as moderate. level 4 severity accounts for 26% of adrs and level 3 in 18% of the adrs (table 4). this type of severity necessitates a change in the drug therapy, specific treatment, or an increase in hospitalization by at least 1 day (38). due to intervention, the majority of adrs were of moderate severity (physicians were controlled the adrs by discontinuing the offending medication. in other cases, clinical treatments were implemented using antihistamines, corticosteroids, and antidotes to relieve symptoms). the relation between the severity of the reported adrs in this study to the gender of the patients was assessed too, and it was found that there is a significant relationship between the severity level of the adrs and the gender of the patients (table 5). the risk for developing moderate adrs was more significant in females than in males (moderate adrs in females correspond to 68.67% of total adrs, while in males it was 47.32%). a pharmacological explanation for this may be due to lower body size, weight in females, in addition to change in absorption, protein binding, and the volume of distribution, clearance, and metabolism of drugs as well as gender-specific hormones(39). regarding relation between the severity of adrs and age of the patients, the severe adrs in patients below-18 years ago were significantly higher and the mild adrs were significantly lower compared to adults and elderly. adverse drug reactions in children can have a relatively more severe effect when compared to adults. thus, the adrs can lead to significant morbidity among children(40). most of the adrs were probably preventable (66%) due to lacking data that makes it hard to assume whether it was preventable or nonpreventable. the findings were like those of another study that looked at the preventability of adrs in four south african hospitals(41), and with a metaanalysis study which also found that approximately half of the adverse drug reactions are preventable(42). just a small percentage of adrs can be avoided, according to some studies (43,44). the high proportion of expected adrs (610 adr that counts for 68 % of total adrs) found in the present study is an anticipated finding since most biologicals’ adverse reactions were labeled and identified in the drug description. as a result, the adrs identified in this study were not mentioned in a significant number of smpcs for suspected drugs. furthermore, the adrs mentioned in the smpcs are not always identified using meddra's exact pt terminology (38). because of this, determining the expectedness of adrs reported in pharmacovigilance databases is difficult. the outcome for biologicals’ adrs was mostly recovered/resolved with 43% of adrs. the data of adrs outcome were missed in 44% of icsrs (table 4). the adrs caused by biological drugs are mainly treatable. premedication with corticosteroids, antihistamines, analgesics, and/or slower infusion rates are usually used to treat these forms of reactions (45). regarding adrs seriousness (i.e., fatal, leading to hospitalization, lifethreatening), it was found that 52.28% of total adrs were serious and 46% were nonserious (table 9). this result may be due to the fact that most biological drugs are used to treat extreme and/or lifethreatening diseases, causing the reporting of reported adrs to switch to more serious adverse events. rituximab yielded the highest percentage of serious adrs (41.28% of total serious adrs). rituximab is a monoclonal antibody to the b-cell marker cd20 that is commonly used (as a single agent and in combination therapy) to treat b-cell lymphoma, lymphoproliferative disorders, and inflammatory conditions that are resistant to standard care, such as rheumatoid arthritis and certain vasculitis. however, a growing number of severe adverse effects are being linked to the use of rituximab, with many patients requiring admission to an intensive care unit (46). cd20-expressing b-cell precursors and mature b cells are rapidly depleted by this drug, and their numbers remain low for 6 to 9 months. many studies have tried to assess immune-mediated consequences in those treated with rituximab because of its peripheral b-cell–depleting effects (47). finally, there are certain drawbacks to the current research that must be accepted. the involvement of confusing causes, such as underlying medical conditions and concomitant medications. aside from the fact that a significant percentage of studies omit critical facts. all of this makes it difficult to determine whether a medication induced a particular adr in several cases, necessitating the development of educational programs to improve the reporting system. conclusions the highest number of adrs was reported for rituximab. most of the reported adrs were of moderate severity, expected, serious, probably preventable and with unknown outcomes. the missing information (especially those related to the outcomes) in the icsrs reports had a clear negative impact on the assessment of the reports which iraqi j pharm sci, vol.31(1) 2022 safety profile of biological drugs 40 necessitates the development of educational programs 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drugs 42 44. rydberg dm, holm l, engqvist i, fryckstedt j, lindh jd, stiller c-o, et al. adverse drug reactions in a tertiary care emergency medicine ward-prevalence, preventability and reporting. plos one. 2016;11(9):1-14. 45. cheifetz a, smedley m, martin s, et al. the incidence and management of infusion reactions to infliximab: a large center\experience. am j gastroenterol 2003;98:1315–24. 46. kasi pm, tawbi ha, oddis cv, kulkarni hs. clinical review: serious adverse events associated with the use of rituximab-a critical care perspective. critical care. 2012 ;16(4):1-0. 47. patel sv, khan da. adverse reactions to biologic therapy. immunol allergy clin north am. 2017;37(2):397-412. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.24(2) 2015 the effect of l-carnitine on the lipid profile 48 the effect of l-carnitine as an adjuvant supplement on lipid profile in iraqi diabetic patients wessam m. shiblawi *,1 and sajida h.ismael ** * ministry of health ,al zahraa hospital, diyala health directory, diyala ,iraq. ** department pharmacology and toxicology, college of pharmacy, university of baghdad,baghdad,iraq. abstract diabetes is a complex set of diseases require continuous medical care, to control blood sugar and prevent complications is .the aim of this research is to determine the effect of administration of l carentin to diabetics on the lipid profile. the research was conducted on sixty diabetic patients were selected from endocrinology and diabetes center / al-rusafa, within selected criteria. the patients divided into 3 groups (control group of healthy people and two groups of patients with diabetes who were on metformin and glibenclamide, one group took a l carnitine in a dose of 1000 mg twice daily and a group dealing with a placebo for a period of 3 months continuously). the study found that patients who took lcarnitine, showed a significant reduction (p <0.05) in the triglyceride level, while no significant changes were observed in the level of cholesterol and hdl and ldl. this study concluded that administration of l carentin improved the lipid profile in type-2diabetic patients. key word: diabetes mellitus (dm), dyslipidemia, l-carnitine (lc). تأثير مكمالث الكارنتين اليساري على صورة شحوم الدم عند مرضى السكري فً العراق احمد شبالوي م محمداوس ,*1 و ساجدة حسين اسماعيل ** * الؼزاق.دَالً ،، ،وسارة الصحت الشهزاء،هسخشفً ** ، الؼزاق.جاهؼت بغذاد،كلُت الصُذلت، فزع األدوَت والسوىم الخالصة اى هزض السكزٌ هى هجوىػت هؼقذة هي االهزاض ححخاج لؼٌاَت طبُت هسخوزة لضبظ سكز الذم و هٌغ حؼقُذاث الوزض الكارًخُي الُسارٌ لوزضً السكز ػلً صىرة شحىم الذم. حن اجزاء البحث ػلً سخُي .الهذف هي البحث هى هؼزفت حأثُز اػطاء هجاهُغ 3هزَضا حن اخخُارهن هي هزكش الغذد الصن و السكزٌ/الزصافت,ضوي هؼُارَت هحذدة فٍ الذراست.حن حقسُن الوزضً الً ىى ػالج الكلبٌكلُوُاَذ و الوُخفىرهُي , حٌاولج )هجوىػت سُطزة هي االشخاص االصحاء و هجوىػخُي هي هزضً السكزٌ هوي َخٌاول اشهز هخىاصلت(. حىصلج 3هلغن هزحاى َىهُا و هجوىػت حخٌاول ػالج وهوٍ لوذة 0111هجوىػت هٌهن كارًخُي َسارٌ بجزػت ذهىى الثالثُت بٌُوا ( فٍ هسخىي الp<0.05الذراست الً اى الوزضً الذَي الذَي حٌاولىا كارًخُي َسارٌ, لىحظ ػٌذهن حغُزاث هؼٌىَت ) حذثج حغُزاث غُز هؼٌىَت فٍ هسخىي الكىلُسخزول و البزوحُي الذهٌٍ الؼالٍ و الىاطئ الكثافت.هذٍ الذراست اسخٌخجج اى اػطاء .الكارًخُي الُسارٌ َفُذ فٍ حٌظُن شحىم الذم الوضطزب ػٌذ هزضً السكزٌ ،الكارنتين اليساري. اضطراب شحوم الدم مرض السكري ،الكلماث المفتاحيت : introduction diabetes mellitus (dm) is a global health issue affecting children, adolescents, and adults. according to the world health organization, approximately 180 million people worldwide currently have type 2 dm (formerly called adult-onset diabetes); over 95% of people with diabetes have this form (1) . world health organization (who) has recently proposed new diagnostic criteria and classification of diabetes mellitus. a major change in diagnostic criteria is lowering of diagnostic fasting plasma glucose level to less than 7mm/l (2) . it is associated with long-term damage, dysfunction, and failure of different organs (3) , diabetes is associated with both micro vascular and macro vascular diseases affecting several organs, including muscle, skin, heart, brain, and kidneys (4) . the terms type 1 and type 2 are used for classification based on etiology. the terms insulindependent and non-insulin dependent are used for pathophysiological staging of diabetes mellitus regardless of the etiology (5) . 1 corresponding author e-mail: wisam.sheblawy@ yahoo.com received: 4/2/ 2015 accepted: 10/11/2015 iraqi j pharm sci, vol.24(2) 2015 the effect of l-carnitine on the lipid profile 49 the prevalence of diabetes for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030. the number of people with diabetes is increasing due to population growth, aging (6) , urbanization (7) , and increasing prevalence of obesity and physical inactivity. l-carnitine is a natural nutrient cofactor required for transport of long-chain fatty acids into the mitochondria, where they undergo beta-oxidation to produce adenosine triphosphate for cellular energy production which is primary fuel source for proper function in many tissues and prevent the toxic accumulation of long-chain fatty acids (8) . role of lcarnitine in the t2dm patients with type 2 diabetes seem to be at elevated risk for carnitine deficiency (9) . lcarnitine short-circuit the randle cycle by sequestering inhibitory acetyl-coa units as acetyl-carnitine and concomitantly increasing free coa levels. lowering of the mitochondrial acetyl-coa: coa ratio would then favor glucose oxidation (16) , l-carnitine mediated sequestering of toxic lipid metabolites may have benefited both mitochondrial performance and insulin signaling (10) , also l-carnitine can play a role in the treatment of type 2diabetics by improving insulin resistance that is caused by post-receptors defect, this means that lcarnitine may be useful for cell membrane repairing and, removal of harmful lipid from the cells may improve or decrease the resistance to insulin action by photoreceptor defect either at the membrane or intracellular level (11) , administration of l-carnitine may shift the metabolic bias of the liver away from esterification and synthesis of triglycerides toward the formation of acetylcarnitines. this could decrease synthesis of triglycerides and vldl cholesterol and likely increase mitochondrial β-oxidation of fatty acids (12) . subjects, materials and methods this study was carried out at the specialized center of endocrinology and diabetes-al-risafa directorate of healthbaghdad. the study was conducted on 60 iraqi subjects 41 male and19 female with age range 40-64 years in nonrandomized method, all volunteers follow inclusion and exclusion criteria. the inclusion and exclusion criteria for volunteers the inclusion criteria for healthy subjects to be free from other chronic disease or drugs, while diabetic patients to be poor controlled type 2dm for at least 5years and more, on metformin and glibenclamide therapy, and had lipid profile disorder, while this study exclude the pregnant, breast feeding or on contraceptive and postmenopausal women, also those with liver disease , kidney disease, epileptic disease, thyroid disease, smokers, and alcohol drinkers also must have no infection or on anti-biotic or any other drug has interaction with l-carnitine. the volunteers are divided into three groups as follows: group (1): includes 20 apparently healthy subjects (16 male and 4 females) as control. group (2): include 20 diabetic patients 10 male and 10 female; this group was taken lcarnitine (1000 mg) tablets two times daily for three months. group (3): includes 20 diabetic patients (15male and 5 female) were treated with placebo for three months. blood samples were taken from all individuals included in this study, at base line time and every 30 days of the study period, blood was collected by venipuncture technique in order to measure serum lipid profile. statistical analysis was performed by using unpaired student ttest between healthy individuals and diabetic patient's ( weather placebo group or on l-carnitine group), and paired student ttest between zero time , after 1st, 2nd and 3rd month in all groups involved in this study. results table(1) shows the effect of lcarnitine on the lipid profile, involving total cholesterol (tc), triglyceride (tg) ,high density lipoprotein (hdl)and low density lipoprotein(ldl) .the comparison between control group with diabetics groups showed a significant difference in the tc,tg and ldl while hdl nonsignificantly affected. concerning the tc, the outcome of trial showed nonsignificant changes observed in the treated group as well as placebo during study period ,meanwhile the data expressed a significant reduction of serum tg from 1 st month in comparison with baseline value, at ( p <0.05) and highly significant at the 2 nd month and 3 rd month respectively. for high density lipoprotein (hdl) the table shows nonsignificant also low density lipoprotein (ldl) showed non-significant changes at the 3 month of the study period weather in treated group or placebo. iraqi j pharm sci, vol.24(2) 2015 the effect of l-carnitine on the lipid profile 50 table (1) the effect of treatment with l-carnitine on lipid profile in t2dm patient (n=20). a,b,c represent statistically significant change (p <0.05) for comparison between healthy group and diabetics ( on placebo or on l-carnitine). * represent significant change (p value<0.05) for patients comparison between pre and post treatment values of treated groups. n= number of individuals. discussion the current study demonstrated a non-significant decline in total cholesterol, the serum hdl-c (high density lipoprotein cholesterol) and ldl-c (low density lipoprotein cholesterol) levels in the treated group after one, two and three months of treatment in comparison with zero time readings, and this may be due to a fact that lcarnitine don’t have direct potent effect on the cholesterol synthesis pathway, or the period of trial not sufficient to observe such a change this observation was consistent with that obtained by gonzalez-ortiz(2008) (12) , golbidi (2011) (13) and roberto(2012) (15) , however, these findings disagree with other studies who observed positive effects of l-carnitine supplementation on total cholesterol (14,16,17) , while irat et al. (2003) suggested that the beneficial effects of l-carnitine treatment partially improve vascular reactivity and antioxidant property beyond its reduction of plasma lipids and it may have an important therapeutic approach in the treatment of diabetic vascular complications (18) also it consider a good adjuvant therapy beside cholesterol-lowering drugs (statin) for its mechanism that reverse the myopathy which is possible side effect of cholesterol lowering drugs and potentiated statin effect (19) also the lc may has an qualitative effect on hdl rather than increase level of the former through improve the integrity that carries important antioxidant enzymes( paroxanase and platelet activating factor acetyl hydrolase) which serves to protect from oxidation and increase half-life of hdl (20) while another study shown lc caused a significant twofold increase in α-tocopherol(vitamin e) content in oxidized ldl and caused a reduction in the level of conjugated dienes, lipid hydro peroxide, malondialdehyde, and dityrosine (21) . the present study found a successful improvement and significant decline in triglyceride level in the treated group in comparison with base line readings, and this may be due to the l-carnitine effects on ffa metabolism, glucose hemostasis, improvement in insulin sensitivity, down-regulated enzymes essential in glycolipid biosynthesis and were up-regulated enzymes involved in fatty acid catabolism (22) or its role in increase fa consumption through increase physical activity. this outcome of the present study disagrees with other studies which find no such difference in tg level after taking lcarnitine (22-24) .while other studies agree with this study outcome (25) . conclusion the present study was conclude that administration of l-carnitine as adjuvant supplement at a dose (1000mg) twice daily for 3 successive months had a benefit effect on the triglyceride level in the t2dm with dyslipidemia meanwhile non-significant changes were observed on the levels of tc,hdl,ldl . references 1. alberti kg 1 , zimmet pz. definition, diagnosis and classification of diabetes mellitus and its complications. part 1: diagnosis and classification of diabetes mellitus provisional report of a who consultation. diabet med. 1998 jul; 15(7):539-53. parameter groups regime total cholesterol mg/dl triglyceride mg/dl hdl mg/dl ldl mg/dl control baseline 183.5±33.1 * 154.7±41.1 * 40.90±5.95 85.7±13.5 a placebo baseline 208.9±30.8 b 173.6 ±49.7 b 39.90±8.23 127.4±27.1 b 1month 206.69±32.5 176.7±51.7 38.9± 5.38 127.3±26.1 2month 208.4±39 177.6 ±49.2 39.98±7.58 126.5 ±25.6 3month 207.37± 21.2 173.2±35.6 40.01±5.25 124.9±17.2 treatment with lcarnitine baseline 201.8±24.1 b 174.6±38.9 b 40.05± 6.6 120.1±22.6 b 1month 202.1 25.9 164.3±30.9 * 39.9 ±5.29 122.1±23.7 2month 201.7±22.4 158± 28.1 * 40.01±5.12 121.2±20.5 3month 200.7 ± 27.4 147.9±26.6* 40.1 ±4.96 120.2±21.2 http://www.ncbi.nlm.nih.gov/pubmed/?term=alberti%20kg%5bauthor%5d&cauthor=true&cauthor_uid=9686693 http://www.ncbi.nlm.nih.gov/pubmed/?term=zimmet%20pz%5bauthor%5d&cauthor=true&cauthor_uid=9686693 http://www.ncbi.nlm.nih.gov/pubmed/9686693 iraqi j pharm sci, vol.24(2) 2015 the effect of l-carnitine on the lipid profile 51 2. metelko z 1 , pavlić-renar i, tomić m, bratanić n. new diagnostic criteria and classification of diabetes mellitus]. lijec vjesn. 2000 may-jun; 122(5-6):99102. 3. kuzuya t1, nakagawa s, satoh j, et al report of the committee on the classification and diagnostic criteria of diabetes mellitus. diabetes res clin pract. 2002 jan; 55(1):65-85. 4. wt cade, pt, phd. diabetes-related micro vascular and macro vascular diseases in the physical therapy setting. phys ther. 2008 nov; 88(11): 1322–1335. 5. ramachandran a, snehalatha c, latha e, et al impacts of urbanization on the lifestyle and on the prevalence of diabetes in native asian indian population. diabetes res clin pract1999; 44: 207– 213. 6. inazu m1, matsumiya t. physiological functions of carnitine and carnitine transporters in the central nervous system. diabetalogia, 2008 jun; 28(3):113-20. 7. cave mc, hurt rt, frazier th et al: obesity, inflammation, and the potential application of pharmaconutrition. nutr clin pract 2008;23:16-34 8. uziel g, garavaglia b, di donato s carnitine stimulation of pyruvate dehydrogenase complex (pdhc) in isolated human skeletal muscle mitochondria, muscle nerve. j biol chem 1998; 11:720–724. 9. koves tr, li p, an j et al. peroxisome proliferator-activated receptor-gamma coactivator 1alpha-mediated metabolic remodeling of skeletal myocytes mimics exercise training and reverses lipidinduced mitochondrial inefficiency. j biol chem 2005; 280:33588– 33598 10. gonzalez-ortiz, hernandez-gonzalez., hernandez-salazar. effect of oral lcarnitine administration on insulin sensitivity and lipid profile in type 2 diabetes mellitus patients. ann nutr metab, 2008; 52(4): 335-338. 11. golbidi, s., ebadi, s.a., & laher, i. (2011). antioxidants in the treatment of diabetes. curr diabetes rev, 2011; 2: 106125. 12. beitullah alipour ; ali barzegar 1; farid panahi et al effect of l-carnitine supplementation on metabolic status in obese diabetic women with hypo caloric diet. health scope, 2014 winter; 2(4):14615. 13. roberto barbosa bazottei; gisele lopesbertolini. effects of oral l-carnitine and dl-carnitine supplementation on alloxandiabetic rat's 2012 braz. arch. biol. technol. 55 (1) : 1516-8913. 14. m. malaguarnera, m. vacante, t. avitabile, l. cammalleri and m. motta, “l-carnitine supplementation reduces oxidized ldl cholesterol in patients with diabetes,” the american journal of clinical nutrition, 2009; 89(1): 2009, 245251. 15. james wa, maya sg, jan t, et al: antioxidant supplementation effects on ldl oxidation for individual with type 2 diabetes mellitus. journal of american college of nutrition.1999; 18:4514. 16. irat am, aktan f, ozansoy g. effects of l-carnitine treatment on oxidant/antioxidant state and vascular reactivity of streptozotocindiabetic rat aorta. j pharm pharmacol 2003; 55: 1389– 1395. 17. arduini a, peschechera a, giannessi f. improvement of statin-associated myotoxicity by l-carnitine. j thrombhaemost 2004; 2: 2270–1. 18. argani h, rahbaninoubar m, ghorbanihagjo a,. effects of l-carnitine on serum lipoproteins and hdl-c subclasses in hemodialysis patients. nephron clin pract 2005;101:174-180 19. agnieszka augustyniak, anna stankiewicz et al .the influence of lcarnitine on oxidative modification of ldl in vitro 2008,toxicology mechanisms and methods 2008;18(6 ):455-462. 20. rahbar ar, shakerhosseini r, saadat n. effect of l-carnitine on plasma glycemic and lipidemic profile in patients with type ii diabetes mellitus, eur j clin nutr. 2005; 59:592–96. 21. rahbar ar1, shakerhosseini r, saadat n, et al. effect of l-carnitine on plasma glycemic and lipidemic profile in patients with type ii diabetes mellitus. eur j clin nutr. 2005 apr;59(4):592-6. 22. vidal-casariego a1, burgos-peláez r, martínez-faedo c et al. metabolic effects of l-carnitine on type 2 diabetes mellitus: systematic review and meta-analysis. exp clin endocrinol diabetes. 2013 apr; 121(4):234-8. 23. beitullah alipour ; ali barzegar ; farid panahi et al effect of l-carnitine supplementation on metabolic status in obese diabetic women with hypocaloric diet. health scope. 2014 february; 3(1): e14615. http://www.ncbi.nlm.nih.gov/pubmed/?term=metelko%20z%5bauthor%5d&cauthor=true&cauthor_uid=11040530 http://www.ncbi.nlm.nih.gov/pubmed/?term=pavli%c4%87-renar%20i%5bauthor%5d&cauthor=true&cauthor_uid=11040530 http://www.ncbi.nlm.nih.gov/pubmed/?term=tomi%c4%87%20m%5bauthor%5d&cauthor=true&cauthor_uid=11040530 http://www.ncbi.nlm.nih.gov/pubmed/?term=tomi%c4%87%20m%5bauthor%5d&cauthor=true&cauthor_uid=11040530 http://www.ncbi.nlm.nih.gov/pubmed/?term=bratani%c4%87%20n%5bauthor%5d&cauthor=true&cauthor_uid=11040530 http://www.ncbi.nlm.nih.gov/pubmed/11040530 iraqi j pharm sci, vol.24(2) 2015 the effect of l-carnitine on the lipid profile 52 24. b. parizadian, m. shams shargh and s. zerehdaran. study the effects of different levels of energy and l-carnitine on meat quality and serum lipids of japanese quail. asian journal of animal and veterinary advances, 2011; 6: 944-952. 25. janine keller, robert ringseis, steffen priebe et al effect of l-carnitine on the hepatic transcript profile in piglets as animal model. nutrition and metabolism 2011; 8:76. . iraqi j pharm sci, vol.23(2) 2014 oxidative stress in cholelihtiasis 57 association of oxidative stress markers with cholelithiasis omer s.sadiem *,1 , mohamed a. taher ** and seenaa s. amin ** * department of clinical laboratory sciences, college of pharmacy, tikrit university, tikrit ,iraq. ** department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad , iraq. abstract oxidative stress markers are of important diagnostic parameters for many disorders including cholelithiasis. this present study has aimed to assess the state of oxidative stress in symptomatic radiographically confirmed (cholelithiasis) patients by measuring two parameters used as oxidative stress parameters which are serum myeloperoxidase (mpo) and superoxide dismutase (sod). this study was carried out on 100 patient diagnosed as (cholelithiasis) patients with 30 age and sex matched healthy controls by measuring serum (mpo) and (sod) by eliza technique .results showed significantly decrease in antioxidant enzyme(sod) and increase in serum level of (mpo) comparing with controls. keywords: cholelithiasis , oxidative stress, mpo, sod. عىامل االجهاد التأكسدي مع حصىة المرارة مصاحبت عمرصالح صديم *،1 ، محمد عباس طاهر ** و سيناء صادق امين ** * . فشع انعهىو انًخخبشَت انسشَشَت ، كهُت انصُذنت ، جايعت حكشَج ،حكشَج ،انعشاق ** . فشع انعهىو انًخخبشَت انسشَشَت ، كهُت انصُذنت ، جايعت بغذاد ،بغذاد ،انعشاق الخالصة انبُاناث انًىجىدة عهً (. حصىة انًشاسة)عىايم االجهاد انخاكسذٌ هٍ يؤششاث قىَت نهعذَذ ين انحاالث انًشظُت بًا فٍ رنك نزنك هذفج انذساست انحانُت انً حقُُى حانت االجهاد . ونكن يحذودة انعالقت بُن حصىة انًشاسة وعالياث االجهاد انخاكسذٌ يحفزة (mpo & sod انخاكسذٌ نذي يشظً حعىة انًشاسة انًشخصُن باالشعت انسُنُت ين خالل عايهُن ين عىايم االجهاد انخاكسذٌ ين االصحاء كًجًىعت سُطشة يطابقت نًجًىعت انًشظً فٍ 30يشَط يشخصُن بحصىة انًشاسة يع 100شًهج انذساست . ) بانًقاسنت يع يجًىعت mpoيع اسحفاع يعنىٌ بًسخىي sodواظهشث اننخائج انخفاض يعنىٌ بًسخىي انزَى , انعًش وانجنس . انسُطشة . mpo ،sodجهاد التأكسدي ،حصىة المرارة ، اال: الكلماث المفتاحيت introduction cholelithiasis is a worldwide disease and it remains to be one of the most common health problems leading to surgical intervention (1,2) . cholelithiasisis is common with the incidence ranging from (10-20)% of the world population (3) ,the prevalence of cholelitheasis varies considerably within population; the highest known prevalence is among american indians with up to (60-70) % in females (4) and (10-15) % in white adults of developed countries (5) .cholelithiasis is classified as either cholesterol stones (cs), pigment stones (ps), or mixed stones (ms) (6) .these categories of gallstones can be identified according to their predominant chemical composition (7) . two thirds of individuals with cholelithiasis is asymptomatic (8) . in these patients, the reasons why symptoms develop are unknown (9) .of these, symptoms will develop in 1% to 4% per year (10, 11) .oxidative stress (os) is a disturbance in the oxidant-antioxidant balance leading to potential cellular damage. most cells can tolerate a mild degree of oxidative stress, because they have sufficient antioxidant defense capacity and repair systems, which recognize and remove molecules damaged by oxidation .the imbalance can result from a lack of antioxidant capacity caused by disturbances in production and distribution, or by an overabundance of reactive oxygen species (ros) from other factors (12) .increased levels of an accelerated generation of reactive oxygen species and toxic degradation products of lipid peroxidation have been reported in the serum of individuals with cholelitheasis (13) .it is well known that, in chronic diseases such as cholelithiasis, the active inflammatory response is induced with neutrophilic infiltration. these neutrophils, macrophages and/or monocytes produce ros which may cause dna damage to the adjacent cells (14, 15) . superoxide dismutase or sod is an enzyme that in humans is encoded by the sod1 gene, located on chromosome 21. sod is one of three human superoxide dismutase (16, 17) . superoxide dismutase (sod) is an enzyme that in humans is encoded by the sod1 gene, located on chromosome 21. sod is one of three human superoxide dismutase (16, 17) . 1 corresponding author e-mail: omer s. sadiem: omer_salah82@gmail.com received:26 /1/2014 accepted: 12/10/2014 mailto:omer_salah82@gmail.com iraqi j pharm sci, vol.23(2) 2014 oxidative stress in cholelihtiasis 58 myeloperoxidase (mpo) is a peroxidase enzyme that in humans is encoded by the mpo gene (18) myeloperoxidase is most abundantly expressed in neutrophil granulocytes (a subtype of white blood cells). it is a lysosomal protein stored in azurophilic granules of the neutrophil. mpo has a heme pigment, which causes its green color in secretions rich in neutrophils, such as pus and some forms of mucus (19) . gallstones and kidney stones are diseases with high prevalence (20) . there has been a marked increase in the prevalence and incidence of these stones within the last 22 years (21) . in spite of different composition and different locations of their formation both types of stones may be associated (22) .obesity, insulin resistance and women with postmenopausal hormone u se are at risk factors for renal stones as well as gallstones (23, 24) . materials and methods subjects this study was conducted at tikrit teaching hospital, tikrit, iraq, and the laboratory investigations were done at tikrit university, department of central laboratories. it is curried from december 2012 to june 2013. a total of 100 patients(group a) were included in the study, among which 68 were females patients and 32 were males in the age group of (20-80) years and mean age (48.38 ± 1.17) , to compare the results of 30 (age and sex matched) healthy controls(group b) (18 females, 12 males) with mean age (47.16 ± 1.5) years were also included. patient's including criteria: all patients were scanned for presence of gallstone by an abdominal ultrasonography (the standard diagnostic test for gallstone), and to confirm the absence of kidney stone. patient's exclusion criteria: 1. detection complicated gallstone diseases like (acute cholecystitis, pancreatitis). 2. patients with liver cirrhosis, viral hepatitis, renal failure and thyroid diseases and any type of cancer. 3. patients with a history of alcohol consumption and smoking. 4. subjects suffered from diseases like asthma. questionnaire subjects were asked to complete a questionnaire that asked for information on name, sex, age, marital state, number of children, pregnancy (for females only), family history of gs ,chronic diseases, urinary tract complications, weight, height, occupation, residence, smoking habits, alcohol consumption, and drugs used. ultrasound for patients with cholelithiasis and nephrolithiasis were checked to confirm the presence of calculi, number of stones and their average diameter. all the laboratory investigations were done in tikrit university, central laboratories department. sample collection after 12 hours of fasting, venous blood samples, about 10 ml were collected from patients before laparoscopic cholecystectomy and from healthy volunteers in plain tubes. after allowing the blood to clot at room temperature for about 15-30 min, blood samples were centrifuged at 3000 rpm for about 15-30 min to obtain serum. the serum were separated in multiple epiendroff tubes (0.5 ml) to prevent thawing of the serum frequently, the serum was stored and frozen at -20ºc until analysis was performed. clinical and laboratory evaluation 1. determination of myeloperoxidase (mpo): the test is based on the immobilisation of highly purified mpo to the solid phase of microtiter strips and subsequent binding of anti-mpo antibodies from patient serum by using elisa technique . the bound antibodies are detected with a peroxidaselabeled secondary antibody that is directed against human igg. after addition of substrate solution, a color appears which intensity is proportional to the concentration and/or the avidity of the detected antibodies. following the addition of stop solution, the colour switches from blue to yellow. the concentration of mpo in the samples is then determined by comparing the optical density of the samples to the standard curve. (25) 2. determination of human superoxide dismutase [cu-zn] (sod1): this assay employs the quantitative sandwich enzyme immunoassay technique (elisa). antibody specific for sod1 has been pre-coated onto a microplate. standards and samples are pipetted into the wells and any sod1 present is bound by the immobilized antibody. after removing any unbound substances, a biotin-conjugated antibody specific for sod1 is added to the wells. after washing, avidin conjugated horseradish peroxidase (hrp) is added to the wells. following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of sod1 bound in the initial step. the color development is stopped and the intensity of the color is measured (26) . iraqi j pharm sci, vol.23(2) 2014 oxidative stress in cholelihtiasis 59 statistical analysis the results were expressed as mean ± standard error of mean (sem). student's t-test was used to examine the degree of significance. p values less than 0.05 was considered significant.the statistical analysis was performed using the statistical package for social sciences (spss 17). table (1): demographic presentation of cholelithiasis patients and control. parameter control n =30 patient n = 100 pvalue age (mean± sd) 48.38 ± 1.17 47.16 ± 1.5 0.724 f : m 18:12 (60%) 32:68 (68%) 0.300 pregnancy 2 (6.6%) 4 (5.8%) 0.485 family history 10 (34%) 55 (55%) 0.092 h.t 4 (4%) 14 (14%) 0.748 d.m 3 (10%) 5 (5%) 0.748 continuous variables presented as mean ± sd, and discrete variables as numbers and frequencies; f:m= female and male ratio; n= number ; h.t= hyper tension; d.m= diabetes mellitus; (*) = significant difference p < 0.05 results demographic presentation of 100 cholelithiasis patients and 30 controls were elucidated in (table 1). frequency matching was successful with patient and control groups having similar age distributions (mean± sd) ages. table 2 showed the result of the parameters for both controls and cholelithiasis patients. the current study demonstrates a significant difference in sod serum level between controls and patients p-value (0.026). so, there was a significant decrease in the serum levels of sod indicates decreasing in antioxidant activity.while for mpo, current study shows also high significant difference between patient and control p-value (0.001). table (2):parameters of the patients and controls parameter s control n = 30 patients n = 100 pvalue sod 0.67 ± 0.51 0.42 ± 0.29 0.026* mpo 7.01 ±1.55 9.27 ±2.66 0.001* value were presented as mean ±sd of mean; (*) = significance difference, (p<0.05) n= no. of individuals in each group. discussion similar finding were shown by correa p. (27) and byungkyu parket.al (28) , they indicate significantly decrease in sod serum levels in patient diagnosed with many gastrointestinal diseases like acute pancreatitis gallbladder cancer and cholelithiasis. also the current study supported by the results of kaur t. and kaur s.2010 (29) found "significant decrease in antioxidant enzymes activities (superoxide dismutase) in patient with gallstone comparing with controls". also ruhidixit,et al (30) and alpaslanterzet al, (31) have the same findings. gallstones can induce inflammation in the gallbladder wall, and the composition of bile changes, at the same time the bilirubin metabolism, which is a potent antioxidant by radical scavenging and reducing activities, may be altered (32) .the changes in bile composition can increase the biliary free radical formation (33) .increased levels of inducible nitric oxide (no) synthesis activity was shown in inflammed gallbladders which has an effect on elevation of oxidative stress and on fluid transport as well (34) .moreover, inflammatory changes of the gallbladder mucosa are associated with granulocyte infiltration; the activated phagocytes can produce reactive oxygen metabolites, and lead to oxidative stress. free radicals and other peroxides derivatives produced physiologically in the human body and increased in many pathological conditions, diffuse into the blood (35) .here, antioxidant components of plasma overwhelm them and they are consumed. total antioxidants; therefore, is detected as being significantly lower in the cholelithiasis patients than in the controls. many antioxidants and oxidants were studiedto determine their effects. in particular, vitamin c, which is a strong external antioxidant, serum ascorbic acid level was inversely related to prevalence of gallbladder disease (36) . bilirubin free radicals can damage the liver cells, which can induce the change of ingredients of the bile by decreasing the amount of bile acids. the abnormal metabolism in hepatocyte can lead to hydrolysis of the conjugated bilirubin, increase in the concentration of free bilirubin, which in turns make bilirubin supersaturated to the bile, and promote the formation of pigment gallstone, during the gallstone formation, bilirubin reacts with the active-oxygen species formed in vivo followed by its polymerization, aggregation, and calcification. it is speculated to be an important step in the formation of pigment gallstones (37) . for the mpo,similar findings of this study were found by (h.ojimaet.al 2005) who iraqi j pharm sci, vol.23(2) 2014 oxidative stress in cholelihtiasis 60 consider serum level of mpo as diagnostic parameter for undifferentiated carcinoma and small cell carcinoma in gallbladder such as extramedullary myeloid tumour (emmt) (38) , also (alfredo menendez 2009)with (shikokuribayashi , et.al. 2004) agree with this finding and used mpo and other oxidative stress markers as effective diagnostic parameters for many bladder dysfunction including cholelithiasis (39, 40) . also in animal models,rege rv, prystowsky jb (41) found a significant elevation in both mpo and il-1 in cholelithiatic animals, and (özgürkasımayet al)2006 who found significant increase in mpo level in gastrointestinal diseases including cholelithiasis (42) . conclusion cholelithiasis patients showed low antioxidant activity, and high mpo serum levels .as shown in this study; 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the anxiolytic role of exercise. physiology in press; 2006. http://aje.oxfordjournals.org/search?author1=cornelia+weikert&sortspec=date&submit=submit http://aje.oxfordjournals.org/search?author1=steffen+weikert&sortspec=date&submit=submit http://aje.oxfordjournals.org/search?author1=matthias+b.+schulze&sortspec=date&submit=submit http://aje.oxfordjournals.org/search?author1=tobias+pischon&sortspec=date&submit=submit http://www.jurology.com/article/s0022-5347(11)04346-1/abstract http://www.jurology.com/article/s0022-5347(11)04346-1/abstract http://www.jurology.com/issues?issue_key=s0022-5347(11)x0013-7 iraqi j pharm sci, vol.24(1) 2015 doxycycline microsphere and diseases of shrimp 33 preparation, characterization and prophylactic study of new microsphere containing doxycycline against diseases of shrimp raheem j. mehsin *,1 ,leaqaa a.raheem * , abdulhussein h. ghazi ** and saba a.khathem ** * department of pharmaceutical chemistry, college of pharmacy, university of basrah,basrah, iraq. ** department of biology, marine science center , university of basrah ,basrah,iraq. abstract therapeutically and prophylactically using microspheres containing doxycycline isolated from shell of shrimp. low molecule weight poly lactic acid was prepared. in this study, poly lactic acid (pla)/ poly vinyl alcohol (pva)/poly ethyleneglycol(peg) loading doxycycline blend solutions was prepared. also poly lactic acid (pla)-tannin blend via solvent evaporation method was prepared. microspheres of chitosan/gelatin microsphere loading doxycycline was prepared by emulsion crosslinking technique. both microsphere and blends were characterized by fourier transform infrared (ftir) spectrophotometer. the ftir spectra were shown distinguish bands. the in vitro release of doxcycline from its matrix at ph 7 was studied. the prophylactic against white spot (ich) disease of shrimp (macrobrachium nipponense) was studied. the results were shown increase of percentage of survival of shrimp in both microsphere and blend compared with control. the highly percentage of survival was shown in the microsphere compare with blends. key words: chitosan, microsphere, polymer blend, shrimp, prophylactic. تحضير وتشخيص و دراسة وقائية للمايكروسفير المحمل بالذوكسيسايكليه المراض الروبيان رحيم جميل محيسه *،1 لقاء عبذالرضا رحيم ، * ، عبذالحسيه حاتم غازي ** و صبا احمذ كاظم ** * انؼزاق.، بصزة، جبيؼت انبصزة،كهيت انصيذنت ،فزع انكيًيبء انصيذالَيت ** ، بصزة، انؼزاق.جبيؼت انبصزة ،يزكش ػهىو انبحبر الخالصـــة يسخخذو قشىر انزوبيبٌ في حصُيغ انًبيكزوسفيز انًحًم ببنذوكسيسبيكهيٍ نالغزاض انىقبئيت وانؼالجيت , نذنك حضز بىسٌ جشيئي واطىء بىني حبيط انالكخيك. في هذِ انذراست أسخخذو طزيقت حبخيز انًذيب نخحضيز يخبنيظ بىنيًزيت يٍ بىني حبيط ثيهيٍ كاليكىل انًحًم ببنذوكسيسبيكهيٍ وكذنك حضز بىني حبيط انالكخيك يغ انًىاد انالكخيك يغ بىني فُيم انكحىل يغ بىني ا . وأسخخذيج طزيقت انًسخحهب انًخشببك في ححضيز انًبيكزوسفيز يٍ انكيخىسبٌ وانجيالحيٍ وانًحًم ببنذوكسيسبيكهيٍ .انخبَيُيت درص ححهم حًزاء نيؼطي انحشو انًًيشة نهذِ انًزكببث .شخصج انًخبنيظ انبىنيًزيت وانًبيكزوسفيز بىاسطت طيف االشؼت ححج ان . وكذنك درص انخبصيت انىقبئيت نًزض انبقؼت انبيضبء انًسببت 7انذواء يٍ انًُظىيت انبىنيًزيت في يحهىل يُظى بذانت حبيضيت انًبيكزوسفيز وانًخبنيظ انبىنيًزيت نهالكبث انزوبيبٌ وقذ اظهزث انُخبئج سيبدة َسبت انبقبء ػهى قيذ انحيبة يٍ انزوبيبٌ في كم يٍ يقبرَت يغ انكُخزول وكبَج َسبت انبقبء االػهى هي نهزوبيبٌ انًؼبيم ببنًبيكزوسفيز . وقائي . ،روبيان ،مخلوط بوليمري ،مايكروسفير ،كيتوسان :الكلمات المفتاحية introduction microsphere plays an important role in the development of dosage forms for the health care industry, because the duration of drug release needs to be extended several days. the microsphere is incorporation of drugs into polymeric materials to control drug release and increase duration of action, the biodegradable polymers were used in drug delivery systems, control the release of therapeutic agents (1) . the polymer must biodegradable and does not require removal from the end of the treatment, because their degradation into physiological occurring compounds that can readily excreted. polymer blends, that is, physical mixture of structurally different polymers which interact with secondary forces such as hydrogen bonding with no covalent bonding. blending of three or more polymers has become an increasingly important technique for preparing materials with tailor made properties different from those of the constituent polymers. blending of polymers may result in reducing their basic cost, improving their processing and maximizing their important properties. the increase inproperties of the blend depend on the degree of compatibility or miscibility of polymers at the molecular level (2) . 1 corresponding author e-mail:raheemjamail@yahoo.com received: 1/10/2014 accepted: 5/4/2015 iraqi j pharm sci, vol.24(1) 2015 doxycycline microsphere and diseases of shrimp 34 the biodegradation of polymer provides significant benefits such as reduction of stress resulting from animal handling and reduction of cost. the natural polymers are the most attractive and commonly used biodegradable polymers such as chitosan, poly (lactic acid) (pla), polyvinyl alcohol (pva),poly ethyleneglycol (peg) and tannin (scheme 1). scheme (1): chemical structure for pla, pva, peg and tannin. the natural polymers are commercially available in different compositions and molecular weights which effect in degradation of the polymer (3, 4) .the degradation designates the process of polymer chain cleavage and loss of molecular weight. degradation induces the fragments of the material which is defined as mass loss of material when polymer chain cleavage (5) . there are two different erosion mechanisms have been proposed for degradation; homogeneous or bulk erosion and heterogeneous or surface erosion (6) . the fish suffer from both diseases and parasites. it defenses against disease which include skin and scales, when the mucus layer secreted by the epidermis that traps microorganisms and inhibits their growth. the fish is developing inflammatory responses that increase the flow rate of blood to infected areas and deliver white blood cells that may destroy the pathogens. the special responses to particular pathogens recognized by the fish's body, is adaptative immune responses (7) . the vaccines have become used in aquaculture and ornamental fish, such as vaccines for furunculosis in farmed salmon and koi, herpes virus,(virus herpes salmon)vhs, ich(white spot) and whirling disease (8,9) . all fish carry pathogens and parasites. usually this is at some cost to the fish. if the cost of pathogens or parasites is high so its. characterized as a disease. the disease in fish is not understood well relates to aquaria fish (10) . disease is affecting in fish mortality, especially in the young fish. the natural droughts or pollution or predators, can precipitate outbreak of disease (11) . there are four general types of fish ailments, bacterial infections, fungal infections, parasitic or protozoan infections, and physical ailments and wounds (12) . the most antibiotics have been used against many intracellular and extracellular disease of fish is chloromycetin or tetracyclins (13-16) . the aim of this study, is synthesis of both microsphere and blends loading doxycycline as drug delivery system to enhance the percentage of survival of shrimp. materials and method materials chitosan was isolated from shrimp shell , doxycycline was supplied from national center of drug evaluation and biological research. all materials were supplied from aldrich company, glutaraldehyde, dichloromethane, nhexan, poly(vinylalcohol), lactic acid, paraffin, gelatin, acetic acid ,hydrochloric acid, sodium hydroxide ,sodium bicarbonate and tween-80. methods extraction and deacetylation of chitin (17) the resources used to extract chitin are shrimp shell. the shells were scraped free of loose tissue, washed, dried, and grounded. demineralization was carried out in dilute hcl solution (7% v/v) at room temperature with stirring and their durations (24-72 hours). the solid fraction was washed with distilled water until neutral ph. the demineralized samples were dried. (10% w/v) naoh at 60 °c for 24 hrs used for deproteinization of chitin. then the solution was washed with water to neutrality, then with hot ethanol. the chitin was dried at 50 °c. 10 gm of chitin was put into (50% w/v) solution of naoh at 60°c for 8 hours to prepare chitosan. the residue was washed with hot distilled water at 60°c for three times. the crude chitosan was drying in oven at 50°c overnight. preparation of poly lactic acid low molecular weight of poly lactic acid (pla) was prepared (18) three necked flask equipped with mechanical stirrer, gas outlet tube, and nitrogen gas tube in bottom of flask, was charged with (0.13 mmole, 10 ml) of l-lactic acid , five drops of diluted 1% v/v hcl was add. the reaction mixture was stirred for 3 hours at 130°c and the nitrogen gas was passed through the mixture to get of the water. then the reaction mixture was cooled and iraqi j pharm sci, vol.24(1) 2015 doxycycline microsphere and diseases of shrimp 35 neutralized by washing with 0.1 % w/v of nahco3, then with water .the product was dried in vacuum-oven for 24 hours at 0.1 mm hg and 40 °c to give a yellow oily product. preparation of chitosan/gelatin microsphere loading doxycycline (19) microspheres containing doxycycline were prepared by emulsion crosslinking technique. the chitosan, gelatin (1:1 by weight) and 0.5 gm (1.133x10 -3 mol) doxycycline were dissolved in 5% acetic acid. a certain amount of tween-80 and liquid paraffin at ratio 1:10 were added to the chitosan/gelatin mixture, under agitation was performed, using mechanical stirrer at 650 rev/min. at 30 °c. after 15 minute, 4 ml of 25 % v/v glutaraldehyde solution was added drop by drop and stirring was continued for 3 hours. the microspheres so obtained were filtered and washed several times with n-hexane, then with distilled water to remove the adhering liquid paraffin and glutaraldehyde, respectively. the microspheres were dried in hot air oven at 50°c . preparation of microsphere by using emulsification-solvent evaporation method (20) 1poly lactic acid (pla) / polyvinylalcohol (pva) /poly ethyleneglycol (peg) microsphere loading doxycycline 7.2 gm (0.1 mol) of (pla) was dissolved in 20 ml dichloromethane. the solution was added drop-wise to a 40 ml aqueous phase solution containing 4.4 gm (0.1mol) (pva), 4.4 gm (0.1 mol) (peg) and 0.5 gm (1.133x10 -3 mol) of doxycycline was dissolved in polymer solution, while the mixture was stirred by an overhead stirrer to form a stable oil/water emulsion system at room temperature (25°c). stirring was continued for overnight to allow the evaporation of both dichloromethane and water to form a solid blend. the blend was dried overnight in oven at 30 °c until no weight loss was observed. the melting point is 75°c. 2polylactic acid (pla)-tannin blend via solvent evaporation method 7.2 gm (0.1mol) of (pla) was dissolved in 20 ml dichloromethane. the solution was then added drop-wise to 20 ml aqueous phase solution containing 30 gm (0.1mol) of tannin and 0.5 gm (1.133x 10 -3 mol) of doxycycline was dissolved in polymer solution. the mixture was stirred at room temperature (25°c), for two hours, to allow formation of solid blend. then evaporation of dichloromethane and the blend was filtered , and dried overnight. the melting point is 210 °c (decomposition). all microspheres and blend products were characterized by using ftir spectro photometer , (table -1). table (1): ft.ir spectral bands of products (cm -1) . products streachin vibration ʋ ch2 streaching vibration ʋ o-h c=0 (dox.) c=0 n-h streaching vibration c-0 ch,ch2 and ch3 bending chitosan/ gelatin microsphere. as. 2923.88 s. 2858.31 3446.56 ------ 1637 3250 1022.2 1458.08 1377.08 chitosan/ gelatindox. microsphere as. 2933.73 s. 2868. 3570.90 1620.04 1656.85 (acetylated chitosan) 3277 1118.71 1460.11 1348.24 pla /tannindox. blend. as.2999.31, s. 2924 as.2954.95, s. 2852.72 3450 1618.28 1759.08 (tannin), 1720.5 poly(lactic acid) 3200 br 1188.15 1456.26 1386.82 pla/pva/pe gdox. microsphere. as 2949. 16 s. 2885.51 and as.2806.43 s. 2800 3616.53 1622 1755.51 (pla) 3245 1114.86 1471.69 1361.74 dox.=doxycycline , as.= asymmetry , s. = symmetry. br. = broad iraqi j pharm sci, vol.24(1) 2015 doxycycline microsphere and diseases of shrimp 36 release studies (21) in vitro drug release from different formulations was invested in phosphate buffer solution (pbs) of ph 7.4. microspheres (50 mg) were dispersed in 400 ml of pbs in c under continuous stirring (60 rpm).at selected time intervals, 5 ml samples were withdrawn and replaced with the same volume of prewarmed fresh buffer solution to maintain a constant volume of the receptor compartment. the samples were analyzed spectrometrically at 347 nm (dilution factor 10). the released drug content was computed from the calibration curve of doxycycline as shown in figure 6. application of both microspheres as prophylactic agents collect the shrimp sample (macrobrachium nipponense) from shat alarab , qarmat ali in 7/4/2014, the small size shrimp was chosen with average weight 0.5±0.1 gm and average height 26± 2 mm. in this experiment we used four plastic aquariums with capacity of 15 liters for each one, in first aquarium treated with chitosan/gelatin microsphere containing doxycycline, and in the second aquarium treated with pla/pva/peg –doxycycline microsphere, while the third one was treated with pla/tannin –doxycycline blend. in the last one, left without antibiotic. the addition of microsphere is 0.25 gm every 48 hours and the water replacement occur in partial manner, during study, the environmental data are recorded through four weeks and the result as follow temperature, in 23-25°c, salt is 1.2-1.5 gm/l, dissolved o25.5-6.4 mg/l . results and discussion microsphere is drug delivery offers an intelligent approach in the medical field. it is encapsulating a drug into a carrier particle (22) . biodegradable microsphere can be prepared from certain synthetic, as well as natural polymers. natural polymers remain attractive primarily because they are natural products of living organisms, readily available, relatively inexpensive and non-toxic. therefore, using natural polymers such as gelatin and chitosan figure (1), have many benefits for developing successful drug delivery systems. the microspher provides sustained release, targeting and stabilization of drug (23-25) . from much research doxycycline application is limited by its relatively low stability in aqueous solution. therefore, to solve this problem by using microsphere to form inclusion compounds within molecules, show great advantages in enhancing the stability of guest molecules (26 ) . figure (1): chemical and 3d-structure of n d-glucosamine(chitosan). the incorporation between drug and polymer was studied by ftir spectroscopy. spectral-data were recorded for drug-loaded microspheres and blend types. table1, illustrate the main characteristic bands in the products. the comparative ftir spectra of doxycycline -loading microspheres and blend types are shown in figures (2-4). peaks are observed around 2900-3000 cm -1 corresponding to aliphatic -ch and -ch 3 of the polymers, chitosan, gelatin, pla and pva. the result indicates the stability of the drug during the microencapsulation process, also showed small deviations of some polymer bands like from 1637 (without drug) to 1656.85 cm -1 , probably due to the presence of doxycycline figure( 2): ft.ir spectrum of chitosan/ gelatin –doxycycline microsphere iraqi j pharm sci, vol.24(1) 2015 doxycycline microsphere and diseases of shrimp 37 figure (3 ): ft.ir spectrum of tannin /pla-doxycycline blend. figure( 4) : ft.ir spectrum of pla/pva/peg-doxycline microsphere. extensive research has been carried out to exploit the use of chitosan as a drug or vaccine carrier (27-29) . the safety of chitosan has been extensively studied, and it was found that it is a biologically compatible polymer with a minimal toxicity (30, 31) . the in vitro release profiles of doxycycline formulations as microspheres, and blend, are shown in figure 5. the results illustrated, that the drug release is fast and requires peroid of time to go completion. this can be explained by the high affinity of the drug towards water, used as a release medium. the release rate is also higher for formulations having pla / tannin -doxycycline blend and pla /pva /peg -doxycyline, than chitosan/gelatin-doxycycline microsphere.the formation of particular aggregates, which extends into the polymer matrix, needs more time to dissolve. the initial release observed for pla/tannin-doxycycline can be explained by the presence of surface associated drug. the aggregates due to the small quantity of drug, leads to fast dissolution into the release medium during the first minutes, especially in the case of hydrophilic drugs (32) . figure (5): release of doxycycline from products. bacterial diseases are usually characterized by red streaks or spots and/or swelling of the abdomen or eye. these are treated by antibiotics such as doxycycline, which has been successfully encapsulated through the microspheres. then investigation both microsphere types, are more effective than blend type, from percentage of survival of shrimp. table 2 shows the high percentage of iraqi j pharm sci, vol.24(1) 2015 doxycycline microsphere and diseases of shrimp 38 survival to the shrimp sample of the treated groups, compared with the control. table (2) percentage of survival to the shrimp sample treatment % of survival chitosan/gelatin – doxycline microsphere 97 pla/peg/pva doxycycline microsphere 90 pla/tannin doxycycline blend 89 control 78 conclusion doxycycline has been successfully encapsulated in microspheres and applied as prophylactic in shrimp culture to control release of doxycycline. the results showed that drug release is fast and only few hours to go to completion were required. this can be explained by the high affinity of the drug towards water used as a release medium. then, the release rate is higher for formulations having the low amount of drug .the conclusion that, at more drug loading, the high chance of particles to interact with each other. this leads to, the formation of particular aggregates, which extends into the polymer matrix, and needs more time to dissolve. references 1. g. schliecker, biodegradable polyesters for veterinary drug delivery systems: characterization , in vitro degradation and release behavior of oligolactides and polytartrate, doctoral dissertation, vom fachbereich der pharmazie der philippssuniversitate marburg, marburg/lahn 2003. 2. bassam i k, study of suspended solution from blending three polymer metal complexes as antimicrobial.chemical and process engineering research. 2014;21: 1-7. 3. li s, vert m. biodegradable polymers: polyesters. in: mathiowitz e. 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6(1) :51-58. 19. thanoo, b.c., sunny, m.c., jayakrishnan, a, crosslinked chitosan microspheres: iraqi j pharm sci, vol.24(1) 2015 doxycycline microsphere and diseases of shrimp 39 preparation and evaluation as a matrix for the controlled release of pharmaceuticals. j. pharm.pharmacol. 1992; 44: 283-286. 20. dinarvand r, moghadam sh, mohammadyari-fard l, atyabi f.preparation of biodegradable microspheres and matrix device containing naltrexone. aaps parmscitech. 2003;4(3):45-54. 21. sankar ch,mishra b,development and in vitro evaluation of gelatin a microspheres of ketorolac tromethamine for intransal administration .j. acta pharm . 2003 ;53:101-110. 22. narasimha rr, sampath km, dileep kk, vineeth p. formulation and evaluation of capcitabine microspheres. int j pharm tech. 2001;3(2):2599-2632. 23. heidi mm, sohn mj, al-ghananeem a, deluca pp. materials for pharmaceutical dosage forms: molecular pharmaceutics and controlled release drug delivery aspects. int j mol sci. 2010;11(9):32983322. 24. sadeghi m. pectin-based biodegradable hydrogels with potential biomedical applications as drug delivery systems. j biomater nanobiotechnol. 2011;2(1):3640. 25. giavaresi g, tschon m, borsari v, daly jh, liggat jj, fini m, bonazzi v, nicolini a, carpi a, morra m, cassinelli c, giardino r. new polymers for drug delivery systems in orthopaedics: in vivo biocompatibility evaluation. biomed pharmacother. 2004;58(8):411417. 26. doxycycline and hydroxypropyl-βcyclodextrin complexin poloxamer thermal sensitive hydrogel forophthalmic delivery zi-xin he, zhou-huawang, haohao zhang, xin pan, wen-ru sud, dan liang, chuan-binwu . acta pharmaceutica sinica b 2011;(4):254-260. 27. dash m, chiellini f, ottenbrite rm, chiellini e. chitosan – a versatile semisynthetic polymer in biomedical applications. prog polym sci. 2011;36(8):981–1014. 28. xu j, mccarthy sp, gross ra, kaplan dl. chitosan film acylation and effects on biodegradability. macromolecules .1996 ;29 (10): 3436–3440. 29. yang ym, hu w, wang xd, gu xs. the controlling biodegradation of chitosan fibers by n-acetylation in vitro and in vivo. j mater sci mater med. 2007;18(11):2117–2121. 30. arai k, kineemaki t, fujita t. toxicity of chitosan. bull tokai reg fish res lab.1968;56:89–94. 31. hirano s, seino h, akiyama y, nonaka i. biocompatibility of chitosan by oral and intravenous administrations. polym mater sci eng. 1988;59:897–901. 32. yoon y, kinam p. control of encapsulation efficiency and initial burst in polymeric microparticle systems. arch of pharm res. 2004; 27(1):1-12. iraqi j pharm sci, vol.22 (2) 2013 synthesis of nsaids and sulfonamide conjugates. 22 synthesis of new conjugates of some nsaids with sulfonamide as possible mutual prodrugs using tyrosine spacer for colon targeted drug delivery shayma l. abdulhadi *,1 , ahlam j. qasir * , and nadeem a. abdul razzak * * department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract the purpose of this research work is to synthesize conjugates of some nsaids with sulfamethoxazole as possible mutual prodrugs to overcome the local gastric irritation of nsaid with free carboxyl group by formation of ester linkage that supposed to remain intact in stomach and may hydrolyze in intestine chemically or enzymatically; in addition to that attempting to target the synthesized derivative to the colon by formation of azo group that undergo reduction only by colonic bacterial azo reductaze enzyme to liberate the parent compound to act locally (treatment of inflammation and infections in colon). key words: mutual prodrug, ester linkage, azo bond, colon targeting تحضيز يشتقاث جذيذة نبعض يضاداث االنتهاب انغيز ستيزويذيت يع انسهفىًَايذ كًقذياث ادويت يحتًهت باستخذاو انتايزوسيٍ كذراع نتىجيهها انى انقىنىٌ شيًاء نؤي عبذ انهادي ،*1 انزساق عبذانستار َذيى عبذ و، احالو جًيم قصيز * . انعزاق، كهيح انصيدنح ، جايعح تغداد، تغداد ، انكيًياء انصيدالَيح فزع الخالصة انهدف يٍ انثحث هى تحعيز يشتقاخ نثعط يعاداخ االنتهاب انغيز ستيزويديح يع يزكثاخ انسهفىًَايد كًقدياخ ادويح يحتًهح ستيزويديح انُاتجح يٍ وجىد يجًىعح انكارتىكسيم انحزج تتحىيهها انى يجًىعح نهتقهيم يٍ انتأثيزاخ انًعىيح نًعاداخ االنتهاب انغيز استيز انتي يٍ انًفتزض عدو تحههها في انًعدج وايكاَيح تحههها في االيعاء كًيائيا او اَشيًيا" تاالظافح انى يحاونح تىجيه انًشتك شال تىاسطح اَشيى االسو ريدكتيش انًتحزر يٍ تكتيزيا انًحعز انى انقىنىٌ يٍ خالل تحعيز اصزج االسو وانتي يحصم نها اخت انقىنىٌ وتذنك يتحزر انًزكة االصهي نيعانج يىظعيا االنتهاب في انقىنىٌ. يقذياث دواء يتبادل، ارتباط االستيز، اصزة االسو، انتىجيه انى انقىنىٌ انكهًاث انًفتاحيت: introduction non-steroidal anti-inflammatory drugs (nsaids) are most commonly prescribed drugs for the treatment of pain, inflammation and fever (1) . however gastric irritation caused by most of the nsaids used today restricts their use. the pharmacological activity of nsaids is related to the blocking of prostaglandin h2 (pgh2) biosynthesis from arachidonic acid by inhibiting the activity of cyclooxygenases (coxs). the cox enzyme exists in two isoforms: a constitutive isoform, cox-1, found in most tissues including stomach, kidney, and platelets, and an inducible isoform, cox-2, expressed at the site of inflammation (2) . classical nsaids, such as ibuprofen, flufenamic acid, diclofenac, and aspirin, preferentially inhibit cox-1, thus suppressing the biosynthesis of prostaglandins that maintain gastric mucosal integrity and leading to gastrointestinal (gi) side effects, including ulceration and hemorrhage (systemic effect) (3,4) .topical irritation by the free carboxylic group of the nsaids is considered an important factor in establishing superficial stomach erosion (5, 6) . since the introduction of specific cox-2 inhibitors, which are less harmful to the gi tract; the use of conventional nsaids have declined. however, the safety profile of cox-2 inhibitors has been questioned due to the risk of ulcer complications in high – risk individuals and to cardiovascular adverse effects (7, 8) . thus, the need for nsaids with improved gi tolerability still exists. one approach that has been used to decrease nsaid induced gi toxicity without adversely affecting their anti-inflammatory activity is to mask the carboxylic acid group by synthesizing the corresponding ester prodrugs (9) . a prodrug is a chemically modified inert drug precursor which upon biotransformation liberates the pharmacologically active parent compound (10) . a major requisite for these prodrugs is that they must be readily hydrolyzed, enzymatically or chemically, after oral absorption to quantitatively release the parent drug (11) . amino acids have been considered the ideal carriers for the development of prodrugs; and some of them have marked antiinflammatory activity of their own (12) .mutual prodrug, where the carrier used is another biologically active drug instead of some inert molecule (13) . 1 corresponding author e-mail:shimmama_hafidh@yahoo.com received: 19/12/2012 accepted: 9/6/2013 iraqi j pharm sci, vol.22 (2) 2013 synthesis of nsaids and sulfonamide conjugates. 23 site specific drug delivery a drug, after its absorption into systemic circulation, gets distributed to target site as well as non-targeted tissues. the distribution of drug to non-targeted tissues may lead to undesirable toxic effects in those tissues and insufficient concentration in the target site to evoke any therapeutic response. if the drug needs a long time to reach to the target site, it may get eliminated without reaching such a site; and even if the drug reaches the targeted area in sufficient concentrations, it may have such a low penetration power that it may not penetrate the target cells at all (14) . targeting the drug to its site of action through prodrug concept has been utilized to overcome these problems. while designing the prodrug, one must take into account the enzymes that are specifically present in that organ or tissue or specific ph of that area which is different from physiological ph so that the prodrug releases the drug only in the targeted organ (15) . colon targeted drug delivery the colon has some unique features, which make this organ attractive for sitespecific drug delivery. there is a considerable interest in the colon specific drug delivery in order to treat diseases of the large intestine, such as colitis, colon cancer, constipation, irritable bowel syndrome, and infectious diseases (16) . to achieve successful colonic delivery, the drug needs to be protected from absorption and /or the environment of the upper gastrointestinal tract (git) and then be abruptly released into the proximal colon, which is considered the optimum site for colon-targeted delivery of drugs (17) . the presence of azo reductase enzyme in colon from colonic microflora, which is not present in the stomach or small intestine, plays an important role in the release of drug from azo bond prodrug. this enzyme causes reduction, and thus cleavage, of the azo bond (18, 19) . sulfonamides are one of the least expensive drugs and this factor largely accounts for their greater extent of use in developing countries. these drugs are considered useful for gastrointestinal (gi) tract infections. an infection always leads to inflammation therefore sulfa drugs can be coupled with nsaids so that these mutual prodrugs can be used for infections as well as for inflammation (20) . materials and methods ibuprofen, naproxen, and sulfamethoxazole were purchased from sdi (iraq); ketoprofen was purchased from (india); boctyrosine was purchased from fluka (switzerland). all chemicals were reagent grade and obtained from commercial sources. elemental microanalysis was performed using chn analyzer (jordan); melting points were measured on barnstead electrothermal melting point apparatus (usa) and are uncorrected; infra red spectra were recorded as kbr disks on ftir spectrophotometer (college of pharmacy, university of al-mustanseriya).uv spectra were done using uv spectrophotometer (college of pharmacy, university of baghdad). chemical synthesis synthesis of compound 1a (diazonium salt formation (21) sulfamethoxazole (2.53g, 10 mmole) was dissolved in a mixture of equal quantities (12.5ml) of each of conc. hcl and water in a suitable beaker; the resulting solution was stirred and cooled by immersing in a bath of crushed ice; throughout the reaction the temperature was kept below 5ºc. a cold solution of (0.75g, 11 mmole) sodium nitrite in(5ml ) water was placed in a dropping funnel which was cooled using crushed ice, then it was added dropwise into the first solution in the ice bath with continuous stirring ; the temperature should not be allowed to rise above 10ºc.the last quantity of the sodium nitrite solution was added more slowly and after stirring for 3-4 minutes, the solution was tested for excess sodium nitrite using potassium iodide-starch paper. a solution of sulfamic acid (1.5ml) of 2% was added and stirring was continued for 20 minutes. the diazonium salt formed was used immediately in the following step. synthesis of compound1b (azo bond formation) ( 21) boctyrosine (2.8 g, 10 mmole) was dissolved in (8 ml) of (10 %) naoh in a suitable beaker immersed in an ice bath. the solution was stirred vigorously and the temperature was kept below 5ºc by the addition of crushed ice. the cold diazonium salt solution from the previous step (compound 1a) was placed in a dropping funnel, then it was added drop by drop to the cooled, stirred boc-tyrosine solution; an orange color was developed and orange crystals soon separated. at the end of the addition the mixture was stirred for 3hours in the ice bath. then the solution was filtered through a buchner funnel with gentle suction, washed well with water, and recrystallized from ethanol: water mixture (1:5) to obtain (37 %) of compound 1b. synthesis of compound 1c (22) to a suspension of compound 1b (2.73 g, 5 mmole) in methanol which was cooled to 10ºc, thionyl chloride (0.7 ml, 10 mmole) was added dropwise with continuous stirring during which the temperature of the reaction mixture iraqi j pharm sci, vol.22 (2) 2013 synthesis of nsaids and sulfonamide conjugates. 24 after completion of the addition, the temperature of the mixture was allowed to rise and kept at 40ºc for 3 hours followed by refluxing for further 3 hours, then left at room temperature overnight. the solvent was evaporated to dryness in vacuum. red powder appeared which was re-dissolved in methanol and evaporated several times to ensure the complete removal of excess thionyl chloride.the residue was then collected and recrystallized from methanol: diethylether mixture (1:5) to obtain (90 %) of compound 1c as brown residue. synthesis of compounds 1d, 2d, 3d ( 23) ibuprofen 1g, 5mmole (or naproxen 1.17g, 5mmole or ketoprofen 1.27g, 5mmole) was dissolved in chloroform (20ml) in a round bottom flask; to it few drops of dmf were added, the mixture was stirred inside an ice bath where the temperature should be below 0ºc. a slight excess of thionyl chloride (0.7 ml, 10 mmole) was added drop wise over a period of 15-20 minutes with continuous stirring. after complete addition of thionyl chloride the temperature was allowed to rise gradually then refluxing for 8 hours. the solvent and the excess thionyl chloride were evaporated under vacuum followed by re dissolving in chloroform and re-evaporation several times. the acid chloride was obtained as yellow oily residue and used immediately in the following step. synthesis of compound i, ii, iii (24) a suspension of compound 1c (2.8 g, 5 mmole) and tea (1.4ml, 10 mmole) in dry thf (100ml) was stirred in an ice bath. followed by a dropwise addition of a solution of compound 1d or 2d or 3d (in dry acetone) (5 mmole) over a period of 1 hour, the temperature of the mixture was kept below 5ºc during the addition. after that the mixture was stirred for 72 hours at room temperature. the solvent was evaporated then the residue was dissolved in chloroform followed by filteration to remove solids. the chloroform layer was shaken with 1m sodium carbonate solution for 15 minutes (3 x 25ml), d.w. (3 x 25ml), 0.05n hcl (3 x 25ml), d.w. (3 x 25ml), and finally with (25ml) brine solution (saturated nacl solution).the chloroform extract was dried over anhydrous magnesium sulfate. the residue, after evaporation of solvent, was collected and recrystallized from chloroform: petroleum ether (40-60) mixture (1:5) to obtain (20 %) yield red residue compound i; (46%) yellow residue compound ii; (60%) yellow residue compound iii. results and discussion primary aromatic amines react with nitrous acid in the presence of hcl (or other mineral acid) at about 0ºc to yield diazonium salts. coupling reaction is an electrophilic aromatic substitution with the diazonium ion acting as the electrophile which reacts at the position of greatest electron availability (the position para or ortho to the electron releasing group) (25) . conversion of acid chloride into ester ; on treatment with the appropriate nucleophile, an acid chloride can be converted to an ester by nucleophilic acyl substitution mechanisms. nucleophilic acyl substitution reactions take place in to steps: 1. addition of the nucleophile. 2. elimination of a leaving group. (26) physical appearance, percentage of yield, melting points, rf values, and the molar extinction coefficients of intermediates and final compounds are presented in table (1). table (1): physical appearance, percentage of yield, melting points, rf values of intermediates and final compounds compound physical appearance yield% melting point (ºc) rf * value a b є at 332 nm 1b orange powder 37% 124-126 0.43 0.26 1c brown powder 90% 152-155 0.82 0.39 i red powder 20% 144-146 0.94 0.54 8555.5 ii yellow powder 46% 139-142 0.96 0.55 3673.9 iii yellow powder 60% 120-123 0.9 0.42 9868.6 * a. chloroform: ethanol (8:2), b. toluene: ethanol (8:2) iraqi j pharm sci, vol.22 (2) 2013 synthesis of nsaids and sulfonamide conjugates. 25 table (2): elemental microanalysis results of the final compounds compound molecular formula molecular weight elemental microanalysis% element calculated observed i c38h45n5o9s 747.86 c h n s 61.03 6.06 9.36 4.29 61.096 6.061 9.456 4.755 ii c39h41n5o10s 771.84 c h n s 60.69 5.35 9.07 4.15 61.505 6.664 9.412 3.721 iii c41h41n5o10s 795.86 c h n s 61.88 5.19 8.80 4.03 62.34 5.312 9.545 4.412 table (3): ft ir characteristic bands of the synthesized compounds compound band (cm -1 ) interpretation compound 1b 3352 3263 3095 2924, 2854 1716 1689 1618,1502 1467 1467,1396 1423 1271 1174, 1342 1174 786,933 n-h stretching of amide o-h stretching aromatic c-h stretching asymmetric and symmetric c-h stretching of ch3 , ch2 carboxylic c=o stretching amide c=o stretching aromatic c=c stretching n=n stretching c-h bending of ch3, ch2 o-h in plane bending c-o stretching of carboxylic acid o=s=o sulfonamide two bands c-o stretching of phenol aromatic c-h bending compound1c 3412 3265 2956, 2858 1749 1616,1502 1464,1396 1464 1278 1396,1170 790, 636 n-h stretching of amide o-h stretching of phenol and n-h stretching of sulfonamide asymmetric and symmetric c-h stretching of ch3, ch2 c=o stretching of ester aromatic c=c stretching c-h bending of ch3 ,ch2 n=n stretching c-o stretching of ester o=s=o sulfonamide two bands aromatic c-h out of plane bending iraqi j pharm sci, vol.22 (2) 2013 synthesis of nsaids and sulfonamide conjugates. 26 table (3): continued ft ir characteristic bands of the synthesized compounds compound band (cm -1 ) interpretation compound i 3298, 3254 3095 2955, 2870 1734 1653 1616, 1512 1589 1464 1464, 1398 1398,1170 1276 785, 638 n-h stretching of amide and sulfonamide aromatic c-h stretching asymmetric and symmetric c-h stretching of ch3, ch2 c=o stretching of ester c=o stretching of amide aromatic c=c stretching n-h bending (amide ii band) n=n stretching c-h bending of ch3 ,ch2 o=s=o of sulfonamide c-o stretching of ester aromatic c-h out of plane bending compound ii 3309, 3215 3063 2958 1739 1643 1612, 1502 1537 1392, 1346 1346, 1172 1271 715, 636 n-h stretching of amide and sulfonamide aromatic c-h stretching asymmetric c-h stretching c=o stretching of ester c=o stretching of amide aromatic c=c stretching n-h bending (amide ii band) c-h bending of ch3 ,ch2 o=s=o of sulfonamide c-o stretching of ester aromatic c-h out of plane bending compound iii 3327 3036 2928, 2850 1743 1627 1573 ,1535 1440 1273 642 n-h stretching of amide aromatic c-h stretching asymmetric and symmetric c-h stretching of ch3, ch2 c=o stretching of ester c=o stretching of amide aromatic c=c stretching and n-h bending (amide ii band) could be in this region n=n stretching c-o stretching of ester aromatic c-h out of plane bending iraqi j pharm sci, vol.22 (2) 2013 synthesis of nsaids and sulfonamide conjugates. 27 s h n n o o o h3c nh2 hcl nano2 s h n n o o o h3 c n n cl + oh 2c ch coo hn c o o naoh s nh n o o o h3c nn oh ch2ch hooc nhc o o diazotization coupling r cooh socl2caboxyl group activation r cocl socl2 ch3ohesterif ication s n h n o o o ch3 n n oh ch2 ch c nh c o o o h3co + s o o h n n o ch3 n n o h2c h c cooch3h n c o o c o r esterif ication ohh 2c ch cooh hn c o o na sulf amethoxazole boc-tyrosine nsaid tea 1a 1b 1d,2d,3d i, ii, iii 1c na scheme of synthesis of compound i, ii, iii determination ofλ max scanning the solutions of compounds i, ii, iii in chloroform (25μg /ml) by uv/visible spectrophotometer at 200-800 nm gave different peaks with λ max at 332nm; see figures (1), (2), (3). the molar extinction coefficient for compounds i, ii, iii were determined at λ max = 332 nm and presented in table (1). iraqi j pharm sci, vol.22 (2) 2013 synthesis of nsaids and sulfonamide conjugates. 28 figure (1): uv spectrum of compound i shows 3 peaks; 246, 332, and 409 nm; λ max is 332 nm. figure (2): uv spectrum of compound ii shows 3 peaks; 241, 332, and 409 nm; λ max is 332 nm. figure (3): uv spectrum of compound iii shows 4 peaks; 236, 255, 332, and 409 nm; λ max is 332 nm. preparation of calibration curve calibration curve of compound iii was constructed in chloroform using different concentration solutions (20, 40, 60, 80, 100 μg/ml) at λ max (332 nm) and presented as straight line; see figure (4). figure (4): the calibration curve of compound iii determination of partition coefficient (27) a drug partition coefficient is a measure of its distribution in a lipophilic/hydrophilic phase system, and is indicative of its ability to penetrate biological multiphase system. the partition coefficient of compound iii was determined in two systems: n-octanol/hcl buffer (ph=1.2) where the value was 35.03 and n-octanol/ phosphate buffer (ph=7.4) where the value was 38.98. this indicates that the compound is highly lipophilic. conclusion the synthesis of the designed compounds has been successfully achieved and the structural formula for these compounds was characterized using ir spectroscopy, elemental microanalysis, melting points, uv spectra, and rf values. from the results compound iii is highly lipophilic. references 1. abhilasha verma, nirupam das, meenakshi dhanawat and sushant k. shrivastava, conjugation of some nsaids with 5-phenyl-2-aminothiazole for reduced ulcerogenicity, thai j. pharm. sci. 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the rearrangement pathway in aqueous media, arch. pharm. chem. life sci. 2007; 340: 32–40. 13. d bhosle, s bharambe, neha gairola, suneela s dhaneshwar, mutual prodrug concept: fundamentals and applications, indian journal of pharmaceutical science,2006; 68(3): 286294. 14. v.s. tegeli, y.s. thorat, g.k. chougule, et al. , review on concepts and advances in prodrug technology, international journal of drug formulation & research, 2010;1(3): 32-57. 15. arun rasheed, i. theja, c.k. ashok kumar, y. lavanya, et al. , synthesis, hydrolysis studies and pharmacodynamic profile of novel colon-specific mutual prodrug of aceclofenac with amino acids, der pharma chemica, 2009, 1(2): 59-71. 16. akhil gupta, anuj mittal, and alok kumar gupta, colon targeted drug delivery systems – a review, international journal of pharmaceutical and clinical research 2010; 2(4): 112-120. 17. m. k. chourasia, s. k. jain, pharmaceutical approaches to colon targeted drug delivery systems, j pharm pharmaceut sci , 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evaluation of amide derivatives of ibuprofen, international journal of chem tech resaerch, 2010; 2(1): 233-238. 24. sarthak jain, susan tran, mohamed a.m. el gendy, et al., nitric oxide release is not required to decrease the ulcerogenic profile of nsaids, journal of medicinal chemistry, 2011; 55: 688-696. 25. francis a. carey, organic chemistry, 5 th ed., the mcgraw hill companies, 2004; p. 944, 950. 26. john mcmurry, organic chemistry, 7 th ed., thomson brooks/cole publishing company, 2008; p. 794-795, 810-811. 27. wang j, hu y, li l, jiang t, wang s, mo f., indomethacin-5-fluorouracil-methyl ester dry emulsion: a potential oral delivery system for 5-fluorouracil, drug dev ind pharm., 2010; 36(6):647-56. http://www.ijpsonline.com/searchresult.asp?search=&author=d+bhosle&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=s+bharambe&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=neha+gairola&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=neha+gairola&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=suneela+s+dhaneshwar&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=deepika+nagpal&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=r+singh&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=neha+gairola&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=sl+bodhankar&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ijpsonline.com/searchresult.asp?search=&author=suneela+s+dhaneshwar&journal=y&but_search=search&entries=10&pg=1&s=0 http://www.ncbi.nlm.nih.gov/pubmed?term=wang%20j%5bauthor%5d&cauthor=true&cauthor_uid=20136491 http://www.ncbi.nlm.nih.gov/pubmed?term=hu%20y%5bauthor%5d&cauthor=true&cauthor_uid=20136491 http://www.ncbi.nlm.nih.gov/pubmed?term=li%20l%5bauthor%5d&cauthor=true&cauthor_uid=20136491 http://www.ncbi.nlm.nih.gov/pubmed?term=jiang%20t%5bauthor%5d&cauthor=true&cauthor_uid=20136491 http://www.ncbi.nlm.nih.gov/pubmed?term=wang%20s%5bauthor%5d&cauthor=true&cauthor_uid=20136491 http://www.ncbi.nlm.nih.gov/pubmed?term=mo%20f%5bauthor%5d&cauthor=true&cauthor_uid=20136491 http://www.ncbi.nlm.nih.gov/pubmed?term=mo%20f%5bauthor%5d&cauthor=true&cauthor_uid=20136491 http://www.ncbi.nlm.nih.gov/pubmed/20136491 http://www.ncbi.nlm.nih.gov/pubmed/20136491 iraqi j pharm sci, vol.23(1) 2014 bioequivalence and pharmacokinetics of amlodipine tablets 60 bioequivalence and pharmacokinetics of two formulations of amlodipine tablets in healthy subjects jaafar j. ibraheem al-tamimi *,1 * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the bioequivalence of a single dose tablet containing 5 mg amlodipine as a test product in comparison to norvasc ® 5 mg tablet (pfizer usa) as the reference product was studied. both products were administered to twenty eight healthy male adult subjects applying a fasting, single-dose, twotreatment, two-period, two-sequence, randomized crossover design with two weeks washout period between dosing. twenty blood samples were withdrawn from each subject over 144 hours period. amlodipine concentrations were determined in plasma by a validated hplc-ms/ms method. from the plasma concentration-time data of each individual, the pharmacokinetic parameters; cmax, tmax, auc0-t, auc0-, cmax/auc0-, z, t0.5, mrt, cl/f and vd/f; were calculated applying non-compartmental analysis. the average values of the above parameters for the test formula were 1.99 ng/ml, 8.3 hours, 82.87 ng.hr/ml, 95.23 ng.hr/ml, 0.0219 hr -1 , 0.018 hr -1 , 38.5 hr, 56.2 hr, 60.9 l/hr and 3483 liters, respectively. the average values of these parameter for the reference formula were 1.92 ng/ml, 7.9 hours, 76.3 ng.hr/ml, 89.31 ng.hr/ml, 0.0225hr -1 , 0.019 hr -1 , 36.7 hr, 59.9 hr, 69.5 l/hr, and 3983.4 liters, respectively. the pharmacokinetic parameters mentioned above were statistically analyzed by anova test. ln-transformed values of the pharmacokinetic parameters used for bioequivalence testing; cmax, auc0-t and auc0- ; were also statistically analyzed by anova, 90% confidence interval (ci) and schuirmann’s two one-sided t-tests. for the tmax, parametric and nonparametric tests were applied. based on fda criteria on bioequivalence, the results of the above statistical tests demonstrated bioequivalence of the two products. keywords: amlodipine, pharmacokinetic, bioequivalence , hplc/ms/ms. دراسة التكافؤ الحٍوي و حركٍة الذواء لمستحضرٌن من اقراص االملودبٍن على متطوعٍن اصحاء جعفر جابر ابراهٍم التمٍمً ،*1 * .،تغداد ،انعزاق جايعح تغداد،كهٍح انصٍدنح ،انصٍدالٍَاخ فزع الخالصة ذى دراسح انركافؤ انحٍوي وحزكٍح اندواء نًسرحضز جٍُس عهى شكم حثوب ٌحروي عهى خًسح يهٍغزاياخ يٍ دواء االيهودتٍٍ دواء االيهودتٍٍ و انًُرج يٍ تانًقارَح يع انًسرحضز انًزجعً َورفاسك عهى شكم حثوب ٌحروي اٌضا عهى خًسح يهٍغزاياخ يٍ شزكح فاٌزر االيزٌكٍح. ذى اعطاء كم انًُرجٍٍ نثًاٍَح وعشزوٌ يٍ انًرطوعٍٍ االصحاء. وقد ذًد اندراسح تاذثاع انرصًٍى انذي ٌشًم عٍُح دو اعطاء اندواء نهًرطوعٍٍ و هى صائًٍٍ ونفرزذٍٍ و تشكم عشوائً ويرقاطع وذفصم تٍٍ انفرزذٍٍ اسثوعٍٍ.ذى سحة عشزوٌ hplc/ms/ms. ساعح ثى حساب ذزاكٍز االيهودتٍٍ نكم يرطوع تواسطح طزٌقح 411يٍ كم يرطوع نفرزج ويٍ خالل ذزاكٍز اندواء نكم يرطوع ذى حساب عوايم حزكٍح اندواء فً انجسى وهً : cmax, tmax, auc0-t, auc0-, cmax/auc0-, z, t0.5, mrt, cl/f ,vd/f :)انًعدل( كًا ٌهً تانُسثح نهدواء انجٍُس وحسة ذسهسم عوايم حزكٍح اندواء انًذكورج اعالِ كًا ٌهًوكاَد انُرائج 1.99 ng/ml, 8.3 hours, 82.87 ng.hr/ml, 95.23 ng.hr/ml, 0.0219 hr -1 , 0.018 hr -1 , 38.5 hr, 56.2 hr, 60.9 l/hr and 3483 liters ج كًا ٌهً:ايا تانُسثح نهدواء انًزجعً فكاَد انُرائ 1.92 ng/ml, 7.9 hours, 76.3 ng.hr/ml, 89.31 ng.hr/ml, 0.0225hr -1 , 0.019 hr -1 , 36.7 hr, 59.9 hr, 69.5 l/hr, 3983.4 liters. دواء و تعد انرحهٍم االحصائً تاذثاع اندسرور االيزٌكً ندراسح انروافز وانركافؤ انحٍوي فقد ذثٍٍ اٌ اندواء انجٍُس يركافؤ حٍوٌا يع ان انًزجعً. hplc/ms/ms الحٍوي، طرٌقة كافؤاملودبٍن , حركٍة الذواء ، الت -الكلمات المفتاحٍة : corresponding author e. mail: drjaafarjaber@yahoo.com received: 4 / 2 / 2014 accepted: 15 / 4 /2014 iraqi j pharm sci, vol.23(1) 2014 bioequivalence and pharmacokinetics of amlodipine tablets 61 introduction amlodipine is a long-acting calcium channel blocker. amlodipine is chemically described as (r.s.) 3-ethyl-5-methyl-1-(2aminoethoxy methyl) 4(2 -chlorophenyl)-1,4-dihydro-6methyl-3,5-pyridinedi-carboxylate benzenesulphonate. its empirical formula is: c20h25cin2o5.c6h6o3s. the molecular weight is 567.1. amlodipine is indicated for the treatment of hypertension, chronic stable and vasospastic angina. absolute bioavailability has been estimated to be between 64 and 90%. the bioavailability of amlodipine is not altered by the presence of food. amlodipine is extensively (about 90%) converted to inactive metabolites via hepatic metabolism with 10% of the parent compound and 60% of the metabolites excreted in the urine. approximately 93% of the circulating drug is bound to plasma proteins. pharmacokinetics of amlodipine are not significantly influenced by renal impairment and patients with renal failure may therefore receive the usual initial dose. after oral administration of therapeutic doses of amlodipine, absorption produces peak plasma concentrations between 6 and 12 hours. elimination of amlodipine from plasma is biphasic with a terminal elimination half-life of about 30-50 hours. the therapeutic dose of amlodipine is 2.5-10 mg (1) . due to wide use of amlodipine in clinical practice, concomitant use with other drugs, high interindividual variation, in addition to, the pharmacokinetic characteristics which involve slow absorption, lone time to peak and long elimination half-life; many investigations were conducted to study the pharmacokinetics, pharmacody-namics, bioavailability, and bioequivalence of amlodipine after different dosage forms, after food/fluid intake, and in different populations (2-17) . determination of amlodipine concentrations in human plasma were achieved by hplc-ms method due to the low therapeutic doses of amlodipine and consequently very low plasma levels (nanograms/ml) (2-17) . bioequivalence studies are considered as pivotal part for registration of generic products since these studies are conducted to show that the rate and extent of bioavailability of the generic product is similar to the brand/innovator product. consequently, the effect(s) and the side effect(s) of the generic product are essentially equivalent to the brand/innovator product, and hence both products are interchangeable in clinical practice. the purpose of this study was to investigate the pharmacokinetics and relative bioavailability (bioequivalence) of two amlodipine formulations; a test product as tablet containing 5 mg amlodipine, in comparison to norvasc ® 5 mg tablet (pfizer usa) as the reference product, after administration to 28 healthy male adult subjects applying randomized crossover design. materials and methods study products information a test product as tablet containing 5 mg amlodipine. the reference product was norvasc ® tablet manufactured by pfizer usa. ethical consideration the study was carried out according to the provisions of the declaration of helsinki (18) and ich guidelines for good clinical practice (19) . the subjects provided informed consent before the commencement of the study. study design a fasting, single-dose, two-treatment, twoperiod, two-sequence, randomized crossover design was applied as recommended by fda guidance for bioavailability and bioequivalence. twenty eight subjects participated in the study. equal number of subjects (14 subjects) were randomly assigned to each dosing sequence of the treatments (test and reference formulations). the treatments were separated by two weeks washout interval between period i and period ii dosing. inclusion criteria twenty eight subjects were selected according to the following inclusion criteria: age between 18-48 years; normal bodymass-index (bmi) =18-28; non smokers; no drug or alcohol abuse; no history of contraindication and/or allergy to the drug and any related compounds; normal physical and clinical examinations including vital signs, hepatic, renal, respiratory, cardiac, gastrointestinal and psychiatric; normal clinical laboratory tests including biochemistry, hematology, routine urine analysis, negative for hiv, hepatitis b and c; no consumption of drugs for two weeks prior the study; no blood donation, hospitalization or participation in any study (clinical, pharmacokinetic, bioavailability or bioequivalence) within the last 2 months prior to the present study. drug product administration and the conditions of the study the drug was administered with 240 ml of water after an overnight fasting of 12 hours. no water was permitted 2 hours before and iraqi j pharm sci, vol.23(1) 2014 bioequivalence and pharmacokinetics of amlodipine tablets 62 after dosing. water was allowed 2 hours after dosing. standard diets (breakfast, lunch and dinner) were administered and were identical in both periods of the study. xanthine containing products were not allowed twelve hours before dosing and then twelve hours after dosing. grapefruit juice or beverages containing grapefruit were not allowed within the past week prior the study and until the completion of the whole study (both periods of the study). the subjects were not allowed to sleep or lie during the first four hours of drug administration, they remained seated upright. blood samples collection seven ml of blood samples were withdrawn via an indwelling cannula placed in the forearm anticubital vein at zero time (one hour before dosing), and then at: 1.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 16.0, 24.0, 36.0, 48.0, 72.0, 96.0, 120.0 & 144.0 hours post dosing. the blood samples were directly transferred to heparinized tubes and then immediately centrifuged for 5 minutes at 4000 rpm. the plasma samples were separated by polypropylene disposable tips and transferred to eppendorf tubes and then immediately stored at -20c until analysis for determination of amlodipine concentrations in plasma. clinical observations vital signs (blood pressure and pulse) of each subject were measured one hour before dosing and then at 3, 6, 9 and 12 hours post dosing. analytical procedure amlodipine’s concentrations in plasma were determined by a modified hplc-ms/ms method obtained from previously published hplc-ms/ms methods (20-23) . the validation of the method was evaluated following fda bioanalytical method validation criteria (24) and glp guidelines (25) . samples were extracted using liquid-liquid extraction technique. in each run, 1ml of plasma was alkalinized using 2 ml of 0.2 m borate buffer then extracted using 6 ml of hexane: ethyl acetate (1:1) extraction solvent. diazepam was used as an internal standard. the samples were then shaken for 20 minutes at 250 rpm and later centrifuged for 5 minutes at 4000 rpm. the upper organic layer was aspirated and placed into another tube and then evaporated to complete dryness under nitrogen stream. the residual samples were reconstituted with 50 µl of 1% acetic acid in methanol: water 1:1 to be ready for direct injection into the hplc. drug quantitation was done using a finnigan lcq duo ion trap mass spectrometer (finnigan thermoquest, usa) equipped with an (esi) source (finnegan) and run by: xcalibur 1.2 software (usa). calibration standard responses were linear over the range of 0.110 ng/ml of amlodipine concentrations in human plasma with a lower limit of quantitation (lloq) of 0.1 ng/ml. the plasma samples were analyzed after the completion of the clinical part of the study as recommended in bioequivalence studies. plasma samples of each subject for both periods were analyzed with their own calibration curve and quality control (qc) samples as one batch in a single run. for each run, six qc samples (dispersed evenly in a low-high and high-low sequence throughout the batch) were analyzed. no determination was done by extrapolation below the lloq and above the upper limit of quantitation (uloq) of the standard calibration curve as recommended by fda bioanalytical method validation guidance (24) . pharmacokinetic analysis kinetica 2000 (v4.0) software was used for all pharmacokinetic analysis of data. a noncompartmental analysis was applied for all pharmacokinetic calculations as recommended by fda and emea guidance in bioavailability and bioequivalence (26, 27) . the pharmacokinetic parameters clearance (cl), apparent volume of distribution (vd), elimination rate contant (k), terminal elimination half-life (t0.5), area under plasma concentration-time curve (auc), area under moment curve (aumc), and mean residence time (mrt) were calculated applying standard methods (28) . statistical analysis kinetica 2000 software (v4.0) was used for the statistical analysis of data. for the purpose of bioequivalence evaluation (26, 27, 29) , analysis of variance (anova), 90% confidence interval and schuirmann’s two one-sided t-test were applied. anova were carried out to account for the effects of the following sources of variation: treatment, period, sequence and subjects nested in sequence; on the pharmacokinetic parameters; cmax, tmax, auc0-t, auc0-, cmax/auc0-, z and t0.5. anova was also executed for the ln-transformed values of the pharmacokinetic parameters; cmax, auc0-t, auc0-, and cmax/auc0-. the difference between the pharmacokinetic parameters of both products were declared statistically insignificant at 5% significance level ( = 0.05) when p  0.05. the 90% confidence interval (ci) for the ratio of the mean test/mean reference (t/r) for the lntransformed values of the pharmacokinetic iraqi j pharm sci, vol.23(1) 2014 bioequivalence and pharmacokinetics of amlodipine tablets 63 parameters; cmax, auc0-t and auc0- were concluded bioequivalent if the lower ci ≥ 80% and the upper ci ≤125%, as recommended by fda guidance in bioavailability and bioequivalence (26, 29) . schuirmann’s two one-sided t-test (30) was also applied for the pharmacokinetic parameters; cmax, auc0-t, and auc0- as a check and further support of bioequivalence between both products. both products were concluded bioequivalent by the schuirmann’s test if the lower-t (tl)  (t0.05 –26 df) and the upper t (tu)  (t0.05 –26df). for the tmax values, the parametric point estimate was measured as the difference between the mean values of the test and the reference products. the acceptance limit for the tmax was within ± 20% of the mean value of the reference product. nonparametric test was also applied for the tmax. anova testing was applied for the pharmacokinetic parameters; mrt, cl/f and vd/f; of the test product versus the reference product. results and discussion clinical observations both test and reference products were well tolerated by all subjects. no incidences of serious side effects or adverse reactions were observed during the study. all the subjects who started the study participated to the end of the study. plasma concentrations the developed lc-ms/ms method presented in this study with lloq of 0.1 ng/ml was rapid, sensitive, precise, accurate and specific for quantitation of amlodipine in human plasma. therefore, the present method can successfully applied to analyze large number of plasma samples for pharmacokinetic, bioavailability and bioequivalence studies of amlodipine in human plasma. no significant differences (p > 0.05) in the plasma concentrations were found in all sampling time points between the test and the reference products. one hour before dosing (pre-dose sample), amlodipine was not detected in plasma samples of any subject indicating the absence of carryover effects and insuring a sufficient washout period. the drug was detected in plasma samples of 21 volunteers and 20 volunteers after 1.0 hour post dosing of the test product and norvasc ® tablets, respectively. this indicates rapid appearance of amlodipine in plasma. figure 1 shows the profiles of the mean amlodipine plasma concentration-time data of the 28 volunteers for each product. this figure indicates that the plasma concentration-time profiles of amlodipine for both products are to a very good extent superimposable. figure 1: mean plasma concentrations (± sd) of amlodipine after a single dose administration of a test product (tablet containing 5 mg amlodipine) and the reference product (norvasc 5 mg tablet) to twenty eight healthy male adult subjects. iraqi j pharm sci, vol.23(1) 2014 bioequivalence and pharmacokinetics of amlodipine tablets 64 table (1) mean (± sd) of pharmacokinetic parameters of amlodipine after a single dose administration of a test formulation (tablet containing 5 mg amlodipine) and the reference formulation (norvasc ® 5 mg tablet) to 28 healthy male adult subjects. pharmacokinetic parameters test formula reference formula mean ± sd mean ± sd cmax (ng/ml) 1.99 0.817 1.92 0.94 auc0-t (ng.hr/ml) 82.87 44.52 76.30 45.68 auc0- (ng.hr/ml) 95.23 46.12 89.31 48.83 cmax /auc0- (hr -1 ) 0.0219 0.0059 0.0225 0.0057 tmax (hr) 8.3 2.19 7.9 2.7 z (hr -1 ) 0.018 0.0057 0.019 0.0048 t0.5 (hr) 38.5 10.07 36.7 11.21 mrt (hr) 56.2 11.78 59.9 15.68 cl/f (l/hr) 60.9 33.54 69.5 42.09 vd/f (l) 3483 1954.8 3983.4 2467.5 cmax = maximum concentration of drug in plasma, obtained directly from the concentration versus time curves of individual volunteers. tmax = the time to attain cmax, obtained directly from the concentration versus time curves of individual volunteers. auc0t = area under the plasma concentration-time curve from time zero to tlast, calculated by trapezoidal rule. auct- = extrapolated area under the plasma concentration-time curve from tlast to infinity, calculated as clast/z. auc0- = total area under the plasma concentration-time curve from time zero to infinity, calculated from the sum of auc0-t + auct-. z = first order terminal elimination rate constant, estimated by linear regression of not less than 3 points of the last points at the terminal phase of the log-concentration versus time curves of individual volunteers. t0.5 = first order terminal elimination half-life, equal to 0.693/z. clast = last measurable concentration which meet or exceed the lower limit of quantitation. tlast = time at which clast occur. mrt = mean residence time, calculated as aumc0-/ auc0- . aumc0- = area under the moment curve from time zero to infinity. cl/f = oral body clearance, calculated as f x dose/ auc0-. dose=5 mg , f=0.7. vd/f = oral volume of distribution, calculated as cloral / z . f = oral bioavailability. statistical evaluation anova tests were performed for all the calculated pharmacokinetic parameters presented in table 1, whereas 90% ci and schuirmann’s two one-sided t-test (table 2) were applied only for the pharmacokinetic parameters cmax, auc0-t and auc0-, since these three parameters are considered as the primary pharmacokinetic parameters for bioequivalence evaluation as recommended by fda guidance (26, 29) . anova tests for the cmax, auc0-t, auc0, tmax, cmax/auc0-, z and t0.5 values and for the corresponding ln-transformed values of cmax, auc0-t, auc0-, and cmax/auc0-, revealed no significant effects (p > 0.05) for the sources of variation: treatment, period and sequence. however, for the subjects nested in sequence, a significant effect (p < 0.05) was found which may be due to the interindividual variation in the above mentioned parameters as shown in table 1. moreover,anova tests for mrt, cl/f and vd/f showed no significant difference (p > 0.05) between the test and the reference formulas. these findings support the similarity in the pharmacokinetic behaviors of the test and the reference formulas. the calculated ranges of the 90% ci (table 2) for the ln-transformed values of cmax, auc0-t and auc0-, were well within the fda bioequivalence acceptance criteria (26, 29) . the ranges of schuirmann’s two-one-sided t-test (table 2) for the above pharmacokinetic parameters were also well within the bioequivalence acceptance criteria (29, 30) . moreover, power calculations iraqi j pharm sci, vol.23(1) 2014 bioequivalence and pharmacokinetics of amlodipine tablets 65 for cmax and auc demonstrated that sample size of 28 subjects is adequate to obtain power above 80% for bioequivalence evaluation of amlodipine tablets. therefore, according to fda guidance on bioequivalence, it is concluded from the results of the above statistical tests that the test product and the reference brand product (norvasc ® tablet) are bioequivalent. table (2) 90% confidence interval and schuiramann’s two one-sided t-tests for the pharmacokinetic parameters of the test versus the reference products. pharmacokinetic parameters t/r geometric mean ratio 90% confidence interval* schuiramann’s two one-sided t-test** lower limit upper limit lower limit upper limit cmax 1.03 95.29 109.82 3.3996 6.7666 auc0-t 1.07 100.3 115.23 2.3308 8.0969 auc0- 1.04 97.76 112.48 2.8818 7.4447 * acceptance criteria = lower limit  80 and upper limit  125.0. ** acceptance criteria = lower limit and upper limit  1.7081. conclusion the present study introduced pharmacokinetic characteristics of amlodipine after therapeutic oral dose to healthy male adult subjects. the pharmacokinetics of the test product are statistically similar to the reference brand product (norvasc ® tablet) produced by pfizer usa. therefore, according to fda guidance on bioavailability and bioequivalence, it is concluded that the test product is bioequivalent to norvasc ® tablet. therefore, both products are interchangeable in therapy with amlodipine, and the test formula can be considered presecribable as norvasc ® tablets produced by pfizer usa. references 1. physician’s desk reference ® (pdr), 68 th edition, 2014; norvasc ® product information, pfizer usa . 2. vincent m, uma k, akula tb, rong w, bharat d. pharmacokinetics of a novel orodispersible tablet of amlodipine in healthy subjects. j bioequiv availab 2013; 5:2. 3. gorain b, choudhury h, halder d, sarkar ak, sarkar p, biswas e, ghosh b, pal tk. a comparative pharmacokinetic study of a fixed dose combination for essential hypertensive patients: a randomized crossover study in healthy human volunteers. drug res (stuttg) 2013; 63 (4): 177-84. 4. noh yh, lim hs, kim yh, choi yh, sung hr, jin sj, lim j, bae ks. pharmacokinetic interaction of telmisartan with s-amlodipine: an open-label, two period crossover study in healthy male volunteers. clin ther 2012; 34 (7): 1625 35. 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establishing bioequivalence. u.s. department of health and human services. food and drug administration. center for drug evaluation and research (cder), january, 2001. 30. shein-chung c and jen-pei l. design and analysis of bioavailability and bioequivalence studies. second edition. revised and expanded. marcel dekker, inc.: new york, 2000. 31. lacey lf, keene on, duquesony c, bye a. evaluation of different indirect measures of rate of drug absorption in comparative pharmacokinetic studies. j pharm sci 1994; 83: (2): 212-5. 32. endrenyi l, tothfalusi l. secondary metrics for the assessment of bioequivalence. j pharm sci 1997; 86 (3): 401-2. iraqi j pharm sci, vol.23(1) 2014 bioequivalence and pharmacokinetics of amlodipine tablets 67 33. duquesony c, lacey lf, keene on, bye a. evaluation of different partial aucs as indirect measures of rate of drug absorption in comparative pharmacokinetic studies. eur j pharm sci 1998; 6 (4): 259-64. 34. flynn jt, nahata mc, mahan jd, portman rj. population pharmacokinetics of amlodipine in hypertensive children and adolescents. j clin phartmacol 2006; 46 (8): 905-16. iraqi j pharm sci, vol.25(1) 2016 serum ferritin in women with breast cancer 23 detection of serum ferritin in women with breast cancer eman s. nassir *, 1 * department of clinical laboratory science, college of pharmacy, university of baghdad ,baghdad, iraq. abstract breast cancer is one of the most common cancers in females. in iraq there are noticeable elevation in incidence rates and prevalence of advanced stages of breast cancer. ferritin is intracellular iron storage protein abundant in circulation and its main application in differential diagnosis of anemia. the level of serum ferritin was found raised in various cancers including breast cancer. the aim of this study was to assess whether the serum ferritin concentration would be altered in iraqi women with breast cancer and it could be related to progression of disease. sixty eight females participated in this study .the mean age of these females was 53.25± 9.52 .the level of serum ferritin was measured in 24 iraqi women of early stage of breast cancer (stage i and ii) and 24 iraqi women of advanced stage (iii and iv). these levels were compared with 20 healthy females as controls. serum ferritin was estimated by using enzyme linked immune sorbent assay method. this serum ferritin was found to be raised in all breast cancer patients (p<0.05) as compared to controls. the rise in ferritin level was significant in advanced stage (p<0.05) as compared to early stage. thus the estimation of ferritin may aid in diagnosis, assessment of severity and monitoring of iraqi women with breast cancer. key words: ferritin, breast cancer, iraq. قياس مستوى الفرتييه في المصل في النساء المصابات بسرطان الثدي ايمان صادق ناصر *،1 * . فشع انعهٕو انًخخبشٚت انسشٚشٚت، كهٛت انصٛذنت، جايعت بغذاد، بغذاد انعشاق الخالصة انثذ٘ ٔاحذ يٍ اْى انسشطاَاث انشائعت نذٖ االَاد فٙ انعشاق ُْاك صٚادة ٔاضحت فٙ َسبت حذٔد انًشض ٚعخبش سشطاٌ ٍ ْٕ انبشٔحٍٛ انًسؤٔل عٍ خضٌ انحذٚذ يٕجٕد داخم انخالٚا ٔاٚضا فٙ انذٔسة انذيٕٚت. ٛٔٔصٕنّ انٗ يشاحم يخطٕسة .انفشحٛ ٍ انًٕجٕد فٙ يصم انذو ٔجذ اَّ ٚشحفع فٙ حاالث انسشطاٌ ٔيٍ ٛسخٕٖ انفشحٛاْى حطبٛقاحّ ْٕ نهخشخٛص انخًٛض٘ الَٕاع فقش انذو. ي يصم انذو ٚخغٛش فٙ انُساء انعشاقٛاث فٙضًُٓا سشطاٌ انثذ٘. انٓذف يٍ ْزِ انذساست ْٕ نخقٛٛى فًٛا ارا كاٌ يسخٕٖ انفشحٍٛٛ ٛت )يشاحم انًشض(.انًصاباث بسشطاٌ انثذ٘ ْٔم اٌ يسخٕاِ فٙ انذو نّ عالقت بخقذو انحانت انًشض ٍ فٙ ٛفٙ ْزِ انذساست حى قٛاط يسخٕٖ انفشحٛ ,25,9 ,259ثًاَٛت ٔسخٌٕ ايشاة شاسكج فٙ ْزِ انذساست ٔيعذل اعًاسْى ايشأة عشاقٛت 92( ٔاٚضا حى قٛاسّ فٙ 9ٔ 1ايشأة عشاقٛت يصابت بسشطاٌ انثذ٘ فٙ انًشاحم االبخذائٛت )يشاحم 92يصم انذو نـ ايشأة عشاقٛت 92( ٔحى يقاسَت انُخائج يع االصحاء باخز عُٛاث دو يٍ 2ٔ 2ٌ انثذ٘ فٙ انًشاحم انًخقذيت )يشاحم يصابت بسشطا نقذ ٔجذ اٌ يسخٕٖ فحص انًُاعٙ انًشحبظ باالَضٚى . حى فٛاط يسخٕٖ انفشحٍٛٛ فٙ يصم انذو باسخعًال طشٚقت غٛش يصابت بانًشض. بسشطاٌ انثذ٘ يقاسَت يٍ انُساء االصحاء ٔاٚضا ٔجذ اٌ يسخٕٖ انفشحٍٛ فٙ انُساء انًصاباث انفشحٍٛٛ يشحفع فٙ انُساء انًصاباث بسشطاٌ انثذ٘ فٙ انًشاحم انًخقذيت اعهٗ يٍ يثٛالحٓى فٙ انًشاحم االبخذائٛت يٍ ْزِ انُخائج َخٕقع اَّ يٍ انًًكٍ االسخفادة يٍ قٛاط انثذ٘ ٔيعشفت يذٖ حقذو انحانت ٔحطٕسْا. نًخابعت انُساء انًصاباث بسشطاٌ يسخٕٖ انفشحٍٛٛ. الفرتييه، سرطان الثدي، العراق. -الكلمات المفتاحية : introduction ferritin is the protein that stored iron (1) . this protein is synthesized in the liver, spleen, myocardium, placenta and other tissues. it is a large macromolecule (450kda) which consists of 24 subunits that form protein shell (apoferritin) around an insoluble core of stored iron. there are 2 types of subunits, the basic l and the acidic h type (2) . although the two subunits share approximately 55% of their amino sequences, in addition to their multihelical three dimensional structures, they are functionally different (3) . the h subunit has ferroxidase activity and it is responsible for the oxidation of ferrous iron into ferric iron, whereas the l subunit contribute to the stable storage of iron in the ferritin core. the efficient storage of iron in ferritin requires cooperation of both (4) . ferritin is an abundant protein in circulation which is known as serum ferritin in addition to its intracellular form. serum ferritin was first detected in 1948 in animals experiencing hepatic cirrhosis or shock (5) .these observation was later confirmed in humans with different types of liver disease (6) . serum ferritin is a dependable indicator of the body’s iron stores. its level is significantly decreased in individuals suffering from iron deficiency anemia or undergoing phlebotomy. 1 corresponding author e-mail: emansadiq1976@yahoo.com received: 4/11/ 2015 accepted: 20/1/2016 iraqi j pharm sci, vol.25(1) 2016 serum ferritin in women with breast cancer 24 in contrast serum ferritin levels are increased in patients with iron over load (hemochromatosis or hemosidriosis), infection or inflammation, malignancies and damage of liver tissues (7, 8) . ferritin has recently been implicated in the pathogenesis of disease including cancer. a number of mechanisms such as pro-oxidant and pro-inflammatory pathways are responsible for this (9) . ferritin has long been disreputable for its association with breast cancer either as cause or result of the disease (10) . breast cancer is the mainly frequent cancer among women, comprising about 23% of all females’ cancer (11) . it is also the most important cause of cancer-related death worldwide, case fatality rates being highest in low resource countries (12) . in iraq, breast cancer is the commonest type of female malignancy accounting for approximately one third of the registered female cancers according to the latest iraqi cancer registry. this shows that the breast is the chief cancer site among the iraqi women (13) . as projected by the world health organization, early detection and screening of the disease, especially when combined with adequate therapy, suggest the most immediate hope for decline in breast cancer mortality (14) . the tumor markers in breast cancer like ca 15-3 and cea are generally useful in followup of patients with metastatic disease with other diagnostic technique like x-rays and ct scan. this disease is still in the need of more precise biomarkers which might help in early detection, assessment of severity and for prediction of therapy response (15) . therefore this study was planned to estimate the level of serum ferritin in patients with breast cancer at different stages (early and advance). the aim was to inspect potential relations between the level of serum ferritin in iraqi women with breast cancer and the progression of disease. material and methods the present study was conducted at the teaching hospital of oncology-baghdad under specialized senior supervision for the period of november 2013july 2014. institutional ethics committee approval was obtained prior to initiation of the study. an informed written approval was recorded from all the study subjects previous to their enrolment in the study. sixty eight females were enrolled for this study. out of these, 48 were patients with breast cancer (age range 3369) and 20 were healthy females (age range 35-67).the patients were staged according to american joint committee of cancer (ajcc) staging 2010 (tnm) (9) . the subjects were divided into three groups: group a: 20 healthy females with mean age 51.9 ± 9.71. group b: 24 patients with histopathologically proven breast cancer in early stage of disease with mean age 53.1± 10.25 (stage i and ii, ajcc-tnm stage). group c: 24 patients with histopathologically proven breast cancer in advanced stage of disease with mean age 55.8 ± 8.80(stage iii and iv, ajcc-tnm stage). inclusion criteria: healthy non anemic females were included in this study as control group .for patients group, subjects with untreated histopathologically proven breast cancer were chosen. exclusion criteria: for control group, subjects with fasting plasma glucose more than 120mg /dl and blood pressure more than130 /85mmhg were excluded from this study. for patients group, subjects with history of liver or kidney damage, acute inflammatory and infectious disease, anemia (hb<10gm%), diabetes and those on medication like iron supplement, or thyroxin or having benign tumor or mass anywhere else in the body are excluded from the study as any of these factors may influence the serum ferritin levels. 5ml fasting blood sample was withdrawn from median cubital vein of each study participant with all necessary aseptic precautions. all samples were allowed to stand for 10 min to obtain serum. after centrifugation, non hemolysed sera were kept at -20 0 c for subsequent analysis as early as possible. ferritin was estimated by using enzyme linked immunosorbent assay method (elisa).the ferritin elisa kit is based on the sandwich principle. this commercially available kit was supplied by monobind inc, usa (accu-bind). serum sample were allowed to incubate in wells coated with specific anti-ferritin antibodies. this was further added with horse-radish peroxidase conjugated anti ferritn antibodies. the amount of bound peroxidase is in direct proportion to the content of ferritin in the sample and the intensity of color formed is proportional to the quantity of ferritin in the samples. later on stopping the reaction, the color intensity of final mixture was measured at 450nm wavelength with micro plate reader (10) . statistical analysis and interpretation of data were done by using student’s unpaired “t” test (for two group comparison) and anova test (for comparison of three groups of variable sample size). all the data have been expressed as mean ± sem [standard error of mean]. the levels of significance were calculated for all iraqi j pharm sci, vol.25(1) 2016 serum ferritin in women with breast cancer 25 three groups. probability value “p” greater than 0.05 was considered as statistically nonsignificant alteration while p less than 0.05 was considered to be statistically significant. all statistical analyses were carried out using spss (version 17) software. results the demographic characteristics of healthy women and patients with breast cancer in the current study were described in table (1). table (1): demographic characteristics of healthy women and patients with breast cancer. age(years) less than 45 more than 45 group a 5(25%) 15(%75) group b 4 (16.6%) 20 (83%) group c 3 (13%) 21 (87%) marital status married single widow/divorced group a 18(90%) 2(10%) group b 18 (75%) 4 (17%) 2 (8%) group c 20 (83%) 3(13%) 1 (4%) family history with breast cancer positive negative group a 20(100%) group b 15 (62.5% 9 (37.5% group c 13 (54%) 11 (46%) bmi normal over weight group a 7(35%) 13(65%) group b 1(4%) 23 (96%) group c 2(8%) 22(92%) bmi: body mass index describes relative weight for height. the healthy weight falls between bmi values of 18.524.9. overweight falls between 25-30 (16) . table (2): ferritin concentrations in healthy and women with breast cancer. parameter group a mean ±sem group b mean ±sem group c mean ±sem ferritin serum level (ng/ml) 44.1±6.19 196.6±27.68 * 248.9±32.32 *,** * significant difference from group a (p<0.05) ** significant difference from group b (p<0.05) in group a, the determined mean of serum ferritin was (44.1) while in group b the determined mean of serum ferritin was (196.6), and in group c, the determined mean of serum ferritin was (248.9). the levels of ferritin were found to be significantly raised in patients with breast cancer (group b and c) as compared to control (group a) as shown in table (2).significant difference (p<0.05) was observed when compared group b and c to group a. statistically significant difference (p<0.05) was observed between group b and c. discussion cancer is the fourth ranked cause of death in the eastern mediterranean region (emr), after cardiovascular diseases, infectious /parasitic diseases and injuries according to world health organization (who) mortality estimates (17) . the major increase in cancer incidence among the who regions in the emr, where breast cancer is recorded as the commonest type of female malignancy in almost all national cancer registers (18) . in iraq in addition to being the most important cancer there are noticeable increase in incidence rates and prevalence of advanced stages at presentation associated with more aggressive tumor behaviors, resulting in larger fatality rates (14) . in this study, breast cancer was diagnosed in 48 females. the percentage of patients with breast cancer increased with increasing age over 45. in table (1) we observed that 83% of the patients in group b are in age over 45 and 87% of the patients in group c are in age over 45. approximately half of cancer cases in emr occur before the age of 55 and the age standardized incidence rates of all cancer in this region is expected to double as risk factor exposure increases as revealed by who estimates (17) . in table (1) high numbers of patients with positive family history observed in this study, 63% of the patients in group b were with iraqi j pharm sci, vol.25(1) 2016 serum ferritin in women with breast cancer 26 positive family history and 54% of the patients in group c were with positive family history. the customary consanguineous marriages which are known to be common throughout this region could be the main cause (14) . weight gain and being overweight are risk factor for breast cancer in women who have gone through the menopause (19) . in this study as we observed in table (1) 96% of the patients in group b were overweight and 92% of the patients in group c were overweight. it is believed that obesity is proinflammatory state as it results in release of inflammatory mediators that promote tumor growth (20) . one of the most vital nutritional elements required in physiological activities in the body is iron. many processes like transport of oxygen, generation of energy and synthesis of dna are dependent on iron (21) . despite the fact that iron is essential for growth and development of cells, it has been proven to be harmful when present in excess amount in the body. in recent times, several lines of evidence have established that the iron storage protein which is termed ferritin is a multi-functional protein that have possible role in proliferation, angiogenesis, immunosuppression and delivery of iron. in the context of cancer ferritin is detected at higher levels in the sera of many cancer patients (7) . in the current study as we observed in table (2) the levels of ferritin were found to be significantly raised in breast cancer patients (in both groups b and c) as compared to control (group a). this increase may either due to increase expression of a tumor derived protein which interferes with iron metabolism or due to nonspecific effect of malignancy on reticule-endothelial iron metabolism (22) . the rise in ferritin levels is reported to be linked with more advanced stage breast cancer (22, 23) . in our study, also the levels in group c were significantly higher as compared to group b (p<0.05). elevation of serum ferritin levels may be attributed to increased iron requirement by malignant cells for growth and for modulation of transferrin receptor. transferrin is considered potential markers for identifying cells undergoing divisional activity and requiring the incorporation of additional iron (24) . in addition to increased synthesis by malignant cells, other causes of raised ferritin levels include presence of inflammation, hepatic necrosis due to metastasis and reduced hepatic clearance of ferritin (25) . hypoxia often associated with solid tumors and it considers one of the factors that encourage production of ferritin (26) .ferritin receptor expression is post transcriptionally regulated by conserved mrna sequence termed iron responsive element (ire), to which a transacting protein called iron regulatory protein (irp) is bound. early studies demonstrated that hypoxia induces the specific and reversible expression of ferritin. this induction occurs primarily on post transcriptional level due to decreased mrna binding capacity of irp (27) . the tumor markers being presently used for breast cancer like ca15 -3 and cea are not specific and are found raised in other disease too. the utility of these markers is more reliable if analyzed in conjunction with other markers (9) . conclusion it is concluded that ferritin may help in assessing the severity and monitoring of breast cancer patients. and it might prove more useful if combined with other biomarkers for breast cancer. further prospective studies, of a large number of subjects, are required to confirm such a statement and to validate the usefulness of ferritin estimation in combination with other tumor markers references 1. marinova m and vladimirova l. atomic absorption assessment of mineral iron quantity in ferritin. bulg j phys. 2009, 36: 139-44. 2. koort am and viljoen m. ferritin and ferritin isoforms: structure, function relationships, synthesis, degradation and secretion. arch physiol biochem 2007; 113:55-64. 3. lawson dm, artymink pj, yewdall sj, smith jm. solving the structure of human h ferritin by genetically engineering intermolecular crystal contacts. nature 1991; 349 (6309): 541-544. 4. al khateeb aa, han b, connor jr. ferritin stimulates breast cancer cell through an iron independent mechanism and is localized within tumor associated macrophages. breast cancer res treat 2013, 137: 733-744. 5. abraham m and ephraim s. hepatorenal factors in cirrulatory homeostasis; the identification of the hepatic vasodepressor substance with ferritin. j. biol chem 1948; 176:771-787. 6. reissmann kr and dietrich mr. on the presence of ferritin in the peripheral blood of patients with hepatocellular disease. j. clin invest, 1956; 33: 588-595. 7. alkhateeb aa and connor j.r. the significance of ferritin in cancer: antioxidant, inflammation and tumorigenesis. biochimica et biophysica acta 2013; 1836:245-254. iraqi j pharm sci, vol.25(1) 2016 serum ferritin in women with breast cancer 27 8. knovich ma. ferritin for the clinician. blood rev. 2009; 23: 95-104. 9. dhankar r .evaluation of ferritin and nitric oxide levels in breast cancer. american journals of cancer science 2014; 3:1-6. 10. norkhede hp. breast cancer and serum ferritin-menopausal status perspective: menopause a fickle determinant. int j res med sci 2014; 2(1): 258-263. 11. alwan na, mualla f.h. promoting clinical breast examination as a screening tool for breast cancer in iraq. iraqi national journal of nursing specialties. 2014; 27(1): 76-82. 12. anderson bo. et al. guideline implementation for breast health care in low income and middle-income countries: overview of the breast health global. initiative global summit. 2007. cancer 2008; 113(8): 2221-43. 13. al-hashimi mm. breast cancer in iraq, incidence trends from 2000-2009. asian pacific journal of cancer prevention 2014; 15 (1): 281-286. 14. alwan na. breast cancer: demographic characteristics and clinical-pathological presentation of patients in iraq. eastern mediterranean health journal 2010; 16(11): 1159-1164. 15. gast mc. clinical proteomics in breast cancer: a review. breast cancer res. treat. 2009; 116: 17-29. 16. whitney e and rolfes sr. understanding nutrition (10 th ed.).thomson learning inc., wads worth, 2005; p. 262-263. 17. revised globle burden of disease (gbd), who 2002 estimate. geneva, world health organization, 2003. 18. who/emro towards a strategy cancer control in the eastern mediterranean region. 1 st ed. cairo, world health organization regional office for the eastern mediterranean 2010. 19. revers gk, pirie k, beral v .cancer incidence and mortality in relation to body mass index in million women study: cohort study bmj 2007, 335 (7630); 1134. 20. key tj, appleby pn, reeves gk. body mass index, serum sex hormones and breast cancer risk in menopausal women. j nat cancer inst, 2003, 95: 1218-1226. 21. jian j. iron and menopause: does increased iron affect the health of postmenopaused women. antioxidant redox signal. 2009; 11(12): 2939-43. 22. shpyleva si, tryndyak vp, kovalchuk o.role of ferritin alterations in human breast cancer cells. breast cancer res. treat 2011, 126; 63-71. 23. mishra s, sharma dc, shamra p. studies of biochemical parameters in breast cancer with and without metastasis. indian j clin biochem 2004, 19: 71-75. 24. elliot rl, elliot mc, wang f. carcinoma and the role of iron metabolism. a cytochemical tissue, culture and ultrasonic study. ann ny acad sci. 1993, 698: 159166. 25. toyokuni s. iron and carcinogenesis: from fenton reaction to target genes. redox rep. 2002, 7: 189-197. 26. torti fm. regulation of ferritin genes and protein. blood 2002; 99: 3505-10 27. .qi y, jamindar tm, dawson gj .hypoxia alters iron homeostasis and induces ferritin synthesis in oligodendrocytes. neurochem 1995; 64: 2458-64. iraqi j pharm sci, vol.29(1) 2020 hrqol among a sample of chronic hepatitis b patients doi: https://doi.org/10.31351/vol29iss1pp33-40 33 health-related quality of life among a sample of chronic hepatitis b patients in al-najaf province /iraq. hanan n. najaf *1 and dheyaa j. kadhim ** .department of clinical pharmacy, college of pharmacy, university of kufa, najaf, iraq* .department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq** abstract the effect of physical and mental health on the feelings of personal welfare are known as health-related quality of life. infection with hepatitis b virus is a major global health problem. health-related quality of life was emerged as an important consideration in the care of patients with chronic hepatitis b infection. the aim of the current study was to measure health-related quality of life among a sample of chronic hepatitis b patients in alnajaf city/iraq. the current study was cross-sectional study carried out on (104) already diagnosed chronic viral hepatitis b patients who attended the gastroenterology and hepatology center/al-sader medical city/najaf during november 2018 to may 2019. in addition, (100) apparently healthy subjects were included as a control group. health related quality of life was measured using the arabic version of the short form 36. the healthrelated quality of life of patients with chronic hepatitis b infection was affected by the disease in a highly significant manner where the median scores for all health-related quality of life domains were significantly lower in patients than in controls. physical functioning score was negatively correlated to age, significantly higher in male patients, significantly higher in highly educated patients, significantly higher in single patients, and significantly higher in patients with no children. role limitation due to physical health score was negatively correlated to age, significantly higher in those with no children. energy/fatigue score was negatively correlated to age, and significantly higher in male patients. social functioning score was significantly higher in male patients. pain score was negatively correlated with age. physical functioning score was negatively correlated to serum albumin. social functioning and general health score were negatively correlated to viral load. in conclusion, chronic hepatitis b patients showed significantly lower health-related quality of life scores in various domains compared to healthy control subjects in al-najaf province /iraq. keywords: health-related quality of life, hepatitis b, short form 36, al-najaf, iraq. في مدينة النجف/ نوع )ب( المزمن لدى عينة من مرضى التهاب الكبد الفيروسي الصحية الحياة جودة العراق **و ضياء جبار كاظم 1*،حنان نجم نجف ،العراق.*فرع الصيدلة السريرية، كلية الصيدلة، جامعة الكوفة، النجف ** فرع الصيدلة السريرية، كلية الصيدلة، جامعة بغداد، بغداد، العراق. الخالصة ب( نوع الكبد الفيروسي بالتهابيُعرف تأثير الصحة البدنية والعقلية على مشاعر الرفاهية الشخصية بجودة الحياة الصحية. تعد اإلصابة مهًما في رعاية المرضى المصابين بعدوى التهاب الكبد الفيروسي المزمن ˝مشكلة صحية عالمية كبرى. برزت جودة الحياة الصحية باعتبارها عامال في مدينة )ب(نوع المزمن الفيروسي قياس جودة الحياة الصحية بين عينة من مرضى التهاب الكبد هو . كان الهدف من هذه الدراسة الحالية )ب(نوع )ب(نوع ضى التهاب الكبد الفيروسي المزمنيض من مر( مر104النجف / العراق. كانت الدراسة الحالية عبارة عن دراسة مقطعية أجريت على ) 2018)تشرين الثاني المشخص بالفعل والذين حضروا الى مركز أمراض الجهاز الهضمي والكبد / مدينة الصدر الطبية / النجف خالل الفترة من (.2019لغاية ايار ( شخص من االصحاء ظاهريا كمجموعة ضابطه. تم قياس جودة الحياة الصحية باستخدام النسخة العربية من 100باإلضافة الى ذلك تم اشراك ) بطريقة )ب(نوع زمن سؤال. تأثرت جودة الحياة الصحية للمرضى المصابين بعدوى التهاب الكبد الفيروسي الم 36االستبيان القصير المكون من ارتبطت درجة األداء البدني بشكل بالغة األهمية حيث كانت النتائج لجميع جوانب الحياة الصحية أقل بكثير في المرضى من المجموعة الضابطة. لمرضى الذين ليس لديهم سلبي بالعمر ، وكانت أعلى بشكل ملحوظ في المرضى الذكور وذوي التعليم العالي ، وغير المتزوجين ، وأعلى بكثير في ا أطفال. ارتبطت درجة الحد من أداء المريض بسبب الصحة الجسدية بشكل سلبي مع العمر ، وهو أعلى بشكل ملحوظ في أولئك الذين ليس لديهم اء االجتماعي أعلى بشكل سلبي مع العمر ، وأعلى بكثير في المرضى الذكور. وكانت درجة األد مرتبطةأطفال. كما كانت درجة الطاقة / التعب سلبا إلى نسبة مرتبطةبشكل سلبي مع التقدم في السن. كما كانت درجة األداء البدني مرتبطةبكثير في المرضى الذكور. و كذلك درجة األلم كانت نسبة الفيروس في الدم. معدرجة األداء االجتماعي والصحة العامة ارتبطتالزالل في المصل. و كذلك االصحاء ةفي العديد من المجاالت مقارنة بمجوع الصحيةفي جودة الحياة ا انخفاًضا ملحوظً نوع )ب( مرضى التهاب الكبد المزمن ، أظهر كنتيجة في مدينة النجف / العراق.الضابطة ، العراق.النجف مدينة ، 36التهاب الكبد الفيروسي نوع )ب(، االستبيان القصير المكون الصحية،جودة الحياة : الكلمات المفتاحية 1corresponding author e-mail: e-mail: han173004@gmail.com received: 9/ 7/2019 accepted: 7/ 9 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp33-40 iraqi j pharm sci, vol.29(1) 2020 hrqol among a sample of chronic hepatitis b patients 34 introduction hepatitis b virus (hbv) infection is one of the widespread and most important public health problems worldwide (1). globally, about 2 billion people have been infected with hepatitis b virus, and about 5% of them have chronic infections (2). in iraq, the prevalence rate of hepatitis b surface antigen (hbsag) in apparently healthy individuals was 1.6% (3). hepatitis b virus infection is transmitted by perinatal, sexual, or percutaneous exposure; sharing of household items such as razors and toothbrushes; and close personto-person contact with open cuts and sores (4). infants born to mothers infected with actively replicating hbv have a 70%-90% risk of acquiring the infection (5). chronic hepatitis b (chb) infection is characterized by the persistence of hbsag and serum hbv-dna levels detectable for more than 6 months (6). there are about 250 million individuals with chb around the world (7). patients with chb have to cope with many recurrent symptoms in their long-term disease histories and are at higher risk of developing end-stage liver diseases such as liver failure, cirrhosis, and hepatocellular carcinoma (hcc) (8). quality of life is a concept that reflects the negative and positive aspects of the life of an individual. the impact of mental and physical health on the feeling of wellbeing is known as health-related quality of life (hrqol) (9). health related quality of life is a multifactorial construct that describes individuals’ perceptions of their psychological, physical, and social functioning. thus, hrqol is a more holistic assessment than clinical parameter, especially in chronic disease in which mortality is not an immediate concern, because it also considers a patient’s functional health and well-being. this is particularly important in chb, in which the natural history is complex and includes a number of phases (6). hrqol was found to be decreased in chronic diseases like diabetes mellitus and hypertension (10). there have been some studies focusing on hrqol of chb patients (11-13). the aim of the current study was to measure hrqol among a sample of chronic hepatitis b patients in alnajaf province /iraq. patients and methods patients the current cross-sectional study was carried out on (104) already diagnosed patients with chronic hepatitis b who were admitted the gastroenterology and hepatology center/al-sader medical city/najaf during november 2018 to may 2019. in addition, (100) apparently healthy subjects were included as a control group. inclusion criteria the inclusion criteria for the current study were: 1-patients having chronic hepatitis b, who were aged 18 years or more of either sex and who were accepted to participate in the study. 2-disease duration (since diagnosis) of at least six months or more. exclusion criteria the exclusion criteria for this study were: 1-patient who had a hearing, speech or cognitive deficits that would impair understanding of the questions. 2-patient who had liver cancer or clinical evidence of decompensated liver cirrhosis (ascites, history of hepatic encephalopathy, or history of variceal bleeding). 3-concomitant medical illness (such as chronic renal failure, chronic lung disease, hypertension, heart disease, dm, stroke). 4-patient who take antidepressant drugs, or being on treatment for any neurological or psychological diseases. 5-pregnant and lactating women. 6-patients providing incomplete information during completion of the questionnaire also will excluded from the study. method the questionnaires in the current study, health related quality of life was measured using the arabic version of the short form 36 (sf-36). the short form 36 (sf-36) is a widely used self-administered questionnaire which provide a reliable assessment of hrqol (14). it includes eight items: general health, role limitations due to physical problems, mental health, physical functioning, vitality, social functioning, role limitations due to emotional problems, and bodily pain. items with higher scores indicate better health conditions (15). administration of questionnaires the data related to the study were collected by the researcher herself. when the patients arrived to the hospital to made their programmed laboratory data and receive their treatment, they were asked if they accept to participate in the study, if they accepted to participate, a complete explanation to the questions in the questionnaire was done and each patient spent about 5 minutes to fill the research questionnaire completely . statistical analysis data were collected, summarized, analyzed using two software programs, statistical package for social sciences (spss) version 23 and microsoft office excel 2010. categorical variables were presented as number and percentage. quantitative variables were initially analyzed for normality distribution kolmogorovsmirnov test. therefore, quantitative variables were described as mean ± standard deviation or median (interquartile range). comparison of mean values between any two groups carried out using independent sample t-test or mann whitney u test in case of normally distributed data or not normally disturbed data, respectively, whereas kruskal wallis test was used to evaluate difference among three groups or more. association between any two categorical variables was done using chi square test. spearman correlation test was used to assess variables. pvalue was considered significant when it is equal or less than 0.05 and highly significant when it is equal or less than 0.001. iraqi j pharm sci, vol.29(1) 2020 hrqol among a sample of chronic hepatitis b patients 35 results demographic characteristics of patients with hepatitis b and control subjects are shown in table 1. while the disease characteristics and investigations in patients with hbv are shown in table 2. table 1. demographic characteristics of patients with chronic hepatitis b infection compared to that of control group. characteristics control hepatitis b p value n mean ± sd n mean ± sd age 100 40.81±11.6 104 42.6±15.8 0.368† ns gender n % n % p value male 53 53 57 54.8 0.796¥ ns female 47 47 47 45.2 education level illiterate 19 19 29 27.9 0.352¥ ns primary 32 32 35 33.7 secondary 32 32 28 26.9 university 17 17 12 11.5 social status single 11 11 10 9.6 0.881¥ ns married 79 79 86 82.7 divorced 4 4 4 3.8 widow 6 6 4 3.8 children yes 83 83 84 80.8 0.679¥ ns no 17 17 20 19.2 residency urban 75 75 75 72.1 0.640¥ ns rural 25 25 29 27.9 data were expressed as either mean ±standard deviation (sd) or number (%); id: iraqi dinar; †: independent samples t-test; ¥: chisquare test; ns: not significant at p ≤ 0.05. table 2. disease characteristics and investigations in patients with chronic hepatitis b infection. characteristic n % characteristic n % biopsy 2 1.9 treatment 10 9.6 other family member 17 16.3 entecavir 28 26.9 mode of transmission interferon 1 1 blood transfusion 19 18.3 interferon + ribavirin 41 39.4 surgery 23 22.1 not treated 24 23.1 tooth extraction 18 17.3 tenofovir 10 9.6 shaving 4 3.8 treatment experience needle 5 4.8 naïve 29 27.9 tattoo 14 13.5 experienced 75 72.1 unknown 3 2.9 hospital admission 5 4.8 vertical 16 15.4 sexual 2 1.9 characteristic minimum maximum median iqr duration of disease 0.50 7.00 1.25 2.50 duration of treatment 0.00 3.50 0.00 1.00 hbv dna (iu/ml) 0.00 200000000. 4140.0 2910000.00 alt (u/l) 5.00 179.00 17.25 15.12 ast (u/l) 3.00 95.00 15.00 13.65 albumin (g/dl) 3.60 39.00 4.64 1.32 inr 1.00 2.30 1.15 0.42 total bilirubin (mg/dl) 0.40 42.10 0.75 0.45 hbv: hepatitis b virus; dna: deoxyribonucleic acid; alt: alanine aminotransferase normal range (up to 12) u/l; ast: aspartate aminotransferase normal range (up to 12) u/l; inr: international normalized ratio (1.0); total bilirubin normal range (0.3-1,2) mg/dl; serum albumin (35-52) g/l. iraqi j pharm sci, vol.29(1) 2020 hrqol among a sample of chronic hepatitis b patients 36 in general, the hrqol of patients with hepatitis b was affected by the disease in a highly significant manner where the median scores for all hrqol domains were significantly lower in patients than in controls (p <0.001) as shown in table 3. table 3. comparison of median values related to short form domain average scores between patients with chronic hepatitis b infection and control group. domain control n = 100 hbv n = 104 p† median score iqr median score iqr physical functioning 100 5 80 45 <0.001 role limitation due to physical health 100 0 0 75 <0.001 role limitation due to emotional problems 100 0 0 33.3 <0.001 energy fatigue 95 25 30 54 <0.001 emotional well being 96 12 20 52 <0.001 social functioning 100 25 50 50 <0.001 pain 100 20 80 51.8 <0.001 general health 90 29 40 35 <0.001 n: number of cases; iqr: inter-quartile range; †: mann whitney u test. pf: physical functioning; rlph: role limitation due to physical health; rlep: role limitation due to emotional problems; ef: energy fatigue; ewb: emotional wellbeing; sf: social functioning; p: pain; gh: general health; * significant at p ≤ 0.05; **significant at p ≤ 0.01. distribution of short form domain scores to demographic characteristics of patients with hepatitis b are shown in table 4. physical functioning score was negatively correlated to age, significantly higher in male patients, significantly higher in high educated patients, significantly higher in single patients, and significantly higher in patients with no children. role limitation due to physical health score was negatively correlated to age, significantly higher in those with no children. energy/fatigue score was negatively correlated to age, and significantly higher in male patients. social functioning score was significantly higher in male patients. pain score was negatively correlated with age. role limitation due to emotional problems, emotional wellbeing and general health scores were unaffected by any of the demographic characteristics of patients with chb infection. correlations between short form domain scores and disease characteristics of patients with hbv are outlined in table 5. patients with previous liver biopsy had significantly higher pain score than those who have never performed liver biopsy. while other domains were unaffected by any of disease characteristics of chb patients. correlations between short form domain scores and laboratory investigations of patients with hbv are shown in table 6. physical functioning score was negatively correlated to serum albumin. social functioning and general health score were negatively correlated to viral load. while other domains were unaffected by any of laboratory investigations of chb patients. iraqi j pharm sci, vol.29(1) 2020 hrqol among a sample of chronic hepatitis b patients 37 table 4.distribution and correlation of short form domain scores and demographic characteristics of patients with chronic hepatitis b infection. characteristic pf rlph rlep e/f ewb sf p gh age r p r p r p r p r p r p r p r p -0.47 <0.001* * -0.220 0.025* -0.033 0.73 9 -0.237 0.016* 0.142 0.150 -0.021 0.832 -0.196 0.047 * -0.074 0.454 gender p € 0.021* 0.545 0.110 0.013* 0.369 0.029* 0.093 0.124 med. score iqr med. score iqr med.sco re iqr med.scor e iqr med.score iqr med.score iqr med.score iqr med.score iqr male 85.00 40.0 0.00 87.5 0.00 100. 45.0 57.5 24.00 56.0 50.00 31.25 90.00 31.25 40.00 35.0 female 70.00 50.0 0.00 75.0 0.00 0.00 20.0 45.0 16.00 40.0 25.00 50.00 80.00 65.00 35.00 40.0 education p € 0.033* 0.190 0.762 0.595 0.544 0.071 0.117 0.863 med.score iqr med. score iqr med.sco re iqr med.scor e iqr med.score iqr med.score iqr med.score iqr med.score iqr illiterate 70.00 57.5 0.00 0.00 16.5 0.00 55.0 35.0 52.00 16.00 50.00 12.50 66.25 70.00 42.50 35.00 primary 75.00 50.0 75.00 0.00 67.0 0.00 65.0 30.0 56.00 24.00 37.50 50.00 55.00 100.0 35.00 35.00 secondary 92.50 27.5 100.0 0.00 33.0 0.00 48.75 35.0 40.00 16.00 21.88 50.00 27.50 90.00 28.75 40.00 university 72.50 37.5 93.75 12.5 91.75 0.00 43.75 20.0 65.00 22.00 50.00 50.00 60.00 80.00 48.75 32.50 social p € 0.016* 0.253 0.922 0.152 0.682 0.821 0.286 0.580 med. score iqr med. score iqr med.sco re iqr med.scor e iqr med.score iqr med.score iqr med.score iqr med.score iqr single 100.0 36.25 87.50 100. 0.00 49.7 5 45.0 57.5 14.00 74.0 50.00 81.25 95.00 70.00 50.00 52.5 married 80.00 40.0 0.00 75.0 0.00 41.5 30.0 55.0 20.00 45.0 50.00 37.50 80.00 50.00 35.00 35.0 divorced 42.50 75.0 0.00 37.5 16.5 83.2 10.0 65. 48.00 86.0 50.00 37.50 40.00 87.50 47.50 62.5 widowed 22.50 70.0 0.00 75.0 0.00 75.0 7.50 33.7 42.00 78.0 25.00 50.00 100.0 67.50 67.50 65.0 children p € 0.007 * 0.021* 0.943 0.208 0.937 0.966 0.283 0.102 med. score iqr med. score iqr med.sco re iqr med.scor e iqr med.score iqr med.score iqr med.score iqr med.score iqr have children 75.00 50.0 0.00 50.0 0.00 33.0 30.0 53.7 24.00 52.0 50.00 50.00 80.00 54.38 35.00 35.0 don’t have 100.0 28.7 75.00 1000 0.00 83.2 40.0 43.7 18.00 62.0 50.00 46.88 95.00 39.38 52.50 47.5 residency p € 0.781 0.723 0.655 0.790 0.439 0.610 0.459 0.928 med. score iqr med. score iqr med.sco re iqr med.scor e iqr med.score iqr med.score iqr med.score iqr med.score iqr urban 80.00 40.0 0.00 75.00 0.00 33.0 35.0 50.00 16.00 52.00 50.00 37.50 90.00 52.50 40.00 35.00 rural 80.00 55.0 0.00 100. 0.00 67.0 25.0 62.5 24.00 50.00 50.00 50.00 80.00 57.50 35.00 42.50 pf: physical functioning; rlph: role limitation due to physical health; rlep: role limitation due to emotional problems; ef: energy fatigue; ewb: emotional wellbeing; sf: social functioning; p: pain; gh: general health; * significant at p ≤ 0.05; **significant at p ≤ 0.01; iqr: inter-quartile range; med: median; r: spearman correlation coefficient; € : kruskal wallis test. iraqi j pharm sci, vol.29(1) 2020 hrqol among a sample of chronic hepatitis b patients 38 table 5. correlations between short form domain scores and disease characteristics of patients with chronic hepatitis b infection. characteristic pf rlph rlep ef ewb sf p gh r p r p r p r p r p r p r p r p duration of disease -0.085 0.389 -0.009 0.927 -0.066 0.502 -0.090 0.363 -0.074 0.454 0.028 0.779 0.074 0.456 0.003 0.978 biopsy 0.067 0.499 0.102 0.304 -0.073 0.460 -0.129 0.192 0.023 0.814 -0.140 0.157 0.213 0.030* 0.130 0.189 other family member -0.027 0.788 -0.147 0.137 0.022 0.826 0.020 0.840 -0.004 0.965 0.002 0.985 0.020 0.839 -0.010 0.923 mode of transmission 0.168 0.088 -0.052 0.602 0.037 0.712 0.073 0.464 0.028 0.776 0.091 0.357 -0.006 0.952 0.162 0.101 treatment -0.114 0.251 -0.046 0.642 -0.024 0.807 -0.061 0.535 0.034 0.733 -0.071 0.472 -0.121 0.220 -0.056 0.574 duration of treatment -0.098 0.321 -0.037 0.712 -0.028 0.775 0.067 0.497 0.000 0.994 -0.027 0.782 0.007 0.945 -0.036 0.716 previously treated -0.123 0.214 -0.057 0.566 -0.047 0.635 -0.022 0.828 -0.002 0.986 -0.087 0.379 -0.014 0.885 -0.136 0.170 hospital admission 0.100 0.314 0.094 0.340 0.041 0.676 0.171 0.083 -0.052 0.601 0.109 0.269 0.104 0.292 0.027 0.785 pf: physical functioning; rlph: role limitation due to physical health; rlep: role limitation due to emotional problems; ef: energy fatigue; ewb: emotional wellbeing; sf: social functioning; p: pain; gh: general health; * significant at p ≤ 0.05; **significant at p ≤ 0.01; r: spearman correlation coefficient. table 6. correlations between short form domain scores and laboratory investigations patients with chronic hepatitis b infection. characteristic pf rlph rlep ef ewb sf p gh r p r p r p r p r p r p r p r p hbv dna 0.038 0.705 -0.183 0.063 -0.174 0.078 -0.175 0.075 -0.136 0.168 -0.234 0.017* -0.131 0.184 -0.291 0.003** alt 0.010 0.916 -0.072 0.470 0.142 0.149 0.034 0.735 0.129 0.193 0.131 0.183 0.047 0.635 0.129 0.191 ast -0.020 0.840 -0.116 0.240 0.050 0.615 -0.031 0.757 -0.008 0.939 -0.030 0.765 0.002 0.986 0.038 0.701 albumin -0.468 0.050* -0.097 0.701 0.101 0.689 0.113 0.655 0.165 0.512 0.150 0.553 -0.118 0.640 0.031 0.903 inr -0.071 0.472 -0.064 0.521 0.035 0.722 -0.116 0.241 -0.078 0.434 0.014 0.887 0.035 0.727 -0.009 0.930 bilirubin -0.068 0.495 0.005 0.957 0.026 0.790 -0.061 0.538 0.040 0.689 0.050 0.617 -0.013 0.897 0.026 0.791 pf: physical functioning; rlph: role limitation due to physical health; rlep: role limitation due to emotional problems; ef: energy fatigue; ewb: emotional wellbeing; sf: social functioning; p: pain; gh: general health; * significant at p ≤ 0.05; **significant at p ≤ 0.01; r: spearman correlation coefficient iraqi j pharm sci, vol.29(1) 2020 hrqol among a sample of chronic hepatitis b patients 39 discussion the impact of the disease and treatment on patient's hrqol has been become an important medical concern. the current study showed that hrqol of patients with chb infection was affected by the disease in a highly significant manner where the median scores for all hrqol domains were significantly lower in patients than in controls (table 3). a study from singapore showed that hrqol was significantly decreased in chb patients and further decreased with disease progression (11). another study from hong kong of china showed that all individuals with chb infection had significantly decreased hrqol compared to general population even among those without any clinical and biochemical abnormalities (16). impairment in hrqol in chb patients (with or without cirrhosis) was also documented in several other countries (12, 13, 17). many other studies have shown hrqol to be impaired in patients with chronic liver diseases (hbv, hcv, alcoholic liver disease), and many physical and psychological factors (depression, anxiety, illness understanding, social stigma, worry about family situation, fear of complications, problems with concentration and memory, and loneliness) have been associated with this impaired hrqol in patients with chronic liver diseases (18-22). although the previous studies from other countries showed impaired hrqol in chb patients (11-13, 16, 17), almost all showed the impairment on some but not all dimensions (11, 13, 16, 17). it may be that the stress of having a serious, and potentially life-threatening illness makes chb patients having reduced hrqol. health related quality of life of chb patients may vary according to their socio-demographic properties. physical functioning, role limitation due to physical health, energy/fatigue and pain scores were negatively correlated to age. similar findings were reported among saudi hepatitis b patients (23). in addition, it was found that physical functioning, social functioning and energy/fatigue score were significantly lower in female in comparison with male. similar finding was reported among turkeys' chb patients (24). it is well known that women, in many countries, receive less social support compared to men if they experience chronic diseases. additionally, women access to medical care is generally delayed compared with men and they are either obliged to work or take over their responsibilities even before they get complete recovery (26). these variations between male and female may cause hrqol to be worse in female patients with chb. regarding education level, the current study showed that physical functioning score was significantly higher in high educated patients. similarly, education level was positively associated to hrqol of chinese chb patients (27). this might be because lower educated patients have limited understanding of hbv, which may in turn lead to poorer disease management and subsequent hrqol. also, similar findings were reported in a study in turkey (25). in addition, physical functioning was significantly higher in single compared to married patients and this disagree with that reported in turkey where married chb patients had better hrqol than singles (24). regarding to children, the current study showed that in patients who do not have children, physical functioning and role limitation due to physical health were significantly higher than in those with children, this may be due to fearfulness of parents from disease transmission when become in contact with their children. while residency do not have any significant effect on hrqol scores and this result disagree with a study in pakistan which showed that rural hbv patients are in worse condition as compared to urban patients (28). also, the study showed that no correlations between disease characteristics and sf-36 domains with exception that patients with previous liver biopsy had significantly higher pain score than those who have never performed liver biopsy. the percentage of patients who had had a liver biopsy in the current study was low (1.9%), which makes us unable to clearly understand the positive correlation between biopsy and hrqol pain score. the current study showed that social functioning and general health scores were negatively correlated to viral load. this might be due that these patients are frequently called back for control visits to check the activity of the disease which may lead to an increase in anxiety among these patients. physical functioning score was negatively correlated to serum albumin. in saudi chb patients, albumin correlated with physical functioning, role limitation due to physical health, social functioning, pain, and general health suggesting that active liver disease or perhaps significant fibrosis was more likely affecting the hrqol domain score rather than the virus itself (23). the current study showed that the duration of disease and the period of therapy did not affect sf-36 scores at all. similar findings were reported among chb patients in turkey (24). showing attention to some limitations of our study regarding the planning process could be helpful for future studies. one limitation is the cross-sectional design of this study rather than prospective which allow studying the effect of treatment on patients' hrqol. future study about the same topic can be conducted as multi-center studies to enroll patients from different centers in iraq. 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licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://www.ncbi.nlm.nih.gov/pubmed/?term=gutteling%20jj%5bauthor%5d&cauthor=true&cauthor_uid=17656809 https://www.ncbi.nlm.nih.gov/pubmed/?term=de%20man%20ra%5bauthor%5d&cauthor=true&cauthor_uid=17656809 https://www.ncbi.nlm.nih.gov/pubmed/?term=busschbach%20jj%5bauthor%5d&cauthor=true&cauthor_uid=17656809 https://www.ncbi.nlm.nih.gov/pubmed/?term=poupon%20re%5bauthor%5d&cauthor=true&cauthor_uid=15368455 https://www.ncbi.nlm.nih.gov/pubmed/?term=chr%c3%a9tien%20y%5bauthor%5d&cauthor=true&cauthor_uid=15368455 https://www.ncbi.nlm.nih.gov/pubmed/?term=chazouill%c3%a8res%20o%5bauthor%5d&cauthor=true&cauthor_uid=15368455 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 synthesis of prodrugs of amoxicillin and metronidazole 1 synthesis and antimicrobial study of possible mutual prodrugs of amoxicillin and metronidazole by direct and indirect coupling through spacer ammar a. ali beg * and ahlam j. qasir **,1 * department of pharmaceutical chemistry, college of pharmacy, university of kufa, najaf, iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract amoxicillin have been conjugated with metronidazole as possible mutual prodrug to get a wider spectrum of activity by acting on aerobic and anaerobic bacteria, have antifungal activity, to provide protection for beta lactam ring of amoxicillin and also to improve patient compliance as it given as a single dose therapy. the structures of the synthesized compound were confirmed and characterized using elemental microanalysis (chn), ir and some physiochemical properties. biological study was done by using disc diffusion method against different bacterial strains which are , staphylococcus aureus , salmonella typhie , pseudomonas aeruginosa , e. coli , klebsiella pneumonia and fungi ( candida albicans) . using nutrient agar medium and 5 mm diameter paper discs. the synthesized compounds showed maintenance or improvement of the antimicrobial activity especially compound (ii) showed superior activity in comparison with compounds (i, iii) . key words: amoxicillin , metronidazole , mutual prodrug . تبادليت لعقاري االموكسيسلين تخليق ودراست الفعاليت المضادة للميكروباث كمقذماث دوائيت زول بطريقت مباشرة وغير مباشرة بواسطت رراعذاوالمتروني عًاس عبذ انعضٌض عهً بٍج * و احالو جًٍم لظٍش **،1 * . انعشاقانكىفت،اننجف،جايعت ،كهٍت انظٍذنت،ًٍاء انظٍذالنٍتٍفشع انك ** . انعشاق،بغذاد،جايعت بغذاد ،كهٍت انظٍذنت،ًٍاء انظٍذالنٍتٍفشع انك الخالصة عماس االيىكسٍسٍهٍن يع عماس انًخشونٍذاصول كًمذياث دوائٍت نكً نحظم عهى فعانٍت راث طٍف أوسع فً هزا انبحذ نمىو بشبظ و ين خالل عًهه عهى انبكخشٌا انهىائٍت و انالهىائٍت , نذٌه فعانٍت ضذ انفطشٌاث , حىفٍش حًاٌت نحهمت انبٍخا الكخاو فً االيىكسٍسٍهٍن نخحسٍن اسخساغت انًشٌض نهعالج . حى حشخٍض انًشكباث انًظنعت باسخخذاو انخحهٍم انذلٍك نهعناطش انًكىنت نها و ين خالل األشعت ححج انحًشاء و بعض انخىاص انفٍضٌائٍت وانكًٍٍائٍت . دساست انفعانٍت انحٍىٌت حًج باسخخذاو طشٌمت االنبعاد انمشطً ضذ بعض staphylococcus aureus , salmonella typhie , pseudomonasخهفت و انفطشٌاث و انخً هً سالالث انبكخشٌا انًخ aeruginosa , e. coli , klebsiella pneumonia and candida albicans . باسخخذاو وسظ يغزي و ألشاص وسلٍت لطش ( حٍذ اظهش فعانٍت iiنمذ أظهشث انًشكباث انًظنعت ححسنا أو حفاظا عهى انفعانٍت انًضادة نهًٍكشوباث وخظىطا انًشكب ) . يم 5 .( i,iiiعانٍت يماسنت بانًشكباث ) .تبادليت مقذماث دوائيت، المترونيذازول ، االموكسيسيلين الكلماث المفتاحيت : introduction anaerobic infections characteristically are polymicrobial (mixed) and include both anaerobic and facultative organisms (1) . the organisms tend to be acquired endogenously. the particular mix of pathogens reflects the combined influence of the complex commensal flora at a specific body site and the unique micro biota of the underlying conditions (2) . because these organisms are generally of low pathogenicity, anaerobic or mixed infections generally develop as a consequence of either structural alterations in the normal mucosal barrier or tissue ischemia with lowered oxidation reduction potential (3) . knowledge of the anatomic location of the primary source of infection and the underlying condition of the host, therefore, is essential in predicting the probable organisms causing anaerobic and mixed infections associated with the indigenous micro flora (4) . microorganism in mixed infections may respond to antimicrobial agents differently than do those in monomicrobial infections, and it may not be necessary to eradicate every bacterial species in mixed infection to achieve cure (5) . in the treatment of mixed aerobic anaerobic infection we use antibiotics that act on both type of bacteria so here we used metronidazole with amoxicillin as there is synergestic effect between them as shown in 1989, by van winkelhoff et al. (6) who showed that mechanical treatment followed by a regimen of metronidazole and amoxicillin for 7 days is effective in eliminating a. actinomycetemcomitans from the infected sites. recently, this was confirmed in a large patient group, in which more than 1 corresponding author e-mail:ahlamqasir@yahoo.com received:28 /1/2013 accepted:15 /6/2013 iraqi j pharm sci, vol.23(1) 2014 synthesis of prodrugs of amoxicillin and metronidazole 2 an explanation for the in vivo efficacy may be the in vitro synergism against 95% eradication of the microorganism was reported (7) . a.actinomycetem comitans not only between metronidazole and amoxicillin but also between metronidazole and its hydroxymetabolite as well as between amoxicillin and the hydroxymetabolite of metronidazole (8) . however, there are potential advantages in giving such coadmi -nistered drugs having comple -mentary pharmacological activities in the form of a single chemical entity. such agents are named as mutual prodrugs which are designed with improved physicochemical property . in the view of this background, the present study was conducted to the design, synthesis, and preliminary biological study of mutual prodrugs of amoxicillin with metronidazole to get antibiotic with broader spectrum of activity and given by single dose. chemistry the synthetic pathways for the designed target compounds (1ac and i, ii and iii) are illustrated in (schemes 1-6). scheme (1) synthesis of mtz-cl scheme (2) synthesis of compound i scheme(3)synthesis of compound 1b scheme (4) synthesis of compound ii iraqi j pharm sci, vol.23(1) 2014 synthesis of prodrugs of amoxicillin and metronidazole 3 scheme (5) synthesis of compound 1c scheme (6) synthesis of compound iii experimental a chemistry all reagents and anhydrous solvents were of analytical grade and were used as received from the commercial suppliers (merckgermany, riedal-dehaen-germany, bdhengland and fluka-switzerland) . amoxicillin and metronidazole were purchased from sdi company, iraq. thin layer chromatography (tlc) was run on kieslgel gf254 (60), merck (germany), to check the purity of the products as well as monitoring the progress of reactions. ft-ir spectra were recorded at college of pharmacy, kufa university by using shimadzu japan spectro photometer and the determination of the spectra were performed by using kbr discs. chno microanalysis has been done at the central laboratory of kufa uniusing euroea shimadzu-japan elemental analyzer . synthesis of 1-(2-chloroethyl)-5-meth -yl-2nitro-1h-imidazole, (1a ) (9) : to a solution of metronidazole ( 5 mmol , 0.858 gm) in thf ( 20 ml ) was added thionyl chloride (6.5 mmol , 0.75 ml) dropwise under stirring. then the reaction mixture was refluxed for 7 hrs. excess thionyl chloride was removed azeotropically. the residue was taken into ethyl acetate (30 ml) and washed with water (10 ml x 3). organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure to provide a brown solid . the percent yield, physical appearance, melting point and rf value are listed in table (1). synthesis of compound (i) (10) : a suspension of amoxicillin trihydrate (1 mmol ,0.4195gm) in 5 ml dmf was prepared and (1 mmol ,0.01 gm) of potassium bicarbonate and [(0.203) ml (2 mmol)] benzaldehyde were added at 0 o c ( to provide protection for amine group0 . the mixture was stirred at 0 o c for 3.5 h. at the end of this time (1 mmol ,0.1 gm) of potassium bicarbonate and (1 mmol ) of compound (1a) were added and stirred at 0 o c for additional 6 hrs. the mixture was poured onto ice water and was extracted with (5 ml x 3) of ethyl -acetate. after washing the organic phase with 10% sodium chloride and drying over anhydrous sodium sulfate, it was evaporated to dryness in vacuo at 20 o c and a yellow oily residue was obtained. the residue was dissolved in 5 ml of acetonitrile. the ph of the solution was adjusted to 2 with 1n hcl and it was stirred for 30 min at 0-5 o c . in order to remove acetonitrile, 6 ml of water was added and the solution was evaporated in vacuo at 20 oc . after washing the aqueous solution with (6 ml x 3) of ethylacetate and saturating with iraqi j pharm sci, vol.23(1) 2014 synthesis of prodrugs of amoxicillin and metronidazole 4 sodium chloride it was extracted with (6 ml x 3) of dichloro -methane. the organic phase was dried over anhydrous sodium sulfate and it was left overnight at 5 o c the residue was filtered and dried . the percent yield, physical appearance, melting point and rf value are listed in table (1) . synthesis of 4– (2– (5– methyl – 2 – nitro 1h -imidazol – 1 –yl ) ethoxy)-4-oxo butanoic acid ( compound ib ) (11) : metronidazole (3 mmol ,0.5135gm) was dissolved in 30 ml of acetonitrile and (3 mmol , 0.3 gm) of the succinic anhydride followed by 4-dimethyl aminopyridine (0.3 mmol , 0.037 gm) were added. the mixture was left at ambient temperature for 48 hrs. until the reaction was completed as evidenced by tlc , the products was precipitated after solvent evaporation and addition of cold water. the percent yield, physical appearance, melting point and rf value are listed in table (1) . synthesis of compound (ii) (12) : compound (ib) (1.133 mmol , 0.303 g ) was dissolved in 10 ml of anhydrous dmso and (1.699 mmol , 0.24 ml ) of tea was added under stirring ; then ( 2.266 mmol , 0.260 gm) of nhs and then (2.266 mmol, 0.4675 gm) of dcc were added . the reaction was let under stirring overnight at room temperature in the dark . dicyclohexylurea ( dcu ) was filtered out and the solution was dropped into 200 ml of diethyl ether. after 4 hrs. at 4 0 c the activated ( ibnhs ) was recovered by filtration and washed with diethyl ether and then dried under vacuum . to ( 1 mmol , 0.4194 gm ) of amoxicillin dissolved in 10 ml dmso , ( 5 mmol ) of ( ib nhs ) dissolved in 6 ml of dmso was added . to the mixture ( 0.02 ml ) of et3n was added and the reaction was let to react over night under stirring at room temperature in the dark . then 5 ml of water was added and the ph adjusted to 7.5 and 5ml of dmf was also added to help phase separation . the ( compound ii ) was extracted from aqueous phase by ethyl acetate ( 50 ml x 4 ) . the organic layer was dried over anhydrous sodium sulphate and evaporate the ethyl acetate to get the compound (ii). the percent yield, physical appearance, melting point and rf value are listed in table (1) . to an ice-cooled solution of metro -nidazole (1.1 mmol , 0.1883 gm) and tea (2.2 mmol , 0.3 ml) in dichloromethane (10 ml) , chloroacetyl chloride (2.2 mmol, 0.18 ml) was added under stirring. the reaction mixture was further stirred at 0-5 o c for 30 min. synthesis of chloro– acetic acid 2– ( 5– methyl–2-nitro – 1 h–imidazol – 1 yl ) ethyl ester ( compound 1c ) (9) : dichloromethane was distilled off and the residue was taken in ethyl acetate (20 ml). the ethyl acetate layer was washed with water (10 ml x 3) and dried over anhydrous sodium sulfate. sodium sulfate was filtered off and washed with ethyl acetate (5 ml x 2). the filtrate was concentrated to give a brown semisolid . the percent yield, physical appearance, melting point and rf value are listed in table (1) . synthesis of compound (iii) (10) : a suspension of amoxicillin trihydrate (1 mmol ,0.4195gm) in 5 ml dmf was prepared and (1 mmol ,0.01 gm) of potassium bicarbonate and [(0.203) ml (2 mmol)] benzaldehyde were added at 0 o c . the mixture was stirred at 0 o c for 3.5 h. at the end of this time (1 mmol ,0.1 gm) of potassium bicarbonate and (1 mmol ) of compound (1c) were added and stirred at 0 o c for additional 6 hrs. the mixture was poured onto ice water and was extracted with (5 ml x 3) of ethyl acetate. after washing the organic phase with 10% sodium chloride and drying over anhydrous sodium sulfate, it was evaporated to dryness in vacuo at 20 o c and a yellow oily residue was obtained. the residue was dissolved in 5 ml of acetonitrile. the ph of the solution was adjusted to 2 with 1n hcl and it was stirred for 30 min at 0-5 o c . in order to remove acetonitrile, 6 ml of water was added and the solution was evaporated in vacuo at 20 o c . after washing the aqueous solution with (6 ml x 3) of ethylacetate and saturating with sodium chloride it was extracted with (6 ml x 3) of dichloro -methane. the organic phase was dried over anhydrous sodium sulfate and it was left overnight at 5 o c the residue was filtered and dried . the percent yield, physical appearance, melting point and rf value are listed in table (1) . iraqi j pharm sci, vol.23(1) 2014 synthesis of prodrugs of amoxicillin and metronidazole 5 table (1) the percent yield, physical appearance, melting point and rf of the intermediate and final products. a =(dichloromethane : ethanol : ethylacetate) (6:1:3) polarity index= 3.7 b=(dichloromethane : ethanol : ethylacetate) (1:6:3) polarity index= 4.75 1-(2-chloroethyl)-5-methyl-2-nitro-1himidazole (1a ) : ir (kbr): 2976 cm -1 [c-h] , 1532 cm -1 [no] , 1460 cm -1 [c=n] , 1365 cm -1 [n-o] , 756 cm -1 [c-cl] . 4-(2-(5-methyl-2-nitro-1h-imidazol-1-yl) ethoxy)-4-oxo butanoic acid (ib ) : ir (kbr): 3181 cm -1 [o-h] , 2962 cm -1 [c-h] , 1732 cm -1 [c=o] of ester , 1531 cm -1 [n-o] , 1476 cm -1 [c=n] , 1266 cm -1 [c-o] . chloro-acetic acid 2-(2-methyl-5-nitro-1himidazol-1-yl)ethyl ester ( compound 1c ) : ir (kbr): 3019 cm -1 [c-h] , 1747 cm -1 [co] , 1529 cm -1 [n-o] , 1471 cm -1 [c=n] , 1262 cm -1 [c-n] , 825 cm -1 [c-cl] . compound (i) : ir (kbr): 3506 cm -1 asy. str. [n-h] of amine , 3444cm -1 sy. str. [n-h] of amine, 3151 cm -1 [o-h] of phenol , 1780 cm -1 [c=o] of lactam , 1738 cm -1 [c=o] of ester , 1697 cm -1 [c=o] of amide , 1550 cm -1 [n-o] of nitro , 1261 & 1174 cm -1 [c-o] . chn calculated: c, 50.96; h, 5.05; n,16.21; o, 21.6; found: c, 51.21; h, 5.13; n, 16.18; o, 21.27. compound (ii) : ir (kbr): 3334 -1 [n-h] of amide , 3230 cm -1 [o-h] of phenol , 2966 cm -1 [c-h] , 1778 cm -1 [c=o] of lactam , 1742 cm -1 [c=o] of ester , 1674 cm -1 [c=o] of amide , 1638 cm 1 [c=n] , 1519 cm -1 asy. str. [n-o] of nitro , 1371 cm -1 sy. str. [n-o] of nitro , 1273 & 1174 cm -1 [c-o] . chn calculated: c, 50.48; h, 4.89; n,13.59; o, 25.86; found: c, 50.67; h, 5.02; n, 13.87; o, 25.46. compound (iii) : ir (kbr): 3511 cm -1 asy. str. [n-h] of amine, 3441 cm -1 sy. str. [n-h] of amine, 3207 cm -1 [o-h] of phenol, 1778 cm -1 [c=o] of lactam , 1751 & 1740 cm -1 [c=o] of ester , 1671 cm -1 [c=o] of amide , 1553 cm -1 [n-o] of nitro , 1275 & 1175 cm -1 [c-o] . chn calculated: c, 49.99; h, 4.89; n,14.58; o, 24.97; found: c, 51.21; h, 5.21; n, 14.03; o, 24.42. b antimicrobial studies antibacterial activity for the synthesized compounds was invest -tigated by disc diffusion method against different bacterial strains and fungi such as, staphylococcus aureus , salmonella typhie , pseudomonas aeruginosa , e.coli , klebsiella pneumonia and candida albicans, using nutrient agar medium and 5 mm diameter paper discs (whatman no. 1). the investigated compounds i.e. the final products, were dissolved in dmso at a concentration of 5000 µg/ml. the filter paper disc were soaked in solutions of the test compounds, dried, and then placed in petri plates previously seeded with the test organisms. the plates were incubated for 24 h at 37 °c and the inhibition zone in the region of each disc was measured . the results obtained are tabulated in table (2) . compounds and intermediates empirical formula molecular weight description % yield melting point o c rf value 1a c6h8cln3o2 189.6 yellow crystals 60.1 77-79 *a=0.33 b=0.42 1b c10h13n3o6 271.23 white crystals 65.3 125-128 a=0.21 b=0.33 1c c8h10cln3o4 247.64 semisolid brown 76.3 a=0.3 b=0.41 i c22h26n6o7s 518.5 faint yellow powder 59.4 189-191 a=0.29 b=0.41 ii c26h30n6o10s 618.6 white powder 66.9 203-205 a=0.25 b=0.37 iii c24h28n6o9s 576.58 off whiteyellow powder 55.3 197-199 d a=0.27 b=0.38 iraqi j pharm sci, vol.23(1) 2014 synthesis of prodrugs of amoxicillin and metronidazole 6 results and discussion the synthesis of the designed compounds has been successfully achieved. characterization and struct -ural formulas of the synthesized compounds were confirmed by melting point determination, rf values, ftir spectroscopy and elemental micro analysis. the antimicrobial study for the synthesized compounds showed maintenance or improvement of the antibacterial activity against both gram negative and gram positive bacteria with activity against fungi . especially compound ii show superior activity in comparison with the other synthesized compounds. table (2) antimicrobial activities (a) of the compounds (i-iii) . (a) growth inhibition diameter (mm) references 1. yoonseon park, jun young choi, dongeun yong , clinical features and prognostic factors of anaerobic infections: a 7-year retrospective study , the korean journal of internal medicine , 2009;24:113 . 2. nagy e. anaerobic infections: update on treatment considerations. drugs. 2010;70 :841-58 . 3. zianabos ao et al : anaerobic infections: general concepts, in principles and practice of infectious diseases, 6th ed, gl mandell et al (eds). elsevier , 2005, pp 2810–2816 4. anthony w. chow, m.d., f.a.c.p, f.r.c.p.c. anaerobic infections acp medicine , (chapter 141) 2006 , pp 1604-1624. 5. brook i: beta-lactamase-producing bacteria in mixed infections. clin. microbiol infect, 2004 ; 10 :777 784 . 6. van winkelhoff, a. j., j. p. rodenburg, r. j. goen6, f. abbas, e. g. winkel, and j. de graaff. metronidazole plus amoxicillin in the treatment of a. actinomycetem comitans associated periodontitis. j. clin. periodontol. 1989 ; 16 :128-131. 7. van winkelhoff, a. j., c. j. tijhof, and j. de graaff. microbiological and clinical results of metronidazole plus amoxicillin therapy in actinobacillus actinomycetemcomitans-associated periodontitis. j. periodontol . 1992 ; 63 :52-57. 8. pavicic m.j. , van winkelhoff a.j.and j.de.groaff, synergistic effects between amoxicillin, metronidazole, and its hydroxyl metabolite against actinobacillus actinomycetem – comitans. aantimicrob. agents chemother. 1991; 35:961 966. 9. lalit kumar,a amit sarswat,a nandlal,a vishnu l. sharma,a, imidazole derivatives as possible microbicides with dual protection , european journal of medicinal chemistry, 2010; 45(2) :817-824 . 10. meltem ceylan ¨unl¨ usoy, nurten altanlar, rahmiye ertan, synthesis and antimicrobial activities of some new flavonyl pro-drug esters of ampicillin , turk j chem 2005 ; 29 : 187 -191. 11. nadia m. mahfouz,tarek aboul-fadl . ahmed k. diab, metronidazole twin ester prodrugs: synthesis, physico -chemical properties, hydrolysis kinetics and antigiardial activity, eur: j. med. chem. 1998; 33: 675-683. 12. pasut g, canal f, dalla via l, arpicco s, veronese fm, schiavon o. antitumoral activity of peg-gemcitabine prodrugs targeted by folic acid. j control release , 2008 ; 127( 3) : 239-248. compound s.aureus escherichia coli pseudomonas aeruginos klebsiella pneumoniae candida albicans control (dmso) amoxicillin 25 22 23 23 metronidazole 6 14 17 i 31 23 32 21 19 ii 29 25 31 29 20 iii 28 24 30 28 19 http://www.ncbi.nlm.nih.gov/pubmed?term=pasut%20g%5bauthor%5d&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=canal%20f%5bauthor%5d&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=dalla%20via%20l%5bauthor%5d&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=arpicco%20s%5bauthor%5d&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=arpicco%20s%5bauthor%5d&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=veronese%20fm%5bauthor%5d&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed?term=schiavon%20o%5bauthor%5d&cauthor=true&cauthor_uid=18346806 http://www.ncbi.nlm.nih.gov/pubmed/18346806 iraqi j pharm sci, vol.22(1) 2013 serum resistin level and acute stemi infarction 90 association of admission serum resistin level with acute st-segment elevation myocardial infarction in iraqi patients dheyaa j.kadhim *,1 , kassim j.al-shamma * and adeeb g. hussein ** * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** ministry of health, al-yarmouk teaching hospital, baghdad, iraq. abstract human resistin is an adipokine, with a possible link to coronary heart disease.a few studies were done about resistin in acute phase of st-segment elevation myocardial infarction (stemi) especially in iraqi patients. accordingly we design a study to investigate the association between resistin concentration and acute phase of stemi in iraqi patients. the present study was carried out at al-yarmouk teaching hospital from december 2011 until june 2012. serum resistin levels were measured in 50 patients with acute stemi (mean age: 58.16 ± 11.73 years) at the first 12 hours of admission and 34 normal controls (mean age: 53.98 ± 15.46 years) matched for age, sex and other risk factors. resistin level in patients with acute stemi (13.08 ng/ml) was significantly higher than that of the control group (5.31 ng/ml) (p < 0.0001). the study revealed a significant negative correlation between serum resistin level and serum adiponectin level among patients. key words: resistin, acute st-segment elevation myocardial infarction, adipokines. العالقة بين هستوى الرسستين في هصل الدم هع احتشاء عضلة القلب الحاد ضياء جبار كاظن *‘1 ، قاسن جليل الشواع * و اديب جادر حسين ** * .فزع اىصٞذىح اىظزٝزٝح ، ميٞح اىصٞذىح ، جاٍعح تغذاد ، تغذاد ، اىعزاق ** .ٗسارج اىصحح ، ٍظرشفٚ اىٞزٍ٘ك اىرعيَٜٞ ، تغذاد ، اىعزاق الخالصة اىرٜ دِضراطاخْٕاىل اىقيٞو ٍِ اه .اىراجٞحِض اىقية ٍزااِض ب ٍحرَيحٕزٍُ٘ اىزطظرِٞ ٕ٘ احذ االدٝث٘ماْٝاخ ٍع عالقح َِضيدْت عِ عُع ِْت اىحادّدجِض اىَزحيح فٜ اىزطظرِٞ ِِض اىَزضٚ فٜ ص٘واًا احرشاء عضيح اىقية ٍِض تِٞىعالقح ا ىرَحزّدٛ دراطحطثقا ىذىل ذٌ ذصٌَٞ ٕذٓ اه. اىعزاقٞٞ ِِض اىَزضٚ فٜٗاحرشاء عضيح اىقية اىحاد اىزطظرِٞ ذزمٞشِض .اىعزاقٞٞ ِْت اىرعيَٜٞ اىٞزٍ٘ك ٍظرشفٚ فٜ ُّعفّدذخْت اىحاىٞح اىذراطح ُِض حرٚ 2011 ااٗهِض ماُّ٘ ٍِض فٜ اىزطظرِٞذٌ قٞاص ٍظر٘ٙ ٕزٍُ٘ .2012 حشٝزا االٗىٚ ٍِ 12فٜ اىظاعاخ اىـ ( طْح 11.73 ±58.16 ٍعذه اىعَز)ٍزٝضا ٍِ ٍزضٚ احرشاء عضيح اىقية اىحاد 50ٍصو اىذً ىذٙ مَجَ٘عح ضاتطح ٍر٘افقح ٍع ٍجَ٘عح ( طْح 15.46 ±53.98ٍعذه اىعَز )شخصا ا ز 34د ٘ىٌٖ اىَظرشفٚ تاالضافح اىٚ قٞاطٔ ىذٙ . زٙ طزِض ٗع٘اٍواىَزضٚ ٍِ ّاحٞح اىعَز ٗاىجْض عيٚ ماُ(ٍو / ّاّ٘غزاً 13,8)ىذٙ ٍزضٚ احرشاء عضيح اىقية اىحاد ٗاىثاىغ فٜ ٍصو اىذً اىزطظرِٞمشفد اىذراطح اُ ٍظر٘ٙ ٕزٍُ٘ ِْت جذاًا . (ٍو / ّاّ٘غزاً 5,31) ٍظر٘آ فٜ اىَجَ٘عح اىضاتطح ٗاىثاىغ ٍِض ًّد طيثٜ إرذثاطٗج٘د عِ َمشفدْت قذ اىذراطحمَا اُ تِٕٞزٍُ٘ اادٝثّ٘ٞنرِٞ ٍٗظر٘ٙ فٜ ٍصو اىذً اىزطظرٍِٞظر٘ٙ ٕزٍُ٘ تِٞ ٕا . اىَزضٚ .رسستين ، احتشاء عضلة القلب الحاد ، اديبوكاينات : الوفتاحيةالكلوات introduction complications of atherosclerosis remain the primary cause of death in most countries despite massive efforts to limit well-documented risk factors such as smoking, hypertension, hyperlipidemia, diabetes mellitus, and obesity. the relationship between obesity and atherogenesis is multifactor, including changes in blood pressure (bp), alterations in the composition and plasma level of lipoproteins, coagulation and inflammatory factors (1) . recent advances in the knowledge of adipose tissue give evidence that it is a secretary organ, producing a variety of adipokines that may be relevant for development or progression of atherosclerotic vascular disease (2) . resistin, the product of the rstn gene, was discovered in 2001 by the group of mitchell lazar as a target gene of the anti-diabetic drug thiozolidinedione (tzd), which was down-regulated in mouse adipocytes upon treatment (3) . 1 corresponding author e-mail:dhia_pharma@yahoo.com received: 20/10/2012 accepted: 16/3/2013 iraqi j pharm sci, vol.22(1) 2013 serum resistin level and acute stemi infarction 91 it was named resistin because of the acquired insulin resistance that mice injected with resistin demonstrated (3-5) . resistin is a ~12.5 kda peptide hormone that belongs to the resistin like molecules (relm) family (also known as adipose tissue specific secretory factor, adsf, or found in inflammatory zone, fizz, family) of cystein-rich secreted proteins (6) . in rodents, resistin is derived almost exclusively from fat cells (3, 7, 8) , whereas in humans resistin is produced by inflammatory cells, primarily macrophages (9) . the relationship between resistin and coronary artery disease (cad) has been controversial (10) . in humans, monocytes and macrophages produce large quantities of resistin, but very little resistin is expressed in adipocytes (11) . resistin has been suggested to be an inflammatory marker in humans, because macrophages are known inflammatory modulators. this suggests a possible link between resistin and cardiovascular (cv) disease via proinflammatory pathways (12) . resistin was suggested to affect endothelial function and the migration of vascular smooth muscle cells (13-15) , which are regarded as key pathophysiological mechanisms of atherosclerosis. further, resistin has been noted to play a vital role in increasing the level of very low density lipoprotein (vldl) and low density lipoprotein (ldl) in an obese person which is directly atherogenic (16-18) . although several studies have been done on resistin and cad, but most of them have been conducted on patients with chronic ischemia and the studies in acute phase of st-segment elevation myocardial infarction (stemi) especially in developing countries are limited and rare. the present study was designed to evaluate the association between admission serum levels of resistin and acute stemi in iraqi patients as well as examining possible associations and correlations between resistin and selected demographic , clinical and laboratory variables among patients with stemi. subjects and method subjects the present study was carried out at alyarmouk teaching hospital from december 2011 until june 2012. the study protocol was approved by the scientific committee in the college of pharmacy, university of baghdad and the medical ethics committee in the ministry of health / republic of iraq. this case-control study was conducted on fifty (50) patients who were treated for acute stemi with the following inclusion criteria: 1 .first experience of acute mi. 2 .absence in the electrocardiogram (ecg) of conditions that might complicate the interpretation of the st segment, such as bundle branch block, preexcitation, atrial fibrillation, atrial flutter, or complete atrioventricular block. 3 .a maximum of 12 hours between the onset of symptoms and initiation of thrombolytic therapy. the diagnosis of acute mi was made on the basis of a history of chest pain lasting for more than 30 minutes that is associated with ecg changes suggestive of st-segment elevation of 1mm or more in at least 2 contiguous leads and is unresponsive to nitroglycerin administration. the diagnosis was subsequently be confirmed by elevation of serum cardiac troponin i (ctni) activity. all the patients involved in the study were followed up clinically during entire hospitalization period to assess them for response to thrombolytic therapy as well as for the development of any complications. in addition thirty four (34) subjects were selected as a control group and matched them with case group for age, sex and other cad risk factors such as hypertension, diabetes mellitus, hyperlipidemia, body mass index (bmi), smoking and renal function. laboratory analysis blood samples were collected from all patients by vein puncture (5ml), at admission before initiation of alteplase and 6-9 hours later to measure the studied parameters. the sample was transferred into clean plain tube, left at room temperature for at least 30 minutes for clotting, centrifuged, then serum separated to be used for measuring the studied parameters. serum resistin level was determined using enzyme linked immunosorbent assay (elisa) (demeditec® diagnostics (germany)) in patients and control groups. in addition, elisa kits were used to determine serum level of ctni (troponin i elisa kit, oxis® international, inc (usa)), leptin (leptin elisa kit, raybio® elisa kits (usa)), and adiponectin (adiponectin elisa kit, demeditec® diagnostics (germany)) in patients and control groups. statistical analysis statistical analysis was performed by spss (version 11; spss, inc., chicago, il). nominal variables are compared using chisquare test. continuous variables were summarized as mean ± standard deviation (sd). continuous variables are tested for normality iraqi j pharm sci, vol.22(1) 2013 serum resistin level and acute stemi infarction 92 using shapiro wilk test. normally distributed variables are compared using t-test. nonnormally distributed variables are compared using mannwhitney u test. spearman correlation was performed to evaluate the relationship between resistin level and the values of other selected clinical variables among patients with stemi. in all cases, a probability value p<0.05 was considered statistically significant. results demographic, clinical characteristics and baseline laboratory variables of the study groups. the demographic and clinical characteristics of the study groups, as well as laboratory variables are shown in table 1. no significant differences were observed between the patients and the control groups in all of these parameters. table 1: demographic, clinical characteristics and baseline laboratory variables of the study groups. patients control p value number of patients 50 34 0.08 a males 41 25 0.74 a females 9 9 0.45 a age (yr) 58.16 ± 11.73 53.98 ± 15.46 0.16 b bmi (kg/m 2 ) 28.05 ± 4.27 27.89 ± 3.50 0.96 c diabetes 15 4 0.11 a hypertension 18 12 0.96 a smoking 26 8 0.08 a s. uric acid (mg/dl) 4.94± 1.32 5.12± 1.24 0.37 c s. creatinine (mg/dl) 0.91 ± 0.31 0.85 ± 0.28 0.37 c s. urea (mg/dl) 39.12 ± 11.16 37.20± 8.06 0.67 c s. total cholesterol (mg/dl) 199.62± 41.09 189.26± 28.35 0.09 c hdl-c (mg/dl) 41.03± 4.79 43.17± 10.56 0.36 c bmi: body mass index, hdl-c: high density lipoprotein cholesterol. a: chi-square test, b: t.test, c: mannwhitney u test. resistin level in the patients and control groups results presented in table 2 showed that serum resistin level in patients with stemi on admission was significantly higher than those in the control group (13.08 ± 2.53 ng/ml vs. 5.31 ± 0.87 ng/ml, p < 0.0001). table 2: admission serum resistin level in stemi patients compared to control. values are presented as mean ± sd; n=number of patients. figure 1: admission serum resistin level in stemi patients compared to control. patients (n=50) control (n=34) p value serum resistin (ng/ml) 13.08 ± 2.53 5.31 ± 0.87 p < 0.0001 iraqi j pharm sci, vol.22(1) 2013 serum resistin level and acute stemi infarction 93 association of admission resistin level with selected clinical variables among patients with stemi studying the association between admission serum resistin level and selected clinical variables among patients with stemi (diabetes, hypertension, sex, location of mi, development of heart failure (hf), development of atrial fibrillation (af), development of ventricular tachycardia (vt) and/or ventricular fibrillation (vf), and achievement of successful reperfusion) revealed a highly significant difference in serum resistin levels between male and female patients (p < 0.001). no significant differences were present for the remaining variables as shown in table 3. table 3: association of admission resistin level to selected clinical variables among patients with stem. values are presented as mean ± sd; n=number of patients. hf : heart failure, af: atrial fibrillation, vf: ventricular fibrillation, vt: ventricular tachycardia. a: t.test, b: mann-whitney u test. ** highly significant (p < 0.001). figure 2: association of admission resistin level to selected clinical variables among patients with stemi. correlation between serum resistin and selected demographic and laboratory variables in stemi patients studying the correlation between admission serum resistin level and selected demographic and laboratory variables among patients with stemi (age, bmi, s. uric acid, s. creatinine, s. urea, s. total cholesterol, hdl-c, serum leptin, serum adiponectin and serum ctni) revealed a significant negative correlation between plasma resistin level and adiponectin level. no significant correlation was found between age, bmi, s. uric acid, s. creatinine, s. urea, total cholesterol, hdl-c, serum leptin, and serum ctni and resistin level (table 4). resistin level (ng/ml) p value yes no 1 diabetes 12.70 ± 3.01 (n=15) 13.24 ± 2.33 (n=35) 0.49 a 2 hypertension 13.76 ± 2.71 (n=18) 12.69 ± 2.34 (n=32) 0.14 a 3 male gender 12.38 ± 2.23 (n=41) 16.24± 0.89 (n= 9) < 0.001** b 4 anterior mi 13.15 ± 2.50 (n=31) 12.95 ± 2.65 (n= 19) 0.77 b 5 development of hf 12.4 ± 2.77 (n=9) 13.23 ± 2.49 (n=41) 0.38 b 6 development of af 13.71 ± 3.37 (n=6) 12.86 ± 2.44 (n=44 ) 0.38 b 7 development of vt and/or vf 13.64 ± 2.68 (n=5 ) 12.89 ± 2.54 (n=45 ) 0.49 b 8 successful reperfusion 13.90 ± 2.83 (n=12) 12.82 ± 2.42 (n=38) 0.14 b iraqi j pharm sci, vol.22(1) 2013 serum resistin level and acute stemi infarction 94 table 4: correlation between serum resistin level and selected demographic and laboratory variables in stemi patients. bmi: body mass index, s. hdl-c: serum high density lipoprotein cholesterol. discussion in this case-control study, we found that the serum resistin values were significantly increased in patients with acute stemi, when compared with controls. these results are in agreement with similar studies done in patients with acute myocardial infarction (1921) . however, there are still controversies about the association of resistin with cad. burnett et al showed that resistin was not independently associated with cad (22) . anyhow, it was reported that resistin levels are substantially higher in human inflammatory cells when compared with human adipocytes (patel et al., 2003;yang et al., 2003; kaser et al., 2003) (9, 11, 23) . elevation of resistin in the acute coronary syndrome (acs) might represent the presence of inflammatory process in mononuclear cells-precede myocardial necrosis. these findings may additionally support the hypothesis that in the conditions of the acs resistin might represent inflammatory rather than a metabolic processes (24) . it has been suggested that resistin may mediate partly its pro-atherosclerotic properties by influencing systemic inflammation (11) .patients with acute coronary syndrome had coronary plaques with more extensive macrophage-rich areas, which was the major source of resistin (23) . inflammatory responses stimulated resistin secretion, and resistin could also promote production of pro-inflammatory mediators such as interleukin-6 (il-6) partially by activation of nuclear factor-κb signaling pathway, hence aggravate the proinflammatory response by a positive feedback (25) . moreover, resistin could affect the functions of vascular cells and exerts direct effects to promote endothelial cells activation by promoting endothelin-1 (et-1) release (26) . resistin has been shown to impair endothelium-dependent dilation of coronary vessels induced by the cardioprotectant bradykinin (dick et al., 2006) (27) . lubos et al. (2007) proposed resistin as a diagnostic marker of mi and future cardiovascular death (21) . despite the fact that resistin exhibits properties commonly associated with cardioprotective agents, based on evidence obtained in animal models of obesity and diabetes, one might expect resistin to exacerbate ischaemia–reperfusion (i/r) injury rather than protect against it (steppan et al., 2001) (3) , particularly as resistin counteracts the beneficial effects of insulin, a recognised cardioprotective agent (hausenloy & yellon, 2009) (28) . indeed, in a recent study in langendorff perfused rat heart, resistin, administered as a preconditioning agent, was found to worsen cardiac i/r injury, as reflected by impaired functional parameters and elevated tissue output of natriuretic peptides, creatine kinase and tumour necrosis factor-α , although infarct size was not determined (rothwell et al., 2006) (29) . regarding the sex difference, the finding of the current study was in agreement with other studies which had been shown that resistin concentrations were significantly higher in women compared to men (tuttolomondo et al., 2010 ; yannakoulia et al.,2003) (30, 31) . however, it remains to be elucidated whether the sexual dimorphism of body fat distribution or differences in sex steroids are responsible for the observed differences in resistin levels. in addition our study revealed significant negative correlation between serum resistin level and adiponectin level in the patients group. adiponectin is a peptide hormone secreted by adipocytes, shown to have a number of beneficial effects, such as antiatherosclerosis and anti-inflammatory properties, and improvement of insulin resistance in the general population (32) .in consistent with current study, a significant inverse correlation between serum adiponectin and resistin levels has also been reported in the literatures (33, 34) . it has been reported that those with highest increases of adiponectin also displayed a trend towards a decline in resistin levels (34) . both hypoadiponectinemia and hyperresistinemia were also positively correlated with hypertension and previous cerebrovascular disease (transient ischemic attack / ischemic stroke) (30) . correlation coefficient p value 1 age 0.236 0.099 2 bmi 0.080 0.580 3 s. uric acid 0.045 0.757 4 s. creatinine -0.040 0.781 5 s. urea 0.195 0.175 6 s. total cholesterol 0.027 0.851 7 s. hdl-c -0.065 0.651 8 serum leptin 0.058 0.688 9 serum adiponectin -0.861 <0.0001 ** 10 serum ctni 0.232 0.105 iraqi j pharm sci, vol.22(1) 2013 serum resistin level and acute stemi infarction 95 furthermore, both hypoadiponectinemia and hyperresistinemia were associated with hypertension (35) and may have prognostic significance for future cardiovascular events in patients with masked hypertension (36) . elevated resistin opposed to adiponectin plasma levels was proposed to be a strong predictive factor for the occurrence of major adverse cardiac events in patients with stable multivessel coronary artery disease over 1-year follow up (37) . thus, the balance of the opposite effects of adiponectin and resistin at the level of the endothelial cell may be an important determinant of endothelial dysfunction, and in turn the progress of atherosclerosis. miyamoto et al. found that resistin may increase the susceptibility of metabolic syndrome by modulating adiponectin secretion from adipocytes (38) . references 1. mazzone t. adipose tissue and the vessel wall. curr drug targets 2007; 8 (november (11)): 1190–5. 2. giannessi d, maltinti m, del rs. adiponectin circulating levels: a new emerging biomarker of cardiovascular risk. pharmacol res 2007; 56 (december (6)):459–67. 3. steppan cm, bailey st, bhat s, et al. the hormone resistin links obesity to diabetes. nature jan 18 2001; 409(6818):307–12. 4. graveleau c, zaha vg, mohajer a, et al. mouse and human resistins impair glucose transport in primary mouse cardiomyocytes, and oligomerization is required for this biological action. j biol chem sep 9 2005;280(36):31679–85. 5. steppan cm, lazar ma. resistin and obesity-associated insulin resistance. trends endocrinol metab jan-feb 2002; 13(1):18– 23. 6. steppan cm, brown ej, wright cm. a family of tissue-specific resistin-like molecules. proc natl acad sci usa jan 16 2001; 98(2): 502–6. 7. kim kh, lee k, moon ys, et al. a cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation. j biol chem apr 6 2001; 276(14):11252–6. 8. rajala mw, qi y, patel hr, et al. regulation of resistin expression and circulating levels in obesity, diabetes, and fasting. diabetes jul 2004; 53(7):1671–9. 9. patel l, buckels ac, kinghorn ij, et al. resistin is expressed in human macrophages and directly regulated by ppar gamma activators. biochem biophys res commun jan 10 2003; 300(2):472–6. 10. hu wl, qiao sb, hou q, et al. plasma resistin is increased in patients with unstable angina. chin med j 2007; 120:871e5. 11. yang, r.z., huang, q., xu, a., et al. comparative studies of resistin expression and phylogenomics in human and mouse. biochem. biophys. res. commun. 2003;310 (3):927-935. 12. bokarewa m, nagaev i, dahlberg l, et al. resistin, an adipokine with potent proinflammatory properties. j immunol 2005; 174:5789e95. 13. calabro p, samudio i, willerson jt, et al. resistin promotes smooth muscle cell proliferation through activation of extracellular signal regulated kinase 1/2 and phosphatidylinositol 3-kinase pathways. circulation 2004; 110: 3335–3340. 14. cohen g, hörl wh. resistin as a cardiovascular and atherosclerotic risk factor and uremic toxin. semin. dial 2009; 22: 373-377. 15. jung hs, park kh, cho ym, et al. resistin is secreted from macrophages in atheromas and promotes atherosclerosis. cardiovasc. res. 2006; 69: 76–85. 16. burnett ms, lee cw, kinnaird td, et al. the potential role of resistin in atherogenesis. atherosclerosis 2005; 182: 241-248. 17. rizkalla j, melone m, zhao a, et al. the pathophysiological role of resistin in impaired lipoprotein metabolism in obesity. circulation 2009; 120: s529. 18. xu w, yu l, zhou w, et al. resistin increases lipid accumulation and cd36 expression in human macrophages. biochem. biophys. res. commun. 2006; 351: 376-382. 19. tarek e , hesham h , eman a , et al. serum resistin in acute myocardial infarction patients with and without diabetes mellitus. postgrad med j. 2011; 87:463-467. 20. songyun c, wenhui d, kang l, et al. plasma resistin associated with myocardium injury in patients with acute coronary syndrome. circ j 2008; 72: 1249–1253. 21. lubos, e., messow, c. m., schnabel, r., et al. resistin, acute coronary syndrome and prognosis results from the atherogene study. atherosclerosis 2007; 193, 121−128 22. burnett ms, devaney jm, adenika rj, et al. cross-sectional associations of resistin, coronary heart disease, and insulin iraqi j pharm sci, vol.22(1) 2013 serum resistin level and acute stemi infarction 96 resistance. j clin 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factors. cardiovasc res 2009; 83, 179−184. 29. rothwell, s. e., richards, a. m., & pemberton, c. j. resistin worsens cardiac ischaemia–reperfusion injury. biochem biophys res commun 2006; 349, 400−407. 30. tuttolomondo a, placa sa, raimondo dd, et al. adiponectin, resistin and il-6 plasma levels in subjects with diabetic foot and possible correlations with clinical variables and cardiovascular comorbidity.cardiovascular diabetology 2010, 9:50. 31. yannakoulia m., yiannakouris n., bluher s., et al. body fat mass and macronutrient intake in relation to circulating soluble leptin receptor, free leptin index, adiponectin and resistin concentrations in healthy humans. j. clin. endocrinol. metab. 2003 , 88: 1730– 1736. 32. matsuzawa y, funahashi t, kihara s, et al . adiponectin and metabolic syndrome. arterioscler thromb vasc biol 2004;24:29– 33 33. wasim h, al-daghri nm, chetty r, et al. relationship of serum adiponectin and resistin to glucose intoleranceand fat topography in south-asians. cardiovascular diabetology 2006, 5:1-5. 34. lewandowski kc, szosland k, o’callaghan c, et al. adiponectin and resistin serum levels in women with polycystic ovary syndrome during oral glucose tolerance test: a significant reciprocal correlation between adiponectin and resistin independent of insulin resistance indices. mol genet metab 2005, 85:61-69. 35. thomopoulos c, daskalaki m, papazachou o, et al. association of resistin and adiponectin with different clinical blood pressure phenotypes. j hum hypertens 2011, 25:38-46. 36. papadopoulos dp, perrea d, thomopoulos c, et al. masked hypertension and atherogenesis: the impact on adiponectin and resistin plasma levels. j clin hypertens 2009, 11:61-65. 37. krecki r, krzeminska m, peruga jz, et al. elevated resistin opposed to adiponectin or angiogenin plasma levels as a strong, independent predictive factor for the occurrence of major adverse cardiac and cerebrovascular events in patients with stable multivessel coronary artery disease over 1-year follow-up. med sci monit 2011, 17: cr26-32. 38. miyamoto y, morisaki h, kokubo y, et al. resistin gene variations are associated with the metabolic syndrome in japanese men. obesity research & clinical practice 2009, 3:65-74. iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 74 design, synthesis, characterization and preliminary anticancer study for methotrexate silibinin conjugates shams a. nadhum * and mohammed h. mohammed *,1 * department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract the spectrum of clinical efficacy of methotrexate (mtx) is broad in that mtx is used in the treatment of certain cancers, severe psoriasis and rheumatoid arthritis.various mechanisms by which cancer cells grown in tissue culture become resistant to anticancer drugs. the use of multiple drugs with different mechanisms of entry into cells and different cellular targets allows for effective chemotherapy and high cure rates. in an efforts to develop effective strategies that increase the therapeutic potential of anticancer drugs with less systemic toxicity ,are being directed towards the investigation of dietary supplements and other phytotherapeutic agents for their synergistic efficacy in combination with anticancer drugs. a promising approach to improve the cancer cell selectivity of methotrexate is the chemical transformation into reversible derivatives which convert the conjugate to the parent drug by virtue of enzyme within cancer tissue. the present study includes the synthesis of two derivatives of methotrexate which are :-schiff base methotrexate-silibinin conjugate ( compound 5), and methotrexate-silibinin conjugate (compound 6).the synthesis of the target compounds was accomplished following multistep reaction procedures. the chemical reactions were followed up and purity of the products was checked by tlc. the structures of the final compounds and their intermediates were characterized and identified by their melting points, infrared spectroscopy, 1 hnmr and elemental microanalysis(c h n s).the anticancer activity of these compounds was investigated by hep-2 cell line(larynx carcinoma), which showed that compounds 5 and 6 have the higher activity than methotrexate or silibinin alone.these are promising data for the discovery of new anticancer agents in future. these compounds may deliver the parent drug selectively into the cancer cells to be hydrolyzed by enzymes that are elevated in tumor tissues compared with normal tissues . keywords: methotrexate, silibinin, cancer treatment resistance, folate receptor, cancer cell targeting. جصميم وجصىيع وجشخيص ودراسة اولية لمضادية السرطان لمقحرن سليبيىيه –ميثىجركسيث واظم شمس عىاد * و محمذ حسه محمذ *،1 .تغذاد، اٌعشاق ،تغذاد اٌصُذٌح، خاِعح اٌصُذالُٔح، وٍُحفشع اٌىُُّاء * الخالصة فٍ عالج أىاع ِٓ اِشاض اٌسشطاْ،واٌصذفُح واٌرهاب السرعّاٌها ذرُّز ِادج اٌّثىذشَىسُد تطُف ِٓ اٌفعاٌُح اٌسشَشَح ورٌه ج اٌّفاصً اٌشوِاذُذٌ. هٕان عذج اٌُاخ اٌرٍ تّىخثها ذّٕى اٌخالَا اٌسشطأُح فٍ اٌزساعح إٌسُدُح واٌرٍ ذىىْ ِماوِح ٌالدوَح اٌّضاد ِخرٍفح َسّح ٌعالج وُُّائٍ ِؤثش وِعذالخ ادوَح ِرعذدج ِع اٌُاخ ِخرٍفح ٌٍذخىي اًٌ اٌخالَا واهذاف خٍىَح اسرعّايٌٍسشطاْ. اْ وٌغشض ذطىَش سرشاذُدُاخ فعاٌح فاْ خهىدا ذثزي ٌزَادج اٌفعاٌُح اٌعالخُح ٌالدوَح اٌّضادج ٌٍسشطاْ ِع الً سُّح ِٓ شفاء عاٌُح تاالذحاد ِع فعاٌُح اوثش ٌرصثح راخخالي ذىخُهها ٔاحُح اٌرحشٌ عٓ اٌّىّالخ اٌغزائُح وعىاًِ االدوَح إٌثاذُح )االعشاب اٌطثُح( االدوَح اٌّضادج ٌٍسشطاْ.وِٓ االهذاف اٌىاعذج ٌرحسُٓ أرمائُح اٌخالَا اٌسشطأُح ذداٖ عماس اٌُّثىذشَىسُد هى اٌرحىي اٌىُُّائٍ ك ِشرمُٓ ٌٍّمذِاخ اٌذوائُح اًٌ اٌعماس االَ )االصً( تحىُ االٔزَُ اٌّىخىد فٍ إٌسُح اٌسشطأٍ. ذرضّٓ اٌذساسح اٌحاٌُح ذخٍُ ٌٍّثىذشَىسُد وهّا : (.5سٍُثُٕٓ )ِشوة –لاعذج شف ي ِمرشْ اٌّثىذشَىسُد (.6سٍُثُٕٓ )ِشوة –ِمرشْ ُِثىذشَىسُد ذمُٕح عّاياٌّشوثاخ اٌرٍ َهذف اٌُها اٌثحث تاذثاع طشائك ِرعذدج اٌخطىاخ وذُ اٌراوذ ِٓ ٔماوج هزٖ اٌّشوثاخ ورٌه تاسر ذحضُشذُ وشوِاذىغشافُااٌصفائح اٌشلُمح.وزٌه ذُ ذشخُص وذىصُف إٌرىاذح إٌهائُح وِىادها اٌىسطُح ِٓ خالي ذعُُٓ دسخاخ أصهاسها واطُاف االشعح ذحد اٌحّشاء وطُف اٌشُٔٓ إٌىوٌ اٌّغٕاطُسٍ ٌٍثشذىْ،واٌرحًٍُ اٌذلُك ٌٍعٕاصش. وهٍ خالَا سشطاْ 2اٌّشوثاخ تاسرخذاَ اٌخٍُح اٌسشطأُح اي هة ٌمذ اثثرد دساسح اٌفعاٌُح اٌّضادج ٌٍسشطاْ ٌهزٖ َّرٍىىْ فعاٌُح اعًٍ ِٓ اٌُّثىذشَىسُد واٌسٍثُُٕٓ و هزٖ إٌرائح ذعرثش واعذج 6واٌّشوة 5اٌحٕدشج واٌرٍ اظهشخ تاْ اٌّشوة عالٖ َرضح تاْ هزٖ اٌّشوثاخ ٌها اٌمذسج عًٍ اَصاي الورشاف ِضاداخ سشطأُح خذَذج فٍ اٌّسرمثً. ووفما ٌهزٖ إٌرائح اٌّثُٕح ا سرىي عاٌٍ فٍ االٔسدح اٌسشطأُح ِماسٔح ِع ّاالدوَح تأرمائىح ٌٍخالَا اٌسشطأُح وتاٌُح ذحشَش ذرضّٓ ذاثُش االٔزَّاخ اٌّىخىدج ت .االٔسدح اٌطثُعُح ةالت الفىليث،اسحهذاف الخاليا السرطاويالكلمات المفحاحية:ميثىجركسيث ،سيلبىيه،مقاومة عالج السرطان،مسحقب 1 corresponding author e-mail: dr.mhassanm666666@yahoo.com received: 17 /1/2015 accepted: 23/5/2015 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 75 introduction the design of cancer chemotherapy has become increasingly sophisticated,yet there is no cancer treatment that is 100% effective against disseminated cancer. resistance to treatment with anticancer drugs results from a variety of factors including individual variations in patients and somatic cell genetic differences in tumors, even those from the same tissue of origin (1) . researchers have been tabulating the various mechanisms by which cancer cells grown in tissue culture become resistant to anticancer drugs. some of these mechanisms, such as loss of a cell surface receptor or transporter for a drug, specific metabolism of a drug, or alteration by mutation of the specific target of a drug, all of which occur for antifolates such as methotrexate (2) . mtx competitively inhibits dihydrofolate reductase (3) . folate receptor a (fra) is a membrane-bound protein with high affinity for binding and transporting physiologic levels of folate into cells. folate is a basic component of cell metabolism and dna synthesis and repair, and rapidly dividing cancer cells have an increased requirement for folate to maintain dna synthesis, an observation supported by the wide spread use of antifolates in cancer chemotherapy (4) . endocytosis is the general mechanism of ( fra) mediated folate uptake. one specialized route, potocytosis , was proposed from the observation that( fra) recycled between an acid-resistant (intracellular) and acid-sensitive (extracellular) pool. proposed that (fra )was concentrated in clusters in invaginations of the cell membrane surface called caveolae, whereby the membrane would transiently close and internalize the folate-bound receptor complex. increased acidification of the internal compartment would dissociate folate from the receptor and move it across the membrane into the cytoplasm of the cell using the energy generated by the acidic gradient (5) . folate receptor-targeted cancer therapies constitute a promising treatment for the approximately one third of human cancers that over express the folate receptor (fr). however, the potencies of all folate-receptor targeted therapies depend on the rate of folate-linked drug conjugate binding to the cancer cell surface, the dose of folate conjugate that will saturate tumor cell surface fr in vivo, the rate of fr internalization, un loading,and recycling back to the tumor cell surface for another round of conjugate uptake, and the residence time of the folate conjugate before its metabolism or release from the cell (6) .linkage of a drug to folic acid can generate a molecular “trojan horse” that can enable tumor-specific delivery of both imaging and therapeutic agents into cancer cells. molecules that have been targeted to tumors by conjugation to folic acid include radiopharmaceuticals (7) , enzymes for prodrug activation (8) ,nanoparticles (9) , peptides, toxic proteins (10) , immunologically potent haptens (11) , antisense oligonucleotides (12) , chemotherapeutic agent , gene therapy vectors (13) , viruses (14) ,and polymeric drug carriers and liposomes (15) ,for example conjugation of methotrexate to poly(l-lysine) increases drug transport and overcomes drug resistance (16) and conjugation of methotrexate with 5fluorouracil (17) . in an effort to develop effective strategies that increase the therapeutic potential of anticancer drugs with less systemic toxicity, more strategies are being directed towards the investigation of dietary supplements and other phytotherapeutic agents (18) .silibinin has shown promising chemo preventive and anticancer effects in various in vitro and in vivo studies (19) .several studies have shown that silibinin and silymarin have anticancer activity against breast, skin, androgen-dependent and independent prostate, cervical, bladder, hepatocellular,colon, ovarian, and lung cancer cells in culture and several in vivo animal cancer model systems (20) . the efficacy of silibinin in inhibiting the growth of different cancer lines is quite different. such differences in the potency of the drug in arresting cell growth may be due to differences in the experimental conditions used, the cell type and potential carcinogenicity of the cell lines (21) . in view of this observation, two derivatives of methotrexate are designed, synthesized and characterized. experimental section general chemicals used in the synthesis were of analytical grade(methotrexate and p-nitro phenyl chloroformate from(hongmao,china)and silibinin(tolbiac s.r.l ,argentina). the melting points of the compounds and their intermediates (uncorrected) were determined by capillary tube method on barnstead electrothermal (usa) and ft-ir spectra were recorded on ft-ir spectrophotometer shimadzu (japan) at the college of pharmacy /university of baghdad. the chns analysis was carried out using euro-vector ea3000a(italy).ascending thin layer chromatography(tlc)was run on silica gel 60 f254 pre-coated aluminum sheets, merck (germany) to check the purity and the reactions progress, 1 h-nmr analysis was performed on 1h-nmr spectroscopy (shimadzu,japan) at university of al-bayt,jordan, and the iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 76 chromatograms were eluted by three solvent systems: a-aceton:chloroform (3:1). baceton:chloroform :acetic acid(3:1: 0.5) (22). the evaluation of cytotoxic activity was done at al-naharin biotechnology research center using rosswell park memorial institute (rpmi1640)which consists from hepes buffer and l-glutamin( sodium bicarbonate ,streptomycin ,benzyl penicillin g,mycostatin ,fetal calf serum) . chemistry the synthetic procedures for the designed target compounds 5 and 6 are illustrated in schemes (1 and 2) . the starting material for the generation of the target compounds 5 and 6 was methotrexate which was refluxed with 2 equivalent benzaldehyde in the presence of 10% sodium hydroxide to give the imines derivative of methotrexate (compound 2). compound 2 was converted to acylchloride derivative by useing thionyl chloride and triethylamine in which one carboxylate group was blocked. the acylchloride derivative was allowed to react with nh2 group of dimethyl ester derivative of cystine in the presence of triethylamine to give the imine-methotrexate cystine derivative (compound 3) which was reacted with paranitrophenylchloroformate to give compound 4. then (compound 4 ) was stirred with silibinin in the presence of sodium hydroxide to give iminemethotrexate silibinin conjugate (compound 5).the methotrexate silibinin conjugate (compound 6 )was obtained by hydrolysis of compound 5.the physical properties of the synthesized compounds are showed in table (1). the structures of the final compounds were characterized by elemental microanalysis (table2) .infrared spectroscopy (table3) and 1 hnmr done for compound 5(table4). methanol socl2 s s oo oo o n n n nh2n nh2 n o h n oh o oho compound1 10% naoh n n n n n nn n h ho o oho o compound 2 2 reflux methotrexatebenzyldehyde l-cystine (sch2ch(nh2)co2h)2 nh3cl nh3cl scheme (1): synthesis of compounds 1and 2. nh3cl nh3cl socl2 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 77 n n n n n nn n h h n o oho o s o o so o hn o o n o o compound 4 n n n n n nn n h ho o oho o s s oo nh3cl nh3cl oo n n n n n nn n h h n o oho o s o o so o compound 3 compound 2 10°c /tea socl2 / -15 °c stirring/tea 1. 2. + reflux o c cl o no2 p-nitrophenylchloroformate nh2 socl2 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 78 silibinin 1n hcl compound 5 2. 5%hcl 1. 5%naoh acetonitrile schiff base hydrolysis compound 6 scheme( 2): synthesis of compounds 3 – 6 . chemical methods: synthesis of 3,3'-disulfanediylbis(1-methoxy1-oxopropan-2-aminium) chloride.(compound 1). a suspension of cystine (41.6mmole,10g) in (150 ml)of absolute methanol,was cooled to -15 ᵒ c then thionyl chloride(99.8 mmole,7.4 ml) was added drop wise ,(the temperature should be keep below -10 ᵒ c) and the reaction mixture was left at 40 ᵒ c for 1hr,then reflux for 4 hr and left at room temperture over night,the excess solvent was evaporated to dryness under vacuum;recrystallize the product from methanol-diethyl ether and collected as hcl salt (23) . synthesis of 2 ( 4 ( ( ( 2 , 4 -bis ( benzylideneamino ) pteridin 6 -yl) methyl ) (methyl) amino) benzamido) pentanedioic acid.(compound2). in 500 ml round bottom flask attached with a reflux condenser, mixture of benzaldehyde (44 mmole,4.47 ml ), methotrexate (11mmole,5 g), and 10% sodium hydroxide (22mmole,0.88 g). add 200 ml of absolute ethanol to the mixture and the final mixture is refluxed for 3 hours, cooled and acidified to ph 5with 5% hcl . the mixture was filtered,the product was washed with cold ethanol and dried (24) . 1n hcl hcl iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 79 synthesis of 4 [ 2 ( 2 amino 2 methoxycarbonyl ethyldisulfanyl ) 1 methoxycarbonyl ethylcarbamoyl ] 2 (4 { [ 2 , 4 bis ( benzylidene amino ) pteridin 6 ylmethyl-methyl amino } benzoylamino ) buty ric acid .(compound 3). a suspension of compound 2 (7.93mmol,5 g) and triethylamine (7.93mmole ,1.10ml ) in acetonitrile (50ml),was coolded down to-15 ᵒ c then thionyl chloride ( 7.93mmol,0.59ml) was added slowly, and the mixture was refluxed for 4hr at( 60-70) ᵒ c with continuous stirring.then evaporate the excess solvent by using rotary evaporater .the viscous liquid was slowly added drop wise into beaker contain compound 1(7.93mmol,2.7g) and triethylamine (15.86mmol, 2.2ml) dissolved in acetonitrile(50ml) for 1 hr in an ice bath ,then stirring over night at room temperature. the obtained suspension was filtered and the filterate was washed with distilled water (20ml),dried over anhydrous magnesium sulfate and the solvent was evaporated by vacuum. the obtained compound was recrystallized from diethyl ether-petroleum ether(4:6)to give compound 3. (25) synthesis of 2-(4-(((2,4bis(benzylideneamino)pteridin-6yl)methyl)(methyl) amino)benzamido)-5-((1methoxy-3-((3-methoxy-2-(((4nitrophenoxy)carbonyl) amino)-3oxopropyldisulfanyl)-1-oxopropan-2yl)amino)-5-oxopentanoic acid.(compound 4). to a stirred solution of compound 3 ( 2.17 mmol,2 g) in toluene(100ml) at -10°c was added triethylamine ( 2.17 mmol,0.61ml). after 5 min, a cooled mixture of paranitrophenylchloroformate (2.17 mmol,0.443 g) in toluene (5 ml)was added drop wise . the mixture was stirred for 2 hr at 10°c and for 15 hr at room temperature, filtered,and evaporated to dryness under reduced pressure.the residual oil was crystallized twice from diethyl ether (150ml) to give a yellow powder (26) . synthesis of15-(4-(((4-benzylideneamino)-2((z)-benzylidene amino ) pteridin 6 -yl ) methyl ) ( methyl ) amino )phenyl ) 1 (4-(3(hydroxylmethyl)-7-(3,5,7-trihydroxy-4oxochroman 2 yl ) -2 , 3 dihydrobenzo [b][1,4]dioxin-2-yl)-2-methoxyphenoxy)-1,10, 15-trioxo-5,6-dithia-2,9,14triazapentadecane-3,8,13-tricarboxylic acid .( compound 5) . to a mixture of silibinin(0.477mmol,0.23 g) and compound 4 (0.477 mmol ,0.5 g)in 30ml distalled water ,add 5%naoh drop wise until the solution become basic at ph 10 and left stirred for 48hr.the mixture was then acidified with hydrochloric acid 5% drop wise to give a precipitate.this precipitate was washed with distalled water thoroughly ,then with ethanol and dried in oven at 50°c,to provide compound 5. (27-29) synthesis of15-(4-(((2,4-diaminopteridin-6yl)methyl)(methyl)amino) phenyl)-1-(4-(3-( hydroxymethyl ) -7 ( 3 , 5 , 7 trihydroxy -4 oxochroman 2 – yl ) 2 , 3 -dihydrobenzo [b][1,4]dioxin-2-yl)-2-methoxyphenoxy)-1 , 10 , 15 – trioxo 5,6-dithia-2,9,14-triaza penta decane-3,8,13-tricarboxylic acid. (compound 6). compound (5) (0.36 mmole, 0.5 g) was dissolved in 2.5 ml of acetonitrile. the ph of the solution was adjusted to 2 with 1n hcl and it was stirred for 1hr at (0-5) ᵒ c. the precipitate was filtered,washed with ether and dried to give compound 6 (30) . cell culture and culture conditions hep2 cell line(passage number( 75) were isolated from human epidermoid larynx carcinoma . this type of cells were maintained in rpmi-1640 medium with 30% bovine calf serum and 10% dmso , then the cell treated with trypsin/versin mixture in order to pursue subculture process . evaluation of cytotoxic activity the in vitro cytotoxic activity (cell viability assay) of these compounds(mtx,silibinin, compounds 5,6) were evaluated by neutral red dye assay; a nonradioactive ,fast assay widely used to quantify cell viability and proliferation . a set of 3 concentrations (0.5,1,2) μg/ml was made for each product and the exposure time of the assay was 24 hours,48hours. the cytotoxicity assay done according to a reported method (31) where the cell exposed to different concentrations of different compounds (0.5,1,2) μg /ml respectively, each compound was added to the cell in triplicate form of each concentration ,rpmi media and cell added as positive control ,only cells incubated with culture media represented the negative control. results and discussion the prepared compounds were characterized by means of physical properties, ftir,chns analysis and 1 h-nmr.the results are illustrated in table 1,2,3 and4,respectively . mechanism of action the compounds(5 and 6) can be considered as prodrugs, when inter the cancer cell can be hydrolysed by reductase enzyme which elevated in cancer cell and lead to hydrolysis of disulfide, and production of two moieties (32) . iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 80 table(1): physical data of the synthesized compounds. compound physical appearance %yield melting point observed ( o c) rf value 1 white powder 59.8 105-107 a= 0.65 b=0.4 2 dark yellow powder 90.9 185 (decomposed) a=0.166 b=0.2 3 yellow powder 52.2 115-118 a=0.71 b=0.78 4 yellow powder 54.28 145-148 a=0.85 b=0.82 5 yellow powder 30 203 (decomposed) a=0.72 b=0.78 6 yellow powder 58.6 170 (decomposed) a=0.78 b=0.9 table (2): elemental microanalysis of the final compounds. elemental microanalysis % empirical formula molecular weight compound 58.15 58.2 c c66h60n10o19s2 1360 5 4.649 4.41 h 10.688 10.29 n 4.48 4.7 s 51.984 52.7 c c52h52n10o19s2 1184 6 4.582 4.38 h 12.141 11.8 n 5.192 5.4 s table (3): the characteristic ir bands of synthesized compounds(1-6). compound characteristic i.r. bands (cm -1 ) 1 (3404-2800broad band n-h stretching vibration of nh3cl,(1745c=o stretching vibration of ester ). 2 (3346n-h stretching vibration of amide), (1668c=o stretching of carboxylic acid),(1655n-h bending of amide),(1600c=n stretching of imine 3 (3346n-h stretching vibration of amide), (1745c=o stretching vibration of ester),(1643c=o stretching vibration of amide),(1602c=n stretching of imine). 4 (3331n-h stretching vibration of amide),(1745 c=o stretching vibration of ester),(1716c=o stretching vibration of carbamate),(1643c=o stretching vibration of amide),(1604c=n stretching of imine),(1521 asymmetric stretching vibration of no2),,(1346symmetric stretching vibration of no2). 5 (3354n-h stretching vibration of amide),(1735c=o stretching vibration of cooh),(1637c=o stretching vibration of amide),(1604c=n stretching of imine). 6 (3346n-h stretching vibration of amide) (1735c=o stretching vibration of cooh),(1637c=o stretching vibration of amide). 1 h-nmr analysis: 1 h-nmr analysis was performed on 1 hnmr spectroscopy (shimadzu,japan) at university of al al-bayt,jordan,using dimethyl sulfoxide(dmso) as a solvent and the results are listed on table (4). iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 81 table (4): 1 h-nmr interpretation of compound (5). n h s s h n n h o oh oho oho o o o o o o o o o oh oh ho ho n n n n nn n 1 3 2 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 25 24 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 h chemical shift (ppm)  functional group 1,3 5.56 aromatic oh 2,4 5.99 1-benzen 6 2.94 alcoholic oh 5,7 5.59 methine 8,9,10,16 6.87 1-benzen 15,17 7.17 1-benzen 13 4 methylene 14 3.7 alcoholic oh 18 3.83 methyl 19,26 8.16 secondary amide 20,25,30 11.47 carboxylic acid 21,24,29 4.68 methine 22,23 2.94 methylene 32,34 7.58 1-benzen 33,35 6.97 1-benzen 36 3.13 methyl 37 4.54 methylene 38 8.63 2-pyrazine 39,45 8.42 ar.protons, benzylidenimin 40,44,46,50 7.79 ar.protons, benzylidenimin 41,42,43,45,47,48,49 7.51 ar.protons, benzylidenimin 1 h-nmr spectrum of compound 5 iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 82 results of cytotoxicity assay the cytotoxic study was done on hep2 cell line(passage number( 75 ) were isolated from human epidermoid larynx carcinoma inhibition rate% the inhibition rate percent (i.r. %)(33) or inhibitory concentration percent (ic %) was estimated, and the result was varied among samples as shown in figure (1,2). figure(1):the cytotoxic activity represented by inhibition rate percent of compounds mtx,silibinin,compound 5,6 at different concentrations (0.5 ,1,2) μg/ml in 24 hrs time of exposure . figure(2): the cytotoxic activity represented by inhibition rate percent of compounds mtx,silibinin,compound 5,6 at different concentrations(2,1,0.5) μg/ml in 48 hrs time of exposure. one cell lines was studied (hep-2) at two times of exposure 24 and 48hr , using a 3 fold dilutions to get concentrations from ( 2 to 0.5 ) μg / ml of our compounds. the produced effect was explained as the following: the cytotoxic effect of mtx on hep – 2 (passage no.75), fig.1 revealed that the high concentration 2 μg/ml gave a significantly high inhibition rate of cells while being low gradually with low concentration 0.5 μg/ml during 24 hr of exposure( fig.1) , while during 48hr the cytotoxic effect high at high concentration 2 μg/ml (figure 2 ) and proliferation develop during lo w concentration due to develop resistant of cell cancer to mtx. the cytotoxic effect of silibinin gave high inhibition rate (33.1%) at high concentration 2 μg/ml and low with low concentration 0.5 μg/ml during 24 hr of exposure (fig.1) while low inhibition rate at low concentration 0.5 μg/ml and proliferation develop as the concentration increase((1,2) μg/ml) during 48 hr of exposure (figure 2). the compound 5 shows high inhibition rate at high concentration 2 μg/ml , in both times and decrease as the concentration decreases( fig.2) , but shows higher inhibition rate at 1μg/ml during 24hr of exposure (fig 1). the compound 6 also shows high inhibition rate (41.2%) at high concentration 2 μg/ml and at 1μg/ml shows higher inhibition rate 44.2% during 24 hr( fig.1), while the inhibition rate decrease during 48 hr( fig.2)and proliferation was develop. an explanation for this behaviour ,that in the design of cell culture experiment , it was important to be aware of growth state of the culture, as well as the quantitative characteristic of cell stain or cell line. culture will vary significantly in many of their properties between exponential growth and stationary phase (34) . also the differences in hep-2 responding to word different treatment might indicate a presence or absence of specific cellular receptor in each type of cell line, making the cell interact at same concentration in different manners. moreover the metabolic pathways in response to each treatment differed from one cell line to another .this fact was mentioned in different studies which investigated at different plant extracts in treating several types of cell line (35,36) . hep-2 resistance might be explained by over expression phenomena through genes responsible for bindingreceptor blockage that prevent the cytotoxic effect of any treatment (37) .from the result we can conclude that the activity of compounds 5,6 better than the activity of mtx or silibinin alone,and these may be attributed to the reduction in the resistance development. conclusions 1. the synthetic procedure for the designed target compounds was successfully achieved and the structural formula for the synthetic compound was characterized using ft-ir spectroscopy, elemental microanalysis, melting points and rf values and 1 h-nmr analysis done for compound 5. 2.the activity of compounds 5 and 6 were studied as anticancer drugs.they were more iraqi j pharm sci, vol.24(1) 2015 synthesis of methotrexate silibinin conjugates 83 effective drugs compared with mtx and silibinin. references 1. gottesman m.m., mechanisms of cancer drug resistance, annu. rev. med., 2002; 53:615–27. 2. longo-sorbello g.s. and bertino j.r., current understanding of methotrexate pharmacology and efficacy in acute leukemias, use of newer antifolates in clinical trials, haematologica,2001 ;86:121–27. 3. smith s. 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prolongs survival of tumor,wearing mice ,am.j. clin. med.,2003;31:857869. 37. kim s. e.,kim y. h. and lee j.j., a new sequiterpene ester from calastrus orbiculatus reversing multi-drug resistance in cancer cells ,j.nat.prod., 1998 ;61: 108-111. iraqi j pharm sci, vol.31(1) 2022 evaluate the detrimental effects of some antiepileptic drugs doi: https://doi.org/10.31351/vol31iss1pp241-245 241 evaluation of the detrimental effects of some antiepileptic drugs on the height and weight of children with epilepsy abdullah shakir mahmood*, aseel s. al mallakhdeer* , muhammad a. alkataan*,1 and mahfoodh s. hasan* * college of medicine, ninevah university ,ninevahm, iraq abstract growth is a multifactorial process influenced by genetic, nutritional, hormonal, psychosocial and other factors including the general health of a child. epilepsy is defined as a chronic condition characterized by recurrent clinical events or epileptic seizures, which occur in the absence of a metabolic or toxic disease the drugs that are used in the treatment of this condition can affect patients’ growth due to their mechanisms of action. this study aimed to evaluate the effect of some antiepileptic drugs on growth (height and weight) in children with epilepsy. this work involved 51 newly diagnosed children with a different form of epilepsy (generalized, absent and partial). patients were collected from the outpatient’s clinic in al salam teaching hospital and private clinic in mosul city from july 2018 to july 2020. patients were divided into three groups of 17 patients each according to the treatment (group one patients on carbamazepine monotherapy with dose mean 13.3 ± 4.8 mg/kg, group two patients on valproic acid monotherapy with a dose of 14.4± 3.3 mg/kg and the last group involve patient on combined therapy carbamazepine 10.8±5.8mg/kg plus 19.7± 8.8 mg/kg of valproic acid. patients ages range from 5-11 years, with an initial bmi range of 12-20. the results of this work showed that carbamazepine monotherapy caused no significant effect on both bmi values after 6 and 12 months of treatment. valproic acid monotherapy significantly elevated bmi after 6 and 12 months of treatment. combined therapy showed no significant effect on bmi. the patient’s centile height significantly elevated after 6 and 12 months of valproic acid compared to the normal growth according to the growth chart. while both carbamazepine and combined therapy showed no significant change in comparison with the normal growth according to the growth chart. in conclusion, children with epilepsy who use antiepileptic drugs need a restricted monitor policy for their growth, especially those on valproic acid. keywords: growth, weight, percentile, carbamazepine, valproic acid. تأثير األدوية المضادة للصرع على بعض متغيرات النمو لدى األطفال المصابين بأنواع مختلفة من الصرع *و محفوظ سليمان حسن 1 ،*طانالقعبد الغفور دمحم *سامي المال خضر اسيل ،*عبد هللا شاكر محمود العراق. نينوى، ، كلية الطب، جامعة نينوى * الخالصة عملية ان أخرى بما في ذلك الصحة العامة معقدة النمو واالجتماعية وعوامل تتأثر بالعوامل الوراثية والتغذوية والهرمونية والنفسية ف الصرع بأنه حالة مزمنة تتميز سام، والتي تحدث في حالة عدم وجود مرض استقالبي أو صرع، متكررة أو نوبات بأعراض سريريةللطفل. يُعرَّ ي عالج هذه الحالة أن تؤثر على نمو المرضى بسبب آليات عملها. هدفت هذه الدراسة إلى تقييم تأثير بعض األدوية يمكن لألدوية التي تستخدم ف طفالا تم تشخيصهم حديثاا بنوع مختلف من الصرع 51المضادة للصرع على النمو )الطول والوزن( لدى األطفال المصابين بالصرع. شمل هذا العمل )المجموعة األولى من المرضى الذين عولجوا بكاربامازيبين وحيداا )معمم وغائب وجزئي(. تم تقسيم المرضى إلى ثالث مجموعات وفقاا للعالج مجم / كجم والمجموعة 3.3± 14.4مجموعة مريضين في العالج األحادي لحمض الفالبرويك بجرعة كجم، مجم / 4.8± 13.3بجرعة متوسط من حمض الفالبرويك. تتراوح أعمار 8.8± 19.7باإلضافة إلى carbamazepine 10.8 ± 5.8 المركباألخيرة تشمل المريض على العالج . وأظهرت نتائج هذا العمل أن العالج األحادي بالكاربامازيبين لم يكن له تأثير 2012مع نطاق أولي لمؤشر كتلة الجسم من سنة، 11-المرضى بين ا من العالج. أدى العالج األحادي بحمض الفالبرويك إلى رفع مؤشر كتلة الجسم بشكل ملحوظ بعد 12و 6كبير على قيم مؤشر كتلة الجسم. بعد شهرا ا من العالج. لم يظهر العالج المشترك أي تأثير كبير على مؤشر كتلة الجسم. ارتفع االرتفاع المئوي للمريض بشكل ملحوظ بعد 12و 6 12و 6شهرا ا بينما لم يظهر كل من العالج بالكاربامازيبين والعالج المركب أي النمو، ويك مقارنة بالنمو الطبيعي وفقا لجدول من استخدام حمض الفالبر 5شهرا معنوي يحتاج األطفال المصابون بالصرع الذين يستخدمون األدوية المضادة للصرع إلى أيون، مقارنة بالنمو الطبيعي وفقاا لمخطط النمو. تغيير . وخاصة أولئك الذين يتناولون حمض الفالبرويك لنموهم، سياسة مراقبة مقيدة . حمض الفالبرويك كاربامازيبين، المئوية،النسبة الوزن، النمو،الكلمات المفتاحية: 1corresponding author e-mail: moh1977729@gmail.com received: 12/8/2021 accepted: 20/10 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp241-245 iraqi j pharm sci, vol.31(1) 2022 evaluate the detrimental effects of some antiepileptic drugs 242 introduction growth is a multifactorial process influenced by genetic, nutritional, hormonal, psychosocial and other factors including the general health of a child (1). deviation from a normal pattern of growth can be the first manifestation of a wide variety of disease processes, including endocrine and non-endocrine disorders and may involve virtually any organ system of the body (2). the rate of linear growth and the physiologic components regulating it varies with age (3). the proliferation of epiphyseal growth plate results from complex interactions between hormones and growth factors, which may directly or indirectly affect the serum levels of calcium and the condition of those cells, leading to final stature (4). the most powerful tool in growth assessment is the growth chart used in combination with accurate measurements of height, weight, head circumference, and calculation of the body mass index (5). the growth trajectory of each stage of growth (fetal, infant (i), childhood (c) and pubertal (p)) can be represented mathematically by the ‘icp growth model’. growth during the first 3 years results from a combination of a rapidly decelerating infancy component and a slowly decelerating childhood component. the latter dominates the midchildhood years but it is altered by the pubertal contribution, which is modelled on a sigmoid curve (2). epilepsy is defined as a chronic condition characterized by recurrent clinical events or epileptic seizures, which occur in the absence of a metabolic or toxic disease or fever (6,7). the international league against epilepsy recognizes two major categories of epileptic seizures, based on clinical and electroencephalographic (eeg) features: focal seizures and generalized seizures. focal seizures originate within neural networks involving one hemisphere of the brain and more or less localized at the onset, whereas generalized seizures start in and rapidly involve networks in both cerebral hemispheres (6). among partial seizures, those in which awareness is impaired are termed complex, whereas those in which awareness is preserved are termed simple (8). for infants and children with their first unprovoked seizure, urgent laboratory studies with neuroimaging should be considered, depending on the history and physical findings. the definitive neuro-diagnostic evaluation, consisting of an eeg and neuroimaging study, of pediatric patients with seizures typically performed on an outpatient basis. the eeg could help in the assessment of the child with a first seizure, especially if an epileptic syndrome is identified; however, a normal eeg does not rule out seizures. although an eeg when performed within 24 hours of the fit, if abnormalities were found, these abnormalities may be due to the postictal condition. typically, the eeg is performed on an outpatient basis within 2 weeks of the first seizure (7, 9). the age and seizure type of the patient are two important factors that should be considered in choosing an anticonvulsant. for generalized tonicclonic or focal onset seizures in infants up to 6 months of age, phenobarbital is often chosen first, given its ease of use, predictable blood levels and modest side-effect profile. for older infants or children with focal onset seizures, oxcarbazepine or levetiracetam is the preferred medication. levetiracetam is commonly used for generalized tonic-clonic seizures at any age (10). divalproex (valproate, divalproex sodium) is a broad-spectrum aed that can be used in all types of generalized epilepsy, including absence, primary gtcs and myoclonic; absence seizures can be treated with ethosuximide, and then when gtcs seizures appear, divalproex or lamotrigine can be used, because ethosuximide does not cover other types than absence. practitioners should monitor the efficacy and side-effects of anticonvulsant therapy, help in dose adjustment of anticonvulsants in patients – especially those receiving single anticonvulsants – and reviewing seizure precautions frequently to encourage healthy and safe living (9,11). discontinuation of aeds can be scheduled when the child is seizure-free for at least 2 years aed therapy should be discontinued gradually; over at least 3-6 months, sudden discontinuation can lead to withdrawal seizures or status epilepticus (1,2). this study aimed to evaluate the effect of some antiepileptic drugs on growth (height and weight) in children with epilepsy. as mentioned above, that antiepileptic agents can affect the proliferation of epiphyseal growth plate, affecting the serum levels of calcium leading to short stature (4). this work will assess growth according to the growth chart. material and methods this work involved 51 newly diagnosed children with different forms of epilepsy (generalized, absent and partial). patients were collected from the outpatient’s clinic in al-salam teaching hospital and private clinic from july 2018 to july 2020. patients were divided into three groups according to the treatment (group one includes 17 patients on carbamazepine monotherapy with a dose mean of 13.3 ± 4.8 mg/kg. group two include 17 patients on valproic acid monotherapy with a dose of 14.4± 3.3 mg/kg, and the last group involve 17 patients on combined therapy carbamazepine 10.8±5.8 mg/ kg plus 19.7± 8.8 mg /kg of valproic acid. the patient`s ages ranged from 5-11 years, with an initial bmi range of 12-20. patients have good physical activity with no other cns disorders. patients with any other health problems were excluded from this work. height was measured using standard human balance rgt.b-200-rt iraqi j pharm sci, vol.31(1) 2022 evaluate the detrimental effects of some antiepileptic drugs 243 (clover-surgicalgermany). bmi was calculated using the following equation: bmi = kg/m2. cdc chart was used as the standard for comparison to patients reading(5). bmi measurement of the patient’s monitors for one year as patients showed no problems with their medication (baseline, 6 months, and 12 months). exclusions were maternal smoking, gestational age outside the normal range, and residence outside the study area. refuse to participate in the study. other exclusions, including an inability to contact family and children who have travelled outside of the area or mental retardation, and cerebral palsy (12). data were presented as mean ±standard deviation. anova used to analyze the data. change with p<0.05 is considered to be statistically significant. excel 2010 software was used in the analysis. results the patient’s centile weight significantly elevated after 6 and 12 months of valproic acid (p<0.01) compared to the normal growth according to the cdc growth chart. while both carbamazepine and combined therapy showed no significant change in comparison with the normal growth according to the growth chart (p>0.05). the results of this work showed (table 1 and table 2) that carbamazepine monotherapy caused no significant effect on both bmi values after 6 and 12 months of treatment (p>0.05). valproic acid monotherapy significantly elevated bmi after 6 and 12 months of treatment (p>0.01). combined therapy showed no significant effect on bmi. no significant effect on waist circumference was noticed when compare valproic acid and combined therapy protocols to carbamazepine protocol. table 1. bmi of children after treatment with carbamazepine, valproic acid and combined therapy. parameter baseline mean ± sd six months mean ± sd twelvemonths mean ± sd p-value carbamazepine (patient no. =17) 15.9± 3.7 16.92±3.72 19.14±3.7 0.54 valproic acid (patient no. =17) 17±4 18.95±2.69 21.12± 1.84 0.01* combined therapy (patient no. =17) 15.78±1.56 16.88±2.78 19.59± 3.25 0.61 * represent significant change (p<0.05). table 2. waist circumference of children after treatment with carbamazepine, valproic acid and combined therapy. parameter baseline mean ± sd six months mean ± sd twelvemonths mean ± sd carbamazepine (patient no. =17) 47.1± 6.3 51.52±17 54.4±6.5 valproic acid (patient no. =17) 48.54±9 52.87±10 54.93± 5.13 combined therapy (patient no. =17) 47.05±4.35 52.48±9.75 54.65± 9.07 iraqi j pharm sci, vol.31(1) 2022 evaluate the detrimental effects of some antiepileptic drugs 244 figure 1. percentile weight of patients after 6 and 12 months of treatment. the patient’s centile height significantly elevated after 6 and 12 months of valproic acid (p<0.01) compared to the normal growth according to the growth chart. while both carbamazepine and combined therapy showed no significant change in comparison with the normal growth according to the growth chart (p>0.05). figure 2. percentile height of patients after 6 and 12 months of treatment discussion this study was conducted to explain the effect of antiepileptic on some growth profile parameters after 6 and 12 months of treatments. carbamazepine had no significant influence on growth, centile-weight, or centile-height. this might be due to the drug's mini effect on resting metabolic rate, as reported by herman et al., (11); however, this does not go with inaloo et al., 2020 (12). in contrast, valproic acid significantly increases the bmi of epileptic patients by increasing body fat due to the elevation of their serum triglycerides and this agrees with inaloo et al, 2020 (13). combined therapy showed no significant effect on bmi and this may be related to significant induction of many cytochromes metabolism and clearance which lead to these effects and this agree with saruwatari et al 2010 (14). valproic acid showed significant elevation in bmi, percentile height and weight after 6 months of therapy and this disagrees with results obtained by lee and his group who described that valproic acid significantly reduced pediatric patient’s longitudinal growth. lee et al., 2013 and might be due to the effect of valproic acid to inhibit proliferation of growing plate chondrocytes but not changing serum calcium. in patients on carbamazepine and combined therapy, the result of this work agree with lee results as he uses oxcarbazepine showed no significant effect on in stature growth of patients (15). the impacts of epilepsy on bone, drugs used to treat epilepsy increased the possibility of bone fracture by different mechanisms. the fracture rate in patients on anti-epileptic drugs is 2–6 times higher than the normal population. this increment in break chance in subjects with epilepsy is comparable to that seen with incessant steroid use as this drugs affects bone mass by affecting bone remodeling factors (16). in conclusion, children with epilepsy who use antiepileptic drugs need a restricted monitor policy for their growth, especially those on valproic acid. references 1. (us) nrc, (us) i of m. influences on children’s health. 2004; 2. pediatric endocrinology e-book mark a. sperling google books. 3. rosenbloom al. physiology of growth. vol. 65, annales nestle. 2008. p. 97–108. 4. emons j, chagin as, sävendahl l, karperien m, wit jm. mechanisms of growth plate maturation and epiphyseal fusion. vol. 75, hormone research in paediatrics. karger publishers; 2011. p. 383–91. 5. kuczmarski rj, ogden cl, guo ss, grummerstrawn lm, flegal km, mei z, et al. 2000 cdc growth charts for the united states: methods and development. department of health and human services, centers for disease control and prevention, national center for health statistics. 2002. 6. beghi e. the epidemiology of epilepsy. neuroepidemiology. 2020 mar;54(2):185–91. 7. epileptic state an overview | sciencedirect topics. 8. pressler rm, cilio mr, mizrahi em, solomon |, moshé l, nunes ml, et al. the ilae classification of seizures and the epilepsies: modification for seizures in the neonate. position paper by the ilae task force on neonatal seizures. epilepsia. 2021;00:1–14. 9. pohlmann-eden b, beghi e, camfield c, camfield p. the first seizure and its iraqi j pharm sci, vol.31(1) 2022 evaluate the detrimental effects of some antiepileptic drugs 245 management in adults and children. bmj br med j. 2006 feb;332(7537):339. 10. goldenberg mm. overview of drugs used for epilepsy and seizures: etiology, diagnosis, and treatment. pharm ther. 2010 jul;35(7):392. 11. brigo f, igwe sc. ethosuximide, sodium valproate or lamotrigine for absence seizures in children and adolescents. cochrane database syst rev. 2017 feb;2017(2). 12. de onis m, onyango a, borghi e, siyam a, pinol a, garza c, et al. enrolment and baseline characteristics in the who multicentre growth reference study. acta paediatr int j paediatr. 2006;95(suppl. 450):7–15. 13. inaloo s, saki f, paktinat m, katibeh p, nemati h, omrani gr. evaluation of the metabolic syndrome criteria and body composition in ambulatory children with epilepsy usingsodium valproate and carbamazepine in southern iran: a case-control study. iran j child neurol. 2020;14(3):47. 14. saruwatari j, ishitsu t, seo t, shimomasuda m, okada y, goto s, et al. the clinical impact of cytochrome p450 polymorphisms on antiepileptic drug therapy. epilepsy and seizure. 2010 apr;3(1):34–50. 15. hs l, sy w, dm s, cc w, sj c, hc f. the impact of the use of antiepileptic drugs on the growth of children. bmc pediatr. 2013 dec;13(1). 16. valsamis ha, arora sk, labban b, mcfarlane si. antiepileptic drugs and bone metabolism. nutr metab. 2006;3:1–11. this work is licensed under a creative commons attribution 4.0 international license. https://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(1) 2012 clinical pharmacy training program in iraq 1 implementation of a clinical pharmacy training program in iraqi teaching hospitals : review article jawad i. rasheed * ,1 and hassan m. abbas* * baghdad teaching hospital ,medical city, baghdad, iraq. abstract pharmaceutical care is a patient-centered, outcomes oriented practice that requires the pharmacist to work in concert with the patient and the patient’s other healthcare providers to promote health, to prevent disease, and to assess, monitor, initiate, and modify medication use to assure that drug therapy regimens are safe and effective. in addition, the presence of clinical pharmacists has led to a higher quality of patient education and provision of complete detailed information for patients. in developed countries pharm d has become the professional degree for practice of pharmacy. the graduates will be enrolled in a pharmacy residency program; admission to the residency programs is available to pharm d graduates of an accredited college of pharmacy. the residency is also designed to prepare the residents to become board certified specialists in their field. in many developing countries three new pharmaceutical education programs have currently been established to serve the pharmaceutical care development. firstly, a six-year curriculum leading to the doctor of pharmacy (pharm. d) degree as the sole professional degree. secondly, pharmacy residency and fellowship training program have been developed to provide intensive training in pharmaceutical care practice to the pharmacists. lastly, the continuing pharmaceutical education program (cpe) has been adopted to ensure the competency of all pharmacists to deliver the best knowledge and skills in pharmaceutical sciences in their specialties. in our opinion we lack for most of these programs, even the program of clinical pharmacy in ministry of health is not residency program and it is short and not subspecialized apart from being not recognized by academic institutes and references. in conclusion, pharmacy profession has to change towards the more responsibility on patient care. new training program has to be adopted by medical education institutes to provide clinical pharmacists as a profession and to prepare board certified clinical pharmacists as specialists to cope with the advances in all medical fields. key words: clinical pharmacy, board certification. لمراجعتل: مقالت تطبيك البرنامج التدريبي للصيدلت السريريت في المستشفياث التعليميت في العراق رشيد* إبراهيمجىاد ،1 و حسن محمد عباس* * ِسخشفً بغذاد اٌخؼٍٍُّ ، ِذَٕت اٌطب ، بغذاد ، اٌؼشاق الخالصة ٔحى اٌّشَط واٌّخىخهت ٔحى إٌخائح اٌخٍ حخطٍب صُذٌٍ سشَشٌ اٌشػاَت اٌصُذالُٔت هٍ اٌّّاسست اٌؼٍُّت اٌّشوضة َؼًّ بخٕاسك ِغ اٌّشَط وِمذٍِ اٌشػاَت اٌصحُت األخشَٓ ِٓ أخً حؼضَض اٌصحت ٌٍىلاَت ِٓ األِشاض واٌششوع باٌّؼاٌدت ِغ حمُُُ صُذٌٍ سشَشٌ َؤدٌ اًٌ إداسة دوائُت وسصذ وحؼذًَ اسخؼّاي اٌذواء ٌعّاْ ٔظُ اٌؼالج باٌؼمالُش بصىسة إِٓت وفؼاٌت. اْ وخىد أوثش أِأا ؤخائح أفعً ٌٍّشظً واسخخذاَ اِثً ٌٍذواء وباٌخاٌٍ خفط حىاٌُف األدوَت. وباإلظافت إًٌ رٌه، أدي وخىدهُ إًٌ اسحفاع د( هٍ -ُذٌٍ ) فاسَفٍ اٌبٍذاْ اٌّخمذِت أصبحج شهادة اٌطبُب اٌص .خىدة حثمُف اٌّشَط، وحىفُش ِؼٍىِاث حفصٍُُت واٍِت ٌٍّشظً اٌذسخت اٌّهُٕت اٌّطٍىبت ٌّّاسست اٌصُذٌت. حُث ٍَخحك اٌخشَدىْ فٍ اٌصُذٌت ببشٔاِح اإللاِت؛ إْ اٌمبىي فٍ بشاِح اإللاِت ِخاحت دة د ِٓ وٍُاث ِؼخّذة فٍ اٌصُذٌُت. وحهذف االلاِت أَعا إًٌ اػذاد صُادٌت ِمُُّٓ ٌُصبحى ِخخصصُٓ ِؤهٍُٓ بشها -ٌخشَدٍ فاسَ فٍ اٌبىسد فٍ هزا اٌّداي. فٍ اٌىثُش ِٓ اٌبٍذاْ إٌاُِت فٍ اٌىلج اٌشاهٓ هٕان ثالثت بشاِح حذسَبُت خذَذة اٌخٍ أٔشأث ٌخذِت اٌخُّٕت أوال، وظغ ِٕهح دساسٍ ٌّذة سج سٕىاث، ٌُؤهً إًٌ دوخىس صُذٌٍ وذسخت ِهُٕت وحُذة، ثأُا حُ حطىَش بشٔاِح اٌشػاَت اٌصُذالُٔت. ت واٌضِاٌت ٌخىفُش اٌخذسَب اٌّىثف فٍ ِّاسست اٌشػاَت اٌصُذالُٔت ٌٍصُادٌت. وأخُشا، حُ اػخّاد بشٔاِح اٌخؼٍُُ اٌصُذالٍٔ اٌّسخّش اإللاِ ِٓ وخهت ٔظشٔا، إٔٔا ٔفخمش ٌّؼظُ هزٖ ٌعّاْ اٌىفاءة ِٓ خُّغ اٌصُادٌت ٌخمذَُ أفعً اٌّؼاسف واٌّهاساث فٍ اٌؼٍىَ اٌصُذالُٔت. فٍ بشٔاِح اٌصُذٌت اٌسشَشَت فٍ وصاسة اٌصحت فهٍ ٌُسج بشاِح الاِت وّا أها لصُشة وٌُس ِخخصصت فعال ػٓ اٌبشاِح، حخً فٍ اٌخخاَ، ِهٕت اٌصُذٌت َدب أْ حخغُش ٔحى اٌّضَذ ِٓ اٌّسؤوٌُت ػًٍ سػاَت .وىٔها غُش ِؼخشف بها ِٓ لبً اٌّؤسساث األوادَُّت الػخّادها ِٓ لبً ِؤسساث اٌخؼٍُُ اٌطبٍ ٌخىفُش اٌصُادٌت اٌسشَشَُٓ وّهٕت وإػذاد اٌّشظً. وّا َدب حبٍٕ بشاِح خذَذ ٌٍخذسَب . اٌصُادٌت اٌّخخصصُٓ بشهادة اٌبىسد اٌسشَشٌ ٌٍخؼاًِ ِغ اٌخمذَ فٍ خُّغ ِداالث اٌطب : الصيدلت السريريت ، شهادة البىرد المفتاحيت الكلماث 1 corresponding author email : jawadnephroclinic@yahoo.com received : 9/10/2011 accepted : 15/2/2012 mailto:jawadnephroclinic@yahoo.com iraqi j pharm sci, vol.21(1) 2012 clinical pharmacy training program in iraq 2 introduction clinical pharmacists started their contributions to medicine in the 1960s and have come to be crucial members of medical teams as seen today in hospitals in developed countries. the presence of clinical pharmacists resulted in a safer medication administration, better patient outcomes, lower use and therefore lower costs of drugs. in addition, their presence has led to a higher quality of patient education and provision of complete detailed information for patients (1-4) .the purpose of this paper is to provide an overview to institutions considering the implementation of a clinical pharmacy program as part of their health care system. we provide a review of the current literatures supporting this program.involvement of clinical pharmacists in patient care in the inpatient hospital setting results in safer and more effective medication use (1) . these pharmacists are typically involved in assuring appropriate prescribing and administration of drugs, monitoring patient adherence to therapy, providing drug information consultation to providers, monitoring patient responses and laboratory values, and providing patient and provider education (5) . data suggest that medication errors are a significant contributor to errors in the emergency department, as well as in the inpatient setting (6) , a study found that 3.6 percent of patients were prescribed an inappropriate medication in the emergency department, and 5.6 percent of patients were prescribed one upon discharge (7) . development of pharmaceutical education far east experience now, as there is an upsurge in clinical pharmacy, many developing countries have expanded their pharmacy curriculum to a 5or 6-year program that issues a doctorate of pharmacy degree. however, it is still to be determined whether these countries are genuinely interested in a practice-based model or simply want their graduates to enroll in the us system (8, 9) . three new pharmaceutical education programs have currently been established in far east to serve the pharmaceutical care development. firstly, a six-year curriculum, leading to the doctor of pharmacy (pharm d) degree as the sole professional degree adopted in 1999 by many colleges of pharmacy. students required to complete 6 clinical clerkship rotations in the final year of the program to assure that they will develop the technical skills, professional judgements and competencies necessary for entry into the pharmacy profession. recently, the pharmacy council of india (pci) decided to introduce pharmd (post-bacclaureate degree) courses for the first time in the country (8, 10) . secondly, pharmacy residency and/or fellowship training programs have been developed since 2000 in order to provide intensive training in pharmaceutical care practice to the pharmacists. prince of songkla university and naresuan university has currently adopted this program under supervision of the college of pharmacotherapy. areas of specialty that are available for training are internal medicine, infectious diseases, cardiovascular diseases, oncology, critical care and pediatrics (11, 12) .lastly, the pharmacy councils have established the continuing pharmaceutical education program (cpe) since 2002 to ensure the competency of all pharmacists to deliver the best knowledge and skills in pharmaceutical sciences in their specialties. the program allows a pharmacist to update his/her knowledge and skills by attending the pre-approved academic meeting or reading the pre-approved article-related to pharmaceutical sciences. at least 10 cpe hours yearly and a total of 100 cpe hours per 5 years must be achieved by a pharmacist prior to continue active status of his/her pharmacist license. currently, al least 70 80% of pharmacists participated in this program (11, 13) . middle east experience interestingly institutions in the middleeast countries like saudi arabia, jordan, and kuwait started 5-6 year pharm d degree courses (14) . egypt too started their clinical pharmacy courses in the curricula of undergraduate, or specializing training courses in this field for postgraduates (15) .the saudi council for health specialties adopted a clinical pharmacy residency program; this council offered a residency program with accredited training for two years and eventually will grant the graduate a board certification of clinical pharmacy after passing the final exam (16) .residency program in clinical pharmacy in jordan was established in 1996 to prepare clinical pharmacists to work in medical teams to provide high quality medical services and to put the clinical pharmacists in the right way for improving their career, at the end they will be eligible for promotion for the job of specialist and consultant in clinical pharmacy. it is four year scientific training program after the b sc pharmacy, through which the pharmacist will be resident working with a medical team and will be in direct contact with the patients. the candidate will prepare daily reports and attend lectures and medical meetings under iraqi j pharm sci, vol.21(1) 2012 clinical pharmacy training program in iraq 3 supervision. he will pass through twelve departments, cardiology, hematology, oncology, neurology, communication skills, and clinical pharmacy departments (17) .in 2006 they improved the program to specialization rather than general clinical pharmacy; accordingly the resident will choose one or two specialties at the final year of residency program. the clinical pharmacist will be specialist after finishing this program. he will be eligible for farther promotion in the profession as senior specialist and consultant in clinical pharmacy depending on the scientific work and research (17) . the clinical pharmacy in developed countries a doctor of pharmacy degree program must have a multidisciplinary curriculum that produces pharmacists with sufficient mental acuity to differentiate their position from that of simply dispensers of drugs to that of providers of pharmaceutical care. the pharm d program in the united states is the epitome of the practice-based model (1821) . the american college of clinical pharmacy’s (accp) 10–15 year vision for the profession is that pharmacists will be health care providers who are accountable for optimal medication therapy in the prevention and treatment of disease. to achieve this vision, the profession must ensure that there will be an adequate supply of appropriately educated and skilled clinical pharmacists (22, 23) . by the beginning of 1990s, the american association of colleges of pharmacy and the american pharmacy professional organizations jointly took an unanimous decision to make pharm d as the minimum requirement for practice of pharmacy in their country. this has positively influenced the pharmacy educational institutions and authorities through out the world to take proper precautions at their countries. all the western / european countries rose to the occasion and took timely decisions to introduce pharm d (, 24, 25) . the cleveland clinic foundation, as a model for implementing the clinical pharmacy program it is an integrated health care system, a one thousand bed tertiary and primary care hospital; it manages over 2 million patient visits annually. the department of pharmacy provides services 24 hours a day. the 200 plus departmental personnel include pharmaceutical care specialists, staff pharmacists, managers, technicians, and other supportive staff. admission to the residency program at cleveland clinic is available to pharm d graduates of an accredited college of pharmacy. the residency is a specialized onetwo year training program which enables the successful resident to develop an expert level of competence in clinical pharmacy. the residency is also designed to prepare the resident to become a board certified specialist (26) . board certified pharmaceutical care specialists the primary purpose of specialization in any health care profession is to improve the quality of care individual patients receive, to promote positive treatment outcomes, and ultimately, to improve the patient’s quality of life. specialties evolve in response to the development of new knowledge or technology that can affect patient care. board certification for health care practitioners is well established as an essential element of quality assurance and professional privileging. future clinical pharmacy practitioners should be board-certified specialists; this is the vision of accp in 20–30 years. accordingly most clinical pharmacy practitioners will be board-certified specialists by adopting a training program (18) . for more than 30 years, the board of pharmacy specialties (bps) has provided specialty-level certification programs for pharmacists – both nationally and internationally. board of pharmaceutical specialties will grant the clinical pharmacist practitioners the specialist degree when they fulfill the requirements: pharm d degree, residency program, fellowship with thesis, and passing the specialty exam (27) . the program of clinical pharmacy in iraq the newly graduate pharmacists can be enrolled in this program for one year under training at teaching hospitals. they will be legally obliged to work as clinical pharmacists for five years according to the need. this program was established at ministry of health. it is a practical initial solution of the problem of clinical pharmacy in iraq. till now it is a successful program. but from the other side, the program is not recognized by ministry of higher education, and it is neither a residency program nor specialized. actually it will not cover the advances in medicine and it is not going parallel with medical upgrading.board certification program is the major step in upgrading of medical profession to improve patients care in iraqi hospitals. adoption of arab board program followed by iraqi board as part of improving patient care and promoting research were very successful expressing the iraqi j pharm sci, vol.21(1) 2012 clinical pharmacy training program in iraq 4 cooperation between ministry of health and ministry of higher education to improve health system in iraq. at the same time, the process of improving pharmaceutical care was limited; there was simple role of educational institutes of ministry of higher education. the role was restricted to ministry of health in the form of one-year program of clinical pharmacy. as a conclusion, the practice of clinical pharmacy, as well as clinical pharmacy education, varies significantly throughout the world. to implement or at least to start such approach; there must be well-planned execution of good clinical pharmacy practice in developing iraqi health system. upgrading of primary pharmacy education will support local pharmacy practice and permits for international recognition of iraqi graduates. this review may recommend application of new postgraduate training programs like board training or fellowships to support the practice of clinical pharmacy as initial step. establishment of (the scientific council of clinical pharmacy) at ministry of higher education to create a recognized professional training program in clinical pharmacy will be the complimentary step in upgrading health system to provide an optimum patient care. upgrading of clinical pharmacy profession through a board certification program will be the logic step in this regard. acknowledgement we thank professor alaa abdulhussain , the dean of college of pharmacy. professor ibrahim adham, for providing the necessary facilities. references 1. horn e and jacobi j. the critical care clinical pharmacist: evolution of an essential team member. critical care medicine 2006;34: s46-s51. 2. kucukarslan sn, peters m, mlynarek m and nafziger da. pharmacists on rounding teams reduce preventable adverse drug events in hospital general medicine units. arch. intern. med. 2003; 163: 2014-2018. 3. buurma h, de smet pa, leufkens hg and egberts ac. evaluation of the clinical value of pharmacists’ modifications of prescription errors. br. j. clin.pharmacol. 2004; 58: 503-511. 4. dooley mj, allen km, doecke cj, galbraith kj, taylor gr, bright j and carey dl. a prospective multicentre study of pharmacist initiated changes to drug therapy and patient management in acute care government funded hospitals. br.j.clin.pharmacol. 2004; 57: 513-521. 5. society of critical care medicine and the american college of clinical pharmacy. position paper on critical care pharmacy services. pharmacotherapy 2000; 20: 1400-1406. 6. hafner, jw, belknap sm, squillante, md, et al. adverse drug events in emergency department patients. ann emerg med 2002; 39: 258-267. 7. chin mh, wang lc, jin l, et al. appropriateness of medication selection for older persons in an urban academic emergency department. acad emerg med 1999; 6: 1232-1242. 8. naushad m. khan ghilzai, arjun p. dutta. india to introduce five-year pharm d program [letter]. american journal of pharmaceutical education 2007; 71(2): article 38. 9. melody ryan, hong shao, li yang, xiao-yan nie, suo-di zhai, lu-wen shi, and william c. lubawy. clinical pharmacy education in china. american journal of pharmaceutical education 2008; 72 (6): article 129. 10. lincy s. lal and padma g. rao. clinical pharmacy education in india. am j health-syst pharm. 2005; 62:1510-1. 11. paveena sonthisombat, sutthiporn pattharachayakul, chalermsri pummangura. development of pharmaceutical education emphasizing on pharmaceutical care: thailand experience .the 4th asian conference on clinical pharmacy 2004. home page. available from www. 4.samford. edu/ schools/pharmacy/ijpe/104/accp04.pdf. accessed may 20, 2011. 12. khurrum ghani, wasif gillani, maria ghani. pharmacy teaching and practices problems in developing countries: review. international journal of pharmacy teaching & practices 2010; 1 (1): 11-17. 13. singapore pharmacy board. a guide to compulsory cpe for pharmacists 2006. home page. available from www.spb.gov.sg.accessed may 20, 2011. 14. mayyada al-wazaify, lloyd matowe, abla albsoul-younes, and ola a. alomran. pharmacy education in jordan, saudi arabia, and kuwait. american journal of pharmaceutical education 2006; 70 (1) article 18. 15. wafaa m.ei. clinical pharmacy in egypt. ijptp, 2010; 1 (2): 18‐19. 16. saudi council for health specialties. pharmacy practice residency program, http://www.spb.gov.sg/ iraqi j pharm sci, vol.21(1) 2012 clinical pharmacy training program in iraq 5 first version 2002. home page. available from www.scohs.org. accessed may 20, 2011. 17. jordanian royal medical services. pharmacy profession evolution at the royal medical services. home page. available from www.jrms.gov.jo. accessed may 1, 2011. 18. american college of clinical pharmacy. future clinical pharmacy practitioners should be board-certified specialists. pharmacotherapy 2006; 26(12):1816– 1825. 19. american college of clinical pharmacy. accp defines clinical pharmacy. accp rep 2005;24(8):1–2. 20. shazia jamshed, zaheer ud din babar, imran masood.the pharmd degree in developing countries. american journal of pharmaceutical education 2007; 71 (6) article 125. 21. accreditation council for pharmacy education. standards and guidelines for the professional program in pharmacy leading to the doctor of pharmacy degree. 2006. available from http://www.acpeaccredit.org/pdf/acpe_revised_pharmd _standards_adopted_jan152006.pdf. accessed may 20,2011 22. american college of clinical pharmacy. the definition of clinical pharmacy. pharmacotherapy 2008;28(6):816–817. 23. american college of clinical pharmacy. interprofessional education: principles and application. a framework for clinical pharmacy. pharmacotherapy 2009; 29(3):145e–164e. 24. american college of clinical pharmacy. a vision of pharmacy’s future roles, responsibilities, and manpower needs in the united states. pharmacotherapy 2000;20:991–1020. 25. american college of clinical pharmacy. the strategic plan of the american college of clinical pharmacy. accp rep 2002;21(10):s1–7. 26. cleveland clinic foundation home page. available from www.clevelandclinic.org. accessed may 20, 2011. 27. board of pharmaceutical specialties. candidate’s guide 2011. home page. available from www.bpsweb.org. accessed may 20, 2011. http://www.jrms.gov.jo/linkclick.aspx?link=249&tabid=159 http://www.jrms.gov.jo/linkclick.aspx?link=249&tabid=159 http://www.jrms.gov.jo/ http://www.acpe-accredit.org/pdf/acpe_revised_pharmd_standards_adopted_jan152006.pdf.%20accessed%20may%2020,2011 http://www.acpe-accredit.org/pdf/acpe_revised_pharmd_standards_adopted_jan152006.pdf.%20accessed%20may%2020,2011 http://www.acpe-accredit.org/pdf/acpe_revised_pharmd_standards_adopted_jan152006.pdf.%20accessed%20may%2020,2011 http://www.acpe-accredit.org/pdf/acpe_revised_pharmd_standards_adopted_jan152006.pdf.%20accessed%20may%2020,2011 iraqi j pharm sci, vol.28(1) 2019 chads2 and cha2ds2-vasc scores in iraqi af patients doi: https://doi.org/10.31351/vol28iss1pp101-105 101 status of chads2 and cha2ds2-vasc scores in predicting risk of stroke and its prevention in iraqi patients with atrial fibrillation mohanad y. al-radeef *,1 *department of clinical pharmacy, college of pharmacy, university of tikrit, tikrit, iraq. abstract atrial fibrillation is associates with elevated risk of stroke. the simplest stroke risk assessment schemes are chads2 and cha2ds2-vasc score. aspirin and oral anticoagulants are recommended for stroke prevention in such patients. the aim of this study was to assess status of chads2 and cha2ds2-vasc scores in iraqi atrial fibrillation patients and to report current status of stroke prevention in these patients with either warfarin or aspirin in relation to these scores. this prospective cross-sectional study was carried out at tikrit, samarra, sharqat, baquba, and alnumaan hospitals from july 2017 to october 2017. chads2 and cha2ds2-vasc scores were manually calculated. one hundred patients were participated, 48 were men and 52 were women. their mean age was 62.56 ± 14.36 years. permanent type of atrial fibrillation, palpitation, and hypertension were the most diagnosed type, symptom and comorbidity recorded in this study respectively. average scores of chads2 and cha2ds2-vasc were 2.34 ± 1.39 and 4.1 ± 2.05, respectively. these scores were not calculated for these patients in hospital setting. aspirin and warfarin were prescribed regardless to these scores. the result of this study indicated that chads2 and cha2ds2-vasc scores were often neglected in hospitals; and aspirin is still widely used as a strategy to minimize the risk of stroke. keywords: atrial fibrillation, chads2, cha2ds2-vasc, aspirin, warfarin. في منها الدماغية والوقاية السكتة خطر توقع في vasc-2ds2cha و 2chads درجات حالة األذيني بالرجفان المصابين العراقيين المرضى 1*،مهند ياسر الرديف الصيدلة السريرية ، كلية الصيدلة ، جامعة تكريت ، تكريت ، العراق .فرع * الخالصة و 2chadsأبسط مخططات تقييم خطر السكتة الدماغية هي درجة يرتبط الرجفان األذيني بزيادة خطر اإلصابة بالسكتة الدماغية. vasc-2ds2cha .ةالفموي التخثر مضاداتللوقاية من السكتة الدماغية في مرضى الرجفان االذيني عادة ما ينصح باستعمال األسبرين و. األذيني بالرجفان المصابين العراقيين المرضى في vasc -2ds2chaو 2chads درجات حالة تقييم كان الهدف من هذه الدراسة هو .الدرجات هذه الى نسبة الوارفارين أو األسبرين باستخدام المرضى هؤالء لدىالدماغية السكتة من الوقاية مستوى وتحديد االول تشرين إلى 2017 تموز من والنعمان وبعقوبة وشرقاط وسامراء تكريت مستشفيات فيالمقطعية المستعرضة الدراسة هذه أجريت .يدويا vasc -2ds2chaو 2chads درجات احتساب تم 2017. كان .سنة5662  3614 كان همأعمار متوسط .امرأة 52 و رجل 48 منهم ،ى الرجفان االذينيمرضمن مئةشارك في هذه الدراسة الدراسة هذه فياالكثر توثيقا المشترك لواالعتال األعراض تشخيًصا، األكثر النوعهم الدم ضغط وارتفاع خفقانال األذيني، الرجفان من الدائم النوع العمل يتم لم .التوالي على  34 ,2 1, 39و vasc -2ds2cha 05 ,2  1 ,4 و 2chads الدرجات متوسط .التوالي على .الدرجات هذه عن النظر بغض والوارفارين األسبرين وصف تمو المذكورة المستشفيات في المرضى لهؤالء الدرجات بهذه نطاق على يستخدم األسبرين يزال الو المستشفيات؛ في تهمل ما غالبا vasc -2ds2chaو 2chads درجاتاظهرت الدراسة ان الدماغية. بالسكتة اإلصابة رخط لتقليل كاستراتيجية واسع , االسبرين, الوارفارين.vasc-2ds2, cha2chadsالكلمات االفتتاحية : الرجفان االذيني, introduction atrial fibrillation (af) is a type of supraventricular arrhythmia that is characterized by uncoordinated activation in trial electricity with irregular and often fast ventricular response triggering hemodynamic compromise (1). roughly, 1 in 100 of general population is thought to develop af, while in elderly cohorts, the prevalence usually exceeds 1 in 10 (2). atrial fibrillation is related with expanded rates of death, thromboembolic events, heart failure and hospitalizations, reduced quality of life, reduced exercise capacity, and left ventricular dysfunction (3). it is unfortunately associated with a five-fold elevated stroke risk, besides it is the most common type of arrhythmia (2,4,5). 1corresponding author: e-mail: mohanadyasir@tu.edu.iq received: 14/10/2018 accepted:9 /12/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp101-105 mailto:mohanadyasir@tu.edu.iq iraqi j pharm sci, vol.28(1) 2019 chads2 and cha2ds2-vasc scores in iraqi af patients 102 identification of numerous stroke risk factors has resulted in development of numerous stroke risk schemes. artificially most of these schemes have classified risk of stroke into high, moderate, and low risk classes (6). chads2 score is the most straightforward risk assessment tool. chads2 [cardiac failure, hypertension, age (>75 years), diabetes, stroke (doubled)] risk index, which is a point system, was developed by the af investigators and stroke prevention in atrial fibrillation (spaf) investigators criteria (7). a newly adjusted scheme that was recommended by european society of cardiology (esc) is cha2ds2-vasc [cardiac failure, hypertension, age ≥ 75 (doubled), diabetes, stroke (doubled), vascular disease, age 65–74, and sex category (female)], that is also a point system (8). according to the guidelines of the american college of cardiology (acc) and the heart rhythm society (hrs) in collaboration with the american heart association (aha) issued in 2014 patients are classified into low (score = 0), intermediate (score =1), and high-risk (score ≥2) groups for a stroke based on their chads2 scores. the same categorization also applies to cha2ds2-vasc (9). for the most of af patients, the backbones of treatment to decrease stroke risk are antithrombotic medicines (antiplatelet and anticoagulants). the effect of these medicines in decreasing stroke risk is well established. aspirin and oral anticoagulants (oacs) are usually suggested for preventing af patients from developing stroke (10). the current study was designed to assess the status of chads2 and cha2ds2-vasc scores in predicting risk of stroke in iraqi patients with af, as well as, to report the status of stroke prevention in these patients with aspirin or warfarin in relation to these scores. subjects, materials, and methods this prospective cross-sectional study was carried out at tikrit, samarra, sharqat, baquba, and al-numaan general hospitals from july 2017 to october 2017. the study protocol was approved by the local ethics committee in the college of medicine, tikrit university, iraq, with verbal informed consent from patients. inclusion criteria involved any patient who introduced to intensive care unit with documented symptoms of af (chest pain, palpitation, etc.) at admission and any of the following stroke risk factors: an age of ≥75 years, a previous stroke or transient ischemic attack, diabetes mellitus (dm), recognized peripheral arterial disease (pad), hypertension, or a left ventricle ejection fraction of 35 – 45% or less. exclusion criteria involve inadequate data, renal failure, and recent symptoms of bleeding. information on demographic characteristics was obtained through patient interview. types and family history of af and the presence of comorbidities including coronary artery disease (cad), hypertension, dm, and heart failure were reported with the aid of patient's specialist. data were collected via face to face structured interview. each patient was given explanation about the purpose of study in details and confidentiality was ensured. for each patient enrolled, chads2 score was calculated by assigning 1 point for cardiac failure, age >75 years, dm, and hypertension and 2 points for a prior stroke, while cha2ds2-vasc score was calculated by assigning 1 point for female gender, cardiac failure, age of 65 – 74 years, dm, hypertension, vascular disease (complex aortic plaque, myocardial infarction, and pad; including amputation because of pad, prior revascularization, or angiographic evidence of pad) and 2 points for a previous stroke and age 75 years. the objective of stroke prevention determination focused on the current antiplatelets and oral anticoagulant therapy that was offered by the specialist to these patients at the time of hospital discharge. therefore, results regarding this issue reported how many patients were receiving aspirin and how many patients were receiving warfarin at time of discharged. statistical analysis was made using the microsoft excel 2010, and continuous variable are expressed as mean ± standard deviation. results one hundred patients were participated in this study (20 participants from each hospital), 48 were men and 52 were women. their mean age was 62.56 ± 14.36 years. current smoking was reported in 42 patients, while the remaining patients were nonsmokers. permanent type of af (abnormal heart rhythm can't be restored) was reported in 32 patients followed by paroxysmal type (symptoms reported occasionally) that was reported in 27 patients, while persistent type (symptoms don't go back to normal on their own and treatment with electrical shock or medications are required) was occurred in 26 patients. only 15 patients were diagnosed to have long-standing persistent type (af is continuous and lasts longer than 12 months) of af. figure 1 summarizes these data. figure (1) types of af recorded in this study. the most common symptom that was associated with af and reported by all patients was palpitations. other symptoms reported were chest iraqi j pharm sci, vol.28(1) 2019 chads2 and cha2ds2-vasc scores in iraqi af patients 103 pain, weakness, lightheadedness, and shortness of breath. many of these symptoms concomitantly occurred in the same patient. figure 2 summarizes all these data. figure (2) symptoms of af reported in this study. many of these symptoms concomitantly occurred in the same patient patient comorbidities are demonstrated in figure 3. hypertension was the most prevalent chronic illness (89% of patients) followed by ischemic heart disease, heart failure, pad, dm and prior stroke attack. many of these comorbidities concomitantly occurred in the same patient figure (3) comorbidities reported in this study. many of these comorbidities concomitantly occurred in the same patient. figure 4 shows chads2 and cha2ds2vasc scores level of utilization in patients enrolled in this study. unfortunately, these scores were often neglected for these patients and clinicians did not use/rely on these scores. figure (4) utilization of chads2 and cha2ds2vasc scores in this study. average scores of chads2 and cha2ds2vasc in this study were 2.34 ± 1.39 and 4.1 ± 2.05, respectively. chads2 score categorized 5% of patients as low risk (score=0), 26% as intermediate risk (score=1), and 69% as high risk (score ≥2). on the other hand, cha2ds2-vasc score categorized 0% of patients as low risk (score=0), 10% as intermediate risk (score=1) and 90% as high risk (score ≥2). distribution of these scores in patients is demonstrated in figure 5. figure (5) distribution of chads2 and cha2ds2vasc scores in this study. figure 6 represents the antithrombotic medications that had being given for these patients at hospital discharge in relation to chads2 score. at score zero, 3 and 2 patients were received aspirin and warfarin respectively. at score one, 24 and 2 patients were received aspirin and warfarin respectively, while at score two and greater, 56 and 12 patients were received aspirin and warfarin respectively. figure (6) antithrombotic medications that had being given for patients at hospital discharge in relation to chads2 score. figure 7 represents the antithrombotic medications that had being given for these patients at hospital discharge in relation to cha2ds2-vasc score. no patients were present at score zero. at score one, 6 and 4 patients were received aspirin and warfarin respectively. at score two and greater, 78 and 12 patients were received aspirin and warfarin respectively. iraqi j pharm sci, vol.28(1) 2019 chads2 and cha2ds2-vasc scores in iraqi af patients 104 figure (7) antithrombotic medication that had being given for patients at hospital discharge in relation to cha2ds2-vasc score. discussion atrial fibrillation is a common type of arrhythmia that can elevate the risk of stroke by fivefold. oacs and aspirin can diminish stroke risk by 64% and 22%, respectively (11). despite the fact that oacs treatment is more effective than aspirin at preventing ischemic stroke, its use is offset by an elevated risk of hemorrhage. hence, the initiation of oacs treatment needs recognizing patients in whom ischemic stroke risk without anticoagulant agents is adequately high to outweigh an elevated risk of major extracranial and intracranial hemorrhage related to oacs treatment (12). as shown in figure 1, permanent type of af was the most common as presented in 32% of patients followed by paroxysmal (occasional) type that was reported in 27% of patients, persistent type was occurred in 26% of patients, and only 15% patients were diagnosed to have long-standing persistent type of af. in study of coppens et al., 52% of patients had permanent af and 28% had paroxysmal af, while 19% had persistent af (12). as shown in figure 2, the most common symptom that was associated with af and reported by all patients was palpitations. other symptoms were chest pain, weakness, lightheadedness, and shortness of breath respectively. in comparison, the chief complaints that were reported by barrett et al. involved palpitations in 41% of patients, chest pain 16%, dyspnea 12%, tachycardia 13%, and syncope in 3% of patients (13). as shown in figure 3, hypertension was the most prevalent chronic illness reported in this study as 89% of patients had it followed by ischemic heart disease, heart failure, pad, dm and prior stroke attack respectively. in a study of mason et al., hypertension was recorded in 56% of the patients followed by cardiovascular diseases (pad and cad), dm and heart failure respectively (14). all these differences may be attributed mainly to different ethnic groups. as shown in figure 4, application of chads2 and cha2ds2-vasc scores, unfortunately, was often neglected for iraqi patients with af. although the chads2 score has been available for many years and is simple to calculate, it does not include several important risk factors and suffers from important limitations (9). for example, a substantial portion of patients are classified as moderate risk, and it is uncertain whether antiplatelet or anticoagulant therapy should be recommended for those patients. furthermore, the scale ignores some potential risk factors for thromboembolism (15). cha2ds2-vasc score overcame many of the limitations of the chads2 score including its ability to reliably identify “truly low risk” patients, who could be managed with no antithrombotic therapy. therefore, the cha2ds2vasc score is now recommended in recent guidelines instead of the chads2 score for stroke assessment in patients with af (9). as shown in figure 6 and 7, aspirin was the most prescribing antithrombotic agent for iraqi af patients regardless to chads2 and cha2ds2vasc scores. the main reason for this strategy is to minimize the risk of bleeding that is related to warfarin treatment as the physicians realize that international normalized ratio (inr) cannot be easily titrated. generally, aspirin is recommended for chads2 score = 0, and oral anticoagulant is highly recommended for a score of ≥ 2, while either aspirin or oral anticoagulant is considered suitable for patients with intermediate-risk at a score =1 (16). on the other hand, the esc guidelines recommend oacs for a cha2ds2-vasc score ≥ 2 and aspirin or oacs for a score = 1, with oacs “preferred” and for cha2ds2-vasc score = 0 no therapy or aspirin (no therapy preferred) (14). completely all guidelines recommend that any patient with chads2 score of ≥ 2 should receive an oac treatment as the ischemic stroke risk outweighs the elevated risk of bleeding that induced by oac treatment. management recommendations were unchanged regardless of cha2ds2-vasc score (12). although oacs have numerous therapeutic benefits, they have certain risks: major bleeding risk that associates with warfarin treatment is expected to be at (1% – 3%) per year, and may be as high as 7% in elderly af patients per year (17). a meta-analysis trial reported that oacs therapy had a relative risk (rr) reduction of 62% (95% ci, 0.48 – 0.72) as compared with placebo or usual care. on the other hand, some trials have compared warfarin with aspirin, and a metaanalysis reports that warfarin has the ability to reduce the risk of stroke (rr reduction, 36%; 95% ci, 0.14 – 0.52). however, warfarin benefit is usually associated with an elevated rate of hemorrhage risk. comparing with aspirin, a metaanalysis reported that warfarin had a risk for intracranial hemorrhage (rr, 2.1; 95% ci, 1.0-4.6) and a double risk for a major extracranial hemorrhage (rr, 2.0; 95% ci, 1.2 – 3.4; absolute risk rise, 0.2% per year) (18). iraqi j pharm sci, vol.28(1) 2019 chads2 and cha2ds2-vasc scores in iraqi af patients 105 this study had several limitations. first, this study was limited by its small sample size from few hospitals. second, it examined patients at only one point in time and patient’s risk scores may change over time. third, it examined the hypothetical effect of these scores and cannot make determination as to which scoring system is best. conclusion chads2 and cha2ds2-vasc scores were often neglected in assessing iraqi af patients in hospitals; in addition to that, aspirin is still widely used as a strategy to minimize stroke risk in patients with af regardless to these scores. therefore, it will be important to determine by a randomized trial whether the use of antiplatelets and/or oacs in relation to these scores will result in improved outcomes, without increased morbidity/mortality due to bleeding complications. acknowledgements deep thanks to pharmacist omer muhammad hamad, ali muhanad ali, wathiq adil mahmood, ahmed muhammad hussein, and husham ataa faris for their valuable efforts and assistance in accomplishing this study. references 1. gutierrez c and blanchard dg. diagnosis and treatment of atrial fibrillation. am fam physician. 2016; 94(6): 442-52. 2. barra s and fynn s. untreated atrial fibrillation in the united kingdom: understanding the barriers and treatment options. j saudi heart assoc. 2015; 27(1): 31-43. 3. atouni m, gunda s, and lakkireddy d. left atrial appendage closure is preferred to chronic warfarin therapy: the pro prospective. in: controversies in electrophysiology. daoud e 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and new oral anticoagulants. ch. 115. in: cardiac electrophysiology: from cell to beside. zipes dp, jalife j, and stevenson wg (eds.). 7th edition. elsevier. 2018; p:1093. https://www.ncbi.nlm.nih.gov/pubmed/?term=friberg%20l%5bauthor%5d&cauthor=true&cauthor_uid=22246443 https://www.ncbi.nlm.nih.gov/pubmed/?term=rosenqvist%20m%5bauthor%5d&cauthor=true&cauthor_uid=22246443 https://www.ncbi.nlm.nih.gov/pubmed/?term=lip%20gy%5bauthor%5d&cauthor=true&cauthor_uid=22246443 https://www.ncbi.nlm.nih.gov/pubmed/22246443 https://www.ncbi.nlm.nih.gov/pubmed/?term=coppens%20m%5bauthor%5d&cauthor=true&cauthor_uid=23018151 https://www.ncbi.nlm.nih.gov/pubmed/?term=eikelboom%20jw%5bauthor%5d&cauthor=true&cauthor_uid=23018151 https://www.ncbi.nlm.nih.gov/pubmed/?term=hart%20rg%5bauthor%5d&cauthor=true&cauthor_uid=23018151 iraqi j pharm sci, vol.28(1) 2019 synthesis of 4-thiazoldinone derivatives of mefenamic acid doi: https://doi.org/10.31351/vol28iss1pp138-146 138 synthesis, characterization and acute anti-inflammatory evaluation of new mefenamic acid derivatives having 4-thiazolidinone nucleus mustafa h. ali alsafi *,1 and muthanna s. farhan ** * ministry of health and environment , baghdad , iraq. **department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract mefenamic acid (ma) is one of the non-steroidal anti-inflammatory drugs, it is widely used probably due to having both anti-inflammatory and analgesic activity, the main side effects of mefenamic acid include gastrointestinal tract (git) disturbance mainly diarrhea, peptic ulceration, and gastric bleeding. the analgesic effects of nsaids are probably linked to cox-2 inhibition, while cox-1 inhibition is the major cause of this classic adverse effects. introduction of thiazolidinone may lead to the increase in the bulkiness leads to the preferential inhibition of cox-2 rather than cox-1 enzyme. the study aimed to synthesize derivatives of mefenamic acid with more potency and to decrease the drug's potential side effects, new series of 4-thiazolidinone derivatives of mefenamic acid were synthesized iva-g. the synthetic procedures for target compounds and their intermediates are designed to be as follows: acylation of secondary amine of mefenamic acid by chloroacetylchloride to produce compound (i), then reaction between compound (i) and hydrazine hydrate to form hydrazine derivative of mefenamic acid (compound ii). after that, schiff base formation by addition of seven benzaldehyde derivatives and finally, cyclization in presence of thioglycolic acid to form 4-thiazolidinone heterocyclic ring. the characterization of the titled compounds has been established on the basis of their spectral ftir, 1hnmr data, and by measurements of their physical properties. in vivo acute anti-inflammatory effect of the synthesized compounds was evaluated in rats using egg-white induced edema model of inflammation. the tested compounds and the reference drug produced significant reduction of paw edema with respect to the effect of dimethyl sulfoxide 10%v/v (control group). compound ive showed more potent effect than mefenamic acid at 240-300 min, while at time 300 min, compounds iva and ivd exhibit more potent anti-inflammatory effect than mefenamic acid (50mg/kg, i.p.) as they reduced paw edema significantly more than mefenamic acid at mentioned intervals (p<0.05) . on the other hand compound ivc exhibited lower anti-inflammatory effect. keywords: mefenamic acid , 4-thiazolidinone, anti-inflammatory -٤لمشتقات جديدة لحمض الميفينامك مع نواة الحاد تصنيع وتشخيص والتقييم المضاد لاللتهاب ثيازوليدينون **و مثنى سعدي فرحان 1*،مصطفى حسين علي الصافي ، بغداد ، العراق . الصحة والبيئةوزارة * الصيدلة، جامعة بغداد، بغداد، العراق. فرع الكيمياء الصيدالنية، كلية** الخالصة من ة.الغير ستيرويدي وهو احد افراد االدوية المضادة لاللتهاب لاللتهابحمض الميفيناميك واسع االستخدام لخواصه المسكنة والمضادة ان االسهال والقرحة الهضمية ونزيف المعدة. حيث ان اهم االثار الجانبية الستخدام حمض الميفيناميك هي اضظرابات الجهاز الهضمي وبشكل رئيسي تثبيط انزيم عن بينما االثار الجانبية هي نتاج 2-ناتج عن كبح انزيم كوكس التاثير المضاد لاللتهاب لالدوية المضادة لاللتهاب الغير ستيرويدية . الهدف من هذه 1-على كوكس 2-ترجيح تثبيط انزيم كوكس . ان اضافة مجموعة الثيازوليدينون ربما يؤدي الى زيادة الحجم و بالتالي1-كوكس ثيازوليدينون لحمض -٤الدراسة هو تصنيع مشتقات لحمض الميفيناميك بفعالية اعلى و اعراض جانبية اقل. حيث تم تصنيع مشتقات ذات نواة الميفيناميك، ان طرق تصنيع المركبات النهائية والمركبات الوسطية تم تصميمها كالتالي: مع (i)، ثم مفاعلة المركب (i)اضافة مجموعة اسيل الى االمين الثنائية لحمض الميفيناميك بمفاعلته مع مركب الكلورواستيل كلورايد النتاج المركب لمركب ا مركب هيدرازين هيدريت لتكوين المركب الوسطي الثاني،بعد ذلك تم تصنيع قواعد شف باضافة سبعة مشتقات للبنزلديهايد و اخيرا تكوين ثيازوليدينون. -٤الحلقي بوجود مركب حمض الثيوجليكولك لتصنيع حلقة تم تشخيص المركبات المحضرة باستعمال مطياف االشعة تحت الحمراء وتحليل االرنين النووي المغناطيسي للبروتون و كذلك قياس الخصائص الفيزيائية للمركبات. ات المصنعة في الجسم الحي باستعمال نموذج االلتهاب المحدث بواسطة حقن بياض البيض تحت الجلد في تم تقييم الفعالية المضادة لاللتهاب للمركب اثير تالجرذان. جميع المركبات المحضرة اضافة الى حمض الميفيناميك ابدوا فعالية ملحوظة في تقليل التورم المحدث في كف الجرذ بالمقارنة مع ( %10الكبريت المذيب المستعمل )ثنائي مثيل اوكسيد 300ابدت فعالية اقوى في الدقيقة iv a,dو كذلك المركبات 300 والدقيقة 240فعالية اقوى من حمض الميفيناميك في الدقيقة iveابدى المركب فعالية اقل كمضاد لاللتهاب مقارنة بحمض ivcملغ/كغ ( بالحقن تحت الغشاء البريتوني للجرذ ، بينما ابدى مركب 50مقارنة بحمض الميفيناميك ) المفيناميك. ثيازوليدينون، مضادات االلتهاب.-٤الكلمات المفتاحية: حمض الميفيناميك، 1corresponding author email: alsafi_mh@yahoo.com received: 30 / 12 /2018 accepted: 26 / 3/2019 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol28iss1pp140-148 https://doi.org/10.31351/vol28iss1pp140-148 mailto:alsafi_mh@yahoo.com iraqi j pharm sci, vol.28(1) 2019 synthesis of 4-thiazoldinone derivatives of mefenamic acid 139 introduction nsaids have become important as an analgesic, antipyretic, and anti-inflammatory medications throughout the world. the main mechanism of nsaids action is inhibition both isoforms of the enzyme cyclooxygenase cox-1 and cox-2 which catalyzes the synthesis of pgh2 from arachidonic acid (1,2). since the gastrointestinal (gi) toxicity, as well as renal toxicity, were identified as the main adverse effect of the chronic use of the known (nsaids), which were mediated by cox-1 enzyme inhibition, attempt was made to develop cox-2 selective inhibitors (coxibs), which possess the therapeutic effect devoid of gastrointestinal (gi) and renal toxicity. (1-6) 3-dimensional structures analysis for the two isomers of cox enzyme demonstrated differences in the hydrophobic channel structure, as well as a rather larger orifice and the presence of an additional pocket lying away from the catalytic site, this makes the selective cox-2 inhibitors to require longer time to fit the cox-2 active site, but when they bind the enzyme, their bond may become permanent. they also may become competitive cox-1 inhibitors when given at high doses (7). well-known nsaids modification into more selective cox-2 inhibitors is considered an interesting maneuver. yet, no general line is followed as a procedure for this purpose (8, 9). thiazolidinones are thiazolidine derivatives possessing at position 1 a sulfur atom, at position 3 a nitrogen atom and at position 2, 4, or 5 a carbonyl group . derivatives of this compound constitute members of the widely investigated agents, and their presence was first documented in penicillin. similarly, 1,3-thiazolidin-4-ones are heterocyclic nucleus that contain a sulfur atom and nitrogen at position 1 and 3, respectively and a carbonyl group at position 4, it has been studied extensively in recent years (10).the 4thiazolidinone skeleton is very flexible and has included in a variety of clinically important medications such as some antitubercular, antimicrobial, anti-inflammatory and antiviral especially anti-hiv agents (11).zarghi et al. reported a novel series of 2,3-diaryl-1,3thiazolidine-4-ones showing cox-2 inhibition properties which was more selective than celecoxib (9). the aim of this work is synthesize and preliminary anti-inflammatory evaluation of 4thiazolidinone derivatives of mefenamic acid in order to obtain a more potent mefenamic acid analogues. materials and methods chemicals used during the synthesis were supplied by hyper-chem (china). completion of reactions and the purity of compounds were monitored by thin-layer chromatography (tlc), using silica gel gf254 (type 60) pre-coated aluminum sheets, merck (germany) exposed to uv254 nm light, five solvent systems were used which include: ethyl acetate:hexane(4:6) (12,13) , ethyl acetate:hexane (6:4) , ethyl acetate: hexane (3:7),ethyl acetate: petroleum ether(5:5) and ethyl acetate: petroleum ether(4:6). melting points were detected by using stuart smp3 melting point apparatus in open capillary tubes, and are uncorrected. the infrared spectra were performed in thin film techginqe, (υ, cm-1), on shimadzu ftir spectrophotometer, (japan). 1hnmr spectra were obtained on bruker model ultra shield 300 mhz spectrophotometer, and bruker model ultra shield 400 mhz spectrophotometer, using deuterated dmso-d6 as solvents and tms as an internal standard, at al-albayt university, ammanjordan. chemical synthesis synthesis of 2-(2-chloro-n-(2,3-dimethylphenyl ) acetamido)benzoic acid (i) (14,15) mefenamic acid (2.0 g, 8.29 mmol) was dissolved in dichloromethane (dcm) 20 ml and in the presence of triethylamine (1.4 ml, 9.97 mmol) the mixture were stirred at 0 oc for10 min. then chloroacetylchloride (1.4 ml,17.6 mmol) was added dropwise. after that stirring at 0 oc for 15 min and at room temperature for one hour ,the end of reaction was detected by tlc , after end of reaction aqueous k2co3( 2 eq.)( 2.5 g) was added to the mixture and the mixture were stirred for 30 min was washed with dichloromethane and water. the aqueous layer was extracted with 3 × 30 ml of ch2cl2. the organic layers were collected and concentrated. the remaining residue was purified by recrystallization from benzene. the percent yield and physical data are given in table (2) ftir: 2920 cm-1(c-h stretching of ch3); 1705 cm 1(c=o of amide) and 1639 cm-1 (c=o of carboxylic acid). 1hnmr (400 mhz: dmsod6) h: 1.91( 3h, s, arch3,ortho mefenamic); 2.09 (3h, s, ar-ch3 meta mefenamic ); 4.89 (2h, s, ch2 chloroacetyl group); 7.47-7.79 (7h, m, arh) synthesis of 2-(n-(2,3-dimethylphenyl)-2hydrazineylacetamido)benzoic acid (ii). (16) a mixture of compound (i) (0.98 g, 3.1 mmol) and hydrazine hydrate 80% (9.4 mmol) in ethanol (20 ml) was heated under reflux for six hours, then cooled and poured on to crushed ice (30 g), then the solid product which formed was filtered off, washed with water and recrystallized from ethanol to give compound (ii) the percent yield and physical data are given in table (2). ftir: 3425 & 3325 cm-1 (nh of primary amine ); 1735 cm-1 (c=o amide)& 1635 cm-1 (c=o of iraqi j pharm sci, vol.28(1) 2019 synthesis of 4-thiazoldinone derivatives of mefenamic acid 140 carboxylic acid), 1hnmr (400 mhz: dmsod6) h: 2.17( 3h, s, arch3,ortho mefenamic); 2.28 (3h, s, ar-ch3 meta mefenamic ); 4.8 (2h, s, co-ch2nh group); 6.49-7.77 (7h, m, arh) synthesis of schiff base(2-(2-(2benzylidenehydrazineyl)-n-(2,3-dimethyl phenyl)acetamido)benzoic acid)compound iii a-g .(17) a mixture of 1.9 mmol of compound ii and 1.9 mmol of the corresponding aldehyde derivatives that mentioned in table (1) in 20 ml of absolute ethanol was stirred at room temperature for 0.5 to 1h, with addition of two to three drops of hydrochloric acid as a catalyst. the end of the reaction was observed by tlc, and the benzyledine hydrazines were isolated by concentrating of the crude product at reduced pressure, the resulting precipitate was filtered, washed with 10 ml water and recrystallized from ethanol. the percent yield and physical data are given in table (2) 2-(n-(2,3-dimethylphenyl)-2-(2-(4-hydroxybenzy lidene)hydrazineyl)acetamido)benzoic acid (iiia) ftir: 3275 cm-1(o-h phenol& n-h hydrazine );1739 cm-1 (c=o amide ); 1654 cm-1 (c=o of carboxylic acid) & 1604 cm-1 (c=n). 2-(n-(2,3-dimethylphenyl)-2-(2-(4-nitrobenzy lidene )hydrazineyl)acetamido)benzoic acid (iiib) ftir: 1739 cm-1 (c=o amide); 1662cm-1 (c=o of carboxylic acid); 1597 cm-1 (c=n); 1519 & 1342 cm-1 (asym. & sym. stretching of no2). 1hnmr (400 mhz: dmsod6) h: 4.9 (2h, s, co-ch2-nh); 6.83-8.43(11h, m, ar-h) ; 9 (1h, s, ch=n); 12.1(1h, s, cooh) 2-(2-(2-(4-chlorobenzylidene)hydrazineyl)-n-(2,3dimethylphenyl)acetamido)benzoic acid (iiic) ftir: 3210 cm-1(nh hydrazine); 1743 cm-1 (c=o amide ); 1647 cm-1 (c=o of carboxylic acid) & 1597 cm-1 (c=n) and 1083 cm-1 (c-cl ). 2-(n-(2,3-dimethylphenyl)-2-(2-(4-fluorobenzy lidene)hydrazineyl)acetamido)benzoic acid (iiid) ftir: 3210 cm-1(nh hydrazine); 1743 cm-1 (c=o amide ); 1647 cm-1 (c=o of carboxylic acid) and 1600 cm-1 (c=n)and 1095 cm-1 (c-f). 2-( 2 ( 2 ( 4 ( dimethylamino ) benzylidene) hydrazineyl ) -n-(2,3-dimethylphenyl )acetamido ) benzoic acid (iiie) ftir: 3210 cm-1(nh hydrazine) ;1743 cm-1 (c=o amide); 1651 cm-1 (c=o of carboxylic acid) & 1600 cm-1 (c=n). 2-(2-(2-(3-chlorobenzylidene)hydrazineyl)-n-(2,3dimethylphenyl)acetamido) benzoic acid (iiif) ftir: 3210 cm-1(nh hydrazine); 1743 cm-1 (c=o amide ); 1647 cm-1 (c=o of carboxylic acid) & 1604 cm-1 (c=n) and 1080 cm-1 (c-cl ) . 2-(2-(2-(3,4-dimethylbenzylidene)hydrazineyl)-n(2,3-dimethylphenyl)acetamido)benzoic acid (iiig) ftir: 3210cm-1(nh hydrazine); 1743 cm-1 (c=o amide ; 1651 cm-1 (c=o of carboxylic acid)& 1600 cm-1( c=n). table (1) aromatic aldehydes name and products no. quantity(gm) r product no. aromatic aldehyde's name no. 0.231 iv a 4-hydroxybenzaldehyde a 0.286 iv b 4-nitrobenzaldehyde b 0.266 iv c 4-chlorobenzaldehyde c iraqi j pharm sci, vol.28(1) 2019 synthesis of 4-thiazoldinone derivatives of mefenamic acid 141 0.235 iv d 4-fluorobenzaldehyde d 0.283 iv e 4-dimethylaminobenzaldehyde e 0.266 iv f 3-chlorobenzaldehyde f 0.254 iv g 3,4-dimethylbenzaldehyde g synthesis2-(n-(2,3-dimethylphenyl)-2-((4-oxo-2phenylthiazolidin-3-yl)amino)acetamido)benzoic acid compound iv a-g.(18) a mixture of benzyledine hydrazine iiia-g (1 mmol) and excess of thioglycolic acid a(0.071 mmol) (5 ml) was heated at 60 oc until reaction complete, as shown by tlc (about 6 h). ethyl acetate (5 ml) was added, the organic layer was washed with water (1 × 10 ml), dried with mgso4, and concentrated to give an oily product, and the final compound was washed with diethyl ether the percent yield and physical data are given in table (2) 2-(n-(2,3-dimethylphenyl)-2-((2-(4-hydroxyphenyl ) -4-oxothiazolidin-3-yl) amino)acetamido)benzoic acid (iva) ftir: 3236 cm-1(oh phenol & n-h hydrazine); 1782 cm-1(c=o thiazolidinone); 1732 cm-1 (c=o amide); 1670 cm-1 (c=o of carboxylic acid) and 1238 cm-1 (c-o). 1hnmr (300 mhz, : dmsod6) h: 3.8-3.9 (2h, d, ch2 thiazolidinone);4.1 (1h,s,ch2-nh-n);4.9 (2h,s,co-ch2-nh);6.1(1h,s,n-chs)(thiazolidinone); 6.7-7.79 (11h, m, ar-h); and 10.1 (1h, s, ar-oh). 2-(n-(2,3-dimethylphenyl)-2-((2-(4-nitrophenyl)4-oxothiazolidin-3-yl)amino)acetamido)benzoic acid (ivb) ftir: 3236 cm-1 (n-h hydrazine); 1778 cm1 (c=o thiazolidinone); 1728 cm-1 (c=o amide); 1681cm-1 (c=o of carboxylic acid) ; 1346 cm-1 (no2 stretching) and 1238cm-1(c-o). 1hnmr (300 mhz, : dmsod6) h: 3.7-3.9 (2h, d, ch2 thiazolidinone);4.1 (1h,s,ch2-nh-n); 4.9 (2h,s,co-ch2-nh); 5.93(1h, s, n-ch s)(thiazolidinone); 6.7-8.25 (11h, m, ar-h) . 2-( 2 ( ( 2 ( 4 – chlorophenyl ) – 4 –oxothiazolidin – 3 – yl ) amino ) – n ( 2 , 3-dimethyl phenyl ) acetamido ) benzoic acid (ivc) ftir: 3236 cm-1 ( n-h hydrazine); 1782 cm1(c=o thiazolidinone); 1728 cm-1 (c=o amide); 1670 cm-1 (c=o of carboxylic acid); 1087 cm-1 (ccl stretching) and 1280cm-1(c-o). 1hnmr (300 mhz, : dmsod6) h: 3.7-3.8 (2h, d, ch2 thiazolidinone);4.1 (1h,s,ch2-nh-n); 4.9 (2h,s,co-ch2-nh); 5.8 (1h, s, n-ch s)(thiazolidinone); 6.7-7.79 (11h, m, ar-h). iraqi j pharm sci, vol.28(1) 2019 synthesis of 4-thiazoldinone derivatives of mefenamic acid 142 2( n ( 2 , 3 – dimethylphenyl ) – 2 ( ( 2 ( 4fluorophenyl) – 4 – oxothiazolidin – 3 -yl) amino ) acetamido ) benzoic acid (ivd) ftir: 3251 cm-1 ( n-h hydrazine); 1782 cm1(c=o thiazolidinone); 1724 cm-1 (c=o amide); 1670 cm-1 (c=o of carboxylic acid) and 1222 cm-1 (c-o). 1hnmr (300 mhz, : dmsod6) h: 3.7-3.8 (2h, d, ch2 thiazolidinone); 4.1 (1h,s,ch2-nh-n); 4.9 (2h,s,co-ch2-nh); 5.8 (1h,s,n-chs)(thiazolidinone); 6.7-7.79 (11h, m, aromatic ch). 2( 2 ( ( 2 ( 4 ( dimethylamino ) phenyl ) 4oxothiazolidin – 3 – yl ) amino ) – n ( 2 , 3dimethylphenyl)acetamido)benzoic acid (ive) ftir: 3251 cm-1 ( n-h hydrazine); 1782 cm1(c=o thiazolidinone); 1728 cm-1 (c=o amide); 1670 cm-1 (c=o of carboxylic acid) and 1238cm1(c-o). 1hnmr (300 mhz, : dmsod6) h: 3.7-3.8 (2h, d, ch2 thiazolidinone); 4.1 (1h,s,ch2-nh-n); 4.9 (2h,s,co-ch2-nh); 5.8(1h,s, n-chs)(thiazolidinone); 6.7-7.79 (11h, m, ar-h). 2( 2 ( ( 2 ( 3 – chlorophenyl ) – 4 – oxothiazolidin – 3 – yl ) amino ) – n ( 2 ,3dimethyl phenyl)acetamido)benzoic acid (ivf) ftir: 3232 cm-1(n-h hydrazide); 1782 cm-1(c=o thiazolidinone); 1724 cm-1 (c=o amide); 1670 cm-1 (c=o of carboxylic acid); 1076 cm-1 (c-cl stretching) and 1242 cm-1(c-o). 1hnmr(300 mhz, : dmsod6) h: 3.7-3.8 (2h, d, ch2 thiazolidinone); 4.1 (1h,s,ch2-nh-n); 4.9 (2h,s,co-ch2-nh); 5.8 (1h, s, n-ch -s)(thiazolidinone); 6.7-7.79 (11h, m, arch). 2-( n ( 2 , 3 – dimethylphenyl ) – 2 ( ( 2-(3 , 4 – dimethylphenyl ) – 4 oxothiazolidin-3yl)amino)acetamido)benzoic acid (ivg) ftir: 3251 cm-1( n-h hydrazide); 1782 cm1(c=o thiazolidinone); 1728 cm-1 (c=o amide); 1670 cm-1 (c=o of carboxylic acid) and 1280 cm-1 (c-o). 1hnmr (300 mhz, : dmsod6) h: 3.7-3.8 (2h, d, ch2 thiazolidinone); 4.1 (1h,s,ch2-nh-n); 4.9 (2h,s,co-ch2-nh); 5.7 (1h,s, n-chs)(thiazolidinone); 6.7-7.79 (10h, m, ar-h). scheme 1.synthesis of the target compounds (iv a-g ) iraqi j pharm sci, vol.28(1) 2019 synthesis of 4-thiazoldinone derivatives of mefenamic acid 143 table (2) the characterization and physical parameters of the target compounds and their intermediates melting point °c % yield description molecular weight molecular formula no. 156-159 81 pale yellow crystals 317 c17h16clno3 i 138-141 80 white powder 313 c17h19n3o3 ii 164-166 69 orange crystals 417 c24h23n3o4 iii a 170-172 67 deep yellow crystals 466 c24h22n4o5 iii b 155-158 45 off white powder 435 c24h22cln3o3 iii c 110-112 63 yellow powder 419 c24h22fn3o3 iii d 105-108 67 orange powder 444 c26h28n4o3 iii e 102-105 85 pale yellow powder 435 c24h22cln3o3 iii f 125-128 80 pale yellow powder 451 c26h27n3o3 iii g 71 orange oil 491 c26h25n3o5s iv a 60 yellow oil 520 c26h24n4o6s iv b 57 yellow to orange oil 510 c26h24cln3o4s iv c 61 yellow to orange oil 493 c26h24fn3o4s iv d 68 brown oil 518 c28h30n4o4s iv e 64 yellow to orange oil 510 c26h24cln3o4s iv f 62 yellow to orange oil 503 c28h29n3o4s iv g 230-233 white powder 241 c15h15no2 m.a preliminary pharmacological studies anti-inflammatory evaluation study the inflammatory model that used to evaluate final compounds (iva-g) for the in-vivo acute anti-inflammatory effects exploited egg-white induced rat paw edema, for comparison with antiinflammatory activity of mefenamic acid. the decrease of paw thickness is the basis of screening of the newly synthesized compounds for their antiinflammatory activity. methods animals albino rats of either sex weighing (250 ± 50 gm) were supplied by biotechnology research center, al-nahrain university, and were housed under standardized conditions in the biotechnology research center, al-nahrain university animal house. commercial chaw was used to feed the animals and they had free access to water ad libitum. animals were brought to the laboratory, one hour before the experiment; animals were divided into nine groups (six rats per group) as follow: group a: injected with the vehicle (dimethyl sulfoxide 10% v/v) and served as a control group. group b: injected with mefenamic acid as reference substance with a dose of 50mg/kg (19), dissolved in dimethyl sulfoxide 10% (v/v). group c-i: injected with the tested compounds (ivag) by doses that are determined below. (dissolved in dimethyl sulfoxide 10% v/v). calculations for dose determination (20) dose of reference compound /mwt. of reference compound = dose of tested compound/ mwt. of tested compound. iraqi j pharm sci, vol.28(1) 2019 synthesis of 4-thiazoldinone derivatives of mefenamic acid 144 table (3) compounds with their molecular weight and dose compounds molecular weight dose mg / kg mefenamic acid 241 50 iv a 491 86.5 iv b 520 94 iv c 510 107 iv d 493 103 iv e 518 93 iv f 510 107 iv g 503 105 experimental design egg albumin was used to induce rat paw edema as acute inflammatory model for studying of the final compounds activity(21). 0.05ml of undiluted ovalbumin was subcutaneously injected into the rats' planter side of the hind paw; preceded by a half hour of intraperitoneal injection of the drugs or their vehicle. electronic lcd digital vernier caliper gauge stainless steel ruler was used for measuring paw thickness at 7 time periods (0, 30, 60, 120, 180, 240, and 300 minutes) after the compounds injection. statistical analysis the mean ± sem was used to report data of this work then student t-test (two sample assuming equal variances) used to calculate data statistical significance between means. anova (two factors without replication) is used to compare between different groups. p-value < 0.05 was assumed significant. results and discussion the anti-inflammatory activity of the tested compounds has been evaluated in comparison with their vehicle (control group) and mefenamic acid. table (4) explains the effect of tested compounds (iva-g) in comparison to control and mefenamic acid. the tested compounds and the reference drug produced significant reduction of paw edema with respect to the effect of dimethyl sulfoxide 10%v/v (control group). all tested compounds significantly limited the inflammation in paw edema, the onset of mefenamic acid and compounds iva,c,e started at time 120 min but ivc became comparable to control at 180 min until the end of the study. while compound ivg started at 180 min and compounds ivb,f started at 240 min. compound ive exhibited more potent anti-inflammatory effect than mefenamic acid (50mg/kg, i.p.) at 240-300 min., while compounds iva,d exhibited higher antiinflammatory effect at time 300 min. however, the effect of all tested compound continued till the end of experiment with statistically significant (p<0.05) reduction in paw edema thickness as shown in figure (1). comparative analysis the comparison explains that at 0-60 min., there are no differences among all groups. compounds (iv a,d,e,f) at time 120-240 minutes show comparable effect to mefenamic acid except iv e which becomes more potent at 240-300 min. at time 300 min compounds iv a,d exhibit more potent effect than mefenamic acid, while ivf remains comparable until the end of the study. although; compounds ivb,g significantly limited the increase in paw edema in comparison to control group, but they are significantly less effective than mefenamic acid and other tested compounds at interval of 120 minutes and after that they exhibit comparable effect to mefenamic acid until the end of the experiment. figure (1) effect of mefenamic acid, dimethyl sulfoxide, compounds iva-g on egg-white induced paw edema in rats. results are expressed as mean ± sem (n = 6 for each group). iraqi j pharm sci, vol.28(1) 2019 synthesis of 4-thiazoldinone derivatives of mefenamic acid 145 table (4) the anti-inflammatory effect of control, mefenamic acid and compounds iva-g on egg-white induced paw edema in rats: compounds time 0 30 60 120 180 240 300 p a w t h ic k n e ss ( m m ) ,n = 6 control 3.98 ±0.1 5.05 ±0.2 6.06±0.1 6 ±0.1 5.8 ±0.1 5.6 ±0.1 5.4 ±0.1 mefenamic 3.89 ±0.2 4.97 ±0.2 5.83 ±0.2 5.5 ±0.2*a 5.1 ±0.1*a 4.8 ±0.05*a 4.43 ±0.06*a iva 3.75 ±0.2 4.9 ±0.1 6.3 ±0.1 5.5 ±0.14*a 5.2 ±0.15*a 4.7 ±0.1*a 4.2 ±0.08*a ivb 3.75 ±0.06 5.1 ±0.1 5.55 ±0.2 6 ±0.1 6 ± 0.1 4.8 ±0.1*a 4.5 ±0.1*a ivc 3.6 ±0.1 5 ±0.2 6.2 ±0.2 5.4 ±0.2*a 5.3 ±0.2 4.8 ±0.3 5 ±0.3 ivd 3.5 ±0.04 5.1 ±0.2 6.3 ±0.14 5.8 ±0.2 5.3±0.14*a 4.7 ±0.1*a 4.1 ±0.04*a ive 3.5 ±0.07 5.1 ±0.2 5.9 ±0.15 5.5 ±0.1*a 4.8 ±0.14*a 4.35 ±0.1*a 4 ±0.05*b ivf 3.6 ±0.1 5 ±0.1 5.9 ±0.25 5.7 ±0.2 5.4 ±0.2 4.9 ±0.2*b 4.3 ±0.1*a ivg 3.5 ±0.1 4.7 ±0.1 6 ±0.1 5.9 ±0.1 5.3 ±0.1*a 4.9 ±0.1* 4.6 ±0.1*a *significantly different compared to control (p<0.05). data are expressed in mm paw thickness as mean ± sem. n= number of animals. time (0) is the time of i.p. injection of mefenamic acid and dimethyl sulfoxide. time (30) is the time of injection of egg white (induction of paw edema). non-identical superscripts (a, b) among different groups are considered significantly different (p<0.05). conclusions a new derivatives of mefenamic acid were successfully synthesized by conventional method and tested for anti-inflammatory activity, acute antiinflammatory study using egg white induced edema model of inflammation revealed that the incorporation of 4-thiazolidinone moiety into a mefenamic acid maintained or enhanced its antiinflammatory activity and also the antiinflammatory study show that compounds (iva , ivd , ive) contain [oh,f,n(ch3)2] groups respectively which are electron donating groups showed superior antiinflammatory activity to mefenamic acid probably due to formation of hydrogen bonding with the target receptor in the body . acknowledgments the authors greatly thankful the college of pharmacy, university of baghdad, for their help and support. references 1. bothara kg. synthesis and investigation of antiinflammatory activity of novel nitric oxide donating hybrid drugs. med chem res 2013;22:3510–3517. 2. saravanan g, alagarsamy v. synthesis , analgesic , anti-inflammatory , and in vitro antimicrobial activities of some novel quinazolin-4 ( 3 h ) -one derivatives. med chem res 2013; 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normal control group received the vehicles; model group received a single dose of cyclophosamide (150 mg/kg, i.p). the other two groups received orally safranal at doses of 50 and 100 mg/kg/day, respectively, for 8 consecutive days prior cyclophosamide injection (at day fifth). the last group received only oral safranol at dose 100 mg/kg/day for 8 consecutive days. this study showed safrenal-pretreatment significantly ameliorated the deterioration of liver function and exerted significant anti-oxidant with a marked decline in malondialdehyde level, and increase in glutathione and nuclear factor erythroid 2-related factor 2 levels in a dose dependent-manner. keywords: liver toxicity, cyclophosamide, safranal, glutathione, malondialdehyde السمية الكبدية الناتجة من استخدام مادة السايكلوفوسامايد ضدالسافرنال تاثير 1**،همناف هاشم زلزلو *فكتوريا سعد كريم وزارة الصحة والبيئة ، دائرة صحة بابل، بابل، العراق . * اقكلية الصيدلة، جامعة بغداد، العرفرع االدوية والسموم، ** الخالصة التي تنشط بواسطة انزيمات الكبد هو العضو الرئيسي للعمليات االيضية للجسم ,السايكلوفوسامايد هو من اقدم انواع العالجات السرطانية ساسية من الكبد لتنتج مادة االكرولين عالية السمية والفوسفوامايد ماسترد لتزيد االكسدة داخل الجسم ,السافرانال هو مستخلص الزيوت العطرية اال تلقت المجموعة النموذجية جرعة ذي ,في هذه الدراسة تلقت مجموعة الكونتروال فقط الماء المغالزعفران ويعمل كمضاد اكسدة ومضاد التهابات , يوم، على /كغم /ملغم 100و 50تلقت المجموعتان األخريان السفرنال عن طريق الفم بجرعات (. كجم /مجم 150)واحدة من السيكلوفوساميد /مجم 100افرانول عن طريق الفم بجرعة تلقت المجموعة األخيرة فقط س(. في اليوم الخامس)أيام متتالية قبل حقن سيكلوفوساميد 8التوالي، لمدة أظهرت هذه الدراسة أن المعالجة المسبقة للسفرينال خففت بشكل كبير من تدهور وظائف الكبد وأظهرت ارتفاع في مضادات أكسدة مع . يوم /كجم .بطريقة تعتمد على الجرعة 2بط باإلريثرويد المرت 2انخفاض ملحوظ في مستوى مالونديلديهيد ، وزيادة في مستويات الجلوتاثيون والعامل النووي . مالونديلديهيد ، سافرنال، لسايكلوفوسامايد ، الكبدية السمية :الكلمات المفتاحية introduction the liver is a critical organ for metabolism and maintain homeostasis in general body function. it also acts as a source of nutrients and a detoxifier of undesirable substances (1) . cyclophosphamid (cp), is one of the earliest chemotherapy drugs that still used today to treat many forms of cancers, cp is a prodrug, it is converted in the liver into acrolein and phosphoramide mustard by the cytochrome p450 system. many previoes studies indicated that during its oxidative metabolism, cp produces reactive oxygen species (ros), and suppresses the antioxidant protection mechanisms of the liver (2) . acrolein could easily react with glutathione (gsh), a thiol-containing protein that serves many essential functions including detoxification. when gsh was depleted, acrolein could react with cellular nucleophiles, such as the thiol groups of cysteine residues in the proteins and the atoms of nitrogen in lysine and histidine, causing protein loss, thus inducing oxidative stress and eventually leading to catastrophic hepatocyte effects (3) .cp interferes with liver oxidant/antioxidant balance, which contributes to reactive oxygen (ros) accumulation, influencing lipid peroxidation and signaling of cytotoxicity pathways (4) .free radicals-induced lipid peroxidation change membrane structure and function. they are also involved in cell defects including mutation and cell death.(5) oxidative stress triggers multiple intracellular signals leading to proinflammatory cytokine up-regulation.(6). moreover, many recent studies found that the oxidative stress, inflammation, and apoptosis are the main pathways leading to cp hepatotoxicity (7). 1corresponding author e-mail: munafzalzala@copharm.uobaghdad.edu.iq received: 6/3/2021 accepted: 6/6/2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp208-213 iraqi j pharm sci, vol.30(2) 2021 safranal effect on cyclophosphamide liver injury 209 glutathione is a thiol-containing tripeptide that helps to minimize oxidative stress by providing a major non-enzymatic endogenous antioxidant. the liver has high glutathione levels because of its active synthesis (8). nuclear related factor 2 (nrf2), is a transcription factor, which is a highly preserved archaic protein that can orchestrate and balance an intracellular oxidative stress response. several studies have shown that the fundamental mechanism of activation nrf2 is based on their negative regulator of kelch-like ech-associated protein 1. nrf2 binds to the antioxidant cisactant (are) reaction element that controls the expression of detoxifying and antioxidant enzymes. nrf2 -stress dependent induction results in induction in the transcription of many antioxidant enzymes directly (9), 10) . most abundant chemicals in saffron plant oils is safranal (2,6,6-trimethyle 1, 3cyclohexadien-1-carbox-aaldehyde). that is composed of 60–70% of dry saffron's volatile fraction. (11), various research groups have documented medicinal properties of safranal with anti-oxidant, anticonvulsant, cardioprotective, antinociceptive, anti-cancer, anti-inflammatory effect and others. the majority of these therapies may be attributed to their ability to quench ros and to minimize the body's oxidative stress.(12) safranal modulates the expression of antioxidant genes and controls mitochondrial antioxidant genes, which produces a less radical mitochondrial oxygen capacity. inflammatory and immune system disease could be effectively affected by safranal, modulating pro-inflammatory cytokines, stress oxidative and immune factors. (13, 14) further, safranal lowered inflammation and decreased tnf-α level, possibly due to its high antioxidant and anti-apoptotic capacity (15, 16) . aim of study the study designed to assess the defensive impact of safranal against liver toxicity induced by cyclophosamide materials and methods cyclophosphamide was purchased from baxter oncology (frankfurt, germany). mda and glutathione rat elisa kit were purchased from sunlong biotech,nrf2elisa kit purchased from mybiosource co. alt and ast kits were purchased from randox laboratories (united kingdom),safranal purchased from sigma aldrich (india) animals rats of wistar were picked from the animal house of the pharmacology and toxicology department of the collage of pharmacy at university of baghdad. adult albino male rat'sweighing (150-250) gm. animals were randomly allocated into five groups (6 rats each). these rats are housed in a temperatureregulated room at 25 ± 2 cº, 50-70 percent humidity and 12 hours of lighting period under probable hygienic conditions. all rats are fed during the experiments with standard pellets and tap water. animals are weighed and their initial weights were registered after 2 weeks of acclimatization, and then weighted every 2 days. the rats are divided into five group as the following: group i (control):six rats received liquid paraffin orally for 8 days and normal saline intraperitoneal (i.p) in the fifth day(3 days before the end of the experiment). group ii (model group): six rats received cyclophosphamide 150 mg/kg intraperitoneal (i.p) in the fifth day (3 days before the end of the experiment). group iii: six rats received safranal orally in a dose of 50 mg/kg/day consecutively for 8 days and cyclophosphamide 150 mg/kg intraperitoneal (i.p) in the fifth day. group iv: six rats received safranal orally in a dose of 100 mg/kg/day consecutively for 8 days and cyclophosphamide 150 mg/kg intra peritoneal (i.p) in the fifth day. group v: six rats received safranal orally in a dose of 100 mg/kg/day consecutively for 8 days biochemical evaluation blood sample is obtained from animal by heart puncture after anesthetized using diethyl ether, the sample left half hour at room temperature then take serum after centrifuging for10minunt at 10000 rpm and stored at -20 °c for determination of liver enzyme, alanine transaminase activity was measured by colorimetric method determination of nrf2, mda, glutathione level in homogenizing tissue a 0.1gm of liver tissue was homogenate using cold phosphate buffer solution (10%w\v) after centrifuging then keep the supernatant at-20 °. estimation of nrf2, mda ,glutathione levels in rat tissues by using the enzyme-linked immuno-sorbent assay (elisa) kit according to sandwich-elisa principle, at 37°c and read the optical density (o.d.) at 450 nm. statistical analysis all the values are presented as means ±standard error of the means (sem) of all experiments. comparisons between different groups were carried out using one-way analysis of variance (anova) the difference was considered significant when p ˂ 0.05. spss software was used to carry out these statistical tests. iraqi j pharm sci, vol.30(2) 2021 safranal effect on cyclophosphamide liver injury 210 results effects of safranal on serum liver microsomal enzymes in cp-induced hepatotoxicity in rats. cyclophosphamide (cp, 150 mg/kg, i.p.) resulted in acute liver damage in rats as evidenced by the significant elevation of serum alanine transaminase (alt) and aspartate transaminase (ast), as compared to the normal control group. (table 1 and 2 ,respectively). table 1. level of alt level among various groups groups serum alt level (u/l) control 18.96 ± 2.16 cp 40.42 ± 3.97 * cp/safranal 50mg/kg 36.21 ± 5.27 * cp/safranal 100mg/kg 23.14 ± 2.09 # safranal 100mg/kg 19.06 ± 1.35 # all values are expressed as mean ±sd * compared with control group, significant different (p<0.05). # compared with model cp group significant different, (p<0.05). table 2. ast level among various groups groups serum ast level (u/l) control 77.78 ± 22.09 cp 169.96 ± 47.39 * cp/safranal 50mg/kg 122.66 ± 34.35 cp/safranal 100mg/kg 80.78 ± 12.88 # safranal 100mg/kg 78.83 ± 16.49 # all values are expressed as mean ±sd * compared with control group, significant different (p<0.05). # compared with model cp group significant different, (p<0.05). effect of safranal on hepatic nrf2 and glutathione level in cp-induced hepatotoxicity in rats. cp induced acute liver damage in rats as evidenced by an decrease in hepatic nrf2 and glutathione content as compared to the normal control group. pretreatment of rats with safranal (50 and 100 mg/kg/day, oral) for five days prior cp injection significantly inhibit the reduction in hepatic nrf2 and glutathione in a dose dependent manner compared to the cp-control group, recording normal levels of nrf2 and glutathione (table 3 and 4 ,respectively). table 3. impact of safranal on nrf2 level among various groups groups hepatic nrf2 level (ng/0.1gm) control 112.95 ± 13.21 cp 66.28 ± 7.98 * cp/safranal 50mg/kg 120.4 ± 26.78 # cp/safranal 100mg/kg 138.82 ± 36.18 # safranal 100mg/kg 143.36 ± 20.06 # all values are expressed as mean ±sd * compared with control group, significant different (p<0.05). # compared with model cp group significant different, (p<0.05). table 4. effect of safranal on glutathione level among various groups groups gsh level (ng /0.1g) control 69.26 ± 14.43 cp 35.74 ± 11.0 * cp/safranal 50mg/kg 73.7 ± 16.02 # cp/safranal 100mg/kg 82.31 ± 27.77 # safranal 100mg/kg 71.84 ± 10.75 # all values are expressed as mean ±sd *different significant variations relative to the control group (p<0.05). #different significant variations relative to the model group (p<0.05) . effect of safranal on hepatic mda in cp-induced hepatotoxicity in rats. cyclophosphamide (cp, 150 mg/kg, i.p.) resulted in acute liver damage in rats as evidenced by a significant increase in hepatic mda as compared to the normal control group. pretreatment of rats with safranal, at 50 and 100 mg/kg, significantly reduced the elevation in liver mda in a dose-dependent manner compared to the cp control group, recording normal levels of mda dose with insignificant difference from normal control group (table 5). ). compared to the control group, there is no statistical difference between the safranal group treated alone (p>0.05). iraqi j pharm sci, vol.30(2) 2021 safranal effect on cyclophosphamide liver injury 211 table 5 . effect of safranal on mda level among various groups groups mda level (ng/0.1g) control 38.39 ± 8.06 cp 56.02 ± 9.14 * cp/safranal 50mg/kg 51.56 ± 6.84 * cp/safranal 100mg/kg 43.19 ± 3.16 # safranal 100mg/kg 45.79 ± 8.4 all values are expressed as mean ±sd *different significant variations relative to the control group (p<0.05). #different significant variations relative to the model group (p<0.05) . discussion cyclophosphamide (cp) is one of the universally used antineoplastic drugs due to its beneficial effects on various cases of cancer, multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus, cp is clinically limited because of adverse effects and toxicity.(18, 17) even at low doses of cp can produce significant hepatotoxicity in humans which often stands as a barrier against its clinical use. cp-induced toxicity is known to have been associated with biochemical pathogenesis with inflammatory cascading and oxidative stress across generations of tissue inflammatory cytokines and free radicals.(19) the degree of cp induced hepatotoxicity has been correlated with elevated levels of ast and alt in patients.(20-22). consequently, it is quite important to develop alternative and safe natural products against liver damage caused by hepatotoxins such as cp. the hepatoprotective agent should restore the normal structure of the liver and protect the normal physiological mechanisms damaged by hepatotoxins. several studies were conducted to define the biological and pharmacological activities of safranal. in the present study, the hepatoprotective, antioxidative and antiinflammatory properties of safranal against oxidative stress and inflammation in cp induced liver damage were investigated. it was reported that safranal stabilized biomembranes in biological systems and reduced unsaturated membrane lipids and reactive oxygen species, effect of safranal on enhanced serum enzymes prevent infiltration of intracellular enzymes.(13) this is consistent with the commonly accepted view that, with the recovery of the hepatic parenchyma and hepatocyte regeneration, serum transaminase levels return to normal.(23) cp therapy lowers the amount of nuclear nrf2 as well as nqo-1 and ho-1 (heme oxygenase-1) expression. the decreased in the level of nuclear nrf2 and the subsequent reduction in enzymes nqo-1 and ho-1 that may be due to an increase in the level of map kinase p38(24) . mapk p38 can phosphorylate nrf2 has been reported to promote the interaction between nrf2 and keap1 proteins, potentially inhibiting nrf2 nuclear translocation and ultimately preventing ho-1 expression.(24) further research found that safranal inhibited kelchlike ech-associated protein, 1 (keap1) expression and promoted elevation in nrf2 translocation. meanwhile, downstream nrf2 antioxidant enzyme genes including glutathione s transferase (gst), glutamate-cysteine ligase catalytic subunit (gclc), nadph-quinone oxidoreductase 1 (nqo1) and glutathione s transferase (gst), nadph-quinone oxidoreductase 1 (nqo1) were also induced by safranal.(26) cyp is activated by cytochrome p450, yielding phosphoramide mustard and acrolein. acrolein inhibits p-450 by alkylating the groups of sulfhydryl. acrolein is metabolized mainly by rapid modification of the groups of glutathione sulfhydryl (gsg), forming mer-capturic acid that is removed in the urine. acrolein directly raises cellular oxidative stress through this process by reducing glutathione levels.(27, 28) cp-induced gsh depletion. not only does acrolein interact with gsh, but also with cysteine, one of gsh's constituent amino acids.(29) safranal has been reported to modulate the expression of antioxidant genes and regulate mitochondrial antioxidant genes, providing a lower radical potential for mitochondrial oxygen.(13) the increase in the cellular gsh content of, which may increase the gsh/gssg ratio and decrease lipid peroxidation due to the effects of safranal.(30) the cp oxidative product responsible for the induction of lpo and the ros under inflammation which will attack normal tissue and disrupt redox cycle, raising lpo(31) and significantly enhance the degree of lipid peroxidation in experimental animal livers with mda as the most popular breakdown product.(32) moreover, safranal exhibited concentration dependent radical scavenging property as proved by its action of donating a hydrogen atom to the diphenylpicryl hydrazine (dpph) radical (33) it also protected cell membrane polyunsaturated fatty acids from damage and promote hepatic cell regeneration. this is revealed by their impact on the lpo terminal product.(34) conclusion in conclusion, safranal is a phytochemical known for its antioxidative properties. this study showed that cyclophosphamide led to the development of or increased lipid peroxidation, free radicals, resulting in hepatic injury. safrenalpretreatment significantly ameliorate the deterioration of liver function and exerted significant anti-oxidant with a marked decline in iraqi j pharm sci, vol.30(2) 2021 safranal effect on 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28. g. cuce et al., “chemico-biological interactions chemoprotective effect of vitamin e in cyclophosphamide-induced hepatotoxicity in rats,” chem. biol. interact., 2015; vol. 232, pp. 7–11. 29. kehrer jp, biswal ss. the molecular effects of acrolein. toxicol sci. 2000 sep;57(1):6-15.. 30. s. samarghandian, r. afshari, and a. sadati, “evaluation of lung and bronchoalveolar lavage fluid oxidative stress indices for assessing the preventing effects of safranal on respiratory distress in diabetic rats,” sci. world j., 2014. 31. j.e. biaglow, m.e. varnes, e.r. epp, e.p. clark, antioxidant and redox enzymes in radioprotection, pharmacology & therapeutics, volume 39, issues 1–3, 1988, pages 275-286. 32. haque r, bin-hafeez b, parvez s, pandey s, sayeed i, ali m, raisuddin s. aqueous extract of walnut (juglans regia l.) protects mice against cyclophosphamide-induced biochemical toxicity. hum exp toxicol. 2003 sep;22(9):473-80. 33. assimopoulou an, sinakos z, papageorgiou vp. radical scavenging activity of crocus sativus l. extract and its bioactive constituents. phytother res. 2005 nov;19(11):997-1000. 34. hussein rm, el-hussieny ea, hassanin la, el-sayed wm (2017) ‘the prophylactic and therapeutic effects of safranal and selenite on liver damage induced by thyrotoxicosis in adult male rats. int j clin pharmacol toxicol. 6(3), 270-279. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 new coumarin derivitives as antibacterial doi: https://doi.org/10.31351/vol30iss1pp249-257 249 synthesis, characterization and antibacterial evaluation of some coumarin derivatives tariq t. abduljabbar*,1 and mohammed k. hadi* *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract coumarin derivatives have shown different biological activities. new coumarin derivatives (hydrazones and an amide) were synthesized through multisteps reactions. all the synthesized target compounds were characterized by ft-ir spectroscopy and 1h-nmr analysis. the compounds then evaluated for their anti-bacterial activity by means of disc-well diffusion against two gram-positive bacteria (staphylococcus aureus and streptococcus pneumoniae) and two gram-negative bacteria (e. coli and pseudomonas aeruginosa). the highest activity was demonstrated by compound k2 which found to be highly active against pseudomonas aeruginosa (containing no2 group which is a strong electron withdrawing group that creates a localized electron deficient sites within molecules) key words: coumarin, hydrazones, amide, antibacterial, well diffusion method. ةمن مشتقات الكومارين الجديد لبعض وتقييم الفعالية المضادة للبكتريا ،تشخيص ،تصنيع طارق طالل عبد الجبار*،1 و محمد كامل هادي* .العراق ،بغداد، جامعة بغداد،كلية الصيدلة ،ةالنيفرع الكيمياء الصيد* الخالصه تم تصنيع مشتقات جديدة للكيومارين )هيدرازونات وامايد( من خالل تفاعالت .أظهرت مشتقات الكومارين أنشطة بيولوجية مختلفة . تم النووي للبرتونالرنين المغناطيسي تحت الحمراء وتحليل مطيافية االشعة جميع المركبات المستهدفة تم تشخيصها بواسطهمتعددة الخطوات. العقدية البكتريا لمكورة العنقودية الذهبية والبكتريا ا ) الموجبهاالنتشار ضد اثنين من البكتيريا تقييم المركبات لنشاطها المضاد للبكتيريا بطريقة والذي k2تم توضيح أعلى فعالية بواسطة المركب ي(. البكتريا اإلشريكية القولونية و البكتريا الزائفة الزنجار ) السالبهواثنين من البكتيريا (الرئوية تعاني من نقص تخلق مواقع لاللكترونات ساحبه قويهوهي مجموعة no2تحتوي على مجموعة )وجد أنه عالي الفعالية ضد الزائفة الزنجارية اإللكترون داخل الجزيئات( مضادات بكتيرية. ،امايد ،هايدروزونات ،الكلمات المفتاحية: هيدرازايد introduction heterocyclic chemistry is one of the main parts in organic chemistry and it is growing quickly. in 1998 heterocyclic synthesis accounted about 60 %. however today the heterocyclic synthesis is much bigger diverse fields such as pharmaceuticals, biochemistry materials and others are the areas which new heterocyclic compounds are published (1 ). the same development is perceived for coumarin. coumarin is a white crystalline solid with a scent like a new mown-hay. the chemical structure of coumarin consist of an aromatic ring fused to an unsaturated cyclic lactone ring (2) . coumarin (which is referred 1,2-benzopyrone or less commonly, as o-hydroxycinnamic acid-8lactone) a natural heterocyclic organic aromatic compound, present in a widespread variability of microorganisms and higher plants (3,4), vogel was first one who insulate coumarin in 1820 by extracting from the tonka beans (dipteryx odorata) specie previously known as coumarona odorata, later the term coumarin. it has been then identified in a huge number of the plants which belong to numerous diverse families(5). coumarin is classified as a member of the benzopyrone family of compounds, all of which consist of a benzene ring joined to a pyrone ring. the benzopyrones may be divided into benzo-α-pyrones (1) to which the coumarins fit and (2) benzo-ᵞ-pyrones named as chromones, of which flavonoids are the main chemical member , the later differs from the earlier only in the position of the carbonyl group in the heterocyclic ring. the various biological activities coumarin derivatives such as anticoagulants and antithrombotics are well known, so that; they are active for the inhibition and treatment of venous and arterial thrombosis (6), a number of coumarin derivatives are also described as antifungal and antibacterial agents (7), antiviral and antitumor agents (8), lipid-lowering agents(9), anti-hiv agents (10), antioxidants and lipoxygenase inhibitor (11). they have also been found to possess antiproliferative, vasorelaxing activities (12), antiinflammatory activity, anthelmintic, hypnotic, insecticidal activities, and diuretic properties (13). the coumarins vary highly in their structure, as a result of the different substitution types in their main structure, which can affect their biological activities . the 4-hydroxycoumarin comprises the structural nucleus of many natural products, drugs and pesticides. 1corresponding author e-mail: tariq_sonic@yahoo.com received: 13/9/2020 accepted:16 /1 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp249-257 iraqi j pharm sci, vol.30(1) 2021 new coumarin derivitives as antibacterial 250 it is the key intermediate for different widely used anticoagulants and rodenticides. as well as drugs used as antithrombotic agents in human (14). coumarin-3-carboxylic acids have anti-tumor and cyto-cell selective effects (15).7-hydroxy-4-methyl coumarin could be used as cardio active drug by inhibition of calcium influx . schiff bases are the compounds carrying imine (–c=n–) functional group, and were first reported by hugo schiff (16-17) schiff bases have a significance in medicinal and pharmaceutical fields as a result of a broad spectrum of biological activities such as anti-inflammatory (18-19) analgesic (20) , antimicrobial (21,22) , anticonvulsant (23) antitubercular (24) , anticancer (25,26) , antioxidant , anthelmintic (27). the nitrogen atom of azomethine may be involved in the formation of a hydrogen bond with the active centers of cell constituents and interferes in normal cell processes more (28) ever ,they are very useful as catalysts, intermediates in organic synthesis, pigments, dyes, polymer stabilizers it was found that the coumarin moiety was introduced with the schiff base, the compound synthesized may have some remarkable pharmacological and microbiological activity corrosion inhibitors (29) . it was found that the coumarin moiety was introduced with the schiff base, the compound synthesized may have some remarkable pharmacological and microbiological activity amide bond creation is one of the most significant chemical reactions. the occurrence of amides can be seen in natural peptides, protien and many amidebond containing biomolecules as well as in several synthetic compounds with varied uses e.g. as pharmaceutically active compounds or synthetic polymers. amides are of great importance in organic chemistry especially because of their high stability and polarity and their conformational variety. coumarin contains amide moiety have drawn considerable amount of interest because of their pharmacological properties and their synthetic derivatives having a significant pharmacological and microbiological activity. mohd et al synthesized new coumarin compounds that screened in vitro for their antibacterial activity against the two bacterial strains, e. coli (gram –ve) and b. cereus (gram + ve) . n-(4-bromo-2-fluorophenyl)-6,8-dichloro-2oxo-2h-chromene-3-carboxamide (20) with dichloro substitution showed maximum growth inhibition in comparison with the rest of the compounds against both bacterial strains (30) . materials and methods coumarin-3-carboxylic acid was bought from hyper-chem. company (china), . solvents (dichloromethane ,ethyl acetate, ethanol, hexane, toluene) (ar) and other reagents that used through reaction were bought from commercial sources. the purity of products and monitoring of the reactions were done by thin layer chromatography tlc(gf254,merkgermany) under uv light (254nm). two solvent systems were used s1 [(toluene: ethyl acetate: ethanol (3:2:1)] and s2 [(hexane:ethyl acetate:ethanol (3:2:1)] (by trial). melting points were uncorrected and detected by using stuart smp3 melting point apparatus in open capillary tubes. ft-ir spectra were done by thin film technique (υ, cm-1), (shimadzu ft-ir spectrophotometer, japan) at college of pharmacy/university of baghdad . 1h-nmr were done, using brucker ultra shield model 500 mhz at (university of tehran ,iran) using dmso as a solvent. chemical synthesis schemes (1), (2), and (3) show the synthesis of targeted compounds. iraqi j pharm sci, vol.30(1) 2021 new coumarin derivitives as antibacterial 251 h1 h2 scheme 1. synthesis of n'-benzylidene-2-oxo-2h-chromene-3-carbohydrazide derivatives compounds (h1h2) scheme 2. synthesis of n,n-diethyl-2-oxo-2h-chromene-3-carboxamide (b). iraqi j pharm sci, vol.30(1) 2021 new coumarin derivitives as antibacterial 252 scheme 3. synthesis of n'-(1-(2-oxo-2h-chromen-3-yl) ethylidene) benzohydrazide derivatives compounds (k1,k2). synthesis of coumarin hydrazide 2-oxo-2hchromene-3-carbohydrazide (a): coumarin-3-carboxylic acid (10 mmol)(1.9g) were dissolved in dichloromethane (dcm)(60 ml) and stirred at room temperature . then, hobt (hydroxybenzotriazole ) (11.84 mmol)(1.6g) was added followed by adding edci (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) (12 mmol)(2.3g). the mixture was stirred overnight at room temperature, which was then gradually added to a solution of hydrazine (60 mmol ,0.64g) and cyclohexene (1 ml) in dcm (5ml) while the temperature was kept between 010°c( using ice bath and check the tempreture by thermometer) . the reaction was completed upon the end of addition. the organic layer wash with 15 ml of 5% sodium carbonate to eliminate hobt and the excess of acid. elimination of the solvents under reduced pressure gave hydrazide which was recrystallized from methanol (31). synthesis of final n'-benzylidene-2-oxo-2hchromene-3-carbohydrazide derivatives compounds (compounds h1-h2) three drops of glacial acetic acid were added to an ethanolic solution of each of the following aldehydes shown in detail in table 1.1 :[ 3,5-dimethoxy-4-hydroxybenzaldehyde , [4hydroxy-benzaldehyde] placed in round bottom flask equipped with magnetic stirrer. compound a ( 5mmole, 1.02g) suspended in absolute ethanol (20ml) was added to a stirred solution of each of the above-mentioned aldehyde separately. then each reaction mixture was refluxed at 80oc for 8 h. at the end of the reaction (monitored by tlc), 50 ml of cold ice water was added to the crude product. the precipitate formed was filtered and dried and recrystallized from ethanol (32) . table 1. aldehydes used in synthesis of (h1,h2) derivitives. no name quantity molecular weight 13,5-dimethoxy-4hydroxybenzaldehyde ( 5 mmole, 0.91g) 182.17 g/mol 24-hydroxy-benzaldehyde (5mmole, 0.61g) 122.12g/mol synthesis of coumarin 3 carboxylic amide (2-oxo2hchromene-3-carboxamide (b): coumarin-3-carboxylic acid (9.9 mmol)(1.9g) were dissolved in dcm (20 ml) stirred at room temperature. then, hobt (11.84 mmol)(1.6g) followed by adding of (9.3 mmol)(1.8g) edci (9.3 mmol)(1.8g) . the reaction was stirred at room temperature . after that a solution of diethyl amine (19.1 mmol)(1.4g) and cyclohexene (1 ml) in dcm (10 ml) was added gradually to above solution while temperature was kept between 0-10 °c during the addition. the reaction mixture stirred overnight (tlc monitored), the mixture was washed with aqueous solutions of 0.1n hcl (15 ml) and 5% sodium bicarbonate (15 ml) to eliminate the excess of amine and acid . the organic layer was evaporated iraqi j pharm sci, vol.30(1) 2021 new coumarin derivitives as antibacterial 253 and the precipitate was dried and recrystallized from ethanol (33) . synthesis of n'-(1-(2-oxo-2h-chromen-3 yl)ethylidene)benzohydrazide derivatives (k1-k2) three drops of glacial acetic acid were added to an ethanolic solution of (5mmole) coumarin 3 acetyl placed in a round flask equipped with magnetic stirrer .both of 2 hydrazides vanalic acid hydrazide ( 5mmole,0.9g mw 182.18 g/mol) and p-nitro –benzylhydrazide ( 5 mmole ,0.9g, mw 122.12 g/mol ) was added separately to above stirred solution . then reaction mixture was refluxed at 80oc for 2 h. at the end of the reaction (monitored by tlc), 50 ml of cold ice water was added to the crude product. the precipitate formed was filtered, dried and recrystallized from ethanol. antibacterial essay disc-well diffusion method has been performed through the use of the bacterial suspension of (1.5x108cfu/ml) obtained from mcfarland standard of turbidity (number 0.50). which has been utilized for inoculation by the swabbing of mueller hinton agar (mha) plates’ surface. the excess liquid has been dried by air under a sterile hood. in every one of the agar plates of the examined bacteria, 4 wells have been made, and (80μl) of every one of the concentrations of the synthesized compound was poured to it. the plates have been incubated for 24h at 37°c. the evaluation of antibacterial activity has been based upon the measurement of the inhibition zone diameter which is formed around the well.(34) results and discussion the synthesis of a and b was done by reaction of coumarin-3-carboxylic acid with hydrazine hydrate and diethyl amine, respectively, in the presence of coupling agent (edci) and catalyst (hobt). schiff base products (h1, h2, k1, and k1) were the results of the reactions between coumarin-3-carboxylic hydrazide with different aldehydes (h1 and h2) or the reactions between coumarin-3-acetyl with two hydrazides (k1 and k2) in ethanol using glacial acetic acid as catalyst. chemistry compound a: (c10h8n2o3), green yellow powder, yield= 86%, m.p: 204-206 0c, rf = 0.68 (s1) ft-ir (ῡ cm-1): 3319, 3273(nh) str. of hydrazide nh2, 3064 aromatic (c-h) str., 1612 (c=o) str. of amide, 1705 coumarin (c=o). compound h1: (c17h12n2o4), yellowish powder, yield: 82%, m.p: (158-160 0c), rf =0.82(s1). ft-ir (ῡ cm-1 ): 3205 (nh) str. of hydrazone, 3159 (oh) str. (broad), 3051 aromatic (c-h) str., 1651 (c=o) str. of amide, 1615 (c=n) str., 1701.22 coumarin (c=o). 1h-nmr:11.58ppm (1h,s,-co-nh), 9.99ppm (1h,s,-oh), 8.89ppm (1h,s,n=c-h), 6.83-8.00ppm (8h,m,ar-h), 8.32ppm (1h,s,c4-h) compound h2: (c19h16n2o6), yellowish brown powder, yield = 70% m.p: (170-1720c), rf =0.78 (s1). ft-ir (ῡ cm-1): 3300-3070 (oh) str. (broad), 3263 (nh) str. of hydrazone, 2989 asymm. str. of ch3, 2839 symm. str. of ch3, 3059 aromatic (c-h) str., 1658 (c=o) str., 1701.22 coumarin (c=o), 1612 (c=n) str. 1h-nmr: 3.81ppm (6h,s,o-ch3), 7.03-8.01ppm (6h,m,ar-h), 8.31ppm (1h,s,c4-h), 8.88ppm(1h,s,n=c-h), 11.64ppm (1h,s,co-n-h). compound b: (c14h15no3), yellowish powder, yield: 82%, m.p.: (158-1600c ) rf =0.82(s2). ft-ir (ῡ cm-1 ): 3020 aromatic (c-h) str., 2970 asymm. str. of ch3, 2939 symm. str. of ch3, 1720.5 coumarin (c=o), 1627 (c=o) amide. 1h-nmr: 1.14ppm (6h,t,-ch3), 3.24ppm (4h,q,ch2), 7.3-7.77 ppm (5h,m,ar-h), 8.15ppm (ih,s,c4-h). compound k1: (c18h13n3o5), white powder, yield: 70%, m.p.: 173 – 1740c , rf=0.84(s1). ft-ir (ῡ cm-1 ): 3255 (nh) str. of hydrazide, 3060 aromatic (c-h) str., 1660 (c=o) str., 1720 coumarin (c=o), 1519 (no2) asymm. str., 1651(c=n) str. 1h-nmr: 2.35ppm (3h,s,-ch3), 7.45-8.35ppm (8h,m,ar-h), 8.27ppm (1h,s,c4-h), 11.13ppm (1h,s,-co-nh), compound k2: (c19h16n2o4 ), yellowish powder, yield: 82%, m.p.: 165-1670c, rf=0.80(s1). ft-ir (ῡ cm-1 ): 3332 (nh) str. of hydrazide, 3051 aromatic (c-h) str., 2939 asymm. str. of ch3, 2835 symm. str. of ch3, 1647 (c=o) str. of amide, 1701.22 coumarin (c=o), 1624 (c=n) str. 1h-nmr: 2.33ppm (3h,s,ch3), 3.84ppm (3h,s,-och3), 6.88-7.85ppm (7h,m,ar-h), 8.2ppm (1h,s,c4-h), 9.7ppm (1h,s,oh), 10.57ppm (1h,s,co-nh). antibacterial evaluation the antibacterial activities of the synthesized derivatives (b,h1,h2,k1,k2) were measured using disc well diffusion method technique, tested on gram +ve and gram -ve bacteria, using amoxicillin as standard . dmso was used as a solvent and as a control, as shown in the following table (2) . iraqi j pharm sci, vol.30(1) 2021 new coumarin derivitives as antibacterial 254 table 2. the antibacterial activity of the target compounds(b,h1-h2,k1,k2). compound no conc. µg/ml pseudomonas aeruginosa staphylococcus aureus streptococcus pneumoniae e.coli inhibition zone(mm) b 103 15 -------12 h1 103 14 -------10 h2 103 12 6 10 15 k1 103 10 6 ---12 k2 103 22 ------14 amoxicilin 103 10 23 20 --- (-)= no activity, slightly active: (inhibition zone in between 5-10 mm), moderately active: (inhibition zone in between 10-15 mm), and highly active (inhibition zone more than15mm) (35) discussion compounds a, b were synthesized by the reaction of coumarin 3 carboxylic acid with hydrazine hydrate (nh2nh2.h2o) or amines. this reaction involves several parts of mechanism. the first part involves two stages that include conversion of the carboxyl group into a good leaving group. the first stage include acid base reaction resulted in the transfer of proton from carboxylic acid to the edci, the second stage involves attacking of rco2 which is formed from the first stage on the edci (conjugate acid). the second part includes the addition of hobt that is supposed to work by primarily reacting with the o-acylsourea to give the obt active ester, which improves the reactivity of the ‘‘activated ester’’ by encouraging/ stabilizing the method of the amine through hydrogen bonding. on the other hand, hobt can produce by-products, thus it catalyses the creation of diazetidine. the third part involves formation of hydrazide/ amides that is due to the last procedure which is the removal of the leaving group (water soluble urea ) .(36,(37). scheme 1 . mechanism of the synthesis of coumarin 3 carboxylic hydrazide and amide. when r group is nh2 the whole structure is hydrazide but if r group is alkyl, aryl or h group the whole structure is amide. iraqi j pharm sci, vol.30(1) 2021 new coumarin derivitives as antibacterial 255 the final compounds (h1, h2, k1 and k2) were synthesized by the reaction of coumarin-3carboxylic hydrazide with different aldehydes and coumarin-3-acetyl (as ketone) with two hydrazides by using glacial acetic acid as a catalyst. aldehydes or ketones react with primary amine of hydrazide to form imines in a two-step reaction . regarding the first step of the reaction; the amine adds to the carbonyl group to form carbinolamine. carbinolamine then undergoes dehydration to give n-aryl-substituted imine. the steps of the reaction are catalyzed by the addition of a few drops of acid (38). . scheme 2. mechanism of the synthesis of coumarin schiff bases. among the synthesized derivatives, compounds (b and h1) show moderate activity against pseudomonas aeruginosa. furthermore, compounds (h2 and k1) show slight activity against staphylococcus aureus, compound k2 show moderate activity against e. coli. the highest activity was demonstrated by compound k2 which found to be highly active against pseudomonas aeruginosa (due to the presence of nitro functional group which is strong electron withdrawing group that creates a localized electron deficient sites within molecules and interact with biological nucleophile present in living system in addition to the formation of two ionic bonds and one hydrogen bond .hydroxy functional group in h1 can form hydrogen bond and its electron donating group ) conclusion new coumarin hydrazones and amide derivatives were synthesized successfully. their chemical structure were characterized using ir spectroscopy and 1-hnmr. the antibacterial activity of target compound (b,h1,h2,k1,k2) was evaluated using disc-well diffusion . the highest activity was demonstrated by compound k2 (ionized containing no2 which is a strong electron withdrawing group) which was found to be highly active against pseudomonas aeruginosa. acknowledgments we're appreciative to the college of pharmacy department of pharmaceutical chemistry university of baghdad, for conducting the research, mr. ahmed college ibn -alhaitham, for pure science, for his polite style and his patience during evaluation of antimicrobial activity, and chemistry analysis center (cac) stuff for their supporting in 1hnmr analysis and evaluation of antibacterial activities of the compounds. iraqi j pharm sci, vol.30(1) 2021 new coumarin derivitives as antibacterial 256 references 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http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32(1) 2022 drug prescription pattern using who prescribing indicators in libya doi: https://doi.org/10.31351/vol32iss1pp266-273 266 evaluation of drug prescription pattern using who prescribing indicators in libya: a cross-sectional study ahmed atia-*,1 , nouran gzllal** and malak gharibe** *department of anesthesia and intensive care, faculty of medical technology, the university of tripoli, libya **department of pharmaceutical sciences, the university of tripoli alahlia, janzur, libya abstract the improper use of drugs due to irrational prescriptions is a common problem in libya. this study aimed to investigate the prescribing practices and extent of rational therapy in primary health care facilities in three districts (east, west, and south) in libya. in this retrospective study, 484 prescriptions were examined. world health organization-recommended indicators for rational use were examined (who): e.g., the percentage of prescriptions covering antibiotics, prescription of injections, and prescription of drugs by a generic name and from a national essential drug list, as well as the average number of drugs per prescription, were all considered. the average number of drugs per prescription was 4.72, with a maximum of 7 drugs in a prescription, and the percentage of prescriptions involving antibiotics and injections was 30.4% and 10.5%, respectively. there were 28.6% drugs prescribed by their generic name and 82.8% were retrieved from the essential drugs list. the most common category of medicines were 18.9% antibiotics, 18.3% antihypertensives, and 15.7% multivitamins and minerals. while the lowest consumed drugs were steroids 2.5%. there was some irrational drug prescribing, particularly with regard to injections and antibiotics. it is suggested that physicians should be participated in continuing education programs on rational prescribing for various medical indications. keywords. antibiotic, rationality, prescription, pattern. introduction nowadays, drug utilization studies (dus) are used as potential tool in the evaluation of healthcare system (1). drug usage studies are effective tools for determining the role of drugs in society. they provide a solid socio-medical and health-economic foundation for making healthcare decisions (2). the world health organization (who) describes drug utilization research as “the advertising, spreading, prescription and use of drugs in a society, with a particular focus on the medical, social, and economic consequences" (3). the evaluation of drug prescription prototype is a significant feature of patient care. it encourages the rational along with estimating the quality and efficacy of the drug and care that provides the rational use of drugs, which is essential for an efficient and disciplined health-care system. on the other hand, irrational drug use considered as a universal risk which exists in all parts of the world includes insufficient dose, poly-pharmacy, improper use of antimicrobial agents, cough and cold preparations. in addition, injections which are considered as powerful and widely overused when oral dosage forms are more applicable (4). medical prescription is a significant document of medico legal value too, that can be kept as proof in medico legal cases in court of law and thus should be cautiously and critically considered (5). more than 50% of all medicines are prescribed, dispensed, or sold improperly and 50% of patients fail to take them properly (5). according to a who report, more than half of all drugs are improperly prescribed in developing countries, where drug monitoring and assessment are in their infancy (6). in addition, poor legibility was found in more than half of the prescriptions (55.3%) examined in a study on patterns of drug prescribing in a hospital in dubai (7). this is lower than, but close to, the situation in saudi arabia (64.3 %) (8) and higher than that reported in the usa seven years ago (15%) (9). in libya, prescription of medications can easily be obtained without prescription from community pharmacies, resulting in potential drug misuse and health hazard (10). it was stated previously that there were overprescribing of certain categories of drugs writing by libyan physicians which necessitating further improvement (11). hence, the current study was aimed to assess the drug use pattern in selected general hospitals: a cross-sectional study in libya. method study design and population a cross-sectional study was conducted in different selected health care centers located in three districts in libya (west, east, and south), involving prescriptions recorded from january to december 2021.of which, randomly selected prescriptions were retrospectively assessed using who prescribing indicators during the study period (1 jan to 31 dec 2021). all patients who visited outpatient departments of selected health care centers in the three selected districts during the study period were used as a target population. 1corresponding author e-mail: ah.atia@uot.edu.ly received: 26/5 /2022 accepted: 3/8 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp266-273 iraqi j pharm sci, vol.32(1) 2022 drug prescription pattern using who prescribing indicators in libya 267 sampling technique and procedure about 414 randomly selected prescriptions recorded during the study period from each health center were included. three well-trained pharmacists were recruited and deployed to assess the prescriptions identified. data from the prescriptions was recorded on a data collection form. the form was thoroughly tested before being modified. patient demographics and the prescribed item information such as dosage forms and the number of medicines per prescription, total number of drugs in the prescription, the average number of drugs prescribed per prescription, prescribed by generic name, and prescribed from the national essential drug list, as well as the category of drug prescribed, and the percentage of antimicrobials prescription were recorded in the data collection form the following drug use indicators were used in the study to determine the prescribing pattern, according to who (12); the average number of drugs prescribed per prescription (evaluate the degree of polypharmacy), the proportion of drugs prescribed under a generic name (measures the costeffectiveness of a health system to use drugs), percentage of antibiotic-prescribed per prescription (measures the level of use of commonly overused antibiotics), the percentage of injectable preparations prescribed per prescription (which measures the level of use of a commonly overused and costly form of drug therapy) and the proportion of drugs prescribed that are on the national essential medicines list. index of rational drug prescribing (irdp) five indexes derived from who drug prescribing indicators make up the index of rational drug prescribing (irdp). the optimal level for each indicator is shown in table 1. for each indicator, the optimal index is 1. drug prescribing is considered more rational when the calculated index value is close to 1. prescriptions containing three or more medications were deemed rational in the current study. the proportion of drugs given by generic name and from the national essential drug list were used to estimate the generic name index and essential medicine index, respectively. the rational antibiotic prescribing index was calculated by dividing the optimal level (30%) by the percentage of prescriptions that included an antibiotic. the prescribing injection index was measured by dividing the optimal level (10%) by the percentage of prescriptions that included the injection. by adding the indices, the irdp with a maximum value of 5 was calculated. table 1. optimal levels of drug prescribing indicators prescribing indicators optimal level (%) optimal index percentage of drugs per prescriptions ≤3 1 percentage of drugs prescribed by generics 100 1 percentage of prescriptions including antibiotics ≤30 1 percentage of prescriptions including injections ≤10 1 percentage of drugs prescribed from national essential drug list 100 1 data analysis the data collected were entered and analyzed using spss version 20, and findings were reported as frequencies and percentages. results characteristics of prescriptions based on our findings in table 2, a total of 484 prescriptions compromising 1957 drugs with an average number of drugs per prescription was 4.72. the maximum of drugs in a prescription was 7, and was from the southern districts of libya. unfortunately, due to the uncompletedness of most of the basic information, patient age, nationality, and gender in some prescriptions, we were unable to further analyze the demographics of the patients. about 43.6% of drugs were tablet dosage form (54.1%, 48.3%, 35.8%; east, west, and south districts, respectively), and the percentage of prescriptions involving injections was 5.3%, 6.4%, 15.6% from the east, west, and south districts, respectively (10.5%; total). most of drugs 82.8% were from the essential drugs list and 28.6% were generic. iraqi j pharm sci, vol.32(1) 2022 drug prescription pattern using who prescribing indicators in libya 268 table 2. analysis of prescriptions using who core prescribing indicators. items east (n=140) west (n=195) south (n=149) total (n=414) % of total number of prescriptions 140(29%) 195(40.3%) 149(30.7%) 484(100%) total number of drugs prescribed 410 621 926 1957 average number of drugs per prescription 3.91 4.78 5.47 4.72 % of prescribed injectable preparation 22(5.3%) 40(6.4%) 145(15.6%) 207(10.5%) % of drug prescribed by generic name 86(20.9%) 134(21.6%) 341(36.8%) 561(28.6%) % of prescription with tablet dosage form 222(54.1%) 300(48.3%) 332(35.8%) 854(43.6%) % of drug prescribed from national essential drug list 360(87.8%) 447(71.9%) 814(87.9%) 1621(82.8%) the results of irrational medicine use are shown in table 3. the overall irdp used as an indicator of rational drug use was 3.91. the overall indicator was made up of the index of drugs per prescription (0.63), generic name (0.28), antibiotic (1.00) , injection (1.00) , and drugs prescribed from the national essential drug list (1.00) (table 3). the index of rational drug prescribing was also calculated for each districts individually. the highest irdp was 3.96, while the lowest was 3.90. all districts had an index of 1 for injection prescription, prescription of antibiotics, and prescription of drugs from the national essential drug list. the indices of drugs per prescriptions and prescription of drugs by generic names varied among the three districts. the highest index for drugs per prescription was 0.76, while the lowest was 0.54. the highest index for prescription of drugs by generic name was 0.36, while the lowest was 0.20. table 3. index of rational drug prescribing (irdp) prescribing indicators optimal level (%) optimal index calculated level (%) irdp percentage of prescription of ≤3 1 4.72 0.63 percentage of drugs prescribed by generics 100 1 28.6% 0.28 percentage of prescriptions including antibiotics ≤30 1 30.4% 1 percentage of prescriptions including injections ≤10 1 10.5% 1 percentage of drugs prescribed from national essential drug list 100 1 82.8% 1 antibiotic prescription rate and pattern out of 414 prescriptions were reviewed from the three selected districts for this study, 126 of them had at least one antibiotic prescription (table 4), giving an overall percentage prescription with antibiotics of 30.4%. antibiotic prescription rate with regards to the districts was as follows: the south (36.2%), > the west (23.6%) > the east (18.6%). because some prescriptions had more than one antibiotic prescribed, a total of 309 antibiotics were prescribed during the study period, with average of 1.81 number of antibiotics per prescription. the percentage of antibiotics prescribed by generic name in all districts was 22%, while prescriptions with brand names comprised 78% of all prescriptions. the overall percentage of drugs prescribed from edl was 99.3, and it was 100% in both east and south districts. table 4. antibiotic use indicators of visited health care facilities antibiotic use indicator east (n=140) west (n=195) south (n=149) total (n=414) antibiotic prescription rate 26(18.6%) 46(23.6%) 54(36.2%) 126(30.4%) total number of antibiotics prescribed 34 89 186 309 av. no of antibiotics per prescription 2.58 3.54 4.31 3.47 % of antibiotics prescribed by generic name 23(67.6%) 70(78.6%) 148(79.5%) 241(78%) % of antibiotics prescribed from edl 34(100%) 87(97.7%) 186(100%) 307(99.3%) iraqi j pharm sci, vol.32(1) 2022 drug prescription pattern using who prescribing indicators in libya 269 antibiotics prescribed and indications for prescription the 309 antibiotics prescribed during the study period belonged to 12 different classes (table 4). the majority (219, 71%) of these were broad spectrum (figure 1) and mostly the penicillins. the most prescribed antibiotics were ceftriaxone (79, 25.5%), followed by metronidazole (70, 22.6%), azithromycin (31, 10.03%), and co-amoxiclav (28, 9.06%) (table 5). we were unable to report the diagnosis subject to antibiotic prescription, as most of prescriptions were recorded as ‘no diagnosis’. table 5. antibiotic category and prescription classes antibiotic spectrum n(%) of times prescribed east west south total penicillins amoxicillin broad 1(0.3%) 4(1.3%) 0 5(1.61%) procaine penicillin narrow 0 4(1.3%) 0 4(1.28%) co-amoxiclav broad 5(1.6%) 16(5.2%) 7(2.3%) 28(9.1%) cephalosporine cefalexin narrow 5(1.6%) 2(0.6%) 0 7(2.26%) ceftriaxone broad 1(0.3%) 7(2.3) 71(22.9%) 79(25.5%) ceftazidime broad 0 0 2(0.6%) 2(0.6%) cefixime broad 5(1.6%) 8(2.6%) 0 13(4.2%) cefotaxime broad 0 0 6(1.9%) 6(1.9%) cefdinir broad 0 1(0.3%) 0 1(0.3%) macrolides erythromycin narrow 0 2(0.6%) 0 2(0.6%) azithromycin broad 3(0.9%) 13(4.2%) 15(4.8%) 31(10%) clarithromycin broad 0 1(0.3%) 2(0.6%) 3(0.9%) spiramycin broad 0 1(0.3%) 0 1(0.3%) tetracyclines tetracycline broad 1(0.3%) 0 0 1(0.3%) doxycycline broad 0 6(1.9%) 0 6(1.9%) quinolone ciprofloxacin broad 6(1.9%) 6(1.9%) 15(4.8%) 27(8.7%) moxifloxacin broad 0 1(0.3%) 0 1(0.3%) levofloxacin broad 1(0.3%) 0 0 1(0.3%) polymyxins narrow 0 1(0.3%) 0 1(0.3%) nitroimidazole metronidazole narrow 5(1.6%) 9(2.9%) 56(18.1%) 70(22.6%) tinidazole broad 0 1(0.3%) 0 1(0.3%) aminoglycoside gentamicin broad 0 1(0.3%) 0 1(0.3%) sulfonamides co-trimoxazole broad 1(0.3%) 0 0 1(0.3%) lincomycin clindamycin broad 0 1(0.3%) 0 1(0.3%) glycopeptide vancomycin narrow 0 1(0.3%) 3(0.9%) 4(1.3%) fusidane fusidic acid narrow 0 2(0.6%) 0 2(0.6%) carbapenem meropenem broad 0 1(0.3%) 9(2.9%) 10(3.2%) total 34(11%) 89(28.8%) 186(60.2%) 309(100%) figure 1. percentage of antibiotics based on the spectrum activities categories of drug prescribed in table 6, the most common category of medicines were 18.9% antibiotics, 18.3% antihypertensives, and 15.7% multivitamins and minerals. while the lowest consumed drugs were steroids 2.5%. among the three districts, the southern districts show the highest percentage of antimicrobial usage 10.2%, while they demand at lower rate with dermatology drugs. multivitamins and minerals was accounted for 7.2% of total prescribed drugs in the western districts as their most frequently prescribed medicine, and the rate was much lower with steroids 3.2%. meanwhile, analgesics was observed in top prescribed medicines of several medical specialties in the eastern areas (3.9%), but the rate was much lower 0.5% with endocrine drugs. iraqi j pharm sci, vol.32(1) 2022 drug prescription pattern using who prescribing indicators in libya 270 table 6. drug category in the prescriptions drug group east (n=140) west (n=195) south (n=149) total (n=414) antimicrobial 68(3.5%) 103(5.3%) 199(10.2%) 370(18.9%) antihypertensive 16(0.8%) 62(3.2%) 280(14.3%) 358(18.3%) multivitamins and minerals 44(2.2%) 140(7.2%) 123(6.3%) 307(15.7%) analgesic 76(3.9%) 109(5.6%) 42(2.1%) 227(11.6%) gastrointestinal tract (git) 36(1.8%) 65(3.3%) 162(8.3%) 263(13.4%) respiratory and antiallergic 68(3.5%) 90(4.6%) 59(3%) 217(11.1%) dermatology 44(2.2%) 66(3.4%) 6(0.3%) 116(5.9%) endocrine 10(0.5%) 20(1 %) 20(1%) 50(2.6%) steroids 18(0.9%) 12(0.6%) 19(0.9%) 49(2.5%) total 380(19.4%) 667(34.1%) 910(46.5%) 1957(100%) discussion in a typical drug use investigation study, the who recommends that at least 30 prescriptions be reviewed per prescriber (12). however, this study reviewed 484 prescriptions from the three selected districts of libya (an average of 161.3 prescriptions per district) within the periods of jan to dec 2021. this was done to get a complete and accurate picture of drug prescribing behaviors among libyan primary health care workers, as well as to rule out the possibility of some prescribing patterns being overlooked. using the who prescribing indicators, the current study explored the drug-prescribing pattern of physicians and identified areas that require intervention. in the present study, the average number of drugs per prescription was 4.79. the value was higher than the who recommended optimum level of 1.6-1.8. studies in libya have reported this index to be 2.85 to 3.00 (11,13). the reported value was similar to study conducted in ghana (14), but higher than that in ethiopia (1.83) (15). similarly, the reported value in our result is higher than the value index that reported in studies conducted in the eastern mediterranean region 2.7 (16), india 2.58 (17), sudan 2.55 (18), egypt 2.5 (19), and saudi arabia 2.4 (20). the possible negative consequences of prescribing a large number of drugs per prescription are increased the occurrences of side effects, drug-drug interactions, patients’ noncompliance with the drug regimen, and raised pharmacotherapeutic expenses as a result of the large number of drugs to be taken. the average quantity of drugs prescribed per prescription is influenced by the prevalence of diseases, the lack of clinical practice guidelines, financial incentives for prescribers, physician incompetence, culture, and other factors (20). as a result, different values have been reported in various parts of libya. unnecessarily prescribed drugs could have a financial impact on the health-care system. conversely, rational prescription can prevent medicine waste and minimize adverse effects on patients while lowering costs (20). the inappropriate use of injections plays a significant role in the transmission of very serious blood-borne infections, resulting in disability and death (11). according to the current study, 10.5% of prescriptions containing injections, mostly consumed in the southern district 15.6%. however, the finding was lower than the who recommendation (13.4-24.1), and lower than the previously reported rate in libya 16% (21,13). similar to our results, studies conducted in nigeria and kenya had shown values of 4% and 13.2%, respectively (22,23). moreover, a study done in the egypt showed that 18.1% prescriptions were injectable (24), 0.66% was revealed in saudi (25), and 27.1% in pakistan (26). people believe that injectable drugs are more effective than oral drugs, which could be the cause of patients’ demand in the use of injections, especially in rural areas, which is considered as irrational drug use (27). this could explain the motivation for injectable drugs in our study. people, particularly in rural areas such as libya's south, believe that injections provide faster and more complete pain relief than oral medications (26). the primary motivation for doctors to prescribe an injectable drug is the patient's demand for faster treatment, and this irrationality could be reduced by interventions such as physician education on safe injection (28). the use of generic name in prescription of drugs is considered very crucial for better communication among healthcare personnel (29). low generic prescribing is seen in this study; from 1957 drugs, only 561 (28.6%) drugs were prescribed by their generic name. this result was quite similar to studies conducted in egypt 16.1% (30), and in bahrain 10.2% (31). however, it was very low comparing to other studies in the literature i-e; the values were 46.3% in sudan (18), 61.2% in saudi arabia (20), 71% sierra leone (29), 64.1% in china (32), and pakistan 71.6% (26). prescription of generic drugs is highly recommended by the who for safety reasons because it allows for easier information exchange and better communication between health care providers (20). in libya, however, prescribing decisions are based on physicians' personal beliefs and pharmaceuticals promotion, which can have an impact on this indicator (33). low generic prescribing iraqi j pharm sci, vol.32(1) 2022 drug prescription pattern using who prescribing indicators in libya 271 may further confuse patients who are already dealing with the burden of polypharmacy. this could result in duplication errors, where patients unknowingly take both the generic and brand versions of the same drug at the same time (33). prescriptions from the national essential drug list are a key indicator of drug use. in the current study, 82.8% of the drugs prescribed were from the list, which is quite close to the ideal value (100%). this was similar to the values reported by other studies; 81.2% in sudan (18), 92% in ethiopia (34), and 67.7% in china (31). the reason for this high value is that, the majority of drugs are imported by companies registered and monitored by the libyan ministry of health, and physicians are required to prescribe medicines from the officially available drugs. according to the current findings, the overall irdp used as an indicator of rational drug use was 3.91, compared to an optimal level of 5. this means that 78% of the rational drug-prescribing criteria were met. our finding was in line with studies reported nearly similar values 3.56 (25) and 3.42 (35). dissimilarly, other study revealed lower values 2.71 (29). moreover, the irdp value in our study for antibiotics was 1, which was higher than values reported from previous studies 0.45 (25), 0.62 (29), and 0.68 (32). the prevalence of antimicrobial resistance in any population is related to the proportion of the population that receives antimicrobial and total antimicrobial exposure. increased antimicrobial use leads to more resistance (29). the average of antibiotic prescription in our study was 30.4% of the total prescriptions which is almost reaches to the upper permissible limit suggested by the who (2026.8%; the cut-off of 30%). this rate of prescription was similar to the percentage of antibiotics prescriptions in nigeria 34.4% (23). however, it was lower than the previously reported antibiotic prescription rate in libya in 2019 (80%) (11). some other studies conducted in sudan, pakistan, and bahrain have also reported higher prescription of antibiotics 54.71%, 51.5%, and 45.8%, respectively, in comparison with the current study (18,26,31). irrational antibiotic use can result in adverse reactions, antibiotic-resistant hospitalizations, and high costs (3, 8, 26). conversely, studies conducted in jordan and the united arab emirates found positive results, with 17.7% and 9.8%, respectively, falling below the cut-off point according to who criteria (36,37). the current study had some limitations. it was only conducted in three districts of libya, so it may not be applicable to other cities. nonetheless, the prescription model is used in all three districts of libya, and the pattern of drug prescription is likely to be consistent throughout the country. the study does not investigate the causes of irrational drug prescribing, but this can be investigated in future studies. conclusion overall, the average number of drugs per prescription and generic prescribing examined in this study were 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. int j clin pharmacol ther. 2017;55(5):425-432. doi: 10.5414/cp202733. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 cytokines and tonsillitis doi: https://doi.org/10.31351/vol31iss1pp194-201 194 some pro and anti-inflammatory cytokines in children with tonsillitis and their correlations with vitamin d deficiency najwan kaisar fakree * , zainab m. hashim*,1, ahmed abdulsamad muhsen** and nuha majeed hashim*** * department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad, iraq ** department of otolaryngology, al-yarmook teaching hospital, baghdad, iraq *** ministry of agriculture, baghdad, iraq abstract inflammation of the tonsils could be described as acute tonsillitis, mainly due to infection. recurrent tonsillitis could be defined as 3-7 episodes during the first 3 years of age. vitamin d, which is a neuro-hormone with pleiotropic biological activities may modulate the immune response by alleviation, and stimulation of th1 and th2 cell proliferation, respectively, that influence the stimulation, synthesis, and secretion of both pro and anti-inflammatory cytokines. in this study we aimed to shed light on the levels of vitamin d in children with different episodes of tonsillitis in association with levels of interleukins (tnfα, il-2, il-4, il-10). blood samples were collected from 48 participants in 3 groups: control, acute tonsillitis (1-2 episodes/year), chronic tonsillitis (more than 7 episodes/year), serum was separated and the levels of vitamin d, tnfα, il-2, il-4 and il-10 were estimated using elisa technique. vitamin d decreased significantly as the episodes of tonsillitis increased, with level of 16.38± 2.41ng/ml in acute and, 14.13± 2.15 ng/ml in chronic tonsillitis as compared to control (30.91± 2.31 ng/ml), while pro-inflammatory cytokines (tnfα and il-2) significantly increased (46.88± 14.05 and 44.55± 9.24, 1267.25± 111.85 and 1191.72± 121.52 ng/ml, respectively) as compared to control (9.45 and 138.48 ng/ml respectively). anti-inflammatory (il-4, il-10) cytokines in control group were (243.08± 28.72 and 24.27± 1.83 ng/ml, respectively), which increased non-significantly in acute and chronic tonsillitis (302.76± 38.93, 290.12± 44.69 and 28.16± 2.01, 26.29± 1.99 ng/ml, respectively). significant direct correlation was observed between the levels of vitamin d and anti-inflammatory cytokines in chronic tonsillitis (p<0.05). in conclusion, deficiency of vitamin d may affect the number of episodes of tonsillitis in children by modulation of the secretion of some cytokines. keywords: tonsillitis, vitamin d, inflammatory markers المحفزة والمضادة لاللتهاب لدى االطفال المصابين بالتهاب اللوزتين وارتباطها مع بعض السايتوكينات نقص فيتامين د *** مجيد هاشم ونهى **، احمد عبد الصمد محسن1*،زينب مجيد هاشم ،*نجوان قيصر فاخر العراق بغداد، بغداد، جامعة الصيدلة، كلية السريرية، المختبرية* فرع العلوم قسم االذن والحنجرة ، مستشفى اليرموك التعليمي ، بغداد ، العراق ** *** وزارة الزراعة، بغداد، العراق الخالصة التهاب اللوزتين او كما يوصف بالتهاب اللوزتين الحاد والذي يحدث على الغالب بسبب العدوى. التهاب اللوزتين المتكرر يعرف بانه االستجابة يحور ان ممكن الجوانبهو هورمون عصبي له فعاليات بيولوجية متعددة dاالولى من العمر. فيتامين نوبات خالل الثالث سنوات 3-7 على تنشيط تركيب وافراز كل من السايتوكينات المحفزة على التوالي والذي يوثر th 2و th1 خاليا تزايد وتنشيط تخفيف خالل من المناعية على مستويات فيتامين والمضادة لاللتهاب . الضوء الى تسليط هدفنا نوبات مختلفة من التهاب عدد لدى االطفال الذين لديهم dفي هذه الدراسة مشارك بثالث مجاميع وهم مجموعة 48تم جمع عينات الدم من (tnfα, il2, il-4, il-10) ناتاللوزتين وربطها مع مستويات االنترليوكي مصل فصلنوبات في السنة( . تم 7نوبة في السنة( ومجموعة التهاب اللوزتين المتكرر)اكثر من 2-1) ومجموعة التهاب اللوزتين الحاد الكونترول .اليزال ا تقنية باستخدام il-10 , il-4, il2 , tnfα, d فيتامين من كل مستويات قياس وتم الدم ( في االلتهاب l / مليلتر منانو غرا 2.41± 16.38)كمستوى يتناقص بصورة معنوية كلما ازدادت عدد نوبات التهاب اللوزتين dلوحظ ان فيتلمين السايتوكيناتبينما (. / مليلتر منانو غرا 2.31± 30.91( بالكنترول مقارنة المتكرر االلتهاب في / مليلتر( منانو غرا 2,15 ±14.13) و الحاد )) il2,tnfa ) لاللتهاب المحفزة معنوية ± 1191.72 , 111.85± 1267.25و 9.24 ±44.55 , 14.0 ±46.88تزداد بصورة ( بالتتابع / مليلتر منانو غرا 138.48و 9.45مقارنة بالكنترول ) )بالتتابع نانو غرام / مليلتر 121.52 1corresponding author e-mail: zainab.atiya@copharm.uobaghdad.edu.iq received: 6/6/2021 accepted: 6/ 10/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp194-201 iraqi j pharm sci, vol.31(1) 2022 cytokines and tonsillitis 195 / مليلتر منانو غرا 1.83 ±24.27و 28.72±243.08في مجموعة الكونترول كانت ) ( il 10و il4السايتوكينات المضادة لاللتهاب وهم ) و 2.01±28.16مقابل 44.69± 290.12و 38.93±302.76والتي تزداد بصورة غير معنوية في التهاب اللوزتين الحاد والمزمن ) بالتتابع( 26.29±1.99 ng/ml لوحظ ان هناك ترابط معنوي مباشر بين مستويات فيتامين د وبين السايتوكينات المضادة لاللتهاب في حالة التهاب )بالتتابع ممكن ان يوثر على عدد نوبات التهاب اللوزتين لدى االطفال من خالل تحوير d. كخالصة ان النقص في فيتامين (p<0.05)اللوزتين المتكرر بعض السايتوكينات. افراز عالمات االلتهاب، dفيتامين اللوزتين،التهاب الكلمات المفتاحية: introduction inflammation of the tonsils could be described as acute tonsillitis, mainly contributed to infection. it ranges from localized tonsil infection to generalized infection of the pharynx, as part of the spectrum of pharangitis. it may affect both sexes at all ages, but mostly it affects younger people, especially in winter season (1). viruses may account to 50-80% of the causative pathogens, as influenza, herpes simplex, epstein-barr virus (ebv), and rhinovirus (2,3). the most common group of bacteria that could be associated with tonsillitis is group a beta-haemolytic streptococci (gas), which cause 536% of cases (4). haemophilus influenzae, streptococcus pneumoniae and neisseria gonorrhoeae, are other types of bacteria that can invade the pharynx and tonsils (5). fungi species such as candida can cause sore throat in immunecompromised patients (6). seven tonsillitis episodes in one year or five tonsillitis episodes in the previous two years and more or three tonsillitis episodes in the previous three years and more, could be defined as recurrent tonsillitis (7,8). development of recurrent tonsillitis, could be associated with many factors, these are including patient incompliance, premature cessation of anti-biotherapy, bacterial tolerance, bacterial load, bacterial biofilms, inadequate antibiotic absorbance, and deficiencies in the immune system (9). two chemical compounds ergosterol, or vitamin d2, and cholecalciferol, or vitamin d3could be associated with the term “vitamin d”. active vitamin d3 (calcitrol) is considered as a neurohormone because of its pleiotropic biological activities (10). in spite of the growing knowledge about the relation between calcium-phosphorus metabolism and vitamin d (vd) and many other roles of vitamin d plays, data suggest that immune cells and a large number of tissue cells, express enzymes that metabolize vitamin d, providing a biologically acceptable mechanism to convert local, auto and paracrine of the native circulating forms, into the active vitamin d3 (calcitrol) (11). vitamin d immune-modulation includes alleviation, and stimulation of th1 and th2 cell proliferation, respectively. primarily, vitamin d3's suppression of th1 development is assumed to be mediated by its influence on apc, which reduces il-2 synthesis, whilst increased expression of th2 specific transcription factors (gata-3 and c-maf) is linked to increased th2 cytokines production (12). correspondingly, vitamin d can stimulate or prohibit the synthesis, secretion, and release of anti inflammatory interleukins (il-10 and il-4) and proinflammatory cytokines (il-1, il-2, tumor necrosis factor alpha (tnf-α), interferon gama (ifn-γ)), respectively (11, 13). as a result, vitamin d appears to reduce inflammation while allowing for a targeted immune response (in other words, bioactive vitamin d may boost innate immunity while suppressing adaptive immunity). this process is essential for normal function of the immune system, and subsequently inadequate or scanty levels of vitamin d could lead to impairment of immune responses (14). in this study, we aimed to shed light on the levels of vitamin d in children with different episodes of tonsillitis in association with levels of interleukins (il-2, tnf-a, il-4, and il-10) . subjects and methods this study was carried out among 48 children between 2 and 7 years of age (mean rang 5.79±0.63) at al-yarmook teaching hospital otolaryngology department in baghdad during the period november 2018 and march 2019. children were classified into 3 groups: group 1(control) which includes16 children with no tonsillitis problem (mean age 7.00 ± 0.7), second group (group 2): include 16 children with mean age (5.62 ± 0.65) and 1-2 episodes per year (acute tonsillitis), while group 3(chronic): include 16 children (mean age 6.25 ± 0.95) who have recurrent tonsillitis with more than 7 episodes per year. tonsillitis was diagnosed depending on physical examination by specialist physician. frequency of tonsillitis in a year is checked by referring the feedback of parents (by asking the parents). the inclusion criteria for the study groups were as follow: children having one or more episode of tonsillitis/year, having symptoms of tonsillitis which developed within last 5 days, not taking antibiotics therapy in the past week and have no vitamin d supplementation. while the exclusion criteria include children with chronic or systemic diseases, condition requiring hospitalization, children taking immunosuppressant drugs and those with previous operations. blood samples were collected centrifuged to separate serum from the three groups. serum levels of 25(oh) d (supplied by calbiotech, cat# 220b), tnf-α (elabscience, cat. no.e-el-h0109), il-2 (elabscience, cat. no.e-el-h0099), il-4 (elabscience, cat. no.e-el-h0101) and il-10 (elabscience, cat. no.e-el-h0103) were estimated using enzyme linked immunosorbent assay (elisa) technique and according to the manufacturer. iraqi j pharm sci, vol.31(1) 2022 cytokines and tonsillitis 196 the study was carried out according to the approval of the research ethical committee in the college of pharmacy, university of baghdad. the statistical analysis systemsas (2012) program was used to compare between control and patient groups in study parameters. student ttest was used to significantly compare between means, (0.05 and 0.01 probabilities). also the correlation coefficient between variables in this study has been investigated. results serum levels of different parameters were measured using elisa technique in all studied groups and the results revealed a significant decrease (p<0.05) in vitamin d level in both acute and chronic group (16.38 and 14.13 ng/ml respectively) as compared to the control group (30.91 ng/ml) (table 1) (figure 1). the levels of pro-inflammatory cytokines, tnfα and il-2 appears to be increased significantly (p<0.05) in patient’s groups (46.88, 44.55 and 1267.25, 1191.72 ng/ml, respectively) as compared to the control group (9.45 and 138.48 ng/ml respectively) (table 1) (figure 2 and 3). an increase in the level of interleukin 4 and 10 was also observed in the both acute and chronic groups (302.76, 290.12 and 28.16, 26.29 ng/ml, respectively) as compared to the control group (243.08 and 24.27 ng/ml, respectively), yet this increase was not significant (p>0.05) (table 1) (figure 4 and 5). table 1. levels of vitamin d, tnfα, il2, il4 and il10 (ng/ml) in different studded groups* vitamin d tnfα il2 il4 il10 control 30.91± 2.31 9.45± 0.918 138.48± 12.09 243.08± 28.72 24.27± 1.83 acute 16.38± 2.41 46.88± 14.05 1267.25± 111.85 302.76± 38.93 28.16± 2.01 chronic 14.13± 2.15 44.55± 9.24 1191.72± 121.52 290.12± 44.69 26.29± 1.99 p value/controlacute 0.0001** 0.012** 0.000** 0.227 0.164 pvalue/controlchronic 0.000** 0.000** 0.000** 0.382 0.462 pvalue/acutechronic 0.49 0.891 0.6507 0.83 0.514 *all values represent mean±se **differences are significant when p<0.05 figure 1. level of vitamin d (ng/ml) in different groups figure 2. level of tnfα (ng/ml) in different groups iraqi j pharm sci, vol.31(1) 2022 cytokines and tonsillitis 197 figure 3. level of il-2 (ng/ml) in different groups figure 4. level of il4 (ng/ml) in different groups figure 5. level of il-10 (ng/ml) in different groups this study, also aimed to find the correlation between the levels of vit.d and levels of both, pro and anti-inflammatory cytokines. vitamin d showed non-significant (p>0.05) inverse correlation with pro-inflammatory cytokines (tnfα and il-2) in all groups. anti-inflammatory cytokines (il-4 and il-10) showed non-significant (p>0.05) positive correlation in both, control and acute tonsillitis, and positive significant (p<0.05) correlation in chronic group (table 2) (fig. 6 and 7) . table 2. correlation coefficient between vitamin d levels and pro-inflammatory cytokines (il-2 and tnfα) and anti-inflammatory cytokines (il-4 and il-10) in different groups. il-10 control il-4 control 1l-2 control tnf control vitamin d control pearson correlation 0.179 0.402 -0.152 -0.126 sig. (2-tailed) 0.508 0.123 0.573 0.642 no 16 16 16 16 vitamin d acute pearson correlation 0.261 0.423 -0.344 -0.107 sig. (2-tailed) 0.329 0.103 0.191 0.692 no 16 16 16 16 vitamin d chronic pearson correlation 0.764** 0.713** -0.147 -0.416 sig. (2-tailed) 0.001 0.002 0.586 0.109 no 16 16 16 16 no: number of samples iraqi j pharm sci, vol.31(1) 2022 cytokines and tonsillitis 198 figure 6. vitamin d in correlation to antiinflammatory cytokine il4 in chronic group figure 7. vitamin d in correlation to antiinflammatory cytokine il-10 in chronic group discussion tonsillopharyngitis consider as one of the leading causes of childhood hospital visits and is characterized as acute tonsil and pharynx infections. the most common causes in etiology are viruses and group a beta-hemolytic streptococci (1) vitamin d deficiency is associated with an increased frequency of upper respiratory tract infections. (16), according to our results there were significantly lower levels of vitamin d in acute and chronic tonsillitis groups comparing to the control. similarly, in the previous studies vitamin d levels were significantly lower in tonsillitis patients as compared to the healthy people suggesting that low vitamin d levels may be significant in the pathogenesis of tonsillar diseases (9, 17) the immune responses to the infection are regulated by a balance that stimulates the synthesis of cytokines for th1 and th2. the th1-derived cytokines (il-2 and tnf-α) induce a response through a cell-mediated immunity. while humoral reaction is induced by th2, involving secretion of il-4, il-5, il-6, il-10, il-15, il-16 (18). it has become quite important to study interleukins as mediators of the immune system that regulate the immunity of mucous membranes. chronic inflammatory infectious diseases of the respiratory tract may involve cytokines as mediator of specific immune response and nonspecific effecter mechanism. moreover, cytokines may assess the clinical course, magnitude and clinical significance of the pathological process (19). in this study, the levels of some pro-inflammatory cytokines (tnfα and il-2) and anti-inflammatory cytokines (il-4 and il-10) in tonsillitis patients and healthy (control) groups has been measured. pro-inflammatory cytokine tnfα, has a vital role in the host defense against parasitic, viral and bacterial pathogens, especially against intracellular bacterial infections (20). in this study tnfα was significantly increased in acute and chronic tonsillitis groups compared to control. these results are consistent with the study of aysha and colleges who showed that patients with chronic and acute tonsillitis have significantly higher tnfα than the control (21). the reason is that tnf-α activates the vascular lining and increases vascular permeability, increasing immune cell entry, complement proteins, and immunoglobulin, including igg and iga. it also increases fluid discharge to the lymph nodes leading to swollen tonsils (22). interleukin 2 (il-2) is pro-inflammatory cytokine secreted by th1 cells and activates t cells to produce tnfα and ifnγ (23, 24). the cytolytic function of natural killer (nk) cells can also increase by il2, thus ensuring their importance in the immune function, and effectually participitate in the pathogenesis of multiple pathological situations like infectious, metabolic, cancer, autoimmune and inflammatory diseases (24). according to our study patients with acute and chronic tonsillitis have significantly higher il-2 than the control group. regulation of inflammation could be mediated by some anti-inflammatory cytokines for example il-4, some of which directed at influencing the activity of macrophage. il-4 inhibit proinflammatory cytokines (tnfα , il-12,and il-1β) and chemokines (ifnγ-inducible protein 10 and macrophage inhibitory factor) (25). our results showed that il-4 non significantly increase in both chronic and acute tonsillitis as compared to control. similar results obtained by sydney et al. who showed that, in both children with acute pharyngotonsillitis induced by b-hemolytic streptococci and control groups, levels of antiinflammatory cytokines, particularly il-4, were identical, indicating an alteration in the immune response regulated by th1 (26). another study shows that the production of th1 type cytokines (tnfα and ifn-γ) was elevated in recurrent tonsillitis as compared to th2 type cytokines production (il-4 and il-6), suggesting the influence of the cellular (th1 type) immune response with respect to the humoral (th2 type) immune response. (22). iraqi j pharm sci, vol.31(1) 2022 cytokines and tonsillitis 199 monocyte, t regulatory cells, nk, dendritic cells (dcs) and macrophages, are the main immune cells that produce anti-inflammatory cytokine il-10 (20). various anti-inflammatory functions by il-10 and its role as a major regulator of none-specific immunity had been reported (27-29). in our study, il-10 non significantly increase in both acute and chronic tonsillitis compared to the control group, because in tonsillitis production of cytokines start with th-1 type including tnfα and ifn-γ and later on secretion of th-2 cytokines (22). there are many lines of evidence to support vitamin d's possible role in improving the balance of anti-inflammatory cytokines. these include studies in which vitamin d has been shown to increase the levels of anti-inflammatory cytokines including beta 1 transforming factor (tgf β1) and interleukin il4 (30) and to inhibit secretion of the proinflammatory ones (il-6, ifn-γ, il-2, and tnfα) (31). it has also been shown that vitamin d is important for normal macrophage functions, while its deficiency has been linked to impaired chemotaxis, phagocytosis, (32) and induce inflammation via up regulation of toll-like receptors, on macrophage cell surface (33). this study investigates the correlation between vitamin d level and inflammatory cytokines in children with tonsillitis. according to our results vitamin d have non-significant negative correlation with pro-inflammatory cytokines (tnfα and il-2) and positive non-significant correlation with anti-inflammatory cytokines (il-4 and il-10) in both control and acute tonsillitis groups, while in chronic tonsillitis vitamin d have significant positive correlation with anti-inflammatory cytokines and non-significant negative correlation with pro-inflammatory cytokines. we conclude that deficiency of vitamin d may affect the number of episodes of tonsillitis in children by modulation of the secretion of some cytokines. the correlation between vitamin d administration and serum levels of inflammatory markers such as cytokines has been investigated previously (34 36). some studies have indicated that there is a direct correlation with ifnγ and il-10 (34), while a significant inverse correlation with serum tnfα, and no correlation with serum il10 or il-6 has been reported (35). some other studies indicate that alteration in the serum levels of pro and anti-inflammatory cytokines was nonsignificant (36). this study has several limitations first; crp level which is considered as best indicator of inflammation was not measured for the studied groups in which low levels of crp exclude the inflammation despite the healthy appearance for the control group (i.e the healthy children may have inflammation due to any causes despite the healthy appearance) second; cytokines have short peripheral half-life and should preferably be "locally" assessed, i.e. in tonsils (37). third; children in the studied groups may have deficiency in other trace elements and vitamins that may affect on the function of the immune system and cytokines production, such as, selenium, zinc, copper, iron, folic acid, vitamins a, e, c, and b6 which are of important significance for response of the immune system (38). fourth; this study investigate the immunological response in tonsillitis patients and its correlation with vitamin d in general regardless of it causes whether virus or bacteria (each one of them may have its immune response), so as recommendation for further studies its better to measure the correlation between vitamin d and inflammatory markers in different types of pathogens. conclusion in this study, the levels of vitamin d were significantly lower, in acute and chronic tonsillitis children. in both groups of tonsillitis, th-1 cytokines production (tnfα and il-2) were higher in comparison to th-2 cytokines (il-4 and il-10). vitamin d has non-significant inverse correlation with pro-inflammatory cytokines and nonsignificant positive correlation with antiinflammatory cytokines in both control and acute tonsillitis groups; while in chronic tonsillitis vitamin d have significant positive correlation with antiinflammatory cytokines and inverse non-significant correlation with pro-inflammatory cytokines. thus, deficiency of vitamin d may affect the number of episodes of tonsillitis in children by modulation of the secretion of some cytokines. further studies are needed to evaluate the correlation between vitamin d and other inflammatory markers in patients with tonsillopharyngitis and whether vitamin d replacement therapy can prevent the recurrent tonsillitis. references 1. macnamara m. acute and chronic pharyngeal infection in: gleezon m ed. scott-brown’s otorhinolaryngology, head and neck surgery. london: edward arnold; 2008:1981-90. 2. parthasarathy r, kumar r, gopal g, amarchand r, et al. incidence and clinical features of viral sore throat among children in rural haryana, india. j family med prim care. 2020;9:5136-41. 3. mriganka de and shahram anari. infections and foreign bodies in ent. surgery (oxf). 2018 oct; 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(2016) ; 27(2): 205-229 this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ 404 not found iraqi j pharm sci, vol.30(suppl.) 2021 rheological investigation of lipid polymer hybrid nano-carriers doi: https://doi.org/10.31351/vol30isssuppl.pp9-15 9 rheological investigation of lipid polymer hybrid nanocarriers for oral delivery of felodipine (conference paper )# hayder kadhim drais*,1 and ahmed abbas hussein** # 9th scientific conference conference sponsored by college of pharmacy , university of baghdad 25-26 august 2021 *ministry of health and environment, babil health directorate, babil, iraq. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the rheological behavior among the factors that are present in stokes law can be used to control the stability of the colloidal dispersion system. lipid/polymer hybrid nanocarriers (lphns) is an interesting colloidal dispersion system that is used for rheological characteristic analysis. the lphns are composed of polymeric components and lipids. this research aims to prepare oral felodipine lphns to investigate the effect of independent variables which are lipid content, surfactant mixture and distilled water, on the rheological behavior of the nanosystem. a microwave-based technique was successfully used to prepare felodipine lphns (h1-h9). the formulations were characterized for particle size and pdi to confirm the colloidal properties of the prepared nanosystem. therefore, rheological evaluation study of described nanosystems was performed using the coaxial rotational digital rheometer. the outcomes showed that all felodipine lphns formulations (h1-h9) had a nanosize and homogenous structure which verified the colloidal features of the nanodispersion systems. the rheogram chart indicated that all felodipine lphns formulations (h1-h9) show pseudoplastic flow (nonnewtonian flow), suggesting a shear-thinning property. the microwave-based method successfully produced felodipine lphns with excellent physical texture that proves its ability as a nanoparticles preparation technique. the pseudoplastic flow behavior of felodipine lphns formulations (h1-h9) suggests that the described nanosystems in this study are physically stable keywords: rheology, felodipine, lipid-polymer hybrid nanocarriers, microwave-based method. الفموي لفيلوديبين لاليصالالتحقيق الريولوجي للحامالت النانوية الهجينة للبوليمر الدهني #) بحث مؤتمر ( **احمد عباس حسينو 1*، حيدر كاظم دريس 2021اب 26 – 25جامعة بغداد ، # المؤتمر العلمي التاسع لكلية الصيدلة .وزارة الصحة والبيئة ، دائرة صحة بابل ، بابل ، العراق* العراق.فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ، ** الخالصة الناقالت النانوية . يمكن استخدام السلوك الريولوجي بين العوامل الموجودة في قانون ستوكس للتحكم في استقرار نظام التشتت الغروي lphnsتتكون . هي نظام تشتت غرواني مثير لالهتمام يستخدم لتحليل الخصائص الريولوجية( lphns)الهجينة من البوليمر الشحمي فيلوديبين عن طريق الفم الستقصاء تأثير المتغيرات المستقلة على السلوك lphnsيهدف هذا البحث إلى إعداد فلوديبين . من مكونات بوليمرية ودهون يف تدخل جميع الصيغ في عملية التوص. بنجاح( lphns h1-h9)تم استخدام تقنية الميكروويف لتحضير فلوديبين. الريولوجي للنظام النانوي للتأكد من الخصائص الغروانية للنظام النانوي المحضر ثم استخدام مقياس االنسياب الرقمي الدوراني المحوري لتقييم pdiلحجم الجسيمات و . نانويلها بنية نانوية ومتجانسة تؤكد السمات الغروانية لنظام التشتت ال( lphns h1-h9)تظهر النتائج أن جميع تركيبات فلوديبين . االنسيابية الذي له ( التدفق غير النيوتوني)تظهر تدفق البالستيك الكاذب ( (lphns h1-h9يشير مخطط الرسم البياني إلى أن جميع تركيبات فلوديبين ًزا ، التي تُظهر نسيًجا فيزيائًيا ممتا( (lphns h1-h9تقوم الطريقة المعتمدة على الميكروويف بإعداد تركيبات فلوديبين . خاصية ترقق القص تدفق البالستيك الكاذب الذي يدعم ( lphns h1-h9)تُظهر جميع تركيبات الفلوديبين . وهذا يؤكد قدرتها كأسلوب لتحضير الجسيمات النانوية .االستقرار المادي للنظام النانوي .طريقة الميكروويفالريولوجيا ، الفلوديبين ، الناقالت النانوية الهجينة للبوليمر الدهني ، : الكلمات المفتاحية introduction rheology is an important physical characteristic in pharmaceutical design and manufacture. creaming, flocculation, and coalescence are responsible for the instability of the colloidal dispersion system. the rheological behavior among factors that are present in stokes law can be used to control the stability of the colloidal dispersion system(1). therefore, the viscosity of colloidal systems is of great importance in scientific pharmaceutical research (1). the coaxial rotational rheometer, also known as couette or concentric cylinder viscometers, is the most common machine used for measuring viscosity. a circular bob inserted concentrically inside a cup contains the tested colloidal dispersion. the process of measurement occurs when fluid will drag on the bob as a result of bob or cup rotation, and a sensor in the machine will record the viscosity at a specific shear rate and shear stress. 1corresponding author e-mail: deera2020@gmail.com received: 22/8/2021 accepted: 6/10 /2021 published online first: 2022-1-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30isssuppl.pp9-15 iraqi j pharm sci, vol.30(suppl.) 2021 rheological investigation of lipid polymer hybrid nano-carriers 10 several readings can be obtained by changing revolutions per minute (rpm). these data can be analyzed to determine the rheological properties of pharmaceutical liquids(2-4).the felodipine lipid/polymer hybrid nanocarriers (lphns) is an interesting colloidal dispersion system that is used as an oral drug delivery system for rheological characteristic analysis(5-7). the lphns compose of polymeric components and lipid matrices that constitute monolithic type colloidal nanocarriers. the felodipine lphns have properties of both polymer nanocarriers and lipid-based nanocarriers constructed from it(8). the polymer content makes felodipine lphns provide more control on felodipine release. whereas, the lipid content enhances felodipine solubility, the biological tissue penetration, and improve absorption in addition to felodipine entrapment efficiency will increase in comparison to lipid based nanoparticles and polymeric nanoparticles alone(9). the process of hybridization between polymer and lipid gives nanoparticles that show: nanoscale particle size, higher drug payload, furiousness, provide sustained drug delivery and more stability in blood circulation and on long term storage of the formulation(10). however, the methods to prepare the lphns in these studies were associated with several limitations such as cost, lower stability and time consuming. recently, a microwave-based method has been used to prepare the lphns(11,12). these reports indicated that the microwave-based method have many merits such as rapid processing, can achieve in both small and large scale, inexpensive, more stable, economical, and absence of impurities(11,12). this research aims to prepare oral felodipine lphns to investigate the effect of independent variables which are lipid content, distilled water content, and surface-active agents:cosurfactant blend on the rheological behavior of the nanosystem. materials and methods materials the lauric acid, felodipine, polysorbate 80, propylene glycol were purchased from nanjing duly biotech co., ltd . china. the peg laurate was purchased from beijing yibai biotechnology co., ltd. china. the cardamom oil was purchased from hemani international kepz, karachi, pakistan. methods preparation of felodipine lphns nine felodipine lphns formulations (h1h9) were prepared using the microwave-based method that was previously described by drais h k and hussein a a(11,12). the hydrophobic mixture was prepared by dissolving chitosan polymer, felodipine, and lauric acid in a blend of cardamom oil and peg-laurate using a magnetic stirrer device at 1000 rpm for 5 minutes. the hydrophilic mixture that contains distilled water, propylene glycol, and polyoxyethylene (20) sorbitan monooleate was prepared under a magnetic stirrer at 1000 rpm for 5 minutes. the final mixture that contains hydrophobic and hydrophilic phases according to the concentrations of each ingredient as shown in table 1, was placed in microwave instrument for less than 15 seconds, then a mixture was subjected to magnetic stirring of 1000 rpm to form solution of the colloidal structure of felodipine lphns. the felodipine lphns formulations were stored in a tightly closed container at 25 oc temperature until the time of the rheological evaluation (11,12). a coaxial rotational digital rheometer (biobase meihua trading co., ltd, china) was used to measure the viscosity of the prepared nanodispersions. the spindle number (1) was used at 25°c. the felodipine lphns formulations were exposed to various rotational velocities (0.1, 0.3, 0.6,1.5, 3, 6, 12, 30, and 60 rpm). the experimental data were collected to analyze the rheological properties of the prepared nanosystems(2,11,12). table 1. the quantitative components of felodipine lphns formulations (h1-h9) for characterization and optimization formulation code felodipine % (w/w) cardamon oil % (w/w) lauric acid % (w/w) chitosan % (w/w) peg-(400) laurate :polysorbate 80: propylene glycol % (w/w) distilled water % (w/w) up to h1 1 4 1 0.1 15:7.5:7.5 100 h2 1 4 1 0.1 17.5:8.75:8.75 100 h3 1 8 2 0.2 17.5:8.75:8.75 100 h4 1 4 1 0.2 20:10:10 100 h5 1 8 2 0.25 20:10:10 100 h6 1 12 3 0.35 20:10:10 100 h7 1 4 1 0.2 22.5:11.25:11.25 100 h8 1 8 2 0.35 22.5:11.25:11.25 100 h9 1 12 3 0.4 22.5:11.25:11.25 100 iraqi j pharm sci, vol.30(suppl.) 2021 rheological investigation of lipid polymer hybrid nano-carriers 11 characterization of the felodipine lphns 1. measurement of particle size the particle size was determined by the nanoparticle analyzer model sz-100 nanopartica series instruments (horiba scientific company, japan). photon correlation spectroscopy (pcs) is a technique that has been used to determine the globular size of the nanosystem. the experiments were performed in triplicate (13,14). 2. measurement of polydispersity index (pdi) pdi is the uniformity parameter. pdi of the prepared nanosystems was measured by the dls (dynamic light scattering) technique. pcs is a technique that has been used to determine pdi. the higher the pdi value indicates the lower particle size uniformity. the measurement was performed in three trials(13-15). 3. atomic force microscopy (afm) the morphological attributes of felodipine lphns formulation can explain by afm angstrom advanced inc. aa3000 usa. the afm measurement was performed by using 1-3 drops of the felodipine lphns formulation onto a glass slide(13). statistical analysis the investigation data was presented as the average of three trials and ± sd (n=3). the microsoft office excel program was used to analyze the data. the analysis of variance (anova) was used where the level at (p<0.05) was kept as significant while the level (p<0.05) was kept as not significant(16). results and discussion particle size and pdi of felodipine lphns the felodipine lphns formulations (h1h9) were successfully prepared by the microwavebased technique according to specified component concentrations as shown in table 1. all formulations were visually clear indicating colloidal properties of the hybrid dispersion system. the results of dls revealed that the mean particle sizes of the felodipine lphns formulations were h1 (87 nm), h2 (70nm), h3 (146 nm), h4 (33 nm), h5 (94 nm), h6 (978 nm), h7 (49 nm), h8 (75 nm) and h9 (809nm).the outcomes show that all felodipine lphns formulations (h1-h9) had nanosize and ascertain colloidal features of the nanodispersion system(17). according to the results, the analysis of variance show null hypothesis refusion that state the correlation between variables of investigation is no significant and accept the alternative hypothesis that state there is a significant correlation between variables of investigation (17). therefore, there is a significant relationship between surface-active agents: co-surfactant mixture, fat content, and distilled water as independent variables and particle size at level (p<0.05)(17). the outcomes of the characterization process for pdi as shown in table 2, were from 0.314 to 0.729. the pdi outcome indicated that felodipine lphns formulations (h1h9) are the homogenous structure that ascertains the stability of felodipine lphns formulations (h1-h9) against physical instability processes of the dispersion system(17). the anova shows a significant correlation between independent variables and pdi at level (p<0.05) (17). table 2. the particle size and pdi results of felodipine lphns formulations (h1-h9) code h1 h2 h3 h4 h5 h6 h7 h8 h9 globule size (nm)* 87.6± 2.26 70.1± 3.38 146.2± 10.095 33.3± 1.216 94.21± 1.587 978.4± 3.004 49.6± 0.953 75± 2.816 809± 2.26 pdi* 0.538± 0.003 0.401± 0.001 0.547± 0.050 0.314± 0.002 0.54± 0.078 0.729± 0.011 0.374± 0.003 0.335± 0.005 0.798± 0.002 *values are expressed as mean ± sd (n=3). rheological study the rheological experiment was performed successfully at different rotational velocities and rheological data were collected which are viscosity, rate of shear, and shear stress. the rheogram chart is obtained by the plot of the rate of shear against shear stress as shown in figure 1. the rheogram chart indicates that all of the felodipine lphns formulations (h1-h9) show pseudoplastic flow (non-newtonian flow) that have shear-thinning property. the pseudoplastic rheological flow improves the stability of felodipine lphns formulations against creaming, flocculation, and coalescence which are responsible for the instability of hybrid dispersion system. pseudoplastic flow behavior reduces the aggregation and settling rates of nanoparticles during a long period of pharmaceutical formulation storage also provide dose uniformity(1). the felodipine lphns formulation (h4) was the selected formula due to has lower particle size and pdi in addition to pseudoplastic flow, in comparison with other present formulations. the afm provides information for the size and shape of nanocarriers and apply for the felodipine lphns formulation (h4). the outcomes show that the morphology of https://www.horiba.com/uk/scientific/products/particle-characterization/particle-size-analysis/details/sz-100-7245/ https://www.horiba.com/uk/scientific/products/particle-characterization/particle-size-analysis/details/sz-100-7245/ iraqi j pharm sci, vol.30(suppl.) 2021 rheological investigation of lipid polymer hybrid nano-carriers 12 felodipine lphns (h4) was approached to spherical appearance with a smooth surface as shown in figure 2, this ascertain the colloidal attributes of optimized felodipine lphns formulation (h4) . figure 1. shear stress against the shear rate of felodipine lphns formulations (h1-h9). figure 2. the afm 3d image of felodipine lphns (h4) where the scanning area is 78 nm * 78 nm table 3. the shear stress results at different shear rates of felodipine lphns formulations (h1-h9) shear stress* shear rate h1 h2 h3 h4 h5 h6 h7 h8 h9 0.0529 506± 0.177 601± 0.645 659± 0.473 521± 0.408 544± 0.296 576± 0.433 506± 0.257 514± 0.295 574± 0.492 0.158 755± 0.961 780± 1.214 810± 0.9 702± 0.816 854± 0.79 877± 1.77 755± 1.237 737± 0.711 789± 0.869 0.317 882± 1.565 933± 1.865 987± 4.972 989± 3.232 1121± 1.731 1178± 3.612 814± 2.215 930± 5.058 987± 1.771 0.793 995± 3.636 1002± 2.776 1046± 8.305 1240± 4.36 1377± 4.43 1386± 5.96 1057± 13.32 1289± 4.017 1320± 5.426 1.587 1205± 8.859 1257± 6.82 1302± 18.39 1499± 8.035 1569± 12.791 1601± 10.865 1347± 13.051 1632± 12.155 1703± 16.322 3.174 1511± 8.22 1634± 25.887 1704± 14.6 1703± 17.986 1820± 18.144 1873± 31.697 1770± 21.542 1877± 22.405 1966± 13.117 6.348 1804± 37.725 2166± 71.064 2369± 37.521 2030± 33.856 2120± 33.737 2389± 34.94 2303± 39.973 2602± 32.793 2698± 35.241 15.871 2703± 102.66 2789± 45.1 2810± 97.328 2987± 118.47 2870± 225.14 2850± 148.59 2945± 77.03 2866± 217.86 2975± 99.141 31.742 3001± 117.80 3020± 124.694 3040± 158.99 3095± 69.18 3012± 82.2 3044± 153.50 3058± 120.10 3055± 107.05 3081± 80.744 *values are expressed as mean ± sd (n=3). table 4. the viscosity results at different shear rates of felodipine lphns formulations (h1-h9) viscosity* shear rate h1 h2 h3 h4 h5 h6 h7 h8 h9 0.0529 8733.45 ±3.347 11361.05 ±12.202 12457.46 ±8.947 9678.63 ±7.722 10283.55 ±5.595 10888.46 ±8.203 9565.21 ±4.863 9716.44 ±5.587 10850.6 6±9.303 0.158 4500 ±6.082 4936.7 ±7.692 5126.58 ±5.699 4443.03 ±5.17 5405.06 ±5 5550.63 ±11.212 4778.48 ±7.831 4664.55 ±4.5 4993.67 ±5.505 0.317 2782.33 ±4.936 2943.21 ±5.883 3113.56 ±15.6851 3119.87 ±10.195 3536.27 ±5.462 3716.08 ±11.397 2567.82 ±6.989 2933.75 ±15.958 3113.56 ±5.587 *values are expressed as mean ± sd (n=3). iraqi j pharm sci, vol.30(suppl.) 2021 rheological investigation of lipid polymer hybrid nano-carriers 13 continued table 4. viscosity* shear rate h1 h2 h3 h4 h5 h6 h7 h8 h9 0.793 1254.72 ±4.586 1263.55 ±3.501 1319.04 ±10.473 1563.68± 5.497 1736.44 ±5.587 1747.79 ±7.516 1332.91± 16.797 1625.47 ±5.065 1664.56 ±6.842 1.587 759.29 ±5.582 792.06 ±4.297 820.41 ±11.588 944.54 ±5.063 988.65 ±8.06 1008.82 ±6.846 848.77 ±8.224 1028.35 ±7.659 1073.09 ±10.285 3.174 476.05± 2.589 514.8±8 .155 536.86± 4.599 536.54±5 .666 573.4±5. 716 590.1±9 .986 557.65±6 .787 591.36± 7.058 619.4±4 .132 6.348 284.18± 5.942 341.2±1 1.194 373.18± 5.91 319.78±5 .333 333.96±5 .314 376.33± 5.504 362.79±6 .297 409.89± 5.165 425.01± 5.551 15.871 170.31± 6.469 175.72± 2.841 177.05± 6.132 188.2±7. 464 180.83±1 4.186 179.57± 9.362 185.55±4 .853 180.58± 13.726 187.44± 6.246 31.742 94.54±3 .711 95.14±2 .408 95.77± 5.008 97.5± 2.179 94.89± 2.589 95.89± 4.835 96.33± 3.783 96.244± 3.372 97.06± 2.543 *values are expressed as mean ± sd (n=3). 1. effect of lipid content on the viscosity of felodipine lphns formulations (h1-h9) it was found that the viscosity will increase as the concentration of lipid content (cardamom oil: lauric acid) increases at a constant concentration of peg laurate: polysorbate 80: propylene glycol. the viscosity values have the following ascending order for h7 < h8 < h9 at 5%(w/w) of lipid content; up to 50%(w/w) of distilled water for h7, 10%(w/w) of lipid content, up to 45%(w/w) of distilled water for h8 and 15%(w/w) of lipid content, up to 40%(w/w) of distilled water for h9, at constant concentration of surface-active agents:co-surfactant blend which is 22.5%(w/w) of peg-laurate and 11.25%(w/w) polysorbate 80 and 11.25%(w/w) of propylene glycol. the viscosity values have the following ascending order for h4 < h5 < h6 at 5%(w/w) of lipid content, up to 55%(w/w) of distilled water for h4, 10%(w/w) of lipid content, up to 50%(w/w) of distilled water for h5 and 15%(w/w) of lipid content, up to 45%(w/w) of distilled water for h6 , at constant concentration of surface-active agents:co-surfactant blend which is 20% (w/w) of peg-laurate and 10%(w/w) polysorbate 80 and 10%(w/w) of propylene glycol. the viscosity values have the following ascending order for h2 < h3 at 5%(w/w) of lipid content, up to 60%(w/w) of distilled water for h2 and 10%(w/w) of lipid content; up to 55%(w/w) of distilled water for h3, at a constant concentration of surface-active agents:co-surfactant blend which is 17.5%(w/w) of peg-laurate and 8.75%(w/w) polysorbate 80 and 8.75%(w/w) of propylene glycol. the h1 shows lower viscosity in comparison to other formulations due to low volume concentration of lipid content 5%(w/w) and higher concentration of continuous phase 65%(w/w) at lower concentration of surface-active agents:cosurfactant blend which is 15%(w/w) of peg-laurate and 7.5%(w/w) polysorbate 80 and 7.5%(w/w) of propylene glycol. the increment in lipid content concentration which is cardamom oil: lauric acid (4:1) increases the viscosity of felodipine lphns formulations at a constant concentration of surfaceactive agents:co-surfactant blend is due to increase in volume concentration of nanocarriers that make colloidal dispersion system more resistant to flow and give pseudoplastic system(1,18).the analysis of variance indicates a significant correlation between the lipid content and viscosity at level p > (0.05). 2. effect of distilled water content on the viscosity of felodipine lphns formulations (h1-h9) the distilled water constitutes the continuous or external phase of the colloidal dispersion systems (h1-h9). it was found that the viscosity will decrease as the distilled water increases at a constant concentration of peg laurate: polysorbate 80: propylene glycol. the viscosity values have the following descending order for h9 < h8 < h7 at 15%(w/w) of lipid content, up to 40%(w/w) of distilled water for h9, 10%(w/w) of lipid content, up to 45%(w/w) of distilled water for h8 and 5%(w/w) of lipid content, up to 50%(w/w) of distilled water for h7 , at constant concentration of surface-active agents:co-surfactant blend which is 22.5%(w/w) of peg-laurate and 11.25%(w/w) polysorbate 80 and 11.25%(w/w) of propylene glycol. iraqi j pharm sci, vol.30(suppl.) 2021 rheological investigation of lipid polymer hybrid nano-carriers 14 the viscosity values have the following descending order for h6 < h5 < h4 at15%(w/w) of lipid content; upto 45%(w/w) of distilled water for h6, 10%(w/w) of lipid content; upto 50%(w/w) of distilled water for h5 and 5%(w/w) of lipid content; upto 55%(w/w) of distilled water for h4 , at constant concentration of surface-active agents:co-surfactant blend which is 20% (w/w) of peg-laurate and 10%(w/w) polysorbate 80 and 10%(w/w) of propylene glycol. the viscosity values have the following descending order for h3 < h2 at 10%(w/w) of lipid content; upto 55%(w/w) of distilled water for h3 and 5%(w/w) of lipid content; upto 60%(w/w) of distilled water for h2, at a constant concentration of surface-active agents:cosurfactant blend which is 17.5%(w/w) of peglaurate and 8.75%(w/w) polysorbate 80 and 8.75%(w/w) of propylene glycol. the h1 shows lower viscosity in comparison with other formulations due to low volume concentration of lipid content 5%(w/w) and higher concentration of continuous phase 65%(w/w) at lower concentration of surface-active agents:cosurfactant blend which is 15%(w/w) of peg-laurate and 7.5%(w/w) polysorbate 80 and 7.5%(w/w) of propylene glycol. the reason for increment in viscosity as distilled water decrease is due to reducing the continuous phase volume that makes felodipine lphns closer to each other that are stabilized by steric forces due to the presence of the outer coat of peg(1,18). the analysis of variance indicates a significant correlation between the continuous phase and viscosity at level p > (0.05). 3. effect of surface-active agents:co-surfactant blend on the viscosity of felodipine lphns formulations (h1-h9) the surface-active agents:co-surfactant blend represent by peg-laurate, polysorbate 80 and propylene glycol. it was found that the viscosity will increase as the concentration of surface-active agents:co-surfactant blend increases at a constant concentration of lipid content. the viscosity values have the following ascending order for h1 < h2 < h4< h7 at 15%(w/w):7.5%(w/w): 7.5%(w/w) of peg-laurate:polysorbate 80 : propylene glycol respectively, upto 65%(w/w) of distilled water for h1. 17.5%(w/w):8.75%(w/w): 8.75%%(w/w) of peg-laurate:polysorbate 80 : propylene glycol respectively, upto 60%(w/w) of distilled water for h2, 20%(w/w):10%(w/w): 10%(w/w) of peglaurate:polysorbate 80 : propylene glycol respectively, upto 55%(w/w) of distilled water for h4. 22.5%(w/w):11.25%(w/w): 11.25%(w/w) of peg-laurate:polysorbate 80 : propylene glycol respectively, upto 50%(w/w) of distilled water for h4, at a constant concentration of lipid content which is 5%(w/w). the viscosity values have the following ascending order for h3 < h5 < h8 at 17.5%(w/w):8.75%(w/w): 8.75%%(w/w) of peg-laurate:polysorbate 80 : propylene glycol respectively, upto 55%(w/w) of distilled water for h3, 20%(w/w):10%(w/w): 10%(w/w) of peglaurate:polysorbate 80 : propylene glycol respectively, upto 50%(w/w) of distilled water for h5. 22.5%(w/w):11.25%(w/w): 11.25%(w/w) of peg-laurate:polysorbate 80 : propylene glycol respectively, upto 45%(w/w) of distilled water for h8, at a constant concentration of lipid content which is 10%(w/w). the viscosity values have the following ascending order for h6 < h9 at 20%(w/w):10%(w/w): 10%(w/w) of peglaurate:polysorbate 80 : propylene glycol respectively , upto 45%(w/w) of distilled water for h6, 22.5%(w/w):11.25%(w/w): 11.25%(w/w) of peg-laurate:polysorbate 80 : propylene glycol respectively, upto 40%(w/w) of distilled water for h8, at a constant concentration of lipid content which is 15%(w/w). the increment in surface-active agents:co-surfactant blend concentration increases the viscosity of felodipine lphns formulations(h1h9) at a constant lipid concentration is due to increase in volume concentration of nanoparticles that create dispersion medium more resistant to flow and improve medium viscosity(1,18). the analysis of variance indicates a significant correlation between the continuous phase and viscosity at level p > (0.05). conclusion the microwave-based method was successful in producing the felodipine lphns (h1h9) that show an excellent physical texture to reflect colloidal features of the hybrid nanosystem that make it the most advanced method for the preparation of nanoparticles. rheological attributes are the main physical characteristics in the pharmaceutical liquid dosage form. from precise viscosity analysis, the type of flow can be determined. the rheogram chart indicates that all of the felodipine lphns formulations (h1-h9) show pseudoplastic flow. the pseudoplastic rheological flow enhances the felodipine lphns formulation's stability against physical instability and provides felodipine dose uniformity. ethical approval the present work does not 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hussein a a. formulation and characterization of carvedilol nanoemulsion oral liquid dosage form. international journal of pharmacy and pharmaceutical sciences. 2015; 7 (12): 209-16. 16. z aigner, hb hassan, o berkesi, m kata, i eros. thermoanalytical, ftir, and x-ray studies of gemfibrozil-cyclodextrin complexes. journal of thermal analysis and calorimetry. 2005; 81(2): 267-72. 17. danaei m, dehghankhold m, ataei s, et al. impact of particle size and polydispersity index on the clinical applications of lipidic nanocarrier systems. pharmaceutics. 2018;10(57):1-17. 18. juntarasakul o, maneeintr k. evaluation of stability and viscosity measurement of emulsion from oil from production in the northern oilfield in thailand. earth environ. sci. 2018; 140:1-6. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 felodipine oral nanoemulsions doi: https://doi.org/10.31351/vol30iss1pp209-217 209 formulation and characterization of felodipine as an oral nanoemulsions sumaya b. hamed* and shaimaa n. abd alhammid** * ministry of health and environment, baghdad, iraq. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract felodipine is a calcium-channel blocker with low aqueous solubility and bioavailability. lipid dosage forms are attractive delivery systems for such hydrophobic drug molecules. nanoemulsion (ne) is one of the popular methods that has been used to solve the dispersibility problems of many drugs. felodipine was formulated as a ne utilizing oleic acid as an oil phase, tween 80 and tween 60 as surfactants and ethanol as a co-surfactant. eight formulas were prepared, and different tests were performed to ensure the stability of the nes, such as particle size, polydispersity index, zeta potential, dilution test, drug content, viscosity and in-vitro drug release. results of characterization showed that felodipine nanoemulsion (f3) with (oleic acid 10%) ,(smix 60% of tween80 :ethanol in a ratio of 3:1), (ddw 30%) was selected as the best formula, since it has a particle size of (17.01)nm, low pdi (0.392), zeta potential (-22.34mv), good dilution without drug precipitation , higher percent of drug content (99.098%) with acceptable viscosity , and complete release of the drug after (45 min.) with significantly higher (p<0.05) dissolution rate in comparison with the pure drug powder. the selected formula (f3) subjected to further investigations as drug and excipient compatibility study by fourier transform infrared spectroscopy (ftir) the outcomes of the (ftir) explain that the distinctive peaks for felodipine were not affected by other components and displayed the same functional group's band with very slight shifting. this indicates that there was no interaction between felodipine and other ne components. therefore, these excipients were found to be compatible with felodipine. in conclusion, the ne was found to be an efficient method to enhance the dispersibility and permeatioins of drugs that have poor water solubility (lipophilic drugs). keywords: nanoemulsion, felodipine, lipid dosage forms. صياغه وتوصيف الفيلودبين كمستحلب نانوي فموي **و شيماء نزار عبد الحميد 1*،حامد سمية بهجت . بغداد،العراق ،الصحة والبيئة ةوزار* . بغداد،العراق بغداد، جامعة الصيدلة، كلية ، الصيدالنيات فرع** الخالصه عقبة رئيسية تقيد استخدامها في اصبحتلعديد من المركبات الصيدالنية الفعالة لديها مشاكل في الذوبان حتى اآلن، والتي ان ا هي ان الصيغ الصيدالنية الدهنية منخفض جدا. هو مانع لقنوات الكالسيوم له ذوبان مائي وتوافر حيوي الفيلودبينان عقار. المستحضرات الصيدالنية الشائعة التي تم استخدامها لحل مشاكل الذوبان في المستحلب النانوي هو واحد من االنظمة وبان وذابة لمثل هذه الجزيئات القليلة الذج صيغ دوائية كخافض للسطح واإليثانول كمضاد للشد 60وتوين 80، توين االوليك اسد باستخدام كمستحلب نانوي الفيلودبين تم تصييغ لقد. العديد من األدوية قياس حجم القطيرات، مؤشر التشتت، المستحلب النانوي مثلتركيبات سائلة وأجريت لها اختبارات مختلفة لضمان ثبات ثمانيهتم تحضير .السطحي أظهرت نتائج التوصيف أن التركيبة رقم لقد. في المختبر ومستوى تحررالعقار مستوى اللزوجة تقدير محتوى العقار، جهد زيتا، اختبار التخفيف، نانومتر،( 17.01) لها حجم قطيرةصيغة كأفضل (60:30:10)بنسب oil: smix (3:1) ddw) والتي تحتوي الفيلودبينلمستحلب )3( ء من محتوى الدوا عاليةترسب الدواء عند اجراء اختبار التخفيف، نسبة مئوية ، عدم(mv -22.34) ، جهد زيتا0.392)مؤشر التشتت ) الصيغة .المقارنة مع مسحوق الدواء النقي عند( >p 0.05) واعلى بكثير دقيقة 45وتحرر العقار الكامل بعد مع لزوجة مقبولة (99.098%) حيث ( ftirبواسطة الفحص باألشعة تحت الحمراء ) المكونات االخرىدراسة التوافق مع مثل( قد خضعت لمزيد من االختبارات f3المفضلة ) عدم وجود النانوي والمكونات األخرى. مما يؤكد على لمستحلباوجود توافق بين ى أن القمم المميزة للـعقار لم تتأثر وهذا يشير إل (ftir)يوضح من النتائج التي تم الحصول لذلك تم التأكد من توافق العقار مع مكونات المستحلب النانوي االخرى . ,العقار ومكونات الصيغة المفضلة تفاعل بين ( وبهذا يمكن اعتباره طريقة جيدة لتحسين الذوبانية المائية لكثير فيلودبينالعليها تبين ان المحلول النانوي وسيلة فعالة لزيادة قابلية الذوبان لعقار ) من االدوية. مستحلب نانوي ، فيلودبين ، الصيغ الصيدالنية الدهنية .الكلمات المفتاحية : introduction biopharmaceutics classification system (bcs) divide the drugs into four categories (depending on drug solubility and permeability) as shown in table (1). table 1. biopharmaceutics classification system (bcs) of the drugs (1) class solubility permeability example class-i high high metoprolol class-ii low high felodipine class-iii high low captopril class-iv low low furosemid. 1corresponding author e-mail: sumayabahjat5@gmail.com received: 22/8/ 2020 accepted:28 /11 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp209-217 iraqi j pharm sci, vol.30(1) 2021 felodipine oral nanoemulsions 210 many drugs belong to class-ii group display low bioavailability due to poor solubility and insufficient dissolution process , to improve the bioavailability of drugs that belong to class ii and obtain a good clinical efficacy , the solubility of these drugs must be increased and the result improved dissolution process and this can be achieved by using several techniques that lead to enhancing the solubility of poorly water-soluble drugs which they are reduction of particle size, ph adjustment, solid dispersion , formation of salt and nanotechnology(2). nanoemulsion is defined as a novel and advance drug delivery system that has a great devotion to the delivery of drugs. nanoemulsions, also is known as submicron emulsions, are submicron sized colloidal particulate systems deliberated as thermodynamically and kinetically stable isotropic dispersions, which consist of two immiscible liquids like water and oil, stabilized by an interfacial film forming agent consisting of a suitable surfactant and co-surfactant to form a single phase. it leads to improve the solubility of poorly soluble drugs (lipophilic drugs) which results in an improvement of the bioavailability of these drugs (3). felodipine (fld) is a dihydropyridine calciumchannel blocker used in the treatment of elevated blood pressure and angina pectoris (m.w. 384.3 dalton, m.p. 145c°, pka 5.39, practically insoluble in aqueous medium, freely soluble in acetone, ethanol, methanol and in methylene chloride). fld is more selective vasodilator and have fewer cardiac action than non-dihydropyridine calciumantagonists. but this advantage is absent due to poor bioavailability of this medicament, which (although the drug is absorbed totally from the git) is simply 15% of the dose is available in blood circulation when it is administered orally. the low bioavailability of felodipine is attributed to its low aqueous solubility and also due to extreme first-pass metabolism (4). this study aimed to prepare and in vitro evaluate of felodine nanoemulsion in order to enhance dispersibility and dissolution rate of drug. materials and methods materials felodipine powder was purchased from baoji guokang biotechnology co.ltdtween 80 was purchased from riedel-de-haen, germany.tween 60 was provided from avonchem, england.dialysis membrane(12000 da) provided from schcuhardt , germany. all other chemicals and solvents were of analytical reagent grade. methods saturation solubility study of felodipine saturated solubility of felodipine was measured in various oils (triacetin, oleic acid, olive oil,corn oil, lavender oil, sunflower oil,lime oil,seassme oil), surfactants (tween 20, tween 60, tween 80) , co-surfactants (peg 400, ethanol and methanol) and dissolution media (0.1 n hcl containing 1% tween80) to ensure sink condition. the measurement of solubility was done as follow: excess amount of felodipine was added to (5ml) of each selected individual oils, surfactants and cosurfactants contained in stoppered vials separately, then was shaken using a water bath shaker for 72 hrs at 25±1°c for the oils, surfactant and co-surfactants and at 37±1°c for the dissolution media to prepare a saturated solution (5). after reaching equilibrium, the mixtures were centrifuged at 3000rpm for 15min, followed by filtration through a 0.45μm millipore filter, samples were suitably diluted with ethanol and analyzed by uv/vis spectrophotometer at λmax of felodipine and the measurements were done in triplicate. pseudo-ternary phase diagrams construction construction of the pseudo-ternary phase diagrams was done by using aqueous titration method. based on the solubility studies, oleic acid was selected as an oil phase, tween 80 and tween 60 were selected as surfactant and ethanol were selected as a co-surfactant, and deionized water (ddw) used as an aqueous phase. the oil: surfactant: co-surfactant (smix) mixed at different ratio. for each phase diagram, oil and smix (at a specific ratios) were mixed gradually at different ratios (ranging between 1:9 to 9:1) in different glass vials (6). smix ratios was 1:3, 1:2, 1:1, 2:1and 3:1 for smix (tween 80/ethanol) and 1:2, 2:1 and 3:1 for smix tween 60/ethanol. preparation of felodipine nanoemulsion different o/w nano emulsion formulations (table 2) were prepared using the smix and oil ratios according to pseudo-ternary phase diagrams. the preparation of primary felodipine nanoemulsion occur through dissolving (5 mg) of the drug in the selected oil, then magnetic stirrer was used then the selected smix added slowly in a fixed proportion until clear solution was gained followed by the addition of deionized distal water dropwise to the clear solution with continuous stirring ((~500 rpm) at room temperature till formation of clear emulsion. the prepared emulsions then were ultrasonicated using a 20 khz sonicator for 10 min to produce very small droplet size nes. (7). iraqi j pharm sci, vol.30(1) 2021 felodipine oral nanoemulsions 211 table 2. composition of different felodipine nanoemulsion ne-f felodipin w/w oleic acid %w/w surfactant cosurfactant smix ratio smix %w/w ddw% w/w f1 0.05w/w 10% tween80 ethanol 1:1 60 30 f2 0.05w/w 10% tween80 ethanol 2:1 60 30 f3 0.05w/w 10% tween80 ethanol 3:1 60 30 f4 0.05w/w 10% tween80 ethanol 1:2 60 30 f5 0.05w/w 10% tween80 ethanol 1:3 60 30 f6 0.05w/w 10% tween 60 ethanol 1:2 60 30 f7 0.05w/w 10% tween 60 ethanol 2:1 60 30 f8 0.05w/w 10% tween 60 ethanol 3:1 60 30 characterization of the prepared nanoemulsion: droplet size and polydispersity index (pdi) the droplet size of ne was determined by analyzing the fluctuations in light scattering due to the brownian motion of the particle using the dynamic light scattering technique (zetasizer nano) nanoemulsion was diluted with distilled water (100fold) and gently stirred (to increase the homogeneity) before measurement (8). while the measurement of (pdi) gives information about the uniformity of droplet size within the formulated ne. the lower pdi value (near zero) indicates a monodisperse droplet population, whereas a pdi value closer to 1 indicates a wide range of droplet size (9). transmittance percentage (%t) the translucence of the prepared nanoemulsions was checked by the turbidity test. by taking 2 ml of each nanoemulsion formula and measuring absorbance at 650 nm (light wavelength) using uv/vis spectrophotometer and distilled water was used as a blank (10). dilution test aqueous dilution test was done, 1 ml of each nanoemulsion formula from (f1-f8) diluted to 50 ml, 100 ml and 500 ml with distilled water at 37° c with constant stirring and was maintained at 50 rpm. turbidity, clarity and the phase separation for each formula was observed visually (11). drug content estimation accurately, 10 ml of each ne formula which contains (5mg) of felodipine was dissolved in 100 ml ethanol, then filtered using 0.45 μm filter syringe and suitably diluted. the contents of felodipine was determined using uv/vis spectrophotometer at the selected λ max (12). zeta potential measurement (ʓ – potential) the droplet charge (zeta potential) of the selected ne formula was determined by using a dynamic light scattering technique (zetasizer nano zs) (13). viscosity measurement viscosity is very important for stability and efficient drug release. nanoemulsion carrier formulations are basically oil-in-water and so in addition to being less greasy than water-in-oil formulations, often possess lower apparent viscosities. they are therefore expected to exhibit faster release of active ingredients (14). the low viscosity of systems shows that it is o/w type and high viscosity shows that it is w/o type system. measurements were performed using viscometer spindle number 2 that was immersed in 100ml sample of each prepared ne formulas and rotated at different speeds (15). in vitro drug dissolution study the in vitro release of felodipine loaded ne occur using usp dissolution apparatus type – ii (paddle method). ten ml of each formula which contains (5mg) of felodipine was poured in the dialysis bag (molecular cut off 12000da), then this bag immersed in 500 ml of dissolution medium. the dissolution medium was (0.1n hcl with 1% tween 80), the dissolution apparatus set at 37 ± 0.5 °c, and the rotation speed was 50 rpm (16). nanoemulsion containing felodipine equivalent to one dose(10ml) was placed in a dialysis bag, and five ml of dissolution medium was withdrawn at 5, 10, 15, 30, 45, 60, 90 and 120 min time intervals and the samples then filtered using a 0.45 μm filter syringe and analyzed by uv/vis spectrophotometer at the λ max of the drug the study was done in triplicate (17). selection of the optimum formula the choice of the optimum formula was accomplished, and this achieved according to the dropletsize, pdi, transmittance percentage, dilution test, drug content, viscosity, and in vitro release studies. evaluation of the selected felodipine optimum formula drug and excipient compatibility study by ftir fourier transform infrared spectroscopy (ftir) to investigate any possible interaction between the drug and the utilized excipients (oleic acid, tween 80, ethanol) in the selected formula. pure drug was mixed with potassium bromide and pressed in a form of a disc. oleic acid, tween 80, ethanol and the selected formula (liquid samples) were analyzed by an ftir device for liquid samples ftir spectroscopy analyzed all the samples from 4000-400 cm-1 (18). iraqi j pharm sci, vol.30(1) 2021 felodipine oral nanoemulsions 212 results and discussion saturation solubility study of felodipine the solubility of felodipine as shown in table below was higher in oleic acid so that oleic acid was used in the formulations to keep the drug in solubilized form, and no precipitation of drug will occur (19,20). regarding surfactants, tween 80 and tween 60 were selected as a surfactant to obtain a one-phase clear solution. considering cosurfactants, ethanol was found to have the higher solubilizing capacity for felodipine,it would increase the miscibility of the aqueous and oily phases due to its partitioning between these phases to reduce the interfacial tension also increase the mobility of the hydrocarbon tail and allow greater penetration of the oil into this region (21) . table 3.saturation solubility study of felodipine in different oils, surfactants, cosurfactants and dissolution media. oil solubility(mg/ml) mean ±sd* sesame oil 27.01633±1.8029 triacetin 29.4836±0.6467 oleic acid 49.733±0.6976 lime oil 4.1796±0.1067 lavender oil 3.78566±0.0784 olive oil 17.35367±1.9459 sun flower oil 4.54633±0.3408 surfactant solubility(mg/ml) mean ±sd* tween 60 36.89827±0.4072 tween 80 47.06033±0.4776 tween 20 27.119±0.3142 co surfactant solubility(mg/ml) mean ±sd* ethanol 48.80167±0.4834 methanol 32.665±0.23926 peg 400 5.071333±0.0475 dissolution media solubility(mg/ml) mean ±sd* 0.1 n hcl (with 1% tween 80) 32.9 ±0.692 *sd standard deviation from the mean, n=3 construction of pseudo-ternary phase diagrams figures 1and 2 showed the pseudo-ternary phase diagram for the o/w nes using oleic acid as an oil phase, tween 80 and tween60 as a surfactant and ethanol as a co-surfactant . figure (1and 2). pseudo-ternary phase diagrams showing the (o/w) nanoemulsion (colored area) regions of oleic acid at different smix ratios. characterization of the prepared nanoemulsions: droplet size and polydispersity index (pdi) table (4) showed the results of droplet size measurement and poly dispersity index. also, in regard to particle size, the results showed that when the concentration of surfactant increased the particles size reduced, since this high surfactant concentration decreases surface tension and stabilizes newly developed surfaces during homogenization and production of smaller particles , these results may be also due to accumulation of surfactant molecules at the interface provides better stabilization against droplet aggregation and helps in lowering the flocculation rate, as well as greater penetration of the oil phase in the hydrophobic iraqi j pharm sci, vol.30(1) 2021 felodipine oral nanoemulsions 213 region of the surfactant, lead to reduction the droplet size ( 22). while the pdi refers to the quality of a polydispersity index and it is not stable. the low value of pdi (0.080.7) is considered to be desirable for uniform distribution, stability and high of the dispersion (23). the higher the value of pdi (>0.7) indicate that the sample has a very broad particle size distribution quality and homogeneity of nanosized droplets within the preparation (24). table 4. particle size and poly pisprersity index of the ne formulas. f code particle size (nm) f code pdi f1 197.6 nm f1 0.298 f2 48.88 nm f2 0.421 f3 17.01 nm f3 0.392 f4 47.40 nm f4 0.395 f5 27.44 nm f5 0.393 f6 31.74 nm f6 0.368 f7 19.70 nm f7 0.367 f8 18.75 nm f8 0.366 figure 3.particle size distribution of felodipine nanoemulsions. transmittance percentage (%t) transmittance percentage of felodioine nanoemulsion formulas demonstrates that all these formulas were translucent, clear and convey the light easily since the values of percentage transmittance closer to 100 % since the reducing droplet sizes to the nanoscale was lead to higher transparency (25(. table 5. percentage of transmittance (%t) of felodipine nanoemulsion f code absorbance %transmittance f1 0.0031 92.6616 f2 0.0244 94.5366 f3 0.0037 99.15166 f4 0.0211 95.2576 f5 0.0107 97.5663 f6 0.0104 97.6337 f7 0.0042 99.0375 f8 0.0116 97.364 dilution test all nanoemulsion formulas (f1-f8) showed fine bluish to clear nanoemulsion indicating o/w type, proved that they could be diluted in gi fluids and maintaining the nanosized character without drug precipitation. thus, it is anticipated that absorption will be enhanced (26). drug content results shown in table (6) indicated that all nanoemulsion formulas agreed with the requirements of the british pharmacopeia range (87.2 % 109.6 %) indicating that, there was no precipitation of drug in any of prepared formulations iraqi j pharm sci, vol.30(1) 2021 felodipine oral nanoemulsions 214 table 6. drug content percentage of the prepared nanoemulsions f code % drug content f1 93.539 f2 94.224 f3 99.098 f4 96.28 f5 95.252 f6 93.881 f7 95.937 f8 95.595 f9 98.336 zeta potential measurement zeta potential values of merely 30 mv or much lower can supply enough stabilization (27) and the zeta potential value of the selected formula as shown in figure (4) was found to be (-22.34), which would be increase the stability of the nanoemulsion as the individual droplets repels each another in order not to coalescence into larger globules. figure 4. zeta potential of formula (f3) viscosity measurements from the figure (5), it was demonstrated as the concentration of the surfactant increased; the viscosity increased this may be due to entrapping of the water molecules in cross-linking surfactants chains and also highest surfactant concentration would make the dispersion medium more rigid (28). the results also showed that the viscosity decreased as the rotation speed increased (shear rate) indicating the pseudoplastic (shear thinning liquids) flow of the preparation (29). figure 5. viscosities data of prepared felodipine nanoemulsion formulas (f1, f2,f3,f4,f5,f6,f7,f8). in vitro drug dissolution study higher and faster the absorption, and hence quicker and greater the drug action can be obtained by smaller the particle size of a drug in the dosage forms (30). the release of felodipine from the formula that contain tween 80 as surfactant (f3) was higher than that contain tween 60 (f8) which could be explained by the smaller droplet size of formulas containing tween 80 as compared to that formulas which contain tween 60 leading to greater rate of dissolution. while the release of formulas that contain tween 80 (f2, f4) is greater than formulas contain tween 60 (f7, f6) due to the higher hlb value of tween 80 which is 15 enhanced the continuous distribution and solubilization of the incorporated lipophilic drug within the system. (31) as shown in figures 5,6. iraqi j pharm sci, vol.30(1) 2021 felodipine oral nanoemulsions 215 figure 6. a comparative dissolution profile of felodipine nanoemulsions (f1, f2,f3,f4 and pure felodipine) in 500ml of 0.1 n hcl (containing 1% tween 80) dissolution medium at 37°c. figure 7. a comparative dissolution profile of felodipine nanoemulsions (f5, f6,f7,f8 and pure felodipine) in 500ml of 0.1 n hcl (containing 1% tween 80) dissolution medium at 37 °c. fourier transform infrared spectroscopy (ftir) the spectrum of the selected formulas) f3) represented in figure (9) reveal presence of main peaks of drug which indicates that there is no considerable interaction between drug and excepeints during preparation of nanoemulsion. figure 8. the ftir spectrum of pure felodipine figure 9 .ftir spectrum of the selected formula (f3) conclusion all the nanoemulsion formulas prepared with oleic acid as an oil phase, tween 80,tween 60 as a surfactant ,ethanol as a co-surfactant provided a 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assessment of health beliefs among iraqi breast cancer patients in baghdad using either tamoxifen or trastuzumab samer imad mohammed* 1, aya thaer sabry*and dania thaer sabry* * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract breast cancer is the most diagnosed form of malignant tumor in iraqi women. tamoxifen and trastuzumab are highly effective adjuvant therapies for breast cancer. the objectives of this study were to define the patient's belief in tamoxifen or trastuzumab when used as adjuvant therapies and to determine the variation in beliefs between the two medications in a sample of iraqi breast cancer patients.the cross-sectional survey was conducted using the belief about the medication-specific (bmq-specific) questionnaire. ninety-seven participants (sixty-seven tamoxifen, thirty trastuzumab) participated in this study. the mean of specific-necessity scale for tamoxifen was (3.7) and for trastuzumab (4). the findings showed a high necessity for both medicines, and there were no significant differences in the scale of necessity between the two treatments. regarding the scale of concern, the trastuzumab group's values are higher (3.35) than tamoxifen (3). most participants who use tamoxifen or trastuzumab strongly agree with or agree with all the questions on the necessity scale with higher percentages for trastuzumab. besides, the concern scale results showed the percentage of patients who agree / strongly agree is higher in the trastuzumab group. for the correlation between the necessity or concern score, the result showed only one significant negative correlation (r=-0.366, pvalue=0.011) between the necessity score and the age of the participants for tamoxifen users. in conclusion, this survey clearly showed a high level of necessity and a high level of concern regarding the use of two effective adjuvant therapies for women with breast cancer, tamoxifen and trastuzumab. furthermore, this study shows that while the level of need for tamoxifen is higher than for trastuzumab, there were no significant variations between them. key words: breast cancer, tamoxifen, trastuzumab, beliefs, necessity, concern. تقييم المعتقدات الصحية بين مرضى سرطان الثدي العراقيين في بغداد المستخدمين لعقاري تاموكسيفين أو تراستوزوماب **دانية ثائر صبري و *اية ثائر صبري ،1،*سامر عماد محمد .بغداد، العراق بغداد،جامعة الصيدلة،كلية السريرية،* فرع الصيدلة العراق.،بغداد ، جامعة بغداد الصيدلة،كلية ، الكيمياء الصيدالنية فرع ** الخالصة سرطان الثدي هو الورم الخبيث األكثر تشخيصا لدى النساء العراقيات. ويعتبرعقار تاموكسيفين وتراستوزوماب عالج مساعد فعال المريض في عقاري تاموكسيفين أو تراستوزوماب عند استخدامه كعالج مساعد كانت أهداف هذه الدراسة هي تحديد اعتقاد .للغاية لسرطان الثدي تم إجراء المسح المقطعي باستخدام استبيان المعتقدات حول .وتحديد االختالف في االعتقاد بين الدواءين في عينة من مرضى سرطان الثدي العراقيين وتسعون مشارًكا )سبعة وستون يستخدمون تاموكسيفين ، وثالثون يستخدمون شارك في هذه الدراسة سبعة .(bmq-specific) الدواء المحدد (. أظهرت النتائج ضرورة قصوى لكال الدوائين ، 4( وللتراستوزوماب )3.7كان متوسط مقياس الضرورة المحددة لتاموكسيفين )و تراستوزوماب( يما يتعلق بمقياس القلق ، فإن قيم مجموعة التراستوزوماب كانت أعلى وال توجد فروق ذات داللة إحصائية في مقياس الضرورة بين العالجين. ف الذين يستخدمون عقار تاموكسيفين أو تراستوزوماب بشدة مع جميع األسئلة المتعلقة ون(. يتفق معظم المشارك3وكسيفين )( من عقار تام3.35) المئوية للمرضى الذين يوافقون / يوافقون بشدة أعلى في مجموعة بمقياس الضرورة. إلى جانب ذلك ، أظهرت نتائج مقياس القلق أن النسبة (p-value = 0.011) ، ( r = -0.366) تراستوزوماب. بالنسبة للعالقة بين درجة الحاجة أو القلق ، أظهرت النتيجة ارتباًطا سلبيًا واحدًا فقط الختام ، أظهر هذا المسح بوضوح مستوى عاٍل من الضرورة ومستوى عاٍل ن درجة الضرورة وعمر المشاركين لمستخدمي عقار تاموكسيفين. في بي عالوة على من القلق فيما يتعلق باستخدام اثنين من العالجات المساعدة الفعالة للنساء المصابات بسرطان الثدي ، عقار تاموكسيفين وتراستوزوماب. ار تاموكسيفين أعلى منه في عقار تراستوزوماب ، لم تكن هناك اختالفات كبيرة ذلك ، تُظهر هذه الدراسة أنه في حين أن مستوى الحاجة إلى عق .بينهما القلق. ،الحاجة،المعتقدات، تراستوزوماب، تاموكسفين ،الكلمات المفتاحية: سرطان الثدي introduction breast cancer is one of the most prevalent cancers among women(1). breast cancer is also the most frequently diagnosed type of malignant tumor in iraqi women and the leading cause of death for women due to malignant neoplasm(2). moreover , metastasized breast cancer, despite extensive studies, remains an incurable condition with an estimated survival time of around two years(3). for breast cancer treatment; surgery, 1corresponding author e-mail: samer.jameel@copharm.uobaghdad.edu.iq received: 25/1/2021 accepted: 15/3 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp113-121 iraqi j pharm sci, vol.30(2) 2021 iraqi breast cancer patients' health beliefs 114 chemotherapy, adjuvant hormones, and radiotherapy may be used(4). in general, several variables , including the pathological features of breast cancer, the demographic and clinical characteristics of patients, and the chances of recurrence decide treatment choice (5,6). as a role, breast cancer therapies such as chemotherapy, radiation, or surgery are usually accompanied by hormonal therapy(7). adjuvant endocrine therapy is adequate and sufficient for almost any woman with positive breast cancer with estrogen receptors (er) and/or progesterone receptor(pr), rendering adjuvant endocrine therapy the most widely prescribed cancer therapy(8). tamoxifen, a selective estrogen receptor modulator (serm), was the standard adjuvant endocrine therapy that can be used for five years, according to the american society of clinical oncology clinical practice(8). furthermore, tamoxifen is used to treat both early and advanced estrogen receptor-positive (erpositive or er+) breast cancer in pre-and postmenopausal women(9). on the other hand, trastuzumab (herceptin; genentech, san francisco, ca) was a monoclonal antibody tested in human clinical trials as a therapy for metastatic breast cancer patients overexpress human epidermal growth factor receptor 2(her2)(10). a variety of studies have shown that additional administration of trastuzumab to adjuvant chemotherapy substantially reduces recurrence rates and that trastuzumab is tolerable for cardiac side effects(11–13). the administration of trastuzumab for one year in addition to adjuvant chemotherapy is standard practice nowadays(13). nevertheless, adjuvant endocrine therapies are noted to have a substantial side effect profile that may negatively impact the quality of life and have been cited as obstacles to enduring care(14). the clinical challenge of adjuvant drug therapy for metastasized breast cancer primarily pursues two goals: first, achieving high effectiveness in progression-free survival and avoiding therapyinduced and disease-related side effects at the same time is essential; second, preserving the best possible quality of life of the patient(15). furthermore, the patient beliefs concerning the use of adjuvant therapy and pain and treatment duration were the most salient obstacles for using these medications(16). adherence to oral endocrine therapy for preventing and treating breast cancer is one of the substantial challenges for health care practitioners(16). since tamoxifen or trastuzumab can only achieve therapeutic benefits when taken as prescribed, it is necessary to examine patient noncompliance with treatments(16). as well-known, patients with breast cancer who do not adhere to adjuvant therapy (i.e., take their medication as directed) and not persist (i.e., take their medication for the required duration) have worse prognoses(17). furthermore, studies of the relationship between demographic data (i.e., age and gender) and medications adherence have shown inconsistent results. however, poor adherence can be attributed to long treatment duration and lifestyle changes such as sleeping routines, social status, or mealtime (17,18). accordingly, it is crucial to study the patient's beliefs about their medication because there is a possible association between beliefs and adherence to adjuvant therapy for treating breast cancer(19). likewise, incorporating health beliefs into research and clinical practice can help to elucidate challenges the patients face while initiating and continuing use of adjuvant endocrine therapy(18). enhancing medication-based beliefs seems especially relevant to the strategy of patients' behavior modification to improve their medication adherence (20). in iraq, tamoxifen is used widely among iraqi patient as an adjuvant hormonal treatment option with er and/or pr positive tumor while trastuzumab, as a biological therapy, was suggested with her2 positive patients(21). the objectives of this study were to define the patient's belief in tamoxifen or trastuzumab when used as adjuvant therapies and to determine the variation in beliefs between the two medications in a sample of iraqi breast cancer patients. patient and method study design a cross-sectional survey was completed by using the bmq-specific questionnaire(22). the research assistant physician does data collection. the collection began in march 2018 and continued until august 2020. patients with breast cancer who have been attending an oncology consultation hospital in baghdad and using either tamoxifen or trastuzumab alone as an adjuvant therapy for a period of between one month and twelve months have been asked to join this study by a research assistant physician. the research assistant briefly clarified the research objectives to the patient and assessed whether the eligibility criteria had been met. if they agreed to participate in the study and since the authors used the english version of the questionnaire, the research assistant physician interpreted the questions to the arabic language with their choices and wrote down the patient choices in the questionnaire paper. the inclusion criteria include 1women with early or metastatic breast cancer treated with trastuzumab alone and not in combination with chemotherapy. iraqi j pharm sci, vol.30(2) 2021 iraqi breast cancer patients' health beliefs 115 2women with early estrogen receptor-positive (er-positive or er+), metastatic and adjuvant breast cancer, both pre-and postmenopausal. 3the treatment period should not be less than one month or longer than twelve months to prevent a disparity in the duration of treatment between the two medications.the total number of patients who met the eligibility requirements and asked to enter this study was 123, and only 97 (67 with tamoxifen and 30 with trastuzumab) were eligible to participate in this study. the response rate is 78.8%. the bmq-specific questionnaire: the bmaq-specific contains two 5-item factors: the specific-necessity measure assessing concerns about prescribed medications based on beliefs about the possibility of dependency and long-term toxicity, and the disturbing effects subscale assessing concerns about prescribed medications based on beliefs about the danger of dependence and long-term toxicity (specific-concerns)(23). respondents rate how much they agree or disagree with each argument using a 5-point likert scale from 1 = strongly disagree to 5 = strongly agree. in sum, the scores for both scales are added up to provide a total ranking. in general, the total point score reflects the score along with the necessity and concern scale from 5 to 25. stronger beliefs suggest higher test scores(23). data analysis the data were analyzed using ibm spss statistics version 26 (ibm corp., armonk, new york, usa) and graphpad prisma version 7.04. descriptive statistics including mean, standard deviation, frequency and percentage have been obtained for both continuous and categorical variables as appropriate. the associations of concern scores, necessity scores with the patient's age and duration of treatment were evaluated using bivariate correlations. two-tailed independent t-tests were used to identify whether there was a significant difference in beliefs between the two groups. pvalues equal or less than 0.05 were considered significant.ethical approval: permissions were obtained from the ethical and scientific committee in baghdad university – college of pharmacy. a verbal consent was taken from all the participants. the data collection questionnaire was anonymous, and data confidentiality was maintained throughout the study. results demographic data for all participants are presented in table 1 showing that for tamoxifen, the sample had a mean age of (53 ±13 years) compared to a mean age of (52 ±13 years) for trastuzumab. most of the participants were married (59.7% for tamoxifen, 70% for trastuzumab), while the remaining groups were either single or divorced/separated, where the latter had the lowest percentage (10.4 for tamoxifen, 6.6% for trastuzumab).regarding tamoxifen participants' education status: almost two-thirds of the participants graduated from high school (34.3%) or awarded the bachelor’s degree (34.3%). only (13.4%) had no schooling. for trastuzumab, one-third of the participants had earned the degree of bachelor whereas only (16.6%) had left full-time education. the participants took tamoxifen for a mean of (8.4±2.3 months) while the mean was (7.6±2.2 months) for trastuzumab. table 1. demographic data for the participants. parameter tamoxifen (n=67) trastuzumab (n=30) age (mean ±sd) 53±13 52±13 duration of treatment (months) (mean ±sd) 8.4±2.3 7.6±2.2 marital status no. (%) single 20 (29.8%) 6 (20%) married 40 (59.7%) 21 (70%) divorce/ widow 7 (10.4%) 2 (6.6%) education no. (%) illiterate 9 (13.4%) 5 (16.6%) primary school 12 (17.9%) 8 (26.6%) secondary school 23 (34.3%) 7 (23.3%) university 23 (34.3%) 10 (33.3%) the findings showed a high necessity for both medicines, as seen in table 2, and there were no significant differences in the scale of necessity between the two medicines. table 2. specific-necessity (bmq-sn) for both tamoxifen and trastuzumab. questions tamoxifen trastuzumab p-value my health, at present, depends on my medicines. 3.820 4.33 0.0876 my life would be impossible without my medicines 3.626 4.2 0.0960 without my medicines, i would be very ill 3.895 3.966 0.1240 my health in the future will depend on my medicines. 3.746 3.933 0.0699 my medicines protect me from becoming worse 3.626 3.733 0.8169 iraqi j pharm sci, vol.30(2) 2021 iraqi breast cancer patients' health beliefs 116 the mean of specific-necessity scale for tamoxifen was (3.7) and for trastuzumab (4) as observed in fig (1). while the mean of specificconcern scale for tamoxifen (3) and trastuzumab (3.35) as indicated in fig (2). figure 1. mean of specific-necessity for tamoxifen and trastuzumab. figure 2. mean of specific-concern for tamoxifen and trastuzumab. regarding the scale of concern, the values in the trastuzumab group are higher. although, except for one question, there were no significant differences between the two medicines as seen in table 3. table 3. specific concern (bmq-sc) for both tamoxifen and trastuzumab. questions tamoxifen trastuzumab p-value having to take medicines worries me. 3.119 3.4 0.4032 i sometimes worry about the long-term effects of my medicines 3.208 3.3 0.3818 my medicines are a mystery to me. 3.358 3.53 0.0421* my medicines disrupt my life. 2.805 3.2 0.2118 i sometimes worry about becoming too dependent on my medicines 2.805 3.33 0.3150 * significant difference between the tamoxifen and trastuzumab groups (p<0.05). table 4 indicates that most participants who use tamoxifen or trastuzumab strongly agree with or agree with all the questions on the scale of necessity. the percentage is higher in the trastuzumab group for all the questions. also, the concern scale results showed that the percentage of patients who agree / strongly agree is higher in the trastuzumab group, as seen in table 5. iraqi j pharm sci, vol.30(2) 2021 iraqi breast cancer patients' health beliefs 117 table 4. percentages of tamoxifen and trastuzumab users agreeing /strongly agreeing with beliefs about medicines questionnaire -necessity statements. necessity scale percentage agreeing or strongly agreeing for tamoxifen group (n = 67) percentage agreeing or strongly agreeing for trastuzumab group (n = 30) my health, at present, depends on my medicines 69% 93% my life would be impossible without my medicines 74% 80% without my medicines, i would become very ill 68% 73% my health in the future will depend on my medicines 64% 77% my medicines protect me from becoming worse 61% 63% table 5. percentages of tamoxifen and trastuzumab users agreeing /strongly agreeing with beliefs about medicines questionnaire -concern statements. concerns scale percentage agreeing or strongly agreeing for tamoxifen group (n = 67) percentage agreeing or strongly agreeing for trastuzumab group (n = 30) having to take medicines worries me 36% 50% i sometimes worry about the long-term effects of my medicines 49% 53% my medicines are a mystery to me 44% 48% my medicines disrupt my life 33% 50% i sometimes worry about becoming too dependent on my medicines 36% 53% regarding the association between the necessity or concern score for tamoxifen users, the result showed only one significant negative correlation (r=-0.366, p-value=0.011) between the necessity score and the age of the participants as shown in the table 6. the concern score for tamoxifen users showed a non-significant positive association with age of participants as shown in the table 6. regarding the association between the length of treatment of tamoxifen and the necessity or concern score, the results showed a non-significant negative correlation (table 6). the study also observed a non-significant positive correlation between the age of trastuzumab users and the requirement score, although the findings showed a non-significant negative correlation between age and concern score or length of treatment for both necessity and concern score (table 6). iraqi j pharm sci, vol.30(2) 2021 iraqi breast cancer patients' health beliefs 118 table 6. the correlation between age, duration of treatment and necessity or concern scale for both tamoxifen and trastuzumab. parameter tamoxifen trastuzumab r p-value r p-value age necessity -0.366* 0.011 0.144 0.449 concern 0.026 0.864 -0.029 0.879 duration of treatment necessity -0.110 0.465 -0.244 0.194 concern -0.077 0.609 -0.236 -0.236 * significant difference between the tamoxifen and trastuzumab groups (p<0.05). discussion it is crucial to improve the treatment of women provided with adjuvant therapy and find ways to prolong the length of therapy to reduce recurrence and mortality(20). compared to socio-demographic variables, patient beliefs about his medication were a powerful predictor of reported adherence(24). moreover, individuals' beliefs about their medicine are modifiable drivers of decision-making about treatment(25). from a current systematic review that looked at the impact of various health beliefs on drug adherence, patients' beliefs and their relationship to drug adherence appear to vary unpredictably across cultures and across populations(26) it is therefore important to examine iraqi patients' beliefs and assess the level of needs and concern for their treatment. the belief about medicine questionnaire (bmq) is a reliable test for measuring patients' beliefs in various populations(27). this study investigates the beliefs concerning tamoxifen and trastuzumab, which are two effective and widely used adjuvant therapies prescribed for breast cancer patients. according to a study conducted in iraq(21) investigating treatment options and follow-up among iraqi breast carcinoma patients, the findings regarding hormone therapy showed that tamoxifen was provided as an adjuvant treatment choice in (63.5%) of patients. in comparison, trastuzumab was recommended in (27.4%) of patients with her2-positive patients(21). the findings of the current study have shown that the participants are primarily between 40-60 years of age and that, in line with previous studies(21,28) conducted in iraq, which showed the peak age frequency for breast cancer patients is between 4060. while there was a higher value of necessity and concern in patients using tamoxifen, the outcome clearly showed a significant higher necessity values, which means the patients' needs for tamoxifen exceed their fear from taking this medicine. the findings are consistent with those of a previous study conducted by elizabeth et al.(29), which shows that patients with strong need for tamoxifen have good adherence. while it is in contrast with rachael et al., study that demonstrated a low necessity for tamoxifen(30). regarding trastuzumab, the value for necessity is additionally significantly higher than the concern value. this study was the first study that assesses the patient's beliefs about trastuzumab, so there were no prior results to compare with. the expected reasons for these results are the well-tolerated side effect profiles and the comorbidities which are also crucial in patient's acceptance for tamoxifen (31) and trastuzumab (32). regarding the difference in necessity score or concern score between tamoxifen and trastuzumab, the current study revealed that there was no significant variation between the two medications. this could be related to the patients' positive experiences with both medications; in addition, most of them were not exposed to an undesirable side effect. the positive experiences were reflected from the high level of necessity with both medications. another factor that may cause this increase in the necessity for both medications is the physicians' prolonged experience with these medications and their knowledge that both medications were approved to be essential to prevent cancer recurrence with a well tolerable history of safety and effectiveness in improving the survival(33). the physicians' confidence could be reflected as a positive overview in their patients as belief is a modifiable variable(34). moreover, both drugs are offered freely to the patients in iraq(21), which may be the reason why the necessity is higher than the concern values. patients' awareness about the side effect of their medication is another factor that can influence the patient response, which can affect the degree of the concern level. one question in concern part of the bmq-specific questionnaire revealed a significant difference in responses between patients: "my medicines are a mystery to me," where there was a higher level of concern about this question in the trastuzumab group. this can be explained by the insufficient information of the medication given by medical staff to the patient. iraqi j pharm sci, vol.30(2) 2021 iraqi breast cancer patients' health beliefs 119 patient's understanding of his drugs and their predicted adverse effects may be attributed to his belief as patients with negative medication beliefs may be vulnerable to misattributing symptoms and stopping medication afterwards(35). a low side-effect profile may explain the low level of concern plus no deleterious influence of trastuzumab(36) or tamoxifen(37) on the healthrelated quality of life has been reported. the only negative association was found between the necessity scale and the patients' age in the tamoxifen group. failure to find a positive or negative correlation might be related to the limited number of patients who participated in this study or short treatment duration. this study was the first study attempted to find a correlation between age, duration of treatment and scale of need or concern, so that no prior studies can be compared. understanding the reasons behind a varying degree of need and concern among patients is crucial in providing optimal health advice and guidance(38). limitations of this study include: firstly, the small number of participants which could be linked to the inclusion – exclusion criteria that excluded many participants such as those with longer medication use (more than one year) or those using combination therapy to prevent any bias that may influence beliefs. secondly, the use of the questionnaire english version since it is a validated version(23). thirdly, the biases that may arise during patient comprehension of the questions may account as another drawback. in addition, the authors did not discuss the quality of contact between the clinician and the patient, which could affect women's awareness, comprehension and beliefs about the two medications. conclusion this survey clearly showed a 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australia. patient prefer adherence [internet]. 2020 [cited 2021 jan 25];14:2163–73. available from: /pmc/ articles/ pmc7648560/ ?report =abstract baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(suppl.) 2022 cupressus sempervirens doi: https://doi.org/10.31351/vol31isssuppl.pp121-130 121 isolation and structural characterization of quercetin 3-o-rhamnoside and essential oil estimation from leaves of iraqi cupressus sempervirens l (conference paper )# amani amer tawfeeq*,1 and shatha h ali ** # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of pharmacognosy and medicinal plants, baghdad college of medical sciences, baghdad, iraq ** department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq abstract cupressus sempervirens l., cupressaceae, which is known as evergreen cypress, mediterranean cypress, and in arabic called “al -sarw. it is evergreen, has a medium-sized, and longevity, and is widely distributed over the world. the plant represents an important member of conifer plants which are characterized by aromatic leaves and cones. cupressus sempervirens have been ethnobotanicals as an antiseptic, relief of cough, astringent, antispasmodic, wound healing, and anti-inflammatory. the aims of this work are phytochemical analysis, isolation, and structural identification of quercetin 3-o-rhamnoside (quercitrin) and essential oil in iraqi c. sempervirens. isolation of quercitrin was performed using a semi preparative hplc from n.butanol fraction and extracted from cupressus sempervirens leaves using ultrasound probe extraction, the structural identification of isolated quercitrin done by tlc, hplc, uv spectrophotometry, ft-ir characterization according to a variety of frequency ranges, and lc-ms/ms revealed a molecular ion at 448 m/z and base peak m/z 301. furthermore, isolation of essential oil using hydro-distillation and estimated by gc-ms shows a good essential oil yield of 0.9% with an interesting concentration of alpha-pinene 44%, carene10%, cedrol 4.86%, and βmyrcene 3.67%. hence, the isolation of a new quercetin-glycoside and 0.9% essential oil yield from leaves of cupressus sempervirens species is considered an important valuable source of quercetin 3-o-rhamnoside and essential oil in the iraqi cypress plant and might prefer the agriculture of this plant and widespread it in different regions. keywords: cupressus, conifers, al sarw, mass, pinene, quercitrin, spectroscopy. الزيت العطريتقدير وquercetin 3-o-rhamnoside سيترينالكويرمركب وتشخيصعزل # ) بحث مؤتمر ( العراقينبات السرو من أوراق المعزولة **شذى حسين علي و 1*،اماني عامر توفيق 2022حزيران 3 – 2# المؤتمر العلمي العاشر لكلية الصيدلة، جامعة بغداد بغداد، بغداد، العراق الصيدلة، جامعهكلية الطبية، رع العقاقير والنباتات ف* بغداد، بغداد، العراق الصيدلة، جامعه ، كليةالسريرية المختبرية**فرع العلوم الخالصة منتشر ر ، ومعم دائم الخضرة ، متوسط الحجم. النبات السرو المتوسطي والسرو دائم الخضرة نبات السرو من عائلة السرويات وايضا معروف ب العطرية. والمخاريط باألوراق تتميز التي الصنوبرية النباتات في مهما عضوا النبات يمثل العالم. أنحاء جميع في واسع نطاق لديهعلى السرو نبات ي التحليل الكيميائي استخدامات نباتية عرقية كمطهر ، وتخفيف السعال ، وقابض ، ومضاد للتشنج ، والتئام الجروح ومضاد لاللتهابات. أهداف هذا العمل ه تم إجراء .المستزرع في العراق الوراق السروالزيت العطري وتحليل هوالكشف عن quercitrinاالكيميائي وتشخيص المركب المعزول النباتي والعزل تشخيص فوق الصوتية ، الموجات باستخدام جهاز نبات السرومن أوراق استخالصه الذي تم n-butanolمن جزء hplcبواسطة quercitrin عزل األيون الجزيئي عند الذي اظهر lc-ms/msتشخيص بواسطه، uv،ft-ir قياس الطيف الضوئي ، tlc, ،hplc بواسطهالمعزول المركب 448 m/z واعلى قمه m/z 301 . عالوة على ذلك ، عزل الزيت العطري باستخدام التقطير المائي وتقديره بواسطةgc-ms . تظهر النتائج عائدا جيدا عزل مركب جديد من ،لذا٪3.67 .٪ ، وميرسين 4.86٪ ، سيدرول 10٪ ، كارين 44 لماده ال باينينتركيز اعلى ٪ مع0.9من الزيت العطري quercetin-glycosideنبات السرو العراقي،وقد تفضل زراعة في مصدر مهم يعتبر من اوراق صنف نبات السرو,إنتاج الزيت العطري ٪ من0.9 و . شاره في مناطق مختلفةهذا النبات وانت التحليل الطيفي ،سيترينكوير، باينين، كتله ، نبات السرو، الصنوبريات ، يات: السروالكلمات المفتاحية introduction medicinal plants are considered an indigenous source of a wide variety of compounds possessing different therapeutic applications (1). sumerian and babylonian civilizations employed clay tablets to document the usage of medical plants in iraq for thousands of years, and they used a variety of treatment methods for medicinal plants (2). the conifers are woody plants formed from varieties of genera and species. the plants are generally characterized by needle-shaped leaves, single-veined, and consist of both female and male unisexual cones categorized with bract scales (3). the conifers consist of eight families, the most common families were cupressaceae, pinaceae, podocarpaceae, cephalotaxaceae, taxaceae, and phyllocladanecea (4). cupressus sempervirens l. (c.s) is an evergreen, aromatic, longevity, and widest extant tree among the cupressaceae family (5). it is usually known as evergreen cypress in different regions, while in arabic called "al -sarw" (6,7). 1corresponding author e-mail: amani.tawfiq1103@copharm.uobaghdad.edu.iq received:2 /7/2022 accepted: 1/11 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp121-130 iraqi j pharm sci, vol.31(suppl.) 2022 cupressus sempervirens 122 the origin of this plant is back to several ancient greek mythology legends according to a man named cypresses, the provence cypress was used as a coating for the wells and the cypress wood was considered as "dowry of the daughter"(8). the previous literature review indicates that various parts of cupressus around the world including wood, cones, leaves, stem and bark different ethnomedicinal applications and have been used as a topical to relieve muscle pain and rheumatoid arthritis, relief of gout, have an expectorant effect, relief whooping cough, asthma (9,10), bronchitis, diabetes and have a diuretic effect. furthermore, the essential oil of coniferous plants is used as an antiseptic for wound healing, as an anti-scarring, and astringent effect (11,12). the major chemotaxonomic secondary metabolites are polyphenols; flavonoids, bi-flavonoids, phenolic acids and terpenes, fatty acids, carbohydrates, and resins (13). amongst the polyphenols reported in the cupressus genus are flavonoid glycosides such as 3,5,7-trihydroxy-4-o-methoxy flavone, quercetin3-d-xyloside, and kaempferol-7 neo-hesperidin side (14,15). chemically, quercitrin is also known as quercitroside or quercetin o-glycoside; figure 1 has a potential role in biological activity such as antioxidant, control of cancer, relief of rheumatoid disorders antiangiogenic, hepatoprotective, and antiproliferative activities (16-18). in addition, these constituents were revealed to have acted as protection from skin ageing through the cytoprotective effect of this constituent on uvbinduced cell injury in humans (19). furthermore, essential oil (eo) is considered one of the major components in c.sempervirens and almost conifer plants. the chemical unit of monoterpenes containing ten carbon atoms with unsaturated carbon atoms (the building unit isoprene) joined head-to-tail and is considered important volatile oil, which is widely spread in medicinal plants with potential biological activity (20,21). moreover, pharmacologic activities of c.sempervirens includes; anti-inflammatory(22), antimicrobial, antiplatelet, and anticoagulant activity (23), and hepatoprotective activity(24). our work aimed to phytochemically investigate iraqi cupressus sempervirens leaves, isolation of quercitrin (quercetin 3-o-rhamnoside ), and structural identification by uv, ft-ir, and lc-ms/ms analysis moreover, gc-ms analysis of estimation of essential oil. figure 1. quercetin 3-o-rhamnoside (16) materials and methods plant material collection and authentication cupressus sempervirens l leaves were collected from baghdad in september 2020. prof. dr khansaa ghazi rasheed identified and authenticated this plant at the iraqi natural history research center and museum -plant and environment departmentbaghdad. leaves were cleaned, air-dried in shade, ground in an electrical grinder to a fine powder, and subsequently used for extraction and phytochemical analysis. equipment and chemicals a rotary evaporator was used in this work from bȕchi rotavapor r-205, swiss). all chemicals used in this study were of high purity the solvents and chemicals were used in analytical grade from bdh, ltd. poole, england, and the reference standard was purchased from chengdu biopurify phytochemicals, china (purity >97). silica gel gf254 tlc plates belonging to merck trademark ltd., india. extraction and isolation of essential oil (eo) the extraction and isolation of (eo) from the leaves of c. sempervirens were performed with the hydro distillation method using the clevenger apparatus. the procedure for the extraction of (e.o) was applied as follows: for every 200 grams of airdried leaves extracted with 1500l water in clevenger-apparatus for 4 -5 hours. a pale-yellow oil was isolated and then it was dried using anhydrous sodium sulfate. the storage was at about 4 °c in an air-tight and dark glass container until analysis by gas chromatography/mass spectrometry (gc/ms) (25). preparation of plant extract and standard ultrasound-assisted extraction (uae) is an efficient, one of the new non-conventional extraction methods, and a favoured technique to isolate phytochemicals from botanical sources. sonication realizes a complete extraction and enhanced extraction yields are obtained quickly (26). ultrasonic extraction was performed using a probe sonicator. 300 gm of powdered leaves of c. sempervirens l was defatted with hexane, then the iraqi j pharm sci, vol.31(suppl.) 2022 cupressus sempervirens 123 defatted leaves powder was extracted with 85 % methanol using ultrasound-assisted extraction (probe) with the following parameters;20 min, solid solvent ratio 1:8mg/ml, temperature 25 0c, and frequency 20 khz;), then concentrated under reduced pressure and suspended with water for fractionation method three times with 150 ml of different solvents polarity in ascending manner (chloroform, ethyl acetate, and n. butanol fraction). the n.butanol fraction was dried and subjected to chemical screening tests for the detection of flavonoids and flavonoid glycosides. on other hand, the target component is determined and isolated by semi-preparative hplc compared with standard. the sample to be analyzed by hplc was dissolved in methanol -hplc grade and filtered through a 0.45μm millipore membrane filter. furthermore, the standard solution; quercitrin was prepared by dissolving 1 mg /1 ml of methanol (analytical hplc grade) to compare with the sample to determine of isolated compound. preliminary phytochemical screening chemical screening tests were used for the detection of flavonoids and to reduce sugar in n. butanol fraction of leaves extract (27-29). alkaline reagent test the alkaline reagent was used for detection of flavonoids, 3ml of the extract was mixed with approximately 1.5 ml of alkaline reagent (5% koh), and the yellow color was considered an appositive indication of flavonoids, and when the addition of a few drops of dilute acid the solution became colourless. ferric chloride test phenolic groups were screened using 1% alcoholic ferric chloride added to 2ml of extract (1:1) the color change to deep green or deep blue color indicates the presence of the phenolic compound. reducing sugar test benedict's reagent was used to screen the reducing sugar in n. butanol fraction. approximately 2 ml of sample and 3 ml of reagent were added in the test tube and then the mixture was heated in a water bath for 10 minutes. the greenish brown precipitate according to the concentration of sugar was an indication of the presence of the reducing sugar. salkowski test salkowski test was used to detection of terpenoids in cupressus sempervirens leaves. 5ml of extracts were dissolved in 1.5 ml of chloroform then added 2 ml of concentrated h2so4 carefully. reddish brown color indicates a positive result. libermann-burchard test sterols and steroids were detected using acetic anhydride (2 ml) and 2ml of h2so4 was added in a test tube with 0.5 gm of extract. the appearance of green or blue color confirmed steroids in the sample. gas chromatography-mass spectrometer (gcms) conditions gc/ms -shimadzu -qp-2010 ultra with scan mode -acq was used at the university of basrah/ college of agriculture/ department food science, for this analysis and adjusted at these conditions: the capillary column that was used to separate constituents was (30 m× 0.25mm, with a thickness of 0.25µm) at a flow rate of 1.0 ml/min. in addition, the carrier gas that was used is helium, the split ratio was 2.0, the injector port was 250˚c, oven temperature: was from 80 º c for i min, then rise to 240 ºc at a rate of 10 ºc /min and the detector was 280º c. electronic impact mode (sei) was used as an ionization mode at 70e (30). structural identification was based on the comparison of component mass spectra with components in the nist mass spectral library. chromatographic analysis for an n. butanol fraction thin-layer chromatography (tlc) in this work, a qualitative analysis of the n. butanol fraction was performed using silica gel 60 f254 plates with 0.25mm thickness and activated at 110 °c (merck), using the mobile phase system etoac/ h2o/ hoac (glacial) (10:10:40) v/v/v (31). the target compound compared with the quercitrin standard was detected under uv light at 254 nm. semi preparative high-performance liquid chromatography in this work, n. butanol fraction of leaves extract was subjected to an analytical hplc system equipped with a fraction collector unit. hplc -uv system, shimadzu, 10av-lc, japan was carried out for separation and isolation of target compound (quercitrin) in n.butanol fraction compared with quercitrin standard the chromatographic separation done by using specific chromatographic conditions (32,33): hplc column: c18reverse phase, 3 nuclear µm particle size (50 x 2.0 mm). the mobile phase consisted of two solvents mixture in gradient mode; (a) 0.1% formic acid in water and (b) methanol with gradient elution program from 10% a to:95% a(v/v). the flow rate was 1 ml /min injection volume was 20 µl and the detection was at ƛ 280 nm -310 nm. iraqi j pharm sci, vol.31(suppl.) 2022 cupressus sempervirens 124 chromatographic analysis for isolated compound (quercitrin) analytical hplc and tlc for identification of isolated compound regardless of hplc, the purity and identification of the isolated compound were directly checked compared with the standard by using the same analytical hplc (without a fraction collector unit) with the observant of the same chromatogram conditions used in isolation of the quercitrin as mentioned above. tlc was considered a simple and also the rapid method used for further screening of purity and simple identification of isolated compound compared with quercitrin standard using the same mobile phase system that was used above; etoac/ h2o/ hoac (glacial) (10:10:40 v/v/v). characterizations for isolated compound (quercetin-3-o-rhamnoside) ultraviolet spectroscopy (uv) the ultra violet spectra analysis was done using a double beam shimadzu-spectrophotometer (uv-1700) at al-mustansirya university college of pharmacy. the isolated compound and quercetin 3-orhamnoside standard were dissolved in methanol and then analyzed between a range of 200 nm to 600 nm. fourier transforms infrared spectroscopy ft-ir spectroscopy was used for structural identification of isolated quercitroside (quercetin 3orhamnoside) and carried out at baghdad university-college of pharmacy. ftir spectra shimadzu were performed using the kbr disk method and the results were recorded in the range of 400–4000 cm-1. liquid chromatography /mass /mass spectrometry (lc/ms/ms) the mass spectrometric analysis was done using api (ion trap mass spectrometer) /3200/ lcms/ms system with ion spray ionization (esi) source ion spray, esi is more frequently used in flavonoid and flavonoid glycosides analysis (34). the liquid chromatographic separation of the target compound was carried out on a phenomenex gemini c18 (250 mm x 4.6 mm i.d.; 5 μm particle diameter), column temperature was kept at 250 c0, and 20µl was injected and a flow rate (fr) was 1 ml/min. full scan spectra were acquired in esi-ms spectra and were carried out in the positive /negative ionization mode. the mass range was measured, m/z 50-900m/z; drying nitrogen gas nebulizing n2 30 psi and the flow rate was 45 ml/min. ms/ms fragmentation experiments were performed on the selected precursor ion and the conditions were; ion spray voltage, 4500 v; data analysis (analyst 1,6.3 version –germany-darmstadt) was used to analyze the mass spectra. results and discussion in this work, the results of phytochemical screening of the c.sempervirens plant indicate the presence of flavonoids, phenolic groups, steroids, reducing sugar, and terpenoids (35). gc-ms analysis of essential oil (eo) of c.sempervirens the chemical components of the essential oil of c.sempervirens leave estimated in this study and identified by gc-ms analysis as in figure 2. in this work, the extraction yield of the eo from the leaves of iraqi cupressus sempervirens l. was 0.9%. table 1 shows the chemical components of the essential oil obtained by gc-ms analysis and revealed 50 compounds;46 compounds identified and 4 unidentified., the results show the essential oil (eo) contains monoterpenes(c10) and sesquiterpenes(c15) as the major constituents in the essential oil at different retention times (rt) and concentrations. hence, the reported previous studies, gcms analysis revealed the essential oil components of c.sempervirens leaves in different geographical regions such as in turkey;α-pinene was; 35.60 %, δ-2-carene concentration was 22.7, 14.9 %, transpino-carveol (5.22 %), α-phellandrene-8-ol percentage was 4.56 %, β-pinene (3.06 %), dlimonene(36); in iran different concentration in this plant that is identified in both parts leaves and fruits such as α-pinene (46.2, 59.2 %) highest one, while its isomer β-pinene was (3.3, 2.8 %), germacrene-d (6.3-2.1 %), in addition, myrcene percentage value was (4.6, 3.4 %), isoterpinolene (3.7, 3.2 %), sabinene (2.2, 2.0 %), limonene (2.8, 2.4 %) as cyclic hydrocarbon, and as ester was αterpenylacetate (2.6, 3.2 %) (37), and in north of tunisia were 𝛼-pinene 47.51%, 𝛿-3-carene7.40,% 𝛼-terpinyl acetate4.11%, 𝛽-caryophyllene, 4.53%, and 𝛼-cedrol4.99% (38). however, these different qualitative and quantitative of volatile oil composition belongs to many factors such as environment, soil composition, and plant organ (39). iraqi j pharm sci, vol.31(2) 2022 cupressus sempervirens 125 figure 2. gc-ms chromatogram of essential oil from iraqi c. sempervirens leaves table 1. essential oil composition from leaves of cupressus sempervirens l. peak no. r. time area% *si name 2 4.488 44.09 95 alpha.-pinene 3 4.752 1.44 93 camphene 5 5.459 7.49 96 thujene , sabinene 6 5.737 3.67 90 beta.-myrcene 7 5.939 10.01 95 3-carene 9 6.146 6.41 90 m-mentha-6,8-diene, (r)-(+)(+)-m-mentha-1(6),8-diene 10 6.437 0.89 95 gamma.-terpinene 11 6.757 2.63 94 (+)-4-carene 12 6.817 0.23 91 alpha.-terpinene 13 6.967 0.18 85 beta.-terpineol 14 7.283 0.41 86 alpha.-campholenal 16 7.519 0.48 90 l-trans-pinocarveol,l-pinocarveol 21 8.163 2.07 94 p-menth-1-en-4-ol, terpinen-4-ol 25 9.820 1.16 95 bornyl acetate 28 10.840 2.64 90 alpha.-terpineol acetate 29 11.464 0.16 91 beta.-elemene, iraqi j pharm sci, vol.31(2) 2022 cupressus sempervirens 126 continued table 1. peak no. r. time area% *si name 30 11.842 0.48 82 beta.-cedrene 31 11.911 1.03 93 caryophyllene 32 11.967 0.23 81 beta.-cedrene 33 12.126 0.40 93 thujopsene, sesquichamene 34 12.434 0.76 95 alpha.-caryophyllene 36 12.804 0.39 87 alpha.-cubebene 37 13.202 0.16 92 alpha.-curcumene 39 13.801 0.25 93 hedycaryol 43 14.646 4.86 92 cedrol 44 14.915 0.19 86 cubenol 46 15.21 0.24 87 beta.-selinenol si: similarity index separation and detection of quercitrin in an n. butanol fraction by tlc thin-layer chromatography was applied for the separation of components in n. butanol fraction and screening of quercitrin in the extract. the result under uv light at 254 nm shows the separation of different components of n. butanol extract (e), and one component shows identical rf values with quercitrin standard(q) at 0.8, as seen in figure 3. figure 3. tlc analysis of n. butanol extract (e)compared with quercitrin standard(q) isolation and identification of (target compound) quercitrin the results of the semi-preparative hplc for separation and isolation of chemical components in n. butanol fraction of c.sempervirens leaves was indicated at three peaks with different retention times and the concentration was at rt 6.44 for the target compound to be isolated compared with quercitrin standard as in figures 4 (a) and (c). after that, the analytical hplc was also performed for identification, and checking the purity of the isolated target compound (quercitrin) was achieved at rt 6.55 min as a single peak as in figures 4 :(b) and (c). figure 4. the hplc chromatogram (a): n. butanol fraction of c. sempervirens leaves; (b): isolated target compound (quercitrin ); (c): quercitrin standard. iraqi j pharm sci, vol.31(2) 2022 cupressus sempervirens 127 furthermore, a thin-layer chromatographic (tlc) method is considered simple and fast screening for separation and simple identification of organic compounds (40). the results revealed the purity of the isolated compound (one spot) and optimized compatibility with the standard at rf value 0.52 and detected under uv light at 245nm as shown in figure 5. figure 5. analytical tlc for identification of the isolated compound and checking the purity: (a)quercitrin standard; (b) the isolated compound. structural elucidations of quercetin-3-orhamnoside for structural characterization, figure 6 shows the uv spectra characteristic of the isolated compound with quercetin-3-o-rhamnoside (quercitrin) at the following spectra of uv max: λ 210, 250, and 350 nm. taken together, these data indicated the binding of quercetin to sugar molecules. the characteristics of ft-ir spectra were in the range of 400–4000 cm −1as seen in figure 7. the results show, that the band at 3160 cm−1and broadband range at 3414 -3340 cm 1 represented the oh stretching vibration of the phenolic o-h and alcoholic o-h due to the intramolecular hydrogen bonding. the strong band observed at 1651 cm 1 assigned to conjugated carbonyl c=o stretching vibration, a prominent peak at 1500 –1454cm 1 represents c=c of an aromatic ring; stretching vibration, the prominent peak at 1056 and 1288 cm -1 represent stretching v. of conjugated ether linkage. the bands near 802 cm -1 indicate the bending vibration of the ar– h group. however, the spectral patterns are in agreement with those data reported previously (41,42). figure 6. uvspectra of quercetin-3-o-rhamnoside compared with standard. figure 7. ftir spectra of the isolated quercetin-3-o-rhamnoside iraqi j pharm sci, vol.31(2) 2022 cupressus sempervirens 128 furthermore, for further characterization, full scan product ion liquid chromatography coupled with negative es ionization spectra was carried out of the isolated quercetin-3-orhamnoside. the scan mode for the identification of isolated compounds was mrm (multiple reaction monitoring). the retention time at 19.11 min and ms-ms of the isolated compound was compared with the standard in library data. figure 8 shows ms/ms spectra of the target compound showing different fragment ions at m/z 448 indicating a precursor ion [m+1] + and base peak at m/z 301 because of loss of c6h11o4 . that represents quercetin. in addition, the characteristic product ions at m/z 177 represent c6h11o4 .-ch2o, and at m/z 194 represent c6h11o4 . – co +h2o and at m/z 181 represent c6h11o5 . -oh (l-rhamnose h2o). however, all the fragmentation patterns are in agreement with those data reported previously (43). figure 8. lc/ms-ms spectra of isolated quercetin 3-o-rhamnoside as a final consideration, the above findings approved that the isolated compound from n. butanol fraction of iraqi c.sempervirens was quercetin 3orhamnoside . this compound was reported in previous studies in conifer plants and in different species of the genus cupressus such as; c. funebris and c. lusitanica, cupressus junebris l, cupressus glabra l., cupressus goveniana, and c. macrocarpa, while it was not detected previously in species c.sempervirens (44,45). however, an important flavonoid glycosyl; quercetin 3o rhamnoside was determined in this species (c.sempervirens )which is cultivated in iraq. conclusion the findings of this work evidenced a new quercetin-glycoside from the species cupressus sempervirens l. cultivated in iraq. quercetin 3-orhamnoside was isolated as a major compound in an n-butanol fraction extracted by ultrasonic extraction. moreover, the study also provides a good concentration of 0.9% essential oil yield. hence, the iraqi cypress plant might be considered a valuable source of quercetin 3-o-rhamnoside and essential oil. conflict of interest the authors declare no conflict of interest. acknowledgements we like to extend our thanks and sincere gratitude to the university of baghdad /college of pharmacy/department of pharmacognosy and medicinal plant for providing a facility to complete this work study. references 1. mujeeb f., p. bajpai, and n. pathak. phytoche mical evaluation, antimicrobial activity , and determination of bioactive components from leaves of aegle marmelos. biomed res. intl. 2 014; 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clinical isolates. antibiotics. 2021;10(8):890. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 protective effects of lutein on bone marrow doi: https://doi.org/10.31351/vol30iss1pp233-239 233 possible protective effects of lutein against ciprofloxacin induced bone marrow toxicity in rats alaa r. khudhair *, 1 and nada n. al-shawi* *department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract ciprofloxacin, which is a second generation of fluoroquinolone and one of the most effective and widely used drugs within fluoroquinolone. unfamiliar adverse effects of ciprofloxacin such as bone marrow (bm) suppression, thrombocytopenia, anemia, agranulocytosis, renal failure, and others observed. lutein, is a xanthophyll (an oxygenated carotenoid), was focused by most studies as it has a strong antioxidant activity in vitro; and also, it has been associated with reducing the risk of the age-related disorders. the current study was designed to describe the role of apoptosis through the measurement of bcl-2 associated x protein (bax) marker, as mechanisms of bone marrow toxicity induced by ciprofloxacin and to find whether lutein may have protective effects on ciprofloxacin-induced toxicity in bone marrow of rats. thirty six sprague-dawley rats were randomly divided into six groups (six animal each): groups ӏ (control), rats received single oral daily dose of liquid paraffin (4ml/kg) for 25 successive days by oral gavage; group ii, (ciprofloxacin-treated), received single oral daily dose of liquid paraffin (4ml/kg body weight/day) for 25 days, and subsequently received 500 mg/kg ciprofloxacin by oral gavage for the last 5 days; groups ӏӏӏ and ӏv, received oral dose of lutein (6mg/kg/day) and (24mg/kg/day), respectively by oral gavage for 25 successive days (lutein-treated); groups v and vӏ, received oral dose of lutein (6mg and 24mg /kg/day), respectively by oral gavage for 25 successive days, and subsequently received 500 mg/kg ciprofloxacin orally for the last 5 days (lutein+ ciprofloxacin). ciprofloxacin (group ii) caused significant (p<0.05) reduction in total rbcs counts and -wbcs, and significantly elevations (p<0.05) bcl-2 associated x protein (bax) in bone marrow (bm) tissues homogenates compared to control (group i) rats. rats that orally received lutein (groups ӏӏӏ and ӏv), each produced nonsignificant differences (p>0.05) in total -rbcs and -wbcs and also produced non-significant differences (p>0.05) in bax levels in bm tissues homogenates with respect to corresponding levels in group ι rats. orallyadministered lutein with ciprofloxacin (groups v and vӏ), resulted in significant elevation (p<0.05) of total rbcs and -wbcs, and significantly reduced (p<0.05) bcl-2 associated x protein (bax) in bone marrow (bm) tissues homogenates caused by ciprofloxacin compared to the corresponding levels in group of rats administered ciprofloxacin (group ii). results of the current research suggested that lutein may be a useful compound that alleviated ciprofloxacin-induced toxicity on bone marrow. keywords: lutein, ciprofloxacin, total rbcs count and total wbcs count. الوقائية المحتملة للوتين ضد سمية نخاع العظم التي يسببها السيبروفلوكساسين في الجرذانالتأثيرات و ندى ناجي الشاوي * 1*،االء راضي خضير فرع االدوية والسموم ، كلية الصيدلة، جامعة بغداد، بغداد،العراق* لخالصةا األدوية األكثر فعالية واألكثر استخداًما في الفلوروكينولون. اآلثار سيبروفلوكساسين ، وهو الجيل الثاني من الفلوروكينولون وواحد من اللوتين ، هو الضارة غير المألوفة للسيبروفلوكساسين مثل تثبيط نخاع العظام ، قلة الصفيحات ، فقر الدم ، ندرة المحببات ، الفشل الكلوي ، وغيرها. ات ألنه يحتوي على نشاط قوي مضاد لألكسدة في المختبر ؛ وأيًضا ، فقد ارتبط بتقليل زانثوفيل )كاروتينويد مؤكسج( ، ركزت عليه معظم الدراس المرتبط bcl-2 (bax) صممت الدراسة الحالية لوصف دور موت الخاليا المبرمج من خالل قياس مؤشر .مخاطر االضطرابات المرتبطة بالعمر لوكساسين وإليجاد ما إذا كان اللوتين قد يكون له تأثيرات وقائية على السمية التي يسببها ، كآليات لتسمم نخاع العظم الناجم عن سيبروف x بالبروتين السيبروفلوكساسين في نخاع عظم الجرذان. )مجموعة ӏقسمت عشوائيا إلى ست مجموعات )ستة حيوانات لكل مجموعة(: المجموعة sprague-dawleyستة وثالثون جرذان يوًما متتاليًا عن طريق الحقن الفموي. 25مل / كجم( لمدة 4التحكم( ، تلقت الجرذان جرعة يومية واحدة عن طريق الفم من البارافين السائل ) ن الجسم / يوم( مل / كجم من وز 4، )المعالجة بالسيبروفلوكساسين( ، تلقت جرعة يومية واحدة عن طريق الفم من البارافين السائل )ӏӏالمجموعة ӏvو ӏӏӏأيام ؛ المجموعات 5مجم / كجم من سيبروفلوكساسين عن طريق الحقن عن طريق الفم خالل آخر 500يوًما ، ثم تلقت بعد ذلك 25لمدة يوًما متتاليًا )المعالجة 25مجم / كجم / يوم( ، على التوالي عن طريق الحقن الفموي لمدة 24مجم / كجم / يوم( و ) 6، تلقت جرعة فموية من اللوتين ) 25مجم / كجم / يوم( ، على التوالي عن طريق الحقن الفموي لمدة 24مجم و 6، تلقيا جرعة فموية من اللوتين ) vӏو vباللوتين( ؛ المجموعتان وتين + سيبروفلوكساسين(. أيام )ل 5مجم / كجم من سيبروفلوكساسين عن طريق الفم خالل آخر 500يوًما متتالًيا ، وبعد ذلك تم تلقي 1corresponding author e-mail: alaa_z520@yahoo.com received: 3/9/2020 accepted:12 /12 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp233-239 mailto:alaa_z520@yahoo.com iraqi j pharm sci, vol.30(1) 2021 protective effects of lutein on bone marrow 234 ( في إجمالي عدد كرات الدم الحمراء و عدد كرات الدم p <0.05تسبب سيبروفلوكساسين )المجموعة الثانية( في انخفاض معنوي ) ( مقارنةً بالسيطرة ) bmفي متجانسات أنسجة نخاع العظام ) x المرتبط بالبروتين bcl-2 (bax) ( في p <0.05البيضاء ، وارتفاع ملحوظ ) ( في إجماليp> 0.05( ، أنتجت كل منها فروًقا غير معنوية )ӏvو ӏӏӏالمجموعة األولى( للفئران. الفئران التي تلقت لوتين فمويا )المجموعات في متجانسات أنسجة نخاع العظام bax( في مستويات p> 0.05اختالفات غير معنوية ) كرات الدم الحمراء و كرات الدم البيضاء وأنتجت أيًضا bm المجموعتان( مع ما يتعلق بالمستويات المقابلة في مجموعة السيطرة للجرذان. أدى تناول اللوتين الفموي مع سيبروفلوكساسينv وvӏ إلى ) bcl-2المرتبط بـ x( بروتين p <0.05عدد كرات الدم البيضاء ، وانخفاض ملحوظ ) ( في إجمالي كرات الدم الحمراء وp <0.05ارتفاع كبير ) (bax( في نخاع العظام )bm المتجانسة لألنسجة التي يسببها السيبروفلوكساسين مقارنة بالمستويات المقابلة في مجموعة الفئران المعطاة ) للسيبروفلوكساسين )المجموعة الثانية(. الحمراء وإجمالي عدد كرات الدم البيضاءلوتين ، سيبروفلوكساسين ، إجمالي عدد كرات الدم الكلمات المفتاحية : introduction the carboxylic acid derivation of fluoroquinolones is ciprofloxacin, which is a second generation of fluoroquinolone and one of the most effective and widely used drugs within fluoroquinolone (1). its mode of action is brought about by inhibiting topoisomerase ii (dna-gyrase) and topoisomerase iv enzymes that are essential for bacterial dna transcription, replication, repair, recombination, and strand supercoiling repair (2). treatment with fluoroquinolones, including ciprofloxacin was reported to cause severe liver failure, elevated intracranial pressure, central and peripheral neuropathy (3). furthermore, unfamiliar adverse effects of ciprofloxacin such as bone marrow (bm) suppression, thrombocytopenia, anemia, agranulocytosis (4). a possible molecular mechanism provided for adverse effects of the ciprofloxacin was mentioned by researchers to be provoked by the inhibition of mitochondrial topoisomerase ii (dna-gyrase) that can lead to impairment of mitochondrial dna transcription and replication, thus has an impact on the cellular differentiation and proliferation (5). hematopoiesis which is the biological process occurs in the bone marrow in which the hematopoietic stem cells (hsc) proliferated into blood cells (6). development of multicellular organism relies on the balance between the cell proliferation and the cell death (that apoptosis). however, the scenario of increasing apoptosis, in long term number of the proliferating cells is decreased and then unable to reach to the normal levels (7). bax is a pro-apoptotic protein of mitochondria, in the intrinsic pathway of apoptosis, that cause release of the cytochrome c, the endoribonuclease g, and other proteins of mitochondria through opening of the mitochondrial outer membrane (8); this release is mediated by the permeability transition pore of the mitochondria (9). this cytochrome release lead to the activation of the initiator caspase 9 and finally caspase 3 that cause apoptosis (8). the nutrients carotenoids are widely distributed in foods, especially in vegetables and fruit (10). carotenoids appear to have antioxidant activity (11). the beneficial effects of carotenoids were associated in many systemic diseases when consumed in large dietary intake (12) and with retinal protection against eye disorders and damage caused by phototoxic light (13). lutein was focused by most studies as it has a strong antioxidant activity in vitro; and also, it has been associated with reducing the risk of the age-related disorders (12). it is a xanthophyll (an oxygenated carotenoid), which in all mammalians including humans, that are unable to synthesize it but deriving it through their diet (14) by absorbing blue light, lutein appears yellow in low concentrations; while in high concentrations, it appears orange-red; moreover, in green vegetables, lutein accounts for about 48% of total xanthophylls (15). furthermore, lutein is absorbed through the gastrointestinal (gi) with fat and it can be transported by lipoproteins; where lutein is transported via apolipoprotein e, and its transference facilitated mostly (52%) via high density lipoproteins (hdls) and (22%) via low density lipoproteins (ldls) (16). objectives this study was designed to describe the role of apoptosis as mechanisms of bm toxicity induced by ciprofloxacin; and to explore the possible protective effects of highand lowdoses of lutein against ciprofloxacin-induced toxicity in bm of rats. materials and methods animals thirty-six adult sprague-dawley rats weighing 150-200g were used; the animals were taken from the animal house of the college of pharmacy / university of baghdad, under controlled and conventional laboratory conditions. rats were housed in cages of stainless steel, at (25°с), relative humidity and natural light/dark cycle. standard laboratory rodent tap water and chow were supplied ad libitum, and the animals adapted for a one-week period prior of the experiment. all animal procedures were approved by ethical committee of college of pharmacy/ university of baghdad. materials the pure powders of ciprofloxacin obtained from shaanxi yuantai biological technology co., ltd. china; and the pure powder of lutein was obtained from xi`an rongsheng biotechnology co., ltd. china. rat bcl-2 associated x protein, bax elisa kit was obtained from sunlong biotech co., ltd, china. iraqi j pharm sci, vol.30(1) 2021 protective effects of lutein on bone marrow 235 animals grouping and tested doses rats were allocated into six groups of six rats each as follows: •group ӏ (control): received single daily oral dose of liquid paraffin (4 ml/kg) for 25 consecutive days by oral gavage. this group served as control. •group ӏӏ (ciprofloxacin-treated): received single oral daily dose of liquid paraffin (4ml/kg body weight/day) for 25 days, and subsequently received 500 mg/kg ciprofloxacin by oral gavage for the last 5 days. •group іӏӏ (lutein-treated): received oral dose of lutein (6mg/kg/day) daily by oral gavage for 25 consecutive days. •group ӏv (lutein-treated): received oral dose of lutein (24mg/kg/day) daily by oral gavage for 25 consecutive days. •group v: received oral dose of lutein (6mg/kg/day) daily by oral gavage for 25 consecutive days, and subsequently received 500 mg/kg ciprofloxacin orally by oral gavage for the last 5 days. •group vӏ: received oral dose of lutein (24mg/kg/day) daily by oral gavage for 25 consecutive days, and subsequently received 500 mg/kg ciprofloxacin by oral route for the last 5 days. twenty-four hours after the end of treatment, all animals were euthanized by diethyl ether anesthesia and from each rat, blood sample were withdrawn from the carotid artery at the neck and collected for the hematological assessments [total -rbcs and -wbcs counts]. furthermore, bm were quickly excised, placed in chilled phosphate buffer solution (pbs) (ph 7.4) at 40 c, blotted with filter paper and weighed. for the preparation of 10% tissues homogenates, 9ml of pbs (ph 7.4) was added to 1gram of bm, then each tissue was homogenized by tissue homogenizer that set at 3 for 1 minute at 4 0c. all preparations were freshly prepared and kept frozen at (-18 c0) unless worked immediately for the measurement of bcl-2 associated x protein (bax) marker in bm tissue homogenates. bax determination in the bm tissue homogenate the principle of rat bcl-2 associated x protein, bax determination in this elisa kit (rat bcl-2 associated x protein, bax elisa kit was obtained from sunlong biotech co., ltd.) is that: this elisa kit uses sandwich-elisa as the method. the microelisa stripplate provided in this kit has been pre-coated with an antibody specific to bax. standards or samples are added to the appropriate microelisa stripplate wells and combined to the specific antibody. then a horseradish peroxidase (hrp) conjugated antibody specific for bax is added to each microelisa stripplate well and incubated. free components are washed away. the tmb substrate solution is added to each well. only those wells that contain bax and hrp conjugated bax antibody will appear blue in color and then turn yellow after the addition of the stop solution. the optical density (od) is measured spectrophotometrically at a wavelength of 450 nm. the od value is proportional to the concentration of bax. you can calculate the concentration of bax in the samples by comparing the od of the samples to the standard curve. statistical analysis data was expressed as the values of mean standard deviation (sd). the data were analyzed by utilizing computerized ibm spss statistics 23.0 program. the statistical significance of the differences among various groups is determined by one-way analysis of variance (anоva). the statistically significant differences were considered when p value less than 0.05 (p<0.05). results and discussion ciprofloxacin (group ii) caused significant (p<0.05) reduction in total rbcs counts (figure. 1), total wbcs counts (figure. 2) each compared to the corresponding levels in control (group i) rats; furthermore, there were significant (p<0.05) elevations in bax contents (figure. 3) in bm tissue homogenates compared to control (group i) rats. groups iii and iv rats that orally received lutein 6mg/kg and 24mg/kg, respectively each produced non-significant differences (p>0.05) in total -rbcs and -wbcs (fig 1 and 2, respectively) and also produced non-significant differences (p>0.05) in bax levels in bm tissues homogenates with respect to corresponding levels in group ι rats (figures 3). administration of lutein at a dose of 6mg/kg body weight, and 24mg/kg each in association with ciprofloxacin (groups v and vi) produced significant (p<0.05) elevation in total rbcs counts (figure. 1), total wbcs counts (figure. 2) each compared to blood counts in group ιι rats; moreover, significant (p<0.05) reduction in bax contents in bm tissues homogenates (figure. 3) was shown compared to the corresponding contents to group ιι (ciprofloxacin-treated) rats. iraqi j pharm sci, vol.30(1) 2021 protective effects of lutein on bone marrow 236 figure 1. effects of various treatments on total rbc counts in rats. data are expressed as mean ± sd, n =6. values with non-identical small letters (a, and b) are significantly different (p< 0.05). values with an identical capital letter (a) are non-significantly different (p> 0.05). figure 2. effects of various treatments on total wbc counts in rats. data are expressed as mean ± sd, n =6. values with non-identical small letters (a, and b) are significantly different (p< 0.05). values with an identical capital letter (a) are non-significantly different (p> 0.05). iraqi j pharm sci, vol.30(1) 2021 protective effects of lutein on bone marrow 237 figure 3. effects of various treatments on bax levels in bm tissues homogenates of rats. data are expressed as mean ± sd, n =6. values with non-identical small letters (a, and b) are significantly different (p< 0.05). values with an identical capital letter (a) are non-significantly different (p> 0.05). dna gyrase which is inhibited by fluoroquinolones, which is an adenosine triphosphate that hydrolyzing topoisomerase ii enzyme for keeping safe and state of the supercoiling in the replicating and the nonreplicating forms of chromosomes of the bacteria also fluoroquinolones has action on topoisomerase iv (17). oridupa a.o., et al. 2013 reported that ciprofloxacin can cause interstitial nephritis, suppression of bm and haemolytic anaemia in humans (18). furthermore, quinolones therapy, including ciprofloxacin can cause severe liver failure, elevated intracranial pressure, central and peripheral neuropathy; also, the occurrence of convulsions after exposure to ciprofloxacin can also be occurred. mechanisms of toxicity caused by ciprofloxacin were reported to be attributed to various causes, including its binding to glycine, n methyl-d-aspartate, and gaba-receptors; furthermore, other explanation to the toxic effects of ciprofloxacin was imputed to the toxic effect on the antioxidant's activity and the similarities of quinolone structure to kynurenic acid and similar compounds' structure which are glutamate receptor endogenous ligands (3). furthermore, adverse reactions of ciprofloxacin, especially in the cns, may be due to free radical-formation reactions; where, this hypothesis was supported by the evidence that ciprofloxacin can cause serious alterations of the glutathione redox status in the brain and the liver tissues of rat (19). moreover, lowes, d.a., et al. 2009 suggested that the toxicity of ciprofloxacin to the mitochondria can be due to oxidative stress (os), topoisomerase inhibition, photosensitization and altered calcium homeostasis (20). additionally, ciprofloxacin can have effects on the cellular growth and differentiation. immediate retardation of the cell division was only present in the cells of functional mitochondria; the retrograde of the signal from the mitochondria to the nucleus; caused either by the impaired of mitochondrial dna replication or by the oxidative stress (5). furthermore, adverse effects of fluoroquinolones including ciprofloxacin can cause tendon rupture, muscle weakness, joint inflammation, epilepsy, peripheral and central neuropathies and psychological symptoms as depression; all these symptoms had been suggested to be due to enhanced the os (21); although, the exact molecular mechanism was unclear yet. altered the topology of mitochondrial dna (mtdna) that cause reduction of mitochondrial transcription and mtdna copy number that may result in serious dysregulation of electron transport chain complexes, as occurring with ciprofloxacin treatment (22), that may lead to respiratory chain dysfunction that may consequently cause the observed increased in the oxidative stress (5). the current study confirms that ciprofloxacin caused bm toxicities, as was evidenced through the significant (p<0.05) reduction in total rbcs counts, and total wbcs a b a a a a a a 0 0.1 0.2 0.3 0.4 0.5 0.6 b a x (b .m ) c o n c . (p g /m l) groups of treated animals group i: liquid paraffin treated group ii: ciprofloxacin treated group iii: 6mg lutein group iv: 24mg lutein iraqi j pharm sci, vol.30(1) 2021 protective effects of lutein on bone marrow 238 counts and elevation in bax contents in bm tissues homogenates (figures 1, 2, 3). the role of apoptosis as a mechanism of toxicity provoked by ciprofloxacin on bm was not previously described; but in this study, such drug caused significant elevation in bax level in bm tissue homogenate (figure 3); as mention previously that (bax) protein cause activation of the cascade of apoptosis reactions by releasing the mitochondrial cytochrome c that helps in the successive activation of the caspases and leads ultimately to cell death (23). and thus, the current study is considered the first that demonstrate the role of apoptosis in bm toxicityinduced by ciprofloxacin. thus, we did not have a chance to compare the results of this study with other reports concerning this respect. studies showed that high concentration of lutein, either by diet intake or as supplementation, has beneficial effects on eye disorders, preventing or even ameliorative both the age-related macular degeneration (amd) (24) and the cataract (12). recently, several studies suggested that lutein might indeed has compatible effects via antiinflammatory effects (25), improving the cognitive functions (26), and reducing the risk of cancers (12), improving cardiovascular diseases (25) and the other systemic conditions (27). also, it has been mentioned that lutein has anti-genotoxic property, and it may attenuate the immunosuppression in mouse models induced by ultraviolet radiation (28). the current study showed that lutein (6mg and 24mg/kg/day) attenuates ciprofloxacin-induced reduction in total -rbcs count, and -wbcs count, and ciprofloxacin-induced elevation in bax contents in bm tissues homogenates of rats (figures 1, 2 and 3). conclusion results of this study suggested that bone 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licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 evaluating the impact of pharmacist asthma counselling doi: https://doi.org/10.31351/vol30isssuppl.pp22-30 22 evaluating the impact of pharmacist counselling for asthmatic children at karbala teaching hospital for children: an interventional prospective study (conference paper )# mohammed zuhair mahdi *,1 and zinah m. anwer* # 9th scientific conference conference sponsored by college of pharmacy, university of baghdad 25-26 august 2021  department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract asthma is one of the most common chronic, non-communicable diseases affecting children worldwide. the estimated prevalence of pediatric asthma in iraq is 15.8%. assessment and monitoring of asthma control can be done by a validated childhood asthma control test (c-act) and asthma control test (act). management plan of asthma must consider asthma counseling and education. patient education accounts for 90% of success and this can be achieved by an active collaboration among health care providers. this study aimed to evaluate the impact of pharmacist counseling on asthma control in children. this was a prospective interventional educational study that recruited 92 asthmatic children aged 2-18 years old attended asthma and respiratory diseases out-patient clinic in karbala teaching hospital for children. the participants were interviewed face-to-face and counseled about asthma, given an educational booklet, examined their inhaler use technique and their act/c-act was calculated. the patients were followed up weekly for one month either by phone or face-to-face to assess asthma control; rescue medication used; signs and symptoms; inhalers use and; any other concerns about asthma. in the fourth week, act/c-act was calculated again. a total of 92 participants were involved. there was a significant increment in act/c-act after providing asthma counseling and weekly follow-up by the interventional pharmacist. the incorporation of pharmacists in the management plan of asthmatic children to provide asthma counseling and education about the proper use of inhalers will improve asthma control in children. keywords: pediatric asthma, patient education, counseling, childhood asthma control test. لتعليمي تقييم فعالية دور الصيدالني في التثقيف حول مرض الربو لدى األطفال في مستشفى كربالء ا #) بحث مؤتمر ( لألطفال: دراسة تداخلية مستقبلية *زينة مظفر أنورو 1*، محمد زهير مهدي 2021اب 26 – 25جامعة بغداد ، # المؤتمر العلمي التاسع لكلية الصيدلة راقعلفرع الصيدلة السريرية، كلية الصيدلة، جامعة بغداد، بغداد، ا* الخالصة ٪. تقييم ١٥.٨االمراض المزمنة غير المعدية عند األطفال حول العالم. تقدر نسبة إصابة أطفال العراق بحدود يعتبر الربو من أكثر جب ان ومراقبة حالة السيطرة على الربو يمكن ان يتم عن طريق استعمال اختبار السيطرة على الربو عند األطفال و اختبار السيطرة على الربو. ي ٪ من نجاح الخطة العالجية على تثقيف المريض والذي يمكن ان يتم ٩٠م المشورة وتثقيف المريض حول الربو. يعتمد تتضمن الخطة العالجية تقدي سيطرة على عن طريق التعاون الفعال بين مقدمي الخدمة العالجية. تهدف هذه الدراسة الى تقييم مدى فعالية دور المشورة التي يقدمها الصيدالني بال سنة و الذين يحضرون الى العيادة االستشارية ١٨-2. هذه الدراسة مستقبلية تداخلية تعليمية تضمنت االطفال الذين تتراوح اعمارهم الربو عند االطفال ائهم لمرضى الربو و االمراض التنفسية بمستشفى كربالء التعليمي لالطفال. تمت مقابلة المشاركين وجها لوجه و مشاورتهم عن مرض الربو, و اعط لمدة تعليمي عن الربو, و اختبار آلية استعمال اجهزة االستنشاق و تسجيل نتيجة اختبار السيطرة على الربو. وتمت متابعة المشاركين اسبوعيا كتيب ستنشاق و استعمال االدوية المنقذة, و العالمات و االعراض, و استعمال اجهزة اال شهر عن طريق الهاتف او وجها لوجه لتقييم السيطرة على الربو, ٩2و االستجابة الي استفسار بخصوص المرض. عند االسبوع الرابع, تم تسجيل نتيجة اختبار السيطرة على الربو مرة اخرى. تضمنت الدراسة المتداخل. مشارك. كان هناك زيادة مؤثرة بنتيجة اختبار السيطرة على الربو بعد تقديم المشورة عن الربو و المتابعة االسبوعية من قبل الصيدالني ى تضمين الصيادلة في الخطة العالجية لمرضى الربو االطفال من اجل تقديم المشورة و االستعمال الصحيح لجهاز االستنشاق سيحسن السيطرة عل الربو عن االطفال. الكلمات المفتاحية: ربو األطفال، تثقيف المريض، المشورة، اختبار السيطرة على الربو لألطفال. introduction asthma is one of the commonest chronic non-communicable diseases of the lower respiratory tract in children (1). pediatric asthma prevalence ranged from 2.1% in indonesia to 32.2% in the united kingdom (2). there are limited numbers of studies about asthma prevalence in iraq. the estimated prevalence of pediatric asthma in baghdad the capital of iraq is 15.8% (3). asthma should be questioned in any child with at least two of the classical symptoms which are cough, shortness of breath, wheezing, and chest tightness. these symptoms frequently develop after viral respiratory tract infection, after workout, 1corresponding author e-mail: ilmilan22@yahoo.com received: 30/8/2021 accepted: 20/10 /2021 published online first: 2022-1-12 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30isssuppl.pp22-30 iraqi j pharm sci, vol.30(2) 2021 evaluating the impact of pharmacist asthma counselling 23 or experience of irritant allergen and subside in response to bronchodilators or anti-inflammatory therapy (4). a combination of inhaled corticosteroids (ics) and a bronchodilator serve as a good option for persistent asthma management (5). the patient and the health care providers must have a mutual understanding about the asthma control (6). asthma control test (act) and childhood asthma control test (c-cat) are validated and two of the numerical tests used to assess asthma control in children aged 12 years and older and 4-11 years old, respectively (7,8). notwithstanding the innovations in health care systems and pharmacological management of asthma, its control is still below the expectation. it is estimated that the prevalence of poor asthma control among the pediatric age group is 46% (9). the main reasons for that are: i) modest medication use; ii) poor medication compliance; iii) inappropriate inhalation technique; iv) exposure to triggers and; v) parents’ phobia of steroids (10,11). pharmaceutical care is a pharmacist’s involvement to optimize the use of medications by individuals. pharmaceutical care may improve adherence, the clinical effectiveness of therapy, and improve the health-related quality of life (12). time spent by physicians with their asthmatic patients is usually limited as well as the available resources for effective counseling and education (13). some studies showed that the health care provider assesses the inhalation technique in only 5% of visits (14). therefore, delegating this task to other health care providers may be a good alternative in providing efficient counseling and education. pharmacists, by their position as a part of health care providers and their contribution as the first and accessible interface, may play a significant role by delivering asthma counseling and inhalers use technique after getting the proper recommendations by the physicians (10,15,16). management of asthma may depend 90% on education and only 10% on medication (11). inspiring health care providers, asthmatic children, and their parents to share their concerns about asthma and medication use will facilitate the communication process and diminish the barriers (17). pharmacist intervention by providing asthma counseling had been shown to bring better asthma control in terms of higher act/ c-act scores, reduced number of exacerbation and systemic steroid use, and better quality of life (18,19). pharmacist integration with the physician to reassure the management of asthmatic patients according to guidelines presented better asthma control (20). the pharmacist-led intervention also brought cost-effectiveness and increased qualityadjusted life years (21). this study aimed at evaluating the impact of pharmacist counseling on asthma control in children following global initiative for asthma (gina) 2020 regarding patient education and involvement in the management plan. methods the study started on the first of january 2021 and ended on the first of may 2021 with a single weekly visit to karbala teaching hospital for children. the visits were on tuesday, which is the day already specified for the asthmatic patient to visit asthma and respiratory diseases outpatient clinic on regular basis to check their asthma and refill their medication. study design this was a prospective interventional educational study that was conducted at karbala teaching hospital for children. it is the main and only hospital for children at karbala where all the referral of pediatric patients occurs. the study was approved by the ethical and scientific committee of the college of pharmacy – university of baghdad and followed the principles of helsinki declaration regarding such sort of studies (22). approval to conduct the study had been taken from karbala teaching hospital for children managerial board verbally. sample size the sample size was determined using the raosoft platform for sample size calculation (23). an accepted margin error of 5% will result in 377 participants needed to conduct the study, however, the sample size couldn’t be collected due to missing data regarding the actual number of asthmatic children who keep regular visits at asthma and respiratory disease outpatient clinic and reduced number of visits to the hospital due to corona virus disease 2019 (covid-19) pandemic worldwide. so that, a convenient sample of patients who were interviewed during the timetable of the study and met the inclusion criteria were involved. patients the patients were first diagnosed with asthma by a specialized physician after paying nominal costs for the specialist's consultation and medications. a sample of those patients had been referred to the interventional pharmacists to be recruited in the study if they met the inclusion criteria. those who did meet the inclusion criteria underwent simple counseling about asthma and inhaler device techniques. the recruited participants aged 4-12 gave their assents and their parents' consent was taken verbally to participate in an asthma educational program and follow up weekly either by phone or face-to-face for one month in an attempt to improve their asthma control and increase their awareness about the disease. inclusion criteria were: age 2-18 years old; asthmatic patients who used controller therapy; regular attendees at asthma and respiratory diseases outpatient clinic; iraqi and resident at karbala; able to understand and complete act\c-act; living iraqi j pharm sci, vol.30(2) 2021 evaluating the impact of pharmacist asthma counselling 24 with anyone who can read and write; have the intent to improve his\her asthma control and; available for following up by phone or face-to-face. exclusion criteria were: patients with any other chronic diseases, pneumonia and missed the weekly follow up at any week. asthma control assessment asthma control had been assessed by act for children 12-18 years old and c-act for children aged 2-11 years old (appendix?). act is a validated numerical test that consists of 5 items to be answered by the patient and then a summation of items represents the total score that ranges 5-25. score less than 19 means the asthma is not well controlled. cact is also a validated numerical test that consists of two divisions. the first division includes four questions to be answered by the child or parents about the symptoms and the reliever medication used. the second division includes three questions related to patient self-assessment of control in the past four weeks. the score of each item was summed to provide a total score of 0-27 with a higher score means better control. act\c-act was calculated at the first face-to-face interview as a baseline and four weeks after the follow-up. data collection a face-to-face interview taking about 10 minutes was conducted either with children aged 1218 years old or the parents for those whose age is 211 years old to collect data by a sheet especially designed for the study which contains four aspects: patients' data; asthma characteristics; inhaler devices technique checklist; weekly follow-up checklist; and act\c-act score. the weekly follow-up checklist had been filled during the follow-up period. asthma counseling after collecting data, the participants or their parents were assessed roughly by the interventional pharmacist for their knowledge about asthma by simple questions (e.g. asthma definition, causes, types of medication, etc.). this assessment provides a good insight into the extent of asthma counseling to be given to them in a 15-minute interview. the interview stressed on asthma definition, signs and symptoms, causes, triggers, inhaler technique, the different types of inhalers, and which one should be used regularly, and which one on need. a short video clip on youtube had been used to show the proper use of different types of inhalers as well as a physical tutorial by one of the interventional pharmacists for participants who use inhalers (24). follow up after the completion of the face-to-face interview, the participants aged 12 years old and older were followed up weekly by phone calls or face to face meetings with them directly for one month, participants aged 11 years and younger were followed up with their parents either by phone or face-to-face meetings for the same period. each week they were asked if there were any symptoms according to a predefined checklist presented in appendix a. at the end of the fourth phone call, the patient underwent an act\c-act again to evaluate the impact of the intervention done by the interventional pharmacists. statistical analyses descriptive statistics (means, standard deviation, frequencies, and percentages) were conducted for all participants. data were analyzed using statistical package for the social sciences (spss) software (version 24). paired samples t-test was used to measure the difference in act\c-act scores of the asthmatic children due to pharmacist intervention. figure 1. the flowchart of the study process iraqi j pharm sci, vol.30(2) 2021 evaluating the impact of pharmacist asthma counselling 25 results one hundred and five participants were enrolled in the study. thirteen participants were excluded (2 participants were excluded due to living outside karbala province, 8 participants were excluded due to missed follow-up, and 3 participants were excluded due to adherence to medication and rejection of the use of inhalers). ninety-two participants who met the inclusion criteria in the study remained. the interventional pharmacist provided asthma counseling to the child’s parent mostly (67.4%). the majority of participants were male (58.7%). more than three-quarters of the consulted parents had secondary school or lower education with low or middle incomes (table 1). table 1. the characteristics of the participants/guardian of the participants and their average pre and post act\c-act score. characteristic subcategories n (%) pre act\c-act score post act\c-act score patient gender female 38 (41.30) 16.16 21.03 male 54 (58.70) 15.59 22.09 parent income low 41 (44.60) 15.54 21.90 middle 42 (45.70) 15.95 21.69 high 9 (9.80) 16.56 20.33 parent education illiterate primary school 47 (51.10) 15.77 21.79 secondary school 31 (33.70) 15.55 21.58 college or higher 14 (15.20) 16.64 21.36 consulted person child 30 (32.60) 16.77 20.63 parent 62 (67.40) 15.37 22.15 the average age of the participants was 9.34. there was an increment in act\c-act score after interventional pharmacist counseling (table 2). table 2.the descriptive characteristics of the participating patients * act = asthma control test. ** c-act = childhood asthma control test sixty-six percent of the participants had uncontrolled asthma at baseline according to their act\c-act score compared to eighty-four percent after interventional pharmacist counseling (table 3). table 3. the descriptive characteristics of asthma control and socioeconomic status pre-intervention post-intervention socioeconomic status % of uncontrolled asthma % of controlled asthma % of uncontrolled asthma % of controlled asthma high 5.43 4.35 2.17 7.60 moderate 32.61 11.96 7.63 39.13 low 28.26 17.39 5.43 38.04 total 66.30 33.70 15.23 84.77 characteristic n minimu m maximu m mean std. deviation age (year) 92 2.0 18.0 9.34 3.83 height (cm) 92 84.0 174.0 131.64 20.52 weight (kg) 92 11.5 88.0 33.82 15.65 pre-counseling act*\c-act** score 92 5.0 26.0 15.83 4.82 post-counseling act*\c-act** score 92 10.0 27.0 21.65 3.54 body mass index (bmi) 92 8.31 32.32 18.46 4.01 iraqi j pharm sci, vol.30(2) 2021 evaluating the impact of pharmacist asthma counselling 26 the majority (61 out of 92) of asthmatic children had familial atopic diseases including asthma and eczema (40.2%), asthma alone (18.5%), allergic rhinitis (3.3%), eczema alone (2.2%), and allergic rhinitis and asthma (2.2%) (figure 2). figure 2. percentage of familial atopic diseases (fad) among participants the interventional pharmacist check listed eight different steps of instructions about the proper use of asthma inhalers. the most common incorrect technique steps were the eighth (44%) and fifth (62%) steps respectively (figure 3). figure 3. steps of an inhaler technique error there was a significant (p-value< 0.05) increment in the act\c-act score after pharmacist counseling to the asthmatic children or their parents about asthma definition, symptoms, types of medication, the proper use of asthma inhalers, and other educational information related to asthma. (figure 4) iraqi j pharm sci, vol.30(2) 2021 evaluating the impact of pharmacist asthma counselling 27 figure 4. the increment in act\c-act score after interventional pharmacist counseling *significant difference (p-value< 0.05) according to paired samples t-test. there was a significant difference in the increment in act\c-act scores due to the pharmacist intervention according to the person who received the counseling (child vs parent) (figure 5) ةؤ figure 5. mean increment in act\c-act scores after pharmacist intervention according to the person who received counseling *significant difference (p-value< 0.05) according to independent t-test discussion the encumbrance of childhood asthma is still increasing worldwide despite the international efforts to manage it according to gina. there are many reasons which could contribute to reduced control including poor patient education, availability of medication, cultural beliefs and challenges in applying guidelines locally (25). this study represents the first prospective interventional educational study for asthmatic children at karbala, iraq. it is a patient-oriented study to evaluate the impact of pharmacist counseling on asthma control in children by increasing awareness about asthma knowledge like definition, causes, triggers, signs and symptoms, types of medication, and the proper use of the inhalers. most of the study participants were male (58.7%) which comes in line with a review article that showed the prevalence of asthma in children is about two-third for boys and one-third for girls with increasing prevalence in girls after adolescence suggesting a role of sex hormone and environmental exposure (26). in this study, most boys were playing and socializing with their siblings in school, neighborhoods, and other places like accompanying their parents at their work which may put them at a higher risk of environmental exposure to asthma triggers while girls had a lower chance of such exposure. these explanations had been made upon the interviews made with parents during data collection. the majority of study participants (90%) was low to medium income families whose children iraqi j pharm sci, vol.30(2) 2021 evaluating the impact of pharmacist asthma counselling 28 had asthma which appears to be similar to that found by davies et al. that stated the prevalence of asthma is greater in low-income families who may have a lower chance of having a good education and optimal academic career (27). a systemic review by didsbury et. al mentioned that children with a chronic disease like asthma and with low income had reduced quality of life when compared to their richer peers (28). this study was conducted at a governmental public hospital which is mainly visited by patients with low to medium socioeconomic status to have their medical visits, investigations, and medications at nominal costs while high socio-economic status patients may prefer to visit private clinics and hospitals to get better quality of service at surely, higher costs. asthma counseling provides better asthma control even for those with limited socioeconomic status as the study showed by the reduction occurred in the percentage of uncontrolled asthma after counseling. the educational degree of parents also contributes to the asthma control level, a lebanese study showed that children whose parents had a low education level are at a greater risk for uncontrolled asthma (29). in this study, half of the participants are either uneducated or have a primary education which appears to be correlated with the poor act\c-act scores before interventional pharmacist’s counseling. another study in sweden showed that higher parental education is positively correlated with asthma control of their children (30). in brazil, roncada c et al published a study in 2018 that examined the association between asthma control and parental knowledge of asthma and found that parents with insufficient asthma knowledge scores had a bad impact on their children's asthma control (31). atopic diseases are considered one of the main risk factors for the development of asthma in these children with the main percentage for those who have a familial history of asthma and eczema. this finding comes in line with a cross-sectional study that included 1601 participants in the kingdom of saudi arabia that showed a significant association between familial atopic diseases and the development of asthma in children aged 7-12 years old (32). other similar results were found in a survey conducted in china in 2014 which showed that atopy is the major risk factor for asthma development (33). atopic diseases running in families can be used to provide primary prevention for the development of asthma or wheeze in young children with breastfeeding in the first month taking the priority as a preventive measure (34–36). inappropriate inhaler techniques were an important issue with asthmatic patients and revisiting that on regular basis with the patient is a key factor for improving their asthma control (11,37,38) . addressing the specific steps at which the techniques are incorrect and ensure proper counseling and education about it results in better asthma control (18,39). simultaneous actuating and breathing in and rinsing mouth after completion were the two commonest steps that appeared to be incorrect techniques and need more attention during dispensing inhaler devices for asthmatic children since pharmacists are usually accessible and represent the only one who are responsible for medication dispensing. the mean act\c-act score was 15.83 at baseline which reflects poor control of asthma despite the optimal medication dispensed for them according to the local practice at karbala teaching hospital for children and availability of medication. interventional pharmacist counseling provided greater act\c-act score that is 21.65 after completing one month of weekly follow up that indicates better control of asthma. the pre-post act\c-act increment was significant in this study which shows the importance of pharmacists counseling with asthmatic children to enhance their knowledge as well as technique of inhaler use. such results had been seen in other studies in jordan and nepal about asthma overview and inhaler technique (19,40,41). the study showed a statistically significant improvement of asthma control according to act\c-act score due to the weekly follow-up check that had been made by the interventional pharmacists. this comes in line with a journal article by mehuys et al. that showed an improvement in the act after pharmacist counseling with the patient about asthma definition, triggers, and medication used to manage it as a complementary process of asthma control (42), however; it needs to be studied extensively in future to get a clear image if the better control was consistent or only during the follow-up period especially that the assessment tool is subjective rather than objective. conclusion pharmacists-led,patient-focused counseling about asthma and its medication will provide better control of symptoms and hence, a better quality of life. this will be a complementary process for the physician diagnosis and prescription of medication. we need more studies that focus on having welltrained pharmacists who can provide smooth counseling and eliciting the patient's hidden concerns and barriers for using the medication regularly and properly. limitations 1. sample size: the reasonable sample size for such a study could not be obtained due to covid-19 restrictions when the parents prefer not to bring their children with them. 2. medication adherence validation: we couldn’t perform the morisky scale for patient’s adherence due to the short time iraqi j pharm sci, vol.30(2) 2021 evaluating the impact of pharmacist asthma counselling 29 invested with the patient in counseling and education about their asthma medication. 3. asthma severity: the severity of asthma was not 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a private hospital of nepal. pulm med. 2019;2019. 41. basheti ia. role of the pharmacist in improving inhaler technique and asthma management in rural areas in jordan. clin pharmacol adv appl. 2019;11:103–16. 42. mehuys e. effectiveness of pharmacist intervention for asthma control improvement. eur respir j. 2008;31(4):790–9. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 h. pylori igg , iga and ca19-9 and cea in gastritis 68 detection of helicobacter pylori igg and iga , serum biomarkers ca19-9 and cea in patients with gastrointestinal diseases shurooq,r. kadhim* ,1 , ishraq,h. alaiwi* and rasha,r.** * department of clinical laboratory sciences, college of pharmacy, al-mustansiriyah university ,baghdad. iraq. ** ministry of health abstract: gastrointestinal diseases and especially chronic gastritis are mainly induced by helicobacter pylori infection, and provides the basis for gastric carcinogenesis and colorectal cancer. the study involved the detection of serum anti-h. pylori igg and iga antibody of and some serum biomarkers ;cea and ca19-9 in patients with gastrointestinal diseases. fifty eight serum samples were collected from 25 males and 33 females .peripheral venous blood was collected from each patient and sera obtained by centrifugation. serum anti-h. pylori igg and iga ,serum cea and ca19-9 were evaluated by enzyme-linked immunoadsorbent assays (elisa).forty eight serum samples were positive for igg (82.7% ) divided into 27 positive samples for females and 21 positive samples for males while ten samples were positive for iga ( 17.2%) divided into eight positive samples for females and two positive samples for males .only three samples were positive for anti igg and serum biomarker ca19-9 .in this study most of positive results of h.pylori iga and igg are between the age 3050 years and low within 1-10 years ,we conclude because most of them are working and eat fast food from different sources. this study concluded that detection of serum igg and iga is effective in monitoring the h. pylori infection in gastrointestinal disorders and chronic infections . keywords: gastrointestinal diseases, gastritis , h.pylori, igg ,iga ,cea,ca19-9. ca19-9و المعلماث البُىلىجُت helicobacter pyloriلبكترَا iga و iggالتحرٌ عن اضداد الجهاز الهضمٍلدي مرضً cea و شروق رَس كاظم * ،1 رشا رحُم** واشراق حسن علُىٌ* ، . انعزاق ،ثغذاد ،انجبيعخ انًسزُصزٌخ ،كهٍخ انصٍذنخ ، السريريةختبرية مفرع العلوم ال* . انعزاق ،ثغذاد ،** ٔسارح انصحخ الخالصة انًعذح ٔانًؤدي انى األصبثخ ثسزطبٌانشيٍ انًعذح يزاض انجٓبس انٓضًً ٔانزٓبةانًسجت انزئٍسً أل helicobacter pyloriرعذ ثكززٌب ٔ ca19-9ٔانًعهًبد انجٍٕنٕجٍخ h. pylori ثكززٌب igg ٔiga انكهٕثٍٕنٍُبد انًُبعٍخ انزحزي عٍرضًُذ ْذِ انذراسخ .ٔانقٕنٌٕ cea عٍُخ يٍ انذكٕر 58ٔرٕسعذ انى انشيٍانًعذح انجٓبس انٓضًً ٔانزٓبة عٍُخ يصم يٍ يزضى 85جًعذ . فً يصٕل انًزضى . فً يصٕل انًزضى ثطزٌقخ األنٍشا ca19-9 ٔceaٔانًعهًبد انجٍٕنٕجٍخ igg ٔ iga عٍُخ يٍ األَبس ٔقٍسذ اضذاد ال 33ٔ عٍُخ نألَبس . ثًٍُب كبَذ انعٍُبد انًٕججخ ألضذاد 52عٍُخ نهذكٕر ٔ 52%.( رٕسعذ ال 55.5)igg عٍُخ اٌجبثٍزٓب ألضذاد ال 85اظٓزد iga (22.5ٕرٕسعذ ثٍٍ ثًبٍَخ عٍُبد نألَبس ٔ اثُبٌ نهذك )%. ألضذاد ال أٔضحذ انُزبئج ظٕٓر ثالس عٍُبد يٕججخ ر igg ٔانًعهى سُخ ألَّ ْذِ انفئخ ًْ األكثز َشبطب ٔاررجبطب 83-33نألعًبر igg ٔ iga.كبَذ َزبئج ْذِ انذراسخ اٌجبثٍخ ألضذاد ca19-9انجٍٕنٕجً سُٕاد ًْ األقم 23-2ثبنعًم ٔاألكثز رعزضب نٓذِ انجكززٌب ألٌ يعظًٓى ٌزُبٔنٌٕ انٕججبد انسزٌعخ ٔيٍ يصبدر يخزهفخ.ثًٍُب كبَذ انفئخ انعًزٌخ ثبسزخذاو انفحص انسٍزٔنٕجً ٔ انذي ْٕ يٍ انفحٕصبد انزٔرٍٍُخ h. pylori جكززٌبن igg ٔ igaاصبثخ . ٌعذ انزحزي عٍ األجسبو انًضبدح ٔانًزٕفزح فعبال فً انزحزي عٍ األصبثخ ثٓذِ انجكززٌب خصٕصب فً األصبثبد انًشيُخ ٔاألضطزاثبد انًعٌٕخ. . السمنالمعدة أمراض الجهاز الهضمٍ والتهاب ، h. pylori، igg ، iga ،ca19-9 ، cea الكلماث المفتاحُت: introduction helicobacter pylori , a spiral shaped pathogenic bacterium found on the human gastric mucosa , was first isolated by warren and marshall (1) in 1982 and soon after was linked with chronic gastritis and peptic ulceration (2) . the world wide prevalence of h. pylori is more than 50% (3,4) . it is more prevalent in developing countries as compared to developed countries (5) . its prevalence in south asia is ranging between 55 to 90% (6) . primary care physicians were reported that 78% of the physicians thought that contaminated water was the source of spread of infection (7) chronic gastritis is mainly induced by helicobacter pylori infection, and provides the basis for gastric carcinogenesis (8) . several prospective studies reported a strong association between h. pylori infection and gastric cancer; hence , h. pylori is now recognized as a group 1 carcinogen for humans (9,10) . however , only a few individuals 1 corresponding author e-mail: shrooq7@yahoo.com received: 20/3/2013 accepted: 22/4/2014 iraqi j pharm sci, vol.23(1) 2014 h. pylori igg , iga and ca19-9 and cea in gastritis 69 infected by h. pylori may eventually develop gastric cancer. the clinical outcome of h. pylori infection may depend on several factors, such as age at the time of first infection, environmental factors, immune response, and genetic characteristics (11) .among the serum tumor markers, cea and ca19-9 are most widely used for the screening and monitoring gastric cancer, because they have been reported to be elevated in some patients with gastric cancer (12,13) . carcinoembryonic antigen (cea) is the most commonly used tumor marker in patients with colorectal cancer, and the tumor marker carbohydrate antigen ca199 is often used in combination with cea to manage patients with colorectal cancer (14,15,16) . the aim of the study is to determine the prevalence of the serum anti-h. pylori immunoglobulin igg and iga antibodies and serum tumor markers, cea and ca19-9 in patients with gastrointestinal diseases . patients and methodes patients and samples: a total of fifty eight patients with abdominal pains, cramps, diarrhea and chronic gastritis whom referred to al-karamah hospital in baghdad from april 2010 to july 2011, were included in the present study . among them 25 were males and 33 were females with gastrointestinal disorders ( 8 persons were considered as control) , aged from 6 to 72 years with a mean age of 55( + )years old. serological tests: blood was sampled twice from patients. enzyme-linked immunoadsorbent assays (elisa) were used to measure the levels of serum anti-h. pylori igg and iga antibodies. the test kits for the detection of anti-h. pylori -igg and antih.pylori iga were purchased by biohit oyi company (finland) . detection of serum markers cea and ca19-9 were also done by elisa technique (biohit oyi company) . results and discussion the results were classed as positive if anti–h pylori immunoglobulin igg titers were >30 u/ml and iga titers positive if they were >20 u/ml. table 1 shows the detective positive rates of serum anti h. pylori igg and iga antibodies of patients in relation to gender and age. the incidence of infection was (43.18 %, 25/58) between the males and (56.8 %, 33/58) the females patients .the h. pylori infection rate was the highest in the ages from 42 to 50 years.also the detected positive rate of h. pylori was not remarkably associated with the age of patients . table (1) age ,gender and number of anti h.pylori iga and igg positive serum samples in females and males. the results showed that anti h.pylori igg positive serum samples in both females and males were elevated in ages 21-50 years as in table 2 and 3. table (2) age distribution of anti h.pylori igg positive serum samples in females age (year) gender numbers of patients 1-10 f 2 11-20 = 3 21-30 = 5 31-40 = 8 41-50 = 7 51-60 = 2 total = 27 table (3)age distribution of anti h.pylori igg positive serum samples in males anti h.pylori iga positive serum samples in males were only 2 between ages 1-30 years as in table 4,but anti h.pylori iga positive serum samples in females were elevated in ages 4150 years as in table 5. gender age (year ) number and percentage of patients with positive anti -iga numbers and percentage of patients with positive anti -igg f 7-60 8(13.7%) 27(46.5%) m 1-58 2(3.4%) 21(36.2%) 10(17.1%) 48(82.7%) age (year) gender numbers of patients 1-10 m 1 11-20 = 1 21-30 = 4 31-40 = 5 41-50 = 7 51-60 = 3 total = 21 iraqi j pharm sci, vol.23(1) 2014 h. pylori igg , iga and ca19-9 and cea in gastritis 70 table (4 ) age distribution of anti h.pylori iga positive serum samples in males age (year) gender numbers of patients 1-10 m 1 11-20 = 21-30 = 1 31-40 = 41-50 = 51-60 = total 2 table ( 5 ) age distribution of anti h.pylori iga positive serum samples in females age (year) gender numbers of patients 1-10 f 11-20 = 21-30 = 1 31-40 = 1 41-50 = 6 51-60 = total 8 table (6 ) age distribution of anti h.pylori igg and iga positive serum samples in females . age (year) gender numbers of positive igg and iga samples 1-10 f 11-20 = 21-30 = 31-40 = 1 41-50 = 3 51-60 = 1 total 5 table (7 )age distribution of anti h.pylori igg and iga positive serum samples in males . table (8 ) age distribution of anti h.pylori igg and iga positive and serum markers cea and ca19-9 in males patients . three specimens of males gave positive result for ca19-9 test but all females specimens gave negative results for ca19-9 test. all females and males specimens gave negative results for cea test. it is now recognized that the clinical detection of serum antibody is effective in monitoring the h. pylori infection and the serology test has been used widely in epidemiologic studies and routine diagnosis of h pylori infection (5,6 ,7,8) .in this study most of positive results of iga and igg are between the age 30-50 years and low within 1-10 years ,we conclude because most of them are working and eat fast food from different sources. serological testing to diagnose h. pylori infection in children is still controversial, although commonly used in clinical practice. a recent study speculate that young children may have a different immune response to h. pylori, with preferable responses to certain antigens, as well as lower titers than adults. the pyloriset test may fail to recognize these specific antibodies (17) .however, most individuals infected with h. pylori do not experience symptoms or have signs of recognizable disease. in most children, the presence of h. pylori infection does not lead to clinically apparent disease, even when the organism colonizing the gastric mucosa causes chronic active gastritis (18) . a recent study was carried in iran and this study showed that igg titers were high in individuals between 40 to 50 years old , anti h. pylori iga was also correlated with increasing age (19) . serological tests are widely available and cheap, and may be helpful-in screening populations or in confirming the presence h. pylori infection in case of equivocal results of the other diagnostic methods due to bleeding ulcers, antibiotic and/or antisecretory treatments. a major advantage of this serologic test is that it age (year) gender numbers of positive igg and iga samples 1-10 m 1 11-20 = 21-30 = 31-40 = 1 41-50 = 51-60 = total 2 iraqi j pharm sci, vol.23(1) 2014 h. pylori igg , iga and ca19-9 and cea in gastritis 71 enables large numbers of subjects to be screened quickly and relatively inexpensively (20) .in table (8) the results showed only three males infected with h.pylori and elevated values of ca19-9 in age between 11-50 years. according to these results our study conclude that these three males may suffered from gastric or other kind of gastrointestinal tumors . a low number of positive results were recorded on elevated values of ca19-9 by kim et al. when they identified only four patients among 1,063 patients with ca 19-9 serum levels >37 u/ml (mean values 50.5±16.8 u/ml) (21) .some times ca19-9 is not used for early screening because it is not present in patients with certain blood types and is often elevated in benign disease. certain changes that occur in the sera of pancreatic cancer patients reflect the high level of inflammation associated with the disease (22) . a recent study on serum ca19-9 mention that it is the most extensively studied and clinically useful biomarker for pancreatic cancer. unfortunately, ca 19-9 serum level evaluation in pancreatic cancer patients is limited by poor sensitivity, false negative and increased false positivity in the presence of obstructive jaundice (10–60%) (23) . we conclude that serum analysis should include ca19-9 and cea for the diagnosis the chronic infections with h. pylori. recommendations: test the specimens of patients with chronic infections with h.pylori. for ca19-9 and cea diagnosis 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rarely occurs however, some groups of people such as elderly and people with intestinal absorption impairment can find it difficult to get adequate vitamin k. recently the interest for vitamin k influence on human health apart from its clotting effect has increased and several studies reported that chronic subclinical deficiency of vitamin k may be a risk factor for many diseases such as osteoporosis, atherosclerosis, cancer, insulin resistance, neurodegenerative diseases and others . the aim of this review is to clarify the role of vitamin k in preventing and treating different aspects of human diseases. keywords: vitamin k, sources of vitamin k , pharmacokinetics of vitamin k , safety and toxicity of vitamin k. تاثير فيتامين ك على صحة االنسان *و هبة هاشم خليل *سبي، سارة هاشم مح 1*، فخري قيصرنجوان العراق .، ، بغداد جامعة بغداد، كلية الصيدلة ، فرع العلوم المختبرية السريرية * الخالصة .ان فيتامين ك 2و فيتامين ك 1نوعين فيتامين ك فيتامين ك هو احد الفيتامينات المهمة الذائبة في الدهون والذي يوجد طبيعيا على وث ضروري في عملية تخثر الدم حيث يلعب دور اساسي في تصنيع بروتينات تخثر الدم في الكبد.على الرغم من ان العوز في فيتامين ك نادر الحد معوي توجد لديهم صعوبة في الحصول على كمية لكن بعض المجاميع من االشخاص مثل كبار السن واالشخاص الذين لديهم اعاقة في االمتصاص ال سات الى كافية من فيتامين ك . حديثا زاد االهتمام حول تاثير فيتامين ك على صحة االنسان بجانب تاثيره على التخثر حيث اشارت العديد من الدرا ثل هشاشة العظام, تصلب الشرايين, السرطان, ان العوز المزمن تحت السريري لفيتامين ك ممكن ان يشكل عامل خطورة للعديد من االمراض م االمراض العصبية التنكسية وغيرها. ان الهدف من هذه المراجعة هو لتوضيح دور فيتامين ك في منع حدوث ومعالجة العديد من مقاومة االنسولين , .االمراض في االنسان .سالمة وسمية فيتامين ك ،حركية فيتامين ك ،مصادر فيتامين ك ،الكلمات المفتاحية: فيتامين ك introduction vitamin k is a fat-soluble vitamin necessary for blood coagulation. the vitamin was discovered by danish research scientist henrik dam in germany in 1929 and was called "k" since the earliest discoveries were published in a german journal, in which the substance was referred to as the " koagulation vitamin." the pure vitamin (k1) was derived from alfalfa in 1939 and it was subsequently realized that a second form (k2) with the more unsaturated side chain was synthesized by the bacteria (1). vitamin k1 and vitamin k2 occur naturally, and are primarily present in vegetables with green leaves. they absorbed from the small intestine. the quantity of fat in the diet and the production of bile acid in the liver are essential for absorption. similar to other lipid soluble vitamins, vitamins k1 and k2 are initially spread in chylomicrons (cms) that go into the circulation via lymphatics. the synthetic type of vitamin k (vitamin k3) is soluble in water and absorbed in the lack of bile acids, directly moving from the mucosal cells of the intestine into the liver portal blood (2). liver consider as the storing of vitamin k. vitamin k is essential for hepatic post synthetic activation of coagulation factor ii (prothrombin), vii, ix and x, in addition to anti-coagulant protein c and s, initially, all are synthesized as non-active precursor proteins by the liver. thus, vitamin k deficiency extends the clotting time. vitamin k deficiency is usually caused by abnormal absorption rather than in the lack of vitamin in food. vitamin k has an essential role for numerous physiological processes as the sole cofactor of γ-glutamyl carboxylase enzyme (ggcx) that catalyzed post translational shift, which lead to vitamin k-dependent proteins (vkdps) activation engaged in the formation of bone, tumor growth prevention, inflammatory reactions and several other biologically important function (3). the goal for this review is to clarify the function of vitamin k in preventing and treating different aspects of human diseases. 1corresponding author e-mail: najwankaisar@yahoo.com received:18 /8/ 2020 accepted:20 /10 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp1-13 iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 2 types and sources of vitamin k there are 3 different vitamin k types: k1 obtained from plants; k2 produced by bacteria or fermentation; and k3 produced synthetically (4, 5). vitamin k naturally occurs as vitamin k1 (phylloquinone, pk) and as vitamin k2 (including several various vitamins, known as menaquinones, mks). menaquinones (vitamin k2) are a subfamily with a length of 1 to 13 isoprene residues in the lateral chain that are all unsaturated. different menaquinones are commonly referred to as mk-n , where n mean the number of the aliphatic side chain isoprene residues . relevant menaquinones include the short chain mk-4, the only mk which formed by a systemically conversion of pk into mk, and the long chain of menaquinones mk-7, mk-8, mk-9 and mk-10, all of which are present in human nutrition while little amounts of mk-6 are contained in different foodstuffs. vitamin k1 is found chiefly in green leafy foods like broccoli, kale and spinach. its viscous oil; yellow color, dissolve in vegetable oil, it is also found in fruit such as avocado, kiwi and grape as well as olive oil and soy oil. vitamin k1 is called phylloquinone since it is a photosynthesis indirectly found in plant leaves where it occurs in chloroplasts and takes part in the l photosynthetic procedure. fascinatingly vitamin k1 is especially sensitive to sunlight (1 hour of exposure destroyed it). although the source of vitamin k2 (menaquinones) seems to be more unclear and while the involvement of bacterial flora in small intestine to vitamin k is still incomprehensible, menaquinones are mainly derived from bacterial flora but the amount of vitamin k2 produced is considered to be minimal. k2 also present in fermented foods (e.g. cheese and the japanese soy product ‘natto’ ) (6,7). furthermore, there is a synthesized form of vitamin k: menadion, or vitamin k3(2-methyl-1, 4-naphthoquinone nucleus), which is a structural precursor to vitamins k1 and k2. menadion supplement is prohibited by fda due to its toxicity (5) (figure 1 , table 1); figure 1. structure of vitamin k1 (phylloquinone) and vitamin k2(menaquinone) menadion (vitamin k3) represent the common k1 and k2 structure menadione is a synthetic vitamin k type. (6) iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 3 table 1. vitamins k types, sources and function (7) type of vitamin k sources of vitamin k function in the human body vitamin k1 green leafy vegetables and some plant oils. takes part in blood coagulation as an enzyme cofactor which catalyzes carboxylication (insertion of a carboxy group-cooh) of some glutamic acid residues to gamma-carboxyglutamate (gla). factors ii, vii, ix and x contain gla protein. vitamin k2, menaquinones-4 (mk-4) (i) butter, egg yolks, pork fat and nutrition based on animals. (ii) synthesized by bacteria flora in the intestine (however, synthesized mk-4 is attached to the bacteria membranes in the intestine and small amount in humans is absorbed). (iii) over-the –counter (otc) medicines (i) osteocalcin (which is synthesized in bones) (ii) matrix gla protein (synthesized in cartilage and in the walls of blood vessel) it is implicated in the transportation of calcium, hindering the accumulation of calcium in the walls of blood vessel, and helps to increase the density of the bone. (iii) mk-4 is short chain type with shorter half-life. vitamink2,menaquinone7 (mk-7) (i) certain types of cheese and fermented foods, extracted from natto (fermented soy) (ii) extracted from natto (fermented soy) as otc medicines function of mk-7like mk-4 .mk-7is long chain type with longer half-life. vitamin k3, menadione. synthetic form of vitamin k considered a provitamin. (i) has been prohibited in the usa by the fda due to possible toxicity (hemolytic anemia). (ii) it recently being investigated as a possible therapy for prostate / hepato cellular cancer and treatment option for the toxicity of skin secondary to kinase inhibitor medication. pharmacokinetics of vitamin k like other lipid-soluble vitamins, vitamin k is mainly absorbed in the proximal intestine. after the absorption, vitamin k emulsified into mixed micelles via bile salts and incorporated into the apoaand apob-48-containing chylomicrons (cms). cm is then secreted into the lymph and goes to blood through lacteals and thoracic ducts. cms in the blood are hydrolyses via lipoprotein lipase on the capillary of endothelial cells surface. after clearance of triglyceride core, chylomicrone remnant (cr) which is smaller cm residues will be produced and circulation will resume after loss most of apoc and apoa, but vitamin k retained in the lipophilic core (8). (figure2 ) iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 4 figure 2. schematic diagram of dietary phylloquinone (k1) and menaquinone-7 (mk-7) absorption, transportation, and cellular uptake (8). uptake of vitamin k by liver in liver cr the classical endocytotic receptor pathway via the low density lipoprotein receptor (ldlr) and receptor-related protein 1 (lrp) is observed. the lipids incorporated into vldl with apob-100 and revert to blood where apoe and apoc are acquired. the consequent hydrolysis of triglycerides within capillaries produces vldl residues known as intermediatedensity lipoproteins (idl) and releases free fatty acids. additional changes to idl include reduction of apoe and apoc and. this way allows the remaining vldl particles to be converted to ldl. vitamin k is still supposed to be in the lipophilic core (8). (figure 2) uptake of vitamin k by bone nutritional vitamin k is supplied by circulating lipoproteins like cr and ldl to the human bone. the lrpi and ldlr are expressed by osteoblasts. study of vitamin k absorption has shown that most osteoblasts obtain phylloquinone by cr way and mk-7 through ldl way (8). excretion of vitamin k vitamin k is metabolized in the liver and excreted in the urine and bile mainly in the form of glucuronide conjugates of two major carboxylic aglycones, with 5–7c side chains, respectively. in study with radio-labeled phylloquinone, 20% of the injected dose was excreted into the urine, while 40% were excreted through the bile in the feces; the proportion was identical despite of either the dose injected is 1-45 mg. there were no equivalent excretion data for menaquinones (8) . mechanism of action of vitamin k the mechanism of action of vitamin k is to add a functional carboxylic acid group to a protein amino acid glutamate (glu) residue, to form a gamma carboxyglutamate (gla). this unusual posttranslation alteration of the protein, referred as the "gla-protein”. within the gamma-carboxyglutamate residue, the existence of two −cooh (carboxyl acid) groups on the same carbon enables it to chelating calcium ions. this way the binding of calcium ions most often affects the action or binding of gla-protein enzymes, like that of the vitamin kdependent coagulation factors. vitamin k is reduced in the cell to a reduced form known as vitamin k hydroquinone, catalyzed by vitamin k epoxide reductase (vkor) enzyme (9) . other enzyme which is called gamma-glutamyl carboxylate or vitamin-kdependent carboxylate then oxidizes vitamin-k hydrochinone, and permit carboxylation of glu to gla (10). the process of carboxylation takes place only if the carboxylase enzyme can simultaneously oxidize vitamin k hydroquinone to vitamin k epoxide. it is said that the carboxylation and epoxidation reactions are linked. vitamin k epoxide is then transformed by vkor into vitamin k. this reduction and consequent reoxidation of vitamin k, iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 5 along with the carboxylation of glu, is named the vitamin k cycle this cycle subsist in the liver cells endoplasmic reticulum (11). people rarely lack vitamin k1, as part of vitamin k1 is continually recycled in cells as seen in figure 3. vkor's activity is inhibited by warfarin and other 4-hydroxycoumarins. this decreases the levels of vitamin k hydroquinone and vitamin k in the tissues, thus rendering the glutamyl carboxylated reaction inefficient. as a consequence, clotting factors with insufficient gla are generated. with lack of gla on the amino ends of these factors, they do not bind efficiently to the endothelium blood vessel and cannot stimulate coagulation to enable clot formation when tissues injury occurs. since it is difficult to estimate the dose of warfarin to suppress coagulation, warfarin must be strictly monitored to prevent overdose (12) . figure 3. vitamin k cycle (13) gamma-carboxyglutamate proteins gamma carboxyglutamate protein (gla proteins) is secretary protein present in the body fluids or extracellular matrix. gla-residues make up a powerful calcium binding group in the proteins to which they bonded, in all situation gla-residues are essential to the functioning of these proteins (14). until recent years , there have been 17 human proteins identified to be gla domains: the blood clotting factor ii (prothrombin), factor vii, factor ix and factor x, the anticoagulant protein s ,protein c, and protein z( factor x-targetting), osteocalcin ( bone gla protein), the matrix gla protein(mgp) which inhibit calcification, growth arrest sequence 6 protein(gas6) which regulate cell growth, and four transmembrane gla proteins(tmgps) the function of these proteins until recent years unknown. the gas 6 protein may have growth factor function that stimulate axi tyrosin kinase receptors and activate cell growth or avoid apoptosis programmed cell death) in some cells (15) (table 3) . table 2. types and functions of 17 gla-proteins (14) name of protein function prothrombin, factors vii,ix,and x haemostasis (procoagulant function) proteins s,c, and z haemostasis (anticoagulant function) matrix gla protein (mgp) inhibit arterial calcification osteocalcin bone metabolism growth arrest sequence 6 protein (gas6) regulation of cell growth periostin bone metabolism, cell migration, angiogenesis gla rich protein (grp) periostin like factor four transmembrane gla protein function unknown functions of vitamin k effect of vitamin k on coagulation vitamin k, which is a coenzyme used to generate coagulation factors, is long considered to be important for controlling blood clotting. blood coagulation is based on the cascading stimulation of a number of coagulation factors (specific proteins) that form blood clots which prevent bleeding. vitamin k acts as an enzyme cofactor which catalyzes carboxylication (insertion of a carboxy group-cooh) of some glutamic acid residues to gamma-carboxyglutamate (gla) in the coagulation factors. vitamin k has critical significance in the iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 6 stimulation of a total of seven clotting factors (16) . gamma-carboxylation based on vitamin k allows these clotting factors for binding to calcium ions (ca2 +) and to activate the mechanism of coagulation. clotting factors ii (prothrombin), x , ix and vii form the coagulation cascade center, whereas protein z tends to increase the function of thrombin, which is the key enzyme in blood coagulation, via raising thrombin binding to cell membranes phospholipids. protein z, protein s, and protein c have inhibitory function of coagulation. these proteins control (coagulation time) and manage the cascade of coagulation. protein s is a subject of fundamental study of vitamin kdependent proteins because of its complexity and variety of functions in the body (17). the human body stores significantly low quantities of vitamin k and quickly exhausts its reservations if there was no periodic dietary intake. a recycle process of vitamin k (vitamin k cycle) enables a little quantity of vitamin k to actively engage many times in the gamma carboxylation of the proteins, thus reducing the requirement for vitamin k from diet. significant deficiencies of vitamin k causes prolonged time of coagulation and rise the hazard of sever bleeding, blood loss, bruise, poor healing of wound and anemia (18). effect of vitamin k on bone health vitamin k and vitamin d are important for the healthy metabolism of the bone. three proteins dependent on vitamin k were isolated from the bone: s protein, matrix gla protein (mgp) and osteocalcin, osteocalcin is synthesize by osteoblasts (cells forming bone) and participates in mineralization of bone, after collagen osteocalcin is the most crucial protein being integrated in the bone matrix. while vitamin d enhances osteocalcin production and increased the amount of calcium, vitamin k activates osteocalcin via vitamin k – dependence carboxylation of 3 of its glutamic acid residues. this activated form of osteocalcin will bind and store calcium (hydroxylated calcium phosphate) in bones. inadequate osteocalcin carboxylation (e.g. due to deficiency of vitamin k) can cause bone density loss. matrix gla protein are located in cartilage, bones, and soft tissue like blood vessels. experimental studies show that mgp hinders cartilage and soft tissue hardening, while it improves normal bone growth and development (19, 20). protein s (which is vitamin k – dependence anticoagulant protein) had been also synthesized via osteoblast but its function in bone metabolism is still unclear. (20) children suffering from inherited deficiency of protein s have increased coagulation and reduced bone density complications (19). postmenopausal women are more susceptible to bone loss and osteoporosis due to reduction in estrogen level after menopause (21). several random trails have evaluated the impact of phylloquinone treatment on bone loss in postmenopausal women (2224). treatment of post menopause women with a large dose of phlloquinone (1000ug/day) plus vitamin d, magnesium, calcium and zinc over a period of 3 years was shown to reduce bone loss on the femoral neck but not on the lumber spine in women between 50 and60 years of age (22). moreover, in a randomized, double blind placebo controlled trail, 5 mg daily supplement of phylloquinone in 440 postmenopausal women suffering from osteopenia for 2 yeas induced more than 50 percent diminish in clinical fracture in comparison with placebo (23). knapen et al. followed healthy postmenopausal women who have obtained either placebo or mk-7 capsules (180 mcg / day) for three years. in the first year, bone loss rates were comparable in both groups, but after 3 years period mk-7 affected bone health positively compared to placebo even after bmi and age adjustment (24). effect of vitamin k on cardiovascular diseases calcification of the blood vessels is an active process causing cardiovascular disease (cvd) which causes the world's largest killing. vitamin k-dependent proteins are known to stimulate the protection mechanism to prevent the occurrence of blood vessels calcification (25). a vitamin k-dependent carboxylation activates matrix gla protein (mgp) and can probably reduce calcium deposit on the blood vessels lining (26, 27) and the elevated blood concentration of inactive, undercarboxylated mgp is considered as a probable indicator for (early) atherosclerosis (24). a case control study found that administrating of 500 micrograms per day of vitamin k1for 3 years could delay the development of early calcification of coronary artery among older men and women (29) .besides from its effect on mgp, vitamin k may inhibit blood vessels calcification via antiinflammatory mechanism. vascular calcification is a process of chronic inflammation through which macrophages are activated causing production of proinflammatory cytokines such as tnfα, oncostatin m, il-6, and il-1β. these proinflammatoty cytokines stimulate osteogenic differentiation of blood vessels smooth muscle cells. vitamin k has anti-inflammatory effect by blocking nf-kb singles transduction, thus inhibits progression of blood vessels calcification (28). in a cross-sectional study conducted with 662 community-dwelling adults from the multi-ethnic study of atherosclerosis (mesa). high levels of blood phylloquinone are inversely correlated with many blood inflammatory markers like crp, il-6 and soluble intercellular adhesion molecule-1 (icam-1) (30). several clinical trials demonstrate vitamin k as an atherosclerosis protective factor and that low concentration of phylloquinone could be correlated with an increased hazard of blood vessels iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 7 calcification (31, 32). it is especially true when older people take antagonists of vitamin k to reduce blood clotting (33). effect of vitamin k on nervous system in the brain tissue, high levels of vitamin k are found and shown being essential for the function of brain. vitamin k could help to prevent old age neurodegenerative diseases by reducing calcification (hardening) of soft tissue. inside the brain, vitamin k is implicated in synthesizing of sphingo-lipids (a group of complex lipids in all mammalian cells where they are crucial parts of the cell membrane) which are found in the central and peripheral nervous systems cells at principally high concentrations (34) . some sphingolipids have a strong correlation with mk-4. inside brain sphingolipids are the main players in important cellular incident like senescence, differentiation, proliferation, interaction between cells and transformation. research in recent years has linked changes in the metabolism of sphingolipids to the process of aging and neurological diseases, like parkinson disease and alzheimer's disease ( ad). there are also two vitamin k – dependent proteins (vkdps), namely gas6 and, to a less degree, protein s which have been strongly linked with nervous system. gas6 is one of a secreting proteins contain 11–12 gla (carboxyglutamic acid) residues. gas6 were implicated in chemotaxis, cell growth, mitogenesis, and myelination in the nervous system. protein s has initially been found to have a role in blood clotting as co-factor for protein c, although limited in range data gathered up to now indicate that protein s is able to protect the nervous system and brain through its antithrombotic and neuroprotective signaling mediating functions (34,35). effect of vitamin k on insulin sensitivity vitamin k may have a beneficial impact on the balance of blood sugar and thus help prevent diabetes. the molecular mechanisms behind vitamin k's beneficial function in the responsiveness of insulin and glucose homeostasis are: 1-osteocalcin activation (by vitamin kdependent carboxylation) : oc can boost proliferation of bcells, expressions and secretion of insulin as well as, enhance expression of adiponectin in the adipocytes , which indicates oc's role in the regulation of glucose metabolism through improvement in the function of b-cells and insulin sensitivity (36. 37, 38). 2-adipokines production regulation: alteration in the levels of circulatory adipocytes derived factors (adipokines) has a significant role in insulin resistance (39, 40). after a year of follow up, people who raise their nutritional phylloquinone consumption showed that inflammatory cytokines such as leptin, interleukin (il)-6, tumor necrosis factor (tnf) and other risk factors of metabolic syndrome which are related to diabetes and insulin resistance, such as visfatin and ghrelin are significantly reduced compared with subjects who do not alter their dietary phylloquinone intake amount. meanwhile it is reported that adiponectin, which is well known adipokines that has positive regulation for insulin sensitivity are significantly increased after dietary dose of phylloquinone (1000mg per day for 4 weeks) in premenopausal women (41). 3-anti-inflammatory function of vitamin k: the most commonly-researched proinflammatory cytokines that induce insulin resistance are tnf-α and il-6 (42, 43). the study has shown an inverse relation between the state of vitamin k and inflammatory circulatory markers. the mechanism behind vitamin k's beneficial function against production of proinflammatory cytokines is uncertain. nevertheless, vitamin k has been shown to inhibit nf-kb leading to decline in production of il-6 and other type’s cytokines (44). effect of vitamin k on cancer vitamin k2 was studied as supplement of cancer treatments in many clinical interventions (45). in vitro experiments, supplementation of k2 was found alone to stop numerous cancer cell lines from growing and metastasizing (46, 47). the mechanisms through which vitamin k2 inhibits cancer growth and metastasis, in brief, vitamin k2 can function in many ways, such as protein kinase c, protein kinase a, steroid, kappa b nuclear , and xenobiotic receptor (48). in addition, there are numerous cases in which k2 addition alongside standard therapy subside the progression of cancer, including some cases in which patients have reached full remission (45,49). the action of vitamin k2 as an anti-cancer agent is not restricted to a definite type of cancer but has been identified in many forms of cancer (48) . effect of vitamin k on kidney the state of dephosphorylateduncarboxylated-mgp (dp-ucmgp) is an established vitamin k deficiency research marker first recorded in chronic kidney disease (ckd) patients (50) . dpucmgp is attributed to ckd progression, since later stage ckd people have high circulating concentrations of dp-ucmgp (51, 52,53,54). supplementation of vitamin k2 shows improved function of renal artery and help stop further progression of calcification in renal artery (55). furthermore, vitamin k2 supplementation has been shown to enhance glomerular filtration in a treatment protocol. in ckd researches the importance of vitamin k2 is excellent and many comprehensive studies are under way using vitamin k addition to treat ckd patients (56, 57) . effect of vitamin k on immune system over the last few years in vivo experiments explained a previously unidentified immunomodulatory function of vitamin k2. firstly, it has been shown that mk-7 modulated il-1alpha, iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 8 tnf alpha, and il-1β expression (58). besides that, k2 lowers t-cell proliferation in healthy subjects, whilst still vitamin k1 has no influence. it has been also confirmed by t-cells from numerous children suffering from atopic dermatitis and healthy children, as well as other study including patients on dialysis (59, 60). both studies have shown that k2 reduces the number and proliferation of activated tcells. accumulated evidence thus shows that k2 has a new role as an immune-suppressive agent. this requires further explanation; till now, a unique physiological mechanism can be speculated by which vitamin k2 can promote immunomodulation but needs further study (58, 61). effect of vitamin k on skin health wound healing considered as a complex and essential physiological process which involves the cooperation between many cell strains and their products. the stages of wound healing include coagulation, epithelization, collagenation and tissue renovating. accelerating the healing of skin injuries is a very important issue for patients and physicians. vitamin k's topical application raises the rate of wound contraction significantly. topical vitamin k was reported to stimulate wound healing, possibly because it can significantly increase the rate of wound contraction, enhance epithelialization duration, build up of fibroblast cells, formation of collagen fibers and blood vessels and increase in hydroxyprolin quantity in the experimental models. in addition, the blood coagulation system is reported to facilitate angiogenesis and wound healing. since vitamin k has an impact on γ -carboxylation of some coagulation factors, its effect upon blood coagulation systems can be the reason of the wound healing action of vitamin k. other studies show that vitamin k is effective antioxidant and it can also improve wound healing on the basis of its antioxidant properties. vitamin k can therefore be used in addition to other established therapies in patients with acute and chronic skin injuries as a supplemental medication (62, 63) . effect of vitamin k on testosterone hormone vitamin k (k) is essential in post translation modifying of blood clotting factors and proteins in the bone matrix (gla proteins). in the previous study, dna microarrays were used and genes whose expression in the k-deficient state of the testis was affected were identified. in the vitamin k deficient group, expression of genes participating in the biosynthesis of cholesterol and steroid hormones was reduced. the mrna levels of cyp11a (a testosterone synthesis rate-limiting enzyme) is positively correlated by menaquinone-4 (mk-4) level in the testis. in addition, the plasma and testis of the k-deficient population had lower testosterone concentrations in comparison with the control and vitamin k-supplemented groups. these findings indicate that k is participated in the production of steroids in the testis via the cyp11a regulation (64) . deficiency of vitamin k the vitamin k requirement depends on age and gender. table 2 displays the average recommended daily quantities in micrograms (mcg). table 3. amount of vitamin k needed in different groups of people (65) . age male female pregnant lactating 0 – 6 months 2.0 mcg 2.0 mcg 7 – 12 months 2.5 mcg 2.5 mcg 1 – 3 years 30 mg 30 mg 4 – 8 years 55 mcg 55 mcg 9 – 13 years 60 mcg 60 mcg 14 – 18 years 75mcg 75mcg 75 mcg 75 mcg 19 – 50 years 120 mcg 90 mcg 90 mcg 90 mcg 51 – 70 years 120 mcg 90 mcg 70 + years 120 mcg 90 mcg deficiency of vitamin k is very rare. many individuals receive adequate vitamin k from their foods. bacteria in the colon also produce some vitamin k which can be absorbed by the body. however, some groups of people can find it difficult to get adequate vitamin k: 1children: osteocalcin is among the most common proteins in the body and is at least 10-times higher during growth than when the peak of bone mass has been achieved. it means that children have a much greater vitamin k need than adults. unfortunately, an rise in snacks and fast-food consumption has led to a decline in child vitamin k intake year after year (66). 2pregnant women. vitamin k is a fat-soluble vitamin that ensures it is not distributed easily through the placenta. however, the growing fetus requires vitamin, which is derived from the bloodstream of the mother. the vitamin k level of mother was shown to decline in particular during the iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 9 third trimester of pregnancy and the addition of vitamin k improved carboxylation of osteocalcin in both mothers and the offspring (cord blood measurement) (67). 3early infancy. there is a short window from conception to 6 months if the human baby is exposed to a minimal but life-threatening hazard of bleeding. late onset vitamin k bleeding (occur at ages between 3-8 weeks) usually leads to death or permanents neurological damage in infants due to intracranial bleeding, the main nutritional danger for infants who've been breastfeeding predominantly (68) 4elderly. osteocalcin carboxylation (as an indicator for generic vitamin k state) has been reported to decrease after 50 years. it can be associated with a decreased intake of food, poorer intestinal absorption or increased demand. it correlates significantly with the beginning of agerelated symptoms such as calcification of blood vessels and increased bone loss. such processes are, of course, multi factorial, but risk factors accumulation typically increases the risk (69). 5people with intestinal absorption impairment. crohn's disease, cystic fibrosis, and galactosemia are all reportedly chronic diseases linked to poor vitamin k status (70) . 6some medicines that can interfere with vitamin k. such examples are as follows: ● warfarin (coumadin ®): vitamin k interacts seriously with warfarin (bloodthinner drug). if warfarin is taken it must ensure that the same amount of vitamin k taken from diet and supplements every day. abrupt alteration in vitamin k consumption may result in harmful bleeding (when less consumed) or blood clots (if more consumed) (71). ● antibiotics may kill good intestinal bacteria. many of these bacteria produce vitamin k. consuming antibiotics for even more than a few weeks may decrease the quantity of vitamin k produced in the intestines and thus the amount accessible for the body to be using (72). ●bile acid sequestrants drugs (like cholestyramine [questran ®] and colestipol [colestid ®] which are used by certain people to reduce cholesterol level in blood. these medicines can reduce body's absorption of vitamin k particularly if were taken for many years (73). ● orlistat (alli ® and xenical ®) is a therapeutic drug that loses weight. this decreased fat absorption in the body and can lowered vitamin k absorption (74) . vitamin k importance in infants transportation of vitamin k over the placenta is low and the hazard of vitamin k deficit in the newborn babies is increased. deficiency in vitamin k may lead to vitamin k deficiency bleeding (vkdb), a disorder previously known as "classic hemorrhagic disease of newborn," within the first few weeks of life. vkdb is linked to umbilical, gastrointestinal, nose, skin, and other sites bleeding. vkdb is described in the first week of existence as a "early vkdb" while " late vkdb" exists at 2-12 weeks of age, particularly in breastfed infant children because of the low level of vitamin k in breast milk or malabsorption trouble in infants (such as cystic fibrosis or cholestatic jaundice) (75). vkdb, especially late vkdb, may also appear as abrupt cranial bleeding which has a high death risk. the american academy of pediatrics recommends a single dose of vitamin k1(0.5 -1 milligram (mg)) given intramuscularly at birth, in order to avoid vkdb (76). safety and toxicity of vitamin k while allergic reactions may be likely, no well-known toxicity linked to high doses of vitamin k1 (phylloquinone) or vitamin k2 (menaquinone). the same would be not correct for synthetic vitamin k3 (menadione) as well as its derivatives. menadion can inhibit the function of glutathione, a natural antioxidant in the body, which contributes to oxidative stress related cell membranes damage. menadione administered by injection caused jaundice, hepatotoxicty, and hemolytic anemia (because of erythrocytes rupture) in babies; thus menadione is not used for deficiency of vitamin k anymore (77). in addition, high doses of vitamin a and vitamin e were establishing to antagonize vitamin k. large dose of vitamin a effect the absorption of vitamin k while vitamin e can suppress vitamin k activity in γ-carboxylation of glutamate residues and influence the cascade of coagulation (78). people who have taken anticoagulatory drugs such as warfarin and those with a deficiency in vitamin k are not allowed to take vitamin e supplements without near medical attention due to increased risk of excess bleeding (haemorrhage) (79;l). conclusion vitamin k is an essential fat soluble vitamin which is important for blood clotting. it serves as a coenzyme which facilitating the gamma carboxylation of calcium binding proteins' glutamic residues. beside its effect on coagulation vitamin k has a number of possible impacts on human health and that low levels of vitamin k are related to different aspects of human diseases. several clinical studies hypothesized that a vitamin k rich diet and / or vitamin k treatment might have numerous health benefits which includes, preventing bone mass loss, delay the development of blood vessels calcification, prevent old age degenerative diseases, improve blood sugar balance, stop numerous cancer lines from growing and metastasizing, enhance function of renal artery, accelerate healing of skin injuries, modulate immune function as well as increase production of steroids in the testis. larger trails should be made to better clarify the detrimental iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 10 long-term impact of vitamin k deficiency, and whether these can be avoided by additional vitamin k institution. references 1. dam, h.; schønheyder, f. the occurrence and chemical nature of vitamin k. biochem. j. 1936, 30, 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61:19-21. iraqi j pharm sci, vol.30(1) 2021 vitamin k and human health 13 74. mcduffie jr, calis ka, booth sl, uwaifo gi, yanovski ja. effects of orlistat on fat-soluble vitamins in obese adolescents. pharmacotherapy 2002; 22:814-22. 75. pichler e, pichler l. the neonatal coagulation system and the vitamin k deficiency bleeding a mini review. wien med wochenschr. 2008; 158:385-95. 76. american academy of pediatrics committee on f, newborn. controversies concerning vitamin k and the newborn. american academy of pediatrics committee on fetus and newborn. pediatrics 2003; 112:191-2. 77. ferland g. vitamin k. in: erdman jr. jw, macdonald ia, zeisel sh, eds. present knowledge in nutrition. 10th ed. ames: wileyblackwell; 2012:230247. 78. olson re. vitamin k. in: shils m, olson ja, shike m, ross ac, eds. modern nutrition in health and disease. 9th ed. baltimore: lippincott williams &wilkins; 1999:363-380. 79. booth sl, golly i, sacheck jm, et al. effect of vitamin e supplementation on vitamin k status in adults with normal coagulation status. am j clin nutr. 2004;80(1):143-148. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 5-fu-plga nanoparticles for the treatment of lung cancer doi: https://doi.org/10.31351/vol31iss1pp130-143 130 development of 5-fu loaded poly lactic-co-glycolic acid nanoparticles for treatment of lung cancer sankha bhattacharya*,1 *department of pharmaceutics, school of pharmacy & technology management, svkm's nmims. deemedto-be university, shirpur, maharashtra 425405, india abstract non-small cell lung cancer (nsclc) accounts for about 84% of all lung cancer types diagnosed so far. every year, regardless of gender, the nsclc targets many communities worldwide. 5-fluorouracil (5-fu) is a uracil-analog anticancer compound. this drug tends to annihilate multiple tumour cells. but 5-fu's most significant obstacle is that it gets very easily metabolized in the blood, which eventually leads to lower anticancer activity. therefore, a perfect drug delivery system is needed to overcome all the associated challenges. in this experiment, an attempt was made to prepare 5-fu loaded poly lactic-co-glycolic acid nanoparticles using solvent evaporation method and subsequently observed the effect of molecular weight of poly lactic-co-glycolic acid, loading of poly lactic-co-glycolic acid, sonication period on the cytotoxic effect of 10 % w/w 5-fu loaded plga nanoparticles against human a549 isogenic cell line. in this experiment, two points are more evident: first, poly lactic-co-glycolic acid has a major impact on 5-fu release due to higher degradation and rate of diffusion in nanoparticle solution; and second, nanoparticles with a larger surface area and smaller particle size have a lower half-maximal inhibitory concentration (ic50) value. the ic50 of all nanoparticles was significantly higher (p=0.0145) than that of the free 5-fu controlled group (8.34nm). the cytotoxicity would be greater if the ic50 value was lower. nanoparticles with an 18-minute sonication time was found to be more cytotoxic than those with plga nanoparticles containing 12% polyvinyl alcohol. in this experiment 10% w/w 5-fu loaded poly lactic-co-glycolic acid nanoparticles was prepared for laboratory research to translational research for the treatment of lung cancer. keywords: non-small cell lung cancer (nsclc), 5-fu, plga, polymeric nanoparticles, a549 isogenic cell line, ic50 value introduction lung cancer has the highest mortality rate of all cancer types worldwide(1-3) . lung cancer is a term used to describe both non-small cell lung cancer (nsclc) and small cell lung cancer (sclc) (4). there are almost 87% of these two nsclc events. it has a very poor prognosis, the greatest problem for nsclc; only 15% of patients live up to 5 years with a greater chance of recurrence (5). chemotherapy and radiotherapy, which also have severe complications, i.e., poor selectivity, resistance, tissue toxicity, myelosuppression, etc., are currently available for any lung cancer treatment (6). the normal iv administration of chemotherapeutic medicaments demands a high dose with the consequence of savior toxicity; the chemotherapeutic drugs eventually kills cancerous cells as well as adjacent healthy cells (7).there are many benefits of encapsulating chemotherapeutic drugs in polymeric nanoparsecs, such as enhancing drug solubility, targeted drug delivery, shielding the drug from bloodstream degradation, low side effects, increasing the duration of drug exposure, and slowing the clearance time from the body (8). many pharmaceutical grades or food and drug administration (usfda) certified polymers are available on the market that could provide the best drug delivery efficiency, i.e. polycaprolactone (pcl) poly lactic-co-glycolic acid (plga) (9). the toxicity of chemotherapeutic drugs may have been decreased by these polymers when encapsulated inside. in addition, proper engineering of these polymers may result in polymeric nanoparticles that may accumulate on the surface of the tumor's body (10). the targeting of tumour cells may be carried out through active or passive targeting (11). the tinny size of the nanoparticles helps them to accumulate in the leaky tumour vasculature environment so that in the presence of a poorly designed lymphatic drainage system they could easily penetrate within the tumour (12).the reticuloendothelial system (res) can easily destroy poorly engineered hydrophobic polymeric nanoparticles or particles that have a particle size below 200 nm, or else they can quickly take over by the liver, spleen, and lungs. this may sometimes contribute to the development of palmar-plantar erythema (13). 1corresponding author e-mail: sankhabhatt@gmail.com received:30 /6/2021 accepted:5 /9/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp130-143 iraqi j pharm sci, vol.31(1) 2022 5-fu-plga nanoparticles for the treatment of lung cancer 131 on the other hand, the bloodstream of those polymeric nanoparticles that have a hydrophilic surface showed an increased circulation time. hydrophilic polymeric nanoparticles are therefore a possible candidate for targeting cancer cells (14). however, nanoparticles must design with particle sizes below 200nm to avoid macrophagic uptake. in preclinical research, docetaxel, a bcs-iv medication, has been shown to have significant similarities compared to traditional formulations with rapid administration time (15).there are many studies suggesting cell lines like., a549 can be targeted with plga nanoparticles' help when the plga surface was engineered with lfc131 peptide and doxorubicin (16). as far as nsclc cells are concerned, it was often found that they produced resistance to chemotherapy and radiotherapy from the stem cells of the said cancer cells; hence, the recurrence rate of nsclc is more (17). in lung cancer treatment, 5-fluorouracil (5fu), a chemotherapeutic hydrophilic agent, is commonly used. 5-fu is an inhibitor of thymidylate synthesis that inhibits excess dna proliferation (18). it has, however, a low biological half-life and insufficient biodistribution, which derives its possible therapeutic significance form (19). usually, this drug is administered using iv route, but due to the inability to act on the target site, this cytotoxic drug produces a lack of site-specificity and adverse effects (10, 20). in the body, 5-fu can sometimes produce reactive oxygen species (ros). the activation of nuclear factor kappa-light-chain-enhancer of activated b cells (nfjb), which prevents ros and ros-induced cytotoxicity, will counterbalance the accumulation of drug-induced ros(21). but the main concern with 5-fu is that the, 5-fu is metabolized to fluorodeoxyridine monophosphate in the liver or intestinal mucosa by dihydrouracil dehydrogenase gets into the blood very easily. a drug delivery system that could safeguard 5-fu from bloodstream metabolism and target nsclc would therefore be a successful translation research system (18). while preparing a stable nanoparticle, this study will examine the various forms of plga and certain process variables. in addition, the in-vitro cytotoxicity of the prepared formula against human a549 isogenic lung cancer cell line (atcc eml4alk fusion,pune,india) will also be tested (22). material and methods materials poly (lactide-co-glycolide) (plga) copolymers of different grades with molecular weight ratios, i.e., expansorb®(lactide:glycolide75:25) mw(100 kda), expansorb® (lactide:glycolide 50:50) mw (50 kda), expansorb® (lactide:glycolide 50:50) mw (20 kda) was purchased from sigmaaldrich (sigma aldrich–merck, bengaluru, india), 5-fu was the gift sample form neon laboratories, mumbai, india. tween 80, methanol (hplc grade), water (hplc grade), polyvinyl alcohol (pva) different molecular weight, sucrose and phosphate buffer saline (pbs ph 7.4), were purchased from sisco research laboratories pvt. ltd. (srl), mumbai, india preparation of 10% w/w 5-fu-loaded plga nanoparticles the solvent evaporation technique was used to make 10 w/w 5-fu loaded plga nanoparticles (this is equivalent to mixing 10 mg of fu with 90 mg of plga). in this experiment, total of twelve formulations have been prepared. among these, three were prepared by considering three different plga, i.e., (1) expansorb® (lactide: glycolide 75:25) dlg 759e; which has molecular weight 100 kda and end terminal ester group. (2)expansorb® (lactide: glycolide 50:50) dlg 50-5a; which has a molecular weight of 50 kda and end terminal -cooh group. (3) expansorb® (lactide: glycolide 50:50) dlg 50-2a; which has a molecular weight of 20 kda and end terminal -cooh group. further, altering pva loading (4%, 8% & 12%), sonication time (6 min, 12 min & 18 min) and pva molecular weight (31 kda, 47 kda & 130 kda) more nine formulations has been prepared considering plga (lactide:glycolide 50:50); dlg 50-2a as a constant class of plga.the 50 mg of 5-fu and 450 mg of plga were dissolved into 30 ml of methanol for this preparation (23). transfer 120 mg of polyvinyl alcohol(pva) to another 50 ml beaker and dissolve it at 45ᵒc in 30 ml of water. thermodynamic stability of such isotropic system was improved by using pva.in addition, the plga-5-fu solution was applied dropwise using an 18g needle to the pva aqueous solution and kept for 60 minutes for stirring (24). the resulting solution was sonicated for 6 minutes at 60% power, then left overnight at 10,000 rpm for 480 minutes, with the supernatant removed. the pellets were resuspended in 30 ml distilled water and centrifuged three times for 30 minutes, with the supernatant removed after each centrifugation. by decantation, supernatant was removed.finally, washed nanoparticles were suspended with 2.5 % d-trehalose solution in 20ml and refrigerated using cryogenic freezer (model no: mr-hf-ir-a3a10, lab freez instruments, chaina) for 24 hours at -85ᵒc and further freeze-dried for 72 hours. until necessary, the dried nanoparticles are stored in refrigeration conditions. table 1 shown the prepared 5-fu-plga nanoparticles formulas. iraqi j pharm sci, vol.31(1) 2022 5-fu-plga nanoparticles for the treatment of lung cancer 132 table 1.the formulation variables and sonication times of the 10% w/w 5-fu loaded poly lactic-co-glycolic acid nanoparticles. the shaded areas indicate the parameter that was varied for that particular group of formulations. formulations variables plga pva loading (%) sonication time (min) pva mw (kda) expansorb® (lactide:glycolide 75:25) mw(100 kda) expansorb® (lactide:glycolide 50:50) mw (50 kda) expansorb® (lactide:glycolide 50:50) mw (20 kda) 4 % pva 8 % pva 12 % pva 6 min 12 min 18 min 31 kda 47 kda 130 kda plga class dlg 75-9e dlg 50-5a dlg 50-2a dlg 50-2a dlg 50-2a dlg 50-2a dlg 50-2a dlg 50-2a dlg 50-2a dlg 50-2a dlg 50-2a dlg 50-2a pva loading (%) 5 5 5 4 8 12 5 5 5 5 5 5 sonication time (min) 10 10 10 10 10 10 6 12 18 10 10 10 pva molecular weight (kda) 75 75 75 75 75 75 75 75 75 31 47 130 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 133 morphology and size of the 10% w/w 5-fu loaded plga nanoparticles using the jsm-it800 field emission scanning electron microscope, scanning electron microscopic images were taken of 10% w/w 5-fu loaded plga nanoparticles (25). during the operation process, a pinch amount of nanoparticles was added to the carbon tab and further coated with gold. the particle size and zeta potential were measured by the delsanano c instrument (beckman coulter, u.s.a.). during the determination of the particle size, 200μl of nanoparticles were suspended in 4ml of deionized water and further sonicated for 5 min to remove suspended air bubbles. the measurement was taken at 25ᵒc. the encapsulation efficiency of the 10 % w/w 5fu-loaded plga nanoparticles to evaluate the encapsulation efficacy of polymeric nanoparticles, 5 mg of lyophilized nanoparticles were dissolved in dichloromethane from each sample (26). the resulting mixture was kept overnight for drying at room temperature. later, the residue is dissolved in methanol and filtered with a 0.22 μl membrane filter (millipore). the filtered samples were further analysed using the rp-hplc process (27) in-vitro release of 10% w/w 5-fu loaded plga nanoparticles the 5mg of lyophilized nanoparticles from each batch (n=5) were placed in the dialysis beg consisting of 1.5% of tween 80 (28, 29). the temperature was kept at 37ᵒc throughout the experiment, and magnetic stirring at 50 rpm was used to keep the dissolution medium uniform.subsequently, the beg was put in a beaker containing 40 ml of 1% w/v polysorbate 80 solutions. each time a 400μl sample was extracted from the beaker at a fixed time interval and a fresh 400μl sample, 1% w/v polysorbate 80 was placed to preserve the sink condition. the experiment went on for fourteen days. the resulting liguid samples were further analysed using the rp-hplc process. determinations of the serum half-life of 5-fu in the 10% w/w 5-fu loaded plga nanoparticles the 5mg of 10% w/w 5-fu loaded plga nanoparticle formulations was drained in 500 ml of human serum albumin (hsa), and the mixture was gently shaken at 37ᵒc and 13000 rpm for fifteen minutes to determine the half-life of the 5-fu (30).the supernatant was extracted and the specified particles were washed three times with 1ml of distilled water. in addition, the particles were dissolved in dichloromethane and left overnight. the resulting residue mixture was further combined with methanol and filtered using a 0.22 μm membrane filter. the filtered samples were also analysed using the rp-hplc process (31, 32). estimation of 5-fu loaded plga nanoparticles stability by rp-hplc method 5-fu 5 mg of each 10% w/w 5-fu loaded plga nanoparticle formulation (n = 5) was considered for the sample preparation. the hplc analysis was performed on 1260 infinity ii prime lc system (agilent, ca) with acclaim™ 120 c18 reversed-phase analytical hplc columns, 3µm (thermo fisher scientific, usa). the mobile phase comprises methanol:water (75:25%). the flow rate was 1.5 ml/min, where else, uv detection was performed at 266nm with an injection volume of 10 µl. after necessary modifications to the anitha, a., et al. procedure, the samples were prepared(33). the cytotoxicity of the 10% w/w 5-fu-loaded plga nanoparticles against human a549 isogenic cell line for in-vitro cytotoxicity analysis, human a549 isogenic cell line was cultured for 48 hr.(5000/well in 96 well flat-bottomed microtiter plates). the cultured cells were exposed with different concentrations, i.e., 0.5nm, 1nm, 5nm, 15nm, 20nm, 25nm 50nm, 75nm, 100nm, 125nm, 150nm of 15% w/w 5-fu loaded plga nanoparticles and further 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium 236 bromide (mtt) assay was performed (34).the native 5-fu was considered as the control group, and the experiment was repeated thrice, and further, ic50 was calculated. statistical analysis statistical analysis was performed using anova (graphpad prism version 8.0). post-hoc comparisons of the mean were performed using tukey's honestly significance test. a significance level of p<0.05 was accepted to denote significance in all cases. results and discussion surface morphology, particle size, and zeta potential of the 10% w/w 5-fu loaded plga nanoparticle the surface morphology of the prepared 10% w/w 5-fu loaded plga nanoparticles were analyzed using sem (figure. 1). iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 134 figure.1. sem image of 10% w/w 5-fu loaded plga nanoparticles it was confirmed from the sem image that the prepared nanoparticles are spherical in shape with a specific smooth surface area without any particular pitting and no characteristic agglomeration or mushroom effects have been observed. the smooth surface of the nanoparticles suggests that; there is little risk that the 5-fu drug leached out of the encapsulated nanoparticles; in other words, there was no such characteristic proof that the drug particles adhered to the outer surface of the nanoparticles. during the process of preparation, the unentrapped 5-fu was washed out. if free 5-fu remains on the nanoparticles' surface, the initial burst effects may be impaired and the encapsulation efficacy subsequently slowed down. pitting in nanoparticles, on the other hand, could promote the nanoparticles' low durability. in addition, plga would become more soluble at altered ph conditions due to pitting, and thus could increase the release of the drug with improved degradation. possible drug release at the target site will then become difficult. it is very difficult to disperse aggregated nanoparticles; often these aggregated nanoparticles become a cause for palmar-plantar erythrodysesthesia. depending on the sonication time and formulation variables, all the particles size of the prepared nanoparticles were obtained in the range of 156.36 nm to 198.34 nm (table 2 & figure. 2). an attempt was made during preparation to prepare nanoparticles with a narrow distribution and smaller particle size (below 200 nm) during preparation, which will eventually help nanoparticles stay stable with less macrophagic absorption in systematic circulation. the reticuloendothelial opsonization process could be prevented by bellow 200 nm particles (35)(36) it was found that different forms of plga had no important effects on the size of the nanoparticles during particle size characterization (p=0.114). however, the particle size of the nanoparticles is significantly reduced by increased pva loading and sonication time (p=0.012). pva molecular weight increases (mowiol®-31 kda, 47 kda, 130 kda) have not been reported to have a major effect on particle size (p=0.136). however, as the molecular weight of pva increases to 130 kda, the particle size decreases significantly (p=0.0118). large increases in higher molecular pva stabilise the nanoparticles' surface by preventing agglomerate from being collected. on the contrary, due to higher shearing stress, increasing sonication time decreases particle size. the increased molecular weight of pva reduces particle size because pvp has less aqueous phase interactions with higher molecular weight. the reduced size of the particle was thus obtained. as far as the surface charge of nanoparticles is concerned, almost all batches of nanoparticles have shown higher electrostatic charges, which means that cationic zeta potential has been observed (+25.56mv to +33.67mv). the nanoparticles' cationic zeta potential has a beneficial impact on binding to the extracellular environment that has been negatively charged (37). it was found that the outer surface of cancer cells has a negative charge; thus, cationic or positive nanoparticles can easily connect with cell membranes that are negatively charged. nevertheless, in contrast to nonionic or anionic nanoparticles, cationic nanoparticles have more penetrating properties with greater cytotoxicity (38, 39). one crucial finding from this research outcome also established that, as such, there is no association between plga's altered molecular weight, and zeta potential. iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 135 table 2.particle size and zeta potential for the 10% w/w 5-fu loaded nanoparticles (mean ± sd) variables plga pva loading (%) sonication time (min) pva mw (kda) dlg 75-9e dlg 50-5a dlg 50-2a 4 8 12 6 12 18 31 47 130 particle size (nm) 169.27± 7.08 168.21± 6.21 166.36± 7.34 198.34± 9.34 171.45± 6.34 164.25± 8.26 175.12± 6.62 166.35± 5.56 158.04± 8.23 193.23± 8.34 187.34± 8.35 156.36± 7.21 zeta potential (mv) 25.27± 6.36 28.27± 3.78 32.71± 3.11 31.56± 4.35 28.51± 2.11 25.719± 8.35 31.29± 2.35 27.15± 4.11 25.56± 2.26 33.67± 2.56 31.56± 2.36 30.22± 1.36 encapsulation efficiency (%) 85.45± 6.37 90.34± 4.56 84.11± 4.62 98.25± 4.81 87.17± 5.78 75.67± 5.12 96.27± 8.29 84.12± 6.36 74.28± 8.34 95.34± 8.45 86.25± 8.48 72.56± 7.37 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 136 d lg 7 59e d lg 5 05a d lg 5 02a 4% 8% 12 % 140 150 160 170 180 190 200 210 220 16 4. 3 17 1. 5 16 6. 4 16 8. 2 16 9. 3 p ar ti cl e s iz e (n m ) plga pva loading ✱ 31 kd a 47 kd a 13 0k da 6 m in 12 m in 18 m in 140 150 160 170 180 190 200 210 220 15 8. 0 16 6. 41 75 .1 15 6. 4 18 7. 2 19 3. 2 p ar ti cl e s iz e (n m ) ✱ pva sonication time (a) (b) figure 2. particle size (a and b) 10% w/w 5-fu loaded plga nanoparticles. * indicates a significant difference the encapsulation efficiency of the 10% w/w 5-fu loaded nanoparticles: the nanoparticles encapsulation efficiency ranged from 72.56±7.37% to 98.25±4.81% on the basis of sonication period and formulation selection. due to the presence of the same polymer ratio (50:50) and the same molecular weight, the selection of the plga polymer had no important effect (p=0.212) on the encapsulation efficacy of the nanoparticles. other experimental results have also shown that plga can affect the product's encapsulation quality, but only when the lactide to glycolide ratio differs. nevertheless, plga's lower molecular weight in an organic solvent would be highly soluble; thus, low encapsulation is probable. in this experiment, it was observed that the encapsulation efficiency of the nanoparticles reduces when plga loading increases (p=0.0345), because of deceased nanoparticle size with increased loading ability of pva. smaller nanoparticles will have less room to encapsulate 5fu; low efficiency of encapsulation can be observed by hance. one hypothesis, however, can also be proposed, i.e. increased pva concentration may bind with 5-fu; thus, the efficiency of 5-fu encapsulation decreases. from the current experiment, it was also observed that encapsulation efficiency (p=0.0345) of the nanoparticles decreases significantly when the molecular weight of pva is increased; due to the size decrease of the nanoparticles. the particle size also decreases with longer sonication time; thus, the efficiency of encapsulation decreases. one theory also indicates that increasing sonication time can assist degradation of plga within the polymeric solution to reduce the loading potential of plga for 5-fu, so that increased plga permeability can occur, allowing the drug to spread out during washing. figure 3 shows the effect of plga, pva loading, pva molecular weight, and sonication time on drug entrapment efficacy (%). samira khaledia et al. (2020)(40) recently developed polymeric nanoparticles for the delivery of 5fluorouracil (5-fu) and chrysin. the polymers used in this experiment were plga and peg. in this study, 5-fu was encapsulated into the polymeric matrix using the double emulsions solvent evaporation method (w1/o/w2). the nanoparticles of 5-fu and chrysin were determined using hplc methods. the encapsulation efficacy of 5-fu was found to be 81.3 % in a dual drug delivery system. however, the highest encapsulation efficiency of the 10% w/w 5-fu loaded nanoparticles was found to be 98.254.81% in the current experiment. in this study, the encapsulation efficacy of 5-fu was improved. iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 137 d lg 7 59e d lg 5 05a d lg 5 02a 4% 8% 12 % 60 65 70 75 80 85 90 95 100 105 110 7 5 .7 8 7 .2 8 4 .19 0 .3 8 5 .5 ✱ pva loading plga e n ca p su la ti on e ff ic ie n cy ( % ) 31 k d a 47 k d a 13 0 kd a 6 m in 12 m in 18 m in 60 65 70 75 80 85 90 95 100 105 110 7 4 .3 8 4 .1 7 2 .6 8 6 .3 e n ca p su la ti o n e ff ic ie n cy ( % ) ✱ pva sonication time (c) (d) figure 3. encapsulation efficiency (c and d) of the 10% w/w 5-fu loaded plga nanoparticles. * indicates a significant difference in-vitro 5-fu release from 10% w/w 5-fuloaded plga nanoparticles figure 4 (a-d) shows the in vitro release of 5-fu from plga nanoparticles over a period of 14 days. the standard diffusion-controlled drug release profile of all nanoparticles is due to the presence of plga. for the release of 5-fu from the nanoparticles, the selection of plga has paramount significance (p=0.0234). the degradation rate of the plga has been found to affect the release profile of nanoparticles. plga-dlg 75-9e has a degradation rate for two weeks, and plga-dlg 50-5a & plga-dlg 50-2a have a degradation rate for more than one month, according to the literature survey and recent research findings (41-44). from figure 4(b) it can be concluded that; upon increases, the concentration of pva (4-12%) would significantly (p=0.0145) increase overall surface area. the increased surface area of the nanoparticles has a significant burst effect for 8% pva loaded nanoparticles at 2nd day. however, it was also observed that increasing pva concentration up to 12% would significantly (p=0.012) decrease the release profiling of 5-fu from the nanoparticle surface; this is due to the formation of high coating over the polymeric nanoparticle surface and the construction of self-conjugation between 5-fu and pva. from figure 4(c), it was confirmed that as such, there were no significant defenses (p=0.345) reported in the release of 5-fu from the nanoparticles from day 1 to 7, containing pva with a molecular weight of 31 kda to 130 kda. however, when the molecular weight of pva increased up to 130 kda, the nanoparticles' release patterns (p=0.0145) increased; this is due to the reduced particle size and higher surface area of the nanoparticles. figure 3(c) shows that increasing sonication time would significantly (p=0.0127) increases 5-fu drug release from nanoparticles. higher sonication time (18min) reduces the size of the nanoparticles and increases overall surface area due to the degradation of plga within the solution. ana cristina de mattos et al.,(2016)(45) prepared prolonged-release pla-peg nanoparticles of 5-fluorouracil (5-fu). the in-vitro release of 5fu was performed in the presence of ph 7.4 phosphate buffer solution and at 37ᵒc. in 30 min, pla nanoparticles release approximately 23% of 5fu, and after 24 h, it removed 39% of 5-fu. after 320 h, it released about 52% of 5-fu from plapeg nanoparticles. according to the researchers, the pronging of the 5-fu release could be caused by an anomalous mechanism of drug release as well as a slow erosion and diffusion process from the plapeg surface of the nanoparticles. however, an in-vitro drug release profile of twelve 10% w/w 5-fu loaded nanoparticles was found to sustain and control releases nature, which could achieve almost 70-90% cumulative drug releases within 14 days. iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 138 0 2 4 6 8 10 12 14 0 20 40 60 80 100 120 140 time (days) 5 -f u r e le a s e ( % ) plga (dlg 75-9e) plga (dlg 50-5a) plga (dlg 50-2a) 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 0 10 20 30 40 50 60 70 80 90 100 time (days) 5 -f u r e le a s e ( % ) 4% pva 8% pva 12% pva (a) (b) 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 70 80 time (days) 5 -f u r e le a s e ( % ) pva mw-31 kda pva mw-47 kda pva mw-130 kda 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 70 80 time (days) 5 -f u r e le a s e ( % ) sonication time (6min) sonication time (12min) sonication time (18min) (c) (d) figure 4. in vitro drug release for the 10 % w/w 5-fu loaded plga nanoparticles prepared using different plga polymers (a), pva loadings (b), pva molecular weights (c), and sonication times (d) the ability of the 10% w/w 5-fu loaded plga nanoparticles to protect 5-fu from degradation in serum: it is important to have extensive accumulation in the environment of cancer cells in an unaltered form for the successful anticancer effect of 5-fu. since the half-life of 5-fu in the human body is only 10-20 minutes, generating an excellent anti-cancer effects on the site of action is a limiting step for 5-fu(46). therefore, for the treatment of nsclc, a drug delivery mechanism that prevents 5-fu from getting metabolised in the bloodstream is required. the literature review reported that plga alone could theoretically increase the half-life of the drug from 2 to 7 hrs(47, 48). it is also essential to know how the choice of plga and sonication time can influence the degradation of nanoparticles within the bloodstream. figure 5 indicating that 5-fu loaded nanoparticles significantly (p=0.0121) elevate the half-life of the 5-fu in serum environment, as compared to a native 5-fu drug, which was acting as a control. it has been confirmed from figure 5(a) that the selection of plga has no effect on the increase in the half-life of 5-fu in the serum. more or less, as compared to the control group, the altered plga grades increased the 5-fu half-life from 25 min to 180min. it was also found that approximately 56% of 5-fu remained in the blood serum after 180 minutes. due to its >200nm particle size, the retention of the nanoparticles in systematic circulation increases. the smaller size of nanoparticles makes the possibility of macrophagic absorption obsolete. the plga coating preserves 5fu for a longer time in serum and increases the halflife of 5-fu. in addition, plga's lactide to glycolide ratio was the same with equal molecular weight, sonication time. thus, the nanoparticles' level of permeability becomes identical and, therefore, the serum protection level of 5-fu being equivalent. on the contrary, the percentage of pva used, and molecular weight has significant (p=0.0118) influence on the extension of 5-fu half-life in the blood serum. the 4%, 8%, and 12% pva loaded nanoparticles enhance the half-life of the 5-fu up to 18 min (figure 5b). similarly, at the different molecular weight of pva (31kda, 47 kda & 130 kda) the half-life of 5-fu increases significantly (p=0.0122). in fact, the increased concentration and molecular weight of pva reduces the size of the particles and thus increases the nanoparticles' surface area. therefore, before it is exposed to 5-fu, iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 139 blood serum will have less time to migrate through the nanoparticles, and the risk of 5-fu degradation will be considerably less. the half-life of 5-fu in the nanoparticles decreases dramatically by increasing the sonication time (p=0.0267) (figure 5c). this phenomenon is due to increasing sonication time (18 min); degradation begins to disperse plga through the solution. therefore, with greater serum exposure time, the resulting nanoparticles will have a higher diffusion rate, which eventually causes 5-fu to have a lower halflife. 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 time (mins) 5 -f u c o n te n t (% ) control plga (dlg 75-9e) plga (dlg 50-5a) plga (dlg 50-2a) 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 time (mins) 5 -f u c o n te n t (% ) control 4% pva 8% pva 12% pva (a) (b) 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 time (mins) 5 -f u c o n te n t (% ) control 31kda 47kda 130kda 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 time (mins) 5 -f u c o n te n t (% ) control sonication time (6min) sonication time (12min) sonication time (18min) (c) (d) figure 5. the influence of plga polymer (a), pva loading (b), pva molecular weight (c), and sonication time (d) on the 10% w/w 5-fu loaded nanoparticles to protect from degradation or engulfment from macrophages in blood serum the cytotoxicity of the 10% w/w 5-fu loaded plga nanoparticles against human a549 isogenic cell line figure 6 demonstrates the in-vitro cytotoxicity of the 10% w/w 5-fu loaded nanoparticles against the isogenic a549 cell. figure 5 indicates that all formulations of nanoparticles exhibit cytotoxic effects against the a549 isogenic cell line. however, the ic50 of all nanoparticles was significantly greater compared to the free 5-fu controlled group (p=0.0145) compared to the free 5fu controlled group (8.34 nm). the plga selection, based on different grades, i.e. dlg75-9e, dlg50-5a, dlg50-2a, had an ic50 of 18.45 nm, 17.28 nm, and 17.11 nm, and had no important impact (p=0.274) on nanoparticles' cytotoxicity (figure 6a). the rise in pva loading, i.e. 4%, 8% & 12% with an ic50 value of 36.58 nm, 18.41 nm & 16.11 nm, significantly (p=0.0156) increases the cytotoxicity of the formulation of the nanoparticle (figure 6b). when the ic50 value for pva mw-31 kda, pva mw-47 kda, and pva mw-130 kda was 28.19 nm, 18.17 nm, and 14.33 nm, respectively, the increased molecular weight of the pva increased the cytotoxicity of the nanoparticles marginally (figure 6c). increased sonification time (6min, 8min & 18min) partly increases nanoparticles' cytotoxicity (figure 6d), with ic50 values of 34.62 nm, 18.44 nm, respectively, & 10.33 nm. another main finding from the cytotoxicity studies was that for almost all formulations, the cell viability (%) of the a549 isogenic cell was found to be stabilised at 75 nm concentration. the linear relationship between particle size and ic50 value can be utilized to determine cytotoxicity. it was also observed that the particles which are having less particle size has low ic50 (figure 6e). iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 140 0 15 30 45 60 75 90 105 120 135 150 0 20 40 60 80 100 120 concentration (nm) v ia b il it y ( % ) control plga (dlg 75-9e) plga (dlg 50-5a) plga (dlg 50-2a) 0 15 30 45 60 75 90 105 120 135 150 0 20 40 60 80 100 120 concentration (nm) v ia b il it y ( % ) control 4% pva 8% pva 12% pva (a) (b) 0 15 30 45 60 75 90 105 120 135 150 0 15 30 45 60 75 90 105 120 concentration (nm) v ia b il it y ( % ) control pva mw-31 kda pva mw-47 kda pva mw-130 kda 0 15 30 45 60 75 90 105 120 135 150 0 20 40 60 80 100 120 concentration (nm) v ia b il it y ( % ) control sonication time (6min) sonication time (12min) sonication time (18min) (c) (d) figure 6. the influence of plga polymer (a), pva loading (b), pva molecular weight (c) sonication time (d) on the cytotoxicity of the 10% w/w 5-fu loaded nanoparticles with compared to native 5-fu controlled. the fundamental relationship between particle size and ic50 value of the 10% w/w 5-fu loaded nanoparticles (e). however, it was also understood that the nanoparticles' permeability could also play an important role, particularly for those prepared under 12 min and 18 min sonication time. at 18 min sonication time, with a 14.33 nm ic50 value, the particle size was 156.36 nm. whereas, the ic50 value was significantly higher (p=0.0134) with nearby particle size (164.25 nm) at 12% pva loading, the ic50 value was significantly higher (p=0.0134) (16.11 nm). the lower the value of ic50, the greater the cytotoxicity would be (49). therefore, as opposed to 12% pva loaded nanoparticles, nanoparticles with 18 min sonication time time will be more cytotoxic. for the nanoparticles, which were sonicated for 18 minutes, the same result was observed. ic50 value was found to be 10.33 nm formulation ic50(nm) control 8.34 dlg 75-9e 18.45 dlg 50-5a 17.28 dlg 50-2a 17.11 4% pva 36.58 8% pva 18.41 12% pva 16.11 pva mw-31 kda 28.19 pva mw-47 kda 18.17 pva mw-130 kda 14.33 sonication time (6min) 34.62 sonication time (12min) 18.44 sonication time (18min) 10.33 iraqi j pharm sci, vol.31(1) 2022 mrsa/vrsa nasal carriage of school students 141 during 18 min sonication time, while ic50 was around 14.33 nm with 156.34 nm particle size for those polymeric nanoparticles containing pva with 130 kda molecular weight. this result clearly shows that excess sonic time increases plga's degradation and infusibility in the solution. ultimately, this results in an adequate amount of a549 isogenic cell line drug (5-fu) diffusion and touch timings; thus, increasing cell death. it is evident from fig.4(d) that the nanoparticles prepared with a sonication time of 18 min had a maximum cumulative drug release. as per yves marc dupertuis et al. (2021)., a combination of long-chain n-3 polyunsaturated fatty acids (n-3 pufas) and 5-fluorouracil (5-fu) had significant anticancer effects on ls174t and ht-29 colorectal cell lines. this research suggests that 5fu and n-3 pufas conjugated nanoparticles felicitate simultaneous drug transport to the cancerous site during tumor metastases (50). the findings of this study strongly suggest that 5-fu has significant anticancer effects against a variety of cell lines, which could explain the current research findings. as per, the recent study conducted by aditya nath pandey et al.,(2020) (51) 5-fu can be encapsulated in plga nanoparticles, which could have potential anti-cancer effects against ht-29 and colo-205 colorectal cancer cell lines. after 48 hours of invitro anti-cancer studies against ht-29 cell lines, 5fu encapsulated plga nanoparticles showing 6.6% cell viability, whereas plga nanoparticles showing16.6% cell viability against colo-205 cell lines after 48 hr of studies. there is a strong correlation between the conclusions reached by these two works and those reached by the current research study. conclusions the research centred primarily on the multiple factors affecting the preparation of 10% 5fu loaded poly lactic-co-glycolic acid nanoparticles. the various factors include different forms of plga (dlg 75-9e, dlg 50-5a & dlg 50-2a), pva loading (4% 8% &12%), pva molecular weight (31 kda, 47 kda & 130 kda) & sonication timings (6 min, 12 min & 18 min) had been involved in this experiment. it was evident from the research results that the impact of different types of plga had no major influence on different characteristics except the percentage of drug release of 5-fu loaded nanoparticles, obviously due to the nature of the plga's bio-degradation. the key finding of this experiment is that an increase in plga concentration, pva loading, plga molecular weight, and sonication time will certainly reduce the size of the particles, eventually leading to surface area increases. surprisingly, the particle size of all the formulations was less than 200 nm, eventually preventing reticuloendothelial opsonization and macrophagic uptake. in addition, this research was enriched with some tangible data, suggesting how to generate 10% w/w 5-fu loaded plga nanoparticles from laboratory research to translational research on full-scale progenitor development. acknowledgments i would like to acknowledge dr. r.s. gaud; director, svkm's nmims, shirpur campus, and dr. ambikanandan misra; directorpharmaceutical research, shobhaben pratapbhai school of pharmacy & technology management, svkm's nmims, shirpur, for providing profound motivation while perusing this project. conflict of interest the author declare that they have no conflict of interest. references 1. torre la, siegel rl, jemal a. lung cancer statistics. lung cancer and personalized medicine: springer; 2016. p. 1-19. 2. pilleron s, soto‐perez‐de‐celis e, vignat j, ferlay j, soerjomataram i, bray f, 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toward cancer and normal cell lines. 2021;141:111895. 50. sharma n, kumari rm, gupta n, syed a, bahkali ah, nimesh sjm. poly-(lactic-coglycolic) acid nanoparticles for synergistic delivery of epirubicin and paclitaxel to human lung cancer cells. 2020;25(18):4243. 51. pandey an, rajpoot k, k jain skjjnj. using 5-fluorouracil-encored plga nanoparticles for the treatment of colorectal cancer: the in-vitro characterization and cytotoxicity studies. 2020;7(3):211-24. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 alkaloids of iraqi echinops 26 phytochemical investigation of alkaloids in the iraqi echinops heterophyllus (compositae) enas j. khadim *,1 , alaa a. abdulrasool ** and zainab j. awad * * department of pharmacognosy, college of pharmacy, university of baghdad,baghdad,iraq. * * chairman of baghdad university, baghdad,iraq. abstract alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms . they are a large family of compounds synthesized by plants in addition to the bacteria, fungi, and animals, they often have pharmacological effects. the aim of this study is to isolate and identified alkaloids in a newly studied, wild iraqi plant named echinops heterophyllus. the medicinal importance of alkaloids, on one hand and the absence of any phytochemical investigation on heterophyllus species of echinops genus on the other hand , acquired this study its importance. three alkaloids (named e1, e2 and e3) were isolated from seed plant part by two chromatographic methods: preparative high performance liquid chromatography (phplc) and preparative thin layer chromatography (ptlc), one of them identified as(1-methyl-2,3-dihydro-4(1h)-quinolinone by different chemical analysis like: ultra violet spectrum analysis (uv spectrum), fourier transforms infrared spectra (ft-ir) , elemental microanalysis (chn) and proton 1 h-nmr and carbon 13 c-nmr analysis. key words: echinops, heterophyllus, quinoline alkaloids. دراسة كيميائية نهقهويدات في نبات شوك انجمم انعراقي ايناس جواد كاظم *،1 عبد انرسول عبد انحسين ، عالء ** زينب جهيم عواد و * * . انؼزاق ،بغذاد ،جايؼت بغذاد،كهٍت انصٍذنت فزع انؼقاقٍز ًاننباث انطبٍت ، .بغذاد ، انؼزاق ** رئٍش جايؼت بغذاد ، الخالصة انقهٌٌذاث ىً يجًٌػت ين انًزكباث انخً حصنغ ين قبم انكثٍز ين اننباحاث ًحهؼب دًرا ييًا فً يؼانجت ً حنظٍى انكثٍز ين االيزاض .انيذف ين ىذه انذراصت ىٌ انفصم ً انخؼزف ػهى انقهٌٌذاث انًٌجٌدة فً نباث ػزاقً بزي ًاصغ االنخشار اصًو echinops . نى ٌذرس صابقا االىًٍت انطبٍت نهقهٌٌذاث ً ػذو ًجٌد ينشٌراث ػهًٍت حخناًل انًكٌناث انكًٍٍائٍت نيذه اننبخت اكضبج ىذه انذراصت اىًٍخيا .حى فصم ,m.p, ft.ir, chn)ثالد قهٌٌذاث ين االجزاء انًخخهفت ين اننباث ً انخؼزف ػهى انخزكٍب انكًٍٍائً نٌاحذ ينيا بٌاصطت قٍاس h 1 nmr and c 13 nmr) : انقهويدات ، نبات شوك انجمم انكهمات انمفتاحية introduction echinops, a genus includes many plants which are individually referred to as globe thistle, is made up of more than 120 species of perennials, annuals, and biennials (1,2) . the genus belongs to the daisy family asteraceae, and its species are found in eastern and southern europe, tropical and north africa and asia (3). in iraq ali al-rawi mention 11 species of echinops in his book (4) among them e. heterophyllus p.h. davis family (compositae) which was chosen for this study. this plant was wildly grown and widely distributed in iraq specially in erbil and sulaimani. in hanara village and surrounding area in wadi bastora and shaklawa in erbil governorate, the plant is called (shakroka). the term (shakroka) is come from that the circle-like part of the plant, before getting harderd in the late spring, is eaten and the taste is sweet, therefore, it is called shakroka: shakr means sugar , shakroka sweet like sugar (oral communication) . the echinopus heterophyllus is a perennial, 40-100 cm high.stems are simple or branching from the base, sparsely cobwebbycanescent. leaves are lanceolate or oblonglanceolate. (5) echinops plant was reported to possess variety of compounds belonging to various classes like: alkaloids, flavonoids, terpenoids, lipids, steroids and polyacetylenes (6) . 1 corresponding author e-mail:enassara@yahoo.com received: 23/1/2013 accepted: 5/2/2014 http://en.wikipedia.org/wiki/chemical_compound http://en.wikipedia.org/wiki/base_(chemistry) http://en.wikipedia.org/wiki/nitrogen http://en.wikipedia.org/wiki/bacteria http://en.wikipedia.org/wiki/fungus http://en.wikipedia.org/wiki/animal http://en.wikipedia.org/wiki/pharmacology iraqi j pharm sci, vol.23(1) 2014 alkaloids of iraqi echinops 27 and many literatures survey revealed different pharmacological activities of echinops plant like, antibacterial activity (7) , antifungal (8) , antioxidant activity (9) , protective effects on testosterone-induced prostatic hyperplasia (10) , hepatoprotective (11) and antiulcerogenic activity (12) . all the alkaloids isolated from different species of echinops related to the quinoline type so, the biosynthetic origin of quinoline alkaloids is the aromatic amine anthranilic (2aminobenzoic) acid involved in the metabolism of the amino acid tryptophan. the skeleton of quinoline alkaloids constitutes a bicyclic system with a fused benzene and pyridine ring (figure 1). the attachment of a furan ring to the pyridine nucleus generates furoquinolines (e.g. furacridone), an important subgroup of quinoline alkaloids. the plant family rutaceae represents the major source of quinoline alkaloids.some of these naturally occurring quinolines have profound medicinal properties while others have served as lead structures and provided inspiration for the design of synthetic quinolines as useful drugs (13) . figure 1: basic structure of quinoline nucleus this study was emphasized on the isolation and identification of alkaloids found in the iraqi species of echinops plant by two methods : preparative high performance liquid chromatography chromatography (phplc) and preparative thin layer chromatography(ptlc) and compare between quantity of these active constituents obtained by both methods. methods and materials plant material the whole plant of echinops heterophyllus of the family (compositae) was collected from nazali, 71km north of erbil . the plant was authenticated by dr. abdul-hussien alkhait specialist in plant taxonomy . the plant seeds were collected during the month of november (2011), while aerial parts (leaves/stem) and roots were collected during the months of may and june (flowering time) and were cleaned, dried at room temperature in the shade then pulverized by mechanical mills and weighed. thin layer chromatography was done on readymade plates of silica gel gf254nm (20x20cm) of 0.25mm thickness (merck), using three different developing solvent systems: s1 = benzene : methanol: (8 : 2) (14) s2 = chloroform: acetone: diethylamine (5 : 4 :1) (14) . s3 = toluene: ethylacetate: diethylamine (70 : 20 : 10) (15) and detected by dragendorffs spraying reagent preparative hplc was done using : acetonitrile : water (65:35) as a mobile phase column: mediterranea c18 , 5 µm 15 x 2.12 cm, flow rate: 5 ml / min injection volume: 1 ml. detection: uv. detector at λ 205 nm. experimental work the experimental work is divided into : 1preliminary phytochemical screening of various secondary metabolites like alkaloids, flavonoids, steroids, tannins, saponins, anthraquinioin, terpenoids and cardiac glycosides) in the different parts of echinops plant. 2 extraction of alkaloids. 3isolation and purification of alkaloids. 4identification and characterization of the isolated compounds. preliminary qualitative phytochemical analysis: chemical tests were carried out using the ethanolic extracts from plants and or the powdered specimens, using standard procedures to identify the active constituents. (16-18) test for alkaloids : wagner’s reagents & mayer’s test for flavonoids : lead acetate test & naoh test tests for steroids: liebermann-burchard test test for tannins : fecl3 solution test test of saponins: foam test tests for anthraquinones: borntrager’s test test for terpenoids naoh test test for cardiac glycoside: keller-kiliani test iraqi j pharm sci, vol.23(1) 2014 alkaloids of iraqi echinops 28 extraction method powdered plant material hexane hexane extract defatted plant material (fat) 80% ethanol in the soxhlet exhausted plant material alcoholic extract (crude extract) 5% hcl / ethylacetate ethylacetate layer aqueous layer (neutral and acids) (alkaloids and water soluble) 5%naoh/ chcl3 aqueous oh layer (f-2) chcl3 layer (water soluble) (f-1) alkaloids figure:2 general scheme for separation of different plant constituents (17) iraqi j pharm sci, vol.23(1) 2014 alkaloids of iraqi echinops 29 results and discussion preliminary qualitative phytochemical analysis: the results of phytochemical screening are given in table (1).the results of preliminary phytochemical screening of plant extracts showed the presence of alkaloids, flavonoids, steroids, tannins and terpenoids in different parts of iraqi species in different percentage, and the absence of, saponins, anthraquinoin and cardiac glycosides in all plant parts. these results can be compared with phytochemical screening of other echinops species for example,: the aerial part of iraqi heterophyllus species was contained traces amount of alkaloids, unlike egyptian species e. spinosissimus, its aerial parts was contained about 11.3% alkaloids (19) , also quinoline alkaloids and flavonoids found in the aerial part of indian e. echinatus with the presence of tannins in the root parts only (20) . many researchers reported that the concentration of secondary metabolites are varying from plant to plant belong to the same genus and even in the different parts of the same plant (21) , this is due to many factors like environmental heterogeneity, since the effect of environmental heterogeneity is highly scaledependent. it may create high niche diversity and hence allow species to coexist at a large spatial scale (22) , also the high complexity and heterogeneity of soil, like( soil structure, texture and depth, moisture retention characteristics, aeration) create a big variation in the chemical constituents even in the same country (23) , good example seen in two iraqi species of echinops plant : e. tenuisectus and e. heterophyllus, phytochemical analysis of e. tenuisectus revealed the presence of high percentage of silymarine in the seeds (0.878%) and aerial parts (0.095) (24) with the absence of this compound in the heterophyllus species. table(1) phytochemical screening of different parts of echinops heterophyllus plant part alkaloids flavonoids steroids tannins saponin anthraquinoin terpenoids cardiac glycoside seeds + + + aerial part traces + + + + roots + + + + + +, represent presence and absence of phytoconstituents respectively. preliminary identification of different echinops parts by tlc thin layer chromatography of fraction 1 (f1) obtained from different parts of the echinops, confirms the following: (a)the presence of three different alkaloids in fraction-1 (named e1, e2 and e3) which is obtained from seeds part and two alkaloids in the same fraction obtained from roots part (e1 and e2) with very traces one compound (e1) in the alkaloidal fraction of aerial plant . figure-3the rf values of these compounds in the different solvent systems were calculated, table(2). table (2) rf values of alkaloids obtained from different plant parts in different developing solvent systems in tlc. compound plant part s1 s2 s3 e1 seed 0.16 .22 0.25 e2 seed 0.58 0.68 0.66 e3 seed 0.75 0.8 0.79 e1 root 0.17 0.25 0.26 e2 root 0.6 0.7 0.67 e1 aerial part 0.15 0.21 0.25 r s a figure 3: tlc of fraction one (f-1) for different echinops parts. ( roots, seeds, aerial parts) using silica gel gf254nm as adsorbent and s1 as a mobile phase. detection by dragendorffs spraying reagent r : roots s : seeds a : aerial part iraqi j pharm sci, vol.23(1) 2014 alkaloids of iraqi echinops 30 isolation and purification of alkaloids: two chromatographic analysis were carried out to isolate in a pure form three alkaloids (named e1, e2, e3) found in the plant which are: preparative hplc and preparative tlc, since seeds contain the largest number and highest quantity of the alkaloids so alkaloids fraction obtained from seeds part was used to separate and isolate these compounds in a pure form. isolation and purification of alkaloids by preparative hplc one gram (1 gm) of f-1 obtained from plant seeds dissolved in a minimum quantity of chloroform was injected in to preparative hplc figure 4: preparative hplc analysis of fraction-1 obtained from seeds plant observing three peaks represent three different compounds, one of them (e2) is a major one. three samples obtained from preparative hplc were weighted and subjected to co-tlc .weight of e1 = 0.07 gm , weight of e2 = 0.5 gm, weight of e3 = 0.16 g isolation and purification of alkaloids by preparative tlc on a 20cm x 20cm glass plates a slurry of 75 gm of silica gel gf 254 suspended in 150 ml of distilled water was applied in 1mm thickness manually by using jobling laboratory division plate coater. the freshly coated plates were left until the transparency of the layer disappears. after 10 minutes, the plates stacked in a dry rack and heated in vertical position for 1 hour at 110 o c with occasional opening of the oven door from time to time in order to allow moisture escape. the completely dried and activated plates were kept in a dry and moisture free container containing adsorbent silica gel one gram (1 gm) of f-1 obtained from plant seeds (highest quantity) dissolved in a minimum quantity of chloroform and applied on a number of preparative tlc plates using s1a solvent system. the solvent was allowed to rise to a height of 15cm from the base line. one major and two minor bands were observed after spraying a side of plates with dragendorffs three band had been scrapped off, eluted with chloroform, then filtered. the filtrate evaporated to dryness, in vacuo to give white crystals, upon re-crystallization out of boiling ethylacetate, a fluffy white crystals of e1, e2 and e3 were obtained. three samples obtained from preparative tlc were weighted and subjected to co-tlc . iraqi j pharm sci, vol.23(1) 2014 alkaloids of iraqi echinops 31 weight of e1 = 0.037 gm , weight of e2 = 0.327 gm, weight of e3 = 0. 063 gm from the above results, the quantity of compounds obtained in a (pure form) by preparative hplc is higher than that obtained by preparative tlc. classical preparative tlc suffers from several drawbacks, the main disadvantage being the removal of purified substance from the plate and its subsequent extraction from the sorbent, other drawbacks include the length of time required for the separation and degree of purity for the separated compounds (25) , compare with preparative hplc, which is consider know, the most powerful and versatile method for purification tasks in the pharmaceutical industry (26) . despite the fact that among the tools used in the large scale purification of pharmaceuticals, preparative hplc is one of the more expensive and solvent-consuming approaches, it yields the highest-purity drug substance. the interest in preparative hplc will continue to grow because of the increasing uncertainty in the market expectations for product purity. its nearly linear scalability makes preparative hplc one of the more viable approaches to compound purification (27) . characterization and identific -ation of the isolated alkaloids (e2): 1melting point: the isolated compound which is named e2 had a sharp melting point of 160-162 ه c 2ultra violate spectra: the isolated alkaloid ( e2 ) show uv absorption near 242nm 3ft-ir spectra: the identification of the unknown alkaloid (e2 ) was further confirmed by using ft-ir spectroscopy figure (5) . the characteristic ir absorption bands showed by this compound are listed in table(3). table (3 )characteristic ir absorption bands( in cm -1 ) of the isolated alkaloids (28) functional group group frequency wave number ( in cm -1 ) assignment n c-h 3306, 3245 2910-2852 n–h stretch (two band for tertiary amine asymmetric and symmetric stretching of ch3 c=o 1590-1750 c=o stretching vibration (conjugated) c-n 1333,1336 c-n stretching bands of tertiary amine ch3 1430,1480 c-h bending vibration c-h 914, 868, 750 c-h of aromatic group out of plane 4elemental micro analysis (chn): elemental microanalysis was performed for unknown isolated compounds ( e2) to confirm their chemical structure. the result of this analysis (table 5) showed that the unknown compounds consist of carbon , hydrogen, oxygen and nitrogen in different percentage. table(4) elemental microanalysis of the unknown isolated alkaloid name c% h% o% n% e2 74.07 6.208 10.25 9.463 5 h 1 and c 13 nmr the e2 compounds presented 13 c nmr spectra (dmso, 75 mhz): with chemical shifts typical of quinoline rings (28) in the ranges of δc 21.12 (c-2), 24.77 (c-3), 170.12(c4),126.987 (c-5), 121.825 (c-6), 127.640 (c7), 114.951 (c-8), 138.26 (c-9), 123.47 (c10), 30.4 (c-11). 1 h nmr (dmso-d6-, 300 mhz) revealed that e2 compound undergo tautomerism which lead to the appearance of chemical shifts of the hydroxyl group at 10.02 at (c-4), 2.4 (3h, as a singlet of the methyl protons ), 2.6 (2h, d, h2),5.09 (1h,s, h-3), 6.84-7.15 (4h, m, h-5 ,h6 , h-7 , h-8). figure (7). depending on the above results, the expected chemical structure for the isolated e2 compound is: n o ch3 1 2 3 4 5 6 7 8 9 10 11 1-methyl-2,3-dihydro-4(1h)-quinolinone, it is a new compound isolated (for the first time) from iraqi echinops heterophyllus plant, it seen to be the hydrogenated form of echinopsine (1-methyl-4(1h)-quinolinone), an alkaloid isolated from 14 species of echinops plant. iraqi j pharm sci, vol.23(1) 2014 alkaloids of iraqi echinops 32 figure 5: ft-ir spectrum of the isolated alkaloid (e2) figure 6 : 13 c-nmr analysis of the isolated e2 compound iraqi j pharm sci, vol.23(1) 2014 alkaloids of iraqi echinops 33 figure 7 : 1 h-nmr analysis of the isolated e2 compound. conclusion phytochemical investigation of a new wild iraqi plant used traditionally for wound healing and snake bit named echinops heterophyllus was done and the results revealed the presence of alkaloids, flavonoids, terpenoids, tannins and steroids in the different plant parts and in a different percentages, aerial parts contain the highest quantity of flavonoids, while seeds contain the highest amount of alkaloids. general schematic procedure of jeffirey b. harborne was used to extract different plant parts using 80% ethanol in soxhlet apparatus.two chromatographic analysis were carried out to isolate in 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http://www.sciencedirect.com.tiger.sempertool.dk/science/article/pii/s1876382012000066 http://www.sciencedirect.com.tiger.sempertool.dk/science/article/pii/s1876382012000066 http://www.sciencedirect.com.tiger.sempertool.dk/science/article/pii/s1876382012000066 iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 11 formulation and evaluation of ezetimibe nanoparticles yasser a.ali * and shaimaa n. abd-alhammid * , 1 * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq, abstract the aim of this study is to formulate and evaluate ezetimibe nanoparticles using solvent antisolvent technology. ezetimibe is a practically water-insoluble drug which acts as a lipid lowering drug that selectively inhibits the intestinal absorption of cholesterol and related phytosterols. ezetimibe prepared as nano particles in order to improve its solubility and dissolution rate. thirty formulas were prepared and different stabilizing agents were used with different concentrations such as poly vinyl pyrrolidone (pvpk-30), poly vinyl alcohol (pva), hydroxy propyl methyl cellulose e5 (hpmc), and poloxamer. the ratios of drug to stabilizers used to prepare the nanoparticles were 1: 2, 1:3 and 1:4. the prepared nanoparticles were evaluated for particle size, entrapment efficiency, dissolution study, fourier transform infrared spectroscopy, differential scanning calorimetry, and atomic force microscopy. the percentage of drug entrapment efficiency of f1-f30 was ranged from 85% ± 1 to 98 % ± 1. on the other hand dissolution rate increasing as the particle surface area is increase due to reduction of particle size to the nano range. the results showed that poly vinyl pyrrolidone (pvpk-30) was found to be the best stabilizer. keywords: ezetimibe, nanoparticles, particle size, poly vinyl alcohol. تيميب بواسطة انجسيمات اننانويةياالز حبيباتوتقييم تصييغ ياسر عبد انصاحب عهي * عبد انحميدنسارشيماء و *،1 * .العراق ،بغداد ،جبمعة بغداد ،كلية الصيدلة ،فرع الصيدالنيبت انخالصة الحرسدددي مددد جكنىلىجيدددب ببسدددحمدا لعقدددبر االميح بيددد نبنىيدددة جسدددي بت وجقيدددي لصددديب ة هدددى الدراسدددة هددد مددد الهدددد أن انحقددددبي بشددددك جثدددد الحدددد علددددً الدددد الدددددهىن وهددددى دواء يع دددد ال ددددبء يددددر ايدددد فدددد دواء هددددى اميح بيدددد . مضددددبد ال دددد ي القببليددددة جحسددددي بغيددددة نبنىيددددة كجسددددي بت أعددددد اميح بيدددد . الصددددلة ات و ال ددددىاد الدهنيددددة األمعددددبء امحصددددبك الكىلسددددحرو مدددد . االمحصبك ومعد لل وببن اللبينيددد ببيروليددددون مثددد ممحللدددة بحراكيددد اسدددحمدمث ممحللدددة اسدددحقرار ببسدددحمدا بدددىلي رات صددديغة ثالثدددي إعدددداد جددد . وبىلىكسددددبمير ،hpmc e5) (السددددليلىم ال يثيدددد بروبيدددد هيدروكسدددد ،(pva) الكحددددى اللينيدددد وبددددىل ،(pvp) ال حعدددددد . 2:1 و 2:1 و 2:1 النبنىية ه الجسي بت إعداد ف ال سحمدمة ال ث حبت إلً الدواء وكبنث نس الشددددك ال يددددبن للححددددرر ودراسددددة انح ددددبد الدددددواء، وكلددددبء مدددد ايددددا الحجدددد الح ي دددد للجسددددي بت " نبنىيددددة جسددددي بت" وقي ددددث النسدددد ة .ال ريددددة القددددى ومجهددددر الحلبضددددل ( ال سدددد وقيددددبد ، الح ددددراء جحددددث األشددددعة مطيبفيددددةوكدددد لا دراسددددة الحىافدددد ) الدددددواي ، %.مدددد نبايددددة 85% الددددً 58هدددد مدددد 13الدددددواء للصدددديي الدواييددددة مدددد الصدددديغة االولددددً الددددً الصدددديغة ال ئىيددددة لكلددددبء انح ددددبد بدددىل أن النحدددبي وأظهدددرت اادددري يددد داد جحدددرر الددددواء كل دددب صدددغر اجددد الجسدددي بت النبنىيدددة ل يدددبد ال سدددباة السدددطحية للجسدددي . نىية.بىلي ر اسحقرار للجسي بت النب هى أفض ( -13pvp k(الليني ببيروليدون .انكحول انفينيم بوني انحبيبي، انحجم انجسيمات اننانوية، إزتيميب، -انمفتاحية : كهماتان introduction solubility is of the most important parameters to achieve the desired concentration of a drug in the systemic circulation for pharmacological response to be shown. a number of methodologies can be adapted to improve solubilization of poor water soluble drug and further to improve its bioavailability include chemical modification , ph adjustment, solid dispersion, complexation, co‐ solvency, and micronization (1) . one of these methods is the nanosuspension which is colloidal dispersions of nano-sized drug particles that are produced by a proper method and stabilized by a suitable stabilizer (19) . the particle size distribution of the solid particles in nano suspensions is usually less than one micron with an average particle size ranging from 200 and 1000 nm (2) . ezetimibe is a member of new class of lipid lowering compounds that selectively inhibit the intestinal absorption of cholesterol and decrease cholesterol absorption (3) . ezetimibe is categorized as a class ii agent (poorly water soluble and highly permeable with a relative 1 corresponding author e-mail: shaimaa-alsamariai@yahoo.com. received: 21/4/ 2015 accepted: 29/6/2015 iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 12 bioavailability range from 35-65 % (4) . the aim of this study is to formulate and evaluate ezetimibe nanoparticles using solvent antisolvent technology. materials and methods materials ezetimibe powder, was purchased from (provizer pharma, gujarat, india). poly vinyl pyrrolidone pvp k-30 (bdh chemicals ltd, liverpool, england). poly vinyl alcohol (riedal de haen ag seelze, hannover, germany), hpmc (provizer pharma, gujarat, india). poloxamer 188 (bdh chemicals ltd, liverpool, england). methanol (gcc analytical reagent, uk). brij35 (riedal de haen ag seelze, hannover, germany). all other chemicals were of analytical grade. methods preparation of ezetimibe nanosuspension nanosuspensions were prepared by the solvent evaporation technique which is also called solvent antisolvet technique (5) , as shown in table (1), (2), (3) that the ezetimibe was dissolved in 10 ml methanol and poured into 100 ml water containing different types of stabilizers (alone and in combination) maintained at a temperature of 50°c and subsequently stirred at agitation speed of 500 revolution per minute (rpm) on magnetic stirrer for 1 hour to allow the volatile solvent to evaporate. the organic solvents which contain 10 mg of ezetimibe were added by means of a syringe drop by drop positioned with the needle directly into stabilizers containing water. the ratios of drug to stabilizers used to prepare the nanosuspension were 1: 2, 1:3 and 1:4. then centrifuge to obtain the nanoparticles. table (1): composition of ezetimibe nanosuspension using different types of stabilizers at drug: stabilizer ratio 1:2. formula no. materials f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 ezetimibe(mg) 10 10 10 10 10 10 10 10 10 10 pvp(mg) 20 10 10 10 pva(mg) 20 10 10 10 hpmc(mg) 20 10 10 10 poloxamer188 (mg) 20 10 10 10 methanol (ml) 10 10 10 10 10 10 10 10 10 10 water (ml) qs 100 100 100 100 100 100 100 100 100 100 table (2): composition of ezetimibe nanosuspension using different types of stabilizers at drug: stabilizer ratio 1:3. formula no. materials f11 f12 f13 f14 f15 f16 f17 f18 f19 f20 ezetimibe(mg) 10 10 10 10 10 10 10 10 10 10 pvp(mg) 30 15 15 15 pva(mg) 30 15 15 15 hpmc(mg) 30 15 15 15 poloxamer188 30 15 15 15 methanol (ml) 10 10 10 10 10 10 10 10 10 10 water (ml) qs 100 100 100 100 100 100 100 100 100 100 iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 13 table (3): composition of ezetimibe nanosuspension using different types of stabilizers at drug: stabilizer ratio 1:4 formula no. materials f21 f22 f23 f24 f25 f26 f27 f28 f29 f30 ezetimibe(mg) 10 10 10 10 10 10 10 10 10 10 pvp(mg) 40 20 20 20 pva(mg) 40 20 20 20 hpmc(mg) 40 20 20 20 poloxamer 188 40 20 20 20 methanol (ml) 10 10 10 10 10 10 10 10 10 10 water (ml) qs 100 100 100 100 100 100 100 100 100 100 evaluation of the prepared nanosuspension particle size determination was done using abt-9000 nano laser particle size analyzer at 25ºc without dilution of the samples. the average particle size (d) was measured for all the prepared formulas at ratios of 1: 2 (f1-f10), 1: 3 (f11-f20) and 1: 4(f21f30). each sample was sonicated for 20 minute before measuring and each sample was measured in triplicate. determination of drug entrapment efficiency (ee) of nanosuspension the freshly prepared nanosuspension of ezitimibe: stabilizer ratio 1:2, 1:3 and 1:4 was centrifuged at 20,000 rpm for 20 minutes using ultracentrifuge. the amount of non incorporated drug was measured by taking the absorbance of the appropriately diluted 25 ml of supernatant solution at 232 nm using uv-visible spectrophotometer. the entrapment efficiency (ee %) was calculated by subtracting the amount of the free drug in the supernatant from the initial amount of drug taken. for each formulation the experiment was repeated in triplicate and the average was calculated (6, 7) . the percentage of drug entrapment efficiency (% ee) could be achieved by the following equation : entrapment efficiency = (weight initial drugweight free drug) / weight initial drug freeze drying of nanosuspension in order to retrieve nanoparticles in dried-powder state from the nanosuspensions, water-removal was conducted through freeze-drying, so that each formula was lyophilized using vacuum freeze dryer at a controlled temperature of (45) ˚c and the pump operating at a pressure of 2.5 × 10 pascal over a period of 48–72 hour. the yielded powders were used for further studies (8) . in-vitro dissolution profile of nanosuspension in vitro dissolution study was performed using usp dissolution test apparatus-ii (paddle assembly). the dissolution was performed using lyophilized powder in 500 ml of 0.1n hcl (ph 1.2) and phosphate buffer solution (ph 6.8) as dissolution mediums containing 2% brij 35 and maintained at 37 °c and 50 rpm for ezetimibe lyophilized powder formulas. the freshly prepared formula f1-f4, f11-f14 and f20-f24 where freeze dried to get the nanoparticles then immersed in dissolution medium. samples (5ml) were withdrawn at regular intervals of 5 minutes for 120 minutes and replaced with fresh dissolution medium to keep sink conditions. samples were filtered through filter paper and assayed spectro photometrically on uv-visible spectrophotometer at 232 nm wave length (9) . fourier transform infrared spectroscopy (ftir) the fourier transform infrared spectroscopy (ftir) spectra were obtained using ftir spectroscope. samples which studied are: pure ezetimibe powder, pvp k30, physical mixture of ezetimibe, and pvp k30 at ratio (1:4); respectively. lyophilized powder (nanoparticles) of the selected formula (f21), microcrystalline cellulose ph 102 (avicel) ® , lactose ,magnesium sulphate. all these samples were grounded and mixed thoroughly with potassium bromide. the iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 14 spectrum obtained was in between the wave number of 4000-400 cm1 (10). differential scanning calorimetry (dsc) dsc can be used to determine the compatibility between the drug and excipients and also used to evaluate the crystalline state of drug especially when converted to nanoparticles. thermal characteristics of the same samples that are studied by ftir were determined also by an automatic thermal analyzer system. therefore accurately weighed samples (5mg) were placed in non hermetically aluminum pans and heated at the rate of 10 ºc/minute against an empty aluminum pan as a reference covering a temperature range of 40 ºc to 300 ºc (11) . atomic force microscopy atomic force microscopy (afm) is capable of scanning the surfaces in controlled environmental conditions, also can measure the particle size of the nanoparticles accurately. the size and surface morphology of ezetimibe nanoparticles in f21 were confirmed by atomic force microscopy after drying of the formula. the optimized formula f21 were lyophilized and dried 15 minutes in desiccators. particle size, 3d-dimension graph and histogram of particle size distribution were obtained (12, 13) . statistical analysis the results of the experiments were given as a mean of triplicate samples ± standard deviation and were analyzed according to the paired t test and one way analysis of variance (single factor anova) at the level of (p < 0.05). results and discussion evaluation of nanosuspension particle size analysis the particle size of formulas f1-f4 at drug: polymer ratio 1:2 was ranged from 95.4 -956.5 nm measured by particle size analyzer, while for f11-f14 at drug: polymer ratio 1:3 the particle size ranged from 66.52-899.1 nm. on the other hand f21-f24 at drug :polymer ratio the particle size of these formula range from 35.3901.2 using pvp k-30 , pva, hpmc and poloxamer 188 as primary stabilizers . the formulations containing pvp k-30, pva , and hpmc as stabilizers had small significant particle size in comparison with the formulation containing poloxamer 188 that gave larger particle size (p<0.05). poloxamer188 (pluronic f68)® is a block co-polymer, responsible for the hydrophobic interaction with the drug molecule ,the crystal growth inhibition is mainly due to the hydrophobic polypropylene oxide group (ppo) in the pluronic polymer , while the hydrophilic polyethylene oxide (peo) chains provide steric hindrance upon aggregation (14) . poloxamer 188 can form a valuable mechanical and thermodynamic barrier at the interface that hinders the approach and coalescence of individual emulsion droplets at their optimum level. although this mechanism of poloxamer 188, but it gave larger particle size in all three ratios 1:2, 1:3 and 1:4 drug: polymer in formulas f4, f14 and f24; respectively. high particle size of f4, f14 and f24 that contain poloxamer188 as a stabilizer may be attributed to the insufficient affinity, of poloxamer188 to ezetimibe, and possess a slow diffusion rate and ineffective adsorption onto the drug particle surface in the water–methanol mixture. however, if there is no affinity between the particle surface and the polymer, the attractive forces between two particles become dominant due to depletion of polymer from the gap of two particles (depletion force) (15) . poly dispersity index values of f1, f2 and f3 ranged from 0.002 -0.005 indicate that these formulas are mono disperse standard. while for f4 that contain poloxamer 188 pdi value was 0.439 which indicate mid range poly dispersity system. the surface area values of the particles in f1, f2 and f3 ranged from 39.2 57.53 (m 2 /g) while for f4 that contain poloxamer 188 particle surface area value was 3.89 (m 2 /g) which has the smallest particle surface area (p<0.05) in comparison with other formulas because it has largest particle size (16) . for drug :stabilizer ratio 1:3, polydispersity index values of f11, f12 and f13 ranged from 0.002 0.033 indicate that these formulas are monodisperse standard, while for f14 that contain poloxamer 188 pdi value was 0.557 which indicates mid range polydispersity system . specific surface area values of the particles in f11, f12 and f13 ranged from 66.21 – 99.23 (m 2 /g) while for f14 that contain poloxamer 188 the ssa value was 4.25 (m 2 /g) which has the smallest ssa (p<0.05) in comparison with other formulas because it has largest particle size. iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 15 on other hand for drug : polymer ratio 1:4 the poly dispersity index values of formulas f21, f22 and f23 range from 0.002-0.031 which indicate that these formulas are mono disperse system , while for f24 that contain poloxamer188 pdi value was 0.811 which indicates mid range poly dispersity system specific surface area values of the particles in f23, f22 and f21 ranged from 122.21 – 557.23 (m 2 /g) while for f24 that contain poloxamer188 the ssa value was 4.25 (m 2 /g) which has the smallest ssa (p<0.05) in comparison with other formulas because it has largest particle size. this difference in values of pdi could be attributed to the efficiency of stabilizers, which cover the organic/aqueous interface of the nano droplets and prevent them from coalescing to each other. from the obtained results, one can conclude that the poloxamer188 is not suitable as a primary stabilizer for nanoparticles because of poor adsorption and poor affinity of poloxamer188 to the ezetimibe molecules. particle size ranged from 35.57 nm for pvpk30 to 999 nm for poloxamer188, which appeared to be affected by relative viscosity of the polymeric dispersion in the presence of stabilizers and followed the trend: pvp ˃ pva ˃ hpmc > poloxamer188. nanosuspension with pvp k-30 as stabilizers possessed the smallest particle size while that containing poloxamer188 had the largest particle size (17) . effect of polymer concentration on the size of ezetimibe nanoparticles the effect of the polymer concentration on the particle size of ezetimibe nanosuspention have been investigated by depending on three ratios of drug : polymer concentration (1:2) in the preparation of f1-f4 and in 1:3 of drug : polymer ratio in the formulation of f11-f14. and in the ratio of 1:4 in the formulas f21f24. polymer concentration affecting on the adsorption affinity of the stabilizers to the particle surface. in general as the concentration of polymers increase the particle size decrease at fixed drug concentration except for poloxamer188, which indicated that the drug particle surface has been sufficiently covered well by the stabilizer molecules (18) . it has been noticed that the particle sizes of f1, f2 and f3 using pvp k-30, pva and hpmc; respectively as stabilizer were decreased when the concentration of polymer increased in the formulas f11, f12 and f13 and they were further decreased in particle size when the concentration of the polymer increased as shown in the formulas f21, f22 and f23 using the same stabilizers, while f4 , f14 and f24 containing poloxamer 188, as the only stabilizer, have maintained within the same value of particle size and not increased sufficiently even when the concentration was increased. it can be interpreted as the fact that poloxamer 188, by itself have poor adsorption properties and affinity to the molecules of ezetimibe that prevent particle agglomeration and this finding did not agree with the reported one (19) . polymer concentration plays a great role in the stabilization of nanoparticles because of too little stabilizer induces agglomeration or aggregation and too much stabilizer promotes ostwald’s ripening (20) .the decrease in the particle size is accompanied by a rapid, highly increase in the surface area. thus, the process of primary coating of the newer surfaces competes with the agglomeration of the uncoated surfaces. hence, an increase in the surfactant concentration in the primary dispersion results in rapid coverage of the newly formed particle surfaces (21) . effect of combination of two polymers on the size of ezetimibe nanoparticles the particle size of f5, containing pva and pvp k-30 as stabilizers combination at drug: stabilizer ratio 1:2 was decreased significantly (p<0.05) from 140 nm to 40.23 nm in f25 at drug: stabilizer ratio 1:4 ratio when the stabilizer concentration increased. poloxamer 188, when used singly was not so effective in reducing the particle size as a stabilizer, rather flocculation was observed. this could be due to its high hydrophilicity (hlb = 29) due to which it may not be undergoing preferential adsorption on the nanoparticle surface. however, poloxamer188 in combination with pvp k-30, pva and hpmc , it worked synergistically therefore the particle size of ezetimibe nanosuspension was drastically reduced. it has been found that the combination of pvpk-30 and poloxamer188 have reduced particle size this mainly due to iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 16 that pvp k-30 is reported to be a protective colloid which is indicative of its greater adsorption potential for the nanoparticles (22) . this is expected as the stabilizers used for preparing the ezetimibe nanosuspensions are either hydrophilic polymers or non-ionic surfactants which stabilize the particles by steric stabilization (22, 23) . determination of drug entrapment efficiency of nanosuspension the percentage of drug entrapment efficiency of f1-f30 was ranged from 85% ± 1 to 98 % ± 1 as shown in figure (11). it is clear that the increase in stabilizer concentration increased the drug entrapment efficiency, but the study revealed that the concentration of stabilizers at ratio 1:2 drug: stabilizer was sufficient to give the optimized entrapment efficiency. f4 containing poloxamer188 as stabilizer had the lowest entrapment efficiency, while f21 containing pvp-k-30 as the only stabilizer had the higher entrapment efficiency. this may be due to the presence of optimum stabilizer and optimum stabilizer concentration (24) . in vitro dissolution study the dissolution profile was done for f1-f4, f11-f14 and f21-24 of drug: stabilizer ratio 1:2, 1:3 and 1:4 respectively. the dissolution of the prepared formulas was carried in 0.1n hcl solution (ph1.2) and phosphate buffer solution (ph6.8) in the presence of 2% brij-35 to get the selected formula that can increase the dissolution rate in these buffers which are simulated to gastric and intestinal fluids. in the dissolution study one notice enhancement of dissolution rate, according to noyes-whitney equation the dissolution rate increasing as the particle surface area is increase due to reduction of particle size to the nano range. superior dissolution of ezetimibe nanoparticles may potentially improve bioavailability and other drug performances. pvpk-30 containing ezetimibe nanoparticles at drug polymer ratio 1:4 with particle size of 35.57 nm showed the highest drug release rate as 100% of drug dissolved in 10 minutes whereas, poloxamer 188 containing ezetimibe nanoparticles at drug polymer ratio 1:2 with particle size of 999 nm showed about 33.9% of drug dissolved within 10 minute of dissolution test in both 0.1 n hcl (ph 1.2) and phosphate buffer (ph 6.8) (25, 26) . figure (1): effect of polymer type on the dissolution profile of ezetimibe nanoparticles from f1, f2, f3, and f4 in 0.1 n hcl solution (ph 1.2) containing 2% brij35 w/v at 50 r.p.m and 37˚c temperature . figure (2): effect of polymer type on the dissolution profile of ezetimibe nanoparticles from f11, f12, f13, and f14 in 0.1 n hcl solutions (ph 1.2) containing 2% brij-35 w/v at 50 r.p.m and 37˚c temperature. figure (3): effect of polymer type on the dissolution profile of ezetimibe nanoparticles from f21, f22, f23, and f24 in 0.1 n hcl solutions (ph 1.2) containing 2% brij-35 w/v at 50 r.p.m and 37˚c temperature. iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 17 figure (4): effect of polymer type on the dissolution profile of ezetimibe nanoparticles from f1, f2, f3, and f4 in phosphate buffer (ph 6.8) containing 2% brij-35 w/v at 50 r.p.m and 37˚c temperature. figure (5): effect of polymer type on the dissolution profile of ezetimibe nanoparticles from f11, f12, f13, and f14 in phosphate buffer (ph 6.8) containing 2% brij-35 w/v at 50 r.p.m and 37˚c temperature. figure (6): effect of polymer type on the dissolution profile of ezetimibe nanoparticles from f21, f22, f23, and f24 in phosphate buffer (ph 6.8) containing 2% brij-35 w/v at 50 r.p.m and 37˚c temperature. drug content in lyophilized powder the drug content result showed that 25 mg of lyophilized powder of the selected formula (f 21) contain 5 mg ±0.1 of ezetimibe when determined by uv-visible spectrophotometer at λmax 232 nm. fourier transforms infrared spectroscopy ftir is one of the most widely reported spectroscopic techniques for solid-state characterization. the characteristics absorption bands of ezetimibe are: 1. o–h stretching band at 3650—2700 cm -1 2. c-o stretching bands of the lactam ring at 1725—1714 cm -1 3. c=c stretching band of the aromatic ring at 1600—1500 cm -1 4. c–f stretching band at 1000—1200 cm -1 5. c–o stretching band at 1300—1000 cm -1 ftir spectra of ezetimibe nanosuspention and tablet show no change in shifting of the position of the major functional groups indicating no major interaction between the drug and the stabilizer pvp k-30 and other excipients. ftir spectra of physical mixture also showed peaks at similar position. hence, it can be conclude that there was no possible interaction between the drug, the stabilizer and the used excipients (27, 28) . figure (9) demonstrate the dsc thermogram of ezetimibe that showed sharp characteristic endothermic peak at 165.30ºc and this agrees with the references. this gives an indication that the drug has crystalline nature with high purity. the dsc thermograms of the ezetimibe of tablet of the selected formula f21, lyophilized powder and physical mixture are shown in figure (10) that show that the drug in the crystal structure have a melting endotherm while ezetimibe molecules in the amorphous state do not exhibit a melting endotherm. as seen in figures (9), and (10) the sharp melting peak of ezetimibe (165.30 °c) is completely disappeared in these figures that’s mean the stabilizer pvp k-30 completely converted ezetimibe particles into amorphous state (29) . iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 18 figure (7): ftir spectrum of eztimibe figure (8): ftir spectrum of ezetimibe nanosuspension differential scanning calorimetry 0.00 100.00 200.00 300.00 temp [c] -10.00 -5.00 0.00 mw dsc 165.30x100c figure (9): dsc thermogram of ezetimibe powder 0.00 100.00 200.00 300.00 temp [c] -2.00 0.00 2.00 4.00 mw dsc 64.82x100c 147.90x100c 214.60x100c figure (10): dsc thermogram of ezetimibe tablet of formula f21 iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 19 evaluation of surface morphology atomic force microscopy study the morphological analysis and particle size of f21 performed by afm showing irregular to spherical shaped nanoparticles with a size of 31 nm as seen in figure (11) as it approved by the histogram of particle size distribution in figure (12) also figure (13) shows histogram of particle size distribution of f21 by atomic force microscopy. the formulation was found to be stable and no aggregation of particles could be observed. the particle size of f21 obtained by afm was comparable to or equal to that measured by abt-9000 nano laser particle size analyzer (35.57 nm) and this in agreement with particle size measurements provide the good size distribution and the stability of ezetimibe nanparticles (31) . figure (11): atomic force microscopy of formula f21 showing cross section and long tudinal section of the nanoparticles surface. figure (12): particle size distribution of f21 by particle size analyzer abt-9000 iraqi j pharm sci, vol.24(2) 2015 ezetimibe nanoparticles 20 figure (13): histogram of particle size distribution of f21 by afm conclusion nano particulate systems have great potentials, being able to convert poorly soluble, poorly absorbed and labile biologically active substance into promising deliverable drugs. references 1. sikarra d, shukla v, kharia aa, chatterjee dp. techniques for solubility enhancement of poorly soluble drugs: an overview, journal of medical pharmaceutical and allied sciences 2012; 1: 1-22. 2. dhanapal r and ratna v. nanosuspensions technology in drug delivery– a review international journal of pharmacy review and research. 2012; 2(1): 46-52. 3. verschuren l, radonjic m, wielinga p, kelder t, kooistra t. systems biology analysis unravels the complementary action of combined rosuvastatin and ezetimibe therapy. journal of pharmacogenetics and genomics 2012;22 (12):837-846. 4. kosoglou t, statkevich p, johnsonlevonas a, paolini j, bergman a. ezetimibe: a review of its metabolism, pharmacokinetics and drug interactions, journal clinical pharmacokinetics.2005 ; 44(5):467–494. 5. mansouri m , pouretedal h, vosoughi v. preparation and characterization of ibuprofen nanoparticles by using solvent/ antisolvent precipitation. the open conference proceedings journal. 2011; 2: 88-94. 6. jassim z. and hussein a. formulation and evaluation of clopidogrel tablet incorporating drug nanoparticles .international 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mohan1 s, khatri h. enhancement of glipizide dissolution rate through nanoparticles: formulation and in vitro evaluation . e-journal of science & technology (e-jst). , 2012; (4)7:19-32. 17. pouretedal h. preparation and characterization of azithromycin nanodrug using solvent/antisolvent method. int nano lett .2014; 4:103. 18. arunkumar n, deccaraman m, rani c, mohanraj k, kumar k. dissolution enhancement of atorvastatin calcium by nanosuspension technology. journal of pharmacy research.2010; 3(8):1903-1906. 19. samar a. afifi, maha a. hassan, abdelhameed a, and elkhodairy k. nanosuspension: an emerging trend for bioavailability enhancement of etodolac. international journal of polymer science. 2015:1-32. 20. sahu b, and das m. optimization of felodipine nanosuspensions using full factorial design. international journal of pharm .tech research. 2013;5(2): 553-561. 21. shetea g, jain h, punja d, prajapat h, akotiya p and bansal a. stabilizers used in nano-crystal based drug delivery 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vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 40 isolation and characterization of iridoid glycoside (gardenoside) present in the leaves of gardenia jasminoides j.ellis cultivated in iraq usama h. abdul wahab *,1 and zainab j. awad * * department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad,iraq. abstract iridoid glycosides are a group of naturally occurring chemical compounds. they are a large family of compounds biosynthesized by plants, they often have pharmacological effects. the aim of this study is to isolate and identified iridoid glycoside in a newly studied, cultivated in iraq named gardenis jasminoides. the medicinal importance of iridoid glycoside, on one hand and absence of phytochemical investigation on leaves of gardenia on the other hand, acquired this study its importance. many compounds were isolated from leaves plant part one of these compounds was identified by different chemical analysis like: melting point (mp), thin layer chromatography (tlc), fourier transforms infrared spectra (ftir) and high performance liquid chromatography (hplc). keywords: gardenia jasminoides , gardenoside,geniposide,genipin. فصل وتوصيف االريدويد كاليكوسايد )الكاردينوسايد( الموجود في اوراق نبات الكاردينيا المستسرع في العراق اسامه حسن عبد الوهاب *،1 و زينة جليل عواد * الخالصة يٍ قبم انكثيز يٍ انُباجات ٔجهؼب دٔرا يًٓا في يؼانجة ٔجُظيى جصُغ األريذٔيذ كاليكٕسايذ ْي يجًٕػة يٍ انًزكبات انحي انٓذف يٍ ْذِ انذراسّ ْٕ فصم ٔجشخيص انكاليكٕسايذات انًٕجٕدِ في انُبات انًسحزرع في انؼزاق اسًّ اٌ انكثيز يٍ االيزاض. (gardenia.) انطبية نألريذٔيذ كاليكٕسايذ ٔػذو ٔجٕد دراسّ ػهًيّ جحُأل انًكَٕات انكيًيائية الٔراق َبات انكارديُيا انًسحزرع ٔنالًْية جى فصم اكثز يٍ يزكب يٍ األريذٔيذ كاليكٕسايذ )انكارديُٕسايذ ٔانجيُيبٕسايذ( يٍ حيث اكحسبث ْذِ انذراسة اًْيحٓا،في انؼزاق .(m.p, tlc, ftir and hplc)انحزكيب انكيًائي نٕاحذ يُٓا بٕاسطة قياس أراق انُبات ٔانحؼزف ػهى .، جنبين ،الجينيبوسايد الكاردينيا ، الكاردينوسايد :لكلمات المفتاحية ا introduction rubiaceae are an easily recognizable family characterized by opposite leaves that are simple and entire, with interpetiolar stipules, tubular sympetalous corollas and an inferior ovary (1) . exceptionally, there are some plants that have only a single leaf at each node, alternating from one side to the other. in these cases, the alternate leaf arrangement is produced through the suppression of one leaf at each node (2) . a wide variety of growth forms are present in the rubiaceae. while shrubs are most common, members of the family can also be trees, lianas or herbs. the flowers, which are usually bisexual, have a 4–5 lobed calyx and generally a 4–5 lobed corolla, 4 or 5 stamens and two carpels (2) . gardenia was named by linnaeus after dr. alexander garden (1730–1791), a scottish physician who immigrated to south carolina and corresponded with linnaeus about american plants; g. jasminoides is botanical latin for ‘jasmine-like'. gardenias are evergreen shrubs and small trees growing 1–5 m tall. the leaves are opposite or in whorls of 3 or 4 cm long and 3–25 cm broad, dark green and glossy with a leathery texture (3) . the flowers are solitary or in small clusters, white, or pale yellow, with a tubular-based corolla with 5–12 petals from 5–12 cm in diameter. many species are strongly scented. the flowers are produced on or at the ends of branches (4) . cultivated forms often have double rose-like flowers that open from large buds with a distinctive whorl of petals. fleshy or leathery berries then follow. gardenias will persist in a wide range of conditions , but if they are not perfectly content, they will tend to look quite awful. they seem to do best in a protected corner of the garden with some morning sun – they don’t like the full glare of the afternoon sun (4) . there are over 200 species of gardenias, but most of them are hybrid varieties. apart from g. jasminoides, the other most common types of gardenia are (5) :  gardenia augusta;  gardenia thunbergia, also known as star gardenia; this can be a shrub or a small tree and grows to about 1.2–1.5 m tall; 1 corresponding author e-mail: usamahasan92@yahoo.com received:8 /12/ 2014 accepted: 28/4/2015 http://en.wikipedia.org/wiki/rubiaceae#cite_note-takhtajan-7 http://en.wikipedia.org/wiki/shrub http://en.wikipedia.org/wiki/tree http://en.wikipedia.org/wiki/liana http://en.wikipedia.org/wiki/herb http://en.wikipedia.org/wiki/plant_sexuality#individual_reproductive_unit_.28a_flower_in_angiosperms.29 http://en.wikipedia.org/wiki/sepal http://en.wikipedia.org/wiki/petal#corolla http://en.wikipedia.org/wiki/stamen http://en.wikipedia.org/wiki/gynoecium http://en.wikipedia.org/wiki/rubiaceae#cite_note-takhtajan-7 http://www.anbg.gov.au/biography/linnaeus.html http://www.somemagneticislandplants.com.au/images/leaves/alternate%20-%20opposite%20leaves.jpg http://www.somemagneticislandplants.com.au/images/samples/whorl.jpg http://www.somemagneticislandplants.com.au/images/samples/corolla.jpg http://www.somemagneticislandplants.com.au/images/samples/berry.jpg http://www.somemagneticislandplants.com.au/images/samples/hybrid.jpg http://www.somemagneticislandplants.com.au/index.php/families/11-plants/871-gardenia-augusta http://www.plantzafrica.com/plantefg/gardenthun.htm iraqi j pharm sci, vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 41  gardenia nitida, a sturdy plant that can reach almost 1.5 m when taken care of properly and well maintained;  gardenia radicans, a dwarf variety that grows to about 45 cm, and produces double blooms. gardenias originated in china and japan, and are now found in africa, asia and australasia. they are attractive landscape subjects in warm climates, and make good container plants. the flowers of some species are used to perfume tea, and others are used to treat influenza and colds in modern chinese herbalism. a yellow dye was made from the fruits (5) . gardenias tend to leach trace elements from the soil. patterning yellow of the leaves may indicate manganese or magnesium deficiencies, and these can be corrected by the addition of appropriate trace elements, or using an enriched fertilizer; but it is quite usual for the lower leaves of healthy plants to turn yellow and fall off as new growth is made at the head of the branches. the root system is shallow and sensitive, so a thick layer of mulch to control weeds is better than cultivating (5) . g. jasminiodes is a smooth, unarmed shrub 1 to 2 meters high. leaves are opposite, elliptic-ovate, 2 to 6 centimeters long, narrowed and pointed at both ends, shining and short petioled, and stipulate. flowers are large and very fragrant, occurring singly in the upper axil of the leaves. calyx is green, with funnel-shaped tube and about 1.5 centimeters long, 5-angled, or winged and divided into linear lobes about as long as the tube (6) . corolla is usually double, white but soon turning yellowish, and 5 to 8 centimeters wide. stamens are as many as the corolla lobes. anthers are linear, sessile. ovary is 1celled; style stout, clavate, fusiform, or 2-cleft, ovules numerous on parietal placentas. fruits are ovoid or ellipsoid, 2.5 to 4.5 centimeters long, 1.5 to 2 centimeters in diameter, yellow, with 5 to 9 longitudinal ridges (7) , as show in figure (1). figure (1):photo of gardenia jasminiodes g. jasminoides is an evergreen flowering plant originated in asia. it is most commonly found growing wild in vietnam, southern china, taiwan, japan, myanmar and india distributed in broad-leaved forests at low to medium elevations (8) . with its shiny green leaves and heavily fragrant white summer flowers, it is widely used in gardens in warm temperate and subtropical climates and as a houseplant in temperate regions (9) .  roots used for fever with delirium.  decoction of roots used for flatulence, dyspepsia, and nervous disorders due to dentition.  decoction of leaves and flowers used for dyspepsia, flatulences, nervous disorders and abdominal pains.  decoction of bark used for menorrhagia and uterine problems.  decoction of flowers used as wash for inflamed eyes.  poultice of leaves for swollen breasts; may be mixed with violeta and other herbs.  antioxidant / crocin: crocin is a water soluble carotenoid found in the fruits of gardenia (gardenia jasminoides) and seems to possess moderately strong antioxidant activity (10) .  diabetes / genipin:study discovered "genipin" from the gardenia extract. genipin blocks the ucp2 enzyme (uncoupling protein 2) that inhibits pancreatic insulin secretion. it suggests a potential for genipin-related compounds (11) .  antiangiogenic activity: the n-butanol fraction of the ethanol extract of gardenia fruit was found to be most effective in the anti angiogenic assay (12) .  anti-cerulein pancreatitis protective activity: a study showed that gardenia jasminoides pretreatment ameliorated the severity of cerulein-induced acute pancreatitis in rats (13) .  sandostatin and gardenia combo / pancreatitis: study showed a combination of sandostatin and gardenia jasminoides can protect pancreatic mitochondria injury in severe acute pancreatitis (14) . iridoids glycosides iridoids are a class of secondary metabolites found in a wide variety of plants and in some animals. they are monoterpenes biosynthesized from isoprene and they are often intermediates in the biosynthesis of alkaloids. chemically, the iridoids usually consist of acyclopentane ring fused to a sixmembered oxygen heterocycle. the chemical structure is exemplified by iridomyrmecin, a defensive chemical produced by http://www.westafricanplants.senckenberg.de/root/index.php?page_id=14&species=759 http://www.nurseriesonline.com.au/pages/gardenia-radicans.html http://en.wikipedia.org/wiki/secondary_metabolite http://en.wikipedia.org/wiki/secondary_metabolite http://en.wikipedia.org/wiki/monoterpene http://en.wikipedia.org/wiki/isoprene http://en.wikipedia.org/wiki/alkaloid http://en.wikipedia.org/wiki/cyclopentane http://en.wikipedia.org/wiki/iridomyrmecin iraqi j pharm sci, vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 42 the iridomyrmex genus, for which iridoids are named as show in figure (1-3). chemical structure of iridomyrmecin figure (2):the basic structure of iridoid (a glygon of glycoside). materials and methods plant materials the leaves of gardenia jasminoides plant family (rubiaceae) was collected from the garden of college of pharmacy, baghdad university during the november, march and april (2013-2014). the plant leaves were cleaned and dried in oven at a temperature (4050 0 c) for (15-20) mints then these leaves were coarsely powered by mechanical grinder and weight. extraction methods of iridoid glycosides: extraction method no.1 (15) a 20gm of the dried powdered leaves of gardenia jasminoides was extracted with three times by reflex with volume (200ml) of 50% ethanol for three hours. after filtration the extract was combined and evaporated to dryness by rotary evaporator at 60 0c and then subjected to identification, as shown in figure (3): dried powder leaves of gardenia jasminoides (20gm) reflex three times with (200ml) of the 50% ethanol for 3 hours. the mixture cool and filter filtrate residue the extract was combined and evaporated to dryness (crude extracts). figure (3):general scheme for method no.1 for extraction iridoid glycosides from the leaves of gardenia jasminoides (15) . extraction method no.2 (16) a 20gm of the dried powdered leaves of gardenia jasminoides was extracted (maceration) with (200ml) of the 90% ethanol for (3-4) days at room temperature, after filtration off the solid parts and evaporation the green solution. the residue is partitioned with water (50ml) and ether (250ml) and separated in a funnel. the aqueous extract was evaporated dryness and then subjected to identification, as shown in figure (4): dried powder leaves of gardenia jasminoides (20gm) maceration with (200ml) 90% ethanol for (3-4) days. filtration filtrate residue (evaporation) the filtrate is partitioned (water: ether) (50:250ml) and separated in a funnel. the aqueous extract was evaporated to dryness (crude extract). figure (4):general scheme for method no.2 for extraction iridoid glycosides from the leaves of gardenia jasminoides (16) . http://en.wikipedia.org/wiki/iridomyrmex http://en.wikipedia.org/wiki/gardenia_jasminoides http://en.wikipedia.org/wiki/gardenia_jasminoides http://en.wikipedia.org/wiki/gardenia_jasminoides http://en.wikipedia.org/wiki/gardenia_jasminoides http://en.wikipedia.org/wiki/gardenia_jasminoides iraqi j pharm sci, vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 43 preliminary identification of iridoid glycosides the preliminary identification of iridoid glycosides of crude extracts of powdered leaves obtained from the extraction method no.1 in result were performed by using thin layer chromatography (tlc) was carried out using the following requirements. ready made plates of silica gel gf 254 (20×20 cm) of 0.25 mm thickness (merck) were used, and then the plates were activated at 110 0 c for 10 min. before used. volume of 100 ml of solvent system was placed in a glass tank (22.5 cm × 22 cm× 7cm), and covered with glass lid and allowed to stand for 45 min. for saturation before use different solvent systems were used for development of iridoid glycosides (gardenoside) (1719) . s1= water: acetic acid: n-butanol (50:10:40). s2= water: n-propanol: n-butanol (60:20:40). s3= water: ethanol: n-butanol (20:20:80). s4= water: n-butanol (50:50). reagent used for detection:  liebermann-burchard reagentis used in this study and prepared as follow (20) add carefully 5 ml of acetic anhydride and 5 ml of concentrated sulfuric acid in to 50 ml of absolute ethanol, while cooling in ice. spray the developed plate and heat it at 100 0 c for 5–10 mints.  vanillin-sulphuric acid reagent (vs) solution i: 5% ethanolic sulphuric acid. solution ii: 1% ethanolic vanillin. the plate is sprayed vigorously with (10 ml) of the (solution i). followed immediately by (510 ml) of the (solution ii), after heating at 110 0 c for (5-10 min) under observation. isolation and purification of gardenoside the dry crude extract obtained from extraction method no.1 of iridoid glycoside was used for isolation and purification of gardenoside and performed as following: fractionation by column chromatography the final residue obtained from the leaves by extraction method no.1 was subjected to column chromatography by using glass column (100 cm x 5 cm) packed with silica gel (0.063-0.200 mm) slurry in (250ml) chloroform, in a ratio of 20 gm of silica gel to each 1 gm of the residue. a dry loading of the sample (residue) was used by dissolving it in small volume of methanol and adsorbing it on small amount of silica gel of the same grade used for packing the column, then dried, grinded and applied to the column in order to prevent clogging. the column was eluted by gradient elution technique using (chloroform: methanol) with an increasing gradually percentage of methanol from zero to 100% and the ratios of (chloroform: methanol) was used (100:0, 90:10, 85:15, 80:20, 70:30, 60:40 and so on till chloroform: methanol 0:100).the column developed by adding 50 ml of each eluent with collecting 5 ml fractions, then monitored by tlc using s3 as mobile phase. a total number of 76 fractions were obtained. those consecutive fractions, which have the same number of spots with the same rf values, were combined and evaporated to dryness to get four major fractions. table (1): major fractions obtained from column chromatography. major fraction no. of collections 5ml each no. of spots f1 22–36 1 f2 37–45 2 f3 46–63 3 f4 64–76 3 preparative tlc plates isolation of iridoid glycosides is carried out by using preparative tlc which was performed by using ready made plates of 20x20cm, which are coated by silica gel gf 254 layers of 1mm thickness, (merck).the major fraction (f3) obtained by column chromatography was applied as a concentrated solution in a row of spots using capillary tube four times on each plate (the spots should dry before the next application).the solvent systems (s1, s2, s3 and s4) was each placed in a glass tank (22.5 cm x 22 cm x 7 cm), and covered with a glass lid and allowed to stand for 45 minutes before use for saturation. the best solvent used from these four solvent is the s3 because in s3 separation is better than other solvent systems (s1,s2 and s4) in preparative tlc plate. the band corresponding to the (gardenoside) standard was scraped out and collected in a beaker, mixed with methanol, stirred and left a side for one hour, then filtered. after evaporation of the solvent, the obtained residue was subjected to chromatography with the available reference standard of (gardenoside) using different mobile phases for identification. qualitative and quantitative estimation of gardenoside using hplc analysis (21) : qualitative and quantitative estimations of (gardenoside) component in the crude extract obtained by all extraction methods was carried out using high performance liquid chromatography hplc) .the identifications was made by comparism the retention time of (gardenoside) (obtained from crude extract with that of authentic standard at identical chromatographic conditions. iraqi j pharm sci, vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 44 hplc conditions of gardenoside (22-24) 1. mobile phase: acetonitrile: 0.1% phosphoric acid in water (30:70). 2. column type: thermo bds hypersil [(c18) 2.4μm]. 3. column dimensions: 100 x 4.6 mm. id. 4. column temperature: ambient. 5. flow rate: 1.3 ml/min. 6. injection volume: 20 µl. 7. injection concentration: 50μg/ml. 8. detection mode and setting: uv detector at λ 238 nm. results two methods of extraction of iridoids glycoside (gardenoside) from dried leaves of g.jasminoides that method no.1 was better, because the percentage yield of crude extract was higher than that obtained from method no.2. in addition quantitative examination by using hplc analysis showed that the amount of gardenoside and obtained by method no.1 was much more compared with that obtained by method no.2 as showed in table (2). table 2: percentage yield of crude extracts obtained from extraction methods no.1 and no.2. identification of iridoid glycoside (gardenoside) by tlc: gardenoside appeared as a single spot in different developing solvent systems (s1, s2, s3, and s4) against gardenoside reference standard and it has the same color and rf values as that of gardenoside reference standard on the tlc plates after visualization by liebermann-burchard spray reagent, as shown in figure (5). results showed that s3 is the best and more efficient for qualitative and quantitative analysis. identification and characterization of isolated gardenoside analytical tlc the tlc plates of the gardenoside showed that after the initial isolation purification using silica gel gf254 plate's detection under uv light at a wave length of 254 or by spraying with reagent gave two spots using the developing solvent system (s3). the spots have the color and rf values to these of gardenoside. (s1) (s2) (s3) (s4) figure (5):-tlc of gardenoside (st.) and the major fraction (f3) was using four solvent systems (s1, s2, s3 and s4) as developing solvent systems, visualization under uv254. extraction method %yield of crude extract %yield of gardenoside method no.1 6.32 2.75 method no.2 4.91 1.34 f3 st. f3 st. st. f3 f3 st. iraqi j pharm sci, vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 45 melting point the crystals of the isolated samples which were obtained from methanol showed a melting point (117-120 0 c) of the isolated gardenoside compared to melting point of (118-120 0 c) of the gardenoside standard. hplc (high performance liquid chromatography) the retention time for the isolated gardenoside was identical to the main peak of the crude extract and standard reference; more over the peaks isolated gardenoside and the standard reference were super imposable as shown in figures (6 and 7). figure (6):hplc analysis of gardenoside standard figure (7):hplc analysis of isolated gardenoside. ftir the ftir spectrum of the isolated sample material and its gardenoside standard reference were identical which confirm that the isolated compounds are gardenoside as shown in figures (3-19) to (3-22). the ir spectra of the isolated gardenoside and its standard reference material (25) , as showed the following absorption bands at cm -1 in table (3) and as shown in figures (8 and 9). table (3):the characteristic ir absorption bands (in cm -1 ) of the isolated gardenoside in comparison with that of gardenoside as reference standard functional group isolated gardenoside gardenoside standard assignment o-h 3367 3371 broad o-h stretching band of alcohol indicate hydrogen bonding c-h 2908 2909 stretching of ch3 and ch2 groups c=o 1689 1688 c=o stretching of lactone (cyclic ester) c=c 1631 1631 stretching of c=c bond c-h 1438,1373 1442,1377 c-h bending of ch2 and ch3, also o-h bending c-o 1292 1311 c-o stretching of ether iraqi j pharm sci, vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 46 figure (8):ftir spectrum of gardenoside standard. iraqi j pharm sci, vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 47 figure (9):ftir spectrum of isolated gardenoside. iraqi j pharm sci, vol.24(1) 2015 gardenoside in the leaves of gardenia jasminoides 48 acknowledgment we acknowledge prof. dr. ali almusawi, university of baghdad for taxonomical i identification of gardenia jasminoides j.ellis . references 1. bremer b "a review of molecular phylogenetic studies of rubiaceae". annals of the missouri botanical garden, 2009; 96: 4–26. 2. takhtajan, armen . "class magnoliopsida (dicotyledons)". flowering plants (second ed.).2009; springer. p. 51516. 3. "genus gardenia". taxonomy. uniprot. retrieved 2010. 4. ellis, john. "an account of the plants halesia and gardenia: in a letter from john ellis, esq; f. r. s. to philip carteret webb, esq.; f. r. s." phil trans r soc: p.p929-935. 5. "gardenia". the plant list. retrieved 2014. 6. gilman, edward f. "fact sheet fps-222: gardenia jasminoides". university of florida: institute of food and agricultural sciences. 2011. 7. yang yifang, chinese herbal medicines comparisons and characteristics, churchill livingstone, london. 2002. 8. bussell, gene a. "gardenias: a fragrance that captivates". southern living. birmingham, al: time inc. lifestyle group. 2012. 9. plantmark . "gardenias". self. retrieved 1 december 2012. 10. thanh quan pham et al antioxidant properties of crocin from gardenia jasminoides ellis and study of the reactions of crocin with linoleic acid and crocin with oxygen j. agric. food chem., 2000; 48 (5) : 1455–1461. 11. wok-seok jung et al ,genipin inhibits ucp2-mediated proton leak and acutely reverses obesityand high glucoseinduced b cell dysfunction in isolated pancreatic islets.2006; 3: 417–427. 12. eun-hee park et al antiangiogenic activity of gardenia jasminoides fruit, phytotherapy 2012; 17; (8), 961 962. 13. wok-seok jung et al gardenia jasminoides protects against cerulein induced acute pancreatitis .world j gastroenterol 2008 october 28; 14(40): 6188-6194. 14. wang yl, liu jy, jing yl, zhang yb, sun-na, wang xj. / sichuan da xue xue bao yi xue ban effects of the combination of sandostatin and gardenia jasminoides ellis on pancreatic mitochondria injury in severe acute pancreatitis rats. 2011 jan; 42(1)37-40. 15. x. wang,y.q. 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recommendation of iraqi pharmacist toward complementary and alternative medicine samer i. mohammed *,1, mohamed i. alshadedi** and ali a. kasim*** * department of clinical pharmacy ,college of pharmacy, university of baghdad, baghdad, iraq. ** ministry of health and environment alrusafa health directorate , pharmacy department, herbal medicines unit, baghdad, iraq. *** department of clinical laboratory sciences ,college of pharmacy, university of baghdad, baghdad, iraq. abstract over the past few decades, the global usage and applications of different kinds of complementary and alternative medicine are greatly exaggerated among the general population, this requires improving the knowledge of all health care provider including pharmacists toward proper and safe use of different complementary and alternative medicine modalities. the current study aims to assess the iraqi pharmacists' knowledge, use, and recommendation toward complementary and alternative medicine .a cross-sectional pilot survey was done on a convenient sample of iraqi pharmacists. data were collected using a pretested questionnaire specifically designed from a previous study with some modification to reflect the work nature in iraq.the most common complementary and alternative medicine modalities used personally by iraqi pharmacists are nutritional supplements (62%) followed by herbal products (47%). on the other hand, nearly half (48%) of pharmacists recommend some herbal products to their patients besides a slightly lower percentage (46%) recommend nutritional supplements. the pharmacists’ knowledge about several complementary and alternative medicine usages was low. the percentage of pharmacists who answered more than 8 questions properly from the 11 questions asked by the questionnaire was (1.2%), while the percentage of pharmacists who answered correct answers for more than 7 questions was (5.6%), and the percentage of pharmacists who have correct answers for more than half of the questions was only (19.8%). this study points outs that despite the widespread use of some alternative and complementary medicine methods by pharmacists themselves or their patients, the level of knowledge is low in this area. therefore, the study recommends the introduction of evidence-based information and updated guidelines for appropriate use of this type of medicine to pharmacy colleges’ curricula to help future pharmacists meet demand from the growing number of alternative and complementary medicine users across iraq. keywords: complementary medicine, alternative medicine, herbal medicines, nutritional supplements, iraqi pharmacists. المعرفة واالستخدام والتوصية من الصيدلي العراقي نحو الطب البديل والتكميلي ***قاسم الحسين عبدعلي و **محمد اقدام الشديدي ،1،*سامر عماد محمد .بغداد، العراق بغداد،جامعة الصيدلة،كلية السريرية،* فرع الصيدلة .، العراقبغداد، االدوية العشبية وحدة الصيدلة،قسم ،صحة الرصافة دائرة ، والبيئةالصحة وزارة ** العراق.،بغداد ،جامعة بغداد الصيدلة،كلية المختبرية،فرع التحاليل *** الخالصة بشكل مبالغ فيه بين عامة و االستخدام والتطبيق العالمي ألنواع مختلفة من الطب التكميلي والبديل تمعلى مدى العقود القليلة الماضية ، مختلفة الطرائق للاالستخدام السليم واآلمن من اجل ضمان ، وهذا يتطلب تحسين معرفة جميع مقدمي الرعاية الصحية بما في ذلك الصيادلة ناسال و التكميلي. استخدامهم وتوصياتهم تجاه الطب البديلمدى معرفة الصيادلة العراقيين ومقدار تهدف الدراسة الحالية إلى تقييم .للطب التكميلي تم تصميمه خصيًصا سابقا وتم إجراء مسح تجريبي مقطعي على عينة مالئمة من الصيادلة العراقيين. تم جمع البيانات باستخدام استبيان تم اختباره شيوًعا والبديل التكميلي الطب طرائق كثر أان الدراسة اظهرت.سة سابقة مع بعض التعديالت لتعكس طبيعة العمل في العراقمن درا يوصي ما يقرب ٪(. من ناحية أخرى ،47٪( تليها المنتجات العشبية )62المستخدمة شخصيًا من قبل الصيادلة العراقيين هي المكمالت الغذائية )و الغذائية. المكمالتب ونيوص ( ٪ 46قليال ) أقل نسبة جانب إلى بعض المنتجات العشبية لمرضاهمب٪( من الصيادلة 48من نصف ) أسئلة بشكل ثمانيةن أكثر من الطبابة البديلة منخفضة. كانت نسبة الصيادلة الذين أجابوا عاستخدامات من العديد حول الصيادلة معرفة كانت من ألكثر الصحيحة إجابات عن أجابوا الذين، بينما بلغت نسبة الصيادلة فقط٪( 1.2) هي االستبيانالتي طرحها الحد عشرصحيح من األسئلة ا تشير هذه . ( فقط %19.8هي ) ٪( ، و النسبة المئوية للصيادلة الذين لديهم إجابات صحيحة عن أكثر من نصف األسئلة5.6) هيئلة اس سبعة ، إال أن مستوى وصفها لمرضاهم أو من قبل الصيادلة انفسهم الطب البديل والتكميلي بعض طرائقالواسع لستخدام االالدراسة إلى أنه على الرغم من المعلومات المستندة إلى األدلة والمبادئ التوجيهية المحدثة لالستخدام المناسب بأدخال توصي الدراسة بهذا المجال .ولهذا المعرفة منخفض فيما يتعلق لمستخدمي الطب البديل والتكميليمتزايد العدد الالصيدلة لمساعدة الصيادلة في المستقبل لتلبية الطلب من اتلمناهج الدراسية لكليلهذا النوع من الطب ل .في عموم العراق .العراقيون الصيادلة, الغذائية المكمالت, العشبية األدوية, البديل الطب, التكميلي بالط: المفتاحية الكلمات 1corresponding author e-mail: samer.jameel@copharm.uobaghdad.edu.iq received: 27/ 7 /2019 accepted: 6/10 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp88-93 iraqi j pharm sci, vol.29(1) 2020 iraqi pharmacist and complementary and alternative medicine 89 introduction complementary and alternative medicine (cam) is defined by the national institute of complementary medicine as “a broad domain of healing resources that encompasses all health systems, modalities, and practices and their accompanying theories and beliefs, other than those intrinsic to the politically dominant health system of a particular society or culture in a given historical period” (1). complementary and alternative medicine is categorized into five main modules which are mind intermediations, body-based manipulation therapies, herbs or biologically based interventions (natural products), and energy based (metaphysical therapies) (2, 3). for many reasons, the use of cam among the general community has been increased over the past few decades. most importantly is the internet that increased the availability and accessibility of information about cam, as well as, contacts with other cultures that have local cam modalities. moreover, the general vision that cam is safer and cheaper than conventional medications (4). complementary and alternative therapies are widely used in many countries of the world, especially among patients with chronic diseases(5). for economic and cultural reasons, cam has been reported as a primary therapy in some populations(6, 7).the global prevalence of cam use is ranging from 9.8% to 76.0% and varies greatly from country to country(8); e.g., 38% among adults and approximately 12 % among children in the united states(9), 51.8% in the united kingdom(10), 68.9% in australia(11), and 74.8% in south korea(12). unfortunately, the prevalence of cam use in iraq is not documented. despite the global widespread use of cam therapies, their clinical efficacy is a subject of debate amongst many medical professionals. this can be attributed to the lack of scientific data regarding many cam modalities, together with disparities in the knowledge level and evaluation of the available evidence about cam amongst medical professionals. it has been shown that about 72% of patients do not reveal the use of cam to their physicians (13). in addition of being in direct contact with patients, pharmacists are one of the most trusted medical professionals (13).hence, pharmacist have professional obligations to deliver quality information and guidance to patients about safe and effective use of all therapies, including cam. taking into consideration that certain cam therapies can interact or reduce efficacy of conventional medicine (14), pharmacists’ knowledge toward cam need to be based on up-to-date evidence based practice to fulfill these obligations. in iraq, the history of the traditional approach of therapy started from the sumerian 5000 bc. (15). nowadays, different cam modalities are used by iraqi patients for diverse diseases (16). information about iraqi pharmacists’ knowledge toward cam is lacking. therefore, the aim of the current study is to assess the iraqi pharmacists’ knowledge, use and recommendation toward cam. subjects and methods a cross-sectional pilot survey performed on a convenient sample of iraqi pharmacists in different governorates of iraq, the study was conducted from november 2018 to january 2019. data were collected using a pretested questionnaire specifically designed from a previous study with some modification to reflect the work nature in iraq (17). the questionnaire consisted of three sections: the first one collected the demographic data (the age, gender, educational level, work experience, and workplace) for the participants, the second section comprises beliefs and practice of participants in any type of cam like [prayer/spiritual healing, herbal medicine, massage, nutritional supplements, cauterization, music, al hujama (cupping), bioelectromagnetic, homeopathy, acupuncture and aromatherapy]. while the third section designed to measure the knowledge of iraqi pharmacists toward cam. statistical analysis the statistics of this study performed using the microsoft excel 2013 program; discrete variables are presented as numbers and frequencies. results the questionnaire was administered to 232 iraqi pharmacists as a hard copy and a soft copy to be filled online, the survey response rate was 100%, and the demographic data for all participants are presented in table 1. iraqi j pharm sci, vol.29(1) 2020 iraqi pharmacist and complementary and alternative medicine 90 table 1. the demographic data for participants. (n= 232) no variable detail of variable n0. (%) 1. gender male 75 (32) female 157 (68) 2. age 23-30 years 163 (70) 31-40 years 40 (17) >40 years 29 (13) 3. qualification bachelor 200 (86) diploma 7 (3) master 11 (5) phd 14 (6) 4. work experience 1-5 years 157 (68) 6-10 years 33 (14) > 10 years 42 (18) 5. workplace pharmacy 57 (25) hospital 131 (56) college 22 (9 ) else 22 (9) the pattern of use and recommendation behaviors of cam among pharmacists in the present study were summarized in table 2. table 2.the usage and recommendation behaviors of cam among pharmacists. (n= 232) cam modalities usage (n) percentage (%) recommendation (n) percentage (%) prayer/spiritual healing 96 41 95 41 herbal medicine 110 47 111 48 massage 91 39 87 38 nutritional supplements 143 62 106 46 cauterization 19 8 42 18 music 70 30 61 26 cuppingal hujama 31 13 77 33 bio-electromagnetic 10 4 31 13 homeopathy 10 4 12 5 acupuncture 21 9 26 11 aromatherapy 32 14 32 14 regarding the pharmacists’ responses to knowledge-based items about various cam uses which shown in table 3, this study showed a low level of knowledge about cam and the percentage of pharmacists who answered more than 8 questions correctly from the 11 questions asked by the questionnaire was (1.2%) which represents only three participants , while the percentage of pharmacists who answered correct answers for more than 7 questions of the questions was (5.6%) which represents thirteen participants, and the percentage of pharmacists who have correct answers for more than half of the questions was only (19.8%) which represents (46) participants . iraqi j pharm sci, vol.29(1) 2020 iraqi pharmacist and complementary and alternative medicine 91 table 3. pharmacists’ responses on knowledge-based items about different cam uses variable pharmacists ( n=232) n (%) true false don’t know alternative medicines are used instead of conventional medicine 85 (37) 65 (28) * 82 (35) the use of ginger, thyme, and green tea in the first trimester of pregnancy is totally safe. 39 (17) 104(45) * 89 (38) ginseng is commonly used as a general health tonic 144(62) * 27 (12) 61 (26) capsaicin relieves arthritis pain 111(48) * 21 (9) 100 (43) hijama is a popular method used in iraq 180(78) * 12 (5) 40 (17) frankincense is used as an antispasmodic 10 (4) * 40 (17) 187 (81) ginger can be used as an analgesic. 46 (20) * 94 (41) 92 (40) it‘s safer to use alternative medicines along with conventional medicine. 32 (14) * 78 (34) 122 (53) herbal medicines are free of side effects. 4 (2) 222(96) * 6 (3) caffeine is used to treat insomnia. 11 (5) 204(88) * 17 (7) peppermint is used as an antimicrobial 62 (27) * 101(44) 69 (30) * percentage of respondents who got the questions correct on each knowledge question. discussion obviously, cam is going to be part of health care for much of the population in the very near future; this is augmented by the rapidly growing research base in this realm.(18) being key healthcare providers, pharmacists have a professional obligation to deliver quality information, advice and guidance to their patients including those using cam. this study was designed to assess the knowledge, use and recommendation of iraqi pharmacists toward cam. most of the participant pharmacists in the study were female (68%), and the predominant age group being between 23 and 30 years (70%); this points short years of practice but more fresh academic information. the results of the current study showed that the most common cam modalities used by iraqi pharmacists were nutritional supplements (62%), herbal medicine (47%) and praying and spiritual healing (41%). while the most common cam modalities recommended were herbal medicine (48%), nutritional supplements (46%) and praying and spiritual healing (41%) other studies showed different pharmacists’ priorities in using or recommending cam modalities. albedah, et al. showed that prophetic medicine including prayer, honey and bee products, herbal medicine and hujama, are the most important cam modalities used by health professionals, including pharmacists, in the riyadh region, saudi arabia.(19) similarly, in iranian study, herbal medicine, hujama and massage therapy were the most frequently recommended cam modalities.(20) the religious values and cultural believes seem to have a significant impact in this regard. in palestine, pharmacists are recommending exercises and nutritional supplements as the two major recommended cam modalities.(21) on the other hand, western researches showed that the most accepted cam modalities were acupuncture, relaxation techniques, chiropractic, and massage therapy.(22-24) lack of knowledge and the inadequate number of trained personnel to use certain cam modalities may be the main barriers toward using or recommending them. regarding the knowledge of iraqi pharmacists toward cam, the results showed that only 19.8% of the participants have answered more than half of the administered questions correctly. this indicates very poor knowledge; taking into consideration that most of the participants had graduated from college within less than seven years period (as discussed earlier), which supposedly means they have considerably fresh academic information. other studies carried out in lebanon(25), palestine(21), and saudi arabia(26) showed fair knowledge of pharmacist toward cam. inadequate level of knowledge may reflect the need to improve the undergraduate curriculum, and to involve pharmacists into well-structured training programs and postgraduation courses of cam. finally, a study conducted in 2003 in singapore among pharmacists attending the 61st international congress of international pharmaceutical iraqi j pharm sci, vol.29(1) 2020 iraqi pharmacist and complementary and alternative medicine 92 federation (fip), more specific those joining the singapore traditional chinese medicine research symposium, showed a good knowledge of pharmacists toward cam.(27) apparently, pharmacists participating in the symposium whom surveyed in the study had good expertise toward cam. the increasing number of cam users in the general population of iraq represents a challenge to iraqi pharmacists. therefore, pharmacists need to integrate more information about the correct use and specialized guidelines about cam into their continuing professional development plan to expand their awareness and skills in dealing with this growing aspect of therapy. ethical statement the study and the questionnaire was validated by the local scientific and ethical committee in college of pharmacy, baghdad university. verbal consent was obtained from all participants included in the study. the authors informed the participants about the purpose of the study at the beginning of each interview. meanwhile, the respondents were informed that their participation was voluntary and they were allowed to withdraw themselves at any point of time during the interview. references 1. national institute of complementary medicine. understanding complementary medicine. 2007 [cited 2019 july 1st.]. available from: http :// www .nicm .edu .au /health_information/information_for_cons umers/. 2. tindle ha, davis rb, phillips rs, eisenberg dm. trends in use of complementary and alternative medicine by us adults: 1997-2002. alternative therapies in health and medicine. 2005;11(1):42-9. 3. roush ra. complementary and alternative medicine.: routledge; 2016. 4. ventola cl. current issues regarding complementary and alternative medicine (cam) in the united states: part 1: the widespread use of 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2008(12):1-23. 10. posadzki p, watson lk, alotaibi a, ernst e. prevalence of use of complementary and alternative medicine (cam) by patients/consumers in the uk: systematic review of surveys. clinical medicine. 2013;13(2):126-31. 11. xue cc, zhang al, lin v, da costa c, story df. complementary and alternative medicine use in australia: a national population-based survey. journal of alternative and complementary medicine. 2007;13(6):643-50. 12. ock sm, choi jy, cha ys, lee j, chun ms, huh ch, et al. the use of complementary and alternative medicine in a general population in south korea: results from a national survey in 2006. journal of korean medical science. 2009;24(1):1-6. 13. eisenberg dm, kessler rc, van rompay mi, kaptchuk tj, wilkey sa, appel s, et al. perceptions about complementary therapies relative to conventional therapies among adults who use both: results from a national survey. annals of internal medicine. 2001;135(5):344-51. 14. elmer gw, lafferty we, tyree pt, lind bk. 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27. koh hl, teo hh, ng hl. pharmacists' patterns of use, knowledge, and attitudes toward complementary and alternative medicine. journal of alternative and complementary medicine. 2003;9(1):5163. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 curcumin-tacrolimus interaction doi: https://doi.org/10.31351/vol31iss1pp246-250 246 effect of curcumin at various doses on the pharmacokinetic profile of tacrolimus in healthy rabbits issam mohammed abushammala*,1, belal mohammed mqat* and abdallah mohammed hamdan* *department of pharmaceutics and industrial pharmacy, faculty of pharmacy, al-azhar university/gaza, palestine. abstract the purpose of present study was to evaluate the effect of co-administration of curcumin (cur) at various doses on the pharmacokinetic (pk) profile of tacrolimus (tac), a cyp 3a4 substrate in healthy male rabbits. healthy male rabbits (n=18) were employed in an in vivo, parallel-randomized study. three groups of rabbits were selected and separated: the rabbits in the first group (control group) received 1 mg/kg tac orally. blood samples (1.5-2 ml) were drawn from rabbits' ear marginal veins at the following time frames: 15.0, 30.0, 45.0, 60.0, 90.0, 120.0, 150.0, 180.0 and 300 minutes after tac administration and analyzed by using a tac chemiluminescent enzyme immunoassay (clia) detection kit. in the second and third groups (test groups), rabbits received tac (1mg/kg) at identical conditions as in the control group with volumes equivalent to (30 and 90 mg/kg/day) of prepared cur suspension in normal saline for seven continuous days. blood samples from the control group were obtained on the eighth day. non-compartmental analysis was used to derive different pk parameters of tac for the three groups. when cur was co-administered at both concentrations, statistically insignificant small changes between the control and testing groups were found. the current results revealed that the differences for the three groups in pk parameters as cmax, tmax, ke, auc0-6 and auc0-∞ were statistically insignificant (p>0.05). in conclusion, it has been found that cur at the experimented doses does not affect the pk of tac. further confirmation of our findings is required before these results can be applied in patient care. keywords: tacrolimus, curcumin, pharmacokinetics لتاكروليموس في األرانب السليمة لتأثير الكركمين بجرعات مختلفة على ملف الحرائك الدوائية * عبدهللا محمد حمدان و *بالل محمد مقاط ،1*،عصام محمد ابو شمالة . غزة، فلسطين –قسم الصيدالنيات والصيدلة الصناعية، كلية الصيدلة، جامعة األزهر * الخالصة ( لعقار pk( بجرعات مختلفة على الخواص الحركية الدوائية )curالكركمين ) أضافة مادةالغرض من هذه الدراسة هو تقييم تأثير في ذكور األرانب السليمة صحيا. في هذه الدراسة تم توظيف أرانب ذكور صحية cyp3a4بواسطة االنزيم الذي يستقلب( وtacالتاكروليموس ) ( في دراسة عشوائية متوازية. تم اختيار وفصل ثالث مجموعات من األرانب: تلقت األرانب في المجموعة األولى )المجموعة الضابطة( 18)ن = مل( من األوردة الهامشية ألذن األرانب في األطر الزمنية المحددة بعد 2-1.5م تم سحب عينات الدم )عن طريق الفم ومن ث tacمجم / كجم من 1 ( المضيئة اإلنزيمية المناعية المقايسة عن الكشف أدوات مجموعة باستخدام تحليلها و التاكروليموس دواء بدواء cliaإعطاء الخاصة ) ختبار الثانية والثالثة )مجموعتي االختبار( ، أعطيت األرانب نفس الجرعة الخاصة بدواء التاكروليموس التاكروليموس. اما فيما يتعلق بمجموعتي اال المحضر في محلول ملحي عادي لسبعة curمجم / كجم / يوم( من معلق 90و 30فى نفس الظروف كما في المجموعة الضابطة بتراكيز تعادل ) للمجموعات الثالث tacعينات الدم كالمجموعة الضابطة وتم تحديد معلمات الحركية الدوائية المختلفة لـ أيام متواصلة. في اليوم الثامن ، تم جمع عن المختبرة والمجموعات الضابطة المجموعة بين إحصائية داللة ذات غير اختالفات هناك كانت ، أنه لوحظ الجزئي. غير التحليل د باستخدام ke, max, tmaxc , ,. كشفت نتائجنا أن الفروق بين المجموعات الثالث في معامالت الحرائك الدوائية مثل في كال التركيزين curاالضافة مع ∞-0and auc 6-0auc ( كانت غير ذات داللة إحصائيةp˃0.05 فى الختام ، لقد وجد ان الكوركمين عند الجرعات المجربة ال يؤثر على .) ن هناك حاجة إلى مزيد من التأكيد على هذه النتائج التي تم التوصل إليها قبل تطبيقها في رعاية المرضى.معامالت الحركية لدواء التاكروليمس ولك تاكروليموس ، الكركمين ، حركية الدواء. المفتاحية: الكلمات introduction tacrolimus (tac), a calcineurin inhibitor, is an important component of the traditional immunosuppressive regimen after renal transplantation. due to its narrow therapeutic index and the fact that various factors intervene with its metabolism (1). tac's pk pathways involve cytochrome p450 (cyp) 3a4 and p-glycoprotein (p-gp), and drugs that interact with these enzyme systems will influence tac blood concentrations (2). because tac covers a restricted therapeutic window and lacks a strong correlation between dosage and serum concentration, it may cause toxicity, subtherapeutic failure, and organ rejection (3). tac interacts with several other treatments used in transplantation therapy that are known cyp3a and/or p-gp inhibitors and/or inducers as a substrate of cyp3a enzymes and p-gp (4). 1corresponding author e-mail: issam.abushammala@uv.es received: 14/8/2021 accepted: 20/10 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp246-250 iraqi j pharm sci, vol.31(1) 2022 curcumin-tacrolimus interaction 247 the cyp450 enzymes are membranebound hemoproteins that play a pivotal role in the detoxification of xenobiotics, cellular metabolism and homeostasis. induction or inhibition of cyp450 enzymes is a major mechanism that underlies drugdrug interactions (5). natural products have shown a promising source of components for the development of new drugs (6,7). one of the most common example is turmeric (curcuma longa linn.), which is a member of the zingiberaceae family (8). the rising importance of natural remedies around the world has sparked concerns concerning herb-drug interactions. these interactions are especially important for drugs with narrow therapeutic indices and may either be pharmacodynamic or pk in nature when used with p-gp and cyp3a4 substrates (9,10). herb-drug interactions are one of the most serious clinical concerns while using herbal remedies and prescribed drugs simultaneously (11). the likelihood of pk medication interactions involves two key challenges: pharmacotoxicity and treatment failure. the earlier can be produced by inhibiting drug metabolism and clearance enzymes, whereas the latter can be triggered by enzymatic stimulation resulting in increased drug metabolism (12). numerous in vitro studies have reported that curcumin inhibited not only p-gp, but also cyp3a4. based on these in vitro results, coadministration of curcumin should increase the oral bioavailability of substrates of p-gp or cyp 3a4 (13). the effect of co-administration of cur at various doses on the pk profile of tac, a cyp 3a4 substrate, was investigated in the present study. materials and methods animals in this study, eighteen new zealand strains of adult male rabbits weighing (3.1-3.4 kg) and aged (8-10) months were used. the research and ethics committee granted permission to the experimental animal care facility, college of pharmacy, alazhar university of gaza (aug), palestine. the rabbits were randomly split into three groups (six for each group). all rabbits were housed in controlled conditions with a 12-hour light/dark cycle at (25°c ± 2°c), pellet feeding, and free access to water (ad libitum). prior to the trial, they also fasted over the night. study design and blood sampling eighteen healthy male rabbits were used in an in vivo, parallel-randomized controlled study. three groups of rabbits were selected and separated: the rabbits in the first group (control group) received 1 mg/kg tac orally from a hard capsule (5 mg, prograf®, astellas) kindly provided by the palestinian ministry of health's medication stores. blood samples (1.5-2 ml) were drawn from rabbits' ear marginal veins at the following time frames: 15.0, 30.0, 45.0, 60.0, 90.0, 120.0, 150.0, 180.0 and 300 minutes following tac administration (14). in the second and third groups (test groups), rabbits were given tac (1mg/kg) at the same conditions as in the control group with volumes equivalent to (30 and 90 mg/kg/day) from prepared cur suspension in normal saline (turmeric® curcumin 550 mg, jamieson) hard gelatin capsules purchased from a local pharmacy in gaza, palestine) for seven continuous days. on the 8th day, tac was given one hour after the last dosage of cur suspension, and blood was collected from the second and third test groups at the same time as the control group. whole blood samples were stored in edta tubes at 2-8°c until they were tested (whole blood sample is stable for up to 3 days). blood samples analysis at the medical relief society-gaza laboratory, whole blood samples were tested to detect tac concentrations using the maglumi 800 system and tac detection kit. the tac detection kit is based on a chemiluminescent enzyme immunoassay (clia). it's utilized in hospitals to check tac doses by doing fast tac assays on whole blood. pk of tac and statistical analysis the pk parameters, cmax, tmax, ke, auc06, and auc0-∞ were determined for the control and cur-treated test groups. the cmax and tmax were accurately observed through plasma concentration versus time curves. the auc0-6 was determined using the linear trapezoidal method. the auc0∞value was calculated using the formula: auc0-∞ = auc0-6 + ct / ke, where ct is the final measured blood concentration at time t and ke is the elimination rate constant. the ke was calculated using semilogarithmic dependency, which corresponds to first-order kinetics, by leastsquares regression of blood concentration-time data points in the terminal region. the pk analysis was determined using a modelindependent method (non-compartmental approach), winnonlin professional software (version 6.3, pharsight corporation, cary, nc), and (graphpad prism version 4.00; san diego, ca, usa). the pk parameters of tac alone (control group) and tac co-administered with cur in the first and second test groups were compared using statistical methods such as descriptive analysis and the mann-whitney test. the data was analyzed by using the spss program (version 22.0). a statistically significant difference was considered when p≤0.05. iraqi j pharm sci, vol.31(1) 2022 curcumin-tacrolimus interaction 248 results cur's effect at various dosages on the pk parameters of tac was explored in vivo. the pk parameters of tac in the control group (first group) were compared to those of the test groups treated with cur 30 and 90 mg/kg/day (second and third groups respectively) and the blood concentrationtime profiles of tac in the first group and in the coadministered with cur (test groups) are shown in figure 1 and table 1. figure 1. plot of tac concentration-time profile with and without cur for the control, first and second test groups. table 1. calculated pk parameters of the control, the first and second test groups (6 for each). pk parameters groups mean ± sd p-values acmax (ng/ml) control group 27.44 ± 5.83 0.668¥ 0.338§ first test group 25.78 ± 7.60 second test group 31.20 ± 4.33 btmax (hr -1) control group 1.50 ± 0.50 0.405¥ 0.690§ first test group 1.90 ± 0.54 second test group 1.65 ± 0.65 cke (hr-1) control group 0.102 ± 0.04 0.736¥ 0.570§ first test group 0.124 ± 0.10 second test group 0.091 ± 0.03 dauc0-6 (ng*hr/ml) control group 97.37 ± 18.06 0.524¥ 0.052§ first test group 112.42 ± 49.14 second test group 130.02 ± 20.54 eauc0-∞ (ng*hr/ml) control group 204.49 ± 75.82 0.564¥ 0.679§ first test group 167.05 ± 71.42 second test group 189.60 ± 25.80 (¥): p-value of the differences between the control and first test group; (§): p-value of the differences between the control and the second test group. *:p≤0.05 statistical significance, sd: standard deviation, amaximum blood concentration, btime to peak concentration, celimination rate constant, darea under the concentration-time profile curve from 0 to 6 hours and darea under the concentration-time profile curve from 0 to infinity. t im e ( h ) t a c ro li m u s ( t a c ) b lo o d c o n c e n tr a ti o n ( n g /m l) 0 .5 1 1 .5 2 2 .5 3 3 .5 4 4 .5 5 5 .5 6 0 1 0 2 0 3 0 4 0 c o n t r o l g r o u p : t a c a l o n e ( 1 m g / k g ) s e c o n d t e s t g r o u p ( t a c 1 m g / k g + c u r 9 0 m g / k g / d a y ) f i r s t t e s t g r o u p ( t a c 1 m g / k g + c u r 3 0 m g / k g / d a y ) iraqi j pharm sci, vol.31(1) 2022 curcumin-tacrolimus interaction 249 discussion tac is an immunosuppressive agent that has emerged as a valuable therapeutic alternative to cyclosporine following solid organ transplantation (15). the most important human cyp isozyme is cyp3a4, which is involved in the metabolism of the majority of therapeutically prescribed drugs (16). tac is a lipophilic compound that is metabolized by the cyp450 3a subfamily and is eliminated after extensive metabolism. because of its low therapeutic index, tac requires blood level monitoring. therefore, metabolic studies, such as drug-drug interaction and metabolite identification studies, are vital and urgent for the development of clinically optimal medication use (17). herb-drug interactions are among the most frequent medical concerns when taking herbs and pharmaceutical prescriptions concurrently (11). in addition, tac bioavailability decreased when concurrently administered with st john’s wort (sjw), cranberry, rooibos tea, and boldo in human models by induction of cyp450 system isoenzyme and/or p-gp efflux pump (18-21). meanwhile, tac bioavailability was enhanced in human and/or animal models when grapefruit juice, schisandra, pomelo, and ginger were given concurrently, presumably due to an inhibitory effect on the cyp450 system or the p-gp efflux pump (22-24). the pk investigations evaluating the interaction between herbal supplements and the bioavailability of various therapeutically monitored drugs, such as digoxin, cyclosporine and carbamazepine revealed that herbal supplements have a clinically insignificant effect on the pk profiles of digoxin and cyclosporine (25-27). our findings, statistical examination of tac pk parameters revealed statistically insignificant differences between the three groups (table 1). the control group's mean cmax decreased slightly from 27.44±5.83 ng/ml to 25.78±7.60 ng/ml in the first test group and a slight increase to 31.20±4.33 ng/ml in the second test group were observed. similarly, liu and his colleagues found comparable results when they investigated the effect of various cur administrations (25 and 50 mg/kg) on the pk parameters of warfarin alone (control group). in this investigation, the increasing in cmax in the herbal treated groups from 1.14±0.33 to 1.49±0.38 and 1.15±0.29 µg/ml was insignificant when compared to the control group (p>0.05). however, in the large dose cur groups (100 mg/kg) were statistically significant (28). also, the current results revealed that the decrease in auc0-∞ from 204.49±75.82 to 167.05±7.10 in the first test group and 189.60±25.80 ng*hr/ml in the second test group respectively, was also similar and statistically insignificant (p>0.05). furthermore, the remaining pk parameters showed insignificant variations, including auc0-6, tmax and ke between the control and tested groups. additional investigations need to be conducted with higher doses of curcumin and over longer periods of time, or recruit a larger number of animals, to support the interpretation of previous research findings. conclusion drug interactions with herbs are critically important for patient safety, especially as herbal remedies become more popular and are used more frequently. the cur had systemically insignificant effect on the pk profile of tac in this investigation at the dosages examined. ethical statement the study was approved and performed under ethical principles laid down by the faculty of pharmacy, al-azhar university-gaza, palestine. conflict of interest no conflicts of interest relevant to this article. acknowledgments the authors wish to express deepest gratitude and thanks to all staff in the faculty of pharmacy at al-azhar university-gaza for their kind support. references 1. schutte-nutgen k, tholking g, and suwelack b, et al. tacrolimuspharmacokinetic considerations for clinicians. current drug metabolism. 2018;19:342-350. 2. van gelder t. drug interactions with tacrolimus. drug safty. 2002;25:707-712. 3. zhai x, chen c, and xu x, et al. marked change in blood tacrolimus concentration levels due to grapefruit in a renal transplant patient. journal of clinical pharmacy and therapeutics. 2019; 44:819-822. 4. christians u, jacobsen w, and benet lz, et al. 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pradhan n, et al. case report: boldo (peumus boldus) and tacrolimus interaction in a renal transplant patient. transplantion proceedings. 2014;46:2400-2402. 22. peynaud d, charpiat b, and vial t, et al. tacrolimus severe overdosage after intake of masked grapefruit in orange marmalade. european journal of clinical pharmacology. 2007;63:721-2. 23. xiao-ling q, jia-li l, and si-han w, et al. coadministration of wuzhi tablet (schisandra sphenanthera extract) alters tacrolimus pharmacokinetics in a doseand timedependent manner in rats. journal of ethnopharmacology. 2020;263:113233. 24. egashira k, sasaki h, and higuchi s, et al. food-drug interaction of tacrolimus with pomelo, ginger, and turmeric juice in rats. drug metabolism and pharmacokinetics. 2012;27:242-247. 25. mauro vf, mauro ls, and kleshinski jf, et al. impact of ginkgo biloba on the pharmacokinetics of digoxin. american journal of therapeutics. 2003;10:247-251. 26. xin hw, wu xc, and li q, et al. the effects of berberine on the pharmacokinetics of cyclosporin a in healthy volunteers. methods and findings in experimantal and clinical pharmacology. 2006;28:25-29. 27. abushammala im, el-shaikh ali fk, abu shammaleh kf, taha mm, miqdad my. effect of panax ginseng on carbamazepine pharmacokinetics in rabbits. turk j pharm sci. 2021;25:18(1):17-20. 28. liu ac, zhao lx, lou hx. curcumin alters the pharmacokinetics of warfarin and clopidogrel in wistar rats but has no effect on anticoagulation or antiplatelet aggregation. planta medica. 2013;79:971-977. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 plant molecules for treatment of tuberculosis doi: https://doi.org/10.31351/vol31iss2pp1-13 1 plant-derived molecules for the treatment of tuberculosis: a review meena yadav*,1 and poonam sharma** *department of zoology, maitreyi college, university of delhi, india **department of zoology, gargi college, university of delhi, delhi, india abstract synthetic anti-tb drugs are being used to treat tuberculosis (tb) as they are effective, however, they are accompanied by many side effects. the disease has remained largely uncured till date. the use of the plant extracts or phytochemicals along with the anti-tb drugs is a very attractive strategy to make the treatment more effective as phytochemicals have no side-effects, are much less toxic than synthetic anti-tb drugs, are safe to use and most importantly, do not produce resistant strains as opposed to synthetic anti-tb drugs. approximately 420,000 plant species have been identified globally and among them only a few have been explored for their therapeutic potential. traditional medicine in different parts of the world has employed crude extracts of several plant species to cure tuberculosis. several anti-tb phytochemicals have been found in the plants that are identified to have therapeutic qualities. these phytochemicals are majorly glycosides, flavonoids, triterpenes, phenolic compounds, alkaloids, diterpenoid, lipids, tannins, sterols etc. they are either antimycobacterial or act synergistically with anti-tb drugs and reduce their adverse effects. phytochemicals ameliorate the symptoms either by reducing the oxidative stress in the afflicted tissues or by regulating the inflammatory response. hence, plant derived molecules have great potential to become a part of the alternative treatment strategy for tb in the future. keywords: tuberculosis, plant-derived molecules, antioxidant, anti-mycobacterial, oxidative stress. introduction tuberculosis (tb) is an infectious disease caused by mycobacterium tuberculosis (m.tb) which affects primarily the lungs in humans. it is widespread and is a serious public health issue that needs to be addressed quickly and effectively. the m.tb lineages are spread worldwide with few having global distribution while few remaining restricted to certain regions. the restricted presence of some strains, in specific regions, had been explained by theory of ‘local adaptation’ in the past, whose latest evidence has been provided by liu et al. (1). wherein they did genome sequencing of hundreds of m.tb strains and confirmed the ‘local adaptation’ theory in tibetan population. the seriousness of the disease may be understood by the fact that in the year 2018, around 10 million people had developed tb and 1.5 million had died from it and by 2020, an estimated 1.7 billion people were infected with m.tb. among the people who get infected with human immunodeficiency virus (hiv), tb is the leading cause of death. approximately half of the tb patients have been reported from eight countries viz. bangladesh, china, india, indonesia, nigeria, pakistan, philippines and south africa and according to latest who update, the success rate of treating multi-drug resistant-tb (mdr-tb) is fiftysix percent (2). various synthetic drugs have been produced to treat tb. who has classified anti-tb drugs into five classes; first-line anti-tb drugs (for drug-susceptible tb) which include rifampicin , isoniazid, pyrazinamide, and ethambutol; secondline anti-tb drugs which include amikacin, capreomycin, kanamycin, and streptomycin; the third-line anti-tb drugs which include fluoroquinolones; the fourth class anti-tb drugs which include cycloserine, ethionamide, paraaminosalicylic acid, prothionamide, terizidone, and thioacetazone and fifth-class anti-tb drugs (drugs with unclear efficacy) which include amoxicillin/clavulanate, clarithromycin, clofazimine, imipenem, and linezolid (3). the m.tb strains that are resistant to at least isoniazid and rifampin (first-line anti-tb drugs) are known as multidrug resistant (mdr) and cause mdr-tb (4) whereas the m.tb strains that are resistant to isoniazid, rifampin, fluoroquinolones and at least one of the three injectable second-line anti-tb drugs are known as extensively drug resistant (xdr) and cause xdr-tb (5). centers for disease control and prevention (cdc) recommends a 6to 9-month treatment schedule for tb. the u.s. food and drug administration (fda) has approved ten drugs for treating tb, out of which four drugs viz. isoniazid (inh), rifampin (rif), ethambutol (emb) and pyrazinamide (pza), form the core of treatment regimen (6) . 1corresponding author e-mail: drmeena.yadav@gmail.com received: 30/7 /2021 accepted:21 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp1-13 iraqi j pharm sci, vol.31(2) 2022 plant molecules for treatment of tuberculosis 2 while national health service, united kingdom recommends inh and rif for six months and emb and pza for first two months of the treatment regimen (7). the synthetic drugs used for treating tb cause varied side effects. hepatotoxicity, nephrotoxicity, ocular toxicity, skin rashes, fever, psychotic alterations are just a few of the adverse effects (8,9,10). the side effects are manifested in the form of symptoms such as gastrointestinal disturbances, arthralgia, hepatitis, psychiatric disorder, hypothyroidism, peripheral neuropathy, dermatological effects, epileptic seizures, ototoxicity etc. (11,12). although, emb has fewer side effects than thioacetazone but it does show some side effects and the most serious of them is retrobulbar neuritis which is manifested as loss of visual acuity and color vision (11). after many trials and testing over the years, who now recommends the use of a fixed dose of myrin®-p forte(a combination drug) for treating tb, which includes rifampicin (13.5 mg/kg), isoniazid (6.75 mg/kg), pyrazinamide (36 mg/kg), and ethambutol (24.8 mg/kg) viz. ripe – for the intensive phase (2 months), followed by continuous rifampicin and isoniazid administration for four-six months. however, these drugs cause hepatic injuries which are mainly caused due to reactive oxygen species (ros)-mediated oxidative damage (13). the current practice of treatment of tb includes a combination of different drugs for a duration of six months for drug-susceptible tb, for 9-20 months for mdr-tb while the duration of treatment can be longer for xdr-tb. the duration can also be longer when the patient is not responding satisfactorily (14). the higher the age of the patients, higher is the risk of severe symptoms of tb. thus, it is a fact that synthetic anti-tb drugs are accompanied by various kinds of side effects, some of which may have serious implications or may even prove fatal. some of the common anti-tb drugs, their side-effects and symptoms have been summarized in table 1. the patients suffering from multi drug resistant tuberculosis (mdr-tb) are advised different treatment regimens than patients with drugsusceptible tb. as a result, the former experience many side effects which are greater in intensity. the patients may experience one or more side effects depending on the dosage of anti-tb drugs (12). table 1. commonly used anti-tb drugs and their side-effects s. no. anti-tb drugs side-effects clinical symptoms references 1 rifampicin (rif) hepatotoxicity hyperbilirubinemia; elevates levels of enzymes like alkaline phosphatase (alp), serum glutamic oxaloacetic transaminase (sgot/ast), serum glutamic pyruvic transaminase (sgpt/alt); allergic reactions jaundice, nausea, vomiting, abdominal pain; allergic reactions include fever, rashes, flu-like symptoms, eosinophilia; sometimes haemolytic anemia, hemoglobinuria, and kidney damage (11) 2 isoniazid (inh) hepatotoxicity; neurologic adverse reactions gastrointestinal symptoms; dermatological manifestations; neuropsychiatric adverse effects like peripheral neuropathy, ataxia and paresthesia (11) 3 pyrazinamide (pza) hepatotoxicity gastrointestinal intolerance; gouty arthritis; non-gouty polyarthralgia (12, 11) 4 ethambutol (emb) hepatotoxicity; ocular toxicity; hypothyroidism gastrointestinal disturbance, psychosis, hypothyroidism (11, 12) 5 streptomycin (sm) otovestibular toxicity; nephrotoxicity permanent deafness, (11) 6 kanamycin (kan) ototoxicity hearing loss (15) 7 capreomycin nephrotoxicity, ototoxicity, electrolyte disturbance loss of balance; hearing loss (16) 8 amikacin renal dysfunction skin rash (3) 9 fluoroquinolones (fqn) less hepatoxicity than firstline anti-tb drugs gastrointestinal problem; allergic reaction; psychosis (12, 17) iraqi j pharm sci, vol.31(2) 2022 plant molecules for treatment of tuberculosis 3 hepatotoxicity is the predominant side effect of synthetic anti-tb drugs. these drugs induce hepatotoxicity in 5-28 percent of individuals, and they are one of the common causes of hepatotoxicity worldwide. the common clinical symptoms of hepatotoxicity include jaundice, nausea, vomiting and abdominal pain. it is also accompanied by higher levels of bilirubin and increased activity of hepatic transaminases. due to these symptoms, many times, therapeutic doses are frequently reduced, which is one of the leading causes of treatment failure. these symptoms are also the biggest reason for discontinuation of the therapy which might be a cause for development of resistant forms of m.tb (14). experts no longer recommend capreomycin, kanamycin, amoxicillin/clavulanate (when administered without a carbapenem), azithromycin, or clarithromycin for treating mdrtb due to serious adverse effects documented after using these anti-tb medications over the years (16). however, other synthetic anti-tb drugs are still largely being used to treat tb as they are effective despite of their adverse side effects and there is no concrete alternative strategy to treat tb more effectively. in spite of several drugs available for treating tb, the disease remains largely uncured. further, there is only one licensed vaccine viz. bacille calmette-guerin (bcg) that can prevent the development of severe tb in children. there is no such vaccine for adults yet (18). exploring plants for molecules with anti-tb properties synthetic anti-tb drugs have numerous side-effects, are less efficient and also cause drug resistance in mycobacteria. hence, there is much need to look for alternative strategies to treat tb. the newer strategies must emphasize improving tb treatment methods, reducing side effects, and maintaining safety of usage. a very good alternative strategy would be to use plant products along with synthetic anti-tb drugs. plant-derived molecules show fewer side effects, have less toxicity, are less likely to develop resistance, and have better efficacy (19), hence could become a good source of alternative medicine for tb. plant-derived molecules as potential anti-tb drugs various studies have shown that the sideeffects of the anti-tb drugs can be ameliorated by using natural products obtained from the plants. the plant-based products are diverse and can provide effective plant molecules or phytochemicals for treating tb. phytochemicals are secondary metabolites of plants and unlike primary metabolites, which are essential for plant growth and development, the secondary metabolites are not directly involved in growth, reproduction or development of plants but they influence plant's survival and defense system (20). plants that are effective against tb are rich sources of alkaloids, flavonoids, glycosides, diterpenoid, triterpenes, lipids, phenolic compounds, tannins, sterols etc., all of which have hepatoprotective function (21). for example, leaf extracts of moringa oleifera contain phytochemicals like alkaloids, flavonoids, carbohydrates, glycosides, saponins, tannins and terpenoids. they repair liver damage caused due to inh, pza, and rif when given orally by restoring normal levels of hepatic enzymes, serum bilirubin, and lipid peroxidation (22). the root extract of cassia auriculata also reduces anti-tb drug induced liver toxicity by significantly lowering the levels of hepatic enzymes like aspartate transaminase (ast), alkaline phosphatase (alp), alanine transaminase (alt) and total bilirubin as well as cholesterol. even the fruits of terminalia chebula have similar functions (23). thus, some phytochemicals reverse the effects of synthetic drugs and reduce the toxic impact on liver (figure 1). figure 1. hepatoprotective role of phytochemicals against synthetic anti-tb drugs(adapted from mangwani et al, 2020) (24) iraqi j pharm sci, vol.31(2) 2022 plant molecules for treatment of tuberculosis 4 like synthetic anti-tb drugs, phytochemicals interfere with cellular mechanisms of mycobacteria and inhibit their growth. however, because exposure to phytochemicals does not lead to development of resistance in mycobacteria (25), there is a negligible risk of drug-resistant strains developing after or during tb therapy using plant molecules. the recent research in this area has given us hope for improving the strategies to combat tuberculosis. several such potential anti-tb phytochemicals have already been identified and their actions have been tested (26,27,28). some of the potential anti-mycobacterial phytochemicals have been listed in table 2. table 2. phytochemicals having potent anti-tb activity s. no. phytochemicals activity isolated from plants references 1 1α-acetoxy-6β, 9βdibenzoyloxy-dihydro-βagarofuran against mdr strain of mtb celastrus vulcanicola donn. sm. (celastraceae) (29) 2 5-hydroxy furanocoumarin against mdr strain of mtb foeniculum vulgare mill. (apiaceae) (30) 3 5-hydroxy-2-(4´hydroxyphenyl)-7methoxy2,3-dihydro-4h-chromen-4one against mtb h37rv phoradendron robinsoni trel. (santalaceae) (31) 4 5,7-dihydroxy-3-methoxy-2(4-methoxyphenyl)-4hchromen-4-one; β,γ-dimethylα,δ-bis (3,4-dihydroxyphenyl) butane and 5,6,7-trihydroxy-3methoxy-2-(4methoxyphenyl)-4h-chromen4-one against mtb larrea divaricata cav. (zygophyllaceae) (31) 5 (14b, 24e)3-oxolanosta7,24-dien-26-oic acid (mic= 64µg/ ml), and (14b, 24e)-3hidroxylanosta-7,24-dien26oic acid against mtb h37rv amphipterygium adstringens (schltdl.) ex standl. (julianaceae). (31) 6 25-hydroperoxycycloart-23en-3β-ol against mdr strain of mtb blepharodon nitidum (vell.) j.f. macbr. (asclepiadaceae) (32) 7 abietane and its derivatives against mdr strain of mtb plectranthus grandidentatus gurke (lamiaceae) (33) 8 aristolactam i, alkaloids, nitro compounds, and triterpenes against mtb h37rv aristolochia brevipes benth. (aristolochiaceae) (34) 9 azorellanes; azorellanol against mdr strain of mtb azorella compacta phil., a. madreporica clos. (apiaceae) (35) 10 beilschmin a against mdr strain of mtb beilschmiedia tsangii merr. (lauraceae) (36) 11 calanolide a, b, and soulatrolide against m. tuberculosis calophyllum brasiliense cambess. (clusiaceae) (37) 12 coumarins, marmelosin, marmin, xanthotoxol, kaempferol 3 orhamnoside and afzelin against mtb h37rv and m. bovis aegle marmelos l (28) 13 dibenzocyclooctadiene lignans polysaccharides inhibit hiv replication; inhibit mtb h37rv schisandra chinensis (38, 39) iraqi j pharm sci, vol.31(2) 2022 plant molecules for treatment of tuberculosis 5 counited table (2) 14 dihydro-β-agarofuran sesquiterpenes (1α-acetoxy6β,9β-dibenzoyloxy-dihydroβ-agarofuran) anti-tb activity against the mdr strain leaves of celastrus vulcanicola donn. sm. (celastraceae) (29) 15 diospyrin against mdr strain of mtb euclea natalensis a.dc. (ebenaceae) (40) 16 diterpene (e)-phytol; triterpenes: cycloartenol, sitosterol, stigmasterol, epidioxysterol; ketosteroids: stigmasta-4-en-3-one and stigmasta-4-22-dien-3-one inhibit mtb h37rv strains morina citrifolia linn. (41) 17 ethyl pmethoxycinnamate against mdr strain of mtb kaempferia galangal. l. (zingiberaceae) (42) 18 eupomatenoid-5, a neolignan inhibit mtb h37rv piper regnellii (14) 19 fargesin; (8r,8 r, 9r)cubebin against mdr strain of mtb aristolochia elegans mast (aristolochiaceae) (43) 20 glabridin, hispaglabridin b antitubercular activity against isoniazid resistant mtb, avirulent h37ra & h37rv glycyrrhiza glabra (44) 21 glycyrrhizin antitubercular activity against isoniazid resistant mtb, avirulent h37ra, h37rv; isoniazid monoresistant and isoniazid polyresistant strains of mtb glycyrrhiza glabra (45) 22 hydroxychavicol acetate, 4allylcatechol, transcaffeicaldehyde inhibit mtb h37rv piper taiwanense (14) 23 isoxazole analogs of curcuminoids against mdr strain of mtb curcuma longa l. (zingiberaceae) (46) 24 licarin a, licarin b and eupomatenoid-7; against mtb h37rv and m. avium; aristolochia taliscana hook. & arn. (aristolochiaceae) (34) 25 oleanolic acid against mdr strain of mtb lantana hispida kunth (verbaceae) (47) 26 oleanane triterpenoid aegicerin against mdr strain of mtb clavija procera b. stahl (theophrastaceae) (48) 27 naphthoquinones, plumbagin and its dimers maritinone and 3,3′-biplumbagin strongest activity against mtb strains diospyros anisandra s.f.blake (ebenaceae) (49) 28 piperine (an alkaloid) inhibits multidrug efflux pump (rv1258c) in mtb piper nigrum l. (50) 29 plumericin against sensitive as well as four mdr strains of mtb plumeria bicolor ruiz & pav. (apocynaceae) (51) 30 silymarin consisting of flavonolignans, mainly silybinin, silydianin and silychristin administered in case of liver injuries silybum marianum (l.) gaernt. (sm) (14) 31 tiliacorinine, 2'nortiliacorinine, tiliacorie against mdr strain of mtb tabernaemontana elegans stapf. (apocynaceae) (52) iraqi j pharm sci, vol.31(2) 2022 plant molecules for treatment of tuberculosis 6 counited table (2) 32 thymoquinone inhibits mtb h37rv and mtb xdr-tb nigella sativa l. (black cumin) (14) 33 ursolic acid, hydroquinone against mdr strain of mtb artemisia capillaris thunb. (asteraceae) (53) 34 ursolic acid against mdr strain of mtb chamaedorea tepejilote liebm. (palmae) (54) 35 ursolic acid, cucurbitacin e20-β-d-glucopyranoside against mdr strain of mtb citrullus colocynthis (l.) schrad. (cucurbitaceae) (55) 36 vasicine acetate; 2-acetylbenzylamine against mdr strain of mtb justicia adhatoda l. (acanthaceae) (56) 37 compounds having lipophilic fatty acid groups against mtb h37rv costus speciosus, cymbopogon citratus and tabernaemontana coronaria (57) lately, sarangi et al. (58) (2021) have listed indian ethnomedicinal plants having potent antimycobacterial and immunomodulatory activity. many of these plants have been mentioned in our ancient texts and among those, the plants having anti-tb activity are allium sativum l, piper sp. l, cinnamomum verum. j. presl, tinospora cordifolia (thunb.) miers and shorea robusta roth. flowers of dodonaea viscosa l. jacq., a native plant to asia, africa and australia, show anti-mycobacterial activity against three strains of m.tb i.e. bg1972, h37rv and bg206(59). another plant that can provide molecules which may be used in managing tb is sansevieria liberica. its rhizomes show activity against m.tb at 1 mg/ml (60). vitellaria paradoxa has been traditionally used in cameroon to treat tb. in vitro study has shown the antimycobacterial activity of bark extracts of v. paradoxa and alstonia boonei against h37rv (61). anti-tb phytochemicals and their mode of action much research has been done to identify phytochemicals which might prove useful in curing or improving the efficacy of tb treatment. phytochemicals might work wonderfully when used with synthetic anti-tb drugs to ameliorate the side effects associated with these drugs. they might do so by disrupting intracellular cell signalling systems. different phytochemicals use different modes of action to reduce the cell damage or abnormal cell response, however, primarily they do so by decreasing oxidative damage and modulating the inflammatory response. phytochemicals are strong antioxidants. they capture free radicals, reduce excessive lipid peroxidation in liver and also lower the activity of enzymes such as alanine transaminase (alt), alkaline phosphatase (alp) and aspartate transaminase (ast), while the antioxidant enzymes activity as catalase, superoxide dismutase and glutathione is increased. all of these activities contribute to the hepatoprotective role of phytochemicals (14,62). it is worth noting that hepatotoxicity caused by anti-tb drugs is one of the causes of tb morbidity and mortality (63). some of the commonly used plants that reduce hepatotoxicity include garlic (alium sativum), onion (alium cepa), terminalia chebula fruit, sylimarin isolated from milk thistle (sylibum marianum) and curcumin. two constituents of garlic viz. diallyl disulfide and diallyl trisulfide upregulate the activity of glutathione-stransferase, a potent antioxidant enzyme. ginger, zingiber (zingiberaceace), has been used traditionally to cure the symptoms associated with tb in uganda and ghana (14,64). bacopa monnieri (brahmi) may be used as a supplement with inh and rif, as anti-tb treatment regimen, as it reduces the oxidative stress caused by anti-tb drugs in the kidneys (10). curcumin is a phytochemical derived from curcuma longa rhizomes. it protects the liver cells from oxidative damage by acting as an antioxidant. it accomplishes this by activating the keap1/nrf2 pathway, suppressing the expression of nadph oxidase in the liver (a potent source of ros), activating the enzymes heme oxygenase-1 and nad(p)h quinone dehydrogenase-1, and lowering the expression of cytochrome p450 2e1 (cyp2e1) and peroxiredoxin 1 (prx1) (increased expression of both is a potent source of ros). keap1/nrf2 pathway is a major regulatory pathway that protects cells from oxidative stress induced by endogenous and external ros, as well as electrophiles (65). nad(p)h quinone dehydrogenase-1 (nqo1) catalyses the conversion of quinones to hydroquinones, which is vital for detoxification (66). quinones are intermediates in the production of reactive oxygen species (ros) (67). the physiological stress, due to anti-tb drugs, triggers the activation of heme oxygenase-1 (ho-1) which iraqi j pharm sci, vol.31(2) 2022 plant molecules for treatment of tuberculosis 7 protects the cells from oxidative damage. other than curcumin, several other plant molecules are potent inducers of ho-1 and consequently cytoprotective when administered with anti-tb drugs. these include quercetin (from fruits and vegetables), resveratrol (from fruits and vegetables), epigallocatechin gallate (egcg) (green tea), garlicderived organosulfur compounds: diallyl sulphide (das), diallyl disulphide (dads), diallyl trisulphide (dats), s-allyl-cysteine (sac) (from allium sativum, mustard and ferula asafoetida) and isothiocyanates (sulforaphane, phenethyl isothiocyanate, allyl isothiocyanate) (from cruciferous vegetables) (68). curcumin is also anti-inflammatory as it downregulates the expression of toll-like receptors (tlr2, tlr4) and nuclear factor kappa light chain enhancer of activated b cells (nf-κb) (69,70). tlr4 and tlr2 are pattern recognition receptors (prrs) which recognize microbial molecules, on pathogen surfaces known as pathogen-associated molecular patterns (pamps). this interaction triggers production of ros and an inflammatory response. during signaling, all the tlrs including tlr4 and tlr2 converge at nf-κb, a pro-inflammatory molecule, and exert their effects (71,72). pamp-prr interaction is a crucial event in innate immunity, and overstimulation caused by anti-tb drugs can result in severe inflammatory responses and its associated symptoms. in vitro studies have shown that demethoxycurcumin and bisdemethoxycurcumin, present in curcumin, also inhibit the growth of m.tb, m. marinum and rifampicin-resistant m.tb strains. the most powerful phytochemical among these is demethoxycurcumin (14). silymarin, the seed extract of silybum marianum, is also known to be hepatoprotective. silymarin is a flavanoid complex which contains silybin, silydianin and silychristin. it protects the liver cell as it stabilizes the membrane potential and restores the functions of the liver enzymes by regulating the aberrant increase of the enzymes like ast and alt. when used with other anti-tb drugs, silymarin significantly reduces liver damage. it acts as anti-inflammatory compound also as it inhibits the expression of inflammatory genes like nf-κb, intercellular adhesion molecule 1 (icam-1) and inteleukin-6 (il-6). similarly, bicyclol, a schisandrin c analogue, from schisandra chinensis protects the liver against anti-tb drug-induced liver damage by lowering increased levels of liver enzymes such as ast, alp, and alt. bicyclol is more powerful of the two (73). piperine is an alkaloid extracted from the plants of piperaceae family. piperine significantly reduces the m.tb bacilli load when co-administered with rifampicin as compared to when rifampicin is administered alone. piperine stimulates killing of mdr-m.tb by inhibiting p-glycoprotein of pathogen. it is a suitable candidate to be considered for developing bioactive drug molecules as it has no related toxicity (14). another phytochemical which may be considered for tb treatment is withanolide, extracted from the plant withania somnifera. it has antioxidant properties and protects the liver cells by reducing hepatocyte necrosis, levels of serum alt and intrahepatic haemorrhage as has been observed under in vitro conditions (21). quercetin is a polyphenolic flavonol found in plants such as vitis vinefera, allium cepa, and camellia sinensis. by altering intracellular signalling pathways such as the nuclear factor erythroid 2-related factor 2/ho-1 (nrf2/ho-1) pathway, it protects hepatic cells from oxidative damage (74). ursolic acid is a triterpenoid and is isolated from plants like mirabilis jalapa, hedyotis corymbosa, calendula officinalis, bouvardia ternifolia and byrsonima crassa. it inhibits the formation of ros by suppressing activation of mitogen-activated protein kinases (mapks), cyp2e1 and nf-κb (21). berberine is another phytochemical isolated from plants such as berberis aristata, coptis chinensis, berberis aquifolium, hydrastis canadensis, berberis vulgaris and hydrastis canadensis. it is an alkaloid which is an antioxidant and also has ameliorative effect on liver. it performs its functions by inhibiting microsomal drug-metabolizing enzymes. it also reduces oxidative stress by suppressing the expression of tumor necrosis factorα (tnf-α), cyclooxygenase-2 (cox-2) and inducible nitric oxide synthase (inos) which results in decreased oxidative stress (21,75). another potential effective anti-tb phytochemical is thymoquinone (tq) which is isolated from seeds of nigella sativa. it is a monoterpenoid and a good antioxidant. it increases the activity of enzymes including glutathione peroxidase (gpx) and superoxide dismutase (sod), which protect liver cells from oxidative damage. it also serves as an anti-inflammatory agent as it inactivates expression of tnf-α, cox-2, inos and il-1β (76,77). stilbenes are extracted from plants like paeonia lactiflora, vitis vinifera and arachis hypogaea and in vitro studies have shown that they possess hepatoprotective properties. resveratrol (trans-3,5,4′-trihydroxystilbene,1), a type of stilbene, repairs the hepatic damage induced by rif and inh. it does so by controlling the expression of sirtuin1 (sirt1), peroxisome proliferator-activated receptors (ppar-γ) and peroxisome proliferatoractivated receptor-γ coactivator-1α (pgc-1α) mrnas (78). high levels of sirt1 increase oxidative stress but a moderate level is protective. sirt1 regulates the acetylation of pgc-1α. pgc-1α is a transcriptional coactivator which binds to ppar-γ, a nuclear receptor, to exert its affects. pgc-1α then activates superoxide dismutase 2 (sod2) and iraqi j pharm sci, vol.31(2) 2022 plant molecules for treatment of tuberculosis 8 glutathione peroxidase, both of which protect the cells from oxidative stress (79, 80). andrographolide, isolated from a. paniculata, is another antioxidant and hepatoprotective phytochemical. it is a diterpenoid which reduces oxidative stress by upregulating the expression of hypoxia-inducible factor-1 alpha (hif-1α), sod-1, ho-1 and glutathione stransferase (gst). it inhibits the notch-1/akt/nfκb signaling pathway and thus acts as antiinflammatory molecule (81,82,83). when it interacts with glutathione (gsh), it induces expression of cyp1a1(81,82). cyp1a1 is a potent enzyme performing xenobiotic metabolism and thus, is involved in detoxification (84). plumbagin is one of the most recent phytochemicals to be discovered. it shows anti-mycobacterial action and was isolated from plumbago indica (also known as chitrak in india). it works by blocking m.tb thymidylate synthase, an essential enzyme for the mycobacterium's survival (85). the chemical structures for selected phytochemicals are demonstrated in figure 2. figure 2. chemical structures of some common phytochemicals phytochemicals and immune system phytochemicals not only act as antimycobacterial agents but also as agents that can boost immunity. most of the studies that confirm the antimycobacterial activities of the phytochemicals have been performed in vitro. the effects of phytochemicals might vary if they are administered in vivo. this is because many factors including the immune system, influence the activity of phytochemicals in vivo. when combined with antitb drugs, phytochemicals or plant extracts work as adjuvants, and the treatment is more effective as they not only reduce anti-tb drugs adverse effects, but also alleviate synthetic drug functions and boost the immune system's ability to fight m.tb (14,86). like other microbial infections, the mycobacterial infection too is accompanied by inflammation in the early stages and later it activates an adaptive immune response. innate immune cells primarily macrophages, monocytes, dendritic cells and neutrophils, play a critical role in the early progression of tb infection. m.tb suppresses immune response against itself. the interaction of m.tb with the body’s immune system is complicated by the anti-tb drugs, which increase the host's susceptibility to reinfection. thus, if antiinflammatory drugs are administered along with the designated treatment regimen for tb it will improve treatment efficacy (14). since there is only one vaccine available for tb, plant products, can be used as immunomodulators to increase immunity, which could help to improve therapeutic efficacy of antitb drugs without too many adverse effects. piperine, quercetin, genistein, plumbagin, caffeic acid, lupeol, ursolic acid, oleanolic acid, stigmasterol, β-sitosterol, and betulinic acid are some of the important phytochemicals with immunomodulatory activity. extracts from plants like aegle marmelos, a. sativum, andrographis paniculata, calophyllum brasiliense, centella asiatica, glycyrrhiza glabra, adhatoda vasica, ocimum basilicum, stachytarpheta cayennensis, withania somnifera and urtica dioica l. show immunomodulatory activity (86). iraqi j pharm sci, vol.31(2) 2022 plant molecules for treatment of tuberculosis 9 alcoholic extract of miana (coleus scutellarioides) leaves has anti-m.tb activity, which is attributed to increased host immunity rather than direct suppression of the mycobacteria. garlic extract has a similar mode of action. allicin present in garlic activates sapk/jnk pathways, causing macrophages to produce less tnfα (a key mediator of the inflammatory response). sylimarin keeps m.tb infection in check indirectly through activating expression of ifn-γ, il-12 and tnf-α, when administered alone or in combination with synthetic drugs. piperine enhances splenocytes as well as humoral and cell-mediated immune responses, when given in combination with rifampicin, by activating th-1, ifn- and il-12. the aqueous extract of phyllanthus niruri activates cells and molecules involved in inflammatory response, killing microbes and lowering mycobacterial load in tuberculosis patients. it does so by activating phagocytic cells like macrophages and mononuclear cells and increasing the levels of ifn-γ and tnf-α (14). the plant extracts also induce expression of surface molecules on macrophages which stimulate immune response. the macrophages release pro-inflammatory cytokines and also forage free radicals. m.tb's intracellular survival is reduced by these actions. curcumin also influences the immune system's ability to fight m.tb. it restores memory immune response and prevents reinfection of m.tb by controlling inflammation and immune response(14). d-pinitol, also known as 3-o-methyl d-chiro inositol, is an antioxidant, antiinflammatory, antimycobacterial, and immunosuppressant derived from acacia nilotica, aegle marmelos, and glycyrrhiza glabra. it acts synergistically with inh and rif (28). thus, using plant-based products in combination with synthetic drugs for the treatment schedule of tb is a better treatment strategy as it increases the efficacy of treatment and also reduces its duration (14). conclusion plants have great potential to be used in the treatment strategies of many diseases including tuberculosis. there have been many studies on plant-derived biomolecules to be used for treating tb. the properties of phytochemicals that make them attractive for tb treatment include 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https://dx.doi.org/10.4062%2fbiomolther.2013.020 https://doi.org/10.1016/j.fct.2015.05.001 https://doi.org/10.2174/2212798410666180221105503 https://doi.org/10.2174/2212798410666180221105503 https://doi.org/10.3390/ijms19041085 https://doi.org/10.1021/np5003143 https://dx.doi.org/10.3390%2fcells9081882 https://doi.org/10.1155/2020/1452696 https://doi.org/10.1155/2020/1452696 https://doi.org/10.1248/bpb.34.1666 https://doi.org/10.1002/jlb.3ma1119-584rrr https://doi.org/10.3892/mmr.2017.6580 https://doi.org/10.1371/journal.pone.0228657 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 7 evaluation of rosuvastatin effect as adjuvant therapy to methotrexate on lipid profile and the possibility of its cardioprotective effect in iraqi patients with active rheumatoid arthritis ehab m. mikhael *,1 , ibrahim a. majeed * , faiq i. gorial ** , mohammad h. al-ossamy ** , ali h. falih ** and dhikra abdulhameed *** * department of clinical pharmacy , college of pharmacy, university of baghdad, baghdad, iraq. ** rheumatology unit, college of medicine, university of baghdad, baghdad, iraq. *** teaching laboratories, baghdad teaching hospital. abstract rheumatoid arthritis (ra) is a common inflammatory disease that associated with increased morbidity and mortality due to accelerated atherosclerosis. rosuvastatin is a unique hydroxy methyl glutaryl co a (hmgcoa) reductase inhibitor that has anti inflammatory effects. the aim of this study was to evaluate the effect of rosuvastatin as adjuvant therapy to methotrexate (mtx) on lipid profile and its possible cardioprotective effect in ra patients. a double blinded placebo controlled clinical trial with 8 weeks follow up periods at which 40 patients with active ra using mtx were randomized into 2 groups to receive either rosuvastatin 10mg or placebo as adjuvant therapy to mtx. in addition to twenty healthy subjects as control group. lipid profile and erythrocyte sedimentation rate (esr) was assessed at the start and at the end of the study. at the start of the study total cholesterol (tc), low density lipoprotein cholesterol (ldlc) and high density lipoprotein cholesterol (hdlc) values were not significantly different between ra patients and control group. at the end of the study rosuvastatin significantly reduced esr, tc and ldlc after 8 weeks of treatment. it can be concluded that mtx has the ability to normalize lipid profile in ra patients. rosuvastatin effectively reduce esr, tc and ldlc; moreover, rosuvastatin might have a possible cardioprotective effect in ra patients. keywords: active rheumatoid arthritis, methotrexate, rosuvastatin. عهً انذهىن وجأثُره انقهبٍ انىقائٍ كعالج مضاف نهمُثىجركسُث جأثُر انروسىفاسحاجُهجقُُم انفعال صابُه بانحهاب انمفاصم انرثىٌانمححمم نهمرضً انعراقُُه انم اَهاب مضر مُخائُم *،1 فائق اَشى كىلاير ، ** ابراهُم ادهم مجُذ ، * انعصامٍ محمذ هادٌ ، ** ، عهٍ حسُه فانح ** ركري عبذ انحمُذ و *** * . انؼشاق ،تغذاد ،خايؼح تغذاد،كهٛح انصٛذنح ،فشع انصٛذنح انسشٚشٚح ** . انؼشاق ،تغذاد ،خايؼح تغذاد ،كهٛح انطة ،انشثٕٚح انًفاصم ٔحذج *** . يسرشفٗ تغذاد انرؼهًٛٙ ،انًخرثشاخ انرؼهًٛٛح الخالصة انرٓاب انًفاصم انشثٕ٘ ْٕ يشض انرٓاتٙ شائغ ٔٚرًٛض تضٚادج َسثح انًشاظح ٔانٕفٛاخ تسثة صٚادج سشػح ذصهة األٔػٛح كًا إٌ نّ خاصٛح يعادج (hmgcoa)انذيٕٚح . ٚؼرثش انشٔسٕفاسراذٍٛ يثثطا فشٚذا إلَضٚى ْاٚذسٔكسٙ يثٛم كهٕذاسٚم كٕ ا٘ نالنرٓاب. اٌ انٓذف يٍ ْزِ انذساسح ْٕ نرقٛٛى فائذج انشٔسٕفاسراذٍٛ كؼالج إظافٙ نهًٛثٕذشكسٛد ػهٗ انذٌْٕ ٔذأثٛشِ انًحرًم نٕقاٚح انقهة ػُذ صم انشثٕ٘ انفؼال ٔانزٍٚ يشظٗ انرٓاب انًفاصم انشثٕ٘ انفؼال.انثحث سشٚش٘ يضدٔج اإلػًاء شًم أستؼٌٕ يشٚعا تانرٓاب انًفا يهغشاو أٔ انؼالج 01ٚسرخذيٌٕ نهًٛثٕذشكسٛد ذًد يشاقثرٓى نًذج أساتٛغ تؼذ ذقسًٛٓى ػشٕائٛا نًدًٕػرٍٛ السرؼًال سٔسٕفاسراذٍٛ انٕاْى. تاإلظافح إنٗ ػششٍٚ شخصا كًدًٕػح يقاسَح. الَرٓاء يٍ ْزِ انذساسح. ػُذ تذء ْزِ انذساسح ذثٍٛ إٌ َسثح ذى قٛاط َسثح انذٌْٕ َٔسثح ذشسٛة كشٚاخ انذو انحًش ػُذ انثذء ٔػُذ ا انكٕنسرشٔل ٔانكٕنسرشٔل يُخفط ٔيشذفغ انكثافح الذخرهف تٍٛ انًشظٗ انًصاتٍٛ تانرٓاب انًفاصم انشٔياذٕٚذ٘ ػٍ َظشائٓى ة كشٚاخ انذو انحًش ٔانكٕنسرشٔل, تانًدًٕػح انًقاسَح ٔػُذ اَرٓاء انذساسح قهم انشٔسٕفاسراذٍٛ ٔتشكم يؼُٕ٘ يهحٕظ يٍ َسثح ذشس ٔانثشٔذٍٛ انًشذثط تانكٕنسرشٔل يُخفط انكثافح. َسرُرح يٍ رنك إٌ نهًٛثٕذشكسٛد قاتهٛح ذؼذٚم يسرٕٖ انذٌْٕ ػُذ يشظٗ انرٓاب انًفاصم انشثٕ٘ انفؼال ٔانشٔسٕفاسراذٍٛ نّ انقاتهٛح ذٍٛ انًشذثط تانكٕنسرشٔل يُخفط انكثافح إظافح إنٗ ذأثٛشِ انٕقائٙ ػهٗ ذقهٛم يسرٕٖ ذشسة كشٚاخ انذو انحًش, انكٕنسرشٔل, ٔانثشٔ انًحرًم نهقهة ػُذ يشظٗ انرٓاب انًفاصم انشثٕ٘ انفؼال. انكهمات انمفحاحُة: انحهاب انمفاصم انرثىٌ انفعال, مُثىجركسُث, روسىفاسحاجُه. 1 corresponding author e-mail: ehab_pharma84@yahoo.com received:3 /11/2012 accepted:21 /9/2013 iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 8 introduction rheumatoid arthritis (ra) is a chronic systemic inflammatory disease of unknown etiology characterized by articular and extra articular features (1) . the atherogenic lipid profile and subclinical atherosclerosis are features of early ra (2). rheumatoid arthritis is associated with increased mortality, which is predominantly due to accelerated coronary artery atherosclerosis (3) . cardiovascular (cv) morbidity and mortality are increased twofold in ra patients compared to those of the general population (4, 5) . this increased cv risk may be due to systemic inflammation and its interplay with traditional cv risk factors like smoking, personal and family history of ischemic heart disease, hypertension, hyperlipidemia, higher body mass index and diabetes mellitus (dm) (6) . the association between lipids and cv risk in ra appears to be more complex than in the general population, with systemic inflammation being a notable contributor to the lipid profile changes (7) . inflammation leads to pro-atherogenic changes of the lipoprotein metabolism and an increased disease activity is associated with lower total cholesterol (tc) levels and even more depressed high density lipoprotein – cholesterol (hdlc) levels and lowered apolipoprotein-a1 (apo-a1) levels (4). beside that active inflammation increases oxidized fatty acids in circulating lipoproteins, promoting low density lipoprotein (ldl) oxidation and hdl dysfunction, thereby increasing atherosclerotic risk (8) . rosuvastatin is a unique hydroxy methyl glutaryl coa (hmgcoa) reductase inhibitor that used to treat dyslipidemia (9) . it also exerts important anti-inflammatory effects in addition to its lipid-lowering actions (10) . aim of the study evaluating the effect of rosuvastatin as adjuvant therapy to mtx on lipid profile and its possible cardioprotective effect in iraqi patients with active ra. methods study design this was an 8-week randomized double blind placebo-controlled single center study conducted at rheumatology unit, baghdad teaching hospital, baghdad, iraq from august 2011 till may 2012. patients were randomly allocated to receive each day either rosuvastatin 10mg tablet or capsule prefilled with glucose as placebo (pbo). rosuvastatin was bought from unipharma company/ syria whereas glucose was bought from sdi/ iraq. methotrexate ampoules from ebewe company/ austria. patients were evaluated at baseline and at week 8. sample selection eligible patients had confirmed to have active ra according to the 1987 american college of rheumatology (acr) criteria and had esr values greater than 20 mm/hr for men and greater than 30 for females(11). additionally all patients included were selected to be users of methotrexate (mtx) regularly for at least 3 previous consecutive months. the exclusion criteria included patients who were taking lipid-lowering therapy, had hypersensitivity to statin, pregnancy, breast feeding, renal and liver impairment, patients younger than 18 years old and those using steroids. additionally 20 healthy individuals with age and sex matched were considered as a control group. informed consent was obtained from all participants and this study was approved by the ethical committee of baghdad university, college of medicine medical department. blood sample collection and laboratory evaluation blood specimen collection: 5 ml of venous blood was obtained from forearm for doing laboratory analysis (at baseline and after 8 weeks). 3 ml was transferred to plane test tube and left to be coagulated then centrifuged at 3000 rpm for 10 minutes and then serum obtained for biochemical measurements of total cholesterol, ldlc, hdlc, and triglyceride (tg) and measuring rheumatoid factor. lipid profile tests were done by specialized laboratory workers who did not participate in this study using specialized kits from randox ® company. total cholesterol was measured by cholesterol oxidase peroxidase aminophenazone (chod-pap) enzymatic colorimetric method (12) . tg estimation was done by glycerol-3 phosphate oxidase – peroxidase (gpo-pap) enzymatic colorimetric method under the principle of bucolo and david 1973 (13) .hdlc was measured by polyethylene glycol (peg) / chod – pap method , according to the principle of lopes (14) . whereas ldlc level was estimated according to the friedewald formula (15) . esr was measured by westergren method and also was done by blinded non interested laboratory worker (16) .rheumatoid factor was measured qualitatively just at start of study by serological agglutination test through antigen antibody reaction using specialized kit from spectrum company/ egypt (17) . iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 9 statistical analysis statistical package for social sciences (spss) version 12, was used for data input and analysis. continuous variables were presented as mean ± standard deviation (sd) and discrete variables were presented as numbers and frequencies. chi square test for independence was used to test the significance of association between discrete variables. continuous variables were tested by a web version of shapiro wilk test to determine if they were normally or abnormally distributed. analysis of variance (anova) test was used to test the significance of difference in the mean of 3 independent samples in normally distributed continuous variables. paired t test was used to test the significance of difference in means of pre and post treatment in normally distributed continuous variables, whereas wilcoxon test was used in case of abnormally distributed continuous variables. unpaired t test was used to test the significance of difference in the mean of two independent samples in normally distributed continuous variables and mann whitney test for abnormally distributed data. all p values used were asymptotic and two sided. findings with p value less than 0.05 were considered significant whereas p values less than 0.01 considered highly significant. statistical power was not calculated since it is a pilot study. results of the 74 patients who were randomized in this double-blind study, only 40 patients completed the 8weeks of treatment (20 from the rosuvastatin group and 20 from the pbo). the two groups did not differ significantly in baseline characteristics. another 20 healthy controls aged and sex matched were also participated (figure1, table 1). baseline lipid profile (table 2) showed that there was a non significant difference at baseline level of tc, ldlc and hdlc between ra patients and control subjects and only tg level was significantly higher in ra patients of pbo group than that in the control group. but unfortunately there was a significant difference at a baseline level of tc, ldlc, and tg between rosuvastatin and pbo group (table 3). additionally baseline esr was higher in ra patients, but with no any significant difference between rosuvastatin and placebo group (table 2, 3). after 8 weeks of starting adjuvant treatment with either rosuvastatin or placebo, tc, ldlc and esr decreased significantly by rosuvastatin (p<0.05) while other parameters showed no any difference between the effect of rosuvastatin and placebo (p>0.05, table 4). figure 1: schematic presentation for patient participating in the study iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 10 table ( 1) demographic data and baseline characteristics of both rheumatoid patients and control subjects parameter rosuvastatin pbo control p value age ( years) 43.35 ± 9.96 44.4 ±13.53 42.95±10.39 0.917 female: male ratio 14:6 (70%) 16:4 (80%) 15:5(75%) 0.766 dose of mtx 13.88± 4.40 13.25 ± 3.54 0.624 smoking 3 2 5 0.431 family hx of cvd n (%) 3 (15%) 2 (10%) 3 (15%) 0.865 hypertension n (%) 5 (25%) 5 (25%) 1 dm n (%) 5 (25%) 3 (15%) 0.429 positive rf n (%) 13 (65%) 12 (60%) 0.743 table (2) comparison in baseline laboratory data of rheumatoid arthritis patients with control subjects parm rosuva control p pbo control p tc (mg/dl) 168.9±38.22 180.05±54.29 0.457 202.85±28.68 180.05±54.29 0.105 ldlc (mg/dl) 99.71±33.24 114.78±52.46 0.285 123.21±19.57 114.78±52.46 0.505 hdlc (mg/dl) 42.25±9.42 43.45±6.06 0.635 43.6±10.34 43.45±6.06 0.956 tg (mg/dl) 134.7±63.91 109.1±43.80 0.148 180.2±51.16 109.1±43.80 0.000 esr (mm/hr) 48.95± 31.1 11.7±3.85 0.000 38.25±19.00 11.7±3.85 0.000 tc = total cholesterol, normal range < 200mg/dl; ldlc = low density lipoprotein cholesterol, normal range < 100mg/dl; hdlc = high density lipoprotein cholesterol, normal range 40 – 60mg/dl; tg = triglyceride , normal range < 150mg/dl; esr = erythrocyte sedimentation rate, normal range < 20 for men and < 30 for females. table (3) difference in baseline laboratory parameters between ra patients in rosuvastatin and placebo group parameter rosuva pbo p tc 168.9±38.22 202.85±28.68 0.003 ldlc 99.71±33.24 123.21±19.57 0.01 hdlc 42.25±9.42 43.6±10.34 0.669 tg 134.7±63.91 180.2±51.16 0.017 esr 48.95± 31.1 38.25±19.00 0.197 table (4) change produced in lipid profile and esr after 8 weeks of treatment with either rosuvastatin or placebo parameter rosuvastatin (%) pbo (%) p value tc -39.3 ± 29.21 (-23.27%) -6.25 ± 17.99 (-3.08%) 0.000 ldlc -36.28 ± 27.10 (-36.39%) -8.49 ± 21.61 (-6.89%) 0.001 hdlc 0.7 ± 8.71 (1.66%) 0.4 ± 4.28 (0.92%) 0.956 tg -18.6 ± 33.11 (-13.81%) 9.2 ± 53.97 (5.11%) 0.096 esr -16.85 ± 28.66 (34.42%) -0.55 ± 17.72 (-1.44) 0.012 iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 11 discussion the results of this study showed a non significant difference in tc, ldl-c and hdlc level between ra patients using mtx and control subjects; the results of other studies were controversial: in one study it was found that both tc and ldl-c level were elevated in ra patients whereas hdl-c level was decreased in patients with early ra, and this atherogenic lipid profile can be improved by initiation of therapy (18) . whereas other studies found that systemic inflammation was a notable contributor to the lipid profile changes. growing evidences suggested that patients with active untreated ra have reduced tc, ldl-c and hdl-c levels (7,19) ; and these abnormal lipid profiles were improved through suppression of inflammation by many disease modifying anti rheumatic drugs (dmards) (20) . any how what ever the baseline level of lipid profile in iraqi patients with ra, it seemed that the use of mtx could improve it. the results of this study showed that there was a significant difference in the baseline level of both tc and ldlc between rosuvastatin and placebo group, which may be resulted by chance in such a randomized controlled trial of small sample size. results of this study showed that rosuvastatin produced a significant reduction in both tc and ldlc level; similar finding was reported in many other studies, comparing the effect of different statins, rosuvastatin (21) , atorvastatin (22) and simvastatin (23) to placebo in patients with ra. rosuvastatin reduced tc by more than 23% and ldl-c by more than 36% which was close to that found in uk survey for use of rosuvastatin in general practice (–28% for cholesterol and – 40% for ldl-c ) (24) . such result can be explained according to the fact that rosuvastatin is one of the hmg coa reductase inhibitors which lower plasma cholesterol due to the inhibition of endogenous cholesterol synthesis in the liver, and subsequently increased expression of ldl receptors, resulting in an up-regulated catabolic rate for plasma ldl (25). moreover, the results showed that rosuvastatin was unable to significantly increase hdl-c despite its greater effect when compared to placebo; similar finding was observed in tara study, at which 40mg atorvastatin failed to improve hdl-c (22) ; however, rosuvastatin 10mg was sufficient to increase hdl-c significantly in ra patients after 1 year of therapy (21) , this mean that short duration of follow up period in this study may be a limiting factor in achieving a real result regarding the effect on hdl-c. the results of the current study showed that tg level was higher in active ra patients than those in the control group, and only those in placebo group were significantly higher than control group; similar finding was observed in patients with early active ra by georgiadis et al (18) . limitation in the current study may be related to the significant difference in baseline level of tg between patients in placebo and rosuvastatin group which may be caused by the small sample size; any how only rosuvastatin significantly reduced tg from baseline level, this effect was not statistically significant when compared to that of placebo; similarly the use of low dose rosuvastatin in patients with mildly active ra failed to achieve significant reduction in tg level (21) . the absence of clinically significant effect on tg level may be attributed to the low dose of rosuvastatin that used in the current study, since ooi et al. found that rosuvastatin effect to reduce tg level was dose dependent (26) . regarding esr which is sensitive for most types of inflammation, but cannot distinguish if the underlying cause is infectious, inflammatory, or paraneoplastic (27) , however it provides a reliable means for discrimination between drugs that provide symptomatic relief only and those with a more profound effect in ra (28). the results of this study showed that esr level in ra patients who participated in this study was significantly higher than that in healthy control subjects, which was similar to the finding in the study of yildirim k et al. (29) since we included only patients with high esr level. more importantly, rosuvastatin (but not placebo) significantly reduced esr level; similarly, in two other studies 40mg atorvastatin have the ability to reduce esr significantly (22,30) . however, a study regarding the effect of 10mg rosuvastatin showed no any benefit, which may be explained in that patients who participated in that study had low initial esr level since they have just mildly active ra disease (21) , whereas this study excluded any patient with mild or inactive ra disease who had low esr values ( less than 20 mm/hr ). myasoedova et al found that inflammatory measures (particularly esr) were significantly associated with the risk of cvd in ra (31) . in addition, it was found that controlling both ra disease activity and dyslipidemia is mandatory for minimizing the cardiac risk in ra patients (32). iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 12 this study showed that rosuvastatin significantly reduced both traditional (tc and ldlc) and non traditional (esr) risk factors for cvd in ra patients which agreed with the recent eular recommendation for using statins for cardiovascular risk management in ra patients (33). conclusions methotrexate has the ability to normalize lipid profile in ra patients. rosuvastatin effectively reduce esr, tc and ldlc with little effect on tg and hdlc in ra patients; moreover, rosuvastatin might have a possible cardioprotective effect in ra patients. reference 1. manole cojocaru, inimioara mihaela cojocaru, isabela silosi, camelia doina vrabie, and r tanasescu . extra-articular manifestations in rheumatoid arthritis: maedica (buchar) 2010; 5(4): 286–91. 2. georgiadis an, voulgari pv, argyropoulou mi, et al. early treatment reduces the cardiovascular risk factors in newly diagnosed rheumatoid arthritis patients. semin arthritis rheum 2008; 38(1):13-9. 3. kaplan mj. cardiovascular disease in rheumatoid arthritis. curr opin rheumatol 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stiffness in rheumatoid arthritis: a randomized controlled pilot trial. scand j rheumatol 2011; 40(6):411-21. 22. mccarey dw, mcinnes ib, madhok r, et al. trial of atorvastatin in rheumatoid arthritis (tara): double-blind, randomized placebo-controlled trial. lancet 2004; 363(9426):2015-21. http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bm%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bim%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20cojocaru%2bim%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20silosi%2bi%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20vrabie%2bcd%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20vrabie%2bcd%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20tanasescu%2br%5bauth%5d 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http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20scalia%2br%5bauth%5d http://onlinelibrary.wiley.com/doi/10.1111/ijlh.2011.33.issue-2/issuetoc http://www.ncbi.nlm.nih.gov/pubmed?term=georgiadis%20an%5bauthor%5d&cauthor=true&cauthor_uid=16646989 http://www.ncbi.nlm.nih.gov/pubmed?term=papavasiliou%20ec%5bauthor%5d&cauthor=true&cauthor_uid=16646989 http://www.ncbi.nlm.nih.gov/pubmed?term=lourida%20es%5bauthor%5d&cauthor=true&cauthor_uid=16646989 http://www.ncbi.nlm.nih.gov/pubmed?term=lourida%20es%5bauthor%5d&cauthor=true&cauthor_uid=16646989 http://www.ncbi.nlm.nih.gov/pubmed/16646989## http://www.ncbi.nlm.nih.gov/pubmed/1295090## http://www.ncbi.nlm.nih.gov/pubmed/1295090## http://www.ncbi.nlm.nih.gov/pubmed/18395771## http://www.ncbi.nlm.nih.gov/pubmed?term=%22tam%20ls%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22li%20ek%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22shang%20q%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=effects%20of%20rosuvastatin%20on%20subclinical%20atherosclerosis%20and%20arterial%20stiffness%20in%20rheumatoid%20arthritis%3a%20a%20randomized%20controlled%20pilot%20trial%20%3a%20scand%20j%20rheumatol%202011 http://www.ncbi.nlm.nih.gov/pubmed?term=effects%20of%20rosuvastatin%20on%20subclinical%20atherosclerosis%20and%20arterial%20stiffness%20in%20rheumatoid%20arthritis%3a%20a%20randomized%20controlled%20pilot%20trial%20%3a%20scand%20j%20rheumatol%202011 iraqi j pharm sci, vol.23(1) 2014 rosuvastatin in rheumatoid arthritis 13 23. kanda h, yokota k, kohno c, et al. effects of low-dosage simvastatin on rheumatoid arthritis through reduction of th1/th2 and cd4/cd8 ratios. mod rheumatol 2007; 17(5):364-8. 24. george k, john r, cathy e, et al. a uk survey of rosuvastatin in general practice: reaching cholesterol targets: br j cardiol 2008;15(2):95-100. 25. igel m, sudhop t, von bergmann k .pharmacology of 3-hydroxy-3methylglutaryl-coenzyme a reductase inhibitors (statins), including rosuvastatin and pitavastatin. j clin pharmacol 2002; 42(8): 835-45. 26. ooi em, watt gf, nestel pj, et al. dosedependent regulation of high-density lipoprotein metabolism with rosuvastatin in the metabolic syndrome . j clin endocrinol metab. 2008;93(2):430-7. 27. bridgen m. the erythrocyte sedimentation rate. still a helpful test when used judiciously. postgrad med. 1998;103(5):257-62. 28. amos rs, constable tj, crockson ra, et al. rheumatoid arthritis: relation of serum c-reactive protein and erythrocyte sedimentation rates to radiographic changes. br med j. 1977; 1(6055): 195–7. 29. yildirim k, karatay s, melikoglu ma, et al. associations between acute phase reactant levels and disease activity score (das28) in patients with rheumatoid arthritis. ann clin lab sci. 2004; 34(4):423-6. 30. ljung, lotta, leirisalo-repo, et al. improvement of cardiovascular risk markers with atorvastatin treatment in rheumatoid arthritis. arthritis rheum 2009; 60 suppl 10: 430. 31. myasoedova e, crowson cs, kremers hm, et al. lipid paradox in rheumatoid arthritis: the impact of serum lipid measures and systemic inflammation on the risk of cardiovascular disease: ann rheum dis 2011; 70(3):482–7. 32. khaled amer, ahmed m. ibrahim, hosni a. younis and mohamed m. ahmed. evaluation of cardiac changes in hyperlipidaemic rheumatoid arthritis patients. journal of american science 2012; 8(3):517-22. 33. peters mj, symmons dp, mccarey d, et al. eular evidence-based recommendations for cardiovascular risk management in patients with rheumatoid arthritis and other forms of inflammatory arthritis. ann rheum dis. 2010;69(2):32531. http://www.ncbi.nlm.nih.gov/pubmed?term=%22kanda%20h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22yokota%20k%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22kohno%20c%22%5bauthor%5d http://jcp.sagepub.com/search?author1=m+igel&sortspec=date&submit=submit http://jcp.sagepub.com/search?author1=t+sudhop&sortspec=date&submit=submit http://jcp.sagepub.com/search?author1=k+von+bergmann&sortspec=date&submit=submit http://www.ncbi.nlm.nih.gov/pubmed/18029469## http://www.ncbi.nlm.nih.gov/pubmed/18029469## http://www.ncbi.nlm.nih.gov/pubmed/9590999## http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20amos%2brs%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20constable%2btj%5bauth%5d http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20crockson%2bra%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed?term=yildirim%20k%5bauthor%5d&cauthor=true&cauthor_uid=15648784 http://www.ncbi.nlm.nih.gov/pubmed?term=karatay%20s%5bauthor%5d&cauthor=true&cauthor_uid=15648784 http://www.ncbi.nlm.nih.gov/pubmed?term=melikoglu%20ma%5bauthor%5d&cauthor=true&cauthor_uid=15648784 http://www.ncbi.nlm.nih.gov/pubmed/15648784## http://www.ncbi.nlm.nih.gov/pubmed?term=myasoedova%20e%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=crowson%20cs%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=kremers%20hm%5bauthor%5d&cauthor=true&cauthor_uid=21216812 http://www.ncbi.nlm.nih.gov/pubmed?term=peters%20mj%5bauthor%5d&cauthor=true&cauthor_uid=19773290 http://www.ncbi.nlm.nih.gov/pubmed?term=symmons%20dp%5bauthor%5d&cauthor=true&cauthor_uid=19773290 http://www.ncbi.nlm.nih.gov/pubmed?term=mccarey%20d%5bauthor%5d&cauthor=true&cauthor_uid=19773290 http://www.ncbi.nlm.nih.gov/pubmed/19773290## iraqi j pharm sci, vol.24(1) 2015 synthesis of new derivatives of cephalexin 85 design, synthesis and preliminary antimicrobial evaluation of new derivatives of cephalexin shakir m. alwan *,1 and sarrah s. jabbar * * department of pharmaceutical chemistry, collage of pharmacy, university of baghdad, baghdad, iraq. abstract there is a continuous and massive need for newer cephalosporins that should have resistance against β-lactamases and can be used orally. an approach of using cephalexin, as a well-studied and potent antibacterial compound is considered to prepare new designed derivatives. these derivatives include the incorporation of amino acid moiety linked through an amide bond with the α-amino group of cephalexin. certain aliphatic amino acids were used, such as glycine, alanine, valine and proline. the chemical structures of these derivatives were confirmed by ir spectroscopy and elemental analyses. all the synthesized compounds were subjected for preliminary evaluation of antimicrobial activity using well diffusion method, against certain microbes. most of the synthesized compounds were found to possess significant antibacterial activities. compound 1 (125 μg and 250μg) showed significant activity against p. aeruginosa. compound 2 (125 and 250μg) exhibited significant activity against p. aeruginosa and bacillus cereus. compound 3 (125 and 250μg) demonstrated very significant activity against e. coli, p. aeruginosa and bacillus cereus and slight activity towards s. aureus. compound 4 (250μg) showed significant activity against p. aeruginosa and no antibacterial activities against e. coli, s. aureus and bacillus cereus, as compared with cephalexin as the standard compound. keywords: cephalosporins, cephalexin, glycine, alanine, valine, proline. اليت المضادة للبكتيريا لمشتقاث جديدةلفعاالولي لتقييم التحضير وتصميم و يهلعقار السيفالكس شاكر محمود علوان ،*1 و سارة ستار جبار * * فزع انكيًيبء انصيذالَيت ، .انعزاق ،بغذاد ،جبيعت بغذاد ،كهيت انصيذنت الخالصة احعح يٍ انذراسبث ٔانبحٕد انعهًيّ ببٌ ُْبنك حبجّ كبيزة نًعبداث حيٕيّ يٍ َٕع انسيفبنٕسبٕريُبث انًمبٔيّ نهبكخزيب انًُخجت الَزيًبث انبيخبالكخًيز ٔانخي لذ حسخعًم عٍ غزيك انفى. اسخخذو انسيفبالكسيٍ نخحعيز يشخمبث جذيذة بٕاسطت ربػ االحًبض االييُيّ عٍ غزيك يجًٕعّ انكبربٕكسيم بًجًٕعت االييٍ االٔنيّ )حزِ( انًٕجٕدِ في جزيئت انسيفبنكسيٍ ٔحكٕيٍ اصزة ايبيذ اسطت ببسخعًبل االحًبض االييُيت االنيفبحيت يثم انكاليسيٍ ,األنُيٍ , فبنيٍ ٔانبزٔنيٍ. حى حشخيص انخزاكيب انكيًيبٔيت نٓذِ انًشخمبث بٕ في )االشعت ححج انحًزاء ( ٔانخحهيم انذليك نهعُبصز )انكبربٌٕ ٔانٓيذدرٔجيٍ ٔانُخزٔجيٍ( ٔكبَج انُخبئج يطببمّ انخحهيم انطي نهخزكيببث انكيًيبٔيّ انًفخزظّ. حًج دراست انفعبنيّ انًعبدة نهبكخزيب ببسخعًبل بعط انًيكزٔببث انًزظيت ٔاظٓزث انُخبئج ببٌ يبيكزٔغزاو( فمذ اظٓز 252-125) 2يّ فعبنيّ يخًيزة ظذ انزائفت انزَجبريت , بيًُب انًزكب يبيكزٔغزاو( نذ 252-125) 1انًزكب يبيكزٔغزاو( اظٓز فعبنيّ جذا يخًيزة 252-125) 3فعبنيّ ظذ انزائفت انزَجبريت ٔانبكخزيب انعصٕيت انشًعيت. في حيٍ اٌ انًزكب يبيكزغزاو( فمذ اظٓز فعبنيّ يخًيزة 252) 4شزيكيت انمٕنَٕيت. ايب انًزكب ظذ انزائفت انزَجبريت ٔانبكخزيب انعصٕيت انشًعيت ٔ اال ظذ انزائفت انزَجبريت ٔنى يظٓز أي فعبنيّ نبميّ انًبيكزٔببث ٔلذ اسخعًم عمبر انسيفبنكسيٍ كًبدة ليبسيت نهًمبرَّ. روليه.فاليه والب ،االلنيه ،كاليسيه ،السيفالكسيه ،الكلماث المفتاحيت:السيفالوسبوريه introduction the wide use of antibiotics in man and animals and their extensive use in areas other than the treatment and prophylaxis of diseases have resulted in a serious problem of drug resistance. more and more bacterial strains have become resistant to available drugs. a relation between the structure of the complexes and their anti-bacterial activity can be observed (1) . in the last two decades, antimicrobial resistance has become one of the greatest health problems, mainly in hospitals (2) . despite the continuous development and introduction of new antibiotics, resistance continues to increase progressively in several microbes (3) . preparation of different semisynthetic derivatives of cephalosporins based on structure-activity relationship has been one of the best approaches. intensive search for new cephalosporins that may have broader antibacterial spectrum and resistance toward β-lactamase–producing bacteria and could be used orally are of great interest (4) . cephalexin is a βlactam antibacterial and has bactericidal action and acts similarly to 1 corresponding author e-mail: shakmawales@yahoo.co.uk received: 17/12/ 2014 accepted: 1/6/2015 mailto:shakmawales@yahoo.co.uk iraqi j pharm sci, vol.24(1) 2015 synthesis of new derivatives of cephalexin 86 benzyl penicillin by inhibiting synthesis of the bacterial cell wall. it is most active against g (+) cocci and has moderate activity against some g (-) bacilli. sensitive g (+) cocci includes both penicillinaseand nonpenicillinase-producing staphylococci, although methicillin-resistant staphylococci are resistant; most streptococci are also sensitive, but not penicillin-resistant streptococci pneumonia; enterococci, which are usually resistant. some g (+) anaerobes are also susceptible. cephalexin is usually inactive against listeria monocytogenes. among g (-) bacteria, cephalexin has activity against some enterobacteriaceae including strains of e. coli, k. pneumoniae, proteus mirabilis, salmonella and shigella spp., but not active against enterobacter, indole-positive proteus and serratia spp. it is also active against moraxella catarrhalis (branhamella catarrhalis) and neisseria spp., though haemophilus influenzae is moderately resistant. bacteroides fragilis and p. aeruginosa are not sensitive and neither, mycobacteria, mycoplasma nor fungi (5) . the incorporation of privileged chemical moieties, such as, amino acids has been found to have great potential in the field of antimicrobial agents. amino acid linked to cephalexin through amide bond can be of great benefits and may add appreciable activity, resistance to β-lactamases and/ or improved pharmacokinetic properties . the proposed compounds may serve for injectable purposes, when prepared as sodium salt or could be used orally due to expected stability in aqueous acidic condition due to the presence of primary amine group at the α-carbon of the acyl side chain. experimental work materials and methods general melting points (uncorrected) were determined using electrical melting point apparatus, electro-thermal 9300, usa. the infrared spectra were performed in kbr disc by ft-ir spectrophotometer/ shimadzu. elemental microanalyses (chn) were performed by euro-vector ea 3000a, italy. checking the purity of the products as well as monitoring the progress of the reaction was achieved by thin layer chromatography using silica gel f254 aluminum sheets, merck, germany. chemicals and reagents ethyl chloroformate (ecf) was purchased from sigma aldrich/ germany, bocglycine; boc-l-alanine, boc-l-valine and boc-lproline were purchased from shanghai world yang chemical/china. trifluoroacetic acid (tfa) was obtained from sigma aldrich/germany. cephalexin monohydrate was from sdi samarra/iraq general procedure for synthesis of the intermediates of cephalexin (1a-d). the new intermediates of cephalexin were synthesized by the mixed anhydride method (7-9) , as shown in (schemes 1and 2). boc-amino acid (11.41 mmol) was dissolved in tetrahydrofuran, thf (20ml) containing tea (11.41 mmol) and this mixture was cooled in an ice bath at (-10°c). ethyl chloroformate, ecf (11.41 mmol) was added drop wise over a period of 10 min and the mixture was continuously stirred for further 30 min. cephalexin (11.41 mmol) in distilled water (10ml), containing tea (11.41 mmol) was cooled to 0 °c and added at once to the above solution and the mixture was stirred for 4 hrs at -10 °c and for 2 hrs at room temperature. the solvent was evaporated and the resultant precipitate was washed with diluted hcl (0.1n) and filtered. the precipitate was collected and was washed with water several times with stirring, then washed with ether, recrystallized from ethanol/ toluene (1:9). synthesis of 1a, 7-(2-(2-((t-butoxy carbonyl)amino)acetamido)-2-phenylacetamido)-3methyl–8-oxo-5-thia-1-azabicyclo {4.2.0}oct-2ene-carboxylate this compound 1a was synthesized, as previously described and as shown in (scheme 1). chemical structure of compound 1a boc-glycine (11.416mmol, 5.75g) in thf (20ml) containing tea (11.416 mmol, 1.153 g) was reacted with ecf (11.416 mmol, 1.24 g). cephalexin (11.416 mmol, 4.16 g) in distilled water (10ml) containing tea (11.416 mmol, 1.153 g) was added. the reaction mixture was treated as described earlier. the physical appearance, percent yield and rf value are listed on table (1). http://www.sdisamarra.com/ iraqi j pharm sci, vol.24(1) 2015 synthesis of new derivatives of cephalexin 87 synthesis of 1b, 7-(2-2-((t-butoxy carbonyl) amino) propanamido)-2-phenylacetamido)-3methyl-8-oxo-5-thia-1-azabicyclo {4.2.0} oct-2ene-carboxylate this compound 1b was synthesized, as follows and as shown in (scheme 1): chemical structure of compound 1b boc-alanine (11.416 mmol, 5.91 g) in 20 ml of thf containing tea (11.416 mmol, 1.153g) was reacted with ecf (11.416 mmol, 1.24 g). cephalexin (11.416mmol, 4.16 g) in distilled water (10ml) containing tea (11.416 mmol, 1.153g) was added. the reaction mixture was treated as previously described. the physical appearance, percent yield and rf value are listed on table (1). synthesis of 1c, 7-(2-2((t-butoxy carbonyl) amino)-3-methylbutanamido)-2-phenyl acetamido)-3-methyl-8-oxo-5-thia-1-azabicyclo {4.2.0} oct-2-ene carboxylate compound 1c was synthesized, as follows and as shown in (scheme 1): chemical structure of compound 1c boc-valine (11.416 mmol, 6.23 g) in 20 ml of thf containing tea (11.416 mmol, 1.153g) reacted with ecf (11.416 mmol, 1.24g). cephalexin (11.416 mmol, 4.16 g) in distilled water (10ml) containing tea (11.416 mmol, 1.153g) was added. the mixture was treated as previously described. the physical appearance, percent yield and rf value are listed on table (1). synthesis of 1d, 7-(2-(1-(t-butoxy carbonyl) pyrrolidin-2-carboxamido)-2-phenylacet amido)-3-methyl-8-oxo-5-thia-1-azabicyclo {4.2.0} oct-2-ene-carboxylate. compound 1d was synthesized, as follows and as shown in (scheme 2): chemical structure of compound 1d boc-proline (11.416mmol, 6.22g) in thf (20ml) containing tea (11.416 mmol, 1.153g) was reacted with ecf (11.416mmol, 1.24g). cephalexin (11.416 mmol, 4.16 g) in distilled water (10ml) containing tea (11.416 mmol, 1.153g) was added. the mixture was treated as previously described. the physical appearance, percent yield and rf value are listed on table (1). scheme (1): synthesis of intermediates 1a-c scheme (2): synthesis of intermediate 1d general procedure for synthesis of the new derivatives of cephalexin (1-4). these compounds were obtained by deprotection (10) of the amino group of compounds 1a-d, to afford the new derivatives of cephalexin 1-4, as follows and as shown in (schemes 3 and 4). compounds 1a-d, (1.984 mmol) was suspended in dichloromethane (dcm) (10ml) and cooled iraqi j pharm sci, vol.24(1) 2015 synthesis of new derivatives of cephalexin 88 to 0 °c in an ice bath and tfa (15ml) was added with continuous stirring for 1hr at 0 o c in presence of anisole (3ml). the completion of the reaction was monitored by tlc using the mobile phase methanol: chloroform (1:1). diethyl ether (100ml) was added to the reaction mixture and the resulting precipitate was collected, suspended in methanol (30) ml and the ph was adjusted to 7 with 5% methanolic solution of naoh. a precipitate was formed after the addition of ether, which was filtered and washed with acetone and recrystallized from ethyl acetate: petroleum ether (9:1). the precipitate was collected and dried in an oven at 50 o c. synthesis of 1, 7-(2-(2-aminoacetamido)-2 phenylacetamido)-3-methyl-8-oxo-5-thia-1azabicyclo{4.2.0}oct-2-ene-carboxylate sodium. compound 1 was obtained by deprotection of the amino group of compound 1a, as follows and as shown in (scheme 3): chemical structure of compound 1 compound 1a (1.984 mmol, 1 g) in dcm (10ml) was reacted with tfa (15ml) in presence of anisole (3ml). the mixture was treated as previously described. a yellowish crystalline powder was collected. the physical appearance, percent yield and rf value are listed on table (1). synthesis of 2, 7-(2-2-amino propan amido)2phenylacetamido)-3-methyl-8-oxo-5-thia-1azabicyclo{4.2.0}oct-2-ene-carboxylate sodium. compound 2 was obtained by deprotection of the amino group of compound 1b, as follows and as shown in (scheme 3): chemical structure of compound 2 compound 1b (1.984mmol, 1.027 g) in dcm (10ml) was reacted with tfa (15ml) in presence of anisole (3ml). the mixture was treated as previously described. the physical appearance, percent yield and rf value are listed on table (1). synthesis of 3, 7(2(2-amino-3-methylbutan amido) 2-phenylacetamido)-3-methyl-8-oxo -5thia-1-azabicyclo {4.2.0}oct-2-ene-carboxylate sodium. compound 3 was obtained by deprotection of the amino group of compound 1c, as follows and as shown in (scheme 3): chemical structure of compound 3 compound 1c (1.984 mmol, 1.083 g) in dcm (10ml) was reacted with tfa (15ml) in the presence of anisole (3ml) and the mixture was treated as previously described. the physical appearance, percent yield and rf value are listed on table (1). synthesis of 4, 7-(2-phenyl-2(pyrrolidin -2 carboxamido) acetamido)-5-thia-1-azabicyclo {4.2.0} oct 2 ene-carboxylate sodium. compound 4 was obtained by deprotection of the amino group of compound 1d, as follows and as shown in (scheme 4): chemical structure of compound 4 compound 1d (1.984 mmol, 1.081 g) in dcm (10ml) was reacted with tfa (15) in presence of anisole (3ml) and the mixture was treated as previously described. the physical appearance, percent yield and rf value are listed in table (1). iraqi j pharm sci, vol.24(1) 2015 synthesis of new derivatives of cephalexin 89 table (1): physical parameters and percent yields of the synthesized compounds. scheme (3): synthesis of the target compounds 1-3 scheme (4): synthesis of the target compound 4 results and discussion the use of two solvent systems a and b was to differentiate between reactants and products and to follow progress of reactions. a-chloroform: methanol (3:1) b-chloroform: methanol (1:3), as illustrated on table (1). the ir characteristic bands of compound 1 are 1543, 1400 (c=o stretching of carboxylate anion), 3060 (n-h stretching 2-amide, 3473, 3397 (n-h stretching of primary amine group), 1656 (c=o stretching 2-amide), 1761(c=o stretching β-lactam), 1543(n-h bending 2amide). the elemental analysis (chn) was calculated for c18h19n4so5na (426); calculated: c; 50.70, h; 4.46, n; 13.145. found c; 48.35, h; 4.684, n; 13.36. the ir characteristic bands of compound 2 are 1549, 1400 (c=o stretching of carboxylate anion), 3061 (n-h stretching 2-amide), 3494, 3399 (n-h stretching primary amine group), 1664 (c=o stretching 2-amide), 1759 (c=o stretching β-lactam), 1527 (n-h bending 2amide). chn analysis was calculated for c19h21n4so5na (440); calculated c: 51.8, h: 4.77, n: 12.72. found c: 49.91, h: 4.56, n: 12.28. the ir characteristic bands of compound 3 are as follows; 1543, 1400 c=o stretching of carboxylate anion, 3063 (n-h stretching of 2amide), 3473, 3397 (n-h stretching of primary amine), 1653 (c=o stretching of 2-amide), 1759 (c=o stretching of β-lactam), 1570 (n-h bending of 2-amide). chn analysis was calculated for c21h25n4so4na (429) calculated; c: 53.846, h: 5.341, n: 11.965. found c: 52.04, h: 5.16, n: 11.71. compound physical appearance % yield m.p. ( o c) rf value 1a white 63.8 172-177 0.60 (a) 1b white 72 182-186 0.44 (a) 1c white 74 160-166 0.55 (a) 1d off white 82 180-185 0.50 (a) 1 pale yellow 95 185 (decomposed) 0.41 (b) 2 pale yellow 44 200 (decomposed) 0.46 (b) 3 off white 70 214 (decomposed) 0.57(b) 4 off white 40 212 (decomposed) 0.55(b) cephalexin off white -182-186 0.11(a) iraqi j pharm sci, vol.24(1) 2015 synthesis of new derivatives of cephalexin 90 the ir characteristic bands of compound 4 are 1550, 1400 c=o stretching of carboxylate anion, 3034.13n-h stretching of 2-amide, 3373, 3317 n-h stretching of primary amine group, 1662.69 c=o stretching of 2-amide, 1755.28 c=o stretching of β-lactam, 1527 nh bending of 2-amide. chn analysis was calculated for c21h25n4so5na (445), calculated; c: 54.663, h: 3.904, n: 12.147. found c: 52.20, h: 3.78, n: 12.68. preliminary antimicrobial evaluation the synthesized compounds were subjected to antimicrobial evaluation by well-diffusion method (11) . the zone of inhibition (mm) was measured in comparison with cephalexin. the antimicrobial activity was performed in nutrient agar medium containing e. coli, p. aeruginosa and bacillus cereus, s. aureus and the compounds used at concentrations (125 and 250 μg/well).the activity was determined after incubation for 24h at 37 o c by the comparison of inhibition of growth of bacteria by cephalexin using dimethyl sulfoxide (dmso) as the solvent. no inhibition zone was observed for the control (dimethylsulfoxide). these compounds were subjected to preliminary antimicrobial evaluation against four types of microbes (e. coli, p. aeruginosa, and bacillus cereus, s. aureus). table ( 2) :the preliminary antibacterial activity of the new derivatives of cephalexin. key to symbols: (-) = no inhibition. the antimicrobial evaluation revealed that the newly synthesized compounds, 1-4 showed reasonable antibacterial activities against g (-) bacteria, such as p. aeruginosa and g (+) bacteria, such as bacillus cereus in comparison with cephalexin, which has no activity against these type of microbes. compound 1 (125 and 250μg) showed significant activity against p. aeruginosa and reduction in antibacterial activities against s. aureus and e. coli as compared with cephalexin. compound 2 (125 μg, 250μg) showed significant activity against p. aeruginosa and bacillus cereus, as compared with cephalexin. furthermore, at concentration (125 μg) compound 2 exhibited no activity against s. aureus and e. coli. however, at concentration of (250 μg) the activity was less against s. aureus as compared with cephalexin. compound 3 (125 and 250μg) showed very significant activity against e. coli, p. aeruginosa and bacillus cereus and slight activity towards s. aureus. this result indicates that compound 3 has a broader spectrum of antibacterial activities, i.e. against both g (+) and g (-) bacteria. compound 4 (125μg) has no antibacterial activities against all strains of bacteria used. however, compound 4 at concentration (250 μg) showed significant activity against p. aeruginosa and no antibacterial activities against e. coli, s. aureus and bacillus cereus as compared with cephalexin. this chemical modification at the acyl side chain positioned at alpha to βlactam ring supposed to provide some protection against β-lactamses. this model contains a primary amino group of the amino acid, which may provide certain stability in the acidic condition of stomach (4). conclusion a series of new derivatives of cephalexin have been synthesized successfully in appreciable yields and screened for their antimicrobial activity using well diffusion method against bacterial strains (e. coli, p. aeruginosa and bacillus cereus, s. aureus). it is concluded that the new derivatives of cephalexin linked with certain amino acids were found to possess moderate antibacterial activities. furthermore, the new derivative of cephalexin linked with valine has a significant activity against p. aeruginosa and bacillus cereus. compound concentration µg/ml escherichia coli. pseudomonas aeruginosa staphylococcus aureus bacillus cereus. dmso -9 10 cephalexin 125 10 12 250 12 14 1 125 11 250 12 2 125 11 6 250 14 8 10 3 125 8 15 9 15 250 9 15 9 17 4 125 iraqi j pharm sci, vol.24(1) 2015 synthesis of new derivatives of cephalexin 91 references 1. brown, a.g. and s.m. roberts, (ed.). recent advances in the chemistry of βlactam antibiotics. the royal society of chemistry, london – 1984. 2. gary a. thiobodeas, kelvin t. patton, elsevier. synthesis and biological evaluation of some novel schiff bases of cephalexin. inter. j. pharmaceut. 2004, 7; 703. 3. furtado g. h. c., perdiz l. b. and medeiros e. a. s., the effect of a 4th generation cephalosporin upon the incidence of multidrug-resistant g (-) bacteria in a non-teaching hospital, am. j. infect. dis., 2008, 4 (4), 267-271. 4. wilson and gisvold´s: text book of organic medical and harmaceutical chemistry , (2013) 12 th ed., 255-256 5. najib nw. suleiman m.s. high performance liquid chromatographic analysis of cephalexin in serum and urine. j. clin. pharm. and therap.; (1987), 12, 419-426. 6. alwan s. m., synthesis and preliminary antimicrobial activities of new arylideneamino-1,3,4-thiadiazole-(thio/ dithio) acetamidocephalosporanic acids, molecules, 2012, 17, 1025-1038. 7. rho, h.s., baek, h.s., kim, d.h. and chang, i.s., a convenient method for the preparation of alkanolamides, bull. korean chem. soc. 2006, 27 (4), 584-586. 8. george, w., joan, e. and francis, m., a re-investigation of mixed carbonic anhydride method of peptide synthesis, j. am. chem. soc., 1967, 89, 5012-5017. 9. vaughan, j. r., and osato, r.l., preparation of peptides using mixed carbonic–carboxylic acid anhydride. j. am. chem. soc., 1952, 74 (3), 676-678. 10. cephalosporin antibiotic. synthesis and antimicrobial activity of cephalosporin derivatives i. csendes, b.w. muller and w. toch. the j. antibiotic, 1983, 36 (8), 1032. 11. fingegold ,s.m.martin , w j.diagnostic microbiology ,6 th ed.:mosby london :p.450;1982 iraqi j pharm sci, vol.24(2) 2015 bioequivalence and pharmacokinetics of er release pentoxifylline tablets 53 bioequivalence and pharmacokinetics comparison of two formulations of extended-release pentoxifylline tablets in healthy subjects after fasting and fed conditions jaafar j. ibraheem al-tamimi *,1 *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the pharmacokinetics and bioequivalence of a newly developed extended-released (er) tablet containing 400 mg pentoxifylline as a test product was compared with the reference brand product trental ® 400 mg er tablet produced by sanofi-aventis. two separate studies were conducted simultaneously. the first study was conducted under fasting condition, whereas, the second study was conducted under fed condition; using the same batches of the test and reference products in both studies. in each study, both products were administered to 32 healthy male adult volunteers applying a singledose, two-treatment, two-period, two-sequence, randomized crossover design with one-week washout period between dosing. twenty two blood samples were withdrawn from each volunteer over 24 hours period. pentoxifylline concentrations were determined in plasma by a validated hplc method according to fda bioanalytical method validation guidance using uv detection and chloramphenicol as internal standard. the lower limit of quantitation of the drug in plasma was 5 ng/ml and the upper limit of quantitation was 500 ng/ml. from the plasma concentration-time data of each individual, the pharmacokinetic parameters; cmax, auc0-t, auc0-, cmax/auc0-, tmax, z & t0.5; were calculated applying non-compartmental analysis. data of the test and reference products were statistically analyzed to test for bioequivalence of the two products, using criteria of fda and emea guidance. the pharmacokinetic parameters mentioned above were statistically analyzed by descriptive statistics, anova test and 90% confidence interval (ci). anova test involved the calculation of the effects of: treatment, period, sequence and subjects nested in sequence. according to the above guidance, the primary pharmacokinetic parameters used for bioequivalence testing, namely cmax, auc0-t and auc0- were also statistically analyzed by anova & ci tests using the corresponding ln-transformed values. the mean values cmax, auc0-t , auc0- , cmax/auc0- , tmax, z & t0.5 for the test formula obtained from the fasting study were; 144.4 ng/ml, 845.4 ng.hr/ml, 868.4 ng.hr/ml, 0.186 hr -1 , 3.29 hr, 0.561 hr -1 and 1.65 hr, respectively; and the mean values of these parameters for the reference formula were; 150.0 ng/ml, 871.1 ng.hr/ml, 893.7 ng.hr/ml, 0.177 hr -1 , 3.70 hr, 0.558 hr -1 and 1.59 hr, respectively. the mean values of the above mentioned parameters for the test formula obtained from the fed study were; 157.8 ng/ml, 826.5 ng.hr/ml, 853.8 ng.hr/ml, 0.198 hr -1 , 5.4 hr, 0.458 hr -1 and 2.06 hr, respectively; and the mean values of these parameters for the reference formula were; 162.1 ng/ml, 869.7 ng.hr/ml, 894.8 ng.hr/ml, 0.196 hr -1 , 4.1 hr, 0.525 hr -1 and 1.80 hr, respectively. based on criteria of fda and emea guidance on bioavailability and bioequivalence, the results of the fasting and fed studies demonstrated bioequivalence of the two products under either condition. accordingly, it is concluded that the newly developed er tablet containing 400 mg pentoxifylline is bioequivalent to trental ® 400 mg er tablet produced by sanofi-aventis. keywords: pentoxifylline, pharmacokinetics, bioequivalence. هقارًة ألتكافؤ ألحيوى وحركية الدواء بيي هستحضريي لحبوب البٌتوكسيفليي طويلة ( في أشخاص أصحاء هع الطعام وبدوى طعام(erألوفعول يلتويواجعفر جابر أبراهين ،*1 * .نعزاق،اتغداد،خايعح تغداد ،كهيح أنصيدنح ،فزع أنصيدالَياخ الخالصة ( ذحٕٖ عهٗ erٔانركافؤ أنحيٕٖ تيٍ يُرح دٔائٗ خديد نحثٕب أنثُرٕكظيفهيٍ طٕيهح أنًفعٕل )ذًد يقارَح حزكيح اندٔاء يهغ نشزكح طُٕفٗ أفيُرض. ذى عًم دراطريٍ فٗ َفض انٕقد. 044يهغ يٍ عقار أنثُرٕكظيفهيٍ يع أنًُرح أنًزخعٗ ذزَرال 044 أنطعاو تاطرعًال َفض أنٕخثح يٍ كم يُرح. فٗ كم دراطّ ذى أعطاء أندراطح أالٔنٗ ذًد تدٌٔ طعاو تيًُا اَدشخ أندراطح أنثاَيح يع 30عيُح دو يٍ كم يرطٕع ٔنفرزج 33ذى طحة يرطٕع تاطرعًال أنرصًيى أنعشٕائٗ ٔتفرزج اطثٕع تيٍ أندزع. 23كم يُرح أنٗ عقار ٔاطرعًال أآليزيكٗ رحهيم أن ٔحظة دطرٕر hplc / uv تٕاططح تانثالسيا أنثُرٕكظيفهيٍ ذزاكيش حظاب طاعح ٔذى 1 corresponding author e-mail: drjaafarjaber@yahoo.com received: 1 /6/ 2015 accepted: 10/11/2015 mailto:drjaafarjaber@yahoo.com iraqi j pharm sci, vol.24(2) 2015 bioequivalence and pharmacokinetics of er release pentoxifylline tablets 54 . يعايم حزكيح أندٔاء فٗ ng/ml 500ٔانحد أآلعهٗ ng/ml 5 دٔاء فٗ أندو نانحد أالدَٗ نرحهيم ا أنكزٔيفُيكٕل كًقياص داخهٗ. ذى حظاتٓا ثى يقارَرٓا تيٍ أنًُرح أندُيض cmax, auc0-t, auc0-, cmax/auc0-, tmax, z & t0.5أندظى ْٔٗ تانُظثح نهدراطح أآلٔنٗ تدٌٔ طعاو فانُرائح نًعدل ٔانًزخعٗ نغزض ذقييى أنركافؤ أنحيٕٖ حظة اندطرٕر أأليزيكٗ ٔأآلٔرتٗ. ٔحظة أنرٕانٗ نهًُرح أندُيض ْٗ : cmax, auc0-t , auc0- , cmax/auc0- , tmax, z & t0.5يعايم حزكيح أندٔاء 144.4 ng/ml, 845.4 ng.hr/ml, 868.4 ng.hr/ml, 0.186 hr -1 , 3.29 hr., 0.561 hr -1 ,1.65 hr. خعٗ ْٗ:ٔنهًُرح أنًز 150.0 ng/ml, 871.1 ng.hr/ml, 893.7 ng.hr/ml, 0.177 hr -1 , 3.70 hr., 0.558 hr -1 , 1.59 hr. أيا نهدراطح أنثاَيح يع أنطعاو فانُرائح نهًُرح أندُيض ْٗ: 157.8 ng/ml, 826.5 ng.hr/ml, 853.8 ng.hr/ml, 0.198 hr -1 , 5.4 hr., 0.458 hr -1 , 2.06 hr.; ٔنهًُرح أنًزخعٗ ْٗ: 162.1 ng/ml, 869.7 ng.hr/ml, 894.8 ng.hr/ml, 0.196 hr -1 , 4.1 hr., 0.525 hr -1 , 1.80 hr. تُاء عهٗ أندطرٕر أأليزيكٗ ٔأألٔرتٗ فاٌ أنُرائح أنًذكٕرِ أعالِ ذدل عهٗ ٔخٕد ذكافؤ حيٕٖ تيٍ أنًُرديٍ عُد ذُأل أندٔاء يع يهغ يٍ 044عهٗ أنًطٕر حديثا أنذٖ يحرٕٖ er)انًًكٍ أألطرُراج تاٌ أنًُرح طٕيم أنًفعٕل )فًٍ أنطعاو ٔتدٌٔ طعاو. نذنك انًُرح يٍ شزكح طُٕفٗ أفيُرض. er)يهغ طٕيم أنًفعٕل ) 044انثُرٕكظيفهيٍ يكافؤ حيٕيا يع ذزَرال بٌتوكسيفليي , حركية الدواء , ألتكافؤ ألحيوى . -الكلوات أآلفتتاحية: introduction pentoxifylline (pentoxiphylline) is a trisubstituted xanthine derivative designated chemically as 1-(5-oxohexyl)-3, 7dimethylxanthine that, unlike theophylline, is a hemorrheologic agent, i.e. an agent that affects blood viscosity. pentoxifylline is indicated for the treatment of patients with intermittent claudication on the basis of chronic occlusive arterial disease of the limbs. after administration of the 400 mg extended-release (er) pentoxifylline tablet, plasma levels of the parent compound and its metabolites reach their maximum within 2 to 4 hours and remain constant over an extended period of time. coadministration of er pentoxifylline tablets with meals resulted in an increase in mean cmax and auc by about 28% and 13% for pentoxifylline, respectively. the usual dosage of er pentoxifylline tablet form is one tablet (400 mg) three times a day with meals (1) . the aim of the present study is to evaluate the bioequivalence of a newly developed extended released (er) tablet containing 400 mg pentoxifylline relative to the reference brand trental ® er tablet containing 400 mg pentoxifylline manufactured by sanofi-aventis. as per fda and emea guidance for bioequivalence evaluation of modified-release products (2-4) , two separate studies are required; a single dose, nonreplicate, fasting study and a single dose, food-effect, nonreplicate study. therefore, the pharmacokinetic parameters cmax, auc0-t, auc0-, cmax/auc0- , tmax, z & t0.5 were calculated in the current investigation for both products after administration to 32 healthy male adult subjects under fasting and fed conditions. materials and methods drug products the test product was a newly developed extended-release (er) tablet containing 400 mg pentoxifylline. the reference product was the brand trental ® er tablet containing 400 mg pentoxifylline manufactured by sanofiaventis. study design as recommended by fda and emea guidance concerning the bioequivalence of modified released drug products (2-4) , two separate bioequivalence studies are required to be conducted to prove bioequivalence of a test product to the reference/brand/innovator product. these studies include a single dose, nonreplicate bioequivalence study under fasting condition, and a single dose, nonreplicate bioequivalence study under fed condition. accordingly, two bioequivalence studies were conducted in the present investigation: a fasting, single-dose, twotreatment, two-period, two-sequence, randomized crossover study, and a fed singledose, two-treatment, two-period, twosequence, randomized crossover study. thirty two subjects participated in each study. in each study, equal number of subjects (16 subjects) were randomly assigned to each dosing sequence of the treatments (test and reference formulations). the treatments were separated by one week washout interval between period i and period ii dosing. inclusion criteria of volunteers the volunteers were selected according to the following inclusion criteria: male, age between 18-45 years, normal body mass index, non-smokers or light smokers (less than 10 cigarettes a day), normal physical examinations, no clinically medical disorders or impairments (hepatic, renal, cardiac, git and psychiatric), no history of contraindication and/or allergy to the drug or any related compounds, no consumption of drugs for two weeks prior the study, otc drugs are allowed as per clinical investigator decision. the volunteers should not have been participated in clinical study (bioavailability, bioequivalence, pharmacokinetics, etc.) studies 3 months prior iraqi j pharm sci, vol.24(2) 2015 bioequivalence and pharmacokinetics of er release pentoxifylline tablets 55 to the present study, no blood donation or hospitalization 3 months prior to the present study, no drugs or alcohol abuse, normal laboratory tests including biochemistry, hematology, hiv (-), hepatitis b and c (-), liver and kidneys function tests and urine analysis. informed consent of volunteers the study was conducted according to ich guidelines for good clinical practice (gcp) and declaration of helsinki (5, 6) . according to helsinki declaration, the informed consent form includes details of the study, benefits and possible risks associated with participation, information regarding the right to withdraw at any time from participation. the informed consent form was provided to each prospective volunteer prior to the start of the study. also, a study-orientation session was held with the volunteers to explain and inform the details of the informed consent form. all the volunteers gave written and signed consent before the study. conditions of the clinical study for fasting and fed studies in case of fasting study; the volunteers were confined at the clinical site 14 hours before dose administration and until 24 hours after dose administration (end of each study period). a standard dinner was served to the volunteers 12 hours before dosing. the drug was administered with 240 ml of water after an overnight fasting of 12 hours. mouth checks and hands checks were performed by the investigators to ensure that the medication is taken by the volunteers as directed. four hours after dosing, a standard lunch was served to the volunteers. food and the time of feeding were identical in all periods of the study. no water was permitted 2 hours before and after dosing. water was allowed as desired 2 hours after dosing. xanthine containing products were not allowed 12 hours before dosing and until 24 hours after dosing (end of each study period). the volunteers were not allowed to sleep or lie during the first four hours of drug administration. grapefruit juice or beverages containing grapefruit were not allowed within the past week prior the study and until the completion of the whole study (both periods of the study). in case of fed study; the same procedure was applied as mentioned above except the volunteers were served standard fatty breakfast before drug intake as per fda and emea guidance (2, 3) . blood sampling from volunteers seven ml of blood samples were withdrawn from each volunteer via an indwelling cannula placed in the forearm anticubital vein. the cannula was kept patent by flushing with 1 ml of heparinized saline (2 iu per ml) after each sample collection. about 0.2 ml of blood was discarded from the cannula before each sampling withdrawal. the blood was sampled from each volunteer at zero time (30 min. before dosing), and then at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 7.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, 20.0 and 24.0 hours post dosing (a total of 22 blood samples were collected from each volunteer). the blood samples were transferred to heparinized tubes and then immediately centrifuged for 10 minutes at 4000 rpm. blood and plasma samples were labeled according to in-house coding system. the labeling system is confidential and the analysts have no key to the labeling system. the plasma was separated by polypropylene disposable tips and transferred to eppendorf tubes and then stored in deep freezer at 20c until the time of analysis for determination of pentoxifylline concentrations. medical observation during and post dosing the clinical staff were available during each study period to conduct the study as per study protocol, and to treat and report any adverse effect if any. vital signs (blood pressure and pulse) of each subject were measured one hour before dosing and then at 1, 2, 3, 6, 9, 12, and 24 hours post dosing (the end of each study period). the volunteers were free to leave the study at any time for any reason. withdrawal to protect the health of the volunteers, if any, was also considered. data analysis for pharmacokinetic (pk) calculations: kinetica software was applied for all pk calculations, and data plotting. the pk parameters; cmax, auc0-t , auct- , auc0- , cmax/auc0-∞ , tmax , z and t0.5 were calculated for each subject and for each period applying non-compartmental analysis (7, 8) . lntransformation of the primary pk parameters used for bioequivalence testing cmax,, auc0-t and auc0- were also computed as recommended by fda and emea guidance (24) . the terminal elimination rate constant (z) was estimated for each subject and for each period via linear regression of the last points (at least three points) of the terminal phase of the log-concentration versus time curve (2-4) . the values of cmax and tmax were obtained directly from concentration versus time curves of each individual. mean drug concentrations in plasma vs. time data for both test and reference products were plotted in rectilinear graph type. iraqi j pharm sci, vol.24(2) 2015 bioequivalence and pharmacokinetics of er release pentoxifylline tablets 56 statistical analysis of the pharmacokinetic (pk) parameters: the same software kinetica that was used for pk calculations was also applied for all statistical analysis. the parameters cmax, auc0-t , auct- , auc0- , cmax/auc0-∞ , tmax , z and t0.5 were statically analyzed to evaluate the differences between the test and the reference products applying descriptive statistics, anova test, and 90% confidence interval (ci). descriptive statistics included arithmetic means, geometric means, ratio of geometric means, and standard deviation. anova test was applied to calculate the effect of the factors; period, subjects nested in sequence, treatment (formulation) and sequence; on the above mentioned pk parameters. anova test was also applied to the ln-transformed values of cmax, auc0-t and auc0- . the two products were declared bioequivalent if the ci of ratio of the test product to reference product (t/r) of the lntransformed parameters cmax , auc0-t and auc0- lie between 80 to 125% (9) . differences are declared statistically not significant at 5% significance level ( = 0.05) when p  0.05. determination of pentoxifylline in plasma a specific high performance liquid chromatographic (hplc) assay using uv detection and chloramphenicol as internal standard was developed in-house for determination of penotxifylline in plasma. the lower limit of quantitation (lloq) of penotxifylline in plasma was 5 ng/ml and the upper limit of quantitation (uloq) was 500 ng/ml. the analytical method was validated according to fda bioanalytical method validation guidelines (10) . all plasma samples of each volunteer obtained from both periods (test and reference products) were analyzed together with quality control (qc) samples (low, medium & high) in one analytical run (batch). a standard curve including blank matrix was generated for each analytical run and was used to determine penotxifylline concentrations in the unknown authentic samples. no determination was done by extrapolation below the lloq or above the uloq of the standard calibration curve. the plasma samples were analyzed after the completion of the clinical part of the study. products assay assay determination of penotxiphylline in the test product and the reference product were carried out to insure that the difference in the content of the drug in the test product and the reference product is not more than 5% as recommended by fda and emea guidance (2-4) . dissolution testing the dissolution testing of the test product and the reference product was carried out to study the similarity in the dissolution profiles between both formulas by calculating the similarity factor f2 as per fda and emea guidance (2, 4, 11) . results and discussions clinical data tables 1and 2 presents the demographic data of the volunteers participated in the fasting study (32 subjects) and the fed study (32 subjects), respectively. table (1): demographic data of 32 volunteers participated in the fasting study. table (2): demographic data of 32 volunteers participated in the fed study. volunteers mean ±sd %cv age (years) 36.1 8.9 24.6 weight (kg) 64.3 9.8 15.2 height (cm) 172.2 7.9 04.6 it is obvious from tables 1 and 2 that the descriptive statistics of the demographic data of the volunteers selected for fasting study was almost similar to the fed study in order to exclude the potential difference in the pharmacokinetics of drug due to age, weight and/or height. all the volunteers started each study (fasting or fed) completed both periods of the study, i.e., no drop out or withdrawal were reported for both studies. both test and reference products were well tolerated by all volunteers. no incidence of side effects or adverse reactions were observed during the entire study. beside, all the volunteers left the study without any change in their base line condition (vital signs). products assay assay of penotxifylline in the test product and the reference product showed that the drug content (penotxifylline) of the test product differs by less than 5% from that of the reference product, thus conform with the fda and emea requirement (2-4) . dissolution testing evaluation of the dissolution data of both products based on fda and emea volunteers mean ±sd %cv age (years) 34.9 7.1 20.3 weight (kg) 65.1 8.4 12.9 height (cm) 173.8 8.9 05.1 iraqi j pharm sci, vol.24(2) 2015 bioequivalence and pharmacokinetics of er release pentoxifylline tablets 57 guidance (2-4) indicate that the dissolution data of both products are similar and insure sameness or equivalence of both products since the similarity factor (f2) value was 95.6%. plasma concentrations of pentoxifylline vs. time data for fasting and fed studies thirty minutes before dosing (at zero time) of each product intake and for both fasting and fed studies, penotxifylline was not detected in plasma in all volunteers and in both periods of the study, which insure the absence of carryover effects. the drug was detected in plasma samples of all the 32 volunteers after 0.5 hour of both products intake and after both fasting and fed studies. this finding indicates rapid absorption of the drug from the test and the reference er form of pentoxifylline tablets. the levels of pentoxifylline were not detected in plasma after 20 hrs (below the lloq 5 ng/ml) post dosing of the test and reference products and in both fasting and fed studies. figures 1 and 2 show mean plasma concentrations of pentoxifylline versus time after a single dose administration of a test formulation and the reference formulation under fasting and fed conditions, respectively. both figures 1 and 2 show a very good agreement between the mean plasma concentration-time profiles of both products. figure (1): mean plasma concentrations of pentoxifylline versus time after a single dose administration of a test formulation (extendedrelease tablet containing 400 mg pentoxifylline) and the reference formulation (trental ® extended-release tablet containing 400 mg pentoxifylline) to 32 healthy male adult subjects under fasting condition. figure(2): mean plasma concentrations of pentoxifylline versus time after a single dose administration of a test formulation (extendedrelease tablet containing 400 mg pentoxifylline) and the reference formulation (trental ® extended-release tablet containing 400 mg pentoxifylline) to 32 healthy male adult subjects under fed condition. using anova test, the plasma concentrations of penotxifylline at each time point for the test product were statistically compared against the corresponding plasma concentrations, at the same time points for the reference product. it appeared from anova test that there is no significant difference in the concentration-time profiles of both products at each time point and in both fasting and fed state. thus, there is good similarity in the concentration-time profiles between the test and the reference formulas, and under both fasting and fed conditions. pharmacokinetic parameters of pentoxi fhylline for fasting and fed studies tables 3 and 4 show the pharmacokinetic parameters of pentoxifylline for both test and reference products under fasting and fed conditions, respectively. for fasting study; the mean values of the pharmacokinetic parameters cmax , auc0-t , auc0- , cmax/auc0- , tmax, z & t0.5 for the test formula were; 144.4 ng/ml, 845.4 ng.hr/ml, 868.4 ng.hr/ml, 0.186 hr -1 , 3.29 hr, 0.561 hr -1 and 1.65 hr, respectively. the mean values of these parameter for the reference formula were; 150.0 ng/ml, 871.1 ng.hr/ml, 893.7 ng.hr/ml, 0.177 hr -1 , 3.70 hr, 0.558 hr -1 and 1.59 hr, respectively (table 3). it is obvious that there is good similarity in the pharmacokinetics of the reference and the test product. concerning the parameter cmax, different values were reported in literature. a study reported a mean cmax of 541.0 ng/ml (12) . other study (13) reported different mean cmax values at two different drug administration times of the day, in which the mean cmax was 326.4 ng/ml following morning (10:00 hr) administration and 266.4 ng/ml following night (22:00 hr) administration (13) . more recent studies (14-17) reported mean cmax values of 166.9 ng/ml, 218.4 ng/ml, 67.9 ng/ml and 132.6 ng/ml, respectively. thus, the reported studies indicate that the mean cmax values are variable among populations and time of drug administration (12-17) . this investigation (table 3) preset mean cmax closer to references (14-17) . regarding the parameter auc, a previous study (12) reported a mean value of 1422.0 ng.hr/ml. other study (13) reported different mean auc values at two different drug administration times of the day, in which the mean auc was 2424.0 ng.hr/ml following morning (10:00 hr) administration and 2124 ng.hr/ml following night (22:00 hr) administration (13) . more recent investigations (14-17) , reported mean values of 1078.2 ng.hr/ml, 1528.9 ng.hr/ml, 1270.0 ng.hr/ml and 1104.1 ng.hr/ml, respectively. the present investigation elucidated mean auc almost iraqi j pharm sci, vol.24(2) 2015 bioequivalence and pharmacokinetics of er release pentoxifylline tablets 58 similar to that reported in the studies (14-17) . thus, similar to the parameter cmax , the parameter auc exhibit variable results between populations and time of drug administration. for the parameter tmax , the reported mean values ranged from 1 to 4 hrs (1, 12, 14-17) . almost similar result was obtained in the present study (table 3). regarding the parameter t0.5, the mean values were found to be 1.32, 2.87 and 1.84 hrs, respectively (12, 14, 16) which is in a good agreement with the mean value found in the present study (table 3). the ratio cmax /auc0- which is considered as an appropriate measure for evaluating drug absorption in bioequivalence testing (18-20) supports the similarity in the absorption rate of both products (tables 3). this in turn suggests similar absorption behavior of the two formulations in the git. for the fed study; the mean values of the parameters cmax , auc0-t , auc0- , cmax/auc0- , tmax, z and t0.5 for the test formula were; 157.8 ng/ml, 826.5 ng.hr/ml, 853.8 ng.hr/ml, 0.198 hr -1 , 5.4 hr, 0.458 hr -1 and 2.06 hr, respectively. the mean values of these parameter for the reference formula were; 162.1 ng/ml, 869.7 ng.hr/ml, 894.8 ng.hr/ml, 0.196 hr -1 , 4.1 hr, 0.525 hr -1 and 1.80 hr, respectively (table 4). the above mentioned data indicate that the pharmacokinetic characteristics of the drug are similar for both products. comparison of these values (table 4) with those observed after fasting condition (table 3) show about 10% increase in cmax and 70% increase in tmax after food intake. whereas, food intake demonstrated no significant changes in other studied pharmacokinetic parameters (tables 3 and 4). a literature (1) reported that coadministration of er pentoxifylline tablets with meals resulted in an increase in mean cmax and auc by about 28% and 13%, respectively. the lloq of 5 ng/ml and uloq of 500 ng/ml applied in the current investigation are quiet enough for pharmacokinetics, bioavailability and bioequivalence studies of pentoxifylline er tablets. beside, 24 hrs blood sampling and one week washout period between dosing used in this study are adequate to ensure almost complete removal of the drug from the body and consequently prevent carryover effects. table (3): pharmacokinetic parameters of pentoxifylline after a single dose administration of a test formulation (extendedrelease tablet containing 400 mg pentoxifylline) and the reference formulation (trental ® extendedrelease tablet containing 400 mg pentoxifylline) to 32 healthy male adult subjects under fasting condition. mean ± sd cmax = maximum concentration of drug in plasma. tmax = time to attain cmax. auc0-t= area under plasma concentration-time curve from time zero to tlast, calculated by trapezoidal rule. auct- = extrapolated area under plasma concentration-time curve from tlast to infinity, calculated as clast/z. auc0- = total area under plasma concentration-time curve from time zero to infinity, calculated from the sum of auc0-t + auct-. z = first order terminal elimination rate constant t0.5 = first order terminal elimination half-life, equal to 0.693/z. clast = last measurable concentration which meet or exceed the lower limit of quantitation. tlast = time at which clast occur. pharmacokinetic parameters test formula reference formula mean ± sd mean ± sd cmax (ng/ml) 144.4 79.2 150.0 94.0 auc0-t (ng.hr/ml) 845.4 481.8 871.1 441.4 auc0- (ng.hr/ml) 868.4 486.9 893.7 445.1 cmax /auc0- (hr -1 ) 0.186 0.058 0.177 0.054 tmax (hr) 03.29 1.895 03.70 2.553 z (hr -1 ) 0.561 0.304 0.558 0.214 t0.5 (hr) 01.65 0.641 01.59 0.654 iraqi j pharm sci, vol.24(2) 2015 bioequivalence and pharmacokinetics of er release pentoxifylline tablets 59 table (4): pharmacokinetic parameters of pentoxifylline after a single dose administration of a test formulation (extendedrelease tablet containing 400 mg pentoxifylline) and the reference formulation (trental ® extended-release tablet containing 400 mg pentoxifylline) to 32 healthy male adult subjects under fed condition. mean ± sd. pharmacokinetic parameters test formula reference formula mean ± sd mean ± sd cmax (ng/ml) 157.8 105.0 162.1 94.0 auc0-t (ng.hr/ml) 826.5 537.0 869.7 468.1 auc0- (ng.hr/ml) 853.8 537.4 894.8 472.7 cmax /auc0- (hr -1 ) 0.198 0.070 0.196 0.078 tmax (hr) 05.4 03.05 04.1 01.89 z (hr -1 ) 0.458 0.223 0.525 0.192 t0.5 (hr) 02.06 0.945 01.80 1.187 statistical analysis of the pharmacokinetic parameters for fasting and fed studies for the fasting study; anova test for the pharmacokinetic parameters cmax , auc0-t , auc0- , cmax/auc0- , tmax, z & t0.5 of the test product versus the reference product exhibited no significant differences (p  0.05). beside, no significant differences (p  0.05) was found in the above mentioned parameters for the fed study between the test and the reference products. the geometric mean ratio and the 90% confidence interval demonstrated bioequivalence of both products under fasting condition (table 5) and fed condition (table 6). therefore, according to fda and emea guidance in bioavailability and bioequivalence (2-4) , it can be concluded that the newly developed pentoxifylline er 400 mg tablets are bioequivalent to the reference brand product trental er 400 mg tablets produced by sanofi-aventis under both fasting and fed states. table (5): geometric mean ratio and 90% confidence interval for the pharmacokinetic parameters of the test versus the reference products in fasting state. pharmacokinetic parameters t/r geometric mean ratio 90% confidence interval* lower limit upper limit cmax 0.94 87.9 111.2 auc0-t 0.95 85.7 106.5 auc0- 0.95 86.4 107.8 *acceptance criteria = lower limit  80 and upper limit  125.0. table (6): geometric mean ratio and 90% confidence interval for the pharmacokinetic parameters of the test versus the reference products in fed state. pharmacokinetic parameters t/r geometric mean ratio 90% confidence interval* lower limit upper limit cmax 0.95 81.7 112.8 auc0-t 0.92 84.1 104.1 auc0- 0.92 85.6 104.2 *acceptance criteria = lower limit  80 and upper limit  125.0. conclusion the present investigation presents the pharmacokinetics of pentoxifylline er 400 mg tablets under fasting and fed conditions. beside, food intake demonstrated no significant changes in the pharmacokinetic characteristics of the drug. plasma concentrations and consequently the pharmacokinetic of the drug can be determined in human applying hplc/uv method. the newly developed pentoxifylline 400 mg er tablets are bioequivalent to the innovator brand product trantal 400 mg er tablets in term of rate and extent of bioavailability. therefore, the newly developed product is interchangeable with trental er 400 mg tablet and can be prescribable in medical practice. iraqi j pharm sci, vol.24(2) 2015 bioequivalence and pharmacokinetics of er release pentoxifylline tablets 60 acknowledgment the author is very grateful and appreciative for the clinical and analytical staff for their help and expertise in conducting this study. special thanks to miss manar altamimi for the technical assistance in editing this article. beside, the author expresses sincere thanks and appreciation to college of pharmacy, baghdad university for their kind help and support. references 1. trental ® product information, sanofiaventis u.s. llc, bridgewater. revised july 2010 ; nj 08807, pet-fspl-sl-jul 10. 2. guidance for industry, bioavailability and bioequivalence studies for orally administered drug products – general considerations, u.s. department of health and human services, food and drug administration, center for drug evaluation and research (cder). march 2003. 3. guidance for industry , food-effect bioavailability and fed bioequivalence studies, u.s. department of health and human services, food and drug administration, center for drug evaluation and research (cder). march 2003. 4. european medicines agency (emea), committee for medicinal products for human use (chmp), guidelines on the investigation of bioequivalence. august 2010. 5. ich guideline for good clinical practice (gcp), e6. 1996. 6. latest wma declaration of helsinki , ethical principles for medical research involving human subjects. october 2013. 7. shargel l and andrew yu. applied biopharmaceutics & pharmacokinetics, sixth edition, appleton & lange, usa. 2012. 8. shein-chung c and jen-pei l. design and analysis of bioavailability and bioequivalence studies, second edition, revised and expanded, marcel dekker, inc., new york. 2000. 9. guidance for industry, statistical approaches to establishing bioequivalence, u.s. department of health and human services, food and drug administration, center for drug evaluation and research (cder). january 2001. 10. guidance for industry, bioanalytical method validation for human studies, u.s. department of health and human services, food and drug administration, center for drug evaluation and research (cder). may 2001. 11. guidance for industry , dissolution testing of immediate release solid oral dosage forms, u.s. department of health and human services, food and drug administration, center for drug evaluation and research (cder). august 1997. 12. pokrajac m, miljkovic b, simic d, brzakovic b, galetin a. pharmacokinetics and bioavailability of pentoxifylline in healthy volunteers a comparative study of three oral formulations. eur j pharm biopharm 1997; 43:193-196. 13. srinivasu p, rambhau d, rao br, rao ym. pharmacokinetics of pentoxifylline after oral administration of a sustainedrelease tablet at two different times of the day. arzneimittelforschung 1999; 49(9):750-3. 14. yuen kh, wong jw, peh kk, julianto t, choywp.comparative bioavailability study of controlledrelease pentoxifylline ta blet preparations. drug dev ind pham 2000; 26(7):803-7. 15. rojanasthien n, kumsorn b,yuen kah h. bioequivalence study of generic pentoxifylline. chiang mai med bull 2003; 42(1):7-16. 16. zeynep s, teksin i, iibeyi a, kadri y. bioavailability of pentoxifylline-chitosan oral matrix tablets in healthy subjects. j of bioequiv availab 2009; 1(4):115-120. 17. zakeri-milani p, ghanbarzadeh s, valizadeh h. comparative in vitro dissolution and in vivo bioequivalence of 2 pentoxifylline sustained release formulations. arzneimittelforschung 2012; 62: 335–339. 19. lacey lf, keene on, duquesony c, bye a. evaluation of different indirect measures of rate of drug absorption in comparative pharmacokinetic studies. j pharm sci 1994; 83: (2):212-5. 20. endrenyi l, tothfalusi l. econdary metrics for the assessment of bioequivalence. j pharm sci 1997; 86 (3): 401-2. 21. duquesony c, lacey lf, keene on, bye a. evaluation of different partial aucs as indirect measures of rate of drug absorption in comparative pharmacokinetic studies. eur j pharm sci 1998; 6 (4): 259-64. http://www.ncbi.nlm.nih.gov/pubmed?term=srinivasu%20p%5bauthor%5d&cauthor=true&cauthor_uid=10514902 http://www.ncbi.nlm.nih.gov/pubmed?term=rambhau%20d%5bauthor%5d&cauthor=true&cauthor_uid=10514902 http://www.ncbi.nlm.nih.gov/pubmed?term=rao%20br%5bauthor%5d&cauthor=true&cauthor_uid=10514902 http://www.ncbi.nlm.nih.gov/pubmed?term=rao%20ym%5bauthor%5d&cauthor=true&cauthor_uid=10514902 http://www.ncbi.nlm.nih.gov/pubmed?term=rao%20ym%5bauthor%5d&cauthor=true&cauthor_uid=10514902 http://www.ncbi.nlm.nih.gov/pubmed?term=yuen%20kh%5bauthor%5d&cauthor=true&cauthor_uid=10872103 http://www.ncbi.nlm.nih.gov/pubmed?term=peh%20kk%5bauthor%5d&cauthor=true&cauthor_uid=10872103 http://www.ncbi.nlm.nih.gov/pubmed?term=julianto%20t%5bauthor%5d&cauthor=true&cauthor_uid=10872103 http://www.ncbi.nlm.nih.gov/pubmed?term=julianto%20t%5bauthor%5d&cauthor=true&cauthor_uid=10872103 http://www.ncbi.nlm.nih.gov/pubmed?term=choy%20wp%5bauthor%5d&cauthor=true&cauthor_uid=10872103 iraqi j pharm sci, vol.22(1) 2013 abo blood group and ulcer 97 the relationship between abo blood group distribution and the incidence of upper gastric and duodenal ulcer in iraqi patients manal k. abdulridha *, 1 * department of clinical pharmacy, college of pharmacy, al-mustansiriyah university, baghdad, iraq. abstract the relationship between blood group antigens and peptic ulcer disease has been widely evaluated in the past, but only one study relating h pylori seroprevalence to abo blood groups among iraqi patients with peptic ulcer disease is available. we aimed to evaluate the frequency of peptic ulcer disease among different abo blood groups in iraqi patients, and we thought it was worthwhile to try to determine whether these components take some part in disease etiology. one hundred and six patients with peptic ulcer disease (pud) (43 male and 63 female; mean age: 48 ± 18 years) who attended baghdad teaching hospital and al yarmouk teaching hospital endoscopy centers were enrolled , and 238 control subjects. finger blood samples were used for abo/rhesus (rh) blood group antigen typing. the abo blood group phenotype frequency in peptic ulcer patients was as follows: 18.9% for blood group a, 15.1% for blood group b, 57.5 % for blood group o and 8.5% for blood group ab. rh positivity was found in 100% of patients. significant higher percentage of patients with both gastric and duodenal ulcer disease are those holding blood group o + compared to other blood group phenotypes (57.5%)( p= 0.003) .the present study show higher incidence of doudenal ulcer( du) in patients with blood group o + compared to gastric ulcer( gu) patients (65.6%vs 54.1%) , although no statistical difference between both diseases was found,( p ˃ 0.05) in respect to other blood group phenotypes. peptic ulcer disease is predominant in patients aged between 50-59 years represents with higher percentage (26.4%) compared to other age groups. patients with blood group o + phenotype presented with a highly significant percentage of peptic ulcer disease, since those individuals may express a higher inflammatory responses to h. pylori with higher levels of lymphocyte infiltration in the gastrointestinal mucosa , and a higher frequency of secretor status . in addition, they do not produce the substance on the surface of blood group o + cells that may protect the lining of the duodenum .according to these results, probably abo/rh blood group (mainly blood group o+) has an important role in patients with peptic ulcer disease as additional risk . the functional significance of abo blood group distribution might be associated with biological behavior of peptic ulcer disease. the impact of blood group on peptic ulcer disease may be a focus for further studies. keywords: abo/ rh blood group system upper gastrointestinal disorders –age & gender distribution تقٍٍن العالقت بٍن فصٍلت الدم هع تقرحاث الوعدة واالثنً عشري عند الورضى العراقٍٍن هنال خالد عبد الرضا *،1 * .العشاق بغذاد،، الجبهعت الوسخٌظشَت ،ولُت الظُذلت ، فشع الظُذلت السشَشَت الخالصة حن فٍ السببك حمُُن العاللت بُي فظُلت الذم الىساثُت واهشاع حمشدبث الوعذة واالثٌٍ عششٌ لىي هٌبن دساست وادذة فمؾ حشبؾ العاللت لهزا َهذف البذث الً حمُُن الخشابؾ والزٌ دسب اعخمبدًب َسخذك .بُي الوسبب البىخُشٌ للخمشدبث وفظُلت الذم عٌذ الوشػً فٍ العشاق 63روش و 43)هشَغ 106حشخول الذساست علً .الوذبولت لوعشفت فُوب ارا وبًج فظُلت الذم عٌذ االًسبى حلعب دوس وعبهل هسبب للوشع وسفٍ هسخشفً بغذاد الخعلُوٍ و هسخشفً الُشهىن هي الوظببُي بمشدت الوعذة واالثٌٍ عششٌ الزَي حن حذىَلهن الً ودذة الٌبظ( اًثً وبًج ًخبئج حىصَع فظُلت الذم . و حن حذذَذ فظُلت الذم هي لطشة دم االبهبم .هي االشخبص الطبُعُُي 238الخعلُوٍ للخشخُض ووزله : الىساثٍ عٌذ هشػً حمشدبث الوعذة واالثٌٍ عششٌ وبالحٍ ووبى جوُع الوشػً َذولىى . .abلفظُلت الذم %8,5و oلفظُلت الذم %57,5و bلذم لفظُلت ا %15,1و aلفظُلت الذم 18,9% دلج الٌخبئج علً ًسبت عبلُت راث هغضي ادظبئٍ لوشع حمشدبث الوعذة واالثٌٍ عششٌ عٌذ الوشػً. الوىجب ( rh)ًىع العبهل الشَسٍ oالذبهلُي لفظُلت الذم + . ( p= 0.003( )%57,5)لببلٍ اًىاع فظبئل الذم همبسًت ببلوشػً االخشَي الذبهلُي corresponding author e-mail: mkar_3564 @yahoo.com received:20/10/2012 accepted:25/3/2013 iraqi j pharm sci, vol.22(1) 2013 abo blood group and ulcer 98 o الذمدة عٌذ هشػً فظُلت ـسح الوعـهشع حك ًسبت عبلُت هي واوذث الذساست الذبلُت علً + عششٌ االثٌٍ اث ـهع حمشح اسًت ـببلوك ( دلج الذساست اَؼب علً اى ًسبت حمشدبث . لىي الٌسبت لن حىي رو هغضي ادظبئٍ ببلومبسًت هع ببلٍ فظبئل الذم ( % 54,1همببل 65,6% دلج الذساست . همبسًت بببلٍ الفئبث العوشَت ( %26,4)سٌت 59-50الوعذة واالثٌٍ عششٌ اوبش عٌذ الوشػً الزَي حخشاوح اعوبسهن بُي o الذبهلُي لفظُلت الذم الوشػً علً اى + َوثلىى ًسبت عبلُت راث هغضي ادظبئٍ َعىد الً لذعشش وهي هشع حمشدبث الوعذة واالثٌٍ داخل جذاس الوعذة واالهعبء ( سبَجاللوفى)واسحفبع ًسبت حجوع الشاص الذم البُؼبء h. pyloriصَبدة الخذسس االلخهببٍ ػذ بىخشَب ببالػبفت الً عذم وجىد هبدة ولبئُت علً سطخ وشَبث الذم الذوشاء عٌذ . ووزله صَبدة افشاصاث الوعذة واالثٌٍ عشش عٌذ هؤالء الوشػً o فظُلت الذم + لهب حبثُش واػخ ( +oالذم وخبطت فظُلت)هي هزٍ الذساست ببى ًىع فظُلت الذم ًسخٌخج.َوىٌهب دوبَت غالف االثٌٍ عشش وهزا . عٌذ الوشػً الوظببُي بخمشدبث الوعذة واالثٌٍ عششَىجضء هي ؽبُعت حىىى الوشع هوب َوىي اعخوبدهب وعبهل هسبب اخش للوشع .الخبثُش َخطلب دساست اعوك لخىػُخ الفىشة . تىزٌع العور -اهراض الجهاز الهضوً العلٍا -abo/ rh فصٍلت الدم : الوفتاحٍت الكلواث introduction the relationship between blood group and the incidence of peptic ulcer disease had been evaluated by several references whom provided a new clue to the etiology of the disease (1, 2, 3) . it is well known that helicobacter pylori (h. pylori) infection and aspirin/(non steroidal antiinflammatory drugs (nsaids)) are the most important factors predisposing peptic ulcer disease in the community (3) . in addition, the possible relationship between genetic factors and the natural history of peptic ulcer has been studied (4) . a number of evidence is in favor of both hereditary (abo blood group) and environmental factors playing a part in the development of bleeding duodenal ulcer (5,6,7) .some reports postulated that overt bleeding from the gastric mucosa, whether aspirininduced or not, may be related to abo blood group and secretor status (8, 9) . the association of blood group o with bleeding from duodenal ulcer was also confirmed (10) . recently it was considered that the life expectancy of persons holding blood group o is less than that of other blood groups , and generally , blood group o holders are more prone to various diseases mainly duodenal ulcer ( 11) . similarly, gastric carcinoma was found to be associated with blood group a, but no explanation for this condition was found (12) . in addition, type o people may be more vulnerable to the bacteria that can cause peptic ulcers, helicobacter pylori (13,14) . many epidemiologic studies had found that non secretors of abo blood group antigens and individuals of blood group o were overrepresented among patients with peptic ulcers (15, 16) . these studies encouraged many researchers to investigate the relation between abo blood groups and their secretor status with peptic ulcer (9, 11) . data concerning the association of abo blood groups among iraqi patients with gastric compared to duodenal ulcer was presented in one study only. we aimed to evaluate this association and we thought it was worthwhile to try to determine whether these components take some part in disease etiology. patients and methods this study include 106 patients with peptic ulcer disease (pud) either gastric ulcer (gu) or duodenal ulcer (du) admitted to baghdad teaching hospital and alyarmouk teaching hospital for gastrointestinal endoscopy, and 238 control subjects. the database was collected from octobor 2011 till march 2012. the demographic and clinical characteristics including age, gender, and abo/ rh phenotype blood group were recorded. the patients were clinically evaluated and the gastrointestinal signs and symptoms were recorded. upper gastrointestinal endoscopy was done according to standard medical procedure. since blood group is not routinely checked in endoscopy clinics, finger blood samples were taken from each patient after endoscopy. abo blood groups and rh phenotype evaluations were carried out by standard rapid slide agglutination method ( using anti abo monoclonal kit) ( 17) . .the control subjects were clinically disease free. statistical analysis data were analyzed using statistical package for social sciences (spss) (student version 13, mcgraw hill company 2006). chi square test was used to detect statistically significant differences among variables. p-values <0.05 were considered significant. http://www.springerlink.com.tiger.sempertool.dk/content/k46j33r4u8130716/fulltext.html#cr2 http://www.springerlink.com.tiger.sempertool.dk/content/k46j33r4u8130716/fulltext.html#cr2 http://www.scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=environmental+factors iraqi j pharm sci, vol.22(1) 2013 abo blood group and ulcer 99 results this study includes a total of 344 subjects 106 (30.8%) patients and 238 (69.2%) controls. the demographics and clinical characteristics is shown in table (1). table 1: demographics and clinical characteristics table (2) (figure 1) show that the abo blood group phenotype distribution in patient and control groups was as follows : 61 (57.5 %) versus 86 (36.1 %) for group o , 20 (18.9 %) versus 71 (29.8 %) for group a, 16 (15.1 %) versus 49 (20.6 %) for group b, and 9 (8.5 %) versus 32 (13.4 %) for group ab respectively. blood group o was found to have highly significant pud frequency in patient group than in the other blood group phenotypes (p= 0.003). the rh positive was in 329 (95.60 %) subjects , and negative was in 15 (4.40%) subjects in all study groups table (1). the rh positivity was 100% in patient group, and 93.6% in the controls. table 2: blood group distribution between patients and controls *pearson chi-square figure 1 : blood group distribution between patients and controls table (3) (figure 2) show that the distribution of endoscopic findings in patient group were 32 (30.2%) for du, and 74 (69.8 %) for gu. the frequencies of abo blood group among endoscopic findings (du vs gu): 21 (65.6 %) versus 40 (54.1%) for group o, 6 (18.8%) versus 14 (18.9%) for group a, 2 (6.20 %) versus 14 (18.9%) for group b, and 3 (9.30%) versus 6 (8.10%) for group ab respectively. the higher incidence of du was in patients with blood group o + compared to gu patients (65.6%vs 54.1%) , although no statistical significance was found between both diseases (p ˃ 0.05) in respect to other blood group phenotypes .table (1) also show that the mean age of control subjects was (39 ±20) years , and in peptic ulcer patients was (48 ±18) . the mean age distribution in patients was as follows : (13.2%) for group 20-29 years , (17.9%) for 30-39 years, (20.8%) for 40=49 years, (26.4%) for 50-59 years , and (21.7%) for 60-69 years. the higher frequency of pud was found in the fifth decade of age. variable total n (%) patients n (%) controls n (%) subjects 344 (100) 106 (30.80) 238 (69.20) gender male female 146(42.40) 198(57.60) 43.(40.6) 63(59.4) 103(43.3) 135(56.7) age (year) 20 – 29 113(32.8) 14(13.2) 99(41.6) 30 – 39 85(24.70) 19(17.9) 66(27.7) 40 – 49 61(17.7) 22(20.8) 39(16.4) 50 – 59 56(16.3) 28(26.4) 28(11.7) 60 – 69 29(8.4) 23(21.7) 6(2.6) blood group subjects n (%) p* p * patients controls a 20 (18.90) 71 (29.80) 0.003 b 16 (15.10) 49 (20.60) ab 9 (8.50) 32 (13.40) o 61 (57.50)* 86 (36.10)* total 106 (100) 238 (100) iraqi j pharm sci, vol.22(1) 2013 abo blood group and ulcer 100 table 3: association between blood group and endoscopic findings *pearson chi-square. figure 2: association between blood group and endoscopic findings discussion the association between the abo blood group and both gastric cancer and peptic ulcers has been studied previously (18-19 ) . this study provide an estimation of the extent of such associations in iraqi patients. in general adult population 22% of all of peptic ulcer (gu&du) disease were idiopathic and almost 40% of duodenal were h.pylori infection. obesity is a risk factor for gastric ulcer as for use of lowdose of aspirin (20,21) . previous studies demonstrated that blood group o is associated with duodenal ulcer disease, while gastric ulcer and gastric carcinoma are associated with blood group a (21,22 , 23) . also it was concluded that gastric ulcer near the pylorus and those occurring with duodenal ulcer were associated with acid hypersecrtion, these cases were marked in patients with blood group o, while gastric ulcer in body of the stomach occurring in patients in which their duodenum was normal, were characterized by acid hyposecretion and this marked in patients with blood group a, the cause that blood type a is most likely to have gastric cancer (24) .romshoo et al. (1997) reported that, the majority of peptic ulcer patients (56%) had blood group o and it though a risk factor for peptic ulcer (25) . in another study, bayan et al. (2009) finding contributes to the positive correlation between group o and upper gastrointestinal bleeding caused by gastroduodenal ulcers and erosive gastropathy and the blood group o which was found to have higher frequency in patient group than in controls ( p = 0.004) (26) . in prospective well-defined cohort study of swedish and danish blooddonors have confirmed that individuals with blood group o have a higher risk of peptic ulcers than those with other blood groups (27) . those findings have been confirmed by many other reports (28,29) . despite epidemiological evidence of increased peptic ulcer disease in abo blood group o subjects, and the evidence that h. pylori adhesion to gastric epithelial cells is mediated by blood group epitopes, no significant association between blood groups and h. pylori serological status was detected ( p˃ 0.05) (30) . jaff ms. (2011) showed a significant association between the o blood group and infection caused by h. pylori (p = 0.01) , the prevalence of seropositivity to h. pylori infection was (64.8%) in symptomatic patients in the kurdistan region of iraq (31) . in the present study, patients with blood group o phenotype presented with a highly significant percentage of pud compared to controls ( 57.5%) vs( 36.1% ) respectively p = 0.003. blood group o individuals express a higher inflammatory responses to h. pylori with higher levels of lymphocyte infiltration in the gastrointestinal mucosa (32) , a lower level of von willebrand’s factor (33,34) , and a higher frequency of secretor status (35) , in addition , they do not produce the substance on the surface of blood cells that may protect the lining of the duodenum (36,37) all these together explain the possible cause of these individuals’ increased susceptibility to peptic ulceration. jaff ms. study support this epidemiological view of gastric susceptibility of o blood group to h. pylori infection which is most probably due to high secretor status (31) . this correlation was supported in many reports (38-40) . bayan et al (2009) showed higher frequency of du among patients with blood group o with significantly higher h. pylori positivity ( p=0.031) compared to other abo phenotype . although no statistical difference was noticed in o blood group distribution between du and gu endoscopic findings (26) .the present study show higher incidence of du in patients with blood group o compared to gu (65.6%vs 54.1%) , although no blood group endoscopic findings n (%) p * gu du a 14 (18.90) 6 (18.80) 0.401 b 14 (18.90) 2 (6.20) ab 6 (8.10) 3 (9.40) o 40 (54.10) 21 (65.60) total 74 (100) 32 (100) http://en.wikipedia.org/wiki/blood_type#cite_note-0 http://en.wikipedia.org/wiki/blood_type#cite_note-0 http://en.wikipedia.org/wiki/blood_type#cite_note-0 http://en.wikipedia.org/wiki/blood_type#cite_note-0 http://en.wikipedia.org/wiki/blood_type#cite_note-0 http://en.wikipedia.org/wiki/blood_type#cite_note-0 http://scialert.net/fulltext/?doi=pjbs.2009.991.993#165541_ja http://scialert.net/fulltext/?doi=pjbs.2009.991.993#165541_ja http://scialert.net/fulltext/?doi=pjbs.2009.991.993#165525_ja http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20jaff%2bms%5bauth%5d iraqi j pharm sci, vol.22(1) 2013 abo blood group and ulcer 101 statistical difference between both diseases in blood group o in respect to other blood groups , p ˃ 0.05. this is probably due to small number of patients enrolled in this study . most studies stated that stomach ulcers are more likely to develop in older people. this is may be because arthritis be prevented by daily use of aspirin / (nsaids) , in addition to age related relaxation of pylorus valve allowing backflow of bile to erode the stomach lining (41) . h. pylori seropositivity increased with age, and was not related to gender (30) . this age – related ulcer development was correlated to abo blood group phenotype in many studies (42,43) , including our study . further studies screening the effect of age on incidence of peptic ulcer disease are warranted to exclude other confounders. according to results, probably abo/rh blood group (mainly blood group o+) has an important role in patients with peptic ulcer disease as additional risk . we can conclude that the functional significance of abo blood group distribution might be associated with biological behavior of pud. the impact of therapeutic strategy with anti-secretary or h. pyloi irradiation protocols on blood group in pud may be a focus for further studies. references 1. mourant ae, domaniewska-sobczak k, kopec ac. blood groups and diseases: a study of associations of diseases with blood groups and other polymorphisms. oxford, united kingdom: oxford university press; 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j infect dis. 1996;174:552-556 . 40. heneghan, m.a., a.p. moran, k.m. feeley, e.l. egan, j. goulding, c.e. connolly and c.f. mccarthy. effect of host lewis and abo blood group antigen expression on helicobacter pylori colonisation density and the consequent inflammatory response. immunol. med. microbiol. 1998; 20: 257266. http://en.wikipedia.org/wiki/international_standard_book_number http://en.wikipedia.org/wiki/international_standard_book_number http://www.ncbi.nlm.nih.gov/pubmed?term=robertson%20ms%5bauthor%5d&cauthor=true&cauthor_uid=12680981 http://www.ncbi.nlm.nih.gov/pubmed?term=cade%20jf%5bauthor%5d&cauthor=true&cauthor_uid=12680981 http://www.ncbi.nlm.nih.gov/pubmed?term=savoia%20hf%5bauthor%5d&cauthor=true&cauthor_uid=12680981 http://www.ncbi.nlm.nih.gov/pubmed?term=clancy%20rl%5bauthor%5d&cauthor=true&cauthor_uid=12680981 http://www.ncbi.nlm.nih.gov/pubmed?term=clancy%20rl%5bauthor%5d&cauthor=true&cauthor_uid=12680981 http://www.ncbi.nlm.nih.gov/pubmed/12680981 http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=search&db=pubmed&term=%20jaff%2bms%5bauth%5d iraqi j pharm sci, vol.22(1) 2013 abo blood group and ulcer 103 41. seyda t, derya c, füsun a, meliha k. the relationship of helicobacter pylori positivity with age, sex, and abo/rhesus blood groups in patients with gastrointestinal complaints in turkey. helicobacter. 2007;12:244–250 42. jaff ms. abo and rhesus blood group distribution in kurds. j blood med. 2010;1:143–146. 43. jafarzadeh a, ahmadi-kahanali j, bahrami m, taghipour z. seroprevalence of antihelicobacter pylori and anti-caga antibodies among healthy children according to age, sex, abo blood groups and rh status in southeast of iran. turk j gastroenterol. 2007;18:165–171. iraqi j pharm sci, vol.25(1) 2016 effect of metformin treatment in women with endometriosis 28 effect of metformin treatment on some blood biomarkers in women with endometriosis. noor a.omer *,1 , muhammad a.taher ** and henan dh skheel aljebory *** * abu-ghraib general hospital, ministry of health , baghdad, iraq. ** department of clinical laboratory sciences college of pharmacy, university of baghdad, baghdad, iraq. *** college of medicine ,al mustansyria university, baghdad, iraq. abstract endometriosis is a common women health disorder that occurs when endometrial-like tissue grows outside the uterus. this may lead to irregular bleeding , pelvic pain, infertility and other complications. metformin, because of its activity to improve insulin sensitivity, it is used for the treatment of diabetes; it also has a modulatory effect on ovarian steroid production and has antiinflammatory properties, all may suggest its possible effect in treatment of endometriosis. this study was planned to determine the effect of metformin on serum levels of interleukineight(il8), tumor necrosis factor-alpha (tnf –α) and estradiol (e2) production , and related symptomatic changes that accompany with endometriosis (pelvic pain, dysmenorrhea and menorrheaga) after three months of study. blood samples were obtained from those taking metformin and measure the serum levels of (il-8) , tnf –α and (e2) were measured before and after three months of taking a metformin .metformin therapy resulted in a significant reduction in the clinical symptoms of endometriosis (pelvic pain and dysmenorrhea) and insignificant changes in menorrhagia. metformin therapy resulted in a significant reduction in the serum levels of il-8, tnf α while insignificant reduction in estradiol e 2 in the study group after 3 months of treatment .in conclusion the results of this study, demonstrated that metformin may be a well-tolerated treatment for endometriosis that relieved pain and reduces menstrual disorders and serum levels of the inflammatory markers (il8 and tnf-α) are decreased in study group treated with metformin after 3 months due to its anti inflammatory effects. keywords: endometriosis, metformin, tnf – a ,il-8, e 2. فعالٍَ دّاء الوٍتفْرهٍي فً بعض الوؤشرات الحٌٍَْ فً الدم على الٌساء الالتً ٌعاًٍي هي داء بطاًَ الرحن الِاجرٍ ًْر اركاى عور *‘1 ، هحود عباس طاُر ** الجبْري ّحٌاى ضاٌع *** * .ٔصاسج انصحح ، تغذاد ، انعشاق ،انعاو يستشفٗ اتٙ غشٚة ** اد ، انعشاق .فشع انعهٕو انًختثٛشٚح انسشٚشٚح ، كهٛح انصٛذنح ، رايعح تغذاد ، تغذ *** . ، تغذاد ، انعشاق حزايعّ انًستسصشٚ،انكهّٛ انطة الخالصــــة انشحى خاسد تًُٕ تطاَح انشحى تشثّ أَسزحَتٛزّ انز٘ ٚحذث ٔ انٓارشِ ْٕ يٍ اكخش االيشاض شٕٛعا نذٖ انُساء تطاَح انشحى . اخشٖ يعاعفاخٔانعقى ٔآالو انحٕض اظطشاب انذٔسِ انشٓشّٚ , ْزا قذ ٚؤد٘ إنٗٔ عهٗ تأحٛش نالنتٓاتاخ ٔ خصائص يعادجٔ نذّٚ أٚعا انحساسٛح نالَسٕنٍٛ انز٘ ٚحسٍ يشض انسكش٘ نعالدٚستخذو انًٛتفٕسيٍٛ انثطاَح انشحى انٓارشِ . احتًال اٌ ٚكٌٕ نّ دٔس فعال فٙ عالد، نٓزا اثٛطانً انستٛشٔٚذ اإلَتاد يهي ٕٚيٛيا نًيذِ 0011 انًٛتفيٕسيٍٛ اسيتخذاو تقٛيٛىل تطاَح انشحى انٓارشِ يٍ خال عهٗ فعاال انًٛتفٕسيٍٛكاٌ استخذاو نذساسّ يا إرا انتيٙ تحيذث فيٙ اععيشاضانتغٛٛيشاخ ٔانذو فٙ انستٛشٔٚذ ٔاَتاد أنفا َخش انٕسو عايمٔ 8 –االَتشنٕكٍٛيستٕٚاخ حالث اشٓش عهٗ .أشٓش يٍ اخز انعالد عسش ٔ غضاسِ انطًج (تعذ حالحح، )أنى انحٕض يع تطاَح انشحى ٚعاٍَٛ يٍ داء تطاَّ انشحى انٓارشِ ٔ ُٔٚقسًٌٕ انٗ: 01تشًم رًع عُٛاخ يٍ َساء يٍ يختهف االعًاس ٔعذدْى ,danazol عقاس انذاَاصٔل, حثٕب يُع انحًم, ْشيٌٕ انثشٔرٛستشٌٔتانًشض ٔٚستخذيٍ انعالد انتقهٛذ٘)َساء يصاتٍٛ -0 ( .gn-rh-agonists يخٛم يحشس ْشيَٕاخ انتُاسم يه يٍ انًٛتفٕسيٍٛ. 0011َساء يصاتٍٛ تانًشض ٚستخذيٍ انعالد انتقهٛذ٘ اظافّ انٗ -2 شٓش يٍ اخز انًٛتفٕسيٍٛ ٔارشاء تعط انتحانٛم انًختثشّٚ عهٗ انعُٛاخ انتٙ تشًم تشًم اخز عُٛاخ يٍ انذو قثم ٔتعذ حالحّ أ ٔيقاسَح e2االستشٔرٍٛ ( ٔ tumor necrosis factor alpha(, عايم َخش انٕسو )interleukin-8)8يستٕٖ االَتشنٕكٍٛ انُتائذ . 1 corresponding author e-mail:noor23.arkan@yahoo.com received: 4/2/2015 accepted: 26 /1/2016 iraqi j pharm sci, vol.25(1) 2016 effect of metformin treatment in women with endometriosis 29 انحٕض ٔآالو عسش انطًج اَخفاض كثٛش فٙ فٙ انتٓاب تطاَح انشحى انًشظٗ انزٍٚ ٚعإٌَ يٍ فٙ انًٛتفٕسيٍٛ انعالد أدٖ يًا ٚشٛش إنٗ .,انعالد أشٓش يٍ 0تعذ نًزًٕعح انذساسح نًستٕٚاخ يصم il-8 ٔ tnf-a فٙ ل اَخفاض كثٛش أسفشخ أٚعا عٍ .انثطاَٙ انشحًٙ عقاس نالنتٓاب كًانهًٛتفٕسيٍٛ اإليكاَاخ انعالرٛح قذ ٚكٌٕ نٓا أَٓا .tnf-a، il-8 ، e2،الوٍتفْرهٍي ،داء بطاًَ الرحن الِاجرٍ الكلوات الوفتاحٍة : introduction endometriosis is a common health problem in women .it got its name from the word endometrium, which is the tissue lining the uterus (1) .during a woman’s menstrual period, this tissue thickens in preparation for a fertilized egg (pregnancy). if there is no fertilization, the tissue breaks down and bleeds with each menstrual period, allowing it to exit the body. in endometriosis, the tissue that normally lines the uterus grows outside the uterus. most commonly, it is found on the ovaries, fallopian tubes, tissue that holds the uterus in place, outer surface of the uterus, or the lining of the pelvic cavity. other sites for growths can include the bladder, bowel, cervix, rectum and vagina, or vulva (2) . the different theories involved in the pathogenesis of endometriosis indicate that the etiology of endometriosis is complex and multifactorial, involving hormonal,genetic immune and environmental components (3) although the etiology of disease is undetermined, four main hypotheses have been circulated as understandable causes (4) : a. sampson’s theory of retrograde menstruation. b. ceolomic metaplasia and induction theories (an extension of the ceolomic metaplasia theory). c. the embryonic rest theory. d. lymphatic and vascular metastasis theories. several theories considering the pathogenesis of endometriosis are shown in table-1 , retrograde menstruation may be one of the initiating steps in the pathogenesis of superficial endometriosis, genetic and micro environmental factors that prevent clearance of ectopic lesions and allow remodeling of peritoneum are essential for the propagation of endometriotic lesions (3,5) . table (1): mechanism of the different theories in the pathogenesis of endometriosis (6) mechanism theory flow of endometrial content into pelvis, allowing implantation of endometrial lesions retrograde menstruation transformation of peritoneal tissue or cells into endometrial tissue through hormonal and/ or immunological factors metaplasia estrogen-driven proliferation of endometrial lesions. resistance to progesterone-mediated control of endometrial proliferation hormones recruitment of immune cells and their production of cytokines that promote endometrial growth oxidative stress and inflammation prevention of eliminating menstrual debris andpromotion of implantation and growth of endometrial lesions immune dysfunction promoting survival of endometrial cells and down regulation of apoptotic pathways apoptosis suppression alteration of cellular function that increases attachment of endometrial cells and evasion of these cells from immune clearance genetic initiation of endometriotic deposits by undifferentiated cells with natural ability to regenerate stem cells iraqi j pharm sci, vol.25(1) 2016 effect of metformin treatment in women with endometriosis 30 tumor necrosis factor-alpha tnf-α is the main pro-inflammatory cytokine known to impair glutathione (gsh) production by several ways, making an environment conducive to the development of oxidative stress(os). this pathogenic cycle of gsh disturbances and enhanced tnf-α production may be active in the female reproductive tract in endometriosis. an in vitro study investigating endometriosisassociated infertility showed that the quality of spermatozoa may be decreased following incubation with tnf-α in a doseand timedependent manner (7). tnf-α secretion is stimulated by il-1 and bacterial endotoxin. when mediated by il-8, tnf-α has been known to promote the growth of endometriotic cells (8) .the mechanisms connecting endometriosis and infertility involved:  distorted pelvic anatomy, including adhesions resulting from endometriosis, which can impair oocyte release or prevent ovum pickup and transport, (9) as well as damaged or plugged fallopian tubes or acquired or congenital uterine defects. (10) .  altered peritoneal function, including increases in fluid volume; concentration of activated microphages; prostaglandins; il-1, il-6, tnf-alpha, igg, and iga antibodies; lymphocytes; an ovum capture inhibitor preventing cumulus -fimbria interaction; (11).  endocrine and an ovulatory disorders, including luteinized unruptured follicle syndrome (luf), luteal phase defect, abnormal follicular growth, and premature as well as multiple luteinized hormone surges. it has been hypothesized that luf may not be a consequence of endometriosis, but, in fact, may be a cause or cofactor in the development of the disease (9) .  impaired implantation, with evidence suggesting that endometriosis may be responsible for reduced expression of the (alpha(v)beta(3) integrin)αvβ3 cell adhesion molecule during the time of implantation (11) . the diagnosis of endometriosis can be substantiated only by direct visualization during laparoscopy or laparotomy confirmed by tissue biopsy (12) larger lesions may be seen within the ovaries as ovarian endometriomas or "chocolate cysts" as they contain a thick brownish fluid, mostly old blood. however, smaller endometriosis implants cannot be visualized with ultrasound technique (12) . surgically, endometriosis can be staged i–iv according to the (revised classification of the american society of reproductive medicine) (13) . in principle the various stages show these findings: stage i (minimal): endometriosis restricted to only superficial lesions and possibly a few filmy adhesions, there are isolated incidents of endometrial tissue growth outside the uterus (14) . stage ii (mild): this diagnosis occurs when there are several small implants and a few small areas of scar tissue or adhesions and some deep lesions are present in the cul-desac. (15) . stage iii (moderate): as in stage ii, plus presence of endometriomas on the ovary and more adhesions. the implants in stage three must be superficial and deep. stage iv (severe): this is the most severe stage of endometriosis. patients with stage iv endometriosis will have many superficial and deep implants as well as large adhesions found. the purpose of medical management is to minimize proliferation/reduce pain, inhibit inflammation, minimize menstrual volume, frequency and oppose e2 action (16) . medical treatment included :initial therapy should include a non-steroidal antiinflammatory drugs (nsaids) such as : anaproxen 500 mg at first then followed by 250 mg orally three times daily, or b-ibuprofen 800 mg as single dose, then 400 mg orally every 6 to 8 hours or cmefenamic acid 500 mg orally then followed by 250 mg every 6 hours. (17) . surgical treatment is highly effective for the alleviation of symptom, pain reduction and can increase fertility in sub-fertile women (18) .medical management is based on hormonal suppression of endometriotic lesions and is particularly effective when amenorrhea occurs via down -regulation of the hypothalamic -pituitary-ovarian axis (19) .as shown in table-2. iraqi j pharm sci, vol.25(1) 2016 effect of metformin treatment in women with endometriosis 31 table (2): hormonal treatment for endometriosis (20) . medication indication dosing comment depot mdpa (depoprovera) r pain relief 150 mg intramuscularly every three months common usage in primary care mdpa (provera) r pain relief 30 to 100 mg daily, given orally common usage in primary care oral contraceptives pain relief 0.02 to 0.03 mg ethinyl estradiol and 0.15 mg desogestrel daily (cyclically) for six months common usage in primary care levonorgestrel intrauterine system (mirena) r pain relief intrauterine system can be placed easily common usage in primary care gonadotropin-releasing hormone analogues (e.g. goserelin [zoladex], pain relief 3.75 mg of leuprolide injected every four weeks or 3.6 mg of goserelin implanted subcutaneously for six months expensive; significant side effects (hypoestrogenic symptoms) nafarelin (synarel) r pain relief 200 mcg intranasally twice daily for six months expensive; significant side effects danazol pain relief 200 mg given orally three times daily 400 mg given orally twice daily for six months significant androgenic side effects gestrinone pain relief 2.5 mg given orally twice weekly for six months hot flashes this study was conducted to: 1investigate the effect of metformin on symptoms of endometriosis and biochemical markers. 2-investigate the effects of metforminon serum interleukin 8 (il-8), tumor necrosis factor alpha (tnf-α )and estradiol (e2)levels at baseline and after 3 months of treatment, compared to baseline values. patients and methods thirty women were included in this study from al-elwiyah maternity teaching hospital and al-yarmok teaching hospital for the period from january 2014 to june-2014. verbal consent was obtained from all women prior to enrollment in the study. inclusion criteria 1women undergo diagnostic laparoscopy for pelvic pain or treated for some causes of infertility. 2women undergo diagnostic laprotomy for ovarian cyst or acute abdominal pain who were found to have endometriosis. 3-women came for follow up for endometriosis. grouping of patients a-control group fifteen patients diagnosed by laparoscopy to have different stages of endometriosis. these patients were complaining of one or more symptoms such as dysmenorrhea, pelvic pain or menorrhagia as seen in table -3. they received classical drugs such as danazol capsules (isoxazolic derivative of a synthetic steroids,17 αethinyl testosterone) , some women treated with zoladex (goserelin acetate implant) ,and some women treated with oral contraceptive pills as seen in table 4. iraqi j pharm sci, vol.25(1) 2016 effect of metformin treatment in women with endometriosis 32 table (3): patient’s characteristics of women with endometriosis. p value (control versus study group) study group control group patient’s characteristics 0.5404 32.93±7.488 34.69±7.476 age in years (mean ± s.d) 0.9415 4.100±1.955 4.045±1.387 duration of infertility in years (mean ± s.d). 0.02 12/15(80.00% ) 6/15(40.00% ) dysmenorrhea 0.14 9/15(60%) 5/15(33.33%) pelvic pain 0.19 2/15(13.33%) 5/15(33.33%) menorrhagia table (4): number of patients treated with danazol , goserelin acetate (zoledax )r , and oral contraceptive for control groups. drugs no. of patients danazol (4)26.66% (goserelin acetate) zoledax r (10)66.66% oral contraceptive pill (1)6.66% b. study group fifteen patients with stages (i-iv) endometriosis diagnosed by laparoscopy or laprotomy and were complaining of one or more symptoms such as pelvic pain or menorrhagia and received metformin at 500 mg every 8 hour in addition to classical drugs as shown in table -5. table (5): numbers of patients treated with danazol, zoledax r , and oral contraceptive pills plus metformin(study group). drugs no. of patients danazol and metformin r (6)40% zoledax r and metformin r (5)33.33% oral contraceptive and metformin r (4)26.66% methods sampling blood samples were obtained for the estimation of the cytokines as (il-8 (21) and tnf-α (22) )in addition to estradiol e2 (23 levels at the start of the study from the fasting patients in morning and also at the follow up visits after three months.estimation of the cytokines il-8, tnf-α level was performed using a commercially available enzyme -linked immunosorbent assay (elisa) kits. estimation of the estradiol e2 was performed using radioimmunoassay. five ml of venous blood was withdrawn aseptically into dry plastic tubes. then the collected blood samples were centrifuged at 3000g for 15 min and the separated serum were stored at −20 °c until time of assay. statistical analysis the collected data were tabulated, compared and proper statistical analyses were performed. a t-test is any statistical hypothesis test in which the test statistic follows a student's t distribution if the null hypothesis is supported. it can be used to determine if two sets of data are significantly different from each other. .when the scaling term is unknown and is replaced by an estimate based on the data, the test statistic (under certain conditions) follows a student's t distribution (24) .frequency, mean and standard deviation (sd) were used to describe data .p value was considered positive and significant if less than 0.05. results and discussion the study sample included (30) patients comprising of 15 female as control groups and 15 female as study groups; endometriosis was diagnosed by laparotomy or laparoscopy as shown in table 6. iraqi j pharm sci, vol.25(1) 2016 effect of metformin treatment in women with endometriosis 33 table(6): endometriosis diagnosis by laparotomy or laparoscopy diagnostic routes causes no. of patients laparoscopy pelvic pain 3 laparoscopy treated some causes of infertility 7 laparotomy ovarian cyst 8 laparotomy acute abdominal pain 6 follow up endometriosis 6 endometriosis classified into stages according to the revised criteria of the american society of reproductive medicine (asrm) (13) table -7 represents the % of stages in endomtrosis were i, ii, iii and iv in 3(10%), 14(46%), 8(27%) and 5(17%) patients. endometriosis can occur in teenagers and adult women of all ages, but most typically it occurs in women ages 23 – 45 for control groups 34.69±7.476.and for study groups are 32.93±7.488 as shown in table-3. table(7): stages of endometriosis. stages of endometriosis frequency minimal (i) 3/30 (10%) mild (ii) 14/30(46%) moderate (iii) 8/30(27%) sever (iv) 5/30(17%) dysmenorrhoea is the most common reported symptom in endometriosis, it occurs in six women in control groups (34.69±7.476.and twelve women in study groups are 32.93±7.488 as shown in table -3. endometriosis is estimated to occur in 5/15(33.33%) of women presenting with pelvic pain for control groups , 9/15(60%) in study groups also showed that 5/15(33.33%) of women presenting menorrhagia in control groups and 2/15(13.33%) in study groups as shown table-3. the serum levels of tnfα , il-8 and e2 in the control and study groups levels in the endometriosis before treatment and after 3 months of treatment. as shown in table -8. table (8): tumor necrosis factor-alpha (tnfα), interlukin-8(il-8) and estradiol (e2) levels in the control and study groups: control group(mean ± s.d) study group (mean ± s.d) p value before(1 st visit) after(2 nd visit) before(1 st visit) after(2 nd visit) tnf-α (pg/ml) 83.90±9.23 78.05±8.88 89.90±11.83 68.39±10.91 p 0.15 0.004 p1 0.23 p2 0.05 il-8 (pg/ml) 41.31±6.376 39.86±6.239 52.70±8.646 44.54 ± 13.94 p 0.09 0.04 p1 0.16 p2 0.01 e2 (pg/ml) 133.2±32.33 114.8±30.43 89.90±11.83 68.39±10.91 p 0.37 0.25 p1 0.92 p2 0.34 p< 0.05 is significant &p> 0.05 is not significant. p: levels at start of the study versus after 3 months. p1: levels at 1start of the study for control versus study group. p2: levels after 3 months of the study for control versus study group. http://www.mefsjournal.org/article/s1110-5690(12)00095-7/fulltext#t0015 iraqi j pharm sci, vol.25(1) 2016 effect of metformin treatment in women with endometriosis 34 in the present study there was a significant reduction in the number of cases with dysmenorrhea and pelvic pain (p< 0.05) after three months of metformin therapy. as seen in table -9. metformin significantly decreased pain and the level of c-reactive protein after 6 months of treatment in patient with endometriosis associated chronic pelvic pain (25) rannou f et al 2007 , study demonstrated that il-6, may be considered as a major regulator of c-reactive protein gene (25) tumor necrosis factor alpha (26) , nuclear factor ĸƀ (27) and transforming growth factor betaone (tgfβ-1) (28) .the inflammation associated with endometriosis, through increased levels of peritoneal fluid vascular endometriosis growth factor (vegf), may promote angiogenesis for progressive growth of endometriosis (29) . table(9): changes in clinical symptoms in endometriosis control and study group before and after three months of treatment . p value study group [n(%)] control group [n(%)] after2 nd visit before1 st visit after2 nd visit before1 st visit after2 nd visit before1 st visit 0.02 0.02 12/15 80.00% 0.02 6/15 40 % 6/15 40.00% 0.46 7/13 53.8% dysmenorrhea p 0.61 0.19 2/15 13.33% 0.09 6/15 40 % 5/15 33.33% 0.88 4/13 30.76% menorrhagia p 0.51 0.14 9/15 60% 0.02 3/15 20% 5/15 33.33% 0.88 4/13 30.76% pelvic pain p n: number of positive cases/total number. thus , endometriosis had a direct effect on adhesion formation (30) . moravek et al (2009) ,provided a preliminary data about the effectiveness of rosiglitazone, an insulin sensitizer, in treating endometriosis -related pain in six patients and concluded that it was effective and promising for usage in endometriosis patients desiring the chance to conceive (31) . the result of this study showed that the level of il-8 was significantly decreased after 3 months of metformin therapy (p< 0.05). il-8 level was significantly higher in the endometriosis groups than in the control groups (p=0.01). table 8. arici et al 1998. reported" that il-8 is produced in the human endometrium in vivo, especially in glandular cells" (32) .il-8 raise the proliferation of endometrial stromal cells as a potential autocrine growth factor (36) . a previous study y. takemura et al (33) has shown that metformin in non decidualized ovarian endometriotic stromal cells could reduce il-1b-induced il-8 production, aromatase activation and proliferation. so far only a reduction of aromatase activation and a reduction of proliferation has been demonstrated in vitro in ovarian endometriotic stromal cells after treatment with metformin (y. takemura et al 2007) (33) .the results of iwabe et al (8) clearly demonstrated that "il-8 is a growthpromoting factor in normal endometrium as well as in endometriotic cells". the study of baracz et al 2012. (34) excluded the primary role of il-8 in the formation of endometriosis -associated peritoneal adhesions and confirmed the role of cytokines in the pathogenesis and progression of endometriosis.the result of the present study showed that there was significant reduction in tnf-α levels in women with endometriosis after 3 months of metformin treatment.the result of iwabe et al 2001 (8) ,proved that it through induction of il-8 gene and protein expression in endometriotic stromal cells ,tnf-α stimulated proliferation of endometriotic stromal cells in a dose dependent fashion. the addition of antitnf-α antibody or anti-il-8 antibody, lead to neutralized the proliferated effect of the endometriotic stromal cells, and the stimulatory effects of tnf-α (34) bedaiwy et al (2002) (35) demonstrated that serum tnf-α had a high degree of sensitivity and specificity so it could be surprisingly used to differentiate between patients with and without endometriosis. the result of the current study showed that there was no significant reduction in level iraqi j pharm sci, vol.25(1) 2016 effect of metformin treatment in women with endometriosis 35 of estradiol e2 in women with endomterosis treated with metformin for periods of 3 months and this disagree with the study of mansfield et al 2003 (36) . also table 8 showed that the significant reduction in estradiol e2 ,fsh ,insulin -stimulated progesterone production in granulosa cells thus, metformin may be effective in treatment endometriosis through suppression both ovarian and local production of estrogen (36) . so the result from this study about serum estradiol e2 may be attributed to the small sample size and short time of study or the dose of metformin was not enough to lower of estradiol e2 level. conclusion from the results of this study, it was found that metformin may be well tolerated treatment for endometriosis that relieved pain and reduced menstrual disorders.serum levels of the inflammatory markers (il-8 and tnf-α) are decreased in study groups with metformin after 3 months indicating that metformin may be have anti-inflammatory effect. references 1. u.s. department of health and human services, office of women’s health 2009. 2. gynaecology treatment adelaide obstetrics & fertility 2011 3. j. gilabert-estelles, l. a. ramon, f. espa˜ na, j. gilabert, r. castello, and a. estelles, “expression of fibrinolytic components in endometriosis,”pathophysiology of haemostasis and throm-bosis, 2006;35,( 1-2):136–140. 4. morin-papunen l, rautio k, ruokonen a, hedberg p, puukka m, tap-anainen j smetformin reduces serum c-reactive protein levels in women with polycystic ovary syndrome. j clin endocrinol metab 2003, 88:4649 – 4654. 5. h.du and h. s .taylor , “reviews :stemcells and female reproduction ,”reproductive sciences,vol.16,no.2, 2009. pp.126–139. 6. samer sourial, nicola tempest, and dharani k. hapangama international journal of reproductive medicine volume 2014, article id p.179515, 9 . 7. said tm, agarwal a, falcone t, sharma rk, bedaiwy ma li l.nfliximab may reverse the toxic effects induced by tumor necrosis factor alpha in human spermatozoa: an in vitro model. fertil steril. 2005;83:1665-73. 8. iwabe t, harada t, tsudo t, nagano y, yoshida s,tanikawa m, et al. tumor necrosis factor-alpha promotes proliferation of endometriotic stromal cells by inducing nterleukin-8 gene and protein expression. j clin endocrinol metab. 2000;85:824-9. 9. practice committee of the asrm. endometriosis and infertility . fertil steril.2006; 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of clinical endocrinology and metabolism. 1998;83:1201-5. 33. y. takemura, y. osuga, o. yoshino, a. hasegawa, t. hirata, y. hirota, et al. metformin suppresses interleukin (il)-1βinduced il-8 production, aromatase activation, and proliferation of endometriotic stromal cells clin endocrinol metab. 2007;92 (8): 3213– 3218 . 34. barcz e, milewski l, dziunycz p, kaminski p, ploski r, malejczyk j. peritoneal cytokines and adhesion formation in endometriosis: an inverse association with vascular endothelial growth factor concentration. fertility and sterility. 2012;97:1380-6 e1. 35. bedaiwy ma, falcone t, sharma rk, goldberg jm, attaran m, nelson dr, et al. prediction of endometriosis with serum and peritoneal fluid markers: a prospective controlled trial. hum reprod. 2002;17:426-31. 36. mansfield r, galea r, brincat m, hole d, mason h. metformin has direct effects on human ovarian steroidogenesis.fertil steril.2003;79:956– 96. iraqi j pharm sci, vol.30(2) 2021 cressa cretica: a review doi: https://doi.org/10.31351/vol30iss2pp31-40 31 cressa cretica pharmacognosy, and pharmacology (a review ) noor s. jaafar*,1, iman s. jaafar** and zainab s. noori* *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmaceutics, college of pharmacy, mustansiriyah university, baghdad, iraq. abstract cressa cretica (shuwwayl) is a halophytic that belongs to convolvulaceae, naturally grown in the middle east including iraq. traditionally the plant is used as a paste for sore treatment, also it is used for fever, jaundice, and other illness. regarding nonclinical use it is used as goat, sheep, and camel feed also as an oil source. flavonoids including quercetin, kamepferol, apigenin, and their glycosides, phenolic acid as chlorogenic acid, and phytosterols mainly β–sitosterol were the most important phytochemicals that were detected in this halophyte. crude ethanolic, methanolic extracts and ethyl acetate fraction of the areal parts were used in clinical studies and demonstrated various effects as hepatoprotective, cytotoxic, and genotoxic effect. in molecular docking studies, 3,5-dicaffeoylquinic acid showed antiviral effect vs sars-cov-2 (sever acute respiratory syndrome corona virus2). the purpose of this review was to clarify and discuss all aspects regarding cressa cretica. keywords: cressa cretica, halophyte, quercetin, 3,5-dicaffeoylquinic acid . cressa cretica لنبات خصائص العقاقيرية والعالجيةال ( مراجعة) *صفاء نوريزينب و **، ايمان صباح جعفر1،*نور صباح جعفر كلية الصيدلة، جامعة بغداد، بغداد،العراق فرع العقاقير والنباتات الطبية ، * ، بغداد،العراقالمستنصرية جامعة الكلية الصيدلة، فرع الصيدالنيات،** الخالصة العراق. تقليديا ، يتم استخدام في الشرق األوسط بما في ذلك ، ينمو بشكل طبيعي حعائلة نجمة الصبا هي نبات ملحي ينتمي إلى الشويل للماعز النبات كعجينة لعالج القرحة ، كما أنه يستخدم للحمى واليرقان وأمراض أخرى. فيما يتعلق باالستخدام غير السريري ، يتم استخدامه كعلف يدات ، وحمض الفينول مثل سبيجينين ، والجليكوأيًضا كمصدر للزيت. الفالفونويد بما في ذلك الكويرسيتين ، والكاميبفيرول ، واألو واألغنام واإلبل سيتوستيرول كانت أهم المواد الكيميائية النباتية التي تم اكتشافها في هذه النبات الملحي. تم –بشكل رئيسي وحمض الكلوروجينيك ، والفيتوستيرول السريرية وأظهرت تأثيرات مختلفة مثل التأثيرات الواقية للكبد في الدراسات وااليثل اسيتيت الخام استخدام المستخلصات اإليثانولية والميثانولية -sars تأثير مضاد للفيروسات مقابل dicaffeoylquinic-3،5والسمية للخاليا والسمية الجينية. في دراسات االلتحام الجزيئي ، أظهر حمض cov-2. بـكان الغرض من هذه المراجعة هو توضيح ومناقشة جميع الجوانب المتعلقة cressa cretica. الكلمات المفتاحية : introduction cressa cretica (convolvulaceae)(1) is obligate halophytes which in demand for saline soil for its growth and development (2)c. cretica is a small dwarf branched shrub or subshrub typically has straight stems with white‐haired, green leaves (3). this subshrub has various activities and used for management of diabetes, asthma, constipation, exerts anthelmintic, and other activities which are related to the presence of different classes of photochemical as flavonoids, sterols and others(4). the taxonomy of cressa cretica is listed below. kingdom plantae phylum angiosperms class magnoliatae subclass asteridae family convolvulaceae tribe zribe cresseae genus cressa species cretica (5-7) in arabic countries cressa cretica is called shuwwayl, molleih (8, 9).rudranti in hindi, rudravanti in bengali (10), bukkan (11). in iran, alaf mourcheh (12). 1corresponding author e-mail: nooraldahan201@gmail.com received:9/5/2021 accepted:11 /7 /2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp31-40 iraqi j pharm sci, vol.30(2) 2021 cressa cretica: a review 32 cressa cretica is grown naturally in the mediterranean, in the middle east including iraq, egypt, syria and, qatar. australia, south america, central and parts of south-east asia. also in south to northern and central africa (11, 13-16) . it is dominant near the arabian sea coastal region(17). this shrub is perennial, hairy in nature, erect from 35-40 cm height. the root is perennial woody, horizontal, geminate, has lateral branches growing upward to give rise to above-ground parts (18-20).the short-lived stems are many, erect initially and then become decumbent, these stems branched at the base (20, 21). the leaves are highly condensed, hairy and stalkless (20-22). typically the leaves are obovate lanceolate to scale-like. flowers are solitary, ovate to obovate imbricate, white or pink in color found in spicate to head like panicles at the apex of the branchlet (5, 20). the one-seeded fruits are ovoid capsules pointed at the tip(5, 21). the seeds are brown, glabrous in nature, and shiny to reticulate (5). cressa criteca grow along the coastal zone in the sandy area and marshes in combination with other halophytes. its life cycle carries on during summer. c.cretica begins to shoot or develop with june onset. meanwhile from the june ending to the august ending flowering and fruiting period is observed(13, 23) in another reference the flowering and fruiting period is all over the year(21). the plant gradually shrivels in september and october and producing seeds. the seeds stay dormant in the immerse soil till the next year june (18, 23, 24). the germination of seeds occurs in spring(18). salinity and humidity content influence the allocation and structure of cressa cretica communities, while soil nutrients deficiency as potassium and nitrogen greatly prohibit plant growth and reproduction(25). cressa crética appears to be one of the most tolerant salt species identified so far(26) tolerating up to 800 mm naci(27). the whole plant and its parts is shown in figure 1 . (a) (b) (c) (d) figure 1. cressa cretica (a): whole plant, (b): stem and leaves, c: upper flowering part, (d): seeds (25, 28). ethno pharmacological action of cressa cretica plants are a rich source of secondary metabolites and have been used since ancient civilization for the management of various illnesses. c. cretica is a well-known medicinal halophyte. in iran, the whole aerial parts of the plant are used topically as liniment intended to produce antibacterial and antifungal effects(12). decoction prepared from the whole plant is anticipated to exert an antioxidant, antiviral and anti-inflammatory effect, while the paste is used topically for sore treatment(29). regarding the folklore utility of this halophyte; it exert expectorant, stomachic, anthelmintic, aphrodisiac actions, and for a urinary problem management. it is used for diabetes, asthma, ulcer, leprosy, constipation, and has certain properties to enrich blood(29, 30). the whole plant is squelched in water with little black pepper and candy and the resultant blend is used to alleviate chronic fever and jaundice (31). in sudan, dry leaves were triturated with sugar and used as an emetic, also aerial parts extracts or menstrum taken as a tonic. a decoction of leaves of vitex doniana with cressa stems used topically for skin eruption(32). iraqi j pharm sci, vol.30(2) 2021 cressa cretica: a review 33 non clinical uses of c. cretica this halophyte is considered suitable biodiesel due to high-level seeds oil contents besides good quality engine parameters. also, it is used as camel, goat, and sheep feed(27, 32). fruits of cressa are considered as probable supply of edible oil and safe for human use since it is free from unwanted or undesirable contents (33). chemical constituents phytochemical alkaloids, phenolic acids coumarins, tannins, and sterols were identified in this plant(16). others like gum, amino acids, proteins, and mucilage were identified in another study(20). different flavonoids were isolated from egyptian aerial parts of c. cretica using column chromatography including; rutin, quercetin, quercetin-3-o-b-glucoside), (kaempferol-3-o-bglucoside) and [kampferol-3-o-a-rhamnosyl(1-6)b-o-glucoside](16). apigenin, apigenin-7glucoside, rutin, quercetin and kaempferol were also detected by hplc in ethyl acetate aerial parts extract of egyptian plant, with rutin being the main flavonoid in this extract while ethanolic extract contains the same flavonoid except apigenin and apigenin-7-glucoside is the main flavonoid in this extract(34). the chemical structures of some flavonoids are shown in figure 2. kaempferol 3-o-βglucoside (astragalin) was also isolated as the main glycoside in ethyl acetate fraction of ethanolic aerial parts extract in iraqi plant(36). flavanol as catechin was also detected by hplc in both ethyl acetate and ethanolic aerial part extract, and it is the major compound in ethyl acetate extract(34). phenolic acids as 3,5 dicaffeoylquinic acid and chlorogenic acids were identified in the plant(38), protocatechuic (3,4-dihydroxybenzoic acid), p-, gentisic acid (2,5-dihydroxy benzoic), chlorogenic , caffeic acid, vanillic acid,ferulic acid, sinapic (3,5-dimethoxy-4hydroxycinnamic acid), p-coumaric, gallic acid, and cinnamic acid were detected by hplc in both ethanol and ethyl acetate extract of aerial parts. chlorogenic acid is the major phenolic acid in ethanolic extract. hydroxybenzoic acid, and gallic acid not detected in ethyl acetate and ethanolic extract respectively(34). chemical structures of phenolic acids (3,5 dicaffeoylquinic acid and chlorogenic acid) are shown in figure 3. figure 2. chemical structures of some flavonoids (35-37). figure 3. chemical structures of phenolic acids ( 3,5 dicaffeoylquinic acid and chlorogenic acid) (39, 40). iraqi j pharm sci, vol.30(2) 2021 cressa cretica: a review 34 lignin as syringaresinol and syringaresinol-h-dglucoside and coumarin as scopoletin(38), umbelliferon and isopimpinellin (furanocoumarin) were detected in the aerial parts(41). the chemical structures of some coumarines (scopoletin and umbelliferone) and lignin are demonstrated in figure 4 and 5 respectively. figure 4. chemical structure of some coumarines (scopoletin and umbelliferon) (42,43) figure 5. chemical structure of syringaresinol (lignin) (44). using gc/ms phytosterols as α–sitosterol, β sitosterol, pentacyclic triterpenoid; β-amyrin, acid, and the terpenoids acyclic diterpene alcohol and phytol were detected in the unsaponifiable matter of the aerial parts (34) , while campsterol, ethyl isoallocholate, and cholestan-3-ol,2-methylene,(3β,5α)were detected in the methanolic extract of leaves(45). the plant also contains ursolic acid(32) . β –sitosterol was the main sterol identified in the oil obtained from aerial parts, other sterols as stigmasterol, .δ⁷-avenasterol and δ⁵avenasterol was detected in equal amounts (46). stigmasterol -3o-β-d-glucoside was also identified (32). the chemical structure of β –sitosterol (phytosterol) and ursolic acid (acidic saponin) are demonstrated in figure 6 and 7 respectively. figure 6. chemical structure of β – sitosterol(phytosterol) (47). figure 7. chemical structure of ursolic acid (pentacyclic triterpenoids)(48). concerning fatty acids the following fatty acids were identified ; octanoic acid; caprylic acid ,14methyl-pentadecanoic acid, 14-methylhexadecanoic acid ,9,12-octadecadienoic acid (linoleic acid) ,5-octadecenoic acid , octadecanoic acid (stearic acid) ,nonadecanoic acid , eicosanoic acid ; arachidic acid , heneicosanoic acid, docosanoic acid , behenic acid , tricosanoic acid ,tetrecosanoic acid ,lignoceric acid , pentacosanoic acid ,hexacosanoic acid, heptacosanoic acid , octacosanoic acid . these acids are detected as fatty acids methyl ester by gc/ms in aerial parts(46). 14methyl-pentadecanoic acid is the major detected acid (34). in the fixed oil obtained from egyptian aerial parts of cressa the majority of the detected fatty acids were unsaturated. palmitic acid is the main saturated fatty acid, while oleic acid and linoleic acid were the major unsaturated fatty acids. regarding hydrocarbons, thirteen hydrocarbons were identified by gc/ms in the unsaponifiable matter, hexadecene, octadecene docosene, tricosane, eicosene, heneicosene tetracosene, tetracosane, pentacosane,hexacosane ,heptacosane , octacosane , nonacosane. heptacosane and nonacosane represent the majority of the identified hydrocarbons(46). 5-methyl-6-phenyltetrahydro1,3oxazine-2thione;adenosine , 4'-methylaminoformy -4'deshydroxymethyl-n-; oxyephedrine; 6carboxypterin; desulphosinigrin; cyclopentanemethylamine 2-isopropylidene n,n,5-trimethyl-; 2,7-diphenyl-1,6dioxopyridazino [4,5:2',3']pyrrolo[4',5'-d]pyridin; . iraqi j pharm sci, vol.30(2) 2021 cressa cretica: a review 35 paromomycin; strychane, 1-acetyl-20α-hydroxy-16 -methylene; 3',8,8'-trimethoxy-3-piperidyl-2 2'binaphthalene-1,1',4,4'-tetra; were the nitrogenous compound which were detected in methanolic leaves extract of cressa using gc/ms (45). 2-isopropyl-4-(1-methyl-dodeca-2,4-dienyloxy)benzene-1,3,5-triol and 11-methyl-dodeca2,4,6,8,10-pentenoic acid 2,3-dihydroxy-5methyl-phenyl ester were isolated by column chromatography from n.butanol fraction of methanolic extract c. cretica and identified by spectral analysis (38). cressa tetracosanoate, cressa tetratriacontanoic acid, creticane cressa naphthacenone and cressa triacontanone were detected in c. cretica(49). minerals as zinc, copper, and manganese were identified by atomic absorption spectroscopy using the dry ashing digestion method(34). aluminum, calcium, iron , magnesium, phosphorous, and sulphur in addition to previously mentioned minerals were detected in c. cretica by atomic absorption spectroscopy and ultraviolet technique(50). pharmacological actions of cressa cretica 1. memory enhancement alzheimer's is a neurodegenerative disease that is related to aging. memory loss and disturbed cognitive function are the main disease manifestations(51) . in the course of in normal aging process, there is a noticeable reduction in learning ability and memory. khare et al (2014) investigated the effect of ethanolic extract of c. cretica on memory and learning in mice. memory loss was produced by scopolamine. orally administered doses of 200 and 400mg/kg were given for 28 days before scopolamine. the dose of 400mg/kg markedly improved the memory and learning abilities through a reduction in acetylcholine esterase activity, the antioxidant action of the plant extract plus remarkable flavonoids content. the nootropic effect of this dose (400mg/kg orally) was analogous to that of piracetam(200mg/kg intraperitoneally ) which was used as a standard or reference nootropic drug(9). 2. hepatoprotective effect c. cretica is known to mediate a variety of effects, hepatoprotective among these. p. thirunavukkarasu et al (2014) examined the in-vitro hepatoprotective effect of different c.cretica fractions that were isolated based on polarity (petroleum ether, ethyl acetate, n.butanol, and aqueous fraction). hepatic damage was induced by ccl4, this damage manifested through an increase in hepatic transaminases, bilirubin, and alkaline phosphatase level. ccl4 causes hepatic necrosis and increased cell permeability. all the previously mentioned fractions exhibit hepatoprotective effect especially n. butanol fraction being the best protective effect. the antioxidant effect, free radical scavenging activity, and restoration on increment in glutathione level involved in the hepatoprotective effect of cressa cretica (41). el-alfy et al (2019) showed the hepatoprotective effect of this halophyte through in vivo study on mice and rats. ccl4 was used to induce a toxic effect on the liver. the used fractions were petroleum ether, ethyl acetate, and 70% ethanolic extract. the dose used for each fraction was 500mg/kg /day for four weeks. animals are divided into nine groups. all these fractions demonstrated hepatoprotective effect shown through perfection in liver actions, liver enzymatic and non-enzymatic antioxidant status, malondialdehyde and nitric oxide and serum transaminases, and liver histopathology. the examined fractions were safe at the used dose and up to 3gm/kg. according to this study the hepatoprotective effect in polar fraction attributed to the presence of flavonoids and phenolic acids, while in petroleum ether fraction the effect attributed to phytosterols as α–sitosterol, β -sitosterol, and to the terpenoid β-amyrin in addition to the presence of trace elements as zn, cu, and mn which play an important role for antioxidant enzymatic reaction(34). 3. antimicrobial effect previous studies demonstrated the antimicrobial effect of cressa extract. mandeel et al (2005) showed the powerful inhibitory effect of ethanolic c. cretica extract against penicillium citrinum followed by candida albicans using agar diffusion method(52). omran et al (2019) prove in a study the antimicrobial effect of three plants including c.cretica, origanum vulgare l. rosmarinus officinalis l. the tested microbes were staphylococcus aureus s. aureus, eschrichia coli e. coli and candida albicans c. albican. crude alkaloidal extracts of these plant were used. c. cretica showed the largest inhibitory effect on microbes than rosemarinus officinalis and origanum. vulgare, the inhibitory effect is proportional to the concentration. the most affected microbe was s. aureus , then e. coli, and c. albicans(53). pirzada et al (2009) evaluated the effect of chloroform, ethyl acetate, ethanol, methanol, and aqueous fractions of c.cretica methanolic extract against aspergillus niger, paecilomyces varioti, aspergillus flavus, trichophyton rubrum and microsporum gypseum. the antifungal effect displayed by all fractions, but chloroform and aqueous fractions were the most effective. trichophyton rubrum was the most affected fungi by c.cretica extracts(50). sunita et al (2012) evaluated the antimicrobial effect of different c. cretica fractions (hexane, ethyl acetate, and methanol) using the agar disk diffusion method. gentamicin, tetracycline were assigned as positive controls for bacteria, and fluconazole and ketoconazole were assigned as a positive control for fungi. among the tested fractions ethyl acetate extract had a pronounced effect than the other iraqi j pharm sci, vol.30(2) 2021 cressa cretica: a review 36 fractions. e. coli and k.pneumoniae were the most affected pathogens. the antibacterial effect of ethyl acetate extract was more noticeable than the antifungal effect. fusarium oxysporum was the least affected fungi as compared to aspergillus fumigates, aspergillus niger. candida tropicalis and candida albicans fill in between(54). 4. antidiabetic effect traditionally c. cretica was used for diabetes mellitus management. verma et al (2014) showed the antidiabetic effect of the methanolic extract of cressa in streptozotocin induced diabetic rats. orally administered dose of 100mg/kg was given for fifteen days. four groups were assigned, group one received distill water, group two diabetic control rats received streptozotocin, group three diabetic rats received methanolic cressa extract, and group four diabetic rats received glibenclamide. at the end of the study period in cressa extract-treated group, the blood glucose concentration markedly reduced to the normal level and the bodyweight recovered probably due to decreasing muscle wasting and regulation of lipid metabolism(55) . kumari et al (2016) proved the anti-hyperglycemic action of methanolic extract of c.cretica in alloxaninduced diabetic wistar rats. doses of 200 mg/kg and 400 mg/kg of extract were given for 28 days. the two groups receiving these doses demonstrated a considerable lowering in glucose concentration, lipid profile, serum transaminases as compared to the control group. at the same time, there was an increase in insulin, glutathione, and high-density lipoprotein(56). rani et al (2020) based on molecular docking study proven the antidiabetic effect of two compounds(2isopropyl-4-(1-methyl-dodeca-2,4-dienyloxy)benzene-1,3,5-triol and 11-methyl-dodeca2,4,6,8,10-pentenoic acid 2,3-dihydroxy-5methyl-phenyl ester ) isolated from c. cretica (49). rani et al (2020) demonstrated in a study the effectiveness of aqueous extract of whole c.cretica plant as antidiabetic preparation in streptozotocininduced diabetic rats. lab data and histopathological studies were the follow-up criteria. doses 200mg/kg, 400mg/kg of the extract and glibenclamide treated groups display a noticeable reduction in blood glucose level. triglyceride, cholesterol & ldl levels were decreased during and at end of the study. the dose 400mg/kg showed almost normal pancreatic cell histology as compared to 200mg/kg dose as there was some damage in these cells (57). 5. cytotoxic effect c. cretica exhibits a cytotoxic effect. mutlag et al (2017) proclaim the cytotoxic effect of ethyl acetate extract of c. cretica through in vivo study on albino swiss mice bone marrow and spleen cells. four groups were categorized each contains 6 animals. group one was the control group received dimethyl sulfoxide (dmso), group two received a single dose of methotrexate (20 mg), group three and four received 100mg/kg and 200mg/kg of ethyl acetate extract respectively for seven days. ethyl acetate extract at both doses results in a remarkable decline in the mitotic index in both cell types (bone marrow and spleen) as compared to (dmso) negative control group. mitotic index is a vital predictive factor that predicts overall survival in addition to the response to chemotherapeutic agents in most types of cancer. ethyl acetate extract effects on mitotic index were more pronounced than that of methotrexate(58). fawzi et al (2019) proved the in vitro cytotoxic effect of ethanolic and ethyl acetate fractions of c. cretica. different concentrations of both extracts were used vs mcf-7 cell line (breast carcinoma cells) and skov-3 cell line (ovarian carcinoma cells). both extracts reveled a considerable cytotoxic effect on both cell lines in a dose-dependent manner(28). 6. genotoxic effect mutlag et al (2017) showed the genotoxic effect of two different doses of ethyl acetate extract of c.cretica . four groups were assigned each one contains six mice. group one was the control group received dimethylsulfoxide(negative control) group two received a single dose of methotrexate (20 mg) (positive control), group three and four received 100mg/kg and 200mg/kg of ethyl acetate extract respectively for seven days. after treatment duration, 1 mg/kg of colchicine was given intraperitoneally for each animal. two hours later the animals were sacrificed and samples were aspirated from femur bone and spleen cells for genotoxic analysis. both examined doses demonstrated a noticeable increment in total chromosomal aberration and chromatid break in bone marrow cells when compared to the negative control group. at the same time showed a significant reduction in total chromosomal aberration and chromatid break in bone marrow cells as compared to the methotrexate receiving group. so c. cretica ethyl acetate fraction has a genotoxic effect in a dose-dependent manner as compared to the negative control. the genotoxicity might be due to the presence of coumarin type of phytochemicals(59). 7. antipyretic, and antinociceptive effects one of the earliest or traditional uses of c. cretica was for fever treatment (31) abdallah et al (2017) demonstrated the antipyretic effect of aqueous aerial parts extract .subcutaneous injection of brewerʼs yeast was used to induce fever in mice which were pronounced 18 hours after injection. paracetamol 150mg/kg was the standard antipyretic drug. 50mg/kg and 100mg/kg of the aqueous extract were given, these doses demonstrated a significant reduction in rectal temperature as compared to iraqi j pharm sci, vol.30(2) 2021 cressa cretica: a review 37 control after 60 and 120minutes of pyrexia induction, but less effective than paracetamol. hot plate test and acetic acid-induced writhing test were used to evaluate the antinociceptive activity of the aqueous cressa extract in mice. the used doses 50mg/kg and 100mg/kg of the aqueous extract showed a valuable reduction in acetic acid-induced abdominal cramps (43% and 48% )in comparison to the control group. for the hot plate test, a significant rise in latency time (66% and 78% ) was observed for both doses(50mg/kg and 100mg/kg) respectively as compared to the control group. c. cretica showed writhing inhibition in acetic acidinduced peripheral nociception of 43% and 48% at doses of 50 and 100 mg/kg, respectively. the same doses increased latency time in a hot plate model of central analgesia by 66% and 78% compared to the control group respectively(60). 8. c. cretica and covid 19 previous studies reported the antiviral effect of c.cretica(58). shah et al.(2021) highlighted the antiviral effect of certain active phytochemicals against the sars-cov-2 virus using molecular docking studies(38). scopoletin, 3,5dicaffeoylquinic acid, quercetin, and syringaresinol from cressa cretica were used in the study. remdesivir was used as a standard drug. the binding potential of these active compounds to serine-like protease of sars-cov-2 was examined followed by an investigation of the vast conformational space of protein–ligand complexes by molecular dynamics simulations. both quercetin and 3,5-dicaffeoylquinic acid demonstrated high docking scores, and through hydrogen bonding and hydrophobic interaction combine with the active site of serine protease which is an important enzyme required for viral replication. 3,5-dicaffeoylquinic acid displayed the best interaction capacity to serinelike protease suggesting this phenolic acid as a potential antiviral agent vs sars-cov-2(38). 13. antidepressant effect herbal medicine is generally utilized to alleviate mental disorders including depression by a variety of pathways(61). khare et al (2016) proved the antidepressant effect of ethanolic extract of c. cretica using forced swim test and tail suspension test. five equal groups of swiss albino mice each group contains six animals. group one received 1ml/100gm of polyethylene glycol, group two and three received 200,400 mg/kg orally of ethanolic extract respectively, and group four and five (positive control) received the antidepressant fluoxetine at a dose of 20mg/kg and imipramine at a dose of 15mg/kg respectively for seven and fourteen days. the immobility period was measured sixty minutes after the last dose. both doses 200 & 400 mg/kg of c. cretica produced considerable antidepressant indicated by a decrease in immobility times of mice in forced swim test and tail suspension test(62). 14. antitussive effect indigenous people of india widely used c. cretica for cough and asthma management. sunita et al show the antitussive effect of methanolic extract (2.5%w/v and 5% w/v) on male guinea pigs using the nebulized citric acid solution and also so2 gas. each group contains 5 animals, 0.1 g/ml of citric acid was aerosolized for 7min. the animals based on their group were exposed to normal saline (group one), 0.03gm/ml codeine solution (group two, positive control), meanwhile group three and four were aerosolized 2.5%w/v, 5%w/v)of c. cretica methanolic extract respectively for 7 minutes. after 10 minutes 0.1 g/ml of citric acid was aerosolized for 7min and the number of coughs was determined. both concentrations and codeine produced a marked antitussive effect as compared to the control group, and at the higher concentration, the antitussive effect being more pronounced. the antitussive effect was also evaluated by using so2 gas to induce cough in mice. c. cretica at a used concentration (100,200,400mg/kg) produced a noticeable antitussive effect in dose -dependent manner. the dose 400mg/kg showed the most powerful effect, but less than that of codeine (63). 15. effect on fertility medicinal plants either improve or deteriorate reproductive function(64) . gupta et al. (2006) demonstrated the inhibitory effect of methanolic c. critica extract on male albino rat fertility. an oral dose of 100mg/kg/day was given for 60 days for one group while the other received distilled water. in c. cretica treated group fertility was reduced by 100%. there was a considerable decline in the testis weight. epididymis, seminal vesicle, and ventral prostate shrink. primary and secondary spermatocytes plus spermatids numbers were reduced. sertoli cell and mature leydig cell counts markedly decrease. glycogen, cholesterol, protein, and sialic acid content of the testis was decline. seminal vesicle fructose also, epididymis sialic acid and protein were considerably decreased. totally all of these observations affects male rat testosterone level and spermatogenesis(65). 16. antioxidant effect pryianka et al (2015) approved the in-vitro antioxidant effect of methanolic c.cretica extract using 1,1-diphenyl-2-picryl hydrazyl (dpph) and hydrogen peroxide assay. the antioxidant effect is dose related, and it is attributed to the presence of polyphenolic compounds and other phytochemicals(66). afshari et al (2017) demonstrated the anti-oxidant effect of c. cretica leaf extract using 1,1-diphenyl2-picryl hydrazyl (dpph), ferric reduction activity potential and total antioxidant capacity assays. aqueous and 70% hydro-ethanolic extract were prepared using different interval for extraction ranging from 3-24 hours. the anti-oxidant effect of iraqi j pharm sci, vol.30(2) 2021 cressa cretica: a review 38 the extract was compared to the synthetic antioxidant bht (butylated hydroxytoluene). bht demonstrated the highest anti-oxidant effect then ethanolic extract after that aqueous extract. also the anti-oxidant effect increase with concentration increment(3). conclusion cressa cretica is an important medicinal plant that has numerous effects. this review represents an updated information specially those concerning the phytochemicals and pharmacological activates. 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sunita p, jha s, pattanayak s. in-vivo antitussive activity of cressa cretica linn. using cough model in rodents. pharmacognosy res. 2009;1(3):157. 64. al-snafi ae. medicinal plants affected male and female fertility (part 1)-a. isor. j. pharm. 2016;6(10): 11-26. 65. gupta r, kachhawa j, khushalani v, tanwar k, joshi y. effect of cressa cretica. methanol extract on testicular function of albino rats. pharm biol. 2006;44(5):382-8. 66. pryianka l, partap s, verma m, jha k. in vitro antioxidant activity of plant extract of cressa cretica. pharm lett. 2015;7:28-32. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.28(1) 2019 etodolac hydrazone derivatives with anti-inflammatory action doi: https://doi.org/10.31351/vol28iss1pp106-112 106 synthesis, characterization and preliminary anti-inflammatory evaluation of new etodolac derivatives omeed m. hassan* and susan w. sarsam*,1 *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq abstract three new hydrazone derivatives of etodolac were synthesized and evaluated for their anti-inflammatory activity by using egg white induced paw edema method. all the synthesized target compounds were characterized by chnmicroanalysis, ft-ir spectroscopy, and 1hnmr analysis. the synthesis of the target (p1-p3) compounds was accomplished following multistep reaction procedures. the synthesized target compounds were found to be active in reducing paw edema thickness and their anti-inflammatory effect was comparable to that of the standard (etodolac). keywords: etodolac hydrazone derivatives, anti-inflammatory, paw edema method. كمضادات لاللتهابلاليتودوالك جديدة مشتقاتوتقييم اولي ل وتشخيص تحضير 1*،سوزان وديع سرسم و *اميد محسن حسن . كلية الصيدلة، جامعة بغداد، بغداد، العراقفرع الكيمياء الصيدالنية، * الخالصة المستحث ببياض المخلب وذمة طريقة باستخدام لاللتهابات المضادة وتم تقييم فعاليتها ثالثة مشتقات هيدرازون جديدة لاليتودوالك تحضير تم ، مطياف األشعة تحت الحمراءوالهيدروجين والنيتروجينلكربون دقيق لذرات العناصر اال بالتحليل شخصت حضرةالم المركبات جميع. البيض وجدت المركبات المحضرة بان لها .باتباع طرق تفاعالت متعددة الخطوات p1-p3وتم تحضير المركبات .جهاز الرنين النووي المغناطيسيو .كفعالية في تقليل سمك ورم المخلب وان تأثيرها المضاد لاللتهابات كان مشابها لاليتودوال المخلب. لاللتهابات، طريقة وذمة مشتقات ايتودوالك هيدرازون، مضادة: كلمات مفتاحية introduction non-steroidal anti-inflammatory drugs (nsaids) are a heterogeneous group of compounds that are used for the treatment of various inflammatory conditions, pain and fever (1). the principal mechanism of action of nsaids involves the inhibition of cyclooxygenase (cox) enzyme also known as prostaglandin-endoperoxide synthase (ptgs). cox is the enzyme that catalyzes the synthesis of prostanoids (thromboxane and prostaglandins) from arachidonic acid(2). coxinhibitors are believed to act as an analgesic (3), antiinflammatory and antipyretic by decreasing prostaglandin synthesis(4). this decrease in prostaglandin synthesis is associated with the occurrence of several unwanted effects accompanied with the use of nsaids, especially gastrointestinal (gi) irritation and ulceration. additionally, several nsaids have a free carboxylic acid group (5); therefore, oral administration is linked with the side effects on the gastric system (6), which are due to direct gi irritation. nsaids can be categorized by the site of action into nonselective (cox) inhibitors which target cox i and cox ii and selective (cox) inhibitors that selectively target cox ii though decrease gastric side effect that comes with cox i inhibitors (7). etodolac (2-(1,8-diethyl-1,3,4,9tetrahydropyrano[3,4-b]indol-1-yl) acetic acid) (figure.1) which is a nsaid, is a derivative of pyrano indoleacetic acid . it is recommended for the treatment of pain and inflammation caused by osteoarthritis and rheumatoid arthritis. figure (1) chemical structure of etodolac the carboxylic acid group of nsaids can be replaced with other groups while these agents still exert a potent anti-inflammatory activity (8). 1corresponding author: e-mail: sarsam14@yahoo.com received: 27/11/2018 accepted: 2/2/2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp106-112 iraqi j pharm sci, vol.28(1) 2019 etodolac hydrazone derivatives with anti-inflammatory action 107 hydrazones are an important class for new drug development and an exceptional class of organic compounds in the schiff base family. they are synthesized by heating an appropriately substituted hydrazide with aldehydes or ketones in solvents like ethanol, methanol, tetrahydrofuran, butanol, with few drops of glacial acetic acid as a catalyst (9). hydrazone have a wide range of biological activities like anti-bacterial, antiviral, antidepressant, cardioprotective activities and anticancer activities (10), alongside with anti-inflammatory action. this study focused on the synthesis of new etodolac hydrazone derivatives and the evaluation of their anti-inflammatory activity. materials and methods etodolac and different aldehydes were bought from hyperchem / china, while other chemicals and solvents (ethanol, methanol, acetone, glacial acetic acid, concentrated h2so4, hydrazine hydrate, n-hexane, petroleum ether, and ethyl acetate) were bought from commercial sources and used without further purification. thin layer chromatography (tlc plates (254f) /merckgermany) was used to check reaction completion and purity of the product under uv light (254 nm).melting points were measured (uncorrected) by using the capillary tube on stuart smp30 electronic melting point apparatus. chn elemental microanalysis was carried out on euro ea elemental analyzer (italy). ir spectra were recorded on ftir-600 spectrophotometer (biotech engineering management, uk)) using kbr disc. 1hnmr spectra were recorded on bruker model ultra shield 300 mhz spectrophotometer using dmso-d6 as a solvent. synthesis of etodolac ester [methyl 2-(1,8-diethyl1,3,4,9-tetrahydropyrano [3,4-b] indol-1-yl) acetate] (compound a) a mixture of etodolac (0.021 moles, 6g) and methanol (40 ml) in a 250 ml round bottom flask was stirred till a clear solution is achieved. the obtained solution was cooled to 0oc by using an ice bath and 3 ml of concentrated sulfuric acid (h2so4) was added dropwise with continuous stirring, then, the mixture was set to reflux with stirring at 75c for 5 h. after completion of the reaction (monitored by tlc (acetone: petroleum ether 5:5)), the solution was cooled down to room temperature, then it was thrown over 75 ml of cold distilled water, followed by the addition of saturated sodium bicarbonate solution (5% w/v) in order to neutralize the excess acid. a yellowish precipitate of etodolac methyl ester was produced. the precipitate was collected by filtration, washed with chilled distilled water and dried, then recrystallized from ethanol. yellowish powder, yield = 67%, m.p. (128-130oc). rf = 0.78 (acetone 5: petroleum ether 5). ir (kbr disc), (v cm-1):3379: (nh) str. of indole, 3062: aromatic (ch) str., 2968: (c-h) asymm. str. of ch3 and ch2, 2875: (c-h) symm. str. of ch3 and ch2, 1709: (c=o) str. of ester, 1236: (c-o-c) str. of ether.1h nmr: (300 mhz, dmsod6 , =ppm): 0.62 (3h, t, -ch2-ch3 at c1), 1.26 (3h, t, -ch2-ch3 at c8), 1.92.11 (2h, m, -ch2-ch3 at c1), 2.5-3.04 (6h, m, ch2-ch3 at c8, -ch2-cooch3 at c1and -ch2 at c4), 3.56 (3h, s, -cooch3), 3.80 (2h, dd, -ch2 at c3), 6.797.00 (2h, m, ar-h5, h6), 7.23 (1h, d, arh7), 10.48 (1h, s, indole, n-h). synthesis of etodolac hydrazide (2-(1,8-diethyl1,3,4,9-tetrahydropyrano[3,4-b] indol-1yl) acetohydrazide) (compound b) to a solution of compound a (0.02 moles, 6 g) in absolute ethanol (70 ml), an excess amount of hydrazine hydrate 80% (0.2 moles, 10ml) was added, and the mixture was refluxed at 80 c for 6 h. at the end of the reflux time, the mixture was left to be cooled down to room temperature (r.t.), then cold distilled water was added to the mixture, a white precipitate was formed which was left overnight. the obtained precipitate was filtered, washed several times with cold distilled water, dried and recrystallized from ethanol. white powder, yield = 88%, m.p. (187-189 oc). rf = 0.41 (acetone 5: petroleum ether 5). ir (kbr disc), (v cm-1): 3354, 3313: (nh) str. of indole and hydrazide, 3062: aromatic (c-h) str., 2970: (c-h) asymm. str. of ch3 and ch2, 2875: (c-h) symm. str. of ch3 and ch2,1655: (c=o) str. of amide, 1620: (nh) bend., 1242: (c-o-c) str. of ether.1h nmr: 0.61 (3h, t, ch2-ch3 at c1), 1.25 (3h, t, -ch2-ch3 at c8), 2.04 (2h, q, -ch2-ch3 at c1), 2.61-2.9 (6h, m, ch2conhnh2, -ch2-ch3 at c8, -ch2 at c4), 3.95 (2h, dd, -ch2 at c3), 4.25 (2h, b.s. nh-nh2),6.80-6.98 (2h, m, ar-h5, h6), 7.22 (1h, d, ar-h7), 8.92 (1h, s, nh-nh2), 10.54 (1h, s, indole n-h). synthesis of aryl hydrazones 2-(1,8-diethyl-1,3,4,9tetrahydropyrano[3,4-b] indol-1yl) acetohydrazide derivatives (p1 p3) three drops of glacial acetic acid were added to an ethanolic solution of each of the following aldehydes (scheme 1): (1) [3,5-dimethoxy-4hydroxybenzaldehyde (0.005 moles, 0.91g)], (2) [4hydroxy-3-nitrobennzaldehyde (0.005 moles, 0.84g)], (3) [2-pyridine carboxaldehyde (0.005moles, 0.6g)], placed in round bottom flask equipped with magnetic stirrer. compound b (0.005moles, 1.55g) dissolved in absolute ethanol (20ml) was added to a stirred solution of each of the above mentioned aldehydes mixtures separately. then each reaction mixture was refluxed at 80 c for 8 h. at the end of the reaction (monitored by tlc), 50 ml of cold ice water was added to the mixture. the precipitate formed was collected, dried and recrystallized from solvents (80% ethanol for p2, 70%, 75% ethanol for p1 and p3 respectively) to get the intended products. iraqi j pharm sci, vol.28(1) 2019 etodolac hydrazone derivatives with anti-inflammatory action 108 (p1) 2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4b]indol-1-yl) n' ( 4 – hydroxyl -3 , 5 dimethoxy – benzylidene ) acetohydrazide. white powder, yield = 81%, m.p. (173175 oc). rf = 0.46 (ethyl acetate 6: n-hexane 4). ir (kbr disc), (v cm-1):3555-3210: (oh) str. broad band, 3377, 3271: (nh) str. of indole and hydrazone,3060: aromatic (c-h) str.,2964: (c-h) asymm. str. of ch3 and ch2. 2875: (c-h) symm. str. of ch3 and ch2, 1641: (c=o) str. of amide.1589: (c=n) str., 1512: ar. (c=c) str., 1213: (c-o-c) str. of ether. 1h nmr: 0.59,0.68(3h, tt, ch2-ch3 at c1), 1.25 (3h, t, -ch2-ch3 at c8), 2.07 (2h, q, -ch2-ch3 at c1), 2.66 (2h, q, -ch2-ch3 at c8),2.77-3.04 (4h, m,-ch2conh,-ch2 at c4), 3.74, 3.79 (6h, 2s, 2–och3), 3.99 (2h, dd, -ch2 at c3), 6.85-7.0, 7.19-7.29 (5h, 2m, ar-h), 7.88, 8.08 (1h, ss, n=ch), 8.85 (1h, bs, oh), 10.50 (1h, s, indole n-h), 11.0, 11.22 (1h, ss, -co-nh).chn: c26h31n3o5, m.wt:465.55, calculated c;(67.08), h; (6.71),n; (9.03). observed c;(68.88), h;(6.483), n; (8.743). (p2) 2-(1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4b] indol-1-yl)-n'-(4-hydroxy-3-nitro-benzylidene) acetohydrazide. pale yellow powder, yield=77%, m.p. (122-125 oc). rf = 0.83 (ethyl acetate 4: n-hexane 6). ir (kbr disc), (v cm-1):3417, 3249: nh str. of indole and hydrazone overlapping with (oh) str. of phenol,3089: aromatic (c-h) str., 2964: (c-h) asymm. str. of ch3 and ch2, 2875: (c-h) symm. str. of ch3 and ch2, 1666: (c=o) str. of amide, 1622: (c=n) str., 1522: ar. (c=c) str. and asymm. (no2) str., 1323: symm. (no2) str., 1257: (c-o-c) str. of ether. 1h nmr: 0.59, 0.66 (3h, tt, -ch2-ch3 at c1), 1.25(3h, t, -ch2-ch3 at c8), 2.10 (2h, q, ch2-ch3 at c1), 2.66 (2h, q, ch2-ch3 at c8), 2.83-2.99 (4h, m, -ch2conh at c1, -ch2 at c4), 3.98 (2h, dd, ch2 at c3), 6.84-6.97 (2h, m, ar-h5, h7), 7.127.28 (2h, m, ar-h6, h5´), 7.83 (1h, dd, ar-h6´), 7.93, 8.08(1h, ss, n=ch), 8.18 (1h, s, ar-h2´), 8.93 (1h, bs, oh), 10.48 (1h, s, indole n-h), 11.17, 11.34 (1h, ss, -co-nh). chn: c24h26n4o5, m.wt:450.50. calculated c; (63.99), h; (5.82), n; (12.44). observed c; (62.634), h; (6.027), n; (12.938). (p3) 2-(1, 8-diethyl-1,3,4,9-tetrahydropyrano[3,4b]indol-1-yl)-n'-(pyridin-2-ylmethylene) acetohydrazide. off-white powder, yield = 54%, m.p. (148-153 oc). rf = 0.61 (ethyl acetate 6: n-hexane 4)). ir (kbr disc), (v cm-1):3298, 3247: (nh) str. of indole and hydrazone, 3053: aromatic (c-h) str., 2964: (c-h) asymm. str. of ch3 and ch2, 2873: (ch) symm. str. of ch3 and ch2, 1672: (c=o) str. of amide, 1606: (c=n) str., 1556: ar. (c=c) str., 1230: (c-o-c) str. of ether. 1h nmr: 0.59, 0.68 (3h, tt, ch2-ch3 at c1), 1.26 (3h, t, -ch2-ch3 at c8), 2.11 (2h, q, ch2-ch3 at c1), 2.61-2.73, 2.82-3.01 (6h, 2m, ch2-ch3 at c8, -ch2conh at c1, -ch2 at c4), 4.06 (2h, dd, -ch2 at c3), 6.82-7.00 (2h, m, ar-h5, h6), 7.23 (1h, d, ar-h7), 7.33-7.45 (1h, m, arh4´), 7.50-7.59 (1h, m, ar-h3´), 7.78-7.92 (1h, m, ar-h2´), 8.03, 8.22 (1h, ss, n=ch), 8.53-8.63 (1h, m, ar-h5´), 10.50 (1h, s, indole n-h), 11.40, 11.51 (1h, ss, -co-nh).chn:c23h26n4o2, m.wt:390.49, calculated c; (70.75), h; (6.71), n; (14.35), observed c; (69.722), h; (7.006), n; (14.868). evaluation of the anti-inflammatory activity (11) albino rats of both sexes weighing (190 ± 10 g) were delivered by the animal house of the college of pharmacy, university of baghdad, and are kept in the same place under consistent conditions. animals iraqi j pharm sci, vol.28(1) 2019 etodolac hydrazone derivatives with anti-inflammatory action 109 were fed commercial chaw and had access to water freely. animals were divided into five groups (each group consists of 6 rats) including standard (etodolac), control (dmso) and p1, p2 and p3 groups. dose determination of the final synthesized compounds (table 1) was done according to the equation below, egg white induced edema model (12) was used to study the anti-inflammatory activity of the target compounds. this was achieved by the administration of an intraperitoneal (i.p) injection of each of the final products, etodolac or control, individually to the five animal groups. thirty minutes after that subcutaneous injection (s.c.) of 0.05 ml of undiluted egg-white was injected into the plantar side of the left hind paw of the rats of each group. vernea was used to measure paw thickness at six-time intervals (0, 30, 60, 120, 180, and 240 min.), where zero time was the time at which the products, standard, and control were administered intra-peritoneally. 𝐃𝐨𝐬𝐞 𝐨𝐟 𝐑𝐞𝐟𝐞𝐫𝐚𝐧𝐜𝐞 𝐂𝐨𝐦𝐩𝐨𝐮𝐧𝐝 𝐌𝐨𝐥𝐞𝐜𝐮𝐥𝐚𝐫 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐫𝐞𝐟𝐞𝐫𝐞𝐧𝐜𝐞 𝐜𝐨𝐦𝐩𝐨𝐮𝐧𝐝 = 𝐃𝐨𝐬𝐞 𝐨𝐟 𝐭𝐞𝐬𝐭𝐞𝐝 𝐜𝐨𝐦𝐩𝐨𝐮𝐧𝐝 𝐌𝐨𝐥𝐞𝐜𝐮𝐥𝐚𝐫 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐭𝐞𝐬𝐭𝐞𝐝 𝐜𝐨𝐦𝐩𝐨𝐮𝐧𝐝 table (1) doses determined for the synthesized final products product no. m.wt. dose mg/kg etodolac (std.) 287.359 10 (a) p1 465.550 16.20 (b) p2 450.495 15.68(b) p3 390.487 13.59(b) (a)the standard dose for etodolac in mg/kg. (b)the determined dose which is equivalent to etodolac dose. multiple comparisons between the synthesized target compounds against control and reference drug were done using one-way anova test, then to see the significance between each pair of compounds, post hoc tukey test was used, which offers an advantage over the use of independent ttest for more powerful accuracy for calculating the p-value. graph pad prism 8.0.0 program was used to carry out the statistical analysis. results and discussion chemistry the synthetic pathways used for the preparation of the target etodolac hydrazone derivatives (p1-p3) are summarized in scheme (1). etodolac methyl ester compound (a) was synthesized by the reaction of etodolac with methanol along with the use of few drops of concentrated h2so4. compound (b) was synthesized by the reaction of etodolac methyl ester with hydrazine hydrate (nh2nh2.h2o). the synthesis of the final etodolac hydrazone derivatives involves the reaction of etodolac hydrazide with different types of aldehydes by using glacial acetic acid as a catalyst. iraqi j pharm sci, vol.28(1) 2019 etodolac hydrazone derivatives with anti-inflammatory action 110 archo: are listed below; scheme (1) the synthesis of target compounds (p1-p3). the structures of all the synthesized compounds were characterized by ft-ir, 1hnmr and chn elemental microanalysis. the infrared spectra of the hydrazone derivatives (p1-p3) showed the characteristic absorption band at (1641-1672) cm-1 due to (c=o) stretching of amide as well as the appearance of bands at (1589-1622) cm-1attributed to the(c=n) stretching of the imine. additionally, two other absorption bands were displayed at (32983417) cm-1 and (3247-3271) cm-1attributed to (n-h) stretching of indole and hydrazone, respectively. the 1hnmr spectra of the target compounds (p1 p3) confirmed the synthesis of hydrazone derivatives. characteristic signals of hydrazone were shown in the region (7.83-8.22 ppm) as two singlets attributed to the azomethine proton n=ch, another characteristic signal of hydrazone due to – co-nh proton was displayed as two singlets resonating at 11.00-11.54 ppm, in addition to the loss of signal at 4.25 ppm for the two hydrazide protons nh-nh2. generally, hydrazones may exist as e and z geometrical isomers (12). this explains the appearance of each of azomethine n=ch and conh proton as two singlets. evaluation of the anti-inflammatory activity comparison of reference drug (etodolac) versus control (dmso) at baseline and after 30 minutes, there was no significant difference between control and etodolac in paw edema reduction, but after 60 minutes the difference becomes significant in which etodolac offer more reduction in the percent paw thickness compared to the control. further reduction was continued significantly at 120 minutes, up to 240 minutes as shown in figure (2) below; figure (2) effect of etodolac (reference), and dimethyl sulfoxide (control) on egg-white induced paw edema in rats measured in percentage. note: time (30) min. is the time of egg-white injection. comparison of the effect of synthesized compounds p1, p2 and p3 versus control no significant difference was found between the target compounds compared to the control at baseline and after 30 minutes. however, compound (p3) produced a significant difference in the reduction of paw thickness at 60, 180 and 240 minutes compared to the control. whereas, a significant difference compared to the control in percent reduction of paw thickness was shown for compound (p2) at 120 and 240 minutes. these results are shown in table (2) and figure (3). iraqi j pharm sci, vol.28(1) 2019 etodolac hydrazone derivatives with anti-inflammatory action 111 table (2) effect of dimethyl sulfoxide (control) and target compounds (p1-p3) on egg-white induced paw edema data are expressed in mm paw thickness as mean ± sem. n= number of animals. time (0) is the time of i.p. injection of tested compounds, and dmso (2ml/kg) time (30) is the time of injection of egg-white (induction of paw edema). significantly different compared to control: p-value *< 0.033 (gp system) comparison of the effect of synthesized compounds p1, p2 and p3 versus etodolac there was no significant difference in the reduction of paw thickness between the synthesized target compounds compared to etodolac at baseline and after 30, 60, 120, 180and 240 minutes. all the synthesized compounds produce reduction in paw thickness which was comparable to the standard (etodolac) as presented in table (3) and figure (3). table (3) effect of etodolac (reference) and target compounds (p1-p3) on egg-white induced paw edema data are expressed in mm paw thickness as mean ± sem. n= number of animals. time (0) is the time of i.p. injection of tested compounds, and etodolac. time (30) is the time of injection of egg-white (induction of paw edema). note: in this case all compounds with no significant difference compared to etodolac figure (3) effect of etodolac, dimethyl sulfoxide (dmso), compounds p1, p2, and p3 on egg-white induced paw edema in rats. results are expressed as mean ± sem & percent. (n=6 for each group). note: time (30) is the time of egg-white injection. time (min) control n=6 p1 n=6 p2 n=6 p3 n=6 p a w th ic k n e ss (m m ) 0 3.77±0.10 3.78±0.13 3.65±0.09 3.70±0.07 30 5.51±0.09 5.24±0.16 4.98±0.10 5.04±0.18 60 6.03±0.18 5.68±0.14 5.53±0.08 5.36±0.14* 120 5.80±0.22 5.56±0.12 5.08±0.08* 5.22±0.13 180 5.52±0.18 5.31±0.11 4.92±0.14 4.85±0.08* 240 5.23±0.12 4.97±0.14 4.57±0.09* 4.58±0.09* time (min) etodolac n=6 p1 n=6 p2 n=6 p3 n=6 p a w t h ic k n e ss (m m ) 0 3.51±0.11 3.78±0.13 3.65±0.09 3.70±0.07 30 4.70±0.09 5.24±0.16 4.98±0.10 5.04±0.18 60 5.33±0.12 5.68±0.14 5.53±0.08 5.36±0.14 120 5.24±0.08 5.56±0.12 5.08±0.08 5.22±0.13 180 5.00±0.17 5.31±0.11 4.92±0.14 4.85±0.08 240 4.64±0.13 4.97±0.14 4.57±0.09 4.58±0.09 iraqi j pharm sci, vol.28(1) 2019 etodolac hydrazone derivatives with anti-inflammatory action 112 conclusion three new etodolac hydrazone derivatives (p1-p3) were synthesized, and their structures were characterized by ft-ir, 1hnmr and chn microanalysis. the compounds synthesized in this study exhibited anti-inflammatory action when tested on rats by using egg white induced paw edema and showed comparable effect as the used standard drug (etodolac) with no significant difference. references 1. mohammed zahd and mh. synthesis of 5fluorouracil derivatives as possible mutual prodrugs with meloxicam and ibuprofen for targeting cancer tissues. iraqi j. pharm. sci. 2011;20(2). 2. matsumura y. chapter 14 – synthesis and pharmacological properties of fluorinated prostanoids. fluorine and health. 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sci. 2013;22(1):120–7. iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 15 impact of different doses of nicorandil-induced ulceration (oral , gastrointestinal tract, and anal) in rats: roles of leptin and prostaglandin e2 asma a. hayder* and tagreed s. altaei**,1 *college of pharmacy , hawler ,medical university. ** college of dentistry , hawler ,medical university. abstract many reports confirm ulcers as an adverse effect of drugs such as nicorandil and aspirin. the exact responsible mechanisms of ulceration have until now not proved. mucosal ulcers associated with the onset of ulcer are manifested by an increase in proinflammatory cytokine, excessive prostaglandin, and up-regulation of endothilin-1 level, which directly impacts the release of leptin. these, released locally within mucosal tissues, have played a role in controlling the extent of local inflammatory responses and processes of mucosal repair. this study was designed to find out the correlation of plasma leptin and prostaglandin levels as a possible mechanism of oral ulcer formation as an adverse effect of nicorandil. the effect of nicorandil for inducing ulceration was assessed. the plasma leptin and prostaglandin e2 for the tested groups in relation to the studied parameters (gender, and daily body weight change) were estimated in albino rats. nicorandil causes mucous membrane damage, inflammation, and ulceration. a significant reduction of plasma leptin level, which was dose-dependent, and a non-significant reduction of serum prostaglandin e2 level. the mechanisms of ulcer induction as an adverse effect of nicorandil can be related to dosedependant leptin and prostaglandin e2 levels, which affects on repair and healing process. keywords: nicorandil, leptin, prostaglandin e2, ulcer. تأثير الجرع المختلفة في استحداث التقرح بواسطة عقار النيكوراندل e2 ندننوالبروستاكالان دور هرمون اللبتين ذ) الفم ، الجهاز الهضمي والشرج ( في الجر 1**،الطائيسهام و تغرند *حيدر اسماء عوني كلية الصيدلة ، جامعة هولير الطبية * ، جامعة هولير الطبية . طب االسنانكلية ** لخالصةا نيكورانديل تؤكد العديد من التقارير السريرية حالة قدرة بعض العقاقير في استحداث القرحة كتأثير ضار سلبي مثل عقار واالسبرين. حتى االن لم تثبت اآلليات الدقيقة المسؤولة للتقرح . ان بداية تكون القرح في الغشاء المخاطي مرتبطة مع زيادة في انتاج شر في ، والذي له تأثير مبا 1_ -سايتوكاين الممهدة لاللتهاب، واالفراط في توليد البروستاجالندين ، وزيادة انتاج ملحوظة في اندوثلين تنظيم تحرير مستوى الهرمونات مثل اللبتين. تلك التي تحررت موضعيا ضمن االنسجة المخاطية، لعبت دورا في السيطرة على مدى االستجابات االلتهابية وعمليات إصالح الغشاء المخاطي. الهدف من الدراسة الحالية لمعرفة وكشف العالقة بين اللبتين و مصل الدم كآلية مسببة ممكنة الستحداث القرحة على انها التأثير السلبي الستخدام قرص نيكورانديل وتمت في e2 ستاكالندينروالب مستويات اللبتين والبروستاجالندين . تم تقييم تأثير النيكورانديل في استحداث القرحة. كذلك تمت دراسة االختالفات بين e2 مقارنة موعة السيطرة وارتباطهم في التحريض على التقرح في االنسجة المختلفة من خالل المعلمات المجاميع قيد الدراسة ومقارنتها مع مج )الجنس، و تغير وزن الجسم يوميا( في الجرذان البيضاء. اظهرت النتائج ان فعالية عقار نيكورانديل تعتمد على كمية الجرعة يؤثر على بنية االنسجة ، مما يؤدي الى التهاب واحتقان في المستخدمة ، وقد كشف الفحص المجهري لالنسجة ان عقار نيكورانديل االوعية الدموية في الغشاء المخاطي وفي تكون القرحة في انسجة الفم ، الجهاز الهضمي ، وفي انسجة الشرج للجرذان البيضاء .عقار نتج من هذه الدراسة ان آليات استحداث نيكوانديل ادى الى انخفاض متوسط تركيز هرمون الليبتين في مصل الدم بنسبة عالية، يست القرحة باعتبارها التأثير السلبي لتعاطي عقار نيكورانديل يمكن اعتباره كآلية ثانية في استحداث القرح وهو االنخفاض الغير ملحوظ . في مصل الدم e2 احصائيا في مستوى البروستاجالندين .قرحة ال، e2 ستاكالندننروالب، اللبتين، نيكوراندنل : الكلمات المفتاحية introduction ulcers are frequent lesions of the mucosa. generally, they are circumscribed round or elliptical lesions surrounded by an erythematous halo and covered with an inflammatory exudate in their central portion, and follow with painful symptoms. oral ulcers are painful and may be single or multiple, symmetric or irregular in shape. once an ulcer forms, it is subject to recurrent irritation from saliva and microflora, and the acute inflammatory stage may be followed by a pattern of chronic inflammation(1). 1corresponding author e-mail:tagreedaltaei@yahoo.com. received: 17/4/2016 accepted: 28/8/2016 iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 16 numerous of the causes and factors involved in the formation of these lesions – including immunological alterations, infections, nutritional deficiency, food repetitive trauma to the mucosa, neoplasms, autoimmune diseases and contact allergies, as well as psychosomatic, genetic and environmental factors (2) were also observed after the use of many drugs such as labetalol, nicorandil, captopril, nsaids (aspirin, paraaminosalicylic acid , diflunisal , ibuprofen, indomethacin, naproxen, rofecoxib, sulindac), as well as after the use of mycophenolate, sirolimus, sodium lauryl sulfate, protease inhibitors (saquinavir, indinavir, ritonavir, lopinavir, nelfinavir, amprenavir), and sulfonamides, and alendronate (3). nicorandil is a potassium channel activator used in the treatment of angina pectoris (4). initial adverse reactions include headaches, nausea and cutaneous erythema. there is a 5% prevalence of oral ulceration in patients on nicorandil, compared with patients on various other anti-anginal medications (5). nicorandil is a cause of life threatening terminal ileum ulceration, while nsaid such as diclofenac and aspirin use has been linked with endoscopic ileitis (6). it is important that clinicians elsewhere be made aware that nicorandil can be a potential inducer of ulcers that may mimic major aphthous ulcers or even carcinoma (7), anal (8), colonic (9), vulval (10), parastomal (11) and intestinal ulceration (12, 13). aspirin could directly damage the gastric epithelium (14). the breaking of the ‘barrier' permitted the back-diffusion of acid into the mucosa, which eventually led to the rupture of mucosal blood vessels. these topical irritant properties were subsequently found to be predominantly associated with those nsaids with a carboxylic acid residue (15). the ability of an nsaid to cause gastric damage correlates with its ability to suppress gastric prostaglandin synthesis; agents that are weak inhibitors of gastric prostaglandin synthesis are less ulcerogenic (16). leptin is known to exhibit a variety of physiological actions on body weight homeostasis (17), lipid metabolism (18), hematopoiesis (19), thermogenesis (20), ovarian function (21), bone formation (22, 23), angiogenesis (24, 25) and wound healing (26-28). the multifunctionality of leptin and the wide distribution of its receptor suggest that leptin plays a variety of physiological roles not only as a systemic hormone but also as a local growth factor. leptin is present in human saliva as well as serum (29, 30). epithelial cells and vascular endothelial cells in oral mucosa are target cells for leptin (31). leptin promotes wound healing by enhancing the epithelial cell proliferation (27, 28). prostanoids, the e type prostaglandins, particularly pge2 derived from arachidonic acid, are most widely produced in the body (32). molecular identification of the e type prostaglandins receptors was achieved by their cdna cloning (33, 34), which revealed that the receptors are g-protein-coupled receptors (gpcrs). this study was designed to study the efficacy of nicorandil for inducing ulcers as adverse effects, by determining any possible correlation between, on one hand, plasma leptin and prostaglandin e2 levels and, on the other, the pathological causes of ulcers in albino rats, and compared to aspirin as a standard ulcerogenic agent. these were performed by: 1-the effect of 0.28, 0.4, 1, and 3 mg/kg of nicorandil on the microscopic appearance and histopathology features of albino rat tissues. 2-effects of different doses of nicorandil mentioned above on the plasma leptin and prostaglandin e2 levels. 3-the correlation analysis of plasma leptin and prostaglandin e2 levels, and with the studied parameters (gender, and body weight). materials and methods the study was carried out at hawler medical university in the experimental animal house of the college of pharmacy, from february to july 2014. it was based on an ethical approved protocol (part of msc study). materials: nicorandil tab 10 mg (merk, germany); soluble in water (4 mg/ml), aspirin effervescent 500 mg (sanofi-aventis, france); ether (diethyl ether=74.12g/mol) from england; normal saline (0.9% sodium chloride) from pioneer sulaymaniyah; formaldehyde, solution 37% w/w, extrapure, pheur, bp, usp, stabilised with approximately 10% methanol (scharlab s.l. spain). for the enzyme-linked immunosorbent assay (elisa), the following kits were used: rat leptin elisa gwb-95c312 40-055-200005 (genway platinum, san diego), and rat high sensitivity prostaglandin e2 enzyme immunoassay (ann arbor, michigan). rats and housing: in the current study, 42 local domestic albino rats, of both sexes weighing 169-409 g and aged 12-16 weeks were obtained and cared for in the animal house of the college of medicine/hawler medical university, erbil, kurdistan, iraq. the animals were kept in polycarbonate cages on a layer of wood shavings, under standard laboratory conditions. the animals were housed in groups of four per cage. all rats used in this study were allowed to adapt to the housing iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 17 conditions for one week prior to commencement of the study. rats were maintained on a 12-hour light/dark cycle at temperature range of 21-28 °c, humidity 10%50%. the animals were kept in standard room conditions and supplied with rodent chow and free access to tap water, in compliance with the institutional animal care and use committee. experimental design: a total of 42 local domestic albino rats, female and male, were randomly divided into six groups; study groups (1-4), and control groups (5, 6) as follows: study groups: nicorandil 10 mg tablet was crushed into powder by mortar and pestle in the biochemistry laboratory of the college of medicine, hawler medical university. the measured amount according to the rats’ weight for each group was administered intraperitoneally (i.p.) as a solution of 1 ml [distilled water (d.w.) was used as a solvent]. four doses of nicorandil were used. the animals were randomised into the following groups: g1: nicorandil 0.28 mg/kg/day. g2: nicorandil 0.4 mg/kg/day. g3: nicorandil 1 mg/kg/day. g4: nicorandil 3 mg/kg/day. control groups: two types of control group were used: positive control: an amount of aspirin measured according to the animal’s weight was dissolved in 1ml d.w. the intraperitoneal dose used was 5 mg/kg/day, which was administered to 7 rats (three females: four males) for 10 days. assigned as g5: aspirin 5mg/kg/day. negative control: intraperitoneally normal saline was administered to 7 rats (three females: four males) for 10 days. assigned as g6: normal saline 1 ml/day. samples preparation: rats were anaesthetized by diethyl ether solution (20 ml of diethyl ether on cotton in jar) to render them unconscious, which required approximately 5 minutes. then intra-cardiac blood (2 ml) was drawn from rats as a baseline using edta tube (35). the blood was immediately centrifuged at 3500 rpm for 10 min and plasma was separated by pipette to eppendorf covered by parafilm and stored at -20 °c (36). rats were weighed daily and then nicorandil was injected i.p. into each of them, except for the control groups which were injected with either aspirin (as +ve control) or normal saline (as -ve control) for 10 days. rats received the corresponding treatment drug according to the stated dose for each group. after 10 days, 24 hr. after the last dose, rats were anaesthetized, then intra-cardiac blood (2 ml) was drawn, and the plasma was separated as mentioned above for further analysis. albino rats were euthanized by 30 ml diethyl ether. after dissection of all animals' groups, the organs and tissues (oral cheek pouches and tongue, git, anal) were removed, dried by filter paper, and fixed in 10% buffered formalin solution for histopathological sectioning. enzyme linked immunosorbent assay rat leptin assay: rat leptin elisa, standards, quality controls, and samples were incubated in microplate wells, which were precoated with anti-rat leptin antibody. after 60 minutes of incubation and washing, biotinlabelled polyclonal anti-rat leptin antibody was added to the wells and incubated with immobilised antibody-leptin complex for 60 minutes. after another washing, streptavidinhrp conjugate was added. after 30 minutes incubation and the last washing step, the remaining conjugate was allowed to react with the substrate solution (tmb). the reaction was stopped by addition of acidic solution and absorbance of the resulting yellow product was measured spectrophotometrically at 450 nm. the absorbance was proportional to the concentration of leptin. a standard curve was constructed by plotting absorbance values against concentrations of standards, and concentrations of unknown samples were determined using this standard curve. rat high sensitivity prostaglandin e2: plasma pge2 was quantitatively measured. standards or diluted samples were pipetted into a clear microtiter plate coated with an antibody rat igg. a pge2-peroxidase conjugate was added to the standards and samples in the wells. after an overnight incubation at 4 °c, the plate was washed and substrate was added. the substrate was reacted with bound pge2-peroxidase conjugate. after 30 minutes incubation, the reaction was stopped and the intensity of the generated colour was detected in a microtiters plate reader capable of measuring 450 nm wave length. histopathology study: fixation and staining: organ specimens were collected from all animals in this study and fixed by immersion in 10% formalin. xylene (clearing agent) was introduced to infiltrate the tissues, and finally paraffin was introduced to complete the tissues embedding process to produce paraffin block (37), and then stained with hematoxylin and eosin (h&e) for microscopic examination (38). statistical analysis: data were analysed using the statistical package for social science (spss) software version 18 (spss inc., 2010). the data represent quantitative observations and were summarized using means ± standard deviations (m±sd). statistical analysis with t test was used to compare between means of iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 18 two groups (comparing means of two different groups or comparing means of one sample in two different occasions), and independent sample t test was used to compare the different average values between two groups. also, oneway analysis of variance (anova) was performed to compare the differences in the means among groups. pearson correlation coefficient was used to assess the correlation between two numerical variables. p≤ 0.05 was considered statistically significant in all the results of the current study. results the effects of nicorandil for ulceration adverse effect: the effect of 0.28, 0.4, 1, 3 mg/kg/day nicorandil was studied, to explore its ability to induce ulcers as an adverse effect in different tissues of albino rats via the examination of the microscopic features, and pathological findings of the oral, gastrointestinal, and anal tissues, as discussed in this section: microscopic appearance and histopathology study: oral tissues of the albino rats: histopathological section of the oral tissues showed that different doses of nicorandil on tongue mucous produced sloughing of the mucous, ulceration with moderate inflammatory cell infiltration, as shown in figure (1). figure (1):section of rat's tongue treated with 3 mg/kg/day nicorandil showing surface ulceration and sloughing, with inflammatory cells infiltration and vascular congestion of mucosal tongue (h&e 400x). gastrointestinal tract tissues of the albino rats: histopathological section of rat’s gastrointestinal tract showed different response in females rather than males group; section from stomach showed gastritis, surface ulceration, granulation tissue formation and mononuclear inflammatory cell infiltration as shown in figure (2). figure ( 2) :section of male rat's stomach treated with 3 mg/kg/day nicorandil showed surface ulceration, granulation tissue and moderate mononuclear inflammatory cell infiltration of lamina proporia (h&e: 400x). in small intestine section showed sloughing, erosion and heavy mononuclear inflammatory cell infiltration of lamina proporia as shown in figure (3). figure ( 3):section of rat's small intestine of male treated with 3 mg/kg/day nicorandil showing sloughing, erosion and heavy mononuclear inflammatory cell infiltration of lamina proporia (h&e: 400x). higher dose of nicorandil (3 mg/kg/day) showed a large size ulcer in the mucosa of the small intestine with heavy mixed inflammatory cell infiltration, granulation tissue formation and fibrosis as shown in figure (4). while section from small intestine of female rats showed heavy mixed inflammatory cell infiltration including eosinophil with erosion and cryptitis as shown in figure (5). the section of the positive control group (g5) treated with aspirin showed gastritis with heavy mixed inflammatory cell infiltration of lamina proporia and granulation tissue formation as shown in figure (6). iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 19 figure (4):section of rat's small intestine of male showing surface ulceration of small intestine, heavy mononuclear inflammatory cell infiltration with lamina proporia (h&e: 400x). figure ( 5 ):section of rat's small intestine of female treated with 3 mg/kg/day nicorandil has heavy mixed inflammatory cell infiltration include eosinophil with erosion cryptitis (h&e: 400x). figure (6):section of rat's stomach treated with 3 mg/kg/day nicorandil showing inflammation (gastritis) h&e: 100x. anal tissues of the albino rats: the histopathological sections showed the damage of anal tissues. there were different responses in the tested groups; in first group; females showed relatively normal, while in male there was erosion of mucosa anal tissue. group 2 showed sloughing, erosion and exudation as shown in figure (7). in higher dose of nicorandil as in groups 3 & 4 section from rectal anal mucosa shows surface ulceration, heavy mononuclear inflammatory cell infiltration, cryptitis and granulation tissue formation as shown in figure (8). control groups (5 and 6) showed non-effect on the anal tissues in both genders. figure (7):section of female rat treated with 3 mg/kg/day nicorandil; anal tissues with sloughing, erosion and exudation (h&e: 400x). figure (8):section of rat's male anal tissue treated with 3 mg/kg/day nicorandil appeared with surface ulcer, heavy mononuclear inflammatory cell infiltration and cryptitis (h&e: 400x). the effects of nicorandil on plasma leptin and prostaglandin e2: estimation of plasma leptin: table (1) showed the effects of treatment with different doses 0.28, 0.4, 1, or 3 mg/kg/day of nicorandil (g1-g4) compared to 5 mg/kg aspirin (g5), and n.s. (g6) groups. the administration of different doses of nicorandil produced a highly significant reduction in plasma leptin concentration; in g2 from 110.81±31.58 to 62.91±46.88, in g3 from 153.86±33.27 to 48.09±16.17, and in g4 from 149.41±58.46 to 56.09±44.43, while in g1 there was a decreased level of plasma leptin from 118.13± 31.58 to 62.91±46.88, but a non-significant difference (p= 0.07). the comparison between baseline and after 10 days of treatment in the aspirin group showed a non-significant increase of plasma leptin levels from 94.01±50.25 to 135.03±88.41, p= 0.161, while the negative control showed a nonsignificant decrease in plasma leptin levels from 86.33±42.30 to 68.38±57.03, with a p value of 0.234. there are significant differences within groups (g1-g6); at baseline p=0.051, and p value within groups after 10 days of treatment was 0.055. iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 20 estimation of plasma prostaglandin e2: there was non-significant reduction of plasma pge2 levels (baseline and after 10 days of treatment) in all tested groups. comparison of significance within baseline, and after 10 days of treatment of all groups showed a nonsignificant difference, with p values of 0.349 and 0.354, respectively, as shown in table (1). table (1):the plasma leptin, and prostaglandin e2 concentrations of all treated groups (baseline and after 10 days of treatment) of albino rats, with p values. plasma leptin concentrations plasma prostaglandin e2 concentration group baseline (pg/ml) after 10 days of treatment (pg/ml) pvalue baseline (pg/ml) m sd after 10 days of treatment (pg/ml) m sd pvalue g1 118.13 31.58 62.91 46.88 0.017 * 32.9912.89 34.1122.68 0.401 g2 110.81 59.04 53.071 52.40 0.017 * 32.9912.89 34.8215.22 0.219 g3 153.86 33.27 48.09 16.17 0.000 * 45.474.54 39.499.41 0.060 g4 149.41 58.46 56.091 44.43 0.002 * 44.1165.78 42.4259.69 0.573 g6 – n.s. 86.33 42.30 68.38 57.03 0.234 * 74.1175.07 31.2422.56 0.211 p value within group 0.051 0.051 0.349 0.354 note: g1: 0.28 mg/kg/day nicorandil , g2: 0.4 mg/kg/day nicorandil , g3: 1 mg/kg/day nicorandil , g4: 3 mg/kg/day nicorandil, g5: 5 mg/kg/day nicorandil , g6: normal saline nicorandil correlation of plasma leptin and pge2 analysis: the correlation of plasma leptin and prostaglandin e2 levels were estimated and analysed according to the studied parameters, gender, and body weight of the albino rats of all tested groups. analysis of leptin, and pge2 with gender: the ratio of female: male albino rats were 18:24, as shown in table (2). the mean plasma baseline leptin (lp-b) concentration of females was 89.31 ± 43.05 pg/ml, while for males it was 140.84 ± 45.87 pg/ml; this represented a highly significant difference of p=0.001. the mean plasma lp-a (after 10 days of treatment) concentrations were 45.02± 37.50 pg/ml, 89.77 ± 66.48 pg/ml for females and males, respectively, which is significantly different (p=0.014). females showed significantly less plasma leptin concentrations (pre and post values). the mean baseline plasma pge2-b concentration was 66.60 ± 56.72 pg/ml, 26.83 ± 13.59 pg/ml, for females and males, respectively, which was more significant in female than male rats (p=0.002). the mean plasma concentrations of pge2 after 10 days of treatment were 39.38 ± 36.38 pg/ml, 23.70 ± 19.92 pg/ml for females and males, respectively (p= 0.08), as shown in table (2). analysis of leptin, and pge2 with body weight of rats: all animals were weighed daily, from day 0 till the end of 10 days of treatment in all studied groups. table (3) showed the mean weight of treated groups, compared to positive and negative controls. the elevation of body weight of albino rats (250.85±59.20 to 265.57± 64.22) that received 3 mg/kg/day nicorandil (g4) was highly significant (p=0.008), and a significant increase of rats’ body weight (260±75.52 to 276.14±87.52) was seen with administration of 1 mg/kg/day nicorandil (g3) (p=0.024), while the second and n.s. group showed a non-significant increase in the rats’ body weights. the aspirin control group showed a significant reduction of albino rats’ body weights (301.28±68.17 to 279.57±69.68) after 10 days of treatment (p= 0.036). there was a weak, non-significant positive correlation between weights and plasma leptin concentrations after treatment; r = 0.272, p=0.08. also there is a weak, nonsignificant negative correlation between rats’ body weights after treatment and plasma prostaglandin e2 concentrations; r = -0.215, p=0.172. the correlation of plasma leptin and prostaglandin e2 levels in albino rats after 10 days of treatment: there was a non-significant negative correlation between plasma leptin and prostaglandin e2 concentrations after 10 days of treatment in all studied groups (g1-6); r = 0.252, p= 0.108. iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 21 table (2):-the relation of plasma leptin and prostaglandin e2 (baseline and after 10 days of treatment) to the rat’s gender. sex no. mean conc. (pg/ml) sd p-value leptin _b m 24 140.8417 45.8725 0.001* f 18 89.3167 43.0592 leptin _a m 24 89.7708 66.4848 0.0014* f 18 45.0267 37.5048 leptin _b m 24 26.83 13.5974 0.002* f 18 66.6 56.7200 leptin _a m 24 23.70 19.9254 0.081* f 18 39.38 36.3802 note: leptin _b : baeline plasma leptin concentration . leptin _a : plasma leptin concentration after 10 days of treatment .pge2 – b : baeline plasma prostaglandin e2 concentration. pge2 – a : plasma prostaglandin e2 concentration after 10 days of treatment . table ( 3 ):-the effect of different doses of nicorandil on rats’ body weights (baseline and after 10 days of treatment) with its relation to plasma leptin and group baseline (day 0) m sd after 10 days of treatment m sd p-value g1 250.42 53.10 242.14 43.17 0.598 g2 269.57 75.52 276.14 87.52 0.024 * g4 230.85 59.20 265.57 64.22 0.008 * g6 – n.s. 270.71 76.73 274.42 80.08 0.531 p value within group 0.738 0.913 discussion all drugs can produce untoward consequences, even when used according to standard or recommended methods of administration. adverse drug reactions can involve every organ and system of the body and are frequently mistaken for signs of underlying disease. systemic medication is also known to have potential adverse side effects on the oral mucosa and the mouth, and many drugs or chemicals can affect associated structures. the results of this study showed for the first time that the dose-dependent effects of nicorandil have the ability to cause ulcerations in oral, git, and anal tissues, which correlated with the plasma levels of leptin and pge2. ulceration has many etiological factors; the oral mucosa is affected by many factors including systemic diseases / conditions such as vascular disease, infection, immunosuppression, and chemotherapy for malignancies. in the presence of these factors, oral lesions often fail to heal adequately, resulting in chronic ulcer formation followed by serious systemic infections (39, 40). nicorandil is generally well tolerated, but more specific adverse effects such as oral ulceration and stomatitis were first reported in 1998 (41), which was subsequently followed by reports of anal ulceration (42, 8). it has emerged that nicorandil can cause very painful chronic ulceration of the colon and small intestine (43, 44). painful parastomal ulceration has been reported in patients with ileostomies or colectomies (10). gastrointestinal ulceration has been reported from the mouth to the perineum, and it may also associate with skin, peri-vulvar and penile ulcers (45-47). oral ulceration is known to occur with an aspirin-like chemical burn if it left to dissolve whilst in contact with the oral mucosa (48). the anti-anginal drug nicorandil is increasingly being recognised as a causative factor for mucous membrane ulceration. the pathophysiology of the ulceration remains unclear. it has been postulated in this study that leptin and prostaglandin e2 may play a role in the formation of ulcers by the administration of nicorandil, and compared to standard ulcerogenic agent aspirin. this may explain the mechanism of mucous membrane damage that led to ulceration. oral ulceration may be due to a more local toxic effect and therefore be dose-related (49). this study agrees with the above-mentioned study, and the effect of nicorandil for inducing ulcers was dosedependent. the evidence of histopathological examination showed that different doses of iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 22 nicorandil have an effect of mild inflammation and inflammatory cells infiltration, and vascular congestion on buccal mucosa, and mucosa of the tongue. stomach section showed moderate inflammatory cell infiltration with vascular congestion of mucosa, gastroenteritis, and small intestinal ulceration. the response of females and males to a dose of 0.28-mg/kg/day nicorandil differed, with a mild erosion in females’ small intestines and gastritis in males. the other tested doses of 0.4 and 1 mg/kg/day showed the same response in both sexes; where, moderate ulceration in the small intestine was observed. the group treated with 3 mg/kg/day nicorandil showed a moderate inflammatory cell infiltration with vascular congestion of mucosa, small intestinal ulceration in oesophagus and stomach for both sexes. the aspirin positive control group showed gastro-intestinal erosion in both sexes. microscopic appearance of anal tissues showed that the damage produced ulcers as well as inflammatory cell infiltration, with different gender responses in the tested groups; females showed normal anal, while there was a basal cell degeneration and erosion in males of the 0.28 mg/kg/day nicorandil treated group. the dose of 0.4 mg/kg/day nicorandil showed anal ulcer in both sexes. increasing doses to 1 and 3 mg/kg/day of nicorandil produced a small anal ulcer in females, and large anal ulceration in males. positive and negative control groups showed no effect on the anal tissues in both sexes of albino rats. the epithelial cells and vascular endothelial cells in oral mucosa are target cells for leptin, which stimulates angiogenesis in the connective tissue beneath the ulcer, and promotes wound healing in the oral mucosa by accelerating the supply of nutrients, oxygen and even some bioactive substances. leptin promotes wound healing in the oral mucosa by accelerating epithelial cell migration as well as enhancing angiogenesis around the wounded area. this explains the physiological function of leptin in saliva promoting wound healing (31). the increased mucosal level of leptin accompanies acute gastric mucosal injury and also characterizes mucosal inflammatory responses to bacterial infection, and exogenous leptin has been demonstrated to exert protective effects against gastric injury induced by ischemia-reperfusion as well as to accelerate the healing of experimentally induced gastric ulcers (50-54). studies with other tissues indicate that the release of adiposederived hormones such as leptin, adiponectin and resistin is regulated by vascular factors such as et-1, a potent vasoconstrictor recognized for its role in normal tissue repair, and play a role in mediation of local leptin release (55-57). this study showed that plasma leptin levels were decreased significantly with the administration of 0.4 mg/kg/day, and highly significantly by the administration of 1 and 3 mg/kg/day, which explains the dosedependent effect of nicorandil. the aspirin positive control group showed a nonsignificant elevation of the plasma leptin concentration. the lowest used dose (0.28 mg/kg/day) and negative control group showed a non-significant reduction of plasma leptin levels. there is a significant difference within groups before and after 10 days of treatment by nicorandil. the ulcer repair process, both in humans and in experimental ulcer models, is mediated by the secretion of growth factors, enzymes and extracellular matrix components (58), which is delayed if prostaglandins are depleted (59). prior work has demonstrated that ulcer induced by 50 mg aspirin showed a notable reduction in the serum levels of tgf-β, while the group in which ulcer was induced by 10 mg nicorandil showed a very slight reduction in the serum levels of tgf-β. this was a nonsignificant statistical difference, when compared to the controls that showed a slight increase in this cytokine level (60) . prostaglandins do not represent a unique pathway to protect the mucosa. nitric oxide (no) has the potential to counteract potentially noxious effects of inhibition of prostaglandin synthesis (61). no has well characterized inhibitory effects on neutrophil activation/adherence demonstrated in various tissues (62). agents that are weak inhibitors of prostaglandin synthesis are less ulcerogenic (63). the mean plasma prostaglandin e2 concentrations assessed in this study through tested doses of nicorandil compared to the positive control (aspirin) and n.s. negative control showed that there was non-significant reduction of plasma pge2 levels when comparing concentrations of baseline and after 10 days of treatment. it may be the case that the short period of observation played a role in this finding. the mean plasma baseline leptin concentration was highly significant (p= 0.001), and after 10 days of treatment was significantly (p= 0.014) less in female than male albino rats. comparison of pre and post plasma leptin values was significantly reduced in both sexes of albino rats. this study agrees with that studied by landt et al. 1998 (64); sex difference is reversed in rats, with male rats having higher leptin concentrations than female rats. this difference is likely to be due to the greater amount of body fat in male rats. iraqi j pharm sci, vol.25(2) 2016 nicorandil effects on leptin and pg e2 23 the mean baseline plasma pge2 concentration was highly significantly higher (p= 0.002), and after 10 days of treatment showed nonstatistically significantly higher pge2 levels in female than male albino rats. the findings of this study agree with those of paul et al. (2011) (65), which found that the mean plasma leptin concentration was significantly higher in males than in females when compared to males before and after treatment. the serum leptin level increased as body mass index (bmi) increased, irrespective of gender. the elevation of body weights of albino rats that received 3 mg/kg/day nicorandil was highly significant (p= 0.008), and a statistically significant increase of rats’ body weight was seen with albino rats administered 1 mg/kg/day nicorandil (p= 0.024), while 0.4 mg/kg/day and negative control groups showed a nonsignificant difference in the rats’ body weights. comparison with the aspirin positive control group, there was a significant reduction of albino rat’s body weights after 10 days of treatment (p= 0.036). the correlation analysis of this study showed that there was a weak, non-significant positive correlation between body weights and plasma leptin concentrations after treatment. for pge2 concentration, there was a weak, nonsignificant negative correlation between rats’ body weights and plasma prostaglandin e2 concentrations after treatment.salivary leptin may have a role in the maintenance of oral health (66). the nicorandil-induced ulceration pathogenesis is unclear. this study showed that plasma leptin concentration was found to play a role in the dose-dependent nicorandilinduced ulceration effects. leptin has emerged as an important regulator of mucosal inflammatory response that may lead to ulceration after administration of different doses of nicorandil in a dose-dependent effect. understanding the method by which nicorandil causes mucous membrane damage, inflammation, and ulceration will help in the development of a prophylactic agent that reduces its toxicity. in conclusion, the present study, clearly demonstrated for the first time that highly significantly decreased plasma leptin level might be one of the mechanisms behind the adverse ulceration effects of nicorandil administration. also, it has been suggested that there is a minimal dose required to induce ulceration, which would point to a dose-dependent effect of nicorandil administration inducing adverse ulceration effects. acknowledgements the authors would like to thanks all that had helped to finish this work. references 1. bruce aj, and rogers iii rs. acute oral ulcers. dermatol clin. 2003; 21: 1-15. 2. o'neill a, and de leon j. bipolar disorders: two case reports of oral ulcers with lamotrigine several weeks after oxcarbazepine withdrawal. 2007; 9: 310313. 3. bertini f, costa nc, brand?o aa, cavalcante as, and almeida jd. ulceration of the oral mucosa induced by antidepressant medication: a case report. j med case rep. 2009; 3: 98. 4. ciantar m, and gibson j. nicorandilinduced oral ulceration: case report. malta med j. 2008; 20: 30-33. 5. marquart-elbaz c, lipsker d, grosshans e, and cribier b. 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105: 1630-1636 64. landt m, gingerich rl, havel pj, mueller wm, schoner b, hale je et al. radioimmunoassay of rat leptin: sexual dimorphism reversed from humans. clinical chemistry. 1998; 44: 565-570. 65. paul rf, hassan m, nazar hsh, gillani s, afzal n, and qayyum i. effect of body mass index on serum leptin levels. j ayub med coll abbottabad. 2011; 23: 40-43. 66. gröschl m, topf hg, kratzsch j, d?tsch j, rascher w and rauh m. salivary leptin induces increased expression of growth factors in oral keratinocytes. journal of molecular endocrinology. 2005; 34: 353366. iraqi j pharm sci, vol.31( 2 ) 2022 neuroprotective effect of vinpocetine doi: https://doi.org/10.31351/vol31iss2pp129-134 129 neuroprotective effect of vinpocetine against lead acetate-instigated neurotoxicity in rats by evaluation tumor necrosis factor-alpha, interleukin-1beta and interleukin-10 manal abdulkhaliq ibrahim*,1, wameed hashim abbas**, muhsin sagheer ghalib*, nada n. al-shawi * ** *department of pharmacology and toxicology, college of pharmacy, university of basrah, basrah, iraq. **department of microbiology, alzahraa college of medicine, university of basrah, basrah, iraq *** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq . abstract lead toxicity elicits neurological damage which is a well-known disorder that has been considered to be a major cause for multiple condition such as behavioral defect; mental retardation; and nerve insufficient activity. this research is designed to estimate potential protective effect of vinpocetine on neurotoxicity stimulated by lead acetate in rats. eighteen adult rats of both sexes were randomly enrolled into three groups. each group includes 6 rats as followings: group irats were given 0.3ml normal saline solution orally; then intraperitoneal injection of 100μl of the normal saline was given 1h later; this group was considered as control. group iirats were given an intraperitoneal injection of 20mg/kg lead acetate for 5 days. group iiirats were orally given 3mg/kg vinpocetine, which was given 1hr before [(the ip injection of pb every 24 hours at a dose of 20mg/kg) for 5 days and continued for 10 days]. on 11th day of the study, the brain of each animal has been surgically cut-out to make homogenate preparation to estimate tumor necrosis factor-alpha (tnf-α), interleukin-1 beta (il-1β), and interleukin-10 (il10) levels. lead significantly elevated tnf-α and il-1beta; while, it significantly decreased il-10 levels. vinpocetine significantly minimized il-1beta and tnf-α; furthermore, vinpocetine significantly raise il-10 levels at (p<0.05). vinpocetine may have a neuro-protective activity against lead-stimulated toxicity brain of rats. keyword: lead, vinpocetine, rats, neuroprotective, cytokines. أسيتات الرصاص في الجرذان عن المستحثة بواسطةالتأثير الوقائي للفينبوسيتين ضد السمية العصبية 10بيتا وإنترلوكين 1طريق تقييم عامل نخر الورم ألفا وإنترلوكين * **ندى ناجي الشاوي و *محسن صغير غالب ،**ميض هاشم عباس ، و ، *منال عبد الخالق ابراهيم بصره ،العراق. ، البصرةجامعة كلية الصيدلة، ، والسموم فرع االدوية * بصره ،العراق. ، البصرة جامعة فرع االحياء المجهرية ،كلية طب الزهراء، ** .بغداد ،العراق جامعة بغداد، كلية الصيدلة، فرع االدوية والسموم ، *** الخالصة الضرررا العصرربن ال اج مي ررمية الر رراة هو حالة معروفة جيداه حيس س ها س رران للعديد مي االلررلرابا معق ال؛ ل الع لن ت تصمي هذا العمق لل؛ح ق فن ال شاط الوقائن للفي بو ي؛يي ملى السمية العصبية ال؛ن تسببها س ي؛ا الر اة . تل األمصاب المشاكق السلوكية : جرذان ل ق م ها مي6 ت ا ررر؛ دام نما ية مشرررر جرذا بالغها مي كس الج سررريي بشررر ق مشررروائن فن نس مجموما م و : اللري ة .فن الجرذان مي محلو ملحن داخرق مي رول؛ر 100 ت ح هرا ب وبعرد ررررامر .مرق مي محلو ملحن مي طريق الف 0.3سملير الجرذان المجمومرة األولى سيام بجرمة 5سملي الجرذان ح ة داخق الصرفاق مي س ري؛ا الر راة لمدة : المجمومة العا ية. تعد هذه المجمومة كمجمومة ريلرة. الصرفاق فن ح ي الر رررراة سيرام قبرق البردء 5لمردة )الفي بو رررري؛يي كج مي /مج 3سملير الجرذان مي طريق الف : المجمومرة العرالعرة. كج /ملج 20 فن اليوم . سيام 5كج لمدة /مج 20 حيس ت إملاؤه قبق رامة واحدة مي ح ي الر راة داخق الصرفاق يومياه بجرمة ( سيام 10واال ر؛مراا لمدة (tnf-α) ل يان مامق ر الوام سلفا الحادي مشرررر مي الداا رررة ، ت ا ررر؛ صرررا دماح كق جرذ جراحيها لعمق تحضرررير م؛جا م وذل اال ؛رلوكيي واحد بي؛ا عبااتفا رربب ا رري؛ا الر رراة بشرر ق مع وي: ال ؛ائج (il -1β)اال ؛رلوكيي واحد بي؛ا و (il-10)اال ؛رلوكيي مشرررةو سريج الدماح. الف بو ر؛يي قلق اال ؛رلوكيي مشررة فن ج ا رة إلى ا فاض مسر؛ويا بي ما ربب الر راة بشر ق مع وي ومامق ر الوام الفا م د اال ؛رلوكيي مشرررة فن ج ا ررة سرريج الدماح فن افع ومامق ر الوام الفا وكان ل دوا مع وي اال ؛رلوكيي واحد بي؛ا مي بشرر ق مع وي (p<0.05) . ا ي؛ا الر اة فن الجرذان تأنير وقائن لد السمية العصبية المس؛حعة بوا لةلف بو ؛يي ل ا: اال ؛ ؛اج . .سايتوكينات,حماية عصبية , الجرذان, فنبوستين ,الرصاص :الكلمات المفتاحية 1corresponding author e-mail: manal.ph2008@yahoo.com received: 18/9 /2021 accepted:7 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp129-134 iraqi j pharm sci, vol.31(2) 2022 neuroprotective effect of vinpocetine 130 introduction exposition to lead (pb) can occur through many routes involving contaminated water, air, food, soil, and other public pathway; and the secure threshold for pb exposure has not been specified, as there is no accurate amount for toxicity of such element (1). pb is available in various forms and is considered as a basic ingredient of different organic compounds, which have been directly penetrated to skin, respiratory, and brain; where, toxic effect of central nervous system is considered as a dominant effect of such element (2). thus; pb can cause significant public health problems, although its concentration in ecosystem has been decreased after several trials (3). researchers published that neurological destruction that stimulated by pb toxicity can urge various disorders like mental defect, alzheimer's disease (ad), behavioral problems, loss of nerve activity, parkinson's disease (pd), and probably schizophrenia (4). pb has the ability to pass through blood-brain barrier (bbb) and can substitute calcium (ca+2) ions. accordingly, such element can interfere with activity of ca+2 on cell functions and perturb several biological actions (5). the pro-inflammatory pathway of neurotoxicity instigated by pb has not been completely evaluated (6,7). vinpocetine is an alkaloid vincamine derivative. in many countries, vinpocetine has been utilized for the treatment of central nervous system disorders such as stroke and dementia for more than 30 years. up to date, it is also obtainable in the market as a nutrition supplement to boost memory and cognizance. the safe and marvelous activity of vinpocetine result in discovering the novel remediation and mechanism actions of it in disease pattern and diverse cell types (8). vinpocetine, has boosting the cognitive function that it has been utilized as a nootropic agent for patients with central nervous system disorder; where, it increases glucose uptake and cerebral blood flow (9). moreover, it can reduce the peril of strokes and temporary ischemic attacks in chronic cerebrovascular insufficiency patients (10). as well as, vinpocetine is an efficacious antioxidant and then inhibit lipid peroxidation (11). furthermore, such drug exhibit memory-protective and memoryboosting properties and potent anti-inflammatory activity (12). besides, vinpocetine is a phosphodiesterase-1 [(pde)-1] inhibitor (13) and a blocker of voltage-gated na+ channels (14). previously, in vitro studies approved that vinpocetine inhibited the blockage of the mitochondrial complexes (ii, iii, and iv) as well as entirely negated the deduction of pyruvate levels and the assemble of free radical-stimulated by noxious concentrations of amyloid peptides in pc12 cells (15). it is a potential choice for the management of various neurodegenerative diseases that related to cognitive improvement properties and the antiinflammatory effect of vinpocetine (16). the current study is designed to estimate probable protective action of vinpocetine against neurotoxicity instigate by pb in rats through the estimation of tumor necrosis factor-alpha (tnfalpha), interleukin-10 (il-10) and, interleukin-1beta (il-1β) levels in brain tissue homogenate. materials and methods experimental animals eighteen male and female albino adult rats (weighing 160-250gm) were selected for this study, rats were obtained from house animal for college of pharmacy, basra university. commercial pellets and tap water ad libitum were dependent in feeding of rats during experiment period. materials lead acetate powder was purchased from fluka chemical, turkey. vinpocetine pure powder was purchased from america medic science (usa). experimental design adult rats were randomly distributed into three equal groups (6 animals for each group) as follows: group ieach rat was given 0.3ml normal saline orally for 10 days,5day before ip injection of normal saline and then 100μl of the normal saline solution injected ip 1hr later for 5day; this group considered as control. group iieach rat was given 0.3ml normal saline orally for 10 days,5day before ip injection of pb acetate which freshly prepared (20 mg/kg/day body wt.) for 5 days (17). group iiieach rat was orally given 3mg/kg/day vinpocetine (dissolve in normal saline for 5 days by oral gavage before starting pb injection and continued for 10 days; where it was given 1hr before pb, which was injected ip every day at a dose 20 mg/kg for 5 days (18). twenty four hour after the end of the treatment duration; i.e.at day six, each animal was euthanized by diethyl ether, and then by cervical dislocation. thereafter, the skull was crushed by surgical scissor and then the brain of each rat has been cutout surgically for homogenate preparation. preparation and estimation of homogenate biochemical parameters the preparation of brain tissue homogenate involved removal of excess blood by rinsing in icecold phosphate buffer saline (pbs,ph=7.4), followed by desiccation using filter paper and then measuring the weight of each brain tissue before homogenization was performed. then each of rats' brain tissue minced to small pieces and put in 15ml plastic test tube containing chilled pbs solution (ph=7.4); where ratio of tissue weight in g to pbs volume in ml is 1:9. homogenization was performed using cell lab homogenizer in icy condition. then, the homogenate was centrifuged for approximately 15 minutes at 2000×g. the supernatant liquid was accurately collected and kept iraqi j pharm sci, vol.31(2) 2022 neuroprotective effect of vinpocetine 131 at -20 ºc until the time for the evaluation of tnf-α, il1β, and il-10 cytokines levels by automated biochemistry analyzer (elabscience, usa) (19). statistical analyses data were explicated as mean±standard error (sem). anova –post hoc test was utilized for estimating the significant difference among groups. differences were statistically considerable for p value less than 0.05 (p<0.05). results effect of vinpocetine against lead acetate on tumor necrosis factor-alpha (tnf-α) in rats' brain tissue homogenate. table 1 and figure 1 showed that rats injected with 20 mg/kg of pb acetate ip every day for 5 days lead to a significant elevation in the level of tnf-α in homogenate tissue of brain compared to those level in control rats. the tnf-α level in brain tissue homogenate were respectively, 307.5±17.1and 83.3±5.3. furthermore, there were significant reduction in tnf-α level in homogenate tissue of brain in group of rats treated with 3mg/kg vinpocetine prior to 20 mg/kg of pb acetate compared to the corresponding level in group of rats injected ip with 20 mg/kg of pb acetate every day for 5 days . the tnf-α level in brain tissue homogenate were respectively, 173.3±4.4 and 307.5±17.1. effect of vinpocetine against lead acetate on interleukin-1beta (il-1β) in rats' brain tissue homogenate. table 1and figure 2 showed that rats injected with pb acetate every 24hours at a dose 20mg/kg for 5 days ip led to significant rising in the level of il-1β in homogenate tissue of brain compared to control rats. the level of such cytokine in homogenate tissue of brain were respectively, 125.5±6.6 and 61.3±4.9. moreover, table 1, and figure 2 showed that, there were significant reduction in il-1β level in homogenate tissue of brain for the groups of rats treated with 3mg/kg vinpocetine prior to 20 mg/kg pb acetate compared to corresponding levels in group of rats injected ip with 20 mg/kg of pb acetate every day for 5 days. the level of il-1β in homogenate tissue of brain were respectively, 84±3.9 and 125.5±6.6. effect of vinpocetine against lead acetate on interleukin-10 (il-10) in rats' brain tissue homogenate. table 1 and figure 3 showed that rats injected with 20 mg/kg of pb acetate every day for 5 days ip, there was a significant reduction in the level of il-10 in brain tissue homogenate compared to those levels in control rats. the level of il-10 in homogenate tissue of brain were respectively, 40±2.8 and 205±7.6. furthermore, there were significant raising in the level of il-10 in homogenate tissue of brain in rats treated with 3mg/kg vinpocetine prior to 20 mg/kg lead acetate compared to corresponding levels in group of rats injected with 20 mg/kg of pb acetate every day for 5 days ip. the level of il-10 in homogenate tissue of brain were respectively, 63.8±3.9 and 40±2.8. table 1. effect of vinpocetine on tnf-α, il-1β, and il-10 levels in brain tissue homogenate of rats after ip injection of lead acetate treatment groups n=6 treatment type tnf-alpha (pg ̸ml) (mean±sem) il-1beta (pg ̸ ml) (mean±sem) il-10 (pg ̸ml) (mean±sem) i negative control/ normal saline 83.3±5.3 61.3±4.9 205±7.6 ii lead acetate (20 mg/kg) 307.5±17.1*a 125.5±6.6*a 40±2.8*a iii 3mg/kg vinpocetine prior to 20 mg/kg of lead acetate 173.3±4.4 b 84±3.9 b 63.8±3.9 b * mean significant-different compared to control rats at p<0.05. different letters mean there are significant different in the same column at p<0.05. iraqi j pharm sci, vol.31(2) 2022 neuroprotective effect of vinpocetine 132 figure 1. effect of vinpocetine on tnf-α levels in brain tissue homogenate after ip injection of lead acetate in rats. figure 2. effect of vinpocetine on il-1β levels in brain tissue homogenate after ip injection of lead acetate in rats. figure 3. effect of vinpocetine on il-10 levels in brain tissue homogenate after ip injection of lead acetate in rats. discussion the present study pointed out on markers of inflammation, [(tnf-α, il-1b), and the anti inflammation marker (il-10)] levels after exposure to pb; and this study furthermore inspected neuroprotective action of vinpocetine on above markers. in developing animals, pb can cross bloodbrain barrier (bbb) and deactivate the basic structural components by damaging the brain glial cells. moreover, such element stimulated devastation that mainly-occur in most area of brain that include cerebellum, cerebral cortex, and hippocampus, that may consequently cause morphological change in the brain (20). the destructive effect of lead may be related to its creation of ros or its confliction with calcium inactivation of protein kinase c (pkcs), which may have a critical role in signal transduction, differentiation and cell development (21). furthermore, lead vies with calcium for prevalent binding sites and is integrated into neurotransmission systems of calcium (22). the major findings of previous studies that approved raising cytokine creation and axonal destruction with astrocytic activation by pb effect in immature rat brain (23). furthermore, researchers mentioned that pb can cause increment in level of inflammatory cytokines (7). glial cells have basic role in local inflammatory processes by creation cytokines such as tnf-α, il-1β, il-6. furthermore, the neuroinflammation was controlled by activation of glial cells which participated in destructive and progression of several disorders. in alzheimer's disease, inflammatory and oxidative induction effect that were created by chronically glial cells activation which result destruction of neurons (24). in this study, ip injection of pb acetate every 24 hours (20mg/kg) for five days to rats in group ii, significantly increase level of inflammatory markers such as tnf-α and il-β levels of these cytokines, and a significant reduction in il-10 level each when compared with control group rats at (p<0.05). results of this work are agreed with previously-mentioned studies (7, 23). furthermore, this study showed the preventive effect of vinpocetine on inflammatory pathway of brain through suppressing the tnf-α and il-1β elevation; where, oral-administration of vinpocetine for 5 days prior to ip injection of lead acetate for 5 days and vinpocetine continued for 10 days in group iii significantly decrease levels of inflammatory markers such as cytokines tnf-α and il-β levels, but with significant increase in il-10 level when compared with those levels in rats ip injected with pb acetate at (p<0.05). researchers reported that, in the cns; where, the pde inhibitor (vinpocetine) can down-regulate the following inflammatory cytokines [tnf-α, il-1, and il-6], however, it can up-regulate the suppressor cytokines such as il-10 (25) due to the effects of lipopolysaccharides. conclusion vinpocetine may have a neuro-protective action against lead-instigate neurotoxicity in rats. iraqi j pharm sci, vol.31(2) 2022 neuroprotective effect of vinpocetine 133 acknowledgements this is the first study to estimate in vivo neuro-protective action of vinpocetine on leadinstigate neurotoxicity in rats. competing interests there are no competing interests to declare. references 1. ahmed m, meki a, and abdraboh n . neurotoxic effect of lead on rats: relationship to apoptosis. international journal of health sciences 2013 ;(7): 192-199 2. ahmed e. abdel moneim & mohamed a. dkhil & saleh al-quraishy. effects of flaxseed oil on lead acetate-induced neurotoxicity in rats. biol trace elem res 2011;144:904–913. 3. rojas-castañeda jc, vigueras-villaseñor rm, rojas p, chávez-saldaña m, gutiérrez-pérez o, montes s, ríos c: alterations induced by chronic lead exposure on the cells of the circadian pacemaker of developing rats. int j exp pathol 2011;4: 243-50. 4. liu j, han d, li y, zheng l, gu c, piao z, au w, xu z, huo x . lead affects apoptosis and related gene xiap and smac expression in the hippocampus of developing rats. neurochem res 2010; 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sci. 91, 113–122. 24. struzynska l ˛bouta b, koza k, sulkowski g. inflammation-like glial response in lead-exposed immature rat brain. toxicological sciences 2007;95(1), 156–162 . 25. yoshikawa m, suzumura a, tamaru t, takayanagi t, sawada m. effects of phosphodiesterase inhibitors on cytokine production by microglia. mult scler 1999; 5(2):126–133. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 treatment of chronic hepatitis c virus genotype 4 doi: https://doi.org/10.31351/vol31iss1pp57-64 57 effect of treatment of ombitasvir with paritaprevir/ritonavir plus ribavirin on naïve patients with chronic hepatitis c virus genotype 4 mohammed abdel-gabbar*, mohammed alkot**,1 and adel abdel-moneim*** *biochemistry department, faculty of science, beni-suef university, p.o. box 62521, beni-suef, egypt. **family medicine department, faculty of medicine, menoufia university, egypt. umm alqura university, saudia arabia. ***physiology division, zoology department, faculty of science, beni-suef university, p.o. box 62521, beni-suef, egypt. abstract direct-acting antivirals (daas) combination therapies from various mechanisms of action and families have been revolutionized the management landscape of chronic hepatitis c virus (hcv). ombitasvir, paritaprevir with ritonavir (obv/ptv/r) ± ribavirin (rbv) is approved to treat hcv genotype 4 (gt4) infections. here, our objective was to delineate the efficacy and safety of obv/ptv/r plus rbv in treating of egyptian naive patients infected with hcv gt4. a cohort of 100 egyptian patients infected with hcv gt4 was allocated and administered orally obv/ptv/r with rbv. the primary endpoint of our study was a sustained virological response (hcv rna < 12 iu/ml) 12 weeks after the cessation of the treatment (svr12). this study is registered with clinicaltrials.gov, number nct04378608. among treatment naive patients with obv/ptv/r+ rbv, svr12 rates achieved 97% (97/100) in overall patients. regarding treatment failure, the regimen recorded 3 % had treatment failure (0 null-responses, 3 relapses). however, the most frequently common adverse events recorded were a headache (28%), fatigue (18%), asthenia (23%), nausea (19%) and dyspnea (14%). the interferon-free regimen combination of obv/ptv/r plus rbv achieved excellent svr12 rates, 97%, with virologic outcome failures 3%, and it was generally safe and well tolerated for treating naïve patients infected with hcv gt4. keywords egyptian naïve, hcv, genotype 4, il-28β, ombitasvir, paritaprevir, ritonavir, ribavirin. abbreviations used. . تأثير عالج أومبيتاسفير مع ريبافيرين وباريتابرفير/ ريتونافير على المرضى الغير معالجين و 4والنمط الجيني سى المصابين بالتهاب الكبد الفيروسى المزمن من النوع *** عادل عبد المنعم و 1 ،**، محمد القط * محمد عبد الجبار بني سويف ، مصر. 62521قسم الكيمياء الحيوية ، كلية العلوم ، جامعة بني سويف ، ص. ب. * جامعة أم القرى ، المملكة العربية السعودية. ، قسم طب األسرة ـ كلية الطب ـ جامعة المنوفية ـ مصر ** *** شعبة الفسيولوجيا قسم علم الحيوان كلية العلوم جامعة بني سويف، ص.ب. 62521 الخالصة المباشر المفعول ذات الفيروسات بمضادات العالجات أحدثت الوبائي (daas) لقد الكبد التهاب فيروس استئصال مشهد في ثورة . (hcv) المزمن . hcv (gt4) لعالج (obv / ptv / r/rbv) لقد تمت الموافقة على استخدام .hcv gt4 في عالج المرضى المصريين المصابين بفيروس obv / ptv / r plus rbv كان هدفنا هنا هو تحديد فعالية وسالمة hcv) عن طريق الفم هذا العالج. تعتبر النتيجة ايجابيةوإعطائهم hcv gt4 مريض مصري مصاب بفيروس 100تم تخصيص مجموعة من rna <12 iu / ml) أسبوًعا من توقف العالج 12بعد. ( في إجمالي 97/100% ) 97معدل svr12 حققت معدالت, وقد .nct04378608 برقم clinicaltrials.gov هذه الدراسة مسجلة في ٪( ، 28٪ فشل العالج. ومع ذلك ، كانت االعراض الجانبية األكثر شيوًعا المسجلة هي الصداع ) 3 المرضى. فيما يتعلق بفشل العالج، سجل النظام نظام العالج الخالي من اإلنترفيرون من، وبذلك يمكن ان نستنتج ان ( ٪14٪( وضيق التنفس )19٪( ، والغثيان )23٪( ، والوهن )18والتعب ) obv / ptv / r plus rbv معدالت ممتازة لـ svr12 ،97 وكان العالج بشكل عام آمنًا وجيد لعالج 3٪ ، مع فشل فيروسي بنسبة ، ٪ hcv gt4 .المرضى المصابين بفيروس ر، ريبافيرين، باريتابرفير/ ريتونافير ب، أومبيتاسفي 28، انترلوكيين 4المرضى المصريين، التهاب الكبد الفيروسى سى، النمط الجينى الكلمات المفتاحية : 1corresponding author e-mail: mohammed_elkott@yahoo.com received: 12/3/2021 accepted: 26/ 7/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp57-64 https://clinicaltrials.gov/ct2/show/nct04378608 iraqi j pharm sci, vol.31(1) 2022 treatment of chronic hepatitis c virus genotype 4 58 introduction hepatitis c virus (hcv) infection affects an estimated 71.1 million people have chronic hepatitis c infection worldwide, is considered the major cause of hepatic morbidity and mortality (1,2). moreover, hcv genotype 4 (gt4) accounts for the prevalence of most hcv infections in areas of the middle east, north africa, and sub-saharan africa, and in some european countries (3). moreover, it has a prevalence up to 20 %, and more than 90% in egypt with subtype 4a predominate (4). importantly, there has been a revolution in the curing of chronic hcv whereby a few different direct-acting antivirals (daas) combination from of action and families have run into advanced to heighten virological outcome, minimize the side effects, devalued the risk of resistance, and reduce the time of the therapy (5). with the emergence of excellent efficacious hcv daas regimen options, the egyptian national committee for control of viral hepatitis began recruiting patients for treatment in september 2014 and by the end of 2017, about 1.5 million egyptians was enrolled for treatment purpose (6). the combination therapy of ombitasvir (obv), paritaprevir (ptv), and ritonavir (r) (obv/ptv/r) has been approved (7), and each of these combined regimens is largely metabolized in the liver, with lower renal clearance; as such, this regimen is not contraindicated in treatment of chronic renal failure patients (7). however, obt/ptv/r regimen is preferred for patients with creatinine clearance less than 30 ml/min. (8). going ahead, the standard of care for chronic hcv patients is a combination of two to four daas (9). thus, a fixed-dose tablet comprising obv/ptv/r plus ribavirin (rbv) is a valuable for the treatment of chronic hcv gt4 infection (10). ritonavir (a cyp3a inhibitor) is added to boost ptv exposure (11), and this combined regimen is considered one of the recommended options for the american association for the study of liver diseases (aasld) and infectious diseases society of america (idsa) guidelines for treatment-naive and experienced patients infected with chronic hcv gt4 (12). the il-28b gene, which is located on chromosome 19q, was discovered in 2003 (13). il28b gene polymorphism is considered as a strong predictor of svr (14). patients who have the il28b cc genotype are more likely to respond to peg-ifn and rbv treatment, whereas patients who have the tt genotype are more likely to be non-responders (15). previous clinical trials indicated that the obv/ptv/r combined therapy had highly efficacious in different scenarios, such as treatment of genotype1a, 1b and 4 chronic hcv as well as in treating naive, experienced, cirrhotic and noncirrhotic patients; all of them achieved svr rates around 90-100% (16, 8). fortunately, obv/ptv/r regimen is generally considered as lower acquisition cost than that of most sofosbuvir-based regimens (17), and that is an extra-beneficial for the developing countries like egypt who approved the free chargebased treatment. however, few dedicated real-life studies with these amazing daa have been done in infected patients with hcv gt4, particularly in egypt. thus, the goal of the present investigation was to study the efficacy and safety of obv/ptv/r plus rbv regimen for 12-weeks treatment of naïve egyptian patients infected with chronic hcv gt4. materials and methods patients’ population treatment-naive patients with hcv gt4 were undergoing treatment in some centers including governmental hospital at beni-suef, egypt. all eligible patients aged 18-70 years and plasma hcv rna level >10,000 iu/ml were enrolled in clinical examination and laboratory investigations. moreover, enrolled patients had liver biochemical markers: albumin<3.5, total bilirubin>1.2 mg/dl, inr>1.2, and platelet count <150,000 mm3. the criteria of exclusion in the current study were based on hepatitis of non-hcv causes, coinfection with other than hcv gt4, hepatitis b or hiv infection, poorly controlled diabetics (hba1c >8) patients, hepatocellular carcinoma, a history of extra-hepatocellular malignancy in the last 5 years, also major severe illness such as congestive heart failure, respiratory failure, evidence of hepatic decompensation. laboratory and blood picture abnormalities such as anemia (hemoglobin concentration of < 10 g/dl) and thrombocytopenia (platelets <50,000 cells/mm3) and (serum albumin <2.8 g/dl, international normalized ratio (inr) of > 2.3, serum total bilirubin concentration of >3.0 mg/dl. a cohort involved 108 patients started treatment from 5 january 2017 to 8 september 2017, 100 patients completed the study and 8 patients lost to follow-up. in addition, a written agreement was gained from enrolled patients. the center's ethical committee approved the study protocol that conformed to egyptian national guidelines, which were performed according to the declaration of helsinki and to good clinical practice guidelines (decision date: 08.11.2016). study design and treatment the regimen was compatible with the protocol of the egyptian national committee for control of viral hepatitis (nccvh). the patients enrolled were treated orally with fixed-dose tablets comprising ombitasvir (25 mg), paritaprevir (150 mg), and ritonavir (100 mg), taken with food once daily. additionally, ribavirin (copegus ®, roche, europe) given oral tablets (total daily dose was dependent on body weight :< 75 kg, 1000 mg; < 75 kg, 1200 mg) and the dose was modified according iraqi j pharm sci, vol.31(1) 2022 treatment of chronic hepatitis c virus genotype 4 59 to patient tolerability. the study included a number of treatment-naive patients who had advanced liver fibrosis (15/108). the existence of advanced liver fibrosis or compensated liver cirrhosis was documented by the histopathological reading of a liver biopsy or liver stiffness measurements ≥9.5 kpa and/or fib-4 score >3.25. efficacy of treatment was expressed by sustained virologic response (svr12), defined as hcv rna level under the quantification level (hcv rna <12 iu/ml) at least 12 weeks after the cessation of treatment. assessment of safety and tolerability all data of adverse events (aes) were recorded throughout therapy administration up to 30 days post the planned eot. safety was assessed by regular clinical examination with physical examination including vital signs, review of any serious adverse events (saes). also, laboratory abnormalities and treatment discontinuations were reported according to who criteria. laboratory investigations including ast (aspartate transaminase), alt (alanine transaminase), serum creatinine, serum bilirubin, prothrombin activity, serum albumin and serum αfetoprotein were done according to the manufacturer's protocol. fib-4 was estimated on the basis of the equation of sterling et al. (18). interleukin28b (il-28b) genotype was estimated by use of pcr, while hcv gt4 genotyping was assessed by the versant-hcv genotype 2.0 assay (lipa) (siemens, germany). quantitative polymerase chain reaction (qpcr) for hcv was estimated by using ampliprep/cobas taqman hcv test version 2.0 (roche diagnostics, branchburg, nj), with a lower detection limit < 12 iu/ml further, hematological parameters were determined using autoanalyzer (micros abx) depending on the manufacturer's protocol. informed consent in studies with human subjects a written agreement was gained from enrolled patients. ethical approval the center's ethical committee approved the study protocol which is conformed to egyptian national guidelines which performed according to the declaration of helsinki and to good clinical practice guidelines, the approval number is (bsu/2016/11/08). the ibr number of beni-suef university is irb00011029. statistical analysis the statistical difference across continuous variables was analyzed using the t-test. statistical package spss statistics 21 (ibm, new york, ny) was used for all data analysis. results are expressed as mean ± standard deviation (sd) or median and number (percentage) for categorical data. in all tests, p<0.05 were considered significant. results overall patient characteristics 100 naive patients completed the treatment therapy, clinical investigations, and analysis, (figure 1). eligible patients were treated 12-weeks with obv/ptv/r plus rbv. concerning interleukin 28, patients with non-cc il-28 β genotype were 77%, while 23% were cc-il-28β genotype patients. further, hcv genotype 4a represents 79% of all gt4 patients (table 1). seven patients had adverse events (anemia, leukopenia, erythrocytopenia, and thrombocytopenia) leading to ribavirin dose modification. no discontinuation occurred due to adverse events related to doses therapy. figure. 1 patients disposition and the study design. obv; ombitasvir, ptv; paritaprevir, r; ritonavir. rbv; ribavirin. iraqi j pharm sci, vol.31(1) 2022 treatment of chronic hepatitis c virus genotype 4 60 table 1. demographics and baseline data for treatment naivepatients with obv/ptv/r plus rbv. bmi: body mass index. hcv, hepatitis c virus, il28b: interleukin 28b; pcr: polymerase chain-reaction. data are presented as mean ± sd or as n (%) antiviral response (efficacy) the current study revealed high rates of virologic responses and very low rates of relapses after 12-weeks treatment-naive patients with obv/ptv/r+ rbv, svr12 rates achieved 97% (97/100) in overall patients. regarding treatment failure, obv/ptv/r + rbv recorded 3 % (3/100) of patients with treatment failure (0 null-responses and 3 relapses) (table 2). moreover, our virologic response was based on "modified intention to treat" analyses (mitt) or per-protocol analysis. table 2. virological response (by modified intention-to-treat (mitt) analysis) note: data are presented as n (%). hcv, hepatitis c virus; eot, end of treatment; svr, sustained virologic response safety assessments concerning safety and tolerability, obv/ptv/r+ rbv regimen, adverse events (aes) occurred in 57 patients (57%) and were generally mild and transient. most frequently aes recorded; a headache (28%), asthenia (23%), nausea (19%), fatigue (18%), dyspnea (14%) and decreased hemoglobin concentration (8%). further, no deaths recorded in the present study, and only 2 patients with serious aes (saes) were reported (patients hospitalization due to bleeding), but not caused discontinuation of the treatment course. while seven patients (7%) had aes leading to ribavirin dose modification (figure 1, table 3). ameliorations were achieved in terms of the significant decreases in sera ast and alt activities (p<0.05) after 12 weeks post-treatment relative to those before the commencement of the study (figure 2). no patients received erythropoietin for managing anemia. parameters overall patients10 0 patients (n) age year (mean ± sd) 48.9 ± 10.7 sex (m/f) 45/55 bmi (mean ± sd) 26.8 ± 4.3 fib-4 score, n (%) <1.45-3.25: none to moderate fibrosis 86(86) >3.25: advanced fibrosis or cirrhosis 14(14) hcv genotype, n (%): 4a 79(79) 4m 5(5) 4n 7(7) 4o 9(9) il28b genotype, n (%): cc 23(23) none-cc 77(77) platelets <150 x 103/l, n (%) 10(10) albumin <3.5 g/dl, n (%) 3(3) hcv pcr, n (%) <800,000 39(39) >800,000 61(61) parameters patients (n = 100) hcv rna, eot, n (%) 100(100) hcv rna, svr12, n (%) virologic failure, n (%) 97(97) 3(3) 1-non-response, n (%) 0(0) 2-relapsers, n (%) 3breakthrough, n (%) 3(3) 0 iraqi j pharm sci, vol.31(1) 2022 treatment of chronic hepatitis c virus genotype 4 61 table 3. adverse events (aes) and laboratory abnormalities in overall patients. side effects patients (n = 100) n (%) any adverse event during treatment 57(57) aes leading to discontinuation 0 aes leading to rbv dose modification serious adverse events 7(7) 2(2) common adverse events headache 28(28) asthenia 23(23) nausea 19(19) fatigue 18(18) dyspnea 14(14) insomnia 9(9) irritability 7(7) laboratory adverse events decrease hemoglobin 8(8) thrombocytopenia 1(1) leukopenia 0 erythrocytopenia 3(3) elevated ast 1(1) elevated alt 0(0) hyperbilirubinemia 1(1) note: data are presented as n (%). abbreviations: alt, alanine transaminase; ast, aspartate transaminase; rbv, ribavirin figure 2. changes in transaminases at 12 weeks post-treatment among treatment-naive patients treated with obv/ptv/r plus rbv. discussion recently, oral daas combination regimens are currently achieving viral outcome more than 90% of hcv patients after treatment of 12 weeks (9). therefore, the strategy of drug combination regimens will become a major idea to prevent treatment failure and relapse. despite the positive clinical trial outcomes, few studies have provided real-life data regarding hcvgt4 patients, especially in egypt which has the highest burden of hcv globally. in the present study, the efficacy outcome clear that treatment-naive patients receiving 12 weeks obv/ptv/r+ rbv achieved 97% (97/100) svr12 rates, and treatment failure was 3 % (3/100) in overall treated patients. in parallel with our results, asselah et al. in agate-i study revealed that 12 weeks administration of obv/ptv/r+ rbv achieved 97% svr12 rate of patients with compensated cirrhosis, and 98% after 16 weeks treatment. moreover, the authors added that obv/ptv/r with rbv achieved 100 % svr12 when administered for 12 weeks to non-cirrhotic naive and experienced patients (19). in other trial, agate-ii, multicenter, phase iii trial performed in treatment-naïve or-experienced egyptian patients infected with chronic hcv gt 4, waked et al. indicated that 12 weeks treatment of obv/ptv/r + rbv achieved 94 % svr12 rate in non-cirrhotic patients, while in cirrhotic cases svr12 was 97% or 93 % after treatment for 12 or 24 weeks, respectively (20). in el kassas et al. study, experienced patients who received 24 weeks of obv/ptv/r/rbv resulted in 100 % svr 12. while the addition of sof to the obv/ptv/r/rbv regimen resulted in 97 % svr after 12 weeks of treatment (21). moreover, flisiak et al. (22), in real-world evidence, have been deduced the antiviral potency of obv/ptv/rdasabuvir (dsv)-rbv in the road of treatment of hcv gt (1&4) infections. additionally, wedemeyer et al. revealed that obv/ptv/r+ dsv± rbv therapy achieved high rates of svr12 in the treatment of hcv-gt (1&4). whereby, overall viral outcome achieved svr12 rates of 96.8% for gt1 and 98.9% for gt4 infection (23). interestingly, the addition of rbv to daas regimens had a significant issue for treating patients with hcv gt4 because gt4 has multiple heterogeneous subtypes (24). on the other side, the multi-targeted regimen of sof plus obv/ptv/r + rbv was well tolerated and achieved excellent svr rates (97%; 109/113) among retreatmentexperienced egyptian patients with prior daa treatments failure (25). moreover, our results cleared that baseline characteristic including gender, age, bmi, hcv-rna levels, gt4 subtype and il-28β genotype, may not impact on the virologic outcome. in the era of oral daas combination therapies, the positive feedback of the cc il-28β genotype varies by type of treatment regimen (26). il-28, which activates interferon-stimulated genes, is the orchestra maestro of innate immunity against hcv burden (27). so, the present study, which achieved svr12 rate of 97% in overall patients, covers the most patients that had non-cc il-28β genotype (77%), suggesting that this host genotype does not have negative feedback on the virological outcome when treated with the current therapy. on the other hand, an alternative interpretation is that the https://www.pubfacts.com/author/h+wedemeyer iraqi j pharm sci, vol.31(1) 2022 treatment of chronic hepatitis c virus genotype 4 62 excellent svr12 was achieved regardless of il28β genotype. it was also revealed that the improved liver disease markers were maintained 3 years post treatment of patients with gt1 (28). on the other hand, duration extension of treatment to 24 weeks did not show boosted efficacy than that of 12 or 16 weeks. (29). the profile of daas combination regimens was favorable and safety, and the tolerability of regimen was mostly mild or moderate in our real-life cohort. common aes reported in our study observed in 57% of patients were headache (28%), fatigue (18%), asthenia (23%), nausea (19%), dyspnea (14%) and decreased hemoglobin concentration (8%). the current data were in line with that reported in agate-i trial; the most of aes were of mild to moderate in severity. whereby the most frequently reported aes were asthenia (18%), fatigue (17%), headache (23%) and anemia (15%) (19). also, in pearl-i study where treatment-naive patients were treated with obv/ptv/r ± rbv, aes were 77 and 88 %, respectively (30). additionally, the most common aes reported by obv/ptv/r plus rbv regimen were headache (41%) and fatigue (35%) in chronic hcv gt4 egyptian patients without cirrhosis in waked et al., study (20). the limitations of our study are the small sample of patients and the absence of treatmentexperienced patients. further, the study includes the absence of baseline resistance tests. conclusion the interferon-free regimen of daas combination, obv/ptv/r plus rbv, achieved high svr12 rates with virologic outcome failures 3% (0% null-responses, 3% relapses). this regimen also achieved high svr regardless of il28b genotype. moreover, the regimen was generally safe and well tolerated with no drug interruptions or discontinuations due to different adverse events in treatment-naive patients with chronic hcv-gt4 infection in egypt. further studies are required to sketch a complete picture describing the relationship between il-28β genotypes and each of ifn-free regimens outcomes. acknowledgment authors are thankful to the staff member of the treatment centers in beni-suef, egypt, especially mohammed ramadan, msc., and omnia a. hasan, md, health administration at beni-suef, to their valuable aids to accomplish this study. conflict of interest we declare that there is no conflict of interest and we did not get any type of funds for this experiment nor for publishing. credit author statement mohammed abdel-gabbar conceptualization; data curation; formal analysis; investigation; methodology; resources; software; validation; visualization; roles/writing original draft; writing review & editing. mohammed alkot conceptualization; investigation; methodology; visualization; roles/writing original draft; writing review & editing. adel abdel-moneim conceptualization; data curation; methodology; project administration; resources; software; validation; supervision; visualization; roles/writing original draft; writing review & editing. references 1. hollande c, parlati l, pol s. micro-elimination of hepatitis c virus. liver int. 2020; 40 suppl 1:67-71. doi: 10.1111/liv.14363. 2. abdel-moneim a, aboud a, abdel-gabaar m, et al. efficacy and safety of sofosbuvir plus daclatasvir with or without ribavirin: large reallife results of patients with chronic hepatitis c genotype 4. hepatol int. 2018;12:348–355. https://doi.org/10.1007/s12072-018-9868-8. 3. messina jp, humphreys i, flaxman a, brown a, cooke gs, pybus og, barnes e. global distribution and prevalence of hepatitis c virus genotypes. hepatology. 2015 jan;61(1):77-87. 4. 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abt450/r-ombitasvir and dasabuvir with ribavirin for hepatitis c with cirrhosis. n engl j med. 2014;370:1973–1982. 27. dawood rm, el-meguid ma, ibrahim mk, et al. dysregulation of fibrosis related genes in hcv induced liver disease. gene, 2018;664:5869, 10.1016/j.gene.2018.04.032 28. poordad f, castro re, asatryan a, et al. longterm safety and efficacy results in hepatitis c virus genotype 1-infected patients receiving ombitasvir/paritaprevir/ritonavir + dasabuvir ± ribavirin in the topaz-i and topaz-ii trials. j viral hepat. 2020;27:497–504. doi:10.1111/jvh.13261 https://doi.org/10.1007/s12072-018-9915-5 https://www.pubfacts.com/author/h+wedemeyer https://www.pubfacts.com/author/a+craxi https://www.pubfacts.com/author/e+zuckerman iraqi j pharm sci, vol.31(1) 2022 treatment of chronic hepatitis c virus genotype 4 64 29. toyoda h. direct-acting antiviral therapy for chronic hepatitis c virus genotype 4 infection: exploring new regimens. health sci rep. 2019;2:e106. doi:10.1002/hsr2.106 30. hezode c, asselah t, reddy k.r, et al. ombitasvir plus paritaprevir plus ritonavir with or without ribavirin in treatment-naive and treatment-experienced patients with genotype 4 chronic hepatitis c virus infection (pearl-i): a randomized, open-label trial. lancet. 2015;385:2502–2509. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 nonalcoholic fatty liver disease (nafld) doi: https://doi.org/10.31351/vol31iss2pp135-143 135 therapeutic effects of vitamin e in non-alcoholic fatty liver disease: an open-labeled clinical trial amal j.fairooz *,1, mohammed y.jamal *, and nawal m.alkhalidi** *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq ** gastroenterology & hepatology teaching hospital, medical city -,ministry of health and environment, baghdad, iraq abstract non-alcoholic fatty liver disease is one of the widespread chronic liver diseases; it is ranging from simple fat buildup in the liver (steatosis) to non-alcoholic steatohepatitis with the presence of inflammation and hepatocyte injury. vitamin e is one of the most potent antioxidants, in addition to the antioxidant effect of vitamin e; it has anti-inflammatory and anti-apoptotic properties. lifestyle modifications are the cornerstone for nonalcoholic fatty liver management to lose weight and reduce hepatic fat content. to assess the effects of vitamin e and lifestyle modifications on the degree of fatty infiltration in the liver, liver enzymes, and lipid profile. this is a prospective pre-post intervention open-labeled clinical trial which was performed in gastroenterology and hepatology teaching hospital in baghdad\iraq, the duration of this study was seven months from january 2021 to july 2021, (39) participants were included after being diagnosed with nafld by the specialized physician depending on ultrasonography findings, they were administered vitamin e 800iu\day for 12 weeks and advised to take low fat, low carbohydrate diet and increase physical activity. steatosis score, liver enzymes, fasting serum glucose, and lipid profile were measured at baseline and repeated at 4 weeks and 12weeks of the study period. anova test was used to compare the measures among baseline, 4 weeks, and 12 weeks of the study, least significant difference lsd was used to find out the significant measures, pearson chi-square test was used to compare steatosis score throughout the study. this study found that there is an association between steatosis score and vitamin e use with lifestyle modifications as the score reduced significantly throughout the study (p=0.0001) whereas 46.2% of the participants turned into a score (0) at 12 weeks of the study. liver enzymes alt, ast, and alp did not show significant differences among baseline, 4 weeks, and 12 weeks (p=0.211, p=0.052, and p=0.352 respectively), however, ast showed significant differences between 4 weeks versus12 weeks (p=0.039) and baseline versus 12 weeks (p=0.032). ldl, vldl, and hdl did not show significant differences among baseline, 4 weeks, and 12 weeks (p=0.569, p=0.195, and p=0.949 respectively) while total cholesterol and triglyceride showed significant difference (p=0.001and p=0.0001 respectively). bmi showed a significant difference between baseline and 12 weeks (p=0.015). fasting serum glucose did not show a significant difference throughout the study (p=0.122). vitamin e with lifestyle modifications has beneficial effects for patients with nafld as steatosis score, total cholesterol, triglyceride, and bmi had reduced significantly during the study period. keywords: nonalcoholic fatty liver disease (nafld), steatosis score, vitamin e في مرض الكبد الدھني غير الكحولي :تجربة سريرية مفتوحة ذات ذراع لفيتامينھ العالجية التأثيرات واحدة ** الخالدينوال مهدي و *محمد ياوز جمال، 1*،آمال جواد فيروز العراق ، بغداد، جامعة بغداد ، كلية الصيدلة ، *فرع الصيدلة السريرية التعليمي ، مدينة الطب ، وزارة الصحة والبيئة ، بغداد ، العراق امراض الجهاز الكبدي والهضميمستشفى ** الخالصة دهني( جمع مرض الكبد الدهني غير الكحولي هو أحد أمراض الكبد المزمنة المنتشرة. وهو يتراوح من تراكم الدهون البسيط في الكبد )ت من أقوى مضادات األكسدة، باإلضافة إلى أنه له تأثير مضاد الكبد. فيتامين هـ خاليا تضرر إلى التهاب الكبد الدهني غير الكحولي مع وجود التهاب و الكبد الدهني غير المسخدمة في عالج أهم الطرق. تعد تعديالت نمط الحياة المبرمج موت الخاليالألكسدة له خصائص مضادة لاللتهابات ومضادة ل إلنقاص الوزن وتقليل محتوى الدهون في الكبد. ولذلك الكحولي .الدهون هيئةوتعديالت نمط الحياة على درجة ارتشاح الدهون في الكبد وأنزيمات الكبد و تقييم تأثير فيتامين هـ بغداد / العراق ، واستغرقت خل ، تم إجراؤها في المستشفى التعليمي ألمراض الجهاز الهضمي والكبد في االتد وبعدقبل سريرية مرتقبةهذه تجربة من لديهم بعد تشخيص مرض الكبد الدهني غير الكحولي مشاركا ٣٩تم تضمين .٢٠٢١زإلى تمو ٢٠٢١هذه الدراسة سبعة أشهر من كانون الثاني وحدة ٨٠٠ هتم إعطاؤهم فيتامين وقبل الطبيب المختص اعتمادًا على نتائج الموجات فوق الصوتية ، الدهني جمعأسبوًعا ونصحهم بتناول نظام غذائي منخفض الدهون وقليل الكربوهيدرات وزيادة النشاط البدني. تم قياس نتيجة الت ١٢دولية يوميًا لمدة أسبوًعا من فترة الدراسة. ١٢أسابيع و ٤بعد و لبدأا عندالدهون هيئةو كلوكوز مصل الدم الصائموإنزيمات الكبد و 1corresponding author e-mail: amalthamer24@gmail.com received: 20/9 /2021 accepted:15 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp135-143 iraqi j pharm sci, vol.31( 2 ) 2022 nonalcoholic fatty liver disease (nafld) 136 أختبار أقل فرق معنوي أسبوًعا من الدراسة ، وتم استخدام ١٢أسابيع ، و ٤، و البدأ المقاييس بين لمقارنة متوسط التباين أالحادي استخدام اختبار تم الدراسة.وجدت هذه الدراسة أن خالل فترة الدهني جمع لمقارنة درجة التمربع كاي ، وتم استخدام اختبارذات الفروقات أالحصائية قراءاتلمعرفة ال ( ٠‚٠٠٠١= pكبير خالل الدراسة )مع تعديالت نمط الحياة حيث انخفضت الدرجة بشكل ه هناك ارتباًطا بين درجة التجمع الدهني واستخدام فيتامين , ناقلة أمين األسبارتات, أسبوًعا من الدراسة. لم تظهر إنزيمات الكبد ناقلة أمين األالنين ١٢( في ٠٪ من المشاركين إلى درجة ) ٤٦‚ ٢ل حيث تحو على التوالي( ٠‚ ٣٥٢= p، و ٠‚٠٥٢= p، ٠‚٢١١= pأسبوًعا ) ١٢أسابيع ، و ٤الفوسفاتيز القلوي فروقًا ذات داللة إحصائية بين البداية ، و ( ٠‚٠٣٢= pأسبوًعا ) ١٢( والبداية مقابل ٠‚٠٣٩= pأسبوًعا ) ١٢أسابيع مقابل ٤ناقلة أمين األسبارتات اختالفات كبيرة بين ، ومع ذلك ، أظهر فروقًا ذات داللة إحصائية بين بروتين الدهني العالي الكثافةو ال, البروتين الدهني منخفض الكثافة جدا بروتين الدهني منخفض الكثافة,.لم يُظهر ال على التوالي( بينما أظهر الكوليسترول الكلي والدهون الثالثية ٠‚٩٤٩= p، و ٠‚١٩٥= p، ٠‚٥٦٩= pأسبوًعا ) ١٢أسابيع ، و ٤البداية ، ( .لم يظهر ٠‚ ٠١٥= pأسبوًعا ) ١٢و بدايةعلى التوالي(. أظهر مؤشر كتلة الجسم فرقًا كبيًرا بين ال ٠‚٠٠٠١= pو ٠‚٠٠١= pفرقًا كبيًرا ) ( مع تعديالت نمط الحياة له آثار مفيدة للمرضى الذين يعانون ه(. فيتامين )٠‚١٢٢= pلوكوز مصل الدم أثناء الصيام فرقًا معنويًا خالل الدراسة )ك والكوليسترول الكلي والدهون الثالثية ومؤشر كتلة الجسم بشكل في الكبد الدهني جمعحيث انخفضت درجة التغير كحولي مرض الكبد الدهني ال من .ملحوظ خالل فترة الدراسة الدھني ، فيتامين ھـ جمع، درجة الت الكلمات المفتاحية: مرض الكبد الدھني غير الكحولي introduction non-alcoholic fatty liver disease (nafld) is one of the widespread chronic liver diseases, it is ranging from simple fat buildup (steatosis) to nonalcoholic steatohepatitis (nash), which is characterized by the presence of steatosis and inflammation, as well as hepatocyte damage(1).in certain cases, nash can develop fibrosis, which can lead to liver cirrhosis. cirrhosis caused by nash is the major cause of hepatocellular carcinoma and the second most common reason for liver transplantation in the united states(2). the prevalence of nafld worldwide was estimated to be 25.2%, with the elevated prevalence was recorded from the middle east and south america (31.8% and 30.4 5, respectively) while africa recorded the lowest rates (13.5% ), as had been observed in a recent meta-analysis(3). there is a strong relationship between metabolic syndrome and nonalcoholic fatty liver disease, furthermore, nafld is identified as a hepatic presentation of metabolic syndrome(4). several risk factors have been linked to nafld, including advanced age, obesity, insulin resistance, type 2 dm, and hyperlipidemia(5,6). insulin resistance is a key component in the onset and progression of nafld. it promotes de novo lipogenesis and lipolysis leading to inflammation and change of lipid metabolism which enhances additional insulin resistance leading to excessive amounts of fatty acids in the liver which in turn causes lipotoxicity, which is the primary cause of hepatocyte impairment and pathogenicity of nafld(7,8). the gold standard for diagnosing nash and the fibrotic stage of nafld is liver biopsy, but it is invasive and expensive so it is not practical to be used in clinical practice(9). several noninvasive diagnostic techniques have been used for nafld diagnosis which include: imaging-based and blood tests(10). there is no single treatment for nafld that has been demonstrated to be completely successful, the mainstays of nafld management are changing dietary habits and increasing physical activity to lose weight, and reducing hepatic lipid contents as weight reduction is favorable and the degree of improvement in liver histology is proportional to the quantity of decreased weight, moreover, even without considerable weight reduction, lifestyle modifications are beneficial for nafld patients, particularly when patients continue the lifestyle modifications program. the majority of researches also showed that changing of lifestyle leads to reductions in risk factors for cardiovascular diseases such as serum lipid values and insulin resistance(11–13). vitamin e (α-tocopherol) has antioxidant, anti-inflammatory, and anti-apoptotic effects. it is considered one of the most potent chain-breaking antioxidants. there are multiple types of tocopherols and tocotrienols, including alpha, beta, and gamma tocopherols; nevertheless, the alpha-tocopherol form is the most common and active in the human body(14). it has been studied in the treatment of nash; due to it is antioxidant property. vitamin e at an 800 iu/day dose was found to be effective more than placebo in the pivens research (pioglitazone versus vitamin e versus placebo for the treatment of non-diabetic patients with nash); it improved liver enzymes as well as steatosis and lobular inflammation but did not affect fibrosis(15). the american association for the study of liver diseases (aasld) and the national institute for health and care excellence (nice) both advocate vitamin e for the management of nafld(16). objectives of the study to assess the effects of vitamin e and lifestyle modifications on: 1. degree of fatty infiltration in the liver. 2. liver enzymes and lipid profile. materials and methods study design and patient recruitment this is a prospective open-labeled study. the participants were recruited from the gastroenterology and hepatology teaching hospital (tertiary center) in baghdad. the study duration was seven months from january 2021 to july 2021. patients were enrolled in this study after being diagnosed by a specialized physician depending on iraqi j pharm sci, vol.31( 2 ) 2022 nonalcoholic fatty liver disease (nafld) 137 ultrasonography and after the screening of inclusion and exclusion criteria and signing of informed consent. inclusion criteria 1. patients of both genders and aged between 18-65 years old. 2. newly diagnosed with nonalcoholic fatty liver disease with any degree of fatty infiltration in the liver. exclusion criteria 1. patients with a history of alcohol consumption. 2. patients with acute liver failure and chronic liver disease. 3. cardiac failure, renal failure, dm type 1and 2, and any severe systemic co-morbidities. 4. previously used vitamin e or has a history of sensitivity to vitamin e. 5. pregnant and lactating women. 6. use of medications has drug-drug interaction with vitamin e or use drugs reported to cause fatty liver as a side effect. 7. patients who did not complete the study. data collection patients, data were collected at baseline and after using vitamin e with lifestyle modifications at 4th weeks and 12th weeks of the study. then measures were compared to assess the effects of vitamin e and lifestyle modifications the following data were obtained from each patient: 1. patient demographic information (name, age, and gender). 2. medical history (liver disease, chronic disease, and chronic medications). 3. patient baseline measures (weight, height, bmi, waist circumference), lab tests (fasting serum glucose, liver enzymes, lipid profile), and the degree of fatty infiltration in the liver. all the participants were advised to take a low-fat, low carbohydrate diet, increase physical activity, vitamin e 800 iu/day was prescribed for 12 weeks, and follow up visits were at 4th week and 12th week of the intervention for measuring of the previous parameters. diagnosis and staging of nafld the degree of fatty infiltration in the liver can be subjectively assessed using ultrasonography imaging. the brightness of the liver, the difference between the liver and the kidney, the us pattern of the intrahepatic vessels, liver tissue, and the diaphragm are all used to grade steatosis. scores were graded as: normal (score 0), mild (score1), moderate (score2) and severe (score3)(17). statistical analysis frequency, percentage, mean and standard deviation were used to present the data. the comparison between baseline, 4weeks, and 12 weeks measures was performed by using of anova test, and then the least significant difference (lsd( test was used to find the significant measures. pearson chi-square test was used to compare the categorical variables. spss-27 was used to analyze the data and statistical significance was evaluated when the p-value ˂ 0.05(18). results sixty patients were enrolled in this study only 39 patients continued the study, the mean age of the participants was 46.2±11.1 years with a range of 25-65 years, and the highest percent (30.8%) was for the age group (40-49) years. female gender was slightly higher than the male (59% and 41% respectively); the mean age of women was 50.1±11.1 with a range of (25-65) years while in men the mean was 40.2±8.6 with a range of (26-57) years. the percentage of housewives was high (43.6%) as the female gender was dominant. hypertension was found in 12.8% of the participants had as demonstrated table 1: table 1. demographic data no % age (years) <30years 2 5.1 30---39 9 23.1 40---49 12 30.8 50---59 9 23.1 =>60years 7 17.9 mean ± sd 46.2±11.1 range 25-65 gender male 16 41.0 female 23 59.0 male age: mean ±sd range 40.6±8.7 26-57 female age: mean ±sd range 50.1±11.1 25-65 occupation governmental-employee 13 33.3 self-employee/worker 9 23.1 housewife 17 43.6 medical history hypertension 5 12.8 no 34 87.2 iraqi j pharm sci, vol.31( 2 ) 2022 nonalcoholic fatty liver disease (nafld) 138 anthropometric measurements of the patients at baseline, obese patients have the highest percent (46.2%) and this percent is still the highest to the end of the study (43.6%) while the percentage of patients with morbid obesity was reduced from (33.3%) at baseline to (12.8%) at 12weeks, the percentage of patients with normal bmi was (12.8%) at 12 weeks. there is a significant association between bmi and vitamin e use with lifestyle modifications (p=0.019) as shown in table 2. table 2. bmi association with lifestyle modifications and vitamin e use at 4weeks and 12weeks of the study as shown in table 3 there are no significant differences in weight and waist circumference means among baseline, 4 weeks, and 12 weeks of the study (p=0.088 and p=0.336 respectively) only bmi showed a low significant difference (p=0.049). there are significant differences in weight and bmi means between baseline and 12 weeks of the study (p=0.029 and p=0.015 respectively) while waist circumference showed no significant difference (p=0.144). table 3. mean ±sd of anthropometric measurements at baseline, 4weeks, and 12weeks of the study liver enzymes and fasting serum glucose as illustrated in table 4 , there is no significant difference in fasting serum glucose levels among baseline, 4 weeks, and 12 weeks of the study (p= 0.122), evermore there is no significant difference between baseline and 12 weeks of the study (p=0.084). there is no significant difference in levels of alt, ast, and alp among baseline, 4 weeks, and 12 weeks of the study (p=0.211, p=0.052, and p=0.352 respectively), whereas ast levels showed significant differences between 4 weeks vs. 12 weeks (p=0.039) and baseline vs.12 weeks (p=0.032) as shown in table 4 . bmi baseline after 4wks after 12wks pearson chi-square test p-value no % no % no % 0.019* normal (18.5-24.9) 5 12.8 overweight (25-29.9) 8 20.5 13 33.3 12 30.8 obese (30-34.9) 18 46.2 15 38.5 17 43.6 morbid obese (=>35) 13 33.3 11 28.2 5 12.8 *significant difference p˂ 0.05. bmi: body mass index. baseline after 4wks after 12wks p-value anov a test lsd test baseline x 4wks 4wks x 12wks baseline x 12wks weight (kg) 91.89±17.47 89.03±15.92 84.14±12.79 0.088 0.417 0.167 0.029 * height (cm) 165.72±10.58 165.72±10.58 165.74±10.59 bmi (kg/m2) 33.42±5.29 32.32±4.89 30.71±4.31 0.049* 0.317 0.145 0.015 * waist circumfere nce (cm) 111.62±11.21 110.26±10.66 108.00±10.71 0.336 0.582 0.361 0.144 *significant difference p ˂ 0.05. bmi: body mass index. iraqi j pharm sci, vol.31( 2 ) 2022 nonalcoholic fatty liver disease (nafld) 139 table 4. mean ± sd of fasting serum glucose and liver enzymes at baseline, 4wks, and 12wks of the study lipid profile there are no significant differences in levels of ldl, vldl, and hdl among baseline, 4 weeks, and 12 weeks of the study (p=0.569, p=0.195, and p= 0.949 respectively) while levels of total cholesterol and triglyceride showed significant differences (p=0.001 and p=0.0001 respectively), furthermore total cholesterol levels showed a significant difference between baseline vs. 4 weeks (p=0.042) and baseline vs. 12 weeks (p=0.0001) whereas levels of triglyceride showed a significant difference between baseline vs. 4 weeks (p=0.009), 4 weeks vs. 12 weeks (p=0.023), and baseline vs.12 weeks (p=0.0001) as showed in table 5: table 5. mean ±sd of lipid profile at baseline, after 4wks and 12wks of the study steatosis score steatosis score was represented as mild, moderate, and severe depending on ultrasonography findings, the highest percentage of patients have mild steatosis (59%) where moderate steatosis and severe steatosis were (33.3% and 7.7% respectively), the percentage of patients who have score 0 (normal liver echogenicity) is (46.2%) at 12weeks. there is a significant association between steatosis score and vitamin e use with lifestyle modifications (p=0.0001) as demonstrated in table 6. baseline after 4wks after 12wks p-value anova test lsd test baseline x 4wks 4wks x 12wks baseline x 12wks fasting serum glucose (mg/dl) 97.18±9.16 96.99±8.89 100.69±8.65 0.122 0.927 0.069 0.084 alt (iu/l) 33.29±16.7 0 32.91±18.2 7 28.50±9.76 0.211 0.344 0.079 0.412 ast (iu/l) 26.38±9.99 26.23±10.4 3 22.08±4.70 0.052 0.938 0.039* 0.032* alp (iu/l) 94.06±31.5 9 93.76±31.8 1 86.18±14.49 0.352 0.962 0.221 0.203 *significant difference p˂ 0.05 level. alt: alanine aminotransferase, ast: aspartate aminotransferase, alp: alkaline phosphatase lipid profile baseline after 4wks after 12wks p-value anov a test lsd test baseline x 4wks 4wks x 12wks baseline x 12wks ldl (mg/dl) 113.21±40.61 104.90±33.94 111.50±34.30 0.569 0.389 0.371 0.974 vldl (mg/dl) 32.01±11.26 28.44±6.49 29.98±7.61 0.195 0.072 0.437 0.303 hdl (mg/dl) 42.19±9.96 42.43±9.68 42.85±7.24 0.949 0.908 0.838 0.750 total cholestero l (mg/dl) 182.53±43.5 6 167.42±25.80 154.87±24.46 0.001* 0.042* 0.091 0.0001* triglycerid es (mg/dl) 166.08±54.5 9 141.29±33.38 119.98±30.48 0.0001* 0.009* 0.023* 0.0001* *significant difference p˂ 0.05 level. ldl: low-density lipoprotein, vldl: very low-density lipoprotein, hdl: high-density lipoprotein iraqi j pharm sci, vol.31( 2 ) 2022 nonalcoholic fatty liver disease (nafld) 140 table 6. steatosis score at baseline, 4wks, and 12wks of the study discussion non-alcoholic fatty liver disease represents the buildup of excess fats in the liver, as observed by imaging or histology in the absence of any secondary etiology and considerable alcohol intake. worldwide urbanization and modernization in the twentieth and twenty-first centuries have been connected to undesirable lifestyle patterns. as a result, the mean worldwide body mass index and occurrence of obesity, which are the main causes of nafld, have increased markedly during the previous three decades. nafld is now the most widespread chronic liver disease in developed countries(11,13,19). in this study the percentage of female patients was slightly higher than male patients. interestingly, the gender-specific predominance of nafld is associated with age: the prevalence of nafld in men rises from youth to middle age, whereas in women it rises approximately 10 years later after men and increases after the age of 50 years(20). however, certain studies revealed that the prevalence in men was higher than in women while others showed vice versa(8). concerning the correlation of the type of occupation with nafld, a cross-sectional study had revealed that working for prolonged time are linked to a greater likelihood of nafld than shorter or standard working times as working for a long time is associated with unhealthy lifestyle such as eating fast food, decreasing of physical activity, and reduction of sleeping hours(21). a cross-sectional study from china found that the prevalence of nafld was higher in women than in men as women were mostly housewives with little physical activity(22), however, this study showed that the percentage of housewives was the highest followed by governmental-employee and self-employed. in this study fasting serum glucose did not change significantly during the study period, even though its mean was increased at 12 weeks of the study but remained within the normal range, however, in a prospective cohort study oral glucose tolerance test was used to detect hyperglycemia in nafld patients, the study showed that older age, elevated bmi, and low hdl levels all of which anticipated the existence of the hyperglycemia, the study recommends the use of oral glucose tolerance test to screen patients with nafld who had one or more of the aforementioned factors to detect and treat impaired glucose metabolism effectively(23). in this study 12.8% of the participants had hypertension, clinical and experimental findings show that nafld may anticipate and/or increase the development of diabetes, hypertension, and cardiovascular disease and the risk of these disorders appears to be linked to nafld severity, as patients with nash and hepatic fibrosis have a higher risk than those with simple steatosis to develop hypertension, type 2 diabetes, and cardiovascular disease(24). the mean weight, bmi, and waist girth was reduced during this study, at 12wks 12.8% of patients were had normal bmi, weight loss has positive effects on nafld patients as exhibited by many studies which demonstrated that weight loss of 5-7% is primarily linked to a reduction in hepatic steatosis, weight loss of 7-10 % increase probability of nash resolution and fibrosis regression while weight reduction ≥ 10% linked to highest chances of nash cure and fibrosis reversion(25,26). according to american and european guidelines, the primary clinical suggestion for the treatment of nafld is lifestyle modifications to obtain progressive weight loss, rising of physical activity, and changing of dietary habits, the type of exercise should be customized according to the decisions of the patients to be sustained over time(27,28). this study showed that the levels of liver enzymes did not change significantly among baseline, 4 weeks, and 12 weeks of the study period since many of the participants had normal liver enzymes at baseline, normal alt levels in nafld\nash patients may be related to genetic causes and personal variations, however, patients with normal alt levels had similar histological and clinical characteristics to patients with high alt levels(29). only levels of ast showed significant difference throughout the study because ast levels were normal in all the participants at 12 weeks. however, recent reviews showed that alt values were within normal ranges in approximately 80% of patients with nafld, and aminotransferase values did not correspond to the extent of liver fibrosis so steatosis score baseline after 4wks after 12wks pearson chisquare test pvalue no % no % no % 0.0001* normal (0) 3 7.7 18 46.2 mild (1) 23 59.0 21 53.8 15 38.5 moderate (2) 13 33.3 13 33.3 4 10.3 severe (3) 3 7.7 2 5.1 2 5.1 *significant difference p˂ 0.05 level. iraqi j pharm sci, vol.31( 2 ) 2022 nonalcoholic fatty liver disease (nafld) 141 increased alt serum levels do not reliably determine disease severity(30,31), whereas a recent systematic review and meta-analysis revealed that 25% of nafld patients and 19% of nash patients had normal alt levels so the alt value as an important metabolic marker does not have enough precision to accurately diagnose nafld and nash, furthermore all protocols agree that normal liver enzyme values may not rule out nafld(29,32). dyslipidemia is common in patients with nafld and it is attributed to insulin resistance in liver and adipose tissue. in addition, the production of vldl is enhanced to compensate for triglyceride buildup in the liver, and the expression of ldl receptors is reduced, resulting in higher amounts of vldl and ldl in the blood(33–35), in this study, many participants had an abnormal lipid profile at baseline especially triglyceride levels, at 12 weeks, levels of total cholesterol and triglyceride showed significant changes while levels of ldl, vldl, and hdl did not. this is because the levels of ldl and vldl remain elevated in some patients who still had steatosis, moreover, ldl and vldl levels were slightly elevated in some participants who had scored (0) at 12 weeks of the study whereas hdl levels were normal in many participants at baseline and remain low in some participants who still had steatosis at the end of the study. however, a longitudinal study from korea found that patients with nafld were less likely to attain their ldl cholesterol goals than those without the disease, even though the predicted percentage of patients who did not reach their ldl cholesterol targets varied over time, it was generally greater in the nafld group than in the normal group(36), furthermore, a retrospective study included 431 patients, revealed that higher cholesterol and triglyceride concentrations, as well as lower hdl concentrations, were presented in more than half of the patients and 81.81% of patients with grade 3 liver steatosis had low hdl levels, which indicates that a greater grade of steatosis raises the risk of hyperlipidemia(37), the results of these studies may explain the reasons of high ldl levels in some patients at the end of the study, and normal levels of hdl at baseline as 59% of participants had mild steatosis while the percentage of participants with severe steatosis was 7. there was an improvement in steatosis score because 46.2% of the study participants had scored (0) at the end of the study and this is consistent with previous studies which found that alt, ast, histological alterations, steatosis, inflammation, and hepatocellular ballooning are largely reduced by vitamin e(38), which is known as a chain-breaking antioxidant because of its capacity to inhibit the progression of lipid peroxidation since the development of fatty liver disorders has been linked to oxidative stress and lipid peroxidation(39,40). besides the antioxidant properties of vitamin e, α-tocopherol levels in plasma are shown to be lower in nafld\nash patients in comparison with healthy controls(41,42). furthermore, a recent systematic review and metaanalysis demonstrated that vitamin e improves both hepatic enzymes and histological markers and these results are independent of bmi therefore patients with nafld who are unable to adhere to lifestyle changes may benefit from vitamin e treatment as a second option(43), as the rates of debility and quit are high reaching 40% and 50% respectively during lifestyle intervention regimens, even though lifestyle alterations are considered as first-line treatment for nafld(16). limitations of this study: it is a single-center study, absence of control group, short time of the study since each participant need follow up for 3 months, small sample size due to loss of the participants during the study and this mainly belong to covid 19 pandemic also this disease is silent so the patients feel they are well and do not need to continue the treatment. conclusions and 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m, et al. systematic review with meta-analysis: the effect of vitamin e supplementation in adult patients with non-alcoholic fatty liver disease. j gastroenterol hepatol. 2021;36(2):311–9. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 toxoplasmosis in parkinson’s disease patients doi: https://doi.org/10.31351/vol30iss2pp99-105 99 seroprevalence of toxoplasma gondii in parkinson’s disease iraqi patients maysoon abdul-zahra merdaw*, ali a.kasim*,1 and mahmood kahtan salih** *department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad-iraq **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad-iraq abstract several studies have addressed the prevalence of toxoplasma gondii (t. gondii), among parkinson’s disease (pd) patients in different countries, and the potential association between the infection and pd; the results of these studies were conflicting. the study aims to investigate the prevalence of toxoplasma infection among sample of iraqi pd patients. also, to examine the potential association of age, pd duration, gender, smoking habit, zone of residence and family history of pd, with the prevalence of toxoplasma infection in pd patients. seventy-four pd patients attaining dr. saad al-witry neuroscience hospital in baghdad/ iraq for routine follow up, from different iraqi governorates, were enrolled in this cross-sectional study. detection of t. gondii was performed by detection of anti-toxoplasma igg and igm antibodies in serum by elisa method. the frequency rate of anti-toxoplasma igg antibodies in iraqi pd patients was 43.2% (32/74); while, none of the participants was seropositive for anti-toxoplasma igm antibody. age, pd duration, smoking habit and zone of residences were not shown to be risk factors for toxoplasma infection in pd patients (p>0.05); meanwhile, female gender and positive family history of pd were shown to have a protective effect; (or, 0.309; 95% ci, 0.099-0.966; p= 0.043) and (or, 0.162; 95% ci, 0.037-0.705; p=0.015); respectively. the prevalence rate of toxoplasma infection in iraqi pd patients is 43.2%, female gender and positive family history of pd might protect against toxoplasma infection in pd patients. key words: iraq, neurodegeneration, parkinson’s disease, prevalence, toxoplasma gondii العراقيين باركنسون داء مرضى في الغوندية المقوسة انتشار **محمود قحطان صالح و 1 *،، علي عبد الحسين قاسم *و اميسون عبد الزهرة مرد فرع العلوم المختبرية السريرية، كلية الصيدلة، جامعة بغداد، بغداد، العراق * ، بغداد، العراق. كلية الصيدلة، جامعة بغداد االدوية والسموم، فرع ** الخالصة مختلفة، والعالقة المحتملة في بلدان ، بين مرضى داء باركنسونأو المقوسة الغوندية التوكسوبالزماتناولت العديد من الدراسات انتشار تهدف الدراسة الحالية إلى معرفة مدى انتشار عدوى التوكسوبالزما . كانت نتائج هذه الدراسات متضاربة .بين عدوى التوكسوبالزما وداء باركنسون ن والجنس والتدخين ومنطقة بين عينة من مرضى داء باركنسون في العراق باإلضافة لفحص االرتباط المحتمل بين العمر ومدة مرض باركنسو . اإلقامة والتاريخ العائلي لمرض باركنسون، مع ايجابية المصل للتوكسوبالزما في مرضى داء باركنسون م تم تسجيل أربعة وسبعين مريضاً من مرضى داء باركنسون في هذه الدراسة المقطعية المستعرضة من مراجعي مستشفى الدكتور سعد الوترى للعلو عن طريق التوكسوبالزما تم إجراء الكشف عن اصابة. العراق الحاضرين للمتابعة الروتينية ، من مختلف المحافظات العراقية /بغداد العصبية في . االمتزاز المناعي المرتبط باالنزيم المضادة للتوكسوبالزما في مصل الدم بطريقة igm و igg الكشف عن األجسام المضادة تواتر معدل المضادةبلغ مرضى igg األجسام في للتوكسوبالزما باركنسون المضادة من (. 32/74)٪ 43.2العراقيين داء أي يكن لم بينما، والتدخين ومنطقة اإلقامة االصابة بداء باركنسون لم يُظهر العمر ومدة. المضادة للتوكسوبالزما igm المشاركين إيجابي المصل لألجسام المضادة ؛ بينما تبين أن للجنس األنثوي والتاريخ العائلي اإليجابي لداء باركنسون (p> 0.05) التوكسوبالزما في مرضى داء باركنسون كعوامل خطر لعدوى تأثير وقائي (or, 0.162; 95% ci, 0.037-0.705; p=0.015)و (or, 0.309; 95% ci, 0.099-0.966; p= 0.043) على التوالي. ٪ ، والجنس األنثوي والتاريخ العائلي اإليجابي لمرض باركنسون 43.2العراقيين هو باركنسون ءداى التوكسوبالزما في مرضمعدل انتشار عدوى . قد يحميان من عدوى التوكسوبالزما في مرضى داء باركنسون العراق، التنكس العصبي، مرض باركنسون، االنتشار، التوكسوبالزما جوندي :الكلمات المفتاحية 1corresponding author e-mail: e-mail: ali.qasem@copharm.uobaghdad.edu.iq received: 6/1/2021 accepted: 15/3 /2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp99-105 iraqi j pharm sci, vol.30(2) 2021 toxoplasmosis in parkinson’s disease patients 100 introduction toxoplasma gondii (t. gondii) is an obligate intracellular parasitic protozoan that causes a zoonotic disease known as toxoplasmosis. t. gondii can infect most worm-blooded mammals including human (1). the infection is transmitted to the humans by consumption of raw or undercooked meat comprising the parasites’ cysts, ingestion of oocysts, and from the infected mother to the fetus (2). moreover, t. gondii is transmitted through blood transfusion and organ or stem cell transplantation (3). it is estimated that over one third of the world population is infected with t. gondii, thus, it is considered the etiological cause of the most prevalent infection in humans (4). the infection is mostly asymptomatic in individuals with competent immune responses, and tissue cysts of the parasite are formed principally in the brain and skeletal muscles (5). the infection remains latent until when the hosts’ immune responses are challenged where tissue cysts rupture causes release of the quiescent parasite that rapidly divide (6). the reactivated infections might result in neurological damage and inflammation (7, 8). parkinson’s disease (pd) is the second most common progressive neurodegenerative disorder, just preceded by alzheimer’s disease. pd affects about 1% of the world population aged 60 years or older (9, 10). the pathological hallmarks in pd involve dopaminergic and non-dopaminergic neurodegeneration and cytoplasmic accumulation of misfolded proteins known as lewy bodies. many pathological mechanisms have been proposed to explain these events (11-17). the prevalence of t. gondii infection in parkinson’s disease patients has been studied in many epidemiological studies in an attempt to investigate whether t. gondii infection is associated with increased risk for parkinson’s disease; and the results have been very inconsistent. ramezani et al. has reported a significantly higher frequency rate of anti‐toxoplasmosis seropositivity in the idiopathic pd patients compared to healthy individuals and patients with other neurological disorders; and has suggested that t. gondii infection contributed to an increased risk of idiopathic pd (18). similarly, miman et al. has considered toxoplasma infection might contribute to the pathogenesis of pd (19). contrariwise, other studies have reported no association between toxoplasma infection and pd (2,20, 21). actually, fallahi et al. has suggested that t. gondii infection could not be a risk factor for pd and that patients with pd are at more risk to acquire toxoplasma infection (2). to date, the prevalence of toxoplasma in iraqi pd patients is not documented. thus, this study aims to investigate the prevalence of anti-toxoplasma antibodies in a sample of iraqi pd patients; and to examine the potential association of age, pd duration, gender, smoking habits, zone of residence and family history of pd, with the prevalence of toxoplasma infection in pd patients. patients and methods this cross-sectional study was conducted at dr. saad al-witry neuroscience hospital in baghdad/ iraq, during the period extending from may to december 2019. the study was approved by the research committee and by the ethics committee of the college of pharmacy/university of baghdad. seventy-four patients with an established diagnosis of pd, according to the united kingdom parkinson's disease society brain bank clinical diagnostic criteria for idiopathic pd (22); who were attending the hospital for routine follow up, were enrolled in the study. patients with other neurological diseases and people with a history of brain surgery were excluded from the study. sociodemographic data regarding patients’ age, gender, zone of residence, and smoking habits; in addition to pd duration and family history of pd were collected by one of the researchers. a consent was obtained from each patient after being informed about all aspects of the study. a five millilitres venous blood samples were collected from the patients and left at room temperature to clot, then the samples were centrifuged at 3000 rpm for 15 minutes to obtain sera. sera were separated and kept frozen at (-20 °c) until assayed. anti-toxoplasma igm and igg levels were assayed using a readily available enzymelinked immunosorbent assay (elisa) kit purchased from (acon laboratories, san diego, usa). calibration curve was drawn to obtain serum antitoxoplasma igg and igm levels from their absorbance. for qualitative assessment of antitoxoplasma igg and igm seropositivity, index value >1.1was interpreted as positive, also according to the manufacturer recommendations. statistical analysis statistical analyses were performed using spss, version 22 (spss inc., chicago, il, usa), software for windows. descriptive statistics, mainly mean values and standard deviation (sd), were presented for numerical variables, and frequencies and percentages were used for categorical variables. independent student t-test was conducted to examine the significance of the difference in the means of numerical variables between antitoxoplasma igg -positive and -negative groups. iraqi j pharm sci, vol.30(2) 2021 toxoplasmosis in parkinson’s disease patients 101 while, chi-square test was used to check the significance of the difference in the frequencies of the categorical variables between anti-toxoplasma igg-positive and -negative groups. univariate and multivariate logistic regression were used to identify risk factors associated with the seropositivity of antitoxoplasma igg in pd patients. variables with pvalues less than 0.05 in the univariate logistic model were included in the multivariate logistic model. the confidence interval was set to 95%, and the default level of statistical significance was based on p < 0.05 results in this study, the mean age of pd patients was 60.65 ± 10.67. forty-three patients (58.1 %) were males and 31 (41.9%) were females. in terms of zone of residence, 60 patients (81.1%) were living rural areas and 14 (18.9%) were living in urban areas. the mean duration of disease was 6.41 ± 3.75 years; only 17 patients (23%) have a family history of pd and only 16 patients (21.6%) were smokers; (table 1). the overall prevalence of toxoplasma in pd patients was 32/74 (43.2%); toxoplasma positive patients were found positive for antitoxoplasma igg antibody, which reflects chronic toxoplasma infections. all pd patients were found as negative for anti-toxoplasma igm antibody. none of the samples was in the equivocal range for anti-toxoplasma igg or igm antibody. the mean serum levels of anti-toxoplasma igg antibody in seropositive pd patients was 3.538 ± 2.164 (table 2). table 1. characteristics of 74 parkinson’s disease patients age (year) 60.65 ± 10.67 pd duration (year) 6.41 ± 3.75 gender [n (%)] male 43 (58.1%) female 31 (41.9%) smoking [n (%)] no 58 (78.4%) yes 16 (21.6%) zone of residence [n (%)] rural 60 (81.1%) urban 14 (18.9%) family history of parkinson’s disease [n (%)] no 57 (77%) yes 17 (23%) table 2. frequency and serum levels of anti t. gondii antibodies in parkinson’s disease patients serum anti-toxoplasma igg antibody serum anti-toxoplasma igm antibody frequency level range frequency level range toxoplasma positive 32 (43.2%) 3.538 ± 2.164 1.30-7.54 0 (0%) ------------ toxoplasma negative 42 (56.8%) 0.473 ± 0.134 0.31-0.79 100 (100%) 0.232 ± 0.048 0.18-0.36 the means of age and pd duration were significantly different between anti-toxoplasma igg antibody seropositive and seronegative patients (p<0.001). moreover, there was a significant difference between the frequency of toxoplasma between male and female pd patients and between those with positive and negative family history of pd (p = 0.036 and 0.015; respectively); (table 3). table 3. patients’ characteristics by prevalence of toxoplasmosis variable toxoplasma positive n=32 toxoplasma negative n=42 p value age (year) 64.31 ± 10.17 57.86 ± 9.87 0.000 pd duration (year) 7.16 ± 4.89 5.83 ± 2.49 0.000 gender male 23 (71.9%) 20 (47.6%) 0.036 female 9 (28.1%) 22 (52.4%) smoking no 23 (71.9%) 35 (83.3%) 0.236 yes 9 (28.1%) 7 (16.7) zone of residence rural 28 (87.5%) 32 (76.2%) 0.218 urban 4 (12.5%) 10 (23.8%) family history of pd no 29 (90.6%) 28 (66.7%) 0.015 yes 3 (9.4%) 14 (33.3%) iraqi j pharm sci, vol.30(2) 2021 toxoplasmosis in parkinson’s disease patients 102 univariate logistic regression analysis showed that the older age, female gender and positive family history of pd are significantly related to anti-toxoplasma igg antibody seropositivity; (table 4). multivariate logistic regression analysis of these variables showed that female gender and positive family history of pd reduce the risk of anti-toxoplasma igg antibody seropositivity in pd patients; (or 0.309, 95% ci: 0.99-0.966, p = 0.043) and (or 0.162, 95% ci: 0.037-0.705, p = 0.015); respectively; (table 4). table 4.multiple logistic regression analysis to predict potential independent risk factors for prevalence of toxoplasmosis in pakinson’s disease patients variable unadjusted adjusted or (95% ci) p value or (95% ci) p value age 1.069 (1.014-1.127) 0.014 1.041 (0.983-1.102) 0.169 pd duration 1.103 (0.968-1.257) 0.141 ------------ gender male (reference group) female 0.356 (0.134-0.948) 0.039 0.309 (0.099-0.966) 0.043 smoking no (reference group) yes 1.957 (0.637-5.991) 0. 240 ------------ zone of residence rural (reference group) urban 0.457 (0.129-1.621) 0.225 ------------ family history of pd no (reference group) yes 1.036 (0.054-0.799) 0.022 0.162 (0.037-0.705) 0.015 discussion latent toxoplasma infection has been shown to be associated with neurodegeneration (23). the exact mechanism by which toxoplasma infection results in neurodegeneration is not well elucidated; however, neuroinflammation has been proposed to be a major contributor (24). in chronic toxoplasma infection, the cysts tend to concentrate in the neurons (25), where they are subjected to persistent local cellular immune reactions (26). neuroinflammation might continue for years if toxoplasma infection is not eradicated (23). many studies have addressed the prevalence of toxoplasma infection among pd patients in different countries, and the potential association between the infection and pd; the results of these studies were conflicting (2,18-21,27,28). except for ramezani et al.(18) and miman et al.(19) who have reported significant difference in seroprevalence of toxoplasma infection between pd patients and healthy control, other studies did not report such finding. in a recent meta-analysis, zhou et al. reported no correlation between pd and antitoxoplasma igg antibody seropositivity (29). this meta-analysis has attributed the positive correlation that has been reported in some studies (18, 19), to the small sample sizes and sampling bias that these studies were conducted in a nearby geographical area. to the best of our knowledge, this is the first study investigating the prevalence of toxoplasma infection and pd in iraq. in the present study, 43.2% of pd patients were seropositive for anti-toxoplasma igg antibody, indicating chronic latent infection. while, all pd patients were seronegative for anti-toxoplasma igm antibody, indicating no acute toxoplasma infection among participants (table 2). these results occur in agreement with studies that have reported seroprevalence, based on anti-toxoplasma igg antibody, in turkey (42.3%)(19), egypt (43.3%)(27), iran (53%)(2). ramezani et al.(18) and mahami et al.(21) have reported higher seroprevelance rates in iran (82.5% and 85%; respectively). low seroprevalence rates has been reported by alvaradoesquivel et al. (9.2%) in mexico(20) and by çelik et al. (18%) in turkey(28). in the present study, the age of seropositive pd patients was significantly higher than their seronegative counterparts (table 3). age has been reported to be a risk factor for toxoplasma infection(30, 31). the increase in toxoplasma seroprevalence with age can be attributed to the increase in the likelihood of contact with the parasites’ oocyts with time, due to occupational factors for example. moreover, the improvement in the knowledge about toxoplasma infection and its routes of transmission might reduce the prevalence iraqi j pharm sci, vol.30(2) 2021 toxoplasmosis in parkinson’s disease patients 103 of toxoplasma infection among younger individuals. the duration of pd in toxoplasma seropositive patients in the present study, was significantly higher than that of their seronegative counterparts (table 3). however, pd duration was not a risk factor for the seroprevalence of toxoplasma in pd patients (table 4). typically, onset and diagnosis of pd occur ages higher than 55 years, thus, the higher pd duration in toxoplasma seropositive pd patients might reflects the higher age of those patients rather than of pointing a pathological significance. regarding gender, the results showed significant difference between males and females of toxoplasma seropositive and seronegative pd patients (table 3), and female pd patients were shown to be at lower risk for toxoplasma infection (table 4), which disagree with mahami et al.(21) who reported no association between gender and toxoplasma infection in pd patients. female gonadal sex hormones in rats have been shown to modulate the dopaminergic actions in the striatum and nucleus accumbens of the brain(32). dluzen et al. has showed that estrogen protects against neurodegeneration of the striatal dopaminergic system, probably by inhibiting the uptake of neurotoxins(33). the toxoplasma-induced dopamine manipulation, which has long been suggested(34), might be inhibited by estrogen, hence, contributing to the lower prevalence and risk of toxoplasma infection in female pd patients reported in the study. the results of the present study showed that pd patients with positive family history of the disease are at lower risk for toxoplasma infection (table 4). up to our knowledge, this is the first study reporting such a relationship and further studies are required for confirmation. if confirmed, this finding may highlight a genetic role in protecting against, toxoplasma infection in pd patients. it is worthy to mention that, several studies have linked genetic polymorphisms with increased(35, 36), or decreased(37) susceptibility to toxoplama infection. finally, smoking and zone of residence were not associated with toxoplasma infection in pd patients that occur in agreement with other studies(20, 21). in conclusion, the study showed that the prevalence rate of toxoplasma infection in iraqi pd patients is 43.2%. moreover, female gender and positive family history of pd are protective 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http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(1) 2012 response to atorvastatin in diabetic 21 therapeutic response of serum lipids to atorvastatin in type ii diabetic patients riyadh m. murtadha* ,1 * department of clinical pharmacy ,college of pharmacy ,university of kabala, kabala, iraq. abstract lipid disorders and cardiovascular disease (cvd) risk are known to be increased in patients with diabetes mellitus. the effects of statins on serum lipid levels are well known; however, previous studies did not compare the effects of statins on serum lipid levels in diabetic patients with non-diabetic patients. to investigate the effects of atorvastatin on serum lipid profiles in hyperlipidemic patients with type 2 diabetes mellitus in comparison with hyperlipidemic patients without diabetes.this study was conducted on 33 type 2 diabetic patients & 34 non-diabetic patients; their age range was 40-80 years, all of them were hyperlipidemic, who had been administered 10, 20, & 40 mg daily of atorvastatin and completed a 6-month follow-up. serum lipids were measured before and after treatment at 1, 3, and 6 months.it was found that the reduction in s. total cholesterol and s. ldl-c were less in diabetic patients than that in non-diabetic patients when they are using the same doses of atorvastatin, while the changes in s. hdl-c and s. triglyceride were nearly similar in both. furthermore, it is noticed that nearly the same responses of s. cholesterol and s. ldl-c reduction were achieved in diabetic patients when they are using doubled doses that are used for non-diabetic patients.so,higher doses of atorvastatin (double doses) are needed to improve serum lipid levels in diabetic patients as compared to non-diabetic patients. key words: diabetic , lipids , atorvastatin. / الىوع الثاوٌ االستجابة العالجَة لمستوى الدهون فٌ مصل الدم عىد مرضي السكرً رٍاض مصطفي مرتضي* ،1 .كشبالء ، انعشاق كهُت انصُذنت ، جايعت كشبالء،فشع انصُذنت انسشَشَت ، * الخالصة ٌّ حأثُشاث عقاس بًا أٌ اظطشاباث انذهىٌ وخطش األيشاض انقهبُت انىعائُت اكثش عُذ انًشظً انًصابٍُ بذاء انسكشٌ، وأ انسابقت نَى حقاسٌ حأثُشاث عقاس انسخاحٍُ عهً يسخىَاث انذهىٌ إال اٌ انذساساث يعشوفتانسخاحٍُ عهً يسخىَاث انذهىٌ فٍ يصِم انذو (atorvastatin)فٍ يصِم انذو عُذ انًشظً انًصابٍُ بذاء انسكشٌ يع غُشانًصابٍُ، وبهذف حَحّشٌ حأثُشاث عقاس االحىسفاسخاحٍُ انزٍَ ًشظً غُش انًصابٍُ بانسكشٌعهً يسخىَاث انذهىٌ فٍ يصِم انذو عُذ انًشظً انًصابٍُ بذاء انسكشٌ بانًقاسَت يع ان يشَط 33يشَط يصاب بذاء انسكشٌ يٍ انُىع انثاٍَ بانًقاسَت يع 33اجشَج هزِ انذساست عهً َعاَىٌ يٍ صَادة انذهىٌ بانذو ، ذيىا قسًىا انً يجايُع نُسخخسُت، 04-34حخشاوح اعًاسهى بٍُ ، نذَهى صَادة يسخىي انذهىٌ بانذو جًُعهىغُش يصاب بانسكشٌ شهىس. حى قُاس يسخىَاث انذهىٌ فٍ يصِم انذو قَبم وبعذ 6يهغ َىيُاً ونُكًهىا فخشة يخابعت يذة 34، و04، 04عقاس احىسفاسخاحٍُ ول واغئ انكثافت فٍ يصم انًعانجت فٍ انشهش االول وانثانث وانسادس.اظهشث انُخائج أٌ انخغُُشاث فٍ انكىنسخُشول انكهٍ وانكىنسخُش فٍ انًشظً غُش انًصابٍُ بانسكشٌ انزٍَ اسخخذيىا َفس انُجَشع يٍ حهك انخٍأقم فٍ انًشظً انًصابٍُ بانسكشٌ يٍ انذو كاَج بًُُا كاَج انخغُُشاث فٍ انكىنسخُشول عانٍ انكثافت وانذهىٌ انثالثُت يخًاثم حقشَباً فٍ ،(atorvastatin)عقاس االحىسفاسخاحٍُ ٌّ االسخجابت كاَج َفسها حقشَباً نهكىنىسخُشول انكهٍ وانكىنسخُشول واغئ انكثافت فٍ انًشظً انًجًىعخٍُ. عالوة عهً رنك َاُلحع بأ َسخُخج يٍ رنك أٌ انًشظً انًصابٍُ بانسكشٌ عُذيا َسخخذيىٌ ُجَشع ُيعاعفت كانخٍ اسخخذيج نهًشظً غُش انًصابٍُ بانسكشٌ. نخحسٍُ االسخجابت انعالجُت ،(atorvastatin)( يٍ عقاس االحىسفاسخاحٍُ انًصابٍُ بانسكشٌ َحخاجىٌ انً ُجَشع أعهً )ُجَشع يعاعفت نًسخىَاث انذهىٌ فٍ انذو بانًقاسَت يع انًشظً غُش انًصابٍُ بانسكشٌ. ورفاستاتَه تالكلمات المفتاحَة : داء السكرً ، الدهون ، اال introduction the increased risk of cardiovascular events in diabetic patients is well established (1–4) . recent studies demonstrate that diabetic patients without a prior coronary artery disease (cad) had approximately similar risk of acute coronary syndrome as non-diabetic patients with prior cad (4,5) . many have demonstrated that cad patients with diabetes have higher mortality following a myocardial infarction than their non-diabetic counterparts (2-5) . although at high risk for future cardiovascular events, patients with cad and diabetes are as likely as those without diabetes to benefit from statins (3-hydroxy-3-methylglutaryl coenzyme a reductase inhibitors) as a lipid lowering treatment. many large trials are consistent in their findings with that in which cad patients with diabetes experienced reductions in relative risk with statin treatment of similar magnitude to the risk reductions for cad patients without diabetes (6,7) . 1corresponding author email : dr_rigadhs@yahoo.com received : 11/4/2011 accepted : 18/12/2011 iraqi j pharm sci, vol.21(1) 2012 response to atorvastatin in diabetic 22 the results from other studies further demonstrate the benefits of statin treatment to reduce the risk of cardiovascular events compared with placebo in type 2 diabetic subjects both with and without cardiovascular disease (8,9) .given their elevated risk and similar lipid management goals, one would expect cad patients with diabetes to be treated at least as aggressively as those without diabetes. nevertheless, cad patients, in general, continue to receive less than optimal lipid management, and those with diabetes may be relatively under-treated compared with those without diabetes (10-12) . statins are highly effective in lowering serum lipid concentrations and preventing ischemic heart disease (ihd); however, we do not know by how much statins at different doses affect serum lipid concentrations in diabetic patients in comparison with non-diabetic hyperlipidemic patients. the aim of the study is to quantify the effects of different doses of atorvastatin on serum lipid concentrations in hyperlipidemic patients with type 2 diabetes mellitus in comparison with hyperlipidemic patients without diabetes. patients and methods this study was conducted in karbala city since february 2009 till february 2010 on thirtythree (33) type 2 diabetic patients (13 males & 20 females; mean age 56.5 ± 8.4) and thirtyfour (34) non-diabetic patients (14 males & 20 females; mean age 56.8 ± 10.6), whom fasting serum lipids concentrations (s.cholesterol, s.hdl-c, s.ldl-c, & s.tg) were measured as a baseline and all of them were having hyperlipidemia.lipid profiles were measured by using “spinreact” enzymatic cholorimetric test. (13) diabetic patients were divided into three subgroups who had been administered 10, 20, & 40 mg daily atorvastatin respectively, while non-diabetic patients were divided into two subgroups who had been administered 10 & 20 mg daily atorvastatin respectively. each one was completed a 6month follow-up period in which serum lipids were measured after 1, 3, and 6 months of treatment. all diabetic patients were on treatment with oral hypoglycemic agents; 5 patients out of 33 (15%) were on sulfonylurea, 10 patients (30.5%) were on metformin, while 18 patients (54.5%) were on sulfonylurea plus metformin. the percentage values are presented as means ± standard deviations. the statistical analysis used for continuous variables was paired student’s t-test. p-value of < 0.05 was considered as significant. results the percentages of changes in serum lipid concentrations in diabetic and non-diabetic patients after treatment with different doses of atorvastatin are shown in tables 1– 4 and figures 1– 4. it is clear from table 1 and fig. 1 that there are significant differences between the changes of serum cholesterol concentrations in diabetic and non-diabetic patients when they are treated with the same doses of atorvastatin (p < 0.05). meanwhile, it is noticed that the changes in s. cholesterol concentrations in diabetics treated with 20mg atorvastatin were near the changes observed in non-diabetics when they are treated with 10mg atorvastatin, and it is also noticed that the changes in s. cholesterol concentrations in diabetics treated with 40mg atorvastatin were near the changes observed in non-diabetics when they are treated with 20mg atorvastatin.the same observations above were also applied to a large extent on the changes of s. ldl-c concentrations shown in table 2 & fig. 2. in that, there are significant differences between the changes of s. ldl-c concentrations in diabetic and non-diabetic patients when they are treated with the same doses of atorvastatin (p < 0.05). meanwhile, diabetic patients responding to a similar degree of s. ldl-c concentration changes with that of nondiabetics when they are treated with double doses used for non-diabetics.regarding the changes in s. hdl-c concentrations which are shown in table 3 & fig 3, it is observed that they are slightly better in non-diabetics than in diabetic patients using the same doses of treatment but without significant difference (p =ns). however; the changes in s. triglyceride concentrations were about to be similar in diabetic and non-diabetic patients using the same doses of treatment as shown in table 4 & fig. 4 (p =ns). iraqi j pharm sci, vol.21(1) 2012 response to atorvastatin in diabetic 23 table 1 : percentage of serum cholesterol reduction after treatment with atorvastatin * period of treatment type ii diabetic patients non-diabetic pts. 10 mg 20 mg 40 mg 10 mg 20 mg 1 month -5.8% ± 2.0 -14.2% ± 6.6 -23.4% ± 3.9 -11.9% ± 6.1 -25.9% ± 4.8 3 months -13.9% ± 4.7 -22.9% ± 8.4 -37.9% ± 6.3 -20.0% ± 7.6 -39.0% ± 7.4 6 months -24.1% ± 8.2 -33.1% ± 10.3 -45.9% ± 8.1 -30.9% ± 9.9 -45.5% ± 9.1 table 2: percentage of serum ldl-cholesterol reduction after treatment with atorvastatin* period of treatment type ii diabetic pts. non-diabetic pts. 10 mg 20 mg 40 mg 10 mg 20 mg 1 month -6.7% ± 3.8 -21.3% ± 4.5 -32.9% ± 4.9 -17.7% ± 4.6 -34.5% ± 8.2 3 months -13.7% ± 5.8 -28.4% ± 8.9 -50.2% ± 9.7 -26.3% ± 6.2 -50.6% ± 12.1 6 months -22.9% ± 6.2 -39.6% ± 9.5 -61.8% ± 12.9 -38.9% ± 11.5 -60.7% ± 13.5 table 3 :percentage of serum hdl-cholesterol elevation after treatment with atorvastatin * period of treatment type ii diabetic pts. non-diabetic pts. 10 mg 20 mg 40 mg 10 mg 20 mg 1 month +3.3% ± 2.4 +5.2% ± 2.8 +5.7% ± 2.9 +7.2% ± 3.0 +7.7% ± 3.7 3 months +6.7% ± 4.5 +8.5% ± 4.6 +9.5% ± 5.2 +10.6% ± 3.9 +10.9% ± 4.3 6 months +9.4% ± 4.5 +10.5% ± 4.8 +11.6% ± 6.0 +11.3% ± 5.7 +11.7% ± 5.8 table 4 :percentage of serum triglyceride reduction after treatment with atorvastatin * period of treatment type ii diabetic pts. non-diabetic pts. 10 mg 20 mg 40 mg 10 mg 20 mg 1 month -12.9% ± 2.7 -15.9% ± 3.7 -17.3% ± 3.9 -11.7% ± 4.6 -13.0% ± 5.6 3 months -18.4% ± 7.3 -24.8% ± 8.5 -28.6% ± 10.3 -20.0% ± 8.6 -22.0% ± 7.8 6 months -24.8% ± 8.8 -32.8% ± 10.2 -38.6% ± 12.8 -27.6% ± 10.7 -31.8% ± 11.7 *values presented as means ± standard deviations iraqi j pharm sci, vol.21(1) 2012 response to atorvastatin in diabetic 24 a b figure 1 :line chart showing percentage of serum cholesterol changes after treatment with atorvastatin in diabetics(a) & non-diabetics(b) a b figure 2 :line chart showing percentage of serum ldl-cholesterol changes after treatment with atorvastatin in diabetics(a) & non-diabetics(b) a b figure 3 : line chart showing percentage of serum hdl-cholesterol changes after treatment with atorvastatin in diabetics(a) & non-diabetics(b) iraqi j pharm sci, vol.21(1) 2012 response to atorvastatin in diabetic 25 a b figure 4 :line chart showing percentage of serum triglyceride changes after treatment with atorvastatin in diabetics(a) & non-diabetics(b). discussion this study provides evidence that the response of lipid profile to statin in diabetic patients differ from that in nondiabetics, in which the changes in s.cholesterol and s. ldl-cholesterol were less in diabetic patients than that in nondiabetic patients when using the same doses of atorvastatin. furthermore, it is noticed that nearly the same changes were achieved in diabetic patients when they are using doubled doses that are used for non-diabetic patients.these findings were agreed by robert et al, when subjects with type 2 diabetes were randomized; mean ldl-c reduction in the atorvastatin group over 4 years was (29%) versus placebo (14) . besides, another trial done by law et al showed that reductions in ldl cholesterol in non-diabetic patients were (40%) with atorvastatin 10mg/day (15) .after treatment, lipid levels for diabetic patients have improved less rapidly than those for nondiabetic patients. mark et al stated that mean non-hdl-c levels, already higher among patients with diabetes, did not decline as rapidly for this group, increasing the gap between them and patients without diabetes (16) .although the mean concentration of ldl cholesterol in diabetic patients is not significantly different from that in individuals without diabetes, qualitative changes in ldl cholesterol may be present. diabetic patients tend to have a higher proportion of ldl particles that are smaller and denser, are more susceptible to oxidation, and may thereby increase the risk of cardiovascular events (17) and may also explain the difference in response of s.cholesterol and s. ldl-c to statin between diabetic and non-diabetic patients.the changes in s. hdl-cholesterol in both diabetic and non-diabetic patients were nearly similar in spite of the less rapid improvement in diabetics; nevertheless, they did not reach what were achieved by other reports such as that was found by klaus et al that hdl-cholesterol increased significantly (+19%) after 4 weeks of atorvastatin therapy (10 mg/day) (18) . that difference may be because our patients are less likely to do exercise to support hdl-c elevation.likewise, the changes in s.triglyceride were also similar in both diabetic and non-diabetic patients which was less than what was reported by klaus et al that fasting triglycerides were reduced by (−43%) after 4 weeks of atorvastatin therapy (10 mg/day) (18) .besides, when reviewing literatures; there is no known drug – drug interaction between atorvastatin and oral hypoglycemic agents (19) to be responsible for that difference in response between diabetic and non-diabetic patients.in conclusion, higher doses of atorvastatin (double doses) are needed to improve serum lipid levels in diabetic patients as compared to non-diabetics. references 1. kannel wb, mcgee dl: diabetes and glucose tolerance as risk factors for cardiovascular disease: the framingham study. diabetes care , 1979;2:120–126. 2. krolewski as, warram jh, valsania p, martin bc, laffel lm, christlieb ar: evolving natural history of coronary disease in diabetes mellitus. am j med 1991;90:56s–61s. 3. sprafka jm, burke gl, folsom ar, mcgovern pg, hahn lp: trends in prevalence of diabetes mellitus in patients iraqi j pharm sci, vol.21(1) 2012 response to atorvastatin in diabetic 26 with myocardial infarction and effect of diabetes on survival: the minnesota heart study. diabetes care,1991; 14:537–543. 4. haffner sm, lehto s, rönnemaa t, pyörälä k, laakso m: mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. n engl j med , 1998;339:229–234. 5. malmberg k, yusuf s, gerstein hc, brown j, zhao f, hunt d, piegas l, calvin j, keltai m, budaj a: impact of diabetes on long-term prognosis in patients with unstable angina and non-qwave myocardial infarction: results of oasis (organization to assess strategies for ischemic syndromes) registry. circulation 2000;102:1014– 1019,. 6. pyorala k, pedersen tr, kjekshus j, faergeman o, olsson ag, thorgeirsson g, the scandinavian simvastatin survival study (4s) group: cholesterol lowering with simvastatin improves prognosis of diabetic patients with coronary heart disease: a subgroup analysis of the scandinavian simvastatin survival study (4s). diabetes care , 1997;20:614–620. 7. the long-term intervention with pravastatin in ischaemic disease (lipid) study group: prevention of cardiovascular events and death with pravastatin in patients with coronary heart disease and a broad range of initial cholesterol levels. n engl j med , 1998;39:1349–1357. 8. collins r, armitage j, parish s, sleigh p, peto r: mrc/bhf heart protection study of cholesterol-lowering with simvastatin in 5963 people with diabetes: a randomized placebo-controlled trial. lancet , 2003;361: 2005–2016. 9. colhoun hm, betteridge dj, durrington pn, hitman ga, neil ha, livingstone sj, thomason mj, mackness mi, charlton-menys v, fuller jh: primary prevention of cardiovascular disease with atorvastatin in type 2 diabetes in the collaborative atorvastatin diabetes study (cards): multicentre randomised placebo-controlled trial. lancet, 2004; 364:685–696. 10. sueta ca, chowdhury m, boccuzzi sj, smith sc jr, alexander cm, londhe a, lulla a, simpson rj: analysis of the degree of undertreatment of hyperlipidemia and congestive heart failure secondary to coronary artery disease. am j cardiol , 1999;83:1303– 1307. 11. pearson t, laurora i, chu h, kafonek s: the lipid treatment assessment project. arch intern med , 2000;160:459–467. 12. massing mw, sueta ca, chowdhury m, biggs dp, simpson rj jr: lipid management among coronary artery disease patients with diabetes mellitus or advanced age. am j cardiol , 2001;87: 646–649. 13. spinreact, s.a.u. ctra.santa coloma, 7 e-17176 sant esteve de bas (gi) spain;www.spinreact.com/en/products/c linical_chemistry/lipids.html. 14. robert hk, michael d, johan gs, stuart j.p: efficacy and safety of atorvastatin in the prevention of cardiovascular end points in subjects with type 2 diabetes. diabetes care, 2006; 29:1478–1485. 15. law m r, wald n j, rudnicka a r: quantifying effect of statins on low density lipoprotein cholesterol, ischaemic heart disease, and stroke: systematic review and meta-analysis. bmj , 2003;326:1-7. 16. mark wm, kathleen af, carla as, mridul c, david pb, charles ma, and ross js.: trends in lipid management among patients with coronary artery disease; has diabetes received the attention it deserves? diabetes care , 2003;26:991-997. 17. haffner sm: dyslipidemia management in adults with diabetes. diabetes care, 2004 ;27 (suppl. 1): s68– s71. 18. klaus gp, ester l and p. hugh rb: effect of atorvastatin on postprandial lipoprotein metabolism in hypertriglyceridemic patients. j lipid res. , 2003;44(6):1192-8. 19. bmj group and rps publishing. british national formulary 58; appendix 1: interactions: , 2009;780. iraqi j pharm sci, vol.32(1) 2022 anti-inflammatory effect of tamsulosin doi: https://doi.org/10.31351/vol32iss1pp283-289 283 study of the anti-inflammatory effect of tamsulosin in rat by evaluating il-4, il-6 and tnf-α: an airway model hala h. abduljabbar *,1 and manal a. ibrahim** *department of pharmacy, al-basrah teaching hospital, basrah, iraq ** department of pharmacology and toxicology, college of pharmacy, university of al-basra, iraq abstract inflammatory airway disease is a known worldwide health problem. current medications are accompanied by serious side effects and it provides only temporary symptom control. aim: to investigate the effect of tamsulosin, on the inflammatory cytokines il-4, il-6, and tnf-α, which are associated with airway inflammation. 30 male, albino rats, weighing 150-250 gm were allocated into 5 groups, each group with 6 rats; group a: normal control group, rats were given commercial pellets and distilled water for 14 days. group b: negative control group, rats were exposed to airway sensitization only. group c: positive control group, rats were treated with prednisolone (4.12mg/kg/d) orally plus airway sensitization. group d: rats were treated with tamsulosin (35 mcg/kg/d) orally plus airway sensitization. group e: rats were treated with tamsulosin (17.5 mcg/kg/d) orally plus airway sensitization. investigation of inflammatory cytokines il4, il-6, and tnf-α in serum samples by enzyme-linked immunoassay (elisa). there was a significant reduction (p-value<0.05) of il4 and tnf-α in serum for tamsulosin treated group (d) and group (e) when compared with the positive control group (b) but only group (d) (35mcg/kg/d) tamsulosin showed significant reduction (p-value<0.05) in il-6 level when compared with the positive control group (b). tamsulosin has an anti-inflammatory effect by reduction of il-4, il-6, and tnf-α in the rat airway model. key words: tamsulosin, airway inflammation, inflammatory cytokines, il-4, il-6, tnf-α. دراسة تأثير عالج التامسولوسين كمضاد أللتهابات المجاري التنفسية المستحدثة في الجرذان *ابراهيم عبد الخالق ومنال 1*،هالة هيثم عبد الجبار العراق. البصرة، التعليمي، مستشفى البصرة الصيدلة، قسم * العراق. البصرة، جامعة الصيدلة، كلية والسموم، فرع االدوية ** خالصهال التهاب المجرى الهوائي هو مشكللة حكةية معروفة في جميأ حاةاا العالم. اودوية المتا ة مصكةوبة بر اا جاابية رةيرة وال روفر كوى و لامل (il-6), ااترلوكيع كتة (il-4) اابعة ااترلوكيع السكيتوكياا التامسكولو كيع للى اولراض. داا كة رث ير لاااالسكيةرة المققتة للى مجمولا ، 5قسكم للى مج 250-150 وزاها، مع الذكوا الرهاب مجرى الهواا. رم ا كتخدام ث و جراا ةالمصكا ب (tnf-α)حلفا-اخر الوام ا. المجمولكة النكاايكة رم 14جراا . المجمولكة االولى مجمولكة السككككيةرة ، حلةيك الفمرا المكاا الماةر لمكدة 6ككل مجمولكة رةتوع للى يومكا ضككافة للى رةسككر باإللع طريق الفم مجم / كجم( 4.12 لبريدايزولو ارعريضككها لتةسككر مجرى الهواا فال. المجمولة النالنة الةي لث باإلضكككافة للى رةسكككر مجرى الهواا. لع طريق الفمميلروجرام / كجم( 35مجرى الهواا. المجمولة الرابعة الةي لث التامسكككولو كككيع باإلضكافة للى رةسكر مجرى الهواا. رم قيات رركيز لع طريق الفمميلروجرام / كجم( 17.5المجمولة الخامسكة الةي لث التامسكولو كيع ااترلوكيع اابعة لك (p-value <0.05) الماايسة االمتصاحية الماالية المرربةة باالازيم. ااخفاض معاوع ةبوا ة السيتوكياا في مصل الجراا ا معاو الرابعة للع المجمولة للمجمولتيع الرابعة والخامسكة لاد الةاا لث التامسكولو كيع. الفا-ومعامل اخر الوام -p) ياافال حظهر ااخفاضكا value <0.05) ااترلوكيع اابعة, ااترلوكيع كتة و ااترلوكيع كتة. لث التامسكولو كيع لأ رث ير مضكاد لثلتهابا لع طريق راليل في مسكتوى .بعد ا تةداث التهاب مجرى الهواا لاد الجراا حلفا-لامل اخر الوام .ألفا-عامل نخر الورم، انترلوكين ستةأربعة، انترلوكين ، سايتوكينات، الهوائيمجرى الالتهاب ، الكلمات المفتاحية: تامسولوسين introduction airway inflammatory diseases, such as asthma and chronic obstructive pulmonary disease (copd), are well-known worldwide health problems that significantly affect the quality of life. the common complaints of affected patients include limited lifestyle activities as well as the economic burden. they usually endure frequent hospital admissions, high treatment costs, and even premature death (1). the most common and effective therapy for the management of acute symptoms and preventing exacerbation are corticosteroids (2). unfortunately, even if they are used as a short course, a wide range of side effects accompany this medical formulation. on the other hand, long term use will lead to a more dangerous impact on human health (3,4). in addition, there is an increase in morbidity and mortality caused by glucocorticoid resistance in asthmatic patients added to the limitation of long-term use of this drug class (4,5). to date, there has been no clinical treatment capable of eliminating the disease or safely controlling it. therefore, finding an effective medical plan by introducing new medication should be a priority. 1corresponding author e-mail: hala.alabdli@gmail.com received: 24/ 6/2022 accepted: 3/8 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp283-289 iraqi j pharm sci, vol.32(1) 2022 anti-inflammatory effect of tamsulosin 284 tamsulosin is an inhibitor of the α1-adrenergic receptor (α1-ar), and the fda has approved it for the management of benign prostatic hypertrophy (bph)(6). drugs belonging to this class are inexpensive, common, and well-tolerated (7-9). nevertheless, tamsulosin adverse events emerged in 1.1% of treated patients in a previous study conducted by martin et al and the reported adverse events included dizziness, nausea, headache, hypotension, dry nose, pruritus, redness of the skin, increased dyspepsia, and insomnia (10). in an animal model of hyper-inflammation on mice, prophylactic treatment of cytokine storm is effective via using α1ar blocker which is performed by obstructing immune responses (11). furthermore, a retrospective study of hospitalized patients admitted for acute respiratory distress syndrome using α1-ar antagonist had a reduced risk of depending on mechanical ventilation and lower mortality rates compared to non-users (12). cytokines have a vital role in regulating the body’s immune reaction to infection (13). il-4 is one of the important cytokines that drive the allergic inflammatory response. it has an important role in initiating the differentiation of cd4 t-cells into t-helper2 calls, and stimulating the release of a great amount of ige from b cells. also, in macrophages, il4 promotes their activation (14,15). another important cytokine is il-6 which serves as a driving force for chronic inflammatory disease of the airways (16. tissue injury and inflammation are the main reasons for the formation and release of il 6. it was initially recognized as b cell stimulatory factor 2 (bsf-2), leading to the differentiation of bcells into antibody-producing plasma cells (13). tumor necrosis factor-alpha (tnfα) participates in the inflammatory process by causing hyper inflammation in the alveolar cells of the lung (17). it is released at the beginning of allergen sensitization and continues to stimulate the immune response further at the effector stage (18). furthermore, tnf α acts as a chemoattractant for neutrophils and eosinophils and improves their cytotoxicity on endothelial cells (19). this research is aimed to investigate the impact of tamsulosin on inflammatory mediators such as il4, il6 and tnf-α that was associated with inflammatory airway disease and comparison with prednisolone. materials and method materials drugs and chemicals that are included in this current study are: tamsulosin (astells pharma, the netherlands), prednisolone (wockhardt, uk), ovalbumin (ova) (chadwll hеath essex, england), al(oh)3 (merck darmstadt, germany), normal saline 0.9% (n/s) (pioneer, iraq) and formaldehyde (37-41%) (s.d. fine chem limited, india). animals thirty healthy, male, rats 150-250 gm were purchased from the college of veterinary medicine/basra university. rats were housed under an optimum temperature 21±4 °c, light-dark photoperiods (12l:12d) and offered a commercial pellet diet with distilled water throughout the experiment. experimental design group a: normal control group, rats were given commercial pellets and distilled water (d/w) for 14 days. group b: negative control group, rats were exposed to airway sensitization only. group c: positive (standard drug) control, rats were given prednisolone ( 4.12 mg/kg/d) orally with airway sensitization (19). group d: treated control , rats were given tamsulosin (35 mcg/kg, equivalent to 0.4 mg tamsulosin for a 70 kg adult patient) orally with airway sensitization (20,21). group e: treated control group, rats were given tamsulosin (17.5 mcg/kg, equivalent to 0.2 mg tamsulosin for a 70 kg adult patient) orally with airway sensitization (21,22). airway inflammation was induced by sensitization (inhalation of ovalbumin) using a modified model by zainab et al (2021) (19). from the 1st to 3rd day, rats were sensitized by 1mg ovalbumin, 100mg al(oh)₃ dissolved in 1ml n/s. on the 6th day, the sensitization dose was increased to 100mg ovalbumin, 100mg al(oh)₃ dissolved in 1ml n/s. challenge started on the 9th day using a glass chamber (30cm × 30cm × 30cm), which was attached to a nebulizer filled with 1% ovalbumin (1 gm ova in 100 ml n/s) for 30 minutes each day; the process was repeated for 6 days. drug doses were administered 60 minutes prior to sensitization in the treated groups. the rats were killed at the 14th day after the first sensitization dose (19). statistical analysis in this study, data were expressed as mean ± (sem). comparison among multiple groups was conducted by analysis of. variance (anova) while significance between two groups was assessed by unpaired. student t-test. concerning this work, pvalues that are less than 0.05 (p-value<0.05) were regarded as significant. results effect of tamsulosin on il-4 in rat serum table 1 and figure 1 elucidate that il-4 levels (mean ± sem) for rats that were exposed to airway sensitization (group b) were significantly elevated (p-value<0.05) when compared to the normal control (group a). serum levels of il-4 for group b and group a were 93.49±9.49 and 50.47±3.93, respectively. furthermore, table 1 and figure 1 shows that levels of il-4 for rats treated with 4.12 mg/kg/d prednisolone (group c), 35 mcg/kg/d tamsulosin (group d) and 17.5 mcg/kg/d tamsulosin (group e) were significantly reduced (p-value<0.05) when compared to the negative control (group b). il-4 levels for groups c, d and e were 42.88±1.87, 49.03±3.53 and 67.87±5.91, respectively. this data iraqi j pharm sci, vol.32(1) 2022 anti-inflammatory effect of tamsulosin 285 indicated a significant reduction (p-value<0.05) in il-4 levels when compared to the negative control (group b). moreover, there was no significant difference (p-value>0.05) in il-4 levels for prednisolone treated group (group c) when compared with tamsulosin 35mcg/kg/d (group d) as illustrated in figure 1. effect of tamsulosin on il-6 in rat serum as shown in table 1 and figure 2, il-6 serum level (mean ± sem) for ova-sensitized rats (group b) was significantly elevated (p-value<0.05) when compared with the normal control (group a). il-6 levels in rat serum for group b and group a were 82.5±1.76 and 41±2.44, respectively. additionally, there was significant reduction (pvalue<0.05) in il-6 levels for rat serum after treatment with 4.12 mg/kg/d prednisolone (group c) (60.5 ± 5.53) and 35 mcg/kg/d tamsulosin (group d) (49.16 ± 5.70) in comparison to the negative control (82.5±1.76). however, tamsulosin 17.5 mcg/kg/d (group e) (75.33 ± 4.1) showed no significant difference (p-value>0.05) in comparison to the negative control (82.5±1.76), (table-1). on the other hand, as shown in table 1 and figure 2, there was no significant (p-value>0.05) difference for prednisolone treated group (group c) when compared with tamsulosin treated group 35mcg/kg/d (group d), table 1. effect of tamsulosin on tnf-α in rat serum table 1 and figure 3 demonstrate that levels of tnf-α (mean ± sem) for rats with induced airway sensitization (group b) were significantly raised (p-value<0.05) compared to the normal control (group a). serum level of tnf-α in groups b and a were 242.23±17.24 and 70.74±4.21, respectively. in addition, table 1 and figure 3 shows that levels of tnf-α for rats treated with 4.12 mg/kg/d prednisolone (group c), 35 mcg/kg/d tamsulosin (group d) and 17.5 mcg/kg/d tamsulosin (group e) were 106.76±26.57, 67.56±20.79& 101.76±11.01, respectively. these changes showed significant reduction (p-value<0.05) in serum tnf-α level compared to the negative control group (group b) 93.49 ± 9.49. table 1. effectiveness of tamsulosin on the serum levels of interlеukin-4 (il4), interlеukin-6 (il6) and tumor necrosis factor-alpha (tnf-α) in rat after airway inflammation. values were represented. as means ± standard error of means (sem). *= significantly different (p-value< 0.05) concerning the normal control group. values with symbol superscript (a) are significantly different (p-value< 0.05) concerning group b. dw = distilled water; ova = ovalbumin. tnf-α (ng/l) (means ±sem) il-6 (pg/ml) (means ±sem) il-4 (ng/l) (means ±sem) type of treatment treatment group n=6 70.74±4.21 41±2.44 50.47±3.93 normal control/dw group a 242.23±17.24* 82.5±1.76* 93.49±9.49* negative control/ ova-sensitization group b 106.76±26.57 a 60.5±5.53 a 42.88±1.87 a positive control/ prednisolone (4.12mg/kg/d) group c 67.56±20.79 a 49.16±5.70 a 49.03±3.53 a tamsulosin (35mcg/kg/d) group d 101.76±11.01 a 75.33±4.10 67.87±5.91 a tamsulosin (17.5mcg/kg/d) group e iraqi j pharm sci, vol.32(1) 2022 anti-inflammatory effect of tamsulosin 286 figure 1.effect of tamsulosin on the serum level of il4 in rat. group a: normal control group, rats given distilled water for 14 days; group b: negative control group, rats exposed to airway sensitization only; group c: positive control, treated with prednisolone (4.12mg/kg/d) orally plus airway sensitization; group d: treated with tamsulosin (35 mcg/kg/d) orally plus airway sensitization; group e: treated with tamsulosin (17.5 mcg/kg/d) orally plus airway sensitization. figure 2 . effect of tamsulosin on the serum level of il-6 in rat. group a: normal control group, rats given distilled water for 14 days; group b: negative control group, rats exposed to airway sensitization only; group c: positive control, treated with prednisolone (4.12mg/kg/d) orally plus airway sensitization; group d: treated with tamsulosin (35 mcg/kg/d) orally plus airway sensitization; group e: treated with tamsulosin (17.5 mcg/kg/d) orally plus airway sensitization. figure 3. effect of tamsulosin on the serum level of tnf-α in rat. group a: normal control group, rats given distilled water for 14 days; group b: negative control group, rats exposed to airway sensitization only; group c: positive control, treated with prednisolone (4.12mg/kg/d) orally plus airway sensitization; group d: treated with tamsulosin (35 mcg/kg/d) orally plus airway sensitization; group e: treated with tamsulosin (17.5 mcg/kg/d) orally plus airway sensitization. discussion sustained inflammation of the respiratory tract arises from the pathogenesis of many chronic pulmonary conditions, including copd and asthma (23). these diseases come with a high case fatality ratio owing to the variable response rates to treatment choices (5). corticosteroids are the cornerstone of treating inflammation according to their major roles as anti-inflammatory and immunosuppressant. these agents also come with a long profile of dangerous side effects, especially with a long-term administration (24). prednisolone is a corticosteroid that is traditionally used as antiinflammatory drug (25). we utilized prednisolone in this research to compare pharmacological action with tamsulosin. inflammatory cytokines have a leading role in orchestrating respiratory injury in asthma and copd, making these mediators very attractive targets in treatment (26). the current study was conducted to evaluate the impact of tamsulosin on selected inflammatory cytokines. the tamsulosin effect was then studied via using the ovasensitized rat model. tamsulosin is commonly prescribed to men as firstline agent for treating benign prostatic hypertrophy (bph) by approximately 80% of physicians. it belongs to the α1-ar inhibitor pharmacological class (6). other members of this class have been found to protect against cytokine storm and hyperinflammation by a previous study (27). prescribing tamsulosin is very common in men, and we aim to investigate other effects this drug may own. in the current study, the prolong ovalbuminsensitization in rats of the negative control (group b) caused airway inflammatory signs in comparison with the normal control (group a). this result is consistent with findings from previous studies (28-30). first, we evaluated il-4, an inflammatory mediator, which was significantly elevated (pvalue <0.05) in rats with ova-sensitization (group b) when compared with the normal control ( group a), as shown in table 1 and figure 1. these results. were in agreement with both bagnasco et al (2016) (31) and zainab et al (2021) (19), studies which showed that il-4 level was considerably high when the rats were exposed to repeated ova-challenge in comparison to the placebo group (19,31). according to table 1, a reverse airway inflammation reflected by significant (p-value <0.05) cytokines level reduction is caused by prednisolone treatment (group c) compared to the negative control (group b), which was also demonstrated by a previous study (28). in this study, both doses of tamsulosin 35mcg/kg/d (group d) and 17.5 mcg/kg/d (group e) had shown the ability to significantly (p-value <0.05) reduce levels of il-4. moreover, there was no significant difference (pvalue >0.05) when comparing treated group of tamsulosin 35 mcg/kg/d (group d) to prednisolone 4.12 mg/kg/d (group c) as seen in table 1 and iraqi j pharm sci, vol.32(1) 2022 anti-inflammatory effect of tamsulosin 287 figure 1. demonstrating that tamsulosin at dose 35 mcg/kg/d had equivalent activity to prednisolone 4.12mg/kg/d regarding the reduction of il-4 in airway model. in addition to il-4, another prοinflammatory mediator il-6 was analyzed, which was produced after ova sensitization in a rat airway model. evidence showed that il-6 has a correlation with the pathogenesis of asthma and is associated with the negative outcome after treatment (32). in the current study, il-6 concentration in serum was significantly reduced (p-value<0.05) by prednisolone treatment when compared to the negative control (group b), which was also observed by a previous study (33). il-6 level in serum was significantly downregulated (pvalue<0.05) in rats treated with tamsulosin (35mcg/kg/d) when compared with ovachallenged rats (group b). this result is in line with another study conducted by grzegorz et al (34). on the contrary, tamsulosin in dose 17.5 mcg/kg/d (group e) showed no statistical difference (pvalue>0.05) in il-6 level compared to the negative control group, figure 2, suggesting this dose may be too low to produce a major reduction in il-6 cytokine. interestingly, tamsulosin 35mcg/kg/d (group d) was found to have an approximate effect to prednisolone (group c) in reducing il-6 level, as there was no significant difference (p-value>0.05) between the two groups, table 1. this anti-inflammatory effect of tamsulosin could be attributed to different mechanisms. in his study, alain et al investigated 26 genes of inflammation markers at mrna level which resulted in an observed downregulation of mean mrna expression by 12/26 (46.2%) in the tamsulosin treated group (20). additionally, lin et al demonstrated. the effect of tamsulosin in blocking the activation of nf-κb, leading to diminishing production of inflammatory cytokines (35). this finding indicated that tamsulosin may have an equivalent mechanism to glucocorticoids in controlling inflammation at the molecular level. prednisolone also acts by repressing the gene transcription by interference with the nf-κb family of nuclear transcription factors to diminish the triggering of several pro-inflammatory cytokines (36,37). moreover, another mechanism is proposed in an animal study which revealed that by blocking α1 ar pathway, catecholamine ability to augment cytokines production is lost, thus improving the capability to survive inflammatory injury in mice (27). the other dominant inflammatory parameter in lung injury is tnf-α (38). in the current study, the level of tnf-α in rat serum of the negative control (group b) was increased significantly (p-value <0.05) compared to the normal control (group a) as shown in table 1. kumar. et al ( 2017) research reported that the tnfα level is elevated in rats with induced airway sensitization compared to the negative control, which agrees with our study (39). treatment with prednisolone 4.12mg/kg/d (group c) showed a significant reduction (p-value <0.05) in tnf-α level, compared to the normal control (group a), as seen in table 1 and figure 3. this result agrees with other studies that corticosteroids are effective agents which are administered for controlling the release of prο-inflammatory cytokines such as tnfα and il-6 (17). in addition, tnf-α was significantly reduced (p-value <0.05) by treatment with tamsulosin 35mcg/kg/d (group d) and this result is in agreement with earlier research by william et al (2018) (6). this result can be explained by the effect of tamsulosin in blocking the activation of nf-κb, therefore reducing the synthesis of inflammatory cytokines (35). additionally, another possible mechanism could be described by a previous animal study in which norepinephrine (ne) was found to control the phosphorylation of mitogen-activated protein kinases (mapk) through ar pathway, which in turn regulated the macrophage production of tnf-α. therefore, after using an α-ar antagonist, ne role in tnf-α production was blocked 40. in another research, sumera et al detected a downregulation in the levels of mrna expression of tnf-α after the administration of α1ar antagonist in rats, suggesting an immunomodulatory potential for α1-ar blockers (41). in the current study, it is interesting to note the presence of a superior reduction of tnf-α in the tamsulosin (35 mcg/kg/d) treated group over the prednisolone 4.12mg/kg/d treated group as shown in table 1 and figure 3. this present study has demonstrated that tamsulosin (17.5 35 mcg/kg/d) reduce and/or prevents certain inflammatory cytokines release in ovalbumin-challenged rats. in this animal model, the anti-inflammatory action of tamsulosin was detected for the first time; nevertheless, previously reported similar anti-inflammatory response was in other α1 ar antagonist drug-class members (27). as a final result, according to table 1, the tamsulosin dose (35mcg/kg/d) had a more pronounced and reliable effect in reducing inflammatory cytokines (il-4, il-6 and tnf-α) than the lower dose (17.5mcg/kg/d). conclusion the current research showed that tamsulosin, an α1 ar blocker had an antiinflammatory effect in airway model that included a reduction in serum concentration of major prοinflammatory cytokines (il-4, il-6 and tnf-α). in the future, more studies with tamsulosin can be conducted for prevention and treatment of other inflammatory lung diseases. iraqi j pharm sci, vol.32(1) 2022 anti-inflammatory effect of tamsulosin 288 acknowledgment this study was abstracted from the master’s study that was submitted. at the department of pharmacology and toxicology, college of pharmacy, al-basrah university. the authors offer their greatest thanks to the department of pharmacology and toxicology, college of pharmacy, al-basrah university. conflict of interest the authors of this work had declared no conflict of interest. references 1. jansen em, van de hei 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mscs in mice with acute lung injury. journal of interferon & cytokine research 2021;41(1):29-36 28. kumar s, joos g, boon l, et al. role of tumor necrosis factor–α and its receptors in diesel exhaust particle-induced pulmonary inflammation. scientific reports 2017;7(1):110 29. grisanti la, woster ap, dahlman j, et al. α1adrenergic receptors positively regulate tolllike receptor cytokine production from human monocytes and macrophages. j pharmacol exp ther 2011;338(2):648-57. 30. qasim s, alamgeer, saleem m, et al. appraisal of the antiarthritic potential of prazosin via inhibition of proinflammatory cytokine tnfα: a key player in rheumatoid arthritis. acs omega 2021;6(3):2379-2388. this work is licensed under a creative commons attribution 4.0 international license http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer doi: https://doi.org/10.31351/vol31iss1pp1-19 1 g protein-coupled receptors: undervalued targets for cancer therapy ismail i. al-janabi*,1 *imprial college “retired academic “, uk. abstract despite the g protein-coupled receptors (gpcrs) being the largest family of signalling proteins at the surface of cells, their potential to be targeted in cancer therapy is still under-utilised. this review highlights the contribution of these receptors to the process of oncogenesis and points to some likely challenges that might be encountered in targeting them. the gpcr-signalling pathways are often complex and can be tissue-specific. cancer cells hijack these communication networks to their proliferative advantage. the role of selected gpcrs in the different hallmarks of cancer is examined to highlight the complexity of targeting these receptors for therapeutic benefit. our increasing knowledge of the mechanisms governing the molecular functions of gpcrs may help to identify new targets to treat specific types of cancers. keywords: g protein-coupled receptor, cancer, signalling pathways, signal transduction, cancer hallmarks, targeting receptors. في معالجه السرطان الحقيقيةلم تقدر بقيمتها أهدافًا g ببروتين المقترنةالمستقبالت 1*،الجنابي ابراهيم اسماعيل * college mpriali "المتحدة المملكة ، "متقاعد كاديمي الخالصة المتواجده االشاره بروتينات من عائله اكبر هي (gpcr بمستقبالت تدعى والتي g ) بروتين مع المقترنه البروتينات ان من الرغم على في المستقبالت هذه مساهمه على الضوء المراجعه هذه تسلط مستغله. غير التزال السرطان عالج في استهدافها امكانيه ان اال الخاليا، سطح على المستقبالت هذه اشارات مسارات ماتكون غالبا دافها.استه في نواجهها قد التي المحتمله التحديات بعض الى وتشير السرطاني الورم تكوين عمليه في التكاثرية. لمصلحتها هذه االتصال شبكات السرطانيه الخاليا تختطف االنسجة. بعض في خصوصياتها لها تكون ان ويمكن الشيء بعض معقده المتزايدة معرفتنا تساعد قد االستهداف. عمليه تعقيد ىمد الدراك للسرطان المميزه السمات في المستقبالت هذه من بعضا دور فحص تم المراجعه هذه السرطانات من معينه انواع لعالج جديده اهداف تحديد في المستقبالت لهذه الجزيئية الوضائف تحكم التي اآلليات حول للسرطان، استهداف المستقبالت المميزة، السمات اإلشارة، السرطان، مسارات االشارات، نقل g مستقبالت بروتين: المفتاحيةالكلمات introduction the guanine nucleotide-binding protein coupled receptors (gpcrs) constitute the largest family of receptors on the surface of human cells that are involved in signal sensing and transmission. these receptors can detect signals in the extracellular environment in the form of chemical molecules to which they bind, undergo conformational changes and pass the message to the inside of the cell for action and outcome. there are around 800 such receptors in humans with half of that number responding to exogenous ligands such as odour molecules and photons which are concerned with physiological functions like smell and vision (1,2). the remaining number of receptors has endogenous ligands, hence referred to sometimes as endogpcrs, and their number is estimated to be 401 (3). these g protein-coupled receptors with endogenous ligands will be the focus of our review as some could be targeted for therapeutic interventions including the treatment of cancer. the endogenous ligands include small molecules, peptides, hormones and neurotransmitters (4,5). one gpcr can interact with more than one ligand. moreover, one ligand can interact with more than one gpcr to create complex and diverse signalling pathways. signals received by gpcrs and relayed to the inside of the cell will regulate a multitude of physiological processes such as behaviour, mood, taste, smell, blood pressure, immune response, neurotransmission, metabolism, cell growth and differentiation (6,7). these important receptors also mediate the effects of around 34% of all the drugs currently on the market (1,8) (table 1.) the gpcrs are also known as seventransmembrane domain receptors (7-tm) because they contain sequence stretches in the shape of αhelices that transverse the bilayer membrane of the cells seven times and exhibit a high degree of calculated hydrophobicity (9,10). 1corresponding author e-mail: ismail.janabi@gmail.com received: 13/4/2021 accepted:4 /7 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp1-19 iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 2 table 1. some common drugs targeting various g protein-coupled receptors (gpcrs)(114,115) common drugs gpcr targeted condition alfuzosin, terazosin α1-adrenergic benign prostate hyperplasia and hypertension bisoprolol, betaxolol, clonidine α2-adrenergic hypertension atenolol, metoprolol β1-adrenergic hypertension nadolol, albuterol β2-adrenergic asthma atropine all muscarinic receptors m1, m2, m3, m4 and m5 bradycardia, hypersalivation, bronchial secretions and poisoning with cholinergic drugs tolterodine m1,m2,m3,m4 and m5 overactive bladder scopolamine m1 motion sickness, diarrhoea cetirizine, loratadine histamine h1 allergies cimetidine, ranitidine histamine h2 heartburns and gastrointestinal ulcers trazodone 5-hydroxytryptamine (serotonin) 5-ht, 5-ht1b depression and anxiety sumatriptan 5-ht1d migraine codeine, fentanyl mu pain oxycodone mu, kappa pain misoprostol prostaglandin e2 gastric ulcers montelukast cyslt1 asthma haloperidol, olanzapine dopamine d2 schizophrenia pramipexol, ropinirole dopamine d2 parkinson’s disease, restless leg syndrome metoclopramide dopamine d2 nausea and vomiting other less widely used names include serpentine receptors, g protein-linked receptors and heptahelical receptors. the seven α-helices are each made up of 23±5 residues and connected by three extracellular and three intracellular loops of varying residue numbers (figure 1.). figure 1. depiction of the general structure of a typical g protein-coupled receptor showing the 7 transmembrane α-helices spanning the cell membrane and linked to the heterotrimeric g protein. each receptor has an extracellular amino-terminal and an intracellular carboxyl domain (11). for a receptor to be classified as a gpcr it will have to satisfy two main requirements. the first is the presence of the seven-transmembrane structure and the second is the ability to interact with a guanine nucleotide-binding protein (g-protein) and together they make up the main features of gpcrs. the crystal structure of a representative gpcr is illustrated in figure 2. iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 3 figure 2. the crystal structure of the human lysophosphatidic acid receptor 1 (a) illustrating the 7 transmembrane nature of these receptors, (b) with its constituents amino acids represented by their international one letter codes a-alanine r-arginine n-asparagine d-aspartic acid c-cysteine e-glutamic acid q-glutamine g-glycine h-histidine i-isoleucine l-leucine k-lysine m-methionine f-phenylalanine p-proline s-serine t-threonine w-tryptophan y-tyrosine and v-valine (c) the receptor being occupied by a ligand, shown as balls, called ono-3080573. baskets in the diagram indicate missing amino acids. the structure was constructed using the visualisation software given in reference 110. there are more than 120 gpcrs with unknown ligands and these are termed orphan receptors (12). both orphan receptors and wellcharacterised receptors have been the subjects of several studies including targeting them for cancer therapy. the g proteins signalling partners of the gpcrs are heterotrimeric proteins tethered to the inside of the cytoplasmic membrane. the three members of the heterotrimer are given the symbols α, β, and ɣ with 21 gα proteins, 6 gβ proteins and 12 gɣ proteins in humans (10,13,). the α member (i.e. gα) being the largest of the trimers and itself is subdivided into four main subclasses based on their primary sequence similarity gαs, gαi, gαq and gα12. despite the large number and diversity of the heptahelical transmembrane receptors, they interact with a relatively small number of g proteins inside the cells (figure 1.). there are currently two classification systems for the gpcrs (14). the first system is based on their sequence identity and classifies the human gpcrs into four main classes and these are: class a (or 1) which are called rhodopsin. class b (or 2) which in turn is subdivided into b1 (secretins) and b2 (adhesions). class c (or 3) which are called metabotropics/glutamates. class f (or 6) which are called frizzleds/smootheneds. classes d (or 4) and e (or 5) are not being relevant for humans. the second system of classification is called the grafs system which is based on the phylogenetic origin of the receptor (15). according to the grafs system, the human gpcr superfamily can be classified into five main families. these five families are the glutamate family (22 members), the rhodopsin family (701 members), the adhesion family (33 members), the frizzled/taste2 family (11 members) and the secretin family (15 members). the rhodopsin family of gpcrs, as can be seen, is by far the largest group and includes receptors for neuropeptide y, oxytocin, endothelin, melatonin, several olfactory receptors and many more. the gpcrdb (a g protein-coupled receptor databases websitesee reference 3) lists 401 endogpcrs ( 289 class a rhodopsin, 15 class b1 secretin, 33 class b2 adhesion, 11 class f frizzled, 25 class t taste and 6 others (3). of these 401 endogpcrs 227 (56%) have not been targeted, 107 (27%) have been targeted with established drugs and 67 (17%) targeted in clinical trials. as can be seen, there is still an enormous potential to exploit the remaining, untargeted, gpcrs for therapeutic benefit including the treatment of cancer. signalling mechanism the signalling pathway detailed below is the commonly observed mechanism although alternative routes were reported. in response to agonist ligand binding to the extracellular domains, gpcr undergoes ligandspecific conformational changes leading to the recruitment and activation of heterotrimeric gproteins composed of three subunits namely α, β and ɣ (16,17,18). antagonist ligands are thought to block the conformational change in the receptor and hence stop the signal transduction (19). in the inactive basal state, the α subunit of the heterotrimer is tethered to the inside of the plasma membrane and bound to guanosine diphosphate (gdp) and the other two subunits of the g protein complex. activation of this complex by its partner gpcr stimulates the exchange of guanosine triphosphate (gtp) instead of guanosine diphosphate (gdp) by the α subunit iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 4 (gα) of the g-protein (20,21,22,23). the gtp-bound α subunit will then dissociate from the complex leaving behind the β and ɣ subunits bound together as a dimer. both the gtp bound gα and the remaining bound β and ɣ subunits (gβɣ) will then go on to modulate several signalling pathways by interacting with several downstream effector molecules (24). the baseline signalling pathway of a gpcr is shown in figure 3. figure 3. baseline signalling pathways of gpcrs. gdp-guanosine diphosphate, gtp-guanosine triphosphate, amp-adenosine monophosphate, camp-cyclic adenosine monophosphate, pi3kphosphatidylinositol-3-kinase, raca member of the rho family of gtpases, pkbprotein kinase b also known as akt., rasfamily of small gtpases proteins, mapkmitogen-activated protein kinase, plcphospholipase c, ip3-ionositol triphosphate (second messenger), dag-diacylglycerol (second messenger), pkc-protein kinase c, rho-ras homologous protein, gef-guanine nucleotide exchange factor, dia1-a forming protein that elongates unbranched actin, rock-rho-associated protein kinase. → (activation) ┴ (inhibition) generally, the type of g-protein recruited by the receptor dictates the signalling pathway. some gpcrs, such as the receptors for lysophosphatidic acid (lpa), can couple with both gi and gq subtypes thus triggering dual signalling cascades (25) and the thyroid-stimulating hormone receptor (tshr) can couple to all four subtypes of the g protein activating multiple pathways (26,27,28,29). other gpcrs can couple to only one g protein subtype e.g. the receptors for sphingosine-1phosphate (s1p) as will be discussed later. the gpcrs can adopt different active conformational states depending on the nature of the ligand and are thus able to select a specific signalling pathway, a phenomenon known as biased signalling (26). a biased ligand can selectively lead to an active receptor state for one particular signalling pathway out of many. during the last two decades, it became apparent that certain gpcrs can also trans-activate receptor tyrosine kinases (rtks) such as the epidermal growth factor receptor (egfr) through either ligand-dependent or ligand-independent mechanisms (30,31). in the ligand-dependent transactivation, ligand precursors for rtks are generated following the activation of gpcrs. however, in the ligand-independent transactivation, the cross-talk between gpcr and rtk creates a complex to trigger an rtk downstream signalling through activated g-protein. cross-talk between acetylcholine muscarinic receptors (machrs) and egfr or platelet derived growth factor receptor (pdgfr) lead to the activation of mitogenic pathways. also, several gpcr ligands like lpa and bradykinin can trans-activate egfr(32,33). physiologically, the activation of the gpcrs must be followed by the removal of the signal when it is not required. gα has an intrinsic guanosine triphosphatase (gtpase) activity that is capable of hydrolysing gtp back to gdp, aided by other proteins, and returning this subunit to its inactive state of being bound to gdp. this gdpbound gα will then re-associate with gβɣ to form the heterotrimeric g-protein once again thus terminating the signal. the ligand-occupied receptor itself will normally undergo desensitisation and internalisation mediated by phosphorylation with g protein-coupled receptor kinases (grks). the phosphorylation facilitates the recruitment of a small family of proteins called β-arrestins to the receptor where it could sterically hinder its conformation and aids the dissociation from the heterotrimeric g-protein. thus β-arrestins, via their interaction with clathrin and the adapter protein apiraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 5 2, target the receptor for vesicle-mediated endocytosis (31). beta (β)-arrestins themselves are also capable of initiating a signalling cascade independent of g-proteins thus creating an alternative (biased) signalling pathway and illustrating the complex role that β-arrestins play not only in the desensitisation of gpcr but also in initiating an independent signalling pathway (34). gpcr signalling is diverse and not only dependent on the gpcr itself but also on the type of g-protein they couple with as well as the ligand, the strength and duration of the signal and the cellular environment. over the past few decades, gpcrs became the target of a variety of drugs from a wide range of clinical applications as can be seen in table 2 and figure 4. table 2. range of drugs targeting selected g protein-coupled receptors (gpcrs)(112-115) gpcr drugs and other molecules that target the listed gpcr thyroid-stimulating hormone receptor (tshr) ergoloid thyroid-stimulating hormone (tsh) thyrotropin alpha thyrotropin smoothened (smo) carbozantinib fluocinonide halcinonide sonidegib vismodegib lysophosphatidic acid receptor 1 (lpar1) none currently frizzled (fzd) none currently melanocortin 1 receptor (mc1r) adrenocorticotropic hormone (acth) afamelanotide chemokine receptor 4 (cxcr4) plerixafor chemokine receptor 5 (ccr5) maraviroc vicriviroc leronlimab α2a-adrenergic receptor (adra2a) adrenaline aminosalicylic acid amitriptyline amoxapine amphetamine apomorphine apraclonidine aripiprazole aspirin atropine benzphetamine bethanidine brimonidine bromocriptin cabergoline carvedilol chlorpheniramine chlorpromazine clonidine clozapine cortisone acetate desipramine dexmedetomidine dicyclomine dihydroergotamine diphenoxylate dipivefrin dobutamine doxepin dronedarone droxidopa echothiophate ephedra epinastine epinephrine ergoloid mesylate ergotamine fenoldopam guanabenz guanfacine hydrocortisone phosphoric acid levonordefrin lisuride lofexidine loxapine lurasidone maprotiline mechlorethamine mephentermine methamphetamine methohexital methotrimeprazine methyldopa mirtazapine moxonidine naphazoline nefazodone noradrenaline nortriptyline olanzapine oxycodone oxymetazoline paliperidone paramethadione pergolide phenoxybenzamine phentolamine phenylpropanolamin pramipexole prazosin propericiazine pseudoephedrine quetiapine quinidine reserpine risperidone ropinirole spironolactone sulfasalazine terguride tizanidine tolazoline trazodone triamcinolone acetonide trihexyphenidyl trimipramine xylometazoline yohimbine ziprasidone zuclopenthixol sphingosine-1-phosphate receptor 1 (s1pr1) fingolimod asfotase alfa adhesion g protein-coupled receptors (agpcr) none currently iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 6 figure 4. (a) the relative percentages of the four major subfamilies of g protein-coupled receptors. (b) the percentages of g protein-coupled receptors that target selected classes of conditions.data were extracted from references 3 and 111. however, targeting these receptors for the treatment of cancer appears to be lagging partly due to the complexity of this disease and the gpcrs’ signalling pathways. due to this complexity, we will restrict our discussion in this review to the targeting of the g protein-coupled receptors themselves rather than the other components of the signalling pathways and how that might affect aspects related to the initiation and progression of cancer. g protein-coupled receptors and cancer enormous progress has been made since the discovery last century that certain viruses can hijack cellular genes, incorporate them into their genome and subsequently transfer them back to the host upon re-infection and potentially cause tumours (35). we understand now that several viruses possess genes encoding gpcrs sharing a degree of homology to the human cytokine receptors with several substitutions conferring constitutive activity and enhanced coupling to g proteins. viral genomes also express agonists and antagonists to cellular gpcrs to promote their survival and replication (36). these ligands can modulate host cellular immune response and resistance to death thus promoting tumorigenesis. kaposi sarcoma-associated herpesvirus (kshv) encodes a gpcr that resemble cytokine receptors cxcr and cxcr2 and the human cytomegalovirus (hcmv) expresses at least four gpcrs. moreover, all herpesviruses of the β and ɣ families have hijacked gpcr genes, almost certainly from a previous infection, and adapted them to their use (37). these genes are then expressed via cellular machinery, following viral integration with host dna, to subjugate the cell to their advantage. it is now recognised that cancer usurps a subset of normal cellular genes and derails their function, through various genetic or epigenetic alterations. many genes participate in cell growth, division, differentiation, programmed cell death, immune function and many more tasks that are exquisitely balanced to keep an overall check and control (38,39,40). however, once that balance is disturbed, e.g. because of a mutation in a relevant gene, tumorigenesis can be initiated and then progressed upon the accumulation of further deleterious mutations. a study in 1986 represented early evidence of the involvement of gpcrs in oncogenesis (41). a gpcr termed mas1 was shown to induce the formation of foci when expressed in nih3t3 fibroblasts (mouse cell line employed widely in research) and lead to the formation of tumours in the nude mouse. this suggested an oncogenic potential for the gpcr encoded by the mas1 gene that is not directly linked to viral infection. subsequent work showed that the amount of ligand available and its binding specificity were key determinants of the oncogenic potential of the receptor (37,41). later on, it was demonstrated that mutations play a key role in turning other gpcrs oncogenic. moreover, the discovery that some gpcrs are over-expressed in a wide range of different cancers highlighted the important role that these receptors play in the initiation and progression of cancer (42,43). iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 7 altered expressions and mutations are now thought to play a pivotal role in converting a normally functioning gpcr into being cancerous. over-expression of gpcrs or their autocrine/paracrine activation by an agonist, released by the tumour or the surrounding stromal cells, represents the most frequent tactic used by tumour cells to stimulate gpcr signalling to their advantage (44,45,46). although many g protein-coupled receptors have been implicated in carcinogenesis when they acquire an oncogenic potential, only a few have been targeted and exploited for the treatment of cancers leaving enormous opportunities for future drug discoveries (47). numerous studies linked aberrant gpcr activation or expression to different types of tumours. tumour initiation, progression and metastasis involve many signalling networks mediated by gpcrs. dysfunctional gpcrs signalling pathways and mutations in gpcr genes can be relevant for tumorigenesis. the understanding of the activation and signalling pathways of g protein-coupled receptors is fundamental to the design of targeted drugs for cancer (48,49). the publication of the six hallmarks of cancer in the year 2000, and the later revision and extension in the year 2001 to ten hallmarks, has provided scientists and molecular biologists with a framework and a context to understand the process of carcinogenesis (50,51). gpcrs impact these hallmarks with considerable overlap of the individual mechanisms involved due to the wide range of effects they exert and the network signalling cascades cross-talks. it is with that in mind that the following sections of this review will try to examine the role of selected gpcr players in the ten hallmarks of cancer. gpcrs and sustaining proliferation cells have to maintain communication for the multicellular organism to have overall control over tissue architecture and function. cells are required to grow, divide and increase in numbers when needed, such as during development, wound healing and to replace lost cells and required to stop when these processes are no longer necessary. to do that, individual cells communicate using chemical signals carrying the instructions to proceed through the cell cycle and proliferate. cancer cells are capable of deregulating these chemical signals and divide out of the strict overall control (51). a few gpcrs are known to be deregulated in various cancer types and the example that i have selected here is the thyroid-stimulating hormone receptor (tshr) (52,53). the tshr is composed of 764 amino acids and encoded by a gene (tshr gene) on chromosome 14. the receptor belongs to the rhodopsin family of gpcrs with over half of its secondary structure located in the n-terminal domain and housing several cognate recognition sites for the thyroid stimulating hormone (tsh). tshr is predominantly expressed by thyroid cells and there is an estimated 5000 such receptors per cell. it responds to ligand binding by coupling to either gαs or gαq of the heterotrimeric g protein. activation of gαs stimulates adenylyl cyclase and camp/protein kinase a (pka) pathway with their well-established mitogenic effects. whereas activation of gαq results in the stimulation of protein kinase b (pkb) and the mitogen-activated protein kinase (mapk) pathway. the net resulting downstream effect is to increase thyroid hormone production. the current strategy in reducing the mitogenic effect of tsh through tshr signalling is to induce a state of systemic hyperthyroidism by the administration of levothyroxine (53). hyperthyroidism will suppress the endogenous production of tsh and hence reduces its tumorigenic effect on the thyroid cells. however, systemic hyperthyroidism is associated with accelerated bone loss and cardiac side effects and alternative strategies are needed to reduce the mitogenic impact of tsh on thyroid cells. the alternative approach in treating certain thyroid cancers might be in the form of the selective targeting of tshr using small molecule inhibitors to block its signalling. several small molecules have been identified and trialled recently but none so far have been found to achieve sufficient specificity and further work is ongoing. other gpcrs involved in sustaining and promoting cancerous growth include the cysteinyl leukotriene receptor 2 (cysltr2) and the metabotropic glutamate receptor 3 (grm3). the cysltr2 was shown to be associated with the development and progression of melanoma (54) while grm3 was implicated in b-cell tumour apoptosis (55). gpcrs and evading suppression the growth signals driving cells to proliferate have to be attenuated to prevent the continuous uncontrolled division and ensure homeostasis. defects in these negative feedback mechanisms can tip the balance in favour of the proliferative signals. g proteins, for example, possess the exchange of gtp for gdp as their negative feedback switch to terminate the transduction of signalling activated by ligand binding to their appropriate coupled 7transmembrane protein. the gpcrs themselves can act as a growth suppressor and mutations disabling that function could lead to cancers as in the case illustrated here by a receptor called smoothened (smo). smoothened is an atypical gpcr with a central role in transducing the famous hedgehog (hh) signalling. it is one of the frizzled family of receptors and consists of 787 amino acid residues (56). the involvement of smo in the hedgehog signal iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 8 transduction pathways has been the subject of several studies (57,58,59,60). in its canonical role, the activation of hedgehog starts when another protein called patched (ptch) releases its suppressive action on smo. this allows smo to activate a complex signalling cascade that leads to the transcription of oncogenes and the development of cancer (60,61). on the other hand, when patched is repressing the action of smo, target oncogenes are not transcribed. as can be seen, this signalling cascade does not involve the coupling with g proteins hence it was labelled atypical gpcr. however, in a separate non-canonical cascade smo associates with heterotrimeric proteins and stimulates rac1 (a member of the rho family of gtpases) and rhoa (a gtpase protein a) leading to enhanced migration of fibroblasts and the consequent influence on the tumorigenesis process (61). mutations in smo that increase its activity are associated with a higher incidence of basal cell carcinomas (bcc) and medulloblastomas. therefore, targeting smo by inhibiting its activity appears to be an attractive therapeutic option. vismodegib became the first smo antagonist to be marketed for the treatment of bcc (62,63) (table 3.). acquired resistance to vismodegib action can be problematic if patients develop mutations in motifs of the receptor that bind to the drug. smo also plays an important role in another hallmark of cancer namely resisting cell death. table 3. drugs targeting g protein-coupled receptors (gpcrs) for the treatment of cancer(111,114,115) drug gpcr target indications degarelix (firmagon®) gnrh1 class arhodopsin advanced hormone-dependent prostate cancer lanreotide (somatuline autogel®) ssr2 class arhodopsin acromegaly neuroendocrine tumours mogamulizumab (poteligeo®) ccr4 class arhodopsin mycosis fungioides (mf) sezary syndrome (ss) plerixafor (mozobil®) cxcr4 class arhodopsin lymphoma multiple myeloma sonidegib (odomzo®) frizzled class ffrizzled basal cell carcinoma vismodegib (erivedge®) smo class f-frizzled basal cell carcinoma gpcrs in resisting cell death the balance between the number of cells dividing and dying at any one time is intricately controlled by programs of cell death, the most important of which is called apoptosis. apoptosis machinery is present in a latent form in all cells and triggered by a variety of stimuli such as dna damage, hypoxia and signal imbalances. many gpcrs play a role in apoptosis among them are the receptors for lysophosphatidic acid (lpa). the lysophosphatidic acids (lpas) are simple lipids involved in virtually all of the hallmarks of cancer. the receptor for lpa (i.e. lpar) was first identified and cloned back in 1996. there are currently six lpars numbered 1 to 6 ranging in amino acid content from between 344 to 372 and the most important of them is lpar1. an important challenge in targeting lpar1 in cancer therapy is the complex array of heterotrimeric g proteins it can couple with to give rise to various outcomes. lpar1 can couple with three types of gα proteins namely gαi, gαq and gα12 which initiates downstream signalling through plc, mapk and rho (see figure 3.). its activation leads to several cellular responses including proliferation, migration, ca++ mobilisation and adenylyl cyclase inhibition (64,65). the role of lpa in protecting epithelial cells from programmed death and apoptosis induced by cisplatin in cervical cancer cells has been investigated (66). although targeting lpa signalling appears to be an attractive option for cancer treatment, it is unlikely that this approach will be effective as a monotherapy. previous studies showed that lpa is a very strong inducer of cell migration, invasion and metastasis but a weak stimulant of proliferation. growth factors such as epidermal growth factor (egf) and insulin-like growth factor (igf) possess strong cell-proliferative effects but weak chemotactic activity. therefore, a combination of these two approaches, i.e. targeting lpar1 and a growth factor, may represent an effective treatment strategy. gpcrs in enabling cell immortality normal cells can replicate an essentially limited number of times and it is defined by two main barriers which are hard-wired into our cells to prevent unnecessary proliferation. the first barrier is senescence, which is usually an irreversible entry into a non-proliferative but viable state, and the second is cell crisis leading to cell death. cancer iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 9 cells, however, have to overcome these barriers to continue to replicate and emerge as “immortal” cells. an example of immortal cells that are commonly used in laboratories is hela cells which were first isolated from the cervical cancer of a woman in 1951 and are still replicating after passing through so many rounds of cell divisions (67). the wnt (a symbol derived originally from wingless integration) signalling pathway is a major player in the enhancement of cell proliferation thus increasing the chance for cells to break the restrictions imposed by the two barriers mentioned earlier (68,69,70). frizzled receptors (fzds) are a family of proteins that serve as receptors for the wnt ligand proteins and as such, they play a crucial role in cell proliferation (71). the fzds family of receptors consists of 10 members numbered 1-10. their cognate ligands, the wnt group of proteins, consist of 19 members. each fdz receptor can interact with several wnt ligands to activate multiple downstream pathways including the canonical wnt/β-catenin cascade. it is possible that dimerization of the frizzled receptor may lead to wnt signalling through the β-catenin path rather than the non-canonical signalling alternatives (71,72,73). members of the frizzled receptors contain amino acids ranging in number from 500 to 700 depending on the subfamily member. they are considered a novel class of gpcrs even though there is a lack of experimental evidence of their interactions with g proteins. nevertheless, there are ample indirect evidence and bioinformatics predictions pointing to the existence of such interactions. in the fzd/β-catenin pathway, wnt stimulation of fzd receptor results in the activation of another protein calls dishevelled (dvl) leading to the inhibition of the constitutively active glycogen-synthase kinase 3 (gsk3) within a destructive complex consisting of adenomatous polyposis coli (apc) and axin which regulates the phosphorylation and destruction of β-catenin. the spared βcatenin is then translocated to the nucleus to aid the transcription of genes involved in cell proliferation (74). over-expression of fzds is often encountered in various cancers pointing to their involvement in carcinogenesis. therefore, targeting fzd receptors will be a potential approach for cancer treatment. targeting an aberrantly overexpressed fzd at the receptor level may enhance the therapeutic advantages in wnt-driven cancers. approaches to target fzd receptors can include small molecules, short peptides and antibodies (table 3.). however, the safety of these molecules needs to be carefully assessed as there are still moderate expression levels of fzds in noncancerous tissues. gpcrs and genome instability genome instability is one of the enabling characteristics of cancer cells to thrive and proliferate. through the deregulation of genes collectively referred to as “caretakers”, neoplastic cells can facilitate their growth and proliferation. to counteract this, our body is endowed with detection and maintenance systems capable of correcting the vast majority of dna damage. one of these important dna-repair mechanisms is nucleotide excision repair (ner). our gpcr example representing this hallmark is the melanocortin1 receptor, mc1r, which is actively involved in the enhancement of ner function (75). the melanocortin receptors are a subclass of the rhodopsin family of gpcrs. there are five melanocortin receptors namely mc1r, mc2r, mc3r, mc4r and mc5r. we will focus on the rest of this discussion on mc1r. mc1r is expressed in melanocytes and leukocytes where its activation promotes uv resistance and anti-inflammatory action in these two types of cells respectively. this receptor is a relatively small protein consisting of 317 amino acid residues and is highly polymorphic. its normal physiological function is to regulate the skin pigmentation and uv-damage responses. loss of mc1r function is associated with having fair and uv-sensitive skin with higher melanoma risk (76). following activation with melanocortin, mc1r will be coupled to gαs and when this gprotein is activated and dissociated from the heterotrimer, it stimulates adenylyl cyclase activity which leaves atp to generate c-amp (see figure 3.). increased levels of c-amp in melanocytes can lead to a host of other events including the increased melanin synthesis and the acceleration of ner activity which together with the enhanced antioxidant defences can provide improved resistance to uv injury (75). inherited mutations in the mc1r gene are common among fair-skinned populations making them more uv-light sensitive and increase their risk of acquiring melanoma (76). improved mc1r signalling can be achieved through the manipulation of c-amp levels in the skin using pharmacological agents such as the topical application of forskolin (adenylyl cyclase activator) or rolipram (a phosphodiesterase inhibitor) for the removal of photoproducts induced by uv light action on the skin. while these interventions are effective, they lack specificity against melanocytes and therefore may induce off-target side effects. targeting mc1r with a degree of specificity to influence its c-amp signalling cascade can be fruitful in the fight against melanoma, human cancer that harbours the most mutations (76). iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 10 gpcrs in avoiding immune destruction our immune system is primed to attack and destroy cancerous cells as they start to make variant proteins which can be interpreted as nonself-due to the acquisition of mutations. therefore, for a tumour to thrive and grow it must be able to overcome the strict immune surveillance imposed by our bodies. cancer cells can achieve that in numerous ways and one of those is through altering the expression of gpcrs with relevant function in the immune system (77). our understanding of the immune system has grown fast in the last three decades through the discovery of chemokine receptors and their crucial role in homeostasis. these receptors cause immune cells, notably leukocytes, to migrate and seek their cognate ligands (chemokine) in a process called chemotaxis (77,78). cancer cells can hijack the function of chemokine for their purpose. tumours consist not only of the mass of cancer cells but also many non-malignant types of cells called stromal cells (or simply stroma). many of these stromal cells are leukocytes making up what is known as leukocyte infiltrate. the identity and number of cells in the leukocyte infiltrate is governed by the chemokine secreted by either the stromal or the tumour cells. there are about 48 distinct chemokines and 19 chemokine receptors in humans. an important member of these chemokines is cxcl12 which acts as a specific ligand for the receptor cxcr4. this receptor, cxcr4, is a gpcr consisting of 352 amino acids and a member of the rhodopsin family. signalling through this receptor plays an important role in the initiation and progression of cancers through the activation of several downstream signalling pathways (79,80). cxcr4, of course, has the additional role of acting as a co-receptor for the human immunodeficiency virus (hiv) but that function is beyond the scope of this review. like many gpcrs, cxcr4 can signal through g protein-dependent or g proteinindependent pathways. however, the g proteindependent pathway appears to delineate the majority of the biological outcomes through the activation of gαi. the activated gαi can inhibit adenylyl cyclase as well as activates the src (short for sarcoma) family of tyrosine kinases while the gβɣ dimer activates phospholipase c (plc) and phosphatidylinositol-3-kinase (pi3k) leading to cell migration (81). cxcr4 is frequently found overexpressed in many cancers such as those of the brain, breast, colon, rectum, lung, prostate, ovary, skin and leukaemia. several inhibitors which target the cxcl12/cxcr4 axis and attenuate the growth and progression of tumours have been trialled (82). these include small molecules, small peptides and antibodies. plerixafor is a small molecule inhibitor of the binding of cxcl12 to cxcr4 which is currently on the market for the treatment of nonhodgkin’s lymphoma and multiple myeloma (tablet 3.). the resistance of some cancer types to the emerging immunotherapies may be due, in part, to the cxcl12/cxcr4 signalling and targeting this axis may enhance the effectiveness of these treatments. gpcrs in promoting inflammation the dense infiltration of many tumours with immune cells which mirrors inflammatory conditions has long been observed. the initial immune cells recruitment might be to fight nascent cancer cells, however, the continuous presence of inflammatory signals can have unintended consequences. chronic inflammation can enhance tumorigenesis, and represents a further hallmark and capability for cancers to progress. the c-c chemokine receptor type 5 (ccr5) is a 352 amino acid chemokine gpcr receptor and gained fame when it was discovered to be acting as another receptor for the entry of the human immunodeficiency virus (hiv) into cells. it binds multiple ligands including ccl3, ccl4, ccl5, ccl8, ccl11, ccl13 and ccl16. the binding of ligands to ccr5 induces conformational changes leading to the coupling of gαi-heterotrimer and the subsequent dissociation of the trimer. gαi inhibits adenylyl cyclase while gβɣ subset activates phospholipase c (plc) which leads to the rapid increase in the cytoplasmic calcium ions, ca++ (83). gβɣ also activates protein kinase c (pkc) and the expression of several genes implicated in inflammation (84,85). additional pathways induced by ccr5 can also lead to cell survival, cell proliferation and immune cell differentiation. owing to the prominent role that ccr5 plays in hiv infection, a few of the drugs that were developed to target that disease have been repurposed for investigating their deployment in the treatment of cancer e.g. maraviroc, vicriviroc and the humanised monoclonal anti-ccr5 antibody leronlimab (see table 2.). additionally, metastatic liver cells secrete ccl5, an important ligand for ccr5. this ligand appears to possess tumourenhancing effects on cancer cells as well as their associated macrophages. blocking ccl5 using an antagonist such as maraviroc leads to the subsidence of the tumour-promoting inflammation in patients with colorectal cancer (85). mediators such as prostaglandins pgs can be another driving force behind carcinogenesis and presenting a further link between gpcrs and inflammation (86). this pathway may start with a type of prostaglandin; the pg2 is the most common one, which is generated by the action of cyclooxygenase enzyme (cox2) on arachidonic acid. pg2 then binds to its cognate g proteincoupled receptor ep1-4 to initiate the inflammation signalling pathway. several studies associated the increased expression of pg2 with gastrointestinal cancers. pg2 and cox2 can also drive the iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 11 expression of chemokines that participate in the process of forming new blood vessels (angiogenesis) and can further promote cancer progression through that route. exploiting the role of gpcrs in chronic inflammation to control tumorigenesis can be challenging due to the complex interplay of several other mediators in this process (84,85). gpcrs in de-regulating metabolism cancer cells are characterised by their ability to re-program their utilisation of glucose even in the presence of adequate oxygen supply. normal cells, in the presence of oxygen, break down glucose to pyruvate by glycolysis and transport most of that pyruvate to the mitochondria to generate adenosine triphosphate (atp). under anaerobic conditions, however, little pyruvate is transported to the mitochondria and most of it is converted to lactate in the cytoplasm. cancer cells exhibit what is known as the "warburg effect” in that they can limit their energy metabolism largely to glycolysis, even in the presence of oxygen, despite this route being around 18 fold less efficient in generating energy. it is thought that this re-programming allows the diversion of the much-needed biosynthesis intermediates to the fast-growing and dividing cancer cells. the gpcrs play an important role as regulators of several aspects of metabolism (87). some metabolic products are often cognate ligands for gpcrs and their binding plays a significant role in cancer development. gpr51 and gpr91 are g protein-coupled receptors, for lactate and succinate respectively that participate in many of the hallmarks of cancer and are found to be elevated in several cancer types (88). the liver is the crucial organ of metabolism and over 50 gpcrs are predicted to be expressed in mouse liver (87). however, our knowledge of the function of these gpcrs is still rudimentary. the α2a-adrenergic receptor (adra2a) is 465 amino acids gpcr that has adrenaline and noradrenaline as its cognate ligands and is widely targeted by other drugs and molecules (see table 2.). it couples primarily with gαi and is associated with several functions including those of the central nervous system, cardiovascular system, insulin secretion and lipolysis (89). the adra2a receptor has been investigated with cervical cancer and was found to be significantly down-regulated (90). elevated expression of adra2a in cervical cancers was demonstrated to suppress cell proliferation and promote senescence and apoptosis through the inhibition of pi3k/pkb/mtor (mechanistic target of rapamycin) axis(90). here we have an example where an increase in the gpcr activity leads to the suppression of cancer. interventions to elevate the targeted expression of adra2a might be worthy of investigation in certain types of cancers. gpcrs in angiogenesis the need for cells to be supplied with nutrients and oxygen and for the removal of their waste metabolic products obliges them to reside close to a capillary blood vessel (within about 1 mm). accordingly, solid tumours cannot grow to more than the head of an average size matchstick without an adequate supply of blood. the formation of a new blood vessel requires the degradation of the extracellular matrix, the increased permeability of the cells, the disruption of cell adhesion and the proliferation and migration of endothelial cells towards the site where the new blood vessel is being constructed. the major mediators of these processes are the vascular endothelial growth factor (vegf) and the fibroblast growth factor (fgf). however, gpcr agonists and signalling pathways have also been documented to have a role in angiogenesis (91). the s1pr1 is a sphingosine-1-phosphate receptor and a member of the gpcr superfamily. the receptor and its ligand, s1p a bioactive lipid, have been shown to have wide biological functions including proliferation, angiogenesis and migration. s1pr1 consists of 382 amino acid residues and signals primarily through gαi to affect downstream targets including ras/erk (extracellular-signalregulated kinase) and rac/pak (p21 activated kinase) (92). transgenic s1pr1-null mice result in embryonic lethality due to deficient formation of blood vessels indicating the essential role this gpcr plays in vascularisation. several investigations have pointed to the different functions of s1prs in both pro-and antisurvival signalling depending on the cancer cell type e.g. activating s1pr1 in glioblastoma leads to a decrease in proliferation while activation in tlymphoblastic lymphoma is linked to increased progression (93). consequently, it becomes necessary to consider the type of cancer tissue involved when targeting this receptor for optimum effect. release of chemokines such as ccl2, ccl3 and ccl5 by cancer cells and the consequent signalling through their gpcrs can act on stromal cells to stimulate the production of matrix metalloproteinases (mmps) to degrade matrix proteins and aid vascularisation (91). targeting gpcrs, particularly in combination with vegf, can be an effective strategy to starve tumours of their increasing need for nutrients. iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 12 gpcrs in activating metastasis mortality from cancer is largely due to the spread of the primary tumour to other sites and as high as 90% of cancer patients die because of metastasis rather than original cancer. it becomes obvious to see why successful intervention in this step of carcinogenesis can be very rewarding. the invasion-metastasis cascade is a complex multistep process involving local invasion, entry of cancer cells into the blood or the lymph vessels (intravasation), transit through these vessels, extravasation and colonisation of the new site (94). gpcrs are active participants in the various steps of the metastatic process. among these is a subfamily of 33 human receptors called adhesion g protein-coupled receptors (agpcrs). they form the second largest subfamily of gpcrs, after the rhodopsin subfamily, with a large number of them still being orphan receptors. emerging evidence suggests that they play a significant role in the adhesion of cells to the extracellular matrix and therefore particularly implicated in the metastasis hallmark of cancer. several signalling pathways have been reported for agpcrs that are cell and receptor-specific. the adhesion g protein-coupled receptors are uniquely identified by their large nterminal fragment (ntf) which gives them the flexibility of containing several domains (95,96). most agpcrs include a gpcr proteolytic site (gps) located in the first transmembrane loop. proteolytic cleavage at this site divides the receptor into two fragments ntf and a carboxylic terminal fragment (ctf). signalling can occur through the ntf, through coupling with g proteins mediated by ctf or through one of several other alternative pathways (95). this makes the targeting of agpcrs for cancer treatment challenging with our limited current knowledge. de-orphansing these receptors could be the first step in improving our understanding of the mechanisms and targets of their signal transduction. there are currently no approved drugs targeting any of the agpcrs (96,97,98). chemokines and chemokine receptors are often aberrantly expressed at the primary site of the tumour and correspondingly at its most preferred metastatic site. for breast cancer, for example, studies have found that the g protein-chemokine receptors cxcr4 and ccr7 are over-expressed at the primary site and their ligands (cxc12 and ccl21 respectively) were also over-expressed at main metastatic sites such as the bones and lungs. cxcr4 is widely expressed in a range of cancer types and interaction with its ligand cxcl12 (also referred to as stromal-derived factor 1, sdf1) initiates several signalling cascades leading to responses such as immune cell mobilisation, cell survival, proliferation and metastasis. tumour cells are guided by their cxcr4 that they express towards the concentration gradient of its ligand cxcl12 which is released by organs that serve as a metastatic site for the primary tumour. activated cxcr4 signalling was also noticed with therapy resistance in the treatment of breast, colon and pancreatic cancers. in basal cell and squamous cell carcinomas, enhanced cxcr4 interaction with its ligand also contributes to tumour progression. targeting the cxcr4-cxcl12 axis was fruitful and led to the marketing of an inhibitor called plerixafor which was shown to be effective against various types of cancers including non-hodgkin’s lymphoma and multiple myeloma (99,100) (refer to table 3) landscape of mutations in gpcrs for investigative targeting sequencing of tumour dna often reveals the presence of several mutations compared to its background normal tissue. not all of the observed mutations are participants in the initiation or progression of cancer. those that are the driving force behind tumorigenesis are termed “driver mutations’ and the remaining gene alterations are neutral, as far as the neoplastic process is concerned, and are called “passenger mutations”. despite a large number of gpcrs and the variety of biological functions they perform, it has been difficult to predict the consequences of some of the observed mutations in g protein-coupled receptors(101,102,103). a tissue-specific pattern of mutations is a further complicating factor in the overall biological function of gpcrs. furthermore, gpcrs copy number variation (cnv) could dramatically alter their expression thus creating a potential complexity in driving oncogenesis (104). however, recent advances in the fields of cancer genomics and bioinformatics have provided us with tools that we could utilise to identify mutated genes and assess their biological significance. using these modern bioinformatics methods, a few studies highlighted the presence of clusters of mutations particularly near the cytoplasmic loop of helix 6 in tshr (thyroid-stimulating hormone receptor) and the dry motif mediating the inactive conformation of class a gpcrs (105,106). the adhesion family of gpcrs is highly mutated in several cancer types. their n-terminal end mediates adhesion to matrix proteins. a member of this family, gpr98, has the highest number of amino acids residues among all gpcrs and is mutated in around 45% of melanomas. other studies linked mutations and over-expression of other members of the adhesion family of gpcrs to different types of cancers (101,102). the glutamate family, which binds glutamate as a ligand, and the taste receptors families of gpcrs are tasked with detecting nutrient availability and when sensing nutrient deprivation they activate the autophagy process thus enabling the survival of cancer cells. therefore, disruption of these gpcrs could lead, albeit indirectly, to tumour iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 13 survival. a subfamily of glutamate, namely grms were found to be increasingly implicated in melanomas (103). mutations in hormone gpcrs such as gper1 (g protein oestrogen receptor 1), fshr (follicle-stimulating hormone receptor), lhr (luteinising hormone receptor), gnhrh (gonadotropin-releasing hormone receptor) and tshr (thyroid-stimulating hormone receptor) have all been connected with cancer initiation and progression. indeed tshr, which was already mentioned above, was found to be highly implicated in many thyroid conditions including thyroid adenomas. oestrogen binding to gper1 activates a canonical cell growth signalling pathway called egfr/mapk to initiate transcriptional activation of growth signals in multiple tissue types. mutations in smoothened (smo), mentioned before with evading suppression, was found to be a possible contributing cause of basal cell carcinoma. mutations in the frizzled family of gpcrs (fzds) were also found to be implicated in various cancer types (72). the frizzled receptor normally mediates another canonical signalling pathway that includes the wnt and the β-catenin. as a family of gpcrs, the frizzled family is collectively mutated in over 15% of colon adenocarcinomas (72,105). the human genome is endowed with a remarkable ability to maintain its integrity against replication errors and a variety of internal and external insults. complex systems are in place to identify and correct the vast majority of errors resulting from dna damage before the cell is released through to cell division. nevertheless, a minority of mistakes may pass the various checks and barriers unnoticed to end up replicating out of control. a few gpcr signalling pathways participate in the response to dna damage surveillance and repair. one of these gpcrs is the receptor for lysophosphatidic acid lpa (bio-active potent mitogen lipid) which was mentioned earlier in connection with resisting programmed cell death. this receptor, lpar, was found to be aberrantly expressed and mutated in several types of cancers suggesting its involvement in a growth advantage for cancer cells. the lpa receptors can couple to several g proteins subtypes which might explain their wide range of effects. their cognate molecules can establish an autocrine loop that further fuels their signalling actions. lpa also promotes stimulatory effects on different types of tumours by trans-activating egfr and triggering a functional cross-talk between its ligands and egfr signalling. various isoforms of lpar such as lpar1 and lpar2 have been shown to mitigate dna damage induced by chemotherapy and radiation treatment particularly in the intestinal epithelium. this tissue consists of relatively fast replicating cells making them more vulnerable to dna damage from chemotherapy and radiation and leading to the frequently encountered sickness and vomiting. although the lpas are rapidly degraded in the gastrointestinal tract, and hence cannot be administered as such orally, synthetic stable derivatives and/or formulations could be investigated for delivery to protect against the devastating side effects of the widely employed chemotherapy and radiation treatment for cancer (64,65). the cxcr4 mentioned previously in connection with metastasis, has another fascinating role in tumour suppression. it was found that blocking the signalling of cxcr4 with a small peptide antagonist promotes ovarian cancer cell death through weakening the checkpoints for dna damage thus precipitating cell crisis and death (107). the intricate balance between cell division on the one hand and cell death on the other keep the cell number of an organism under unified overall control. cancer develops as a result of tipping the balance in favour of cell division with fewer cells dying compared to those produced by cell division. facilitating the death of cancerous cells is a mainstay strategy for treating this disease. gpcr signalling has been implicated in driving the resistance to some cancer therapy. basal cell carcinoma (bcc), for instance, results from an overactive hedgehog signalling through the smoothened (smo) gpcr axis. an inhibitor of smo was found and marketed under the name vismodegib. however, resistance to this therapy soon developed and appears to be common. it is thought that bcc largely relies on this signalling pathway for its survival and this over-reliance can drive the evolution of bcc into further activation of the hedgehog signalling leading to the observed resistance to vismodegib. other gpcrs were also implicated in treatment resistance to melanomas (62,63). hyperactive gpcrs are often behind the initiation and progression of cancer highlighting their proto-oncogenic characteristics (eight out of the ten gpcrs featured in figure 5 are protooncogene in that sense). however, a growing body of evidence points to a tumour-suppressive role of some gpcrs in certain types of cancer. an orphan gpcr called gpr 56 inhibited prostate cancer and melanoma progression and its expression were inversely correlated with malignancies of latter cancer suggesting a tumour-suppressor role for this orphan receptor. s1pr1 is another example of a gpcr that can mediate both proliferative and antiproliferative effects on growing cancer cells. iraqi j pharm sci, vol.31(1) 2022 targeting gpcrs in cancer 14 figure 5. depiction of the influence of selected gpcrs on the various ten hallmarks of cancer.(tshrthyroid-stimulating hormone receptor, s1pr1-sphingosine-1-phosphate receptor 1, agpcrs-adhesion g protein-coupled receptors, cxcr4-chemokine receptor type 4, ccr5-chemokine receptor type 5, smosmoothened, lpar1-lysophosphatidic acid receptor 1, mc1r-melanocortin 1 receptor, adra2a-alpha2a adrenergic receptor, and fzds-frizzled receptors). the gpcrs offer significant opportunities to intervene in their signalling functions for the treatment of cancer using small molecules or biologics (108,109). further comprehensive studies of how these gpcrs are de-regulated should reveal more targets for rational drug design. repurposing existing drugs to target gpcrs for the treatment of cancer is a further investigative avenue to follow particularly given a large number of such drugs available on the market for indications other than cancer. concluding remarks the large number of the different types of gpcrs available and the variety of oncogenic signalling pathways they participate in offering a great opportunity for therapeutic intervention for the treatment of cancer. the tissue-specific and complex signal transduction network must be carefully investigated and assessed when targeting gpcrs to 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http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 heliotropium europaeum doi: https://doi.org/10.31351/vol30iss2pp158-166 158 extraction and identification of phenolic compounds from the iraqi heliotropium europaeum l. plant walaa h. jasim *,1 and maha n. hamad* **department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq. abstract the heliotropium europaeum l. (boraginaceae) are well-known to contain toxic pyrrolizidine alkaloids in addition to other secondary metabolites. its spread in the mediterranean area, southern europe, central asia, and iraq. the present study included extraction of the aerial parts of iraqi heliotropium europaeum with methanol (soxhlet apparatus), fractionation, screening the active constituent, and identification by chromatographic techniques. the extract was suspended in distilled water and partitioned with chloroform, ethyl acetate, and nbutanol. the hydrolysis step was done for the two fractions (n-butanol and ethyl acetate). phytochemical screening and identification of bioactive substances of the plant was done for the two fractions. a qualitative and quantitative analysis of the fractions was carried using a high -performance liquid chromatography technique and twenty–one reference standards. the outcomes of this study were the identifications of new six phenolic compounds (kaempferol (1), silybin (2), caffeic acid (3), genistein (4), apigenin (5), in addition to syringic acid (6)) from heliotropium europaeum ethyl acetate fraction. key words: heliotropium europaeum, soxhelt, genistein, syringic acid, high -performance liquid chromatography. من النبات العراقي حشيشة العقربالمركبات الفينولية وتشخيص فصل * مها نوري حمد و 1*،والء حامد جاسم .،العراق بغداد ، بغداد جامعة ، الصيدلة ،كلية الطبية والنباتات العقاقير فرع* الخالصة على قلويدات بيروليزيدين السامة باإلضافة إلى نواتج معروف باحتوائه (boraginaceae) نبات حشيشة العقرب أو التنووم من عائلة الدراسة الحالية على استخالص تتضمن وجنوب أوروبا وآسيا الوسطى والعراق. األيض الثانوية األخرى. ينتشر في منطقة البحر األبيض المتوسط التجزئة ، وفحص المكون النشط ، والتعرف على التقنيات تم اجراء عملية ، و(وكسلتجهاز ساألجزاء الهوائية من الهليوتروبيوم العراقي بالميثانول ) بيوتانول. تم إجراء خطوة التحلل المائي -ن الكروماتوجرافية. تم تعليق المستخلص في ماء مقطر وتم تقسيمه باستخدام الكلوروفورم ، أسيتات اإليثيل ين. تم إجراء تحليل نوعي جزئاء فحص كيميائي نباتي وتحديد المواد النشطة بيولوجًيا في النبات للاإليثيل(. تم إجر اسيتاتبيوتانول و-ن) جزئينلل باستخدام تقنية كروماتوغرافيا سائلة عالية األداء وواحد وعشرون معياًرا مرجعيًا. كانت نتائج هذه الدراسة هي تحديد ستة مركبات الجزاءوكمي ل (( من جزء أسيتات 6، باإلضافة إلى حمض الحقن )أبجنن (5)، جنستين (4)( ، 3، حمض الكافيك ) (2)ليبينس، (1) كامفيرولفينولية جديدة ) .لنبات حشيشة العقرب اإليثيل السائل عالية االداء . كروماتوجرافيا , جينيستين ، حمض الحقن,سوكسيلت ,هيليوتروبيوم يوروبيوم الكلمات المفتاحية: introduction large number of plants of heliotropium genus belong to boraginaceae family which is biggest family of plant that includes around 250-300 species in the world. these plant species are widespread in temperate and tropical parts of both hemispheres (12) figure 1. the name “heliotrope” is taken from the fact that these plants move their leaves toward the sun (3,4). the phytochemical studies about heliotropium europaeum are very limited and majority of these studies are concentrated on pyrrolizidine alkaloids (pas) (5,6). the toxicity of this type of alkaloids are concentrated usually in the flowering parts of the plants and in the seeds of the boraginaceae (all genera) (7). some pyrrolizidine alkaloids-bearing plants are used as ornamental plants, ground cover, soil improvers, and can contaminate feeds of animal (8). risks of exposure to pyrrolizidine alkaloids, in the large countries, originate from so called herbal remedies, folk medicines and herbal teas (7). pyrrolizidine alkaloids (pas) are produce by plants and regarded as one of the widespread toxins of natural origin (9, 10). they are esters produced from esterification of various amino alcohol bases called necine and mono or dicarboxylic acids (11). toxicological studies displayed the reason behind such toxicity of h. europaeum is the alkylation that take place by some biological nucleophiles such as nucleic acids (enzymes, dna and rna) and proteins via 1,2-unsaturated pas metabolites (12,13). the importance of this plant lead to screen the chemical constituents especially the phenolic compounds. 1corresponding author e-mail: walaa.hamid@yahoo.com received: 30/1/2020 accepted: 9/5 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp158-166 iraqi j pharm sci, vol.30(2) 2021 heliotropium europaeum 159 figure 1. the iraqi heliotropium europaeum materials and methods plant materials the aerial parts of heliotropium europaeum l. were collected from the bank of tigris river in the periphery of tikrit city in august 2019. the collected plant purified carefully then identified, authenticated by dr. khansaa rasheed at iraq natural history research center and museum plant and environment department / university of baghdad. the aerial parts were cleaned, dried in shade, and pulverized in a mechanical grinder to a fine powder extraction and fractionation of the different active constituents 100 g of powdered plant was first defatted with hexane (250 ml) for 24 hr. at room temperature (about 25 ℃) then filtered. the residual plant was dried on air and packed in the thimble of soxhlet apparatus. the extraction was carried with 750 ml of methanol till exhaustion. the extract was filtered and the solvent was evaporated by applying a reduced pressure by a rotary evaporator. a dark greenish residue was obtained and then suspended in 150 ml distilled water and partitioned with three solvent chloroform, ethyl acetate, and n-butanol (3x150 ml) for each solvent. the chloroform and ethyl acetate fraction were dried with sodium sulfate (anhydrous) then filtered and evaporated to dryness. hydrolysis of ethyl acetate and n-butanol fractions the hydrolysis step was done for the two fractions by reflux for 10 hr. using 5% hcl (200 ml). the cooled solution was partitioned by extraction with ethyl acetate (3x200 ml). the layers were combined, dried and filtered. the identification of n-butanol and ethyl acetate fractions were carried out by thin layer chromatography (tlc) and hplc technique. identification of phenolic compounds in heliotropium europaeum plant extract 1. preliminary phytochemical analysis by chemical tests preliminary qualitative phytochemical analysis for the screening and identification of some important chemical constituents of heliotropium europaeum plant under study was carried out on crude extract, and fractions using the standard procedures as previous reported (14) . test for phenolic compound: the plant extract (or fractions) (500 mg) was dissolved in 80% methanol (20ml) and filtered. the filtrate was used for the following tests: (a) naoh test: the alkaline reagent test for flavonoids was performed by placing 0.5 mg of the crude alcoholic extract in a test tube followed by addition of diluted solution of 10% sodium hydroxide drop wise. the dissolved flavonoids formed an intense yellow color due to formation of sodium phenolate salt as precipitate. this color was changed to faint yellow upon the addition of diluted solution of hydrochloric acid hcl. it was noted that any further addition of diluted solution of hcl to the resulted solution, turns the solution to colorless that indicate the flavonoids presence (15, 16). (b) ferric chloride test: the plant extract (25 mg) dissolves in distilled water (10ml) and filter. aqueous ferric chloride (1% fecl3) solution was added to the filtrate. the appearance of intense green, black or blue color indicates the of phenolic acid presence in the sample due to formation of ferric phenolate salt as precipitate. 2. thin layer chromatography using readymade plate aluminum coated tlc sheet g/f254, 0.20 mm stationary phase used was silica gel, using uv light for detection of the spot. different mobile phases were tried for the detection of plant constituents (phenolic compounds) and it was used for the identifying of phenolic compounds of heliotropium europaeum l that found in ethyl acetate and n-butanol fractions of the plant. the p1 mobile phase (chcl3: acetone: formic acid (75:16.5:8.5) (17) proved to be the best for separation of the fractions, and it was adopted for separation. after drying the developed plates were sprayed with 5% ethanolic koh. 3. the hplc technique the hplc conditions for analyzed fractions hplc system instruments were supplied by knauer, germany. iraqi j pharm sci, vol.30(2) 2021 heliotropium europaeum 160 table 1. hplc system components. component model or version company and origin 1 binary highpressure gradient pump p6.1l knauer, germany 2 diode array detector dad 2.1l knuaer, germany 3 sample loop (20 µl) and injector d1357 knuaer, germany 4 analyses and system control software claritychrom, v 7.4.2.107 dataapex, czech republic in this method a c18 column was used for separation with internal diameter of (250 4.6 mm). the particle size is 5 µm with a pore size of 80 å (18) . 1% aqueous acetic acid, solvent a, and acetonitrile, solvent b, were the mobile phases. moreover 1 ml/min was the flow rate. the column was thermostatically controlled at 28℃ and the injection volume was fixed at 20 μl. the technique was employed was gradient elution with continuous change of b solvent proportionally to a solvent. table (2). the instrument is equipped with photo diode uv detector at the following wave lengths (272, 280 and 310 nm) in order to scanned the resulted chromatogram. table 2.the gradient program used in separation by hplc system. time (min) mobile a (%) mobile b (%) flow rate ml/min 0 90 10 1ml/min 28 60 40 1ml/min 39 40 60 1ml/min 60 10 90 1ml/min the detection of each metabolite was performed by matching retention time and absorbance spectrum of the standards. for quantitative analysis the concentration components were calculated by calibration curve in which serial concentrations of reference standards was plotted against their equivalent peak area, the calculation was done by using the straight line equation y= ax +b, slop = y/x (y and x: are holding the place of coordinates (x, y) of any point that lies on the line, a: slop, b: yintercept), where r2: regression factor. twenty-one standards were used in this study and they were: quercetin, apgenin, luteolin, chlorogenic acid, kaempferol, caffiec acid, genistein, ferullic acid, cinnamic acid, silybin, oleuropein, caffeine, myricetin, salicylicacid, sinapicacids, curcumin, demethoxycurcumin, bisdemethoxycurcumin, daidzein, syringic acid, gallic acid. preparation of stock solutions for standards compounds and the examined samples for hplc stock solutions used in hplc analysis were prepared from 0.04 mg of each standard in 1ml methanol (hplc grade). the operator data were obtained from the database of the instrument. the examine samples were subjected to ultrasonication (baransonsonifier, usa) at 60% duty cycle at 25˚c for 30 minutes. the resulted suspension was centrifuge (at 7500rpm) 20 min. the clear upper layer of sample solution was separated then evaporated under vacuum. the residue was dissolved in 1ml methanol using vortex mixer followed by filtration through disposable filter (2.5 μm). the clear solution temperature adjusted to 4˚c. the used volume for injection of the sample was 20 µl. results and discussion in spite of the importance of h. europaeum as a toxic plant, the phytochemical studies concentrated on the alkaloids secondary metabolites especially pyrrolizidine type. therefore, the chosen of phenolic compounds in this study is the main target because they have different pharmacological activities. in this study, hot extraction method was done by absolute methanol to extract the active constituent depend on the nature of these active constituents. the hydrolysis process was done to remove the glycoside moiety from the compounds by cleavage the ether linkage (glyosidic bond) producing the free a glycon. the screening of these sample revealed that they contain various phytochemical constituents by using p1 solvent system (chloroform: acetone formic acid) (75:16.5:8.5) which is best mixture of solvents used in the seperation of phenolic compound from fraction and 254 nm and 366 nm uv light. identification of phenolic compounds in heliotropium europaeum plant extract 1preliminary phytochemical investigation like chemical tests were carried on the h. europaeum aerial parts and showed the following results as shown in table 3 . table 3. qualitative analysis of phytochemical constituents in crude extract of heliotropium europaeum l. name of test naoh test ferric chloride test fraction ethyl acetate +ve +ve n-butanol +ve +ve 2preparative thin layer chromatography analysis for the ethyl acetate and nbutanol extract. iraqi j pharm sci, vol.30(2) 2021 heliotropium europaeum 161 preparative thin layer chromatography technique was used for separation of small amount constituent by using the same separation condition that was followed previously in ordinary tlc technique. the results of preparative tlc were more than one bands in each fraction which represent that the sample contain many of chemical constituents which can be separated easily. figure (2-3). figure 2. preparative thin layer chromatography on silica gel gf254 for fractions (1) fraction of ethyl acetate before hydrolysis (2) fraction of n-butanol before hydrolysis. using solvent system p1 (chcl3:ch3och3: hcooh) in a ratio (75:16.5:8.5). the detection under uv light 365 and 254 nm, p1 which is best mixture of solvents used in the separation of phenolic compound from fractions. figure 3. preparative thin layer chromatography on silica gel gf254 for fractions (1) fraction of ethyl acetate after hydrolysis (2) fraction of n-butanol after hydrolysis. using solvent system p1(chcl3:ch3och3: hcooh) in a ratio (75:16.5:8.5). the detection under uv light 365 and 254 nm. high-performance liquid chromatography (hplc) hplc technique can provide a lot of information about the content of the extract. it is also used for qualitative identification of extract constituents by making a comparison of their retention time and the shape of the uv spectrum of the detected compound by the detector of the instrument with that for authenticated reference standards at identical chromatographic conditions. iraqi j pharm sci, vol.30(2) 2021 heliotropium europaeum 162 in this study, the sample was analyzed by this technique, ethyl acetate fraction after hydrolysis (eta). the resulted chromatogram from fraction was compared with twenty-one standards at the same condition. figure (4) shows the hplc chromatogram of ethyl acetate fraction (eta) matched with six of the used standards. figure 4. hplc chromatogram for ethyl acetate fraction (eta) matched with six detected standards chromatograms. the matching between the spectra of the detected compounds in ethyl acetate fraction with corresponding standards spectra as shown in figures (5-10) respectively. these figures show an excellent fitness between the detected compounds and the six standards. these results concluded that heliotropium plant ethyl acetate might contains syringic acid, silybin, kaempferol, apigenin, caffiec acid and genistein. figure 5. uv spectrum of fraction of ethyl acetate matched with syringic acid standard uv spectrum. iraqi j pharm sci, vol.30(2) 2021 heliotropium europaeum 163 figure 6. uv spectrum of fraction of ethyl acetate matched with silybin standard uv spectrum. figure 7. uv spectrum of fraction of ethyl acetate matched with kaempferol standard uv spectrum. figure 8. uv spectrum of fraction of ethyl acetate matched with genistein standard uv spectrum. iraqi j pharm sci, vol.30(2) 2021 heliotropium europaeum 164 figure 9. uv spectrum of ethyl acetate fraction matched with caffiec acid standard uv spectrum. figure 10. uv spectrum of fraction of ethyl acetate matched with apigenin standard uv spectrum. it is important to refer that the ethyl acetate fraction chromatogram has at least three significant compounds did not match with the used standards. this reflects the need to extend study by using additional standard. because in hplc technique, the area under the peak of a certain compound is proportional to its concentration therefore calibration curves of the six matched standards were constructed for quantitive analysis and the peak areas of the detected compounds in fraction of ethyl acetate were used to determine the concentration of each one in the fraction, according to the first line equation y= ax +b, slop = y (area under the curve)/x (concentration mg/ml). the quantitative concentration of the six phenolic compounds in ethyl acetate fraction revealed that kaempferol has the highest concentration while genistein concentration was the least.as shown in table 4. iraqi j pharm sci, vol.30(2) 2021 heliotropium europaeum 165 table 4. the hplc quantitative analysis for ethyl acetate fraction. ethyl acetate fraction peak area (y) µg/ml (x) mg/ml kaempfero l 5327.83 5 111.39857 0.11039 9 silybin 3695.17 64.995163 0.06499 5 caffeic acid 1977.47 2 65.970708 9 0.06597 1 genistein 1209.13 1 6.2299365 2 0.00623 apigenin 446.711 19.853822 2 0.01985 4 syringic acid 7812.71 2 89.880837 1 0.08988 1 from the above finding, heliotropium europaeum is a promising plant as it contains different secondary metabolites especially phenolic compounds that was detected novelty in this study conclusion little attention was reported about the toxic h. europaeum plant and its secondary metabolites especially its phenolic compounds. ethyl acetate and nbutanol fractions of this plant were prepared and their phenolic compounds were studied. the technique used was high performance liquid chromatography equipped with uv-vis facility and group of standard phenolic compounds. six phenolic compounds were isolated and identified for the first time from ethyl acetate extract of this plant. the isolated compounds (syringic acid, silybin, kaempferol, apigenin, caffiec acid and genistein) were evaluated qualitatively and quantitatively. the study revealed that kaempferol has the highest concentration while genistein concentration was the least. the results obtained from h. europaeum study provide a good scientific base to examine the pharmacological effects of this plant in the future. references 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flavonoids and ascorbic acid in four different solvent extracts of two wild edible leaves, sonchus arvensis and oenanthe linearis of north-eastern region in india. journal of applied pharmaceutical science. 2016 ;6(2):157-66. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(suppl.) 2021 adherence &beliefs of breast cancer patients doi : https://doi.org/10.31351/vol30isssuppl.pp31-39 31 adherence and beliefs to adjuvant hormonal therapy in patients with breast cancer: a cross-sectional study (conference paper )# anwar h. saad*,1, ihsan s. rabeea**and haider n. salih*** # 9th scientific conference conference sponsored by college of pharmacy , university of baghdad 25-26 august 2021 *kufa technical institute, al-furat al-awsat technical university, najaf, iraq **faculty of pharmacy, university of kufa, najaf, iraq ***middle euphrates cancer center, najaf, iraq abstract breast cancer is the most common cancer among women over the world. to reduce reoccurrence and mortality rates, adjuvant hormonal therapy (aht) is used for a long period. the major barrier to the effectiveness of the treatment is adherence. adherence to medicines among patients is challenging. patient beliefs in medications can be positively or negatively correlated to adherence. the aims of the study were to investigate the extent of adherence and factors affecting adherence, as well as to investigate the association between beliefs and adherence in women with breast cancer taking aht. the method was a cross-sectional study included 124 iraqi women with breast cancer recruited from middle euphrates cancer center. morisky medication adherence scale (mmas) and beliefs about medication questionnaires (bmq) are used to assess adherence and beliefs respectively. as a result, 25% of women were fully adherent (mmas = 8). 83.06% of all women developed side effects from medications received. side effects and unemployment state were significantly associated with nonadherence. additionally, there is no significant association between beliefs in medications and adherence. the conclusion of the study was that high percent of poor adherence caused by side effects suggests the need for interventions by educating patients about the importance of their treatment and how to overcome side effects. keywords: adherence, beliefs, breast cancer, adjuvant hormonal therapy. مقطعية دراسة: الثدي سرطان مرضى لدى المساعد الهرموني بالعالج والمعتقدات االلتزام #) بحث مؤتمر ( *** صالح نعمان حيدر و ** صالح ربيع إحسان ، 1*، حبيب سعد أنوار 2021اب 26 – 25جامعة بغداد ، # المؤتمر العلمي التاسع لكلية الصيدلة .العراق ، النجف ، التقنية األوسط الفرات جامعة ، التقني الكوفة معهد* .العراق النجف، الكوفة، جامعة الصيدلة، كلية** .العراق ، النجف ، األوسط الفرات سرطان مركز *** الخالصة استخدام العالج سرطان الثدي هو أكثر أنواع السرطانات شيوعاً بين النساء حول العالم. لتقليل عودة السرطان ومعدل الوفيات، يتم كما يمكن أن الهرموني المساعد لفترة طويلة. العائق الرئيسي لفعالية العالج هو االلتزام بالعالج حيث يمثل االلتزام باألدوية بين المرضى تحديًا. م النساء باألدوية ومعرفة العوامل المؤثرة ترتبط معتقدات المريض باألدوية ارتباًطا ايجابياً أو سلبيًا بااللتزام. الهدف من الدراسة هو قياس مدى التزا هرموني على التزامهن، باإلضافة إلى قياس مدى االرتباط بين المعتقدات وااللتزام لدى النساء المصابات بسرطان الثدي الالتي يتلقين العالج ال ية مصابة بسرطان الثدي حيث استخدم مقياس امرأة عراق 124المساعد. أقيمت الدراسة في مركز الفرات األوسط لألورام السرطانية وشملت ( من %83,06٪ من النساء ملتزمات تماماً، ) 25لتقييم االلتزام والمعتقدات على التوالي. النتيجة: كان مورسكي ومقياس المعتقدات حول الدواء حالة انعدام العمل بشكل كبير مع عدم االلتزام باألدوية جميع النساء ظهرت عليهن آثار جانبية من األدوية التي تم تلقيها. ارتبطت اآلثار الجانبية و ر الجانبية إلى الحاجة بينما ال توجد عالقة ذات داللة إحصائية بين المعتقدات في األدوية وااللتزام. تشير النسبة العالية من قلة االلتزام الناجم عن اآلثا على اآلثار الجانبية. إلى دراسات تثقيفية للمرضى حول أهمية عالجهم وكيفية التغلب الكلمات المفتاحية: االلتزام، المعتقدات، سرطان الثدي، العالج الهرموني المساعد. introduction breast cancer is the most common cancer among women over the world in 2020 and accounting for 24.5% of all cancer cases worldwide while it is accounting 24% of overall malignancies in iraq (1). approximately two-thirds of all breast cancer cases have hormone receptor-positive breast cancer [estrogen receptors positive (er+) or estrogen receptors positive plus progesterone receptors positive (er+ plus pr+)]. after primary treatment (surgery, radiation, and chemotherapy), adjuvant hormonal therapy (aht) is a standard therapy prescribed for most hormone receptorpositive breast cancer. the most common two types of aht used are selective estrogen receptor modulators (serm) such as tamoxifen and aromatase inhibitors (ai) that inhibit estrogen production such as anastrozole (2). the use of any type depends on the menopause state of the woman. guidelines recommended the use of tamoxifen for 5 years in premenopausal women while ai is the recommended type in postmenopausal women as 1corresponding author e-mail: anwar.habeebsaad@gmail.com received: 24/8/2021 accepted:15 / 11/2021 published online first: 2022-1-12 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30isssuppl.pp31-39 iraqi j pharm sci, vol.30(suppl.) 2021 adherence &beliefs of breast cancer patients 32 primary or extended after using tamoxifen for 5 years and sometimes it is recommended to use ai as sequential (a combination with tamoxifen for 3 years followed by 2 years of ai) (3). long-term management by aht for five years or more is highly effective in reducing recurrence, mortality rates and improve overall survival (4,5). however, to obtain these benefits, women should take medication as prescribed. studies found that adherence to aht was suboptimal, only 50% of women with breast cancer were adherent to aht and about two-third discontinued aht before completing recommended period (6,7). a systematic review of psychosocial motivators and barriers of adherence to oral anticancer used in breast cancer revealed that the barriers of adherence are patients feeling discomfort toward taking aht, concerns about side effects of ai such as joint pain and gynecological symptoms while good beliefs about medication and good patient-physician relationship are positively correlated with adherence (8). the nonadherence is also associated with sociodemographic factors such as younger or older age (less than 40 and more than70), ethnicity, unmarried women, and those with lower income (9). many studies measured adherence for chronic disease in the arabic population but few of them focusing on the association between beliefs and adherence in breast cancer survival (10–12). therefore, this study aims to investigate the extent of adherence among the breast cancer population, predictors for non-adherence, as well as to investigate the association between beliefs and adherence in women with breast cancer. method study design and subjects a cross-sectional study was conducted among 124 women recruited from middle euphrates cancer center\najaf governorate\ iraq from the 15th november 2020 to the 30th april 2021. the study was approved by the scientific committee of researches of najaf health department (reference number 30599). women were eligible to participate if they were aged ≥ 18, diagnosed with breast cancer, taking tamoxifen or anastrozole for at least one month, and completed all primary treatment (surgery, radiation, and chemotherapy). we excluded women who have metastatic breast cancer and receiving chemotherapy, have a history of recurrence, and women with a psychological problem. tools sociodemographic and clinical variables were collected from patients including age, marital status, income categorized as following: good income >1,000,000 iqd; medium income 500,0001,000,000 iqd and; poor income <500,000 iqd (13), smoking, years since diagnosis, type of aht used, drug side effects, and others summarized in (table 1). medication adherence the assessment of adherence was done by using the morisky medication adherence scale (mmas) (14). it is a validated 8 items scale from previously published 4-items mmas-4 (15). mmas8 that used in this study was a copy of mmas-8 used in the previous study (16). this scale includes 8 items, where the first seven questions are answered by yes or no and the eighth one is a five likert scale answered either by never/rarely, once in a while, sometimes, usually, and all the time. the score of mmas ranged from 0 to 8 and in this study scores < 6 were considered to be poor adherence, scores ≥ 6 < 8 represent medium adherence, and scores = 8 represent high adherence. women with poor or medium adherence are considered non-adherent (17). medication beliefs women's beliefs about aht are measured by the beliefs about medication (bmq) scale which is widely used in chronic disease (18). bmq proven to be used in women taking aht (19). the validated arabic version with proven validity and reliability is used in a previous study (20). the scale consists of 18 items included in two sections which are either general or specific. specific-bmq (questions specific for aht medications) has two 5-item subscales, the specific-necessity subscale to assess the necessity of the prescribed aht medication and the specific-concern subscale to assess concern about the negative effect of prescribed aht medication. general-bmq (questions about medicines in general) has 8 items also subdivided into two 4-item subscales, general-harm that to assess the beliefs about the harmful effect of the any medication and general-overuse to assess beliefs of medication overuse by doctors. each item is a fivepoint likert scale (strongly disagree, disagree, uncertain, agree, and strongly agree to have a score of 1, 2, 3, 4, and 5 respectively). answers are recorded and a high score (agree and strongly agree answers) indicates stronger beliefs in the concepts of each subscale. procedure face-to-face interview was chosen because it is the most effective way of capturing a wide range of perspectives and allow interviewers to explore deeply for responses and clarify any ambiguity. furthermore, during the interview, patients' sensitive topics concerning their daily and social lives can be discussed, making participants feel more comfortable providing this information privately in a one-to-one setting and when compared to telephone interviews or self-reporting questionnaires, face-to-face interviews are known to have a greater response rate (21). women who visited middle euphrates cancer center to refill the prescription were asked to participate in the study. iraqi j pharm sci, vol.30(suppl.) 2021 adherence &beliefs of breast cancer patients 33 after informed consent was taken from women, face-to-face interview by the researcher with each woman for about 20 min. firstly, sociodemographic data and clinical information were taken from patients. accordingly, mmas and bmq questionnaires were filled. data analysis the results were analyzed by using a statistical package for social sciences (spss) version 23. descriptive analysis was expressed as mean ± standard deviation (sd) and median (lowerupper quartiles [q1-q3]). categorical variables were represented as numbers and percentages. correlation between adherence levels and demographic and clinical data was analyzed by chisquare test for categorical variables, while unpaired t-test for continuous normally distributed variables and mann whitney test for continuous non-normal distributed variables were used to compare between groups. mann-whitney test and wilcoxon z score were used for an association between bmq and adherence levels. logistic regression was performed to investigate predictors of non-adherence. the level of significance was set as a p-value < 0.05 using the adherent group as reference. results characteristic of study participants and adherence a total of 165 women interviewed to participate in the study, 124 women met the inclusion criteria and completed the questionnaires. the mean age was 50.00±9.383 years (range 30 78). most of them were married (83.06%), unemployed (79.84%), medium-income (48.39%), and (79.84%) women with primary school education. the median of aht duration was 18 months (6-36). 41.94% of women could not buy their medications from private sectors if they are unavailable in the oncology center, since they may stop their treatment until being available in the center. 54.84% of women had chronic diseases, where hypertension (41.94%) and diabetes mellitus (25.81%) were the most frequently reported among the study sample. there is a significant association between non-adherence and unemployed (p = 0.002), retired women (p = 0.002) and the presence of side effects (p = 0.04). sociodemographic and clinical characteristics are summarized in table 1. table 1. sociodemographic and clinical characteristics of participants variables overall number (n)=124 adherence level p value adherers (n =31 ) non-adherers (n =93 ) age (years), mean ± sd 50.00±9.383 52.74±11.04 49.09±8.638 0.06 residency, n (%) urban rural 100(80.65) 24(19.35) 25(80.65) 6(19.35) 75(80.65) 18(19.35) >0.9 employment status, n (%) employee unemployed retired 20(16.13) 99(79.84) 5(4.03) 5(16.13) 24(77.42) 2(6.45) 15(3.22) 75(80.65) 3(16.13) 0.02* 0.02* marital status, n (%) married single widow divorced 103(83.06) 6(4.84) 14(11.29) 1(0.85) 26(83.87) 1(3.23) 4(12.90) 0(0) 77(82.79) 5(5.37) 10(10.75) 1(1.07) 0.64 0.79 income, n (%) good >1,000,000 iqd medium 500,000-1,000,000 iqd poor <500,000 iqd 13(10.48) 60(48.39) 51(41.13) 1(3.23) 20(64.52) 10(32.26) 12(12.90) 40(43.01) 41(44.09) 0.06 0.31 smoking, n (%) no yes 121(97.58) 3(2.42) 30(96.77) 1(3.23) 91(97.85) 2(2.15) 0.66 educational level, n (%) <secondary school ≥secondary school 99(79.84) 25(20.16) 24(77.42) 7(22.58) 75(80.65) 18(19.35) 0.69 type of aht tamoxifen anastrozole 69 (55.65) 55 (44.35) 13 (41.94) 18 (58.06) 56 (60.22) 37 (39.78) iraqi j pharm sci, vol.30(suppl.) 2021 adherence &beliefs of breast cancer patients 34 continued table 1. duration of aht (month), median (q1‑q3) 18 (6 36) 24(10-38) 15(5.5 36) 0.2 years since diagnosis, mean ± sd 3.589±2.346 3.79±2.78 3.52±2.197 0.58 can buy medication yes no/by finance help 72(58.06) 52(41.94) 16(51.61) 15(48.39) 56(60.22) 37(39.78) 0.4 family remind you to take medication? n (%) yes no 27(21.78) 97(78.22) 4(12.90) 27(87.10) 23(24.73) 70(75.27) 0.17 do medication affect your daily activity? n (%) yes no 116(93.55) 8(6.45) 29(93.55) 2(6.45) 87(93.55) 6(6.45) >0.9 side effects, n (%) no yes 21(16.94) 103(83.06) 9(29.03) 22(70.97) 12(12.91) 81(87.09) 0.04* chronic disease, n (%) no yes 58(46.77) 66(53.23) 13(41.94) 18(58.06) 45(48.39) 48(51.61) 0.53 number of chronic illness, n (%) 1 ≥2 39(59.09) 27(40.91) 9(50) 9(50) 30(62.50) 18(37.50) 0.36 diabetes mellitus, n (%) no yes 92(74.19) 32(25.81) 22(70.97) 9(29.03) 70(75.26) 23(24.74) 0.64 hypertension, n (%) no yes 72(58.06) 52(41.94) 14(45.16) 17(54.84) 56(61.54) 35(38.46) 0.11 ischemic heart disease, n (%) no yes 120(96.77) 4(3.23) 30(96.77) 1(3.23) 90(96.77) 3(3.23) >0.9 number of other medication, n (%) no 1 ≥2 63 29 32 14 8 9 49 21 23 0.58 0.53 * =p< 0.05 indicate statistically significant differences. adherence of the study participants overall, mmas score in patients was 6.55 ± 1.35. only 31 (25%) study individuals were high adherent, whereas 93 (75%) women were nonadherent. 40.32% of women forgot to take their medication. 24.19% of them did not take their medication at least once in the last 2 weeks. 5.65% of women stopped taking their medications because they felt worse when taking them. 9.68% of women forgot to take their aht medication when they have a trip. most of them (93.55 %) answered that they take their medication yesterday. 0.81% (one individual) of women answered yes as she stopped taking her medication when became in a good health. 35.48% of women were hassled about the treatment plan. about half of women (46.77%) did not forget to take their medication (see table 2). iraqi j pharm sci, vol.30(suppl.) 2021 adherence &beliefs of breast cancer patients 35 table 2. self-reported medication adherence behavior of study participants as determined by the mmas8. mmas-8: morisky medication adherence scale -8 items. side effects of aht and the impact on adherence the total number of women who had side effects was 103 participated women (83.06%) with a significant proportion in non-adherent women (87.09%) when compared with adherent women (70.97%). the most frequent side effects reported were hot flush (79.61%), joint pain (76.70%), night sweating (39.81%), vaginal discharge (22.33%), fatigue (11.65%) and depression (10.69%) (see table 3). table 3. number (n) and percentage (%) reporting side effects, for total patients as well as for adheres and non-adheres patients. bmq and impact on adherence the majority of women supported the necessity of aht by answering agree or strongly agree that their medications have protected them from being worse and their state improved by their medications. the concerns about aht were also reported. many women answered agree or strongly agree that they were not familiar with their medications and they were worried from a long term effect of their medication. approximately, all women answered that the doctors are reliable on medicines and if doctors had more time with their patients they would prescribe fewer medicines. the item number and percentage (%) of female patients who answered yes do you sometimes forget to take your breast cancer oral medication? 50 (40.32) people sometimes miss taking their medications for reasons other than forgetting. thinking over the past two weeks, were there any days when you did not take your breast cancer oral medication? 30 (24.19) have you ever cut back or stopped taking your breast cancer oral medication without telling your doctor because you felt worse when you took it? 7 (5.65) when you travel or leave home, do you sometimes forget to bring along your breast cancer oral medication? 12 (9.68) did you take your breast cancer oral medication yesterday? 116 (93.55) when you feel like your breast cancer is under control, do you sometimes stop taking your medication? 1 (0.81) taking medication every day is a real inconvenience for some people. do you ever feel hassled about sticking to your breast cancer treatment plan? 44 (35.48) how often do you have difficulty remembering to take all your medications? never/rarely once in a while sometimes usually always 58 (46.77) 43 (34.68) 15 (12.09) 7 (5.65) 1 (0.81) side effect total, n (%) adherence level adheres, n (%) non-adheres, n (%) any 103/124(83.06) 22/103(21.36) 81/103(78.64) hot flush 82/103(79.61) 17/22(77.27) 65/81(80.25) night sweating 41/103(39.81) 8/22(36.36) 33/81(40.74) joint pain 79/103(76.70) 17/22(77.27) 62/81(76.54) fatigue 12/103(11.65) 1/22(4.55) 12/81(14.81) vaginal discharge\dryness 23/103(22.33) 7/22(31.82) 16/81(19.75) depression 11/103(10.69) 2/22(9.09) 9/81(11.11) iraqi j pharm sci, vol.30(suppl.) 2021 adherence &beliefs of breast cancer patients 36 mean rank of total belief scores of each section showed that adherent women had non-significant high specific‑necessity belief, low specific‑concern belief, high general‑harm belief and low general‑overuse belief. in terms of bmq items, there were no significant differences between adherent or non-adherent women. (table 4) table 4.response to bmq items and participants’ scores for each item by adherence level bmq: beliefs about medication scale. item total, agree/strong ly, agree answers n (%) adherence level wilcoxon z scores p-value adherers non-adherers mean rank specific‑necessity bmq necessity score, as a total 65.13 61.62 -0.476 0.634 1-my life would be impossible without medicine 76(61.29) 42.61 37.13 -0.974 0.330 2-without medicine i'll be very ill 81(65.32) 43.57 40.10 -0.602 0.547 3-my health, at present, depending on my medicine 87(70.16) 49.29 42.32 -1.131 0.258 4-my medicine protected me from becoming worse 89(71.77) 50.45 43.32 -1.133 0.257 5-my health in the future depends on my medicine 86(69.35) 50.97 41.52 -1.468 0.142 specific‑concern bmq concerns score, as a total 58.16 63.95 -0.779 0.436 6-i sometimes worry about the long term effect of my medicine 73(58.87) 43.47 35.18 -1.396 0.163 7-having to take medicine scares me 75(60.48) 43.44 36.53 -1.137 0.256 8-i sometimes worry about becoming too dependent on my medicine 43(34.67) 25.85 20.83 -1.123 0.262 9-my medicine disrupt my life 17(13.71) 8.25 9.41 -0.467 0.641 10-my medicines are mystery to me 79(63.70) 46.39 38.62 -1.156 0.248 general‑harm bmq harms score, as a total 62.38 61.88 -0.068 0.946 11-people who take medicines should stop their treatment for a while every now and again 7(5.65) 3.50 4.08 -0.255 0.799 12-most medicines are addictive 39(31.45) 19.40 20.21 -0.197 0.844 13-medicines do more harm than good 49(39.52) 23.91 25.32 -0.295 0.768 14-all medicines are poison 68(54.84) 32.26 35.25 -0.547 0.585 general‑overuse bmq overuse score, as a total 59.68 63.44 -0.512 0.609 15-doctors use too many medicines 15(12.09) 9.83 7.54 -0.808 0.419 16-doctors place too much trust on medicines 117(94.35) 60.04 58.67 -0.188 0.851 17-if doctors had more time with their patients they would prescribe fewer medicines 122(98.38) 58.42 62.55 -0.569 0.569 18-natural remedies are safer than medicines 32(25.80) 16.63 16.46 -0.044 0.965 iraqi j pharm sci, vol.30(suppl.) 2021 adherence &beliefs of breast cancer patients 37 multivariate analysis of factors predicting nonadherence the factors included in this test were age, education, side effects, and bmq sections (specific necessity, specific-concerns, general-harm, and general-overuse). no one of these variables was a significant predictor of non-adherence (table 5). table 5. factors predicting non-adherence or: odd ratio, ci: confidence interval (lower-upper), bmq: beliefs about medication scale. discussion: in the current study, the adherence was suboptimal and the majority of women (75%) were non-adherent to aht. the results of this study were in agreement with those of previous studies that examined adherence to aht (17,22,23). the level of adherence to aht is greatly below the level investigated in the study conducted by karbala/iraq who found that (62.38%) of postmenopausal women are highly adherent to ais (16). another study in sudan found revealed that high percent of patients (93%) were adherent to aht (24). this confliction may be attributed to the type of method used to measure adherence such as face to face interviews, self-reported questionnaires, pill count, electronic monitoring devices, and medical records. there is no standard method to measure medication adherence precisely (25). non-adherence is a multifactorial problem that affects therapy outcomes and lead to disease recurrence, re-hospitalization, and decrease survival rate (26). several studies found that socioeconomic status and clinical data including older age, single, low income and out-of-pocket cost of aht (26–31), comorbidity, treatment side effects, depression (32–34) are associated with non-adherence. in this study, non-adherence is significantly associated with side effects of aht and the employment state of women. this finding was supported by several studies investigated the factors associated with non-adherence indicating that side effects are a potential contributing factor (35). unemployed and retired women were significantly non-adherent. in contrast, a previous study in the uk found that in-paid employment was more likely to be non-adherent to aht (32). these differences imply that these results needed further investigation. patients' beliefs about their medications were observed to correlate with medication adherence. a positive association between high score specific-necessity and adherence while a high score of specific-concern showed significantly in patients with poor adherence. many studies of chronic diseases and cancer revealed these associations (10,12,17,36) but in our study, binary logistic regression test found no positive or negative significant association between each part of bmq and adherence. this is similar to previous studies that investigated no effects of beliefs on adherence (37–39). this may be due to our small sample size and may have insufficient strength to find a significant effect. the limitations of this study, are limited to one center and does not represent all iraq population, many variables are related to the adherence were not included in this study such as self-efficacy, perceived social support, patient-physician communication, and severity of symptoms, the data presented here reflects only the aht adherence using a questionnaire through face to face interview. in our opinion, this method is more accurate than obtaining data from self-reported questionnaires, prescription and pharmacy records and medical claims methods. still, all those observational methods remain limited (40). a confirmation method that measures the medications or related markers in blood is more accurate (41) and recommended in future studies. conclusion this study provides important information about the adherence extent of iraqi women with breast cancer to their aht and factors leading to poor adherence. a majority of women were nonadherent to their treatment. side effects and unemployment women were the only factors investigated to be associated with non-adherence. there was no positive or negative association between beliefs and adherence. variable or (95% ci) p value age 1.037 (0.989-1.088) 0.135 education 1.523 (0.381-6.084) 0.551 employment 1.011 (0.2094.895) 0.989 side effect 0.423 (0.143-1.252) 0.120 bmq-specific-necessity 1.009 (0.926-1.101) 0.833 bmq-specific-concerns 0.935 (0.853-1.023) 0.144 bmq general-harm 1.051 (0.904-1.221) 0.518 bmq-generaloveruse 0.930 (0.738-1.172) 0.541 iraqi j pharm sci, vol.30(suppl.) 2021 adherence &beliefs of breast cancer patients 38 competing interests the authors confirm that there is no conflict of interest. funding the authors did not receive any external funding for this work. availability of data and materials: the databases used and/or analyzed for the present study are accessible from the corresponding author on reasonable request. references 1. cancer today. international agency for research on cancer. 2021. 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treatment factors on adherence to adjuvant endocrine therapy in breast cancer. oncol nurs forum. 2014;41(3):274–285. 39. kimmick g, edmond sn, bosworth hb, peppercorn j, marcom pk, blackwell k, et al. medication taking behaviors among breast cancer patients on adjuvant endocrine therapy. the breast. 2015;24(5):630–636. 40. lam wy, fresco p. medication adherence measures: an overview. vol. 2015, biomed research international. hindawi publishing corporation; 2015. 41. hansen la. impact of nonadherence to cancer therapy [internet]. 2021. available from: https://jhoponline.com/ton-online-first/3639ton-3639 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases doi: https://doi.org/10.31351/vol31iss1pp109-118 109 using phone calls to promote community pharmacist counselling during covid-19 pandemic in baghdad, iraq susan tawfiq hameed*,1 and zinah mudhafar anwer* *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract drug consultation is an important part of pharmaceutical care. a mobile phone call or text message can serve as an easy, effective, and implementable alternative to improving medication adherence and clinical outcomes by providing the information needed significantly for people with chronic illnesses like diabetes and hypertension particularly during pandemics like the covid-19 pandemic. this study aimed to estimate the use of phone calls to promote pharmaceutical counseling and explore the commonest question asked by patients and do the socio-demographic or disease characteristics play any role regarding such questions. a prospective, interventional, clinical study was conducted during the period (from 5th of november 2020 to 21st of february 2021). a total of 246 patients were enrolled in the study. the mean age was 40.93 years (±15.84). the majority were female (62.6%) and age group (35-54) years (44.3%). a total of 507 questions were asked by patients, the researcher provided pharmaceutical consultations in response to 47 % of the question. there were significant associations between socio-demographic characteristics and some of the domains. in conclusion, sociodemographic characteristics influence the type of question asked by patients. most of the patients got educational advice and some of them were referred to physicians. key words: phone counselling, community pharmacist. في المجتمع دالنيةإلرشاد الصيا إليصالستخدام المكالمات الهاتفية ا العراق ، في بغداد 19خالل جائحة كوفيد *مظفر أنور و زينة 1*، توفيق حميدسوزان العراق بغداد، بغداد، جامعة الصيدلة، كلية السريرية، *فرع الصيدلة الخالصة هي جزء مهم من الرعاية الصيدالنية. يمكن أن تكون المكالمة الهاتفية أو الرسائل النصية بمثابة بديل سهل بخصوص الدواءاالستشارة من أمراض وفعال وقابل للتنفيذ لتحسين االلتزام باألدوية والنتائج السريرية من خالل توفير المعلومات الالزمة بشكل كبير لألشخاص الذين يعانون استخدام المكالمات الهاتفية ييمهدفت هذه الدراسة إلى تق .19-كوفيد وارتفاع ضغط الدم خاصة أثناء األوبئة مثل وباءمزمنة مثل مرض السكري مرضية لتعزيز االستشارة الصيدالنية واستكشاف األسئلة األكثر شيوًعا التي يطرحها المرضى وهل تلعب الخصائص االجتماعية والديموغرافية أو ال (. تم تسجيل 2021 شباط 21إلى 2020 تشرين الثاني 5خلية أجريت خالل الفترة )من اق بهذه األسئلة. دراسة سريرية مستقبلية تدأي دور فيما يتعل 35٪( والفئة العمرية )62.6سنة(. وكانت الغالبية من اإلناث ) 15.84سنة )± 40.93العمر مريضا في الدراسة. كان متوسط 246ما مجموعه . 1وقدمت الباحثة استشارات دوائية المرضى، أسئلة من قبل 507٪(. تم طرح ما مجموعه 44.3( سنة )54 االستشارة الهاتفية ،صيدلي المجتمع الكلمات المفتاحية : introduction hypertension (ht) can be defined as a condition in which blood pressure (bp) is elevated to a level likely to lead to adverse consequences. there is no clear-cut blood pressure threshold separating normal blood pressure from high blood pressure(1). diabetes mellitus (dm) is a group of metabolic disorders characterized by hyperglycemia and abnormalities in carbohydrate, fat, and protein metabolism(2). drug consultation is an important part of pharmaceutical care offered by the pharmacist. this service not only promotes optimal medication use, which helps enhance health outcomes, but it also exemplifies an opportunity for pharmacists to hone their professional skills. it is particularly convenient for patients and pharmacists to provide these consultation services via telephone(3). today, with the technological advances of the past ten years and the broadening of pharmacy services to include direct patient care, mobile phone usage has drastically increased, irrespective of region or country, urban area or rural area, and literacy or illiteracy, evidence suggests that a mobile phone call or text message can serve as an easy, effective, and implementable alternative to improving medication adherence and clinical outcomes by providing the information needed. counseling by telephone might improve patient counseling(4). 1corresponding author e-mail: susantofiqhameed@gmail.com received: 21/6/2021 accepted: 22/8 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp109-118 iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases 110 the advantages of using phone calls in pharmaceutical services include enhancement of the clinical role for pharmacists significantly for people with chronic illnesses particularly during pandemics (like the covid-19 pandemic), where many people have various medicines-related concerns(5). secondly, it increases patients’ awareness about their conditions(6), improved patient adherence(7), supporting rational medicines use(8), enhance access to healthcare services in remote and rural communities(9), and lastly, the counseling via telephone ensures greater satisfaction of patients with regard to the pharmacist counseling and time required to obtain medication(10). the main role of community pharmacists during covid-19 include supporting rational use of medicine, promotion medication adherence, medication review and follow-up, information and communication about covid-19, and triaging at the community level for suspected covid-19 cases(11). at the health system level, the implementation of telepharmacy services in hospitals and primary health care centes expanded hours of service, improved the speed of processing of physician medication orders, increased clinical pharmacy services, and cost avoidance(12). aim of the study 1. to evaluate the use of phone calls to promote pharmaceutical counseling 2. to explore the frequently asked questions by patients about their medications and their relation to their sociodemographic or disease status. subjects and method study type and design this is a prospective, interventional, clinical study, which includes interviewing and enrolling the subjects during the predetermined period of four months (5th of november 2020 to 21st of february 2021). inclusion criteria patients with any age who were diagnosed as hypertensive and/or diabetic by their physicians and continue to refill their medications. exclusion criteria 1. abnormal cognitive behaviors or unable to understand the questions. 2. patients asking questions not related to ht or dm. sampling all patients who received the identification cards through randomly selected sixty pharmacies, social media, or by snowball sampling and met the inclusion criteria were enrolled in the study. data collection to gather the information from the participants, the researcher used one thousand cards distributed by sixty community pharmacies in baghdad and through social media groups on (instagram, facebook, whatsup, telegram, and viber) with the assistant of the admins of these groups to enroll a larger number of participants. the cards included the phone number of the researcher and types of counseling like laboratory investigation follow-up, treatment follow-up, side effects. a special sheet was prepared in advance to be used to gather the information from the patients via phone call. its purpose was to acquire detailed information, including socio-demographic data, contact information, disease history, disease characteristics, co-morbidities, treatments being received for the ht, dm, or any other medications which are being taken concurrently. during the phone calls, the researcher introduced herself, took verbal consent from patients, and asked a number of questions regarding the patient's disease and management and answered the questions of the patients regarding the disease or previously prescribed treatments. the patient's questions were distributed into twelve domains that were prepared by the researchers and accepted by the scientific committee of the clinical pharmacy department. domain 1. related to side effects domain 2. related to disease domain 3. related to complications of the disease. domain 4. related to drug-drug interaction. domain 5. taking a drug in relation to food. domain 6. drug-food interaction. domain 7. how to decrease a side effect. domain 8. how to increase the drug’s action. domain 9. warning about dangerous actions while using the drug (smoking, drinking alcohol, driving a car) domain 10. storage of drugs (ex. insulin) domain 11. use of a substance (herbs, supplements... etc.) domain 12. give other consultations like which test is more accurate, home or lab test for dm, which lab data should be done continuously for diabetic patients, mechanism of drug action. the researcher answered the patient’s questions depending on trusted references (clinical pharmacy and therapeutics, 6th edition and pharmacotherapy principles and practice, 4th edition). if the question was related to pharmaceutical practice, the researcher gave a decision. still, if the question was related to the physician, the researcher will give advice (suggested recommendations) to the treating physician. the physician's response was studied if it is accepted and adopted, accepted without adapted, rejected. iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases 111 ethical approval the study has been proposed and subsequently approved by the scientific committee of the college of pharmacy/ baghdad university. fully informed consent was obtained from the patients verbally after explaining the aim of the study thoroughly and clearly. all the information and questions were communicated to the patients with honesty and transparency in an objective manner to avoid bias as much as possible. statistical analysis the collected data were analyzed using microsoft excel software, version 2016, and statistical package for the social sciences (spss), version 22. the descriptive analysis focused on frequencies and percentages, while the chi-square test, fisher's exact test were utilized to determine the mean differences between groups. a p-value of less than 0.05 was considered statistically significant. results a total of 246 patients were enrolled in the study. the mean age was 40.93 years (±15.84). the majority were female (62.6%) and age group (3554) years (44.3%). more than half (65.5%) had either an intermediate or secondary school degree, as shown in table 1. all participants had chronic disease (s), diabetes (75.2%), hypertension (9.8%), or both (15%), as shown in table 2. table 1. sociodemographic characteristics of participants characteristics n % gender female 154 62.6 male 92 37.4 age group <15* 13 5.3 15*-24 29 11.8 25-34 38 15.4 35-44 59 24.0 45-54 50 20.3 55-64 45 18.3 ≥65 12 4.9 education primary school or less 43 17.4 intermediate school 70 28.4 secondary school 68 27.6 college or higher 65 26.4 occupation employed (government employee, self-employee) 100 40.7 unemployed (housewife, out of a job, retired) 146 59.3 resident urban 193 78.5 rural 53 21.5 *the interviewees were conducted with a child parent or an adult guardian/relative. total n=246. table 2.medical characteristics of the patients regarding to the 507 questions asked by the participants, 92 (37.3%) were related to the diseases, 72 (29.3%) were related to complications, and 113 (45.9%) were related to the need for other consultations as shown in table 3. characteristics n % number of medications <4 194 78.9 ≥4 52 21.1 chronic disease diabetes 185 75.2 hypertension 24 9.8 diabetes and hypertension 37 15.0 duration of disease <one year 38 15.4 one-five years 124 50.4 >five years 84 34.1 iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases 112 table 3.the domains of the participant questions questions n % 1. related to drug side effects 40 16.3 2. related to diseases 92 37.4 3. related to complications of the diseases 72 29.3 4. related to drug-drug interaction 6 2.4 5. taking a drug in relation to food 28 11.4 6. drug-food interactions 9 3.7 7. how to decrease drug side effects 41 16.7 8. how to increase the drug’s action 20 8.1 9. warning about dangerous actions while using the drugs (smoking, drinking alcohol, driving a car) 6 2.4 10. storage of drugs (ex. insulin) 50 20.3 11. use of substances (herbs, supplements... etc.) 30 12.2 12. give other consultations 113 45.9 total 507 100.0 the pharmacist (researcher) received 507 questions from 246 patients. she provided pharmaceutical consultations in response to 47 % of the questions, consultations and refer in response to 44 % of the questions, while referred patients to physicians in response to 9 % of them as shown in figure 1. figure 1. the pharmacist response to patient questions a total of 271 questions were referred to physicians, of those, 92 (34%) accepted the pharmacist recommendations and consulted their physicians as shown in figure 2. figure 2. physician access to pharmacist referral recommendations the physicians accepted and adopted 43.5% of the pharmacist referral recommendations as shown in figure 3. figure 3. physician responses regarding pharmacistreferral recommendations there were no significant associations between the gender and the type of asked questions to the pharmacist (table 4). iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases 113 table 4.the relationships between the patient gender and the study domains domains gender p-value male female domain 1 no 79 (38.3%) 127 (61.7%) 0.484 yes 13 (32.5%) 27 (67.5%) domain 2 no 58 (37.7%) 96 (62.3%) 0.912 yes 34 (37.0%) 58 (63.0%) domain 3 no 67 (38.5%) 107 (61.5%) 0.577 yes 25 (34.7%) 47 (65.3%) domain 4§ no 92(38.3%) 148 (61.7%) 0.060 yes 0 (0.0%) 6 (100.0%) domain 5 no 84 (38.5%) 134 (61.5%) 0.305 yes 8 (28.6%) 20 (71.4%) domain 6§ no 90 (38.0%) 147 (62.0%) 0.338 yes 2 (22.2%) 7 (77.8%) domain 7 no 82 (40.0%) 123 (60.0%) 0.059 yes 10 (24.4%) 31 (75.6%) domain 8 no 87 (38.5%) 139 (61.5%) 0.232 yes 5 (25.0%) 15 (75.0%) domain 9§ no 89 (37.1%) 151 (62.9%) 0.674 yes 3 (50.0%) 3 (50.0%) domain 10 no 69 (35.2%) 127 (64.8%) 0.159 yes 23 (46.0%) 27 (54.0%) domain 11 no 77 (35.6%) 139 (64.4%) 0.128 yes 15 (50.0%) 15 (50.0%) domain 12 no 53 (39.8%) 80 (60.2%) 0.389 yes 39 (34.5%) 74 (65.5%) *significant (p-value <0.05) according to pearson chi-square. § fisher's exact test. yes ( the patient’s questions fell within this domain). no (the patient’s questions did not fall within this domain). the level of patient education significantly influenced the patients asking of two domains of the questions (domains 1 and 12) (table 5). in other words, people with high education (high school/college degree or higher) asked significantly more questions related to domain 1 (about drug side effects) than patients with lower education (table 5).the employment of the patients had no significant influence on the type of asked questions except in domain 12 (table 6). table 5.the relationships between the patient education level and the study domains domains education level pvalue college or higher high school middle school primary or less domain 1 no 49 ) 23.8% ( 54 )26.2% ( 64 )31.1% ( 39 )8.9% ( 0.032 * yes 16 )40.0% ( 14 ) 35.0% ( 6)15.0% ( 4 )10.0% ( domain 2 no 40 (26.0%) 44 (28.6%) 43 (27.9%) 27 (17.5%) 0.977 yes 25 (27.2%) 24 (26.1%) 27 (29.3%) 16 (17.4%) domain 3 no 43 (24.7%) 34 (19.5%) 45 (25.9%) 34 (19.5%) 0.205 yes 22 (30.6%) 9 (12.5%) 25 (34.7%) 9 (12.5%) domain 4§ no 64 (26.7%) 67 (27.9%) 69 (28.8%) 40 (16.7%) 0.211 yes 1 (16.7) 1 (16.7%) 1 (16.7%) 3 (50.0%) domain 5§ no 52 (23.9) 64 (29.4%) 63 (28.9%) 39 (17.9%) 0.085 yes 13 (46.4%) 4 (14.3%) 7 (25.0%) 4 (14.3%) domain 6§ no 60 (25.3%) 67 (28.3%) 69 (29.1%) 41 (17.3%) 0.169 yes 5 (55.6%) 1 (11.1%) 1 (11.1%) 2 (22.2%) domain 7 no 49 (23.9%) 58 (28.3%) 62 (30.2%) 36 (17.6%) 0.209 yes 16 (39.0%) 10 (24.4%) 8 (19.5%) 7 (17.1) iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases 114 continued table 5. *significant p-value <0.05 according to pearson chi-square. § fisher's exact test. yes ( the patient’s questions fell within this domain). no (the patient’s questions did not fall within this domain) table 6. the relationships between the patient employment status and the study domains domains employed p-value yes count (%) no count (%) domain 1 no 80 (38.8%) 126 (61.2%) 0.188 yes 20 (50.0%) 20 (50.0%) domain 2 no 66 (42.9%) 88 (57.1%) 0.362 yes 34 (37.0%) 58 (63.0%) domain 3 no 74 (42.5%) 100 (57.5%) 0.351 yes 26 (36.1%) 46 (63.9%) domain 4§ no 98 (40.8%) 142 (59.2%) 0.712 yes 2 (33.3%) 4 (66.7%) domain 5 no 88 (40.4%) 130 (59.6%) 0.801 yes 12 (42.9%) 16 (57.1%) domain 6§ no 94 (39.7%) 143 (60.3%) 0.105 yes 6 (66.7%) 3 (33.3%) domain 7 no 84 (41.0%) 121 (59.0%) 0.816 yes 16 (39.0%) 25 (61.0%) domain 8 no 93 (41.2%) 133 (58.8%) 0.591 yes 7 (35.0%) 13 (65.0%) domain 9§ no 99 (41.3%) 141 (58.8%) 0.226 yes 1 (16.7%) 5 (83.3%) domain 10 no 84 (42.9%) 112 (57.1%) 0.163 yes 16 (32.0%) 34 (68.0%) domain 11 no 85 (39.4%) 131 (60.6%) 0.266 yes 15 (50.0%) 15 (50.0%) domain 12 no 67 (50.4%) 66 (49.6%) 0.001* yes 33 (29.2%) 80 (70.8%) *significant (p-value <0.05) according to pearson chi-square. § fisher's exact test. yes ( the patient’s questions fell within this domain). no (the patient’s questions did not fall within this domain). domains education level p-value college or higher high school middle school primary or less 65 (28.8%) 39 (17.3%) 0.954 yes 6 (30%) 5 (25%) 5 (25%) 4 (20%) domain 9§ no 65 27.1% 66 27.5% 66 (27.5%) 43 (17.9%) 0.134 yes 0 (0.0%) 2 (33.3%) 4 (66.7%) 0 (0.0%) domain 10 no 54 (27.6%) 56 (28.6%) 55 (28.1%) 31 (15.8%) 0.504 yes 11 (22.0%) 12 (24.0%) 15 (30.0%) 12 (24.0%) domain 11 no 55 (25.5%) 62 (28.7%) 60 (27.8%) 39 (18.1%) 0.581 yes 10 (33.3%) 6 (20.0%) 10 (33.3%) 4 (13.3%) domain 12 no 47 (35.3%) 31 (23.3%) 30 (22.6%) 25 (18.8%) .002* yes 18 (15.9%) 37 (32.7%) 40 (35.4%) 18 (15.9%) iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases 115 the number of scheduled medications significantly influences two types of question domains (no. 3 & 9). patients with polypharmacy (≥ 4 medications) had a significantly higher number of questions related to “the warning about dangerous actions while using the drugs” compared to patients taking a smaller number of medications (< 4) (table 7). table 7. the relationships between the number of scheduled medications and the study domains domains number of medications p-value < 4 count (%) ≥ 4 count (%) domain 1 no 164 (79.6%) 42 (20.4%) 0.513 yes 30 (75.0%) 10 (25.0%) domain 2 no 120 (77.9%) 34 (22.1%) 0.640 yes 74 (80.4%) 18 (19.6%) domain 3 no 144 (82.8%) 30 (17.2%) 0.020* yes 50 (69.4%) 22 (30.6%) domain 4§ no 191 (79.6%) 49 (20.4%) 0.080 yes 3 (50.0%) 3 (50.0%) domain 5 no 175 (80.3%) 43 (19.7%) 0.130 yes 19 (67.9%) 9 (32.1%) domain 6§ no 187 (78.9%) 50 (21.1%) 0.935 yes 7 (77.8%) 2 (22.2%) domain 7 no 165 (80.5%) 40 (19.5%) 0.162 yes 29 (70.7%) 12 (29.3%) domain 8§ no 180 (79.6%) 46 (20.4%) 0.389 yes 14 (70.0%) 6 (30.0%) domain 9§ no 193 (80.4%) 47 (19.6%) 0.002* yes 1 (16.7%) 5 (83.3%) domain 10 no 153 (78.1%) 43 (21.9%) 0.543 yes 41 (82.0%) 9 (18.0%) domain 11 no 170 (78.7%) 46 (21.3%) 0.871 yes 24 (80.0%) 76 (20.0%) domain 12 no 109 (82.0%) 24 (18.0%) 0.197 yes 85 (75.2%) 28 (24.8%) § fisher's exact test. yes ( the patient’s questions fell within this domain). no (the patient’s questions did not fall within this domain). patients with dm had a significantly higher number of questions of six domains (3,4,5,6, 10 & 12) compared to patients with hypertension or hypertension and dm. these six domains included questions related to complications of the diseases, drug-drug interaction, taking a drug in relation to food, drug-food interactions, drug storage, and other consultations (table 8). table 8. the relationships between the patient chronic disease type and the study domains domains chronic disease type p-value dm count (%) ht count (%) dm & ht count (%) domain 1 no 160 (77.7%) 18 (8.7%) 28 (13.6%) 0.126 yes 25 (62.5%) 6 (15.0%) 9 (22.5%) domain 2 no 115 (74.7%) 17 (11.0%) 22 (14.3%) 0.649 yes 70 (76.1%) 7 (7.6%) 15 (16.3%) domain 3§ no 130 (74.7%) 22 (12.6%) 22 (12.6%) 0.025* yes 55 (76.4%) 2 (2.8%) 15 (20.8%) domain 4§ no 185 (77.1%) 21 (8.8%) 34 (14.2%) 0.000* yes 0 (0.0%) 3 (50.0%) 3 (50.0%) domain 5§ no 168 (77.1%) 22 (10.1%) 28 (12.8%) 0.041* yes 17 (60.7%) 2 (7.1%) 9 (32.1%0 *significant (p-value <0.05) according to pearson chi-square. § fisher's exact test. dm=d=diabetes mellitus; ht=hypertension. yes ( the patient’s questions fell within this domain). no (the patient’s questions did not fall within this domain). iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases 116 continued table 8. *significant (p-value <0.05) according to pearson chi-square. § fisher's exact test. dm=d=diabetes mellitus; ht=hypertension. yes ( the patient’s questions fell within this domain). no (the patient’s questions did not fall within this domain). discussion the use of information and telecommunication technologies has expanded rapidly, which strongly influences healthcare delivery in many countries(13). to our best knowledge, this is the first study to assess the use of phone calls in pharmaceutical counseling during covid-19 in iraq. most of the participants were female, identical results were obtained in other studies in the united states 2018(14) and iran 2012(15). in contrast, males were predominant in al-blooshi et al. study in the united arab emirate(uae) 2020(16). many explanations related to these results as females are more worry about their diseases and complications, mark et al found that diabetic women were substantially more concerned about weight increase, which was linked to the majority of the negative outcomes including lower self-rated health, poorer psychological well-being, poorer reported regimen adherence, and more diabetes-related distress(17) another study postulated that men appeared to underestimate problems related to diabetes more than women. they were less concerned about long-term consequences and hypoglycemia (18), in agreement with this postulation, elke found that 24% of the participant men and 23% of the participant females reported non-adherence to anti-diabetic medication(19). this might explain the predominant female participation in this study and most other similar studies as women have a higher desire to know more about their disease, treatment, and complications. the largest age group was (35-44), in comparing to another study, (15-25) years were the largest age group in a similar study in the kingdom of saudi arabia 2015(20). unemployed, those with intermediate school graduation and patients with monthly income less than one million iqd constituted the largest part of the sample. in contrast, most participants in another study in uae 2020 were employed with college graduates (16). in terms of the estimated family monthly income, over twofifths of the sample (41.7%) reported the lowest monthly income, of which less than 5000 saudi riyal (1,333 us$) was observed in another study in the kingdom of saudi arabia, 2015(20), this agreement with the current study results. the low cost of phone counseling and its availability at home may be the reason behind these results. about one-fifth of the participants lived in the urban areas, this is related to the area where the study was done, and the urban people are more aware about the use of phone calls in medical and specifically pharmaceutical consultations. the most frequently asked questions were related to domain12 (related to other consultations like which test is more accurate, home or lab test for dm), which lab data should be done continuously for diabetic patients, mechanism of drug action). as postulated by other studies, most iraqi diabetic and hypertensive patients have good knowledge about their conditions, that the major parts of the current study participant asked about more advanced issues of their disease. esraa et al revealed that the majority of the patients had strong beliefs in the necessity of anti-diabetic treatment for controlling their illness(21), in erbil, a study was done there revealed most diabetic participants had acceptable to good knowledge(22). another study was done in baghdad revealed that hypertensive patients in our domains chronic disease type p-value dm count (%) ht count (%) dm & ht count (%) dm count (%) domain 6§ no 185 (78.1%) 20 (8.4%) 32 (13.5%) 0.000* yes 0 (0.0%) 4 (44.4%) 5 (55.6%) domain 7§ no 156 (76.1%) 18 (8.8%) 31 (15.1%) 0.513 yes 29 (70.7%) 6 (14.6%) 6 (14.6%) domain 8§ no 171 (75.7%) 22 (9.7%) 33 (14.6%) 0.804 yes 14 (70.0%) 2 (10.0%) 4 (20.0%) domain 9§ no 182 (75.8%) 24 (10.0%) 34 (14.2%) 0.070 yes 3 (50.0%) 0 (0.0%) 3 (50.0%0 domain 10§ no 142 (72.4%) 24 (12.2%) 30 (15.3%) 0.028* yes 43 (86.0%) 0 (0.0%) 7 (14.0%) domain 11§ no 162 (75.0%) 21 (9.7%) 33 (15.3%) 0.962 yes 23 (76.7%) 3 (10.0%) 4 (13.3%) domain 12§ no 100 (75.2%) 19 (14.3%) 14 (10.5%) 0.007* yes 85 (75.2%) 5 (4.4%) 23 (20.4%) iraqi j pharm sci, vol.31(1) 2022 using phone calls for pharmaceutical counseling for chronic diseases 117 community have relatively good knowledge and attitude(23). conclusion in conclusion, socio-demographic characteristics influence the type of question asked by patients, most questions related to consultation regarding the mechanism of drug action or laboratory diagnosis and questions about the disease. most patients got educational advice and some of them were referred to physicians. most patients got education and advice regarding their questions to resolve their conditions, increase the awareness of the patients about their disease, and adhere 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http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract doi: https://doi.org/10.31351/vol29iss1pp94-114 94 uplc-esi-ms/ms and various chromatographic technique for identification of phytochemicals in populus euphratica oliv. leaves extract mohammed a. ezghayer*,1 and enas j. kadhim** *department of pharmacognosy and medicinal plants, college of pharmacy, university of tikrit, tikrit, iraq. *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq abstract the aim of this study is to screen the phytochemicals found in populus euphratica leaves since this type of trees are used traditionally by many villagers as treatment for eczema and other skin disease and also this plant is poorly investigated for their phytochemicals especially in iraq. phytochemical screening of the extracts obtained from the n-hexane and chloroform fraction of leaves of populus euphratica was done by thinlayer chromatography and various spraying reagents to test if alkaloids, sterols and other compounds are present. uplc-electrospray ionization –tandem mass spectroscopy along with gc-ms and hptlc are used to identify the phytochemicals present in the plant leaves.uplc-esi-ms/ms method 20 compounds have been identified in various fractions among which are protopine alkaloids. salicin, salicortin, tremulacin. gc-ms showed that the observed data obtained are matched with that in nist library and confirmed the presence of hexadecanoic acid trimethylsilyl ester in 43.80% beta-sitosterol in 37.14% and diisooctyl phthalate 11.46%.uplc-esi-ms/ms is a powerful method for the identification of compounds in mixture based on comparison of their molecular, weight retention time and ms/ms fragmentation. protopine alkaloid is identified for the first time in populus euphratica and genus populus. gc-ms is a valuable method for both qualification and quantification of various phytochemicals that are volatile in nature keywords: populus euphratica, gc-ms, phytochemical, uplc-esi-ms/ms تقنيات كروماتوغرافيا كشف المواد الكيميائية النباتية في مستخلص اوراق نبات الغرب باستخدام يف الكتلة المتتالي والتأين برذاذ الكهرباء كرموماتوغرافيا السائل فائقة األداء بوجود ط متعددة و **ايناس جواد كاظمو 1*،محمد علي ازغير العراق. تكريت، تكريت،جامعة الصيدلة،كلية الطبية،قسم العقاقير والنباتات * بغداد ، العراق. بغداد،جامعة الصيدلة،كلية الطبية،والنباتات لعقاقيرارع ق** الخالصة ا تهدف هذه الدراسة إلى اكتشاف المواد الكيميائية النباتية الموجودة في أوراق نبات الغرب، ألن هذه األنواع من األشجار تستخدم تقليدي دراسات قليلة حول المركبات الكيميائية الموجودة في هذ النبات خاصة من قبل العديد من القرويين كعالج لألكزيما وأمراض الجلد األخرى وهناك في العراق. للتأكدالغرب وذلك ألوراقالفحص الكيميائي النباتي للمستخلصات التي تم الحصول عليها من اجزاء الهكسان والكلوروفورم تم اجراء كروماتوغرافيا السائل فائقة االداءطبقة الرقيقة وكواشف الرش المختلفة. من وجود قلويدات ، ستيرول ومركبات أخرى بواسطة كروماتوغرافيا ال وكروماتوغرافيا مطياف الكتلة -كروماتوغرافيا الغاز إلى جنب مع الشامل جنباستخدام التحليل الطيفي وا (uplc-esi)مع التأين برذاذ الكهرباء مركب ا 20تم تشخيص ،uplc-esi-ms / msباستخدام طريقة .لنباتية الموجودة في النباتالطبقة الرقيقة عالية االداء لتحديد المواد الكيميائية ا أن البيانات المرصودة التي تم الحصول gc-msبينها قلويد البروتوبين ، الساليسين ، الساليكورتين ، التريميوالسين . أظهرت المستخلص منمن وبيتا سيتوستيرول في ٪، 43.80ميثيل سيليل أستر حامض الستاديك في وجود ثالثيدت وأك nistعليها تتزامن مع تلك الموجودة في مكتبة هو وسيلة قوية لتحديد المركبات الموجودة في خليط معين بواسطة المقارنة uplc-esi-ms / ms٪. 11.46٪ و ديسوكتيل فثاليت 37.14 فيات بين وزنها الجزيئي ووقت االحتفاظ وتفتيت طيفها الكتلي المتتالي، تم تحديد قلويد البروتوبين ألول مرة في نبات الغرب وفي جنس الصفصا gc-ms الخاصية الطيارةمن المواد الكيميائية النباتية ذات هي طريقة قيمة لمعرفة الماهية الكمية والنوعية للعديد. مع التحليل الطيفي ءالكهربامع التأين برذاذ كروماتوغرافيا السائل فائقة االداء ،نبات الغرب ،مطياف الكتلة -كروماتوغرافيا الغاز الكلمات المفتاحية: .المواد الكيميائية النباتية ، (uplc-esims/ms)الشامل 1corresponding author e-mail: ph.techno.lno@gmail.com received: 2/ 7/ 2019 accepted: 20/ 10/2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp94-114 mailto:ph.techno.lno@gmail.com iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 95 introduction populus euphratica tree is a member of the salicaceae family, which abundant of phenols and its glycosides such as salicin and populin(1). previous studies on populus euphratica have reported that some phenolic compounds (2) have been identified and volatile oils (3) have been detected in this plant. w. wei et al. (2015) undertook a detailed chemical investigation of the leaves of populus euphratica that afforded 13 compounds, among which 6-o-ciscinnamoylsalicin and 6-obenzoylsalicortinol were new compounds. the spectral data of 6-o-trans-cinnamoylsalicin and salicortinol were reported for the first time. meanwhile, nine known compounds were characterized as follows: salicin (4) benzyl-o-b-dglucopyranoside tremulacin(5), salireposide (6) cinnamrutinose a (7), and saligenin (8). rahimi,et-al (2008) have concluded that the smoke of burnt leaves of the populus euphratica tree was curative in about 66% of patients with warts and there was a low recurrence rate of about 4%. (9) . populus euphratica extract has been used traditionally by some villagers in some areas of iraq in the treatment of eczema and various skin condition. so in this study we will investigate the phytochemicals found in the leaves of this naturally abundant tree found in every city in iraq and trying to resonate this traditional use. the goal of this study is investigating and screening the phytochemicals and their proportions in hexane fraction of leaves of populus euphratica tree naturally grown in iraq since there is no phytochemical study had been done previously in iraq and trying to resonate its traditional use in the treatment of eczema. material and methods plant material collection leaves of populus euphratica oliv. was collected from the bank of the tigris river in the periphery of tikrit city in april 2018, authenticated in iraq natural history research center and museum by dr. khansaa rasheed. leaves were cleaned dried in shade at room temperature and then pulverized and stored for further use. method of work 100gm of air-dried powder of the leaves is weighted and defatted with petroleum ether overnight to get rid of chlorophyll and waxy material then extracted in soxhelt with 90% methanol for 9hr, the extract is combined and dried by rotary evaporator the dry extract is weighted and the yield of extraction is calculated to be 37gm. the dry extract is dissolved in 100ml of distilled water. then partitioned with 100ml of nhexane using a separatory funnel for three times all the upper layers are combined together and dried using rotary evaporators, the dried hexane fraction was weighted to be 7.5 gm. the aqueous part is partitioned with chloroform 100ml 3 times to get 17.2gm of chloroform extract. again the aqueous part is partitioned with butanol to get butanol fraction (12g) symbolized b.b fraction. thin-layer chromatography and the spraying with liebermannburchard reagent were used to identify the hexane extract containing phytosterols. general identification test for alkaloid has been done with dragendorff's reagent and mayer's reagent both gave positive results for alkaloid (11) . b.b fraction is hydrolyzed by reflex for 6 hrs. using 5% hcl to get butanol after hydrolysis fraction symbolized b.a. readymade tlc pre-coated plate of 1mm gf254 for isolation and 0.25 mm gf254 was used for purification of isolated compounds. acid-base extraction of alkaloids from the crude extract part of the crude extract is suspended in nhexane and partitioned with water to remove pigment and other non-polar compounds then the aqueous part is treated with nh4oh to ph10 to liberate free alkaloid then equal volume of chloroform is added to separatory funnel partitioned and the lower organic layer was acidified with 5% h2so4 to ph2 , and again partitioned with equal volume of water , the aqueous layer now contain all alkaloid as salt, to this layer add nh4oh to ph10 and partitioned with chloroform the chloroform layer now contain free tertiary alkaloids, this fraction is symbolized as cha fraction (12). high-performance thin-layer chromatography (hptlc) examination of cha b.b fraction the presence of flavonoids and phenolic glycoside and alkaloids in the analyzed fractions were confirmed by using the modern technique of hptlc using eike-reich/camag-laborator/ switzerland, by comparing retention factor of analyzed sample with that of standards. method for uplc-esi-ms/ms esi-ms positive and negative ion acquisition mode was carried out on a xevo tqd triple quadruple instrument waters corporation, milford, ma01757 u.s.a, mass spectrometer column: acquity uplc beh c18 1.7 µm 2.1 × 50 mm column flow rate: 0.2 ml\min solvent system: consisted of (a) water containing 0.1 % formic (b) methanol containing 0.1 % formic acid the sample (100 μg/ml) solution was prepared using high performance liquid chromatography (hplc) analytical grade solvent of/meoh, filtered using a membrane disc filter (0.2 μm) then subjected to lc-esi-ms analysis. samples injection volumes (10 μl) were injected into the uplc instrument, sample mobile phase was prepared by filtering using 0.2 μm filter membrane disc and degassed by sonication before injection. the parameters for iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 96 analysis were carried out using negative ion mode as follows: source temperature 150 °c, cone voltage 30 ev, capillary voltage 3 kv, desolvation temperature 440 °c, cone gas flow 50 l/h, and desolvation gas flow 900 l/h. mass spectra were detected in the esi between m/z 100–1000. the peaks and spectra were processed using the maslynx 4.1 software and tentatively identified by comparing its retention time (rt) and mass spectrum with reported data. results and discussion analysis of fractions by high-performance thinlayer chromatography (hptlc) hptlc is one of the most advanced forms of tlc, efficient for qualitative and quantitative analysis. table 3-1 show hptlc results of standard flavonoids, alkaloids, phenolic glycoside, and the analyzed fractions. table 1. phenolic glycoside, flavonoids and alkaloid content of different extract fraction detected by hptlc and their max rf value. fractions mobile phase isolated compounds reference standard rf of compound rf in fraction cha m5 c1 protopine 0.73 0.73 b.b m4 s1 salicin 0.29 0.28 m4 neohesperidinn1 0.17 0.17 m5 s1 salicin 0.56 0.56 m5 neohesperidin. n1 0.60 0.60 hptlc of b.b fraction figure1. hptlc plate analyzed fraction with reference standard detection under uv light at 256nm (b.b=butanol before hydrolysis fraction, sst=salicin standard ,n1= neohesperidin standard , s1 = isolated compound from b.b fraction) developed in [chloroform : methanol : acetic acid( 70:20:10)] solvent system. symbolized m4. hptlc of cha and b.b fractions figure 2 .hptlc of analyzed fraction and reference standard at 256nm (pst=protopine standard sst=salicin standard, nst=neohesperidin standard, p1=c1=isolated compound from cha fraction, aq.f=aqueous fraction after butanol hydrolysis, s1=isolated compound from b.b, cha=acid-base purification of chloroform fraction. developed in [ethyl acetate: acetic acid: formic acid: water (70:10:10:10)] solvent system symbolized m5. iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 97 uplc-esi-ms/ms for tentative identification of compounds in fractions twenty phytochemicals have been putatively characterized by uplc-esi-ms/ms in populus euphratica leaves based on comparison of mass fragmentation pattern, lc retention time and molecular weight with previous study and the literature in phytochemicals mass library such as respect (http:// spectra.psc .riken. jp/ menta .cgi/ respect/search/keyword?page=1), mona (http: //mona .fiehnlab .ucdavis .edu / ) , gnps (https://gnps.ucsd.edu/proteosafe /gnpslibrary.jsp?library=gnps-nist14matches), meltin (https://metlin.scripps.edu/ landing _page .php?pgcontent=batch_search). comparing the results of mas fragmentation with the previous study also helpful in confirming the result. sometime, the precursor ion is not as the molecular weight this is due to adduct formation which is common in esi – mode especially with phenolic glucoside (11). all the analysis have done, using xevo tqd triple quadruple instrument (milford, ma01757 usa, mass spectrometer) figure3. uplc chromatogram of isolated c1 figure 4 . uplc chromatogram of isolated s1. http://mona.fiehnlab.ucdavis.edu/ https://gnps.ucsd.edu/proteosafe%20/gnpslibrary.jsp?library=gnps-nist14-matches https://gnps.ucsd.edu/proteosafe%20/gnpslibrary.jsp?library=gnps-nist14-matches https://gnps.ucsd.edu/proteosafe%20/gnpslibrary.jsp?library=gnps-nist14-matches https://metlin.scripps.edu/%20landing%20_page%20.php?pgcontent=batch_search https://metlin.scripps.edu/%20landing%20_page%20.php?pgcontent=batch_search iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 98 figure 5. uplc chromatogram of isolated aq1 compound. mass 1 of isolated compounds. figure 6. mass 1 of isolated c1 compound in positive ion mode showing m and m+h iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 99 figure7. mass 1 of isolated s1 compound in negative ion mode showing m and m-h. figure 8. mass 1 of predicted s1 in b.b fraction in negative ion mode showing m and m-h without adduct formation. iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 100 figure 9. mass 1 of isolated aq1 compound in negative ion mode showing m and m-h with adduct formation. table 3 .uplc-esi-ms/ms of the isolated compounds. a, b these precursor ions are due to adduct formation presumably arising from combination with the formic acid present in the lc mobile phase which is common. in negative esi mode especially with phenolic glycoside, the unmodified molecular ion is also present but with small intensity(11). figure10. uplc chromatogram of chloroform fraction peak no. rt compound sample m.w m+h, [m+fa h], m-h ms/ms fragments ref. 2 23.34 c1 353.1 354 148,163,159,188,190, 275,247,354 (12)(13) 2 2.33 s1 286 331a 121,123, 124,207,93,144 (14) (11) 6 7.59 aq1 424 469b 423,317,316,299,285, 155,123,111,121 (11)(16) 2 8.72 n1 610 609 301,300,609,489,283,34 3,257, (17)(18) iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 101 table 4. identification result by uplc-esi-ms/ms fragmentation of chloroform fraction (number in bold line represent base peak) r e fe r e n c e m s /m s fr a g m e n ts m + h m .w r t c o m p o u n d c la sse s c o m p o u n d n a m e p e a k n o (1 2 )(1 3 )(2 0 ) 1 4 8 ,1 6 3 ,1 5 9 ,1 8 8 ,1 9 0 , 2 7 5 ,2 4 7 ,3 3 6 ,3 5 4 3 5 4 3 5 3 .1 2 3 .6 7 b e n z y liso q u in o lin e a lk a lo id p ro to p in e 2 0 (1 3 )(2 1 ) 1 4 8 , 3 2 4 , 2 3 3 ,1 1 2 , 1 7 8 , 9 1 , 8 9 , 1 6 5 , 3 1 0 3 2 6 3 2 5 8 .8 4 b e n z y liso q u in o lin e a lk a lo id c h e ila n th ifo lin e 3 (2 2 )(2 3 ) 4 1 4 ,3 9 6 ,2 7 1 ,2 5 2 ,1 0 4 ,1 5 7 4 1 4 4 1 3 1 0 .7 8 g ly c o a lk a lo id so la so d in e 4 (2 4 )(2 5 ) 1 1 9 ,2 9 5 ,1 0 7 ,1 0 9 ,5 7 ,2 1 9 ,3 4 4 ,9 1 ,8 1 4 1 5 4 1 4 8 .0 5 s te ro id s b -sito ste ro l 2 iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 102 (2 6 )(2 7 ) 6 1 6 ,4 5 4 ,4 7 0 ,3 0 8 ,1 6 3 ,2 9 2 ,1 4 7 ,2 2 0 ,2 2 1 ,2 0 4 6 1 6 6 1 5 1 1 .7 6 p o ly a m in e a lk a lo id s d i-c a ffe o y l-c o u m a ro y l sp e rm id in e 6 (2 8 )(2 9 ) 2 2 0 ,2 1 9 ,1 3 6 ,1 4 8 , 2 0 2 ,2 0 5 ,1 3 5 , 3 8 2 3 8 1 2 5 .1 0 a m in o p u rin e z e a tin -9 -g lu c o sid e 2 4 (2 6 )(2 7 ) 4 8 3 ,4 5 4 ,2 9 2 ,1 4 7 ,1 6 3 , 2 0 4 ,3 1 8 ,3 1 9 ,4 3 8 6 0 0 5 9 9 1 1 .8 2 p o ly a m in e a lk a lo id s c a ffe o y l d i_ c o u m a ro y l sp e rm id in e 7 figure 11. uplc chromatogram of b.b fraction iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 103 continue to figure11. uplc chromatogram of b.b fraction . table 5 .uplc-esi-ms/ms of b.b fraction. table 5 .uplc-esi-ms/ms of b.b fraction. r e fe r e n c e s tr u c tu r e m s /m s fr a g m e n ts m -h & [m + f a -h ] m .w c o m p o u n d c la ss c o m p o u n d n a m e r t p e a k n o . (3 0 ) 2 8 4 .9 ,2 4 0 .9 , 2 6 6 .9 , 2 5 6 .9 , 2 4 2 .9 , 2 1 6 .9 , 1 9 8 .9 , 1 7 4 .8 , 1 5 0 .8 , 1 3 2 .9 4 4 7 4 4 8 .3 8 f la v o n o id g ly c o sid e s l u te o lin 7 -o -b e ta -d -g lu c o sid e 8 .4 2 1 2 (1 5 ) (1 1 ) 4 2 3 ,3 1 7 ,3 1 6 ,2 9 9 ,2 8 5 ,1 5 5 ,1 2 3 ,1 1 1 , 1 2 1 4 6 9 a 4 7 0 [m + f a h ] 4 2 4 m p h e n o lic g ly c o sid e s s a lic o rtin 7 .7 5 1 1 iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 104 (1 8 ) (1 7 ) 3 0 1 ,3 0 0 ,6 0 9 ,4 8 9 ,2 8 3 ,3 4 3 , 2 5 7 6 0 9 6 1 0 f la v o n o id g ly c o sid e s n e o h e sp e rid in 8 .7 2 1 3 (1 4 ) (1 1 ) 2 6 7 ,1 2 1 ,1 2 3 , 1 2 4 ,2 0 7 ,9 3 , 1 4 4 3 3 1 b 2 8 6 p h e n o lic g ly c o sid e s s a lic in 2 .6 6 2 (3 1 )(3 2 ) 4 7 7 ,3 1 5 ,2 7 1 ,2 9 9 ,3 5 7 ,2 8 5 , 2 4 3 4 7 7 4 7 8 f la v o n o id g ly c o sid e iso rh a m n e tin -3 -g lu c o sid e 9 .7 1 1 8 (3 4 ) (3 5 ) 2 7 3 ,2 7 4 ,2 2 9 ,1 6 7 ,4 3 5 ,1 2 3 ,1 2 5 4 3 5 4 3 6 f la v o n o id o -g ly c o sid e s p h lo re tin -2 '-o -g lu c o sid e 1 0 .4 9 2 0 (3 6 )(3 7 ) 1 9 1 ,1 9 1 ,1 7 6 ,1 7 9 ,1 3 3 ,3 3 7 ,3 2 5 ,1 4 8 ,2 5 5 3 5 3 3 5 4 c o u m a rin g ly c o sid e s sc o p o lin 5 .4 4 5 iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 105 a,b these precursor ions are due to adduct formation presumably arising from combination with the formic acid present in the lc mobile phase which is common in negative esi mode especially with phenolic glycoside, the unmodified molecular ion is also present but with small intensity (11). figure12. uplc chromatogram of b.a fraction iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 106 table 6. uplc-esi-ms/ms of b.a fraction. r e fe r e n c e s tr u c tu r e m s /m s fr a g m e n ts m -h m .w c o m p o u n d c la ss c o m p o u n d n a m e r t p e a k n o . (3 0 )(3 8 ) 1 9 0 .9 ,1 7 8 .8 ,1 2 6 .8 ,8 4 .9 ,1 7 2 .9 ,1 1 0 .8 ,9 2 .9 , 1 0 8 .8 3 5 3 3 5 4 p h e n o lic a c id c h lo ro g e n ic a c id 2 .2 3 3 (3 9 )(1 1 ) 1 3 7 ,2 9 9 ,2 4 3 ,1 3 8 ,1 2 3 ,9 3 3 7 3 ,8 5 4 0 5 4 0 6 p h e n o lic g ly c o sid e s a lic y lo y lsa lic in 1 3 .0 7 3 3 (3 0 ) (4 0 ) 1 9 1 ,1 6 2 .9 ,1 7 3 ,1 2 7 ,1 7 1 ,8 5 3 3 7 3 3 8 p h e n o lic a c id (5 -p -c o u m a ro y lq u in ic a c id ) 6 .5 8 1 0 (4 1 )(3 9 ) (1 1 ) 4 0 5 ,1 5 5 ,5 2 7 ,1 3 7 ,1 2 1 5 7 3 c 5 2 8 p h e n o lic g ly c o sid e s tre m u la c in 1 2 .5 0 3 0 iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 107 (4 2 )(4 3 ) 3 1 1 ,4 6 1 ,2 0 5 ,1 8 9 ,1 0 9 ,4 4 3 ,1 6 3 5 0 8 d 4 6 2 p h e n y lp ro p a n o id s a n d p o ly k e tid e s n c g c 0 0 3 4 7 6 7 8 -0 2 !7 -(3 ,4 -d ih y d ro x y p h e n y l)-1 -(4 h y d ro x y p h e n y l)-5 -[(2 s ,3 r ,4 s ,5 r )-3 ,4 ,5 -trih y d ro x y o x a n 2 -y l]o x y h e p ta n -3 -o n e 1 5 .5 3 6 (4 4 )(4 5 ) 1 3 3 ,1 2 2 ,1 3 7 , 1 7 6 ,1 4 8 1 9 1 1 9 2 s c o p o le tin c o u m a rin s 0 .9 0 1 identification of isolated compounds compound c1: is isolated from fraction cha and give positive results with alkaloidal identification results such as dragendorff´s, mayer´s, and hager´s. so it is alkaloid. the uplcesi-ms/ms method is powerful method for identification of phytochemicals it revealed that the molecular weight of this compound is 354 da and fragmentation pattern as in figure 13 during search in various mass libraries such as respect, mona, gnps. the only alkaloid that matches both the molecular weight and fragmentation pattern is protopine alkaloid and to get 100% identification conformity protopine standard is compared with the isolated compound in hptlc so both gave the same rf 0.75 value as in figure 2(12,13). figure13. mass 2 fragmentation of protopine . iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 108 the protopine alkaloids can fragment by the rda reaction, and, it also can undergo another characteristic fragmentation pathway. selecting protopine as an example, fragment ion at m/z 206.0823 and 149.0603 in the ms/ms spectrum is generated by rda c ring-opening (figure 14 ) but given the presence of hydroxyl groups, the product ion at m/z 336.1209, m/z 188.0721 are probably formed by loss of h2o from the molecular ion and from the m/z 206.0823 ion (27). figure 14 . ms/ms fragmentation pathway of protopine. (27) . compound s1: is isolated from butanol before hydrolysis fraction this compound gave positive results with koh so it is glycoside and also gave gray violet color with vanillin –glacial acetic acid reagent (vga) this reagent gave gray violet color with salicin and its derivatives (46) the isolated s1 gave the same rf value 0.56 with standard in hptlc analysis as in figure 2, in uplc-esims/ms it gave molecular ion 331 instead of 285 of salicin, this precursor ion (331) is due to adduct formation presumably arising from combination with the formic acid present in the lc mobile phase which is common in negative esi mode especially with phenolic glycoside [ m+fa-h] (11). compound s1 has displayed the fragment ions at m/z 267, 123 (base peak), 121 and 93 in accordance with the loss of [m–h-18]−, [m–h-162]−, and [m–h-164]−, [m– h-192]−. a comparison of these ms/ms ions with the literature, moreover, this compound has previously been reported in this plant, according to these data s1 is salicin (14) (11) . as in fragmentation pattern (figures 15 and 16). figure 15 .mass fragmentation of salicin iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 109 figure 16. salicin fragmentation pathway. compound aq1 : it is isolated from aqueous fraction (aqf) it gives positive result with koh, it is also gave positive result to vga reagent which confirm that it is from salicin derivatives, hplcesi-ms/ms gave precursor ion 469 and mass fragmentation as that of salicortin and again due to adduct formation with formic acid salicortin (m.w 424 ) had result this molecular ion [m+fa-h] m/z 469 and mass fragment as in figure17(11,15) . figure17 .mass fragmentation pattern of salicortin. compound aq1 has displayed fragmentation ion m/z 423 in the mass1 as in figure9 due to loss of format adduct [m-h-hcooh] [46946=m/z 423] the fragment ions at m/z 285,155,137,123 and 120 in accord with the loss of [m–h(m/z423) -138]−, [m–h-268]−, and [m–h286]−, [m–h-300]−, [m–h-303]−, a comparison of these ms/ms ions with the literature, moreover, this compound has previously been reported in this plant, according to these data compound s2 has been identified as salicortin (47) . figure 18. fragmentation pathway of salicortin iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 110 phytochemicals identified in hexane fraction of populus euphratica leaves. the n-hexane fraction was analyzed using gc-mas spectroscopy and revealed the following compound as shown below. figure 19.gc-ms of n-hexane fraction of populus euphratica leaves figure20. ( a1) mass spec. of peak 1 figure 20. (b1) mass spec. of heptadecanoic acid trimethylsilyl ester (palmitic acid trimethylsilyl ester) from nist library as shown in the figure 20complete match of mass spec. between a1 and b1 it indicates the peak 1 is palmitic acid trimethylsilyl ester. iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 111 figure 21 . (a2) mass spec. of peak 3 figure21 . (b2) mass spec. of diisooctyl phthalate from nist library from mass spec. of the figure 21 complete match of mass data between a2 and b2 indicate that peak 3 is diisooctyl phthalate. figure 22. (a3) mass spec. of peak 4 figure 22. (b3) mass spec. of beta-sitosterol peak 5 is beta-sitosterol due to the exact mass pattern between a3 and b3mass charts. iraqi j pharm sci, vol.29(1) 2020 phytochemicals in populus euphratica oliv. leaves extract 112 conclusion based on the result the following point may be concluded 1. uplc-esi-ms/ms is a powerful method for identification of compounds in mixture base on their molecular weight, retention time and ms/ms fragmentation 2. twenty compounds have been tentatively identified with this method as in tables 3.8,3.9 and 3.10 3. protopine alkaloid is identified for the first time in populus euphratica and genus populs. 4. salicinoids are major compounds in populus species 4 of them have been identified, salicin, salicortin, salisaoylsalicin, and trmulacin. 5. salicin, neohesperidin, protopine, and salicortin have been isolated from different fractions and identified by various methods. 6. hexane fraction of leaves of populus euphratica is rich in beta-sitosterol with 37.14% of total hexane fraction, this may be the cause that extracts of this plant is used traditionally by farmers for various skin conditions like dermatitis and eczema, so the leaves of this tree may be exploited as a rich source for beta-sitosterol 7. spermidine derivatives, zeatin-9-glucoside, and solasodine have been identified for the first time in this plant. references 1. dembitsky vm. astonishing diversity of natural surfactants: 5. biologically active glycosides of aromatic metabolites. lipids. 2005;40(9):869–900. 2. luo j, jiang h, zhao y, zhou j, qian j. components of the heartwood of populus euphratica from an ancient tomb. 2008;44(1):7–9. 3. wei w, rena k, yang xw. new salicin derivatives from the leaves of populus euphratica. j asian nat prod res. 2015;17(5):491–6. 4. itoh a, tanahashi t, ikejima s, inoue m, nagakura n, inoue k, et al. five phenolic glycosides from alangium c hinense. j nat prod. 2000;63(1):95–8. 5. ishikawa t, nishigaya k, takami k, uchikoshi h, chen i-s, tsai i-l. isolation of salicin derivatives from homalium c ochinchinensis and their antiviral activities. j nat prod. 2004;67(4):659–63. 6. belyanin ml, stepanova e v, ogorodnikov vd. first total chemical synthesis of natural acyl derivatives of some phenolglycosides of the family salicaceae. carbohydr res. 2012;363:66–72. 7. li x-d, kang s-t, li g-y, li x, wang j-h. synthesis of some phenylpropanoid glycosides (ppgs) and their acetylcholinesterase/xanthine oxidase inhibitory activities. molecules. 2011;16(5):3580–96. 8. fisher th, chao p, upton cg, day aj. one‐ and two‐dimensional nmr study of resol phenol—formaldehyde prepolymer resins. magn reson chem. 1995;33(9):717–23. 9. rahimi ar, emad m, rezaian gr. clinical trial smoke from leaves of populus euphratica olivier vs . conventional cryotherapy for the treatment of cutaneous warts : a pilot , randomized , single-blind , prospective study. 2008;393–7. 10. ultra performance liquid chromatography tandem mass spectrometer (uplc-ms/ms) [internet]. wageningen. 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9th scientific conference conference sponsored by college of pharmacy , university of baghdad 25-26 august 2021 *department of pharmaceutics, college of pharmacy, uniersity of kerbala, kerbala iraq ** department of pharmaceutics, college of pharmacy, university of alqadisiyah, iraq ***department of pharmaceutics, college of pharmacy, university of baghdad abstract the idea of this study is to improve transdermal permeability of methotrexate using eucalyptus oil, olive oil and peppermint oil as enhancers. eucalyptus oil (2% and 4%), peppermint oil (2% and 4%) and olive oil (2% and 4%) v/v all used as natural enhancers to improve transdermal permeability of methotrexate via gel formulation. the gel was subjected to many physiochemical properties tests. in-vitro release and permeability studies for the drug were done by franz cell diffusion across synthetic membrane, kinetic model was studied via korsmeyer-peppas equation. the results demonstrate that safe, nonirritant or cause necrosis to rats' skin and stable till 60 days gel was successfully formulated. methotrexate permeability without enhancer was only about 20%, while with enhancers, it reached to 85%, 99% and 90% with eucalyptus oil 4(v/v %), peppermint oil 4(v/v %) and olive oil 4(v/v %) respectively after 24 hours. methotrexate transdermal gel was prepared and evaluated fruitfully in-vitro with a good permeation across semipermeable membrane. the results indicated that using of peppermint oil as enhancer has superiority to enhance the transdermal permeation of the methotrexate. keywords: permeability, methotrexate gel, eucalyptus oil, peppermint oil and olive oil. تعزيز نفاذية جل الميثوتريكسات عبر الجلد باستخدام زيت الكافور وزيت النعناع وزيت الزيتون #) بحث مؤتمر ( ***و موفق محمد غريب *، حسين محمد محسن **، بسام وفاء مهدي * ، جنان محمد1*،جمال علي عاشور 2021اب 26 – 25جامعة بغداد ، # المؤتمر العلمي التاسع لكلية الصيدلة فرع الصيدالنيات ، كلبة الصيدلة ، جامعة كربالء ، كربالء ، العراق * ، العراق القادسية ، القادسية فرع الصيدالنيات ، كلبة الصيدلة ، جامعة ** ، العراق بغداد، بغدادفرع الصيدالنيات ، كلبة الصيدلة ، جامعة *** الخالصة فكرة هذه الدراسة لتحسين نفاذية دواء الميثوتريكسات جل عبر الجلد باستخدام زيت األوكالبتوس وزيت الزيتون وزيت النعناع كمعززات .للنفاذية ٪( كسوائل مذيبة في سوائل )ح/ح ( جميعها طبيعية تستخدم ٤٪ و ٢٪( وزيت الزيتون )٤٪ و ٢، زيت النعناع )٪( ٤٪ و ٢زيت األوكالبتوس ) تم إجراء تجارب خضع الجل للعديد من اختبارات الخصائص الفيزيائية والكيميائية. كمعززات لتحسين نفاذية دواء الميثوتريكسات عبر الجلد. .بيبباس-لدواء عن طريق خلية فرانز عبر الغشاء االصطناعي ، ودُرس النموذج الحركي عبر معادلة كورسميرالحركية والنفاذية في المختبر ل كانت نفاذية دواء الميثوتريكسات بدون يوًما. ٦٠أظهرت النتائج أن المادة الجيالتينية آمنة وغير مهيجة أو تسبب نخًرا لجلد الفئران ومستقرة حتى ِّن للنفاذية حوالي نات إلى ٢٠ ُمحس )ح/ح ٪( ٤وزيت النعناع ( ٪ )ح/ح ٤٪ بزيت األوكالبتوس ٩٠٪ و ٩٩٪ و ٨٥٪ فقط ، بينما وصلت مع الُمحس .ساعة ٢٤ح/ح ٪( على التوالي بعد ) ٤وزيت الزيتون ت النتائج إلى أن استخدام أشار تم وضع جل الميثوتريكسات عبر الجلد وتقييمه في المختبر مع نفاذ جيد عبر الغشاء شبه القابل للنفاذ. .زيت النعناع كمحسن له تفوق في تعزيز نفاذ دواء الميثوتريكسات عبر الجلد .زيت النعناع وزيت الزيتون ، زيت األوكالبتوس، الميثوتريكساتالكلمات المفتاحية : نفاذية ، جل introduction methotrexate (mtx) is an anti-metabolite that is a synthetic chemical analogue of folic acid and is commonly prescribed to treat psoriasis. although it is authorized for the treatment of mild / moderate psoriasis with oral and parenteral dosing, long-term usage causes diarrhea, mucosal ulcers, stomatitis, bone marrow suppression, hepatic fibrosis, and cirrhosis. as a result, patients might benefit from topical mtx treatment to prevent its serious side effects. unfortunately , current commercial topical mtx formulations have limited penetration into the stratum corneum, which has been linked to its hydrophilicity and significant dissociation at physiological ph. (1, 2). 1corresponding author e-mail: jamal.ali@uokerbala.edu.iq received: 30/8/2021 accepted: 12/10 /2021 published online first: 2022-1-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30isssuppl.pp16-21 iraqi j pharm sci, vol.30(suppl.) 2021 methotrexate transdermal gel 17 transdermal medication delivery is a method of delivering a drug formula via the skin for systemic absorption. it is completely painless technique that begins with drug penetration from the surface of the skin (stratum coronium) to the deeper layers: epidermis and dermis, to be available for systemic absorption. (3) transdermal drug delivery system offers several advantages include self-administered and consistent, sustained release of drug, and with steady plasma levels throughout the entire time of administration, hepatic first pass effect and enzymatic degradation of gastrointestinal tract (git) is avoided without git side effects, and it can be used as an alternate route for patients who are unable to take their pills orally, furthermore, the medication administration terminates with the formula being removed from the surface of skin. (4, 5) gel is a semi-rigid substance made up of at least two components: a base, which is a smoothly flexible liquid in which the dispersion medium (water) is entirely absorbed. the combination of these two components is what gives the gel its unique properties. (6) penetration enhancers can improve the drug's penetration through the skin. permeation enhancers come in a variety of forms, the majority of which are surfactants that can harm cells. natural enhancers, such as oils and fatty acids, are favored since they are not dangerous to cells like surface active agents and may not interfere with calibration techniques.(7) several physical and chemical techniques have been employed to improve mtx skin permeability, like iontophoresis, liposomes, chemical enhancers as well as microneedles. (8) these methods had limited success and some imperfections remain. in this work, mtx transdermal permeability was enhanced by adding and increasing the content of eucalyptus, peppermint and olive oils by forming the product as a gel using (carbopol 940) as gelling agent. materials and methods materials mtx was bought from yibai biotechnology (china), carbopol 940 was purchased from sigma ch. (usa), eucalyptus, peppermint and olive oils were provided from bar-sur-loup (france), triethanolamine (tea) was bought from merck (germany). franz cell was purchased from ses gmbh (germany). methods preparation of methotrexate gel the gel was prepared by dissolving the gelling agent 2 (w/v) % of carbopol 940 in deionized water by stirring until a homogeneous mixture was obtained. then, the mtx was dissolved in mixture of (1 ml dimethyl sulfoxide and 4ml acetone) and this mixture was left for about 10 min to ensure that completely dissolved in the solution. the mtx solution was added step by step to the homogenous mixture of carbopol 940 (9) . an enhancer (eucalyptus, peppermint and olive oils) with different concentration was added to six different formulas, for ph adjustment (10) a solution of triethanolamine (tea) was added in drops and mixed separately with each formula. eventually, the final volume was made up to 25 ml by adding the sufficient quantity of deionized water, with stirring until a homogeneous gel was made. different concentration of each component explained in table 1. table 1. formulation of mtx 0.1% (w/v) transdermal gel. formulas mtx (w/v) % carbopol 940 (w/v) % tea (v/v) % enhancer deionized water eucalyptus oil(v/v) % peppermint oil(v/v) % olive oil(v/v) % f1 0.1 2 1 0 0 0 up to 25ml f2 0.1 2 1 2 0 0 up to 25ml f3 0.1 2 1 4 0 0 up to 25ml f4 0.1 2 1 0 2 0 up to 25ml f5 0.1 2 1 0 4 0 up to 25ml f6 0.1 2 1 0 0 2 up to 25ml f7 0.1 2 1 0 0 4 up to 25ml evaluations of methotrexate gel organoleptic characteristics the organoleptic properties of the mtx gel including liquefaction, color, phase separation and homogeneity were investigated by visual inspection at different intervals of 1st, 7th, 14th,21th, 28th, and 60th days. (9) skin irritation test this test was done by applying sufficient quantity of the gel to small region of rats' skin under supervision (at time of application and after one hour) for any hypersensitivity reaction in the skin (redness, irritation) or any necrosis could be made iraqi j pharm sci, vol.30(suppl.) 2021 methotrexate transdermal gel 18 by the gel, the number of rats has been used are seven. (10) changes in ph the ph of different formulas was measured using ph meter device at 1st, 7th, 14th, 21th, 28th, and 60th days after formulation. (11) drug content a gel amount of 50 mg from each produced formula was taken, then dissolved with 50 ml of ph 7.4 phosphate buffer with stirring until a completely soluble mixture obtained. the mixture then filtered with syringe filter. the concentration of mtx was measured spectrophotometrically by using equation of the drug calibration at 303nm. (12) viscosity the viscosity of the formulated gel was determined by using brookfield . viscometer at 37 °c. the apparatus spindle was rotated at 10 rpm. (13) in vitro drug permeability and release kinetic study all formulas permeation were studied by using franz diffusion cell. the donor compartment was supplied by 1gm of mtx gel and the receptor compartment with 100ml of phosphate buffer ph 7.4. the donor and receptor parts separated by synthetic semipermeable membrane (mw 8000 da). the media was stirred at 100 rpm and the temperature kept at 37 c. 5ml sample of mtx gel was withdrawn from the receptor which was replaced by the same volume of buffer to maintain the sink condition. the quantity of mtx was determined spectrophotometrically at 303nm by using the phosphate buffer as the blank. the release of the drug determined by applying the result data on korsmeyer peppas model kinetic. (14) in vitro the release mechanism of mtx determined by using the following model equation mt/ m∞ = k*tn where mt/m∞ is the fractional mtx re lease from the gel into the receptor media, k is a drug delivery constant whereas (n) is diffusion coefficient and its value indicates the mechanism of the drug release in the solvent. the (n) value of 0.5 that indicates quasifickian diffusion mechanism, while if (n>0.5) then anomalous or non-fickian diffusion mechanism exists and if it is (=1) then the zero order release one exists. (15) statistical analysis one-way analysis of variance (anova) was applied in this research and if p<0.05 the difference was considered statistically significant. results and discussion the general properties of all prepared formulas were good from preparation till two months, so the homogeneity of all formulas was acceptable with no particles was made. the color of all formulas was yellow (attributed to mtx color) and without change in color till two months. (16) the formulas were stable without liquefaction at 2% of carbopol 940 so this indicates a good percentage of gelling agent. no phase separation was made at all time to the all formulas. the more important character which was skin irritation (irritancy test), redness and necrosis not observed at rats' skin after apply sufficient quantity of mtx gel (17). the drug content data obtained was shown in table 2. the drug content was between 95.2 % to 97.1%. this indicates the good content of the drug formulas and good uniformity of the preparation. the viscosity of the preparations was decreased with increasing the concentration of the enhancer, that are between 17112 cp to 18655 cp. as shown in the table 2 and figure 1 (18). table 2. organoleptic properties and skin irritation test of mtx transdermal gel no. parameters f1 f2 f3 f4 f5 f6 f7 1 color yellow yellow yellow yellow yellow yellow yellow 2 liquefaction no no no no no no no 3 phase separation no no no no no no no 4 homogeneity good good good good good good good 5 skin irritation test no no no no no no no 6 drug content 99.1% ±1.2 95.6% ±1 96.1% ±2.1 96.3% ±2.1 97.1% ±1.3 95.2% ±1.5 95.6% ±1.9 7 viscosity 19775 ±43 18655 ±36 17323 ±22 17745 ±45 17112 ±18 18221 ±29 17342 ±32 iraqi j pharm sci, vol.30(suppl.) 2021 methotrexate transdermal gel 19 figure. 1 rats' skin at time of application (left) and after one hour (right) no redness, irritation or any necrosis could be seen after application of mtx gel. changes in ph the ph of all formulas are within the normal range of skin till to 60 days, so no irritation or burning sensation to the skin could be occurred. figure 2 shows ph as y axis and duration of storage as x axis. (19) figure.2 ph change during 60 days after formulation. all formulas represented by ph±sd, where the f1 gel without enhancer, where other formulas with enhancer. in vitro mtx permeability and release kinetic study the permeability and release of the mtx study by using franz cell membrane explained in figure 3, according to the results; using the gel without enhancer had poor (20%) permeability after 24 hours, the addition of eucalyptus oil as enhancer at 2% and 4% (f2, f3) respectively, result in significantly (p<0.05) increasing the permeability to 79% and 85% respectively after 24hr, the increasing in permeability due to increasing in enhancer concentration. in formulas with peppermint oil (f4 and f5) the permeability is 92% and 99% with increasing in concentration of enhancer from 2% to 4% respectively. in the f6 and f7 which had olive oil as enhancer with concentration 2% and 4% the permeability reaches 80% and 90% respectively. so, from the data, the permeability of the mtx gel was maximum with peppermint oil due to the solubility of the mtx is higher than in eucalyptus oil and in olive oil. the suggested mechanism for the oils to work as penetration enhancers is either disintegration of the intracellular highly ordered lipid structure or induce conformational modification in the intracellular proteins or might be due to increasing the partitioning of mtx into stratum cornium. (20) iraqi j pharm sci, vol.30(suppl.) 2021 methotrexate transdermal gel 20 carbopol 940 was used to control the release of mtx up to 24hr as it acts as a controlling rate polymer. so, according to krosmeyer-peppas model, the kinetic release of drug was obtained. and the mechanism of release was non-fickain and super case ii diffusion which is demonstrated in figure 3. (21) table 3. korsmmeyer-peppas kinetic model for mtx transdermal gel through synthetic membrane figure3.percent of mtx permeated across synthetic semipermeable membrane at ph 7.4 buffer solution, where n=3. conclusion methotrexate gel was successfully prepared and evaluated in-vitro with a good permeation across the synthetic membrane. the previous results showed the using of the peppermint oil as permeation enhancer at 4(v/v) % concentration give worthy transdermal gel of mtx with respectable permeation across semipermeable membrane consequently for stratum corneum. moreover, the formula had good drug content, viscosity, ph as an end result suitable for the skin. references 1. murtadha s. jabur, mohammed r. i. ghazal, hayder r.al-hamamy. topical methotrexate for treatment of psoriasis: formulation and clinical implications. fac med baghdad 2010; vol. 52, no. 1 2. gillian s. leslie singka, nor abu samah, mohd h. zulfakar, aysu yurdasiper, charles m. heard. enhanced topical delivery and antiinflammatory activity of methotrexate from an activated nanogel. european journal of pharmaceutics and biopharmaceutics 76 (2010) 275–281 3. nawaz a, jan su, khan nr, hussain a, khan gm. formulation and in vitro evaluation of clotrimazole gel containing almond oil and tween 80 as penetration enhancer for topical application. pak j pharm sci. 2013;26(3):61722. 4. m a-h. enhancement effect of almond oil on transdermal penetration of flurbiprofen topical gel. journal of kerbala university. 2015;13:160. 5. jawad a at, jiyauddin k, s.budiasih, m kaleemullah, samer a.d, eddy y, and fadi a. fabrication and evaluation of a stable flurbiprofen hydrogel. ijpar journal. 2014;3(3):284-90. 6. corbo m, schultz t, wong g, van buskirk g. development and validation of in vitro release testing methods for semisolid formulations. pharmaceutical technology. 1993;17(9):112-. 7. finnin bc, morgan tm. transdermal penetration enhancers: applications, limitations and potential. j pharm sci 1999;88:955-8. 8. zhao yc, zheng hl, wang xr, zheng xl, chen y, fei wd, zheng yq, wang wx, zheng ch. enhanced percutaneous delivery of methotrexate using micelles prepared with novel cationic amphipathic material. int j nanomedicine. 2020;15:3539-3550 9. helal da, el-rhman da, abdel-halim sa, elnabarawi ma. formulation and evaluation of fluconazole topical gel. int j pharm pharm sci. 2012;4(5):176-83. 10. alaayedi m, mahmod, hasanian, saeed, ashti. the enhancement effect of castor oil on the permeability of flurbiprofen as transdermal gel. international journal of applied pharmaceutics. 2018;10(1):140-4. 11. maryam h. alaayedi, et al. (2017). "effect of hydroxy propyl methyl cellulose (hpmc) on amoxicillin floating tablet." karbala journal of pharmaceutical sciences(13): 311-319. 12. güngör s, bergişadi n. effect of penetration enhancers on in vitro percutaneous penetration of nimesulide through rat skin. die pharmaziean international journal of pharmaceutical sciences. 2004;59(1):39-41. formulas r2 nvalue type of release f1 0.9594 0.74 non-fickain diffusion f2 0.876 2.75 super caseii transports f3 0.8906 2.97 super caseii transports f4 0.9023 3.57 super caseii transports f5 0.8879 3.98 super caseii transports f6 0.8945 2.89 super caseii transports f7 0.9159 3.26 super caseii transports iraqi j pharm sci, vol.30(suppl.) 2021 methotrexate transdermal gel 21 13. hasanain sh. mahmood et al / the enhancement effect of olive and almond oils on permeability of nimesulide as transdermal gel. international journal of pharmaceutical research oct dec 2019 vol 11. 14. hirva s, jenisha p. bicelle: a lipid nanostructure for transdermal delivery. j crit rev 2016;3:17-22. 15. keleb e, sharma rk, mosa eb, aljahwi az. transdermal drug delivery system-designand evaluation. int j adv pharm sci 2010;1:201-11. 16. li dx, han mj, balakrishnan p, yan yd, oh dh. enhanced oral bioavailability of flurbiprofen by combined use of micelle solution and inclusion compound. arch pharm rev 2010;33:95-101. 17. syed nhs, mahboob r, muhammad fa. study of percutaneous absorption of diclofenac diethylamine in the presence of cetrimide through hairless rabbit skin. j res sci 2006;17:45-51. 18. rambharose s, kalhapure rs, jadhav m, govender t. exploring unsaturated fatty acid cholesteryl esters as transdermal permeation enhancers. drug delivery transl res 2017;7:333–45. 19. hussain a, khan gm, shah su, rahim n. development of a novel ketoprofen transdermal patch: effect of almond oil as penetration enhancer on in vivo and ex-vivo penetration of ketoprofen through rabbit skin. pak j pharm sci 2012;25:227-32. 20. qiudong jiang, yeming wu, hui zhang, pei liu, junhong yao, peijunyao, jun chen & jinao duan .development of essential oils as skin permeation enhancers: penetration enhancement effect and mechanism of action, pharmaceutical biology. 2017; 55:1, 15921600. 21. dash s, murthy pn, nath l, chowdhury p. kinetic modeling on drug release from controlled drug delivery systems. acta pol pharm. 2010;67(3):217-23.22. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( suppl. ) 2022 knowledge, attitudes, and practices towards covid-19 doi: https://doi.org/10.31351/vol31isssuppl.pp111-120 111 knowledge, attitudes, practices, and online distance learning experience of malaysian university students towards covid-19: a cross sectional study (conference paper) # siew chin ong*,1 and ehab mudher mikhael** # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *school of pharmaceutical sciences, universiti sains malaysia, penang, malaysia ** department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract some new norms need to be adapted due to covid-19 pandemic period where people need to wear masks, wash their hands frequently, maintain social distancing, and avoid going out unless necessary. therefore, educational institutions were closed to minimize the spread of covid-19. as a result of this, online education was adapted to substitute face-to-face learning. therefore, this study aimed to assess the malaysian university students’ adaptation to the new norms, knowledge and practices toward covid-19, besides, their attitudes toward online learning. this study was conducted from january to february 2021 and included a sample of 500 malaysian university students. for data collection, all students were asked to fill in a questionnaire that was developed based on previous literature, using google forms. 498 students completed the questionnaire (response rate 99.6%). malaysian ministry of health was the main source (83.73%) that students refer to when looking for information on covid-19. only 40% of the participants had good overall knowledge about covid-19; such knowledge was influenced by the students' field of study. the current practice towards covid-19 was good only by 26.1% of participating students; such practice was influenced by the ethnic groups. additionally, 60% of participated students agreed that covid-19 can be successfully controlled. about one-third of participants had positive attitudes toward online learning. the major challenges facing students during online learning include distraction of the learning environment (80%), unstable internet connectivity (75%), lack of motivation (70%), limited technical skills (41%), and limited broadband data (34%). in conclusion, the knowledge and practice toward covid-19 was good in less than half of malaysian university students. attitudes to the controlling of covid19 were positive, while the attitudes toward online learning were neutral among most of the malaysian university students. challenges toward online learning are diverse and include both technical and student-related problems. keywords: covid-19; distance learning; university students. المعرفة والمواقف والممارسات وتجربة التعلم عن بعد عبر اإلنترنت لطالب الجامعات الماليزية # ) بحث مؤتمر ( : دراسة مقطعية19-كوفيد تجاه *مضر ميخائيل ايهاب و 1* ،اونكسيو جن 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي . بينانغ ، ماليزيا ماليزيا، جامعة سينز الصيدالنية، كلية العلوم * الصيدلة، جامعة بغداد، بغداد، العراق. كلية ، فرع الصيدلة السريرية** الخالصة كورونا جائجة التباعد على والحفاظ متكرر، بشكل اليدين وغسل ، لكماماتأ رتداءكإ الجديدة المعايير بعضب االلتزام يجببسبب لذلك، تم االعتماد نتيجة. 19-كوفيد انتشار لتقليل التعليمية المؤسسات إغالق تم لذلك، . ضروريًا ذلك يكن لم مامن المنزل الخروج وتجنب االجتماعي، المعايير مع الماليزية الجامعات طالب تكيف تقييم إلى الدراسة هذه هدفت .(لوجه وجًهانظام التعلم الحضوري ) محل ليحل اإلنترنت عبر التعليمعلى 500 من العينات ألخذ نظام العينة المالئمة استخدام تم .اإلنترنت عبر التعلم تجاه مواقفهم جانب إلى ، 19-كوفيد تجاه الجديدة والممارسات والمعارف الطالب جميع من ُطلب البيانات، لجمع. االجتماعي التواصل وسائل عبر 2021 شباط ولغاية كانون الثاني خالل الفترة من ماليزي جامعي طالب ٪(. 99.6 االستجابة معدل) االستبيان طالبًا 498 لاأكم. اشات النتائج الى كوكلنماذج باستخدام السابقة، األدبيات على بناءً تطويره تم استبيان ملء من فقط ٪40. 19-كوفيد حول معلومات عن البحث عند الطالب إليه يشير الذي٪( 83.73) الرئيسي المصدر الماليزية الصحة وزارة كانت بنسبة فقط جيدة 19-كوفيد تجاه الحالية الممارسة كانت. الطالب دراسة بمجال المعرفة هذه تأثرت ؛ 19-ن كوفيدع جيدة عامة معرفة لديهم المشاركين إمكانية على المشاركين الطالب من٪ 60 وافق ذلك، إلى باإلضافة. العرقية بالمجموعات الممارسة هذه تأثرت المشاركين؛ الطالب من٪ 26.1 تواجه التي الرئيسية التحديات تشمل. اإلنترنت عبر التعلم تجاه إيجابية مواقف المشاركين ثلث حوالي لدى كان. بنجاح 19-كوفيد على السيطرة المهارات ومحدودية (، ٪70) الحافز ونقص (، ٪75) باإلنترنت المستقر غير واالتصال (، ٪80) التعلم بيئة إلهاء اإلنترنت عبر التعلم أثناء الطالب إن ٪(.34) االنترنيت ومحدودية (، ٪41) التقنية ذلك من الجامعات طالب نصف من أقل لدى جيدة 19-كوفيد تجاه والممارسة المعرفة نستنتج الجامعات طالب معظم بين محايدة اإلنترنت عبر التعلم تجاه المواقف كانت بينما إيجابية، جائحة كورونا على السيطرة تجاه المواقف كانت. الماليزية . بالطالب المتعلقةتلك و التقنية المشاكل وتشمل متنوعة اإلنترنت عبر التعلم تواجه التي التحديات. الماليزيين corresponding author e-mail: siewchinong@usm.my received: 11/7 /2022 accepted: 23/10 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp111-120 iraqi j pharm sci, vol.31( suppl. ) 2022 knowledge, attitudes, and practices towards covid-19 112 introduction in an effort to mitigate the outbreak of covid-19, many countries including malaysia, have imposed drastic lockdown, movement control, or shelter in place orders on their residents. some new norms need to be adapted due to covid-19 pandemic period where people need to wear masks, wash their hands with soap or hand sanitizer frequently, maintain social distance with others, and avoid going out unless to buy the necessities (1). however, the effectiveness of these mitigation measures is highly dependent on the cooperation and compliance of all members of society (2). due to covid-19 pandemic and to minimize its spread, there is a need to close educational institutions like universities, schools, tuition centres, and nurseries (3). students ranging from primary to tertiary level cannot go back to their respective educational institutions to learn as they usually did. as a result of this, the education system and learning methods have been reshaped. the method of having online learning with technologies and devices as a mediator of communication in order to substitute the physical face-to-face learning has been adapted by most malaysian education sectors (4). this is currently the most popular and effective way to continue the learning and teaching process and at the same time stop the spread of covid-19. the platforms used for online classes include google meet, zoom, microsoft teams, and free conference call. in addition, university students also sit for their examinations online and submit their answers via the university’s designated platform. despite the pros of this new teaching and learning method, it has its cons as it may affect all walks of life, especially the students and educators (5). there were a few studies conducted in malaysia to assess the knowledge, attitudes, and practices among the malaysian general public towards covid-19 (2, 6). however, to the best of our knowledge, no study was conducted among university students. the knowledge, attitudes, and practices among university students toward the disease play an integral role in determining a society's readiness to accept behavioural change measures from health authorities. besides, observance of students to the prevention measures is essential for controlling the spread of covid19 in universities where most students are staying in dormitories or hostels. therefore, the aim of this study is to assess malaysian university students’ adaptation to the new norms, knowledge levels, attitudes, and practices toward covid-19, besides their attitudes toward online learning. methods study design and sampling method a cross sectional study was conducted from january to february 2021. although random samples produce more informative and less biased results than convenient ones, covid-19 may call for desperate actions and serves as an excuse for using less stringent criteria in choosing survey samples (7). therefore, a non-probability convenience sampling technique was used to recruit malaysian university students in this study. university students from all over malaysia were approached to participate in the study. due to covid-19, data were collected only using google forms. social media such as whatsapp, facebook, instagram, and twitter were used to disseminate and promote the survey to university students. the inclusion criteria of this study were malaysian university students, aged 18 years old and above from both genders who were able to read and understand english. study ethical approval was obtained from the institutional human ethics committee at the universiti sains malaysia with the number 20120638 at 11th january 2021. written informed consent was taken from the participants prior to data collection. sample size estimation the sample size was calculated according to a 95% confidence level, 80% power of the study (1𝛽) and 5% margin of error (α value or type i error). according to the free online raosoft sample size calculator, the required sample size was 384. in case of missing data, an additional 30% was added and the required sample size became 500. research instruments the research questionnaire was adapted from the previous studies (2, 6). the questionnaire consists of six sections. section a is comprised of demographic information. section b is to ascertain the respondents’ online distance learning experience. section c is to assess respondents’ knowledge and understanding of covid-19. scoring of 12 points or less was considered as poor knowledge about covid-19, while scores more than 12 was considered as good knowledge. attitude and practice towards covid-19 are assessed in sections d and e. scoring of 5 points or less was considered as poor practice, while a score of more than 5 was considered as a good practice. attitudes towards online learning and challenges of online learning can be found in section f. data analysis data were entered and analysed with spss version 26. descriptive statistics were used to summarize the socio-demographic characteristics of the subjects. categorical data was presented as percentages, while continuous data was presented as mean ± standard deviation. median of knowledge and practice scores was used as a cut-off point to discriminate between good and poor knowledge and practice, respectively (8). chi square test was used to assess the difference between categorical variables. p values less than 0.05 were considered significant. iraqi j pharm sci, vol.31( suppl. ) 2022 knowledge, attitudes, and practices towards covid-19 113 results only 498 students completed the questionnaire (response rate 99.6%). the average age of participants was 19.62 years. most participants were females (80.92%), with malay ethnicity (59.04%), living in the central region of malaysia (28.11%), and studying sciences (46.59%) online in their parent's house (90.96%). malaysian ministry of health (moh) was the main source (83.73%) that students refer to when looking for information on covid-19. on the other hand, social media such as instagram, facebook, twitter, and tv were the main media that students use to look for information about covid-19. further details are given in table 1. table 2 showed the participants' knowledge about covid-19. participants’ knowledge about seriousness, transmission, symptoms, and treatment of covid-19 was good in 95.58%, 78.92%, 89.76%, 85.14% of the participants, respectively. only 40% of the participants had good overall knowledge about covid-19. the mean knowledge score was 11.82 ± 1.98. regarding the practices toward covid19, about 70% of the participated students adhered to covid-19 protective measures (avoidance of crowded places, wearing face masks, and proper hand hygiene) in the week before the movement control order (mco) had been started; thereafter, such practices were adopted by about 95% of participated students. on the other hand, the current practice towards covid-19 was good only by 26.1% of participating students. further details are shown in table 3. table 4 showed that knowledge about covid-19 was influenced only by the student's field of study, whereas the practice was influenced by the ethnic groups, in which indian students had better practice than students from other ethnic groups. regarding the attitudes toward covid-19, table 5 showed that about 60% of the respondents agreed that covid-19 can be successfully controlled. additionally, only a minority (4.4%) of students disagreed with the ability of malaysia to win the battle against covid-19. furthermore, more than half of participating students trusted the ability of the malaysian government to handle the covid-19 health crisis in a good way. on the other hand, most study participants agreed on the effect of covid-19 on national security (72%) and on personal daily life (88%), besides the need of citizen cooperation to overcome such dangerous disease (88%). regarding the effectiveness of online learning (table 6), half of participating students were neutral, one-third had negative attitudes, and one-sixth had positive attitudes toward online learning. more than half of the participants agreed on the benefit of coursemates and teammates while studying online. on the other hand, 45% of students thought the lecturers were helpful for students during the online studying period. regarding the challenges in online learning (figure 1), more than 80% of participating students found the distraction of the learning environment renders focusing during online lectures difficult. on the other hand, as high as about three-fourths of the participants suffered from unstable internet connectivity. other challenges during online learning included lack of motivation (70%), limited technical skills (41%), and limited broadband data (34%). table 1. demographic data of participants parameter value age mean±sd 19.62±3.33 gender female n(%) 403 (80.92) male n(%) 95 (19.08) ethnicity malay n(%) 297 (59.64) chinese n(%) 118 (23.69) indian n(%) 46 (9.24) others* n(%) 37 (7.43) region** central n(%) 140 (28.11) east n(%) 108 (21.69) north (penang, perlis, perak) n(%) 117 (23.49) south (negeri sembilan, melaka, johor) n(%) 49 (9.84) sabah and sarawak n(%) 84 (16.87) field of study medical n(%) 18 (3.61) sciences n(%) 232 (46.59) engineering n(%) 72 (14.46) education n(%) 47 (9.44) business n(%) 36 (7.23) others n(%) 93 (18.67) iraqi j pharm sci, vol.31( suppl. ) 2022 knowledge, attitudes, and practices towards covid-19 114 continued table (1) parameter value academic level 1st year n(%) 397 (79.72) 2nd year n(%) 67 (13.45) 3rd year n(%) 15 (3.01) 4th year n(%) 7 (1.41) 5th year n(%) 4 (0.80) postgraduate n(%) 8 (1.61) staying place during online active period parent's house n(%) 453 (90.96) in campus n(%) 32 (6.43) others (rental house, kampung) n(%) 13 (2.61) the main sources that students refer to when looking for information on covid-19 ministry of health (moh) n(%) 417(83.73) majlis keselamatan negara (mkn) n(%) 296 (59.44) world health organization (who) n(%) 210 (42.17) family and friends n(%) 159 (31.93) othersˆ n(%) 2 (0.40) the main media that students use to look for information on covid-19 tv n(%) 295 (59.24) online n(%) 291 (58.43) mobile applications# n(%) 286 (57.43) instagram n(%) 229 (45.98) facebook n(%) 209 (41.97) portal n(%) 109 (21.89) youtube n(%) 102 (20.48) twitter n(%) 76 (15.26) mysejahtera n(%) 12 (2.41) *others: bumiputra, siam, melanau, mixed, bajau. **central: selangor, kedah, wilayah persekutuan; east: kelantan, terengganu, pahang; north: penang, perlis, perak; south: negeri sembilan, melaka, johor. ^others: mysejahtera. #mobile applications: whatsapp and telegram. table 2. participants' knowledge about covid-19 question good knowledge, n (%) poor knowledge, n (%) is covid-19 more critical or severe than normal flu? 476 (95.58) 22 (4.42) the main clinical symptoms of covid-19 are fever, fatigue, dry cough, and body aches. 447 (89.76) 51 (10.24) unlike the common cold, stuffy nose, runny nose, and sneezing are less common in persons infected with the covid-19 virus. 218 (43.78) 280 (56.22) there is currently no effective cure for covid-19, but early symptomatic and supportive treatment can help most patients recover from the infection. 424 (85.14) 74 (14.86) with immediate treatment, is it possible for person infected with covid-19 has higher chance for survival? 390 (78.31) 108 (21.69) not all persons with covid-19 will develop to severe cases. only those who are elderly and/or have chronic illnesses are more likely to have a severe case. 344 (69.08) 154 (30.92) eating or touching wild animals would result in the infection by the covid-19 virus. 217 (43.57) 281 (56.43) persons with covid-19 cannot transmit the virus to others if they do not have a fever. 393 (78.92) 105 (21.08) iraqi j pharm sci, vol.31( suppl. ) 2022 knowledge, attitudes, and practices towards covid-19 115 continued table 2. question good knowledge, n (%) poor knowledge, n (%) the covid-19 virus spreads via respiratory droplets of infected individuals. 420 (84.34) 78 (15.66) the covid-19 virus is airborne. 296 (59.44) 202 (40.56) ordinary residents can wear face masks to prevent the infection by the covid-19 virus. 450 (90.36) 48 (9.64) it is not necessary for children and young adults to take measures to prevent the infection by the covid-19 virus. 403 (80.92) 95 (19.08) to prevent the infection by covid-19, individuals should avoid going to crowded places and avoid taking public transportations. 474 (95.18) 24 (4.82) isolation and treatment of people who are infected with the covid-19 virus are effective ways to reduce the spread of the virus. 451 (90.56) 47 (9.44) people who have contact with someone infected with the covid-19 virus should be immediately isolated in a proper place. in general, the isolation period is 14 days. 484 (97.19) 14 (2.81) overall knowledge good knowledge >12 points poor knowledge ≤12 202 (40.56) 296 (59.44) overall knowledge score mean ± sd (median) 11.82 ± 1.98 *good knowledge was defined as knowledge score >12, whereas poor knowledge was defined as knowledge score ≤12. table 3. participant's practice towards covid-19 practice good practice, n (%) poor practice, n (%) practice in the week before the movement control order (mco) had been started did you avoid going to crowded places such as weddings? 361 (72.49) 137 (27.51) did you wear a face mask when leaving home? 329 (66.06) 169 (33.94) did you practice proper hand hygiene by frequently washing your hands & using hand sanitizer? 373 (74.90) 125 (25.10) current practice do you avoid going to crowded places such as weddings? 471 (94.58) 27 (5.42) do you wear a face mask when leaving home? 492 (98.8) 6 (1.20) do you practice hand hygiene by frequently washing your hands & using hand sanitizer? 474 (95.18) 24 (4.82) i put a distance from people suspected with covid-19 only. 302 (60.64) 196 (39.36) i limit my movement and not going out except for important business. 467 (93.78) 31 (6.22) do you keep yourself updated about covid-19 cases every day? 259 (52.01) 239 (47.99) overall current practice good practice > 5points poor practice ≤ 5 points 130 (26.1) 368 (73.9) overall practice mean ± sd 4.95 ± 0.87 *good practice was defined as practice score >5, whereas poor practice was defined as practice score ≤5. iraqi j pharm sci, vol.31( suppl. ) 2022 knowledge, attitudes, and practices towards covid-19 116 table 4. the effect of different demographic data on students' knowledge and practice about covid-19 demographic no. of participants knowledge practice knowledge score mean ± sd good (>12) n (%) poor (12 or less) n (%) p value practice score mean ± sd good (>5) n (%) poor (≤5) n (%) p value gender male 95 11.69±2.01 35 (36.84) 60 (63.16) 0.411 4.78±0.97 18 77 0.077 female 403 11.85±1.98 167 (41.44) 236 (58.56) 4.99±0.84 112 291 ethnicity malay 297 11.93±1.70 119 (40.07) 178 (59.93) 0.391 5.0±0.83 82 215 0.002 chinese 118 11.71±2.37 50 (42.37) 68 (57.63) 4.73±0.96 18 100 indian 46 11.91±2.22 22 (47.83) 24 (52.17) 5.15±0.89 20 26 others 37 11.22±2.36 11 (29.73) 26 (70.27) 5.0±0.75 10 27 study field medical 18 12.22±1.70 9 (50) 9 (50) 0.001 4.83±0.71 3 15 0.519 sciences 232 12.28±1.65 116 (50) 116 (50) 4.96±0.89 62 170 engineering 72 11.39±2.15 20 (27.78) 52 (72.22) 4.82±1.01 18 54 education 47 10.96±2.24 13 (27.66) 34 (72.34) 4.91±0.75 9 38 business 36 11.39±2.21 10 (27.78) 26 (72.22) 4.94±0.79 8 28 others 93 11.56±2.17 34 (36.56) 59 (63.44) 5.08±0.84 30 63 source of information 1 source 101 11.39±2.36 36 (34.65) 65 (63.35) 0.484 4.93±0.89 27 74 0.742 2 sources 204 11.94±1.70 80 (39.22) 124 (60.78) 5.0±0.85 55 149 3 sources 142 11.92±2.11 63 (44.37) 79 (55.63) 4.92±0.93 38 104 4 sources 51 11.98±1.79 23 (45.1) 28 (54.9) 4.84±0.78 10 41 region of residency north 117 11.70±2.15 46 (39.32) 71 (60.68) 0.796 4.97±0.92 33 84 0.967 east 108 12.06±1.59 46 (42.59) 62 (57.41) 4.94±0.83 26 82 central 140 12.03±1.75 61 (43.57) 79 (56.43) 4.99±0.82 37 103 south 49 12.02±1.39 19 (38.78) 30 (61.22) 5.0±0.79 13 36 sabah & sarawak 84 11.24±2.65 30 (35.71) 54 (64.29) 4.83±0.98 21 63 iraqi j pharm sci, vol.31(suppl.) 2022 knowledge, attitudes, and practices towards covid-19 117 table 5. attitudes and perceptions of the participated students toward covid-19 question strongly agree agree neutral disagree strongly disagree do you agree that covid-19 will be successfully controlled? 68 (13.65) 227 (45.58) 175 (35.14) 23 (4.62) 5 (1.00) do you agree that malaysia can win the battle against the covid19 virus? 123 (24.70) 230 (46.18) 123 (24.70) 16 (3.21) 6 (1.20) the government of malaysia is handling the covid-19 health crisis very well. 88 (17.67) 180 (36.14) 170 (34.14) 45 (9.04) 15 (3.01) covid-19 outbreak is difficult for me to handle. 44 (8.84) 190 (38.15) 215 (43.17) 42 (8.43) 7 (1.41) covid-19 outbreak needs my cooperation as a citizen to overcome it. 259 (52.01) 180 (36.14) 52 (10.44) 3 (0.60) 4 (0.80) covid-19 outbreak can threaten national security. 128 (25.7) 232 (46.59) 121 (24.30) 12 (2.41) 5 (1.00) covid-19 outbreak can affect the comfort of daily life. 271 (54.42) 169 (33.94) 51 (10.24) 4 (0.80) 3 (0.60) table 6. attitudes and perceptions of students toward learning during covid-19 question strongly agree agree neutral disagree strongly disagree do you agree that online learning is effective to you? 15 (3.01%) 69 (13.86%) 249 (50%) 132 (26.51%) 33 (6.63%) do you agree that your lecturers are helpful while you are studying online? 59 (11.85%) 165 (33.13%) 205 (41.16%) 54 (10.84%) 15 (3.01%) do you agree that your coursemates are helpful while studying online? 97 (19.48%) 187 (37.55%) 152 (30.52%) 42 (8.43%) 20 (4.02%) do you agree that your teammates are helpful while working on project online? 74 (14.86%) 193 (38.76%) 149 (29.92%) 55 (11.04%) 18 (3.61%) figure 1. challenges faced by students in online learning iraqi j pharm sci, vol.31(suppl.) 2022 knowledge, attitudes, and practices towards covid-19 118 discussion the results of the current study showed that most malaysian students relied on the ministry of health to get the recent information about covid19. meanwhile, most students also obtained such information through searching social media like facebook, instagram, and twitter. this finding was similar to that obtained among people living in mosul, iraq (9). this may be because of increased popularity and easy access to social media, especially among young people (10). regarding knowledge about covid-19, more than 95% of participants realized the seriousness of covid-19 and protection methods from it. a similar result was also found among pakistani individuals (11). the good knowledge of malaysian students about the seriousness of covid-19 and its protection methods was mainly attributed to the frequent awareness programs adopted by universities and malaysian moh (12). despite this good knowledge about some covid-19 related topics, the overall knowledge about covid-19 was good only among 40% of malaysian universities students. this poor knowledge may have resulted from the presence of a huge amount of information in social media and internet websites, most of which were inaccurate and/or false (13, 14). the overload of information may have caused confusion and difficulty in ascertaining the correct information. this explanation is further confirmed by the result of the current study that showed the knowledge about covid-19 was influenced only by the students’ field of study regardless of students' gender, age, or ethnicity. in this regard, students of the medical and scientific field had the highest knowledge scores due to the nature of their study, which enable them to recognize the accurate information about covid-19. the present study showed that the average knowledge score among the participated malaysian university students was 11.82 ± 1.98. this score was close to that among the malaysian general public (2). this finding is somewhat less than expected because of a good educational level among university students as compared to the general population. however, it was found that knowledge score was not correlated with the individual’s educational level, instead, it was directly correlated with the individual’s risk perception of contraction and complications from the covid-19; such perception was found to be higher among individuals older than 50 years (15). the results of the present study showed a positive effect of the movement control order (mco) in malaysia to increase university students’ adherence to covid-19 protective measures (avoidance of crowded places, wearing face masks, and proper hand hygiene). this result was consistent with that found in other studies conducted in malaysia (16, 17). the current practice to reduce the risk of covid-19 was good only in 26.1% of participating university students. this level of practice was close to that found in ethiopia (18) but much less than that in china (19). this variation in practice may be related to the differences in governmental strategies that influence the covid-19 prevention practice and the desire of individuals to adhere to such practices. furthermore, the present study showed that ethnicity had a significant effect on the students’ practice of covid-19 preventive measures. in this regard, university students from indian ethnicity were more adhere to covid-19 preventive measures. the higher knowledge about covid-19 among indian students than students from other ethnic groups (20) may be the main reason for such better adherence to covid-19 preventive practices (21). the results of this study showed positive attitudes of most participating university students toward the ability of the malaysian government to win the battle against covid-19 and handle the health crisis in a good manner. these positive attitudes were also detected among the malaysian general public (2). on the other hand, most participants agreed on the threat of covid-19 on national security. similar attitudes toward pandemic influenza were detected by lay participants in france (22). additionally, participants of the present study felt that covid-19 strongly affect their daily life, such perception was similar to that found in studies conducted in many other countries (23-25). this perception is highly expected because of the necessity for travel restriction, social distancing, and closure of many facilities except primary and essential services (26). the present study showed that half of the participants had neutral attitudes toward the effectiveness of online learning. this finding was not surprising since online learning is a new mode of learning in most developing countries (27). on the other hand, most participating university students agreed on the great help from coursemates and teammates while studying online. this type of cooperation between students is reasonable because electronic learning methods provide tools that help in building online communities for students and also facilitate collaboration between students (28). despite this advantage for electronic online learning, the present study showed many problems with such mode of learning including device and studentrelated problems. the device-related problems include limited broadband data, slow devices (e.g. laptop or smartphone), and unstable internet connectivity. these problems are common in other developing countries and may result from the limited infrastructure for online learning (29, 30). meanwhile, student-related problems for online learning are diverse and include lack of technical skills, lack of motivation during online lectures, difficulty in understanding the subject contents, and learning environment that render focusing to lecture iraqi j pharm sci, vol.31(suppl.) 2022 knowledge, attitudes, and practices towards covid-19 119 difficult. most of these problems are linked to the absence of face-to-face contact during the online lectures (31); thus, such problems can be partly solved by interactive mode of online learning (32). in conclusion, the knowledge and practice toward covid-19 was good in less than half of malaysian university students. attitudes about the controlling of covid-19 were positive, while the attitudes toward online learning were neutral among most malaysian university students. challenges toward online learning are diverse and include both technical and student-related problems. conflict of interest the authors declare that there are no conflicts of interest. funding this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. references 1. umair s, waqas u, faheem m. covid-19 pandemic: stringent measures of malaysia and implications for other countries. postgrad med j. 2021;97:130-32. 2. esposito s, principi n. school closure during the coronavirus disease 2019 (covid-19) pandemic: an effective intervention at the global level? jama pediatr. 2020;174:92122. 3. krishnan 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dev using inf commun technol. 2017;13. 31. alsaaty fm, carter e, abrahams d, et al. traditional versus online learning in institutions of higher education: minority business students’ perceptions. bus manag res. 2016;5. 32. zheng m, bender d, lyon c. online learning during covid-19 produced equivalent or better student course performance as compared with pre-pandemic: empirical evidence from a school-wide comparative study. bmc med educ. 2021;21:495. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines doi: https://doi.org/10.31351/vol31iss1pp72-86 72 understanding the experience of hospital pharmacists with the effectiveness, safety, adverse drug reaction reporting and interchangeability of biopharmaceutical medicines hiba leith fahmi*,1 and ali azeez al-jumaili* * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq abstract the study objectives were to 1) explore the real-world experience of hospital pharmacists with the differences in effectiveness safety, and interchangeability between biosimilar medicines and their reference biological counterparts, 2) reveal pharmacist recommendations to enhance the safety of biopharmaceutical medicines in public hospitals. the study has a mixed-method design where the core component was qualitative (interviews) and the supplemental component was quantitative (adverse drug reaction, adr, reports). this qualitative component included semi-structured (mostly face-toface) interviews involving hospital pharmacists from different hospitals with experience with biological or biosimilar medicines. the interviews were conducted from nov 2020 through feb 2021. thematic analyses were used to analyze qualitative data generated from the interviews. the study sample included 25 pharmacists from ten governmental hospitals in baghdad, iraq. the pharmacists were 21 women and 4 men. because most pharmacists had a short experience with biosimilar medications, they were unsure about their effectiveness and safety. most pharmacists preferred reference biological over biosimilar medicines because of their effectiveness. however, they believed that initial prescribing and switching between a reference and counterpart biosimilar rely on their availability. the pharmacists tended to underreport biopharmaceutical adrs. the non-sustainable supply of the same biopharmaceutical medicines in public hospitals negatively impacted pharmacist evaluation of the effectiveness and safety of biosimilar medicines. both pharmacist interviews and the iraqi pharmacovigilance center (iqphvc) data showed under-reporting of biopharmaceutical adrs. medicine procurement in healthcare settings should focus on sustainably securing high-quality biopharmaceuticals rather than looking only at costs to enhance patient clinical outcomes. providing pharmacist training, electronic reporting, promoting documentation, and following up with patients is pivotal to prevent, monitor and treat their adrs. keywords: biological medicines, biosimilar medicines, biopharmaceutical medicines, hospital pharmacists, effectiveness, safety, interchangeability, perceptions, qualitative study. فهم تجارب صيادلة المستشفيات مع فعالية وسالمة والتبليغ عن التفاعالت الدوائية الضارة والتبديل لالدوية البايولوجية والبدائل الحيوية *عزيز الجميلي علي و 1*،هبه ليث فهمي .العراق، جامعة بغداد ، كلية الصيدلة، السريريةفرع الصيدلة * خالصةال المرجعية، البيولوجية نظيراتهاو استقصاء الخبرة الواقعية لصيادلة المستشفيات حول االختالفات في الفعالية والسالمة بين األدوية الحيوية عن توصيات الصيدلي لتعزيز سالمة األدوية الصيدالنية الحيوية في المستشفيات العامة الكشف نوعي جزء رئيسي وبشكل المختلطة الدراسة هذه بالمقابالت تضمنت الجانبية متمثل التفاعالت تقارير هو الثاني لألدوية والجزء بالت كانت في غالبيتها وجًها لوجه شملت صيادلة من مستشفيات مختلفة ممن لديهم خبرة المرسلة الى المركز العراقي لليقظة الدوائية. المقا البيولوجية . واستخدمت التحليالت المواضيعية لتحليل 2021 وشباط 2020أجريت المقابالت بين تشرين الثاني لحيوية.في األدوية البيولوجية و / أو األدوية البيانات النوعية الناتجة عن المقابالت رجال. وبسبب أن 4وامرأة 21. كان عدد الصيادلة قبغداد، العراصيدلياً من عشرة مستشفيات حكومية في 25الدراسة ينةتضمنت ع المرجعية البيولوجية الصيادلة االدويةلم يكونوا متأكدين من فعاليتها وسالمتها. يفضل معظم معظم الصيادلة لديهم خبرة قليلة مع األدوية البايوسملر ، توفرها. االدوية الحيوية . ومع ذلك ، االعتقاد أن الوصف االول والتبديل بين االدوية البايولوجية ونظائرها االدوية الحيوية يعتمد على مدى على عن التفاعالت الدوائية السلبية لالدوية الحيوية الصيدالنية يميل الصيادلة إلى عدم اإلبالغ يجب أن يركز شراء األدوية من قبل وزارة الصحة في أماكن الرعاية الصحية على تأمين األدوية الحيوية عالية الجودة بطريقة مستدامة من النظر فقط إلى التكاليف لتعزيز النتائج السريرية للمريض. يعد تعزيز الت وثيق و اإلبالغ اإللكترونية ومتابعة التأثيرات االخاصة باالدوية بدالً البايولوجية أمًرا محوريًا لمنع ورصد وعالج التفاعالت الدوائية الضارة . االعتقاد. ، السالمة ،الفعالية ، صيادلة المستشفيات ، االدوية الحيوية ، الكلمات المفتاحية: االدوية البايولوجية introduction since the development of recombinant human insulin in 1982, the use of biological medications (also called therapeutic proteins) has continued to overgrow (1). recombinant dna (rdna) molecules are introduced into different living systems to express proteins of therapeutic interest, termed biopharmaceuticals (2). 1corresponding author e-mail: ali.baraak@copharm.uobaghdad.edu.iq received: 10/6/2021 accepted: 14/ 8/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp72-86 iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 73 biopharmaceutical (reference biological and biosimilar) medicines are revolutionized medicines used to treat many serious diseases across different specialties such as cancer, rheumatoid arthritis, colitis, diabetes mellitus, crohn’s disease, anemia, immunologic diseases, osteoporosis, and other diseases (3). originator ( also known as reference biological) medicine is a single product of the innovating company (innovator) and approved by regulatory authorities based on a full complement of safety and effectiveness data (4). after the patent of reference, biological medicines have expired, biosimilars (similar biological medicinal products) can be authorized (3, 4). a biosimilar is a biologic treatment manufactured by other producers from other cell lines (5). biosimilar medicines should be equivalent to an authorized reference product in terms of quality, safety, and effectiveness (3, 6), but they are not identical (7). although they have been very effective in treating certain medical conditions, many biologics are expensive. in 2019, the global biologics market was approximately $269,152.8 million, with a 12.6% increasing in a compound annual growth rate (cagr) since 2015 (8). biosimilar medications may result in price reductions for reference biological medicines as they open the market to competition (9). the manufacture of biosimilar agents is complicated by the requirement for their production in biological systems, slight variations that can influence the biosimilar products' structure, activity, and metabolism (11). altering the molecule structure of biopharmaceutical medicines can impact their effectiveness and safety by changing biological activity and causing immunogenicity, leading to a loss of effectiveness (12). the published literature has raised concerns regarding the use of biosimilar medicines (13). these concerns include a lack of confidence that biosimilar medicines can provide equivalent clinical outcomes for patients (13, 14), their indications are approved based on extrapolation of clinical data for the reference medicine, in addition to perceptions of risk in “switching” of patients already receiving a reference biologic medicine to a biosimilar medicine (13). iraq is one of the middle eastern countries that has authorized its biosimilar approval guidelines recently (2019) through the biologics and biosimilars registration committee (bbrc), which mainly relies on the european medicines agency (ema) guidelines (10). according to biosimilar guidelines, the iraqi ministry of health (moh) approved 18 biosimilar medicines within a short period (10). the switching to biosimilar medicines could save the iraqi health system more than 50 million dollars over several years (10). although there is increasing support for biosimilar medicines by the moh, there is scarce information about whether hospital pharmacists accept these medicines and support movement toward replacing reference medicines with their biosimilar counterparts. exploring the experience of hospital pharmacists with biopharmaceutical medications can help to evaluate their safety and effectiveness in the iraqi public healthcare sector and send feedback to health officials to assess their policy regarding these medications. the study objectives were to 1) explore the realworld experience of hospital pharmacists with the differences in efficacy, safety, and interchangeability between biosimilar medicines and their reference biological counterparts, and 2) reveal pharmacist recommendations to enhance the safety of biopharmaceutical medicines in public hospitals. methods study design the study has a mixed-method design where the core component is qualitative (interviews) and the supplemental component is quantitative (iqphvc adr reports) (15). the study had two sources of information: qualitative data from pharmacist interviews and the iqphvc reports of biopharmaceutical adrs. the reason for using two methods is “complementarity to seek elaboration, enhancement, illustration, clarification of the results” from the qualitative method with the results from the quantitative method. (15) qualitative source the primary source of information included face-toface or phone semi-structured interviews involving hospital pharmacists (from different wards) who have experience with biological and/or biosimilar medicines. the interviews were conducted from november 2020 through february 2021. the sample size relied on saturation point, which is an essential role in such studies. in other words, the researcher stopped data collection after reaching a point when new participants were repeating the same previous answers. researchers usually define data saturation as "the point when no new information or themes are observed in the data."(16) semi-structured interviews were conducted by an msc candidate who is a pharmacist with 10 years of hospital experience. settings pharmacists from ten public hospitals in baghdad participated in the study. twenty-one interviews were conducted face-to-face in public hospitals, while four interviews were conducted via phone. only three of them agreed to audio-record their interviews. each interview lasted about 30-60 minutes. inclusion criteria hospital pharmacists have experience with biological and/or biosimilar medications (have dispensed them) in addition to the agreement for participation in the interviews. iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 74 participant recruitment two sampling techniques were used to recruit the participating pharmacists: purposive and snowballing. initially, a purposive sampling of hospital pharmacists within public hospitals in baghdad province was used. purposive sampling was used to select "individuals that are especially knowledgeable about or experienced with a phenomenon of interest "(17, 18). the contact information of some participants was obtained from the iqphvc. additionally, the researchers used snowballing technique which means the researcher asked the participants about other pharmacists interested in participating and meeting the inclusion criteria. the interviews were arranged either inperson or via phone calls and conducted by face-toface researcher meetings at the hospitals or over the phone. the researcher targeted the pharmacists working in the included 10 hospitals, but not all of them had experience with biopharmaceutical medicines. additionally, some who met the inclusion criteria denied participation due to their workload during working hours. the interview guide was sent to the participants via whatsapp before the interview. the interviews continued until we reached saturation in the information. verbal consent was obtained from interviewees before the interviews and the comments were deidentified to keep their confidentiality. the audio recording of the interviews was voluntary. the vast majority of the face-to-face interviews have not consented to audio-record the interview. thus, notetaking and hand-reporting were conducted by the interviewer. the interview was semi-structured with open-ended questions. each interview was conducted in a quiet place and lasted for about 3060 minutes. the interviews were conducted in english and arabic (according to the participants’ convenience), and then the interview transcripts were translated to english by two bilingual authors. thematic analyses thematic analyses were used to analyze qualitative data, which was generated from the interviews. the researchers extracted qualitative data from the participant comments to identify and generate themes. the researchers followed the six phases of thematic analysis described by braun and clarke, 2006 which include familiarizing with data (comments), generating initial codes, searching for themes, reviewing themes, defining and naming themes, and producing the report(19). the research team cross-checked the comments. inductive analytic methodology (datadriven) was used, and the constructivist paradigm was followed (17). this means we did not rely on a previous framework to develop the themes, but we constructed the themes from the common trends emerging from the participant comments. the data item was each sentence within the participant comments. finally, to enhance the credibility and trustworthiness of the findings, peer checking/debriefing was performed two times to validate the qualitative analysis. interview guide the interview guide included four sections: 1) the participant's professional characteristics, 2) their experience with the effectiveness and safety (adrs) of biopharmaceutical medications, 3) experience with the iqphvc role and its regulations about reporting adrs of biopharmaceutical medications, and 4) pharmacist recommendations to enhance medication safety of biopharmaceutical medications in public hospitals. the interview schedule started with introducing the researcher and the research objective. the inclusion question was:" do you have experience with biological/ biosimilar medications? (if they answered no, the interview was discontinued). ethical consideration verbal consent was obtained from the participant before launching the interviews. the participation was voluntary and the interview recording was optional. the interviewees were anonymous (without names) to keep participants confidentiality. no incentive was offered to the participants. the study received ethical approval from both the central scientific committee at the university of baghdad college of pharmacy and the ethical committee at the moh before starting data collection. quantitative source the iqphvc shared with the researchers the reports of adrs received from 2014 through april 2021. these reports were mainly submitted from healthcare providers (hcps) at public hospitals across the country to the iqphvc, ministry of health (moh). the adr data from the iqphvc reports were used as a source of comparison between the reported biopharmaceutical-adrs and the pharmacists' perceptions about adrs and the reporting process. results the qualitative (interviews’) findings the study interviewed 25 pharmacists from 10 governmental hospitals in baghdad. the participating pharmacists included 21 women and four men. table 1 shows the demographic and professional characteristics of the participating pharmacists. iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 75 table 1. demographics characteristic of pharmacists. code gender degree professional title years of experience workplace ph1 female bs pharm chief pharmacist, outpatient pharmacy 19 years 1. al-kadhemia teaching hospital ph2 female bs pharm practicing pharmacist/ internal pharmacy 5 years 2. al-amal oncology hospital ph 3 female bs pharm trainee pharmacist 1.5 year 3. yarmouk teaching hospital ph 4 female bs pharm practicing pharmacist 5 years al-amal oncology hospital ph 5 female bs pharm resident pharmacist 3 years al-amal oncology hospital ph 6 female master clinical pharmacology specialist clinical pharmacist, pharmacovigilance 10 years yarmouk teaching hospital ph 7 female bs pharm trainee pharmacist 1.5 years yarmouk teaching hospital ph 8 female bs pharm practicing pharmacist 5 years 4. kidney diseases and transplant center/ medical city ph 9 female bs pharm trainee pharmacist 1.5 years 5. gastroenterology and hepatology hospital /medicine citybaghdad ph 10 male master clinical pharmacy specialist clinical pharmacist 5 years gastroenterology and hepatology hospital /medicine citybaghdad ph 11 female bs pharm practicing pharmacist, pharmacovigilanceا,ou tpatient pharmacy 4 years 6. oncology teaching hospital/ medical city ph 12 female bs pharm practicing pharmacist, internal pharmacy 6 years kidney diseases and transplant center/ medical city ph 13 male bs pharm practicing clinical pharmacist/pharmaco vigilance 6 years 7. al-kindi teaching hospital ph 14 female bs pharm trainee pharmacist 2 years kidney diseases and transplant center/ medical city ph 15 female msc in pharmaceutical chemistry specialist pharmacist 8 years yarmouk teaching hospital ph 16 female bs pharm practicing pharmacist 6 years kidney diseases and transplant center/ medical city ph 17 female bs pharm chief pharmacist 16 years oncology teaching hospital/ medical city ph 18 female phd. clinical pharmacy specialist pharmacist 12 years oncology teaching hospital/ medical city ph 19 female master in clinical pharmacy specialist clinical pharmacist 9 years yarmouk teaching hospital ph 20 female bs pharm trainee pharmacist 1 year kidney diseases and transplant center/ medical city ph 21 female bs pharm practicing pharmacist, pharmacovigilance 14 years 8. dr. saad al watri hospital for neurosciences ph 22 female bs pharm practicing clinical pharmacist 10 years 9. ibn al haytham hospital for ophthalmology ph 23 female bs pharm practicing pharmacist pharmacovigilance , 9 years ibn al haytham hospital for ophthalmology ph 24 male bs pharm clinical pharmacist 8 years baghdad teaching hospital ph 25 female bs pharm chief pharmacist, practicing clinical pharmacist 7 years 10. baghdad teaching hospital bs pharm=bachelor of pharmacy. practicing pharmacist=done with rotation, training (at least has 3 years of experience). trainee pharmacist=within the first 3 years of employment. iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 76 the effectiveness of reference biological medications there was a general agreement among the participating pharmacists that reference biological medications are effective and give good results according to their indications. most participating pharmacists had adequate experience with reference biological medications. they treat many diseases, including different types of cancers, inflammatory bowel diseases, autoimmune diseases, retinal diseases, and renal diseases. "biological medicines are very effective in decreasing disease symptoms and making patients more committed to treatment because they receive treatment in hospital before they return home" (ph. 9). “they are effective drugs and used in many diseases like multiple sclerosis (ms), gut disease, rheumatology arthritis (ra). most patients have a good response to biological treatment” (ph-24). the adverse reactions of reference biological medications most of the participating pharmacists confirmed that reference medications have manageable adverse effects such as allergic reactions and rash at the injection site. the pharmacists believed that no serious adverse effects had been reported with biological medicines used in various disciplines when used with caution, proper indications and patients are follow-ups by physicians and pharmacists. “no adverse events associated with biological medications; they are generally safe” (ph 7). “[they have] well-tolerated adverse events” (ph 18). “mabthera (retuximab) can cause allergic reactions in the administration site”(ph 14). "mabthera (rituximab) can cause an allergic reaction, and gilenya (fingolimod) can cause severe hypotension"( ph 24). dispensed biosimilar medications fifteen out of 25 of the participating pharmacists dispensed biosimilar medications like remsima (infliximab), bevacizumab (suivant), aryotrust (trastuzumab), retacrit (epoetin alfa epbx). according to the availability, not all hospitals had biosimilar medications. the pharmacists preferred biosimilar medications licensed by the u.s. food and drug administration (fda) or european medical agency (ema). it is worth mentioning; the majority mentioned that biosimilar medicines were available for a short period in their hospitals. “ yes, i have approximately 3 months experience with remsima (infliximab)” (ph 3). “ yes, [i have experience with ] eprex (human erythropoietin ), and retacrit (epoetin alfa epbx )” (ph 16). “yes [i have experience with] aryotrust (trastuzumab), and stivant (bevacizumab)”(ph 17). “the biosimilar medicine (bevacizumab) was available for two weeks only in the hospital, and not all doctors agreed to dispense it because it was bevacizumab (stivant) for iranian company (cinnagen) and did not have fda/ema approval” (ph 4). effectiveness of biosimilar medications compared to biological medications the pharmacists' perceptions regarding the effectiveness of biosimilar medicines widely varied. the majority (10) of the pharmacists believed that reference biological medicines are more effective than biosimilar medicines. at the same time, the rest either had not enough experience with biosimilars (10) or believed they are comparable (3). only two pharmacists believed that biosimilar medications were more effective. “biological medications are more effective than biosimilar medications” (ph 2, ph 4, ph 5, ph 10, ph 12, ph 14, and ph 24). “there is no comparison because the effectiveness of biosimilar drugs is much lower than biological drugs; according to the lectures held in the hospital, after the fifth dose of the biosimilar treatment for patients, a telescope was made for them, and no evidence for mucosal healing” (ph 9). adverse reactions of biosimilar medications compared to their biological counterparts the pharmacists' perceptions regarding the adrs of biosimilar medicines widely varied. the majority of the pharmacists have not had adequate experience with biosimilar medications due to their short availability. a pharmacist who had 12 years of experience and practice in oncology teaching hospital, expressed: “the biosimilar was available for the insufficient period, and all patients took only one dose, and some of them did not take any dose, so i cannot compare" (ph. 18). some pharmacists believed that biosimilar medicines either have the same or more adverse reactions compared to the originators. “both have same side effects” (ph-12). “biosimilars have more adverse events” (ph-11) documentation inpatient medical records varied among hospitals. the availability of adequate data about the biopharmaceutical medicines inpatient medical records has varied among hospitals. six departments at different hospitals had adequate documentation about the effectiveness and adrs of biopharmaceutical medicines. only one hospital (for ophthalmology) had electronic health records. in contrast, there was no adequate documentation in the other five departments/hospitals. iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 77 “the patient medical records in the hospitals cannot give enough information to measure the effectiveness and safety of biological or biosimilar medicines"(ph 26). “there is inadequate documentation about the biopharmaceutical medicines which is insufficient to measure the effectiveness and adverse reactions of these medicines. there are a small number of biopharmaceutical doses given for a short period and not enough to be used for evaluation” ( ph 18). interchangeability between biological and biosimilar medications relies on availability. the pharmacists either disagreed with interchangeability between reference biological and biosimilar medications or agreed, depending on the availability. the common theme was the switching by the prescriber can be recommended in case of unavailability, which frequently happens in public hospitals. thus, the switching (by a physician) was not optional in most cases. a pharmacist 5 years of experience and practicing in al-amal national hospital for oncology, disagreed with the interchangeability when the biosimilar medicines do not have u.s. fda approval. “i disagree if the biosimilar medications do not have fda approval such as bevacizumab (suivant) of cinnagen company (iran), but i agree if the biosimilar medications have fda approval and the same effectiveness of biological medications”( ph. 4) “i agree according to the medicine availability because patients suffer from severe anemia (hemoglobin level reaches 3 g / l), so i prefer giving patients the available treatment rather than keeping them untreated” (ph 8). “no, because the safety and the efficacy of the products cannot be tracked if the patient switches many times between biological and biosimilar products” (ph 25). the future role of biosimilar products to optimize therapy most pharmacists supported the use of biosimilar medicines and their potentially positive role in their field future, particularly those having international certifications. eleven pharmacists said “yes” and six said “maybe” for the future role of biosimilar medicines. “the use of biosimilar medications depends on their manufacturer" (ph 18). “if a biosimilar medication works well and with a good price, it can invade the field and if it is less effective, it cannot” (ph 12). “it depends on the prescriber’s experience with the biosimilar” (ph 26). under-reporting of biopharmaceutical adverse reactions to the iqphvc according to the interviews, only four out of 25 pharmacists reported adrs of biopharmaceutical medicines. most of the interviewees believed there are no serious adrs associated with biopharmaceutical medicines. in general, there was underreporting of biopharmaceutical adrs to the iqphvc, which mainly relies on healthcare provider (hcp) reporting. “ no, i have not reported adverse effects because they are reversible and do not occur with all patients, and there is a pharmacist responsible for drug monitoring, and these are his duties” (ph 25). “no, i did not write any report on the adverse events of biological or biosimilar medications because there is a pharmacist in charge for these tasks” (p 17). most pharmacists relied on a medication safety pharmacist who is responsible for reporting any adrs to the iqphvc. “there is a pharmacist dedicated to making reports on the adverse events of medications linked to the iraqi pharmacovigilance center” (ph 18). “yes, i reported adrs of biologics medications like rituximab (mabthera) related to roche company” (ph 8). pharmacist unawareness of the iraqi pharmacovigilance center (iqphvc) regulations about reporting biopharmaceutical adrs most pharmacists were unaware of the iqphvc regulations. indeed, only 7 out of 25 pharmacists were aware of iqphvc regulations. hence, most pharmacists (20) have not changed their adr reporting behavior after the 2019 iqphvc reporting regulations. therefore, some pharmacists who were aware of the iqphvc regulations have not changed their reporting behavior. “ i am unaware of the iqphvc recent regulation about reporting adrs of biological and biosimilar medications” (ph 1). “i am not aware of the recent regulations of the moh about reporting adrs of biological medications, but i do not send any report before referring it to our division official who determines the report ’s format according to the letter received from the iqphvc”( ph 9). it seems that the current regulations of the iqphvc did not reach most of the participating pharmacists. the participants believed that the iqphvc does not have an influential role in most iraqi hospitals related to reporting the adverse reactions of biopharmaceutical medicines since most hcps were not aware of its reporting regulations. iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 78 barriers to reporting biopharmaceutical adverse reactions to the iqphvc the majority (19) believed no barriers are preventing adr reporting. "no, there are no barriers, but no adverse events to report" (ph 19). however, some pharmacists listed a few barriers to report biopharmaceutical adrs such as inadequate time and high workload. the main barriers were the over workload and lack of time. ph 16, who had 6 years of experience practicing in kidney diseases and transplant center, medical city, revealed: “yes, there is no enough time for preparing all reports of adverse drug events" (ph 16) “yes [there are barriers], the website is down for now” (ph 10). the pharmacist recommendations to the iqphvc about reporting biopharmaceutical ades the pharmacists had some recommendations for the iqphvc, such as following up with hospitals, providing education to pharmacists, and adopting electronic means for adr reporting. “the iqphvc should follow up with the centers to ensure the pharmacists understanding about their recommendations for biological / biosimilar medicines in order to report properly any adr” (ph 25). “conducting courses for pharmacists about biological and biosimilar medications in order to monitor their effectiveness and side effects” (ph 6). “providing means of communication via any quick electronic means in order to send reporting adrs from health institutions to the iqphvc and to receive instructions or notifications from the iqphvc to health institutions” (ph 15). most of the participating pharmacists were unaware of the iqphvc functions and regulations due to a lack of contact between health institutions and the center. consequently, they under-report biopharmaceutical adrs. the pharmacist recommendations to the moh about procuring biopharmaceutical medications most pharmacists recommended that the moh should provide reference biological medicines to public hospitals in a sustainable manner to avoid treatment interruption. additionally, the moh should consult the prescriber (physician) before replacing references with biosimilar medicines. this is because procuring biosimilar medicines to replace reference biological medicines is not always acceptable by physicians from an effectiveness and safety point of view. “providing an adequate and large quantity of expensive biological medicines for patients because most patients cannot buy medicines to avoid interruption of treatment for the patient such as avastin (bevacizumab)” (ph 7). “i prefer the moh to buy biological medications, especially the simulect (basiliximab) that belong to novartis company because it has very good and effective results.” (ph 8). buying biological medications in large quantities in order to cover the largest possible number of patients. for example, avastin is used for metastatic cancer, and mabthera is used for lymphoma" (ph 15). "the moh should take the prescribers' opinion before buying the biosimilar in order not to switch from the biological to the biosimilar suddenly for all patients" (ph 25) because the pharmacists had little experience with biosimilar medications, most participating pharmacists were afraid of a biosimilar. however, most pharmacists agreed to use biosimilar medications if the fda or ema has approved them or are from reputable international companies. the pharmacist recommendations for physicians to enhance the medication safety of biopharmaceutical medications the participating pharmacists recommended that physicians following up with patients taking biopharmaceutical medicines to monitor adrs. monitoring can be done by ordering lab data before and after prescribing biopharmaceutical medicines. follow-up, the adrs of biopharmaceutical medications, is critical to enhancing patient safety. some pharmacists recommended that physicians should educate patients about the common adrs. e “physicians should follow-up with a patient after each [biopharmaceutical] dose, and it is preferable to have a physician present during treatment, especially the first dose"(ph 9). “physicians should order liver function test (lft), kidney function test (kft), complete blood culture (cbc), x-ray before prescribing biopharmaceutical medications” (ph 9). “physicians should inform patients about the common adrs since they will be in touch with patients in the first and follow-up visits” (ph 21). iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 79 the participant recommendations for hospital pharmacists to enhance the medication safety of biopharmaceutical medications the participating pharmacists had different recommendations for hospital pharmacists to enhance medication safety. they recommended promoting pharmacists' knowledge about biopharmaceutical adrs, following up on the adrs in patients taking biopharmaceutical medicines, and educate patients about their adrs. “the pharmacists should have all information about the safety of medications and reporting any side effects” (ph 5). “organizing a follow-up form for patients who take biological or biosimilar medications after taking each dose to follow up on the adverse events of treatment, if any, as well as the effectiveness of the treatment for comparison and transferring information to physicians"(ph 9 ). “follow up and educate patients about the effect and adverse effects” (ph 2). quantitative findings the biopharmaceutical-adrs reported to the iqphvc from 2014 through april 2021, the iqphvc received 282 reports of adrs about 11 biopharmaceutical medicines; 43% of them were serious (figure 1a). the most common adrs were related to five biopharmaceutical medicines: infliximab (65, 23.0%), rituximab (59, 20.9%), interferon (56, 19.9%), etanercept (48, 17.0 %) and trastuzumab (34, 12.1%) (table 2). the adrs afflicted more females (62%) compared to males (38%). the reports of the biopharmaceutical adrs have not specified whether the causative drugs were reference or biosimilar medicines. patients were aged 18-44 years and 45 – 64 years had the highest adr experience rates, 53.2% and 30.2%, respectively (table 3). the adrs led to different levels of consequences: medically important conditions (34.8%), prolonged hospitalization (29.5%), life-threatening conditions (22.0%), disabling (7.6%), and death (6.1%)(figure 1b). pharmacists (41.7%) and physicians (26.5%) were the most common reporters of biopharmaceutical-adrs to the iqphvc (figure 1c). the most common reported adrs: general and administration site disorders (20.4%), cns disorders (11.2%), skin disorders (11.0%), respiratory disorders (10.5%), and gastrointestinal disorders (6.5%)(table 4). the last three years had the highest number of adr reports: 2018 (64), 2019 (114), and 2020 (61) (figure 1d). table 2. the number of adrs reported to the iqphvc regarding each biopharmaceutical medicine biopharmaceutical n % 1 infliximab 65 23.0 2 rituximab 59 20.9 3 interferon 56 19.9 4 etanercept 48 17.0 5 trastuzumab 34 12.1 6 epoetin alfa 8 2.8 7 adalimumab 7 2.5 8 pertuzumab 2 0.7 9 bevacizumab 1 0.4 10 binocrit 1 0.4 11 erythropoietin 1 0.4 total 282 23.0 table 3. the patient and the reported biopharmaceutical-adr characteristics character subcategories n % gender male 98 38.0 female 160 62.0 age categories 0-27 days 1 0.4 2-11 years 4 1.7 12-17 years 7 3.0 18-44 years 125 53.2 45 – 64 years 71 30.2 65 – 74 years 22 9.4 ≥ 75 years 5 2.1 iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 80 table 4. the reported adverse drug reactions to the iqphvc for each biopharmaceutical medicine from 2014 through april 2021 adalimumab bevacizumab binocrit erythropoietin etanercept epoetin alfa infliximab interferon pertuzumab rituximab trastuzumab total general and administration site condition 4 1 12 3 11 36 20 5 92 cns disorders 19 19 9 5 52 skin disorders 1 10 1 4 4 1 19 8 48 respiratory disorders 5 1 4 4 1 19 14 48 gastrointestinal disorders 1 1 1 9 9 1 8 4 34 immune system disorders 2 2 1 10 9 1 25 infections 10 2 4 4 2 22 musculoskeletal disorders 2 6 8 3 3 22 investigations 1 6 1 2 2 1 7 20 injury and procedural disorder 5 3 1 9 1 19 iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 81 continued table 4. note: one adr report may include more than adrs for the same patient due to biopharmaceutical medicine adalimumab bevacizumab binocrit erythropoietin etanercept epoetin alfa infliximab interferon pertuzumab rituximab trastuzumab total cardiac disorders 1 1 1 2 4 9 18 psychiatric disorders 1 7 7 15 vascular disorders 1 1 1 6 9 renal disorders 1 4 2 1 1 9 eye disorders 3 1 3 1 8 hepatobiliary disorders 2 1 1 4 reproductive system 1 1 1 3 blood and lymphatic disorders 1 1 1 3 surgical 1 1 iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 82 figure 1. the descriptions of the reported biopharmaceutical adrs to the iqphvc a. the seriousness of the reported biopharmaceutical-adrs b. the percentages of adr consequences c. the percentages of biopharmaceutical adr reporters d. the number of reported adrs to the iqphvc across several years discussion using two sources of information in this study helped integrate the interviews' findings, and the adr reports from the iqphvc. the study compared the reported adrs to the iqphvc and the pharmacist perceptions about the biopharmaceutical adrs and reasons for the under-reporting process. the study findings can also help assess the effectiveness of the iraqi pharmacovigilance center (iqphvc) regulations regarding biopharmaceutical medications. since the reporting of adrs is voluntary and mainly relies on healthcare providers (hcps), both pharmacist interviews and the iqphvc data showed under-reporting over the last several years. most participants in the interviews were female pharmacists because they represent most pharmacy college students and, consequently, most pharmacists in public hospitals (22). iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 83 effectiveness of reference biological medications most participating pharmacists had adequate experience with reference biological medications since they were available for several years. the pharmacists believed that their effectiveness depends on the use within the approved indications and have manageable adrs if patients take precautions. similarly, a study in the u.s. found that biological medications are helpful in the management of a variety of illnesses. however, their costs, which are typically higher than smallmolecule drugs, strain the healthcare system. (23). adverse reactions of the reference biological medications the participants indicated that reference biological medicines have manageable adrs. however, the iqphvc reports showed some serious and fatal biopharmaceutical-adrs. on the other hand, a study in columbia found comparable biological adr reports to the iqphvc with respiratory system disorder (16.8%), skin disorder (15.6%), body -general disorder (10.3%), and gastrointestinal disorder (7%)(24). a brazilian study found that more than 43.0% of patients with rheumatoid arthritis and psoriatic arthritis taking biological medicines had one or more adverse effects, with an average of 1.6 events per patient. additionally, 25% of them had one or more serious adrs, with the majority requiring hospitalization (25). thus, pharmacists should follow up reports about adrs of biological medications. effectiveness of biosimilar medications compared to biological medications. most pharmacists needed to have more experience with biosimilar medicines by making them permanently available in hospitals. similarly, a study conducted in poland found that 87% of hospital pharmacists believed that the essential benefit of biosimilars is that they are less expensive compared to reference medicines. however, 88% of them were concerned about biosimilar medicines in terms of immunogenicity and pharmacokinetics. (26). similarly, the u.s. health leaders and stakeholder representatives believed that the lack of long-term data on biosimilar medicines could deter their use despite the potential benefits of cost-saving (23). adverse reactions of biosimilar medications compared to their biological counterparts most participants had inadequate experience with biosimilar medicines due to short term of use. in contrast, a polish study found that 65.6% of hospital pharmacists were very familiar with biosimilars, whereas 32.8% were somewhat familiar with these medications (26). again, the moh should provide biopharmaceutical medicines in sustainable supply to avoid treatment interruptions and help hcps assess their effectiveness and safety. the hospital patient medical records in general, there was inadequate documentation in public hospitals, which is probably because most hospitals do not have trained physician assistants to help in medical data entry and a high workload on physicians. in other words, medical records need skilled data entry personnel who are not available in almost all iraqi hospitals. additionally, most iraqi public healthcare settings do not implement electronic health records(20). in the united states, several existing systems allow for post-marketing pharmacovigilance surveillance of biopharmaceutical medicines, including voluntary adr reporting, the fda's sentinel system, and the academy of managed care pharmacy's (amcp) edossier system. the sentinel system is a national electronic system for monitoring the safety of fdaregulated medical products, including biopharmaceutical medicines. the amcp's edossier system is a web-based tool that provides qualified health care decision-makers the opportunity to quickly review, evaluate, and compare products to make an evidence-based evaluation (23). in iraq, post-marketing pharmacovigilance surveillance relies on voluntary reporting from hcps and patients (27). since the documentation is critical to follow up with the safety and effectiveness of the medications, our hospitals or health officials should work to have adequate documentation about biopharmaceutical medications. interchangeability between biological and biosimilar medications for the same patient in iraqi hospitals, the interchangeability between biological and biosimilar medications is recommended when biological medicines are not available in hospitals to avoid leaving patients without treatment. according to a european article, if interchangeability between reference biological and biosimilar medicines having the same active ingredient is done according to the eu legislation, the switching does not expect to trigger immunogenicity (28). the future role of biosimilar products to optimize therapy the majority expected that biosimilar medicines will enhance treatment options by introducing these affordable cutting-edge medicines likewise, a study in the u.s. concluded biosimilars have great opportunities in the future, but they need a long time to ensure their wide availability in the market. they firmly believe that understanding the concept of biosimilars is crucial for oncologists(29). the number of biosimilar drugs is expected to increase in the coming years due to the economic situation and the patent expiration for several biological drugs. iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 84 under-reporting of biopharmaceutical adrs among the hospital pharmacists adverse drug reaction (adr) reporting is essential to enhance medication safety and helps identify rare adrs (5). the iqphvc is responsible for post-marketing surveillance for all medicines in both public and private sectors, which relies mainly on hcp reporting (30, 31). in 2019, public secondary and tertiary healthcare facilities served 62,958,897 outpatient visits (20, 21). however, the reported pharmaceutical-adrs over seven years were 282 cases only; pharmacists submitted 126 reports (18 reports per year) across 15 provinces. the iqphvc received the highest number of biopharmaceuticaladr reports in 2019, probably because the iqphvc sent an official letter to all public healthcare settings emphasizing the reporting of adrs and requiring the name of the biopharmaceutical company. therefore, yearly routine reminders from the iqphvc can encourage healthcare facilities to submit their adr reports. similarly, there was a noticeable underreporting of biopharmaceutical adrs among the participating pharmacists. the participating pharmacists claimed there were no serious adrs to report. in fact, the iqphvc reports showed 133 serious biopharmaceutical-adrs over the last seven years. most pharmacists had the misconception that they do not have to report non-serious adrs. some pharmacists indicated they do not have to report adrs because a safety pharmacist (focal point) connects with the iqphvc. although there is such a safety pharmacist in each hospital, all other pharmacists should notify him/her about any adr incidents in their wards (30). therefore, both interviews and the iqphvc data showed the underreporting of biopharmaceutical adrs. according to a systematic review of 37 studies from 12 western countries, the median under-reporting rate was 94% (interquartile range 82–98%) [12]. factors contributing to underreporting among hcps include inadequate awareness, negative attitudes, lack of time and motivation, uncertainty about the real cause of adrs, and difficulty accessing the reporting form [12, 13]. to increase the safety of biopharmaceutical medicines, adrs should be reported. to facilitate reporting of biopharmaceutical adrs to the iqphvc, training courses for pharmacists and electronic reporting systems are needed. furthermore, the pharmacovigilance center in every hospital should take a more significant role in following up the circulation of any regulations about reporting adrs to all departments. the pharmacist recommendations to the moh about procuring biopharmaceutical medications most of the 10 public hospitals in baghdad suffered from interruptions in the availability of treatment, whether biological or biosimilar medications or both for periods, which negatively impacts patient clinical outcomes. additionally, the procuring of medicines should rely on medical costs and physician experience-based recommendations. the pharmacist recommendations for physicians to enhance biopharmaceutical safety despite the effectiveness of biological drugs in treating many diseases, they have adrs, similar to other medications. severe adrs may become dangerous if the patient is not monitored and treated. surprisingly, several pharmacists indicated that patient adr monitoring and followup are the responsibility of physicians. that means they did not admit these are sharing responsibilities between pharmacists and physicians. the participant recommendations for hospital pharmacists to enhance the biopharmaceutical safety one of the crucial roles of the clinical pharmacist is to monitor adrs of treatment and notify the physicians if any adrs occur. hospital pharmacists should also review prescribed medicines to identify any potential drug-drug interactions (32). furthermore, any adrs of the biosimilar or biological medications need to be reported to protect the patient's life and avoid these symptoms from occurring with other patients. the study had some limitations. the quantitative component covered two parts which were the biopharmaceutical safety and reported adrs. the qualitative phase was limited mainly to one province (baghdad), which has the largest hospitals in the country regarding biopharmaceutical medicines availability. additionally, most pharmacists declined audio-recording of the interview. conclusions most of the 10 public hospitals in baghdad have been experiencing non-sustainable supplies of biosimilar medicines. inadequate recording of the biopharmaceutical effectiveness and safety information in the patient medical record was common. most participating pharmacists had adequate experience with the reference biological medications and believed they are effective with manageable adrs. in contrast, most pharmacists had a short experience with biosimilar medications and were unsure about their effectiveness and safety. most pharmacists preferred reference biological over biosimilar medicines because of their effectiveness. however, they believed that initial prescribing and switching between a reference and counterpart biosimilar relies on their availability in public hospitals. they preferred biosimilars with the fda or ema approval over other biosimilar medicines. both pharmacist interviews and the iqphvc data showed under-reporting of biopharmaceutical adrs. an electronic application can be implemented to send the adr reports quickly and easily. iraqi j pharm sci, vol.31(1) 2022 pharmacist experience with biopharmaceutical medicines 85 additionally, the iqphvc should send regular notifications to encourage healthcare facilities to report their adr incidents. medicine procurement in healthcare settings should focus on securing biopharmaceuticals having the fda or ema approval in sustainable supply rather than looking only at costs to avoid treatment interruptions and enhance patient clinical outcomes. providing pharmacist training, electronic reporting, promoting documentation, and following up on biopharmaceutical effects are pivotal to prevent, monitor, and treat biopharmaceutical adrs. acknowledgment the authors appreciate the help of dr. manal m. younus in providing the iqphvc adr reports and recruiting some participants. the authors would also thank all the participating pharmacists for sharing their experiences. references 1. ebrahim gj. pharmaceutical biotechnology: concepts and application gary walsh (ed). journal of tropical pediatrics. 2008;54(3):214. 2. santos sb, sousa lobo jm, silva ac. biosimilar medicines used for cancer therapy in europe: a 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http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 lavender oil and olive oil massage https://doi.org/10.31351/vol30iss1pp163-168 163 comparing the effects of lavender oil and olive oil massage on pain due to muscular cramp during hemodialysis mohsen saeedi abo-s-haghi *, asad imani **,1 mohammad alidadi***3 elham shafiei**** *boroujen faculty of nursing, shahrekord university of medical sciences, shahrekord, iran **department of nursing, faculty of nursing and midwifery, ilam university of medical sciences, ilam, iran ***shohaday lordegan hospital, shahrekord university of medical sciences, shahrekord, iran ****clinical research development unit, shahid mostafa khomeini hospital, ilam university of medical sciences, ilam, iran abstract pain due to muscular cramp during hemodialysis is one of the most common problems experienced by patients undergoing hemodialysis, and is associated with poor outcomes of patients. the main aim of this study was to compare the effects of lavender oil and olive oil massage on pain due to muscular cramp during hemodialysis. in this randomized clinical trial, 60 hemodialysis patients were selected randomly and the samples were randomly divided into two groups of 30.. the intervention included flora massage on the lower leg muscles so that the first group received lavender oil massage (10 drops) and the second group received olive oil massage (10 drops) for four weeks. massage duration was 5 minutes and three times within hemodialysis sessions. the collected data was analyzed in spss (v.22) using repeated measure anova. pain due to muscular cramp in the lavender group was significantly less than that of the olive oil group in the 2nd (p<0.001), 3rd (p<0.001), and 4th (p<0.001) weeks of intervention. pain due to muscular cramp in hemodialysis patients can be attenuated with lower leg massage using olive oil and lavender oil. lavender oil was more effective than olive oil. keywords: massage, hemodialysis, lavender oil, olive oil, pain introduction more than 80 per cent of patients with chronic kidney disease use hemodialysis for treatment (1). in patients, pain due to muscular cramp during hemodialysis is one of the debilitating and common symptoms with a prevalence rate of 50%– 60% (2). the chronic and debilitating nature of cramp pain decreases self-care measures and increases dependence on health care for daily activities (3). literature review showed that limb ischemia is the common cause of muscular cramp pain (4). muscular cramp pain control in hemodialysis patients includes pharmacological and non-pharmacological approaches. since, the majority of methods are pharmacological and the majority of pharmacological metabolites are discharged by the kidneys, these methods are featured with risk of kidney intoxication (5). therefore, popularity of complementary and none-pharmaceutical treatments has been increased in health systems (6). one of the non-pharmacological methods of preventing and alleviating pain is massaging that attenuates pain through lowering muscle sensitivity and tone and improving blood flow that leads to attenuation of pain and relaxation (7). although there are some previous studies about the effect of massage with olive oil or lavender oil on outcomes of patients in different diseases (8-10), but there is no earlier study on comparing the effects of lavender oil and olive oil massage on pain due to muscular cramp in hemodialysis patients. there is lavender contains linalool, ketone alcohol, asters, and aldehyde. ketones in lavender are effective in alleviating pain and inflammation. asters prevent muscle spasm and lower tension (11). on the other hand, olive contains antioxidants that improves cells’ resistance to oxidation and in return increases perfusion and lowers pain and fatigue (12). the main aim of this study was to compare the effects of lavender oil and olive oil massage on pain due to muscular cramp during hemodialysis. methodology study design and participants this study was a clinical trial with two groups. 60 patients were selected using random allocation software. after justifying the patients to participate in the study and obtain written informed consent; the samples were randomly divided into two groups of 30. 1corresponding author e-mail: imani.nursing@gmail.com received: 28/9/2020 accepted: 14/11 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp163-168 iraqi j pharm sci, vol.30(1) 2021 lavender oil and olive oil massage 164 first group, massage with lavender oil, lavender oil massage was used with 10 drops of 1/5% lavender oil. the second group, was used with 10 drops of olive oil. prior to the intervention, the baseline assessment was performed for two weeks and during this phase, the demographic information questionnaire as well as the pain severity and frequency of cramps were recorded. the research setting is the dialysis wards of lordegan and borujen hospitals (affiliated to shahrekord university of medical sciences, shahrekord, iran). in this study, the participants in the study (patients), nurses working in the hemodialysis departments and the statistical advisor (data analyst) were kept blind. the sample size at 95% confidence level and 80% test power was calculated 54 patients according to below formula, considering probability of attrition of the samples, the maximum sample size was 60 cases in total and 30 in each group. 𝜎1 2 = 𝜎2 2 = 3.8 𝛿=𝜇2−𝜇1 = 2.5 n=total 60 and 30 in each group 𝜎𝐴 2 = 𝜎𝐵 2 = 𝜎 2 𝑛 = (𝜎 2 + 𝜎 2)(𝑧 1− 𝛼 2 + 𝑍1−𝛽 ) 2 𝛿 2 𝑛 = 2𝜎 2(𝑧 1− 𝛼 2 + 𝑍1−𝛽 ) 2 𝛿 2 𝜑 = 1 three participants left the study due to immigration (one patient), reluctance to continue the study (one patient), and traveling (one patients). (fig.1) inclusion and exclusion criteria inclusion criteria: 1. chronic kidney failure with at least one year of hemodialysis history 2. age range between 30 and 60 years 3. having physical and mental ability 4. lack of cutaneous lesions, scarring and limb redness exclusion criteria: 1. catabolic diseases such as cancer 2. diabetic neuropathy 3. use of pain relieve medicines, hypnotics, narcotic and psychotropic drugs 4. lower limb skeletal disorders, neuromuscular disorders and arthritis 5. alcohol abuse 6. allergy to lavender or olive oils. figure. a flow diagram of the protocol intervention flora massage was performed on the lower leg muscles so that the first group received lavender oil massage (10 drops) and the second group received olive oil massage (10 drops) daily. they are popular, accessible and commonly used among iranian populations, but probable allergy to lavender oil or olive oil was checked by using them on the subjects 'arm. the subjects were checked for any skin allergy after two weeks. none of the subjects demonstrated skin allergy symptoms. the intervention continued for four weeks and during each hemodialysis sessions. massage duration was 5 minutes and three times: one hour after dialysis, two hours after dialysis, and half an hour to the end of dialysis-most cramps occur at this time-(15 minutes total). in this study, all patients need hemodialysis session 2 or 3 times weekly that distributed into groups randomly. we used a female colleague to massage female participants due to cultural restrictions. therefore, male and female participants received massage by different individuals. to compensate, the assistants practiced the massage on a model to made the massages as identical as possible. iraqi j pharm sci, vol.30(1) 2021 lavender oil and olive oil massage 165 the visual analog scale for pain (vas pain) would be filled out for each patient at the end of every week. the massaging method was flora massage so that the nurses’ fingers would cover the lime by articulating a c shape and perform the massage. since the most common pain in dialysis patients happens on the back of legs, this spot was selected for performing the massage (13). instruments data gathering tool including a demographics form and the vas pain were distributed among the participants. the demographics form included age, gender, marital status, education, history of hospitalization, and hemodialysis treatment duration. the vas pain consists of a straight line from 0-10 with the endpoints defining extreme limits such as zero score or ‘no pain’ and ten score or ‘worse pain’(14). this tool is scored by patients and the level of pain is interpreted as following, 0-3 score means ‘mild pain’, 4-7 score means ‘moderate pain’ and 8-10 score means ‘sever pain’. this vas pain is valid and reliable test; simple use and accessibility are the main specifics of it (15). the collected data was analyzed in spss (v.22) and after ensuring consistency of the inserted data with the collected data, descriptive statistics (mean, sd, frequency, and percentage) and repeated measure anova, were used to analyze the data. normal distribution of the data was checked using kolmogorov smirnov (ks) test (p=0.05). ethical considerations this study was approved by ethics committee from shahrekord university of medical sciences, shahrekord, iran with an ethical code (ir.ssu.medeine.rec.1395.103). a written informed consent which was prepared based on declaration of helsinki was obtained from all participants. this study was registered in the iranian registry of clinical trials (irct20190528043741n1). results mean age of the patients participating in this study was 48.56 ± 5.66 years and mean duration of dialysis treatment was 5.45±3.11 years. the majority of participants were men (n=31; 54.4%) and 26 participants were women (45.6%). the majority of participants were married (n=52; 91.2%). there is normal distribution about demographic data between two groups (p>0.05). (table1) repeated measure test showed that pain score was significantly lower in the 2nd, 3rd, and 4th weeks in the lavender oil group compared to the olive oil group. also, there is a significant difference in vas pain score before and after the intervention between two groups. (tables 2) table 1. the demographic data for participants. variable detail of variable no (%) mean±standard deviation gender male 31(54.4%) female 26 (45.6%) marital status single 5 (8.8%) married 52 (91.2%) age ≤40 year 15 (26.64%) 48.56 ± 5.66 >40 year 42 (73.68%) duration of dialysis treatment <5 year 34 (45.33) 5.45±3.11 5-10 year 16 (28.07) > 10 year 7 ( 26.6) variable detail of variable no (%) mean±standard deviation gender male 31(54.4%) female 26 (45.6%) marital status single 5 (8.8%) married 52 (91.2%) age ≤40 year 15 (26.64%) 48.56 ± 5.66 >40 year 42 (73.68%) duration of dialysis treatment <5 year 34 (45.33) 5.45±3.11 5-10 year 16 (28.07) > 10 year 7 ( 26.6) iraqi j pharm sci, vol.30(1) 2021 lavender oil and olive oil massage 166 table 2. mean score of pain in the two groups of intervention at different times repeated measure anova test. f= 133.07, df= 4 discussion whereas the majority of hemodialysis cases occur following to chronic conditions, so patients with ages more than 50 years reported in different studies (16-18), also in the present study results showed that the mean age of participant were 48.56 ± 5.66 year. in our study, the lower leg massage using olive oil and lavender oil decreased pain in the subjects; this result is consistent with different studies. some studies examined the effect of complementary therapies on pain in hemodialysis patients. a study showed that menthol and rosemary can alleviate severity and frequency of recurrence of musculoskeletal pain in hemodialysis patients (5). in a systematic review and meta-analysis showed that aromatherapy with the use of complementary oils absorbed through the skin or olfactory system can successfully treat pain when combined with conventional treatments (19). a randomized control trial concluded that lavender aromatherapy significantly reduced pain and anxiety in hemodialysis patients (10). olive oil can be used as effective complementary oil in the treatment of pain due to constipation in patients undergoing hemodialysis (20). massage with two oils appeared effective because through lowering sensitivity and rigidness of muscles, improves perfusion and alleviation of pain. also, the increase in blood flow in the massaged spot improves circulation and remove of wastes like acid lactic, which results in energy discharge, alleviation of pain, and removal muscles spasm (13). although massage lead to muscular pain relief but several factors such as the age of patients and their underlying disorders, the duration and method of massage, patient’s psychological conditions, or the amount of oil used might be contributed to controversies on the effects of lavender or olive oil. for instance, fismer and pilkington (21) only used one drop of lavender for one night or in hashemi et al (9) study, massage followed for three weeks. however, our intervention was repeated frequently with 10 drops within each hemodialysis sessions and continued for four weeks. also, these two oils are accessible and popular in iranian population. accordingly, examining the main hypothesis of our study results showed that pain in the 2nd, 3rd, and 4th weeks in the lavender oil group was less than that in the olive oil group. lavender is a safe herb and no toxicity has been reported and it seems that lavender contains linalyl acetat and linalool with sedative effects (21). on the other hand, lavender stimulates the limbic system and releases neurotransmitters such as enkephalin, endorphins, serotonin, and in addition to creating a sense of calm and reducing anxiety, also reduces pain perception (22). this comparison between lavender oil and olive oil was done for the first time in the present study. literature review shows that there is no previous study in earlier research, for confirmation of this finding; future studies are suggested with increased sample sizes and longer interventional duration. another study showed that postoperative lavender oil aromatherapy did not significantly affect pain score (23). the findings of mentioned study are inconsistent with our study. it is necessary to note that massage duration and cycle in this study only short term at 5, 30, and 60 minutes postoperatively. conclusion pain due to muscular cramp in hemodialysis patients can be attenuated with lower leg massage using olive oil and lavender oil. in the present study, results showed that there is a significant difference between the two groups, and pain score in the lavender oil group was significantly lower than olive group. therefore, this economic, safe, and simple procedure is recommended as a complementary therapeutic approach. after intervention fourth week third week second week first week before intervention group variable 3.94±0.59 3.65± 0.69 3.96± 0.7 4.35± 0.81 4.81± 0.9 5.31 ± 0.83 massage with lavender oil pain 4.56±23 4.28± 0.8 4.4±0.86 4.61±0.82 4.98±0.86 5.35±0.88 massage with olive oil <0.0001 0.019 0.002 0.024 0.125 0.76 p value iraqi j pharm sci, vol.30(1) 2021 lavender oil and olive oil massage 167 limitation one of the limitations in the present study was the necessity of asking a female colleague to massage female participant due to cultural restrictions. and also one of the strengths of the study was the novelty approach in controlling pain in hemodialysis patients without any complications. acknowledgment we are deeply grateful for the patients who participated in this study. conflict of interest none declared references 1. complications of arteriovenous fistulas for hemodialysis. med princ pract. 2013;22(3):220-8. doi: 10.1159/000343669. 2. brkovic t, burilovic e, puljak l. prevalence and severity of pain in adult end-stage renal disease patients on chronic intermittent hemodialysis: a systematic review. patient prefer adherence. 2016;10:1131-50. doi: 10.2147/ppa.s103927. 3. jaime-lara rb, koons bc, matura la, et al. a qualitative metasynthesis of the experience of fatigue across five chronic conditions. j pain symptom manage. 2019. 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.07.009. 21. fismer kl, pilkington k. lavender and sleep: a systematic review of the evidence. european journal of integrative medicine. 2012 ;4 (4) :e436 -e47. 22. koulivand ph, khaleghi ghadiri m, gorji a. lavender and the nervous system. evid based complement alternat med. 2013;2013:681304.doi: 10.1155/2013/681304 23. kim jt, wajda m, cuff g, et al. evaluation of aromatherapy in treating postoperative pain: pilot study. pain pract. 2006;6(4):273-7. doi:10.1111/j.1533-2500.2006.00095.x. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://dx.doi.org/10.1155%2f2013%2f681304 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.25(2) 2016 renal function tests among painters in sulaimani 35 relationship between renal function tests and the levels of mda, zinc, and cadmium among painters in sulaimani zheen a. ahmed *,1 * department of pharmacology and toxicology, faculty of medical sciences, school of pharmacy, university of sulaimani, kurdistan region, iraq. abstract kidney damage in workers within environments of highly expected exposure to toxin, including heavy metals, could be a primary marker to expect hazards in population exposed to low levels of many environmental pollutants. the present study was designed to evaluate the possible effect of environmental exposure to cadmium and zinc on renal function among painters in sulaimani city. cross sectional study was performed on 37 male painting workers in sulaimani city. each worker was interviewed using structured questionnaire. twenty five non-exposed healthy subjects were included as control group. venous blood samples (10 ml) were obtained by vein puncture from both subjects and utilized for estimation of serum urea, creatinine levels, serum levels of cadmium (cd) and zinc (zn), and for the estimation of malondialdehyde. the results show that serum urea levels were significantly elevated in painters; meanwhile, no significant difference was reported in serum creatinine levels. the results indicated that both serum levels of (cd) and (zn) were not significantly elevated in painting workers; however, mda levels were significantly elevated in painters compared to control group. in conclusion, the association of cadmium and zinc levels with the disturbance in renal function is not clear in painters working in sulaimani city. keywords: painting workers, cadmium, zinc, renal function, oxidative stress العالقة بُه وظائف الكلُتُه ومحتىي الذم مه الخارصُه والكادمُىم والمالىوذَالذَهاَذ لذي عُىة مه الصباغُه فٍ السلُماوُة أورحمان أحمذزَه ،*1 * فزع األدّيت ّالسوْم، كليت الصيذلت، جاهؼت السليواًيت، كزدسخاى/الؼزاق . الخالصة هي اكثز االػضاء حضزرا بالوْاد الساهت الوْجْدة في هحيظ الؼول كالوؼادى الثقيلت لذٓ الؼاهليي في هٌِت الصباغت. خييحؼخبز الكلي ُذٍ الذراست صووج لوؼزفت هذٓ حاثيز الخؼزض لكل هي الكادهيْم ّالزًك ػلٔ فؼاليت الكلٔ لذٓ الصباغيي الؼاهليي في هذيٌت السليواًيت. شخصا كوجوْػت سيطزة بْاسطت طزيقت الوقابلت هغ اسخخذام اسخبياى خاص 52هال في هٌِت الصباغت ّ ػا 73اجزيج الذراست ػلٔ بالذراست. حن اخذ ػيٌاث هي دم الوشاركيي لغزض قياس ًسب كل هي الكزياحٌيي ّ يْريا الذم باالضافت الٔ قياس هسخْٓ الكادهيْم ّالزًك اليْريا في دم الؼاهليي احصائيا اػلٔ هي ًسبخَ في دم هجوْػت السيطزة بيٌوا ًسب الكزياحٌيي لن ّالوالًْالذيِايذ. اظِزث الٌخائج اى هسخْٓ ا. كوا ّكشفج الذراست ػي ارحفاع هسخْٓ الوالًْالذيِايذ في دم الؼاهليي هغ ػذم ّجْد حغييز احصائي في هسخْياث كل ظيظِز حغيزا هلحْ ائف ظقارًخِن هغ هجوْػت السيطزة. اسخٌخجج الذراست باًَ الحْجذ ػالقت ّاضحت بيي حخلخل ّهي الكادهيْم ّالزًك في دم الؼاهليي اثٌاء ه .في هذيٌت السليواًيت الكلٔ ًّسب كل هي الكادهيْم ّالزًك في دم الؼاهليي في هٌِت الصباغت . التأكسذٌ االجهادوظاىف الكلً ، العاملُه فٍ مهىة الصباغة ، الكادمُىم ،الزوك ،الكلمات المفتاحُة : introduction occupational kidney diseases provide models for understanding the impact of pollutants on kidney functions. kidney damage in workers within environments of highly expected exposure to toxin, including heavy metals, could be a primary marker to expect hazards in population exposed to low levels of many environmental pollutants (1,2) . the toxic effect of many heavy metals usually involves an interaction between the metal ion and specific target protein, resulting in modified protein structure and function (3) . cadmium and zinc are among the metals that have etiological role in kidney diseases. nephrotoxicity caused by cadmium has been described in settings of industrial exposure and environmental pollution. 1 corresponding author e-mail: mail: zheen.ahmed@univsul.edu.iq received:10 /7/2016 accepted:22 /11/2016 iraqi j pharm sci, vol.25(2) 2016 renal function tests among painters in sulaimani 36 cadmium, a metal ordinarily obtained as a byproduct of zinc refining, is used industrially in plating of steel, paints, plastics, alloys, and nickel-cadmium batteries, and in nuclear and electronic engineering (4-6) . furthermore zinc is an essential trace element that can cause symptoms of deficiency and can be toxic when exposures exceed physiological needs. the relationship between intake and health is affected by physiological factors (homeostasis) and by extrinsic factors that affect the availability of zinc for absorption and utilization or that interfere with the metabolism of zinc and biochemical processes that require zinc (7) . many clinical studies have confirmed the major role played by oxidative stress in renal dysfunction, and malondialdehyde (mda) has been suggested as one of the best predictors of renal damage in hemodialysis patients (8,9) . in painting workers, part of the toxicities appeared on painters could be due to the exposure to organic solvents and some heavy metals present in the paints. the possible role of organic solvent exposure in the development and/or the progression of chronic renal failure are still a controversial scientific issue (10) . accordingly the present study was designed to evaluate the possible effect of environmental exposure to cadmium and zinc on renal function among painters in sulaimani city. materials and methods this cross sectional study was performed on 37 male painting workers in sulaimani city during the period from february to june 2015. their age ranges was 20-49 years, and have work experience range of 1-15 years. each worker was interviewed using structured questionnaire that includes personal data, clinical signs and symptoms, duration of exposure to paint products and the whole time they spend in this profession. all subjects were apparently healthy at enrolment time. twenty five non-exposed healthy subjects, their age matched with that of workers, were included as control group. each subject signed informed consent before enrollment and the study protocol was approved by the local ethical committee of the school of pharmacy, university of sulaimani. venous blood samples (10 ml) were obtained by vein puncture from the workers and control group subjects; 5 ml was kept in a plain tube. after clot formation, the samples were centrifuged at 3000 rpm for 15 min to obtain the serum, which was utilized for estimation of serum urea and creatinine levels using autoanalyzer based method (11) , serum levels of cadmium (cd) and zinc (zn) using icp atomic absorption spectrophotometer (12) , and for the estimation of malondialdehyde (mda)as oxidative stress indicator (13) . statistical analysis all values were expressed as mean±s.d; statistical analysis was performed using graph pad prism software (version 6.0). unpaired student's t-test was utilized to evaluate the difference between means. pearson’s correlation was utilized to evaluate the relationship between variables. p values less than 0.05 indicated significant differences. results in figure 1, 27 (73%) of painters were cigarette smokers while 27% of them were nonsmokers. moreover, figure 1 indicates that 6 workers (16.2%) were exposed to painting products from 1 to 5 years, while 5 workers exposure time range was 6-10 years; the remaining number of enrolled workers (26; 70.3%) were exposed for more than 10 years. figure 2 shows that serum urea levels were significantly elevated (14.2%; p=0.028) compared with that reported in control group; however, these values are still within the normal range that exclude impaired renal function. meanwhile, figure 3 indicates that no significant difference was reported in serum creatinine levels compared with control (0.78 vs. 0.81 mg/dl; p= 0.28). figure (1): distribution of painters (%) according to smoking habits and duration of exposure to painting products in the work environment. iraqi j pharm sci, vol.25(2) 2016 renal function tests among painters in sulaimani 37 figure (2): changes in serum urea levels of painting workers in sulaimani city. n: number of subjects; p<0.05: significant difference compared with control group. figure (3): changes in serum creatinine levels of painting workers in sulaimani city. n: number of subjects; p>0.05: non-significant difference compared with control group. the results indicated that both serum levels of cd and zn were not significantly elevated in painting workers group compared with that reported in controls (9.8 vs. 9.9 μg/l; p= 0.64 for cd, and 2532 vs. 2502 μg/l; p= 0.27 for zn) (figures 4&5). in figure 6, serum mda levels were significantly elevated (15%; p= 0.012) in painting workers compared with that of controls. utilizing pearson’s correlation method, evaluation of the relationship between the age of workers and their mda serum levels indicates that they are poorly and non-significantly correlated (r= 0.18 and p= 0.29) (figure 7). figure (4): serum cadmium (cd) levels of painting workers in sulaimani city. n: number of subjects; p>0.05: non-significant difference compared with control group. figure (5): serum zinc (zn) levels of painting workers in sulaimani city. n: number of subjects; p>0.05: non-significant difference compared with control group. figure (6): serum malondialdehyde (mda) levels of painting workers in sulaimani city. n: number of subjects; p<0.05: significant difference compared with control group. iraqi j pharm sci, vol.25(2) 2016 renal function tests among painters in sulaimani 38 figure (7): correlation between the age of painters and their serum levels of mda. n= 37 subjects; r: pearson’s correlation coefficient. discussion occupational exposure of humans to heavy metals may predispose to long-term deleterious effects in many vital organs, including the renal function (14) . many epidemiological reports demonstrate that low environmental exposure to cadmium is associated with impaired renal functions (15) . accordingly, monitoring renal functions in highly expected subjects with environmental exposure to cadmium may be vital as occupational safety measure. among those subjects, painting workers are highly vulnerable to heavy metals toxicity, especially in inappropriate work environments, and cadmium could be potential hazard. cadmium is one of the common pollutants and it poses occupational hazards to different individuals which include painters (16) . however, the present study did not show clear evidence for the involvement of cadmium as a risk of renal impairment; however, the increase in oxidative stress marker (mda) could be an early sign of expected toxicity. moreover, the reported duration of exposure to paint products in the present study is relatively short, and may be not enough to associate with remarkable changes in renal function, even when serum urea shows significant elevation in those workers. because the biologic half-life of cadmium is long (>30 yr), prolonged low level exposure is required to produce excessive accumulation in certain tissues, especially the kidney (17) . in this regard, clinical manifestations of chronic poisoning with cadmium include nasorespiratory signs, mild anemia, tooth discoloration, osteomalacia, and occasional impairment of renal function which may not be easily remarkable due to high compensatory reserve of the kidney. meanwhile, cadmium-related nephrotoxicity is multifactorial, with potential influence of age, time of exposure, dietary and smoking habits and associated co-morbidity (18) , these factors are not fully covered in the present study which may be considered and important limitation in addition to small subjects sample involved in the study. environmental cadmium exposure occurs mostly in subjects living in proximity to potential industrial pollution (19) and also in heavy smokers (19) , since tobacco smoke has a high cadmium contents (21) . in the present study, the high prevalence of smoking habit among the included painters could be a potential risk behind the reported changes in oxidative stress marker and blood urea levels. cadmium is a redox-inactive metals that challenges antioxidant defenses by binding to thiols in many cellular components, including antioxidant defense systems (22,23) , which may be associated with disturbance in the concentrations of trace metals that have redox potential like copper and iron. moreover, the involvement of zinc in this cycle cannot be excluded, since it is potentially involved in the activity of metallothionines involve in the handling and elimination of cadmium (24) . in conclusion, within the limitations of the present study, the association of cadmium and zinc levels with the disturbance in renal function is not clear in painters working in sulaimani city. larger epidemiological study is highly suggested to cover all the associated factors in this respect. acknowledgement the author thanks university of sulaimani for supporting the project. references 1. ehrenreich t. renal disease from exposure to solvents. ann clin lab sci 1977; 7(1):616. 2. wedeen rp. occupational and environmental renal disease. semin nephrol 1997; 17(1):46-53. 3. he w, guo w, qian y, zhang s, ren d, liu s. synergistic hepatotoxicity by cadmium and chlorpyrifos: disordered hepatic lipid homeostasis. mol med rep 2015; 12(1):303-308. 4. adams rg, harrison jf, scott p. the development of cadmium-induced proteinuria, impaired renal function, and osteomalacia in alkaline battery workers. q j med 1969; 38:425-443. 5. kawasaki t, kono k, dote t, usuda k, shimizu h, dote e. markers of cadmium exposure in workers in a cadmium pigment iraqi j pharm sci, vol.25(2) 2016 renal function tests among painters in sulaimani 39 factory after changes in the exposure conditions. toxicol indust health 2004; 20(1-5):51-56. 6. nordberg gf, jin t, wu x, lu j, chen l, lei l, hong f, nordberg m. prevalence of kidney dysfunction in humans: relationship to cadmium dose, metallothionein, immunological and metabolic factors. biochimie 2009; 91(10):1282-1285. 7. barregard l, fabricius-lagging e, lundh t, molne j, wallin m, olausson m, et al. 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health 2005; 13:149-152. 20. bachelet m, pinot f, polla ri, francois d, richard mj, vayssier-taussat m, et al. toxicity of cadmium in tobacco smoke: protection by antioxidants and chelating resins. free radic res 2002; 36:99-106. 21. landsberger s, larson s, wu d. determination of airborne cadmium in environmental tobacco smoke by industrial neutron activation analysis with a compton suppression system. anal chem 1993; 65:1506-1509. 22. stohs sj, bagchi d. oxidative mechanism in toxicity of heavy metals. free radic biol med 1995; 18(2):321-336. 23. ercal n, gurer o, aykin b. toxic metals and oxidative stress. part 1: mechanisms involved in metal-induced oxidative damage. curr topics med chem 2001; 1(6):529-539. 24. nordberg g, jin t, wu x, lu j, chen l, liang y, lei l, hong f, bergdahli a, nordberg m. kidney dysfunction and cadmium exposure--factors influencing dose-response relationships. j trace elem med biol 2012; 26(2-3):197-200. iraqi j pharm sci, vol.31( 2 ) 2022 terpinen-4-ol on amastigote forms of leishmania tropica doi: https://doi.org/10.31351/vol31iss2pp233-236 233 a study of the inhibitory effect of terpinen-4-ol on amastigote forms of leishmania tropica within macrophages of mouse in vitro abdulazim hami*,1 , shaza al laham* * pharmacology and toxicology department faculty of pharmacydamascus university abstract it was recorded that terpinen-4-ol has an anti-parasitic activities, so that it can be noteworthy to intensify further studies about such compound. the present study aimed to test the effectiveness of terpinen-4-ol on amastigote forms of leishmania parasite in macrophages. the effect was studied by adding of the increasing concentrations of terpinen-4-ol in the culture wells containing mouse's macrophages that were previously incubated with the promastigote forms of the parasites for 24 hours .then, they were incubated for another 24 hours with increasing concentrations of terpinen-4-ol. parasites were enumerated into macrophages in wells either treated with terpinen-4-ol or in control wells. treatment with terpinen-4-ol at concentrations of (0.01%, 0.02%, 0.05%, 0.1%) in (v/v) decreased the viability of the amastigote forms inside macrophages (24.02%), (32.74%), (66.72%) and (100%) respectively, compared to control wells (distilled water). the present study showed the activity of terpinen-4-ol against amastigote forms of leishmania tropica in vitro with a minimal inhibitory concentration (mic50) level which was 0.0416% (v/v). keywords: terpinen-4-ol , amastigote forms, promastigote forms, macrophages, viability. المثبطة لليشمانات الليشمانيه المدارية في المختبر terpinen-4-olدراسة فعالية * و شذى اللحام1، *عبد العظيم حامي سورية دمشق، دمشق، جامعة الصيدلة، كليّة في والسموم علم تأثير األدوية قسم* الخالصة تهدف هذه الدراسة إلختبار فعالية , مما أستدعي لتكثيف الدراسات حول هذا المركب. terpinen-4-ol لـُسِجلت خواص مضادة للطفيليات ل terpinen-4-ol تمت هذه الدراسة بإضافة تراكيز متزايدة من مركب .العمعلى ليشمانات طفيلي الليشمانيه داخل البterpinen-4-ol حفر إلى -terpinen-4ساعة. بعد ذلك ُحِضنت مع تراكيز متزايدة من 24قات الطفيليات لمدة شي فأرية والتي ُحِضنت مسبقاً مع مُ العمالزرع الحاوية على ب ol على حاويةالزرع ال حفرفي مالعساعة أخرى. ثم تم تعداد الطفيليات داخل الب 24لمدة terpinen-4-ol الشاهدة. لحفروفي ا بنسبة العمالليشمانات داخل البحيوية ()حجم/حجم( إلى تناقص في %0.01 ,%0.02 ,%0.05 ,%0.1بتراكيز) terpinen-4-olأدت المعالجة بـ هذه الدراسة فعالية ثبتتأ الشاهدة التي تحتوي على ماء مقطر فقط. لحفر( على التوالي بالمقارنة مع ا %24.02, 32.74% ,66.72% ,100%) ol-4-terpinen دنى مثبطأعلى ليشمانات الليشمانيه المدارية في المختبر عند تركيز (50mic )0.0416% .حجم/حجم terpinen-4-ol.ليشمانات طفيلي الليشمانيه, مشيقات الطفيليات, بالعات فأرية و : الكلمات المفتاحية introduction leishmaniasis is a parasitic disease that is caused by leishmania tropica. it leads to intracellular infection in human after being bitten by a female of sand-fly. the insect belongs to one of more than 30 species of the heart family, which spread in the old and new world. it is considered as the main vector of the disease(1). leishmaniasis is endemic in 88 countries(2). 95% of the cases are found in south and central america, the mediterranean basin, the middle east, and central asia(3). according to its clinical forms, leishmaniasis can be divided into three main types: visceral, cutaneous and mucocutaneous(4). cutaneous leishmaniasis is one of the most important current public health problems. according to the world health organization (who), about 1.3 million new cases of cutaneous leishmaniasis are reported annually(5). in syria, the manifestations of cutaneous leishmaniasis vary from spontaneous healed lesions to permanent disfigurement(6). since 1960, cutaneous leishmaniasis cases are concentrated in aleppo and damascus. this is corresponded to the registration of 2,300 new cases annually(7). the infection rate has significantly increased by the beginning of 2014, reaching 41,000 new cases annually(8). leishmaniasis is a hetero-host parasite, that can colonize different hosts(9). the life cycle of the parasite is divided into two forms: the amastigote form and the promastigote form. the first form is found in humans, while the promastigote form occurs in the sand-fly(10) 1corresponding author e-mail: abdhami089@gmail.com received: 9/9 /2021 accepted: 23/1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp233-236 iraqi j pharm sci, vol.31(2) 2022 terpinen-4-ol on amastigote forms of leishmania tropica 234 an infected human can carry some types of leishmaniasis for a long time without showing any symptoms, and the incubation period for cutaneous leishmaniasis varies from 1-2 weeks to several months, and sometimes it takes several years(11). leishmania major (l.major) and leishmania tropical (l.tropica) are the main two species of leishmanial parasite that can cause cutaneous leishmaniasis. the incubation period is less than two months for l. major and between 2 to 8 months for l. tropica(12). australian tea tree oil (tto) has antibacterial, antiviral, antifungal activity (including yeast)(13). tto has anti-parasitic activity when applied on the skin and mucous membranes(14). studies have confirmed that the main criteria for tto are that terpinen-4-ol should be at least 30%. it has antifungal and antibacterial activity(15), therefore this study was designed to evaluate the effectiveness of this substance as an anti-parasitic agent against amastigote forms of leishmania parasite within macrophages. materials and methods preparation of the terpinen-4-ol solution a concentrated solution of terpinen-4-ol (4%) was prepared by diluting 0.04 ml of terpinen-4-ol solution (purchased from sigma) using distilled water. the final volume was 1 ml, and it was called the stock solution. the stock solution was used to prepare solutions with concentrations of (2%, 0.8%, 0.4%) v/v. adding 25 µl of the tube containing terpinen-4-ol at a concentration 0. 4% to get the final concentration of terpinen-4-ol equal 0.01% in culture wells containing 1 ml of culture medium. obtaining mouse’s macrophages six to eight week-old mice (balb/c) were used as a source of macrophage. 5 ml of rpmi-1960 medium was injected into the mouse peritoneum and then withdrew the fluid after 10 minutes. the macrophages were counted using neubauer counting chamber to determine the number of live cells in 1 ml of the previous suspension. and the number of macrophages were calculated according to the following equation: the number of macrophages in 1 ml = average number of macrophages in 4 white squares×10×1000(16). testing the efficacy of terpinen-4-ol on amastigote form of leishmania tropica inside macrophages the effect was studied by adding mouse's macrophages to culture wells. then, promastigote forms taken from a farm in the process of cultivation were added at a rate of 5 parasites per macrophage, they were previously incubated at 37co for 24 hours(16). then, terpinen-4-ol was added at final concentrations of 0.01, 0.02, 0.5 and 0.1%, while the same volume of distilled water was added to the control wells and the culture wells were incubated at 37co for an additional 24 hours. parasites were counted into 100 macrophages in each well of wells treated with terpinen-4-ol and in control wells(16). the calculation of minimum inhibitory concentration(mic50) ic50 was calculated using the linear deviation equation, where the concentrations of terpinen-4-ol were plotted against the number of amastigote forms inside macrophages on the x-y axes using the same statistical program and the equation were as follows: y= a× x+ b(16). statistical analysis statistical analysis was performed using graph pad prism, version 8.0. anova test to compare more than two groups. the results were considered statistically significant when p>0.05. results treatment with terpinen-4-ol at concentrations of 0.01%, 0.02%, 0.05% and 0.1% (v/v) led to a decrease in the viability of the amastigote forms within the macrophages by 24.02%, 32.74%, 66.72% and 100% it respectively as a comparison with the control group. figure1. the ic50 and ic90 of terpinen-4-ol on the viability of amastigote forms in vitro. figure 2. viability of amastigote forms after treatment it by different concentrations of terpinen-4-ol. iraqi j pharm sci, vol.31(2) 2022 terpinen-4-ol on amastigote forms of leishmania tropica 235 table1. the inhibitory effect of terpinen-4-ol on the proliferation of amastigote forms of parasites in mouse macrophages. concentration% of terpinen-4-ol the number of parasites in 100 macrophages mean ±sd inhibition of parasites proliferation% within treated macrophages compared with the control mean ±sd 0 1298 ±12 0 0.01 1021 ±6.6 24.02 ±3.02 0.02 873 ±4.6 32.74 ±1.02 0.05 432 ±1.2 66.72 ±0.6 0.1 0 100 discussion several recent studies support the antiinflammatory activity of australian tea tree oil (tto). they have shown that tto affects many inflammatory factors both in vitro and in vivo. terpinen-4-ol plays the main role in this effect(17). terpinen-4-ol is a strong bactericidal agent. it has anti-fungal properties and an inhibitory activity against staphylococcus aureus. it has been reported that the combination of this natural substance and traditional medicines may help in the treatment of yeast resistance and bacterial infection(18). many recent reports have praised that terpinen-4-ol has antitumor effects by selectively inducing cell death through cell cycle arrest in human melanoma cells. furthermore, terpinen-4-ol has been shown to induce a dose-dependent cytotoxic response against large cell lung cancer cells(18). terpinen-4-ol reduce the infection rate of host cells, and decrease protozoa survival rate. also, they increase phagocytic and lysosomal activities and nitric oxide levels in treated host by preventing bioenergetics imbalance in the spleen of treated host and they modulate immune activity by increasing igg and pro-inflammatory cytokine (tnfα, ifn-γ, il-1, il-4, and il-6) levels and decreasing antiinflammatory il-10 level in treated host(19). the results of this study declared a decrease in the number of leishmania parasites inside macrophages with an increase in the concentrations of terpinen4-ol with ic90 of 0.08%. francesca and colleagues recorded similar results to the results obtained in this study when studying the activity of terpinen-4-ol against azole-susceptible and resistant human pathogenic candida species in vivo, asic90 was 0.06%(20). the present study was differed from the study of mikus and his colleagues in the value ic90 which was 335.9 µg/ml(21), while in our study it was 837µg/ml. the reason for this difference may be due to the difference in the incubation period. which was 72 hours in the study of mikus and his colleague(21), while in the current study it was 24 hours. according to our knowledge this study could be the first one that record the effect of terpinen-4-ol on amastigote forms of l. tropica within macrophages. conclusion the present study was concluded that terpinen-4-ol has activity on amastigote forms of leishmania tropica in vitro. acknowledgments the authors would like to thank the staff of the department of pharmacology and toxicology in the faculty of pharmacy – damascus university and the staff of the leishmania center of epidemiological and biological studies – damascus university, especially, prof. chadi soukkarieh and dr. hassan alkhouri for providing research facilities. references 1. torres-guerrero, e., et al., leishmaniasis: a review. 2017. 6. 2. meireles, c.b., et al., atypical presentations of cutaneous leishmaniasis: a systematic review. 2017; 172: 240-254. 3. ergen, e.n., a.h. king, and m.j.c. tuli, cutaneous leishmaniasis: an emerging infectious disease in travelers. 2015. 96(4): e22-e26. 4. norouzinezhad, f., et al., cutaneous leishmaniasis in iran: results from an epidemiological study in urban and rural provinces. 2016;6(7): 614-619. 5. salam, n., w.m. al-shaqha, and a.j.p.n.t.d. azzi, leishmaniasis in the middle east: incidence and epidemiology. 2014; 8(10): e3208. 6. abdrebbi, s.b., et al., cutaneous leishmaniasis: endemic regions and epidemiological characteristics of cases declared at university hospital center of tlemcen, algeria. 2019; 7: 249-254. 7. haddad, n., et al., cutaneous leishmaniasis in the central provinces of hama and edlib in syria: vector identification and parasite typing. 2015; 8(1): 1-8. 8. hayani, k., a. dandashli, and e.j.a.d.-v. weisshaar, cutaneous leishmaniasis in syria: clinical features, current status and the effects of war. 2015; 95(1): 62-66. iraqi j pharm sci, vol.31(2) 2022 terpinen-4-ol on amastigote forms of leishmania tropica 236 9. dantas-torres, f., et al., detection of leishmania infantum in rhipicephalus sanguineus ticks from brazil and italy. 2010; 106(4): 857-860. 10. barrera, c.a.c., et al., seco-limonoids and quinoline alkaloids from raputia heptaphylla and their antileishmanial activity. 2011;59(7): 855-859. 11. fever, d., kala-azar, black fever, dumdum fever, oriental sore, tropical sore, uta, chiclero ulcer, aleppo boi, pian bois; espundia. 2009. 12. habif, t.p.j.a., netherlands: elsevier health sciences, clinical dermatology e-book: a color guide to diagnosis and therapy. 2015. 13. mertas, a., et al., the influence of tea tree oil (melaleuca alternifolia) on fluconazole activity against fluconazole-resistant candida albicans strains. 2015. 14. pazyar, n., et al., a review of applications of tea tree oil in dermatology. 2013;52(7): 784790. 15. mondello, f., et al., in vitro and in vivo activity of tea tree oil against azole-susceptible andresistant human pathogenic yeasts. 2003;51(5): 1223-1229. 16. naddaf, n. and s.j.j.o.p.d. haddad, apigenin effect against leishmania tropica amastigotes in vitro. 2020;44: 574-578. 17. nogueira, m., et al., terpinen-4-ol and alphaterpineol (tea tree oil components) inhibit the production of il-1β, il-6 and il-10 on human macrophages. 2014; 63(9): 769-778. 18. sobral, m.v., et al., antitumor activity of monoterpenes found in essential oils. 2014. 2014. 19. lam, n.s., et al., melaleuca alternifolia (tea tree) oil and its monoterpene constituents in treating protozoan and helminthic infections. 2020;130: 110624. 20. mondello, f., et al., in vivo activity of terpinen4-ol, the main bioactive component of melaleuca alternifolia cheel (tea tree) oil against azole-susceptible and-resistant human pathogenic candida species. 2006; 6(1): 1-8. 21. mikus, j., et al., in vitro effect of essential oils and isolated mono-and sesquiterpenes on leishmania major and trypanosoma brucei. 2000; 66(04): 366-368. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 study of microbial contamination of some commercial contact lenses' solutions doi: https://doi.org/10.31351/vol30iss2pp214-218 214 study of microbial contamination of some commercial contact lens solutions faris ali muhammed al-hilli *,1 * department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad. iraq. abstract microbial contamination is very common among contact lens users whether contamination involves the solutions or the lens. many bacteria cause eye infections through this route and some may lead to serious outcomes to the eye. most isolated bacteria from previous studies on the subject belong to gram negative bacteria and few gram positive bacteria. our study was aimed at identifying microbial contamination of contact lens solutions from students at the college of pharmacy/ university of baghdad. the lenses used were varied from medical to cosmetic types. both used and unused solutions were subjected for culturing to diagnose any microbial contamination. thirty six (60%) of used samples showed bacterial growth. pseudomonas aeruginosa accounts for the highest number of isolates (25%) followed by e. coli (21%), staphylococcus epidermidis (6.6%), pseudomonas fluorescence (5%) and proteus mirabilis (1.6%) respectively. only one unused (sealed) sample showed growth of p. fluorescence. fungal growth was absent in both used and unused lenses. the bacterial contamination is likely to come from bad personal hygiene and improper or misuse of the solutions where these bacteria especially p. aeruginosa are frequently found in various environments from skin to solid materials and surfaces and are known to thrive in harsh environments. no relation was found between eye associated diseases and solution contamination among contact lens users. special care should be paid in maintaining aseptic solutions and proper handling to avoid transmitting harmful bacteria to the eye where it may lead to serious eye infections. keywords: contact lens solution, microbial contamination, eye infections, pseudomonas aeruginosa, staphylococcus epidermidis سات الالصقة التجاريةدراسة التلوث المايكروبي لبعض محاليل العد 1*،فارس علي محمد الحلي العراق .، ، بغداد جامعة بغداد، كلية الصيدلة ، فرع العلوم المختبرية السريرية * الخالصة يعتبر التلوث الميكروبي من االمور الشائعة عند مستخدمي العدسات الالصقة بغض النظر اذا كان التلوث يحدث في المحاليل الخاصة ام للعين. العدسات نفسها. يوجد العديد من البكتريا التي تسبب عدوى للعين عن طريق هذا المسلك من االصابه و قسم منها قد تؤدي الى عواقب خطيرة ام.تهدف اشارت الدراسات السابقة في هذا المجال الى ان اكثر البكتريا المعزوله هي سالبة لصبغة كرام و القليل من المجاميع الموجبة لصبغة كر ن عينة من محاليل العدسات وتم جمع ستالدراسة الى تشخيص التلوث الميكروبي لمحاليل العدسات الالصقه بين طالب كلية الصيدلة في جامعة بغداد. أوساط في وضعهام ت. تتنوع أنواع العدسات المستخدمة من الطبية إلى التجميلية. جامعة بغداد /الالصقة المتوفرة تجارياً من طالب كلية الصيدلة أظهرت ست . زرع المايكروبيخاضعة لل (الجديدة)المستعملة وغير المستعملة المحاليلكانت . لتشخيص أي تلوث ميكروبي داخل المحاليل زرعية أكبر عدد من العزالت pseudomonas aeruginosaتمثل . فطري نمو أي يظهر ولمنمًوا بكتيريًا ، المستعملةمن العينات ( 60%)وثالثون proteus% )و e. coli (21،%( ,staphylococcus epidermidis (6.6 pseudomonas fluorescence (5%)% )تليها( 25%) mirabilis (1.6 نمو ( جديدة) مستعملةأظهرت عينة واحدة غير . على التواليpseudomonas fluorescence . من المحتمل أن يكون هذا التلوث في بيئات مختلفة من الجلد p. aeruginosaحيث توجد هذه البكتيريا وخاصة المحاليل مالبكتيري ناتًجا عن النظافة الشخصية السيئة وسوء استخدا وث المحلول لم يتم العثور على عالقة بين األمراض المرتبطة بالعين وتل. إلى المواد الصلبة واألسطح ومن المعروف أنها تزدهر في البيئات القاسية يجب إيالء عناية خاصة في الحفاظ على المحاليل المعقمة والتعامل المناسب لتجنب انتقال البكتيريا الضارة إلى . مستخدمي العدسات الالصقة ضمن .العين حيث قد تؤدي إلى التهابات خطيرة للعين pseudomonas aeruginosa ،staphylococcus epidermidisالعين، الكلمات المفتاحية: محلول العدسات الالصقة، التلوث المايكروبي، اصابات introduction contact lenses are thin plastic lens worn between the eye and eyelid that may be used instead of eyeglasses. actors, models, and others wear them for appearance, and athletes use them for safety and convenience. contact lenses may also be used to correct certain abnormalities of the eye that cannot be corrected by regular glasses. it was invented in 1887 but was not used until 1938 when the first plastic contact lens was introduced. in 1950, the corneal contact lens was introduced. it covered only the cornea of the eye, floated on the tears of the wearer, and could be worn all day without difficulty. recent improvements include flexible lenses that shorten the initial period of adjustment for the wearer and porous lenses that do not have to be removed each day. today, contact lenses that "breathe" have become popular. they allow oxygen to get to the cornea, preventing blurred vision due to the corneal exhaustion syndrome (1,2). 1corresponding author e-mail: scorpionc3po2000@gmail.com received: 26/11/2020 accepted:20 /6 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp214-218 iraqi j pharm sci, vol.30(2) 2021 study of microbial contamination of some commercial contact lenses' solutions 215 the solutions that preserve contact lenses are subject to contamination by microorganisms such as bacteria and fungi and may lead to serious infections through the invasion of the cornea and conjunctiva(3). staphylococcus aureus, pseudomonas, bacillus, proteus, haemophilus, enterobacter, serratia, and klebsiella spp. are the most common isolated bacteria from eye infections related to contaminated medications(4). these bacteria can damage important functional structures which might lead to vision loss and blindness (5). bacterial keratitis and endophthalmitis are some of these serious infections that were found to be associated with the use of contaminated topical medications (68). the contamination rate of in-use ophthalmic solutions is variable according to published researches and ranges from 0.07% to 35.8 %(9). most eye preparations may possess antimicrobial effect and if not they are provided with antimicrobial substances to prevent contamination. these substances prevent microbial growth to avoid infection of the eye and drug degradation (10). in a study by tsegawa et.al on 100 ophthalmic solutions used by patients and eye care workers cultured for microbial contamination, it was found that 11% were contaminated with various bacterial species and the significant findings of this study were the isolation of resistant staphylococcus aureus and coagulase negative staphylococcus both were methicillin resistant among other species(4). the current study focused on screening for microbial contamination in commercially eye lens solution used by students at college of pharmacy/ university of baghdad. materials and methods specimen collection the samples were collected from october 2018 until may 2019. sixty samples were collected form students at college of pharmacy/university of baghdad. sample characteristic 1the samples were collected from 58 females and 2 males. 2fifty eight samples were extended wear, and 2 samples were daily wear. 3five samples have been used as medical lenses, while 55 samples have been used as cosmetic lenses. 4associated eye disease: one sample has a previous history of toxoplasmosis retinitis infection in the right eye. while the other samples have astigmatism as diagnosed by ophthalmologists. 5the manufacturing companies were the most common available in the iraqi market and they were; bellasoft, neoplus , fresheye, safe & sound, hd, my choice, anastasia, desio, cute, aquasoft, heidi , freshlook and they were manufactured mainly in korea, syria and france. 6the collected solutions were divided into two groups: 20 new and 40 used solutions for comparison and culturing. culture media the samples were cultivated on nutrient agar, macconkey agar, mannitol salt agar and blood agar to isolate the bacteria. sabaraud dextrose agar was used to isolate the fungi. all media were sterilized in autoclave at 121⁰c for 15 min. prior for sub culturing. duplicate cultures were made for each sample. identification of microorganisms nutrient agar was used to identify gram positive bacteria, while macconkey agar was used to identify gram negative bacteria. hydrogen peroxide solution (3%) was used to distinguish between staphylococcus and streptococcus. mannitol salt agar was used to differentiate staphylococcus aureus growth from staphylococcus epidermidis. gram negative bacteria were isolated by macconkey agar, oxidase test and growth at 4⁰c/42⁰c(both were used to identify pseudomonas species) and triple sugar iron agar test also done for confirmation of the identification. the api 20e test system was used for the performance of 20 standard biochemical tests that differentiate various species of enterobacteriace & other gram negative bacteria. (11). results and discussion results of the total 60 samples collected and cultured, all new 20 solutions (not used with contact lenses) showed negative growth except one sample showed growth of pseudomonas fluorescence while the used solutions showed that 36 out of 40samples were contaminated with bacteria. no fungal growth was observed in all samples. table1shows the types of bacterial pathogens isolated from the samples with number and percentage of contamination analyzed through spss program. table1. microorganisms isolated from contaminated used lens solutions samples pathogen no. of samples % pseudomonas aeruginosa 15 41.6 escherichia coli 13 36.1 staphylococcus epidermidis 4 11.1 pseudomonas fluorescence 3 8.3 proteus mirabilis 1 2.7 total 36 samples 100 iraqi j pharm sci, vol.30(2) 2021 study of microbial contamination of some commercial contact lenses' solutions 216 discussion pseudomonas aeruginosa was the most frequent isolated bacteria from samples that included in this study. contamination with pseudomonas aeruginosa may be due to many reasons, as bad storage of the solution, long time of the solution storage without replacing it frequently, bad hygiene means, when the lens removed from the eye and inserted directly into the solution bags without rinsing or washing the lenses with the solution. sometimes, the eye was infected before wearing the contact lenses. contact lens with the persistence eye diseases may worse the condition (12). in the past, hard contact lenses were sterilized in boiling water. when they were replaced by soft lenses, boiling water sterilization was discarded due to the damage and deterioration that it causes to theses soft ones. furthermore, this type of sterilization causes allergic diseases (13). care system bottles can be easily contaminated and become a source of microbes that contaminate the lens storage cases (14,15). the results of the current study revealed that all new solutions showed no growth except one sample which was contaminated by pseudomonas fluorescence. all types of care solutions, including hydrogen peroxide, have been reported to contain microorganisms even in experienced users or unopened factory sealed bottles (16,17). it may be possible that contamination occurred during packaging in the factory. no gram positive bacteria or fungus were found in the cleaning solutions. gram negative bacteria have the best opportunity to contaminate lens care solutions due to their ability to adapt to various environments even with minimum nutritional requirements (18). the reason for such findings might be due to the fact that the moist tips of solutions might serve as a reservoir for microbial contamination, and this will put the risk of contaminating the eye drop with bacteria as a result of touching the dropper tips while opening it for usage, contact of dropper with the ocular tissues, and environmental factors(4). lens care solutions are provided with preservatives and some disinfectants such as alexidine dihydrochloride, polyhexamethylene biguanide, oxychlorite complex (sodium chlorite and hydrogen peroxide), benzalkonium chloride and other compounds. these are added to maintain low levels of microorganism during the product’s life cycle whilst having minimal impact on the ocular surface (19). narayana et.al observed in their study that the antimicrobial activity of different solutions varies with respect to time of incubation, and also there was a marked difference in the activity of some solutions against some pathogens like pseudomonas aeruginosa and staphylococcus aureus and they concluded that the users should store their lenses in solutions for longer periods of time (20). the stock solutions are intended to prevent microbial growth due to their antimicrobial efficacy however, common pathogens of the eye such as staphylococcus aureus and pseudomonas aeruginosa are becoming increasingly resistant to some commercial solutions(12,21). gram negative bacteria, especially pseudomonas aeruginosa has the ability to form biofilms on various materials both living and nonliving surfaces and the biofilm formation is an obstacle concerning the uses and design of ocular devices, such as contact lenses because it shelters the bacteria from harmful factors like antibiotics and the immune system thus leading to infection of the eye (22). oliver et.al mentioned in his study that biofilms of p. aeruginosa resist multipurpose contact lens solutions and cause contamination but ozonized water and chlorohexidine reduced biofilm formation by reduction in bacteria numbers with ozonized water much more preferable as it doesn’t depose any toxic residues like chlorohexidine (23). escerichia coli are the 2nd most contaminant isolated in our study. it has been isolated from contact lens solutions from previous studies and cause a problem in daily wear lens users(24,25). as mentioned by demirbilek and evrenin their study on multipurpose contact lens solutions, 5% povidon iodine was more effective in elimination of this bacteria (26). as for the single isolate of pseudomonas fluorescence, this bacterium is common in soil and it is known to cause infections in immuncompromised persons such as cancer patients through blood transfusions and saline solutions contamination (27). it is likely that this bacterium contaminated the solution during the manufacturing and filling of the product within the factory limits as it was found in new contact lens solution sample (sealed). while staphylococcus epidermidis is a common commensal inhabitant of the skin at various sites, this bacteria is now widely studied and under monitoring due to increasing nosocomial infections caused by it. some authors call it "the accidental pathogen" where it infects immune-compromised hosts and introduced to the system via instrument like urethral catheters. there is also an increasing resistant among nosocomial infections with these bacteria (28). the isolation of this bacterium among contact lens solutions most likely came from hands or contaminated bottle tips from the environment where these bacteria are commonly found. correa et.al mentioned that s. epidermidis was one of the isolated bacteria from silicone hydrogel contact lens if they were used incorrectly or maladapted and lead to eye infection. also, this may lead to iraqi j pharm sci, vol.30(2) 2021 study of microbial contamination of some commercial contact lenses' solutions 217 contamination of solutions if they were bacteriocidally inefficient in eliminating them (29). proteus mirabilis is well known as one of the most frequent causative agent of urinary tract infections as well as causing wound infection, sepsis and pneumonia in hospitalized patients (30). it also can contaminate any material due to its actively swarming movement on solid surfaces. lipener et.al mentioned in his study that p. mirabilis was the second most frequently isolated bacteria along with p. aeroginosa from contact lens saline solutions with a percentage of 60% (31). it is likely that these bacteria contaminated the solutions via hands with poor hygiene or improper handling of the solutions. there were no relation between eye associated diseases and solution contamination as both healthy and disease associated eyes showed contamination of contact lens solutions by their users. conclusion contact lens solutions users should pay more attention to restrictions and medical guidelines in maintaining aseptic solutions free from microbial contamination. lens solution could be a source of eye infection if aseptic procedures were ignored. college students are likely to get contamination and infections due to the crowded environment where they study and that there are many sources and material that provide these microbes. acknowledgement the author would like to thank pharmacist marwa mohammed for her assistance in sample collection from students and also special thanks to college of pharmacy/ university 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zamboni fj, lewinski r, kwitko s, uras r . bacterial contamination in soft contact lens wearers. clao j. 1995apr;21(2):122-4. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(suppl.) 2022 upper and lower urinary tract infections in al-diwaniya doi: https://doi.org/10.31351/vol31isssuppl.pp86-91 86 determining the prevalence of upper and lower urinary tract infections’ pathogens and their antibiotic susceptibility profile for adult patients in al-diwaniya, iraq (conference paper) # bashar g. al-fatlawi *,**,1 and ali l. jasim * # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** al diwaniyah health directorate, ministry of health, al qadisiyyah, iraq abstract until today, one of the leading predominant infections is urinary tract infection (uti). it exerts a huge burden on health systems worldwide each year. treating uti empirically with antimicrobials improves morbidity rates. this study aims to assess the prevalence of uti associated bacteria in adult patients and to determine their antibiotic susceptibility profile. a retrospective study was conducted for adult outpatients who visited aldiwaniya tertiary hospitals from january 2020 till february 2022 to review their medical and lab records in addition to sociodemographic data. a total of 256 patients’ records included of which 204 (79.7%) belong to females and 52 (20.3%) were males with average age of 39.22±17.10 years. the predominant organisms’ isolates were staphylococcus spp. found in 100 records (39.1%), escherichia coli (e. coli) demonstrated in 90 records (35.2%), and klebsiella spp. revealed in 23 records (9%). staphylococcus spp. showed full resistance to cefepime and high resistance to ampicillin (92.9%) followed by ceftazidime (87.5%), and were highly sensitive to vancomycin. the higher resistance profile of e. coli was to ampicillin (97.9%) and ceftriaxone (81.3%) while was highly susceptible to meropenem (97.9%) and amikacin (97.6%). additionally, klebsiella spp. was highly susceptible to nitrofurantoin (78.6%), while was completely resistant to ampicillin. this study presents staphylococcus spp. as the most prevalent gram-positive uropathogen and e. coli as the most prevalent gramnegative bacteria with multidrug resistance profile to commonly used antimicrobials which is an alarming situation to implement an immediate effective stewardship program. keywords: urinary tract, uti, uropathogen, antimicrobial susceptibility, iraq. لدى وشكل تحسسها للمضادات الحيويةالعليا والسفلى تحديد انتشار مسببات عدوى المسالك البولية # ) بحث مؤتمر (المرضى البالغين في مدينة الديوانية، العراق *لطيف جاسم علي و ***,بشار غازي عبد الشهيد 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي فرع الصيدلة السريرية، كلية الصيدلة، جامعة بغداد، بغداد، العراق. * دائرة صحة الديوانية، وزارة الصحة، القادسية، العراق. ** الخالصة تعد عدوى المجاري البولية من أحد من األمراض البكتيرية الواسعة االنتشار حتى يومنا هذا. تسلط هذه العدوى كل سنة عبئ كبير جدا الى على األنظمة الصحية حول العالم. ان عالج عدوى المجاري البولي بالمضادات البكتيرية تجريبيا يقلل معدالت المراضة. تهدف هذه الدراسة لمرضى البالغين الذين املفات على تراجعيةتم تنفيذ هذه الدراسة ال هذه الممرضات في المرضى البالغين ومدى تحسسها للمضادات البكتيرية. تحديد يناير من للفترة الثالثية الديوانية مدينة مستشفيات في الخارجية العيادات شباط 2020زاروا الديموغرافية ل 2022وحتى المرضى بيانات جمع ± 39.22ذكور وكان معدل العمر تعود لل( %20.3) 52ناث وتعود لال( %79.7) 204سجل طبي للمرضى منه 256. تم جمع الطبية والمختبريةو ( على %9، %35.2 ، %39.1)القولونية والكلبسيال واالشريكيةسنوات. كانت أكثر الممرضات المعزولة انتشارا هي المكورات العنقودية 17.10 ( على %87.5، %92.9عالية لمضاد االمبسلين، السفتازيديم )تامة لمضاد السيفيبيم ومقاومة بكتريا المكورات العنقودية ذات مقاومة كانت التوالي. (، السفترايكزون %97.9كانت اعلى مقاومة لبكتيريا االشريكية القولونية ضد مضاد االمبسلين )والتوالي ولكنها حساسة جدا لمضاد الفنكمايسين. ( تقريبا. كانت بكتيريا الكلبسيال مقاومة تماما لمضاد %97(، بينما كانت مضادات الميروبنيم واالميكاسين ذات فعالية عالية ضدها بمقدار )81.3%) فعاال كان النايتروفيورانتوين مضاد لكن ) االمبسلين ا%78.6ضدها المجاري لعدوى انتشارا األكثر المسبب بان الدراسة هذه بينت هي لبولية (. المضادات ألغلبوأنها مقاومة ضمن البكتريا السالبة لصبغة غرام القولونية المكورات العنقودية ضمن البكتريا الموجبة لصبغة غرام واالشريكية البكتيرية شائعة االستعمال وهي حالة تستوجب وقفة جدية للتطبيق الفوري لبرامج االشراف على وصف المضادات الحياتية. العراق. البكتيرية،الكلمات المفتاحية: المجاري البولية، عدوى المجاري البولية، الممرض البولي، الحساسية للمضادات introduction one of the most prevalent bacterial infections is urinary tract infection (uti) (1). a uti diagnosis costs more than $6 billion usd yearly and affects 150 million people worldwide (2). uncomplicated cystitis to severe infections like pyelonephritis and other consequences make up the spectrum of uti disorders (3). since the female urethra is structurally less effective in blocking bacterial entry, uti is often more common in women than in men (4). 1corresponding author e-mail: bashar.dr79@gmail.com received: 15/7/2022 accepted:5 /9 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp86-91 iraqi j pharm sci, vol.31(suppl.) 2022 upper and lower urinary tract infections in al-diwaniya 87 in addition, a number of variables, including age, past antibiotic treatment, hospitalization, and catheterization, affect the frequency rate of uti. it is well known that a single bacterial species is responsible for more than 95% of urinary tract infections. the organism that causes acute infections most frequently is e. coli (5,6). some iraqi studies revealed that the most prevalent pathogenic organisms causing uti were staphylococcus spp., e. coli, and klebsiella pneumonia, all of which were resistant to the most popular medicines (7–10). early uti therapy with first-line antibiotics lowers the rate of morbidity (11). it is essential to understand the principal bacteria producing urinary tract infections and their respective antibiotic susceptibility patterns in order to deliver an effective empirical therapy (12). determining bacteria and their patterns of antibiotic susceptibility permits effective treatment outcomes, regulates the rise in antimicrobial prescription, and aids in the management of antimicrobial resistance, a global public health issue. this study aims to assess the prevalence of uti associated bacteria in adult patients and to determine their antibiotic susceptibility profile. patients and methods this study was a descriptive quantitative retrospective study. records for 256 adult outpatients who visited al-diwaniya tertiary hospitals (diwaniya teaching hospital and gynecology teaching hospital) starting from jan 2020 till feb 2022 were reviewed and patients’ sociodemographic (age, gender) and laboratory data (urine sample culture and antibiotic susceptibility test) were collected. the inclusion criteria for the current study are records of patients aged ≥ 18 years old presented with upper or lower utis and had a urine culture and susceptibility profile, while exclusion criteria are records of patients < 18 years old or ≥ 18 years but with missing data or culture with no growth. ethical approval this study got approval from the scientific and ethical committee at college of pharmacy, university of baghdad and the scientific and ethical committee at iraqi ministry of health. statistical analysis microsoft office excel 2016 (microsoft corporation, redmond, washington, united states) was used to code, enter, and analyze the data. mean and standard deviation (sd) were used to express age of patients, while numbers, frequencies and percentages were used to express the rest of data. results two hundred and fifty-six patients’ records that match the inclusion criteria were collected in this study, with majority of female patients (204, 79.7%). the average age of them was 39.22±17.10 years. the predominant age group was (18-30) years old (108, 42.2%). more than three quarters of the patients’ records (82.0%) were from al diwaniyah teaching hospital while the rest were from gynecology teaching hospital (table 1). table 1. characteristics of patients characteristic value n (%) age mean ±sd (years) 39.22±17.10 (minimum-maximum) (years) (18 85) age groups 18 – 30 108 (42.2) 31 – 40 54 (21.1) 41 – 50 26 (10.2) 51 – 65 45 (17.6) > 65 23 (9.0) gender male 52 (20.3) female 204 (79.7) hospital al diwaniyah teaching hospital 210 (82.0) gynecology teaching hospital 46 (18.0) total valid record no. = 256, n: number, %: percentage. from the collected patients’ records, 142 (55.5%) isolates revealed gram-negative bacteria, and 114 (44.5%) revealed gram-positive bacteria (table 2). the predominant isolated organisms were staphylococcus spp. 100 (39.1%), e. coli 90 (35.2%), klebsiella spp. 23 (9%), and both enterococcus spp. and pseudomonas spp. 10 (3.9%) (table 2). iraqi j pharm sci, vol.31(suppl.) 2022 upper and lower urinary tract infections in al-diwaniya 88 table 2. prevalence of uropathogens detected in the isolates (n= 256) uropathogen value n (%) gram-negative bacteria 142 (55.5) escherichia coli 90 (35.2) klebsiella spp. 23 (9.0) pseudomonas spp. 10 (3.9) proteus mirabilis 5 (2.0) serratia fonticola 5 (2.0) enterobacter spp. 4 (1.6) acinetobacter baum. 1 (0.4) aeromonas hydrophilia 1 (0.4) burkholderia cepacia 1 (0.4) cronobacter sakazakii 1 (0.4) raoultella ornithinolytica 1 (0.4) gram-positive bacteria 114 (44.5) staphylococcus spp. 100 (39.1) enterococcus spp. 10 (3.9) aerococcus viridans 1 (0.4) diphtheroid spp. 1 (0.4) micrococcus spp. 1 (0.4) streptococcus agalactiae 1 (0.4) total 256 (100) n: number, %: percentage the evaluation of the antibiotic resistance profile of common uropathogens to various antimicrobial agents is summarized in tables 3. staphylococcus spp. were the prevalent gram-positive uropathogens with full resistance to cefepime and high resistance to ampicillin (92.9%) followed by ceftazidime (87.5%), and norfloxacin (83.3%). furthermore, around 97% of the isolated staphylococcus spp. were sensitive to vancomycin. the higher resistance profile of e. coli, the most prevalent gram-negative uropathogen, was to ampicillin (97.9%), ceftriaxone (81.3%), ceftazidime (79.5%) and ciprofloxacin (64.5%). e. coli was highly susceptible to meropenem (97.9%), imipenem (97.3%) and amikacin (97.6%). k. spp. were highly susceptible to nitrofurantoin (78.6%), imipenem (72.2%), and amikacin (69.6%), while were completely resistant to ampicillin. additionally, pseudomonas spp. were highly resistant to norfloxacin (100%), nitrofurantoin (85.7%) and levofloxacin (83.3%), while they were highly sensitive to meropenem (87.5%). enterococcus spp. was fully resistant to norfloxacin, amikacin, piperacillin-tazobactam, ceftazidime, and cefepime while was sensitive to vancomycin (87.5%). other uropathogens’ resistance rates to commonly used antibiotics are showed in table 3. table 3. antibiotic resistance profile of common uropathogens. antibiotics number of resistance isolates n (%) gram-negative gram-positive e. coli k. spp. p. spp. proteus mirabilis s. fonticola e. spp. staph. spp. enterococcus spp. ampicillin 47 (97.9) 10 (100) 0 1 (50) 1 (100) 0 13 (92.9) 3 (75) tmp/smx 48 (61.5) 10 (47.6) 0 0 1 (20) 0 29 (51.8) 0 nitrofurantoin 7 (11.7) 3 (21.4) 6 (85.7) 3 (100) 0 2 (66.7) 19 (28.4) 3 (50) ciprofloxacin 49 (64.5) 8 (42.1) 5 (62.5) 2 (66.7) 3 (60) 3 (100) 52 (63.4) 6 (85.7) levofloxacin 46 (66.7) 5 (33.3) 5 (83.3) 1 (33.3) 1 (50) 1 (50) 28 (45.2) 4 (57.1) norfloxacin 28 (70) 9 (75) 8 (100) 1 (100) 0 1 (50) 35 (83.3) 5 (100) vancomycin 0 0 0 0 0 0 2 (3.4) 1 (12.5) amikacin 2 (2.4) 7 (30.4) 7 (77.8) 1 (20) 0 1 (25) 20 (30.8) 5 (100) gentamycin 38 (54.3) 8 (44.4) 7 (70) 0 (0) 1 (20) 1 (25) 30 (44.8) 5 (71.4) imipenem 2 (2.7) 5 (27.8) 4 (50) 3 (100) 0 1 (33.3) 2 (18.2) 1 (50) meropenem 1 (2.1) 5 (41.7) 1 (12.5) 0 0 0 1 (33.3) 1 (50) piperacillintazobactam 14 (23) 6 (35.3) 0 1 (25) 1 (20) 1 (33.3) 0 1 (100) ceftriaxone 52 (81.3) 14 (70) 0 3 (75) 2 (50) 1 (100) 12 (75) 1 (50) ceftazidime 58 (79.5) 11 (64.7) 3 (37.5) 2 (50) 2 (40) 2 (66.7) 7 (87.5) 1 (100) cefepime 48 (70.6) 10 (55.6) 4 (44.4) 3 (75) 2 (40) 2 (66.7) 4 (100) 1 (100) n: number, %: percentage. discussion e. coli is regarded as the most frequent uropathogen involved in community-acquired uti (being implicated in more than half of all uti cases), which is one of the most common diseases in the world (1,13). regional variation in uropathogens’ antibiotic resistance profiles is likely due to different prevention and treatment strategies against utis or iraqi j pharm sci, vol.31(suppl.) 2022 upper and lower urinary tract infections in al-diwaniya 89 misuse of drugs and self-medication by the population in different geographic regions (14,15). this variation urges the need for continuous monitoring to provide updated information to optimize the therapeutic selections. data analysis, in this study, revealed that females still the majority among utis cases compared to males which is supported predominantly by previous studies (10,16–19). the finding supports that female continue to be more vulnerable to contracting uti due to their basic anatomy, whereby their urethra is closer to the anal opening and shorter than men’s urethra (20,21). again, women’s hormonal fluctuations across the menstrual cycle and the possible genetic factor that tends to run in families may also be fueling their vulnerability (14,17,20). the majority of studies found that gram-negative bacteria (10,16,17) were a common cause of uti cases rather than gram-positive (8,9,22– 24), which are in line with the present study. possible reason for that is the presence of special virulence factor in gram-negative bacteria like the presence of unique structure and adhesion proteins in gramnegative bacteria, which facilitates attachment to the uroepithelial cell, resulting in high prevalence in utis (25). analyzing the data of detected uropathogens, our results revealed that e. coli was the most frequent isolate among the isolated gramnegative pathogens. high prevalence of e. coli could be due to the fact that it belongs to the normal flora of the human intestine, and therefore, it easily colonizes the urinary tract and can exhibit multidrug resistance (10). furthermore, several literatures from different regions concluded that e. coli was the predominant gram-negative bacteria causing utis (8–10,17,23,26–29). however, the resistance pattern of e. coli to antibiotics has been very different in various studies. in a study conducted in iran 2006, sharifian et al. found the highest susceptibility rate of e. coli to ceftriaxone (97.8%) and cefotaxime (95.2%) (14), while in iraq another pattern found. other studies carried out in iraq reported that e. coli was highly sensitive to imipenem and meropenem while has a high resistance profile toward cephalosporines including the third generation (9,10,30). the iraqi reports go parallel with this study results in addition to high sensitivity to amikacin. these results also supported by national antimicrobial resistance surveillance done by ministry of health in 2020 (31). the preserved effectiveness of these agents may be explained by the fact that they are usually used only under medical supervision and not used in community without prescription only seldomly. however, the high resistance of e. coli to ampicillin, quinolones, cefepime, ceftriaxone, and ceftazidime can be explained partially by the high rate of antibiotics abuse and overuse in the region. in the current study, klebsiella. spp. also found among uropathogens cause uti and was susceptible to nitrofurantoin, imipenem, while was fully resistant to ampicillin. other studies found that klebsiella. spp. was among the causes of uti (9,10,31). in the case of gram-positive bacteria, staphylococcus spp. were the predominant grampositive uropathogens that cause uti. in line with this finding, other studies stated that staphylococcus spp. as the most common gram-positive uropathogens causing utis (10,12,17,32,33). they showed a high sensitivity toward vancomycin followed by imipenem, while presented a high resistance profile which exceeding 50% toward third and fourth generation cephalosporin and ampicillin. these results supported by other researches globally (8,10,34–37). other study yielded different results that showed full resistance to vancomycin (34). the rising in resistance rate to previously effective antibiotics may be due to uncontrolled usage of prescription only medication including antibiotics and steroids in private settings (15,38,39), and even due to irrational antibiotics prescribing practice (40–44). one of the limitations of the current study is being retrospective which will limit the flexibility to control study variables and data collected. other limitation is the inclusion of only public settings data without private settings which causes a loss of vast amount of data. this is because private laboratories in al diwaniyah city have no archived patients’ records for long periods of time which limits retrospective data collection needed for our research. carrying out the study in only one city also may be considered as a limitation because it does not reflect a national outcome about utis prevalent pathogens and their susceptibility profile. so that, further studies needed to overcome these limitations. conclusion this study presents staphylococcus spp. as the most prevalent gram-positive uropathogens and e. coli as the most prevalent gram-negative uropathogen. it also spots the light on the emerging multidrug resistance profile for those pathogens to commonly used antimicrobials such as ampicillin, cephalosporines, and even quinolones. this requires a serious effort to implement the stewardship iraqi j pharm sci, vol.31(suppl.) 2022 upper and 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under a creative commons attribution 4.0 international license https://moh/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 oroslippery tablets irbesartan and hydrochlorothiazide doi: https://doi.org/10.31351/vol31iss2pp91-100 91 preparation and evaluation of oroslippery tablets contain irbesartan and hydrochlorothiazide combination yaseen t. khalaf *, yasir q. almajidi1,**, naeem m. shalan*, israa h. alani* and wael a. abu dayyih*** *department of pharmaceutics and pharmaceutical technology, faculty of pharmacy, al-ahliyya amman university, jordan. ** department of pharmaceutics, baghdad college of medical sciences, baghdad, iraq *** faculty of pharmacy, mutah university, jordan. abstract oro slippery tablets (osts) is a technique used to improve swallowing of tablets for patients with dysphagia. the aim of this study was to formulate irbesartan, a hypotensive agent and hydrochlorothiazide, a duiretic as oroslippery tablets (ost) containing 150 mg irbesartan and 25 mg hydrochlorothiazide designed for dysphagia patients. a simple and rapid method of analysis was developed and validated according to the ich guideline using hplc with uv detector. tablets were prepared by direct compression and then coated with the slippery coat of three different concentrations of the slippering substance “xanthan gum’ (2%, 3% and 4%) in opadry colorcone® and evaluated according to usp. slipperiness test was performed using albino rabbits. results showed that 2% xanthan gum gave the shortest swallowing time. also, disintegration time was increased by the coat significantly with the increase of the gum’s concentration in the coat. the release kinetics study of the tested formulations (uncoated versus coated with 2% gum) gave the highest correlation for the "first-order release model" for both drugs in the absence and presence of the slippering agent which indicates that the coating did not interfere with the release kinetics of both drugs. in a conclusion, 2% xanthan gum as slippering agent was the optimum concentration used to promote easy ingestion of this tablet. keywords: dysphagia, hydrochlorothiazide, irbesartan, oroslippery tablets (ost), and slipperiness test, hplc, ich guideline. زلقة التي تحتوي على مزيج اربيسارتان وهيدروكلور ثايازايد نوتقييم األقراص الفموية الم تحضير * إسراء حامد العاني ،*شعالن مصطفى نعيم ،1**،ياسر قاسم الماجدي ،*ياسين طه خلف *** أبوديةأحمد وائل و االردن -قسم الصيدالنيات والتقنية، كلية الصيدلة، جامعة عمان االهلية* العراق -قسم الصيدالنيات، كلية بغداد للعلوم الطبية، بغداد ** ا األرد -كلية الصيدلة، جامعة مؤتة *** الخالصة ( المنزلقة تحفيز ostsاألقراص عامل دمج تحضيرها يتضمن البلع. عسر لمرضى األقراص بلع لتحسين تستخدم تقنية هي ) وهو مدرر والهيدروكلورثايازايدوالذي هو عامل مخفض لضغط الدم االنزالق في مادة الطالء. تهدف هذه الدراسة الى صياغة عقار االربيسارتان لمرضى عسر البلع مصممة ملغم هيدروكلورثايازايد 25لغم اربيسارتان وم 150على شكل أقراص فمويه منزلقة تحتوي على ( باستخدام التحليل السائل عالي ichالمؤتمر العالمي للتوافقية ) تم تطوير طريقة بسيطة وسريعة للتحليل والتحقق من صحتها وفقًا إلرشادات الكفاءة مع كاشف االشعة فوق البنفسجية. ٪ 3٪ ، 2تم تحضير األقراص عن طريق الضغط المباشر ثم تم تغطيتها بطبقة زلقة من ثالثة تراكيز مختلفة من المادة المنزلقة "صمغ الزانثان" ) الغالف "كولوركون" ثم تقييمها حسب دستور االدوية االمريكي. ٪( في4و ٪ أعطى أقصر وقت للبلع. كما زاد وقت تفكك الطبقة بشكل 2ائج أن صمغ الزانثان أظهرت النتو تم إجراء اختبار االنزالق باستخدام أرانب ألبينو . في الغالف الصمغكبير مع زيادة تركيز ( أعلى ارتباط لـ "نموذج اإلطالق من الدرجة األولى" لكال الزانثان٪ صمغ2أعطت دراسة حركية اإلطالق للصيغ المختبرة )غير المطلية مقابل . وجود عامل االنزالق مما يشير إلى أن الطالء لم يتدخل مع حركية إطالق كال العقارينالعقارين في غياب و هذه االقراص. ٪ صمغ الزانثان كعامل انزالق ، هو التركيز األمثل المستخدم لتعزيز سهولة تناول2 فإن في الختام ، رشادات ، االتحليل السائل عالي الكفاءة ، اختبار االنزالق ، األقراص الفموية المنزلقة ، اربيسارتان ، هيدروكلور ثايازايد، عسر البلع لكلمات المفتاحية:ا المؤتمر العالمي للتوافقية 1corresponding author e-mail: almajidiyasir@gmail.com received: 29/9 /2021 accepted: 5/12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp91-100 iraqi j pharm sci, vol.31(2) 2022 oroslippery tablets irbesartan and hydrochlorothiazide 92 introduction the term “dysphagia” is widely used to describe a symptom that is manifested as subjective perception of swallowing difficulty during the passage of a liquid or a solid bolus from the oral cavity to the stomach or obstruction perception during swallowing (1-3). oropharyngeal dysphagia is a common type of dysphagia that refers to an oral preparatory, pharyngeal and/or oral swallowing disturbance (4). various dosage forms are developed to combat dysphagia for which medications are administered into the oral cavity even without requirement for chewing to create a systemic or local effect. effervescent and lozenges tablets, as well as sublingual (sl), buccal, orodispersible (odts), and the newly developed oroslippery (os) tablets, are among them (5-8). dysphagia is a serious problem requires continuous treatments; new formulations will be helpful in controlling the progression of this problem and offer comfort to the patients. modification of the available dosage forms especially the widely used oral tablets is an ongoing process in pharmaceutical industry. one new modification is by the introduction of a material that induce a slipperiness characteristic to the tablet coat which facilitate easy smooth swallowing of the tablet (9,10) hypertension (htn) affects over one billion persons globally, with a systolic bp of higher than the 140 mmhg and/or the diastolic bp lesser than the 90 mmhg. htn is a serious health issue that is a common risk factor of cardiovascular disease (cvd) and death (11). more than 50% of hypertensive patients have additional cardiovascular risk factors. the most common additional risk factors are diabetes (15%– 20%), lipid disorders (elevated low-density lipoprotein-cholesterol [ldl-c] and triglycerides [30%]), overweight-obesity (40%), hyperuricemia (25%) and metabolic syndrome (40%), as well as unhealthy lifestyle habits (e.g., smoking, high alcohol intake, sedentary lifestyle). the presence of one or more additional cardiovascular risk factors proportionally increases the risk of coronary, cerebrovascular, and renal diseases in hypertensive patients (12). irbesartan is administered orally and competitively antagonize the angiotensin ii receptors that is being used to manage the hypertension and the kidney impairment in hypertensive and diabetic individuals. among humans’ smooth muscle vascular cells, it shows high affinity to the receptor of at1, causing a decrease of peak response for angiotensin ii (13). irbesartan is a solid crystalline powder slightly soluble in water with m.wt. 428.5 and pka(s) 4.08, 4.29 (14). hydrochlorothiazide is a diuretic of the thiazide form that has been available for more than 5 decades for clinical use (14). hydrochlothiazide hydrochloride is a thiazide derivative with mwt (334.2 g/mol). it is a white crystalline powder, slightly soluble in water and freely soluble in sodium hydroxide solution.(15) irbesartan is available in a fixed-dose combination formulation with hydrochlorothiazide, to achieve an additive antihypertensiveeffect. irbesartan/hydrochlorothiaz ide combination preparations are marketed under various brand names. a 25mg of hct with 300mg irb can exhibit synergistic antihypertensive efficacy in such a dose-dependent way with excellent tolerability in a variety of patient classes. in individuals who are not managed neither irb nor hct alone, the mixture lowers the bp considerably. the combination of these drugs had a beneficial impact. irb seemed to attenuate hypokalemia, uric acid and the total cholesterol side effects that caused by hct in many experiments (16). oroslippery tablets (osts) aim to facilitate the swallowing of the tablets by the modification of the coat to offer slippery smooth passage of the tablet especially that of large size through pharynx and esophagus. very few studies are available and many factors that affect the efficiency of the coat are still in need to be studied. a study of mahdi and marie in 2015 described coating of valsartan tablet with kollicoat immediate release (ir) (film former) and xanthan gum(slipperiness inducer) at different coating levels.(10) opadry colorcone is a coating mixtute powder introduced by colorcone. it is one-step film coating system which combines polymer, plasticizer and pigment, as required, in a dry concentrate. different types of opadry are available depending on the type of polymer if it produces immediate release, controlled release or enteric coating. (17) xanthan gum is an extracellular polysaccharide secreted by the microorganism xanthomonas campestris. commercially it is manufactured by a fermentation process. the xanthan gum polysaccharide consists of a backbone of β‐(1→4) linked d‐glucose molecules. every second glucose molecule is substituted at c3 with a trisaccharide side chain consisting of β‐d‐mannose‐(1→4)‐β‐d‐glucuronic acid‐(1→2)‐α‐d‐mannose. it has many uses as food additives, viscosity builder and pharmaceutical uses. (18) the aim of this study is to formulate a combination of irbesartan and hydrochlothiazide as oroslippery tablets by formulation of this combination as core tablets then coating with https://en.wikipedia.org/wiki/fixed-dose_combination https://en.wikipedia.org/wiki/hydrochlorothiazide iraqi j pharm sci, vol.31(2) 2022 oroslippery tablets irbesartan and hydrochlorothiazide 93 opadry colorcone immediate release tablets with different ratios of xanthan gum as slippery agent and evaluation of the tablets. materials and methods materials irbesartan was purchased from (purity 99.5%, jiangsu yew pharmaceuticals co. limited, china) hydrochlorthiazide (purity 99.6% provizer pharma, india), sodium starch glycolate, avicel (ph102), aspartame, mannitol, peppermint flavor, aerosol (hydrophilic grade), na stearate, xanthan gum and opedry ® colorcon were all given as gift from dar al dawa pharmaceutical/jordan. methanol (merck) hplc grade, tetrahydrofuran, sodium acetate (sigma), acetic acid (sigma), sodium hydroxide (sigma). methods chromatographic condition high performance liquid chromatography (hplc) was used as a method of analysis to detect and quantitate irb and hct in tablet intended to be prepared. after several trials the chromatographic conditions showed in table 1 were decided and followed. the elusion was isocratic were the composition and flow rate of mobile phase was constant along the run time. table 1. chromatographic conditions used in method development component type hplc server (lc-thermo) with by lc solution software detector uv detector. wavelength 265 nm mobile phase methanol-tetrahydrofuran: acetate buffer ph 6.5 (47:10:43) v/v/v. flow rate 1ml/min injection volume 20 µl total run time 9 min column supelcosil c18 (150 mm × 4.6 mm, 5 nm part..size) column temp. room temp (25oc). the method was validated by measuring linearity, precision, accuracy and recovery for both drugs measured simultaneously. linearity of irb was measured by measuring concentrations range (10, 20, 40, 80, 140, 180) μg /ml and of hct (1, 12, 24, 36, 44, 50) μg/ml. regression and correlation coefficient was calculated. inter and intra-day precision of the method were determined by the analysis of 6 samples (calibration conc.) with 3 replicates and calculation of percent coefficient of variation (%cv). while recovery of drugs was calculated by analysis of powder mixture (before compression). conc. of irb used were (35, 50, and 65 μg/ml) and for hct also, (31, 45, and 58 μg/ml) μg/ml) using different degrees of dilution. all tests were performed and evaluated according to the ich guideline. formulation of core tablet a standard formula was prepared as in table 2. one hundred tablets were prepared and evaluated. then other batches were prepared for coating table 2. composition of irb-hct combination core tablet ingredient amount (mg) /tab. irbesartan (irb) 150 mg hydrochlorothiazide (hct) 25 mg sodium starch glycolate 20 mg avicel (ph102) 150 mg mannitol 47 mg aerosil (hydrophilic grade) 4 mg na stearate 4 mg total weight 400 mg one hundred (100) tablets batch of the formula was prepared. all ingredients were weighed, and mixing process was performed manually by wide mouth conical flask. first all materials were sieved through mesh no. 36 to get rid of any particulate materials. then all constituents except the lubricant were put the flask and rotated manually in one direction for 20 min. after that the lubricant was sieved in 60 mesh size and added to the mixture and mixed for 1-2 min (19). the obtained powder blend was directly compressed into tablets using tablet press (erweka, single punch compression machine) using oval shape upper punch and die (20). one hundred tablets of the formula were compressed evaluation of the prepared tablets uniformity of weight weight variation of the prepared tablets were performed by weighing, randomly selected, twenty tablets individually from each formula using electrical sensitive balance (shimadzu, japan) and the average weight ±sd was calculated (21). tablet thickness thickness of tablets was measured using thickness caliper (mitutoyo® cd-15b, england) for 10 randomly selected tablets. average thickness in mm ±sd was recorded (22). iraqi j pharm sci, vol.31(2) 2022 oroslippery tablets irbesartan and hydrochlorothiazide 94 hardness and friability the breaking force (hardness) was measured using hardness taster (dr. schleuniger pharmatron 8m, switzerland). the test was performed in the beginning, during and at the end of the tablet production to ensure a fixed hardness over the production process. friability of the tablets was determined using (erweka® ta 100 friability tester, germany). twenty tablets were initially weighed (wt1) and transferred into friabilator. the friabilator was operated at 25 rpm for 4 mins. the tablets were weighed again (wt2). the percentage (%) of friability was then calculated using equation 7: percentage of friability = 𝑾𝟏−𝑾𝟐 𝑾𝟏 x 100 % where w1 is weight of 20 tablets before placing in friabilator and w2 is the weight of 20 tablets after taking out of friabilator (23). disintegration time disintegration time was determined using the disintegration apparatus usp (electrolab, bangalore, india) in d.w maintaining the temperature at 37 ± 1°c. six tablets were used. each tablet was put in each vessel and time of disintegration was recorded in minutes± sd (24). assay of apis in the tablets twenty tablets were taken randomly from the prepared tablets and crushed to fine powder. a powder weight was taken equivalent to 150 mg irb and to 25 mg hct. mobile phase of the validated method was used to solubilize the drug each time to 50 ml total volume. solution was filtered through 0.45 µm membrane filter to get clear solution. suitable dilutions were made to get concentration of irb 50 µg/ml and 40 µg/ml hct and each drug content was calculated from its calibration curve(25). coating process tablets were coated using opadry colorcone® to which added the slippering agent in different concentrations and other additives. table 3 shows the coating formulations. three liters of each coating formula was prepared. solid materials were all weighed and put aside. a mixture of solvent system (60:40) water: ethanol (90%) was prepared in 3500 ml hard glass beaker and put under electrical stirrer. 2750 ml solvent system was put first then gradually adding the solid material while starting low speed stirring until the whole quantity is added. the stirring speed was then increased until fixed onto 1000 rpm for 40 min. then the speed was gradually decreased to avoid air bubbles and stopped. volume was completed to 3 l and additional 15 min of stirring were added to ensure homogenization. the coating dispersion was freshly prepared before each coating run (17). due to small number of tablets prepared, the coating pan was loaded with placebo tablets (made of compressed avicel) to make tablets rolling over and the optimization of coating parameters easier (17). the tablets prepared for the animal study were smaller in size (50 mg wt.) in addition to the placebo bulk. the bed was pre-heated to 40oc using dryer and thermometer, the coating pan was loaded with the tablets and switched on. few tablets were taken after 60, 80 and 100 min for inspection and weighing. the coating process was stopped after 100 mins. where the weight gain was 3 % (average 11-14 mg) (26). table 3. the experimental design and formulas of the slippering coat. material control formula (cf)(%) coating formula 1 (cf1)(%) coating formula 2 (cf2)(%) coating formula 3 (cf3)(%) opadry 12 12 12 12 xanthan gum (slippering agent) 0 2 3 4 peppermint flavor 0.5 0.5 0.5 0.5 aspartame (sweetener) 0.1 0.1 0.1 0.1 total solid concentration 12.6 14.6 15.6 16.6 evaluation of the coated tablets weight gain by coat weight uniformity test was repeated on coated tablets and the average weight gain by coating process was calculated.(27) thickness of coated tablets thickness of coated tablets was measured to check the increase in tablet thickness due to coating process. 10 tablets were chosen randomly, and the thickness was measured by (mitutoyo® cd-15b, england). average thickness ±sd was recorded and compared with the thickness of the uncoated tablet statistically. (22) disintegration time disintegration time was repeated for the coated tablets, average time ±sd was recorded for iraqi j pharm sci, vol.31(2) 2022 oroslippery tablets irbesartan and hydrochlorothiazide 95 each formula and compared statistically to the standard uncoated formula. (24) slipperiness test four albino rabbits were used in this test. (ethical approval dec. 6/7 20-21, al-ahliyya amman university). each one received 3 tablets of the specified concentration of the slippering agents. rat 1 received tablets coated with opadry without slippering agent, rat 2 received coated tablets with 2% slippering agent, rat 3 received tablets of 3% slippering agent and rat 4 received tablets of 4% slippering agent. the rabbits were given water to drink immediately prior the test to avoid dry mouth. then a tablet is put on his tongue and time is recorded as zero time, then the hand is put gently on his throat to sense the swallowing of the tablet. this moment is recorded as time from zero. three readings were taken for each tablet tested, average ±sd was estimated.(10) drug release and dissolution the dissolution test conditions were chosen according to the usp monographs and published works of both drugs. usp app ii with 0.1 n hcl (900 ml) as a dissolution media, 75 rpm stirring rate and the temperature of 37±0.5 oc were chosen as dissolution conditions. time points: 5, 10, 15, 20, 30, 45, 60 min. six (6) tablets were put each in a jar and 5 ml samples were withdrawn and replaced by fresh media to keep sink condition, samples were taken at time points; 5, 10, 15, 20, 30, 45, and 60 min., filtered through 0.45µm and suitably diluted by mobile phase and analyzed using the validated method developed and data was reported as average conc. ±sd of each reading. then percent drug dissolved vs time was plotted as the dissolution profile. dissolution test was performed on the uncoated formula and the coated formulas. (28) results and discussion method development and validation irb absorbs at 270 nm (29) and hct has maximum absorption at 238 and 271 nm (30). these values differ slightly according to the method. here, 265 nm was chosen as suitable λ for best results where a good absorption of both occurs and both peaks appeared in the same chromatogram clearly using the specified mobile phase. hct was eluted first with rt = 3.0 min, then next irb with rt= 6.5 min. figure 1 shows one chromatogram of irb and hct and the clear separation of the two apis. figure 1. chromatogram showing peaks of hct (30 µg/ml) and irb (80 µg/ml) irb and hct showed linearity in the specified concentrations with correlation coefficient equals to 0.999 for both drugs and relative standard deviation (rsd) less than 2.00. calibration curves are shown in figure 2 (a and b). 4 a b figure 2. calibration curves of irbesartan (a) and hydrochlorothiazide (b) inter and intra-day precision results gave the mean value of percent accuracy between 97.7 101.1% for both irb and hct and the %rsd is below 2%, which means an accepted criterion according to the ich guideline. recovery of irb from the formula was 99.3% with rsd 0.46 and that of hct was 98.0 with rsd equals to 0.9. formulation design and powder preparation the master formula was prepared using sodium starch glycolate as disintegrant, avicel and mannitol as bulking agents. the powder mix was iraqi j pharm sci, vol.31(2) 2022 oroslippery tablets irbesartan and hydrochlorothiazide 96 prepared, and no segregation of materials was detected. this may be related to close particle size achieved by sieving before mixing process. no clogging or attachment of powder to the flask walls was observed except minor dusting. physical evaluation of the prepared tablets the obtained tablets were inspected visually. tablets were good shaped with sharp edges, shiny, smooth with bright white color. no cracks, no capping or peeling were observed. considering uniformity of weight, the average weight of ten tablets was measured individually according to the specification of the usp (21). tablets weigh less than 180 mg might have less than ±10 % of weight variation. tablet’s weight 180-325 mg would accept ±7.5 % while tablets weigh more than 325 mg would accept ± 5 % of its weight variation. no tablet should weigh more or less than double the allowed percent variation. average theoretical weight of the tablets is 400 mg, and it follows the third category all tablets were in the usp specifications, and this reflects the efficiency of compression process. results are shown in table 8. thickness of tablets are shown in table 8. coating will add to the tablets and would be measured again to detect how much thickness the coat would add. hardness and friability hardness of tablets should be enough to handle coating process and satisfy the properties of immediate release tablets and allow disintegration and dissolution. friability of tablets should not exceed 1% according to the usp which is like regular tablets. after several trials in compression of the formula, the hardness was kept on 5 kg and friability was below1%. results of weight uniformity, hardness, friability and disintegration test are shown in table 5. disintegration time of the uncoated tablets was 5.6 ± 0.8 min, this result is compatible with the specification of usp of immediate release tablets. table 5. results of physical evaluation of the uncoated tablets test results (average ± sd) weight uniformity test (mg) n= 20 403±6.2 hardness (kg) 5.0 ± 0.1 friability (%) 0.3 disintegration time (min) 5.6 ± 0.8 min assay of active ingredient in the tablets results of assay of apis in the prepared tablets are presented in table 6. percent drug recovery of irb and hct was high and close to 100% knowing that the guideline allows 90-110% drug content in the dosage form. table 6. results of assay of irb and hct in the prepared uncoated tablets tablets apis theoretical concentration (µg/ml) average area measured n=3 actual concentration (µg/ml) percent content (%) precision (rsd) irb 50.0 21047.42 48.90±1.2 97.8 0.8 hct 40.0 37735.6 41.1±0.9 102.75 0.7 tablet coating the coated tablets with the slippering coat were prepared as described earlier. the 3 batches were re-evaluated physically before performing the slipperiness test. the coated tablets were examined visually. they showed good appearance, uniform white color with no peeling or mottling. tablets are shown in figure 3. weight gain by coating film coating should not add much weight to the tablets. weight gain was checked during or before finishing the process. however, reweighing the tablets after coating and drying was done. results gave increase in the weight about 3.25%. thickness was also increased due to the coating process. results showed increase in thickness about 1.3 % as illustrated in table 7. figure 3.the coated tablets and the animal model iraqi j pharm sci, vol.31(2) 2022 oroslippery tablets irbesartan and hydrochlorothiazide 97 disintegration time disintegration test was repeated for the coated tablets to investigate effect of coat and slippering agent on the physical characteristics of the tablets. disintegration time was elongated by coating due to extra wetting time needed by the coat. increasing the percent of xanthan gum resulted in increase in disintegration time as shown in table 7. xanthan gum is an extracellular bacterial exopolysaccharide synthesized by xanthomonas campestris. the unique properties of xanthan gum make it widely used in different applications. it is highly soluble in water, stable over a wide range of temperature, acidic and alkaline conditions (31). xanthan gum is a stabilizer used in suspensions and cosmetics that helps “binding” materials together. possibly this “net” formed by the gum within the film resulted in increase in disintegration time of the coated tablets, although all values are still accepted according to the usp. statistically, disintegration time increased significantly (p<0.05) of all coated formulas compared to the uncoated formulas. and the disintegration time was increased significantly in cf1, cf2 and cf3 compared to cfe (without xanthan gum). table 7. weight gain and disintegration time of the coated formula. formula code average weight (mg) disintegration time (min: sec) uncoated formula s 402 ± 3.2 1:00 ± 2 sec cf (0%) e 406.8 ± 2.5 1:10 ± 2 sec cf1 (2%) 408 ± 3.2 1:30 ± 3 sec cf2 (3%) 407.2 ±3.1 2:20 ±4 sec cf3 (4%) 405.5 ±2.3 3:00 ± 3 sec slipperiness test results of slipperiness test are listed in table 8. the properties that enable the application of xanthan gum in pharmaceutical industries are emulsifying, thickening, stabilizing, film forming and gelling nature (31). here, xanthan gum is used as “slippering agent”. this characteristic is obtained due to its nature of fast wetting and forming a thin stable layer within the film that have that slippery nature when mixed with saliva. the coated tablet without slippering agent (cf) took the longest time in swallowing and swallowing was difficult and the animal was helped to swallow it. all coated formulas with the slippering agent gave significantly shorter time of swallowing than cf (p<0.05). cf1 gave the shortest slippering time among the three concentrations used (30 ± 1 sec) and it was significantly shorter than cf2 (37 ±2 sec) and cf3 (40±2 sec) (p<0.05). increasing the concentration of xanthan gum from 2% to 3 and 4 %, did not enhance the slipperiness of the tablets, on the contrast it resulted in elongation of time. although xanthan gum is described as (non-gelling agent), other studied described xanthan gum as gel inducing agent (16) but increasing concentration was observed to form “over wet” sticky tablet which in turn faced a slight difficulty in swallowing it. figure 5 show the performance of the test. in conclusion, 2% xanthan gum with opadry immediate release coat is the best concentration used to enhance swallowing of this tablet.(10) table 8. results of slipperiness test on rabbit formula code percent of slippering agent slippering time (sec) cf 0% 80±5 cf1 2% 30±1 cf2 3% 37±2 cf3 4% 40±2 figure 4. performing slippering test in albino rabbit dissolution of irb and hct figures 5 and 6 show the dissolution rate profile of the uncoated formula and cf1 which gave the best result in slipperiness test in animal. both apis were released from the uncoated ablets (>75 % in 45 min). when they were coated, the dissolution profile was highly like that of the uncoated (also >75 % in 45 min). taking the 30 min as a comparative point, non-significant iraqi j pharm sci, vol.31(2) 2022 oroslippery tablets irbesartan and hydrochlorothiazide 98 difference (p>0.05) was observed between the release of both apis from the coated formula cf1 with respect to the coated formula (table 9). this indicates that the coat didn’t have a negative effect on the drug release. it neither enhanced nor delayed drug release. the only effect was an elongation in the disintegration time which resulted in these slight differences in amount of drug released with time. mahdi and maraie, 2015 showed also that 0.3% xanthan gum dis not affect the release of valsartan from ost using different ratios of coating materials (9). studying the release kinetics of irb and hct from the tested formulas (uncoated and cf1) gave the highest correlation (r) of “first order release model” for irb (uncoated): 0.987, hct (uncoated): 0.975 and for cf1, irb 0.979 and hct 0.982. this indicates that the coat also did not interfere with release kinetics of both apis and the idea of “immediate release tablet dosage forms figure 5.dissolution profile of irb and hct of uncoated tablets at 0.1 n hcl, 37oc and 75 rpm showing t3. figure 6.dissolution profile of irb and hct of formula cf1 at 0.1 n hcl, 37oc and 75 rpm showing t30. table 9. t30 of release of irb and hct from the uncoated formula and cf1 t30 (percent api released in 30 min) percent api released in 30 min t(30%) t-test result irb from uncoated tablet 70 ± 2.0 % irb from cf1 68 ±3.0 % p> 0.05, non-significant compared to the uncoated hct from uncoated tablet 77 ±2.0% hct cf1 77 ±2.5.0% p> 0.05, non-significant compared to the uncoated conclusion a successful, simple, valid method of analysis for simultaneous determination of irb and hct was developed using hplc with uv detector. all coated formulas with the slippering agent gave significantly shorter time of swallowing than cf (p<0.05). cf1 gave the shortest slippering time among the three concentrations used (30 ± 1 sec) and it was significantly shorter than cf2 (37 ±2 sec) and cf3 (40±2 sec) (p<0.05). increasing the concentration of xanthan gum from 2% to 3 and 4 %, did not enhance the slipperiness of the tablets, on the contrast it resulted in the elongation of time. in conclusion, 2% xanthan gum with opadry immediate release coat is the best concentration used to enhance swallowing of this tablet. iraqi j pharm sci, vol.31(2) 2022 oroslippery tablets irbesartan and hydrochlorothiazide 99 acknowledgment the authors would like to thank alahliyya amman university, amman-jordan for supporting this work. thanks are extended to dar al dawa pharmaceuticals in jordan for their help and support. authors contributions all the authors have contributed equally conflict of interests declared none references 1. khan a, carmona r, traube m. dysphagia in the elderly. clin geriatr med 2014; 30: 43–53. 2. abdel jalil a a, katzka d a, & castell do. approach to the patient with dysphagia. am j med 2015;128(10): 17-23. 3. chang mc, lee c, park, d. validation and inter-rater reliability of the modified videofluoroscopic dysphagia scale in dysphagic patients with multiple etiologies. j clin med. 2021; 10(13): 2077-0383. 4. vieira jm, andrade cc , santos tt, okuro pk , garcia st, rodrigues mi, vicente aa, cunha rl. flaxseed gum-biopolymers interactions driving rheological behaviour of oropharyngeal 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and validation of uvspectrophotometric method for simultaneous estimation of amlodipine besylate and hydrochlorothiazide in combined dosage form including stability study. j pharm sci biosci res 2015; 5: 487–493. 31. lopes b, lopes l, silva b, & carvalho m, schnitzler e, lacerda, l. xanthan gum: properties, production conditions, quality and economic perspective j food nutr res 2015; 54:185-194. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 anti-inflammatory activity of gingko biloba extract doi: https://doi.org/10.31351/vol31iss1pp184-193 184 anti-inflammatory activity of gingko biloba extract in cotton pelletinduced granuloma in rats: a comparative study with prednisolone and dexamethasone ahmed azad kareem *, tavga ahmed aziz*,1, zheen aorahman ahmed*, hemn hassan othman * and saad abdulrahman hussain ** * department of pharmacology and toxicology, college of pharmacy, university of sulaimani, sulaimani, iraq ** department of pharmacy, al-rafidain university college, baghdad, iraq. abstract the current study was designed to evaluate the anti-inflammatory effect of gkb in the rat model of granulomatous inflammation. thirty rats were distributed into five groups: the first group served as negative control group that received distilled water (dw) only without inducting inflammation, positive control group; treated with dw with the induction of inflammation and they were assigned to cotton pellet-induced granuloma, ginkgo biloba (gkb) treated group (200mg/kg/day), dexamethasone-treated group (1mg/kg), and prednisolone treated group (5mg/kg). all the treatments were given orally for seven consecutive days. on day eight, the rats were anesthetized and the pellets together with granulation tissue were carefully removed and made free from extraneous tissue. the weight and the percent of the exudate and granuloma were determined and samples of the tissues were sent for histopathological examination. blood samples were collected by cardiac puncture and used for the analysis of the inflammatory markers: tnf-alfa, il10, vcam-1, and hs-crp. the study revealed a significant reduction in the weight and the percent of exudate (p-value = 0.019), (17%) and granuloma (p-value = 0.013), (20%) by gkb which was comparable to that produced by prednisolone. all the treatment groups showed a significant reduction in serum tnf-α, vcam-1, and hs-crp concentration compared to the positive control. the histopathological finding revealed pronounced improvement. in the current study, gkb was effective in attenuating the level of inflammation by decreasing the exudate, granuloma, and inflammatory markers. the underlying mechanisms could be the inhibitory effect on the expression of the inflammatory cytokines and endothelial adhesion molecule. these findings suggest gkb as a good contender to be tested in the treatment of inflammatory diseases. keywords: gkb extract, dexamethasone, prednisolone, granuloma, inflammatory markers. في نموذج الورم الحبيبي المستحدث في الجرذان: التاثير المضاد لاللتهابات لمستخلص الجينكوبيلوبا دراسة مقارنة مع كل من الدكساميثازون والبريدنيزولون احمد ازاد كريم * ، تافكة احمد عزيز *،1 ، زين اورحمان احمد * ، هيمن حسن عثمان*و سعد عبد الرحمن حسين ** العراق سليمانية، سليمانية، جامعة الصيدلة، كلية والسموم، فرع االدوية * العراق. ، الجامعة، بغدادكلية الرافدين قسم الصيدلة، ** الخالصة في نموذج الجرذان لاللتهاب الحبيبي. تم تقسيم ثالثين جرذاً إلى خمس gkb تم تصميم الدراسة الحالية لتقييم التأثير المضاد لاللتهابات لـ قطر مجموعات: مجموعة السيطرة السلبية: عولجت بالماء المقطر فقط بدون تحريض لاللتهاب ، مجموعة السيطرة اإليجابية. تم عالجهم بالماء الم مجم / كجم / يوم( ، والمجموعة 200) (gkb) مجموعة المعالجة بالجنكوبيلوبافقط وتم تعيينهم على الورم الحبيبي الناجم عن حبيبات القطن ، وال مجم / كجم(. أعطيت جميع العالجات عن طريق الفم لمدة سبعة 5مجم / كجم( ، والمجموعة المعالجة بالبريدنيزولون ) 1المعالجة بالديكساميثازون ) لة الكريات مع األنسجة الحبيبية بعناية وجعلها خالية من األنسجة الدخيلة. تم تحديد الوزن والنسبة أيام متتالية. في اليوم الثامن ، تم تخدير الجرذان وإزا المئوية لإلفرازات والورم الحبيبي وأرسلت عينات من األنسجة للفحص التشريحي المرضي. تم جمع عينات الدم عن طريق ثقب القلب واستخدمت االلتهاب عالمات المئوية .hs-crp و vcam-1 و il10 و tnf-alfa :لتحليل والنسبة الوزن في ملحوظ انخفاض عن الدراسة كشفت . gkb ٪( بواسطة20( ، )0.013٪( والورم الحبيبي )القيمة االحتمالية = 17( ، )0.019لإلفرازات )القيمة االحتمالية = hs-crp و vcam-1 و tnf-α الجية انخفاًضا كبيًرا في تركيزوهو ما يقارن بتلك التي ينتجها بريدنيزولون. . أظهرت جميع المجموعات الع في تقليل االلتهاب gkb في الدم مقارنةً بمجموعة السيطرة اإليجابية. لوحظ تحسن كبير في النتائج النسيجية المرضية. في الدراسة الحالية ، نجح على التعبير عن السيتوكينات عن طريق تقليل اإلفرازات والورم الحبيبي وعالمات االلتهاب. يمكن أن تك ون اآلليات األساسية هي التأثير المثبط .مرشح جيد لعالج األمراض االلتهابية gkb االلتهابية وجزيء االلتصاق البطاني. تشير هذه النتائج إلى أن ، ورم حبيبي ، عالمات التهابية ، ديكساميثازون ، بريدنيزولون gkb مستخلص الكلمات المفتاحية: 1corresponding author e-mail: avga.aziz@univsul.edu.iq received: 30/7/2021 accepted: 22/9 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp184-193 iraqi j pharm sci, vol.31(1) 2022 anti-inflammatory activity of gingko biloba extract 185 introduction inflammation is a part of the body's defense mechanism against any harmful stimuli such as tissue injury and infections (1). it is a beneficial process by which the host’s immune system eliminates these harmful agents. the inflammatory process initially starts to get rid of the causative agent and the process will be followed by repair mechanisms on the molecular and tissue levels initiating the healing process and restoring homeostasis (2). disrepair occurs when the body's defense mechanisms fail to control inflammation, it will eventually result in complications and longlasting damage (3). there are two types of inflammation: acute and chronic. acute inflammation is the initial fast response of the body to foreign agents, it is rapid in onset, starting within minutes to hours, and resolving within hours to few days after the offending agent has been eliminated successfully. while chronic inflammation is a slow response of the body to foreign stimuli, it is slow in onset, starting after days, and has a long duration of years (2). there are a bunch of medications for the management of inflammatory reactions like steroids, nonsteroidal anti-inflammatory drugs (4). steroids are broadly used medications in various diseases for their anti-inflammatory effects. in the biological system, during inflammation, a high level of unbound cortisol will be available since the affinity for binding to their receptor will be attenuated giving more chance to the free form to fight against inflammation (5). despite the great effectiveness of steroids in inflammatory diseases; unwanted effects may halt their uses such as musculoskeletal, metabolic, and endocrine adverse effects, infections, cardiovascular, neuropsychiatric, git, and dermatological adverse reactions (6). therefore, we need to seek new medications that possess anti-inflammatory activity with minimum unwanted reactions. growing evidence proved the role of medicinal plants in the management of many diseases (7). ginkgo biloba (gkb) is broadly used as nutraceutical in the developed countries. this plant possesses antioxidant, immune-modulating, and anti-inflammatory activities. additionally, ginkgo biloba extract is also used in many neurological disorders (8), cardiovascular disease (9), diabetes mellitus (10, 11), and gastrointestinal diseases such as ulcerative colitis and acute pancreatitis (1214). furthermore, gkb is effective in ameliorating hippocampal neuronal death secondary to ischemia through its anti-inflammatory and antioxidant properties (15). another study proved the effectiveness of gkb in the chronic inflammatory condition in colons of mice by suppressing the macrophage activation and down regulating the inflammatory mediators (16). all of the previous studies suggest gkb to be evaluated for the anti-inflammatory activities; accordingly, the current study was intended to test the anti-inflammatory activities of gkb in the rat model of granulomatous inflammation. methodology experimental animals thirty wistar rats with age range 8-10 weeks age of both genders weighing (150-200g) were purchased from the college of medicine/ hawler medical university and housed in the college of pharmacy/university of sulaimani from march to september 2020. the rats were kept in well-ventilated plastic cages, temperature (25±2c°), and 12h light-dark cycle. they had free access to food and water and were fed standard pellet chaw. the experiment was approved by the ethical committee of the college of medicine, university of sulaimani (certificate number 193 on september 20th, 2020). the study was performed following the canadian council on animal care (ccac) guidelines, 1998. study design thirty rats were used and allocated into the following groups (six rats in each group): 1. negative control group: treated with dw without induction of inflammation. 2. positive control group: treated with dw with the induction of inflammation and they were assigned to cotton pellet-induced granuloma. 3. gkb group: treated with gkb (200mg/kg/day) orally for the study of anti-inflammatory activity of gkb in rat model cotton pellet-induced granuloma. 4. dexamethasone treated group: treated with dexamethasone (1mg/kg) orally as a standard antiinflammatory agent, in a rat model of cotton pelletinduced granuloma. 5. prednisolone treated group: treated with prednisolone (5mg/kg) orally as a standard antiinflammatory agent, in a rat model of cotton pelletinduced granuloma. the method used for the induction of inflammation was winter and porter method (17, 18). in this method of inducing inflammation, (10±1mg) cotton pellets were used after sterilization for 30 minutes in an autoclave at 120°c, four pellets were implanted subcutaneously into the ventral region of the anesthetized rat, two on each side. ginkgo biloba (200mg/kg), dexamethasone (1mg/kg), prednisolone (5mg/kg), and vehicle distilled water (0.2ml/100gm body weight) were given one hour before the induction and the treatment sustained for seven successive days. on eighth day, the animals were anesthetized and the cotton-pellets together with granulation tissue were sensibly removed and made free from unnecessary tissue. iraqi j pharm sci, vol.31(1) 2022 anti-inflammatory activity of gingko biloba extract 186 the weight of the wet cotton-pellets was determined, and then dried using an incubator for 18 hr at 60°c, the dried pellets were weighted again; and the amount of exudate was calculated by subtracting the constant weight of dried pellets from the weight of wet pellets. the weight of granulation tissue was calculated by subtracting the weight of cotton pellets (10mg) from the weight of dried pellets. the following equations were used to calculate the percentage of inhibition of exudate and granulation tissue formation (19): exudate inhibition (%) = {1 ‒ exudate in treated group / exudate in controls} c x 100. granuloma inhibition (%) = {1 ‒ granuloma in treated group / granuloma in controls} x 100. histotechnique procedure the histological protocol was started at the endpoint of the experiment. initially, animals were fasted for at least 10 hours before sacrificing and then euthanized in a humane practice. successively, after animal sacrificing necropsy started by collecting tissue samples for histological preparation. tissue sections were stained with harris's hematoxylin and eosin and then viewed under a bright field light microscope. in brief, granuloma skin samples were immobilized into tissue cassettes then fixed with 10% neutral buffered formaldehyde solution for 48 to 72 hours. then, sections were dehydrated via passing through series of ascending ethanol alcohol (60%, 70%, 90%, and 100%), followed by three steps of xylene clearance. thereafter, skin tissues were infiltrated and embedded in melted paraffin blocks using an automated wax embedder at (60 -70ᵒc). paraffinized tissues were sectioned to 4 µm using a semiautomated rotary microtome (leica-germany). after that, tissue sections were hunted on glass slides and dried with the aid hot plate tissue holder. later on, the mounted tissue sections were deparaffinized and cleaned with xylene solution for 30 minutes and air-dried. finally, harris's hematoxylin and eosin solution was used for staining tissue sections; cleaned with xylene, and then coverslipped. histological semi-quantitative measurement a semi-quantitative measure of granuloma histological sections from the skin of each animal was measured in µm and statistically evaluated as a mean percentage. moreover, inflammatory exudate, area of granuloma and proliferated fibrous connective tissue were estimated in four fields tissue sections under medium power magnification (200x), then the mean average was measured in µm and calculated statistically in percentage. tissue sections were examined under the light microscope (novel xsz-n107, china) using an image analyzer software (amscope ver. 3.7) with aid of a microscope digital camera (mu300, 2019). the mean percentage of the calculated values were expressed as the following lesion scores (score 010% as no lesions; score 10-50% as mild; score 5075% as moderate; score 75-100% as severe lesions). statistical analysis the data were analyzed by using graphpad prism 7.00 software (graph pad software inc., san diego, ca, usa). unpaired t-test and one-way anova followed by tukey’s multiple comparisons test were utilized for statistical evaluation of the differences between the means. p<0.05 were considered statistically significant. results effect of gkb extract on the weight and percent of inhibition of exudate and granuloma in cotton pellet-induced granulomatous inflammation ginkgo biloba extract inhibited exudate formation in a rat model of granulomatous inflammation (figure 1a), achieving (17%) with the use of 200mg/kg gkb; while prednisolone produced (22%) inhibition but it was still less than that produced by dexamethasone 1mg/kg which was (33%). figure 1b shows that gkb significantly reduced the weight of exudate (p-value = 0.019) compared to the positive control, which was less than that produced by prednisolone (p-value = 0.006) while the greatest reduction in weight of exudate produced by dexamethasone 1mg/kg (pvalue < 0.001). iraqi j pharm sci, vol.31(1) 2022 anti-inflammatory activity of gingko biloba extract 187 figure 1: effect of gkb extract on the percent (a) and weight (b) of exudate in cotton pellet -induced granulomatous inflammation. values were presented as mean ± s.d (n= 6 animals in each group); values with (*) are significantly different from the positive control using anova and post hoc test (* p < 0.05), (** p < 0.01), and (*** p < 0.001). ginkgo biloba also inhibited granuloma formation. figure 2a shows that gkb 200mg/kg dose resulted in (20%) inhibition, while prednisolone 5 mg/kg inhibited granuloma formation by (23%), meanwhile, dexamethasone 1mg/kg significantly reduced the formation of granuloma by (55%). figure 2b indicates that gkb significantly reduced the weight of granuloma when compared to a positive control (p-value = 0.013) which was comparable with that produced by prednisolone 5mg/kg (p-value = 0.04) but, still significantly less than that produced by and dexamethasone 1mg/kg (p-value = 0.0001). figure 2: effect of gkb extract on the percent (a) and weight (b) of granuloma in cotton pellet-induced granulomatous inflammation. values were presented as mean ± s.d (n= 6 animals in each group); values with (*) are significantly different from the positive control using anova and post hoc test (* p < 0.05), (** p < 0.01), and (*** p < 0.001). effects of ginkgo biloba on the inflammatory markers in granuloma model of inflammation in the granuloma model of inflammation, figure 3a shows a significant attenuation (p = 0.02) in the level of serum tnf-α compared with the positive control (22.43 ± 3.098 vs 37.98 ± 4.867) was achieved by the use of gkb 200 mg/kg. dexamethasone and prednisolone also resulted in significant (p = 0.02) and (p = 0.004) reduction in serum tnf-α level (22.73 ± 1.93 vs 37.98 ± 4.867) and (18.43 ± 3.287 vs 37.98 ± 4.867) respectively. figure 3b revealed a non-significant reduction in the level of il-10 in the positive control group when compared to the negative control. the use of gkb, dexamethasone, and prednisolone produced no significant change when compared to the positive control. iraqi j pharm sci, vol.31(1) 2022 anti-inflammatory activity of gingko biloba extract 188 figure 3. effects of gkb extract on the inflammatory markers tnf-alfa (a) and il10 (b) in granuloma model of inflammation. values were presented as mean ± s.d (n= 6 animals in each group); values with (*) are significantly different from the positive control using anova and post hoc test (* p < 0.05), and (** p < 0.01). the result presented in figure 4a shows that the level of vcam-1 in the positive control group was non-significantly increased when compared to the negative control. ginkgo biloba 200 mg/kg revealed a significant (p = 0.006) reduction in the level of vcam-1 compared to the positive control (1.94 ± 0.2731 vs 3.367 ± 0.286); more significant (p = 0.0005) and (p = 0.0001) reduction was observed with the use of dexamethasone and prednisolone (1.617 ± 0.1887 vs 3.367 ± 0.286) and (1.533 ± 0.1174 vs 3.367 ± 0.286) respectively. figure 4b displays a significant (p = 0.029) increase in the level of hs-crp in the positive control in comparison with the negative control (28.33 ± 4.773 vs 15 ± 2.236) and the use of gkb, dexamethasone and prednisolone resulted in resulted in a significant (p = 0.005), (p = 0.003), and (p = 0.015) decrease compared with the positive control (8.02 ± 1.98 vs 28.33 ± 4.773), (10 ± 0 vs 28.33 ± 4.773) and (10 ± 0 vs 28.33 ± 4.773) respectively. figure 4.effects of gkb extract on the inflammatory markers vcam-1 (a) and hs-crp (b) in granuloma model of inflammation. values were presented as mean ± s.d (n= 6 animals in each group); values with (*) are significantly different from the positive control using anova and post hoc test (* p < 0.05), (** p < 0.01), and (*** p < 0.001). iraqi j pharm sci, vol.31(1) 2022 anti-inflammatory activity of gingko biloba extract 189 histopathological findings primarily, table 1 reveals morphometric assessment and lesion scoring of foreign body granuloma indued with cotton pellets inserted subcutaneously. generally speaking, animals in group g2 (granuloma positive control) show significant augmentation in the area of granuloma in comparison to g1 (control negative group) with no granuloma lesion, demonstrated by profound deposition of eosinophilic inflammatory exudate together with diffuse infiltration of inflammatory cells. moreover, a small area of dystrophic calcification can be seen within the granuloma lesion. on the other hand, animals treated with prednisolone 5mg/kg (g5) and dexamethasone (g4) 1mg/kg reveal a statistically significant and obvious reduction in the area of granuloma and inflammatory exudates together with an increasing amount of proliferated fibrous connective tissue in comparison to g2, indicating the process of healing since it was more prominent in g5 animals treated with prednisolone. additionally, and remarkably, animals in g3 who received 200mg/kg ginkgo biloba demonstrated a significant p<0.05 decrease in the extent of granuloma lesions evident by dropping in the amount of inflammatory exudates and proliferation of more collagen fibers. therefore, accordingly, treatment with 200mg/kg of ginkgo biloba show actual therapeutic effect against experimentally induced granuloma lesion, however, it is more effective in animals treated with 5mg/kg prednisolone and 1mg/kg dexamethasone, respectively. all the results were shown in table 1 and figure 5. table 1 .histological evaluation of granuloma lesion experimental groups n=5 area of granuloma * (mean %)** inflammatory exudate (mean %)** connective tissue thickness * (mean %)** lesion scoring (0 -100%) lesion grading (g1) cng† 0.1 % a# 1.6 % a 8.4 % a 0-10 % no lesion (g2) grc 96.2 % e 89.3 % e 78.5 % d 75-100 % severe (g3) gr+ gkb 200mg 62.3 % b 64.7 % b 68.1 % c 50-75 % moderate (g4) gr+ dex 1mg 56.3 % d 53.4 % d 62.8 % d 50-75 % moderate (g5) gr+ prd 5mg 52.8 % c 51.4 % c 73.5 % c 50-75 % moderate notes: *area of granuloma, inflammatory exudate and connective tissue thickness were estimated by (µm). **each value represents mean percentage (n=6). #statistical comparison among groups: mean values with different capital letters have significant differences at (p < 0.05). †g1: control negative group; g2: granuloma positive control; g3: granuloma and ginkgo biloba 200mg/kg; g4: granuloma and dexamethasone 1mg/kg; g5: granuloma and prednisolone 5mg/kg. iraqi j pharm sci, vol.31(1) 2022 anti-inflammatory activity of gingko biloba extract 190 figure 5.photomicrograph of skin from groups; (g1) control group, show distinctive structural arrangement of the epidermis (ep) and dermis (d), with the presence of several sebaceous glands (sg) attached to hair follicles together with some muscle tissue (mt) seen in the skin section. (g2) granuloma positive group, demonstrate the area of granuloma (gr) infiltrated with significant amount of inflammatory exudate (if) mixed with inflammatory cells (ic), together with the presence of several longitudinal sections of cotton pellets (cp). slight deposition of calcium crystals within the granuloma area (yellow arrows) indicates local tissue destruction. (g3) granuloma with ginkgo biloba (gkb) 200mg/kg group, reveal significant reduction in the area of granuloma (gr) together with the number of inflammatory cells and exudate (yellow arrows). granuloma area is replaced with deposition of eosinophilic connective tissue (ct) consisting of many proliferated collagen fibers (cf) and inflammatory cells. (g4) granuloma with dexamethasone 1mg/kg group, show significant reducing in the amount of inflammatory exudate (yellow arrow), the granuloma area (gr) is contained by eosinophilic deposition of ground substance (gs) together with proliferated collagen (cf) and connective tissue (ct). (g5) granuloma with prednisolone 5mg/kg group, show significant reduction of inflammatory exudate within the area of granuloma (gr), in addition to, deposition of connective tissue (ct) and proliferation of more collagen fibers (cf) which infiltrate the granuloma. various sections of cotton pellet (yellow arrows) can be seen within the granuloma given section, along with some amount of adipose tissue (at). h&e. scale bars: 4 mm. discussion the effectiveness of steroids is welldocumented in inflammatory diseases (20, 21), however, prolong steroid therapy could be associated with numerous undesirable effects (6, 22). the reason behind choosing dexamethasone and prednisolone as standard steroids for comparison; was the fact that each of the aforementioned steroids has been used in similar cases of inflammation. dexamethasone has been proven to be effective in reducing formalin-induced paw edema and granulomatous inflammation in various animal studies (18, 23, 24), and prednisolone appeared to have a successful role in reducing the size and extent of the lesion in patients with idiopathic granulomatous mastitis (25) and patients with solitary cysticercus granuloma (26). ginkgo biloba extract for the first time in the current study tested in granulomatous inflammation and was efficacious in producing a significant reduction in the inflammatory response. nutraceuticals are increasingly being used as an alternative medicine for a variety of diseases, (27) since they contain many pharmacologically active constituents (28), such as vitamin e, carotenoids, and polyphenols. additionally, the presence of dietary supplements like proteins, vitamins, and minerals, iraqi j pharm sci, vol.31(1) 2022 anti-inflammatory activity of gingko biloba extract 191 besides these phytochemicals render these nutraceuticals to become very effective in the prevention and treatment of chronic inflammations (27, 29), ginkgo biloba extract contains flavonoids including quercetin, kaempferol, isorhamnetin, and glycoside that act as free-radical scavengers preventing lipid peroxidation and oxidative stress. it also contains terpenoids that inhibit platelet aggregation and improves memory (30). the data in the current study shows the effectiveness of gkb extract in decreasing both the amount of exudate and granuloma although the effect was less than that produced by dexamethasone and prednisolone; the anti-inflammatory effect of gkb was more obvious on the inflammatory markers. the significant decrease in the level of tnf-α produced by gkb mainly contributed to the radical scavenging properties, down-regulating some of the inflammatory mediators and inflammatory responses through the modulatory effect on the expression of many inflammatory mediators (8, 14). targeting tnf-α may augment the down regulation of the immune and inflammatory responses, because of its pivotal role in the stimulation of ros production, expression of other pro-inflammatory mediators, activation of leucocytes, and eventually amplification of the inflammatory cascades (14, 16). ginkgo biloba was also effective in producing a significant decrease in the level of vcam-1 compared with the positive control which could be attributed to the antioxidant properties that in turn modulate the redox-sensitive transcription pathways and minimize the expression of endothelial adhesion molecule including vcam-1 via suppressing tnfα–induced expression of vcam-1 and icam-1 (31). in contrast to the study that showed no association between taking supplements contain ginkgo biloba and the level of hs-crp (32): the current study revealed that the level of hs-crp has been attenuated by the use of gkb and the two steroids used in the study. this finding was in tune with other studies done on patients with metabolic syndrome where the use of ginkgo biloba as adjuvant therapy was shown to be effective in reducing the level of hs-crp (33). ginkgo biloba extract has a crucial role in dropping oxidative stress and inflammatory response (34,35). furthermore, another underlying mechanism for the anti-inflammatory effects of gkb could be attributed to the inhibitory action on the expression of inflammatory cytokines through suppressing the inflammatory signaling pathway toll-like receptor 4/ nuclear factor kappa-light-chain-enhancer of activated b cells (tlr4/nf-κb) (36). conclusion in conclusion, gkb was effective in diminishing inflammation as reported by decreasing the exudate, granuloma, and inflammatory markers. the underlying mechanisms could be the inhibitory effect on the expression of the inflammatory cytokines and endothelial adhesion molecule. these findings suggest gkb as a good contender to be tested for the treatment of inflammatory diseases. funding the current study did not receive any financial support. conflicts of interest the authors report no conflicts of interest in this work. references 1. hussain t, tan b, yin y, blachier f, tossou mcb, rahu n. oxidative stress 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environment, baghdad, iraq. *department of pharmaceutics college of pharmacy, university of baghdad, baghdad, iraq. abstract meloxicam (mlx) is non-steroidal anti-inflammatory, poorly water soluble, highly permeable drug and the rate of its oral absorption is often controlled by the dissolution rate in the gastrointestinal tract. solid dispersion (sd) is an effective technique for enhancing the solubility and dissolution rate of such drug. the present study aims to enhance the solubility and the dissolution rate of mlx by sd technique by solvent evaporation method using sodium alginate (sa), hyaluronic acid (ha), collagen and xyloglucan (xg) as hydrophilic natural polymers. twelve formulas were prepared in different drug: polymer ratios and evaluated for their, percentage yield, drug content, water solubility, dissolution rate, crystal lattice using powder x-ray diffraction (pxrd) and fourier transform infrared spectroscopy (ftir) for determination the drug-polymer interaction. all the prepared formulas showed improvement of drug solubility except that prepared with ha. the best result was obtained with formula sd1 (mlx: sa 1:1) that showed a high percentage yield (97%), high drug content (97.4±0.05%) and increase in solubility compared to solubility of pure mlx with improved dissolution rate. the pxrd study revealed the conversion of the drug to amorphous form without chemical interaction according to ftir results keywords: meloxicam, solid dispersions, sodium alginate, hyaluronic acid, collagen, xyloglucan. حامية للمعدةمال بوليمرات طبيعية تعباسالمنتشر لصلب خارج الجسم للميلوكسيكام اتقييم صييغ وت *و ايمان بكر حازم الخضيري 1، *هبة راضي محسن الحسني .فرع الصيدالنيات ،كلية الصيدلة ،جامعة بغداد،بغداد،العراق* الخالصة الصلب المنتشر هو يذوب في الماء ذو نفاذيه عاليه يعتمد سرعه امتصاصه على سرعه تحلله في الجهاز الهضمي. الالميلوكسيكام عقار قليلة الذوبان الدويةفعالة لتعزيز قابلية ومعدل الذوبان لطريقة الجينات الصوديوم, حيث تم استخدام .باستخدام تقنية الصلب المنتشر الميلوكسيام وسرعة تحرر الهدف من هذه الدراسة هو تحسين قابلية الذوبان للماء.بوليمرات طبيعية محبة كحامض الهايلورونك, الكوالجين و الزايلوكلوكان ، سرعة تحرر قابلية الذوبان في الماء ، محتوى الدواء ،االنتاجية النسبة وتم تقيمها حسب, ( يوليمر) دواء:مختلفة بنسب اثنا عشرة صيغةتم تحضير .الناقل والدواءشعة تحت الحمراء لتحديد التفاعل الحاصل بين شعة السينية و مطياف اال، والهيكل البلوري باستخدام تقنية حيود االالدواء عدا تلك المحضرة باستعمال حامض الهايلورونك ولقد اظهرت النتائج تحسن في قابلية الذوبان للدواء في جميع الصيغ المحضرة بتقنية الصلب المنتشر ( و محتوى دواء %97نسبة انتاجية عالية ) التي اظهرت (1:1جينتات الصوديوم ال: ميلوكسيكام) sd1 نتيجة بالصيغة وتم الحصول على افضل . كما ان الدواء فقد خاصية التبلور مع تحسن في سرعة تحرر الدواء النقي الميلوكسيكامزيادة في الذوبانية مقارنة بذوبان و( %0.05±97.4)عالي .الحمراء تحت االشعة مطيافبدون تفاعل كيميائي مع البوليمر حسب ما اظهره السينية االشعة حيودحسب نتائج .زايلوكلوكان ،كوالجين ،حامض الهايلورونيك ،الجينات الصوديوم، ر، الصلب المنتشميلوكسكامالكلمات المفتاحية: اال introduction drug product solubility can be defined as being both quantitative and qualitative. the concept of quantitative solubility is the number of gram of solute particles required to make a saturated solution at certain temperature. while qualitative solubility is defined as where two phases are mixed together to form a homogeneous solution ( 1). it is one of the essential parameters for achieving desired drug concentration in systemic circulation for the desired pharmacological response. low aqueous solubility is the main problem associated with the development of formulations of new chemical entities. solubility is a major challenge for formulation scientist. any drug to be absorbed must be present in the form of solution at the site of absorption (2). solid dispersion (sd) is one formulation strategy used to improve the bioavailability of compounds with poor solubility. it can be defined as a pharmaceutical form in which the drug is dispersed in a biologically inert matrix, generally to improve the oral bioavailability of the drug by improving its dissolution rate in aqueous media (3). 1corresponding author e-mail: hiba.radi91@gmail.com received: 23/8/2020 accepted: 21/ 11/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp169-178 iraqi j pharm sci, vol.30(1) 2021 meloxicam solid dispersion 170 natural polymers have been used in different applications due to their desirable characteristics, such as abundant availability, biodegradability, and renewability. these polymers are known to produce less toxic effects when compared with synthetic polymers and they versatile areas of their applications makes them attractive for research and development to make them suitable for different applications in food and pharmaceutical (4). many natural polymers were used as carriers in the preparation of sd to enhance the dissolution rate of drug as they are easily soluble in water (5). natural polymers such as collagen, alginates, hyaluronic acid xyloglucan are commonly used in wound healing because they have beneficial properties for this function (6). collagen is a complex protein; the biodegradable properties of collagen are of critical importance in the natural process of healing wounds. collagen prompts the formation of fibroblasts and accelerates endothelial cell migration contact with infected tissue (7). hyaluronic acid (ha) is a natural linear polysaccharide (8). it is used in medicine for the treatment of osteoarthritis, eye and plastic surgery, tissue engineering. it is also used for drug delivery (9,10). sodium alginate (sa) is the sodium salt of alginic acid a polyanionic polysaccharides of microbial origin it can protect the git lining due to the gel formation depending on the ph of the environment. in the stomach, water penetrates the dosage form, initiating surface hydration, leading to the formation of a gel layer, which may protect the stomach lining (11,12). the tamarind seed xyloglucan (xg) is a natural neutral hemicellulose (hydrophilic polysaccharide), characterized by film-forming protective barrier properties, it is a safe nonpharmacological alternative for the management of different diseases such as gastrointestinal disorders (13,14) . meloxicam (mlx) chemically known as ” 4-hydroxy-2-methyl-n-(5methyl-2-thiazolyl)-2h1,2-benzothiazine-3-carboxamide 1,1-dioxide” , c14h13n3o4s2 as empirical name , molecular weight of 351.4. mlx has structural formula described in figure 1. figure 1.structural formula of mlx (15) meloxicam is a non-steroidal anti inflammatory drug of the oxicam class that acts by inhibiting the prostaglandin synthesis, by inhibiting prostaglandin synthetize (cyclooxygenase (cox) ) mainly cox-2 isoform of cyclooxygenase thereby exerting anti-inflammatory, anti-exudative, analgesic and antipyretic effects, it is also emerging as a promising drug for the treatment of alzheimer’s disease and cancer. a primary advantage of the oxicam family of drugs is their long half-life which permits it to be used once daily (16). the drug is practically insoluble in water; (17). it has log p (octanol/water) equals 3.43 and dissociation constants pka of 1.1 and 4.2 and its absolute bioavailability is about 89% (15). mlx may result in gastrointestinal toxicity and bleeding can occur at any time during use and without signs of warning. elderly patients are at greater risk of serious gastrointestinal problems (16). the aim of this research was to prepare solid dispersion of mlx using sodium alginate, hyaluronic acid, collagen and xyloglucan as natural polymers with expected gastro-protective effect and study their effect on the solubility and dissolution rate of mlx. materials and methods materials meloxicam (mlx), hyaluronic acid (ha), collagen, xyloglucan (xg) were purchased from hangzhou, hyperchem. china. sodium alginate (sa) was purchased from himedia laboratories, india method preparation of solid dispersion of mlx solid dispersion of mlx was prepared by a solvent evaporation method using different natural polymers sa, collagen, xg and ha as shown in table 1. the polymers were dispersed in 20 ml of 20% v/v of methanol separately by utilizing a magnetic stirrer, until a clear solution was obtained, mlx was then added and stirring was continued for 10 min. different drug: polymer ratio (w:w) were utilized depending on the viscosity of the polymeric solution. after that, the solvent was allowed to evaporate using oven at a temperature of 40°c for 24 hours. the resultant dried mass was pulverized and sieved through sieve no. 20 to get uniform particle size then stored in a desiccator for further investigations (18,19). iraqi j pharm sci, vol.30(1) 2021 meloxicam solid dispersion 171 table1. composition of different sd formulas of mlx formula code mlx (g) sa (g) collagen (g) xg (g) ha (g) sd1 1 1 … … … sd2 1 2 … … … sd3 1 3 … … … sd4 1 … 0.5 … … sd5 1 … 1 … … sd6 1 … 2 … … sd7 1 … … 0.5 … sd8 1 … … 1 … sd9 1 … … 2 … sd10 1 … … … 0.5 sd11 1 … … … 1 sd12 1 … … … 2 preparation of physical mixture (pm) the pm was prepared by uniform mixing of drug and polymer in the same ratio as that of the best sd formula. the mixed powder was passed through a sieve no.20 to get uniformly sized particles evaluation of sd of mlx determination of percentage yield (py %) of the prepared sd of mlx the percentage yield for each type of sd was determined by measuring the ratio of the actual weight of the obtained sd on the theoretical weight of sd which was calculated using the equation below (20). practical yield (%)= mass of sd recovered ( practical mass) mass of polymer and drug used in formulation (theoretical mass) ×100 determination of drug content of the prepared sd of mlx a precisely weighed quantity of sd equivalent to 15 mg of mlx was taken and dissolved in a 20 ml of 0.1n naoh, with stirring for 10 min, after that, the contents were filtered using a filter paper. then after suitable dilution the solution was assayed for drug content using uv spectrophotometer by determining the absorbance at 363nm using 0.1n naoh as blank (21). the percentage of drug content in the sd was measured by using the following equation (22). 𝐃rug content (%) = actual amount of drug in solid dispersions theoretical amount of drug in solid dispersions × 100 determination of saturation solubility an excess amount of mlx and sd were added to 10ml of distilled water separately, the samples were incubated in water bath shaker for 48 hr at 25 ˚c. after that, the samples were removed from the shaker water bath. the solutions were filtered via a 0.45μm filter syringe and diluted when necessary. the concentration of mlx was analyzed by uv spectrophotometer at 362nm, which represents the solubility of mlx. the procedure was performed in triplicate (23). in-vitro dissolution studies of pure mlx and sd of mlx in-vitro dissolution studies of pure mlx and sd were performed by placing an amount equivalent to 15 mg mlx in 900 ml phosphate buffer (ph 7.5) as dissolution medium using usp type 2 apparatus for two hours. the temperature of the dissolution media was kept at 37 ± 0.5 ˚c, with an operating speed of 75 rpm (24,25). five milliliters were withdrawn and then replaced by fresh media of dissolution at regular time intervals, filtered and analyzed spectrophotometrically at 361nm. solid dispersion formulas with highest solubility were subjected to this test. this test was performed in triplicate for all samples. the results acquired from the dissolution studies were statistically validated using a similarity factor (f2). the f2 was used to consider similar dissolution profiles (equation below). f2 = 50 × log {[1 + 1 𝑛 ∑ |𝑅𝑡 − 𝑇𝑡| 2 𝑛 𝑡=1 ] −0.5 × 100} where (n) is the number of dissolution time points. (rt), (tt) are the reference and test dissolution values at time t respectively. the two dissolution profiles consider similar when f2 values greater than 50 (50– 100); otherwise, the profiles are not similar (26) . powder x-ray diffraction (pxrd) powder x-ray diffraction was utilized to analyse the crystallinity of the pure mlx and selected formula. measurement was performed under the following conditions: the target metals cu, filter kα, 45kv voltage, 30ma current. samples iraqi j pharm sci, vol.30(1) 2021 meloxicam solid dispersion 172 scanned over a 2(θ) range of 5-80° at a step size of 0.02° (27). fourier transforms infrared spectroscopy (ftir) ftir spectroscopy of mlx, selected polymer, pm and the selected sd formula was performed to investigate drug-polymer intermolecular interaction. the samples were compressed with kbr and pressed in disk form (13 mm in diameter). the ftir spectroscopy analyzed the disk from 4000-400 cm-1 (28). results and discussion percentage yield (py %) the prepared sd calculation of the py% of the prepared formula is important to determine the efficiency of the preparation method. high py was obtained from all the prepared sds formulas ranged between (90.5100%) as shown in table3. this relatively small amount of loss occurred due to some steps of preparation or sieving of the formula. this result indicated the suitability of the method (solvent evaporation method) with the used material for such preparations. drug content of the prepared sd of mlx the results of drug content in all formulations, was found to be within (91-104%) w /w, which is consistent with usp requirements (90105%) (29). suggesting no loss of mlx during the preparation and uniform distribution of mlx particles in all the prepared formulas. the results of drug content are shown in table 2. table2. percentage yield and drug content of sd. formula code percentage yield (py %) drug content (w/w) (%) (mean±std) (n=3) formula code percentage yield (py %) drug content (w/w)(%) (mean±std) (n=3) sd1 97 97.4±0.05 sd7 90.5 99.92 ±0.42 sd2 91.98 97.47 ±0.12 sd8 95 104.07 ±0.06 sd3 98.5 104.36 ±0.32 sd9 95 98 ±0.05 sd4 92 96.79 ±0.3 sd10 97.5 92.1 ±0.1 sd5 99.40 99.94 ±0.06 sd11 97 99.1 ±0.01 sd6 99 99.89 ±0.11 sd12 99 95.09 ±0.08 saturation solubility of mlx sds results of saturation solubility studies of mlx sds are shown in table 3. table 3.the saturation solubility of pure mlx and sds formulas using different natural polymers with different drug: polymer ratio in distilled water at 25˚c it was found that there was significant difference (p<0.05) between sa, collagen, xg in enhancing solubility of mlx. the solubility enhancing effect of the above polymers can be ranked in the following order sa > collagen > xg, whereas no significant (p>0.05) enhancing in solubility was obtained by using ha the above sequence may be explained to be due to different properties of the used polymers; sa has high hydrophilic property, and its particles are hydrated at a faster rate when the solid dispersion mixture comes into contact with water. therefore, sa wetting effect may explain the improved solubility of mlx from solid dispersion even with the least amount as there was no significant difference (p>0.05) between the three different used ratios. this result may be attributed to the increase in the viscosity of the solution by increase in polymer concentration, which hinder further increase in solubility (30). the second polymer, collagen although it is insoluble polymer, it consists of three polypeptides chains: glycine, proline, and hydroxyproline, arranged as a triple helix, the hydrophilic groups (–oh, –cooh, and –nh) contained in these amino acids have the ability to formula code polymer ( drug : polymer ) ratio saturation solubility mg/ml mean ±std (n=3) mlx 0.00981±0.001 sd1 sa (1:1) 0.340±0.001 sd2 sa (1:2) 0.351±0.06 sd3 sa (1:3) 0.347±0.05 sd4 collagen (1:0.5) 0.0222±0.0005 sd5 collagen (1:1) 0.0663±0.025 sd6 collagen (1:2) 0.0401±0.016 sd7 xg (1:0.5) 0.0228±0.0035 sd8 xg (1:1) 0.0276±0.0018 sd9 xg (1:2) 0.0276±0.003 sd10 h.a (1:0.5) 0.0185±0.0009 sd11 h.a (1:1) 0.0166±0.01 sd12 h.a (1:2) 0.015±0.007 iraqi j pharm sci, vol.30(1) 2021 meloxicam solid dispersion 173 absorb water which may enhance the wettability and hence solubility of mlx. when the collagen sds formulas dispersed in water, these hydrophilic groups which located within the inner parts of the structure may not make contact with water, and consequently, this reduce the water absorption, therefore this polymer has lower solubility enhancing effect than sa (31,32). the solubility enhancing effect obtained by xg can be attributed to its water solubility that led to its application as a solubility enhancing polymer in preparation of sd. (33,34). this result is in agreement with that obtained by babu et (35). increasing the amount of xg up to1:2 (drug: polymer) did not show increase the solubility over that of 1:1 ratio which may be due to the formation of a viscous layer in the form of an aqueous solution that hinder the hydration of mlx particles (33). on the other hand, the no significant (p>0.05) enhancement in solubility of mlx by ha may be due to the formation of very viscous solution by large water soluble hyaluronic acid molecule event at low concentration (36). this viscosity increased by increasing in polymer concentration, which could be linked to the increasing polymer chain entanglement and higher chances of hydrogen bonding at higher polymer concentration, resulted in further decrease in the solubility of mlx (37). in-vitro dissolution studies in the present study, the sd1, sd2 and sd3 (prepared with s.a as a polymer with different ratio 1:1, 1:2 and 1:3, respectively) were selected to study the effect of drug: polymer ratio on the dissolution of mlx, since the highest solubility of mlx was obtained by these formulas. figure 2 demonstrates that all sd1, sd2 and sd3 have enhanced dissolution rate in phosphate buffer (ph7.5) in comparison to pure drug (f2 = 7.33), (f2 = 8.91) and (f 2=11.50), respectively. sd1 exhibited the highest the percent of mlx release by (95%) after 30 minutes as compared with sd2 (84%), sd3 (75%) and pure mlx (2%). the enhanced dissolution of the drug from sds can be attributed to reduced particle size of the drug within sds, also it is assumed that the higher the wettability and dispersibility of a drug in a hydrophilic polymer, the better the chances of achieving an increase in drug dissolution profile (6,38). on the other hand, similar dissolution profile (f2= 62.03) was obtained by comparing sd1 to sd2, while dissimilar dissolution profile (f2=44.21) obtained by comparison of sd1 with sd3. from the previous results, it was found that the increasing the amount of sa resulted in lowering the rate of release of mlx. this result can be explained to be due the formation of viscous layer with increased thickness that acted as a barrier for the dissolution medium thereby retarding the diffusion of drug from the sd causing retardation in the dissolution of the drug (39,40). this result is in accordance with noyes – whitney equation which expresses the dissolution process as follow (41): 𝑑𝑚 𝑑𝑡 = 𝐴 𝐷 𝑑 (𝐶𝑠 − 𝐶b) ..eq (4). where dm/dt = solute dissolution rate m = mass of dissolved material t = time a = surface area of the solute particle d = diffusion coefficient, which is related, in part, to the viscosity of the solvent d = thickness of the of the diffusion layer cs = particle surface (saturation) concentration cb = concentration in the bulk solvent/solution. so decreasing d (due to increase viscosity) and increase d resulted in decrease dissolution rate figure (2). effect of drug: sa ratio of mlxsolid dispersion on the in-vitro dissolution profile of mlx in phosphate buffer ph 7.5 at 37°c. evaluation of the optimum formula comparative dissolution study an improvement in the dissolution was obtained by sd1 and by pm in comparison with pure drug (f2 = 7.33, f2=13.94 respectively) as shown in figure 3, which can be ascribed to enhance drug wettability, by the hydrophilic property of sa. on the other hand, the enhanced dissolution obtained by sd1 compared to the pm (f2=35.69) can be explained to the efficiency of the solid dispersion technique that may cause particle size reduction and formation of amorphous form of drug, which required further investigation by xrd (42). iraqi j pharm sci, vol.30(1) 2021 meloxicam solid dispersion 174 figure 3. comparative in-vitro dissolution profile of the pure mlx, sd1 and physical mixture of sd1 (pm) in phosphate buffer (ph7.5) at 37±0.5 °c powder x-ray diffraction (pxrd) the pxrd diffractogram of pure mlx, pm and sd1 are shown in figure 4 respectively. the diffraction pattern of the pure mlx showed characteristic high intensity peaks at 6.06 °, 13.74°, 15.89°, 19.32°, 21.48°, 21.55°, 24.7° and 26.57°, which indicates that the drug is present in the crystalline form. these values approach the values of the previous studies (43,44). physical mixture’s pattern showed the characteristic peaks of mlx with lower intensity compared to the pure drug. this result can be explained to be due to the dilution effect of the polymer. in contrast, further decrease of the characteristic peaks of mlx was observed with sd1, indicating that an amorphous form mostly exists in sd1 (45). this amorphous form may contribute to solubility improvement since this form is more easily soluble than the crystalline form. figure 4. xrd diffractograms of mlx, sa, pm and sd1 fourier transform infrared (ftir) the ftir spectrum of pure mlx, sa, pm and sd1 are shown in figures (5-8) respectively. the spectrum of pure mlx showed characteristic peaks at 3288 cm-1 (n-h stretching vibrations), 1620 cm-1 (c=n stretching vibrations), 1159 cm-1 (s=o stretching vibrations) respectively (28). the obtained results were in accordance with the previous studies (43, 44). in the case of pure sa, a sharp broad absorption peak occurs at 3413 cm−1, referring to the presence of –oh groups bonded with hydrogen. the major sa absorption peaks occurring at 1603 and 1429 cm1 related to coo– groups, while the peak occurring at 2925 cm−1 is due to c h stretching. sa's characteristic peak (na–o) is shown at 849 cm−1 (28). these results were in agreement with previous studies (46, 47). the spectra of pm figure 7 and that of selected sd1 formula figure 8 showed all the characteristic peaks of the drug, indicating that there was no chemical interaction between mlx and sa. the decreased intensity of the pecks in the above figures may be due to dilution effect of the polymer. iraqi j pharm sci, vol.30(1) 2021 meloxicam solid dispersion 175 figure 5.ftir spectrum of mlx figure 6. ftir spectrum of sa iraqi j pharm sci, vol.30(1) 2021 meloxicam solid dispersion 176 figure 7. ftir spectrum of pm figure 8. ftir spectrum of sd1 conclusions an improvement in solubility and dissolution rate of mlx was obtained by preparing it as solid dispersion by solvent evaporation method using sa as a natural hydrophilic polymer in a ratio of 1:1. drug: polymer. this improvement was due to increased wettability and reduced crystallinity of the drug. future work clinical studies are required to confirm whether the sodium alginate protects the stomach lining from irritation due to the chronic use of mlx acknowledgement the authors are grateful to acknowledge the college of pharmacy -university of baghdad for iraqi j pharm sci, vol.30(1) 2021 meloxicam solid dispersion 177 providing the necessary facilities to carry out this study. references 1. patrick j. sinco. martin's physical pharmacy and pharmaceutical science. 6th ed. lippincot williams and wilkins 2011; p.182. 2. karen d h, prajapti p. h., chaudhary j i. bcs 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nanoparticles. rsc advances .2017 ;7(84) :53422 -53432. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 antioxidant activity of echinops polyceras leaves doi: https://doi.org/10.31351/vol31iss1pp270-277 270 phytochemical screening and free radicals scavenging activity of leaves of echinops polyceras boiss. grown in syria issa al-assaf*,1 and mays khazem* * department of pharmacognosy in faculty of pharmacy, damascus university, damascus, syria abstract free radicals are reactive compounds, their excessive production is considered to be an important cause of oxidative damage in biomolecules causing degenerative diseases. polyphenols are one of the most important groups of secondary metabolites of plants, which have an antioxidant activity depending on their properties as hydrogen donors. echinops polyceras boiss. (asteraceae) is one of echinops genus species that spread in syria, lebanon, and palestine. phytochemicals found in this species leaves have been extracted with gradient polarity solvents, and primary screening of the secondary metabolites was established. the phenolic compounds and flavonoids contents were determined. the free radicals scavenging activity was evaluated for all extracts with dpph• in a 96-well microplate. the specificity study indicates that ascorbic acid was absent, and reducing sugars were exist in the aqueous extract. the identification tests showed the presence of polyphenols like flavonoids and coumarins. the methanolic extract of the e. polyceras leaves was the most effective scavengers of free radicals (90.22% in 30 min) with phenolic compounds content 682.5 mg gae/g of dried extract (de) and flavonoids content 194.5 mg qe/ g de. the chloroform extract was the least effective as free radical scavenging (60% in 30 min) as the phenolic compounds content was 278.5 mg gae/g de and flavonoids content 94 mg qe/ g de. in conclusion, the phenolic compounds and flavonoids from echinops polyceras boiss. are effective in free radicals scavenging and protecting from diseases caused by oxidative stress. keywords: antioxidants, polyphenols, flavonoids, free radicals, and echinops polyceras boiss تحّري الُمركبات الفعّالة والفعاليّة الكاسحة للجذور الُحّرة ألوراق نبات echinops polyceras boiss. النامي في سورية ميس خازم* و 1،عيسى العّساف* * قسم العقاقير في كليّة الصيدلة، جامعة دمشق، دمشق، سورية الخالصة مؤدّية لحدوث أ في الجزيئات الحيويّة ُمتفاعلة بشدّة، ويُمكن أن يُسبب فرط إنتاجها أذيّاٍت تأكسديّة ُمركبات مراٍض تُعدّ الجذور الُحّرة ُمضادة لألكسدة بسبب خص تمتلك فعاليّة مجموعات الُمستقلبات الثانويّة في النباتات، والتي الفينول إحدى أهّم عديدات المانحة تنكسيّة. تُعدّ ائصها في سورية ولبنان echinops وهو أحد أنواع جنس (asteraceae الفصيلة النجميّة) .echinops polyceras boiss للهيدروجين. ينتشر نوع إج ذلك بعد تّم وقد القطبيّة، ُمتدّرجة ُمحّّلٍت باستخدام النوع هذا أوراق في الموجودة الفعّالة الُمكّونات استُخلصت عن وفلسطين. أولّي تحّرٍ راء المدروسة باستخدام كاشفالُمستقلبات الثانويّة. تّم تحديد الُمحتوى الفينولّي والفّلفونوئيدّي، ومن ثّم تقييم الفعاليّة الكاسحة للجذور الُحّرة للخّلصات dpph• المائيّة على سكاكر ُمرجعة، بينما خلت هذه حفرة. أظهرت نتائج دراسة االنتقائيّة احتواء الخّلصات 96وذلك باستخدام طبٍق للزرع ذو ُمستخلص الخّلصة من حمض األسكوربيك. كما أظهرت نتائج الكشوفات األوليّة وجود عديدات الفينول مثل الفّلفونوئيدات والكومارينات. وقد أبدى ال ( وبتقدير الُحّرة الجذور كسح في نشاط أفضل بلغ 30خّلل % 90.22الميثانولّي حيث الفينولّي دقيقة( mg gae/g de 682.5الُمحتوى دقيقة( حيث بلغ الُمحتوى 30خّلل %60، وكانت أدنى فعالية ُمضادة لألكسدة لُمستخلص الكلوروفورم )mg qe/ g de 194.5والفّلفونوئيدّي يُمكن تلخيص ما سبق بأن الُمركبات الفينوليّة وخاصةً الفّلفونوئيدات .mg qe/ g de 94والفّلفونوئيدّي mg gae/g de 278.5الفينولّي .وبالتالي الوقاية من األمراض الناتجة عن الشدة التأكسديّةتمتلك فعّاليّة في كسح الجذور الُحّرة echinops polyceras boiss الكلمات المفتاحيّة: ُمضادات األكسدة، عديدات الفينول، الفالفونوئيدات، الجذور الُحّرة و introduction free radicals are reactive compounds that tend to capture electrons from stable biological molecules in order to stabilize themselves (1). the excessive production of free radicals is considered to be an important cause of oxidative damage in biomolecules, such as proteins, lipids, and dna, this damage leads to numerous degenerative diseases (2), such as cancer, atherosclerosis, gastric ulcer, and other conditions (3). polyphenols are one of the most important groups of secondary metabolites of plants, they are widely distributed in the plant kingdom and can be obtained directly from plants, foods or drug-supplements. these compounds are important agents in the prevention of several diseases. polyphenols function as antioxidants depends on their properties as hydrogen donors. these hydrogen atoms are accepted by reactive radicals to yield much less reactive radicals and non-radical species (4). the mechanism of the protective action of phenolic compounds in plants rely on the antioxidant activity that scavenges free radicals, protection of lipid peroxidation, and the chelation of toxic metals (5). 1corresponding author e-mail: issa.alassaf.92@gmail.com received: 15/8/2021 accepted: 3/11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp270-277 iraqi j pharm sci, vol.31(1) 2022 antioxidant activity of echinops polyceras leaves 271 echinops polyceras boiss. is a perennial herb, 40-60 cm, sometimes with very fine and short whitish glandular hairs in the lower part. basal leaves congested, oblong-lanceolate, pinnatipartite into short lobes armed with short yellow spines. heads generally abundantly, with about 4.5-5 cm in diameter (not including cornigerous bracts). partial involucre of non-cornigerous headlets about 2 cm, pale green. corolla is white to pale bluish, anthers are greyish-violet, and the flowering time is june – july (6). this species spread in syria and lebanon and palestine (7). the aim of this study was to evaluate the phytochemicals and the free radicals scavenging activity of echinops polyceras boiss. leaves since no previous studies have distinguished the chemical constituents and the biological effects of this plant. materials and methods chemicals gallic acid (was purchased from avonchem), quercetin (from sigma-aldrich), 2,2-diphenyl-1-picrylhydrazyl dpph (from tci), distilled water, absolute ethanol (from merck), absolute methanol (from sigma-aldrich), ethyl acetate (from sham lab), chloroform (from merck), glacial acetic acid (from bdh), hydrochloric acid (from himedia), sulfuric acid (from himedia), toluene (from bdh) , formic acid (from bdh), folin ciocalteu (from sigma-aldrich), sodium carbonate (from scharlau), aluminum chloride (from scharlau), potassium acetate (from merck), ascorbic acid (from panreac quimica slu), magnesium metal turnings (from chem-lab), ferric chloride (from panreac), potassium iodide (from eurolab), bismuth nitrate (from himedia) and mercuric chloride (from himedia), picric acid (from panreac eu), iodine crystals (from honeywell), 3,5dinitrobenzoic acid (from titan biotech), gelatin, and tlc silica gel 60 f254 (from merck). apparatus 96-well microplate reader (biotek), rotary evaporator, tlc plate heater (camag), and tlc scanner (camag). plant material the whole plant of flowering echinops polyceras boiss. was collected from ma'aret sednaya, rif-dimashq, syria in july 2019, and authenticated by dr. imad alkadi at the department of plant biology, damascus university, syria. the leaves were separated from the rest of the plant parts, then dried in shade and powdered. extraction of the plant leaves dried and powdered leaves (20 g) of echinops polyceras boiss. were extracted with 200 ml of each of gradient polarity solvents: distilled water, ethanol 50%, methanol, methanol + ethyl acetate (1:1) and chloroform, at room temperature with shaking for three days. the five extracts were evaporated with a rotary evaporator, and the extraction yield was calculated by the equation: yield% = (weight of evaporated extract/ weight of leaves powder) ×100 phytochemical identification echinops polyceras leaves extract was assessed for the existence of flavonoids, coumarins, tannins, anthraquinones, alkaloids, saponins and cardiac glycosides. test for flavonoids flavonoids were identified using uv (366 nm) fluorescence after aluminum chloride (5%) in ethanol was added (8). to 5 ml of ethanolic extract, 0.5 g of magnesium metal and 1 ml of concentrated hcl were added. the pink or red color formation indicates the presence of flavones (shinoda test). test for coumarins ethanolic extract (5 ml) was evaporated then the residue was dissolved in 2 ml of hot distilled water. few drops of this solution were put on a clean filter paper and the fluorescence under uv light (366 nm) was examined. an intense blue fluorescence indicates the presence of coumarins. test for tannins ferric chloride test to 1 ml of ethanolic extract, 2 to 3 drops of 10% of ferric chloride (fecl3) solution were added, and observed for a dark green (hydrolysable tannins) or dark blue (condensed tannins) coloration (9). gelatin test to 1 ml of tested extract, 2 drops of 1 % gelatin solution with 10% sodium chloride were added. the formation of white precipitate indicates the presence of tannins (10). test for anthraquinones borntrager test e. polyceras leaves powder (1 g) was extracted with 10 ml of chloroform for 10 minutes and filtered, then 2 ml of ammonia were added. the formation of red color in the aqueous layer indicates the presence of free anthraquinones (9). modified borntrager test boil 1g of the plant material with 2ml of dilute sulphuric acid and 2ml of 5% aqueous ferric chloride solution for 5 minutes and continue the reaction as borntrager test. the formation of red color in the aqueous layer indicates the presence of anthraquinone glycosides (10). test for alkaloids ethanolic extract (20 ml) was evaporated, and the dry residue was dissolved in 5 ml of hcl (2n) and filtered. then, few drops of mayer, dragendroff, wagner and hager reagents were added. the formation of white, orange, reddishbrown and yellow precipitates respectively indicate the presence of alkaloids (9, 10). iraqi j pharm sci, vol.31(1) 2022 antioxidant activity of echinops polyceras leaves 272 test for saponins to 0.25 g of the aqueous extract, 15 ml of hot water were added into a test tube, the tube was shaken vigorously. the formation of a stable foam indicates the presence of saponins (9). test for cardiac glycosides keller killiani test to 2 ml of the ethanolic extract, 1 ml of glacial acetic acid and one drop of 5% fecl3 and 1 ml concentrated h2so4 were added. the formation of reddish-brown color at the junction of the two liquid layers, and the bluish-green color at the upper layer indicate the presence of cardiac glycosides (11). kedde’s test evaporate the chloroform extract of the leaves, then add one drop of 90% alcohol and 2 drops of the reagent (2% 3,5-dinitro benzoic acid in 90% alcohol), an alkaline solution (20% sodium hydroxide solution) was added. purple color is produced in the case of the presence of β unsaturated-o lactones (10). determination of total phenolic content (tpc) total phenolic content was determined by a micro colorimetric method described by ainsworth & gillespie (12): 200 mg of each extract were dissolved with 2 ml of methanol 95% (vol/vol). 100 µl of each sample were transferred to 2 ml microtubes and were mixed with 200 µl of 10% (vol/vol) folin–ciocalteu reagent, the mixture was vortexed thoroughly. then 800 µl of na2co3 (700 mm) was added into each tube, and the assay tubes were incubated at room temperature for two hours. 200 µl of samples, standard (gallic acid), and blank were transferred to a clear 96-well microplate, and the absorbance of each well was read at 765 nm. gallic acid calibration curve the calibration curve was established with nine dilutions of gallic acid standard at concentrations of (12, 24, 36, 48, 60, 84, 96, 108, 120) mg/l. then the absorbance was read at 765 nm using the microplate reader. tpc of the extracts total phenolic content was calculated as gallic acid equivalents (gae) in 1 g of dried extract using the regression equation between gallic acid standard concentrations and the absorbance at 765 nm (a765). specificity the specificity of folin–ciocalteu method was checked by detection of the presence of some reducing compounds like reducing sugars and ascorbic acid. detection of reducing sugars using fehling's test (9): 1 ml of the ethanol extract was diluted with 1ml of water in a test tube, then 20 drops of boiling fehling’s solution (a and b) was added. the formation of a precipitate red-brick in the bottom of the tube indicates the presence of reducing sugars. detection of ascorbic acid using a spectrophotometric method determination of λmax of ascorbic acid (13) 0.1 g of ascorbic acid was dissolved with distilled water in a volumetric flask (100 ml), then 1 ml of this solution was transferred into another 100 ml volumetric flask with the addition of 10 ml of 0.1 n of hydrochloric acid, then distilled water was used to complete the rest volume to 100 ml. the λmax was determined by a spectrophotometric scan between 200-300 nm. scanning of extract solution (13): 1g of the aqueous extract was also dissolved with distilled water in a volumetric flask (100 ml), then 1 ml of the solution was transferred into another 100 ml volumetric flask with 10 ml of 0.1 n of hydrochloric acid, then distilled water was used to complete the rest volume to 100 ml. the absorbance was detected at the λmax of ascorbic acid. determination of total flavonoids content (tfc) flavonoids content was determined according to chang et al. protocol (14): 200 mg of each extract were mixed with 1.5 ml of 95% ethanol, 100 µl of 10% alcl3 (w/v) solution, and 100 µl of 1 mol/l potassium acetate solution were added, and the assay tubes were incubated at room temperature for 30 min. 200 µl of samples, standard quercetin and blank were transferred to a clear 96well microplate and read the absorbance of each well at 420 nm. quercetin calibration curve the calibration curve was established with seven dilutions of quercetin standard at concentrations of (6, 12, 24, 30, 36, 48, 60) mg/l. then the absorbance was read at 420 nm using the microplate reader. tfc of the extracts total flavonoids content was calculated as quercetin equivalents (qe) in 1 g of dried extract using the regression equation between quercetin standard concentrations and the absorbance at 420 nm (a420). evaluation of free radicals scavenging activity (rsa) the dpph• radical scavenging activity was evaluated according to cheung et al. method (15) with some modification by choi et al., 160 µl of 0.2 mm dpph• in methanol were mixed with 40 µl of the extracts or standards (ascorbic acid, gallic acid, and quercetin) in a 96-well microplate. the mixtures were left to stand at room temperature, and the absorbance was measured at 520 nm against methanol as a blank after 10 and 30 min. free radicals scavenging activity (rsa) was determined by the equation: iraqi j pharm sci, vol.31(1) 2022 antioxidant activity of echinops polyceras leaves 273 rsa% = 100 × 𝐀𝟎−𝐀𝐬 𝐀𝟎 where: a0: absorption of dpph • solution as: absorption of dpph• solution after 10 and 30 min of the sample addition. qualitative and quantitative determination of quercetin in the ethanolic extract quercetin standard (qs) and ethanolic extract of leaves (eel) solutions were prepared at concentrations of 40 mg/l and 5 g/l respectively, by dissolving 4 mg of quercetin and 500 mg of the extract in 100 ml ethanol with ultrasound assistance for 10 min at room temperature. 15 µl of quercetin and the ethanolic extract solutions were applied to the tlc plate, toluene: ethyl acetate: formic acid (50:40:2) was used as a mobile phase in a glass chamber saturated with the mobile phase. the plate was dried using a tlc plate heater at 90 c°, and then it was visualized under uv light at 254 nm. after that, they scanned at 254 nm using a tlc scanner. the quercetin percentage in the ethanolic extract was determined according to the equation: 𝐐𝐮𝐞𝐫𝐜𝐞𝐭𝐢𝐧% = 𝐀𝐔𝐂𝟏 × 𝐂𝟏 𝐀𝐔𝐂𝟐 × 𝐂𝟐 × 𝟏𝟎𝟎 where: auc1: area under the curve of the extract auc2: area under the curve of quercetin c1: the concentration of the extract c2: the concentration of quercetin results extraction yield the extraction yield% of the five extracts is shown in table (1). aqueous extract showed the highest yield (13.25 %) followed by hydroethanolic 50%, methanol, methanol: ethyl acetate (1:1), and chloroform extracts 11.25, 8.5, 6.5, and 2%, respectively. table 1.extraction yield extracts yield% dh2o 13.25 etoh 50 11.25 meoh 8.5 meoh+etoac 6.5 phytochemical identification the results of the identification tests are shown in table (2). table 2. phytochemical identification of e. polyceras roots flavonoids aluminum chloride + shinoda test + coumarins fluorescence + tannins ferric chloride test + gelatin test anthraquinones borntrager test modified borntrager alkaloids mayer dragendroff wagner hager saponins foam test + cardiac glycosides keller killiani test kedde’s test gallic acid calibration curve gallic acid concentrations and their absorbances are shown in table (3). also, the linearity and the regression equation are shown in figure (1). table 3. gallic acid concentrations and their absorbances figure 1.gallic acid calibration curve tpc of the extracts: total phenolics content in the extracts was presented in table (3). the methanolic extract showed the highest content of phenolic compounds (682.5 mg gae/g de), followed by dh2o, meoh: etoac (1:1), etoh 50%, and chcl3 extracts, concentration (mg/l) ā 765 0 0 12 0.174 24 0.317 36 0.428 48 0.595 60 0.721 84 1.011 96 1.13 108 1.223 120 1.378 iraqi j pharm sci, vol.31(1) 2022 antioxidant activity of echinops polyceras leaves 274 respectively. the tpc results are shown in figure (2). table 3. gallic acid concentrations and their absorbances figure 2. tpc in 1 g of the dried extracts .all results of tpc are presented as the mean of three replicates ± sd (p < 0.05) specificity detection of reducing sugars: fehling's test indicates that the extract of e. polyceras leaves contain reducing sugars, because of the formation of a precipitate red-brick. detection of ascorbic acid using a spectrophotometric method determination of λmax of ascorbic acid ascorbic acid solution showed a maximum absorbance at 240 nm, according to figure (3). figure 3. the scanning of ascorbic acid solution scanning of extract solution the scanning of the extract solution is shown in figure (4), it indicates that the extract of e. polyceras leaves is free of ascorbic acid. figure 4.the scanning of leaves extract solution quercetin calibration curve the concentrations of quercetin and there absorbances are shown in table (4). also, the linearity and the regression equation are shown in figure (5). table 4. quercetin concentrations and their absorbances figure 5. quercetin calibration curve tfc of the extracts flavonoids content in the extracts is also presented in table (5). as the methanolic extract showed the highest phenolic amount, it contained the highest flavonoid content (38.9 mg qe/ g de), followed by meoh: etoac (1:1), etoh 50%, dh2o and chcl3 extracts, respectively. the tfc results are shown in figure (6). concentration (mg/l) ā 765 0 0 12 0.174 24 0.317 36 0.428 48 0.595 60 0.721 84 1.011 96 1.13 108 1.223 120 1.318 concentration (mg/l) ā 420 0 0 6 0.058 12 0.109 24 0.196 30 0.235 36 0.285 48 0.363 60 0.456 iraqi j pharm sci, vol.31(1) 2022 antioxidant activity of echinops polyceras leaves 275 table 5.tpc, tfc and rsa% of the standards and the extracts all results of tfc are presented as the mean of three replicates ± sd (p < 0.05) figure 6. tfc in 1 g of the dried extracts rsa of the extracts dpph• assay revealed that the methanolic extract was the most effective in free radicals scavenging after 30 min (90.22 %), compared with the other extracts: meoh: etoac (1:1), etoh 50%, dh2o and chcl3, they scavenge 82.65, 80.76, 77.6 and 60.09 % of dpph• radical, and the scavenging activity after 10 and 30 min for the standards and studied extracts is shown in figure (7). figure 7.rsa% of standards and studied extracts after 10 and 30 min the dpph• scavenging activity after 10 and 30 min, total phenolic and flavonoid contents of the extracts is shown in table (5). qualitative detection and quantitative determination of quercetin in the ethanolic extract (16) tlc plate used for detection of quercetin in the ethanolic extract under uv light at 254 nm is shown in figure (8), and the rf value of quercetin was 6.2 the quantitative determination of quercetin depends on the auc of the chromatogram of quercetin in the standard solution and the sample solution figure (9). figure 8. tlc plate of quercetin and ethanolic extract samples yield% tpc samples yield% 10 min 30 min ascorbic acid 91.21 93.85 gallic acid 83.52 87.54 quercetin 85.71 89.59 dh2o 13.25 618 ±0.177 155.5 ±0.282 72.84 77.6 etoh 50 11.25 603 ±0.223 167.5 ±0.475 75.98 80.76 meoh 8.5% 682.5 ±0.135 194.5 ±0.475 85.4 90.22 meoh+etoac 6.5% 606 ±0.541 176.5 ±0.206 78.02 82.65 chcl3 2% 278.5 ±0.28 94 ±0.207 52.28 60.09 iraqi j pharm sci, vol.31(1) 2022 antioxidant activity of echinops polyceras leaves 276 figure 9. chromatogram of quercetin in the standard solution (a) and the sample solution (b) according to the table obtained from the tlc scanner: auc1 = 4428.39, auc2 = 16554.55 and c1 = 40 mg/l, c2 = 5000 mg/l. then, quercetin percentage was: 0.214 % of dried ethanolic extract of leaves and 0.024% of dried leaves. statistical analysis all experiments were accomplished in triplicate. the results were expressed as the mean± standard deviation (sd). one-way analysis of variance (anova) was carried out to identify significant differences between experimental groups, using microsoft excel 2019. differences were considered significant (p < 0.05). correlation ratio was calculated between: total phenolic content and total flavonoids content of the leaves extracts, it was ≈ 97%. free radicals scavenging activity and total phenolic content, it was ≈ 96%. free radicals scavenging activity and flavonoid content, it was ≈ 99% discussion neither the chemical composition nor the biological effects of echinops polyceras boiss. have been studied previously. in this study, the chemical constituents have been identified, the polyphenols and flavonoids content has been determined and the free radicals scavenging activity has been evaluated. the results of extraction yield of e. polyceras boiss. leaves (h2o> etoh 50%> meoh 100% > meoh+ etoac (1:1) > chcl3) -which descended according to the polarity of the solventmay refer to the polarity of the extracted materials, or to the presence of the secondary metabolites as glycosides more than aglycons because the solvents have a crucial role in the type of the secondary metabolites found in the extracts (17, 18). the phytochemical screening revealed the existence of polyphenols as a major component in the extract, especially flavonoids and coumarins, because of that the content of total phenols and flavonoids were determined. phenolic compounds especially flavonoids have a notable antioxidant and free radicals scavenging activities. a structure activity relationship study of flavonoids such as quercetin indicates that the hydroxyl groups, the 3,4-catechol structure in the bring, the 2-3 double bond and 4-oxo function [19] which found in the quercetin structure are key factors for the antioxidant activity. quercetin was identified qualitatively and determined quantitively in the dried ethanolic extract (0.214 %), so this extract showed the highest activity. ascorbic acid and reducing sugars are reducing compounds that may interfere with the antioxidant activity of the extracts (20), so, they were identified in the specificity tests, which indicates the presence of reducing sugars and the absence of ascorbic acid. the methanolic extract showed the highest content of total phenols (683 gae/ g de) and flavonoids (195 qe/ g de), while the chloroform extract showed the lowest contents 279 gae/ g de and 94 qe/ g de respectively. based on these results, the examined extracts have significant antioxidant and free radicals scavenging effects, the most effective extract among them was the methanolic extract (90.2% in 30 minutes), while the chloroform extract was the less effective (60% in 30 minutes), this may be explained by the content of phenols and flavonoids in the extracts. according to the correlation ratio, the most of the phenolic compounds in the extracts were flavonoids, and these compounds were responsible of the most of the activity. the phenolic content may contribute directly to the antioxidant activity (21). the aqueous extract which can use in traditional medicine as an infusion (22), showed a good effect in scavenging of free radicals (≈78 in 30 minutes). all the results of this study showed that e. polyceras leaves can be a valuable source of polyphenols such as flavonoids, which have a crucial role as antioxidants and free radicals scavengers, this can predict the ability to use this species in the treatment of oxidative stress illnesses. this plant needs more studies about the safety, the toxicological effects and the determination of the therapeutic dose. conclusion this study is the first report about echinops polyceras bois. leaves, where primary identification tests of secondary metabolites were established, and the phenolic compounds and flavonoids contents were determined in different extracts, using micro methods in a 96-well microplate. also, the scavenging activity of free radicals was evaluated using the dpph• radical. our in vitro results were 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patch doi: https://doi.org/10.31351/vol29iss1pp184-194 184 formulation and evaluation of iornoxicam as dissolving microneedle patch adeeb r. alkhiro *,1 and mowafaq m. ghareeb** *department of pharmaceutics, college of pharmacy, al-nahrain university, baghdad, iraq **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the objective of the study was to develop microneedle (mn) patch, with suitable properties to ensure the delivery of a therapeutic level of lornoxicam (lxm) in a period suitable to replace parenteral administration in patients, especially those who fear needles. the used polymers were cold water-soluble polyvinyl alcohol (pva) and polyvinylpyrrolidone (pvp) of low molecular weight with peg 400 as plasticizer and tween 80 (to enhance the release) using micro molding technique. patches were studied for needle morphology, drug content, axial fracture force measurement and drug release while the optimized formulas were further subjected to ph measurement, folding endurance, ex vivo permeation study, histopathology study, stability study and compatibility study. the patch with 11:1 ratio of pva to pvp, 30% solid content, 5% peg 400 and 3% tween 80 resulted in axial needle fracture force value of (1.35 n) which is suitable for skin penetration. the release was fast with almost 100% of drug released in 60 minutes. the permeation was enhanced significantly with a steady state flux of about 3.1 times that of the solution. the lag time of mn is shorter in comparison with ordinary patch. histopathology studies demonstrated the safety of the formulation, both stability studies and compatibility studies showed the suitability of the formulation. the results indicated that lxm microneedle patch could enhance drug permeation while achieving fast and painless administration. keywords: microneedle patch, polyvinyl alcohol, polyvinylpyrrolidone, fracture force, lornoxicam. باستخدام بوليمرات مختلفة كرقعة مجهرية اإلبر قابلة للذوبانصياغة وتقييم لورنوكسيكام **موفق محمد غريب و1*،و اديب رغيد الخير .العراقالصيدلة ، جامعة النهرين ، بغداد ،قسم الصيدالنيات ، كلية * .العراق ، بغداد ، بغداد جامعة ، الصيدلة كليه الصيدالنيات، فرع** الخالصة بمستوى عالجي في فترة مناسبة (lxm)مجهرية ذات خواص مناسبة لضمان دخول لورنوكسيكام تهدف الدراسة إلى تطوير رقعة إبرية ء البارد الستبدال اإلعطاء بالحقن للمرضى ، وخاصة أولئك الذين يخشون من زرق اإلبر. البوليمرات المستخدمة هي كحول البولي فينيل الذائب بالما (pva( والبولي فينيل بيروليدون )pvp ذو الوزن الجزيئي المنخفض مع )peg 400 )كمادة ملدنة( وtween 80 ) )لتعزيز تحريرالدواء دواء في باستخدام تقنية الصب. تمت دراسة شكل اإلبرللرقع المحضرة باإلضافة الى دراسة المحتوى الدوائي ، قياس قوة الكسر العامودية و تحرر ال اسة الثابتية الدوائية ودراسة ، النفاذية خالل جلد حي، دراسة التشريح المرضي ، در حين أن التركيبة المحسنة خضعت لقياس درجة الحموضة tween80٪ 3و peg 400٪ 5محتوى صلب ، ٪30و ذات 1: 11بنسبة pvpإلى pvaالتوافق الكيميائي. نتج عن التركيبة التي تحتوي على دقيقة. 60٪ تقريبًا من الدواء في 100نيوتن( وهو مناسب الختراق الجلد. كان التحرر الدوائي سريعًا مع تحرر 1.35أحدثت قوة كسر مقدارها ) صر مع أضعاف محلول الدواء. كان وقت التأخير بعبور الدواء من خالل الجلد أق 3.1تم تعزيز النفاذية بشكل كبير مع تدفق بصورة مستقرة حوالي الرقعة اإلبرية المجهرية. أظهرت دراسات التشريح المرضي سالمة النسيج ، وأظهرت كل من دراسات االستقرار ودراسات التوافق مدى مالءمة التركيبة. ستعزز الرقع االبرية الميكروية للورنوكسيكام تغلغل الدواء مع تحقيق إعطاء سريع وغير مؤلم. ، البولي فينيل بيروليدون ، قوة الكسر ، لورنوكسيكام. ، الكحول البولي فينيل برية المجهرية: الرقعة اإلالمفتاحية الكلمات introduction the largest organ of the body is the skin making it potentially a highly accessible organ of the human beings. but the stratum corneum (sc) represents the main barrier of the skin and its penetration is very difficult. invasive methods by employing conventional and hypodermic needles were able to bypass this barrier, but it may cause pain, possibility of contamination, infections and fear. transdermal drug delivery using patches can overcome the drawbacks of hypodermic needles, but the conventional patch system has many limitations, mainly limited to drugs that can diffuse through the skin barriers of which the sc represents the main challenge and only drugs with low molecular weight <500 da and satisfactory lipophilicity can successfully penetrate it to result in successful administration. improving permeation can be achieved only by reversibly disrupting the molecular construction of these barriers (1). 1corresponding author e-mail: adeebalkhiro@gmail.com received: 6/ 9 /2019 accepted:12 /1 /2020 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp184-194 iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 185 promising technique to facilitate permeation of drug through the skin barriers are microneedles (mn), which are collections of arrays of micron measurement projections that enhances permeability of the sc by reversibly creating channels of micron size in the skin layers, thereby the skin penetration by a relatively impermeable molecules is enabled. mn devices are minimally invasive, due to avoiding the activation of sensory nerve fibers. the main types that have been developed include, “solid, coated, dissolving and hollow mns”. the type that received major attention was “dissolving mns” because of its potential advantages that overcomes the other types, which are: higher drug loading capacity contrasting both coated mns and hallow mns, as these mns dissolve with no sharp biohazard waste, scaling up to mass production of “dissolving mns” is possible as the polymers used are inexpensive and the method of fabrication is based on micro molding (2). hydrophilic polymers that fit the requirements for fabricating polymeric, dissolvable mns include pva and pvp (3). they were chosen due to the complementary properties of each other, as pva imparts strength while pvp imparts flexibility and increases the solubility of the polymeric mixture (4). lxm is a member of the oxicam class of nsaids. it is commercially available in both oral and parenteral dosage forms. lxm oral dose ranges from 4-8 mg which is usually well tolerated, but still causes some side effects mainly gastrointestinal side effects. lxm plasma half-life is relatively short about 3 to 5 hr. and has good permeability, which make it a good candidate for transdermal delivery (5). the objective of this study is to prepare dissolvable mn patch for lxm, with better penetration ability by bypassing the sc and to investigate the different materials required to optimize the formula. materials lxm was purchased from chemshuttle, usa, water soluble pva from central drug house (p) ltd, india, pvp k30 from urchem, china, na2hpo4 from thomas baker, india, potassium dihydrogen phosphate (kh2po4) from merck, germany, triethanolamine and ethanol from sigmaaldrich, germany, tween 80 from riedel-de-haën, germany, lastly blue silica gel powder from om chemicals, india. methods preparation of lornoxicam microneedle patches preparation of lxm mn patches was accomplished by using different ratios of pvp k 30, pva and 5% w/w polyethylene glycol 400 (peg 400) by micro molding techniques as shown in table (1). first, lxm was dissolved in certain amount of solvent mixture composed of 75 % phosphate buffer, 20% ethanol and 5% triethanolamine using magnetic stirrer. then adding and dispersing pvp then pva in different ratios to the drug solution mentioned above, then placing the whole mixture in a sonicater for 1.5 h. after the whole mixture was dissolved, calculated amount of the dispersion was added to the polydimethylsiloxane (pmds) microneedle mold (micropoint, singapore) with a patch size of 6.25 cm2, array size of 15 x 15, needle height of 500 µm and a needle base of 200 µm. then the filled mold is sonicated to remove air bubbles and finally the mold is placed in a desiccator under vacuum for one day and then in the oven at 40 °c for 24 hours to ensure drying and needle formation, as shown in figure (1) (6). aluminum foil was used as backing layer for the formulated patches (7). figure 1. fabrication process of pva: pvp microneedle patch (6). table 1. composition of prepared micro needle patches using different ratios of pva and pvp. iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 186 formula no. drug (mg) pva (g) pvp (g) peg 400 % w/w solvent (ml) f1 40 1 1 5 10 f2 40 1.5 1 5 10 f3 40 1.25 0.75 5 10 f4 40 1.5 0.5 5 10 f5 40 2 0.5 5 10 f6 40 2.5 0.5 5 10 f7 40 3 0.5 5 10 f8 40 3.5 0.5 5 10 f9 40 2.75 0.5 5 10 f10 40 2.75 0.25 5 10 preparation of lornoxicam ordinary patch ordinary patches were made by preparing the same solution mixture of lxm and polymers in same amounts as in preparation of lxn mn. the mixture was casted in petri dish with 8.5 cm diameter. dried in an oven (memmert oven, germany) at 40°c for 24 h. after complete drying, the patch was cut to smaller patches with same amount of drug and same surface area as mn patches (6.25 cm2) that resulted from mn mold. evaluation of lxm mn patch microscopic and visual inspection the prepared micro needle patches were inspected visually for any defects and by using digital microscope (depstech, china) to inspect the needles morphology for any defects. drug content the analysis was determined by immersing one patch in 100 ml ph 7.4 phosphate buffer. then, the sample was diluted and filtered, the absorbance of prepared solution was measured at 376 nm using uv visible spectrophotometer. the percentage drug content was calculated the process was done in triplicate (8). in vitro drug release studies a modified dissolution method was used. mn patches were placed on a mesh wire with sieve opening of approximately 800 micrometers, fastened with an elastic band on the open end of tube with the patch placed in center (9, 10). the tube was immersed in the dissolution jar of the dissolution apparatus (pharma test, germany) containing 500 ml of phosphate buffer ph 7.4 which is enough to ensure sink condition according to the used drug loading 8 mg. the apparatus speed was set at 50 rpm (11) and temperature was maintained at 35± 0.50 °c (12). the study continued for 1 hour and samples were withdrawn every 5 minutes. a 5 ml sample was withdrawn from the beaker and replaced with fresh 5 ml of buffer, finally the cumulative drug release percent was calculated. tween 80 was added in 3% concentration to the formula that did not achieve 100% drug release during 1h (13). measurement of axial needle fracture force axial needle fracture force is defined as “minimum force, applied parallel to the microneedle axis, required to deform or break the microneedle (needle failure)”. axial needle fracture force was measured using texture analyzer (ta.xt stable micro systems, uk). the microneedle patch was fixed on a fixed cylindrical platform using double sided adhesive band. the instrument was programmed to axially compress the single microneedle on each occasion by a spherical probe (diameter: 1 inch) travelling at a speed of 0.05 mm/s to a distance of 2 mm (14). evaluation of the selected formula ex vivo permeation study a modified drug permeation apparatus similar to that used in the release study with same parameters was utilized (figure 2). the volume of dissolution media was 750 ml to ensure sink condition for the specified drug loading. a fresh abdominal rat skin was used in this study, the area was shaved using an electric clipper, after sacrificing the animal the skin was extracted and the subcutaneous layer was removed and kept in saline phosphate buffer ph 7.4 at low temperature (4°c) and used the next day with sc facing the donor chamber, while the other end faces the receptor chamber. applicator device (mpatch mini microneedle applicator from micropoint, singapore) was used to ensure the insertion of mn patch, the applicator speed was 1 (mm/ sec) (9). 5 ml samples were withdrawn at different time intervals and replenished by equal volume of buffer. the withdrawn samples were analyzed for lxm. the study continued for 6 hours for 22 mg drug loading (15). the microneedle patch lxn mn was compared to lxm solution (22mg of lxm in 10 ml composed of phosphate buffer ph7.4 with 5% triethanolamine) and lxm ordinary patch with the same composition of polymers and amount of lxm as that of mn patch. iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 187 dd solver was used in determining release kinetics of the final selected mn patch. f2 similarity factor function of dd solver was used to determine the degree of similarity between the permeation profiles of selected mn formula with the above-mentioned comparators (16). figure 2. modified drug permeation apparatus. kinetics of drug permeation permeation kinetics were examined using dd solver to fit the permeation data utilizing four popular release models such as zero-order, firstorder, higuchi and korsmeyer peppas equations and n value, which have been described in the literature (16). histopathology study histopathology study was done on the abdominal skin used in the permeation study of the selected mn patch loaded with drug compared with unexposed abdominal rat skin to determine the pathological changes. both tissues were fixed in 10% formalin, routinely processed, and embedded in paraffin. paraffin sections were cut on glass slides and stained with hematoxylin and eosin to examine the morphological changes to the tissue via blinded study (7). ph measurement it was determined by immersing the patch for 30 min with 100 ml of de-ionized water, then the electrode of a ph-meter was placed on the insert surface and the ph was recorded. the process was done in triplicate (17). weight and thickness uniformity five patches were selected randomly from each batch and weighed individually using a digital balance; their thickness was measured using a digital vernier (neiko, usa), the mean weight, thickness and standard deviation were calculated (18). folding test folding endurance is “the number of folds that can be achieved without breaking of the patch when repeated folding at the same position is done”. folding endurance of three patches of the selected formula was carried out (8). compatibility study using ftir lxm ftir spectrum was obtained by using the pressed-disk technique. a small amount of drug was ground with potassium bromide powder then the mixture was pressed into a disk. the ftir spectroscopy was used to investigate the prepared disc at wave number range of 4000-400 cm-1 . the ft-ir technique was performed on both the drug alone and the formulated mn to investigate any chemical interaction (7). stability study stability studies of the optimized formulation was carried out by storing the replicates of the patches at 40 ± 0.58 °c and 75± 5% relative humidity for 3 months. samples were withdrawn every two weeks to measure percent drug content as stability indicating parameter (19). statistical data analysis the results of the experiments were given as a mean of triplicate samples ± (sd) and were analyzed according to the one way analysis of variance (anova) to determine if the changes in the applied factors are statistically significant at level of (p ≤ 0.05) and non-significant at level of (p > 0.05). results and discussion microscopic and visual inspection inspection was done visually and by digital microscope depstech 1600x, images are illustrated in table (2). formulas f7 and f8 failed from visual inspection due to the high viscosity of the polymer matrix created a challenge to fill the mold micro cavities by vacuum application (20). the other formulas show clear needles. the pyramidal shape of mn donates a superior mechanical strength, thus increasing the chances of effective skin penetration (21). table 2. microscopic images of all prepared microneedle patches. iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 188 formula code microscopic images formula code microscopic images f1 f7 f2 f8 f3 f9 f4 f10 f5 f10tw3% f6 f9 tw3% drug content iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 189 the mean percent drug content of mn is illustrated in table (4). the results demonstrated drug content of the formulations was within the limits of usp38/nf33 (2015) (22). table 4. the percent of drug content of mn patches (mean ±sd) n=3 formula code drug content % f4 99.51 ± 0.038 f5 98.67 ± 0.125 f6 98.58 ± 0.121 f9 97.33 ± 0.172 f10 98.12 ± 0.233 f9 3% tween 80 98.17 ± 0.221 f10 3% tween 80 98.92 ± 0.156 in vitro drug release studies in vitro cumulative percent of lxm release at different sampling time is shown in the figures (3). formulas with successful mn formation were chosen to study the effect of pvp: pva ratio on drug release. the release of (f4, f5 and f6) was 100% in 60 min, so they were categorized as fast release formulations, and this is believed to be the result of a high pvp: pva ratio. it is shown that increasing the pvp concentration, reduces the time needed for dissolution and generated holes in the mns with water resulting in an enhanced dissolution rate. the actions of pvp was attributed for its both hygroscopicity and moisture adsorption property, which resulted in the hydration and the burst effect made the release of drug from patches containing higher amount of pvp faster (4, 23). while f10 and f9 showed 78% and 83% respectively, during 1 hour of release time, due to a lower pvp: pva ratio. tween 80 was chosen as the surfactant of choice due to its availability and studies identifying its effects to be greater than tween 60 and 20 in improving solubility (24). tween 80 was added for both f10 and f9 at 3% w/w concentration to improve their drug release. fast release with 100% release was achieved with 3% tween 80 addition for both formulas as shown in figure (4). the improvement in dissolution rate caused by tween 80 was due to the reduction in the interfacial tension which increased the wetting of the polymer to a higher degree thus increasing the erosion of the polymer (25). figure 3. in vitro release studies of lxm mn formulations in phosphate buffer ph 7.4 and at a temperature of 35± 0.50 °c. figure 4. in vitro release studies of f10 and f9 with and without 3 % tween 80 in phosphate buffer ph 7.4 and at a temperature of 35± 0.50 °c. measurement of axial needle fracture force interspacing between mn plays an important role in the force of insertion and when it is increased, the force is decreased which indicates a higher stress on the mns at small interspacing and lower stress at wider interspacing. there is a reduction in the resistance to mn insertion in a form of stress on the tips which signifies that the mns face less resistance when the needles are spaced at a greater area (26).so the mold used in this study was selected with an interspacing of 1500 µm to reduce the required force of insertion. the accepted axial needle fracture force value is 0.0856 n/mn array for interspacing of 600 µm and above when the velocity of insertion is 1 mm/s iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 190 (which is the velocity of the applicator used in this study) (26). for 15 array mn patch, the calculated insertion force for this research mn patch is 1.284 newton (n). therefore; all the patches with axial fracture force below 1.284 n was considered as failed formulations. the results of the fracture force are shown in table (3). fracture force test showed that when pvp concentration increased, the elasticity increased while fracture force reduced, thus the applied force in n required to break the needles decrease (27). pva polymer enforces hardness and rigidity, thus increasing pva concentration results in a harder needles and higher force should be applied (n) to break the needles (28). the results show only f10 has exceeded this insertion force and with an axial fracture force of 1.72 n. as it has the highest ratio of pva: pvp (11:1) among other formulas. tween 80 was added to f9 and f10 at 3% (w/w) concentration to improve their release profile. the addition of tween 80 increased the flexibility of the polymers thus reducing the mechanical strength of the formed mn patches (29), this was shown with both f10 and f9 as the fracture force was 1.35 n and 0.82 n respectively. the selected formula was f10 + 3% tween 80 (f10%+3%t80) as its fracture force exceeds the required insertion force. table 3. peak force and the time required for fracture of some of lxn mn formulas using texture analyzer. formula peak force “hardness” (n) time (s) f1 0.52 2.10 f2 0.69 2.52 f3 0.71 2.78 f4 0.96 2.50 f5 1.03 3.00 f6 1.20 4.90 f9 1.23 4.33 f10 1.72 6.39 f9 + 3%(w/w)tween 80 0.82 2.52 f10 + 3%(w/w)tween 80 1.35 5.00 ex vivo permeation study microneedle patches of f10+3%t80 showed the greatest cumulative amount of drug permeated which reached 67% during 6 hr., while ordinary patch only 24% of lxn permeated, while lxm solution permeated only 19% in the permeation study. the study states flux rate (ssfr), which was calculated as the slope from the straight portion of permeation graph, for the three arms showed the superiority of mn patch with ssfr of 3.98 µg/cm2 /min, while ordinary patch 1.46 µg/cm2 /min and the lxm solution resulted in 1.27 µg/cm2 /min. the overall improvement with respect to flux rate was obtained with lxm mn as it increased by 3.1 folds when mn patch is compared to lxm solution. also the lag time for mn patch was the shortest with only 10 minutes, while ordinary patch lag time was 50 minutes, and lxm solution had initially faster drug permeation when compared to ordinary patch as the drug was already in solution overcoming the time required to dissolve the polymer matrix which is essential for solubilizing the drug. but the ordinary patch had a higher cumulative amount of drug permeated, this is probably due to the surfactant and solubilizing nature of both pva and pvp which aided in higher amount of drug to be permeated (30). the mn patches improved permeation due to the pores created by the needles breaching the sc allowing the drug to permeate at a higher rate, the created holes are estimated to remain open during the time of application of the mn patch by the action of high polymer concentration in the holes preventing their closure, this is true when the polymer matrix act as the backing layer (31). in addition, the hydration of the drug reservoir in the polymer matrix was enhanced by the fluid from the skin entering form channels created by mn which improves drug diffusion (26). figure 5. collective ex vivo permeation study of mn f10+3%t80, ordinary patch f10+3%t80 and lxm solution in phosphate buffer ph 7.4 and at a temperature of 35± 0.50 °c. kinetics of permeation by using dd solver excel add-in program the kinetics of drug release was deduced by calculating the r2 value of zero order, first order and iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 191 higuchi models also the n value in korsmeyer peppas models. the r2 was the highest for higuchi model with a value of 0.991, while the results were 0.955 and 0.681 for first order and zero order respectively. the n value from korsmeyer peppas model was 0.531 which indicated an anomalous (non-fickian) transport that implies the release mechanism was governed by both diffusion and relaxation or erosion (32). histopathological study histopathology of the abdominal tissue (figure 6) indicated the absence of tissue damage and toxicity. both lxm loaded microneedle patch treated and untreated abdominal skin tissues showed no effect on the microscopic structure of the skin. no cell necrosis was detected after application of lxm mn patch and saline buffer ph 7.4 as comparator. figure 6. histopathological evaluation of sections of rat abdominal tissues. a. rat abdominal tissue incubated in the diffusion chamber with lxm loaded microneedle patch showing the created channels, b. rat abdominal tissue incubated in phosphate buffer ph 7.4. ph measurement the ph of the selected formula was 7.7 ± 0.2, this result may be due to the effect of triethanolamine which has a ph adjusting activity, and has the ability to increase and maintain ph level about 7.8 which is known to have no skin irritating effects (33). weight and thickness uniformity five patches were selected randomly from each batch; the thickness of each patch was measured at five positions using a digital vernier caliper (12). the mean thickness and standard deviation were calculated (1 mm ±3%). batches with a variation of more than ±5% from the mean were rejected (34). the mean weight for tested patches was 0.913 ± 0.043g. folding test the selected formula f10 + 3% t 80 withstood breakage for 210 ± 2 folds. which indicates its high suitability for application and strength. compatibility study using ftir the ftir spectrum of lxm and the selected formula (f10+3%t80) is shown in the figure (7). lxm peaks at 3468.18 cm-1 indicates oh stretching, intense bands at 3064 cm-1 indicates nh stretching, 1594 cm-1 indicates c=o stretching of amide, 1037 cm-1 indicates s=o stretching. these peaks were also observed in ftir spectrum of the final formula, suggesting that there was no interaction with drug and polymer and all bands were compared with reference (7). iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 192 figure 7. compatibility ftir, ir spectrum of a: lornoxicam, b: pvp, c: peg 400, d: pva and e selected formula. a b c d e iraqi j pharm sci, vol.29(1) 2020 lornoxicam microneedle patch 193 stability test effect of storage condition on stability indicating parameters, folding endurance and percent drug content are presented in table (5). insignificant decrease was observed in percent drug content and folding endurance, indicating the stability of the developed formulation . table 5. stability data of optimized microneedle patch (f10 3% t80) stored at 40 ± 0.58 °c and 75± 5% relative humidity. time of sample folding endurance drug content% at the time of preparation 211 98.33 after two weeks 211 98.08 after four weeks 211 97.82 after six weeks 209 97.14 after eight weeks 208 96.62 after ten weeks 207 95.92 after twelve weeks 205 95.54 conclusion based on the results obtained from this study, it is concluded that lxm mn patch was successfully prepared using pvp and pva. all the formulations were found to have the desired amount of drug content. the apropriate ratio of pva to pvp will result in the desired fraction force with rapid dissolution and good permeability profile.the optimized formula f10 3% tween 80 showed drug release of 100% within 1 h. ex-vivo studies on rat abdominal skin showed permeation of 67% within 6h. the selected formula exhibited diffusion by highuchi model. the mechanism of release of the selected formula showed both diffusion and erosion mechanism. the optimized formula was found to be safe, since no histopathological changes were observed after incubation with the 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cytotoxicevaluation of bis-(2mercaptoacetate) gold (iii) chloride 46 design, synthesis, characterization and comparative cytotoxic evaluation of bis-(2mercaptoacetate) gold (iii) chloride nohad a. alomari *, 1 * department of pharmaceutical chemistry, college of pharmacy, university of mosul, mosul, iraq. . abstract in recent years , the interest in gold (iii) species have gained more and more attention for cancer chemotherapy , this was stimulating by the possibility to develop new agents with mode of action and clinical profile different from the established platinum metalodrugs. with this frame, recently new square planar au(iii) complexes (au(l)(l')n); where l=sch2coo ; l'=hsch2coo had been synthesized with s/o – donor ligands. in this article and by the aim to replace, one of (l') ligand by anion chloride ligand (which supposedly more relevant for the biodistribution of the compound than for its pharmacodynamic effects), new complex (au(l')2 cl) was synthesized : (h (aucl4) + 2 hsch2coo na + au(l')2 cl) the prepared complex was characterized by elemental analysis, ftir, h'nmr, uv, molar conductance and magnetic susceptibility and its anticancer activity was tested against hep-2 cell line. the comparative figure of the cytotoxic activities of all the synthesized complexes against hep-2 cell line , showed cytotoxic profile variant to the wellknown anticancer drug cisplatin , indicated that there is no direct correlation exists between the nature of the ligands (l),(l') and (cl ) , their different arrangements around central metal core with their collectives cytotoxic results. key words: s/o – donor ligands. , gold (iii), cytotoxic للمعقدتشخيص وتقييم ضد سزطاني و مقارن , تصميم ,تحضيز bis –(2-mercapto acetate) gold (iii) chloride الوهاب العمري نهاد عبد ،*1 * ، انعزاق .يٕصم ، انًٕصم ، كهيت انصيدنت ، خايعت كيًياء انصيدالَيتانفزع الخالصة خالل ححضيز ٔ ( في انسُيٍ األخيزة باْخًاو أكثز ٔأكثز كعالج ضد انسزطاٌ َٔخح ْذا يٍ iiiنمد حضج يزكباث انذْب) احخًانيت ظٕٓر يزكباث خديدة حًخاس بفعانيت ٔصفاث سزيزيّ يخخهفت نًا اثبج سابما نًزكباث انبالحيٍ. ٔيٍ ْذا اإلطار فمد حى يؤخزا" l=sch2coo(,عُديا n('au (l)lبصيغت ) au( iiiححضيز يعمداث انذْب ) ,hsch2oo =l' ياَحت يٍ َٕع اثبهيكاَد .s/o بايٌٕ انكهٕريد ) ٔانًمخزذ أٌ يكٌٕ نّ عاللت باالَخشار انبايٕنٕخي أكثز يٍ 'l) ٔبٓدف اسخبدال , ٔاحدة يٍ نيكاَد )في ْذا انبحث ٔكًا يهي: au(l')clحأثيزِ اندٔائي(, ٔعهيّ فهمد حضز انًعمد h (aucl4) + 2 hsch2coo na + au(l')2(cl) ت انًغُاطيسيت أيا فعانيخّ انمابهي, انخٕصيهيت انًٕالريت ٔ ,ftir, hnmr, uvحى حشخيص انًعمد انًحضز بانخحهيم اندليك نهعُاصز .انسزطاَيت hep-2 ضد انسزطاٌ فمد اخخبزث ضد خاليا فعانيت يخخهفت نهدٔاء انًعزٔف سيُزبالحيٍ ن, أظٓزث صيغت hep-2أٌ انشكم انًمارٌ نفعانيت ضد انسًيت انًعمداث ضد خاليا ٔحزحيبٓى انًخخهف حٕل انُٕاِ (cl)أٔ 'l)(, )(lٔانًسخخدو ضد انسزطاٌ, ْٔذا يؤشز اَّ اليٕخد عاللت بيٍ طبيعت انهيكاَد سٕاء كاٌ ."يع انُخائح ضد انسًيت يدخًعت au(iii)انًزكشيت .s/o ليكاندات واهبة ،،الدراسة السمية (iii) الذهب الكلمات المفتاحية: introduction cancer is a major health problem. numbers are evident: 10 million cases are diagnosed each year (1) , and in 2020, new cancer cases are predicted to have doubled to a 20 million per year (2). in the past, inorganic compounds were applied in an empirical fashion with little attempt to design the compounds used and with little or no understanding of the molecular basis of their mechanism of action. the development of modern medicinal inorganic chemistry has been made easy by the inorganic chemist’s extensive knowledge on coordination and redox properties of metal ions. then, systematic consideration of specific properties of metal ions, their patterns of tissue uptake and distribution in organisms 1 corresponding author e-mail: nohad.alomari@gmail.com received: 2/6/2013 accepted: 29/10/2013 iraqi j pharm sci, vol.22(2) 2013 cytotoxicevaluation of bis-(2mercaptoacetate) gold (iii) chloride 47 and their preferred coordination in complexes has depened up the possibility for inorganic chemists to contribute to the health and wellbeing of man. the unexpected success of cisplatin in treating a fairly wide variety of cancers, however, was slightly obscured by the evidence of serious kidney toxicity and other side effects, natural and acquired resistance to cisplatin and the reduced therapeutic indexes that could be used considering toxicity limitations (3) . consequently the development of novel metallodrugs with pharmacological profile different from that of the platinum drugs in the focus of modern medicinal chemistry and drug design. gold therapies have been historically used to treat rheumatoid arthritis which led to the discovery that such treatments also lowered the risk of several types of cancer, and subsequently to increased interest in their application as anticancer therapies (4) . gold (iii) compounds, in particular, are being explored because they are isoelectronic to the pt(ii) ion found in cisplatin (5) . thus it was originally thought that structural analogues of cisplatin would demonstrate a similar reaction mechanism, however, it has since been demonstrated that gold(iii) coordination complexes tend to show a much lower binding affinity to dna as compared to cisplatin (6-7) . despite this, gold compounds appear to be a particularly strong avenue to pursue, since gold complexes tend to have strong cytotoxic potential in the micromolar or even nanomolar ranges (8) . indeed, gold complexes appear to be more cytotoxic than other analogous, isoelectronic metalcomplexes (9) . hence, gold species might give access to a class of non – platinum metal compounds with non – cisplatin – like pharmacodynemic and pharmacokinetic properties, which are major goals of bioinorganic and bioorganometallic medicinal chemistry research (10) . the renewed interest toward gold(iii) compounds as potential antitumour agents, that started at the beginning of 1990s, has resulted, until now, in the identification of several gold(iii) and organogold (iii) compound characterized by a significant structural variety and by encouraging in vitro pharmacological properties . notably, these gold (iii) compounds constitute today an interesting family of new cytotoxic agents that undoubtedly deserve more extensive pharmacological testing and a careful analysis of their mechanism of action (11) . many gold(iii) complexes have been evaluated against an in vitro panel of human tumour cell lines comprising cells of different tissue types and different responses to cisplatin. initial in vitro studies indicated that the carcinoma cell line are sensitive to these compounds (12-14 ) . a rational design of new potential organometallic drugs, with increased biological activities and the potential to overcome resistance, selectivity issues and toxicity, is possible nowadays due to the large diversity of structure and bonding modes (πcoordination, metal-carbon multiple bonds, etc) that can be tuned . the next stage in drugs design has to be the development of highcomplex drugs that deal successfully with transport (though membranes), survival in the cell, binding to dna and excretion mechanisms with minimum side effects where both metal coordination and hydrogen bonding more likely are the key factors (15) . however, developing drugs with metals incorporated in the structure is not an easy mission. it is important to specify which parts of the complex are essential for activity : the metal itself , the ligands , or the entire complex. recently, we had synthesized a novel au (iii) complexes of thioglycolate and mercaptoglycolate ligands .the cytotoxic evaluation revealed that the hep-2 cell line differ in its sensitivity toward the selected complexes compared to cisplatin (16) . in order to better understand the role of ligand or the metal core involvement of biological action of this type of metal-based anticancer compounds , or at least to establish some structure activity relationship that could be applied in the design of more effective drugs, it is paramount involved in the biochemical interaction , in addition the drug design strategy for this novel gold (iii) complex was motivated by the aim to replace one of the mercaptoglycolate l' = hs ch 2 coo -, by chloride ligand (good leaving group) which is supposedly more relevant for the biodistribution of the complex than for its pharmacodynemic effects . the prepared complex (au(l')2cl) is also designed with a two monodentate ligands ( thioglycate and chloride) that could be hydrolyzed and thereby make more than one site for substitution . the potential interest lies mainly in the facility of modifications of the ligand moiety , which could help in the tuning of the biological properties. it is also of major iraqi j pharm sci, vol.22(2) 2013 cytotoxicevaluation of bis-(2mercaptoacetate) gold (iii) chloride 48 importance to consider the expensive and highly demanding and time consuming clinical trials of a researcher is truly serious about developing realistically useful drug. the mentioned complexes essentially correspond to a square planar geometry , since its diamagnetic (au(iii) d 8 ), while octaand tetrahedral are paramagnetic (au(iii) and au 0 are d 10 ) ,as this confirmed by electronic spectra assigned to 1 a1g 1 a2gand 1 a1g 1 eg transition ; specifically related to square planar but not octa or tetrahedral geometry. these complexes have different sets of donor atoms. in all cases, of the previous compounds and the new analogue, the remaining donors are o/s – ligands – now it is clear that multidisciplinary research is needed to define the main factors involved in the structure-activity relationship of all drugs that later will help in an increasingly purposeful design of new and more effective metal-based therapeutics. the potential interest lies mainly in the facility of modifications of the ligand moiety, which could help in the tuning of the biological properties. thus, the main goal of this article is to enlarge the scope and repertoire of gold (iii) complex of potentially interest as anticancer agents through characterization of their chemical structures and preliminary comparative cytotoxic studies. . figure (1) : the chemical structures of the previously synthesized complexes (c1-c4)(16) and the target complex of this article (au(l')2 cl). experimental chemistry all chemicals were of reagent grade quality and were purchased from commercial sources (bdh and fluka). they were used without further purification. ir spectra were recorded on brucker tensor 2710 (ftir) spectrophotometer in the 4000-250 cm -1 range using csi disc. electronic spectra were recorded on shimadzu uv 160 spectrophotometer for 10 -3 m solution of the complexes in dimethylformamide using 1 cm quartz cell. the 1 hnmr spectra were recorded on brucker/ hims university, syria. spectrophotometer in dmso-d6 at room temperature conductivity measurements were made on conductivity meter 4070 jenway. the magnetic measurements were carried out at 25 o c on the solid state by faraday's method using bruker bm6 instrument. metal content analyses were made on shimadzu aa670 atomic absorption spectrophotometer. elemental analysis (c h s ) were carried out using perkin elmer 2400 in al bait’s university / jordan. preparation of disodium 2thioglycolate (l') the ligand was prepared according to the following general method. the reaction of an equivalent amount of naoh (4.00g, 0.01 mol) and mercaptoacetic acid sodium salt (1. 12 g, 0.01 mol) in 30 ml ethanol. the mixture was boiled under reflux for 3h. preparation of (au(ococh2sh)2cl)= (au(l')2cl) a solution of the ligand mercaptoacetic acid sodium salt (0.22g, 0.002 mol.) in 15 ml ethanol was mixed with a solution of h(aucl4) (0. 34 g, 0.001 mol.) in 15 ml ethanol in (1:2) molar ratio. the reaction mixture was refluxed for 2 h., the mixture was left 24 h. at room temperature to give a brown precipitate, which was filtered off, washed with ethanol and diethyl ether and then dried under vacuum for several hours. stability in buffer the stability test was run on the synthesized compound. a minimum amount of dimethyl sulfoxide (dmso) was used to dissolve the complex, which was then diluted in phosphate buffer (ph 7.4), a solution of concentration 5.0 * 10 -5 m was made and c ch2 s o o c ch2 cl au oo sh h au o o h s h2c c o ch2 c o s ss o o h2c c o o ch2 au cna au s o o o ch2 c o c c o o hs hs naau h s o o c ch2 o oc c o o hs hs c1 c2 c3 c4 [au(l')2 cl] iraqi j pharm sci, vol.22(2) 2013 cytotoxicevaluation of bis-(2mercaptoacetate) gold (iii) chloride 49 observed daily for a period of 7 days. the sample allows to stored in a dark environment throughout the 7-day period. there appears to be no significant shift in the absorption maxima at 320 nm, which is the absorption that arises due to the gold(iii) metal ion. cytotoxic study preliminary cytotoxic test human larynx epidermoid carcinoma (hep-2) was kindly provided by the iraqi center for cancer and medical genetics research iccmgr). this human cells grew rapidly ,doubling themselves in 2-3 days and were shown to be extremely resistant to ultraviolet rays (17) . passages 227-229 were used throughout this study and rpmi-1640 was used in maintaining the cells since they had been adapted at the iraqi center for cancer and medical genetics research to grow on this medium,& were grown in rosswell park memorial institute (rpmi) 1640 medium (gibco ,usa) , which was prepared as follows  rpmi 1640 medium powder was dissolved in approximately 600 ml of double distilled water (ddw) and then the other components added :  0.5 ml ,streptomycin (1g/5ml)  sodium bicarbonate (4.4%) 15 ml to give the final ph of 7.2 sodium bicarbonate solution was prepared by dissolving 4.4 g. in 100 ml d.w. the solution was autoclaved at 121 o c for 15 minutes and stored at 4 o c(18)  0.5 ml benzyl penicillin g (600 i u /5ml)  2.5 mg amphotericin b.  100ml fetal calf serum. the volume was completed to one liter with ddw. then the mixture was sterilized using seitz filter and filtration repeated using in a sterile environment, then stored at 4 o c for direct use. all antibiotics were freshly prepared. the growth medium was decanted off and the cell sheet washed twice with phosphate buffered saline (pbs), composed of:  sodium chloride (nacl) 8 g.  disodium hydrogen phosphate (na2hpo4) 0.9 g.  potassium dihydrogen phosphate (kh2po4) 0.2 g. after dissolving all components, the solution was autoclaved at 121 o c for 15 min and then stored at 4 o c prior to any usage, pbs was warmed to 37 o c. cells were regularly subcultured when monolayers were confluent. two to three ml of warm trypsin-versene (prepared by mixing 20 ml of trypsin solution, 10 ml of versene solution and 370 ml pbs and stored at 4 o c). were added to the sheet and the flask rocked gently (18) . preparationof3-(dimethylthiazol-2-yl)-2,5diphenyltetrazoliumbromid(mtt) solution (sigma ,usa) fifty milligram per ml of mtt dye was used as a final concentration ( 19) .the solution was filtered through 0.22µ syringe filter to remove any blue formazan product (20) , and then stored in sterile , dark ,screw –capped bottles at 4 o c. the solution was used within no longer than 2 weeks of preparation. cytotoxic assay on hep-2 cell line: this step must be prepared under aseptic condition. the complex was prepared for microtitration assay by dissolving 2 mg. of each compound in 2 ml of solvent (0.2 ml dmso & 1.8 ml ddw, the stock concentration is 1000µg/ml)and filtered by 0.22 µ millipore filter .serial dilutions of each compound (2.50, 1.25, 0.625 & 0.313) µ mol./ml under assay in sfm were added to the well. three replicates were used for each concentration of either four tested complexes in addition to cisplatin (ebewe, austria europe) as a reference (d: positive control) when the cells are exactly in the exponential phase in the population doubling time (pdt) , then the cells in full of their activity , the cells were collected after adding trypsin /versin (2-3 ml) not more than 10 min. ,then concentrated into known volume with sfm. afterwards, 0.2 µ ml of cells in growth medium were added to each well of sterile 96well micro titration plate. the plate was sealed with a self adhesive filer, placed on co2 incubator at 37 o c for not more than 24 hrs. (for cell adherence) .after cells attachment, the plate was checked–out for contamination and the media were removed. serial concentrations were added and three replicates were used to each concentration and negative control (cell with sfm only), the exposure time was 72hrs. after the exposure time was finished , the mixtures of analogues and media were removed and a fresh sfm was added to all wells ,and incubated for 24 hrs at 37 o c to give chance if the affected cells and not damaged being repaired by self repairing system. then the media was removed from the plate and washed pbs. a 0.2 ml of mtt working solution dye was added to each well and incubated at 37 o c for 3hrs. at the end of last incubation period the dye was removed from the plate and the well washed with warm pbs twice, then 0.2 ml dmso was added to each well to dissolve the iraqi j pharm sci, vol.22(2) 2013 cytotoxicevaluation of bis-(2mercaptoacetate) gold (iii) chloride 50 mtt–formazan crystals, during that we added 25 µl of glycine buffer to each well containing dmso. finally the plate became readily for reading by elisa reader at 570 nm. statistical analysis experimental data were analyzed using statistical software spss 17.0 for windows. significance between control and samples was determined using students’t-test. a p value ≤ 0.05 was considered statistically significant. the results were expressed as percentage of viability which was calculated as the percentage of the mean of absorbance compound to the control. the ic50 , which is the lowest concentration that kill 50% of cells (21) was calculated according to wilson (22) . results and discussion chemistry the thioglycolate ligands form stable, colored solid and acts as monodentate (o), chloride and bidentate (o/s) with au(iii) ion. the complexe is thermally stable and insoluble in organic solvents. however, fair solubility was attributed in dmf and dmso. the 10 -3 m solution in dmso display molar conductance equal to value 11 ohm -1 cm -2 mol -1 indicating non electrolytic (neutral) nature of thecomplexe (23) .this is consistent with stoichiometry for the complexes on the basis of analytical data. table( 1) : physical properties of the complex complex colour m.p ( o c) yield % elemental analysis% found/(calc.) λoh m -1 µeff (b.m) c h s au (au(hsch2coo)2cl) brown 360 d 75 11.51 (11.58) 1.42 (1.45) 15.41 (15.44) 47.50 (47.53) 11 dia table( 2 ): electronic and infrared specification of the complex uv. vis λmax (cm -1 ) ir bands (cm -1 ) ν assy (coo) v sym (coo) δv (vas-vsym) v (c-s) v (au-o) v (au-s) v (au-cl) 30000, 27248, 44050 1580s 1415s 165 845s 486m 340m 290m the most important diagnostic feature of ir spectra of the complex was listed in table 2. the most significant information on the geometry of this complex came from the analysis of caboxylate and thioether absorption region. stretching frequencies of these functional groups are closely related to the way in which they are coordinated to the metal ion (24) .the ir spectra of the complex showed broad and intense bands ranging between 1580 and 1415 cm -1 assigned for asym ν(coo -1 ) and for sym. ν (coo -1 ) respectively.(table 2). the magnitude of ∆ ν (∆ν = ν asym cooν symcoo ) were in the range 150-180 cm -1 suggested monodentate bonding of carboxylic group to metal ion (25) . further support for this argument came from the ir of the complex which showed a new band at 486 cm -1 attributable ν (au-o). the (c-s) band of the ligand was observed at 845 cm -1 , upon coordination with metal ions in complexes it was shifted to lower frequency values (table 2).they also showed a band in the region 290 cm-1 , which may due to ν (au-cl) vibration frequency (26) . the (c-s) band of the ligand was observed at 860 cm-1, upon coordination with metal ion in complex, it was shifted to lower frequency value (table 2) . further support for this coordination has provided from the appearance of new band in the 340cm -1 ranges which are tentatively attributed to ν(au-s) (27) . the 1 hnmr spectra of the complex were recorded in dmso d6 and show the signal of the coordinated ligand. for the tested complex, it shows a band at 3.01-3.89 ppm which can be attributed to sch2co group of each ligand and sh proton of 1.67 and 1.71 ppm. also two bands can be assigned to each oh2 in the sch2co at 2-258 ppm and the thiol protons of 1.07 and 1.21 ppm,. the diamagnetic nature of the au(iii) complex is consistent with normal squareplanar geometry around au(iii) ion (28) . electronic absorption spectra of the complexes in dmso are listed in table2. in the spectrum of the ligand the ππ* transition were observed at 36232 and 33200 cm -1. the spectrum of the complex shows new bands at 2500-26666 and 27248-32845 cm -1 assigned to iraqi j pharm sci, vol.22(2) 2013 cytotoxicevaluation of bis-(2mercaptoacetate) gold (iii) chloride 51 1 a1g 1 a2g and 1 a1g 1 eg transition respectively (29) , these bands correspond fairly well to a square planar geometry around the au(iii) ion. also the band at 40050 cm -1 is tentatively assigned as ligand charge transfer transition. similar results were found in pt (ii) and au(iii) complexes of the (m(diimine) (dithiolate)) type (30-34) . cytotoxic study methylthiazoletetrazolium (mtt) assay was employed to assess cell viability. mtt assay was based on the ability of the viable cells to reduce soluble yellow mtt to blue formazan crystals .in this assay, optical density (od) values represented the absorption of formazan dissolved by dmso at 570 nm (35) . the comparative cytotoxic properties of synthesized au(iii) complex (au(l')cl) and previously synthesized (c1-c4) were tested against hep-2 cell line ; well-known resisted to cisplatin (36) . in most cases the investigated compounds showed relevant in vitro anticancer properties with ic50 values generally falling in the low µm concentration, additionally, these compounds turned out to overcome largely resistance of hep-2 cell line to cisplatin ( figure 2 and figure 3). the application of t-test, show that the potency parameter (ic50) of the tested complexes (c1-c4) , although not significant , it represent high potency compared to the reference compound (table 3), the lack of cross-resistance suggests that gold (iii) induce cytotoxicity through different mechanism. so, s-ligands (l) & (l') are crucial both in stabilizing the au (iii) center and in carrying the metal to its cellular targets (37) .i.e. to pharmacokinetic. table( 3 ): ic50 of c1 – c4 compared to cisplatin ( au(l')2cl) d (cisplatin) c4 c3 c2 c1 0.56 0.325 0.125 0. 45 0.45 0.39 figure 2: % viability of the[ au(l')2cl] iraqi j pharm sci, vol.22(2) 2013 cytotoxicevaluation of bis-(2mercaptoacetate) gold (iii) chloride 52 figure (3) : comparative cytotoxicities (% viability)of the synthesized complexes (( au(l')2cl)).(c1-c4) with cisplatin (positive control) it may be ruled out that there is no correlation between different arrangement of ligands (l) and (l') whether monodentate or bidentate, planar or pentacyclic , and neutral or charged around central metal,. such a finding allows us to state that the presence of h in sulfur-donor ligand is not an essential requirement for cytotoxicity in gold (iii) complexes, i.e. to pharmacodynemic (16) . since, the cytotoxic profile of synthesized complexes is largely differ from cisplatin , it may come in agreement with many literatures stated that au (iii) were found to perturbs greatly the mitochondrial function (38-39) . the overall of this study pointed out that the cytotoxicity profile of the 2-mercaptoacetate (l), mercaptoglycolate (l') and chloride derived complexes was related to the presence of the gold (iii) central atom , the activity was not related to good leaving group , no direct correlation of the antiproliferative effects and the strong stabilization of the gold (iii) center (c1 and c2) . cross anticancer activity against the cisplatin resistant cell line was found for one of the complexes c3. the effect of the targeted complex may related to one of the following mechanisms: (i) formation of monofunctional or bifunctional coordination bonds at the two (liable) cis positions of the gold(iii) center ; (ii) intercalation of the ligand moiety. in some cases these compounds exhibit a cytotoxic activity that counteract the hep-2 cell resistance to cisplatin & being more sensitive . it may be ruled out that there is a direct correlation between the type of the ligand (halide)& the function of the complex , this finding disagree with the literature (40) . this may come agree that the synthesized complex with mono halide ligand and superior cytotoxic effect compared to the others au (iii) ( c1-c4) detection of in vitro cytotoxicity has represented, for these metal – based agents, the primary screening criterion in order to assess their potential anticancer properties. ic50 values of 10 -5 m or lower were used to indicate a promising, or at least acceptable, antitumor efficacy (41) . analysis of the cytotoxicity data (% viability) permits formulation of some preliminary structure/function relationships that are summarized below: a. the cytotoxicity of these gold (iii) complexes is strictly related to the presence of the gold(iii) centre , c1-c4 and (au (l')2cl) are significantly more cytotoxic than the corresponding platinum compound. b. the presence of hydrolysable chloride atoms in the gold(iii) centre or, in general, of good leaving groups, does not represent an essential requirement for cytotoxicity compared to c1c4 but showed very pleased unexpected effect compared to cisplatin. c. attachement of chloride as a ligand may lead to an improvement in their biological activity as a consequence of the hydrophilic nature of chloride that may increase their transportation into biological system. d. the amount of gold (iii) that enters in the cells is roughly proportional to the exposure time, at least during the first hours and increased with increase the concentration except for c3. conclusion one of our main duty as medicinal chemists, to provide a complete chemical study of each system that is designed and developed, with the aim to accurately predict the reactivity of these compounds under physiological conditions and more over to predict the reactivity in the biological systems. unfortunately this is not always possible, because most of the studies are life-time experimental projects. tissue targeting is a highly desirable goal for metal-based therapeutics or diagnostics, but it is not always feasible and more specific targeting ligands must be found. not only the right ligand, but also the right metal-ligand combination, is important. when studying the biological properties of the coordination compounds, it should be stressed the need of performing biological tests even for the free ligands. this protocol has been omitted often in the biological studies iraqi j pharm sci, vol.22(2) 2013 cytotoxicevaluation of bis-(2mercaptoacetate) gold (iii) chloride 53 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(camb) , 2003;21: 1718. 38. m. coronello ,e. mini, b. caciagli ,m.a. cinellu, a. bindoli , c. gabbiani , l. messori, j.med. chem. 2005;48:6761. 39. y. wang q. y. he, c.m. che , j.f. chiu , proteomics , 2006 ;6:131-42. 40. b. bruni, a. guerri, g. marcon, l. messori and p. orioli, structure and cytotoxic properties of some selected gold(iii) complexes , croatica chemical acta , 1999; 72 (2-3) :221-229. 41. casini, ch. hartinger , ch gabbiani, e. mini, p j. dyson, b k keppler, l messori, gold (iii) compounds as anticancer agents : relevance of goldprotein interactions for their mechanism of action . j. of inorg. biochemistry. 2008; 102 :564-575. the relationship between uric acid concentration and some of plasma lipids in patients with c iraqi j pharm sci, vol.18(2) 2009 uric acid and some of plasma lipids 68 the relationship between uric acid concentration and some of plasma lipids in patients with c. v. disease in general hospital of al-nasseriya hiba a. al-hussein hassan *,1 * medical laboratory science technology. college of health and medical technology ,baghdad , iraq abstract cardiovascular disorders are refer to the class of diseases that involve the heart or blood vessels (arteries and veins). while the term technically refers to any disease that affects the cardiovascular system. cholesterol is classified as a sterol (a contraction of steroid and alcohol).although cholesterol is essential for life; high levels in circulation are associated with atherosclerosis. triglyceride (more properly known as triacylglycerol , tag or triacylglyceride) is a glyceride in which the glycerol is esterified with three fatty acids. it is the main constituent of vegetable oil and animal fats.high-density lipoproteins (hdl) is one of the 5 major groups of lipoproteins (chylomicrons, vldl, idl, ldl, hdl) which enable lipids like cholesterol and triglycerides to be transported in the blood stream. in healthy individuals, about thirty percent of blood cholesterol is carried by hdl. uric acid (or urate) is non-protein nitrogen compound with the formula c5h4n4o3. this study aimed to determine the relation between uric acid, cholesterol, triglyceride and hdl-cholesterol concentration in cardiovascular disorders patients. serum sample were collected from 84 cardiovascular disorders patients attended to the general hospital of alnasseriya. all samples were analyzed for uric acid , cholesterol, triglyceride and hdl-cholesterol concentration by enzymatic-colorimetric method kits. the data showed a highly significant increased in the serum concentration of uric acid , cholesterol and triglyceride in cardiovascular disorders patients compared with healthy control (p<0.01) .in addition , there was no significant increased in the serum concentration of hdl-cholesterol in cardiovascular disorders patients compared with healthy control (p>0.05) .the data of this study strengthen the possibility that high concentration of uric acid may act as a risk factor in cardiovascular disease. key words: high density lipoprotein (hdl-cholesterol),very low density lipoprotein(vldl), low density lipoprotein(ldl),intermittent lipoprotein(idl), hyperuricemia. الخالصة انً صُف ايشاض انقهب او االوعُت انذيىَت )انششاٍَُ واالوسدة(بًُُا حقُُا حشُش انً أي ااِلظطشاباث انقهبُت انىعائُت حشُش َصُف انكىنىسخُشول كأسخُشول)إَذياج االسخُشوَذ وانكحىل(. بانشغى يٍ أٌ انكىنُسخُشول يشض َؤثش عهً اندهاص انقهبٍ انىعائٍ . ٌُ يذي انحُاة، حشحبط يسخىَاحت انعانُت فٍ انذوسة انذيىَت بًشض حصهب انششاٍَُ.حشاَكهسشاَذ)غانبا انًعشوف بخشاٌ أسم ظشوس ٍُ نهضَج tag كهُسشول, أو حشاٌ أسم كهُسشاَذ(هى كهُسشاَذ انزٌ فُت انكهُسشول يؤسخش بثالثت احًاض دهُُت. وهى انًكىٌ انشئُس ٌِ انحُىاَُِت. (وانخٍ هٍ واحذة يٍ خًست يدايُع يٍ hdlفٍ االفشاد االصحاء انبشوحُُاث انذهُُت راث انكثافت انعانُت )انُباحٍ وانذهى ٍ حًكٍ انذهىٌ يثم انكىنُسخُشول و انخشاَكهُسشاَذ يٍ ( انخchylomicrons, vldl, idl, ldl, hdlانبشوحُُاث انذهُُت) ًُىُل ِيٍ قِبم ٍْ كىنُسخُشوِل انذوِّ َيْح .حايط انُىسَك )أو انُىساث( هى ُيشّكُب hdlاالَخقال فٍ يدشي انذو، حىانٍ ثالثىٌ بانًائت ِي ٍْ انًشكباث انُخشوخُُُت غُش انبشوحُُُت راث انصُغت ٌُ ِي انذساست نهخحشٌ عٍ انعالقت بٍُ حشكُض اخشَج هزة c5h4n4o3 .ععى كىنُسخُشول نذي يشظً ااِلظطشاباث انقهبُت انىعائُت حُث خًعج hdlحايَط انُىسَك وحشكُض انكىنُسخُشول , حشاَكهسشاَذ و نعُُاث فحصج اسبع وثًاَىٌ عُُت يصم يٍ يشظً ااِلظطشاباث انقهبُت انىعائُت انزٍَ ادخهىا انً يسخشفً انُاصشَت انعاو . خًُع ا اإلَضًَُت, أظهشث انُخائح فشوق يعُىَت عانُت فٍ حشاكُض حايط انُىسَك, انكىنُسخُشول و انخشاَكهُسشاَذ فٍ –بطشَقت انخحانُم انهىَُت hdlيشظً ااِلظطشاباث انقهبُت انىعائُت يقاسَت يع يدًىعت انسُطشة. باالظافت انً رنك نى حظهش انُخائح فشوق يعُىَت فٍ حشكُض كىنُسخُشول فٍ يشظً ااِلظطشاباث انقهبُت انىعائُت يقاسَت يع يدًىعت انسُطشة. َخائح هزة انذساست دعًج احخًانُت انخشكُض انعانٍ نحايِط انُىسَك قَْذ َكىٌ انعايم انخطِش فٍ ايشاض األوعُت انقهبُِت. 1 corresponding author e – mail : hebaabdul@yahoo.com received : 17 / 5 / 2009 accepted : 27 / 10 /2009 http://en.wikipedia.org/wiki/heart http://en.wikipedia.org/wiki/blood_vessel http://en.wikipedia.org/wiki/artery http://en.wikipedia.org/wiki/vein http://en.wikipedia.org/wiki/circulatory_system http://en.wikipedia.org/wiki/circulatory_system http://en.wikipedia.org/wiki/sterol http://en.wikipedia.org/wiki/steroid http://en.wikipedia.org/wiki/alcohol http://en.wikipedia.org/wiki/atherosclerosis http://upload.wikimedia.org/wikipedia/en/5/5d/triglyceride.ogg http://upload.wikimedia.org/wikipedia/en/9/97/triacylglycerol.ogg http://en.wikipedia.org/wiki/glyceride http://en.wikipedia.org/wiki/glycerol http://en.wikipedia.org/wiki/ester http://en.wikipedia.org/wiki/fatty_acid http://en.wikipedia.org/wiki/vegetable_oil http://en.wikipedia.org/wiki/animal_fat http://en.wikipedia.org/wiki/lipoprotein http://en.wikipedia.org/wiki/chylomicrons http://en.wikipedia.org/wiki/vldl http://en.wikipedia.org/wiki/intermediate_density_lipoprotein http://en.wikipedia.org/wiki/ldl http://en.wikipedia.org/wiki/hdl http://en.wikipedia.org/wiki/lipids http://en.wikipedia.org/wiki/cholesterol http://en.wikipedia.org/wiki/triglycerides http://en.wikipedia.org/wiki/blood http://en.wikipedia.org/wiki/chylomicrons http://en.wikipedia.org/wiki/vldl http://en.wikipedia.org/wiki/intermediate_density_lipoprotein http://en.wikipedia.org/wiki/ldl http://en.wikipedia.org/wiki/hdl iraqi j pharm sci, vol.18(2) 2009 uric acid and some of plasma lipids 69 introduction cardiovascular disorders is any disorder that affects the heart's ability to function normally (1) . cholesterol is a waxy material that is found in many food items including milk, cheese ,eggs, butter ,fish, beet, pork, chicken, goat meat, it is naturally occurring, fat-like substance with a complex chemical formula, and is used to build cells and make hormones.itis present in plasma mainly esterified with fatty acids. the body can not break down the sterol nucleus, so cholesterol is either excreted unchanged in bile or converted to bile acids and then excreted. the effects of cholesterol on the heart may involve more than just one the arteries. there are some evidences unhealthy levels may affect the heart muscles and increase the risk for heart failure (2,3) . triglycerides are composed of fatty acid molecules and are the basic chemicals contained in fats in both animals and plants (4) . high-density lipoproteins (hdl), the smallest and most dense. (referred to as the "good" cholesterol) it is formed in the liver and walls of the small intestine, while maturing in blood stream, it obtains cholesterol from the surrounding tissues.the blood circulation then transports the hdl back to the liver , from where the cholesterol is excreted in the bile (5) . uric acid is the major product of purine metabolism and is formed from xanthine by the action of xanthine oxidase. the normal limits of serum uric acid are between 387 and 416 µmol/l in men and lower than 327 µmol/l in women (6) .hyperuricemia is usually defined as a serum uric acid level of 416 µmol/l or higher in men, and 357 µmol/l or higher in women (7,8) . methods studied group this study included (84) patients from general hospital of al-nasseriya. patients’ ages ranged between (30-70) years . sample collection from each patients included in this study blood samples were collected to obtain the serum during the period between may/2008 until november/2008. all cardiovascular disorders patients were diagnosed by consultants of cardiologist in hospital of alnasseriya. procedure enzymatic -colorimetric method for detection of uric acid , cholesterol, triglyceride and hdl-cholesterol concentration in serum : uric acid , cholesterol, triglyceride and hdlcholesterol: enzymatic –colorimetric method kits provided by (bio lab). estimation of uric acid , cholesterol, triglyceride and hdlcholesterol concentration in serum by enzymatic –colorimetric method .this method depend on that the chromogenic enzyme is binding with substrate. after incubation for 5min. at 37°c . the intensity of color developed is proportional to the concentration of the sample (9) . statistical analysis the suitable statistical methods were used in order to analyze and assess the results, includes the following (10) : 1descriptive statistics a) statistical tables including observed frequencies with their percentages. b) summary statistic of the readings distribution(mean,minimum&maximum). 2 – inferential statistics these were used to accept or reject the statistical hypotheses, they include the followings: a) chi-square (χ 2 ), b) repeated student test (t-test). note: the comparison of significant (pvalue) in any test were: s= significant difference (p<0.05). hs= highly significant difference (p<0.01). ns= non significant difference (p>0.05). 3 computer & programs all the statistical analysis was done by using pentium-4 computer through the spss program (version-10) and excel application. results the distribution of patients according to age groups is listed in table (1) shows that the age group between 51-60 years and 30-40 years is more percentage of cardiovascular disorders patients when compared with the healthy control give a non significant differences(p>0.05 ). table (2) shows a non significant differences (p> 0.05 ) in the distribution of studied groups according to gender with predominance of the percentage of male patients than female patients . data illustrated by table (3) clearly shows a highly significant increased (p<0.01) between the mean concentration of cholesterol and triglyceride in patients when compared with the mean concentration of cholesterol and triglyceride in healthy control, while no significant difference noticed (p>0.05) in between the mean concentration of hdlcholesterol in patients and healthy control .in addition , the study shows a highly significant increased (p<0.01) in the mean of uric acid in cardiovascular disorders patients when the results compared with healthy control , as shown in table 4 iraqi j pharm sci, vol.18(2) 2009 uric acid and some of plasma lipids 70 table 1: distribution of studied groups according to age / year . studied groups total healthy control patients a g e g r o u p s (y e a r ) 30-40 n % 19 38.0% 22 26.2% 41 30.6% 41-50 n % 17 34.0% 20 23.8% 37 27.6% 51-60 n % 10 20.0% 24 28.6% 34 25.4% 61-70 n % 4 8.0% 18 21.4% 22 16.4% total n % 50 100.0% 84 100.0% 134 100.0% value df p-value chi_square 6.958 3 0.073 ns table 2: distribution of studied groups according to gender. studied groups total healthy control patients g e n d e r male n % 26 52.0% 52 61.9% 78 58.2% female n % 24 48.0% 32 38.1% 56 41.8% total n % 50 100.0% 84 100.0% 134 100.0% value df p-value chi_square 1.246 1 0.261 ns table 3 : mean distribution of (cholesterol, triglyceride & hdlcholesterol) concentration in the studied groups . n mean std. deviation t-test (p-value) sig. minimum maximum serum cholesterol (mmol/l) healthy control patient total 50 84 134 3.964 5.383 .824 1.185 2.5 1.3 5.1 7.0 0.00 hs serum triglycerides (mmol/l) healthy control patient total 50 84 134 1.318 2.252 .349 .948 .8 .9 2.0 5.5 0.00 hs s.hdl cholesterol (mmol/l) healthy control patient total 50 84 134 1.273 1.200 .391 .433 .9 .8 2.0 2.2 .512 ns table 4: mean distribution of uric acid concentration of studied groups. serum uric acid(µmol/l) n mean std. deviation range t-test (p-value) sig. minimum maximum healthy control patient total 5 8 13 291.33 375.14 108.17 88.59 137 120 416 520 .000 hs iraqi j pharm sci, vol.18(2) 2009 uric acid and some of plasma lipids 71 discussion generally , this study showed the concentration of cholesterol and triglyceride was highly elevated in cardiovascular disorders patients compared with the healthy control groups. these findings are in a good agreement with other studies (11-15) who found that effects of increased cholesterol deposition in the arterial wall, enhanced foam-cell formation , generation of oxygen free radicals in monocytes, promotion of smooth-muscle–cell proliferation, and induction of monocyte chemotactic activity in endothelial cells .while hypertriglyceridemia may represent a procoagulant state mediated by increased levels of factors i,vii, viii, and x and plasminogen activator inhibitor-1, as well as by reduced tissue plasminogen activator activity (16) .triglyceride-rich lipoproteins may also be directly atherogenic (17) . researchers have suggested that the higher uric acid levels in subjects with cardiovascular disorders might be a compensatory response designed to counteract excessive oxidative stress (18,19) . this theory has a strong rationale in the biochemical characteristics of uric acid as anti-oxidant and is supported by pre-clinical studies performed in vitro and in experimental animals (20) . however, the role of uric acid in humans is still uncertain. the above results agreed with the results obtained by skinner 1998 who observed that serum uric acid has antioxidant properties and contributes to free radical scavenging activity in human serum. when uric acid interacts with peroxynitrite to form a stable nitric oxide donor, vasodilatation increases and the potential for peroxynitriteinduced oxidative damage decreases (21) . thus, uric acid can be protective against oxidative stresses, but it can also lead directly or indirectly to vascular injury (22,23) .others has been reported that uric acid promotes vascular smooth muscle proliferation and upregulates the expression of platelet-derived growth factor and monocyte chemoattractant protein-1 (24,25) .hypoxanthine is converted to uric acid via xanthine. this reaction can be catalyzed by xanthine hydrogenase and xanthine oxidase, the latter of which produces uric acid and superoxide. thus, it is possible that, in certain diseased conditions, hyperuricemia is accompanied by the increased production of reactive oxygen species, which may result in the modulation of vascular contractility (26,27) . another possible explanation is that hyperuricemia may induce endothelial dysfunction by decreasing the production of nitric oxide in the vascular endothelial cells (28) . adenosine synthesized locally by vascular smooth muscle in cardiac tissue is rapidly degraded by the endothelium to uric acid , which undergoes rapid efflux to the vascular lumen due to low intracellular ph and negative membrane potential (29) . uric acid synthesis is increased in vivo under ischemic conditions, and therefore elevated serum uric acid may act as a marker of underlying tissue ischemia. in human coronary circulation, hypoxia, caused by transient coronary artery occlusion, leads to an increase in the local circulating concentration of uric acid (30) . in conclusion, raised serum uric acid concentrations are a powerful predictor of cardiovascular risk and poor outcome, although the underlying mechanisms remain unclear. several potential explanations have been put forward to explain the apparent association between hyperuricaemia and cardiovascular risk. studies have demonstrated mechanisms by which uric acid could be directly injurious to the endothelium and to cardiovascular function. paradoxically, uric acid elevation could be expected to confer protective antioxidant effects in the cardiovascular system, but these potential benefits may be obscured by detrimental effects elsewhere. the effects of raising or lowering serum uric acid on endothelial function, autonomic 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"uric acid and oxidative stress" curr pharm des , 2005,11: 4145–4151. 21. skinner ka, white cr, patel r et al. “nitrosation of uric acid by peroxynitrite. formation of a vasoactivenitric oxide donor” j biol chem, 1998,273:2491– 2447. 22. reyes a j “significance of uric acid for the heart and vessels”eur .heart j.,august 1, 2006 , 27(15): 1886 1886. 23. ishizaka n, ishizaka y, toda ei et al. “higher serum uric acid is associated with increased arterial stiffnessin japanese individuals” atherosclerosis, 2007, 192(1):131-7. 24. kanellis j, watanabe s, li jh et al. “uric acid stimulatesmonocyte chemoattractant protein-1 productionin vascular smooth muscle cells via mitogen-activatedprotein kinase and cyclooxygenase-2” hypertension, 2003,41: 1287–1293. 25. ruggiero c, cherubini a, ble a, bos a j.g., et al. “uric acid and inflammatory markers” european heart journal 2006 ,27(10):1174-1181. 26. white cr, brock ta, chang ly et al. “ superoxideand peroxynitrite in atherosclerosis” proc natl acadsci usa, 1994,91: 1044–1048. 27. berry ce, hare jm. “xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications” j physiol 2004; 16: 589–606. 28. khosla um, zharikov s, finch jl et al. “hyperuricemiainduces endothelial dysfunction” kidney int, 2005,67: 1739– 1742. 29. kroll k, bukowski tr, schwartz lm, knoepfler d,bassingthwaighte jb. “capillary endothelial transportof uric acid in guinea pig heart” am j physiol, 1992, 262 (2 part 2): h420–h431. 30. de scheerder ik, van de kraay am, lamers jm et al.“myocardial malondialdehyde and uric acid releaseafter short-lasting coronary occlusions during coronaryangioplasty: potential mechanisms for free radicalgeneration” am j cardiol, 1991,68: 392–395. iraqi j pharm sci, vol.32( 1 ) 2023 university students and life style medications doi: https://doi.org/10.31351/vol32iss1pp185-193 185 awareness and frequency of using lifestyle medications among university students in kurdistan region-iraq ,*othman kawa zhinya ,** saeed hama ghafour ahmed ,1,* marouf hassan bushra ***ahmed jaza mohammed roya and *gharib m othman sava ,*karim arkan sima iraq sulaimani ,sulaimani of university pharmacy, of college ,toxicology and pharmacology of department * iraq sulaimani ,sulaimani of university pharmacy, of college , pharmacy clinical of department ** iraq , sulaimani, sulaimani of university ,medicine of college *** abstract life style medications (lsms) are used for the improvement of lifestyle of an individual. these drugs are being taken to modify a non-medical or non-health-related purpose. this study aimed to investigate the extent of using lsms among university students, reasons for using them, and identify the types, adverse effects to provide helpful information for justification and prevention of this phenomenon. a descriptive observational cross-sectional study was conducted. a questionnaire was designed to target undergraduate medical and pharmacy students at three universities of sulaimani (uos), hawler medical university (hmu), and university of duhok (uod) in sulaimani, hawler, and duhok cities-kurdistan region-iraq respectively. student knowledge, awareness of the use of lsms, the motivations for using these medications were assessed by addressing these aspects in different sections of the questionnaires. the number of respondents was 209 from which, the number of students who were using lsms was 149 (71.3%). the source of information on lsms among those who had aware of using lsms was advertisement 25(12%), family 28(13.4%), friends 51(24.4%), medical needs 51(24.4%), internet 115(55%) and the pharmacies 4(1.9%). the most frequent agent that has been used by the highest number of the students was caffeine 71(47.7%), followed by dietary supplement 63(42.3%) then cosmetics 48(32.2%). 135 (64.6%) students did not agree on the prevention of the use of lsms, while the rest 74 (35.4%) encouraged the prevention of lsms intake by providing many strategies to prevent this phenomenon. in conclusion, prevalence of using lsms among university students is high and tendency for medicalization of healthy individuals in the aim of better academic performance and improve quality of life is increasing. the prevention of lsms intake by providing many strategies to prevent this phenomenon was raised by 74 (35.4%) of the participants. key words: life style medications, lsms, motives, prevention strategies, university students العراق-الكردستان االقليم في الجامعات الطلبة بين الحياة النمط األدوية تناول و تكرارالوعي ،*غريب محمد عثمانه ساڤ ،*كريم اركان سيما عثمان، كاوة ژينيا ،**سعيد حمة غفور احمد ، 1* معروف حسن بشرى *** احمد جزا محمد رۆيا العراق ، سليمانيةال السليمانية، جامعة الصيدلة، كلية والسموم، االدوية فرع* العراق ، سليمانيةال السليمانية، جامعة الصيدلة، كلية السريرية، الصيدلة عفر ** العراق ، سليمانيةال السليمانية، جامعة الطب، كلية *** الخالصة بحالة متعلق وغير طبي غير غرض لتحسين األدوية هذه تناول يتم الفرد. حياة نمط لتعزيز تستخدم التي األدوية من الحياة نمط ادوية تعتبر أنواعها على والتعرف استخدامها ودوافع الجامعات طلبة بين الحياة نمط األدوية استخدام مدى معرفة هو الدراسة هذه إجراء من الهدف إن المرضية. الجامعات. طلبة بين تناولها من والوقاية الضوابط لوضع قيمة بمعلومات الجامعات تزويد أجل من الموضوع هذا على المفيدة البيانات لجمع السلبية وتأثيراتها في والصيدلة الطب الكليات وطالبات طالب منها الطبية المجموعة طلبة استهدف الذي استبيان تصميم خالل من مستعرضة مقطعية وصفية دراسة أجريت لألدوية الطالب معرفة مدى تقييم تم التوالي. على دهوك و أربيل السليمانية، التالية، المحافظات في دهوك جامعة و هوليرالطبية جامعة و السليمانية جامعة يستخدمون كانوا الذين الطالب عدد طالبا. 209 لالستبيان استجاب االستبيان. في المختلفة األقسام في الموجودة األسئلة خالل من االدوية هذه الستخدام ووعيهم ينالذ أولئك بين االدوية لهذه المعلومات مصدر بان تبين االطالق. على االدوية هذه يستخدموا لم ٪(28.7) 60 بينما ٪(71.3) 149 هو الحياة نمط أدوية ٪( 24.4) 51 الطبية األحتياجات من ، ٪(24.4) 51 األصدقاء ، ٪(13.4) 28 األسرة ٪(،12) 25 اإلعالنات خالل هومن االدوية لهذه علم لديهم كانوا 63 الغذائي المكمل يليه ، ٪(47.7) 71 الكافيين هو الطالب قبل من استخدامه شيوعا األكثر المادة ٪(.1.9) 4 والصيدليات ، ٪( 55) 115 االنترنيت من ، 74 الباقون أيد بينما الحياة نمط أدوية استخدام منع على المشاركين طالب من ٪(64.6) 135 يتفق لم ٪(.32.2) 48 التجميل المستحضرات ثم ٪(42.3) نمط أدوية استخدام نسبة بان الدراسة هذه من نستنتج . الظاهرة هذه لمنع االستراتيجيات من عديد وضع خالل من االدوية هذه تناول منع على ٪(35.4) االزدياد. في الحياة نوعية وتحسين األكاديمي أدائهم تحسين لغرض االصحاء األشخاص قبل من االدوية تناول الى والميل جدا عالية الجامعات طلبة بين الحياة . الظاهرة هذه لمنع ٪( 35.4) 74 المشاركين من نسبة قبل من االستراتيجيات من عديد االقتراح تم لقد الجامعات. طلبة الوقاية، طرق االستخدام، دوافع الحياة، نمط أدوية المفتاحية: الكلمات 1corresponding author e-mail: bushra.marouf@univsul.edu.iq received: 18/ 3/2022 accepted: 31/ 5/2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp185-193 iraqi j pharm sci, vol.32( 1 ) 2023 university students and life style medications 186 introduction the term lifestyle medication (lsm) has multifaceted definition and various descriptions are frequently used interchangeably for this term as there is incomplete consensus on its definition, types, indications and its access. (1,2) however, general consensus has agreed on the medications that are intended for improvement of lifestyle of an individual. these drugs are being taken to modify a non-medical or non-health-related purposes. it can be used to alter the appearance as well as the physical and mental capabilities of the individual such as improving academic performances, changing and/or improving physical appearance. these drugs as named as non-essential drugs that used to manage non-serious medical issue which is non-life threatening and non-painful status such as indicated for baldness, erectile dysfunction or impotence, postpone menstruation, muscle building, physical, mood alteration, acne, and wrinkle. (1,3) there are many medications and pharmaceutical products that are considered as lsms, these include drugs that are utilized for hair loss prevention such as: minoxidil, finasteride or hair tonics; orlistat for losing weight; bupropion for smoking cessation, sildenafil for erectile dysfunction, onabotulinumtoxin a for wrinkle and aging marks; melatonin as a sleep aid; cyproheptadine as appetite enhancer and dietary supplements that may improve physical appearance. (4) these medications are taken in an attempt to improve personal life quality rather than to manage a medically identifiable and well-defined disease (5) today, the use of lifestyle medications is increasing due to easy accessibility and persuasive advertisement of pharmaceutical products such as cosmetics, dietary supplements, and weight-loss products which increases the concerns of misuse of these medications. (6) additionally, disease mongering which is defined as maximizing minor illness into severe medical issue, treating mid illness as serious one, all these have increased the medicalization of these conditions. (7) on the other hand, university student’s living environment, socio-economic factors, modernized life and peer pressures are considered as the factors that make students to be at high-risk for using lsms. (8) the issue of lsms usage have been investigated in many studies and there have been some reports about the prevalence of lsms usage among students and healthy people. (4,9) rahman et al, 2010 stated that lifestyle has itself become an object of medical attention and the impact of these medications on the society need to be more elaborated (1). moreover, the majority of the students who were lsms users confessed that most of lsms were unnecessarily used. (4) unfortunately, these lsms are not without adverse effects thus the development of preventive strategy and educational awareness for reducing the use of lsms requires information on the most commonly used medications among students. furthermore, factors for using these drugs and prevalence of the usage of these medications in the university environment should be addressed. (9) therefore, the aims of this study were to investigate the extent of using lifestyle medications among university students, motives for using them, and identify the types, adverse effects and other measurable outcomes to provide helpful information for justification and prevention of this phenomenon. methods study design and development of the questionnaire this was an anonymous descriptive crosssectional on-line survey where a multiple-choice with single word answer questionnaire was designed to target undergraduate medical and pharmacy students at the university of sulaimani, hawler medical university, and university of duhok in sulaimani, hawler, and duhok cities-kurdistan region-iraq respectively from june 2020 to january 2021. a self-administered questionnaire was used in this study, which was designed after extensive literature review. (4,10) the questionnaire was divided into four sections and it was utilized for collection of sociodemographic characteristics, knowledge on lsms, the most commonly used lsms and preventive measure for lsms practice. a pilot study was conducted among ten medical and pharmacy students to assess the understandability, reliability, and clarity of the questionnaire. validation of the questionnaire was performed by face validity based on an expert panel from pharmacists and biostatisticians. the purpose of the research has been clarified for the students and the researchers assured them of their information confidentiality. the students also have been informed on how to fill this questionnaire. demographic information included age, gender, university and colleges and the current academic year of the student. the questionnaire was sent out via email to the students at the targeted colleges through the college representative member. student knowledge, awareness of the use of lsms, motives for using these medications were assessed by addressing these issues in different sections of the questionnaires. objectives of the study was explicitly described at the beginning of the questionnaire form to each participant. iraqi j pharm sci, vol.32( 1 ) 2023 university students and life style medications 187 inclusion and exclusion criteria the sampling scheme included undergraduate university students of both gender, age of 18 and above years, residing at the university campuses in the three cities; sulaimani, hawler, and duhok cities-kurdistan region-iraq. the questionnaire has sent to the levels from 2nd year to the last year of the academic study. the survey included students who were using prescription and nonprescription medications without a medical diagnosis. postgraduate students and students who were identified as users of these medications to treat serious and chronic diseases have been excluded. ethical consideration the project of the study was registered and approved by the ethics and research registration committee of the college of pharmacy–university of sulaimani with registration number (ph-17-20 in 15.11.2020). consenting students was also assured for confidentiality of the volunteered information; thus, the study designed as anonymous on-line survey without name and personal details of the students. statistical analysis analysis and graph of the data were performed in graphpad prism version 9.3.1. descriptive statistics; frequency, percentage, confidence intervals, minimum and maximum range, graphs, were conducted for data presentation. results basic characteristics of the participants the recommended sample size for the current study was calculated based on a formula that was described in a previous research with modification. (11) the number of responses was 215 which is 66.5% of the recommended sample size, six responses have been excluded as they were postgraduate students and were using prescribed medications. the included students from the three universities were 209. basic characteristics of the participants is illustrated in table 1. half of the responses 104(49.8%) were obtained from university of sulaimani while 79(37.8%) were from university of duhok and 26 (12.4%) students were from hawler medical university. the majority of the respondents were female (70.3%). most of the respondents were from third and fourth grade (56%, 66%) respectively. nearly three quarters of the students 152 (72.7%) were with the age range between (18-21), 50(24%) students were with (2224) and 7(3.3%) were 25 years and above. about two-third of the students 161 (77%) were living inside the cities while the rest 48 (23%) were from rural areas. table 1. demographic characteristics of the participants n=209 frequency % universities of the participants university of sulaimani 104 49.8 hawler medical university 26 12.4 university of duhok 79 37.8 gender female 147 70.3 male 62 29.7 age (year) 18-21 152 72.7 22-24 50 24.0 25 and above 7 3.3 year of the study 2nd 47 22.5 3rd 56 26.8 4th 66 31.6 5th 40 19.1 place of living urban area 161 77.0 rural area 48 23.0 knowledge, source of information and student’s attitude on lsms majority of the students 144(68.9%) were aware of lsms intake, while around one third 65(31.1%) had never heard about lsms. the source of information on lsms among those who had aware of using lsms was advertisement, family, friends, medical needs and rarely from the pharmacies 4(1.9%) while highest number of the students 115(55%) were obtained information on lsms from internet. table 2 shows the number of students who were using lsms. the frequency of using lsms and number of the drugs per students was also calculated as shown in table 2. the majority of the participants 112(53.5%) were using one lsms, and 39(18.6%) were not using any lsms. the current study also elaborated on the purpose and benefit of using lsms among students by providing multiple options in the questionnaire. the perception of the students on the purpose of lsms usage varies and the majority of the students were using lsms as a tonic for improving their health (26.3%) and to alleviate stress (21.5%). furthermore, some of them use lsms as cosmetics (13.4%), to increase immunity (9.6%) , treat minor condition (8.1%), for improvement of physical appearance and as body builder (1.4%)(figure.1). iraqi j pharm sci, vol.32( 1 ) 2023 university students and life style medications 188 table 2. awareness and information on lsms among university students n=209 knowledge on lsms frequency % students have information on lsms 144 68.9 students have never heard about lsms 65 31.1 sources of information on lsms advertisement 25 12.0 family 28 13.4 friends 51 24.4 medical needs 51 24.4 internet 115 55.0 pharmacy 4 1.9 students use lsms number of students using lsms 149 71.3 number of students not using lsms 60 28.7 number of lsms used by the students 0 39 18.6 1 112 53.5 2 42 20.0 3 10 4.8 more than 3 6 2.9 figure 1. purpose and benefit of using lsms. the most frequently used lsms, adverse effects associated with the use of lsms this study has recorded the frequency of the drugs that have been taken by all the students during their lifetime. table 3 has summarized the most frequently used lsms used by the students in the studied colleges. table 3. the most frequently used lsms by the university students life style medications (lsms) number % 95% ci alcohol 5 3.4 (0.46, 6.25) dietary supplement 63 42.3 (34.35, 50.21) antioxidants 23 15.4 (9.63, 21.24) benzodiazepines 7 4.7 (1.3, 8.1) caffeine (caffeinated products) 71 47.7 (39.63, 55.67) beta-blockers for alleviate anxiety and social phobia (propranolol, atenolol) 18 12.1 (6.85, 17.31) bupropion “stop smoking” 3 2.0 (0, 4.27) nicotine replacement therapy 5 3.4 (0.46, 6.25) cosmetics (skin depigmenting agents) 48 32.2 (24.71, 39.72) cyproheptadine (appetite stimulant) 11 7.4 (3.18, 11.58) hair loss prevention (alopecia) agents 25 16.8 (10.78, 22.78) melatonin 20 13.4 (7.95, 18.9) norethisterone or contraceptive pill as a “period delay pill” like for ramadan or any other private occasion 6 4.0 (0.87, 7.18) orlistat “any other weight reduction pills” 15 10.1 (5.24, 14.9) stimulants (amphetamines, modafinil, methylphenidate) 10 6.7 (2.69, 10.73) vitamin c 3 2.0 (0, 4.27) anabolic steroid 3 2.0 (0, 4.27) fluoxetine 1 0.7 (0, 1.98) iraqi j pharm sci, vol.32( 1 ) 2023 university students and life style medications 189 the most frequent agent that has been used by the highest number of the students was caffeine 71(47.7%), followed by dietary supplement 63(42.3%) then cosmetics (skin depigmenting agents) 48(32.2%). only one (0.7%) student was using a prescription drug without medical diagnosis as lsms for alleviating stress. the other common lsms are summarized in table 3. furthermore, the adverse effects associated with lsms intake is summarized in table 4. headache and/or mood changes, dryness of mouth, drowsiness or insomnia as well as nausea and vomiting are the most repetitive adverse effects that have been experienced and recorded by the students. in another part of the study, opinion of the student on the effect of lsms on drug-drug interaction and possibility of side effect potentiation have been recorded as shown in table 5. half of the students 106(50.7%) believed that lsms lead to drug-drug interaction and the possibility of side effects are increased. table 4. the most common adverse effects associated with the use of lsms experienced by the university students adverse effects number % 95%cl constipation and/or stomach upset 15 10.1 (5.24, 14.9 ( diarrhea 24 16.1 )10.2, 22.01 ( dizziness or agitation 20 13.4 )7.95, 18.9 ( drowsiness or insomnia 34 22.8 )16.08, 29.56 ( dryness of mouth 38 25.5 )18.5, 32.5 ( facial hair growth and/or hormonal disturbance 24 16.1 )10.2, 22.01 ( fatigue and/or sedation 18 12.1 )6.85, 17.31 ( headache and/or mood changes 43 28.9 )21.58, 36.13 ( loss of appetite 30 20.1 )13.7, 26.57 ( nausea and vomiting 32 21.5 (14.88, 28.07) steatorrhea 15 10.1 (5.24, 14.9) stinging and/or dermatitis 5 3.4 (0.46, 6.25) weight gain 20 13.4 (7.95, 18.9) table 5. perception of the students for the impact of lsmsdrug interaction and potential side effects type of response number % 95% cl students believed that lsms may lead to drug-drug interaction 84 40.2 (48.41, 64.34) students believed that lsms do not lead to drug-drug interaction 19 9.1 (7.4, 18.11) student believed that lsms definitely lead to drug-drug interaction 106 50.7 (63.87, 78.42) motives or factors enhancing the use of lsms among students the highest response on the motives that are enhancing the use of lsms was associated with the treatment of non-serious minor condition 82 (39.2%) then social media or tv advertisement 49 (23.4%). the other factors that boost the use of these medications have illustrated in figure 2. figure 2. motives enhance the use of lsms among students students perception on the effects of lsms on quality of life table 6 depicts the students’ perception about the effects of using lsms on the individual’s quality of life. the majority of the students 107 (51.2%) were disinterested upon the impact of lsms on quality of life, while 67 (32.1%) and 13(6.2%) agreed and strongly agreed on the intake of lsm respectively, because they believed that lsms can improve the quality of life of the individuals. the rest either strongly disagreed or disagreed on the use of lsms for the purpose of quality of life improvement. iraqi j pharm sci, vol.32( 1 ) 2023 university students and life style medications 190 table 6. perception of students toward using lsms to improve quality of life type of response number % 95% ci agree 67 32.1 (36.98, 52.95) disagree 14 6.7 (4.71, 14.08) neutral 107 51.2 (64.59, 79.04) strongly agree 13 6.2 (4.19, 13.26) strongly disagree 8 3.8 (1.75, 8.99) in the last part of the survey, the preventive measure for lsms intake was elaborated therefore, students’ opinion on prevention of lsms has been collected. 135 (64.6%) students did not agree on the prevention of the use of lsms, while the rest 74 (35.4%) encouraged the prevention of lsms intake by providing many strategies to prevent this phenomenon. among those methods self-education about their side effects 83(39.7%), providing college counsellors 33(15.8%), establishing university oncampus counseling centers 32(15.3%), social media and tv advertising regulation 31(14.8%) and mass media campaigns 15(7.2%) have been mentioned by the students as shown in figure 3. figure 3. prevention strategies for the use of lsms among university students discussion the change in lifestyle gave rise the emergence of various medications that address the aspects of quality of life and lifestyle of individuals, therefore the use of these medications without medical diagnosis or as off-labeled drugs raises a public, ethical and safety concerns. (12) they also have a significant unfavorable socioeconomic and safety impact on public health and society especially in developing countries. (10) university students are a diverse group of relatively healthy individuals who are subjected to lifestyle and physical change. within the first year of university, students report an increase in body weight and body fat due, in part, to the changes in their diet and the stress of the new university environment (13) thus, they might be more prone to use lsms. the present study demonstrated the prevalence of using lsms among university students in three universities. it has revealed that three quarter of the participants were using those medications during their lifetime with the intention to either improve their immunity and wellbeing or to decrease stress for better academic performance. these purposes were also stated by a study conducted in tokyo on the japanese students who were registrants of macromill inc and their age was between 18-24 years old. the students experienced high prevalence of consumption of these medications. (14) clear differences in the prevalence of lsms between and within the countries have been reported for example a study in an australian university revealed that 75.5% of the students used stimulants to enhance academic performance while us data from a systematic review displayed a range between 5% and 35% of non-medical use of the stimulants by university students (15) . in the present study, the most common life style substances used among the participants was caffeine (47.7%) followed by dietary supplement (42.3%) and cosmetics (32.2%). the finding of the present study in the context of caffeine consumption is consistent with other studies which conducted in some universities such as egypt, lebanon and united state. (16,17,18) caffeine is the most consumed psychoactive drug in the world, as a psychostimulant. it shows all the pharmacological properties of classical psychostimulants, such as cocaine and amphetamine. an epidemiological study on caffeine withdrawal and dependence indicated that regular caffeine intake creates dependence, which in part depends on withdrawal symptoms. (19) perceptions for consuming caffeine among university students in the previous studies were feeling of alertness, improved performance, attention in task performance, concentration, improved long-term memory and faster locomotors speed (20,21) feeling of energy and capability to do work for long periods of time after taking caffeine. the perceptions for not taking caffeine were irritability, increasing heart rate, bad feelings, and insomnia. (22) the popularity of consumption of the caffeinated products associated with the food related social behaviors, individual habits and hereditary traits. (18) literature suggests that the consumption of caffeine among young people is significantly high all over the world and different caffeinated products are easily accessible. (23) approximately 90% of the college students in united states are using caffeine (18) and in another study conducted among students of dutch university a daily consumption of caffeine iraqi j pharm sci, vol.32( 1 ) 2023 university students and life style medications 191 was 87.8%. (24) in addition, the consumption of caffeine products among canadian young people was found to be 73.6% and approximately one in six consumers had exceeded the usual guidance for maximum daily consumption thus potentially increasing their risk of experiencing adverse effects. (25) moreover, the intake of caffeine was reported to be 97% in india (26) and 98.5% in united arab emirates. (27) additionally, the prevalence of the use of dietary supplements has globally increased and dietary supplements are extensively consumed worldwide despite unproven efficacy. in the present study, the consumption of dietary supplement among the college student; which are intended to provide the diet with additional nutrients, were also high. approximately less than half of the participants were using dietary supplement during their lifetime which is consistent with the studies conducted in different countries including japan, the united states and european countries (14,28,29,30,31) the main issues of using dietary supplements as a lsms are associated with lack of enough knowledge on its properties, active ingredients, beneficial effects as well as its adverse effects such as nephrotoxicity, hepatotoxicity, immunotoxicity. (32,33) the present study reported that some students had experienced adverse events with the use of these lsms which is impart might related to the use of dietary supplements such as constipation and/or stomach upset, dizziness or agitation, loss of appetite, weight gain, facial hair growth and/or hormonal disturbance, headache and/or mood changes. cosmetics and dermatological products consumption were also demonstrated in the responses of the participants. it has been found that one-third of the students were using cosmetics including skin depigmenting agents and products for female hirsutism which were quite close to the data recorded by other researchers in different countries such as nigeria where the prevalence of use of skin lightening products among female of undergraduate medical students was quite high. (34) moreover, skin toning practices have been reported among young women in ghana and considerable number of university students in saudi arabia who use topical steroids on the face without knowing its nature. (35,36) stressful environment and workload on the medical university students also led to administration of bblockers such as propranolol or atenolol especially during examination. this practice is addressed in many other studies shown among medical and dental students at king saud university in riyadh where inappropriate use of beta-blockers has been used for relieving their anxiety and stress during examinations, and most of them were selfprescribed. (37) in another section of the study the motives and the factors influencing the use of lsms among the students have been investigated. the treatment of non-serious minor condition then social media or tv advertisement and peer pressure or friend influence were considering as the main motives for usage of lsms among university students while reasons and motives for using these medications in another study was mostly peer pressure and to alleviate stress and anxiety (4) . advertisement of pharmaceutical companies and their impact in medicalizing healthy life (7) have a crucial role in influencing the students to use these medications. in the present study, the prevention of lsms usage among university students has been suggested only by one-third of the students. they provided useful strategies for awareness of lsms usage among students to decrease this phenomenon. their suggestions were parallel with the previous reports which stated that providing appropriate education on dietary supplement among university students particularly pharmacy and medical students is crucial for their health and for their future profession as a pharmacist and physician. (38) the present study has certain limitations, despite the good response rate that was received, the sample size was relatively smaller for cross sectional studies on awareness, attitudes, and use. as this is a cross-sectional study design, thus factors affecting student’s responses cannot be studied over time. another limitation was the number of universities, i.e., uos, hmu and uod which cannot be generalized to other universities of iraq. therefore, more studies are required from different universities which might have different opinions. in conclusion, prevalence of using life style medications among university students is high and tendency for medicalization of healthy individuals in the aim of better academic performance and improve quality of life is increasing. advertisement of pharmaceutical companies on a particular product has an important role in influencing the university students to use these medications for non-serious medical states. the prevention of lsms intake by 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iraq abstract certain cyclic amine derivatives of metronidazole via acetate spacer were prepared. cyclic amines used are piperidine and piperazine to improve the physicochemical properties and reduce some of metronidazole side effects. this is believed to be done by modification of its structural features to get prodrugs with improved properties over that of metronidazole. the present work includes esterification of metronidazole with choroacetic acid, n-alkylation of the cyclic amines by the halogenated ester and characterization of their structures by spectral(uv and ir) and elemental(chn)analysis.the melting points, degree of solubilities and partition coefficients were also determined. both metronidazole-piperazine and metronidazole-piperidine derivatives have different structural and physicochemical properties that may be excellent to diminish problems associated with metronidazole. key words: metronidazole, chloroacetate, prodrug. الخالصة ميّ نهًخشَٔيذاصٔل يٍ خالل جسش انخالث. اٌ االييُاث انحهميت في ْزا انبحث َمٕو بخحضيش بعغ انًشخماث االييُيّ انحه انًسخعًهتْي انببشاصيٍ ٔانببشيذيٍ ٔانغشع يٍ ْزا انبحث ْٕ ححسيٍ انخٕاص انفيضيأيت ٔانكيًيأيت ٔحمهيم بعغ انخأثيشاث يمذياث ادٔيّ راث خظائض افضم يٍ انجاَبيت نهًخشَٔيذاصٔل .اٌ ْزا يخى بأجشاء ححٕيش في حشكيبت انًخشَٔيذاصٔل نهحظٕل عهى كهٕسٔ حايغ انخهيك ثى حعٕيغ انًشكب اعالِ عهى رسة َخشٔجيٍ -انًخشَٔيذاصٔل .يشًم انجاَب انعًهي اسخشة انًخشَٔيذاصٔل يع انفا حهيم انذليك االييُاث انحهميت ٔيٍ ثى حشخيض ْزِ انًشكباث بٕاسطت انخحهيالث انطيفيّ ) ححج انحًشاء ٔفٕق انبُفسجيت (ٔكزنك انخ َخشٔجيٍ(.كزنك فأٌ دسجاث االَظٓاس,لابهياث انزٔباٌ ٔانخٕصع بيٍ سائهيٍ غيش يًخضجيٍ لذ -ْيذسٔجيٍ-نهعُاطش انًكَّٕ نٓا)كاسبٌٕ حى ححذيذْا ايضا ٔكهٓا اثبخج حظٕنُا عهى يشخميٍ نهًخشَٔيذاصٔل يًخهكاٌ خٕاص فيضيأيت ٔكيًيأيت ٔحشكيبيّ حخخهف عٍ ٔلذ حكٌٕ افضم يٍ حيث حمهيم انجٕاَب انسهبيت نّ.انًخشَٔيذاصٔل introduction metronidazole is one of the nitroimidazole derivatives that are mostly used in treating many infections nowadays. it is effective against wide range bacterial, protozoal and parasitic infections (1a) .it has little more effect over mebendazole against giardia intestinalis as well as intestinal nematodes (2) .intravaginal metronidazole is effective in the treatment of bacterial vaginosis (3) .metronidazole,as benzoyl form,could be used as supportive , suppressive and/or synergistic /additive drug in treatment of african trypanosomiasis (4) . metronidazole can be considered as a prodrug in the sense that it requires metabolic activation by sensitive microorganisms in which the nitro group is reduced to hydroxylamine and covalently bind the dna of the microorganism so that triggering the lethal effect (1a,5) . metronidazole is rapidly and completely absorbed when given orally. it has a limited plasma protein binding, but can attain very favorable and rapid tissue distribution, including cns and csf (6) . resistance to metronidazole can be attributed to decreased activity of pyruvate: ferredoxin oxidoreductase which reduces metronidazole, induction of oxidative stress mechanism including superoxide dismutase and peroxiredoxin (7) ,mutation of genes rdxa (8) or lower activities of oxidases and reductases (9) .increasing the dose and duration of treatment with acid suppression can overcome h.pylori resistance to metronidazole (10,11) . in therapeutic dose, the side effects of metronidazole include metallic bitter taste and cns symptoms (dizziness, headache and sensory neuropathies). it also has a strong reported teratogenic effect (6) . in case of piperazine, it has the ability to dissolve uric acid, and so might be useful in gout and other rheumatic diseases. it is used as an effective anthelmentic in citrate form for the treatment of pinworms as well as round worms (1b) . piperazine moiety was introduced to certain antibacterial drugs to increase activities of norfloxacin and ciprofloxacin against some bacteria (1c) . 1 corresponding author email : maadhqm@yahoo.com received : 14 / 9 / 2008 accepted : 9 / 2 / 2009 iraqi j pharm sci, vol.18(2) 2009 cyclic amines derivatives of metronidazole 2 it was found that parenteral combination of ciprofloxacin with metronidazole enhances the antimicrobial spectrum of quinolone derivatives against anaerobic organisms and gram-positive bacteria (12) . in cetrizine (h1antihistaminic drug) the piperazine moiety participate in formation of zwitterions of the drug to prevent penetration into the brain and in turn minimizes cns effects (13,14) . on the other hand piperidine is a cyclic amine and can be used as a moiety for the synthesis of many drugs such as the antihistamine, diphenylpyraline, which is structurally related to cetrizine (15, 1d) . several of the functionalized piperidines have been shown to exhibit diverse biological activities through some enzyme inhibition such as glucosidase (16,17) , tryptase (18) and some serine protease (19) . both piperidine and piperazine moieties in fibrates structures were found to have superior activities in lowering serum trigiycerides, cholesterol and sugar in mice and rats (20) .also many opioid receptors ligands are piperazine or piperidine-based agents (21) . prodrugs of metronidazole; to reduce or abolish some of its side effects as well as improving its activities; like some amino acids (22,23,24) , organic acids (25) and others (26,27,28) were synthesized. this field requires more investigations to get the most effective and suitable derivatives. accordingly, we synthesized two derivatives of metronidazole using piperazine and piperidine linked via acetate bridging in order possibly to get a new suitable mutual ester prodrug with piperazine diacetate or piperidine-acetate moiety; and these prodrugs (especially compound iii) were supposed to be hydrolyzed to generate the parent drugs that may have gastrointestinal or systemic anthelmentic effects as in using diethyl carbamazine for ascaris and filareasis. this will occur in parallel to metronidazole release exhibiting its anti-protozoal and anti-anaerobic action; or a new derivative of possible better physicochemical and antibacterial properties since the presence of piperazine moiety might contribute in improving activity, as shown in ciprofloxacin antipseudomonal activity (1c) . materials and methods metronidazole was supplied by sdi, iraq; piperazine hexahydrate and methylene chloride (e. merck ag, germany); piperidine, chloroacetic acid and pyridine (fluka, switzerland); absolute ethanol 99% and thionyl chloride (bdh, england), dioxan (riedel-dehean ag "seelze-hannover", germany); methanol and chloroform (gcc, uk);while cyclohexane by (chem supply, south australia). methods of synthesis used were the conventional procedures (scheme 1) (22) , melting points and tlc (rf) values of intermediates and products were used to follow up the reactions, spectral analysis (uv and ir spectra) and elemental analysis (chn analysis) to characterize the products were conducted. partition coefficients and water solubilities were also determined. the synthesis started by activation of the carboxyl group of chloroacetic acid(15 gm,158.73 mmole) with 15 ml thionyl chloride (socl2) in 50 ml dried chloroform to produce chloroacetyl chloride, which(11.37 gm,120 mmole) was reacted with metronidazole(15 gm,87.66 mmole) in pyridine(7.95 gm,100.63 mmole) and methylene chloride(125 ml) to give metronidazole chloroacetate ester as an intermediate (compound ii). rf values and chn analysis were given in tables (2) and (3) respectively. metronidazole piperazine derivative (compound iii) was prepared by reaction of compound ii (9.9 gm , 40 mmole) dissolved in 125 ml dry dioxin with piperazine hexahydrate(1.942 gm,10 mmole) dissolved in dry methanol. compound iii was then purified by washing with saturated sodium chloride solution, dried over anhydrous sodium sulphate and recrystalized from chloroformcyclohexan mixture. the percent yield, physical description and melting point are given in table (1). rf values and chn analysis are given in tables (2) and (3) respectively. metronidazole-piperidine (compound iv) was synthesized by the same method as for compound iii except piperidine was used instead of piperazine solution and its properties were also shown in tables (1), (2)and(3). the partition coefficients of both compounds (iii) and (iv) were determined by shaking 20 ml of 0.00012 m chloroform(organic solvent) solution of either compound with equal volume of distilled water for 30 min and their concentrations were measured spectrophotometrically at  max 310nm. a standard curve using series of concentrations of each compound in chloroform was constructed. the partition coefficients of the prepared compounds were calculated and shown in table (4) (29) . iraqi j pharm sci, vol.18(2) 2009 cyclic amines derivatives of metronidazole 3 ch2 ccl oh o socl2 ch2 ccl cl o n n o2n ch3 ch2ch2oh ch2 ccl cl o n hn nh n n n n o2n ch3 ch2ch2 h2cc cl o chloroacetic acid chloroacetyl chloride metronidazole + metronidazole chloroacetate ester compound ii piprazine 1 mole compound iii compound ii 2 moles + nh n piperidine 1 mole compound iv compound ii 1 moles + o n n o2n ch3 ch2ch2 h2cc o o n n no2h3c h2ch2cch2 c o o n n o2n ch3 ch2ch2 h2cc o o n n o2n ch3 ch2ch2 h2cc cl o o n n o2n ch3 ch2ch2 h2cc cl o o scheme 1: synthesis of compounds iii and iv. table 1: physical description, melting points and percent yield of metronidazole and compounds i, ii, ill and iv. compound empirical formula molecular weight (gm/mole) description % yield m.p. oc (observed) metronidazole c6h9o3n3 171.1 yellow crystals -- 159-161 compound i c2h2ocl2 113.0 colorless-faint yellow oily liquid 93 -- compound ii c8h10o4n3cl 247.5 yellowish brown crystals 76 70-72 compound iii c20h28o8n8 508.5 off white powder 61 201-203 (dec.) compound iv c13h20o4n4 296.0 deep yellow crystals 78 106-109 iraqi j pharm sci, vol.18(2) 2009 cyclic amines derivatives of metronidazole 4 table2: rf values of metronidazole, compound ii, iii and iv. solvent system: a = chloroform : ethanol (4:1), b = chloroform : methanol (6:1), c = methanol alone. table 3: elemental microanalysis of metronidazole and compounds ii, iii and iv. compound molecular weight (g/mole) empirical formula element elemental analysis % calculated % observed metronidazole 171,1 c6h9o3n3 c h n 42.10 5.26 24.56 41.96 5.190 24.43 compound ii 247.5 c8h10o4n3cl c h n 38.78 4.040 16.96 38.65 3.980 16.84 compound iii 508.5 c20h28o8n8 c h n 47.24 5.510 22.04 46.98 5.330 21.86 compound iv 296.0 c13h20o4n4 c h n 52.70 6.750 18.91 52.51 6.580 18.63 table 4: partition coefficients and water solubilities of metronidazole and compounds iii and iv. compound (p) log (p) water solubility (mg/ml) metronidazole 0.677 ~ -0.169 10 compound iii 98.26 ~ 1.99 0.13 compound iv 6.800 ~ 0.832 0.59 compound rf values a b c metronidazole 0.662 0.851 0.611 compound ii 0.822 0.864 0.641 compound iii 0.39 0.54 0.269 compound iv 0.794 0.826 0.723 iraqi j pharm sci, vol.18(2) 2009 cyclic amines derivatives of metronidazole 5 results and discussion the products obtained were analyzed by measurement of physical properties like, melting points, rf values, partition coefficients, solubilities, as well as spectral methods, including uv and ir and were supported by significant analysis of the empirical formula of metronidazole, compounds ii, iii and iv as shown in tables 1, 2 and 3 respectively. the uv spectra shown in figures (1 and 2) showed that compounds ii, iii and iv differ from metronidazole in their chemical constitutions. the higher partition coefficient and lower water solubility of products iii and iv indicate a higher lipophilicity and in turn might enhance the absorption through biological compartments, even the blood-brain barrier, since log (p) values occur within the optimum value of (03) (30) . with respect to ultraviolet spectra using equal molar concentrations of these compounds in chloroform show the same  max with just small variations within 5nm ( max 313-317 nm). changing metronidazole to compounds ii, iii and iv has no effect on the extent of conjugation as shown in figures (1 and 2) (31) . this follows the principle stating that, when two or more chromophoric groups are present in a molecule and they are separated by two or more single bond, the effect on spectrum is usually additive and there is little electronic interaction between isolated chromophoric groups (32,33) . figure 1: shows nearly equal max (313 – 317 ) nm for metronidazole (b),compound ii(c) and compound iii(d) using equal conc.(0.0008m) of the three compounds (a) for solvent . figure 2 : shows nearly equal max (313 – 317) nm for metronidazole (b),compound ii(c) and compound iii(d) using equal conc.(0.00012m) of the three compounds (a) for solvent . this is also seen in the extent of absorption intensity. the intensity of compound iii(having molar absorbitivity  =2.72 x 10 4 mole -1 .l.cm -1 ) is nearly twice that of metronidazole and compound ii(=1.61 x 10 4 mole -1 .l.cm -1 ), since compound iii has double concentration of chromophore of metronidazole and compound ii as shown in figure (1); whereas in figure (2), the intensities of absorption of metronidazole, compound ii and iv(=1.03 x 10 4 mole -1 .l.cm -1 ) are nearly equal, since they have the same concentration of chromophore. the comparison of the ir spectra for piperazine, piperidine, metronidazole and compound ii with those for compounds iii and iv showed the following differences :  strong bands at (1715-1720) cm -1 and (1750) cm -1 due to ester (c=o) stretching vibration for compounds ii , iii and iv respectively.  two asymmetrical coupled bands at (1265 and 1180) cm -1 , (1245 and 1155) cm -1 and (1225 and 1193) cm -1 for (c-c(=o)-o) and (o-c-c) groups were appeared in compounds ii, iii and iv respectively.  two bands at (2870 and 2780) cm -1 due to asymmetrical and symmetrical stretching vibrations and two bands within fingerprint region at (1440 and 1380) cm -1 due to (c-h) bending vibrations of methyl group.  the disappearance of a single weak-band in lower than (3550-3200) cm -1 region which is due to (n-h) stretching of secondary amine in spectrum of compounds iii and iv. iraqi j pharm sci, vol.18(2) 2009 cyclic amines derivatives of metronidazole 6  disappearance of the strong-broad band (3550-3200) cm -1 ; which corresponds to hydroxyl (oh) stretching vibrations ; in compounds ii, ill and iv spectra.  a strong broad band between(910-712)cm -1 and medium broad band between (820-710) cm -1 in piperazine and piperidine spectra respectively arisen from secondary amine (n-h) wagging. compounds iii and iv spectra have no this band.  in the lowest frequency region of the spectra, which is sensitive region, there is a prominent variation in spectra of compounds iii and iv from that of compound ii which shows (c-c1) stretching (850-550) cm -1 , and the (ch2) wagging band of (ch2cl) group at (1300-1150) cm -1 . conclusion cross-linking metronidazole 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kenneth, l. and rinehart, j.r. (eds.), englewood cliffs, n.j., prentice-hall, inc., 1965; pp. 11. 33. willard, h.h.; merritt, l.l.; dean, j.a. and settle, f.a.: ultraviolet and visible absorption methods, instrumental methods of analysis (6 th ed.). d.van nostrand company, new york. 1981; pp. 94. iraqi j pharm sci, vol.30(2) 2021 synergistic effects of s. striata extract and antibiotics doi: https://doi.org/10.31351/vol30iss2pp219-224 219 evaluation of synergistic antibacterial effect of combined scrophularia striata extract and antibiotics against pseudomonas aeruginosa and methicillin -resistant staphylococcus aureus shabnam pourmoslemi*, shirin moradkhani**, pari tamri***,1and sahar foroughinia* *department of pharmaceutics, school of pharmacy, medicinal plants and natural products research center, hamadan university of medical sciences hamadan, iran **department of pharmacognosy, school of pharmacy, medicinal plants and natural products research center, hamadan university of medical sciences hamadan, iran ***department of pharmacology &toxicology, school of pharmacy, medicinal plants and natural products research center, hamadan university of medical sciences hamadan, iran abstract scrophularia striata from scrophulariacea family has been used in iranian folk medicine for the treatment of infectious diseases. in this study we evaluated the synergistic effect of s. striata hydroalcoholic extract (sse) and commercially available antibiotics against pseudomonas aeruginosa and methicillinresistant staphylococcus aureus (mrsa) bacteria. the resazurin-based microdilution method was used to determine the minimum inhibitory concentration (mic) values of plant extract and standard antibiotics. the interaction between standard antibiotics and scrophularia striata extract was evaluated by using the checkerboard method. the results of this study revealed that sse enhances the antibacterial activity of antibiotics. the combination of sse and vancomycin had synergistic to additive effects against mrsa. sse in combination with gentamicin had synergistic to additive effects against p. aeruginosa. the interaction between ceftazidime and sse was additive against p. aeruginosa. the best result was the synergistic effect between sse and piperacillin-tazobactam against p. aeruginosa. in conclusion this research indicated that s. striata has the potential to enhance the antibacterial activity of antibiotics and could be a source to the designing new compounds with synergistic effect in combination with standard antibiotics. keywords: scrophularia striata, pseudomonas aeruginosa, methicillin resistance staphylococcus aureus, synergy, antibiotics introduction antibiotic resistance has become a serious public health problem worldwide (1) . methicillin resistant staphylococcus aureus (mrsa) and pseudomonas aeruginosa are two of the more problematic antibioticresistant pathogens encountered over the past decade (2) . mrsa infection is the main cause of nosocomial infections and usually is associated with mortality, morbidity and cost burden (3). resistance to methicillin has occurred in s. aureus by penicillin binding protein mutation, a chromosomal mutation(4) . the rate of mrsa infections is increasing rapidly throughout the world and more importantly, in the past decades the prevalence of community acquired mrsa infections has notably increased (5). the most common mrsa infections are skin and subcutaneous tissue infections or invasive infections such as meningitis, pneumonia, osteomyelitis, lung abscess, bacteremia and infective endocarditis (6). several antibiotics including clindamycin, cotrimoxazole, vancomycin and daptomycin are being used to treat mrsa infections (7) . however, the increasing resistance of pathogens to these medicines and their side effects have led to poor therapeutic outcomes and increased mortality (8). pseudomonas aeruginosa is a gram negative bacillus commonly found in soil, water and the environment (9) . p. aeruginosa, as an opportunistic pathogen is a major cause of hospital acquired infections, especially in patients with underlying conditions (10). p. aeruginosa has the ability to survive on minimum nutritional necessities and to tolerate different environmental conditions, allowing this organism to persist in both hospital and community setting(9). it has become difficult to eradicate p. aeruginosa due to its high capacity to resists antibiotics (11) . a number of antibacterial agents such as piperacillin-tazobactam, ceftazidime, cefepime, ciprofloxacin and imipenemcilastatin are used to treat p. aeruginosa infections but a limited number of these agents have reliable activity against p. aeruginosa isolates (9). thus, it is necessary to find new ways to overcome the resistance of mrsa and p. aeruginosa to antibiotics. combination therapy using two or more 1corresponding author e-mail: ptamri@gmail.com received: 17/4/2021 accepted: 20/6 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp219-224 mailto:ptamri@gmail.com iraqi j pharm sci, vol.30(2) 2021 synergistic effects of s. striata extract and antibiotics 220 antibacterial agents is an important strategy to overcome antibioticresist organisms (12). however, combining antibiotics result in more antibiotics adverse effects and drug interactions. many previous studies have shown the antibacterial activity of plant constituents and some studies have proved the synergistic antibacterial effect of the combination of antibiotics and phytochemicals (13, 14, 15) . scrophularia striata (scrophulariaceae) is an herbaceous plant that grows wild in the west regions of iran (16). in traditional medicine, it has been used for the treatment of the inflammatory and infectious diseases (17) . several studies have shown biologic activities of s. striata, including antibacterial (18), anti-inflammatory (19), antioxidant (20), anticancer (21) and healing effects (22). the aim of this study was to investigate the synergistic effect between sse and commonly used antibiotics against mrsa and p. aeruginosa. materials and methods materials and strains nutrient agar (na) and muller hinton broth (mhb) culture media were obtained from merck (darmstadt, germany) and used for growing the bacteria and antibacterial activity tests throughout the study. standard strains of mrsa (atcc 33591) and p. aeruginosa (atcc 27853) were obtained from persian type culture collection in iranian research organization for science and technology (irost), tehran, iran. isolates were maintained in tryptic soy broth (tsb) containing 15% glycerol at -80 ° c until use. bacterial inocula were prepared from 24 h culture of the organisms grown on nutrient agar (na) plates. the organisms were harvested, and suspended in normal saline (ns) to produce a macfarland 0.5 (turbidity equivalent to 108 colony forming units (cfu) (23). preparation of s. striata extract the aerial parts of s. striata were collected from the west parts of iran (ilam province). the authentication of herb material was performed at the department of pharmacognosy, school of pharmacy, hamadan university of medical sciences. the plant was dried and grounded to a fine powder. the plant hydroalcoholic (ethanol/distilled water 7/3 v/v) extract was prepared by using maceration method (24) . preparation of stock and standard solutions a sse stock solution of 32mg/ml was prepared by accurately weighing and dissolving the extract in sterile dimethyl sulfoxide (dmso). aliquots of the stock solution were brought to 10 ml volume using sterile 0.9% (w/v) normal saline to obtain further dilutions. commercial parenteral dosage forms of antibiotics [co-trimoxazole (ctx), clindamycin (cld) vancomycin (van), piperacillin + tazobctam (pip-tazo), gentamicin (gen) and ceftazidime (cef)] were used for preparing antibiotic solutions. whole content of one vial or ampule was dissolved and further diluted in normal saline to obtain antibiotic solution with the intended concentration determination of minimum inhibitory concentration (mic) mic of s. striata extract against mrsa and p. aeruginosa were determined on sterile 96 well microdilution plates according to the clinical and laboratory standards institute (clsi) guidelines (25). sse solutions in the range of concentrations of 32 0.015 mg/ml were prepared through two-fold serial dilution of the stock solution. 100 µl of each solution was mixed with 100 µl of mueller hinton broth (mhb) medium inoculated by bacterial suspension (containing 106 cfu/ml) in three wells row of microdilution plate. four wells without adding the extract were used to show maximum growth for each microorganism and four others containing uninoculated medium were used as negative control to show the aseptic technique. mic of the antibiotics against corresponding microorganisms were determined using the same method explained above. after incubation of the plates at 37° c for 24 h, the lowest concentration at which no growth was observed was determined as mic (26). visual inspection was used to determine any signs of bacterial growth and turbidity. for more accurate determination of mic, 50 µl of 0.002% w/v sodium resazurin solution was added to the wells and color change was investigated after 1 h incubation at 37 °c. change of color from blue to purple or red was considered as a sign of bacterial growth (27). the test was performed in three separate experiments, each one in three replicates. quantities determined as mic in at least two experiments were reported as the final mic. determination of minimum bactericidal concentration (mbc) mbc was determined by transferring 100 µl culture from the wells exhibited no growth on na plates and incubated at 37° c for 24 h. the lowest concentrations of sse and antibiotics that show no colony growth on na were reported as mbc. this test was repeated in three separate experiments. quantities determined as mbc in at least two tests were reported as the final mbc. investigation of antibacterial activity of combined s. striata extract and antibiotics the antibacterial activity of combined sse and antibiotics was investigated using the checkerboard dilution test that is one of the methods used for evaluation of in vitro synergy for multiple drugs (28). this test determines the impact on antibacterial activity of the combination of iraqi j pharm sci, vol.30(2) 2021 synergistic effects of s. striata extract and antibiotics 221 antibacterial agents in comparison to their individual activities. fractional inhibitory concentration (fic) index value was used to evaluate the interaction of the two agents tested. fic is determined according to the following equation (eq. 1), where a and b are the mic for each antibacterial agent when combined in a single well, and mica and micb are the mic for each agent individually. fic index = fica+ficb= a/mica + b/micb fic index values are interpreted as follows: fic index ≤ 0.5, synergistic, 0.5 ≤ fic index ≤ 1, synergistic to additive, 1 ≤ fic index ≤ 4, additive, and fic index ≥ 4, antagonistic(29). checkerboard test was performed for combinations of the sse with ctx, cld and van against mrsa and with pip-tazo, gen and cef against p. aeruginosa. an 8-by-8 well configuration on sterile 96 well microdilution plates was utilized. final concentrations of the sse and antibiotics in the wells ranged from 1/8 × mic to 4 × mic. the wells contained mhb medium inoculated with 106 cfu/ml of the respective microorganism. positive and negative controls containing inoculated and uninoculated mhb were set on every plate. after incubation of the plates at 37° c for 24 h, bacterial growth was investigated by visual inspection followed by adding 50 µl resazurin solution to observe the color change. the lowest fic index of all the non-turbid wells along the turbidity/nonturbidity interface was used for interpretation of the results. this test was performed in triplicate and results observed in at least two replicates were reported. statistical analysis microsoft excel 2016 was used to calculate mean and variance of data. results antibacterial activity the results of the evaluation of the antibacterial activity of sse showed that this extract has low activity against mrsa (mic=8 mg/ml) (figure. 1) and p. aeruginosa (mic= 4 mg/ml, mbc= 8mg/ml) (figure 2). the sse had no bactericidal activity against mrsa at the concentrations of 32-0.015 mg/ml. the mrsa strain was resistant to clindamycin and cotrimoxazole. the mic and mbc of sse and standard antibiotics are shown in table.1. figure. 1. determination of mic for vancomycin and scrophularia striata against methicillin resistance staphylococcus aureus (atcc 33591). figure. 2. determination of mic for ceftazidime and gentamicin against p. aeruginosa (atcc 27853) iraqi j pharm sci, vol.30(2) 2021 synergistic effects of s. striata extract and antibiotics 222 table 1. the antibacterial activity of antibiotics and s. striata extract mrsa p. aeruginosa sse ctx cld van sse pip gen cef concentrations 0.01532 mg/ml 0.252000 µg/ml 0.015-256 µg/ml 0.031-64 µg/ml 0.01532 mg/ml 0.031-64 µg/ml 0.03164 µg/ml 0.031-64 µg/ml mic 8 mg/ml nd* nd 2 µg/ml 4 mg/ml 2 µg/ml 0.25 µg/ml 0.062 µg/ml mbc nd nd nd nd 8 mg/ml nd 1 µg/ml 0.5 µg/ml * not determined in the concentration ranges study of synergistic effect between antibiotics and sse the results of interaction between sse and antibiotics expressed in fici are shown in table. 2. the combination of sse and vancomycin had synergistic to additive effect against mrsa. the combinations of sse and pip + tazo showed synergism against p. aeruginosa and in the case of the combination of sse and gentamicin the interaction was synergism to additive. the interaction between sse and ceftazidime was additive (figure 3). the combination of sse and pip-tazo showed the best synergistic capacity against p. aeruginosa. table 2. the interaction between antibiotics and s. striata extract bacteria antibiotics + sse fici interpretation mrsa van 0.75 synergistic to additive p. aeruginosa pip 0.3 synergism gen 0.75 synergistic to additive cef 1.5 additive figure 3. checkerboard test for determination of combined antibacterial activity of gentamicin and scrophularia. striata extract against p. aeruginosa (atcc 27853), a) after incubation, b) after adding the resazurin solution and c) after incubation of resazurin discussion as the results of this study showed, the sse in combination with standard antibiotics has good synergistic and additive effects and has the potential to be used as an adjunct therapy in the treatment of infections caused by resistant microorganisms such as p. aeruginosa and mrsa. the mechanism of sse to enhance the antibacterial activity of antibiotics is unknown. in addition to the direct antibacterial activity of plants, one of the possible mechanisms for the synergistic antibacterial effect of plants extract and antibiotics is the modifying and inhibiting the acquired resistance in bacterial cell and thus enhance the antibiotic antibacterial activity (13). the main compounds that isolated and characterized from sse were flavonoids such as quercetin, nepitrin and isorhamnetin-3-o-rutinoside (30). flavonoids may affect cellular membrane, inhibit nucleic acid synthesis, and energy metabolism. iraqi j pharm sci, vol.30(2) 2021 synergistic effects of s. striata extract and antibiotics 223 additionally, flavonoids may interrupt cell membrane and cell wall synthesis (31). according to the results of this study the sse has better antibacterial activity against p. aeruginosa (mic= 4 mg/ml) comparing to mrsa (mic= 8 mg/ml) and in combination with piptazobactam has a synergistic effect against p. aeruginosa. in addition, sse enhanced the gentamicin antibacterial activity against this microorganism. the resistance of p. aeruginosa against antibiotics may be intrinsic, acquired or adaptive. this microorganism has a low permeable outer membrane, expresses an efflux pump and produces antibiotic-inactivating enzymes to resist antibiotics, intrinsically. the acquired resistance of this organism may be due to mutation changes or horizontal gene transfer. previous studies indicated that the phenolic compounds and flavonoids initially change the permeability of cell membrane and this leads to the leaking of cellular content or disrupt the membrane structure by interfering with membrane proteins (32, 33). therefore, sse may increase the entry of antibiotics into bacterial cells by increasing the permeability of bacterial cell membrane. sse also enhanced the vancomycin antibacterial activity against mrsa. vancomycin is a bactericidal antibiotic that inhibits bacterial cell wall synthesis by binding to dala-d-ala peptide and following that preventing peptidoglycan crosslinking by transpeptidation and eventually inhibit the cell wall biosynthesis and bacterial cell death (34). vancomycin has been widely used for the treatment of mrsa infections and it has led to the emergence of vancomycin resistant s. aureus (35). the augmentation of antibacterial activity of vancomycin by sse could be a result of sse antibacterial activity or modifying the mechanism of acquired resistance. conclusion in conclusion, our findings in this study revealed the synergistic and additive activity of sse combined with standard antibiotics against p.aeruginosa and mrsa. antibiotics resistance is a growing problem and the perspective of antibiotics effectiveness in the future is uncertain. plants are important sources of biologically active compounds with antibacterial activity. the antibacterial effect of plants can be due to their direct activity against bacteria or their synergistic activity with antibiotics. s. striata could be a source of new antibacterial compounds. however, the further studies are needed to explore the mechanism underlying its synergistic effects. in addition, the toxicity, antibacterial activity and bioavailability of the sse should be studied in vivo. conflict of interest the authors declare there is no conflict of interest. acknowledgement the study was funded by vice-chancellor for research and technology, hamadan university of medical sciences under grant (number: 980127289). references 1. ventola cl. the antibiotic resistance crisis: part 1: causes and threats. p t 2015(40): 277283. 2. holland tl, arnold c, fowler vg, jr. clinical management of staphylococcus aureus bacteremia: a review. jama , 2014 (312), 1330-1341 3. kavanagh kt, abusalem s,calderon le. the incidence of mrsa infections in the united states: is a more comprehensive tracking system needed? antimicrob. resist.infect. control. 2017; 6: 34. 4. siddiqui ah,koirala j. methicillin resistant staphylococcus aureus (mrsa). i: statpearls. treasure island (fl): statpearls publishing copyright © 2020, statpearls publishing llc; 2020. 5. arjyal c, kc j,neupane s. prevalence of methicillin-resistant 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antimicrobial plant metabolites: structural diversity and mechanism of action. curr. med. chem. 2013; 20: 932-52. 33. cowan mm. plant products as antimicrobial agents. clin microbiol rev. 1999; 12: 564-82. 34. biondi s, chugunova e,panunzio m. chapter 8 from natural products to drugs: glycoand lipoglycopeptides, a new generation of potent cell wall biosynthesis inhibitors. i: atta ur r, editors. stud. nat. prod. chem. elsevier; 2016. p. 249-97. 35. shariati a, dadashi m, moghadam mt, et al. global prevalence and distribution of vancomycin resistant, vancomycin intermediate and heterogeneously vancomycin intermediate staphylococcus aureus clinical isolates: a systematic review and meta-analysis. sci rep. 2020; 10: 12689. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ comparative study between oral hypoglycemic drugs repaglinide, glibenclamide and rosiglitazone on some biochemical parameters in type 2 diabetic patients iraqi j pharm sci , vol.18(2) , 2009 comparative study between oral hypoglycemics 23 comparative study between oral hypoglycemic drugs repaglinide, glibenclamide and rosiglitazone on some biochemical parameters in type 2 diabetic patients sardar kader* , kassim j.al-shamma** ,1 , ibrahim a. majeed** and abdulla t. al-ani*** * college of pharmacy, hawler medical university, iraq. ** department of clinical pharmacy ,college of pharmacy, university of baghdad, baghdad, iraq. *** faculty of pharmacy and medical sciences, al-ahliyya amman university , jordan. abstract type 2 diabetes mellitus is often characterized by hyperglycemia as a result of increased insulin resistance in hepatic/peripheral tissues and pancreactic b-cell dysfunction. approximately 92% of patients with type 2 diabetes mellitus demonstrate insulin resistance, however hyperglycemia is always a consequence of insulin deficiency. this study was done on 120 patients newly diagnosed diabetes type 2 characterized by dyslipidemia that is increased triglycerides and decreased hdl. hypoglycemia and weight gain are common problem with oral sulfonyl urea drugs. in this work three different oral hypoglycemic drugs repaglinide and glibenclamide (insulin secretagogues) and rosiglitazone (insulin sensitizer) were used for treatment of patients with type 2 diabetes mellitus. the effect of these drugs on fasting and postprandial blood glucose levels, lipid profiles, alanine aminotransferase and serum creatinine were studied.three groups of newly diagnosed type 2 diabetic patients ,group 1 (40 patients) were subjected to treatment with repaglinide (2 mg three time daily) ,group 2(45 patients) were subjected to treatment with glibenclamide (5 mg once daily) ,group 3,(35 patients) were subjected to treatment with rosiglitazone (4 mg twice daily) . fasting and postprandial blood glucose levels, lipid profiles (tc, tg, hdl and atherogenic index), alanine aminotransferase and serum creatinine were analyzed for these patients before and 4 and 8 weeks after oral hypoglycemic drug treatment. the same parameters were recorded for 40 normal individuals as control.the results demonstrated that repaglinide has a greater postprandial glucose regulator effect than glibenclamide and rosiglitazone. in addition the hypoglycemic episode and weight gain were less in patients treated with repaglinide than those treated with glibenclamide. repaglinide produces greater percent reduction with respect to fasting blood glucose levels, postprandial blood glucose levels and atherogenic index compared to glibenclamide and rosiglitazone. key words: oral hypoglycemic, repaglinide, glibenclamide, rosiglitazone. الخالصة كالياَذ و روسَكهُخاسوٌ عهً بعط انخحهُالث انكًُُائُت انحُاحُت نًزظً انسكزٌ يٍ ُدراست يقارَت نخأرُزاث رَباكهُُاَذ , كهُب انسكز فٍ انذو انُاحش عٍ انًشيُت وانذٌ َخصف بارحفاع فٍ يسخىي األيزاضيضًىعت إنًَُخًٍ 3يزض انسكزٌ َىع .3َىع حى وصف . نألَسىنٍُ% يٍ يزظً انسكزٌ َبذوٌ يقاويت ٢3 حقزَبا ،خهم فٍ كهُهًا أو األَسىنٍَُشاغ أو إفزاسوصىد قصىر فٍ أصزٌ هذا عٍ غزَق انفى نًعانضت انُىع انزاٍَ يٍ يزض انسكز . أخذهاكالياَذ و روسَكهُخاسوٌ وانخٍ حى ُرَباكهُُاَذ , كهُب أدوَتعذة رالد يضايُع وكًا إنًحىسَعهى عشىائُا ىوانذٍَ حى حشخُصهى حذَزا وح3يزَعا يصابا بًزض انسكزٌ يٍ َىع ٠3١انبحذ عهً يزَعا ( حى ٠٤يهغى رالد يزاث َىيُا , انًضًىعت انزاَُت )3يزَعا ( حى يعانضخهى بعقار رَباكهُُاَذ ٠١) األونًَهٍ , انًضًىعت يهغى ٠يزَعا ( حى يعانضخهى بعقار روسَكهُخاسوٌ 2٤انًضًىعت انزانزت ) أيايهغى يزة واحذة َىيُا , ٤كالياَذ ُر كهُبيعانضخهى بعقا يسخىي , بعذ انصىو انذو يسخىي انكهىكىس فٍ كًضًىعت سُطزة . حى قُاص األصحاءشخصا ( يٍ ٠١يزحٍُ َىيُا كًا حى اخخُار ) , حزاَكهُسُزاَذ , كىنسخزول انبزوحٍُ ٍقُاص انشحىو فٍ انذو ) كىنسخزول انكه حى ٍ يٍ وصبت انغذاء كذنكانكهىكىس فٍ انذو بعذ ساعخُ و ٠ويسخىي انكزَاحٍُُُ فٍ انذو قبم وبعذ altانكبذ وإَشًَاثانكزافت , كىنسخزول انبزوحٍُ انشحًٍ عانٍ انكزافت واغئانشحًٍ نذي يزظً انسكزٌ وانذٍَ حًج يعانضخهى بعقار رَباكهُُاَذ اَخفاظا يعُىَا واظحا فٍ انُخائش أظهزثيٍ بذء انعالس , أسابُع٨ انعالقت كاَج عانُت بٍُ أٌكالياَذ و روسَكهُخاسوٌ كًا وصذ ُيٍ كهُب أكززيسخىي انكهىكىس فٍ انذو بعذ ساعخٍُ يٍ انىصبت انغذائُت انذو بعذ ساعخٍُ يٍ وصبت انطعاو نذي انًزظً انًعانضٍُ بعقار رَباكهُُاَذ يسخىي انكهىكىس فٍ انذو بعذ انصىو ويسخىي انكهىكىس فٍ يٍ انعالس كًا كاٌ االَخفاض فٍ يسخىي انسكز فٍ انذو وانشَادة فٍ انىسٌ اقم حذورا نذي انًزظً أسابُع٨و ٠و روسَكهُخاسوٌ بعذ يٍ انعالس . أسابُع٨و ٠كالياَذ بعذ ُانذٍَ حًج يعانضخهى بعقار رَباكهُُاَذ يقارَت يع كهُب 1 corresponding author e-mail : drkassim_alshamaa @ yahoo.com received : 8 / 2 /2009 accepted : 26/4 /2009 iraqi j pharm sci , vol.18(2) , 2009 comparative study between oral hypoglycemics 22 introduction diabetes mellitus is a metabolic disorder characteriszed by hyperglycemia, abnormal lipid and protein metabolism along with specific long term complication affecting the retina, kidney and nervous system (1) . hyperglycemia is an important factor in the development and progression of the complications of diabetes mellitus (2) . diabetes mellitus may be categorized into several types, but the two major types are type i and type 2 (3) .type 2 diabetes is the commonest form of diabates and is characterized by disorder of insulin secretion and/ or insulin resistance (4) . in the non pharmacological treatment, weight management and exercise are the initial focus because insulin resistance can be dramatically improved with minimal weight loss (10: of body weight) and drug therapy is rement (5) . in sulfonylureas, oral hypoglycemic agent, glibenclamide is a second generation which is more potent than first generation sulfonylureas (6) . it is valuable in the treatment of non obese patient with type 2 diabetes, who fails to respond to dietary measures alone. it stimulates the secretion and enhances the utilization of insulin by appropriate tissues (7) .repaglinide (non sulfonylureas) is abenzoic acid derevative of the meglitinide family which is reported to be more potent insulinotropic agent than glibenclmide and other sulfonylureas (8) . rosiglitazone (thiozolidinediones) is introduced in 1999 and widely used as monotherapy or in fixed dose combination with either metformin or glimepiride (9) . it is a potent hypoglycemic agent (10, 11) .the present work was conducted to investigate the effects of repaglinide, glibenclamide and rosiglitazone on fasting, 2hr postprandial blood glucose, lipid profile, alt enzyme and serum creatinine in type 2 diabetic patients. evaluation of the correlation between fasting and postprandial blood glucose in patients treated with repaglinide, glibenclamide and rosiglitazone separately were also conducted. materials and methods patients the study included 120 patients (50 male and 70 female) of newly diagnosed type 20 diabetes mellitus and 40 healthy subjects (20 male and 20 female) as control. patients were interviewed according to the patient information sheets. all newly diagnosed type 2 diabetic patients enrolled in the study were treated and followed by specialized physician.patients were randomized into three groups. group i included 40 patients (16 male and 24 female), their ages range between 40-69 years (49.3 ± 5.8) treated with repaglinide 2mg three times daily before meal. group 2 included 45 patients ( 20 male and 25 female) theirs ages range between 45 – 65 years (51.2 ± 7.6) treated with glibenclamide 5mg once daily before meal. group 3 included 35 patients (14 male and 21 female), their ages range between 38 – 64 years (53.8 ± 6.5) treated with rosiglitazone 4 mg twice daily after meal. all patients were followed after 4 and 8 weeks of treatment. control forty healthy subjects (20 male and 20 female) with ages range between 40 – 60 years (42.6 ± 6.8) were enrolled in the study and served as a control group. drugs used were as followed drugs drug dose supplier repaglinide 2mg tablet novo nordisk/danmark rosiglitazone 4mg tablet pharmasyer/ syria glibenclamide 5mg tablet hikma/ jordan blood samples before drug treatment, eight ml of venous blood was drawn from each patient of newly diagnosed type 2 diabetes mellitus who was fasted at least for 8 hours. using sterile disposable syringe 23g, the blood was transferred into disposable plain tube and let stand for 30 minutes to clot. serum was separated by centrifugation at 3000 rpm for 5 minutes, which was collected in plain tube and kept frozen unless analyzed immediately. the serum was utilized for determination of total cholesterol (tc), triglyeride (tg), high density lipoprotein cholesterol (hdl-c) low density lipoprotein chelestrol (ldl-c), glucose, alanine aminotransferase (alt), and serum creatinine (scr). the same procedurs were carried out in blood samples from patients after 4 and 8 weeks of treatment. commercial kits for biochemical analysis, the following kits were used: kits supplier kit for determination of serum glucose biolabo sa/ france kit for determination of serum cholesterol biolabo sa/ france kit for determination of serum hdl cholesterol biolabo sa/ france kit for determination of serum triglycerides biolabo sa/ france kit for determination of serum creatinine biomeriex/ france kit for determination of serum alt biomeriex/ france iraqi j pharm sci , vol.18(2) , 2009 comparative study between oral hypoglycemics 2٠ statistical analysis all values were represented as mean ± sd. analysis of variance (anova) were used to find the differences among groups.duncan test was used to find the factor effect. p values less than 0.05 was considered significant. correlation coefficient (r) was used to determine the relationship between fasting and two hours postprandial blood glucose for each diabetic group who was treated by specific drug. results table 1 showed the effect of repaglinide on fasting and 2hr postprandial blood glucose levels in diabetic patients treated with repaglinide (group 1) at 4 and 8 weeks of treatment. significant reduction in fasting and postprandial blood glucose levels were observed in treated patients after 4 and 8 weeks of treatment when compared to zero level values (before treatment). however, the fasting and postprandial blood glucose levels after 8 weeks of treatment were higher than the control (healthy) but statistically insignificant. the same table also showed that repaglinide significantly increases hdl-c, and decreased tg after 4 weeks of treatment when compared to zero level values. reduction of atherogenic index was also observed in treated patients when compared to zero levels values, while these values after 4 and 8 weeks of treatment were similar to the values of control individuals. alt values were significantly reduced in treated diabetic patients compared to zero level values. table 1: effects of repaglinide on serum fasting blood glucose, 2hrs postrandial glucose, total cholesterod, low density lipoprotein cholesteros, high density lipoprotein cholesterol, triglycerides, alanine aminotransferase and serum creatinine after 4 and 8 weeks of treatment. parameters control (n=40) before treatment (n=40) 4 weeks after treatment (n=40) 8 weeks after treatment (n=40) fbg mmol/l 5.64±2.22 a 9.86±1.98 b 7.30±1.77 c 6.17±1.76 a 2hr ppg mmol/l 7.94±3.10 a 15.22±2.21 b 9.90±2.38 c 8.10±2.21 a tc mmol/l 4.88±1.89 a 5.06±1 a 4.99±1.60 a 4.95±1.13 a ldl-c mmol 2.45±0.94 a 2.76±0.77 b 2.59±0.58 a 2.55±0.40 a hdl-c mmol 1.52±0.43 a 0.99±0.34 b 1.40±0.42 a 1.41±0.53 a tg mmol 1.94±0.90 a 2.90±0.90 b 2.19±0.93 a 2.14±0.78 a atherogenic index tc:hdl 3.21 a 5.11 b 3.56 a 3.51 a atherogenic index ldl:hdl 1.61 a 2.78 b 1.85 a 1.80 a alt (iu) 16.82±5.31 a 27.32±4.37 b 18.05±3.63 a 18.18±5.09 a s cr mmol/l 62.73±7.73 a 63.84±6.07 a 63.86±6.10 a 63.89±6 a data represented by mean ± sd. n= number of subjects values with non-identical superscript (a, b, c) for the same parameter indicate significant difference at level p< 0.05 table 2 showed the effects of glibenclamide on fasting and 2hr postprandial blood glucose levels in diabetic patients treated with glibenclamide (group 2) at 4 and 8 weeks of treatment. significant reduction in fasting and postprandial blood glucose levels were observed in treated patients when compared to zero level values. however, the reduction in postprandial blood glucose levels with repaglinide were significantly higher than that with glibenclamide after 8 weeks of treatment. other parameters were similar to that observed with repaglinide.table 3 showed the effects of rosiglitazone on fasting and 2hr postprandial blood glucose levels in diabetic patients treated with rosighitazone (group 3) at 4 and 8 weeks of treatment.significant reduction in fasting and postprandial blood glucose levels were observed when compared to the zero level values. the reductions were similar to that observed with glibenclamide, however the hdl-c, ldl –c and tc were significantly increased at 4 and 8 weeks of treatment when compared to zero level values.table 4 showed the percentage decrease of fasting and postprandial blood glucose in diabetic patients treated with repaglinide, glibenclamide and rosiglitazone after 4 and 8 weeks of treatment. iraqi j pharm sci , vol.18(2) , 2009 comparative study between oral hypoglycemics 2٤ table 2: effects of glibenclamideon the serum fasting blod glucose , 2hrs-postprandial glucose , lipid profile ,alanine aminotransferase , and serum creatinine after 4 and 8 of weeks of treatement . data represented by mean ± sd. n= number of subjects values with non-identical superscript (a, b, c) for the same parameter indicate significant difference at level p< 0.05 table 3: effects of rosiglitazone on the serum fasting blod glucose , 2hrs-postprandial glucose , lipid profile ,triglycerides , alanine aminotransferase , and serum creatinine after 4 and 8 of weeks of treatment data represented by mean ± sd. n= number of subjects values with non-identical superscript (a, b, c) for the same parameter indicate significant difference at level p< 0.05 table 4 : percentages decrease of fasting and 2 – hours postprandial blood glucose in group1 , 2 and 3 of diabetic patients after 4 and 8 weeks of treatment drugs decrease of fasting blood glucose % decrease of two hour post prandial blood glucose after 4 weeks after8 weeks after 4 weeks after 8 weeks repaglinide 26.16% 37.61% 34.95% 46.78% glibenclamide 25.07% 36.23% 28.43% 36.33% rosiglitazone 22.59% 31.58% 25.87% 33.56% parameters control (n=40) before treatment (n=45) 4 weeks after treatment (n=45) 8 weeks after treatment (n=45) fbg mmol/l 5.64±2.22 a 9.77±1.70 b 7.32±1.92 c 6.23±1.78 a 2hr ppg mmol/l 7.94±3.10 a 14.56±2.3 b 10.42±2.53 c 9.27±2.11 a tc mmol/l 4.88±1.89 a 5.01±1.25 a 4.93±1.60 a 4.90±1.32 a ldl-c mmol 2.45±0.94 a 2.68±0.81 a 2.57±0.68 a 2.54±0.63 a hdl-c mmol 1.52±0.43 a 0.99±0.29 b 1.38±0.37 a 1.39±0.49 a tg mmol 1.94±0.90 a 2.93±0.81 b 2.16±0.81 a 2.12±0.62 a atherogenic index tc:hdl 3.21 a 5.06 b 3.57 a 3.52 a atherogenic index ldl:hdl 1.61 a 2.70 b 1.86 a 1.84 a alt (iu) 16.82±5.31 a 26.55±6. 7 b 18.02±4.47 a 18.30±5.26 a s cr mmol/l 62.73±7.73 a 63.11±6. 76 a 63.07±6.91 a 63.32±6.95 a parameters control (n=40) before treatment (n=35) 4 weeks after treatment (n=35) 8 weeks after treatment (n=35) fbg mmol/l 5.64±2.22 a 9.56±1.76 b 7.40±0.76 c 6.54±1.93 a 2hr ppg mmol/l 7.94±3.10 a 14.30±2.23 b 10.60±2.53 c 9.50±2.20 a tc mmol/l 4.88±1.89 a 5.03±1.65 a 5.52±1.57 b 6.14±1.48 c ldl-c mmol 2.45±0.94 a 2.63±0.90 a 3.13±0.80 b 3.54±0.83 c hdl-c mmol 1.52±0.43 a 1.07±0.40 b 1.46±0.35 a 1.64±0.63 c tg mmol 1.94±0.90 a 2.91±0.73 b 2.07±0.71 a 2.10±0.54 a atherogenic index tc:hdl 3.21 a 4.70 b 3.78 a 3.78 a atherogenic index ldl:hdl 1.61 a 2.45 b 2.14 a 2.15 a alt (iu) 16.82±5.31 a 28.52±6. 55 b 18.22±3.96 a 18.72±4.59 a s cr mmol/l 62.73±7.73 a 65.80±6. 7 a 65.69±5.37 a 65.83±5.37 a iraqi j pharm sci , vol.18(2) , 2009 comparative study between oral hypoglycemics 23 it is obvious from the table that repaglinide produces higher percentage decrease in fasting and postprandial blood glucose level after 4 and 8 weeks of treatment when compared to glibenclamide and rosiglitazone.in table 5 the percentage of changes in atherogenic index (tc:hdl) is higher in diabetic patients treated with repaglinide and glibenclamide, while diabetic patients treated with rosiglitazone showed no changes after 4 and 8 weeks of treatment. percent atherogenic index ldl: hdl showed higher reduction than the atherogenic index tc:hdl in diabetic patients treated with repaglinide, glibenclamide and roiglitazone after 4 and 8 weeks of treatment.table 6 showed high percent of adverse effects (hypoglycemia 15% and headache 11%) in glibenclamide treated patients compared to repaglinide and rosiglitazone treated patients. table 5 : percentage changes of atherogenic index tc : hdl and ldl :hdl in groups 1,2 and 3 of diabetic patients after 4 and 8 week of treatment drugs % change of atherogrnic index tc:hdl % change of atherogenic index ldl:hdl after 4 weeks after 8 weeks after 4 weeks after 8 weeks repaglinide 30.33% ( ) 31.31%( ) 33.45% ( ) 35.25( ) glibenclamide 29.44%( ) 30.43%( ) 31.11%( ) 31.85( ) rosiglitazone 19.57%( ) 19.57% ) ( 12.6%( ) 12.2%)(   = increase lowering atherogenic .   = decrease lowering atherogenic  = unchanged atherogenic table 6 adverse – ettects in the three groups of diabetic patients treated with repaginide ,glibenclamide and rosiglitazone after 8 weeks of treatment discussion the present study demonstrates that fasting blood glucose and post prandial blood glucose levels were significantly reduced in type 2 diabetic patients treated with repaglinide after 4 and 8 weeks of treatment. this result is quite similar to that reported by goldberg etal 1998 (12) , who found that repagtinide is highly effective in controlling both fasting and postprandial blood glucose levels. the effect of repaglinide on the postprandial blood glucose level is significant because of the rapid onset of action and the short half life of bypoglycemic effect, which makes repaglinide an ideal drug for controlling postprandial hyperglycemia (13) . glibenclamide showed significant reduction in fasting and postprandial blood glucose after 4 and 8 weeks of treatment. this result is in agreement with the observation of kolterman etal 1984 (14) , and rosak etal 2002 (15) who found that glibenclamide significantly reduced blood glucose levels . both glibenclamide and repaglinide have the same mechanism of action in lowering blood glucose. repaglinide, like sulfonylureas, acts by linide stimulating the release of insulin from the bcell of the pancreas by inhibiting atp-sensitive k channels, thereby activating the ca++ channel with increase in intracellular calcium to release insulin (16) . however, repaglinide acts on a different binding site than that of sulfonylureas (17) .the present work showed that fasting blood glucose and postprandial blood glucose in diabetic patients treated with rosiglitazone were significantly decreased after 4 and 8 weeks of treatment. this result is compatible with that reported by miyazaki etal 2001 (18) , and tan etal, 2005 (19) who found that rostiglitazone significantly reduced both fasting and postprandial blood glucose in type 2 diabetes mellitus.postprandial glucose level is an important factor in the development of diabetic complications and increase risk factor for cardiovascular diseases marfella etal (20) adverse effects group1 repaglinide group 2 glibenclamide group 3 rosiglitazone hypoglycemia (mild ) 10% 15% 0% hypersensitivity 1 tolerability good good good headache 5% 10% 2.5% iraqi j pharm sci , vol.18(2) , 2009 comparative study between oral hypoglycemics 23 found that hyperglycemia has been implicated as a cause of endothelial dysfunction in normal as well as diabetic subjects. hyperglycemia, particularly postprandial hyperglycemia has great effect on the development of diabetic complications (21) . postprandial hyperglycemia is an important risk factor associated with the development of macrovascular and microvascular complications, especially coronary heart diseases (13) . moreover it appears that the management of postprandial plasma glucose level, rather than fasting plasma glucose level, is important to prevent complications associated with type 2 diabetes (22) . therefore repaglinide is a noval and superior to glibenclamide and rosiglitazone in controlling postprandial glucose profile in type 2 diabetic complications.regarding lipid profile in type 2 diabetic patients treated with repaglinide and glibenclamide, there were no significant changes after 4 and 8 weeks of treatment. these results were supported by ykijarvinan 2004 (23) study, where lipid profile changes as a result of improved glycaemic control are not uniform findings associated with antidiabetic therapy (insulin secretagogues). however diabetic patients treated with rosiglitazone showed significant increase in hdlc, ldl-c and total cholesterol after 4 and 8 weeks of treatment compared with the base line. this result is similar to that reported by raskin etal 2000 (24) who showed that total cholesterol and ldl-c were significantly increased in patients treated with rosiglitazone.concerning other parameters like a therogenic index, alt, and serum creatinine, no significant changes were reported in diabetic patients treated with repaglinide compared to rosiglitazone and glibenclamide.the results showed that the the correlation (r2 values) between fasting and postprandial blood glucose in diabetic patients treated with repaglinide was 0.743 in comparing with r2 values in diabetic patients treated with glibenclamide 0.624 and rosiglitazone 0.499. these results indicated that correlation is more significant in repaglinide treated group.the results indicated that the incidence of hypoglycemia was about 15% with glibenclamide, while it was 10% with repaglinide and 0% with rosiglitazone. this finding is consistent with the finding of damsbo etal 1999 (25) who indicates that repaglinide therapy was associated with less frequent hypoglycemic events compared with glibenclamide.the results also indicated that diabetic patients developed weight gain which was least in diabetic patients treated with repaglinide and higher in diabetic patients treated with glibenclamide and rosiglitazone. there results are similar with that reported by marbury etal 1999 (26) who showed that patients given repaglinide may gain less weight than those treated with glyburide. this weight gain may be explained by increased adipocyte differentiation, increased appetite or water retention (27) . generally, adverse effects of rosiglitazone include weight gain.in conclusion repaglinide regulates postprandial blood glucose in diabetic patients greater than glibenclamide and rosiglitazone, while the weight gain was less in patients treated with repaglinide than glibenclamide and rosiglitazone. the correlation between fasting and postprandial blood glucose in diabetic patients treated with repaglinide was greatert than that with glibenclamide and rosiglitazone. references 1. david, m.n., james m, daniel e. the epidemiology of cardiovascular disease in type 2 diabetes mellitus. lancet; (1997),350: 514-519. 2. luzi l. pancreas transplantation and diabetic complication. n eng jmed; (1998), 339: 115-117. 3. zimmet p, cowie c, ekoe jm, et al. classification of diabetes mellitus and other categories of glucose intolerance. international text book of diabetes mellitus chapter 1, 3 rd ed, (2004), pp 3-14. 4. defranzo ra. bondonna rc, ferrannini e. pathogenesis of niddm. in albert kgmn, zimmet p, defronzo ra (eds). international text book of diabetes mellitus 2 nd ed. chichester: wiley, (1997), pp, 635-712. 5. franz m. maximizing the role of nutrition in diabetes management alexandria, va: (1994), american diabetes association. 6. lebovitz. e, feinglos mn. the oral hypoglycemic agents. in: diabetes mellitus, theory and practice, 3 rd ed. (m. ellenberg and h. rifkin eds.) medical examination publication, new hyde park, new york, (1993), pp. 591-610. 7. long jw in: the essential guide to prescription drugs, harper and row new york, (1990) ,pp. 505-509. 8. malaisse wj. stimulation of insulin release by non-sulfonylurea hypoglycemic agents: the meglitinide family. horm metab res; (1995), 27:263-266. 9. steven e. nissen, m.d., kathy wolski m.p. effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. n engl j medic; (2007),356: 2457-2471. 10. patel j, miller e, patwardhan r. rosiglitazone 011 study group, iraqi j pharm sci , vol.18(2) , 2009 comparative study between oral hypoglycemics 2٨ rosiglitazone (brl49653) monotherapy has significant glucose lowering effect in type 2 diabetic patients. diabetes; (1998), 47: 17-25. 11. young w, buckle r, cantello c et al. identification of highaffinity binding sites for the insulin sensitizer rosiglitazone (brl 49653) in rodent and human adipocytes using a radioiodinated ligand for peroxisomal proliferators-activated receptor gamma. j pharmacol exp ther; (1998), 284: 751-759. 12. goldberg rb, einhorn d, luca cp, et al. a randomized placebocontrolled trial of repaglinide in the treatment of type 2 diabetes. diabetes care; (1998), 21: 1897-1903. 13. ambavane v, patil r, ainapure ss. repaglinide: a short acting insulin secretagogue for postprandial hyperglycaemia. j postgrad med; (2002), 48:246. 14. kolterman og, gray rs, shapiro g et al. the acute and chronic effects of sulfonylurea therapy in type ii diabetic subjects. diabetes; (1984), 33 (4): 346354. 15. rosak c, haupt e, walter t et al. the effect of combination treatment with acarbose and glibenclamide on postprandial glucose and insulin profiles: additive blood glucose lowering effect and decreased gypoglycaemia. diabet nutr metab; (2002), 15: 143-151. 16. gromada j, dissing s, et al. effects of the hypoglycaemic drugl repaglinide and glibencamide on atp-sensitive potassium –channels and cytosolic calcium levels in beta tc3 cells and rat beta pancreatic cells. diabetologia; (1995), 38: 1025-32. 17. fuhlendorff j, rorsman p, et al. stimulation of insulin release by repaglinide and glibenclamide involves both common and distinct processes. diabetes; (1998), 47: 345-51. 18. miyazaki y, glass l, triplitt c, et al. effect of rosiglitazone on glucose and non esterified fatty acid metabolism in type 2 diabetic patients. diabetologia; (1999), 44: 2210-2219. 19. tan g, fieding b, currie j, et al. the effect of rosiglitazone on fatty acid and triglyceride metabolism in type ii diabetes. diabetologia; (2005), 48: 83-95. 20. marfella r, verrazzo g, acampora r, et al. glutathione reverses systemic hemodynamic changes by acute hyperglycemia in healthy subjects. am j physiol; (1995), 268: 1167-1173. 21. ceriello a. the possible role of postprandial hypoglycemia in the pathogenesis of diabetic complication. diabetologia; (2003), 46 (1): 9-16. 22. hanefeld m, fischer s, julius u, et al. risk factors for myocardial infarction and death in newly detected niddm: the diabetes intervention study, 11-year follow-up. diabetologica; (1996), 39: 1577-83. 23. yki-jarvinen h. thiazolidinediones. n engl j med; (2004), 351: 1106-18. 24. raskin p, rappaport eb, cole st, et al. rosiglitazone short-term monotherapy lowers fasting and post-prandial glucose in patients with type ii diabetes. diabetologia; (2000), 43: 278-284. 25. damsbo p, clauson p, marbury tc, et al. a double-blind randomized comparison of meal-related glycemic control by repaglinide and glyburide in wellcontrolled type 2 diabetic patients. diabetes care; (1999), 22:789-794. 26. marbury t, huang wc, strange p. repaglinide versus glyburide: a one-year comparison trial. diabetes res clin pract; (1999), 43(3): 155-66. 27. phillips ls, grunberger g, miller e, et al. rosiglitazone clinical trials study group. once-and twice-daily dosing with rosiglitazone improves glycemic control in patients with type 2 diabetes. diabetes care; (2001), 24: 308-315. iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives doi: https://doi.org/10.31351/vol31isssuppl.pp141-152 141 knowledge, attitude, and practices of iraqi community pharmacists toward emergency contraceptives (conference paper )# juan majid shaukat* and basma zuheir al-metwali**,1 # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 * ministry of health and environment , aladel sector primary health care centre, baghdad ,iraq. ** department of clinical pharmacy , college of pharmacy, university of baghdad, baghdad, iraq. abstract emergency contraceptives (ecs) are indicated for preventing the chance of unintended pregnancy that follows unprotected sexual intercourse in cases of incorrectly used regular contraceptives and in sexual assault. it is considered a safe choice to prevent pregnancy than abortion which is considered life threating. the aim of this study was to assess knowledge, attitude, and practices (kap) of community pharmacists towards emergency contraceptives and their association with sociodemographic variables. this study was a cross sectional study conducted between august and september 2021 on a convenient sample of community pharmacists from iraq. the survey tool was an online, selfadministered questionnaire, in english language and a paper-based copy of the questionnaire was delivered face-to-face to some of the study participants. the questionnaire consisted of four parts, sociodemographic characteristics, knowledge, attitude and practices of community pharmacists toward emergency contraceptives. a total of 212 community pharmacists participated in the study. of the study participants, 67.9% were in the age range of (24-29) years, 61.8% were females,60% had less than 5 years of experience, the majority (73.1%) were from baghdad. this study showed that the majority of community pharmacists (74%) had good knowledge and 95% of them had a very positive attitude. however, 51% of the participants had poor practice where 62.5% of the pharmacists did not make counselling during dispending on mechanism of action. this study results have shown no significant relationship between demographic characteristics and kap of participants towards emergency contraceptives. the majority of the study participants had good knowledge and very positive attitude too, whereas poor practice was observed in more than half of the participants. educational programs and training sessions are required to raise knowledge about the importance of emergency contraceptives and also to improve the dispensing practice of these products. keywords: emergency contraceptives, knowledge, attitude, practice, community pharmacists. معرفة و مواقف وممارسات صيادلة المجتمع في العراق نحو استخدام وسائل منع الحمل #) بحث مؤتمر ( الطارئة ** زهير المتولي بسمة و 1*،تجوان ماجد شوك 2202حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي األولية ، بغداد ، العراق قطاع العدل للرعاية الصحية والبيئة، زارة الصحةو* ، بغداد ، العراق جامعة بغداد ، كلية الصيدلة السريرية، فرع الصيدلة ** ةالخالص موانع الحمل الطارئة تستخدم لمنع فرصة الحمل غير المقصود الذي يتبع الجماع غير المحمي في حاالت االستعمال غير الصحيح لوسائل منع تقييم معرفة راسة هيالحمل االعتيادية و األعتداء الجنسي. يعتبر منع الحمل خيارا امنا مقارنة باإلجهاض الذي يعتبر خطرا على الحياة. كانت أهداف هذه الد ارة عن دراسة و مواقف و ممارسات صيادلة المجتمع تجاه موانع الحمل الطارئة و عالقتها مع المتغيرات الديموغرافية األجتماعية. كانت هذه الدراسة عب انت أداة المسح عبارة عن استبيان على عينة مالئمة من صيادلة المجتمع من العراق. ك 2021استقصائية مقطعية أجريت في الفترة من آب الى ايلول عام من أربعة أجزاء: الكتروني يدار ذاتيا باللغة األنكليزية و تم تسليم نسخة ورقية من االستبيان وجها لوجه لبعض المشاركين في الدراسة. يتكون األستبيان ه وسائل منع الحمل الطارئة. شارك في الدراسة ما مجموعه الخصائص األجتماعية الديموغرافية, و المعرفة, و المواقف, و ممارسة صيادلة المجتمع تجا ( من %73.1سنوات , و األغلبية ) 5من المشاركين لديهم خبرة أقل من % 60إناث, % 61.8( سنة, 29-24في الفئة العمرية ) % 67.9صيدلي مجتمع, 212 منهم لديهم مواقف جيدة جدا. و % 95ة عن وسائل منع الحمل الطارئة و أن ( لديهم معرفة جيد%74بغداد. أظهرت هذه الدراسة أن غالبية صيادلة المجتمع ) لم يقدموا المشورة بشأن آلية عمل وسائل منع الحمل. باإلضافة لذلك, % 62.5من المشاركين كانت لديهم ممارسات ضعيفة حيث أن %51مع ذلك, فإن ين الخصائص الديموغرافية و المعرفة و المواقف و الممارسات تجاه وسائل منع الحمل أظهرت نتائج هذه الدراسة عدم وجود عالقة ذات داللة أحصائية ب في حين لوحظت ممارسات تطبيقية ضعيفة في أكثر من نصف الطارئة،الطارئة. كان لدى غالبية المشاركين معرفة و مواقف جيدين تجاه وسائل منع الحمل لتحسين ممارسة الصيادلة لصرف وكذلك يبية لرفع مستوى الوعي حول أهمية وسائل منع الحمل الطارئة تدر ودوراتالمشاركين. يقترح وضع برامج تعليمية هذه المنتجات. . صيادلة المجتمع ، ممارسات،مواقف ،معرفة ،الطارئةالكلمات المفتاحية: وسائل منع الحمل introduction emergency contraceptives (ecs) are indicated for preventing the chance of unintended pregnancy that follows unprotected sexual intercourse in cases of contraceptive failure and sexual assault. the ecs are considered as a last choice since they cannot be used as a regular method of contraception and they cannot provide protection against sexually transmitted diseases (stds)(1) . 1corresponding author e-mail: basma.naji@copharm.uobaghdad.edu.iq received: 13/7/2022 accepted: 23/10 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp141-152 iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 142 ecs do not terminate the pregnancy if it has already occurred. also, they have no harmful effects neither on the fetus nor on the mother compared to methods used in abortion which have adverse effects like maternal death and infertility(2). unintended pregnancy and induced abortion can be a significant problem in the community, both from the health and economic point of view. a cross sectional study conducted on 500 pregnant women in erbil/ iraq has shown that about 39.4% of women had unintended pregnancy which was significantly higher in those aged ≥35 years and those with insufficient income(3). another study carried out in mosul/iraq on 1302 married women showed that 13.5% of women tried to induce abortion by physical activity, herbal remedies or pharmacological preparations(4). this can impose a significant risk on the mother’s health. the same study had shown that induced abortion was significantly higher in women with unemployed husbands, those who were housewives and those who were not using contraceptives (4). therefore, the use of ecs can reduce both the health and economic consequences of unintended pregnancy and subsequent induced abortion. there are four types of ecs which vary in their mechanisms of action and efficacy. available types of ecs include emergency hormonal contraceptives (ehc) pills, also known as morning after pill, which include two major types: ulipristal acetate (ella)® and levonorgestrel (plan b) ®. in addition, many types of combined oral contraceptives (coc) that contain progesterone and ethynyl estradiol can be used as ecs. other non-hormonal ecs include copper intrauterine device (iud). ulipristal acetate (ella)®, a progesterone receptor modulator, acts by delaying or preventing the ovulation process. it is effective until five days (120 hours) of unprotected sexual intercourse, however, as a general principle for all types of ec, it is best to be given as soon as possible (5) . the possible side effects of ulipristal acetate include abdominal pain, headache, dizziness, nausea, vomiting and dysmenorrhea. other less common side effects include bloating and uterine cramping(6).levonorgestrel is a progesterone only tablets. it should be taken within 72 hours after unprotected intercourse(7). the mechanism of action of levonorgestrel is inhibiting or delaying the ovulation process by inhibiting rupture of follicle and release of ova that prevents fertilization and pregnancy (8). the possible side effects related to levonorgestrel include heavy or light menstruation, nausea, vomiting, abdominal pain, most women show their cycle within few days with expected date(9). combined oral contraceptives containing an estrogen and a progestin can also be used for emergency contraception in method called yuzpe regimen. it should be taken within three days (72 hours) after unprotected sexual intercourse (8) .the mechanism of action of cocs as ecs is by inhibiting implantation of a fertilized egg, delaying or suppressing ovulation, interfering with corpus luteum function and making changes in the endometrium that prevents implantation(8) . the possible related side effects of cocs include nausea, vomiting in higher proportion than ulipristal or levonorgestrel, headache and abdominal cramping (8). copper intrauterine device (iud) is the most effective method of ec. it is used within five days of unprotected intercourse. although copper iud has high efficacy, many studies showed that it had low recommendation from physician related to its practice and the presence of oral emergency contraceptives(10).the mechanism of action of copper iud involves releasing copper ion from this device which can have toxic effects on both sperm and ova, so preventing fertilization and preventing implantation if fertilization has occurred. the possible related side effects of iud include uterine cramping, dysmenorrhea and increasing duration of menstruation (7). the role of the pharmacists is very important in dispending ec. this involves providing consultation about the use and side effects and answering related questions on ec use. different studies were conducted to assess knowledge, attitude and practice of pharmacists toward ec. a study conducted in kathmandu/nepal on a sample of community pharmacists has shown that about 65% of participant pharmacists had good knowledge, about 93% of them had positive attitude and 75% of them good practice in dispending ecs (11) . in ethiopia, a study conducted on pharmacy professionals found that overall participants had very good knowledge, attitude and dispensing practice regarding ecs (12). in iraq, knowledge about ecs was investigated among primary healthcare doctors (obstetrics and gynecology specialists, general practitioners and family physicians) in baghdad. the results showed that there was a defect in primary healthcare physicians’ knowledge which led to ec underuse (13). in addition, knowledge, attitude, and practice about ec among women in primary healthcare centers was investigated in baghdad. the study has shown that participants had little knowledge about ecs and that only 12% of them used ec to prevent unwanted pregnancy(14). to our knowledge, there is no study conducted in iraq that has evaluated the level of knowledge, attitude and dispensing practices of ecs among community pharmacists. the aim of this study was to assess knowledge, attitude, and practices of community pharmacists towards emergency contraceptives and their association with sociodemographic variables. iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 143 subjects and methods the current study was approved by the ethical and scientific committee at college of pharmacy/university of baghdad. the pharmacists were informed of the research objectives and confidentiality of their responses. this was a cross sectional study conducted between august and september 2021. the inclusion criteria were pharmacists of both genders who were working in community pharmacies and consented to participate in this study. community pharmacists who were excluded from this study were those who did not consent to participate in the study and those who provided incomplete responses. the survey tool was an online selfadministered questionnaire in google form distributed through medical groups on social media, in english language, that was used to collect study data from eligible participants. in addition, a paperbased copy of the questionnaire was delivered faceto-face to some of the study participants due to limited time of study and also small sample size who responded to the online questionnaire. the questionnaire was adopted from a previous study with some modifications that were made according to the local practice(11). the questionnaire consisted of four sections. the first section included sociodemographic questions, whereas the other three sections included questions about knowledge, attitude, and practices; respectively. the questionnaire was first pretested on ten community pharmacists to ensure the clarity of the questions. then it was distributed to other community pharmacists. the statistical analyses were performed using microsoft excel (2010) and statistical package for social sciences (spss) version 15 (spss inc., chicago, il, usa). descriptive statistics were used to summarize the characteristics of the study population. the categorical variables were expressed as frequencies and percentages. for the assessment of knowledge and practice scores, each correct response was given a score of one, whereas each incorrect response was given a score of zero. then the cumulative and mean scores were calculated. participants whose scores above the mean score were considered to have good knowledge and good practice, whereas those who had scores below mean score were considered to have poor knowledge and poor practice (11) . the attitude of participants was calculated using fivepoint likert scale ranging from strongly agree with a score of 5, to strongly disagree with a score of 1, while a reverse scoring was used in questions one, three and six. then, both of the cumulative and median score were calculated. those participants who had scores equal and above median score were considered to have positive attitude while those who had scores below the median score were considered to have negative attitude (11). the data was not normally distributed. therefore, for the detection of the relationship between sociodemographic variables and the participant knowledge level and practice level, chi-square and fischer exact tests were used where relevant. additionally, for the assessment of the relationship between the attitude level and the sociodemographic variables mann whitney u and kruskal-wallis h tests were used where relevant. a pvalue of less than 0.05 was considered to be significant. results sociodemographic characteristics of the study population a total of 217 responses were received form the participants. five responses were excluded because of incomplete answers and 212 responses were included in the final analysis. there were 162 online and 50 paper-based responses received from the study participants. table (1) presents the sociodemographic characteristics of community pharmacists who participated in the study. most of the participants (67.9%) were in the age group (2429), were females (61.8%), had bachelor degree (79.2%), had less than 5 years of experience (60.8%) and were from baghdad (73.1%). most of the participants (83%) had another work in addition to the community pharmacy from whom 48.1% were working in a hospital. table 1. sociodemographic characteristics of the study participants. characteristic percentage distribution of respondents, number (%) age (years) 24-29 144 (67.9%) 30-35 40 (18.9%) 36-41 16 (7.5%) 42> 12 (5.7%) iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 144 continued table 1 characteristic percentage distribution of respondents, number (%) gender male 79 (37.3%) female 131 (61.8%) missing 2 (0.9%) degree of education bachelor 168 (79.2%) diploma 8 (3.8%) master 27 (12.7%) phd 9 (4.2%) years of experience: <5 129 (60.8%) 5-10 48 (22.6%) >10 35(16.5%) governorate in which pharmacy is located baghdad 155(73.1%) babil 10(4.7%) karbala 9(4.2%) others a 38(17.9%) do you have additional work? yes 176 (83.0%) no 36 (17.0%) if yes please specify: hospital 102 (48.1%) hospital, medical representer 2 (0.9%) primary health care center 26 (12.3%) academia 24 (11.3%) others b 18 (8.5) missing 4 (1.8) location of the pharmacy near the hospital 24 (11.3) near gynecology clinic 37 (17.5) none of them 151 (71.2) a : al-anbar, al-najaf, al-qadisiya, basra, dhi qar, diyala, erbil, , kirkuk, maysan, nineveh, saladin, sulaimaniya, wasit. b : medical representer, supervisor. knowledge of the community pharmacists towards emergency contraceptives table (2) shows the participants responses about knowledge questions. regarding the mechanism of action of ecs, 59.4% of the participants stated that ecs act by preventing or delaying ovulation. only 39.6% of the community pharmacists indicated that they received information about ecs more than once, with about half of those who received information (51.8%) stated that they received it from undergraduate/postgraduate study. in contrast, 22.6% of them indicated that they did not receive any information in previous years. more than one-third of the participants (37.7%) indicated that levonorgestrel should be taken within 72 hours, and more than half of them (56.6%) stated that levonorgestrel is the major ecs constituent. only 48.6% of all participants showed that ec have no harmful effects on fetus development. most of the participants (83.9%) stated that condom leak was the main cause of ecs use. the majority of the study participants (90.9%) indicated that ecs do not iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 145 protect from sexually transmitted diseases (stds). the most commonly reported side effects of ecs were nausea and vomiting (figure 1). regarding the knowledge level of the community pharmacists, results showed that the mean knowledge score was 0.55 and that about 74% of the participants had good knowledge, while 26% had poor knowledge (figure 2). table 2. knowledge of community pharmacists towards emergency contraceptives knowledge questions number (%) mechanism of action of ecs: prevent or delay ovulation (release of ovum from an ovary) 126 (59.4%) induce abortion 9 (4.24%) prevent an already established pregnancy 73 (34.4%) don’t know 15 (7.07%) how many times in the past years have you received information about ecs? yes, once 80 (37.7%) yes, more than once 84 (39.6%) no 48 (22.6%) sources of information: undergraduate/postgraduate study 110 (51.8%) medical representative 16 (17.5%) textbook 47 (22.1%) training 39 (18.3%) internet 212 (38.2%) within how many hours after unprotected sexual intercourse should the levonorgestrel pills be taken? 120 9 (4.2%) 72 80 (37.7%) 48 22 (10.3%) 24 36 (16.9%) don’t know 14 (6.6%) mention the constituents of ecs. levonorgestrel 120 (56.6%) levonorgestrel plus ethinyl estradiol 17 (8.01%) cupper iud 22(10.3%) don’t know 5 (2.3%) ecs can harm a developing fetus yes 58 (27.4%) no 103 (48.6%) don’t know 51 (24.0%) situations where ecs can be used missed injection due date and had unprotected sex 140 (66.3%) condom leaked/slipped 178 (83.9%) victims of sexual assault 172 (81.1%) intercourse without any family planning method 143 (67.4%) don’t know 16 (7.5%) do you know the side effects of ecs? yes 92 (43.3%) no 89 (41.9%) not sure 31 (14.6%) does ec protect from sexually transmitted infections (sti)? yes 6 (2.83%) no 191 (90.09%) don’t know 15 (7.07%) iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 146 figure 1. community pharmacists’ responses on side effects. figure 2. the knowledge level of the study participants. relationship between sociodemographic characteristics and the knowledge level of the community pharmacists results of the current study showed that none of the sociodemographic characteristic of the participants had a significant effect on the knowledge level (p > 0.05) (table 3). however, a higher knowledge level was found in the age group of (30-35) (82.5%), those with master degree (81.5%), and those with 5-10 years of experience (79.2%). table 3. association between sociodemographic characteristics and community pharmacists’ knowledge level. variables good knowledge n(%) poor knowledge n (%) p value age 24-29 104 (72.2%) 40 (27.8%) a 0.144 30-35 33 (82.5%) 7 (17.5%) 36-41 13 (81.3%) 3 (18.8%) 42> 6 (50%) 6 (50%) gender male 58 (73.4%) 21 (26.6%) b 0.983 female 96 (73.3%) 35 (26.7%) degree of education bachelor 123 (73.2%) 45 (26.8%) a 0.485 diploma 6 (75.0%) 2 (25.0%) master 22 (81.5%) 5 (18.5%) phd 5 (55.6%) 4 (44.4%) years of experience <5 93 (72.1%) 36 (27.9%) a 0.174 5-10 38 (79.2%) 10 (20.8%) >10 25 (71.4%) 10 (28.6%) location of the pharmacy near the hospital 14 (58.3%) 10 (41.7%) a 0.174 near gynecology clinic 29 (28.4%) 8 (21.6%) none of them 113(74.8%) 38 (25.2%) a: fishers exact test b: chi-square test iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 147 attitude of the community pharmacists toward emergency contraceptives table (4) shows the attitude of the community pharmacists towards ecs. results showed that 46% of participants expressed their disagreement on giving easy access for teenagers in using ecs, and 50.7% showed their agreement that adolescents should be discouraged to use ecs. also, 78.9% showed their agreement that routine information on ecs should be included in the ecs counselling, and 31% of participants agreed that they were uncomfortable with ecs dispending for moral or religious reasons. in addition, 76.1% showed their agreement on the importance of formal training in enabling pharmacists to appropriately dispense ecs and 55.4% agreed that ecs without prescription will promote unsafe sexual intercourse. table 4. attitude of community pharmacists towards emergency contraceptives. attitude questions strongly disagree n (%) disagree n (%) neutral n (%) agree n (%) strongly agree n (%) adolescents (teenagers) should be given an easy access to ecs. * 45 (21.1) 53 (24.9) 42 (19.7) 51(23.9) 20 (9.4) adolescents (teenagers) should be discouraged to use ecs. 14 (6.6) 45 (21.1) 45 (21.1) 68 (31.9) 40 (18.8) ecs can be used as a regular oral routine contraceptive method. 73 (34.3) 78 (36.6) 21 (9.9) 28 (13.1) 12 (5.6) ecs discourage regular contraceptive method use among youth. 16 (7.5) 49 (23.0) 56 (26.3) 73 (34.3) 17 (8.0) routine information about ecs should be included in contraceptive counseling. 5 (2.3) 7 (3.3) 32 (15.0) 103(48.4) 65 (30.5) are you uncomfortable dispensing ecs for moral or religious reason? * 29 (13.6) 42 (19.7) 75 (35.2) 50 (23.5) 16 (7.5) formal training is needed to enable the dispensers to appropriately dispense ecs. 2 (.9) 12 (5.6) 36 (16.9) 89 (41.8) 73 (34.3) ecs without prescription will promote unsafe sexual intercourse. 13 (6.1) 30 (14.1) 51 (23.9) 71 (33.3) 47 (22.1) *reverse scoring was adopted. results of the participants’ attitude showed that about 95% of them to have positive attitude, while only 5% of them to have negative attitude (figure 3). figure 3. attitude of community pharmacists toward ecs. relationship between sociodemographic characteristics and attitude level. in the current study, none of the sociodemographic characteristics of the study population was found to have a significant relation with attitude (table 5). however, a higher positive attitude was found in participants aged more than 42 years (83.3%), female pharmacists (79.4%), those with phd and bachelor qualification (77.8%) and (77.4%) respectively, those who have more than 10 years of experience (82.9%), and where the location of their pharmacies being near a hospital (83.3%). iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 148 table 5. association between sociodemographic characteristics and community pharmacists’ attitude level. variables positive attitude negative attitude p value age 24-29 136 (94.4%) 8 (5.6%) a 0.7 30-34 38 (95.0%) 2 (5.0%) 35-41 16 (100.0%) 0 (0.0%) 42> 12 (100.0%) 0 (0.0%) gender male 73 (92.4%) 6 (7.6%) b 0.5 female 128 (97.7%) 3 (2.3%) degree of education bachelor 160 (95.2%) 8 (4.8%) b0.1 diploma 7 (87.5%) 1 (12.5%) master 26 (96.3%) 1 (3.7%) phd 9 (100%) 0 (0.0%) years of experience <5 125 (96.9%) 4 (3.1%) a 0.2 5-10 43 (89.6%) 5 (10/4%) >10 34 (97.1%) 1 (2.9%) location of the pharmacy near the hospital 23 (95.8%) 1 (4.2%) a0.07 near gynecology clinic 36 (97.3%) 1 (2.7%) none of them 143 (94.7%) 8 (5.3%) a: kruskal-wallis h test b: mann-whitney u test practice of community pharmacists toward ecs table (6) shows the responses of participants towards the practice of ecs. most of the participants (79.2%) stated that they have dispensed ecs in their pharmacy. more than half of them (56.1%) thought that ecs should not be categorized as otc drugs whereas 43.3% stated that they should be. for the participants who dispensed, 71.4% of them indicated that levonorgestrel (ipill) ® tablet was the most sold brand of ecs. in addition, 90.4% of the community pharmacists indicated that dispensing ecs was upon their recommendation and 75% of them stated that they have provided ecs for girls under 18 years old. regarding counseling about ecs 70.2% of the pharmacists stated that they counsel all ecs users while dispensing, whereas 62.5% of them indicated that they do not make counselling on ecs mechanism of action. also, 86.9% of the pharmacists indicated that they make counselling on the time ecs should be taken, on other hand, about 61.3% of the pharmacists stated that they provide counselling about ecs side effects. table 6. the practice of the community pharmacist toward ec practice questions percentage distribution of respondents, number (%) have you ever dispensed emergency contraceptives? yes 168 (79.2%) no 44 (20.7%) do you feel ecs should be categorized under over the counter (otc) drug? yes 92 (43.4%) no 119 (56.1%) don’t know 1 (0.47%) which brand of ecs is sold the most? levonorgestrel (i pill) 120 (71.4%) ulipristal (ella one) 17 (10.11) combination oral contraceptives 29 (17.2%) iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 149 continued table 6. practice questions percentage distribution of respondents, number (%) intrauterine device (iud) 5 (2.9%) most often the products are sold on: patient request 2 (1.1%) patient approaches with prescription 79 (47.02%) on your recommendations 152 (90.4%) to whom you have provided ecs: girls aged under 18 years 126 (75%) victims of sexual assault 15 (8.9%) women whose partner’s barrier contraception method fail 5 (2.9%) women who did not use any contraception method 70 (41.6%) do you counsel all ecs users while dispensing? yes 118 (70.2%) no 50 (29.8) %) do you counsel on mechanism of action of ecs? yes 63(37.5%) no 105 (62.5%) do you counsel the time at which the ecs should be taken? yes 146 (86.9%) no 22 (13.09%) do you conceal side effect of ec? yes 103(61.3%) no 65(38.7%) figure (4) shows the practice level of the community pharmacists based on their responses. the mean practice score was 0.6, and 82 (48.8%) of the participants were found to have good practice while 86 (51.1%) of them were found to have poor practice. association between sociodemographic characteristics and practice level table (7) shows the results of the relationship between sociodemographic characteristics of the study population and the practice level. no significant relationship was found between any of the sociodemographic and practice level of the participants. however, a good practice was found within the age group (24-29) (51.4%), participants with diploma degree (83.3%), those with 5-10 years of experience (57.6%), and those who had location of the pharmacies near hospital (58.8%). figure 4. the community pharmacists’ practice level table 7. association between sociodemographic characteristics and community pharmacists’ practice level. variables good practice poor practice p value age 24-29 56 (51.4%) 53 (48.6%) a 0.4 30-35 17 (50.0%) 17 (50.0%) 36-41 6 (37.5%) 10 (62.5%) ≥42 2 (25.0%) 6 (75.0%) iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 150 continued table 7. variables good practice poor practice p value gender male 30 (44.8%) 37 (55.2%) a 0.4 female 47 (48.0%) 47 (48.0%) degree of educating bachelor 62 (48.8%) 65 (51.2%) b 0.3 diploma 5 (83.3%) 1 (16.7%) master 11 (42.3%) 15 (57.7%) phd 3(37.5%) 5 (62.5%) years of experience <5 47 (48.0%) 51 (52.0%) a 0.3 5-10 20 (57.6%) 18 (47.4%) >10 14 (45.2%) 17 (54.8%) location of the pharmacy near hospital 10 (58.8%) 7 (41.2%) a 0.3 near gynecology clinic 10 (37.0%) 17 (63.0%) none of them 61 (49.6%) 62 (50.4%) a: chi-square test b: fishers exact test discussion community pharmacists play an essential role in dispensing of ecs. in addition, looking at the significant numbers of unintended pregnancies and induced abortions reported in iraq (3), it has become very imperative to conduct a study to evaluate the knowledge, attitude and dispensing practice of community pharmacists towards these medications. the results of this study have shown that 74% of community pharmacists had good knowledge about ecs. other studies have shown that pharmacists had very good knowledge (12) or had good knowledge (11) about ecs. in comparison, another study has shown a low level of the knowledge among community pharmacists (15). having good knowledge could be attributed to receiving information about ecs from various sources. this was shown in the results of this study where the majority of participants indicated that they had received information about ecs, with about half of them had received the information during their undergraduate/postgraduate studies. this reinforces the important role of the undergraduate/postgraduate curriculum to provide essential information to pharmacists. the overall good knowledge level obtained in this study came from the participants’ responses. the majority of participants provided correct responses about the major ecs constituents, situations where ecs can be used and whether ecs can protect from stds. however, a fewer number of participants have provided correct information about the mechanism of action of ecs, the time within which ecs should be used after sexual intercourse, whether ecs can harm a developing fetus, and side effects of ecs. additionally, some participants gave inaccurate side effects of ecs. this may be related to receiving information from internet (38.2%) which may be inaccurate or not receiving any information about ecs (22.6%). possessing correct information about ecs, particularly that regarding the mechanism of action, timing of use and side effects is very essential for the community pharmacists to enable them to properly dispense these medications. for example, giving inaccurate information about the side effects of ecs may result in fear of women from using these products and using other risky ways to terminate pregnancy. the currents study’s results had shown that there was no significant relationship between sociodemographic characteristics of the community pharmacists and knowledge score. however, a higher knowledge level was observed in the age group of (30-35) and those with 5-10 years of experience. this may implicate that as pharmacists had more years of work, they gain more experience and knowledge about ecs. this came in agreement with a study conducted in iran which found nonsignificant association between pharmacists’ knowledge and their demographic variables (15). on the other hand, the study of ethiopia found a positive relationship between knowledge and years of practice (12) . also, in nepal study pharmacists’ age had a significant effect on level of knowledge (11) . regarding the attitude of community pharmacists towards ecs, this study’s results have shown that 95% of participants had very positive attitude towards ecs. similarly, the studies conducted in turkey and ethiopia have shown that pharmacists had positive attitudes towards ecs(12,16) . in addition, the study done in nepal showed that high percentage of pharmacists (93.4%) had positive attitude towards ecs (11). this level of positive attitude may have come from the responses obtained from the participants. half of the study participants (50.7%) agreed that adolescents should be iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 151 discouraged from using ecs, and about half (46%) of them expressed their disagreement in giving an easy access for teenagers in using ecs as this may be decrease the use of regular contraceptive methods among youth (42.3%), also may encourage some teenagers on illegal relationships. attitudes about the use of ecs by adolescents and youngest people may be subject to cultural backgrounds. the study of nepal has shown that 48.9% of participants disagreed that adolescents should have an easy access to ecs (11) whereas studies of turkey and ethiopia have shown that 40% and 63.3% of participants, respectively, thought that teenagers and youngest people can take responsibility of using ecs(12,17). another positive attitude was shown when the majority of the participants indicated the importance of including routine information on ecs during counselling and felt that formal training is important to enable pharmacists to appropriately dispense ecs. besides, more than half of the participants believed that dispensing ecs without prescription will promote unsafe sexual intercourse. therefore, 56.1% of them felt that ecs should not be categorized under over-the-counter drugs. the current study’s results showed nonsignificant relationship between sociodemographic characteristics and attitude score. that coincides with nepal study where the relationship between sociodemographic characteristics and attitude score was non-significant (11) . regarding the dispensing practice of community pharmacists towards ecs, the current study’s results have shown that only 48.8% of the participants had good practice. these findings contradict the results that showed that most of the study participants had good knowledge and very positive attitude. having good knowledge and attitude does not necessarily mean applying it in practice. this could be due to the finding that some participants did not dispense ecs in their pharmacies or the dispensing was improper as will be discussed later. similar findings were obtained from a study conducted in united arab emirates where the dispensing practice of ecs was rated as suboptimal (18), and the turkish study which concluded that some aspects of the dispensing practice of ecs needs to be improved (17). however, in nepal study, 74.9% of pharmacists had good practice (11) and in ethiopia study, the practice level was described to be very good (12).. in the current study, only 79.2% of the participants indicated that they had dispensed ecs which may indicate underuse of ecs in our community. this was shown in a study conducted in baghdad/iraq where only 12% of women used ecs to prevent unwanted pregnancy(3). in the current study, dispensing of ecs was mostly based on the pharmacists’ recommendations. this may reflect the level of knowledge of customers who request ecs, where it has been shown that women had limited information about ecs in the study done in baghdad (14). an interesting finding in the current study where more than half of the participants stated that they have provided ecs to girls under 18 years old, which was contradicting to their attitude where about half of them disagreed that adolescents should have easy access to ecs. regarding counseling during dispensing, only 70.2% of the participants indicated that they counsel all ec users while dispensing. this is comparable to the finding that 79% of the participants thought that providing information on ecs during counselling is essential. counselling was mostly about the time of taking ecs, and the ecs’ side effects, with only a minority of participants who counselled about the mechanism of action of ecs. counseling is a very essential role of the pharmacist and it enables women to make the right choice of which ec product to use and the proper way to use it. providing inadequate counseling may be attributed to the lack of time due to the work load or lack of private areas in the pharmacies to discuss such sensitive issue. similarly, only 58.2% of the pharmacists in the study done in united arab emirates provided spontaneous counseling about the use of ecs(18). in contrast, the nepal study has shown that 70% of the participants provided counseling to all ec users(11) .additionally, 85% of the respondents in ethiopia study stated that they counsel all women while dispensing ecs (12). this study’s results showed that there was non-significant relationship between demographics variables and practice level. in comparison, the results of the nepal study showed a significant association between age, years of experience and location of the pharmacy with the dispensing practice (11). on the other hand, the ethiopian study results showed that dispensing practice had positive relation of years of experience (12). the limitation of this study was the small sample size due to the short time of data collection. conclusions this study detected that community pharmacists in iraq had good knowledge and positive attitude but poor practice especially about counselling about ecs during dispending. additionally, the sociodemographic variables had non-significant relationship with the knowledge, attitude and dispensing practice of ecs. recommendations of the current study were: 1. future educational programs are very important like regular training programs by the pharmacists syndicate. 2. necessity of inserting the subject of ecs in the continuing education system in ministry of health to the pharmacists especially in general hospitals, and primary health care centers in which family planning department is present. iraqi j pharm sci, vol.31(suppl.) 2022 emergency contraceptives 152 3. necessity of educating the community education about the importance of ecs and their safe and effective use. conflict of interest authors of the 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community pharmacists in the united arab emirates: a simulated patient study. pharm pract (granada). 2019;17(2):8–14. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ occupational hazards facing healthcare providers iraqi j pharm sci, vol.30(2) 2021 doi: https://doi.org/10.31351/vol30iss2pp41-49 41 occupational toxicity and health hazards of the healthcare providers at healthcare facilities in sulaimani city, iraq tavga ahmed aziz*,1, renas raouf hama amin** , zheen aorahman ahmed* hazhan jalal sleman **and bahez hassan aziz ** *department of pharmacology and toxicology, college of pharmacy, university of sulaimani, sulaimani city, iraq, **directorate of health, sulaimani city, iraq. abstract the present study aimed to evaluate the occupational health hazards that face health care providers in sulaimani city.a cross-sectional study conducted utilizing quantitative data collection methods. it involved 159 respondents including physicians, pharmacists, medical assistants, laboratory instructors and nurses who worked in 8 major health facilities in sulaimani city, kurdistan region, iraq. nurses were the most susceptible group to sharp related injuries (13.84%), cuts and wounds (10.69%) than the others and they were more experiencing verbal abuse in the workplace (15%). laboratory instructors represent the most exposed group to contaminated specimens/biohazards (17.6%) and blood borne pathogens (13.84%), while the physicians represent the most prone group to acquire infectious diseases (15.1%) and both the physicians and the nurses were equally exposed to airborne diseases (11.32%). furthermore, physicians were the most group that suffered from work related stress (13.8%); and medical assistants were the most susceptible to radiation (3.1%). meanwhile, laboratory instructors were the most exposed group to physical distress (15.1%), falls (5%), unsafe staffing (13.8%), chemical spills (8.8%) and noise (5.4%). healthcare providers in these settings experienced various types of occupational hazards in their workplaces, which became a dominant issue among the health care providers. interventions should be established to alleviate these hazards. keywords: occupational hazards biological, non-biological, healthcare providers, iraq, sulaimani city. العراق -السليمانية دينةم في الصحية لمرافقا في لصحيةا الرعاية مقدميل المهنية المخاطر **ليمانس جالل زان هه ،*احمد ورحمنا زين ،** امين حمه رؤؤف ريناس ،1*،عزيز احمد تافكه ** عزيز حسن هيز به و العراق ،انيةالسليم محافظة السليمانية، جامعة الصيدلة، كلية والسموم، االدوية فرع * العراق ،السليمانية محافظة الصحة، دائرة ** الخالصة الدراسة اجريت السليمانية. محافظة في المستشفيات يف عملهم اثناء الصحية لكوادرا تواجه للتىا المهنية لمخاطرا تقييمل ممتص لدراسةا هذه كانوا اللذين التمريضي الكادرو االطباء مساعديو لمختبراتا يف لعاملينا و دلةوالصيا االطباء من كل شمل ذيوال صحي كادر خمسينو وتسع مائة على الفئات اكثر التمريضي الكادر بان الدراسة اظهرت لدراسة.ا بهذه خاص استبيان ستخداما بواسطة لسليمانيةا محافظة يف ستشفياتم مانيث يف يعملون لمختبراتا في لعاملينأ .اما%51 نسبةب اللفظية لإلساءات عرضة واالكثر %10.69 نسبةب وحالجر و %13.84 بنسبة لحادةا بالمواد لالصابة عرضة هم االطباء ان الدراسة وبينت كما .%13.84 بنسبة الدم طريق نع المنقولة وللعدوى %17.6 نسبةب الملوثة لعيناتا من الصابةل رضةع الكثرا لفئةا فهم بنسبة الهواء طريق عن المنقولة للعدوى رضةع االكثر ونهمك لتمريضيا لكادرا عم يتشاركون وهم %115. بنسبة المعدية لالمراض عرضة االكثر تعرضا االكثر فهم االطباء مساعدي اما .%3.81 بنسبة المهنة ضغوطات ناحية نم الفئات اكثر مه االطباء نا يضاا لدراسةا اظهرتو .هذا11.32% بنسبة االمن غير والتوظيف %5 السقوط وحوادث %5.11 الجسدية للمحن عرضات االكثر مه لمختبراتا يف ملينلعاا بينما %3.1 بنسبة االشعاع لمخاطر .%5.4 بنسبة ءالضوضا رومخاط %8.8 بنسبة الكيمياوية والمواد 13.8% سليمانية.ال ةمدين العراق، الصحية، الكوادر البيولوجية، غير و البيولوجية ،المهنية المخاطر المفتاحية: الكلمات introduction the healthcare workforce constitutes 12% of the working population around the world(1). according to national institute for occupational safety and health (niosh), healthcare providers are facing expanding numbers of occupational hazards including wounds and ailments, with rates having increased significantly during the previous decade (2). it has been assessed by the international labour organization (ilo) that 160 million people in the world suffer from occupation-related illnesses such as musculoskeletal diseases and psychiatric problems. meanwhile, 270 million lethal and non lethal work-related accidents resulted in more than 350, 000 victims and over two million work-related deaths each year were reported and indorsed to occupational hazards (3). 1corresponding author e-mail: tavga.aziz@univsul.edu.iq received: 6/11/2020 accepted:14 / 1/2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp41-49 mailto:aziz@univsul.edu.iq occupational hazards facing healthcare providers iraqi j pharm sci, vol.30(2) 2021 42 ealthcare providers are exposed to a wide range of work related hazards; these include biological and non-biological hazards. biological hazards refer to organisms or organic matters produced by these organisms that are harmful to human health. these include parasites, viruses, bacteria, fungi and protein. generally, there are three major routes of entry for these micro organisms into our body, i.e. through the respiratory system, transmission through contact with body fluids of the infected person or contact with contaminated objects(4). regarding non-biological hazards it includes chemical, physical, psychological hazards, noise, stress and others (5). only few hospitals have sorted out extensive work related surveillance frameworks and most of these hospitals do not have a definitive plan. hospitals incorporate various "mini-industries" inside their partitions, and health workers regularly move from one department to another during their shift. the threats mainly come from exposure to chemicals, radioactive substances, infectious agents, mechanical agents, latex, violence, and mental, psychosocial, and physical stressors (6, 7). a few of these threats have been managed, for instance exposure to blood borne pathogens, ethylene oxide, and formaldehyde. substances such as glutaraldehyde and hazardous drugs are not being addressed as biohazards however, it represent a danger to the workers wellbeing. in addition to managed substances and those regulated by niosh and different agencies, risks specifically to health workers incorporate exposures to natural rubber latex, infectious diseases, anesthetic gases, ergonomic stressors, hazardous drugs, and psychological, psychosocial, and physical stress. health care-specific hazards must be considered while developing a surveillance program for healthcare workers (8). there are only a few existing researches focusing on the wellbeing of workers in the healthcare settings. studies that have concentrated on worker’s injury in healthcare facilities recommend that poorly controlled work environment and high workloads are directly correlated with the increments in health care worker’s injuries, including needle stick wounds and near-misses to medical nurses (8,9). another important point is that till now most of the studies focused on patient safety-related outcomes and sets procedures to improve patient safety, without paying attention to the hazards that faces health workers daily during performing their work (10,11). some reports on patient-related adverse events and patients mortality showed that hospitals with better safety climate overall had lower relative incidence of adverse events in hospitals (12,13).accordingly, the present study was designed to shed a light on the daily occupational hazards that face healthcare providers while performing their work. study design and setting this cross-sectional study utilized quantitative data collection methods. it was carried out in sulaimani city, kurdistan region, iraq. the study was conducted between november 2019 and february 2020. the study protocol was approved by the research ethics committee of the college of pharmacy, university of sulaimani (certificate no.(1) in september 3, 2018), in accordance with the declaration of helsinki revised in 2000. sampling eight major hospitals were targeted during the study. these were purposively selected based on size and patient capacity. the selection ensured a combination of governmental and private facilities. these included sulaimani teaching hospital, shar hospital, central laboratory, sarwari healthcare center, piramerd dental center, pediatrics teaching hospital and shorsh hospital as public facilities in addition to some of private clinics and laboratories. study population the study population comprised a range of healthcare workers working in the selected health facilities. to select the respondents, sampling proportionate to size was used to determine the number of healthcare workers to be interviewed from each hospital. at the hospital, all healthcare workers who were presented at the facility were considered. among 200 healthcare workers interviewed, one hundred and fifty nine (159) responded. the selection was made according to their occupation in the healthcare facility, and they were categorized into five groups as follow: (a) physicians (b) pharmacists (c) medical assistants (d) laboratory instructors and (e) nurses. data collection all participants were interviewed by the researchers using a structural questionnaire designed and validated for this study (appendix i). it included demographic data, duration of exposure, use of personal protective equipment (ppe), health status, and working condition; and whether they have been trained to protect themselves against different types of work place-related health hazards. consensual agreement is signed by the subjects at the end of each questionnaire. each interview consumed approximately 35–45 minutes. occupational hazards facing healthcare providers iraqi j pharm sci, vol.30(2) 2021 43 appendix i. questionnaire occupational toxicity and health hazards at the healthcare facilities of sulaimani city, iraq this is where i describe the study and let people know that their participation is voluntary and that their data are anonymous and confidential. name gender male female contact number age weight hight marital status single married divorced cadre of health worker (a) physician b)pharmacist (c) medical assistant (d) lab. instructor (e) nurse 8. monthly income (a) > 500,000 id (b) < 500,000id 9. type of health care facility: (a) public (b) private 10. duration of service (a) > 5 years (b) < 5 years 11. description of the current job: (a)part time (b)full time 12. have you had training course about hazards and safety of your job? (a) yes (b) no 13. what protective measures do you use? answer protective measures (a) yes (b) no work clothes (a) yes (b) no goggles (a) yes (b) no gloves (a) yes (b) no mask (a) yes (b) no 14. do you drink alcohol? (a) yes (b) no 15. do you smoke? (a) yes (b) no 16. do you wash your hands before and after work? (a) yes (b) no 17. do you wear your work clothes at home? (a) yes (b) no 18. do you have frequent exercise? (a) yes (b) no 19. daily hours of sleep (a) > 8 hrs (b) < 8 hrs 20. do you face pressure from your job? (a) yes (b) no 21. have you noticed any negative change in your health during your work? (a) yes (b) no 22. do you take the necessary vaccines required from your job? (a) yes (b) no 23. what types of hazards do you expose to at the workplace? a. biological hazards answer sharp related injury (such as needle sticks) (a) yes (b) no cuts and wounds (a) yes (b) no direct contact with contaminated specimens /biohazards materials (a) yes (b) no airborne diseases (a) yes (b) no infectious diseases and/or infection (a) yes (b) no others (a) yes (b) no b. non-biological hazards answer stress (a) yes (b) no physical, psychological, sexual, and/or verbal abuse (a) yes (b) no musculoskeletal injuries (slips, trips, falls and/or fractures) (a) yes (b) no unsafe staffing (a) yes (b) no others (chemical spills, noise, burns, and radiation) (a) yes (b) no occupational hazards facing healthcare providers iraqi j pharm sci, vol.30(2) 2021 44 24. do you have periodic follow-up examination tests to ensure health safety? (a) yes (b) no 25. do you have preventive or risk control measures in the place of work? (a) yes (b) no 26. does your work provide health care protection when you face injury? (a) yes (b) no 27. has there been any accidental death in your workplace? (a) yes (b) no 28. has your work been supervised by institutions like directorate of health (doh)? (a) yes (b) no i agree to give the above information and for that purpose i am signing……………………… statistical analysis data analysis was performed using the statistical package for social science (spss) for windows using the linear regression model and bivariate correlation to analyze and test the relationship between variables. results a total of 159 interviews were conducted with the participants and they were categorized in a descending manner as follow: laboratory instructors, table 1. demographic data of the study sample physicians, medical assistants, nurses, and pharmacists. most respondents were female (91) and the number of males were (68), about 76.1% were married in the age range 20–40 year old. the majority were with body mass index (bmi) of 25-35 kg/m2. more than half of the participants were on part time working and in the public sector. most of them had a work experience of more than 5 years. maximum number of the participants kept hand hygiene and more than half of them had more than 8 hours of sleep a day (table1). demographical data of healthcare providers percentage demographical data of healthcare providers percentage gender male 42.77% public 58.49% female 57.23% private 2.51% age (years) both 38.99% 20-40 64.78% duration of service 40-60 35.22% less than 5 years 24.53% bmi (kg/m2) more than 5 years 75.47% 15-25 32.70% description of the current job 25-35 69.30% part time 52.20% marital status full time 47.80% single 23.90% personal behavior of healthcare providers married 76.10% drinking alcohol 4.40% cadre of health workers smoking 9.43% physician 20.75% washing hands before and after work 93.08% pharmacist 16.35% wearing work clothes at home 1.26% medical assistant 20.12% frequent exercise 35.85% lab. instructor 23.90% daily hours of sleep nurse 18.87% < 8 hr 67.29% type of health care facility > 8 hr 32.70% safety measurements of the study sample in the current study the results of the safety measures followed by the study sample were as follow and in a descending manner, from the highest percent to the lowest: supervision from directorate of health (doh) , necessary vaccines , periodic follow-up examination to ensure health status, preventive or risk control measures in the place of work, training course about work-place hazards and work safety, and health care protection when facing injury (table 2). occupational hazards facing healthcare providers iraqi j pharm sci, vol.30(2) 2021 45 table 2. safety measurements of the study sample safety measurements percentage highest % lowest % training course about hazards & work safety 38.99% nurses 10.6% pharmacists 4.4% necessary vaccines 72.33% physicians 20.12% pharmacists 4.4% periodic follow-up examination tests to ensure health 61.01% lab instructors 15.7% pharmacists 8% preventive or risk control measures in the place of work 60.38% nurse 16.9% pharmacists 7.54% health care protection when facing injury 18.24% medical assistants 5% pharmacists 2.5% accidental death in the work place 0% supervision from doh 93.08% lab instructors 23.89% physicians 18.23% types of biological and non-biological hazards exposure in the study sample nurses were most susceptible to sharp related injuries, cuts and wounds than the other groups. laboratory instructors represent the most exposed group to contaminated specimens/biohazards and blood borne pathogens, while the physicians represent the most prone group to acquire infectious diseases and both the physicians and the nurses were equally exposed to airborne diseases (table 3). table 3. biological hazards in different healthcare providers health care provider biological gender m/f sharp related injury cut and wound contact with contaminated specimen / biohazard airborne disease infectious disease blood borne pathogen physician 20.12% 19/ 14 11.32% 6.29% 11.95% 11.32% 15.10% 10.10% pharmacist 8.17% 9/4 0.63% 0.63% 1.26% 7.55% 5.66% 0.63% medical assistant 13.21% 9/12 6.92% 5.66% 5.66% 5.66% 5.03% 6.92% lab instructor 23.90% 11/27 12.58% 10.10% 17.61% 4.40% 10.70% 13.84% nurse 18.24% 9/20 13.84% 10.69% 7.55% 11.32% 8.80% 6.29% total 83.64% 57/77 45.29% 33.37% 44.03% 40.25% 45.29% 37.78% regarding the non-biological hazards, the results showed that physicians were the most likely group to suffer from work related stress; and medical assistants were the most susceptible to radiation. meanwhile, laboratory instructors were the most exposed group to physical distress, falls, unsafe staffing, chemical spills and noise. furthermore, nurses were more prone to experience verbal abuse in the workplace (table 4). table 4. non-biological hazards in different healthcare providers. h e a lt h c a r e p r o v id e r n o n b io lo g ic a l g e n d e r m /f s tr e ss p h y si c a l p sy c h o lo g ic a l v e r b a l a b u se f a ll f r a c tu r e u n sa fe s ta ff in g c h e m ic a l s p il ls n o is e r a d ia ti o n physician 18.9% 17/13 13.8% 9.4% 0.0% 9.4% 1.8% 1.3% 9.4% 1.9% 2.3% 1.3% pharmacist 13.2% 14/7 5.0% 2.5% 0.6% 3.8% 3.1% 0.0% 5.0% 2.5% 1.5% 0.0% medical assistant 19.4% 13/17 11.3% 9.4% 0.6% 9.4% 3.1% 0.0% 10% 1.9% 2.3% 3.1% lab instructor 23.3% 11/26 10.0% 15.1% 1.9% 9.4% 5.0% 1.2% 13.8% 8.8% 5.4% 1.9% nurse 18.3% 8/21 11.9% 12.6% 0.0% 15% 1.3% 0.0% 12.5% 1.2% 3.9% 0.6% total 93.1% 63/84 52% 49.0% 3.1% 46.9% 14.3% 2.5% 50.7% 16.3% 15.4% 6.9% occupational hazards facing healthcare providers iraqi j pharm sci, vol.30(2) 2021 46 correlation between vaccination and incidence communicable diseases the results of this study indicated that taking the required vaccines by the healthcare providers can reduce the exposure to blood borne diseases (p= 0.016), but do not significantly reduce the risk of infectious (p= 0.74) and air borne diseases (p= 0.80) (table 5). table 5. correlation between vaccination and communicable diseases. s u b je c t v a c c in a ti o n in fe c ti o u s d is e a se a ir b o rn e d is e a se b lo o d b o rn e d is e a se yes/no yes % yes/no yes % yes/no yes % yes/no yes % physicians 32/1 97 24/9 73 18/15 54 16/17 48 pharmacists 7/19 27 9/17 35 12/15 46 1/25 4 medical assistants 22/10 69 8/24 45 9/23 18 11/21 58 lab instructors 32/6 84 17/21 25 7/31 28 22/16 34 nurses 22/8 73 14/16 47 18/12 60 10/20 33 p value 0.7439 ns 0.7988 ns 0.0156* r2 0.000415 0.000682 0.0367 *: p ≤ 0.05, fair evidence against the h0. ns: p > 0.05, not significant. correlation between training course and biological/non biological hazards the study revealed that there is no significant difference between the healthcare providers who have had training course about occupational safety and those who did not, regarding exposure to biological (p= 0.06) and non-biological hazards (p= 0.85) (table 6). table 6. correlation between training course and exposure to biological/non biological hazards. subject training course biological hazards non-biological hazards yes/no yes % yes/no yes % yes/no yes % physician 14/19 42 32/1 97 30/3 91 pharmacist 7/19 27 13/13 50 21/5 81 med.assis. 12/20 37 21/11 66 31/1 97 lab.inst. 12/26 31 38/0 100 37/1 97 nurse 17/13 57 29/1 97 29/1 97 p value 0.0604 ns 0.8541 ns r2 0.02228 0.000216 ns: p > 0.05, not significant. summary of exposure to biological /non biological hazards table 7 summarizes the exposure to biological and non-biological hazards among the studied subjects, where exposure to sharp materials (45.9%) was the major biological hazard and stress represents the highest rate (52.2%) of non-biological hazards among the studied sample. occupational hazards facing healthcare providers iraqi j pharm sci, vol.30(2) 2021 47 table 7. summary of biological/non-biological hazards within the study sample hazards experienced by health workers frequency (𝑁= 159) yes (%) biological hazards 133 (83.64) sharp related injuries (such as needle sticks) 73 (45.29) cuts and wounds 53 (33.37) direct contact with contaminated 70 (44.03) specimens/biohazardous materials airborne diseases 64 (40.25) infectious diseases and/or infections 72 (45.29) blood borne pathogens 60 (37.78) non-biological hazards 148 (93.1) stress 83 (52) physical 78 (49) psychological 5 (3.1) verbal abuse 76 (46.9) falls 23 (14.3) fractures 4 (2.5) unsafe staffing 81 (50.7) chemical spills 26 (16.3) noise 20 (15.4) radiations 13 (6.9) discussion occupation related health injuries, wounds and sicknesses cause a lot of human suffering and bring about high costs, both for those affected individuals and for the society in general14. there are limited studies that focus on the safe environment in the workplace. research that have concentrated on healthcare employee injury outcomes endorse that bad organizational status and high workloads are associated with increased injuries, including needle stick injuries and near misses to practicing nurses.15 additionally, most of the efforts on hospital and healthcare safety climate to date are patient oriented rather than employee oriented. these studies focused mainly on patient safety-related strategies to improve patient safety rather than employee safety. previous studies investigating medical institution and healthcare safety state exhibit fewer patient-associated injury events and mortality associated with superior work place protection climates 16,12. in the current study, health care providers who have participated in training courses on the safety and hazards exposure were less exposed to biological hazards yet statistically non-significant. a study conducted by sarbaz et al. concluded that training had a remarkable effect and reported substantial decrease in the number of exposure events 17. the present study revealed that the majority of respondents had experienced both types of hazards. lab instructors were the most exposed category and the least were the pharmacists; these findings were highly comparable with those reported in other studies that reported women are the majority of health care providers, exposed to risks of different types of hazards like infection, violence, musculoskeletal injuries 5,16. in the current study, the nurses were the most healthcare personnel who follow the safety control measures, through taking training courses on hazards and work safety while the pharmacists are the least in this respect. meanwhile, taking necessary vaccinations was mostly reported in the physicians and least among pharmacists. regarding the periodic follow up examinations, they are mostly observed among lab instructors and the least among pharmacists. additionally, the presence of preventive or risk control measures in the place of work was mostly accessible by the nurses and least by the pharmacists. healthcare protection when facing injuries are mostly reported by the medical assistants and least by pharmacists. an interesting finding in our study is that healthcare providers who had taken the necessary vaccines related to their occupation were significantly less exposed to blood occupational hazards facing healthcare providers iraqi j pharm sci, vol.30(2) 2021 48 borne diseases with no significant changes in infectious diseases and air borne diseases, compared to those who did not take the suitable vaccinations. nurses were found to be more prone to sharp related injuries and cut/wound in the hospitals compared to other professionals; this can be attributed to the nature of their work and handling of sharp equipment when practicing their job. similar finding was reported by ilhan et al. who claimed that the percentage of nurses facing a sharp or needle stick injury during their professional life was high (18). the present study also showed that lab instructors are more exposed to contact with contaminated specimens/biohazards and air borne diseases, which may be due to the fact that they are dealing with the biological specimens in public and private health sectors during practicing their daily work. unsafe disposal of medical wastes is a major challenge in developing countries as they contributes largely to occupational injuries and infections (19, 20). additionally, in the present study, other hazards such as psychological, chemical spills and fall although are uncommon at healthcare settings (5), but lab instructors are still relatively more prone to such types of hazards 21. lab instructors, nurses, medical assistants, physicians, and pharmacists are exposed to the risks of noise, in descending order. furthermore, medical assistants are most at risk of radiation in the work place due to dealing with radiating machinery at the hospitals. regarding the risks of exposure to infectious disease, physicians are on the top of the list, since they are more prone to acquire infections among other healthcare providers that mainly resulted from their close contact with infected patients in both public and private clinics (22). moreover, physicians are also the most exposed group to stressful conditions compared to others and the study reported two cases of fractures which could be probably due to 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2006;56(5):563-568. doi:10.1111/j.1365 2648.2006.04041.x 19. patwary ma, thomas o’hare w, sarker mh. assessment of occupational and environmental safety associated with medical waste disposal in developing countries: a qualitative approach. 2011. doi:10.1016/j.ssci.2011.04.001 20. nsimba sed, gesase ap, massele ay. dangers of injections overuse in developing countries with a high hiv/aids prevalence: a review on hiv risk hazards, traumatic effects and other blood borne infections. asian pacific j trop dis. 2011;1(2):158-163. doi:10.1016/s2222-1808(11)60057-1 21. board on chemical sciences and technology. prudent practices in lab.; 2011. www.nap.edu. 22. albert nienhaus ck, wendeler d, dulon fh and m. infectious diseases in healthcare workers – an analysis of the standardised data set of a german compensation board. j occup med toxicol. 2012;7:8. https://www.ncbi.nlm.nih.gov/pmc/articles/pm c3474162/pdf/1745-6673-7-8.pdf. 23. tomioka k, morita n, saeki k, okamoto n, kurumatani n. working hours, occupational stress and depression among physicians. occup med (chic ill). 2011;61(3):163-170. doi:10.1093/occmed/kqr004 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://www.nepjol.info/index.php/ http://www.nap.edu/ http://www.ncbi.nlm.nih.gov/pmc/articles/pm http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.22(1) 2013 preparation of bisoprolol fumarate tablet 32 effect of different diluent and binder types on the preparation of bisoprolol fumarate as tablet dosage form heba a. fatohy *, 1 and alaa a. abdul-rasool ** * department of pharmaceutics, college of pharmacy, hawler medical university, iraq. ** chairman of baghdad university, baghdad, iraq. abstract hypertension is one of the main causes of heart disease; betablockers play a crucial role in the management of patients with essential hypertension. bisoprolol is one of the widely used drugs for the treatment of hypertension. bisoprolol tablets were prepared by two methods (direct and wet) using different proportion and types of diluents, different binder types and forms, then evaluated for, weight variation, hardness, friability, disintegration time and dissolution rate. the results were compared with a reference bisoprolol tablet. both methods of preparation wet and direct compression method gave good results, which are consistent with the requirements of british pharmacopeia and united states pharmacopeia. it was found that the type of diluent (mannitol and lactose), binder type (polyvinyl pyrrolidone and acacia) and the presence of starch as a disintegrant affect the hardness, disintegration and the dissolution rate of the tablet. while liquid binder (polyvinyl pyrrolidone solution) gave longer disintegration time (12 min) with higher hardness compared with the powdered binder (polyvinyl pyrrolidone powder) that gave (5 min). the results indicate that formula f1 which consists of [mannitol (65mg)/ avicel 102 (26 mg), starch and magnesium stearate] showed the best results as it has lower disintegration time (2.7 min), faster dissolution rate (100% release within 20 min) and prepared by a low cost method (direct method). key words: bisoprolol, diluent, binder, direct compression, wet granulation. جأثير مخحهف أوواع انممذدات و انعوامم انرابطة عهى جحضير مضغوطات انبيزوبرونول فيوماريث هبة أوطوان فحوحي *،1 عالء عبذ انحسيه عبذ انرسول و ** * . انعشاق ،أستٍم،خايعح هىنٍش انطثٍح، كهٍح انصٍذنح ،فشع انصٍذالٍَاخ ** . انعشاق ،تغذاد،/سئٍس خايعح تغذاد الخالصة ذهعة دوس حاسى فً عالج انًشضى انًصاتٍٍ تاسذفاع ضغظ انذو يعىلاخ انثٍرا ادوٌح.اسذفاع ضغظ انذو هى احذ اسثاب ايشاض انمهة انهذف يٍ هزِ انذساسح هى ذحضٍش حثىب انثٍضوتشونىل .يٍ اكثش االدوٌح انشائعح نعالج ضغظ انذو" ٌعرثشانثٍضوتشونىل واحذا .االساسً , يٍ انهىاصك وذمٍٍى انرشاكٍة يٍ حٍث اخرالف انىصٌ و اشكال يخرهفح تطشٌمرٍٍ يخرهفرٍٍ تاسرعًال اَىاع وَسة يخرهفح يٍ انًخفف انطشٌمح انشطثح )كال طشٌمرً انرحضٍش".خاسٌاولد انرفكك وانزوتاٍَح ويماسَح انُرائح يع حثىب انثٍضوتشونىل انًرىفشج خ, انهشاشح, انصالتح ( انًاٍَرىل و انالكرىص(و لذ وخذ اٌ َىعٍح انًخفف.اعطد َرائح خٍذج و انرً هً يرىافمح يع يرطهثاخ انًصادس انشسًٍح( و انكثس انًثاشش يٍ حٍث ) ي روتاٍَرها كزنك َىعٍح انًفكك كاٌ نها ذاثٍش عهى صالتح، ولد ذفكك انحثح وتانرال( انثىنٍفٍُم تاٌشونٍذوٌ و االكٍشٍا)وانالصك . كاٌ نها ذاثٍش عهى ذفكك انحثح( وخىد انُشا او ال يع صالتح اعهى يٍ ( دلٍمح 12)اعطد ولد ذفكك اطىل( انثىنٍفٍُم تاٌشونٍذوٌ انسائهح)انًحضشج تانًادج انالصمح انسائهح انحثىب (.دلائك 5)فً ( وٌ تاودسانثىنٍفٍُم تاٌشونٍذ)انحثىب انًحضشج تانشواتظ اندافح اعطد احسٍ انُرائح ( انًاٍَرىل وانًاٌكشوكشٌسرالٌٍ سهٍهىص وانُشا وانًغٍُسٍىو سرٍاسٌد)و انًؤنفح يٍ 1انُرائح اوضحد اٌ ذشكٍثح سلى نرً ذعطً الم وكاَد يحضشج تانطشٌمح ا( دلٍمح 20خالل % 100)وسشعح عانٍح فً انزوتاٌ( دلائك 2.7)كاٌ نها ولد لصٍش نهرفككحٍث .انركانٍف . نححضير انحبيبات انطريقة انرطبة, انكبس انمباشر, انهواصق, انمخفف, انبيزوبرونول :انكهمات انمفحاحية introduction oral route is the most common route of drug administration. the most popular way of delivering a drug for oral use are tablets (1) .tablets can be defined as solid preparations each containing a single dose of one or more active ingredients and usually obtained by compressing uniform volume of particles (2) . tablets are convenient for the patients and are usually easy to handle and identify (1,3).tablets are commonly manufactured by one of the following manufacturing processes: 1 corresponding author e-mail:hiba_antoan@yahoo.com received: 20/10/2012 accepted:21/1/2013 iraqi j pharm sci, vol.22(1) 2013 preparation of bisoprolol fumarate tablet 33 direct compression , wet granulation, dry granulation methods (4) . substances included in the manufacturing process other than the active ingredient called excipients e.g: diluents, disintegrants, binders, lubricants, flavoring agents and etc. (5) , they are added to perform different functions, they may be used to enhance stability (antioxidants), modify drug release (disintegrants), provide essential manufacturing technology functions (binders, lubricants), enhance patient acceptance (flavoring agents) (6) . treatment of hypertension both decreases morbidity and prolongs life expectancy. βblockers are one of the drugs used to treat hypertensive patients (7) . bisoprolol is one of cardioselective beta blockers used in the management of hypertension, the usual dose of bisoprolol is 5 to 10 mg as a single daily dose tablet (8) , it is 2propane l1 [ 4 [ [ 2 ( 1 methylethoxy) ethoxy] methyl] phenoxy]-3-[(1-methylethyl) amino] as fumarate (9) . bisoprolol has white crystals, very soluble in water, freely soluble in alcohol, its melting point about 100 0 c, its pka is 9.5 (10) . the goal of this study is to prepare bisoprolol tablet having faster disintegration time, faster dissolution rate and cheapest way of preparation than that of the reference one. materials and methods materials bisoprolol (merk), mannitol (scharlau, spain), avicel 102 (microcrystalline cellulose 102) (awamedica drug industry, iraq), polyvinyl pyrrolidone (sigma, germany), acacia, magnesium stearate (riedal-de-haen ag seelze, germany), single tablet compression machine (erweka), electrical melting point apparatus (uk), sensitive balance (germany), disintegration tester (erweka), hardness tester (pharma), friability tester (pharma), dissolution apparatus (pharma ) and oven (rostfrei). methods bisoprolol tablet was prepared using different formulas by two different methods (direct compression and wet granulation) then evaluating the prepared bisoprolol tablets and compared with the reference bisoprolol tablet (merk) to find the best formula and method to prepare tablet comply with the pharmacopeial properties. preparation of calibration curve of bisoprolol the calibration curve of bisoprolol in distilled water was constructed by preparing series of diluted solutions of the drug from a stock solution (100 µg/ ml). the absorbance was measured at its λmax (225 nm) then plotted against the concentration (11) . preparation of bisoprolol tablet dosage form table (1) shows the compositin of different formulas used to prepare bisoprolol tablets. formulas f1, f2, f5 and f7 were prepared by direct compression through mixing all the ingredients together for 15 minutes except magnesium stearate (lubricant), then the lubricant was added and mixed for 2 minutes, the mixture then was compressed into tablets (12) . table 1: different formulas of bisoprolol tablet iraqi j pharm sci, vol.22(1) 2013 preparation of bisoprolol fumarate tablet 34 on the other hand f3, f4 and f6 formulas, were prepared using wet granulation method by weighing and mixing the ingredients except the lubricant and the binder, then a damp mass was prepared by adding a liquid binder to the powder mixture to facilitate adhesion of the powder particles. the wet mass was screened into granules, after drying the lubricant was added, mixed and the whole formula was compressed into tablets (13) . flowability measurement of prepared bisoprolol formulas granules mixtures by determination of angle of repose the angle of repose (θ) is a relatively simple technique for estimating the flow properties of a powder. it can easily determined by allowing a powder to flow through a funnel and fall freely onto a surface. the height and diameter of the resulting cone are measured and the angle of repose was calculated by the following equation (13) : tan (θ) =h/r where h and r are the height and radius of powder cone respectively. evaluation of physical properties of bisoprolol prepared tablets this was assessed under standard laboratory lighting, each of ten tablets per formula were examined for color, odor, capping and lamination, texture and appearance (14) . weight variation test the united states pharmacopeia (usp) weight variation test for all prepared formulas and the reference bisoprolol tablet was studied by weighing twenty tablets individually, calculating the average weight and comparing each individual tablet weight with the average. the tablets meet the usp requirements if no more than two tablets are out of the percentage limit (10%) and if no tablet differs by more than two times the percentage limit (20%) (3) . hardness test tablet should be sufficiently hard to resist breaking during normal handling and soft enough to disintegrate properly after swallowing (13) . this test was done using hardness tester by which the hardness of three tablets of all prepared formulas and the reference bisoprolol tablet was determined and expressed in kg, then the mean readings was calculated (12) . friability test this test was done for the prepared formulas and the reference bisoprolol tablet, by taking twenty tablets from each formula removing the dust from them by using soft brush. tablet samples were weighed and placed in the friabilator, after the given number of rotations (100 rotations/ 4 min.), dust was removed again, the tablets were weighed and compared with the initial weight, the value is expressed as a percentage, a maximum weight loss of not more than 1% of the weight of the tablets being tested during the friability test is considered generally acceptable. the percentage of friability was determined by using the following equation (15,16) : friability% =(initial weight –final weight) x100 initial weight disintegration test the tablet units were placed in a basket type usp disintegration apparatus. distilled water at 37 0 c ± 0.5 used as a disintegration medium. the time required for complete disintegration of six tablets was recorded (10) . this test was done for all prepared formulas and the reference bisoprolol tablet. effect of diluent the effect of diluent proportion in f1 [mannitol (65)/ avicel 102 (26)] with f2 [mannitol (26)/ avicel (65)] and diluent type in f1 (mannitol as major diluent) with f7 (lactose as major diluent) on the hardness and disintegration of tablet was studied. effect of binder type the effect of binder type polyvinyl pyrrolidone (pvp) and acacia on the hardness and disintegration of the tablet was studied using formulas f3 and f4, respectively. effect of method of preparation by fixing the ingredients used and changing the method of preparation from direct compression using f5 to wet granulation using f6, the effect of method on the hardness and disintegration time of the tablet was studied. effect of type of excipient this factor was studied by utilizing of starch as a disintegrant in f1, f2 (containing starch with same % but with different diluent proportion) and f5 (without starch), using the same method of preparation. dissolution test the dissolution study for the selected formulas (f1, f3and f5) and a reference bisoprolol tablet was performed using a usp paddle ii at75 rpm rotation speed at 37 o c ± 0.5. the dissolution medium was 900 ml distilled water; 5 ml sample was withdrawn at different iraqi j pharm sci, vol.22(1) 2013 preparation of bisoprolol fumarate tablet 35 time intervals and replaced by equal volume of dissolution medium. the samples were filtered and the absorbance of the samples was measured spectrophotometrically at the drug λmax225 nm (17) . stability study within one month the effect of different temperatures on the degradation of bisoprolol tablet was studied by storing samples of bisoprolol tablet f1 under 40, 50 and 60 o c for one month; samples were taken at certain time intervals to determine percent drug remaining versus time. results and discussion the calibration curve of bisoprolol was plotted using the absorbance versus the concentration of serial dilutions of the drug; a straight line was obtained as shown in figure (1). figure 1: calibration curve of bisoprolol in distilled water table (2) shows that all formulas have angle of repose within (25 o -30 o ) which considered to be a good flowability, that means the use of different type of excipients did not make large change in flowability of the formulas. this good flowability for formulas prepared by direct compression method f1, f2, f5 and f7 was due to the presence of avicel 102 that has good flow property and improve the flowability of the whole formula because it is in granule form 3 , while for the formulas that prepared by wet granulation method their good flowability was due to conversion of powder mixture to more flowable granules form during preparation steps by wet granulation method 3 . formulas f3 and f6 gave good flow behavior due to the using of pvp in liquid form as a binder which is consistent with the results found that wet granulation with povidone results in hard granulates with excellent flow properties (18) . table 2: angle of repose of the prepared powder and granule mixtures all formulas showed no change in color and odor and no capping and lamination occur. the effect of different binders was studied using f3 which contains pvp liquid binder and f4 which contains acacia mucilage binder both prepared by wet granulation method, their results showed that the presence of acacia in f4 gives higher disintegration time (4 min) compared with f3 (3.6 min), this result is consistent with acacia which can produce tablets with a prolonged disintegration time19, nevertheless both fulfill the official b.p requirements. the results are shown in figure (2). figure (3) shows the effect of type of method of preparation on the hardness and disintegration time of the tablet for f5 which was prepared by direct compression method and f6 which was prepared by wet granulation method. figure 2: effect of different binder on the hardness and disintegration time of formulas f3 (pvp) and f4 (acacia) bisoprolol tablets. formulas angle of repose (θ) f1 27.56 f2 26.69 f3 28.43 f4 28.26 f5 29.72 f6 28.52 f7 29.61 conc. (µg/ml) iraqi j pharm sci, vol.22(1) 2013 preparation of bisoprolol fumarate tablet 36 figure 3: effect of method of preparation on the hardness and disintegration time of formulas f5 (direct method) and f6 (wet method) of bisoprolol tablets . the results revealed that formula f6 had longer disintegration time (12 min) than formula f5 (5 min), the reason behind the difference in the time of disintegration of them is due to the way of introducing the binder in the formula either as powder or liquid form, the liquid binder have higher binding tendency which gives harder granules compared to powdered binder. this is in agreement with the reported where binders are much more effective when they are added as solutions in the preparation of granulations than when they are added dry to a direct compression formula 3 . this will affect the disintegration time, that’s why f6 took more time for the tablet to disintegrate which in turn is consistent with that, the bond promoting properties of binder may, however, counteract rapid disintegration 20 . however both f5 and f6 are within the required range. the effect of diluent type on the hardness and disintegration time was studied depending on the comparison between formulas f1 (mannitol as major diluent) and f7 (lactose as major diluent), as seen from their results f7 which used lactose is harder (4.8 kg) and has longer disintegration time (4 min) than f1 which contains mannitol (4.4 kg hardness and 2.7 min disintegration time), this result agrees with the reported one about the effect of lactose on the disintegration of tablets when used as a major component as it hinders the development of disintegration force and tends to dissolve rather than disintegrate 21 . these results are shown in figure (4). figure 4: effect of different type of diluent on the hardness and disintegration time of formulas f1 (mannitol) and f7 (lactose) bisoprolol tablets . the effect of diluent proportion on the hardness and disintegration time was studied using f1 and f2 where both formulas prepared by the same method of preparation under same conditions but with different diluent proportion [mannitol (65)/avicel 102 (26)] and [mannitol (26)/ avicel 102 (65)] respectively. as seen from figure (5), the different proportion of the diluent gave around the same results of hardness (4.4 kg and 4.6 kg respectively) and lower disintegration time( 2.7 and 3 min respectively), this is because of using combination of soluble hydrophilic diluent (mannitol) with avicel 102 that has the property of disintegration in addition to the diluent property 3 . figure 5: the effect of diluent proportion on hardness and disintegration time of formulas f1 (mannitol 65 mg/ avicel (102) 26 mg) and f2 (mannitol 26 mg/ avicel (102) 65 mg) bisoprolol tablets. iraqi j pharm sci, vol.22(1) 2013 preparation of bisoprolol fumarate tablet 37 while comparing the results of f5 (without starch) with those of f1 and f2 (containing starch with different diluent proportion) which are all prepared by direct compression method showing that f5 has longer disintegration time (5 min) than f1 (2.7 min) and f2 (3 min) , this result is consistent with both capsules and tablets which are disintegrated rapidly due to the presence of starch disintegrant in the formulations 22 , as shown in figure (6). figure 6: effect of excipient type on disintegration time of formula f1 (starch), f2 (starch) and f5 (without starch) bisoprolol tablets . the weight variation test showed that all values are within the limited ranges allowed by usp, this indicates a good process of preparation and uniform distribution of the powder within the prepared tablets. the hardness and friability tests results of all prepared formulas and the reference bisoprolol tablet were determined and illustrated in table (3); it was found that all are within the acceptable limits (4.4-6.4kg) 13 . on the other hand the friability data give indication on the mechanical resistance to loss fine particles from their surfaces 23 . for all prepared formulas and the reference bisoprolol tablet the friability percentage was (0.3-0.4%) as shown in table (3), being in the acceptable range recommended by official references which should be less than 1%, indicating that the tablet surfaces are strong enough to withstand mechanical shock and attrition during storage and transportation 7 . table 3: the hardness, friability and disintegration results of the prepared bisoprolol tablets in this study all prepared formulas and reference bisoprolol tablet having disintegration time range (2.7-12 min) which fulfills the official requirements (within 15 min) for tablet disintegration 4 . the reason behind the lower disintegration time for formulas f1-f4 and f7 (2.7-4 min) which contain starch as a disintegrant comparing with formulas f5 and f6 (5-12 min) which formulated without starch, was the presence of starch as a disintegrant in them 22 , and they have lower disintegration time (2.7-4 min) compared with the reference bisoprolol tablet (6 min) as seen from table (3). formulas (f1, f3 and f5) having the faster disintegration from all groups used and/or using the cheapest excipients so they were selected to study their dissolution and comparing their results with the reference bisoprolol tablet using usp paddle apparatus at speed of 75 rpm in 900 ml of distilled water at 37 o c. the results are illustrated in figure (7). it appears that f1 has faster and higher % of drug release (100% within 20 min) as compared with f3 (99% within 30 min), f5(99% within 40 min) and the reference bisoprolol tablet(99% within 40 min), this is due to the presence of hydrophilic soluble diluent (mannitol) which undergoes faster dissolution in addition the disintegration time of f1 is lower than the other (2.7 min), as it is generally known that decreasing the disintegration time leads to increase in the dissolution rate 24 because faster disintegration of tablets delivers a fine suspension of drug particles resulting in higher surface area and faster dissolution 25 . as a result the dissolution properties of tablets are affected by the type of excipient. on the other hand, f1 (starch) f2 (starch) f5 (without starch) 2.7 3 5 0 1 2 3 4 5 6 d is in t e g r a t io n ( m in ) formulas no. hardness mean (kg) friability % disintegratio n time (min) f1 4.4 0.32 2.7 f2 4.6 0.37 3.0 f3 5.0 0.35 3.6 f4 5.5 0.4 4.0 f5 5.1 0.4 5.0 f6 6.4 0.3 12.0 f7 4.8 0.35 4.0 the reference 5.5 0.36 6.0 iraqi j pharm sci, vol.22(1) 2013 preparation of bisoprolol fumarate tablet 38 stability study of bisoprolol in the prepared tablet f1 did not undergo any degradation and stay stable with 100% of drug remaining under different temperatures (40, 50 and 60 o c) within one month indicating a stable preparation for f1. figure 7: effect of method of preparation and presence of starch on the cumulative release of bisoprolol fumarate in a comparison with concor as a refrence in distilled water at 37 0 c temperature. conclusion both direct compression and wet granulation method gave tablets with good evaluation results as compared with the reference bisoprolol tablet. the type of the diluent, binder type and the presence of starch affect the hardness, disintegration and the dissolution rate of the tablet. formula f1 can be chosen to prepare bisoprolol as tablet dosage form as it prepared by easier, simplified and economical method of tablet manufacturing with better results of disintegration(2.7 min) and dissolution rate( 100% release within 20 min). references 1. winfield a.j., pharmaceutical practice, 3 rd ed. new york : churchill livingstone, 2004: 230-231. 2. aulton m.e., pharmaceutics the design and manufacture of medicine, 3 rd ed., chuechill livingstone, 2007: 107, 441-482, 661. 3. lachman l. and lieberman h.a., the theory and practice of industrial pharmacy, indian edition, 2009: 293-345. 4. david j., pharmaceutics –dosage form and design, 1 lambeth high street, london se1 7jn, uk, 2008: 203-243. 5. gupta a.k., introduction to pharmaceuticsi, 3 rd ed., india, 1994: 239-274. 6. baldrick p., pharmaceutical excipient testing: regulatory and preclinical perspective. in:james s. (editor). encyclopedia of pharmaceutical technology. 3 rd ed. usa:informa health care, 2007 inc.: 2771-2782. 7. lipincott w.and wilkins., remington the science and practice of pharmacy, 21 st ed., london, 2005, vol.3: 1350-1370, 10251036. 8. sweetman s.c., martindale, the complete drug reference, 34 ed., london, 2005 : 8091029. 9. madhusudhanareddy i., bhagavan r. m. and rajendra p. y.,validated and stability indicating liquid chromatography method for quantification of bisoprolol fumarate tablet dosage form, international journal of pharmacy, 2012, vol.2(1): 64-70. 10. british pharmacopeia (bp) (2007).electronic edition, crown inc.london. 11. shirkhedkar a.a., throve r. r. and surana s. j., simultaneous spectrophotometric estimation of bisoprolol fumarate and hydrochlorothiazide in tablet dosage form, pak. j. pharm. sci, 2008, vol. 21(4): 366-69. 12. shahla s.s., preparation and evaluation of lamotrigine water dispersible tablets. msc. thesis. hawler medical university, college of pharmacy. iraq, 2011: 41, 45, 46, 89, 57. 13. ansel h.c., allen l.v. and popovich n.g., ansel's pharmaceutical dosage forms and drug delivery systems, 8th ed.,philadelphia, 2005: 97,227-259. 14. reveng a. a., formulation and evaluation of bisoprolol fumarate as an orodispersible tablet. msc.thesis. hawler medical university, college of pharmacy. iraq, 2012: 51. 15. naz j.i., design and evaluation of sustained release bilayer tablets of oxcarbazepine. msc.thesis. hawler medical university, college of pharmacy. iraq, 2011: 34, 39, 41, 42. 16. bushra r., shoaib m.h., aslam n., hashmat d.and rehman m.u., formulation development and optimization of ibuprofen tablets by direct compression method, journal of pharmaceutical science, april 2008, vol.21, no. 2: 113-120. 17. usp 29-nf24 (2006), united states pharmacopeia online. 18. hubertus f. and anisul q., polyvinylpyrrolidone (pvp) – one of the most widely used excipients in iraqi j pharm sci, vol.22(1) 2013 preparation of bisoprolol fumarate tablet 39 pharmaceuticals, drug delivery technology , june 2008, vol 8 no 6: 22-27. 19. raymond c.r., paul j. s. and sian c. o., handbook of pharmaceutical excipients, 5 th ed., london u.k., 2006: 1. 20. mattsson s., bredenberg s. and nyström c., formulation of high tensile strength rapidly disintegrating tablets.evaluation of the effect of some binder properties, s.t.p. pharma. sci., 2001, 11 (3): 211-220. 21. ferrari f., bertoni m., bonferoni m.c., rossi s., gazzaniga a., conte u. and caramella c., influence of porosity and formula solubility on disintegrant efficiency in tablets, s.t.p. pharma sci., 1995, 5 (2): 116-121. 22. michael u., uhumwangho and rolannd s.o., a comparative study of the dissolution characteristics of capsule and tablet dosage forms of melt granulations of paracetamol diluent effects, acta poloniae pharmaceutica drug research, 2007, vol. 64 no. 1: 7379. 23. puttewar t.y., kshirsagar m.d., chandewar a.v.and chikhale r.v., formulation and evaluation of orodispersible tablet of taste masked doxylamine succinate using ion exchange resin., jornal of king saud university (science), 2010, 22: 229-40. 24. takeuchi h., tanimura s., nagira s., yamamoto h. and kawashima y., tabletting of solid dispersion particles consisting of indomethacin and porous silica particles, chem. pharm. bull., 2005, 53(5): 487-491. 25. mallikarjuna s. c., prasad d. v. and gupta v. r., development of fast dispersible aceclofenac tablets: effect of functionality of superdisintegrants, ind. j. pharm. sci., 2008, 70(2): 180-185. iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica doi : https://doi.org/10.31351/vol30iss1pp41-55 41 a review on active constituents and pharmacological effects of eriobotrya japonica lindl. (loquat) ruaa m. ibrahim*,1 *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq. abstract eriobotrya japonica lindl., named as loquat, is a subtropical fruit tree of the family rosaceae which is well known medical plant originated in japan and china. loquat portions, like leaves, peels and fruits have been shown to possess various health usefulnesses. in chinese classical medicine, it is vastly utilized in many illnesses, like gastroenteric disorders, diabetes mellitus, pulmonary inflammatory diseases and chronic bronchitis. loquat plant contains many active constituents, such as carotenoids, vitamins, polyphenolic compounds, others that have many biological effects like anti-tumor, anti-diabetic, anti-inflammatory, anti-mutagenic, antioxidant, antiviral, antitussive, hepatoprotective and hypolipidemic activity. keywords: loquat, polyphenolic compounds, pharmacological effects of eriobotrya japonica . (دنيا يلينك) lindl eriobotrya japonica .الدوائية ل والتأثيرات الفعالة للمكونات مراجعة 1*، مابراهيرؤى محمد .العراق بغداد، بغداد، جامعه الصيدلة، كلية ،والنباتات الطبية العقاقير فرع* الخالصة eriobotrya japonica lindl. ، معروف طبي نبات الوردية وهي العائلة من استوائية شبه فاكهة شجرة هي ، لينكي دنيا بـ المسمى ، الكالسيكي الصيني الطب في. الصحية الفوائد من العديد تمتلك ، والفاكهة والقشور األوراق مثل ، إسكدنيا أجزاء أن ثبت. والصين اليابان في نشأ والتهاب الرئوي االلتهاب وأمراض ، السكري وداء ، الهضمي الجهاز اضطرابات مثل ، األمراض من العديد في واسع نطاق على استخدامه يتم وغيرها ، البوليفينول ومركبات ، والفيتامينات ، الكاروتينات مثل ، النشطة المكونات من العديد على اسكدنيا نبات يحتوي. المزمن الهوائية الشعب ، للطفرات ومضادة ، االلتهابات ومضادات ، السكري ومضادات ، األورام مضادات مثل ، البيولوجية التأثيرات من العديد لها التي المركبات من .الدم شحميات نقص نشاط ، الكبد ووقاية ، والسعال ، للفيروسات ومضادة ، األكسدة ومضادات أثيرات الدوائية للينكي دنيا، الت البوليفينول مركباتلينكي دنيا ، الكلمات المفتاحية : introduction the genus eriobotrya, distributed in subtropical and tropical southern and eastern asia, comprises about 26 species (1). eriobotrya japonica (thunb.) lindl. is a green fruit tree of medium sized belong to family rosaceae. it is usually named as loquat. it has been implanted for more than two thousands of years, and is native to japan and china but recently implanted commercially worldwide for over 30 countries, such as japan, iraq, turkey, spain, italy, syria and other. the local english name of the plant (loquat); arabic (beshmelah); german (loquate); french (bibassier); chinese (luju,biba), etc.it attains up to 6 meters or more height, with thick and evergreen oval-oblong leaves. fruit yellow to orange, pearshaped, with seeds 3 to 4, long 4 centimeters, and sweet test as in figure (1) (2). among other fruits, unusually loquat flower in early winter or autumn, and its fruit ripen in early spring or late winter (3). currently loquat has been utilized in the jam, chutney and jelly preparation, in addition to it is eaten as a fresh fruit (4). loquat is implanted mostly to fruit production and in the chinese classical medicine; also its leaf has been utilized (5). figure 1. photo of eriobotrya japonica plant taxonomical classification (6): kingdom = plantae subkingdom = viridaeplantae division = tracheophyta subdivision = spermatophytina class = magnoliopsida order = rosales family = rosaceae genus = eriobotrya species = eriobotrya japonica (thunb.) 1corresponding author e-mail: mnrsyrg@yahoo.com received: 3/9/2020 accepted: 28/11 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp41-55 iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 42 traditional uses eriobotrya japonica is known widely medicinal plant in china and japan (1) and for 1000 of years, used in folk medicines (2). the fruits are used as sedative and helpful in alleviated thirst and vomiting and used for wound treatment in china (7). the flowers considered an expectorant (2), and they are a good source of honey. traditionally, loquat fruits and leaves are used as expectorant and for treating coughs (8). eriobotrya japonica leaves were utilized in the therapy of stomach and lung illness, diabetes mellitus, skin diseases and inflammatory problem (9) and also efficient in dysmenorrhea, phlegm, asthma, headache, chronic bronchitis, and lower back pain (10). also leaves of loquat have been considered digestive, antipyretic, and diuretic agents in japan (4) and used to alleviated nausea time for treating diseases in the, ethno medicine (11). the leaf decoction was locally put on wounds, cancers and ulcers (1). loquat tea is formed from leaves that are roasted for 30 minutes at 350°c, and according to the folk customs, it is commonly used as beverage (12) . chemical constituents: broad range of important phytochemicals had been detected in loquat, like phenols, alkaloids, cardiac glycosides, flavonoids, mucilage, gums and phytosterol (8). many active compounds present in leaves and fruits that are responsible for various biological activity of loquat plant (13). fruit enrich with active compounds such as vitamins, flavonoids, phenols, carotenoid and others (14). caffeic acid, 4-o-caffeoylquinic acid, neochlorogenic acid, chlorogenic acid, together with 4-hydroxybenzoic acid, protocatechuic acid, ocoumaric acid, ellagic acid and ferulic acid have been identified in mature fruits of loquat as major phenolic compounds (15). the kernel of loquat is rich with tannins, starch, minerals and proteins (16,17). phytochemical study reported that the e. japonica flowers are good sources of flavonoids and phenolic compounds (18). loquat leaf contain phenolic acids like p-coumaric, gallic, caffeic, and ellagic acid and flavonoids like quercetin, epicatechin and catechin (19), in addition to tannins, sesquiterpenes, triterpenes and megastigmane glycosides (18,20). oleanolic and ursolic acids (triterpenoid compounds) have been found in flowers and leaves of loquat with different contents based on the type and developmental stage of cultivar (21). in addition to ursolic and oleanolic acids, e. japonica leaves contain maslinic acid, tormentic acid, and corosolic acid (22). tormentic acid possess normoglycemic, antiatherogenic, hypoglycemic, anticancer, anti-inflammatory properties; and it decreases the expansion of vascular smooth muscle cell (23). four flavonoids, kaempferol 3-o-β-glucoside, quercetin, quercetin 3-o-α-rhamnoside, naringenin and three triterpene acids, ursolic, corosolic and oleanolic acids were the bioactive components of stems of e. japonica (18). catechin, β-sitosterol, oleanolic acid, cinchonain iib, β-sitosterol-3-o-β-d-glucopyranoside, lyoniresinol and lyoniresinol 2-a-o-β-dxylopyranoside are identified and separated from loquat stem barks (1). by using high performance liquid chromatography, flavones like quercetin derivatives (likely rutin or similar quercetin glycoside) and hydroxycinnamic acid derivatives like chlorogenic acid with other pcoumaric acid and caffeic acid derivatives, have been identified in leaves, fruit, flower of loquat but chlorogenic acid has been the main phenolic compound. flowers possess higher content of quercetin and chlorogenic acid derivatives than new and old leaves. the new leaves possess higher pcoumaric and caffeic acid derivatives contents than flowers and old leaves (5). pharmacological activities of eriobotrya japonica cytotoxic activity bushra alwash (2017) reported that the fruit juice of loquat shown a significant anticancer effect on two cell lines of cancer, rhabdomyo sarcoma (rd) and human cervical cancer (hela) cell. but the cytotoxic effect of fruit juice was higher on hela than on rd cell line while on rat embryogenic fibroblast (as a normal cell), the effect of juice was lower than on rd and hela cancer cell lines (2). loquat juice contained active constituents such as terpenoids, phenols, tannins, and flavonoids that have ability to effect on cells. studying the anticancer effect of loquat juice on cell lines of cancer demonstrated the highest cell lines sensitivity by stimulation of some glutathione-s transferase enzymes (gsts) by many compounds, mainly the polyphenolic compounds in plant extract (24). the gsts considered as an antioxidant and by inducing their combination with reduced glutathione causing cellular detoxification and leading to the cells of cancer toward death and apoptosis (24). so the utilization of e. japonica fruit juice to protective the body of human against cancer and free radicals better than use of the extraction methods, in which some effective compounds will be lose during the process of extraction and the poisonous of solvent will be get rid (2). the ethanol and aqueous leaf extracts of e. japonica suppressed 7, 12dimethylbenz[α]anthracene -stimulated breast https://www.researchgate.net/profile/bushra_alwash2 iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 43 cancer and inhibited the cancer development of breast by inhibiting the cancer cells proliferation and initiation in rats. but water extracts displayed a higher anticancer activity (25). four triterpenic acids, ursolic , betulinic, δoleanolic, and 3-o-(e)-p-coumaroyl tormentic acid (fig.2), separated from methanol loquat leaf extract exerted cytotoxic effect on human hl60 cells and also shown potent inhibition of dna topoisomerase i. in hl60 line, 3-o-(e)-p-coumaroyl tormentic acid stimulated apoptotic death of cell mainly through the mitochondrial pathway, considered as key substance for human leukemia therapy (26). figure 2. chemical structures of four triterpenic acids isolated from loquat (26) in meth-a-fibrosarcoma-bearing mice, aqueous extracts of loquat (by hot method) also exhibited anticancer effect through immunomodulatory effect, as specified by factors like interleukin-17, interferon-gamma, and transforming growth factor beta 1 (9). the ethyl acetate fraction of leaf (27) and the methanol extracts of seed and leaf of loquat (by maceration, 48h) (28) displayed anti-metastatic effects by suppressing the invasion and migration of b16f10 melanoma cells and mda-mb-231 human cancer cells of breast that were partly by suppressed the matrix metalloproteinase-9 (mmp-9) and also mmp-2 (27, 28). 2α-hydroxyursolic acid and ursolic acid separated from the ethyl acetate e. japonica extracts significantly prevent mmp-9 and mmp-2 effects and considered as key active compounds (27). the polyphenols such as proanthocyanidins, flavonoids and their glycosides have anticancer effect on human oral tumor cells (10). anti-diabetic activity the diabetes mellitus known as a metabolic illness with increasing in the blood glucose levels and also characterized by raised the rate of basal metabolism, abnormalities of lipoprotein, high oxidative stress caused injury to beta cells pancreas and trouble in reactive oxygen species (ros) scavenging enzymes (11,29). shafi et al. reported (2019) that in streptozotocin induced diabetic rat, the ethanol loquat fruit extract (by maceration, 48h) shown good anti-diabetic effect. (11) the anti-diabetic effect may be related to insulin sensitivity enhancement that will raise tissue glucose uptake or related to induce insulin secretion from the pancreatic beta cells (30). the flavonoids in the ethanol extract report to have antidiabetic effect (11). combination of summer-harvested leaf of green tea and leaf of loquat produced new fermented tea product. in maltose-loaded sprague–dawley rats, this product exhibited reduction of blood levels of glucose and a corresponding decreasing in serum secretion of insulin (31). aqueous leaf extracts of loquat led to attenuate the elevation in serum glucose, triglyceride and total cholesterol levels caused by feeding the diet of high cholesterol content in a hypercholesterolemic zebrafish model (32). the loquat leaf extracts were shown hypoglycemic effects by utilizing the model of high-fat dietstimulated diabetic c57bl/6j mice (33,34). iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 44 ethanol loquat cell suspension extract (by hot method, 8h) include many pentacyclic terpenoids like corosolic acid, maslinic acid, also ursolic acid, and oleanolic acid. in the high-fat diet mice, administration of such cell suspension cultures of loquat produced the inhibition of the elevation of the blood glucose levels, insulin and leptin levels (34). in 45% high-fat diet c57bl/6j mice, ethanol leaf extract of loquat containing maslinic acid and corosolic acid (fig.3) significantly improved the hyperinsulinemia, hyperglycemia, and hyperleptinemia (33). figure 3. chemical structures of maslinic acid and corosolic acid (26) glucocorticoids are critical metabolic processes regulators involving in gluconeogenesis and increased glucocorticoids level has been accompanied with type2 diabetes, hyperglycemia and resistance of insulin. 11β-hydroxysteroid dehydrogenase 1 (11β-hsd1) stimulates the transformation of 11ketoglucocorticoids (inactive) into 11βhydroxyglucocorticoids (active). therefore, inhibition of such glucocorticoids-activating enzyme is a good curative base for antidiabetic drugs (35,36). the leaf of loquat preferentially suppressed 11βhsd1 among 6 traditional hypoglycemic medicinal plants (35). the 2-α hydroxy-3-oxo urs-12-en-28-oic acid, 3-epicorosolic acid methyl ester, tormentic acid methyl ester (fig.4), corosolic acid (fig.3), and ursolic acid (fig.2) are identified as low micromolar 11β-hsd1 inhibitors (36). figure 4. chemical structures of 3-epicorosolic acid methyl ester (a), tormentic acid methyl ester (b), and 2-α hydroxy-3-oxo urs-12-en-28-oic acid(c) (36) iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 45 as for hyperglycemia and its problem, the study revealed the certain plant phenolic compounds that are able to inhibit the digestive enzymes such as αglucosidase and α-amylase contributory in the carbohydrates breakdown related to the ability of these enzyme to bind to the protein active site (37). ahumada et al. (2017) reported that both methanol flowers and leaves extracts (by cold method) of e. japonica shown inhibitory impact on both enzymes, suggesting a potential hypoglycemia activity, but flowers extract showed the strongest antihyperglycemia effect because the levels of flavonoids such as quercetin derivatives and levels of chlorogenic acid (fig.5) are higher in flowers than leaves (5). figure 5. chemical structure of chlorogenic acid (39) so the inhibitory effect of these enzymes were directly related to the contents of quercetin derivative and chlorogenic acid and with the antioxidant effect indicated that flowers and leaves have hypoglycemia effect in vitro. loquat fruits extract not shown inhibitory impact on α-glucosidase or α-amylase due to the derivatives of quercetin and chlorogenic acid that detected only in flowers and leaves (5). toshima et al. (2010) reported that fermented tea product shown good inhibitory activity against the α-glucosidase enzyme than that formed from fermented tea leaves only (38). oboh et al. (2015) exhibited that quercetin derivatives and chlorogenic acid play a significant role on the suppression of α-glucosidase and αamylase enzymes (39). anti-hyperlipidemic activity shafi et al. (2019) reported that in streptozotocin stimulated diabetic rat, the ethanol fruit extract of eriobotrya japonica (by maceration, 48h) showed the significant hypolipidemic activity (11). in high-fat diet mice, the cell suspension culture of pentacyclic terpenoids ethanol extracts of loquat exhibited hypolipidemic activity because it decreased white adipose tissue (wat) weights (include visceral fat, mesenteric, perirenal and epididymal wat), body weight gain, content of hepatic triacylglycerol and adipocytes size in the visceral depots (33,34). fermented tea product (leaves mixture of both green tea (camellia sinensis) and loquat) displayed antiobesity and hypotriacylglycerolemic effects by inhibit the synthesis of hepatic fatty acid and postprandial hypertriacylglycerolemia through pancreatic lipase inhibition (40). the aqueous extract of leaf of loquat exhibited anti-atherosclerotic effect in a hypercholesterolemic zebrafish model and in cellular assays (32). anti-inflammatory activity the inflammation is a first system of physiological defense and induced macrophages to generate prostaglandins (pge2) (41), cytokine, nitric oxide (no) and pro-inflammatory enzymes like secreted phospholipase a2 (42) that stimulate the membrane phospholipids hydrolysis to produce lysophospholipids and free arachidonic acid. the secreted phospholipase a2 group iia has been firstly found in patients with rheumatoid arthritis in synovial fluid (43) and play a role in inflammatory process (44). many inhibitors of phospholipases a2 have been found out and confirmed as a therapy of inflammatory disorders (45). maher et al. study (2015) reported the ethyl acetate -ethanol (1/2) extract and the dichloromethanemethanol (0:1) fraction of loquat suppressed the pig secreted phospholipase a2 group ib (pg-ib) and the human secreted phospholipase a2 group iia (hg-iia) but ch2cl2/meoh 0:1 extract was the better one to suppressed especially hg-iia related to the existence of flavonoid and phenolic compound (46). the common experimental model for antiinflammatory research is lipopolysaccharide (lps)stimulated inflammation. the n-butanol fraction of leaves of loquat were demonstrated antiinflammatory property by inhibited the expression of nitric oxide synthase and production of no by attenuating the activation of nuclear factor-κb (nfκb) and also down-regulated cyclooxygenase-2 expression and pro-inflammatory cytokines secretion such as interleukin-6 and tumor necrosis factor-α (tnf-α) in the lps-activated murine peritoneal macrophage model (47). seong et al (2019) stated that in lipopolysaccharideinduced raw 264.7 macrophage cells, the ethanol loquat leaf extract (by reflux, 4h) has demonstrated anti-inflammatory effect by suppressing tnf-α production and no expression (48). zar et al. (2013) reported that in lipopolysaccharideinduced raw 264.7 cells, loquat tea water extract of leaves (by boiling for15minutes at100°c) exerted anti-inflammatory effect via inhibiting pge2 and cox-2 production that stimulated by lipopolysaccharide by using elisa and western blot assay, respectively. the new bioactive phenolic compounds in loquat tea may be responsible for its anti-inflammatory potential (12). iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 46 in mice, the triterpenes of methanol extract of loquat leaves (by reflux, 3h) inhibited 12-otetradecanocylphorbol-13-acetate-stimulated inflammation (49). the total triterpenes decreased the inflammatory cytokines production by suppressing nf-κb stimulation from alveolar macrophages in chronic bronchitis rat model (50). the another model that is utilized to estimate the anti-inflammatory property of eriobotrya japonica extract is a mouse paws edema model (51,52) in the mouse macrophage-like raw 264.7 cells, loquat tea water extract (by boiling for15 minutes at100°c) shown inhibitory effects on the expression of tnf-α, interleukin-6, nitric synthase, and no through the downregulation of pathways of the tgf-β-activated kinase-mediated nf-κb and mapk (51). the other mechanism of anti-inflammatory properties of loquat might be antioxidant activity. tormentic acid (fig.6) of ethanol cell suspension extract of loquat may increase the activities of glutathione peroxidase, superoxide dismutase and catalase in the tissue of liver and decreased paw edema of mice (52) figure 6. chemical structure of tormentic acid (36) in rat ear, by using a model of dinitrofluorobenzenestimulated allergic dermatitis, ethanol seed extracts (by stirring with mixer, 1week) of eriobotrya japonica also displayed in vivo anti-inflammatory activity and inhibited allergic dermatitis development, where improved th1/th2 balance and lower serum levels of immunoglobulin e and thickness of ear were shown (53). renal activity diabetes mellitus was known as metabolic disease differentiated by hyperglycemia lead up to several complications, such as retinopathy, neuropathy, angiopathy, nephropathy and others (29). shafi et al. (2018) reported that in alloxanstimulated diabetic rats, ethanolic extract (50%) of seeds and fruits of eriobotrya japonica (by maceration, 48h) displayed renal effect and this effect was evaluated by estimating the tests of kidney function like levels of serum total proteins, creatinine and urea. the result reported that nonsignificant elevated in serum level of total protein and reduced in levels of serum creatinine and urea. the ethanolic extract of loquat fruits has proper effects on levels of serum glucose (54). antitussive and expectorant activity wu et al. (2018) reported that the ethanol and aqueous extracts of loquat fallen and growing leaves (by hot method, 2h) shown expectorant and antitussive effects. but the ethanol extracts of fallen leaves shown better antitussive effect (relieve cough) that may be related to the higher triterpenoids content of it, like tormentic acid (fig.6), corosolic acid, maslinic acid (fig.3), ursolic acid (fig.2) and other, while aqueous extracts of growing leaves had higher expectorant effect (reduce airway mucus secretion), which may be due to their higher flavonoid content like quercitrin, isoquercitrin, hyperoside, rutin and others (fig.7) (55). iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 47 figure 7. chemical structures of some flavonoids present in loquat(58) the major reason of phlegm and coughing, airway mucus hypersecretion, affects the function of lung and closely accompanied with the development and occurrence of chronic inflammation of airway (56). in the aqueous leaves extract of loquat, some of the identified flavonoids show anti-inflammatory effect via nf-κb and signal transducer and activator of transcription 1 inhibition (57). nf-κb plays an important role in proinflammatory cytokines production. in severe cough cases, inflammation is playing an essential role. in eosinophilic bronchitis, an inflammation of lower airway can alleviate sensitivity and severity of cough (59). the anti-inflammatory activity of loquat triterpenoids might be due to suppressed signal transduction of mapk (52, 60). anti-melanogenic activity seong et al (2019) stated that ethanol loquat leaf extract (by reflux, 4h) show antimelanogenic activity through its anti-inflammatory and anti-oxidant activities. ethanol extract can protect the skin of human from inflammation and also from oxidative stress, related to the higher content of quercetin (fig.8) and polyphenol that exhibit the inhibition of melanin synthesis (48 ). figure 8. chemical structure of quercetin (18) ethanol extract can regulate production of melanin since melanin plays an important role versus production of uv-stimulated ros; ethanol extract has shown potent anti-inflammatory and anti-oxidant activities (48,61). new studies have found that by different inflammatory skin disorders (like dermatitis, eczema), the hyperpigmentation is shown (62, 63). in b16 melanoma cells, the methanol leaf extract of eriobotrya japonica exerted dose-depending melanogenesis suppression (64). in addition, 70% and 30% ethanol loquat leaf extracts (by several processes of extraction) inhibited mushroom tyrosinase for whitening effects (65). effect on eye and skin seong et al (2019) stated that ethanol loquat leaf extract (by reflux, 4h) did not exert any irritation or induce toxicity in eye and skin by using animal alternative test like het-cam, bcop assay ( irritation test of eye ) and rhe model (irritation test of skin) (48). the aim of het-cam test is to recognize agents which induce irritation of conjunctiva and the bcop test is to recognize substances which induce damage of cornea (66). therefore, ethanol extract can be used for skin improvement as a cosmetic ingredient. hepatoprotective activity shahat et al (2018) revealed that the methanol extract (80%) of leaves of loquat and its butanol, aqueous, and ethyl acetate fractions were displayed hepatoprotective activity in rats with carbon tetrachloride (ccl4)-induced hepatotoxicity (67). the methanol extract or its fractions administration of this plant were significantly reduce biochemical parameters levels in rats like aspartate transaminase (ast), gamma-glutamyl transferase, alanine aminotransferase (alt), bilirubin and alkaline phosphate levels but did not influence lipid profiles (67). the elevation of these enzyme levels specified the loss function of cell membrane and cellular injury (68). the methanol extract and its fractions also significantly suppressed the ccl4-stimulated increasing level of malondialdehyde (mda) which was proved by decreasing in histopathological injuries (67). mda, an important ccl4-induced toxicity indicator in rats, is a final product of peroxidation of lipid in liver (69). treatment with ccl4 also increased levels of serum triglyceride, low density lipoprotein, cholesterol and also decreased levels of high density lipoprotein that is inverted the weakness of the ability of the liver cells to convert cholesterol to bile acid for excretion and to metabolize lipids. the fractions and extract of loquat suppressed these effects (67). on the endoplasmic reticulum, ccl4 induces disassociation and disruption of polyribosomes that leads to reduce biosynthesis of protein (70). administration of butanol and ethyl acetate fractions significantly suppressed ccl4-stimulated the depletion of total protein and the reduction levels of nonprotein sulfhydryl groups (np-sh) , but aqueous fraction and methanol extract administration did not. butanol and ethylacetate fractions are more effective in the protection from liver damage than aqueous fraction and methanol extract (67). the hydroalcoholic flower extracts of e. japonica (by shaking) shown hepatoprotective activity in mercuric chloride treated rats (71). by hplc, phytochemical analysis of hydroalcoholic flower extracts of loquat has indicated that polyphenols like gallic acid (fig.9) iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 48 and hesperetin (fig.10) as main antioxidants are present (71). figure 9. chemical structure of gallic acid (58) figure 10.chemical structure of hesperetin long-term alcohol consumption may cause alcohol related hepatic disease development. the ethanol stimulated cytochrome p-450 2e1and cause oxidative stress. the ethanol extracts of loquat leaf (by hot method, 3h) displayed hepatoprotection by reducing the intracellular production of ros and improving the antioxidant effect in hepg2 cells overproduction cytochrome p-450 2e1. as a result, in manner of concentration-dependent, leaf extract elevated the viability of hepg2 cell and exhibited protective effect in hepg2 cells against ethanolstimulated toxicity (72). in rats with non-alcoholic steatohepatitis, ethanol seed extracts of this plant (by stirring with mixer, 7 days) displayed protective effect. the ethanol seed extract (70%) strongly suppressed the elevation in the levels of ast, alt and fatty droplets forming in the rats liver. the inhibition of fibrosis and fatty liver may result from increasing activity of antioxidant enzyme which may relieve oxidative stresses (73). antimicrobial activity rashed et al. (2014) reported that methanol extract (80%) of stems of loquat (by maceration) was demonstrated antimicrobial effect versus strains of fungi and bacteria related to the presence of triterpenes and flavonoids. it was more effective against candida albicans indicated that in the treatment of fungal infections, it can be utilized and has no impact on the other strains of fungi and bacteria (18). methanol extract of loquat stems due to the presence of flavonoids (74), tannins (75) and also kaempferol 3o-β-glucoside (76) (fig.11) which exhibited a good antioxidant and antimicrobial effects. figure 11. chemical structure of kaempferol 3o-β-glucoside (18) antiosteoporosis activity methanol leaf extract of loquat shows antiosteoporosis effect in the model of ovariectomized mice (77). ursolic acid (fig.2) that isolated from loquat leaves is displayed inhibitory effect on osteoclast differentiation that means inhibited osteoclastogenesis. the elimination of the multinucleated osteoclasts which contributing to resorption of bone is one of the traditional strategy of osteoporosis treatment. ursolic acid (fig.2) suppresses the differentiation of osteoclast via targeting exportin 5 (xpo5) as nuclear exporter protein (78). antifibrosis activity triterpenic acids of ethanol extract of leaves of loquat (by cold method, 2h) displayed antifibrosis activity via alleviating fibrosis and improving the structure of lung in a rat model of bleomycin-induced pulmonary fibrosis. in the macrophage of alveolar of pulmonary fibrosis, the production of tgf-β1and tnf-α both at the level of mrna and level of protein were decreased in rats. the triterpene acids such as α-hydroxyoleanolic, euscaphic, arjunic, oleanolic (fig.12), and ursolic acids (fig.2) are present in such loquat extracts (79). iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 49 figure 12.chemical structures of other triterpene acids present in loquat (26,79) antioxidant activity bushra alwash (2017) reported that e. japonica fruit juice exhibited antioxidant property by using dpph assay (2). the juice plant has ability to scavenge dpph free radicals by donating their h atom (80,81) . the phenolic compounds especially flavonoids which present in e. japonica fruit juice have ability to achieve this reaction (13) and effective as antioxidant rely on position and number of oh groups on the basic flavonoids structure; an antioxidant activity is directly correlated with increasing of hydroxyl groups number (1). delfanian et al study's (2015) reported the ethanol extract of loquat pulp and ethanol-water extract of loquat fruit skin (by shaker, 48h at room temperature) shows the higher antioxidant effect in the rancimat method the β-carotene bleaching and dpph assay. in stabilization of soybean oil, protective effects of extracts were checking and compared to synthetic antioxidant such as tert-butyl hydroquinone. the treatment of soybean oils with skin extract revealed the strong antioxidant property than the extract of pulp, but soybean oil containing tert-butyl hydroquinone shows the best protection effect. in general, in soybean oil, extracts of fruit pulp and peel displayed good antioxidant effect, so to raise the oil shelf life; these can be used for synthetic antioxidants as a substitute (3). few studies reported that this effect of its fruits was related to the existence of cyanidine glycoside, derivatives of benzoic and hydroxycinnamic acids (82). ahumada et al (2017) revealed that methanol loquat flower and leaf extracts (by cold method) had higher antioxidant effect and also higher total phenolic contents than extract of fruit by using 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid , oxygen radical absorbance capacity (orac) and dpph assay. but the extracts of flower displayed the good antioxidant effect than that of leaves, indicating that the strong antioxidant effect of flowers is directly correlated to their high quercetin derivatives and chlorogenic acid contents. in addition, higher caffeic acid derivatives contents were likely related to higher values of orac. loquat fruits have lower antioxidant and total phenolic contents than leaves and flowers (5). rajalakshmi et al (2017) stated that methanol fruit and seed extract of loquat (by stirring in shaker, 38h at room temperature) exhibited antioxidant activity by using scavenging action against superoxide free radical. but the methanol extract of e. japonica seed shown the highest antiradical activity than the extract of fruit (8). https://www.researchgate.net/profile/bushra_alwash2 iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 50 maher et al. (2015) reported that the ethyl acetate ethanol (1/2) extract of leaves of loquat displayed an antioxidant effect by using scavenging action against dpph free radical. but the dichloromethane methanol (0:1) fraction exhibited a high antioxidant effect and the rich one on phenolic compounds and flavonoids (46). there is a relation between antioxidant activity and the content of flavonoid and phenolic of loquat extracts that mean these compounds might be contribute for its antioxidant effect (83). nawrot-hadzika et al. (2017) reported that the ethyl acetate leaves fraction of loquat (crude extract get by ultrasonic bath at 30°c) shown significant antioxidant effect by phosphomolybdenum, linoleic acid tests and dpph that mean such extract shown the strongest capability to reduce metal ions, to scavenge dpph, and to prevent the linoleic acid oxidation. to localize antioxidants, hplc-based activity profiling is applying and reported that flavonoid glycosides like isoquercitrin, hyperoside, quercetinrhamnoside (fig.7), kaempferol glycosides, cinchonain iib (fig.13), and two temporarily specified derivatives of protocatechuic acid (fig.14), considered as a main compound in charge for good antioxidant effect (20). figure 13.chemical structure of cinchonain iib (1) figure 14. chemical structure of protocatechuic acid (58) zar et al. (2013) reported that loquat tea (made from roasted leaves of loquat) shows higher antioxidant effect compared to its fresh leaves by inhibiting cellular ros and scavenging dpph. in lipopolysaccharide (lps)-activated raw 264.7 cells, loquat tea extract suppressed ros production. new bioactive phenolic compounds that found in loquat tea are responsible for its antioxidant effects (12). seong et al (2019) revealed that ethanol loquat leaf extract (by reflux, 4h) displayed good anti-oxidant activity by using abts, dpph and a superoxide dismutase (sod) assay (48). rashed et al (2014) stated that methanol stems extract (80%) of loquat (by maceration) was demonstrated the excellent antioxidant effect by the teac and orac assays related to the presence of active constituents as triterpenes like ursolic (fig.2), corosolic (fig.3) and oleanolic acids (fig.12) and flavonoids like kaempferol 3-o-β-glucoside (fig.11), quercetin (fig.8), quercetin 3-o-αrhamnoside (fig.7), and naringenin (fig.15) (18). figure 15. chemical structure of naringenin (18) ursolic acid (fig.2) that separated from loquat was displayed a strong antioxidant effect and good scavenging activity against dpph radical at different degrees (84). fouedjou et al. (2016) reported that methanol extracts (crud extracts) and their fraction (etoac and n-buoh extracts) and compounds that isolated from e. japonica stem bark shown antiradical activity by using ferric (fe3+) reduction antioxidant power, anti-hemolytic and dpph assays and the most active fraction among them was the etoac fraction of e. japonica. it can be reported that antioxidant effect increases with the crude extracts fractionation. amongst the isolated compounds of e. japonica, cinchonain iib (fig.13), catechin (fig.16), lyoniresinol 2-a-o-β-d-xylopyranoside (fig.17) were identified as the most important antioxidant ones. the antioxidant effect that exhibited by e. japonica were related to their high phenolic compounds content (1). iraqi j pharm sci, vol.30(1) 2021 eriobotrya japonica 51 figure 16. chemical structure of catechin (1) figure 17. chemical structure of lyoniresinol 2a-o-β-d-xylopyranoside (1) conclusions in this review we focused on the main pharmacological activities identified and the main bioactive compounds reported in different parts of loquat. phytochemical studies with loquat have reported that tannin, quercetin, chlorogenic acid, ursolic acid, oleanolic acid and caffeic acid, consider the major constituents. different kinds of pharmacological activities have been reported in eriobotrya japonica such as antioxidant, antibacterial, hypolipidemic, anti-diabetic activity and other. for researchers, the wide scope range is available to evaluate and explore the medicinal uses of the drugs. acknowledgements i am highly thankful to the college of pharmacy / university of baghdad to providing me the facilities and also opportunity to achieve my review. reference 1. fouedjou rt, nguelefack-mbuyo ep, ponou bk, nguelefack tb, barboni l and tapondjou la. antioxidant activities and chemical constituents of extracts from cordyline fruticosa (l.) a. chev. 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5(1):12–21. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(1) 2022 lornoxicam-loaded cubosomes doi: https://doi.org/10.31351/vol31iss1pp144-153 144 lornoxicam-loaded cubosomes: preparation and in vitro characterization. rasha s. younus alkwak*,1 and nawal a. rajab** * ministry of health and environment , , baghdad, iraq . **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract cubosomes are nano-sized structures self-assembled materials used for controlling the release of the entrapped drug molecules . lornoxicam (lxm) is a potent analgesic nonsteroidal anti-inflammatory (nsaid) drug with a short half-life (3-4) hours. the present study aims to prepare lxm-loaded cubosomes with welldefined morphology, small particle size, low pdi, high entrapment efficiency (ee), sustained drug release, and acceptable zeta potential value. twelve formulas of lxm-loaded cubosomal dispersions were prepared by a solvent dilution method using glyceryl monooleate (gmo) as polar lipid with different stabilizers as pluronic® f127 or tween 80 and different types of hydrotrope as ethanol or propylene glycol. these formulas were evaluated for their particle size analysis and pdi, entrapment efficiency (%ee), and in vitro drug release to select a group of the optimum formulas, that further characterized by transmission electron microscopy (tem) and zeta potential analyzer to select the optimum dispersion. the obtained results indicated that f3, composed of gmo, pluronic® f127, ethanol, drug, and phosphate buffer solution ph 7.4 at concentration of 7.28%, 1.82%, 8%, 2%, and 80.9% w/w, respectively, prepared in 20min agitation period, as the optimum formula for its high ee (94.30±0.002%), small particle size (16.3±0.19nm), low pdi (0.06±0.02), and high zeta potential value (-65.9±0.05mv), and well-defined cubic structure. this study's conclusion illustrated that lxm-loaded cubosomal dispersion could be considered a promising nano-carrier for drug delivery. key words: lornoxicam, cubosomes, glyceryl monooleate. لورانكسكم: تحضيير وتقييم الب المحّملة الكوبوسومات **و نوال عياش رجب 1*، يونس رشا سعدي ، العراق ، بغدادوزارة الصحة والبيئة * فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق ** الخالصة الدواء الكوبوسومات تحرير في للتحكم تستخدم نانوية هياكل قصير) .هي نصف عمر له فعال مسكن دواء هو ( 43اللورانكسكام .ساعات. تهدف الدراسة إلى تحضير المكعبات المحملة با للورانكسكم تم تحضير اثنا عشر صيغة بواسطة طريقة تخفيف المذيب ثم التقييم المختبري لها من حيث حجم الجسيمات , نسبة تحرر الدواء الفحص المجهري اإللكتروني , جهد زيتا. و بفر فوسفات ذي و ايثانول و لورانكسكم 127اثبتت النتائج ان الصيغة الدوائية الثالثة المكونة من جليسريل مونوليت و بلورونيك ف على التوالي هي االفضل حيث اظهرت النتائج االتية : نسبة %89.9و %2, %8, %1.82, %7.28بنسبة 7.4االس الهيدروجيني م ف( باالضافة 65.9 ( قيمة جهد زيتا العالية )0.06نانومتر( معامل التشتت )16.3( حجم الجسيمات الصغيرة ) %94.3كفائة عالية) كل مكعب واضح الى هي استنتجت الدراسة أن الكوبوسومات المحّملة بلورانكسكم تعتبرناقل نانوي واعد لتوصيل األدوية. كلسرين مونواوليت. ,الكلمات المفتاحية: الكوبوسومات , لورانكسكم introduction nanoparticles are those particles upon minimizing their size to the nanometer range; tend to show different characteristics from those of the original larger ones. (1, 2) various types of nanocarriers are used for drug delivery. it offers many advantages over conventional drug delivery systems as modifying the solubility of hydrophobic materials, achieving controlled or sustained release, promoting drugs' stability, and finally targeted therapy to the site of action that increases efficacy and minimizes side effects, nanoparticles (n.p.s) can effectively deliver drugs across the skin due to their unique characteristics. following topical administration of nano-particulate formulations, active compounds can enter the skin via intercellular, transcellular or trans-appendageal pathways. nanoparticles can either remain intact or degrade near the skin surface releasing active substances to penetrate skin layers. (1, 2) 1corresponding author e-mail: rashasy991@gmail.com received: 22/10/2020 accepted: 6/ 9/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp144-153 iraqi j pharm sci, vol.31(1) 2022 lornoxicam-loaded cubosomes 145 cubosomes are nanostructured lyotropic liquid crystalline particles. their size ranges from 10-500nm in diameter, made of specific amphiphilic lipids (in definite proportions) in an aqueous media where suitable surfactants are used, known as biocompatible carriers in drug delivery. luzzati and husson revealed that these structures appeared square in shape while being slightly spherical dots using x-ray scattering. (3) lornoxicam (lxm), also known as chlortenoxicam, is a nonsteroidal anti-inflammatory drug (nsaid) of the oxicam class. (4) it has analgesic, anti-inflammatory and antipyretic properties. (5) lornoxicam is highly bound (99%) to plasma proteins (almost exclusively serum albumin), which results in a low apparent volume of distribution (0.2 l/kg). (6) lornoxicam undergoes extensive hepatic metabolism in humans, with negligible amounts of unchanged drug being detected in the urine. as with other nsaids. (6) the cytochrome p450 (cyp) 2c subgroup of isoenzymes [possibly (cyp) 2c9] appears to play a significant role in the oxidative metabolism of lxm. (7) the present study aims to prepare cubosomes with well-defined morphology, small particle size, low pdi, high entrapment efficiency, sustained drug release, and high zeta potential value. materials lornoxicam (chemshuttle/ usa), ethanol absolute, triethanolamine (chem. lab./ belgium), kh2po4 and naoh (pellet), glyceryl monooleate and 2% w/v phosphotungstic acid (sigma-aldrich/ germany), tween 80 (alpha chemika/ india), pluronic® f127 (industrial department actico/jordan), deionized water (janeen for chemical and lab materials/ iraq), ortho_phosphoric acid 85% (merck kga/ germany), propylene glycol (thomas baker/ india). preparation of lxm loaded cubosomal dispersion lornoxicam loaded cubosomal dispersions were prepared by a solvent dilution method using a vortex. (8) by melting lipid and stabilizer (tween 80 or pluronic® f127) at 45 ± 2°c using a water bath to produce the oil phase mixture. the precise amount of lxm (2 gm) was weighed and dissolved in the hydrotrope to prepare a drug solution. this drug solution was then added to a previously prepared oil phase mixture to form a bicontinuous lipid bilayer subjected to vortex at high speed for 3 min to ensure a low-viscosity preparation homogenous melted mixture. finally, the low-viscosity homogenous melted mixture was injected into an excess phosphate buffer solution ph 7.4 that was preheated at the same temperature as the homogenous melted mixture. the final mixture was then subjected to vortex at high speed for 20 min to prepare the lxmloaded cubosomal dispersion, as shown in table (1), and stored at ambient temperature until required. table 1. composition of lxm-loaded cubosomal dispersions a g ita tio n tim e (m in .) aqueous phase hydrotrope stabilizer oil (o il: sta b iliz e r ) m ix tu r e drug f o r m u la c o d e p h o sp h a te b u ffe r s o lu tio n p h 7 .4 (g m ) p r o p y le n e g ly c o l (g m ) e th a n o l (g m ) t w e e n 8 0 (g m ) (g m ) p lu r o n ic ® f 1 2 7 (g m ) g m o (g m ) l o r n o x ic a m (g m ) 20 86.4 … 2.5 … 1.82 7.28 4:1 2 f1 20 83.9 … 5 … 1.82 7.28 2 f2 20 80.9 … 8 … 1.82 7.28 2 f3 20 80.9 8 … … 1.82 7.28 2 f4 20 80.9 … 8 1.82 … 7.28 2 f5 10 80.9 … 8 … 1.82 7.28 2 f6 20 86.4 … 2.5 … 3.03 6.07 2:1 2 f7 20 83.9 … 5 … 3.03 6.07 2 f8 20 80.9 … 8 … 3.03 6.07 2 f9 20 80.9 8 … 3.03 6.07 2 f10 20 80.9 … 8 3.03 … 6.07 2 f11 10 80.9 … 8 … 3.03 6.07 2 f12 iraqi j pharm sci, vol.31(1) 2022 lornoxicam-loaded cubosomes 146 variables affecting formulation different variables were studied to investigate their effects on the prepared lxmloaded cubosomal dispersion properties. effect of stabilizer concentration two lxm-loaded cubosomal dispersions (f1 and f7), were prepared to assess the effect of the stabilizer concentration on the cubosomal dispersion properties, where pluronic® f127 was used in the disperse phase in a concentration of 1.82% and 3.03% w/w, with different oil: stabilizer ratio (4:1, and 2:1), respectively effect of ethanol concentration six lxm-loaded cubosomal dispersions were prepared using different ethanol concentrations as 2.5% w/w (f1 and f7), 5%w/w (f2 and f8) and 8 % w/w( f3 and f9) with different oil: stabilizer ratio (4:1, and 2:1) were used to assess its effect on cubosomal dispersion properties effect of hydrotrope types two lxm-loaded cubosomal dispersions (f4 and f10) were prepared using 8% w/w propylene glycol with different oil: stabilizer ratios (4:1 and 2:1) instead of ethanol, to assess the effect of hydrotrope type on the cubosomal particle size. effect of stabilizer types two lxmloaded cubosomal dispersions (f5 and f11) were prepared using (1.82% and 3.03%) w/w tween 80, with different oil: stabilizer ratio (4:1, and 2:1), respectively, instead of pluronic® f127 to assess the effect of stabilizer types on the cubosomal dispersion properties. effect of agitation time two lxm-loaded cubosomal dispersions (f6 and f12) were prepared by 10 min. agitation using vortex with different oil: stabilizer ratio (4:1 and 2:1), instead of 20min., to assess the effect of decreasing the agitation time on the cubosomal dispersion properties. characterization of lxm-loaded cubosomal dispersion particle size and pdi determination triplicate measurements of the mean particle size (mean diameter) and polydispersity index (size range of particles) were done using a dynamic light scattering method. the light scattering fluctuations were examined; this fluctuation is due to brownian motion of dispersion particles. (9) drug content accurately, one gram of each lxm-loaded cubosomal dispersion was transferred to a volumetric flask of 250 ml; initially, only 200 ml phosphate buffer ph 7.4 was added; then, after sonication for 30 minutes, a clear solution was obtained. finally, volume was completed with phosphate buffer ph 7.4 to 250 ml. subsequent dilution was made to determine the percentage of lxm content spectrophotometrically using the uvvisible spectrophotometer at 375 nm (10) . entrapment efficiency for the determination of the loading capacity (entrapment efficiency), each of the lxmloaded cubosomal dispersions was subjected for 30 minutes to centrifugation at 15000 rpm. the supernatant liquid was collected, diluted appropriately with the phosphate buffer ph 7.4 and estimated using u.v. visible spectrophotometer at 375nm. (11) the following equation calculated the entrapment efficiency ee %: % ee = (total drug free drug) / total drug × 100 …… (eq.1). in vitro drug release the release of lxm from the cubosomal dispersion formulas was done by using a dialysis membrane (mwco 12-14 kda). (12) rotating paddle dissolution apparatus type ii was used to measure the in vitro drug release from all prepared formulas. sink condition was provided throughout the experiments. the sealed dialysis bag containing one gram of the lxm-loaded cubosomal dispersion formula (equivalent to 20 mg of lxm) was sunken in 900 ml phosphate-buffered ph 7.4 (dissolution media) with a speed of 50 rpm, and the temperature of the medium was maintained at 32±0.5 ᵒc. these conditions were used, since cubosomes may be incorporated in a tdds in the future work. at predetermined time points of 0.5, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 32, and 33h. five mls were withdrawn and replaced with fresh medium. the withdrawn sample's released drug concentration was determined spectrophotometrically using a uv-visible spectrophotometer at the selected λ max 375nm. (13) selection of the optimum formula the choice of the best formula was achieved according to the following tests: particle size analysis and pdi, drug content, ee %, and in vitro lxm release studies from the lxm-loaded cubosomal dispersions. the selected formulas were exposed to further investigations to select the optimum formulation, such as zeta potential measurement and morphology determination by (tem). statistical data analysis the results of the experiments were given as a mean of triplicate samples ± standard deviation. they were analyzed according to the one-way analysis of variance at the level of pvalue equal to 0.05 statistically significant is considered at the level of (p-value ≤ 0.05) and non-significant at the level of (p-value > 0.05). (14) iraqi j pharm sci, vol.31(1) 2022 lornoxicam-loaded cubosomes 147 result and discussion the particle sizes and pdi of lxm-loaded cubosomal dispersion the particle size and pdi of the prepared lxm-loaded cubosomal dispersion formulas (f1f12) were characterized by dynamic light scattering (dls) using the abt-9000 nanoparticle laser analyzer. (15) table (2) shows the particle size of the lxm-loaded cubosomal dispersion formulas. the smallest one was (12.3±0.27 nm), and the highest one was (112.2±0.32 nm). on the other hand, the mean pdi values for the drug-loaded formulations varied in the range of 0.07±2.12 to 0.01±0.00. particle size measurement was carried out to confirm that all the dispersion particles were in the nanometer size range; the small particles might be attributed to many reasons; such as, the bottomup approach has a unique formation mechanism of cubosomes where the stabilizers are homogenously dispersed onto nanostructured particles' surface and it needs less energy input than the top-down approach because the bottom-up avoid severe fragmentation. also, hydrotrope will coat the cubosomal particles and imparts a net negative charge to the cubosomal system. hence, it confers some degree of electric stabilization that may finally reduce cubosome size. the index represents the particles' homogeneity and uniformity in the formulations and width of the size distribution. pdi's low value is considered desirable for uniform distribution and high stability of the dispersion. (16) table2. the particle size of lxm-loaded cubosomal dispersions (mean ±sd) n=3. drug content the mean percent of drug content of the formulations were within the range (97.52±0.085 101.724±0.053%), as shown in table (3). the results were in inconsistent with the usp requirements (17), indicating high adequacy of the preparation method. (17) entrapment efficiency as shown in table (3), the entrapment efficiency of the lxm-loaded cubosomal dispersions was within the range (89.41±0.022%95.08±0.002%), indicating that most of the lxm was encapsulated in cubosomes. due to the strong affinity between lxm and the gmo in the cubosomes nanoparticles, the high internal area of cubosomal nanoparticles was 'grabbed' in the liquid crystal structure, and thus, the obtained result matches the result of the entrapment efficiency of the tropicamide-loaded cubosomes for ocular delivery. (18) the results showed that particle size and entrapment efficiency of cubosomes formulation could be significantly affected by varying the ethanol and lipid concentration and keeping other variables constant. table 3. the drug content and entrapment efficiency per cents of lxm-loaded cubosomal dispersions. (mean ±sd) n=3. (o il : st a b il iz e r) f o rm u la c o d e particle size in nm pdi (o il : st a b il iz e r) f o rm u la c o d e particle size in nm pdi 4:1 f1 35.5±0.45 0.07±0.04 2:1 f7 25.7±0.21 0.07±0.00 f2 25.6±0.2 0.05±0.02 f8 20.9±0.71 0.01±0.00 f3 16.3±0.19 0.06±0.02 f9 12.3±0.27 0.06±8.49 f4 112.2±0.32 0.02±0.00 f10 101.24±0.2 0.01±0.16 f5 39.3±0.21 0.01±0.00 f11 30.3±0.25 0.01±0.00 f6 56.4±0.38 0.01±0.00 f12 48.2±0.42 0.01±0.00 formula code drug content% ee % formula code drug content% ee % f1 99.808±0.012 93.28±0.002 f7 100.1±0.001 93.16±0.002 f2 100.308±0.005 93.52±0.003 f8 100.502±0.002 93.34±0.005 f3 97.856±0.013 94.30±0.002 f9 99.52±0.085 94.05±0.001 f4 99.824±0.07 94.13±0.002 f10 97.74±0.012 93.94±0.001 f5 100.05±0.05 90.39±0.003 f11 100.52±0.025 89.41±0.022 f6 98.524±0.012 93.68±0.001 f12 101.724±0.053 93.61±0.006 iraqi j pharm sci, vol.31(1) 2022 lornoxicam-loaded cubosomes 148 influence of formulation variables on particle size and ee% effect stabilizer concentration stabilizers used to modify cubosome’s surface properties and hence impart stability. the stabilization is due to shielding of the lipid nanostructure from the surrounding aqueous medium. (1921) the results showed that both of the particle size and ee% of cubosomes were indirectly proportional to the increase in pluronic® f127 concentration. upon reducing pluronic® f127 concentration, significantly (p-value<0.05) larger size cubosomes nanoparticles with higher ee% were formed, particle size results may be attributed to the reduced interfacial stability that resulted from an insufficient amount of stabilizer, leading to aggregation of cubosomal nanoparticles. (22) these results were in agreement with the work of barauskas et al. (23) while the increased cubosomal ee% may be explained by the hydrophobic nature of the drug provide strong attraction with the hydrophobic domain in the cubic phase bilayer, facilitate a high drug entrapping in the cubic system. also, reducing pluronic® f127 concentration combined with increasing the amount of lipid resulted in faster solidification of the cubosomal nanoparticles due to the increased viscosity of the medium. moreover, this would prevent drug diffusion to the external phase of the medium. in addition ethanol will in the aqueous dispersion medium, assumed to stabilize the formed cubosomal nanoparticles by forming a coat over them. (24) effect of ethanol concentration ethanol is used to dissolves the lipid and enhance the miscibility between hydrophilic and lipophilic phases that helps bind the two phases. the effect of increasing organic phase volume seems conflicting. some studies showed that it causes a decrease in the particle size (25) while others showed the opposite phenomenon. (26) the results showed that the elevation of the ethanol concentration cause reduction of the particle size and elevation in ee% of cubosomal nanoparticles. the particle size of lxm-loaded cubosomal dispersion that contains 2.5% ethanol (f1 and f7) was significantly decreased with increase in the ee% (p-value<0.05) by increasing the concentration of ethanol to 5% w/w as in (f2 and f8) then further significant decreased in particle size and increased ee% (p-value<0.05) by increasing the concentration of ethanol from 5% w/w to 8% w/w as in (f3 and f9). the results showed the particle size decreased as the total concentration of both stabilizer and hydrotrope increased, which could be explained by the combined effects of the pluronic® f127 and ethanol in providing a high degree of electric stabilization that may finally lead to a significant decrease in the cubosomes average particle size (p-value<0.05). (27, 9) this might be because ethanol in the aqueous dispersion medium is assumed to stabilize the formed cubosomal nanoparticles by forming a coat over them. the formed coat could retain an excess lxm amount so increasing its entrapment. (18, 9) since the lxm-loaded cubosomal dispersions with 8% w/w ethanol showed a smaller particle size than those containing (2.5% or 5%) w/w ethanol, they were used to study the effect of other factors. effect of hydrotrope types the results showed the cubosomal particle size and ee% increased significantly (p-value<0.05) by replacing the ethanol with 8% propylene glycol as in the formulas (f4 and f10). limayem blouza et al. investigation showed that an increase in polymer molecular weight generally increases particles' size. (28) the same effect was obtained after using a more viscous organic solvent (propylene glycol). these findings were explained by an increase in the organic phase's viscosity, which hindered solvent diffusion more difficult and thus led to larger nanoparticles' size and an increase in the ee%. (29) effect of stabilizer types the results showed that the mean particle size of the dispersions (f5 and f11), that prepared with tween 80, was significantly (p-value<0.05) larger than that seen with (f3 and f9) in which pluronic® f127 was used for stabilizing the cubosomes. (28) while the cubosomal ee % decreased significantly (p-value < 0.05) by replacing the pluronic® f127 with tween 80. the result may be explained by the increased solubility of lxm in an aqueous solution when tween 80 was used as a stabilizer. (30) effect of agitation time the results showed that by decreasing agitation time from 20 min to 10 min, the cubosomal particle size of the dispersions (f6 and f12) increased significantly (p-value<0.05) in the other side the cubosomal ee % decreased significantly (p<0.05). this result may be because 20min agitation had provided enough time for greater penetration of the oil phase and hydrophobic region of the pluronic® f127 and, for ethanol to coat the cubosomal particles and confers some degree of electric stabilization that may finally lead to a size reduction of the cubosomes, by reducing particle aggregation risk and enhance the retention of drug in cubosomal particles thus increase the cubosomal ee%. (31-33) in-vitro drug release to diffuse the entrapped drug through the dialysis membrane, it should release from the vesicles to the surrounding liquid medium. (34) the sustained release of a drug from nanoparticles is an essential factor for the successful iraqi j pharm sci, vol.31(1) 2022 lornoxicam-loaded cubosomes 149 development of nanoparticle formulations. as shown in figure (1), the release of lxm from all cubosomal formulations (f1-f12) had an initial burst release, and then the drug release was sustained for several hours. initial burst release of all cubosomal dispersions was approximately between (4.778-21.268%) in the first hour after that the drug release was sustained with approximately (62.25989.954%) of the entrapped lxm within 32 hours; these results were in agreement with zeng et al., who stated that the release pattern of cubosomes characterized by initial burst release followed by sustained release. (33) the initial burst release may be explained by the weakly adsorbed drug on the surface of cubosomes or located just at or beneath the nanoparticles' surface. (34) while lxm entrapped inside the nanoparticles contributed to the following sustained release, which can be attributed to the unique structure of the cubosomal nanoparticles. (35, 36) figure 1.in-vitro release profile of lxm-loaded cubosomal dispersions (f1-f12), divided into two (oil: stabilizer) groups, in phosphate buffer solution ph 7.4 at 32 °c. as shown in figure (1), the release comparison of cubosomal dispersion according to stabilizer concentration, where f1 (pluronic® f127 (1.82%w/w)) released only 86.18% of the lxm within 33hr. in contrast f7 (pluronic® f127 (3.03%w/w)) released about 89.95 % of the lxm within 33hr. the data shows that the f2 similarity function results of lxm-loaded cubosomal dispersions with different stabilizer concentration > 50 which mean that the in-vitro dissolution profiles were similar. as shown in figure (1) after 33 hrs. the released lxm from the cubosomal dispersions containing 2.5% ethanol (f1and f7) was 86.18% and 89.95%, respectively, which were the highest among other formulas, while by increasing ethanol concentration to 5% w/w (f2 and f8), only 81.999% and 82.986% of lxm were released, respectively; and by further increasing ethanol concentration to 8% w/w (f3 and f9), only 73.406% and 72.116% of lxm were released, respectively. the data shows that the f2 similarity function results of lxm-loaded cubosomal dispersions with different ethanol concentration were > 50, which mean that the invitro dissolution profiles were similar. in addition the lxm released from the cubosomal dispersions with 8% w/w ethanol (f 3and f9) were 73.40623% and 72.11593%, respectively, while the lxm released from cubosomal dispersions with 8% w/w propylene glycol (f4 and f10) where only 70.0967% and 69.341%, released respectively. the data shows that the f2 similarity function results of lxm-loaded cubosomal dispersions with different hydrotrope types were >50, meaning the in-vitro dissolution profiles were similar. in addition, the lxm released from the cubosomal dispersions that contain pluronic® f127 (f3 and f9) were 73.40623% and 72.11593%, respectively, while the cubosomal dispersions that contain tween 80 (f5 and f11) where 76.964% and 81.883%, released respectively. the data shows that the f2 similarity function results of lxm-loaded cubosomal dispersions with different stabilizer types were >50, which means the in-vitro dissolution profiles were similar. furthermore, the lxm was released from the cubosomal dispersion prepared within 20 min. agitation (f3 and f9) were 73.40623% and 72.11593%, respectively, while the cubosomal dispersions prepared within 10 min agitation (f6 and f12) were only 62.259% and 65.181%, released respectively. the data shows that the f2 similarity function results of lxm-loaded cubosomal dispersions with different agitation time were >50, which mean the in-vitro dissolution profiles were similar. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4556290/figure/f1-ijn-10-5459/ iraqi j pharm sci, vol.31(1) 2022 lornoxicam-loaded cubosomes 150 selection of optimized formulas of lxm-loaded cubosomal dispersion it was previously reported that nanoparticles with a diameter of 300 nm or less could deliver the drug to some extent to the skin's deep layers. moreover, if the particles' diameter was decreased to 70 nm or less, this leads to maximum deposition of the drug in the epidermis and the viable dermis. (37-40) that means the cubosomal particle size plays an essential role in drug delivery systems. the results of the study showed that f9 had the smallest particle while, f3 showed maximum ee %, and the in-vitro release study of (f3 and f9) showed the drug's sustained release in both of the formulations. so the selected group of lxm-loaded cubosomal dispersion (f3and f9) exposed to further investigations to select the optimum formula. particle surface charge (zeta potential) a high zeta potential absolute value indicates a high electric charge on the cubosomal surface, which can cause strong repellent forces among particles and prevent aggregation of the cubosomes in a buffer solution. (41, 42) as shown in table (4) and figure (2), cubosomal formulations f3 showed higher zeta potential value and maximum stability than f9; this might be attributed to the higher concentration of the used fatty acid, gmo. (43) table 4.zeta potential values of lxm-loaded cubosomal dispersions (mean ±sd) n=3. formulas code (oil: stabilizer) zeta potential value in mv f3 4:1 -67.2±0.05 f9 2:1 -63.5±0.11 figure 2. zeta potential values of lxm-loaded cubosomal dispersions. transmission electron microscopy the external morphology images of the selected group of lxm-loaded cubosomal dispersion (f3 and f9) were taken using transmission electron microscopy (tem). as in figure (3), the transmission electron micrographs show that the cubosomal dispersion f3 was cubic and well separated from each other and in the nano-size, which confirms particle size measurement results. while f9 showed vesicular particles over the formation of the desired particles of cubic structure, which can be explained to be due to high pluronic® f127 concentrations which resulted in formation of smaller particles, they also promote vesicular formation particles. 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creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 papaver rhoeas l doi: https://doi.org/10.31351/vol30iss2pp78-85 78 isolation of alkaloids from papaver rhoeas (papaveraceae) wildly grown in iraq amenah ayad lafta*,1 and maha n. hamad* *department of pharmacognosy and medicinal plant, college of pharmacy, university of baghdad, iraq abstract the plant papaver rhoeas ,which belongs to family papaveraceae and known as common poppy is wildly grown in iraq .it was used in traditional medicine in wide range of diseases including inflammation, diarrhea, sleep disorders, treatment of cough, analgesia, and also to reduce the withdrawal signs of opioid addiction. the project provide the first comprehensive research done in iraq to study the phytochemical and the methods of extraction and separation of alkaloids from papaver rhoeas wildly grown in iraq .the plant was harvested in april 2019 from zurbatiya is an iraqi town located at the northeast of waist province in iraq.the collected plant was washed thoroughly, dries under shade, and grounding in a mechanical grinder to fine powder. the plant was extracted by hot extraction method using methanol then fractionation was done to separate alkaloids from chloroform fraction by tlc and ptlc .the alkaloids were isolated and purified by ptlc then subjected to various analytical techniques for alkaloids identification such as uv, lc mass and ir .the result was indicated of three alkaloids (dihydrocodien, chelidonine and papaverrubine c) in papaver rhoeas plant. keywords: papaver rhoeas, dihydrocodien, chelidonine, papaverrubine c. نبات الخشخاش المنثور البري في العراقمن القلويداتعزل *و مها نوري حمد 1*,آمنه اياد لفته العراق . ،بغداد ،جامعه بغداد ،كلية الصيدله ،*فرع العقاقير والنباتات الطبيه الخالصه نبات الخشخاش والمعروف بالخشخاش المنثورالذي ينمو بريا في العراق ويستخدم في الطب الشعبي على مدى واسع لمعالجة االلتهابات ت الموجوده ,االسهال ,مشاكل النوم ,معالجة السعال,مسكن اآلم وتقليل اعراض االدمان .يعتبر هذا البحث اول بحث شامل في العراق لدراسة االلكلويدا وتم 2019ي نبات الخشخاش البري في العراق وطرق استخالصها وفصلها . تم جمع النبات من قضاء زرباطيه في محافضة واسط في شهر شباط ف ويدات غسل وتجفيف النبات في الظل ثم طحنه بالمطحنه الميكانيكيه.تمت عملية االستخالص ب الطريقه الحاره ثم تمت عملية التجزئه لفصل االلكل الكتلي واالشعه والطيف السائل وكروماتوغرافيا التحضيرية الطبقه وكروماتوغرافيا الرقيقة الطبقه كروماتوغرافيابقه الكلوروفورم بواسطه من ط ( في نبات cالكيميائيه وجود القلويدات )الدايهايدروكواديين والجليدونين وبابافروبين الكشوفات نتيجة وكانت تحت الحمراء واالشعه فوق البنفسجيه .الخشخاش .cبابافروبين ،الجليدونين ،الدايهيدروكودايين ،المفتاحية : الخشخاش المنثور الكلمات introduction poppy (papaver rhoeas l.) figure 1 is a temperate native with a very wide distribution area, from africa to temperate and tropical asia andeurope(1). it grows in fields, beside roads, and on grassland .papaver rhoeas is a variable, erect annual, forming a long-lived soil seed bank that can germinate when the soil is disturbed. in the northern hemisphere it generally flowers in late spring (between may and october but if the weather is warm enough other flowers frequently appear at the beginning of autumn. it grows up to about 70 cm (28 in) in height 2).the stems hold single which are large and showy, 5–10 cm (2–4 in) across with four petals that are vivid red, most commonly with a black spot at their base(3).the plant has been used for medicinal proposes a long time ago for treatment of a wide range of diseases including inflammation, diarrhea, sleep disorders, treatment of cough, analgesia, and also to reduce the withdrawal signs of opioid addiction (4)furthermore, it is known to claim intestinal and urinary irritation and to be useful in various conditions such as bronchitis, pneumonia, and rash(5). pharmacological studies have shown that the plant extract may have some radical scavenging properties(6).investigations also indicated that the papaver rhoeas extract also possess properties of anti-ulcer genic (7), antinociception (8), anti in flammatory effect (9) and stress amelioration effect (10). this study was conducted for identification of alkaloids that extracted from papaver rhoeas. 1corresponding author e-mail: nnuna714@gmail.com received: 1/11/2020 accepted: 1/ 3/2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp78-85 iraqi j pharm sci, vol.30(2) 2021 papaver rhoeas l 79 figure 1. iraqi papaver rhoeas. materials and methods collection of plant material the arial parts of papaver rhoeas l. (papaveracea) are collected from zurbatiya which is an iraqi township in the north east of wasit province in iraq , a border crossing with iran in april (2019) the plant authenticated by dr.sukiana abbas alewi in college of sciences, university of baghdad. the plant parts cleaned and dried in shade for two weeks then the dried plant material was coarsely powdered using electrical grinder and weighed. extraction of alkaloids about 200gm of the dried plant material was defatted by maceration in hexane for 24 hours then filtered and the dried defatted plant was placed in a soxhlet, and a sufficient amount of 85% methanol (1 l) was added to the apparatus for 14 hours until complete exhaustion was achieved. the alcoholic extract was filtered by filter paper and the filtrate was evaporated to dryness using rotary evaporator to obtain 50gm dark-greenish residue. the residue was suspended in about 70ml of 6% hcl (ph 4), and partitioned with chloroform (70ml x 3). the upper aqueous acidic layer was separated and basified by ammonium hydroxide (23-25%) added gradually by a dropper in room temperature with stirring until getting ph 10 then the basified aqueous layer partioned three times with equal volume of chloroform in separatory funnel. the lower chloroform layer was collected, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. the steps of alkaloids extraction were shown in the following scheme. figure 2. steps of alkaloids extraction. iraqi j pharm sci, vol.30(2) 2021 papaver rhoeas l 80 identification, isolation and purification of alkaloids from papaver rhoeas plant 1. preliminary phytochemical screening of alkaloid compound for the methanolic plant extract using dragendroff and mayer reagent 2. thin –layer chromatography (tlc):few milligrams from the extracted alkaloids was re suspended with 1 ml absolute methanol then applied on an analytical tlc plate pre coated with silica gel 0.25mm using the mobile phase: cyclohexane: chloroform: diethyl amine (70:20:10)(12) . 3-isolation and purification of alkaloid compounds by preperative thin layer chromotagraphy :a readymade pre coated silica gel glass plate with 0.5mm thickness was placed in oven for 5 minute for activation at 100c then the plate was placed into glass jar contained 100 ml mobile phase then closed tightly, and left for saturation for about one hour far from sunlight and air current, after development, the plates were taken out of the jar then left at room temperature to dry then the bands were determined and scrubbed by needle under uv light using a wavelength of 254. 5lc mass (liquid chromatography–mass spectrometry):the analytical lc-ms was performed using agilent system joined to an applied bio systems api 2000 mass spectrometer. 6ft-ir (fourier-transform infrared spectroscopy):-fourier transform infrared spectroscopy is a technique for material analysis it offers an qualitative analysis of the sample. ftir identified chemical bands in molecules, the range of scanning 4000-400 cm-1. 7uv (ultraviolet–visible spectroscopy): identification of isolated compound was done by measuring the absorbance by measuring their uv absorption at 240nm results preliminary identification of alkaloid n papaver rhoeas plant by preperative thin layer chromatography isolation and purification of alkaloids was carried out by using preparative tlc, in jar contains: cyclohexane: chloroform: diethyl amine (70:20:10) as mobile phase, figure 3. preparative tlc chromatogram for papaver rhoeas alkaloids on silica g f254 plate using mobile phase cyclohexane: chloroform: diethyamine (70:20:10) under u.v light identification of the isolated compounds by lcmass technique identification of compound a lc mass (liquid chromatography–mass spectrometry) for compound a are shown in figure 4 below figure 4. lc mass for compound a. https://en.wikipedia.org/wiki/ultraviolet%e2%80%93visible_spectroscopy iraqi j pharm sci, vol.30(2) 2021 papaver rhoeas l 81 the molecular ion peak at m/z 304 [m]+ was nearly correspond to a molecular formula of dihydrocodeine (c18h23no)which is 301 , also the abundance of peak 304 is (80538) which is the second highest one between other ions as as shown in figure 4 above. depending on the analysis above, the expected chemical structure for the isolated compound a is demonstrated in figure 5 below figure 5.chemical structure of compound a (dihydrocodeine). the ʎ max spectrum and ftir chart for compound a were shown in figure 6, 7 and table 1 figure 6. the ʎ max spectrum for dihydrocodeine. the ʎ max spectrum for the extracted alkaloid was 211 nm which is a typical spectrum for dihydrocodeine alkaloid 211nm(13). figure 7. ir spectrum for compound a table 1 .the ftir spectrum regions indicated the major functional groups in compound a ir band of compound a interpretation 3308 ,2971 o-h starching of phenol and carboxylic. 2877 asymmetric and symmetric stretching of ch2 1378 o-h bending of phenol 1273 c-o-c stretching of ether 1066,1043 c-h bending of aromatic (in plane) 887,802,654 c-h and c=c bending of aromatic in and out and in-plane iraqi j pharm sci, vol.30(2) 2021 papaver rhoeas l 82 finally, the data obtained from ir, uv, lc/ms of the isolated compound a were identical with the data of dihydrocodeine, which indicate that compound a could be dihydrocodeine alkaloids(14). identification of compound b the lc mass (liquid chromatography– mass spectrometry) for compound a are shown in figure 8 figure 8. lc mass for compound b. the molecular ion peak at m/z 370 [m]+ and 371 which represent m and m+h respectively that correspond to a molecular formula of papaverrubine c (epiporphyroxine) 370 , also the abundance of peak 370 is (75756) which is the second highest peak between other ions . depending on the analysis above, the expected chemical structure for the isolated compound is demonstrated in figure 11. figure 9. chemical structure of compound b indicated papaverrubine c (epiporphyroxine). the ʎmax spectrum and ftir chart for compound b were shown in figure 10, 11 and table 2 . figure 10. the ʎmax spectrum for papaverrubine c compound. the ʎmax spectrum for the extracted alkaloid was 232 and 285nm which is a typical spectrum for papaverrubine alkaloid 285 nm(15). iraqi j pharm sci, vol.30(2) 2021 papaver rhoeas l 83 . figure 11. ir spectrum for compound b table2 .the ftir spectrum regions indicated the major functional groups in compound b ir band of compound b interpretation 3280, 2971 o-h starching of phenol and carboxylic 2877 asymmetric and symmetric stretching of ch2 1648 c=c starching of alkene 1379 o-h bending of phenol 1068.1043 c-h bending of aromatic (in plane) 879,803,681 c-h and c=c bending of aromatic in and out and inplane finally, the data obtained from ir, uv, lc/ms of the isolated compound b were identical with the data of papaverrubine c (epiporphyroxine), which indicate that compound b could be papaverrubine c (epiporphyroxine) which is rhoeadines/ papaverrubines type of alkaloids(16,17). identification of compound c the lc mass (liquid chromatography– mass spectrometry) for compound c are shown in figure 12. figure 12. lc mass diagram for compound c. the molecular ion peak at m/z 353 [m] + and 354 which represent m and m+h respectively that that correspond to a molecular formula of chelidonine (c20h19no5, also the abundance of peak 353 is (65582) which is the highest peak between other ions .depending on this analysis, the expected chemical structure for the isolated compound is demonstrated in figure 13 figure 13. chemical structure of chelidonine. the ʎmax spectrum and ftir chart for compound c were shown in figure 14, 15 and table 3. iraqi j pharm sci, vol.30(2) 2021 papaver rhoeas l 84 figure 14. the ʎ max spectrum for compound c the major absorption maxima are 201 which is the same that also observed for chelidonine at 204. figure 15. ir spectrum of chelidonine. table 3 .the ftir spectrum regions indicated the major functional groups in compound c. ir band of compound c interpretation 3318,2971 o-h starching of phenol and carboxylic 2878 asymmetric and symmetric stretching of ch2 1652 c=c starching of alkene 1378,1325 o-h bending of phenol 1273 c-o stretching of alkyl aryl ether 1086,1043 c-h bending of aromatic (in plane) 878,802,662 c-h and c=c bending of aromatic in and out and inplane finally, the data obtained from ir, uv, lc/ms of the isolated compound c were identical with the data of chelidonine, which indicate that compound c could be chelidonine which is isoquinoline type of alkaloid. conclusion phytochemical investigation of wild iraqi plant papaver rhoeas was done to the whole plant and the results include the presence of different type of alkaloids[ dihydrocodeine ( morphinans ),papaverrubine c (rhoeadines/ papaverrubines ), chelidonine (isoquinoline)] and these types detected by lc mass,uv,ir . acknowledgements the authors are grateful to acknowledge the college of pharmacy -university of baghdad for providing the necessary facilities to carry out this study. references 1. philips, roger; rix, brian (1996). perfect plants. london: macmillan. p. 298. isbn 0333653416. 2. reader’s digest field guide to the wild flowers of britain. reader's digest. 2018. p. 30. isbn 9780276002175. 3. blamey, m.; fitter, r.; fitter, a (2003). wild flowers of britain and ireland: the complete iraqi j pharm sci, vol.30(2) 2021 papaver rhoeas l 85 guide to the british and irish flora. london: a & c black. isbn 978-140817950. 4. choe, s., kim, s., lee, c., yang,w., park, y., choi, h., chung, h., lee, d., hwang, b. y., species identification of papaver by metabolite profiling. forensic. sci. int. 2016, 211, 51–60. 5. valnet, j., 1992. phytotherapie, sixth ed. maloine, paris, france. 6. schaffer s, schmitt-schillig s, müller we, eckert gp. antioxidant properties of mediterranean food plant extracts: geographical differences. j physiol pharmacol. 2005; 56(suppl 1):115-24. 7. gürbüz i, ustün o, yesilada e, sezik e, kutsal o. anti-ulcerogenic activity of someplants used as folk remedy in turkey. j ethnopharmacol. 2003; 88(1):93-7. 8. sahraei h, fatemi sm, pashaei-rad, faghih monzavi z, salimi sh, kamalinegad m.effect of papaver rhoeas extract on the acquisition and expression of morphine induced conditioned place preference in mice. j. ethnopharmacol. 2006; 103:420-4. 9. mcewen bs, de leon mj, lupien sj, meaney mj. corticosteroids, the aging brain and cognition. tem 1999; 10:92-96. 10. seed-abadi s, ranjbaran m, jafari f, najafiabedi a, rahmani b, esfandiari b, delfan b, mojabi n, ghahramani m, sahraei h. effects of papaver rhoaes (l.) extract on formalin-induced pain and inflammation in m ice. pak j biol sci. 11. ibrahim. e.: isolation and characterization of pyrrolizidine alkaloids from echium glomeratum poir (boraginaceae) (doctoral dissertation, thesis (m.sc. in applied chemistry). faculty of graduate studies, jordan university of science and technology); 2007. 26 p 12. stahl e.: thin layer chromatography hand book, 1999 13. cowan, d. a., woffendin, g., & noormohammadi, a. (1988). two assays for dihydrocodeine in plasma and in urine and their use to determine the bioavailability of a controlled-release product. journal of pharmaceutical sciences, 77(7), 606–609. doi:10.1002/jps.2600770711 14. küppers, fjem, salemink ca, bastart m and paris m. alkaloids of papaver bracteatum: presence of codeine, neopine and alpinine. phytochem. 1976; 15: 444 5. 15. hughes, d. w., kühn, l., & pfeifer, s. (1967). the isolation and structure of papaverrubine c (epiporphyroxine). j. chem. soc. c, 0(0), 444– 446. 16. guggisberg a, hesse m, schmid h, bohm h, ronsch h and mothes k. uber papaver bracteatum lindl. iv. mitteilung, zur struktur des alkaloids e. helv. chim. acta 1967; 50:621 4. 17. pfeifer s and banerjee sk. cber rotfarbungs alkaloide der gattung papaver, 3. mitteilung. pharmazie. 1964; 19: 286 9. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ abstract iraqi j pharm sci, vol.21(1) 2012 protective effect of ginger extract 27 protective effect of ginger extract against cisplatin-induced hepatotoxicity and cardiotoxicity in rats. ahmed m. attyah* and sajida h. ismail* ,1 *department of pharmacology and toxicology,college of pharmacy,university of baghdad, baghdad,iraq. abstract the protective effect of ginger extract against cisplatin-induced hepatotoxicity and cardiotoxicity was evaluated in 30 albino white rats(weighing 200-300 gm ) classified into 5groups (6 rats per each group). the rats were treated with 0.5g/kg/day or 1g/kg/day ginger extract orally 5 successive days before and 5 successive days after induction of toxicity with intraperitoneal (ip) injection of (10mg/kg ) cisplatin, resulted in a significant reduction in the levels of aspartate aminotransferase (ast), alanine aminotransferase (alt) , total serum billirubin(tsb) , lactate dehydrogenase (ldh) and creatine kinase(ck) enzymes in comparison with the cisplatin treated animals; ginger extract also improves the histological changes produced by cisplatin in the liver cells and cardiac muscle fiber cells in comparison with the control . it is concluded that , ginger extract when used concomitantly with cisplatin protects the liver and heart against the toxicity induced by this cytotoxic drug. key wards :ginger, cisplatin,oxidative stress. تأثيراث الحمايت لمستخلص الزوجبيل ضد التسمم الكبدي والقلبي المستحدث بعقار السزبالتيه في الجرذان احمد محمد عطيت* و ساجدة حسيه اسماعيل* ،1 ٔانسًٕو ، كهٛت انصٛذنت ، جايعت بغذاد ، بغذاد ، انعشاق األدٔٚت* فشع الخالصة بالحٍٛ زش انٕلائٙ انًحخًم نًادة يسخخهص انزَجبٛم ضذ انخهف انز٘ حسببّ يادة انسانخأثٛانٓذف يٍ ْزِ انذساست ْٕ حمٛٛى إٌ جشراٌ( ٦خًست يجايٛع ٔنكم يجًٕعت ٔلسًج انٗ جشر يٍ انُٕع االيٓك٠٣)عضهت انمهب فٙ انجشراٌ خالٚا فٙ خالٚا انكبذ ٔ نًذة خًست اٚاو نهجشراٌ يٍ خالل انفى ( ٕٚيٛا ى غى/كغ١ٔا غى/كغى٣٫٠) أظٓشث انُخائج اٌ اعطاء يادة يسخخهص انزَجبٛم ٔبجشعت انٗ اَخفاض يعُٕ٘ بًسخٕٚاث رنك دٖأعٍ طشٚك انبشٚخٌٕ ( /كغىىيهغ١٣بالحٍٛ )زانسيٍ ٔاحذة جشعت اعطاء بعذخًست اٚاو لبم ٔ . بالحٍٛ فمظزانسجشعت ب احى عالجٓ خٙانجشراٌ ان يجًٕعتيماسَت بًسخٕٚاحٓا عُذ ( , ck,tsb,ast ,alt ldh) االَزًٚاث حى عضهت انمهب نذٖ يجًٕعت انجشراٌ انخٙ خالٚا انزَجبٛم فٙ ححسٍٛ انخهف انحاصم فٙ َسٛج خالٚا انكبذ ٔ سخخهصيكزنك اسٓى يٍ سخُخجا بالحٍٛ فمظ .ٔلذزجشعت انسحى عالجٓا ببالحٍٛ يماسَت بخهك انخٙ زجشعت يسخخهص انزَجبٛم لبم ٔبعذ جشعت انسعالجٓا ب عضهت انمهب يٍ خالٚا بالحٍٛ لذ ٕٚفش انحًاٚت انكافٛت نُسٛج خالٚا انكبذ ٔزانس عماسيادة يسخخهص انزَجبٛم يع اٌ اسخعًال انذساست .بالحٍٛزانخهف انز٘ ٚسببّ عماس انس .جهد التأكسد السزبالتيه ، :الزوجبيل،الكلماث المفتاحيت introduction cisplatin is a platinum-based drug (1) , which is one of the most effective antineoplastic agents used for treatment of testicular, ovarian, bladder, cervical, lung, and neck cancers (2) . the cytotoxic effect of cisplatin is believed to result mainly from its interaction with dna, via the formation of covalent adducts between certain dna bases and the platinum compound (3) , despite its clinical usefulness,cisplatin treatment has been associated with several toxic side effects including nephrotoxicity (4) , hepatotoxicity and cardiotoxicity (5) . cardiac events have been reported in many case reports as well including electrocardiographic changes , arrhythmias , myocarditis, cardiomyopathy and congestive heart failure (3) . it has been reported that oxidative stress through the generation of reactive oxygen species , decreases antioxidant defense system including antioxidant enzymes and non enzymatic molecules , reduced glutathione, are major alterations in the cisplatin toxicity (6) . ginger belongs to a tropical and sub-tropical family-zingiberaceae, it has been cultivated for thousands of years as a spice and for medicinal purposes (7) . for centuries, it has been an important ingredient in chinese, ayurvedic and tibb-unani herbal medicines for the treatment of rheumatism, gingivitis, toothache, asthma , stroke, nausea, vomiting and diabetes (8) . extracts of the ginger are rich in shagaols and gingerols which exhibit anti-inflammatory , anti-oxidant and anti-carcinogenic proprieties under ‘‘in vitro’’ and ‘‘in vivo’’systems (9) . this work was designed to assess the protective effect of orally administered ginger extract against cisplatin-induced hepatotoxicity and cardiotoxicity in rats . 1 corresponding author email : ph.sajida@yahoo.com received : 27/9/2011 accepted : 18/12/2011 mailto:ph.sajida@yahoo.com iraqi j pharm sci, vol.21(1) 2012 protective effect of ginger extract 28 materials and methods preparation of ethanolic ginger extract the ethanolic extract of ginger was made according to the method of ajith et al. (10) , by washing of fresh rhizomes of zingiber officinale several times with water. the 500 g of rhizome was cut into small pieces and juice was prepared in 70% ethanol. the extract was prepared by heating the juice at 50–60 0 c for 24 hr with intermitted shaking , filtration made, then centrifuge the extract at 2500g ,supernatant layer were pooled.the solvent was evaporated completely at 50–60 0 c with the help of rotary vacuum evaporator. the residue thus obtained (6.5 g w/w) was used for this study. animals and experimental protocol thirty white albino rats of both sexes, weighing 200300 gm were used in this study; they were obtained from and maintained in the animal house of the pharmacy college, university of baghdad under conditions of controlled temperature. the animals were fed commercial pellets and tap water/ad libitum. the animals were divided into five groups of six animals and treated as follow: group i received single ip dose of normal saline . the animals were sacrificed after 5 days , this group served as a negative control . group ii received single ip dose of cisplatin (10mg/kg) and animals were sacrificed after 5 days, this group served as positive control. group iii pretreated with oral dose of ginger extract alone (1g/kg/day) and animals were sacrificed after 10 days . group iv and vanimals pretreated with oral dose of ginger extract (0.5g and 1g /kg/day respectively) for 5 successive days before and 5 successive days after single ip cisplatin (10mg/kg) . animals were sacrificed after 10 days . animals have been anesthetized by ether, blood was collected directly from the heart by (intracardiac puncture) and poured into plain tubes , the clot was dispersed with glass rod and then centrifuged at 3000 rpm for 15 minute ; the serum was used within 2 days for the estimation of ast (11 ) , alt (11 ) ,tsb ( 12) , ldh ( 13) and ck ( 14) . histological sections of the hepatic and myocardial tissues were prepared according to the method of junqueira lc. et al in 1995 ( 15) for evaluating the histopathological changes with ordinary microscope, using paraffin sections technique , tissues was cut into 3 millimeter pieces , fixed in 10% formaldehyde solution , processed and embedded in paraffin , blocks were cut by microtome into 5micrometer ,thick sections , washed in water bath and left in the oven for dewaxing ,then stained with haematoxyline and eosin stain and then examined under light microscope . the data presented as mean+sd . the significance of differences between the mean values were calculated using unpaired student's t-test. p -values less than 0.05 were considered to be significantly different. results table (1) indicated that serum levels of ast, alt and tsb were significantly elevated in the gp.ii in comparison with the gp.i (p<0.05). while in gp. iv and gp.v the serum levels of ast, alt and tsb were significantly reduced , (fig.1,2&3). histological examination of tissue sections from the liver showed certain degeneration and necrosis of hepatocytes with inflammatory cells infilteration around portal area with sinusoidal dilatation were observed in the gp.ii (fig.5). while in gp.v the hepatic tissues was protected against the cisplatin-induced damage and shows normal liver tissues with no significant degenerative changes when compared with the gp.i liver tissue slide ( fig. 4&6). table 1 : the effect of ginger extract on the serum levels of ast, alt and tsb of the treated animals. each value represents mean ± sd. * p < 0.05 in respect to the gp i . s = significant difference in respect to the gp ii. n = number of animals ginger (1g/kg) + cisplatin ( grp.v) ginger (0.5g /kg) + cisplatin ( grp.iv) ginger (1g/kg) alone ( grp.. iii) cisplatin (10mg/kg) ( grp.ii) control (grp.i) groups n=6/each group 8.5 ± 1.64 s 52.78 % 9.17 ± 1.94 s 49 % 5.33±1.37 18% 18± 2.28* 177 % 6.5± 1.05 serum ast level (u/l) and percent of change 5 ±1.41 s 50.84 % 5.17 ±1.17 s 49.1 % 3.67±1.37 18.4% 10.17 ± 2.99* 126 % 4.5± 0.55 serum alt level (u/l) and percent of change 2.25 ± 0.21 s 63.4% 2.55 ± 0.15 s 58.5 % 1.38 ± 0.15 1.47% 6.15 ± 0.83* 352 % 1.36±0.29 tsb level (u/l) and percent of change iraqi j pharm sci, vol.21(1) 2012 protective effect of ginger extract 29 table(2) indicated that serum levels of ldh and ck were significantly elevated in the gp.ii in comparison with the gp.i (p<0.05), while in gp.iv and gp.v the serum levels of ldh and ck were significantly reduced in comparison to the gp.ii (fig.7& 8).histological examination of tissue sections from the heart muscle clearly showed degeneration and necrosis of cardiac muscle fiber cells with fibrous tissue reaction were observed in the gp.ii (fig.10). while in gp.v the cardiac tissues was protected against cisplatin-induced damage and shows normal tissue with no significant degenerative changes when compared with the gp.i heart tissue slide ( fig. 9&11). table (2):the effect of ginger extract on the serum levels of ldh and ck of the treated animals. each value represents mean ± sd. * p < 0.05 in respect to the gp i. s= significant difference in respect to the gp ii. n = number of animals figure 1 : effect of ginger extract on the serum levels of aspartate aminotransferase (ast) in rats with hepatotoxicity induced by cisplatin . *p<0.05 in comparison with the gp i. s = significant difference in respect to the gp ii. figure 2 : effect of ginger extract on the serum levels of alanine aminotransferase (alt) in rats with hepatotoxicity induced bycisplatin. *p<0.05 compared with the gp l. s=significant difference in respect to the gp ii. figure 3: effect of ginger extract on the serum levels of total serum billirubin (tsb) in rats with hepatotoxicity induced by cisplatin *p<0.05 compared with the gp i. s= significant difference in respect to the gp ii. figure 4 : section showing the normal liver tissue of rats as control group. magnification :100x , staining : haematoxylline &eosin. ginger (1g/kg) +cisplatin ( grp.v) ginger (0.5g /kg) + cisplatin ( grp.iv) ginger (1g/kg) alone ( grp. iii) cisplatin (10mg/kg) ( grp.ii) control (grp.i) groups n=6/each group 156 ± 30.2 s 43.37 % 176.7 ± 32.8 s 36 % 96.7 ± 14.5 1.86% 275.6 ± 29.26* 179.58 % 98.6± 18.04 serum ldh level (u/l) and percent of change 96.57 ± 3.59 s 35.13 % 128.71±7.93 s 13.54 % 75.73± 6.21 3.81% 148.87± 10.4* 89 % 78.73± 5.73 serum ck level (u/l) and percent of change iraqi j pharm sci, vol.21(1) 2012 protective effect of ginger extract 30 figure 5 : section showing morphological alteration of liver tissue for cisplatin –treated rats. blue arraw represents hepatocyte degeneration. yellow arraw represents hepatocyte necrosis. red arraw represents inflammatory cells infilteration around portal area (white arraw ) . green arraw represents sinusoidal dilatation . magnification :200x, staining :haematoxylline &eosin. figure 6: section showing near normal structural appearance of hepatocytes in gp vby administration 1g/kg/day ginger extract against cisplatin-induced liver damage. blue arraw represents normal hepatocyte.yellow arraw represents normal central vein. magnification : 200x, staining :haematoxylline & eosin. 0 50 100 150 200 250 300 gp i gp ii gp iii gp iv gp v ldh levels(u/l) * s s figure 7: effect of ginger extract on the serum levels of lactate dehydrogenase (ldh) in rats with cardiotoxicity induced by cisplatin . *p< 0.05 compared with the gp i. s= significant difference in respect to the gp ii. 0 20 40 60 80 100 120 140 160 180 gp i gp ii gp iii gp iv gp v ck levels u/l s s * figure 8 : effect of ginger extract on the serum levels of creatine kinase (ck) in rats with cardiotoxicity induced by cisplatin . *p<0.05 compared with the gp i. s= significant difference in respect to the gp ii. figure 9: section showing the normal cardiac muscle fiber cells of rats as control group . magnification :200x, staining :haematoxylline &eosin. figure 10: section showing morphological alteration of cardiac muscle fiber cells from cisplatin-treated rats( gp ii). yellow arraw represents degeneration and necrosis of myocardial fibers cells. blue arraw represents fibrous tissue reaction. magnification :200x, staining : haematoxylline &eosin. iraqi j pharm sci, vol.21(1) 2012 protective effect of ginger extract 31 figure 11 :section of heart from rats treated by ginger extract 1g/kg/day( gp v). showing near normal like structure appearance of cardiac muscle fiber cells (blue arraw). magnification : 200x, staining :haematoxylline &eosin. discussion the cytotoxic effect of cisplatin is enhanced by the elevation of the dose, however,at higher doses, the less common toxic effects, such as hepatotoxicity, may arise (16) .it has been suggested that oxidative stress is an important mechanism of cisplatin-induced toxicity possibly due to depletion of reduced glutathione gsh (17) , also many studies reported that there were a significant elevation in the hepatic malonaldehyde( mda) and reduction in the level of antioxidant enzymes in rats treated with cisplatin (18,19) . transaminases are the most sensitive biomarkers directly implicated in the extent of cellular damage and toxicity because they are cytoplasmic in location and are released into the circulation after cellular damage (20) , elevation of the serum levels of the hepatic enzymes and bilirubin are the indicators for impaired liver functions (21) . in this study,the hepatotoxicity of cisplatin was clearly observed through an a significant elevation of serum ast,altand tsb levels in cisplatin-treated rats compared with the control (fig.1,2&3) as it had been previously reported that cisplatin administration causes deteriorations of liver function tests such as serum alt, ast, ldh and tsb revealed hepatic dysfunction, which could be a secondary event following cisplatin-induced liver damage with the consequent leakage from hepatocytes (19,21&22) . several reports (23,10) which showed the protective effects of ginger extract or its constituents, through their antioxidant properties and improve the hepatic dysfunctions and hepatic damage that induced by hepatotoxicants, ccl4 and acetaminophen. results of this study demonstrated that ginger extract improve the elevated levels of the serum ast , alt and tsb when compared to the cisplatin treated group (p<0.05), and these may be attributed to ginger components which may stabilize hepatocytes plasma membrane and prevent delivery of ast and alt to the extracellular fluid (10) . histopathological changes observed in the present study including necrosis and degeneration of hepatocytes with inflammatory cells infilteration around portal area with sinusoidal dilatation ( fig.5) are consistent in general with the other reports (6, 24& 25) , and these changes were nearly normalized, when ginger extract in dose of 1g/kg/day was co-administered with cisplatin (fig. 6). in addition to antioxidant effects , ginger may also exert its hepatoprotective effect by means of different ways. for example , in the mechanism of cisplatin toxicity it was shown that cisplatin induces liver cells apoptosis by cytochrome-c release and caspase 3 release activation and causes hepatotoxicity by increasing messenger ribonucleic acid( mrna) expression of nuclear factor-kappa b (nf-ĸb) dependent cyclo-oxygenase (cox-ii) and inducible nitric oxide synthase (inos) (26) . however, ginger shows anti-inflammatory action by direct inhibition of cox activity (27) , also exhibits greater inhibitory activity toward the evolution of pro-inflammatory signaling compound prostaglandin-e2( pg-e2) from cox-ii in lipopolysaccharide-activated macrophages (28) , also cisplatin hepatotoxicity was shown to be exacerbated by elevated expression of cytochrome p450-2e1 enzyme (29) , on the other hand foster et al. (2003) demonstrated that ginger components showed significant inhibition of cytochrome p450 mediated metabolism of marker substrates (30) and also ginger prevents bromobenzene-induced hepatotoxicity by blocking the enzyme cytochrome p4502e1 (31) .in this study ,the group administered a single ip dose of cisplatin(10mg/kg) revealed significant elevation of serum ldh and ck levels compared to the control rats (fig.7&8), are consistent with those observed in other studies (3,32) which were reported that cisplatininduced cardiotoxicity could be a secondary event following cisplatin-induced lipid peroxidation of cardiac membranes with the consequent increase in the leakage of ldh and ck from cardiac myocytes . concerning the histological changes ,the cardiac damage produced by cisplatin revealed degeneration and necrosis of cardiac muscle fiber cells with fibrous tissue reaction,(fig.10), are consistent in general with findings observed by al-majed et al. in 2006 which showed degenerative changes, vacuolated cytoplasm of many muscle cells and blood vessels are engorged with blood (32) .kidney damage induced by cisplatin iraqi j pharm sci, vol.21(1) 2012 protective effect of ginger extract 32 may lead to inhibition of carnitine synthesis and also inhibition of carnitine reabsorption by the proximal tubule of the nephron consequently leading to carnitine deficiency . this marked decrease (78%) of carnitine level in cardiac tissue after treatment of cisplatin was parallel to the marked increase in ldh and ck and the degenerative changes in cardiac tissues, which may point to the possible consideration of carnitine deficiency as a risk factor in cisplatin-induced cardiomyopathy (32) . cisplatin elevates serum cardiotoxicity enzymatic indices (ldh and ck) and causes severe histopathological lesions in cardiac tissues,the effect could be a secondary event following cisplatin-induced lipid peroxidation of cardiac membranes with the consequent increase in the leakage of ldh and ck from cardiac myocytes (32) . there are many evidences deal with the administration of antioxidants may be effective in ameliorating cisplatin-induced cardiotoxicity, acetyl-l-carnitine, dl-α-lipoic acid and silymarin, which have been proven to possess antioxidant potentials, appear to be potential candidates to ameliorate cardiotoxicity associated with cisplatin use in rats (33) . the cardiac protection of ginger is very well evident in this study, where increasing the dose consequently reflected in better protection; the reduction of serum enzymes levels and reversing the histological changes revealed by low incidence of degeneration and necrosis in addition diminishing fibrous tissue reaction as shown in figures 7,8&11.mansour et al. in 2008 showed that 6-gingerol act as apotentially selective cardioprotective agent , against cardiotoxicity induced by doxorubicin by augmentation of endogenous myocardial antioxidants activities (33) . the extent of cardioprotection offered by ginger is associated with a significant attenuation of serum ldh ,ck ,ast and alt levels , a possible explanation is that, ginger , via its effect against lipid peroxidation, causes stabilization of cardiac membranes and prevents the leakage of cardiac enzymes, also may be due to amelioration of renal functions and inhibition of suppression of carnitine levels and antioxidant enzymes such as catalase and superoxide dismutase (34) .in conclusion oxidative stress plays a major role in cisplatininduced toxicities during the normal clinical regimens of treatment. antioxidants have proven to be effective in ameliorating cisplatininduced toxicity in many preclinical and few clinical interventions. ginger extract is a potent antioxidant which is reported to have antitumor effect and to enhance the effect of many known anticancer agents in addition to reducing their toxicities as well.ginger extract prior and co-administration with cisplatin provided near complete protection in terms of plasma biochemical changes and organs histological changes . references 1. ali, b.h., al moundhri, m.s., agents ameliorating or augmenting the nephrotoxicity of cisplatin and other platinum compounds: a review of some recent research. food chem. toxicol. 2006; 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[6]-dehydroshogaol, a minor component in ginger rhizome, exhibits quinone reductase inducing and antiinflammatory activities that rival those of curcumin. food research international. 2010; 43: 2208–2213. 29. a.a. caro, a.i. cederbaum,. oxidative stress, toxicology, and pharmacology of cyp2e1, annu. rev. pharmacol. toxicol. 2003; 44: 27–42. 30. foster, b.c., vandenhoek, s., hana, j., krantis, a., akhtar, m.h., bryan, m., budzinski,j.w., ramputh, a., arnason, j.t., in vitro inhibition of human cytochrome p450-mediated metabolism of marker substrates by natural products.phytomedicine. 2003; 10: 334–342. 31. el-sharaky as, newairy aa, kamel ma, eweda sm., protective effect of ginger extract against bromobenzene-induced hepatotoxicity in male rats. food chem. toxicol. 2009; 47: 1584–90. 32. al-majed, a.a., sayed-ahmed, m.m., alyahya, a.a., aleisa, m.a., al-rejaie, s.s., al-shabanah, o.a. propionyl-l-carnitine prevents the progression of cisplatin induced cardiomyopathy in a carnitine-depleted rat model. pharmacol. res. 2006; 53:278–286. 33. m. a. mansour, s. a. bakheet, a. m. aleisa, s. s. al-rejaie,a. a. al-yahya, m. elameen and o.a.al-shabanah. protective effect of 6-gingerol against cardiotoxicity induced by doxorubicin. the open pharmacology journal.2008; 2: 20-23. 34. ansari, m.n., bhandari, u., pillai, k.k., ethanolic zingiber officinale r. extract pretreatment alleviates isoproterenol-induced oxidative myocardial necrosis in rats. indian j. exp. biol. 2006 ; 44: 892–897. iraqi j pharm sci, vol.32( 1 ) 2023 protective effects of n-acetylcysteine on 5-fluorouracil intestinal toxicity in rats doi: https://doi.org/10.31351/vol32iss1pp40-44 40 the protective effects of n-acetylcysteine against 5-fluorouracil induced intestinal toxicity in albino rats muna z.al-hamdany*,1and abduljabar y. al-hubaity* *department of anatomy, college of medicine, university of mosul, mosul, iraq. abstract 5-fluorouracil (5-fu) is a pyrimidine analogue widely used in the treatment of various malignancies. it belongs to the antimetabolites family that acts during the s-phase of the cell cycle thus it prevents dna synthesis. n-acetylcysteine (nac) is a nutritional complement that acts as antioxidant. the purpose of the current study is to investigate whether there is a protective role of n-acetylcysteine against intestinal toxicity induced by 5fluorouracil in albino rats. eighteen healthy adult male rats were distributed into 3 groups of 6 rats for each. group a is a control group. group b, rats injected with 5-fu (20 mg dissolved in 2ml normal saline per kilogram body weight intraperitoneally for 7 successive days. group c, rats received n-acetylcysteine 200 mg per kilogram body weight 24 hour prior to 5-fu injections for 7 consecutive days. the animals were sacrificed one day after the last injection; specimens of the intestine (colon) tissue of the three groups were removed and prepared for light microscopic examination. results showed an increase in the depth of the colonic crypts in group b rats as compared to the control group, mucinous degeneration of the intestinal mucosal cells along with necrosis, and inflammatory cells infiltration in the lamina propria. the appearance of the crypts is nearly normal in group c with reduction in the depth and normal columnar epithelium lining the crypts the study concluded that 5-fu seriously affects the structure of the intestinal tissue and pretreatment with nac protects the intestinal tissue against the toxic effects provoked by 5-fu. keywords: n-acetylcysteine, 5-fluorouracil, intestinal, toxicity, rats. خمسة ضد السمية المعوية التي يحدثها عقار ان اسيتايلسيستين لعقارالمحمية التأثيرات البيض في الجرذانفلورويوراسيل *الجبار ياسين الحبيطي وعبد 1*،منى زهير الحمداني العراق الموصل، الموصل، جامعة الطب، التشريح، كليةفرع * الخالصة الدراسة هذهفي البيض في الجرذانفلورويوراسيل خمسة ضد السمية المعوية التي يحدثها عقار ان اسيتايلسيستين لعقارالمحمية التأثيرات ( غرام , تم تقسيمها الى ثالث مجاميع 250-200( أشهروأوزانها مابين )3-2.5البيض البالغة تتراوح اعمارها مابين ) انذالجرثمان عشرة من ذكور وهي مجموعة السيطرة فقطمحلول الملح القياسي aحيوانات.أعطيت المجموعة 6كل مجموعة تضم cو مجموعة bمجموعة aمتساوية مجموعة 200أعطيت )cاسبوع, المجموعة الثالثة لمدة من وزن الجسم(يوميا مليغرام لكل كيلوغرام 20) عقار خمسة فلورويوراسيلأعطيت b,المجموعة اسبوع .أظهر الفحص لمدةل و عقار خمسة فلورويوراسيستين بيوم واحد قبل اعطاء كل جرعة من ( من عقار ان اسيتايل سيمليغرام لكل كيلوغرام منطقة بؤرية للنخر محاطة بترسب انحالل فجوي للخاليا الكأسية المبطنة للتجاويف الغدية اضافة الى b النسيجي لالمعاء في المجموعة الثانية ة فقد لوحظ استعادة التركيب الطبيعي للخاليا المبطنة لتجاويف القولون الغدية نسيجا طالئيا سليما في الغشاء الخاليا األلتهابية أما في المجموعة الثالث فلورويوراسيل يحدث تغييرات واضحة في نسيج االمعاء والتي يمكن تقليلها باستخدام خمسة المخاطي السطحي.نستنتج من هذه الدراسة ان عقار تخدم كمكمل غذائي لمرضى السرطان ويستخدمون العالج الكيمياوي. والذي يس اسيتايلسيستين عقار جرذان ،سمية ،معوية ، فلورويوراسيل خمسة ، ان اسيتايلسيستينالكلمات المفتاحية: introduction chemotherapeutic drugs have been used worldwide for the treatment of a variety of neoplasm given as a single or combined treatment protocol (1). 5-fluorouracil (5-fu) is pyrimidine analogue that belongs to the family of antimetabolites. it is s-phase specific drug which principally inhibits thymidine synthase (ts) enzyme resulting in a decreased dna synthesis. moreover, it interferes with rna processing and protein synthesis (2).the cytotoxic effects of 5-fu might be exerted by the generation of reactive oxygen species ( ros ) , upward suggestion implies that stem cells of the tumor may increase stemness of cancer cells causing 5-fu resistance (3). 5-fu has a half-life of approximately 10 minutes; just about 15-25% of the administered dose is excreted in urine (4). 5-fu is corrupted by the hepatic dihydropyridine dehydrogenase (dpd), which is the initial and rate limiting enzyme in 5-fu catabolism (5). it is used in the management of advanced colorectal cancer, breast cancer, carcinoma of the stomach and in ophthalmic surgery (6). some adverse effects include stomatitis, mucositis and diarrhea (7). 1corresponding author e-mail: mza@uomosul.edu.iq received: 29/12 /2021 accepted: 27/2 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp40-44 iraqi j pharm sci, vol.32( 1 ) 2023 protective effects of n-acetylcysteine on 5-fluorouracil intestinal toxicity in rats 41 extensive investigations have been conducted on the 5-fu induced hepatotoxicity, cardiotoxicity and neurotoxicity and pulmonary toxicity (8).however, few accounts on the mechanism of intestinal toxicity caused by 5-fu. n-acetylcysteine (nac) is the nacetyl derivative of the amino acid l-cysteine, nac is the drug of choice in acetaminophen overdose which is used frequently in self-poisoning (10). it exhibits direct antioxidant effect through its free sulphydryl (thio) group, nac exerts an indirect antioxidant effect as a precursor of glutathione (11). formerly, there is some evidence supporting the use of n-acetylcysteine as an adjunctive therapy for covid-19. further randomized clinical trials studies are justified to establish the optimal dosage and path of administration, and to determine the efficiency and safety of n-acetylcysteine in the management of covid-19(12). the aim of present work is to assess the defensive role of nac against damage induced by 5-fu in the colon of albino rats. materials and methods agreement from the medical research ethics committee in the college of medicine, university of mosul has been obtained. lethal dose, pilot studies and related literatures were taken into account and the accurate doses of 5-fu and nac were calculated (13, 14). eighteen adult healthy male albino rats their age between (2.5-3 months) and their body weight ranges from 200 to 250 grams were obtained from the college of veterinary medicine, university of mosul. the animals were housed in a standard condition. the body weight of each rat was recorded at the beginning of the experiment before the injection of 5-fu and recorded again at the end of the experiment just before killing of the animals. the animals were randomly divided into 3 groups (6 animals each). group a (control group): each animal of this group was given 2 ml /kg body weight /day of normal saline by intraperitoneal injection for 7 consecutive days and served as a control group. group b (5-fu recipient group): each animal of this group was given 5-fu in a dose of 20mg/kg/day by intraperitoneal injection for 7 consecutive days. group c (nac+5-fu recipient group): each animal first received nac in a dose of 200 mg/kg body weight by intraperitoneal injection as a single dose or for 7 days and 6 hours after each injection of nac, each animal was given 5-fu in a dose 20mg/kg/day by the intraperitoneal route for 7 consecutive days. one day after the last injection of the 3 groups, the animals were sacrificed and dissected under light ether to collect the colon specimens and were immediately fixed in 10% neutral buffered formalin solution for 24 hours. the histological sections were prepared according to bancroft et.al, 1994 (15) in which small pieces of about 4-5 mm in thickness were cut from each colon and dehydrated in ascending grades of ethanol then cleared by xylene and embedded. serial sections of about 5 microns in thickness were obtained and stained with harris haematoxylin and eosin stain and masson's trichrome stain then examined microscopically. statistical analysis was performed by spss version 20 for windows software. data were presented as mean ± sd and were analyzed using one-way analysis of variance (anova). the limit point for statistical significance was set at 0.05 thus ≤ 0.05 reflect a significant and> 0.05 reflect a nonsignificant value. results the animals of the control group remained alive, active with good appetite whereas the treated group became less active and grouped at one place of the cage. some rats had frequent diarrhea and loss of hair. the data of body weight were expressed as mean±sd. the substantial significant drop (p =0.001) of the animals' mean body weight was observed in the treated group (5-fu recipient group) compared with the control group)(table 1). microscopic findings the control group showed normal mucosal lining with intact surface epithelium & closely packed simple straight tubular intestinal glands (colonic crypts) in the lamina propria (figure.1). the mucosal surface epithelium and the epithelial lining of the crypts were formed of simple columnar cells with oval nuclei basally located & numerous goblet cells (figure 2). the group which received 5fu showed increasing in the depth of the crypts with vascular degeneration in the serosa (figure 3), mucinous degeneration of the goblet cells, which is nearly obliterating the lumen of the crypts with inflammatory cells in the lamina propria (figure 4), desquamation of the surface epithelium lining the mucosa with some necrosis (figure 5). the group which received nac then 5-fu showed nearly normal appearance of the crypts which are covered by columnar epithelium with only few mononuclear cells infiltration between the goblets (figure 6). iraqi j pharm sci, vol.32(1) 2023 protective effects of n-acetylcysteine on 5-fluorouracil intestinal toxicity in rats 42 table 1. the mean changes of the body weights at the beginning and the end of the experiment groups n body weight (mean ± sd) at the beginning of the experiment body weight (mean ± sd) at the end of the experiment statistical significance among the groups pvalues a control 6 152.00 ± 16.43 155.50 ± 17.70 a vs. b=(vhs) a vs. c = (ns) 0.001** 0.1 b 5-fu 6 160.50± 69.72 106.60 ± 39.39 b vs. a = (s) b vs. c = (ns) 0.02 0.4 c nac+5 -fu 6 185.00 ± 21.67 159.20 ± 28.10 c vs. a = (ns) c vs. b = (ns) 0.6 0.3 sd= standard deviation; s=significant (p≤0.05); ns=non-significant (p>0.05); vhs= very high significant (p<0.01); vs. =versus; a=control group; b=5-fu recipient group; c= nac+5-fu group. figure1. micrograph of the colon of group a (control groups) showing surface epithelium (black arrows), intestinal glands in the lamina propria (white arrows) (h&e x 100). figure 2.micrograph of colon of group a (control groups) showing intestinal glands lined by simple columnar cells with basal oval nuclei (black arrows) and goblet cells in between them (arrow heads), a blood vessel in the lamina propria (bv) (h&e x 400). figure 3. micrograph of colon of group b showing elongation of colonic crypts occupying most of the mucosa (black arrows) (h&e x 40). figure 4. micrograph of colon of group b showing mucinous degeneration of goblet cells nearly obliterating the colonic crypts (white arrows), inflammatory cells infiltration in the lamia propria (yellow arrows)(h&e x150). figure 5. micrograph of colon of group b showing desquamation of the surface epithelium lining the colonic mucosa (white arrows) (h&e x150). figure 6. micrograph of colon of group c showing the columnar epithelium lining the colonic crypts (white arrows) with few mononuclear cells infiltration in between the crypts(black arrows)(h&ex150). iraqi j pharm sci, vol.32(1) 2023 protective effects of n-acetylcysteine on 5-fluorouracil intestinal toxicity in rats 43 discussion in the present study, the structural changes in the colon induced by 5-fu, as degenerative changes with elongation of the colonic crypts which are lined by degenerated goblet cells observed in the 5-fu recipient group could be attributed to the epithelial damage induced by oxidative stress provoked by 5-fu which contribute to intestinal mucositis, in addition to the alteration of epithelial function mediated by chronic inflammatory cells as mast cells (16). the mucinous degeneration of the goblet cells might be a consequence of stem cells damage, which suppresses the renewal of goblet cells (17). the inflammatory cells infiltration observed in the mucosa and submucosa of colon induced by 5-fu is similar to the intestinal mucositis which precedes gut dysbiosis in the mouse model following the exposure to irradiation (18). previous studies suggested that 5-fu might increase the release of pro-inflammatory cytokines like prostaglandins, interleukins and tumor necrosis factors and it also increases the release of 5hydroxytryptamine (5-ht) from chromaffin cells of the intestinal mucosa (19). desquamation and necrosis of the epithelium lining the colon observed in 5-fu recipient group could be attributed to stromal edema and mucosal damage mediated by oxidative damage similar observation previously noticed in the rats treated with methotrexate (20). partial occlusion of the lumen of colonic crypts observed in the 5-fu recipient group might be due to proliferation of epithelium lining the crypts, such finding is in agreement with those reported in the intestinal mucositis induced by chemotherapeutic drugs due to inflammation in the mucosa arising from stem cell apoptosis and disturbed cellular renewal and maturation processes (21). the appearance of nearly normal epithelial lining the colonic crypts with few mononuclear infiltration in group c indicates the defensive effect of nac against the colonic mucosal damage induced by 5-fu. this is in agreement with what has been reported when dietary supplement with nac can alleviate colitis induced by acetic acid in a pig model (22). such antioxidant role of nac has been reported by shahripour et.al, 2014 (23), who observed a protective action of nac & improved the clinical status in chronic neurological disorders and similarly the damaged intestinal mucosa induced by 5-fu in rats was markedly minimized by the administration of curcumin probably by the same antioxidant mechanism as that of n-acetylcysteine (24). conclusion the use of 5-fu for the treatment of some tumors seriously affects the structure of the intestinal tissue causing increase in the depth of the colonic crypts, mucinous degeneration of the intestinal mucosal cells along with necrosis , and inflammatory cells infiltration. pretreatment with nac protects the intestinal tissue against the toxic effects provoked by 5fu. thus, nac may be considered as a useful dietary supplement for patients taking antineoplastic drugs like 5-fu. acknowledgment we would like to precise our deepest appreciation to all staff members in the department of anatomy in the mosul college of medicine for their support. a special thanks to all the staff of college of veterinary medicine for their aid in the histopathological assessment of the sections. conflict of interest the author declares that there are no conflicts of interest regarding the publication of this manuscript. references 1. falzone l, salomone s, libra m.evolution of cancer pharmacological treatments at the turn of the third millennium.front pharmacol 2018; 9(1): 1300. 2. dchar m, fenouil t, machon c, vincent a, marcel f, mertani h, bouvet 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exploring the capability of the hospital pharmacists in conducting pharmacy practice research: a study from malaysia ali qais blebil*,1, juman abdulelah dujaili*,**, ali haider mohammed*, ahmed awaisu***, mohamed azmi ahmad hassali****, bassam abdul rasool hassan*****, abdulrasool m. wayyes***** *school of pharmacy, monash university malaysia, selangor, malaysia **swansea university medical school, swansea university, swansea, uk *** college of pharmacy, q.u. health, qatar university, doha, qatar **** discipline of social and administrative pharmacy, school of pharmaceutical sciences, university sains malaysia, penang, malaysia ***** department of pharmacy, al rafidain university college, baghdad, iraq abstract the engagement of pharmacists in research activities is pivotal in the advancement of the pharmacy practice. the study aims to evaluate the confidence and competence of malaysian hospital pharmacists in conducting clinical and practice-based research. a cross-sectional study was carried out between september 2019 and april 2020 using an online survey. pharmacists from eight different hospitals in malaysia were involved in the study. the survey link was sent to all pharmacists of the included hospitals via email. data were analysed using spss version 25. a total of 226 pharmacists participated in this study, and their average age was 28 years old. about 82 % of the participants reported that they did not have any previous research experience, and around 62% of them indicated that the research training during their undergraduate study was inadequate. at least 60% of the participants reported inadequate competence and/or confidence in developing research protocols, critically appraising the literature, undertaking and applying appropriate statistical techniques, and interpreting research findings. there is an urgent need to reinforce undergraduate and postgraduate research training in the institutions among potential and current pharmacists to build competence in research techniques such as literature reviews and scholarly participation. keywords confidence, competence hospital pharmacists, pharmacy practice research ماليزيا من دراسة :الصيدالنية الممارسة أبحاث إجراء على المستشفيات صيادلة قدرة استكشاف *** عويسو أحمد ، *محمد حيدر علي ، * *، *الدجيلي اإللهعبد جمان ،1*،بليبل قيس علي ***** ويس محمود الرسول عبد و *****حسن الرسول عبد بسام ، ****حسالي أحمد عزمي محمد ماليزيا ، سيالنجور ، ماليزيا موناش جامعة ، الصيدلة كلية* المتحدة المملكة ، سوانزي ، سوانسي جامعة ، سوانسي بجامعة الطب كلية ** قطر ، الدوحة ، قطر جامعة ، الصحية قطر جامعة ، الصيدلة كلية *** ماليزيا ، بينانج ، ماليزيا سينز جامعة ، الصيدالنية العلوم كلية ، واإلدارية االجتماعية الصيدلة تخصص **** العراق ، بغداد ، ةالجامع الرافدين كلية ، الصيدلة قسم ***** الخالصة صيادلة وكفاءة ثقة تقييم إلى الدراسة تهدف .الصيدلة بممارسة النهوض في محوريًا أمًرا البحثية األنشطة في الصيادلة مشاركة تعد . والممارسة السريرية البحوث إجراء في الماليزية المستشفيات في مختلفة مستشفيات ثمانية من صيادلة .اإلنترنت عبر استطالع باستخدام 2020 وأبريل 2019 سبتمبر بين مقطعية دراسة إجراء تم البيانات تحليل تم .اإللكتروني البريد عبرالمشاركة المستشفيات في الصيادلة جميع إلى االستبيان رابط إرسال تم .الدراسة في شاركت ماليزيا .25االصدار االجتماعيةالحزمة اإلحصائية للعلوم برنامج باستخدام أي لديهم ليس أنه المشاركين من ٪82حوالي أفاد .عاًما 28 أعمارهم متوسط وكان ، صيدليًا 226مجموعه ما الدراسة هذه في شارك المشاركين من األقل على ٪ 60 أفاد . كافياً يكن لم الجامعية دراستهم أثناء البحثي التدريب أن إلى منهم ٪62 حوالي وأشار ، سابقة بحثية خبرة وتفسير ، المناسبة اإلحصائية التقنيات بتطبيق والقيام ، نقدي بشكل األدبيات وتقييم ، البحث بروتوكوالت تطوير في الثقة أو / و الكفاءة كفاية بعدم البحث. نتائج الكفاءة لبناء والحاليين لمستقبليينا الصيادلة بين المؤسسات في والخريجين الجامعيين للطالب البحثي التدريب لتعزيز ملحة حاجة هناك . العلمية والمشاركة األدبيات مراجعات مثل البحث تقنيات في الصيدالنية الممارسة بحوث ، المستشفيات صيادلة ، الكفاءة ، الثقة : المفتاحية الكلمات 1corresponding author e-mail: aliblebil@yahoo.com received: 28/11/2021 accepted: 15/11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp71-82 iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 72 introduction in recent years, the professional roles of practising pharmacists have evolved in many parts of the world, including malaysia (1). as a result, there is increasing recognition of existing and emerging pharmacy services, roles, and models of practice geared toward improving patient care (2, 3). to support this paradigm shift, practicing pharmacists should be equipped with essential skills to conduct pharmacy practice research (4-6), thus improving the overall healthcare outcomes (2, 79). likewise, pharmacy practice in malaysia and other asian countries has evolved toward direct patient care and advanced pharmacy services. however, sufficient evidence has demonstrated that pharmacists' involvement in practice research is limited globally (10). several studies have reported that hospital pharmacists are ill-equipped in terms of practicebased research skills (5, 11-16). these studies also reported a lack of training on research as one of the main barriers to participation in practice research. in addition, pharmacists have also reported a lack of competence and skills to undertake high-quality research. therefore, this highlights the need for research capacity building to strengthen hospital pharmacists' research competencies and capacities. research capacity building is a driving force to developing and enhancing research culture and improving research skills within pharmacy practice (17). in malaysia, the culture of conducting research related to pharmacy practice and services has progressed at a faster pace in recent years. the malaysian government, via the pharmaceutical services programme, recognized scientific research as fundamental to aid policy decision-making and achieve the pharmacy programme strategic plan (18). despite the increasing attention among pharmacists regarding the preparation and competencies required to seek and succeed in a research career (7-9, 19), few pharmacists have the opportunity to join formal graduate programs to expand their research capacity, especially those who practice in a busy hospital environment. in a recent study undertaken by tan and hatah (20), malaysian pharmacists with less involvement in research activities had low to moderate utilisation of research evidence in their practices. this suggests the implication of research skills on evidence-based practice among practising pharmacists. to date, information regarding the competence and ability of hospital pharmacists related to practice-based research is limited and not widely documented in malaysia. it is important to evaluate if the malaysians hospital pharmacist’s workforce adequately trained and prepared to face the current challenges of and quest for cutting-edge health-related research as it is part of their competent skills that they should equipped in the delivery of pharmaceutical care. hence, the current study aimed to assess malaysian hospital pharmacists' confidence and competence in conducting clinical and practice-based research. methods study design, setting and recruitment procedure a multi-centered cross-sectional, online survey using surveymonkey® software was conducted to assess the malaysian hospital pharmacists' confidence and competence in conducting clinical and practice-based research. pharmacists working in public and private hospitals across kuala lumpur, selangor, putrajaya, and perak states in malaysia were approached. eight hospitals that provided approval from their administration were included. the link to the survey was shared with the research officers of the respective hospitals. the survey link was then sent to all pharmacists of the included hospitals via email. reminders were sent out every two weeks on several occasions over seven months. all information collected from the study was anonymized and kept confidential. participants and sampling registered pharmacists or provisionally registered pharmacists (prps) practicing in malaysia during the study were included in the data collection period. the data were collected between september 2019 and april 2020. the survey included electronic participant information and a consent form. participants were asked to confirm their willingness and consent to participate by clicking a checkbox. those who opted not to participate were automatically signed out of the online survey and could not proceed further. the minimum required sample size for the study calculated using the raosoft calculator was approximately 267. however, all pharmacists in the approved sites were approached to participate in the study due to the limitations associated with the low response rate of the online survey reported previously by other studies (21-23). study instrument the questionnaire adapted from a previous study (17, 24) comprised of four sections as follows: (1) respondents' demographics; (2) background in research activities; (3) pharmacists' competence and confidence in planning and conducting research and; (4) pharmacists' preferences for capacity building and formal postgraduate training. the questionnaire consists of 72 items (24). cronbach's alpha coefficients for the competence and confidence domains were determined as 0.96 and 0.98, respectively. data analysis the data were analyzed using ibm statistical package for social sciences (ibm spss software), version 24. all categorical variables, iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 73 including respondents' sociodemographic and professional characteristics, items assessing competence and confidence in research, and other attitudinal items, are expressed as counts and percentages. ethical approval ethical approval was obtained from the medical research ethics committee (mrec) of the ministry of health, malaysia (nmrr-19-65746520-iir) and monash university human research ethics committee (ref. no: 2019-2135434143). additional approval was acquired from each hospital included in the study before commencing the study. results demographic and professional characteristics of the study participants although more than 1000 hospital pharmacists were contacted to increase the response rate, only 226 agreed to participate in the study. the majority of the participants (73.45%) were female. the pharmacists in this study can be grouped distinctly according to the type of hospitals they were practicing in. around 92% of the participants were working in public hospitals. in terms of age, the largest age group was 21-30 years old, which constituted 57.96% of all respondents. about 83% of the pharmacists attained their first professional pharmacy degree from malaysia, while 16.81% obtained their degrees from non-malaysian institutions such as the u.k. and new zealand. the highest degree obtained by the majority (79.65%) was a bachelor of pharmacy degree. more than half of the pharmacists (53%) who responded to the survey had been practicing in a hospital pharmacy setting for five years or less (table 1). table 1. demographic and professional characteristics of malaysian hospital pharmacists who responded to the survey (n = 226) characteristic* frequency (%) gender male 60 (26.55) female 166 (73.45) age (years) mean (sd) : 28 ± 7.82 21-30 years old 131 (57.96) 31-40 years old 85 (37.61) 41-50 years old 9 (3.98) 51 years and above 1 (0.45) country from which they obtained their first professional degree malaysia 188 (83.19) non-malaysian country 38 (16.81) highest level of education completed bachelor’s degree (bpharm) 180 (79.65) master’s degree (ms, msc, mpharm, mba) 43 (19.03) doctor of philosophy (phd) 3 (1.32) number of years spent in pharmacy practice 5 years or less 119 (52.65) 6-10 years 62 (27.43) 11-15 years 36 (15.93) more than 15 years 9(3.99) number of years spent as hospital pharmacist in malaysia 5 years or less 142 (62.83) 6-10 years 52 (23.01) 11-15 years 25 (11.06) more than 15 years 7 (3.10) type of hospital they are working at public hospital 208 (92.04) private hospital 18 (7.96) research background and the interests of the hospital pharmacists who participated in the study the majority of the pharmacists who participated in the study (82.35%) indicated that they did not have previous experience in conducting research, and over a third of the pharmacists (48.04%) had received training by attending workshops. probing was done to check the pharmacists' perception of the adequacy of the research training they received during their undergraduate studies. less than two-thirds of them (61.76%) reported that their training during their undergraduate study was inadequate compared to only 23.53%, who indicated that the training was adequate (table 2). further probing was done to check the pharmacists' perception of the research training adequacy they received during their postgraduate studies. only 16.66% of the respondents stated that the training they received during their postgraduate study was adequate. concerning pharmacists' perception of the research training adequacy during their career, iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 74 more than half of the participants (52.94%) stated that their training was inadequate compared to 24.51% who stated that their training during their job career was adequate. this was followed by another question to assess pharmacists' interest in conducting pharmacy practice or related research. again, the responses were fairly distributed; less than 10% were highly interested, while 4.41% were totally not interested. most of the respondents reported either never or sometimes to involve in pharmacy or healthcare-related research activities as a research investigator or co-investigator. less than 4% of respondents reported that they were always involved in these researches. items were listed to identify the barriers and challenges that hinder the pharmacists' involvement in research activities. lack of time was the most common barrier to research identified by 86.27% of the participants. furthermore, 71.57% of the pharmacists in this study reported a lack of adequate training. nearly half of the respondents (53.43%) acknowledged that the lack of interest hinders their participation in research activities. interestingly, only 0.98% stated that there were no research barriers, as shown in figure 1. table 2 . research background and interests of malaysian hospital pharmacists who responded to the survey (n= 226) parameter † frequency (%) previous research experience (as investigator, co-investigator, research assistant or associate) yes 168 (82.35) no 36 (17.65) previous research related training during your undergraduate, or postgraduate, or working career (participants were allowed to choose more than one answer) no training obtained 48 (23.53) workshop 98 (48.04) seminar 70 (34.31) specialized short course (1-6 months) 41(20.10) others 15 (7.35) research training received while being in undergraduate pharmacy school not applicable 30 (14.71) inadequate 126 (61.76) adequate 48 (23.53) research training received while being in postgraduate pharmacy school not applicable 141 (69.12) inadequate 29 (14.22) adequate 34 (16.66) research training received during job career not applicable 46 (22.55) inadequate 108 (52.94) adequate 50 (24.51) interest in learning more about conducting pharmacy practice or related research extremely interested 36 (17.65) interested 65 (31.86) somewhat interested 61 (29.90) not very interested 32(15.69) not interested at all 10(4.90) interest in conducting pharmacy practice or related research extremely interested 19 (9.31) interested 67 (32.84) somewhat interested 64(31.37) not very interested 45 (22.06) not interested at all 9 (4.42) involvement in pharmacy or healthcare related research activities as a respondent or subject always 9 (4.41) usually 21 (10.29) often 31 (15.20) sometimes 132 (64.71) never 11 (5.39) overall ability to design and conduct pharmacy practice or related research currently excellent 1 (0.49) very good 15 (7.35) good 47 (23.04) fair 100 (49.02) poor 41 (20.10) iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 75 continued table 2 . parameter † frequency (%) involvement in pharmacy or healthcare related research activities as a research investigator or co-investigator always 8 (3.92) usually 14 (6.86) often 19 (9.31) sometimes 115 (56.37) never 48 (23.54) number of peer-reviewed journal articles published within the last 5 years 0 118 (57.84) 1–3 80 (39.21) ≥4 6 (2.95) number of peer-reviewed posters and/or abstracts in local/regional conference within the last 5 years 0 123 (60.29) 1–3 70 (34.31) ≥4 11(5.40) number of peer-reviewed posters and/or abstracts in international conference within the last 5 years 0 168 (82.35) 1–3 28 (13.73) ≥4 8 (3.92) †22 participants skipped these items figure 1. pharmacists identified barriers to pharmacy practice-based research (n= 226). confidence and competence level of the hospital pharmacists in planning and conducting research the pharmacists were asked to rate their competence and confidence in performing different aspects of designing, conducting, and analyzing research. the relevant data are presented in tables 3 and 4. based on the findings, most hospital pharmacists (not less than 84%) rated themselves as moderately to not competent and confident enough to conceptualize the research ideas. likewise, many of the respondents (more than 70%) believed that they were competent and confident in searching the literature efficiently, writing a research proposal or developing a protocol, collecting relevant data using pre-planned data collection forms, interpretation the findings and determining the significance of obtained results, summarizing the data in tables and/or charts, and preparing an oral or a poster presentation. iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 76 table 3. self-perceived competence of hospital pharmacists in planning and conducting research (n= 226). research competence domain† frequency (%) extremely competent very competent moderately competent not very competent not competent at all conception of research idea 4 (2.13) 25 (13.30) 72 (38.30) 68 (36.17) 19 (10.1) searching the literature efficiently 3 (1.60) 34 (18.09) 92 (48.94) 47 (25.00) 12 (6.37) critically reviewing research literature 4 (2.13) 18 (9.57) 87 (46.28) 60 (31.94) 19 (10.08) formulating research hypotheses and research questions 5 (2.66) 23 (12.23) 79 (42.02) 63 (33.51) 18 (9.58) proposing appropriate study designs or methods 4 (2.13) 20 (10.64) 64 (34.04) 73 (38.83) 27 (14.36) writing research proposal or developing a protocol 4 (2.13) 22 (11.70) 75 (39.89) 64 (34.04) 23 (12.24) defining target population, sample and eligibility criteria 4 (2.13) 31 (16.49) 73 (38.83) 59 (31.38) 21 (11.17) determining appropriate sample size 3 (1.60) 18 (9.57) 64 (34.04) 73 (38.83) 30 (15.96) choosing an appropriate sampling technique (e.g. random sampling 3 (1.60) 24 (12.77) 56 (29.79) 77 (40.96) 28 (14.88) determining outcome measures (variables to measure) 2 (1.06) 25 (13.30) 68 (36.17) 68 (36.7) 25 (12.77) ethical considerations 13 (6.91) 32 (17.02) 71 (37.77) 52 (27.66) 20 (10.64) outlining detailed statistical plans to be used in data analyses 3 (1.60) 20 (10.64) 60 (31.91) 69 (36.70) 36 (19.15) designing a data collection form 9 (4.79) 28 (14.89) 78 (41.49) 51 (27.13) 22 (11.70 ) developing and validating a study instrument (e.g. questionnaire) 3 (1.60) 20 (10.63) 62 (32.98) 66 (35.11) 37 (19.68) collecting relevant data using preplanned data collection forms 11 (5.85) 36 (19.15) 74 (39.36) 50 (26.60) 17 (9.04) managing and storing data including data entry into a database 12 (6.38) 40 (21.28) 65 (34.57) 53 (28.19) 18 (9.58) statistical analyses using software (e.g. stata, spss, epiinfo) 2 (1.06) 20 (10.64) 61 (32.45) 65 (34.57) 40 (21.28) iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 77 continued table (3) choosing and applying appropriate ‘‘inferential’’ statistical tests and methods 1 (0.53) 16(8.51) 53 (28.19) 71 (37.77) 47(25.00) summarizing data in tables or charts 9 (4.79) 39 (20.74) 78 (41.49) 47 (25.00) 15 (7.98) interpretation of the findings and determining the significance of obtained results 3 (1.60) 30 (15.96) 75 (39.89) 56 (29.79) 24(12.76) preparing a presentation (oral or poster) 10 (5.32) 36 (19.15) 76 (40.43) 49 (26.06) 17 (9.04) writing a manuscript for publication in a scientific journal 4 (2.13) 17 (9.04) 58 (30.85) 82 (43.62) 27 (14.36) †38 participants skipped these items table 4 self-perceived confidence of hospital pharmacists in planning and conducting research (n= 226). research confidence domain† frequency (%) extremely confidence very confidence moderately confidence not very confidence not confidence at all conception of research idea 6 (3.45) 20 (11.49) 78 (44.83) 54 (31.03) 16 (9.20) searching the literature efficiently 4 (2.30) 26 (14.94) 85 (48.85) 46 (26.44) 13 (7.47) critically reviewing research literature 3 (1.72) 22 (12.64) 75 (43.10) 57 (32.77) 17 (9.77) formulating research hypotheses and research questions 4 (2.30) 17 (9.77) 76 (43.68) 59 (33.91) 18 (10.34) proposing appropriate study designs or methods 4 (2.30) 17 (9.77) 69 (39.66) 63 (36.20) 21 (12.07) writing research proposal or developing a protocol 3 (1.72) 20 (11.49) 73 (41.95) 58 (33.33) 20 (11.51) defining target population, sample and eligibility criteria 4 (2.30) 23 (13.22) 73 (41.95) 55 (31.61) 19(10.92) determining appropriate sample size 1 (0.57) 21 (12.07) 71 (40.80) 59 (33.91) 22 (12.65) choosing an appropriate sampling technique (e.g. random sampling 2 (1.15) 23 (13.22) 60 (34.48) 68 (39.08) 21 (12.07) determining outcome measures (variables to measure) 4 (2.30) 22 (12.64) 6665 (37.93) 63 (36.21) 19 (10.92) ethical considerations 9 (5.17) 26 (14.94) 64 (36.78) 53 (30.46) 22(12.64) outlining detailed statistical plans to be used in data analyses 3 (1.72) 17 (9.77) 58 (33.33) 68 (39.08) 28 (16.09) designing a data collection form 6 (3.45) 30 (17.24) 66 (37.93) 52 (29.89) 20 (11.49) developing and validating a study instrument (e.g. questionnaire) 4 (2.30) 22(12.64) 62 (35.63) 61 (35.06) 25 (14.37) iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 78 collecting relevant data using preplanned data collection forms 9 (5.17) 32 (18.39) 70 (40.23) 50 (28.74) 13 (7.47) managing and storing data including data entry into a database 10 (5.75) 31 (17.82) 69 (39.66) 47 (27.01) 17(9.76) statistical analyses using software (e.g. stata, spss, epiinfo 1 (0.57) 17 (9.77) 59 (33.92) 60 (34.48) 37 (21.26) choosing and applying appropriate ‘‘inferential’’ statistical tests and methods 1 (0.57) 17 (9.77) 51 (29.37) 64 (36.78) 41 (23.56) summarizing data in tables or charts 10 (5.75) 32 (18.39) 72 (41.38) 43 (24.71) 17 (9.77) interpretation of the findings and determining the significance of obtained results 6 (3.45) 21 (12.07) 74 (42.53) 51 (29.31) 22 (12.64) preparing a presentation (oral or poster) 10 (5.75) 27 (15.52) 78 (44.83) 41 (23.56) 18 (10.34) writing a manuscript for publication in a scientific journal 2 (1.15) 19 (10.92) 61 (35.06) 63 (36.21) 29(16.66) †52 participants skipped these items pharmacists’ interest in pursuing postgraduate study nearly two-thirds of the respondents were interested in pursuing postgraduate studies. the majority of the hospital pharmacists (87.20%) indicated they were not interested in any postgraduate program. of those who were interested and enrolled, 7.56% were pursuing their master degree. an overwhelming majority of the hospital pharmacists (84.21%) showed an interest in clinical pharmacy and practice research, while only 15.79% were interested in pharmaceutical science. their interests lay majorly in the direct patient care for those interested in the clinical pharmacy research field (34.46% out of the participants). while for those interested in pharmaceutical science, over half (50.58%) were interested in pharmacology (table 5). table 5 .pharmacists’ interest in postgraduate studies (n= 226) interest in postgraduate studies† frequency (%) not interested 149(87.13) pharmd 1(0.58) residency and/fellowship 4(2.35) masters 13(7.60) phd 4(2.34) area of interest in clinical pharmacy‡ pharmacoepidemiology and drug safety research 2(1.41) pharmacoeconomics research 22(15.49) pharmacotherapeutics research 17(11.97) social and behavioral aspects of pharmacy research 20(14.08) clinical and outcome research 32(22.54) direct patient care 49(34.51) area of interest in pharmaceutical science§ pharmaceutics 11(18.64) pharmacokinetics 7(11.86) pharmacogenetics 5(8.47) medicinal chemistry 5(8.47) pharmacology 30(50.87) pharmacognosy 1(1.69) †90 missing data; ‡107 missing data; §190 missing data. iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 79 discussion this study is one of the initial studies which examined the pharmacists’ confidence and competence in conducting clinical and practicebased research within the malaysian context. the findings suggested that most hospital pharmacists in our sample expressed limited interest in participating in research activities. in addition, the pharmacists expressed facing barriers to performing pharmacy practice research which dates back to their undergraduate studies. this study's respondents were hospital pharmacists, predominantly from public hospitals, and the majority indicated that they received their bachelor’s degree from malaysia. over half of the respondents indicated that they feel the research training they received in their undergraduate studies was inadequate. while several workshops, seminars, and specialized short courses teach research skills to undergraduate students, their frequency is often limited as they would be done to avoid interruption with the regular lecture schedules. moreover, when these research training programmes are carried out, they are often made optional or carried out in an environment where they cannot retain much information. consequently, this would result in students having limited exposure to research activities and being incompetent in research activities by the time they graduate. therefore it is necessary to reinforce the importance of engaging in research activity from the undergraduate study (25). pharmacists' research skills need to be taught and developed from the early stages of their study before they proceed to postgraduate study. previous studies also support the importance of training in research from the early days to develop researchrelated proficiency enculturation (26, 27). furthermore, several reports across different disciplines show that engaging in an undergraduate research experience can enhance disciplinary skills, such as research design, data collection and analysis, information literacy, and scientific communication (28-30). this would suggest that for competence and confidence in research activities to be developed in the hospital pharmacists, it would be initiated from their undergraduate studies. the hospital pharmacists also indicated that the research training which they received through their job career was inadequate. while the job career is an ideal environment for research training to be conducted, it is often filled with work activities that leave minimal research training time. therefore, the pharmacists would have to rely on the research skills which they would have acquired before commencing their jobs. this is consistent with the study of amjad et al (2018), which indicates that involvement in clinical activities for hospital pharmacists creates a challenge when they do not have the prerequisite skills (31). this would decrease the interest in participating in research activities as it would expose their incompetence. therefore, instead of participating in these clinical activities, the hospital pharmacists would only concentrate on their prescribed roles (32). according to this study, career progression and efficiency in the pharmaceutical career are enhanced by continuous research participation. the pharmacy degree's objectives clearly state that progression occurs through research activities; thus, it is inherent for the respondents to be willing to participate (33). however, a considerable number of respondents indicated no interest at all, partial interest, and indifference. the other proportion was willing to participate in the research study, but considerably a small group relating to the entire population. this leads to the necessity of probing into the hindrances of undertaking research and how they can be alleviated to lead to increased interest and participation rates. this is supported by talsma’s study, which notes that the key to increased and competent participation rates in the pharmaceutical study stems from identifying barriers and resolving them before reinforcing the importance of the research activities and culture (34). despite the higher proportion of the pharmacists having experience in prior research activities, the results display a limited selection of training methods used. based on alqahtani et al., the methods used in training have a bearing on the efficacy and competency skills that the participants have training initiatives such as workshops are effective, especially when they are interactive sessions. however, they have their limitation in terms of location and time limits (35). therefore, the burden is on the facilitators to evaluate the training methods used to ensure that the participants gain skills. this would help optimize the research training methods to address the low level of confidence that hospital pharmacists have in designing and conducting research activities despite their prior experiences (36). moreover, the current study showed that most participants had low involvement in research activities that are often related to low levels of participation at the international level in research collaborations, participating in conferences and peer reviews. however, scholarly peer reviews are doing well at the local and regional levels but do not often progress to international levels. therefore, it would necessitate identifying why academic publications are not prominent at the international level. according to the literature, collaborative efforts at the international level often lead to more information sharing, skills transfer, and widened insight into areas of interest in the iraqi j pharm sci, vol.31(2) 2022 hospital pharmacists’ ability to conduct research 80 pharmaceutical field, facilitating more significant innovation and productivity in the industry (15, 37). at the center of this study lies the issue of the self-perceived competence of hospital pharmacists. by having a high level of selfconfidence and competence in undertaking research activities, one would be proactive and productive in their work. in undertaking research-related activities such as literature review, formulation of hypotheses and writing up their findings, approximately half of the pharmacists disclosed that they were moderately competent. simultaneously, their remaining counterparts were less competent and confident in undertaking the aforementioned research activities. therefore, the self-evaluation exercise indicated moderate to low confidence and competence levels, which tallies with the low participation and completion of research by the hospital pharmacists in malaysia (14). additionally, the respondents demonstrated the highest competency levels in data-related skills, including designing a data collection form and managing and storing data within a database. in contrast, they indicated the least competence in choosing and applying appropriate statistical tests and methods. this is similar to iorga’s study, which emphasised that identifying, designing, and analysing an appropriate research instrument is considered one of the essential skills a pharmacist should have to do more research within the pharmaceutical field (38). consequently, high confidence in conducting pharmacy practice research should be built gradually through optimal educating and training. hence this can be proposed as a plausible solution for the hospital pharmacists (39). limitations despite this study providing insight into hospital pharmacists' self-perception towards confidence and competence in research activities, this study has its limitations. there was a low response rate, as the number of respondents who participated was less than the sample size. however, this reflects the extent of the malaysian hospital pharmacist's reluctance to participate in research activity. in addition, due to the nature of the research model, where the respondents have to provide the inputs of their skills, the concept of self-bias is evident. give that some of the research questions required the respondents to recall information from several years ago, thus resulting in recall bias. conclusion the hospital pharmacists in malaysia indicated moderate to low levels of confidence and competence in undertaking research activities. there is a need to reinforce undergraduate and postgraduate research training in the institutions to build competence in research techniques such as literature reviews and scholarly participation. there is also a need to modify appropriate training methods to help the pharmacists plan research instruments, implement and effectively collect the data while being competent in utilizing the statistical tools available. as a result, this would provide the necessary skills and techniques to build confidence and competence in 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http://creativecommons.org/licenses/by/4.0/ the effect of long term use of glibenclamide on serum and urinary sodium and potassium level in type 2 dm patients iraqi j pharm sci, vol.19(1) 2010 effect of glibenclamide on sodium and potassium level 58 the effect of long term use of glibenclamide on serum and urinary sodium and potassium level in type 2 dm patients ali a. ali* ,1 *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract long-term use of sulfonylureas including chlorpropamide, is known to potentiate the antidiuretic action of arginine vasopressin (avp), predisposing to hyponatremia.the present study was designed to evaluate the effect of long term use of glibenclamide on serum and urinary levels of sodium and potassium in type 2 dm patients in iraqi dm centers. ninety eight patients with type 2 dm who were maintained on different doses of glibenclamide for at least 1 year, attending the centre for diabetes and endocrinology in al-rusafa, baghdad, were enrolled in the study, in addition to 15 normal healthy subjects. patients were allocated into three groups according to the dose of glibenclamide that they received. blood and urine samples were obtained for evaluation of sodium and potassium levels in these samples by flamephotometry. the results indicated that glibenclamide use resulted in significant elevation in serum levels of sodium and potassium compared to controls, while urinary excretion of these cations was not significantly changed. stratification of patients according to the dose of glibenclamide revealed that this effect on sodium and potassium was not dose dependent. in conclusion, long term use of glibenclamide impairs normal values of sodium and potassium independent of the administered dose. key words: glibenclamide, sodium, potassium. الخالصة ) غٍز اىَعخَذ عيى اُ دواء اىنيىربزوباٍاٌذ وهى ٍِ ٍدَىعت اىظيفىٍّو ٌىرٌا ٌظخخذً ىعالج ٍزضى اىظنزي ٍِ اىْىع اىثاًّ و ٌعخقذ اُ هذا اىذواء ٌعَو ٍباشزة عيى واىذي ٌظبب ّقص اىصىدٌىً االّظىىٍِ( وهى ٍعزوف بخحفٍشٓ ىعَو اىهزٍىُ اىَضاد ىيبىه فً هذا اىبحث ّىد دراطت حاثٍز دواء اىنيٍبٍْنالٍاٌذ وهى ٍِ ّفض ٍدَىعت اىنيىربزوباٍاٌذ )أي .ىيبىه ٍظخقبالث هزٍىُ اىَضاد طاعت فً اىَزضى 42اىَدَىع خاله ه اىبىاىظيفىٍّو ٌىرٌا(عيى حزمٍش اىصىدٌىً واىبىحاطٍىً فً اىذً )اىَصو( ومذىل فً وقذ 4009ٍِ عاًاىثاًّ وىغاٌت شهز ماّىُ 4002اىَصابٍِ بذاء اىظنزي اىْىع اىثاًّ وقذ اخزٌج هذا اىذراطت فً شهز اٌيىه هْاك ٍزٌض خارخً ٍِ ٍزمش اىغذد اىصٌ واىظنزي فً اىزصافت ببغذاد باىخعاوُ ٍع اىطبٍب االطخشاري 92شارك فً هذٓ اىذراطت حزمٍش اىصىدٌىً وقذ حٌ قٍاصطاعت فً اىَْشه( 42خاله اىَدَىع ىبىها) ىبىهشخص طيٌٍ وقذ اخذث عٍْاث ٍِ اىَصو وا 51و ٌؤثز بشنو ٍيحىظ عيى حزمٍش اىصىدٌىً اىنيٍبٍْنالٍاٌذواظهزث اىْخائح اُ قٍاص االىىاُ فً اىيهب واىبىحاطٍىً بىاططت خهاس 42اىَدَىع خاله ىبىهفً ا واىبىحاطٌٍ ذً ٍقارّت ٍع اىَدَىعت اىقٍاطٍت اٍا حاثٍزٓ عيى حزمٍش اىصىدٌىًواىبىحاطٍىً فً ٍصو اى ٍيحىظ. طاعت ىٌ ٌنِ بشنو introduction glibenclamide is a first line option for treating patients with type 2 diabetes mellitus (dm) who are not overweight or who can not take metformin (1) . it is mostly used when diet control and exercise have failed to achieve tight control of plasma glucose level; it also can be used alone or in combination with other hypoglycemic agents to provide better glycemic control (2) .sodium is the important electrolyte in the extracellular space while potassium is the essential one in the intracellular space, this asymmetrical distribution of the electrolytes across the cell membrane requires the active exchange of both cations by the na+/k+-atpase . (3) potassium is the principle electrolyte (cation) of intracellular fluid and the primary buffer within the cell itself. ninety percent of potassium is contain in blood and bone. damaged cells release potassium into the blood .(4) using free flow micropuncture technique in rats, intravenous infusion of glibenclamide (3mg/hr) evoked a natriuresis and diuresis; while potassium excretion remained unchanged. it has been reported that glibenclamide impaires sodium reabsorption in one or more of the nephron segment that comprise the loop of henle (5) .moreover, other hypothesis indicates that the natriuretic effect of glibenclamide is a consequence of blockade of potassium channels in the apical membrane of the thick ascending part of henle loop; or it may inhibited a small secretory potassium flux in the proximal tubule (6) .the present study was designed to evaluate the effect of long term use of different doses of glibenclamide on serum and urinary levels of sodium and potassium levels of iraqi patients with type 2 dm. 1corresponding author email : aliaziz_1977@yahoo.com received : 27/6/2009 accepted : 9/3/ 2010 iraqi j pharm sci, vol.19(1) 2010 effect of glibenclamide on sodium and potassium level 59 subjects and methods ninety eight patients (54 female and 44 males) with type 2 diabetes mellitus, were referred to the specialized center for endocrinology and diabetes, baghdad during the period from september 2008 to january 2009 were enrolled in the study; their age range was 52.26 ± 8.65 years and diagnosed to have type 2 dm for at least five years( average duration of type 2 dm 5.5 ± 0.4 years) and maintained on glibenclamide 5 mg , 10 mg or 15 mg at least 1 year with adequate glycemic control (average fasting blood sugar = 6.0 ± 0.94 mmole/l) and no detectable secondary complications .all patients were inflamed about the nature of the study and their signed consents were obtained before participation. additionally, 15 healthy subjects with comparable age to that of patients were utilized as control. after overnight fasting, venous blood samples were obtained from all subjects without using tournique to avoid leakage of cellular potassium, and serum was prepared for evaluation of serum levels of sodium and potassium using flamephotometer (the flamephotometer used is fp 20 seac from seag radim company-italy). moreover, 24 hours urine sample was collected for evaluation urinary excretion of sodium and potassium using flamephotometry. for 24 hours urine collection, we instructed the patients to void at 8 am and discard the specimen. then collect all urine including the final specimen voided at the end of the 24-hour collection period (i.e, 8 am the next morning) (7). for evaluation of the effect of glibenclamide on serum and urinary levels of sodium and potassium, all patients data were compared with those of controls; meanwhile ,for the evaluation of the effect of glibenclamide dose ,patients were classified in three groups, those who are treated with 5 mg/day(n=56), those treated with 10 mg/day(n=29) and those treated with 15 mg/day(n=13). all data were expressed as mean ± standard deviation; un-paired students t-test between patients and control and anova test was performed for evaluation of differences between groups and p-values < 0.05 were considered significant. flame photometry based on atomic emission method for the routine detection of metal salts, principally na, k, li, ca and ba. quantitative analysis of these species is performed by measuring the flame emission of solution containing the metal salts.solution is aspirated into the flame. the hot flame evaporates the solvent, atomizes the metal, and excites a valence electron to an upper state. optical filters are used to select the emission wavelength monitored for the analyte species. comparison of emission intensities of unknowns to either that of standard solutions, or to those of an internal standard, allows quantitative analysis of the analyte metal in the sample solution .(8, 9) results in the present study, table 1 shows that serum sodium concentration in total number of patients is significantly different (p<0.05) compared to that in control group. table 1 also shows that both urine sodium and potassium concentrations in total number of patients are not-significantly different (p>0.05) compared to that in control group. in table 2, the average urinary excretion of sodium and potassium was not significantly different (p>0.05) among patients who were using different doses of glibenclamide. table 1 : effect of glibenclamide on serum and urinary levels of sodium and potassium in type 2 patients. parameter control subjects (n=15) dm patients treated with glibenclamide (n=98) serum sodium (meq/l) 139.95 ± 3.859 142.29 ± 6.67* serum potassium (meq/l) 4.13 ± 0.56 4.71 ± 0.663* urine sodium (meq/l) 103.14±26.25 100.64 ± 48.8 urine potassium (meq/l) 62.02±33.2 51.76 ± 25.12 values were expressed as mean ± sd; n= number of subjects; * = significantly different from control (p< 0.05). iraqi j pharm sci, vol.19(1) 2010 effect of glibenclamide on sodium and potassium level 60 table 2: effect of 5 mg /day , 10 mg/day and 15 mg/day glibenclamide on serum and urinary sodium and potassium levels in type 2 dm patients. parameter patients treated with 5 mg /day glibenclamide n=56 patients treated with 10 mg /day glibenclamide n=29 patients treated with 15 mg /day glibenclamide n=13 serum sodium ( meq/l) 142.3 ± 4.5 a 142.7 ± 7.8 a 141.53±4.33 a serum potassium ( meq/l) 4.746 ± 0.659 a 4.648 ± 0.748 a 4.73 ± 0.496 a urine sodium ( meq/l) 109.08 ± 52.28 a 96.4 ±41.58 a 73.75 ± 46.89 a urine potassium (meq / l) 55.39 ± 21.59 a 41.85 ±24.77 a 54.75 ± 35.964 a values were expressed as mean ± sd; n= number of subjects; values with identical superscripts (a ) were considered non significantly different (p>0.05) (anova). discussion systemic administration of glibenclamide inhibits sodium reabsorption in the loop of henle (6) ; this extra-pancreatic effect of glibenclamide reveal natriuretic activity (10) ,which is attributed to elevation of functional sodium delivery to the distal tubules. moreover ,glibenclamide impairs sodium reabsorption in one or more of the nephron segments that comprise the loop of henle (5) .in the present study, long term use of glibenclamide by dm patients resulted in significant elevation in serum levels of sodium and potassium (table 1) ;this effect may be attributed to enhancement of free water excretion induced by glibenclamide in patients with dm (11) .in clinical practice, administration of 5 mg single oral dose of glibenclamide in patients with type 2 dm did not essentially affect sodium and potassium excretion (12) .several agents belong to sulfonylureas group ,including glibenclamide, have diuretic action in well hydrated normal subjects (11) , and glibenclamide enhances water excretion in patient with diabetes insipidus (13,14) , and diabetes mellitus (11) .in this respect, the reported elevation in serum levels of sodium and potassium (in the present study),without affecting urinary levels, may be explained on the bases of improper hydration status of enrolled patients, especially when associated with excessive of water excretion. the data presented in table 2 indicated that there is no dose-related effect for glibenclamide on sodium and potassium homeostasis; this observation may indicate that the reported elevation in serum levels of the cation is beyond the pharmacodynamic activity of glibenclamide .moreover, the limitation of patients' sample, duration of follow up multifactorial pathophysiology may have potential interference in this respect. accordingly, larger patients sample with proper selection criteria could be a proper approach for clear definition of the problems. conclusion in this study we conclude that glibenclamide dose not satisfy syndrome of inappropriate antidiuretic hormone secretion (siadh) criteria because no hyponatremia occure (siadh criteria are hyponatremia and urinary sodium concentration >20mmol/l) (15, 16) .also we conclude that long-term use of different doses of glibenclamide in type 2 dm may impaire sodium and potassium homeostasis unrelated to the administered dose. acknowledgments i would like to thank dr.khalid ibraheem , specialist of endocrinology, centre of diabetes and endocrinology in al-rusafa-baghdad . also i am grateful to the staff of quality control department central laboratories /baghdad. references 1. hensen j; haenelt m; gross p. water retention after oral chlorpropamide is associated with an increase in renal papillary arginine vasopressin receptor.eur.j endocrinolo 1995 apr;132(4): 459-64. 2. who model list of essential medicines, 15th edition, word health organization, p.21.retrieved on 2007-11-19. 3. thomas l: water balance and fluid compartments; sodium, clinical lab diagnostics, 1st edition, 1998, vol.1, 289. 4. frances fischbach, potassium, a manual of laboratories and diagnostic test, sixth edition, 2000, chapter 6, 349. 5. baily m.a and s.j. walter: renal effects of glibenclamide: a micropuncture study: the journal of pharmacology and experimental thrapeutics; may 1998, vol.285, issue 2,464-467. iraqi j pharm sci, vol.19(1) 2010 effect of glibenclamide on sodium and potassium level 61 6. bailey m.a., shirley d.g., stocking cj. jonathan m. slater, stephen j. walter . the natriuretic effect of glibenclamide:evidence for a non-luminal site of action. european journal of physiology. vol.444, number 6/ september, 2002, 777-784. 7. harrington jt and cohen jj," measurement of urinary electrolytesindications and limitations", n engl j med, 1975,293(24):1241-3. 8. sawyer, heineman, beebe, chemistry experiments for instrumental methods, wiley, new york, 1984. 9. d. c. harris quantitative chemical analysis 4th ed., w. h. freeman and company, new york 1995 chapter 21. 10. clark ma, humphrey sj ,smith mp, ludens jh. unique natriuretic properties of the atp-sensitive k1 channel-blocker glyburide in conscious rats. j pharmacol exp ther 1993; 165:933-937. 11. moses am, howantiz j, miller m. diuretic action of three sulfonylurea drugs. ann intern med 1973,78:541— 544,. 12. ylitalo p, oksala h, pitkäjärvi t. comparison of acute and prolonged effects of glibenclamide and chlorpropamide in patients with non insulin-dependent diabetes. pubmed. arzneimittelforschung. 1985; 35 (10) : 1596-9. 13. rado jp; borbely l.: glybenclamide enhancement of polyuria in patients with pituitary diabetes insipidus. endocrinology 1972, 59:397—402,. 14. rado jp; borbely l; szende l; fiscner j; tako j.: investigation of the diuretic effect of glibenclamide in healthy subjects and in patients with pituitary and nephrogenic diabetes insipidus. horm metab res , 1974,6:289—292. 15. rose,bd,post,tw. : clinical physiology of acid-base and electrolyte disorders, 5 th ed,mcgraw hill,new york ,2001 , 707-701. 16. vrohr, cerny t, toss ra, brunner kw. the syndrome of inappropriate adh secretion (siadh) in small cell lung cancer. schweiz med wschr, 1991; 121; 1271-82. http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22ylitalo%20p%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22oksala%20h%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22pitk%c3%a4j%c3%a4rvi%20t%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'arzneimittelforschung.'); iraqi j pharm sci, vol.25(2) 2016 flavonoids of calendula officinalis 1 detection and isolation of flavonoids from calendula officinalis (f.asteraceae) cultivated in iraq maha n. hamad *,1 * department of pharmacognosy and medicinal plants , college of pharmacy, university of baghdad, baghdad, iraq abstract calendula officinalis l. (asteraceae) known as marigold is known to have several pharmacological activities and used for the treatment of several diseases as measles, jaundice, constipation and several inflammations. marigold flowers contain several chemical constituents mainly flavonoids, triterpenoids and essential oil. in this study marigold flowers cultivated in iraq had been investigated for its flavonoids content. the study revealed the presence of quercetin and kaempferol glycosides and the absence of myricetin glycosides. the flowers were extracted with ethanol 70% fractionated with different solvent and the flavonoids were isolated by preparative hplc. the isolated flavonoids were identified by measuring melting points, uv, ir, analytical hplc and tlc. keywords: marigold, flavonoids, kaempferol, preparative hplc. عسل وتشخيص الفالفىوىيدات مه وبات االقحىان ) العائلة : استيراسيا ( المستسرع في العراق حمدمها وىري ،*1 * .فشع انعقاقٍش ٔانُباحاث انطبٍت ، كهٍت انصٍذنت ، جايعت بغذاد الخالصة َباث األرسٌٌٕ )انعائهت أسخٍشاسٍا( انًعشٔف باسى انقطٍفت أ االقحٕاٌ انزي ٌعشف بانعذٌذ يٍ األَشطت انذٔائٍت ٌٔسخخذو نعالج ٕس انقطٍفت ححخٕي عهى انعذٌذ يٍ انًكَٕاث انكًٍٍائٍت أساسا انعذٌذ يٍ األيشاض يثم انحصبت ٔانٍشقاٌ، ٔاإليساك، ٔانخٓاباث عذة. صْ انفالفٌَٕٕذاث، انخشبٍُاث ٔانضٌٕث. فً ْزِ انذساست اسخخذيج صْٕس َباث انقطٍفت انًسخضسع فً انعشاق نذساست يضًَّٕ يٍ ٔجٕد جهٍكٕسٍذاث يٍشٌسٍخٍٍ. حى انفالفٌَٕٕذاث. كشفج انذساست عٍ ٔجٕد جهٍكٕسٍذاث انكٌٕشسخٍٍ ٔجهٍكٕسٍذاث انكاًٌبفشٔل ٔعذو % ثى جضأ انًسخخهض يع انًزٌباث انًخخهفت ٔحى عضل انفالفٌَٕٕذاث باسخخذاو حقٍُت 07اسخخالص انضْٕس يع اإلٌثإَل انكشٔياحٕغشافٍا انسائهت راث االَجاص انعانً. ٔحذدث انفالفٌَٕٕذاث انًعضٔنت بقٍاط دسجت االَصٓاس،ٔطٍف األشعت فٕق انبُفسجٍت األشعت ححج انحًشاء ٔكشٔياحٕغشافٍا انسائهت راث االَجاص انعانً ٔكشٔياحٕغشافٍا انطبقت انشقٍقت.ٔ االقحىان ، فالفىوىيدات، كامبفيرول ، كروماتىغرافيا السائلة ذات االوجاز العالي .الكلمات المفتاحية: introduction calendula officinalis linn (asteraceae) known as marigold is native to central and southern europe, western asia and usa. (1) marigold flowers were used for the treatment of measles, small pox, jaundice, constipation and suppression of menstrual flow (2) . it has anti-inflammatory, cytotoxic, antitumor activity (3,4) , antibacterial and antifungal activity (5,6) , the flowers contain several chemical constituents as flavonoids and other phenols, triterpenoids, essential oil, polysaccharides (7,8) , saponins, carotenoids, mucilage and phytosterols (9,10) . various terpenoids have been reported from the petroleum ether extract of c. officinalis flowers as β-sitosterol, stigmasterol (11) faradiol, calendulodiol, lupeol (12) , volatile oil (13) and also carotenoids as neoxanthin, luteoxanthin, anthroxanthin, and lutein (14,15) . calendula flowers also contain coumarins as scopoletin, umbelliferon and esculetin (16) , flavonoids as quercetin, isorhamnetin, narcissin, calendoflavoside and other flavonoids glycosides (17,18) in addition to other constituents as amino acids, bitter constituents and n-paraffin (19,20) . several flavonoids had been isolated from marigold using column chromatography on polyamide sorbent (21) or on column using silica gel followed by polyamide column (17) or by preparative tlc on silica gel plates (22) . quantitative estimation of total flavonoids in marigold was done using one and twodimensional tlc or by hplc on silica gel precoated aluminum plates (23, 24) . in our study two flavonoids aglycones were isolated using preparative hplc and one flavonoid glycoside was detected by analytical hplc and tlc. materials and methods plant material: marigold flowers were collected from the garden of medicinal plants at the college of pharmacy/university of baghdad in march and dried in shade at room temperature. 1 corresponding author e-mail: manoha_1957@yahoo.com received: 11/ 6 /2016 accepted: 3/ 2/ 2016 iraqi j pharm sci, vol.25(2) 2016 flavonoids of calendula officinalis 2 extraction: dried flowers of marigold (5gm) were extracted by maceration with 70% ethanol (150 ml) for 24h, filtered. the procedure was repeated two times; the filtrates were combined together and concentrated under reduced pressure. the obtained extract was fractionated by partitioning with different organic solvents, petroleum ether, chloroform, ethyl acetate, n-butanol (50ml x3 for each solvent) (23) . the first three fractions were dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure, while the n-butanol fraction was evaporated to dryness directly. the yields of the fractions were: petroleum ether= 0.17gm chloroform = 0.23gm ethyl acetate = 0.31gm n-butanol = 0.60gm hydrolysis of n-butanol fraction part of the n-butanol fraction was hydrolyzed by reflux using 30ml of 10% hcl, cooled and partitioned with ethyl acetate (30ml x3) the ethyl acetate layers were combined together, dried over anhydrous sodium sulfate, filtered, and evaporated to dryness under reduced pressure. hplc analysis ethyl acetate, n-butanol and hydrolyzed nbutanol fractions were analyzed by hplc using hyperclone odcc c18 column and a mixture of methanol: water (70:30) as a mobile phase with a flow rate of 0.5ml/min and detected at 280 nm. thin layer chromatography ethyl acetate and hydrolyzed n-butanol fractions using silica gel gf254 pre coated aluminum plates developed with following mobile phases: ichloroform: methanol 9:1 iichloroform : acetone : formic acid 75:16:8 iiitoluene: chloroform: acetone 40:25:35 the flavonoids contents of these fractions were compared with standards of quercetin (flukaaustria), rutin and kaempferol (sigma-aldrich, usa). isolation of flavonoids by preparative hplc two aglycones (i & ii) were isolated from the hydrolyzed n-butanol fraction by preparative hplc using nf 22561/techno korma column and methanol: water (70: 30 ratio) as a mobile phase and with a flow rate of 10ml/min, detection at uv 280nm and injection volume of 2 ml the isolated aglycones were recrystallized from boiling ethanol and compared with standard quercetin and kaempferol using the previously mentioned mobile phases. tlc of n-butanol fraction n-butanol fraction was analyzed by tlc using the silica gel gf254 pre-coated layers developed in the following mobile phases: i: methanol: water: formic acid 40: 57: 3 (v/v/v) ii: ethyl acetate: glacial acetic acid: formic acid: h2o (100:11:11:25) iii: ethyl acetate: acetic acid: water 7:1.5: 1.7 (v/v/v) vi: toluene: ethyl acetate: methanol: formic acid 32:14: 12:5. spiking of n-butanol fraction part of n-butanol fraction used for analysis in hplc was mixed with standard rutin and the resultant mixture was re-analyzed by hplc. results two flavonoid aglycones were isolated from the hydrolyzed n-butanol fraction and one flavonoid glycoside was detected in the un-hydrolyzed n-butanol fraction. flavonoid aglycone i: m.p. 314-317 0 c, uv λmax (meoh) 252, 291, 371 nm, ir (kbr) 3418, 3375 (o-h), 1678 (c=o), 1610s, 1562s, 1508, 1451 (aromatic). tlc results of isolated aglycon i are shown in table (1) and hplc chromatogram of standard quercetin is shown in figure (1). flavonoid aglycone ii: m.p. 275-278, uv λmax (meoh) 250, 270 sh, 301, 372sh. ir (kbr) 3321(o-h), 1705 (c=o), 1602, 1612s, 1558s, 1508, 1450 (aromatic) tlc results of isolated aglycon ii compared with standard kaempferol are shown in table(1) and hplc chromatogram of standard kaempferol, rutin, and myrecetin are shown in figure(1). iraqi j pharm sci, vol.25(2) 2016 flavonoids of calendula officinalis 3 table (i):rf values of standard quercetin and kaempferol compared with isolated aglycons i&ii in different mobile phases. mobile phase no. rf value of standard quercetin rf value of isolated aglycon i rf value of standard kamepferol rf value of isolated aglycon ii i 0.43 0.41 0.62 0.60 ii 0.39 0.40 0.5 0.48 iii 0.77 0.74 0.86 0.87 figure (1):hplc of standards quercetin, kampferol, rutin and myrecitin detection of rutin the presence of rutin in n-butanol fraction was detected by hplc and tlc. hplc chromatogram of standard rutin is shown in figure 1 tlc of nbutanol fraction compared with standard rutin results are shown in table(2). table (2):rf values of standard rutin compared with the n-butanol fraction. mobile phase no. rf value of standard rutin rf value of n-butanol fraction i 0.16 0.14 ii 0.42 0.39 iii 0.48 0.50 iv 0.11 0.09 hplc of std quercetin rt 4.382 hplc of std kampferol rt 5.957 hplc of std myrecitin rt 3.772 hplc of std rutin rt 3.064 iraqi j pharm sci, vol.25(2) 2016 flavonoids of calendula officinalis 4 hplc chromatograms of ethyl acetate, nbutanol and hydrolyzed n-butanol fractions are shown in figures 2,3 and 4 respectively. figure (2): -hplc of ethyl acetate fraction. figure (3):hplc of n-butanol fraction. figure (4):hplc of hydrolyzed n-butanol fraction figure (5):hplc of spiking of n-butanol fraction with standard rutin. the presence of rutin in n-butanol fraction was further confirmed by random spiking with standard rutin & the result is shown in figure (5). discussion detection of the presence of quercetin and kaempferol glycosides was confirmed after hydrolysis of the n-butanol fraction which iraqi j pharm sci, vol.25(2) 2016 flavonoids of calendula officinalis 5 leads to the liberation of these aglycones which could not be detected in the ethyl acetate fraction only traces of kaempferol. the aglycones are flavonols and their uv absorptions band i appear at 300-350 nm range arise from ring b while band ii in the 240-285 nm ranges arise from ring a (24). melting points, rf values and ir coincide with that reported for quercetin and kaempferol (25) and the rf values are identical with the standard. rutin (flavonol ghycoside) presence in nbutanol fraction was confirmed by analytical hplc and by spiking with standard rutin and by analytical tlc and co-chromatography with standard rutin in different mobile phases. rutin is aglycoside of quercetin (quercetin 3rhamnoglucoside) therefore acid hydrolysis of rutin produces quercetin and this was approved by the isolation of quercetin from the hydrolyzed n-butanol fraction. myricetin could not be detected in any fraction of marigold neither as aglycone nor as glycoside. conclusion quercetin and kampferol are mainly found in calendula officinalis as their glycosides not as aglycones, one of quercetin glycosides detected was rutin (quercetin-3rhamnoglucoside) while myricetin could not be detected neither as aglycone nor as glycoside. acknowledgment the author is deeply grateful to the college of pharmacy/university of baghdad for supporting this project. references 1. pdr for herbal medicine, 2 nd edition, montvale, thomson-medical economic 2003, p497-500. 2. bashir s, janbaz kh, jabeen q & gilani ah, studies on spasmogenic and spasmolytic activities of calendula officinalis flowers. phytother. res., 2006; 20:906-910. 3. ukiya m, akihisa t, yasukava k, tokuda h, suzuki t, kimura y, antiinflammatory, antitumor-promoting and cytotoxic activities of constituents of marigold (calendula 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https://doi.org/10.31351/vol32iss1pp1-13 1 pharmacological aspects of borago officinalis (borage): a review ruaa mohammed ibrahim*,1 and dhuha abdul saheb alshammaa* *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq. abstract borago officinalis is an interesting nutritional and medicinal sources attributed to high. the plant is characterized by bright blue star-shaped inflorescence naturally growing globally and is usually known as borage. the borago phytochemical analysis using chromatographic showed the presence of alkaloids, tannins, flavonoids, phenolic acids, essential oil, and vitamins. borage is cultivated all over the world and used in traditional medicine as a demulcent, diuretic, emollient, tonic, expectorant, for the treatment of coughs, inflammation and swelling, and other diseases. in herbal medicine, borage seed oil (bso) has been utilized progressive illnesses. the bso include p-cymene-8-ol, β-caryophyllene, mono-, sesquiterpenes, alpha-linoleic, gamma-linoleic, and linoleic acids. the bso is famed to be the richest vegetable origin of gamma (γ)-linoleic acid (gla). this plant has antiinflammatory, cytotoxic, antioxidant, and wound healing properties due to flavonoids, phenolic. it anxiolytic polyphenols and gla. borage possess other pharmacological properties and hepatoprotective effects high gla content and keywords: borage, gamma-linoleic acid, pharmacological effects of borago officinalis ) لسان الثور ( : مراجعة borago officinalisدوائية للـــ الالجوانب *ضحى عبد الصاحب الشماع و 1،*إبراهيم رؤى محمد . العراق بغداد، بغداد، جامعه الصيدلة، كلية ، والنباتات الطبية العقاقير فرع* الخالصة من المصادر الغذائية والطبية التي تُعزى إلى المحتوى العالي من المركبات المفيدة. يتميز النبات بإزهار borago officinalisيعتبر باستخدام تقنيات كروماتوغرافية للسان الثورتحليل ال على شكل نجمة بشكل طبيعي على مستوى العالم ويعرف عادةً باسم لسان الثور. ةالمع زرقاء وزي gs-msو lc-ms / msو hplc مثل مختلفة وفيتامينات. ةعطري وتأظهرت وجود قلويدات وعفص وفالفونويد وأحماض فينولية ، لعالج السعال ملطفًا و للبول ومطريًا ومنشًطا ومقشعًا وااللتهاب يُزرع لسان الثور في جميع أنحاء العالم ويستخدم في الطب التقليدي باعتباره ( في العديد من األمراض كعامل عالجي. تشتمل مركبات bsoب ، تم استخدام زيت بذور لسان الثور )والتورم وأمراض أخرى. في طب األعشا . acids linoleicو gamma-linoleicو alpha-linoleicو sesquiterpenesو -monoو β-caryophylleneو p-cymene-8-olعلى ) bsoتشتهر غاما نباتي أغنى ) -( γبكونها ومضادات (. glaاللينوليك للخاليا وسامة لاللتهابات مضادة خصائص على النبات هذا يحتوي البوليفينول األكسدة والتئام الجروح بسبب وجود مركبات الفالفونويد والمركبات الفينولية والستيروالت. كما أن له تأثيرات مزيلة للقلق بسبب وجود ايمكن أن تعزى إلى محتواهالتي التأثيرات الواقية للكبد ووى بما في ذلك تحسين الذاكرة . يمتلك لسان الثور أوفيسيناليس خواًصا دوائية أخرglaو ووظيفة إزالة الجذور الحرة ، باإلضافة إلى التأثيرات المضادة للسمنة والتأثيرات المسكنة. glaالعالي من لسان الثور ، حمض كاما لينوليك ، التأثيرات الدوائية للسان الثور :الكلمات المفتاحية introduction medicinal plants have been utilized for many purposes, as nutritional, medicinal, flavorings, cosmetics, fragrances, beverages, dyeing, and other applications. at present time, medicinal plant extracts are important not only in phytotherapy and phytopharmacology but also in industry (1). borago officinalis l. (family: boraginaceae) is a substantial plant nutritional and medicinal values. the plant demonstrated industrial and pharmaceutical uses (2,3). it is usually named starflower or borage (4,5). borage emerges in the mediterranean region; and has been diffused to europe, asia minor, south america, and north africa. it was initially cultivated for medicinal and culinary uses (6). borage could grow wildly in many areas and different soil types. it is harvested during the flowering period before (7) and germinates through november to january. it extends to 70 100 cm in height (6, 8). the parts are excreting extensive fresh cucumber-like aroma and there are with pinkto-blue and rarely white (9,10). the plant is for oil and seed collection; the remainder of the plant is oftentimes treated like trash material and sold as herbal tea. since the plant contain different types of active constituents, so that it can be used for purposes (11). the flowers and the leaves are generally used as food (12), and can be utilized as an economic healthy products source (13).the plant seed showed the highest concentration of the gla, the reason why is utilized as a dietary supplement for several illnesses management (14). 1corresponding author e-mail: mnrsyrg@yahoo.com received: 17/10 /2021 accepted:19 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp1-13 iraqi j pharm sci, vol.32(1) 2023 pharmacological aspects of borage 2 in addition, the bso has proven activity in management of various pathologies such as rheumatoid arthritis (15), acute respiratory distress syndrome (3, 16), atopic dermatitis, menopauserelated symptoms, and diabetic neuropathy (17). former studies on bso demonstrated bone health improvement, anti-inflammatory actions (18), and regulation of lipid metabolism (19), there are other studies reporting skin moisturizing, eczema, and uses in dermatitis (20). interesting note, the medicinal values of bso are attributed to the high gla content (15-22%) (21). figure 1. photo of borago officinalllis plant (4) taxonomical classification (9): kingdom : plantae subkingdom: tracheobionta superdivision: spermatophyta division: magnoliophyta class: magnoliopsida subclass: asteridae order: lamiales family: boraginaceae genus: borago l. species: borago officinalis l. chemical composition several metabolites were reported including resins, tannins, ascorbic acid, niacin, betacarotene, riboflavin, silicic acid, thiamine, choline arabinose, polyphenolics, and unsaturated pyrrolizidine alkaloids (amabiline, lycopsamine and supinidine) (8,22-25). using high-performance liquid chromatography (hplc), six phenolic acids were detected in the borage seed extract, namely chlorogenic, trans-cinnamic, gallic, rosmarinic, pcoumaric, and syringic acids while rosmarinic acid comprises the majority of the polyphenolics(26). other components like p-hydroxyphenyl lactate, phydroxybenzoic, and caffeic acid were also reported in the methanolic borage seed extract (27,28). analysis of the fresh leaves and flowering tops of borage showed high concentrations of palmitic acid in the leaves, and α-linoleic acid in the flowers' fixed oils fractions. the leaves were devoid arachidonic acid; however, carotenoids, flavonoids, essential oils, ascorbic acid, and chlorophyll a and b were detected. other lipids including, oleic, stearic, elaidic, linolelaidic, linoleic, lauric, and myristic acids were identified (29) newly reported metabolites including, oleuropein (secoiridoid), and other ten flavonoids detected with liquid chromatography-mass spectrum (lc-ms-ms) analysis of the borage leaf extract (30). seven flavonoids (isoquercetin, quercetin, catechin-7-o-glucoside, luteolin 7-oglucoside, isovitexin, vitexin and naringenin ohexoside) was identified in the ethanolic extract and three flavonoids (kaempferol 3,7,40-trimethyl ether, naringenin o-hexoside, and uteolin 7,30,40trimethyl ether) in the mother liquor (30). the phenolic compounds (cinnamic, ferulic, syringic, coumaric, and sinapic acid) were detected in the methanolic borage leaf extract (31). the reverse phase-hplc analyses revealed that the methanolic extract of borage flowers contains flavonoids (rutin, myricetin), daidzein (isoflavonoid), and phenolics (pyrogallol, gallic acid, caffeic acid, salicylic acid) (32). the watersoluble vitamins (ascorbic acid, thiamin, riboflavin, and niacin) and two alkaloids (berberine and sanguinarine) were identified in borage flowers with hplc (33). iraqi j pharm sci, vol.32(1) 2023 pharmacological aspects of borage 3 the bso 95% triacylglycerol composed of c16-c20 fatty acids, while minor components 5% consist of flavonoids, tocopherols, phospholipids, free fatty acids, di-and monoacylglycerols, sterols (34) and small quantity of erucic acid (21, 35). numerous unsaturated fatty acids including, linoleic, palmitic, gamma (γ)-linolenic (gla), and stearic acids were found in the bso (36, 37). borage seeds are regarded as one of the best gla sources of several important biomolecules like prostaglandin e1 (pg e1) and its derivatives (38). gas-chromatography–mass spectrometry analysis (gs-ms) of the essential oil (eo) of borage seed revealed sixteen volatile compounds. hexanol and nonadecane were the minor components while p-cymene-8-ol and b-caryophyllene were the major. a previous study showed that the composition of essential oil was recognized by higher oxygenated monoterpenes abundance, then followed by sesquiterpenes (26). another study utilized gc-ms identified seven molecules in borage eo including spathulenol, trifluoromethyl, thymol, verrucarol, globulol, guaiol, gamma-himachalene and the major component of these eo was spathulenol(39). traditional use borage above the ground parts and the seed oil are utilized in folk medicine (14). b. officinalis infusions are used for bronchitis, urinary tract infections, colds, skin rashes, and rheumatism (40). traditionally, borage extracts are utilized to help in respiratory, cardiovascular, and hyperactive gastrointestinal disorders (3). the aerial parts of borage have been utilized in iran folk medicine since ancient times as a tranquillizer, and for treatment of sore throat, cough, pneumonia, inflammatory and edema (14). borage leaves are used as demulcent, diuretic, expectorant, and emollient (41). the borage flowers are used in traditional medicine as a bronchodilator (14) and sedative (42). pharmacological activity antioxidant activity the seedcake extract of borago officinalis (using ethanol 50% at 50°c for 48 hr) has a high phenolic content with significant antioxidant effect, and this extract is utilized in agricultural, cosmetic, food, and pharmaceutical applications as natural antioxidant additives. the antioxidant effect was evaluated using 2,2'-azino-bis (3ethylbenzothiazoline-6-sulfonic acid (abts) and (2,2-diphenyl-1-picrylhydrazyl) (dpph) methods (28). in an earlier study, the polyphenolic rich extract from seedcakes of borago officinalis possess freeradical scavenging, transition metal ion chelating, and antioxidant activities (43). essential oil and flavonoid extract of borage shown antioxidant effect by using the ferric reducing and dpph methods. this activity appears to be low for essential oil and high for flavonoids of borage (39). the antioxidant capacity of the borage flowers' methanolic crude extract and its partitions was examined with several techniques including, ferric ion (fe3+) reducing power (frap), ferrous ion (fe2+) metal chelating, and dpph while hydrogen peroxide (h2o2) was excluded by peroxidase (pox) and catalases (cat) activities (33). the potent antioxidant capacity of borage extracts is attributed to ability to inhibit peroxidation and scavenge different reactive oxygen species (ros). to scavenge the possible damage in cat and pox, these extracts possess active ingredients that to a free radical (33). the antioxidant effect of borage leaves and flowers was determined using dpph; frap assay, and folin’s method (measuring the content of polyphenolics). flowers of borage had higher ferric reducing ability and more content of polyphenols than leaves. in addition, the remaining unreduced dpph radical content in the flower was higher (29). an antioxidant capacity comparison involved the wild versus cultivated species of borage revealed further activity in the favor of the wild species, which also showed higher polyphenolic content (44). a comparative study showed more antioxidant and superoxide scavenging capacity when ethanol was used as a diluent compared to distilled water, owing to more solubility of the phenolics in ethanol (30). about ten flavonoids and an (figure 2) were reported in the borage leaves' extract for the first time in 2019 which explained the potent antioxidant action of this species (30). figure 2. chemical structure of oleuropein (30,45) three borage flowers' extracts were evaluated using dpph, nitric oxide (no) scavenging activity, and frap assays. the water, ethanolic and methanolic extracts exhibited the lowest, moderate, and strongest antioxidant effects, respectively. the methanolic extract of borage showed stronger antioxidant properties in all the assays compared to both water and ethanolic extracts. the antioxidant properties of borage extract were less than that of reference antioxidants (butylated hydroxytoluene and vitamin c). all the reported studies attributed the antioxidant potentials of borage extracts to the presence of high concentrations of important bioactive molecules like, flavonoids, and unsaturated fatty acids (figure 3), while in some other mentioned studies, the antioxidant activity was related to the existence of polyphenolic compounds iraqi j pharm sci, vol.32(1) 2023 pharmacological aspects of borage 4 exemplified by chlorogenic, gallic, trans-cinnamic, syringic, rosmarinic, sinapic, cinnamic, coumaric, and ferulic acids, in addition to the unsaturated fatty acids like, oleic, gamma-linolenic, linoleic, and palmitic acids(47,48). figure 3. chemical structures of some fatty acid in borage (26,45) antimicrobial activity borage possesses potent antimicrobial activity, which excuses its possible use in infection. the phytochemical investigation of the essential oils and flavonoid extracts shows the existence of spathulenol as the main component of the essential oils, figure 4 while the flavonoids of quercetin and rutin(39) . figure 4. chemical structure of spathulenol (46) the antimicrobial effect of flavonoids of borage extract is more significant than essential oil and this can be related to antimicrobial components in both extracts. the isolated spathulenol from the essential oil extract demonstrate low antibacterial effect, this compound is more regarded as an antifungal agent. quercetin and rutin (figures 5 and 6) considered antimicrobial agents. the essential oils of this plant more activity against gram-negative than gram-positive bacteria unlike flavonoids which show activity against both types, and extended activity against resistant respiratory strains (39). figure 5. chemical structure of quercetin (46) figure 6. chemical structure of rutin (46) in another study, the borage leaves aqueous extract (ae) exhibited antibacterial potential against enterobacteria and staphylococci to different extents. the ae showed no bacterial inhibition against listeria monocytogenes, however some activity was recorded against salmonella enterica (49). using various solvents for extraction of the borage flower metabolites demonstrated in the antimicrobial effect when using distilled water, ethanol, and methanol respectively. the methanol extract exerts higher antibacterial capacity due to more flavonoids obtained when compared to distilled water and ethanol. the borage extracts are considered a promising source for natural antimicrobials and could be beneficial in food preservation and drug (47). among gram-negative bacteria, micrococcus luteus was the most resistant, and bacillus cereus was the most sensitive strain. while for gram-positive bacteria, pseudomonas aeruginosa was the most resistant, and escherichia coli was the most sensitive. the antimicrobial potentials of the borage flower extracts were related to the presence of flavonoids, fatty acids, and other polyphenolics (47). other studies stated that the borage leaf extracts showed antibacterial potential versus several pathogens such as enterobacter spp., salmonella enterica, staphylococcus aureus, listeria monocytogenes, and bacillus subtilis (50, 51). wound healing activity borago officinalis can wound healing process, attributed to significantly elevated mononuclear cells number at the wound site, these properties may be due to phenolic compounds that found in hydroethanolic extract and these cellular changes specifies the starting of the proliferative phase and lessening of the inflammatory phase at once (52). iraqi j pharm sci, vol.32(1) 2023 pharmacological aspects of borage 5 epliptogenic activity one of the main content of borage oil is gla, figure 7 (22). some research proposed that there is a potent correlation between epilepsy and the gla increased level (53). figure 7. chemical structure of gama-linoleic acid (gla) (4) a dose-dependent lidocaine-simulated convulsion assay was performed using borage ethanolic extract, and exhibited a potent epileptogenic activity in mice; the epilepsy features had become more prominent as the dose is increased. this result may be related to the active ingredient role which affects lidocaine metabolism by which borago stimulates lidocaine (54). hypoglycemic activity a methanolic extract obtained by soxhelation of b. officinalis exhibited hypoglycemic properties against alloxan induced diabetes in rat model (55). the plant understudy is rich in gla that utilized as a drug to treat different diseases like arthritis, local eczema, diabetes, heart disease, multiple sclerosis, and cyclical mastalgia (56, 57). anti‑inflammatory activity inflammation is a normal defensive response stimulated by infection or tissue damage to remove damaged or dead host cells and to fight invaders in the body (foreign objects, and microorganisms). the excessive pro-inflammatory mediators and cytokines production, such as tumor necrosis factor-α, interleukin-6 (il-6), il-1, prostaglandin e2 (pge2), and nitric oxide (no) play a serious role in inflammatory disease development (58). anti-inflammatory agents reduce inflammatory response and pain via inhibiting the production of prostaglandin (pg) suppressing the activity of cyclooxygenase1 (cox1) and cox 2 enzyme . the anti-inflammatory effect of aqueous, ethanolic and methanolic borage extracts tested using lipopolysaccharide (lps)/ gamma interferon (inf-γ) induced murine macrophage cell line raw (ralph and william’s cell line) 264.7 in murine raw 264.7 macrophage cells, the extracts of borage flowers exerted a low antiinflammatory effect and this effect was related to the existence of phenolic compounds. the ethanolic and methanolic extracts could suppress the production of nitric oxide similar to nitro-l-arginine methyl ester (l-name) as anti-inflammatory agents (47). a previous study showed that the seed oil of b. officinalis has strong anti-inflammatory activity on carrageenan-stimulated paw inflammation. this effect was tested by determining the rates of edema and inhibition using a plethysmometer test. the seed oil of borage may participate in the inhibition of pg synthesis (32). interleukin 1 beta (il-1 beta) can be regarded as an important inflammatory factor produced by both macrophages and monocytes (59). borago officinale has shown a protective effect on il-1 beta protein and hippocampus gene in amyloid 𝛽 (a𝛽)-stimulated inflammation in the rats. in the hippocampus, the rate of the il1-beta protein and gene production get a significant rise that confirmed the inflammation production after a𝛽injection. after the administration of the borago alcoholic juice, the rate of il1-beta protein and gene in the hippocampus were significantly decreased. borage consumption attenuates the elevation of the produced inflammatory factors (il-1 beta) and reduces the inflammation in the alzheimer model which is formed by a𝛽 in the hippocampus. therefore, borage may be useful in alzheimer treatment (60). borago officinale oil causes inflammation reduction due to increasing the cyclic adenosine monophosphate (c-amp) and pg e synthesis and the presence of gla (61). the gla inhibits releasing of the il-1 beta and its production by monocytes which in turn causes inflammation reduction. the gla also stimulates the pg e secretion and this factor reduces tumor necrosis factor-beta (tnf-𝛽) production and finally, the inflammation will be reduced (62). cytotoxic activity in vitro cytotoxic effects of aqueous, ethanolic , and methanolic borage extracts were tested using mtt assay against the colon (ht-29), prostate (lncap), and human liver (hpg2) cancer cells. the extracts of borage flower exhibited weak cytotoxic effects on human hepatic, colon, and prostate cancer cells. but the methanolic extract with a higher polyphenolic contents higher cytotoxic effects compared to the aqueous and ethanolic extracts (47). the edible parts (petioles and leaves) of cultivated and wild b. officinalis exhibit anticarcinogenic effects and dna protection as do their main phenolics mixtures (sinapic, rosmarinic, and syringic acids) (63). iraqi j pharm sci, vol.32(1) 2023 pharmacological aspects of borage 6 figure 8.chemical structures of some phenolic acid in borage (83) hepatoprotective activity the bso exhibited hepatoprotective activity against gamma (γ-) radiation stimulated disturbances in rat liver models (64, 65). oral bso administration significantly ameliorated lipids profile, serum liver enzymes levels, as well as the levels of hepatic and serum malondialdehyde (mda) and reduced glutathione (gsh). the significant decrease in hepatic and serum levels of reduced glutathione (gsh) associated with increases in hepatic and serum levels of malondialdehyde (mda) relate to radiation exposure (64). the elevated levels of mda might be related to the free radicals and polyunsaturated fatty acids interaction in the portion of phospholipids of cellular membranes (66). the reduced levels of gsh might be related to its exhaustion through the oxidative stress stimulated by ionizing radiation (67). rats that were exposed to γradiation and treated with borage exhibited a significant elevation of hepatic gsh levels with a significant decrease in the hepatic mda (65). after γirradiation exposure, the increased serum enzymatic activity of gamma-glutamyl transferase (ggt), alanine aminotransferase (alt), and aspartate aminotransferase (ast), could suggest hepatic injury occurrence (65). the increase in the liver enzymes level may be a result of an injury in liver membrane (68). impairment of intrahepatic and extrahepatic bile flow, hepatobiliary injury, or erythrocyte destruction stimulated by irradiation (69), and oral borage administration efficiently attenuated the increasing serum biomarkers levels such as alt, ggt, and ast. the significant decline in the liver enzymes levels as a result of borage administration could be related to the borage's ability as a potent antioxidant to suppress hepatic injury by keeping the plasma membrane integrity, thereby preventing the enzymes leakage into the serum (65). also, irradiation stimulated hyperlipidemia elevating the serum total cholesterol (tc), lowdensity lipoprotein cholesterol (ldl-c), and triacylglycerols (tg) levels and reducing the highdensity lipoprotein cholesterol (hdl-c) levels (64, 70). the stimulation of liver enzymes by γirradiation is responsible for the fatty acids biosynthesis and fat mobilization to the bloodstream from adipose tissues result in a hyperlipidemic state (71). the low glucagon or the high insulin levels induces the triacylglycerol synthesis in both liver and adipose tissues, which is associated with the accelerated fatty acids mobilization to the blood from the fat deposits (72). the lipid markers improvement by bso may be related to gla that has been recognized to improve the abnormal profile of lipids and metabolism of insulin-mediated glucose (73). the γ-irradiation pathological damage, swelling and liver injury in rats. these results may be attributed to the fact that radiation exposure induces vascular damage with necrosis and hepatic parenchymal degeneration (74). the bso showed a protective activity against γ-irradiation induced pathological changes, and the amelioration was more obvious in irradiated bso pretreated than post-treated group and this may be related to the composition of fatty acids of bso, which considers the beneficial protector versus the free radicals production stimulated by γ-irradiation (74). the bso demonstrated hepatoprotective activity lessening the production of proinflammatory mediators (75). the bso mechanisms that are behind the prevention of hepatotoxicity may be clarified by mda inhibition, prevention of depletion of gsh, and its antioxidant effect due to its high gla content. oral bso administration significantly reduces hepatotoxicity and γirradiation stimulated oxidative damage in rats. so bso may be utilized as a useful supplement for subjects through radiotherapy treatment (64). the b. officinalis ethanolic crude extract show hepatoprotective activity against carbon tetrachloride (ccl4)-stimulated chronic liver damage in, rats which may be due to the antiinflammatory and antioxidant activities that prevent cellular damage and inhibit the formation of ccl4 free radical derivative. oral ccl4 administration exhausted gsh and stimulated lipid peroxidation of the liver. administration of ccl4 caused overproduction of the nuclear factor kappa-b (nfκb) and tumor necrosis factor-alpha (tnf-α) protein levels (the inflammatory markers) and a significant elevated in levels of serum liver biomarker. rat treatment with ethanolic extract produced significant raise in the liver gsh content. ethanolic extract administration exhibited liver protection by significantly decreasing the elevated serum ast, alt, lactate dehydrogenase (ldh) levels and this proved that ethanolic extract inhibited lipid peroxidation. the ethanolic extract significantly decreased the nfκb and tnf-α protein production (76). the ethanolic extract has anti-inflammatory activity by suppressing nfκb and tnf-α proteins synthesis (77). iraqi j pharm sci, vol.32(1) 2023 pharmacological aspects of borage 7 in the ccl4-treated mice liver, the level of thiobarbituric acid-reactive substance (tbars) (main reactive aldehyde produced from the polyunsaturated fatty acids (pufas) peroxidation (78)) was notably elevated, suggesting that exposure to ccl4 stimulated oxidative stress. in the ccl4treated rat liver homogenates, therapy with ethanolic extract reduced the production of tbars which may be attributed to its potent free radical scavenging and antioxidant effects (79). the ethanolic extract could at least partially reduce oxidative stress by reducing lipid peroxide and ros levels in ccl4treated rats. leaf extract of borage has a strong antioxidant effect related to their high polyphenolic content including kaempferol 3-o-β-dgalactopyranoside, and officinalioside (figures, 9 and10) (80). figure 9. chemical structure of kaempferol 3-o β-d-galactopyranoside (80) figure 10. chemical structure of officinalioside (80) analgesic activity in herbal medicine, the flower of borage is recognized as a sedative in a study involved formalin-induced pain in rat models, the borage hydroalcoholic extract showed antinociceptive properties. hydroalcoholic extract administration acute and chronic pain and this may be related to its antioxidant activity that possibly ameliorated the damaged cells stimulated by injection of formalin or suppressed the depolarization trigger in pain sensory neurons (42). the bso has strong analgesic activity. this effect was evaluated using the writhing test (determine the peripheral analgesic effect) and tail immersion test (determine central analgesic effect) in mice. the analgesic activity of the bso may be done by a peripheral mechanism including inhibition of pgs, substance p, and bradykinins synthesis, or by a central mechanism including dopaminergic descending noradrenergic, serotonergic, opiate systems (32). anxiolytic activity the anxiolytic effect of borage flowers' extract was determined by using an elevated plus-maze test (epm) in male rats. borago officinalis extract elevated both the entries percentage into the open arms of the maze and time spent percentage in the open arms of the maze that means the extract was capable to produce anxiolytic activity in rats. on the number of closed arm entries, the borage extract had no action (2). the increase in anxiety was been positively connected with elevated levels of ros. by a non-pharmacological method, the oxidative stress induction resulted in anxietylike manners in rats (81). borage extracts have an antioxidant effect, due to their phenolic compounds content (82, 83). borage use has become more common since it is a source of γ-linolenic acid (7). several studies determined the associations between γlinolenic acid content and the antioxidant property of borage extract (84, 85). the existence of flavonoids, polyphenols, and γ-linolenic acid in flowers extract of borage promotes anxiolytic activities. anti-obesity activity the bso showed anti-obesity actions in a diet-stimulated obesity rat model. the bso supplementation significantly decreases energy efficiency and body weight gain and prevents the accumulation of white adipose tissues without affecting the food intake. the bso improves insulin-resistance, promotes downregulation of cebpa (adipogenesis-related gene), and elevates serum high-density lipoprotein (hdl) levels (86). memory improvement the borage extracts revealed protective activity on amyloid β (aβ)-stimulated memory impairment. alzheimer’s disease (ad) is a neurodegenerative disease causing memory impairment and dementia. the memory and learning abilities were tested with morris water maze (mwm) and the passive avoidance tasks in the rats, while they used frap for determination the” antioxidant activity. the intrahippocampal (ihp) a𝛽 injection stimulated a significant disturbance of learning in the mwm and passive avoidance tasks in the rat. administration of borage attenuated the abstimulated impairment of memory in both the mwm and passive avoidance tasks. the ab stimulated a marked reduction in hippocampal frap value (antioxidant effect) and borage prohibited the reduction in antioxidant status of the hippocampus. so borage may ameliorate the oxidative damage and learning impairment in hippocampus tissues after ab treatment. borage could be a helpful tool in the treatment of patients with memory impairment (87). iraqi j pharm sci, vol.32(1) 2023 pharmacological aspects of borage 8 the ad starts with synaptic function impairment before advancing into later neural loss and neurodegeneration. the borago officinalis extract showed protective effects on ab-stimulated long-term potentiation (ltp) disruption in gyrus (dg) of hippocampus in male rats. in performant dg synapses, the ltp was tested using the electrophysiology method, and population spike (ps) and field excitatory post-synaptic potential (fepsp) slope amplitude was determined (88). in the hippocampus, ab administration produced an alteration in ltp which resulted in memory impairment and cognitive dysfunction in rodents (89). the ihp ab injection reduced ps and epsp slope amplitude while administration of borage extract elevated these parameters. the ab stimulated a marked reduction in the hippocampal total sulfhydryl (sh) groups and borage extract suppressed the lowering of total sh content of the hippocampus. the ab can effectively suppress ltp in the dg granular cells in the hippocampus, and following ab treatment in dg, supplementation of borage inverse the synaptic plasticity, and that consumption of borage may result in improvement in ad-stimulated cognitive dysfunction (88). borage oil could ameliorate ab-stimulated ltp deficit and memory impairment. the protective effect of this plant on ltp and memory can be attributed to its high gla content and its scavenging free radicals function (90). the borage oil neutralizes free radicals, inhibits, inhibits the effects of inflammatory proteins, prevents the inhibitory potential of ab on ltp and learning, and might be involved in the preservation of ab-stimulated neurotoxicity (87, 88). decreasing the opioid withdrawal symptoms the hydroalcoholic extract of borage flower demonstrated a significant reduction in opioid withdrawal symptoms in animal models precipitated by naloxone, and this included abdominal twitching and scratching (91). a coand post-treatment with borage flower extract produced a significant decline in the frequency of blinking, jumping, ptosis; paw trembling, scratching, and bowel movement (91). the polyphenolic components of the herb suppress the cholinergic outflow through cholinesterase inhibition, causing a symptomatic from opioid withdrawal symptoms (92). asthma symptoms improvement the borage hydroalcoholic flower and leaves extracts revealed beneficial considerable symptomatic in asthmatic patients without pathophysiological changes in moderate persistent asthma. oral administration of borage crude extract (3times daily) for one month was capable to prevent main clinical asthma findings involving dyspnea, cough, and airway hyper-responsiveness. physical exam showed noticeable wheezing improvement. borago extract also improved gastro-esophageal reflex-associated symptom. the borago extract was able to decrease the acute asthmatic attack frequency with lower invasive treatment. physiological parameters involving fractional exhaled nitric oxide test, spirometry, and also sputum cytology involving neutrophil and eosinophil were not changed. as the plant extracts presented temporary symptomatic relieve, it showed benefit as an adjunct for asthmatic patients who needs herbal alternative medicine (93). improvement of cyclical mastalgia the most common mastalgia type is cyclical mastalgia, with a reported spreading of up to third of all women at reproductive age (94). though its cause is not known, cyclical mastalgia is correlated with the menstrual cycle and hormonal changes especially estrogen, originating as a result of breast tissues proliferation coinciding with ovulation (95). borago extract was shown to ameliorate both emotional and physical symptoms of premenstrual syndrome (pms) (96). the borago extract was effective and safe in the cyclic mastalgia treatment among the treated patients. the reported positive results may be related to the higher content of the gla in the borage extract (97). the mechanism of action of this fatty acid is supposed to produce a downregulation of pge2, which mediate fast gla conversion to dgla (dihomo-γ-linolenic acid). this conversion elevates the production of pge1, and increases the levels of intracellular camp, which in turn suppresses phospholipase, and reduces the release of arachidonic acid (98). conclusion borago officinalis is a valuable medicinal mediterranean herb. previous reports showed that the borage areal parts crude extracts embrace valuable bioactive metabolites as, acid, quercetin, rutin, spathulenol, and several other metabolites. the isolated metabolites show promising benefits among which, antioxidant, memory improvement, antinociceptive, hepatoprotective properties. the reported beneficiaries suggest further analysis of the secondary metabolites to find new lead molecules with promising therapeutic benefits. the author/s of this review appreciates the help 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creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 chenopodium murale effect on atopic dermatitis on mice doi: https://doi.org/10.31351/vol32iss1pp84-91 84 evaluation the effect of phytosterol fraction of chenopodium murale in comparison with tacrolimus on mice induced atopic dermatitis zahraa y. hassan1*.1, tuka y. hassan** and ahmed r. abu raghif *** * ministry of health and environment, al-imamain al-kadhimain medical city, baghdad, iraq. ** ministry of health and environment, public health directorate, baghdad,iraq. *** department of pharmacology and therapeutics, college of medicine, al-nahrain university, baghdad, iraq. abstract atopic dermatitis (atopic eczema), is a common familial chronic inflammatory skin disease, determined by xerosis, itching, scaly and erythematous skin lesions, and high serum levels of ige. between 10 to 20% of children and 1 to 3% of adults worldwide affected by it and has negative medical and social effect on patients and their families. to evaluate the effectiveness of phytosterol fraction of chenopodium murale on dncb-induced atopic dermatitis (ad) of mice; forty mice were included in the study, divided in to four groups (10 mice/group): apparently healthy, induced ad without treatment, induced ad treated with tacrolimus 0.1% ointment, and induced ad treated with phytosterol fraction of chenopodium murale cream 3% topically. examination of histopathology was done and skin homogenates levels also measured using mann whitney u test to determine mean±sd. levels of wbc, eosinophil, skin tissue homogenate of il-13 and il-4, serum ige, and histopathological scores were significantly increased among induced non treated ad group in comparison with control group. comparisons of non-treated induced ad group with chenopodium murale or tacrolimus treated groups; shows a significant reduction in the levels of all studied parameters’ (wbc, eosinophil, skin tissue homogenate of il4and il-13, serum ige, observational severity score, and histopathological scores) after the application of tacrolimus 0.1% ointment or chenopodium murale cream 3% topically. the comparison between the effect of topical application of tacrolimus and phytosterol fraction on the studied variables shows that the level of wbc and thickness of epidermis and inflammatory cells were significantly lower after tacrolimus treatment, while high significant reduction was founded in parakeratosis and score of observational severity among chenopodium murale treated group in comparison with tacrolimus treated group. in conclusion, topical application of phytosterol fraction of chenopodium murale seems to be effective in treatment of atopic dermatitis through their abilities to decrease wbc, eosinophil, s. ige, skin tissue homogenate of il4, and il13; as well as improving histopathology picture and reducing observational severity score. the use of phytosterol fraction of chenopodium murale that target ige, il4, and il13 could be promising in the treatment of atopic dermatitis. key words: phytosterol fraction, chenopodium murale, atopic dermatitis, tacrolimus, interleukin-4, interleukin-13 تقييم فعالية جزيئات الفايتوستيرول لكينوبوديوم ميوريل بالمقارنة مع تاكروليموس على التهاب الجلد التحسسي المستحث في الفئران المختبرية ** رغيف أبو و أحمد **، تقى يونس حسن 1*،زهراء يونس حسن العراق. بغداد، ، االمامين الكاظمين الطبيةوزارة الصحة والبيئة ، مدينة * .الصحة العامة ، بغداد ، العراق وزارة الصحة والبيئة ،دائرة** .كلية الطب ، جامعة النهرين ، بغداد ، العراق*** الخالصة ارتفاع ، و احمرار الجلد وتقشرهبالجفاف ، والحكة ، سمالتهاب الجلد التأتبي )األكزيما التأتبية( ، هو مرض جلدي التهابي مزمن عائلي شائع ، يت واجتماعي صحي به وله تأثير يصابون ٪ من البالغين في جميع أنحاء العالم 3إلى 1٪ من األطفال و 20إلى 10االجسام المضادة أي في الدم. ما بين ى مستو . تم تضمين أربعين المختبرية على التهاب الجلد التأتبي في الفئران ميوريل وبوديوملكين الفايتوستيرول جزيئاتلتقييم فعالية سلبي على المرضى وعائالتهم. فئران / مجموعة(: صحية ، ُمحفَّزة بالتهاب الجلد التأتبي دون عالج ، ُمحفَّزة بالتهاب الجلد التأتبي ُمعالج 10فأًرا في الدراسة ، مقسمة إلى أربع مجموعات ) ٪ . تم إجراء فحص التشريح 3 ميوريل كينوبوديوممن كريم الفايتوستيرول جزيئاتُمحفَّزة بالتهاب الجلد التأتبي معالج ب ٪ مرهم ، و0.1بتاكروليموس واالجسام 4و االنترلوكين 13و في االنترلوكين الخاليا الحمضية و كريات الدم البيضاء المرضي وقياس مستويات تجانس الجلد. وجد زيادة في مستويات في الدم ونتائج األنسجة المرضية بشكل ملحوظ بين المجموعة المستحثة غير المعالجة بالمقارنة مع المجموعة الضابطة. دة أيالمضا ب المعالجة المجموعات مع المعالجة غير التأتبي الجلد بالتهاب الُمحفَّزة المجموعة بين يُظهر ميوريل كينوبوديوممقارنات ؛ تاكروليموس أو أظهرت المقارنة بين تأثير التطبيق الموضعي س.وتاكروليم مرهم كريم الفايتوستيرول او ًرا في مستويات جميع المعلمات المدروسة بعد وضعانخفاًضا كبي كانت و الخاليا المضادة لاللتهاب سماكة البشرة كريات الدم البيضاء و على المتغيرات المدروسة أن مستويات الفايتوستيرول جزيئاتو لتاكروليموس عقار ال أقل بشكل و درجة شدة المالحظة نظير التقرن ىبين المجموعات المدروسة ، بينما كانت مستو للتاكروليموس الموضعي التطبيقأقل بشكل ملحوظ بعد الفايتوستيرول لجزيئات الموضعي التطبيق أنك نستتج من ذلبين المجموعات المدروسة. ميوريل لكينوبوديوم الفايتوستيرول جزيئاتملحوظ بعد تطبيق 4االنترلوكين و 13 االنترلوكين و الحمضية الخاليا و كريات الدم البيضاء تقليل على قدرته خالل من التأتبي الجلد التهاب عالج في فعال ميوريل لكينوبوديوم ميوريل لكينوبوديوم الفايتوستيرول جزيئات استخدام يكون لهذا, قد. المالحظة شدة درجة وتقليل سماكة البشرة تحسين وكذلك ؛ الدم في أي المضادة واالجسام . التأتبي الجلد التهاب عالج في واعدًا 13، إنترلوكين 4كينوبوديوم ميوريل ، التهاب الجلد التأتبي ، تاكروليموس ، إنترلوكين ، : جزيئات الفايتوستيرول المفتاحيةالكلمات 1corresponding author e-mail: zahraahassan793@gmail.com received: 14/1 /2022 accepted: 6/4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp84-91 iraqi j pharm sci, vol.32(1) 2023 chenopodium murale effect on atopic dermatitis on mice 85 introduction atopic dermatitis (ad) is an inflammatory skin condition with pruritus; erythema; dryness; and scaly skin. asthma presented in 30% of patients with ad. it is tending to be chronic with relapsing and remission periods. patients can be cured during puberty; or persist for long life. (1, 2) bacterial and viral infections of skin may be a consequencies to ad. herpetic eczema due to herpes simplex virus is frequently observed among ad patients. most of patients with ad have staphylococcus aureus infection. (3) restoring skin barrier is the gold standard in the management of ad, often through skin moisturizing, decrease itching and inflammation. (4) tacrolimus, a macrolide lactone of fungal origin (streptomyces tsukubaensis), is an immunosuppressive drug commonly used in humans. tacrolimus has been shown to inhibit the granules release of preformed mediators from skin mast cells and basophils and to down regulate the expression of ige by mast cells, basophils, and langerhan cells.(5) its small size make its ability to penetrate skin more powerful; therefore, it can be used in severe cases of ad and improve control of acute attacks and prevention of new ones due its mechanism of action as immune regulation.(6, 7) tacrolimus has side effects, such as skin burning and itching. (8) accordingly, effective therapy with fewer side effects is required for treatment of ad. the world health organization encourages, promotes and facilitates effective herbal health programs. (9) extracts from the leaves of various plants have various pharmacological activities, among these antioxidant activities due to their redox properties, which permit them to act as reducing agents, hydrogen donors, and single oxygen quenchers. the crude extract of various plants and its fractions were examined previously against different human pathogens including, escherichia coli, klebsiella pneumonia, bacillus subtilis, and salmonella typhus, by agar well diffusion method (anti-bacterial activity). phytosterol fraction seemed to be a potential nutraceutical tool for some diseases such as gastrointestinal inflammatory disease. in addition, combining metabolic systematic and local anti-inflammatory effects (anti-inflammatory activity). previous research showed that plant also useful as an anthelmintic, stomachic, antispasmodic, diaphoretic, sweaty, for amenorrhea pain, for abortion and for the relief of asthma, cold, and migraine. (10-12) although the currently used medications in the treatment of ad are effective in managing the disease; adverse reactions may decrease their usefulness (8). pharmacological activities of chenopodium murale: antioxidant activity, antibacterial activity, anti-inflammatory and skin disease, previous research showed that plant also useful as an anthelmintic, stomachic, antispasmodic, diaphoretic, for amenorrhea pain, for abortion and for the relief of asthma, cold, and migraine. (11, 13) this study was carried out to evaluate the effectiveness of phytosterol fraction of leafs of chenopodium murale on treatment of induced atopic dermatitis mice model through their effect on wbc, eosinophil, serum ige, tissue homogenate of il4 and il13, observational severity score, and histopathological score. the study also aimed to compare the anti-inflammatory effect of phytosterol fraction of chenopodium murale with tacrolimus on induced atopic dermatitis mice model. materials and methods a randomized prospective, controlled animal study was carried out. this study was conducted from december 2020 june 2021, in the department of pharmacology-college of medicineal nahrain university. the protocols for the animal experiment used were carefully reviewed for ethical and scientific care procedures and approved by al nahrain university – college of medicine review council (approval number 857 in 28/9/2020). experimental animals and design of study this study included 40 healthy adult male albino mice weighted 25-30g. they were housed in animal house in a good ventilated isolated place; with a room temperature of 20-24°c. the animals were left for seven days to acclimatize to the animal room conditions and allowed free access to water and ad libitum feeding. the animals were housed in animal house, at college of veterinary, and kept light for 12 hours. the practical part of the study was directed at college of veterinary medicine, university of baghdad, baghdadiraq. ten mice were chosen randomly and considered as a (healthy control) group and compared with other induced groups. thirty mice treated with 1-chloro-2, 4dinitrobenzene (dncb) induced ad (14) and randomly divided into three groups 10 mice/group (without using anesthetic medication in the ad induction period) (15-17).induced ad mice non treated (negative control), induced ad mice treated with tacrolimus 0.1% ointment (positive control), and induced ad mice treated with phytosterol friction of chenopodium murale cream 3% topically (test compound).(18) topical treatment was applied once daily at 9:00 am for 21 days induction mouse model of 1-chloro-2, 4-dinitrobenzene induced atopic dermatitis mice described ad skin through shaving hair from dorsal of skin then 150 μl of 1% dncb in 3:1 (v/v) acetone/olive oil solution was topically applied once to the exposed skin, then after four days, 0.2% dncb dissolved in an acetone: olive oil mixture (3:1 vol/vol) was applied to the same dorsal skin (150 μl) three times a week for 3 weeks. after iraqi j pharm sci, vol.32(1) 2023 chenopodium murale effect on atopic dermatitis on mice 86 the visual confirmation of skin sensitization, mice were treated with test samples. (14) figure 1. figure 1. normal skin lesion without induction (a), induced atopic dermatitis skin lesion (b) and (c). plant material chenopodium murale plant was identified and authenticated by prof. dr. ibrahim s. aljubori /department of pharmacognocy/ college of pharmacy/ al-mustansryiah university .the extraction of herb was executed in pharmacognocy department, collage of pharmacy, al-mustansryiah university (iraq). leaves were washed thoroughly, dried under shade, and ground in a mechanical grinder to coarsely powder. the aerial parts of chenopodium murale were extracted and authenticated in november, 2020 by department of pharmacognocy and medicinal plants / college of pharmacy/ al-mustansiriya university (iraq). extraction and fractionation of phytosterol fraction of chenopodium murale 1. shade-dried coarsely powdered leaves (250g) plant will extracted with 90% ethanol (500ml) in soxhlet apparatus until complete exhaustion. the alcoholic extract was evaporated under reduced pressure at a temperature not exceeding 40 °c to give a dark green color designated as crude fraction. 2. crude fraction was acidified with hydrochloric acid (5%) to ph2 and partitioned (three times) with equal volume of ethyl acetate to get two layers. aqueous acidic (f1, f2) was left and get ethyl acetate layer. 3. the ethyl acetate layer of the original alcoholic extract (crude fraction) was evaporated to dryness under reduced pressure and basified with 300ml of sodium hydroxide 5% to ph 10 and extracted with chloroform in the separator funnel to get two layers, the aqueous basic layer (f3) was left and chloroform layer was collected. 4. chloroform layer was also separated and evaporated to dryness under reduced pressure then two types of solvents: methanol 80% and petroleum ether were added to chloroform layer to obtain phytosterol in petroleum ether layer (fraction f4, used in the study) (15) . preparation of phytosterol fraction 3% cream three gm of phytosterol fraction extracted from chenopodium murale was weighted and dissolved in 3 ml of alcohol and shaking it for 4 minutes until it dissolved completely and became clear, after that we complete the weight to 100 gram with aquasoft cream (ajanta company) and shake the combination for 5 minutes by spatula (16) . preliminary qualitative phytochemical analysis chemical tests were carried out using ethanol extracts using standard procedures to identify the phytosterol fraction of chenopodium murale (15) (i) liebermann-burchard test: extract (3ml) was treated with chloroform, acetic anhydride and drops of sulphuric acid were added. the formation of dark pink or red color indicates the presence of steroids. (ii) h2so4 test: the development of a greenish color was considered as indication for the presence of steroids, when 2 ml of the organic extract was treated with sulphuric and acetic acids). qualitative and quantitative estimation of phytosterol fraction of chenopodium murale using high performance liquid chromatography (hplc) (19) high performance liquid chromatography was used for identification of quantitative and qualitative estimation of phytosterol fraction in the plant. the identifications will made by detection of retention time obtained at identical chromatographic conditions of steroid fraction and the standards. experimental condition of hplc • mobile phase: ethyl acetate: water (7:30 ratio) • column: hyper clone odcc c18 v-25cm ods c18 • column temperature: 25ºc • flow rate: 0.5ml/min • injection concentration 0.5mg/1ml. • injection volume: 20μl • detection wavelength: 280 nm treatment protocols, parameters, and animal sacrificing the topical applications of tacrolimus 0.1% ointment(17) and phytosterol fraction of chenopodium murale cream 3% (18) were applied on atopic dermatitis area of animal for 21 days once daily at 9 am starting from the fifth day of induction. parameters are used to compare the results were wbc, eosinophil, serum ige, il4 il13, and histopathology of ad skin lesion and compared with those of controls, and then we determine the observational severity score. after 21 days of treatment, we took whole number of mice from each study groups and anesthetized through a piece of cotton socked with ether put with the mouse inside a closed jar for few minutes to ensure be anesthetized by inhalation. before sacrificed; blood sample collected (1ml) in edta tube for cbc and serum ige, then sacrificed iraqi j pharm sci, vol.32(1) 2023 chenopodium murale effect on atopic dermatitis on mice 87 by cervical dislocation; (blood sample collected by cardiac puncture (one ml) by using (three ml syringe) in edta tube for cbc and serum ige. after that cervical dislocation for the mice was done, then atopic dermatitis skin area was cut by sharp blade (no.15). this skin wound was dissection into two equal pieces one for the histological analysis and the second for the preparation of skin homogenate. the remaining mice from each group were subjected to the same procedure at the 21th day of the treatment. dorsal skin samples were collected from each animal in study groups and fixed in 10% formaldehyde paraffin embedded and cut into 6 μm sections. deparaffinized sections were stained with ordinary hematoxylin and eosin (h&e) to determine inflammatory degree and histological changes associated with atopic dermatitis (20). histopathological follow-up procedures were used for the skin samples taken from each group on the 21 days of treatment. histopathology of skin of each specimen were evaluated and scored by semi quantitative scoring systems. histopathology included epidermal thickness, hyperkeratosis, parakeratosis, erosion, inflammatory cell infiltration, and extracellular edema, each scored from 0 to 3 (0 no abnormality, 1+ slight, 2+ mild, and 3+ moderate)(14), the sections examined by pathologist and carried out in histopathology department /ibn sina university of medical and pharmaceutical sciences to observe the changes in tissues. skin tissue homogenate preparation the second piece of skin obtained were washed with normal saline, and rinsed with chilled phosphate buffer saline (1x pbs), with filter paper and weighed. each 100 mg of skin wound tissue was homogenized with 1 ml of (1x pbs) with the aid of tissue homogenizer (21) for 1 minute at 4 °c, and must be stored overnight at 20°c. two freeze-thaw cycles must be performed to break the cell membrane; the homogenate were centrifuged for 10 minute at 2000 rpm at 2-8 °c. the supernatant was obtained and stored at –20°c to the assay of il-4 and il-13 levels in the tissue. serum ige: the enzyme-linked immunosorbent assay) (elisa) kit for the estimation of ige was obtained from cusabio\china kit. specific different antibodies can be measured quantitatively by the enzymelinked immunosorbent assay (elisa). after incubating the tested serum in an antigen-coated polystyrene plat or tube, enzyme specifically labeled anti-immunoglobulin is then added and the remaining in the plate after washing will gives a measure to the quantity of specifically related antibody in the serum. the procedure depends on the insolubilization of specific antigens by passive adsorption to a solid phase (plate), example polystyrene phase (22). skin tissue homogenate of il4 and il13: elisa kit for the estimation of il4 and il13 was obtained from cusabio\china kit was established on the base of sandwich enzyme-linked immunosorbent assay technology. antiil4 and antiil13 antibodies were pre-coated onto 48-well plates. and as detection antibodies, the biotin conjugated antiil4 and antiil13 antibodies were used. we added; the standards, test samples and biotin conjugated detection antibodies to the wells subsequently, and washed with wash buffer. hrpstreptavidin was added and unbound conjugates were washed away with wash buffer. the concentration of il4 and il13 in each sample was expressed in pg/ml for comparison of results with those of controls concentration (23). assessment of observational severity score the severity of ad on the dorsal area was evaluated for each group on the 21th days of treatment. the evaluation of erythema, dryness, erosion and edema scored as 0 (none), 1 (mild), 2 (moderate), and 3 (severe). clinical skin score was defined as the summation of each individual scores, range from 0 to 12 (24) . statistical analysis microsoft excel 2016 and spss 24 were used for data entry and analysis. numerical variables were expressed as mean ± sd while categorical variables were expressed by frequencies and percentages, then represented by figures and tables. all statistical comparisons were made using one -way anova test. p <0.05 was considered statistically significant. results high performance liquid chromatography (hplc) for examination of phytosterol fraction of chenopodium murale qualitative and quantitative estimations of active constituents of chenopodium murale fraction was done, in which identifications was made by comprising the retention times obtained at identical chromatographic conditions of analyzed samples; the results show the presence of beta sitosterol as a major constituent. table 1. table 1. retention time of standard and sample of beta sitosterol of chenopodium murale subject retention time of stander/min retention time of sample/min area of sample beta sitosterol 2.547 2.517 822409 iraqi j pharm sci, vol.32(1) 2023 chenopodium murale effect on atopic dermatitis on mice 88 the comparison between all study groups (healthy control, negative control, test compound and positive control) regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13, histopathology changes and scores in comparison between the effect of topical tacrolimus and phytosterol fruction among all study groups, the level of wbc, eosinophil. ige, il4 were significantly lower after tacrolimus treatment among studied groups, (p<0.05) table 5. the effect of positive control (topical tacrolimus) on the epidermal thickness, extracellular edema, and inflammatory cells were significantly reduced, p<0.05. while high significant reduction in il3, parakeratosis, erosion and score of observational severity was observed among test compound (chenopodium murale treated group). p<0.001 and p=0.028 respectively. hyperkeratosis shows high significant reduction among both positive control and test compound (tacrolimus and chenopodium murale treated groups), p<0.001. table 2, figure 2, 3 and 4. table 2 . comparison between all study groups (healthy mice, negative control, test compound, and positive control treated groups) regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13, histopathological changes and score variables groups (mean±sd) p* healthy control negative control test compound positive control wbc (x109 /l) 3.3 ± 2 10±2.1 7.06± 2.01 6.03± 2.02 0.01* eosinophil (x109 /l) 0.0 2.5±0.02 0.025±0.09 0.020± 2.02 0.001* ige(ng/ml) 15.59±8.65 26.62±5.15 16.50±6.61 16.0±6.08 0.003* il13 (pg/ml) 22.3±68.76 57.8±10.53 31.63±12.31 31.82±21.3 <0.001* il4 (pg/ml) 6.68±3.01 22.11±6.21 9.68±2.88 9.05±4.03 <0.001* epidermal thickness 0.0 3.50±0.52 2.20±0.78 1.20±1.22 <0.001* hyperkeratosis 0.0 3.00±0.81 1.60±0.51 1.60±0.51 <0.001* parakeratosis 0.0 3.40±0.69 1.00±0.003 1.20±0.78 <0.001* erosion 0.0 1.50±0.52 0.20±0.35 0.20±0.42 <0.001* inflammatory cell 0.0 2.60±0.51 1.80±0.42 1.70±0.42 <0.001* extracellular edema 0.0 2.50±0.52 1.23±0.42 1.20±0.51 <0.001* observational severity score 0.0 10.00±0.81 3.50±0.97 4.50±1.08 <0.001* *one way anova test where p significant at < 0.05 and high significant at <0.001 iraqi j pharm sci, vol.32(1) 2023 chenopodium murale effect on atopic dermatitis on mice 89 figure 2. histopathology changes in negative control group (b) in comparison with healthy controls (a), test compound group (c), and positive control group (d) (10x): ordinary hematoxylin and eosin stain. figure 3 comparison between negative control, healthy controls, test compound, and positive control groups regarding wbc, eosinophil, serum ige, and skin tissue homogenate of il-4 and il-13 in mice; results are expressed as mean ± sd by one way anova test, p is significant at < 0.05. figure 4. comparison between negative control, healthy controls, test compound, and positive control groups regarding histological changes and observational score in mice; results are expressed as mean ± sd by one way anova test, p is significant at < 0.05 discussion atopic dermatitis (ad) is the most common chronic inflammatory and chronically relapsing skin disease. the disease leads to a significantly reduced quality of life. the pathogenesis of ad is not well understood but appears to be associated with the activation of innate immune responses, including inflammation (25, 26). apparently, ad induced untreated group shows significant inflammation signs and significant increase in thickness and in the level of observational severity score among ad induced untreated group. similarly; a study reported that significant increase in all types of wbc was found among ad induced untreated group (27). histopathology changes and observational scores after application of 3% cream topically of phytosterol fraction show a significant decrease when compared with untreated induced ad group. this indicates that the anti-atopic effect of βsitosterol (hplc for examination of phytosterol fraction of chenopodium murale was done, and the results show the presence of beta sitosterol as a major constituent) is similar to the effect of tacrolimus. β-sitosterol is attributable to the regulation of inflammatory mediator as in study concluded that the anti-inflammatory activities of βsitosterol could be attributed to the inhibition of inflammatory cytokine in ad-like skin lesion and reported the effect of β-sitosterol as a therapeutic use in inflammatory skin diseases such as ad. (28) a study on animals have indicated that β-sitosterol reduces the secretion of pro-inflammatory cytokines, as well as edema and increases antiinflammatory cytokines (29). similar to the effect of phytosterol fraction of chenopodium murale topical treatment, application of tacrolimus 0.1% topically was significantly associated with reduction; as compared with non-treated induced ad group, in the levels of wbc, eosinophil, skin tissue homogenate of il4 a b c d iraqi j pharm sci, vol.32(1) 2023 chenopodium murale effect on atopic dermatitis on mice 90 and il-13, serum ige, observational severity score, and histopathological changes. a significant improvement in erythema, pruritus, sleep pattern, and quality of life was reported by 4 week tacrolimus treatment study (30). in addition to its ability in to preventing, delay, and reduce the occurrence of disease exacerbation in adult and children. (31, 32) the levels of parakeratosis and observational severity score after phytosterol fraction of chenopodium murale treatment were lower than after tacrolimus treatment. while wbc, epidermal thickness and inflammatory cells shows more significant decrease among tacrolimus treated group. (18, 28) these results indicate that phytosterol fraction of chenopodium murale that targeting ige, il4, and il13 resemble the effectiveness of previously approved drug tacrolimus ointment 1%; therefore it may be promising a good treatment for atopic dermatitis. conclusion topical application of phytosterol fraction of leafs of chenopodium murale seems to be effective in treatment of atopic dermatitis through their abilities to decrease wbc, eosinophil, s. ige, skin tissue homogenate of il4, and il13; as well as improving histopathology picture and reducing observational severity score. the use of phytosterol fraction of chenopodium murale that target ige, il4, and il13 could be promising in the treatment of atopic dermatitis. references 1. fuxench zcc. atopic dermatitis: disease background and risk factors. in management of atopic dermatitis. springer, cham, 2017;1027:11-19. 2. spergel jm. epidemiology of atopic dermatitis and atopic march in children. immunol allergy clin north am. 2010;30(3):269-280. 3. nutten s. atopic dermatitis: global epidemiology and risk factors. ann nutr meta, 2015; 66(1):8-16. 4. tanei, r. atopic dermatitis in older adults: a review of treatment options. drugs aging 2020:37(3):149-160. 5. astellas pharma us. patient information sheet: protopic 0.1% (tacrolimus), ndc 2006; 04695202-30. 6. lee jh, son sw, cho sh. a comprehensive review of the treatment of atopic eczema. allergy asthma immunol res, 2016; 8(3):181– 190. 7. nghiem p, pearson g, langley rg: tacrolimus and pimecrolimus: from clever prokaryotes to inhibiting calcineurin and treating atopic dermatitis. j am acad dermatol, 2002; 46 (2):228-241. 8. kang s, lucky aw, pariser d, lawrence i, hanifin jm. long-term safety and efficacy of tacrolimus ointment for the treatment of atopic dermatitis in children. j am acad dermatol , 2001;44(1):58–64 9. singh kp, dwevedi ak, dhakre g, evaluation of antibacterial activities of chenopodium album. ijabpt, 2011;2(3):398-401. 10. ahmed a a, abu-raghif a r. effect of topical phytosterol fraction of chenopodium murale on induced hypertrophic scar in rabbits. journal of global pharma technology, 2020;12(02):115124 11. aldini r, micucci m, cevenini m, fato r, bergamini c, nanni c, et al. anti inflammatory effect of phytosterols in experimental murine colitis model: prevention, induction, remission study. plos one, 2014;9(9), e108112. 12. ahmad b, jan q, bashir s, nisar m, shaheen f, ahmad m. pharmacological and biological investigations of chenopodium murale linn. asian journal of plant sciences, 2003;2(15-16), pp.1107-1111. 13. batcha o, gnatoulma k, gérard t, laura l, efui g, manuel r, et al. anti-inflammatory, antibacterial and antioxidant activities of chenopodium ambrosioides l. 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work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 knowledge and perception towards biosimilars doi: https://doi.org/10.31351/vol30iss1pp226-232 226 knowledge and perception of iraqi pharmacists towards biosimilar medicines ashwaq j. mohammed*,1 and dheyaa j. kadhim** * ministry of health and environment , iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract biosimilars are non-innovative versions of biologic medicines which are proven to be clinically equivalent to, as effective, and as safe as their reference biologics. biosimilars create opportunities for cost savings for payers, governments, and patients compared to the reference products. pharmacists play an essential role in developing biosimilars from manufacturing to post-marketing pharmacovigilance monitoring. the aim of the current study was to explore the level of knowledge and perception of a sample of iraqi pharmacists towards biosimilars. the current study was a cross sectional and was carried out during may 2020. a total of 264 pharmacists )143 male, 121 female) were involved in this study. a web-based self-administered questionnaire was used for data collection. regarding pharmacists’ knowledge of biosimilar medicines, the results showed that two questions received the highest percentages of adequate answers: biosimilar medicine requires preclinical and clinical studies (58.0%) and biosimilar medicines require more comprehensive data compared to generic drugs (56.1%). in contrast, marketing authorization of biosimilar medicines is granted on the sole investigation of pharmacokinetic bioequivalence received the lowest percentage of adequate answers (21.6%). in addition, the current study showed that more years of experience and male gender were associated with better knowledge. with respect to perceptions of pharmacists to biosimilars, two statements received the highest percentage of pharmacist agreements: biosimilar medicines are tested in terms of efficacy and safety (64.4%) and biosimilar prescription allows for reducing costs (64.4%). at the same time, 40.2% of the participating pharmacists agreed with pharmacist replacing a reference biologic medicines with its biosimilar product. in conclusion, the majority of the pharmacists included in the survey had insufficient knowledge about biosimilars. the study highlighted that iraqi pharmacists needed more accurate and comprehensive information about biosimilars. keywords: biosimilars, pharmacists, iraq, knowledge, perception. معرفة وتصور الصيادلة العراقيين تجاه المشابهات الحيوية **و ضياء جبار كاظم 1،*شواق جاسم محمدأ العراق.والبيئة ، وزارة الصحة * .بغداد، بغداد، العراق الصيدلة، جامعة السريرية، كلية الصيدلة فرع ** الخالصة االحيائية االحيائية التي ثبت علميًا أنها مكافئة سريريًا ، وفعالة وآمنة مثل األدوية هي نسخ غير مبتكرة من األدوية الحيوية المشابهات يلعب الصيدلي .في التكاليف للدافعين والحكومات والمرضى ، مقارنة بالمنتجات المرجعية توفيرفرًصا لتحقيق الحيوية المشابهاتتخلق . المرجعية .بدًءا من التصنيع حتى دراسات اليقظة الدوائية وما بعد التسويق الحيوية المشابهاتدوًرا مهًما في تطوير الدراسة الحالية .الحيوية المشابهاتمن الصيادلة العراقيين تجاه لعينةتصورات استكشاف مستوى المعرفة والكان الهدف من الدراسة الحالية هو تم استخدام استبيان عبر (. أنثى 121ذكًرا و 143)صيدليًا 264شارك في هذه الدراسة .2020 عبارة عن دراسة مقطعية أجريت خالل شهر ايار النسب المئوية ، أظهرت النتائج أن سؤالين تلقيا أعلى الحيوية المشابهاتبفيما يتعلق بمعرفة الصيادلة . المتعلقة بالدراسةاإلنترنت لجمع البيانات تتطلب بيانات أكثر شموالً مقارنة الحيوية المشابهاتو ( ٪58.0)تتطلب دراسات ما قبل السريرية والسريرية الحيوية المشابهات: لإلجابات الكافية بناًء على تحقيق وحيد للتكافؤ الحيوي الدوائي حصل على أقل الحيوية المشابهاتفي المقابل ، يُمنح ترخيص تسويق ( . ٪56.1)دوية الجنيسة باأل باإلضافة إلى ذلك ، أظهرت الدراسة الحالية أن المزيد من سنوات الخبرة وجنس الذكور مرتبطان بمعرفة (. ٪21.6)نسبة من اإلجابات الكافية من الحيوية المشابهاتتم اختبار :، حصل بيانان على أعلى نسبة من اتفاق الصيادلة الحيوية المشابهاتفيما يتعلق بتصورات الصيادلة حول . أفضل المشاركين ٪ من الصيادلة 40.2في الوقت نفسه ، اتفق (. ٪64.4)يسمح بخفض التكاليف الحيوية المشابهاتووصف ( ٪64.4)حيث الفعالية واألمان غالبية الصيادلة الذين شملهم االستطالع كنتيجة فأن .الحيوية المشابهاتاالحيائية المرجعية بمنتج من على امكانية قيام الصيدالني باستبدال األدوية المشابهاتمات دقيقة وشاملة عن سلط المسح الضوء على الحاجة إلى تزويد الصيادلة العراقيين بمعلو. الحيوية المشابهاتبلم تكن لديهم معرفة كافية .الحيوية تصور. ، معرفة، العراق ،الصيادلة، الحيوية المشابهات الكلمات المفتاحية: 1corresponding author e-mail: winterice1010@gmail.com received: 5/10/2020 accepted: 5/12 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp226-232 iraqi j pharm sci, vol.30(1) 2021 knowledge and perception towards biosimilars 227 introduction the introduction of biologic medicines represented a significant development in therapy for many serious diseases. (1) despite their important value, biologic medicines have become a big financial problem to the health care systems. (2) therefore, availability of “generic” copy of biological medicines would be essential as this could save billions of dollars yearly. (3) the u.s. food and drug administration (fda) defines a biosimilar product as highly similar but not identical to an already licensed biologic product in terms of quality, safety, and efficacy. (4) costs of biosimilars are about 15–30% less than the originator prices so their use creates opportunities for cost savings for payers, governments, and patients compared with the reference products since. (5) biologic medicines are big molecules that are mostly proteins. the manufacturing of biologic medicines is very complex which required living cells; so variation in the final product is a concern. post-translational modifications such as oxidation and glycosylation and even a small variation in the manufacturing process can change the structure of biologic medicines, with possible changing in pharmacological activity, safety profile, efficacy, and immunogenicity. accordingly, the manufacturing of biologic medicines must be closely monitored so that consistency of batch to batch will be ensured. consequently, it is almost difficult to synthesize a biologic medicine that is identical to its originator. (6) in iraq, a specialized committee called the ‟biologics and biosimilars registration committee (bbrc)” to register biosimilars was established by the national regulatory authority (nra). the bbrc established the first version of guideline for biosimilars and submitted it to the medicines policy committee for approval on april 28th of 2019. the iraqi biosimilars guidelines were adapted mainly from the european medicines agency (ema) guidelines, since ema was the pioneer in the biosimilars' field. (7) pharmacist plays an important role in developing biosimilars medicines from synthesis to dispensing to post-marketing pharmacovigilance monitoring. in many countries, pharmacists must be well educated about any new regulations concerning biosimilars. in addition, pharmacists are involved in educating physicians and patients about similarity, efficacy and safety of biosimilar medicines. (8) the aim of the current study was to explore the level of knowledge, behaviors and practices of a sample of iraqi pharmacists towards biosimilar medicines. subjects and method study population the current study was a cross sectional study carried out during may 2020. a total of 264 iraqi pharmacists (143 males, 121 females) were involved in this study. inclusion criteria any iraqi pharmacist willing to participate in the study were included. data collection a web-based, self-administered, questionnaire was used for data collection. the questionnaire investigates knowledge, behaviors and practices of pharmacists regarding biosimilar medicines. it consisted of three parts, the first part was about the sociodemographic and background characteristics of the participants. the second part addressed the knowledge of pharmacists about biosimilar medicines, this included 9 statements about quality, safety, efficacy and marketing authorization. knowledge domain have response categories of (yes, no, and don’t know). the third part (6 questions) were used to investigate pharmacists perceptions about biosimilar medicines, implementation of biosimilar medicines, substitution, and cost saving. the agreement of participants and attitude domains have five likert scale response categories. (9) ethical consideration the current study gained its approval from the ethical and scientific committee at the college of pharmacy/university of baghdad. all participants were informed about the study objectives and confidentiality of their answers. statistical analysis analyses were conducted using the statistical package for the social science (spss, version 22, ibm, new york, usa). descriptive statistics (categorical variable) were described using frequencies and percentages while, mean and standard deviation (sd) were used to describe (continuous variables) of the participants. independent t-test was used to measure the difference between the means of accurate answer scores according to gender. we excluded “not sure” group from the analysis. the total score of correct answers for each participants was calculated by giving one point for each correct answer. spearman correlation was used to measure the correlation between the pharmacist experience years and the mean of total score of correct answers to questions about biosimilar medicines. a p-value of less than 0.05 was considered to be statistically significant. iraqi j pharm sci, vol.30(1) 2021 knowledge and perception towards biosimilars 228 results the study recruited 264 pharmacists with average age of (30.16 ±6.94 years) and more than half (54.2%) were men. the average years of pharmacists' experience in field of pharmacy were (6.38±6.04 years). approximately, three-quarters (75.0%) of participants had bachelor’s degree in pharmacy (bsc). most of them lived in baghdad (58.7) as shown (table 1). table 1. demographic characteristics of participating pharmacists item subcategory frequency (n) % gender female 121 45.8 male 143 54.2 degree bachelor 198 75.0 diploma 18 6.8 master 40 15.2 ph.d. 8 3.0 province baghdad 155 58.7 anbar 28 10.6 diyala 16 6.1 karbala 12 4.5 najaf 11 4.2 others 42 15.9 item minimum maximum mean std. deviation age 22 68 30.16 6.94 experience years 0 33 6.38 6.04 when the respondents were asked about the basic information regarding biosimilar medicines, the results showed that two questions received the highest percentages of adequate answers: the requirement of biosimilar medicine for preclinical and clinical studies (58.0%) and the requirement for more comprehensive data compared to generic drugs (56.1%)ˮ . in contrast, the lowest percentage of adequate answers (21.6%) was for question regarding the role of bioequivalence study in marketing authorization of biosimilar medicines (table 2 and figure 1). table 2. pharmacists’ knowledge about biosimilar medicines statement adequate n % 1 «a biosimilar medicine is structurally identical to its reference medicinal product» no 106 40.2 2 «a biosimilar medicine is similar to a reference medicinal product that has gone off-patent» yes 114 43.2 3 «a biosimilar medicine has no meaningful differences from a reference medicinal product in terms of quality» yes 117 44.3 4 «a biosimilar medicine has no meaningful differences from a reference medicinal product in terms of safety» yes 142 53.8 5 «a biosimilar medicine has no meaningful differences from a reference medicinal product in terms of efficacy» yes 137 51.9 6 «a biosimilar medicine has the same dosage and route of administration compared to its reference medicine» yes 146 55.3 7 «a biosimilar medicine is a drug for which marketing authorization is granted on the sole investigation of pharmacokinetic bioequivalence with its reference medicine» no 57 21.6 8 «a biosimilar medicine is a drug for which assessment of biosimilarity requires more comprehensive data compared to generic drugs» yes 148 56.1 9 «a biosimilar medicine requires preclinical and clinical studies» yes 153 58.0 iraqi j pharm sci, vol.30(1) 2021 knowledge and perception towards biosimilars 229 0 1 0 2 0 3 0 4 0 5 0 6 0 b s m h a s t h e s o l e i n v e s t i g a t i o n o f p h a r m a c o k i n e t i c b i o e q u i v a l e n c e . b s m i s s t r u c t u r a l l y i d e n t i c a l t o i t s r e f e r e n c e m e d i c i n e s b s m i s s i m i l a r t o a r e f e r e n c e m e d i c i n a e s b s m h a s n o d i f f e r e n c e s i n q u a l i t y v s r e f e r e n c e . b s m h a s n o d i f f e r e n c e s i n s a f e t y v s r e f e r e n c e . b s m h a s n o d i f f e r e n c e s i n e f f i c a c y v s r e f e r e n c e . b s m h a s t h e s a m e d o s a g e a n d a d m i n i s t r a t i o n r o u t e o f r e f e r e n c e b s m r e q u i r e s m o r e c o m p r e h e n s i v e d a t a c o m p a r e d t o g e n e r i c s . b i o s i m i l a r m e d i c i n e r e q u i r e s p r e c l i n i c a l a n d c l n i c a l s t u d i e s % o f p h a r m a c i s t s w i t h a d e q u a t e a n s w e r s figure 1. the percentage of pharmacist with correct answers about biosimilar medicines (bsm). the number of participants who adequately answered to the questions about biosimilar medicines is normally distributed. approximately 41% of the pharmacist answered half of the questions adequately, 60 (22.7%) and 48 (18.2%) participants correctly answered five and four questions respectively. only 14 (5.3%) had all wrong answers, while eight (3.0%) had all nine correct answers (table 3). table 3. frequency distribution of participants according to number of correct answers with respect to knowledge. correct answers n % 1 nine correct answers 8 3.0 2 eight correct answers 10 3.8 3 seven correct answers 25 9.5 4 six correct answers 36 13.6 5 five correct answers 60 22.7 6 four correct answers 48 18.2 7 three correct answers 31 11.7 8 two correct answers 23 8.7 9 one correct answer 9 3.4 10 no correct answer 14 5.3 the spearman correlation shows there is significant (p< 0.05) positive correlation between the pharmacist years of experience and total score of the correct answers (table 4). in other words, longer experience years gives more correct answers. according to the independent samples t-test, male pharmacists had significantly higher mean of total score of correct answers compared to female pharmacists (table 5). table 4. correlation between the score of adequate answer and pharmacist experience years correlation coefficient (r) 0.138 p-value 0.025* n 264 * correlation is significant at the 0.05 level (2tailed). significant positive correlation using spearman correlation iraqi j pharm sci, vol.30(1) 2021 knowledge and perception towards biosimilars 230 table 5. the difference between the means of accurate answer scores according to gender. gender n mean std. dev. mean difference p-value total score female 121 3.926 2.122 -1.025 .0001* male 143 4.951 1.962 * significant difference (p-value < 0.05 level). a total of 6 questions were used to investigate pharmacists' perceptions about biosimilar medicines, and the results were outlined in (table 6). two statements received the highest percentage of pharmacist agreements: biosimilar medicines are tested in terms of efficacy and safety (64.4%) and biosimilar prescription allows for reducing costs (64.4%). at the same time, 40.2% of the participating pharmacists agreed replacing a reference biologic medicines with its biosimilar product by pharmacist. table 6. pharmacists’ perceptions about biosimilar medicines item strongly disagree n (%) disagree n (%) neutral n (%) agree n (%) strongly agree n (%) «i am in favor with the implementation of biosimilar medicines» 24 (9.1) 14 (5.3) 114 (43.2) 90 (34.1) 22 (8.3) «biosimilar medicines are tried and tested in terms of efficacy and safety» 19 (7.2) 31 (11.7) 44 (16.7) 123 (46.6) 47 (17.8) «biosimilar medicines are not only pharmacist’s concern» 33 (12.5) 27 (10.2) 50 (18.9) 107 (40.5) 47 (17.8) «i approve a pharmacist substitution of a reference biological medicine to its biosimilar product» 33 (12.5) 54 (20.5) 81 (30.7) 73 (27.7) 33 (12.5) «i approve a pharmacist substitution of a reference medicine product to its generic product» 31 (11.7) 34 (12.9) 79 (29.9) 80 (30.3) 40 (15.2) «biosimilar medicines prescription allows for reducing healthcare costs» 25 (9.5) 22 (8.3) 47 (17.8) 99 (37.5) 71 (26.9) discussion the current study provided a snapshot of iraqi pharmacists’ knowledge, and perceptions about biosimilars. regarding pharmacists’ knowledge of biosimilars, about half of respondents (52.6%) have answered 5 or more questions (out of 10 questions) correctly. comparing the results of the current study with that of one french study revealed a significant gap. for example, more than 90% of french pharmacists answered adequately regarding the statements " a biosimilar has no meaningful differences from a reference in terms of quality" and " a biosimilar medicine has no meaningful differences from a reference in terms of efficacy " compared to (44.3%) and (51.9%) in the current study, respectively. all french pharmacist adequate answers for the remaining of the nine questions were more than 70.0% except for the first one " a biosimilar medicine is structurally identical to its reference medicinal product" which was (59.4%). (9) in europe union, france is the first country establishing a regulation for biosimilar substitution and creation of a french directory for biosimilars. (10) although the exact means of biosimilar education were not stated, van overbeeke et al. suggested a positive relationship between biosimilar education and biosimilar knowledge. (11) the current finding showed that there is a strong need for pharmacistdirected education to enhance knowledge about biosimilars which might increase acceptance of biosimilars as safe and effective therapeutic options. in addition, the current study showed that more experience years associated with better knowledge. when there is little education about biosimilars in the undergraduate study, it is expected that practical experience will be an influential factor. also, the current study showed that male gender was associated with better knowledge. possible explanation may be that, compared to female, male iraqi pharmacists are more involved in drug promotion and marketing where the term " biosimilars" frequently encountered. a total of 6 questions were used to investigate pharmacists' perceptions about biosimilars. two statements received the highest iraqi j pharm sci, vol.30(1) 2021 knowledge and perception towards biosimilars 231 percentage of pharmacist agreements: biosimilar are tested in terms of efficacy and safety (64.4%) and biosimilar prescription allows for reducing costs (64.4%). regarding efficacy and safety, various clinical trials gave evidence-based results to confirm that there are no essential differences between a reference biological medicines and their biosimilars in terms of efficacy, and safety. (12, 13) concerning cost reduction, biosimilars are not only lower in cost to biologic therapies, but also introduce price competition leading to a decrease in the price of reference product. (6) pharmacist-led biosimilar substitution was another main issues raised in current survey where about 40.0% of pharmacists have approved a pharmacist substitution of a reference biologic medicine to its biosimilar counterpart. this proportion is relatively comparable with the rate of pharmacists who agreed with the substitution of a reference chemical medicines by its generic one (about 45.0%). however; it is lower than that reported in french study where about 53.0% of pharmacists approved pharmacist substitution of a reference biologic medicine to its biosimilar. (9) iga pawłowska et al. in their study detected that most polish pharmacists (about three-quarters) were worried about pharmacist-led substitution and only 23% of pharmacists thought that the reference medicine could be substituted by a biosimilar during therapy (14). in ireland, joan ocallagan et al. found that more than half of irish pharmacists (58%) were comfortable in changing patients from reference medicine to biosimilars in agreement with the prescriber and only (14%) preferred to make their own decision regarding suitable substitution. (15) pharmacists must be included in any policy making or regulations concerning biosimilars since they have close contact with both physicians and patients. there are continuous debates about the right of interchangeability, substitution and extrapolation of indications. while the innovative manufacturers are interested to keep their market share large and avoid any competition, biosimilar advocates are more interested in increasing share of biosimilars in the biologic market. pharmacists are in a good position to keep the balance between the two groups by looking for this issue from the patients’ side to provide biologic products readily accessible and at acceptable prices. (8) conclusions the majority of the surveyed pharmacists had insufficient knowledge towards biosimilar medicines. the study highlighted that iraqi pharmacists needed more comprehensive information concerning biosimilar medicines. limitation of the study the study had some limitations. first, a small sample size of the participants. hence, the results of the current study cannot be generalized to the whole iraqi pharmacists. second, the survey was short and detailed information was not obtained. nevertheless, the short form of the questionnaire may increase the willingness of pharmacists to complete the survey. third, the study was crosssectional done at a particular time point, and the perceptions of pharmacists may change over time. so, it will be valuable to repeat a similar survey several years later to know whether perceptions have changed or not. references 1. stevenson jg, popovian r, jacobs i, hurst s, shane lg. biosimilars: practical considerations for pharmacists. annals of 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practice in iraq.dovepress.2019;11:1-9. 7. al-kinani k.k , ibrahim mj, al-zubaidi rf , younus mm, ramadhan sh, kadhim jk, et al. iraqi regulatory authority current system and experience with biosimilars. regulatory toxicology and pharmacology.2020;117:1-5. 8. kunter i̇, balogun ho, şahin g. the role of the pharmacist from development to pharmacovigilance of biosimilars. marmara pharm. j . 2018; 22(4): 469-473. 9. beck m, michel b, rybarczyk-vigouret mc, levêque d, sordet ch, sibilia j, et al. knowledge, behaviors and practices of community and hospital pharmacists towards biosimilar medicines: results of a french webbased survey. mabs .2017;9 (2) :384-391. 10. adéa a , bourdon o, bussières j f. a survey of pharmacists knowledge and views of biosimilars in quebec and france. ann pharm fr.2017;75(4):267-275. 11. van overbeeke e, de beleyr b, de hoon j, westhovens r, huys i. perception of originator https://www.amgenbiosimilars.com/media/themes/amgen/amgenbiosimilars-com/amgenbiosimilars-com/pdf/2019-trends-in-biosimilars-report-electronic-version---usa-bio-80182.pdf https://www.amgenbiosimilars.com/media/themes/amgen/amgenbiosimilars-com/amgenbiosimilars-com/pdf/2019-trends-in-biosimilars-report-electronic-version---usa-bio-80182.pdf https://www.amgenbiosimilars.com/media/themes/amgen/amgenbiosimilars-com/amgenbiosimilars-com/pdf/2019-trends-in-biosimilars-report-electronic-version---usa-bio-80182.pdf https://www.amgenbiosimilars.com/media/themes/amgen/amgenbiosimilars-com/amgenbiosimilars-com/pdf/2019-trends-in-biosimilars-report-electronic-version---usa-bio-80182.pdf https://www.amgenbiosimilars.com/media/themes/amgen/amgenbiosimilars-com/amgenbiosimilars-com/pdf/2019-trends-in-biosimilars-report-electronic-version---usa-bio-80182.pdf iraqi j pharm sci, vol.30(1) 2021 knowledge and perception towards biosimilars 232 biologics and biosimilars: a survey among belgian rheumatoid arthritis patients and rheumatologists. biodrugs. 2017;31(5):447-59. 12. park w, hrycaj p, jeka s, kovalenko v, lysenko g, miranda p, et al. a randomized, double-blind, multicentre, parallel-group, prospective study comparing the pharmacokinetics, safety, and efficacy of ctp13 and innovator infliximab in patients with ankylosing spondylitis: the planetas study. ann rheum dis. 2013; 72(10):1605-12. 13. yoo dh, hrycaj p, miranda p, ramiterre e, piotrowski m, shevchuk s, et al. a randomized, double-blind, parallel-group study to demonstrate equivalence in efficacy and safety of ct-p13 compared with innovator infliximab when coadministered with methotrexate in patients with active rheumatoid arthritis: the planetra study. annrheum dis 2013; 72(10):1613-20. 14. pawłowska i, pawłowski l, krzyzaniak n, kocić i . perspectives of hospital pharmacists towards biosimilar medicines: a survey of polish pharmacy practice in general hospitals. bio drugs. 2019;33(2):183-91. 15. o'callaghan j, bermingham m, leonard m, hallinan f, morris m, moore u, et al. assessing awareness and attitudes of healthcare professionals on the use of biosimilar medicines: a survey of physicians and pharmacists in ireland. regulatory toxicology and pharmacology.2017;88:252-261. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 exploring aspirin derivatives doi: https://doi.org/10.31351/vol31iss2pp14-32 14 aspirin derivatives exploration: a review on comparison study with parent drug azni izwati hamdan*, dike dandari sukmana*and norsyafikah asyilla nordin*,1 *faculty of pharmacy, university of sultan zainal abidin, besut campus, 22200, besut, terengganu, malaysia abstract in recent decades, drug modification is no longer unusual in the pharmaceutical world as living things are evolving in response to environmental changes. aspirin as one of the non-steroidal anti-inflammatory drugs (nsaid) is a common over-the-counter drug due to its analgesic, antipyretic and anti-inflammatory activity. this review article highlights on the recent derivatives of aspirin, which were developed either to reduce the risk of side effects or to obtain better physicochemical properties. aspirin derivatives can be synthesized in various pathways and have been reported to give better biological activities compared to the parent drug. nitric oxide (no)-aspirin gives a potent anticancer drug as it able to inhibit lung and prostate cancer cells. meanwhile noshaspirin that release hydrogen sulphide (h2s) and no moiety is a potent anti-inflammatory agent that stimulate the gastric and colonic secretion, prevent the penetration of acid in gastrointestinal. it also has anticancer action that is effective in hindering the proliferation of pancreatic and colon cancer cells. aspirin-thiourea has been studied its antimicrobial activity. still, it resulted in poor inhibition due to steric hindrance of the compounds and influence its penetration into the enzyme’s active site. however, aspirin-amide has managed to inhibit the bacterial and fungal, and compound with halogen substituents is reported with the highest inhibition. aspirin derivative linked with chalcone has poor antibacterial and antioxidant due bulky structure of the compounds, but it has a superior anticancer that induce cancer cells apoptosis by reactive oxygen species (ros) treatment. the modification of azo-aspirin has more potential in antibacterial activity compared to ampicillin especially when the presence of halogens substituents is involved. overall, these aspirin derivatives are safe to be considered as potential pharmaceutical agents. keywords: aspirin, aspirin derivatives, biological activities, chemical modification, nsaids introduction drug modification is vital in drug discovery and development process as it is usually done by altering the molecular structure of the formerly characterized lead compound to improve drug potential for treatment of diseases. some of the chemical alterations are done either by the specification of a particular body target site, modification of time course in the body, or by increasing the rate and degree of absorption. it is also able to improve the potency of drugs, provide the desired feature by decreasing the toxicity or changing the physical as well as chemical properties. aspirin is one of the most common overthe-counter drugs, which has been widely known as a fever reducer and anti-inflammatory drug for years. at low dose (75-100 mg), aspirin is selective to inhibit cox-1 activity. as a result of that interaction, aspirin can promote antithrombotic purpose and suppressing platelet aggregation without damaging vascular endothelial cell function which express cyclooxygenase enzyme(1). thus, it can be used prophylactically in patient with heart attack, high cardiovascular risk, and stroke by taking it in daily low dosage(2–4). however, prolonged use of the aspirin may result in vomiting, major gastrointestinal (gi) bleeding (5), and other side effects such as hypertension, renal or gi toxicity due to dosage-related(3). therefore, aspirin derivatives are being explored in order to get better biological activity. the presence of significant moieties such as nitric oxide (no), nosh, thiourea, azo, amide, and chalcone on the modified aspirin plays an important role in achieving desired biological activities. the addition of the halogen in the modification has also become a preference among researchers as it also affects the actions due to its ability to hinder bacterial activity (6,7).. aspirin aspirin or acetylsalicylic acid (fig. 1) is an nsaids approved by the fda to be used as antipyretic, antiplatelet, and analgesic agents(8,9). aspirin can inhibit the synthesis of prostaglandin by blocking the cyclooxygenase (cox) which contributes to its properties such as antiinflammatory, antipyretic, antiplatelet, etc. aspirin is also being considered as a chemopreventive agent because of its antithrombotic effects through the cox’s inhibition, (10,11) and its antioxidant action that inhibit the cancer cells growth by donating their electron to the free radicals that cause proliferation, induce apoptosis or necrosis to the cells(12,13). however, the prolonged use of aspirin can cause heartburn, ulceration, and gastro-toxicity in children and adults. 1corresponding author e-mail: azni64@gmail.com received: 17/10 /2021 accepted:23 /1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp14-32 iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 15 it contains an aromatic ring with carboxyl functional groups. carboxyl group plays many important roles in pharmaceuticals like acting as solubilizer or cell permeation for antibiotic or antihistaminic drug class, prodrug and/or bioprecursor that activated at specific conditions to act as an antihypertensive, antithrombotic or antiviral(14). aspirin is prepared by reacting acetic anhydride and salicylic acid in the presence of acid catalyst (h2so4/h3po4) (scheme 1). the hydroxyl group of salicylic acid is converted to an ester, with acetic acid as a byproduct(15). figure1. structure of aspirin scheme 1. synthesis of aspirin in the body, aspirin mainly absorbed in the stomach and upper part of small intestine after oral administration. it reacts with water in the plasma, liver and within the cells, by esterases to give salicylic acid and acetic acid (scheme 2) (16,17). the plasma half-life of salicylic acid is 15-20 min. in the liver, most of salicylic acid is metabolized into salicyluric acid, salicylic acid and phenolic glucuronides, and a small part of it is metabolized into genistic acid. these metabolites are mainly discharge by the kidneys to the urine(18). scheme 2. hydrolysis of aspirin however, aspirin lead to gi side effects by reducing mucosal prostaglandin synthesis that affects leukocyte adherence and decrease in bicarbonate, mucus secretion, and blood flow(19). mucus is mainly secreted from the surface epithelial cell and foveolar cells. the mucus bicarbonate is being used to regulate the ph gradient in the gi tract(20). it protects the stomach from a highly acidic environment. aspirin will inhibit the synthesis of the prostaglandins and decrease gastric mucus secretion. thus, mucosal blood flow not maintained effectively, stomach epithelium can be damaged as mucus layer is disrupted(21). figure. 2. mechanism of action of aspirin iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 16 aspirin is an irreversible-cox inhibitor that causes the inhibition of prostaglandin synthesis(22). aspirin can inhibit both cox-1 and cox-2 (figure. 2). as it is being administered, the aspirin transfers its acetyl group to a serine residue in the cyclooxygenase (cox) active site, making it unable for arachidonic acid to becoming prostaglandin h2, resulting in cyclic prostanoid (beckoning the molecules to mediate inflammation and other immune response) not to be synthesized(23). the pharmacological activity of aspirin is proven to be antiplatelet by inhibiting thromboxane a2 and anti-inflammatory by preventing prostaglandin i2, e2, d2, and f2a. turning off the cox-1 enzyme can upset the stomach and cause ulcers or gi bleeding. aspirin bearing nitric oxide moiety (no) no-aspirin (fig. 3) is one of gaseous mediator prodrug that is synthesized to improve the efficacy of parent aspirin, and to decrease the side effect that is associated with gi bleeding or ulcer(10). no is needed to regulate the physiological pathways, particularly regarding the homeostasis of the gi tract. it is usually formed in esophageal, gastric, and intestinal mucosa via the enzymatic activity of no synthases; neuronal (nnos), endothelial (enos), and inducible (inos) (24). figure 3. nitric oxide aspirin (no-aspirin) in various clinical conditions, no-aspirin is a potential therapeutic agent and typically synthesized by esterification of a no-releasing moiety to the nsaids (25). in addition, it has related parts in cancer biology, such as anti-inflammatory and anti-tumor properties, mainly exerted by noactivated apoptotic pathways(26). the summary of the synthesis of noaspirin can be seen in scheme 3. the halide from salicylic acid derivative react with hydroxybenzylalcohol in the presence of base, giving 2-(hydroxymethyl) phenyl 2acetoxybenzoate continue reaction with nitric acid in organic acid. it is recrystallized using selected solvent to form final product, no-aspirin(27). scheme 3. synthesis of no-aspirin no-aspirin hybrids as a promising anticancer activity one of the research reviews has indicated that the biological analysis by using no-aspirin derivative (figure. 3) was associated with reduced gi risk and could be consider as a potentially an anticancer agent (28). no-aspirin exhibited lower ic50 value (1 µm) in comparison to aspirin alone (>1000 µm). it was reported that proliferating cell nuclear antigen expression was reduced to 54.5%; meanwhile, at g0/g1 phases, over 83.9% of tumor cells were blocked after being treated with no-aspirin (29). no has dual role in anticancer activity depending on the type of cancer, the tumor microenvironment, the type of no synthase and the concentration of no itself. a low-rate no donor will end up with tumorigenesis, whereas a high-rate no can cause death to cancer cells (29). as an anticancer agent, no has ability to sequester iron into iron-nitrosyl complexes, resulting in a loss of intracellular iron and the inhibition of mitochondrial respiration and dna synthesis in the tumor cells. meanwhile, aspirin is also known for its ability to bring cell cycle arrest, apoptosis, and lead to cell proliferation suppression(30,31). it can be concluded that the hybrid of aspirin and nitrate ester-based on no donor is significantly potent anti-proliferate and apoptosis induction against the colon tumor cells compared to the aspirin itself(28,32). noaspirin as a potential anti-lung cancer no-aspirin has been studied as a highly potent in preventing lung cancer within high-risk populaces(10). when no-aspirin (fig. 3) was administered, the antiproliferative and apoptotic effect of erlotinib (an epidermal growth factor receptor (egfr) tyrosine kinase inhibitor) considerably increased. the study also indicated that no-aspirin managed to inhibit inflammationinduced lung tumorigenesis in mice (10). the drug was used to inhibit the proliferation of tumorigenic bronchial cell line (1170), non-small cell lung cancer (nsclc), and colony formation by nsclc cells. effect of noaspirin on the 1170 and nsclc cells was deduced by mtt (3-(4, 5-dimethylthiazolyl-2)-2, 5diphenyltetrazolium bromide) assay, annexin v/propidium iodide apoptosis assay, colony formation assay, and tumor bioassay using mice (10). iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 17 cell viability assay was used to determine the antiproliferative outcome of no-aspirin against 1170 and nsclc cells. the result showed that the proliferation of cells had been reduced in dosedependent by 30%, 56%, and 71% after being compared to treatment-free cells. furthermore, the apoptosis effect of cells increased when exposed to no-aspirin in dose-dependent manor as 8%, 18%, and 24% using flow cytometry-based analysis of annexin v and pi stained cells (10). aspirin inhibited cox and platelets activation, which caused the anticancer effect. activated platelets were not only able to activate the expression of cox-2 in epithelial cells but also capable of repressing t-cell immunity on cancer (33). it has been suggested another potential mechanism of anticancer since no-aspirin is unable to balance the cox-2 level in mouse lung tumor tissue. though, it was clear that phosphorylation of egfr and the downstream effectors akt, erk, and stat3 in 1170 and nslc cells had been restricted by the presence of no-aspirin (10). there is also a study that found no moiety caused cell growth, apoptosis, and cancer invasion mostly over phosphorylation transition proteins, pi3k/akt pathway, and downstream proteins (34). no-aspirin as a potential anticancer agent for metastatic prostate cancer one of the most prevalent malignant tumors identified in men is prostate cancer (35,36). most cancer-related death is caused by metastatic castrate-resistant prostate cancer (crpc) (37). based on the phase of prostate cancer, either surgery, androgen deprivation or chemotherapy can be alternatives for the treatment (38). the influence of no-aspirin inducing apoptosis in metastatic castration-resistant prostate cancer (crpc) (pc3) cell via hydrogen peroxide (h2o2)-mediated oxidative stress has been reported (38). the reactive oxygen species (ros) or oxygen radical is comprised of both radical and nonradical depend on its reactivity (39). radicals are the species which contain at least one unpaired electron in the shells around the atomic nucleus and are capable of independent existence, such as superoxide radical (o2•–), hydroxyl (oh), nitrogen monoxide (no), nitrogen dioxide (no2–), and etc. while non radical species are not free radicals but can easily lead to free radical reactions in living organisms, for examples hydrogen peroxide (h2o2), hypochlorous acid (hocl), ozone (o3), and etc. (38,40). in order to regulate normal physiological functions that are required in development, ros is crucial. however, excessive levels of ros harms proteins and membranes, which results in apoptosis or cell death (41). compared to normal cells, cancer cells are more high-level in ros, thus causing oxidative stress. oxidative stress is an inconsistency between the output of ros in the body that interrupts its ability to purify reactive immediate or restore the damage to the organ and cellular systems initiated by ros (42). the free radical-induced oxidative stress can damage cellular, tissue, and organ systems, leading to several diseased conditions such as cardiovascular, asthma, and various cancers (colorectal, lung, prostate) (43,44). the majority of chemotherapeutic drugs display anticancer mechanisms by bringing free radicals into cancer cells (38,40). for example, nitric oxide as a free radical conjugated with aspirin in order to have anticancer properties to reduce the chance of proliferation of prostate cell cancer. the pc3 cells viability had been tested for antiproliferative effect by using mtt (3-(4, 5dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay, by treating it with numerous concentrations of no-aspirin and parent compound aspirin. untreated cells were used as control and incubated according to condition, read under spectrometer, and percent cell viability was recorded (38). anticancer activity was investigated using three methods; colony formation assay, annexin vfitc/propidium iodide assay, and cell cycle analysis by flow cytometry (38). mtt assay showed that no-aspirin almost completely inhibited pc3 cells at 100µm, compared to aspirin at 100mm. as anticipated, no-aspirin is more practical in hindering pc3 cell viability compared to aspirin as an anticancer agent (38). the phosphatidylserine in the annexin fitc/pi staining gives eat-me signals, making the identified and phagocytosis of dying cancer cells. thus, apoptotic cells can be elucidated (45). histogram of cell cycle analysis indicated that noaspirin induced g0 phase arrest at almost 90% concentration compared to untreated cells. the presence of high concentration of h2o2 also leads to cancer cell apoptosis. no-aspirin has induced oxidative stress via no group, which turned into h2o2, resulting in cell cancer pc3 apoptosis (38). this concluded that no-aspirin has an anticancer effect on colon, lung, and prostate cell cancer. nosh–aspirin as anti-inflammatory and anticancer agent nosh-aspirin is developed as a substitute aspirin with broader application ranges to decrease the risk of hemorrhage stroke in aspirin users (46). it is a novel hybrid between hydrogen sulphide (h2s) and no moiety covalently bonded at 1, 2 positions of aspirin (ortho-nosh-aspirin) (47,48) which is also known as nbs-1120 (fig. 4) (19). the synthesis summary of the ortho nosh-aspirin can be seen as scheme 4 below(48–50). iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 18 fig. 4 structure of nosh-aspirin releasing h2s and no aspirin and nosh-aspirin had been evaluated the effect on rats’ stomachs when the drugs were being administered orally. after being treated with aspirin, the rats’ stomach showed ulceration and bleeding while nosh-aspirin-treated was free from ulceration (47). while aspirin regulates prostaglandin, the no and h2s donors have the same properties as prostaglandins that protect gastric mucosa (51,52). the gastric mucosa defense mechanism requires mucus to block the penetration of acid and pepsin by creating a viscous gel layer that assists a ph gradient in the epithelial surface of the stomach, thus blocking the penetration of acid and pepsin. no, and h2s donors improve barrier function by stimulating the gastric and colonic secretion, which leads to reduce gi toxicity (51,53). both donors also play a protective role in reducing oxidative stress, which is good in preventing cancer (54). nosh-aspirin is a potent anti-inflammatory agent compared to aspirin parent by using carrageenaninduced inflammation on rat paw. inflammation is usually linked with cancer (55). as anti-inflammatory agents are capable to hinder with tumor development, they are significant in the prevention and treatment of cancer (56). accordingly, there was a study mentioned that nosh-aspirin showed 5 times more potency in targeting mouse model of colon cancer, which it lessens the cell proliferation and cell cycle arrest leading it to apoptosis (47). the latest study on nosh-aspirin also stated that it was highly potent in inhibition of tumor growth in pancreatic cells. this was due to the ability of the drug to arrest cells in the g0/g1 phase transition and caused apoptosis in vitro (57). scheme 4. the synthesis of the ortho nosh-aspirin (nbs-1120) aspirin-thiourea bearing alkylated amine derivatives as antimicrobial agents fig. 5. thiourea structure thiourea (fig. 5) is an organosulfur compound with the formula of s=c(nh2)2. this compound and its derivatives, in particular, have showed various pharmacological activities such as anti-fungal (58,59), antiviral (58,60), anticancer (61), antituberculosis (62), antimicrobial (63) and antiinflammatory (64–66). thiourea has gained significant values since being studied for their application in commercial and industrial applications; plastics, textiles dyes, elastomer, herbicides, pesticides, rodenticides and catalytic (65,67,68). fig. 6. structure of aspirin-thiourea bearing alkylated amine iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 19 a study showed that compounds with two or more thiourea moiety hold better antimicrobial activity (67). moreover, it gained much attention from researchers as it contains carbon, nitrogen, hydrogen, and sulfur elements (69). at acidic conditions, c=s and n-h functional groups can be protonated, which gives the thiourea ideal potential site for electrostatic binding on the bacterial surface, which consist of carboxyl and phosphate group (anionic), thus complementing its biological activities (67,70,71). aspirin-thiourea (fig. 6) with alkylated amine derivative as potential antimicrobial agents has been reported. the synthesis of aspirin-thiourea by reacting acetoxybenzoyl isothiocyanate with series of methyl, methoxy anilines, and alkylated anilines had been prepared through williamson esterification (scheme 5) scheme 5. synthesis of aspirin-thiourea derivatives the modification of alkylated amine on aspirin-thiourea gave various results on antibacterial activity against e. coli and s. aureus. the presence of c=o, c=s, and nh group, had increased the activities of antibacterial activities through the interaction on bacterial surface that contained carboxyl and phosphate group (72). the synthesized compounds 1-12 were studied on their antibacterial activity. however, it was found that compounds 4, 6, 10, 11, and 12 did not give any inhibition towards e. coli and s. aureus. iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 20 table 1. results on the alkylated amine on aspirin-thiourea based on their substituents. r’ compounds result (4) the presence of methyl (4) and methoxy (6) groups in the structure have reduced the biological activity due to steric hindrance (75).. (6) (10) as the alkyl chain increased from the compound (10), (11) and (12), a parabolic effect had been displayed (71) longer the alkyl chain (>10), gave higher chance to hinder the cell membrane penetration, which prevents inhibition on bacterial growth(63). (11) (12) by comparing the result of the biological testing of the prepared derivatives on e. coli and s. aureus, it was found that e. coli was easier to be inhibited (63). this is due to the characteristic of s. aureus that is hard to penetrate because of its thick peptidoglycan layer that increases cell wall rigidity (73). nphenylcinnamideaspirin for antimicrobial and antifungal activity amide functional group contains (r-nc=o) has been chemists’ choice since it has a lot of potentials. there are amide derivatives reported to be potent anticancer (74), anti-inflammatory (75), antioxidant (76), antibacterial, antifungal, antimalarial (77,78) and etc. as aspirin also has a lot potential, the modification of aspirin with amide is being studied, especially the antibacterial and antifungal activity. the synthesis of n-phenylcinnamamide derivatives that linked with aspirin (scheme 6), started with dissolving the aryl aldehyde, giving substituted acetanilide chalcones compounds. the compounds were linked with aspirin by using mixed anhydride method, producing nphenylcinnamamide-aspirin and continued with antimicrobial screening and antifungal assay (79). the final product of aspirin-n-phenylcinnamamide had three phenyl rings but was able to inhibit s. aureus and e. coli. antimicrobial screening found that the compound 2c gave the highest inhibition against e. coli, 19 mm but 16 mm for the s. aureus, meanwhile 2a gave the highest inhibition of s. aureus, 18 mm, but against e. coli, 16 mm. as for the antifungal assay, 2c gave the largest inhibition of c. albicans, 18 mm, while 2a only gave 10 mm (79). iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 21 scheme 6. synthesis of n-phenylcinnamamide-aspirin of 2a and 2c table (2) results on the antibacterial and antifungal of n-phenylcinnamamide-aspirin (2a & 2c). compounds antibacterial anti-fungal e. coli s. aureus c. albicans 2a 16 mm 18 mm 10 mm 2c 19 mm 16 mm 18 mm although the aspirin with substituted amide (2a) has good antibacterial activity, the presence of –cl substituent in the compound (2c) is slightly higher. it was determined that its high lipophilicity which penetrated the bacterial cell wall has contributed to the higher success rate of inhibition (79). this may caused by the lipophilic characteristic of n-phenylcinnamide due to interaction of its active site to the bacterial cell membrane and gain access to its target and restrained the bacteria (80,81). a study reported that halogenation also affect c. albicans virulence activity due to their steric effect that provides best fitting of small molecule to conquer the target’s binding site. the – cl substituent also found to be the most stable halogen that tolerates a steady docking on c. albicans. as the result on these studies, -cl substituent on the compound was found to give most stable derivatives (82). aspirin-chalcone derivatives fig. 7. chalcone structure chalcone (fig. 7) comprises of two aromatic rings that are highly interconnected by three-carbon α,β-unsaturated ketones that contribute to the pharmacological activity (83,84). in medicinal chemistry, chalcone is a simple scaffold that originates in countless naturally occurring compounds and is being used widely as an efficacious model for drug discovery (85). it is stated that chalcones have many benefits, for instance, low interaction with dna and low-risk of mutagenicity (86). chalcone is known as the precursor for the synthesis of flavonoids, which is practical as antiplatelet(83), anticancer(84), anti-inflammatory, antioxidant, anti-diabetic, and antimicrobial (87–90). researches on chalcone derivatives reported that chalcones have high antioxidant activity (91,92) .since antioxidants have the ability to donate electron, it can neutralize the free radical and prevent any damage to biological compound in the body (93). as chalcones are known as minor flavonoids (94), they can scavenge free radicals (92). excess of free radicals and ros (reactive oxygen species) in human body may cause diseases like cancer, cardio, and cerebrovascular due to damage to lipids, proteins, and nucleic acids (95). that is the reason why the proper physiological function depend on the balance between free radicals and antioxidants (96). the accumulation of ros in the body can be influenced by several factors including endogenous factors such as by-products of mitochondrial activity, exogenous factors including ultraviolet radiation, and even the lack of antioxidant agents in the body such as glutathione, vitamins a, c, and e (39). the accumulation of oxidative damage can be prevented by avoiding the excessive ros formation through optimal functioning of oxygen metabolism and avoidance of environmental pollutants, as well as increasing the neutralization of ros by having appropriate antioxidant intake (96). therefore, a study regarding aspirin-chalcone with antibacterial effect was done. aspirin-chalcone with antibacterial and antioxidant activities the synthesis of aspirin-chalcone was done by the reaction of aspirin and chalcone derivatives by esterification, giving aspirin-chalcone (scheme 7) (97). iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 22 scheme 7. synthesis of aspirin chalcone a-g the antibacterial evaluation of the aspirinchalcone derivatives was analyzed against e.coli and s. aureus. however, the result indicated that most derivatives gave no inhibition against e. coli and s. aureus when compared to ampicillin (97). the similar result had been also conducted by ngaini et al. on the previous research., in which aspirin chalcone derivatives gave no inhibition against e. coli for antibacterial assay (98). there is a high possibility it occurs due to e. coli is easier to accumulate resistance genes, making it more resistance toward older antibiotics like phenicols, sulfonamides, and trimethoprim (99). the asymmetric lipopolysaccharide (lps)-phospholipid bilayer of the outer membrane of e. coli causes a weaker permeable barrier for both hydrophobic and hydrophilic compounds (100). nevertheless, it was discovered that the aspirin-chalcone displayed poor antioxidant properties on 2,2-diphenyl-1-picrylhydrazyl (dpph) assay in comparison to ascorbic acid was due to low phenolic pharmacophore and steric hindrance which also cause bulky structures (97). the phenolic group was necessary for getting high antioxidant activity (101). the presence of phenyl ring in the compound may also contribute to the bulkiness and cause steric hindrance, making it harder for penetration into phospholipid bilayer of s. aureus and e. coli (102,103). aspirin-chalcone with anticancer activity the studies of the aspirin-based drug for anticancer effect have been discovered and encouraged since a while back (104), including breast cancer that is highly prevalent among women worldwide (105). anticancer drugs usually kill these cancer cells by inducing ros generation since the high level of ros causes cell damage as well as apoptosis, autophagic and necrotic cell death (106,107). cancer cells have abnormal metabolism; thus, they have higher ros compared to normal cells, making them more susceptible to ros-induction treatment (107). chalcone derivatives have received significant attention as they exhibited potent anticancer activity against some cancer cell lines, such as naphthalene-chalcone derivatives that displayed potent antiproliferative activity against breast cancer cells (mcf-7) (108). chalcone inhibited proliferation in mcf-7 by inducing apoptosis and hindering cell cycle development (109) by increasing ros (110). aspirin also prevents breast tumor cell growth through induction apoptosis (111). thus aspirin-conjugated chalcone polymeric micelles for anti-breast cancer activity is an interest (112). polymeric micelle unsurprisingly is a decent delivery system for anticancer drugs with lower water solubility. at the size of 10-100 nm, it is able to elongate circulation time of the drugs in the blood (112). aspirin also compromised the condition of chalcone which is a hydrophobic polyphenol with poor aqueous solubility (110). it has been studied that aspirin-conjugated chalcone derivative-loaded nanoparticles (asdk143-loaded np) as potential chemotherapy agents with anticancer efficacy. synthesis of asdk143, which is also known as (e)-2-(2,3dimethoxyphenyl)acrylol)-4-methoxyphenyl-2acetoxybenzoate, started with preparation on one of the chalcone derivatives from the previously reported study by the same author, which is known as (e)-3-(2,3-dimethoxyphenyl)-1-(2-hydroxy-5methaoxyphenyl)prop-2-en-1 or dk143 (113). the method of the synthesis can be seen in scheme 8. then the process continued with the preparation of as-dk143 polymeric micelles by thin-film hydration method. the as-dk143 undergoes cell viability and animal studies for testing the anticancer effect towards nude mice (110). iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 23 scheme 8. synthesis of as-dk143-loaded-np as-dk143 was synthesized and characterized using 1h nmr and ir spectroscopy, and the nanoparticles were tested in 4t1 cell viability. the chalcone-based compound can be used as a potent anticancer agent as it induces cancer cell apoptosis by increasing ros production (106,110). however, due to its non-polar properties, they increased the bioavailability by interlinking –oh in dk143 with the polar group of aspirin, –cooh in the form of nanoparticles (110). as-dk143 showed significant reduction of 4t1 cell viability to 25.97%, ±5.69% and 11.02% ± 0.01%. it was also found that the ic50 of aspirin and as-d143 gave 4955µm and 39.61µm. thus proving that the modification of aspirin-chalcone derivative (as-dk143) against 4t1 cell gave greater anticancer effect at the lowest concentration compared to chalcone derivative (dk143) itself. azo-aspirin with antibacterial properties in order to increase the antibacterial activity, it is recommended to introduce azo moiety into the structure as –n=nis important in bactericidal activities (114) the study for antibacterial properties of aspirin derivatives continued on pursuing on azo-aspirin as it gives good biological activity. the synthesis started with a phenol and aniline derivatives and produced azo derivatives. the product reacted with aspirin, becoming azo-aspirin. further explanation on synthesis can be seen in scheme 9 (102). scheme 9. synthesis of azo and azo-aspirin derivatives iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 24 e. coli and s. aureus had been chosen for the antibacterial activity of azo-aspirin. presumably, halogen being chosen as substituent, it is due to its high reactivity, which can be deadly to bacteria in a sufficient amount. the ortho-fluorine aspirin-azo derivatives (compound 2d) gave better antibacterial activity against e. coli (156.3 ppm), meanwhile metafluorine aspirin-azo (compound 2b) gave better results against s. aureus (194.1 ppm) in comparison to parent aspirin (>220 ppm). the result indicated that –f substituted compounds showed superior antibacterial activity compared to –cl substituted compounds (102). this is because of larger atomic radius of –cl atom, which creates a larger steric hindrance than –f. in other hand, although its electronegativity is less than –f atom (pauling electronegativity of 4.0), –cl (pauling electronegativity of 3.2) can form very strong noncovalent interactions. however, these compounds are not superior antibacterial agents compared to the ampicillin (115). ampicillin disrupt the bacterial cell wall synthesis during active replication and kill the bacterial, making it one of the chosen antibacterial agents used in medicine (116,117). few years later, another journal published by the same author reported on halogenated azo aspirin with additional procedures, diazotization followed by coupling reaction (scheme 10). the presence of halogens, –br and –i played a significant role in raising the antibacterial activities of the derived compounds compared with the aspirin and ampicillin (114). scheme 10. the synthesis of azo-aspirin by diazotization and coupling reaction the –i substituent at ortho position gave the highest inhibition with mic value, 74 ppm against e. coli and 64 ppm against s. aureus. surprisingly, it showed a far superior result as antibacterial agents compared to the ampicillin. nevertheless, –br at ortho position also gave high mic value, 89 ppm for both e. coli and s. aureus (114). comparing the result from previous journals, the presence of the halogens affects the inhibition of bacteria in the rate of -cl < -f < -br < -i (102,114). although the –i gave the highest inhibition, it also needs to be considered whether it will affect the other cells or not. the released of –i substance need to be regulated as it can iodinate the lipids that are main component of the cell membrane, and will oxidize various cellular components. therefore, it can be dangerous towards human skins, or cell as well if the released of the substance is not controlled. however, it does not change the fact that halogens can be strong oxidizing substances that damage the cell wall or membrane of microorganisms which contribute to the bacteriostatic effect (118). the presence of the halogen also improved the lipophilic tendency of azo-aspirin to penetrate the microorganisms’ cell walls. the increasing levels of lipophilicity can enhance the ability of compounds to penetrate the cell membranes of gram-negative bacteria which are hydrophobic (119). that is one the reasons on why halogens are being considered to improve antibacterial effect of drugs modification. meanwhile, the presence of –n=n(azo) moieties that can be protonated under acidic condition and reacted with phosphate group on the peptidoglycan layer; can hinder the cell wall formation. then hydrogen bonding will form between the azo-aspirin compound and the active site of the enzyme, causing the bacteriostatic effect (113). iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 25 table (3) summary of the aspirin derivatives and their related biological activities: modification authors methods aim results/finding no-aspirin ding et al. (28) anticancer • over 83.9 % of the tumor cells are blocked at g0/g1 phase song et al. (10) • mtt (3-(4, 5-dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide) assay, • annexin v/propidium iodide apoptosis assay, • colony formation assay, • tumor bioassay using mice anti-lung cancer • the proliferation of the tumor cells is reduced meanwhile the frequency rate of cells’ apoptosis is increased chinnapaka et al. (38) • mtt (3-(4, 5-dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide) assay, • colony formation assay, • annexin v-fitc/propidium iodide assay, • cell cycle analysis by flow cytometry anti-prostate cancer • 90% of the tumor cells are inhibited at g0 phase nosh-aspirin kashfi et al.(47) • carrageenan-induced inflammation on rat paw anti-inflammation & anti-colon cancer • it is 5 times more potent in targeting mouse model of colon cancer, then reduce the cell proliferation and cell cycle arrest leading it to apoptosis chattopadhyay et al.(57) • cell growth inhibition assay, • cell proliferation, • flow cytometry of phase distribution in the cell cycle • detection of apoptosis anti-inflammation & anti-pancreatic cancer • it reduces gastric mucosa and arrests cells in the g0/g1 phase transition which caused apoptosis in vitro. aspirin-thiourea nordin et al. (63) • synthesis using williamson esterification, • antibacterial screening against e. coli and s. aureus antimicrobial/ antibacterial • the presence of c=o, c=s and n-h give good inhibition, however och3 and ch3 contribute to steric hindrance, and long alkyl chain (>10) showed parabolic effect iraqi j pharm sci, vol.31(2) 2022 exploring aspirin derivatives 26 aspirin-amide alwash et al. (79) • synthesis of nphenylcinnamamide using claisen-schmidt condensation linked with aspirin, • in-vitro antibacterial and antifungal screening against e. coli, s. aureus and c. albicans antimicrobial & antifungal • -cl substituted compound gave the highest inhibition of antibacterial and antifungal. 19 mm, 16 mm and 18 mm of e. coli, s. aureus, and c. albicans • –cl substituent also found to be the most stable halogen that tolerates a steady docking on virulence-related target aspirin-chalcone nordin et al. (97) • synthesis of hydroxylated chalcone • synthesis of aspirin chalcone • antibacterial screening against e. coli and s. aureus • antioxidant evaluation using dpph antibacterial & antioxidant • poor inhibition against bacterial and fungal activity. • bulky structures and lack of phenolic pharmacophore contribute to poor antioxidant activity lee et al. (110) • thin-film hydration method • cell viability assay anti-breast cancer • the cell viability of as-dk143 against 4t1 cells reduced to 25.97%, ±5.69% and 11.02% ± 0.01%. • the ic50 of aspirin and as-d143 gave 4955µm and 39.61µm. azo-aspirin ngaini and ho (102) • synthesis of azo • synthesis of aspirin-azo derivatives • antibacterial screening against e. coli and s. aureus antibacterial • the ortho-fluorine gave better antibacterial activity against e. coli, meanwhile meta-fluorine aspirin-azo gave better results against s. aureus. • larger atomic radius of –cl atom creates larger steric hindrance than –f, making –f substituted compounds good antibacterial agent ngaini and mortadza (114) • synthesis of azo • synthesis of aspirin-azo derivatives diazotization followed by coupling reaction • antibacterial screening against e. coli and s. aureus antibacterial • the –i substituent gave the highest inhibition with mic value, 74 ppm against e. coli and 64 ppm against s. aureus. • –br also 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boonpangrak s, sinthupoom n. derivatives ( halogen , nitro and amino ) of 8-hydroxyquinoline with highly potent antimicrobial and antioxidant activities. biochem biophys reports . 2016;6:135–41. available from: http:// dx.doi. org/10.1016 /j.bbrep .2016.03.014 this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.19(1) 2010 bioequivalence of two formulations of amoxicillin 14 bioequivalence of two formulations of amoxicillin in human healthy volunteers on (hplc) technique alaa k. jabbar alhamd *, 1 * department of medicinal sciences, college of pharmacy, al-mustansirya university, baghdad , iraq abstract amoxicillin is commercially available in the form of capsules and tablets containing 250mg or 500mg for oral administration. it is also available in the form of suspension containing "25mg/ml‖ . amoxicillin is presently used as the most common antibiotics .ten healthy human volunteers were characterized respected to their pharmacokinetic and bioavailability of two formulations of amoxicillin from two sources of industrial companies after a single dose administration was given orally. a procedure is described for determination the concentration levels of amoxicillin in human plasma of healthy volunteers using high performance liquid chromatography (hplc) with reversed-phase isocratic column at low wave length of uv-visible detection "230nm". an efficient drug extraction procedure was used for the separation of amoxicillin after simple extraction with cold methanol using ods-c18-db column. the pharmacokinetic 500mg of amoxicillin capsule orally administrated treatment through 10 hours has been examined. the amoxicillin was eluted for "10.0 minutes" at flow rate "1.5ml/min." and temperature equal to 298 k .the retention time of amoxicillin was observed at 7.0 minutes. the mean absolute recovery of amoxicillin in blood plasma of all healthy volunteers were 94.1% at 1.0ppm, 102% at 5.0ppm, 103% at 10.0ppm 102% at 20ppm, 99.3% at 40ppm and 104% at 50ppm respectively. the assay showed excellent relationships between area under the curve ratios and drug concentration levels (p>0.002) .oral amoxicillin administration in ten healthy volunteers gave maximum concentration peak plasma at two hours and decline through ten hours. treatment with iraqi formulation amoxicillin produced higher area under the curve ―auc‖ and maximum concentration ―c (max)‖ of amoxicillin than indian formulation. key word: amoxicillin, bioequivalence, ods -db column. الخالصة يهٍ غشاو 322يهٍ غشاو وانثبَُت عهً 032َخىفش االيىكسُهٍُ حدبسَب عهً شكم كبسىالث و حبىة ححخىٌ االونً عهً اندشعت عٍ طشَق انفى وَعخبش االيىكسهٍُ انًضبد زيهٍ غشاو / يهٍ نخش حُث حىخ 03كًب وَخىفش عهً شكم يحهىل عبنق بخشكُض انحُىٌ االكثش أسخخذايب . دسسج حشكُت انذواء وانخىافش انحُىٌ عهً حىنُفخٍُ يٍ كبسىالث االيىكسهٍُ نششكخٍُ يخخهفخٍُ طبقج نخٍ حسبج فُهب يسخىَبث يهٍ غشاو اخزث عٍ طشَق انفى . حىضح انطشَقت ا 322عهً عششة يٍ انُبط االصحبء انًخطىعٍُ بدشعت حشاكُض االيىكسُهٍُ فٍ بالصيب انذو نعذد يٍ انُبط االصحبء وانًخطىعٍُ ببسخخذاو خهبص كشويىحىغشفُب انسبئم راث االداء انعبنٍ (hplc( عهً ًَبرج بالصيب انذو اسخخهص يُهب االيىكسُهٍُ ببنًُثبَىل انًبشد عهً عًىد يٍ َىع ) ods-c18-db). عُُج يسخىَبث حشاكُض االيىكسُهٍُ عهً خهبص كشويىحىغشافُب انسبئم راث االداء انعبنٍ بضيٍ قذسِ عشش دقبئق وبسشعت طىس , حسبج انُسبت انًئىَت دقبئق 7دسخت كهفٍ و وقج االحخدبص ناليىكسُهٍُ 092يهٍ نُخش / دقُقت وبًحُظ حشاسٌ 5.3يخحشك , 99.1, 520, 521, 520, 92.5بٍ بٍ أو (عهً انخىانٍ ) 32, 22, 02, 52, 3, 5كُض)نالسخشخبع فٍ بالصيب انذو نهخشا ( اشبسث انُخبئح ببٌ اعهً حشكُض فٍ بالصيب انذو حكىٌ خالل سبعخٍُ يٍ وقج اندشعت انًأخىرة p>0.002 % ( بًسخىي ثقت )522 ة يٍ وقج اندشعت كًب واٌ انُخبئح اشبسث ببٌ سهىك انخكبفىء انحُىٌ عٍ طشَق انفى ثى َبذأ انخشكُض ببنُضول حخً انسبعت انعبشش يٍ انخىنُفت انهُذَت . نهخىنُفخٍُ غُش يشببّ واالخخالف فٍ انًسبحت وانخشكُض االعهً نهخىنُفخٍُُ الٌ انخىنُفت انعشاقُت اعهً حىافش حُىٌ introduction amoxicillin capsules and amoxicillin suspensions were analyzed for their drug content as described in the united state pharmacopeia (1) . amoxicillin is the most commonly prescribed antibiotic for the treatment of phyaryngitis in the us. (2) pharmacokinetic and bioavailability may be vital to ensure successful protocol in the clinic as well as in researches. often the clinical evaluation of drugs is carried out on the basis of some secondary response because of the non existence of directly measurable parameter which is related to the treatment of disease by the drugs (3) .many times no response is measured at all, and the clinician attempts to make objective and subjective assessment of the patients general welfare. a dosage regimen for a new drug may in fact be based on such an evaluation and may not include a comparison with a standard drug or analog, a dosage regimen based upon such studies can be only a rough approximation at best. this point is well illustrated if one compares drug dosage regimens (e.g. sulfonamides) calculated 1corresponding author email : prof_alaaalhamd@yahoo.com received : 11 /3 /2009 accepted : 28/12/2009 iraqi j pharm sci, vol.19(1) 2010 bioequivalence of two formulations of amoxicillin 15 from pharmacokinetic data to those commonly used in the clinic. the same importance from this point of view does not concern only medicines but all other compounds including pharmaceuticals which are introduced to the organism for the diagnostic purposes. there is a through pharmacokinetic examination of a diagnostic agent which is extremely important from the point of view of decreasing of the possible risk (4) .pharmacokinetics are the studies of the movement of drugs in the body through the time course; it must not be hidden in mathematics itself. it is extremely important for everybody who is interested in pharmacotherapeutics in drug researches and in drugs productions to understand what the pharmacokinetics really means (5) . pharmacokinetic study of children that assessed the single-dose administration of an investigational oral amoxicillin sprinkle designed to sequentially deliver an immediaterelease and multiple delayed-release pulses of amoxicillin to provide prolonged plasma concentrations of amoxicillin (6) . a new pharmacokinetically enhanced formulation of amoxicillin has recently become available (7) . the new formulation can maintain the main amoxicillin serum concentration about 49% of the dosing interval in contrast with only 34% for three times daily regimen (7,8) . the pharmacodynamic and pharmacokinetic properties of the new formulation predict high rates of success against respiratory tract pathogens (8) . amoxicillin (amo) is oral semisynthetic penicillin structurally related to ampicillin as shown below: the present of benzyl ring in the side chain extends the antibacterial activity to gramnegative bacteria (9) . amoxicillin presents as a highly absorption after oral administration and is not altered by the concomitant ingestion with food (10) . amoxicillin exhibits low binding with plasma proteins and is quickly distributed through the body. it has an elimination half life of one hour (11) . amoxicillin pharmacokinetics was obtained across pregnancy states. oral clearance and renal clearance were higher while the half life was shorter during pregnancy, these changes suggest that the amoxicillin exposure will be less while pregnancy that maintenance of trough concentrations will be difficult (12) . anew per oral amoxicillin \ clavulanate therapeutic system was developed and evaluated by in vivo bioavailability study (13,14,15) . amoxicillin was effective in reducing oral micro organism level up to 12 hour post dose (16) . the treatment with amoxicillin for 3 to 7 days had similar clinical efficiency and also similar selection of oral streptococci with reduced susceptibility to amoxicillin (17) . amoxicillin is commercially available in the form of capsules and tablets (250 or 500 mg) for oral administration and also available in the form of suspensions containing "25 or 50 mg / ml". amoxicillin is one as the most commonly used as antibiotic. to understand the bioavailability and pharmacokinetic behavior of this drug in human which is needed reliable qualitative and quantitative methods. there are several high performance liquid chromatography (hplc) methods were used for separation and determination of amoxicillin in body fluids .some of these methods were developed to use direct uvvisible detection at low wave length (225229nm) (18—22) . the bioavailability of two brands of melixican (7.5mg and 15mg) tablets and to obtained pharmacokinetic parameters of this molecules on mexican population using modified and validated high performance liquid chromatography ―hplc‖ technique pharmacokinetic parameters auc , c(max) , and t(max) were determined from plasma concentration levels of both formulations that the results indicated in a c(max) 120% larger and t(max) 65% faster than those reported (23,24) . others were used fluorimetric detection (25—28) . some others special techniques have been used to enhance the sensitivity and selectivity such as ion-pairing reagents and post column derivatization (21, 28, 29) . there are several different methods used to preparation the sample that have been applied prior to chromatographic analysis mostly based on iraqi j pharm sci, vol.19(1) 2010 bioequivalence of two formulations of amoxicillin 16 liquidliquid extraction (28) , deproteinization by precipitation (19,25) and solid phase extraction (18,30,31) . plasmatic amoxicillin concentration levels were determined by combined reversed-phase liquid chromatography and mass spectrometry with positive ion electro spray ionization using the selective ion monitoring technique (32, 33) . the paper presents the favored approach of clinical studies involved in qualitative and quantitative assay of pharmacokinetics of two amoxicillin formulations firstly, iraqi formulation of capsule containing 500mg amoxicillin compared with indian formulation capsule containing 500mg amoxicillin. after evaluation of the various condition of the (hplc) assay, a suitable and simple assay for the determination of amoxicillin in human plasma of healthy volunteers was developed using reversed-phase isocratic (hplc) at direct low wave length of uv-visible detection (230nm) and temperature equal to 298 k for subsequent study of pharmacokinetics and bioavailability. experimental materials and methods chemicals and drugs all chemicals used in this study were the highest analytical grade purchased from commercial sources and used without any further purification. the deionized distilledwater was used for all preparation. methanol (absolute meoh) and acetonitrile (absolute acn) ―hplc grade‖ were purchased from (fluka). amoxicillin capsules from two sources one from iraq (s d i, iraq ) and the other from india(micro labs limited ,bangalore ,india) potassium dihydrogen phosphate (kh2po4), dipotassium hydrogen phosphate (k2hpo4), phosphoric acid and 1octane sulphonic acid sodium salt were purchased from (bdh, england). sepelcoods-c18-db column (250 x 4.6mm i.d.) was purchased from (sepelco, united kingdom). standard solutions amoxicillin (1.0 mg) was dissolved in 100ml of freshly prepared mixture of water: methanol (95:5) ―10000 ppm‖. the standard solution was filtered, degassed and stored at 253 k for further use. the standards were prepared freshly every month. stock solution of amoxicillin (1mg/ml) was prepared in a mixture of (water: methanol) (95: 5). the applied standard solutions were prepared from stock solution by sequential dilution with the same mixture to produce final concentrations (1, 5, 10, 20, 40, 50ppm). the stock and applied solutions were protected from light and stored at 253 k. calibration standard curve were performed to achieve the concentration of (0.1, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 and 12.0ppm) figure-1. figure 1: linearity of different concentration levels of amoxicillin using hplc technique on ods-db column extraction of amoxicillin blood sample (3--5ml) were drawn from vein by syrings in to hyparinized blood tubes, then transferred immediately into polyproplene tubes and centrifuged within 5 min. at 500g for 15 min. one milliliter of sodium metabisulphate, (ph equal to 8.0) was added for each one milliliter of plasma. amoxicillin was extracted from human plasma samples by deproteinization using precipitation process. a 500µl aliquot form each plasma sample was transferred to a 5.0ml polypropylene tube. one milliliter of cold methanol was added. after slightly vortex mixing, the tubes were centrifuged for 15min. at 500g. a 100µl aliquot of the supernatant was transferred to the injection vials and 10µl were injected into chromatographic system. all samples from volunteers were analyzed on the same day in order to avoid inter-assay variation. plasma solutions were protected from the light and stored in a deep freezer at (203 k). hplc instrumentation this study was performed on shimadzu instruments model lc-6a hplc system. the unit was operated in the isocratic model using solvent reservoirs fitted with 0.22 µm stainless steel filter at the end of polytriflouroethylene (ptfe) tubes, transferring the mobile phase from reservoirs to the pump, the system also involved an injector with 50µl sample loop iraqi j pharm sci, vol.19(1) 2010 bioequivalence of two formulations of amoxicillin 17 model ( reseadyre 7125 ) , column in type of ods-c18-db "250 x 4.6mm i.d.", thermostatic oven model cto-6a shimadzu, uv-visible detector model "spd" and chromatopac unit model r4-6a shimadzu. hplc operation condition for routine hplc analysis of amoxicillin use the following estimation condition. the mobile-phase was phosphate buffer conc. 10mm that containing 0.1mm 1octane sulphonic acid sodium salt: methanol (95:5) (v/v), ph buffer equal to six, column temperature 298 k, flow rate equal to (1.5ml/min.) and uv-visible detection at 230nm. the typical chromatograms of standard solution and blood plasma samples of amoxicillin are shown in fig-2 and fig-3 respectively. figure 2: typical chromatogram of hplc analysis of amoxicillin on ods-db column figure 3: typical chromatogram of hplc analysis of plasma amoxicillin on ods-db column pharmacokinetics and statistical analysis the observation of maximum plasma concentration levels (c(max)) and time consuming to reach it (t(max)) were obtained from drug concentration versus time curves. the area under the curve ‖auc‖ of the amoxicillin concentration levels versus time from 5.0 minute to ten hours were estimated from "figure-4". figure 4: pharmacokinetics of iraqi formulation and indian formulation amoxicillin in blood plasma of healthy volunteers results the isocratic reverse-phase hplc technique described and used here for estimation of drug provides the appropriated sensitivity, specificity and high sample accuracy for bioavailability and pharmacokinetic studies. fig.1 shows the retention time of amoxicillin standard solution that under described chromatographic condition. the retention time of amoxicillin was 7.0 minutes. the optimal chromatogram of analysis was given an ideal shape, symmetrical, and good resolution of peak. fig.-2 shows the typical chromatogram of amoxicillin in blood plasma samples of healthy volunteers which was appeared no endogenous interfering peaks at the retention time of interest compound. the mean absolute recovery of amoxicillin in blood plasma was 94.1% at 1.0ppm, 102% at 5.0ppm, 103% at 10.0ppm 102% at 20ppm, 99.3% at 40ppm and 104% at 50ppm respectively. the calibration curve was linear with regression coefficient r 2 =0.989 (table-1). the analytical precision and accuracy values was obtained from assays of six quality control (1, 5.0, 10.0, 20.0, 40.0, and 50.0 ppm) are shown in table-1 .the accuracy were 94.1% , 102% , 103% , retention time (minute) retention time (minute) iraqi j pharm sci, vol.19(1) 2010 bioequivalence of two formulations of amoxicillin 18 102%, 99.3% and 100% respectively and there is not significant degradation of amoxicillin was observed during the period of storage. table 1: the linearity, precision and accuracy of blood plasma amoxicillin samples. spiked concentration (ppm) 1.0 5.0 10.0 20.0 40.0 50.0 recovered average con. (ppm) 0.94 5.1 10.3 20.4 39.7 50.2 slope r 2 p.v 1134 0.989 0.001 discussion the hplc technique presented in this study decreases the lower limit of quantitation of amoxicillin to about 0.1ppm. it was appeared that is more sensitive than many other assays. the low limit of the estimation of the plasma concentration of amoxicillin was sufficient to perform the pharmacokinetics study of drug. amoxicillin plasma concentration levels were measured by several methods in combination with uv-visible detection. the lowest plasma concentration levels of amoxicillin was obtained by uvvisible detection which was 0.05ppm but the process was consumed long time that was 30min. (10,14) .charles et al were described a procedure for determination of amoxicillin in urine (8) . other complicated procedures for extraction and estimation of plasma concentration levels of amoxicillin by using solid-phase extraction has been also reported (7, 9, 18, &19) . nevertheless, solid-phase extraction (spe) procedures are laborious and require spe cartridges, increasing the cost of analysis. in order to improve the sensitivity, a column ion-pair hplc with post column derivatization has been used (16) . where the low limit of quatitation was 0.01ppm, this procedure was more complicated due to the more step of post column derivatization and their retention time that will be longer than 10min.which is compared with our procedure that the retention time of amoxicillin peak was 7min. and the full time of process not greater than 10min. . also these procedures cannot be used in pharmacokinetics studies in human where a large number of samples were analyzed.the pharmacokinetics study was done in "10 hours" and the results indicate that the iraqi formulation has higher bioavailability compared to the indian formulation depending on the area under the curve auc and c(max) .our technique was evaluated and produced the best results in terms of selectivity and sensitivity consideration the fact that the present technique involves a shorter running time and a simple sample preparation process. conclusion our hplc technique was employed here proved to be fast, simple, precise, selective and sensitive enough to be used in clinical pharmacokinetic and bioavailability study for amoxicillin in plasma human. the auc and c(max) of iraqi formulation are higher than indian formulation of amoxicillin and the t(max) of both two formulations are similar which is shown in table-2 and the relative bioavailability of indian to iraqi formulation was estimated equal to 64.11% . table 2 : the pharmacokinetic parameters “ cmax , tmax and auc” for the iraqi and indian formulations of amoxicillin . pharmacokinetic parameter indian formulation ”a” iraqi formulation “b” c(max) ―ppm‖ 8.9 11.5 t(max) ―hour‖ 2 2 auc 30.25 47.18 acknowledgment i wish to thank miss. kafa q. al-obydee for carrying out the experiments that associated with preparation of samples and kolood i. mohamad for their technical assistance. references 1. us pharmacopeial ,(2005), usp 28\nf 23 .the united states pharmacopeial convention, inc, rockville, md , usa. 2. ims american ltd, (2003) , national disease and therapeutic index data junuary -2003 , ims america ltd. ambler, pa. 3. notari r. e. , pharmaceutics and pharmacokinetics , 2 nd edition , new york (1975). 4. david l. nelson , michal m. cox , lehninger principles of biochemistry , 4 th edition,new york (2005). 5. gibaldi m. , pharmacokinetic aspect of drug metabolism ann. y. acd. sci. , (1979) 197 : 19--31 . 6. m. e. pichichero ,j. r. casey, s. l. block et al , pharmacodynamic analysis and clinical trials of amoxicillin sprinkle iraqi j pharm sci, vol.19(1) 2010 bioequivalence of two formulations of amoxicillin 19 administrated one daily for 7 days compared to pencillin v potassium administrated four times daily for 10 days in the treatment of tonsillopharyngitis to streptococous pyrogenes in children . j. antimicrobial agents chemother. 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(2002) 774: 141-148. iraqi j pharm sci, vol.22(1) 2013 rowatinex and tamsulosin for ureteral stone 1 comparison between rowatinex and tamsulosin as a medical expulsion therapy for ureteral stone ghassan s.ahmed * , kassim j. al-shamma **,1 and majeed m.asghar *** * department of pharmaceutics and clinical pharmacy, college of pharmacy, basra university, basra, iraq. ** department of clinical pharmacy , college of pharmacy , baghdad university ,baghdad, iraq. *** department of clinical urology, college of medicine, basra university, basra, iraq. abstract the objective of this study is to evaluate the efficacy and safety of rowatinex and tamsulosin in the treatment of patients with ureteric stone. forty patients with ureteric stone ranged (412) mm, were included in this study. they were randomized into two groups where the first group includes twenty patients treated with rowatinex three times daily (group 1), and the second group includes twenty patients treated with tamsulosin 0.4mg/day (group 2). all patients were randomly assigned to receive the designed standard medical therapy for a maximum of 3 weeks. each group was given an antibiotic as prophylaxis and an injectable non-steroidal anti-inflammatory drug used on demand. at the outpatient clinic all subjects were assessed by ct-scan at baseline and evaluated every 7 days by physical examination, plain abdominal x-ray (kub), and abdominal ultrasonography. data were analyzed by using student t-test method to compare the results; differences in the success rate between treatments were compared with the chi-square test for 2× 2 tables. the results showed that tamsulosin significantly increases the expulsion rate, and reduce expulsion time of ureteric stone when compared with rowatinex group. tamsulosin results in a better control of renal colic pain, and decreases in endoscopic procedures performed to remove the stone. key words: ureteric stone, tamsulosin, rowatinex, medical expulsion therapy. تأثيرات التامسولوسيه ، الفارديالفيل والراواتنكس كعالج طبي طارد لحصوات الحالب غسان صالح احمد * ، قاسم جليل الشماع **،1 اصغر محمد و ماجد *** * .فزع انصٍذالٍَاخ وانصٍذنح انسزٌزٌح ، كهٍح انصٍذنح ، ظايؼح انثصزج ، تصزج ، انؼزاق ** .انصٍذنح ، ظايؼح تغذاد ، تغذاد ، انؼزاق فزع انصٍذنح انسزٌزٌح ،كهٍح *** .انثىنٍح ، كهٍح انطة ، ظايؼح انثصزج ، تصزج ، انؼزاق انعزاحح فزع الخالصة .إٌ انهذف يٍ هذِ انذراسح هى ذقٍٍى فؼانٍح وساليح انزواذُكس وانرًسىنىسٍٍ فً ػالض انًزضى انذٌٍ ٌؼاَىٌ يٍ حصاج انحانة نقذ ذى ذىسٌؼهى تصىرج . يهى قذ شاركىا فً هذِ انذراسح( 12-4)ٌؼاَىٌ يٍ حصاج انحانة انرً ذزاوغ حعًها أرتؼىٌ يٍ انًزضى انذٌٍ وذضى , (انًعًىػح األونى)يزٌضا ػىنعىا تانزواذُكس شالز يزاخ ٌىيٍا 20ػشىائٍح إنى يعًىػرٍٍ حٍس ذضى انًعًىػح األونى ذى ذىسٌغ كم انًزضى ػشىائٍا نرهقً انؼالض نًذج (. انًعًىػح انصاٍَح)يهغى تانٍىو 0.4يزٌضا ػىنعىا تانرًسىنىسٍٍ 20انًعًىػح انصاٍَح .شالشح أساتٍغ كحذ أقصى ذى ذقٍٍى ظًٍغ انًزضى فً . أػطٍد كم يعًىػح انًضاداخ انحٍىٌح كىقاٌح وأدوٌح يضادج نالنرهاب غٍز سرٍزودٌح كًسكٍ ػُذ انحاظح أٌاو تىاسطح انفحص انثذًَ واألشؼح 7وذى ذقٍٍى انًزضى كم , قطؼً انًحسىب فً تذاٌح انذراسحانؼٍادج انخارظٍح تىاسطح انرصىٌز انى .انسٍٍُح نهثطٍ وانًىظاخ انفىق صىذٍح نهثطٍ أظهزخ انُرائط اٌ انرايسىنىسٍٍ أحذز سٌادج يؼُىٌح فً يؼذل طزد حصاج انحانة وذقهٍم انىقد انالسو نطزد انحصاج ػُذ يقارَرها يغ واَخفاض فً ذُفٍذ انؼًهٍاخ انًُظارٌح إلسانح , فضال ػٍ ذحسٍٍ انسٍطزج ػهى أالو انًغص انكهىي, وػح انرً ػىنعد تانزواذُكسانًعى . انحصاج .تامسولوسيه ، فارديالفيل ، راواتنكس :الكلمات المفتاحية introduction urolithiasis is a common disease worldwide and its incidence in western countries is growing (1) . urolithiasis has been considered as a significant source of morbidity , affecting all geographical,cultural, and racial groups. the risk of developing urolithiasis throughout lifetime is about 10 – 15% in the developed countries, but can be as high as 20 – 25% in the middle east. 1 corresponding author e-mail:drkassim_alshamaa@yahoo.com. received: 19/5/2012 accepted: 1/12/2012 iraqi j pharm sci, vol.22(1) 2013 rowatinex and tamsulosin for ureteral stone 2 the increased risk of dehydration in hot climates, coupled with a diet that is 50% lower in calcium and 250% higher in oxalates compared to western diets, accounts for the higher net risk in the middle east .(2) stones <3 mm in diameter have a better chance to pass spontaneously in the majority of cases, whereas stones>6 mm in the ureter are unlikely to pass in most situations (3) . many factors are involved in the interaction between the ureter and stones, therefore it is useful to understand mechanisms involved in the contraction and relaxation of the ureter. these mechanisms would possibly lead to discovery of new drugs that might facilitate stone passage, relieve symptoms and act as an adjunctive treatment to existing conventional modalities (4) . minimal invasive treatment strategies such as extracorporeal shockwave lithotripsy and ureteroscopy are frequently applied procedures in ureteral stone disease. however, indications for watchful waiting might be extended by addition of so-called ‘medical expulsive therapy’(met). met developed from several physiologic and pathophysiologic premises. alpha 1 adrenoceptors (ar) have also been reported to mediate contractile responses in the ureter, and there is evidence that α-1 receptors predominate in ureteral smooth muscle (5,6) . alpha-adrenoceptors (ar) -1a,-1b and -1d subtypes were found to be localized in human ureter irrespective of location. (7) . sympathetic nerve bundles were distributed throughout the entire ureter. therefore, a1-adrenergic receptor antagonists may act on the entire ureter, reducing its tonus. as a result, these antagonists may be useful for improving the stone freeing rate and inhibiting pain attacks (8) . several studies have demonstrated that lower tract ureteral stones can be treated efficiently with different types of alpha1blockers with a low incidence of side effects (9) . of the available alpha1-blockers, we chose tamsulosin because it is a combined alpha1a and alpha1d-selective adrenergic antagonist and the existence of alpha1a and alpha1d-adrenoceptor subtypes have been demonstrated in the smooth muscle cells of the human ureter (10) . rowatinex is a special terpenes combination and is considered to have diuretic, antiinflammatory and analgesic properties. in a randomized clinical trial, rowatinex improved stone-free rates and reduced symptoms during stone passage (11) . rowatinex is considered to be a traditional therapy for ureteral stones, and selected to be used in this study as comparative drug. this study was conducted to evaluate the efficacy and safety rowatinex and tamsulosin in the treatment of patients with ureteric stone. patients and methods this study was conducted prospectively at albasra general hospital from october 2011 till the end of april 2012. forty patients with ureteric stone ranged from 4mm to12mm (mean 8 ± 2.3), were randomized into 2 groups. patients age ranged from 18 to 65 year (mean 35.27 ± 14.7), of which 31 patients (77.5%) were males and 9 patients (22.5%) were females. the first group (group 1) was treated with rowatinex three times daily for 3 weeks, and the second group (group 2) was treated with tamsulosin 0.4 mg once daily for 3 weeks. in the first group the percentage of the proximal, middle, and distal ureteric stones were found to be 45%,30%, and 25% respectively, while, in the second group they were found to be 55%, 30%, and 15% respectively. both groups were given an antibiotic as prophylaxis during the medical expulsive therapy period and an injectable nonsteroidal anti-inflammatory drug used on demand. at the outpatient clinic all subjects were assessed by ct-scan at baseline and evaluated every 7 days by physical examination, plane abdominal x-ray (kub), and abdominal ultrasonography. data were analyzed by using student t-test method used to compare the results and these data were represented as mean ± standard error of the mean (se), differences in the success rate between treatments were compared with the chi-square test for 2× 2 tables . results the data in table (1) showed that expulsion rate in patients treated with tamsulosin ( group 2) was significantly higher (p<0.05) than that of patients treated with rowatinex (group1) for the same course time. table 1: the effects of rowatinex and tamsulosin on the expulsion rate of ureteric stone. * significantly different at (p<0.05) expulsion rate number of subjects groups 40 20 group 1 (rowatinex) 85* 20 group 2 (tamsulosin) iraqi j pharm sci, vol.22(1) 2013 rowatinex and tamsulosin for ureteral stone 3 significant reduction (p<0.05) in expulsion time was obtained from treatment with tamsulosin when compared with results of treatment with rowatinex as shown in table (2). table 2: the effects of rowatinex and tamsulosin on expulsion time of ureteric stone. values expressed as mean + standard error of mean. * significantly different at (p<0.05). as shown in table (3), the percent of patient experience colic episode was significantly lower (p<0.05) in patients treated with tamsulosin when compared to the percent of patient experience colic episode in patients treated with rowatinex. at the same time, the total number of colic episodes in the group of patients treated with tamsulosin was significantly lower (p<0.05) than that of patients treated with rowatinex. table 3: the effects of rowatinex and tamsulosin on the incidence of colic episodes in patients with ureteric stone. * significantly different at (p<0.05). * significantly different at (p<0.05). table (4) represents the total number of ureteroscopic (urs) procedures performed to remove the stone, the data showed that the total number of ureteroscopy in the group treated with rowatinex was significantly higher (p<0.05) as compared to those of group treated with tamsulosin. table 4: effect of treatment with rowatinex and tamsulosin on the total number of ureteroscopic procedures performed to remove the ureteral stone. * significantly different at (p<0.05). discussion tamsulosin, a selective α-1a-, α -1d adrenergic antagonist, which has been recently used to treat ureteral stones, achieving unexpected and startling results. the rationale for its use for this pathological condition was taken from several studies. sympathetic nerve fibers seem to be distributed throughout the entire ureter, and therefore, α-1-blockers would seem to be effective for elimination of ureteral stones irrespective to their locations (7,12,13) . in this study, tamsulosin 0.4mg/d shows a significant advantage on the expulsion rate of ureteric stone over rowatinex. the data in table (1) showed that expulsion rate in patients treated with tamsulosin was significantly higher (p<0.05) than that of patients treated with rowatinex for the same course time. the results also demonstrated that patients treated with tamsulosin were associated with a greater tendency to reduce the expulsion time when compared with rowatinex. these results were consistence with those obtained by m. dellabella et.al. (14) who found that , the use of tamsulosin in treatment regimen produced stone expulsion in almost all cases in a short time, allowing complete home patient treatment. tables (3,4), represent that patients treated with tamsulosin showed better control of renal colic pain as well as demonstrated by the fact that less patients in this group was urgently hospitalized during the study period. the causes of pain related to ureteral colic are the strain on muscular nerve endings and mucosa, which is caused by the increase in ureteral intraluminal pressure resulting from lithic obstruction and the production of lactic acid due to smooth muscle spasm. the stimulus is transferred to the spinal expulsion time (days) ± sem number of subjects groups 12.13 ± 1.94 20 group 1 (rowatinex) 7.69* ± 1.02 20 group 2 (tamsulosin) no. of colic episode % of patient experie nce colic episode number of subjects groups 14 35 20 group 1 (rowatinex) 4* 10* 20 group 2 (tamsulosin) total no. of ureteroscopy (%) number of subjects groups 9 (45) 20 group 1 (rowatinex) 2 (10)* 20 group 2 (tamsulosin) iraqi j pharm sci, vol.22(1) 2013 rowatinex and tamsulosin for ureteral stone 4 cord through type a slow fibers and type c fast fibers, and then to the cerebral centers. α-1 receptor blockade results in the reduction of visceral pain and it has been assumed that this α adrenergic blockade occurs in c-fibers (15,16) . therefore, according to our clinical results it would be possible to suppose a double action of tamsulosin on the control of pain associated with ureteral colic, that is a first action on smooth muscles, preventing spasm, and a second action on c-fibers or sympathetic postganglionic neurons, which also blocks pain conduction to the central nervous system. our results clinically supporting the validity of the hypothesis of the role of α-adrenergic receptors in the physiology of ureteral motility and the pathophysiology of renal colic .(17,18) . references 1. pak cyc: kidney stones. lancet 1998; 51:1797–1801s. 2. reilly jr. rf. nephrolithiasis 2005; chapter 13: pp. 192–207. 3. simon j,roumegueret,vaessen c, schulman cc: conservative management of ureteric stones. acta urol belg 1997; 65: 7–9. 4. davenport k, timoney a, keeley fx. a comparative in vitro study to determine the beneficial effect of calcium-channel and alpha (1) adrenoceptor antagonism on human ureteric activity. bju int 2006; 98:651–5. 5. obara k, takeda m, shimura h, kanai t, tsutsui t, komeyama t alpha-1 adrenoreceptor subtypes in the human ureter: characterization by rt-pcr and in situ hybridization. j urol 1996;155(suppl):472a 6. hernandez m, prieto d, simonsen u, rivera l, barahona mv, garcia-sacristan a. noradrenaline modulates smooth muscle activity of the isolated intravesical ureter of the pig through different types of adrenoceptors. br j pharmacol 1992, 107:924 7. hyoung keun park · eun young choi , byong chang jeong · hyeon hoe kim, byoung kwon kim. localizations and expressions of _-1a, _-1b and _-1d adrenoceptors in human ureter. urol res 2007; 35:325–329. 8. kazunari ohki, yosihiro ohno , kazuhiro suzuki. the investigation of ureteral sympathetic innervation, using semi-serial sections: why does the a1-adrenergic receptor antagonist work well for ureteral stones? int urol nephrol ;2010; 42:113–117. 9. dellabella m, milanese g, and muzzonigro g: efficacy of tamsulosin in the medical management of juxtavesical ureteral stones. j urol 2003; 170: 2202–2205,. 10. sigala s, dellabella m, milanese g, et al: alpha1 adrenoceptor subtypes in men juxtavesical ureters: molecular and pharmacological characterization. eur urol 2004; suppl 3: 119. 11. djaladat h, mahouri k, khalifeh shooshtary f, ahmadieh a. effect of rowatinex on calculus clearance after extracorporeal shock wave lithotripsy. urol j 2009;6:9–13. 12. dellabella, m., milanese, g. and muzzonigro, g.: efficacy oftamsulosin in the medical management of juxtavesical ureteralstones. j urol (2003);170: 2202. 13. rang hp, dale mm, ritter jm, flower rj 2007. "ch. 11", rang and dale's pharmacology. elsevier churchill livingstone, pp179-180. 14. marco dellabella, giulio milanese and giovanni muzzonigro. randomized trial of the efficacy of tamsulosin, nifedipine and phloroglucinol in medical expulsive therapy for distal ureteral calculi. . j urol 2005;174, 167–172. 15. ishigooka, i., nakada, t., hashimoto, t., iijima, y. and yaguchi, h.: spinal substance p immunoreactivity is enhanced by acute chemical stimulation of the rat prostate. urology 2002; 59: 139. 16. kinnman, e., nygards, e. b. and hansson, p.: peripheral alphaadrenoreceptors are involved in the development of capsaicin induced ongoing and stimulus evoked pain in humans. pain 1997; 69: 79, 17. hernandez, m., prieto, d., simonsen, u., rivera, l., barahona, m. v. and garciasacristan, a.: noradrenaline modulates smooth muscle activity of the isolated intravesical ureter of the pig through different type of adrenoceptors. br j pharmacol 1992; 107: 924. 18. morita, t., ando, m., kihara, k. and oshima, h.: function and distribution of autonomic receptors in canine ureteral smooth muscle. neurourol urodyn 1994; 13: 315. iraqi j pharm sci, vol.31( 2 ) 2022 synthesis, molecular docking study of some quinazolinone derivatives doi: https://doi.org/10.31351/vol31iss2pp283-296 283 synthesis, molecular docking study and cytotoxicity evaluation of some quinazolinone derivatives as nonclassical antifolates and potential cytotoxic agents mohammed abdulameer oleiwi *,1 and munaf h. zalzala** *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract new 4(3h)-quinazolinone derivatives (s1-s4) were synthesized and characterized by ftir, 1hnmr, and 13cnmr. their cytotoxic activity against a set of human cancer cell lines mcf-7 (breast) and a549 (lung) was evaluated using mtt assay. to detect their selectivity toward cancer cells, the compounds were also tested against epithelial cells derived from normal human fibroblast (nhf). methotrexate (mtx) was used as a positive control. all the tested compounds (s1-s4) exhibited toxicity against the normal cells lower than cancer cells. with the exception of compounds (s3 and s4), the tested compounds showed no siginificant differences from mtx regarding their inhibition rate against the normal cells (nhf) in all concentrations. all tested compounds displayed higher cytotoxicity against the lung cancer cell line (a549) than mtx with the most potent one is being compound s2 (ic50: 5.73 µm). among the tested compounds, compound s1 exhibited the highest cytotoxic activity against the breast cancer cell line (mcf-7) (ic50: 3.38 µm) compared to mtx (ic50: 27.32 µm). the binding modes of the synthesized compounds with the target proteins (dhfr and ts) were investigated by molecular docking studies using gold software. molecular docking study showed that both compounds s1 and s2 displayed partially a similar binding mode with the active site of dhfr in comparison with the co-crystallized ligand (mtx), whereas compound s1 showed a different binding mode with the active site of ts when compared with the co-crystallized ligand (raltitrexed). keywords: 4(3h)-quinazolinone, dhfr, 1,3,4-thiadiazole, thymidylate synthase. الجزيئي وتقييم السمية الخلوية لبعض مشتقات الكينازولينون كمضادات التراصف تصنيع ودراسة محتملة سمية خلوية ذات عوامل غيرتقليدية للفوالت و ** زلزلة ، مناف هاشم 1*محمد عبد األميرعليوي العراق. بغداد، الكيمياء الصيدالنية ، كلية الصيدلة ، جامعة بغداد، فرع* فرع االدوية والسموم ، كلية الصيدلة ، جامعة بغداد، بغداد ،العراق** . الخالصة طيف االشننعة ت ا ال مراء م تاننصيصننوا بواسنن ةوت (s1-s4) الجديدة كيعازوليعون (3h) 4تم تصننعيس سننلسننلة ما مانن ا ا )الرئة( a549 )الثدي( و mcf-7 وه كما تم ت ييم فعالي وا السنمية دند مجمو ة ما الصايا السنرطانية اليانريةوالرنيا العووي المغعاطيسن ا دند الصايا الاوارية المان ة ما الصايا اللي ية اليانرية ال ييعية mtt م ايسنةباسن صدام مدى للكانف ا (nhf) ، تم اخ يار المركيا أيضنا أظور جميس المركيا المص يرة سمية دد الصايا ال ييعية ععصرت كم ايجاب .ك ان ائي وا تجاه الصايا السرطانية .تم اس صدام ارالميثوتريكسيا ما (a549) أقل ما خ وط الصايا السننرطانية المص ارة كما أظور جميس المركيا سننمية خلوية أ ل دنند خ خايا سننرطان الرئة بدرجة (7دنند خ خايا سننرطان الثدي ما بيا المركيا ال تم اخ يارها . ic)5.73µm)50: هواال ل فا لية s2الميثوتريكسننيا وكان المرك -(mcfالمركن اظورs1 ال نا لينة اال ل ) :3.38 µm50(ic 27.32 :كسننننينايبنالم نارننة مس الميثوتر µm) 50(ic كمنا تم ف أودنننناع . gold الجزيئ باسن صدام برنام ال راصنف ما خا دراسنا (ts) و (dhfr) ااالرتياط للمركيا المصنععة مس اليروتيعا المسن ودفة بالم ارنة مس ليغاند dhfrجزئيا ودننس رب مما ل مس الموقس العانن اظورا s2و s1الجزيئ أن كا المركييا ال راصننف أظور دراسننة ليغنانند الم يلور المانننن ر بنالم نارننة مس tsودننننس رب مص لف مس الموقس العانننن s1أظور المركن بيعمنا ,(mtxالم يلور المانننن ر ) (raltitrexed) . .سينثيز ثيميدالت , -1,3,4,مختزل ثنائي هيدروفوالت, ثياديازول -(3h)4كينازولينون المفتاحية: الكلمات introduction the major problems associated with the current chemotherapeutic drugs are represented by multidrug-resistant tumours and severe systemic toxicity (1). compounds that are designed to hit a single biological target often have limited clinical benefit in the treatment of complex diseases such as cancer, therefore the strategy of designing a single drug with multiple targets is considered as an alternative therapeutic approach (2). 1corresponding author e-mail: amir_pharm82@yahoo.com received: 9/ 11/ 2021 accepted: 12/2 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp283-296 iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 284 the dependence of rapidly dividing cells on the availability of nucleotide precursors provides an attractive therapeutic target for the development of new anticancer agents, the most widely utilized among these targets is the folate biosynthesis pathway that leads to thymidine synthesis (3). dihydrofolate reductase (dhfr) and thymidylate synthase (ts) are the key enzymes in folate metabolism which is necessary for the synthesis of dna, rna, and proteins (4). antifolates are structurally related to folate derivatives, their antitumor activity is attributed to their ability to inhibit the folate-dependent enzymes (5). antifolates are divided into two classes; classical antifolates that possesses a glutamate tail and must be actively transported into the cell utilizing a carrier known as reduced folate carrier (rfc), and non-classical antifolates, these are lipophilic inhibitors that cross the cell membrane by passive diffusion (3).the cytotoxic activity of classical antifolates is highly dependent upon the glutamate component, which is necessary for the active uptake into cells via the reduced folate carrier. this component is further polyglutamated by an enzyme called folylpolyglutamate synthetase (fpgs) (6). cancer cells develop resistance to classical antifolates such as raltitrexed, which depend on polyglutamylation for their antitumor activity by producing defective folylpoly-γ-glutamate synthetase (fpgs) enzyme or reducing its synthesis (7). therefore, to overcome these potential disadvantages associated with classical antifolates, nonclassical antifolates have been designed and synthesized (8). these lipophilic nonclassical antifolates lack the polar glutamate moiety and, therefore, do not require fpgs for their antitumor activity (figure 1) (9). figure 1. classification of antifolate drugs. 4(3h)-quinazolinone scaffold has drawn much attention in the field of drug design and developments due to its wide spectrum of biological activities, mainly its cytotoxic potential (10).the anticancer activity of quinazolinone derivatives depend on their ability to inhibit many enzymes essential in cell division such as thymidylate synthase (ts) (11, 12) and dihydrofolate reductase (dhfr) enzymes (13). on the other hand, 1,3,4thiadiazole scaffold was found to possess a promising antitumor activity against many cancer cell lines through the inhibition of many molecular targets, such as tyrosine kinase and histone deacetylase (hdac) (14). the aim of this research was to synthesize some quinazolinone derivatives as nonclassical antifolates and possible inhibitors of dihydrofolate reductase and thymidylate synthase enzymes. materials and methods all reagents, chemicals, and solvents used in the chemical synthesis were used as obtained from their suppliers. 6-(bromomethyl)-2-methyl4(3h)-quinazolinone was purchased from henan tianfu chemical co. (china). 5-methylamino-1,3,4thiadiazole-2-thiol was purchased from apollo scientific (uk), methotrexate was purchased from (mylan) company (france).the progress of the reactions and purity of the products were checked by thin layer chromatography (tlc) on pre-coated aluminium sheets silica gel 60 f254 (merck) and the spots were visualized under a 254 nm uv lamp. melting points were determined by open capillary method using the stuart smp3 apparatus (uk). fourier transform infrared (ftir) spectroscopy was done utilizing shimadzu iraffinity-1 spectrometer (japan) and specac® atrdiamond type (uk). 1hnmr and 13cnmr analysis were performed using the bruker and varian model ultrashield spectrometer at 500 mhz for 1hnmr and 125 mhz for 13cnmr with tetramethylsilane (tms) as the internal standard and d6-dmso or cdcl3 as the solvent. chemical shift values are expressed in the δ (ppm) scale and the signals are described as s (singlet), d (doublet), m (multiplet), and b (broad) whereas coupling constants (j) are expressed in hertz. cytotoxicity assay was performed at the iraqi biotech. research centre (baghdad, iraq) using fetal bovine serum (fbs), trypsin/edta and roswell park memorial institute iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 285 (rpmi)1640 medium (capricorn, germany), cell culture plates and dimethylsulfoxide (dmso) (santacruz biotechnology, usa), (mtt) stain (bioworld, usa) ), microtiter reader (gennex lab., india ), laminar flow hood and co2 incubator (cypress diagnostics, belgium). the crystallized structures of dhfr (pdb: 3eig) (15) and ts (pdb: 5x5q) (16) were downloaded from the protein data bank (pdb, www.rcsb.org) (17). general synthesis of compounds (a1-a4) the synthesis of compounds (a1-a4) was done according to the following procedure (18). to a stirred solution of aniline or substituted anilines (20 mmol) in 25 ml of dry benzene, tea (20 mmol) was added, the mixture was stirred on the ice bath, then chloroacetylchloride (24 mmol) (dissolved in 20 ml of dry benzene) was added drop wise in 30 min. the mixture was then refluxed for 3 h. the excess of solvent (benzene) was evaporated under vacuum and the precipitate was washed with sodium carbonate (2%), hcl (5%), and distilled water, then dried and recrystallized from ethanol. 2-chloro-n-phenylacetamide (a1) light yellow powder, yield 78%, m.p (1311330c). rf =0.74 (n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ, cm−1), 3267(nh) str. of sec. amide, 3097 ar (c-h), 1666 (c=o) str. of amide ,748 (ccl) str. 1hnmr (dmso‐d6, 500 mhz, δ=ppm):4.23(s,2h,ch2-cl),7.07(t,1h, arh),7.31(m,2h,ar-h), 7.57 (d, 2h ,j= 8.47, arh),10.27(bs,1h, nh).13cnmr (dmso‐d6, 125mhz): 44.03, 119.79 ,124.27,129.29,138.91,165.04. 2-chloro-n-(4-chlorophenyl) acetamide (a2). white crystals, yield 86%, m.p (1721740c), rf =0.83 (n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ, cm−1),3263(nh) str. of sec. amide, 3086 ar(c-h),1666 (c=o) str. of amide, 775 (c-cl) str. 1hnmr(dmso‐d6, 500mhz ,δ= ppm) :4.24(s,2h,ch2-cl),7.37(d, 2h, j= 8.50hz, ar-h), 7.60 (d, 2h, j=8.50hz, ar-h) ,10.39(bs,1h,nh).13cnmr (dmso‐ d6,125mhz):43.95, 121.35, 127.88, 129.21 , 137.86,165.22. 2-chloro-n-(4-methoxyphenyl) acetamide (a3). gray powder, yield 94%, m.p (116-1180c), rf =0.81(n-hexane: ethyl acetate: metanol 5:3:2). ftir (υ, cm−1),3294(nh) str. of sec. amide, 3074 ar(c-h ),1662 (c=o) str. of amide, 1246 (c-o) str., 786 (c-cl) str. 1hnmr (dmso‐d6, 500mhz, δ=ppm): 3.71(s,3h,o-ch3), 4.19 (s, 2h,ch2-cl), 6.89 (d,2h ,j=8.37hz ,ar-h),7.48(d, 2h,j=8.37hz, ar-h), 10.11 (bs,1h,nh). 13cnmr (dmso‐d6, 125mhz): 48.72, 60.41, 119 .17, 126.18, 136.76, 160.84, 169.32. 2-chloro-n-(4-nitrophenyl) acetamide (a4). green powder, yield 88%, m.p (1791810c), rf =0.78 (n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ, cm−1), 3275(nh) str. of sec. amide, 3070ar(c-h) ,1685(c=o) str. of amide ,1566,1334 asymmetric and symmetric str. of (no2), 748 (ccl) str. 1hnmr (dmso‐d6, 500mhz, δ=ppm): 4.32 (s,2h,ch2-cl),7.82 (d,2h, j=7.28, ar-h),8.23(d, 2h, j=7.28, ar-h),10.82(bs,1h,nh).13cnmr (dmso‐d6,125mhz): 44.01,119.48,125.38,143.02,145.00 ,165.98 . general synthesis of compounds (b1-b4). the synthesis of compounds (b1-b4) was done according to the following procedure (19). to a stirred suspension of 5-methylamino-1,3,4thiadiazole-2-thiol (1.7 mmol) in 10 ml distilled water, tea (2 mmol) was added and the mixture was stirred until the compound completely dissolve, then one of the compounds (a1-a4) (1.7 mmole) was dissolved in (3 ml) of dmf and added gradually to the aqueous solution with stirring which was continued for 3 h. (monitored with tlc). the product was collected by filtration, washed with distilled water (3x50ml), dried, and recrystallized from ethanol. 2-((5-(methylamino)-1,3,4-thiadiazol-2-yl)thio)-nphenylacetamide (b1). light brown powder, yield 68%, m.p (1471490c), rf =0.61(n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ,cm−1),3290 (nh) str .of sec. amine ,3267 (nh) str.of sec. amide, 3086 ar (c-h) ,1685 (c=o) str. of amide ,1600 (c=n) str. 1hnmr (dmso‐d6, 500mhz, δ=ppm) :2.86(s,3h,nhch3),3.98(s,2h,ch2-s-) ,7.05(t,1h,arh),7.30(m,2h,ar-h), 7.56(d,2h,j=8.06,ar-h), 7.70 (bs, 1h, nh, sec.amine),10.20(bs,1h,nh,sec. amide).13cnmr(dmso‐d6, 125mhz): 31.52, 39.34, 119.63, 124.01, 129.24, 139.21, 149.35, 166.19, 171.12. n-(4-chlorophenyl)-2-((5-(methylamino)-1,3,4thiadiazol-2-yl) thio)acetamide (b2). light yellow powder, yield 77%, m.p (2032050c), rf =0.58 (n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ,cm−1),3282 (nh) str. of sec. amine, 3255(nh) str.of sec. amide, 3059 ar(ch),1685(c=o) str. of amide, 1608 (c=n) str. 1hnmr(dmso‐d6, 500mhz, δ=ppm):2.88(s,3h, nh-ch3),4.01(s,2h, ch2-s-), 7.36 (d,2h, j=8.60hz, ar-h),7.58(d,2h, j=8.60 hz, arh),7.84(bs ,1h,nh ,sec. amine) ,10.34 (bs ,1h, nh, sec. amide) .13cnmr (cdcl3, 125mhz):36.29, 44.03, 125.93 ,132.35 ,133.93 ,142.93 ,153.97,171.16,175.88. n-(4-methoxyphenyl)-2-((5-(methylamino)-1,3,4thiadiazol-2-yl)thio)acetamide (b3). off-white powder, yield 76%, m.p (1511530c), rf =0.56 (n-hexane: ethyl acetate: methanol 5:3:2). ftir(υ, cm−1),3363(nh)str. of sec. amine, 3217 (nh) str .of sec. amide, 3001 ar (c-h), 1654 (c=o) str. of amide ,1608 (c=n) str., 1234(c-o) str.1hnmr (dmso‐d6, 500mhz, δ=ppm) :2.83 (s,3h ,nh-ch3)3.70(s, 3h ,o-ch3),3.93(s,2h, ch2-s-),6.87(d, 2h , j= 8.55 hz, ar-h) ,7.45 (d,2h, http://www.rcsb.org/ iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 286 j =8.55 hz , ar-h),7.69 (bs,1h, nh, sec. amine ),10.06 (bs, 1h, nh, sec. amide) . 13cnmr (dmso‐ d6, 125mhz): 31.51, 39.24, 55.61, 114.37, 121.15, 132.35, 149.97, 155.86, 165.65, 171.09. 2-((5-(methylamino)-1,3,4-thiadiazol-2-yl)thio)-n(4-nitrophenyl)acetamide (b4). beige powder, yield 73%, m.p (2192210c), rf =0.53 (n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ, cm−1),3332 (nh) str. of sec. amine, 3282 (nh) str. of sec. amide, 3089 ar (c-h) str.,1697 (c=o) str .of amide, 1616 (c=n) str. ,1554,1330 asymmetric and symmetric str. of (no2). 1hnmr(dmso‐d6, 500mhz, δ=ppm): 2.85(s,3h, nh-ch3), 4.04 (s,2h, ch2-s-), 7.70 (bs, 1h, nh, sec. amine),7.80(d,2h, j=9.14hz, ar-h), 8.22 (d,2h, j=9.14hz , ar-h),10.82 (bs, 1h,nh,sec.amide).13cnmr(dmso‐d6,125mhz): 31.52 ,39.27 ,119.32 ,125.51 ,142.86,145.31 ,148.87,167.33,172.45. general synthesis of compounds (s1-s4) the synthesis of the target compounds (s1s4) was done according to the following procedure (20,21). a solution of 6-(bromomethyl)-2-methyl-4(3h)quinazolinone (1.48 mmol) in dry dmf (10 ml) was added gradually to a mixture of one of the compounds (b1-b4) (1.48 mmol) and dry tea (1.48 mmol) in dry dmf (15 ml) and the reaction mixture has been stirred at 80 0c overnight. the solvent was evaporated under reduced pressure; the residue was triturated with acetone, dried, and recrystallized from (methanol: dmf) mixture (4:1). 2-((5-(methyl((2-methyl-4-oxo-3,4dihydroquinazolin-6-yl)methyl)amino)-1,3,4thiadiazol-2-yl)thio)-n-phenylacetamide(s1). yellow powder, yield 71%, m.p (2152170c), rf =0.23 (n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ, cm−1), 3479, 3375 (nh) str. of sec. amides, 3096 ar (c-h) str., 1670 (c=o) str. of amide, 1612 (c=n) str.1hnmr(dmso‐d6, 500mhz, δ=ppm): 2.51(s,3h,ch3,quinazoline),2.89(s,3h, n-ch3), 4.22 (s, 2h, -ch2-s-), 4.89 (s,2h,ch2-n-), 7.06 (t,1h ,ar-h) ,7.30 (m,2h, ar-h), 7.56 (d,2h ,j=8.65, ar-h),7.75 (d,1h, j= 8.36, ar h), 8.02 (d,1h, j= 8.36, ar-h) ,8.25 (s,1h, ar-h), 10.48 (b s,1h,nh),12.86 (b s,1h,nh).13cnmr (dmso‐d6, 125 mhz): 19.63, 31.79, 52.51, 58.37, 119.57, 119.96, 121.80, 124.03, 127.43, 129.26, 137.41, 138.74, 139.21,151.08,160.01,166.03,170.41. n-(4-chlorophenyl)-2-((5-(methyl ((2-methyl-4oxo-3,4-dihydroquinazolin-6-yl) methyl) amino)1,3,4-thiadiazol-2-yl)thio)acetamide (s2). light brown powder, yield 66%, m.p (207-2090c), rf =0.21(n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ, cm−1), 3479,3290 (nh) str. of sec amides ,3078 ar(c-h), 1670 (c=o) str. of amide, 1612(c=n) str.1hnmr(dmso‐d6, 500mhz, δ=ppm ): 2.38 (s,3h,ch3,quinazoline) ,3.19 (s,3h,n-ch3) ,4.00 (s,2h,-ch2-s),4.64(s,2h,ch2-n-),7.37(d,1h, j=8.55, ar-h) ,7.61 (d,1h, j= 8.55, ar-h), 7.67(d, 2h, j=8.47,ar-h), 7.86 (d,2h,j=8.47, ar-h) ,8.24 (s,1h,ar-h). 10.46 (bs,1h,nh) ,12.41 (bs, 1h,nh) .13c-nmr (dmso‐ d6, 125 mhz) : 22.04, 31.51, 52.56, 59.42, 121.16, 121.22, 125.74, 127.78, 129.20, 130.95, 138.28, 149.26, 150.35, 156.43, 161.86, 166.44, 171.10. n-(4-methoxyphenyl)-2-((5-(methyl((2-methyl-4oxo-3,4-dihydroquinazolin-6-yl)methyl)amino)1,3,4-thiadiazol-2-yl)thio)acetamide (s3). yellow powder, yield 68%, m.p (1751780c), rf =0.18 (n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ, cm−1), 3475,3379 (nh) str. of sec amides, 3037 ar(c-h) ,1670 (c=o) str. of amide,1612 (c=n) str., 1226 (c-o) str. 1hnmr(dmso‐d6,500mhz ,δ= ppm ): 2.38 (s,3h,ch3, quinazoline), 3.21 (s,3h,n-ch3),3.71 (s,3h,o-ch3), 3.97(s,2h,-ch2-s-),4.66 (s,2h, ch2n-), 6.88 (d,2h, j=8.50, ar-h),7.49 (d, 2h, j=8.50, ar-h),7.67(d,1h, j=8.38, ar-h), 7.87(d,1h ,j=8.38, ar-h), 8.24(s,1h, ar-h) ,10.18 (b s ,1h ,nh) ,12.42 (b s,1h,nh). 13cnmr (dmso‐d6, 125 mhz): 22.05, 31.48, 52.55, 55.63, 59.39, 114.35, 121.12, 12.19, 125.76, 127.75, 130.93, 132.39, 138.30, 149.48, 150.31, 155.83, 156.41, 161.84, 165.68, 171.03. 2-((5-(methyl((2-methyl-4-oxo-3,4dihydroquinazolin-6-yl)methyl)amino)-1,3,4thiadiazol -2-yl)thio)-n-(4nitrophenyl)acetamide (s4). dark brown powder, yield 73%, m.p (1611630c), rf =0.17 (n-hexane: ethyl acetate: methanol 5:3:2). ftir (υ, cm−1), 3471,3336 (nh) str. of sec amides, 3078 ar (c-h) str. ,1670(c=o) str. of amide,1612 (c=n) str.,1496,1334 asymmetric and symmetric str. of (no2). 1hnmr (dmso‐d6, 500mhz, δ=ppm): 2.38 (s,3h,ch3, quinazoline),3.21 (s, 3h,n-ch3) ,4.09 (s,2h,-ch2s-), 4.65 (s, 2h, ch2-n-),7.67(d,1h, j=8.35, ar-h ),7.79(s,1h,ar-h) , 7.85 (m, 3h, ar-h), 8.23 (d,2h, j=8.56, arh),10.95(bs,1h,nh),12.42(bs,1h,nh).13cnmr (dmso‐d6 ,125mhz): 22.04, 31.49, 52.55, 59.38, 119.32, 121.19 ,125.53, 125.75, 127.76, 130.93, 138.29,146.12,151.09,156.42,161.84,167.37, 171.03. in vitro cytotoxicity study maintenance of cell cultures human breast cancer cell line (mcf-7), human lung cancer cell line (a549), and normal human fibroblast (nhf) cell line were grown in rpmi-1640 media supplemented with (fbs, 10%), penicillin (100iu/ml), and streptomycin (100 mg/ml). the cells were treated with the trypsinedta and re-seeded at 50% confluence twice a week then incubated at 37 °c (22). iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 287 cytotoxicity assay to determine the cytotoxic effect, a cell viability assay was done using 96-well plates. cell lines were seeded at 1 × 104cells/well. when a confluent monolayer was achieved, cells were treated with the target compounds (s1-s4) separately at different concentrations (3.125, 6.25, 12.5, 25, 50, 100 µm) using methotrexate as a positive control. cell viability was measured after 72h. of treatment by removing the medium, adding 28 µl of the mtt solution (2 mg/ml) and incubating the cells for 1.5 h. at 37 °c. after the removal of mtt solution, the remaining crystals were solubilized by the addition of 130 µl of dmso and incubation for 15 min. at 37 °c with shaking (23). the absorbency was measured at 492 nm using a microplate reader, the assay was performed in triplicate. the percentage of cytotoxicity (rate of cell growth inhibition) was calculated using the following equation (24,25): 𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 (%) = 𝐴 – 𝐵/ 𝐴 × 100 where a refers to the control optical density and b refers to the sample optical density. molecular docking study the chemical structures of the ligands were drawn by the chembiodraw program and then changed over into a 3d structure with the chembio3d computer program, energy minimization of the ligands was done by using the mm2 job and saved in the form of mol2 files, only the chain to which the co-crystallized ligand is bound was kept and all other chains were deleted. water molecules, unnecessary ions, and other ligands were deleted also, hydrogen atoms were added and gasteiger charges were assigned to simulate the in vivo conditions, the prepared protein was then saved as a (mol2) file. the co-crystallized ligands were selected and only the extracted ligands were used to define the binding sites. all protein atoms within 10 å of each selected ligand were used for the binding site definition. the ligands were docked into the binding sites of the target enzymes using gold algorithm software (26). the reliability of the docking process was validated using the x-ray structures of the co-crystallized ligands namely methotrexate for dhfr and raltitrexed for ts in the active sites of the target enzymes by redocking them into the corresponding binding sites, at the end of the docking run, the conformations of the ligands were ranked by their plp-fitness scores, and the interactions were visualized in the discovery studio (27) . in silico adme prediction swissadme website makes it possible for researchers to compute physicochemical descriptors as well as pharmacokinetic parameters and drug-like properties of the designed molecules to support drug discovery (28). drug likeness and lipinski rule descriptors of the designed compounds are listed in (table 4). results and discussion chemistry the synthesis of the intermediates and final compounds is shown in scheme 1. the purity of the products was confirmed by tlc in which a single spot for each compound was obtained. n-acylation of aniline or p-substituted anilines using chloroacetylchloride afforded compounds (a1-a4) in good yields, the reaction is carried out in presence of triethylamine, which acts as a base to neutralize hcl formed. the ir spectra of the synthesized compounds showed the disappearance of asymmetric and symmetric stretching vibration bands for nh2 of the starting compounds (aniline and p-substituted anilines) and the appearance of new absorption band at 3263-3294 cm-1 for nh stretching vibration of amide, the appearance of characteristic bands at 1662-1685 cm-1 for c=o stretching vibration of amide (amide i band) and at 748-786 cm-1 for c-cl stretching vibration. compounds a1-a4 were confirmed also via 1hnmr by the presence of a singlet peak for the amidic proton (conh) at 10.11-10.82 ppm in addition to the singlet peak of (ch2cl) at 4.23-4.32 with an integration of 2 protons. compound a3 was confirmed also by the presence of a characteristic singlet peak of 3 protons at 3.71 ppm due to (och3). characteristic peaks were also found at 165.04169.32 ppm in the 13cnmr spectra attributed to the amide carbonyl group (conh). compounds (b1b4) were prepared by s-alkylation of 5methylamino-1,3,4-thiadiazole-2-thiol with the chloroacetamide derivatives (a1-a4) in an alkaline aqueous medium (green reaction) in the presence of tea to deprotonate the thiol group (sh) of 5methylamino-1,3,4-thiadiazole-2-thiol and convert it to a thiolate anion (s-) which acts as a strong nucleophile that attacks the carbon in the haloalkyl side chain of chloroacetamide intermediates to get the sulfide derivatives. the ir spectra of these compounds showed the disappearance of the absorption band of the sh group in the starting thiol compound at 2773 cm -1 , in addition to the presence of bands due to the secondary amine stretching vibration at 3282-3363cm-1 and absorption bands at 1600-1616cm-1due to c=n stretching vibration of the thiadiazole ring. 1hnmr spectra of compounds (b1-b4) were characterized by the presence of a sharp singlet peak signified for 3 protons due to ch3 of (nhch3) at 2.83-2.88 ppm in addition to a broad singlet peak at 7.69-7.84 due to secondary amine proton (nhch3). the final compounds (s1-s4) were prepared by n-alkylation of the secondary amine of 5-methylamino-1,3,4-thiadiazole-2-thiol with the alkyl bromide (6-(bromomethyl)-2-methyl4(3h)-quinazolinone) in the presence of tea to pick up the released hbr. the ir spectra of the final compounds showed the disappearance of the iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 288 absorption bands due to the secondary amine stretching vibration at 3282-3363cm-1 and the appearance of new absorption bands in the range 3471-3479cm-1 of nh stretching vibration of the quinazolinone amide group (conh) which are shifted to higher frequency region. 1hnmr spectra of the final compounds are characterized by the disappearance of the singlet peak at 7.69-7.84 due to secondary amine proton (nhch3), in addition to the appearance of all other signals which are characteristics of the substituted 4(3h)quinazolinone group that includes sharp singlet peak signified for 3 protons at 2.38 ppm due to the quinazolinone methyl group, sharp singlet at 4.644.66 ppm signified for 2 protons due to (ch2-n-), multiplet peaks at 7.37-8.25 ppm attributed to the three protons of the fused benzene ring and broad singlet peak at 12.38-12.42 ppm due to quinazolinone amidic group (conh). scheme 1. synthetic route of the target compounds (s1-s4). reagents and condition: a. chloroacetylchloride, tea, dry benzene, reflux 3h. b. 5-methylamino-1,3,4-thiadiazole-2-thiol,distilled water ,tea, room temperature 3h. c. 6-(bromomethyl) -2-methyl-4(3h)-quinazolinone, tea, dry dmf, stirring overnight at 800c. in vitro cytotoxicity screening the synthesized compounds were evaluated for in vitro cytotoxic activity against various cell lines such as mcf-7 (breast cancer) and a549 (lung cancer) by the mtt assay method. to detect the selectivity of the tested compounds against cancer cells, epithelial cells derived from normal human fibroblast (nhf) were used also. the relationship between drug concentration and growth inhibition was plotted to obtain the survival curve of all cell lines mcf-7, a549, and nhf. figure (3).the in vitro cytotoxic activities of the tested compounds are summarized in table (1), whereas figures (4 and 5) compare between the cytotoxicity of all compounds in relative to the positive control (mtx).the ic50 values were used as a response parameter which refers to the concentration required to get 50% inhibition of cell viability (29) . all the compounds exhibited cytotoxic effects against the cancer cells higher than the normal cells, giving hope of the ability of these compounds to target cancer cells with a high degree of selectivity. the inhibition of growth exhibited by the tested compounds against the selected cell lines was parallel and in a concentration dependant manner. among the tested compounds, s1 exhibited maximum cytotoxic activity against the mcf-7 cell line ( ic50 3.38 µm) and is about eight folds higher activity than methotrexate (ic50 27.32 µm).the cytotoxic activity against a549 cells showed that all the tested compounds displayed higher potency than methotrexate. the most potent one is compound s2 (ic50 5.73µm) compared to methotrexate (ic50 259.4 µm). apart from s2, compound s1 was also found to be highly active (ic50 9.04 µm). the structure activity relationships of the tested compounds related to mcf-7 revealed that compound s1 (with no substituent at the para position of the anilide moiety) was the most potent one, compound s3 (with the electrondonating methoxy group at the para position of the anilide moiety) was more potent (ic50 11.37 µm) than s2 (ic50 57.1 µm) and s4 (ic50 14.96 µm) with electronwithdrawing groups (cl and no2) respectively. conversely, the presence of electron donating group at the para position of anilide moiety reduced the cytotoxicity against a549 cells , hence, compound s3 was found to be less potent (ic50 29.12 µm) than both compounds s2 and s4 (ic50 5.73 and 16.75 µm) respectively . iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 289 figure 2. morphology of the cell lines after treatment with compounds (s1-s4) at their ic50 concentrations. the cells were observed under an inverted microscope at 100× magnification. iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 290 figure 3. concentration vs. growth inhibition curve of compounds (s1-s4) against breast cancer cells (mcf-7) (blue), lung cancer cells (a549) (orange) and healthy fibroblast cells (nhf) (gray). (values are represented by the mean ± sem of triplicate measurements). figure 4. histogram showing the concentration (µm) versus growth inhibition of the tested compounds: compound s1 (a), compound s2 (b), compound s3 (c), and compound s4 (d) against breast carcinoma cell line (mcf-7). (values are represented by the mean ± sem of triplicate measurements). iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 291 figure 5. histogram showing the concentration (µm) and growth inhibition of the tested compounds: compound s1 (a), compound s2 (b), compound s3 (c), and compound s4 (d) against lung carcinoma cell line (a549). (values are represented by the mean ± sem of triplicate measurements). table 1. in-vitro cytotoxicity evaluation of the synthesized compounds against the selected cancer cells. compound ic50 (µm) mcf-7 cell line a549 cell line s1 3.38 9.04 s2 57.1 5.73 s3 11.37 29.12 s4 14.96 16.75 mtx 27.32 259.4 molecular docking study the docking study was carried out on compounds (s1-s4) as the ligands, where the complexes of dhfr and ts structures with their cocrystallized ligands (methotrexate and raltitrexed) respectively were selected as the docking models. for each docking, the parameters studied and compared were the plp fitness score, number and length of hydrogenbonds between ligand atoms and amino acid residues located in the binding site of the enzymes. all results are summarized in tables (2 and 3). captured images representing these ligand poses as well as hydrogen-bond interactions between the ligand and the enzymes are shown in figures (6 and 7). results of the docking study showed that all compounds exhibited good interactions with plp fitness scores ranging from 93.21 to 95.93 against dhfr target protein and from 71.33 to 81.97 against ts enzyme. the docking study against dhfr enzyme revealed that the most potent compound on the mcf-7 cell line (s1) exhibited a hydrogen-bonding interaction with the active site residue arg 70 with a distance of 2.681å through the carbonyl oxygen atom of the quinazolinone moiety and a hydrogenbonding interaction between the carbonyl oxygen of the anilide and ser59 residue with 2.52 å distance (figure 6 (b)). the most potent compound against lung cancer cell line (s2) displayed different binding modes at the dhfr cavity in which it formed two hydrogen-bonding interactions between quinazolinone carbonyl oxygen atom and arg31 residue with distances of 2.907 å and 2.984 å and two hydrogenbonding interactions between the two thiadiazole nitrogen atoms and arg31 residue with distances of 2.86 å and 3.021 å in addition to one hydrogen bonding interactions between the anilide nh group and glu35 residue with a bond length of 2.942 å (figure 6 (c)). docking study of methotrexate on the dhfr enzyme showed that the polar glutamate tail was involved in the formation of four hydrogen bonds with arg31 and arg70 residues (figure 6 (a)). from the above results, it could be concluded that both compounds s1 and s2 displayed partially the same binding pattern of methotrexate with dhfr target protein. this confirms that binding interactions with both arg31 and arg70 residues were essential for dhfr inhibition and improving the cytotoxic activity against the two cancer cell lines. docking study on ts enzyme revealed that compound s1 formed only iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 292 one hydrogen-bonding interaction with the active site residue leu221 with a distance of 2.909 å through the nh group of the anilide moiety (figure 7 (b)), while compound s2 formed a hydrogenbonding interaction between the carbonyl oxygen of the anilide and asn112 residue at 3.067 å distance (figure 7 (c)). in comparison with raltitrexed, it was shown that the most potent compound against lung carcinoma cells (s2) displays in part a similar binding pattern with raltitrexed since both of them showed hydrogen bonding interactions with asn112 (figure 7 (a)). table 2. docking study data showing amino acid interactions and the hydrogren bond lengths of target compounds and methotrexate on dhfr (pdb: 3eig). compound no. chem plp score no. of hbonds atoms of compound forming h-bond amino acid residues forming h-bonds (h-bond length in å) s1 93.21 2 quinazolinone c=o (hbond acceptor). anilide c=o (h-bond acceptor). arg70 (2.681) ser59 (2.520) s2 94.97 5 quinazolinone c=o (2 bonds) (h-bond acceptors). thiadiazole n (2 bonds) (h-bond acceptors). anilide nh (h-bond donor). arg31 (2.907) arg31 (2.984) arg31 (2.860) arg31 (3.021) glu35 (2.942) s3 93.88 1 quinazolinone c=o (hbond acceptor) arg31 (2.630) s4 95.93 2 anilide c=o (h-acceptor). anilide nh (h-bond donor). arg31(2.864) glu35 (2.954) methotrexate 99.41 9 pteridine nh2 (2 bonds) (hbond donors). pteridine nh2 (hbond donor). n-ch3 (h-bond acceptor). amide c=o (h-bond acceptor). glutamate α cooh (2 bonds) (h-bond donor and acceptor). glutamate γ cooh (2bonds) (hbond donor and acceptor). val115(2.712) ile7(2.640) glu30(2.753) ser59(2.580) asn64(2.685) arg70(2.957, arg70(2.964) arg31(2.886) arg31(3.027) iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 293 table 3. docking study data showing amino acid interactions and the hydrogen bond lengths of target compounds and methotrexate on ts (pdb: 5x5q). compound no. chem plp score no. of hbonds atoms of compound forming h-bond amino acid residues forming h-bonds (h-bond length in a° ) s1 71.33 1 anilide nh(h-bond donor) leu221(2.909) s2 74.28 1 anilide c=o (h-bond acceptor) asn112(3.067) s3 78.28 2 och3(2 bonds) (hbond acceptor) arg78(2.997) arg78(2.989) s4 81.97 4 quinazolinone nh(hbond donor) anilide c=o (h-bond acceptor) no2 (2 bonds) (h-bond acceptors). arg78(3.068) asp218(3.057) arg50(3.022) tyr258(2.778) raltitrexed 78.43 5 glutamate α cooh (2 bonds) (h-bond donor and acceptor). glutamate γ cooh (3 bonds) (1h-bond donor and 2h-bond acceptors). arg50(2.818) asn112(2.913) arg215(2.894) arg215(3.04) arg50(2.867) figure 6. comparison of binding mode of methotrexate (a), compound s1 (b), and compound s2 (c) into the binding site of dhfr (pdb: 3eig). the key amino acid residues are represented by black sticks. hydrogen bonds are shown as green dashes. iraqi j pharm sci, vol.31(2) 2022 synthesis, molecular docking study of some quinazolinone derivatives 294 figure 7. comparison of binding mode of raltitrexed (a), compound s1 (b), and compound s2 (c) into the binding site of ts (pdb: 5x5q). the key amino acid residues are represented by black sticks. hydrogen bonds are shown as green dashes. in silico adme prediction the physicochemical properties of drugs play an important role in their biological effects, one of the most critical parameters is the partition coefficient (clog po/w) which gives an expectation about the way of drug transport within the body (30). clog po/w values of all the synthesized compounds were less than five. the compounds also displayed an acceptable bioavailability scores (0.55).with the exception of compound s3, the designed compounds were also seemed not to be substrates for the multidrug resistance protein (p-glycoprotein). table 4. drug likeness and lipinski rule descriptors of the target compounds. comp . m.wt no. of hbond acceptors no. of hbond donors tpsa* c log po/w lipinski violation pgp substrate bioavailability score s1 452.55 5 2 157.41 2.93 0 no 0.55 s2 486.99 5 2 157.41 3.54 0 no 0.55 s3 482.58 6 2 166.64 2.98 0 yes 0.55 s4 497.55 7 2 203.23 2.23 1 no 0.55 *tpsa: total polar surface area iraqi j pharm sci, vol.31(2) 2022 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reticulum stress-mediated apoptosis in human non-small cell lung cancer cells: synthesis, docking study, and anticancer activity. pharmacia 2021; 68(3): 679–692. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ lipid peroxidation and antioxidant status in β-thalassemic patients: effect of iron overload iraqi j pharm sci, vol.18(2) 2009 antioxident in thalassemla 8 lipid peroxidation and antioxidant status in β-thalassemic patients: effect of iron overload bassm n. aziz *,1 , mohammad a. al-kataan ** and wasan k. ali *** * department of anaesthesis, mosul technical institute, mosul, iraq. ** department of clinical pharmacy, college of pharmacy, mosul university, mosul, iraq. *** department of chemistry, college of science, mosul university, mosul, iraq. abstract to study the effect of iron overload due to continuous blood transfusions on peroxidation products, such as malondialdehyde (mda) and peroxynitrite, with evaluation of some antioxidants like, glutathione (gsh), superoxide dismutase (sod), vitamin a, vitamin c, vitamine e, ceruloplasmin, uric acid and albumin in thalassemia patients. forty patients with thalassemia major, aged 5 to 15 years, were carried out in abn-alatheer teaching hospital in mosul city, during the period from october 2007 to april 2008. they were on chelation therapy with desferrioxamine. they were divided into two groups, the first one without iron overload (90,97±12.92), and the second one with iron overload (157.75±7.57). all the patients were received whole blood. blood samples were collected before and after blood transfusion. the results showed that there were significant increase in mda and peroxynitrite in patients with iron overload five days before and after blood transfusion in compared with groups having normal iron level. on the other hand, glutathione, superoxide dismutase activity, vitamin a, vitamin c, vitamin e, albumin and ceruloplasmin were significantly decreased whereas, uric acid was increased significantly. it is concluded that, iron over load due to continuous blood transfusion in thalassemia causes increase in oxidative tissue damage with a changes in antioxidants status. key words: beta-thalassemia, lipid peroxidation, antioxidants, malondialdehyde, iron الخالصة فزغ ذحًُم انحذَذ َرُدح إعطاء انذو انًسرًز فٍ َىاذح انثُزوكسذج انًرًثهح تانًانىَذَانذَهاَذ وَرزاخ انثُزوكسُذ, نذراسح ذأثُز eوفُرايٍُ cوفُرايٍُ aعاداخ األكسذج كانكهىذاثاَىٌ وانسىتز اوكساَذ دسًُىذُش وكم يٍ فُرايٍُ يع قُاص تعط يٍ ي يزَعا يٍ انًصاتٍُ تانُىع انزئُسٍ نًزض ٨٤وانسُزوتالسيٍُ وحايط انُىرَك واألنثىيٍُ عُذ يزظً انثالسًُُا. أخرُز سُح ويٍ انزاقذٍَ فٍ يسرشفً أتٍ ٥٩−٩ذزاوحد أعًارهى يٍ انثالسًُُا, وانهذٍَ هى ذحد عالج عقار انذسفُزوكسايٍُ. حُث يدًىعرٍُ, األونً اذصفد تعذو إنً. ذى ذقسًُهى ٦٤٤8ونغاَح َُساٌ ٦٤٤٧األثُزانرعهًٍُ تانًىصم, خالل انفرزج يٍ ذشزٍَ األول عذها ذى إعطاء انذو نكم أفزاد (. ت٧٫٩٧±٥٩٧٫٧٩( وانثاَُح تىخىد فزغ ذحًُم نهحذَذ )٥٦٫٠٦±٠٤٫٠٧وخىد فزغ ذحًُم نهحذَذ ) انعُُح انًذروسح, وأخذخ عُُاخ انذو قثم وتعذ عًهُح َقم انذو. أظهزخ َرائح هذِ انذراسح وخىد سَادج يعُىَح فٍ يسرىَاخ حذَذ انًانىَذاَانذَهاَذ وَرزاخ انثُزوكسُذ فٍ انًزظً انذٍَ َعاَىٌ يٍ فزغ ذحًُم انحذَذ يقارَح تاِخزٍَ انذٍَ َكىٌ يسرىي ان aعُذهى غثُعٍ. يٍ َاخُح أخزي, فقذ اَخفعد يعُىَا يسرىَاخ انكهىذاثاَىٌ وفعانُح أَشَى انسىتز اوكساَذ دسًُىذُش وكم يٍ فُرايٍُ فزغ وقذ أسرُرح يٍ انذراسح تأٌ واألنثىيٍُ وانسُزَىتالسيٍُ, تًُُا ارذفع يعُىَا يسرىي حايط انُىرَك. eوفُرايٍُ cوفُرايٍُ ٌ يع اخرالف فٍ يسرىَاخ يعاداخ ذَذ انُاذح يٍ َقم انذو انًرىاصم نًزظً انثالسًُُا قذ سثة سَادج شذج انكزب انراكسذحًُم انحذ . األكسذج introduction increased level of lipid peroxidation and decreased level of antioxidants play important roles in the pathogenesis of anemias 1 . it is well documented that disturbances of oxidant antioxidant balance occur in hemoglobinopathies, especially in thalassemia 2 . in beta-thalassemia, decreased or impaired biosynthesis of beta-globin leads to accumulation of unpaired alpha globin chains 3 . excess presence of the alpha-globin chains primarily 3 and also iron overload, as a result of multiple transfusions, are the main reasons for the cellular oxidative damage in thalassemias 4 . iron overload is still a major concern in homozygous β-thalassemia. under physiological conditions, iron ions are not available to catalyze the conversion of molecular oxygen to highly reactive radical species by fenton reaction, because ferric iron is bound to proteins, preventing it from participating in reactions that could lead to cell injury 5 . under various pathological conditions associated with iron overload, including thalassemia, due to blood transfusion used for treatment of thalassemia. there is evidence of an increase in iron in both serum and cells 6 . 1 corresponding author email : bassam_alwakeel@yahoo.com received : 26 / 11 / 2008 accepted : 23 / 6 / 2009 mailto:bassam_alwakeel@yahoo.com iraqi j pharm sci, vol.18(2) 2009 antioxident in thalassemla ٠ this increases generation of free radicals 7 , and promotes peroxidative damage to cell and organelle membranes in organs that accumulate excess iron, including liver, pituitary gland, pancreas, and heart 8 . this study evaluates the total antioxidant potential and several individual antioxidants, as well as parameters of peroxidative stress, including malondialdehyde (mda) (the breakdown product of lipid peroxidation), in serum of patients with β-thalassemia major, transfusiondependent, and under regular iron chelation therapy with or without signs of iron overload, before and after transfusion. subjects and methods experimental design this study was conducted at abnalatheer teaching hospital in mosul city, from october 2007 to april 2008. forty patients with β-thalassemia major, aged 5 to 15 years (mean, 7.3±3.7), were divided into two groups. the first group included twenty patients without signs of iron overload as revealed from the mean value of serum iron (90,97±12.92), referred as control, while the second group included twenty patients with clinical and biochemical signs of iron overload (157.75±7.57). both groups were under continuous and regular blood transfusion program (one transfusion process/ month). chelation therapy with desferrioxamine (dfo) was administered to each of the patients (by pump, five days a week, 40-60mg/kg/day, 12 hours infusion) 9 . all of the patients were examined regularly once a month in pediatric hematology department of this hospital. sample collection and clinical chemistry analysis ten milliliters of blood samples were obtained from each patient five days before and after blood transfusion process. after clotting, serum was separated by centrifugation and divided in several aliquots. the analytical determinations described below were either performed immediately, or serum was stored at -20°c and used within 72 hours. the readings of the measured parameters were done at clinical biochemical laboratory in the college of science. clinical laboratory examinations on serum including, mda and peroxynitrite as an oxidant indicator and the total antioxidant capacity like glutathione, sod, vitamin a, vitamin c, vitamin e, ceruloplasmin, uric acid, and albumin levels were evaluated. the level of serum mda was determined by a modified procedure using the thiobarbituric acid reaction substance (tbars) methods 10 , and the activity of sod levels in blood serum was determined using photochemical method described by brown and goldstein 11 . this methods depends on an indirect approach to determine the sod activity through the change in formazene absorbance formed from the reduction of o2 •¯ , which is produced by radiating the sample of serum with light) for nitroblue tetrazolum (nbt) dye. decreased difference in formazene absorbance means increased sod activity. serum glutathion is determined by a modified procedure utilizing ellman`s reagent 12 .serum vitamin a 13 , vitamin c 14 and vitamin e 15 were measured spectrophotometrically. ceruloplasmin, peroxy nitrite activity were measured by modified method described by menden et al. 16 and vanuffelen et al. 17 respectively. the level of uric acid 18 and serum albumin 19 were measured. statistical analysis all data were compared by t-test between patient groups in spss 10.0 program. the values within the tables were given as mean ± standard deviation. statistical significance was considered at p<0.05 20 . results the effects of blood transfusion in thalassemic patients, without iron overload, represented with normal serum iron (90,97±12.92), on lipid peroxidation and antioxidant status are presented in table (1). glutathione, vitamin a, vitamin c vitamin e, and albumin increased significantly, while mda, peroxynitrite, sod activity, ceruloplasmin, and uric acid did not changed significantly after blood transfusion in comparison with the control group (before transfusion). on the other hand, with the exception of peroxynitrite, sod activity, albumin and ceruloplasmin other parameters like glutathione, vitamin a, vitamin c, and vitamin e were increased significantly, while mda and uric acid were decreased significantly in thalassemic patients, with iron overload, after receiving blood transfusion as shown in table (2). the effects of iron overload in thalassemic patients, represented with increased serum iron (157.75±7.57), before blood transfusion, on lipid peroxidation and antioxidant status are presented in table (3). mda, peroxynitrite, and uric acid were found to be higher in thalassemic patients with iron overload when compared with non-iron overload patients. on the other hand, glutathione, sod activity, vitamin a, vitamin c, vitamin e, ceruloplasmin, and albumin were significantly decreased in the same group. moreover, after blood transfusion, all parameters were altered significantly in iron overload group in comparison with thalassemic iraqi j pharm sci, vol.18(2) 2009 antioxident in thalassemla ٥٤ patient without iron overload as shown in table (4). glutathione, sod activity, vitamin a, vitamin c, vitamin e, albumin and ceruloplasmin were decreased significantly. in contrast, mda, peroxynitrite, and uric acid were shown to be increased significantly. table 1: effects of blood transfusion in thalassemic patients, without iron overload, on lipid peroxidation and antioxidant status.  values are expressed as means ± sd from 20 subjects per group.  (ns): not significant *significantly different from the control (p<0.05) ** significantly different from the control (p<0.01) *** significantly different from the control (p<0.001) table 2: effects of blood transfusion in thalassemic patients, with iron overload, on lipid peroxidation and antioxidant status.  values are expressed as means ± sd from 20 subjects per group.  (ns): not significant *significantly different from the control (p<0.05) ** significantly different from the control (p<0.01) *** significantly different from the control (p<0.001) table 3: effects of iron overload in thalassemic patients, before blood transfusion, on lipid peroxidation and antioxidant status.  values are expressed as means ± sd from 20 subjects per group.  (ns): not significant *significantly different from the control (p<0.05) ** significantly different from the control (p<0.01) *** significantly different from the control (p<0.001) iraqi j pharm sci, vol.18(2) 2009 antioxident in thalassemla ٥٥ table 4: effects of iron overload in thalassemic patients, after blood transfusion, on lipid peroxidation and antioxidant status.  values are expressed as means ± sd from 20 subjects per group.  (ns): not significant *significantly different from the control (p<0.05) ** significantly different from the control (p<0.01) *** significantly different from the control (p<0.001) discussion it has been postulated that the biochemical and metabolic changes of thalassemic patients are associated with a constant oxidative stress within the red cell caused by precipitation of excess alpha-globin chains, and release of free iron 21 . the measurement of the peroxidation products, together with the evaluation of the antioxidants may be the simple measurement of iron overload due to blood transfusion in thalassemia 9 . increased plasma mda level, which is measured by the thiobarbituric acid reaction substance (tbars) methods, was found in beta-thalassemia patients 22 , where mda is a good indicator of oxidative damage. the increased in serum mda levels in patient with thalassemia in our study, as shown in tables (3 and 4) can be compared with those obtained by other investigators 9 . in addition, peroxynitrite, was measured in the present study. serum levels of this pro-oxidants was increased significantly in thalassemic patient with iron overload compared with other without iron overload.. nitric oxide (no ∙ ) contains unpaired numbers of electrons and are therefore free radicals. it was first recognized as a distinct gas in 1772 by joseph priestley 23 . it can be produced by vascular endothelium. it can react with another endogenous free radical, superoxide, to produce a reactive intermediate, peroxynitrite (onoo − ), which is a powerful oxidant, able to damage many biological molecules, and can decompose at acid ph to release small amounts of hydroxyl radicals. 24 onoo − + h + ∙ oh + no2 as a result of continuous blood transfusions, our patients might be subjected to peroxidative tissue injury by the secondary iron overload 4 . these finding might support the idea of iron overload in beta-thalassemia leads to an enhanced generation of reactive oxygen species and oxidative stress. iron is also an important nutritional metal for many physiological functions 25 . however, persons receiving multiple transfusions as part of the treatment for thalassemia, are faced with problem of iron overload and consequent metabolic dearrangements. under normal circumstances there is virtually no free iron. all irons are protein bound. in patients with thalassemia, the irons liberated from the haemoglobin saturate the transferrins, and the transferrins transfer the iron on to a storage iron protein called apoferritin. when the ferritin is saturated, another storage iron protein called hemosiderin is formed. 7 on this bases, iron overload, in the present study, was not created immediately until multiple blood transfusion were performed. for this reason, mda level was not altered significantly in patients after blood transfusion was achieved immediately.during the 1984, the cambridge chemist h.j.h. fenton described a reaction between iron salts and h202 that caused oxidative damage to organic molecules such as tartaric acid 26 . the fenton reaction is widely represented as in the following eqs.. fe 2+ + h2o2 fe 3+ + oh − + ∙ oh fe 3+ + h2o2 fe 2+ + ho2 ∙ + h + overall reaction iron salt + 2 h2o2 2h2o + o2 in the process of changing from the ferrous to the ferric state, an electron is transferred from iron to oxygen to make superoxide as shown in in the following eq. 5. fe 2+ + o2 fe 2+ o2 ↔ fe 3+ o2 − fe 3+ + o2 ∙ − iraqi j pharm sci, vol.18(2) 2009 antioxident in thalassemla ٥٦ therefore, the presence of iron complexes stimulate peroxidation by peroxide decomposition of unsaturated fatty acids generating alkoxyl (lo ∙ ) and peroxyl (lo2 ∙ ) radicals 27 . oxidative stress is a prominent contributor to the premature destruction of rbc as well as anemia in thalassemia. the oxidative status within red blood cells is maintained by the balance between oxidative systems, such as reactive oxygen species (ros), and antioxidative systems, like reduced glutathione (gsh) 28 . glutathione is a lowmolecular-mass, thiol-containing tripeptide, it is synthesized endogenously in the human cell, it acts to protect the body against the production of free radicals 29 . as a result, increased production of h2o2 in thalassemia major induces glutathione peroxidase, an enzyme that lead to decreased glutathione concentration 30 . on this basis, a significant decrease in serum glutathione concentration was noticed in the present work in patients with iron overload, serving as oxidative stress, versus those from non-iron overload group. during the course of metabolism, a superoxide anion is produced. normally the superoxide anion is converted by the enzyme sod to produce h2o2 31 , which in turn is converted to innocuous compounds by the action of catalase and peroxidase. but if free ferrous iron is available it reacts with h2o2 to produce hydroxyl radical which is an extremely reactive species leading to depolymerisation of polysaccharide 32 . the production of free radicals due to thalassemia was associated with a significant decrease in enzymatic antioxidants activity like sod as shown in the present study in tables (3 and 4). it can be compared with other research of şimşek, et al., 9 in which erythrocyte sod (a preventive antioxidant) levels was found to be higher in beta-thalassemic patients than healthy children. moreover, marked changes in the other antioxidant pattern were also observed in all patients. evidence is presented of a net drop in the concentration of vitamin a, vitamin c, and vitamin e in all patients with iron overload when compared with those without iron overload, as shown in tables (3 and 4). on the other hand, a significant increase of nutrient antioxidants was observed in all patients received blood transfusion in comparison with the same patient before receiving blood transfusion as shown in tables (1 and 2). as vitamin c is essential to maintain vitamin e status and function, depletion of vitamin c, in turn, contributes to further exacerbate the depletion of vitamin e 33 . although efficient antioxidants such as uric acid and bilirubin are high, they cannot compensate for lipid-soluble antioxidants, so that tissue lipid compartments are not suitably preserved. the observed depletion of serum levels of vitamin e and vitamin a can be explained by impairment of liver function and peroxidative processes causing a substantial reduction of serum lipids, producing a concurrent reduction of serum vitamin e and vitamin a 34 . a significant drop in nutrient antioxidants obtained in the present work can be compared with another research made by livrea et al., 4 who observed a significant decline in the concentration of vitamin a, vitamin c, and vitamin e in all patients affected with thalassemia due to continuous blood transfusions. another antioxidant parameter involves uric acid and albumin was included in the present study. uric acid was increased significantly in opposite to albumin which decreased significantly in patients with iron overload. similarly, livrea et al., 4 showed an increase of uric acid whereas serum albumin was in the normal range in all thalassemic patients.uric acid provides an excellent example of the adaptation of the organism to oxidative stress. it is a cellular waste product originating from the oxidation of hypoxanthine and xanthine by xanthine oxidase and dehydrogenase. high uric acid levels may provide efficient antioxidant activity for the organism. urate, the physiological state of uric acid, reacts with hydroxyl radicals producing a stable urate radical that can be regenerated by ascorbate. this compound can act with peroxyl radicals, superoxide dismutase, ozone, nitrous oxide ∙ , and other nitrogen-oxygen radicals. urate also protects protein from nitration; it can chelate metal ions, such as copper and iron, and prevent them from participating in redox cycling 35 . ceruloplasmin, is a chain breaking antioxidant, it can protect the body against the deleterious effects of oxygen free radical (ofr) 36 . the antioxidant property of ceruloplasmin is through its oxidase activity, which is directed towards ferrous ions (ferroxidase activity). it also inhibits ferrous ion stimulated lipid peroxidation and is known to be involved in the decomposition of lipid peroxides 37 . serum ceruloplasmin was significantly lower in the iron-overload group compared to the non iron-overload patients, as shown in tables (3 and 4). the inverse relationship between serum ceruloplasmin and the level of iron is indicating the antithalassemic nature of this antioxidant. conclusion these results point out that the ironinduced oxidative stress in thalassemia may play a major role in the depletion of most iraqi j pharm sci, vol.18(2) 2009 antioxident in thalassemla ٥٧ antioxidants, including lipid-soluble antioxidants. our results suggest that the measurement of peroxidation products, matched with evaluation of antioxidants, may be a simple measure of oxidative stress in thalessemia. references 1. mchiu d, kuypers f, lubin b: lipid peroxidation in human red cells. semin hematol 1989; 26: 257-276. 2. kattamis c, kattamis ac. oxidative stress disturbances in erythrocytes of βthalassemia. pediat hematol oncol 2001; 18: 85-88. 3. scott md, van 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(1), london, pp. 222-225, 553-555. 16. menden ee, boiano jm, murthy l, petering hg. "modification of phenylene diamine oxidase method to permit nonantomated ceruloplasmin determination in batches of rat serum or plasma micro samples. analytical. 1977; 10: 197-204. 17. vanuffelen be, van derzec j, dekoster bm. biochem j. 1998; 330: 719. (cited by al-zamely et al., 2001). 18. al-umary, mohammad ramzi. (1986). practical clinical chemistry, dar altikani for publishing and edition, technical institution. (in arabic) 19. varley h, gowenlock ah, bell m. “practical clinical biochemistry” 1980; vol. (1), london, pp. 222-225, 553-555. 20. steel, r.g.d. and torrie, j.h., 1980. principles and procedures of statistics. 2 nd ed. new york: mcgraw-hill book company, inc. 1960: pp.87-80, 107-109, 125-127. 21. kattamis c, kattamis ac. oxidative stress disturbances in erythrocytes of βthalassemia. pediat hematol oncol 2001; 18: 85-88. 22. tesoriero l, d’arpa d, butera d, et al. oral supplements of vitamin e improve measures of oxidative stress in plasma and reduce oxidative damage to ldl and erythrocytes in beta-thalassemia intermedia patients. free radical res 2001; 34(5): 529-540. (cited by şimşek et al., 2005) 23. moncada s, higgs ea. endogenous nitric oxide: physiology, pathology and clinical relevance (review). eur j clin invest 1991; 21: 361-374. 24. beckman js, beckman tw, chen j, marshall pa, freeman ba. apparent hydroxyl radical production by iraqi j pharm sci, vol.18(2) 2009 antioxident in thalassemla ٥٨ peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. proc natl acad sci u s a 1990; 87: 16201624. 25. conrad me. introduction: iron overloading disorder and iron regulation. seminars in hematol. 1998; 35 (1): 1-4. (cited by goswami et al., 2005). 26. fenton hjh. oxidation of tartaric acid in presence of iron. j chem soc 1984; 65: 899-909. 27. gutteridge jmc, kerry pj. detection by fluorescence of peroxides and carbonyls in samples of arachidonic acid. br j pharmacol 1982; 76: 459-61. 28. fibach e, amer j, rachmilewitz e, guy e, rivella s. oxidative stress of rbc in a murine model of beta-thalassemia can be reversed by treatment with antioxidants. am soc hematol 2005; 106: 3643. (abstract). 29. kohen r and nyska a. invited review: oxidation of biological systems: oxidativestress phenomena, antioxidants, redox reactions, and methods for their quantification. toxicol pathol 2002; 30 (6): 620–650. 30. beutler e, matsumoto f, powars d and warner j. increased glutathione peroxidase activity in alpha-thalassemia. am soc hematol 2008; 50: 647-655. from www.bloodjournal.org. 31. mccord jm. (1992). superoxide production and human disease. in jesaitis a and dratz e. (eds.) : molecular basis of oxidative damage by leukocytes. boca raton. fl. crc, pp. 225-239. 32. mccord jm. free radicals and inflammation: protection of synovial fluid by superoxide dismutase. science 1974; 185: 159-163. 33. rice-evans c, baysal e: iron-mediated oxidative stress in erythrocytes. biochem j 1987; 244: 191. 34. jordan p, brubacher d, moser u, stahelin hb, gey kf. vitamin e and vitamin a concentrations in plasma adjusted for cholesterol and triglycerides by multiple regression. clin chem 1995; 41: 924. 35. ames bn, cathcart r, schwiers e, hochstein p. uric acid provides an antioxidant defense in humans against oxidantand radical-caused aging and cancer: a hypothesis. proc natl acad sci usa 1981; 78: 6858–6862. 36. mateeseu m, chahine r, roger s, atanasiu r. protection of myocardical tissue against deleterious effects of oxygen free radicals by ceruloplasmin. arzneim forsch/drug res 1995; 45, 476480. 37. osaki s, johnson d, frieden e. the possible significance of the ferrous oxidase activity of ceruloplasmin in normal human serum. j biol chem 1966; 241: 2746-2751. . http://www.hematology.org/meetings/abstract_rights.cfm http://www.bloodjournal.org/ iraqi j pharm sci, vol.32( 1 ) 2023 salmonella typhi and o-antigen encoded genes doi: https://doi.org/10.31351/vol32iss1pp156-159 156 isolation and identification of salmonella typhi from clinical samples with molecular detection of o-antigen encoded genes huda j. *mohemmad*,1, semma h.shalal*and hadeel a.ghajiri** *department of pharmaceutical sciences, college of pharmacy, thi-qar university, iraq. **national university of science and technology, thi-qar, iraq. abstract treatment of s. typhi is difficult as compared to treatment of acute infection. antibiotic susceptibility test carried against s. typhi by using 6 kinds of antibiotics from different classes, their results showed that all isolates were high resistance to ampicillin (99%), gentamicin (98%), amikacin (79%) and less resistances to trimethoprim (55%), imipenem (60%) and ceftriaxone (66%).the present study focused on the molecular detection of wzx flippase and wzy polymerase genes in some salmonella typhi isolates, samples were collected from typhoid patients by classical lab work. antibiotics susceptibility was investigated using the disc diffusion method. the dna and molecular wzx flippase, wzy polymerase were implemented using specific primers. the results showed that there was 33.33% had wzy gene. the wzx gene did not observe in any salmonella isolates. the present study concluded that there was an importance of the genetic diversity of o-antigen encoded genes included wzx and wzy, which may be effective in typhoid diagnosis and treatment. keywords: molecular identification, wzx flippase, wzy polymerase genes salmonella typhi oمن العينات السريرية مع الكشف الجزيئي للجينات المشفرة للمستضد s. typhi عزل وتشخيص ** غجيريهديل علي و *سيماء حسن شالل ،1، *هدى جاسم محمد العراق قار، ذي ، قارجامعة ذي كلية الصيدلة، فرع العلوم الصيدالنية، * ** كلية طب االسنان، الجامعة الوطنية للعلوم والتكنولوجيا ، ذي قار، العراق . الخالصة 6باستخدام s.typhiتم تحديد المضادات الحيوية ضد . أن العالج بالمضادات الحيوية من التيفوئيد صعب مقارنة بعالج العدوى الحادة لألمبيسيلين مقاومة عالية ، وأظهرت نتائجها أن جميع العزالت كانت ذات مختلفة الجنتاميسين ( ٪99)أنواع من المضادات الحيوية من فئات ، (. ٪66)ياكسون ، سيفتر( ٪60)، إيميبينيم trimethoprim (55 ٪ )وأقل مقاومة( ٪79)، أميكاسين ( 98٪) في بعض عزالت السالمونيال التيفية ، وتم wzy polymeraseو wzx flippaseركزت الدراسة الحالية أيضا على الكشف الجزيئي لجينات . تم فحص حساسية المضادات الحيوية باستخدام طريقة االنتشار القرصي. بري التقليديت جمع العينات من مرضى التيفوئيد عن طريق العمل المخ لم . wzy٪ لديهم جين 33.33أن وأظهرت النتائج. باستخدام بادئات محددة wzy polymeraseالجزيئي ، wzx flippaseتمت دراسة وجود المتضمنة oاستنتجت الدراسة الحالية أن هناك تنوًعا جينيًا مهًما في الجينات المشفرة لمستضد . في أي من عزالت السالمونيال wzxيالحظ جين wzx وwzy والتي قد يكون لها تأثير في تشخيص التيفوئيد وأنواع العالج. بكتريا التيفوئيد , wzx , wzyلكلمات المفتاحية : التشخيص الجزيئي , جينات ا introduction salmonella typhi is an enterobacteriaceae member responsible for typhoid fever, its gram negative and serological positive to lipopolysaccharide antigens o9 and o12 in addition to polysaccharide capsular antigen vi (1,2). the diversity of antigen –o in bacterial strains regarding to the genetic polymorphisms in the rfp cluster that coded the enzymes involved in the synthesis of construction of o-antigen (3), these genes included the enzymes that contributed in sugar synthesis, transferase for construction sugar and o subunit, and genes encoded proteins involved in construction o antigen via assembly subunits, these genes included wzx and wzy that encoded to o-antigen transporter or flippase and o-antigen polymerase respectively (4,5,6). the wzx proteins in salmonella enterica the o-antigen transport mechanism is dependent on the wzx proteins (8(. the o-antigen variability which basis on the shigella serotypes schemes, there are 12 o-antigen clusters includes (2, 10, 12, 23, 25, 26, 3234, 66, 75 and 76) these clusters located between rep and aqpz genes on chromosomes (4,9). bacteria: the salmonella typhi isolates were collected from patients suffering from typhoid fever who attended to the hospital centers. after clinical and serological diagnostic tests, collected samples using conventional methods by using blood agar, xld agar and ssa agar the antibiotic susceptibility detected using disc diffusion method (ampicillin, gentamicin, amikacin, trimethoprim, imipenem and ceftriaxone). the multi-resistance isolates were used for molecular detection of wzx flippase and wzy polymerase genes. 1corresponding author e-mail: huda.jassim@utq.edu.iq received: 9/1 /2022 accepted: 10/5 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp156-159 iraqi j pharm sci, vol.32( 1 ) 2023 salmonella typhi and o-antigen encoded genes 157 pcr experiment bacterial dna was extracted from all strains using the appropriate kit. the molecular detection of wzx flippase and wzy polymerase genes were implemented using the following primers (5). wzx (f): ccg ggt ttc gat ttg tga agg ttg, (r): cac aac agc cac tac tag gca gaa wzy (f): gaa att atg cca tct tgg cga gcg (r): cat gtg aag cct gaa ggc aaa ctc the following conditions were used for pcr. 5 min 94˚c, 35 cycles consist of 30 s 94˚c, 30 s 59˚c, 30 s 72˚c, and 10 min at 72˚c for both primers in mono-plex reactions (10). pcr products were visualized in 1% agarose, tbe 0.5 x for 1 hour, 70 v and 20 ma under uv trans-illuminator. results bacterial isolation: strains of s. typhi were recovered from blood samples of outpatients confirmed by conventional method plus vitek identification (table1) table 1. bacterial isolation during the study sample no. bacteria widal test conventional and vitak 1 salmonella typhi + + 2 salmonella typhi + + 3 salmonella typhi + + 4 salmonella typhi + + 5 salmonella typhi + + 6 salmonella typhi + + 7 salmonella typhi + + 8 salmonella typhi + + 9 salmonella typhi + + antibiotic susceptibility test the results of the present study show that, there were 9 isolates were multi-resistance to antibiotics in different manner, figure1. figure 1. multi drug resistances salmonella typhi isolates (n=9) molecular detection of wzx and wzy genes genomic dna was extracted from bacterial cells using a dna extract kit. figure2. figure 2. gel electrophoresis 2% of dna extracted from salmonella typhi isolates polymerase chain reaction were applied for detection of wzx and wzy genes the molecular detection of wzx and wzy were showed that some isolates of s. typhi had wzy gene while no isolates detected that have wzx gene. figure 3. figure 3. pcr product of salmonella typhi isolates carrying wzy gene with molecular weight of 451bp visualized on 1% agarose (conditions). percentage of wzx gene was 33.33% from all samples while the percentage of presence of wzy was 0%. table 2 and figure 4. iraqi j pharm sci, vol.32( 1 ) 2023 salmonella typhi and o-antigen encoded genes 158 table 2. presence of wzy and wzx gene in different isolates of salmonella typhi. sample no. bacteria wzy gene wzx gene 1 salmonella typhi + 2 salmonella typhi 3 salmonella typhi 4 salmonella typhi + 5 salmonella typhi 6 salmonella typhi + 7 salmonella typhi 8 salmonella typhi 9 salmonella typhi figure 4. the percentage of wzy and wzx genes in salmonella typhi. discussion in the present research, the resistance to common antibiotics used in the treatment of s. typhi was tested and appeared that all the persisting isolates of s. typhi obtained from patients who suffered typhoid fever showed high resistance to gentamycin, ampicillin amikacin, and ceftriaxone; these results agree with andualem et al. and alaarajy et al. (11, 12) they were found that all isolates were resistant to four or more classes of antibiotics as antimicrobials. the present study implemented to identifywzx and wzy genes in salmonella typhi isolates that have an important role in typhoid diagnosis via o-antigen, although of the low sensitivity and specificity of widal test in typhoid diagnosis, it is still used in some labs (11), on the other hands the false negative results (13, 14) may be associated with genetic diversity of o-antigen genes which formed diversity in o-antigen proteins. the evidence found that according to the kauffmann-white scheme there are 46 somatic o antigens of salmonella, thus the diversity of o-antigens encoded genes can be used in the pcr development test to molecular serotypes identification. the negative results in the present study of wzx gene and in wxy (66.66%) maybe because of genetic diversity in target loci such as duplication, formation of the pseudogene, deletion, and insertion of the bacteriophage elements that occur ubiquitously through serogroups (15). the present finding needs more investigation to detect the genetic diversity of o-antigen in s. typhi which may have an important role in serotypes identification and development of knew molecular tests for typhoid fever diagnosis. the detection of these genes in s. typhi was poor in the last decades, yara et al (13) in a study dealing with the pangenome of salmonella o-antigens, they found high metabolic and genetic differences within and across o-antigen groups in salmonella strains. in iraq, there were high multi-resistance bacterial isolates in the last decade which contributed to the health problems of diseases infection and this need to focused efforts for developing a new method for diagnosis and treatments based on the new finding like o-antigen encoded genes. conclusion the analysis of the drug susceptibilities of the isolates was observed that all isolates were multidrug-resistant (mdr). besides, salmonella typhi is shown to persist chronically within the patients and molecular methods are more rapidly than the conventional method. the present study concluded that there was an importance of the genetic diversity of o-antigen encoded genes including wzx and wzy which may be effective in typhoid diagnosis and treatment types. references 1. falcao, d. p., trabulsi, l. r., hickman, f. w., & farmer 3rd, j. j. unusual enterobacteriaceae: lactose-positive salmonella typhimurium which is endemic in sao paulo, brazil. journal of clinical microbiology.1975;2(4):349-353. 2. ashurst jv, truong j, woodbury b. salmonella typhi.in: statpearls. treasure island (fl): statpearls publishing; jan2020. 3. tarr pi, schoening lm, yea yl, ward tr, jelacic s, et al. acquisition of therfb-gndcluster inevolution ofescherichia colio55 and o157. j bacteriol . 2000; 182: 6183 – 6191. 4. samuel g, reeves p biosynthesis of o-antigens: genes and pathways involved in nucleotidesugar precursor synthesis and o-antigen assembly. carbohydr res .2003; 338: 2503–2519. 5. patel, k. b., toh, e., fernandez, x. b., hanuszkiewicz, a., hardy, g. g., brun, y. v., bernards, m. a., & valvano, m. a. functional characterization of udpglucose:undecaprenylphosphate glucose-1-phosphate transferases of escherichia coli and caulobacter crescentus. journal of bacteriology.2012;194(10):2646– 2657. 6. vinés, e. d., marolda, c. l., balachandran, a., & valvano, m. a.. defective o-antigen iraqi j pharm sci, vol.32( 1 ) 2023 salmonella typhi and o-antigen encoded genes 159 polymerization in tola and pal mutants of escherichia coli in response to extracytoplasmic stress. journal of bacteriology.2005;187(10): 3359–3368. 7. liu, d., r. a. cole, and p. r. reeves. an oantigen processing function for wzx (rfbx): a promising candidate for o-unit flippase. j. bacteriol. . 1996 ; 178:2102-2107. 8. fitzgerald, c., sherwood, r., gheesling, l. l., brenner, f. w., & fields, p. i. molecular analysis of the rfb o antigen gene cluster of salmonella enterica serogroup o:6,14 and development of a serogroup-specific pcr assay. applied and environmental microbiology. 2003; 69 (10) , 6099–6105. 9. liu b, knirel ya, feng l, perepelov av, senchenkova sn, reeves pr, wang lstructural diversity in salmonella o antigens andits genetic basis. fems microbiol. rev. 2014;38: 56-89. 10. al-terehi m, khazaal s, muhammed h, behjet r. molecular detection of wzx1 and wzy genes in multi drugs resistance e. coli isolates. international journal of pharmaceutical quality assurance 2018; 9(3); 304-307 11. andualem, g., abebe, t., kebede, n., gebreselassie, s., mihret, a., & alemayehu, h. a comparative study of widal test with blood culture in the diagnosis of typhoid fever in febrile patients. bmc research notes.2014; 7: 653. 12. al-aarajy, a. nabeel. assessment of silver nanoparticles as antisalmonella agent: phenotypic, genotypic and histological study .thesis degree of doctorate of philosophy of college of scienceuniversity of anbar2020. 13. alobaidi ahmed flyyih; mohammed n. t.; abdulrazzaq mohammed and basim basima.molecular detection of salmonella typhi isolated from diarrheal patients in alnajaf governorate .annals of tropical medicine & public health . 2019; 22(9). 14. olopoenia la, king al. widal agglutination test 100 years later: still plagued by controversy. postgrad med j. 2000;76:80–84. 15. yara seif, jonathan m. monk, henrique machado, erol kavvas, bernhard o. palsson systems biology and pangenome of salmonella o-antigens, ecological and evolutionary science .2019; 10 (4) : e01247-19. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 ondansetron hydrochloride transdermal patches doi: https://doi.org/10.31351/vol32iss1pp147-155 147 preparation and in vitro evaluation for different types of ondansetron hydrochloride transdermal patches karima abd allatif *,1 and jameela ali hasian** department of pharmaceutical technology, damascus university, syria.  department of pharmaceutics, faculty of pharmacy, damascus university, syria. abstract this research aims to develop transdermal patches of ondansetron hydrochloride (osh) with different types of polymers as ethyl cellulose and polyvinyl pyrrolidone k30 in a ratio (3:0.5,3:1,3:2,2:1,1:1) with propylene glycol 20%w/w as a plasticizer by the solvent evaporation technique. prepared transdermal patches were evaluated for physical properties. the compatibility between the drug and excipients was studied by differential scanning calorimetry (dsc), where there is no interaction between the drug and polymers. from the statistical study, there is a statistical difference between all the prepared formulations p<0.05. in-vitro release study of transdermal patches was performed by using a paddle over the disc. the release profile of osh followed korsmeyer peppas model in p2 (ec:pvp 3:1, p4 ec:pvp 3:2) formulations and, higuchi model in p1, p3, p5 formulations. the best formulation p6 carrying ec:pvp in ratio1:1 released 96.47% of ondansetron hydrochloride during 12 hr. the release profile of p6 followed the higuchi model and correlation coefficient (r2 = 0.9815) keywords: ondansetron hydrochloride, transdermal patches, ethyl cellulose, propylene glycol, polyvinyl pyrrolidone. تحضير وتقييم في الزجاج ألنماط مختلفة من اللصاقات الجلدية من مادة األوندانسيترون هيدروكلورايد **جميلة حسيانو 1، *كريمة عبد اللطيف قسم الصيدالنيات والتكنولوجيا الصيدلية, كلية الصيدلة, جامعة دمشق . * جامعة دمشق. -كلية الصيدلة قسم الصيدالنيات والتكنولوجيا الصيدلية ** ةالخالص لتحضير البحث هذا نسب الصقات يهدف باستخدام هيدروكلورايد األوندانسيترون مادة تحوي الماتريكس نمط من من جلدية مختلفة . تم w/w%20لماء والبولي فينيل بيروليدون المحب للماء باستخدام البروبلين غليكول كملدن بنسبة المحب لغير ايتيل سيللوز إمزيج البوليميرات ل المسح الحراري تمت دراسة التوافق بين السواغات والدواء من خال. الصقات األوندانسيترون هيدروكلورايد من ناحية الخواص الفيزيائيةتقييم التفاضلي حيث ال يوجد تداخل بين البوليميرات المستخدمة والدواء. أظهرت الدراسة اإلحصائية وجود فارق إحصائي بين جميع الصيغ المحضرة عة حيث سا 12لمدة paddle over the diskتم اجراء اختبار التحرر في الزجاج باستخدام جهاز المجداف فوق القرص p<0.05 . حيث كانت الصيغ الصيغ p2, p4تان حررت وحررت , بيباس ماير كورس نمط من الصيغة p1, p3, p5الدواء كانت هيغوتشي. نمط من p6الدواء . 0.98152r=هيغوشي وكانت قيمة ساعة بنمط تحرر 12خالل من الدواء %96.47قد حررت 1:1بنسبة ec: pvpالمكونة من بروبيلين غليكول ,بولي فينيل بيروليدون, ايتيل سيللوز, اللصاقات الجلدية, نسيترون هيدروكلورايداألونداالكلمات المفتاحية : introduction chemotherapy-induced nausea and vomiting (cinv) cause unpleasant side effects that affect the quality of life in patients and their compliance to chemotherapy(1). serotonin receptor blockers are used in the treatment of nausea and vomiting caused by chemotherapy in the acute phase, which extends from 0 to 24 hr(1). ondansetron hydrochloride (osh) is a firstgeneration competitive serotonin receptor blocker used in the treatment of postoperative or chemotherapy-induced nausea and vomiting(2). orally administered of ondansetron hydrochloride (osh) undergoes extensive metabolism in the liver by cytochrome enzymes p450, which explains a short biological half-life and reduced bioavailability(3). the bioavailability of ondansetron hydrochloride is 60% after oral administration, and the half-life is 3-4 hr. ondansetron hydrochloride has a low molecular weight(293.36 da) and good transdermal penetration, so it has ideal properties to be introduced into the transdermal drug delivery system(4). transdermal therapeutic systems are defined as discrete dosage forms applied to intact skin where the drugs are delivered through the skin to the systemic circulation(5). there are three types of transdermal patches: single-layer drug-inadhesive, the adhesive layer contains the drug and is responsible for releasing the drug. reservoir, the drug layer is a liquid in a separate compartment in the form of a solution or suspension. the matrix system is a semi-solid matrix that contains the drug in the form of a solution or suspension, where the adhesive layer surrounds the drug layer(6). 1corresponding author e-mail: karimaabd523@gmail.com received: 15/2 /2022 accepted: 10/5 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp147-155 iraqi j pharm sci, vol.32( 1 ) 2023 ondansetron hydrochloride transdermal patches 148 the first generation is designed for drug delivery at low doses, the second generation has enhanced transdermal drug penetration with chemical permeability enhancers. the skin is the most common organ that receives one-third of the blood supply to all parts of the body. the skin was not known to be a systemic drug delivery route until the late twentieth century(7). transdermal drug delivery systems deliver the drug into systemic circulation at a controlled rate to maintain constant drug concentrations in the plasma for long periods(8). transdermal drug delivery systems avoid the first hepatic passage of drugs, reduce gastrointestinal side effects, and increase patient compliance(8). the first transdermal system containing scopolamine was approved in the united states in 1979; the us food and drug administration (fda) approved nicotine patches in 1984. a decade later, transdermal patches for pain relief, analgesic activity, contraception, and hormone replacement therapy were fda approved and marketed (9). materials and methods materials ondansetron hydrochloride supplied from mahrshee laboratories pvt ltd panoli, india. ethyl cellulose and polyvinyl pyrrolidone were obtained from sigma-aldrich. chloroform and propylene glycol were obtained from warehouse chemicals of the college of the pharmacy/ university of damascus. methods solubility study the solubility of ondansetron hydrochloride was determined using different ph grades from a phosphate buffer ph 5.8, 6.8, 7.4 at 37° c. a saturated solution was prepared from ondansetron hydrochloride by taking 1 g in 10 ml of each solution and placing it in a 50 ml plastic tube in a mechanical shaker for 24 hours. the solution was filtered, 1 ml was taken, and the absorbance was measured with a spectrophotometer at a wavelength of 310 nm(10,11). spectrophotometric analysis determination of λ max of ondansetron hydrochloride: the λ max was determined in phosphate buffer ph 5.8 by spectrophotometric scanning of osh solution with a spectrophotometer in the range 200-400 nm. preparation of standard solution: a stock solution of 1000 mcg/ml was prepared by taking 100 mg of the drug in a calibrated 100 ml volumetric flask and dilute it with a phosphate buffer with ph = 5.8. 1 ml was taken from the first stock solution and extended to 10 ml with a 10 ml calibrated volumetric flask to obtain a solution with a concentration of 100 mcg /ml(10), from this solution standard series with concentrations ranging from 5-30 mcg/ml was prepared and the absorption was measured with a spectrophotometer at a wavelength of 310 nm.the graph was drawn between the concentrations and the absorbance in phosphate buffer solution at ph 5.8. preparation of backing film for the transdermal patch an aqueous solution of 4% polyvinyl alcohol was prepared by taking 4g of polyvinyl alcohol and add it to 100 ml of distilled water. it was placed in a water bath at 80 c° with stirring for two hr until a clear solution was obtained. after cooling the solution, 4 ml of the solution was poured into an aluminum foil mold and dried at room temperature for 24 hr (12). figure 1. aluminum foil mold preparation of transdermal patches the polymers were weighed as in table 1, added to 10 ml of chloroform, and kept aside until the polymers were completely dissolved for 12 hr. then propylene glycol was added with stirring for 30 min, then 16 mg of osh was added with stirring for another 30 min. the polymer solution was poured over the supporting layer and dried at room temperature for 24 hr. the molds were covered with inverted glass funnels to control the evaporation of the solvent(13). table 1. formulations of osh transdermal patches p1 p2 p3 p4 p5 p6 ec 0.3g 0.3g 0.3g 0.3g 0.3g 0.3g pvp 0.05g 0.1g 0.15g 0.2g 0.25g 0.3g pg 0.067ml 0.076ml 0.086ml 0.096ml 0.10ml 0.11ml osh 16mg 16mg 16mg 16mg 16mg 16mg solvent chloroform chloroform chloroform chloroform chloroform chloroform up to 10ml up to 10ml up to 10ml up to 10ml up to 10ml up to 10ml *ec-ethyl cellulose; pvp-polyvinyl pyrrolidone; osh-ondansetron hydrochloride; pg-propylene glycol. iraqi j pharm sci, vol.32( 1 ) 2023 ondansetron hydrochloride transdermal patches 149 evaluation of transdermal patches physical appearance the prepared transdermal patches were evaluated for morphology, transparency and, flexibility of the film(14). folding endurance three transdermal patches were evaluated, so the patch was folded into a specific piece several times until it broke. foldability is determined by the number of times the patch is folded without breaking(15). uniformity of weight ten transdermal patches were weighed from each formulation individually on a sensitive scale and the standard deviation from the mean was calculated(16). the thickness of the patch the thickness of the film was determined by using a micrometer screw gauge device at several points of the film, then the average readings and standard deviation were calculated(17). moisture uptake(14) three transdermal patches were selected from each formulation. each patch was weighed before starting the test and then placed in a desiccator containing a saturated solution of potassium chloride, which gives relative humidity of 84% w/w. the weight of the patches was then taken for 3 days, then the uptake percentage was calculated from the following relationship: m%= [final weight – initial weight / initial weight] × 100. moisture content(18) three transdermal patches were selected from each formulation, where each patch was weighed before the start of the test, then it was placed in a desiccator containing activated calcium chloride. the weight of the patches was taken for 3 days and moisture content was calculated from the relationship: m%= [initial weight – final weight / final weight] × 100. drug content determination: one cm2 of the film was weighed and dissolved in 10 ml of mixture of methanol and ethanol, then filled up with phosphate buffer solution to 100 ml. it was stirred for 24 hr, then the solution was filtered and the absorbance was measured at wavelength 310 nm maximum absorbance. the drug concentration was determined by the absorption equation from the standard graph(19). in vitro release study: the test was performed using a usp apparatus v (paddle over the disc). 500 ml of phosphate buffer solution was used at ph = 5.8, the device was set at a temperature of 32 + 0.5 ° c and a paddle speed of 50 rpm. the film was fixed on a circular glass disc by a stainless steel mesh and stainless steel clips then placed into the dissolution medium, so that it was 2.5 cm away from the paddle according to the requirements of the american pharmacopeia. 5 ml were withdrawn after the following intervals of 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12 hr and replaced with phosphate buffer ph=5.8. the absorbance of samples was then measured using a spectrophotometer at wavelength 310 nm(20). figure 2. fixed transdermal patch figure 3. paddle over the disc statistical analysis the statistical study was performed using a one-way anova analysis through the statistical analysis program graph pad prism 9 to study the effect of polymer and its percentage on the release of the drug. after applying the test, the p-value was calculated and the results were interpreted. post-hoc tukey-hsd (honestly significant difference) test was performed where p< 0.05. differential scanning calorimetry (dsc) study thermal differential scanning of ondansetron hydrochloride, ethyl cellulose, polyvinyl pyrrolidone and, the physical mixture of the drug with the excipients was performed to determine any incompatibility between them. a device (dsc131, sitaram, france) was used, and the samples taken with a quantity of 5 mg were placed in an open aluminum chamber against iraqi j pharm sci, vol.32( 1 ) 2023 ondansetron hydrochloride transdermal patches 150 another aluminum chamber used as a reference, then the temperature was raised from (25 to350) c° at a rate of 10 degrees per min(21). results and discussions solubility study ondansetron was soluble within the pbs buffer ph 5.6. the solubility results are shown in table 2. table 2. solubility of ondansetron hydrochloride. n=3 the solubility of ondansetron hydrochloride exhibits high solubility at low ph (ph 5.8, this degree is considered close to the skin’s ph so that the patch does not cause irritation to the skin ) at 37°c because of ondansetron hcl is a weakly basic drug belongs to bcs class-ii and it is showing distinct ph-dependent solubility ; however, it exhibits poor solubility at higher ph (6.8 ph phosphate buffer) at 37°c. these results were parallel with the findings of anilkumar et al where the solubility in phosphate buffer ph=6.8 was found to 36 µg/ml (22) . spectrophotometric analysis standard graph of osh in pbs ph 5.8 the estimated λ max is 310nm that's agreed with the reported values figure. 4. figure 5 showed the calibration curve within the involved media.the standard graph showed good linearity with r2 =0.999 which indicates that it obeys “beerlambert’s” law. figure 4. λ max of osh in phosphate buffer ph=5.8 figure 5. standard curve of osh in phosphate buffer ph=5.8 evaluation prepared transdermal patches a smooth, non-broken and, thin matrix was obtained. table 4 shows the results of physical tests for the prepared formulations, where the results of the foldability test ranged between (22100) times(4), which indicates that the use of propylene glycol as a plasticizer gave the unbroken film. for the weight test, there is no specific constitutional value for weights of transdermal patches. the weights of the patches ranged between (0.32-0.54) g, and we notice a small difference in the standard deviation values ranging between (0.003-0.01), which indicates uniformity of weight. for drug content uniformity test, results were found in the range (92-99) %, which indicates a homogeneous distribution of the drug within the patch. table 3. physical evaluation of ondansetron hydrochloride films folding endurance ±sd thickness (mm) ±sd drug content (%)±sd weight variation (g) ±sd patch code 22±2.16 0.1mm 98.49±0.7 0.32±0.004 p1 26±1.24 0.2mm 94.55±0.95 0.433±0.004 p2 46±1.24 0.2mm 97.89±0.24 0.443±0.012 p3 71±1.35 0.3mm 92.88±1.02 0.495±0.005 p4 97±2.05 0.3mm 96.67±0.56 0.545±0.003 p5 106±1 0.3mm 99.21±1.78 0.565±0.007 p6 n=3, sd= standard deviation. solubility(µg/ml) time duration(hr) solubility medium 515±0.01 µg/ml 24 phosphate buffer ph=5.8 23±0.12 µg/ml 24 phosphate buffer ph=6.8 insoluble 24 phosphate buffer ph=7.4 iraqi j pharm sci, vol.32( 1 ) 2023 ondansetron hydrochloride transdermal patches 151 moisture uptake and moisture content table 5 shows the results of the moisture uptake test for all prepared formulations. the formula p1 is the lowest among all formulations due to the decrease in the amount of pvp in the formula, which amounted to 3.29±0.05, while the moisture uptake percentage increases with the increase in the amount of pvp in the formula, and this is explained by its hygroscopic nature. in addition, the plasticizer works to separate the polymer chains from each other, which facilitates the absorption of water into the matrix, these results are in agreement with sheba et al where pg containing patches have higher percentage of moisture content and water absorption (> 1)(3). the moisture content test showed an increase in moisture content with the increase in the amount of pvp in the formula. p6 gave the largest value of 6.82±0.18 after72 hours because it contained the largest amount of pvp unlike formula p1, which has the lowest value. table 4. moisture uptake and moisture content of osh films. patch code moisture uptake 72hr±sd moisture content 72hr±sd p1 3.29±0.05 4.45±0.16 p2 5.21±0.48 4.83±0.05 p3 5.6±0.19 5.14±1 p4 6.17±0.15 5.52±1.82 p5 7.01±0.13 6.7±1.82 p6 7.21±0.48 6.82±0.18 *72 h: test after 72 hr, sd= standard deviation. in vitro release study the cumulative % of ondansetron hydrochloride release were shown in table 5, and release curves of ondansetron hydrochloride were shown in figure 6. there is an initial release from all formulations after 30 min and it lasted for 12 hr. the release of the drug from formulations p1-p6 gradually increases with the increase in the amount of pvp while the amount of ec remains constant, this is explained by the hydrophilic nature of pvp which facilitates absorption of water to the matrix enhances the release of the drug, in addition, the pvp works to form pores in the matrix structure that facilitates drug particles leave to the dissolving medium(3). furthermore, the presence of pvp in patches with chloroform as casting solvent reduce the crystalline state of the drug, which leads to an increase in drug release. these results are in agreement with mutalic et al where the formulation with ec:pvp (3:2) exhibited the greatest percentage of drug release value (71.25 %±8.55) compared to the lowest value observed with the formulation containing ec:pvp (4.5:0.5) (47.65%±9.55)(23). . formulation p6 gave the highest release rate when using ec and pvp with a ratio of 1:1. oneway anova analysis produced statistically significant differences among all formulations where p=0.001, (p<0.05). table 5. in vitro drug release from p1 to p6. time hr p1 p2 p3 p4 p5 p6 0 0 0 0 0 0 0 0.5 16.2±0.75 24.25±0.68 35.9±0.75 33.46±0.12 34.31±0.14 32.92±0.39 1 20.23±1.97 28.8±0.21 41.73±1.09 41.4±0.02 42.53±0.47 37.40±0.02 2 29.34±0.96 34.71±1.72 56.44±0.16 46.84±0.23 52.09±0.66 47.30±0.19 3 34.33±0.11 37.46±1.28 57.11±0.35 48.53±0.67 59.85±0.09 54.55±0.96 4 37.01±0.18 41.77±0.18 59.63±0.82 52.86±0.82 63.24±0.31 60.77±0.1 5 40.01±0.42 44.1±0.48 60.01±0.55 55.36±0.77 66.06±0.45 66.23±0.56 6 41.47±1.29 45.17±1.31 60.61±0.35 56.93±0.43 67.56±0.37 69.79±0.54 7 41.96±0.91 47.39±0.68 60.97±0.55 58.51±0.14 68.65±0.96 74.03±0.03 8 45.94±0.27 51.77±0.79 63.31±0.67 63.79±0.37 73.25±0.85 79.02±0.35 10 50.15±0.76 59.4±0.68 64.94±0.79 73.45±0.36 80.1±0,45 88.81±0.39 12 53.93±0.47 65.82±0.77 68.72±0.63 83.21±0.10 87.18±0.83 96.47±0.23 n=3, mean ±sd= standard deviation. iraqi j pharm sci, vol.32( 1 ) 2023 ondansetron hydrochloride transdermal patches 152 figure 6. release curves of ondansetron hydrochloride films. kinetic modeling of drug release several models describe the kinetics of drug release from the pharmaceutical form, such as korsmeyerpeppas, higuchi, zero order and first order. table 7 shows the value of correlation coefficient r2 for the release models of zero order, first order, higuchi and korsmeyerpeppas. the formulations (p2, p4) gave a release from the korsmeyerpeppas model, where the value of the correlation coefficient r2 was maximum and the value of the exponential coefficient n was calculated, it was less than 0.5, this indicates that the mechanism of drug release is diffusion from a non swellable matrix. the formulations (p1, p3, p5, p6) also gave release from the higuchi model, where the value of the correlation coefficient was greater compared to the rest of the types, that is, the release of the drug from the matrix is proportional to the square root of the time. it also indicates that the process of diffusion through the matrix is the main mechanism to release the drug. table 6. kinetics study of prepared transdermal patches korsmeyer peppas higuchi first order zero order patch code r2 n r2 slope r2 slope r2 slope 0.9167 0.1772 0.972 14.689 0.861 0.0454 0.8205 3.6506 p1 0.9576 0.0381 0.954 16.222 0.9301 0.0336 0.8315 4.0967 p2 0.7154 0.0217 0.7772 16.069 0.7154 0.0217 0.5299 3.5891 p3 0.9447 0.0325 0.9142 19.264 0.9447 0.0325 0.7808 4.8155 p4 0.8835 0.0338 0.9269 21.72 0.8835 0.0338 0.7443 5.2648 p5 0.9555 0.0439 0.9815 25.449 0.9555 0.0439 0.8636 6.4571 p6 table 7.value of n and drug release mechanism(24). release exponent (n) drug transport mechanism rate as a function of time drug release mechanism n< 0.5 quasi – fickian diffusion tn non swellable matrix diffusion 0.5 fickian diffusion t0.5 0.5 < n < 1.0 anomalous (non -fickian transport) tn-1 for both diffusion and relaxation ( erosion ) 1 case ii transport (time – independent) zero order release higher than 1 super case ii transport tn-1 (relaxation / erosion ) differential scanning calorimetry (dsc) study the dsc thermogram of ondansetron hcl showed a sharp endothermic peak at 187.47c with a starting point of 180.97c and an endpoint of 240.06c as shown in figure7a which corresponds to the melting point of the substance and indicates that the substance is pure and has a crystalline shape. the dsc thermograms of ethyl cellulose and pvp k30 showed endothermic peaks at 348.66°c as shown in figure 7b and 87.04°c as shown in figure 7c, respectively. the dsc thermograms of physical mixture osh +pvpk30 as shown in figure 7d and physical mixture of osh+ ec as shown in figure 7e, showed endothermic peaks separately, not overlapping with each other and without changing the melting point of the active substance, which indicates that there is no interaction between ondansetron and used excipients. iraqi j pharm sci, vol.32( 1 ) 2023 ondansetron hydrochloride transdermal patches 153 figure 7a. dsc of ondansetron hydrochloride. figure 7b. dsc of pvp k30 figure 7c. dsc of ec -25 -20 -15 -10 -5 0 5 10 15 0 50 100 150 200 250 300 350 400 450 temperture/cº onset point:64.87cº endset point:139.04cº peak 1 top: 100.90cº enthalpy / j/g :293.97 onset point:182.12cº endset point:231.33cº peak 1 top: 187.47cº enthalpy / j/g :153.7799 -12 -10 -8 -6 -4 -2 0 0 50 100 150 200 250 300 350 400 450 temperture/cº onset point:43.09cº endset point:125.45cº peak 1 top: 87.04cº enthalpy / j/g :336.1625 -6 -5 -4 -3 -2 -1 0 0 50 100 150 200 250 300 350 400 450 temperture/cº onset point:28.30cº endset point:88.83cº peak 1 top: 53.79cº enthalpy / j/g :29.7998 onset point:272.50cº endset point:301.24cº peak 1 top: 286.38cº enthalpy / j/g :31.2577 onset point:218.25cº endset point:249.06cº peak 1 top: 237.51cº enthalpy / j/g :17.5069 iraqi j pharm sci, vol.32( 1 ) 2023 ondansetron hydrochloride transdermal patches 154 figure 7d. dsc of osh+pvp k30. figure 7e. dsc of osh+ec conclusion ondansetron hydrochloride transdermal patches were prepared using two different types of polymers, polyvinyl pyrrolidone and, ethyl cellulose . formula p6 gave the highest release rate among the prepared formulas, reaching to 96% w/v (cumulative drug release) after 12 hr. the release kinetics of all formulations were studied and the p6 formula followed the higuchi model. formula p6 can be nominated for further studies in animal models and pharmacokinetic studies. references 1. smith c, smith m, cunningham r, davis s. recent advances in antiemetics: new formulations of 5-ht3 receptor antagonists in adults. cancer nurs. 2020;43(4):e217–28. 2. huddart r, altman rb, klein te. pharmgkb summary: ondansetron and tropisetron pathways, pharmacokinetics and pharmacodynamics. pharmacogenet genomics. 2019;29(4):91–7. 3. david srn, rajabalaya r, zhia es. development and in vitro evaluation of selfadhesive matrix-type transdermal delivery system of ondansetron hydrochloride. trop j pharm res. 2015;14(2):211–8. 4. bhardwaj s, bhatia s, singh s. transdermal delivery of ondansetron hydrochloride in the management of hyperemesis gravidarum. int j pharma sci res. 2020;11(8):206–14. 5. shayeda d, ayesha n. development of tizanidine hcl transdermal patches: in-vitro and ex-vivo characterization. j drug deliv ther. 2019;9(1-s):295–300. 6. saeedi 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concepts and applications. int j pharm res technol. 2019;8(1):12–20. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 targeting of epithelial to mesenchymal transition &cell cycle in mcrpc by the fxr & fmd doi: https://doi.org/10.31351/vol32iss1pp115-124 115 role of fasting mimicking diet in farnesoid x receptor for suppressing epithelial-to-mesenchymal transition, cell cycle progression, and viability of prostate cancer cells wrood s. al-khfajy, inam sameh arif, basma t. al-sudani *department of pharmacology and toxicology, college of pharmacy, mustansiriyah university, baghdad. iraq abstract the systemic and resistant nature of metastatic castration-resistant prostate cancers (mcrpc) renders it largely incurable even after intensive multimodal therapy. proliferation, survival, and epithelial-mesenchymal transition (emt) are three fundamental events that are deeply linked to carcinogenesis. hence, it is necessary to find a new combination of several therapies, targeting those vital mechanisms without causing side effects. significant research works have shown differential low expression of the metabolic farnesoid x receptor (fxr) in primary and metastatic prostate cancer suggesting their importance in prostate pathogenesis. obticholic acid (int 747), a potent fxr agonist is widely used in primary biliary cholangitis, and fasting mimicking diet (fmd) both were drastically showed effects on different cancer progression. the purposes of the present study were to test the hypothesis that fxr agonist int 747 is a novel therapeutic target for prostate cancer progression and metastasis. besides, to assess the synergistic effects of fasting-mimicking diet with int 747 on pc-3 cells. in the present work, the anti-proliferative and cytotoxic effects of fxr and fmd were analyzed by the mtt, colony formation and tumor spheroidal assays. while, the cell cycle distribution was performed by using fluorescent dyes propidium iodide (pi) and then analyzed by flow cytometry, the nuclear morphology of apoptotic cells stained with crystal violet were detected by phase contrast microscopy. besides, this study conducted a series of in vitro experiments to investigate most of the biological phenotype steps involved in the metastasis such as wound healing (scratch) assay and matrigel invasion assay. finally, this work analyzed gene and protein expression by rt-qpcr and western blotting to elucidate how int 747 and /or fmd functions in prostate cancer cell line. the results of the present study showed that int 747 treatment caused apoptotic morphological changes and significantly reduced the survival of human prostate cancer cell line (pc-3) cells incubated in normal medium. furthermore, this study showed that the combination of the int 747 and fmd was much more harmful to cancer cells than the treatment with int 747 or fmd alone. moreover, our study showed that int 747 either alone or combined with fmd robustly induced cell cycle arrest at the g1 phase. interestingly, the combination treatment on human prostate cancer cell line (pc-3) cells not only showed several lines of evidence of apoptotic cells death, but also inhibited carcinogenic potential and metastasis capacity as evaluated by impairment of spheroid formation capacity, delayed wound healing and matrigel invasion assays. mechanistically, fxr agonism was also capable of withdrawing the molecular variations associated with epithelial to –mesenchymal transition, which a crucial mechanism governing cancer cell migration and invasion, as was showed by reduced vimentin (a mesenchymal marker) expression. this anti carcinogenic effect facilitated by fxr was also complemented by decreased mrna gene expression of matrix metallopeptidase 9 (mmp-9), which destroy the extracellular matrix. furthermore, fxr activation resulted in proteolytic activation of procaspase -3 protein. the present study further revealed that fmd alone could restrained growth and metastasis of androgen refractory prostate cancer cells, but when combined with fxr synergistically augmented all the anti-cancer effects of fxr against these cells. in summary, our findings suggest that activation of fxr and improving its function through fmd could be a hopeful treatment option for mcrpc. keywords: fxr, fmd, epithelial-mesenchymal transition, cell cycle, migration, invasion. االنتقال لقمع مستقبالت فارنيسويد إكس األيضيمفاعل زيعزلت للصيام المحاكي الغذائي لنظامادور الخبيث البروستاتا رطانالس ورمواالنتشار وتطور دورة الخاليا في * بسمة طالب السوداني , *عارف انعام سامح , 1* الخفاجي ورود سالم فرع االدوية والسموم ، كلية الصيدلة ، الجامعة المستنصريه ، بغداد ، العراق * الخالصة غير قابل للشفاء إلى حد كبير حتى بعد العالج المكثف بالعقاقير المتعددة. يعد االنتشار هو احد أنواع السرطان ال سرطان البروستات المقاوم ات تستهدف والبقاء واالنتقال الثانوي ثالثة أحداث أساسية ترتبط ارتباًطا وثيقًا بالسرطنة. وبالتالي، من الضروري إيجاد مزيج جديد من عدة عالج جانبية. أظهرت األعمال البحثية الهامة انخفاض التعبير التفاضلي لمستقبالت فارنيسويد إكس األيضي في تلك اآلليات الحيوية دون التسبب في آثار 1corresponding author e-mail: pharm.wroodsalim@uomustansiriyah.edu.iq received: 6/ 2/ 2022 accepted: 19/4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp115-124 iraqi j pharm sci, vol.32( 1 ) 2023 targeting of epithelial to mesenchymal transition &cell cycle in mcrpc by the fxr & fmd 116 وهو أحد منبهات القوية ك، البروستات األولي والثانوي مما يشير إلى أهميتها في التسبب في أمراض البروستات. يستخدم حمض االوبتي كولسرطان ان لمستقبالت فارنيسويد إكس األيضي ويستعمل على نطاق واسع في التهاب األقنية الصفراوية األولي. اضافة الى لك قد أظهرت دراسات متعددة على تطور السرطان المختلف. في هذه الدراسة، افترضنا أن لمستقبالت فارنيسويد إكس األي النظ كبيًرا له تأثيًرا ضي ام الغذائي المحاكي للصيام لخاليا، و اوالنظام الغذائي المحاكي للصيام قد يمنعان االنتشار والنمط الظاهري النقلي في خاليا سرطان البروستات. تم إجراء تحليالت لتكاثر ونم و/ أو حمض االوبتي كولكوتوزيع دورة الخلية، ومقاسات الهجرة والغزو، وتحليل التعبير الجيني والبروتيني في هذا العمل لتوضيح كيفية وظائف لوجيا سببها ادى الى تغيرات مورفو حمض االوبتي كولكالنظام الغذائي المحاكي للصيام في خط خاليا سرطان البروستات. هذه الدراسة كشفت ان ظهرنا موت الخاليا المبرمج وقلل بشكل كبير من بقاء خاليا خط خاليا سرطان البروستات البشرية المحتضنة في الوسط الطبيعي. عالوة على ذلك، أ ذلك، أظهرت دراستنا كان أكثر ضرًرا للخاليا السرطانية من العالج مفردا. وإضافة الىحمض االوبتي كولك والنظام المحاكي للصيام أن الجمع بين . ومن المثير لالهتمام، صناعة الحمض النوويكان إما بمفرده أو مدمًجا سبب لتوقف دورة الخلية المستحث بقوة في المرحلة حمض االوبتي كولكأن يا المبرمج، بل أدى أيًضا أن العالج المركب على خاليا خط خاليا سرطان البروستات البشرية لم يُظهر فقط عدة خطوط من األدلة على موت الخال الخاليا حركة وتأخر السرطان، كرويات تكوين قدرة ضعف خالل من تقييمه تم كما االنبثاث على والقدرة السرطان انتشار إمكانية تثبيط إلى يث والمضادة للبقاء من خالل تقليل وفحوصات االنتشار الخلوي والغزو السرطاني. ميكانيكيًا، تم التوسط في الغالب في التأثيرات المضادة للورم الخب . عالوة على ذلك، أدى إلى تنشيط بروتين بروكاسباس . باختصار، تشير النتائج التي الفايمنتينوبروتين المعدني البروتين صفوفةتعبير كال من م ًرا توصلنا إليها إلى أن تنشيط مستقبالت فارنيسويد إكس األيضي وتحسين وظيفته من خالل مرض النظام المحاكي للصيام بانه يمكن أن يكون خيا .سرطان البروستات النقلي والمقاومعالجيًا مأمواًل لـ توقف دورة الخلية،تغيير نمط الخلية ،النظام المحاكي للصيام،مستقبالت فارنيسويد إكس األيضي الكلمات المفتاحية: introduction prostate cancer (pca) is the most prevalent invasive cancer in men worldwide and the secondleading cause of cancer-related death among men (1). in 2020, there were 1,898,160 cases and 608,570 deaths globally. the androgen-dependent stage of prostate cancer may be effectively cured with androgen-deprivation therapy (adt)(2). however, prostate cancer mortality happens due to reversion from androgen-dependent to androgen-independent prostatic growth (3). as a result, the prostate cancer changed to metastatic castration-resistant prostate cancer (mcrpc) associated with high mortality risk, and short survival of only 16–18 months (4). failure of therapy in the androgen-independent is due to metastasis to adjacent tissues and chemo resistance of cancer cells. based on that, urgent research is needed to explore new efficient therapeutic agents interfering with novel signaling pathways during the treatment of mcrpc(3, 5). the epithelial-mesenchymal transition (emt) has been identified as a complex molecular and cell process involved in tissue reconstruction that plays essential roles in cell invasion, migration, and chemoresistance in many cancer types including prostate cancer(6). during emt, cells undergo transformation from epithelial to mesenchymal state (4). the acquired mesenchymal features are cell mobility, invasiveness, acquiring stem cell characteristics, and protective resistance to apoptosis (7). obticholic acid (int 747), a potent fxr agonist is widely used in primary biliary cholangitis, can inhibit different diseases disorders by downregulation or upregulation of transcription of its target genes such as shp , oatp , srebp-1c , pparα , pepck and glucose-6-phosphatase which play an important role in metabolism (8, 9). furthermore, many epidemiological studies have shown that the use of int 747 is associated with a reduction in the incidence of different types of cancers. most notably in cholangiocarcinoma, hepatocellular cancer and colon cancer (10-12). however, whether there is activity against prostate cancer remains to be shown. fasting mimicking diet has fewer adverse effects, but the same benefits as traditional fasting, such as a drop in insulin-like growthffactor-1 (igf1) levels (13). fmd has been shown to improve the therapeutic efficacy of anticancer drugs. (14, 15). moreover, previous studies showed that chronic caloric restriction reduces and delays cancer incidence, and inhibits tumor progression and metastasis (16). consequently, our aim in this study is to assess the potential synergy of the treatment of int 747 and fmd and determination their potential impact on proliferation, survival, and metastasis of castration -resistant prostate cancer cell line. this research evaluated theses potential effects in vitro by the cell viability assay, spheroid formation assay, morphological assay, flow cytometry assay, and migration and invasion assays in prostate cancer cells. materials and methods chemicals and their sources obeticholic acid (int 747) was obtained from medchemexpress (south brunswick, nj)). all chemicals used were of analytical grade. cell culture and fasting-mimicking condition in this study pc-3 (human adenocarcinoma prostate cancer cells) was used as a model for mcrpc that is not caused by androgen (17). compared to other models of prostate cancer cell lineage, pc3 cells have a high metastatic potential (17). human pc-3 cells purchased from the atcc, usa. pc-3 cells were cultured in various media containing penicillin and streptomycin and kept at 37 degrees celsius in a humidified environment containing 5% co2. the control media (control) consisted of 2g/l glucose rpmi 1640 medium (euroclone, italy) supplemented with 10% fetal bovine serum (fbs) and dmso (sigma-aldrich). fasting was emulated by incubating cells in this medium for 48 h, as described elsewhere, using glucose-free rpmi 140 (euroclone, italy) iraqi j pharm sci, vol.32( 1 ) 2023 117 supplemented with glucose 0.5 g / l (santa cruz, ca) and 1% fbs. (di tano et al. (2020). cell viability assay pc-3 cell lines were seeded in 96-well culture plates and incubated overnight at 37°c in a humidified incubator. int-747 was purchased from medical chemical express. cells were treated with various concentrations of int 747 for 24, 48, and 72 hours or left untreated in the control group. int 747 (was added either alone or in combination with fmd. the number of viable cells was determined by mtt assay using promega celltiter 96™ nonradioactive cell proliferation assay (mtt). absorbance was measured by using plate reader. the optical density(od) of 530 and 630 nm. the percentage of cell viability was calculated as the following equation as described , where od is the measured optical density at 530 and 630 nm (18): 𝑪𝒆𝒍𝒍 𝒗𝒊𝒂𝒃𝒊𝒍𝒊𝒕𝒚 = 𝑶𝑫𝟓𝟑𝟎,𝟔𝟑𝟎 (𝒔𝒂𝒎𝒑𝒍𝒆) 𝑶𝑫𝟓𝟑𝟎,𝟔𝟑𝟎 (𝒄𝒐𝒏𝒕𝒓𝒐𝒍) × 𝟏𝟎𝟎. cell morphological analysis to visualize morphological changes, pc-3 cell lines at a density of 2.0 × 105 were seeded into sterilized coverslip mounted in a 6-well plate and exposed to int 747 treatment with or without fmd for 48 h. subsequently, the cells were washed with cold pbs and then fixed in 1% formaldehyde in dpbs for 10 minutes at room temperature. next, the cells were stained with a mixture of 0.5% crystal violet in 20% methanol for 30 minutes. then, the cells were washed two times with water and visualized using a phase-contrast microscope optika, im-3 (italy) at 400× total magnification. cell cycle analysis pc-3 was exposed to int 747 treatment (8 μm) with or without fmd for 48 h. next, the cells were trypsinzed and recoverd by centrifuging at 1000×g for 5 min. then, the cells were washed by dpbs, fixed in 70% ethanol at 4 °c overnight and subsequently re-suspended in 400μl buffer containing pi and rnase for 30 min. the rates of cell cycle distribution were determined by flow cytometry (bd facsversetm) and the pattern of cell distribution was determined using, bd facsdiva™ software (usa). in vitro assessment of cell migration and wound closure scratch assay it is an in vitro methodology widely used to estimate the healing capacity of different drugs(19). in the current study, 150,000 cells/well of pc-3 cells were seeded in 24-well tissue culture plates at 37°c and 5% co2 for 48 h. once the cells were a nearly confluent cell monolayer, the culture medium was removed, and a wound was created using a sterile 200 μl tip and the debris was removed by washing with pbs. next, int 747 and/or fmd were added to corresponding wells then incubated for 48 h. the cell migration and wound closure were detected at diverse time intervals under an inverted microscope, and a digital camera captured images. the space area caused by cell migration at different time intervals was measured with the imagej software. the rate of wound closure was expressed as the ratio of wound closure (the wound area at 0 hours is set to 100%) and it measured through the following equation (20): 𝐖𝐨𝐮𝐧𝐝 𝐜𝐥𝐨𝐬𝐮𝐫𝐞 (%) = (𝐖𝐨𝐮𝐧𝐝 𝐚𝐫𝐞𝐚 𝐚𝐭 𝟎 𝐡 – 𝐖𝐨𝐮𝐧𝐝 𝐚𝐫𝐞𝐚 𝐚𝐭 𝐭𝐢𝐦𝐞) 𝐖𝐨𝐮𝐧𝐝 𝐚𝐫𝐞𝐚 𝐚𝐭 𝟎 𝐡) × 𝟏𝟎𝟎 matrigel invasion assay to investigate the effects of int 747 and/ or fmd on invasiveness’ features of pc 3 cells, cytoselect™ 24-well cell invasion assay (cba100-c, cell biolabs, ca, usa) was used. a total of 300,000 cells/well (300 μl/well) suspended in either normal or fmd medium were added to the upper chamber, and 500 µl rpmi 1640 supplemented with 10% fbs was added to the lower chamber. cells were immediately treated with the indicated concentration of int 747 and incubated at 37°c. cells were allowed to invade for 48h. the medium of the insert was aspirated and non-invasive cells were removed with a cotton-tipped swab. cells were stained with cell stain solution, rinsed in dh2o, and dried. invaded cells were visualized and counted from five random fields with an inverted microscope (optika, im-3, italy). then, the optical densitometry was measured at a wavelength of 530 nm using a microplate reader. the cell images were analyzed using imagej software (imagej software, nih, usa). tumor sphere formation assay spheroids were produced using a previously described agarose-overlay method (21, 22). in short, a non-adhesive agarose plate was prepared by solidifying agarose solution (0.5% agar dissolves in a complete culture medium) into 24 well cell culture plates. next, the pc-3 cells were suspended in the indicated media (normal or fmd) at a density of 50 cells/well and treated with int 747 where indicated. then the cell suspension was added to the solid agarose and incubated at 37 c at 5% co2. after 10 days of culture, tumor-spheroids were visualized, counted, and sized using light microscopy. the results of the tumorsphere formation assay were displayed as a relative fold decrease in size of tumorspheres formed compared to control and analyzed by imagej software (21). quantitative real-time pcr (qrt-pcr) the rneasy micro kit (qiagen) was used to extract total rna from cells that had been treated or not treated with int 747 for 48 hours, according to the manufacturer's recommendations. 1 g rna was applied to each sample for cdna synthesis using the easyscript one-step gdna removal and cdna synthesis supermix kit, as directed by the manufacturer. the perfectstart green qpcr iraqi j pharm sci, vol.32( 1 ) 2023 118 supermix was used for the amplification stage in quantitative rt-pcr. all reactions were carried out.the rneasy micro kit (qiagen) was used to extract total rna from cells that had been treated or not treated with int 747 for 48 hours, according to the manufacturer's recommendations. 1 g rna was applied to each sample for cdna synthesis using the easyscript one-step gdna removal and cdna synthesis supermix kit, as directed by the manufacturer. the perfectstart green qpcr supermix was used for the amplification stage in quantitative rt-pcr. all reactions were carried out in duplicate using the particular primers specified below, and all results were standardized to gapdh, the housekeeping gene. table.1 sequences of primers used in qpcr pcr primers forward reverse mmp9 ttgacagcgacaagaagtgg gccattcacgtcgtccttat gapdh tgcac caccaactgcttagc ggcatgga ctgtggtcatgag western blots the pc-3cells were lysed with ripa lysis buffer, complemented by a cocktail of protease and phosphate inhibitors. (elabscience). next, the cell lysates (30µg protein per well) in 5× loading buffer were resolved by 12% sds-page and transferred on polyvinylidene fluoride membranes (pvdf). after blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary rabbit antibodies against gapdh (1:2000, elabscience), vimentin (1:1000, elabscience), procaspase-3 (1:1000, elabscience) overnight at 4 °c. the primary antibodies were detected with peroxidaseconjugated goat anti-rabbit igg (h + l) secondary antibody (1: 5000). the blots were incubated with enhanced chemiluminescence substrate (elabscience) and the signals were quantified using a bio-rad western blot analysis system (chemdoc xrs plus, bio-rad, usa) with imagelab software (bio-rad laboratories). statistical analysis the data are presented as means with standard deviations (s.d.). microsoft excel and graphpad prism 7 were used to conduct statistical analyses (graphpad software inc., san diego, ca, usa). the student's t-test or one-way anova was used to compare the differences between the experimental and control groups, and statistically significant differences are indicated by asterisks as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001. results fxr activation and fmd inhibits pc-3 viability and induce apoptotic morphological changes the effect of fxr and fmd on the growth and proliferation of prostate cancer cells was measured. in this study, pc-3 cells were treated by int 747(8μm) in either, normal or fasting media conditions for 48 h of incubation, then cell viability was determined by mtt assay. as shown in (figure 1. a), int 747 reduced the degree of cell viability in the aggressive prostate cancer cells (pc-3), as compared to the corresponding control. in pc 3 cells treated with int 747 in normal media (ncm), the cell viability was 67.17± 0.89, compared to untreated control cells. interestingly, the incubation of pc-3 cells with int 747 in fmd had a more noticeable effect (about a 3-fold decrease) on cell viability (34.9±0.32). these effects are visible by inverted microscopy (figure 1b). although cell shape and number are influenced by the int 747 or fmd used only, combined treatment is much more powerful. the apoptotic phenotype included rounded cells with cytoplasmic blebbing, cells not attached well, and their number was reduced as compared to pc-3 cells treated with either int 747 or fmd alone. figure 1 . inhibition of viability by int 747 in prostate cell carcinoma (pc-3) cells. (a) pc-3 cells were treatd with 8 μm int 747 747 in either normal medium (ncm) or fmd for 48 h. the cell viability was monitored by mtt assay. (b) representative photomicrograph shows morphological changes of prostate cancer cells and imaged by inverted phase-contrast microscope (total magnification 400x). arrows indicate (green) control cell with intact nuclei, (blue) condensed nuclei, (red) cell shrinkage, (yellow) membrane blebbing( black) loss of cell-cell contact, (orange) nuclei are cracked into two or multiple apoptotic bodies. iraqi j pharm sci, vol.32( 1 ) 2023 119 fxr activation inhibits proliferation in prostate cancer cell lines in vitro by inducing g1 phase arrest and apoptosis next, this study investigated whether fxr and fmd might effect on cell cycle distribution and mediating the growth and proliferation inhibition on pc-3 cells. cells treated with int 747 and/or fmd were stained with pi and then analysed by flow cytometry. in the present study, as presented in figure 2, there was a significant difference between the int 747 incubated in normal media and untreated control in the cell cycle analysis. int 747in normal media caused a significant increase in the cells in the g1 phase (p < 0.029), and decreased the percentage of s phase cells by 6.20 folds (p< 0.0011) as compared to control. whereas in fmd alone group, cell growth was significantly arrested at the g1 phase where accumulated cells reached 75% as compared to cells incubated in control normal media(p=0.0004). remarkably, int 747 combined with fmd has also caused a more significant increase (p < 0.0001) in the percentage of cells in the g1 phase in the pc-3 cell line and high increase in the sub-g1 apoptotic phase. figure 2. int 747 induces g1 phase arrest of prostatic cancer cells. (a) cell cycle detection of pc-3 cells following treatment with int 747 at different media conditions. the percentages of each phase of the cell cycle were obtained by flow-cytometry analysis. statistically significant differences between the control group and the treated groups were danalyzed using graphpad prism 7 software and differences between the groups were considered to be significant at*p<0.05, **p<0.01, and ****p<0.0001. (b) quantification of sub-g1, g0/g1, s, and g2/m phase cells in cells fxr and fmd inhibit the initiation of prostatic cancer spheroid formation a 3-dimensional spheroidal formation assay was performed to determine whether fxr and /or fmd inhibits the initiation of cancer spheroids formation. the ability of a cell to grow and colonize in agarose indicates that the cells have an aggressive phenotype in vitro that contributes to its invasiveness, metastatic, and resistance to therapy(21). as shown in figure 3, inhibition of tumorspheres by both int 747 and fmd was markedly impaired compared to untreated cells. specifically, the spheroid formation was almost totally impaired by int 747 combined with fmd in comparison with the control group, (p<0.0001). these results indicated that metastatic progression iraqi j pharm sci, vol.32( 1 ) 2023 120 of the pc-3 cells was suppressed by fxr activation and under tumor starvation. figure 3. int 747 treatment impedes prostate cancer cells spheroid formation. (a) pc-3 cells were grown in low adherent plates (prepared by precoating with 0.5% agarose in rpmi 164 media), and treated with int 747 and/or fmd. after 10 days, the spheroids were photographed. (b) the relative spheroid size was counted by imagej and performed bar diagram. fxr activation and fmd suppresses in vitro cell migration of prostate cancer cells the wound-healing assay was performed to investigate the effect of fxr on prostate cancer cell migration, a key event in carcinogenesis. in this study, pc-3 cells were imaged following treatment with 8 μm int747 with or without fmd at the same marked site. the difference in wound width was measured at three-time points (0 h, 24 h, and 48 h). as shown in figure 4, the group treated with int 747, demonstrated significantly impaired wound healing ability after 24 and 48 hours (fig. 4 b, p<0.001) compared with controls. the cell-free area increased by approximately two folds after 24 h compared to int 747-free pc-3 cells. this inhibition effect was even stronger after 48 h when cells were treated with int 747at the same dose (figure 4 a.b). interestingly, int 747 in combination with fmd highly significantly reduced cell migration of pc3 compared to int 747 alone. moreover, fmd alone was as effective as int 747 treatment in inhibiting cell migration. these data indicated that fxr and /or fmd negatively regulated prostate cancer cell migration. figure 4. int 747 inhibits prostate cancer cell migration in vitro (a) inverted microscopy (at lower magnification lens 4 x) shows that the wound closure area of pc-3 cells control monolayers is nearly closed after 48 h. in cells treated by different conditions of the int 747 and/or fmd, the closure of the wounded area is calculated. (b) the images were analyzed using the image j software to evaluate the scratch area after 24, 48 h respectively. the diagram shows the average value and standard error of the three experiments in quadruples. test anova followed; ***p < 0.001. scale bar in (a): 200 µm. fxr activation and starvation synergistically inhibit prostate cancer cell invasion an invasion assay was performed to further explore the role of fxr in the invasiveness’ capacity of prostate cancer. in this study, pc-3 cells treated with int 747 in the presence and absence of fmd for 48 hours. as shown in figure 5, treatment of pc-3 cells with int 747 considerably reduced cellular invasion by about more than 2.5 folds (p = 0.0045 vs. untreated pc-3 cells). likewise, significant differences were also noted for pc3 cells incubated in fmd alone (p > 0.0425 vs. untreated pc-3). furthermore, fxr activation in cells incubated in fmd largely reduced cellular invasion by more than 8 folds (p = 0.0009 vs. untreated pc-3 cells). to determine the possible causes of reduced cancer cell invasion and migration iraqi j pharm sci, vol.32( 1 ) 2023 121 during activation of fxr, the expression of mmp9 was analyzed by rt–pcr. as shown in figure 5, we observed that mmp9 mrna expression was relatively very low in pc-3 cells treated with either int 747 or fmd alone. remarkably and consistent with our result, int 747combined with fmd highly decrease mmp9 expression level ( p > 0.0056 vs. untreated pc-3). figure 5. the combined inhibitory effect of fxr and fmd on the invasion of pc-3 cells. (a) images of the invaded pc-3 cells that were treated with int 747 (8 µm) in the presence or absence of normal or fmd. (b) quantitative analysis was performed by destaining and reading the od at 530 nm. (c) the levels of gene expression were estimated with the relative qrt-pcr method (a fold-change of untreated control samples normalized to gapdh). values are presented as means± s.d. from three independent experiments performed in triplicates. (*p < 0.05, **p < 0.001, ***p < 0.0001 vs control). scale bar=100 µm. fxr inhibits epithelial-to-mesenchymal transition and survival of pc-3 cells via vimentin suppression and procaspase-3 proteolytic activation epithelial-to-mesenchymal transition (emt) and apoptosis are two of the most important processes in enabling cancer cells to have increased survival, migration, and invasive capacity (7, 23). therefore, key biomarker molecules of these signaling pathways were evaluated. according to functional studies, the inhibitory effects of fxr and/ or fmd on survival and emt are consistent with western blotting analysis. which indicated a significant decrease in the mesenchymal marker; vimentin in the pc-3 cell incubated with int 747 (fig.6). remarkably, our results further showed that the protein levels of full-length procaspase in pc-3 cells significantly decreased upon int 747 treatment, which is decreased due to proteolytic cleavage and activation to promote the apoptotic cell death. moreover, we also found that fmd alone also significantly decreased the protein levels of vimentin and procaspase3 as compared to the pc-3 cells in the control group. interestingly, we observed that both vimentin and procaspase-3 expression were relatively very low in pc-3 cells treated with the combination treatment as compared with control (p > 0.008, p > 0.0001 respectively). figure 6. the effects of int 747 and/or fmd on both emt and apoptosisrelated protein expression in pc-3 cells. (a) cells were treated with int 747 and/or fmd for 48h. cell lysates were analysed by western blot. here, gapdh is detected as internal control. (b) protein bands were quantified by imagej software, standardized with gapdh levels, and expressed as normalizations of control. the significance was determined by one way anova (*p < 0.05, **p < 0.001, ***p < 0.0001). discussion in many patients with metastatic castrationresistant prostate cancer (mcrpc), the hormonal therapy can be ineffective due to the high resistance of cancer cells to various anticancer treatments. thus, innovative non-ar-dependent treatment approaches should be explored in the future. in several studies, the induction of cell cycle arrest and apoptosis have been emphasized as a crucial targets of controlling the infinite growth of cells(3). fxr is a bile acid orphan nuclear receptor (nr), which is important in the homeostasis of bile acids, glucose metabolism and lipid metabolism(24, 25). recent evidence also suggests that fxr plays an important role in apoptosis and cancer. (26, 27).the inhibitory effects of fxr agonists have been investigated in some other tumors, such as liver, colon, breast cancer (11, 28, 29). in the present study, we further iraqi j pharm sci, vol.32( 1 ) 2023 122 validated the above results by assessment its inhibitory role on prostate cancer. furthermore, a drug combination study was utilized to identify whether the fasting mimicking diet (fmd) act synergistically with int 747 in prostate cancer cells. pc-3 cell cultures was used that recapitulate the typical features of metastatic human androgen independent adenocarcinoma, including high representation of csc markers and emt traits, and high cancerogenic potential in vitro and in vivo (30). in this study, we first showed that int 747 had a significant inhibitory effect on the viability of pc-3 cells (fig. 1). in particular, an 8 µm int 747 treatment was sufficient to inhibit pc-3 cell growth approximately half that of non-treated cells at 48 h after treatment. in the next step, the effect of fxr on the cell cycle was studied. as shown in figure 2, flow cytometry indicated that before inducing cellular apoptosis, int 747 also arrest cell cycle by increases the number of cells in the g1 phase and decreases the portion of those in the s1 phase cells. cells subsequently accumulated in the sub-g1 phase of the cell cycle, suggesting that int 747 mediated sequential cell cycle arrest and apoptosis. the morphological and cell cycle quantitative assessments in this study further suggested that int 747 treatment-induced cell death and eventually cell cycle arrest. the cell cycle is the basis for cell proliferation (31). our results further show that int 747 increases the fraction of cells in the g1 phase of the pc-3 cells, illuminating that some cells are blocked at the g1 phase. cell arrested of cell cycle progression provide an opportunity for cells to either undergo repair mechanisms or follow the apoptotic pathway (32). when apoptosis is initiated, it ultimately leads to a repair failure of dna damage; this result is catastrophic for cell proliferation (33). the apoptotic cell death is also confirmed by the decreased protein levels of procaspase-3 due to proteolytic cleavage and activation. however, on using an antibody specific for procaspase-3, which detects the full-length 32-kda form which according to the manufacturer’s instructions; therefore, we could not detect the cleaved form. caspase-3 plays an important in the execution of apoptosis and leads to the characteristic of morphology changes of the final event of apoptosis such as nuclear condensation and fragmentation (34). in agreement with other studies, fxr agonists induced procaspase-3 proteolytic cleavage in many cancer cells (11, 35). these findings suggest int 747 modulates cell cycle regulatory machinery leading to s cell cycle arrest, and it prompts the caspase-3 activation triggering apoptotic pathway. the combination of int 747 plus fmd exerts synergistic tumor inhibition in pc-3 human prostate cancer in vitro and in vivo by cooperatively induced cell cycle arrest and cell apoptosis. these findings confirm those of earlier studies, such as, fasting has been shown to be multi-functional on tumor progression (36-38). epithelial-mesenchymal transition (emt) is identified to play an important role in cancer development, metastasis and drug resistance (39). this research demonstrated that fxr and fmd activation inhibited the migratory and invasiveness capacity of pc-3 cells. based on the cell migration photomicrographs, pc-3 cells incubated with approximately 8 μm of int 747 have displayed wide wound gaps between cells compared to untreated cells after 48 h. however, wider wound gaps were noted as pc-3 cells incubated in combination of both int 747 and fmd, meanwhile, the untreated cells filled the entire wound gaps at 48 h.. similar to the cell migration data, the combination of int 747 and fmd produced a decrease in cellular invasion of pc-3 cell line equal to the calculated additive effects of int 747 or fmd used alone. accordingly, the anti-invasiveness additive effect of the combination of int 747 and fmd was also confirmed by a synergistic loss of matrix metalloproteinase9 (mmp9) and vimentin expression in pc-3 cells. many previous studies showed that both mmps and vimentin in general have been involved with the emt and other stages of malignancy, including primary tumor growth, angiogenesis, invasion of the basement membrane and stroma (4, 17). consequently, this study is consistent with earlier studies, which showed that fxr activation by int 747treatment impaired the invasive and migratory potential of colon cancer cells by arresting emt through vimentin repression (29). another earlier study also found that int 747 abrogated the induction of il-6-induced emt in an intrahepatic cholangiocarcinoma in vivo model and thereby inhibit their metastasis potential (28). moreover, may scientific works also showed that fmd suppressed the invasion of various cancer cells including liver and breast cancer cells (38, 40). very few studies have investigated int 747 anti-cancer effects in cancer therapy. in this context, we demonstrate that int 747combined with fmd has an additive impact on prostate cancer cell viability, spheroid formation and invasion, and synergistically decreases cell migration. conclusion this study demonstrated for the first time the fxr ligand int 747 that is commonly used to improve bile excretory function; becomes potentiated in their anticancer activity by fasting mimicking diet (fmd) in prostate cancer cells. the present paper documents the antiproliferative, antiemt and anti-metastatic capabilities of int747 and/or fmd and lists the mechanisms that may cause these effects in prostate cancer cells. at cellular level, these effects are mediated mainly by suppressing vimentin and mmp-9 expression; promote apoptosis and cell cycle arrest, which all iraqi j pharm sci, vol.32( 1 ) 2023 123 further supports the notion that fxr agonist could be a negative regulator of androgen refractory prostate tumorigenesis. this work also demonstrated that fmd synergistically enhanced the efficacy with which int 747 blocks cell cycle signaling and consequently affects crucial events in tumor survival, invasion, and metastasis. these results are particularly important as they provide new possibilities for specially designed fastingmimicking diet programs in combination with fxr agonists in oncology. it could replace much more toxic and less effective chemotherapy treatments. acknowledgment the authors are grateful to the establishments of mustansiriyah university/ college of pharmacy for providing the necessary facilities to carry out this work, conflict of interest the authors declare that there are no conflicts of interest. references 1. kimura t, egawa s. epidemiology of prostate cancer in asian countries. international journal of urology. 2018;25(6):524-31. 2. sung h, ferlay j, siegel rl, laversanne m, soerjomataram i, jemal a, et al. global cancer statistics 2020: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca: a cancer journal for clinicians. 2021;71(3):209-49. 3. westhofen t, chaloupka m, herlemann a, todenhöfer t, stief cg, kretschmer a. 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[prostate cancer: when to treat, which treatment options by stage?]. mmw fortschr med. 2021;163(7):3640. 37. de groot s, lugtenberg rt, cohen d, welters mjp, ehsan i, vreeswijk mpg, et al. fasting mimicking diet as an adjunct to neoadjuvant chemotherapy for breast cancer in the multicentre randomized phase 2 direct trial. nature communications. 2020;11(1). 38. salvadori g, zanardi f, iannelli f, lobefaro r, vernieri c, longo vd. fasting-mimicking diet blocks triple-negative breast cancer and cancer stem cell escape. cell metabolism. 2021;33(11):2247-59.e6. 39. paul cd, mistriotis p, konstantopoulos k. cancer cell motility: lessons from migration in confined spaces. nat rev cancer. 2017;17(2):131-40. 40. li w, wang y, zhou x, pan x, lü j, sun h, et al. the anti-tumor efficacy of 20(s)protopanaxadiol, an active metabolite of ginseng, according to fasting on hepatocellular carcinoma. journal of ginseng research. 2022;46(1):167-74. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 b2 ,b12 against myelosuppression induced by cyclophosphamide doi: https://doi.org/10.31351/vol29iss1pp134-142 134 evaluating the effects of different doses of vitamin b2 and single dose of vitamin b12 against myelosuppression induced by cyclophosphamide in experimental rats waleed k. ghanim*,1 and nada n. al-shawi** * department of pharmacology and toxicology, college of pharmacy, university of basra, basra, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract cyclophosphamide is chemotherapeutic agent that is utilized for the treatment of different malignancies; however its’use can be associated with numerous adverse effects. vitamin b2 and vitamin b12 suggested having myeloprotective effect. this work is designed to investigate the myeloprotective effect of both vitamins against myelosuppression induced by cyclophosphamide. one hundred adult rats of both sexes were used in this study. the animals were randomly enrolled into ten groups of 10 rats per each. group i: control group. group ii: cyclophosphamide-treated. group iii and group iv orally-administered of vitamin b2 (10, and 40 mg/kg/day), respectively alone for 7 days. group v: orally-administered vitamin b12 (0.1 mg/kg/day) alone for 7 days. group vi and group vii: orally-administered vitamin b2 (10, and 40 mg/kg/day), respectively for 7 days and a single intraperitoneal injection of cyclophosphamide (150 mg/kg) at day 7. group viii: orally-administered vitamin b12 (0.1 mg/kg/day) for 7 days and a single intraperitoneal injection of cyclophosphamide (150 mg/kg) at day 7. group ix: orally-administered a combination of vitamin b2 (10 mg/kg/day) and vitamin b12 (0.1 mg/kg/day) for 7 days and a single intraperitoneal injection of cyclophosphamide (150 mg/kg) at day 7. group x: orally-administered a combination of vitamin b2 (40 mg/kg/day) and vitamin b12 (0.1 mg/kg/day) for 7 days and a single intraperitoneal injection of cyclophosphamide (150 mg/kg) at day 7. on day eight, animals were sacrificed and blood collected for complete blood counts and femur bone were extracted for bone marrow histological examination. vitamin b2 and vitamin b12 significantly (p<0.05) increase complete blood counts; and the combination of vitamins produce -a significant (p<0.05) increase in complete blood counts compared to corresponding counts in other groups, and -improve histopathological changes compared to group ii rats. in conclusion both vitamins may have myeloprotective effects against cyclophosphamide-induced myelosuppression. key words: cyclophosphamide, vitamin b2, vitamin b12, myelosuppression, rats. ضد كبت 12والجرعة الواحدة من فيتامين ب 2الجرع المختلفة من فيتامين ب تأثيرتقييم نخاع العظم الناجم عن عقار سايكلوفوسفامايد في الجرذان المختبرية **الشاويندى ناجي و 1،*وليد خالد غانم ، البصرة ، العراق جامعة البصرة ،كلية الصيدلة،* فرع االدوية والسموم ، بغداد ، العراق جامعة بغداد، كلية الصيدلة ، ** فرع االدوية والسموم لخالصةا يستخدم لمعالجة انواع مختلفة من السرطان لكن قد يصاحب استخدامه اضرار جانبية كثيرة. فيتامين عقار السايكلوفوسفامايد 12و ب 2قد يمتلكان القدرة على حماية نخاع العظم. لذلك كان الهدف من هذه الدراسة هو تقييم تأثير فيتامين ب12وفيتامين ب 2ب جرذ بالغ من كال الجنسين حيث قسمت الى عشر مجاميع وكل 100استخدام ضد سمية عقار سايكلوفوسفامايد على نخاع العظم. تم ملغم/كغم 150أيام, المجموعة الثانية: حقنت ب 7جرذان. المجموعة االولى: حقنت ب محلول ملحي لمدة 10مجموعة تحتوي على ايام, المجموعة 7تباعا عن طريق الفم لمدة 2ملغم/كغم فيتامين ب 40و 10سايكلوفوسفامايد, المجموعة الثالثة و الرابعة: اعطيت ملغم/كغم 40و 10ايام, المجموعة السادسة و السابعة: اعطيت 7عن طريق الفم لمدة 12ملغم/كغم من فيتامين ب 0.1الخامسة: اعطيت 0.1ع, المجموعة الثامنة: اعطيت ملغم/كغم سايكلوفوسفامايد في اليوم الساب 150ايام وحقنت ب 7تباعا عن طريق الفم لمدة 2فيتامين ب ملغم/كغم سايكلوفوسفامايد في اليوم السابع, المجموعة التاسعة: 150ايام وحقنت ب 7عن طريق الفم لمدة 12ملغم/كغم فيتامين ب لوفوسفامايد ملغم/كغم سايك 150ايام وحقنت ب 7عن طريق الفم لمدة 12ملغم/كغم فيتامين ب 0.1و 2ملغم/كغم فيتامين ب 10اعطيت ايام وحقنت 7عن طريق الفم لمدة 12ملغم/كغم فيتامين ب 0.1و 2ملغم/كغم فيتامين ب 40في اليوم السابع, المجموعة العاشرة: اعطيت ملغم/كغم سايكلوفوسفامايد في اليوم السابع. في اليوم الثامين تم التضحية بالجرذان لغرض جمع الدم لمعرفة تعداد كريات الدم 150ب انتجا زيادة ملحوظةفي حساب 12و ب 2وجمع العظم للحصول على نخاع العظم ودراسة التغيرات النسيجية.بينت النتائج ان فيتامين ب كريات الدم والمزج بينهما انتج زيادة معنوية في حساب كريات الدم بالمقارنة مع المجموعة الثانية فضال عن انتاج تحسن ملحوظ لنسيج القدرة على حماية نخاع العظم ضد سمية عقار سايكلوفوسفامايد على نخاع العظم 12و ب 2يكون لكل من فيتامين بنخاع العظم. قد , تثبيط العظم, الجرذان.12, فيتامين ب 2الكلمات المفتاحية: سايكلوفوسفامايد, فيتلمين ب 1corresponding author e-mail: e-mail: ph.wkg81@yahoo.com received: 16/ 9 /2019 accepted: 23/11 /2019 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp134-142 iraqi j pharm sci, vol.29(1) 2020 b2 ,b12 against myelosuppression induced by cyclophosphamide 135 introduction cyclophosphamide (cpa) among the most widely used chemotherapeutic drug to kill cancer cells (1). such drug is used alone or in combination with other chemotherapeutic agents for the treatment of wide variety of malignant diseases such as breast cancer, multiple myeloma, hodgkin’s disease, furthermore, cpa is used as immunosuppressant agent and in organ transplantation, either alone or in combination with corticosteroids (2). myelosuppression was reported to be a dangerous condition that’s related to defect in the blood cell-forming process, which affects body functions of patients, including their quality of life (3). it has been reported that, compensating myelosuppression in chemotherapy is so hard (4). vitamin b2 (riboflavin) is a water soluble vitamin (5) which found in different food sources. it is well recognized that such vitamin can participate in different redox reactions which is important to human metabolism; furthermore, vitamin b2 is a source for cofactors flavin mononucleotide (fmn) and flavin adenine dinucleotide (fad) that act as electron carriers (6). different steps in the oxidation of fatty acids are depending on flavin as electron acceptors in the mitochondria (7). it was found that the effect on oxidation of fatty acids is thought to be responsible for the altered fatty acid profile in hepatic lipids in severely riboflavin-deficient mice which seems to be independent of the dietary source of lipid (8). the effect of riboflavin-deficiency on fatty acid profiles may reflect an overall reduction in the oxidation of fatty acids; while the essential fatty acids are present in the diet accumulate (9). vitamin b12 is a generic name for a specific group of cobalt-containing corrinoids with important biological activities for humans (10). vitamin b12 has been reported to be required for the conversion of methylmalonic acid to succinyl-coa (11, 12). the aim of the study was to evaluate the effects of different doses of vitamin b2 and fixed dose of vitamin b12 on myelosuppression induced by cyclophosphamide in experimental rats. materials and methods experimental animals one hundred healthy adult albino rats of both sexes, three months old, weighing 180220gm were used in this study; they were obtained from and maintained in the animal house of the college of pharmacy, baghdad university under conditions of controlled temperature. the animals were fed commercial pellets and tap water ad libitum throughout the experiment period. the study was approved by the scientificand the ethicalcommittees of the college of pharmacy/university of baghdad. drugs cyclophosphamide vial (500 mg) was purchased from baxter, usa. vitamin b2 capsule (400 mg) was purchased from amazing nutrition, usa. vitamin b12 tablet (1 mg) was purchased from tq pharma, japan. experimental protocol the healthy rats were randomly divided into ten groups (10 animals/group) as follows: group i: ip injected 1ml/kg/day normal saline for 7 days; this group served as control. group ii: ip injected with single dose of cyclophosphamide (150 mg/kg). group iii: orally-administered vitamin b2 at a dose of (10 mg/kg/day) for 7 days. group iv: orally-administered vitamin b2 at a dose of (40 mg/kg/day) for 7 days. group v: orally-administered vitamin b12 at a dose of (0.1 mg/kg/day) for 7 days. group vi: orally-administered vitamin b2 at a dose (10 mg/kg/day) for 7 days and a single ip injection of (150 mg/kg) of cyclophosphamide at day 7. group vii: orally-administered vitamin b2 at a dose (40 mg/kg/day) for 7 days and a single ip injection of (150 mg/kg) of cyclophosphamide at day 7. group viii: orally-administered vitamin b12 at a dose (0.1 mg/kg/day) for 7 days and a single ip injection of (150 mg/kg) of cyclophosphamide at day 7. group ix: orally-administered a combination of vitamin b2 at a dose (10 mg/kg/day) and vitamin b12 at a dose of (0.1 mg/kg/day) for 7 days and a single ip injection of (150 mg/kg) of cyclophosphamide at day 7. group x: orally-administered a combination of vitamin b2 at a dose (40 mg/kg/day) and vitamin b12 at a dose of (0.1mg/kg/day) for 7 days and a single ip injection of (150 mg/kg) of cyclophosphamide at day 7. twenty-four hour after the end of the treatment duration (i.e. at day 8), rats were euthanized by diethyl ether and then by intra cardiac puncture, 8 ±1 ml of blood was obtained by cardiac puncture for complete blood counts(cbcs) (total wbc, lymphocytes, neutrophils and rbcs). bone marrow tissue sample femur bone was obtained by making incision in abdomen then extends the incision down to leg, remove skin; soft tissue and connective tissue attached to femur and tibia iraqi j pharm sci, vol.29(1) 2020 b2 ,b12 against myelosuppression induced by cyclophosphamide 136 then dislocate shift bone and remove tissue attached to it (16). bone marrow isolation isolation of bone marrow was performed according to sarah r et al (2016) (13). histological examination bone marrow of each animal was prepared for histological examination according to the method of junqueira (14, 15). statistical analysis data were expressed as the mean values, mean± standard error of the mean (sem). unpaired student t-test was used for testing the significant difference between two groups. the statistical significance of the differences among various groups was determined by one-way analysis of variance (anоva). differences were considered statistically significant for p-value less than 0.05. results table 1 showed that there were nonsignificant differences (p<0.05) in total number of wbcs in the groups of rats orallyadministered of different doses of vitamin b2 each alone for one week (groups iii, and iv) and fixed dose of vitamin b12 for one week (group v) each compared to the corresponding numbers in control (group i) rats. mean±sem of total number of wbcs were respectively, 7.03 * 109/l±0.094, 6.93 * 109/l±0.113, 6.97 * 109/l±0.115 and 6.99 * 109/l±0.113. furthermore, rats ip injected with cpa at day 7 (group ii) produced significant reduction (p<0.05) in the total number of wbcs compared to the corresponding numbers in control (group i) rats. mean±sem of total number of wbcs were respectively, 1.31*109/l±0.073 and 6.99*109/l±0.113. moreover, there were significant elevation (p<0.05) in total number of wbcs in groups treated with different doses of vitamin b2 each alone for one week (groups vi, and vii), vitamin b12 for one week (group viii), and combination of different doses of vitamin b2 with vitamin b12 (group ix and group x) each for one week prior to ip injection of cpa compared to the corresponding numbers in (group ii) rats ip injected with a single dose of cpa. mean±sem of total number of wbc were respectively;1.81*109/l±0.099, 2.29*109/l±0.099, 2.68*109/l±0.122, 3.32*109/l±0.091, 3.89*109/l±0.073, and 1.31*109/l±0.073. furthermore, table 1 showed that there were significant elevation (p<0.05) in total number of wbcs in groups treated with combination of vitamin b2 with vitamin b12 prior to cpa (group ix) and vitamin b2 with vitamin b12 (group x) for one week prior to ip injection of cpa compared to the corresponding total number of wbcs to either use of vitamin b2 or vitamin b12 alone (groups vi, vii and viii). mean±sem of total number of wbcs were respectively, 3.32* 109/l±0.091, 3.89* 109/l±0.073, 1.81* 109/l±0.099, 2.29* 109/l±0.099 and 2.68* 109/l±0.122. table 1 showed that there were non-significant differences (p<0.05) in lymphocytes number in the groups of rats orally administered different doses of vitamin b2 each alone for one week (groups iii, and iv) and vitamin b12 for one week (group v) each compared to the corresponding numbers in control (group i) rats. mean±sem of lymphocytes number were respectively; 2.05 * 109/l±0.016, 2.06 * 109/l±0.022, 2.01 * 109/l±0.023 and 2.02* 109/l±0.02. furthermore, rats ip injected with cpa at day 7 (group ii) produced significant reduction (p<0.05) in the lymphocytes number compared to the corresponding number in control (group i) rats. mean±sem of lymphocytes number were respectively, 0.14 * 109/l±0.016 and 2.02 * 109/l±0.02. moreover, there were significant elevation (p<0.05) in lymphocytes number in groups treated with different doses of vitamin b2 each alone for one week (groups vi, and vii), vitamin b12 for one week (group viii), and combination of vitamin b2 with vitamin b12 (group ix) and vitamin b2 with vitamin b12 (group x) each for one week prior to ip injection of cpa compared to the corresponding numbers in (group ii) rats ip injected with a single dose of cpa. mean±sem of lymphocytes number were respectively, 0.22 * 109/l±0.013, 0.31 * 109/l±0.017, 0.39* 109/l±0.01,0.48 * 109/l±0.013, 0.58 * 109/l±0.013, and0.14 * 109/l±0.016. furthermore, table 1 showed that there were significant elevation (p<0.05) in lymphocytes number in groups treated with combination of vitamin b2 with vitamin b12 prior to cpa (group ix) and vitamin b2 with vitamin b12 (group x) for one week prior to ip injection of cpa compared to the corresponding lymphocytes number to either use of vitamin b2 or vitamin b12 alone (groups vi, vii and viii). mean±sem of lymphocytes number were respectively, 0.48 * 109/l±0.013, 0.58 * 109/l±0.013, 0.22 * 109/l±0.013, 0.31 * 109/l±0.017and 0.39* 109/l±0.01. meanwhile, table 1 showed that there were nonsignificant differences (p<0.05) in neutrophils number in the groups of rats orally administered of different doses of vitamin b2 each alone for one week (groups iii, and iv) and vitamin b12 for one week (group v) each compared to the corresponding numbers in control (group i) rats. mean±sem of neutrophils number were iraqi j pharm sci, vol.29(1) 2020 b2 ,b12 against myelosuppression induced by cyclophosphamide 137 respectively, 3.14 * 109/l±0.016, 3.13 * 109/l±0.021, 3.16 * 109/l±0.031 and 3.22 * 109/l±0.047. furthermore, rats ip injected with cpa at day 7 (group ii) produced significant reduction (p<0.05) in the neutrophils number compared to the corresponding numbers in control (group i) rats. mean±sem of neutrophils number were respectively, 0.51 * 109/l±0.048 and 3.22 * 109/l±0.047. moreover, there were significant elevation (p<0.05) in neutrophils number in groups treated with different doses of vitamin b2 each alone for one week (groups vi and vii) and vitamin b12 for one week (group viii), and combination of vitamin b2 with vitamin b12 (group ix and group x) for one week prior to ip injection of cpa compared to the corresponding numbers in (group ii) rats ip injected with a single dose of cpa. mean±sem of neutrophils number were respectively; 0.77 * 109/l±0.021, 1.01 * 109/l±0.023, 1.25 * 109/l±0.022, 1.45 * 109/l±0.017, 1.76 * 109/l±0.034, and 0.51 * 109/l±0.048. furthermore, table 1 showed that there were significant elevation (p<0.05) in neutrophils number in groups treated with combination of vitamin b2 plus vitamin b12 prior to ip injection of cpa (group ix) and vitamin b2 with vitamin b12 (group x) for one week prior to ip injection of cpa compared to the corresponding neutrophils number to either use of vitamin b2 or vitamin b12 alone (groups vi, vii and viii). mean±sem of neutrophils number were respectively, 1.45 * 109/l±0.017, 1.76 * 109/l±0.034, 0.77 * 109/l±0.021, 1.01 * 109/l±0.023 and 1.25 * 109/l±0.022. table 1 also showed that there were nonsignificant differences (p<0.05) in rbcs number in the groups of rats orallyadministered of different doses of vitamin b2 each alone for one week (groups iii and iv), and vitamin b12 for one week (group v) each compared to the corresponding numbers in control (group i) rats. mean±sem of rbc numbers were respectively, 4.29 * 1012/l±0.064,4.36 * 1012/l±0.081, 4.41 * 1012/l±0.060 and 4.3 * 1012/l±0.082. furthermore, rats ip injected with cpa at day 7 (group ii) produced significant reduction (p<0.05) in the rbcs number compared to the corresponding numbers in control (group i) rats. mean±sem of rbc numbers were respectively, 2.36 * 1012/l±0.022 and 4.3 * 1012/l±0.082. meanwhile, there were significant elevation (p<0.05) in rbcs number in groups of rats treated with different doses of vitamin b2 each alone for one week (groups vi, and vii), vitamin b12 for one week (group viii), and combination of different doses of vitamin b2 with vitamin b12 (group ix and group x) each for one week prior to ip injection of cpa compared to the corresponding numbers in (group ii) rats ip injected with a single dose of cpa. mean±sem of rbcs numbers were respectively, 2.62 * 1012/l±0.025, 2.92 * 1012/l±0.029, 3.16 * 1012/l±0.034, 3.40 * 1012/l±0.021, 3.70 * 1012/l±0.030, and 2.36 * 1012/l±0.022. furthermore, table 1 showed that there were significant elevation (p<0.05) in rbcs numbers in groups treated with combination of vitamin b2 plus vitamin b12 prior to ip injection of cpa (group ix), and vitamin b2 with vitamin b12 (group x) for one week prior to ip injection of cpa compared to the corresponding rbcs numbers to either use of vitamin b2 or vitamin b12 alone (groups vi, vii and viii). mean±sem of rbcs numbers were respectively, 3.40 * 1012/l±0.021, 3.70 * 1012/l±0.030, 2.62 * 1012/l±0.025, 2.92 * 1012/l±0.029 and 3.16 * 1012/l±0.034. iraqi j pharm sci, vol.29(1) 2020 b2 ,b12 against myelosuppression induced by cyclophosphamide 138 table 1.effects of different doses of vitamin b2 and single dose of vitamin b12 each alone and in combination on cbcs (total wbc, lymphocytes, neutrophils and rbc) after ip injection of cyclophosphamide (cpa) in rats each value represents mean ± standard error of means (sem). values expressed in small letters (a, b, c, d, e, f, and g) are significantly different (p<0.05). number of animals in each group=10. histological examination of rats' bone marrow tissue rats ip injected 1ml normal saline (group i, control group), orally-administered different doses of vitamin b2 (group iii and group iv), and orally-administered vitamin b12 (group v) each for 7 days shows normal bone marrow section; where, normal appearance of reticular area and leukocyte production area with notice of megakaryocyte are observed in figures (1-a, b, c, d), respectively. the bone marrow section from (group ii) exposed to ip injection of cpa showed massive cell death with adipose tissue, fibroid area distribution and reduction in erythrocyte genesis and leukocyte genesis, with pyknotic nuclei with massive apoptotic cells are observed in figure 1-e. the bone marrow section from (group vi) orally-administered vitamin b2 for 7 days prior to ip injection of cpa at day seven showed that histological changes include abnormal fibroid area with adipose tissue with pyknotic nuclei with numerous apoptotic cells as shown in figure 1-f. the bone marrow section from (group vii) orally-administered vitamin b2 for seven days prior to ip injection of cpa at day seven showed that abnormal fibroid area with adipose tissue with pyknotic nuclei with numerous apoptotic cells as shown in figure 1-g. the bone marrow section from (group viii) orally-administered vitamin b12 for seven days group/treatment total number of white blood cells* 109/l lymphocytes number*109/l neutrophils number*109/l rbc number * 1012/l group i/control (normal saline) 6.99±0.113a 2.02±0.02a 3.22±0.047a 4.3±0.082a group ii/ cyclophosphamide (150mg/kg) 1.31±0.073g 0.14±0.016g 0.51±0.048g 2.36±0.022g group iii/vitamin b2 (10 mg/kg/day) 7.03±0.094a 2.05±0.016a 3.14±0.016a 4.29±0.064a group iv/vitamin b2 (40 mg/kg/day) 6.93±0.113a 2.06±0.022a 3.13±0.021a 4.36±0.081a group v/vitamin b12 (0.1 mg/kg/day) 6.97±0.115a 2.01±0.023a 3.16±0.031a 4.41±0.060a group vi/vitamin b2 (10 mg/kg/day) with a single ip injection of cpa 1.81±0.099f 0.22±0.013f 0.77±0.021f 2.62±0.025f group vii/ vitamin b2 (dose 40 mg/kg/day) and a single ip injection of cpa 2.29±0.099e 0.31±0.017d 1.01±0.023e 2.92±0.029e group viii/vitamin b12 (dose 0.1mg/kg/day) and a single ip injection of cpa 2.68±0.122d 0.39±0.01d 1.25±0.022d 3.16±0.034d group ix/a combination of vitamin b2 (10 mg/kg/day) and vitamin b12 (0.1 mg/kg/day) and a single ip injection of cpa 3.32±0.091c 0.48±0.013c 1.45±0.017c 3.40±0.021c group x combination of vitamin b2 (40 mg/kg/day) and vitamin b12 (0.1 mg/kg/day) and a single ip injection of cpa. 3.89±0.073b 0.58±0.013b 1.76±0.034b 3.70±0.030b iraqi j pharm sci, vol.29(1) 2020 b2 ,b12 against myelosuppression induced by cyclophosphamide 139 prior to ip injection of cpa at day seven showed abnormal fibroid area with adipose tissue with pyknotic nuclei and numerous apoptotic cell as shown in figure 1-h. however the bone marrow sections from (groups ix and x) orally-administered vitamin b12 with different doses of vitamin b2 respectively for 7 days prior to ip injection of cpa at day seven showed mild abnormal fibroid area and pyknotic nuclei with replacement of normal bone marrow area with limited number of apoptotic cells as shown in figures 1-i and 1-j respectively. figure 1. histopathological section of bone marrow in various experimental rats' groups; (hematoxylin and eosin; x40). discussion in the present study ip injection of (150 mg/kg) cpa at day 7 (group ii) produce significant reduction in cbcs including (total wbcs, lymphocytes, neutrophils, rbcs and hb) (p<0.05) compared to control rats (group i); authors reported that the myelosuppression induced by cpa occurred due to different mechanisms, which include: 1induction of apoptosis: inappropriate and excessive spontaneous and activation-induced apoptosis can lead to myelosuppression and result in myelodysplasia, thrombocytopenia, leukopenia and lymphopenia (16). 2induction of hematopoietic stem cell (hsc) senescence: cells undergo senescence after extensive replication or exposure to a genotoxic or oncogenic stress although senescent cells metabolically active, they are no longer capable of dividing (17). the senescent hscs induced by cpa have diminished clonogenic activity and express increased levels of sa-β-gal, p16ink4a and arf (18). 3damage to bm stroma has been observed after treatment with cpa (19). in general bm stroma is more resistant to the chemotherapy compared to the effect of chemotherapy on hematopoietic progenitor cells and hematopoietic stem cells; however treatment with cpa can produce damage to bm stroma (20). iraqi j pharm sci, vol.29(1) 2020 b2 ,b12 against myelosuppression induced by cyclophosphamide 140 results of the current study concerning the effects of cpa on cbcs are coincide with those of qing-yu et al (2018) who found that cpa induced myelosuppression in mice bone marrow mediated by genotoxic mechanism (20). recently, syeda et al (2019) also found that cpa can induce myelosuppression via oxidative stress (os)-mediated dna damage (21). similarly, kartick et al (2019) found that cpa-induced myelosuppression and hepatic os as evident by lipid peroxidation and activity assays of antioxidant enzymes such as sod (22). moreover, results of this study shows that -vitamin b2 in dose-dependent manner,fixed dose of vitamin b12, and -combination of vitamin b2 with vitamin b12 prior to cpa produce a significant increase in cbcs compared to corresponding counts in rats of group ii these effects could be explained that riboflavin possess antioxidant property and it considered as an important precursor for fmn and fad, which served as coenzymes for several enzymes particularly antioxidant enzymes including sod and catalase (23). also, riboflavin was reported to play important role in conversion oxidized glutathione (gssg) to the reduced form (gsh), which plays important role as antioxidant defense factor (24); these roles of riboflavin make it capable to reduce myelosuppression induced by cpa. furthermore; authors reported that vitamin b12 is also required for the synthesis of methionine and s-adenosyl methionine, which is a common methyl donor required for the maintenance of methylation patterns in dna that determine gene expression and dna conformation (25). so that a reduction in the level of vitamin b12 may lead to elevation in dna damage and alter dna methylation and elevation in the level of homocysteine (26). furthermore, hornung et al (2004) explored that vitamin b12 may have myeloprotective effect; where, it played an effective role in patients with rheumatoid arthritis (ra) (27). effects on rats' bone marrow (bm) histology in the present study, histopathological examination of bm section of rats ip injected with cpa at a dose (150 mg/kg) at day 7 (group ii) confirmed the myelosuppression induced by such drug compared to control (group i) rats; where bm section of cpatreated rats under light microscope showed massive fibroid tissue replacement of bm with clear vacoulation in addition to distribution of adipose tissue with massive apoptosis were observed figure (1-e). these findings are coinciding with the work of sun c et al (2018) (28). effect of vitamin b2 (10 and 40 mg/kg) (groups vi and vii) and combination with (0.1 mg/kg) vitamin b12 (groups ix and x), orally-administered prior to cpa showed that there were improvement of the histopathological bm lesions in abovementioned groups, figures (1-f, 1-g, 1-i and 1-j) compared to (group ii) rats (cpatreated) figure (1-e). in this study, results are in agreement with zhaoli et al (2015); where, a protective effect of vitamin b2 against bm toxicity was observed by histopathological examination (29). also the effect of (0.1 mg/kg) of vitamin b12 orally-administered prior to cpa (group viii) showed that there were improvement of the histopathological bm lesions figure (1-h) compared to (group ii) (cpa-treated) figure (1-e). in this study, results are in agreement with demet et al (2019); where, a protective effect of vitamin b12 against myelosuppression was observed by histopathological examination (30). acknowledgments this article was abstracted from ph.d. thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the authors gratefully thank family, friends in the college of pharmacy. references 1. fereshteh talebpour amiri, maedeh hamzeh, saeed yaghubi beklar, seyed jalal hosseinimehr. anti-apoptotic and antioxidant effect of cerium oxide nanoparticles on cyclophosphamide induced hepatotoxicity. erciyes med j 2018; 40: 148-54. 2. nouran k. olama, medhat taha, hagar y. rady. the potential protective role of coenzyme q10 on the cyclophosphamideinduced lung toxicity in adult male albino rats: a histological and ultrastructural study. int j sci rep 2018; 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40: 2534. 29. zhaoli dai, woon-puay koh. b-vitamins and bone health–a review of the current evidence. nutrients 2015; 7: 3322-3346. 30. demet cekdemir, fatma behice serinkan, birsen aydemir, nilgun dilaveroglu, yasin ertug cekdemir, mehmet gunduz, et al. effects of immune complexes on holotranscobalamine assay of vitamin b12 deficiency in myeloproliferative disorders. international journal of hematology and oncology 2019; 29: 31 38. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://www.ncbi.nlm.nih.gov/pubmed/?term=sun%20c%5bauthor%5d&cauthor=true&cauthor_uid=29077519 https://www.ncbi.nlm.nih.gov/pubmed/?term=yang%20j%5bauthor%5d&cauthor=true&cauthor_uid=29077519 https://www.ncbi.nlm.nih.gov/pubmed/?term=pan%20l%5bauthor%5d&cauthor=true&cauthor_uid=29077519 https://www.ncbi.nlm.nih.gov/pubmed/?term=guo%20n%5bauthor%5d&cauthor=true&cauthor_uid=29077519 https://www.ncbi.nlm.nih.gov/pubmed/?term=li%20b%5bauthor%5d&cauthor=true&cauthor_uid=29077519 https://www.ncbi.nlm.nih.gov/pubmed/?term=yao%20j%5bauthor%5d&cauthor=true&cauthor_uid=29077519 https://www.ncbi.nlm.nih.gov/pubmed/29077519 https://www.ncbi.nlm.nih.gov/pubmed/29077519 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 effect of simvastatin, omega-3 and their combination on ucp1 doi: https://doi.org/10.31351/vol31iss2pp101-112 101 anti-obesity effect of simvastatin and/or omega-3 on obese male wistar rats rasha aljuboury*,1and nada n. al-shawi** *department of technical affairs, ministry of health and environment, baghdad-iraq. **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad-iraq. abstract strategies to reduce obesity have become main priority for many health institution and health staff around the world, as the prevalence of obesity has risen and exacerbated in most of the world mainly because of the modern life style which tend to be more sedentary with an increase eating unhealthy fast western food. many years ago, the lipid-lowering drug simvastatin; and omega-3 were considered as a traditional lipid-lowering drug that have been well-documented to possess anti-inflammatory, cardioprotective and triglyceride-lowering properties; and their co-administration may demonstrate a complementary effect in lowering patients' triglycerides and total cholesterol to treat atherosclerosis. many previous studies have been found other beneficial effects for simvastatin, and omega-3; since, simvastatin can be used for the treatment of alzheimer's disease; and for prevention of prostate cancer; while omega 3 can reduce the risk of sudden cardiac death in addition for preventing obesity that has been documented by recent studies. but, the effect of simvastatin alone or its combination with omega-3 as potential anti-obesity therapy and /or protection against obesity is not yet known through their effects on thermogenic factors. the purpose of the current study is to evaluate the effect of simvastatin and omega-3 on thermogenic genes including (ucp1) using quantitative real time pcr, and the expression of uncoupling protein 1 (ucp1) protein was detected in ibat and iwat adipocyte by immunohistochemistry. one hundred and twenty (120) male wistar rats at age five to six week, and weighing 100-150g were allocated into five groups, group i is the obese rat that receive high fat diet only. group ii rat receive simvastatin 9 mg/kg/day; group iii rats treated with 18mg/kg/day simvastatin, group iv rat receive omega-3 (1ml/day); group v rat receive mixed treatment (i.e., simvastatin 9mg +omega -3 (1ml/day). treatments were given along the eight weeks. three rats from each group were weekly-authenticated along the 60 days interscapular brown adipose tissue (ibat) and inguinal white adipose tissues (iwat) were obtained. simvastatin and omega-3 have an obvious activation of ucp1genes; this reflects an increase in thermogenic process in adipose tissue in obese high fat diet rats and their combination exert a synergistic increase in the thermogenic mechanism when compared to simvastatin 9mg/ kg /day alone. our results give a hope for the utilization of simvastatin either alone or in combination with omega-3 as anti-obesity therapy; through their enhancement of thermogenic in white and brown adipose tissues with a consequent weight loss. keywords: obesity, high-fat diet, simvastatin, omega-3, brown adipocyte, beige adipocytes, thermogenesis, ucp1. وتركيبها على فئران ويستار الذكور ذات الطراز 3 اآلثار المضادة للسمنة للسيمفاستاتين واألوميغا السمين ** ي ندى ناجي الشاوو 1*،رشا عبد اللطيف عبد الحسين الجبوري ،بغداد، العراق. الفنيةاألمور دائرة، والبيئة الصحةوزاره * فرع االدوية والسموم، كلية الصيدلة، جامعة بغداد، بغداد، العراق. ** الخالصة أصبحت استراتيجيات الحد من السمنة أولوية رئيسية بالنسبة للعديد من المؤسسات الصحية والموظفين الصحيين في جميع أنحاء العالم، مع ويرجع ذلك أساسا إلى أسلوب الحياة الحديث الذي يميل إلى أن يكون أكثر استقرارا العالم، حيث أن انتشار السمنة قد ارتفع وتفاقم في معظم أنحاء عقارات تقليدية لخفض 3-وأوميغازيادة تناول األغذية الغربية السريعة غير الصحية. قبل سنوات عديدة، كان الدواء المخفض للدهون سيمفاستاتين تهم المشتركة وقد تظهر إدار الجليسيريدات؛ الدهون تم توثيقه توثيقا جيدا المتالكهم خصائص مضادة لاللتهابات وحماية القلب وخفض الدهون ثالثية وإجمالي نسبة الكوليسترول لعالج التهاب األوعية الدموية. كذلك وجدت العديد الجليسيريدات للمرضىتأثيرا تكميليا في خفض نسبة الدهون ثالثية والوقاية الزهايمر؛ الج مرض . إضافة الى ذلك يمكن استخدام السيمفاستاتين لع3-واألوميغا للسيمفاستاتين، من الدراسات السابقة تأثيرات مفيدة أخرى يمكن أن يقلل من خطر الوفاة القلبية المفاجئة باإلضافة إلى الوقاية من السمنة التي وثقتها الدراسات omega 3في حين أن البروستاتا؛ من سرطان ة من السمنة غير معروف حتى اآلن من خالل كعالج محتمل ضد السمنة و/أو الحماي 3-تأثير السيمفاستاتين وحده أو توليفه مع أوميغا الحديثة. ولكن pcr الحرارية بواسطة الكيمياء المناعيه على الجينات 3واالوميكا تقييم تأثير السيمفاستاتين هذه الدراسة تهدف الى آثارها على العوامل الحرارية. ستة أسابيع من -)تم تخصيص خمسةذكر ( 120وعشرين ) مائة في ال الشحوم البيضاء والشحوم البنية. ucp1 ذلك التعبير البروتيني ل بما في واألوميغا أن السيمفاستاتين تأظهر . 3-غ( في خمس مجموعات: معالجة بجرعتين مختلفتين من السيمفاستاتين واألوميغا 150-100العمر ووزنها الدهون التي تعتبر مجموعة مراقبة. تم إعطاء العالج باإلضافة إلى مجموعة غذائية عالية المختلط، والعالج ucp1geneلديهما تنشيط واضح من 3 البنية بمنطقة بين يوما وقد تم الحصول على األنسجة الدهنية 60ثالثة فئران من كل مجموعة على مدى بأسبوعيا ضحية لمدة ثمانية أسابيع. تم الت االكتاف واألنسجة الدهنية البيضاء بالمنطقة االربية. 1corresponding author e-mail: rashaaljuboury@gmail.com received: 30/9 /2021 accepted: 5/12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp101-112 iraqi j pharm sci, vol.31( 2 ) 2022 effect of simvastatin, omega-3 and their combination on ucp1 102 ية وهذا يعكس زيادة في العملية الحرارية في األنسجة الدهنية في فئران الحمية ذات الدهون العالية السمنة وتركيبها يؤدي إلى زيادة تآزر القتران مع عطي األمل في استخدام السيمفاستاتين إما بمفرده أو باتهذه الدراسة .. كغ/يوم فقط/9mgفي اآللية الحرارية عند مقارنتها بالسيمفاستاتين .من خالل تعزيزها الحراري في األنسجة الدهنية البيضاء والبنية مع ما يترتب على ذلك من فقدان الوزن للسمنة؛ كعالج مضاد 3-أوميغا ucp1 ، وتوليد الحرارة ،الحبيبيةواألدبوكيّات ، البنيواألديبوكايت ،3-واألوميغا والسيمفاستاتين، الدهون،والحمية عالية السمنة، الكلمات المفتاحيه: introduction obesity is a complex, multifactorial, disease, affecting, over a third of the all-world’s population (1). it is typically defined as an increased energy intake combined with less energy expenditure (2). the change in life style from highly active, hardworking jobs to sedentary life results in imbalance in weight gain (3) and as a result obesity occurs; with increase in the risk of morbidity due to chronic diseases mainly type 2 diabetes and cardiovascular disease (4, 5), with increasing in mortality scales (6). adipose tissue has emerged as a dynamic organ and plays an important role in the pathogenesis of obesity and its associated metabolic disorders (4, 7). it is classified into white adipose tissue (wat) and brown adipose tissue (bat), which are visibly distinguishable according to tissue color however they differ in shape, size, and the intracellular structure of their organelles (8). the bat is mainly proposed to maintain thermal homeostasis through dissipating the energy in the form of heat (9) by specific protein located in the mitochondria known as uncoupling protein1 (ucp1) (10). moreover, wat can play a role in thermogenesis through its browning resulting in what is known as beige/brite cells (11), which have increased mitochondrial proteins and ucp1(12). many studies observed that activation of bat and increasing the beige/ brite cells in wat may exert greater metabolic benefits and are associated with improvement in many physiological parameters such as a reduction in blood glucose levels and weight reduction (13), through increasing resting energy expenditure with a subsequent anti-obesity effect (14, 15). studies hypothesized that ucp1 gene can be enhanced through activating many transcriptional complexes of thermogenic genes mainly pr domain containing 16 (prdm16) and peroxisome proliferator-activated receptor-gamma coactivator alpha (pgc-1α) coactivators at thermogenic and their binding with dna. studies hypothesized that prdm16 might be recruited to the enhancer region of the ucp1 gene through the interaction with pgc-1α and a very large increase in the uncoupled fraction of respiration (16). a previous study showed that simvastatin could potentially help to ameliorate metabolic abnormalities associated with a long-term olanzapine treatment partly via activating the function of bat. these findings support a potential mechanism of simvastatin in ameliorating olanzapine-induced weight gain through the mediation of energy expenditure (17). omega-3 fatty acids [ω-3 long chain polyunsaturated fatty acids (ω-3 pufas)], composed of docosahexaenoic acid (dha) and eicosapentaenoic acid (epa). animal studies suggested that increased consumption of the longchain omega-3 can protect against the development of obesity in animals exposed to an obesogenic diet and reduce body fat when already obese (18) (19). a recent study suggested that epa can activate thermogenic transcription factors in brown fat, namely, prdm16, pgc1α, and pparγ. this led to increased expression of ucp1 in bat which may subsequently contribute to energy expenditure and possibly reduced obesity and metabolic disorders (20). the aim of the present work is to evaluate the effect of simvastatin and omega-3 and there combination as anti-obesity drugs through their effect on the most common thermogenic gene marker “ucp1“using quantitative real time pcr in white and brown adipocyte of obese male wistar rat model where the obesity had been induced using high fat diet. material and methods animals and experimental design the research protocol and animal care procedures were approved by the local research ethics committee, college of pharmacy, university of baghdad, iraq, and in accordance with the standard requirements for the care and the use of experimental animal reported elsewhere. one hundred and twenty (120) male wistar rats, age of fiveto six-week-old weighing 100-150g were obtained from the local bred of the animal house, department of pharmacology and toxicology, university of baghdad and housed under light/dark cycle (12/12 hr) and controlled room temperature (24cº±2) with standard chow and drinking water ad libitum. after a week of acclimatization, all animals were fed for eight weeks with a high fat diet [(hfd) (standard chow contains 30% lard)] especially prepared for this purpose (21) in order to induce obesity and create an obese rat model. since the standard chow consist of carbohydrate 48.8%, protein 21%, and fat 3%, calcium 0.8%, phosphorus 0.4%, fiber 5%, moisture 13%, and ash 8% (22). after that, when rats get obese according to their body weights and body mass index (bmi) and body parameter. rats were randomly-allocated into five groups (each contains twenty-four rats), four receiving test compounds and one group continues feeding with hfd without any treatment and considered as a control (group i). twelve rats were housed /cage in order to reduce their activity inside the cage and reduce energy expenditure. rats groups were treated as follows: group ii: obese (hfd) rats administered pure simvastatin powder [ph. eur. artemis biotech (9 mg /kg /day)] via 14 inch oral iraqi j pharm sci, vol.31( 2 ) 2022 effect of simvastatin, omega-3 and their combination on ucp1 103 gavage needle within 2cc running water (17); group iii: obese hfd rats given double dose of pure simvastatin powder (18 mg/kg/day) via 14 inch oral gavage needle within 2cc running water (17). group iv: obese, hfd rats, given omega-3 fatty acids (oral dose 1 ml/day) [green field nutrition’s, inc. chicago, il.60625 u.s.a. fish oil =1000 mg, epa = 180 mg, dha =120 mg] with 14-inch oral gavage needle, and the group v: obese, hfd rats, given a combination of simvastatin (9mg/kg) and omega-3 fatty acids (1 ml/day) with 14-inch oral gavage needle (23, 24). in this experiment, normal diet group were excluded since the main target is to focus on the reduction in body weights in obese rats receiving test compounds in order to know if there is a hope to use such test compounds in obese human in the future clinical design. moreover, the design of this experiment depended on two different doses of simvastatin which is the main drug target required to be studied for its anti-obesity effect; for this reason 9mg/kg /day dose were selected from previous study (17) and duplication of this dose were taken into account in order to know if there is a difference in the onset of effect, in the potency, and in the possibility of occurring adverse effect in such high dose. while omega-3 dose which is (1ml/day) has been selected according to previous study (24) where they depend in their experiment on giving each rat 1ml of omega-3 rather than calculating the dose on kg. in order to ensure standardized dosage of eicosapentaenoic acid (epa) 180 mg and docosahexaenoic acid (dha)120 mg. three rats were sacrificed from each group [which is the minimum statistically acceptable rat group and has been performed in many previous studies to limit the variation (25-27)] (and this experiment have five group) in each week i.e., 15 rats euthanized weekly by diethyl ether (romia pure chemistry/ cambridge/uk). so, along the eight weekswhich is the total duration of this experiment -all the total number of rats i.e., 120 rats have been killed and the interscapular brown adipose tissue (ibat), and inguinal adipose tissue (iwat) were dissected. this present study has different module than any other experiment modules since the accumulated effect of test compound from week one till week eight must be measured in order to detect the exactly week that the onset of weight reduction with the expression of thermogenic gene ucp1 has been occurred. the ucp1 thermogenic genes expression was measured using quantitative real time pcr and triplicate pcr amplification has been done to reduce bias. moreover, uncoupling protein 1 (ucp1) protein in brown and brite adipocyte was detected using immunohistochemistry. statistical analysis data were analyzed using spss/ibm version 24. the numeric data were expressed as mean ± standard error of the mean (sem). the statistical significance of each group in comparison with the obese/high fat diet control group was determined by post hoc analysis in addition to independent ttest to confirm the results; while comparison among all groups were tested by one wayanova test. p-values less than 0.05 (p<0.05) were considered significant for all data presented in this study. results detection of ucp1 gene in brown adipose tissue results showed that a clear significant increase in (group ii) at fifth week (154.86± 8.46, p<0.05), while (group iii) gave significant increase in ucp1 expression from the first week of therapy (57.1667±1.74, p<0.05) moreover, (group iv) gave significant increase in ucp-1 expression in week two (76.19±2.79, p<0.05), while (group v) gave significant expression in ucp-1 from the first week of treatment (70.93±3.92, p<0.05) as shown in table 1. furthermore, there was significant increase (p<0.05) in ucp-1 expression among all groups from the first week of treatment by using one-way anova test. table 1. effects of two-different doses of simvastatin, omega-3, and combination of simvastatin and omega3 on the ucp1 gene expression level in brown adipose tissue (bat) weekly. hfd obese s 9 mg s18 mg omega-3 o+s group i group ii group iii group iv group v week_1 40.667±4.029 43.58±3.2 57.1667±1.74* 48.06±5.411 70.93±3.92* week_2 17.69±2.98 64.86±6.11 140.74±10.207* 76.19±2.79* 201.6±26.15* week_3 73.533±8.897 108.39±10.7 140.63±14.65* 166.24±18.409* 291.33±10.47* week_4 92.933±10.22 127.47±11.15 222.66±16.43* 261.26±11.01* 396.23±45.48* week_5 54.966±3.653 154.86±8.46* 326.3±12.77* 296.83±24.98* 512.11±48.79* week_6 42.92±6.33 131.056±10.194* 302.5±22.33* 626.71±27.63* 977.03±41.4* week_7 58.45±5.1 183.466±6.78* 585.86±30.99* 901.17±22.72* 929.66±44.78* week_8 59.8±1.5 195.3±11.113* 885.18±31.76* 759.23±37.32* 939.93±28.39* values are expressed as mean ± sem; n=24 rats in each group; (*) refers to significant difference in groups (p<0.05) compared to group i; time represent treatment in each week. iraqi j pharm sci, vol.31( 2 ) 2022 effect of simvastatin, omega-3 and their combination on ucp1 104 figure 1. effects of two-different doses of simvastatin, omega-3, and combination of simvastatin and omega3 on ucp 1 genes in brown adipose tissue (bat). detection of ucp1 gene in white adipose tissue (wat) results showed that significant increase started from the fifth week in (group ii) when compared to obese hfd (group i) rats (400.34±9.5, p<0.05); while, obvious significant increase occurs from the fourth week in (group iii) rats (372.91±32.41, p<0.05); and from the third week in (group iv) (412.67±34.42, p<0.05), and the third week in (group v) (412±11.05, p>0.05) as shown in table 2. moreover, the anova test gave a significant increase among all groups from the first week of treatment which explained activation of ucp1 gene in bat from the first week of therapy. figure 2 demonstrated effects of two-different doses of simvastatin, omega-3, and combination of simvastatin and omega3 on ucp-1 genes in white adipose tissue (wat). table 2. effects of two-different doses of simvastatin, omega-3, and combination of simvastatin and omega3 on the ucp1 gene expression level in white adipose tissue (wat) weekly. hfd obese s 9 mg s18 mg omega-3 o+s group i group ii group iii group iv group v week_1 180.36±16.799 64.097±7.24 107.313±2.819 97.065±4.79 191.667±6.53 week_2 213.29±22.8 162.136±2.017 131.19±10.04 195.93±2.74 191.8±11.85 week_3 285.33±16.36 270.136±8.978 229.48±15.67 412.67±34.42* 412±11.05* week_4 135.72±13.49 176.23±8.03 372.91±32.41* 508.58±35.03* 728.62±35.5* week_5 191.51±7.522 400.34±9.5* 445.78±50.2* 774.0667±119.6* 1557.18±91.31* week_6 191.12±9.11 792.97±50.57* 885.014±137.93* 1622.3±139.98* 3082.5±94.01* week_7 166.1667±25.05 811.713±91.6*9 1757.13±34.142* 1997.67±162.46* 2684.9±55.38* week_8 173.1916.569 1221.82±123.89* 2992.8±61.28* 2826.03±99.3* 3424.28±151.041* values are expressed as mean ± sem; n=24 rats in each group; (*) refers to significant difference in groups (p<0.05) compared to group i; time represent treatment in each week. iraqi j pharm sci, vol.31( 2 ) 2022 effect of simvastatin, omega-3 and their combination on ucp1 105 figure 2. effects of two-different doses of simvastatin, omega-3, and combination of simvastatin and omega3 on ucp 1 genes in white adipose tissue (wat). detection of ucp1 protein using immunohistochemistry this study showed the ability of simvastatin and omega-3 to induce weight loss by activation of bat in rodents with the recruitment of brown fat in wat depots as shown in the image 1. (a, b, c, d, and e in white adipocyte) and (a, b, c, d, and e in brown adipocyte). rat weight measurements body weight measurements is the corner stone in this experiment, for this reason, body weight has been weekly measured in all rat groups and results showed that there was significant decrease (p<0.05) in rat body weight at the fifth week in (group ii), and from the fourth week in (group iii); while in (group iv); the decrease in body weight occurs at the week three of therapy; moreover, (group v) gave significant decrease in body weight from first week as in table 3 and figure 3, 4, 5, and 6. while one way anova test results showed a significant decrease in body weights started from the first week of therapy (p<0.05) among treated groups table 3. values of body weight of male wistar rats through eight weeks reduction of body weights. hfd obese s 9 mg s18 mg omega-3 o+s group i group ii group iii group iv group v week_1 311±5.47 377.6±7.8 395.4±5.31 339±3.6 345.45±8.25* week_2 348.14±5.89 367.38±7 366.2±7.7 330.5±7.8 320.8±8.35* week_3 369.5±6.54 376±8.13 359.7±5.7 329.9±5.4* 333.8±9.5* week_4 371.6±8.4 356±5.89 350.8±3.9* 321.2±3.9* 321.8±5.5* week_5 372.9±6.72 337±3.46* 336.66±4.16* 316.5±5.49* 317.4±4.3* week_6 387.3±10.1 331.5±2.93* 333.55±5.05* 315.4±6.2* 312.8±4.9* week_7 401.33±7.62 330.8±4.85* 327.8±5.5* 315.16±7.02* 310.5±7* week_8 417.67±4.33 255.6±8.68* 255.6±8.68* 292±4.61* 280±5.7* values are expressed as mean ± sem; n=24 rats in each group; (*) refers to significant difference in groups (p<0.05) compared to group i; time represent treatment in each week. iraqi j pharm sci, vol.31( 2 ) 2022 effect of simvastatin, omega-3 and their combination on ucp1 106 a. detection of ucp1 protein in iwat after eight weeks treatment with simvastatin 9 mg/kg/day c. detection of ucp1 protein in iwat after eight weeks treatment with omega-3 alone. b. detection of ucp1 protein in iwat after eight weeks treatment with simvastatin 18 mg/kg/day d. detection of ucp1 protein in iwat after eight weeks treatment with omega-3 and simvastatin 9mg/kg/day e. iwat in obese high fat diet a.detection of ucp1 protein in ibat after eight weeks treatment with simvastatin 9mg/kg/day b. detection of ucp1 protein in ibat after eight weeks treatment with omega-3 and simvastatin 9mg/kg/day c. detection of ucp1 protein in ibat after eight weeks treatment with simvastatin 18 mg/kg/day image 1. detection of ucp1 protein by immunohistochemistry in iwat after eight weeks treatment(a) with simvastatin 9mg/kg/day, (b) with simvastatin 18 mg/kg/day, (c) with omega-3 alone, (d) with omega-3 and simvastatin 9mg/kg/day, compared to (e). iwat in obese high fat diet. and detection of ucp1 protein by immunohistochemistry in ibat after eight weeks treatment, (a) with simvastatin 9mg/kg/day, (b) with omega-3 and simvastatin 9mg/kg/day, (c) with simvastatin 18 mg/kg/day, (d) with omega-3 alone compared to (e) ibat in obese high fat diet. iraqi j pharm sci, vol.31( 2 ) 2022 effect of simvastatin, omega-3 and their combination on ucp1 107 d. detection of ucp1 protein in ibat after eight weeks treatment with omega-3 alone. e. ibat in obese high fat diet continued image 1. figure 3. demonstrate the effect of preand post-treatment with simvastatin 9 mg on body weights in male wistar. figure 4. demonstrate the effect of preand post-treatment with simvastatin 18mg on body weights in male wistar. iraqi j pharm sci, vol.31( 2 ) 2022 effect of simvastatin, omega-3 and their combination on ucp1 108 figure 5. demonstrate the effect of preand post-treatment with omega-3 (1ml/day) on body weights in male wistar. figure 6. demonstrate the effect of preand post-treatment with omega-3 and simvastatin on body weights in male wistar rats. images no. 1 (a, b, c, d and e) clearly demonstrated the differences in body fat accumulation in the abdominal region of obese rats treated with two different doses of simvastatin (group ii and iii) and omega-3 (group iv) and the co-administration of omega 3 and simvastatin (group v) compared to hfd (group i) rats. iraqi j pharm sci, vol.31(2) 2022 effect of simvastatin, omega-3 and their combination on ucp1 109 aimage represents an obese high fat diet rat without treatment (group i) bimage represents an obvious weight loss after eight weeks in rats receiving 9 mg simvastatin. (group ii). cimage represents an obvious weight loss after eight weeks in rats receiving 18mg simvastatin. (group iii). dimage represents an obvious weight loss after eight weeks in rats receiving omega-3 treatment (group iv). eimage represents an obvious weight loss after eight weeks in rats receiving omega-3 and simvastatin co-treatment (group v). images no 1. a, b, c, d and e; represent a clear difference in body profile of male wistar rat after eight weeks of treatment comparing to obese rat without treatment. iraqi j pharm sci, vol.31(2) 2022 effect of simvastatin, omega-3 and their combination on ucp1 110 discussion this study focused on the role of simvastatin that utilized in two different doses (9mg/kg/day) and its double dose (18mg/kg/day) on the level of thermogenic genes mainly ucp1 gene in brown and white adipocyte, in an attempt to upregulation of a thermogenic program using simvastatin, omega-3 and its co-treatment; with a subsequent protection against weight gain for this aim, the induction of obesity in male rats was done with hfd (group i) for eight weeks before starting the experiment. male rat is preferred in such experimental model more than female, because induction of obesity is more easier in male, since estrogen hormones in female rats found to protect against fat adipose tissue accumulation through its action on estrogen α (erα) and estrogen β (erβ) (28). studies found that, once precursor cells are induced to differentiate into brown/brite adipocytes by growth factors or hormones, a whole network of transcriptional modulators are activated to regulate the expression of specific genetic programs which are responsible for the acquisition of their unique phenotype and function (19, 20). early studies indicated that both white and brown/brite adipocytes share a transcriptional cascade that controls the adipogenic process, including the transcriptional factors ccaat/enhancer binding protein c/ebpβ, c/ebpα and pparγ that act sequentially to control the hundreds of genes involved in the common adipogenic program (29). however, the differentiation of precursors cells into brown/brite adipocytes requires specific transcriptional regulators that are responsible for the acquisition of their differential “thermogenic” feature including prdm16 protein which has been revealed as a key element for the differentiation of both brown and brite adipocytes and the peroxisome proliferator activated receptor gamma co-activator 1α (pgc1α) (20, 30). however, according to previous study; the expected thermogenesis mechanism could be postulated as the following; ppar γ recruits prdm16 transcription factor to form a core transcription complex (31) and ppar γ/ prdm16 complex recruits pgc-1α (32), pgc-1α, which considered as a transcriptional co-activator , activates camp dependent pka, responsible for lipolysis and thermogenesis in bat through β3 receptor control under ne (33); and this leads to increased expression of ucp 1 in brown/beige adipose tissue which may subsequently contribute to energy expenditure with a possibility to reduce obesity and metabolic disorders (20, 34, 35). brown adipose tissue (bat) considered as an important thermogenic tissue; the thermogenic mechanism occurs when free fatty acids are burned in brown adipocytes during uncoupling respiration when uncoupling protein 1 (ucp1) are inserted in the inner mitochondrial membrane to produce the heat necessary for maintenance of the euthermic state (36). omega-3 fatty acids supplements provides various health benefits in a tissue-specific manner; since many studies have been shown that administration of omega-3 results in increase energy expenditure in muscle (37). furthermore, the metabolic benefits of pufa derived from fish oil resemble the adaptive metabolic responses upon brown/beige fat activation through activation of adaptive thermogenesis process. for this reason, n3 pufa intake has gained attention as a dietary regimen to promote thermogenesis (38). therefore, omega-3 can exert dual benefits in obesity by reducing lipid accumulation in wat with a consequent activation of thermogenesis and reducing lipogenesis in bat (39). omega-3 polyunsaturated fatty acids induce brown and beige adipocyte differentiation and thermogenic activation (18), and these effects require gpr120. gpr120 activation results in activation of thermogenic transcription factors in brown fat, namely, prdm16, pgc-1α, and pparγ. this leads to increase expression of ucp 1 in brown adipose tissue with a consequence increase in energy expenditure and reduction in body weight (4, 40). this study gave a significant increase in ucp1 expression level in bat from fifth week of simvastatin 9mg/kg/day treatment (group ii), while simvastatin treatment 18mg/kg/day (group iii) and omega-3 combined with simvastatin (9mg/kg/day) (group v) gave significant increase in ucp1 expression from the first week of therapy, moreover, treatment with omega-3 (group iv) gave significant increase in ucp-1 expression from second week when compared to obese hfd (group i) rats. results in white adipose tissue (wat) demonstrate a significant up-regulation in ucp1 gene expression level from the fifth week after treating with simvastatin 9mg/kg/day treatment (group ii), and from fourth week of simvastatin at18mg/kg/day therapy (group iii). however, treating with omega-3 (1ml/day) (group iv) results from increase ucp1 gene expression from third week of therapy, also in case of mixed dose of simvastatin and omega-3 (group v) rats where ucp1 gene expression level from increased from third week of therapy (4). to our knowledge no study was conducted to study the effect of simvastatin, omega 3 and its cotreatment. thus, we cannot have a chance to compare results of this study with others. moreover, the current study opens the door for further research in this field. conclusion this study gives a hope for the utilization of simvastatin and omega-3and its combination has been shown to have the ability to turn on a thermogenic gene program in brown fat and activate a “browning” of 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adipocytes. nat commun. 2016;7:13479. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(1) 2012 effect of iv fluids on postoperative pain 34 effect of administration of crystalloid iv fluids preoperatively on postoperative pain # zeena m. al-nema* ,1 , samer i. mohammed* and fadia t. ahmed* *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq abstract pain is a sensory and emotional experience that is influenced by physiologic, sensory, affective, cognitive, socio-cultural and behavioral factors. postoperative pain is the commonest reason for delayed discharge and unanticipated hospital admission after ambulatory surgery. our objective is to test the hypothesis that administration of 2 ml/kg/hr preoperative iv fluids may attenuates postoperative pain.the study was carried out in the baghdad teaching hospital, al-yarmok teaching hospital and al-karama teaching hospital from 12 may till 17 june 2009. the total number of patients was 120 (35 males and 85 females) with their age ranged between 10-90 years. the patients were divided into two groups according to administration of preoperative iv fluids, group a (65 patients) did not receive iv fluids and group b (55 patients) received iv fluids preoperatively. regarding group a, the pain scale was ≤ 5 in 15.3% of patients, and it was >5 in 84.7% of patients and these results obtained within 0-5 hours after awaking from anesthesia. whereas in group b, the pain scale was ≤5 in 29.09% of patients and was >5 in 70.9% of patients. we have demonstrated that the preoperative administration of 2ml/kg/hr iv fluids (crystalloid) to patients who had fasted from fluids decreased the severity of postoperative pain, and the need for postoperative analgesia. we report for the first time that administration of large volume preoperative iv fluids significantly reduce the incidence and severity of pain in patients at high risk for pain. key words: i.v fluids, pain, surgery. بعد العمليتاأللم تأثير اعطبء محبليل وريديت جسيئيت قبل العمليت على شدة زينت مظفر النعمت* ،1 ، سبمر عمبد محمد* و فبديت ثبمر احمد* .* فشع انصٍذنت انسشٌشٌت ، كهٍت انصٍذنت ، جامعت بغذاد ، بغذاد ، انعشاق الخالصة بانعىامِم انسهىكٍِت وانثقافٍِت واالجخماعٍت واإلدساكٍِت وانعاطفٍِت وانحّسٍِت وانفسهجٍِت األنم حجشبتُ حّسٍتُ وعاطفٍتُ انخً حُخأثّشُ اس .األنم ما بعذ انجشاحت انمسبُب انمشخشُك نإلطالِق انمخأخِش ودخىِل انمسخشفى انغٍش مخىقّعِ بعذ انجشاحِت انمخىقهِت.هذفىا كان الخخب انذساست وُفّزْث فً ساعت( قبم انجشاحِت قَْذ حُخفُّف أنَم ما بعذ انجشاحتَ.\كغم\ممِٕت )بمقذاس انفشضٍِت انخً حفٍذ بأن اعطاء سىائم وسٌذٌ ًّ 2ٌٕٓٓىوٍى/حزٌشاِن 1ٔحخى ٕٔمسخشفى بغذاد انخعهٍمً، مسخشفى انٍشمىك انخعهٍمً، ومسخشفى انكشامت انخعهٍمً ِمْه . انعذد انكه َ٘ٙسىَت. انمشضى قُّسمىا إنى مجمىعخٍه انمجمىعِت انف ) 2ٓ-ٓٔشاوحْج بٍه أوثى( بأعماس حَ 5٘ركش و ٖ٘) ٕٓٔنهمشضى َكان ( اسخهمىا سىائم وسٌذٌت قبم انجشاحت.بخصىص انمجمىعت انف ، ِمقٍاس األنَم ٘٘مشٌض( نَْم حُسخهْم آي محانٍم وسٌذٌت وانمجمىعت باء ) ساعاث بعذ ٘ٓشضى وهزي انىَخائِج حم انحصىل عهٍها خالل % ِمْه انم 5ٗ.1فً ٘% ِمْه انمشضى، وَكاَن< ٘ٔ. ٖفً َ٘كاَن> % ِمْه مشضى 1ٓ.2فً ٘% ِمْه انمشضى وَكاَن< 2ٓ.2ٕفً ٘انَصحىة ِمْه انخخذٌِش بٍىما فً انمجمىعت باء ، ِمقٍاس األنَم َكاَن> ا مه وقض بانسىائم قذ قهم مه شذَّةَ األنِم ما بعذ بٍَّىَّا بأنَّ حقذٌم سىائم وسٌذٌت بكمٍاث كبٍشة ما قبم انجشاحت نهمشضى انزٌه عاوى ساعت( ما قبم انجشاحِت \كغم\ممٕانجشاحِت، وانحاجت نمسكىاث األنم ما بعذ انجشاحِت.وَْزكُش نهمشة األونى بأّن حقذٌم سىائم وسٌذٌِت )بمقذاس ًِ نألنمِ حُخفُّض بشكم مهحىظ حذود األنم وشذَّةَ األنِم فً انمشضى انمعشضٍه نهخطِش انعا .ن : محبليل وريديت ، األلم ، الجراحت المفتبحيت الكلمبث introduction pain is a sensory and emotional experience that is influenced by physiologic, sensory, affective, cognitive, socio-cultural, and behavioral factors (1) .postoperative pain is the commonest reason for delayed discharge and unanticipated hospital admission after ambulatory surgery (2) .pain causes an increase in the sympathetic response of the body with subsequent rises in heart rate, cardiac work and oxygen consumption. prolonged pain can reduce physical activity and lead to venous stasis and an increased risk of deep vein thrombosis and consequent pulmonary embolism. in addition, there can be widespread effects on gut and urinary tract motility which may lead, in turn, to postoperative ileus, nausea, vomiting and urinary retention. these problems are unpleasant for the patient and may prolong hospital stay (3) . # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : zena_alnema@yahoo.com received : 5/3/2011 accepted : 7/1/2012 mailto:zena_alnema@yahoo.com iraqi j pharm sci, vol.21(1) 2012 effect of iv fluids on postoperative pain 35 the site of the surgery has a profound effect upon the degree of postoperative pain a patient may suffer. operations on the thorax and upper abdomen are more painful than operations on the lower abdomen which, in turn, are more painful than peripheral operations on the limbs. however, any operation involving a body cavity, large joint surfaces or deep tissues should be regarded as painful. in particular, operations on the thorax or upper abdomen may produce widespread changes in pulmonary function, an increase in abdominal muscle tone and an associated decrease in diaphragmatic function. the result will be an inability to cough and clear secretions which may lead to lung atelectasis (collapse of lung tissue) and pneumonia. matters are made worse by postoperative bowel distension or tight dressings (1) .post-operative pain increases the possibility of post-surgical complications, raises the cost of medical care, and most importantly, interferes with recovery and return to normal activities of daily living. management of post-surgical pain is a basic patient right. when pain is controlled or removed, a patient is better able to participate in activities such as walking or eating, which will encourage his or her recovery. patients will also sleep better, which aids the healing process (4) . preoperative correction of intravascular volume deficits effectively reduces postoperative pain in high risk patients presenting for ambulatory surgery (5) .studies clearly demonstrate the potential for large volume iv fluids (2ml/kg/hr crystalloid) to markedly reduce postoperative pain. 2ml/kg/hr iv fluids (crystalloid) markedly reduced both the mean severity of pain and the worst pain scores in these patients, and the need for supplemental parenteral opioid and oral analgesia (5) .they demonstrated a marked trend toward less opioid and nonsteroidal antiinflammatory use in the patients who received 2ml/kg/hr iv fluids (6) .a further novel finding is that the treatment effect was prolonged, and was still present at 72 hours postoperatively (5) .in fact, 2ml/kg/hr crystalloid iv fluids significantly reduced the combined need for analgesic and antiemetic medication in those studies (7) .other study reported that few patients who received preoperative iv fluids complained of moderate or severe postoperative pain (8) .in summary, the potential for preoperative iv fluid regimens to modulate the severity of postoperative pain and analgesic requirements and the demonstration of the analgesic potential of preoperative large volume crystalloid iv fluids is clear (9) . methodology the study was carried out in the baghdad teaching hospital, al-yarmok teaching hospital and al-karama teaching hospital from 12 may till 17 june 2009.the total number of patients was 120 (35 males and 85 females) with their age ranged between 1090 years.patients were divided into two groups according to the administration of preoperative iv fluids, group a (65 patients) did not receive crystalloid iv fluids and group b (55 patients) received crystalloid iv fluids preoperatively.patients were referred to the surgical wards and underwent different types of laparotomies: intestinal obstructions, caesarian sections, appendicectomies… others). all patients were suffering from postoperative pain.for each patient a short clinical notes were recorded which include: type and duration of surgery, type and quality of iv fluids prescribed, associated diseases, drugs used, type and dose of postoperative analgesia.every patient was interrogated to specify the degree of postoperative pain according to the comparative pain scale of the mayo clinic (10) .some patients were difficult to communicate with, while others were uncooperative because of the surgery and some tried to exaggerate the pain which may affect scoring of pain.this needed an extra work to explain the type of pain and to get the exact description of the pain related to the scale used.pain was measured within 5 and 24 hr after the patient was fully awake from anesthesia according to the comparative pain scale of the mayo clinic (10) .statistics were performed using standard statistical program (spss inc, version 15). demographic data were analyzed by using chi-square test. all p values ≤0.05 were considered significant. results most of the patients in group a (who did not receive preoperative iv fluids) underwent appendectomy, cholecystectomy and caesarian section, whereas, most of the patients in group b (who received preoperative iv fluids) underwent caesarian section and correcting intestinal obstruction, (table 1).this is important regarding the severity of pain. regarding group a, the pain scale was ≤5 in 15.3% of patients, and it was >5 in 84.7% of patients and these results obtained within 0-5 hours after awaking from anesthesia.whereas in group b, the pain scale was ≤5 in 29.09% of patients and was >5 in 70.9% of patients, (table 2). table 3 shows that only 10.1% of patients in group a did not use postoperative analgesia while 90% of them used either iraqi j pharm sci, vol.21(1) 2012 effect of iv fluids on postoperative pain 36 tramadol (77.9%) or diclofenac sodium (22.0%). whereas 18.18% of patients in group b did not use postoperative analgesia while 81.82% of patients used either tramadol (58.18%) or diclofenac sodium (23.64%).the results in table 3 show that the use of analgesics was significantly reduced (p<0.05) in group b when compared with group a and this reflects that pain is less in group b when compared with group a. in group a 64% of the patients have no associated disease, and the rest (36%) with associated disease mainly hypertension alone (56%) and other have hypertension with ischemic heart disease, or with diabetes mellitus, in addition to diabetes mellitus or asthma alone, (table 4). whereas 65% of the patients in group b have no associated disease, and (35%) with associated disease, hypertension (52%) and other have hypertension with diabetes mellitus, or diabetes mellitus, anemia, or asthma alone. in both groups (84%) of the patients were nonsmoker. seventy percent of the patients were females. eighty percent of the patients underwent long surgeries lasting more than one hr. table 5 demonstrates that the overall incidence of pain in the 1 st 5 hours was significantly (p<0.05) reduced in group b compared with patients in group a. table 1: relation between type of surgery and number of patients in group a (who did not receive preoperative iv fluids) and b (who received preoperative iv fluids) table 2: postoperative pain within 5 hours in group a and b pain scale of the mayo clinic (10) number of patients group a % of patients group a number of patients group b % of patients group b less or equal 5 10 15.3 16 29.09 more than 5 55 84.6 39 70.9 total 65 100 55 100 table 3 : use of analgesics in group a and b use of analgesics number of patients group a % of patients group a number of patients group b %of patients group b without analgesic 6 10.1 10 18.18 tramadol 46 77.9 32 58.18 diclofenac sodium 13 22.0 13 23.64 total 65 100 55 100 type of surgery no .of patients in group a % .of patients in group a no. of patients in group b % .of patients in group b colectomy 1 1.5 appendectomy 19 29.2 3 5.4 cholecystectomy 11 16.9 4 7.2 ovarian cyst 1 1.5 hysterectomy 4 6.1 2 3.6 perforated ulcer 1 1.5 4 7.2 splenectomy 2 3.0 intestinal obstruction 3 4.6 15 27.27 hernia 5 7.9 2 3.6 caesarian section 15 23.0 15 27.27 pyeloplasty 1 1.5 perforated colostomy 1 1.5 hydatid cyst 1 1.5 2 3.6 colostomy 1 1.8 gastrectomy 2 3.6 curitage 1 1.8 removal of anus 1 1.8 peritoneal wash 1 1.8 closure jejunectomy 1 1.8 abdominal trauma 1 1.8 iraqi j pharm sci, vol.21(1) 2012 effect of iv fluids on postoperative pain 37 table 4: comparison between the presence of associated diseases in group a and b. associated diseases % of patients in group a %of patients group b without an associated disease 64 65 with an associated disease 36 35 table 5: comparison between the incidence of pain within 1 st 5 hours in group a and b. group b group a pain scale 29% 23% less than 5 70.9% 77% more than 5 discussion postoperative pain continues to be a common and distressing complication of surgery. we have demonstrated that the preoperative administration of 2ml/kg/hr crystalloid iv fluids to patients who had fasted from fluids decreased the severity of postoperative pain, so minimizes the need for postoperative analgesia, by correction of intravascular volume deficit (5) .the study clearly demonstrates the potential for 2ml/kg/hr crystalloid iv fluids which markedly reduce postoperative pain, and the need for supplemental parenteral opioid and oral analgesia, this fact was supported by other studies like that done by ali et al (6) .a further finding is that the treatment effect was prolonged for 72 hours postoperatively. in fact, a close examination of other studies of preoperative fluid regimens does reveal evidence for reduced postoperative pain with 2ml/kg/hr preoperative iv fluid therapy, although this has received little attention. ali et al demonstrated a marked trend toward less opioid and nonsteroidal antiinflammatory use in the patients who received the 2ml/kg/hr iv fluids (6) . in a study done by yogendran et al, concluded that less patients in the large volume group required postoperative morphine (9) . cook et al reported less codeine and acetaminophen use in the large volume group of their study (7) . it is reported that large volume iv fluids significantly reduced the combined need for analgesic and antiemetic medication (7) . ooi et al (8) reported that few patients who received preoperative iv fluids complained of moderate or severe postoperative pain. in the study by moretti et al comparing the effect of crystalloids and colloids, colloid was used for intra-operative resuscitation in 90 patients undergoing elective non-cardiac surgery; they found that the incidence of nausea and vomiting, severe pain, periorbital oedema, double vision and the use of rescue antiemetics was significantly reduced in patients receiving colloids (11) .the finding that large volume iv fluids decreased postoperative pain and opioid requirements raised the possibility that this could alone account for the decreased postoperative nausea and vomiting (12) . and this in turn will reduce the postoperative pain because it will reduce the tension and abdominal muscles produced by the vomiting. since women experienced more severe postoperative pain and required greater dose of analgesics than men in the immediate postoperative period, as reported by aubran in 2005, therefore we decided that female patients in this study are greater in number than male patients in order to achieve better estimation of the results (13) . in addition to the effects of analgesics and iv fluids for postoperative pain, preoperative nursing intervention for pain has positive effect for patient undergoing abdominal surgery (14) .in summary, the potential for preoperative iv fluid regimens to modulate the severity of postoperative pain and analgesic requirements is clear. the current study provides clear demonstration of the analgesic potential of preoperative large volume (2ml/kg/hr) iv fluids. the mechanism underlying these analgesic effects remains to be determined. in conclusion the study reports for the first time the potential for large volume (2ml/kg/hr) preoperative iv administration of a balanced salt solution to significantly reduce the incidence and severity of pain in patients at high risk for pain. the potential for large volume (2ml/kg/hr) iv fluids to produce a sustained reduction in pain is novel due to its ability to reduce postoperative pain and opioid and simple analgesic requirements. we recommend the administration of a volume of 2 ml/kg for every hour of fasting to high risk patients undergoing ambulatory surgical procedures. references 1. crews j c. multimodal pain management strategies for office-based and ambulatory procedures. jama 2002; 288: 629-632. 2. caumo w , schmidt a , schneider c, bergmann j , iwamoto c , adamatti l , bandeira d , ferreira m. preoperative predictors of moderate to intense acute postoperative pain in patients undergoing abdominal surgery. acta anasthesiol scand 2002; 46(10) : 1265. 3. schecter w p, bongard f s, gainor b j, weltz d l, horn j k. pain control in iraqi j pharm sci, vol.21(1) 2012 effect of iv fluids on postoperative pain 38 outpatient surgery. j am coll surg 2002; 195: 95-104. 4. mccaul c, moran c, o'cronin d, et al. intravenous fluid loading with or without supplementary dextrose does not prevent nausea, vomiting and pain after laparoscopy. can j anaest6. ali sz, taguchi a, holtmann b, kurz a. effect of supplemental pre-operative fluid on postoperative nausea and vomiting. anaesthesia 2003; 58: 780–4. 5. maharaj ch, kallam sr, malik a, hassett p, and j. g. laffey, preoperative intravenous fluid therapy decreases postoperative nausea and pain in high risk patients . anesth analg 2005 ,100(3): 675-682. 6. ali sz, taguchi a, holtmann b, kurz a. effect of supplemental preoperative fluid on postoperative nausea and vomiting. anaesthesia 2003; 58: 780–4. 7. cook r, anderson s, riseborough m, blogg ce. intravenous fluid load and recovery: a double-blind comparison in gynaecological patients who had day-case laparoscopy. anaesthesia 1990; 45: 826– 30. 8. ooi lg, goldhill dr, griffiths a, smith c. iv fluids and minor gynaecological surgery: effect on recovery from anaesthesia. br j anaesth 1992; 68: 576–9. 9. yogendran s, asokumar b, cheng dc, chung f. a prospective randomized double-blinded study of the effect of intravenous fluid therapy on adverse outcomes on outpatient surgery. anesth analg 1995;80:682–6. 10. gold bs, kitz ds, lecky jh, neuhaus jm. unanticipated admission to the hospital following ambulatory surgery. jama 1989;262:3008–10. 11. moretti ew, robertson km, el-moalem h, gan tj.intraoperative colloid administration reduces postoperative nausea and vomiting and improves postoperative outcomes compared with crystalloid administration. anesth analg 2003;96 : 611-7. 12. apfel cc, laara e, koivuranta m, et al. a simplified risk score for predicting postoperative nausea and vomiting: conclusions from cross-validations between two centers. anesthesiology 1999; 91: 693700. 13. aubrun f, salvi n, coriat p, riou b. sex and age related differences in morphine requirements for postoperative pain relief. anesthesiology 2005;103 (1): 156-60. 14. lin l, wang r. abdominal surgery pain and anxiety: preoperative nursing intervention. j adv nurs 2005 ; 51(3) : 252-60. iraqi j pharm sci, vol.31(suppl.) 2022 doxorubicin combination with baicalein and resveratrol doi: https://doi.org/10.31351/vol31isssuppl.pp92-99 92 cytotoxic evaluation of doxorubicin combination with baicalein and resveratrol against hct116 and hepg2 cancer cell lines (conference paper) # wafa naji shnaikat*, ekbal h. al-khateeb**, nawfal am. numan*,***,1 manal m. abbas**** and ashok k. shakya* # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *faculty of pharmacy, al-ahliyya amman university, amman, jordan **college of pharmacy, alkitab university, kirkuk, iraq *** department of pharmacy, al-marrif university college, anbar, iraq **** faculty of allied medical sciences, al-ahliyya amman university, amman, jordan abstract the combination of natural polyphenolic compounds with chemotherapeutic agents is recently being a novel strategy in cancer therapy research owing to their potential antioxidant and anti-inflammatory properties that modulate several intracellular signaling pathways. baicalein and resveratrol are well known polyphenolic compounds that belong to flavone and stilbene subclasses, respectively. this study aims to investigate the possible enhancement effect of baicalein and resveratrol when combined with doxorubicin using a different combination ratio and applied on two cancer cell lines: hct116 (colorectal cancer cells) and hepg2 (hepatocellular cancer cells). it also investigates the possibility of such natural compounds to provide a protection effect on cardiocyte (h9c2) when baicalein and resveratrol treatment followed by doxorubicin is used. the two cancer cell lines were treated with different combination groups, including the combination between doxorubicin and baicalein or resveratrol and the combination between the three compounds using a different combination ratio for both treatment groups (i.e., two drugs or three drugs combination). treatment applied on cells, using cell density of 7000 cells /well and incubation time was 48 hrs. mtt test was performed to assay the cell viability. the results obtained showed that the cytotoxicity of doxorubicin in the two cancer cell lines has increased when combined with baicalein and resveratrol. doxorubicin ic50 decreased from 4.99 µg/ml to 0.3657 µg/ml and from 7.3 µg/ml to 0.676 µg/ml on hct116 and hepg2 cells, respectively, using constant combination ratio (1:1:1). the combination of doxorubicin, baicalein, and resveratrol has resulted in a less cardiotoxic effect compared to treatment with doxorubicin alone. this decrease was obviously seen when the three compounds were combined using a low concentration range and with a constant combination ratio.conclusion: combinations of baicalein and resveratrol with doxorubicin chemotherapeutic drug in-vitro had enhanced the cytotoxic activity of such a chemotherapeutic drug, while simultaneously eliminating its cardiotoxicity side effect. key words: doxorubicin, baicalein, resveratrol, ic50, combination index, cardiotoxicity. تقييم سميه الدوكسوروبسين في مزيج البايكلين والريزفيراترول ضد خطوط #) بحث مؤتمر (مختلفةخاليا سرطانيه *اشوك كومار شاكيهو ****، منال محمد عباس 1،**،** ، نوفل عبد المنعم نعمان **، اقبال حسن الخطيب *كاتيوفاء ناجي شن 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي األردن عمان، كلية الصيدلة، جامعة عمان االهلية، * العراق كركوك، الكتاب، جامعة الصيدلة، كلية ** العراق االنبار، قسم الصيدلة، كلية المعارف الجامعة، *** األردن. االهلية، عمان، جامعة عمان المساندة، كلية العلوم الطبية **** الخالصة الطبيعية وعوامل العالج الكيميائي استراتيجية جديدة في أبحاث عالج السرطان نظًرا لخصائصها المحتملة يعتبر الجمع بين مركبات البوليفينول المضادة لألكسدة والمضادة لاللتهابات التي تعدل العديد من مسارات اإلشارات داخل الخاليا. baicalein وresveratrol تي عائل هي مركبات بوليفينول معروفة جيدًا تنتمي إلى flavone وstilbene .الفرعية ، على التوالي مع دوكسوروبيسين باستخدام نسبة توليفة مختلفة ماعند دمجه resveratrolو baicalein لـ تهدف هذه الدراسة إلى التحقيق في تأثير التعزيز المحتمل درس إمكانية ت)الخاليا السرطانية الكبدية(. كما hepg2يم( و )خاليا سرطان القولون والمستق hct116وتطبيقه على سطرين من الخاليا السرطانية: ( القلب لخاليا وقائي تأثير لتوفير الطبيعية المركبات هذه مثل بالh9c2وجود العالج استخدام عند متبوًعا resveratrolو baicalein ـ ( عالج .doxorubicinبال بمجموعات سطرين تم السرطانية بين مختلطةالخاليا الجمع ذلك في بما ، و baicaleinو doxorubicinمختلفة resveratrol ين أو ثالثة أدوية(. تم تطبيق العالج على الخاليا باستخدام ءوالجمع بين المركبات الثالثة باستخدام نسب مختلفة لكال مجموعتي العالج )أي دوا لفحص صالحية الخلية. mttساعة. تم إجراء اختبار 48لية / بئر ومدة الحضانة خ 7000كثافة الخاليا ل الخلوية السمية أن عليها الحصول تم التي النتائج مع doxorubicinأظهرت دمجها عند زادت قد السرطانية الخاليا سطري و baicaleinفي resveratrol انخفض .doxorubicin ic50 ميكروغرام / مل إلى 7.3ميكروغرام / مل ومن 0.3657إلى ميكروغرام / مل 4.99من . 1corresponding author e-mail: ashokshakya@hotmail.com received: 15/7 /2022 accepted: 28/9 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp92-99 iraqi j pharm sci, vol.31(suppl.) 2022 doxorubicin combination with baicalein and resveratrol 93 (. 1: 1: 1، على التوالي ، باستخدام نسبة تركيبة ثابتة ) hepg2و hct116ميكروغرام / مل على خاليا 676 وحده. لوحظ هذا doxorubicinإلى تأثير أقل على القلب مقارنةً بالعالج بال resveratrolو baicaleinو doxorubicinأدى الجمع بين resveratrolو baicaleinإن توليفات من المركبات الثالثة باستخدام نطاق تركيز منخفض وبنسبة تركيبة ثابتة.النقص بشكل واضح عندما تم الجمع بين للعالج الكيميائي في المختبر قد عززت النشاط السام للخاليا لمثل هذا الدواء العالجي الكيميائي ، مع التخلص في الوقت نفسه من doxorubicinمع عقار . اآلثار الجانبية السامة للقلب . ، المؤشر المركب ، السمية القلبية doxorubicin ،baicalein ،resveratrol ،ic50الكلمات المفتاحية: introduction colorectal cancer (crc) is ranked in the third order of the most common cancers over the world and it becomes an emanating health problem and a leading cause of death among all types of cancer in both men and women (1). hepatocellular carcinoma (hcc) also is considered the third leading cause of death cases related to cancer worldwide. the number of cases diagnosed each year is more than half million. the hcc is characterized by its poor prognosis due to the lack of early observed symptoms and limited therapeutic options (2). doxorubicin (dox) is one of the most effective anticancer drugs in treating various forms of cancers; however, dox-induced cardiotoxicity (dic) limited its therapeutic effectiveness. the exact molecular mechanisms of (dic), include mechanisms dependent on mitochondrial dysfunction such as dox influence on the mitochondrial electron transport chain, redox cycling, oxidative stress (os), calcium dysregulation, and apoptosis pathways (3,4). the resistance of cancerous cells to chemotherapeutic agents is one of the main challenges in cancer treatment (5). due to such challenges and due to drawbacks and severe toxicity of chemotherapeutic agents used in cancer therapy, several approaches are being explored in cancer research that aim to overcome such problems. improving efficacy and providing less toxicity are the target of such investigations (6). the use of natural products/herbal medicines is one of such attractive investigations in cancer research (7). the combination of natural compounds with the conventional treatment of cancer such as chemotherapy and radiotherapy were found to enhance the efficacy and meliorate the side effects of such treatment modalities (8). polyphenolic compounds are the most abundant group of all phytochemicals (9). the primary characteristic that leads to their chemopreventive and therapeutic actions on cancer is their powerful antioxidant effect (10). baicalein (ba) and resveratrol (rsv) are polyphenolic compounds; where, ba is a flavone which is subclass of flavonoid (11) and is derived from chinese herbal medicine scutellaria baicalensis georgi (12); and rsv is a polyphenolic compound that is the most common example of stilbenes (13, 14) and is found in a variety of natural sources; it is highly abundant in red grapes; mainly in the skin, in berries, peanuts, red wine, pineapple and others. the present study aims to explore the possible enhancement effect of baicalein and resveratrol when combined with doxorubicin using a different combination ratio on two different cancer cell lines: hct116 (colorectal cancer cells) and hepg2 (hepatocellular cancer cells). it also investigates the possibility of such natural compounds to provide a protection effect on cardiocyte (h9c2) when baicalein and resveratrol treatment followed by doxorubicin. materials and method materials baicalein (ba), rsv and dox were obtained as reference standards with purity > 98% from adooq bioscience (canada). dulbecco's modified eagle medium (dmem) (high glucose and low glucose) and other cell culture materials were obtained from euroclone (italy). fetal bovine serum was obtained from biowest (france). dimethylsulfoxide (dmso, analytical grade) was obtained from gcc (uk). the viability of cells treated with dox, ba, and rsv was determined by 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (mtt kit) from promega corporation (usa). cell culture hct116, hepg2 cells & h9c2 rat myoblast cells were obtained from ecacc (uk). cells of hct116 and h9c2 were maintained in dmem (high glucose) medium supplied with 10% fetal bovine serum (fbs), 1% penicillin, and 1% streptomycin whereas hepg2 was maintained in dmem (low glucose) medium supplied with 10% fetal bovine serum (fbs), 1% penicillin, and 1% streptomycin. cells were incubated in a co2 incubator at 37c°, with 5% co2 and 95% filtered air. all the studies were conducted at faculty of pharmacy and medical sciences, al-ahliyya amman university, amman, jordan. drug combinations baicalein (ba), rsv & dox were prepared in 10 serial dilutions starting from 100 µg/ml. a stock solution of each agent was prepared in dmso with concentration not exceeding 0.5%, while the dilutions were obtained using a suitable medium. hct116 & hepg2 cells were treated with either fixed combination ratio (1:1:1) from the three agents over the concentration (conc) range 0.195 – 100 µg/ml or with non-fixed ratio using approximating values for the ic50 of ba & rsv and concentration range 0.0195 – 100 µg/ml of dox. agents were added in consecutive manner iraqi j pharm sci, vol.31(suppl.) 2022 doxorubicin combination with baicalein and resveratrol 94 starting always with ba followed by rsv and ends with dox. the h9c2 cells were treated with only one combination of the three agents, which is 1:1:1 in the conc range 0.0195 – 100 µg/ml. cell viability assay effect of ba, rsv & dox combination on hct116, hepg2 and h9c2 was evaluated by mtt assay. cells were seeded into 96-well plate at a density of 7000 cells/well in suitable medium. after 24hrs of incubation at 37 °c, cells were treated with these agents and their combination in various combination ratios and various conc for 48 hrs. the mtt was then carried out using mtt kit where 15µl of the reagent was added to each well and incubated for 4hrs at 37°c followed by the addition of 100µl of the solubilization stop/mix solution and incubation for 1hr before measuring the absorbance using microplate reader [enzyme linked immunosorbent assay (elisa)] at 590nm. data analysis the toxicity versus concentration curves (dose-response curves) of compounds and their combinations were obtained. the half-maximal inhibitory concentration (ic50) was determined for each compound and the combination of the three agents in a fixed ratio on each type of cell using systat software version (12.0). the effects of the combination were calculated for each experimental condition using the combination index (ci) method based on the median-effect analysis of chou and talalay,1984 (15) and by using compusyn software (version 1.0). ci<1, ci=1, and ci>1 indicates synergy, additive, and antagonism, respectively. statistical analysis all data were obtained from at least three independent experiments and expressed as mean±standard deviation (sd). comparisons of the different groups were performed using two-ways univariate analysis of variance (anova), graphpad prism software (version 7.0) was used. the p<0.05 was considered the minimal level of significance. results the ic50 for each compound (ba, rsv & dox) and for certain combinations of the three compounds and for each type of cells was determined (table 1) after using a range of doses (concentration) & 48hrs incubation time followed by mtt assays that were performed to assess cell growth inhibition. the ic50 then was used to generate fixed and non-fixed ratios for subsequent combination studies and for calculation of combination indices (cis).combination of the three compounds (ba, rsv & dox) using fixed ratio (1:1:1) over the range 0.195–100 µg/ml or nonfixed ratio where ic50 of the two natural compounds was used with dox that has concentration over the range 0.195–100 µg/ml. dose-response curves were obtained for the combination of the three compounds (fixed and nonfixed ratio) on hepg2 & hct116 respectively (figures 1&2) and on cardiocyte (figure 3). combination indices were calculated also for these combinations on the three types of cells (hct116, hepg2 and h9c2) (tables 2, 3 & 4). table 1. ic50 values of individual drugs and their combination treatment on hct116 and hepg2 cell lines. treatment type ic50 value (µg/ml) hepg2 cells hct116 cells doxorubicin 7.3 4.99 baicalein 59.4 50.87 resveratrol 27.8 35.15 doxorubicin + baicalein 0.287 0.175 doxorubicin + baicalein ic50 na na doxorubicin + resveratrol 0.49 3.34 doxorubicin + resveratrol ic50 na na doxorubicin+ baicalein + resveratrol 0.676 0.3657 doxorubicin+ baicalein ic50+resveratrol ic50 na na na: not applicable iraqi j pharm sci, vol.31(suppl.) 2022 doxorubicin combination with baicalein and resveratrol 95 figure 1. log concentration versus toxicity of dox , dox+ba+rsv (1:1:1) and dox+ba.ic50+ rsv.ic50 (non-fixed ratio) against hepg2 cell line. figure 2. log concentration versus toxicity of dox , dox+ba+rsv (1:1:1) and dox+ba.ic50+ rsv.ic50 (non-fixed ratio) against hct116 cells. figure 3. log concentration versus toxicity of dox and dox+ba+rsv (1:1:1) combination against h9c2 cells. iraqi j pharm sci, vol.31(suppl.) 2022 doxorubicin combination with baicalein and resveratrol 96 table 2. combination index (ci) for the combination of doxorubicin (dox), baicalein (ba) and resveratrol (rsv) using a constant ratio (1:1:1) against hct116 and hepg2 cell lines. doxorubicin + baicalein + resveratrol combination combination index (ci) at constant ratio (1:1:1) concentration (µg/ml) hepg2 hct116 rsv ba dox 0.80575 * 6.45071 100 100 100 0.66906 * 2.51902 50 50 50 0.67603 * 1.39832 25 25 25 0.19925 * 0.75036 * 12.5 12.5 12.5 0.37898 * 0.39897 * 6.25 6.25 6.25 0.23337 * 0.26753 * 3.125 3.125 3.125 0.10534 * 0.16359 * 1.562 1.562 1.562 0.12871 * 0.08182 * 0.781 0.781 0.781 0.19875 * 0.09682 * 0.391 0.391 0.391 0.42671 * 0.10021 * 0.195 0.195 0.195 * indicates synergism table 3. combination index (ci) for the combination of doxorubicin (dox), baicalein (ba) and resveratrol (rsv) using a non-constant ratio against hct116 and hepg2 cell lines. doxorubicin + baicalein + resveratrol combination combination index (ci) at non-constant ratio concentration (µg/ml) hepg2 hct116 rsv ba dox 0.58628 * 3.18083 30 50 100 0.62880 * 1.91412 30 50 50 0.60467 * 1.96734 30 50 25 0.50938 * 1.11004 30 50 12.5 0.40431 * 1.60200 30 50 6.25 0.22792 * 0.96704 * 30 50 3.125 0.45588 * 1.04398 30 50 1.562 0.44342 * 1.02333 30 50 0.781 0.53132 * 1.14895 30 50 0.391 0.48068 * 1.03185 30 50 0.195 * indicates synergism table 4. combination index (ci) for the combination of doxorubicin (dox), baicalein (ba) and resveratrol (rsv) using a constant ratio (1:1:1) on h9c2 cells. doxorubicin + baicalein + resveratrol combination combination index (ci) at constant ratio (1:1:1) concentration (µg/ml) h9c2 rsv ba dox 0.94673 100 100 100 0.33128 50 50 50 23.8670 ** 25 25 25 5.08621 ** 12.5 12.5 12.5 2.96175 ** 6.25 6.25 6.25 8.16311 ** 3.125 3.125 3.125 8.89197 ** 1.562 1.562 1.562 6.65993 ** 0.781 0.781 0.781 1.82591 ** 0.391 0.391 0.391 2.36383 ** 0.195 0.195 0.195 ** indicates antagonism. iraqi j pharm sci, vol.31(suppl.) 2022 doxorubicin combination with baicalein and resveratrol 97 discussion natural products baicalein (ba) and resveratrol (rsv) combinations along with doxorubicin (dox) at constant ratio (1:1:1) produced significant increase in dox toxicity compared to treatment with dox alone in both types of cancer cell line. the significant reduction in ic50 of dox was yielded from this combination (from 4.99µg/ml to 0.3657µg/ml and from 7.3µg/ml to 0.676µg/ml dox) on hct116 & hepg2, respectively. the interaction between the three compounds produced a synergistic effect that was predominant at a lower concentration of dox (12.50.195 µg/ml) in hct116 cells, while the synergistic interaction between the three compounds in hepg2 was independent of dose and was predominant at all concentration levels indicating that diversity in mechanisms of action of the three agents yielded better antiproliferative and anticancer activity on such cancerous cells. the cytotoxic effect produced by this combination in hepg2 cells was greater compared to hct116 cells, indicating higher sensitivity and better response of these cells to this combination at all concentration levels, however, the results conversely changed at lower concentration levels (0.78–0.195 µg/ml) where the response of hct116 to this combination was become more than that of hepg2 cells. the combination of non-constant ratios between the three compounds (dox + ba.ic50 + rsv.ic50) produced a greater enhancement in dox toxicity compared to treatment with dox alone in both cell lines where additive effects were obviously seen at a lower concentration of dox on hct116 cells and synergistic interaction at all concentration levels on hepg2 cells. the behavior or cell response to the cytotoxic action of this combination seems to be similar between the two cell lines (hct116 & hepg2) with minor variation at certain concentration levels. the results obtained from the combination of three drugs using the non-constant ratio (dox+ba.ic50+rsv.ic50) showed a greater cytotoxic effect compared to the combination between the three compounds and using the combination of constant ratio (1: 1: 1) (dox + ba + rsv) on both types of cell lines. such combinations between the three compounds and the use of two combination criteria are being studied for the first time, revealing a better cytotoxicity profile than the use of dox alone and on both types of cell line. comparing all previous results obtained with two or three drugs’ combinations revealed that three drugs combination and particularly the using of nonconstant ratio produced the best toxicity and also the best enhancement in dox toxicity in all cancerous cell line used in this study compared to all other drugs combinations, however combination between dox and ba using non-constant ratio (dox + ba.ic50) has the best cytotoxic activity among all combinations on hct116 cells. regarding h9c2 cells, dox showed sever toxic effect on such cells whereas ba showed a true safety profile at all concentration levels, when used on rat cardiomyocytes h9c2, similar behavior was reported with rsv except the moderate toxicity produced at the highest concentration (100µg/ml). significant cardio-protection and decrease in the dox toxicity achieved when the two naturally occurring compounds were combined with dox. best decrease in dox toxicity on this type of cells was obviously-noticed at lower concentration levels of the three compounds, which is being evaluated for the first time. this is truly-explained by the ci between the three compounds that was >1 indicating antagonistic interaction leading to decrease efficacy (toxicity) of dox on such cells. such finding agreed with previous studies that evaluate each compound (ba or rsv) separately from the other on the toxicity of dox. the ba when combined with dox produces a significant reduction in dox cardiotoxicity. the underlying mechanism was through the antioxidant mechanisms in the heart of mice that were initially reduced by dox. owing to its potent antioxidant properties, ba decreases reactive oxygen species (ross) generation and the apoptosis induced by dox in chick cardiomyocytes (16, 17). the rsv also decreases dox toxicity as previously revealed using the same cells of the h9c2, where activation of sirt-1 pathway leads to protection from the upregulation of p53 induced by dox along with weakening of the cascade of events such as cytochrome c release and over expression of bax induced by dox in such cells (18, 19). combination of both ba and rsv with dox provides additional advantage over the combination of either ba or rsv with dox by giving a variety of molecular mechanisms in cardio-protection and enhancing the safety profile of dox. compared to another study, which indicated that the use of cynarine, a natural polyphenolic compound, was found to improve hct-116 and hep-g2 and it was found to decrease the cardiotoxicity of dox (20). the development of these combinations between polyphenolic compounds with a wide range of anticancer activities (ba & rsv) and the wellknown, potent chemotherapeutic drug, dox, which resulted in a significant improvement in the cytotoxicity profile of dox, may also offer an additional benefit by reducing the side effects of dox. this is explained by the fact that the negative effects of dox, particularly cardiotoxicity, a wellknown and dose-dependent side effect, will be diminished when combined with such natural chemicals due to the considerable reduction of dox concentration (ic50) (22). similar findings were iraqi j pharm sci, vol.31(suppl.) 2022 doxorubicin combination with baicalein and resveratrol 98 obtained from previous studies, which indicated that ba or rsv enhanced the cytotoxicity of dox (23, 24). additionally, the addition of dox to those two compounds improves the safety profile of the chemotherapeutic agent in the rat heart cells and offers additional protection to the cardiocytes. low concentrations of these natural polyphenols' anticancer effects may hasten the nonmutagenic repair of dna damage, and their therapeutic potential may interfere with dna repair mechanisms (25). the findings of this study also lend credence to a revolutionary method of treating cancer that involves combining natural substances with chemotherapy and other standard cancer treatments to increase their efficacy and lessen their adverse effects (8). however, the detailed information on the mechanisms of action at molecular levels of such combinations is required and needs further studies on both types of cell lines (hct116 & hepg2) to investigate the actual mechanisms that stand behind the synergetic, additive and all interactions between those compounds. several combinations using ba, rsv, and dox were tested on two cell lines (hct116 & hepg2 cells) in an attempt to evaluate their effect on the enhancement of the anticancer activity of doxorubicin. promising results were obtained from the combination of dox with ic50 of ba and ic50 of rsv (dox + ba.ic50 + rsv.ic50) 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https://doi.org/10.31351/vol31iss1pp285-292 285 the formulation and evaluation of high-fat pellet on lipid profiles and body mass index of male wistar rats rosnah rosnah*,1, nurpudji a. taslim**, andi makbul aman**, irfan idris**, suryani as’ad**, agussalim buchari**, burhananuddin bahar**, aminuddin**, elly wahyudin*** and gaga irawan nugraha**** *department of nutrition, poltekkes kemenkes kendari, indonesia **faculty of medicine, universitas hasanuddin, makassar, indonesia ***faculty of pharmacy, universitas hasanuddin, makassar, indonesia ****faculty of medicine, universitas padjadjaran, bandung, indonesia abstract this study aims to explore the manufacturing of high-fat pellets for obesity induction diets in male wistar rats and determine their effects on lipid profiles and body mass index. it was conducted using an experimental laboratory method with posttest with control group. the formulation and evaluation of the physico-chemical characteristics of the high-fat pellets (hfd) were conducted in september 2019. in this study, the 28 male wistar white rats used were 2 months old with 150-200 g bodyweight. the rats were acclimatized for 7 days and divided into 2 groups, namely the p0 group which was fed with standard pars cp594 confeed as many as 7 rats (p0) and the p1 group which was fed high-fat diet (hfd fii) as many as 21 rats, each at 30 g/head/day for 8 weeks.. the result showed that the mean fat content of formula ii pellets (hfd fii), 25.44% ± 0.16 was higher than formula i pellet (hfd fi) (22.55% ± 0.16) and 3% standard feed. furthermore, the mean of body weight and bmi of obesity induction rat groups (p1) were significantly higher than the standard rat group (p0) (p <0.05). the feed consumption in the rat fed with hfd fii pellets was also higher than the standard group (p0), indicating that rats preferred the hfd fii pellets. the lipid profile of the obesity induction group showed higher total cholesterol, triglycerides, and ldl, while the hdl levels were significantly lower compared to the standard feed group (p0). therefore, giving hfd fii pellets, which are a source of fat from butter, full cream milk powder, and eggs of purebred chickens for 8 weeks can make male wistar rats obese and dyslipidemic. keywords: physio-chemical characteristics of pellets, high-fat diet, body mass index, lipid profiles, wistar rats introduction obesity and related metabolic diseases are currently a priority of the study area. the increase in its global prevalence at various ages has profound economic and health impacts. several factors such as genetic, environmental, and diet plays an important role in developing obesity. meanwhile, scientific evidence shows that increased fat intake is associated with weight gain, leading to the disease(1). for example, metabolic syndrome, impaired lipid metabolism and represents a risk factor for type 2 diabetes mellitus (dm), cancer, hypertension, hypercholesterolemia, coronary heart disease, heart failure, stroke, and osteoarthritis(2,3). moreover, white rats (rattus norvegicus) are widely used as experimental animals in medicine, pharmacy, medicinal plants, nutrition, and other fields of science to study the effects of drugs, toxicity, metabolism, embryology, and behavior (4,5). the induction of obesity in experimental animals can be mediated through neuroendocrine, genetic, and diet manipulation. however, the high-fat diet (hfd) induction method is often used due to its closeness to the human model (6). a high-fat (atherogenic) diet is a food formula with high saturated fat (total fat> 10%) and cholesterol content of 0.28 mg/calorie of energy7. in america and europe, the atherogenic diet for animal models is made from the main ingredients of egg flour or pure crystalline cholesterol, but it is not easily obtained in indonesia due to a relatively high price. therefore, there is a need to make a breakthrough through the use of local ingredients. this is possibly achieved from atherogenic diets made with local raw materials at affordable prices, sourced from cholesterol in purebred chicken egg yolk, coconut oil, which is 86% high in saturated fatty acids, and beef tallow. pellets are a mass form of feed or ration material formed by pressing and compacted through a mold hole mechanically. the use of pellet-shaped feed has several advantages, such as ensuring the balance of feed nutrients, being more durable, not producing dust-like mash feed, and reducing the amount of feed wasted(8). 1corresponding author e-mail: rosnahgunawan71@gmail.com received:12 /9/2021 accepted: 2/11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp285-292 iraqi j pharm sci, vol.31(1) 2022 physicochemical characteristics of high-fat fellet 286 furthermore, its use is also more practical without any prior preparation, and available in ready-to-use packages. currently, the manufacturing of pellets for atherogenic diets in wistar rats generally uses lard, coconut oil, beef fat, goat fat, and egg yolk as a source of fat9. meanwhile, the use of lard in the manufacturing of high-fat diet feed is generally as a suspension, where the process uses water as a solvent medium for easy rancidity. rancid odors arise from contact between fats and oxygen (oxidation), water molecules (hydrolysis), or metals to form hydroperoxides. this further becomes susceptible to more oxidation and degradation of secondary reaction products such as aldehydes, ketones, acids, and alcohols (10, 11). compared to the regular diet, the rancid aroma of the atherogenic diet gave a lower intake but higher fat content, affecting changes in body weight and lipid profile of the rat(12). therefore, the preference of feeding on fresh feed with a fragrant aroma by livestock affected its palatabilit and acceptance(13). the manufacturing of hfd pellets using lard also poses a particular problem for muslim researchers due to its unavailability as other ingredients. furthermore, high-fat feed for the obesity induction and atherogenicity for wistar rats in the form of instant pellets is not yet available in the local indonesian market, even though the need for its use in the fields of medicine, pharmacy, medicinal plants, nutrition, and others is relatively high. therefore, this study used butter, chicken eggs, and full cream milk powder as sources of saturated fat to manufacture a high-fat (atherogenic) diet for male wistar rats as experimental animals. the foods of animal origin such as fatty meat, cheese, butter, and milk cream, which contain saturated fatty acids and cholesterol, were used. moreover, cheese raises less cholesterol at the same fat content as butter, whole milk is rich in saturated fat, cholesterol, and increases serum cholesterol(14-16). butterfat is generally recognized for its consistent increase in plasma cholesterol concentrations, especially in hypercholesterolemic subjects(17). previous study has shown an epidemiological link between butterfat consumption and cardiovascular mortality as the main cause of a decrease in the intake of full-cream milk fat(18). moreover, dairy products are rich in myristic and palmitic acids, which increase serum cholesterol(19). the dominant effect of saturated fatty acids is increased total cholesterol and ldl cholesterol levels(20). several studies on human have shown that butter, which contains 66% of saturated fatty acids in hypercholesterolemic compared to other fat sources (21, 22). the intake of this saturated fat had been associated with an increased risk of cardiovascular disease (cvd)(23), which is mediated primarily by high ldl cholesterol concentrations. in the united states, the primary dietary sources of saturated fatty acids are total fat dairy products and red meat. an egg is a source of high-quality protein, which provides all essential amino acids for humans, especially in the white part. furthermore, its yolk is a source of fat, containing 65.5% triglycerides, 28.3% phospholipids, and 5.2% cholesterol. egg also contains many active lipid components such as phospholipids, choline, and carotenoids(24). monounsaturated fatty acids (mufas) and polyunsaturated fatty acids (pufas) weighted 2.0 g and 0.7 g, respectively, and one medium egg contains 1.6 g saturated fatty acids. moreover, egg consumption is positively associated with an increase in serum total cholesterol (tc), ldl-c, and a high incidence of coronary heart disease mortality(25,26). its intake and cholesterol were also related to all causes of higher cvd and cancer deaths (27), while increased mortality is influenced mainly by cholesterol intake. therefore, it is advisable to limit cholesterol intake and replace whole eggs with egg whites/substitutes or other alternative protein sources. this study aims to develop hfd pellets for dietary induction of obesity in experimental wistar rats. the formulation was based on materials derived from halal fats, practical use, ready to use, relatively low prices, and easy to obtain in the local market such as butter, chicken eggs, and full cream milk. materials and methods this study was conducted using a laboratory experimental method with a post-test design a post-test design with control group. it obtained ethical approval from the research ethics commission of the hasanuddin university faculty of medicine, makassar, number 1156 / un4.6.4.5.31 / pp36 / 2019. meanwhile, this study was carried out in 3 stages, namely (1) the formulation and manufacturing of high-fat pellets (hfd), with physico-chemical characteristics investigations in september/ 2019 at the animal food chemistry laboratory, department of nutrition and food, faculty of animal husbandry, hasanuddin university. (2) maintenance of experimental animals and the induction of obesity by giving selected hfd pellets, formula ii hfd pellets (hfd fii) in male wistar rats, for eight weeks from january to march 2020, at the pharmacy laboratory of the faculty of pharmacy, university of muslim indonesia (umi) makassar. subsequently, anthropometric index measurement included body weight, length, and bmi was carried out every seven days, while pellet consumption was measured daily. (3) lipid profile examinations such as total cholesterol, triglycerides, ldl, and hdl were carried out at the laboratory of molecular biology and immunology, faculty of medicine, hasanuddin university makassar, in may 2020. iraqi j pharm sci, vol.31(1) 2022 physicochemical characteristics of high-fat fellet 287 high fat diet (hfd) pellet formulation the hfd feed was made in pellets based on the study method with a modification7. the formulation consisted of two treatments, namely formula i (hfd fi) and formula ii (hfd fii) as shown in table 1. table 1. high-fat pellet formulation for male wistar strain white rat (rattus novergicus) material formula i (hfd fi) formula ii (hfd fii) % amount (g) % amount (g) confeed pars cp594 (standard feed) 40 400 20 200 wheat flour 20 200 20 200 butter 20 200 30 300 chicken eggs (whole) 10 100 20 200 full cream powdered milk 10 100 10 100 total 100 1000 100 1000 furthermore, the composition of the ingredients in one kg of confeed pars cp594 (standard feed) consisted of corn, bran, fish meal, soybean meal, coconut meal, broken wheat, peanut meal, leaf flour, and canola. the tools for making hfd pellets were a digital scale, electric oven, blender, pellet molding device (pelleter), water measuring device, 80 mesh filter, mixing bowl, spoon, airtight plastic packaging, and vacuum packaging device. the preparation was ccarried out initially by weighting each ingredient according to the formula's proportion (table 1). the standard feed of confeed pars cp594 was mashed in a blender, filtered using an 80-mesh sieve, and mixed evenly with flour and full cream powdered milk (ingredient a). subsequently, butter, eggs (yolks and whites) are evenly mixed and ingredient a was added into the mixture and mixed continually to form smooth dough, which was weighted and molded using a pelleter. it was baked in the oven at 160°c for 60 minutes, pellets were removed, allowed to cool, and weighted for yield calculation. the pellet was further packed with airtight plastic using a vacuum packaging tool and stored in an undamped place until its use for physical analysis and chemical composition of hfd pellets. the results of the highfat pellet formulation with better chemical and physical characteristics were used as feed for obesity induction diets in male wistar rats. chemical characteristics of hfd pellets the duplo analysis of the chemical composition of hfd pellets carried out included moisture content (gravimetric method, sni 012891-1992 item 5.1), ash content (aoac 2005. 942.05), crude protein by kjeldahl method (aoac 2005.2000.11), crude fat in soxhlet method (aoac 2005.2003.06), carbohydrates (aoac 2005), crude fiber (sni 01-2891-1992 item 11), calcium (aas method, aoac 2005.968.08), and phosphorus (aas method, aoac 2005.965.17). physical characteristics the physical characteristics of hfd fi and hfd fii pellets were measured using weight, length, and diameter parameters. the pellets were weighted using analytical scales, while the length and diameter were with a caliper. the measurement of yield was according to the formula below: yield (%) = pellet dry weight(gm) pellet wet weight(gm) 100 maintenance and induction of obesity in male wistar rats the 2 months old 28 male wistar white rats, which were 150-200 g body weight, were from pt. indoanilab bogor (laboratory animal development facility, faculty of veterinary medicine, ipb university, bogor). the rats were acclimatized for 7 days, placed in individual cages, light/dark cycle for 12 hours, temperature 26-29 °c, humidity 60-70%, and given standard feed and drinking water ad libitum. meanwhile, the cage was in form of a plastic box with a wire cover of 30 cm x 20 cm x 12 cm, covered with rice husks, and cleaned regularly two times a week to keep the cage dry and healthy. after acclimatization, the body weight and length were estimated, and the remaining feed was measured daily, while the rats' body weight and length were measured every 7 days. the experimental animals were divided into 2 groups, namely the p0 group which was fed with standard pars cp594 confeed as many as 7 rats (p0) and the p1 group which was fed high-fat diet (hfd fii) as many as 21 rats, each at 30 g/head/day for 8 weeks, while drinking water was given ad libitum. the amount of consumption was calculated from the amount of feed given minus the remaining feed. after eight weeks, the rats were examined for their level of obesity and were declared obese when the bmi value was ≥ 0.68 g/cm2. at the 8th week after treatment, rats were fasted for 12 hours and kept drinking before aseptic blood was drawn through the tails' lateral veins or ventral arteries. a total of 1 ml of blood was collected into an eppendorf tube and left for 60 minutes. the blood serum appeared differently from the blood clot and was further separated by centrifugation 15 minutes, speed 3000 rpm. after iraqi j pharm sci, vol.31(1) 2022 physicochemical characteristics of high-fat fellet 288 centrifugation, it was isolated with a single-use syringe and transferred to a new eppendorf tube, labeled, wrapped in plastic, and stored in the freezer at –20 ºc upright until the serum was ready to be analyzed. lipid profile examination (total cholesterol, triglycerides, ldl, and hdl) the total cholesterol levels were measured by the elisa sandwich method using the rat total cholesterol (tc) elisa kit cat. no: mbs2600008. subsequently, hdl and ldl levels were examined by a homogeneous enzymatic (diasys) photometric test of cholesterol oxidase, phenol amino phenazon (chod-pap). triglyceride levels were measured by the enzymatic colorimetric test method for glycerol-3phosphatase oxidase-paminophenazone (gpopap) using the system diagnostic kit (diasys) based on the kit procedure. statistical analysis processing and data analysis were carried out using the ibm spss program, while the data were presented as means ± standard deviations. chemical and physical characteristics data of hfd fi and hfd fii pellets were analyzed descriptively. furthermore, the anthropometric data such as bw, bl, bmi, average feed intake, lipid profile such as total cholesterol, triglycerides, ldl, and hdl were tested for normality by the shapiro-wilk test and data variance with the homogeneity of variance test. the parametric hypothesis test used was independent t test at 95% confidence level (α = 0.05). results and discussion chemical characteristics of hfd pellets according to table 2, the 25.44% ± 0.16 average fat content of hfd formula ii (hfd fii) pellets was higher than hfd formula i (hfd fi) pellets of about 22.55% ± 0.16 and 3% standard feed. the difference in fat content was due to the higher percentage of butter and eggs in the hfd fii formula, 30%, and 20%, compared to 20% and 10% in hfd fi. furthermore, the protein, carbohydrate, fiber, calcium, ash, and phosphorus content of hfd fii pellets was lower than hfd fi pellets. meanwhile, the standard dietary fat was from different meals such as fish, soybean, coconut, and peanut, and vegetable. the moisture content of hfd fii pellets (7.87% ± 0.09) was lower than that of hfd fi pellets (8.47% ± 0.03). moreover, the water content in an ingredient greatly affects the quality of feed ingredients. when the water content of a material does not meet the requirements, there will be physical and chemical changes identified by the growth of microorganisms which makes them unfit for consumption29. in addition, the reduction in water content also decreases the ration weight, which makes packaging easier(8). based on the physicochemical analysis of high-fat pellet formulations, hfd fii pellets had higher fat content and yield of 25.44% ± 0.16; 81.01% and lower moisture content of 7.87% ± 0.09 compared to hfd fi pellets. therefore, hfd fii pellets were selected as obesity induction feed for male wistar rats to obtain animal models of obesity and dyslipidemia physical characteristics of hfd pellet yield of hfd fi = 800.49 1000 x 100% = 80.05% yield of hfd fii = 810.08 1000 x 100% = 81.01% table 3 shows the initial weight of each hfd fi, and the hfd fii formula was 1000 g. after drying, hfd fi and hfd ii pellets had a weight reduction of 190.51 g and 189.20 g, respectively. however, the diameter and length of each pellet formulation did not change, while the yield on hfd fi was 80.05% and hfd fii 81.01%. table 2. chemical composition of hfd fii pellets and standard feed nutrients (%) pellet hfd fi (%) pellet hfd fii (%) standard feed cp594* (%) fat 22.55 ± 0.16 25.44 ± 0.16 3 protein 19.57 ± 0.35 19.37 ± 0.01 17.5 – 19.5 carbohydrate 45.48 ± 0.26 44.51 ± 0.21 48.7 fiber 6.34 ± 0.64 5.31 ± 0.49 8 ash 6.08 ± 0.12 5.39 ± 0.09 7 water 8.47 ± 0.03 7.87 ± 0.09 13 calcium 0.85 ± 0.00 0.82 ± 0.01 0.9 phosphor 0.17 ± 0.01 0.15 ± 0.01 0.9 source: * pt. pokphand (feed producer) iraqi j pharm sci, vol.31(1) 2022 physicochemical characteristics of high-fat fellet 289 table 3. physical characteristics of hfd fi and hfd fii pellets formula wet drying weight (g) length (cm) diameter (cm) weight (g) length (cm) diameter (cm) hfd fi 1000 1 0.5 800.48 1 0.5 1000 1 0.5 800.50 1 0.5 mean 1000 1 0.5 800.49 1 0.5 hfd fii 1000 1 0.5 810.10 1 0.5 1000 1 0.5 810.06 1 0.5 mean 1000 1 0.5 810.08 1 0.5 maintenance and induction of obesity in male wistar rats after 8 weeks of obesity induction, the results of the independent t-test on body weight and bmi of wistar rats obtained a p-value <0.05. this showed that there is a significant difference between body weight and bmi of the rat group fed with hfd fii pellets (p1) and the standard feed group (p0). the mean body weight and bmi of 21 rats in the obesity-induced group were significantly higher than those in the standard diet (p0). table 4. anthropometric parameters and intake of male wistar rats receiving standard feed and hfd fii pellets parameter groups mean ± sd p value weight (gr) p0 198.0 ± 2,82 0.000* p1 271.9 ± 4,52 length (cm) p0 18.3 ± 0,55 0.336 p1 18.0 ± 0,37 bmi (gcm-2) p0 0.49 ± 0,03 0.000* p1 0.71 ± 0,03 feed intake (g) p0 21.8 ± 0,42 0.000* p1 23.7 ± 0,76 * independent t test; *significantly different (p<0.05). in table 4, based on the results of independent t test, it is known that there are significant differences in mean body weight, bmi and feed intake, while body length does not differ significantly between groups p0 and p1. lipid profiles of wistar rats lipid profile examination was carried out after 8 weeks of treatment. the obesity induction rat group (p1) was obese (imt > 0.68 g.cm-2), while the average body mass index for the standard feed group was within the normal range (imt= 0.48 g.cm-2). at the beginning of this study, the rats and the obesity induction were 8 weeks old; therefore, the lipid profile was examined when the wistar rats were 16 weeks old. reference values for the lipid profiles of 16-week-old male albino rats are as follows: serum total cholesterol 109.72 ± 3.67 (100.00–133.33) mg/dl, serum hdl cholesterol 45.46 ± 2.74 (36.36– 54.55) mg/dl, serum triglycerides 92.67 ± 5.77 (72.00–130.00) mg/dl and ldl cholesterol 45.73 ± 3.54 (25.45–59.98) mg/dl. the results of the lipid profile of rats' measurements are shown in table 5. table 5. lipid profile of wistar rats after the standard feed and high-fat pellets formula ii (hfd fii) parameter groups mean ± sd p value cholesterol total (mg/dl) p0 112, 96 ± 3,22 0,000* p1 178, 73 ± 6,73 triglyceride (mg/dl) p0 96, 12 ± 5,41 0,000* p1 156,10 ± 26,77 ldl (mg/dl) p0 47,79 ± 2,64 0,000* p1 108,04 ± 5,45 hdl (mg/dl) p0 48,44 ± 2,47 0,000* p1 33,16 ± 1,78 the results of the independent t test showed that the levels of total cholesterol, triglycerides, ldl in the p0 group were significantly lower and hdl levels were significantly higher than those in the p1 group (p<0.05) (table 5). meanwhile, food compositions that are commonly used to induce obesity are mostly rich in cholesterol/fats rather than carbohydrates and proteins, which help increase animal body weight and lipid levels(28). iraqi j pharm sci, vol.31(1) 2022 physicochemical characteristics of high-fat fellet 290 obesity status in the rat can be assessed using the parameters of body mass index (bmi) and lee's index(30). normal bmi in adult male wistar rats ranged from 0.45 to 0.68 g/cm2, therefore, rats were declared obese when the bmi was ≥ 0.68. moreover, it is assumed that body fat and obesity in rats are better estimated in bmi than in lee's index. changes in bmi were associated with a profile of dyslipidemia and oxidative stress in rat serum and are used to predict the adverse consequences of obesity in rats. such naturally available foods and their mechanisms maintain a healthy body weight, which reduces the morbidities and mortalities of obesity (31). maintaining a healthy weight is important for overall health, which helps prevent and control many of diseases (32). the weight gain in the rats fed with hfd fii was significantly different from those given standard feed (p0). this showed that feeding on high fat for 8 weeks can significantly increase the body weight of the rat. this occurred due to the significant difference in feed intake between the standard feed group (p0) and the groups receiving hfd fii pellets (p1). in addition, there was a variation in the percentage of fat from the pellets where the fat content of hfd fii pellets was higher (25.44% ± 0.16) than the 3% standard feed. the source of fat from hfd fii pellets was from butter, chicken eggs, and full cream powdered milk, while in standard feed, the majority was vegetable fats such as soybean, coconut, and fish. however, feeding the standard 3% fat content did not have a significant increase in rat body weight and fat storage. fat is the largest source of energy where each gram produces 9 kcal, while protein and carbohydrates only produced 4 kcal per gram. at the exact weight of feed ingredients, fat production was higher than carbohydrates and protein. previous studies showed that the consumption of a high-fat (hf) diet causes obesity and metabolic disorders in rodents that mimic metabolic syndrome in humans(33, 28, 34) .although the weight gain in experimental animals can be assessed after 2 weeks, it is more noticeable after 4 weeks of hfd administration. in this study, prolonged feeding on a fat-rich diet-induced weight gain in the susceptible rat ranged from 10% to 20% compared to controls fed standard diets. this showed that obesity induction is most effective when the diet is started at a younger age and continues for several weeks. providing a high-fat diet at early stages also increases the number of adipocyte cells. after an extended period, an enlargement of the adipocyte cell diameter due to the accumulation of fat associated with the risk of metabolic disorders was discovered(35). the feed intake in the rat-given hfd fii pellets group was higher than the group fed with standard feed (p0), showing that the rat preferred the hfd fii pellets. this was because the high fat in hfd fii pellets gave a tasty and crunchy effect; therefore, it was preferred by rats. however, the more rancid smell of the atherogenic diet than the regular diet led to a lower intake of the rats' feed(12). obesity can cause lipid metabolism disorders characterized by abnormalities in the plasma of the lipid profile such as high total cholesterol and triglyceride levels, and low highdensity lipoprotein (hdl) cholesterol levels. since it is the key risk factor in the natural history of other chronic non-communicable diseases, its prevention strategies offer a cost-effective approach in avoiding other chronic non-communicable diseases(36). meanwhile, the administration of a high-fat diet causes an increase in the concentration of chylomicrons in plasma, while high triglyceride levels lead to increased ldl formation(37). it was assumed that the high-fat diet causes triglyceride and ldl levels in the obesity-induced diet group to be higher than the standard feed group. a study about intervention for 4 weeks was conducted in a group of healthy men and women in the general population, which consume 50 g per day of either these different dietary fats, namely extra virgin coconut, butter, or extra virgin olive oil. the results showed that the ldl-c concentration increased significantly in the buttered group than coconut and olive oil. furthermore, cholesterol synthesis was lower during a diet rich in coconut fat and oil, which was safer than a diet rich in butter. this occurred due to lower production levels of lipoproteins containing apo-b. in the obesity induction group, the hdl levels were lower than in the standard feed group (p0). a significant reduction in hdl cholesterol levels in wistar rats after a high-fat diet showed that a high-fat diet affected serum cholesterol levels. generally, an increase in hdl concentration is known as a protective cardio-protein because the lipoprotein functions to absorb excess cholesterol to the liver for excretion. however, a low hdl concentration is a diagnostic marker for metabolic syndrome (mets) and cvd. recent studies have shown that metabolic syndrome is associated with an increase in the liver enzymes such as alt, ast, ggt, and albumin(38). similarly, epidemiological and clinical studies showed the inverse correlation between serum high-density lipoprotein cholesterol (hdl-c) concentration and the risk of atherosclerotic diseases(39). also, a strong inverse relationship exists between hdl and cvd concentrations at the epidemiologic level, which makes it a significant marker for assessing cardiometabolic health (40). the limitation of this study is that the total cholesterol and trans fatty acid content of the highfat pellets produced were not examined. also, the texture of the pellets was still rather fragile and easily crushed, which could affect the amount of feed intake in the experimental animals. iraqi j pharm sci, vol.31(1) 2022 physicochemical 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hassan n, shahwan m, alkhoujah s, jairoun a. association between serum liver enzymes and metabolic syndrome among type 2 diabetes mellitus patients. research journal of pharmacy and technology. 2021 feb 16;14(2):1050–4. 39. ibrahim s, khayat mi, awama ma. comparative study for the measurement of hdl cholesterol between a direct assay and a precipitation method. research journal of pharmacy and technology. 2018 mar 30;11(3):1035–8. 40. tuteja s, rader dj. high-density lipoproteins in the prevention of cardiovascular disease: changing the paradigm. clinical pharmacology and therapeutics. 2014;96(1):48–56. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ synthesis and preliminary pharmacological study of sulfonamide conjugates with ibuprofen and indomethacin as new anti-inflammatory agents iraqi j pharm sci, vol.18(2) 2009 sulfonamide conjugates 93 synthesis and preliminary pharmacological study of sulfonamide conjugates with ibuprofen and indomethacin as new anti-inflammatory agents bader s. salem * , monther f. mahdi **,1 and mohammed h. mohammed ** * college of pharmacy, university of basrah. , basrah, iraq ** department of pharmaceutical chemistry,college of pharmacy,university of baghdad.,baghdad, iraq abstract 4-aminobenzenesulfonamide conjugates of ibuprofen (compound 10) and indomethacin (compound 11) have been designed and synthesized by the reaction of sulfanilamide (compound 7) with 2-(4-isobutylphenyl) propanoic acid (ibuprofen) and 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl1h-indol-3-yl)acetic acid (indomethacin) for the evaluation as potential anti-inflammatory agents with expected selectivity against cox-2 enzyme. in vivo acute anti-inflammatory activity of the synthesized final compounds (10 and 11) was evaluated in rats using egg-white induced edema model of inflammation in a dose equivalent to (10mg/kg) of ibuprofen and (2mg/kg) of indomethacin. the tested compounds produced significant reduction of the rat paw edema with respect to the effect of propylene glycol 50% v/v (control group). moreover, compound (10) exhibited comparable antiinflammatory activity to diclofenac (3mg/kg) (reference group) while compound (11) exhibited less anti-inflammatory activity than diclofenac. the result of this study indicate that the incorporation of the 4-aminobenzenesulfonamide pharmacophore into ibuprofen and indomethacin maintained their antiinflammatory activity & may increase their selectivity toward cox-2 enzyme which can be confirmed in future by assessing cox-2:cox-1 inhibitory ratio. key words: cyclooxygenase; anti-inflammatory activity; ibuprofen; indomethacin; nsaids. الخالصة ( عوٍ رزٌو 00( ى نالَدىيٍثاسوٍٍ يزبو م01نالٌبويبزىيٍٍ ميزبو ]ايٍُيبُشٌٍ سهفيَاياٌد-4[يقتزَات تى تصًٍى ى تحضٍز -2-يٍثيبسوً-5-بهيرىبُشىٌوم(-4م-0م-2اٌشىبٍيتٍم يٍُم( بزىباَيٌك اسٍد ماالٌبيبزىيٍٍ( ى -4م2( يع 7تفاعم انسهفاٍَالياٌد ميزب ٌم(اسووتٍك اسووٍد ماالَدىيٍثاسووٍٍ( نتقًٍٍوًووا بايايووم يضووادي نالنتوووام لدٌوودي يووع اضتًانٍووي اَتقا ٍووي يضووادي الَووشٌى -9-لاَوودى-0h-يثٍووم ( يوً انجوزب باسوتمداو 00ى 01الزي تقٍٍى انفاانٍي انًضادي نالنتوام يً انجسى انحوً نهًزببواٌ انُوا ٍواٌ م. (cox-2)انكيبس انثاًَ يهغى/بغوووى( ى بجزعوووي يكايفوووي نالَدىيٍثاسوووٍٍ 01بٍوووي تحووود انجهووود بجزعوووي يكايفوووي نالٌبووويبزىيٍٍ مسالل انبوووٍس يسوووتحدةي ىبيوووي انتوا ( propylene glycol%م51.انًزببووات انًمتبووزي اَتجوود اَمفووال يووةةزا نهيبيووي بانًقارَووي يووع انبووزىبهٍٍ بالٌكوويل بغى(/يهغووى2م يهغى/بغووى( بًجًيعووي 9( م diclofenacنهووداٌكهييٍُا) م( ياانٍووي يضووادي نالنتوووام يقارَووي 01م بًجًيعووي بووابقي.نقد ااوووز انًزبوو ( ااوز ياانٍي يضادي نالنتوام اقم يٍ انداٌكهييٍُا).تشٍز َتٍجي هذِ اندراسي انى اٌ اَدياج انجشء اناقواقٍزي 00يزلاٍي بًٍُا انًزب م ضوادي نالنتووام يوع اضتًوال سٌوادي اَتقا ٍتوًوا تجواِ ياانٍتوًا انً قد ضايظ عهىايٍُيبُشٌٍ سهفيَاياٌد يع االٌبيبزىيٍٍ ى االَدىيٍثاسٍٍ -4 . 0-انى انكيبس 2-اَشٌى انكيبس انثاًَ ىانذي ًٌكٍ اٌ تثبد يستقبال بتحصٍم انُسبي انًثبقي نهكيبس introduction prostaglandins (pgs) are active mediators of inflammatory responses and also provide cytoprotection in the stomach and intestine. the key enzyme of their biosynthesis is prostaglandin h2 synthase (pghs) or called cyclooxygenase (cox) (1) . it is now well established that three distinct cox isoforms exist: the constitutive form cox-1 is expressed virtually in all tissues and is involved in the regulation of physiological functions such as in maintaining platelet aggregation and homeostasis of the gi tract and the kidney (2) . cox-2 is rapidly induced in inflammatory cells in response to cytokines such as tumor necrosis factor-α (tnf-α), interleukines, growth factors, and so on (3) . recently a third full active isoform, cox-3, and two partial isoforms, pcox1a and b, were reported to be found in the cerebral cortex and in human heart (4, 5) . non-steroidal antiinflammatory drugs (nsaids) continue to be one of the more widely used groups of therapeutic agents, which inhibit cox-1, cox-2, and tromboxane synthase with a varying degree of selectivityresearchers have recently focused on selective cox-2 inhibitors which are believed to reduce inflammation without influencing normal physiologic functions of cox-1. the first cox-2 selective nsaid approved by food and drug administration (fda) was celecoxib (1), which was followed by introduction of rofecoxib (2) and valdecoxib (3). 1 corresponding author e-mail : dmfalameri @yahoo.com received : 8/2/2009 accepted : 10/6/2009 iraqi j pharm sci, vol.18(2) 2009 sulfonamide conjugates 41 a common structural backbone of most cox2 selective inhibitors consists of two aryl groups linked to adjacent atoms of a central ring which can be homocyclic or heterocyclic, one of the aryl groups is substituted in the para position with an aminosulfonyl (so2nh2) group (6) . since their introduction, cox-2 specific inhibitors have become a rapidly growing segment of prescription drug market, especially for osteoarthritis and rheumatoid arthritis patients. however, recently there is a controversy regarding the hepatic toxicity of nimusulide (4) and for the cardiovascular complications of rofecoxib. fda has banned the use of nimusulide in pediatric patients and rofecoxib in both adults and children. in spite of these facts, there is a growing need for the development of safer cox-2 selective inhibitors (7) . therefore, in view of the above facts, we report the synthesis and preliminary pharmacological evaluation of an ibuprofen_ and an indomethacin_ sulfonamide conjugate as nsaids with expected cox-2 inhibitors. (1) (2) (3) (4) chemistry the general routes outlined in schemes 1, 2 and 3 were used to synthesize all compounds described here.as shown in scheme 1; 4-aminobenzenesulfonamide (7) was prepared as described previously (vogel) (8) starting from acetanilide. iraqi j pharm sci, vol.18(2) 2009 sulfonamide conjugates 40 scheme 1: synthesis of compound 7 scheme 2: synthesis of compounds 8, 9, 10 and 11 iraqi j pharm sci, vol.18(2) 2009 sulfonamide conjugates 42 experimental all reagents and solvents were of analar type and generally used as received from the commercial supplier (e merck-germany, reidel-dehean-germany, sigma-aldrichgermany & bdh-england). ibuprofen and indomethacin were supplied from safa company-iraq.melting points were determined by capillary method on gallenkamp apparatus (england) and ascending thin layer chromatography (tlc) was run on silica gel to check the purity and progress of reaction. the identification of compounds was done using iodine vapor and the chromatograms were eluted by: ethyl acetate: methanol: ammonia (85:10:5). ir spectra were recorded on shimadzu ftir spectrophotometer (japan) as a kbr film in the college of science/university of basrah. chn microanalysis was done by using euro vector ea3000a elemental analyzer, italy, in the alalbait university /jordan. synthesis of 4-acetamidobenzene-1-sulfonyl chloride (5) acetanilide (1.35g, 10mmol) was placed in a 100ml flask and melted in it over a free flame that caused the compound to solidify over the lower part of the flask when swirling the liquid formed after immersion in an ice bath momentarily. chlorosulfonic acid (3.5ml, 53mmol) was added all at once with continuous shaking, and then the reaction mixture was heated on a water bath for 90 minutes. the mixture was cooled and poured with stirring onto crushed ice (or ice water), and after stirring for about 5 minutes an even suspension of the granular solid was obtained. this suspension was filtered off under vacuum, washed with a little cold water, and pressed dried to give compound (5) which was immediately used in the next step without further purification (8) . synthesis of n-(4-sulfamoylphenyl) acetamide (6) compound (5) (2.34g, 10mmol) was added to a mixture of concentrated ammonia solution (20 ml) and water (20 ml). the contents of the flask were heated with occasional swirling to just below the boiling point for about 20 minutes. the formed suspension was cooled in an ice bath and then acidified with diluted sulfuric acid and the formed precipitate was filtered under vacuum then washed with cold water to give the intermediate product (6) (8) . (53% yield) as a faint yellow crystals. m.p. 213-215 o c. rf =0.61. synthesis of 4-aminobenzenesulfonamide (7) compound (6) (2.14g, 10mmol) was transferred to a flask containing mixture of concentrated hydrochloric acid (10 ml) and water (30 ml). the mixture was boiled gently under reflex for 90 minutes. cooled to room temperature, then activated charcoal (2 gm) was added. the mixture was heated to boiling and filtered with suction through hardened filter paper. the filtrate was placed in a beaker and sodium bicarbonate was added in portions with stirring until the suspension become neutral to litmus. the mixture was cooled in ice bath and filtered by suction and dried to give compound (7) (8) , (45% yield) as white crystals. m.p. 160-161 o c. rf =0.75. ir 3477& 3375 (n-h) stretching of primary amine, 3367&3296 (n-h) stretching of sulfonamide, 1593, 1562&1502 (c=c) stretching of aromatic, 1301&1149 cm -1 (so2) stretching. synthesis of 2-(4-isobutylphenyl) propanoic anhydride (8) ibuprofen (2.06g, 10mmol) was dissolved in tetrahydrofuran (thf) (10 ml), and then dicyclohexyl carbodiimide (dcc) (1g, 5mmol) was added. the reaction mixture was stirred continuously at room temperature for 3.5 hours, where a white precipitate of dicyclohexylurea (dcu) was formed which was then removed by filtration. the solvent was evaporated under vacuum to yield compound (8) (9) , (80% yield) as a white powder. m.p. 208-213 o c. rf =0.67. synthesis of 2-(1-(4-chlorobenzoyl)-5methoxy-2-methyl-1h-indol-3-yl)acetic anhydride (9) indomethacin (3.58g, 10mmol) was dissolved in tetrahydrofuran (thf) (10 ml), and then dicyclohexyl carbodiimide (dcc) (1g, 5mmol) was added. the reaction mixture was stirred continuously at room temperature for 3.5 hours, where a white precipitate of dicyclohexylurea (dcu) was formed which was then removed by filtration. the solvent was evaporated under vacuum to yield compound (9) (9) , (77% yield) as an oily substance. synthesis of 2-(4-isobutylphenyl)-n-(4sulfamoylphenyl) propanamide (10) compound (8) (3.94g, 10mmol), compound (7) (1.72g, 10mmol), zinc dust (0.010g), glacial acetic acid (1.1ml, 20mmol) and dioxan (30ml) were placed in a flask equipped with reflux condenser. the reaction mixture was refluxed gently for 90 minutes, the solvent was evaporated under vacuum, the residue was dissolved in ethyl acetate and washed with 5ml. nahco3 (10%, 3x), hcl (1n, 3x), and distilled water (3x),then filtered over anhydrous magnesium sulfate. the filtrate iraqi j pharm sci, vol.18(2) 2009 sulfonamide conjugates 49 was evaporated under vacuum to give compound (10). the recrystallization was carried out by dissolving the compound in ethyl acetate and addition of petroleum ether on the filtrate until turbidity occurred and kept in cold place over night. the mixture was filtered while it was cold and the precipitate was collected to give compound (10) (10) . (63% yield) as an off white crystals. m.p. 128-130 o c. rf =0.8. ir: 3296 (n-h) stretching of sulfonamide, 1662 (c=o) stretching of secondary amide, 1593, 1523&1456 (c=c) stretching of aromatic & 1328&1159 cm 1 (so2) stretching. chn calculated (c19h24n2o3s): c, 63.31; h, 6.71; n, 7.77; s, 8.9. found: c, 63.11; h, 6.93; n, 7.36; s, 8.78. synthesis of 2-(1-(4-chlorobenzoyl)-5methoxy-2-methyl-1h-indol-3-yl)-n-(4sulfamoylphenyl)acetamide (11) compound (9) (6.97g, 10mmol), compound (7) (1.72g, 10mmol), zinc dust (0.010g), glacial acetic acid (1.1ml, 20mmol) and dioxan (30ml) were placed in a flask equipped with reflux condenser. the reaction mixture was refluxed gently for 90 minutes, the solvent was evaporated under vacuum, the residue was dissolved in ethyl acetate and washed with 5ml. nahco3 (10%, 3x), hcl (1n, 3x), and distilled water (3x), then filtered over anhydrous magnesium sulfate. the filtrate was evaporated under vacuum to give compound (11). the recrystallization was carried out by dissolving the compound in ethyl acetate and addition of petroleum ether on the filtrate until turbidity occurred and kept in cold place over night. the mixture was filtered while it was cold and the precipitate was collected to give compound (11) (10) . (50% yield) as an oily substance. ir 3325 (n-h) stretching of sulfonamide, 1687 (c=o) stretching of secondary amide, 1600, 1523 & 1475 (aromatic) & 1361&1151 cm -1 (so2) stretching. chn calculated (c25h22cln3o5s): c, 58.65; h, 4.33; n, 8.21; s, 6.26. found: c, 57.89; h, 4.1; n, 8.16; s, 6.35. pharmacology rats weighing (160 ±10g) were supplied by the college of veterinary medicine/ university of basrah and were housed in the animal house of the same college. animals were fed commercial chaw and had free access to water. animals were brought to the laboratory, and were divided into four groups (each group consist of six rats) as follow: group a: six rats served as control; and treated with the vehicle (propylene glycol 50% v/v); group b: treated with diclofenac (reference agent) in a dose of 3mg/kg suspended in propylene glycol 50% v/v (11) ; group c: treated with tested compound 10 in a dose equivalent to 10 mg/kg of ibuprofen as suspension in 50% v/v propylene glycol (12) ; group d: treated with tested compound 11 in a dose equivalent to 2 mg/kg of indomethacin as suspension in 50% v/v propylene glycol (13) . anti-inflammatory activity the anti-inflammatory activity of the tested compounds was studied using egg-white induced edema model. acute inflammation was produced by a subcutaneous injection of 0.05ml of undiluted egg white into the planter side of the left hind paw of the rats; 15 minutes after i.p. administration of the drugs or the tested compounds or their vehicle. the paw thickness was measured by vernea at six time intervals (0, 1, 2, 3, 4 and 5 hours) after drug administration. the results were analyzed for statistical significance using paired student ttest for comparisons between mean values with respect to their baseline, while comparisons between different groups were made using analysis of variance (anova) test. p value of less than 0.05 was considered significant. results and discussion among the many methods used for screening of anti-inflammatory drugs, one of the most commonly employed techniques is based upon the ability of such agents to inhibit the edema produced in the hind paw of the rat after injection of an irritant agent (14) . the intra-planter injection of egg white into rat hind paw induces a progressive edema, which was reached maximum (measured by millimeters) after 2 hours of injection. table 1 showed the effect of tested compounds (10&11) in respect to control group. all tested compounds were effectively limited the increase in paw edema, with the effect of compound 10 started at 1 hour (significantly different compared to control), while compound 11 started at 3 hours, which mean it has slower onset of action than the other tested compounds. however, the effect of all tested compounds continued till the end of the experiment with statistically significant (p>0.05) reduction in paw edema. iraqi j pharm sci, vol.18(2) 2009 sulfonamide conjugates 44 table 1: effect of control, compounds 10 & 11 on egg-white induced paw edema in rats. treatment group paw thickness (mm) time control (n=6) compound 10 (n=6) compound 11 (n=6) baseline 4.48±0.08 4.35±0.07 4.45±0.16 0 5.41±0.11 5.40±0.04 5.41±0.11 1 hr. 6.03±0.08 5.47±0.04 *a 5.81±0.07 b 2 hrs. 6.62±0.18 5.69±0.11 *a 6.37±0.29 b 3 hrs. 5.87±0.11 5.28±0.06 * 5.36±0.09 * 4 hrs. 5.65±0.11 4.97±0.05 * 5.00±0.23 * 5 hrs. 5.33±0.11 4.90±0.08 * 4.81±0.28 * non-identical superscripts (a & b) among different tested compounds are considered significantly different (p<0.05) * significantly different compared to control (p<0.05). table 2 showed the effect of tested compounds (10 & 11) in respect to reference group (diclofenac). as seen in this table; at baseline and time 0 there are no differences among different groups; at time 1 and 2 hours compound 11 has significantly lower effect than diclofenac and compound 10. however, it appears that all the tested compounds had a comparable effect to that of diclofenac at times of 3-5 hours of experiment. table 2: effect of diclofenac & compounds 10&11 on egg-white induced paw edema in rats. treatment groups paw thickness (mm) time diclofenac (n=6) compound 10 (n=6) compound 11 (n=6) baseline 4.42±0.14 4.35±0.07 4.45±0.16 0 5.43±0.14 5.40±0.04 5.41±0.11 1hr. 5.51±0.10 5.47±0.04 a 5.81±0.07 *b 2hr. 5.71±0.12 5.69±0.11 a 6.37±0.29 *b 3hr. 5.43±0.27 5.28±0.06 5.36±0.09 4hr. 5.14±0.13 4.97±0.05 5.00±0.23 5hr. 4.78±0.08 4.90±0.08 4.81±0.28 non-identical superscripts (a & b) among different tested compounds are considered significantly different (p<0.05) *significantly different compared to diclofenac (p<0.05). conclusion in vivo, the anti-inflammatory study showed that the incorporation of 4 aminobenzenesulfonamide into well known anti-inflammatory drugs (ibuprofen & indomethacin) retained the anti-inflammatory activity, when compared with that of diclofenac, compound (10) showed a comparable effect, whereas that of compound (11) showed a lower effect. similar work in future will 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of the aqueous extract of camellia sinensis against methotrexate-induced liver damage in rats ahmed h. jwied *,1 * department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract methotrexate (mtx) is a folate antagonist widely used in the treatment of neoplastic diseases; its biotransformation in the liver produced active metabolites that promote hepatotoxicity. the present study was designed to evaluate the hepatoprotective effect of aqueous extract of camellia sinensis (green tea) against mtx-induced liver damage in rats. a model of liver injury in rats was induced by intraperitoneal injection of 20mg/kg mtx as a single dose followed by saline and 1.25% and 2.5% aqueous extract of green tea (gte) were orally administered 7 days prior and 5 days after mtxintoxication as a sole source of drinking water. after killing the animals, blood samples were obtained for evaluation of serum levels of alanine and aspartate aminotransferases (alt and ast) and alkaline phosphatase (alp) activities, while liver tissue homogenate was prepared to evaluate tissue levels of glutathione (gsh) and malondialdehyde (mda). additionally, liver tissue sections were prepared and stained with hematoxylin and eiosin for histological evaluation. the results showed that administration of green tea extract (gte) significantly decreased the elevated levels of alt, ast and alp activities in the serum compared to mtx-treated group. treatment of animals with gte 7 days before and 5 days after mtx also elevates gsh levels and decreases mda levels significantly compared to mtxtreated group, this was associated with improving histological features that already impaired due to exposure to mtx. in conclusion, treatment of rats with gte protects hepatic tissue against mtxinduced liver damage in dose dependent manner. key words: green tea, hepatotoxicity الخالصة يثٕحشكضٚج ْٕ َظٛش نهفٕنٛج رٔ اسخؼًال ٔاسغ فٙ ػالج االيشاض انسشغاَٛت, ٚخحٕل انًٛثٕحشكضٚج بؼذ اٚعّ داخم انكبذ انٗ camelliaَاحح ساو ٚؤد٘ انٗ حسًى انكبذ.صًًج ْزِ انذساست نخقٛٛى فؼانٛت خشع يخخهفت يٍ انًسخخهص انًائٙ نهشا٘ االخعش) sinensisًاٚت كبذ اندشراٌ انًخخبشٚت يٍ انًثٕحشكضٚج.حى اسخحذاد انخسًى انكبذ٘ نهدشر بٕاسطت حقُّ داخم انغشاء ( فٙ ح -% ٢,,٠يهغى /كهغى (يٍ يادة انًٛثٕحشكضٚج نًذة خًست اٚاو يخخانٛت ٔرنك ٚخى بانخخابغ يغ يؼاندت اندشر ب) ٠٢انبشٚخَٕٙ ب ) غشٚق انفى كبذٚم ػٍ ياء انششب نًذة خًست اٚاو ياقبم ٔ سبؼت اٚاو يا بؼذ انحقٍ %( يٍ يسخخهص انًائٙ انشا٘ االخعش ػٍ ٠,, ٔ االسباسحٛج ايُٕٛحشاَسفٛشٚض alanine ) بانًٛثٕحشكضٚج.بؼذ قخم انحٕٛاٌ اخزث ػُٛت يٍ يصم انذو نقٛاط يسخٕٖ فؼانٛت االالٍَٛ) (aspartate aminotransferases) و انكًٛت انُسٛدٛت يٍ , يٍ خّٓ اخشٖ َاخز يسخخهص اَسدت ِِ ِِ ِِ ِِ ِِ ِِ ِٙ انكبذ ٔ َق alkaline)ٔاالنكالٍٚ فٕسفخٛض malondialdehyde) ٔ انًانَٕذاٚانذْاٚذ ) glutathione) ) اندهٕحاثإٌٚ phosphatase) هٍٛ.اظٓش ححهٛم باالظافت انٗ اخز خضء يٍ َسٛح انكبذ نهفحص انُسٛدٙ بٕاسطت صبغّ بًٕاد االٕٚسٍٛ ٔ انًٓٛاحٕكس انُخائح اٌ اػطاء يسخخهص انشا٘ االخعش ٚقهم ٔ بشكم يهحٕظ يسخٕٚاث فؼانٛت كم يٍ االالٍَٛ , االسباسحٛج ايُٕٛحشاَسفٛشٚض ٔ ( ٔرنك بانًقاسَت يغ اندشراٌ غٛش انًؼاندت بانشا٘ االخعش ٔ انخٙ حى حقُٓا alkaline phosphatase االنكاالٍٚ فٕسفخٛض) ٔادٖ اٚعا انٗ صٚادة يسخٕٖ اندهٕحاثإٌٚ ٔخفط يسخٕٖ انًانَٕذٚانذْاٚذ بصٕسة يهحٕظت بانًقاسَت يغ انحٕٛاَاث بانًٛثٕحشكضٚج انخٙ اػطٛج انًٛثٕحشكسٛج ٔ كاٌ ْزا ٔاظحا يٍ ححسٍ انشكم انُسٛدٙ نهكبذ ٔ انز٘ كاٌ يخعشسا يٍ يادة انًٛثٕحشكضٚج .كاسخُخاج انشا٘ االخعش ححًٙ اَسدت انكبذ يٍ انعشس انُاحح ػٍ انًٛثٕحشكضٚج. ,انًؼاندتانًبكشة باندشع انًؼخًذة يٍ introduction methotrexate (mtx) , a folate antagonist is widely used in the treatment of neoplastic diseases . it has also been used successfully as anti-inflammatory and immunosupressive agent in non neoplastic diseases such as psoriasis, arthritis biliary cirrhosis and reiter's syndrome (1, 2) . methotrexate is actively accumulated in the liver where it is metabolized and stored in polyglutamated form. the major side-effect of chronic methotrexate administration is hepatotoxicity, which is characterized by fatty infiltration, inflammation, cellular necrosis and apoptosis, steatosis, fibrosis and cirrhosis (3, 4). the mechanisms of methotrexate induced hepatotoxicity are not fully understood. from the results of in vitro experiments it has been suggested that increased oxidative stress contributes to methotrexate hepatotoxicity (5, 6) , both through increased reactive oxygen species activity and impaired anti-oxidative defense via depleted intrahepatic glutathione depots (7) . 1 corresponding author e – mail :ahemd ataimish76@yahoo.co.uk received : 16 / 6 / 2009 accepted : 27 / 10 / 2009 iraqi j pharm sci, vol.18(2) 2009 green tea in hepatotoxity 74 typical histopathological findings in mtx induced liver disease include nuclear atypia, vacuolization, and mild fatty metamorphosis. flavonoids have been found to play important roles in the non-enzymatic protection against oxidative stress (8, 9) , especially in case of cancer. flavonoids are group of polyphenolic compounds that occur widely in fruit, vegetables, tea, cocoas and red wine (10, 11, 12) .fresh tea leaves are rich in flavanol monomers known as catechins such as epicatechins (13) , which are 13.6 g/100 g in green tea and 4.2 g/100 gm dry weight in black tea (14) .in animal studies, it has been revealed that green tea may protect liver and brain cells against sequelae of oxidative stress induced by ethanol intoxication (15, 16, 17) . supplementation of green tea extract (gte) attenuates cyclosporine a-induced oxidative stress in rats (18) . catechins derived from tea leaves are natural, safe for consumption, and have been proved to be very effective antioxidants. the present study was designed to evaluate the protective effect of gte against mtx-induced hepatotoxicity in rats. material and methods preparation of aqueous green tea extract the aqueous extract of green tea was made according to the method of maity et al (1998) (19) by soaking for 10 minutes 1.25 gm and 2.5 gm of green tea leaves respectively in 100 ml of distilled water at 90 o c. solutions of gte were freshly prepared on daily bases, and then filtered to obtain final concentrations of 1.25% and 2.5% respectively. these solutions were used as substituent for water as the sole source of drinking fluid. experimental protocol twenty eight sprague-dawley rats (150250 g) of both sexes were housed in the animal house of the college of pharmacy, university of baghdad under controlled conditions of temperature and humidity and fed standard chow died and drinking water ad libitum. the animals were allocated into 4 groups and treated as follow: group i: seven animals received normal saline by i.p. injection for 12 days, sacrificed by cervical dislocation on day 13 and served as controls. group ii: seven animals were injected with single 20 mg/kg i.p. mtx followed by saline for 5 days (20, 21) . the animals were sacrificed by cervical dislocation on day 6 and served as positive controls. groups’ ιιι and iv: seven rats in each group treated with either 1.25% or 2.5% gte for 7 days before induction of hepatoxicity and 5 days after, and then the animals were sacrificed by cervical dislocation on day 13. after sacrification of animals by cervical dislocation, blood samples were obtained by thoracic section and serum was prepared for the evaluation of the activities of alanine aminotransferases (alt), aspartate aminotransferases (ast) and alkaline phosphatase (alp). moreover, liver were quickly excised, placed in chilled phosphate buffer solution (ph 7.4) at 4 0 c, blotted with filter paper and weighed. one gram of liver was then taken to prepare 10% tissue homogenate using the same buffer solution utilizing tissue homogenizer (22) for 1 minute at 4 0 c. all preparations were freshly prepared and kept frozen at -18 0 c unless worked immediately. tissue homogenate levels of gsh and mda were evaluated using standard procedures (23, 24) . liver tissues were prepared for histological examination using paraffin sections technique (25) . blocks were cut by microtome into 5 mm thick sections, stained with hematoxyline and eosin and then examined under light microscope. all data were expressed as mean ± s.e. unpaired t-test was carried out to compare populations using graph pad prism software (san diego, usa). a 0.05 level of probability was used as the criterion for significance. results significant increase in alt, ast and alp was observed in the serum of rats treated with mtx compared to control group. after treatment of rats with green tea extract 7 days prior & 5 days after mtx, a significant improvement in the levels of alt, ast, and alp was reported, their levels were significantly decreased (p<0.001) compared to mtx-intoxicated rats (figures 1, 2 and 3). 6 figure 1: effect of different treatment approaches on the serum activity of alanine aminotransferase (alt) in rats. each value represents mean ± s.d. -*significantly different with respect to control. -values with non-identical superscripts (a,b,c) with each parameter are significantly different (p<0.05). iraqi j pharm sci, vol.18(2) 2009 green tea in hepatotoxity 75 figure 2. effect of different treatment approaches on the serum activity of aspartate aminotransferase (ast) in rats. each value represents mean ± s.d. * significantly different with respect to control. -values with non-identical superscripts (a,b,c) with each parameter are significantly different (p<0.05) figure 3: effect of different treatment approaches on the serum level of alkaline phosphatase in rats. each value represents mean ± s.d. -* significantly different with respect to control. -values with non-identical superscripts (a,b,c) with each parameter are significantly different (p<0.05). rats treated with mtx resulted in a significant increase (p<0.05) in hepatic lipid peroxidation measured by the amount of mda formed, associated with significant decrease (p<0.05) in the liver tissue gsh levels. however, treatment of rats with gte for 7 days prior & 5 days after mtx, led to a significant decrease in mda levels (p<0.001) and elevation in gsh level (p<0.001) compared to mtxtreated group (figures 4 and 5). concerning the histological finding (figure 6), uses of mtx produces several pathological figure 4. effect of different treatment approaches on the malondialdehyde (mda) contents in rats liver homogenate. each value represents mean ± s.d. -* significantly different with respect to control. -values with non-identical superscripts (a,b,c) with each parameter are significantly different (p<0.05). figure 5. effect of different treatment approaches on the glutathione (gsh) level in rats liver homogenate. each value represents mean ± s.d. -* significantly different with respect to control. -values with non-identical superscripts (a,b,c) with each parameter are significantly different (p<0.05). changes in liver tissues, including fatty infiltration, macrovascular degeneration, pleomorphism, ballooning degeneration and hypertrophied hepatocytes (figure 7), while the liver section from rats treated with 1.25% of gte for 7 days prior & 5 days after mtx showed moderate fatty change, mild apoptosis and moderate collapse of the structure (figure 8) and livers of animals treated with 2.5%gte for 7 days prior & 5 days after mtx showed mild fatty changes of the mid zone, absence of fatty changes and preserved periventricular structures (figure 9). effect on alk.ph. 0 10 20 30 40 50 60 contrpl mtx g.t.1.25%+mtx g.t.2.5%+mtx effect on mda 0 1000 2000 3000 4000 5000 6000 7000 8000 contrpl mtx g.t.1.25%+mtx g.t.2.5%+mtx effect on gsh 0 5 10 15 20 25 30 contrpl mtx g.t.1.25%+mtx g.t.2.5%+mtx iraqi j pharm sci, vol.18(2) 2009 green tea in hepatotoxity 76 figure-1:control liver section showed portal area(pa),central vein(cv),mid-zone. cv pa midzone hepatic plates sinusoids figure 6. control section showed normal rat’s hepatic tissue with normal portal (pa); cental vein (cv) and mid zone(x 400). figure 7. liver section from rats treated with mtx showed magnification of periportal area, moderate fatty changes, macrovascular degenaration (blue arrows), pleomorphism, different cellular shapes (brown arrows), ballooning degeneration, hypertrophied hepatocytes (large size greenyellow arrows) (x 800). figure 8. liver section from rats pretreated with 1.25%gte and challenged with mtx showed closer view of periportal area, revealing moderate fatty change ( blue thin arrows), mild apoptosis (black arrows), moderate collapse of structure (wide blue arrow)(x 400). figure 9. liver section from rats pretreated with 2.5%gte and challenged with mtx showed preserved periventricular (cv) structure, absence of fatty changes, while noticing mild fatty changes of the mid zone (mz) (arrow) (x 400). figure-33 mt;liver section showed magnification of perpiportal area moderate fatty changes,macrovesicular degeneration(blue arrows),pleomorphism(different cellular shapes(brown arrows),ballooning degeneration; hypertrophied hepatocytes (larger size)(green-yellow arrows)(x800). figure-59 mv1;liver section showed preserved perivenular(cv) structure,absence of fatty changes ,while noticing mild fatty changes of the midzone(mz)(arrow)(x400). cv mz cv iraqi j pharm sci, vol.18(2) 2009 green tea in hepatotoxity 77 discussion epicatechins (antioxidant present in green tea) scavenge a wide range of free radicals including the most active hydroxyl radical, which may initiate lipid peroxidation. it prevents the loss of lipophilic antioxidant αtocopherol by repairing tocopheryl radicals and protection of the hydrophilic antioxidant ascorbate (26) . therefore, it may decrease the concentration of lipid free radicals and terminate initiation and propagation of lipid peroxidation (27) . the data presented in this study demonstrated the implication of oxidative stress in hepatic tissue induced by mtx treatment (fig. 3), manifested by increase in mda contents in liver tissue. epicatechins are effective scavengers of physiologically active reactive oxygen and nitrogen species including superoxide (28) , peroxyl radical (27) , peroxynitrite (29) and hypochlorous acid (30) . it was reported that, epicatechines can react with superoxide radical via one electron transfer mechanism or by a hydrogen abstraction mechanism to form the corresponding semiquinone (31) . epicatechins may chelate metal ions, especially iron and copper, which, in turn inhibit generation of hydroxyl radicals and degradation of lipid hydroperoxides which causes reactive aldehyde formation (32) . the liver damage was determined by measuring serum levels of alt and ast while level of tbars in liver was used as an indicator of lipid peroxidation. the levels of the antioxidant thiol in liver homogenates (gsh) was significantly improved upon treatment of mtx-intoxicated rats with 2.5%gte (fig. 4) which inhibited mtx-induced hepatic injury and thereby the level of oxidative stress, as it can decrease lipid peroxidation and enhance antioxidant enzyme activities, whereas the level of mda was significantly decreased comparable to mtx-intoxicated group. in agreement with the results obtained in this study, administration of green tea to ethanol-intoxicated rats resulted in the normalization of lipid peroxidation as well as glutathione concentration and alt activity in liver (17) . damaging liver tisse after mtx exposure is a well-known phenomenon, and the obvious sign of hepatic injury is the leakage of hepatic enzymes into plasma. there is no doubt that both the histological appearance and biochemical parameters supported a diagnosis of liver damage. the increased levels of serum enzymes such as alt, ast and alp have been observed in mtx-treated animals, which indicate the increased permeability, damage or necrosis of hepatocytes. green tea extract gave a high hepatoprotective effect by reversing these changes produced by mtx (fig.1, 2 and 5). the observed decrease in the serum activities of these enzymes showed that gte, to some extent, preserved the structural integrity of the liver from the toxic effect. it is well known that gte is effective scavengers of reactive oxygen species and may also function indirectly as antioxidants through their effects on transcription factors and enzyme activities (33, 34) . green tea extract, water-soluble antioxidants, has been demonstrated to inhibit iron-induced oxidation of synaptosomes by scavenging hydroxyl radicals generated in the lecithin/lipoxidase system (35) . on the one hand, gte can penetrate the lipid bilayer, decreasing free radicals concentration or influencing antioxidant capability in biomembranes (36, 17) . on the other hand, they could reduce the mobility of free radicals into the lipid bilayer as well. moreover, gte can interact with phospholipid head groups, particularly with those containing hydroxyl groups, so they could decrease the fluidity in the polar surface of phospholipid bilayer (37) . in addition, gte can prevent the loss of the lipophilic antioxidant _-tocopherol, by repairing tocopheryl radicals, and protection of the hydrophilic antioxidant ascorbate (38, 39) . liver is the major site for synthesis of gsh and detoxification of different drugs and xenobiotics in the liver may involves use of this tripeptide (40) . glutathione plays a common role in cellular resistance to oxidative damage as a free radical scavenger and by generation of ascorbate or tocopherol in liver (41) . the decreased hepatic gsh in mtx-intoxicated rats could be as a result of hexose monophosphate (hmp) shunt impairment due to mtx, thereby nadph availability is reduced and the ability to recycle gssg to gsh is decreased (42) . by blocking oxidative damage through lipid peroxidation and protein oxidation, green tea extract prevent the loss of membrane permeability and dysfunction of cellular proteins and decreases the endogenous level of hydroxyl radical and gsh (40) . in conclusion, green tea has hepatoprotective activity against 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45:703-710. 41. mark, d., ip, s., li, p., poon, m. and ko, k. alterations in tissue glutathione antioxidant system in streptozotocininduced diabetic rats. mol. biochem 1996; 20:153-158. 42. lu s. regulation of hepatic glutathione synthesis: current concepts and controversies. faseb j. 1999; 3:11691183 iraqi j pharm sci, vol.19(1) 2010 detection of ca19-9 in benign cases 62 detection of carbohydrate antigen ca19-9 levels in sera and tissues' homogenate of breast and thyroid benign cases gheid h. alubaidi *,1 , zeyan a. ali ** and abdulrahman r. mahmood * * department of chemistry,college of education ibn-alhaitham,university of baghdad,baghdad,iraq. ** department of chemistry, science education college, university of salahaddine,erbil, iraq. abstract the aims of the present study are to evaluate the levels of ca19-9 in sera and tissues' homogenate of breast and thyroid benign patients in order to assess its use as an early diagnostic parameter in differentiation between malignant and benign cases. the study was conducted on 8 patients with breast benign tumor and 8 patients with thyroid benign tumor, by the enzyme linked immunosorbent assay (elisa) technique. the results of ca19-9 levels in sera were (15 ±1.58 and 10.67 ±2.08)u/ml respectively compared with serum ca19-9 levels of control group which was 7.74 ±4.92 u/ml, the results were found to be highly significantly in breast tumor patients and non significantly in thyroid tumor patients than control group. the results of ca19-9 levels in tissues' homogenate were (356.2 ±173.75 and 20 ±14.4)u/ml respectively. the results were found to be highly significant in tissues' homogenate of breast tumor patients and non significant in thyroid tumor patients of higher compare with the it's serum levels of the same patients groups. key words: carbohydrate antigen ca19-9, benign cases and ca19-9, breast tumor and ca19-9, thyroid tumor and ca19-9. الخالصة فً مصىل وأنطجت مرضى أورام الثدي والغدة الدرقٍت الحمٍدة ca19-9الغرض من الدراضت الحالٍت هى قٍاش مطخىٌاث 8لدراضت على اجرٌج ا لغرض ححدٌد امكانٍت اضخخدامه كمؤشر حشخٍصً مبكر ٌمكن من خالله الخمٍٍس بٍن الحاالث الحمٍدة والخبٍثت. .(elisaمصابٍن باورام الغدة الدرقٍت الحمٍدة بىاضطت حقنٍت االمخساز المناعً االنسٌمً ) 8مرضى مصابٍن باورام الثدي الحمٍدة و مقارنت مع مطخىٌاحه فً مجمىعت الطٍطرة u/ml(2.08±10.67و 1.58±15فً المصىل ) ca19-9وقد كانج النخائج لمطخىٌاث , و أظهرث النخائج قٍمت معنىٌت عالٍت جدا فً مصىل اورام الثدي وقٍمت معنىٌت غٍر عالٍت فً اورام u/ml(4.92±7.74والخً كانج) و 173.75±356.2اما بالنطبت لمجانص انطجت المرضى فقد كانج مطخىٌاحه ) الغدة الدرقٍت مقارنت بمجمىعت االصحاء. 20±14.4)u/ml لٍت جدا فً مجانص انطجت اورام الثدي وقٍمت معنىٌت غٍر عالٍت فً اورام بالخعاقب. أظهرث النخائج قٍمت معنىٌت عا الغدة الدرقٍت مقارنت مع مصىل نفص المرضى. introduction the term "polyp" is clinical description of any evaluated tumor which may be found in tissues as a projection in the lumen as polyp referred as benign tumor. (1) tumor markers in general are substances present in or produced by a tumor or by the tumor host in response to the tumor's presence that can be used to differentiate a tumor from normal tissue or to determine the presence of a tumor based on measurements in the blood or secretions. (2) although increased levels of serum tumor markers are often associated with the presence of cancer, marker concentrations may also rise in a number of benign conditions. (3) chemically, ca19-9 is a tetrasaccharide derived from lewis blood group antigens that are not exclusive to erythrocytes, but they can be found in different tissues and organs. carbohydrate antigen ca19-9 after its discovery was first described as tumor associated antigen and used as tumor marker for colon and pancreas caner. (4) in addition, a previous study demonstrated that the level of serum ca19-9 is dependent on the severity of the bile duct obstruction and the degree of cholangitis. an increase in the serum level ca19-9 can be detected even in benign bile duct diseases. (5) it has been reported that ca19-9 was elevated in breast malignant cases only, while the level of ca19-9 was within normal value (37u/ml) in the benign cases. (6) a recent study claimed that ca19-9 levels in sera of thyroid cancer was found to be significantly higher than that for control group. (7) the aims of the present study are to investigate ca19-9 levels in sera and tissues homogenate of patients with benign breast and thyroid tumors by enzyme linked immunosorbent assay elisa technique, then to assess the differences in the levels of ca199 in the serum of benign tumor patients and control, also to correlate the serum and tissue homogenate levels of ca19-9 in patients with breast and thyroid, such determinations might be useful to predict whether or not the tumor marker is related to the benign tumor. 1corresponding author email : dr.gheid_2008@yahoo.com received : 29/12/2009 accepted : 6 / 4/ 2010 iraqi j pharm sci, vol.19(1) 2010 detection of ca19-9 in benign cases 63 materials and methods patients the patients included in this study with age ranged 17-75 years, were classified into three groups as follows: 1. the first group included 8 patients with benign breast tumor (g1). 2. the second group included 8 patients with benign thyroid tumor (g2). 3. the last group included 30 healthy subjects considered as control group (g3). the patients were selected, according to the histopathological investigation. they were admitted for treatment at medical city hospitals (baghdad teaching hospital and nursing home hospital) and all surgical operations for all patients were carried out under the supervision of surgeons. preparation of blood samples five milliliters (mls) of venous blood were drawn from each patient by veinpuncture just before surgery, left to clot, and then centrifuged at 4000 r.p.m. for 30 min. serum was separated and stored at -20˚c until time of analysis. collection of specimens the tumor tissue was surgically removed from patients. the specimens were immediately kept in normal saline solution and stored at -20˚c until the time of homogenizing process. homogenization of tumor tissues the frozen tissue was sliced finely scalped in petridish standing on ice, and then homogenized with three fold volumes of phosphate buffer ph7.4 by the homogenizer. the homogenate was filtered through nylon gauze to eliminate fiber connective tissues. the filtrate was centrifuged at 4000 r.p.m for 30 min at 4˚c in order to precipitate the remaining intact cells and the intact nucleus. the supernatant and precipitate fraction were separated and frozen at -20˚c until use. determination of (ca19-9) antigen using elisa assay a basic elisa work follows simple steps according to included manufacturer’s instructions of ca19-9 kit supplied by dia metra company, italy. statistical analysis comparison between variables was performed by using student's t-test. p values ≤ o.o5 considered significant and ≤ o.oo1 considered highly significant. results and discussion the mean ± standard deviation (sd) for serum levels of ca19-9 of control group was found to be 7.74 ± 4.92 u/ml from table ( 1 ) table 1: ca19-9 values in sera of patients groups group mean ± sd p value breast benign patients 15 ±1.58 0.0027 thyroid benign patients 10.67 ±2.08 0.32 control 7.74 ± 4.92 a highly significant elevation in the level of ca19-9 in sera of benign breast patients, but a nonsignificant elevations were found in sera of thyroid patients compared to control was observed.the results are in agreement with other reported data stated that elevated serum ca19-9 levels were found in 61% of benign cases, while ca19-9 serum levels remain within normal range in 50% of malignant cases. (8) a study performed on 91 adults to determine validity of ca19-9 among other cancer antigens as tumor markers, which claimed that ca19-9 showed statistical differences in the serum and ascitic fluids both of malignant and benign compared with control. (9) a research conducted to evaluate the etiology of elevated ca19-9 serum level in 353 subjects, 2.8% were diagnosed with malignancies, 27.5% with benign and 69.7% were non tumor patients, the authors concluded that ca19-9 should not be used as a screening tool, also in cases of a persistently elevated ca19-9 levels, further work-up for determining the etiology should be done. (10) a recent study has confirmed the role of estrogens as the cause of endometrial thickening through hormonal imbalance leading to elevated ca19-9 among other tumor markers in the serum of benign gynecological conditions may be a source of misdiagnosis of malignant diseases. (11) table 2 shows the results of the mean ± sd of serum and tissue homogenate of breast and thyroid benign patients. a highly significantly elevation were found in tissue homogenate compared to serum in both studied groups.a study conducted on patients with different malignant diseases and benign conditions postulated that ca19-9 may be used as fairly reliable diagnostic tool, but cannot be used to predict survival. (12) some authors have reported that the elevation in ca19-9 could be provided by differences in the cellular and humoral immune response to hydatidosis. the theories about these findings include substance that may be synthesized by the host in response to infection which lead then to conclude that the possibility of a hydatidosis should be born in mind in iraqi j pharm sci, vol.19(1) 2010 detection of ca19-9 in benign cases 64 differential diagnosis of palpable mass and elevated ca19-9 levels in the breast especially in endemic areas. (13) table (2):ca19-9 value in sera and tissues of patients groups group serum mean ± sd tissues mean ± sd p value breast benign patients 15 ±1.58 356.21 ±173.75 4.7*10 -4 thyroid benign patients 10.67 ±2.08 20 ±14.4 0.187 conclusion a conclusion could be drawn that the tumor marker ca19-9 was elevated in benign conditions like breast and thyroid benign tumors, so it couldn't be used as peculiar detective, screening and diagnostic tool for malignant cases. references 1. schwartz f c, bunicardi md, and andersen dm, philadelphia, "small intestine colon, rectum and anus principles of surgery'', (2005), 8 th ed, 1038-1083. 2. bishop ml, fody ep, schoeff l, philadelphia, "clinical chemistry", (2005), 3 rd ed, 608-614. 3. guo q, zhang b, dong x, xie q, guo e, huang h and wu y, elevated levels of plasma fibrinogen in patients with pancreatic cancer: possible role of a distant metastasis predictor" pancreas, (2009), 38(3): e75-79. 4. ugorsks m and laskowsk a, sialy lewis(a): a tumor associated carbohydrate antigen involved in adhesion and metastatic potential of cancer cells, acta bioch polonica, (2002), 49(2): 303310. 5. leelawwt k, sakchinabut s, narong s and wannaprasert j, detection of serum mmp7 and mmp-9 in cholangiocarcinoma patients: elevation of diagnostic accuracy, bmc gastroenterology, (2009), 9: 30-38. 6. shitrit d, zingerma b and kramer mr, diagnstic value of cyfra 21-1, cea, ca19-9, ca15-3 and ca125 assay in pleural effusions: analysis of 116 cases and review of the literature, the oncologist, (2005), 10: 501-507. 7. aldujaili ah, altaei wf, turkey km, alubaidi gh, comparative study of ca199 levels as tumor marker in sera and tissues homogenate of breast, prostate and thyroid cancer patients, ibn al-haitham j for pure and appl sci, (2009), 22(1): 8896. 8. harrelli d, caruso s, pedrazzani c and roviello f, ca19-9 serum levels in obstructive jaundice: clinical value in benign and malignant conditions, am j surg, (2009), 16-19. 9. tuzun y, celik y, bavan k and canoruc f, correlation of tms in ascitic fluid and serum: are measurements asitic tms a futile attempt?, j int med res, (2009), 37(1): 79-86. 10. kim bj, lee kt, moon tg and rhee j c, how do we interpret an elevated carbohydrate antigen 19-9 levels in asymptomatic subject?, dig liver dis, (2009), 41(5): 364-369. 11. cecchi e, lapi f, vannacci a and mugelli a, increase levels of ca125 andca19-9 serum tumor markers following cyclic combined hormone replacement therapy, j clin pharm ther. (2009), 34(1): 129-132. 12. sandlom g, granroyh s and rasmussen ic, tps, ca19-9, vegf-a and cea as diagnostic and prognostic factors in patients with mass lesions in the pancreatic head, ups j med sci, (2008), 113(1): 57-64. 13. yuksel bc, ozel h, akin t, avsar fm and hengirmen s, primary hydatid cyst of the breast with elevated ca19-9 levels, am j med hyg, (2005), 73(2): 368-370. iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata doi: https://doi.org/10.31351/vol29iss2pp48-61 48 isolation, structural characterization and identification of major constituents in ephedra foliata naturally growing in iraq by tlc, gcms and uplc-esi-ms/ms ahmed s. khaleefa*,1 and maha n. hamad** * ministry of health and environment, baghdad,iraq. **department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad. iraq abstract the aerial part of ephedra foliata family ephedraceae have long been used in traditional medicine and now ephedra species have medicinal, ecological, and commercial value. the variety of pharmacological actions of this plant is due to its chemical constituents. ephedrine and related alkaloids; are the new potential medicinal value of ephedra supplements for weight loss or performance improvement. other pharmacological actions like antibacterial and antifungal effects of the phenolic acid compounds, the immunosuppressive action of the polysaccharides, and the antitumor action of flavonoids. the genus of this plant wildly distributed throughout asia, america, europe, and north africa. the study is aimed at screening the phytochemical constituents due to the importance of pharmacological actions of this plant. that is done by maceration the aerial part of ephedra foliata with 80% ethanol for 9 days and fractionated by n-hexane, chloroform, ethyl acetate, and n-butanol. the n-hexane, chloroform, n-butanol fractions, and isolated compounds were analyzed by gas chromatography-mass spectrometry, thin layer chromatography; ultra-performance liquid chromatography coupled with electrospray ionization mass/ mass spectroscopy. the various chromatographic and spectroscopic results indicate the presence of a different type of phytochemicals like ephedrine, 6-hydroxy kynurenic acid, vicenin 2 and quercetin 3-sophoroside-7-rhamnoside. these active constituents of ephedra foliata have been identified play a crucial role in our life due to its pharmacological actions. keywords: ephedra, gas chromatography, mass spectrometry, ultra-performance liquid chromatography electrospray ionization mass/ mass , 6-hydroxy kynurenic acid. نمو بشكل طبيعي في ي ذيال نبات العلندىالمكونات الرئيسية في و تشخيص التوصيف الهيكلي ,العزل كروماتوغرافيا الغازو و كروماتوغرافياو الرقيقهبواسطه كروماتوغرافيا الطبقه العراق من خالل عالية االداء السائله **و مها نوري حمد 1*،احمد سعدي خليفة العراق ،وزارة الصحة والبيئة، بغداد* ، العراق .فرع العقاقير والنباتات الطبية ، كلية الصيدلة ، جامعة بغداد** الخالصة مجموعة .الهوائية لنبات االفيدرا في الطب القديم واالن نبات االفيدرا لديها فوائد دوائية وبيئية و تجاريةمنذ فترة طويلة تستخدم االجزاء قلويدات ذات صلة بها؛ لديها القيمة الطبية االفيدرين وال .متنوعة من االستعماالت الدوائية لهذا النبات ويرجع ذلك إلى مكوناته الكيميائية المختلفة االستخدامات الدوائية األخرى مثل التأثيرات المضادة للبكتيريا والفطريات لمركبات .ا لمكمالت اإليفيدرا لفقدان الوزن أو تحسين األداءالمحتملة حديث في جميع أنحاء آسيا بكثرةجنس هذا النبات الموزع .حمض الفينول ، المثبط للمناعة من السكريات ، والعمل المضاد للورم من مركبات الفالفونويد يتم ذلك .الدوائية لهذا النبات ستعمالتالدراسة إلى فحص المكونات الكيميائية النباتية بسبب أهمية اإل هذة تهدف .وأمريكا وأوروبا وشمال إفريقيا الهكسان ، والكلوروفورم ، وخالت بواسطةتجزيئة من ثمأيام و 9٪ لمدة 80مع اإليثانول بنسبة نبات االفيدرامن هوائيةء الاجزاال تنقيععن طريق و كروماتوغرافيا الطبقه الرقيقه الهكسان ، الكلوروفورم ، بيوتانول ، والمركبات المعزولة من قبلاجزاءوقد تم تحليل وتانول.باإليثيل ، و من المواد ة نوع مختلفاتشير النتائج الكروماتوجرافية والطيفية المختلفة إلى وجود .كروماتوغرافيا عالية االداء السائلهالغازو و كروماتوغرافيا . تم التعرف على سيفروسايد -7رامنوسايد -3ستين ور، والكي 2هيدروكسي ، والفينسينين -6الكيميائية النباتية مثل اإلفيدرين ، وحمض الكينورينيك .الدوائية فعاليتهالعب دورا حاسما في حياتنا بسبب ت نبات االفيدرا التي هذه المكونات النشطة من هيدروكسي.-6حمض الكينورينيك ، كروماتوغرافيا عالية االداء السائله ،الغازو كروماتوغرافياالكلمات المفتاحية: اإلفيدرا ، 1corresponding author e-mail: ameerzayona88@gmail.com received: 14/10 /2019 accepted: 10/3 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp48-61 iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 49 for at least five thousand years, ephedra plants have been used in traditional medicines in which dry stems are used for symptoms derived from the common cold, flu, asthma, bronchitis, nasal congestion and hay fever(1). the ephedra plant is also used for the treatment of arthritis, fever, hives, dyspnea, headache, joint and bone pain, wheezing and hypotension(2). ephedra corresponds to a genus of gymnosperms including over 50 species of tropical and subtropical, small, much-branched shrubs founds in the dry regions of both hemispheres(3). it is related to the gnetophyta division of gymnosperms plants and is related to the conifers(4). the plant species are short, evergreen and virtually leafless shrubs that grow about (60to90cm) tall. the stems are green in color, slender, erect, small ribbed and channeled, about (1.5 mm) in diameter and commonly terminating in a sharp point. nodes are (4to6 cm) apart, and small triangular leaves appear at the stem nodes which are usually reddish brown(5). these species grow in dry weather over wide parts of the northern hemisphere including north america, europe, north africa, and southwest and central asia(6). the chemical constituents and pharmacological actions of ephedra species the aerial parts of various plant species first of all, ehedrine-type alkaloids, usually have from (0.02% to 3.4%) of six optically active alkaloids as shown in figure 1,(−)-ephedrine (eph) is the major one including 30–90% of the total alkaloids, (+)-pseudoephedrine (pse), the diastereomer of (−)-eph, (−)-n-methylephedrine, (−)-norephedrine , (+)-n-methylpseudoephedrine and (+)-norpseudoephedrine (7) . secondly, nonephedrine alkaloids and amino compounds in ephedra species. ephedroxane(8), ephedradine a(9), cyclopropyl-α-amino acid(10), maokonine(11), 6methoxykynurenic acid(12), nmethylbenzylamine(13), tertmethylpyrazine(14), and 6-hydroxykynurenic acid(10). thirdly, miscellaneous non-alkaloidal natural constituents of ephedra: trans-cinnamic iacid, icatechin, isyringin, iepicatechin, symplocoside, ikaempferol i3-orhamnoside i7-o-glucoside, iisovitexin i2-orhamnoside, iherbacetin i7-o-glucoside, iand ipollenitin ib iand iherbacetin 7-oneohesperidoside (15). ephedra species have numerous pharmacological actions for instance antiinflammatory due to the inhibition of prostaglandin e2 biosynthesis(8), antibacterial and antifungal (16), anti-cancer activity(17)(18), cns stimulant and perhaps thermogenic effects (19), antiviral activity(20) and finally antioxidant and hepatoprotective activity(21). this study was designed to investigate the phytochemicals and their proportions of the aerial part of ephedra foliata naturally growing plants in iraq. figure 1. ephedrinetype alkaloids material and methods collection of plant materials: ephedra foliata was collected during march – june 2018 from tikrit province, iraq. this plant was authenticated by dr. khansaa rasheed / iraq natural history research center and museum plant and environment department / university of baghdad. the stems and aerial parts were, dried in a shed, rendered into a coarse powder. extraction extraction by maceration then fractionation according to active constituents. about 650 grams of aerial part the powdered plant material was soaked in 2500ml (1:7) 80% ethanol, with regular shaking, at room temperature. after 3 days, the ethanol extracts are filtered, repeat the process 3 times for 9 days. the filtrate was evaporated to dryness under vacuum using a rotary evaporator, to get dried extract. the dark greenish residue was suspended in 250ml h2o and partitioned successively with n-hexane, chloroform, ethyl acetate, and n-butanol until reaching a clear layer for each fraction. the first three fractions are dried over anhydrous sodium sulfates, filtered, and evaporated to dryness(22). iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 50 phytochemical investigation of chemical constituents of iraq ephedra foliata: preliminary identification by chemical test: 1-test for alkaloids:  mayer’s reagent.  wagner’s reagent 2-test for flavonoids about 5% alcoholic potassium hydroxide is added and then a few drops of 5% hydrochloric acid are added. 3-test for phenols few imilligrams of ethanol plant extract are treated with ifew drops of 1% ferric chloride(23). purification of crude alkaloidal extract: the chloroform fraction was acidified by adding hydrochloric acid (5%). this solution was then placed in a separatory funnel and partitioned with equal volume of chloroform (four times). the upper aqueous acidic layer was separated and basified with ammonium hydroxide (25% nh4oh) to ph 10 using ph meter. after the basification process, the solution becomes warm and allowed to stand for 2 hours. then partitioned with an equal volume of chloroform in a separatory funnel (three times). the chloroform ilayer iwas iseparated, dried with anhydrous sodium sulfate, filtered and evaporated under reduced pressure then tested with dragendorff and mayer’s reagents(24). isolation of some phytochemicals by using preparative tlc thin-layer chromatography was used to determine phytochemical compounds by using different solvent systems like chloroform; methanol (90: 1), chloroform: acetone: formic acid (75: 16.5: 8.5) and ethyl acetate: formic acid: acetic acid: water (80:5: 6: 10) for n-butanol fraction(25). while toluene-chloroform-ethanol-methanol (20:50:30:10), ethyl acetate-isopropanol-ammonia (100:2:1) and cyclohexane-ethanol-dietllyamine (80:10:10) for chloroform fraction that were tried for identification to get the best separation and the largest number of spots (26).  as1 compound was isolated from n-butanol fraction using readymade preparative tlc silica gel gf254 plates and mobile phase ethyl acetate: formic acid: acetic acid: water (80:5: 6: 10) (25). detection of the as1 compound was done by examination under uv light with wavelengths, 254 and 366 nm.  as2 compound tertiary amine alkaloid was isolated from purified chloroform fraction after basification using readymade aluminum oxide on tlc-glass plates and mobile phase toluenechloroform-ethanol-methanol (20:50:30:10 ) (26). detection of the as2 compound was done using dragedorff,s spray reagent is detected as a brown zone.  the purity of each bands are verified by analytical tlc until a single point are obtained on the tlc plates for identification. identification and structural characterization of isolated compounds and phytochemicals in fractions were done by i-gc-ms analysis the conditions used in the gc / ms analysis are compatible with the thermal desorption system (td-20), gc / msqp / 2010 plus (shimadzu, japan) composed of an automatic sampler. the mass spectrometer instrument was connected. column rtx-5ms (30 mm × 0.25 mm × 0.25 µm), operating in electronic printing mode at 70 ev. in this instrument, (99.99%) of helium gas is used as a carrier gas with a movement frequency of (1.2 ml / min). the initial temperature of the column oven is 80 ° c (isothermal for four minutes) with a constant increase from (5 ° c / min to 310 ° c), flow rate of (1.21 ml / min) and column pressure of 81, 7 kpa in the scanning interval of 0.50 s, the mass spectrum is prepared with a mass scan of (40to650) m /z(27). ii-ultra performance liquid chromatographyelectrospray ionization mass/ mass spectrometry (uplc-esi-ms/ms) analysis electrospray ionization mass spectrometry in negative and positive ions acquisition mode is performed in xevo tqd triple quadruple instruments. water corporations, milford, ma01757 usa uu. the sample solution (100 μg / ml) is prepared using high-performance liquid chromotography (hplc) analytical grade methanol, the filtrate uses a membrane disk filter (0.2 μm), then subjected to lc / esi / ms. the sample injection volume (10 μl) is injected into the uplc instruments equipped with c-18 reverse phase columns (acquitys uplc / beh c18 particle size of 1.7µm-2.1 ×50mm column). the mobile phase is prepared by filtration using a 0.2μm filter membrane disk and degassed by sonication before injections. the elution of the mobile phase is carried out with a flow rate of 0.2 ml per minute using a mobile gradient phase which includes two eluents: the eluent a is acidified in water with 0.1% of hcooh and the eluent b is methanol acidified with 0.1% of hcooh. the elution is performed using the gradient. the parameters for the analysis are performed using the negative ion mode as follow:150° c source temperature, 30ev cone voltage, 3kv capillary voltage, desolvation temperature about (440 ° c, 50l / h) cone gas flow and desolvation gas flow of (900l / h)(28). mass spectra are detected in electrospray ionization between m/ z (100–1000). peaks and spectra are processed using maslynx (4.1) software and are tentatively identified by comparing their retention times and masses spectra with the reported data (27). https://www.sigmaaldrich.com/catalog/product/sial/90066?lang=en&region=iq https://www.sigmaaldrich.com/catalog/product/sial/90066?lang=en&region=iq iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 51 results phytochemical investigation of chemical constituents of ephedra foliata: 1-preliminary identification by chemical test: various qualitative phytochemical screening tests were done to establish the chemical composition of each extract shown in table1. tabe1. phytochemical screening tests for crude extract phytochemical test type of phytochemical results mayer’s alkaloids + wagner’s alkaloids + koh flavonoids + fecl3 phenols + 2-thin layer chromatography tlc (analytical and preparative): according to tlc results which are shown below a1 and a2 were found the best mobile phases for separation and isolation of as1 and as2 respectively as result shown below. figure 2. tlc for chloroform fraction before basify (1), after basify (2) and pseudoephedrine standard (s) developed with a2 solvent system, at 254 nm and after dragedorff,s spray reagent. figure 3. preparative tlc for isolated as2 from chloroform fraction after basify with developed the a2 solvent system with dragendorff reagent. figure 4. tlc of n-butanol fraction before hydrolysis with different titration under uv 253nm and 366nm. iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 52 figure 5. preparative tlc of n-butanol fraction before hydrolysis with different titration under uv366 nm to isolate as1. figure 6. preparative tlc of n-butanol fraction before hydrolysis with different titration under uv 254 nm to isolate as1. 3-gas chromatography mass spectrometry gc/ms: a.gc/ms of n-hexane fraction: identification of phytochemical compounds in n-hexane fraction by gas chromatography mass spectrometry. figure 7. gc/ms of n-hexane fraction. table 2. compounds identified in n-hexane fraction by gc/ms. no. of peaks retention time (r.t) name base peak 1 31.725 1-octadecyne 41.00 2 34.885 n-heptadecanol-1 43.10 3 34.948 hexadecanoic acid, ethyl ester 88.05 4 35.966 hexadecanoic acid, trimethylsilyl ester 73.00 5 38.430 1-methyl-1-(2-tridecyl)oxy-1 silacyclopentane 143.15 6 38.681 1-octadecene 43.05 7 44.870 di-n-octyl phthalate 149.00 8 52.711 17-pentatriacontene 43.00 9 56.446 gamma.-sitosterol 43.05 10 59.144 stigmast-4-en-3-one 43.00 b. gc/ms of chloroform fraction: identification of phytochemical compounds in chloroform fraction by gas chromatography mass spectrometry. iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 53 figure 8. gc/ms for chloroform fraction. table 3. compounds identified in chloroform fraction gc/ms. no. of peak retention time name m.wt base peak 1 20.663 3,4-dimethyl-5-phenyl-2 oxazolidinone 191 57.05 2 20.958 1-undecene 154 41.05 3 21.175 ephedrine 165 58 4 24.257 phenol, 3,5-bis(1,1-dimethylethyl)206 191.05 21 34.537 aziridine, 1,2-dimethyl-3-phenyl-, trans 147 146 33 38.194 linoleic acid ethyl ester 196 57.05 58 48.991 squalene 410 69 c. gc/ ms for isolated as2: identifiation of isolated as2 compound form chloroform fraction by gas chromatography mass spectrometry. figure 9. gc ms for isolated as2 compound from chloroform fraction after basify. iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 54 figure 10. fragmentation and structure elucidation of isolated as2 compound by gc/ms. table 4. isolated as2 compound identified by gc ms(29-30) no. of peak retention time name area % m.wt base peak 2 20.818 ephedrine 80.7 165 58.05 4ultra-performance liquid chromatography electrospray ionization mass/ mass ((uplc-esims/ms): identification of the results from uplc-esi-ms / ms depended on molecular weight, retention time and mass fragmentation through different sites specialized in the identification and confirms the result of a search with previous studies. a. uplc for isolated as2: identifiation of isolated as2 compound form chloroform fraction by ultraperformance liqiud chromatography figure 11. uplc for isolated as2 from chloroform fraction after basification. iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 55 figure 12. first mass for isolated as2 peak 1 at retention time 4.1min with major molecular ion [m+h]+ 166.093. figure 13. mass fragmentations for isolated as2 compound. table 5. uplc esi ms/ms for isolated as2 compound peak no. of ms1 r.t [m+h] peak no. of ms2 r.t base peak name reference 1 4.1 166 14 3.93 148.0392 ephedrine (31-32) iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 56 figure 14. structural elucidations of as2 fragmentations (31)(32). b. uplc for isolated as1: identifiation of isolated as1 compound form n-butanol fraction by ultra performance liqiud chromatography figure 15. uplc for isolated as1 compound from n-butanol fraction before hydrolysis at peak 3. figure 16. first mass for isolated as1 compound peak 3 at retention time 3.92 min with major molecular ion [m-h]204.0892 iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 57 figure 17. mass fragmentation for isolated as1 compound. table 6. uplc esi ms/ms for isolated as1 compound figure 18. structural elucidations of as2 fragmentations(33). c. uplc n-butanol fraction: identification of phytochemical compounds in n-butanol fraction by ultra-performance liqiud chromatography figure 19. uplc for n-butanol fraction. peak no. of ms1 r.t1 [m-h] peak no. of ms2 r.t2 base peak name reference 3 3.92 204.0892 10 4.04 159.9218 6-hydroxykynurenic acid (33-10-34-35) iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 58 figure 20. first mass for peak 6 at retention time 7.29 min with molecular ion [m-h] 593.1935 from nbutanol. figure 21. first mass for peak 9 at retention time 7.99 min with molecular ion [m-h] 771.2651 from nbutanol fraction. table 7. identified compounds by uplc-esi-ms/ms fragmentation of n-butanol fraction: peak no. compound name class rt.1 m.w ms1 m-h rt.2 ms/ms references 6 vicenin 2 flavonoid glycosides 7.29 594 593.19 31 7.58 593,575, 565,533, 503,475,459,445,431,38 2,353, 311,105,87,73 (36-37-38) 9 quercetin 3sophoroside7rhamnoside flavonoid glycosides 7.99 772 771.26 51 7.3 771,505,461,447,341,30 1,299,271,253,179,161,1 47,133,103,73,59,43 (39-40-41) 3 6-hydroxy kynurenic acid quinoline-2carboxylic acid 3.32 205 204.08 92 3.8 204,176,159.9, 158,132.9,117.9 (33-10-34-35) iraqi j pharm sci, vol.29(2) 2020 identification of major constituents in ephedra foliata 59 discussion natural products have always been a preferred choice of all as it plays a great role in discovering new medicines. the hexane fraction of the plant was investigated by gc-ms which revealed the presence of gamma.-sitosterol and stigmast-4-en-3-one, the chromatogram showed peaks with retention times (56.446 and 59.114) respectively and corresponding to the molecular ion peaks in comparison with nist database as shown in (figure7, table 2). the chloroform fraction of the plant was investigated by tlc, gc ms and uplsesims/ms which showed the presence of a different type of secondary metabolites like alkaloids and triterpene. as2 compound isolated from chloroform fraction after basify by alumina tlc plates investigated as ephedrine due to its results. first of all, analytical tlc gives a brown zone with dragedorff,s spray reagent as shown in (figure 2-3). furthermore, the gc ms result showed the presence of ephedrine in chloroform fraction at peak 3 (figure 8 and table 3) also, isolated as2 investigate by gc ms as ephedrine according to its retention time, molecular weight [165] and base peak [58] depending on nist database (figure 9,10 ,table 4). finally, upls-esims/ms characterized as2 compound as ephedrine according to its retention time, molecular ion peak at m/z 166 [m+h]+ and mass fragmentation show loss of water [m+h-h2o] to give 148(base peak), then [m+h-ch3] yield m/z 132 and loss of methyl group from nitrogen atom yield m/z 117 (figure 1112-13-14, table 5)(31). the n-butanol fraction was investigated by tlc and upls-esims/ms which showed the presence of flavonoid glycosides which play important anticancer activity (figure 19-20-21, table7). as1 compound was isolated from nbutanol fraction by preparative tlc recognized as 6-hydroxykynurenic acid since it is given under ultraviolet light at 254 nm reddish-white fluorescence and 366 nm strong fluorescence and a very small amount could be detected(42). besides upls-esims/ms results of as1 compound and n-butanol fraction predicted 6-hydroxykynurenic acid depending on its retention time, molecular ion peak at m/z 204.0892 [m-h]and mass fragmentation suggesting the loss of 44 da [m h -44] to give m/z 159.9(base peak) in comparison with mass bank database. beside, 6-hydroxykynurenic acid was previously isolated from ephedra foliata (figure 15 1617-18, table 6) (33-10-34-35). conclusion the results of the current study showed isolate ephedrine from chloroform fractions after purification. while 6-hydroxykynurenic acid presence in n-butanol fraction due to its acidity. the active components of e. foliata have been identified play a crucial role in our life due to its 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ayob jaccob**,1 and muhsin s.g. al-moziel*** * ministry of health and environments, maysan center for cardiac diseases and surgery. maysan, iraq. ** department of pharmacology and toxicology, college of pharmacy, basrah university, basrah, iraq ** department of pharmacology and toxicology, college of pharmacy, basrah university , basrah, iraq abstract one of the most efficient aminoglycosides is amikacin. yet, it has been linked to unwanted renal toxicity, which has resulted in negative alterations in various biochemical indicators, particularly those related to oxidative stress, kidney function, and inflammation. the goal of this research was to see how citrus bergamot extract affected hemato-biochemical, inflammatory, and oxidative stress parameters in male rats triggered by acute amikacin toxicity. a total of 30 male rats were divided into five equal groups, each with six rats. the control group received 1 ml dw orally for ten days. group 1 was given 1 ml of dw orally for ten days, and i.p. ak (1.2 g/kg) on day seven. group 2 was given 100 mg/kg citrus bergamot extract orally for ten days. groups (3) and (4) were given 100 mg/kg and 200 mg/kg of citrus bergamot extract, respectively, orally for ten days. on the seventh day of the experiments, i.p. ak (1.2 g/kg) was given to the test groups. at the end of the trial, blood samples were used to assess oxidative stress, renal function, inflammatory markers, and some hemato-biochemical parameters. significantly higher levels of serum mda, il-6, urea, and creatinine, and adversely affected lipid profile, are indications of ak-induced nephrotoxicity. supplementation of cbe attenuated ak-induced change in these biomarkers. it was concluded that cbe supplementation protects against the nephrotoxic effects of ak because of its antioxidant, anti-inflammatory, and hypolipemic properties. keywords: amikacin, nephrotoxicity, oxidative stress, inflammation, circus bergamot. التي واألكسدة وااللتهابية البيوكيميائية اإلجهاد عوامل على الحمضيات برغموت مستخلص تأثير .الجرذان ذكور في ألميكاسين الحادة السمية تسببها *** غالب صغير نمحس و1**،يعقوب ايوب اسامة ،*ضاري فريد فاطمة العراق ميسان، ، القلب وجراحة ألمراض ميسان مركز والبيئة، الصحة وزارة* العراق. البصرة، البصرة، جامعة الصيدلة، كلية والسموم، االدوية فرع ** العراق البصرة، البصرة، جامعة الصيدلة، كلية والسموم، االدوية فرع *** الخالصة أدى مما ، فيه المرغوب غير الكلوي بالتسمم ربطه تم فقد ، ذلك ومع كفاءة. لألمينوكليكوسيد الحيوية المضادات أكثر هوأحد االميكاسين هذا من الهدف كان وااللتهابات. ، الكلى ووظائف ، التأكسدي باإلجهاد المتعلقة تلك سيما ال ، مختلفة حيوية كيميائية مؤشرات في سلبية تغيرات إلى الناتجة الجرذان ذكور في واألكسدة تهاباتلواال الحيوي الكيميائي اإلجهاد عوامل على الحمضي البرغموت مستخلص تأثير كيفية معرفة هو البحث السيطرة مجموعة تلقت .جرذان ستة منها كل ، متساوية مجموعات خمس إلى الجرذان ذكور من 30 إجمالي تقسيم تم .لألميكاسين الحادة السمية عن ( كغ / غم 1.2) و ايام عشرة لمدة الفم طريق عن المقطر الماء من مل 1 (تلقت 1) المجموعة .ايام عشرة لمدة الفم طريق عن المقطر الماء من مل 1 إعطاء تم أيام. عشرة لمدة شفويا المستخلص من كغ / مغ 100 تلقت (2)المجموعة اما السابع. اليوم في البيريتوني التجويف في االميكاسين من التجارب من السابع اليوم في أيام. عشرة لمدة الفم طريق عن ، التوالي على ، المستخلص من كجم / مجم 200 و غك /غم 100 (4 ,3) المجموعات اإلجهاد لتقييم الدم عينات استخدام تم ، التجربة نهاية في االختبار. لمجموعات البيريتوني التجويف في االميكاسين من (غك / مغ 1.2) إعطاء تم ، من كل مستويات بارتفاع الدراسة هذه اضهرت للدم. الحيوية الكيميائية المتغيرات وبعض ، االلتهاب وعالمات ، الكلى ووظيفة ، التأكسدي السمية على مؤشرات تعتبر التي ، الدهون على سلبي تأثير إلى باإلضافة ، والكرياتينين ، اليوريا ، ((6il-6االنترلوكين ، ((mdaالمالونديالدهايد التحسن بعض مع الحيوية المؤشرات هذه في االميكاسين عن الناجم التغيير بتقليل المعالجةبالمستخلص سببت حين االميكاسين.في يسببها التي الكلوية بسبب االميكاسين بسبب للكلية السامة التأثيرات من حميي الحمضي البرغموت مستخلص انه الدراسة هذه تالدهون.استنتج تعريف ملف في . الدم دهون نسبة تقليل و لاللتهابات والمضادة لألكسدة المضادة خصائصها .الحمضي البرغموت ، االلتهاب ، التأكسدي اإلجهاد ، الكلوية السمية أميكاسين، : المفتاحية الكلمات 1corresponding author e-mail: ausama.jaccob@uobasrah.edu.iq received: 26/3 /2022 accepted: 20/6 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp219-226 iraqi j pharm sci, vol.32( 1 ) 2023 protective effect of citrus bergamot extract against amikacin toxicity 220 introduction aminoglycosides (amgs) are one of the earliest antibiotics. they were first made in the 1940s and used to treat various infectious diseases caused by gram-negative aerobic bacteria and grampositive staphylococcus aureus. amgs inhibit the synthesis of proteins and induce changes in the integrity of the bacterial cell membrane, which leads to bactericidal cell death (1). the semi-synthetic aminoglycoside derivative amikacin (ak) has the broadest spectrum among semi-synthetic aminoglycoside derivatives. it is produced by the acetylation of kanamycin, a natural medication (2). amikacin is the most preferred and frequently used to treat severe hospital-acquired diseases caused by multidrug-resistant bacterial strains for its beneficial features like intense bactericidal activity, limited resistance to many bacterial enzymes, synergistic effects with lactam antibiotics, and low cost (3). dose-dependent and reversible nephrotoxicity is the most significant and life-threatening side effect of ak, which limits its usage (4). many pathophysiological pathways are involved in akinduced nephrotoxicity, involving inflammation, transporter blockade, oxidative stress, and vascular alterations (5). it also forms a connection with mitochondrial fe2+, which stimulates the formation of free radicals (6).ak accumulates most prominently in the renal cortex, where it produces free radicals that destroy unsaturated fatty acids in the cell membrane, leading to an increase in lipid peroxidation, an increase in mda levels, and a decrease in glutathione levels in renal tissue (7).amgs-induced nephropathy is linked to biochemical and structural changes in the renal cortex, which include phospholipase c inhibition associated with increased phospholipid content in the cortex and the aggregation of myeloid bodies in the tissue (8) .according to salem et al., 2011, gentamicin (gm) therapy raised blood tc, tg, and ldl levels while decreasing serum hdl values (9). rashid et al., 2005, discovered that after gentamicin exposure, blood cholesterol raised significantly in rats and caused significant damage to renal tissue (10). it was also demonstrated that an increase in blood cholesterol levels had a major effect on the progression of gm-induced nephrotoxicity (11).natural remedies have gained increasing popularity for treating various illnesses, and they may be the primary ingredients employed by the pharmaceutical industry. plants contain a wide spectrum of compounds that are beneficial to one's health. various phenolic compounds found in fruits and vegetables have antioxidant, anti-inflammatory, and antibacterial activities (12–14). citrus fruits have been shown to have health-promoting characteristics, owing to their high flavonoid content, which has a wide range of biological characteristics, including anti-oxidant, metal ion chelating, vasoprotective, hepatoprotective, antiinflammatory, anti-cancer, and anti-infective activity (15,16). among these, bergamot has recently received a lot of attention due to its unique characteristics. bergamot (citrus bergamia rissoetpoiteau) is a rutaceae plant that grows mostly in southern italy, especially in calabria (17). around 1660, bergamot was initially employed as a pain reliever, and later as a perfume. bergamot has been a vital component of the calabrian economy for a long time, especially because it is the main source of essential oil used in the cosmetics sector (18). bergamot has a unique set of flavonoids and glycosides, like neohesperidin, neoeriocitrin, poncirin, naringin, and, rutin that make it different from other citrus fruits. not only for the composition of its flavonoids but also for its high content (12,13). recent scientific data have established the powerful anti-oxidative, anti-inflammatory (19) and beneficial effect on the lipid profile of citrus bergamia in animals and human studies (20), shining new light on its usefulness as a nutraceutical accordingly, this study attempts to look at how ak affects some hemato-biochemical parameters and the possible ameliorative role of cbe in male albino rats who were exposed to acute amikacin toxicity. materials and methods chemicals and drugs the drug amikacin (amikozit®, 500 mg/2 ml vial) was bought from sanofi (istanbul, turkey). a citrus bergamot extract supplement (cbe 500 mg capsule) was bought from double wood supplements (usa). almost all of the biochemical analysis kits were bought from abbott company (usa). il-6 and mda elisa kits came from shanghai yl biotech company (china). the other chemicals and instruments we used in our research were of the best quality that could be found. experimental animals in this study, 30 adult healthy albino male rats weighing 150 to 230 grams (aged 2-3 months) have been used. the rats were purchased from the university of al-qadisiyah, college of veterinary medicine, iraq. after getting permission from the ethics committee at the university of basra to do this experimental research. this study was conducted in the college of pharmacy on october 2021, 3/5/293. the animals were kept in the animal house of the college of pharmacy at the university of basra under clean and standard conditions (25℃± 2°c, moderate humidity, and a 12-hour light/dark cycle). as part of the study, the rats were kept in large indoor plastic cages with wood chips as flooring. they also had ad libitum access to essential rat food and fresh water. iraqi j pharm sci, vol.31(2) 2022 protective effect of citrus bergamot extract against amikacin toxicity 221 experimental design after 14 days of acclimatization, thirty rats were divided into five experimental groups, each with six rats. the normal control group received 1 ml of dw gavaged orally for 10 days. group (1) was given 1 ml of dw gavaged orally for ten days, and i.p. ak (1.2 g/kg) on day seven. for group (2), 100 mg/kg citrus bergamot extract was gavaged orally for ten days. groups 3 and 4 represent the test groups were given 100 mg/kg and 200 mg/kg of citrus bergamot extract, respectively, and were gavaged it orally for ten days and they represent the test groups. on the seventh day of the experiments, i.p. ak (1.2 g/kg) was given to the test groups. on day 7, the control group and group (2) were given dw intraperitoneally to mimic a rat model of ak intraperitoneal injection. the doses were chosen in agreement with prior studies(21–23). for nine days, all administered doses were provided once a day in the morning. the weights of the rats were measured three times during the experiment: at the start, five days later, and before the animals were sacrificed. [relative kidney weight (rkw) = (kidney weight/ rat weight)* 100] calculated for comparison.(24). hemato-biochemical assessment twenty-four hours after the final dosage was administered, blood samples from the posterior vena cava were obtained for examination of hematobiochemical parameters after the rats entered profound unconsciousness produced by breathing chloroform. two tubes were used to collect blood samples. the blood in the first tube, was kept at room temperature for a brief period to enable clotting, and serum samples were obtained following centrifugation at 10,000 rpm for 20 minutes. samples of clear serum were stored frozen at -20 °c until they were utilized for biochemical analysis. while the other tube contained an anticoagulant (na2 edta) for hematological parameter testing. for both the experimental as well as control groups, elisa kits were used to check the levels of both mda and il-6 markers in serum according to kit instructions. the spectrophotometric method was used to analyze kidney function tests (serum urea and creatinine) and lipid profiles (ldl, vldl, hdl, cholesterol, and triglycerides). a diagnostic automated laboratory analyzer (abbott architect 4000c, usa) was used to test all of these parameters, except nonhdl-c, which was calculated by subtracting hdlc from the total cholesterol and adding it back. the hemoglobin levels were estimated using animals' whole blood by a semi-auto chemistry analyzer following the manufacturer's instructions (mindray ba-88a, china). statistical analysis for comparison, one-way anova was utilized among groups. turkey’s posthoc analysis test for further assessment was used. data were expressed as mean± sem with p<0.05significance. data was analyzed using graphpad prism software (version 6.0). results the effects of amikacin on the body weights and relative kidney weight in the current research, no significant changes were documented p>0.05 when compared and correlate the changes in body weights in the control and treated groups with respect to the number of days of the experiments (table1). increase in body weights appear to be comparable in all groups. in contrast, the calculation of relative kidney weights suggests significant increase in rkw in both group1 and group3 compared to control. table 1. changes in rat body weights with respect to days of the experiments. *indicate significant difference if we compare it to control p<0.05. rat weight (g) p-value relative kidney weight days 1 5 10 control 166.16±3.35 169.5±2.7 182.5±2.97 0.1688 0.732± 0.023 group1 171.5±7.68 189.16±8.9 197.8±8.62 0.1648 0.89±0.028* group2 180.5±7.48 189.3±7.12 195.5±7.58 0.1059 0.649±0.021 group3 181±5.89 193±4.788 198.6±6.38 0.1707 0.871±0.045* group4 180±6.61 191.5±6.87 197.16±5.02 0.1641 0.861±0.033 iraqi j pharm sci, vol.31(2) 2022 protective effect of citrus bergamot extract against amikacin toxicity 222 the influence of citrus bergamot extract (cbe) on serum malon dialdehyde (mda) levels after high dose intraperitoneal amikacin administration to rats experimental rats exposed to toxic dose of amikacin had significant rise in mda levels compared to control and remaining groups (figure1). this value reflects oxidative stress associated with ak toxicity. high pharmacological dose (200 mg/kg of cbe) ameliorates and counteracts oxidative damage rendering ak less toxic. compared to group1, the levels of mda dropped significantly in all of the groups that were treated with cbe and the normal control group (intoxicated groups). figure 1. influence of citrus bergamot extract (cbe) on serum malon dialdehyde (mda) levels after high dose intraperitoneal amikacin administration to rats. *represent significant difference p<0.05 between groups. the influence of citrus bergamot extract (cbe) on seruminterleukin-6 (il-6) levels after high dose intraperitoneal amikacin administration to rats figure (2) clearly shows a significant increase in il-6 level in group1 treated with ak alone compared to control and remaining groups. in both doses, cbe lowers the level to similar levels of the control group. the lowest level was found in group 2. figure 2. influence of citrus bergamot extract (cbe) on serum interleukin-6 (il-6) levels after high dose intraperitoneal amikacin administration to rats. *indicate significant difference p<0.05 among groups. the influence of citrus bergamot extract (cbe) on renal biomarkers levels after high dose intraperitoneal amikacin administration to rats serum concentrations of urea and creatinine were significantly elevated in group1 as seen in figures 3 (a and b). rats in group1 injected with ak associated with clear increment in renal biomarkers mostly occur in renal injury. administration of pharmacological doses of cbe decrease both creatinine and urea values with greater reduction associated with dose 200mg/kg of cbe were normalization of renal biomarkers values resemble to control group. group2 administered cbe alone show normal values of both urea and creatinine. figure 3. influence of citrus bergamot extract (cbe) on renal biomarkers levels after high dose intraperitoneal amikacin administration to rats. a: represent serum urea concentrations, b: represent serum creatinine concentrations. **represent significant difference p<0.001 among groups. *represents significant difference p<0.05 among groups. the influence of citrus bergamot extract (cbe) on lipid profile after high dose intraperitoneal amikacin administration to rats fig. 4 showed serum concentrations of ldl, vldl, cholesterol, tg, uhdl and non-hdl in the control and treated groups of rats after cbe oral gavage and intraperitoneal amikacin administration. serum ldl significantly increased in all groups administered ak, serum vldl significantly increased in group1 associated with a bad effect on lipid profile. oral treatment with cbe iraqi j pharm sci, vol.31(2) 2022 protective effect of citrus bergamot extract against amikacin toxicity 223 in both doses reduced serum vldl elevation and led to normalization towards serum vldl level in the control and group3. it was found that serum levels of cholesterol and tg in the studied groups expressed in resemble manner to ldl and vldl respectively. serum cholesterol levels still elevated in spite of cbe oral administration for 10 days. on the other hand, serum tg levels in cbe treated groups decrease significantly as documented in figure4. regarding serum uhdl the highest value observed in group2 this level indicates the good effect of cbe on lipid profile. actually, the nonhdl levels in all groups administered ak increased significantly indicated it is bad effect on lipid profile. figure 4. influence of citrus bergamot extract (cbe) on lipid profile after high dose intraperitoneal amikacin administration to rats. * indicate significant difference p<0.05 among groups in each lipid profile panel. influence of citrus bergamot extract (cbe) on hemoglobin levels (hb) after high dose intraperitoneal amikacin administration to rats hemoglobin levels were evaluated in order to obtain complete idea about the effect of cbe and ak on hb serum concentration. there was no significant difference between the tested and control groups in this study. all groups well matched as seen in figure 5. figure 5. influence of citrus bergamot extract (cbe) on hemoglobin levels (hb) after high dose intraperitoneal amikacin administration to rats. discussion aminoglycoside antibiotics, , specifically ak, have long been recommended as a first-line antimicrobial treatment option for severe gramnegative infectious diseases (25). despite their benefits, aminoglycosides have considerable oto and nephro-toxicity, which are limiting considerations for their clinical usage, in which the oxygen free radicles have been involved in mechanistic toxicity (4). the results of the existing work showed anon-significant correlations regarding the effect of times on changes in body weight of the tested and control groups. this finding can be explained by single dose administration of ak on day7 of the treatment schedule appear not enough to induce a significant change in body weight during 10 days’ period of the study. the present findings are in line with those of noori et al., 2019, who showed that the body weight of rats was not affected after amikacin administration (26). opposite to our findings, fatima et al., 2021, discovered that when rats that given aminoglycosides, their body weights change when compared to control groups, however, ak induced nephrotoxicity in rats evidenced by significant elevation in relative kidney weight in the ak-treated groups compared to the control group. these findings may be attributed to the fact that injection iraqi j pharm sci, vol.31(2) 2022 protective effect of citrus bergamot extract against amikacin toxicity 224 of nephrotoxic substances caused an increase in kidney weight and swelling of renal tissue, these findings are supported and came in line with a large number of articles explained the nephrotoxic effect of aminoglycoside(26). in this study, rats that had been given ak had higher levels of mda in their serum than rats in normal control group. this elevation can be explained by excess ros production by the accumulation of amgs in renal proximal tubular cells, which causes an increase in lipid peroxidation (mda) and the destruction of unsaturated fatty acids in the cell membrane (7). this is supported by other investigations which revealed .the involvement of oxidative stress in nephrotoxicity as indicated by elevation in mda levels in animals (27–29).today, researchers are focus on finding natural supplements that potentially protect the body from the oxidative stress caused by drugs and chemicals. in this research, cbe administration to rats significantly lowers mda levels as compared to ak-treated rats (group 1). these finding highlights that bergamot extract possesses a robust antioxidant activity. in tune of our results, zhang et 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http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 topical letrozole nanoemulsion for breast cancer doi: https://doi.org/10.31351/vol29iss1pp195-206 195 preparation and characterization of topical letrozole nanoemulsion for breast cancer ihab d. hammodi*,1 and shaimaa n. abd alhammid* * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract letrozole (lzl) is a non-steroidal competitive aromatase enzyme system inhibitor. the aim of this study is to improve the permeation of lzl through the skin by preparing as nanoemulsion using various numbers of oils, surfactants and co-surfactant with deionized water. based on solubility studies, mixtures of oleic acid oil and tween 80/ transcutol p as surfactant/co-surfactant (smix) in different percentages were used to prepare nanoemulsions (ns). therefore, 9 formulae of (o/w) lzl ns were formulated, then pseudo-ternary phase diagram was used as a useful tool to evaluate the ns domain at smix ratios: 1:1, 2:1 and 3:1. all the prepared formulae were characterized for thermodynamic stability studies, zeta potential, droplet size, polydispersity index (pdi), % transmittance estimation, ph, % drug content, electro-conductivity and in vitro drug release. ns-7 (compose of 5% oleic acid, 45% smix of 1:9 ratio and 50% water) was chosen as an optimum prepared formula for many reasons. initially, it has a lower pdi (0.054), optimum droplet size (75.9 nm), highest transmittance percent (99.89±0.015%), high drug content (99.88%±0.03%), acceptable ph (5.96±0.025), highest electro-conductivity (230±1 μs/cm) and optimum drug release% (99.58±1.92) after 75 min in phosphate buffer (ph 6.8) compared to other ns formulations.one can conclude that preparation of lzl ns is an effective method for improving the permeation of lzl throught the skin. keywords: letrozole, nanoemulaion, permeation and in vitro release. تحضير ودراسة خواص الليترزول على هيئة مستحلب نانوي دقيق **عبدالحميدنزارشيماء و 1*، ايهاب دحام حمودي ،العراق ،بغداد بغداد ،جامعة الصيدلة كلية، فرع الصيدالنيات* الخالصة من خالل lzlتحسين نفوذية ال هو الدراسة هذه من الهدف.الستيرويد غير تنافسي أروماتيز إنزيم امنظ مثبط هو( lzl) ليتروزول / السطحي للشد كخافض ترانسكيوتول بي/ 80 وتوين األوليك حمض زيت من مخاليط استخدام تم ، الذوبان دراسات على بناء . الجلد لـ (o / w) صيغ 9 صياغة تمت ، لذلك(. ns) النانوية المستحلبات عدادإل مختلفة مئوية بنسب( smix) السطحي للشد مساعد خافض lzl ns ، مجال لتقييم مفيدة كأداة الكاذب الطور مخطط استخدام تم ثم ns نسب عند smix 1:1 ، 1:2 1: 3 و. ،( pdi) االنتشار تعدد مؤشر ، القطرة حجم ، زيتا جهد ، الحراري الديناميكي الثبات لدراسات المحضرة كانت قد خضعت الصيغ كل كصيغة ns-7 اختيار تم.التوصيل الكهربائي وتحرير الدواء في المختبر ،٪ الدواء محتوى ، phالحموضة درجة ،٪ النفاذية وتقدير نفاذية نسبة أعلى ،( نانومتر 75.9) األمثل القطرة وحجم ، pdi (0.054) اقل على يحتوي ، البداية في. أسباب لعدة مثالية معدة مقبولة phالحموضة درجة ،٪( 0.025166± ٪ 99.88333) الدواء من عالية نسبة ،٪( ±0.015275 99.89333) دقيقة 75 بعد (1.92±99.58٪ ) تحرير للدواء وأسرع( s / cm/ 1± 230)اعلى توصيل كهربائي ،( ±0.025166 5.956667) .األخرى ns تركيبات مع مقارنة (6.8 الحموضة درجة) للفوسفات المؤقت المخزن في .من خالل الجلد lzlنفوذية ال لتحسين فعالة وسيلة هو lzl ns تحضير أن نستنتج أن يمكن .النفوذيه وتحرير الدواء في المختبر ، مستحلب نانوي ، ليتروزول: الرئيسية الكلمات introduction lzl is one of the most effective aromatase inhibitors present nowadays for management of breast cancer. lzl has gained attention since it has demonstrated high safety and effectiveness profile in comparison to tamoxifen. its chemical structure (4, 40-[(1h 1, 2, 4-triazol-1-yl) methylene] bisbenzonitrile) as seen in figure 1(1, 2). figure 1. chemical structure of lzl(3) 1corresponding author e-mail: e-mail: commandosteam2005@yahoo.com received: 13/ 9/2019 accepted: 12/ 1 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp195-206 iraqi j pharm sci, vol.29(1) 2020 topical letrozole nanoemulsion for breast cancer doi: https://doi.org/10.31351/vol29iss1pp195-206 196 lzl practically insoluble in water (3). to improve the solubility either crystal modification, self-emulsification, particle size reduction, amorphization, or ph modification, are considered to be effective. moreover, nss are emulsion of submicron sized that are under extensive investigation act as carriers of drug for enhancing the therapeutic agent delivery. also ns can be defined as “oil-in-water (o/w) emulsions with mean droplet diameters ranging from 50 to 1000 nm”. depending on the droplet size, it can be divided into groups of milky (up to 500 nm) and the transparent or translucent (50–200 nm) (4).they can exist either as oil in water (o/w) or water in oil (w/o) emulsion forms, where the core of the internal particle is either oil or water, respectively. the aim of this study was to formulate lzl as ns in order to improve its permeation through the skin. materials and methods materials letrozole powder was obtained from baoji guokang bio-technology co., limited, china. cremophore el 200 (polyoxyl 35 castor oil), transcutol®p (highly purified diethylene glycol monoethyl ether), peg 200 (polyethylene glycol 200) and span 20(poly sorbate 20) were bought from shanghai ruizheng chemical tech co., ltd, china. methanol was bought from sigma-aldrich, germany. oleic acid oil, tween 80(poly sorbate 80) and olive oil were supplied from central drug house (p) ltd. (cdh®), new delhi, india. peg 400 (polyethylene glycol 400) was from scrc, china. all other solvents and chemicals were of analytical grade. experimental methods solubility screening of excipients excess quantity of lzl was dissolved in 2 ml of different oils, surfactants and cosurfactants individually(4). the oils that were selected for solubility testing of lzl were: oleic acid, sesame oil, olive oil, peppermint oil, sunflower oil, glycerol mono-oleate (gmo) and corn oil. the surfactants and co-surfactants that were selected for solubility testing were tween 40, tween 20, span 20, tween 80, tween 60, span 80, peg 200, peg 300, peg 400, peg 600, and transcutol p. besides, vortex mixer was used for mixing in order to get uniform disperse system (5). shaking mixtures in a water bath at 25 ± 0.5 ºc for 72 hrs was then performed. this was followed by 20 min centrifugation at 5000 rpm. a filter syringe of 0.45 µm was used to filter the supernatant. the concentration of drug was determined spectrophotometrically at its λ max (240 nm) after dilution of the supernatant with methanol. the unknown concentration of lzl dissolved in certain surfactant or oil was determined by formerly established calibration curve(4). phase diagrams depending on the lzl solubility and phase diagram result, various components including oil, surfactant and co-surfactant were scanned and ns was formulated with the aim of construct the diagrams. the titration method was employs as follows: mixed the surfactant and co-surfactant completely in ratios of fixed quantity (1:1, 2:1, 3:1) and then smix were mixed with the oil at ambient temperature. for each ratio of smix, phase diagram, the oil ratio to the smix were differed as follow, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1. during titration, water had added drop by drop to the oil–smix combination that was under stirring vigorously, then kept aside for visual assessment for clarity or turbidity(6). chemix school software v 7.00 (arne standnes , bergen, norway) was used to draw these diagrams. drug loaded ns preparation letrozole required amount was first dissolved in the selected oil, and then followed by the smix addition. both of them (the oil and the smix) were properly mixed by vortex and then eventually titrated with deionized water to get ns. the adding excipients order is extremely crucial to formulate the ns(7). evaluation of the produced lzl ns thermodynamic stability studies heating/cooling cycles/studies six cycles between two temperature of 4°c (refrigerator temperature) and temperature 45°c with storage and at each high temperature not less than two days were conducted (8). centrifugation test the ns system was centrifuged for ten minutes at 5000 rpm to determine the signs of phase separation or creaming by examine the formula visually for its appearance (5, 9). freezing/thawing cycles test deep freezing at −21 ◦c in a freezer (vest frost, india) and at 25±2 ◦c (room temperature) for 48 h were performed (three cycles each). measurement of globule size and polydispersity index scattering of light had been observed at room temperature and at a scattering angle of 90°. the ns (1–1.5 ml) was placed into a cuvette of disposable polystyrene with the micropipette help. the mean droplet size was measured in triplicate. the ns dilution has no effect on their size of globule. the mean polydispersity index and particle size were measured at room temperature by using a malvern nanosizer (malvern instruments, usa)(10, 11). https://doi.org/10.31351/vol29iss1pp195-206 http://www.chemix-chemistry-software.com/school/author-information.html http://www.chemix-chemistry-software.com/school/author-information.html iraqi j pharm sci, vol.29(1) 2020 topical letrozole nanoemulsion for breast cancer doi: https://doi.org/10.31351/vol29iss1pp195-206 197 measurement of zeta potential zeta potential study was often used as an indicator of the droplet stability in ns. nss were put into clear and disposable zeta cells and zeta potential that points out the developed nss surface charge. it was measured by zetasizer at 25°c (12). ph measurement the ph of the formulated ns was measured by dipping directly the ph meter electrode into a sample of 10 ml at ambient temperature(12). all measurements were obtained in triplicates and mean calculated. electro-conductivity measurement the ns conductance is determined by a conductometer by immersing its probe in 10 ml of the formula (at 25 ºc) then the instrument measure the conductivity in μs/cm. the apparatus probe composes from two metal electrodes 1 cm apart one from the other. these two electrodes immersed in the ns sample and then a constant voltage was appertained across these electrodes. an electrical current flowed via the aqueous sample only. drug content uniformity it was measured as lzl percent found in each formula in comparison with a theoretical amount of the formulation. one ml of the ns was diluted in suitable volume of methanol then mixed and sonicated for complete extraction the drug. finally centrifuged for 15 min at 3000 rpm to separate and exclude the undissolved excipients. the supernatant was taken and filtered via 0.45µm syringe filter(13). then diluted to be analyzed by uv-vis spectrophotometer at its λ max (240 nm). drug content = (measured content / theoretical content) ×100 measurement of transmittance percent transmittance was made to obtain information regarding the internal phase globule size as well as developed ns stability. the formulated ns percent of this test was calculated at 650 nm wave length using uv– visible spectrophotometer and d.w was used as a blank(14). in vitro drug release study using dissolution apparatus type ii, a 0.9 l of freshly prepared dissolution medium. the in vitro drug release for all the produced ns formulas was performed using dialysis membrane and has pore size of 2.4 nm with approximately 8000-14000 kda molecular weight (it was treated previously by soaking it into the medium of dissolution for 24 hours only prior to beginning of the test experiment). the phosphate buffer of 6.8 ph was used as release medium (15).the study was done at 37±1 °c and 50 rpm stirred for 2 hr. the ns involving 2.5mg of lzl was put in the bag. the bag should be closed properly at both end of bag to avoid leakage. a sample of 5 ml of release medium should be drawn at a time interval of 5, 10, 15, 30, 45, 60, 75, 90, 105, 120 min for the ns and 5 ml of fresh medium was added in sake to ensure the sink condition maintenance. these samples were then filtered via syringe filter of 0.45 μm and lzl quantitative estimation at its λ max of 240 nm (16, 17). selection of optimum lzl nanoemulsion formula choosing the optimum formula had depended on the results and data of transmittance percent, electro-conductivity, ph, smix concentration, drug content, and in vitro drug dissolution. basically, the optimum nanoemulsion formula is preferred to have particle size, pdi and zeta potential as <100 nm, <0.3 and more than ±30 nv, respectively(7). morphology evaluations of optimum lzl nanoemulsion field emission scanning electron microscopy (fe-sem) the shape, size and morphology of the surface can be examined by using fe-sem. a small quantity of ns without dilution was spread above a piece of slab and left to dry. using platinum to coat the sample, then in all fields the results obtained of particle size of the particles were calculated by using sem software operated as 5.0kv (18). fourier-transform infrared spectroscopy (ftir) it is important to explore the purity and compatibility of lzl with components of nanoemulsion. using potassium bromide substance to be packed with the pure drug as a disc (using special cuvette to measure the liquid sample) then setting the range of scanned wavelength to be (400 to 4000 cm-1) then registered the shown spectrum data to be analyzed and record if there any incompatibility between the samples or not (19). statistics the one way anova test was used to compare results gained from data of our study with 95% confidence interval set for the variation in the mean of samples (20). results solubility screening the solubilization of lzl in the oils was with a maximum concentration in oleic oil (76.57 mg/ml) as illustrated in figure 2. additionally, the value of hlb (hydrophilic/lipophilic balance) is required to be higher than 10 to obtain o/w ns since the greater value leads to faster formation of the oil in water droplets which meaning fast dispersion https://doi.org/10.31351/vol29iss1pp195-206 iraqi j pharm sci, vol.29(1) 2020 topical letrozole nanoemulsion for breast cancer doi: https://doi.org/10.31351/vol29iss1pp195-206 198 production. according to the solubility study in figure 3, tween 80 which is a water soluble non-ionic surfactant has a superior solubilizing concentration (132.5577±0.00295mg/ml). it has a good hlb =15 which is essential consideration for o/w ns preparations(21, 22). figure 2. chart of solubility results of letrozole in various oils figure 3. letrozole solubility in different surfactants and co-surfactants phase diagrams construction they were created from a mixture of different ratios of oleic acid as oil, tween 80 and transcutol p as surfactant and co-surfactant, respectively. figure 4 involves the obtained diagrams which were prepared using various smix; r1: smix (1:1), r2: smix (2:1), r3: smix (3:1) with an existence field of ns (specific coloured area). in general, changing smix ratios lead to obtain different ns areas. additionally, when the size of ns field is large that is meaning obtaining the more efficiency of ns system. the ns region has been increased when the concentration of tween 80 increased. therefore, an increasing the amount of tween 80 which employed as surfactant relative to the amount of transcutol p that used as cosurfactant resulted in improvement the ns region. it seems that the largest size of ns region was reached as smix of 3:1. there are many studies and debates have presented that the region of ns are basically affected by the smix ratio as well as the type and concentration of the oil used. accordingly, smix 3:1 has associated with high emulsification efficacy which was resulted in diminishing the interfacial tension to the small level and optimum tween 80/transcutol p with forming high flexible coherent film at oleic acid oil/deionized water interface. figure 1. pseudo-ternary phase diagrams of the quaternary containing oleic acid / tween 80/ transcutol p®/ water as oil/surfactant/co-surfactant/aqueous phase, respectively at different smix ratios; r1: smix (1:1), r2: smix (2:1), r3: smix (3:1) https://doi.org/10.31351/vol29iss1pp195-206 iraqi j pharm sci, vol.29(1) 2020 topical letrozole nanoemulsion for breast cancer doi: https://doi.org/10.31351/vol29iss1pp195-206 199 preparation of lzl loading in nanoemulsion according to pseudo-ternary results a titration method by low energy process was used to prepare different formulations of lzl loaded ns. firstly, added 2.5 mg of drug to a volumetric flask and poured a specific amount of oleic acid oil then placed in sonicator to be mixed and dissolved at room temperature for 10 minutes. secondly, the supposed amount of smix was added to the prepared mixture and moved the volumetric flask to the vortex mixer. finally, deionized water was added drop to form the formula. continues the dropping till obtain clear and transparent liquid formulas. then, the formulas were moved in tightly closed containers in ambient temperature till apply for further characterization, details of the formulas in table 1(23). using smix (surfactant and co-surfactant) more than 50% would decrease the drug release because high amount of mixture could prevent the dissolution media from getting in contact with the internal phase of the ns as it acts as barrier. table 1. composition of lzl loaded nanoemulsion deionized water w/w % smix ratio oil: smix ratio smix tween 80: transcutol p w/w % oleic acid w/w % ns 50 1:1 1:9 45 5 ns-1 40 1:1 2:8 50 10 ns-2 45 1:1 3:7 45 10 ns-3 45 2:1 1:9 50 5 ns-4 35 2:1 2:8 55 10 ns-5 30 2:1 3:7 60 10 ns-6 50 3:1 1:9 45 5 ns-7 35 3:1 2:8 55 10 ns-8 30 3:1 3:7 60 10 ns-9 evaluation of prepared lzl-ns formulations thermodynamic stability studies of lzl-ns formulations there was no cracking or creaming or even phase separation appeared in tested samples(24, 25). therefore, the formulae were examined at different conditions using heating-cooling, centrifugation and freeze-thawing then the samples were taken to be studied for dispersion test as described in table 2. table 2. thermodynamic stability studies and dispersibility grades results of prepared lzl nss. ns h-c/c cf/t ft/t dsg et (sec.) fr ns-1 √ √ √ a 33 passed ns-2 √ √ √ a 45 passed ns-3 √ √ √ a 41 passed ns-4 √ √ √ a 20 passed ns-5 √ √ √ a 41 passed ns-6 √ √ √ a 32 passed ns-7 √ √ √ a 20 passed ns-8 √ √ √ a 29 passed ns-9 √ √ √ a 37 passed h-c/c= heating-cooling cycles, cf/t= centrifugation test, ft/t=freezing-thawing cycles, dtg= dispersibility test grade, et= emulsification time, fr= final result. particle size distribution and polydispersity index determination fo lzl-ns formulations the most valuable consideration to distinguish the microemulsion from ns is to determine its droplet size that should be in the nano range (6). for that reason, all prepared formulation ought to be examined and should have nano size particle to pass this test. as shown in table 3, it was observed that formula (ns-6) showed the maximum average droplet size (396.8) nm. on the other hand, formula ns-7 has the smallest particle size (75.9 nm). generally, using tween 80 as a surfactant can extremely reduce the particle size of formulations better than others because of its https://doi.org/10.31351/vol29iss1pp195-206 200 low molecular weight (1,310 g/mol). on another hand, to depict the degree of nonuniformity of particle size distribution the term polydispersity. it is a measurement of the distribution of particle size within a prepared formulation. the acceptable value of dispersity ranges from (zero to 1.0); the first range is for a uniform sample while the second is for multiple particle size populations with highly polydispersed sample (26-28). generally, equal to 0.2 or below is deemed most acceptable value for nanoparticle formulations since it refers to narrow droplet size distribution. the small polydispersity values lead to better stability against ostwald ripening (destabilization phenomena)(29, 30). as shown in table 3, values of polydispersity for lzl-ns formulations are small and within excellent rang (0.054-0.305) and indicates uniformity of droplet size within each formulation. table 3.results of particle size distribution and pdi for lzl nanoemulsion formulationm. pdi particle size (nm) ns pdi particle size (nm) ns 0.305 396.8 ns6 0.095 105.1 ns1 0.054 75.9 ns7 0.301 334.5 ns2 0.085 98.3 ns8 0.262 370 ns3 0.123 198.1 ns9 0.204 145.7 ns4 0.121 214.2 ns5 measuring of zeta potential (ζ-potential) of prepared nanoemulsion formulations according to the rule of thumb, ζpotential values more than 30 mv is a good indication of ensuring physical stability of prepared formulation of ns. table 4 includes the main values of zeta potential scale and the their indications for nanoparticles (31). table 4. zeta potential (zp) distribution results of lzl nanoemulsion ns zp ns zp ns-1 -39.09 ns-6 -109.46 ns-2 -83.45 ns-7 -113.76 ns-3 -87.47 ns-8 -70.65 ns-4 -90.43 ns-9 -102.55 ns-5 -111.82 measurement of ph of lzl-ns formulations the results obtained after dipping the ph meter inside the solution that the formulation are suitable to be used topically since they are between 4.97±0.092 and 5.99±0.091 as presented in table 5. additionally, there is no significance difference in the ph measurement (p>0.05). table 5. measurement of ph of lzl nanoemulsions; (mean ± sd);n=3 ns ph ns ph ns-1 4.93±0.005 ns-6 5.19±0.074 ns-2 5.28±0.028 ns-7 5.99±0.091 ns-3 4.97±0.092 ns-8 5.95±0.391 ns-4 5.11±0.098 ns-9 5.83±0.037 ns-5 4.91±0.122 measurement of electro-conductivity (σ) of lzl-ns formulations the target of this test is to explore the nature of the external phase whether it is aqueous (o/w nss) or oily (w/o nss) since the first has highly conducted (more than 10 µs/cm) because of there are more freedom for moving of ions in compared to the latter which has the water in internal or dispersed phase. the data in table 6 involves the results of lzl-ns formulations were measured by conduct meter pen, they have a range from 77 to 229 µs/cm. there is no significant variation (p>0.05) in conductivity among lzl-ns formulations. table 6. electro-conductivity prepared lzl ns; (mean±sd); n=3 ns σ (µs/cm) ns σ (µs/cm) ns-1 200±2.081 ns-6 77±1.008 ns-2 130±2.093 ns-7 229±0.569 ns-3 160±3.009 ns-8 101±2.881 ns-4 177±3.921 ns-9 79±2.987 ns-5 121±0.931 drug content measurement according to the requirements of usp, the acceptable range of drug content is between (85-%115%)(32). table 7 presents the result of this test for lz-ns formulation and all of them are within the acceptable range. that means the high content uniformity and no signs of precipitation too. also, there is no significance variation (p>0.05) among the samples. 201 table 7. drug content percent measurement of lzl-ns; (mean±sd); n=3 ns drug content %±sd ns drug content %±sd ns-1 98.11±0.019 ns-6 98.83±3.009 ns-2 99.19±0.82 ns-7 99.89±0.987 ns-3 96.81±1.903 ns-8 99.71±1.009 ns-4 97.68±3.491 ns-9 98.69±0.944 ns-5 99.74±3.334 transmittance percent measurement the samples are considered as transmit light easily, clear and transparent in nature when the percent transmittance is more than 99% (31).the highest percent value was equal to 99.98% for ns-7 and the lowest transmittance value was found to be 97% for ns-6. it seems from table 8, that the results of transmittance % increase with lessen the globule size of the formulations of lz-ns. nevertheless, there is no significant difference among the results of this test (p>0.05). table 8. transmittance percent measurement of lzl-ns formulations; (mean±sd); n=3. ns transmittance % ns transmittance % ns1 99.64±0.30 ns6 98.89±0.09 ns2 98.96±0.04 ns7 99.89±0.02 ns3 99.52±0.21 ns8 99.74±0.03 ns4 99.30±0.22 ns9 99.35±0.30 ns5 99.70±0.12 in vitro drug release study the dissolution of lzl from the prepared ns samples is shown in figures 5, 6 and 7 in 6.8 ph media. the shortest dissolution time was 30 min for ns-4 which was 100.42±2.16%. it is clear that might be due to improved solubility of lzl in the oleic acid, existence of tween 80 and in particular, small particle size of molecules of ns. generally, the small droplet size with a large surface area promotes rapid release of the drug from the formulations (9). figure 5. a dissolution profile of lzl nss (ns-1, ns-2 and ns-3) in 900 ml of 6.8 ph dissolution medium at 37 °c, all the results represent mean ± sd, where n=3 figure 6. a dissolution profile of lzl nss (ns-4, ns-5 and ns-6) in 6.8 phosphate buffer ph at 37 °c, all the results represent mean ± sd, where n=3. figure 7. a dissolution profile of lzl nss (ns-7, ns-8 and ns-9) in 900 ml of 6.8 ph dissolution medium at 37 °c, all the results represent mean ± sd, where n=3. selection of optimum lzl nanoemulsion formula many factors should be considered to select the optimum formula and ns-7 was chosen as an optimum prepared ns formulation for many reasons. initially, it has lower pdi (0.054) and optimum droplet size (75.9 nm) as seen in figure 8 and zeta potential (-113.76) as seen figure 9, highest transmittance percent (99.89±0.02%), high drug content (99.88±0.03), ph (5.96±0.03) and optimum drug release (99.58±1.92) after 75 min compared to other ns samples. 19 figure 8. globule size distribution and pdi of ns-7 figure 9. zeta potential of optimum ns-7 morphology examination of optimum lzl ns-7 nanoemulsion formula microscopic examinations are fundamental method to obtain actual range of data regarding the morphology of optimum ns formulation. fourier transforms infrared spectroscopy (ftir) there was no significant variation on position and shape of the obtained peaks between the pure drug and optimum formula diagrams (figure 10 and 11(. lzl pure powder in figure 10 illustrated major peaks at 3010 cm−1 for sp2 ch stretching 2250 cm−1 for c≡n stretching and 690-900 cm−1 for out-of-plane ch bending (as shown in table 9). no significant difference in shape and position of the absorption peaks of drug has been observed between the spectra. table 1. the ft-ir characteristic functional groups of materials materials functional group reference peaks (cm-1 ) result peaks (cm1) letrozole c≡n stretching 2250 2229 sp2 ch stretching 3010 3055 out-of-plane ch bending 690 900 790 oleic acid oil o-h bond 2674 3006 2524 (-ch2-) asymmetric and the symmetric stretching 2925 2856 2854 c=o stretching 1714 1706 c-o elongation 1284 1284 c-o-h bond 1412 1415 plan of o-h bond 939 949 tween 80 (–ch3) 2920 2904 –ch2-stretching 2864 2858 c=o 1735 1735 stretching of c–o–c 1095 1090 203 figure 10. the ft-ir spectrum of pure lzl powder. figure 11.ft-ir spectrum of optimum formula ns-7 field emission scanning electron microscopy (fe-sem) fe-sem is microscopy examined which can approve the particle size of optimum formula ns-7. as it clear from figure 12 the microscopy could investigate the nano sized particles of ns-7 formulation. the average range is 43.7-79.23 nm with spherical and not adherent shape. 204 figure 12. fe-sem of optimum ns-7 formulation conclusion nss is a promising new system that can improve the permeation of drugs through skin by using low energy of emulsification method. oleic acid, tween 80 and transcutol p were chosen as oil, surfactant, and cosurfactant, respectively. one can conclude that preparation of lzl ns is an effective method for improving the permeation. in future, choose a suitable gelling agent to increase the viscosity of ns to be more adhesive and studying their properties and evaluations. also, a deeper understanding and studying is necessary in order to develop the topical lzl ns in pharmaceutical applications by undergoing to in vivo test to assess the clinical performance of the formulated dosage form references 1. bhatnagar as. the discovery and mechanism of action of letrozole. breast cancer research and treatment. 2007;105(1):7-17. 2. vosooghi m, firoozpour l, rodaki a, pordeli m, safavi m, ardestani sk, et al. design, synthesis, docking study and cytotoxic activity evaluation of some novel letrozole analogs. daru journal of pharmaceutical sciences. 2014;22(1):83. 3. bansal t, mustafa g, khan zi, ahmad fj, khar rk, talegaonkar s. solid selfnanoemulsifying delivery systems as a platform technology for formulation of poorly soluble drugs. critical reviews™ in therapeutic drug carrier systems. 2008;25(1). 4. hosny km, banjar zm. the formulation of a nasal nanoemulsion zaleplon in situ gel for the treatment of 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acta pharmaceutica. 2007;57(3):315-32. 30. acharjya s, bhattamisra s, muddana b, bera r, panda p, panda b, et al. development of a highperformance liquid chromatographic 206 method for determination of letrozole in wistar rat serum and its application in pharmacokinetic studies. scientia pharmaceutica. 2012;80(4):941-54. 31. singh kgask. review of nanoemulsion formulation and characterization techniques. indian journal of pharmaceutical sciences. 2018;80(5):781-9. 32. saeed a, alaayedi mh, alshohani adh. new combination suppositories of lornoxicam and aloin for rheumatoid arthritis. asian j pharm clin res. 2018;11(2):308-12. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening doi: https://doi.org/10.31351/vol30iss2pp1-15 1 an overview of some plant based products with hepatoprotective activity(a review) s d labhade*,1 , sarvesh paliwal *, swapnil sharma* and shivani desai** * department of pharmacy, banasthali vidyapeeth, banasthali, rajasthan, india ** department of pharmacology, dr. d. y. patil institute of pharmaceutical sciences and research, pune, india abstract in folk medicine there are various medicinal amalgamation possessing hepatoprotective activity. toxins may cause liver insult as well. hence, many pharmaceutical companies are targeting herbal medicines for the treatment of liver abnormalities and towards evolving a safe and effective formulation with desired route of administration. review focused on the studies showing hepatoprotective effect using marine compounds and plant derived compounds. liver disorder, a global health problem, usually include acute or chronic hepatitis, heptoses, and cirrhosis. it may be due to toxic chemicals and certain antibiotics. uncontrolled consumption of alcohol also affects liver in an unhealthy way. to cure liver disorders several formulations of medicinal plants are being used. it is observed that hepatoprotective effect of plant is mostly due to flavonoids, alkaloids, terpenoids, steroids, and glycoside. a single drug cannot be useful for all the types of liver disorders. therefore, several plant extracts for liver illness resulting fromdifferent causes such as poisonous chemicals, viruses, extra alcohol consumption, and repeated administration of medication is to be considered. by using standards of protection and efficacy, manufacture of plant products need to be taken into consideration. current review provides an understanding of ethnopharmacology and toxicology of several medicinal plants manifesting hepatoprotective potential. despite of varied database analysis new discoveries and their probabilities, evidences on viral hepatitis treatment and/or liver cirrhosis are inadequate. further information about phytotherapy, toxicology, quality control studies shall be endorsed. further in depth studies are required to discover quality trait like structure activity relationship, mechanism of action, safety and toxicity and therapeutic potential of phytoconstituents in clinical settings. aim: the phytoconstituents studied for their protective effect in liver diseases are reviewed. keywords: liver disease, hepatoprotective herbs, phytoconstituents . introduction the liver is a crucial organ that regulates various functions in the body such as, detoxifying, storage, secretion, and metabolism. distortion of some of these functions is usually associated with hepatic damage caused by various agents and environmental factors. most of the hepato-toxic agents act by generating oxidative stress, reactive oxygen species,oxidative damage in proteins, dna, and reducing atp. notably, protecting the liver from hepato-toxic agents and their harmful effects i.e. altering the anti-radical defensive mechanism is called hepatoprotection(1). the persistence of toxins in liver tissue results in liver scarring which is known as fibrosis. this fibrosis results in impaired blood flow in the liver and influences its structure and capacity to function legitimately commonly characterized as called cirrhosis. this condition if remains untreated, causes accumulation of blood in the spleen and the digestive organs to cause portal hypertension including loss of blood and ascites (build-up of fluid in the abdomen)(2). further, these pathological conditions diminish the liver's capacity to store and process supplements required for survival. also, the inability of the liver to remove toxins from the bloodstream eventually leads to mental confusion and even coma (hepatic enteropathy) and death. according to who reports, liver diseases lead to approximately 2.4 million deaths per year. for instance, over 900 drugs have been accounted as the sole reason for liver injury from which 50% of acute liver failures, 10%cases of acute hepatitis, 5% of hospitalizations, cirrhosis, and chronicliverdisease(3). despite the presence of several advancements in the modern era, the incidence of the hepatic disease has not reduced and on the contrary, an exponential increase is observed. numerous plants were studied for their ethno pharmacological activity for liver illnesses. but it is tough to recover the damaged liver from toxicity. natural products containing active phytoconstituents were significantly used showing the high recovery of liver injuries. accordingtothe well-searched scientific articles, it is observed that herbal medicines showing liver protectionexerted their activity through properties related to antioxidants(4). materials and methods materials and metho ds in this article, several resources were spotted through editorial books, articles, indexed and non-indexed journals. other databases mainly google scholar, pubmed, scifinder, science-direct, 1corresponding author e-mail: sonalilabhade16@gmail.com received: 5/12/ 2020 accepted: 1/3 /2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp1-15 mailto:sonalilabhade16@gmail.com iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 2 medline were used to collect all the pertinent appropriate findings to the literature articles published on hepatoprotective action of medicinal plants. some books like charaka samhita, sushruta having traditional records of ancient medicines were also exploited. several common names like hepatitis, lipid peroxidation, hepatoprotective potential, antioxidants, herbal medicines, ethnopharmacology were the search tools. patents, conferences proceedings, case reports were not included in the study as from a scientific point of view these were considered unconvincing. several non-indexed resources were exploited through health websites, international health agency reports. due focus is given on plants with a descriptive explanation of hepatoprotective potential.studies like tumor cell lines and tumor-bearing animals have not been considered while doing a literature survey for this article. extra motivation to prohibit such examinations was conflicted utilization of hcc cell lines for examining both cytotoxic and cytoprotective impacts of tested compounds, bringing about disputable outcomes. pathophysiology of liver the largest internal organ in the body is liver. it is located below the diaphragm in the upper right quadrant of the abdominal cavity. its weight is 1.6 kg in men and 1.4 in women. it consists of two lobes. the right lobe is much greater in size than the left lobe. they are again divided into smaller lobules. there are millions of parenchymal cells also known as hepatocytes which are known to be metabolic cells of the liver as shown in figure 1(5).it is highly vascular. from hepatic portal veins, most of its blood supply (around 80%) comes from which delivers the blood with essential nutrients to the small intestine. these huge veins further partition into vessels to provide blood to each one of the lobules(6,7). figure 1. diagram depicts 4 major liver cell types (parenchymal cells/ hepatocytes, stellate cells, kupffer cells, endothelial cells) in normal liver and in liver injury. reasons for hepatic diseases viral infections, alcohol consumption, genetic disorders, immunological disorders, non alcoholic fatty liver disease, excessive medications, malignancy, abnormalities in structures like biliary arteries are very common causes of chronic liver disease.such conditions are common indicationsforliver transplantation(8, 9). oxidants level decrease/ antioxidants level increase antioxidants, at moderately low concentrations, can rival other substrates and lead to hinder the oxidation of those substrates(10).it is apparent that few phytoconstituents can instigate microsomal enzymes either by quickening the discharge of the hepatotoxin or by hindrance of lipid peroxidation initiated by it. saponins, flavonoids, triterpenoids, alkaloids are wellknown to have hepatoprotective activities(11). they are expected to exert their antioxidant activity by scavenging free radicals that leads to lipid peroxidation(12). there are few enzymes which help in inducing protection from oxidants either by causing neutralization of ros formation or inhibition like super oxide dismutase, peroxidase, and catalase(12). cyp’s inhibition is known to be cause by terpenoids present in plants as one of the essential phytoconstituents by conjugation mechanism. as major metabolic activities occur in liver hepatic cells, variety of enzymes are involved in it, which include, aspartate aminotransferase (ast), alanine transaminase iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 3 alt, alkaline phosphatase (alp). the raised activities of these enzymes lead to hepatic cell damage further causing functional integrity and cellular leakages. there are several agents which harm liver in cellular breakdown process and are known as hepatotoxins, which is associated with raised levels of alp,alt, bilirubin, triglycerides, and cholesterol in serum. hepatotoxins produce changing degrees of harm to the liver(13). oxidative degradation of lipids and free radicals it is reported that free radicals inhibit lipid peroxidation (14). as there is increase in lipid peroxidation due to ethanol, there are more chances of development of liver cirrhosis. due to lesser toxicity, plant-based medicines are preferred as hepatoprotective agents. this has lead to increase in the research activities based on hepatoprotective effects of phytoconstituents. according to hartmut jaeschke, 2011,livercell death is induced by stress such as ischemia-reperfusion, cholestasis, and drug toxicity. these factors can trigger a sterile inflammatory response with activation of innate immune cells through release of damage-associated molecular patterns (damps). a similar inflammatory response can be induced by pathogenassociated molecular patterns (pamps), such as endotoxin. both damps and pamps activate through toll-like receptors the resident macrophages (kupffer cells) and recruit activated neutrophils and monocytes into the liver. central to this inflammatory response is promotion of reactive oxygen species (ros) formation by these phagocytes. ros are the principal toxic mediators by which inflammatory cells kill their targets, e.g. bacteria during host defense but also hepatocytes and other liver cells. the mechanism of rosinduced cell killing during inflammation involves the promotion of mitochondrial dysfunction through an intracellular oxidant stress in hepatocytes leading mainly to oncotic necrosis and less apoptosis. although there is satisfactory progress in interpretation of ros role, more study is needed to explore the exact mechanism of working of ros in acute liver inflammation and progress with clinical therapeutic effect that successfully hit the harmful effect due to oxidative stress with intransigency to essential function of reactive oxygen species in host defense. liver disease and alcohol according to wahid a, alcohol dehydrogenase converts ethanol to acetate that generates ros via cytochrome p4502e1. this process causes oxidative stress in liver and consequently leads to hepatic damage and disturbs the rigidity of structure of liver cell membranes due to which in blood stream cytosolic enzymes are exuded. hence, concentration of ast and alt in mitochondria and blood stream is escalated. due to this mechanism the bilirubin level in serum also becomes high which in turn causes increase in erythrocyte sedimentation rate(16). a) d) c) b) figure 2. brief overview of a) functions of liver, b) in-vivo studies carried out to understand the iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 4 hepatotoxicity, c) various factor effecting liver health and their effect in change in levels, d) stages of liver impairment. figure 3. a) depicts fever methods of screening of hepatoprotective agents, b) blood flow in liver (from and to body) correlation of growth factors and hepatoprotection by the folk medicines insulin-like growth factor one of the main factors that contribute to malnutrition in cirrhotic patients is decreased hepatic production of insulin–like growth factor (igf-i). it has wide range of anabolic activities and is produced under the stimulus of growth hormones located in the hepatocytes (16,8).studies have revealed the effect of igf – i on histopathologic changes on liver of rat with ccl4 induced cirrhosis, where free radicals are the prime cause of hepatotoxicity which leads to cell damage. the evolved oxidative stress causes lipid peroxidation, dysfunction of mitochondria and also atp depletion(8).antioxidants scavenge the free radicals and can regulate the gene expressions associated with fibrosis, lipogenesis, and inflammation. hepatocyte growth factor(hgf) hgf is also called scatter factor. regeneration drug injury and liver repair are the two key roles which hgf possesses. it forms a complex network of signaling pathway which activates the cellular redox control, liver survival, and repair function. it happens when hgf binds to c-met receptor after autophoshyrlation which induces varied signal transduction proteins(17).however, more research is need to identify the exact mechanism of intervention in hgf activation of signals and c-met receptors. role of phytoconstituents in hepatic disorder hepatic disorders are prominently prevalent in india(18).many allopathic drugs, such as triclabendazole, pembrolizumab etc., are extensively used in the treatment of these liver diseases but they are associated with several adverse effects like abdominal pain, decreased appetite, headache, urticaria, mucoskeletal chest pain etc. moreover, these medicines are liable to cause socioeconomic burden(19). due to these concerns, extensive work on alternative medicine is needed. some herbal plants are also screened for their hepatoprotective potential; however, their synergistic effects have not been studied yet. moreover, toxicity studies of some plants have not been performed which might be toxic at certain extent. hence, there is a need to develop some alternative cost effective therapies which can be beneficial in the effective management of severe liver injuries or diseases. the indian ancient literature mentions various medicinal herbs that may be useful for liver diseases; however, they lack proper validation. thus, there is a growing need to focus on medicinal plants as hepatoprotective agents and establish their safety as well as efficacy in the treatment of liver diseases. nature is a storage facility of various restorative herbs containing dynamic bio-active constituents which are considered as potential source of medicines and play a key role in the management of various diseases. a single drug cannot be effective against all types of liver diseases(30). notwithstanding the significant approval of several folk medicines conventionally and for liver diseases in particular, they are still unsatisfactory treatment methods to liver diseases. the factors responsible for their occurrences are lack of: 1) toxicological evaluation 2) randomized and controlled clinical trials iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 5 3) active ingredient identification 4) herbal drugs standardization a large group of folk medicines are reported to show hepatoprotective activities. various phytoconstituents and plants, within india as well as in other geographical continents, possess liver protecting ability and some of the patented formulations are available in market (60). hepatoprotective medicaments the folk medicines are expected to be safe and not possessing serious adverse response, as they are derived from nature and are effortlessly accessible. table 1. summarized overview of plants along with their botonical names/family, parts used for their therapeutic effect, extract studied, inducing agents, histopathological and biochemical parameters showing hepatoprotective activity, chemical constituents. name of plant (source/family) plants part used extract studied hepatotoxic inducing agent biochemical and histopathologica l parameters studied chemical constituents abutilon indicum ( malvaceae) (21) whole plant aqueous ccl4, paracetamol activates antioxidative enzymes carbohydrates, glycosides, steroids, tannins, phenolic compounds and flavonoids acacia catechu (leguminosae)(22) powdered pale catechu ethyl acetate carbon tetrachloride sgot, bilirubin content, sgpt, sap taxifolin, quercetin, catechin, rutin and isorhamnetin adhitoda vasica (acanthaceae) (23) leaves aqueous ccl4 reduced elevated levels of sgot,sgpt alkaloids, tannins, flavonoids, terpenes, sugars, and glycosides alchornea cordifolia(euphorbi aceae) (24) leaf methanol ccl4 decreases alt, ast value steroids, flavonoids, terpenoids allium cepa (liliaceae)(27) bulb extract aqueous cadmium, paracetamol, acetaminop hen sgot, sgpt, alkaline phosphatase, direct and total bilirubin carbohydrates, proteins, flavonoids potassium, sodium and phosphorus amaranthus spinosus (amaranthaceae) (25,26) whole plant ethanol ccl4 mda, hydroperoxides, gsh, sod and cat alkaloids, flavonoids anogeissus latofilia(combretac eae) (28) bark hydroalco holic ethanol, ccl4 reduces alt,ast,alp levels and lipid peroxidation tannins, gallic acid, ellagicacd, lutin and quercetin apium graveolens (apiaceae) (27) seeds methanol, pet. ether, acetone paracetamol, thioacetami de reduces raised serum transaminases, alp,total protein and albumin flavonoids, anthrons, xanthons, tannins arachniode sexilis (dryopteridacea) (29) rhizome ethanol ccl4 reduces levels of sgpt and sgot polyphenols iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 6 continued table 1. name of plant (source/family) plants part used extract studied hepatotoxic inducing agent biochemical and histopathologi cal parameters studied chemical constituents azadiracta indica(meliaceae) (30) leaves aqueous, alcoholic, ethyl acetate and petroleu m ether paracetamol, carbon tetrachloride glutathione peroxidase (gpx), gst, sod and cat quercetin-3-o-βd-glucoside (ii) quercetin-3-o-α l-rhamnoside, (iii) myricetin – 3-o-rutinoside (iv)kaempferol3-o-rutinoside (v) quercetin-3o-rutinoside (vi) kaempferol-3-oβ-d-glucoside baliospermum montanum (euphorbiaceae)(3 1) roots alcohol, chlorofor m extract paracetamol got and gpt flavonoids, quercitin boerhaavia diffusa(nyctaginac eae)(32) roots aqueous thioacetamid e aspartate amino transferase, reduced glutathione levels, amt, sod, glutathione peroxidase, catalase and glutathione-stransferase alkaloids, flavonoids, steroids, terpinoids, safonine butea monosperma fabacea (33) flowers aqueous thioacetamid e prevents from oxidative potential by inducers butein, butin, isobutin, isomonospermoside byrsocarpus coccineus (connaraceae) (34) leaf aqueous ccl4 rich in antioxidants and strongly inhibitlipid peroxidation reduces the ast,alt,alp flavonoids and polysaccharides cassia fistula fabacea (35) leaves n-hexane paracetamol facilitates in lowering the serum transaminases, bilirubiln and lap phenolic compounds, cyaniding b2, biflavonoids, triflavonoids iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 7 continued table 1. name of plant (source/family) plants part used extract studied hepatotoxic inducing agent biochemical and histopathologi cal parameters studied chemical constituents cochlospermumpla nchoni(coclosperm aceae)(36) rhizomes aqueous ccl4 total bilirubin, alkaline phosphatase and alanine aminotransferas e flavonoids, sterols, lignans cordiama cleodii(boraginacea e) (37) leaves ethanolic ccl4 sgot, gpt flavonoids crataeva nurvala (capparaceae) (38) stem bark ethyl acetate ccl4 scavenges peroxyl radicals by facilitating the levels of enzymes system which have antioxidant properties lupeol, lupeol linoleate crossandrainfundib uliformis (acanthaceae) (39) leaf pet . ether ccl4 decreases heptocyte peroxidation and lipoprotein lipase in liver phytosterols, phenolic compounds, flavonoids curcuma longa (zingiberaceae)(40) rhizome aqueous ccl4 and taa sod, cat enzymes flavonoids, steroids, tumerone, atlantone, and zingiberene cyathea gigantean (cyatheaceae) (41) leaves methanol paracetamol reduces the raised level of sgot,sgpt,a lp,tb triterpenes, sterols, saponins, flavonoids daucus carota (apiaceae) (42) seeds methanol paracetamol, isoniazid, alcohol decreases sgot,sgpt,a lp flavonoids enicostemmaaxillar e(gentianaceae) (43) whole plant ethanolwater dgalactosamine, paracetamol decreases the lipid peroxidation secoiridoid glycoside fumaria indica(papaveracea e) (44) whole plant ethanolwater carbon tetrachloride, paracetamol and rifampicine reduces the elevated levels of serum transaminases (sgot,sgpt) narceimin, (-) tetrahydrocoptisin e, bicuculine and fumariline iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 8 continued table 1. name of plant (source/family) plants part used extract studied hepatotoxic inducing agent biochemical and histopathologi cal parameters studied chemical constituents gardenia gummifera(rubiace ae) (45) roots methanol paracetamol su.resses the raised levels of serum alt ast, mad alp, ldh phenols, flavonoids ginkgo macrophylla (ginkgoaceae) (46) dried extract ethanolic ccl4 , lantadenes sgot, serum glutamic pyruvate transaminase, sap and bilirubin content polyphenols glycyrrhiza glabra (fabaceae) (47) powdered form of root powdered root mixed with animal feed carbon tetrachloride lipid peroxidation triterpene, saponins, glycyrrhizin/glycc yrrhizic acid and glycyrrhetic acid graptopetalumpara guayense(crassulac eae) (48) whole plant aqueous ethanol, ccl4 ast, alt, ldh, sod, gpx, catalase at, and gst anthocyanins, phenolic compounds heterothecainuloide s ( asteraceae) (49) whole plant methanol, acetone ccl4 inhibits lipid peroxidation stigmasterol, quercetin, bsitosterol, cadalen-15-oic acid, kaempferol hoslundia opposita (lamiaceae)(50) stem methanol and ethyl acetate carbon tetrachloride aspartate amino transferase and alanine amino transferase and bilirubin saponins, alkaloids, tannins, sterols/triterpenes, acidic compounds, luminetzeraracemo sa(combretaceae) (51) bark ethanol, water acetaminophe n cat, sod, and gst flavonoids, alkaloid, polyphenol lycium chinense ( solanaceae) (52) fruit ethyl acetate ccl4 blocked the release of sgpt free radical scavenging property cerebrosides and pyrrole derivatives , flavonoids iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 9 continued table 1 . name of plant (source/family) plants part used extract studied hepatotoxic inducing agent biochemical and histopathologi cal parameters studied chemical constituents mallotus japonicas ( euphorbiaceae)(53) whole plant water d-galatosamine prevents the elevation of mda and glutathione content in the liver bergenin, gallic acid, quercetin melothria heterophyll(cucurbi taceae) (54) aerial plants ethanol ccl4 ast, alt, alp, total bilirubin and protein. in liver homogenate varied antioxidant enzyme activities were studied and lipid peroxidation product b-sitosterol,, glycosides, saponin, flavonoids moringa oleifera (moringaceae) (55) stem bark pet. ether, ccl4 cadmium ast, alt, alp, significant (p≤0.01) increase of lpo and decrease in sod phenolic content and flavonoids ocimum sanctum( lamiaceae)(56) whole plant aqueous paracetamol, ccl4, lead albumin globulin ratio, serum proteins, apt, histopathology of liver rosmarinic acid, β caryophyllene, oleanolic acid, eugenol, ursolic acid, carvacrol, germacrene β elemene, linalool, phyllanthus niruri(euphorbiace ae) (57-59) leaves and fruits methanoli c and aqueous carbon tetrachloride , paracetamol (gpt) glutamate pyruvate transaminase, glutamate oxaloacetate transaminase (got) six phenolic compounds; epicatechin, (+) gallic acid, (-) epigallocatechin, (-)gallocatechin, (-) epigallocatechin 3-o-gallate , epicatechin, 3-ogallate and (-) amariin, lignans iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 10 continued table 1 . name of plant (source/family) plants part used extract studied hepatotoxic inducing agent biochemical and histopathologi cal parameters studied chemical constituents piper longum (piperaceae) (60) fruit milk extract carbon tetrachloride sgt, sgpt, bilirubin piperine (1piperoyl piperidine) pleurotuseryngii ( pleurotaceae) (61) dried fruits water alloxan, ccl4, thioacetamide, ethanol, diethyl nitrosamine, dimethyl nitrosamine, deltametrin increases antioxidant enzymes activities, cat, sod, gsh and prevents uncontrolled lipid formation in liver lipids, polysaccharides, peptides, dietary fibre and sterols scoparia grandiflora(scorphu lariaceae)(62) whole plant methanol, diethyl ether and petroleum ether carbon tetrachloride alanine amino transferase (amt), total bilirubin and alkaline phosphatase ketones, g sitoterol, alkaloids, flavanoids, diterpenoids, hexacosonol, spirulina platensis (spirulinaceae) (63) spirulina microalgae lead n gsh content, and ldh, ache, sod, cat and gst enzymes vitamins, minerals, carbohydrates, carotenoids, xanthophyll, and γ-linolenic acid terminialiacatappa (combretaceae) (64) leaves chlorofor m, aqueous ccl4 prevents the mitochondrial disruption intramitochondrial ca+2 overload and su.resses ca+2 atpase activity flavonoids (keam.ferol, quercitin), tannins (punicalin, punicalagin, tercatin), saponins, phytosterols trianthemadecandr a (aizoaceae) (65) leaves aqueous ccl4 alanine amino transferase, amt and bilirubin flavonoid, fats, terpenes, carbohydrates, tannins, and alkaloids trianthemaportulac astrum (aizoaceae) (66) whole plant ethanol paracetamol, thioacetamide stimulates hepatic regeneration saponin and punarnavine tridaxprocumbens (asteraceae) (67,68) leaves ethanolic extract paracetamol , dgalactosamine glutathione, superoxide dismutase and catalase flavonoids, alkaloids, tannins, carotenoids iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 11 continued table 1 . name of plant (source/family) plants part used extract studied hepatotoxic inducing agent biochemical and histopathologi cal parameters studied chemical constituents trigonella(69) leaves methanoli c carbon tetrachloride, deltamethrin serum bilirubin level, sgot, sgpt polysaccharides, saponins, fibers, flavonoids and alkaloids like trigonelline, trigocoumarin, choline trigonella foenumgraecum (fabaceae) (70,71) seed polypheno lic thioacetamide alkaline phosphatase, γglutamyl transferase, serum gamma glutamyl transferase (ggt), lipid peroxidation (lpo), glutathione reductase and peroxidase, xanthine oxidase (xod) polyphenolic compounds tylophora indica(asclepidacea e) (71) leaves methanoli c carbon tetrachloride sgot, serum glutamic pyruvate transaminase, total bilirubin alkaloids, steroids, saponins, triterpenes, steroids v. trifolia (verbenaceae) (72) leaves water and ethanol carbon tetrachloride total protein, amt, alanine amino transferase flavonoids , triterpenoids conclusion the herbal medicine popularity is being increasing for many decades with regards to liver diseases. hepatic disorders may be caused by toxic chemicals and certain drugs. uncontrolled consumption of alcohol also affects liver. several formulation of medicinal plants are used to cure liver disorders. it is observed that hepatoprotective effect of plant is mostly due to flavonoids, alkaloids, terpenoids, steroids, glycoside. a single drug cannot be useful in position to all types of excessive liver problems. several plant extracts for liver illness results from poisonous chemicals, viruses, extra alcohol consumption and repeated administration of medication. well modified and updated metholodologies and clinical trials are needed to study the hepatoprotective mechanism of folk medicines. this approach will lead to several other discoveries which will enable the researchers to come up with numerous dosage forms in ayurvedic medicine. however,herbal remedies are not well documented and hence are not much prescribed. an attempt has been made in this review article to highlight various mechanism of hepatoprotection of some plants. this article extends a help to the scientists, researchers, and scholars who are working in the therapeutic field to develop a cure for liver diseases. acknowledgement i would like to express my sincere gratitude to dr. s. sharma, dr. s. paliwal, dr. s. s. chitlange for always supporting and motivating me for to complete the review article. i wish to thank my iraqi j pharm sci, vol.30(2) 2021 hepatoprotective drugs screening 12 parents and my husband for their support and encouragement throughout my 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paracetamol induced hepatotoxicity in male albino rats. adv stu biol. 2010;2:105-2. 69. bhanger mi, bukhari sb, memon s. antioxidative activity of extracts from a fenugreek seeds (trigonellafoenumgraecum). pakistan journal of analytical & environmental chemistry. 2008;9(2):6. 70. kaviarasan s, viswanathan p, anuradha cv. fenugreek seed (trigonellafoenumgraecum) polyphenols inhibit ethanol-induced collagen and lipid accumulation in rat liver. cell biologyand toxicology. 2007;23(6):373-83. 71. gujrati v, patel n, rao vn, nandakumar k, gouda ts, shalam md, kumar ss. hepatoprotective activity of alcoholic and aqueous extracts of leaves of tylophoraindica (linn.) in rats. indian journal of pharmacology. 2007;39(1):43. 72. manjunatha bk, vidya sm. hepatoprotective activity of vitextrifolia against carbon tetrachloride-induced hepatic damage. indian journal of pharmaceutical sciences. 2008;70(2):241. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 16-https://doi.org/10.31351/vol29iss2pp8 :doi 8 cyp2d6 genotype in relation to liver toxicity due to tetrabenazine in iraqi patients with hyperkinetic movement disorders zainab a. abbood*,1, shatha h ali** and nawfal m sheaheed** * department clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** department of clinical and laboratory science, college of pharmacy, university of baghdad, baghdad, iraq *** ministry of health and environment, baghdad teaching hospital, medical city, baghdad, iraq. abstract the common types of movement disorders are; dystonia which is a syndrome of repetitive muscle contractions. while, huntington disease is autosomal dominant progressive neurodegenerative disorder, which is characterized by involuntary movements (“chorea”). tetrabenazine therapy has been shown to effectively control these movements compared to placebo. the most commonly reported side effects of tetrabenazine increase liver enzymes and these side effects are dose dependent. this is a prospective case controlled study was carried on 50 patients whom divided into 2 groups: group 1 involved 25 chorea patients, and group 2 included patients with dystonia, whom treated with (tetrabenazine) for three months. in addition to control group involved 25 healthy subjects to estimate the prevalence of genetic polymorphism of cyp 450 2d6 enzyme in related to tetrabenazine efficacy and toxicity. blood samples were collected at the beginning to perform a genotyping assay for cyp 450 2d6 enzyme by pcr and to assess liver function and after three months of treatment to assess liver and measuring the plasma concentration of tetrabenazine , alpha and beta dihydrotetrabenazine by hplc. the results show a significant cyp 450 2d6 enzyme polymorphism. and elevations of liver enzymes in the patient indicate hepatotoxicity of tetrabenazine and its metabolites. keyword:dystonia, chorea, tetrabenazine, alpha and beta dihydrotetrabenazine , cyp 450 2d6 enzyme polymorphism, liver function. نالعراقيي اضطرابات الحركة المفرطةلدى مرضى بسمية الكبد تهوعالق cyp2d6 النمط الوراثي ل التترابينازين الذين يتعاطون عالج **نوفل ماضي شهيد و **، شذى حسين علي 1*،زينب علي عبود فرع الصيدلة السريرية، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق. * فرع العلوم المختبرية السريرية ، كلية الصيدلة ،جامعة بغداد ، بغداد ،العراق.** ، مستشفى بغداد التعليمي، مدينة الطب ، بغداد، العراق. وزارة الصحة والبيئة *** الخالصة تقلصات الى من بين األنواع الشائعة الضطرابات الحركة ؛ خلل التوتر العضلي ، وهو متالزمة اضطراب الحركة العصبية التي تؤدي .العضالت المستمرة. في حين أن مرض هنتنغتون هو في المقام األول اضطراب وراثي جسدي ، ويتميز بحركات ال إرادية )"رقص"( هي ارتفاعتيترابينازين لل اآلثار الجانبية شيوًعا.ان أكثر نازين يتحكم بشكل فعال في هذه الحركات مقارنةً بالعالج الوهميلقد ثبت أن عالج التيترابي ..الجرعة ةعلى زيادإنزيمات الكبد وهذه اآلثار الجانبية تعتمد ريًضا يعانون من مرض الرقص ، والمجموعة الثانية م 25مريضاً تم تقسيمهم إلى مجموعتين: المجموعة األولى شملت 50 أجريت الدراسة على شخص 25شملت مرضى مصابين بخلل التوتر العضلي ، الذين سيتم عالجهم بالتترابينازين لمدة ثالثة أشهر. باإلضافة الى مجموعة السيطرة وبعد ثالثة أشهر من الدراسة cyp 2d6جيني إلنزيم. باإلضافة الى تعدد األشكال الالكبد صحي. تم جمع عينات دم في بداية الدراسة لتقييم وظيفة .مع وظائف الكبد للمرضى تم قياس تركيز تيترابينازين ، ألفا وبيتا تيترابينازين في البالزما مقارنة مع ضىأنزيمات الكبد في المر في . مع ارتفاع كبير cyp 2d6بنسبة كبيرة إلنزيم الجينية األشكالبهذه الدراسة توضح أن هناك تعدد . تيترابينازين و مستقلباته ال بفعل دواءتشير إلى تسمم الكبد مجموعة المقارنة والتي .ظائف الكبدو، cyp 2d6هايدرو تترابينازين ، تعدد االشكال الجيني هنتنغتون ، تترابينازين، ألفا و بيتا داي الخلل التوتر العضلي،: الكلمات االفتتاحية 1corresponding author e-mail: zainabaa87@yahoo.com received: 28/11 /2019 accepted:15 /2 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp8-16 iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 9 introduction the term "movement disorders" refers to a group of nervous system (neurological) conditions that cause abnormal increase in the movements, which may be voluntary or involuntary(1) .common types of movement disorders include: dystonia is a syndrome of neurological movement disorder in which constant or repetitive muscle contractions result in twisting and repetitive movements or abnormal fixed postures(2).dystonia is often intensified or aggravated by physical activity, and symptoms may progress into adjacent muscles (3) huntington disease is primarily an adult-onset hereditary autosomal dominant progressive neurodegenerative disorder, which is characterized by involuntary movements (“chorea”), psychiatric symptoms, and cognitive dysfunction that can lead to dementia (4). presently, the backbone of treatment for movement disorders is symptomatic and supportive care –– no drugs are available to stop or prevent the progression of hyperkinetic movement disorders)5(,)6(. tetrabenazine therapy (tbz), a benzoquinoline derivative, is an oral monoamine-depleting agent with selectivity for dopamine. it specially prevents the presynaptic monoamine storage by inhibiting the vesicular monoamine transporter type-2, a presynaptic transporter found mainly in the central nervous system )7(, has been shown to effectively control chorea and dystonia symptoms compared with placebo. by using pharmacogenomics, the pharmacotherapy can be optimized, thereby increasing efficacy and decreasing the incidence of adverse events. the cytochrome p450 (cyp2d6) enzyme metabolizes about 25% of clinically used drugs for many different drug classes including antidepressants, antipsychotics, antihypertensive, and analgesics. the cyp2d6 is a highly polymorphic gene locus with more than 75 allelic variants, and subjects can be classified into poor metabolizers (pm), intermittent metabolizers (im), and extensive metabolizers (em), or ultra-rapid metabolizers(um)(8,9) pharmacogenomics, aiming to clarify the relationship between human genome sequence variations and drug responses, has focused on genes whose affecting absorption, distribution, metabolism, and elimination of a given drug (pharmacokinetic pathways), as well as on identifying potential drug targets (pharmacodynamic pathways) )10(. pharmacogenetic considerations are particularly relevant to tetrabenazine, as the drug is metabolized via the cytochrome p450 enzyme system (cyp). the two metabolites of tetrabenazine, α-htetrabenazine and β-h tetrabenazine, are metabolized in the liver primarily by cyp2d6(11). the most commonly reported side effects of tetrabenazine increase liver enzymes(alp, alt, and ast))(12). side effects of tetrabenazine are dose dependent. this study aimed to predicted phenotypes of cyp2d6: poor metabolizers, intermediate metabolizers, and extensive metabolizers or ultra-rapid metabolizers among iraqi subjects. and to investigate whether cyp2d6 gene polymorphism effect the tetrabenazine, and it is two metabolites :, α-htetrabenazine and β-h tetrabenazine and their relation to liver toxicity. patients and methods patient selection and study design fifty movement disorder patients participated in the current prospective case controlled study where divided into 2groups: group 1 involved 25 patients suffer from chorea (12 males and 13 females) with mean age 41.36±2.39 years, and group 2 includes patients with dystonia (8males and 17 females) with mean age 38.04± 2.62 years, treated with (tetrabenazine) for three months. the starting dose of tetrabenazine was 12.5 mg once daily in the morning; titrate up at weekly intervals by 12.5 mg/day; doses of 37.5 to 50 mg was divided into 3 doses. ethical approval was obtained from the department of clinical pharmacy at the college of the pharmacy. the study is carried out in baghdad medical city during the period from september 2018 to june 2019. patients undergoing clinical examination by measuring unified huntington's disease rating scale (uhdrs) and unified dystonia rating scale(udrs) in the movement disorder unit of the hospital as well as in private clinic. ten millimeter blood sample were collected at the beginning of the study to measure, liver function tests (serum alp , ast, and alt) and genetic polymorphism of cyp 450 2d6 enzyme for both group and after three months of the study to measure the plasma concentration of tetrabenazine , alpha and beta tetrabenazine and liver function tests with the clinical examination for side effects. iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 10 genotyping of cyp2d6 gene after dna isolation from venous blood by wizard genomic dna purification kit (promega,usa). genotyping of cyp2d6 gene performed by polymerase chain reaction (pcr) conventional (allele specific method) according to method described by taimour langaee, issam hamadeh, arlene b. chapman, et al)13(. the following primers was used for pcr amplification. 1.cyp2d6 *2 (2850 c>t rs16947) forward 5’ ggcccctgcactgtttcc 3’ reverse 5’ aaggggaaccctgagagc 3’ 2. cyp2d6 *4 (1846 g>a) rs3892097 forward 5’ tgccgccttcgccaaccact 3’ reverse 5’ gcagagactcctcggtctctc 3’ 3. cyp2d6 *10 (100 c>t rs1065852) forward 5’ tgtccagaggagcccattt 3’ reverse 5’gtcgaagcagtatggtgtgttct 3’ measure plasma level of tetrabenazine by hplc about (20 µ1) of patient plasma was injected into the liquid chromatographic system consisting of a cl8 µ bondapak column and fluorescence detector. the mobile phase was acetonitrile-1% acetate buffer, ph 4.5 (50: 50) at a flow-rate of l_ml/min. the fluorescence of the eluent was quantified using an excitation wave length of 265 nm and an emission filter (kv418)(14),(15). biochemical assay for liver function serum alp level was measured by cobas c 311 chemistry analyzer made by roche diagnostics in cooperation with hitachi high-technologies corporation (16).while serum ast, alt , and tsb level evaluated using a ready-made kit for this purpose, according to the method of kirsch jf , et al (17), kim wr, et al (18), and kao tw, et al (19) respectively. statistical analysis data will be analyzed by using sas (statistical analysis system) (version 24.0) program (spss inc., chicago, illinois, usa) and minitab version 17 software. in all comparisons, a p-value <0.05 was considered statistically significant. results distribution and demographic data of the patients fifty patients completed the course of study successfully, there were non-significant differences between all parameters at baseline as shown in (table 1). table 1. demographic data and baseline characteristics of the patients. p-value group 2 group 1 data 0.468 38.04± 2.62 41.36±2.39 age (yrs.) -----25 25 no. of subjects -----17 females 8 males 13 females 12 males gender 0.374 5.400± 0.580 6.148±0.599 disease duration(yrs.) 0.259 22.760±0.751 21.120±0.590 bmi 0.909 0.64± 0.060 0.61 ± 0.052 serum total bilirubin(mg/dl) 0.570 58.26± 4.44 60.03± 3.59 serum alkaline phosphatase u/l 0.435 30.48±1.70 28.96± 1.49 serum aspartate transaminase u/l 0.666 41.89± 2.17 42.72± 2.05 serum alanine transaminase u/l data are expressed as mean±sem. distribution of patients with (cyp 450 2d6 )gene polymorphism seventy four percent (37) of patients have cyp 450 2d6*2 (normal allele) while 26% (13) of patients have allele polymorphisms (cyp 450 2d6*10) . no patients were observed with cyp 450 2d6 *4 polymorphisms in this study(figure 1) . iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 11 figure 1. histogram showing the distribution of patients with (cyp 450 2d6 )gene polymorphism plasma concentration of tetrabenazine, alpha and beta dihydrotetrabenazine for patients with cyp 450 2d6 polymorphism versus the patients without cyp 450 2d6 polymorphism in patients using tetrabenazine: tetrabenazine, alpha and beta dihydrotetrabenazine concentration were increased significantly in the patients with cyp 450 2d6*10 polymorphisms compared to patients with cyp 450 2d6*2 using tetrabenazine in chorea and dystonia (figure 2, 3) . figure 2. plasma concentration of tetrabenazine, alpha and beta dihydrotetrabenazine for patients with cyp 450 2d6 polymorphism versus the patients without cyp 450 2d6 polymorphism in chorea patients using tetrabenazine. figure 3. plasma concentration of tetrabenazine, alpha and beta dihydrotetrabenazine for patients with cyp 450 2d6 polymorphism versus the patients without cyp 450 2d6 polymorphism in dystonia patients using tetrabenazine. iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 12 effect of tetrabenazine on liver enzymes patients was subdivided into a subgroup (patients with normal allele and b subgroup (patients with allele polymorphism).there was a non-significant difference in serum alkaline phosphatase (alp), alanine transaminase (alt), aspartate transaminase (ast), and total bilirubin (tsb) among treated subgroups at pretreatment. while after three months of treatment with tetrabenazine, the study showed that was a significant elevated in serum alp, alt, and ast while non-significant difference tsb in both subgroup a and b compared to subgroups at pretreatment, which is highly increased in liver enzymes in subgroup b as showed in ( table 2) . table 2. effect of tetrabenazine on liver enzymes in patients with chorea and dystonia after three months of treatment. p-value dystonia pvalue chorea liver enzyme after treatment pretreatment after treatment pretreatment 0.047* 74.02±4.67* 59.35±5.36 0.014* 80.11±4.71 63.93±4.09 a alp (u/l) 0.004* 91.67±5.75* 54.80±7.93 0.001* 98.57± 7.53 49.99±6.25 b 0.007* 54.55± 3.08 42.99±2.58 0.049* 50.92± 2.18 44.02±2.58 a alt (u/l) 0.001* 67.55± 4.77 38.40±3.87 0.001* 61.74±4.22 39.40±2.95 b 0.001* 39.47± 1.89 29.95±1.83 0.045* 33.72± 1.17 29.50±1.63 a ast (u/l) 0.005* 52.50± 3.45 32.17±4.35 0.015* 39.86± 2.35 27.57±3.46 b 0.826 0.679± 0.06 0.658±0.08 0.583 0.557±0.033 0.594±0.06 a tsb (mg/dl) 0.876 0.5833±0.06 0.567±0.08 0.820 0.614±0.059 0.643±0.11 b data are expressed as mean±sem; ;*significantly difference compare to pretreatment within the same group (p<0.05). a (patients with normal cyp 450 2d6 gene) and b (patients with cyp 450 2d6 polymorphism). the correlation of plasma concentration of alpha dihydrotetrabenazine and plasma concentration of tetrabenazine with liver enzymes (alp, ast, alt, and tsb) in patients group: the study showed a significant correlation between tetrabenazine and alpha dihydrotetrabenazine plasma concentrations and serum ast (figure 4) and alt (figure 5) and (figure 6) . the alp, and tsb serum concentration and was non-significant with serum alp and tsb as in (table 3) in chorea patients . table 3. summarizes the relationship between tetrabenazine and alpha dihydrotetrabenazine plasma concentration and serum concentration of alp, ast, alt, and tsb in chorea patients. concentration alp ast alt tsb tbz r _value 0.344 0.399 0.591 0.173 p_value 0.092 0.048* 0.002* 0.409 alpha dtbz r _value 0.303 0.343 0.599 0.189 p_value 0.141 0.093 0.002* 0.366 * significant difference iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 13 figure 4. plasma concentration of tetrabenazine related to serum ast in chorea patients. figure 6. plasma concentration of alpha dihydrotetrabenazine related to serum alt in chorea patients. while in dystonia the study showed a non-significant correlation between tetrabenazine and alpha dihydrotetrabenazine plasma concentrations and the alp, ast, and tsb serum concentration and only was significant between the tetrabenazine plasma concentrations and serum ast and alt as in (table 4) and (figure 7, 8) . figure 5. plasma concentration of tetrabenazine related to serum alt in chorea patients. table 4. summarizes the relationship between tetrabenazine and alpha dihydrotetrabenazine plasma concentration and serum concentration of alp, ast, alt, and tsb in dystonia patients. concentration alp ast alt tsb tbz r _value 0.210 0.460 0.430 -0.158 p_value 0.314 0.021* 0.032 * 0.450 alpha dtbz r _value 0.235 0.288 0.249 -0.108 p_value 0.259 0.162 0.230 0.609 figure 7. plasma concentration of tetrabenazine related to serum ast in dystonia patients. figure 8. plasma concentration of tetrabenazine related to serum alt in dystonia patients. 654321 50 45 40 35 30 25 tbz plasma con (ng\ml) a s t ( u \l ) r=0.399, p=.048 654321 70 60 50 40 30 20 tbz plasma con (ng\ml) a s t ( u \ l ) r=0.460, p=0.021 654321 90 80 70 60 50 40 30 tbz plasma con (ng\ml) a l t ( u \ l ) r=0.430, p=0.032 7060504030 80 70 60 50 40 alpha dtbz plasma con (ng\ml) a l t ( u \l ) r=0.599, p=0.002 iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 14 discussion distribution of patients with (cyp 450 2d6 ) gene polymorphism figure (1) shows that the number (percentage) of patients with cyp 450 2d6*2 was 37 (74%), whereas, patients with cyp 450 2d6 *10 was 13 (26%). no patients were observed with cyp 450 2d6 *4 polymorphisms in this study. to avoid tetrabenazine side effects, genotyping tests before the initiation of therapy could identify patients with unacceptable mortality and morbidity risks. the cyp2d6 activity ranges considerably within a population and includes ultrarapid metabolizers (ums), extensive metabolizers (ems), intermediate metabolizers (ims) and poor metabolizers (pms). the allele *10 give rise to substrate-dependent decreased activity. it is clear that alleles *3, *4, *5, *6 and *7 have no enzyme activity(20). no patients were observed with cyp 450 2d6 *4 polymorphisms in this study. plasma concentration tetrabenazine, alpha and beta dihydrotetrabenazine for all patients: there has been promoting from monitoring a drug level in plasma of tetrabenazine and its metabolites for hyperkinetic movement disorders and take with precaution in the polymorphism of cyp 450 2d6 enzyme. the plasma concentration of tetrabenazine after one and half hour for therapeutic efficacy suggested as less than 2.5 ng/ml and alpha and beta dihydrotetrabenazine 40.5 ng/ml and 25.7 ng/ml respectively (21). tetrabenazine is rapidly metabolized via first-pass metabolism into two main metabolites known as alpha and betadihydrotetrabenazine (dtbz). of these two compounds, alpha-dtbz is pharmacologically active, whereas beta-dtbz is pharmacologically inert. peak plasma concentrations of alpha-dtbz and beta-dtbz are achieved within one to 1.5 hours following an oral dose, and these compounds have a halflife of approximately 10 hours. in contrast, tbz exhibits a half-life of about six hours. dtbz is further metabolized by cyp2d6 to odealkylated dtbz, which is subsequently excreted via the urine and feces. the metabolites are primarily renal eliminated(22). there was a significant increase in plasma concentration of alpha and betadihydrotetrabenazine in patients with cyp2d6 polymorphism in comparison with those with non_mutant gene-phenotype that is due to the low activity of the enzyme (31.91± 0.56 vs. 52.69± 3.7) (19.96± 0.34 vs. 25.01± 0.97) respectively for chorea patients, while (33.59± 0.58 vs. 52.5± 4.7) (18.25± 0.87 vs. 24.85± 1.1) for dystonia patients, which results in elevation in the plasma concentration of tetrabenazine (2.467±0.086ng/ml vs. 4.657± 0.37) for chorea and (1.932± 0.10 vs. 4.325±0.34) for dystonia . effect of tetrabenazine on liver enzymes the results regarding alp, alt, and ast as shown in (table 2) indicate that a significant elevation in liver enzymes in both groups. moreover, a non-significant difference in serum tb among groups post-treatment. the present results indicate that a highly significant effect in both subgroup a and b of both group compared to pretreatment, and statistically significant difference observed after three months between subgroups this increment in groups b related to cyp 450 2d6 polymorphism which lead to increase the drug concentrations. in this study, highly significant elevations of liver enzymes in the patients compared to pretreatment, indicating hepatotoxicity.. a positive non-significant correlation between tetrabenazine plasma concentrations and alp in both chorea and dystonia were observed , while it was significantly correlated with alt and ast in both chorea and dystonia. regarding alpha dihydrotetrabenazine concentration, a (positive) non-significant correlation was found with alp, alt , and ast in both groups. consequently, there was no significant correlations were detected between plasma concentrations of tetrabenazine and alpha dtbz levels and tsb level in both chorea and dystonia conclusions according to the data of the present study, we can conclude that: 1. there was high incidence (26%) of cyp 450 2d6 gene polymorphisms in iraqi patients with movement disorders and mainly cyp 450 2d6*10 which is intermediate metabolizers (ims) . 2. higher plasma concentration of tetrabenazine among patient with cyp 450 2d6*10 polymorphism in comparison with patient without cyp 450 2d6 polymorphism. 3. positive significant correlations were detected between serum levels of tetrabenazine and its metabolites with liver function parameters indicating hepatic toxicity due to tetrabenazine metabolites. iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 15 acknowledgement the present work was abstracted from phd thesis submitted to the department of clinical pharmacy, college of pharmacy, university of baghdad. the authors are greatly thankful to baghdad teaching hospital, medical city and al-zahraa teaching hospital for supporting this project. reference 1. fahn s, jankovic j. principal & practice of movement disorders,1st ed., churchill livingstone, elsevier, philadelphia, usa; 2007:p. 1-652. 2. 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tablet dosage form. chemical methodologies. 2017 oct 1;1:136-44. 17. patton c. j, crouch s .r. spectrophotometric and kinetics investigation of berthelot reaction for the determination of ammonia . annual. chem 1977; 49(3):464-469 18. kirsch jf, eichele g, ford gc, et al. mechanism of action of aspartate aminotransferase proposed on the basis of its spatial structure. journal of molecular biology. 1984;174(3):497-525. 19. kim wr, flamm sl, di bisceglie am, bodenheimer hc. serum activity of alanine aminotransferase (alt) as an indicator of health and disease. hepatology. 2008 ;47(4):1363-1370. iraqi j pharm sci, vol.29(2) 2020 tetrabenazine 16 20. kao tw, chou ch, wang cc, et al. associations between serum total bilirubin levels and functional dependence in the elderly. internal medicine journal. 2012;42(11):1199-1207. 21. shu-feng zhou. polymorphism of human cytochrome p450 2d6 and its clinical significance part ii. clinical pharmacokinetics. 2009; 48(12): 761-804. 22. scherman d, jaudon p, henry jp. characterization of the monoamine carrier of chromaffin granule membrane by binding of [2–3h]dihydrotetrabenazine. proceedings of the national academy of sciences. 1987;80(2):584-8. 23. kenney c, jankovic j. tetrabenazine in the treatment of hyperkinetic movement disorders. expert review of neurotherapeutics. 2006;6:7-17. creative commons attribution is licensed under a bijpsbaghdad iraqi journal pharmaceutical sciences by .university of baghdad -copyrights© 2015 college of pharmacy .international license 4.0 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 40 formulation and evaluation of bilayer tablets containing immediate release aspirin layer and floating clopidogrel layer nawar m.toma *,1 and yehia i.khalil * * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract aspirin and clopidogrel are considered the most important oral platelets aggregation inhibitors. so it is widely used for treatment and prophylaxis of cardiovascular and peripheral vascular diseases related to platelets aggregation .in this study aspirin and clopidogrel were formulated together as floating bilayer tablet system. three different formulas of 75 mg aspirin were prepared by wet granulation method as immediate release layer; different disintegrants used to achieve rapid disintegration. formula with crosscarmellose as disintegrant achieve rapid disintegration was selected for preparation of bilayer tablet. different formulas of 75 mg clopidogrel were prepared as sustained release floating layer by wet granulation (effervescent ) method ;the physical and floating properties for compressed clopidogrel matrix were studied in addition to study the effect of polymer concentration(hpmc) ,and its combination with ethyl cellulose and carbapol ,effect of different diluents and effect of increasing sodium bicarbonate amount on the release from compressed matrix . formula prepared with hpmc and ec in a ratio of 1:1 was capable to retard the release of clopidogrel for 6 hours in addition to its good floating behavior and therefore selected to prepare bilayer tablets in combination with selected aspirin layer. the prepared bilayer tablets were further subjected to evaluation of their physical, floating properties and release behavior. finally the kinetic study reflects acceptable shelf life for aspirin and clopidogrel. key words: aspirin, clopidogrel, bilayer tablet, floating tablet. تصييغ وتقييم الحبوب الثنائية الحاوية االسبرين كطبقة فورية التحرر واللكوبيدوكريل كطبقة طافية نوار ميخائيل توما *،1 خليل يحيى اسماعيل و * * .خايؼح تغذاد ، تغذاد ، انؼزاق فزع انظٛذالَٛاخ ، كهٛح انظٛذنح ، الخالصة نذا فٓٙ ذسرخذو تشكم ٔاسغ نهٕلاٚح ٔانؼالج ,ٚؼرثز االسثزٍٚ ٔانكهٕتٛذٔكزٚم يٍ اْى يثثطاخ ذدًغ انظفائح انذيّٕٚ انًؼطاج ػثز انفى ذظٛٛغ االسثزٍٚ ٔانكهٕتٛذٔكزٚم فٙ ْذِ انذراسّ ذى .يٍ ايزاع انمهة انٕػائّٛ ٔااليزاع انًحٛطّٛ انًزذثطّ تردًغ انظفٛحاخ انذيّٕٚ ذى ذحضٛز ثالز طٛغ يخرهفّ نالسثزٍٚ تطزٚمّ انرحثٛة انزطة كطثمّ سزٚؼح انرفكك تاسرخذاو . ػهٗ شكم حثٕب طافّٛ تُظاو ثُائٙ انطثمّ خ اسزع ٔلد نهرفكك نمذ اظٓزخ انُرائح اٌ انظٛغّ انرٙ ذحٕ٘ انكزٔسكاريهٕس كًفكك حمك.يفككاخ يخرهفّ يٍ اخم ذحمٛك انرفكك انسزٚغ ذى دراسح ,(انفٕارِ)ذى ذحضٛز انؼذٚذ يٍ انظٛغ انًخرهفّ نهكهٕتٛذٔكزٚم تطزٚمح انرحثٛة انزطة .ٔذى اخرٛارْا نرظُٛغ انحثٕب ثُائّٛ انطثمح انٓاٚذرٔكسٙ )خذو انخظائض انفٛشٚأّٚ ٔخٕاص انطٕفاٌ نهحثٕب انًحضزِ كمانة تاالضافح نذراسح ذاثٛز انرزاكٛش انًخرهفّ نهثٕنًٛز انًسد ٔذاثٛز ديدّ يغ تٕنًٛزاخ اخزٖ يثم االثٛم سٛههٕس ٔانكارتاتٕل ٔذاثٛز اسرخذاو يخففاخ يخرهفّ اضافّ نذراسح ذاثٛز (تزٔتٛم يثٛم سٛههٕس .سٚادج كًّٛ تٛكارتَٕاخ انظٕدٕٚو ػهٗ ذحزر انكهٕتٛذٔكزٚم نٓا انماتهّٛ ػهٗ اػالح ذحزر انكهٕتٛذٔكزٚم 1:1ٔس ٔاالثٛم سٛههٕس تُسثّ نمذ ٔخذ اٌ انظٛغّ انًحضزِ يٍ انٓاٚذرٔكسٙ تزٔتٛم يثٛم سٛهم نسد ساػاخ اضافّ اليرالكٓا خٕاص طٕفاٌ خٛذِ نذا فمذ ذى اخرٛارْا نرحضٛز انحثٕب ثُائٛح انطثمح تانذيح يغ انطثمّ انًُرماج يٍ اظٓزخ .انفٛشٚأّٚ ٔخٕاص انطٕفاٌ ٔانرحزر خارج اندسى نمذ ذى اخضاع انحثٕب ثُائٛح انطثمّ انًحضزِ الخرثاراخ انخٕاص .االسثزٍٚ .دراسح حزكّٛ انذٔاء ػًز رف يمثٕل تانُسثح نكال االسثزٍٚ ٔانكهٕتٛذٔكزٚم .،الحبوب الطافية ثنائية الطبقة االسبرين ،الكلوبيدوكريل ، حبوب :الكلمات المفتاحية introduction the oral route remains the most considered one for administration of drugs and tablets of various types still the ruling dosage form since years. multilayered tablets are a form of modified release tablets (1) and they are designed for many reasons:  to control the delivery rate of either single or two different active pharmaceutical ingredient(s).  to separate incompatible active pharmaceutical ingredients from each other, to control the release of active pharmaceutical ingredients from one layer by utilizing the functional property of the other layer (such as, osmotic property). 1 corresponding author e-mail:nawarelias2004@yahoo.com received: 19/12/2011 accepted: 23/1/2013 iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 41  to modify the total surface area available for active pharmaceutical ingredients layer either by sandwiching with one or two inactive layers in order to achieve swell able /erodible barriers for modified release.  to administer fixed dose combinations of different active pharmaceutical ingredients, prolong the drug product life cycle, fabricate novel drug delivery systems such as chewing device, buccal/mucoadhesive delivery systems, and floating tablets for gastro-retentive drug delivery (2). one of the most important aspects of layered tablets is gastro retentive system which is shown to improve bioavailability, reduces drug waste, and improves solubility for drugs that are less soluble in a high ph environment. it has applications also for local drug delivery to the stomach and proximal small intestines. gastric retention helps to provide better availability of new products with new therapeutic possibilities and substantial benefits for patients (3) . aspirin and clopidogrel are a well known platelets aggregation inhibitors; the combination of both drugs was shown to be synergistic because they have different mechanism of action. aspirin has a significant effect on platelets this action is mainly due to decrease in production of thromboxane a2 which consider the main promoter of platelets aggregation; aspirin in low doses (60-80mgdaily) can irreversibly inhibit thromboxane production (4), while clopidogrel interfere with the binding of adenosine diphoshate adp to its receptors, adp is the second most important trigger to platelets aggregation behind thromboxane a2 (5). it appears that rapid release formulation of aspirin should be preferred in anti platelets therapy either alone or in combination with other anti platelets drugs (6). clopidogrel solubility is strongly ph dependent and it is very soluble and stable at ph value <3 (7). in addition to that clopidogrel has low oral bioavailability (50%), undergoes extensive first pass metabolism (85%) and frequent high doses are required to maintain the therapeutic level as a result, dose related toxic effects developed (8). the goal of this study is to utilize bilayer tablet approach to administer aspirin as immediate release layer and clopidogrel as sustained release floating layer in an attempt to improve bioavailability and to get maximum therapeutic benefits by patients that need the combination of aspirin and clopidogrel in cases of in acute coronary syndromes, including acute myocardial infarction and unstable angina, and in coronary stenting. materials and methods aspirin supplied by "sdi, iraq", carbapol (goodrich,usa), clopidogrel powder (zhejiang menovo pharmaceutical co.,ltd,china), croscarmellose (hekma drug industry,jordan), ethylcellulose (ec) ( bdh chemicals ,ltd, england) , explo tab(bdh chemicals, ltd, england) hydrochloric acid (bdh chemical ltd, england) , hypromellose usp(metolose 90sh-4000sr) (hpmc4000) ( shin-etsu chemicals co.ltd., japan) , lactose(riedeldeltaen /germany), microcrystalline cellulose (avicel ph 101)( whatman intrnational , england), polyvinylpyrrolidone (pvp)( riedel de haen ag seelze, honnover, germany), sodium bicarbonate(riedel-deltaen/germany), starch(afco ,india), talc(afco ,india). table (1) summarizes three formulas to prepare aspirin immediate release layers by non aqueous wet granulation method. .a known weight of granules were mixed with calculated amount of talc powder for 5minutes then compressed to first layer of tablet using 9mm flat face punch tabletting machine. table 1: different formulas of aspirin immediate release layer. ingredients in mg formula no. aspirin lactose explo tab croscarmellose 10% alcohol starch paste talc total wt .(mg) aasspp11 7755 5577 ----------- --------------------- 1155 33 115500 aasspp22 7755 5544 33 --------------------------- 1155 33 115500 aasspp33 7755 5544 ----------- 33 1155 33 115500 iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 42 evaluation of compressed aspirin immediate release layer the different prepared formulas of immediate release layer were compressed and their disintegration time was recorded. the disintegration time of the prepared tablets was measured using the disintegration apparatus and method as described in the usp, a disintegration media of 0.1 n hcl held at 37ºc. the in vitro release study of each formula were conducted in usp dissolution apparatus (paddle)in 900 ml 0.1 n hcl solution as dissolution media at 37˚c, samples of 5ml were withdrawn at different time intervals ,filtered and measured at the uv of maximum absorbance which is 230 nm . preparation of clopidogrel floating sustained release layer different formulas of clopidogrel floating controlled release layer were prepared as shown in table (2) using non aqueous wet granulation method. a known weight of granules was mixed with calculated amount of talc powder for 5minutes then compressed using 9mm flat face punch tabletting machine. table2: different formulas of clopidogrel tablet as sustained release layer. evaluation of compressed clopidogrel floating sustained release layer the prepared formulas were subjected to following tests: friability test the friability test was done for the prepared tablet using roche friabilitor ,the friability was calculated as the percent weight loss. hardness the hardness of six tablets from each of the prepared formulas was measured using monsanto hardness tester. content uniformity test content uniformity test was done as described in usp, the amount of clopidogrel was determined by employing uv absorption at the wave length of maximum absorbance at about 270nm. determination of floating lag time and floating duration the floating lag time and floating duration was determined by placing tablets in a 100-ml beaker containing 0.1 n hcl . dissolution test the in vitro release study of each formula were conducted in usp dissolution apparatus (paddle)in 900 ml 0.1 n hcl(ph1.2)solution as dissolution media at 37˚c, samples were measured at the uv of maximum absorbance which 270 nm. ingredients in mg formula no clo hpmc ec carbapol sodiumbicar. mcc lactose dcp pvp talc total wt.(mg) cl-1 75 70 17.5 156 17.5 14 350 cl-2 75 140 17.5 86 17.5 14 350 cl-3 75 210 17.5 16 17.5 14 350 cl-4 75 157.5 52.5 17.5 16 17.5 14 350 cl-5 75 105 105 17.5 16 17.5 14 350 cl-6 75 157.5 52.5 17.5 16 17.5 14 350 cl-7 75 105 105 17.5 16 17.5 14 350 cl-8 75 210 33.5 17.5 14 350 cl-9 75 210 8.75 24.75 17.5 14 350 cl-10 75 140 17.5 86 17.5 14 350 cl-11 75 140 17.5 86 17.5 14 350 iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 43 variables affecting release profile from clopidogrel floating matrix tablets effect of polymer concentration formulas cl-1, cl-2, cl-3, were used to study the effect of polymer concentration on the release profile .these formulas (cl-1, cl-2, cl3) contains hpmc in concentration of 20%, 40%, 60%w/w respectively. effect of polymer combination and ratio formulas cl(4,5,6,7) were used to study the effect of polymer combination and how the ratio of these combination will affect the release from the floating matrix tablet . formulas cl4,cl-5 contain ec in a ratio of 3:1and 1:1 respectively while formulas cl-6,cl-7, contain carbapol in a ratio of 3:1and 1:1 respectively. effect of the amount of sodium bicarbonate different amounts of sodium bicarbonate were used in formulas cl(3,8 ,9) ,formula 8 has no sodium bicarbonate in its composition ,formula 3 contain the standard amount used in the study which is 5%,while formula 9 contain half the amount present in formula 3.t he effect of increasing the concentration of sodium bicarbonate on the release were studied. effect of diluent type formulas cl(2, 10, and 11) were used to study the effect of different types of diluents on the release profile of the prepared matrix tablets. all three formulas have the same concentration of diluents but different types of diluents used .these fillers are microcrystalline cellulose ,lactose ,dibasic calcium phosphate. bilayer tablets preparation optimized formula of aspirin and clopidogrel was selected for formulation of bilayer tablet. the required weight from each layer which is equivalent to active ingredients of both aspirin and clopidogrel were individually weighed, the clopidogrel layer manually filled into the 9mm die and compressed slightly so that flat rough surface required for adhesion of the aspirin layer was created . then aspirin granules was poured into the die above the clopidogrel layer; both layers finally were subjected to the final compression; bilayer tablet of aspirin and clopidogrel was ejected from the die. evaluation of bi-layer tablet determination of friability, hardness, floating lag time and floating duration were done as per the procedures previously mentioned in the evaluation of clopidogrel floating controlled release layer. content uniformity test:hplc analytical method the assay was done by high pressure liquid chromatography (hplc) method according to the following condition. column: c18 250x4.6mm, 5µm: mobile phase: was acetonitrile: 50mm potassium dihydrogen phosphate buffer: methanol (50:30:20v/v, ph3). flowrate: 1.5ml/min, detection: uv, 240nm. drug release study the in vitro release study of the prepared bilayer tablet were conducted in usp dissolution apparatus (paddle)in 900 ml 0.1 n hcl (ph1.2)solution as dissolution media at 37˚c,samples were analayzed by hplc method. kinetic study the effect of temperature on the degradation rate of aspirin and clopidogrel in the optimized floating bilayer tablet was studied at three different temperatures: 40˚c, 50˚c, 60˚c for 16 weeks .samples were taken at different time intervals and analyzed for aspirin and clopidogrel content by hplc method. results and discussion evaluation of compressed aspirin immediate release layer the disintegration times of the compressed aspirin immediate release layers shown in table (3); all the prepared formulas show fast disintegration time but formula asp3 is the fastest one and it is selected as first layer for preparation of bilayer tablets. table 3: the disintegration time of the compressed aspirin immediate release layer disintegration time (min) formulas 12 asp1 7 asp2 4 asp3 all the prepared formulas of aspirin immediate release layer show rapid and complete release of aspirin within short period of time ;formula asp3 showing rapid disintegration and complete release of aspirin within 30 minutes therefore it is selected for preparation of bilayer tablets ,the results of dissolution test are shown in figure (1). iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 44 figure1: the effect of different disintegrants on the dissolution profile of aspirin layer in 0.1n hcl and 37˚c . evaluation of compressed clopidogrel floating sustained release layer the results of friability, hardness , content uniformity , floating lag time and floating duration are summarized in table(4). all the prepared formulas showing acceptable results regarding to friability, content uniformity, while hardness was kept constant. formula (1)shows bad floating properties and disintegrate rapidly leading to fast release of tablet in addition to that the tablet loss its integrity ;the evolved co2 causes rapid disintegration of the tablet ;also the concentration of polymer used in this formula was not enough to form proper matrix that can swell upon hydration and form coherent gel capable of entrapping liberated co2 (9). with the increase in the polymer concentration in formulas 2and 3, the viscosity of the gel layer around the tablet increases, thereby limiting the release of active ingredient. the higher concentration of polymer also helps to retain the generated carbon dioxide for a longer period thereby conferring good floating properties on the formulations (10). this lead to prolongation of floating lag time and total floating duration. table 4: evaluation parameters of clopidogrel floating sustained release layer floating duration(hrs) floating lag time(seconds) content uniformity% hardness(kg) friability(%) formulas <1 1 97.5 5 0.56 cl-1 20 5 92.2 5 0.39 cl-2 >24 30 96.8 5 0.27 cl-3 >24 42 94.7 5 0.36 cl-4 >24 60 99 5 0.3 cl-5 14 200 99.3 5 0.28 cl-6 12 240 92.9 5 0.46 cl-7 12 480 99.4 5 0.44 cl-8 20 160 98.1 5 0.42 cl-9 >24 20 97.7 5 0.42 cl-10 >24 45 94 5 0.39 cl-11 as the ethylcellulose was added in formula (4and5) floating lag time and floating duration was increase by increasing concentration of ethylcellulose; ethylcellulose has a well known release retardant effect due to its hydrophobic nature so it can retard the diffusion of dissolution medium to the matrix and this will delay the reaction between the dissolution medium and sodium bicarbonate ; generation of co2 will affected and hence floating time will be prolonged (11) .incorporation of carbapol in formulas (6and7) lead to prolongation of floating lag time and decrease in total floating duration .carbapol has a well known negative effect on floating behavior of the delivery system. this can be explained by the fact that carbapol has a much higher moisture absorption compared to hpmc. this results in a dramatic increase in the density of the floating system which, in turn, iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 45 shows a corresponding decrease in the floating capacity of floating system (12). formula cl-8 was prepared without addition of sodium bicarbonate while formula cl9 contains half the standard amount used in all formulas ,floating lag time was decreased with increasing the amount of sodium bicarbonate and the total floating duration was prolonged (13). formulacl(10, 11) contains lactose and dicalcium phosphate respectively .lactose is a well known water soluble filler while although dicalcium phosphate is hydrophobic in nature, but soluble in acidic solution (9) this property facilitates the penetration of medium to matrix lead to floatation of the tablet in a short period. variables affecting release profile from the floating matrix tablets effect of polymer concentration formulas cl1,2and 3 were prepared to show the effect of different amount of hpmc used on the release profile; formula cl1 failed to control the release of clopidogrel because the concentration of polymer used which is 20%w/w was insufficient to maintain the matrix integrity so rapid disintegration of the tablet was shown (9) by increasing the amount of polymer used to 40% and 60% w/w in formulas 2and 3 respectively it was found that there is a decrease in the amount released with increasing the amount of polymer ;in general the greatest percentage of polymer corresponds to a lower porosity of the matrix, which achieves slower drug release rates (14). the results are shown in figure 2. figure2: effect of hpmck4m concentration on the release of clopidogrel in 0.1 n hcl and 37˚c. effect of polymer combination and ratio formulas (4, 5) and (6, 7) were designed to study the effect of incorporation of ethylcellulose and carbapol respectively on the release profile of clopidogrel. addition of ethylcellulose to formulas 4and 5 lead to decrease in the release of clopidogrel from the matrix tablet in comparsion with formula 3 which contain no ethylcellulose; the retardation in the release from formulas containing ethylcellulose is related to hydrophobic nature of ethyl cellulose which restrict the penetration of medium inside the matrix and also restrict the formation of gel layer around the matrix as compared to the hydrophilic hpmc (15). the results are shown in figure 3. figure 3: the effect of ec concentration on the of clopidogrel in 0.1 nhcl and 37c release. incorporation of carbapol in the formulas 6and 7 lead to decrease the clopidogrel released from the matrix ;although at acidic phs carbapol forms a weak gel not capable of controlling the drug release but addition of sodium bicarbonate which elevates ph may improve their retarding effect in acidic media by making the matrices form a stronger polymer network (16) also combination of anionic polymer ( carbopols) with nonionic (hpmc) produces a synergistic increase in viscosity. the results are shown in figure 4. iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 46 figure4:the effect of carbapol concentration on the release of clopidogrel in 0.1nhcl and37˚c. effect of the amount of sodium bicarbonate formulacl8 andcl9 were designed to study the effect of decreasing the amount of sodium bicarbonate on the release of clopidogrel from compressed matrix .the results obtained indicate that there is a direct relationship between the amount of sodium bicarbonate incorporated in the formula and the amount released of clopidogrel. the rate of drug release was found to increase with increasing weight ratio of sodium bicarbonate. this is the direct result of the porous nature of the sodium bicarbonate containing tablet; the high amount of gas generating agent (sodium bicarbonate), which creates path for drug release by increasing pore size of matrix and increasing gas pressure inside the matrix (17). the results are shown in figure 5 . figure5:the effect of sodium bicarbonate amount on the release of clopidogrel in 0.1n hcl and 37˚c. effect of diluent type the effect of different types of diluents on release profile was studied using three different diluents differ in their properties each one can affect the release in different manner .microcrystallineellulose (mcc) was used as diluents in formulacl2,in formula cl-10 mcc was replaced by lactose and in formula cl-11 dicalcium phosphate(dcp) was utilized as a diluent. replacement of mcc with lactose in formulacl10 and by dcp in formula cl11lead to acceleration of release. lactose is well known water soluble filler; so incorporation of lactose leads to increases in the hydration rate and relaxation of the polymer chains, resulting in more dissolved drug diffusing out from the matrix. also when the drug solubility increases, the enhanced osmotic stress accelerates water penetration into the matrix resulting in a higher degree of polymer swelling and formation of more micro-cavities; therefore lactose, by its water-soluble and hydrophilic nature, facilitates gel formation and shortens the penetration time of the dissolution medium into the matrix (18). the results are shown in figure6. figure6: the effect of diluent type on the release of clopidogrel in 0.1n hcl and 37˚c bilayer tablets preparation formula asp3 (fast disintegration and rapid release) was chosen as optimized formula for rapidly disintegrating first layer while formula cl-5 was chosen as optimized formula for floating sustained release layer(prolonged release for 6 hours with good floating properties). 350 mg of clopidogrel floating sustained release layer was manually poured into 9mm die and compressed slightly .150 mg of immediate release aspirin layer was poured into the die above the clopidogrel layer and finally subjected iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 47 to final compression . bi layer tablet of aspirin and clopidogrel was ejected from die. evaluation of bi-layer tablet the prepared bilayer tablets were subjected to friability, hardness, floating lag time ,floating duration and content uniformity test tests and the results are shown in table (5). table 5: evaluation parameters of bilayer tablets hard -ness friability floating duration floating lag time content uniformit y% 5kg 0.4 >24 hr 80 sec aspirin98 %,clopid ogrel99.3 % dissolution study dissolution study was performed for prepared bilayer tablet ;.there is no significant difference in the release of clopidogrel from bilayer tablet in comparison with compressed clopidogrel matrix alone . rapid and complete release of aspirin was occurred with 30 min of test .the results are shown in figure 7 and figure 8. figure7:dissolution profile of selected aspirin formula from bilayer tablet in 0.1n hcl and 37˚c. figure8:dissolution profile of selected clopidogrel formula from bilayer tablet in 0.1n hcl and37˚c kinetic study the stability of of aspirin and clopidogrel bilayer tablets were studied at three different temperatures;40˚c,50˚cand 60˚c.the degradation of aspirin and clopidogrel followed first order kinetics because straight lines were obtained when logarithm of percent remaining of both drugs were plotted versus time .figure 9and10 show the degradation curves of aspirin and clopidogrel at 40˚c,50˚cand 60˚c. the slopes of these lines were determined and from them we can calculate rate constant (k) .arrhenius plot was constructed as shown in figure 11and 12 by utilizing arrhenius plot we can determine rate of degradation at lower temperature. the shelf life can be calculated at 25˚cand it was about 3 years for aspirin and was about 2.8 years for clopidogrel. figure9: first order plot for the degradation of aspirin in bilayer tablet at different temperature figure10:first order plot for the degradation of clopidogrel in bilayer tablet at different temperature. iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 48 figure11: arrhenius plot of aspirin for estimation of shelf life figure12: arrhenius plot of clopidogrel for estimation of shelf life references 1. ansel h., allen l., popovich n. modified release dosage form and drug delivery systems.8thed. lippincot, williams, and wilkins. 2005; p.264-267. 2. rohan d. deshpande, d. v. gowda. bi-layer tabletsan emerging trend: a review. international journal of pharmaceutical sciences and research 2011; vol2 (10):2534-2544 3. narang n., an updated review on: floating drug delivery system .international journal of applied pharmaceutics2011; vol 3, issue 1. 4. richard d., mary j., lippincott's 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drugs from sustained release hydrophilic matrices. journal of controlled release 154 (2011) 2–19 15. khandai m., chakraborty s., development of propranolol hydrochloride matrix tablets: an investigation on effects of combination of hydrophilic and hydrophobic matrix formers using multiple comparsion analysis. international journal of pharmaceutical sciences review and research 2010; volume 1, issue 2. iraqi j pharm sci, vol.22(1) 2013 bilayer tablets of aspirin and clopidogrel 49 16. mehrganh., mortazavis. the release behavior and kinetic evaluation of diltiazem hcl from various hydrophilic and plastic based matrices. iranian journal of pharmaceutical research (2005) 3: 137146 17. santha sheela n. b., damodharan n., formulation and evaluation ofclarithromycingastroretentive dosage form. international journal of pharmacy and pharmaceutical sciences, 2010; vol 2, issue 3 18. tukaramb., rajagopalani., the effects of lactose, microcrystalline cellulose and dicalcium phosphate on swelling and erosion of compressed hpmc matrix tablets: texture analyzer. iranian journal of pharmaceutical research (2010), 9 (4): 349-358. iraqi j pharm sci, vol.31( 2 ) 2022 determination of isoxsuprine hydrochloride in pharmaceutical forms doi: https://doi.org/10.31351/vol31iss2pp83-90 83 sensitive cloud point extraction method for the determination of isoxsuprine hydrochloride in pharmaceutical forms using spectrophotometry wasan a. al-uzri*, hind hadi*,1 and mariam jamal** * department of chemistry, college of science, university of baghdad, baghdad, iraq ** ministry of education, educational rusafa directorate ii abstract a simple and highly sensitive cloud point extraction process was suggested for preconcentration of micrograms amount of isoxsuprine hydrochloride (isx) in pure and pharmaceutical samples. after diazotization coupling of isx with diazotized sulfadimidine in alkaline medium, the azo-dye product quantitatively extracted into the triton x-114 rich phase, dissolved in ethanol and determined spectrophotometrically at 490 nm. the suggested reaction was studied with and without extraction and simple comparison between the batch and cpe methods was achieved. analytical variables including concentrations of reagent, triton x-114 and base, incubated temperature, and time were carefully studied. under the selected optimum conditions, the linearity ranges of calibration curves were 1-9 and 0.5-8 µg/ml with detection limits of 0.26 and 0.09 µg/ml of isx for batch and cpe methods respectively. a relative standard deviation (rsd %) best than 1.98 and 2.67 % with the percentage recoveries range 100.14 and 99.63 % were obtained for both methods respectively. the proposed methods were successfully used in routine analysis of isx in pharmaceutical forms with high accuracy and reproducibility. keywords: isoxsuprine hydrochloride, triton x-114, cloud point extraction, sulfadimidine, diazotization reaction. التقدير الطيفي لهيدروكلوريد االيزوكسوبرين في األشكال الصيدالنية بطريقة االستخالص بنقطة الغيمة الحساسة ** ومريم جمال 1*،وسن االزري، هند هادي قسم الكيمياء، كلية العلوم، جامعة بغداد، العراق * مديرية تربية الرصافة الثانيةوزارة التربية، ** الخالصة تم اقتراح طريقة االستخالص بنقطة الغيمة كطريقة بسيطة وعالية الحساسية للتقدير المسبق لكميات بالمايكروغرام من دواء هيدروكلوريد السلفاديميدين المؤزوت في الوسط القاعدي لتتكون مع isx بعد االزوتة واالزدواج لل ة.في النماذج النقية والصيدالني (isx ) االيزوكسوبرين ، يتم اذابتها في االيثانول و تقديرها طيفياً عند الطول x-114 صبغة االزو والتي يتم استخالصها كمياً الى الوسط الغني بعامل الشد السطحي ترايتون ه ومقارنة بسيطة تم عملها بين طريقتي الدفعة واالستخالص بنقطة تم دراسة التفاعل المقترح باستخدام االستخالص وبدون نانومتر. 490الموجي وكذلك القاعدة ودرجة الحرارة x-114 التحليلية بعناية متضمنةً تركيز الكاشف، وعامل الشد السطحي ترايتون اجريت دراسة للمتغيراتالغيمة. و 0.26مل مع حدود كشف \مايكروغرام 0.5-8و 1-9المعايرة والوقت. تحت الظروف المحددة المثلى حيث كانت المديات الخطية لمنحنيات ( اللذي rsdبطريقتي الدفعة واالستخالص بنقطة الغيمة عالتوالي. تم الحصول على االنحراف القياسي النسبي )% isxمل لل \مايكروغرام 0.09 يقتين على التوالي. استخدمت الطرق المقترحة بنجاح لكال الطر %99.63و 100.14مع نسبة استعادية بالمدى % 2.67و 1.98كان افضل من في االشكال الصيدالنية بدقة ومصداقية عاليتين. isxفي التحليل الروتيني لل ،استخالص نقطة الغيمة، سلفاديميدين، تفاعل االزوتة. x-114الكلمات المفتاحية: هيدروكلوريد االيزوكسوبرين، ترايتون introduction isx, chemically named 4-hydroxy-α-[1[(1-methyl-2-phenoxy-ethyl) amino] ethyl] benzene methanol hydrochloride (1), is a pharmaceutical compound that causes relaxation of uterine smooth muscles and dilatation of vessels wall, therefore it used in cases of premature labor (2). several methods were suggested for assay of isx in pharmaceutical and biological samples included spectrophotometry (3), cyclic voltammetry (4), high performance liquid chromatography (hplc) (5, 6), sequential injection spectrophotometry (7), and ultrahplc-tandem mass spectrometry (8, 9). spectrophotometric methods are still wieldy used for routine analysis of drugs, because of its simplicity and low cost compared with other analytical techniques. cloud point extraction (cpe) technique, is a simple extraction process based on used of micelles systems for preconcentration traces amount of different organic and inorganic compounds. 1corresponding author e-mail: hindhadi13@yahoo.com received: 22/ 9/ 2021 accepted:28 /11 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp83-90 iraqi j pharm sci, vol.31(2) 2022 determination of isoxsuprine hydrochloride in pharmaceutical forms 84 in aqueous solutions, surfactants molecules tend to form micelles and at a suitable temperature named "cloud point temperature", the solution become turbid. above this temperature, the turbid solution separates into a surfactant-rich phase and aqueous phase. cpe method has several characteristics feature over other extraction methods, such as high efficiency, sensitivity and enrichment factor, in addition of use safe aqueous medium rather than toxic organic solvents (10). in the present work, isx drug was extracted and then estimated by diazotization-coupling reaction with diazotized sulfadimidine (also a drug) as a safe and low cost reagent in alkaline medium. the azo dye product was extracted by cpe process, dissolved and measured spectrophotometrically. a simple comparison was performed between batch and cpe methods (with and without extraction). experimental equipment all the absorption spectra and absorbance measurements of the samples were performed by digital single beam spectrophotometer (shimadzu uv–vis 1260/japan) equipped with matched quartz cells (50 μl). for cpe process, a thermostatic water bath expert (england) was used for proving range of incubation temperature, in addition a centrifuge (hettich, eba 21) supplied with calibrated centrifuge tubes (50 ml) were used for separation the two phases. reagents and materials all the materials used in this work were of analytical grade. isx (purity 99.9%) was supplied from the state company for drug industries (sdi/samara-iraq). pharmaceutical tablets containing isx (duvilaneisoxsuprine® 10 mg, asia pharmaceutical-aleppo, syria) were purchased from local pharmacies and subjected to the method of analysis. • isoxsuprine solution (1000 µg/ml): stock standard solution of isx was prepared by dissolving 0.1 g of pure isx in distilled water, and completed the volume of 100 ml calibrated flask with the same solvent. • sulfadimidine sodium (0.02 m): the reagent solution was prepared by transferred 1.8 ml of standard solution of 333 mg/ml of sulfadimidine sodium to 100 ml calibrated flask and completed with distilled water. • sodium nitrite (0.02 m): a 0.3450 g of nano2 was dissolved in 250 ml calibrated flask with distilled water. • triton x-114 (10% v/v): a 10 ml of triton x114 (99.9%, fluka) was dissolved and diluted with 100 ml of distilled water. solutions of pharmaceutical tablets crush thirty tablets of pharmaceutical applications containing commercially available isx. a quantity of the tablets powder equivalent to 100 mg of isx was accurately weighed and dissolved in 100 ml distilled water. later the solution was shaked well and filtered to produce a stock solution (1000 µg/ml). more diluted solutions of medical tablets were prepared after dilution with distilled water. general procedure of batch method (without extraction) into 10 ml calibrated flasks, equimolar of 1 ml nano2 solution (0.02 m) and 1 ml of sulfadimidine sodium (0.02 m) with 1 ml of 1m hydrochloric acid solution were transferred and in ice-bath with cooling to 10 ºc. then increasing volumes ranged from 0.1-0.9 ml of 100 μg/ml of isx (covered the ranged of concentrations 1-9 μg/ml) and 1 ml of 2m of sodium hydrixide were added. the contents of flasks were shaken well and diluted with distilled water, after that left for 10 min for detection spectrophotometrically at 430 nm. general procedure of extraction method (cpe) into 10 ml calibrated flasks, equimolar (1 ml of 0.02 m) of nano2 solution and sulfadimidine sodium with 1 ml of 1m hydrochloric acid solution were transferred and in ice-bath (cooling to 10 ºc). then increasing volumes ranged from 0.05-0.8 ml of 100 μg/ml of isx (covered the ranged of concentrations 0.5-8 μg/ml), 1 ml of 2m of naoh and 1ml of triton x-114 (10% v/v) were added. the contents of flasks were mixed and diluted with distilled water and transferred to a centrifuging tube (10 ml) which equilibrated at 60̊ c for 30 min in the thermostatic bath. the tubes were then centrifuged for 10 minutes at 3000 rpm to isolate the two phases. after that the tubes were cooled (using ice bath) to assisted the separation process and the aqueous phase was decanted while the surfactant-rich phase (micelles surrounding the azo-dye) was dissolved with 1 ml ethanol and estimated spectrophotometrically at 490 nm. results and discussion absorption spectra and the mechanism of reaction in order to investigate the maximum wavelength for the coloured product, the absorption spectra of azo-dye resultant from diazotization coupling of diazotized sulfadimidine (dsd) with isx in alkaline medium was shown in figure 1. absorption spectra for dye product and the blank were documented between 300 and 700 nm and the maximum absorption band situated at 490 nm demonstrating the formation of a complex between the reagent and drug. the molar ratio of the reactants (dsd and isx) was assessed by continuous variation method (job’s method) (11) using equimolar concentrations (0.0029 m) of isx and dsd, and the results indicated the 2:1 ratio product (dsd: isx) was formed (fig. 2). the primary step in reaction mechanism involved diazotization process of iraqi j pharm sci, vol.31(2) 2022 determination of isoxsuprine hydrochloride in pharmaceutical forms 85 sulfadimidine (also drug compound) using sodium nitrite/hydrochloric. in alkaline medium isx, phenolic compound, converted to more reactive form “phenoxide” which is easy coupling with dsd and formed azo-dye product as shown in scheme 1. figure. 1. absorption spectra of 8 μg/ml of isx treated dsd with and without cpe recorded against reagent blank, and the blank. dependent on job’s method data, the conditional stability constant of the azo-dye product was estimated by the following equation (12): 𝐾𝑓 = 𝐴/𝐴𝑚 (1 − 𝐴/𝐴𝑚) 𝑛+1𝐶𝑛𝑛𝑛 where a and am are demonstrated to the maximum value of absorbance and the absorbance analogous to the intersection of the two tangents of the job’s method curve respectively (fig. 2), n: stoichiometric constant, and c is the isx concentration at the highest absorbance. the obtained value of kf was equal to 6.7×108 l2/mole2, point to the stability of the product. gibbs free energy (δg) was calculated using equation: δg = 2.303 rt logkf (r, 8.314 j/mol deg and t, 298 k). the negative value of calculated δg (-50.36 kj/mole) indicated the spontaneously of the reaction. figure. 2: job’s method of the reaction between isx and dsd scheme 1. coupling reaction between isx and dsd iraqi j pharm sci, vol.31(2) 2022 determination of isoxsuprine hydrochloride in pharmaceutical forms 86 selected optimum variables for batch and cpe methods in order to enhance the sensitivity of the azo-dye product, the variables that affect the reaction product were carefully studied for batch and cpe methods. different conditions such as the sulfadimidine, sodium hydroxide, and triton-x114 concentrations and extraction conditions like incubation temperature and time were studied by changing one variable with the time, and keeping the others constant. a 8 μg/ml of isx was used in all optimization experiments, with measuring absorbance at 430 and 490 nm against the blank for both methods respectively. study of the chemical variable influence of concentration of acid according to the previous published researches, the diazotization reaction usually carried out by using sodium nitrite and acid to produce nitrous acid which is converted the amino group of reagent to diazonium salt. different types of acids used for diazotization reaction were examined but only hydrochloric acid give a good result. the influence of different concentrations of hcl (0.050.3 m) using different volumes of 1m of acid ranged 1 to 3 ml was examined. the results indicated that 0.1 m of the acid (1 ml of 1 m in 10 ml total volume) gave the best response for both methods (figure 3). figure. 3. influence of concentration of hydrochloric acid for assay 8 µg/ml of isx (conditions for cpe: incubation time, 30 min; temperature 60 ºc; surfactant volume (10%v/v), 1 ml; sulfadimidine and nitrite, 1ml of 0.02 m) influence of concentration of sulfadimidine sulfadimidine as a sulfa drug, it is used as new and safe reagent in diazotization coupling reactions. amino group of sulfadimidine is easy to covert to daizonium salt under diazotization conditions. various concentrations of reagent (equimolar with nitrite) ranged from 1-6 mm were studied to investigate its effect on the sensitivity of the azo-dye product. the results shown in figure 4 indicated that maximum analytical signal was obtained when use 2 mm of sulfadimidine (i.e. 1 ml of 0.02 m in 10 ml final volume) for both methods. therefore, this concentration of reagent and nitrite was designated as the optimum and used in further work. figure. 4. effect of concentration of sulfadimidine (conditions for cpe: incubation time, 30 min; volume of surfactant (10%v/v), 1 ml; hcl, 0.1 m) influence of type and concentration of base solution the preliminary experiments indicated that the suggested coupling reaction must be carried out in alkaline medium. in order to activate the phenolic group of isx, the alkaline medium improves and promotes the coupling reaction by transforming isx to phenoxide species. various alkalies (nh4oh, naoh, and na2co3) were considered and the results displayed that sodium hydroxide gave the best analytical response (fig.5a). the influence of series of naoh concentrations ranged from 0.06-0.4 m on absorbance of azo-dye was examined. maximum response was obtained when used 0.1 m of naoh (i.e. 0.5 ml of 2 m in 10 ml final volume) with and without extraction (fig.5b). iraqi j pharm sci, vol.31(2) 2022 determination of isoxsuprine hydrochloride in pharmaceutical forms 87 figure. 5. (a) effect of type of base, (b) influence of the concentration of sodium hydroxide solution study and optimized cpe parameters influence of surfactant concentration and extraction temperature among different types of surfactant, triton-x114 considered to be the most reactive and efficient surfactant that used for extraction different organic and inorganic species. the surfactant amount is typically affected the efficiency of separation process. to study this effect, different volumes of 0.5-2 ml of 10% (v/v) surfactant was examined by extraction different samples of 4 µg/ml of isx. figure 6a showed that maximum sensitivity was attained with 1 ml of surfactant and was chosen for further use. more amount of surfactant would not significantly change the response; this may be due to complete separation process. to attain complete extraction and separation, optimum incubation temperature must be investigated. the temperature effect on the extraction of the azo-dye was estimated in the range 40-80 oc during the 30 min incubation time. the best analytical signal was obtained at 60 ºc (fig. 6b), so this temperature was selected as optimum. figure. 6. study the effect of (a) volume of triton x-114 (b) incubation temperature (cpe conditions: 4 µg/ml of isx; incubation time, 30 min; sulfadimidine, 2 mm; naoh, 0.1 m influence of incubation time the extraction efficiency and the equilibrium between two phases is mainly affected by the incubation time. so the incubation time need for completing separations was studied in the range of 10-40 min at 60 ºc. the results (fig. 7) showed that the absorbance of extracted azo-dye was enhanced with elevating the incubation time up to 30 min then slightly decreased, so 30 min was selected as the best time to achieve quantitative extraction. the centrifugation time was examined from 2-10 min, and completed separation was performed at 5.0 min, so it was chosen for further use. figure. 7. study of the incubation time (cpe conditions: 4 µg/ml of isx; sulfadimidine, 2 mm; naoh, 0.1 m; triton x-114, 1% (v/v); temp., 60 ºc) iraqi j pharm sci, vol.31(2) 2022 determination of isoxsuprine hydrochloride in pharmaceutical forms 88 selected optimum variables all the investigated chemical and physical conditions that may affected the extraction efficiency and sensitivity of azo-dye product are listed in table 1 for batch and cpe methods. table 1. summary of studied variables with optimum values for assay of isx using batch and cpe methods variable studied range selected value batch cpe concentration of hcl (m) 0.05-0.3 0.1 0.1 concentration of sulfadimidine (mm) 1-6 2 2 concentration of naoh (m) 0.06-0.4 0.1 0.1 volume of 10%(v/v)surfactant (ml) 0.5 –2 --1.0 incubation temperature (ºc) 40-80 --60 incubation time (min) 10-40 --30 methods validation using the previous optimum variables for batch and cpe methods summarized in table 1, which were used for estimation of isx, the calibration curves for both methods were constructed. for batch and cpe the linearity ranges were 1-9 and 0.5-8 µg/ml of isx respectively. in addition, the detection limit was estimated from lod=3sd/b (where sd is standard deviation of 10 replicate of the blank and b the slope of calibration curve) were of 0.26 and 0.09 µg/ml for suggested reaction with and without extraction respectively. the low values of lod especially for cpe method indicated the sensitivity of the methods. the small values of the analytical characteristics (standard deviation of the residual (sy/x), the intercept (sa) and the slope (sb)) also showed a small scattering of the calibration points and the accuracy of the present methods. the enrichment factor value “the ratio of the slope of the calibration curve of method with extraction to that of without extraction” was calculated to be 3.4. accuracy and repeatability the accuracy and repeatability of both suggested methods were investigated. two different concentrations of isx solutions were assay using batch and cpe methods (with and without extraction) in five replicates. the low values of error (good recoveries values) with acceptable values of relative standard deviation documented in table 3 indicated the high accuracy and repeatability for both suggested methods. table 2. analytical characteristics for suggested method with and without extraction parameter value batch method cpe method λmax (nm) 430 490 regression equation y = 0.0515x + 0.0077 y = 0.1754x + 0.0068 correlation coefficient, r 0.9982 0.9996 range of linearity (µg/ml) 1-9 0.5-8 detection limit (µg/ml) 0.26 0.09 quantification limit (µg/ml) 0.86 0.29 molar absorptivity (l/mol cm) 1.74×104 5.93× 104 sandell’s sensitivity (µg/cm2) 0.0194 0.0057 slope (ml/μg) 0.0515 0.1754 intercept 0.0077 0.0068 preconcentration factor 10 - enrichment factor 3.4 - sy/x 0.0092 0.0152 sb 0.0012 0.0020 sa 0.0067 0.0096 iraqi j pharm sci, vol.31(2) 2022 determination of isoxsuprine hydrochloride in pharmaceutical forms 89 table 3. accuracy and repeatability for batch and cpe methods method conc. of isx (µg/ml) recovery% erel% rsd% (n=5) present found batch 3 3.02 100.67 0.67 1.94 4 3.98 99.50 -0.50 1.32 cpe 2 1.98 99.00 -1.00 2.68 4 4.01 100.25 0.25 1.56 effect of interferences with the aim of assessment, the selectivity and efficiently of the suggested methods in routine assay of isx drug in commercial tablets, the interference of some excipients (lactose, magnesium stearate, polyvinylpyrolidone, and starch) which may be added to the drug compound for manufacturing requirements, were tested. twentyfold of each interference (40 µg/ml) was added individually to the 2 µg/ml of isx solution and analysed according to batch and cpe procedures. the ranged of rec.% of 98-101% referred to insignificant effect of excipients in the analysis of isx in medical forms. assay of isx in pharmaceutical samples both suggested methods were applied efficiently for estimation isx with and without extraction in commercial medical tablets (table 4). good recoveries and variation values obtained from both methods indicated the competence and applicability of these methods in routine analysis of isx in pharmaceutical forms. in addition, the achieved recoveries were compared with those estimated by applying the hplc standard method (1). two common tests were used to perform a statistical comparison between methods (t-and ftests at a confidence level of 95 percent) (13). the t and f values measured were lower than the tabulated values, suggesting that there was no major variance in accuracy and applicability between the methods used in the isx assay of its pharmaceutical tablets. table 4. estimation of isx in tablets using batch and cpe methods compared with standard method pharmaceutical form proposed methods standard method batch cpe conc.(µg/ml) rec. (%)* mean rec.(%) rsd (%)* conc.(µg/ml) rec. (%)* mean rec.(%) rsd (%)* mean rec.(%) taken found taken found duvilane (isoxsuprine hydrochloride® tablet 4 4.02 100.50 101.00 1.29 4 4.03 100.75 101.38 3.38 100.20 6 6.09 101.50 3.25 6 6.12 102.00 2.93 pure isx 100.14 99.63 99.50 t (4.303)c f (161.4)c 1.299 1.509 0.695 6.250 * average of four determinations; ** theoretical value; conc., concentration; rsd = relative standard deviation. (n1 –1) = 1,(n2 –1) = 1, (n1+ n2 – 2) = 2 conclusion the present work involves rapid, highly sensitive and green methods for estimation and extraction a micrograms amount of isx in pharmaceutical applications. a diazotization reaction based on use another drug compound (sulfadimidine) as a green reagent instead of poisonous and expensive reagents. using triton x114, the cloud point extraction method was used to extract the azo-dye resulting from the diazotization reaction. without requiring poisonous solvents or advanced techniques, a combination of cpe with spectrophotometry provided an easy and low-cost method for isx estimation. a simple comparison established between batch and cloud point extraction methods indicated the accuracy and repeatability of methods. the presented methods have been successfully implemented with acceptable accuracy for the assay of isx in pharmaceutical tablets. financial disclosure: no financial disclosure. conflict of interest: none to declare. references 1. “british pharmacopoeia on cd-rom”, version 5, 3rd ed., vol. 1,copyright by system simulation ltd, the stationery office ltd., london, 2001. 2. d.s. tatro, a–z drug facts, facts and comparisons, st. louis, 1999 3. t. kalsang, b. kanakapura, d.r. hosakere, b.v. kanakapura, talanta., 2010; 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six rats in each group. the second group involved 36 hypertensive rats, were divided into six subgroups, each of six rats. the first subgroups served as a positive control, the second, third and fourth sub-groups were received 25, 50, 100 mg/kg of p. farcta fruit extract respectively. the fifth and sixth subgroups were received 5 mg/kg spironolactone, and 25 mg/kg thiazide orally for a week. p. farcta extract produced a significant increase in urine flow, sodium excretion rate, egfr and urinary creatinine level. in addition there were significant reduction in heart rate, and serum creatinine and blood urea. conclusion: p. farcta fruit extract has mild diuretic activity in normal and hypertensive rats that resemble the potassium sparing diuretics. keywords: prosopis farcta, diuretics, thiazide, spironolactone, antihypertensive. الجرذان عند وهيدروكلوروثيازيد سبيرونوالكتون مع بالمقارنة للبول كمدر الينبوت فعالية الدم بضغط المصابين **يى دزه فاريق كاوة و 1،* رؤوف محمد ازان السليمانية بوليتكنيك جامعة* الطبية هولير جامعة ** الخالصة و البكتيرية. والعدوى والسكري والكلى الدموية واألوعية القلب أمراض مثل األمراض من العديد في تقليديا الينبوت استخدم وسبيرونوالكتون الثيازيد (بالعَقّار الينبوت لمستخرج المختلفة الجرعات فعالية ومقارنة لتقييم الدراسة هذه تصميم تم .للبول مدر نشاط له على تقسيمها تم جرذا، ٤٨ على الدراسة أجريت .الدم الضغط رتفعي المو طبيعي ضغط ذات الجرذان في البول إدرار على وتأثيرها) مصاب الغير والمجموعة الضابطة المجموعة لتمثيل ،الدم ضغط في بارتفاع امصاب جرذا ١٢ من تتألف األولى المجموعة التالي؛ النحو تشمل الثانية والمجموعة .مجموعة كل في جرذان ٦ ،الينبوت فواكه مستخرج من كلغ/ملغ ٥٠ بجرعة زودت التي الدم ضغط بارتفاع الفرعية المجموعات زودت جرذان. ٦ من منها كل وتتكون فرعية، مجموعات ٦ الى تقسيمها تم الدم، ضغط بارتفاع مصابا جرذا ٣٦ المجموعتين وزودت :التوالي على الفم طريق عن الينبوت المستخرج من كلغ/ملغ ١٠٠ ،٥٠ ،٢٥ بجرعات والرابعة والثالثة الثانية تأثير لها الينبوت فواكه مستخرج أن.الثيازيد بدواء كلغ/ملغ ٢٥ و ، سبيرونوالكتون بدواء كلغ/ملغ م ٥ والسادسة الخامسة الفرعيتين هناك كان ذلك، إلى باإلضافة .البولي الكرياتينين ومستوى الكلوي الترشيح معدل ، الصوديوم إفراز ومعدل ، البول تدفق في ملحوظ .الدم ضغط بارتفاع المصابة غير الجرذان في الدم ويوريا الدم في والكرياتينين القلب ضربات ومعدل الدم ضغط في انخفاض الجرذان في للبول مدر خفيف نشاط له الينبوت فاكهة المستخرج أن الجرذان على أجريت التي الدراسة هذه في أوضحنا لقد :الخالصة للبوتاسيوم المستبقية البول مدرات ويشبه كثيرا الدم ضغط يخفض وال الدم، ضغط بارتفاع مصابة غير .للضغط ،الخافضة سبيرونوالكتون ، الثيازيد ، البول مدرات الينبوت، فاكهة :المفتاحية الكلمات introduction nowadays, several medications are either used as a single agent or in combination to manage cardiovascular among these medications are diuretics. diuretic frequently prescribed in a clinical setting to control blood pressure and fluid overload (1). 1corresponding author e-mail: zanamuhamad@hotmail.com received:16/ 11/2019 accepted: 25/ 1 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp226-235 iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 227 generally, this type of medication is affordable, easily accessed, with minimum side effects, and can be used alone or with other antihypertensive drugs to have a blood pressure lowering, as a result avoiding resistant and complications (2). there are several plants have been reported to show significant diuretic activity (3). remarkably, there is an increase in the popularity of herbal medicine among the general population; this made it possible to increase in the number of herbal drug manufacturers (4). in traditional folk medicine, many plants have been used for diuretic purposes, such as prosopis farcta fruit (p.f.f.), matricaria chamomilla, mangifera indica, mimosa pudica, lipidium sativum, and achyranthes aspera and doradilla . in addition p. farcta particularly had been studied comprehensively in cardiovascular diseases and have been successful in lowering blood pressure (5,6) . the genre of p. farcta classified to as many as 50 species, the tree of p. farcta is spiny and grow in the hot and arid climate, which is naturally grown and has been reported to benefit for many cardiovascular diseases, cancer diabetes, skin disorder (7). this study is designed to explore the potential diuretic effect of p. farcta fruit decoction in normotensive and hypertensive rat model.to this point, as far as the scientific research engine concern in english language there is not known study on diuretic properties of p. farcta fruit extract. material and methods plant material prosopis farcta fruit was obtained from the sulaimani province in the kani panka area. the taxonomic classification of the collected plant samples was confirmed by expertise in botany and plant sciences at the faculty of agriculture in bakrajo. the fruit was washed and dried then crushed to a fine powder by the grinder, 10 g powder boiled in 200 ml of water for 15 minutes (8). after cooling the solution was passed through a filter paper, and the resulted solution was freshly used. animals all the experiments used a total of 48 adult rats (200 to 250 g), from the animal facilities of the university of sulaimani. they were acclimated under a temperature of 23 ± 2 °c, and 12 hours light/12 hours dark cycle. food and water were given. all experiments followed the experimental protocols previously approved by the ethics commission, and the animals were handled according to internationally accepted standard guidelines for animal use. the experiment was terminated when the scientific aims and objectives have been reached. during the experimental study, we ensured that pain and distress were minimized or relieved. experimental designs after two weeks of adaptation, 48 rats were allocated into two groups. the first group (a) was twelve normotensive rats; six of them in group (a1) accounted for a healthy or control group, and the other group (a2) of six was normotensive rats receiving 50mg /kg of p. farcta fruit extract (decoction) by oral gavage for one weeks; this was a normal treated group. on the other hand, the second group (b) was 36 hypertensive rats induction carried out by administration of fructose in the feeding bottle for 2 weeks (9) ; as mentioned in the hypertension induction section. the hypertensive group was divided into six subgroups; each group consisted of six rats. the first subgroup was dedicated for a positive control (b1), the second (b2), third (b3) and fourth (b4) subgroup were administered 25mg/kg, 50mg/kg, and 100 mg/kg of the decoction extract of p. farcta fruit respectively by oral gavage for one weeks. the fifth (b5) and sixth (b6) subgroup was given 5mg/kg spironolactone (spl), and 25/kg mg of hydrochlorothiazide (hct) respectively by oral gavage for 1 week; this was according to the pilot study carried out before the study ,it was found that 1 week is sufficient to show the diuretic effect of p. farcta, and other drugs. after 1 week when the administration by oral gavage of the fruit extract of p. farcta, and drug has ended, the blood collection by direct cardiac puncture (this was an accessible procedure to obtain the required amount of blood after sacrifice ) had carried out to investigate the aforementioned parameters. blood pressure measurements the rats were placed in a special tube restrainer before starting the blood pressure measurement cuff technique for 15-20 minutes for 2 weeks for adaptation purposes, in order to avoid distress as a result of wrong reading of the bp. iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 228 throughout the procedure the rats were warmed for half an hour at 28°c in a thermostatically controlled heating pad for better detection of tail artery pulse, and they were warmed for 30 min at 28°c where the tail was passed through a cuff and a tail-cuff sensor, the cuff were automatically inflated and deflated at each measurement by this bp measured (10). in addition to the bp (both systolic and diastolic pressure), heart rate of the conscious rats were measured by the same machine at the same time as the bp recording during the experiment. later the mean arterial blood pressure (map) was calculated using the following formula: map = dbp + 1/3(sbp – dbp) (11) map: mean arterial pressure dbp: diastolic blood pressure sbp: systolic blood pressure mean arterial pressure is the average pressure in arteries during one cardiac cycle, and it is considered as a better indicator to perfusion of coronary arteries, brain, and kidneys. hypertension inductions fructose was obtained from merck kgaa-germany chemical to be used in the experiment for the purpose of inducing hypertension. the rats were housed 2 per cage on 12 hours light/12 hours dark cycle and allowed to free access to standard food and drinking water. fructose was prepared in the form of a solution in a feeding bottle freshly after dissolving 10 % of the fructose in tap water then given ad libitum every day, and the bottles were changed every other day for 2 weeks. during that time of feeding the animal was checked to record a blood pressure increase and be used in the experiments. this type of hypertension induction is reversible once the rats has provided with a normal and healthy regimen (12). urine collection at the start of the experiment, each rat was placed in a specific metabolic cage for 2 weeks from time to time for adaptation purposes. after such period each rat was allocated in an individual metabolic cage attached with a clean urine collection tube. after 24 hours period, the tube was collected, and fresh urine volume, potassium concentration, urea, creatinine, egfr, were measured. glomerular filtration rate the urine collected from different groups was used to calculate egfr by the renal creatinine clearance (13, 14) using the following calculation egfr≃creatinine clearance = (urine creatinine/serum creatinine ) × urine volume sodium excretion rate calculation sodium excretion rate is the product of urine flow and sodium concentration; meaning sodium excretion rate = urine flow × urine sodium concentration (15) blood collection and sampling at the end of experimental procedures after 3 weeks ;the blood samples were collected from each allocated group of rats within the standard obtained by the ethical committee. the blood samples were collected by direct cardiac puncture and sent to the scientific laboratory to carry out urine volume, urine and serum electrolytes concentration, urea and creatinine concentration. the blood parameters were measured by the ise module of the roche, hitachi cobas system. statistical analysis all data are expressed as mean ± standard error mean (m ± sem) and the statistical analysis was carried out by using (spss version 22). data analysis was made using one-way analysis of variance (anova). the comparison among the groups done by using duncan test and student t-test. p≤0.05 considered as statistical significance. ethical considerations statistical analysis an approval was taken officially from the ethical committee of the faculty of medical science/ school of medicine at sulaimani university. results effects p. farcta fruit decoction (50mg/kg) on blood pressure of normotensive rats (n=12) there were no significant differences of heart rate and blood pressure between normal (control) rats (a1) and the rats which received 50mg/kg of the p. farcta extract. (table 1). table 1. effects p. farcta fruit decoction (50mg/kg) on blood pressure of normal rats (n=12) parameters normal/cont rol (a1) (n=6) received 50 mg of p. farcta (a2) (n=6) pvalue systolic bp (mmhg) 107±1.72 105±0.88 0.23 diastolic bp (mmhg) 80±0.93 79.67±0.67 0.828 mean bp (mmhg) 87.8±1.62 88.17±0.48 0.84 heart rate (bpm) 400±11 428±11 0.167 values are expressed as mean ± sem p-value of 0.05 or less was considered to be statistically significant iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 229 effects of p. farcta fruit decoction (50mg/kg) on urine flow, sodium excretion rate, and egfr in normotensive rats remarkably, the urine flow of the normal rats treated with 50mg decoction of p. farcta fruit was significantly higher (100% increase) than the normal rats that did not receive the plant decoction (table 2). sodium excretion rate was significantly increased (>100% increase) in normal rats received fruit decoction of p. farcta (table 2). there was (57%) increase of estimated glomerular filtration rate in the rats treated with p. farcta fruit in comparison to non-treated normal rats. table 2. effects of p. farcta fruit decoction 50mg/kg on urine flow, sodium excretion rate, and egfr in normal rats parameters normal (a1) (n=6) received 50 mg of p. farcta (a2) (n=6) pvalue urine flow (ml/kg/hr) 1.26±0.08 2.55 ±0.15 0.001 sodium excretion rate (mml/hr/kg) 0.22±0.03 0.55±0.05 0.001 egfr (ml/hr/kg) 486±85 760±35 0.014 values are expressed as mean ± sem p-value of 0.05 or less was considered to be statistically significant effects of p. farcta fruit decoction on serum electrolytes, serum urea and creatinine concentration in normal rats. serum sodium (na+) concentration of healthy rats treated with p. farcta fruit extract was decreased by 4.8%, whereas serum potassium (k+) level was significantly increased by 68% (table 3). daily administration of p. farcta fruit had no significant effect on serum creatinine, however, serum urea concentrations were decreased by 36% in rats treated with 50mg of p. farcta fruit extract (table 3). table 3. effects of p. farcta fruit decoction (50mg/kg )on serum electrolytes, serum urea and creatinine concentration in normal rats. parameters normal (a1) treated (a2) pvalu e serum sodium (meq/l) 144±0.37 139±0.49 0.01 serum potassium (meq/l) 4.46±0.21 7.52±0.19 0.01 serum urea (mg/dl) 44.93±0.95 28.17±1.01 0.01 serum creatinine (mg/dl) 0.49±0.01 0.51±0.01 0.71 values are expressed as mean ± sem p-value of 0.05 or less was considered to be statistically significant table 4. comparison between blood pressure parameters in normal and hypertension group after fructose 10 % provision for 3 weeks. parameters normal/control (a1) hypertensive rats (b1) p value systolic bp (mm.hg) 107±1.72 149±1 0.01 diastolic bp (mmhg) 80±0.93 126±3.05 0.01 mean bp (mmhg) 87.8±1.62 134±2.23 0.01 heart rate (bpm) 400±11 475±23 ns values are expressed as mean ± sem , p < 0.05 indicate significant difference ns not significant iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 230 effect of different doses of p. farcta fruit decoction, spironolactone (5mg/kg), and hydrochlorothiazide (20mg/kg) on hypertensive rats different doses of p. farcta have significantly decrease both systolic and diastolic blood pressure. however, there was no significant change in the heart rate exception seen in the 100 mg of the used plant (table 5). in addition, the reduction in the blood pressure was significant for both hydrochlorothiazide and spironolactone. however, heart rate was slightly reduced when hydrochlorothiazide was administered, whereas the reduction in heart rate were significant when spironolactone were used (table 5) blood pressure parameters in normal/control rats (a1) , and fructose induced blood pressure (b1) feeding normal rats with 10 % of the fructose for 2 weeks has lead to a rise in the blood pressure , this is shown in the table 4. table 5. effect of different doses of p. farcta fruit decoction ,spironolactone (5mg/kg), and hydrochlorothiazide (20mg/kg) on hypertensive rats: parameters hypertensive rats (b1) 25 mg/kg p.f.f.e. (b2) 50 mg/kg p.f.f.e (b3) 100 mg/kg p.f.f.e (b4) spl 5mg/kg (b5) hct 20mg/kg (b6) systolic bp (mm.hg) 149±1 a 143±1.80 b 144±1.43 b 142±1.35 b 111±0.67 d 137±2.26 c diastolic bp (mmhg) 126±3.05 a 115±2.5 b 115± 2.29 b 103±1.77 c 86±1.82 d 111±0.76 b mean bp (mmhg) 134±2.23 a 124±2.09 b 125±1.83 b 116±0.82 c 94±1.31 d 120±0.99 bc heart rate (bpm) 475±23 a 486±10 ab 452±25 ab 418±19 b 343±4 c 453±5 ab p.f.f.e. :prosopis farcta fruit extract spl:spironolacton hct: hydrochlorothiazide values are expressed as mean ± sem , different letters indicate significant differences at p < 0.05. effects of different doses of p. farcta fruit decoction, spironolactone (5mg/kg) and hydrochlrothiazide (20 mg/kg) on urine flow, sodium excretion rate, gfr and in hypertensive rats. urine flow of hypertensive rats receiving 50mg/kg decoction of p. farcta fruit was slightly and nonsignificantly increased. whereas in hypertensive rats that have received spironolactone or hydrochlorothiazide, urine flow was significantly increased (table 6). urinary sodium excretion rate of the hypertensive rats receiving p. farcta fruit decoction was non-significantly changed, while spironolactone or hydrochlorothiazide significantly reduced sodium excretion rate (table 6). glomerular filtration rate in hypertensive rats receiving prosopis farcta fruit decoction or spironolactone were significantly changed, as shown in ( table 6). however, egfr in hypertensive rats receiving hydrochlorothiazide was not significantly changed. iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 231 table 6. effets of diffèrent doses of p. farcta fruit decoction, spironolactone(5mg/kg) and hydrochlrothiazide (20 mg/kg) on urine flow, sodium excrétion rate, egfr and in hypertensive rats. p.f.f.e.:prosopis farcta fruit extract , spl:spirnolactone , hct: hydrochlorothiazide, different letters indicate significant differences at p < 0.05. values are expressed as mean ± sem, effects of different doses of p. farcta fruit extract,spironolactone (5mg/kg) and hydrochlorothiazide(20mg/kg) on serum electrolytes, serum urea and creatinine concentration in hypertensive rats serum na+ concentrations of hypertensive rats treated with p. farcta fruit decoction or spironolactone or hydrochlorothiazide were significantly lower than non-treated hypertensive rats (table 7). serum k+ concentration of the hypertensive rats received 50mg p. farcta fruit decoction was significantly increased , other doses of the fruit decoction were slightly increased serum k+ concentration, however in spironolactone and hydrochlorothiazide treated rats , serum potassium concentration was significantly changed (table 7). serum urea concentration in hypertensive rats treated with p. farcta decoction was significantly decreased. this significant decrease was also apparent for spironolactone, and hydrochlorothiazide treated groups. (table 7). serum creatinine concentration in hypertensive rats that received prosopis farcta decoction, spironolactone, and hydrochlorothiazide was significantly higher than the non-treated group. noticeably, the doubling dose of p. farcta fruit decoction to 100 mg did not lead to further reduction of serum na+ concentrations neither to further increase in serum k+ concentration in comparison to rats treated with 50 mg of p. farcta fruit decoction. parameters hypertensive rats (b1) 25 mg p.f.f.e. (b2) 50 mg p.f.f.e (b3) 100 mg p.f.f.e (b4) spl 5mg/kg (b5) hct 20mg/kg (b6) urine flow (ml/min/kg) 1.85±0.19 a 2±0.33 a 2.59±0.27 a 2.16±0.13 a 3.74±0.46 b 3.48±0.33 b sodium excretion rate (μeq/min/kg) 0.78±0.03 a 0.22±0.09 ab 0.25±0.05 ab 0.2±0.06 ab 0.33±0.77 b 0.39±0.06 b gfr (ml/hr/kg) 804±252 a 233±31 b 271±54 b 250±66 b 391±71 b 548±81 ab iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 232 table 7. effects of different doses of p. farcta fruit extract, spironolactone (5mg/kg) and hydrochlorothiazide(20mg/kg) on serum electrolytes, serum urea and creatinine concentration in hypertensive rats( n=36) parameters hypertensiv e rats (b1) 25 mg/kg p.f.f.e. (b2) 50 mg/kg p.f.f.e (b3) 100 mg/kg p.f.f.e (b4) spl 5mg/kg (b5) hct 20mg/kg (b6) serum sodium (meq/l) 143±1.12 a 140±0.37 b 136±0.56 c 139±1.41 b 135±0.73 c 139±0.67 b serum potassium k+ (meq/l) 6.76±0.25 b 7.17±0.17 b 9.98±0.34 c 8.19±0.89 b 8.93±0.94 c 3.98±0.08 a serum urea (mg/dl) 59±1.23 a 25±0.6 c 38±2.62 b 35±4.2 b 20.16±0.70 c 26±1.56 c serum creatinine (mg/dl) 0.39±0.008 a 0.43±0.01 2 b 0.47±0.02 4 b 0.56±0.06 b 0.43±0.008 b 0.42±0.01 b p.f.f.e.:prosopis farcta fruit extract spl :spirnolactone hct: hydrochlorothiazide values are expressed as mean ± sem different letters indicate significant differences at p < 0.05. discussion this is the first systematic study to investigate the effect of prosoips farcta fruit extract (p.f.f.e) in comparison to spironolactone and hydrochlorothiazide in animal model in both control (healthy) rats and hypertension rats induced by oral administration of freshly prepared fructose. interestingly, none of the rats in the experiment died as a result of oral gavage of the prosoips farcta fruit extract, spironolactone and hydrochlorothiazide in our study which support the concept of safety and nontoxic effect of the extract. in this study, urine flow of normal rats was significantly increased after receiving the extraction of p. farcta fruit. the possible mechanisms could explain this increase in urine flow; is an inhibition of sodium reabsorption (16), as the consequence urinary sodium concentration and urinary excretion rate of sodium is increased significantly. the study investigated the serum concentration of sodium and potassium. there was a reduction of serum sodium concentration by 4.8%, in healthy-control rat however there was a marked rise of serum potassium level by 68% that accounts for almost a double in value was seen for serum potassium concentration, which could cause hyperkalemia. the diuretic activity of the p.f.f.e. is not resemble to the mode of action of the thiazide group because the fruit extract increased sodium concentrations significantly that is responsible for producing both diuresis and natriuresis. eventually causing hyperkalemia, opposite to the hypokalemia result from usage of thiazide (17). furthermore; in term of onset of action ; p.f.f.e diuretic properties can not be attributed to the blockage of aldosterone secretion because the fruit decoction diuretic activity was observed after two hours, in contrast to the aldosterone antagonists in which their diuretic effect usually appears after more extended period, because aldosterone antagonists compete aldosterone for mineralocorticoid receptor, which is an intracellular receptor of the nuclear receptor family located in the kidneys, it modulates dna transcription, causing the synthesis of protein mediators as the mechanism of gene transcription, thereby inhibiting distal sodium retention and potassium secretion (18). it can be suggested that the diuretic activity of prosopis farcta fruit extract could be resemble the directly acting potassium sparing diuretic; inhibiting sodium ions reabsorption by blocking luminal sodium channels and decreasing potassium ions excretion. iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 233 this brings about a significant increase in the distal tubular concentration of sodium, reducing the high intensity of the surrounding interstitium, and less water reabsorption in the collecting duct. this result is in agreement with other studies when an aqueous extract of urtica dioica studied in rats and rabbits (19). as further study will be required to find out the exact mechanism of the ingredients present in the fruit extract of p. fracta ,there are suggestion that the fruit extract and their metabolites could work by one of these possible mechanisms; stimulating regional blood flow or initial vasodilatation, or by producing inhibition of tubular reabsorption of water and electrolytes, or by increasing renal circulation and thus the rate of glomerular filtration which finally result in diuresis (20,21). in this study hypertension was induced by administration of freshly prepared fructose. in addition there are many mechanisms explain the effect of fructose on hypertension in animal studies (22), for instance, high-fructose diets upregulation of sodium and chloride transporters, which result in a state of salt overload, hence increases blood pressure. besides, excess fructose has also been found to activate vasoconstrictors, inactivate vasodilators, and over-stimulate the sympathetic nervous system. in recent years there was research to understand the cause of such high blood pressure, for example, increased salt absorption, endothelial dysfunction, and chronic stimulation of the sympathetic nervous system (23). in our study, the urine flow of hypertensive rats receiving the extract of p. farcta fruit was slightly but not significantly increased, this indicates that p. farcta fruit decoction has mild diuretic properties in the hypertensive rats. also there was change in sodium ion concentration but not significant as far as spironolactone and hydrochlorothiazide concerns, there was a substantial increase in their blood flow. hydrochlorothiazide function by inhibition of the sodium chloride ion co-transporter in the renal distal convoluted tubule helps the absorption of sodium from the distal tubules. by decreasing sodium reabsorption, acutely result in an increase in fluid loss to urine; this eventually leads to a reduction in blood pressure (24). however, spironolactone is a competitive aldosterone antagonist around the junction of the distal convoluted tubule and collecting duct where it inhibits sodium-potassium exchange. this is responsible for the increases in sodium excretion while reducing potassium loss at the distal renal tubule. it has a gradual and prolonged action. also, spironolactone can work on receptors in the arterioles, where it antagonizes aldosteroneinduced vasoconstriction (25). glomerular filtration rate of the hypertensive rats receiving p. farcta fruit extract was significantly lowered. this was witnessed in the renal impairment, which is known as fructoseinduced metabolic syndrome (26). in the current study, there was a significant lowering in egfr when spironolactone administered; this was similar to the lowering effect produced by the fruit of the prosopis farcta. on the other hand, the egfr of the thiazide was not significantly decreased. we have noted that, hypertensive rats received p. farcta fruit decoction significantly decreased serum sodium concentration, and it was visible from the beginning of the experiment. this effect is related to inhibition of sodium reabsorption in the renal tubules. on the other hand, serum potassium concentration was significantly increased when a dose of 50 mg of p. farcta administered, which was similar to the significant increase of the spironolactone. however, other treatments were slightly but not significantly increased serum k ions concentration. serum creatinine concentrations of hypertensive rats receiving p. farcta fruit decoction were significantly increased; this is considered a routine change; this indicates that fruit extract is safe during renal disorders (27). however ,serum urea concentration of hypertensive rats receiving p. farcta fruit extract were significantly decreased ,this is due to an increase in urine volume. the serum creatinine of hypertensive rats treated with spironolactone and hydrochlorothiazide was almost similar to the fruit extract of p. farcta. the study had shown that different doses of p. farcta fruit decoction has significantly decreased both systolic and diastolic blood pressure; this is not due to diuretic activity, this could be explained by unknown mechanisms, and binding to different receptors. however, there was not a significant change in the heart rate, and this is not a significant factor for a reduction in blood pressure (28) . in the context of hypertension, the reduction in blood pressure were seen for both systolic and diastolic after administration of hydrochlorothiazide and spironolactone; however, heart rate was slightly reduced when hydrochlorothiazide was administered, whereas the reduction in heart rate was significant when spironolactone was provided. iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 234 conclusion in this animal study we demonstrated that p. farcta fruit extract has mild diuretic activity in normal rats and without significant blood pressure reduction that resemble the potassium sparing diuretics. references 1. khan yh. fluid overload and diuretics prescribing in chronic kidney disease patients. j. value in health. 2018 sep 1;21:s69. 2. mohammadi r, jain s, agboola s, palacholla r, kamarthi s, wallace bc. learning to identify patients at risk of uncontrolled hypertension using electronic health records data. amia summits on translational science proceedings. 2019;2019:533. 3. sarafidis pa, georgianos pi, lasaridis an. diuretics in clinical practice. part i: mechanisms of action, pharmacological effects and clinical indications of diuretic compounds. expert opinion on drug safety. 2010 mar 1;9(2):243-57. 4. agbodjogbe wk, aïkpe aj, ayedoun ma, assogba fm, dansou ph, gbenou jd. diuretic and natriuretic activities from ten medicinal plants used in south benin. journal of chemical and pharmaceutical research. 2015 ; 7(12) : 1145 – 52 . 5. verma s, singh sp. current and future status of herbal medicines. veterinary world. 2008 nov 1;1(11):347. 6. al-jeboory a, dizaye kf. cardiovascular effects of vitexin isolated from prosopis farcta. iraqi journal of pharmacy. 2006;6(1):14-9. 7. omidi a, ghazaghi m. prosopis farcta beans increase hdl cholesterol and decrease ldl cholesterol in ostriches (struthio camelus). tropical animal health and production. 2013 feb 1;45(2):431-4. 8. prabha ds, dahms hu, malliga p. pharmacological potentials of phenolic compounds from prosopis spp.-a. j coastal life med. 2014;2(11):918-24. 9. sánchez-lozada lg, tapia e, jiménez a, bautista p, cristóbal m, nepomuceno t, soto v, ávila-casado c, nakagawa t, johnson rj, herrera-acosta j. fructoseinduced metabolic syndrome is associated with glomerular hypertension and renal microvascular damage in rats. american journal of physiology-renal physiology. 2007 jan;292(1):f423-9. 10. dizaye k, ali rh. effects of neprilysinrenin inhibition in comparison with neprilysin-angiotensin inhibition on the neurohumoral changes in rats with heart failure. bmc pharmacology and toxicology. 2019 dec;20(1):23. 11. demers d, wachs d. physiology, mean arterial pressure. instatpearls [internet] 2019 feb 24. statpearls publishing. 12. dai s, mcneill jh. fructose-induced hypertension in rats is concentration-and duration-dependent. journal of pharmacological and toxicological methods. 1995 apr 1;33(2):101-7. 13. aziz rs, dizaye k. diuretic effect of adiantum capillus and its chemical constituents in hypertensive rats. international journal of pharmaceutical research. 2019 jul;11(3). 14. thakar s, paller ms. sodium metabolism in chronic kidney disease. in chronic renal disease 2020 jan 1 (pp. 633-641). academic press. 15. rennke hg, denker bm. renal pathophysiology: the essentials. lippincott williams & wilkins; 2019 jan 14. 16. thakar s, paller ms. sodium metabolism in chronic kidney disease. in chronic renal disease 2020 jan 1 (pp. 633-641). academic press. 17. dizaye kf, otraqchy aa.diuretic efficacy of matricaria chamomilla in normotensive and salt-induced hypertensive rats.the second international conference college of medicine;22nd 24th november 2017;erbil -iraq.h. m.u;2018 feb 18. 18. gomez‐sanchez e, gomez‐sanchez ce. the multifaceted mineralocorticoid receptor. comprehensive physiology. 2011 jan 17;4(3):965-94. 19. dizaye kf, alberzingi bo, sulaiman sr. renal and vascular studies of aqueous extract of urtica dioica in rats and rabbits. iraqi journal of veterinary sciences. 2013;27(1):25-31. 20. negri g, tabach r. saponins, tannins and flavonols found in hydroethanolic extract from periandra dulcis roots. revista brasileira de farmacognosia. 2013 nov 1;23(6):851-60. 21. gupta vk, arya v. a review on potential diuretics of indian medicinal plants. j chem pharm res. 2011;3(1):613-20. iraqi j pharm sci, vol.29(1) 2020 diuretic efficacy of prosopis farcta in hypertensive rats 235 22. klein av, kiat h. the mechanisms underlying fructose-induced hypertension: a review. journal of hypertension. 2015 may;33(5):912. 23. xu l, gaizun h, masahiro k, osamu i. a4283 high fructose-induced hypertension and renal dysfunction exaggerate in dahl salt-sensitive rats. journal of hypertension. 2018 oct 1;36:e34. 24. duarte jd, cooper-dehoff rm. mechanisms for blood pressure lowering and metabolic effects of thiazide and thiazide-like diuretics. expert review of cardiovascular therapy. 2010 jun 1;8(6):793-802. 25. mccormick ja, ellison dh. distal convoluted tubule. comprehensive physiology. 2011 jan 17;5(1):45-98. 26. bratoeva k, stoyanov gs, merdzhanova a, radanova m. manifestations of renal impairment in fructose-induced metabolic syndrome. cureus. 2017 nov;9(11). 27. gounden v, jialal i. hypoalbuminemia. instatpearls [internet] 2018 aug 25. statpearls publishing. 28. poulter nr, wedel h, dahlöf b, sever ps, beevers dg, caulfield m, kjeldsen se, kristinsson a, mcinnes gt, mehlsen j, nieminen m. role of blood pressure and other variables in the differential cardiovascular event rates noted in the anglo-scandinavian cardiac outcomes trial-blood pressure lowering arm (ascot-bpla). the lancet. 2005 sep 10;366(9489): 907-13 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 cardiovascular drug and analgesics refusal doi : https://doi.org/10.31351/vol30iss2pp71-77 71 the difference in contributing factors and costs associated with outpatient refusal to accept cardiovascular medications or analgesics during dispensing process. m v mohamed koya*, saiful nizam *,1, abd rahman hazirah*and radzuan nurul syahida* * department of pharmacy, jerantut hospital, malaysia. abstract activities during dispensing process is vital in reducing antihypertensive and analgesics waste. however, study looking into refusal to accept cardiovascular disorders (cvd) medicines or analgesics activities during dispensing process is lacking. to determine differences in factors and costs associated with refused cvd medicines or analgesics during dispensing process this study was approved by medical research and ethics committee (mrec) (registration number: nmrr-20-177-53153(iir)). participants receiving cvd medicines or analgesics during dispensing process were recruited via convenience sampling technique between february and march 2020 at the outpatient pharmacy department of jerantut hospital, malaysia. refusal to medications and its reasons were asked based on the questionnaire developed by the researchers. overall, 175 patients participated in this survey and cvd drugs contributed toward 58.9% of the refused medicines. those who refused cvd drugs and analgesics were significantly different in terms of gender, medications dosing frequency, refusal reasons namely side effects, medications use, intentionally skipping dose and skipping the dose when feeling well. no associations were found between forgetfulness and age with refusal to cvd drugs or analgesics. those who refused cvd medicines had a significantly higher total daily medicines, total daily pill burden, and total number of medicines refused per prescription compared to those who refused analgesics. cost of cvd medicines refused per prescription was significantly higher compared to analgesics, median united states dollar (usd) 2.58 (iqr, usd 3.69) versus median usd 1.47 (iqr, usd 3.69), p=0.01. refusal to cvd medicines and analgesics was associated with several medication’s and patient’s factors. however, higher cost of refused medication was observed for cvd medicines. keywords: cardiovascular disease, analgesics, dispensing, wastage introduction today, healthcare system around the globe is burdened with increasing healthcare cost due to struggling economy and the responsibility to provide accessible medical care to the populations. (1,2) despite this, occurrence of medication wastage is still at large. for instance, arabian gulf countries reported usd 150 million worth of medications were wasted annually in that region alone (3). usually, various factors could contribute toward medication waste such as patients’ poor medication adherence and change in medical regime (2). other possible factors include oversupply of medication due to lack of interactions between pharmacists and the patients during dispensing process.1 this can occur under certain scenarios such as patients are continued to be dispensed with pro re nata (prn) drugs such as analgesics by the pharmacists even though these patients are not using them (4). in order to reduce drug wastage, a more proactive steps should be taken by the pharmacists at various stages of medication supply. for instance, pharmacists can play a role during prescribing process by giving advice and recommendation related to polypharmacy intervention4 and deprescribing of inappropriate medicines to the prescribers (5). another step to reduce medication waste involves discussion between pharmacists and their patients regarding the quantities of medication needed during dispensing process, so that patients would not keep excess medication at home (6). effective communication during the dispensing process is vital since research has shown that pharmacists strongly believe that activities undertaken during dispensing process is crucial in reducing medication waste (6). once the medications are released to the patients, the only initiatives to reduce medication waste involves collection of unused drug by the pharmacists for re-use (1). however, pharmacists believed that activities involving re-dispensing unused medications returned by patients as the least importance stage and impractical in medication waste reduction process (6). 1corresponding author e-mail: enlightened1683@hotmail.com received: 23/12/2020 accepted: 22/ 2/2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp71-77 iraqi j pharm sci, vol.30(2) 2021 cardiovascular drug and analgesics refusal 72 this is due to the fact that only around 25% of returned medications were usually eligible for redispensing (7). there are also issues involving safety, appropriateness and cost effectiveness related to this practice (1) . previous studies reported that the trends in the quantity of medication wasted or returned unused to the pharmacies varied across different countries. for example, research in taiwan2, united kingdom (7) and the united states (8) reported higher cardiovascular disease (cvd) drugs wasted compared to muscoskeletal disorder drugs. on the contrary, quantities of muscoskeletal disorder drugs wasted were higher compared to cvd drugs in austria (9), malta (10) and another part of the united states (11). generally, available studies focused mainly on amount and cost of drugs returned to or disposed by pharmacies (2,7,8,9,10) and factors associated with unused drugs such as improved health conditions, forgetfulness, medication’s side effects (2,11) .other research have also looked into the cost saving activities involving polypharmacy interventions (4) and de-prescribing medications during prescribing stage (4,5) .study has shown that 55% and 10% of deprescribed drugs in patients prone to fall were contributed by cvd drugs and tramadol analgesics respectively (12) . cvd drugs and analgesics are of particular interest to the current setting since we have observed that cvd drugs and analgesics are commonly refused or rejected by the patients during dispensing process. in addition, cvd drugs were commonly returned unused to our pharmacy. however, there is paucity in the studies that investigate the involvement of factors associated with refusal to cvd drug. . thus, this study aimed 1) to determine differences in factors associated with outpatient refusal to some dispensed cardiovascular disorders (cvd) medicines or analgesics and 2) to measure the cost difference between cvd drugs or analgesics refused by patients during dispensing process. method this cross-sectional study was conducted during dispensing process using convenience sampling method at outpatient pharmacy department of jerantut hospital, malaysia from february to march 2020. this is a public health facility and the cost for the medications are provided free of charge by the government of malaysia. this study was approved by medical research and ethics committee (mrec) (registration number: nmrr20-177-53153(iir)). this study involved patients who collected cvd medications or prescription-only analgesics at our outpatient pharmacy department. data collection involved several steps that was agreed between researchers prior to the initiation of the study. the principal and co-investigators have working experience of 12 years and one year respectively. data collection by the co-investigators are supervised by the principal investigator during the dispensing process, medications were dispensed to the patients by the researchers according to the current facility standard operating procedure. researchers also provided counselling related to the importance of medication adherence, health complications that arises due to medication nonadherence for their specific conditions, medications side effects, medication administration, storage of medication and how to examine medication expiry dates. as part of an initiative to reduce medication wastage during dispensing process (1,6), patients were also enquired if they kept excess stock of medications at home. if they did, they were advised to finish remaining medication at home before opening the recent medication supply. finally, patients were also informed regarding the study and invited to participate. after signing the consent form, participants were enquired if there were any of the previously dispensed medications that they wished to refuse. medication refusal referred to patient’s act of rejecting medications at the dispensing counter before the end of the dispensing process. researchers would also enter the participants’ details such as gender, age, name of medications refused, total daily medicine (tdm) and total daily pill burdens (tdpb) into the data collection form. the participants were also asked to choose the reasons for their medication refusal. these questions were developed by the researchers based on the factors associated with medication waste reported from the previous research. (2,11) participants were asked if 1) they experienced any medication’s side effects, 2) they felt that they did not need the medication, 3) they ever forget to take the dose prescribed and 4) they intentionally missed the dose prescribed and 5) they ever skipped the dose when they felt well. the tdm referred to count of different oral medications prescribed to patients (13). the tdpb referred to total number of pills patients had to consume on daily basis (13). for prn oral medications, its pill burden was estimated from the pharmacy information system (phis). this is an online prescription system that stores patients’ medication details and the price for the drugs used at current facility. percentage of medicines refused during dispensing process referred to the total numbers of medicines refused per total numbers of medicines prescribed on the same prescription. regularly dosed drugs referred to drugs that were prescribed to be taken regularly on daily basis by the prescribers (14) . prn dosing referred to drugs that were prescribed to be taken as required or when necessary (14) . medications refused by the participants were grouped accordingly for data analysis. cardiovascular medications refer to combinations of any type of antihypertensive (calcium channel iraqi j pharm sci, vol.30(2) 2021 cardiovascular drug and analgesics refusal 73 blockers (ccb), beta blockers (bb), angiotensin converting enzyme inhibitors (acei), angiotensin receptor blockers (arb), diuretics, alpha blockers) and anti-anginal drugs (glyceryl trinitrate (gtn), trimetazidine and isosorbide dinitrate, antiplatelet (aspirin, clopidogrel) and anti-hypercholesterolemia (simvastatin, atorvastatin, pravastatin, gemfibrozil, fenofibrate)) (5) .analgesics include prescriptiononly drugs from nonsteroidal anti-inflammatory drugs (nsaids) such as celecoxib or meloxicam and/ or opioids tramadol that are available at current facility. total cost for every prescription and price for every item refused was generated from the phis. inclusion criteria of this study were: all patients who refused the prescribed cvd medications or prescription-only analgesics (oral celecoxib or oral meloxicam and/or oral tramadol) during dispensing process, aged more than 18 years old and collected medication by themselves. exclusion criteria were patients from emergency department, newly started on cvd medicines or prescription-only analgesics treatment on the day of the data collection and did not refuse the prescribed cvd medicines or prescription-only analgesics. statistical package for social sciences (spss) version 21.0 was used for the statistical analysis. mann whitney u test and chi-square test were used for continuous and categorical variables respectively. continuous variables were expressed as median with interquartile range (iqr) where applicable. a p < 0.05 was considered statistically significant. result between february and march 2020, 175 patients agreed to participate in this survey. most of the participants were male, 56% (n=98). median age of the participants were 62.0 years old (iqr, 15.0). the medians tdm and tdpb for the participants were 6.0 (iqr,4.0) and 7.0 (iqr, 6.0) respectively. the medians for treatment cost was usd 17.69 (iqr,usd 19.41). medians number and the percentage of medicines refused per prescription during dispensing process were 1.0 (iqr, 1.0) and 20.0% (iqr, 19.1%) respectively. medians for cost of medicine refused during dispensing process per prescription was usd 2.13 (iqr,usd 3.48). cvd drugs and analgesics contributed toward 58.9% (n=103) and 41.1% (n=72) of the refused medicines respectively. the most commonly refused cvd drugs were calcium channel blockers group, 28.2% (n=29). for analgesics, 76.4% of the refused drugs were tramadol (n=55). data are shown on table 1. table 1. demographic and treatment characteristics parameter median (iqr) age (years) 62 (15.0) total daily medicines 6.0 (4.0) total daily pill burden 7.0 (6.0) treatment cost (usd) 17.71(19.41) total medicines refused 1.0 (1.0) median percentage of medicines refused (%) 20.0 (19.1) cost of medicines refused (usd) 2.13 (3.48) percentages for cost of medicines refused (%) 13.9 (19.8) type of medicines refused (n, %)* cardiovascular drugs 103 (58.9) analgesics 72 (41.1) groups of cvd drugs refused (n,%)* gtn 11 (10.7) calcium channel blockers 29 (28.2) beta blockers 21 (20.4) angiotensin converting enzyme inhibitors (acei) 5 (4.9) angiotensin ii receptor blockers 6 (5.8) diuretics 6 (5.8) antihyperlipidemia 10 (9.7) antiangina 15 (14.6) groups of analgesics refused (n,%)* tramadol 55 (76.4) nonsteroidal anti-inflammatory drugs 17 (23.6) data are expressed as median (iqr) except where indicated *data are expressed (n,%) age for the participants who refused cvd medicines was not significantly different compared to those who refused analgesics, median 69.0 years old (iqr, 19.0) versus median 63.5 years old (iqr, 14.0), p=0.136. however, refused cvd drugs were mostly contributed by male participants (67%) while refused analgesics were mostly contributed by female participants (59.7%), (x2=12.272, df= 1, p<0.05). majority of the refused cvd drugs were prescribed as regular dosing (89.3%) while refused analgesics were mostly prescribed as a prn dosing (59.7%), (x2=47.772, df= 1, p<0.05). majority of the refused cvd drugs were due to side effects (82.5%) compared to only 18.1% of the refused analgesics, (x2=71.481, df= 1, p<0.05). however, most of the refused analgesics were due to ‘does not need’ (77.8%) compared to 27.2 % of the refused cvd drugs, (x2=43.458, df= 1, p<0.05). forgetfulness to take the dose did not differ between iraqi j pharm sci, vol.30(2) 2021 cardiovascular drug and analgesics refusal 74 refused cvd drugs or analgesics, (x2=0.001, df= 1, p=0.981). majority of those prescribed with analgesics (76.4%) intentionally skipped their drug compared to those prescribed with cvd drugs (23.3%) (x2=48.228, df= 1, p<0.05). around 86.1% of patients who were prescribed analgesics were associated with skipping the dose when feeling well compared to 12.6% of those were prescribed cvd drugs (x2=93.454, df= 1, p<0.05). patients who refused cvd medicines reported a significantly higher tdm compared to those who refused analgesics, median 7.0 (iqr, 3.0) versus median 4.0 (iqr,3.0), p<0.05. patients who refused cvd medicines also reported a significantly higher tdpb compared to those who refused analgesics, median 9.0 (iqr, 6.0) versus median 5.0 (iqr,5.0), p<0.05. median treatment cost for participants who refused cvd medicines was usd 23.09 (iqr, usd18.92) versus usd 14.13 (iqr, usd14.49) for analgesics, p<0.05. total number of cvd medicines refused per prescription was significantly higher compared to the total numbers of analgesics refused per prescription, median 1.0 (iqr, 1.0) versus median 1.0 (iqr, 0), p<0.05. however, percentage of cvd medicines refused per prescription was significantly lower compared to percentage of analgesics refused per prescription, median 20.0% (iqr, 10.7%) versus median 25.0% (iqr, 30.5%), p<0.05. the cost of cvd medicines refused per prescription was significantly higher compared to analgesics, median usd 2.58 (iqr, usd 3.69) versus median usd 1.47(iqr, usd 3.69), p=0.01. data are shown on table 2. table 2. difference in factors and cost between refused cvd medications and analgesics cvd medicine analgesic x2 df p-value age (years) 60.0 (19.0) 63.5 (14.0) 0.136 gender (n,%) * male 69 (67.0) 29 (40.3) 12.272 1 <0.05 female 34 (33.0) 43 (59.7) dosing (n, %)* regular 92 (89.3) 29 (40.3) 47.772 1 <0.05 prn 11 (10.7) 43 (59.7) reasons for refusing side effects (n, %)* yes 85 (82.5) 13 (18.1) 71.481 1 <0.05 no 18 (17.5) 59 (81.9) does not use the medicine (n, %)* yes 28 (27.2) 56 (77.8) 43.458 1 <0.05 no 75 (72.8) 16 (22.2) forgetfulness to take dose (n, %)* yes 90 (87.4) 63 (87.5) 0.001 1 0.981 no 13 (12.6) 9 (12.5) intentionally missed the dose (n, %)* yes 24 (23.3) 55 (76.4) 48.228 1 <0.05 no 79 (76.7) 17 (23.6) skipped dose when feeling well (n, %)* yes 13 (12.6) 62 (86.1) 93.454 1 <0.05 no 90 (87.4) 10 (13.9) total daily medicines 7.0 (3.0) 4.0 (3.0) <0.05 total daily pill burden 9.0 (6.0) 5.0 (5.0) <0.05 treatment cost (usd) 23.09 (18.92) 14.13 (14.49) <0.05 total numbers of medicines refused 1.0 (1.0) 1.0 (0) <0.05 percentage of medicines refused 20.0 (10.7) 25.0 (30.5) <0.05 cost of medicines refused (usd) 2.58 (3.69) 1.47 (3.69 0.010 percentage for cost of medicines refused 12.9 (18.3) 15.8 (22.0) 0.690 data are expressed as median (iqr) except where indicated *data are analysed using chi-square test iraqi j pharm sci, vol.30(2) 2021 cardiovascular drug and analgesics refusal 75 discussion to our knowledge, this is the first study to compare refusal to cvd medicines and analgesics during the dispensing process. current study recorded 58.9% medication refusal involving cvd medicines and this could be related to the fact that unused medicines returned to the pharmacies were commonly prescribed by cardiology division (2) . factors associated with cvd drugs and analgesics refusal age in current study, there was no association found between refused cvd medicines or analgesics with age. this finding is parallel with that of previous study that reported medication returned from various therapeutic groups was not associated with age.7 similar to their study7, the age for the participants in our study who refused cvd drugs and analgesics were also revolved around those in their 60s. they also reported that the most commonly returned drugs were prescribed for cvd. gender in current study, refusal to cardiovascular drugs were higher in male compared to females. this could be related to the fact that hypertensive males were less adherent to their medication compared to females due to factors such as busier lifestyle and heavier work pressure (15) .besides, females were more likely to make lifestyle changes and were more committed toward their hypertension management (15 ).on the contrary, relationships between analgesics’ adherence (16) or consumptions (16,17,18 )with gender varied across different studies. for instance, studies conducted in norway (17 ) and brazil (18) found that the use of non-prescription, over the counter (otc) analgesics such as paracetamol, aspirin, ibuprofen (17,18) naproxen (18) and diclofenac (18) were significantly common in females. however, higher percentages of females refusing prescription-only analgesics in current study could be an indication of females’ unwillingness to use these categories of analgesics. medication dosing most of the prn medication prescribed in the care home (35.3%) came from analgesics groups.19 however, utilisation of prescription-only analgesics prescribed for prn use in outpatient hospital setting might be minimal. this was reflected by the fact that 59.7% of analgesics refused in current study were prescribed for prn use compared to only 10.7% of the cvd medicines. this finding was consistent with studies from australia and the united kingdom that reported, commonly returned prn medicine for disposal by the community pharmacies were analgesics and gtn (7,24) . meanwhile, regularly dosed drugs such as statin, perindopril, telmisartan/amlodipine and frusemide were in the top 20s of the commonly returned unused to the pharmacy (24) . reasons for refusal apparently, majority of those participants who refused analgesics stated that they did not need the drugs prescribed, intentionally missed the dose and skipped the dose when feeling well. current study indicates that discussion for the need of the prescribed analgesics between pharmacists and the patients has potential to minimize wastage since medications provided for occasional symptomatic relief such as acute pain or for conditions that have already resolved contributed toward 27.5% of unused medicines.2 current study also reported that, forgetfulness was common among those refusing cvd medicines or analgesics and was documented as one of the leading causes for medication being unused.11 on the contrary, the discussion between pharmacists and the patients refilling their cvd medicines have uncovered that refusal to the cvd drugs might have stemmed from the side effects of the prescribed drugs. tdm and tdpb the occurrence of side effects or patient’s unwillingness to take the prescribed drugs can be due to inappropriate polypharmacy (4) .apparently, patients who refused cvd medicines reported significantly higher total daily medicines (tdm) and total daily pill burden (tdpb) compared to those who refused the prescribed analgesics. present of higher tdm also indicates occurrence of polypharmacy, defined as taking five or more medications per day (20).polypharmacy was associated with several side effects such as deterioration of renal function (23) and orthostatic hypotension (22) .the high tdm of 7 among those prescribed with medication for cvd compared to only four in those prescribed with analgesics also posed as risk factors for medication wastage. this is because half of patients with tdm of only even four per day did not comply to their treatment regimen (23). besides, high tdpb were also commonly associated with poor medication adherence (13). this in turn results in excessive supply of medication at home and greater chances of medication wastage among those prescribed with chronic medication(2,9,11). numbers and cost of medications refused polypharmacy intervention led by pharmacists has resulted in cost saving mostly via reduction of analgesics prescribed for prn basis compared to their counterpart prn cvd drugs (14) .in contrast, enquiry of the needs for the dispensed drugs by the pharmacists during the dispensing process indicated that the drug’s cost refused per prescription for cvd drugs was almost doubled of that refused analgesics. possible explanation for this observation is that, our participants with cvd medicines refused between one to two drugs during dispensing process compared to only one type in those prescribed with analgesics. nonetheless, the iraqi j pharm sci, vol.30(2) 2021 cardiovascular drug and analgesics refusal 76 high refusal to 20% of cvd medicines per prescription is a worrying observation. while polypharmacy intervention by pharmacists result in no reduction in the numbers of regularly dosed cvd (14), yet majority of the refused cvd drugs in current study involved regularly dosed drugs. this indicates potential activities related to medication and behavioral interventions in order to minimize factors causing medication refusal or non-adherence in those prescribed with cvd medicines. on the other hand, refusal to 25% of analgesics per prescription indicates the need to assess the necessity of the prescribed analgesics in those diagnosed with muscoskeletal disorder. this is important in order to reduce unnecessary analgesics wastage at public sector hospital where medications are provided to the patients without any charges limitations firstly, medications collected by patients’ representatives were excluded from the survey. thus, refusal behavior and the cost associated with medication refusal might be slightly underor overestimated. secondly, recall bias during refusal might occur and resulted in patients refusing the medications they actually using or not using. thirdly, medication adherence was not assessed. thus the benefits of refusal toward reducing drugs wastage could not be confirmed. fourthly, the balance of medication at home was not assessed hence the medication use behavior could not be assessed. fifthly, while refusal to pick-up medicines may cause waste of income in addition to return to stock time if it occurs in private pharmacies, yet this effect was not investigated in this studysince this study was conducted in governmental pharmacy where the medicines are subsidized. finally, the study was conducted at our pharmacy alone. hence the generalizability of the findings might be limited to our setting alone. conclusions in conclusion, discussion on the refusal to cvd medicines or analgesics during dispensing process has uncovered several issues related to medication use in our patients. refusal to these medications apparently were associated with several medications and patient’s factors. even though refusal to accept medication during dispensing process might prevent wastage, yet pharmacists have a responsibility to advise patients regarding issues related to side effects and other medicationrelated issues such as good adherence in those with cvd in order to prevent further health complications. in contrast, the needs for analgesics should be discussed for patients with muscoskeletal disorder since they might not always wish to refill it. this activity if conducted during dispensing process might have a potential to optimise medication use. conflict of interests the authors declared that they have no conflicts of interest to disclose. funding this study did not receive any funding. acknowledgements none references 1. trueman p, taylor dg, lowson k, bligh a, meszaros a, wright d, et al. evaluation of the scale, causes and costs of waste medicines. report of dh funded national project, 2010. york and london:york health economics condortium and the school of pharmacy, university of london 2. wang tc, ku, pj, lu hl, hsu kc; trezise d, hong yh. investigation and analysis of medication disposal in hospitals and community pharmacies in taiwan. sustainability 2020;12:11. 3. abou-huda, h.s. an economic assessment of the extent of medication use and wastage among families in saudi arabia and arabian gulf countries. clin. ther.2003;25:1276-1292 4. bryant e, claire k, needham r. reducing inappropriate polypharmacy in primary care through pharmacy-led intervention. the pharmaceutical journal. 2019;303:7932 5. mecca mc, thomas jm, niehoff km, hyson a, jeffery sm, sellinger j, et al. assessing an interprofessional polypharmacy and deprescribing educational intervention for primary care post-graduate trainees: a quantitative and qualitative evaluation. j gen intern med.2019;34:1220–1227. 6. bekker cl, gardarsdottir h, egberts acg, bouvy ml, van den bemt bjf. pharmacists' activities to reduce medication waste: an international survey. pharmacy (basel). 2018;6(3):94. 7. mackridge aj, marriott jf. returned medicines: waste or a wasted opportunity? j public health. 2007;29:258-62 8. schuh jl, hewuse aj. the cost of unused medications. fed pract. 2015;32:14-18. 9. vogler s, de rooij rhpf. medication wasted contents and costs of medicines ending up in household garbage. res social adm pharm. 2018;14:1140-1146. 10. vella v, west lm. analysis of disposed unused medications at a village community pharmacy. pharmacy (basel). 2019;7:45. 11. law av, sakharkar p, zargarzadeh a, tai bw, hess k, hata m, et al. taking stock of medication wastage: unused medications in us households. res social adm pharm. 2015;11:571-578. 12. 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polypharmacy among urban communitydwelling older adults in multi-ethnic malaysia. plos one. 2017;12(3):e0173466. 21. sakamoto ji, shikata t, ito s, kimura t, takamoto k, manabe e, et al. polypharmacy is associated with accelerated deterioration of renal function in cardiovascular outpatients. cardiol res.2020;11:15-21. 22. cohen a, vidal js, roca f, rananja h, hernandorena i, coude du foresto l, et al. feasibility and determinants of orthostatic hypotension self-measurement at home in an elderly community-dwelling population. am j hypertens. 2019;32:824-832. 23. mair a, wilson m, dreischulte t. addressing the challenge of polypharmacy. annu rev pharmacol toxicol. 2020;60:661-681. 24. bettington e, spinks j, kelly f, wheeler aj. returning unwanted medicines to pharmacies: prescribing to reduce waste. aust prescr. 2018;41:78-81 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 mitochondrial copies number in type 2 diabetic on metformin doi: https://doi.org/10.31351/vol31iss2pp33-3 33 mitochondrial copies number and some renal function biomarkers in type 2 diabetes mellitus on metformin samara sameer yonus*,1 and muhammad a. ahmed** * college of pharmacy, university of mosul, mosul , iraq ** colleges of pharmacy and medicine, university of nineveh, mosul, iraq abstract one of the most common metabolic illnesses in the world is diabetes mellitus. this metabolic disease is responsible for a large percentage of the burden of kidney damage and dysfunction. this study aims to explain mitochondrial copies number changes in relation to both glycemic index and renal status in t2dm treated with metformin monotherapy visiting al-wafaa diabetes research center in mosul. during the period 1st january to 30 may 2021, 47 patients with t2dm (mean age 50.48 ± 7.74 years) were participate in this case-control study. these patients' results were compare to a control group of 47 seemingly healthy people (mean age 45.89 ± 9.06 years). all participants' demographic and medical histories were acquire through the delivery of a questionnaire. blood samples collected and tested for the ubiquinone oxidoreductase chain 1 gene, hba1c, uric acid, urea, and creatinine, among other things. in diabetics, there were extremely significant increases in blood hba1c, serum urea, and creatinine (p < 0.001, 0.003, and 0.043, respectively) when compared to the control group. in diabetic group, serum uric acid levels did not change significantly. hba1c and uric acid had a strong negative correlation (r = -0.045 and p˂0.05, respectively). in diabetic individuals, the number of mitochondrial copies was substantially lower than in the control group (p < 0.001). in comparison to non-diabetic controls, diabetic patients treated with mono-metformin treatment had a lower mitochondrial copy number and moderate renal impairment. keywords: mitochondria, h-ubiquinone oxidoreductase chain 1, hba1c, creatinine, diabetes mellitus, kidney dysfunction, urea, uric acid المؤشرات الحيوية للوظيفة الكلوية عند المصابين عدد نسخ الميتوكوندريا وبعض داء السكري من النوع الثاني على الميتفورمين ** محمد عبد الغفور احمد القطان و 1*، يونس سمارة سمير ، الموصل ، العراق جامعة الموصل، كلية الصيدلة * ، الموصل ، العراق جامعة نينوى، كلية الطب ** الخالصة على كبير بشكل األيضي االضطراب هذا يوثر العالم. أنحاء جميع في شيوًعا الغذائي أمراض التمثيل أكثر أحد السكري مرض يعد مؤشر نسبة السكر في الدم والحالة الكلوية في وظائف الكلية. عدد نسخ الميتوكوندريا فيما يتعلق بكل من هذه الدراسة إلى شرح تغييرات تهدف t2dm 2021اذار 30كانون الثاني إلى 1للفترة من هم بالميتفورمين .الذين يزورون مركز الوفاء لبحوث مرض السكري في الموصلتم عالج . نتائج هؤالء t2dm سنة( تم تشخيصهم بالفعل من مرضى 7.74± 50.48مريض )بمعدل اعمار 47. تتضمن دراسة الحالة والشواهد هذه عاًما(. البيانات الديموغرافية وكذلك التاريخ 9.06± 45.89شخًصا بصحة جيدة )بمعدل اعمار 47طرة المكونة من المرضى مقارنة بمجموعة السي لجين الدم عينات وتحليل األنثروبومترية القياسات أخذ تم المشاركين. لجميع استبيان إدارة خالل من عليها الحصول تم التي mt-nd1 الطبي وجوهر اليوريك وجوهر البول والكرياتينين أظهرت النتائج وجود زيادات معنوية عالية في النسبة المئوية للسكر التراكميوحامض hba1cو على التوالي( في مرضى السكري مقارنة بمجموعة السيطرة. في حين لم يظهر حامض اليوريك ,p <0.001 0.043, 0.003) البول والكرياتينين انخفض .على التوالي( 0.05و r =-0.045وحامض البوليك ) hba1c السكري. كان هناك ارتباط سلبي معنوي بينأي تغير معنوي في مرضى .(p <0.001)السيطرة عدد نسخة الميتوكوندريا بشكل كبير في مرضى السكري مقارنة بمجموعة مع ضعف خفيف في وظائف الكلى، الطاقة عدد نسخ بيوت الخالصة كان مرضى السكري الذين تم عالجهم بالميتفورمين فقط يعانون من انخفاض في .غير المصابة بالسكري السيطرةمقارنة بمجموعة حامض اليوريك البول،التراكمي، الكرياتينين، السكري، ضعف الكلى، جوهر السكر nd1-gene ,،الطاقةبيوت الكلمات المفتاحية: introduction diabetes mellitus (dm) is a metabolic disease characterized by hyperglycemia of carbohydrates , lipids, and proteins(1) .globally, dm affects around 415 million individuals of all ages, with the number predicted to grow to 642 million by 2040. diabetes has become a pandemic in poor countries, with low and middle-income economy nations accounting for 75 percent of dm cases. furthermore, diabetes affects working age in low and middle-income countries(2). 1corresponding author e-mail: dr.smart1999@gmail.com received: 9/ 7/ 2021 accepted: 10/9 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp33-38 iraqi j pharm sci, vol.31(2) 2022 mitochondrial copies number in type 2 diabetic on metformin 34 diabetes mellitus classified into two types: type 1 (juvenile) and type 2(adult) diabetes mellitus. diabetes mellitus type 2 is the most common. t2dm is characterized by hyperglycemia, insulin resistance, and insulin insufficiency (4). diabetes is a long-term hyperglycemia that affects organs such as the heart, kidneys, eyes, nerves, and blood vessels (5) .t2dm is caused by a mix of genetic variables linked to insulin resistance and decreased insulin synthesis, as well as environmental influences such obesity, overeating, inactivity, stress, and aging (6) .metformin, a biguanide derivative, is one of the most often prescribed medications for the treatment of type 2 diabetes (t2d) and has been in use for almost sixty years (7) . while it has a minimal risk of hypoglycemia, it has a higher risk of gastrointestinal side effects and is not recommended for persons with renal failure8.because renal function declines with age, elderly metformin users should be evaluated on a regular basis.(9) metformin is contraindicated or should be taken at reduced dosages depending on renal function, according to clinical recommendations in the united kingdom, canada, and australia.(10) metformin usage has also been linked to an increased risk of lactic acidosis, but this has not been widely documented.(11) diabetic nephropathy (dn), one of the most prevalent and dangerous complications of diabetes mellitus, affects approximately one-third of diabetic patients and is associated with increased morbidity and mortality in diabetic patients. dn occurs during long periods of inactivity, which can last several years. the biggest risk factors for developing dn are high blood pressure and persistent hyperglycemia. poor treatment outcomes are the result of a difficult and insufficient knowledge of the pathogenesis of dn.(12) in addition to excreting waste products and poisons such as urea, creatinine, and uric acid, the kidneys controlled the volume of extracellular fluid, osmolality of the serum, electrolyte concentrations, and the production of hormones such as erythropoietin, 1, 25dihydroxyvitamin d, and renin. the findings of renal function tests determine how persons with kidney disease or diseases that impair renal function are treated. renal function tests are useful for detecting renal disease, assessing the kidneys' response to therapy, and determining the course of renal sickness. the national institutes of health estimate that 14% of people have chronic kidney disease (ckd).(13-16) the best indicators of progression and prognosis are serum creatinine and blood urea nitrogen levels, instituting dietary restrictions in the renal disease in type 2 dm.(17) in the degradation of purine nucleotides, uric acid is the final enzymatic product. high uric acid serum concentration is a risk factor for t2dm, according to a major epidemiological research on adult japanese males. despite the fact that hyperuricemia has been identified as a risk factor for t2dm in studies, there are conflicting views on the relationship between the two.(18) mitochondria are the power houses, due to ability to produce highenergy compound inform of atp and any change in mitochondrial function will reflect on metabolic status. t2dm patients have high blood sugar and insulin resistance and both condition associated with elevated ros formation that intern significantly affect mt-dna copies number.(19 ) this study aimed at explaining the mitochondrial copies number changes in relation to both glycemic index and renal status in t2dm on metformin monotherapy. subjects and method the al-wafaa center for diabetes management and research in mosul conducted this case-control research, between first of january to 30 may 2021. screening and management of patients were done as per american diabetes association guidelines (ada,2018).(20) subjects with hba1c 6.5% were considered as cases, and hba1c 4.75.4% were also considered as controls. study included 47 subjects (23 males and 24 females) which were diagnosed type 2 diabetes mellitus with age range between (25-65) years, on metformin therapy with dose range between (500mg/day 1000mg/day) with durations from two to six months. a control group of 47 apparently healthy participants (20 males and 27 females) with ages ranging from (30-60) years old and no illness or treatment was included. for all of the patients, demographic information such as age, height, weight, and body mass index (bmi) were recorded. body mass index (bmi) was computed according to the following equation: bmi (kg/m2) is calculated by dividing body weight (kg) by the square of body height in meters (m2).(21) documented renal or hepatic illnesses, a history of alcohol use, hepatitis b and c virus infection hbsag positive and hcv antibody positive, pregnant women, patients with autoimmune disorders were also excluded from the study. blood sample collection after an eight-hour overnight fasting, five milliliters (5ml) of venous blood were taken from patients on metformin monotherapy and healthy controls using a disposable syringe. the blood was then split into two aliquots, one for clotting in a gel tube at room temperature and the other for mt-dna extraction and hba1c testing in an edta tube. the serum was tested for urea, creatinine, and serum uric acid using a multi-chemical fully automated chemistry analyzer and the giesse diagnostics manufacturer's kit (italy). biohermes kit was used to test hba1c (china), hemoglobin a1c was determined by the boronate affinity chromatography method.22 urea, by the urease-hypochlorite method, creatinine by alkaline picrate method , and uric acid, by the uricaseiraqi j pharm sci, vol.31(2) 2022 mitochondrial copies number in type 2 diabetic on metformin 35 peroxidase method. mitochondrial genome extracted using addprep genomic dna extraction kit (china). 100ng/µl sample used for q-pcr analysis with go-taq qpcr master mix from promega (a6000).23 homo sapiens mt-nd1primers was design using ncbi program. forward sequence, (attatcgccccaaccctctc) and reverse sequence (gctcgtaggg ctccgaatag). gapdh gene used as housekeeping gene with forward sequence (cgggtctttgcagtcgtatg) and reverse sequence (ctgtttctggggacta ggg g). software for eco studies analyzes data. the mean and standard deviations are used to depict the data in this investigation. all statistical analyses were performed using excel 2010. the differences in parameters between the two groups were investigated using the student's t-test. a p-value of 0.05 or less is regarded as statistically significant. results according to this study, the number of mitochondrial copies significantly decreased in t2dm patients by 32.8 times compared to the control group (p 0.01). the mean level of hba1c was significantly higher in the diabetic subjects when compared with control group (7.76 ± 1.94 vs 5.18 ± 0.49; p=0.001). the mean level of urea and creatinine was significantly higher in the diabetic subjects when compared with the control group. (28.31 ± 8.24 vs 24.27 ± 5.81; p=0.003 and 0.94 ± 0.20 vs 0.88 ± 0.08; p=0.043, respectively). whereas no significant changes in uric acid levels (4.91 ± 0.82 vs 4.67 ± 1.34; p=0.252), there was also no significant difference in their body mass index, (p = 0.542). in our study as shown in (table 1; figures 1–5), in addition, blood hba1c and serum uric acid had a significant negative association (r = -0.045, p˂0.05). uric acid and urea have a significant positive association (r = 0.019, p˂ 0.05). uric acid and creatinine had a substantial negative association (r = -0.00048, p˂ 0.05). in our study as shown in (table 2). table 1. comparison of mean values of variables in t2dm and control groups (n=94) variables mean ± sd t2dm n=47 control n=47 p value age (years) 7.74 ± 50.48 9.06 ± 45.89 0.011 * bmi (k/m²) 33.5 ± 6.33 21.87 ± 1.82 0.542 hba1c (%) 7.76 ± 1.94 5.18 ± 0.49 0.001 * urea (mg/dl) 28.31 ± 8.24 24.27 ± 5.81 0.003 * creatinine (mg/dl) 0.94 ± 0.20 0.88 ± 0.08 0.043 * uric acid (mg/dl) 4.91 ± 0.82 4.67 ± 1.34 0.252 table 2. correlation of parameters in diabetic patients. parameters r-value p-value hba1c vs uric acid -0.054 ˂ 0.05 uric acid vs urea 0.019 ˂ 0.05 uric acid vs creatinine -0.00048 ˂ 0.05 figure 1. blood hba1c percentage levels in t2dm and control individuals, the red color bar as t2dm and blue as control. figure 2. serum urea levels in t2dm and control individuals, the red color bar as t2dm and blue as control. iraqi j pharm sci, vol.31(2) 2022 mitochondrial copies number in type 2 diabetic on metformin 36 figure 3. serum creatinine levels in t2dm and control individuals, the red color bar as t2dm and blue as control. figure 4. serum uric acid levels in t2dm and control individuals, the red color bar as t2dm and blue as control. figure 5. measurement of mitochondrial dna copies number (mtdna) in control and in t2dm group on metformin monotherapy. n = 47; *p < 0.01. the red color bar as t2dm and blue as control. discussion this study found that the number of mitochondrial copies in t2dm patients was 32.8 times lower than in the control group (p<0.01). this is consistent with the findings of (cho et al.,2017), who discovered a substantial reduction in mitochondrial dna (mtdna) copy number in individuals with type 2 diabetes (p < 0.01).(24) in this work results showed that there were highly significant increases in the mean of hba1c (p ˂ 0.001), urea (p = 0.003) and creatinine (p =0.043) in diabetic population when compared to control as shown in (table 1) hba1c (p=0.0001) was significantly higher in t2dm patients compared to controls in a research by kashinakunti, s. et al. (25) additionally, mishra et al discovered that diabetics had substantially higher levels of blood urea and serum creatinine compared to controls (p 0.001).(26) the serum uric acid levels in type 2 diabetes patients were not significantly higher than in healthy control subjects, according to this study. various other studies, such as those done by fazlani et al (27), kumari and sankar narayana(28) showed conflicting results which indicated that patients with type 2 diabetes have higher serum uric acid levels than healthy people.(27,28) the results of a previous work found that serum uric acid negatively correlation to hba1c in both male and female groups (male: r=-0.224, p=0.000; female: r=-0.245, p=0.000). in the current study, there is a significant negative correlation between hba1c and uric acid in the patient group (r = -0.045, p0.05 serum uric acid decreased by 3.868 units in the male group and 6.036 units in the female group when hba1c increased by one unit after adjusting for age, bmi, sbp, dbp, tg, and creatinine. the current study's findings contrasted with those of babikr et al (30), who reported that blood uric acid levels in diabetics were positively connected with hba1c (r=0.135, p=0.026). in healthy controls, there was a statistically insignificant positive relationship between uric acid and hba1c (r=0.037, p= 0.106). (30) as demonstrated in the graph, there was a substantial negative association between uric acid and creatinine (r = -0.00048, p<0.05) and a significant positive correlation between uric acid and urea (r = 0.019, p<0.05) in the current study (table 2). according to hu et al (31), serum uric acid levels were favorably associated with blood urea levels. serum creatinine (r = 0:23, p <0:0001; r = 0:35, p <0:0001) in male and female, respectively. 31 the major limited of this work was the small sample size. conclusion t2dm patients treated with monometformin therapy were experiencing low mitochondrial copies number with mild kidney dysfunction, compared to non-diabetic controls in relation to renal function. iraqi j pharm sci, vol.31(2) 2022 mitochondrial copies number in type 2 diabetic on metformin 37 references 1. fagninou a, tougan up, nekoua m, ruffine f, akadiri y. diabetes mellitus: classification, epidemiology, physiopathology, immunology, risk factors, prevention and nutrition. 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j. 2016;9(3):1005-1008. 31. hu y, li q, min r, deng y, xu y, gao l. the association between serum uric acid and diabetic complications in patients with type 2 diabetes mellitus by gender: a cross-sectional study. peerj. 2021;9:e10691. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 wild leek against cisplatininduced nephrotoxicity doi: https://doi.org/10.31351/vol30iss2pp177-184 177 ameliorative effects of the aqueous extract of allium porrum (wild leek) against cisplatin-induced nephrotoxicity in rabbits hs mohammed*, mhs ahmida**,1 mf madi * and a a abdel-gayoum*** department of nutrition, faculty of public health, university of benghazi, benghazi, libya. department of clinical laboratory sciences, faculty of applied medical sciences, libyan international medical university , benghazi, libya ***department of clinical laboratory sciences, faculty of applied medical sciences, university of hail, hail, saudi arabia. abstract the aim of the present study was to investigate the nephroprotective, hypolipidemic and hypoglycemic effects of allium porrum (leek) in rabbits with cisplatin nephrotoxicity. forty adult male new zealand rabbits were divided randomly into four groups (ten rabbits in each group) as follows: group i: (negative control) (c) received oral daily dose of distilled water for 15 successive days. groups ii: (leek) (l) received oral daily dose of aqueous leek extract (500mg/kg/day) for 15 successive days. group iii: (positive control) [cisplatin (cp)] received oral daily dose of distilled water for 15 successive days, and subsequently administered single dose of cisplatin (3.5mg/kg/day) by intraperitoneal injection from day 10 for five days. groups iv: (leek and cisplatin) (lcp) received oral daily dose of aqueous leek extract (500 mg/kg/day) for 15 successive days with subsequently administered single intraperitoneal dose of cisplatin (3.5 mg/kg/day) from day 10 for five days in association with aqueous leek extract. all animals were fasted overnight then sacrificed. serum urea, creatinine, glucose, lipids, renal histology, tissues glutathione, lipid peroxidation (thiobarbituric acid reactive substance), catalase and superoxide dismutase was measured. cisplatin intoxication exhibited significant (p˂0.001) elevations in serum creatinine and urea with marked renal tubular injury. whereas, treatment of aqueous leek extract prior to cisplatin intoxication significantly caused (p˂0.001) reductions of serum creatinine and urea levels with moderation of renal histology. cisplatin intoxication also showed significant (p˂0.01) reductions in renal glutathione and activities of catalase and superoxide dismutase and increased lipid peroxidation accompanied with increases in serum glucose, total cholesterol, triglycerides, low density lipoproteins cholesterol, very low density lipoproteins cholesterol and decreased high density lipoproteins cholesterol compared to controls. however, administration of aqueous leek extract prior to cisplatin intoxication showed significantly (p<0.001) elevated glutathione and activities of superoxide dismutase and catalase and significant reduction in thiobarbituric acid reactive substance, reductions in glucose, triglycerides, low density lipoproteins cholesterol, very low density lipoproteins cholesterol and increased high density lipoproteins cholesterol. cisplatin treatment impaired the kidney function of rabbits with marked renal injury. this was accompanied with increased cortical lipid peroxidation and reduced antioxidant system. cisplatin also induced dyslipidemia and hyperglycemia. all deranged parameters were reversed by co-administration of the leek extract. keywords: allium porrum, cisplatin, dyslipidemia, nephrotoxicity, wild leek بعقار المستحثة الكلوية السمية ضد البري الكراث لنبات المائي للمستخلص الوقائية التأثيرات االرانب في السيسبالتين الخالصه ونقص الكلوية السمية ضد البري الكراث لنبات المائي للمستخلصالوقائية التأثيراتالهدف من الدراسة هو التحقق من . تم استخدام أربعون من ذكور األرانب النيوزيالندية االرانب في السيسبالتين بعقار المستحث شحميات الدم ونقص السكر في الدم البيضاء وتم تصنيفها الى اربع مجموعات تحتوي كل منها على عشر ارانب على النحو التالي: المجموعة األولى: )المراقبة السلبية( من واحدة يومية جرعة تلقت: الثانية المجموعةمتتاليا. يوًما 15 لمدة المقطر الماءتلقت جرعة يومية واحدة عن طريق الفم من . المجموعةمتتاليا يوًما 15 لمدة االنبوبية التغذية بواسطة الفم طريق عن( يوم/ كغم/ ملغم 500) البري للكراث المائي المستخلص 1corresponding author e-mail: mmadi772007@yahoo.com received: 12/1/2021 accepted:24 /5 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp177-184 iraqi j pharm sci, vol.30(2) 2021 wild leek against cisplatininduced nephrotoxicity 178 مغلم 3.5)وكذلك تلقت عقار السيسبالتين متتاليا يوًما 15 لمدة المقطر الماءااليجابية(: تلقت جرعة يومية واحدة عن طريق الفم من .)المراقبة: الثالثة متتالية بالتزامن مع الماء المقطر. المجموعة الرابعة: تلقت جرعة أيام خمسة لمدةو العاشر اليوم نعن طريق الحقن داخل الصفاق م( / يوم مغك / وكذلك تلقت متتاليا يوًما 15 لمدةعن طريق الفم بواسطة التغذية االنبوبية ( يوم/ مغك/ ملغم 500) البري لكراثل المائي مستخلصيومية واحدة من ال المائي مستخلصمتتالية بالتزامن مع ال أيام خمسة لمدةو العاشر اليوم ناخل الصفاق معن طريق الحقن د( / يوم مغك/ ملغم 3.5)عقار السيسبالتين .النسيجية الفحوصات لغرض الكلى واخذت الكيموحيوية التحاليل إجراء لغرض منها الدم وسحب الحيوانات قتلت التجربة نهاية في .البري لكراثل في تراكيز وانخفاضوالجلوكوز والدهون في الدم الكرياتنينو اليوريا مستوى في معنوي ارتفاع حدوث عليها الحصول تم التي النتائج أظهرت المجموعة في االنسجة في superoxide dismutase الوcatalase ال ات وكذلك انخفاض نشاط انزيماتبيروكسيدال مستوى إرتفاعالغلوتاثيون السيسبالتين بعقار يوم( وحقنت/ كغم/ ملغم 500) بتركيز البري للكراث المائي بالمستخلص المعالجة ةالمجموع أما فقط. السيسبالتين عقارب المحقونة الغلوتاثيونيات مستو تحسنو الدم في والدهون والجلوكوز الكرياتينينو اليوريا مستوى في معنوي انخفاض لوحظ فقد (يوم/ كغم/ ملغم 3.5) بجرعة مقارنة بالمجموعة االنسجة في superoxide dismutase والcatalase ال انزيمات نشاطالبيروكسيدات وكذلك استعادة مستوى وانخفاض و في تمثلت مرضية نسيجية تغيرات حدوث فقط السيسبالتين بعقار المحقونة للمجموعة الكلى لنسيج المجهري الفحص نتائج بينت كذلك .الضابطة المجموعات جرعت وعندما االلتهابية، الخاليا تجمع مع الكلوية الكبيبات بعض وضمور انكماش وحدوث القشرة، منطقة في شديد دموي نزيف وجود تشير الى ان النتائج. اوترتيبه شكلها حيث من الكلى أنسجة في واضح تحسن لوحظالبري للكراث المائي بالمستخلص السيسبالتين بعقار المحقونة من خالل االستفادة من اثاره االرانب في السيسبالتين بعقار المستحثة الكلوية السميةكان فعاال في تقليل البري للكراث المائي مستخلصال استخدام اإلضافية المفيدة المتعددة كمضاد لألكسدة وااللتهابات الكراث البري. ،الكلوية السمية، الدم شحميات عسر، السيسبالتين، الكلمات المفتاحية : نبات الكراث introduction tablecisplatin is an anticancer drug with a high preference; commonly used as a first line therapy against solid tumors such as the head and the neck, ovarian, and testicular carcinoma. unfortunately, the clinical use of the cisplatin is limited due to the severe renal toxicity that may accompany its therapy (1) which was shown to occur in about 36% of patients treated with the drug (2). due to its therapeutic efficiency as an antineoplastic agent, there is a pressing need for ways to prevent the renal toxicity associated with cisplatin therapy. mechanisms of cisplatin-induced nephrotoxicity are believed to involve the generation of oxygen free radicals and oxidative stress (3). this is known to induce the dna damages and produces inflammatory cytokines that proceed into renal injury (4). thus, several antioxidant agents have been tested to alleviate the cisplatin nephrotoxicity. several medicinal plants are rich in potent antioxidant agents, and many have been tested as protective agents against drug-induced nephrotoxicity (5,6). allium ampeloprasum var. porrum (also known as allium porrum family, liliaceae), and commonly named leek is a plant used as food that contains excellent amounts of polyphenols, trace minerals and phytochemicals, and the plant has hypolipidemic, hypoglycemic and antimicrobial properties(7). however, from literature survey little is known about its nephroprotective activity. therefore, the present study was planned to investigate the possible protective effects of the aqueous extract of wild leek against the cisplatin induced nephrotoxicity and dyslipidemia in the rabbit. materials and methods experimental animals forty adult male new zealand white rabbits (average body weight 1100-1300 grams), were obtained from the animal house of the faculty of medicine, university of benghazi, benghazilibya. the animals were maintained under natural lighting conditions (12 h light and 12 h dark cycle) with temperature of 22-25 °c and 50% relative humidity. the animals were divided randomly into four equal groups and housed in well-ventilated stainless steel cages with free access to a standard pelleted diet and tap-water. all procedures performed in the studies involving experimental animals were in accordance with the ethical standards of the institutional ethical committee for experimental animals (institutional approval number eaau-26/17) and in accordance with the helsinki declaration for experimental animals. the leek (allium porrum) plant was collected from its habitat in the surrounding area of sedi-khalifa; an area located 15 km east of benghazi, libya, between february, 15 and march 1st 2016. the collected plants were washed, dried at room temperature, ground under liquid nitrogen and stored in a refrigerator awaiting use within one week. weighed amounts of the powder was extracted with distilled water at the concentration of 500 mg/ml and filtered through a cloth filter. protocol of experiment the experimental groups were treated as follows: animals of the group i received distilled water orally by gastric intubation (1ml/kg) for 15 days, and were injected intraperitoneally (i.p) with normal saline from day 10 to the day 15; this group served as negative control (c). group ii (l) were given leek extract (500mg/kg/day) orally by gastric intubation for 15 consecutive days, and injected i.p with normal saline from day 10 to the day 15. animals of the group iii received distilled water orally by gastric intubation (1ml/kg) for 15 days and iraqi j pharm sci, vol.30(2) 2021 wild leek against cisplatininduced nephrotoxicity 179 dosed with single dose of cisplatin (3.5mg/kg/day) i.p from day 10 and continued for five consecutive days, and served as positive control (cp). group iv (lcp): rabbits received oral daily dose of aqueous leek extract (500mg/kg/day) for 15 successive days with subsequently administered single intraperitoneal dose of cisplatin (3.5mg/kg/day) from day 10 for five days in association with aqueous leek extract. after 24 h of the end of the experimental period (15 days) all animals were sacrificed by decapitation under light anesthesia with diethyl ether. the animals were weighed before treatments and before killing. blood samples were collected from the heart into plain tubes then centrifuged at 1000 g for 15 minutes and, the serum was used for biochemical analysis. the kidneys were excised immediately and weighed. one kidney was homogenized in ice-cold saline (10 % w/v) and used for the assay of catalase (cat) and superoxide dismutase (sod) enzymes activities, reduced glutathione (gsh) and lipid peroxidation levels. the other kidney was fixed in 10 % (v/v) formal saline for histopathological examination. biochemical analysis the concentration of serum glucose, serum creatinine, urea, total cholesterol (tc), high density lipoprotein cholesterol (hdl-c) and triglyceride (tg) were measured by cobas integra 400 using commercial kits (roche diagnostics, in, usa) according to the manufacturer’s instructions. the very low density lipoprotein (vldl) and low density lipoproteins (ldl) were estimated based on the friedewald equation (ldl-c (mg/dl) = tc (mg/dl) − hdl-c (mg/dl) − tg (mg/dl)/5) (8). the protein concentration was determined using commercial kit from (thermo fisher scientific,uk) (9). the activities of cat and sod enzymes were assayed as described by goth (10) and spitz and oberley (11), respectively. the renal cortical gsh was measured by the method of sedlak and lindsay (12) and the amount of lipid peroxidation was measured as thiobarbituric acid reactive substance (tbars) according to the method of walter et al.(13). histopathologic examination the fixed kidneys were embedded in paraffin wax, cut into 5 μm sections and stained with hematoxylin and eosin. five coded slides from each group were examined by a histopathologist unaware of the treatments under a light microscope. intensity of the tubular injury was assessed as follows: grade 0 (normal) no cell necrosis; grade i (mild): rare necrotic cells (less than 1% of the outer cortical tubules involved); grade ii (moderate): necrosis involved in less than half of the cortical tubules; grade iii (marked): exhibiting total necrosis in more than half of the tubules; grade iv: total or subtotal outer cortical tubular necrosis (14). statistical analyses data are expressed as mean±sem. the comparison between the means was performed by the one way analysis of variance by spss statistical software version 21. the significance of differences between the means was assessed by the tukey– kramer multiple comparison test, and p < 0.05 was considered significant. results in a pilot study the aqueous extract of allium porrum was found to be nontoxic up to the dose of 3000mg/kg showing no deaths or altered kidney functions. the cisplatin-treated rabbits were less active and three of the animals died during the course of experiment. body weight and kidney weight was assessed at the end of the study. as shown in table 1, cp treated rabbits showed a significant loss in body weight when compared with control rabbits. there was no significant change in the body weight of leek treated rabbits (l) when compared with control rabbits. the co-administration of cisplatin with leek extract showed a significant gain in body weight (p < 0.001) as compared to cp treated rabbits. moreover, substantial growth in kidney weight (p < 0.001) was observed in cp treated rabbits, as compared to control rabbits. co-administration of cisplatin with leek extract restored the kidney weight (p < 0.01) to control rabbit's kidney weight. table 1. the changes in body weights and kidney weights in different groups of rabbits. c l cp lcp change in body weight (g) + 62.80 ±16.70 + 61.00 ±15.50 -120.00 ±14.70 a ‡ a‡ -31.60 ±10.40 a ‡ a ‡ kidney. weight (g) 0.60 ± 0.03 0.63 ± 0.03 1.10 ± 0.10 a*b** 0.77 ± 0.06 a**b** c :(negative control), l(leek), cp: (positive control) and lcp (leek and cisplatin) ,n=40 (10 rabbits in each group).data expressed as means ± sem.** p<0.01 ‡ p<0.001 (a):significantly different from c (b): significantly different from l.(+): weight gained ,(-):weight lost figure 1, summarizes the changes in serum creatinine and urea levels measured as renal function parameters. the serum creatinine concentration in the cp animals showed a significant (p˂0.001) elevation by 3.21-fold compared to control, whereas, animals of lcp exhibited a significant (p˂0.01) reduction by 67.2% compared to cp group, but was still higher than that of group c by iraqi j pharm sci, vol.30(2) 2021 wild leek against cisplatininduced nephrotoxicity 180 40.52%. similarly, animals of cp group showed a significant increase (p˂0.001) in serum urea concentration by 10.99-fold compared to control, whereas, that of lcp had significant reduction (p˂0.001) in the serum urea levels by 74.83% compared to cp group. figure 1. serum creatinine and urea concentrations in different groups of rabbits. c : (negative control), cp: (positive control), l (leek) and lcp (leek and cisplatin) column and vertical bars represent means ± sem. * p<0.05, ** p< 0.01, ŧ p<0.001, a significantly different from c; b significantly different from l; c significantly different from cp. antioxidant changes cisplatin treated group exhibited significant reductions in the renal cortical gsh content and activities of cat and sod by 32.20%, 73.86% and 41.82%, respectively and a significant (p˂0.01) increase in the cortical lipid peroxidation by 89.64% compared to control. however, the lcp animals showed significant increases in the gsh content and the activities of cat and sod by 36.07%, 2.19-fold and 63.88%, respectively and a significant decrease in the tbars by 42.43% compared to cp as shown in (table 2). table 2. the cortical gsh, cat and sod enzyme activities and tbars in different groups of rabbits c l cp lcp gsh (µmol/g protein) 29.19 ± 2.018 28.84 + 1.29 19.79 ± 1.15 a** b** 26.93 ± 1.76 cŧ cat (u/mg protein) 13.20 ± 1.68 13.09 ± 1.07 3.45 ± 0.2 aŧ bŧ 10.97 ± 1.7 cŧ sod (u/mg protein) 263.70 ± 12.01 261.00 ± 17.7 153.4 + 22.7 a** b** 251.4 ± 8.83 cŧ tbars (nmol/mg protein) 2.51 ± 0.53 2.62 ± 0.76 4.76 ± 1.12 a** b** 2.74 ± 1.39 c** c : (negative control), l(leek), cp: (positive control) and lcp (leek and cisplatin) ,n=40 (10 rabbits in each group). data expressed as means ± sem.** p<0.01, ŧ p<0.001, (a): significantly different from c ,(b): significantly different from l(c): significantly different from cp. changes in fasting serum glucose and lipid profiles fasting serum glucose concentration in cp animals was significantly elevated by 2.35-fold as compared to negative control (c) animals; whereas, that of lcp group there was a significant (p˂0.001) reduction in fasting serum glucose by 50.85% compared to that of cp group, but was still higher than that of control (c) by 49.90% (table 3). on the other hand, serum levels of tc, tg, ldl-c and vldl-c in the cp group exhibited significant elevations by 1.91-fold, 1.25-fold, 3.36-fold and 2.21-fold, respectively; and a significant (p˂0.01) decrease in the hdl-c by 41.97% compared to that in (c) group of animals. whereas, in the lcp animals there were significant (p˂0.001) reductions in the serum tc by 48.8%, tg by 45.7%, ldl-c by 57.5%, vldl-c by 45.6% , and a significant increase in hdl-c by 45.6%, respectively compared to corresponding concentrations in group (cp) rabbits. however, in lcp group animals, the serum tc was still higher than that in control (c) by 48.8%, the tg by 21.1%, and ldl by 50.4%, whereas, the hdl-c and vldl-c were not significantly different (p>0.05) compared to that of control group (c) rabbits. iraqi j pharm sci, vol.30(2) 2021 wild leek against cisplatininduced nephrotoxicity 181 table 3. the fasting serum glucose and lipid profiles in different groups of rabbits lcp cp l c 7.84 ± 0.17 aŧ bŧ cŧ 17.76 ± 0.22 aŧ bŧ 5.18 ± 0.29 5.23 ± 0.28 serum glucose (mmol/l) 4.20 ± 0.06 aŧ bŧ cŧ 8.21 ± 0.15 aŧ bŧ 2.65 ± 0.11 2.82 ± 0.17 serum total cholesterol (tc) (mmol/l) 1.24 ± 0.05 a* b* cŧ 2.30 ± 0.03 aŧ bŧ 1.03 ± 0.06 1.02 ± 0.06 serum triglycerides (tg) (mmol/l) 3.09 ± 0.13 aŧ bŧ cŧ 7.28 ± 0.37 aŧ bŧ 1.61± 0.11 1.81± 0.08 serum low density lipoprotein cholesterol (ldl-c) (mmol/l) 0.86 ± 0.03 cŧ 0.47 ± 0.07 aŧ bŧ 0.84 ± 0.04 0.81± 0.02 serum high density lipoprotein cholesterol hdl-c (mmol/l) 0.25± 0.01 cŧ 0.46 ± 0.01 aŧ bŧ 0.20 ± 0.02 0.20 ± 0.01 serum very low density lipoprotein cholesterol vldl-c (mmol/l) c :(negative control), l(leek), cp: (positive control) and lcp (leek and cisplatin) , n=40 (10 rabbits in each group). data expressed as means ± sem. * p<0.05 ŧ p<0.001 (a): significantly different from c (b): significantly different from l (c): significantly different from cp histopathologic changes kidney sections of groups (c) and group (l) animals had a normal morphology (grade 0), whereas, kidney sections from group (cp) animals showed a grade iii histology with widespread degeneration of tubular architecture and tubular necrosis as shown in (figure 2). however, kidney sections from group (lcp) had moderate kidney injury with sparse tubular injury (grade i). figure 2. the histopathology of renal cortex from different groups of rabbits (a) negative control, (b) leek (500 mg/kg/ day) orally for 15 days, (c) cisplatin (3.5 mg/kg) injected i.p., (d) leek +cisplatin. observe the normal renal glomeruli and normal tubules in both (a) and (b), shrinkage and atrophy of some renal glomerular with disappearance of bowman’s space and intensive tubular necrosis in (c) and improvement in the kidney tissues was showed in (d)the slides were stained with hematoxylin and eosin. (×40 mm). iraqi j pharm sci, vol.30(2) 2021 wild leek against cisplatininduced nephrotoxicity 182 discussion cisplatin treatment is known to be associated with depletion of the renal antioxidant defense system which is implicated as the main causes of cisplatin induced nephrotoxicity (15). allium porrum is a medicinal plant rich in polyphenols, flavonoids and contains significant amounts of carotenoids and has been studied for its antioxidative and anti-inflammatory effects (7). in the present study, injection of the rabbits with cisplatin (3.5 mg/kg) for five days resulted in severe nephrotoxicity that is evidenced by the marked reduction in body weights, elevation of serum urea and creatinine concentrations and the necrotic appearance of the renal cortices figures (1 and 2); and these were in accordance with reports from several studies (1-4). reduction in body weights in animals treated with cisplatin could be due to the drug-induced gastrointestinal (gi) toxicity and decrease in food intake or due to direct renal toxicity causing reduction in renal tubular water reabsorption that can lead to dehydration and body weight loss (16). cisplatin-treated animals also exhibited significant increase in the kidney to body weight ratio compared to the controls. this finding is also consistent with reports from previous studies (1-4, 16). these changes were suggested to be due to edema of renal parenchyma caused by cisplatininduced renal tissue inflammations (17). moreover, all animals exposed to cisplatin exhibited significantly depleted cellular gsh, elevated tbars and diminished activities of cat and sod. it has been shown that cisplatin accumulates in the proximal tubular epithelial cells and is transformed into toxic metabolites (18-20). cellular gsh provides the first line of defense against oxidative damage and toxic compounds beside its role in several metabolic processes. thus, depletion of the cellular gsh is believed to be the first step in the process of drug toxicity (18-20). the oxidative stress develops as a result of an imbalance between cellular generation of free radicals and its antioxidant defense system (19). cisplatin is believed to produce oxidative stress through reduction in the activities of the antioxidant enzymes such as catalase, glutathione peroxidase and superoxide dismutase leading to failure of the antioxidant defense system against the free radical challenge (20). the overwhelming cellular oxidative stress causes per oxidation of vital macromolecules such as membrane lipids, proteins, and nucleic acids ending up in cellular damage (21). the observed decrease in sod activity following the cisplatin exposure may be attributed to the depletion of the enzyme`s essential cofactors, copper and zinc, known to be wasted during cisplatin treatment (22). interestingly however, the coadministration of the leek extract caused a significant increase in the cellular antioxidant status and alleviated lipid peroxidation. the nephroprotective property of the leek extract seems to be due to its potent antioxidant activity due to the high polyphenols and flavonoid contents (7). the polyphenols are potent reducing agents and have a strong free radical scavenging activity. this probably explains the potent ameliorative effects of leek against the cisplatin nephrotoxicity. on the other hand, supplementation of the animals with the leek extract reversed the body weight loss caused by cisplatin alone and reduced the kidney to body weight ratio back to the control levels. this could be attributed to the anti-inflammatory effects of leek (23). taken together, allium porrum could be a promising candidate for clinical use in protecting the kidneys against cisplatin toxicity in patients undergoing cisplatin chemotherapy. in the present study, animals administered cisplatin alone (group iii) developed significant fasting hyperglycemia. this was in congruence with findings of previous studies in animals (24). the cisplatininduced hyperglycemia is believed to be due to the associated impaired insulin action (25). szilvassy and coworkers (26) observed that guinea pigs treated with cisplatin developed insulin resistance with hyperinulinemia and produced a significant decrease in the insulin-stimulated cellular glucose uptake. however, co-administration of leek extract reversed the cisplatin-induced hyperglycemia back to the control levels. the hypoglycemic activity of leek is believed to be attributed to the presence of several sulfurcontaining compounds and flavonoids that are believed to reduce the rates of glycogenolysis and gluconeogenesis and delay the intestinal glucose absorption (7). on the other hand, the cisplatin treated animals exhibited marked elevations in the serum levels of total cholesterol, triglyceride, ldl, and vldl-c fractions with reduced hdl-c particles. those results of the current study were in agreement with those of previous reports (27 and 28). furthermore, results of this study indicated that about 80% of the circulating cholesterol was associated with ldl particles. this is probably the result of the ldl oxidation triggered by the increased cellular free radicals produced by cisplatin. the oxidative modification of ldl particles is believed not to be recognized by the ldl receptors causing impairment of their cellular engulfment by endocytosis (29). cisplatin is believed to reduce the expression and activity of lipoprotein lipase enzyme, which is responsible for the clearance of vldltriglyceride (26, 29); this could probably explain the elevated serum tg levels in the cisplatin treated animals (table 3). however, the co-administration of leek extract (group iv) reversed the altered levels of tc, tg, ldl and hdl by cisplatin back to the control levels (table 3). these results were consistent with those of previous study (23). the hypolipidemic effects of leek was suggested to be due to the high contents of flavonoids (30, 31) and the organic sulfur compounds known to inhibit the iraqi j pharm sci, vol.30(2) 2021 wild leek against cisplatininduced nephrotoxicity 183 hydroxy methyl glutaryl-coa (hmg-coa) reductase enzyme (hmg-coa reductase), which is the regulatory enzyme in hepatic cholesterol biosynthesis (30, 31). also, leek is rich in ferulic acid, which was shown to decrease the fatty acid synthesis by suppressing the gene expression of fatty acid synthase enzyme complex and reduce the hepatic tg accumulation (30). references 1. nasri h, baradaran-ghahfarokhi m, and rafieian-kopaie m. cisplatin-induced renal toxicity: a short review. life sci j. 2014; 11: 55-63. 2. tezcan s, izzettin fv, and sancar m, et al. nephrotoxicity evaluation in outpatients treated with cisplatin-based chemotherapy using a short hydration method. pharmacol pharm. 2013; 4: 296-302. 3. alipour f and wankhade v. ethanolic extract of ficus religiosa prevents cisplatin toxicity by enhancing antioxidant status in mice. bull env, pharmacol life sci. 2014; 3: 16-21. 4. trujillo j, molina-jijon e, and medina-campos on, et al. curcumin prevents cisplatin-induced decrease in the tight and adherens junctions: relation to oxidative stress. food funct. 2016; 7: 279–293. 5. abdel-gayoum aa, al-hassan aa, and ginawi ia, et al. the ameliorative effects of virgin olive oil and olive leaf extract on amikacin-induced nephrotoxicity in the rat. toxicol. report. 2015; 2: 1327-1333. 6. kamel km, fawzy hm, and metwally sa, et al. protective effects of onion oil and selenium against cisplatin-induced nephrotoxicity and oxidative stress in rats. egypt j hosp med. 2015; 58: 18-25. 7. mohamed sm, abdel jaleel ga, and abdallah hmi, et al. hypoglycemic, hypolipidemic and antioxidant activities of allium porrum leaves extract in streptozotocin-induced diabetic rats. int j pharm tech res. 2016; 9: 187-200. 8. friedewald, wt., robert il, and donald sf. estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. clin. chem. 1972; 18 (6): 499-502. 9. bradford mm. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. analytical biochem. 1976; 7; 72(1-2): 248-254. 10. goth l. a simple method for determination of serum catalase activity and revision of reference range. clin chim acta. 1991; 196: 143–151. 11. spitz dr and oberley lw. an assay for superoxide dismutase activity in mammalian tissue homogenates. anal biochem. 1989; 179: 8-18. 12. sedlak j and lindsay rh. estimation of total protein-bound and non-protein bound sulfhydryl groups in tissues with ellman reagent. anal biochem. 1968; 25: 192–205. 13. walter mf, jacob rf, and jeffers b, et al. serum levels of thiobarbituric acid reactive substances predict cardiovascular events in patients with stable coronary artery disease: a longitudinal analysis of the prevent study. journal of the american college of cardiology. 2004; 44(10):1996-2002. 14. tavares mb, almeida md, and martins rt, et al. acute tubular necrosis and renal failure in patients with glomerular disease. renal failure. 2012; 34(10):1252-1257. 15. ahmida mh, abdel-gayoum aa, and elfakhri mm. effect of spironolactone on cisplatin-induced nephrotoxicity in rabbits. human & experimental toxicology. 2001; 20(9):453-459. 16. palipoch s and punsawad c. biochmical and hoistological study on rat liver and kidney injury induced by cisplatin. j toxicol pathol. 2013; 26: 293-299. 17. nematbakhsh m, ashrafi f, and nasri h, et al. a model for prediction of cisplatin induced nephrotoxicity by kidney weight in experimental rats. j res med sci. 2013; 1: 370373. 18. ghanbari a, zare f, and khazaei m, et al. tribulus terrestris hydroalcoholic extract effect on cisplatin-induced apoptosis in mice kidney. int j morphol. 2016; 34: 713-718. 19. soliman am, desouky s, and marzouk m, et al. origanum majorana attenuates nephrotoxicity of cisplatin anticancer drug through ameliorating oxidative stress. nutrients. 2016; 8: 264-273. 20. taguchi t, nazneen a, and abid mr, et al. cisplatin-associated nephrotoxicity and pathological events. contrib nephrol. 2005; 148: 106–120. 21. salama am, bassiouny k, and el-sayed ih, et al. protective effect of solanum nigrum on nephrotoxicity in animals treated with antitumor drug (cisplatin). int j adv res. 2016; 4: 1013-1019. 22. el-beshbishy ha, aly ha, and fakher ha. abrogation of cisplatin-induced nephrotoxicity in mice by alpha lipoic acid through ameliorating oxidative stress and enhancing gene expression of antioxidant enzymes. eur j pharmacol. 2011; 668: 278–284. 23. nasir as. hepatoprotective and some haematological parameters effect of allium ampeloprasum against carbone tetrachloride induced liver toxicity in albino rats. kufa j vet med sci. 2012; 3: 117-126. 24. karafakıoglu ys, bozkurt mf, and hazman o, et al. efficacy of safranal to cisplatin-induced iraqi j pharm sci, vol.30(2) 2021 wild leek against cisplatininduced nephrotoxicity 184 nephrotoxicity. biochem j. 2017; 47: 1195– 1203. 25. sheena n, ajith ta, and janardhanan kk. prevention of nephrotoxicity induced by the anticancer drug cisplatin, using ganoderma lucidum, a medicinal mushroom occurring in south india. current sci. 2003; 85: 478-482. 26. szilvassy j, sziklai i, and sari r, et al. neurogenic insulin resistance in guinea-pigs with cisplatin-induced neuropathy. euro j pharmacol. 2006; 531: 217-225. 27. portilla d, li s, nagothu kk, and megyesi j, et al. metabolomic study of cisplatin-induced nephrotoxicity. kid int. 2006; 69: 2194-2204. 28. bano n, najam r, and qazi f. adverse cardiac manifestations of cisplatin a review. inter j pharm sci. rev res. 2013; 18: 80-85. 29. li s, nagothu k, ranganathan g, et al. reduced kidney lipoprotein lipase and renal tubule triglyceride accumulation in cisplatinmediated acute kidney injury. am j physiol renal physiol. 2012; 303: 437-448. 30. son mj, rico cw, and nam sh, et al. influence of oryzanol and ferulic acid on the lipid metabolism and antioxidative status in high fatfed mice. j clin biochem nutr. 2010; 46: 150– 156. 31. dey p and khaled kl. an extensive review on allium ampeloprasum a magical herb. int j sci res. 2013; 4: 371-377. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 microneedle patch characterization doi : https://doi.org/10.31351/vol30iss1pp66-75 66 microneedle array patches: characterization and in -vitro evaluation zainab a. sadeq*,1 *department of pharmaceutics, collage of pharmacy, university of baghdad, baghdad,iraq abstract patches in transdermal drug delivery system (tdds) have been used to overcome the hypodermic needles drawback, however, these patches also have absorption limitation for hydrophilic drugs and macromolecule like peptides and dna. therefore, micronized projections which have the ability for skin penetration named as microneedles were developed. microneedle array patches can penetrate the stratum corenum to form condutis in the epidermis for drug delivery, avoiding nerve fibers and blood vessels contact to minimize pain sensation and bleeding. there are types of microneedles such as solid, coated, hallow, dissolving and hydrogel microneedles with different methods of microfabrication. keyword: microneedle array, transdermal delivery, penetration enhancement techniques, microneedles application. االبرية المصفوفة المجهرية : الوصف والتقييم الخارجياللواصق 1*،زينب احمد صادق فرع الصيدالنيات ، كلية الصيدلة ،جامعة بغداد ، بغداد، العراق * الخالصة محددة للمواد اللواصق الجلدية التي تعطى من خالل الجلد تستخدم للتغلب على عقبات الحقن التقليدي ولكن هذه اللواصق لديها امتصاصية لذلك تم تطوير نتوءات مصغرة لها قابلية اختراق الجلد تدعى الصقات ابرية مصفوفه مجهرية. dnaالمحبة للماء والجزيئات الكبيرة مثل الببتيدات و تجنبه االلياف العصبية طبقات الجلد العليا لتكون قنوات في طبقه البشرة لتوصيل الدواء م قاللواصق االبرية المصفوفة المجهرية تستطيع اخترا هالم مائي مع واالوعية الدموية للتقليل من االحساس باأللم والنزف. هناك انواع عديدة من هذه اللواصق مثل الصلبة , المغلفة , المجوفة , الذائبة و طرق عديدة للتشكيل. االبر المجهرية. تطبيقات ، نة لالختراقتقنيات محس ، التوصيل عبر الجلد، الكلمات المفتاحية : مصفوفه االبر المجهرية introduction transdermal drug delivery system (tdds) is delivery of a therapeutic agent through the skin for systemic effect. when compared to oral administration it has many advantages like avoiding the 1st pass effect of the liver (1). when it is compared with hypodermic needles, it has no pain and in addition it provides the advantages of no risk of transmitting disease that produced by the reusable of needle especially in developing countries(2). transdermal drug delivery system is generally considered as a self-administering noninvasive sustained release drug delivery system. human skin consists of three layers: epidermis, dermis and the subcutaneous fatty layer. epidermis represents the outer layer of skin and it is a 100-150 µm in thickness and the stratum corenum which is the upper layer of epidermis exhibiting a formidable barrier towards transdermal delivery due to its composition that prevents foreign substances from entering and water to exit from human skin. transdermal drug delivery system is also an alternative way for solving the issues of enzymatic degradation of drugs, or poorly absorption after oral administration. however, it shows some restrictions regarding the inability of large and hydrophilic molecules to penetrate through the skin (3,4). therefore, several penetration enhancement strategies were used to facilitate drug delivery through the skin including passive and active penetration. factors affecting transdermal permeation physicochemical properties of drug (5-7) 1. the drug molecular weight must be less than 500da. 2. the drug melting point must below 200 °c 3. partition coefficient: if the drug having high partition coefficient, it is not readily leaving the skin lipid part. while, drug with low partition coefficient is not able to permeate. so that, the optimum partition coefficient is necessary for better action, log p (1-3). 4. aqueous solubility, ideally the aqueous solubility of the drug should be equal or greater than 1 mg/ml. 5. the effective daily dose of the drug should be in the range of 10-40 mg/day . 1corresponding author e-mail: albaraznchi1@gmail.com received: 8/11/2020 accepted: 13/2 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp66-75 iraqi j pharm sci, vol.30(1) 2021 microneedle patch characterization 67 biological properties of drug (8, 9) 1. must not produce irritation or allergic effect. 2. has short half-life. 3. potent with low dose. 4. the drug which is inactivated by hepatic enzyme or degraded by git fluid is good candidate for transdermal delivery. active physical penetration enhancement techniques iontophoresis to increase the topical drug permeability to the skin, the electrical current used in which the same charge of electrode as the therapeutic agent is placed on the site of application while the opposite electrode is placed on the counter side of the body. the electrode presents over the drug will repel and force the therapeutic agent into the skin (10). heat many studies indicated that the change in temperature around 5°c was sufficient to produce changing in cell membrane permeability. in addition, it produces changes in transdermal patch physicochemical properties. sweating lead to increase hydration and improving permeation of drug through the skin (11). sonophoresis this method includes using of ultrasonic energy at low frequencies rather than high frequencies so that, skin penetration of the active ingredient will increase (12). magnetophoresis it includes a magnetic field application as a driving force which leads to altering skin structure which results in an increase in drug penetration into the skin (13). radiofrequency it involves the formation of microchannel in the cell membrane by using heat followed by skin exposure to high frequency 100khz of an alternating current. the drug delivery rate depends on the deepness and a number of microchannels formed which in turn influenced by the microelectrode used throughout the process (14). electroporation(1517) the mechanism of drug penetration is by formation of transient pores by electrical pulses (pulses of very high voltage 50-100 volts ) resulting in macromolecules passage from extracellular to intracellular region. thus, drug penetration is done by two processes which are diffusion and electrophoresis. many large macromolecules were delivered by electroporation like vaccines, insulin and oligonucleotides. microportion this achieved by using sharp micron sized projection applied on the skin that improves skin penetration called microneedles (18). microneedles they consist of different shape micro projection arrays ranging from10 to1800µm in length which are connected to the base that support them. microneedles can be fabricated from different techniques such as etching, lithographic and molding using different materials such as plastic, ceramic, silicon and polymers. following microneedles application, the skin is punctured to produce transient aqueous micro-channels which facilitate the transport of drug molecules. these micro-channel having a diameter larger than macromolecules which allow the transport of hydrophilic and supermolecule substances (19,20). advantages of microneedle array patches the following are the advantages of microneedle array patches (21-22) 1. minimally invasive, painless technique because they penetrate the skin superficial layer where the nerve receptor density is low. 2. they can provide an accurate complex drug release enhancement of the biological stability of drug and the local delivery can be achieved by drug storing in micro volume that can be controlled precisely. 3. rapid healing at the site of injection. 4. because the microneedles puncture only the epidermis so that the microbial penetration is low when compared with hypodermic needle. 5. increasing patients’ compliance because the elimination of the fear from administration of needle. disadvantages of microneedles array patches the following are the disadvantages of microneedle array patches (23-24) 1. irritation of skin may occur due to sensitive or allergic skin 2. they have small and thin needle than hair diameter, so that the tips of microneedle could be broken and left under the skin. 3. less dosage accuracy when compared with hypodermic needle. 4. there is a variation of stratum corenum thickness between individual, so that the depth of particles penetrations could vary too. types of microneedles solid microneedles they made from metal, silica or polymers with sharp tips. they act by creating micro sized pores in skin which facilitate the direct travel of drug molecules within skin. this approach of drug delivery is called poke and patch (25). poke and patch approach this approach consists of two steps (26) : 1. insertion of solid microneedles into the skin, the skin pierced and microchannels are created for drug transport. iraqi j pharm sci, vol.30(1) 2021 microneedle patch characterization 68 2. the application of the patch (drug formulation) to microneedle pre-treated skin area for diffusion through the created channels. the rate of drug delivery depends on size of pores created and on concentration of drug in the formulation. coat and poke approach coat approach depends on drug diffusion that coat microneedle after its dissolution. after the microneedle insertion to the skin, the drug locally diffuses and distribute to the body circulation. the layer thickness that covering microneedle is considered the main limitation for the rate of drug delivery (27). hallow microneedles hallow microneedles consist of hallow lumen and solid microneedle which have the ability to increase loading of large molecules of drug and its drug delivery depend on pressure driven convection transport. the method by which the drug is delivered to the skin is called poke and flow, by which the drug delivery occurs after needle injection. then, it flows throughout the lumen. in this approach, the fabricated hole inside the solid microneedle center allows the molecules of drug for reaching the skin inner layer (28). dissolving microneedles water-soluble substances are used in dissolving microneedle fabrication like pvp, pva, maltose, sodium hyaluronate and dextran(29).the substances used are water-soluble, biocompatible and completely dissolved in skin, so that protect both patient and health care by no formation of any stabbing edge after their use. poke and release approach is considering the method of delivery of drug from dissolving microneedles(30). figure 1 shows the type of microneedles drug delivery (31). hydrogel forming microneedles this new developed microneedle type in which a super swelling polymer is used for microneedle creation. these polymers have the ability taking up water in large quantity in to their three dimensions’ network and swell when contact with skin interstitial fluid, resulting in channel creation between drug patch and blood vessels (32). figure 1 microneedle drug delivery: (a) ‘poke and patch’ (b) ‘coat and poke’ (c) ‘poke and release’ (d) ‘poke and flow ( 31). techniques used in microfabrication micromolding based fabrication this technique is used for lab scale work which depends on duplicate the master structure by using the mold. the wieldy using substance is poly dimethyl siloxane (pdms)which provides both master mold’s reproducibility and plasticity. in addition, the mold can be reused for another fabrication process after its cleaning(33).following that, the microneedles are produced by pouring the blend of polymer and drug manually with the use of vacuum or centrifuge to fill any microcavities, and then drying of the solution in the mold using moderate temperature (34). master structure fabrication techniques lithography this method involves two-steps; drawing lithography and soft lithography as shown in figure 2 (35) . the melted polymer distributed over a plate, following formation of elongated pillars by the movement of the plate in upward movement. the viscosity of polymer increased due to cooling till the polymer reaching the glass transition temperature, further cooling lead to polymer solidification to a strength that piercing the skin. the advantage of this iraqi j pharm sci, vol.30(1) 2021 microneedle patch characterization 69 method is low polymer wastage and rapid fabrication (36). while, soft lithography is done by polymer film coupling with micro cavity mold then passed through out a heated nip. after that, the filled mold put on a flexible plate and re-pass again throughout the heated nip (37). laser-based fabrication this method uses a monochromatic, coherent wavelength with low angle of divergence. the beam of laser can focus on small spot diameter. the amount of removed material depends on material type, intensity of length and laser light (3839) . injection molding this is widely used in thermoplastic, thermoset molding and elastomer. it is expensive and complex method. in this method, a molten polymer is injected at high temperature and pressure in to a mold. after that, it is cooled to solidify (40). figure2. manufacturing methods of microneedle array patch (35). mechanism of drug delivery from microneedles microneedle patch prepared by hundred microneedles arrangement in arrays form to supply a suitable drug quantity, thus producing a prerequisite therapy responses. after the stratum corenum punctured by microneedles, the drug directly delivered to epidermis or dermis upper layer and then absorbed to systemic circulation to produce its therapeutic activity (41-42). materials used in microneedles fabrication metallic materials due to its advantages of toughness and mechanical strength, they are widely used in transdermal drug delivery. metallic materials are used for solid microneedle, hallow and coated microneedle creation. the delivery of a drug form these materials is affected by pore density, size and shape of microneedles (43). inorganic materials many inorganic materials such as glass, silicone and ceramic are used for microneedle fabrication. inorganic materials can be used for solid microneedle, hallow and coated microneedles creation, but broken silicon and biocompatibility are the main factors affecting the inorganic microneedle application (44). polymeric materials they are used for dissolving, solid, coated and hallow microneedles creation. many polymeric materials are used for microneedle preparation such as hyaluronic acid, pvp, pva, pga (poly glycolic acid), plga (poly lactide –coglycolide acid). pvp and cmc are wieldy used in dissolving microneedle fabrication because they rapidly dissolve in skin (45). microneedle application due to the convenience associated with the use of microneedles including their invasiveness, pain-free administration and improvement of patients’ compliance in addition to their higher efficiency in drug delivery. nowadays, microneedles can be used in diagnosis, cancer therapy, administration of anti-inflammatory/ analgesic drugs, oligonucleotides delivery, vaccine therapy and cosmetics (46). diagnosis the microneedles can be inserted through the skin for biomarkers harvesting from interstitial fluid of skin’s blood vessel by capillary force. the interstitial fluid of skin is considered as biomarkers source for the diagnosis of diseases. microneedles consist of crosslinked methacrylate hyaluronic acid were used for skin interstitial fluid extraction, in which the microneedle patch provided iraqi j pharm sci, vol.30(1) 2021 microneedle patch characterization 70 the advantages of easy application, no pain and the ability of skin interstitial fluid extraction in short time. after that, the skin interstitial fluid removed by centrifugation from microneedle patch for analysis(47). for chronic health conditions monitoring, microneedle can be used. mohan et al. developed sensor array of microneedles to alcohol monitoring. alcoholism is considered the main causative factor for many diseases and traffic fatalities. therefore, it is necessary for the development of a new method to monitor the consumption of alcohol blood testing is considered an accurate method for this task, but it is painful so that, microneedle arrays can be used for continuous monitoring of alcohol from skin interstitial fluid (48). microneedles can be used indirectly in monitoring glucose level by using silicone microneedles that give a proper precision in which an appropriate penetration of needle helped for interstitial fluid extraction with minimum pain, commercial device for glucose monitoring is kumertrix® that made from silicone microneedles (49). birchall et al developed a system containing therapeutic agents on the tip and the indicator substances is beyond its on microneedles, at the moment the drug delivered, the indicator give indication for the drug delivery successfully to the site of action (50). cancer therapy according to the world cancer report from world health organization (who), cancer in the world is considered the deadliest disease. it exceeds twenty million by 2025 (51). human epidermal and breast cancer which belong to superficial cancer are considered the public health problem so that, many efforts made for superficial cancer therapy (52). microneedles can be used for various delivering of anticancer therapy, for melanoma treatment, selfdegradable microneedles were used for delivery anti-pd-1(apd1) with glucose oxide loaded ph sensitive dextran nanoparticles(53).the permeability of 5fluorouracil topical cream that used in basal cell carcinoma will be increasing up to 4.5 times when the cream used on skin pretreated with solid microneedles (54). bhatnagar et al investigate the tamoxifen and gemcitable chemotherapeutic agents through microneedles for breast cancer treatment (55). treatment subcutaneous cancer 5-aminolevulinic acid is coated to tips of sodium hyaluyonate fast dissolving microneedles , this leading to maximizes the utilization of drug and reducing of drug residue (56). delivery of anti-inflammatory and analgesic drugs pain divided into acute and chronic according to its duration. acute pain produced from tumor, infection trauma, metabolic and endocrine diseases. chronic pain may last for three to six months without biological value apparent. transdermal administration in pain relieving producing lower systemic side effects than oral administration of analgesics (57) . microneedle array patches are regarded as one of more important technologies for pain management. microneedles act by delivering peptide locally, anti-cgrp (calcitonin gene –related peptid) produces selective anti-hypersensitivity throughout peripheral cgrp receptor antagonism by microneedle dissolving in neuropathic pain area. in this study, the anti-cgrp peptide delivered directly to the painful area by dissolvable microneedles. thus, reducing both systemic exposure and its side effects (58). in addition, a microneedle patch was used for lidocaine delivery for the management of acute and chronic pain. this microneedle patch was designed with high drug loading for lidocaine delivery into the skin to relieve acute and chronic pain (59). baek et.al design microneedles tips coated with lidocaine that showed improvement in the drug delivery within 2 min so that provided pain free and faster local anesthesia(60).polymeric microneedles loaded with meloxicam was prepared by using polydimethylsilicone molds, it was seen there is improvement in transdermal flux and the in-vitro permeation will increase about 2.58 times when compared to a free drug solution (61). oligonucleotides delivery they are short rna or dna molecules, oligonucleotides delivery to intra site of action is very difficult , so that an attempt for using microneedles to deliver oligodeoxynucleotid by using solid microneedles made from titanium or stainless steel through using the poke with patch approach. it was found that more drug amount reaching to the action site as compared to the intact skin (62). vaccine therapy is a biological preparation of a weakened or a killed form of diseases causing microorganism. vaccine therapy act by the immune system stimulation and providing the protection against the micro-organism encounter. in the delivery of dna vaccine, the microneedles approach will be effective by better immunity responses than normal injection (63). there is attempt for developing microneedles patch to be use in influenza vaccine administration (64). administration of rabies and anthrax vaccine also studies by using hallow microneedles, it was found a less dose of drug is required as compared to intramuscular injection (65). cosmetics microneedles used for skin blemishes ,scares treatment and for skin appearance improvement. marketed product used for scars and wrinkles treatment is dermaroller (66).there is an attempt for deliver cosmetic ingredients such as retinyl retinote , eflornithine , ascorbic acid by using microneedles approach(67). melanin pigment iraqi j pharm sci, vol.30(1) 2021 microneedle patch characterization 71 incorporated into nanoliposome showed increasing the solubility in the lipids and it was found that the pigment amount reaching deeply near the hair structure will be more when applied by e-roller (68). evaluation of microneedle array patches microneedle patch morphology digital microscope is used to study the dimension and morphology of prepared patch including the length, width, height of patch and microneedle patch interspace. after that, it analyzed and the result compared with the master mold (69). weight variation three patches are selected and weigh individually by using electrical balance to calculate the average weight value (70). thickness uniformity this test is done by using digital venire caliper. three patches are selected randomly to measure their thickness at five points to find the average point (71). drug content uniformity the drug content can be measured by immersing the prepared microneedle patch in 100 ml phosphate buffer ph 7.4 for 30 min. after that, the resulted solution is filtered and analyzed for drug content (72). folding test folding test can be defined as the number of folds that obtained with no patch breaking, when the folding repeated at same place. this test is done in triplicate, to measure the mean value (73). ph measurement the patch is immersed in a glass container containing 10 ml of deionized water. the ph is recorded by placing the ph electrode in contact with patch surface for one minute for equilibrium (74) percentage of moisture loss (pml) three patches are weighed individually and placed in a desiccator containing anhydrous calcium chloride for three days at room temperature. after that, the patch is reweighed again to calculate the difference between the weights. finally, the percentage of moisture loss is calculated according to the following equation: (75). pml = [(𝐖0−𝐖t)/w0]× 100 w0= initial weight, wt = final weight percentage of moisture absorbed (pma) three patches are weighed individually and placed in a desiccator containing saturated solution of potassium sulphate for three days at room temperature. after that, the patch is reweighed again to calculate the difference between the weights, finally the percentage of moisture absorbed is calculated according to the following equation (76). percent moisture absorbed = [(𝐖t−𝐖0)/w0 ]×100 w0= initial weight, wt = final weight measurement of axial needle fracture force the axial needle fracture force can be defined as the minimum force that applied parallel to microneedle axis which causes microneedles breakdown. this test measures using texture analyzer ta. xt apparatus. in this test, a microneedle patch is placed on affixed cylinder platform and the instrument programed to compress axially the microneedle patch. the maximum force that immediately apply before patch deflection is considered the axial needle fracture force and the sudden force drop recorded as a needle failure (77). in-vitro skin permeation study in vitro skin permeation study was done by using a franz cell diffusion apparatus with an effective diffusion area and an appropriate receptor volume. animal skin is placed between the donor and receptor in which the subcutaneous layer placed in the face of donor cell. the medium of receptor was stirred to produce regular drug distribution throughout the experiment, certain sample volume withdrawal from receptor fluid and replaced by equal volume at certain time interval. finally, the profile of cumulative permeation for microneedle treated and untreated skin was compared (78). histological study the microneedle patch efficiency for stratum corenum penetration is studied by using rat skin through applying microneedle patch on the skin for 1minby using gentle thumb pressure, after that the skin immersed in 10% formalin solution and left for one day. then the tissue imbedded with paraffin and by using of microtome, a thin section is cut and fixed on the slide and stain by haematoxylin and eosin pigment (79). in-vitro release study by using franz cell diffusion, a microneedle patch is mounted between the donor and receptor compartment of diffusion cell. the receptor filled with certain volume of phosphate buffer ph 7.4 and the temperature adjusted at 32°c, the liquid of receptor is stirred by magnetic stirrer. sample withdrawals at certain time interval and replaced with same volume of phosphate buffer ph7.4. after that, analyzed at certain wave length after a certain dilution, the test done in triplicates then take the mean value (80). conclusion drugs administration by microneedles array patch permit the dug molecules to cross the stratum corenum of skin layer. microneedles array patch improve patient compliance, selfadministration, providing rapid onset of action and increasing drug permeation. also, there are some limitations of their uses like allergy to sensitive skin, skin irritation, redness and breaking of microneedles tips and if it remains within the skin may cause inflammation but these limitations are rare and can iraqi j pharm sci, vol.30(1) 2021 microneedle patch characterization 72 overcome by the selection of advanced materials for microneedles fabrication. microneedle and ultrasound combination are studied to further increasing drug permeability. modification of conventional microneedles may be used to administer hundred milligrams protein to go 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evaluation of transdermal drug delivery of ondasteron hydrochloride. latin american journal of pharmacy 2012;31(7):86-92. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32 ( 1 ) 2023 impact of omega 3 on the genotoxicity of irinotecan doi: https://doi.org/10.31351/vol32iss1pp53-58 53 impact of omega 3 on the genotoxicity of irinotecan on bone marrow and spleen of rats: in-vivo study alaa radhi khudhair*,1, nada n. al-shawi* and ali faris hassan* *department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract the present study is designed to explore the genotoxicity of irinotecan through measurement of mitotic index in the bone marrow and the spleen cells and to describe the protective actions of omega 3 against irinotecan induced genotoxicity in the bone marrow and the spleen of rats. twenty four (24) rats (sprague-dawley) of both sexes were randomly-divided into four groups: group ӏ, rats received a single oral daily dose of distilled water (2 ml/kg) for 25 days (negative control); group ӏӏ/induced group (by irinotecan), received a single daily oral dose of (2 ml/kg) distilled water for 25 days by the oral gavage and subsequently-received irinotecan (50mg/kg) on days: 5, 10, 15 (total dose=150 mg/kg) by intraperitoneal injection; group ӏӏӏ, received an oral dose of omega-3 fish oil (600mg/kg/day) daily for 25 successive days by an oral gavage (omega-3 fish oil-treated); group ӏv (omega-3 fish oil + irinotecan), received oral dose of omega-3 fish oil (600mg/ kg/ day) given daily for 25 successive days by oral gavage and received subsequently irinotecan (50mg / kg body weight) on days: 5, 10, 15 (total dose=150 mg/kg) by intraperitoneal injection. mitotic index in the bone marrow and the spleen cells were shown to be significantly-decreased (p<0.05) in rats intraperitoneally-injected with irinotecan (group іі) compared to corresponding levels in the negative control (group i) rats; orally-administered omega-3 fish oil with a total cumulative dose of irinotecan (group iv), resulted in significant elevation (p<0.05) of the mitotic index in the bone marrow and the spleen cells compared to corresponding levels in rats treated with irinotecan (group іі). results of the current study suggested that the administration of omega-3 fish oil could be useful supplement that may alleviate irinotecan induced genotoxicity through the elevation of mitotic indices in the bone marrow and the spleen cells of the rats; but, at a mild level. keywords: omega-3 fish oil, irinotecan, bone marrow, spleen, mitotic index. : الجرذانرينوتيكان على نخاع العظم وطحال لإل السمية الجينيةعلى 3تأثير أوميغا دراسة في الجسم الحي * حسن ارسعلي ف و *الشاوي ناجيندى ، 1*،خضير ضيرا االء العراق بغداد ، والسموم ، كلية الصيدلة ، جامعة بغداد ، االدوية فرع * لخالصة ا لإلرينوتيكان من خالل قياس مؤشر االنقسام في نخاع العظم وخاليا الطحال. والستكشاف صممت الدراسة الحالية لوصف السمية الجينية .الجرذانفي طحال الرينوتيكان في نخاع العظم والضد السمية الجينية التي يسببها إ 3ألوميغا لالتأثيرات الوقائية المحتملة ، تلقت الجرذان ӏة قسمت عشوائيا إلى أربع مجموعات: المجموعمن كال الجنسين sprague-dawley ( جرذ24أربعة وعشرون ) ( المعالجة باإلرينوتيكان) ӏӏيوما )مجموعة المراقبة السلبية( ؛ المجموعة 25( لمدة ممل / كغ 2جرعة يومية واحدة من الماء المقطر عن طريق الفم ) ، 10، 5( في األيام: غمم / كلغم 50اإلرينوتيكان ) تيوًما وتلق 25( لمدة ممل / كغ 2مقطر عن طريق الفم )جرعة يومية واحدة من الماء ال تتلق 3جرعة فموية من زيت السمك أوميغا تلقت الجرذان، ӏӏӏ ملغم / كغم( عن طريق الحقن داخل الصفاق ؛ المجموعة 150)الجرعة اإلجمالية = 15 + إرينوتيكان( 3ميغا الوأ) vi ( ؛ المجموعة3زيت السمك بأوميغا ب معالجة اليوًما متتاليًا ) 25م / يوم( يومياً عن طريق الفم لمدة غم / كلغم 600) يوًما متتاليًا ، وتلقت اإلرينوتيكان 25لمدة الفمق م / يوم( يوميًا عن طريغم / كغمل 600) 3جرعة فموية من زيت السمك أوميغا تلقت الجرذان، .م( عن طريق الحقن داخل الصفاقغم / كلغم 150)الجرعة اإلجمالية = 15، 10، 5م من وزن الجسم( في أيام : غم / كغمل 50) المعالجة باإلرينوتيكان )المجموعة الجرذانفي (p <0.05) معنوياأظهر مؤشر االنقسامية في نخاع العظم وخاليا الطحال انخفاًضا عن طريق الفم مع 3مقارنة بالمستويات المقابلة في المجموعة السلبية )المجموعة األولى( من الجرذان؛ أدى تناول زيت السمك أوميغا ( الثانية في مؤشر االنقسام في نخاع العظم وخاليا الطحال (p <0.05) معنويلي الجرعة التراكمية من إرينوتيكان )المجموعة الرابعة( إلى ارتفاع إجما (.الثانية مقارنة بالمستويات المقابلة في الفئران المعالجة باإلرينوتيكان ) المجموعة يخفف من السمية الجينية التي يسببها اإلرينوتيكان من خالل لقد يكون مفيدًا 3زيت السمك أوميغا مركب إلى أن تناول ةالحالي الدراسةتشير نتائج متوسط . ارتفاع المؤشرات االنقسامية في نخاع العظم وخاليا الطحال في الجرذان ؛ لكن بمستوى .لعظم ، الطحال ، ومؤشر االنقسام، إرينوتيكان ، نخاع ا 3الكلمات الرئيسية: زيت السمك أوميغا 1corresponding author e-mail: alaa_z520@yahoo.com received: 16/1 /2022 accepted: 27/ 3/2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp53-58 iraqi j pharm sci, vol.32( 1 ) 2023 impact of omega 3 on the genotoxicity of irinotecan 54 introduction irinotecan, which is a semisynthetic anticancer drug derived from the natural alkaloid chinese plant camptothecin, was approved in 1996, by the food and drug administration, for colorectal cancer treatment (1). such a drug which is a prodrug for sn-38 the active metabolite is widely used for pancreatic, lung, and colorectal cancer treatment (2). researchers reported that irinotecan showed considerable effects against the refractory-5-fluorouracil colorectal cancer; and this lead to a comprehensive program to evaluate the use of irinotecan as a single agent and also as a part of the combination therapies (3). irinotecan is an anticancer agent which belongs to the topoisomerase i enzyme interactive class, targets the complex of dna-topoisomerase i, lead to prevent the reannealing of nicked dna strands resulting in the arrest of the dna replication (1) and consequently might lead to fast-proliferating cell death as in the intestinal basal cells and the bone marrow cells; and the main dose-limiting toxicities associated with irinotecan treatment were the gastrointestinal (gi) and the hematologic toxicities; severe delayed diarrhea and sever neutropenia (4). the spleen acts as a phagocytic filter through the removal of the senescent and the damaged cells, and it also produces antibodies by initiating an immune response versus the encapsulated bacteria so the removal of the spleen is associated with a large reduction of the igm memory b cells (5). dietary fish oil shows to have a beneficial effect on some chronic degenerative diseases as rheumatoid arthritis (6), cardiovascular disease (7,8), other autoimmune diseases (9,10), diabetes (11), and cancer (12,13). these beneficial effects of the fish oil are due to the high level of 𝜔-3 polyunsaturated fatty acids (pufas) as the docosahexaenoic acid (dha) and the eicosapentaenoic acid (epa) (14). all three omega 3 fatty acids; docosahexaenoic acid (22:6), eicosapentaenoic acid (20:5), and α linolenic acid (18:3); are directly-inhibit the arachidonic acid production from the linoleic acid (15). omega-3 pufa dietary supplements have many beneficial effects if giving prior to or during the cancer therapy including reversing the drug resistance of the tumor cell, reducing haematological, cardiac or gi side effects of different chemotherapeutic agents, protection from alopecia, and reduce the cancer cachexia (16). objectives the present study was designed to explore the genotoxicity of irinotecan through measurement of mitotic index in the bone marrow and the spleen cells and to describe the protective actions of omega 3 against irinotecan induced genotoxicity in the bone marrow and the spleen of rats. materials and methods reagents: absolute methanol, bdh (england); giemsa stain, fluka (switzerland); glacial acetic acid, fluka (switzerland); potassium chloride powder fluka, switzerland; and glycerin fluka (switzerland). drugs: irinotecan 100mg vials obtained from fresenius kabi, india; colchicine tablet, aventis (france); and omega-3 fish oil capsule 1000mg/ml, natrol (usa). animals and experimental design twenty-four (24) adult sprague-dawley rats of both sexes, each weighing 150-200gm taken from the animal house of college of pharmacy/ university of baghdad. duration of the study from april to october 2021. the institutional animal care and the use committee of the college of pharmacy/ university of baghdad approved the protocol of this study and the conduction of the work was performed under the controlled-conditions of the conventional laboratory ethics. experimental rats are kept in stainless steel cages, in temperature (25°с), natural light/dark cycle and a relative humidity. standard laboratory rodent tap water and chow supplied ad libitum, and animals adapted for one week period before the experiment. the animals were divided into four groups of six rats each as follows: •group ӏ: rats administered a single oral daily dose of distilled water (2ml/kg) for 25 successive days by oral gavage. this group served as control. •group ӏӏ (irinotecan/induction group): rats received a single oral daily dose of (2 ml/ kg) distilled water for 25 successive days by oral gavage, and subsequently ip injected with irinotecan (50mg per kg body weight) on days: 5, 10, 15 (total dose=150 mg/kg) (17). •group ӏӏӏ (omega-3 fish oil-treated): rats orally-administered omega-3 fish oil at a dose of (600mg/kg/day) (18) daily for 25 successive days by oral gavage. •group ӏv (omega-3 fish oil-irinotecan): rats received oral dose of omega-3 fish oil (600mg/kg/day) daily by oral gavage for 25 successive days, and subsequently-injected with irinotecan (50mg per kg body weight) on days: 5, 10, 15 (total dose=150 mg/kg) by intraperitoneal injection. twenty-four (24) hours after the end of treatment that is to say at day 26, all the rats used in the experiment were euthanized by diethyl ether anesthesia and then samples of bm and spleen cells were taken for cytogenetic analyses. iraqi j pharm sci, vol.32( 1 ) 2023 impact of omega 3 on the genotoxicity of irinotecan 55 preparation of solutions colchicine solution: the colchicine solution was prepared by dissolving 2 tablets of (1mg) colchicine in 4ml distilled water to prepare (0.5mg/ml) solution (19). hypotonic solution, potassium chloride (kcl), for the chromosomal aberration: potassium chloride in a concentration of 0.075m prepared through adding kcl salt (5.75gm) to be dissolved in 1000 ml distilled water; then sterilized by the autoclaving and then stored in the refrigerator (at 4ºc) (20). fixative solution: the fixative solution is freshly prepared by adding three parts of the absolute methanol to one part of the glacial acetic acid (21). preparation of the chromosome using the somatic cells of the bm and the spleen of rats this procedure is done as follows (21): a. first 1ml of colchicine are injected into the rats intraperitoneally, the concentration of colchicine is (1mg/2ml), two hours before their scarification. b. on day 26 the animals were sacrificed. c. by using a plate of anatomy each experimental rat was fixed so that the animal abdomen swabbed with its thigh region with ethanol 70%. d. the animal femur bone was taken and then cleaned from tissues and other muscles, by using forceps the bone gapped vertically from the middle over a test tube edge, using a sterile 5ml syringe of pbs that was injected in the test tube to drop the bm in it. e. then in petri dish extract the spleen of the rat and inject it by pbs in various spots. f. by centrifuging the test tubes that spanned at 2000 rpm for about 10min. g. removing of the supernatant layer then adding 5ml of kcl (0.075m) as a hypotonic solution, then put the tubes in the water bath for 30 min. at 37ºc and every 10 min. shaking the tubes. h. after that the tubes were put in the centrifuging for 10min (at 2000 rpm). i. remove the supernatant layer with the addition of 5ml fixative solution drops by drops on the test tube in siding walls with shaking continuously. then well shaking the test tube. j. cells are fixed by keeping tubes at 4ºc for about 30min. k. these tubes were put for centrifuging for 10min (at 2000 rpm). this was repeated three times then in 2ml fixative solution the cells are suspended. l. from three feet height by using a pasteur pipette few drops dropped vertically on the chilled slide at 4-5 drops rate for giving chance for well spreading of the chromosomes. then dried the slides at room temperature. m. stained the slides by the giemsa stain, 15min left the slides and washed by d.w. n. two slides were prepared for the cytogenetic studies. assaying of the mitotic index the following slides (by using the light microscope) were examined through the 40x power, count (1000) cells of both divided and non-divided. the only dividing cells are calculated by the percentage according to the following equation (22): 𝐌𝐢𝐭𝐨𝐭𝐢𝐜 𝐢𝐧𝐝𝐞𝐱 (%) = 𝐍𝐮𝐦𝐛𝐞𝐫 𝐨𝐟 𝐝𝐢𝐯𝐢𝐝𝐞𝐝 𝐜𝐞𝐥𝐥𝐬 𝐓𝐨𝐭𝐚𝐥 𝐜𝐨𝐮𝐧𝐭 (𝟏𝟎𝟎𝟎) × 𝟏𝟎𝟎 statistical analysis the data were expressed as values of the mean ± the standard deviation (sd); and analyzing was performed utilizing the computerized program of statistical package for social sciences (spss) (version 23). statistical significance of differences among various groups was determined by the one way-analysis of variance (one way-anоva). the statistically-significant differences between the groups were considered when the p value was less than 0.05 (p<0.05). results irinotecan (group ii rats), caused a significant-reduction (p<0.05) in the mitotic index in bone marrow cells (figure 1), and in the spleen cells (figure 2) as compared to corresponding levels in the control (group i) rats. furthermore, in groups iii rats/orally received omega-3 fish oil (600mg/kg/day) produce non-significant differences (p>0.05) in the mitotic index in the bone marrow cells (figure 1), and spleen cells (figure 2) respecting to the corresponding levels in group ι/control rats. administration of omega-3 fish oil (600mg/kg/day) in association with irinotecan (groups iv) rats, there was a significant-elevation (p<0.05) of the mitotic index in bone marrow cells (figure 1), and in the spleen cells (figure 2) compared to corresponding index in rats’ tissues of group ιι. rats pretreated with 600mg/kg of omega-3 fish oil for 25 successive days with the induction of genotoxicity by irinotecan administration (group іv), there was a significant decreased (p<0.05) in the mitotic index in the bone marrow cells (figure 1), and in the spleen cells (figure 2) compared to corresponding index in rats’ tissues of group ι. additionally, rats pretreated with 600mg/kg of omega-3 fish oil for 25 successive days with the induction of genotoxicity by irinotecan administration (group іv), there was a significant decrease (p<0.05) in the mitotic index in the bone marrow cells (figure 1), and the spleen cells (figure 2) compared to such index in rats’ tissues pretreated with 600mg/kg of omega-3 fish oil for 25 successive days (group ιιι). iraqi j pharm sci, vol.32( 1 ) 2023 impact of omega 3 on the genotoxicity of irinotecan 56 figure 1. the effects of omega 3 fish oil and irinotecan intoxication on the mitotic index in bone marrow cells. data expressed as mean±sd, n =6. (*): significantly different with respect to group ι (p<0.05). values with non-identical superscripts (a, b and c) were significantly different (p<0.05). s superscript: significant difference with respect to the irinotecan treated group. figure 2. the effects of omega 3 fish oil and irinotecan intoxication on the mitotic index in spleen cells. data expressed as mean±sd, n =6. (*): significantly different with respect to group ι (p< 0.05). values with non-identical superscripts (a, b and c) were significantly different (p<0.05). s superscript: significant difference with respect to the irinotecan treated group. discussion it has been reported that, myelosuppression caused by irinotecan treatment resulted in neutropenia, which can be considered as the predominant hematological adverse effect of such chemotherapeutic drug (23). in animals treated with irinotecan showed a significant reduction in the wbc count and in the rbc count (24). moreover, a link between irinotecan treatment and steatohepatitis development was mentioned by authors (4). a recent study confirmed that irinotecan results in liver toxicities, as was evidenced through the elevations in mda content; in addition, there were reduction in serum total antioxidant capacity (taoc) and elevation in serum ast, and alt (17). researchers reported that treatment with irinotecan can cause activation of the cascade of many apoptotic-related signaling pathways via breakages of the double strands dna; thus, ultimately resulting in apoptotic-cell death (17; 25). previous studies indicated that omega 3 fatty acids decrease the total myeloid progenitor cell frequencies and also promoted the differentiation of the specific progenitor cell types in mice bone marrow (15). omega 3 fatty acids resulted in the inhibition of proliferation, induction of apoptosis, and promotion of differentiation in many cancer types by mechanisms including the regulation of the signaling pathways and the gene expression through the peroxisome proliferator receptor activator γ (pparγ), that omega 3 fatty acids are one of its natural ligands (26). other mechanisms of action of the omega 3 fatty acids include the inhibition of cyclooxygenase 2 (cox2) unregulated in different cancer types and it is known to have antiapoptotic and proliferative effects (27). the phospholipase a2 (pla2) enzyme liberate the arachidonic acid (aa) from phospholipids of the membrane, then the cox2 transfer the aa to the pro-inflammatory mediators, so through the inhibition of pla2 and the cox2 enzymes, omega-3 fatty acid decreased the pro-inflammatory mediators (28). moreover, both the activation of the pparγ and the inhibiting of cox2 expression were shown to inhibit the proliferation and also induce apoptosis in pancreatic cancer (29). also, researchers reported that omega-3 fatty acid possesses a beneficial therapeutic action in patients who were suffering from neurological disorders as epilepsy (28). furthermore, omega 3 fatty acids have inhibitory effects on the immune system that led to the usage of fish oils for the management of different autoimmune and inflammatory diseases (30). the current study showed that omega 3 fatty acids (600mg/kg/day) attenuate irinotecaninduced reduction in mitotic index in bone marrow cells (figure 1), and the spleen cells (figure 2). iraqi j pharm sci, vol.32( 1 ) 2023 impact of omega 3 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abstract a direct, sensitive and efficient spectrophotometric method for the determination of nitrofurantoin drug (nit) in pure as well as in dosage form (capsules) was described. the suggested method was based on reduction nit drug using zn/hcl and then coupling with 3-methyl-2-benzothiazolinone hydrazone hydrochloride (mbth) in the presence of ammonium ceric sulfate. spectrophotometric measurement was established by recording the absorbance of the green colored product at 610 nm. using the optimized reaction conditions, beer ’ s law was obeyed in the range of 0.5-30 µg/ml, with good correlation coefficient of 0.9998 and limits of detection and quantitation of 0.163 and 0.544 µg/ml, respectively. the accuracy and precision of the proposed method represented by recovery and relative standard deviation were satisfactory; about 99.33% and 1.16%, respectively. the proposed method was applied for determination of nit in its pharmaceutical forms and the results compared successfully with those obtained by standard method (british pharmacopeia method). keywords: nitrofurantoin , mbth , oxidative coupling reaction. باستخذام ةفي المستحضرات الصيذالني نتوينتروفيورايالتقذير الطيفي لذواء الن mbth ككاشف ازدواج هنذ هادي عبذهللا ،*1 مروه مؤيذ عبذالواحذو * * انكًٍٍاء ، كهٍة انعهوو ، جايعة بغذاد ، بغذاد ، انعشاق .لسى الخالصه ( بشكهّ انُمً وكزنك فً انًسححضشات nitجى وصف طشٌمة طٍفٍة يباششِ, حساسة وفعانة نحمذٌش دواء انٍُحشوفٍوساَحوٌٍ ) بواسطة انضَك فً انوسظ انحايضً ثى بعذ رنك االلحشاٌ يع nitانصٍذالٍَة )كبسوالت(. جسحُذ انطشٌمة انًمحشحة عهى اخحضال دواء ( وبوجود كبشٌحات االيوٍَوو . حٍث لذست انمٍاسات انطٍفٍة بواسطة mbthبُضوثٍاصونٍُوٌ ٍْذساصوٌ ٍْذسوكهوسٌذ )-2-يثم -3 5-36د لاَوٌ بٍشَاَويٍحش. باسحخذاو ظشوف انحفاعم انًثهى كاٌ حذو 616جسجٍم االيحصاصٍة نهُاجج االخضشانهوٌ عُذ ياٌكشوغشاو/يم عهى انحوانً. 6.544و 6.163وبهغ حذ انكشف وحذ انكًٍة 0....6ياٌكشوغشاو/يم يع يعايم اسجباط جٍذ بحذود جى حساب انذلة وانًصذالٍة نهطشٌمة انًمحشحة عٍ طشٌك َسبة االسحشجاع واالَحشاف انمٍاسً انًعٍاسي وكاَث يمبونة بحذود فً انًسححضشات انصٍذالٍَة وجى يماسَة انُحائج يع انطشٌمة nitى انحوانً. طبمث انطشٌمة بُجاح فً جمذٌش % عه1.16% و 33... انمٍاسٍة )طشٌمة انذسحوس انذوائً انبشٌطاًَ(. . ، تفاعالت ازدواج تاكسذيmbthالكلمات المفتاحية: نيتروفيورانتوين، introduction nitrofurantoin (figure 1), is 1-(5-nitro2-furfurylidene)-1-amino hydration (1) with a molecular weight of 238.16 g/mol. it is a lemon-yellow, odorless fine powder, very slightly soluble in water and alcohol, but it is soluble in dimethyl formamide(2). figure (1):structure of nitrofurantoin nitrofurantoin , a nitro furan derivative, is an antibiotic, that is preferable choice of oral use for wide spectrum of infections especially for urinary tract infections (3). the mechanism of action in killing the germs is not known accurately, however, it is well absorbed in the intestines and up to very high concentrations in the urine(4). a survey of the literature exposed that numerous techniques have been reported for analysis of nit in both body fluids and pharmaceuticals, among them a spectrophotometic (5), hplc(6-8), liquid chrom tandem mass spectrometry (9), square wave voltametric (10, 11), and flow injection chemiluminescence methods (12). 1 corresponding author e-mail: hindhadi13@yahoo.com received: 20 /4/2016 accepted: 29/6/2016 o2n o n n nh o o iraqi j pharm sci, vol.25(2) 2016 spectrophotometric determination of nitrofurantoin 8 unfortunately all the mentioned techniques are expensive and complicated. the sensitive uvvisible spectrophotometric methods for the determination of the drug are limited and literature contains only few methods for the determination of nit in pure and dosage forms. this work describes the development of simple and rapid method based on colorimetric reaction for the quantitative determination of nit in pure and pharmaceutical forms. the proposed method carried out at room temperature is based on the reduction of the nitro group of nit, followed by coupling with mbth in the presence of ammonium ceric sulphate as oxidizing agent. experimental apparatus a digital double beam shimadzu uv–vis 260 spectrophotometer (shimadzu, kyoto, japan) was used for all measurements (spectral and absorbance). the absorbance measurements were carried out using matched 1cm quarts cells. reagents and standards  all the chemicals and reagents were of analytical grade and the freshly prepared solutions were always used throughout this work.  nitrofurantoin (nit) solution(500 μg/ml) (13) : the reduction solution of nit was prepared by dissolving 0.0500 g of nit in 50ml of ethanol. this solution was transferred into 125 ml beaker and 20 ml of distilled water, 20 ml of concentrated hydrochloric acid, and 3.0 g of zinc powder were added. in order to complete the reduction process the beaker was allowed to stand for 15 min at room temperature (25°c), then the solution was filtered into 100 ml volumetric flask, and the volume was diluted to the mark with distilled water to obtain 500 μg ml -1 of nit reduction solution then transferred in a brown bottle. working solution was prepared daily by appropriate dilution using distilled water.  methylbenzothiazoline2one hydrazone hydrochloride (0.3%w/v) (14) , sigma-aldrich) the acidic solution of this reagent was prepared by dissolving 0.3000g of the reagent in 100 ml of 0.2 m hcl. this solution should be freshly prepared. working solution was prepared daily by appropriate dilution using the same solvent.  ammonium ceric sulphate, acs (0.03m, bdh) this acidic solution was prepared by dissolving 1.9789 g of the oxidant in 100ml of 1% h2so4 solution. working solution was prepared daily by appropriate dilution using the same solvent.  hydrochloric acid (merck, germany) 0.2m aqueous solution.  sulphuric acid (merck, germany) 1% v/v aqueous solution.  pharmaceutical preparations the different pharmaceutical preparations were purchased from the commercial source in the local market. 1. nitrofurantoin capsules, 100 mg (uvamin retard, switzerland). 2. furantil capsules, 50mg ( bio active t-uk). solutions of pharmaceutical preparations ten and/or fifteen capsules of commercial nit (labeled to contain 100 and/or 50mg of nitrofurantoin per capsule) were accurately weighted and finely powdered. an amount of the powder equivalent to 50 mg of nit was dissolved in 30 ml of ethanol, then the solution was filtered into a 50 ml volumetric flask. and the volume was diluted to the marked with the same solvent to obtain 1000 μg ml -1 of nit. this solution was transferred into 125 ml beaker and was reduced as previously described. further appropriate solutions of pharmaceutical capsules were prepared using distilled water. for the proposed method, the content of a tablet was calculated using the corresponding regression equation of the appropriate calibration graph. general procedure (constructing the calibration curve) an increasing volumes of standard drug solution in the range (0.01-0.6) ml of 500µg/ml reduce nit were transferred into a series of 10 ml volumetric flasks to obtain the concentration range of 0.5-30 µg/ml. to each flask, 0.5 ml of mbth reagent (0.3% w/v) and 0.5 ml of ammonium ceric sulfate (0.03m) were added. after 5 min the contents were diluted to the mark with distilled water and mixed well. the absorbance of the colored product was measured after 15 min at 610 nm against the corresponding reagent blank prepared similarly omitting the drug content. stoichiometry of the coupling reaction using job’s method and mole ratio method (15) the stoichiometry of oxidative coupling reaction was calculated using equimolar of reduced nit and mbth (2mm) at constant oxidant concentration adopting job’s method of continuous variation, and mole ratio methods. job’s method of continuous variation of equimolar solutions was employed: a 2mm of standard solution of reduced nit and mbth were used. a series of solutions was prepared in which the total volume of the nit and mbth was kept at 10 ml. the drug and reagent were mixed in varying complementary iraqi j pharm sci, vol.25(2) 2016 spectrophotometric determination of nitrofurantoin 9 proportions (0:5, 1:4, 2:3….5:0, inclusive) and completed as directed under the recommended procedure. the absorbance of the resultant product was measured at optimum wavelength. in mole ratio method an increasing volume of mbth (0.5, 1, 1.5, 2…..4ml) was added to 1 ml of reduced nit at constant oxidant concentration. in varying proportions of both drug and reagent, the solutions were mixed and diluted with distilled water in 10ml volumetric flask, then the absorbance was measured at optimum wavelength and under optimal time and temperature against a reagent blank. results and discussion nitro group containing drugs are very important kinds of drugs, but the spectrophotometric determination of these drugs (especially nit) is not easy because of the poor affinity of these drugs to react directly with other coupling reagents. in order to increase this reactivity an attempt to convert the nitro group to a more reactive group (amino group) was done. the present work depends on a simple oxidative coupling reaction between reduced nit and mbth in the presence of an efficient oxidant (acs). a green colored product was formed and have a maximum absorbance at 610nm. the absorption spectra of the reaction product and the reagent blank are presented in figure 2, and it was used in all subsequent experiments. figure (2) :absorption spectra of the product obtained by the reaction of mbth with 25 µg/ml of reduced nit in presence of ammonium ceric sulphate versus reagent blank, and the reagent blank versus distilled water. optimum reaction conditions the proposed method was optimized to achieve complete reaction formation, highest sensitivity and maximum absorbance. all the experimental parameters were optimized using 25 µg/ml of nit. effect of reaction time the color intensity reached a maximum after the reduced drug (25µg/ ml) solution had been reacted after 10 min with mbth and acs in aqueous medium and became stable after 15 min and remained stable for at least 50 min (figure 3). this experiment was repeated after optimized all the reaction parameters and the results were the same. figure ( 3):stability time optimization of the experimental conditions mbth is an efficient coupling reagent for many drugs. in order to study the effect of the amount of reagent, different volumes in the range (0.1-3.0)ml of 0.3%w/v mbth was examined in the presence of 0.5ml of ammonium ceric sulfate (0.03m). the results (figure 4) show that 0.5 ml of the mbth solution was enough to obtain a maximum absorbance, and it was used in the subsequent experiments. different oxidants were studied to accomplish the coupling reaction such as sodium preiodate, ferric chloride, n-brome succmimide(nbs), potassium thiosulphate and acs. the maximum absorbance was obtained only when using acs as oxidant. the optimum volume of the used oxidant used (acs) was examined. this study was carried out using different volumes of 0.03m of acs in the range (0.1-2.0) ml. an increase in absorbance was obtained up to 0.5 ml of acs as shown in the figure 5. beyond this volume the absorbance was decrease with increasing the amount of oxidant because of the increasing the absorbance of blank, therefore 0.5 ml of acs was chosen, and was used in the subsequent experiments. iraqi j pharm sci, vol.25(2) 2016 spectrophotometric determination of nitrofurantoin 10 figure (4):effect of the reagent figure ( 5 ):effect of the oxidant the effect of order of addition is also studied under the obtained optimum results. according to the results, it was found that the order of addition of reagents (drug + mbth + acs) as shown in figure 6, gave the maximum absorbance and stability in measurement. figure (6):the effect of the order of addition (d=drug (nit), ox= oxidant (acs) , r=reagent(mbth) the effect of temperature on the oxidative coupling reaction was also studied using three different temperatures (5, 25, 65)c. figure 7 shows that a maximum absorbance and a good stability were obtained when the formed product developed at ambient temperature (25 0 c). figure (7):effect of temperature the structure of the product was adopted based on the mole ratio and continuous variation methods (15) , using equimolar solutions (2 mm) of reduced nit and mbth reagent. the results obtained in figures (8 and 9) show that 1:2 ratio reduced nit to reagent was formed. figure (8): the mole ratio method figure (9):the job method an electrophilic intermediate was formed when mbth loses two electrons and one proton under oxidation process, which can be substituted on reduced nit under the reaction conditions to form a green colored product (16) . therefore, the mechanism of formation of the product may be suggested according to the following equation: iraqi j pharm sci, vol.25(2) 2016 spectrophotometric determination of nitrofurantoin 11 conditional stability constant (kf) of the product and gibbs free energy of the reaction the conditional stability constant of the green color product was calculated from the continuous variation data using the following equation (17) : where: a and am are the maximum absorbance of the continuous variation curve and the absorbance corresponding to junction of the two tangents of the continuous variation curve respectively (fig. 8). n is the number of molecules of the reagent in the reaction product (the stoichiometric constant). c is the molar concentration of nit at the maximum absorbance. kf was found to be equal to 6.084×10 5 l 2 mole -2 . this indicates a stable reaction product. the gibbs free energy of the reaction δg was also calculated adopting the following equation: δg=-2.303rtlogkf where, r is the universal gas constant (8.314 j mole -1 deg -1 ). t is the absolute temperature (273+25°c), kf is the formation constant of the reaction. the value of δg was found to be -33 kj/mole. the negative value of δg refers to the spontaneity of the reaction. kinetic of the reaction the initial rates of the reaction were determined by measuring the slopes of the initial tangents the absorbance time curves for the first 10 min (fig. 10). furthermore, logarithmic analysis of the reaction rate (r) was plotted against the logarithm of concentration of the drug (fig. 1). figure (10):-absorbance versus time graphs figure (11):log (rate) versus log [nit] graphs the rate of reaction was also found to be dependent on nit concentrations. the rates were followed at room temperature (25˚c) with various concentration of nit in the range of (2.08×10 -5 to 8.39×10 -5 m) keeping mbth and acs concentrations constant. the reaction rate was found to obey the following equation: rate = k' where k' is the pseudo-order rate constant and n is the order of the reaction. the rate of the reaction may be estimated by the variable-time method (18) (differential initial rate method) as ∆a/∆t, where a is the absorbance and t is the time in minutes .taking logarithms of rates and concentration, the previous eq. is transformed into iraqi j pharm sci, vol.25(2) 2016 spectrophotometric determination of nitrofurantoin 12 : log (rate) = log(δa/δt) = log k' + n log [nit] regression of log (rate) versus log [nit] gave the regression equation: log (rate) = 3.4945 + 1.2944log c (r 2 = 0.9980) hence k' = 3122.4 min -1 = 52 sec -1 and the reaction is first order (n = 1.2944)with respect to nit concentration. validation of the proposed method after optimized all the reaction conditions mentioned above, the calibration graph was plotted between the absorption intensity with the corresponding concentration of nit. regression analysis and the statistical parameters are calculated from the calibration graph using least-square method. the slope, correlation coefficient, intercept for the calibration data and the sensitivity parameters (molar absorptivity and sandell sensitivity), are summarized in table 1. a high correlation coefficient (r=0.9998) with the small intercept value of the regression equation were confirmed the linearity of calibration curve. the small values of the statistical parameters calculated from regression equation point out to the high precision of the proposed method and low scattering of the points of the calibration curve and high accuracy (table 1). table(1):summary of optical characteristics and statistics for the proposed metho sensitivity limit of detection (lod) and limit of quantitation (loq) were calculated according to the 3.3s/k and 10s/k criterions, respectively (19) , where s(0.001506 ), is the standard deviation of the response of the blank or the standard deviation of intercepts of regression lines and k is the sensitivity, namely the slope of the calibration curve. the lod and loq values were 0.179 and 0.544 μg/ml respectively. accuracy and precision in order to evaluate the accuracy and precision of the proposed method, solutions containing three different concentrations of nit were prepared and analyzed in five replicates. the analytical results of this investigation are summarized in table 2. the low values of percentage relative standard deviation (% rsd) as precision and percentage relative error as accuracy of the proposed method were calculated. table ( 2 ): accuracy and precision of the proposed method amount of nit taken,(µg/ml) found* (µg/ml) %relative error* %(recovery ± sd)* %rsd* 6.00 5.95 -0.83 99.17±0.23 1.07 8.00 7.81 -2.38 97.63±0.53 1.96 20.00 20.07 0.35 100.35±0.27 0.45 *average of five determinations, rsd, relative standard deviation. effect of interferences in order to examine the usefulness of the method, the studied drug (nit) was determined in the presence of diluents, excipients and additives which often accompany nit in its dosage forms such as poly vinyl pyrrolidone, lactose, starch and magnesium stearate. the experiment accomplished by measuring the absorbance of solution containing 25 µg/ml of nit in the presence of tenfold of excipient (250 µg/ml). the good percentage recoveries were obtained indicating no interference was observed from any of these excipients and additives, and a parameter value λmax (nm) 610 color green regression equation y = b x + a; y = absorbance, x = concentration(μg/ml) y=0.0277x+0.0568 correlation coefficient, r 0.9998 linearity percentage, r 2 % (r 2 % = r 2 × 100) 99.9891 dynamic range (μg/ml) 0.5-30 molar absorptivity, ε (l/ mol cm) 6.5970x10 3 slope, b (ml/μg) 0.027742 intercept, a (a = y– b x) 0.056758 standard deviation of the residuals, sy/x 0.004377 standard deviation of the slope, sb 1.364x10 -4 standard deviation of the intercept, sa 2.003 x10 -3 sandell’s sensitivity, s (μg/cm) 3.61016x10 -2 iraqi j pharm sci, vol.25(2) 2016 spectrophotometric determination of nitrofurantoin 13 high selectivity for determining the nit in its dosage forms (table 3). table (3):analysis of nit in presence of common interferences by batch method excipent (250 µg/ml) conc. of nit, μg/ ml %(recovery ± sd)* present found poly vinyl pyrrolidone 25.0 25.69 102.76±0.67 lactose 24.50 98.00±0.13 starch 24.19 96.76±0.43 magnesium stearate 24.28 97.12±0.71 *average of five determinations. analytical applications the proposed method was successfully applied to determine nit in pharmaceutical preparations by the analysis of three different concentrations of pharmaceutical preparations using the analytical procedure directly and using standard addition methods. the results are given in table 4 and 5. for evaluating the competence and the success of the proposed method, the results obtained were compared with those obtained by standard bp method (1) . the same pharmaceutical preparations for nit were analyzed by standard bp method. the results obtained by the two different methods as show in table 4, were statistically compared using the student t-test and variance ratio f-test at 95% confidence level when degree of freedam(n=3) (15) . in all cases, the calculated values were less than the theoretical one, which indicate that there is no significant difference between either methods in accuracy and precision in the determination of nit in pharmaceutical preparations. table (4 ):-application of the proposed method to the determination of nit in different dosage forms using standard addition method table ( 5 ):application of the proposed and official methods to the determination of nit in different dosage forms directly. dosage form proposed method official method[hplc] taken conc. (µg/ml) found conc. (µg/ml) rec. (%) a rsd (%) a taken conc. (µg/ml) found conc. (µg/ml) rec. (%) b rsd (%) b nitrofurantoi n (cap.100 mg), uvamin retard, switzerland 5.00 5.15 102.94 0.97 25.0 25.31 101.22 0.05 10.00 10.27 102.69 0.73 15.00 14.87 99.15 1.34 furantil(cap. 50mg), bioactivetuk 5.00 5.01 100.09 1.60 25.0 24.89 99.55 0.03 10.00 9.85 98.48 1.01 15.00 14.74 98.27 0.67 t (2.776) c f (19.000) c 0.134 1.069 a, (n=5); b, (n=3); c theoretical value; conc., concentration; rsd, relative standard deviation. conclusions the proposed method showed good sensitivity , and low detection limit. in addition, the data given above reveal that the proposed method was accurate and sensitive with good precision and accuracy; it can be successfully applied to the routine estimation dosage form taken conc. (µg/ml) proposed method pure drug added conc. (µg/ml) total found conc. (µg/ml) (%recovery± sd) n=5 nitrofurantoin (cap.100 mg), uvamin retard, switzerland 5.00 5.00 10.01 100.14±0.010 10.00 14.81 98.12±0.020 furantil (cap.50mg), bioactivet-uk 5.00 5.00 10.03 100.55±0.003 10.00 14.44 94.40±0.005 iraqi j pharm sci, vol.25(2) 2016 spectrophotometric determination of nitrofurantoin 14 of nit in bulk and in pharmaceutical preparations. the values of relative standard deviation were satisfactorily low (less than 2%) with good recoveries which indicate the high reproducibility and accuracy for the proposed method. the reaction was adopted to suggest a new flow injection method for the determination of nit in a separate work send for publication by the same authors. references 1. british pharmacopoeia on cd, 2005, pp 110. 2. the usp pharmacists pharmacopeia text , 2008 ; section 13 , pp 118 3. j. m ritter, l. d a. lewis, t. g. mant, and a. ferro , “ a textbook of clinical pharmacology and therapeutics “, 2008 ;3 rd , hodder arnold , uk , chapter 1 ,pp51 . 4. j. m. beale, jr. and j. h. block ,” organic medicinal and pharmaceutical chemistry”, 2011; 12 th , lippincott williams & wilkins, a wolters kluwer business, china, chapter 6 pp 213. 5. m. tubino, l. f. bianchess, m. palumbo, and m. m. d. c. vila, “green and simple uv-visible diffuse reflectance and transmittance methods for the determination of nitrofurantoin in pharmaceutical preparations”, 2011; eclectic quimica, issue 0100: 4670, vol 36, pp 62-67. 6. i. t. hanoon and h.h.kharnoob, “determination of nitrofurntion in drug formulations by high performance liquid chromatography”, kerbala journal of pharmaceutical secinces, 2013; 6: 164178. 7. m. d. ashraful alam, a. m. yeasinia t. m. waliullah, s. ahmed, and m. m. mia,”a comparison of confirmatory method development and validation of antibiotic: nitrofurantoin metabolites (amoz, aoz, and sem) in fish and shrimp matrix amony some lc-ms/ms systems (hplcquattro micro api, uplc, quattro premier xe and uplc, tqd”, word journal of pharmaceutical world, 2015;4(5): 2392-2416. 8. k. pietruszka, m. olejnik and b. sell, “development and validation of a liquid chromatography method for the determination of nitrofurans in water”, bull vet instpulawy2007; 51: 267-270. 9. d. s. patel, n. sharma, m. c. patel, b. n. patel, p. s. shrivastav, and m. sanyal, “quantitation of nitrofurantoin in human plasma by liquid chrom tandem mass spectrometry”, acta pharm2013; 63: 141158. 10. p. neta, a. n. correia, r. r. portela, m. d. s. juliao, g.f. l. junior, and j. e. s. lima, “sugare wave voltametric determination of nitrofurantoin in pharmaceutical formulation on highly borandoped diamond electrodes at different boron – doping contents” talanta, 2010; 80: 1730-1736. 11. z. krejcova, j. barek and v. vyskocil, “voltammetric determination of nitrofurantoin at mercury meniscus modified silver solid amalgam electrode”, electro analysis, 2015; 27: 185-192. 12. p. thongsrisomboom, b. liawruangrath, s. liawruangrath, and s. satienperakul, “determination of nitrofurans resided in animal feeds by flow injection chemilumiescence procedure”, food chemistry, 2010; 123: 834-839. 13. r. s. abdulsattar, “spectrophotometric determination of nitrazepam in pharmaceutical tablets using flow injection analysis” journal of university of anbar for pure science, 2010; l 4, no1. 14. a. el-emam, f. f. belal, m. a. moustafa, s. m. elashry, d. t. el -sherbiny, s.h. hansen, “spectro photometric determination of propranolol in formulation via oxidative coupling with 3-methylbenzothiazoline-2-one”, farmaco(societa chimica italiane: 1989), 2003; 58(11): 1179-1186. 15. david harry, “modern analytical chemistry spectroscopy method of analysis”, 2000, the mc-gram-hill companies, usa, chapter 10, pp 404-406. 16. b. ramachandra, n. venkatasubba naidu, “ spectrophotometric method for the determination of furazolidone in pharmaceutical formulations and human blood samples with mbth” international journal of science and research (ijsr), 2013; 4:1026-1031. 17. j. inczedy, analytical application of complex equilibria, wiley, budapest, p. 101, 1976. 18. d. p. bendito, and m. silva,“kinetic methods in analytical chemistry”, ellis horwood, chichester, 1988; through ref. 578. 19. j.n. miller, j. c. miller, statistics and chemometrics for analyt-ical chemistry, fifth ed., prentice hall, england, 2005. iraqi j pharm sci, vol.31( 2 ) 2022 comparison between black and white desert truffles doi: https://doi.org/10.31351/vol31iss2pp184-192 184 comparison of phenolic contents and antioxidant activities for black and white desert truffles spread in syria abdullah zuhair al-atassi*,1, racha aboualkher al-khatib** and mohammad abdulraouf othman*  department of pharmaceutics and pharmaceutical technology, faculty of pharmacy, damascus university, damascus, syria.  department of pharmacognosy, faculty of pharmacy, damascus university, damascus, syria. abstract desert truffle is considered as a type of syrian wild fungi that spreads heavily, and it occupies important rank in folk medicine, where its aqueous extract is used for the treatment of some eye and skin illnesses, and people prefer the use of black truffle. this work interested in studying of the most available species; terfezia claveryi (black) and tirmania pinoyi (white). the extracts of the two species of truffle were prepared by maceration with water, methanol, and ethanol 70%. their total phenolic contents (tpc) and total flavonoid contents (tfc) were analyzed using folin-ciocalteu and aluminum chloride methods respectively, and their antioxidant activities was tested using 2,2-diphenyl-1-picrylhydrazyl (dpph) and ferric reducing antioxidant power (frap) methods, after microscopic examination and detection of phytochemical components. then, phenolic profile of ethanolic 70% extract of black truffle t.claveryi was studied by using lc-ms/ms. the values of tpc were between 25.3-43.6 mg gae/g dry extract and tfc were between 2.5-6.8 mg qe/g dry extract. the values of dpph (ic50) and frap were between 5.6-9.0 mg/ml and 90.1-153.4 µmol aae/g dry extract respectively. there is a great similarity in content and activity of two species, also the aqueous extract is similar to other extracts in content and activity, and this means that the method of extract preparation in traditional medicine is reliable. it has been predicted about 14 phenolic compounds in the extract; as p-hydroxy benzoic acid, syringic acid and trans-cinnamic acid. as a result, both truffle species are a new rich resource of antioxidant compounds which are usable in nutritional, cosmetic, and therapeutic applications. keywords: dpph, flavonoids, frap, lc-ms/ms, polyphenols, truffle. السوداء والبيضاء المنتشرة الصحراوية كمأةل المحتوى الفينولي والفعالية المضادة لألكسدة لمقارنة في سوريا * ومحمد عبد الرؤوف عثمان و **ورشا أبو الخير الخطيب 1،*األتاسيعبد هللا زهير . سوريادمشق، كلية الصيدلة، جامعة دمشق، ، قسم الصيدالنيات والتكنولوجيا الصيدلية * . سوريادمشق، كلية الصيدلة، جامعة دمشق، عقاقير والنباتات الطبية، قسم ال ** ةالخالص ، حيث تُستخدم في الطب الشعبي وهي تشغل منزلة قيّمةالسورية، في البادية بكثرةالفطور المنتشرة نواعالصحراوية أحد أتعد الكمأة السوداء الكمأة استخدام الناس ويفضل والجلدية، العينية األمراض بعض عالج في المائية توافراً . خالصتها األكثر النوعين بدراسة البحث اهتم terfezia claveryi (و )السوداء tirmania pinoyi ،)رت خالصات نوعْي الكمأة إذ )البيضاء ، %70نقيع بالماء والميتانول واإليتانول بالت ُحّضِّ سودُ فعاليتها اختبار تم، وعلى الترتيب سيوكالتو وكلوريد األلمنيوم-بطريقتي فولين tfc الكلي والفالفونوئيدي tpcالكلي الفينولي محتواها رِّ دراسة الكشف عن المجموعات الكيميائية. ثم تم مجهري وال الفحصبعد ، frapوالمرجعة للحديد dpphالمضادة لألكسدة الكابحة للجذر الحر 43.6-25.3بين tpc. تراوحت قيم lc-ms/msباستخدام تقنية t.claveryiلنوع الكمأة السوداء %70التركيب الفينولي للخالصة اإليتانولية تراوحت ملغ مكافئ من الكيرسيتين لكل غرام خالصة جافة. 6.8-2.5بين tfcمن حمض العفص )الغاليك( لكل غرام خالصة جافة، وملغ مكافئ لكل غرام خالصة جافة على ميكرومول مكافئ من حمض األسكوربيك 153.4-90.1ملغ/مل، و 9.0-5.6بين frapو 50ic (dpph)قيم صات، وهذا الترتيب. ُوجد تشابه كبير بين النوعين من حيث المحتوى والفعالية، إضافةً إلى تشابه محتوى الخالصة المائية وفعاليتها مع بقية الخال الفينولية التي تم التحّري عنها في مركباً من المركبات 14تبيّن وجود يعني أن طريقة تحضير الخالصة في الطب الشعبي يمكن االعتماد عليها. أهمها من غنيّاً . trans-cinnamic acidو syringic acidو p-hydroxy benzoic acidالخالصة، جديداً مصدراً بنوعيها الكمأة تشكل بالمركبات المضادة لألكسدة والتي يمكن االستفادة منها بالتطبيقات العالجية والتجميلية والغذائية. . dpph ،frap ،lc-ms/msالكمأة، عديدات الفينول، الفالفونوئيدات، المفتاحية: الكلمات 1corresponding author e-mail: a111atassi@gmail.com received: 30/10 /2021 accepted:19 /1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp184-192 iraqi j pharm sci, vol.31(2) 2022 comparison between black and white desert truffles 185 introduction truffle is one of the wild edible hypogeous macrofungi, and it belongs to the phylum ascomycota that do not have cap, gills and stalk unlike other fungi such basidiomycetes (1). truffle has hundreds of species. the species that grow in arid and semi-arid areas in mediterranean basin and middle east are called “desert truffle”, and are classified under genus of “tirmania” and “terfezia” that belong to pezizaceae family (2). some species were identified in syria as terfezia claveryi, terfezia boudieri, tirmania pinoyi and tirmania nivea (3), where they grow naturally in the desert plains such as the region between homs and deir ezzor. the season of gathering is between march and may. the quantity of truffles is usually varied from season to season, and it is dependent on rainfall and temperature, so truffles absent some years when weather conditions are not suitable (4,5). truffles have high values of proteins, minerals, unsaturated fatty acids, and antioxidant compounds (6,7), that candidates it to be as complements and complete food. bedouins use boiled truffle extract in treatment of trachoma and illnesses of eyes and skin, and they consume truffle as meat alternative (8). the application of truffle extract on infected eyes was advised by prophet mohammad (peace be upon him). species of truffle have wide biological activities such as antioxidant (9), antimicrobial (10), anti-inflammatory (11), anticancer (12), and antidepressant (13). types of wild and cultivated mushrooms are well known to contain various polyphenolic compounds which are considered as an excellent antioxidants (14,15). some articles studied the antioxidant activity and the antioxidant compounds of the two species of desert truffle (terfezia calveryi and tirmania pinoyi). the same species of desert truffles from different geographical regions may not exhibit the same chemical composition, because many environmental factors such as amount of rain, soil types and climatic changes have influence on the chemical profile (16). polyphenols are considered as secondary metabolites, and they are wide group of compounds that differ in structure and resources. they help the organism to defend against oxidant stress and free radicals which damage the cells (17). the use of modern technics in extraction of polyphenols increases efficacy, save time, and decrease amounts of solvents, furthermore they are convenient for thermo-sensitive compounds (18). dpph method is considered as one of the most common methods to determine the antioxidant activity according to hydrogen atom or single electron transfer, and it depends on a stable organic radical (2,2-diphenyl-1picrylhydrazyl) which changes from purple to yellow color when interacts with antioxidants (19). ferric reducing antioxidant power (frap) is another method to determine the antioxidant activity according to single electron transfer, and it measures reducing of fe (iii) to fe (ii) by antioxidants (20). desert truffle is famous fungi that has crucial value in traditional medicine, and there is a lack of information about chemical components and the importance of antioxidant activity of truffles that are harvested in syria. this study aims to compare between the most two abundance species according to their phenolic contents and antioxidant activities. materials and methods chemicals 2,2-diphenyl-1-picrylhydrazyl (dpph) and 2,4,6-tri-2-pyridinyl-1,3,5-triazine (tptz) were from (cayman chemical, usa). folin-ciocalteu's phenol reagent (fc) was from (sigma-aldrich, switzerland). l(+)-ascorbic acid (aa), ferric chloride hexahydrate, sodium acetate anhydrous, and sodium carbonate anhydrous were from (panreac, spain). gallic acid (ga) was from (titan biotech, india). quercetin (q) was from (sigmaaldrich, usa). aluminium chloride hexahydrate was from (riedel-de haen, germany). all other chemicals were of analytical grades. collection and preparation of fungi ascocarps were collected from desert region in the east of homs (palmyra and al-sukhna) during april 2020. they were intact and showed no sign of spoilage. the identification of two species was made advising with dr. fawaz al-azmah and dr. hijazi mando from faculty of agricultural engineering, damascus university, by macroscopic and microscopic (olympus bx41, japan) observations for fresh ascocarps and asci as described in the literature (21,22). ascocarps were cleaned, peeled, and sliced. the slices were dried in an oven at (40°c) and the size was reduced mechanically, then stored at room temperature in dry place away from light until use. determination of chemical composition these reactions were conducted according to standard methods (23–25). two species of truffle were evaluated before extraction to determine the presence of alkaloids, anthraquinones, saponins, coumarins, flavonoids, tannins, steroid and triterpenoid glycosides. the resulting solutions were observed for color change and/or precipitate formation to indicate positive results. preparation of extracts three extracts of each type were prepared. namely, powder of dried truffle was extracted by maceration with water, methanol and ethanol 70% separately (10 g of dried truffle/100 ml of solvent) at room temperature, and the extracts were exposed to ultrasonication (phylo ush-10d, italy) for 15 min at the beginning of extraction. after 24 h, the extracts were centrifugated and filtrated, then the solvent was evaporated by the rotary evaporator (great wall, rotary evaporator r-1001-vn, china) iraqi j pharm sci, vol.31(2) 2022 comparison between black and white desert truffles 186 at (45°c). finally, the dried extracts were stored in a refrigerator. determination of total phenolic content (tpc) total phenolic content was estimated by folin–ciocalteu method adapted from (al-laith, 2010) (9) with a slight modification. briefly, 20 µl of the extract, standard and the blank were added to 1.58 ml h2o, followed by the addition of 100 µl concentrated fc reagent. after 3 min standing at room temperature, 300 µl of sodium carbonate buffer (200 g/l) was added. the mixture was incubated at (40°c) for 30 min. the absorbance was measured at 736 nm (pg instruments uv-vis specrtophotometer t80+, uk). the phenolics concentration was determined by comparing with the standard calibration curve of gallic acid (y=0.0009x+0.00177, r2=0.9961), and results were reported as mg of gallic acid equivalent (gae)/ g of dry extract. determination of total flavonoid content (tfc) total flavonoid content was estimated by aluminium chloride colorimetric assay adapted from (sembiring et al., 2018) (26) with a slight modification. briefly, 0.5 ml of extract or standard were added to 100 μl of the 10% aluminium chloride solution and followed by 1.5 ml of 96% ethanol. finally, 100 μl of (1 m) sodium acetate was added to the mixture. ethanol 96% was used as blank. the mixture was incubated at room temperature for 40 min protected from light. the absorbance was measured at 415 nm. total flavonoid content was determined by comparing with the standard calibration curve of quercetin (y=0.0106x+0.077, r2=0.9986), and results were reported as mg of quercetin equivalent (qe)/ g of dry extract. dpph radical scavenging activity dpph˙ scavenging activity of the extracts was estimated according to (gouzi et al., 2013) (27) with a slight modification. various concentrations of extracts or positive control (ascorbic acid) (0.1 ml) were added to 2.9 ml of methanolic solution containing dpph radical (0.1 mm). the absorbance was measured at 515 nm after the mixture was left to stand at room temperature for 30 min in a dark place. the methanol was used as a blank. dpph free radical scavenging activity was calculated according to the following equation: dpph radical scavenging activity (%) = [(abs0 – abs1)/abs0] × 100 where abs0 was the absorbance of the negative control (methanol), and abs1 was the absorbance of the extract or the positive control. the extract concentration that scavenges 50% of dpph radical (ic50) was calculated from the graph that plots the percentage of radical scavenging activity against the extract concentration, also ic50 was calculated for the positive control (ascorbic acid). ferric reducing antioxidant power (frap) assay the reducing power of extracts was estimated according to (benzie et al., 1996) (28) with a slight modification. the frap reagent contained 2,4,6-tripyridyl-s-triazine (tptz) solution (10 mm) in hcl (40 mm), ferric chloride solution (20 mm), and acetate buffer (0.3 m) at ph 3.6 in the ratio 1:1:10 respectively. 100 μl of the extract, or standard, or blank was mixed with freshly prepared frap reagent (3 ml). the mixture was incubated at (37°c) for 30 min. the absorbance was measured at 595 nm. the results of frap assay were determined by comparing with the standard calibration curve of ascorbic acid (y=0.002x+0.0067, r2=0.9991), and were reported as micromoles of ascorbic acid equivalent (aae)/ g of dry extract. lc-ms/ms analysis of selected phenolics ethanolic 70% extract of t.claveryi was selected to qualitative detection of some phenolic compounds in it. this detection was carried out using liquid chromatography-mass spectroscopy (agilent technologies lc-ms/ms, usa) according to procedure of (orcˇic et al., 2014) (29) and (kıvrak, 2015) (6). data were acquired in multiple reaction monitoring (mrm) mode, using the optimized compound specific parameters (precursor ion, product ion, fragmentor voltage, collision voltage). nineteen phenolic compounds which were detected are the following: (apigenin, catechin, chlorogenic acid, trans-cinnamic acid, p-coumaric acid, 3,4dihydroxybenzaldehyde, eugenol, ferulic acid, gallic acid, gentisic acid, hesperidin, homogentisic acid, p-hydroxy benzoic acid, protocatechuic acid, pyrocatechol, rutin, syringic acid, vanillic acid, and vanillin). statistical analysis the results were expressed as a mean of three frequencies followed by standard deviation. statistical analysis was performed using spss software (version 25). one-way anova (tukey’s hsd) was used for comparison between means. values were considered significantly different when p-value<0.05. results and discussion macroscopic and microscopic identification of truffles, based on morphological characteristics of ascocarps and asci, reveals two species: terfezia claveryi (black truffle) locally called “obaidi”, and tirmania pinoyi (white truffle) locally called “zubaidi” (figures 1-4). iraqi j pharm sci, vol.31(2) 2022 comparison between black and white desert truffles 187 according to alsheikh (21,22), the ascocarp of two studied species have lobed shape and diameters of 4 to 10 cm. the studied species t.claveryi and t.pinoyi present different characteristics. t.claveryi has a brownish-yellow peridium and a gleba with fleshy appearance and yellow-pinkish color. asci are subglobose and contain 8 globose spores ornamented with warts. t.pinoyi has a white peridium, and a gleba with fleshy appearance and white color. asci are pearshaped and contain 8 smooth globose spores which have double outer layer. figure 1. an ascocarp of terfezia claveryi and a cross section of it. figure 2.an ascocarp of tirmania pinoyi and a cross section of it. figure 3. an ascus of terfezia claveryi which contains 8 globose spores ornamented with warts (colored with alcoholic iodine, magnification ×40). figure 4.an ascus of tirmania pinoyi which contains 8 smooth globose spores (colored with alcoholic iodine, magnification ×40). the results of detection about phytochemical components for dried t.claveryi and t.pinoyi indicate the probability of containing alkaloids, coumarins, flavonoids, phenols, and steroid and triterpenoid glycosides. t.claveryi also may contain little of saponins (table 1). this is the first time that determination of chemical composition to truffle was published. table 1. detection of phytochemical components for two species of truffle: terfezia claveryi and tirmania pinoyi. note: = absent, + = present phytochemical constituents tests results terfezia claveryi tirmania pinoyi alkaloids dragendorff + + mayer + + hager anthraquinones borntrager saponins foam + coumarins alcoholic fecl3 + + fluoresence + + flavonoids shinoda + + naoh + + ammonia + tannins/phenols ferric chloride + + lead acetate + + gelatin deposition matchstick + steroid and triterpenoid glycosides salkowski + + iraqi j pharm sci, vol.31(2) 2022 comparison between black and white desert truffles 188 the extraction’s yields of for both studied species of truffle were in the following arrangement (aqueous> ethanolic 70%> methanolic). the aqueous extracts had the highest yield (44.2%, 43.5%) for t.claveryi and t.pinoyi respectively. when we compare the yields of two species for each solvent, we observe high similarity (table 2). table 2. yields of extraction, phenolic and flavonoid contents, and antioxidant activities of terfezia claveryi and tirmania pinoyi extracts. type of extract yield of extraction (%) total phenolic content (mg gae/g dry extract) total flavonoid content (mg qe/g dry extract) dpph ic50 (mg/ml) frap (µmol aae/g dry extract) ac 44.2±9.4 a 43.6±9.6 a 3.2 ± 0.4 a,b 9.0 ± 1.8 a 126.1 ± 25.2 a,b mc 16.7±7.3 b 28.7±4.4 a,b 2.5 ± 0.4 a 8.6 ± 0.6 a 136.3 ± 14.8 a,b ec 21.9±3.0 b 32.7±0.4 a,b 3.7 ± 0.1 b 6.1 ± 0.7 a,b 153.4 ± 12.4 a ap 43.5±1.4 a 25.3±4.6 b 3.9 ± 0.3 b 8.8 ± 1.4 a 90.1 ± 26.6 b mp 11.5±0.9 b 30.4±7.1 a,b 5.0 ± 0.6 c 8.5 ± 0.4 a,b 134.8 ± 20.0 a,b ep 23.8±0.7 b 33.3±2.8 a,b 6.8 ± 0.2 d 5.6 ± 0.8 b 135.1 ± 14.7 a,b where ac, mc, and ec: aqueous, methanolic, and ethanolic 70% extracts of terfezia claveryi respectively. ap, mp, and ep: aqueous, methanolic, and ethanolic 70% extracts of tirmania pinoyi respectively. ic50: inhibitory concentration to 50% of substrate. values are means ± sd of three measurements. means within each column with same letters don’t differ significantly (p-value>0.05) according to tukey’s test. to evaluate the phenolic profile of the truffle’s extracts, the total phenolic and flavonoid contents were determined. the total phenolic contents (tpc) for the analyzed truffle extracts which were evaluated by the fc method are shown in (table 2). tpc values were between 25.3-43.6 mg gae/g dry extract. aqueous extract of t.claveryi (ac) was found to have the highest phenolic content, on the other hand, the lowest phenolic content was found in aqueous extract of t.pinoyi (ap). statistical study demonstrated that there were no significant differences between three extracts of the same species in tpc values. boufeldja et al. (30) found that tpc for methanolic 80% extract of t.claveryi was 15.5 mg gae/g dry weight, and this value resembles to present finding for aqueous extract. regarding to t.pinoyi, tpc for methanolic extract (30.4 mg gae/g dry extract) has been obviously above the values of (stojkovic et al., 2013) (31) and (gouzi et al., 2013) (27) (13.19 and 2.1 mg gae/g dry extract) respectively. depending on previous results, the tpc values for the two studied species of truffle were higher than tpc values for foods which are classified as rich in polyphenols such strawberry and cherries. this high level of polyphenols assists truffle to overcome severe stress condition in deserts (17,32). values of total flavonoid contents (tfc) for the analyzed truffle extracts were between 2.56.8 mg qe/g dry extract [methanolic extract of t.claveryi (mc) and ethanolic 70% extract of t.pinoyi (ep) respectively] (table 2). the flavonoid contents for ethanolic 70% extracts were the highest among other extracts for both species of truffle. values of tfc for t.pinoyi were approximately two fold the values of t.claveryi. kivrak (6) found that tfc for ethyl acetate extract of t.claveryi was 4.71 mg qe/g dry extract, and this value is slightly higher than the value of ethanolic 70% extract in this study (3.74 mg qe/g dry extract). tfc of t.pinoyi had not been studied previously. to extensively characterize the antioxidant potential of truffle extracts, two antioxidant assays were applied. the combination of more than one method helps in understanding the mechanism of action for antioxidants. with regard to ic50 values of dpph (table 2), they were between 5.6-9.0 mg/ml [ethanolic 70% extract of t.pinoyi (ep) and aqueous extract of t.claveryi (ac) respectively]. there were no significant differences between two species of truffle in ic50 values of dpph. ethanolic 70% extract of t.pinoyi (ep) exhibited the highest scavenging activity with the lowest ic50 (5.64 mg/ml). the reported scavenging activity for all extracts is too lower than the one showed by ascorbic acid (positive control, ic50= 0.11 mg/ml), but the comparison between pure compounds and extracts should be avoided, because they are individual/purified compounds and not mixtures, where the concentration of each individual compound is actually too lower. it seems that the ic50 value for methanolic extract of t.claveryi (8.6 mg/ml) was comparable to that was reported by (neggaz et al., 2015) (2) (8.6 mg/ml) for an extract which was prepared using soxhlet, by applicating same protocol, but the value of maceration extract was (22.16 mg/ml). gouzi et al. (27) and stojkovic et iraqi j pharm sci, vol.31(2) 2022 comparison between black and white desert truffles 189 al. (31) found that ic50 for methanolic extracts of t.pinoyi were (2.51 and 6.41 mg/ml respectively), and these values are lower than ic50 in this study (8.5 mg/ml), by applicating same protocol. the values of frap assay for investigated extracts were between 90.1-153.4 µmol aae/g dry extract [aqueous extract of t.pinoyi (ap) and ethanolic 70% extract of t.claveryi (ec) respectively] (table 2). there were no significant differences between the extracts of both truffle’s species, also between three extracts of the same species in frap values. our results were in agreement with those reported by (dahham et al., 2018) (33) where the values of reducing power for examined extracts of t.calveryi were also in this arrangement (ethanolic 70%> methanolic> aqueous). the value of frap for methanolic extract of t.pinoyi in this study (134.8 µmol aae/g extract) was higher than that in the work of (gouzi et al., 2013) (27) (126.8 µmol aae/g extract). these results demonstrate that the reducing power of truffle was high, and it was among the best foods in this field. the results of lc-ms/ms revealed the presence of 14 from 19 phenolic compounds which were detected in ethanolic 70% extract of t.claveryi, and they are listed in (table 3) starting by the most intensity. figure 5 presents the chromatograms of some founded phenolics in the extract. table 3.phenolic compounds that are founded in ethanolic 70% extract of t.claveryi (starting by the most intensity). where mw: molecular weight, rt: retention time. rt (min) product ion [mh](m/z) precursor ion [mh](m/z) total mw compound name 6.457 93 137 138.12 p-hydroxy benzoic acid 1 8.015 155.1 199 198.17 syringic acid 2 5.598 103 147 148.16 trans-cinnamic acid 3 15.844 119 163 164.16 p-coumaric acid 4 7.275 125 169.1 170.12 gallic acid 5 3.955 123 167 168.15 homogentisic acid 6 2.543 109 153 154.12 protocatechuic acid 7 6.542 92 151.1 152.15 vanillin 8 3.079 134 193 194.18 ferulic acid 9 6.376 301 609 610.5 rutin 10 5.099 107.9 166.9 168.15 vanillic acid 11 2.115 151 269 270.24 apigenin 12 8.002 245 289 290.27 catechin 13 15.209 303 611 610.6 hesperidin 14 iraqi j pharm sci, vol.31(2) 2022 comparison between black and white desert truffles 190 figure 5.chromatograms of some phenolics in ethanolic 70% extract of t.claveryi by lc-ms/ms. where (a): hydroxy benzoic acid, (b): trans-cinnamic acid, (c): vanillin, (d): vanillic acid kıvrak (6) found that p-hydroxy benzoic acid was the most abundance in the same studied truffle, and this resemble present results, but there is no existence of three compounds (gentisic acid, pyrocatechol, and 3,4-dihydroxybenzaldehyde) as a result of this study, also there are no existence of two phenolics (chlorogenic acid and eugenol) which are founded by vahdani et al (34). this variation in phenolic profile for studied samples of the same species may be due to variation of synthesis of these compounds as a result to differences in climate conditions, soil and geographical regions, or the difference in the method of extraction and the differences in the parameters of analytical methods. the prediction of the presence of these phenolic compounds supports medical, cosmetic and nutritional applications of truffle. conclusion the results of this work reveal that two studied species of desert truffle are rich in phenolic compounds, and all of their extracts showed appreciable dpph free radical scavenging activity and reducing power with frap assay, so they can act as antioxidant by donating a hydrogen or an electron. there is great similarity in content and activity between two studied species. the water can be use continually for extracting the phenolic compounds as it is used in traditional medicine, instead of other non-safe polar solvents, because the values of tpc are similar in the two cases. more profound studies of truffle and fungi generally which exist heavily in syrian environment should be performed, that may provide a great economic and medical benefits. 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nutritional, antioxidant and antibacterial properties of tirmania nivea, a wild edible desert truffle from tunisia arid zone. med aromat plants. 2016;5(4). 33. dahham ss, al-rawi ss, ibrahim ah, majid asa, majid amsa. antioxidant, anticancer, apoptosis properties and chemical composition of black truffle terfezia claveryi. saudi j biol sci. 2018;25(8):1524–34.34. 34. vahdani m, rastegar s, rahimzadeh m, ahmadi m, karmostaji a. physicochemical characteristics, phenolic profile, mineral and carbohydrate contents of two truffle species. j agr sci tech. 2017;19:1091–101. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ introduction: iraqi j pharm sci, vol.19(1) 2010 chronic renal failure on thyroid hormones 65 the effect of chronic renal failure on thyroid hormones layla k. ali *,1 *nursing department , koya technical institute abstract chronic renal failure (crf) affects thyroid function in multiple ways, including low circulating thyroid hormone concentration, altered peripheral hormone metabolism, disturbed binding to carrier proteins, possible reduction in tissue thyroid hormone content, and increased iodine store in thyroid glands.the target of study is to find a relationship between chronic renal failure and thyroid function.in addition, we tried to study the effect of crf on serum creatinine dependent on the level of thyroid hormones (t3 and t4) and thyroid stimulating hormones(tsh). forty patients with chronic renal failure (20 male, 20 female) were enrolled in this study in addition to forty healthy individual as control group (20 male, 20 female). the age ranged from (25 -65) years. t4, t3, tsh, urea, uric acid and creatinine were measured in each of the two groups. the results revealed statistically significant reduction in t3 and t4 while there is elevation in tsh, urea,uric acid and creatinine in the patients group compared to the control group. key word : chronic renal failure,thyroid hormones. الخالصة لٍت الهزموواث الدرقٍت بطزق مخعددة حخضمه قلت فً حزكٍش هزمون الغدة الدرقٍةت فةً الةد ٌؤثز القصور الكلوي المشمه على فعا حبةةدأ ت حغٍةةز اٌةةم الهزمةةون فةةً ايويةة ت الماٍطٍةةت تترحبالةةق بةةالبزتحٍه الىاقةةس خمالٍةةت اولدةةاة ميةةخو الهزمووةةاث الدرقٍةةت فةةً الدرقٍةت الهةدم مةه الدرااةت اٌ ةاد عالقةت بةٍه القصةورالكلوي المةشمه ايوي ت الماٍطٍت تالبالسما تكذلك سٌادة خةشن الٍةود فةً الغةدة تالغدة الدرقٍت بايضافت الى دراات حاثٍز الدشس الكلوي المشمه اعخمةادا علةى ميةخوٌاث الهزمووةاث الدرقٍةت تالهزمةون المادةش للدرقٍةت " مةه ايحةااك كم موعةت اةٍطزة اعمار الم ةامٍ شلصةا 04مزٌضا" مصابا" بالدشس الكلوي بايضافت الةى 04جمعج الىمادج مه ( اةةةىت حةةة قٍةةةاص هزمووةةةاث الغةةةدة الدرقٍةةةت ثالثةةةً ٌودٌةةةد الاةةةاٌزتوٍه تالااٌزتكيةةةٍه( الهزمةةةون المادةةةش 52 -52حخةةةزاتي بةةةٍه توٍه تالااٌزتكيةٍه للدرقٍت الٍورٌا امم الٍورٌك تالكزٌاحىٍه بٍىج الىخائج تجةود اولدةاة معىةوي فةً كةس مةه ثالثةً ٌودٌةد الاةاٌز فً ٍه تجد ارحداع ملاوظ فً كس مةه الهزمةون المادةش للدرقٍةت الٍورٌةا ةامم الٍورٌةك تالكزٌةاحىٍه فةً مزضةى القصةور الكلةوي مقاروت بايحااك introduction the thyroid gland produces two major related hormones thyroxine and triiodothyronine, commonly called t4 and t3, respectively, which play essential roles in the complete lack in the processes of metabolism, growth and development in most vertebrate tissue. complete lack of thyroid secretion usually causes the basal metabolic rate to fall 40 to 50 percent below normal and extreme excesses of thyroid secretion can increase the basal metabolic rate to 60 to 100 percent above normal [1, 2] . thyroid secretion is controlled primarily by thyroid stimulating hormone (tsh) secreted by the anterior pituitary gland. small amount of reverse triiodothyronine (rt3) and other compounds are also found in venous blood [1] . the synthesis of the thyroid hormones requires (150 -200 µg) of iodine daily. most dietary iodide is reduced to iodine before absorption. iodine after conversion to iodide in stomach is rapidly absorbed from the gastrointestinal tract and distributed in the extra cellular fluid [3] .there are three thyroid hormones binding proteins in human plasma: albumin, with high capacity and low affinity; thyroxine binding globulin (tbg), with low capacity and high affinity, and transthyretin (ttr) with an intermediate capacity [4] . thyroid hormone levels are under strict control; this is achieved mainly by feed-back inhibition through: hypothalamic-pituitary – thyroid axis (hpta); thyrotrpin – releasing hormone (trh).l.thyroid hormone regulation ,thyroid stimulating hormone (tsh) secretion or negative – feed back system on pituitary secretion of (tsh); and factors altering (tsh) secretion ,such as somatostatins dopamine and /or glucocorticoids [5] . some changes in thyroid function test results are observed in most of the acute and chronic illnesses (i.e. renal diseases, cardiovascular diseases, inflammatory conditions and pulmonary diseases). alteration in thyroid function test findings may reflect changes in production of thyroid hormone by effects on the thyroid itself, on the hypothalamic pituitary thyroid – axis, on peripheral tissue metabolism of the hormones, or a combination of these effects [6] . 1corresponding author email : nergzkarem@yahoo.com received : 13/12/2009 accepted : 27/4/2010 iraqi j pharm sci, vol.19(1) 2010 chronic renal failure on thyroid hormones 66 a general conviction exists that patients with thyroid function test abnormalities do not have hypothyroidism despite the low serum hormone levels in blood and low t3 in most of the tissues. many patients with nonthyroid illness (nti) also receive drugs-that affect thyroid hormone regulation and metabolism. this discussion does not consider pharmacological interference an intrinsic part of the spectrum of changes in hypothalamic – pituitary thyroid function that occur in (nti) [6] . assessment of thyroid function in patients with nonthyroid illness is difficult. many of them have low serum concentrations of both t4 and t3 and their serum (tsh) concentration may also have been low. previously, these patients were thought to be eurthyroid, and the term eurthyroid – sick syndrome was used to describe the laboratory abnormalities. it is possible that the changes in thyroid function during severe illness are protective in that they prevent excessive tissue catabolism [7] . there are two important general principles in laboratory assessment of thyroid: thyroid function should not be assessed in seriously ill patients unless there is strong suspicion of thyroid dysfunction. when thyroid dysfunction is suspected uncritically ill patients, measurement of serum(tsh) alone is inadequate for the evaluation of thyroid function [7]. chronic kidney disease is defined as either kidney damage or a decreased kidney glomerular filtration rate(gfr) of less than 60 ml / min / 1.73 m 2 for 3 or more months [8] . the causes of the chronic kidney disease could be due to primary and secondary glomerular disease, tubulointerstial disease and vascular disease [9] . previous studies on thyroid function tests indicate lower thyroid hormone concentration (t3, t4) with normal tsh in heaemodialysed patients compared with normal subjects [10] . thyroid gland produces t4 but only 20% of the most metabolically active thyroid hormone t3 and 5% to 8% as the calorgenically inactive reverse t3 (rt3) hormone and t4 in tissues such as liver,kidneys and muscles [11] . haemodialysis employs the process of diffusion across a semi permeable membrane to remove toxic products and excess fluid from the blood, while adding desirable components [12] .the aim of this work is to evaluate thyroid gland function in chronic renal failure patients as an attempt to find a relationship between chronic renal failure and thyroid dysfunction. materials and methods -selection of subjects &blood collection forty patients with chronic renal failure (20 male, 20 female) were enrolled in this study in addition to forty healthy individual as control group (20 male, 20 female). the age ranged from (25 -65) years.to compare the significance of the difference in the mean values in comparison groups, student t test was applied; p ≤ 0.05 was considered statistically significant. patients with ischemic heart disease, diabetes mellitus and thyroid disease (such as hypothyroidism, hyperthyroidism and goiter) were excluded [13] . five ml of venous blood were aspirated from control group and crf patients at 8:00 9: 00 am. blood samples were collected into plain test tubes and centrifuged after 30 minutes of collection for 10 minutes at 3000 rpm.serum was frozen at -20 c 0 till used in determination of t3, t4, tsh ,urea , uric acid and creatinine. determination of t3, t4 and tsh total triiodothyronine (tt3), total thyroxin (tt4) and thyroid stimulating hormone (tsh) were evaluated using vidas (t3) ref, 30403, vidas (t4) ref 304041 and vidas (tsh) ref. 30400 from biomerix (france) respectively.the principle of the quantitave determination of t3, t4 and tsh combines an enzyme immunoassay competition method with a final fluorescence detection (eifa). -determination of urea urea concentration levels were determined in serum by using enables end point enzymatic (urease – modified berthelot reaction) in which urease hydrolyzes urea in an alkaline medium.the ammonium ions react with the salicylate and hypochlorite to form a green colored indophenol.the reaction is catalyzed by the sodium nitroprusside [4] . -determination of uric acid uric acid concentrations were determined by using uricaseperoxidose chromogen sequence in which hydrogen peroxide is formed and reacts as tinder type reaction [4] . -determination of creatinine creatinine levels were evaluated according to jaffes method.the production of orange color after the addition of alkaline picrate. the color is proportional to the concentration of creatinine [4] . iraqi j pharm sci, vol.19(1) 2010 chronic renal failure on thyroid hormones 67 results and discussion table (1) shows the (mean ± sd) of t3, t4, tsh, urea, uric acid and creatinine concentrations in sera of patients with chronic renal failure and control group,in which p ≤ 0.05 was considered significant . table 1: levels of t3, t4, tsh, urea, uric acid and creatinine concentrations in sera of patients with renal failure and control group. this study shows highly significant reduction in t3 and t4 concentration in patients serum with crf compared to control group (p ≤0.05) . intensive studies revealed that renal insufficiency affects thyroid function in multiple ways, including altered peripheral hormone metabolism, disturbed binding to proteins, reduction in tissue thyroid hormone content, and iodine accumulation in thyroid gland [14,15] .another explanation is that the reason for the decrease in t4 could be attributed to multiple factors such as deficiency of thyroxine binding globin (tbg) [16,17] . in the present study serum tsh was measured in crf and control groups.crf group had tsh above normal range. some authors interpret this tsh elevation as a sign of recovery from a hypothyroid state despite the distortion of tsh in some euthyroid patients with nti, who have significant elevation of tsh due to underlying primary hypothyroidism [18] . highly significant elevation was found in urea, uric acid and creatinine in serum of crf patients compared to control group.increase in urea in renal failure are caused by impaired ability to excrete proteinaceous catabolites because of marked reduction in glomerular filtration rate(grf).increases in serum creatinine are also a result of decreased renal excretion [19] . references 1. guyton a.c. and hall j. e.: "text book of medical physiology",11 th ed, sanders, elsevier inc 1600 john f. kenedy blvd, philadelphia,2006, 2-9. 2. iwasaki y., morishita m., asai m. , onish: a., yoshida m., oiso y. and inoue k. :effects of hormones targeting nuclear receptors on transcriptional regulation of the growth hormone gene in the mtt/s rat somatotrope cell line . neuroendocrinology, 2004, 79, 229 – 236. 3. andreoli, bennett, carpenter:" cecil essentials of medicine", 4 th ed, w. b. saunders company, new york,1997,111116. 4. benvenga s.: "the thyroid, fundamental of clinical text books", 9 th ed. le,utiger rd(eds),lippincot williams and willkins, philadelphia ,2005, 97. 5. zoeller r. t., tan s.w. and tyl r. w:"general background on the hypothalamic-pituitarythyroid(hpt)axis", crit rev toxical. ,2007 ,37 (1-2),11 53. 6. serhat aytug md., lawrence e and shapiro md :euthyroid sick syndrome,emedicine specialties,endocriology,thyroid,2007,18 ,55. 7. douglas s. ross:thyroid function in non –thyroid illness,: up to date cme, 2007,134,1-7. 8. levey as, coresh j and bake e.:national kidney foundation practice guidelines for chronic kidney disease:evaluation,classification,and stratification, ann intern med.,2003, 139(2),137-147. 9. chobanian av, bakris gl, black hr, cushman wc: green la and izzo jl:the seventh report of the joint national committee on prevention, detection, evaluation and treatment of high blood pressure, jama,2003, 289 (19),2560 – 2572. 10. goffin e., oliveira dbg, raggatt p. and evans db:assessment of the thyroid function of patients undergoing regular haemodialysis, nephron.,1993,65, 568– 572. 11. shamsadini s. , darvish and abdollah h.:blood urea nitrogen and thyroid hormone levels before and after haemodialysid, health journal,2006,12,55. 12. hakim rm and lazarus jm. :medical aspects of haemodialysis,in:brenner bm,rector fc,eds. the subjects control (n=40) patients (n=40) t -test t3(ng/ml) 1.55 ± 0.54 0.63±0.05 p ≤ 0.05 t4(µg/ml) 9.13±2.84 3.32±1.99 p ≤ 0.05 tsh ( µiu/ml) 2.41± 0.52 6.22±3.31 p ≤ 0.05 urea (mg/dl) 35.27±5.94 120.81±20.62 p ≤ 0.05 uric acid (µmol/l) 300.7± 85.05 1220.6±24.1 p ≤ 0.05 creatinine (mg/dl) 0.94 ± 0.19 7.42 ±1.94 p ≤ 0.05 iraqi j pharm sci, vol.19(1) 2010 chronic renal failure on thyroid hormones 68 kidney.philadelphia, saunders ,1986 ,5,1791. 13. lisandro lrizarry md. , nadine a. and jeffry g.:toxicity,thyroid hormone ,medicine specialties, emergency medicine,toxicology,2008,65,44-50. 14. lim vs:thyroid function in patients with chronic renalfailure, am j. kidney discase,2001,38(1), s80 – s84. 15. kaptein em , quion – verde h., chooljian cj, tany ww , friedman pe , rodriquez hj and massry sg:the thyroid in end –stage renal diseases, medicine (baltimore),1988,67,187 – 197. 16. biff f. palmer : metabolic disturbances in chronic renal failure,saudi journal of kidney disease and transplantation,2002,13(3),273-280. 17. boelen a, mass ma, lowik cw., platvoet mc. and wiersinga wm:induced interleukin-6 (il-6) knockoutmice:a causal role of il-6 in the development of the low 3,5,3triiodothyronine syndrome, endocrinology,1996,137,5250 – 5254. 18. michalak m. , vagenakis ag. , and makri: m:dissociation of the early decline in serum (t3) concentration and serum il-6 rise and tnfalpha in nonthyroidal illness syndrome induced by abdominal surgery,j. clin. endocrinol metab.,2001,86 (9),4198 – 4205. 19. shivananda nayak b.: "manipal manual of clinical biochemistry", 3 rd ed., medical publishers ltd., new delhi,2007,182 – 186. prevalence of β-thalassemia carriers among a cohort of university students in hawler province of iraqi kurdistan iraqi j pharm sci, vol.18(2) 2009 β-thalassemia in iraqi kurdistan 15 prevalence of β-thalassemia carriers among a cohort of university students in hawler province of iraqi kurdistan abdulkadir a. alnakshabandi *,1 and huda a. muhammad** * department of clinical analysis, college of pharmacy, hawler medical university, kurdistan region, iraq. **hematologist in pediatric hospital, ministry of health, kurdistan region, iraq. abstract a representative sample of a thousand volunteer university students was screened for evidence of thalassemia minor.complete blood counts using automated blood cell analysers and blood smears were examined. patients having anemia, abnormal red cell indices or morphological features of thalassemia minor like hypochromia, microcytosis, target cells erythrocytosis and family history of thalassemia were then investigated for determination of hba2 & hbf levels. estimation of hemoglobin a2 was performed by micro-column chromatography while hbf was done using alkali denaturation. seventy seven out of the thousand samples tested positive for thalassemia minor. they all showed a hemoglobin a2 of more than 3.6 percent and higher, associated in most of the cases with mild anemia, erythrocytosis and hypochromic microcytic red cells. we reached to the conclusion that the prevalence of thalassemia minor in our community, represented at college students at fertile age, to be 7.7%. we hope that similar figures could be made available in the future for the rest of kurdistan and the bigger iraq so that a national figure could be presented to the world literature. key word : β-thalassemia , hemoglobin a2 , hemoglobin f الخالصة الٚجاد َسثة اَتشاس أ ( طانة ٔطانثّ جايؼّٛ فٙ يحافظة استٛم ٔتى اجشاء انًسح 0111فٙ ْزِ انذساسّ تى اخز ػُّٛ يؤنفّ يٍ ) انكايم نًكَٕات انذو ٔاجشاء فحص فهى انذو ٔقذ تى فشص تفشٙ حًهة صفة فقش انذو انثحش٘ فٙ ْزِ انؼُّٛ . نقذ تى اجشاء فحص انتحهٛم انحاالت نهطالب انزٍٚ ٚؼإٌَ يٍ فقش انذو كزنك حٍٛ ٚكٌٕ حجى انكشٚات انحًشاء أ َسثة تشثؼٓا تانخضاب اقم يٍ انحذ انطثٛؼٙ ، كزنك انة تاسٚخ ػائهٙ نفقش انذو ، نكم ْزِ حٍٛ ٚكٌٕ ػذد انكشٚات انحًشاء اكخش يٍ انحذ انطثٛؼٙ , ٔخصٕصاً حٍٛ ٚصاحة رنك نذٖ انط ( طانثاً يٍ انؼُّٛ انًذسٔسة ٚحًهٌٕ انصفة انٕساحّٛ 77( . نقذ تثٍٛ اٌ )f( َٕٔع )a2انحاالت اجش٘ فحص تحذٚذ َسثة خضاب انذو َٕع ) خفٛف ٔاصدٚاد ػذد %( ٚصاحثّ فٙ يؼظى انحاالت فقش دو 6.3اكخش يٍ ) a2نفقش انذو انثحش٘ ٔجًٛؼٓى اظٓشٔا َتائج خضاب انذو ثة انكشٚات انحًشاء يغ قهة حجى انكشٚات ٔقهة تشثؼٓا تانخضاب. َستُتج يٍ ْزا اٌ اَتشاس حًهة صفة فقش انذو انثحش٘ فٙ يجتًؼُا يًخهّ تطه اء كٕسدستاٌ %( َٔأيم اٌ تتٕفش دساسات ٔاسقاو يًاحهّ نٓزِ انذساسّ فٙ انًستقثم انقشٚة نكافة اَح7.7انجايؼة حٛج ػًش انخصٕتّ ْٕ ) . ٔانؼشاق ٔيٍ حى انحصٕل ػهٗ اسقاو ٔطُٛة تقذو نهثحٕث انؼانًّٛ introduction the thalassemias are characterised by reduced synthesis of one or more of the globin chains that form the oxygen-carrying hemoglobin molecules found in red blood cells (18) . hemoglobins are tetrameric molecules, with 2 α-like and 2 β-like globin polypeptide chains, each associated with a heme group (21) . in normal adult, hba (α2β2) accounts for around 97.5% of the hemoglobin in erythrocytes; there is another component called hba2 (α2δ2), which normally constitutes <2.5% of total hemoglobin. fetal hemoglobin or hbf (α2γ2) is the major hemoglobin synthesised before birth, but normal adult have< 1% hb f (1) . β-thalassemia constitutes one of the most serious health problems worldwide, accounting for a major number of childhood deaths per year primarily in regions of the world endemic for malaria (19) . it is an autosomal recessive disorder characterized by microcytosis and hemolytic anemia. it results from a variety of molecular defects that reduce (β + -thalassemia) or abolish (β 0 -thalassemia) the synthesis of the β-globin chains of hemoglobin (2) . β-thalassemia mutations differ greatly in their phenotypic effects. these could range from the extremely mild mutations, which are both clinically and phenotypically silent (3,20) , to those which are rare and produce 1 corresponding author e – mail : abdulqader.aziz@hawlermu.org received : 16 / 12 / 2008 accepted : 18/ 5 /2009 iraqi j pharm sci, vol.18(2) 2009 β-thalassemia in iraqi kurdistan 16 phenotype of thalassemia intermedia, even in the heterozygous state, due to the inheritance of a single copy of the abnormal gene (4) . between these two extremes lie the majority of βthalassemia mutations whose carriers are asymptomatic, whereas homozygotes and compound heterozygotes suffer from a transfusion-dependent anemia (5) . in iraq, there is a definite need for a carrier screening program. it is really hard to reach a consensus regarding the time of screening due to lack of education and public awareness about the disease. the stigma attached to being a carrier for thalassemia gene usually creates reactions against blood testing for this public health problem. there have been reports for the incidence of thalassemia minor in different provinces and cities of iraq with varying results, but usually ranging between “3.7% to 6.5%”. it was reported as 6.5% in mosul (6) ; 4.6% in basrah (7) ; 4.4% in baghdad (8) and 4.1% in sulaimaniyah (9) . our aim is to find the prevalence in erbil city to complement data already available. hopefully, in the future a national survey will utilize athe data available to provide a consensus figure for the prevalence of this extremely important genetic problem. subjects and methods one thousand university students were randomly involved in this study, including (368) females and (632) males, aged 18-49 years (s.d±5.4). students were included in this study on a voluntary basis. participants were requested to give information regarding personal and health data. after having all the requested permissions, sample collection started on december 12 th 2007. samples were collected in the colleges after giving a brief talk to the students regarding the nature of the disease and concentrating on the way to prevent it by population and premarital screening programmes. one thousand university students were screened. samples collection ended on may 2008. a sample of 2 ml venous blood was obtained by venipuncture from each participant and collected into edta tube. a complete blood count (cbc) using electronic blood analyzer that was coulter counter model (act diff beckman with eighteen parameters) including (hb, wbc count, platelet count,packed cell volume(pcv),mean cell volume(mcv), mean cell hemoglobin(mch),mean cell hemoglobin concentration(mchc),red blood cell(rbc) count. those with low red cell indices (mcv, mch and mchc) and high rbc count were further investigated for determination of hemoglobin a2(hba2) and hemoglobin f (hbf) levels. cbc was done within one hour of sample collection. for red cell morphology; freshly prepared, leishmann stained blood films were used. any individual is considered as a carrier when in addition of having low red cell indices has an elevated hba2 (to more than 3.5% ) with or without elevation of hbf. hba2 estimation was done chromatographically using commercial kit (beta-thal hba2 quik column) from (helena laboratories). hemoglobin f estimation was done by alkali denaturation method (10). results a total of one thousand students were studied, they included 632 males (63.2%) and 368 females (36.8%) correspond to a male: female ratio of 1.7:1. ages of screened students ranged between 18-49 years with a mean age of 22.7 years (s.d±5.4). seventy five percent of them were below 24 years. figure 3 shows the age and sex distribution of studied students. six hundred ninety eight students were from erbil city, the rest were from koya, soran, khabat, dashti hawler and shaqlawa fig 2 (all these towns related to erbil) . figure 1: age and gender distribution of studied students. 252 190 292 120 60 26 17 16 0 50 100 150 200 250 300 350 400 450 18-21 22-25 26-29 30-33 34-37 38-41 >41 age group female male area 698 95 69 60 40 31 7 0 100 200 300 400 500 600 700 800 erbil koya soran khabat dashty hawler shaqlawa choman series1 population sample figure 2: residence of screened students. iraqi j pharm sci, vol.18(2) 2009 β-thalassemia in iraqi kurdistan 17 ccording to the results of the cbc and hemoglobin fractions pattern; subjects were divided into three groups: group i: individuals with normal cbc. this group included 857 students. group ii: individuals with low red cell indices and high hba2 levels with or with out elevation of hbf levels, these individuals were considered to be β-thalassemia carriers. this group included 77 students; this figure makes the frequency of β-thalassemia trait in this sample (7.7%). group ш: individuals with low red cell indices but normal hemoglobin fractions, these were labeled as having anemia with low indices this group included 66 students. figure 3 shows prevalence of β-thalassemia minor among the studied sample. the various haematological parameters of studied groups are summarized in table (1) figure 3 :distribution of students in different groups .table1: statistical summary of hematological and clinical parameter of studied subjects. variable group number mean standard deviation p value age normal 857 22.7 3.7 0.615 tha. minor 77 23.1 3.5 h.ch.anemia 66 22.9 4.2 total 1000 22.7 3.8 *hb gm/dl normal 857 14.4 1.6 0.000 tha. minor 77 12.7 1.8 h.ch.anemia 66 12.5 2.4 total 1000 14.1 1.8 pcv% normal 857 42.7 5.6 0.000 tha. minor 77 39.9 5.2 h.ch.anemia 66 38.7 6.7 total 1000 42.2 5.8 mcv fl normal 857 87.9 3.8 0.000 tha. minor 77 69.1 8.3 h.ch.anemia 66 75.3 6.3 total 1000 85.6 7.3 mch pcg normal 857 29.7 1.7 0.000 tha. minor 77 22.1 3.2 h.ch.anemia 66 24.4 2.9 total 1000 28.8 3.1 mchc g/dl normal 857 34.1 0.9 0.000 tha. minor 77 31.5 1.5 h.ch.anemia 66 31.9 1.7 total 1000 33.8 1.3 rbc×10ˆ 6 /ul normal 857 4.9 0.5 0.000 tha. minor 77 5.8 0.8 h.ch.anemia 66 5.1 0.8 total 1000 5.0 0.6 *hemoglobin f was elevated to more than 1% in 33% of carriers. no significant differences in total hb, hba2 level and other red cell indices were noted between carriers with elevated hbf and those with normal hbf level. 857 77 66 normals thal.minor anemia with reduced indices iraqi j pharm sci, vol.18(2) 2009 β-thalassemia in iraqi kurdistan 18 discussion among our studied subjects male predominated. out of a thousand people we studied, 632 were male and 368 were female, with a male to female ratio of 1.7 to 1. 77 subjects were β-thalassemia carriers, giving it a prevalence arte of 7.7%. of the carriers, 52 (8.2%) were male and 25 (6.8%) were females. the mean age of the normal subjects was 22.7 years, while mean age of the thalassemia carriers was 23.1 years, this result is not significant. the residence distribution of the studied students showed that 698 were from erbil center, 95 subjects from koya, 69 subjects from soran, 60 from khabat and the rest were from the other provinces of hawler shown in figure 1. the bulk of carriers were from erbil center (56) which means that 72.7% of carriers were from erbil center. the second populous city studied was koya; there were 95 subjects tested and seven of them were carrier, that is (9%) of carriers are from this town. according to the number of subjects tested, the third town was soran, with sixty nine person tested and one carrier = (1.29%) of all carriers. then khabat from which we have 60 subjects, eight of them were carriers (10.3%) of all the carriers. then shaqlawa from which we have 31 student and four of them (5.2%) were carriers. in iraq, few studies has been done on this topic, in baghdad a study have been done (8) , on 500 pregnant ladies and it revealed a percentage of 4.4% βthalassemia carriers. another study was done in basra and the prevalence was 4.6% (7) , in duhok it was 3.7% (6) , sulaimani was 4.14% (9), this study was done on couples as premarital test and in mosul it was 6.5% (6) , and this result is comparable to the relatively high prevalence of β-thalassemia minor as documented in this study from erbil. erbil region is a region located in the north of iraq on (14.428) square kilometer with an estimated population of 1,392,093 (mopdc/undp, 2005) mainly kurdish muslims, this region includes seven main towns, these in turn includes hundreds of small towns and villages, hawler province is about 400 meter above the sea level. malaria was endemic throughout iraq including erbil. it would be expected to find thalassemia genes prevalent in erbil and we are now in the control phase of eradication of malaria in hawler. regarding the countries near or neighbouring iraq, the estimated prevalence of β-thalassemia minor was, in qatar 28% (11) , saudia arabia 3% (12) , lebanon 2 to 3% (13,14) , jordan 3-3.5% (15) , prevalence in turkey is ranging between 3.4 in east anatolia to 11% in western thrace and antalya (16,17) , these results are comparable to our results. the mean difference between the means of mcv for the three groups were 88, 69 and 75 fl respectively; here there is an obvious difference of 19 fl between the mcv of the normal students in comparison with the carriers. the mch values were 29.7, 22.1, 24.4 pcg respectively with a p value of less than 0.001which is highly significant difference. 7.6 pcg was the difference between the mch values of the carriers and the normal students(table 1). mean cell hemoglobin concentration (mchc) values were 34.1, 31.5 and 31.9 g/dl respectively; there is a significant difference of 2.6 g/dl between the normal subjects and carriers with a p value of < 0.001. finally rbc counts were notably elevated among β-thalassemia carriers as compared to the normal. rbc counts were 4.9, 5.8, 5.0 ×10ˆ 6 /ul with 0.9 ×10ˆ 6 /ul difference between carriers and normal subjects and p value was < 0.001(tabe 1). during this study complete blood count (cbc) was performed on a total of one thousand university students. it was the cornerstone to determine the subjects on whom hba2 had to be estimated. reduced mcv or mch values in the majority of heterozygous βthalassemia has been used as a basis for population screening for these disorders (18,,22) , and although cut-off values for the mcv and mch of 80 fl and 27 pcg respectively may involve a relatively large number of confirmatory hba2 estimation it would detect virtually all affected cases. hemoglobin a2 estimation was done for 143 students with hypochromic microcytic parameters. seventy seven of these were β-thalassemia carriers based on elevated hba2 levels. no association could be noticed between the severity of the anemia and the hba2 level. it was noted that increasing level of hba2 above 6% was negatively associated with mcv. , when mcv values were less than 72 fl, among 11 carriers with hba2 more than 6% we have 9 individual (81.8%) whom mcv values were less than 72 fl. same thing when applied to mch we have 81.8% of carriers having mch values of (22 pcg) and less, no such relation could be found between hba2 level and rbc count. conclusions 1. this study revealed that the prevalence of thalassemia minor or thalassemia carrier state in our community is 7.7%. 2. thalassemia carriers can be detected through clinical examination and complete blood count. the cardinal feature of thalassemia carrier state is elevated hba2 level . iraqi j pharm sci, vol.18(2) 2009 β-thalassemia in iraqi kurdistan 19 references 1. kanavakis e, traeger-synodinos j, editor (2006),molecular basis of thalassemia syndromes ,schrier sl, disorders of iron homeostasis, erythrocytes, erythropoiesis. 2. angastiniotis m, modell b. global epidemiology of hemoglobin disorders. ann n y acad sci; (1998) , 850:251-9 3. rosatelli, m.r., pischedda, a., meloni, a., saba, l., pomo, a., travi, m.,fattore, s., & cao, a. (1994) ”homozygous βthalassemia resulting . 4. thein, s.l., “dominant β-thalassemia: molecular basis and pathophysiology,” british journal of hematology, (1992), 80, 273-277. 5. weatherall, dj., clegg, jb., higgs, d.r.,& wood, w, g., “the hemoglobinopathies,”in cr, scriver, al, beaudet, ws, sly, and d, volle (editors) the metabolic basis of inherited disease, (1989) , 2281-1339, mcgraw-hill, usa. 6. al-allawi nas , jubrael jms, anwar a.,fariq f.(2007) service indicators from a regional hemoglobinopathy preventive program in duhok-iraq. proceedings of the 2 nd panaarab human genetics conference, dubai, cags, 20-22 2nd november pp78 7. hassan mk, taha jy, al-noama lm, widad nm, jasim sn frequency ofhemoglobinopathies and glucose-6 phosphate dehydrogenase deficiency in basra. east. mediterr. health j; (2003), 9(1/2): 1. 8. yahya hi, khalel kj, al-allawi nas, ferial hilmi f, thalassemia genes in baghdad-iraq. east. mediterr. health j ; 2 (2):315-319 9. jalal sd, al-allawi nas, faraj ah, ahmed nh, prevalence of hemoglobinopathies in sulaimani-iraq dohuk med. j. 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(1991) ,11;373-6 16. aksoy m, dincol, g, erdem s, “survey on hemoglobin variants, βthalassemia, glucose -6-phosphate dehydrogenase deficiency and haptoglobin types in turkish people living in manavgat, serik and boztepe (antalya),” human heredity, (1980) , 30, 3-6. 17. aksoy m.,kutlar, a., kutlar f., dincol g.,erdem, s.,and bastesbihci, s., “survey on hemoglobin variants, β+-thalassemia, glucose-6phosphate dehydrogenase deficiency and haptoglobin types in turksfrom western thrace, journal of medical genetics. (1985),22, 288-290. 18. weatherall dj, editor. disorders of globin chain synthesis, williams hematology, 7 th edition, mcgraw-hill medical ,(2006), 1, 651. 19. centis f, tabellini l, lucarelli g, the importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with βthalassemia major. blood ; (2000) , 96 :3624-9. 20. weatherall dj editor hoffbrand av, catovsky d, tuddenham egd hemoglobin and inherited disorders of globin chain synthesis in postgraduate hematology, 5 th edition, blackwell scientific pub (2005),pp 85-90. 21. kong y , zhou s, kihm aj,loss of alphhemoglobinstabilizingprotein impairs erythropoiesis and exacerbates beta thalassemia. j. clin. invest; ,(2004) , 114:1457-1466. 22. klee g g. role of morphology and erythrocyte indices in screening and diagnosis. the automated hematology group (cbc) as a screen for thalassemias and hemoglobinopathies , in hemoglobinopathies and thalasasemias, labo ratory methods and clinical cases (ed v f fair banks). decker newyork, (1980), p.38. iraqi j pharm sci, vol.30(2) 2021 the anti-asthmatic activity of guggulsterone doi: https://doi.org/10.31351/vol30iss2pp64-70 64 study the anti-asthmatic activity of guggulsterone in ovalbumin-induced asthma in rat zainab h. ahmed *,1 and munaf h zalzala** * department of pharmacology and toxicology, college of pharmacy, university of basra, basra, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract asthma is a chronic inflammatory respiratory disease associated with the changes of asthmatic airway structural that result from interact remodeling and inflammatory processes lead to obstruction of airway. guggulsterone (gs) is a bioactive compound and plant steroid present in guggul gum of commiphora wightii, which has anti-inflammatory and antioxidant activities. this study designed to investigate of anti-inflammatory activity of gugglsterone in improvement of asthma. forty eight healthy albino male rats divided to six groups, group i: control group (distal water), group ii: positive control group (distal water) with sensitization, group iii: guggulsterone (25 mg/kg/day) with sensitization, group iv: guggulsterone (50 mg/kg/day) with sensitization, group v: prednisolone (4.12 mg/kg/day) with sensitization, group vi: guggulsterone (50mg/kg/day) without sensitization. rats were sacrificed and blood samples were collected to prepare of serum samples that used in elisa kits for measuring of il-4, il-5, il-33, tnf-α, & ige. in addition, wbc counts in bronchoalveolar lavage fluid. all parameters (il-4, il-5, il-33, tnf-α, & ige) levels for rats of treated groups with gugglsterone were significant (p<0.05) reduced in compared to sensitized group. similarly, wbc count for rats treated groups with guggulsterone was significantly (p<0.05) fewer than sensitized group. in conclusion, our results provide a clue that guggulsterone has a potent anti-inflammatory activity that improved ova-induced asthma and is useful for the preventive of allergic airway disease in rodents. keywords: anti-inflammatory activity, asthma, guggulsterone, ovalbumin. دراسه فعاليه المضاده للربو للجوجلستيرون لعالج ربو الجرذان المحفز بااللبومين زينب هارون أحمد *،1 ومناف هاشم زلزلة ** فرع الدوية والسموم ،كلية الصيدلة، جامعة البصرة ، البصرة ، العراق* والسموم ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراقفرع الدوية ** الخالصة هو احد االمراض االلتهابات المزمنه وعند حصوله يكون مصحوب بتغيرات في هيكليه المجاري التنفسيه ناتجه عن عمليات الربو ج الصمغي االلتهاب وتغيرات تصمميه تؤدي الى تضيق المجاري التنفسيه، جوجلستيرون احد المركبات النباتيه االستيروديه الفعاله موجوده في الراتن لمنتشره في الهند،وهذا المركب يمتلك فعاليات مضاده لاللتهاب واالكسده.الغايه من هذه الدراسه لمعرفه فعاليه مضاده لاللتهاب للشجره شائكه ا جرذان البينو الذكور السليمه مقسم الى ستة مجاميع، المجموعه االولى اعطيت ماء نقي، المجموعه الثانيه اعطيت 48للجوحلستيرون في عالج الربو. يوم مع تحسس ، 14مغم /كغم/يوم( لمده 25يوم(، المجموعه الثالثه اعطيت الجوجلستيرون ) 14نقي مع تحسست لتهيج الربوبماده البومين )ماء مغم /كغم/يوم( 4.12يوم مع تحسس ، المجموعه الخامسه اعطيت بريزيلون ) 14مغم /كغم/يوم( لمده 50المجموعه الرابعه اعطيت الجوجلستيرون ) تم انهاء حياة الجرذان للحصول 15يوم ، وفي اليوم 14مغم /كغم/يوم( لمده 50يوم مع تحسس.المجموعه السادسه اعطيت الجوجلستيرون ) 14لمده وكذلك حساب كريات الدم البيضاء في سائل الرئوي. واظهرت النتائج على عينات الدم واستخالص السيروم لقياس عدة متغيرات بجهاز االاليزا، قويه و ، لذلك نستنتج بان الجوحلستيرون يمتلك فعاليه مضاده لاللتهاب ملحوظ للمتغيرات وكذلك كريات الدم البيضاء عن المجموعه الثانيه نقصان الناتج من التحسس ومنع التهاب المجاري التنفسيه للقوارض. مفيده في معالجه الربو .البومين جلستيرون،، الربو، جولتهابفعاليه مضاده لال :الكلمات المفتاحية introduction asthma is a chronic inflammatory respiratory disease, associated with changes of asthmatic airway structures that result from remodeling and inflammatory processes.(1) in the inflammatory process, common features as exudation, vascular congestion, and inflammatory cell agglomerate in the interstitial tissue. the chronic changes of inflammatory stage develop epithelial-mesenchymal interactions.(2) the fibrosis of subepithelial layer is one of important features of airway remodeling that initiated from simple thickening to prolong fibrosis.(3) 。the formation of pro-inflammatory cytokines like il-4, il-5, can maintain and initiate the features of disease pathophysiology. whereas il-4 is crucial for ige production and allergic sensitization, and il-5 is important for eosinophil survival in the lungs.(4) il33 is a cytokine of the il-1 family. 1corresponding author e-mail: zha_23980@yahoo.com received: 13/10/2020 accepted: 22/2/2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp64-70 iraqi j pharm sci, vol.30(2) 2021 the anti-asthmatic activity of guggulsterone 65 after tissue injury, il-33 produces as an alarm signal and stimulates other immune cells to release of il-5 and 13. also il‑33 induces ige synthesis and b-cell expansion leading to il4 secretions by innate cells.(5, 6) furthermore, tnf is a major inflammatory mediator, which has two major forms including tnf-α and -β, the inflammatory mechanisms of tnf-α including tnf-α–stimulated ahr, chemoattractant for eosinophils and neutrophils and up regulation of adhesion molecules lead to migration of inflammatory cells to the respiratory tract.(7) however, the other inflammatory marker is ige, which is stimulating the produce of transcription of cytokines, vasoactive mediators, and de novo synthesis of leukotrienes and prostaglandins on mast cell. in the lung these mediators are rapidly evolve mucus production, edema of the bronchial mucosa, and constriction of the smooth muscle and lastly induce an inflammatory infiltrate that lead to inflammatory responses of asthma.(8) . guggulsterone is a steroid plant (polyphenol) present in guggul gum of commiphora wightii, which belongs to burseraceae family and it has two bioactive isomers eand z-guggulsterone. both isomers of guggulsterone have antiinflammatory activities by inhibit lipopolysaccharide-induced inflammation through inhibiting nf-κb activation and iκb-α degradation.(9) moreover, guggulsterone drastically curbed tnf-induced promoter bounce of cox-2, which is responsible for degradation of arachidonic acid to thromboxane and prostaglandins that lead to inflammatory responses. on other hand, guggulsterone has antioxidant activity due to present of ch3, h, and o bonds in the steroidal structure that helps in reducing free radicals (singlet oxygen and hydroxyl ions) that lead to inhibit the production of lipid peroxides, therefore gugglsterone is preventing oxidative stress.(10) all these gugglsterone activities may contribute for reducing inflammatory responses associated with asthma. materials and methods animals forty-eight healthy albino male rats weighing 150-300 gm, were brought from animal house of the college of pharmacy / university of baghdad. rats were housed under controlled temperatures and photoperiods (12:12-hours light/dark cycle). during the experiment period, these animals were fed commercial pellets. the local research ethics committee in college of pharmacy, university of baghdad, approved the research protocol. chemicals and kits guggulsterone powder was purchased from (xi’an geekee biotech, china) and was prepared as suspension (5mg/ml). ovalbumin was purchased from (chadwll heath essex, england), predinsoline (syrup, 15mg/5ml) was taken up from (pioneer, iraq). also il4, il5, il33, tnf & ige kits were bought from (mybiosource, usa). study design rats were randomly divided into six groups (8 rats in each). the dose of guggulsterone (25 & 50 mg/kg)(11), (12) according to the previous studies and predinsoline dose (4.12 mg/kg) depending on a simple practice guide for dose conversion between animals and human.(13) ld50 of guggulsterone e and z was (1600 mg/kg p.o.) in rats.(14) . group i: rats were administrated distal water orally (daily dose) without sensitization for 14 days as control group. group ii: rats were administrated distal water orally (daily dose) with sensitization for 14 days as positive control group. group iii: rats were administrated (25 mg/kg) guggulsterone orally with sensitization for 14 days as treated group. group iv: rats were administrated (50 mg/kg) guggulsterone orally with sensitization for 14 days as treated group. group v: rats were administrated (4.12 mg/kg) prednisolone orally with sensitization for 14 days as treated group. group vi: rats were administrated (50 mg/kg) guggulsterone orally without sensitization for 14 days as treated group. inflammatory sensitization method for ovainduced asthma of sensitized rats by modified protocol of manal et al (2013), tong et al (2008), and michael et al (1999) studies. the experimental groups were sensitized intraperitoneally (i.p.) with 1 mg ova adsorbed on 100mg al(oh)3 gelatinous and dissolved in1ml of pbs on days 1, 2, and 3. then on the 6th day , the rats were challenged with a 100mg ova adsorbed on 100mg al(oh)3 and dissolved in 1ml of pbs. after that, the experimental animals were challenged on the 9th day by glass chamber with volume (20 cm × 20 cm × 20 cm) , connected to a nebulizer with 1% ova (1gm ova in 100ml pbs) for 30 minutes daily for 6 days.(15-17) wbc count and differentiation after blood collection, the chest was opened, and the trachea with the heart–lung package was excised from the thorax; the left main bronchi were clamped. a cannula was inserted into the trachea in situ, the right lung was lavaged three times with 5ml pbs solution, and bal fluid was collected and centrifuged (6000 rpm/min for 15 minutes) to separate whole cells as pellets. after the supernatant was removed, the pellet of whole cells was dissolved with 1 ml of normal saline. wbc count was measured and differentiated by coulter cellular analysis system (brea, usa). iraqi j pharm sci, vol.30(2) 2021 the anti-asthmatic activity of guggulsterone 66 preparation of serum samples blood samples were collected in plane tube and kept in room temperature for 30 minutes. then, they were centrifuged for 15 min. (1000 x g). after that, serum was removed and stored at (-20oc or 80oc). then, parameters (il-4, il-5, il-33, tnf-α &ige) were measured by elisa kits. principle of kit this kit was depended on sandwich (enzyme linked immune-sorbent) assay method. plate (96 wells) was coated with polyclonal antibody. polyclonal antibody was connected with the biotin as detection antibodies. the test samples (serum and tissue hemogenate), standards, and biotin connected detection antibody were added to the plate subsequently, and wash buffer used for washing the wells. then, abc was added and wash buffer was washed unbound conjugates. tmb substrates were utilized for visualizing enzymatic reaction hrp (blue color). after that stop solution (acidic solution) was added to change the color of reaction (yellow color), that read by a microplate reader (450 nm). statistical analysis data expressed as means, standard error mean and percentage. where, unpaired student ttest was used for testing the significant difference between two groups. on other hand, one-way anova analysis was used for testing the significant difference between three or more than groups. statistically, significant differences were considered for p-value < 0.05. results and discussion in asthma, there is cellular stimulation and production of inflammatory mediators by degranulation of mast cell and vacuolation of eosinophil. the pro-inflammatory cytokines, interlukines-3, 4, 5, 9, & 13 and gm-csf (granulocyte–macrophage colony-stimulating factor), which lead to the ige, eosinophilic and mast cell responses which are produce the distinctives of allergic asthma(18) . 1-the effect of guggulsterone on wbc counts in bronchoalveolar lavage fluid (balf) of ovainduced asthma in rats as shown in figure 1,wbc count (mean ± std. error) in balf for rats of group ii (positive control, induction group) was highly significant elevated (p<0.001) compared to group i (control group). moreover, there was highly significant reduction about 78 % (p<0.001) of wbc count in balf for rats of group iii, 73 %(p<0.001) reduction in group iv, & 51 % (p<0.001) reduction in group group v in compared with group ii (positive control). furthermore, there was a significant reduction (p<0.05) in wbc count in balf in rats of group iv (104.3 ± 14.9) compared to group v (192.1 ± 39.6). likewise, there was a significant reduction (p<0.05) in wbc count in balf in rats of group iii (86.1 ± 16.9) compared to wbc count in balf in rats of group v (192.1 ± 39). figure 1. the effect of guggulsterone on wbc counts in bronchoalveolar lavage fluid of ovainduced asthma in rats. values are indicated as means ± std. error (n=8) for each group. group i: control group, group ii: positive control group (with sensitization), group iii: guggulsterone (25 mg/kg) with sensitization, group iv: guggulsterone (50 mg/kg) with sensitization, group v: predinsoline (4.12 mg/kg) with sensitization, group vi: guggulsterone (50 mg/kg) without sensitization. *** symbol referred to significant different (p<0.001) compared to control group, unpaired test student t-test. groups with nonidentical letters are significantly different (p<0.05). series 1 referred to means of wbc counts in balf for each group. wbc count in bal was significantly increased in sensitized group (ii) compared to control group (i), the previous study, monteseirín (2009) revealed to significant elevation in wbc especially neutrophils in asthma due to platelet activating factor (paf) more in asthmatic patients than a healthy group that lead to induce chemoattractive of neutrophils.(19) on other hand, wbc count in bal fluid for treated groups with guggulsterone and predinsoline have significant reduction because of gugglsterone has antiinflammatory activity by inhibiting the activation of nfκb and reduce of gene expression of chemokines that lead to reduce of eosinophils and neutrophils migration (20). 2-the effect of guggulsterone on il4 levels in the serum of ova-induced asthma in rats: as shown in figure 2, highly significant (p <0.001) elevation in serum levels of il4 was observed in rats of group ii compared to group i. furthermore, in rats of treated groups with (iii, iv & v), the serum levels of il4 were highly significant reduced by 71 % (p <0.001), 67% (p <0.001) and iraqi j pharm sci, vol.30(2) 2021 the anti-asthmatic activity of guggulsterone 67 39% (p <0.001), respectively compared to group ii. however, non-sensitized gugglsterone-treated rats of group vi showed no significant changes in serum levels of il4. figure.2. the effect of guggulsterone on il4 levels in the serum of ova-induced asthma in rats. values are indicated as means ± std. error (n=8) for each group. group i: control group, group ii: positive control group (with sensitization), group iii: guggulsterone (25 mg/kg) with sensitization, group iv: guggulsterone (50 mg/kg) with sensitization, group v: prednisolone (4.12 mg/kg) with sensitization, group vi: guggulsterone (50 mg/kg) without sensitization. *** symbol referred to significant different (p<0.001) compared to control group, unpaired test student t-test. groups with nonidentical letters are significantly different (p<0.05). series 1 referred to means of wbc counts in balf for each group. in current study, il-4 levels in serum for sensitized group are significantly greater than control group. the other studies like afshari et al (2007) & gour et al (2015), appeared that in asthma, il-25, il-33, thymic stromal lymphopoietin (tslp) and leukotrienes are activated t helper cell, mast cell and basophil and released il-4, which has the role in regulating th2 cell survival and proliferation and ige synthesis that lead to the initiation of humoral and airway allergic responses associated with high serum il-4 level.(8)&(21) on other hand, guggulsterone-treated groups have significant reduction in il-4 levels in serum than sensitized group due to gugglusterone has glucocorticiodmediated effect that lead to inhibit gene expression of proinflammatory mediater including ils-4, 5, 8 and 13 that lead to reduce inflammatory events associated with asthma(22) . 3the effect of guggulsterone on il-5 levels in the serum of ova-induced asthma in rats as shown in figure 3, a highly significant (p <0.001) elevation in serum levels of il5 was observed in rats of group ii compared to group i. furthermore, treated groups (iii & iv), the serum levels of il5 were highly significant reduced by 34 % (p <0.001), and 54% (p <0.001), respectively compared to group ii. furthermore, there was a significant reduction by 47% (p<0.05) in serum il 5 levels in group iii compared with group v. figure3.the effect of guggulsterone on il-5 levels in the serum of ova-induced asthma in rats: values are indicated as means ± std. error (n=8) for each group. group i: control group, group ii: positive control group (with sensitization), group iii: guggulsterone (25 mg/kg) with sensitization, group iv: guggulsterone (50 mg/kg) with sensitization, group v: prednisolone (4.12 mg/kg) with sensitization, group vi: guggulsterone (50 mg/kg) without sensitization. ** symbol referred to significant different (p<0.001) compared to control group, unpaired test student t-test. groups with nonidentical letters are significantly different (p<0.05). series 1 referred to means of wbc counts in balf for each group. the other an important inflammatory mediater is il-5, in serum for rats of sensitized group ii is higher than control group i. the role of il-5 in the pathogenesis of asthma is supported by previous studies, garcia et al (2013), dunican et al (2015),when il-5 release by mast cells, th2lymphocytes and eosinophils and occupy to the specific subunit receptor, il-5rα. il-5/il-5rα complex stimulate eosinophil differentiation, proliferation, maturation, and migration to tissue sites with survival, as well as forbidding of eosinophil apoptosis.(23), (24) furthermore, guggulsterone has anti-inflammatory activity by inhibiting the activation of nfκb and reduce of gene expression of il-5(22) . 4the effect of gugglsterone on il 33 levels in the surem of ova-induced asthma in rats figure 4 appeared that highly significant elevation (p <0.001) in serum levels of il33 was observed in rats of group ii compared to group i. furthermore, in treated groups (iii, iv &v), the serum levels of il33 were highly significant reduced by 36% (p <0.001), 41% (p <0.001) and 49% (p iraqi j pharm sci, vol.30(2) 2021 the anti-asthmatic activity of guggulsterone 68 <0.001), respectively compared to group ii. likewise, there was significant reduction (p<0.05) in il33 in serum for rats of group iii compared to il33 in serum for rats of group v. figure.4. the effect of gugglsterone on il 33 levels in the surem of ova-induced asthma in rats. values are indicated as (means ± std. error (n=8) for each group. group i: control group, group ii: positive control group (with sensitization), group iii: guggulsterone (25 mg/kg) with sensitization, group iv: guggulsterone (50 mg/kg) with sensitization, group v: prednisolone (4.12 mg/kg) with sensitization, group vi: guggulsterone (50 mg/kg) without sensitization. *** symbol referred to significant different (p<0.001) compared to control group, unpaired test student t-test. groups with nonidentical letters are significantly different (p<0.05). series 1 referred to means of wbc counts in balf for each group. recently, many studies are suggested that difference in genes encoding il-33 and il-1rl1 is present with asthma. il-33 levels in serum for rats of sensitized group ii is significantly more than control group i. li et al (2015) & momen et al (2017) studies, showed that il-33 levels were higher in asthmatic group than normal group because of il33 induces of intracellular molecules by map kinase and nf-κb signaling pathway, which is an important role in initiate allergic inflammation in asthma.(25), (26) on other hand, il-33 levels in serum for rats treated with guggulsterone has significant reduction in compared with sensitized group due to gugglsterone reduce the activation of transcriptional factors, such as ap-1 and nf-κb, inhibit translation of target genes and/or mrna stabilization that that lead to inhibit inflammatory responses associated with asthma(27) . 5the effect of gugglsterone on tnfα levels in the serum of ova-induced asthma in rats figure 5 showed that highly significant elevation (p <0.001) in serum levels of tnfα was observed in rats of group ii compared to group i. also, there was highly significant reduction (p<0.001) in tnfα levels in serum for treated groups (iii, iv & v), the serum levels of tnfα was highly significant reduced by 43 % (p <0.001), and 39% (p <0.001), & 54% (p <0.001), respectively compared to group ii. on other hand, treating nonsensitized animals with gugglsterone showed no significant changes in serum levels of tnfα. figure.5. the effect of gugglsterone on tnfα levels in the serum of ova-induced asthma in rats. values are indicated as means ± std. error (n=8) for each group. group i: control group, group ii: positive control group (with sensitization), group iii: guggulsterone (25 mg/kg) with sensitization, group iv: guggulsterone (50 mg/kg) with sensitization, group v: prednisolone (4.12 mg/kg) with sensitization, group vi: guggulsterone (50 mg/kg) without sensitization. *** symbol referred to significant different (p<0.001) compared to control group, unpaired test student t-test. groups with nonidentical letters are significantly different (p<0.05). series 1 referred to means of wbc counts in balf for each group. moreover, the results of the current study appeared tnf levels in serum are significantly elevated with sensitized group ii in compared to normal group i, nakae et al (2007), & brightling et al (2008) revealed that tnf is higher with asthmatic group in compared to normal group due to tnf-α/ tnf-α receptor complex that produces intracellular signaling and phosphorylation of iκba and stimulation of nf-κb, which is induces the expression of proinflammatory genes like il-1b, il6, il-8, and tnf-α itself and contributes to the inflammatory airway responses.(28), (29) on other hand, guggulsterone with anti-inflammatory steriod activity that inhibit tnf inflammatory pathway through inhibit the activation of pro-inflammatory transcription factors, ap-1 and nfκb that lead to suppress the gene expression of proinflammatory cytokines including tnf-α and reducing cellular and molecular evets associated with asthma(30) . 6the effect of guggulsterone on ige levels in the serum of ova-induced asthma in rats: iraqi j pharm sci, vol.30(2) 2021 the anti-asthmatic activity of guggulsterone 69 in figure 6 appeared that highly significant elevation (p <0.001) in serum levels of ige was appeared in rats of group ii compared to group i. also ige levels in serum for rats of treated groups (iv & v), were significantly elevated by (p<0.05), (p<0.01), respectively compared to ige levels in serum for rats of group i. furthermore, in treated groups (iii, iv & v), the serum levels of ige were significantly reduced by 23 % (p <0.001), 33% (p <0.001) & 51% (p<0.001), respectively compared to group ii. but, treating non-sensitized rats with guggulsterone observed non-significant different in serum levels of ige. figure 6. the effect of guggulsterone on ige levels in the serum of ova-induced asthma in rats. values are indicated as means ± std. error (n=8) for each group. group i: control group, group ii: positive control group (with sensitization), group iii: guggulsterone (25 mg/kg) with sensitization, group iv: guggulsterone (50 mg/kg) with sensitization, group v: prednisolone (4.12 mg/kg) with sensitization, group vi: guggulsterone (50 mg/kg) without sensitization. *** symbol referred to significant different (p<0.001) compared to control group, unpaired test student t-test. groups with nonidentical letters are significantly different (p<0.05). series 1 referred to means of wbc counts in balf for each group. ige antibody is immune marker associated with asthma, which is play a an important role in asthmatic pathway. ige levels in serum of rats of sensitized group ii are significantly higher than normal control group i. in the same line, afshari et al (2007) & froidure et al (2016) studies, also showed that higher level of ige in asthmatic group than control group. il4 is cytokine that produce switching in b-cells and responsible in elevated ige synthesis. ige occupied to the high-affinity of fcεri (ige receptor) on mast cells and stimulated the produce of transcription of cytokines, vasoactive mediators, and de novo synthesis of leukotrienes and prostaglandins, which are increasing airway inflammation. but guggulsterone treated groups are significant reduce ige levels through the ability of guggulsterone for suppression of gene expresion of proiflammatory cytokines specifically il-4 and reducing the release of ige from inflammatory cells in the lung.(8) & (22) conclusion guggulsterone has anti-inflammatory activity on ova-induced asthma in rats as evident by significant reducing in wbc count in balf, proinflammatory cytokines (il-4, il-5, il-33 & tnfα) and immune marker ige antibody in serum. further studies are required for future work to investigate the beneficial anti-inflammatory activity of guggulsterone for treatment of other inflammatory diseases. references 1. fehrenbach h, wagner c, wegmann m. airway remodeling in asthma: what really matters. cell tissue res. 2017;367(3):551-69. 2. kudo m, ishigatsubo y, aoki i. pathology of asthma. frontiers in microbiology. 2013;4:263. 3. matsumoto h, niimi a, takemura m, ueda t, minakuchi m, tabuena r, et al. relationship of airway wall thickening to an imbalance between matrix metalloproteinase-9 and its inhibitor in asthma. thorax. 2005;60(4):277-81. 4. lloyd cm, hessel em. functions of t cells in asthma: more than just t(h)2 cells. nature reviews immunology. 2010;10(12):838-48. 5. schmitz j, owyang a, oldham e, song y, murphy e, mcclanahan tk, et al. il-33, an interleukin-1-like cytokine that signals via the il-1 receptor-related protein st2 and induces t helper type 2-associated cytokines. immunity. 2005;23(5):479-90. 6. komai-koma m, brombacher f, pushparaj pn, arendse b, mcsharry c, alexander j, et al. interleukin-33 amplifies ige synthesis and triggers mast cell degranulation via interleukin4 in naive mice. allergy. 2012;67(9):1118-26. 7. brightling c, berry m, amrani y. targeting tnf-alpha: a novel therapeutic approach for asthma. the journal of allergy and clinical immunology. 2008;121(1):5-12. 8. afshari j, hosseini r, farahabadi s, heydarian f, boskabady mh, khoshnavaz r, et al. association of the expression of il-4 and il-13 genes, il-4 and ige serum levels with allergic asthma. iranian journal of allergy, asthma, and immunology. 2007;6:67-72. 9. sairkar p, sharma a, shukla n. estimation of guggulsterone e and z in the guggul based commercial formulations using high performance thin layer chromatography (hptlc). j pharm bioallied sci. 2016. 10. shah r, gulati v, palombo e. pharmacological properties of guggulsterones, the major active components of gum guggul. phytotherapy research : ptr. 2012;26:1594-605. iraqi j pharm sci, vol.30(2) 2021 the anti-asthmatic activity of guggulsterone 70 11. sirisha k. pharmacokinetic and pharmacodynamic interactions of atorvastatin with guggulsterone in hyperlipidemic rats. international journal of pharmaceutical sciences and research. 2014;5:4262-8. 12. gebhard c, stämpfli sf, gebhard ce, akhmedov a, breitenstein a, camici gg, et al. guggulsterone, an anti-inflammatory phytosterol, inhibits tissue factor and arterial thrombosis. basic research in cardiology. 2009;104(3):285-94. 13. nair ab, jacob s. a simple practice guide for dose conversion between animals and human. j basic clin pharm. 2016;7(2):27-31. 14. dar m, masoodi h, mohi-ud-din r. guggulipid as an adjuvant therapy for 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sociedad latinoamericana de alergia e inmunología. 2009;19:340-54. 20. liu t, zhang l, joo d, sun s-c. nf-κb signaling in inflammation. signal transduction and targeted therapy. 2017;2:17023. 21. gour n, wills-karp m. il-4 and il-13 signaling in allergic airway disease. cytokine. 2015;75(1):68-78. 22. lawrence t. the nuclear factor nf-kappab pathway in inflammation. cold spring harb perspect biol. 2009;1(6):a001651-a. 23. dunican em, fahy jv. the role of type 2 inflammation in the pathogenesis of asthma exacerbations. ann am thorac soc. 2015;12 suppl 2(suppl 2):s144-s9. 24. garcia g, taille c, laveneziana p, bourdin a, chanez p, humbert m. anti-interleukin-5 therapy in severe asthma. european respiratory review : an official journal of the european respiratory society. 2013;22(129):251-7. 25. li r, yang g, yang r, peng x, li j. interleukin-33 and receptor st2 as indicators in patients with asthma: a meta-analysis. int j clin exp med. 2015;8(9):14935-43. 26. momen t, ahanchian h, reisi m, shamsdin sa, shahsanai a, keivanfar m. comparison of interleukin-33 serum levels in asthmatic patients with a control group and relation with the severity of the disease. international journal of preventive medicine. 2017;8:65. 27. newton r, shah s, altonsy mo, gerber an. glucocorticoid and cytokine crosstalk: feedback, feedforward, and co-regulatory interactions determine repression or resistance. the journal of biological chemistry. 2017;292(17):7163-72. 28. nakae s, ho l, yu m, monteforte r, iikura m, suto h, et al. mast cell-derived tnf contributes to airway hyperreactivity, inflammation, and th2 cytokine production in an asthma model in mice. the journal of allergy and clinical immunology. 2007;120:48-55. 29. brightling c, berry m, amrani y. targeting tnf-alpha: a novel therapeutic approach for asthma. the journal of allergy and clinical immunology. 2008;121(1):5-10; quiz 1-2. 30. burris tp, montrose c, houck ka, osborne he, bocchinfuso wp, yaden bc, et al. the hypolipidemic natural product guggulsterone is a promiscuous steroid receptor ligand. mol pharmacol. 2005;67(3):948-54. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ 2 iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 21 formulation of metoprolol bilayer tablets as an oral modified release dosage form mohammed s. jabbar* ,1 and yehia i. khalil** *department of pharmaceutics, college of pharmacy, university of basrah, basrah ,iraq. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract metoprolol is a β1 adrenergic blocker used in treatment of heart diseases. metoprolol (100mg) tablets was formulated as a modified release oral system utilizing the concept of bilayer system, first layer contained (30mg) as immediate release and the other (70mg) in the sustained release matrix. the immediate release layer consisted of lactose or microcrystalline cellulose as diluents with sodium starch glycolate or sodium croscarmellose as disintegrants. the result showed that the layer contains microcrystalline cellulose and 2% sodium starch glycolate gave disintegration time similar to that of conventional metoprolol tartrate tablet. this result was subjected in the subsequent preparation of the bilayer tablet. the sustained release layer was prepared using three polymers: ethylcellulose (ec), hydroxypropyl methylcellulose (hpmc) and hydroxyl ethylcellulose (hec) as retardant materials. it was found that the combination of ec with hpmc in ratio of 2:1 in f11 was best formula because of it’s release profile and the tablet integrity and dimensions were conserved for the period of the test, but according to similarity factor (f 2), f15 (which contained ec:hpmc in ratio 2:1 with polyvinyl pyrrolidone (pvp) as a binder) was the best formula showed higher (f2) among all other formulas and equals to 72.3 comparing to reference product. key words: metoprolol, bilayer tablet, immediate release, sustained release. الخالصة كُظبو يغ(111حجىة انًُزىثزونىل ) َسزخذو فٍ عالج أيزاض انقهت. رى رصُُغ 1ُزىثزونىل هى حبصز أدرَُبنٍُُ ةانً يغ( 01ال )ركىٌ يجبشزح انزحزر وو يغ(31رحىٌ ) دوائٍ فًىٌ يزغُز انزحزر ثإسزخذاو يجذأ َظبو ثُبئٍ انطجقخ، انطجقخ األونً يدهزٌ انزجهىر كًخففبد يع صىدَىو نسهُهىسانزحزر رزكىٌ يٍ كم يٍ انالكزىس أوا انطجقخ يجبشزح قبنت ثطُئ انزحزر. فٍ االخزي % يٍ 2يدهزٌ انزجهىر يع نسهُهىسَشأانكالَكىنذ أو صىدَىو انكزوسكبريهىس كًفككبد. رجٍُ انُزبئح ثإٌ انطجقخ انزٍ رحىٌ عهً ا ُذَخ ونذنك اٌسزخذيذ فٍ انزحضُزاد انالحقخ نهحجىة ثُبئُخ صىدَىو َشأانكالَكىنذ رعطٍ وقذ رفكك يشبثه نحجخ انًُزىثزونىل انزقه انطجقخ. ُحضزد انطجقخ ثطُئخ انزحزر ثإسزخذاو ثالثخ ثىنًُزاد هٍ االثُم سهُهىس ،انهبَذروكسٍ ثزوثُم يثُم سهُهىس و انهبَذروكسٍ هى 11فٍ انصُغخ 1:2ُم سهُهىس ثُسجخ وخذ إٌ خهُط يٍ االثُم سهُهىس و انهبَذروكسٍ ثزوثُم يث اثُم سهُهىس كًثجطبد رحزر. إعزًبدا" عهً عبيم خالل فززح اإلخزجبر، نكٍ ىيحبفع عهُه كبٌ أفضم صُغخ ثسجت انشكم اندُذ نهزحزر وهُكهُخ انحجخ وأثعبدهب بَزونُذوٌ كًبدح يع انجىنٍ فُُم ث 1:2)انزٍ رحىٌ االثُم سهُهىس و انهبَذروكسٍ ثزوثُم يثُم سهُهىس ثُسجخ 11انزشبثه، انصُغخ يقبرَخ ثبنًُزح انًزخعٍ. 02.3راثطخ( كبَذ أفضم صُغخ وأظهزد اعهً عبيم رشبثه وهى introduction tablets may be defined as solid pharmaceutical dosage forms containing drug substances with or without suitable diluents and have traditionally prepared by either compression, or molding methods. recently, punching of laminated sheets, electronic deposition methods, and three-dimensional printing methods have been used to formulate tablets (1) . modified-release tablets are coated or uncoated tablets that contain special excipients or they are prepared by special procedures, or both, designed to modify the rate, place or time of release of the active substance(s) (2) . layered tablets, type of modified release, are prepared by compressing several different granulations fed into a die in succession, one on top of another, in layers. each layer comes from a separate feed frame with individual weight control to form twoor threelayered tablets, depending on the number of separate fills. each layer may contain a different medicinal agent, separated for reasons of chemical or physical incompatibility, staged drug release, or simply for unique appearance of the layered tablet (3) . some layers may be formulated to exert certain functions. rahmanz z. et al, designed a bilayer floating tablet of captopril in which one layer responsible for floating of the tablet for prolongation the gastric residence time with a controlled release mechanism (4) . the bioavailability of propranolol was enhanced by formulating a bilayer and multilayer buccal mucoadhesive tablet in which one layer adhere to the buccal mucosa so localized the drug in the oral cavity and avoid hepatic first pass metabolism (5) . 1corresponding author email : mohamedsattar2@yahoo.com received : 1 /8 /2009 accepted : 28/12/2009 iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 22 the bioavailabilty of rosiglitazone was improved by developing a bilayer and floatingbioadhesive drug delivery system exhibiting a unique combination of floatation and bioadhesion to prolong residence of rosiglitazone in the stomach (it is highly soluble in 0.1 mol/l hcl) so improve its bioavailability (6) .bilayer tablet is suitable for sequential release of two drugs in combination, with one layer of drug for immediate release while the second layer designed to release drug in an extended release manner. bhavesh s. et al developed bilayer tablet for treatment of migraine consisting from ibuprofen in the sustained release layer and metoclopramide (to enhance the absorption of non-steroidal antiinflammatory drug) in the immediate release layer (7) .the most important application of the bilayer is a quick/slow release system provides an initial burst of drug release followed by a constant rate (ideally) of release over a defined period of time. this type of system is used primarily when a maximum response needs to be achieved quickly, and it is followed by a sustained release phase to avoid repeated administration. when a single constant rate for drug release does not entirely satisfy the therapeutic objective, the quick/slow delivery system may be an interesting alternative. this biphasic release system can be achieved by the application of an immediate release layer to the conventional layered matrix tablet (8) .suitable candidate drugs for this type of administration include nonsteroidal anti-inflammatory drugs (nsaids) and antihypertensive, antihistaminic, and anti-allergic agents (9) . metoprolol (mt) is a cardioselective β1blocker exert equilibrium-competitive antagonism of the actions of catecholamines and other adrenomimetics at β-receptors, used in treatment of hypertension, angina pectoris, myocardial infarction, cardiac arrhythmias and heart failure (10) . peak plasma concentrations are achieved within 1.5 to 2 hours after a single oral dose, but because of the drug’s short half-life (3 to 4 hours), the therapeutic plasma concentration can be maintained only if the metoprolol is administered frequently (11) . these characteristics make metoprolol a suitable candidate for administration by a quick/slow delivery system. the purpose of this study is to design a quick/slow delivery dosage form as biphasic bilayer tablet in which one layer containing superdisintegrant like sodium starch glycolate or sodium croscarmellose responsible for immediate release of the drug and the other is matrix layer of retardant polymer like ethylcellulose (ec), hydroxypropyl methylcellulose (hpmc) and hydroxyethylcellulose (hec) responsible for sustained action. materials and methods materials metoprolol tartrate, hydroxypropyl methylcellulose and colloidal silicon dioxide (cabosil) from sigma, germany. metoprolol tartrate 50mg tablet: uk limited, england. metoprolol tartrate 100mg sustained release: ratiopharm, germany. ethylcellulose from acros organics, united states. hydroxyethylcellulose from merck, germany. lactose from riedel-dehaen, germany. microcrystalline cellulose (avicel ph 102) and croscarmellose from hekma drug industry, jordan. starch and talc from afco, india. polyvinylpyrrolidone, sodium starch glycolate (explotab) and magnesium stearate from bdh laboratory, england. methods preparation of immediate release (ir) granules different formulas (table 1) were prepared using wet granulation method to achieve most acceptable pharmacopial requirements and consider as comparable test with the reference one (metoprolol tartrate uk limited 50 mg) tablet.after weighing the drug and the excipients enough to prepare 40 tablets in dried form, and then blend the ingredients using mortar and pestle, the powders were mixed with binding solution (10% alcoholic starch paste) gradually until proper ball test consistency was resulted.the wet mass was screened through sieve (10 mesh) and dried in pre warmed oven at 60 ºc for one hour. the dried granules then reduced in size by screening them through 12 mesh size sieve. a known weight of granules equivalent to the stated dose was mixed with calculated amount of magnesium stearate and talc powder for five minutes. table 1: different formulas of metoprolol granules as immediate release (ir) layer iinnggrreeddiieenntt ((mmgg)) iirr11 iirr22 iirr33 iirr44 iirr55 iirr66 mmeettoopprroollooll ttaarrttrraattee 30 30 30 30 30 30 mmiiccrrooccrryyssttaalllliinnee cceelllluulloossee ((mmcccc)) 103 100 100 llaaccttoossee 103 100 100 ssooddiiuumm ssttaarrcchh ggllyyccoollaattee 3 3 ccrroossccaarrmmeelllloossee 3 3 1100%% aallccoohhoolliicc ssttaarrcchh ppaassttee 15 15 15 15 15 15 mmgg sstteeaarraattee 1 1 1 1 1 1 ttaallcc 1 1 1 1 1 1 ttoottaall wwtt 150 150 150 150 150 150 iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 23 preparation of sustained release (sr) granules different formulas (table 2) were prepared using wet granulation method like immediate release granule to achieve most acceptable pharmacopial requirements. after weighing the drug, polymer(s) and diluent enough to prepare 40 tablets in dried form, and then blend the ingredients using mortar and pestle, the powders were mixed with binding solution gradually until proper ball test consistency was resulted. the wet mass was screened through sieve (10 mesh) and dried in pre warmed oven at 60 ºc for one hour. the dried granules then reduced in size by screening them through 12 mesh size sieve. a known weight of granules equivalent to the stated dose was mixed with calculated amount of magnesium stearate, cabosil and talc powder for five minutes. table 2 : different formulas of metoprolol granules as sustained release layer vv aa rr ii aa bb ll ee ff oo rr mm uu ll aa ss mm ee tt oo pp rr oo ll oo ll tt aa rr tt rr aa tt ee ee cc hh pp mm cc hh ee cc ll aa cc tt oo ss ee mm ii cc rr oo cc rr yy ss tt aa ll ll ii nn ee cc ee ll ll uu ll oo ss ee 11 00 %% aa ll cc oo hh oo ll ii cc ss tt aa rr cc hh pp aa ss tt ee pp oo ll yy vv ii nn yy ll pp yy rr rr oo ll ii dd oo nn ee mm gg ss tt ee aa rr aa tt ee cc aa bb oo ss ii ll tt aa ll cc tt oo tt aa ll ll aa yy ee rr ww tt mm gg eeffffeecctt ooff eecc ff11 70 70 172 35 1 1 1 350 ff22 70 140 102 35 1 1 1 350 ff33 70 210 32 35 1 1 1 350 eeffffeecctt ooff hhppmmcc ff44 70 70 172 35 1 1 1 350 ff55 70 140 102 35 1 1 1 350 ff66 70 210 32 35 1 1 1 350 eeffffeecctt ooff hheecc ff77 70 70 172 35 1 1 1 350 ff88 70 140 102 35 1 1 1 350 ff99 70 210 32 35 1 1 1 350 eeffffeecctt ooff eecc- hhppmmcc ff1100 70 70 70 102 35 1 1 1 350 ff1111 70 140 70 32 35 1 1 1 350 eeffffeecctt ooff eecc- hheecc ff1122 70 70 70 102 35 1 1 1 350 ff1133 70 140 70 32 35 1 1 1 350 ddiilluueenntt ttyyppee ff1144 70 140 70 32 35 1 1 1 350 bbiinnddeerr ttyyppee ff1155 70 140 70 32 35 1 1 1 350 preparation of metoprolol bilayer tablet the quantities of material required to produce the tablets were individually weighed. the sr layer was manually poured into the 10 mm die and was gently precompressed so that a flat surface was created. then the other ir layer was poured into the die to create the second layer. the layered granules were then compressed to a specified maximum compression. evaluation of bilayer tablet disintegration test the different formulas of ir layer were compressed alone at the same compression forced and their disintegration times were compared with the reference conventional tablet (metoprolol 50 mg).the disintegration time was determined in 0.1n hcl (ph 1.2) (2) . iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 24 friability test this test was done for 20 tablets, starting by weighing them and then operating the friabilator at 25 rpm for 4 minutes, reweighing the tablets to determine the loss in their weight (12) . tensile strength the tensile strength of five tablets of each prepared formula was determined from diametrical compression tests, which were performed using a universal hardness tester to measure the maximal diametrical crushing force (f) accurately. together with the measured diameter (d) and thickness (t) of the tablets (by micrometer), the tensile strength (σ t) is then calculated according to the following equation (13) : dt f t   2  uniformity of dosage units the content uniformity was performed for the prepared metoprolol bilayer tablet by taking five tablets and assayed individually. the requirement for this test is met if the amount of ingredient in each of the five tablets lies within the range of 90-110% of the label claim (14) . dissolution behavior a suitable dissolution tests were carried out to demonstrate the appropriate release of metoprolol, the dissolution studies were carried out using the usp basket method at 37 ºc ± 0.5 ºc and rotation speed 100 rpm as follows (14) : one tablet of each prepared formula was running out in a dissolution jar containing 900ml of 0.1 n hcl (ph 1.2) for two hours, then in 500ml of phosphate buffer (ph 6.8) for the rest of the experiment. samples of 5ml were withdrawn at specific time intervals (first at 5, 10 and 15 min then at 1,2,3,4,5,6,7,8,9,10,11 and 12 hr), the volume of samples were replaced with the same buffer solution. samples were filtered and diluted, analyzed using uv spectrophotometer at 223 nm, the procedure was triplicated for each run test. some variables that affect the dissolution behavior had been studied like retardant polymer type, polymer concentration, polymer – polymer ratio, type of diluent, type of binder and ph of dissolution media (1.2, 4.6 and 6.8). swelling and erosion study the swelling and erosion studies were performed for optimized formulation. briefly, weighed tablets with watch glass [h1] and placed in dissolution vessel, containing 0.1 m hydrochloric acid for first 2 hr followed by 6.8 ph phosphate buffer for 4 hr using paddle method at stirring speed of 100 rpm. at selected time interval (1 to 6 hr) the tablets were withdrawn. the watch glass and tablets were blotted dry to remove water and weighed [h3]. the axial and radial swelling was measured using micrometer. the wetted tablets were dried in an oven at 110 °c for 24 hr, cooled and dried in a dessicator and finally weighed as dry weight [h2] (15) : the percent absorption was calculated as a%=100[h3-h2]/h2 the percent erosion was calculated as e%=100[h1-h2]/h1 fourier transform infrared studies (ft-ir) the ft-ir spectra of metoprolol tartrate, physical mixtures and granules of immediate release and sustained release layers were obtained after making potassium bromide discs in the range of 4000 cm -1 to 500 cm -1 to detect drug–excipients interaction, if any (16) . statistical analysis the results of the experiments are given as a mean of triplicate samples ± standard deviation and were analyzed according to the one way analysis of variance (anova) at the level of (p < 0.05). results and discussion evaluation of bilayer tablet disintegration test all the disintegration times of the prepared formulas are shown in table (3) are within the pharmacopial requirements but the ir3 is the closest one to the standard metoprolol tartrate 50 mg conventional tablet so it was used for the further bilayer formulation. table (3) the disintegration time of the prepared immediate release (ir) layer friability test and tensile strength as shown in table (4), all the prepared formula had acceptable weight loss (not more than 1%) and high tensile strength this is mainly due to the composition of each layer. microcrystalline cellulose used as diluent in the immediate release layer, is well known to ffoorrmmuullaass ddiissiinntteeggrraattiioonn ttiimmee ((mmiinn)) iirr11 1166±±22 iirr22 55±±22 iirr33 ((1111±±11..55)) iirr44 33±±11 iirr55 33..55±±00..55 iirr66 22..55±±00..55 ssttaannddaarrdd ttaabblleett ((mmeettoopprroollooll ttaarrttrraattee 5500mmgg ttaabblleett)) ((1100±±11)) iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 25 deform in a predominantly plastic manner which is a direct result of the presence of slip planes or dislocations and is thought to be an important factor affecting the compressibility of microcrystalline cellulose (17) and result in the consolidation of granules and increased intraparticulate bonding during compaction in addition to the homogenous distribution of bonds in the compact (18) . on the other hand, all the sustained release layers containing a considerable amount of the retarding polymer (20% to 60% of tablet weight) which improve the compressibility of the matrix and this elevated with increase the polymer concentration (19) . the formulas (f7, f8 and f9) have lower strength than that of other formulas this may be attributed to the better capability of the granulating solvent to dissolve the polymeric support of ec (and not to hec), thus improving the strength properties of tablets (20) . table (4) physical properties of the prepared bilayered tablet uniformity of dosage units table (4) shows that, all the prepared formulas complied with the united state pharmacopial specification which is 90-110% of metporolol content in each individual tablet (14) . variables affecting the dissolution profile of metoprolol bilayer tablets the effect of retarding polymer type the release of metoprolol from formulas f2, f5 and f8 which contain (1:2) drug: polymer ratio for ec, hpmc and hec respectively is shown in figure (1). a significant difference (p < 0.05) was found among the cumulative amount of metoprolol released (after the first hour) with time depending on the nature of each polymer. ec is hydrophobic polymer and the drug release occurs by dissolution of the active ingredient through capillaries composed of interconnecting drug particle clusters and the pore network as drug release continues, the interconnecting clusters increase the pore network through which interior drug clusters can diffuse causing eventually rapture of the matrix system and rapid release (21) . hpmc and hec are hydrophilic polymers on first contact of theirs matrix system with aqueous fluid rapid uptake of water occurs, chains gradually uncoil and extend but never form into linear coils, as the water content increases, the polymer becomes hydrated and the layer takes on the full characteristics of a gel. this is followed by retardation of water uptake by the rest of the tablet due to the formation of this gel layer. the major difference in the release profile of these two polymers due to the difference in the rate of hydration resulted from type of substitution on the cellulosic backbone (22) . 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 12 time (hr) % m t r el ea se d f2 (ec) f5 (hpmc) f8 (hec) ph 1.2 ph 6.8 figure 1: the effect of polymer type at (1:2) drug: polymer ratio on the cumulative release of mt at 37 ºc. the effect of retarding polymer concentration the effect of polymer concentration on metoprolol release from the bilayer tablets was studied for each type of polymers and the result illustrated in the figure (2). formulas f1, f2 and f3 were prepared utilizing ec at ratio ffoorrmmuullaass ffrriiaabbiilliittyy ((%%)) tteennssiillee ssttrreennggtthh kkgg //ccmm 22 mmeettoopprroollooll ccoonntteenntt ((%%)) ff11 00..4455 1133..11 9999±±44..55 ff22 00..3388 1133..33 9933±±22..99 ff33 00..2255 1155..66 9977±±55..99 ff44 00..5511 1100..44 9999±±44..88 ff55 00..4444 1122..99 9988±±44..22 ff66 00..3355 1144..33 9955±±22..11 ff77 00..9900 55..99 9988±±44..99 ff88 00..7799 66..99 9955±±44..44 ff99 00..5511 77..66 9933±±22..22 ff1100 00..3377 1166..00 9955±±11..99 ff1111 00..2255 1166..44 9988±±44..22 ff1122 00..5577 1122..88 9922±±11..22 ff1133 00..5500 1133..55 9966±±55 ff1144 00..2200 1155..00 9933±±44..99 ff1155 00..1155 1177..44 9922±±33..55 iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 26 of 1, 2 and 3 (in respect to the metoprolol) respectively. the same ratios were used for hpmc in formulas f4, f5 and f6, and for hec in formulas f7, f8 and f9 respectively. it appears that there is a significant difference (p < 0.05) in the release of metoprolol from matrices of ec (f 1-3), hpmc (f 4-6) and hec (f 7-9) when the polymer concentration was increased. in case of ec the results can be attributed to the decrease in the porosity with a concomitant increase in the tortuosity of matrix (19) . the most important factor affecting the rate of release from hydrophilic matrices is the drug:polymer ratio. the increase in polymer concentration causes an increase in the viscosity of the gel and formation of a gel layer with a longer diffusional path leading to a decrease in the drug release (23) . ec polymer 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 time (hr) % m t r e le a s e d f1 (1:1) f2 (1:2) f3 (1:3) ph 1.2 ph 6.8 hpmc polymer 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 12 time (hr) % m t r e le a s e d f4 (1:1) f5 (1:2) f6 (1:3) ph 1.2 ph 6.8 hec polymer 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 time (hr) % m t r e le a s e f7 (1:1) f8 (1:2) f9 (1:3) ph 1.2 ph 6.8 figure 2: the effect of varying drug:plymer ratio on the cumulative release of mt at 37ºc. the effect of polymer-polymer ratio four formulas (f10, f11, f12 and f13) were prepared by mixing hydrophobic ec with hpmc and hec (both hydrophilic polymer) in different ratios 1:1 and 2:1 and the result shown in figure (3).as shown in the figure (2) ec alone was not able to sustained the release of metoprolol beyond 6hr even with 60% of the polymer used (as in f3) in spite of excellent granular and tablet properties this is mainly due to high solubility of the metoprolol that lead to rapid ingress of water and loss the integrity of the matrix (24) . the use of hydrophilic matrix alone for extending drug release for highly water soluble drugs is restricted due to rapid diffusion of the dissolved drug through the hydrophilic gel network. for such drugs it becomes essential to include hydrophobic polymers in the matrix system (25) . so the mixing of hydrophobic with hydrophilic polymer to improve the retardant ability resulting in good release profile and the tablet integrity and dimensions were conserved for the period of the test. 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 12 time (hr) % m t r e le a s e f10: ec:hpmc (1:1) f11: ec:hpmc (2:1) ph 6.8ph 1.2 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 12 time (hr) % m t r e le a s e f12: ec:hec (1:1) f13: ec:hec (2:1) ph 6.8ph 1.2 figure 3: the effect of ec: hydrophilic polymer ratio on the on the cumulative release of mt at 37 ºc. iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 27                        100 0.5 n 1t 2 t t t r n 1 1log50 2 f the effect of diluent type to study the effect of diluent, f11 which contain lactose as a diluent compared with the f14 which contain microcrystalline cellulose as in figure (4). although both lactose and microcrystalline cellulose consider as hydrophilic filler, there was retardation in the release when changing from lactose to microcrystalline cellulose (from 84% to 75% at 8hr). this may be due to the high solubility of lactose so that it will act as channeling agent, permitting a rapid ingress of dissolution medium into the matrix tablets, thus facilitating drug release (26) . while the presence of a swelling, insoluble filler like microcrystalline cellulose changes the release profile due to a change in the rate of swelling at the tablet surface (27) . 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 12 time (hr) % m t r e le a s e d f11 (lactose) f14 (mcc) ph 1.2 ph 6.8 figure 4: the effect of diluent type on the cumulative release of mt from formulas f11 (lactose) and f14 (mcc) at 37 ºc. the effect of binder type to study the effect of binder, f11 which contain starch as a binder compared with the f15 which contain polyvinylpyrrolidone (pvp) as in figure (5). it was seen that slight retardation in the release of metoprolol from matrix when the starch was replaced by pvp (from 84% to 77% at 8hr). it was hypothesized that this may have been resulted from a change in the properties of the ‘pseudo-gel layer’ surrounding the tablet, which controls the drug release rate through intimate mixing during hydration of the dosage form and through the formation of strong hydrogen bonds between pvp and hpmc (28) . the effect of the ph of the dissolution media three tablets of f11 were running out separately in dissolution jars containing 900ml of ph 1.2, 4.6 and 6.8 the procedure was triplicated for each run test and the result of the drug released compared with that obtained normally (2 hr at ph 1.2 then at 6.8) as in figure (6). there was no significance differences (p> 0.05) among the release of metoprolol at different ph media this mainly due to two reasons. first, the metoprolol is highly soluble drug and its solubility dose not affected by the ph (29) . second, the mechanism of the drug release is by swelling the polymer to form barrier gel that resist the diffusion and the hpmc swelling property not affected by the ph of the media (22) . 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 12 time (hr) % m t r e la s e d f11 (starch) f15 (pvp) ph 1.2 ph 6.8 figure 5: the effect of binder type on the cumulative release of mt from formulas f11 (starch) and f15 (pvp) at 37 ºc. 0 10 20 30 40 50 60 70 80 90 0 1 2 3 4 5 6 time (hr) % r e le a s e d f11 (2hr at ph 1.2 then 6.8) f11 (at ph 1.2) f11 (at ph 4.6) f11 (at ph 6.8) figure 6: the effect of ph variation on the total amount of metoprolol release at 37 ºc. selection of the best formula to select the most promised formula in this study, reference product equivalent to 100 mg of metoprolol as a sustained release tablet (metoprolol ratiopharm® 100 sustained release) was utilized as a standard one, this was carried out using similarity factor (f 2) introduced by moore and flanner as shown in equation (3). a difference not exceeding 10% at any sampling time point between reference and test products may be acceptable and f 2 value of 50-100 indicates similarity in the dissolution profiles (30) : …… (3) iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 28 where n is the number of dissolution time points, rt and tt are the reference and test dissolution values at time t. f15 (which contain ec:hpmc in ratio of 2:1 and pvp as a binder) showed higher (f 2) among all other formulas and equals to 72.3 as shown in figure(7). 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 time (hr) % m t r e le a s e d f15 st ph 1.2 ph 6.8 figure 7: the dissolution profile of selected formula f15 versus reference standard (st) with (f ¬2¬) =72.3 at 37 ºc. swelling and erosion swelling and erosion had been studied to the best formula f15 and the result shown in figure (8) revealed that the tablets were swollen and retained their physical integrity till the end. although there is an increase in both percentage water absorbed and percentage erosion of the tablet, but the percentage water absorbed was more dominant than erosion indicating that the release was biphasic mechanism mainly by diffusion than erosion (31) . 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 1 2 3 4 5 6 time hr % s w e ll in g 0.00 0.50 1.00 1.50 2.00 2.50 % e ro s io n swelling erosion ph 1.2 ph 6.8 figure 8: swelling-eroding behavior of optimized selected formula f15. ft-ir studies the ft-ir spectra of metoprolol tartrate, physical mixtures and granules of immediate release and sustained release layers are shown in figures (9) and (10). the o-h stretching vibration peak of metoprolol is seen in 3458 cm -1 and the c-n peak is seen at 2872 cm -1, n-h bending vibration at 1590 cm -1, at 1112 cm -1 the c-o stretching bond, at 1458 cm -1 the c=c, and at 1244 cm -1 the c-o stretching bond are obvious (32). similar peaks are observed in the corresponding physical mixture and granules. furthermore, the broadening of o-h stretching vibration peak mainly due to hydrogen bonding of the drug with the polymers and other excipients, also the absence of significant shifts in the wave numbers of the ir peaks of the granules compared with physical mixture indicates lack the possibility of interaction between metoprolol tartrate and excipients used in this bilayer tablets (16) . figure 9: ft-ir spectra of metoprolol, physical mixture and granules of the ir layer. iraqi j pharm sci, vol.19 (1) 2010 metoprolol bilayer tablets 29 figure 10: ft-ir spectra of metoprolol, physical mixture and granules of the sr layer. references 1. remington: the science and practice of pharmacy, 21 st ed., lippincot, williams, & wilkins; u.s.a, (2006), 889. 2. british pharmacopoeia bp (cd). the stationary office, crown copyright, london, 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pharm sci, vol.31( 2 ) 2022 molecular docking programs accuracy doi: https://doi.org/10.31351/vol31iss2pp160-168 160 a comparative study for the accuracy of three molecular docking programs using hiv-1 protease inhibitors as a model twana salih1,* *department of pharmacognosy & pharmaceutical chemistry, college of pharmacy, university of sulaimani, iraq. abstract flexible molecular docking is a computational method of structure-based drug design. this method is used to evaluate binding interactions between receptor and ligand and identify the ligand conformation within the receptor pocket. currently, various molecular docking programs are extensively applied; therefore, realizing the accuracy and performance of the various docking programs could have a significant value. in this comparative study, the performance and accuracy of three widely used non-commercial docking software (autodock vina, 1click docking, and ucsf dock) was evaluated through investigations of the predicted binding affinity and binding conformation of the same set of small molecules (human immunodeficiency virus-1 protease inhibitors) and a protein target hiv-1 protease enzyme. the tested sets are composed of eight receptor-ligand complexes with high-resolution crystal structures downloaded from the protein data bank website. molecular dockings were applied between approved hiv-1 protease inhibitors and the hiv-1 protease using autodock vina, 1-click docking, and dock6. then, docking poses of the top-ranked solution were realized using ucsf chimera. furthermore, pearson correlation coefficient (r) and coefficient of determination (r2) between the experimental results and the top-scored docking results of each program were calculated using graphpad prism v9.2. after comparing the saquinavir top-scored binding poses of each docking program with the crystal structure, various conformational changes were observed. moreover, according to the relative comparison between the top-ranked calculated δgbinding values against the experimental results, the r 2 value of autodock vina, 1-click docking, and dock6 were 0.65, 0.41, and 0.005, respectively. the outcome of this study shows that the top-scored binding free energy could not produce the best pose prediction. in addition, autodock vina results have the highest correlation with the experimental results in comparison with the other programs. keywords: molecular docking accuracy, comparative study, 1-click docking, autodock vina, ucsf dock, binding free energy, hiv-1 protease inhibitors. لاللتحام الجزيئي باستخدام مثبطات البروتييز لفيروس نقص المناعة دراسة مقارنة لدقة ثالثة برامج كنموذج 1البشرية 1*،الحتوانا ص العراق ، جامعة السليمانية الصيدلة، كلية الصيدالنية، قسم العقاقير والكيمياء * ةالخالص االلتحام الجزيئي المرن هو طريقة حسابية لتصميم دواء قائم على المستقبالت لتقييم تفاعالت الربط بين المستقبل والجزيئة وتحديد شكل داء قة واألالترابط داخل جيب المستقبل. حاليًا ، يتم تطبيق العديد من برامج االلتحام الجزيئي على نطاق واسع ؛ لذلك ، يمكن أن يكون لتحقيق الد غير تجارية مستخدمة على نطاق واسع لمختلف برامج االلتحام قيمة كبيرة. الهدف من هذه الدراسة هو تقييم أداء ودقة ثالثة برامج لاللتحام الجزيئي ثمانية مجمعات مستقبالت (. تتكون المجموعات المختبرة منجامعة كاليفورنيا سان فرانسيسكولاللتحام و االلتحام )االلتحام الذاتي فينا و نقرة واحدة يني فيروس نقص ليجند مع هياكل بلورية عالية الدقة تم تنزيلها من موقع بنك بيانات البروتين. تم تطبيق االلتحام الجزيئي بين مثبطات األنزيم البروت لاللتحام و االلتحام جامعة فينا و نقرة واحدة االلتحام الذاتي باستخدام 1-المعتمدة و البروتييز فيروس نقص المناعة البشرية 1-المناعة البشرية اب معامل كاليفورنيا سان فرانسيسكو. بعد ذلك ، تم تحقيق وضعيات االلتحام للحل األعلى مرتبة باستخدام برنامج کايميرا. عالوة على ذلك ، تم حس لة لكل برنامج باستخدام منشور كرافباد. بعد مقارنة أوضاع الربط ارتباط بيرسون ومعامل التحديد بين النتائج التجريبية وأعلى نتائج االلتحام المسج ذلك ، وفقًا المسجلة بأعلى درجات ساکوينافير لكل برنامج االلتحام الجزيئی مع الهيكل البلوري ، لوحظت العديد من التغييرات التوافقية. عالوة على و نقرة واحدة ٠٬٦٥ل النتائج التجريبية ، كانت قيمة معامل التحديد لاللتحام الذاتي فينا للمقارنة النسبية بين قيم الربط المحسوبة األعلى مرتبة مقاب . تظهر نتيجة هذه الدراسة أن الطاقة الحرة الملزمة ذات أعلى الدرجات ال يمكن ٠٬٠٠٥ جامعة كاليفورنيا سان فرانسيسكوو االلتحام ٠٬٤١لاللتحام لى ذلك ، فإن نتائج االلتحام الذاتي فينا لها أعلى ارتباط بالنتائج التجريبية. أن تنتج أفضل تنبؤ للوضع. باإلضافة إ الطاقة الملزمة ، جامعة كاليفورنيا سان فرانسيسكولاللتحام، االلتحام الذاتي فينا، االلتحام نقرة واحدة، دراسة مقارنة، دقة االلتحام الجزيئي: الكلمات المفتاحية . 1-فيروس نقص المناعة البشرية ل زيالبروتي مثبطات ، الحرة 1corresponding author e-mail: twana.salih@univsul.edu.iq. received:13 /10 /2021 accepted:20 / 12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp160-168 iraqi j pharm sci, vol.31(2) 2022 molecular docking programs accuracy 161 introduction one of the most crucial steps in the drug discovery process is lead identification; various approaches are available to identify a lead compound, such as reliable computational approaches (1). in silico design of ligands based on the knowledge of the 3d chemical structures of receptors is known as structure-guided or structure-based drug design (sbdd). this method is predicting small-molecule ligands that are geometrically and physicochemically complementary to a receptor-binding site (2). regardless of the design method, prediction of complementarity between ligand and receptor involves two discrete but interdependent steps, firstly, docking of the 3d encoded ligand into a 3d model derived from the structural coordinates of the receptor structure (binding poses), and then, an assessment of the goodness of fit in terms of interaction energies (δgbinding) (3, 4). receptor-ligand docking (molecular docking) is one of the most recommended approaches in sbdd of the drug discovery projects, as it could design new small molecule against the specified macromolecule receptors and screen the library of compounds to find the most appropriate molecule as a novel compound to activate or inhibit (as required) the target receptor (5). this method can identify the ligand conformation within the receptor binding site (pocket) and predict the binding affinity between a ligand and a target protein. hence, a successful docking experiment is the result of two piers, which are the right pose (sampling) and the binding affinity estimation (scoring function) (6). nowadays, the scientific community use diverse molecular docking programs, such as autodock, autodock vina, ucsf dock, ligandfit, glide, gold, moe dock (7), and mcule (8, 9); which are either commercial or free for academics (10). according to pagadala et. al. (2017), the most commonly used programs are autodock vina, gold, and moe-dock (11). each of the docking programs has advantages and limitations regarding their accuracy and time consumption due to applying diverse sampling approaches and scoring functions (12). however, the issues related to sampling efficiency (pose prediction) and speed could be fixed to a large extent owing to a significant development of computer hardware and using supercomputers (13), while estimation of actual and comparative binding affinity of small molecules is still a critical concern. despite much research on the efficiency and relative accuracy of the docking programs in the past three decades, it is still challenging to decide on certain software for a specific project. therefore, investigations of the advantages and downsides of these programs are essential to select the most appropriate program and improve the drawbacks (14). various metric approaches can be implemented to define the accuracy of molecular docking programs and assist users to select the most appropriate program (15, 16). one of the global human health threats is the suppression of immune functions due to human immunodeficiency virus (hiv). this disease is known as acquired immunodeficiency syndrome (aids). currently several drug classes are available to treat hiv/aids, such as non-nucleoside reverse transcriptase inhibitors and hiv-1 protease inhibitors. in addition, many studies have been achieved to develop novel anti-hiv agents from the natural sources like coumarin-based compounds (17, 18). the hiv-1 protease inhibitors are a class of antiviral drugs that are widely used to treat hiv/aids and hepatitis c. these protease inhibitor drugs prevent viral replication by selectively binding to hiv-1 protease and blocking proteolytic cleavage of protein precursors, which are necessary to produce infectious viral particles (19). numerous classes of substrate-based and symmetry-based inhibitors have been designed, synthesized, tested, and crystallized with the enzyme. the indispensable role of hiv-1 protease in viral maturation makes it a popular target for drug design (20). many solved hiv-1 protease structures can significantly facilitate the design of new and improved inhibitors. presently, various hiv-1 protease inhibitors are approved by the medicinal regulatory authorities, which include amprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir (fig. 1) (21). iraqi j pharm sci, vol.31(2) 2022 molecular docking programs accuracy 162 figure 1. structures of some of the hiv-1 protease inhibitors. the aim of this study is to realize conformations of the best energy scores and identify the most accurate and promising program through docking various approved hiv-1 protease inhibitor molecules against the hiv-1 protease enzyme with a known experimental binding affinity of the relevant protein-ligand complexes. methods the hiv-1 protease enzyme as a target protein of this study dockings is an essential element for viral maturation in the hiv life cycle. it is a homodimeric aspartyl protease; each monomer is composed of 99 amino acid residues with a catalytic asp25 residue. the active site is not fully exposed, being covered by two flexible β-hairpin flaps. the flaps need to open to allow the substrates to access the active site. blocking the active site can inhibit the hiv-1 protease enzyme activity. inhibition of this key enzyme proved highly effective at reducing viral burden, specifically (fig. 2) (22). figure 2. the crystal structure of the hiv-1 protease-inhibitor ligand complex (23). amprenavir atazanavir darunavir indinavir lopinavir nelfinavir ritonavir saquinavir iraqi j pharm sci, vol.31(2) 2022 molecular docking programs accuracy 163 this project was performed to compare the relative accuracy of predicted binding free energy of three non-commercial docking software (autodock vina, ucsf dock, and 1-click docking) by testing eight of the fda-approved protease inhibitor drugs against the target hiv-1 protease enzyme. the details of the applied programs are summarized in table 1. table 1. the outlines of the applied docking programs. program outline url autodock vina designed and implemented by dr oleg trott in the molecular graphic lab at the scripps research institute. the current version is v.1.2.0. (2021) (24). http://vina.scripps.edu/ ucsf dock developed by the kuntz group in the department of pharmaceutical chemistry, university of california san francisco. the current version is dock6 (25). http://dock.compbio.ucsf.edu/ 1-click docking a web-based server developed by robert kiss group in gedeon richter plc (8). https://mcule.com/apps/1-clickdocking/ protein preparation the started coordinate of the wild-type hiv-1 protease enzyme bound with saquinavir (solved at 1.16 å resolution) was downloaded from protein data bank (pdb) (pdb id: 3oxc) (26) website (https://www.rcsb.org) (27). after downloading the three-dimensional structure of the complex, all water molecules, formic acid, sulfate ions, and the ligand (saquinavir) were removed, then, hydrogen atoms were added using pymol molecular graphic system (28) to prepare the protein for molecular docking. furthermore, partial charges were added by chimera using amber ff14sb (14). ligand preparation all the eight ligands were produced for both autodock vina and ucsf dock6 through using a suite of applications known as marvin (http://www.chemaxon.com/products/marvin) (29). all the marvin tools were accessible from the marvinsketch application. the two-dimensional structures of the ligands were generated using the import name of marvinsketch. after that, the structures were modified to the three-dimensional models of the molecules using molecular dynamics (md) and energy minimization algorithm to calculate a new position of the ligand’s atoms. lastly, hydrogen atoms were added. all the compounds were saved in tripos mol2 file format. besides, ucsf chimera was used to assign partial charges of each ligand by adding gasteiger charges for non-standard residues (30). contrarily, production of the 3d ligands for 1-click docking was achieved using inchikey formulae obtained from pubchem substance and compound database (https://pubchem.ncbi.nlm.nih.gov) (31). 1-click docking software as a part of mcule platform were assigned both hydrogen atoms and gasteiger charges to the ligands automatically through using autodock tools (32). molecular dockings after the preparation of the target protein and the ligands, docking of the hiv inhibitor ligands over hiv protease enzyme achieved using autodock vina (http://vina.scripps.edu) (33), ucsf dock (http://dock.compbio.ucsf.edu) (25) and 1click docking (https://mcule.com/apps/1-clickdocking) (8). all the dockings performed were automated computational algorithms (virtual screening) to select and score the potential molecules matching the shape and chemical complementarity of the hiv-1 protease enzyme and the inhibitor molecules according to the poses. the magnitude of the search space was determined for each of the applied programs with the hiv-1 protease binding centre was x= 20.568, y= -0.979, z=15.127, and the dimension was x= 21.033, y= 16.539, z= 16.445 in angstrom (å) (34, 35). the output files were ranked according to their binding mode from the highest to the lowest binding free energy. visualization and analysing programs pymol molecular graphics system was implemented to remove water and other nonrequired molecules and add hydrogen atoms to the target protein (28). both pymol and ucsf chimera 1.6 were used to visualize interactions, measure distances between the ligands and the hiv-1 protease enzyme for the selected atoms, and investigate the conformational changes (36). graphpad prism v9.2 (graphpad software inc., san diego, ca; www.graphpad.com) was applied to evaluate the binding affinity results of the various programs (37). results and discussion nowadays, the field of molecular docking as a part of sbdd is a fundamental technique in the drug discovery and development process (38). the components of docking programs are considered as search algorithms to identify the poses of the protein-ligand complexes and scoring function http://vina.scripps.edu/ http://dock.compbio.ucsf.edu/ https://mcule.com/apps/1-click-docking/ https://mcule.com/apps/1-click-docking/ https://pubchem.ncbi.nlm.nih.gov/ http://vina.scripps.edu/ http://dock.compbio.ucsf.edu/ https://mcule.com/apps/1-click-docking https://mcule.com/apps/1-click-docking http://www.graphpad.com/ iraqi j pharm sci, vol.31(2) 2022 molecular docking programs accuracy 164 (binding affinity) based on the generated poses (39). in this study, the prime focus was on the performance and challenges of three noncommercial programs (autodock vina, ucsf dock, and 1-click docking) by examining the accuracies of binding pose estimation (power of sampling) and the binding free energy prediction (power of scoring). the test set was composed of eight globally approved inhibitors of hiv-1 protease. each molecule bound with hiv-1 protease enzyme. all of them have a high-resolution crystal structure (< 2.5 angstroms) and reliable binding free energy or dissociation constant (kd) (table 2). the original ligand-receptor binding conformation in the x-ray crystal structures was compared with the topranked solutions produced by each of autodock vina, ucsf dock, and 1-click docking programs. moreover, the predicted scoring functions of each program were compared with the experimental values to reveal the relative accuracy of each platform. table 2. experimental and calculated binding free energy using autodock vina, 1-click docking, and ucsf dock6 programs (40-42). hiv-1 inhibitor drug experimental δgbinding (kcal/mol) autodock vina δgbinding (kcal/mol) 1-click dock δgbinding (kcal/mol) dock6 grid score (kcal/mol) amprenavir -12.10 -8.90 -8.50 -90.03 atazanavir -12.81 -10.00 -9.60 -90.22 darunavir -12.00 -8.30 -9.70 -93.21 indinavir -11.90 -10.10 -7.70 -89.28 lopinavir -13.63 -10.20 -10.40 -90.60 nelfinavir -13.05 -10.40 -10.30 -92.11 ritonavir -12.37 -8.60 -8.80 -106.77 saquinavir -12.98 -10.50 -10.40 -106.30 as illustrated in table 2, the highest binding free energy of the experimental results is lopinavir (-13.63 kcal/mol). the binding free energy rank of saquinavir is considered as one of the highest ones (-12.98 kcal/mol) after lopinavir and nelfinavir. however, the observed results were showed that the highest binding free energy of autodock vina is saquinavir (-10.5 kcal/mol), 1click docking is lopinavir and saquinavir (-10.4 kcal/mol) and ucsf dock is ritonavir and saquinavir (-106.77, and -106.3 kcal/mol, respectively). ostensibly, all three programs were confirmed that saquinavir has the highest or one of the highest binding free energies. in addition, this ligand is the first discovered hiv-1 protease inhibitor (43). therefore, the crystal structure conformation and the molecular docking program’s top-scored poses were analyzed. a model of saquinavir-hiv-1 protease complex, determined by x-ray crystallography to a resolution of 2.30 å as described by krhon et al. in 1991, where saquinavir is referred to as ro 31-8959 (43). the structure has been taken from the protein data bank (pdb) website (http://www.rcsb.org, access code 1hxb.pdb) (27). to investigate this crystal structure and saquinavir-hiv-1 protease enzyme docking results, ucsf chimera was used (36). the top-scored binding poses of saquinavir with hiv-1 protease enzyme in each docking program and the x-ray crystal structure (26) are illustrated in fig. 3. both autodock vina and 1-click docking poses have a significant similarity and a moderate difference with the crystal structure conformation, nevertheless, all the conformations are occupying the substrate-binding cavity of hiv-1 protease. however, the ucsf dock pose is shown a significant change; the ligand’s conformation in the enzyme’s binding site is upturned and the quinoline group of saquinavir is positioned further towards out of the pocket. as shown in fig. 4, the essential binding interactions between saquinavir and hiv-1 protease enzyme in the crystal structure and the docking results were realized. x-ray crystal structure of the ligand-protein complex was revealed four binding interactions between saquinavir and hiv-1 protease. the hydroxyl group of saquinavir (d) (fig. 2) is an essential functional group to interact with the receptor. it was produced a hydrogen bond (h-bond) with both carboxylic acid side-chains of asp25a (2.9 å) and asp125b (2.7 å) in the active site of hiv-1 protease. in addition, the ligand’s carbonyl oxygen of both amide groups (g and h groups) were interacted with asp129b through the main-chain n-h group, with distances 3.4 å and 3.1 å, respectively. lastly, the nh2 group of the amide at g position was produced h-bond with the carboxylic acid side-chain of asp130b (fig. 4a). then, the docking pose of the highest calculated δgbinding using autodock vina was investigated. as shown in fig. 4b, four binding interactions are available between saquinavir and hiv-1 protease. the ligand’s hydroxyl (d) and nh2 (g) groups hbonding interactions were conserved, however, with a weaker binding interaction of the hydroxyl with asp25a (4 å) and the stronger interaction of the nh2 group with the main-chain carbonyl of gly48b (2.9 å) rather than asp130b. moreover, interactions between the ligand and both asp125b and asp129b were abolished. on the other hand, two new weaker iraqi j pharm sci, vol.31(2) 2022 molecular docking programs accuracy 165 hydrophilic interactions were formed. the first was between the main-chain n-h group of asp29a and the ligand’s oxygen of amide carbonyl attached to the decahydroisoquinoline group (b) (4.1 å). the second interaction was between the carboxylic acid side-chain of asp29b and the carbonyl oxygen of an amide group adjacent to the quinoline group (h) (4.5 å). figure 3. saquinavir-hiv-1 complex conformation of the top-scored docking results and the crystal structure. a) xray crystal structure of the hiv-1 protease active binding site bound with saquinavir. the enzyme shows in grey when the ligand is entirely in its pocket, b) autodock vina conformation of the enzyme pocket (blue) and saquinavir (pink), c) 1-click docking conformation of the hiv-1 protease binding site, which is in violet and the ligand, d) ucsf dock6 conformation of the hiv-1 protease binding site and upturned saquinavir in the receptor’s pocket. figure 4. binding interactions between saquinavir and hiv-1 protease in the crystal structure and the topscored docking solutions. a) x-ray crystal structure of saquinavir-hiv-1 protease, b) autodock vina topscore docking solution, b) 1-click docking top-score docking solution, d) ucsf dock6 top-score docking solution. a b c d a b c d iraqi j pharm sci, vol.31(2) 2022 molecular docking programs accuracy 166 after realizing the conformation of the topscored 1-click docking results, five interactions were identified between the ligand and receptor. despite maintaining a h-bond between the ligands’ hydroxyl group and the carboxylic acid side-chain of asp25a (3.1 å), the nh2 group at g position was interacted with the same functional group of asp25a (2.8 å). moreover, the amide carbonyl of the b group was created h-bond with the backbone n-h group of gly48b (2.9 å). furthermore, two new favourable hydrophilic interactions were observed; carbonyl oxygen atom of h group interacted with guanidine side-chain of arg8a (3.0 å) and the nitrogen of quinoline group (i) interacted with the hydroxylic side-chain of asp29b (3.9 å) (fig. 4c). the last docking experiment was ucsf dock6. in this pose, four bonding interactions were explored. this conformation is remarkably different from the crystal and other programs docking poses. hydroxyl group of the ligand produced an interaction with the backbone n-h group of ile50a (3.1 å). the next interaction was between the amide oxygen atom of the h group with both the main-chain n-h group (2.8 å) and the sidechain carboxylic group (3.3 å) of asp29a. the last h-bond interaction was between the amide nh2 g group and the backbone n-h group of asp30a (3.0 å) (fig. 4d). due to the invisibility of hydrogen atoms in the crystal structure, hydrogen atoms were removed from the docking results to be consistent. hence, approximately 1.0 å should be reduced from each of the measured distances. for example, if the distance between the hydroxyl group (d) and asp30a side-chain is 3.0 å, after subtracting 1.0 å, the distance will be 2.0 å. this study denoted diverse predicted binding free energies of each hiv-1 protease inhibitor for each program owing to the differences in the docking poses. the top-ranked predicted binding modes were compared relatively with the experimental observed results. what can be highlighted in fig. 5 is the differences in accuracy of the docking programs using top-scored values, when the most accurate results were produced by autodock vina, the coefficient of determination (r2) is 0.65 and the pearson correlation coefficient (r) is 0.80. figure. 5. correlation between δgbinding results and experimental results of hiv-1 protease inhibitor drugs. (a) correlation between experimental results and δgbinding results using 1-click docking. (b) correlation between experimental results and δgbinding results using autodock vina. (c) correlation between experimental results and δgbinding results using ucsf dock6. although the autodock vina correlation was moderate (r2 is 0.65), the program was considered as the most accurate one in this study owing to the highest correlation between the predicted and experimental values compared with the other two programs. after that, 1-click docking was ranked as the second program in accuracy, as the r2 value is 0.41(weak correlation) (44). however, the dock6 program’s correlation between the calculated binding free energies and the experimental values was insignificant i.e., no correlation (r2 is 0.005). the issues of correlation between the predicted and experimental results could not only be due to the conformational changes but also be related to the preferences of non-covalent interactions between the ligand and the receptor molecule (45). conclusion the best-scored energy of the molecular docking programs could not correspond to the preferred pose of the ligands within 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https://books.google.iq/books?id=aw61ygaac aaj. 45. li x, li y, cheng t, liu z, wang r. evaluation of the performance of four molecular docking programs on a diverse set of protein-ligand complexes. j comput chem. 2010;31(11):2109-2125. doi: 10.1002/jcc.21498. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32(1) 2023 the optimization of bisanthraquniones production doi: https://doi.org/10.31351/vol32iss1pp160-166 160 optimization of bis-anthraquinones production from endophytic fungi diaporthe sp. gnbp-10 listiana oktavia *, evana evana*, reza fahardita** and andria agusta*,***, 1 *research center for pharmaceutical ingredients and traditional medicine, national research and innovation agency republic of indonesia (brin), indonesia **department of pharmacy, national institute of science and technology (itsn), indonesia. ***faculty of medicine, malahayati university, indonesia abstract bis-anthraquinones with a unique molecular backbone, (+)-2,2’-epicytoskyrin a (epi) and (+)-1,1′bislunatin (bis), were produced by endophytic fungi diaporthe sp. gnbp-10 associated with gambir plant (uncaria gambier roxb. rubiaceae). epi and bis possess robust antimicrobial activity toward various pathogens. this study focus on knowing the optimum condition of epi and bis production from endophytic fungi diaporthe sp. gnbp-10. a series of culture media with various nutrient compositions was investigated in epi and bis production. the content of epi and bis was determined by measuring the area under the curve from tlcdensitometric (scanner) experiment. the linear regression analysis was then applied to obtain the results. the optimized media to produce the highest extracted mass is media 7, while epi and bis were greatly produced in liquid media 3. the nutrient content of media 3 is potato starch and dextrose with the amount of epi component produced 0.484 mg and bis content is 0.163 mg. the presence of carbohydrates, whether simple sugar or carbohydrate complex, plays an essential role in the bis-anthraquinones production from diaphorthe sp. gnbp10 culture. the presence of minerals and excessive protein sources did not significantly affect bis-anthraquinones production. keywords: bis-anthraquinone, diaporthe, endophyte media, regression, tlc. introduction endophytic fungi are colonized and detected within the healthy host plant tissue (1). endophytes build a symbiotic relationship with the host plants, majorly defined as mutualism constitutive and inductive mutualism (2,3). endophytic fungi are believed to be a biological protector for the host plants from phytopathogens (2). the endophytes could express secondary metabolites against the foreign intruders (4). the released metabolites stimulate the activation of induced systemic resistance that is related to the host defense mechanism (5). furthermore, the exerted metabolites can promote plant growth via phytohormones (6). the produced metabolites also help the host plant to degrade xenobiotics (7) and improve the resistance to soil contaminants (8). due to the unique relationship between endophytic fungi and host plants, endophytic fungi can produce similar secondary metabolites as their host plant (9).the interaction between endophytes and the host leads to the co-production of bioactive compounds(10). that phenomenon is hypothesized due to horizontal gene transfer between endophytes and the host plant (11). a considerable amount of invaluable therapeutic compounds were discovered from endophytic fungi such as paclitaxel/taxol (anticancer), and camptothecin (anticancer) (12), ergoflavin (antiinflammatory) (13), cyclosporine (antiviral) (14), etc. the endophytic fungi diaporthe sp. gnbp-10, which is associated with the tea plant (camellia sinensis (l.) o. kuntze) and gambir plant (uncaria gambier roxb.), generated two bisanthraquinones (+)-2,2’-epicytoskyrin a (epi) (figure 1a) and (+)-1,1′-bislunatin (bis) (figure 1b) (15–17). the formed bis-anthraquinones, epi, were reported to have displayed strong in vitro antimicrobial activity against s. aureus (mic: 0.06 µg ml-1), b. subtilis (2 µg ml-1 ), and k. pneumonia (4 µg ml-1) (16). meanwhile, bis exhibited moderate activity against b. subtilis, s. aureus, e. coli, m. luteus, p. vulgaris, and p. mirabilis with a mic value (64 µg ml-1) (17). epi and bis retain a robust invitro antimycobacterial activity against m. tuberculosis h37rv, as indicated by mic values 0.844 µm and 0.422 µm, respectively (18). additionally, the oral toxicity assays showed that both metabolites have low acute toxicity profiles (19).due to their immense biological activities, a certain amount of pure epi and bis compounds is needed to meet sufficient criteria for further research. the cultivation method that optimized the production of epi and bis has not been yet investigated. the growth medium has a vital role in natural product production. 1corresponding author e-mail: andr005@brin.go.id received: 8/ 2/ 2022 accepted: 11/5 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp160-166 iraqi j pharm sci, vol.32(1) 2023 the optimization of bisanthraquniones production 161 a previous study demonstrated that various conditions and nutrients significantly contribute to the diversity and quantity of the expressed secondary metabolites (20). culture conditions variations are applied to optimize selected metabolites expressionby the endophytic fungi (12,21). potato dextrose broth (pdb) and potato dextrose agar (pda) were utilized in the cultivation of diaphorthe sp. associated with the tea plant (15). ethyl acetate was used as a diluent to get the endophytic extracts. for the same amount of media culture volume, pdb (525 mg) was reported to have a higher extracted mass of endophytic fungi than pda (80 mg). the production of epi and bis from pdb cultured-endophytic fungi were 47 mg and 10.3 mg, respectively. when the endophytic fungi cultured in the pda generated 17.5 mg and 5.5 mg for both components (15). the previous research showed a media influence in the production of bioactive compounds isolated from endophytic fungi. the purpose of this study is to investigate the effect of different media on the production of bis-anthraquinones from endophytic fungi diaphorte sp. gnbp-10. the media selection will particularly be elaborated in optimizing the production of epi and bis, which are structurally depicted in figures 1a and b. figure 1.(a) (+)-2,2-epicytoskirin -a (epi) ( mw: 574.494 g/mol). (b) 1,1 bislunatin (bis) (mw : 570.462 g/mol) materials and methods the optimized bioproduction of bisanthraquinones was obtained via a selection of culture media from a series of culture medium variations representing a wide range of nutrient content. in general, this study contained three major workflows: cultivating endophytic fungi diaporthe sp. gnbp-10 from various culture media, extraction of secondary metabolite, and determining bisanthraquinones content using tlc scanner. the identification of endophytic fungal diaporthe sp. gnbp-10 and the optimization of fungal growth were previously reported by ilyas et al (2009) (22). endophytic fungi cultivation in various culture media the culture media was prepared in erlenmeyer flasks. the composition of each media is listed in table 1. a volume of 200 ml culture media was used in this assay. the endophytic fungi, diaporthe sp. gnbp-10 was isolated from the gambir plant (uncaria gambier roxb.: rubiaceae). the fungi culture growth was obtained after 14 days of incubation at room temperature (25 – 28 oc) with shaking at 120 rpm in the orbital shaker (newsbrunwich scientific). table 1. the nutrient’s content and compositions versus each media applied in the assay media id nutrient content composition gl-1 h2o media id nutrient content composition gl-1 h2o 1 potato starch table sugar (sucrose) 4.0 20.0 8 peptone (bactotm) yeast extract (bactotm) malt extract (bactotm) dextrose (merck milipore) 2.0 2.0 2.0 20.0 2 potato starch glucose (merck milipore) 4.0 20.0 9 pdb (himedia®) dextrose (merck milipore) 0.48 19.6 3 potato starch dextrose (merck milipore) 4.0 20.0 10 pdb (himedia®) glucose (merck milipore) 0.48 19.6 4 yeast extract malt extract glucose (merck milipore) 1.0 1.0 20.0 11 glucose (merck milipore) yeast extract peptone k2hpo4 mgso4.7h2o feso4.7h2o caco3 20.0 1.0 5.0 0.5 0.5 0.01 1.0 (a) (b) iraqi j pharm sci, vol.32(1) 2023 the optimization of bisanthraquniones production 162 continued table 1. 5 pdb (bd difco™) 24 12 dextrose (merck milipore) yeast extract peptone k2hpo4 mgso4.7h2o feso4.7h2o caco3 20.0 1.0 5.0 0.5 0.5 0.01 1.0 6 pdb (bd difco™) 12 13 potato infusion dextrose (merck milipore) agar 4.0 20.0 7 peptone (bactotm) yeast extract (bactotm) malt extract (bactotm) glucose (merck) 5.0 3.0 3.0 200.0 secondary metabolites extraction the grown fungi mycelium was transferred from the media and crushed using a laboratory blender. a solvent mixture from ethyl acetate and acetone 7:1 ratio (v/v) (merck millipore) was added to the crushed media and left to extract the secondary metabolites. the crushed mycelium was macerated for 3x 6 hours at room temperature. the liquid-liquid solvent fractionations were implemented to separate the organic and aqueous fractions from culture media. the ethyl acetate fraction was vacuum dried at 35 oc using a rotary evaporator (ika® rv 8), weighted, and reserved for further analysis. determination of bis-anthraquinones content by thin layer chromatography (tlc) scanner a calibration curve was constructed to determine the epi and bis content in the extracts. a stock solution (1 mg/ml) from epi and bis was prepared by dissolving the preserved sample with ethanol (merck millipore). a serial dilution was carried out to obtain100, 500, 1000, 2500, 5000, and 7500 ng/ml standard solutions. a spot of 7.5 µl aliquot from the sample extract was applied to thinlayer chromatography (tlc) silica plate (merck millipore, tlc silica gel 60g f254 plate) by pinpointed capillary tube and subsequently dried before being put to the tlc chamber. the applied spot was developed on the chromatogram with dichloromethane-methanol-acetic acid (merck milllipore) (10:1:0.1 ) v/v/v in a tlc chamber saturated environment with the mobile phase vapor. the air-dried chromatogram tlc plate was then placed into a tlc scanner (shimadzu) and the area under the curve was measured for both epi and bis components at 433 and 481 respectively. the calibration curve was established by plotting the measured area under the curve. the calibration curves are shown in figures 2a and 2b. each endophytic extract obtained from different culture media was solvated with ethanol to form 1 mg/ml solutions. the volume of 7.5 µl sample specimens from the extracts was tlc assayed and scanned using the same conditions mentioned above. the calibration of the area under the curve was carried out for all the samples using the below equation and the anthraquinones contents in each extract were obtained. linear regression analysis was used to determine the quantity of both epi and bis in the extract samples. the original extract epi and bis components were calculated by multiplying with the aliquote factor when the sample was deposited into the tlc plate as described in equation 1, equation 1: 𝑂𝐶: ( 𝑎 1000 µ𝑔 𝑥 100%) 𝑥𝑏 a: bis-anthraquinones (epi or bis) obtained from measurement (in µ𝑔) b: mass of extract sample (µ𝑔) oc: concentration of bis-anthraquinones on the initial extract results and discussion gambir plant (uncaria gambier roxb) consisted of various types of endophytic fungi.. one of the endophytic fungi known for its biological activity is diaporthe sp. gnbp-10 (22). taxonomic identification of diaporthe sp. gnbp-10 was conducted through observation of macroscopic and microscopic characters based on several references as described comprehensively in ilyas et al (2009) (22). the macroscopic appearance of gnbp-10 is presented as dark-yellow thick colonies, with mycelium characterized as immersed, branched, septate, hyaline (22). anthraquinones produced by diaporthe sp. gnbp-10 in various culture media linear regression analysis. the retention factor (rf) of epi after the elution was 0.4 demonstrated by was iraqi j pharm sci, vol.32(1) 2023 the optimization of bisanthraquniones production 163 measured with yellow spots on the chromatogram. the bis compound was moved by the mobile phase to 0.6 rf appearing as a red spot on the chromatogram. the tlc scanner was used to evaluate the content targeted samples quantitively. this instrument combines the uv chamber equipped with a specific wavelength with a detector that can detect the area of the separated sample and convert it into a particular curve pattern (23). the tlc scanner was chosen due to its quick analysis time, lack of complex sample preparation, low solvent consumption, which makes it more environmentally friendly, and ability to analyze multiple samples simultaneously. according to the calibration curve, each standard of epi and bis concentration was linear over this concentration range. the correlation coefficient (r2) for epi and bis is 0.9944 and 0.9888, respectively, as shown in figures 2a and 2b. the linear concentration versus absorbance of the standard anthraquinones relation was applied to determine the concentrations of the metabolites in the samples while the correlation coefficient (r2) for both authentic samples epi and bis were 0.9779 and 0.9893 respectively, figure 2a and 2b. figure 2. (a)the calibration curve for epi. (b) calibration curve for bis. the nutrient variation of the culture media greatly affects the obtained extract mass, as shown in table 2 and figure 3a. the malt extract induces higher cell mass production, indicating the highest extract mass (159.4 mg) obtained from the cultivation of media 7 (figure 3a). the differences in the weight of the extracts could also be influenced by the extraction process such as the maceration step, liquid extraction step, and filtrations. the difference in composition and the diversity of secondary metabolites might contribute to the differences in extracted mass from various culture media (20). as shown in figures 3a and 3b, the optimized extract mass was obtained from media 7, while the targeted metabolites were found high in media 3. this finding indicates that the promoted robust fungal growth media and a high amount of the extract may not always produce high amount of the targeted compounds. the previous study discussed pd (potato dextrose) and yesd (soy peptone, dextrose, yeast extract, h2o) media facilitated a large amount of g24 extract but failed to produce high content of the targeted compound (20). the availability of simple sugar improves the production of bis-anthraquinones. media 1 containing the disaccharides showed lower epi expression than media 2 and 3 containing monosaccharides (glucose and dextrose). dextrose displayed a better nutrient than glucose for epi production in diaporthe sp. gnbp-10, as shown by higher epi production in media 3 (0.484 mg) than in media 2 (0.3705 mg). dextrose is d-glucose, while glucose contains both l-glucose and d-glucose. carbohydrates, energy, minerals, and vitamins (especially thiamin) are key nutrients for fungal growth (24). dextrose, glucose, and potato starch are the source of carbohydrates (25). though simple sugars are vital for endophytic fungi growth, the ultimate glucose content in the culture media was reported to induce oxidative stress in yeast. it can negatively affect important cellular components like dna, lipid, and proteins (26). potato dextrose media containing potato infusion and dextrose have been considered the main media for fungi cultures (25). serving as a carbohydrate and energy source. moreover, potato is a material full of nutrients such as nitrogen, enzyme, vitamins, and minerals needed for fungal growth (27). malt extract is a carbohydrate source that consists primarily of maltose. both malt extract and potato dextrose are the carbohydrates and energy sources for the growing fungi. in this case, potato starch exhibited better performance to support the production of metabolites, as shown by all media containing potato (1,2, and 3) having higher bis-anthraquinones content than media with malt extract (4,7,8). the acidic environment provided by the malt extract (28) probably influences the biosynthetic pathway of both bis-anthraquinones in diaporthe sp. gnbp-10. figure 3a depicts the comparison of the extracted mass, while figure 3b represents bis-anthraquinones production from endophytic fungi diaporthe sp. gnbp-10. regarding the production of the bis metabolite, the highest production rate was attained from media 3, showing the number of bis content in the extract is 0.1621mg. the bis component production was undetected in diaporthe sp. gnbpiraqi j pharm sci, vol.32(1) 2023 the optimization of bisanthraquniones production 164 10 cultured in media no, 4,5,7,8, 11,12, and 13. there is no distinct content in the nutrient from the mentioned media. media number 1 showed the optimized culture media to produce bis compound. another study is needed to examine the induction capacity of the culture media composition on bis compound production in more regarding the effect of culture media composition to induce bis production in diaporthe sp. gnbp-10. the peptone and yeast incorporation as nitrogen sources showed an insignificant effect on the production of both anthraquinones from diaporthe sp. gnbp-10. media 11 (table 1) demonstrated the presence of various minerals seemed not to substantially contribute to the improvement of bis anthraquinone production. though the culture media contain relatively complete minerals, protein sources, and simple sugars, the absence of complex carbohydrates such as potato starch appeared to decrease the production of bis-anthraquinone in media 11. table 2 and figure 3b present the production of bis-anthraquinones was better in the manually prepared media compared to in the commercially available media (pdb himedia and pdb difco). both pdb difco and himedia contain potato starch and dextrose. table 2. the measurement results of bis-anthraquinone from endophytic fungi cultured with various nutrient composition culture media id extract sample (mg) epi bis area under curve measured content (µg/ml) content in the initial extract (µg)* area under curve measured content (µg/ml) content in the initial extract (µg)* 1 47.3 8117.37 2.51 118.723 5762.68 1.94 91.762 2 139.3 8571.91 2.66 370.538 1973.12 0.7 97.51 3 113.9 13405.05 4.25 484.075 4226.79 1.43 162.877 4 92.6 1367.9 0.29 26.854 ud ud ud 5 106.7 2108.52 0.53 56.551 ud ud ud 6 53.8 7069.69 2.17 116.746 2962.14 1.02 54.876 7 159.4 ud ud ud ud ud ud 8 90.1 317.63 < 0.1 < 1.0 ud ud ud 9 28.2 3613.99 1.03 29.046 1185.16 0.44 12.408 10 16 3334.65 0.94 15.04 699.67 0.28 4.48 11 53.4 222.84 < 0.1 < 1.0 ud ud ud 12 31.7 ud ud ud ud ud ud 13 139.3 307.7 < 0.1 < 1.0 ud ud ud *calculation refer to equation 1, ud: undetected a b figure 3. the comparison of (a) production of extract mass and (b). bis-antraquinones produced from endophytic fungi diaporthe sp. gnbp-10 is cultured in various media. iraqi j pharm sci, vol.32(1) 2023 the optimization of bisanthraquniones production 165 this study shows the optimized culture media composition for the production of bisanthraquinones from endophytic fungi diaporthe sp. gnbp-10. this finding can be used for optimum production of epi and bis components to meet a sufficient amount of epi and bis to further biological evaluation of both bis-anthraquinones. conclusion this study is a cultured compositions optimization assay for the endophytic fungi diaporthe sp. gnbp 10 (associated with gambier plant) growth and metabolites expression. the optimized fungal extract was obtained from media no. 7 (159.4 mg), consisting of malt extract as a carbohydrate source. meanwhile, the optimum epi and bis components production was observed in media no. 3 with the content of epi and bis in the respective extract are 0.484 mg and 0.1628 mg. the manually prepared culture media showed a better environment for producing the targeted anthraquinones than the commercially available pdb media. potato starch provides a higher production level for bis-anthraquinones matched to the malt extract. the presence of glucose and dextrose is important to the production of bisanthraquinones. hopefully, this finding can be useful to produce the optimum amount of epi and bis compounds, 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sulaiman * , saad a. hussain *,1 , kasim m. jum'ma ** and assad h. sigar * * department of pharmacology and toxicology,college of pharmacy,university of baghdad,baghdad,iraq ** department of pharmacy, baquba general hospital, diyala, iraq abstract the antidiabetic thiozolidinediones (tzds) a class of peroxisome proliferators-activated receptor (ppar) ligands has recently been the focus of much interest for their possible role in regulation of inflammatory response. the present study was designed to evaluate the anti-inflammatory activity of pioglitazone in experimental models of inflammation in rats. the present study was conducted to evaluate the anti inflammatory effect of tzds (pioglitazone 3mg/kg) on acute, sub acute and chronic model of inflammation by using egg-albumin and formalin–induced paw edema in 72 rats, relative to reference drugs dexamethasone 5mg/kg and piroxicam 5mg/kg. in each inflammation model, 24 rats were allocated into four subgroups, each containing six rats representing control, two standards, and test groups. all treatments were given (i.p) 30 minutes before induction of inflammation and the increase in paw edema was measured at certain time intervals by using vernier caliper. pioglitazone produced nonsignificant reduction (p>0.05) of egg albumin-induced acute inflammation of the rat hind paw, while significantly produced time-related reduction of formalin-induced sub-acute and chronic inflammation of the rat hind paw. in conclusion, pioglitazone possesses anti-inflammatory activity in the animal models of sub-acute and chronic inflammations. key words: pioglitazone, ppar-γ, anti-inflammatory activity الصةالخ أخذ األهخًاو َشداد فٍ اِوَت األخُزة بدراست انفعانُت انًحخًهت انًضادة نألنخهاا نًتاخكاث انزاَىسونُدَُاداَىٌ وانًداخ ديت اانُاا انخضزَبُات بتكم فعال فٍ يعانضت داء اندكزٌ. حى حصًُى اندراست انحانُت نخكُُى انفعانُت انًضادة نألنخها نًاادة بُىلهُخااسوٌ فاٍ انًُاا س فااٍ اااا ث يهغى/لغااى يااٍ يااادة بُىلهُخاااسوٌ ۳نألنخهااا نضز اات يكاادارها نألنخهاباااث فااٍ انضااز اٌت اُااذ حًااج دراساات انخاا رُز انًضاااد وححااج انحااادة وانًشيُاات انًدااخحدرت بىاسااطت انفىريااانٍُ فااٍ أقااداو انضااز اٌ وقُاااص تت س ل انبااُ طاألنخهااا انحااادة انًدااخحدرت بىاساا لبااث انكُاساُت زويكارَت يزام هاذا انخا رُز ياك ناي اناذٌ حدابب انً تيت انًخكىَت قبم وبعد اسخ داو انًزلب قُد اندراستيدخىي حكىٌ انى صاز ا نكام ًَاى س ياٍ ًَاا س األنخهاا وحاى حكداًُها اناً أربعات ٤٢ انًضادة نألنخها يزم انبُزولداُكاو واندَكداايُزاسوٌ. حاى اساخ داو ت اُاذ حاى ا طااء ندُطزةت يضًى خاا انًزلبااث انكُاساُت انًضاادة نألنخهاا ويضًى ات انبُىلهُخااسوٌلم يٍ يضًى ت ا يضًى اث حًزم صًُااك انًزلباااث ااٍ لزَااف انااشرت فااٍ انبزَخااىٌ قباام اسااخحداد األنخهااا بزارااٍُ دقُكاات ويااٍ رااى قُاااص اضااى انى ياات انًخكىَاات بىاسااطت لهُخااسوٌ هاً انحاد ياٍ حكاىٌ انى يات وبفاارت يعُاىٌ َعخًاد هاً انفخازة انىرَُت فٍ فخزاث سيُُات يحاددة. أرهازث انُخااقش يكادرة انبُى انشيُُاات بعااد األ طاااء فااٍ اااا ث األنخهااا ححااج انحااادة وانًشيُااتت أيااا فااٍ اااا ث األنخهااا انحااادة فهااى َ هااز يزاام هااذا انخاا رُز. ًَكااٍ نخهاباث ححج انحادة وانًشيُت انًدخحدرت فٍ انضز اٌ.األسخُخاس ب ٌ نهبُىلهُخاسوٌ فعانُت يضادة نألنخها فٍ انًُا س انخضزَبُت نأل introduction inflammation is a complex biological set of interactions between soluble factors and cells that can arise in any tissue due to disturbed homeostasis in response to traumatic, infectious, post-ischemic, toxic or autoimmune injuries (1) . the inflammatory process is often viewed as being comprised of three closely linked phases: initiation, propagation and resolution. it is a protective attempt by the organism to remove the injurious stimuli as well as initiate the healing process for the tissue (2) ; however, if the targeted destruction and assisted repair are not properly phased, inflammation can lead to persistent tissue damage contributing to the pathogenesis of common chronic inflammatory diseases such as atherosclerosis, arthritis, inflammatory bowel disease and multiple sclerosis (1,3,4) . the current antiinflammatory therapies designed to limit or interrupt the synthesis or action of mediators that drive the host's response to injury i.e. limit the initiation and propagation phases (2) . however, it is increasingly recognized that therapies aimed at enhancing the resolution phase will be important in limiting the damage associated with inflammation-based disease (5) . recently, the modulatory role of peroxisome proliferator-activated receptors (ppars) has been proposed in the inflammatory response of different tissues and organs (6) . 1 corresponding author e-mail : saad_alzaidi@yahoo.com received : 3/1/2009 accepted : 8/3/2009 iraqi j pharm sci , vol.18 (suppl.) , 2009 anti – inflammatory effect of pioglitazone ٤ three different isoforms of this receptor have been recognized; pparα, pparδ and pparγ (7) , the later is predominantly detected in adipose tissue, intestine and macrophages. pparγ activators, such as pioglitazone, are a new class of oral antidiabetic drugs that ameliorate insulin resistance with an improvement in glucose control (8,9) . next to their anti diabetic properties, these drugs were shown to exert variety of anti-inflammatory and vasoprotective effects in diabetic and non diabetic subjects (10,11,12) . these recent findings provide opportunities for the potential therapeutical application of these drugs in chronic inflammatory diseases with fewer side effects than traditional anti-inflammatory drugs.the present study was carried out to evaluate the anti-inflammatory effect of pioglitazone relative to commonly used antiinflammatory drugs piroxicam and dexamethasone. materials and methods the present study was carried out on 72 sprague-dawley rats of both sexes weighing 180-250 gm, selected from the animal house of the college of pharmacy, university of baghdad. the animals were maintained on normal temperature, humidity and light/dark cycle. they fed standard rat pellet diet and had free access to water until the night of the day of investigation. the animals were allocated into three main groups each of 24 animals for evaluation the anti-inflammatory effect of pioglitazone on acute, sub acute and chronic inflammation models. for egg albumininduced acute inflammation, after an overnight fasting 24 animals were allocated into four subgroups (each of six rats), the control group was treated with dimethylsulfoxide 2 ml/kg (vehicle), the two standard groups were treated with piroxicam 5mg/kg, and dexamethasone 5mg/kg respectively, while the test group was treated with pioglitazone 3mg/kg. all drugs were administered intraperitoneally. thirty minutes after drug treatment, inflammation was induced by injecting 0.1 ml of fresh egg albumin (13,14) into the dorsal surface of the right hind paw. the increase in paw edema as a result of inflammation was measured using vernier caliper method (15) , where thickness was measured by vernier before and 1hr, 2hr, 3hr and 4hr after induction of inflammation. the difference in paw thickness after and before induction of inflammation was calculated and determined as mean increase in paw thickness (mm). the ability of antiinflammatory drug to suppress paw inflammation was expressed as percentage of inhibition of paw edema (16) .in formalininduced sub acute inflammation, the test group was treated with pioglitazone 3mg/kg and the two standard groups were treated with piroxicam 5mg/kg and dexamethasone 5mg/kg, while the control group was treated with dimethylsulfoxide 2ml/kg. all drugs were administered intraperitoneally 30 minutes before induction of inflammation and the paw thickness was measured by vernier caliper (15) immediately prior to drug administration (at zero time) and then at 1.5hr, 24hr, and 48hr after formaldehyde injection. mean increase in paw thickness and the percentage of inhibition then calculated as mentioned previously. chronic inflammation was induced by injection of 0.1ml of 2% formalin into sub planter area of the right hind paw of rat (17) . all treatments were administered 30 minutes prior to formalin injection and continued for seven consecutive days. the increase in paw thickness was measured by vernier caliper method (15) before and six days after induction of inflammation. the mean increase in paw thickness and the percentage of inhibition was calculated as in previous models. all data were expressed as mean ±sem. comparisons between groups were performed by anova and student's t-test to evaluate the statistical differences. the p value < 0.05 was considered significant. results the anti-inflammatory effect pioglitazone on acute inflammatory model was illustrated in table 1 and figure 1. treatment with dexamethazone and piroxicam significantly reduced egg albumin-induced paw edema (p<0.05) compared to control group after 1hr, 2hr, 3hr and 4hr after induction of inflammation, while treatment with pioglitazone results in non-significant reduction (p>0.05) in paw thickness compared to control group all over the period of investigation. figure 1. effect of pioglitazone on egg albumin-induced acute inflammation in rats. iraqi j pharm sci , vol.18 (suppl.) , 2009 anti – inflammatory effect of pioglitazone ۳ table 1. effect of pioglitazone on egg albumin-induced acute inflammation in rats. % of inhibition mean increase in paw thickness (mm) treatment groups 4 h 3 h 2 h 1 h 4 h 3 h 2 h 1 h _ _ _ _ 1.52  0.16 1.95  0.18 2.47  0.20 3.07  0.24 dimethyl sulfoxid 62 51 43 19 0.58 ± 0.03 * a 0.95 ±0.04 * a 1.40 ±0.11 * a 2.47 ±0.11 * a dexamethazone 50 35 33 19 0.76 ± 0.14 * a 1.27 ±0.05 * b 1.65 ±0.09 * a 2.48 ±0.05 * a piroxicam 33 22 20 15 1.02 ±0.19 * a 1.53 ±0.17 * a 1.98 ±0.15 * a 2.62 ±0.22 * a pioglitazone data were expressed as mean ± sem; number of animals = 6 in each group; * p< 0.05 with respect to control group; values with non-identical superscripts (a, b) are considered significantly different (p<0.05). the suppressive effect of pioglitazone in formalin-induce sub-acute inflammation was shown in table 2 and figure 2; all drug treatments significantly reduced the paw edema during the whole time of assessment compared to control group at 1.5 hr, 24 hr and 48 hr (p<0.05). pioglitazone (3 mg/kg) showed significant reduction in paw thickness (p<0.05) compared to piroxicam and dexamethazone over all the time of assessment. table 3 demonstrated the effect of pioglitazone on formalin-induced chronic inflammation; all treatments significantly reduced the paw edema induced by formalin (p<0.05) compared with control group. both pioglitazone and piroxicam produced comparable effect on formalin-induced chronic inflammation and no significant differences were detected between them; while their effect was significantly different compared to that produced by dexamethazone (p<0.05), which produced the greatest effect. figure 2. effect of pioglitazone on formalininduced sub-acute inflammation in rats. table 2. effect of pioglitazone on formalin-induced sub-acute inflammation in rats % of inhibition mean increase in paw thickness (mm) treatment groups 48 h 24 h 1.5 h 48 h 24 h 1.5 h _ _ _ 2.68 ±0.20 2.87 ±0.19 3.12 ±0.17 dimethyl sulfoxide 64 63 35 0.97 ±0.10* a 1.07 ±0.09* a 2.03 ±0.15* a dexamethasone 51 44 38 1.32 ± 0.05* b 1.62 ±0.06* b 1.93 ±0.05* a piroxicam 36 28 20 1.72 ±0.07* c 2.08 ±0.11* c 2.50 ±0.06* b pioglitazone data were expressed as mean ± sem; number of animals = 6 in each group; * p<0.05 with respect to control group; values with non-identical superscripts (a, b, c) are considered significantly different (p<0.05). iraqi j pharm sci , vol.18 (suppl.) , 2009 anti – inflammatory effect of pioglitazone ٢ discussion egg albumin-induced paw edema in rats an in vivo model of inflammation (18), which has long accepted as a useful tool to study and evaluate drugs with anti inflammatory activity in acute inflammation (19,20) . the degree of swelling in paws injected with egg albumin was maximal 1hr after injection and then decreased with time. in the present study, the effect of pioglitazone was evaluated on egg albumininduced edema as a model for acute inflammation, where the data (table 1; figure 1) revealed no significant reduction in paw edema compared to control group; this could be explained by the fact pparγ ligands regulate gene expression (21,22) , which consequently produce their expected effects after a characteristic lag time that may extend to several hours. so, the improvement of the induced pathological state not occur immediately, but require enough time which represent that required for the synthesis of new signaling protein (23) . in sub-acute and chronic models of inflammation, injection of formaldehyde in the hind paw of rats produced pain and peripheral tissue inflammation (24) , which is biphasic and includes a phase of inflammatory response, where histamine, 5ht, pgs and bradykinin are involved (25) . in the model of sub-acute inflammation, pioglitazone (3mg/kg) produced significant reduction (p<0.05) in paw thickness along the period of investigation compared to control group, and the level of inhibition is found to be less than that produced by standard antiinflammatory drugs (piroxicam and dexamethazone) as shown in table 2 and figure 2; this effect may be attributed to repression of synthesis of many inflammatory mediators. many studies have demonstrated that pparγ agonists inhibit the production of several inflammatory cytokines (26,27) , including those that plays an important role in the nociceptive and inflammatory responses induced by formaldehyde, moreover, inhibition of the production of ecosanoids and no has also been demonstrated after treatment with ppars agonists ( 28,29, 30) . in chronic inflammation, this represents a continuous inflammatory state that could be driven by the development of an immune response to an endogenous antigen (31) . the effect of pioglitazone on formalininduced paw edema, as a chronic inflammatory model, was assessed by vernier caliper method. pioglitazone significantly reduced paw thickness (p<0.05) and the level of inhibition was found to be higher than that of piroxicam but less than dexamethazone inhibitory effect as shown in table 3. this result may provide an indication about the possible usefulness of pioglitazone in the management of chronic inflammation of many diseases. recently, pioglitazone was tested in different chronic inflammatory diseases including neurological, cardiovascular and gastrointestinal diseases. it significantly accelerates ulcer healing in experimental animals due to hyperemia at ulcer margin and the anti inflammatory action including suppression of pro inflammatory cytokine, down regulation of cyclooxygenase-2 (cox2) and inducible nitric oxide synthase (inos) at the level of mrna and protein synthesis (32) . also pioglitazone has been observed to ameliorate pancreatic damage associated with cerulin-induced pancreatitis (cip) by inhibiting the production and release of il-1β (33) . it effectively provides neuroprotection against lps insult in dopaminergic neurons through the inhibition of jnk-nf-kb pathways as well as suppression of cox-2 activity and decreased pge2 production (34) .in conclusion, pioglitazone showed reproducible anti-inflammatory activity in sub-acute and chronic models of inflammation in rats within the therapeutic dose utilized to increase sensitivity to insulin, which is comparable to that produced by piroxicam and less than that produced by dexamethasone. table 3. effect of pioglitazone on formalin-induced chronic inflammation in rats. data were expressed as mean ± sem; number of animals = 6 in each group; p < 0.05 with respect to control group; values with non-identical subscription (a, b) are considered significantly different (p<0.05). % of inhibition mean increase in paw thickness (mm) after 6 days treatment groups  3.30 ± 0.15 dimethyl sulfoxide 64 1.18 ± 0.14* a dexamethazone 37 2.08 ± 0.13* b piroxicam 44 1.85 ± 0.09* b pioglitazone 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pc, dembinski a, warzecha z, et al. pioglitazone, a specific ligand of ppar-γ, protects pancreas against acute cerulein-induced pancreatitis. world j gastroenterol 2005; 11(40): 6322-6329. 34. xing b, liu m, bing g. neuroprotection with pioglitazone against lps insult on dopaminergic neurons may be associated with its inhibition of nf-κb and jnk activation and suppression of cox-2 activity. j neuroimmunol 2007; 192: 8998. iraqi j pharm sci, vol.32( 1 ) 2023 l-carvone and acute lung injury doi: https://doi.org/10.31351/vol32iss1pp125-132 125 anti-inflammatory effect of l-carvone on lipopolysaccharide-induced acute lung injury samah mahde*,1 and sarmad haishm kathem* department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract acute lung injury is among the most serious conditions that affect the lung which is characterized by an exacerbation of inflammatory response that can result from a severe lung infection. the enantiomers of carvone (l and d) are found in various plants species each with special pharmacological effects; where, the levo (l)carvone is chiral monoterpenoid ketone present in the essential oils of dill, caraway, and spearmint and possess many pharmacological properties like antioxidant, anti-inflammatory, antimicrobial, antidiabetic, and anticonvulsant effects. in a previous study, l-carvone inhibited mucositis induced by irinotecan. this study is the first to evaluate the lung anti-inflammatory protective effects, and potential mechanism of action of l-carvone in acute lung injury induced by lipopolysaccharide by measuring the gene expression level of inflammatory mediators (tumor necrosis factor-alpha, cyclooxygenase-2 and nuclear factor-kappa of activated b cells) by conducting real-time quantitative polymerase chain reaction test. fifty adult mice were allocated into 5 groups as follows: control group (mice received normal saline, group i). mice in the -induction group received (lipopolysaccharide 10mg/kg/day intraperitoneally, group ii) and were euthanized 2 hours later. group iii (vehicle group, mice received corn oil 0.1 ml + lipopolysaccharide 10 mg/kg). mice in the -treatment groups received either [(50mg/kg/day), group iv] or [(100mg/kg/day), group v] of oral l-carvone for 5 consecutive days before lipopolysaccharide injection. pretreatment with l-carvone (50mg/kg/day) (group iv) markedly attenuated pro-inflammatory cytokines as observed by significant (p<0.05) reduction in mrna expressions of tumor necrosis factor-alpha (7.56 ±1.195 vs 29.20±4.9) and cyclooxygenase 2 (5.72±0.329 vs.10.58 ±0.777) in mice’s lung tissue compared to those in the induction group, non-treated mice (lipopolysaccharide model group) (group ii). increasing the dose of l-carvone to 100mg/kg/day (group v) also resulted in a significant reduction in mrna expressions of tumor necrosis factor-alpha (7.84±1.4 vs 29.20±4.9) and cyclooxygenase2 (4.589± 0.946 vs 10.58±1.641) compared to that expression in the induction group, non-treated mice (lipopolysaccharide model group) (group ii); however, the attenuating effect is dose-independent. furthermore, the results revealed that nuclear factorkappa of activated b cells mrna gene expression was significantly lowered by l-carvone 50mg/kg /day (group iv) (5.01±0.826 vs 11.88±1.227) and 100mg/kg /day (group v) (6.81±1.362 vs 11.88±1.227) compared to induction group, non-treated mice (lipopolysaccharide model, group ii). conclusion this study clearly revealed that l-carvone exerted anti-inflammatory and lung-protective effects on lipopolysaccharide-induced acute lung injury. the observed effects were dose-independent and resulted from hampering of the nf-κb signaling pathway. keywords: acute lung injury, lipopolysaccharide, tnf-α, cox2, l-carvone. المضاد لأللتهاب لليفو كارفون في تلف الرئة الحاد المستحث بواسطة عديد السكاريد الدهنيالتأثير *سرمد هاشم كاظم و 1*،سماح مهدي . العراق، بغداد، بغداد جامعة، الصيدلةكلية ، والسموم*فرع االدوية الخالصة تعد أصابة الرئة الحادة من أخطر الحاالت التي تصيب الرئة حيث تتصف بتفاقم االستجابة األلتهابية التي يمكن ان تنتج من التهاب هو كيتون دوائية خاصة . ليفو كارفون تأثيرات منها لكل مختلفة نباتية أنواع في (l and dله نوعان مصاوغ مرآتي ) كارفونال.رئوي حاد .لديه العديد من الخصائص الدوائية مثل مضاد األكسدة والنعناع والكراوية الكمون تفي الشب األساسية أحادي التربينيدات موجود في الزيوت الليفو كارفون ثبط التهاب الغشاء المخاطي واأللتهاب ومضاد للميكروبات وتأثير مضاد لمرض السكر ومضاد لألختالج. في دراسة سابقة، فان الليفو كارفون ضد المستحث بواسطة عقار االيرينوتيكان. هذه الدراسة هي األولى لتقييم االثار الوقائية المضادة اللتهاب الرئة واآللية المحتملة لعمل α لتعبير الجيني للسيتوكينات المؤيدة لأللتهاب عامل نخر الورمتلف الرئة الحاد المستحث بواسطة عديد السكاريد الدهني. عن طريق قياس مستوى ا ) (tnfوالعامل النووي 2وانزيم سايكلوأوكسي جينيزκ للخاليا البائيةb في الكمي المتسلسل البلمرة تفاعل اختبار المنشطة عن طريق اجراء (.األولى المجموعة ، طبيعيًا محلواًل الفئران تلقت) التحكم مجموعة -: التالي النحو على مجموعات 5 إلى بالغًا فأًرا خمسين تقسيم تم .الفعلي الوقت 1corresponding author e-mail: samahmahde@gmail.com received: 16/2 /2022 accepted: 24/4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp125-132 iraqi j pharm sci, vol.32(1) 2023 l-carvone and acute lung injury 126 . ساعتين بعد القتل الرحيم وتم( الثانية المجموعة ، الصفاق داخل يوم/ كغم/ ملغم 10 الدهني السكاريد عديد ) الحث مجموعة في الفئران تلقت العالج مجموعات في الفئران تلقت(. كغم/ ملغم 10 الدهني السكاريد عديد+ مل 0.1 الذرة زيت الفئران تلقت ، المذيب مجموعة) الثالثة المجموعة قبل متتالية أيام 5 لمدة الفموي الكارفون من[ الخامسة المجموعة ، ( يوم/ كغم/ ملغم 100]) أو[ الرابعة المجموعة ، ( يوم/ كغم/ ملغم 50]) إما الدهني. السكاريد عديد حقن األلتهابية كما مالحظ من انخفاض بشكل كبير في التعبير الجيني ملغم/كغم /يوم )المجموعة الرابعة ( قلل بشكل كبيراألحداث 50لعالج بالليفو كارفون ا ( 0.777± 10.58مقابل 0.329±5.72)2( وأنزيم سايكلوأوكسي جنيز 4.9± 29.20مقابل 1.195±7.561في أنسجة الرئة لعامل نخر الورم ) المعالجة غير بالمجموعة المجموعمقارنة , الدهني السكاريد عديد )مجموعة الحث (,مجموعة الثانية الى الجرعة زيادة وعند ة كارفون لليفو ( و أنزيم سايكلو 4.9±29.20مقابل 1.4±7.84ملغم/كغم/يوم )الجموعة الخامسة ( أيضا قلل بشكل كبير التعبير الجيني لعامل نخر الورم )100 الحث )مجموعة عديد السكاريد الدهني , ( مقارنة بالمجموعة الغير معالجة ,مجموعة 10.58± 0.777مقابل 0.946±4.589) 2أوكسي جنيز امل النوويالمجموعة الثانية(. مع ذلك فان تأثير الليفو كارفون لم يعتمد على الجرعة .وأيضا اظهرت النتائج أنخفاض كبير في التعبير الجيني للع κ للخاليا البائيةb ( وجرعة 1.227±11.88مقابل 1.362 ±6.81عة ( )ملجم/كغم/يوم)المجموعة الراب 50المنشطة عند جرعة الليفو كارفون ( مقارنة بمجموعة الحث , المجموعة الغير معالجة )مجموعة 1.227±11.88مقابل 0.826± 5.01ملغمم/كغم/يوم ) المجموعة الخامسة ( )100 عديد السكاريد الدهني , المجموعة الثانية ( كارفون له تأثير مضاد لأللتهاب ووقائي في أصابة الرئة الحادة المستحثة -الليفواألستنتاج :كشفت هذه الدراسة بوضوح أن bللخاليا البائية κبعديد السكاريد الدهني، حيث ان التأثيرات كانت غير معتمدة على الجرعة ونتجت عن أعاقة مسار أشارات العامل النووي المنشطة . . كارفون، ليفو 2الفا ، سايكلو اوكسي جنيز -،عامل نخر الورمديد السكاريد الدهني ع،الكلمات المفتاحية :التهاب الرئة الحاد introduction acute lung injury (ali) and its more severe form, acute respiratory distress syndrome (ards), is a serious inflammatory condition affecting the lung with a high percentage of morbidity and mortality (1). the ali is caused by neutrophil leakage, production of a huge amount of pro-inflammatory cytokines, injury to airway epithelium and alveolar endothelium which lead to pulmonary fluid accumulation, and the impairment in gases exchange. lipopolysaccharide (lps) is a component of the gram-negative bacterial cell wall, which is a glycolipid that consists of many disaccharide units and polar lipid head group and can be employed to create an ali model in the mice lung which mimicked its characteristic and pathological features in the human (2,3). previous studies have demonstrated that lps could cause ali by activating the toll-like receptor 4/nuclear factorkappa b (tlr4/nf-κb) signaling pathway, which controls the transcription of pro-inflammatory cytokines, such as interleukin 1β (il-1β), interleukin-6(il-6), and tumor necrosis factor-alpha (tnf-α). these cytokines are responsible for the activation of the innate immune system which results in injury in the lung tissue (4–6). carvones are chiral monoterpenoid ketones (5-isopropenyl-2-methyl-2-cyclohexenone) that are abundant in many plants including caraway, angelica, and spearmint. in recent years it attracts attention as alternative medicine. it is present in nature in two enantiomers (+, d) mainly present in caraway carum carvi and (-, l) carvone mainly present in mentha spicata. many studies evaluate pharmacological effects like anti-inflammatory, antioxidant effects, antibacterial, antidiabetic, anticonvulsant, and antinociceptive effects. according to the previously mentioned effects of lcarvone, the anti-inflammatory and possible lung protective effect in ali induced by lps was investigated in an animal model in this study (7–10). the anti-inflammatory effect of l-carvone was previously reported through the inhibition of myeloperoxidase activity (mpo), reduction of prostaglandin e2 (pge2), interleukin-1β (il-1β), tumor necrosis factor (tnf-α), nitric oxide (no˚), inducible nitric oxide synthase (inos) expression, cyclooxygenase e2 (cox2), an increase in glutathione levels (gsh) (10). moreover, l-carvone was able to produce a protective effect on irinotecan-induced intestinal mucositis in mice by reducing pro-inflammatory cytokines tnf-α production and diarrheal score (11). materials and methods chemicals and kit levo-carvone (l-carvone) was obtained from sigma aldrich/usa, lps o55:b5was purchased from sigma aldrich/ germany, nf–кb, gapdh, cox 2, and tnf α primers were purchased from macrogen / south korea, rna extraction kit was purchased from dong sheng biotech /china, trans start® green qpcr supermix was purchased from transgen biotech/ china, easy script® one-step gdna removal and cdna synthesis. animal selection fifty (50) albino male mice weighing (20-30) grams were divided into five groups were brought from and maintained in the animal facility of the college of pharmacy, university of baghdad, under conditions of the controlled temperature, humidity, and light periodicity (12-hour light/dark cycle). mice had free access to a standard diet and water during the experimental period. experimental protocol this study was approved by the scientific and ethical committees of the college of the pharmacy/university of baghdad. mice employed in this study were randomly-divided into five groups of ten mice each, as follows: iraqi j pharm sci, vol.32(1) 2023 l-carvone and acute lung injury 127 groupⅰ. mice received 0.1ml intraperitoneal injection of normal saline for five constitutive days and were considered as the normal group (negative control group). group ⅱ (lipopolysaccharide model group). mice received a single dose of intraperitoneal lps in dose (10mg/kg) and were euthanized after two hours. this group served as the acute lung injury model group(induction group) (12). group ⅲ (corn oil+lps). mice received corn oil (0.1ml) orally for five consecutive days. on day 5, mice received lipopolysaccharide (10mg/kg) and were euthanized after 2 hrs. the purpose of using corn oil+lps was to verify if the corn oil has an antiinflammatory effect or not (as corn oil is used to dissolve l-carvone). group ⅳ(l-carvone treatment). mice received lcarvone solution in dose (50mg/kg/day) orally for five consecutive days (13). on day 5, mice received lipopolysaccharide (10mg/kg) and were euthanized after 2 hrs. group ⅴ(l-carvone treatment). mice received lcarvone solution (100mg/kg/day) orally for five consecutive days (13). on day 5, mice received lipopolysaccharide (10mg/kg) and were euthanized after 2 hrs. administration of l-carvone is done by oral gavage at 8:00 am daily from day 1 through day 5. euthanization is done by diethyl ether followed by cervical dislocation determination of lung tissue gene expression of tnf-α, nf–кb, and cox2 the right lung was isolated and 50-100 mg of it was placed in a tube containing 1 ml of trizol (rl solution) and frozen for later use. analysis and calculation of gene expression levels of tnfα, nf –κb, cox 2, and glyceraldehyde-3phosphate dehydrogenase (gapdh), depend on mrna concentration after converting it to complementary dna. the process includes total rna extraction and purification using (dong sheng biotech /china), complementary dna (cdna) synthesis using random primers (transgen biotech / china), the sybr green pcr master mix (taqdna polymerase, syber green1, dntps, pcr enhancer and stabilizer) was used for real-time pcr analysis. the cycle time values of the interested genes were first normalized with gapdh(housekeeping gene which is a reference gene used to achieve accurate normalization for the rt-qpcr of the same sample, the changes in the mrna expression in all the groups were calculated by the comparative method of 2-∆∆ct. the primer pairs of the expected products were as follows (forward and reverse, respectively): nf-κb (5`aagacaaggagcaggacatg-3`and5` agcaacatcttcacatccc-3), tnf– α(5`tagcccacgtcgtagcaaac 3` and 5` acaaggtacaacc catcggc-3`), cox2(5` gctcagccaggcagcaaatc-3` and 5` cacc atagaatccagtccggg-3`) and gapdh(5` cgggttcctataaatacg gactg-3`and5`ccaatacggccaaatccgttc-3`) primers were purchased from macrogen /south korea, real-time pcr was performed using corbett research rtqpcr device according to the manufacturer instructions (14,15). statistical analysis data presented as mean± standard error of the mean (sem). statistical package for the social sciences (spss, version 25) was used for data analysis and student t-test was used. one-way anova and tukey used for comparison among groups were (p˂0.05) considered significant. results effect of l-carvone on tnf-α mrna level analysis of the data in the table (1) revealed that, the level of tnf-α mrna is significantly elevated (p<0.05) in the lps model (group ii) compared to corresponding level in the normal control (group i) (29.20±4.9 vs. 1.141±0.31). furthermore, administration of lcarvone in dose 50mg/kg/day (group ⅳ, l-carvone treatment group) significantly (p<0.05) attenuated the tnf-α mrna level in lung tissue (29.20 ±4.9 vs. 7.56±1.195) compared to those levels in the nontreated mice (group ⅱ, lps model group) (figure1). similarly, increasing the dose of lcarvone to 100mg/kg/day (group ⅴ, l-carvone treatment group) also resulted in significant (p<0.05) attenuation of the tnf-α mrna level (29.20 ±4.9 vs. 7.84±1.40) compared to non-treated mice (group ⅱ, lps model group). in addition, data of table 1 and figure 1 pointed out that there is a non-significant (p>0.05) difference between the (group ⅲ, corn oil + lps) and that in (group ⅱ, lps model group) in terms of tnf-α mrna level which revealed that corn oil used as a vehicle in the study has no effect on the tnf-α mrna gene expression level (which prove corn oil had no antiinflammatory effect). further, data analysis reveals that there is a non-significant (p>0.05) difference in the tnf-α mrna expression level between (group iv) mice treated with 50mg/kg/day and (group v) mice treated with 100mg/kg/day l-carvone. table (1) and figure (1). iraqi j pharm sci, vol.32(1) 2023 l-carvone and acute lung injury 128 table 1. effect of l-carvone on tnf-α, cox2, and nf-κb mrna gene expression levels. groups tnf-α gene expression level (mean ± sem) cox2 gene expression level (mean ± sem) nf-κb gene expression level (mean ± sem) g i: negative control (normal saline treated group) 1.141818± 0.318204 1.111382 ± 0.294431 1.15711 ± 0.369087 g ii: lps group (ali model group) 29.2048± 4.767602# 10.58144 ± 0.777646# 11.88348 ± 1.575529# g iii: (corn oil + lps group) 28.81076± 4.501522a 10.78121±2.751145a 11.31629 ± 0.684981a g iv: l-carvone treatment group 50mg/kg/day 7.838699± 0.399415*b 5.721759 ± 0.329006*b 6.810089 ± 1.719718*b g v: l-carvone treatment group 100mg/kg/day 7.560717± 1.155811*b 4.589894 ± 0.946045*b 5.005296 ± 0.800742*b #: significant (p<0.05) differences compared to the normal (negative) control group i mice. a: non-significant (p>0.05) differences compared to the lps model group ii mice. *: significant (p<0.05) compared to the lps model group ii mice. b: non-significant (p>0.05) differences between groups iv and v mice. figure 1. bar chart showing the effects of lcarvone on tnf-α expression in lps-induced acute lung injury (ali). rt-qpcr analysis of tnf-α mrna expression level in lung tissue of mice treated with l-carvone 50mg/kg/day and 100 mg/kg/day for 5 days and euthanized 2 hrs. after lipopoly-saccharide (lps) administration (n=10 mice in each group). data represent mean ±sem. #: significant (p<0.05) differences compared to the normal (negative) control group i mice. a: non-significant difference (p>0.05) (group iii, corn oil+lps) compared to the lps model group ii mice. *: significant difference (p<0.05) of l-carvone groups (iv and v) each compared to the lipopolysaccharide (lps) model group (ii) mice. b: non-significant (p>0.05) difference between groups iv and v mice. effect of l-carvone on cox2 mrna level results of table (1) showed that the cox2 mrna gene expression level was significantly-elevated (p<0.05) in lps model group (group ii) compared to the corresponding gene expression level in normal control group (10.58±0.777 vs. 1.1± 0.29); and the administration of l-carvone in a dose of 50mg/kg/day (group iv) significantly (p<0.05) attenuated the cox2 mrna gene expression level in lung tissue (10.58±0.777 vs.5.72±0.329) compared to non-treated animals (group ii, lps model group) (figure 2). moreover, doubling the dose of l-carvone to 100mg/kg/day (group v, lcarvone treatment) also resulted in significant (p<0.05) attenuation of cox2 mrna level (10.58±0.777 vs. 4.589± 0.946) compared to such level in non-treated mice (lps model group in addition, data of table (1) and figure (2) pointed out that there was a non-significant (p>0.05) difference in the cox2 mrna expression level between the (group ⅲ, corn oil + lps) and non-treated animals (group ii, lps model group), which can indicate that the utilization of corn oil as a vehicle in the study has no effect on the cox2 mrna gene expression level (in other word, corn oil had no antiinflammatory effect). in addition, data analysis reveals that there is a nonsignificant (p>0.05) difference in the cox2 mrna expression level between (group iv) mice treated with 50mg/kg/day and (group v) mice treated with 100mg/kg/day l-carvone. table (1) and figure (2). iraqi j pharm sci, vol.32(1) 2023 l-carvone and acute lung injury 129 figure 2. bar chart showing the effect of lcarvone on cox2 expression in lps-induced acute lung injury (ali). rt-qpcr analysis of cox2 mrna expression level in lung tissue of mice treated with l-carvone 50 mg/kg or 100 mg/kg/day for 5 days and euthanized 2 hrs after lps administration (n=10 mice in each group). data represent mean ±sem. #: significant (p<0.05) differences compared to the normal (negative) control group i mice. a: non-significant difference (p>0.05) (group iii, corn oil+ lps) compared to the lps model group ii mice. *: significant difference (p<0.05) of l-carvone groups (iv and v) each compared to the lipopolysaccharide (lps) model group (ii) mice. b: non-significant (p>0.05) difference between groups iv and v mice. effect of l-carvone on nf-кb mrna level: results shown in table (1) revealed that levels of nf-κb mrna gene expression were significantly-elevated (p<0.05) in the lps model group compared to the corresponding gene expression level in the normal control group (11.88±1.227 vs. 1.15 ± 0.369), while in the mice’s group received l-carvone 50mg/kg/day (group iv) showed that there was significant (p<0.05) downregulation of nf-κb mrna gene level (11.88±1.227 vs. 6.81±1.362) compared to nontreated mice (lps model group) (figure 3). furthermore, administration of l-carvone 100mg/kg/day (group v) to mice also resulted in a significant (p<0.05) reduction in nf-κb mrna gene expression level (11.88±1.227 vs. 5.01±0.826) compared to such level in non-treated mice (lps model, group ii). in addition, data pointed out in table (1) and figure (3) there were non-significant (p>0.05) differences in nf-κb mrna gene expression level between the corn oil group (corn oil + lps group iii) and lps model group which can indicate that corn oil used as a vehicle in the study has no effect on the nf-κb mrna gene expression level. also, results of table (1) and figure (3) revealed that there was a non-significant difference (p>0.05) in the nf-κb mrna gene expression level between the treatment groups with l-carvone 50mg/kg/day (group iv) and 100mg/kg/day (group v). figure 3. bar chart showing the effect of lcarvone on nf-κb expression in lps-induced acute lung injury (ali). rt-qpcr analysis of nf-kb mrna expression level in lung tissue of mice treated with l-carvone 50mg/kg/day or 100 mg/kg/day for 5 days and euthanized 2 hrs. after lps administration (n=10 mice in each group). data represents mean ±sem. #: significant (p<0.05) differences compared to the normal (negative) control group i mice. a: non-significant difference (p>0.05) (group iii, corn oil+ lps) compared to the lps model group ii mice. *: significant difference (p<0.05) of l-carvone groups (iv and v) each compared to the lipopolysaccharide (lps) model group (ii) mice. b: non-significant (p>0.05) difference between groups (iv and v) mice . discussion acute lung injury (ali) is a serious inflammatory condition with a high mortality rate in the intensive care unit around the world. it is caused by many factors which affect the lung either directly or indirectly (16), (17). one of the causes of ali is the lipopolysaccharide (lps), which is a component of the gram-negative bacterial cell wall. in the present study, the expression of pro-inflammatory mediators tnf-α, cox2, and nf-κb were significantlyelevated in mice intraperitoneally-injected with 10mg/kg lps to induce ali (group ii mice); results of this study are consistent with those of others (18– 21). pretreatment with l-carvone at doses of 50mg/kg/day or 100mg/kg/day for 5 days (groups iv and group v), respectively each decreased the gene expression levels of tnf-α, cox2, and nfκb in lung tissue of treated mice and each compared to those expression levels in lps model (ali, group ii) but the effects are not significant (p>0.05) (dose-independent effect). table 1 and figures (1, 2, and 3). iraqi j pharm sci, vol.32(1) 2023 l-carvone and acute lung injury 130 levo (l)-carvone can reduce the mrna gene expression levels of tnf-α, cox2, and nfκb, this may indicate the anti-inflammatory effect of l-carvone that has been supported by other studies (22–24) ; furthermore it has recently been reported that, l-carvone can inhibit the elevated tnf-α level and diarrhea score in intestinal mucositis induced by irinotecan (11). results of many in vitro studies have shown that the anti-inflammatory effect of lcarvone is related to the suppression of tnf-α induced neutrophil adherence; moreover, studies showed that l-carvone inhibited tnf-α production by macrophage cell line raw264.7 stimulated with lps which further proved the anti-inflammatory effect of l-carvone also in this study; moreover, lcarvone inhibited nitric oxide (no˚) and interleukin1β (1l-1β), interleukin-1α (il-1α), and nf-κb which are critical inflammatory mediators associated with severe acute and chronic inflammatory diseases (22–24). furthermore, a study of others revealed that the inflammatory cytokines tnf-α, il-1β, il-6, cox2 and nf-κb play critical roles in the development of ali; thus, inhibiting these cytokines is of significant concern in attenuating ali (25). tumor necrosis factor–α (tnf-α) is a crucial endogenous mediator that is mostly-produced by monocytes and macrophages; it can activate the inflammatory response, and plays a major role in the regulation of the inflammatory process in the lung, and causes damage to the cells of vascular endothelium (26). furthermore, the cox2 enzyme is involved in converting arachidonic acid (aa) to inflammatory prostanoids, which are involved in the development of early and late phase endotoxemia; moreover such enzyme is a critical component in the inflammatory response downstream of the nf-κb signaling pathway; where, it can be stimulated by several proinflammatory factors such as il-6 and tnf-α, oxidative stress (os), and growth factors (23, 24). macrophages, dendritic cells, and neutrophils are the major component of the innate immune system involved in inflammation; furthermore, these cells express pattern recognition receptor (prrs) that detect various microbial components, which is called (pathogen-associated molecular patterns) (29). moreover, vidya mk et al (2018) reported that lps activated the prrs receptor (toll-like receptor 4), which is a classical pathway that initiates intracellular inflammatory signal transduction which stimulates macrophages to produce proinflammatory cytokines production (30), and it is essential for inflammatory m1 macrophage polarization and inflammatory cytokines production (31). also, its activation resulted in the activation of nf-κb through myeloid differentiation 88 dependent (myd88 dependent) and myd88 independent pathway (32); furthermore, the nf-κb is a crucial transcription factor of m1 macrophages and is required for the induction of a large number of inflammatory genes, such as tnf-α, il-1β, il-6, and cox-2 (29). moreover, os can contribute to the pathogenesis of ali by the activation of transcription factor nf-κb and the downstream pro-inflammatory cytokines (33). levo (l)-carvone showed an antioxidant effect in a previous study by reducing the superoxide-free radical (o2· −); and, the antioxidant treatment in oxidant-induced lung injury has been widely observed to suppress nf-κb activation and the outspread neutrophilic lung inflammation (34). in the current study, the lung-protective effects of lcarvone could either be due to its direct inhibition of nf-κb signaling pathway or indirect effect by inhibition of os/ nf-κb pathway which is been shown in table (1) and figure (3). furthermore, l-carvone was able to exert a protective effect in ali induced by lps by hampering nf-κb activation and its downstream pro-inflammatory cytokines (tnf-α, cox2) production; the results of the present study are consistent with the results of other, which revealed that downregulation of nf-κb resulted in ameliorating lung injury in mice (35); in addition, lcarvone induced the production of the antiinflammatory factor interleukin-10 (il-10) which further prove its anti-inflammatory effect (22). conclusion based on the observed effects, l-carvone (in dose-independent effect) has lung-protective and anti-inflammatory effects on lps-induce ali; and its anti-inflammatory effect is ensured by hampering nf-κb gene expression and its downstream proinflammatory cytokines tnf-α and cox2, thus protecting the lungs from acute inflammatory damage. acknowledgment the data of this article were abstracted from the m.sc. thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the authors are extremely grateful to the college of the pharmacy/university of baghdad for supporting this work. references 1. vázquez-medina jp, tao jq, patel p, bannitzfernandes r, dodia c, sorokina em, et al. genetic inactivation of the phospholipase a2 activity of peroxiredoxin 6 in mice protects against lps-induced acute lung injury. am j physiol lung cell mol physiol. 2019;316(4):656–68. 2. matthay ma, mcauley df, ware lb. clinical trials in acute respiratory distress syndrome: challenges and opportunities. lancet respir med. 2017;5(6):524–34. 3. schultz c. 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cui y, et al. the therapeutic effects of jaceosidin on lipopolysaccharide-induced acute lung injury in mice. j pharmacol sci. 2019;140(3):228–35. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus doi:https://doi.org/10.31351/vol29iss2pp202-213 202 detection and isolation of some flavonoids and aromatic acid from iraq cultivated in colymusscynara head(capsule) of zaineb h. ajeel*,1 and maha n. hamad** * ministry of health and environment ,iraq. **department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq abstract the target of this study was to study the natural phytochemical components of the head (capsule) of cynara scolymus cultivated in iraq. the head (capsule) of plant was extracted by maceration in70% ethanol for 72 hours, and fractioned by hexane, chloroform and ethyl acetate. preliminary qualitative phytochemical screening was performed on the ethyl acetate fraction for capsule was revealed the presence of flavonoid and aromatic acids. these were examined by (high -performance liquid chromatography) (hplc diodarray), (high—performance thin-layer chromatography)(hptlc). flavonoids were isolated by preparative layer chromatography and aromatic acid was isolated by preparative high-performance liquid chromatography hplc from the ethyl acetate fraction of capsule. then identified by high performance thin layer chromatography hptlc, high performance liquid chromatography hplc diode array , ultraviolet diode array uv-diode array and liquid chromatography /mass spectroscopy lc/ms. the chloroform fraction from the capsule was evaluated by gas chromatography//mass spectrometer(gc/ms). the different chromatographic and spectroscopic techniques revealed the presence of luteolin, apigenin and cinnamic acid in capsule of cynara scolymus, also 9-octadecanoic acid (oleic acid), oxalic acid, allyl tetradecyl ester, limonene, in chloroform of cynara scolymus the results of the current study proved the presence of luleolin, apigenin, and cinnamic acid in the ethyl acetate fraction of cynara scolymus capsule. keywords: cynara scolymus, flavonoids, aromatic acid , gas chromatography //mass spectrometry (gc/ms) ,highperformance thin -layer chromatography(hptlc), high-performance -layer chromatography(hplc) and liquid chromatography//mass spectroscopy(lc/ms). كشف وعزل الفالفونويد والحامض العطري من نبات الخرشوف المستزرع في العراق **و مها نوري حمد 1*،عجيل زينب حسين وزارة الصحة والبيئة ، العراق . * * بغداد،العراق. ، بغداد جامعة، الصيدلة كلية، الطبية والنباتات العقاقير فرع الخالصة تم استخالص الجزء .الهدف من الدراسة هو دراسة المكونات الكيميائية للجزء العلوي )الكبسولة (لنبات الخرشوف المستزرع في العراق باالهكسان ,الكلوروفورم وبعدها تم تجزئة المستخلص اإليثانولي ساعه72ايثانول لمدة %70بنسبة نقع الباردالعلوي لنبات الخرشوف بواسطة عملية ال مستخلص خالت و على أولي نباتي كيميائي فحص إجراء تم العطريخالت األثيل. والحمض الفالفونويد وجود كشف وتم ، وكذلك اإليثيل الطبقها تم عزل الفالفونويدات بواسطة كروموتوغرافيhplc) )السائل عالي االداء اوكروموتوكرافي( (hptlcالرقيقه لطبقها كروموتوكرافيا في الكبسولة و تم خالت االثيل (من جزء hplcالسائل عالي االداء ) اوالحامض العطري بواسطه كروموتوغرافي ((plcلتحضيري الرقيقه ا مطياف و hplc )ثنائي الصمام( السائل عالي االداء اكروموتوغرافيو hptlc الرقيقه الطبقها المفصوله بواسطة كروموتوغرافي المواد تحديد (.gc / msالغاز / مطياف الكتلة )ا ( تم تحليل جزء الكلوروفورم من الكبسولة باستخدام كروماتوجرافي(lc/msالكتلة كروموتوكرافيا ، كروموتوكرافيا الطبقة الرقيقة، مطياف الكتلة ، كروموتوغرافيا الغاز، فالفنويد,حامض عطري، الخرشوف الكروي الكلمات المفتاحية : . مطياف الكتلة، كروموتوغرافيا السائل ، السا ئل عالي االداء 1corresponding author e-mail: zainebhusain5@gmail.com received: 5/ 1/2020 accepted: 19/7 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp202-213 iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 203 introduction medicinal plants commonly used as raw materials for extraction of active ingredients used in the production of different drugs (1) the therapeutic effectiveness of a medicinal plant is due to the presence of some bioactive constituents (2) herbal medicine around the globe, is based on traditional medicine, pharmacological screening and exploration of the chemical constituents of the plants may provide us the basis for developing a lead molecule through herbal drug discovery. in modern medicine, the very important life-saving drugs have been provided by herbs (3) this awareness in iraq was explained and enhanced by the arab physicians (4-6) in many republics in the world, traditional medicine remains accordingly important to the formal health system (7) the arab countries including iraq are among these countries (8). artichoke (cynara scolymusl. belong to asteraceae familly) , the asteraceae (compositae, alternate name) with its approximately 1,620 genera and more than 23,600 species is the major family of flowering plants(9). the family is distributed worldwide except for antarctica but is especially diverse in the tropical and subtropical regions of north america, eastern brazil, the andes, southern africa, the mediterranean region, central asia, and southwestern china. the globe artichoke in appearance is like a large, blue thistle (10). globe artichoke (cynarascolymusl.), a perennial species of this family is grown for its big fleshy immature inflorescences flower heads(11).the globe artichoke (cynara scolymus) is a unique vegetable, having fibrous, fleshy rhizomes with buds that develop into several tomentose and branched stems. the most vigorous varieties may reach 1.20-1.30 m in height. the bearing stem is erected, ribbed, and rounded in cross-section, ending with a floral head (capitulum), capitulum it is composed of several tubular and bluish-violet fertile florets opening from the outside inwards(12).this plant an medicinal plant and golden harvest and, the therapeutic possible of which was known to the ancient egyptians, greeks and roman (13), it contains (cynarin and chlorogenic acid) caffeoylquinic acid derivatives) and flavonoids (apigenin and luteolin) , as well as the anthocyanidins such as 2-(4-hydroxy-3methoxyphenyl) chromenylium-3,5,7tirol (pending),2-(3,4,5 trihydroxyphenyl) chromenylium -3,5,7tirol (delphinidin) and 2-(3,4-dihydroxyphenyl) chromenylium-3,5,7-tirol (cyanidin), these have been isolated in only in the capsule of artichoke (14,15) the above flavones luteolin and apigeninhas been identified in capsule and leaves of the plant in the form of rutinosides and glucosides and, whereas anthocyanin pigments are present only in capsule, in form of sophorosides and glucosides(14). pharmacological activities of cynara scolymus, antioxidant ntimicrobial activities(16),antiatherogenic and hypoglycemic effect(17) antispasmodic activity(18) ,cardiovascular protection (19,20)choleretic effects (stimulation of bile secretion(21,22), antifungal activity(23), anti-metabolic syndrome(24,25) and anti-cancer effect(26).the dominant study was studying the natural phytochemical components of the head(capsule) of cynara scolymus cultivated in iraq. figure 1. fresh head(capsule) of cynara scolymus . material and methods plant material plant material of cynara scolymus capsule (head) was obtained from university of baghdad/ the college of pharmacy during june/2018.the plant was identified and authenticated by d.r khansaa. al-joboury in iraqi natural history center museum in baghdad university. all parts that were obtained were washed thoroughly, dried in the shade, followed grinding by an electrical grinder to a fine powder. iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 204 extraction of plant 100 grams of the powdered plant material was extracted by maceration in 70% ethanol for 72hours with frequent shaking, at room temperature, the extract was filtered off, this procedure was repeated three times. the filtrates were mixed together and evaporated under vacuum by a rotary evaporator. there mains (100ml) were partitioned successively with hexane, chloroform and ethyl acetate (3x100). the hexane, chloroform and ethyl acetate fraction were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness using a rotary evaporator. phytochemical examination for fractions of plant phytochemical analysis for screening and identification of bioactive chemical constituents in the medicinal plants as described (27). test for flavonoids: (a) 0.5 gm of fractions were suspended in ethanol mixed with few drops of 1% aluminum chloride in methanol in a test tube, and the color was observed. formation of yellow color indicates the presence of flavonoids. (b)0.5 gm of fractions were suspended in ethanol mixed with few drops of 1% potassium hydroxide in a test tube, and the color was observed. a dark yellow color indicated the presence of flavonoids. test for phenols: 0.5 gm of each fractions were suspended in ethanol in a test tube, then few drops of 5% ferric chloride was added and a deep green to black color was observed for formation. examination of ethyl acetate fraction (capsule) by high performance thin layer chromatography (hptlc)(28) the presence of phenolic compounds in the analyzed fractions was confirmed by using a modern technique of hptlc, using eike reich/camaglaborator/ switzerland. ►samples ethyl acetate fraction of the capsule ►standards 1.apigenin 2.luteolin 3.cinnamic acid ►preparation of standards and samples for hptlc: the standards (1mg) and samples (few milligrams) were prepared by dissolving them individually in 1 ml of absolute methanol. ►developing solvent system: the mobile phase used was composed of: chloroform: methanol: formic acid (16:3.5:0.5) ►detection: detection was done under uv light at 254 nm . -examination of ethyl acetate fraction by high performance liquid chromatography (hplc) hplc conditions for analyzed fraction: show in table 1. table 1 . conditions of analytic hplc(29). stationary phase c18 (250x10) 5 µm particles size. mobil phase solvent (0.05% tfa in hplc grad water) and solvent (acetonitrile). standard cinnamic acid. sample ethyl acetate fraction. detection monitoring on 225 nm injected volume sample loop (200 µl) and injector. flow rate 3ml/min. isolation of flavonoids and aromatic acid from ethyl acetate fraction from the capsule of plant -isolation of flavonoids were done by preparative layer chromatography (plc), from the ethyl acetate fraction of cynara scolymus capsule the conditions of isolation show in table 2 table 2. conditions of preparative layer chromatography ( plc) stationary phase silica gel gf254 mobil phase chloroform: methanol: formic acid(16:3.5:0.5) standards apigenin and luteolin sample ethyl acetate fraction detection u v 254nm isolation of aromatic acid by preparative (hplc) from an ethyl acetate fraction of cynara scolymus capsule: conditions of isolation of hplc show in table 3 table 3. conditions of isolation of hplc(29). stationary phase c18 (250x10) 5 µm particles size. mobil phase solvent (0.05% tfa in hplc grad water) and solvent (acetonitrile). standard cinnamic acid. sample ethyl acetate fraction. detection monitoring on 225 nm injected volume sample loop (200 µl) and injector. flow rate 3ml/min. identification of separated compounds identification of flavonoids (a4)(1)and (a6)(3) isolated by(plc)and aromatic acid(a1) isolated by preparative (hplc): hptlc: the sample was prepared by dissolving 0.5 mg of the isolated compound in 1 ml of absolute iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 205 methanol and examined under same previously mentioned conditions. hplc(diodarray): the sample was prepared by dissolving 0.5 mg of the isolated compound in 0.5 ml of absolute methanol and examined under same previously mentioned conditions. uv-diodarray: 0.5 mg was dissolved individually in 1 ml absolute methanol, and the uv absorbance was scanned from 200-400 nm. lc/ms mobil phase solvent acetonitrile and water colum 0.19 mm external diameter (75 mm i.d)and 200mmlength wave packed with thermo scientific hypersil gold c18 with 5mmpartical size. sample were run under the following condition /z rang was 250 to 1000.200k resolution, top 5 configuration with one m/s scan and five ms/ms scans, and dynamic exclusion set to 1 with a limit of 90 second.150 femtomole of angiotensin standard mix from micron biosciences was loaded on column per injection .a 2.5 hour lc/ms separation was used for all blank and standard sample..(30) gc/ ms analysis of the chloroform fraction for capsule gc/ ms analysis of the chloroform fraction for capsule was done using agilent gc-ms model with the below conditions: agilent 190915433ui, hp-5ms ultra inert, in front ssz inlet he, out msd, initial 60c, pressure 7.037 psi, flow 0.9ml/min, average velocity 34772 cm/sec, holdup time 1.4379 min results and discussion the results of preliminary phytochemical analysis in different fractions of the plant shown in table 4. table 4. phytochemical analysis for fractions of c.scolymus fractions phenols flavonoids hexane + + ethyl acetate + + the present study done for the cynara scolymus cultivated in iraq showed the presence of medicinally active constituents phytochemical active compounds were qualitatively analyzed and the results are presented in table 4 the positive results based on the presence or absence of color change. in this screening process, flavonoids and phenols give positive (+) results. analysis of fraction and standards by highperformance thin layer chromatography (hptlc) hptlc chromatography for standards and ethyl acetate fraction. hptlc is a valuable tool for reliable identification because it can provide chromatographic finger prints that can be visualized and stored as electronic images (31). hptlc chromatography show max rf value for standards (apigenin ,luteolin and cinnamic acid) and ethyl acetate fractions shown in figure2. figure 2. hptlc chromatography show max rf value for standards and fraction. iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 206 analysis of ethyl acetate fraction by highperformance liquid chromatography (hplc) the hplc results of the weight of main compounds in ethyl acetate fraction (capsule) shown in table 5: weight of main compounds in ethyl acetate fraction by calibration curve table 5. weight of main compounds in ethyl acetate fraction name of compounds ethyl acetate fraction of (capsule) w.t (μg / ml ) cinnamic acid 15.939 luteolin 69.6949 apigenin 103.71325 isolation compounds by preparative layer chromatography (plc) from the ethyl acetate fraction preparative layer chromatography(plc) was done utilizing ethyl acetate fraction of maceration method was developed in mobile phases12: chloroform: methanol: formic acid(16:3.5:0.5).2 bands separated symbolized as (a4)(1) and (a6)(3) shown in figure3. figure 3. preparative layer chromatography of ethyl acetate fraction developed in mobile phase (s12) chloroform: methanol: formic acid(16:3.5:0.5) observed at 254 nm. hptlc for isolated compounds a4(1) and a6(3) isolated by (plc)and a1 isolated by (hplc) and standards. hptlc results shown in figure4: (a4)(1)and apigenin(standard),(a6)(3) and luteolin(standard) and (a1) and cinnamic acid (standard). hplc for isolated compounds a4(1) and a6(3) isolated by (plc) and a1 isolated by (hplc) and standards. the hplc diode array results of analyzing separated compounds and standards are demonstrated in table 6.the identification of compounds by hplc is usually performed by comparing the obtained retention times with the ones of related standards. table 6. retention time of the standards and the isolated compounds retention time of isolated compounds ( min) isolated compounds retention time of standards( min) standards apigenin (17.16) a4(1) (17.15) luteolin (16.18 ) a6(3) (16.18) cinnamic acid (16.8) a1 16.7) ) iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 207 figure4. hptlc for isolated (a4)(1) standard(apigenin),(a6)(3) and standard( luteolin) and (a1) and and standard(cinnamic acid). hplc for isolated compound a4(1) and apigenin standard hplc results show in figures ( 5 -6) figure 5.analytic apigenin standard and isolated compound a4(1) iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 208 figure 6. uv spectrum for apigenin standard and isolated compound a4(1) hplc for isolated compound (a6) (3) and. luteolin standard hplc results show in figures (78): figure 7.analytic luteolin standard and isolated compound a6(3) figure 8. uv spectrum for luteolin standard and isolated compound a6(3). iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 209 hplc for isolated compound (a1) and cinnamic acid standard hplc results show in figures (9-10): figure 9.analytic cinnamic acid standard and isolated compound(a1). figure 10. uv spectrum for cinnamic acid standard and isolated compound(a1). lc/ms of isolated compounds a4(1) and a6(3) isolated by (plc) and a1 isolated by (hplc). lc/ms of isolated compoundsa4(1): the result show in figure11. figure 11.lc/ms for isolated compound a4(1) lc/ms for isolated(a6)(3): the result show in figure12: iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 210 figure 12:lc/ms for isolated compound a6(1) lc/ms of isolated compound (a1): the result show in figure13. figure 13.lc/ms for isolated compound a1 the compounds detected in chloroform fraction for capsule of cynara scolymus show in table7 and figure 14: table 7. gc/ms analysis of the chloroform fraction of cynara s colymus molecular formula molecular weight g/mol compounds name c19h34o4 326 oxalic acid, allyl tetra decyl ester c18h34o2 282 oleic acid $$ 9-octadecenoic acid c10h16 136.23 limonene iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 211 figure 14.gc/ms chromatography of chloroform fraction of plant discussion the preliminary phytochemical analysis confirmed the presence of flavonoids and aromatic acid. the hplc results show the occurrence of flavonoids and aromatic acid in the capsule of cynara scolymus, such as luteolin, apigenin, and aromatic acid cinnamic acid ,apigenin more concentration, then luteolin and finally cinnamic acid. the consequences of the present study show the isolation of flavonoids (apigenin and luteolin) from ethyl acetate fraction by plc and aromatic acid (cinnamic acid) by preparative hplc .hptlc results revealed the presence of apigenin, luteolin and cinnamic acid lc/ms systems facilitate the analysis of samples that traditionally have been difficult to analyze. iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 212 liquid chromatography (lc) separates the sample components and then introduces them to the mass spectrometer (ms). the ms creates and detects charged ions. the lc/ms data may be used to provide information about the molecular weight. electrospray is a soft ionization technique that produces a large number of molecular adduct ions. adduct ions are typically protonated parent ions [m+h]+.(32). lc/ms results give m/z for apigenin isolated compound (271) molecular weight of apigenin standard (270), m/z for luteolin (287), molecular weight of luteolin standard (286) and m/z for cinnamic acid (149), molecular weight of cinnamic acid standard(148) .so from these all data, isolated compound could be identified conclusions based on the results, the following points may be concluded: 1. phytochemical screening of cynara scolymus cultivated in iraq demonstrates the presence of flavonoids and aromatic acid which were separated from head (capsule)of plants according to differences in their chemical nature. 2 . in this study, two chromatographic analyses were carried out to isolate in a pure form: one: preparative tlc for flavonoids (apigenin and luteolin) apigenin more concentration than luteolin isolate from head (capsule) and second : preparative(hplc) for isolated aromatic acid(cinnamic acid). references 1. hasan mm, hossain sa, ali ma, et al. medicinal plant diversity in chittagong, bangladesh: a database of 100 medicinal plants. journal of scientific and innovative research. 2014;3(5):500-514. 2. wuthi-udomlert m, kupittayanant p, gritsanapan w. in vitro evaluation of antifungal activity of anthraquinone derivatives of sennaalata. journal of health research. 2010;24(3):117-122. 3. handa ss. 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resistance. in advances in virus research, academic press.2012 ;1 ( 84) :1-29. 11. loebenstein, gad, hervélecoq. viruses and virus diseases of vegetables in the mediterranean basin. academic press, 2012. 12. rossi v, de paoli g. micropropagation of artichoke (cynarascolymus). inhigh-tech and micropropagation iii 1992 :118-134. 13. lattanzio v, kroon pa, linsalata v, et al. "globe artichoke: a functional food and source of nutraceutical ingredients. journal of functional foods . 2009; 1(2):131-144. 14. rieseberg lh., raymond o., rosenthal dm., et al. major ecological transitions in wild sunflowers facilitated by hybridization. science. 2003;301(5637):1211-1216. 15. sabnis rw. handbook of biological dyes and stains: synthesis and industrial applications. john wiley & sons; 2010. 16. vamanu e, vamanu a, nita s, et al. antioxidant and antimicrobial activities of ethanol extracts of cynarascolymus (cynarae folium, asteraceae family). tropical journal of pharmaceutical research. 2011;10(6):777-783. 17. mocelin r, marcon m, santo gd, et al. hypolipidemic and antiatherogenic effects of cynarascolymus in cholesterol-fed rats. revistabrasileira de farmacognosia. 2016; 26(2):233-239. 18. emendörfer f, emendörfer f, bellato f, et al. antispasmodic activity of fractions and cynaropicrin from cynarascolymus on guineapig ileum. biological and pharmaceutical bulletin. 2005;28(5):902-904. 19. lupattelli g, marchesi s, lombardini r, et al. artichoke juice improves endothelial function in hyperlipemia. life sciences. 2004;76(7):775782. 20. li h, xia n, brausch i, et al. flavonoids from artichoke (cynarascolymus l.) up-regulate endothelial-type nitric-oxide synthase gene expression in human endothelial cells. journal of pharmacology and experimental therapeutics. 2004;310(3):926-932. 21. kraft k. artichoke leaf extract—recent findings reflecting effects on lipid metabolism, liver and gastrointestinal tracts. phytomedicine. 1997;4(4):369-378. 22. matuschowski p, gumbinger hg, nahrstedt a, et al. "testing of cynarascolymus in the isolated perfused rat liver." 43rd ann congrsoc med plant res.1996;10: 103-107. iraqi j pharm sci, vol.29(2) 2020 flavonoids and aromatic acid from head(capsule) of cynara scolymus 213 23. zhu xf, zhang hx, lo r. antifungal activity of cynarascolymus l. extracts. fitoterapia. 2005; 76(1):108-111. 24. villiger a, sala f, suter a, et al. in vitro inhibitory potential of cynarascolymus, silybummarianum, taraxacumofficinale, and peumusboldus on key enzymes relevant to metabolic syndrome. phytomedicine. 2015;22(1):138-144. 25. mileo am., di venere d., abbruzzese c., et al. long term exposure to polyphenols of artichoke (cynarascolymus l.) exerts induction of senescence driven growth arrest in the mdamb231 human breast cancer cell line. oxidative medicine and cellular longevity. 2015;2015. 26. miraj s, kiani s. study of therapeutic effects of cynarascolymus l.: a review. der pharmacia lettre. 2016;8(9):168-173. 27. ben sm, affes h, athmouni k, et al. chemicals compositions, antioxidant and antiinflammatory activity of cynarascolymus leaves extracts, and analysis of major bioactive polyphenols by hplc. evidence-based complementary and alternative medicine. 2017. 28. seal t. quantitative hplc analysis of phenolic acids, flavonoids and ascorbic acid in four different solvent extracts of two wild edible leaves, sonchusarvensis and oenanthelinearis of north-eastern region in india. journal of applied pharmaceutical science. 2016;6(2):157-166. 29. bashir ak, abdalla aa, wasfi ia, et al. flavonoids of limoniumaxillare. international journal of pharmacognosy. 1994;32(4):366372. 30. sánchez f, jáuregui o, raventósr, et al.identification of phenolic compounds in artichoke waste by high-performanceliquid chromatography–tandem mass spectrometry. journal of chromatography a.2003;1008(1): 57–72. 31. morlock g. man mohan srivastava (ed.): high-performance thin-layer chromatography (hptlc). analytical and bioanalytical chemistry. 2011;401(8):2331-2332. 32. saibaba sv, kumar ms, pandiyan ps. mini review on lc/mstechniques .worldgournal of pharmacy and pharmaceutical sciences. 2016;5(4):2381-2395. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(suppl.) 2022 floating oral in situ gel doi: https://doi.org/10.31351/vol31isssuppl.pp162-167 162 preparation and in-vitro evaluation of floating oral insitu gel of montelukast sodium (conference paper) # manar adnan tamer*,1, roaa a. nief *, shaimaa nazar abd-al hameed* and haneen abdulhadi kharaba* # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of pharmaceutical, college of pharmacy, university of baghdad, baghdad, iraq abstract a polymeric drug delivery liquid system of oral in situ gel of montelukast sodium has been formulated by using natural ingredients like gellan, sodium alginate and pectin. there were several goals in this study, the most important of which was to formulate an oral liquid preparation (ion sensitive floating oral in situ gel system for sustained delivery of montelukast sodium for pediatric patients) that would make administration easier, provide the correct therapeutic dose, and allow the drug to be released more slowly into the gastrointestinal tract (git) for better control. montelukast sodium in situ gels at different concentrations (w/v) of natural polysaccharides, gellan gum and sodium alginate and natural polymer pectin were formulated and characterized in the terms of appearance, viscosity and in vitro release. as the concentrations of ion-sensitive gel-forming components, gellan gum, sodium alginate, and pectin, and gasgenerating ingredients increased, the viscosity of formulations in solution was increased. in vitro release study showed that the release of montelukast sodium from these gels was characterized by an initial phase of high release (burst effect), followed by a more gradual release in the second phase. the formulated oral in situ gel system of montelukast sodium can be regarded as a promising approach for oral delivery of montelukast sodium to pediatrics. keywords: oral sustained drug delivery, floating in situ gel, montelukast sodium, gellan gum. #) بحث مؤتمر (تحضير وتقييم مختبري للهالم الفموي العائم موضعيا لعقار المونتيلوكاست صوديوم * خرابة يعبد الهادحنين ،*شيماء نزار عبد الحميد ،*عبد الحميد نايف رؤى ،1*،منار عدنان تمر 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي ، بغداد ، العراق جامعة بغداد ، كلية الصيدلة الصيدالنيات ، فرع * الخالصة مو العائم الفموي الهالم تحضير المونتيلوكاست ضتم لعقار بوليميري هيئةعلى صوديوم عيا الجيالن سائل كصمغ طبيعية مواد وباستعمال عيا حساس ضمو نظام هالم عائم)هو تطوير مستحضر سائل عن طريق الفم اهمها الدراسةناك العديد من االغراض لهذه هوالجينات الصوديوم والبكتين. تم والسماح للدواء ان يتحرر ببطء الى القناة الهضمية لتحكم افضل للدواء،مما يزيد من راحة اإلعطاء واعطاء الجرعة العالجية الصحيحة (لألطفاللأليونات ( وألجينات الصوديومجيالن الصمغ )من متعدد السكريات الطبيعي ( حجم /وزن )كيز مختلفة ابتر مونتيلوكاست الصوديوملل عياضعائم موال هالم ال تحضير حيث زادت لزوجة التركيبات في المحلول مع زيادة تركيزات صمغ .المختبر داخل والتحررواللزوجة رلمظها من حيثتقييمها وتم ( بكتين)والبوليمر الطبيعي وأظهرت دراسة التحرر في ( كاربونات الصوديوم) الغازوتركيز عامل توليد ( ألجينات الصوديوم والبكتين جيالن،صمغ )مكونات الهالم الحساسة لأليونات المرحلة الثانية من اإلطالق تليها( تأثير االنفجار)الصوديوم من هذه المواد الهالمية تميز بمرحلة أولية من اإلطالق العالي المختبر أن إطالق مونتيلوكاست صوديوم عن طريق الفم مونتيلوكاستعقار ال مونتيلوكاست الصوديوم أسلوبًا واعدًا إلعطاءل ل عياضمو يمكن اعتبار نظام الهالم العائم .المعتدل التدريجي .لألطفال . جيالنالصمغ الصوديوم،مونتيلوكاست هالم موقعي عائم في الموقع، الفم،توصيل األدوية عن طريق :الكلمات المفتاحية introduction traditional oral dosage forms, which have low bioavailability because of the quick gastric transition from the stomach and necessitate higher dosing frequency, can be overcome using floating oral in situ gel. as a result, pharmaceutical researchers are highly interested in finding ways to boost the therapeutic benefits of this floating oral insitu gelling system. the floating oral in situ gel has the ability to overcome the drawbacks of conventional oral dosage forms, which have a limited bioavailability to small intestine . because of its controlled release and site – specific drug administration, the floating oral in situ gelling system is of tremendous interest to pharmaceutical researchers (1). the development of polymeric drug delivery systems has been the subject of extensive study. last years have seen a significant increase in interest in the development of in situ gel systems (2). before administration, the in situ gel dosage form is a liquid, but when it comes into contact with gastric contents, it transforms into a gel. as soon as the gastric fluids come into contact with the lowviscosity solution, the polymeric conformation of the viscous gel changes, and it floats on the surface of the gastric fluids (3, 4). 1corresponding author e-mail: mannar.adnan@copharm.uobaghdad.edu.iq received: 15/7/2022 accepted: 6/11 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp162-167 iraqi j pharm sci, vol.31(suppl.) 2022 floating oral in situ gel 163 for prolonged drug delivery, in situ gel forming polymeric delivery systems have been extensively studied. this is in addition to other advantages of in situ forming polymeric delivery systems such as simplicity of administration, lower dose frequency, enhanced patient compliance and comfort (5). an in situ gel undergo a sol-to-gel phase transition when certain physicochemical factors, including ionic strength, temperature, or ph, are altered (6). when compared to a traditional liquid dosage form, a gastroretentive in situ gelling technology can help boost medication bioavailability. polymer-based gels, which are lighter than gastric fluids, float over the stomach contents or stick to the gastric mucosa, resulting in increased gastric retention of dose forms and a longer period in the gastrointestinal tract of the drug (7). the formulation of floating in situ gelling solution may sustain and prolong drug action, enhance patient compliance, and lower the frequency with which the drug is administered. because of the system decrease in density, it initially sink in the stomach before absorbing water, swelling, and finally floating (8). polysaccharides and polymers of various sorts, as well as their gel-forming mechanisms, are being studied as part of this effort to build a stomachspecific gelling system. gellan gum is an anionic deacetylated, exocellular polysaccharide secreted by pseudomonas elodea with a tetrasaccharide repeating unit. double-helical junction zones are formed in the gelation process, followed by aggregation of the double-helical segments into a three-dimensional network via complexation with cations and hydrogen bonding with water (9). because it works as a gelling agent, sodium alginate can produce a wide range of gel textures in the finished product. as a result of the free ca2+ ions being trapped in sodium alginate polymeric chains, the polymer chains cross link to form a matrix structure. the gelation process begins with the production of double helical junction zones, which are then re-aggregated to create a three-dimensional network through complexation with cations and hydrogen bonding with water (10). water-soluble and ph-sensitive anionic acidic pectin polysaccharide isolated from fruit. because of its natural gelling, stabilizing, and thickening properties, it is an excellent drug delivery vehicle. an efficient delivery system can be achieved by using pectin polysaccharide-based medication carriers (11). montelukast sodium (mk) is a selective leukotriene receptor antagonist. chronic asthma, exerciseinduced asthma, and other leukotriene-related disorders like capsular contracture and obstructive sleep apnea can all benefit from this medication (12). there is only a 10 mg/day dose for adults and a 4–5 mg/day dose for children with mk. acute asthma attacks should not be treated with this medication (13). mk bioavailability after oral administration is around 62%, and its half-life in the human body is approximately 6.7 hours (14). gellan, sodium alginate or pectin solution was proposed with calcium carbonate and sodium citrate, which complexed free calcium ions so that they could only be liberated in a stomach acidic environment. the formulation was kept liquid until it was swallowed, at which point it immediately turned into a gel. there were several goals in this study, the most important of which was to formulate an oral liquid preparation (ion sensitive floating oral in situ gel system for sustained delivery of mk for pediatric patients) that would make administration easier, provide the correct therapeutic dose, and allow the drug to be released more slowly into git for better control. materials and methods materials montelukast sodium was purchased from hangzhou hyper chemical limited, china. sodium alginate was purchased from himedia laboratories, india. gellan gum and pectin were purchased from hangzhou hyper chemical limited, china. only commercially analytical-grade reagents were used for the experiment. preparation of in situ gel in situ gel formulations were made using the ingredients listed in table (1). gellan gum and sodium alginate were dissolved in distilled water containing 0.17 percent w/v sodium citrate and heated to 90°c using a heating magnetic stirrer (labtech, korea) while stirring to produce solutions of 0.25, 0.50, and 1.0 w/v percent. before dissolving the calcium carbonate 0.75% w/v and mk in the solution, it was cooled down to the desired temperature of less than 40°c (15). afterward, the solution was cooled to room temperature and the stirring was halted. distilled water was added up to 100 ml which was the volume of the final formulation (16). evaluation appearance a black-and-white background was used to evaluate the clarity of each formulation (17). rheological study of in situ gel brookfield viscometer dv-iii (brookfield, usa) spindle number 21, cup and bob setting at 50 rpm was used to measure the viscosity of formulations after gelling in artificial gastric fluid (14). each sample's viscosity was measured three times, with each measurement taking about 30 seconds (18-19). ph measurement a calibrated digital ph meter (schott gerate, germany) was used to measure the ph levels of the produced mixtures (16). iraqi j pharm sci, vol.31(suppl.) 2022 floating oral in situ gel 164 in vitro floating study the time the formulation took to emerge on the medium surface (floating lag time). time in seconds was used to calculate the floating lag time, which was calculated by introducing 5 ml of formulation into a beaker containing 100 ml of 0.1n hcl, ph 1.2, at 37°c (20). the time the formulation constantly floated on surface of the dissolution medium (duration of floating) were recorded. ten milliliters of the formulation were added to the container of 100 milliliters (0.1n hcl, ph 1.2) at 37 ○ c for the invitro floating study and the time it remained on the surface (duration of floating) were documented (21). in-vitro gelation study for in vitro gelling evaluation, 10 ml of each formulation was added to 100 ml of 0.1n hydrochloric acid (hcl, ph 1.2) at 37°c in a beaker with gentle agitation that avoided breaking the gel. the ability to gel in vitro was divided into three categories based on the stiffness of the generated gel, the time it took to gel, and the length of time the gel stayed firm as such. (+) gels after few minutes, dispersed rapidly (++) gelation immediate remains for few hours (+++) gelation immediate remains for an extended period (22). determination of drug content for 1 hour, a magnetic stirrer was used to stir 5 ml of the solution into ph-1.2 simulated stomach fluid 0.1n. once the solution was filtered and sufficiently dilute with simulated gastric fluid, a uv-visible spectrophotometer (biotecheng. uv9200, uk) at 357 nm was used to measure the drug concentration in comparison with an appropriate blank solution by using calibration curve equation (23). measurement of drug release rate from gels dissolution test apparatus (pharma-test, germany) usp type ii (paddle method) was used to perform an in vitro release investigation in the laboratory. the formulations' in vitro dissolution rates were measured using the procedure outlined below. the dissolution of mk from the formulations was measured using a paddle stirrer at 50 rpm in a usp type ii dissolution test device. a 900 ml solution of (0.1n hcl, ph 1.2) was employed as the dissolution media, and the temperature was maintained at 37°c. a 0.1n hcl solution was carefully added to the dissolution vessel without disturbing the petri plate after 10 ml of the formulation had been placed in it to prevent breaking of the gel formulation. dissolution medium was pipetted out and refilled at regular intervals to ensure that the samples were accurately quantified. the uv-spectrophotometer (shimadzu uv – 2201, japan) was used to measure the aliquot's montelukast sodium concentration spectrophotometrically at 357nm (24). using a graph with cumulative drug release on the y-axis and time on the x-axis, the raft formulation's dissolving profile may be plotted (25-26). table 1.floating in situ gel formulations of montelukast sodium formulation code montelukast sodium (mg) gellan gum (%w/v) sodium alginate (%w/v) pectin (%w/v) caco3(%w/v) sodium citrate (%w/v) f1 4.2 0.25 0.75 0.17 f2 4.2 0.5 0.75 0.17 f3 4.2 1.0 0.75 0.17 f4 4.2 0.25 0.75 0.17 f5 4.2 0.5 0.75 0.17 f6 4.2 1.0 0.75 0.17 f7 4.2 0.25 0.75 0.17 f8 4.2 0.5 0.75 0.17 f9 4.2 1.0 0.75 0.17 f10 4.2 1.0 1.5 0.17 * all formulations were made as 100 ml volume with d.w where each formula contain 4.2 mg of montelukast sodium in each 5 ml. results characteristic of in situ gel as a result, the in situ gels that were created were completely transparent. an in situ gelling system can be achieved by developing formulations that behave like a fluid yet harden when exposed to gastric acid (figure 1). calcium carbonate in the formulation was dissolved and released carbon dioxide when it came into contact with the stomach's acid. the calcium ions that had been released in situ formed a gel that floated. measurement of viscosity of in situ gel table 2 shows a significant rise in viscosity with increasing polymer concentration. ph measurement oral medication is necessary to avoid throat discomfort. iraqi j pharm sci, vol.31(suppl.) 2022 floating oral in situ gel 165 everything in this recipe has a neutral or slightly alkaline ph. according to table 2, a range of 7.17.86 for the ph of the formulations was obtained. floating behavior in (0.1n hcl, ph 1.2) solution, the floatability of the produced formulations was tested. the floating lag time. a thick gel with a good floating tendency was formed when the formulation floated for 12 hours on the surface of the dissolving medium (duration of floating) (27). a b c figure 1. photographs taken during the in vitro floating study of formulae (a: f3, b: f2, c: f1) in 100 ml 0.1 n hcl (ph 1.2) in-vitro gelation study drug content for each formulation, a percentage of the total drug content was calculated and is given in table 2. the drug content was found to being the range of 90.2-94.4% for all the formulations indicating, indicating a consistent distribution of the medicine. in vitro drug release figure 2 depicts the impact of polymer type and concentration on in vitro drug release from in situ gels. mk was shown to be released from these gels in phases, with the initial phase being characterized by significant release rates (burst effect) followed by a second phase of moderate release. matrix diffusion kinetics has a unique biphasic pattern of release. in addition, the polymer type and concentration had an effect on the release rate. release rates from f1-f9 formulations showed that the release of mk was different when different polymer types were used, and this release was ranked in the following order: pectin > sodium alginate > gellan: polymer concentrations range from 0.25 to 1.5%, with the longest sustained release achieved by 1% gellan containing formula f3. figure 3 depicts the impact of in vitro drug release from in situ gels of various concentrations of gasgenerating agent (caco3). formulae f3 and f10, for example, show a slower release of the drug when the concentration is increased from 0.75% to 1.5%. discussion three distinct polymer kinds and concentrations were used to make in situ gels in this investigation. in situ gelling systems must have optimal viscosity and gelling capability as prerequisites (speed and extent of gelation). the ideal viscosity for the formulation is one that is easily swallowed as a liquid, and then rapidly transforms into a gel due to ionic contact. because they will be administered orally, the solutions' rheological qualities are critical. in order to explain the correlation between viscosity and concentration, it has been hypothesized that as the polymer concentration increased, the polymer chains got closer and the number of contacts between the polymer chains increased, leading to a denser 3-d network structure (28). depending on the formulation parameters, the floating lag time changed significantly (p < 0.05). when calcium carbonate is added to a formulation, the amount of co2 that may be trapped in the gel increases, resulting in a rising in the time it takes for buoyancy. as a gas producing agent and a source of cations for gelation, calcium carbonate was included in the formulation. in formulae f1-f9, 0.75 percent caco3 was employed to achieve the required floating duration, however larger concentrations were used in an effort to provide a more retarded release of the medication (29). to achieve the necessary floating time, the formulas comprising 0.75 and 1.5 percent caco3 were used, however larger concentrations were used in an effort at a more delayed drug release, as illustrated in figure 3. formulas having 1.5% of caco3 released drugs more slowly than those containing less of the compound (23). in other words, as the formulation's calcium carbonate concentration grew, the drug's release reduced. due to the fact that cross-linking occurs when calcium ions are present in higher concentrations, this behavior can be linked to a stronger gel being formed, which results in a more restricted and slower release of the drug. iraqi j pharm sci, vol.31(suppl.) 2022 floating oral in situ gel 166 table 2.mk in-situ gelling compositions' properties. formulation code drug content (%) ph graded gel response floating lag time (sec) duration of floating (hr) viscosity (cp) f1 90.20±0.22 7.26±0.02 +++ 8±0.12 > 12 hr 244.86±1.2 f2 93.45±0.43 7.10±0.11 +++ 8.5±0.16 > 12 hr 326.62±0.21 f3 92.40±0.24 7.33±0.22 +++ 14±0.26 > 12 hr 522.34±0.44 f4 90.35±0.35 7.34±0.02 +++ 9.5±0.42 > 12 hr 210.55±0.24 f5 94±0.12 7.22±0.18 +++ 8.4±0.04 > 12 hr 287.32±0.1 f6 91.8±1.2 7.10±0.36 +++ 12±0.05 > 12 hr 386.99±0.13 f7 92.6±0.3 7.32±0.4 ++ 10.5±0.11 > 12 hr 205.24±0.5 f8 90.3±1.1 7.24±0.21 ++ 12±±0.02 > 12 hr 264.76±0.35 f9 92.8±0.16 7.16±0.02 ++ 15±0.03 > 12 hr 354.66±0.24 f10 94.4±0.42 7.30±0.24 +++ 5±0.02 > 12 hr 725.35±0.33 figure 2 a. formulations containing gellan gum figure 2 b. formulations containing sodium alginate figure 2 c. formulations containing pectin. figure 2. f1 to f9 dissolution patterns were compared by an in vitro release test. figure 3. comparison of two caco3 concentrations, 0.75% and 1.5%, in f3 and f10, respectively, by an in vitro release test. conclusion mk in situ gelling liquid oral formulations were developed in this investigation. it was found that the release rate could be changed by altering variables including the kind and concentration of ion-sensitive polymer and the concentration of the gas-generating agent (caco3). the changes were non-significant (p > 0.05). drug release from mk floating in situ gel may be sustained while it is still in the stomach. as a stomach-specific delivery method for montelukast sodium, it appears to be promising in the treatment of persistent asthma, prevention of exercise-induced asthma, and treatment of other illnesses in children. reference 1. bapurao ps., v. n deshmukh. floating oral insitu gel: a review. j. emerg. tech. innov. res. 2022; 9(2): c274-c287. 2. peppas na, langer r. new challenges in biomaterials. science 1994; 263:1715-20. 3. asmaa eh., afaf ra., asmaa em. development and 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carrier for nasal administration of mometasone furoate. int. j. pharma. 2009; 365.1-2: 109–115. 10. singh bn, kim kh. floating drug delivery systems: an approach to oral controlled drug delivery via gastric retention. j control release. 2000; 63: 235–59. 11. li, d-q., li, j., dong, h-l., li, x., zhang, j-q., ramaswamy, s., et al. pectin in biomedical and drug delivery applications: a review. int. j. biol. macromolecules 2021; 185: 49–65. 12. huang ck, handel n. effects of singulair (montelukast) treatment for capsular contracture. aesthet surg j. 2010; 30:404–8. 13. goldbart ad, greenberg-dotan s, tal a. montelukast for children with obstructive sleep apnea: a double-blind. placebo-controlled study. pediatrics. 2012; 130(3):e575–80. 14. zhao jj, rogers jd, holland sd, larson p, amin rd, haesen r, et al. pharmacokinetics and bioavailability of montelukast sodium (mk-0476) in healthy young and elderly volunteers. biopharm. drug dispos. 1997; 18:769–77. 15. xu h, shi m. a novel in situ gel formulation of ranitidine for oral sustained delivery. biom. therap. 2014; 22(2):161. 16. siraj et al. design, development and evaluation of gastric floating in-situ gel of piroxicam. int. j. pharma. sci. res. 2020; 11(7):3409-3416. 17. dalapathi gugulothu et al. design and evaluation of glipizide loaded in-situ gel formulation using natural mucilages for improved bioavailability. int. j. pharma sci. res. 2021; 12 (2):41-49. 18. s arvapalli, b kiranmayi, v sushma, j divya and ch. krishnaveni. a novel in-situ gel approach for sustained release of eplerenone. the pharma innov. j. 2020; 9(3): 527-534. 19. singh garima s. et al. formulation, development and evaluation of intragastric floating in-situ gel of brivaracetam. indo amer. j. pharm. sci. 2021; 8 (4):182-198. 20. islam t et al. study of release-retardant polymers in formulation development of cinnarizine-hcl sustained release oral suspension using raft technology. dhaka univ. j. pharm. sci. 2020; 19(1): 15-24. 21. parthy et al., formulation development and characterization of eplerenone in-situ oral gels. int. j. indig. herbs drugs 2021; 6(3):69-78 22. nagaraj b. formulation and evaluation of stomach specific in-situ gel of urapidil hydrochloride. int. j.of pharmacy bio. sci. 2019; 9 (1): 1431-1439. 23. rohith g, sridhar bk, srinatha a. floating drug delivery of a locally acting h2-antagonist: an approach using an in situ gelling liquid formulation, acta pharm 2009; 59(3): 345–354. 24. preetha pj, karthika k, rekha nr, elshafie k, formulation and evaluation of in situ ophthalmic gels of diclofenac sodium. j. chem. pharm. res. 2010; 2(3): 528-535. 25. suraj, s.; gunjan, s.; bhupendra, s. in-situ fast gelling formulation for oral sustained drug delivery of paracetamol to dysphagic patients. int. j. biol. macromol 2019; 134: 864–868. 26. rani, k.r.v. et al. the effect of polymers on drug release kinetics in nanoemulsion in situ gel formulation. polym. 2022; 14(3): 427. 27. remya pn, damodharan n, venkata ma, oral sustained delivery of ranitidine from in-situ gelling sodium-alginate formulation, j. chem. pharm. res. 2011; 3(3): 814821. 28. nickerson, m. t. and paulson, a. t. rheological properties of gellan, k-carrageenan and alginate polysaccharides: effect of potassium and calcium ions on macrostructure assemblages. carbohydr. polym. 2004; 58: 1524. 29. thomas, l. m. formulation and evaluation of floating oral in-situ gel of metronidazole. int. j. pharm. pharma. sci. 2014; 6(10): 265-9. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 5 efficacy, safety and cardiovascular disease risk lowering ability of ace inhibitors, β-blockers and combination antihypertensive drug regimes in iraq dlveen m. sulaiman * , kassim j.al-shamma **, 1 and ansam n.al-hassani * * college of pharmacy, hawler medical university, hawler , iraq. ** department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract hypertension is a major health problem throughout the world because of its high prevalence and its association with increased risk of cardiovascular diseases. it is defined as systolic blood pressure ≥ 140 mmhg and/or diastolic blood pressure ≥ 90 mmhg. the aim of this study was to compare the efficacy, safety and cardiovascular disease risk lowering ability, of three antihypertensive drug regimens. a retrospective study was carried out on 66 hypertensive patients, divided in to three groups based on their antihypertensive drug regimens (ace inhibitors, β-blockers treated and combination antihypertensive therapy, the combination therapy consist of two or more of the following antihypertensive drugs ace inhibitor diuretic, ccbs β-blockers), the study also included 22 healthy individuals. duration of treatment was 2-10 years. blood pressure and pulse rate were measured and blood sample was collected, and the serum processed for the measurement of lipid profiles, fasting blood glucose, liver function test, kidney function test, electrolytes, and c-reactive protein. cardiovascular disease risk lowering ability have been assessed by cardiovascular risk assessor computer program. the results shows that systolic and diastolic blood pressure in the three antihypertensive drug regimens treated group, were significantly higher than systolic and diastolic blood pressure in control healthy individuals indicating that these antihypertensive drug regimens were unable to reach hypertension treatment target, although ace inhibitors and combination antihypertensive drugs reach minimal hypertension treatment target. ace inhibitors regimen did not show any significant adverse effects on lipid profiles and blood glucose, while β-blockers regimen adversely affected it. most predominant adverse effects that appear, in ace inhibitors treated group were dry cough and taste disturbances, in β-blockers treated group were bradycardia and sleep disturbances while in combination therapy treated group were according to the combination used. in combination containing thiazide diuretics, disturbed lipid profiles and hyperurecemia were predominant and in combination containing calcium channel blockers constipation and peripheral edema were predominant. coronary heart disease and stroke risk percentage in all three antihypertensive drug regimens were significantly higher compared to control healthy individuals group, and all three antihypertensive drugs regimens have the same cardiovascular risk lowering ability. in conclusion the results indicated that all three antihypertensive drug regimens used were not efficient enough to reach hypertension treatment target, the combination therapy and ace inhibitors regimens were only capable to reach minimal hypertension treatment target which is ≤150/90 mm hg. key words: ace inhibitors, b blockers, hypertension. ضغط انذو ويقارَة سالية هذِ انُظى انذوائية يقارَة فعانية ثالثة َظى دوائية تستخذو نًعانجة فزط وقذرتها عهى تقهيم خطز االصابة باأليزاض انقهبية وانىعائية دنفيٍ يىسى سهيًاٌ * ، قاسى جهيم انشًاع **،1 و اَساو َاجي انحسُي *** * . ، اٌؼشاق استيًوٍيح اٌصيذٌح ، جاِؼح هىٌيش اٌطثيح ، ** .فشع اٌصيذٌح اٌسشيشيح ،وٍيح اٌصيذٌح ، جاِؼح تغذاد ، تغذاد ، اٌؼشاق الخالصة أرشاسها اٌىثيش واسذثاغها ِغ صيادج خطش االصاتح تاالِشاض فشغ ظغػ اٌذَ هي ِشىٍح صحيح سئيسيح في جّيغ أحاء اٌؼاٌُ تسثة اٌهذف االساسي ٌّؼاٌجح . ٍُِ صئثمي 90 ≤و ظغػ اٌذَ االٔثساغي ٍُِ صئثمي ا  140ويؼشف تعغػ اٌذَ االٔمثاظي. اٌمٍثيح اٌىػائيح . ظغػ اٌذَ هى ذحميك اٌحذ االلصً ِٓ ذمٍيً خطش االصاتح تاألِشاض اٌمٍثيح اٌىػائيح واٌىفياخ ػًٍ اٌّذي اٌطىيً ِشيط فشغ 1 corresponding author e-mail:drkassim_alshamaa@yahoo.com received: 10/3/2012 accepted: 12/12/2012 iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 6 ػًٍ واٌهذف ِٓ هزٖ اٌذساسح هى ِماسٔح فؼاٌيح ثالثح ٔظُ دوائيح ذسرخذَ ٌّؼاٌجح فشغ ظغػ اٌذَ وِماسٔح سالِح هزٖ إٌظُ اٌذوائيح ولذسذها ج اٌذوائيح اٌّعادج ِشيط ِصاب تفشغ اٌذَ خعؼىا ٌٍّؼاٌج 66اجشيد هزٖ اٌذساسح ػًٍ . ذمٍيً خطش االصاتح تاألِشاض اٌمٍثيح واٌىػائيح 22ِٕهُ ذّد ِؼاٌجرهُ تىاسطح ِثثطاخ االٔضيُ اٌّحىي ٌؤلٔجيىذٕسيٓ و 22ٌفشغ ظغػ اٌذَ ٌّذج ذرشاوح تيٓ سٕريٓ اًٌ ػشش سٕىاخ ، ء ِٓ االفشاد االصحا 22ِشيط تىاسطح ٔىػيٓ ِٓ االدويح اٌّعادج ٌفشغ ظغػ اٌذَ وايعا 22ِشيط تىاسطح ِثثطاخ ِسرمثالخ اٌثيرا و . شاسوىا في اٌذساسح اٌىىٌيسرشوي ، ) وذّد ِؼاٌجح اٌّصً ٌمياط ِسرىي اٌذهىْ في اٌذَ ولذ ذُ لياط ظغػ اٌذَ وِؼذي إٌثط وجّؼد ػيٕاخ ِٓ اٌذَ ، ast,alt,alp and) ، لياط ِسرىي اٌسىش اٌصياِي في اٌذَ ، اخرثاس وظيفح اٌىثذ ( اٌذهىْ اٌثالثيح وتشوذيٓ شحّي ِشذفغ اٌىثافح ggt ) ًٍلياط ِسرىي االيىٔاخ ( حاِط اٌيىسيه ، اٌيىسيا ، اٌىشياذيٕيٓ ) اخرثاس وظيفح اٌى ، (na,ca,mg,cl ) ولياط اٌميّح إٌىػيح وتي ولذ ذُ ذمييُ ِذي لاتٍيح هزٖ إٌظُ اٌذوائيح ػًٍ ذمٍيً خطش االصاتح تاألِشاض اٌمٍثيح اٌىػائيح تىاسطح تشٔاِج حاط. اٌرفاػٍي cٌثشوذيٓ . ٌرمييُ خطش االصاتح تاألِشاض اٌمٍثيح اٌىػائيح إٌرائج ذثيٓ اْ ظغػ اٌذَ االٔمثاظي واالٔثساغي في اٌّجّىػاخ اٌثالز اٌري اسرخذِد ٔظُ ِخرٍفح ٌّؼاٌجح فشغ ظغػ اٌذَ واْ اٌذوائيح اٌثالز اٌّسرخذِح ٌّؼاٌجح اػًٍ تىثيش ِٓ ظغػ اٌذَ االٔمثاظي واالٔثساغي ػٕذ االشخاص االصحاء ، وهزا يثيٓ اْ هزٖ إٌظُ ٌُ ذسطغ اٌىصىي اًٌ هذف ِؼاٌجح فشغ ظغػ اٌذَ اًٌ اْ اٌّؼاٌجح تاالدويح اٌّثثطح ٌالٔضيُ اٌّحىي ٌؤلٔجيىذٕسيٓ وإٌظاَ فشغ ظغػ اٌذَ . اٌّىىْ ِٓ ٔىػيٓ ِٓ ِعاداخ فشغ ظغػ اٌذَ تٍغد اٌحذ االدًٔ ِٓ هذف ِؼاٌجح فشغ ظغػ اٌذَ يح اٌّثيطح ٌؤلٔضيُ اٌّحىي ٌؤلٔجيىذٕسيٓ ٌُ يظهش ايح ذأثيشاخ ػىسيح وثيشج ػًٍ ِسرىي اٌذهىْ واٌسىش في اٌذَ في حيٓ اْ ٔظاَ االدو اٌرأثيشاخ اٌؼىسيح اٌسائذج اٌري ظهشخ في اٌّجّىػح اٌري اسرخذِد ِثثطاخ االٔضيُ اٌّحىي . ِثثطاخ ِسرمثالخ اٌثيرا اثشخ ػىسيا ػٍيها اٌسؼاي اٌجاف واظطشاتاخ اٌّزاق ، اِا في اٌّجّىػح اٌري اسرخذِد ٔىػيٓ ِٓ االدويح اٌّعادج ٌفشغ ظغػ اٌذَ ٌآلٔجيىذٕسيٓ وأد ٔان فىأد اٌرأثيشاخ اٌؼىسيح تحسة اٌرشويثح اٌّسرخذِح ، ففي اٌّجّىػح اٌري اسرخذِد اٌرشويثح اٌّحرىيح ػًٍ ِذساخ اٌىي اٌثياصيذيح واْ ٖ في اٌذَ واسذفاع ِسرىي حاِط اٌيىسيه في اٌذَ اِا في اٌّجّىػح اٌري اسرخذِد اٌرشويثح اٌّحرىيح ػًٍ ذأثيش سٍثي ػًٍ ِسرىي اٌذهىْ ٔسثح خطش االصاتح تّشض اٌششياْ اٌراجي واٌسىرح . ِثثطاخ لٕىاخ اٌىاٌسيىَ فىاْ اٌرأثيش اٌؼىسي اٌظاهش هى االِسان واٌىرِح اٌطشفيح خذِد ٔظُ ِخرٍفح ٌّؼاٌجح فشغ ظغػ اٌذَ وأد اػًٍ تىثيش ِماسٔح ِغ ِجّىػح االفشاد االصحاء اٌذِاغيح في اٌّجّىػاخ اٌثالز اٌري اسد ج ، ولاتٍيح إٌظُ اٌذوائيح اٌثالز اٌّسرخذِح ٌّؼاٌجح فشغ ظغػ اٌذَ ػًٍ ذمٍيً ٔسثح خطش االصاتح تّشض اٌششياْ اٌراجي واٌسىرح اٌذِاغي . وأد ِرشاتهح سرخذِح ٌّؼاٌجح فشغ اٌذَ اٌّسرخذِح ٌّؼاٌجح فشغ اٌذَ ٌُ ذىٓ فؼاٌح تذسجح وافيح اٌذوائيح اٌثالز أٌُسرٕرج ِٓ إٌرائج اْ إٌظُ ٌٍىصىي اًٌ هذف ِؼاٌجح فشغ ظغػ اٌذَ واْ اٌّؼاٌجح تاألدويح اٌّثثطح ٌؤلٔضيُ اٌّحىي ٌآلٔجيىذٕسيٓ وإٌظاَ اٌّىىْ ِٓ ٔىػيٓ ِٓ . ٍُِ صئثمي 150/ 90 ≤دف ِؼاٌجح ظغػ اٌذَ ِعاداخ فشغ ظغػ اٌذَ تٍغد اٌحذ االدًٔ ِٓ ٖ .، فزط ضغط انذو يثبطات االَزيى انًحىل نألَجيىتُسيٍ ، يثبطات يستقبالت انبيتا :انكهًات انًفتاحية introduction hypertension is defined as systolic blood pressure (sbp) ≥ 140 mmhg and /or diastolic blood pressure (dbp) ≥ 90 mmhg and /or the use of antihypertensive medication (1) . although hypertension may occur secondary to other disease processes, primary or essential hypertension is more common occurring in 9095 % of the hypertension population (2) , a disorder of unknown origin affecting the blood pressure regulating mechanism (3) . the primary goal of treatment of the hypertensive patient is to achieve the maximum reduction in the long-term total risk of cardiovascular morbidity and mortality, as well as treatment of the raised bp (4) . blood pressure goal recommendations are based on results from randomized, controlled studies and recommendations from guidelines committees (table 1) (5) . table 1: recommended target bp goals nkf indicates national kidney foundation; ada, american diabetes association. tod, target organ damage; jnc, joint national committee; cvd, cardiovascular disease. *jnc vi bp goal also recommended for those with tod or clinical cvd. guideline uncomplicated not tod or clinical cvd; at least 1 cv risk factor excluding diabetes diabetes * jnc vi nkf ada <140/90 mm hg <140/90 mm hg <130/85 mm hg ≤130/80 mm hg ≤130/80 mm hg iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 7 treatment involves non-pharmacological measures, followed by the staged introduction of drugs, starting with those of proven benefit and least likely to produce side effects (6) . essential hypertension is a very heterogeneous disease and different pressor mechanisms might interact to increase bp, therefore it is not surprising that antihypertensive drugs, given as monotherapy, normalize bp in only a fraction of hypertensive patients (7) . the jnc 6 recommendations acknowledge evidence from clinical trials, demonstrate that most patients with hypertension require at least 2 antihypertensive drugs to reach target bp levels. the addition of a second antihypertensive agent with a different mechanism of action should be initiated when adequate doses of an initial agent fail to achieve target bp goals (8) .furthermore, combination therapy should be considered as initial therapy for patients who are more than 20 mmhg above their sbp target and more than 10 mmhg above their dbp target; one agent should be a thiazide-type diuretic unless otherwise indicated (9) . british hypertension society (bhs) guidelines recommend ace inhibitors as firstline agents for younger, non-black patients (10) , and recommended to start treatment with either an ace inhibitors or an angiotensin receptor blockers (arb) in patients who are likely to have normal or raised plasma renin (i.e. younger white people), and with either a thiazide diuretics or a calcium channel blockers (ccb) in older people and people of african origin (who are more likely to have low plasma renin). if the target bp is not achieved but the drug is well tolerated, then a drug of the other group is added, it is best not to increase the dose of any drug excessively, as this often causes adverse effects (6) . national institute for health and clinical excellence (nice) stated that the decision not to recommend β-blockers for first line therapy is based on the evidence suggests that they perform less well than other antihypertensive drugs, particularly in the elderly, and the increasing evidence that the most frequently used βblockers at usual doses carries an unacceptable risk of provoking type 2 diabetes. recent clinical studies have suggested that antihypertensive agents that inhibit the renin angiotensin system (ras) may reduce risk for new-onset type 2 diabetes (11) . the aim of the study was to compare the effectiveness of three antihypertensive drug regimens used to treat hypertension in dohok city in northern iraq, to compare the adverse effects of these drugs, and the extent to which each regimen have the ability to decrease the cardiovascular disease risk. patients and methods the study was carried out in duhok governorate from 15 th of december 2010 to the end of june 2011. sixty six hypertensive patients, 20 males and 46 females, with an age range from 29-75 years, the mean age was 51.88 years, they were divided into three groups each group included 22 patients according to their antihypertensive drug regimen. group one: this group included 22 hypertensive patients, 6 males and 16 females, with an age range from 29-75 years, the mean age was 54.77 years, 8 of them in addition to ace inhibitors were treated with 3-hydroxy-3methylglutaryl coenzyme a (hmg-coa) reductase inhibitors for dyslipidemia and 9 patients were diadetic hypertensive. group two: this group included 22 hypertensive patients, 6 males and 16 females with an age range from 42-74 years, the mean age was 55.04 years, 8 of them in addition to β-blockers, were treated with hmg-coa reductase inhibitors for dyslipidemia and 5 patients were diabetic hypertensive. group three: this group included 22 hypertensive patients, 8 males and 14 females with an age range from 34-70 years, the mean age was 54.27 years, 9 of them in addition to combination antihypertensive therapy were treated with hmg-coa reductase inhibitors for dyslipidemia and 7 patients were diabetic hypertensive,( the combination therapy consist of two or more of the following antihypertensive drugs, ace inhibitor, diuretics, ccbs, β-blockers). control group: the control group included 22 healthy subjects, free from hypertension, lipid disorders, diabetes mellitus, cvd and renal disease, 8 males and 14 females and their ages ranged from 31-57 years, the mean age was 43.45 years. inclusion criteria include: 1. essential hypertensive patients. 2. age range between 25-80 years old the exclusion criteria included patients with: 1. cardiovascular disease. iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 8 2. renal disease. 3. liver disease. 4. smokers. 5. pregnant women. patients have been informed about the aims of the study and the parameters that will be taken to assess the efficacy and safety of the treatment. each patient have been asked to attend the hospital or the health center at three months interval for follow-up. systolic and diastolic bp were the primary efficacy parameters, they were measured by electronic bp measuring device and cuff appropriate for arm size, the same device was used to measure pulse rate, bp measurements were taken during first and second study visit, after participant had been seated for at least 5 minutes. safety was evaluated by asking patients about possible adverse effects, recorded during first and second study visit, and laboratory biochemical analysis of lipid profiles (tc, tg, hdl, ldl and vldl), fasting serum glucose, liver function test (aspartate aminotransferase, ast, alanine aminotransferase, alt, alkaline phosphatase, alp and ggt), kidney function test (uric acid, urea and creatinine), electrolytes (ca, mg, k, na and cl). cardiovascular risk lowering ability have been assessed by cardiovascular risk assessor computer program, the program compute coronary heart disease (chd) and stroke as the percentage likelihood of an event over a period of 10 years for e.g. a risk of 30% means that there is a 30 in 100 chance of an event in the next 10 years, and laboratory biochemical assessment of c-reactive protein (qualitative) , and pulse rate measurement. all data were analyzed using the statistical package for social science (spss) version 14; using one way analysis of variance (anova), the comparison among groups were done using least significant difference test (lsd). all the results were expressed as mean ± standard error (se) of mean. the level of significance was set at p≤ 0.05. results results are shown in the following tables: table 2: sbp and dbp in control healthy individuals and hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. table 3: lipid profiles (tc, tg, hdl, ldl and vldl) in control healthy individuals and hypertensive patients treated with different antihypertensive drug regimens and hmg-coa reductase inhibitors. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. a p ≤ 0.05 significant difference from ace inhibitors treated group. group n sbp (mmhg) dbp (mmhg) control 22 121.27 ± 2 78.81 ± 1 ace inhibitors 22 146.18 ± 3 * 90.27 ± 2 * β-blockers 22 151.13 ± 3 * 89.31 ± 2 * combination therapy 22 144.54 ± 3 * 88.40 ± 2 * group n tc (mg/dl) tg (mg/dl) hdl (mg/dl) ldl (mg/dl) vldl(mg/dl) control 22 179.36 ± 6 102.45 ± 7 49.36 ± 1 96.04 ± 3 22.59 ± 2 ace inhibitors 8 188.50 ± 5 140.25 ± 22 42.75 ± 1 101.87 ± 3 25.12 ± 3 β-blockers 8 195.87 ± 12 173.00 ± 17* 43.00 ± 2 106.37 ± 6 32.62 ± 2 * combination therapy 9 188.77 ± 14 184.33 ± 18 *a 43.66 ± 2 99.88 ± 6 37.22 ± 4 *a iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 9 table 4: lipid profiles (tc, tg, hdl, ldl and vldl) in control healthy individuals and hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. a p ≤ 0.05 significant difference from ace inhibitors treated group. table 5: fasting serum glucose in control healthy individuals and non diabetic hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. group n pulse rate (beat/min) control 22 79.72 ± 1 ace inhibitors 22 81.36 ± 1 β-blockers 22 70.31 ± 1 *ab combination therapy 22 76.04 ± 2 a table 6: serum ast, alt, alp and ggt in control healthy individuals and hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. group n tc (mg/dl) tg (mg/dl) hd(mg/dl) ldl(mg/dl) vldl(mg/dl) control 22 179.36 ± 6 102.45 ± 7 49.36 ± 1 96.04 ± 3 22.59 ± 2 ace inhibitors 14 182.07 ± 4 152.25 ± 14 43.21 ± 2 * 110.71 ± 5 33.14 ± 4 β-blockers 14 200.71 ± 9 * 208.35 ± 27 * 37.00 ± 1 * a 122.50 ± 8 * 37.35 ± 4 * combination therapy 13 189.69 ± 9 192.38 ± 29 * 41.69 ± 2 * 111.30 ± 10 34.00 ± 4 * ggt (u/l) alp (u/l) alt (u/l) ast (u/l) n group 25.81 ± 1 233.90 ± 7 24.86 ± 1 30.31 ± 1 22 control 25.13 ± 1 251.18 ± 15 23.07 ± 1 29.37 ± 2 22 ace-i 27.59 ± 1 236.86 ± 7 26.27 ± 2 30.50 ± 1 22 β-blockers 29.77 ± 3 248.63 ± 14 24.34 ± 1 27.49 ± 1 22 combination therapy iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 10 table 7: serum uric acid, serum urea and serum creatinine in control healthy individuals and retrospective hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. table 8: serum electrolytes (ca +2 , mg +2 , k +, na + , cl ) in control healthy individuals and hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. table 9: c-reactive protein qualitative value in control healthy individuals and hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. serum creatinine (mg/dl) serum urea (mg/dl) serum uric acid (mg/dl) n group 0.84 ± 0.1 27.13 ± 1 3.72 ± 0.2 22 control 0.83 ± 0.02 27.90 ± 1 4.21 ± 0.2 22 ace inhibitors 0.82 ± 0.03 29.23 ± 1 4.57 ± 0.3 * 22 β-blockers 0.85 ± 0.21 29.21 ± 1 4.60 ± 0.2 * 22 combination therapy cl (mmol/l) na (mmol/l) k(mmol/l) mg(mg/dl) ca (mg/dl) n group 100.09 ± 0.66 141.81 ± 0.94 4.27 ± 0.10 1.77 ± 0.02 9.25 ± 0.15 22 control 100.31 ± 0.69 139.04* ± 0.52 4.13 ± 0.08 1.79 ± 0.03 8.99 ± 0.11 22 ace inhibitors 101.31 ± 0.49 140.95 ± 0.71 4.18 ± 0.12 1.82 ± 0.02 9.11 ± 0.12 22 β-blockers 100.04 ± 0.53 140.50 ± 0.81 4.15 ± 0.08 1.83 ± 0.03 9.01 ± 0.14 22 combination therapy % positive % negative n group 0.00% 0 100% 22 22 control 27.27% 6 72.72% 16 22 ace inhibitors 31.81% 7 68.18% 15 22 β-blockers 31.81% 7 68.18% 15 22 combination therapy iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 11 table 10: pulse rate in control healthy individuals and hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. a p ≤ 0.05 significant difference from the ace inhibitors treated group. b p ≤ 0.05 significant difference from the combination therapy treated group. table 11: chd risk % and stroke % based on sbp in control healthy individuals and non diabetic hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. table 12: chd risk % and stroke % based on sbp in control healthy individuals and retrospective diabetic hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. pulse rate (beat/min) n group 79.72 ± 1 22 control 81.36 ± 1 22 ace inhibitors 70.31 ± 1 *ab 22 β-blockers 76.04 ± 2 a 22 combination therapy stroke risk % chd risk % n group 0.52 ± 0.08 2.72 ± 0.5 22 control 3.56 ± 1 * 11.25 ± 2 * 13 ace inhibitors 3.07 ± 0.5 * 11.55 ± 1 * 17 β-blockers 2.32 ± 0.3 * 8.92 ± 1 * 15 combination therapy stroke risk % chd risk % n group 0.52 ± 0.08 2.72 ± 0.5 22 control 4.77 ± 1 * 14.13 ± 2 * 9 ace inhibitors 4.34 ± 1 * 16.02 ± 3 * 5 β-blockers 4.60 ± 0.7 * 14.27 ± 1 * 7 combination therapy iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 12 table 13: chd risk % and stroke % based on dbp in control healthy individuals and non-diabetic hypertensive patients treated with different antihypertensive drug regimens. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. table 14: chd risk % and stroke % based on dbp in control healthy individuals and retrospective diabetic hypertensive patients treated with different antihypertensive drugs. each value represents mean± standard error of mean. * p ≤ 0.05 significant difference from the control. discussion the study compared the efficacy and safety of three antihypertensive drug regimens used to treat high bp in duhok city, and the extent to which each regimen have the ability to decrease the cvd risk. the efficacy of antihypertensive group of drug regimens as shown in table (2) mean systolic and diastolic bp in control healthy individuals group were normal according to the bhs ' s classification of blood pressure (12) . mean sbp and dbp in ace inhibitors treated group were significantly higher than mean systolic and diastolic bp in control healthy individuals, which might indicate that we could not reach the normal systolic and diastolic bp in this group of patients. similar results were reported by heran etal, 2009, evaluating the bp lowering ability of 14 different ace inhibitors in 12,954 participants. the study followed participants for approximately 6 weeks, the bp lowering effect was modest, and most of the bp lowering effect (about 70%) achieved with the lowest recommended dose of the ace inhibitor drugs (13) (14) . mean systolic and diastolic bp in β-blockers treated group were significantly higher than sbp and dbp in control healthy individuals group. this could indicate low effect of β-blockers when used as mono-therapy, similar results have been found in 10 randomized controlled studies in 16,164 patients, who were treated with either a diuretic or a β-blocker (atenolol), bp was normalized in two-thirds of diuretic-treated patients but only one-third of patients treated with atenolol as mono-therapy, diuretic therapy was superior with regard to all end points, and βblockers were found to be ineffective except in reducing cerebrovascular events (15) . stroke risk % chd risk % n group 0.54 ± 0.1 2.83 ± 0.6 22 control 2.92 ± 1 * 11.01 ± 2 * 13 ace inhibitors 2.29 ± 0.4 * 10.56 ± 1 * 17 β-blockers 2.28 ± 0.4 * 9.04 ± 1 * 15 combination therapy stroke risk % chd risk % n group 0.54 ± 0.1 2.83 ± 0.6 22 control 5.27 ± 1 * 15.62 ± 3 * 9 ace inhibitors 3.76 ± 0.9 * 16.90 ± 3 * 5 β-blockers 3.77 ± 0.5 * 13.82 ± 2 * 7 combination therapy iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 13 mean systolic and diastolic bp in group treated with either two or three antihypertensive drugs i.e combination therapy were significantly higher than sbp and dbp in control healthy individuals group, but on the basis of ontreatment analysis patients whose bp below 150/90 mmhg were also not bad (16) . in similar combination study, the addition of hydrochlorothiazide (or bisoprolol) to therapy with bisoprolol (or hydrochlorothiazide) produced an incremental reduction in bp, dosages of hydrochlorothiazide as low as 6.25 mg/d contributed a significant antihypertensive effect (17) . the effect of antihypertensive group of drug regimens on lipid profiles in present study, as shown in table (3) lipid profiles in ace inhibitors treated group were not significantly different from mean of the same profiles in control healthy individuals group. in this group of patients we cannot indicate the effect of ace inhibitors on lipid profiles because this group of patients also treated with hmgcoa reductase inhibitors. mean lipid profiles in β-blockers treated group (tc, hdl and ldl), were within normal range and not significantly different from mean of the same profiles in control healthy individuals group and other treated groups (ace inhibitors and combination therapy); however this did not indicate that β-blockers had no effect on tc, ldl and hdl, but we could not observe these effects because of the action of hmg-coa reductase inhibitors. mean tg and vldl were higher than normal and significantly higher than mean tg and vldl in control healthy individuals group. this could indicate the bad effect of β-blockers on tg and vldl in spite of the use of hmg-coa reductase inhibitors. similar results were reported in a study compared the effects of propranolol, pindolol, and atenolol given as a single daily dose for the control of hypertension, they observed small but significant increase in fasting plasma tg levels after 4 weeks of treatment. these rises were not accompanied by changes in plasma cholesterol (18) . mean lipid profiles in combination therapy treated group tc, hdl and ldl were not significantly different from healthy individuals group and other treated groups (ace inhibitors and β-blockers). again this did not indicate that combination therapy have no effect on tc, ldl and hdl, but hmg-coa reductase inhibitors could normalize antihypertensive effects on these parameters. regarding mean tg and vldl, they were significantly higher than mean tg and vldl in control healthy individuals group, which could indicate the bad effect of β-blockers and thiazide diuretics included in combination therapy on tg and vldl, in spite of the use of hmg-coa reductase inhibitors. table (4) showed lipid profiles in ace inhibitors treated group tc, ldl, vldl and tg were within normal range and did not significantly differ from the mean of the same profiles in control healthy individuals group. this could indicate that ace inhibitors had no effect on lipid profiles. similar study indicated that the ace inhibitors appear to have no important effect on plasma lipids (19) , however another study have been concluded that fosinopril therapy for 6 months resulted in a reduction in lipid profiles (20) . regarding hdl it was significantly lower than hdl of healthy individuals mean lipid profiles in β-blockers treated group were significantly different from lipid profiles in control healthy individuals group. this could indicate that β-blockers increased tc, ldl, vldl and tg levels; and decreased hdl. level of hdl was also significantly lower than hdl in ace inhibitors group. this could give an indication that ace inhibitors are better than β-blockers for decreasing the risk of cvd by keeping lipid profiles normal. in one study they have been found that antihypertensive treatment with β-blockers decreases hdl parameters, whereas treatment with ace inhibitors appears to decrease tc and ldl-related parameters (21) . β-blockers have little effect on cholesterol levels but lead to an approximate 10% fall in cardioprotective hdl cholesterol and a 20 to 40 % rise in tg (22) . a study evaluated 45 patients with non-insulin-dependent diabetes mellitus and hypertension who were randomized to therapy with β-blockers, associated with a 5% reduction in hdl and a 12 % elevation in tg (19) . mean lipid profiles in combination therapy treated group tc and ldl were not significantly different compared to control healthy individuals group and other treated groups. this could indicate that combination therapy have no effect on tc and ldl. the effect of combination therapy in most cases appears to reflect the sum of the effect of the individual drugs. a recent meta-analysis of over 450 published studies found that thiazide therapy raised the plasma cholesterol concentration by about 5 mg/dl (0.13 mmol/l) (23) . iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 14 mean tg was significantly higher in the combination therapy treated group than mean tg in control healthy individuals group. hdl was significantly lower than hdl in control healthy individuals group and vldl was higher than vldl in control healthy individuals group. this could indicate the increased effects of βblockers, thizide diuretics and other antihypertensive drugs contained in combination therapy on tg and vldl and decreased effect on hdl. antihypertensive treatment with hydrochlorothiazide alone, or in combination with a β-blockers, was associated with increased tg and decreased hdl; this was not so for patients treated with an arb alone or in combination a ccb (24) . the effect of three antihypertensive drug regimens on fasting serum glucose table 5 showed fasting serum glucose levels in non-diabetic hypertensive patients. mean fasting serum glucose in 13 ace inhibitors treated patients was not significantly different from mean fasting serum glucose in control healthy individuals group. this could give an indication that treating hypertensive patients with ace inhibitors for long period did not affect blood glucose level. in contrast to our finding one study showed that captopril increased the insulin-mediated disposal of glucose, as compared with placebo, it had no effect on the basal insulin concentration, but it decreased the late (30to 90-minute) insulin response to glucose and increased the early (2to 6-minute) insulin peak this finding may be explained by an increase in insulin sensitivity with captopril (25) . mean fasting serum glucose in 17 β-blockers treated group was significantly higher than fasting serum glucose in healthy individuals group and also significantly higher than fasting serum glucose in ace inhibitors treated group. this could indicate that β-blockers increase serum glucose level and that ace inhibitors are better for treating hypertension and not adversely affect serum glucose level. the diuretics and β-adrenoreceptor antagonists further decrease insulin sensitivity. the mechanisms by which β-blockers treatment exert its disadvantageous effects on serum glucose are not fully understood but several possibilities exist, alterations in insulin clearance and insulin secretion (26) . long term use of metoprolol and atenolol causes metabolic abnormalities that may be related to the increased incidence of diabetes in patients with hypertension who are treated pharmacologically. these results may help to explain why the two drugs (metoprolol and atenolol) have failed consistently to reduce the incidence of chd in several large scale studies (27) . in combination therapy treated group (15pateints), fasting serum glucose was significantly higher than fasting serum glucose level in healthy individual. this could indicate the effects of βblockers and thiazide diuretics in increasing serum glucose, about 6 patients out of 15 their combination therapy contain β-blockers and about 10 patients out of 15 their combination therapy contain thiazide diuretics. these results could indicate that β-blockers and thiazide diuretics have increasing effect on serum glucose level and that ace inhibitors are better in keeping serum glucose normal. hydrochlorothiazide decreased the insulinmediated disposal of glucose, as compared with placebo. it increased the basal insulin concentration and the late insulin response to glucose this may be explained by a decrease in insulin sensitivity with hydrochloro-thiazide (25) . cardiovascular risk lowering ability of antihypertensive group of drugs regimens table 9 showed qualitative estimation of crp in hypertensive patients, crp are predictors of cvd (28) , the risk of ihd and cerebrovascular disease was increased in persons who had crp levels above 3 mg per liter, as compared with persons who had crp levels below 1 mg per liter (29) . in ace inhibitors treated group 6 out of 22 patients showed positive crp value, while in βblockers and combination therapy treated groups 7 out of 22 patients showed positive value. this could give an indication that ace inhibitors are probably better in lowering cardiovascular risk in hypertensive patients. in one study ace inhibitor treatment was associated with lower 2.6 fold median crp levels and with a reduced 2 year cardiovascular risk compared with a different bp lowering regimen (30) . table 10 showed pulse rate, in ace inhibitors treated group was not significantly different from mean pulse rate in healthy individual. this could indicate that ace inhibitors have no effect on pulse rate, however it was significantly higher than mean pulse rate in β-blockers treated groups and combination therapy treated group this is because of βblocking activity of β-blockers in both βblockers treated group and in combination therapy treated group which also contain βblockers. mean pulse rate in β-blocker treated group was significantly lower than mean pulse rate in healthy individual, and in ace inhibitors iraqi j pharm sci, vol.22(1) 2013 risk lowering ability of ace inhibitors 15 treated group and combination therapy treated group, this indicate the β-blocking effect of these drugs and could indicate the cardiovascular risk lowering effect of β-blockers, because the reduction of pulse rate by β-blockers is accompanied by a decrease in peripheral bp with consequently reduced cardiac oxygen consumption and a longer diastolic filling time allowing for increased coronary perfusion. βblockers have consistently been shown to reduce cardiovascular mortality, sudden cardiac death, and reinfarction in patients recovering from previous infarction (31) . table 11 and 13 showed chd risk % and stroke % based on sbp and dbp, respectively in non diabetic hypertensive patients treated with different antihypertensive drugs. chd risk % and stroke risk % based on sbp and dbp in all three treated groups of patients showed higher risk % compared to control healthy individual group. this could indicate that antihypertensive drugs used were not efficient enough to decrease the % of chd risk and stroke risk percentage. this may be explained by the inability of these drugs to reach the normal bp and adverse effect of some drugs cause increase tc and decrease hdl values. the causal role of an elevated serum cholesterol level in the genesis of atherosclerosis and its clinical sequelae, particularly ihd, is now well established. the recognition of this role has been the impetus for numerous studies designed to test the hypothesis that a reduction in the cholesterol level will lead to a reduction in morbidity and mortality from cvd. most of these studies have indeed demonstrated a reduction in the incidence of ischemic cardiac events, and some have also shown a reduction in mortality from cvd (32) . randomized controlled studies indicates that an average reduction of 12-13 mmhg in sbp over 4 years of follow-up is associated with a 21% reduction in chd, a 37% reduction in stroke risk, a 25% reduction in total cardiovascular mortality, and a 13% reduction in all-cause mortality (33) . a 5 mmhg lower dbp is associated with about a one-third lower risk of stroke whereas a 10 mmhg lower dbp is associated with more than a one-half lower risk of stroke. the strength of these associations was not clearly different in men and in women (34) . table 12 and 14 showed chd risk % and stroke % based on sbp and dbp, respectively in diabetic hypertensive patients treated with different antihypertensive drugs. chd risk % and stroke risk % in all three treated groups of patients showed higher % risk compared to control healthy individual group and to non diabetic patient group. this could indicate that antihypertensive group of drugs used were not efficient enough to decrease the % of chd risk and stroke risk percentage. this may be explained by the inability of these drugs to reach the normal bp and adverse effect of some drugs caused increase tc and decrease hdl values and high blood glucose level. however inability of the antihypertensive drugs used in our study to decrease chd risk% and stroke risk% was incompatible with the overview of placebo-controlled studies of aceinhibitors that revealed reductions in stroke (30%) chd, (20%), and major cardiovascular events (21%). also the overview of placebocontrolled studies in which ccbs showed reductions in stroke (39%) and major cardiovascular events (28%) (35) . there was no significant difference in chd risk % and stroke risk % between different antihypertensive group of drug regimens used, similar result have been found in placebocontrolled study, no significant differences in total major cardiovascular events between regimens based on ace inhibitors, ccb, or diuretics or β blockers, although ace inhibitorbased regimens reduced bp less (36) . conclusions 1. the study indicated that the antihypertensive regimens used were not able to reduce bp to the target level, but the combination therapy and ace inhibitors regimens were only capable to reach minimal bp target which is ≤150/90 mm hg. 2. in view of the results of this study ace inhibitors are better than β-blockers and combination therapy containing both β-blockers and/or thiazid diuretics, they did not adversely affect lipid profiles and blood glucose. 3. the study indicated that all three antihypertensive drug regimens have the same cardiovascular risk lowering ability, more specific evaluation is required by excluding other cardiovascular risk factors. 4. the study suggested that ace inhibitors may act to decrease the c-reactive protein level and as a consequence lowering 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http://www.bmj.com/search?author1=t.+pollare&sortspec=date&submit=submit http://www.bmj.com/search?author1=h.+lithell&sortspec=date&submit=submit http://www.bmj.com/search?author1=i.+selinus&sortspec=date&submit=submit http://stroke.ahajournals.org/search?author1=francesca+papa&sortspec=date&submit=submit http://www.ncbi.nlm.nih.gov/pubmed?term=%22he%20j%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22whelton%20pk%22%5bauthor%5d iraqi j pharm sci, vol.32( 1 ) 2023 pharmaceutical sales as a future career doi : https://doi.org/10.31351/vol32iss1pp177-184 177 pharmaceutical sales and marketing as a future career for pharmacy students in iraq: perception and barriers. ahmad k. al-jalehawi *,1, mohammed d. al-rekabi* and furqan hashim** college of pharmacy, university of al-kafeel, najaf, iraq. college of dentistry, university of al-kafeel, najaf, iraq. abstract the exposure of pharmacy students to fieldwork experience throughout the early stages of their education would assist in preparing them for their future careers. many students feel working in community pharmacies does not give a profitable wage when compared to other opportunities influences their perceptions and opinions and chooses the promotional pharmaceutical field. there is a lack of studies that focused on these points in iraq. therefore, the present study aims to assess students' perceptions of the pharmaceutical promotional sector as a potential future profession as well as the obstacles they might face. this is a cross-sectional study targeting students of pharmacy in iraq through an online questionnaire to assess their perceptions and to determine the influencing factors. the majority of students work for improving their skills and for saving money for the future. regarding the barriers that students face in pursuing a career in pharmaceutical marketing, the existence of unethical actions, as well as a lack of the required skills and knowledge, were cited as barriers by the highest percentage of respondents. in conclusion, generally, there is a favorable view of working in this field and there is an apparent need for colleges to focus more on the aspects of this career. keywords: pharmaceutical promotion, pharmacy students, pharmacy career, student perception, pharmaceutical sales. و العوائق كمهنة مستقبلية لطالب الصيدلة في العراق: التصور االعالم الدوائي ** فرقان هاشمو *محمد الركابي، 1*، الجليحاوي احمد العراق ، النجف ، الكفيل الصيدلة، جامعةكلية * ، العراق النجف ، جامعة الكفيل االسنان، كلية طب ** الخالصة يشعرالعديد الميداني طوال المراحل األولى من تعليمهم سيساعد في إعدادهم لمستقبلهم المهني.ن تعرض طالب الصيدلة لتجربة العمل ا مع على اختيارهم. تهدف هذه الدراسة إلى مما قد يؤثر مربًحا مقارنة بالفرص األخرى رفة من الطالب أن العمل في الصيدليات االهلية ال يكون كم الصيدالني الترويج لقطاع الطالب طالب تصورات تستهدف مقطعية دراسة هذه يواجهونها. التي العقبات إلى باإلضافة محتملة مستقبلية هنة ت وتوفير الصيدلة في العراق من خالل استبيان الكتروني لتقييم تصوراتهم ومعرفة العوامل المؤثرة. تعمل أكبر نسبة من الطالب على تحسين المهارا مهنة ضمن مجال التسويق الصيدالني ، فإن وجود إجراءات غير أخالقية ، الالتي يواجهها الطالب في ممارسة المال للمستقبل. فيما يتعلق بالعقبات وهناك ا المجالفضالً عن االفتقار إلى المهارات والمعرفة الالزمة ، هي اكثرالعوائق المذكورة . بشكل عام ، هناك وجهة نظر إيجابية للعمل في هذ .يز أكثر على جوانب هذه المهنةحاجة واضحة للكليات للترك التسويق الصيدالنيادراك الطالب ، ، مهنة الصيدلة ، طالب الصيدلة، الصيدالني الترويج الكلمات المفتاحية: introduction around $15.7 billion was spent on pharmaceutical promotion and marketing in the united states in 2000, representing approximately 20–30 % of total sales turnover, which is two to three times the amount spent on pharmaceutical research and development (1). pharmacists perform critical roles in the healthcare field, including clinical pharmacy services, drug discovery and research, community pharmacy, hospital pharmacy, pharmaceutical-related industrial employment, and others. the exposure of pharmacy students to fieldwork experience throughout the early stages of their education would assist prepare them for their future careers and entry into the labor market once they graduate (2) . about three quarters of the undergraduate students at kuwait university claimed having experienced at least one pharmaceutical promotional activity(3), whereas 95% of the students at the universities of the united states(4)and finland reported such occurrences(5). an international survey has shown that even where the topic of drug promotion is addressed in formal classes, it is part of the core curriculum in 72% of cases, but it is rarely discussed in class for longer than a single two-hour lecture or small-group exercises, and often takes place through presentations. (6)some medical schools in the usa and abroad have made efforts to limit student 1corresponding author e-mail: ahmad.k.pharm@gmail.com received:10 /1 /2022 accepted: 21/ 5/2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp177-184 iraqi j pharm sci, vol.32( 1 ) 2023 pharmaceutical sales as a future career 178 interaction with drug detailers before they have gotten some basic training in ethical problems the harvard medical school barred pharmaceutical representatives from contacting first and secondyear students in 1991(7)this is in contrast to iraq’s situation as there are no strong laws and standards to regulate student labor in the promotional area, and students' ethical and regulatory understanding is weak. after 2003, the iraqi government allowed multinational businesses to engage in the iraqi private pharmaceutical industry, making it one of the middle east's most promising and expanding industries. in august 2020, the iraqi ministry of health (moh) registered 4665 medications from both domestic and international manufacturers. it also registered approximately 1800 pharmaceutical firms (8). iraq's pharmaceutical promotion and marketing industry is dominated by male employees; approximately one-third of the workers are undergraduate students and the majority are the last years (fourth and fifth) pharmacy college students; the majority were medical representatives and earn less than $1,000 per month(9). as in saudi arabia, the scenario in iraq might become comparable to that seen in that country, which is problematic given the restricted job possibilities for pharmacists in hospitals as a result of the saturation of the ministry of health and governmental institutions. consequently, the situation of pharmacists being out of work might deteriorate further, which has already been a major source of worry in the kingdom of saudi arabia, in addition, many students feel working in community pharmacies does not give a profitable wage when compared to other opportunities in the pharmacy profession(10)which resemble the iraqi situation. these factors might strongly influence students to choose to work in the promotional pharmaceutical field. high earned salary, financial security, interest in the sciences, and the will to help people are among the important factors influencing students' decision to major in pharmacy(11,12). this study aimed to determine the perceptions of pharmacy students toward the pharmaceutical promotional sector as a potential future profession and also to investigate the factors that influence their perceptions as well as the obstacles they face. method study design and sample size this was a cross-sectional study conducted between 9th april to 26th may 2021, among undergraduate pharmacy students at different colleges in iraq. raosoft; an online calculator, was used to calculate the sample size through. the required sample size was 384 to achieve a 5% margin of error (alpha) and 95% confidence interval. at the end of the period, 743 responses were collected, and eventually, 737 were considered (6 excluded: 2 were not pharmacy students, 2 duplicated responses, and 2 invalid responses). development of the questionnaire the online questionnaire has been developed by authors and the face and content validity were reviewed by three experts with ph.d. degrees before being circulated through various pharmacy college social media groups. a pilot study was performed and a sample of students was asked about their comments and the clarity of the questionnaire. all comments were considered and the items were edited before the distribution of the final version of the questionnaire. the first portion inquired about the students' demographics and characteristics, while the second section included 12 items to assess students' perceptions, questions on a five likert scale (1=strongly disagree,2=disagree,3=neutral,4=agree, and 5= strongly agree). the perceptions were evaluated to determine what factors influenced them, such as gender, year of study, family income, university, and marital status. the remaining sections investigate the reasons for choosing this sector for employment and as a potential career, while the final section focuses on the obstacles to working in the promotional industry. the reliability test shows that all sections had cronbach alpha of >0.8. the study approval this study was approved by the scientific committee of the college of the pharmacy – alkafeel university. there was no intervention taken place and the survey was collected under the free will of the participant. statistics and analysis microsoft excel 2019 was used to collect and arrange the data. mean, standard deviation (sd), and percentages were used to construct descriptive statistics. statistical package for the social sciences (spss) version 25 was used to assess the significance of affecting factors such as gender, family income, study year, and marital status using parametric or non-parametric tests (kruskal wallis test or mann whitney u test) when applicable. the significance level was set at p<0.05. results as seen in table 1, of the total of 737 respondents, males constituted 59.8 %, while females constituted 40.2 %. according to the survey results, the majority of respondents were in the fifth year of pharmacy college, followed by their fourthyear students (42.33 and 22.93 %, respectively). in addition, the majority of respondents' families had an income of 7001000 usd each month. the great majority of students were single (83.18 %), with married students accounting for 10.58 %. when comparing private colleges to public universities, 62.14 % of students were enrolled in private colleges, compared to 32.84 % in public universities. 5.02 % of students were enrolled in iraqi j pharm sci, vol.32( 1 ) 2023 pharmaceutical sales as a future career 179 public colleges, although they were required to pay for their education (parallel study). najaf accounted for 45.7 % of the total, with baghdad accounting for 16.6 %. table 1. demographic characteristics of participants (n=737). the experience of the respondents is shown in table 2. it was shown that61.33 % of the students did not work in the promotional sector at all. less than a quarter of respondents (21.03 %) currently work in this field. the bulk of the personnel (77.78 %) were medical representatives. 58.05 % of those polled had no prior experience in the promotional area. the majority of working students have less than a year of experience. 51.97 % stated that they have never obtained any type of training in this profession. only 25.64 % of those polled stated their college education addressed the ethical elements of the pharmaceutical promotional activity. it was found that there is a significant difference between the previous experience in this field with whether they paid for their college study, in which 66.3% of whom worked were paying for their study. table 1. promotional field experience of the participants item no. % do/did you work in pharmaceutica l promotion n=737 no 452 61.33 yes 285 38.67 what is/was your position n=261 medical representativ e 203 77.78 team leader 31 11.88 supervisor 23 8.81 others 4 1.53 years of experience in the pharmacy business n=727 non 422 58.05 < 1 161 22.15 1-2 61 8.39 2-3 27 3.71 3-4 24 3.3 >4 32 4.4 did you receive any kind of training regarding working in the pharmaceutica l promotion field? n=737 no 383 51.97 yes 354 48.03 did your study in the college cover the ethical aspects of the promotional field n=737 somewhat 293 39.76 no 255 34.6 yes 189 25.64 do you pay for your study? do/did you work in pharmaceutical promotion pvalue no (%) yes (%) no 146 (32.3) 96 (33.7 ) <0.00 1 yes 306 (67.7) 189 (66.3 ) characteristics no. % gender male 441 59.8 female 296 40.2 year of study first 56 7.6 second 85 11.53 third 115 15.6 fourth 169 22.93 fifth 312 42.33 estimated family income (usd) <$400 104 14.11 $400$700 172 23.34 $700$1000 186 25.24 $1000$1500 152 20.62 >$1500 123 16.69 marital state single 613 83.18 married 78 10.58 engaged 43 5.83 divorced 3 0.41 type of the university public 242 32.84 private 458 62.14 parallel 37 5.02 university alkafeel 191 26.3 kufa 128 17.7 alzahrawi 54 7.4 al-safwa 42 5.8 baghdad 32 4.4 islamic 32 4.4 others 246 33.9 provenance najaf 335 45.7 baghdad 122 16.6 karbala 59 8.0 qadisiya 54 7.4 babil 52 7.1 others 111 15.1 iraqi j pharm sci, vol.32( 1 ) 2023 pharmaceutical sales as a future career 180 the students' perceptions of working in the pharmaceutical promotional area are summarized in table 3. the two areas of most agreement were that pharmaceutical promotions need specific training and abilities and that respondents believe this profession enhances their social relationships, with mean (sd) values of 4.17 (0.78) and 4.1 (0.86), respectively. the greatest disagreement of the students was seen about their preference for pharmaceutical promotion work over community pharmacy employment, with a mean of 3.02, followed by their impression of continuing to work in the promotional area, with a mean of 3.2. the significance of different variables such as level of study, gender, family income, and type of the university was examined over the perception items as shown in table 4. the adjusted significant (bonferroni correction) was calculated and it was seen that first stage students encourage their colleagues to work during the study compared to students in the fifth stage. third-year students see that pharmaceutical promotion might be their future career more than fifth-year students. while fourthyear students see this field improve their social relations more than second-stage students. it was seen that fourth-year students had higher concern than second-year students and the fifth year had more concern than first and third-year students regarding the ethical and religious aspects of the pharmaceutical promotion work. on the other hand, gender showed a significant difference in favor of males regarding choose of this field as a future career and increasing future opportunities, improving the skills and the social relations. also seeing this job as difficult and requiring special training in addition to preferring it over community pharmacy showed significant differences with gender variables. family income also showed some significant differences as demonstrated in the table while the type of the university showed insignificant differences across the perception items except in encouraging their colleagues to work after graduation were parallel studying students who pay for the study in the public universities showed higher score than public university students whom not pay for their study. table 2. perception of the student toward working in pharmaceutical promotion n=736 no. items strongly disagree disagree neutral agree strongly agree mean (s.d) 1 it would be better to study the pharmaceutical promotion aspects in the college 11 (1.5%) 33 (4.5%) 163 (22.1%) 293 (39.8%) 237 (32.2%) 3.96 (0.93) 2 i encourage my friends and colleagues to work in pharmaceutical promotion during my study 33 (4.5%) 92 (12.5%) 246 (33.4%) 261 (35.5%) 104 (14.1%) 3.42 (1.02) 3 i encourage my friends and colleagues to work in pharmaceutical promotion after graduation 19 (2.6%) 63 (8.6%) 181 (24.6%) 269 (36.5%) 204 (27.7%) 3.78 (1.03) 4 i see that pharmaceutical promotion might be my future career 48 (6.5%) 126 (17.1%) 206 (28.0%) 259 (35.2%) 97 (13.2%) 3.31 (1.1) 5 the pharmaceutical promotion is the job that improves my skills 12 (1.6%) 61 (8.3%) 178 (24.2%) 313 (42.5%) 172 (23.4%) 3.78 (0.95) 6 the pharmaceutical promotion is the job that improves my social relations 9 (1.2%) 27 (3.7%) 106 (14.4%) 337 (45.8%) 257 (34.9%) 4.1 (0.86) 7 the pharmaceutical promotion is the job that increases my future career opportunities 10 (1.4%) 26 (3.5%) 153 (20.8%) 350 (47.6%) 197 (26.8%) 3.95 (0.86) 8 pharmaceutical promotion is the job that i will continue to work in 48 (6.5%) 144 (19.6%) 237 (32.2%) 220 (29.9%) 87 (11.8%) 3.2 (1.09) 9 i have some ethical/religious concerns about working in the pharmaceutical promotions 29 (3.9%) 77 (10.5%) 259 (35.2%) 275 (37.4%) 96 (13.0%) 3.45 (0.98) 10 the pharmaceutical promotions work is a difficult job 8 (1.1%) 38 (5.2%) 243 (33.0%) 307 (41.7%) 140 (19.0%) 3.72 (0.87) 11 the pharmaceutical promotions work needs special training and skills 4 (0.5%) 13 (1.8%) 104 (14.1%) 351 (47.7%) 264 (35.9%) 4.17 (0.78) 12 i prefer working in the pharmaceutical promotion field to working in the community pharmacy 81 (11.0%) 162 (22.0%) 227 (30.8%) 191 (26.0%) 75 (10.2%) 3.02 (1.15) iraqi j pharm sci, vol.32( 1 ) 2023 pharmaceutical sales as a future career 181 table 3. perception items across different variables (expressed in p-values) items level of study** gender* family income** university type** 1 it would be better to study the pharmaceutical promotion aspects in the college 0.127 0.117 0.288 0.445 2 i encourage my friends and colleagues to work in pharmaceutical promotion during my study 0.008 § first>fifth (0.039) 0.118 0.419 0.418 3 i encourage my friends and colleagues to work in pharmaceutical promotion after graduation 0.141 0.555 0.209 0.047 § parallel>publ ic 4 i see that pharmaceutical promotion might be my future career 0.011§ third>fifth (0.047) 0.012§ 0.095 0.086 5 the pharmaceutical promotion is the job that improves my skills 0.115 0.036§ 0.553 0.480 6 the pharmaceutical promotion is the job that improves my social relations 0.010§ fourth>second (0.006) <0.001§ 0.237 0.426 7 the pharmaceutical promotion is the job that increases my future career opportunities 0.143 0.019 § 0.106 0.064 8 pharmaceutical promotion is the job that i will continue to work in 0.302 0.681 0.055 (<400$)>($70 0-$1000) 0.077 9 i have some ethical/religious concerns about working in the pharmaceutical promotions 0.000 § fourth>second (0.001) fifth>first (0.036) fifth>third (0.002) 0.121 0.930 0.154 10 the pharmaceutical promotions work is a difficult job 0.139 0.008§ 0.001 § (>1500$)>(<$ 400) ($1000$1500)>(<$4 00) ($400$700)>(<$40 0) 0.395 11 the pharmaceutical promotions work needs special training and skills 0.136 0.006 § 0.000§ (<$400)< other income 0.688 12 i prefer working in the pharmaceutical promotion field to working in the community pharmacy 0.431 0.010 § 0.257 0.919 *mann–whitney u test **one way anova.§ significant different as shown in table 5, there are several reasons why students choose to work in this sector, with the greatest percentage being 21.5 % for improving skills and 21.1 % for saving money for the future. only 17% of people say they enjoy working in marketing. iraqi j pharm sci, vol.32( 1 ) 2023 pharmaceutical sales as a future career 182 table 4. potential causes for the student to work in the promotional field. cause no. % improve my skills 425 21.5% to save some money for future 418 21.1% increase my knowledge 365 18.5% i like to work in this field 337 17.0% a new field that i want to explore 244 12.3% to solve my current financial issues 195 9.9% from table 6, less than one-third believe they will continue and grow in this area, whereas 26.9 % believe they may consider working in it. on the other hand, nearly one-fifth (19.8 %) said they would never consider working in the promotional area. table 5. promotional field as a feature career items no. % i will continue to work in this field and grow 217 29.4% i am thinking to work in this field in future 198 26.9% i consider it, a short term business 176 23.9% i never think to work in this field 146 19.8% concerning the barriers that students face in pursuing a career in pharmaceutical marketing, 18.5 % stated that they believe the job is tough. the existence of unethical actions, as well as a lack of needed skills and knowledge, were cited as barriers by a greater percentage of respondents (18.4 % and 18.3 % , respectively). gender was also mentioned as a limitation to working in this area by 201 students, 84.6% of whom were female. (table 7). table7. potential barriers for students to work in the promotional field barriers no. % difficult job 256 18.5% non-ethical behaviors 254 18.4% my skills and knowledge 253 18.3% my gender 201 14.6% interact with religious beliefs 156 11.3% my family and friends 153 11.1% my look 105 7.6% living location 3 0.2% discussion even though iraqi legislation only allows licensed pharmacists to operate in pharmaceutical promotion (13), this study found that a high number of pharmacy students are working in this field. despite the huge number of graduates, shortages in employment may occur, necessitating the hiring of students. in comparison to the scenario in saudi arabia, where an insufficiency of pharmacists owing to a lack of graduates and pharmacy schools necessitated the recruitment of a considerable number of foreign pharmacists(14). the majority of participants were from three cities (najaf, baghdad, and karbala), which have a large number of private universities. the existence of a greater number of private colleges may result in a higher proportion of students working in this profession compared to public colleges. the percentage of saudi students who consider the pharmaceutical sales and marketing industry as a future career is 46.7% (15) compared to this study’s findings of 48.4% for both agreed and strongly agreed on responses collectively. in another saudi study half of the students expressed an unfavorable view of pharmacists who work in the community and considered the work environment to be unsatisfactory. in the same study, roughly onefifth of students said they would consider working in a community pharmacy in the future. maybe it's because there isn't enough representation and a supportive work environment for women in saudi arabia. the majority of students preferred community pharmacy work to promotional employment, according to this study. for many students, community pharmacy is less profitable than hospitals, regulatory agencies, or academic institutions. (10) in contrast to what occurs in many developed countries, where the majority of newly graduated pharmacists work in community pharmacy settings(16), iraqi graduated pharmacists may seek more profitable careers as the major causes to choose this career was improving their skills and save money for the future, that resemble the saudi situation in which community pharmacy may not be as encouraging because the public's image of community pharmacists as healthcare professionals is unclear. (17) a study in iraq found that most employees were satisfied, with salary and position being the two most important factors determining satisfaction. (9). financial incentives are regarded as the main objective for students working in community pharmacies in other nations like the united states. (18) in addition to the difficulty of the promotional job, the presence of unethical promotional behaviors was the second major barrier to the participants in this study. there is still an ethical or moral problem with promotions because iraqi j pharm sci, vol.32( 1 ) 2023 pharmaceutical sales as a future career 183 there are no local norms in iraq and restrictions are not strictly enforced. interactions between medical representatives and physicians in iraq frequently result in irrational prescription practices, which can have a detrimental impact on patients' health while also increasing the cost of drugs(19). it is expected that inadequate regulatory and monitoring procedures have a significant impact on medical practitioners' prescribing behaviors(20). similar to what was found in iraq, the disparity in perceptions about the acceptability of promotional gifts in kuwait may be attributed to the absence of industry ethical norms and/or government regulations(3). conclusion over one-third of pharmacy students work in the pharmaceutical promotion area. in general, there is a favorable view and good perception of working in this industry. due to the present scenario in iraq, there may be an agreement regarding this sector being considered a future job; however, there is an apparent need for colleges to focus more on the aspects of this career. among the most important factors influencing the decision to choose this work are the desire to improve one's abilities and save money for the future. the students see the difficulty of the job in general, as well as the existence of unethical practices, as major hurdles to working in this sector, in addition to their insufficient knowledge and skills. recommendations it is recommended that colleges focus more on the issues that students will face in the future regarding career choice and development in terms of preparing them with the required skills in addition to the knowledge they gain from studying different subjects, especially in the presence of a high number of graduated pharmacists and the risk of a lack of job opportunities. lectures and instruction on the ethical and moral aspects of promotional activity are extremely important and essential. limitation of the study this study based on an online form questionnaire and in-depth face-to-face interview might reflect a more accurate result. this survey was made during the covid-19 pandemic which might affect the perception and some answers. conflicted of interest statement the authors state that no commercial or financial relationships that might be regarded as a possible conflict of interest were present throughout the research. references 1. national institute for health care management. 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19. mikhael, e. m. evaluating the effect of medical representative on physician prescribing pattern in iraq. asian journal of pharmaceutical and clinical research.2014;7(1) : 222-3 . 20. ra r. breaking the bureaucracy : drug registration and neocolonial relations in egypt. soc sci med. 1998;46(11):1487–94 . this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ abstract iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 46 synthesis and biological evaluation of two new analogues of gonadotropin releasing hormone (gnrh)d-alanine 8 and d-alanine 10 kawkab y. saour *.1 * departement of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract so far synthesis of gonadotropin releasing hormone (gnrh) analogues reported in the literature has clarified some aspects of structural activity of the naturally released gnrh. as a part of continuing efforts for further understanding of this relationship, the present investigation was undertaken which involved synthesis and biological evaluation of two gnrh analogues, firstly, by replacement of the amino acid l-argenine in the 8 th position at the backbone structure of the natural hormone by the amino acid d-alanine; and secondly, by replacement of the amino acid l-glycine in the 10 th position by d-alanine also at the backbone structure of the nature hormone, to obtain the following analogues respectively: pglu-his-trp-ser-tyr-gly-leu-dala-pro-gly-nh2 (analogue i: d-alanine 8 gnrh), pglu-his-trp-ser-tyr-gly-leu-arg-pro-dala-nh2 (analogue ii: d-alanine 10 gnrh), which were synthesized by applying conventional solution method. peptides were purified by several recrystallization using appropriate solvent and proved to be homogenous. conformity of the synthetic procedure was achieved by applying different physico-chemical analyses including melting point (mp), thin layer chromatography (tlc.), infrared spectroscopy (ir), elemental analysis (chn), amino acid analysis (aaa), and nuclear magnetic resonance (h 1 nmr).preliminary biological activity of the two analogues was determined by testing their effects of parenteral administration on ascorbic acid depletion from the ovary of pseudopregnant mice and compared with that of natural gnrh hormone. analogue ii showed significant ascorbic acid depletion as compared to the native hormone while the percentage in ascorbic acid depletion after administration of analogue i were not significant as compared to the native hormone. key words: gonadotropine releasing hormone, peptide synthesis, biological activity of gnrh الخالصة نمذ كشفج يًبثالث هىسيىٌ gonadotropin releasing hormone (gnrh) انًُشىسة فٍ األدبُبث انعبنًُت ُُت انجزَئُت نههىسيىٌ انطبُعُت، وكجزء يٍ يسبعُُب بهزا االحجبِ وانشايُت انً االسخزادة بعضب يٍ جىاَب انعاللت نهفعبنُت انحُىَت وانب -يٍ انخفهى األعًك واألشًم نهذِ انعاللت فمذ حى حُبول هزِ انذساست وانخٍ حشًم ححضُش يًبثهٍُ نههىسيىٌ انطبُعٍ ورنك عٍ طشَك: l-arginine بذالً يٍ انحبيط األيٍُُ احالل انحبيط األيٍُُ -d-alanine 1 فٍ انًىلع 8 l-glycine ٍُُفٍ انًىلع 10بذال يٍ انحبض األي d-alanine احالل انحبيط األيٍُُ -2 -عهً انخىانٍ وكًب َهٍ: pglu his trp ser tyr gly leu dala pro gly-nh2 (analogue i: d-alanine 8 gnrh) pglu his trp ser tyr gly leu larg pro dala-nh2 (analogue ii: d-alanine 10 gnrh) وانخٍ خهمج ببسخخذاو طشق انًحهىل انًعهىدة وحى حُمُت انًًبثالث انًحضشة عٍ طشَك اعبدة انبهىسة ببسخخذاو يزَببث يالئًت. أيب انخٍ انسخخذيج بهذف انخىصم نهخىاص انًًُزة نهًًبثالث انًحضشة فمذ كبَج كشويبحىكشافُب انطبمت انخمُُبث انفُزَبوَت وانكًُُبوَت انشلُمت، لُبس دسجت االَصبس، يطُبف األشعت ححج انحًشاء، االسخذاسة انبصشَت ححهُم انعُبصش ويٍ ثى ححهُم انحىايط األيُُُت انببَىنىجٍ انبذائٍ حُث اسخُذ عهً حمذَش حأثُش انهىسيىٌ انًصُع عهً يعذل ويطُبف انشٍَُ انُىوٌ انًغُبطُسٍ. حى اجشاء انخمُُى اَخفبض حشكُز حبيط االسكىسبُك فٍ يببَط انفئشاٌ كبربت انحًم. نمذ أدي حمٍ انًًبثهٍُ انًحضشٍَ انً حصىل اَخفبض يعُىٌ فٍ يعذل حشكُز حبيط األسكىسبُك فٍ انًببَط ( فٍ يعذل حشكُز حبيط األسكىسبُك بًُُب أظهش انًًبثم األول اَخفبضiiنهًًبثم ) َسبت نهفعبنُت انببَىَىنىجُت انمُبسُت نههىسيىٌ انطبُعٍ. introduction the hypothalamus releases gonadotropin releasing hormone (gnrh), a decapeptide that stimulates the anterior pituitary to secrete leutinzing hormone (lh), and follicular stimulating hormone (fsh) respectively. this peptide controls and regulates both male and female reproduction system. gnrh is a modified decapeptide: pyroglu-his-trp-sertyr-gly-leu-arg-pro-gly-nh2 (figure1). the pyroglutamte at the n terminus and the cterminal amide distinguish this peptide from unmodified decapeptides (1-3) . 1 corresponding author e-mail : dr.ksaour@yahoo.com received : 9/2/2009 accepted : 23/6/2009 mailto:dr.ksaour@yahoo.com iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 47 figure1: basic structure of gn-rh hormone the gonadotropins have a close functional relationship to estrogen, progesterone, and testosterone. they are called gonadotropins because of their action on the gonads. they control ovulation, spermatogenesis, and development of sex organs, and they maintain pregnancy. included in this group gnrh, lh, fsh, chorionic gonadotropin (cg; hcg is human gonadotropin), a glycoprotein produced by the placenta; its pharmacological actions are essentially the same as those of lh (4-9) . gnrh interacts with high-affinity receptors on the gonadotropes in the anterior pituitary, leading to the biosynthesis and release of the gonadotropins lh and fsh. the pulsetiming and concentration levels of gnrh are critical for the maintenance of gonadal steroidogenesis and for normal reproductive function (10-12) .because of its simple structure there has been an enormous amount of interest in development of analogues as medicinal agents. over 3000 gnrh analogues have been synthesized and studied in an attempt to evaluate their structure activity relationship (3) .the structure activity data reviewed in the previous works provide that arg 8 was identified as being critical for high affinity binding to mammalian receptors alsoa number of early studies results showed that d-arg, gln, leu, ornithin, diaminobuteryl substitution for arg 8 result in substantial decrease in activity while lys retained most of the activity, it has been shown also that replacement of glynh2 at position10 by ala resulted in mild reduction in activity, therefore we attempted to replace d-ala at position8 and position10 respectively in the back bone structure of the native hormone in the hope of gaining more information of the structure activity relationship of the gnrh hormone (23). this paper reports the synthesis and preliminary biological evaluation of d-ala 8 and d-ala 10 gnrh analogues using conventional solution method by stepwise elongation manner. the products obtained could provide enough materials for further chemical and physical characterization and for biological evaluation and for future work, especially for broad biological testing of the hormone, which is indicated because of its possibly far-reaching in clinical medicine (13) . materials and methods all the chemicals used in this study were analar grade purchased from sigma company. due to the presence of numerous complicated amino acids, some difficulties for the synthesis of the decapeptide should be expected. we therefore applied intermediates, which supposedly could be purified easily. pyro glutamic acid has been prepared according to budavari (14) . c-terminal protecting esters of amino acids also prepared according to huber and brenner (15) . they are histidine, serine, tyrosine, leucine, proline, glycine, and dalanine ethyl esters by dissolving 2 gm (1 mol.) of the amino acid in 15 ml. ethanol. then the mixture was cooled to -10 o c, thionyl chloride was added gradually with stirring. the mixture was left for one hour at this temperature, then left over night at 40 o c. the mixture was refluxed in water bath at 55-60 o c for three hours. purification was done under vacuum using ether to obtain the precipitate c o h n ch c ch2 o n nh h n ch c ch2 o hn h n ch c ch2 o oh h n ch c ch2 o oh h n ch c h o h n ch c ch2 o ch ch3 ch3 h n ch c ch2 o ch2 ch2 nh c nh2 nh n c o h n ch c h nh2 o fig. basic structure of gn rh hormone o 1 2 3 4 5 6 7 8 9 10 pglu his trp ser tyr gly leu arg pro gly -nh2 iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 48 n-terminal protection benzyloxy carbonyl amino acids (z) were prepared according to zervas and bergmann and baily (16) . they are z tryptophan, zd-alanine, z-glycine, and zarginine. conventional solution method was applied as a coupling method between the protected amino acid for peptide bond formation using dicyclohexyl carbodiimide dcc in the presence of 1-hydroxy benzotriazole hbt and n-methyl morpholine (25) nmm. deprotection of cterminal protecting groups (saponification) was performed using 1.5 equivalent of sodium hydroxide (naoh) solution (1 n). table 1 showed the physico-chemical properties of these esters. deprotection of the n-terminal protecting groups was performed using hydrobromic acid (hbr) in glacial acetic acid (equimolar). table 2 showed the physicochemical properties of these z protected groups (16) . the intermediates and segment peptides had been purified by repeating recrystalization several times (4 times) using different solvents like diethyl ether, petroleum ether (40-60 o ), ethyl acetate, absolute ethanol, distilled water (d.w.), chloroform. ascending thin layer chromatography was run on kieslgel gf 254 type (60) merck, for checking the purity of the prepared compounds as well as monitoring the reaction process. spots were revealed by reactivity with iodine vapour or irradiation with u.v. light or by ninhydrine (15) spraying reagent 2% in absolute ethanol chromatograms were eluted by the following systems: acetic acid 30% methanol chloroform a 20 45 60 ammonia methanol chloroform b 20 45 60 water acetic acid butanol c 45 10 40 table 1: physico-chemical properties of the prepared amino acid esters name of amino acid ester rfa mp(c o ) νcm-1 1. histidine methyl ester 0.42 205-207 3180 for primary amine, 1750 for (c=o) of ester. 2. serine methyl ester 0.46 163-165 3400 for primary amine, 1750 for (c=o) of ester,1260 for (c-o)of ester. 3. leucine methyl ester 0.62 190-192 as in (2.) 4. proline methyl ester 0.49 150-153 as in (2.) 5. glycine methyl ester 0.58 69-72 as in (2.) 6. d-alanine methyl ester 0.66 172-174 as in (2.) benzyloxy carbonyl n-protected amino acids (z) was produced as precipitate except for zargenine which was produced as oily residue using 0.2 mol. and 0.2 mol. of benzyl chloroformate, 4 volumes of 1-n sodium hydroxide. table 2: physico-chemical properties of the nprotected amino acids by benzyloxy carbonyl prepared (z) in this work name of zprotected amino acid rfa mp(c o ) cm -1 1 . z-tryptophan 0.84 114-121 3400 for secondary amine, 1500 secondary amide, 1680 for (c=o). 2 . z-glycine 0.82 124-129 3310 secondary amine, 1680 for (c=o). 3 . z-argenine 0.78 oily as above 4 . z-d-alanine 0.69 118-122 as above iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 49 scheme i and scheme ii showed the pattern followed to obtain analogue i and analogue ii respectively. m.p., tlc., optical rotation, ir,chn, and nmr were the analytical techniques utilized to the chemical evaluation of the different coupling procedures between amino acids and peptide intermediates and to confirm the success of the synthetic process for both analogues. all intermediates and peptides showed optical activity, and purity as revealed by tlc, and acceptable ir, chn, h 1 nmr, and amino acid analysis as well. scheme i: synthetic steps of analogue i scheme ii: synthetic steps of analogue ii. biological activity of the prepared analogues the potency of each analogue was estimated by comparing their effect in treated mice with 0.5,1.0,2.0 mcg/0.1 ml of buffered plasma albumin (bpa)/mouse, on ascorbic acid depletion as compared with standard gnrh preparation according to bogdauove and gay (17) modified procedure published in british pharmacopoeia (1988).female rats approximately 21 days old has been chosen of approximately equal weights with the range 12-13 gm, and then randomly distributed into four groups 5each.group1 control group2-4 test group. the mice were hormonally treated as follows all pseudo pregnant mice were subcutaneously injected with 50 units of human chorionic gonadotrophin (hcg) at the first ,third & fifth day of the starting time of the experiment and after 6 days 5 mice in the groups 2-4 were injected with 0.5,1.0,2.0 mcg of d-alanine 8 gnrh/0.1 ml bpa subcutaneously and respectively while the mice in the first group were injected with bpa for comparison after 3 hours all mice were killed, their ovaries are removed ,weighed and placed immediqately in ice bath to avoid losses and dryness ,the ascorbic acid concentration was measured by homogenizing the 2 ovaries of each mice in 10 ml of metaphosphoric acid allowing the homogenate to stand for 30 minutes & centrifuge ,then 7 ml of the clear supernatant liquid was added to a freshly prepared mixture of 7 ml of acetic acid (ph=7) 3 ml distilled water and 2 ml of the dye 2,6 dichlorophenol indophenol standard solution ,30 seconds after the mixing ,the absorbance of the resulting solution was measured at the maximum at about 520 nm (17) .the result of the assay was calculated by standard statistical method using complete random design (crd) (18) .the biological activity of d-alanine 10 analogues has been estimated by same manner.table 3and 4 show the percent of depletion of ascorbic acid in the ovaries of the treated group. iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 50 table 3: average weight of pseudo pregnant mice ovaries, concentration of ascorbic acid, and percent of depletion of ascorbic acid in treated groups by analogue (ii): group number concentration of d-alanine 8gnrh analogue mcg/mouse average weight of mice * (gm) average weight of ovaries/mg concentration of ascorbic acid mcg/ovary % of depletion of ascorbic acid 1 control 12.11 117.03 65.49±0.156 2 0.5 12.34 117.38 61.109±0.195a 6.809 3 1.0 12.13 118.03 60.01±0.156b 8.37 4 2.0 12.17 117.52 59.18 ±0.372c 8.68 table 4: average weight of pseudo pregnant mice ovaries, concentration of ascorbic acid, and percent of depletion of ascorbic acid in the treated groups by analogue (i): group number concentration of d-alanine 8gnrh analogue mcg/mouse average weight of mice (gm) average weight of ovaries/mg concentration of ascorbic acid mcg/ovary % of depletion of ascorbic acid 1 control 12.16 117.05 65.54 2 0.5 12.32 117.36 3.41 3 1.0 12.133 118.001 4.17 4 2.0 12.175 117.63 4.301 * five mice in each group. a, b, and c indicate significant difference at percentage of error 0.01. results and discussion the results of physico-chemical evaluation of intermediate peptides were good indication of the success of the synthetic methodology using m.p, tlc, and ir as shown in table 5. elemental analysis(chn), optical rotation, and sometimes aaa have been applied to add conformity to the success of the synthetic procedure: 1. for the dipeptide p glu-his-oh: 25 ][ d  = -25.9 (c2 dmf) this indicates that this peptide is optically active. elemental analysis (chn); calculated: c 49.61, h 5.76 n 21.05, found: c 49.65 h 5.29 n 21.11. 2. for dipeptide trp-ser: 25 ][ d  = -46.4 (c2 dmf) this indicates the optical purity of the peptide, chn analysis; calculated: c 59.97 h 5.73 n 14.94, found: c 60.06 h 5.77 n 15.53. ] 3. tetrapeptide p glu-his-trp-ser: 25 ][ d  = 44 (c2 dmf), chn analysis; calculated: c 55.64 h 5.42 n 18.18, found c 55.98 h 5.66 n 18.63. 4. pentapeptide p glu-his-trp-ser-tyr: 25 ][ d  = -39.1 (c2 dmf), chn analysis; calculated: c 58.1 h 5.45 n 15.85, found: c 58.78 h 5.66 n 16.23. 5. peptide z-gly-leu: 25 ][ d  = -40.6 (c1 dmf). chn analysis; calculated c 51.3 h 8.61 n 14.96, found c 51.76 h 8.81 n 51.03. 6. for completion of analogue i the following steps have been done peptide d ala-prooch3: 25 ][ d  = -14.0 (c1 dmf). chn analysis; calculated: c 53.97 h 8.06 n 13.99, found: c 54.3 h 8.23 n 14.44. 7. tripeptide d ala-pro-gly-och3: 25 ][ d  = -12 (c1 dmf). amino acid analysis was d-ala 1.01, pro 0.99, gly 1.03. 8. pentapeptide z gly-leu-d ala-pro-glyoch3: c 57.73 h 7.00 n 12.47 o 22.80, found: c 58.32 h 7.38 n 12.86 o 23.11. 9. z deprotected pentapeptide gly-leu-d ala-pro-gly-och3: 25 ][ d  = -33 (c=2 dmf). chn analysis; calculated: c 53.37 h 7.78 n 16.39 o 22.46, found: c 53.91 h 7.99 n 16.83 o 22.71. 10. decapeptide pglu-his-trp-ser-tyr-glyleu-dala-pro-gly-och3: chn analysis; calculated: c 57.22 h 6.26 n 16.38 o 20.15, found: c 57.49 h 6.57 n 14.01 o 20.65. 11. aminolysis of the above decapeptide using ammonia and tri ethyl amine pgluhis-trp-ser-tyr-gly-leu-dala-pro-glynh2 (analogue i). 12. for completion of analogue ii the following steps have been done: dipeptide zarg-pro-och3: chn analysis; calculated: c 57.25 h 6.97 n 16.70 o iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 51 19.09, found c 57.89 h 6.23 n 16.91 o 19.52. 13. z deprotected arg-pro-och3: chn, calculated: c 50.49 h 8.13 n 24.55 o 16.83, found: c 51.01 h 8.43 n 25.03 o 17.22. 14. tripeptide zarg-pro-dala-och3: aaa arg 0.89, pro 0.93, d-ala 1.09. 15. zgly-leu-arg-pro-dala-och3: chn analysis; calculated: c 56.33 h 7.33 n 16.99 o 19.38, found: c 57.01 h 7.45 n 17.23 o 19.58. 16. z deprotected zgly-leu-arg-pro-dalaoch3: chn analysis; calculated: c 52.44 h 8.04 n 21.28 o 18.23, found: c 52.71 h 8.29 n 21.57 o 18.43. 17. decapeptide pglu-his-trp-ser-tyr-glyleu-arg-pro-dala-och3: chn analysis; calculated, c 56.50 h 6.49 n 18.51 o 18.50, found: c 61.01 h 6.84 n 18.91 o 18.79. 18. aminolysis of the above decapeptide using ammonia and tri ethyl amine pgluhis-trp-ser-tyr-gly-leu-arg-pro-dalanh2. table 5: some physico-chemical properties of the synthesized intermediate peptides (percent yield, melting points, rf values, and characteristic ir spectra). characteristic ir band (ν cm -1 ) rf value melting point percent yield amino acid residues peptide sequence 3320 stretching vibration of secondary amine, 1630 stretching vibration of amide band. 0.73b 109-112 55% p glu-his-och3 1-2 3320 stretching vibration of secondary amine disappearance of ester band at 1730, 1630 stretching vibration of amide band. 0.64a 214216 80% p glu-his-oh 1-2 3220 stretching vibration of primary amine, 1660 stretching vibration of carbonyl of amide 1 band. 0.18a 128130 73% trp-ser-och3 3-4 3370 stretching vibration of secondary amine, 1640 stretching vibration of carbonyl of amide 1 band. 0.89b 198200 66% pglu-his-trp-ser-och3 1-4 1590 c-o vibration of carboxyl group. 0.81a 172-177 62% pglu-his-trp-ser-oh 1-4 3370 stretching vibration of secondary amine interfered with phenol stretching vibration at 3000, 1630 stretching vibration of carbonyl of amide 1 band. 6.78c 204206 64% pglu-his-trp-sertyr-och3 1-5 3600-3300 (broad) of phenol group of tyrosine, c=c-h stretching vibration of aromatic ring 3100-3030, 760 out plane bend of aromatic c c 0.78c 221-226 58% pglu-his-trp-sertyr-oh 1-5 3380 stretching vibration of secondary amine, 1640 stretching vibration of amide 1 band 0.48c 101103 81% zgly-leu-och3 6-7 2970-2920 and 2860-2830 stretching vibration of ch3 and ch2, disappearance of ester absorption. 0.53a 99-103 77% zgly-leu-oh 6-7 completion of analogue i dala 8 gnrh 1630 stretching vibration of carbonyl of amide 1 band. 0.68a 181186 64% zd alapro-och3 8-9 3500 stretching vibration of carboxyl group interfered with secondary amine. 0.33c 162-165 60% zd ala-pr-oh 8-9 1680 stretching vibration of carbonyl of amide 1. 0.55c 170174 72% zd ala-pro-gly-och3 8-10 0.86c 126128 59% d ala-pro-gly-och3 8-10 1660 stretching vibration of carbonyl of amide 1 band, 1550 stretching vibration of secondary amine. 0.72b 152-156 67% z gly-leu-d ala-pro-glyoch3 6-10 1650-1620 c=o stretching vibration of amide band. 0.61b 143-146 55% gly-leu-d ala-pro-glyoch3 6-10 1650-1680 n-h bend, 1460, 1380 c-h bend of ch3 and ch2, 1240 o-h bend. 0.89b 218-222 75% pglu-his-trp-ser-tyr-glyleu-dala-pro-gly-och3 1-10 (aminolysis of the decapeptide (analogue i) was performed using ammonia in tri ethyl amine) pglu-his-trp-ser-tyr-gly-leu-dala-pro-gly-nh2. yield 63%. 1-10 completion of analogue ii dala 10 gnrh 1675 stretching vibration of amide band. 0.68a 181183 79% z arg-pro-och3 8-9 1660-1630 c=n stretching vibration of arginine, disappearance of ester band. 0.55a 173-176 66% z arg-pro-oh 8-9 3450 n-h stretching vibration of secondary amine, 1650 stretching vibration of carbonyl of amide 1, 1730 stretching vibration of carbonyl of ester group, 700 bending vibration of benzyl ring. 0.48a 169172 71% z arg-pro-d ala-och3 8-10 0.76b 116118 53% argprod ala-och3 8-10 3500 n-h stretching vibration of terminal primary amine, 1630 stretching vibration of carbonyl of amide 1 band. 0.72a 147150 82% z glyleuargprod alaoch3 6-10 0.59a 140-146 60% gly-leu-arg-pro-d alaoch3 6-10 0.87a oily 72% pglu-his-trp-ser-tyr-glyleu-leu-arg-pro-d alaoch3 1-10 aminolysis of the decapeptide (analogue ii) d alanine 10 gnrh was performed using ammonia in tri ethyl amine. pglu-his-trp-ser-tyr-gly-leu-leu-arg-pro-d ala-nh2. yield 68%. 1-10 iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 52 chemical evaluation of analogue i (dala 8 gnrh): melting point ( o c) = 212214. rf = 0.21a, 0.65b. c=1; -37 25 d ][ optical rotation in dimethyl formamide elemental analysis amino acid composition of acid hydrolysates of the analogue was: tyr ser trp his glu 1.03 0.98 1.1 0.99 1.01 pro d-ala 2gly 0.9 0.89 2.03 h 1 nmr resolution at 300 mhz in deteriuarated dimethyl sulfoxide using tetramethyl silane as a standard showed the following characteristic chemical shifts represented in ppm starting from glycine amide ending with pyroglutamine as follows: nh2 7.21 prim(s). amide, nh 9.04 sec(s). amide, ch2 3.15;3.41 pyrrolidine (m), ch 4.40 pyrrolidine(m) , ch2 2.34;2.09 pyrrolidine(m) , ch2 2.02;1.92 pyrroline (m), nh 8.32 sec(s). amide, ch2 1.75 methylene(d) , ch 4.53 methine (m), nh 8.32 sec. (s) amide, h 4.09 methylene (d), ch2 4.09 methylene, oh 9.83 aromatic(s) c-oh, ch 6.95 1-benzene(s) , ch 6.68 1-benzene(s), ch 6.68 1-benzene(s) , ch 6.95 1-benzene(s) , nh 8.32 sec. amide(s) , ch2 4.16;3.91 methylene(d) , ch 4.62 methine(m) , ch 7.58 3-indole(s), nh 8.32 sec. amide(s), ch 7.66 imidazole(d) , nh 13.4 imidazole (d), ch 8.73 imidazole (d), nh 8.32 sec. amide(s) ,nh 7.79 pyrrolidin-2-one(s) , ch2 2.28;2.18 pyrrolidin-2-one(m) , ch2 2.46;2.21 pyrrolidin-2-one(m) . chemical evaluation of analogue ii dala 10 gnrh melting point ( o c) = 218220 rf = 0.19a, 0.36b c=1; -28 25 d ][ optical rotation elemental analysis amino acid analysis tyr ser trp his glu 1.09 1.11 0.89 0.98 1.08 d-ala pro arg leu gly 1.02 1.09 1.01 0.96 0.99 h 1 nmr resolution at 300 mhz in deteriuarated dimethyl sulfoxide using tetramethyl silane as a standard showed the characteristic chemical shifts represented in ppm starting from d-alanine amide and ending with pyroglutamine as follows: nh2 7.21 prim. amide(s), ch3 1.48 methyl(d), ch 4.71 methine (m), nh 8.32 sec. amide (s) , ch2 3.51;3.41 pyrrolidine (m), ch 4.40 pyrrolidine(m) , ch2 2.34;2.09 pyrrolidine (m), ch2 2.02;1.92 pyrrolidine (m), nh2 6.63 amine, nh 2.0 amine(m), ch 4.53 methine(m) , nh 8.32 sec. amide(s) , ch2 1.75 methylene(d) , ch 4.53 methine(m) , nh 8.32 sec. amide (s), h 4.09 methylene, ch2 4.09 methylene, nh 9.04 sec. amide(s), oh 9.83 aromatic (s), (s) c-oh, ch 6.68 1benzene, ch 6.68 1-benzene(s) , ch 6.95 1benzene(s) , ch2 3.17;2.92 methylene(d) , ch 4.92 methine (m), oh 4.78 alcohol(s) , ch2 4.16;3.91 methylene (d), ch 4.62 methine (m), ch 7.34 3-indole (s),nh 10.85 3-indole(s) , ch 7.18 3-indole, ch 4.92 methine, nh 8.32 sec. amine(s), ch 7.66 imidazole (d), nh 13.4 imidazole (d),ch 8.73 imidazole (d), ch2 3.17; 2.92 methylene, ch 4.92 methine, nh 8.32 sec. amide(s) , nh 7.79 pyrrolidin-2-one(s) , ch2 2.28;2.18 pyrrolidon-2-one (m), ch2 2.46;2.21 pyrrolidin-2-one (m). so, the synthetic approaches according to the step wise manner of this study were proved to be effective for the synthesis of homogeneous analogues as indicated from m.p., tlc., optical rotation, chn, ir, and nmr. these analogues were preliminary estimated for gnrh activity, dalanine 10 gnrh (analogue ii) was found to posses significant activity as shown in table 3 while d-alanine 8 gnrh (analogue i) showed lower activity as shown in table v. chang et al (22,23) described the importance of basicaty of arginine moiety at position (8) and the influence of it is multi structural function for biological activity there for injection of the analog dalanine 10 gurh in the mice lead to significant depletion (1>0.01) in ascorbic acid concentration in the ovaries after 3hrs of the injection time as indicated in table 3 the percent of decrease in ascorbic acid concentration was 6.089, 8.37, 8.68 % for the group. 2-4 respectively compared with the control. this is similar to that obtained by gay and bogudonve (17) who n h c 17.88 6.25 56.91 calculated 17.91 6.55 56.56 found n h c 19.91 6.49 56.21 calculated 19.99 6.71 56.29 found iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 53 mentioned that the percent of decrease in ascorbic acid concentration in the ovary was 10% after 3hrs of the injection time 1.35 mcg of standard gnrh preparation in the mice. this is a clear indication of the presence of significant gnrh activity of the prepared dalanine 10 gnrh in stimulating the release of leutenizing hormone (lh) which in its turn affect the yellow body cells in the ovaries of pseudo pregnant mice (pretreated with hcg) which lead to the consume of ascorbic acid content in these cell will be to stimulate consumed peroxidase enzyme. this will lead to the oxidation of ascorbic acid and in this oxidation state ascorbic acid can be utilized for the production of very important steroid (progesterone) which is released from the yellow body cells, other probable mechanism of lh function in decreasing ascorbic acid content in the ovary is by increasing the excretion of ascorbic acid, as the venous blood exuded from the ovary (19, 21) . dalanine 10 gnrh ascorbic acid depletion >dalanine 8 gnrh. the result of this preliminary biological study may indicate that gly at cooh terminal is not essential for biological activity since its replacement by d-ala which has a reverse stereochemistry showed no reduction in the potency of the native hormone and may be the most important finding from this study is that arg at position 8 seems to be critical for high affinity binding to mammalian receptors because the substitution of arg8 by d-aala cause a marked reduction in the biological activity and this could be based from the suggestion that arg8 of gnrg may interact with acidic moieties on the receptors (24) . acknowledgement the author would like to acknowledge the college of agriculturedepartment of animal resources for their help in carrying the biological activity and supplying hcg hormone. references 1. fink .g gonadotropin secretion and its control.ln : knobil e , neill .j (eds) the phsyiology of reproduction. raven press, new york, 1988. pp 1349-1377. 2. casper rf clinical uses of gonadotropinreleasing hormone analogues . can med assoc j, 1991, 144:153-158. 3. schneider j.s. and rissman e.f.: gonadotropin-releasing hormone ii :a multi-purpose neuropeptide. integr.comp.biol., november 1,2008; 48 (5) : 588-595. 4. conn pm, crowley jr wf gonadotropinreleasing hormone and its analogues. n enggl j med, 1994, 324:93-103. 5. moghissi ks clinical applications of gonadotropin-releasing hormone in reproductive disorders. endocrinol metab clin north am, 1992, 21:125-140 6. barbieri rl clinical applications of gnrh and its analogues. trends endocrinol metab, 1992, 3:30-34. 7. emons g, schally av the leuteinzing hormone releasing hormone agonists and antagonists in gynaecological cancers. hum reprod, 1994 , 9:1364-1379. 8. derijk, r.h., schaaf, m., and de 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budavari,s.;mecrk index.an encyclopedia of chemicals,drugs and biological.merck and co. inc. usa, 1989. 15. brenner, m and huber,w.herstullung von alpha-aminosaureestern duch alkoholyse der methyl ester. helv.chem. acta,1953,36,1109-1111. 16. baily, l.:techniques in protein chemistry, expanded ed. elsevier publishing company, amsterdam, 1967. 17. bogadonve, e.m., and gay, l.v.: enhancement of ascorbic acid depletion response during estrogen-prolonged pseudopregnancy. endocrinology 1967, 81: 1104-1117. iraqi j pharm sci, vol.18(2) 2009 synthesis and biological evaluation of two new gnrh analogues 54 18. steel, r., and torrie, j.: prinicples of procedures of statistics. mcgraw-hill book co., new york, 1960. 19. niswender, g.d., and nett, t.m.: biological assay of gonadotropic and gonadal hormones. in reproduction indomestic animals, 3rd ed. cole. h.h. and p.t. copps academic press, chicago. usa, 1976. 20. agrawal, p. and lalloraya, m.m.: induction of peroxidase in corpora lutea of rat ovary by lutropin. biochem. j. 1977 66:,205-208. 21. yanaihara, n., yanaihara, c., sakagmi, m., tsuji, k., and hashimoto, t.: synthesis and biological evaluation of lh and fsh releasing hormone and its analogs. j. med. chem1973,. 15: 373-377. 22. chang, j.k., sivertssam, h., currir, b.l., bogentoft, c., and folkers, k.: synthesis of lhrh of the hypothalamus and the 8lysine analog. j. med chem. 1972,15: 23. chang, j.k., williams, r.h., and humphries, a.j.: lhrh synthesis and arg-analogs, and conformation-sequenceactivity relationships. biochem-biophys. res. commun. 1972,47: 727-732. 24. fujino m, kobayashi s, obayashi m, shinagawa s, fukuda t structure activity relationship in the c-terminal part of the luteinizing hormone releasing hormone(lh-rh). biophys res commun 1972 49:863-869 . 25. rich,dh.singh,j.the carbodiimide: the peptides, analysis,synthesis, biology 2002,1 st edition newyourk academic center, vol. 1,241-261 iraqi j pharm sci, vol.30(2) 2021 role of nutraceuticals against cognitive impairment process doi: https://doi.org/10.31351/vol30iss2pp135-142 135 ameliorative role of nutraceutical quercetin and its derivatives against cognitive impairment process induced by lead exposure in drosophila melanogaster(fruit fly) b. s. katsayal *,1, a. a. lema**, k. jibrin*, w. nuraddeen***, e. m. alexander****and n.h mohammed***** *department of biochemistry, ahmadu bello university, zaria, kaduna state, nigeria **department of biotechnology, university sultan zainal abidin, kuala terengganu, malaysia ***department of biological sciences, al-qalam university, katsina, katsina state, nigeria ****department of zoology (applied entomology & parasitology), university of jos, plateau state, nigeria *****department of plant biotechnology, faculty of bioresources and food industry, university sultan zainal abidin, terengganu malaysia abstract cumulative lifetime lead (pb) exposure has been associated with accelerated declines in cognition through free radical generation and epigenetic effects.several literature has established the link between lead exposure and neurodegenerative disorders.harwich strain of drosophila melanogaster were exposed to lead acetate for two weeks and the changes in impulse transmission through acetylcholinesterase and systemic redox state were assessed. in addition, molecular docking studies of acetylcholinesterase against quercetin was carried out. in silico toxicity, pharmacokinetics studies on quercetin were also carried-out. the data obtained showed alteration in function of antioxidant enzymes and molecules such as catalase, glutathione-s-transferase and glutathione. up regulation of acetylcholinesterase activity was observed following treatment with quercetin. molecular docking studies revealed quercetin to bind to both active and peripheral pocket of acetylcholinesterase. pharmacokinetic studies show moderate solubility, high therapeutic index, excellent absorption capacity, hepatoprotective and non-mutagenic properties. therefore, quercetin alongside other antioxidant molecules can play a vital role in preventing the onset of alzheimer and antioxidant related disorders. keywords: alzheimer; antioxidant; acetylcholinesterase; neurodegeneration; oxidative damage. introduction neurodegenerative disorders are agerelated neurological disorders associated with the progressive loss of neurons' structure and function (1,2). commonly known among these disorders include alzheimer's disease, parkinson's disease, the foam of dementia, and huntington's disease(3). alzheimer's disease is the most predominant among the elderly and fully characterized by extracellular plaques of β-amyloid, hyperphosphorylated tau protein, and loss of cholinergic neurons leading to a behavioral loss and cognitive function (4). several hypotheses have been put forward to explain this disorder, but the cholinergic hypothesis, which described the involvement of acetylcholinesterase and butyrylcholinesterase and oxidative stress hypothesis, were more prominent (3,5). evidence has proven the neuroprotective role of acetylcholinesterase inhibitors in the clinical management of alzheimer's diseases. some of these inhibitors reported include donepezil, galantamine, tacrine, neostigmine, pyridostigmine, and physostigmine, among many other compounds and plant extracts (6,7). the role of oxidative damage in the pathophysiology of alzheimer's disease and other neurodegenerative disorders has been fully documented (8). quercetin is a natural flavonoid found abundantly in almost all edible vegetables and fruits such as red onion, common onion, cranberry, blueberry[figure 1] (8). intake of a quercetin-rich diet is highly encourage and is positively correlated with health improvement(10). it can also be taken as dietary supplement with daily recommended doses of 200–1200 mg as well as a nutraceutical through functional foods with a concentration range of 10– 125 mg per serving (11). several studies suggest quercetin therapeutic potential and its derivatives to prevent and treat various chronic diseases, including cardiovascular, neurodegenerative, and cancer (12-14). figure 1. structure of quercetin lead (pb) is one of the most abundant heavy metal pollutants in the environment and it’s considered to be one of the most hazardous chemicals for humans and animals health (15). 1corresponding author e-mail: ssskatsayal@gmail.com received: 6/12/ 2020 accepted: 22/3 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp135-142 iraqi j pharm sci, vol.30(2) 2021 role of nutraceuticals against cognitive impairment process 136 lead exposure has been attributed to several clinical symptoms including arthritis, renal dysfunction, birth defects, mental retardation, psychosis, hyperactivity, autism and brain damage (16). and, several neurodegenerative disorders such as huntingtons syndrome, parkinson and alzheimer diseases models were simulated in drosophila (17-19), the organism has contributed significantly to the understanding of processes affecting metal toxicity (20). it is therefore considered to be a useful model species for the investigation of biological reactions to toxic chemicals (21,22,15), with its genes having many structurally and functionally preserved homologies to humans (23). this study aimed to determine the effect of short-term exposure to acetylcholinesterase activity in relation to oxidative damage associated with alzheimer's disease pathogenesis and the role of functional foods in improving these oxidative processes. materials and methods the randox protein kit was purchased from medicom, state of jos plateau. sigma aldrich purchased 1-chloro-2, 4-dinitrobenzene (cdnb), and 5-dithiobis (2-nitrobenzoic acid)(dtnb) (st louis, mo). harwich strain flies were collected from jos, plateau state, african center of excellence for phytomedicine research and development. grouping and treatments the flies were cultured on the standard drosophila culture medium consisting of corn flour, brown sugar, yeast, agar, and propionic acid as a mould inhibitor. the culture was maintained at 25±2°c in controlled 12 hour light and darkness. lead acetate (300 µl) was used as model pollutant and the flies were divided into four groups. group one was treated with normal feed containing normal distilled water, group two with quercetin (100 µl ), group three lead acetate (300 µl) and group four with the combination of lead acetate and quercetin in stated concentration. the flies were anesthetized to ice and homogenized to 100 mm phosphate buffer saline (ph7.4) in 1:10 volumes. the flies were centrifugated with a cold centrifuge at 4000 rpm. determination of total thiol content the total thiol content was calculated using ellman's process [24]. the reaction mix comprised 510 μl potassium phosphate buffer (0.1 m, ph 7.4), 25 μl sample, and 30 μl dtnb (10 mm). the reaction mixture was incubated for 30 min at room temperature, the optical density was taken at 412 nm and total thiol content was expressed in mmol/mg protein. standard glutathione was used for standard calibration. according to the manufacturers' instructions, the protein concentration of homogenates was determined with the total protein kit (randox). the data were measured with blank and blank samples, and the results were all corrected with protein content. determination of glutathione-s-transferase (gst) activity activity of glutathione-s-transferase (gst; ec 2.5.1.18) was calculated using habig and jacoby method [25], with 1-chloro-2, 4dinitrobenzene (cdnb) as substrate. the reaction mix consist solution a 600 μl, (20 μl 0.25 m potassium phosphate buffer, ph 7.0 with 2.5 mm edta and 510 μl 0.1 m gsh at 25°c), 60 μl sample (1:5 dilution) and 30 μl 25 mm cdnb. optical density was calculated at 340 nm at 10 s intervals for 2 min (jenway). the data were expressed in mmol/min using the 9.6 mm1 cm1 molar extinction coefficient of the gst-formed gs– dnb conjugate. determination of catalase (cat) activity the activity of catalase (cat; ec 1.11.1.6) was monitored using a process of aebi [26]. reaction mix containing 100 ml of potassium phosphate buffer (ph 7.0), 194 ml of solution a (300 mm h2o2). about 10 µl of sample was added to 590 µl of solution a and the clearance of h2o2 was monitored using 240 nm wavelength at 25°c. catalase activity was expressed as in mmol of h2o2 consumed/min. determination of acetylcholinesterase activity acetylcholinesterase activity was monitored following the method described by ellman et al., (27). the reaction mixture contain 285 l of distilled water, 180 µl of 100 mm potassium phosphate buffer (ph7.4), 60 µl of 10 mm dtnb, and 15 µl of sample, 60 µl of 8 mm acetylthiocholine were added. the change in absorbance was monitored at 412 nm for 2 min at 10 s intervals, using a uv spectrophotometer. the enzyme activity was expressed as micromole/min/mg of protein. molecular docking studies the crystal structure of acetylcholinesterase was obtained from the protein database file 4exy. the file was prepared by extracting solvent molecules, co-crystallized ligands (donepezil) and configured using chimera v 1.1 to model physiological conditions (28). added polar hydrogen and allocated partial charges to the regular residue using gasteiger partial charge means all the hydrogen atoms are specifically represented. however, the most favorable binding interactions were ascertained using autodock vina. the docking complex interactions were analyzed visually using discovery studio 2017 r2 client (v17.2.0.16349). autodock vina uses conformation-dependent algorithms to rate interactions binding ligands: c = ∑ 𝑓𝑡𝑖𝑡𝑗 𝑛 𝑖<𝑗 (𝑟𝑖𝑗 ) (1) in silico pharmacokinetic and toxicity studies some admet properties of quercetin was evaluated using admetlab platform iraqi j pharm sci, vol.30(2) 2021 role of nutraceuticals against cognitive impairment process 137 (http:/admet.scbdd.com/webserver/admetpredicti on) as defined by (29) jie et al. (2018). the physicochemical properties, distribution, and toxicity evaluation was performed using the canonical smile format of the respective compounds derived from the pubchem database. the study is based on a thorough exploration of a systematic database of 288,967 entries from different sources, including peer-reviewed journals, chembl, epa, and drugbank databases. all data were divided into six groups (basic, a, d, m, e, and t) and a set of subclasses by their endpoint meanings. molecular operating environment (moe, version 2016) has tested and evaluated the corresponding necessary details and experimental values of these entries, forming the basis for predicting a new compound based on computational similarity check. statistical analysis the findings were shown as mean ± sem. graphpad prism 5 software was used for statistical analysis. the disparity in treatment groups was examined using one way anova, and the difference was considered significant at p<0.05. microsoft office excel 2007 used to plot graphs. results antioxidant biomarkers treatment with lead acetate induced a sharp decrease in total thiol content [figure 2]. coadministration of lead acetate and quercetin was able ameliorate the decrease in total thiol content observed in a group treated with lead acetate alone. the activity of glutathione-s-transferase and catalase was also shown to be compromise by administration of lead acetate [figure 3 and 4]. quercetin alone as well as in co-administration with lead acetate improve the decline in activity of both glutathione-s-transferase and catalase. figure 2. effects of quercetin on total thiol concentration in lead treated drosophila.* = significant is when compared to normal control group. figure 3.effects of quercetin on glutathione-stransferase activity in lead treated drosophila melanogaster. * = difference significant when compared to normal control group. figure 4. effects of quercetin on catalase activity in lead treated drosophila melanogaster. * = difference significant when compared to normal control group. acetylcholinesterase activity the activity of acetylcholinesterase was drastically reduce due to lead acetate exposure, while co-administration with quercetin resuscitate its activity to optimum [figure 5]. the activity of the enzyme has also improved after administration of quercetin alone. figure 5.effects of quercetin on acetylcholinesterase activity in lead treated drosophila melanogaster. * = difference significant when compared to normal control group. iraqi j pharm sci, vol.30(2) 2021 role of nutraceuticals against cognitive impairment process 138 in silico pharmacokinetic and molecular docking studies pharmacokinetic showed quercetin to confer moderate aqueous and non-polar solubility, high therapeutic index, non-mutageni c and hepatotoxic properties as depicted in table 2. it also demonstrated excellent permeability based on epithelial colorectal adenocarcinoma cell-line (caco2-), while compound could remain in the body due to their low clearance rate. molecular docking studies reveal very motivating binding energy and inhibition constants of different interactions, as shown in table 3. the interactions included the polar and non-polar amino acid residue present in the acetylcholinesterase binding pockets [figure 68]. furthermore, there were hydrogen, hydrophobic, and other non-conventional interfaces between the compounds and the intended receptor. table 2. in silico toxicity and pharmacokinetic prediction profile on quercetin and its. category property (unit) predicted result inference / reference range qcn dpl basic physicochemical property logp (partition coefficient ) (log mol/l) 1.99 2.59 logp <0: poor lipid bilayer permeability. logp >3: poor aqueous solubility. logd7.4 (distribution coefficient d) (log mol/l) 0.14 0.79 <1: high solubility; 1 to 3: moderate solubility; ≥3: low solubility. absorption papp (caco-2 permeability) (cm/s) -6.17 -5.12 optimal: higher than -5.15 or -4.70 distribution ppb (plasma protein binding) (%) 94.9 87.3 90%: significant with drugs that are highly protein-bound and have a low therapeutic index. bbb (blood brain barrier) (%) 0.24 0.17 ≥0.1: bbb positive. <0.1: bbb negative. excretion clearance (ml/min/kg) 2.05 1.89 range: >15 high; 5< cl <15: moderate; <5: low. t1/2 (half life) (h) 0.20 1.08 range: >8h: high; 3h< cl <8h: moderate; <3h: low. toxicity h-ht (human hepatotoxicity) 0.56 0.64 >0.5: hht positive <0.5: hht negative ames (ames mutagenicity) 0.74 0.08 >0.5: positive <0.5: negative qcn = quercetin and dpl = donepezil table 3. binding energy and inhibition binding constant of quercetin against acetylcholinesterase receptor bound to donepezil (4exy). quercetin binding energy (µm) inhibition binding constant(kcal/mol) quercetin -10.1 0.981 donepezil -10.6 0.981 iraqi j pharm sci, vol.30(2) 2021 role of nutraceuticals against cognitive impairment process 139 figure 8. amino acids interactions and 3d conformations of quercetin within the binding pocket of acetylcholinesterase. qcn = quercetin, dpl = donepezil. discussions the function of oxidative stress and cholinesterase activity is one of the most verified hypotheses and try to explain the pathophysiology associated with neurodegeneration (30,31). oxidative radicals have been documented to play a critical role in promoting cellular damage, particularly in fatty tissues, and have been involved in neurodegenerative disease pathogenesis (32). intensifying evidence indicates that oxidative radicals may play a crucial role in the brain of patients with neurodegenerative diseases (33,34). upregulation of antioxidant biomolecules due to administration of quercetin observed in this study could be explained from two viewpoint. the first point could be linked to quercetin ability as an excellent antioxidant/metal chelator to donate a proton thereby quenching oxidative chain reaction (35). and the second reason is that quercetin might possess the ability to induce gene expression for the production of catalase, thiol and glutathione-stransferase. hydroxyl, carbonyl groups and resonance activity in flavonoids and other phytocompounds were detailed to be responsible for their antioxidant and chelation properties (36, 37). marija et al., (11) asserted that in most cases the capacity for antioxidants is directly proportional to the number of free hydroxyl groups. acetylcholinesterase (ache) is an enzyme of the α/β hydrolase-fold superfamily with a critical role in synaptic neurotransmission (38, 39). it is responsible for terminating the nerve impulses in cholinergic and neuromuscular synapses by separating the acetylcholine neurotransmitter into choline and acetate (40, 41). this influence results in downstream dpl qcn iraqi j pharm sci, vol.30(2) 2021 role of nutraceuticals against cognitive impairment process 140 modulation of striatal and hippocampal neurons of significance to motion, learning, and memory (42). hence given the aforementioned global advocacy for expanded use of nutraceuticals in managing various diseases, including neurodegenerative disorders, this study was conducted to highlight the efficacy of certain food-based phytocompounds; with alzheimer's disease, the activity level of ache enzyme declines and the level of butyrylcholinesterase activity increases in the brain (3). dual inhibition strategy on these enzymes has been reported most recently to increase the effectiveness of the treatment rather than to restore the initial balance. in this attempt, like in many current approach, we attempt to focus on restoration of the initial balance between acetylcholinesterase and butyrylcholinesterase by reactivation of acetylcholinesterase activity. finding in this work revealed quercetin to confer the potentials to induce the activity of acetylcholinesterase. this finding is in agreement with many works reported in the earlier studies (43). ulrike et al., (44) affirmed that development of bivalent ligands that occupy both the active and the peripheral site of acetylcholinesterase might be more beneficial for treatment of alzheimer´s disease than simple inhibition of the acetylcholine hydrolysis. molecular docking studies revealed the structures of quercetin could be potential acetylcholinesterase reactivators based on their binding site and activation constant. the activation binding process was validated by identifying the co-crystallized ache inhibitor donepezil. donepezil bind to acetylcholinesterase through different kinds of interactions which essentially involved phe295 and many other amino acid residues such as trp286, trp341, trp81, val294, gly121, arg296, phe297, tyr124, his447, gly448, glu202 and ser203, respectively. quercetin bind to acetylcholinesterase through various amino acid residue including those involved in its interaction with donepezil. omamuyovwi and augustine [38], shows kolaviron may be a novel herbal, medicinal product to treat neurodegenerative disorders associated with dysregulated cholinergic neurotransmitter systems. these compounds were further predicted to safe and physicochemically promising bioactive molecules. this finding correlate with the work reported by aliyu et al.,(45) , who predicted a great pharmacokinetic potentials of some phytocompounds. interestingly, quercetin could be transferred efficiently to their different targets via the bloodstream as proof of plasmabinding abilities. conclusion based on the present work, quercetin shows potentials ability to restore acetylcholinesterase activity in lead induced damage in drosophila melanogaster species. it’s further demonstrated the ability of up regulate the production of biological antioxidant molecules. by correlating the docking outcomes with experimental data obtained, it can be suggested that presence of quercetin alongside many other phytocompounds in food could be reasonable in the management of alzheimer’s diseases and related neurodegenerative disorders. further studies should be performed on other phytocompounds for the treatment of neurodegenerative disorders. declaration of interest the authors declared no competing interest with regards to the publication of this paper. funding no funding was received from any funding bodies. acknowledgement the authors wish to acknowledge the contributions of all technologies in biochemistry department ahmadu bello university zaria for their assistance and guidance towards successful completion of this work. we also wish to appreciate mrs. maryam aliyu of biochemistry department bayero university kano for improving the quality of this manuscript. reference 1. du, x., wang, x., geng, m., alzheimer’s disease hypothesis and related therapies. transl. neurodegener. 2018. 7 (1), 2 2. lleo a, greenberg sm, growdon jh: current pharmacotherapy for alzheimer’s disease. ann. rev. med. 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(2020). https://doi.org/10.1007/s40203-02000057-8 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://doi.org/10.1007/s40203-020-00057-8 https://doi.org/10.1007/s40203-020-00057-8 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 treatment regimens for covid 19 in iraq doi: https://doi.org/10.31351/vol31iss2pp223-232 223 highlighting the treatment regimens used in covid-19 epidemic in iraq with special regards to vitamin d zinah m. anwer*,1, ruaa natiq yahya* and noor mubdir khalf* * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract corona virus disease 2019 (covid-19) is a flu-like infection caused by a novel virus known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). severe cases of covid 19 may lead to organ failure and even death. the aims of this study are highlighting the management protocols and the supportive therapy (especially vitamin d), and manifesting the severity of the clinical symptoms of patients in iraq. a retrospective cross sectional study was conducted among 200 patients and their descriptive parameters for data were calculated to analyze the results. the mean age was 42.56±17.49 years and the majority of patients had mild to moderate symptoms (78%). there were many different pharmacological treatment regimens and different doses and durations of vitamin d. in conclusion, non-specific treatment protocols were used without compliance to the national guidelines for the treatment of covid-19 patients in iraq, and a wide range of pharmaceutical agents was administered without monitoring their safety and efficacy. vitamin d was administered in different doses and durations without depending on the basal serum concentration. key words: covid-19, vitamin d, treatment protocol, iraq. مع اهتمام خاص بفيتامين د 19تسليط الضوء على نظم العالج المستعملة خالل وباء كوفيد زينة مظفر أنور*،1، رؤى ناطق يحيى* و نور مبدر خلف العراق فرع الصيدلة السريرية ، كلية الصيدلة، جامعة بغداد، بغداد، * الخالصة المتالزمة 2( عدوى شبيهة باإلنفلونزا ناتجة عن فيروس جديد يُعرف باسم فيروس كورونا 19)كوفيد 2019فيروس كورونا يعد مرض ( الوخيمة الحادة تؤدي sars-cov-2التنفسية كوفيد (. من الشديدة الموت. الى 19الحاالت او األعضاء كوفيد تتمفشل مرضى 19معالجة الداعمة العالجات على ياستعمال الضوء تسليط إلى الدراسة هذه تهدف للمرض. نهائي عالج اليوجد ولكن فعالة تكون أن يُتوقع التي واألدوية أجريت العراق. في المرضى لدى السريرية األعراض شدة واستعراض د فيتامين وخاصة الداعمة العالجات عن فضال العالجات بروتوكوالت سنة 17.49± 42.56العمر وتم حساب المعلمات الوصفية للبيانات وتحليل النتائج. كان متوسط مريض 200على قطعية بأثر رجعيدراسة م ٪(. هناك العديد من أنظمة العالج الدوائي المختلفة وتم أخذ جرعات عشوائية على 78وغالبية المرضى عانوا من أعراض خفيفة إلى متوسطة ) تنتج من هذه الدراسة انه تم استخدام بروتوكول عالجي غير محدد للمرضى دون االمتثال لإلرشادات فترات مختلفة من فيتامين د من قبل المرضى. نس ولقد ,في العراق مع إعطاء مجموعة واسعة من العالجات الدوائية التي تتطلب المراقبة من أجل سالمتها وفعاليتها 19الوطنية لعالج مرضى كوفيد ت مختلفة دون االعتماد على تركيزه االبتدائي في مصل الدم.تم إعطاء فيتامين د بجرعات وعلى فترا البروتوكول العالجي، العراق.، فيتاميد د، 19الكلمات المفتاحية: كوفيد introduction the coronavirus disease of 2019 (covid19), caused by sars-cov-2, has caused a major impact on the global health and economics since its emergence at the end of 2019. on 29 october 2021, more than 245 million cases and over 4.9 million deaths have occurred globally since the start of the pandemic. in iraq 2,052,123 cases and 23,083 deaths have been reported till the date mentioned above(1). for mild cases, supportive treatment is used by providing continuing hydration, nutrition, vitamins, and antipyretic; whereas oxygen therapy was used for hypoxic patients in advanced stages and it required multiple pharmacological therapies(2). most of the pharmacological medications used for covid-19 were obtained from previous experience of drugs used during the (sars-cov) or middle east respiratory syndrome coronavirus (mers-cov) pandemics due to their expected activity with no definitive success of treatment yet(2). the united states food and drug administration (fda) advocated the therapeutic effect of many antiviral therapies(3,4). favipiravir, a polymerase inhibitor attracted attention for covid19 treatment due to its large spectrum of antiviral properties. as an oral drug, it is easy to be administered to asymptomatic or mild cases. early administration was found to be effective in reducing viral load, as well as improving the clinical and radiological outcomes(5,6). 1corresponding author e-mail: zina.ahmed@copharm.uobaghdad.edu.iq received: 12/9 / 2021 accepted:19 /1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp223-232 iraqi j pharm sci, vol.31(2) 2022 treatment regimens for covid 19 in iraq 224 remdesivir, and rebavirin proved their activity against covid 19(7,8). lopinavir, in combination with ritonavir, reported antiviral activity against sars-cov by reducing the viral load, improve the clinical symptoms and halt the progression of lung damage. they were used as an emergency treatment for covid-19 patients in usa, japan and other countries(9). chloroquine (cq) and hydroxychloroquine (hcq) are antimalarial drugs that have been proposed to be effective against covid-19(10). later, results from systematic review and meta-analysis studies on the two drugs indicated their low efficacy in reducing mortality or lowering the risk of hospitalization in outpatients(11). a study conducted by the who demonstrated that about 70% of covid-19 patients received antibiotics, although 8% only had an evidence of superimposed bacterial infections(12). at the beginning of the pandemic, unverified researches recommended the combined administration of chloroquine / hydroxychloroquine with the macrolide antibiotic azithromycin(13). another factor that encourages the use of macrolide antibiotics is the potential immunomodulatory effects causing down regulation of pro-inflammatory cytokines by an unknown mechanism(14). nowadays, the clinical course of disease, laboratory tests, and imaging are required to assess if the covid-19 patient has bacterial coinfection and requiring empirical antibiotic therapy(15). advanced disease may cause the release of high levels of pro-inflammatory cytokines (interleukins (ils), tumor necrosis factor (tnf)-α, c-reactive protein (crp), ferritin, and d-dimer. this is known as the cytokine storm, which requires the use of immunomodulatory drugs and can lead to sepsis, shock, respiratory failure, multi-organ dysfunction, and death(14). tocilizumab is a recombinant human monoclonal antibody and is considered as a therapeutic option for severely ill patients with extensive lung lesions and high il-6 levels(16). interferons (ifns) are cytokines with antivirals and immunomodulatory activity causing direct inhibition of viral replication and supporting an immune response to clear virus infection(17). however, more clinical studies are needed to prove their significance.(18). the plasma collected from healthy donors after recovery from a recent infection (convalescent plasmacp) contains high neutralizing antibody titer (nat), these expected to provide immediate passive short-term immunity and could prevent the infection without major safety concerns(19). on 24th march 2020, the fda approved the use of cp for patients with serious or immediately life-threatening covid-19 infections(20). many studies were conducted to assess the benefit of anticoagulation in covid‐19 patients and show that using low molecular weight heparin (lmwh) resulted in higher lymphocyte and lower il‐6 levels compared to control patients, indicating an improvement in coagulation parameters and normalization of immunity(21). supplements (like vitamin a, c, d, copper, selenium, zinc) are of great importance in the prevention of symptoms after viral or bacterial infections(22). a special concern was given to vitamin d due to its valuable impact on both innate and adaptive immunity, in addition to its role in the treatment of acute respiratory tract infections and other viral infections(23). yet, there is insufficient evidence on the association between vitamin d levels and covid-19 severity and mortality (24). it was observed that the covid-19 patients with vitamin d deficiency had poorer outcomes with longer stay in the intensive care unit (icu) and higher mortality rates(25). many reasons for vitamin d deficiency such as sun exposure time regarding to occupation, sun exposure surface area, old age and diet(26). old people also have comorbid diseases and take more than five medications and found to be prescribed a potentially inappropriate medication(27). although the exact level of vitamin d for its immunomodulatory action is unknown, however, it has been proposed that > 30 ng/ml is required to decrease the covid-19 severity and mortality(28). the aims of the study is highlighting the pharmacological treatments and supportive therapy (especially vitamin d) used by covid-19 patients in iraq, and manifesting the severity of the clinical symptoms presented during the course of the disease. patients and methods a retrospective, cross sectional, observational study was conducted among 200 patients who have recovered from covid-19 recently. the data collection was conducted from february till april 2021. convenience sampling technique was used in the study. patients from both genders at any age were included, they used vitamin d during the treatment of active disease and agreed to participate in the study. exclusion criteria were patients who did not take vitamin d during the infection, did not respond to phone calls, pregnant women, and those whose information were incomplete. the study protocol was approved by the scientific committee of the department and the college counsel, and it was conducted in accordance with the 1994 declaration of helsinki. verbal consent was also taken from each patient, in addition to ethical approval (no. 2709 in 14th jan. 2021) from the primary health care sector (aladel sector primary health care, directory of health alkarikh, ministry of health). data collected include patients’ characteristics, pharmacological regimens used, and the dosing of vitamin d supplement. in addition to information about covid 19 symptoms and iraqi j pharm sci, vol.31(2) 2022 treatment regimens for covid 19 in iraq 225 complications after the recovery were collected and used to classify disease severity as: patient with no symptoms, mild to moderate, severe and critical cases . data related to the infection (symptoms, and treatments) were obtained from patient data, and was recorded by the treating physicians in the primary care center and from the patients through phone calls (duration of sun exposure in minutes per day, and the complications after the recovery). the researchers from primary health care centers in baghdad / alkarikh and baaquba teaching hospital conveniently selected patient data sheets. all the collected data was assembled and analyzed using an excel spreadsheet. study findings were demonstrated by descriptive statistics. percentages and frequencies were used for the categorical variables, while the mean ± standard deviation was calculated for the continuous variables. results the characteristics of the patients are demonstrated in table-1. the study involved 200 patients [97 (48.5%) male, 103 (51.5%) female] and the mean age was 42.56±17.49 years (range from 4 to 89). about half of the patients (44.8%) had a body mass index (bmi) of 25-29.5, and 26.5 % of them had a second disease in addition to covid-19 and had a medication history. sun exposure for less than 15 minutes/day had the highest percentage between the patients (39%) and the rest was equally divided between the patients with sun exposure from 15-30 minutes/day and the patients with sun exposure ˃ 30 minutes/day. table 1. patients’ characteristics variable category frequency (n) percentage (%) age (years) 4-30 31-50 51-70 >70 58 66 52 24 29 33 26 12 body mass index 18.5-24.9 25-29.5 30-34.5 35-39.5 >40 43 87 49 14 1 22.2 44.8 25.3 7.2 0.5 comorbidities no yes 147 53 73.5 26.5 medication history no yes 147 53 73.5 26.5 diseases asthma copd heart failure renal failure hypertension diabetes mellitus cerebrovascular accident myocardial infarction anemia arrhythmia chronic bronchitis hypothyroidism sinusitis left bundle branch block rickets chronic tonsillitis 5 4 3 1 18 14 2 1 1 1 1 1 1 1 1 1 2.5 2 1.5 0.5 9 7 1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 sun exposure (minutes/day) <15 15-30 >30 78 59 63 39 29.5 31.5 disease severity was classified according to signs and symptoms as: patient with no symptoms, mild to moderate, severe and critical cases (figure 1) as mentioned in the patients methods section. the majority of patients (78%) had mild to moderate symptoms, and only 2.5% of them were asymptomatic. patients with severe symptoms constituted 12.5% of patients sample while those with critical disease were only 7%. iraqi j pharm sci, vol.31(2) 2022 treatment regimens for covid 19 in iraq 226 figure 1. severity of patients’ symptoms. pharmacological protocols used by patients are shown in table-2. different therapies were used and all of them included vitamin d3. other treatments used include: antipyretic (paracetamol), antibiotics (either: azithromycin, levofloxacin, meropenem, metronidazole, vancomycin, teicoplanin, amoxiclav, ceftriaxone or ceftazdime), antivirals (either: oseltamivir, favipiravir or remidisver), anticoagulants (either: enoxaparin or rivaroxaban), steroids (either: dexamethasone, prednisolone or hydrocortisone) and antimalarial (hydroxychloroquine) and their frequencies and percentages are presented in table2. information about vitamin d dose and duration is prsented in table-3. about half of the patients (51%) used vitamin d in a dose of 5,00010,000 iu/day. some patients used vitamin d as a single dose (9%), more than the half used it daily (55%) and the rest (36%) used it on weekly basis. vitamin d was used for more than two weeks among less than half of the patients (44.5%) and some (6.3%) continued to use it even after recovery. table 2. pharmacological protocols used for the patients. percentage (%) frequency (n) medications no. 12.5 25 vitamin d3 +vitamin c +zinc+ antipyretic 1 17 34 vitamin d 3+vitamin c +zinc+ antibiotics 2 9 18 vitamin d3+antibiotics +antiviral 3 6.5 13 vitamin d3+ antibiotics +aspirin 4 6 12 vitamin d3+vitamin c +zinc +ivermectin 5 6.5 13 vitamin d3+ antibiotics +antiviral +anticoagulant 6 4.5 9 vitamin d3+ antibiotics +anticoagulant +steroids 7 8 16 vitamin d3+antimalarial +antiviral 8 5.5 11 vitamin d3+aspirin +ivermectin 9 7 14 vitamin d3+ antibiotics +antimalarial 10 8.5 17 vitamin d3+antibiotic +steroids 11 5 10 vitamin d3+ antimalarial +ivermectin 12 4 8 vitamin d3+antiviral +antibiotic +antimalarial +anticoagulant 13 iraqi j pharm sci, vol.31(2) 2022 treatment regimens for covid 19 in iraq 227 table3.vitamin d3 dose and duration. patients dose (x 1000 iu) single daily weekly 10 200 300 < 5 5 10 ≤ 10 15 30 50 no. (%) 4 (2) 5 (2.5) 9 (4.5) 8 (4) 102 (51) 5 (2.5) 6 (3) 9 (4.5) 52 (26) duration single dose 1 – 2 weeks ˃ 2 weeks continuous no. (%) 18 (9) 80 (40) 89 (44.5) 13 (6.5) table-4 shows the complications presented by the patients. duration of symptoms for more than 10 days was experienced by about half of the patients (56%) and about the same percentage (56.6%) for those undergoing complication of the disease. the predominant complication was fatigue (20.5%) and there were rare symptoms of visual, rheumatologic, and hematological, reported in the study sample. table4. complications of the patients. type of complication frequency (n) percentage (%) fatigue 41 20.5 hair loss 18 9 hormonal complications 6 3 respiratory complications 26 13 visual impairment 2 1 reduce smell sensation* 11 5.5 reduce taste sensation* 6 3 gastrointestinal complications 8 4 rheumatological complications 5 2.5 cardiovascular complications 8 4 metabolic complications 11 5.5 hematological complications 2 1 depression 12 6 total 113 56.5 * long standing complication after recovery discussion management of covid-19 depends on the stage and severity of the disease, and since the virus replication is the highest before or soon after symptom appearance, antiviral medications are more effective when used early(29). hyperinflammation and coagulopathy are likely the cause of clinical complications. so, in later cases, anti-inflammatory, immune-modulators, anticoagulants, or combination of these treatments may be more effective than the antivirals. although, there is no approved therapy for covid-19, some medications have been shown to be beneficial(30). the results of this study showed that male to female ratio was about 1:1, while in another study it was found to be about 2:1(31). in most countries, data were reasonably similar, whereas in some countries the infection rate was higher in men than women(32). however, the available data from previous studies provides differences in severity and death rate being a little higher in men versus women. this could be attributed to many reasons like higher smoking rate, suppression of immune response by testosterone, and higher rate of respiratory tract infection in male(32). the highest rate of infected patients was found in age group ranging from 31-50 years. usually younger patients showed less symptoms or may be asymptomatic(33). additionally, the lower number of older patients who participated in this study may explain this rate. younger patients showed less susceptibility to infection due to high level of neutralizing antibodies, low levels of ace2 receptors in nasal epithelium, immature b and t cells and lower cytokines production, in contrast to that found in elderly patients which make them more susceptible to covid-19(34). the most prevalent comorbidities in this study sample were hypertension and diabetes mellitus seen in 9% and 7% of the patients respectively, while other comorbidities were distributed in lower percentages. a meta-analysis of six studies found that 17.1% of patients with covid-19 were hypertensive, 16.4% had cardiac/cerebrovascular iraqi j pharm sci, vol.31(2) 2022 treatment regimens for covid 19 in iraq 228 disease, and 9.7% were diabetic. these comorbidities are risk factors for infection and bad prognosis(34). most of the patients had mild to moderate symptoms (78%) and few patients were asymptomatic while those with severe and critical symptoms were 12.5% and 7% respectively. according to the iraqi national guidelines for management and treatment of covid-19 released in august-september 2020, the initiation of a treatment depends on clinical presentation of the patients and clinical judgment by the doctors(35). for mild cases, the treatment is mainly supportive with early starting with favipiravir, while the use of other antivirals or antibiotics depends on the patient case. moderate cases with manifestation of pneumonia required the early initiation of antiviral therapy (favipiravir or lobinavir/ritonavir) with consideration for possible administration of supportive therapy, antibiotics, prophylactic anticoagulants, and plasma. interferon and ribavirin initiation depends on clinical judgment. severe cases were managed by antiviral therapy of remdesivir, favipiravir or triple antiviral therapy (lobinavir/ ritonavir + ribavirin+ interferon). plasma therapy was considered at this stage and immuno-modulatory drug tocilizumab can be used for cytokine storm. empirical antibiotics, anticoagulant therapy and corticosteroid use should be well considered in severe cases. critical cases required mechanical ventilation or evidence of cytokine storm. critical cases managed as severe cases with ventilatory support and close monitoring(35). many different pharmacological protocols were used for treating the patients in the current study, in addition to supportive therapy (antipyretics, vitamin d, c and zinc). antivirals were used in about half of the regimens since the majority of patients in the study sample had moderate infection. oseltamivir is best initiated as early as possible and is useful in lowering the duration of fever when combined with antibacterial(36). favipiravir was included in the iraqi guideline for the management and treatment of covid-19 patients with mild disease even without the evidence of pneumonia. results from a metaanalysis study showed clinical and radiological improvements but no significant differences on viral clearance, oxygen requirement and side effect profile from other antivirals, and additional clinical studies are required to give a definite judgment on whether the treatment with favipiravir is the best option or not(37). remdesivir was considered as a promising agent for treating covid-19 patients, as it further provides a new clinical insight into an efficient and adequate treatment for covid-19 patients with the consideration of disease severity(38). an iraqi study emphasized that its early administration is accompanied with higher recovery rate, shorter residency in hospital and had a role in prevention of cytokine storm(39). there were 48 patients using the antimalarial hydroxychloroquine. it was used as an off-label drug from the beginning of the pandemic due to its anti-inflammatory activity in addition to decreasing of lupus anticoagulant levels and platelet activation that is responsible for thrombotic events associated with covid-19(40). on the other hand, a meta-analysis study focused on its gastrointestinal, ophthalmic and cardiac adverse effects which may lead to treatment discontinuation(41). “a strong recommendation against hydroxychloroquine for inpatients with covid-19 of any severity" was included in the who living guidance on 31 march 2021(42). taking a deep look at the results, antibiotics were used by more than half of the patients (63%) with different antimicrobial agents have been prescribed. extensive use of antibiotics could be due to the suspected bacterial pneumonia and evidence of super-infection that was accompanied with covid-19(43). however, inappropriate antibiotic prescribing can lead to bacterial resistance and other adverse effects. similarly, inappropriate antibiotic prescription is highly associated with low age of patients, less disease severity, dry cough, bilateral interstitial infiltrates, and increased c reactive protein. therefore, it is necessary to integrate an antibiotic use optimization program for covid-19 patients (43). ivermectin was found in the treatment regimen of 33 patients in the current study despite that it is not included in the treatment protocol of covid-19 patients in iraq. it is an antiparasitic agent known from the 1970s and was discovered later to have an antiviral activity and was effective against certain flavivirus and chikungunya virus. recently, it was assumed to be effective against coronavirus by inhibiting importin α/β mediated transport system of viral proteins demonstrated in vitro studies which lead to off-label use for covid19(44). some studies showed that it was associated significantly with lower covid-19 deaths(45,46), especially in patients with severe pulmonary involvement (47). however, the who living guideline on 31 march 2021 recommended its use only in the context of clinical trials, since there is little evidence about the effects of ivermectin on mortality, mechanical ventilation, hospital admission, duration of hospitalization and viral clearance. data collected about vitamin d administration in the current study showed its unplanned use. this wide range of doses used should be taken into consideration and in required readjustment to meet the body needs and prevent toxicity from overdose. vitamin d intake is recommended in many countries and the who iraqi j pharm sci, vol.31(2) 2022 treatment regimens for covid 19 in iraq 229 recommended a daily intake of 5 μg (200 iu) for adults but rising to15 μg (600 iu) over 65 years. an upper limit of 4000 iu /day in adults is relevant to european food standards agency, uk scientific advisory committee on nutrition and us institute of medicine(48). older age, especially those with little sun exposure require a daily intake of 10–20 μg (400–800 iu /day) since they may have a lower response. it is critical to determine a baseline vitamin d status and target serum concentration to identify the appropriate supplementation dose (48). vitamin d level of >50 nmol/l is recommended by the us institute of medicine and achieved by 1000 iu/day dose and it is adequate even for obese individuals while it should be between 3000 4000 iu/day to achieve serum concentration of >75 nmol/l(49). it was suggested that long-term exposure to doses of 25 μg (1000 iu) may be well tolerated, and higher exposures such as 45 μg (1800 iu) may be tolerated for short-term and under medical supervision, while a toxicity threshold of daily intakes ranges between 250 μg 1000 μg ((10,000 iu40,000 iu)/day(50). vitamin d deficiency is a well-known global health problem and has negative impact on respiratory viral diseases such as covid-19 in both children and adults. vitamin d supplementation could potentially be effective in the treatment and prevention of covid-19 particularly in those with risk factors and chronic diseases(51).the role of vitamin d in covid-19 has been hypothesized since it may prevent acute respiratory infections (aris) by activating the innate and adaptive immune system, antioxidant activity and inhibition of renin-angiotensin system. these proposals lead to several randomized controlled trials trying to prove them but are still not convincing(50). in this study, the results could not find a direct link between the dose of vitamin d and disease prognosis since it was not the sole therapy used, and the improvement in symptoms and recovery could be due to antiviral, immunomodulatory drug or other supportive therapy involved in the treatment protocol. another explanation is the different regimen of vitamin d. results from previous studies were ambiguous. two meta-analysis studies found there were significant relations between vitamin d concentration and covid-19 infection, severity and mortality (52,53). in contrast, another meta-analysis that could not approve the association between vitamin d deficiency and greater covid-19 severity reveal that no causal relationship has been tested until now and blood vitamin d levels had not been evaluated in covid-19 patients (54). the most predominant complication among the study sample was fatigue, respiratory complications and hair loss. more than half of the patients (56%) had symptoms for more than 10 days, which may reflect the same proportion of patients who experienced complications (56.5%). some patients (even those who had mild versions of the disease) continue to experience symptoms after their initial recovery, and they may describe themselves as "long haulers" and the conditions have been called post-covid-19 syndrome or "long covid19."(55) by a variety of mechanisms, the lungs are the organs most affected in covid-19(56), however non-respiratory complications have also been reported in the current study sample. conclusions a wide, non-specific treatment protocols are used for the patients without compliance to the national guidance for management and treatment of covid-19 patients in iraq with administration of a wide range of pharmaceutical agents that usually requires monitoring for their safety and efficacy. vitamin d administered in different doses and duration without depending on the basal serum concentration and after initiation of the treatment. limitation of the study patients’ vitamin d serum levels were not measured, while measuring them could be helpful to explain the wide range of doses used by the patients and to determine their true requirement to achieve better outcome and to overcome overdose administration. the evidence is currently inconsistent and insufficient due intensive care unit admission, inflammation, and pneumonia; further studies are required to evaluate the role of vitamin d supplements on treatment and management of covid-19 patients. acknowledgment the authors would like to express their thanks to abdul-hakeem bassam for his support in implementing this study. the authors would like also to thank all the health professionals and patients for providing the necessary information reported in this study and giving precise insight into the situation of covid 19 treatment in iraq. conflict of interest: none funding and support: none references 1. hannah ritchie, edouard mathieu, lucas rodés-guirao, cameron appel, charlie giattino, esteban ortiz-ospina, et al. max (2020–2021). 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*ministry of health and environment, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract cataract, which is the opacity inside clear ocular lens of eye, result in the scattering of visible light as it passes via the lens and consequently deterioration in optical image. the aim of the present study was to investigate whether safranal, an active constituent of crocus sativus l. stigmas, has a protective effect on the cataract in the rat's pups. the animals were randomly divided into five groups, each of which consisted of 7 rat pups. group i served as normal control (vehicle administration). for testing cataract induction, animals of groups ii, iii, and iv were administered a single subcutaneous injection of sodium selenite on postpartum day 12. after sodium selenite intoxication, group ii served as control selenite, groups iii-iv received intraperitoneal safranal at doses of 200, and 300 mg/kg, respectively from the 11th day through the 17th day, while group v receive only safranal (300 mg/kg). on postpartum day 30, the rat pups were examined for cataract formation, and the lenses were isolated for further analysis. this study found that selenite caused significant (p < 0.05) cataract formation. through the effects of selenite on the level of lipid peroxidation (mda) which was upregulated. furthermore, the antioxidant enzymes levels gsh levels and nuclear factor erythroid 2-related factor 2 (nrf2) protein were downregulated. in contrast, treatment with safranal could significantly (p < 0.05) ameliorate cataract formation and oxidative damage in the lens. moreover, safranl administration significantly increased the protein expressions of nrf2 and the gsh level, in addition to reducing the level both the mda and the level soluble proteins in the lens. taken together, safranal is a prospective anti-cataract agent that probably delays the onset and progression of cataracts induced by sodium selenite. keywords: cataract, safranal, sodium selenite, oxidative damage التأثير الوقائي للسفرانال ضد تولد الماء االبيض في العين المستحث بواسطة السيلينيت في الجرذان و مناف هاشم زلزلة 1*،فرح عبد الكريم مهدي وزارة الصحة والبيئة ، العراق * ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . االدوية والسموم ** فرع الخالصه تدهور وبالتالي العدسة عبر مروره أثناء المرئي الضوء تشتت عنه ينتج ، الصافية العين عدسة داخل التعتيم وهو ، العين عدسة إعتام في الساد على وقائي تأثير له ، لنبات الزعفران النشط المكون ، كان السفرنال إذا ما معرفة هو الدراسة هذه من الهدف كان. البصرية الصورة في كعنصر األولى المجموعة خدمت الجرذان صغار 7 من تتكون منها كل ، مجموعات خمس إلى عشوائي بشكل الحيوانات تقسيم تم. الفئران صغار اليوم في الصوديوم سيلينيت من الجلد تحت واحدة حقنة والرابعة والثالثة الثانية المجموعات حيوانات إعطاء تم ، الساد تحريض الختبار. مراقب على كجم/ مجم 300و 200المجموعة الثانية خدمت كنموذج للساد. المجموعة الثالثة والرابعة تلقت جرعة من السافرنال .الوالدة بعد عشر الثاني ، الوالدة بعد 30 اليوم في(. كجم/ مجم 300) السفرنال فقط الخامسة مجموعةال تتلقى بينما ، عشر السابع اليوم حتى عشر الحادي اليوم من التوالي العين عدسة إعتام تكوين في تسبب السيالنيت أن الدراسة هذه وجدت. التحليل من لمزيد العدسات عزل وتم ، الساد لتشكيل الفئران صغار فحص تم مستويات تقليل تم ، ذلك على عالوة (.المالوندي الديهايد) الدهون بيروكسيد مستوى على السيلينيت تأثيرات خالل من(. p <0.05) كبير بشكل باستخدام العالج يؤدي أن يمكن ، ذلك من النقيض على الكلوتاثيون والعمل النووي المتعلق بالكريات الحمر ومستويات األكسدة مضادات إنزيمات في كبيرة زيادة إلى أدى تناول السفرانل ، ذلك على عالوة(. p <0.05) كبير بشكل العدسة في التأكسدي والضرر الساد تكوين تحسين إلى السفرنال ومستوى المالوندي الديهاي من كل مستوى تقليل إلى باإلضافة الكلوتاثيون، العمل النووي المتعلق بالكريات الحمر ومستوى فعالية مستويات ظهور يؤخر أن المحتمل من والذي العين عدسة إلعتام مضاد محتمل عامل هو فان السفرنال ، معًا أخذنا إذا. العدسة في للذوبان القابلة البروتينات الصوديوم. سيلينيت عن الناجم العين عدسة إعتام وتطور مضادات االكسده. ،صوديوم سلينيت ،سفرنال ، الكلمات مفتاحيه:اعتام عدسة العين)الماء االبيض( introduction cataract, which is the opacity inside clear ocular lens of eye, result in the scattering of visible light as it passes via the lens and consequently deterioration in optical image (1). cataract is the world's leading blindness condition. who has valued that by the year 2020 there will be 54 million blind individuals aged 60 years or older (2). it's causing a huge socio-economic burden (3). while cataract surgery has become one of the most common cataract therapies, many risks and complications still occur (4). if the onset of clinical cataract can be delayed for 10 years, it is estimated that half of the cataract surgery will not be necessary (5). 1corresponding author e-mail: munafzalzala@copharm.uobaghdad.edu.iq received: 24/ 9 /2020 accepted: 24/ 11 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp196-203 iraqi j pharm sci, vol.30(1) 2021 safranal and cataract 197 the most powerful initiating factor for cataract development is the damage caused by the generation of reactive oxygen species (ros), and exacerbated by oxidative stress / endoplasmic reticulum stress (6, 7). lens distortion is a direct result of oxidative stress, when aging occurs. abnormalities in the normal state of redox cells can cause toxic effects by generating peroxides and free radicals which damage all cell components, including proteins, lipids and dna, the redox cycles are important for maintaining lens transparency (8) . nuclear factor erythroid 2-related factor 2(nrf2), a main antioxidant protection manager, can monitor gene expression of antioxidant; nrf2 translocates a nucleus from cytoplasm and thus attaches to antioxidant response elements (ares), stimulated by exogenous oxidative stress. a process that causes enhances the expression of antioxidant product, which finally strengthens the antioxidant defense mechanism intracellular. the development of lens opacification is evidently linked to the lack of action on antioxidant enzymes. when the development of the antioxidant enzyme increases, cataractogenesis may be stopped or postponed (9). the sodium selenite is a cataractogenic agent widely used since 1978 in animal models (10). this causes cataract development when administered to young rats at nearly 16 days of age before the completion of the essential lens maturation duration (11). biochemical processes, including modified epithelial metabolism, accumulation of calcium, proteolysis caused by calpain, precipitation of crystalline, phase transition, loss of the cytoskeleton , and nrf2 suppressors which inhibition the nrf2-dependent antioxidant protection in lenses occur during the cataract development can be induced by selenite (11, 12). the reaction of selenite with reduced gsh has proven oxygen active production (13). various antioxidants such as vitamin c prevent this oxidative stress and the cataract induced by selenite (14). safranal (2,6,6 –trimethyl -1 ,3 – cyclohexadiene -1 carboxaldehyde) is one of the organic compounds that isolated from saffron(crocus sativus)(15). crocus sativus l. (saffron) is utilized as a coloring and flavoring agent in the food preparation in numerous areas of the world, also as healing agents against several medicine disorders (16). safranal has reported to have several pharmacological activities involving antioxidant, anti-inflammatory, anti-apoptotic, antiischemic, anticonvulsant, antidepressant, and anxiolytic effects (17). safranal as an antioxidant in biological environments has benefited from its ability scavenge of free radical and reduce the oxidative stress of unsaturated phospholipid membrane (18). in this study, we evaluated if intraperitoneal safranal administration avoids cataract development in a selenite-induced experimental cataract rat's model, and the status of gsh as antioxidant and malondialdehyde (mda) as a lipid peroxidation indicator in extracted rat lenses. materials and methods chemical and kits all chemicals used within the experimental were available in the highest degree of purity. safranal and sodium selenite obtained from (sigma chem. co., st. louis), diethylether and liquid paraffin from (bdh chemicals /england). the kits used in this study include [rat glutathione (gsh) and malondialdehyde (mda) elisa kit] were bought from (sunlong biotech co. /china) while rat nuclear factor –e2 related factor 2(nrf2) elisa kit from (my biosource /usa), and bradford protein assay kit from (gene copoeia /usa). animal and experimental model neonatal rat's pups of wister albino strain each one weighting 12-16 gm on postpartum 8 days. the pups were housed in polypropylene cages in rooms held at 25±1°c along with their mother. the rats were maintained on a standard laboratory animal diet, providing water ad libitum during the experimental period and a daily 12h cycle during the test period: 12 h mild-dark cycle. the animals were handled according to regulations approved by ethical committee in university of baghdad for using the animals in research. a suckling rat pups and their mother were divided in to 5 group one control group and four experimental groups, each group consist of 7 animals as follows: group i (control group): rat pups injected subcutaneously with normal saline at day ten postpartum with intra-peritoneal injection of liquid paraffin at day 8 postpartum for 30 days. group ii (model group): rat pups injected subcutaneously with single dose of sodium selenite at day ten postpartum with daily intra-peritoneal injection of liquid paraffin at day 8 postpartum for 30 days. group iii: rat pups injected subcutaneously with single dose of sodium selenite at day ten postpartum with daily intra-peritoneal injection of safranal 200 mg/kg at day 8 postpartum for 30 days group iv: rat pups injected subcutaneously with single dose of sodium selenite at day ten postpartum with daily intra-peritoneal injection of safranal 300 mg/kg at day 8 postpartum for 30 days group v: rat pups injected subcutaneously with normal saline at day ten postpartum with intraperitoneal injection of safranal 300 mg/kg at day 8 postpartum for 30 days. iraqi j pharm sci, vol.30(1) 2021 safranal and cataract 198 morphological evaluation of rat pups lenses: using an ophthalmoscope or with a naked eye, the lenses were visualized starting from the day they opened their eyes (14-16 days after birth). the ophthalmoscope assessed opacity gradation weekly. the pupils were dilated to induce mydriasis using a topical tropicamide ophthalmic eye drop. the lens opacity score (using ophthalmoscope grading criteria) was performed classification(19): grad 0: clear normal lens; grad 1: subcapsular opacity; grad 2: nuclear cataract; grad 3: strong nuclear cataract with perinuclear area opacity; grad 4: matured nuclear cataract. preparation of lenses samples on postpartum day 30, all animals were anesthetized and euthanized by cervical rupture. lenses were isolated intracapsulay via incision 2mm posterior to limbus, lenses without their capsules were weight then separated pair of lenses were homogenized ten times their mass with phosphate buffer ph (7.4) by homogenizer. the supernatant was obtained by 15 minutes centrifugation of homogenate at 5000 rpm. supernatant was stored for analysis of gsh, mda level and nfr2 activity and protein content at -80 c in aliquots. all these assays were done in standard condition (10). statistical analysis all the values in this study are recorded as the mean ± standard deviation, and the statistically significant differences have been determined by unpaired student’s t-test for comparisons between groups by usage of the statistical package for social sciences spss (version 26.0) the program (spss, chicago, il, usa). p-values below 0.05 have been considered statistically significant. results effect of safranal on the opacification and morphology of the lenses in the sodium selenite induced cataractogenesis morphological examination of both eyes of each rat pup was done by ophthalmoscopically observation on the 30th day after birth (figure 1). all pups of rats in the cataract model group (which obtained s.c. sodium selenite injections) demonstrated an advanced opacification of the lenses (opacity grade 4). interestingly, the study revealed that 4 out of 7 (57.1%) of rat pups pretreated with 300mg/kg of safranal decreases the number of rat pups with lenses opacification to 2 out of 7 (28.5%). those two pups showed moderate lenticular opacification grade 1 and grade 2; while the other five pups' lenses were appearing regular (grade 0). on the other hand, only 4 out of 7 (57.1 percent) in group iii rat pups (which provided i.p. safranal 200mg/kg injections + sodium selenite) showed lenticular opacification grade 1(n=2), grade 2 (n=1), and grade 3(n=1) , while the other three pup lenses looking regular (grade 0, n=3). moreover, all pups in group i (which provided normal saline as control) showed full transparency of the lens (grade 0) as show in (table 3-1). all lenses were transparent in control group (group i). however, all lenses were developed cataract in model group (group ii). no cataract occurred in the remaining lenses of the treated group as shown in (table 1). table 1. morphological examination on the opacification of rat lenses. number of pups with lenticular appearance the number of pups with varying grads of opacification pups number group of experimental grads 4 3 2 1 0 0 7 _ _ _ _ 7 group i (control) (100%) all 7 pups _ _ _ _ 7 7 group ii (sodium selenite) 4 out of 7 (57.1%) 1 1 2 3 7 group iii (sodium selenite + safranal 200mg/kg) 2 out of 7 (28.5%) _ _ 1 1 5 7 group iv (sodium selenite + safranal 300mg/kg) 0 7 _ _ _ _ 7 group v (safranal 300mg/kg) group i rat pups received only saline; group ii rat pups received only selenite; group iii rat pups received selenite and safranal (200 mg/kg); group iv rat pups received selenite and safranal (300 mg/kg); group v received safranal only. the degree of opacification was graded as follows: grade 0: was normal clear lens; grade 1: subcapsular opacity; grade 2: nuclear cataract; grade 3: strong nuclear cataract with perinuclear area opacity; grade 4: mature dense opacity involving the entire lenses. iraqi j pharm sci, vol.30(1) 2021 safranal and cataract 199 figure 1. figure represent pictures were taken at week 4 (1 day before sacrifice) of experiment. panel a: clear lens with no identifiable opacity panel b: lenses from na2seo3 group exhibited mature cataracts panel c: lenses from safranal (200mg/kg) + na2seo3 group exhibited lower grade of cataracts. panel d: lenses from safranal (300 mg/kg) + na2seo3 group exhibited lower grade of cataracts. panel e: lenses from safranal group exhibited similar results as that of control, with no detectable cataracts. effect of safranal on the gsh and mda level in the sodium selenite induced rat's cataract model. in this study, we measured the activities of the antioxidant enzyme gsh and mda levels in the lenses, and the results are shown in figures 2 a and b. glutathione is an important nonenzymatic antioxidant in the detoxification pathway that downgrades the reactive toxic metabolites of selenite. the value of gsh in the lenses of selenitetreated group were significantly lower (p < 0.05) than in the normal control group. conversely, treatment with safranal at doses of 200 mg/kg or 300 mg/kg increased the percentages of gsh significantly by 32% and 56% respectively, compared with the selenitetreated group (figure 2a). moreover, safranal only treatment at doses of 300 mg/kg significantly increase the level of gsh in rat's pubs by 14 % compared to the control group (p < 0.01). (figure 2a). additionally, the mda is the general biomarker that appears during the lipid peroxidation of polyunsaturated fatty acids in the biological membrane. the results of the mda examinations of the lenses are also shown in figure 2b. the mda concentrations in the selenite-treated group were significantly higher than in the control group (p < 0.001). however, the administration of safranal significantly reduced selenite-induced lipid peroxidation in the lenses. the mda concentrations in both groups of safranaltreated with a dose of 200 mg/kg or 300 mg/kg were at least 25% and 31 % respectively; which are highly significantly lower than they were in the selenite-treated control group (p < 0.001). these results suggested that the lipid peroxidation indicator induced in the lens were effectively inhibited by safranal. a. b. figure 2. effects of safranal on lens gsh and mda in lenses of rat pups treated with sodium selenite.. (a) gsh (b) mda. data are presented as the mean ± standard error of the mean for 7 rat pups. * p < 0.05 compare with control group; # p < 0.05 compare with sodium selenite-treated group. iraqi j pharm sci, vol.30(1) 2021 safranal and cataract 200 effect of safranal on the nrf2 level in the sodium selenite induced rat's cataract model. next, we studied the nrf2 level after safranal treatment in the sodium selenite induced rat's cataract model. the nrf2‐keap1 system is known as one of the main cellular defense mechanisms against oxidative stresses. as shown in figure 3, the value of nrf2 in the lenses of selenite-treated control group were significantly lower (p < 0.05) than in the normal control group. conversely, treatment with safranal at doses of 200 mg/kg or 300 mg/kg increased the percentages of nrf2 significantly by 18 % and 19% respectively, compared to the selenitetreated control group (p < 0.05). (figure 3). figure 3. effects of safranal on lens nrf2 in lenses of rat pups treated with sodium selenite. data are presented as the mean ± standard error of the mean for 7 rat pups. * p < 0.05 compare with control group; # p < 0.05 compare with sodium selenite-treated group. effect of safranal on the soluble protein level in the sodium selenite induced rat's cataract model at the end of the experiment, the level of soluble protein was measured. as shown in figure 4, a significant decrease (p < 0.05) in the amount of soluble protein in selenite treated group was observed, as compared to the normal control. furthermore, treatment with safranal (200 mg/kg or 300 mg/kg) in the sodium selenite intoxicated rat pups significantly increased (p < 0.05) the soluble protein content by 9% and 10 % respectively. figure 4. effect of safranal on the soluble protein level in lenses of rat pups treated with sodium selenite. data are presented as the mean ± standard error of the mean for 7 rat pups. * p < 0.05 compared to control group; # p < 0.05 compared to sodium selenite-treated group. discussion the lens is extremely sensitive to attack from ros. a number of researches indicate that antioxidant-potential medicinal herbs may protect the system against ros toxicity. previous findings suggest ros is responsible for the formation of selenite cataracts (20). the crystalline of the lens contains a huge amount of gsh, which prevents oxidant degradation of the lens and impairment in redox cycle. the level of gsh reduces in nearly all forms of cataracts. it can, therefore be proposed that gsh shows an essential impact in maintaining the clarity and consistency of the lens and this relates to both in the defense against the protein sulfhydryl groups from oxidation and it preserves cytoskeletal proteins and avoids the crosslinking of soluble crystalline (21, 22). approaches have been investigated in recent years to determine the possible positive effects of safranal in ophthalmology, despite the increasingly growing literature on the neuroprotective effect of safranal in retinal degeneration through inhibiting photoreceptor cell degeneration (23, 24), no studies have been performed to determine its potential effects on cataract development. in this study, morphological analysis showed that the development of cataracts induced by selenite administration had been attenuated by safranal administration, indicating its potential for treatment of cataract. in addition, biochemical analyzes were carried out to support the clinical study. the depletion of gsh in the selenite cataract is a non-enzymatic reaction of gsh with selenite forming a derivative, selenodiglutathione (gs-se-sg), superoxide anion is produced as an intermediate in oxidation (25). maintenance of gsh level in lens may help to maintain its defensive iraqi j pharm sci, vol.30(1) 2021 safranal and cataract 201 ability against oxidative damage and delay the onset of cataract (26). in the current study, the gsh content was significantly lower in lenses of rat pups that exposed to sodium selenite compared to the control. in comparison, rat pups treated with safranal showed significantly higher gsh content in their lenses than untreated pups (figure 2). these findings show that safranal reduces selenite-induced cataractogenesis in rat pups, suggesting that safranal pretreatment maintains the lens gsh at acceptable rates and attenuates the production of superoxide and more oxidative against oxidative stress. consistent with these data, previous studies found safranal elevates the plasma gsh level which could increase the ratio of gsh / gssg and decline lipid peroxidation in different tissues (27-31). in addition, cataractogenesis involves oxidative stress-mediated lipid peroxidation, because the accumulated peroxidation products caused fragmentation of the soluble lens protein and damage the structure of membrane, correlating with increased opacity of the lens (32, 33). accordingly, both mda (the principal metabolite formed by lipid oxidation) and soluble proteins levels were evaluated next in the current study. as shown in (figure 2 b), the level of mda was significantly increased in the selenite cataract group as compared to control group. that may be related to the lipid membrane degradation induced by sodium selenite. interestingly, in both of the safranal treated group, amount of mda was reduced in a dose dependent manner suggesting its defensive effect against catractogenesis. our finding are compatible with the previous studies that stated safranal was seen as preventing both a decline in antioxidant enzyme activity and lipid peroxidation in different disease models (34-37). subsequently, our data indicates that safranal maintained the lens' architectural and thus avoided opacification of lenticular. furthermore, the soluble protein content of the lens was found to be significantly lower in rat pups that were exposed to sodium selenite compared rat pups in the control, as shown in figure 3, because sodium selenite responsible for the formation of insoluble high molecular weight misfolded proteins that suggest an intermediate level of water-soluble conversion to water-insoluble proteins. in contrast, rat pups that treated with safranal showed significantly more soluble protein content in its lenses than selenite induced group, pre-treatment with safranal will prevent the oxidation of these proteins and protein aggregates in a dose dependent manner, so cataract progression can be delayed (34). furthermore, the current study evaluate nrf2 level because it is a central factor in nuclear transcription that regulates over 200 genes related to stress which include 20 antioxidant genes. therefore, nrf2 inducer may also serve as an anticataract compounds (38). as shown in figure 3, mean levels of nrf2 significantly lower in rat pups that exposed to sodium selenitegroup, compared to rat pups in the control. however, the safranal treatment showed a significant raise of the protein expression of nrf2 in a dose dependent manner compared to sodium selenite induced cataract group, suggesting that safranal could maintain the activity of gsh directly or indirectly via nfr2 expression and kelch-like ech-associated protein 1(keap( modification in sodium selenite-damaged lens. previous findings from previous research found that safranal defends against rotenoneinduced neurotoxicity via nrf2 signaling pathway and assumed that safranal may serve as a potent therapeutic drug in the treatment parkinson's disease (39). accordingly, the current study shows that nrf2 induced by safranal helps maintain glutathione levels, which act as a buffer to accumulate reactive oxygen species during the unfolded protein response. conclusion this study revealed that safranal possess a potent anticataractogenic effect, which may be due to its excellent antioxidant ability. this preliminary analysis is promising, however further researches are needed to 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international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsion doi: https://doi.org/10.31351/vol29iss1pp143-153 143 formulation and characterization of isradipine as oral nanoemulsion noor a. sadoon*,1 and mowafaq m. ghareeb** * ministry of health and environment, baghdad, iraq. * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract isradipine related to dihydropyridine (dhp) class of calcium channel blockers (ccbs). it is used to treat hypertension, angina pectoris, as well as parkinson disease. it goes under the bcs class ii drug (low solubilityhigh permeability). the drug will experience extensive first-pass metabolism in liver, thus, oral bio-availability will be approximately15 to 24 %. the aim of the study is preparing stable oral oil in water (o/w) nanoemulsion of isradipine to promote the colloidial dispersion of isradipine in the nano range, so that it may be absorded by intestinal lymphatic transport in order to avoid hepatic first-pass metabolism (israpidine undergoes 15-24% first pass metabolism) and increase drug bioavailability. the solubility study was carried out in various vehicles for selecting best solvent for dissolving isradipine. pseudo-ternary phase diagrams is formed at (1:1, 1:2, 1:3, 1:4 and 2:1) ratios related to smix (cosurfactant and surfactant). there are 11 nano-emulsion was prepared through the use of many concentrations of ( transcutol, tween20, and triacetin). all formulations assessed for (in-vitro drug dissolution, ph measurement, viscosity, drug content, polydispersity index, particle size distribution, thermodynamic stability, dye test, and light transmittance). it is indicated that the extent as well as the rate of release regarding all the prepared formulations have considerably higher in comparison to that of crude drug powder. results of characterization were explained that isradipine nano emulsion (ne9) with s mix(1:4) : oil : deionized water (40: 5: 55) ratio was the optimized formula that has droplet size range (177.1nm), the lowest value of polydispersity index (0.12), the highest percent of drug content (99.7%) typical ph value for oral administration (5.21) , good percent of light transmittance (99.86 %), the range of viscosity (65.12 -25.2 m pas. sec.) was suitable for oral administration, also the isradipine’s in vitro release has been considerably higher. it can be concluded that nano emulsion drug delivery system (dds) can be considered as an encouraging method for improving the stability, dissolution and solubility of formulation. keywords: nanoemulsion, isradipine , peudoternary phase diagrams. يعطى عن طريق الفم دقيق نانوي سائل كمستحلبأسرادبين وتقييم إعداد **موفق محمد غريب و 1*،سعدون عبد هللانور .العراق ،، بغدادالعراقيةوزاره الصحة والبيئة * العراق، بغداد بغداد، جامعة الصيدلة، كلية الصيدالنيات، فرع** الخالصة .ينتمي لألدوية الشلل االرتعاشي،الصدرية الذبحة الضغط،يستخدم لعالج يريدين مغلق لقنوات الكالسيومايدروبايهاد 4,1رادبين هو سا وبان في الماء .المصنفة من الدرجة الثانية )الذوبانية قليله ,النفاذية العالية( حسب نظام تصنيف الصيدالنيات البيولوجي ,لذلك فانها غير قابله للذ الذوبان، وهذا لتعزيز نانوي االستحالب سائل نظام بشكل أيزرادبين إعداد الى نهدفوبالتالي .%24-15التوافر البيولوجي الفموي هو ميقارب السواغات أفضل لتحديد مختلفة مركبات في الذوبان قابلية دراسة أجريت للدواء جيد امتصاص على الحصول سيتم لذلك الحل، معدل زيادة إلى يؤدي من نسبة الفاعل بالسطح و المادة الخافضة للتوتر ( 1:2و 4: 1 3: 1، 2: 1، 1: 1في)الزائفة الثالثيةالمخططات الطورية تم بناء .أسرادبين وترانسكيوتول . تم تقييم جميع التركيبات المعدة 20مختلفة من زيت الترايستين , توين عشر تركيبات باستخدام تراكيز ىتحضير أحدالسطحي. تم الصبغة مع الماء ، ، مقياس درجة امتزاج، اللزوجةالثيرموديناميكي, االستقرار توزيع حجم الجزيئات، مؤشر التشتت المتعدد، محتوى الدواء،) , خليط %5زيت الترايستين ( الذي يتكون من )(ne9 اديبينسراإل النانوينتائج دراسة الخواص توضح بان المستحلب .الضوء نفاذية, الحموضة ,هي الصيغه االمثل حيث تمتلك %55وماء منزوع االيونات %40سطحي على تقليل الشد ال والمادة المساعدةالتي تقلل الشد السطحي المادةمن (, نسبه %99.7محتوى الدواء ) (5.2عن طريق الفم) لإلعطاء(, قيمه الرقم الهيدروجيني 1.21مؤشر التشتت ),( نانومتر177.1حجم كروي ) عن طريق الفم وكان التحرر المختبري لإلعطاء( مناسبا ثانية. باس م 25.265.12) (,نطاق اللزوجة%99.86مئويه جيده من نفاذية الضوء) قابلية الذوبان واالنحالل واستقرار هو نهج واعد لتحسين يخلُص إلى أن نظام توصيل الدواء بالمستحلب النانو التحرر،لدواء االسرادبين عالي .التركيبة الثالثية الزائفة المرحلة مخططمستحلب سائل نانوي, أسرادبين المفتاحية:الكلمات 1corresponding author e-mail: noorabdallah9089@yahoo.com received: 21/ 9 /2019 accepted: 23/ 11 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp143-153 iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsions 144 introduction low dissolution rate and partial absorption will be generally seen in drugs with low permeability and low water solubility (1). pharmaceutical researches focus on two fields to improve the oral bio-availability, which are: (i) enhance the dissolution rate as well as the solubility for drugs with low water-solubility, (ii) enhance the permeability related to drugs with low permeability (2). solubility can be defined as a major parameter used for the purpose of achieving the required drug’s concentration in a systemic circulation for pharmacological response (3). several methods were modified for resolving the drug’s poor aqueous solubility (4). there is high permeability and poor water solubility in the class ii drugs according to bsc (5,6). distinctive approaches for the drug’s solubilization are hydrotropy, complexation, ph adjustment, chemical modification, micronization, co-solvency, solid dispersion, and micellar solubilization. nano emulsions can be defined as new which might occur as oil-in-water (o/w) or water-in-oil (w/o) structure, in which the molecule’s centre is oil or water, respectively with droplet diameters of range (50-1000nm), the average size of the droplet range between (100 and 500nm) (7). the nano-emulsions drug deliveryare the best routes because of their capacity for dissolving large quantities of the low soluble drugs, also due to their common compatibility and ability for protecting drugs from hydrolysis and enzymatic degradation (8). the small size of the nano-emulsion’s droplet are more clear transparent appearance which are definitely different from the milky white color related to coarse emulsion (in which the micron-sized droplets are factors in the multiple light scattering)(9). nano emulsions are kinetically stable but thermodynamically unstable, with ostwald ripening being the main factor of their instability (10, 11, 12) isradipine belongs to dhp class of ccbs, and it binds to the calcium channels with specificity and affinity to inhibit the calcium flux into cardiac and arterial smooth muscle cells (13). it is used in prophylactic treatment of angina pectoris, treatment of hypertension, some currently studies have indicated that isradipine in the treatment of parkinson’s disease (14,15). isradipine is considered to be freely soluble in methylene chloride, acetone, chloroform; soluble in ethanol; insoluble in water (<10 mg/l at 37°c)(16). it is mainly absorbed from gastrointestinal tract following oral adminstration, experiences some extensive first-pass metabolism;therefore bio-availability is (15 to 24%)(17). materials and methods isradipine, triacetin,cremphore el and transcutol were purchased from hyper chem company,china, tween 80 and tween 20 have been bought from thomas baker (chemicals) pvt ltd, india. castor oil obtained from evans medical ltd, united kingdom. olive oil supplied by pomace olive oil oilex, s.a. spain, coconut oil,cinnamon oil obtained from shaanxi guanjie technology co, ltd, china. liquid paraffin obtained from merck, germany. poly ethylene glycol 400,200 supplied by m/s provizer pharma. india.. methanol supplied by avantor performance materials, norway. hydrochloric acid from grin land chemical comp, united kingdom. na2hpo4, kh2po4 and deionized water were supplied by janeen for chemical and laboratory materials, baghdad, iraq. methods differential scanning calorimeter (dsc) the thermal properties of drug powder samples were examined via the use of dsc /ta-60thermal analysis controller with the intercooler-2 cooling system(dsc-60, shimadzu, japan). sample heating was run for each sample set for 50-250°c at the rate of 10°c/min, using nitrogen as blank gas(18). solubility study of isradipine the solubility of isradipie measured in different oils which are coconut oil, liquid paraffin, cinnamon oil, castor oil, olive oil, triacetin, the surfactants which are tween 80, tween 20 and cremphore el and co-surfactants which are glycol polyethylene glycol 200,400 and transcutol. excess amounts of the isradipine powder was added to 5ml of each oil, cosurfactants and surfactant in small plain tubes. then, these tubes were tightly closed and placed in isothermal shaker water bath at 25 ± 0.5 °c for 48 hr. after this time, samples were centrifuged at 3000 rpm for 20 minutes, then the supernatant layer for each sample filtered by using filter membrane (0.45 µm). after filtration, samples were diluted with methanol, the solubility evaluated at λ max at 326nm through the use of uv-visible spectrophotometer and the measurement carried out in triplicate (19,20). construction of pseudoternary phase diagrams components related to the pseudoternary phase diagrams consist of oil, mixture of surfactant and co-surfactant (s mix) as well as deionized water was prepared through the iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsions 145 use of aqueous titration approach(21).cosurfactant and surfactant were mixed in various ratios (1:1, 1:2, 1:3, 1:4, 2:1). with regard to each one of the phase diagrams, oil as well as the s mix were blended in various weight ratios until obtaining the maximum ratio of s mix and oil, there are 11 different combinations related to s mix and oil have been prepared, such combinations were slowly titrated with aqueous phase (deionized water), each mixture was titrated with deionized water under gentle magnetic stirrer without heating. the concentration of water at which transparent-toturbid changes occurred was measured as the endpoint of the titration (22). thermodynamic stability studies(23) all formulations that were prepared were exposed to various thermodynamic stability tests (freeze-thaw cycle, heatingcooling cycle and centrifugation) for overcoming choosing metastable formulation. centrifugation test all the prepared nanoemulsion formulations were centrifuged at 3500 rpm for (2030minutes) and assessed for cracking, creaming, and phase separation. heating-cooling cycle (h/c cycle) there are 6 cycles between (4-45° c) with storage at each one of the temperatures for 48hr. formulations, that are considered stable at such temperatures, were subjected to freezethaw cycle . three freeze-thaw cycles between (-21 and +25 ° c) with storage at each one of the temperatures for 48hr determined for all the prepared nano-emulsion formulations. droplet size measurement an amount of 0.1 ml of each formula was dispersed in fifty milliliters of deionized water in volumetric flask, after that subjected to mixing process via inverting the flask. globule size has been assessed via particle size analyzer which analyzed fluctuations in the light scattering because of particle’s brownian motion. light scattering has been inspected at 25° c at a 90° angle ( 24). polydispersity index (pdi) this assay is used to measures uniformity related to the globules size in nanoemulsion. it could be obtained by abt-9000 nanolaser particle size analyzer. higher polydispersity value indicates lower uniformity of globules size of nano-emulsion(25). measurement of ph the ph of all prepared nano-emulsion formulas was determined with the use of digital ph meter utilizing (20-30ml) sample placed in 50-ml capacity beaker (26). all the measurements have been repeated 3 times. the ph value is of high importance to determine the nano-emulsion’s stability since the ph alteration mean the occurance of the chemical reactions which could damage the final product’s quality. percent of light transmittance measurement (%t) measurement of the %t was achieved for eleven control nano-emulsions at 650nm, while the distilled water kept as blank (27). viscosity determination the viscosity of sample was determined without dilution with the use of ndj-5s digital viscometer and spindle number (2), the spindle has been inserted in (30-40ml) of the prepared ne sample in graduated beaker and revolved at distinctive speeds which have been (6, 12, 30 and 60 rpm)(28). dye test water-soluble dye (methyl orange) was mixed with each one of the isradipine ne formulations. in the case when the dye is mixed with nano-emulsion homogenously without precipitation, this will indicate that the continuous phase is water and nano-emulsion structure is o/w (29). drug content drug content was determined via uv visible spectrophotometer. formulation diluted with methanol to the needed concentrations, and absorbance was determined at wave length against solvent blank. drug content was calculated by employing the following equation(30) . drug content = analyzed content theoretical content × 100 in vitro dissolution study in vitro drug release of all prepared nano-emulsion formulations were assessed via the use of usp dissolution apparatus-ii (paddle approach). dissolution medium, on the basis of monograph of isradipine in usp, is 0.1n hcl as dissolution media (500ml), at 37±0.5 °c and 50 rpm(31) through the use of dialysis bag technique (molecular cut off 12000 da) (31), formula that contain isradipine equivalent to single dose has been placed in dialysis bag, also 5ml of the dissolution medium has been taken. the time intervals for drawing out aliquots have been 5, 10, 15, 30, 45, 60, 90 and 120 min (32). zeta potential measurement zeta potential can be defined a a parameter that is applied for measuring surface charge properties and as a sign of aphysical stability related to the nano-emulsions(33). iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsions 146 fourier transform infrared spectroscopy (ftir) compatibility of drug and formulation was determined by ftir. the spectrum was obtained for(drug, tween 20, transcutol, triacetin, and selected formula). it is used for identifying the functional groups with their meas of attachment, and the fingerprint of the molecule. the sample can be prepared by employing a suitable method such as potassium bromide pellet method, nujol mulls, and then the sample is scanned in ftir at a moderate scanning speed between( 4000 400 cm-1) ( 34). field emission scanning electron microscope (fesem) isradipine nanoemulsions were also analyzed by field emission scanning electron microscope (fesem).it is apparatus that used for an image surface roughness analysis, used to explain the shape, size droplets within formulated nanoemulsion of isradipine (35). statistical analysis the results of the study ingicate the average of triplicate readings for each sample. analysis of variance test (anova) were applied to investigate that the results of study considered as significant results when (p< 0.05) and the results were considered not significant when (p> 0.05). results and discussion study of differential scanning calorimetry (dsc) the measured melting point via dsc is used to confirm the purity of the drug used in the study . the sharp peak appeared in figure 1 refered to melting point of the drug and indicated the crystalline drug in nature (36). figure 1. differential scanning calorimetry of isradipine. solubility study selection of the main constituents (cosurfactant, surfactant and oil) in preparation of nanoemulsion has been considered as an essential factor in formulation development, since the nano-emulsion ability of maintaining drug in solubilized form has been considerably impacted via the drug’s solubility in oil phase(37). the isradipine’s solubility was indicated to be highest in triacetin (81mg/ml) in comparison to other oils in table 1. with regard to the smix part, high drug solubility in transcutol(75.1mg/ml) and tween20(20.101mg/ml) in table 2 (38). although the drug solubility considered also high in peg200,peg400, transcutol was selected as a cosurfactant due to its higher solubility, better miscibility and give good nanoemulsion region in pseudo ternary phase diagram compared with peg200,peg400. table1.saturation solubility of isradipine in different oils oil solubility (mg/ml) mean ±sd* castor oil 4.135± 0.15 coconut oil 12.744± 0.04 cinnamon oil 7.112± 0.03 triacetin oil 81.000± 0.05 olive oil 4.102± 0.01 liquid paraffin 1.123 ±0.12 table2. saturation solubility of isradipine in different surfactant/ cosurfactant surfactant/ cosurfuctant solubility (mg/ml) mean ±sd* tween 20 20.101± 0.28 tween 80 5.601± 0.02 cremphore el 12.100 ± 0.03 peg 200 40.10 ± 0.09 peg 400 35.013± 0.05 transcutol 75.101± 0.154 pseudoternary phase diagram construction the components of the pseudo-ternary phase plot are (oil, deionized water and smix surfactant/co-surfactant, which considered as a variable component due to that, it presents in various (surfactant /co-surfactant) ratio such as 1:1, 2:1, 1:2,1:3 and 1:4. in the pseudo-ternary phase plot, the shaded area represents the area of nanoemulsions while unshaded area represents the area of the emulsion. pseudo-ternary phase diagram plot for different s mix ratio tween 20: transcutol are shown in figure ( 2) . in smix 2:1when concentration of surfactant was increased as related to cosurfactant, nano-emulsion area reduced in comparison to the smix ratio1:2. cosurfuctant concentration at smix(1:2,1:3,1:4) rises in the nanoemulsion region more than in smix ratio (2:1)(39). iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsions 147 figure 2. pseudo ternary phase diagrams for different s mix ratio (tween 20: transcutol 1:1, 1:2, 1:3,1:4 and 2:1) preparation of isradipine loaded nanoemulsion the quantity of isradipine(0.025) gram that required for preparation of (100) gram formula dissolved in the specialized quantity of triacetin, then s mix ratio was added to the oil and isradipine mixture and the whole mixture mixed with the use of vortex mixer for 5 minutes , then deionized water titrated drop by drop up to 100 gm which is the final weight of the formula,so nine formulas of nanoemulsion (o/w) were produced .all of the nanoemulsion formulation were clear and transparent . table 3. formulation of different isradipine loaded nanoemulsion formula no. s mix ratio % w/w of a component of nanoemulsions smix( surfactant: cosurtactant) oil water drug s mix(tween20,transcutol) tricetin deionzed water isradipine n1 1:1 40 10 50 0.025 n2 55 5 40 0.025 n3 1:2 40 5 55 0.025 n4 45 10 45 0.025 n5 1:3 40 5 55 0.025 n6 45 10 45 0.025 n7 1:4 35 5 60 0.025 n8 30 10 60 0.025 n9 40 5 55 0.025 n10 45 5 50 0.025 n11 2:1 35 5 60 0.025 iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsions 148 thermodynamic stability studies all of the prepared nanoemulsions formulations passed the thermodynamic stability test, that is (freeze-thawing test, heating-cooling test, and centrifugation test) in order to get thermodynamic stable formulations of nanoemulsions that have no creaming, cracking and phase separation which may present in the macroemulsion(39). the thermodynamic results of the prepared formulation were shown in table 4. table 4 .thermodynamic stability of isradipine loaded nanoemulsion formul a code centrifugati on test heating cooling cycle test freeze thawin g cycle test ne1 pass pass pass ne2 pass pass pass ne3 pass pass pass ne4 pass pass pass ne5 pass pass pass ne6 pass pass pass ne7 pass pass pass ne8 pass pass pass ne9 pass pass pass particle size measurement(40) it has been indicated that the systems which consist of 10 percent triacetin created nano-emulsion with larger particle size in comparison with the systems consisting of five percent triacetin. high surfactant ratios enabled interfacial film to stabilize and condense, while the film will increase with the increase in cosurfuctant concentration. small mean droplet size might be related to penetration of the co-surfactant molecules into surfactant film. this might reduce the interfacial film’s surface, which might lower radius of curvature of droplets and forming transparent systems. therefore, relative proportion of surfactant to cosurfuctant has different impact on droplet size. polydispersity index (pdi)( 40) polydispersity of all the formulations has been not more than (0.5)), which indicates uniform and narrow globule size distribution, as can be seen in table(3) determination of ph the ph related to all the formulations have been determined via ph meter in triplicate at 25 ± 1 °c and indicated to be in range of ( 4.9-5.21 ) as can be seen in the table (3) . the higher ph value in the formulations of isradipine nanoemulsions was (5.21) for ne9 and this ph value is suitable for oral administration.( 41,44). measurement of light transmittance values of transmittance percentage shown in table 5 are close to 100% specified that all formulations were transparent, clear, and transmit light efficiently (42). table 5. characterization of isradipine loaded nanoemulsion where,mean droplet size,polydispersity , ph and %t (mean ± sd, n= 3) formula code mean droplet size polydispersity ph measurement light transmittance ne1 297.7 ±0.01 0.345±0.00 5.120± 0.091 97.145± 0.1321 ne2 315.7 ±0.01 0.363±0.00 4.910± 0.0652 98.298± 0.0235 ne3 98.4 ±0.011 0.217±0.00 5.071± 0.008 98.512± 0.0041 ne4 358.2 ±0.21 0.271±0.00 4.811± 0.0026 97.223± 0.008 ne5 260.0 ±0.01 0.333±0.00 4.8143± 0.008 99.617±0.0046 ne6 357.8 ±0.01 0.005±0.00 4.912± 0.0087 98.387± 0.0043 ne7 255.0 ±0.01 0.215±0.00 5.111± 0.003 98.411± 0.0076 ne8 186.2 ±0.21 0.213±0.00 4.517± 0.001 99.125± 0.15 ne9 177.1 ±0.01 0.121±0.00 5.214± 0.009 99.878± 0.035 ne10 287.1 ±0.12 0.341±0.00 5.140± 0.067 98.690± 0.034 ne11 413.0 ±0.10 0.301±0.00 4.910± 0.0013 97.121± 0.061 iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsion 149 viscosity measuremen the results of the viscosity of isradipine nanoemulsions were found to be in range (25.026 – 300.782 mpa.sec.). it was observed that all formulations have low viscosity and this important to ensure pourability, packing, especially if the nanoemulsion dedicated for oral use (43).when the rotation speed increased (velocity ), the viscosity decreased indicating the pseudoplastic (shear thinning liquids) flow of the preparation (44) . dye test methyl orange is an azo dye miscible with water. following the addtion of methyl orange dye, it has been shown that the dye miscible with all the isradipine ne formulations; no cumulative or cluster was formed(44). drug content estimation the drug content related to all prepared isradipine nano-emulsions was more than 95% and there has been no considerable difference between different formulations (p > 0.05), which meet british pharmacopia requirement and were within an acceptable range (95.0%-105.0%), which indicates that there has been no precipitation of drug in any of prepared formulations(40). in vitro drug release pure drug showed (40.45%) drug release at end of sixty minute because of its low aqueous solubility. with nanoemulsion formulation, it was noticed as the concentration of transcutol increase, the release is high reach 100% at the end of dissolution test , the reason behind this is due to the effect of co-surfactant which are good solubilizing and facilitating dissolution process of the pure drug (46, 47). at the same time, ne9 showed highest release; end of sixty minutes, that could be the result high content of transcutol . co-surfactant role in nano-emulsion systems decrease interfacial tension and increases the interface’s fluidity. also, it increases the hydrocarbon tail’s mobility and enable greater penetration of the oil in nanoemulsion region(48 figure3. effect of smix:oil ratio on release profile where (a) release of ne1, ne2 and pure drug (b) release of ne3, ne4and pure drug (c) release of ne5, ne6 and pure drug, (d) release of ne7, ne8 ne9,ne10 and pure drug (e) release of ne11and pure drug iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsion 150 figure 4. effect surfactant:cosurfuctant ratio on release profile of isradipine nanoemulsion (ne1,ne3,ne5,ne9,ne11) and pure isradipine . selection of optimized formula of isradipine nanoemulsions according to the characterization study of the prepared nanoemulsions,there was an indication that the ( ne9) is the optimized formula, because it is characterized by good droplet size (177.1), low pdi (0.120), best ph value for oral use (5.214), good percent transmittance (99.96), percent of drug content was higher (98.9), accepted viscosity range for oral use (25.026 – 65.12 mpa.sec) and highest release of isradipine from the formula. the optimized formula would be subjected to further studies such as zeta potential, drug – excipient compatibility, field emission scan electron microscope (fesem). zeta potential measurement the value of optimized formula was (-18.3) . figure 5. zeta potential for ne9 formula studies of dru – excipient compatibility study by fourier transformed infrared spectroscopy (ftir) pure isradipine exhibited characteristic peaks at 3,348 cm–1, (n-h stretching), 2,945cm–1, (c-h stretching), 2,358 cm–1, (o-h stretching), 1,701 cm–1, (c=o stretching), and 1,448 cm–1 ,(c=c stretching) (figure5). all these peaks had appeared in ftir spectrum of nanoemulsion formulation .this indicates that there was drug – excipient compatibility between all components of prepared nanoemulsion. figure 6. ftir spectrum of isradipine powder. iraqi j pharm sci, vol.29(1) 2020 isradipine nanoemulsion 151 figure7. ftir spectrum of optimized formula (ne9) field emission scan electron microscope(fesem) figure 7 shows that oil droplets of the optimized formula are spherical in shape and there are accumulation of smaller oil droplet, however, there was no main changes in shape and size of the oil droplets upon accumlation (35). figure 8. fesem of optimized formula(ne9). conclusions from this study, it is concluded that nanoemulsion provided an important dosage form for the oral water-insoluble drug. ne that was prepared from triacetin oil, tween 20 and transcutol was encouraging method for improving dissolution rate and solubility of isradipine. the present study may serve as a prototype approach for the formulation development of other hydrophobic drugs nanoemulsion drug delivery system. acknowledgements the 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talegaonkar nanocarrier for the transdermal delivery of an antiparkinsonian drug aaps pharmscitech, 2009;10 (4):1093-1103. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 56 some hormonal changes in women with primary hypothyroidism under the effect of thyroid hormone replacement therapy zainab s. jabbar* , kassim j. al-shamma*,1 and mohammad a. taher** *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. **department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad, iraq. abstract hypothyroidism has been associated with disorders of glucose and insulin metabolism..the present study was designed to evaluate the possible change in some hormones (free testosterone, estradiol, prolactin, insulin), glucose and homeostasis model assessment of insulin resistance (homa-ir) in women with primary hypothyroidism under thyroid hormone replacement therapy .this cross-sectional study was carried on 62 hypothyroid patients׳ women and 22 healthy women as control group at the specialized center for endocrinology and diabetes, al-rasafa directorate of health baghdad, with age range(15-60 years), diagnosed as having primary hypothyroidism on thyroxine replacement therapy with duration not less than four months. each of the selected patients women and the healthy control were distributed into two groups, normal cyclic and postmenopausal. blood samples were collected to measure thyroid stimulating hormone(tsh), total thyroxine (tt4) , total triiodothyronine (tt3), free thyroxine(ft4), free testosterone (ft), estradiol(e2) ,prolactin(prl), insulin , fasting blood glucose( fbg), homeostasis model assessment-insulin resistance(homa-ir). the results showed that the majority of hypothyroid women (older than 40 years and obese) had high levels of tsh in normal cyclic and postmenopausal patients women. a significant increase in f t4 and tt4 in postmenopausal patients women when compared with postmenopausal control group f t4 and tt4. a significant increase in free testosterone and fbg and significant decrease in tt3 and e2 levels in normal cyclic patients women when compared with normal cycle control group. high prolactin levels were found in the normal cyclic patients women in comparism with control group. higher levels of insulin were found in normal cyclic and postmenopausal patients women as compared with control groups, however insulin was not statistically different. significant increase in homa-ir of normal cyclic patients women compared with control group. in conclusion, elevation of tsh levels in postmenopausal patients women were less than in normal cyclic patients women this explain the increase levels of thyroxine hormone(t4)in this group of patients as compared with both control group and normal cyclic patients women. some hormonal changes were found in normal cyclic hypothyroid patients women as compared with control group.the alteration of these hormones disappear when euthyroid state restored, so adjustment of thyroxine therapy is required in these patients. key words: hypothyroidism , thyrid replacement therapy. بعض الخغيراث الهرمىنت في النساء المصاباث بقصىر الغدة الدرقيت االبخدائي ححج حأثير عالج الهرمىن الدرقي البديل جبار زينب سخار * ، قاسم جليل الشماع *،1 و محمد عباس طاهر ** * .فشع انصٛذنت انسشٚشٚت ، كهٛت انصٛذنت ، خايؼت بغذاد ، بغذاد ، انؼشاق ** .كهٛت انصٛذنت ، خايؼت بغذاد ، بغذاد ، انؼشاق فشع انؼهٕو انًخخبشٚت انسشٚشٚت ، الخالصة صًًج ْزِ انذساست نخقٛٛى انخغٛش انًسخًم فٙ .قصٕسانغذة انذسقٛت يغ اظطشاباث فٙ انخًثٛم انغزائٙ نهدهٕكٕص ٔاألَسٕنٍٛاسحبط ،خهٕكٕصانذو نهصائى ٔ يقأيت االَسٕنٍٛ ( اسخشادإٚل، انبشٔالكخٍٛ، االَسٕنٍٛ انخسخٕسخٛشٌٔ انسش، االسخشٔخٍٛ)بؼط انٓشيَٕاث (homa-ir ) ْزِ انذساست انؼشظٛت .انغذة انذسقٛت االبخذائٙ حسج حأثٛشػالج انٓشيٌٕ انذسقٙ انبذٚمقصٕسنذٖ انُساءانًصاباث بًشض أيشأة سهًٛت نًدًٕػت انسٛطشة فٙ انًشكض انخخصصٙ نهغذد انصى ٔانسكش٘،دائشة 22أيشأة يصابت بقصٕس انغذة انذسقٛت ٔ 62اخشٚج ػهٗ سُت، حى حشخٛصٓى باالصابت بًشض قصٕس انغذة انذسقٛت بُاءا ػهٗ انخقٛٛى انخاسٚخٙ 60-15حخشأذ بٍٛ صست بغذاد انشصافت، باػًاس خًٛغ انُساء انًصاباث . ْٕػادة يشخص نالصابت بانًشض( tsh)إٌ صٚادة حشكٛضانٓشيٌٕ انًسفض نهغذة انذسقٛت . ٔانسشٚش٘ ٔانًخخبش٘ إٌ انُساء انًصاباث انالحٙ حى اخخٛاسٍْ ٔانُساء انسهًٛاث حى حٕصٚؼٓى انٗ يدًٕػخٍٛ .سػٕندٕا بانثاٚشٔكسٍٛ نًذة ال حقم ػٍ اسبؼت اشّ . ،رٔاث انذٔسة انطبٛؼٛت ٔيا بؼذ سٍ انٛأط 1 corresponding author e-mail:drkassim_alshamaa@yahoo.com received: 14/3/2012 accepted: 16/3/2013 iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 57 انٓشيٌٕ انًسفض :انًشٚعاث ٔانسهًٛاث نقٛاط انخغٛشاث انًسخًهت نهًؤششاث انًذسٔست ٔانخٙ حخعًٍحى خًغ ػُٛاث انذو يٍ انُساء ، انبشٔالكخٍٛ، (اسخشادإٚل)انخسخٕسخٛشٌٔ انسش، االسخشٔخٍٛ ،(f t4)، انثاٚشٔكسٍٛ انسش tt4 ،tt3، انثاٚشٔكسٍٛ انكهٙ tshنهغذة اظٓشث انُخائح اٌ اغهبٛت انُساء انًصاباث بقصٕسانغذة انذسقٛت ٍْ .(homa-ir)ٍٚ االَسٕنٍٛ ، خهٕكٕصانذو نهصائى ٔ يقأيت االَسٕل فٙ انُساء انًشٚعاث رٔاث انذٔسة tshاسخُخدج انذساست ٔخٕد اسحفاع فٙ يسخٕٚاث قٛاط . سُت ٔبذُٚاث 40يًٍ حضٚذ اػًاسٍْ ػٍ ٚت فٙ انثاٚشٔكسٍٛ انكهٙ ٔانثاٚشٔكسٍٛ انسش فٙ انُساء انًشٚعاث كزنك ٔخذث صٚادة يؼُٕ. انطبٛؼٛت، ٔفٙ انُساء انًشٚعاث بؼذ سٍ انٛأط بُٛج انُخائح ٔخٕد صٚادة يؼُٕٚت . بؼذ سٍ انٛأط بانًقاسَت يغ انُساء انسهًٛاث بؼذ سٍ انٛأط نهثاٚشٔكسٍٛ انكهٙ ٔانثاٚشٔكسٍٛ انسش فٙ انُساء ( االسخشادإٚل)ٔ ْشيٌٕ االسخشٔخٍٛ tt3ٚتبانخسخٕسخٛشٌٔ انسش ٔخهٕكٕص انذو نهصائى ٔ َقصاٌ يؼُٕ٘ بٓشيٌٕ انغذة انذسق اسحفاع يسخٕٖ ْشيٌٕ انبشٔالكخٍٛ فٙ انُساء . انًشٚعاث رٔاث انذٔسة انطبٛؼٛت بانًقاسَت يغ انُساء انسهًٛاث رٔاث انذٔسةانطبٛؼٛت االَسٕنٍٛ فٙ انُساء انًشٚعاث رٔاث انذٔسة اسحفاع يسخٕٚاث ْشيٌٕ . انًشٚعاث رٔاث انذٔسة انطبٛؼٛت بانًقاسَت يغ يدًٕػتانسٛطشة صٚادة . ،ازصائٛا الٕٚخذ فشق يؼُٕ٘ فٙ يسخٕٖ االَسٕنٍٛ انطبٛؼٛت ٔ انُساء انًشٚعاث بؼذ سٍ انٛأط بانًقاسَت يغ يدًٕػاث انسٛطشة إٌ أغهبٛت ٔبزنك ًٚكٍ االسخُخاج.بانًقاسَت يغ يدًٕػت انسٛطشة يؼُٕٚت بًقأيت االَسٕنٍٛ فٙ انُساء انًشٚعاث رٔاث انذٔسة انطبٛؼٛت فٙ انُساء tshٔخذ اٌ اسحفاع يسخٕٖ ْشيٌٕ . سُت ٔبذُٚاث 40انُساء انًصاباث بقصٕس انغذة انذسقٛت ٍْ يًٍ حضٚذ اػًاسٍْ ػٍ ْشيٌٕ انًشٚعاث بؼذ سٍ انٛأط اقم يٍ اسحفاػّ فٙ انُساء انًشٚعاث رٔاث انذٔسة انطبٛؼٛت ْٔزا ٕٚظر انضٚادة انساصهت فٙ ٔخذث بؼط . انثاٚشٔكسٍٛ فٙ ْزِ انًدًٕػت يٍ انًشٚعاث بانًقاسَت يغ يدًٕػت انسٛطشة ٔ انُساء انًشٚعاث رٔاث انذٔسة انطبٛؼٛت اٌ حغٛش ْزِ انٓشيَٕاث ٚخخفٙ ػُذ اػادة .انخغٛشاث انٓشيَٕٛت فٙ انُساء انًشٚعاث رٔاث انذٔسة انطبٛؼٛت بانًقاسَت يغ يدًٕػت انسٛطشة .انطبٛؼٛت نهذسقٛت ،نزنك ٚخطهب حُظٛى ػالج انثاٚشٔكسٍٛ نخهك انًشٚعاث انٕظٛفت .قصىر الغدة الدرقيت ، الهرمىن الدرقي البديل :الكلماث المفخاحيت introduction hypothyroidism has been associated with disorders of glucose and insulin metabolism, involving defective insulin secretion in response to glucose, hyperinsulinemia, altered peripheral glucose disposal, and ir (1) . the mechanisms linking hypothyroidism with ir in general and in skeletal muscles (sm) in particular are still under investigation. insulin resistance in hypothyroidism is associated with a negative regulation of one or more intracellular enzymes involved in glucose catabolism (2) . an impaired translocation of glucose transporter 4 (glut4) on the plasma membrane has been also observed in the monocytes of subjects with clinical and subclinical hypothyroidism, in relation to a decreased insulin-mediated glucose uptake (imgu) (3) . an effect of thyroid hormones on insulin receptors has been suggested, but the existing data are rather conflicting, supporting either no relationship between thyroid status and the affinity of insulin receptors or diminished high affinity insulin receptors (hairs) in hypothyroidism (4) . in patients with primary hypothyroidism, increased levels of thyroid releasing hormone(trh) can cause elevation of prolactin levels (5) . many studies supported the existence of interplay between prolactin and insulin along with the influence of dopamine in the regulation of insulin secretion (6) . hyperinsulinemia increases the production of androgens in the ovaries and of insulin-like growth factor(igfs) in the liver (7) .the direct effect of insulin and igf-1 is increased 17hydroxylase activity in the ovaries, causing an excessive production of androgens, particularly androstenedione and testosterone and its precursor, 17-hydroxyprogesterone(17-ohp) (8) . free testosterone had been found to be positively correlated with insulin resistance (9) ,as well as with components of the metabolic syndrome, including fasting plasma glucose, adiposity (10) , fasting and post challenge insulin levels (11) , insulin-to-glucose ratio (12) , and blood pressure (13) . high levels of free testosterone (11) have been shown to predict incident type 2 diabetes in women, giving the relation between androgens and insulin sensitivity. the aim of the study was to determine whether there is any change in serum levels of free testosterone, estradiol, prolactin, insulin, glucose and homeostasis model assessment of insulin resistance (homa-ir) in women with primary hypothyroidism under thyroid hormone replacement therapy. materials and methods this study was performed on 62 hypothyroid patient women at the specialized center for endocrinology and diabetes, alrasafa directrate of health-baghdad with age range(15-60 years) with mean duration of hypothyroidism of 4 months.the patients women were distributed into two groups: normal cyclic and postmenopausal. these were compared with 22 healthy women as control group with the same age range of patients, and also distributed as normal cyclic and postmenopausal.the hypothyroid women were diagnosed as hypothyroid by a senior physician through historical, clinical, and laboratory assessment. the demonsetration of an elevated tsh concentration is usually diagnostic (14) . iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 58 table 1: characteristics of control women and hypothyroid patients women. the data are expressed as mean ± sd. exclusion criteria the study exclude any patient women with: secondary hypothyroidism, pcos, inherited tbg disease, heart disease , renal disease , liver disease ,pregnancy, lactation, diabetic disease, smoking and any drugs effect thyroid function except thyroxine. blood specimen collection and preparation blood was taken after 12 hr fasting and at the early follicular phase (in women with normal cycle) for measurement of thyroid function tests (tsh,tt4,tt3,ft4),fasting blood glucose(fbg)levels, fasting insulin levels, free testosterone levels, estradiol levels and prolactin levels.tsh,tt4 and tt3 were measured, radioimmunoassay(ria kits) while f t4,insulin, free testosterone, estradiol and prolactin immunoassay readymade-kits(elisa) were had been used. statistical analysis the following statistical data analysis approaches were done through the statistical package of social sciences (spss) program(version-10) and excel application and used in order to analyze and assess the results of the study: i . descriptive data analysis: statistical tables (frequencies, percents), mean value, standard deviation, standard error, (95%) confidence interval or population mean values, two extreme values (min. and max.) respondents. ii inferential data analysis: student t-test for equality of means coincidence testing in two independent samples. results the results showed that an increase incidence of hypothyroidism in women with age older than 40 years old, while frequency of patients as the following : between (15-19)years old were 2(3.2%),between(20-29)years old were 7(11.3%),between (30-39)years old were 12(19.4%) , between (40-49)years old were25(40.3%) and between (50-60)years old were 16 (25.8%). the majority of hypothyroid patients were obese, in the normal cyclic patients women group were 22 (52.4%) as well as in the postmenopausal patients women were 14 (70%). the results in table(1,2,3) showed high levels of tsh were found in normal cyclic patients women (10.37±3.03, 95% c.i=4.24-16.49 µiu/ml ) and in postmenopausal patients women( 4.23±1.12, 1.89-6.57 µiu/ml)were observed. a significant increase in f t4 (14.51±0.85 , 12.72-16.30 pmol/l),and tt4 (117.8±8.34,100.31-135.23 nmol/ml ) in postmenopausal patients women when compared with postmenopausal control group f t4 (10.58±0.41, 9.64-11.52 pmol/l) and tt4 (89.71±4.19, 80.24-99.1 nmol/ml). a significant increase in the plasma free testosterone(1.65±0.26, 1.11-2.18 pg/ml) and fbg (5.36±0.13, 5.09-5.64 mmol/l)and significant decrease in tt3(1.57±0.07, 1.42-1.71 nmol/ml ) and e2(50.63±4.20, 42.15-59.11 pg/ml) levels in normal cyclic patients women when compared with normal cycle control group ,free testosterone (0.76±0.16,0.41-1.11 pg/ml), fbg(4.78±0.19, 4.36-5.20mmol/l) ,tt3 (1.90±0.06, 1.77-2.03nmol/ml )and e2(76.80±9.79, 55.25-98.35pg/ml). high prolactin levels were found in normal cyclic patients women (22.57±4.43, 13.63-31.52 ng/ml) as compared with control group(17.51±1.68, 13.82-21.20 ng/ml(. higher levels of insulin were found in normal cyclic and postmenopausal patients women as compared with control groups ((15.86±1.47 , 12.89-18.84 versus 11.34 ±1.28, 8.52-14.16 ) , (15.47±1.92, 11.46-19.49 versus 9.90±1.41, 6.71-13.1)µiu/ml) repectively, however insulin was not statistically different. significant increase in homa-ir of normal cyclic patients women (3.83±0.39,3.05-4.62) when compared with control group (2.41±0.29,1.79-3.04). variable hypothyroid patients women(mean ±s.d) healthy control women(mean ±s.d) number (total) 62 22 normal cyclic women age(years) bmi(kg/m²) 42 37± 9.04 31.7 ± 5.39 12 36 ± 6.68 29.9 ± 2.73 postmenopa usal women age(years) bmi(kg/m²) 20 52 ± 5.40 32.3 ± 5.33 10 50 ± 4.27 30.5 ± 3.03 iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 59 table 2: distribution of thyroid hormones levels in patients and control groups a: control(normal cycle)group, b:control(postmenopause)group, c:patients(normal cycle)group, d:patients(postmenopause)group. s: significant, ns: non-significant, hs: high significant. l.b: lower bound, u.b: upper bound. c.i: confidence interval. parameters groups n mean ± std.e. 95% c. i. for mean min. max. c.s p-value l.b. u.b. tsh (µiu/ml) control (normal cycle) a 12 1.48 ± 0.18 1.09 1.87 0.30 2.3 a×c p=0.061 ns b×d p=0.655 ns c×d p= 0.118 ns control (postmenopause) b 10 1.75 ± 0.20 1.31 2.19 0.80 2.7 patients (normal cycle ) c 42 10.37 ± 3.03 4.24 16.49 0.05 78.0 patients (postmenopause) d 20 4.23 ± 1.12 1.89 6.57 0.04 17.9 ft4 (pmol/l) control (normal cycle) a 12 11.26 ± 0.40 10.38 12.15 9.0 13.9 a×c p=0.299 ns b×d p=0.001 hs c×d p= 0.010 hs control (postmenopause) b 10 10.58 ±0.41 9.64 11.52 8.9 12.0 patients (normal cycle ) c 42 12.31 ± 0.50 11.29 13.33 7.2 24.6 patients (postmenopause) d 20 14.51 ± 0.85 12.72 16.30 8.2 24.9 tt4 (nmol/ml) control (normal cycle) a 12 88.53 ± 2.07 83.98 93.08 75.9 99.8 a×c p=0.161 ns b×d p=0.017 hs c×d p= 0.05 s control (postmenopause) b 10 89.71 ± 4.19 80.24 99.19 71.7 113.7 patients (normal cycle ) c 42 102.3 ± 4.92 92.32 112.20 52.3 183.5 patients (postmenopause) d 20 117.8 ± 8.34 100.31 135.23 58.3 206.9 tt3 (nmol/ml) control (normal cycle) a 12 1.90 ± 0.06 1.77 2.03 1.6 2.2 a×c p=0.017 hs b×d p=0.314 ns c×d p= 0.355 ns control (postmenopause) b 10 1.62 ± 0.10 1.39 1.85 1.1 2.0 patients (normal cycle ) c 42 1.57 ± 0.07 1.42 1.71 0.7 2.7 patients (postmenopause) d 20 1.46 ± 0.10 1.25 1.66 0.8 2.4 iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 60 table 3: distribution of (free testosterone, estradiol and prolactin) hormones in patients and control groups parameters groups n mean ± std.e. 95% c. i. for mean min. max. c.s p-value l.b. u.b. ft (pg/ml) control (normal cycle) a 12 0.76 ± 0.16 0.41 1.11 0.13 2.1 a×c p=0.047 s b×d p=0.491 ns c×d p=0.044 s control (postmenopause) b 10 1.26 ± 0.32 0.53 1.99 0.03 2.7 patients (normal cycle ) c 42 1.65 ± 0.26 1.11 2.18 0.04 7.2 patients (postmenopause) d 20 0.90 ± 0.16 0.57 1.23 0.01 2.6 e2 (pg/ml) control (normal cycle) a 12 76.80 ± 9.79 55.25 98.35 32.48 137.8 a×c p=0.002 hs b×d p=0.623 ns c×d p=0.001 hs control (postmenopause) b 10 30.76 ± 7.82 13.06 48.45 6.29 83.9 patients (normal cycle ) c 42 50.63 ± 4.20 42.15 59.11 14.66 120.2 patients (postmenopause) d 20 25.88 ± 3.10 19.39 32.36 7.94 52.8 prl (ng/ml) control (normal cycle) a 12 17.51 ± 1.68 13.82 21.20 9.59 26.7 a×c p=0.464 ns b×d p=0.287 ns c×d p=0.045 s control (postmenopause) b 10 19.65 ± 2.55 13.88 25.42 9.93 32.4 patients (normal cycle ) c 42 22.57 ± 4.43 13.63 31.52 3.59 163.0 patients (postmenopause) d 20 10.91 ± 1.38 8.03 13.79 4.05 29.0 a: control(normal cycle)group, b:control(postmenopause)group, c:patients(normal cycle)group, d:patients(postmenopause)group. s: significant, ns: non-significant, hs: high significant. l.b: lower bound, u.b: upper bound. c.i: confidence interval. iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 61 table 4: distribution of of insulin, fasting blood glucose and homa-ir levels in patients and control groups. parameters groups n mean ± std.e. 95% c. i. for mean min. max. c.s p-value l.b. u.b. insulin (µiu/ml) control (normal cycle) a 12 11.34 ± 1.28 8.52 14.16 4.54 17.87 a×c p=0.100 ns b×d p=0.088 ns c×d p=0.863 ns control(postmenopausal )b 10 9.90 ± 1.41 6.71 13.10 4.05 16.83 (normal cycle ) patients c 42 15.86 ± 1.47 12.89 18.84 3.80 44.00 postmenopausal patients d 20 15.47 ± 1.92 11.46 19.49 4.35 35.61 fbg (mmol/l) control (normal cycle) a 12 4.78 ± 0.19 4.36 5.20 3.83 5.68 a×c p=0.025 s b×d p=0.835 ns c×d p=0.764 ns control (postmenopausal)b 10 5.37 ± 0.21 4.90 5.83 4.45 6.52 (normal cycle ) patients c 42 5.36 ± 0.13 5.09 5.64 4.10 8.20 postmenopausal patients d 20 5.43 ± 0.15 5.10 5.75 3.60 6.96 homa-ir control (normal cycle) a 12 2.41 ± 0.29 1.79 3.04 0.77 3.90 a×c p=0.05 s b×d p=0.099 ns c×d p=0.950 ns control (postmenopausal)b 10 2.37 ± 0.37 1.55 3.20 0.80 3.90 (normal cycle ) patients c 42 3.83 ± 0.39 3.05 4.62 0.70 11.40 postmenopausal patients d 20 3.80 ± 0.52 2.70 4.89 0.89 8.60 a: control(normal cycle)group, b:control(postmenopause)group, c:patients(normal cycle)group, d:patients(postmenopause)group. s: significant, ns: non-significant, hs: high significant. l.b: lower bound, u.b: upper bound. c.i: confidence interval. iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 62 discussion the results of an increase incident of hypothyroidism in women with age older than 40 years old were in agreement with the results of yamada et al.(1984) (15) , who referred this to increase in the level of production of antithyroglobulin antibodies , antiperoxidase antibodies and increase level of tsh hormone ,also the mean of trh hormone increase with advance age specially in women. subtle elevation of tsh is associated with measurable deficiency in resting energy expenditure and increased body weight. fox et al.(2008) (16) noted that modest increases in serum tsh concentration within the reference range may be associated with weight gain , women gained more weight than men did , although both sexes gained.the result of this study confirmed previous results that hypothyroidism and obesity frequently co-exist in varying degree of severity. most patients in this study showed persistent elevation of tsh levels, despite they were under treatment for hypothyroidism. the elevation of tsh levels in postmenopausal patients women is less than in normal cyclic patients women, this may explained by the decreased clearance of thyroxine (t4) in postmenopausal patients women and this may confirmed by the significant higher level of thyroxin(ft4, tt4) in postmenopausal patients women when compared with both control group and normal cyclic patients women (17) . in most patients the circulating concentration of tsh serves as a reflection of thyroid hormone effect upon the pituitary and thereby as an effective marker of the adequacy of the replacement dose (18) . triiodothyronine represented the active form of thyroid hormone for it easy separation from binding protein and easy binding to the receptors in the cells of target organ ;while t4 is the less active biologically than t3 ,and t4 represent the stored spare in the blood. this explain the presence of a significant difference in t3 level between patients women group and control group, because it is in continueous use (19) . this finding is confirmed in this study by the presence of significant decrease in t3 levels in the normal cyclic patients women in comparison with control group. there is a significant elevation of free testosterone levels in normal cycle of patients women in comparism with control group. estrogen deficiency leads to reduced sex hormone binding globulin(shbg) production in the liver (20) . testosterone has a high affinity for shbg.with decreasing shbg concentration, less testosterone is bound to shbg and the free testosterone in the blood increases, resulting in a relative hyperandrogenemia. the present work demonstrated that estradiol level is decreased in both groups of patients women (normal cyclic and postmenopausal) when compared with their control groups. estradiol is produced by both the interconversion of estrone (21) and the aromatization of testosterone. the main source of estrogen in postmenopausal women appears to be the aromatization of plasma androstenedione to estrone (22) . women with hypothyroidism had decreased metabolic clearance rates of androstenedione and oestrone and an increase in peripheral aromatization (23) . the present study showed a high levels of prolactin hormone in normal cyclic patients women as compared with control group. hyperprolactinaemia is not seen in all patients with hypothyroidism, but it has been reported to occur in 0-40% of hypothyroid patients (24) . prolactin hormone levels return to normal with appropriate l-thyroxine treatment. there is a significant increase in fbg levels and homa-ir in the normal cyclic patients women when compared with control group. obesity is a predictor of impaired fasting glucose( ifg), and pre-diabetic state. insulin resistance is increased by abdominal obesity, and fasting hyperinsulinemia farming a risk factor for the development of impaired fasting glucose (25) . the loss of t3 within the cells leads to increase in the level of tsh and the reduction of the activity of glut-4 in the insulin sensitive tissue such as skeletal muscles and adipose tissues, thus contributing to the stimulation of insulin resistance , this was observed in high percentage among obese individuals (26) .the results of the present study were in agreement with study by singh et al.(2010) (27) . the study concluded that patients with hypothyroidism demonstrated insulin resistance as observed by the higher homa-ir level as compared to controls. maratou et al.(2009) (3) ,had concluded that insulin resistance is associated with comparable homa-ir values in overt hypothyroidism and subclinical hypothyroidism. the study showed that insulin resistance in hypothyroidism is associated with a negative regulation of one or more intracellular enzymes involved in glucose catabolism. conclusions the majority of hypothyroid patients women were older than 40 years old and obese. the elevation of tsh levels in postmenopausal iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 63 patients women were less than in normal cyclic patients women, this indicate good compliant with treatment in postmenopausal group and this explain the increase levels of thyroxine hormone(t4)in this group of patients. some hormonal changes were found in normal cyclic hypothyroid patients women as compared with control group. the alteration of these hormones disappears when euthyroid state restored, so adjustment of thyroxine therapy is required in these patients. further research, is needed to optimize thyroid disease monitoring therapy, adjust dosages and encourage compliance. references 1. diaz g. b, paladini a.a, garcia m.e, gagliardino j.j. changes induced by hypothyroidism in insulin secretion and in the properties of islet plasma membranes. archives internationales de physiologie, de biochimie et de biophysique,1993;101(5):263–269. 2. czech m. p, malbon c. c, kerman k, gitomer w, pilch p. f. effect of thyroid status on insulin action in rat adipocytes and skeletal muscle. j clin invest 1980;66(3):574–582. 3. maratou e, hadjidakis d. j, kollias a, et al. studies of insulin resistance in patients with clinical and subclinical hypothyroidism. euro j endocrinolo 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m, milli b, ugolotti d,prampolininr,maggiom, ceresini g. thyroid disease in elderly: sex-related differences in clinical expression.j endocrinolo invest 2005 ; 28 (suppl 11):1014. 17. sawin c.t, herman t, molitch m.e, london m.h, kramer s.m. aging and the thyroid. decreased requirement for thyroid hormone in older hypothyroid patients. am j med 1983;75:206-9. 18. morris j. c. how do you approach the problem of tsh elevation in a patient on high-dose thyroid hormone replacement? clinical endocrinology 2009;70(5): 671–673. 19. ibrahim s. r. the relation between hypothyroidism and insulin resistance. iraqi j pharm sci, vol.22 (1) 2013 hormonal changes in women with primary hypothyroidism 64 academic [dissertation]. baghdad university 2008; pp: 71-79. 20. sluijmer a.v, heineman m.j, de-jong f.h, evers j.l. endocrine activity of the postmenopausal ovary: the effects of pituitary downregulation and oophorectomy. j clin endocrinol metab 1995; 80: 2163–7. 21. longcope c, tait j.f. validity of metabolic clearance and interconversion rates of estrone and 17 -estradiol in normal adults. j clin endocrinol metab 1971; 32:481–490. 22. grodin j.m, siiteri p.k, macdonald p.c. source of estrogen production in postmenopausal women. j clin endocrinol metab 1973; 36:207–214. 23. longcope c, abend s, braveman l.e, et al. androstenedione and estrone dynamics in hypothyroid women. j clin endocrinol metab 1990; 70:903-908. 24. raber w, gessl a, nowotny p, vierhapper h. hyperprolactinaemia in hypothyroidism: clinical significance and impact of tsh lee s, chun k, kim d. does abdominal obesity accelerate the effect of hypertriglyceridemia on impaired fasting glucose? yonsei med j 2010; 51(3): 360-366. 25. mentuccia d, proictti-ponnunzi l, tanner k, bacci v, pollin t. l, poehlmen e. t, shuldiner a. r, celi f. s. association between a novel variant of the humans type 2 deiodinase gene thrqzala and insulin resistance. ann internat med 2002;139:802809. 26. singh b. m, goswami b, mallika v. association between insulin resistance and hypothyroidism in females attending a tertiary care hospital. indi j clinic biochemis 2010; 25(2):141-145. iraqi j pharm sci, vol.32 (1) 2023 renoprotective effect of lipoic acid and bosentan doi: https://doi.org/10.31351/vol32iss1pp274-282 274 evaluation of renoprotective effect of lipoic acid and bosentan against diclofenac-induced acute renal failure lina b. qasim*,1, ghaith a. jasim* and ihsan s. rabeea** pharmacy, mustansiriyah university, baghdad, iraq.pharmacology and toxicology department, college of * pharmacology and toxicology department, college of pharmacy, kufa university, najaf, iraq.** abstract acute renal failure is also known as acute kidney injury (aki) is a complex health condition related to significant morbidity and mortality. non-steroidal anti-inflammatory drugs (nsaids) such as diclofenac have potential risk of renal injury. the direct effect of diclofenac-induced renal injury depends on the formation of reactive oxygen species (ros) resulting in oxidative stress. secondly, diclofenac inhibit renal prostaglandin production, limiting renal afferent arteriole vasodilation; thus glomerular filtration rate will decrease resulting in acute kidney injury. alpha-lipoic acid (ala) acts as an antioxidant and anti-inflammatory micronutrient. bosentan is a competitive antagonist with dual endothelin-1 (et-1) receptors. in present study, we investigated the effect of α-lipoic acid and bosentan in diclofenac-induced acute renal failure in male rats. measurement of serum levels of urea, creatinine, were done by colorimetric technique. on the other hand, serum levels of malondialdehyde, superoxide dismutase-1, kidney injury molecules-1, transforming growth factor β1, fibronectin and collagen type i were measured by enzyme linked immunosorbent assay technique. the receiver operating characteristic (roc) curve analysis was used for evaluating accuracy of biomarkers in diagnosis aki. area under the curve (auc) is a metric that summarize the diagnostic accuracy of the test. we observed that diclofenac increased serum levels of urea, creatinine, malondialdehyde, kim-1, tgfβ1 and fibronectin significantly (p >0.05) in the induction group compared to control group. while, sod1 significantly (p >0.05) reduced in the induction group compared to control group. both of α-lipoic acid and bosentan alone did not significantly protect against diclofenac induced aki. however, the combination group showed a significant protection against aki. pearson correlation analysis showed a significant positive correlation between (urea and kim-1) and between (creatinine and kim-1) (r2=0.792 and r2=0.677 respectively). furthermore, there was a significant positive correlation between fibronectin and urea (r2= 0.498, p>0.01) and fibronectin and creatinine (r2=0.356, p>0.05). interestingly, kim-1 showed a significant positive correlation with fibronectin (r2=0.536, p>0.01). the auc for kim-1 was 0.986 and for fibronectin was 0.829. we concluded that combination therapy of α-lipoic acid and bosentan showed a significant protective effect against diclofenac-induced aki. in addition, fibronectin could be a promising biomarker for detection and diagnosis of acute kidney injury. keywords: diclofenac, oxidative stress, alphalipoic acid, endothelin-1, bosentan. الحاد الناجم عن الديكلوفيناكحمض الليبويك والبوسنتان ضد الفشل الكلوي ل الوقائيتقييم التأثير لينا بهجت قاسم , غيث علي جاسم 1و احسان صالح ربيع 2 *فرع االدوية والسموم، كلية الصيدلة، الجامعة المستنصرية، بغداد، العراق **فرع االدوية والسموم، كلية الصيدلة، جامعة الكوفة، النجف، العراق الخالصة الحاد الكلوي الفشل )يُعد الحادة الكلى إصابة باسم أيًضا الكبيرة.akiالمعروف والوفيات بالمراضة تتعلق معقدة صحية حالة ان ( مثل م الستيروئيدية غير االلتهاب منمخاطر لها ديكلوفيناك الضادات يسببها اص محتملة التي الكلوية لإلصابة المباشر التأثير يعتمد الكلى. ابة الكلوي، يمنع الديكلوفيناك إنتاج البروستاغالندين ثانيًا، ( مما يؤدي إلى اإلجهاد التأكسدي. rosاألكسجين التفاعلية )أنواع تشكيلديكلوفيناك على يعمل (.(akiالحادة للكلى مما يؤدي إلى اإلصابة معدل الترشيح الكبيبي وبالتالي سينخفض الوارد، مما يحد من توسع األوعية الشرياني الكلوي ( ليبويك ألفا يعتبر alaحمض لاللتهابات. ومضاد لألكسدة كمضاد مستقبالت البوسنتان ( مع تنافسيًا في . المزدوجة endothelin-1مضادًا ل الكلوي الحاد الناجم عن الديكلوفيناك في ذكور الجرذان. تم قياس مستويات درسنا تأثير حمض ألفا ليبويك والبوسنتان في الفش الحالية، الدراسة ناحية من األلوان. قياس بتقنية الدم في والكرياتينين من أخرى، اليوريا الدم مصل مستويات قياس malondialdehyde ، superoxideتم disutase-1 الكلى إصابة جزيئات المحول 1-، النمو عامل ،β1 المرتبط المناعي الفحص بتقنية األول النوع من والكوالجين الفبرونكتين ، باإلنزيم. ( المستقبل تشغيل خاصية منحنى تحليل استخدام تشخيص rocتم في الحيوية المؤشرات دقة لتقييم )aki المنحنى تحت الواقعة المنطقة . (auc هي ) أن ديكلوفيناك زاد من مستويات المصل من اليوريا والكرياتينين والمالونديالديهايد الالختبار. الحظنمقياس يلخص الدقة التشخيصية في الدم sod( في مجموعة الحث مقارنة بمجموعة التحكم. بينما انخفض معدل p >0.05والفيبرونكتين بشكل ملحوظ ) tgfβ1و kim-1و اصابة لم يحميا بشكل كبير من على حده( في مجموعة الحث مقارنة بمجموعة التحكم. كال من حمض الليبويك والبوسنتان p >0.05بشكل كبير ) الناجم عن الديكلوفيناك. ومع ذلك ، أظهرت المجموعة المركبة حماية كبيرة ضد القصور الكلوي الحاد. الكلى الحادة 1corresponding author e-mail: lina6bahjat@gmail.com received: 4/4 /2022 accepted:3 /8 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp274-282 iraqi j pharm sci, vol.32(1) 2023 renoprotective effect of lipoic acid and bosentan 275 = r2 )و kim-1 ((r2 = 0.792) و creatinine (وبين( kim-1أظهر تحليل ارتباط بيرسون عالقة ارتباط موجبة معنوية بين )اليوريا و ( والفيبرونيكتين r = 0.498 ، p >0.01كان هناك ارتباط إيجابي معنوي بين الفبرونيكتين واليوريا ) ذلك، على التوالي. عالوة على ( 0.677 (.0.356r = ، p >0.05والكرياتينين ) إلى أن العالج المركب لحمض استنتجنا(. r = 0.536 ، p >0.01ارتباًطا إيجابيًا معنويًا مع الفبرونيكتين ) kim-1أظهر لالهتمام، ومن المثير مؤشر يمكن أن يكون الفبرونيكتين ذلك، الناجم عن ديكلوفيناك. باإلضافة إلى اصابة الكلى الحادةليبويك والبوسنتان أظهر تأثيًرا وقائيًا كبيًرا ضد عن إصابة الكلى الحادة وتشخيصها. واعد للكشف حيوي بوسنتان ،1-إندوثيلين ليبويك،حمض ألفا التأكسدي،اإلجهاد ديكلوفيناك، :األساسيةالكلمات introduction nonsteroidal anti-inflammatory drugs (nsaids) are considered one of the most commonly prescribed medicines in the world, with over 30 million people taking them every day(1). diclofenac as a nsaids has gained special attention over the past few years due to the potential risk of renal injury (2). nsaids can cause two different types of acute renal failure (arf) which also known as acute kidney injury (aki); hemodynamically mediated (pre-renal injury and/or acute tubular necrosis) and immune mediated (acute interstitial nephritis (ain)) (3). the direct effect of diclofenac-induced renal injury depends on targeting the mitochondria of the kidney, and formation of reactive oxygen species (ros) leading to oxidative stress (4). in addition, diclofenac inhibits renal prostaglandin production, limiting renal afferent arteriole vasodilation, increase afferent resistance; consequently the glomerular capillary pressure will drop below normal values with diminished gfr resulting in aki (2). diclofenac (2-[(2,6-diclorophenyl) amino] phenyl acetate) is a derivative of phenyl acetic acid that exhibits antipyretic, pain-relieving, antirheumatic and anti-inflammatory activities with inhibitory effect on prostaglandin biosynthesis (5). it is used to treat gout and ureteric colic, as well as rheumatoid arthritis, osteoarthritis and prescribed postoperatively(6). alpha-lipoic acid (ala) is also called thiotic acid (7) , is chemically known as 1,2 dithiolane-3-pentanoic acid (c8h14o2s2)(8). ala has beneficial effects on prevention or relief of symptoms of oxidative stress-related diseases. in which, a recent study reported by oktan m et al. (2020) evaluated that ala could be an effective strategy for the management of nephrotoxicity induced by colistin (9). it acts as antioxidant and dehydrogenase enzymes cofactor, which is involved in metabolism of mitochondrial macronutrients and energy production (10). ala can act as antioxidant in lipophilic and hydrophilic environments (11), it also acts as a free radical scavenger of ros such as (hydrogen peroxide, hydroxyl radical) and reactive nitrogen species (rns) such as (peroxynitrite, nitroxyl and nitrogen dioxide)(7). in addition, ala has metal chelating activity and its able to regenerate endogenous antioxidants such as vitamin e, vitamin c (12). ala has anti-inflammatory activity through inhibiting nuclear factor kappa beta (nf-κb), a transcription factor involve in regulation of gene expression of many pro-inflammatory cytokines such as tumor necrosis factor alpha (tnfα), interlukine-1 (il-1) & interlukine-6 (il-6) (13). in addition, ala has anti-inflammatory effect through inhibition of intercellular adhesion molecule-1 (icam-1) and vascular cells adhesion molecule-1 (vcam-1) expression by central nervous system endothelial cells (14). bosentan is a competitive an antagonist of endothelin-1 receptors, it acts as antagonist of both endothelin-a receptor (etar) and endothelin-b receptor (etbr)(15). bosentan was the first endothelin receptor antagonist (era) approved for treatment of patients with pulmonary arterial hypertension (pah) (16). it is chemically designed as monohydrate of 4tert-butyl-n-[6-(2hydroxyethoxy)-5-(2-methoxy-phenoxy)-[2,2´]bipyrimidin-4-yl]-benzenesulfonamide (17). endothelin-1 (et-1) is a 21 amino acid peptide (18) , involved in activation of many transcription factors particularly nf-κb and expression of pro-inflammatory cytokines including tnf-α, il-1, and il-6 (19). in addition, et-1 up-regulates expression of adhesion molecules on vascular endothelial cells and induces the aggregation of polymorphonuclear neutrophils (pmns) contributing to inflammation and endothelial dysfunction (20). renal vascular et-1 system is upregulated under many pathophysiological situations, therefore using of bosentan as competitive an antagonist of endothelin-1 receptors could decrease the inflammation that involved in aki. the recent study reported by caires et al (2017) showed that bosentan could reverse cyclosporine-induced changes in renal function (21). therefore, aki induced by diclofenac could be avoided while using ala and/or bosentan as renal protective agents. although , a rise in serum creatinine and urea are current hallmarks for diagnosing aki (22), they are insensitive, nonspecific, biomarkers(23). many biomarkers have been shown to indicate the onset of aki before serum creatinine and urea rise such as kidney injury molecule-1 (kim-1)(24). after ischemia or nephrotoxic injury, the transmembrane glycoprotein kim-1 is upregulated in proximal tubular cells (25). renal ischemia represents the main trigger of ros resulting in oxidative stress. oxidative stress can be assessed by indirect methods which can measure the stable iraqi j pharm sci, vol.32(1) 2023 renoprotective effect of lipoic acid and bosentan 276 by-products of ros activity on biomolecules (lipid, protein, dna) like malondialdehyde (mda). many human and animals models are focusing on modifying aki with the help of antioxidants (26). the main antioxidants fighting oxidative stress and can be used as biomarkers in aki are superoxide dismutase (sod), catalase, and glutathione peroxidase(27). acute kidney injury may also involve glomerular injury, which can detect by measuring mesangial extracellular matrix (ecm) secretions such as fibronectin, and collagen type i that undergo upregulation upon mesangial cells proliferation (28). materials and methods animals thirty adult male albino rats weighing between 200 and 250g were used in the present study. these animals were supplied by the animal house of iraqi center for cancer research and medical inheritance / mustansiriyah university and was accepted by the ethics committee for animal experimentation of college of pharmacy / mustansiriya university, where the study took place. the animals were separated into six rats in each sterilized cage which was installed with an artificial light/dark 12/12 cycle at an appropriate temperature (22±2°c). they left for 14 days for acclimation with free access to water and normal chow pellets. drugs materials were collected from following sources: diclofenac sodium (olfen ®) was purchased from acino pharma ag, liesberg, switzerland. alpha lipoic acid (lipoic forte®) was purchased from america medic & science, usa. bosentan was purchased from cipla ltd, india. lipoic acid and bosentan were suspended in distilled water immediately before oral administration. experimental design thirty male wistar albino rats were randomly divided into five groups (n=6 for each group). control group, rats received distilled water (5 ml/kg, p.o.) for 11 days, on 5th day they received an intraperitoneal injection of normal saline (5 ml/kg). induction group, rats received distilled water (5ml/kg, p.o.) for 11 days, on the 5th day they received an intraperitoneal injection of diclofenac sodium (100mg/kg). lipoic acid group, rats received lipoic acid 200mg/kg by preparing a stock solution of 600mg of α-lipoic acid in 10ml distilled water (each 1ml contains 60mg of α-lipoic acid as suspension) delivered orally by oral gavage for 11 days, on the 5th day they received an intraperitoneal injection of diclofenac sodium (100mg/kg). bosentan group: rats received bosentan 100 mg/kg by preparing a stock solution of 125mg of bosentan in 10ml distilled water (each 1ml contains 12.5mg of bosentan as suspension) delivered orally by oral gavage for 11 days, on the 5th day they received an intraperitoneal injection of diclofenac sodium (100 mg/kg). combination group: rats are treated with a combination of lipoic acid (100mg/kg p.o.) and bosentan (100mg/kg p.o.) for 11 days, on the 5th day they received an intraperitoneal injection of diclofenac sodium (100mg/kg). on 12th day ketamine (alfasan woerden-holland) (90mg/kg) and xylazine (kepro-holland) (10mg/kg) were used to anesthetize the laboratory animals. blood samples were collected by cardiac puncture and were allowed to drain in sterile serum separatory tubes (cmc medical devices & drugs s.l, malaga-spain), and then centrifuged for 7 min at 1500rpm at room temperature. finally the supernatant layer was isolated in eppendorf tubes (2ml) and kept at −20°c to be evaluated later. determination of serum creatinine and serum urea by semiautomated biochemistry analyzer serum samples were processed according to the manufacturer’s instructions (spinreact, s.a.u. ctra santa coloma, spain) and (linear chemicals, s.l.u. joaquim costa 18 2ª planta, spain) to evaluate serum urea and creatinine levels respectively as indicators of acute renal failure (29). determination of serum biomarkers by sandwich enzyme-linked immunosorbent assay (elisa) technique determination of serum oxidative stress biomarkers (superoxide dismutase, malondialdehyde), selective acute kidney injury biomarker (kidney injury molecule-1), and fibrosis biomarkers (transforming growth factor-β, collagen-1, fibronectin) were performed according to manufacturer’s protocols (mybiosource company, usa) (30). statistical analysis the data were provided in the form of means ± standard error (m±sem) by spss-16.0 statistical program. the analysis of variance (anova) was used to determine the significance of various means. when p-value was less than 0.05, a statistically significant difference was reported. pearson correlation analysis also was used in present study to evaluate whether there is an association between the serum biomarkers levels in aki cases (31). the receiver operating characteristic curve (roc) analysis was also used for evaluating accuracy of biomarkers in diagnosis of aki. area under the curve (auc) is a metric that summarizes the diagnostic accuracy of the test (32). results the effect of lipoic acid, bosentan, and the combination of both on serum levels of kidney function biomarkers (urea and creatinine) in rats induced acute renal failure by diclofenac following diclofenac-induced acute renal failure, mean serum creatinine and urea levels in the induction group were significantly elevated (p > 0.05) compared to control group as shown in table iraqi j pharm sci, vol.32(1) 2023 renoprotective effect of lipoic acid and bosentan 277 (1). in addition, mean serum creatinine and urea levels in both lipoic acid and bosentan groups showed a non-significant difference in comparison to the induction group. finally, mean serum creatinine and urea levels in the combination group were significantly reduced (p>0.05) when being compared to the induction group to achieve similar levels to control group as illustrated in the table (1). table 1. the effect of lipoic acid, bosentan, and the combination of both on mean serum levels of creatinine and urea in rats induced acute renal failure by diclofenac. data are mentioned as means± sem (sem: standard error of mean) different small letters (a, b) indicate significant difference between groups (p > 0.05) the effect of lipoic acid, bosentan, and the combination of both on serum levels of oxidative stress biomarkers (mda and sod1) & kim-1 in rats induced acute renal failure by diclofenac in the present study the mean serum levels of mda and kim-1 in the induction group were significantly rise (p >0.05) compared to control group. however, the mean serum mda and kim-1 levels in lipoic acid and bosentan groups showed a non -significant difference when being compared to the induction group. the combination group showed a significant decrease (p>0.05) in mean serum mda and kim-1 levels when compared to the induction group as clarified in table (2). on other hand, the mean serum sod1 levels in the induction group were significantly decreased (p > 0.05) compared to control group. both lipoic acid and bosentan groups showed a non -significant difference in mean serum sod1 levels when being compared to the induction group. meanwhile, the mean serum sod1 levels in combination group were markedly elevated (p>0.05) when compared to all other groups as illustrated in table (2). table 2. the effect of lipoic acid, bosentan, and the combination of both on mean serum levels of mda and sod1 in rats induced acute renal failure by diclofenac data are mentioned as means± sem (sem: standard error of mean) different small letters (a,b) indicate significant difference between groups (p > 0.05) the effect of lipoic acid, bosentan and the combination of both on serum levels of fibrosis biomarkers (tgf-beta1, fibronectin, and collagen type i) in rats induced acute renal failure by diclofenac the mean serum tgf-beta1 and fibronectin levels in the induction group were significantly rise (p >0.05) when compared to control group. on other hand, the lipoic acid group and bosentan group showed a non-significant difference in mean serum tgf-beta1 and fibronectin levels among all other groups. the combination group showed a significant decrease (p>0.05) in mean serum tgf-beta1 and fibronectin levels compared to the induction group as illustrated in table (3). finally, the mean serum collagen type i levels in the induction group did not differ significantly among all other groups as showed in table (3). groups number of rats per group (n=6) mean serum creatinine level (mg/dl) mean serum urea level (mg/dl) control 6 0.6333±0.04216 a 40.33±1.85a induction 6 1.0167±0.0872 b 127±5.955 b lipoic acid 6 0.9±0.08165 b 121±6.255 b bosentan 6 0.933±0.088433 b 123.5±9.59 b combination 6 0.65±0.06191 a 52.67±4.01 a groups number of rats per group (n=6) mean serum mda level (nmol/ml) mean serum sod1 level (u/ml) mean serum kim-1 level (pg/ml) control 6 0.2168±0.0165 a 35.0868±3.5 a 122±6.09 a induction 6 0.493±0.0617 b 20.7957±2.15 b 229±6.21 b lipoic acid 6 0.446±0.0764 b 24.4232±1.835 b 200±11.94 b bosentan 6 0.417±0.0651 b 29.0912±2.78 b 198±12.6 b combination 6 0.221±0.031 a 48.5875±4.21 c 118±9.03 a iraqi j pharm sci, vol.32(1) 2023 renoprotective effect of lipoic acid and bosentan 278 table 3. the effect of lipoic acid, bosentan and the combination of both on mean serum levels of tgfbeta, fibronectin, and collagen type i in rats induced acute renal failure by diclofenac groups number of rats per group (n=6) mean serum tgf beta level (pg/ml) mean serum fibronectin level (ng/ml) mean serum collagen level (ng/ml) control 6 71.14±3.312 a 72.5768±2.322 a 0.0885±0.0334 a induction 6 86.626±3.054 b 84.2857±2.45 b 0.0933±0.049 a lipoic acid 6 78.48±2.34 a b 75.4767±2.25 ab 0.1090±0.0424 a bosentan 6 80.429±2.07a b 78.303±2.12 ab 0.1322±0.0541a combination 6 72.47±2.166 a 70.1548±1.67 a 0.0751±0.0378 a data are mentioned as means± sem (sem: standard error of mean) different small letters (a, b) indicate significant difference between groups (p > 0.05) correlations between the biomarkers in pearson correlation analysis, data showed a significant (p>0.01) positive linear relationship between urea and fibronectin as demonstrated in figure (1). also, urea has a significant (p >0.01) moderate positive linear relationship with kim-1 as clarified in figure (2) .on the other hand, creatinine has a significant (p>0.01) positive linear relationship with kim-1 biomarker as illustrated in figure (3). figure (4) showed a significant (p >0.05) positive linear correlation between fibronectin and creatinine biomarker. finally, a significant (p>0.01) positive linear relationship appeared between fibronectin and kim-1 biomarkers as shown in figure (5) figure 1. weak positive relationship between urea and fibronectin, pearson correlation coefficient (r) = 0.498. figure 2. moderate positive relationship between urea and kim-1, pearson correlation coefficient (r) = 0.792. figure 3. moderate positive relationship between creatinine and kim-1, pearson correlation coefficient (r) =0.677. figure 4. weak positive relationship between creatinine and fibronectin, pearson correlation coefficient (r) = 0.365. iraqi j pharm sci, vol.32(1) 2023 renoprotective effect of lipoic acid and bosentan 279 figure 5. moderate positive relationship between fibronectin and kim-1, pearson correlation coefficient (r) =0.536. receiver operating characteristic curve (roc) analysis in the present study, receiver operating characteristic curve analysis was performed for kim-1 biomarker as clarified in figure (6). in which the sensitivity (true positive rate) was (94.4%) and specificity (false positive rate) was 91.7%. in addition, roc for fibronectin biomarker also was done as demonstrated in figure (7). in which, the sensitivity (true positive rate) was 83.3% and specificity (false positive rate) was 66.7%. figure 6. roc of kim-1 biomarker, area under the curve (auc) =0.986. figure 7. roc curve of fibronectin biomarker, area under the curve (auc) =0.829 discussion the present study emphasized that diclofenac induced aki, as observed by elevated biomarkers of kidney injury, oxidative stress, and fibrosis(4). the first effect of diclofenac in aki is to reduce prostaglandins synthesis, which leads to afferent arteriole vasoconstriction, ischemia and decreased gfr (33).as a result, serum creatinine and serum urea levels elevated significantly (p > 0.05) in the induction group when compared to control group. lipoic acid group showed nonsignificant reduction in both serum urea and creatinine levels when compared to induction group. in addition, serum urea and creatinine levels in bosentan group did not differ significantly (p>0.05) in comparison to induction group. this results disagreed with previous study reported by sharma et al (2020) that induced aki by arsenic, which decrease endothelium nitric oxide synthase that was attenuated by bosentan(34). the combination group showed a significant (p>0.05) reduction in serum creatinine and urea levels when compared to induction group. this could be related to additive effect of lipoic acid and bosentan. after ischemia, transmembrane glycoprotein kim-1 is up-regulated in proximal tubular cells (25) which was consistent with our findings that showed increased serum levels of kim-1 significantly (p>0.05) in the induction group compared to the control group. lipoic acid group showed a slight decrease in serum levels of kim-1 compared to the induction group. this outcome disagreed with a recent study that stated ala alleviate kidney damage by decrease kim-1 serum levels significantly (p>0.05) in the induction group compared to control. this could be due to ala ameliorate folic acid-induced aki by increase iron storage and decrease fenton reaction resulting in decrease oxidative stress(35). meanwhile, present study showed a non-significant difference in serum kim-1 levels in bosentan group when compared to the induction group. on the other hand, the combination group showed a nonsignificant difference in kim-1 serum levels when compared to the control group. as a result, this lead us to believe that there could be an additive effect of lipoic acid and bsentan. the second cause of aki caused by diclofenac is a mitochondrial dysfunction that leads to oxidative stress. the main biomarkers measured in the present study that indicated occurrence of oxidative stress are malondialdehyde (mda) and superoxide dismutase (sod), serum mda levels were significantly elevated in the induction group when compared to control levels. on the other hand, serum sod levels were significantly reduced. meanwhile, serum levels of mda in lipoic acid group were slightly decrease compared to the induction group whereas, sod serum levels slightly increase. these findings were consistent iraqi j pharm sci, vol.32(1) 2023 renoprotective effect of lipoic acid and bosentan 280 with previous study that stated ala didn’t significantly affect serum levels of mda and sod (9). similar results were obtained from the bosentan group. the mda serum levels in combination group were significantly reduced compared to the induction group. meanwhile sod serum levels were significantly elevated. these findings might be explained by the fact that α-lipoic acid decrease nadph oxidase 2 (nox2) and nadph oxidase 4 (nox4) as major sources of free radicals(36).in addition, bosentan increase endothelial nitric oxide synthase (enos) through phosphatidylinositol 3 (pi3) kinase /aki activation(32). as a result, combination therapy can outperform each separate agent. many fibrotic mediators can be activated in aki such as tgfβ1 (37), the serum levels of tgfβ1 in induction group significantly elevated when compared to control group. whereas, lipoic acid group showed a slight reduction in tgfβ1 levels compared to the induction group. the tgfβ1 serum levels in bosentan group didn’t differ significantly when compared to lipoic acid group. finally, significant reduction in serum tgfβ1 levels were observed in combination group when compared to the induction group. high levels of circulating active tgfβ1 produce progressive renal illness such as glomerulosclerosis, and tubuleinterstitial fibrosis which is characterized by mesangial enlargement and extracellular matrix protein like fibronectin build up(38). this fact was consistent with another present study that indicated a significant elevation of serum levels of fibronectin in the induction group when compared to control group. on the other hand, lipoic acid showed a non-significant difference in serum levels of fibronectin when compared to the induction group. these results disagreed with a previous study that stated ala alleviate mesangial cells damage significantly by inhibiting map-kinase phosphorylation resulting in attenuating tgfβ1 and fibronectin levels(39). meanwhile, fibronectin serum levels in bosentan group didn’t differ significantly in comparison to induction and lipoic acid groups. in addition, combination group showed a significant decrease in fibronectin serum levels when compared to induction group. finally, the serum levels of collagen type i in the present study didn’t show significant differences between the all studied groups and further studies needed in concern to these results. pearson correlation test showed a significant positive correlation between fibronectin and urea (r2= 0.498, p >0.01) and between fibronectin and creatinine (r2=0.356, p>0.05). these results agreed with previous study that found prefibrogenic markers associated positively with creatinine and urea levels in short term induction of renal failure(40). interestingly, kim-1 showed a significant positive correlation with fibronectin (r2=0.536, p>0.01). these findings highlighted future perspective on the possibility of using these biomarkers for diagnosis of aki. in the present study, roc curve analysis showed that kim-1 was with high sensitivity and specificity and auc of 0.986 and cut off value of 163.65 pg/ml. this result resemble a recent study that indicated an auc for kim-1 was 0.968 (41). in addition, fibronectin roc analysis recorded an auc of 0.829 and cut off value of 73.87 ng/ml suggesting a promising predictable biomarker for aki diagnosis. this data came in line with another study that indicated the prefibrogenic biomarkers can be utilized to predict aki, increasing the opportunities of more interventions in treatment of renal conditions (40). it should be mentioned that our study has some limitations. as with most experimental studies the research was limited to the measures used. moreover, experimental animal sample size was small. conclusion alpha-lipoic acid and bosentan as combinative therapy have a role in improving kidney functions by decreasing serum urea and creatinine levels significantly compared to using each treatment alone. oxidative stress that occurred in diclofenac induced aki was prevented by reducing serum mda levels and increasing serum sod-1 levels significantly in combinative treatment. ischemic tubular injury was reversed in combinative treatment through reducing serum kim-1 levels significantly compared to the induction group. the combinative treatment showed a significant decrease in fibrosis events through decreasing serum tgfβ1 and fibronectin levels significantly when compared to the induction group. therefore, alpha-lipoic acid and bosentan as combinative therapy could have an ameliorate role against diclofenac induced aki. fibronectin could be a promising biomarker for detection and diagnosis of aki. acknowledgement this work would not have been possible without the support that made by mustansiriyah university / college of pharmacy /pharmacology and toxicology department. references 1. r. vaishnavi pr, gaikwad n, dhaneria sp. assessment of nonsteroidal anti-inflammatory drug use pattern using world health organization indicators: a cross-sectional 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medicinal plants, college of pharmacy, al-mustansiriyah university, baghdad, iraq. abstract lignans are natural products widely distributed in the plant kingdom. they are composed of two ββ-linked phenylpropane (shikimate-derived biogenetic subunits). although the backbone of lignans is composed of phenylpropane units, there is enormous diversity in the structure of lignans leading to different classes of lignans, such as γ-butyrolactone derivatives, eg. hymatairesinol, bicyclooctadiene derivatives, e.g. pinoresinol, tetrahydrofuran derivatives e.g.lariciresinol, di-arylbutandiol derivatives, e.g. secoisolariciresinol. introduction of a further carbon –carbon linkage leads to a class of lignans collectively known as cyclolignans such as tetrahydro-naphthalene derivatives, for example podophyllotoxin. lignans have a broad range of biological activities; many of them show significant antitumour, antimitotic, and antiviral effects. they also have cardiovascular effects, antimicrobial and insecticidal activities. the efflux mechanisms of bacteria to some antibiotics and resistance of bacteria to many antibiotics led to search for antibacterial compound of plant origin to overcome these problems. the antibacterial activity of hmr lignan was determined using the disc diffusion method and the mic of the isolated compound was tested by the broth micro-dilution method. hmr lignan showed activity against staphylococcus epidermidis(17 mm), candida albicans (13mm), proteussp(12mm) and klebsiella sp (12mm). key words: lignans, hydroxymatiaresinol, antimicrobial activity. هيدروكسي ميتارزينول نكنان كمضاد نهمايكروبات امم انعانيوداد مصطفى ك ،*1 و فتوة منور عسيس * اىعشاق . بغذاد،فشع اىعقاقُش واىْباتاث اىطبُت ، ميُت اىصُذىت ، اىجاٍعت اىَستْصشَت ، الخالصة . هْاك phenylpropaneاىينْاُ ٍشمباث طبُعُت راث اّتشاس واسع فٍ اىطبُعت وهٍ تتنىُ فٍ اىْباتاث ٍِ استباط جضئُتُِ ٍِ بُىىىجُتوغُشها . اىينْاُ ٍشمباث راث فعاىُت hmrتْىع مبُش فٍ تشمُب اىينْاُ عيً اىشغٌ ٍِ اّها تتشابه فٍ هُنيها اىشئُسٍ ٍثو . اصداد ومزىل ىها فعاىُت فٍ عالج اٍشاض اىقيب واألوعُت اىذٍىَت واىفُشوطيسشطاُ وأخشي ٍضادة ىيبنتشَا ٍتعذدة فبعضها ٍضادة ى اهتَاً اىباحثُِ فٍ اىسْىاث االخُشة فٍ اىبحث عِ ٍشمباث ٍضادة ىيبنتشَا راث اصو ّباتٍ طبُعٍ ورىل الصدَاد ظاهشة اىَقاوٍت ٍِ قبو اىبنتشَا ىيَضاداث اىحُىَت ومزىل ظهىس سالالث ٍِ اىبنتشَا ىها قابيُت عيً سحب اىذواء وسٍُه خاسج اىخيُت ٍَا َؤدٌ اىً عذً ضذ اىبنتشَا بطشَقت االّتشاس. اظهش اىَشمب ّشاط ضذ اىبنتشَا ٍتَثال بىجىد ٍْع َّى hmr. َهتٌ اىبحث بتحذَذ ّشاط اه فعاىُته اىبنتشَا حىه ّقطت تىاجذ اىَشمب اىْباتٍ. . مفتاح انكهمات : انهكنان، هيدروكسي ميتاريسينول، مضادات انمايكروبات introduction lignans are natural products widely distributed in the plant kingdom. they are composed of two β-β-linked phenylpropane (shikimate-derived biogenetic subunits). lignans are biosynthesized by the coupling of two conifer (l) alcohol units through a one electron oxidative coupling process (figure 1).lignans have a broad range of biological activities; many of them show significant antitumour, antimitotic, and antiviral effects. they also have cardiovascular effects, antimicrobial and insecticidal activities (2 ).in the early 1980s, it was suggested that a diet rich in hmr lignan may help prevent breast cancer through conversion to enterolactone (enl) (3) .an epidemiological study indicated that enl found in the serum of women with no previous history of breast cancer but not in the serum of women who have such a history. this study led to the suggestion that a diet high in lignans may have chemopreventive effects (4) . hmr lignans usually occur in the form of glycosides in the fiber of cereals and some fruit like apricot (5) .the knots of the norway spruce, from finland, are considered to be a rich source of hmr. concentrations of hmr up to 20% have been found in some knots, making the norway sprucea source of hmr for experimental studies (6) .the precipitation of (-)hmr by potassium acetate as a potassium a adduct from a concentrated ethanolic extract, by freudenberg, is a superior method for largescale production (7) . # based on oral presentation in the fourteenth professional and scientific conference of the syndicate of iraqi pharmacists 19-21 march 2013 1 corresponding author e-mail:wmkalani@yahoo.com received: 4/5/2013 accepted: 9/6/2013 iraqi j pharm sci, vol.22(2) 2013 antimicrobial activity of hydroxymatairesinol lignan 31 figure(1):biosynthesis and example of lignans hmr had stronger lipid peroxidation inhibition capacity than any other lignan or flavonoid tested (8) .hmr was compared to the well-known antioxidants trolox (a watersoluble vitamin e derivative), butylated hydroxyanisole, and butylated hydroxytoluene in terms of their ability to inhibit lipid peroxidation, inhibit ldl oxidation, and scavenge superoxide and peroxyl radicals. hmr was found to be the strongest antioxidant, more effective than butylated hydroxyanisole or butylated hydroxytoluenein all assays and stronger than trolox in all assays except in the lipid peroxidation inhibition assay in which the compounds were almost equally active (9) . the oestrogenic activity of hmr was investigated by measuring their effects on growth and apoptotic markers in the human oestrogen-sensitive cell line mcf-7 in comparison to oestradiol (e2). hmr and enl targeted the same intracellular mechanisms acted upon by e2 but had a milder effect. this weak oestrogenic activity was blocked by the anti-oestrogenic drug tamoxifen (10) .it was found that hmr decreases flushing in women during menopause, which indicates that this compound mimics oestrogen activity (11) . searching for antimicrobial natural products have been acquired a considerable importance nowadays due to the development of multiple drug resistance and efflux mechanisms of bacteria to many antibiotics (12) .side effects associated with use of synthetic antibiotics are frequently more reported than that of natural products (13) . three lignans were isolated and characterized from larreatridentata and compounds were tested against 16 bacterial species/strains. results showed that: dihydroguaiaretic (4) acid had activity towards methicillin resistant (mr) staphylococcusaureusand multidrug-resistant (mdr) strains of mycobacteriumtuberculosis (14) . the relationship between the stereochemistry and antimicrobial activity of butane-type lignans was clarified by satoshi et al. all stereoisomers of dihydroguaiaretic acid (4) showed both antibacterial and antifungal activity. no activity of any stereoisomer of secoisolariciresinol (5) was apparent (15) . 4 5 lignans secoisolariciresinol 5, pinoresinol 6 and lariciresinol 7, were isolated from meoh extract from araucaria araucana wood for the first time in this species and their structures determined with spectroscopic methods. the antimicrobial activities of these compounds were determined for the bacteria citrobacter sp., bacillus subtilis, escherichia coli, micrococcus luteus, staphylococcus aureus,and pseudomonas aeruginosa, and for the white rooting and staining fungi mucor miehei,paecilomyces variotii, ceratocystis pilifera, trametes versicolor, and penicillium notatum, in addition, the meoh extract was evaluated against aspergillus niger, candida albicans,fusarium moniliforme, f. sporotrichum and trichophyton mentagrophytes. the most sensitive bacteria against pinoresinol were the gram-positive. however, secoisolariciresinol exhibited antifungal activity against wood rooting (16) . iraqi j pharm sci, vol.22(2) 2013 antimicrobial activity of hydroxymatairesinol lignan 32 6 7 materials and methods general hmr potassisum adduct from linea r company was converted to free hmr lignan by dissolving in phosphate buffer ph 4.5. free hmr from potassium adducts the food supplement hmr potassium adducts (1 g) was dissolve in minimum amount ofaqueous ethanol. phosphate buffer ph 5(100 ml) was added and the ph was adjusted to 4.5. the mixture was stirred for 15 minutes and the free hmr was extracted with ethyl acetate (3 x 100 ml). the combined ethyl acetate layers were dried with anhydrous magnesium sulphate, evaporated under vacuo to give free hmr (7.8 g, 78% yield). the purity of the lignan was tested by tlc (dcm / ethanol, 7%). screening of antibacterial activity hmr was dissolved in methanol to obtain final concentrations of (100, 50, and 25) mg / ml and sterilized by filtration through a 0.45 μm membrane filter. the muller-hinton agar was inoculated with one of the tested bacteria. methanol was used as a control to eliminate solvent effect. ciprofloxacin (5 μg/ disc) was used as a positive control. the agar diffusion method was used to determine antimicrobial activity of the tested lignan (hmr).six mm diameter wells were punched in to the agar. inocula were obtained from overnight cultures on nutrient agar slants at 37 o c and diluted in sterile saline solution to a final concentration of 10 (6) colony forming uints (cfu)/ ml (adjusted according to the turbidity of 0.5 mc farland scale tube). results in this study hmrlignan showed an efficient antimicrobial activity against most of the bacteria and fungi used except pseudomonasaeruginosa .this bacteria showed resistance to all concentration used. the most sensitive microorganism was staphylococcus epidermidis (inhibition zone, 17, 15, 8 mm) and candida albicans showed (inhibition zone, 13, 12, 10 mm). klebsiella sp and proteus sp. also showed sensitivity to all concentrations. escherichia coli was sensitive to a high concentration only (100 mg/ ml). the result of our study revealed that hmr lignan posses antimicrobial properties as antibiotic principles (table 1) table (1): inhibition zones of bacterial growth produced by hmr lignan methanol as a solvent showed no antimicrobial activity against the bacteria, while hmr lignan showed a good inhibition zone for both (staphylococcus epidermidis) and klebsiella sp (figure 2). microorganisms concentrations (mg/ml)/ inhibition zone in mm 100 mg/ml 50 mg/ ml 25 mg/ ml solvent (c) ciprofloxacin proteus sp. 12 9 7 35 pseudomonasaeruginosa 27 escherichia coli 8 32 staphylococcus aureus 8 7 19 candida albicans 13 12 10 klebsiella sp 12 9 7 staphylococcus epidermidis 17 15 8 iraqi j pharm sci, vol.22(2) 2013 antimicrobial activity of hydroxymatairesinol lignan 33 figure (2): inhibition zone for staphylococcus epidermidis discussion natural products have been found to be an efficient antimicrobial compounds especially those with phenolic group. it is necessary to investigate the activity of the diphenolic hmr lignan to improve the quality of health care. many organisms develop resistance to antibiotics which lead to search for novel antimicrobial agents. references 14 and 15 focus on the antimicrobial activity of butane type lignan therefor hmr is also a good example for those butane type antimicrobial lignans. the diameter of inhibition zone of the antibacterial agents the lignans and ciprofloxacin were different according to the kinds, concentrations and purity. 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javascript:al_get(this,%20'jour',%20'pharmacol%20res.'); http://www.ncbi.nlm.nih.gov/pubmed?term=%22kangas%20l%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22saarinen%20n%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22mutanen%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22ahotupa%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22hirsinummi%20r%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22unkila%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22per%c3%a4l%c3%a4%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22soininen%20p%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22laatikainen%20r%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22korte%20h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22santti%20r%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'eur%20j%20cancer%20prev.'); http://www.ncbi.nlm.nih.gov/pubmed?term=favela-hern%c3%a1ndez%20jm%5bauthor%5d&cauthor=true&cauthor_uid=22422605 http://www.ncbi.nlm.nih.gov/pubmed?term=garc%c3%ada%20a%5bauthor%5d&cauthor=true&cauthor_uid=22422605 http://www.ncbi.nlm.nih.gov/pubmed?term=garc%c3%ada%20a%5bauthor%5d&cauthor=true&cauthor_uid=22422605 http://www.ncbi.nlm.nih.gov/pubmed?term=rivas-galindo%20vm%5bauthor%5d&cauthor=true&cauthor_uid=22422605 http://www.ncbi.nlm.nih.gov/pubmed?term=camacho-corona%20mr%5bauthor%5d&cauthor=true&cauthor_uid=22422605 http://www.ncbi.nlm.nih.gov/pubmed?term=camacho-corona%20mr%5bauthor%5d&cauthor=true&cauthor_uid=22422605 http://www.researchgate.net/researcher/38105719_satoshi_yamauchi http://www.researchgate.net/researcher/38355017_toshiya_masuda http://www.researchgate.net/researcher/38796736_takuya_sugahara http://www.researchgate.net/researcher/35703514_yuya_kawaguchi http://www.researchgate.net/researcher/35703514_yuya_kawaguchi http://www.researchgate.net/researcher/31505916_maya_ohuchi http://www.researchgate.net/researcher/39658111_tatsushi_someya http://www.researchgate.net/researcher/35429168_shiori_tominaga http://www.researchgate.net/researcher/39577362_manami_yamawaki http://www.researchgate.net/researcher/39671077_taro_kishida http://www.researchgate.net/researcher/39303878_koichi_akiyama http://www.researchgate.net/researcher/39568939_masafumi_maruyama http://www.researchgate.net/researcher/39568939_masafumi_maruyama iraqi j pharm sci, vol.22(2) 2013 antimicrobial activity of hydroxymatairesinol lignan 34 16. carlos l. cespedesa, j. guillermo avilab, ana m. garcıab, jose becerrad, cristian floresd, pedro aquevequed, magalis bittnerd, maritza miguel martinezc, and mario silvad, z. naturforsch., 2006; 61: 35. 17. conner, d.e. and beuchat , sensitivity of heat stressed yeasts to essential oils of plants, applied environ. microbiol. 1984; 47: 229. 18. willfore, s., reunanen, m., eklund , b., sjoholm, r .kronberg, farminm , p.; pietarenen, s. and holmbom, b., holzforschu, 2004;45: 345 . iraqi j pharm sci, vol.31( 2 ) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits doi: https://doi.org/10.31351/vol31iss2pp260-270 260 role of topical ritodrine hydrochloride in experimentally induced hypertrophic scar in rabbits haitham mahmood kadhim*, fouad kadhim gatea**,1, ahmed r. aburaghif** and kholod a. ali*** *department of pharmacology and toxicology college of pharmacy al-nahrain university, baghdad, iraq. **department of pharmacology and therapeutics, college of medicine, al-nahrain university, baghdad, iraq ***department of dermatology and venereal diseases, college of medicine, al-nahrain university, baghdad, iraq abstract hypertrophic scars are fibroproliferative illnesses caused by improper wound healing, during that, excessive inflammation, angiogenesis, and differentiated human dermal fibroblast (hdf ) function contribute to scarring, whereas hyperpigmentation negatively affects scar quality. over 100 million patients heal with a scar every year. to investigate the role of the beta 2 adrenergic receptor (β2ar); ritodrine, in wound scarring, the ability of beta 2 adrenergic receptor agonist (β2arag) to alter hdf differentiation and function, wound inflammation, angiogenesis, and wound scarring was explored in hdfs, zebrafish, chick chorioallantoic membrane assay (cam), and a porcine skin wound model, respectively. a study identify a β2ar-mediated mechanism for scar reduction. β2arag significantly reduced hdf differentiation, via multiple camp and/or fibroblast growth factor 2 or basic fgf (fgf2)-dependent mechanisms, in the presence of transforming growth factor betaβ1, reduced contractile function, and inhibited mrna expression of a number of profibrotic markers. β2arag also reduced inflammation and angiogenesis in zebrafish and cams in vivo, respectively. in red duroc pig full-thickness wounds, β2arag reduced both scar area and hyperpigmentation by almost 50% and significantly improved scar quality. indeed, mechanisms delineated in vitro and in other in vivo models were evident in the β2arag-treated porcine scars in vivo. both macrophage infiltration and angiogenesis were initially decreased, whereas df function was impaired in the β2arag-treated porcine wound bed. this data reveal the potential of β2arag to improve skin scarring. the purpose of this study was to assess the therapeutic effect of topical ritodrine hydrochloride on hypertrophic scars in rabbits. thirty-two healthy male albino rabbits that divided in to 4 groups were included in the study (healthy; induced untreated hypertrophic scars; induced hypertrophic scars treated with 0.1% triamcinolone acetonide (tac) as a standard drug; and induced hypertrophic scars treated with 0.5% ritodrine hcl gel twice daily for 21 days. histopathology of skin sections, transforming growth factor beta1 tgfβ-1 level, and collagen iii alpha1 in skin tissue were all used as outcome measures. compared to the induced hypertrophic scar group; treatment with ritodrine significantly reduced means of tgf β1 and collagen iii (p ≤0.01); significantly reduce mean score of inflammation (p ≤0.001), significantly lowered scar size (p ≤ 0.001), and significantly lower mean scar height (p≤0.001), but no significant decrease in sei (p>0.05). therapy of induced hypertrophic scar with topical ritodrine was successfully effective in rabbits. it reduced the immunological score (tgf-β1, collagen iii), inflammation, and scar size in a substantial way. this effect was comparable (except in terms of sei) to topical triamcinolone acetonide efficacy. keywords: hypertrophic scar, ritodrine hydrochloride, scar elevation index, collagen iii. هيدروكلوريد الموضعي في الندبة الضخامية المستحثة تجريبياً في األرانب دور الريتودرين ***علي عباس خلودو **رغيف ابو علي رحمة احمد،1**، كاطع كاظم فؤاد، *كاظم محمود هيثم * .،العراق بغداد النهرين، جامعة الصيدلة، ،كلية والسموم االدوية فرع .العراق بغداد، النهرين، جامعة الطب، ،كلية والتداوي االدوية فرع** .العراق بغداد، النهرين، جامعة الطب، ،كلية والزهرية الجلدية االمراض فرع*** الخالصة والذي يتميز بزيادة أو انخفاض في تنظيم بعض عمليات التئام الندبات المتضخمة هي أمراض تكاثرية ليفية ناتجة عن التئام الجروح غير السليم ، ٪ 67٪ إلى 32المتقدم كل عام. يصيب التندب الضخامي مليون شخص في العالم 100الجروح. تؤثر األنسجة المرتبطة بالندبات على ما يقرب من ٪ بعد إصابة الحروق ، اعتمادًا على 91لمصطبغة وتصل إلى ٪ عند األطفال والشباب وذوي البشرة ا75من األشخاص في الدراسات ، وترتفع إلى ، وهو سيتوكين بروتيفي ، مفرط في الخاليا الليفية الناشئة عن الندوب الضخامية ، باإلضافة إلى تعبير مطول 1عامل النمو المحول بيتا عمق الجرح ومستقبالت األدرينالية بسبب نشاط األرومة 1عامل النمو المحول بيتا ذات الصلة. تفاعل مسارات تأشير1عامل النمو المحول بيتا عن مستقبالت 1corresponding author e-mail: dr.fouadk@yahoo.com received: 10/11 /2021 accepted:31 /1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp260-270 iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 261 . األدرينالية. تم تقييم دور نظام اإلشارات األدرينالية في إصالح الجروح الجلدية مؤخًرا ووجد أن تنشيط 2تثبطه ناهضات مستقبالت بيتا الليفية الذي باإلضافة لجرح.مستقبل بيتا األدرينالية يقلل بشكل ملحوظ انتقال الخاليا الكيراتينية ، وهي خطوة أساسية في إعادة االندمال بتشكل النسيج الظهاري ل األدرينالية لديه القدرة على تقليل تندب 2إلى ذلك ، أفادت دراسة أن االنخفاض في تكوين األوعية الدموية للجرح الطبيعي بوساطة مستقبالت بيتا . الجرح وبالتالي قد يكون مفيدًا سريريًا ، ال سيما في الندبات الضخامية ، والمعروف أن لديها األوعية الدموية المنظمة .كان الغرض من هذه الدراسة هو معرفة كيفية تأثير الريتودرين هيدروكلوريد الموضعي على األرانب ذات الندوب الضخامية غير معالجة ؛ مجموعات في الدراسة )صحية ؛ ندوب تضخمية مستحثة 4تم تضمين اثنين وثالثين من ذكور األرانب البيضاء التي تم تقسيمها إلى ٪ 0.5كدواء قياسي ؛ والندوب الضخامية المستحثة التي تم عالجها باستخدام ٪ تريامسينولون أسيتونيد0.1ندوب تضخمية مستحثة تم عالجها بـ أنسجة الجلد المستحثة. ياس, وتم ق1الفا 3 و والكوالجين 1محول بيتا و اليوماً. تم قياس عامل النم 21ريتودرين هايدروكلوريد جل مرتين يومياً لمدة مركبات من كبير بشكل يقلل بالريتودرين العالج ان وجد , المعالجة غير المستحثة الضخامية الندبات بمجموعة النمو مقارنة عامل دبة بشكل ملحوظ، انخفاض متوسط حجم الن (p <0.001) درجة االلتهاب بشكل ملحوظ؛ تقليل متوسط (p<0.001) 3والكوالجين 1المحول بيتا (p< 0.001) ارتفاع الندبة بشكل ملحوظ، وانخفاض متوسط (p<0.001) ( ولكن لم يحدث انخفاض كبير في مؤشر تقييم الندبة ،p>0.05). عامل النمو المحول بيتا من درجة كان فعاالً بنجاح في األرانب. لقد قلل , 1عالج الندبة الضخامية المستحثة بالريتودرين الموضعي ر ، وااللتهاب ، وحجم الندبة بطريقة كبيرة. كان هذا التأثير مشابًها الى فعالية عالج التريامسينولون أسيتونيدالموضعية بأستثناء مؤش 3والكوالجين تقييم الندبة. .، مؤشر ارتفاع الندبة ، الكوالجين الثالث الكلمات المفتاحية: ندبة تضخمية ، ريتودرين هيدروكلوريد introduction hypertrophic scars are fibroproliferative illnesses caused by improper wound healing, which is characterized as an increase or reduction in the regulation of certain wound healing processes (1). during wound healing, excessive inflammation, angiogenesis, and differentiated human dermal fibroblast (hdf) function contribute to scarring, whereas hyperpigmentation negatively affects scar quality. over 100 million patients heal with a scar every year. to investigate the role of the beta 2 adrenergic receptor (β2ar) in wound scarring, the ability of beta 2 adrenergic receptor agonist (β2arag) to alter hdf differentiation and function, wound inflammation, angiogenesis, and wound scarring was explored in hdfs, zebrafish, chick chorioallantoic membrane assay (cam), and a porcine skin wound model, respectively. a study identifies a β2ar-mediated mechanism for scar reduction. β2arag significantly reduced hdf differentiation, via multiple camp and/or fibroblast growth factor 2 or basic fgf (fgf2)-dependent mechanisms, in the presence of transforming growth factor betaβ1, reduced contractile function, and inhibited mrna expression of a number of profibrotic markers. β2arag also reduced inflammation and angiogenesis in zebrafish and cams in vivo, respectively. in red duroc pig fullthickness wounds, β2arag reduced both scar area and hyperpigmentation by almost 50% and significantly improved scar quality. indeed, mechanisms delineated in vitro and in other in vivo models were evident in the β2arag-treated porcine scars in vivo. both macrophage infiltration and angiogenesis were initially decreased, whereas df function was impaired in the β2arag-treated porcine wound bed. these data collectively reveal the potential of β2arag to improve skin scarring. (2) it is elevated, red, inflexible, and causes serious functional and esthetic issues. collagen type iii aligned parallel to the epidermal surface with many collagen nodules is the main component. hypertrophic scars are also characterized by nodular formations including alpha smooth muscle actin expressing myofibroblasts and smaller vessels (3). pathological scarring is a difficult to predict and prevent post-operative consequence (4). scar-related tissues affect roughly 100 million persons in the developed world each year (5). hypertrophic scarring affects 32 percent to 67 percent of people in studies, rising to 75 percent in children, young adults, and those with pigmented skin (6) and up to 91 percent after a burn injury, depending on the depth of the wound (2). scar formation's underlying mechanisms are complex, and they can be influenced by a variety of circumstances (7). in adult tissue, the physiologic reaction to wounding is the creation of a scar, which can be divided into three separate phases: inflammation, proliferation, and remodeling (8). according to recent study, combination therapies of steroids (especially triamcinolone) should be recommended for the treatment of pathological scars. these therapies have the advantage of good curative effects and fewer side effects. but not useful for patients who cannot tolerate the side effects. (9) there are multiple interactions between fibrotic and anti-fibrotic growth factors, cells, extracellular matrix (ecm) components, and other enzymes within these stages, which often overlap (10). transforming growth factor beta 1 (tgf-β1) is a family of growth factors thought to be the master regulator of fibrosis, and its effects on collagen deposition, cell proliferation, immunological regulation, apoptosis, differentiation, and several other processes have been well documented in hypertrophic scar (11). tgf-β is released in three isoforms (tgf-β1, 2, and 3) as inactive latent precursors that must be activated before binding to tgf β receptors (12). tgf-β signaling appears to be altered in hypertrophic generated fibroblasts (due to increased phosphorylation of the receptor smad proteins) and lower expression of the inhibitory smad 7 in hypertrophic scar derived fibroblasts (13). the majority of wound-healing cells produce tgf-β in iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 262 an inactive state that actively stimulates fibroblast chemotaxis to the site of injury (3). tgf-β1, a profibrotic cytokine, was found to be overexpressed in fibroblasts originating from hypertrophic scars, as well as a prolonged expression of the related tgf-β receptors (14). the interaction of the tgf-β1 and adrenergic receptor signaling pathways due to fibroblast activity inhibited by beta 2 adrenergic receptor agonists (β2-arag).(15) β-arags are g protein-coupled receptors (gpcrs) for the endogenous catecholamines, adrenaline and noradrenaline. there are three β-ar subtypes: β1-ar, β2-ar, and β3-ar, which differ in their protein sequences and respond differently to their catecholamine ligands. (16) β-arags can all couple to gαs activating the membrane effector enzyme adenylate cyclase (ac) which generates the secondary messenger molecule cyclic adenosine monophosphate (camp) by catalysing the conversion of adenosine triphosphate to camp. (17) in dermis, β2-arag promote fibroblast migration and proliferation via rous sarcoma oncogenemediated transactivation of the epidermal growth factor receptor and the camp-mediated activation of protein kinase a (pka), respectively, in twodimensional assays in vitro (18). the authors are evaluating the role of the adrenergic signaling system in cutaneous wound repair and recently found that β2-adrenergic receptor (β2-ar) activation markedly decreases keratinocyte migration, an essential step in wound reepithelialization. (19) in addition, a study reported that the reduction in normal wound angiogenesis mediated by β-arag have the potential to reduce wound scarring and may thus be useful clinically, particularly in hypertrophic scarring and keloids, known to have upregulated vasculature. (20) similarly; isoxsuprine (β-arag) is a drug with the ability of direct relaxation of uterine and vascular smooth muscle fibers, stimulation of beta adrenoceptors, production of positive chronotropic and inotropic effects, and dilatation of blood vessels and in particular those supplying skeletal muscles. there are three principal mechanisms that induce the pharmacodynamics of this drug. the first is the stimulation of beta adrenoceptors, the second is the inhibition of α-adrenoceptors, and the third one is the direct papaverine-like spasmolytic of smooth muscles. (21) the observation that beta blockers induce the formation of skin pathology through the enhancement of angiogenesis,(22, 23) we suggest that the use of beta agonist, such as isoxsuprine, may counter act this mechanism, resulting in reduction of scar size and resultant disfigurements. excessive wound inflammation contributes to scarring. a zebra fishtail wound model was used to visualize neutrophil guidance to wounds in real time. (24) β2-arag reduced neutrophil recruitment by 60% after 6 hours. although angiogenesis is essential for wound repair, reduced angiogenesis is linked with improved healing (25) and less angiogenesis occurs in non-scarring oral wounds (26) and scarless fetal wounds. (27) β2-arag significantly reduced angiogenesis in the chick chorioallantoic membrane assay (cam) by 29%. (2, 28) the goal of this study was to investigate the activity of β2-arag (ritodrine hydrochloride) in the treatment of hypertrophic scar in rabbits. materials and methods the present study included 32 healthy male albino rabbits between the ages of 6 and 12 months. the animals were given 48 hours to acclimate to the animal room conditions of controlled temperature (28–30°c) and free access to water and food before beginning the work. al nahrain university college of medicine's institute review board accepted the current study's protocol. ketamine (45 mg/kg) and xylazine (5 mg/kg) injections were used to anesthetize rabbits in the hypertrophic scar model. on the first day, surgical wounds were created using an 8 mm biopsy punch. on the ventral surface of one ear, four injuries were precisely made down to cartilage. after achieving homeostasis with manual pressure, the perichondrial layer was removed, and the wounds were bandaged with sterile gauze for 1 day. on the 30th day, the scars were detected (29). preparation of gels formulations gels formulations of chemicals were prepared as following: first, in order to prepare base gel from hydroxypropyl methyl cellulose (hpmc) approximately 3 g of gelling agent hpmc was weighted and added to75ml of warm distilled water (70 ºc) then stirred with magnetic stirrer for 2hours to obtain homogeneous gel (solution a). second, solution b of chemicals was prepared as following: 1. one hundred milligram (0.1g) of triamcinolone acetonide was weighted then dissolved in 10 ml of absolute ethanol alcohol to prepare (solution b) as slandered drug. 2. five hundred milligram (0.5g) of ritodrine hydrochloride was weighted then added to 10 ml absolute ethanol with the purpose of make (solution b) as suspected active drug. solution a and b were mixed thoroughly and the final weight was made up to 100 ml (30). all the samples were allowed to equilibrate for at least 24 h at room temperature (31). treatment groups the treatment groups are as follows (each with eight animals): group i: healthy animals; group ii: hypertrophic scars were induced and the animals were left untreated (only base gel); group iii rabbits with induced hypertrophic scars were given 0.1 percent triamcinolone acetonide (tac) as a standard drug; group iv rabbits with induced hypertrophic scars were given 0.5 percent ritodrine hcl. drugs were iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 263 given as a formulated topical gel twice a day for 21 days. collection and preparation of samples after anesthetizing the animals at the end of the experiment (51 days), samples were taken using an 11 mm punch biopsy with a margin of more than 3 mm of adjacent skin (32) and submitted for histological and immunohistochemical investigation. each wound sample was preserved in a 10% formaldehyde solution processed in section for histopathological and immunohistochemistry examinations. preparation of formalin-fixed paraffin-embedded tissues the fixative volume was 20 times that of the tissue on a weight-per-volume basis, and the tissue was fixed for at least 48 hours at room temperature before being treated with gentle agitation (33) tissues were subsequently embedded in paraffin blocks. tissue sectioning and slide preparation using a microtome, serial sections of 3–5 μm thickness were produced, and 105 slides were made from each wound paraffin block. to prevent tissue sections from folding during the mounting method, sections were mounted on ordinary slides (for hematoxylin and eosin (h&e) staining) and positively charged slides (for immunohistochemistry) using a water bath at 45°c. each slide was labeled with a pencil to carry the same number on its paraffin block. (34) assessment of histopathological changes in skin sections (height of the scar, scar elevation index, and scar size) the scar elevation index (sei) is calculated as the ratio of the highest vertical height of the scar region between the perichondrium and the skin surface to the highest vertical height of the normal area around the scar between the perichondrium and the skin surface. a blinded examiner used a calibrated ocular reticule to measure each wound; histopathological scores reflecting scar size (35). inflammation was assessed by an expert pathologist and graded as mild, moderate, and severe. mild inflammation was given a score of 1, moderate inflammation was given a score of 2, and severe inflammation was given a score of 3; while a score of 0 was given for no inflammation(36). immunohistochemistry ihc detection and procedure of collagen iii, tgf-β1 (i) anti-collagen iii antibody: rabbit polyclonal antibody to collagen iii (code number: mbs822102) (mybiosource, usa). (ii) antitgf-β1antibody: rabbit polyclonal antibody to tgf-β1 (code number: ab190503) (abcam, uk). on positively charged slides, five-micrometer thick sections were cut, and the staining treatment was carried out according to the manufacturer's instructions with the (ab80436 staining kit). collagen iii alpha1 and tgf-β1 immunohistochemistry kits are employed for detection. evaluation of ihc results under x20 light microscopy, the expression of tgf-β1 and collagen protein was measured. the extent of the immunohistochemical reactivity of ecm proteins like collagen was determined by ranking signal intensities on a scale of – (absence), + (mild), ++ (moderate), and +++ (marked) (37). tgf-β1 immunoreactivity was determined by examining stained slides. a scoring system was established, with the average intensity of the expression being recorded as the score: absence of immunoreactivity received a value of zero, mild immunoreactivity received a score of one, moderate immunoreactivity received a score of two, and strong immunoreactivity received a score of three (38). statistical analysis two statistical software packages were used to gather, summarize, analyze, and present data: the statistical package for the social sciences (spss version 22) and microsoft office excel 2013. all data are presented as means ± standard deviation. the mann–whitney u test and the unpaired t-test were used to compare mean values between the two groups. kruskal–wallis test was used to analyze data for multiple comparisons. p ≤0.05 was considered significant. results healing rate as illustrated in figure 1, the normal healing process of the untreated induced hypertrophic scar involves three overlapping phases: inflammation (0–3 days), cellular proliferation (3– 12 days), and remodeling (3–6 months). as a result, inflammation could be seen in group ii on the 1st day in all animals, with partial wound closure b on the 4th day and severe fibrosis formation (100 percent induction) starting on the 30th day. in the triamcinolone-treated group, healing signs appeared immediately after treatment, with disappearance of inflammatory signs. figure 2a shows complete wound closure and scar thickness reduction after 21 days of treatment. after administration of ritodrine gel (group iv), signs of wound healing gradually appeared. at the end of the 21-day period, there was a decrease in inflammatory signs, wound edges converging and closing, and a partial reduction in scar thickness (figure -3). iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 264 figure 1. gross morphological features of healing rate in the induced hypertrophic scar of rabbits during 30 days figure 2.treatment with triamcinolone acetonide (g3). a. application of topical gel on induced model b. after 21 days of treatment a b iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 265 figure 3. treatment with ritodrine (g4).a. application of topical gel on induced model b. after 21 days of treatment immunohistochemical results tables 1 & 2 demonstrates immunohistochemical results for tgf-β1 and collagen iii. according to the healthy control and the induced hypertrophic scar group recruited in the current investigation, there was a highly significant increase in mean immunohistochemistry scores of tgf-β1 and collagen iii among induced hypertrophic scar group (p≤ 0.001). compared to the induced hypertrophic scar group, treatment with triamcinolone acetonide and ritodrine significantly reduced ihc expression scores for tgf-β1 and collagen iii (p ≤0.01). table 1 & 2. table1. mean tgf-β1 scores in control and study groups: groups mean ± sd p-value healthy control (g1) 1.13±0.35 <0.001* induced hypertrophic scar (g2) 3.0±0.0 0.1%tac steroid (g3) 2.0±0.54 0.002* 0.5%ritodrine (g4) 1.75±0.46 <0.001* kruskal-wallis test. sd standard deviation; p indicate the level of significance at (p≤0.05); * indicate a significant difference between induced hypertrophic scar and the other groups table2. mean collagen iii scores in control and study groups groups mean ± sd p-value healthy control (g1) 1.0 ±0.0 <0.001* induced hypertrophic scar (g2) 3.0 ±0.0 0.1%tac steroid (g3) 2.13 ±0.64 0.01* 0.5%ritodrine (g4) 2.0 ±0.54 <0.002* kruskal-wallis test.sd: standard deviation; p indicate the level of significance at (p≤0.05); * indicate a significant difference between induced hypertrophic scar and the other groups histological results inflammation as shown in table 3, the histopathological score reflecting the scar in the experimentally generated hypertrophic scar was very high and significantly increased in the untreated induced hypertrophic group compared to the healthy control (p<0001). in comparison to the induced hypertrophic scar group, treatment with triamcinolone acetonide and ritodrine resulted in a significant reduction in mean score of inflammation (p ≤0.001). a b iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 266 table3. mean inflammation score among study groups groups mean ± sd p-value healthy control (g1) 0±0 <0.001* induced hypertrophic scar (g2) 2.75 ±0.46 0.1%tac steroid (g3) 0.75±0.46 <0.001* 0.5%ritodrine (g4) 1.13±0.35 <0.001* kruskal-wallis test.sd: standard deviation; p indicate the level of significance at (p≤0.05); * indicate a significant difference between induced hypertrophic scar and the other groups scar size in the untreated induced hypertrophic scar group, histopathological scores reflecting scar size were significantly higher (p <0.001) than in the healthy group, with mean (3.0±0.0) compared to (0.0±0.0) in the healthy group . in comparison with the induced non-treated groups, both triamcinolone acetonide and ritodrine treatment resulted in a significant reduction in scar size (p <0.001) (table 4) table 4.mean scar size score in control and study groups groups mean ± sd p-value healthy control (g1) 0±0 <0.001* induced hypertrophic scar (g2) 3.0 ±0.0 0.1%tac steroid (g3) 0±0 <0.001* 0.5%ritodrine (g4) 0.13±0.0 <0.001* kruskal-wallis test.sd: standard deviation; p indicate the level of significance at (p≤0.05); * indicate a significant difference between induced hypertrophic scar and the other groups height and scar elevation index (sei) according to the healthy control and induced untreated groups, there was a highly significant difference in mean height and scar elevation index (p≤0.001). the mean scar height and scar elevation index in the triamcinolone acetonide group were significantly lower than that of induced untreated group (p≤0.001). in addition to; ritodrine treated group was compared to the induced untreated group and there was a significant reduction in scar height (p=0.047) but no significant decrease in sei (p>0.05). table 5 and figure 4 table 5. scores evaluation and scores among study groups parameters g1 n=8 g2 n=8 g3 n=8 g4 n=8 height of scar mean 0±0 756.25 285.0 687.5 sd 0±0 40.7 27.91 76.5 p-value <0.001* <0.001* 0.047* scar elevation index mean 0±0 8.03 3.03 7.3 sd 0±0 0.87 0.42 1.07 p-value <0.001* <0.001* 0.156* kruskal-wallis test.sd: standard deviation; p indicate the level of significance at (p≤0.05); * indicate a significant difference between induced hypertrophic scar and the other groups figure4. represent height of scar from perichondrium to skin surface(x4) a) normal skin (110µm) b) induced hypertrophic scar tissue (700 µm), (c) treated hypertrophic scar of 0.1% tac steroid gel (320µm), (d) treated induced hypertrophic scar of 0.5% ritodrine gel (700 µm) *(10x, 4x): ordinary hematoxylin and eosin stain. b a c d iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 267 discussion scarring following surgery or injury is difficult to predict, and both physicians and their patients are highly concerned with minimizing scar appearance and value as clinically meaningful even small improvements in scarring. despite a plethora of various in vivo and in vitro studies, to date only limited information is available on the exact cause of hypertrophic scar and keloid formation. knowledge of the cellular and molecular mechanisms implicated in the development of these fibroproliferative disorders remains relatively poor because of the lack of representative and wellrecognized animal models of human hypertrophic scar formation.(39) some herbs have also been found to be helpful in the treatment of hypertrophic scars. in a rabbit ear model, phytosterol 0.3 percent extract of chenopodium murale reduced scarring and was nearly as effective as triamcinolone acetonide. (40) in a rabbit ear model of hypertrophic scar, a phytosterol fraction derived from fumaria officinalis significantly reduced transforming growth factor beta 1 (tgf-β1) on hts. (41) tgf-β1 controls the expression of fibrosis-related proteins such as type i and iii collagens (10). it can also stimulate the transformation of fibroblasts into myofibroblasts, which are important cells in the formation of hypertrophic scars (hts) and are characterized by enhanced collagen synthesis and cytokine up regulation (42). in the current work, hts in the rabbit's ear model was successful since there were substantial changes in cellular response to growth factor (tgf-β1) between induced hts and normal skin, which is consistent with a previous study. (43) after 21 days of treatment, topical triamcinolone acetonide significantly reduced tgf-β1 compared to the untreated group (p<0.001), which is consistent with (44), which demonstrated substantial variations in pro-inflammatory cytokines tgf-β1 and collagen iii in a rabbit ear model after treatment with topical triamcinolone acetonide. in the current study, ritodrine administration for 21 days in the induced hypertrophic scar rabbit model resulted in a significant reduction in immunohistochemical expression of the proinflammatory cytokine tgf-β1, which is consistent with previous study, which found that salbutamol and formoterol; which are beta2 adrenergic receptor agonists (β2-arag), reduce tgf-β1 gene expression (in vitro). (28) in addition to a drop in tgf-β1 after treatment with triamcinolone acetonide, one possible mechanism for collagen distribution in the ecm is the influence on plasma protease inhibitors, allowing collagenase to breakdown collagen (45). in addition, 7 days of topical olodaterol (β2-arag) treatment reduced tgf-β mediator by 50 to 70% in bleomycin-treated mice, which is consistent with our findings (46). ritodrine's exact mechanism, as well as how camp interferes with the tgf-β1 signaling cascade, are unknown. the interference of camp with tgf-β1 specific smad3/4-dependent gene expression is one of the proposed reasons. additionally, camp may suppress fibrotic responses by inhibiting tgf-β1 stimulated erk1/2 and jnk activation via the pka or epac pathways (47). collagen type iii is primarily found parallel to the epidermal surface in hypertrophic scars (1), and the current study findings showed that collagen iii expression is elevated in the induced hts group, which is consistent with oliveira et al (48). this study also discovered a considerable reduction in collagen iii in triamcinolone acetonide, which is consistent with other literature (49). ritodrine, a (β2-arag) medicine, reduced collagen iii in mice wounds, which is analogous to previous research that described the effect of salbutamol a (β2-arag) in mice wounds and reported a significant decrease in collagen iii after 5 and 10 days of follow-up. (50) furthermore, salbutamol and formoterol (β2arag) during wound healing resulted in a significant reduction in collagen synthesis, which is consistent with the findings of this study. (28) in terms of inflammation, the current study found that triamcinolone acetonide considerably reduced the inflammation and had anti-inflammatory activity after 14 days of therapy in a rabbit wound model, which was essentially identical to previous findings (51). ritodrine also had an effect on the inflammatory process after 21 days of treatment, resulting in a significant drop in inflammation, which is consistent with a study reported that a β2-arag reduced neutrophil recruitment in zebrafish wounds within hours of wounding. there was also a decrease in macrophage in the induced scar of the porcine model after 7 days, with a minor increase after 14 days, and no difference after 21 days. (28) β2-arag has been reported to have antiinflammatory effects in addition to their effects on smooth muscle relaxation in the airways. they have been shown to inhibit the expression of inflammatory mediators and to reduce capillary permeability and formation of plasma exudate and tissue edema (52, 53). also; it was reported that β2arag reduced carrageenan-induced paw edema in rats and that effect was attenuated when the β2receptors were blocked by a non-selective βreceptor antagonist (54). another study found that β2arag inhibited the production of tnf in macrophages and carrageenan-induced paw edema was reduced by β-receptor antagonist in rats (54). showing that β2-arag have anti-inflammatory effects in vitro and in vivo. (55) iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 268 topical triamcinolone acetonide reduced scar size significantly, which is consistent with another study reported that a reduction in scar size of 82.3 percent in the steroid group after 4 weeks.(56) in comparison to the generated hypertrophic scar, the ritodrine group showed a significant reduction in scar size. salbutamol (β2-arag) reduces scar area in red durocs by 50% in previous study, which consistent the findings of the current study. (28) the standard medicine triamcinolone acetonide caused a significant decrease in height and sei, which agrees with a previous study. (27) after 21 days of therapy with ritodrine, there was a significant reduction in height and no change in sei in the rabbit ear model. salbutamol causes a 34 percent reduction in height in red durocs, which is consistent with our findings. (28) the lack of a significant decrease in sei following ritodrine treatment may be due to other factors affecting ecm proliferation and disposition with no net reduction in scar index, or that the 3-weeks treatment time was too short to detect a considerable reduction in scar hypertrophy. β2-arag as a regulator of wound healing/scarring. there are currently no clinically tested or licensed interventions/pharmaceuticals available to reduce wound scarring/fibrosis or to improve scar hyperpigmentation. topical salbutamol significantly improved acute skin scarring in vivo and could have significant potential as a treatment. future work will address the potential to improve hypertrophic scarring, keloid formation, and organ fibrosis.(28) therefore, there are still shortcomings in ritodrine, and its conclusions need to be further confirmed by well-designed and rigorous rcts. conclusion induced hypertrophic scar therapy with topical ritodrine proved successful in rabbits. it reduced the immunological score (tgf-β1, collagen iii), inflammation, and scar size in a substantial way. this effect was comparable (except in terms of sei) to topical triamcinolone acetonide efficacy. references 1. lee hj, jang yj. recent understandings of biology, prophylaxis and treatment strategies for hypertrophic scars and keloids. int j mol sci 2018;19:e711. 2. le provost gs, pullar ce. β2-adrenoceptor activation modulates skin wound healing processes to reduce scarring. journal of investigative dermatology 2015;135(1):279288 3. wolfram d, tzankov a, pülzl p, piza-katzer h. hypertrophic scars and keloids-a review of their pathophysiology, risk factors, and therapeutic management. dermatol surg 2009;35:171-81. 4. o’leary r, wood ej, guillou pj. pathological scarring: strategic interventions. eur j surg 2002;168:523-34. 5. krakowski ac, totri cr, donelan mb, shumaker pr. scar management in the pediatric and adolescent populations. pediatrics 2016;137:e20142065. 6. bombaro km, engrav lh, carrougher gj, wiechman sa, faucher l, costa ba, et al. what is the prevalence of hypertrophic scarring following burns? burns 2003;29:299-302. 7. ren ht, hu h, li y, jiang hf, hu xl, han cm, et al. endostatin inhibits hypertrophic scarring in a rabbit ear model. j zhejiang univ sci b 2013;14:224-30. 8. niessen fb, spauwen ph, schalkwijk j, kon m. on the nature of hypertrophic scars and keloids: a review. plast reconstr surg 1999;104:1435-58. 9. yang s, yujia j. luo yj, luo c. network metaanalysis of different clinical commonly used drugs for the treatment of hypertrophic scar and keloid. frontiers in medicine. 2021;8:691628 10. dakhil as. association of serum concentrations of proinflammatory cytokines and hematological parameterspatients. j pharm sci res 2017;9:1966-74. 11. luo l, li j, liu h, jian x, zou q, zhao q, et al. adiponectin is involved in connective tissue growth factor-induced proliferation, migration and overproduction of the extracellular matrix in keloid fibroblasts. int j mol sci. 2017 may 12. 18 (5). 12. abdou ag, maraee ah, al-bara am, diab wm. immunohistochemical expression of tgf-β1 in keloids and hypertrophic scars. the american journal of dermatopathology 2011;33(1):84-91. 13. zhang t, wang x, wang z, lou d, fang q, hu y, et al. current potential therapeutic strategies targeting the tgf-β/smad signaling pathway to attenuate keloid and hypertrophic scar formation. biomedicine & pharmacotherapy 2020;129: 110287 14. raktoe rs, rietveld mh, out-luiting jj, kruithof-de julio m, van zuijle pp, van doorn r, et al. exon skipping of tgfβri affects signalling and ecm expression in hypertrophic scar-derived fibroblasts. scars burn heal. 2020;6:2059513120908857. published 2020 may 28. doi:10.1177/2059513120908857 15. pollard cm, desimine vl, wertz sl, perez a, parker bm, maning j, et al. deletion of osteopontin enhances β₂-adrenergic receptordependent anti-fibrotic signaling in cardiomyocytes. int j mol sci. 2019 mar 20;20(6):1396. doi: 10.3390/ijms20061396. pmid: 30897705; pmcid: pmc6470638. iraqi j pharm sci, vol.31(2) 2022 ritodrine hydrochloride on hypertrophic scar in rabbits 269 16. hall ra. β-adrenergic receptors and their interacting proteins. semin cell dev biol. 2004;15:281–288. 17. hamm he. the many faces of g protein signaling. j biol chem. 1998;273:669–672. 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pmid:22665951 55. keränen t, hömmö t, hämäläinen m, moilanen e, korhonen r. anti-inflammatory effects of β2-receptor agonists salbutamol and terbutaline are mediated by mkp-1. plos one 2016;11(2): e0148144. https://doi.org/10.1371/journal.pone.0148144 56. yang sy, yang jy, hsiao yc. comparison of combination therapy (steroid, calcium channel blocker, and interferon) with steroid monotherapy for treating human hypertrophic scars in an animal model. annals of plastic surgery, 2015;74, s162-s167. this work is licensed under a creative commons attribution 4.0 international license. https://doi.org/10.1371/journal.pone.0148144 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ 404 not found iraqi j pharm sci, vol.31( 2 ) 2022 alginate/ natural polymers synergy doi: https://doi.org/10.31351/vol31iss2pp150-159 150 characterization of alginate with natural polymers combination for drug encapsulation viviane annisa*, teuku nanda saifullah sulaiman**, akhmad kharis nugroho** and agung endro nugroho**,1 *faculty of pharmacy, universitas gadjah mada, yogyakarta, indonesia, 55281 **departement of pharmaceutics, faculty of pharmacy, universitas gadjah mada, yogyakarta, indonesia, 55281 abstract alginate is one of the natural biopolymers that is widely used for drug formulations, combination of alginate with other polymers, such as gum acacia, pectin, and carrageenan can increase mechanical strength, therefore, can reduce leakage of the encapsulated active pharmaceutical ingredient from the polymer matrix. interaction of alginate and these polymers can occur via intermolecular hydrogen bonds causing synergism, which is determined from the viscosity of polymer mixture. alginate was combined with gum acacia/pectin/carrageenan in different blending ratios (100:0, 75:25, 50:50, 25:75, and 0:100) with and without addition of cacl2. the synergism effect is obtained from the design of experimental (doe), and calculation the percentage value of viscosity deviation viscosity synergism index, then the strength of gel was analyzed. the interaction between two polymers was observed using ftir spectroscopy. in distilled water, the synergistic effect was found in the combination of alginate-carrageenan at ratios 25:75 and 50:50. otherwise, in cacl2 solution, synergistic effect appears in alginate-gum acacia (75:25), alginate-pectin (50:50 and 75:25), and alginate-carrageenan (50:50 and 75:25). the synergistic effect and strength of gel polymers increased, with the addition of cacl2. keywords: synergistic interactions, alginate, pectin, carrageenan, polymer viscosity introduction the combinations of alginate with some electronegative polymers such as pectin, gum acacia, carrageenan, etc. have been widely used in drug formulation. to encapsulate active ingredients to prevent degradation (1), improve thermal and chemical stability (2–5), reduce toxicity (6,7), increase the effectiveness of active substances (7,8), control the release of the active substance (9–12), improve mechanical properties of microbeads (13), and as a carrier for drug targeting (14). alginate as biopolymer material does not have sufficient mechanical properties, and this makes it difficult to use in specific products. to overcome this, it can be resolved by combining alginate with other polymers. alginate can dimerize to form bridges with other chains and produce hydrogel networks (15). alginate and other polyelectronegative polymers will interact via intermolecular hydrogen bonds (16,17). the combination with polymers, such as gum acacia, pectin, and carrageenan can increase their synergistic effect compared to the single polymer. the synergistic ability of polymers can be characterized through changes in viscosity that occur when polymers are combined (18). the determination of synergy effect is very useful for the fabrication of the matrix used for drug encapsulation. an approach using experimental design has been studied by jadhav, et al (2018) to identify polymeric synergy. the benefit of using the design of experiment tool is an experiment can be more effective with a smaller sample (19). marimuthu et al (2017) have calculated the synergism index to determine the synergy effect on the combination of carrageenan with several natural polymers (18). on the other hand, nkenmogne et al (2020) have calculated using percentage deviation calculation the synergy effect of a combination of alginate and hydrocolloids from tropical vegetal species (20). the synergism between two polymers can be determined from experimental design or mathematical equations. the objective of this study was the characterization of alginate combination with pectin, gum acacia, or carrageenan with and without the addition of calcium chloride (cacl2) solution. the interaction of two polymers combination would be observed using calculation of synergy effect ability. synergism will be determined using viscosity data obtained from an experimental design, and mathematical equation including viscous synergism index and percentage deviation of viscosity. ftir spectroscopy was used to show the bands of interaction in the polymer’s mixture. 1corresponding author e-mail: nugroho_ae@ugm.ac.id received:26 /9 /2021 accepted: 15/12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp150-159 iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 150 materials and methods materials sodium alginate alg was manufactured by shandong jiejing group corporation (china), gum acacia ga was manufactured by spectrum chemical mfg corporation (california), pectin pc was manufactured by danisco usa inc (usa), kappa-carrageenan cr was manufactured by top p&p co (china), cacl2 was manufactured by pt. smart-lab indonesia and deionized water was supplied from cv. alfa kimia. preparation of polymer mixture solution a polymer solution with a concentration of 1% was prepared by weighing 1.0 g and dissolved slowly into 100 ml deionized water then stirred until homogenous. the solution of alginate (alg) mixed with each gum acacia (ga), pectin (pc), or carrageenan (cr) that weighted with a total weight of 50 g (w/w). the ratio of alg-ga, alg-pc, and alg-cr were 100:0, 75:25, 50:50, and 0: 100. the aqueous solution of combination alg with other polymers was mixed under stirred 500 rpm for 30 minutes together continuously until polymer mixture solution is completely mixed. then, the polymers mixture solution was added to 50 ml 0.2% w/v cacl2 solution. design of experimental (doe) the simple lattice design (sld) software design expert version 10 was applied to determine the ratio of mixture between two polymers then be used to identify the combination synergistic polymers. the sld is one of the types of doe that have been used to address formulation exercises and follow two major constraints, equality, and nonnegatively. the model f ratio was found statistically significant (a<0.0001) indicating that there is only a 0.01% chance that an f-value this large could occur due to noise. the p-value for lack of fit was not significant because a>0.05 indicates the model is good to fit. the sld can describe mixtures with proportions ranging from zero to 100% for the components under study (21). in this study, the ratios were obtained from sld software. the independent factors are the ratio of alginate (x1) and pectin/gum acacia/carrageenan (x2), which are 100:0, 75:25, 50:50, 25:75, and 0:100. the dependent factor is viscosity as a response (y) from polymers mixture (total concentration 1%). the total runs for each combination were 8 runs conducted with 2 replicates. the equation for sld is described as follows: 𝑌 = 𝑏_1 𝑋_1 + 𝑏_2 𝑋_2 + 𝑏_12 𝑋_1 𝑋_2 + 𝑏_12 𝑋_1 𝑋_2 (𝑋_1– 𝑋_2 ) + 𝑏_12 𝑋_1 𝑋_2 (𝑋_1– 𝑋_2 )^2 (equation 1) characterization of polymers mixture determination of viscosity and rheological analysis viscosity was measured using a viscometer brookfield at 50°c±5°c following stirring with spindle no. 02 or 03, at 100 rpm for 15 s for each mixture solution. the data was determined in triplicate in mpa.s. equation 2 described the synergistic effect of combination polymers, but it doesn’t predict the viscosity of the mixture solution. equation 3 was first used by miller and mann (1994) to calculate the power requirements for agitation of mixtures of immiscible liquids and predict a geometric mean viscosity which provided a better approximation to the experimental value for the viscosity of polymer mixture (22). 𝜂𝑚𝑖𝑥 = 𝑋𝐴𝜂𝐴 + 𝑋𝐵 𝜂𝐵 (equation 2) 𝜂𝑚𝑖𝑥 = 𝜂𝐴 𝑋𝐴 × 𝜂𝐵 𝑋𝐵 (equation 3) the determination of percentage deviation was calculated by equation 4. the negative value indicates an antagonistic effect, while the positive value indicates a synergy effect that shows the interaction of coupled network form (20). the theoretical viscosity in this equation is used from equation 1. %𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 = ( 𝜂𝑒𝑥𝑝 𝜂𝑡ℎ𝑒𝑜𝑟𝑖 ()) (equation 4) the viscous synergism index was calculated by equation 5. index value between 0-0.5 means there is antagonistic interaction. index value = 0.5 means no interaction occurs. index value between 0.5-1.00 or > 1 indicates interaction in mixture solution is higher than the sum of two polymers, hence referring to the synergistic effect (18). 𝑆𝑦𝑛𝑒𝑟𝑔𝑖𝑠𝑚 𝑖𝑛𝑑𝑒𝑥 = 𝜂𝑚𝑖𝑥 𝜂𝐴+𝜂𝐵 (equation 5) gel strength gel strength was measured using texture analyzer ametek with type ta1 in duplicates. fourier transforms infrared (ft-ir) spectroscopy analysis samples were dried by freeze dryer to powder and analyzed as kbr pellets using ft-ir spectroscopy thermo scientific nicolet is10 (usa). spectral scanning was measured between the wavelength region 4000 to 400 cm-1. result and discussion design of experimental the simple lattice design was selected to calculate of combination polymer in with and without cacl2 solution with viscosity as a response change (table 1). iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 151 table 1. design of experimental for combination polymers run ratio viscosity (mpa.s) without cacl2 with cacl2 alg:ga alg:pk alg:cr alg:ga alg:pk alg:cr 1 0:100 12.4 8.8 32.8 10.4 9.2 17.2 2 0:100 11.6 8.4 36 11.2 11.6 17.2 3 25:75 26 22 188.8 34 36 39 4 40:60 42 48.4 164 5 50:50 43.2 38.8 145.2 78 210 263 6 75:25 52.4 70.4 75.2 216 229 332 7 100:0 152.8 146.4 146.4 202 209 209 8 100:0 146.4 150.8 150.8 209 234 202 9 100:0 150.8 152.8 146.4 234 202 234 the result of the equation of experimental was represented in table 2. we need additional data for the combination of alginate-gum acacia and alginate-pectin due to the model did not show a lack of fit value. therefore, to obtain a lack of fit value, we fabricated an addition ratio of polymers combination which is aimed at a 40:60 ratio. the model could determine the synergistic of polymer combination. a positive sign of ab refers to a synergistic effect, while a negative sign of ab refers to an antagonistic effect. the polymer combination that showed synergies effect only alginatecarrageenan in distilled water. whereas, in cacl2 solution, the synergy effect has been found not only in alginate-carrageenan, but also in alginate-pectin. the determination of synergism with the design of experimental did not know specific at ratio specifically, to found out the ratio we need equation methods such as viscosity deviation and viscosity index. table 2. model design of experimental mixture lack of fit equation r-squared model alginate : gum acacia without cacl2 0.4762 yviscosity = +150.012 x a + 11.973 x b – 145.915 x ab – 231.753 x ab (a-b) – 304.351 x ab (a-b)2 0.9982 quartic with cacl2 0.9189 yviscosity = +215.008 x a + 10.781 x b – 135.93 x ab + 422.971 x ab (a-b) + 804.437 x ab (a-b)2 0.9848 quartic alginate : pectin without cacl2 0.3989 yviscosity = +149.894 x a + 8.442 x b – 169.073 x ab – 119.074 x ab (a-b) 0.9983 cubic with cacl2 0.5486 yviscosity = +215.052 x a + 10.284 x b + 411.622 x ab + 464.414 x ab (a-b) -1208.3 x ab(a-b)2 0.9825 quartic alginate : carrageenan without cacl2 0.9087 yviscosity = 147.879 x a + 34.4184 x b + 217.089 x ab – 908.428 0.9987 cubic with cacl2 0.0644 yviscosity = +213.42 x a + 14.830 x b + 481.763 x ab – 1033.09 x ab (a-b) 0.9648 cubic viscosity synergism index and percentage deviation of viscosity combination of alginate with other anionic polymers could form an intermolecular association via hydrogen bonding. the synergies effect were shown by viscosity characterization as a result of multicomponent gel (23). alginate gave a major contribution to increase the viscosity of polymer combination. the synergies effect in both polymer combinations could be known from percentage deviation (equation 4) and viscous synergism index (equation 5). if alginate was combined with another polymer, a semi-interpenetrating polymer network (s-ipn) could be formed which increased the mechanical strength of the mixture gel (24) . iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 152 figure 1. the viscosity of mixture solution polymer with presence and absence cacl2. (a) alginate with gum acacia; (b) alginate with pectin; and (c) alginate with carrageenan. data shown are the average values and standard deviation (n=3). in distilled water, the highest viscosity of polymers combination would be the highest alginate composition ratio (fig. 1a and 1b). however, viscosity in the combination of alginate-carrageenan (fig. 1c) would increase if the composition of carrageenan increases. the dispersion of carrageenan could be able to form gel briefly. this gelling ability leads to increase in the viscosity of combination carrageenan with alginate. in cacl2 solution, the viscosity could increase at all polymers combination and their ratio specifically compared with using distilled water without cacl2 (fig. 1). the cation (ca2+ from cacl2) can cross-link with alginate so that an egg-box network is formed. this could increase the viscosity in the combined solution, proportional to an increasing composition ratio of alginate. generally, viscosity from experimental and theoretical did not always have the same value, which concludes the mixture was not much fit (table 3 and 4) due to specific interaction such as synergism. table 3. estimation of the apparent viscosity of alginate mixture without cacl2 ratio gum acacia (mpa.s) pectin (mpa.s) carrageenan (mpa.s) exp. eq. 1ab eq. 2a exp. eq. 1ab eq. 2a exp. eq. 1ab eq. 2a 100:0 150.0 150 150 150 150 150 150 150 150 75:25 52.4 115.6 80.4 70.1 114.7 73.5 76.8 121.4 104.8 50:50 43.7 81.2 43.1 39.1 79.3 36.1 142.3 92.9 73.2 25:75 26.0 46.8 23.1 22.4 44 17.7 190.8 64.3 51.1 0:100 12.4 12.4 12.4 8.7 8.7 8.7 35.7 35.7 35.7 ap > 0.05 indicate no significant difference in mixture ratio that compared to exp. group bp > 0.05 indicate no significant difference in mixture ratio that compared to equation 2 iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 153 table 4. estimation of the apparent viscosity of alginate mixture with cacl2 ratio gum acacia (mpa.s) pectin (mpa.s) carrageenan (mpa.s) exp. eq. 1ab eq. 2a exp. eq. 1ab eq. 2a exp. eq. 1ab eq. 2a 100:0 215 215 215 215 215 215 215 215 215 75:25 265.3 163.8 100.5 212.3 163.7 99.8 341.67 165.55 114.3 50:50 72.3 112.6 47 256.3 112.5 46.4 284 116.1 60.8 25:75 29.7 61.4 22 33.3 61.25 21.5 40.67 66.65 32.3 0:100 10.3 10.3 10.3 10 10 10 17.2 17.2 17.2 ap > 0.05 indicate no significant difference in mixture ratio that compared to exp. group bp > 0.05 indicate no significant difference in mixture ratio that compared to equation 2 figure 2. viscosity deviation of alginate in combination with gum acacia/pectin/carrageenan. (a) in distillate water and (b) in cacl2 solution. in distilled water, combination of alginate with carrageenan at 25:75 and 50:50 have a positive value (fig. 2a). meanwhile, combination alginate with gum acacia and pectin at all weights the ratio showed a negative value, which means no synergy effect occurs. alginate and pectin were able to form a synergistic mixed gel at low ph even in the absence of cacl2. however, in this study, no synergistic effect was found in the combination of alginate and pectin due to the degree of esterification (de) of pectin being categorized as high de. synergism between alginate and pectin depended on a heterogeneous association between galacturonic acid region of alginate and degree of esterification of pectin. both regions could form twofold crystalline arrays (13). the more the degree of esterification, the higher carboxylic groups that were methylated and the lower available carboxylic groups so that the binding pectin with the region of alginate was limited. the presence of cacl2 could make an egg-box network on each pectin and alginate that could enhancement viscosity in mixed combination, so synergies effect occurs. in cacl2 solution, the positive value of viscosity deviation was found in combination of alginate and gum acacia at ratio 75:25, alginate and pectin at ratio 50:50 and 75:25, also alginate and carrageenan at 50:50 and 75:25 (fig. 2b). the synergies effect in cacl2 solution was more than in distilled water due to the cation interaction effect from ca2+. the synergistic effect result in cacl2 solution was a combination of alginate-gum acacia (75:25), alginate-pectin (75:25 and 50:50), and alginate-carrageenan (75:25 and 50:50). otherwise, only a combination of alginate-carrageenan (50:50 and 25:75) had good synergy distilled water. there was no different result from the viscosity index equation compared with the deviation index (table 5). therefore, we could use one of the equations to determine the synergistic effect, either deviation index or viscosity index. table 5. viscous synergism index of alginate mixture in with and without cacl2 ratio viscosity index gum acacia pectin carrageenan without cacl2 a with cacl2 without cacl2 a with cacl2 without cacl2 a with cacl2 100:0 75:25 0.32 1.18 0.44 0.94 0.41 1.47 50:50 0.27 0.32 0.25 1.14 0.77 1.22 25:75 0.16 0.13 0.14 0.15 1.03 0.18 0:100 ap > 0.05 indicate no significant difference in mixture ratio that compared to a solution with cacl2 iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 154 gel strength the combination of the polysaccharide was an effective method to improve the physical properties and interactions of these biomacromolecules(25). the combination of alginate with other polyelectrolyte polymers could create strong complex bonds in gels forming with the addition of divalent ions, such as ca2+ from cacl2 solution. the mechanical strength and chemical stability of alginate beads would result (26). the hydrophilic effect in these natural polymers leads to formation of a viscous gel structure due to their capability to hydrate in water. this is advantageous in encapsulating the drug and forming a viscous layer around the drug hindering the diffusion and causing a prolonged release of the drug, which is proportional with the viscosity and molecular weight of the polymer (11). the gel strength is shown in fig. 3. figure 3. the gel strength of mixture solution of alginate with gum acacia/pectin/carrageenan. a: alginate with gum acacia; b: alginate with pectin; and c: alginate with carrageenan. data shown are the average values and standard deviation (n=2). the structure created by the interaction of ca2+ with alginate is known as the egg-box (fig. 4) (27,28). the ca2+ ion interacts with a guluronic acid residue of alginate and carboxyl group groups of alginate and gum acacia (about 17% glucuronic acid) which were negatively charged ions. the ca2+ ion could not form an egg-box structure like alginate with gum acacia (4), this could be represented by the viscosity value of gum acacia alone that did not change on the addition of cacl2. figure 4. egg-box alginate cross-linking with cacl2 however, in polymer combination of gum acacia with alginate, the higher the alginate fraction, the greater the strength of gel (fig. 5). this interaction occurred via the ionotropic gelation technique by cross-linking between hydroxy groups of both polymers (9). the addition of gum acacia into alginate in cacl2 solution could reduce side-by-side aggregation that can lead to alginate swelling due to electrostatic repulsive force between carboxylate anions (4). iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 155 figure 5. interaction between alginate and gum acacia in cacl2 solution pectin could form an egg-box structure in the presence of ca2+ ions as well as alginate. therefore, increasing gel strength after addition of cacl2 (fig. 3b). gel formation was preferable if pectin had a low degree of esterification (de) instead of a high de. the lower de, the more carboxylic groups that were not methylated so they could bind more ca2+. the pectin that was used in this study had a high de, which was 70%. therefore, the addition of ca2+ did not significantly affect gel strength. the higher the alginate fraction in the mixture solution, the greater gel strength, because ca2+ did not have a significant influence on pectin. the interaction between ca2+ with carboxyl groups of alginate and pectin may cause crosslinking that could reduce electrostatic repulsion between polymers. the character of the gel formed depends on the degree of esterification of pectin and guluronic acid residue of alginate. the synergistic interaction between pectin and alginate in mixed gels was not fully known (fig. 6) (29). figure 6. interaction between alginate and pectin in cacl2 solution carrageenan could undergo ionic gelation with monovalent or divalent ions, such as k+ or ca2+ ions. the interaction of carrageenan with ca2+ ion underwent electrostatic attractions which then formed an intramolecular bridge between oxygen and -oso3 groups of carrageenan. after that, the cross-linking network between carrageenan was formed. cation could induce conformation in the polymer through the coil-helix transition, then the aggregation of helix form a gel (30). in this study, the gel strength in a solution of sole carrageenan with the presence of cacl2 was lower than without cacl2. this was probably because divalent ion did not have a major effect on kappa-carrageenan, but had a major influence with iota-carrageenan which has two sulfate groups (31). in the combination of alginate and carrageenan, the addition of cacl2 could increase the gel strength along with the increase in the alginate ratio. the interaction was formed between the carboxyl groups of alginate and the sulfate and carboxyl groups of carrageenan (fig. 7) (32). iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 156 figure 7. interaction between alginate and carrageenan in cacl2 solution ft-ir analysis ft-ir spectra determine intermolecular interactions and specific vibrations of each functional group in the sample of alginate and other polymers. before ftir, the samples of the hydrogel mixture have been freeze-dried. this step could also initiate or reinforce the potential interactions between the two polymers (33). the ftir spectra of alginate and gum acacia showed similar bands. fig. 8a presents spectra of alginate and gum acacia. in mixture solution, the sharp peaks at 1420 cm-1 along with increasing alginate ratio. the absorption band of the oh moiety has shifted to lower wavenumbers. this change could occur due to there being the interaction of intermolecular hydrogen bonding between alginate and gum acacia(34). the specific peak at region 1035-1072 cm-1 showed a difference on all combinations of the ratio of alginate and gum acacia, depending on the ratio of each polymer, which could be useful to distinguish all ratios. in mixture solution alginate/gum acacia 25:75, the intensity absorption bands at 1635 cm-1 decreased with decreasing of alginate ratio. in mixture solution alginate/gum acacia 75:25, the absorption bands at 1420 cm-1 showed low intensity due to the ratio of gum acacia decreased. the spectra of alginate showed the bands at 1622 cm-1 and 1417 cm-1 that correspond to asymmetric and symmetric c=o stretching vibrations of the carboxylate groups, respectively (9,34). this result described that all mixed ratios of alginate and gum acacia were successfully blended. ftir spectra of sole alginate with the presence of cacl2 (fig. 8b) showed a sharper peak than absence due to carboxylate groups being affected. the sole gum acacia showed the sharper peak at 1073-1036 cm-1 that represented c=o stretching vibrations that attributed to glycosidic linkages. that impact to all ratio combination showed more intense peak than without presence cacl2. spectra of alginate and pectin in distillate water were presented in fig. 8c have specific absorption bands between 1000-1200 cm-1 range correspond to ring vibrations overlapping with the stretching vibrations of c-oh side groups and c-oc glycosidic bond vibration (35). the region between 1600-1800 cm-1 was specific interest that did not show in alginate to compare pectin sample with other polymers (13). the combination of alginate and pectin in cacl2 solution (fig. 8d) showed the typical spectra with some differences compared with a single polymer. although the differences were not significant. in mixture solution, alginate/pectin 75:25 showed two bands between 1077 and 1144 cm-1 similar with bands in single alginate (13). the absorption bands at 1600 cm-1 increased with increasing alginate ratio due to alginate having more carboxylate groups than pectin. in mixture, solution alginate/pectin 25:75 showed the intensive absorption at 1144 cm-1 that correspond to glycosidic pectin vibration. the spectra at 1738 cm1 increased with increasing pectin ratio due to the presence of methyl-esterified carboxyl group (35). this result described that all mixed ratios of alginate and pectin were successfully blended. the ftir spectra of alginate and carrageenan showed similar bands. fig. 8e presents spectra of alginate and carrageenan. the specific peak of carrageenan showed at 1381 cm-1 and 1234 cm-1 assigned to the stretching vibration band of sulfate presence (s-o) (24,36). ftir spectra of sole alginate with the presence of cacl2 (fig. 8f) showed a sharper peak than absence due to carboxylate groups being affected. the sole carrageenan showed a sharper peak at 1149 cm-1 that correspond to glycosidic linkage. that impact to all ratio combination showed more intense peak than without presence cacl2. in mixture solution alginate/carrageenan 75:25, there were two spectra similar with bands in single alginate. the intensity absorption bands of single carrageenan at 1421 cm-1 decreased with increasing alginate ratio due to alginate have more carboxylate groups than carrageenan. in mixture solution alginate/carrageenan 25:75, the absorption bands at 1637 cm-1 showed low intensity, otherwise, the iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 157 intensity would be high following the increased ratio of alginate. this result described that all mixed ratios of alginate and carrageenan were successfully blended. figure 8. ft-ir spectra in cacl2 solution (a) alginate with gum acacia (c) alginate with pectin (e) alginate with carrageenan, in absence cacl2 (b) alginate with gum acacia (d) alginate with pectin (f) alginate with carrageenan. conclusion the result of synergic effect in combination polymers used the design of experimental in distilled water have been found in alginate-carrageenan. whereas, in cacl2 solution, the synergy effect has been found not only in alginate-carrageenan, but also in alginate-pectin. the determination of synergism using the design of experimental cannot show the specific ratio of polymer combination that have synergic effect, so that the synergism should be determined by mathematical equation including viscous synergism index and percentage deviation of viscosity too. determination of synergy effect from percentage value of viscosity deviation and viscous synergism index equation have a similar result. in distilled water, the synergies effect was found in the combination of alginate-carrageenan at ratios 25:75 and 50:50. on the other hand, the addition of cacl2 to alginate-gum acacia (75:25), alginate-pectin (50:50 and 75:25), and alginate-carrageenan (50:50 and 75:25) showed more synergistic effect. the synergistic effect is confirmed with data of strength of gel polymers that increased with the addition of cacl2. conflict of interest the authors have no conflicts of interest regarding this investigation. iraqi j pharm sci, vol.31(2) 2022 alginate/ natural polymers synergy 158 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qureshi a ur r, mahmood ha. kinetics and controlled release of lidocaine from novel carrageenan and alginate-based blend hydrogels. international journal of biological macromolecules, 2020; 147:67–78. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication doi: https://doi.org/10.31351/vol29iss2pp152-160 152 medication safety in patients under 18 years old; a retrospective study based on iraqi pharmacovigilance center database hani gh. jawad*,1, eman s. saleh**, manal m. younus*** * ministry of health and environment, medical city, gastroenterology and hepatology specialized hospital, baghdad, iraq. ** department of clinical laboratory science, college of pharmacy , university of baghdad ,baghdad, iraq. *** head of iraqi pharmacovigilance center, directorate of technical affairs, ministry of health, baghdad,iraq. abstract medication safety is an important part of the comprehensive patient safety term. medication safety is gaining more attention as the world health organization set the goal of decreasing medication harm by (50%) for the next 5 years when launching the third global challenge. studying medication safety in the risk groups such as young ages, children are crucial to learn more about the effect of medicines in this risk group since they are not included in the clinical trials. adverse drug reaction is defined as any harm resulted from the drug itself during medical process journey, while medication errors are any harm resulted from the treatment process rather than the drug or it is the result of the failure in a step of the treatment process and by that it came clear that adverse drug reaction in non-preventable event while medication error is preventable one. the objectives of this study are to find the preventable medication errors from the iraqi database of adverse drug events in ages from neonatal to adolescent age. this study is a retrospective descriptive study conducted using the iraqi pharmacovigilance center database. the study included reports that were received by the iraqi pharmacovigilance center from 1st of january 2014 until the 1st of january 2020. in this study, the total number of reports included was 2344. the type of medication involved, type of adverse event, type of error and phase of error were reviewed. the results show that (50.47%) of the adverse event following administrating medication to the pediatric population were medication errors while adverse drug reaction represented the remaining (49.53%). the prescribing phase was the one of greatest error (59.43%) and the least one was dispensing (0.17%). most reports were general skin and subcutaneous tissue. the most common type of encountered error was self-medication with the non-over-the counter drug by (44.28%). the result of the present study revealed that medication errors contribute significantly to adverse drug event among patients under 18 years old. this study highlights the importance of monitoring medication safety specifically in the risk groups. keywords: medication errors, adverse drug reaction, medication safety, iraqi pharmacovigilance center, pediatric. , دراسة بأثر رجعي طبقا لبيانات المركز العراقي لليقظة عاما 18السالمه العالجية للمرضى دون الدوائية ***و منال محمد يونس **صالح سعدي ، ايمان1,هاني غسان جواد * مدينة الطب، مستشفى الجهاز الهضمي والكبد، بغداد، العراق. وزارة الصحة والبيئة ،* ،كلية الصيدلة، جامعة بغداد، بغداد، العراق. العلوم المختبرية السريرية** فرع العراق. بغداد ، *** مدير المركز العراقي لليقظة الدوائية، دائرة االمور الفنية، وزارة الصحة، الخالصة السالمة العالجية هي جزٌء مهم من مصطلح سالمة المريض. بدأت السالمة العالجية بجذب االنتباه أكثر بعد أعالن منظمة الصحة العالمية ( خالل الخمس سنوات القادمة وذلك خالل المؤتمر العالمي الثالث لها. دراسة السالمة العالجية في %50عن هدفها بتقليص الضرر الطبي بنسبة ) وعة حرجة كمجموعة األطفال هو أمٌر ضروري لمعرفة تأثير األدوية في هذه المجموعة خصوصاً وأنه ال يتم تضمينهم في التجارب السريرية. مجم ليس من األعراض الجانبية هي أي ضرر ينشأ من الدواء نفسه خالل المرحلة العالجية بينما األخطاء العالجية هي أي ضرر ينشأ من العملية نفسها و دف العالج أو هي فشل في أي خطوة من الخطوات العالجية وهكذا فهي حدث يمكن منعه بينما األعراض الجانبية للعالج فهي حدث ال يمكن منعه. ه . لى بالغرضيعٍ إهذه الدراسة هو أيجاد األخطاء العالجية التي يمكن منعها ضمن قاعدة بيانات المركز العراقي لليقظة الدوائية لمن هم أعمارهم بين المركز هذه دراسة دراسة رجعية وصفية لتقارير الحاالت ذات األخطاء العالجية بأستخدام قاعدة بيانات المركز حيث تناولت الحاالت المرسلة إلى الطبي تقرير تم تضمينهم في البحث وتمت دراسة نوع الدواء ونوع الحالة العرضية ونوع الخطأ 2344. 2020\1\1إلى غاية 2014\1\1منذ ( من األعراض الدوائية تعود لألخطاء الطبية بينما األعراض الجانبية الدوائية كانت %50.47وبأي مرحلٍة حدث الخطأ.أظهرت النتائج أّن ) عظم (. م%0.17( بينما أقل مرحلٍة كانت هي مرحلة صرف العالج بنسبة )%59.43(. مرحلة كتابة الوصفة كانت األكثر غالبيةً بنسبة )49.53%) التقارير كانت بصورة عامة لمشاكل في الجلد. وكان خطأ أخذ المريض الدواء غير قابل للصرف بدون وصفة بنفسه هو األكثر عدداً بنسبة ة سنة. هذه الدراس 18(. من النتائج أعاله نستنتج بأن األخطاء العالجية تسبب بشكٍل كبير بالتأثيرات الجانبية للمرضى لمن هم دون ال 44.28%) تجذب األضواء نحو أهمية مراقبة السالمة العالجية في المجاميع الحرجة. تأثير عالجي سلبي، المركز العراقي لليقظة الدوائية، أطفال. سالمة العالج، : أخطاء طبية،المفتاحية لكلماتا 1corresponding author e-mail: hani.pro55@gmail.com received: 5/ 3/ 2020 accepted: 14/6 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp152-160 iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication 153 introduction medication safety is a crucial part of any medical management and any adverse event that is preventable could compromise it. medication safety is the application of various strategies in order to reduce the occurrence of adverse drug events during any stage of the medical process. medication safety is an umbrella term that involves both medication error (me) and adverse drug reaction (adr). medication safety may also refer to adverse drug event (ade) which is defined as any harm that occurs during providing health services to patients regardless to the complexity of the disease and by that the definition of both (me) and (adr) become clear. adr is defined as any harm resulted from the drug itself during medical process journey, while me is any harm resulted from the treatment process rather than the drug or it is the result of the failure in a step of the treatment process and by that it came clear that adr in non-preventable event while me is preventable one (1). one study in england indicates that me cost up to £750 million yearly and its one of the most encountered ade with a great potential to cause patient harm (2). ghadah. et. al, did a systematic review on the epidemiology of me worldwide and concluded that the incidence of preventable ades was estimated to affect 15 people from every 1000 people yearly and that the prevalence of preventable ade as (0.4%) (3). errors that happened but didn’t reach the patient are called “near misses”, “close calls” and “potential ade”. accordingly, not all medication errors cause harm, some are harmless and some do not reach the patient “near misses” (4). in order to detect and reduce the incidence of these ade, every country must establish its own pharmacovigilance system. according to world health organization (who), pharmacovigilance is defined as “the science and activities relating to the detection, assessment, understanding, and prevention of adverse effects or any other medicine-related problem” (5). one of the most priority of each pharmacovigilance system is to develop systems that identify and rule out the root cause of medication error and how to decrease its incidence (6). most agencies categorize the treatment process into 4 phases which include: prescribing, dispensing, administering and monitoring (1). each error must be located in only one of these phases to be identified and corrected in the future and it is a sequence process as one error may lead to the subsequent error also, but for example, if the error started in the prescribing phase and lead to other errors in dispending and other phases, then the fault one must be attributed to prescribing only (1). pediatric patients are prone to medication error at a higher rate than adult patient, this may be attributed to age difference between them and the wide difference in weight between them which make urge to calculate specific dose for each patient individually based on their own weight (7,8). the objective of this study was to measure the prevalence of medication errors, which gender and age group affected more, and at which phase did the medication error occurs (prescribing, dispensing, administration, monitoring) in patients under 18 years depending on the individual case safety reports (icsr) data base of the iraqi pharmacovivelence center. methods this study is a retrospective descriptive study for identifying medication error cases among the reports that contain ade reported to the iraqi pharmacovigilance center from 1st of january 2014 until the 1st of january 2020. official approvals were obtained from the authorities of the university of baghdad\college of pharmacy, the iraqi ministry of health and iraqi pharmacovigilance center prior to the start of the study in order to get an account and log in access to the database of the center. vigiflow was used in order to extract the data of reports received to the center. vigiflow is a data management system created by the uppsala monitoring center (umc). the data of ade were collected, identified and studied. inclusion criteria are any ade case reports for patients under 18 years old and include the use of medicines, herbals, and complementary medicines. exclusion criteria were reports of adult patients and the reports of vaccines (9). in order to distinguish between cases of adr from me, the p method was implied. the p method is adopted by who and it is based on several factors that if met, means that the case is a medication error case and it is preventable. factors included 20 criteria that are related to various personnel involved in the treatment process. from these criteria 1 to criterion 16 are related to health care providers, criteria 19 and 20 are related to the patient himself while criteria 5, 6, 17 and 18 are related to drug product quality. the criteria 5 and 6 are related to both health care providers and drug product quality. the p method requires answering by “yes” whenever one of the criteria is met and that case would be medication error case and it is preventable which means that the root cause for error is known and it can be corrected in the future, or answering by “no” whenever none of the criteria is met which implies that the case is adr case and it is nonpreventable, or by answering “unknown” when the data available is not enough to assess the situation and by answering “not assessable” means that the case does not fit for the criteria to be decided on. the “unknown”, “not assessable” will not be detected in this type of study, as the data available does not help in distinguishing these types of answering and this is summarized in table 1 below (1). iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication 154 table 1. criteria for the assessment of the preventability of ades(1). factors related to preventability criteria yes no uk na professional practice “pr” 1. incorrect dose? 2. incorrect drug administration route? 3. incorrect drug administration duration? 4. incorrect drug dosage formulation administered? 5. expired drug administered? 6. incorrect storage of drug? 7. drug administration error (timing, rate, frequency, technique, preparation, manipulation, mixing)? 8. wrong indication? 9. inappropriate prescription according to characteristics of the patient (age, sex, pregnancy, other)? 10. inappropriate prescription for patient’s clinical condition (renal failure, hepatic failure …), or underlying pathology? 11. documented hypersensitivity to administered drug or drug class? 12. labeled drug-drug interaction? 13. therapeutic duplication? (prescription of 2 medicines or more with similar ingredient) 14. necessary medication not given? 15. withdrawal syndrome? (due to abrupt discontinuation of treatment) 16. incorrect labora tory or clinical monitoring of medicine? product/drug “pd” 17. poor quality drug administered? 18. counterfeit drug administered? patient “pa” 19. non-compliance? 20. self-medication with non-over-the counter drug? age classification of patients was done according to the classification style adopted by the fda which categorizes the age group into neonates, infants, children and adolescents (10) as shown in table 2 below. table 2. age groups as suggested by fda (10). group age neonates birth up to 1 month infants 1 month up to 2 years children 2 up to 12 years adolescents 12 years up to 18 years the medication error was classified into the phases at which the error occurs; prescribing, dispensing, administration, monitoring (1). for classification of drugs and herbals, the anatomical therapeutic chemical (atc) classification system was used, in which the active substances are divided into different groups according to the organ or system on which they act and their therapeutic, pharmacological and chemical properties (11). system organ class (soc) was used for the classification of the adverse event which is the highest level of the hierarchy that provides the broadest concept for data retrieval (12). iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication 155 results the total number of icsr included in the current study was 2344 after the exclusion criteria were implied as shown in figure 1. figure 1. number of cases included and excluded in the study. ade: adverse drug events; aefi: adverse events following immunization the most gender of which aes reported was males by 1237 (52.77%), while aes for females was 984 (41.98%), while 123 reports did not specify the gender of the patient. the most age group of which aes reported was infants by 1239 (52.86%), while aes for children were 763 (32.55%), adolescents 234 (9.98%) and neonates 106 (4.52%). two reports did not specify the age group of the patient. the most province that contributes to reports of aes was baghdad (52.39%), followed by diyala (10.49%). in addition, (10.45%) of the reports were from diwaniyah as shown in table 3. table 3. demographic distribution of individual case safety reports. gender icsr count (%) male 1237 (52.77%) female 984 (41.98%) n\a 123 (5.25%) age of each group years (%) infants 1239 (52.86%) children 763 (32.55%) adolescents 234 (9.98%) neonates 106 (4.52%) n\a 2 (0.09%) city no. of reports received (%) baghdad 1228 (52.39%) diyala 246 (10.49%) diwaniyah 245 (10.45%) n\a 153 (6.53%) nineva 142 (6.06%) najaf 100 (4.27%) basrah 89 (3.80%) anbar 43 (1.83%) erbil 30 (1.28%) karbala 23 (0.98%) kirkuk 20 (0.85%) babylon 16 (0.68%) thiqar 4 (0.17%) wasit 4 (0.17%) maysan 1 (0.04%) grand total 2344 (100.00%) n/a: not available it was found that anti-infective for systemic use drugs had the greatest number of reports by 1141 (48.68%) followed by alimentary tract and metabolism drugs by 596 (25.43%). the third-most reports were for antineoplastic and immunomodulating agents by 153 (6.53%) as shown in table 4. 14930 • total number of report in the database 12814 • 2116 case of aefi were excluded 2344 • 10,470 case of ade related to adult patient were excluded iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication 156 table 4. classification of drugs involved in the individual case safety reports. type of atc classification ae count (%) anti-infective for systemic use 1141 (48.68%) alimentary tract and metabolism 596 (25.43%) antineoplastic and immunomodulating agents 153 (6.53%) nervous system 130 (5.55%) respiratory system 92 (3.92%) systemic hormonal preparations, excluding reproductive hormones and insulins 57 (2.43%) blood and blood-forming organs 45 (1.92%) musculoskeletal system 43 (1.83%) various atc structures 43 (1.83%) dermatological drugs 17 (0.73%) cardiovascular system 11 (0.47%) antiparasitic products, insecticides, and repellents 7 (0.30%) sensory organs 5 (0.21%) genitourinary system and reproductive hormones 4 (0.17%) grand total 2344 (100%) after performing the p method for extraction of me cases from adr cases, the number of adr cases was 1161 (49.53%) while me cases were 1183 (50.47%). regarding the phases at which reaction occur, prescribing was the phase of greatest error (59.43%), followed by administration phase (39.98%), while monitoring phase errors were (0.42%), and in the last dispensing phase errors were (0.17%) as shown in table 5. table 5. adverse drug event type and phases of medication errors. adverse drug events count % medication error 1183 50.47% adverse drug reactions 1161 49.53% grand total 2344 100.00% phase of medication error count % prescribing 703 59.43% administration 473 39.98% monitoring 5 0.42% dispensing 2 0.17% grand total 1183 100.00% for the type of errors according to p method, selfmedication with non-over-the counter drug error is the most encountered one by (44.28%), followed by documented hypersensitivity to the administered drug or drug class error by (34.85%) and incorrect dose error by (10.69%) as shown in table 6. iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication 157 table 6. types of errors according to the p method. types of error count % self-medication with the non-over-the counter drug? 526 44.46% documented hypersensitivity to the administered drug or drug class? 414 35.00% incorrect dose? 127 10.74% wrong indication? 31 2.62% inappropriate prescription according to characteristics of the patient (age, sex, pregnancy, other)? 26 2.20% poor quality drug administered? 20 1.69% drug administration error (timing, rate, frequency, technique, preparation, manipulation, mixing)? 20 1.69% counterfeit drug administered? 5 0.42% necessary medication not given? 5 0.42% inappropriate prescription for patient’s clinical condition (renal failure, hepatic failure …), or underlying pathology? 2 0.17% labeled drug-drug interaction? 2 0.17% non-compliance? 2 0.17% incorrect drug administration route? 1 0.08% incorrect drug dosage formulation administered? 1 0.08% incorrect storage of drugs? 1 0.08% total 1183 100% the most common ade was related to skin and subcutaneous tissue disorders (28.23%), followed by gastrointestinal disorders (25.87%). in addition, (10.82%) of the ade were for metabolism and nutrition disorders as shown in table 7. from the skin and subcutaneous tissue disorders, about (47.83%) were related to penicillin allergy. table 7. adverse drug event resulted from medication error classified by system organ class soc type of adverse event (soc) count % skin and subcutaneous tissue disorders 334 28.23% gastrointestinal disorders 306 25.87% metabolism and nutrition disorders 128 10.82% immune system disorders 105 8.88% respiratory, thoracic and mediastinal disorders 81 6.85% general disorders and administration site conditions 78 6.59% injury, poisoning, and procedural complications 48 4.06% nervous system disorders 32 2.70% cardiac disorders 16 1.35% infections and infestations 14 1.18% renal and urinary disorders 6 0.51% hepatobiliary disorders 5 0.42% musculoskeletal and connective tissue disorders 5 0.42% psychiatric disorders 5 0.42% congenital, familial and genetic disorders 4 0.34% investigations 4 0.34% vascular disorders 4 0.34% blood and lymphatic system disorders 3 0.25% eye disorders 3 0.25% ear and labyrinth disorders 1 0.08% gastrointestinal signs and symptoms 1 0.08% grand total 1183 100.00% soc: system organ class. iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication 158 discussion once a drug is approved to be sold in the market, it will be used by thousands of patients rather than those numbers used during the trials. thus, the information gained during the trials considered incomplete and more information about the effect of these products will appear once it is used by a lot of patients and this creates a powerful tool for reaching the ultimate goal of safe and effective use of drugs(4). the results of the current study revealed that males and infants encountered the most reports by (52.77%) and (52.86%) respectively as shown in table 3. in one iindian study, more than half of the ade reports were for male (58.5%) and infants (53.2%)(13). as for the provincese contribution to reports, it was found that baghdad provinces contribute to the most of reports (52.39%) as compared to others as shown tin table 3, this is expected as baghdad is the capital of iraq and represents the center of most health care institutes and have the largest number of populations among the other provinces. regarding the atc classification system, anti-infective for systemic use was (48.68%) of the total cases in this study as shown in table 4. a study in turkey revealed that the rate of antibiotic use was (70.8%) in pediatric patients, from it, unnecessary antibiotic prescription was the most common cause for inappropriate antibiotic use (51.9%) (14). in two studies conducted in south carolina and nigeria, both revealed that the most reported therapeutic agent that has been associated with hospital me in children is antimicrobials (between (22.9%)(15) and (50.3%)(16)) respectively. the prevalence of medication errors that were found in this study was (50.47%) which are preventable in comparison with (49.53%) that of adrs cases that are non-preventable as shown in table 5 and this draws big attention to supply recommendations to reduce these preventable incidences to improve health and safety. in one ethiopian study that included 1251 patients, it was found that the prevalence of medication errors was (62.7%) (17). prescribing phase error were the most prevelance one according to the result of current study as shown in table 5. richard. et. al, found that education should be targeted to all doctors who have prescribing responsibility for children. because so many doctors care for children as part of their job, this should begin at the undergraduate level and continue in specialty training and as part of induction processes (18). according to p method, self-medication with non-over-the counter drug error was the most encountered one by (44.28%) as shown in table 6, as this error involve any case of patients took medications without counselling physician. from these errors, (sagwa) poisoning was in the lead and it was considered to be fit in this category for this study. sagwa is a folk remedy prepared from a mixture of boiled animal parts of urchin, tar, drug (diphenoxylate and atropine), rose water, and cow feces (19). one study in iraq concluded that there is a significant morbidity and mortality caused by (sagwa) use in children with acute gastroenteritis (20). the most comon type of ade due to me was related to skin and subcutaneous tissue disorders which include rash, erythema, urticaria as related to allergy and mostly because they did not perform sensitivity test. from these, about (47.83%) were related to penicillin allergy. infants must be tested for penicillin allergy before administering of penicillin for the first time. one study in rochester which is a city in the u.s., children were more likely to have a positive penicillin skin test (p < .0001) compared with adults (21). priyadharsini. et. al, found in their study of adr in pediatric patients that rashes and urticaria were the most common type of adr (37%) followed by fever, anaphylactic shock, vomiting, chills, and rigors (22). also, a study in saudi arabia found that skin associated adrs were most frequent in both retrospective and prospective studies (37%) and (42.9%) followed by gastrointestinal tract (33.3%) and (24.5%) respectively (23). the limitation of this study was the lack of completed report information like the absence of patient history in many reports, . also, the accuracy of many reports was not accurate. finally, lower reporting rate for health care professionals other than pharmacists. conclusions and recommendations the result of the present study revealed that medication errors contribute significantly to adverse drug event among patients under 18 years old. further investigations must be done and there must be a periodic analysis of the reports that are submitted to the iraqi pharmacovigilance center. activities to decrease medication errors must be adopted including establishing programs and workshops in pediatric hospitals about diseases and their first-line treatment, providing a chart for physicians and pharmacists that include the recommended doses in relation to the weight and height of patients, encouraging the pharmacists to participate in medication decision during morning tours, and encourage the pharmacist to educate parents and medical staff about the correct way of administrating the medications. iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication 159 references 1. benabdallah g, 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alezzi j, j kadhim t. morbidity and mortality associated with community used herbal (sagwa)use in children with acute gastroenteritis in diyala governorate. diyala j med. 2019;17(2):92–106. iraqi j pharm sci, vol.29(2) 2020 medication errors related to pediatrics medication 160 21. tanzi mg. new recommendations released for allergic, respiratory conditions. pharm today [internet]. 2017;23(2):22-23. 22. priyadharsini r, surendiran a, adithan c, sreenivasan s, sahoo fk. a study of adverse drug reactions in pediatric patients. j pharmacol pharmacother . 2011;2(4):277-280. 23. khan lm, al-harthi se, saadah oi. adverse drug reactions in hospitalized pediatric patients of saudi arabian university hospital and impact of pharmacovigilance in reporting adr. saudi pharm j. 2013;21(3):261-266. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma doi: https://doi.org/10.31351/vol29iss1pp154-165 154 evaluating the clinical outcomes of three medication regimens for treating a sample of iraqi persistent asthmatic patients ali l. jasim *, 1, eman s. saleh ** and mustafa n. abd ali *** *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. *** department of medicine, baghdad college of medicine, university of baghdad, baghdad, iraq abstract asthma is a complex disease defined by chronic airway inflammation and airflow limitation causing variable respiratory symptoms which include shortness of breath, wheezing, chest tightness and cough. asthma guidelines recommend adding a second long acting bronchodilator to medium doses of inhaled corticosteroids rather using high doses of inhaled corticosteroid alone to control moderate to severe persistent asthma. the aim of this study was to evaluate the clinical outcomes of three medication regimens indicated for treating a sample of iraqi patients suffering from persistent asthma. this study was interventional randomized clinical study conducted on a sample of adult iraqi asthmatic patients in baghdad city. the study composed of three visits distributed over eight weeks; baseline visit followed by first follow up and second follow up visits after four and eight weeks respectively. the study enrolled 78 adult patients with moderate to severe persistent asthma as diagnosed by specialist physician according to patient history and baseline pulmonary function test and allocated them randomly to three groups (each group included 26 patients) to receive equivalent medium doses of budesonide inhaler in addition to either formoterol inhaler, oral modified release aminophylline tablets or tiotropium inhaler (first, second and third group respectively). sixty four patients completed this study. the mean ages of patients were above 35 years with slightly more male predominance. the study groups developed significant increase of peak expiratory flow rate and forced vital capacity values at the first follow up visit compared to baseline values (p<0.001). thereafter, the first and third groups achieved significant higher values at the second follow up visit compared to first follow up visit (p<0.001), while second group produced no change. all the groups developed significant improvement of mini asthma quality of life questionnaire scores and percentage of symptom free days at first follow up visit and continued further significant improvement at the second follow up visit (p<0.001). generally, between groups comparison according to extent of change of study parameters revealed that third group produced the greatest improvement over the entire study period followed by the first group, whereas the second group was associated with the least extent of improvement. this study concluded that all groups caused significant improvement in study parameters compared to baseline values and also concluded that the third group which consisted of budesonide and tiotropium inhalers was associated with the highest extent of improvement followed by first group, while the second group was the least. key words: persistent asthma, tiotropium inhaler, pulmonary function test, mini asthma quality of life questionnaire. تقييم المخرجات السريرية لثالثة انظمة دوائية في عالج عينة من مرضى الربو المزمن العراقيين تقييم المخرجات السريرية لثالثة انظمة دوائية في عالج مرضى الربو العراقيين ***و مصطفى نعمة عبد علي **و ايمان سعدي صالح 1*،علي لطيف جاسم * فرع الصيدلة السريرية، كلية الصيدلة، جامعة بغداد، بغداد، العراق ** فرع العلوم المختبرية السريرية، كلية الصيدلة، جامعة بغداد، بغداد، العراق *** فرع الطب، كلية الطب، جامعة بغداد، بغداد، العراق الخالصة وتقييد مزمن في تدفق الهواء مما يسبب اعراض تنفسية متفاوتة تشمل الربو مرض معقد يعرف بالتهاب مزمن يصيب المجاري التنفسية صعوبة التنفس و صفير اثناء الزفير وضيق الصدر وسعال. توصي القواعد االرشادية باضافة دواء ثان مديد الفعالية موسع للقصبات الى جرع ويدات منفردة بجرع عالية في عالج الربو متوسط الى شديد الدرجة. متوسطة من الكورتيكوستيرويدات المستنشقة بدال عن استعمال الكورتيكوستير .الهدف من هذه الدراسة هو لتقييم المخرجات السريرية لثالثة انظمة دوائية مختلفة في عالج عينة من مرضى عراقيين مصابين بالربو المزمن 1corresponding author e-mail: alilateef2010@gmail.com received:15 /10 /2019 accepted:23 /11 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp154-165 iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 155 الدراسة الحالية هي دراسة تداخلية عشوائية سريرية اجريت على عينة من مرضى الربو العراقيين في مدينة بغداد وتكونت من ثالث اسية ن الزيارة االسزيارات موزعة على ثمانية اسابيع: الزيارة االساسية و زيارة المتابعة االولى و زيارة المتابعة الثانية بعد اربعة و ثمانية اسابيع م مريضا بالغا مصابا بالربو المزمن متوسط الى شديد الدرجة حسب تشخيص الطبيب االختصاصي المبني على 78على التوالي. سجلت الدراسة مريضا في كل مجموعة ليتم عالجهم 26تاريخ المريض و فحص وظائف الرثة االساسي. تم تقسيم المرضى عشوائيا الى ثالثة مجاميع عالجية بواقع بخاخ رع متوسطة متكافئة من بخاخ البودسونايد باالضافة الى بخاخ فورموترول أو أقراص االمينوفيللين الفموية ذات التحرير المديد للدواء أوبج 35 مريضا. تجاوز معدل المرضى 78تايوتروبيام )المجموعة االولى و الثانية والثالثة بالتتابع(. أكمل اربع و ستون مرضا الدراسة من مجموع عاما مع غلبة قليلة للمرضى الذكور. انتجت مجموعات الدراسة زيادة معنوية في القيم المقاسة لكل من سرعة جريان الزفير القصوى و السعة الحيوية القسرية خالل زيارة نويا في زيارة المتابعة الثانية بالمقارنة مع المتابعة االولى بالمقارنة مع الزيارة االساسية. بعد ذلك حققت المجموعتان االولى والثالثة قيما أكبر مع ر الربو على جودة زيارة المتابعة االولى بينما لم تنتج المجموعة الثانية اي تغيير. انتجت المجاميع الثالثة تحسنا معنويا في االستبيان المختصر لتاثي المتابعة االولى واستمر التحسن المعنوي المسجل في زيارة المتبعة الثانية. الحياة و النسبة المئوية اليام االسبوع الخالية من اعراض الربو في زيارة ر التحسن خالل اجماال, بينت المقارنة بين المجاميع على اساس مقدار التغير في معالم هذه الدراسة ان المجموعة الثالثة انتجت الدرجة االكبر في مقدا نما كانت المجموعة الثانية صاحبة الدرجة االقل في مقدار التحسنفترة الدراسة الكلية متبوعة بالمجموعة االولى بي ستنتجت الدراسة ان جميع المجموعات سببت تحسنا معنويا في معالم الدراسة مقارنة مع قيمها االساسية قبل الدراسة كما واستنتجت ان ا ر االكبر من هذا التحسن متبوعة بالمجموعة االولى في حين ان المجموعة المجموعة الثالثة المكونة من بخاخي التايوتروبيام و البودسونايد سببت المقدا الثانية كانت االقل مقدارا الكلمات المفتاحية: الربو المزمن, بخاخ التايوتروبيام, فحص وظائف الرئة, االستبيان المختصر لتاثير الربو على جودة الحياة introduction "asthma is a complex disease defined by chronic airway inflammation causing the characteristic history of respiratory symptoms which include shortness of breath (sob), wheezing, chest tightness and cough that vary in intensity and frequency together with variable expiratory airflow limitation" (1). the variable nature of airflow obstruction is the principle cause of variable asthma symptoms which resolve spontaneously or in response to certain asthma medications forming the reversible pattern of asthma (2). asthma is an important cause of health resources utilization, lessened activities and impaired quality of life (qol) of the asthmatic patients (3). the major mechanism responsible for the fluctuating pattern of chronic or persistent asthma is the chronic inflammation leading to functional and structural abnormalities of respiratory airways (4). the parasympathetic neurotransmitter, acetylcholine (ach), is a key pathophysiologic factory involved in precipitating multiple changes in asthmatic air passages; it stimulates airway smooth muscle spasm, mucus hpersecretion in addition to airway remodeling through its effects on many types of muscarinic (m) receptors which are m1, m2 and m3 receptors (5). the most important goal of persistent asthma treatment is to achieve the maximum possible extent of asthma control by decreasing the frequency and severity of asthma symptoms (6), enhance patients' qol (7) status and improve the pulmonary function test (pft) parameters which represent objective measure of airway obstruction (8). inhaled corticosteroids (ics.s) are the most effective and are considered the first line anti-inflammatory drug for controlling moderate or severe persistent asthma when co-administered with one or more controller medication; the dose requirement of ics increases as severity worsens (9). because risk of ics induced systemic side effects increases when their doses increase beyond the daily dose of 800 µg of budesonide or its equivalent, guidelines of asthma therapy advocate adding a second long acting bronchodilator to lower doses of ics to control moderate to severe persistent asthma instead of high doses of ics monotherapy(10). long acting β-2 agonists (laba.s) are inhaled bronchodilators indicated for long term control of persistent asthma when used in combination with ics since they have a long duration of action of 1224 hours. the most commonly prescribed labas are salmeterol and formoterol (11). unfortunately, it was noted that patients may experience reduced response to labas as well as reliever short acting β-2 agonist (saba) up on long term regular administration of labas which was attributed to tachyphylaxis (12). oral methyl xanthine bronchodilators [theophylline and its derivative aminophylline] represent a wellknown class of bronchodilators which was used for long time in the management of persistent asthma (13), but their use declined due to narrow therapeutic index which can be overcome by prescribing relatively lower doses of theophylline in the range of 400-600 mg/day (14). the inhaled long acting muscarinic antagonist (lama), tiotropium, is approved recently for the control of persistent asthma based on findings of many studies that demonstrated its clinical efficacy (15) and excellent safety profile (16). tiotropium should co prescribed with ics to avoid the possible increase mortality associated with lama monotherapy (17). this study aimed to assess the clinical efficacies of three medication regimens which consist of a corticosteroid inhaler with a controller bronchodilating medication for treatment of a sample of iraqi patients suffering from moderate to severe persistent asthma . patients and methods patients this study was interventional open label randomized eight weeks clinical study conducted from september -2018 till june 2019 on adult iraqi iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 156 asthmatic patients in two centers in baghdad city; the first center was dowaly private hospital (respiratory clinic) and the second center was alzahra center of asthma and allergy. the study enrolled 78 adult patients and allocated them randomly to three groups (each group included 26 patients) to receive one of the following treatment regimens: first group: budesonide 160 µg / formoterol 9 µg (bud/for) combined in one dry power inhaler (dpi) as two puffs every 12 hours second group: budesonide 200 µg dpi + modified release aminophylline 225 mg tablet (bud/ami): budesonide was administered as two puffs every 12 hours and aminophylline tablets were administered as one tablet every 12 hours after meals. third group: budesonide 200 µg dpi + tiotropium 18 µg dpi (bud/tio): budesonide was administered as two puffs every 12 hours and tiotropium was administered as single capsule inhaled via the dpi at evening inclusion criteria 1adult male and female patients between 18 – 70 years old with symptomatic moderate – severe persistent asthma as diagnosed by the specialist physician 2history of asthma symptoms or diagnosis of asthma for at least 6 months. in both situations, the status of asthma symptoms and severity should continue for at least one month prior to baseline visit 3use of inhaled or nebulized salbutamol for quick relief of asthma attacks for at least 4 weeks before baseline visit 4nonsmoker or ex-smoker of less than 10 pack years that stopped smoking at least one year before enrollment visit exclusion criteria 1pregnant and breast feeding women 2current significant respiratory or cardiac diseases 3regular administration of asthma controlling medications within 4 weeks of the baseline visit 4respiratory tract infections or asthma exacerbation which was treated by systemic steroids within four weeks of the enrollment visit methods this study composed of three visits; the baseline visit which was set for recruiting patients and recording their baseline data, while the remaining visits after four and eight weeks respectively were set for assessing the patients' responses to the study medication regimens (first and second follow up visits). patients performed pft maneuver and recorded their qol level and percentage of symptom free days (sfds) at each visit. the pft procedure was performed using spirometers under the supervision of well-trained technician. the procedure was performed three times and the best results of the pft parameters were recorded. the qol status was estimated using the mini asthma quality of life questionnaire (miniaqlq) which is a simple tool used to measure the impact of asthma and its treatment on patient qol during the past two weeks (18). this questionnaire consists of fifteen elements; each element can be measured from 1-7 numbering scale where one reflects worst impairment, while seven reflects no impairment. a change of 0.5 point from the mean score represents the minimal clinically important difference (mcid) (19). the english format of miniaqlq was used in this study and patients took about 4-6 minutes to complete it by the support of researcher. the percentage of sfds was calculated roughly according to the evaluation of the patients themselves during the last seven days preceding each visit. to calculate percentage of sfd; number of days with no symptoms were divided by seven and then multiplied by 100 %. statistical analysis continuous variables were presented using their means ± standard deviation (sd), whereas discrete variables were presented using their numbers and percentages. for the analysis of discrete variables, chi square test or fisherfreeman-halton exact test was applied. trend (or repeated measure) anova was applied to test the differences of means within each individual group using the measured values in the three visits, while one way anova was used to analyze the differences among the groups using the means of values at baseline visit and then used the means of changes (increment or decrement) of values produced during the first and second four weeks of study. thereafter, if the overall comparison revealed significant differences, post hoc tukey test was applied to analyze the significance difference between each pair of means. if data did not follow normal distribution, kruskal wallis test was performed instead of one way anova, whereas friedman anova was used instead of trend anova. statistical package for the social sciences (spss) version 23.0.0 (chicago, il), graphpad prism version 8.0.0 for windows, graphpad software, san diego, california usa, and software package was applied to conduct the statistical analysis. the level of difference was chosen to be significant when p value was less than 0.05. results sixty-four patients completed this study; twenty-one patients in each of first and second group, while twenty-two patients completed the study in the third group. fourteen patients discontinued prematurely the medication regimens due to different reasons and their data were excluded. the socio-demographic data of the recruited patients were well balanced between the studied groups as shown in table 1; the mean ages of patients were older than 40 years in the first group, whereas they iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 157 were 35 40 years in the remaining two groups. the first and second groups included slightly more male patients, while the third group included equal numbers of both genders. all patients were overweight and their bmi values were in the range of 25-30 kg/m2. marital status showed predominance of married patients in the groups. about two thirds of patients were urban residents and the remaining third were rural. nearly 70% of patients never smoked and the majority of them were unemployed. educational status revealed higher proportion of secondary level followed by college, primary and illiterate levels respectively. table 1. comparison of socio-demographic data between the study groups. group first group (bud/for) second group (bud/ami) third group (bud/tio) p-value number of patients 21 21 22 age (y), mean ± sd 41.6±15.2 35.4±10.4 36.3±10.9 0.218a gender, n (%) 0.893b female 9 (42.9%) 10 (47.6%) 11 (50.0%) male 12 (57.1%) 11 (52.4%) 11 (50.0%) bmi (kg/m2), mean ± sd 27.0±3.6 27.7±3.1 28.5±2.7 0.305a marital status, n (%) 0.904 b married 13 (61.9%) 14 (66.7%) 15 (68.2%) unmarried 8 (38.1%) 7 (33.3%) 7 (31.8%) residency, n (%) 0.691 b urban 15 (71.4%) 14 (66.7%) 13 (59.1%) rural 6 (28.6%) 7 (33.3%) 9 (40.9%) smoking, n (%) 0.619b non-smoker 17 (81.0%) 16 (76.2%) 15 (68.2%) ex-smoker 4 (19.0%) 5 (23.8%) 7 (31.8%) occupation, n (%) 0.782c student 3 (14.3%) 3 (14.3%) 3 (13.6%) unemployed 12 (57.1%) 12 (57.1%) 12 (54.5%) employed 4 (19.0%) 6 (28.6%) 4 (18.2%) retired 2 (9.5%) 0 (0.0%) 3 (13.6%) education level, n (%) 0.782c illiterate 4 (19.0%) 1 (4.8%) 2 (9.1%) primary 5 (23.8%) 4 (19.0%) 5 (22.7%) secondary 7 (33.3%) 9 (42.9%) 9 (40.9%) college 5 (23.8%) 7 (33.3%) 6 (27.3%) bud/for: budesonide/formoterol bud/ami: budesonide/aminophylline bud/tio: budesonide/tiotropium y: years sd: standard deviation n: number bmi: body mass index with unit of kilogram per square meter a: one way anova b: chi square test c :fisher-freeman-halton exact test asthma history and baseline symptom characteristics of recruited patients were shown in table 2; patients randomized to first, second and third groups were diagnosed to have asthma before 9.8, 9 and 6.7 years respectively, while the duration of disease deterioration before the baseline visit was 3.3, 3.1 and 3.3 months respectively. shortness of breath and wheezing were the most commonly encountered symptoms at time of presentation (each symptom was developed by 61 patients). the other less commonly presented symptom was chest tightness and the least one was coughing. iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 158 table 2. comparison of baseline asthma characteristics between the study groups. group first group (bud/for) second group (bud/ami) third group (bud/tio) p-value number of patients 21 21 22 duration of asthma (y), mean ± sd 9.8±5.1 9.0±4.3 6.7±3.5 0.064a duration of asthma deterioration (month), mean ± sd 3.3±1.5 3.1±1.4 3.3±1.5 0.842a asthma symptoms sob, n (%) 20 (95.2) 20 (95.2) 21 (100.0) 0.542b cough, n (%) 17 (81.0) 17 (81.0) 16 (72.7) 0.806b wheezing, n (%) 21 (100.0) 21 (100.0) 19 (86.4) 0.096b chest tightness 17 (81.0 19 (90.5) 18 (81.8) 0.755b bud/for: budesonide/formoterol bud/ami: budesonide/aminophylline bud/tio: budesonide/tiotropium y: years sob: shortness of breath n: number a: one way anova b: fisher-freeman-halton exact test the effect of study regimens on patients' peak expiratory flow rate (pefr), a pft parameter, was illustrated in table 3. all groups developed significant increase of pefr values at the first follow up visit compared to the baseline visit (p<0.001). thereafter, the measured values of first and second groups at the second follow up visit were significantly higher than their counterpart first follow up values (p<0.001), unlike the second group which produced no change between the first and second follow up visits (p=1.0). table 3. effect of study regimens on pefr group first group (bud/for) second group (bud/ami) third group (bud/tio) p-value number of patients 21 21 22 pefr (l/sec) baseline 2.7±0.7 3.1±1.1 3.3±0.9 0.111 a first follow up (after 4 weeks) 3.1±0.8 * 3.6±1.4 * 4.0±1.0 * 0.041 a second follow up (after 8 weeks) 3.5±0.9 *, ¥ 3.6±1.2 * 4.3±1.08 *, ¥ 0.073 a p-value <0.001b <0.001b < 0.001b bud/for: budesonide/formoterol bud/ami: budesonide/aminophylline bud/tio: budesonide/tiotropium pefr: peak expiratory flow rate l/sec: liters/seconds a: one way anova b: trend anova (repeated measure anova) *: significant difference compared to baseline values ¥: significant difference compared to first follow up values iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 159 comparison between the three groups according to the extent of change of pefr revealed no significant difference among the groups during the first four weeks of treatment (p=0.093), although numerically third group produced the highest change as shown in figure 1. figure 1. comparison of groups according to extent of pefr change during the first four weeks of study. bud / for (first group): budesonide/formoterol bud /ami (second group):budesonide / aminophylline bud / tio (third group): budesonide /tiotropium . both first and third groups maintained significant higher increments of pefr than the second group during the second four weeks of study (p=0.002, p=0.003 respectively). meanwhile, there was no significant difference between the first and second groups (p=0.97) as demonstrated in figure 2. figure 2. comparison of groups according to extent of pefr change during the second four weeks of study. *: significant difference compared to second group (bud/ami group) bud/for (first group): budesonide/formoterol bud/ami (second group): budesonide/ aminophylline bud / tio (third group): budesonide / tiotropium . the impact of medication regimens on the patients' forced vital capacity (fvc), another pft parameter, was demonstrated in table 4. the fvc values measured in the first follow up visit were significantly increased in comparison with pretreatment values in all groups (p<0.001). the fvc continued to increase further in first and third groups and attained new significant higher values in the second follow up visit compared to the first follow up visit (p<0.001), while the second group demonstrated similar values in the first and second follow up visits (p=0.201). table 4. effect of study regimens on fvc. group first group (bud/for) second group (bud/ami) third group (bud/tio) p-value number of patients 21 21 22 fvc (liters) baseline 2.3±0.4 2.9±0.7 3.2±0.9 <0.001 a first follow up (after 4 weeks) 2.5±0.4 * 3.1±0.7 * 3.6±0.9 * <0.001 a second follow up (after 8 weeks) 2.7±0.4 *, ¥ 3.1±0.7 * 3.7±0.8 *, ¥ <0.001 a p-value <0.001b <0.001b <0.001b bud/for: budesonide/formoterol bud/ami: budesonide/aminophylline bud/tio: budesonide/tiotropium fvc: forced vital capacity a: one way anova b: trend anova (repeated measure anova) *: significant difference compared to baseline values ¥: significant difference compared to first follow up values * * iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 160 conducting comparisons between the groups revealed that extent of fvc improvement over the first four weeks of study was significantly better in the third group compared to first and second groups (p=0.026, 0.004 respectively) and revealed no significant difference between the first and second groups (p=0.79) as shown in figure 3. figure 3 comparison of groups according to extent of fvc change during the first four weeks of study *: significant difference compared to first and second groups bud/for (first group): budesonide/formoterol bud/ami (second group): budesonide / aminophylline bud / tio (third group): budesonide/tiotropium. both first and third groups created significant higher increment in fvc values compared to the second group during the interval between fourth and eighth week of study (p=0.001), while there was no significant difference between these two groups (p=0.99) as illustrated in figure 4. figure 4.comparison of groups according to extent of fvc change during the second four weeks of study *: significant difference compared to second group bud/for (first group): budesonide/formoterol bud/ami (second group): budesonide /aminophylline . bud/tio (third group): budesonide/tiotropium . the effect of study medication regimens on the percentage of sfds was explained in table 5. the percentages of sfds were significantly increased in the first follow up visit compared to baseline visit and continued the significant increment in the second follow up visit compared to the first follow up visit within all groups of study (p≤0.001). between groups comparison based on the degree of sdfs increment during the first four weeks of study revealed no significant differences among the three groups (p=0.365) as shown in figure 5. table 5. effect of study regimens on percentages of sfds group first group (bud/for) second group (bud/ami) third group (bud/tio) p-value number of patients 21 21 22 sfd% baseline 7.5±10.7 3.4±7.7 3.9±6.5 0.335c first follow up (after 4 weeks) 32.7±13.7 * 22.5±16.1 * 26.6±17.2 * 0.365 c second follow up (after 8 weeks) 48.97±13.96 *, ¥ 36.1±14.0 *, ¥ 50.0±22.8 *, ¥ 0.013 c p-value <0.001d ≤0.001d <0.001d bud/for: budesonide/formoterol bud/ami: budesonide/aminophylline bud/tio: budesonide/tiotropium sfd%: percentage of symptom free days c: kruskal wallis h test d: friedman anova *: significant difference compared to baseline values ¥: significant difference compared to first follow up values iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 161 figure 5. comparison of groups according to extent of change in percentages of sfds during the first four weeks of study bud/for (first group): budesonide/formoterol bud / ami (second group): budesonide / aminophylline bud/tio (third group): budesonide/tiotropium significant difference was noted among the groups during the 4th – 8th weeks of study; the third group produced significantly the best increment in the percentage of sfds compared to the remaining groups (p=0.01 each). although the first group developed numerically larger extent of sfds increment, there was nonsignificant difference observed between first and second groups (p=0.75) as shown in figure 6. figure 6. comparison of groups according to extent of change in percentages of sfds during the first four weeks of study *: significant difference compared to first and second groups bud/for (first group): budesonide/formoterol bud/ami (second group): budesonide/aminophylline bud/tio (third group): budesonide/tiotropium the impact of medication regimens on the patients' miniaqlq scores was demonstrated in table 6. all study groups showed significant improvement at the first follow up visit compared to pretreatment levels and continued their significant improvement in the second follow up visit compared to the first follow up scores (p<0.001) . comparing groups regarding extent of improvement of miniaqlq scores over the first four week of administering study regimens showed no significant differences among the studied groups (p=0.645) as explained in figure 7. table 6. effect of study regimens on patients' miniaqlq scores group first group (bud/for) second group (bud/ami) third group (bud/tio) p-value number of patients 21 21 22 miniaqlq scores baseline 3.2±0.4 3.2±0.5 3.6±0.6 0.044 a first follow up (after 4 weeks) 4.3±0.4 * 4.3±0.5 * 4.7±0.4 * 0.645 a second follow up (after 8 weeks0 4.9±0.5 *, ¥ 4.6±0.6 *, ¥ 5.6±0.34 *, ¥ <0.001 a p-value <0.001b <0.001b <0.001b bud/for: budesonide/formoterol bud/ami: budesonide/aminophylline bud/tio: budesonide/tiotropium miniaqlq: mini asthma quality of life questionnaire a: one way anova b: trend anova (repeated measure anova) *: significant difference compared to baseline values ¥: significant difference compared to first follow up values iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 162 figure 7. comparison of groups according to extent of change in the miniaqlq scores during the first four weeks of study bud/for (first group): budesonide/formoterol bud/ami (second group): budesonide/aminophylline bud/tio (third group): budesonide/tiotropium the second four weeks revealed that the third group produced significantly higher improvement in miniaqlq scores compared to first and second groups (p=0.004, p<0.001 respectively). also, the first group was associated significantly with larger extent of improvement compared to the second group (p<0.001) as shown in figure 8. figure 8. comparison of groups according to extent of change in the miniaqlq scores during the second four weeks of study *: significant difference compared to second group #: significant differences compared to first and second groups bud/for (first group): budesonide/formoterol bud/ami (second group): budesonide/aminophylline bud / tio (third group): budesonide/tiotropium discussion to the authors' knowledge, there were no comparative studies conducted to evaluate the efficacies of asthma controller medications in iraqi patients, so this study was the first study that aimed to compare between different controller medication regimens in such patients and the results of this study may be explained by comparison with other studies conducted in different societies. the baseline socio-demographic data and clinical characteristics of asthma were well balanced across the groups of this study. moreover, the doses of inhaled budesonide in the groups were chosen to be in the clinically equivalent range of 640-800 µg/day (20). therefore, the two components of each regimen may be responsible for the changes within each group, but any difference in the clinical outcomes among the groups after administering the medication regimens can be attributed to the second controller medication which was different among these groups. the pefr is the maximum rate obtained during forceful expiratory phase after deep maximum inspiration. it is considered as a primary outcome for evaluating efficacies of different asthma controllers. this spirometric parameter is primarily concerned with evaluating airflow in large and medium sized airways (21). a previous study aimed to compare efficacies of tiotropium/ics and salmeterol/ics regimens according to many outcomes including pefr found that these two regimens caused significant improvements of pefr after 8 weeks of treatment compared to their pretreatment values with non-significant difference between them (1.93±0.88 vs. 1.91±0.68 respectively) and concluded that these two regimens were comparable in treating persistent asthma (22). another randomized clinical study conducted by wang et al., to compare between efficacies of laba combined with ics and those of oral long acting theophylline combined with ics in patients with moderate to severe persistent asthma demonstrated that both groups produced significant elevation of pefr values after eight weeks of treatment compared to pretreatment values (from 3.43±1.62 to 4.37± 2.00 l/sec in ics/laba group and 3.96 ±1.38 to 4.68 ±1.7 l/sec in ics/theophylline group) and also demonstrated no significant difference between the two groups regarding their extents of pefr changes (p=0.6) although ics/laba regimen was associated with larger numerical increment (23) iraqi j pharm sci, vol.29(1) 2020 evaluating three medication regimens for asthma 163 the fvc is considered essential pft parameter for describing asthma severity and assessing disease response to the controller medications especially bronchodilators(24). it was recommended that fvc should be considered in evaluation process because some patients with severe asthma responded to bronchodilators with significant elevation of fvc instead of other pft parameters (25). it was shown that both laba and tiotropium caused significant increment in fvc compared to their pretreatment levels when added to an ics. also, it was observed that fvc improvement produced by the laba was not significantly different from that produced by tiotropium, although numerically laba was associated with higher values than tiotropium (0.121 versus 0.95 liter respectively)(26). in agreement with the current study, adachi et al. noted that combination of salmeterol and fluticasone produced progressive significant increment of fvc over four and then eight weeks compared to the pretreatment values, while the slow release theophylline preparation caused less increment of fvc at the 4th week followed by decrement to values approaching those of baseline visit at the 8th week (27). symptom free days are considered important indicators of successful asthma treatment for patients as well as their physicians (28). a previous study was performed to assess the outcomes of adding laba or tiotropium for 16 weeks in asthmatic patients poorly controlled on ics alone showed that both medications caused improvement of common baseline sfd value of 1.4 days per week to post treatment values of 2.4 and 2.2 days per week in the ics/laba and ics/tiotropium respectively with non-significant differences between the two groups (29). assessing the preand post-randomization outcomes of administering either sustained release oral theophylline/ics or salmeterol/ics regimens for eight weeks in patients with moderate persistent asthma revealed that all patients were presented with daily symptoms for one week before enrollment (percentage of patients with sfd=0.0). the percentage of patients who felt symptom free during the last week of study showed significant increment in response to study medications compared to baseline values and the response the ics/salmeterol was significantly better than that to ics/theophylline (percentage of patients with sfd was 18.2 versus 9.5 respectively) (27). mini asthma quality of life questionnaire is a shortened and simplified mode of the original asthma quality of life questionnaire (aqlq). these two formulas can be considered comparable to each other and the mcid was 0.5 for both (18). a previous study aimed to investigate the clinical outcomes of laba versus tiotropium when added to ics for 16 weeks in persistent asthmatic patients found that the changes of miniaqlq scores after 16 weeks of receiving treatment in both groups neither met the mcid nor significantly differed from baseline values [differences from baseline were 0.28 (p=0.064), 0.131 (p=0.068) for laba and tiotropium groups respectively] (29). concordant with the current study, wang et al. noted that both oral theophylline/ics and laba/ics regimens started to cause significant improvement of aqlq scores after 4 weeks of receiving treatment compared to baseline values (4.36±0.45 compared to 3.94±0.67 p<0.05, 4.26±0.133 compared to 3.82±0.98 p<0.05 respectively), while the mcid was recorded at the 12th week of study (0.53 versus 0.54 respectively). the important observation was that extent of improvement of aqlq scores from baseline was not different between the two groups (23). conclusions this study concluded that using inhaled formoterol, inhaled tiotropium or modified release oral aminophylline tablets in addition to equivalent doses of inhaled budesonide in a sample of iraqi persistent asthmatic patients produced significant control of asthma status and significant improvement in pulmonary function test measures and quality of life levels compared to pretreatment values. also, the study concluded that medication regimens of formoterol or tiotropium inhalers with inhaled budesonide were associated with significant better extents of improvement in all study parameters compared to oral aminophylline/inhaled budesonide regimen and that inhaled tiotropium/inhaled budesonide regimen caused higher values of improvement in most of study parameters compared to inhaled formoterol/inhaled budesonide regimen. acknowledgments the authors would like to thank the specialist physician, ibtisam s. hassan zwaylif, at al-zahra center of asthma and allergy for her great and valuable role in clinical evaluation of patients. also, authors thank the clinical pharmacist hayder a. fawzi at baghdad teaching hospital for his help in statistical analysis. references 1. al-moamary ms, alhaider sa, idrees mm, al ghobain mo, zeitouni mo, et al. the saudi initiative for asthma 2016 update: guidelines for the diagnosis and management of asthma in adults and children. annals of thoracic medicine 2016; 11: 3-42 2. ohta k, ichinose m, nagase h, yamaguchi m, sugiura h, et al. japanese society of allergology. japanese guideline for adult asthma 2014. allergology international 2014; 63: 293–333. 3. doz m, chouaid c, com-ruelle l, calvo e, brosa m, et al. the 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sj, icitovic n, moore wc, et al. predictors of response to tiotropium versus salmeterol in asthmatic adults. journal of allergy and clinical immunology 2013;132:1068-74. 29. bateman ed, kommann o, schmidt p, pivovarova a, engel m, et al. tiotropium is noninferior to salmeterol in maintaining improved lung function in b16-arg/arg patients with asthma. journal of allergy and clinical immunology 2011;128:315-22. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report doi: https://doi.org/10.31351/vol30iss2pp269-277 269 the students’ experience of hybrideducation model at the university of baghdad college of pharmacy: lessons and future directions kawther khalid ahmed *, ali azeez al-jumaili **, salema sultan salman *** and sarmed hashem kathem **** ,1 * faculty and examination committee member, college of pharmacy, university of baghdad, baghdad, iraq. ** faculty member, college of pharmacy, university of baghdad, baghdad, iraq. *** faculty and ibn sina unit for e-learning head, college of pharmacy, university of baghdad, baghdad, iraq. ****1 dean of the college of pharmacy, university of baghdad, baghdad, iraq. abstract the impact of covid-19 pandemic on education models was mainly through the expansion of technology use in the different educational programs. earlier impact of covid-19 was manifested in the complete and sudden transition to distance education regardless of institution preparedness status. gradually, many institutions are moving back to on-campus face-to-face education. however, others including all higher education institutions in iraq are adopting the hybrid education model. this report presents part of the end of semester evaluation survey conducted at the university of baghdad college of pharmacy for the spring 2021 semester. the survey aims to address points of strength and weakness associated with the hybrid education model and specifically the virtual content delivery aspect of hybrid education. the outcomes of the end of semester evaluation will shape a better experience for upcoming years and guide distance education implantation in the program. keywords: e-learning, covid-19, hybrid education, student satisfaction, pharmaceutical education, iraq. الدروس والتوجهات المستقبلية: في كلية الصيدلة بجامعة بغداد مدمجالتعليم الالطالب مع تجربة 1،****و سرمد هاشم كاظم ***سليمه سلطان سلمان، **علي عزيز الجميلي ، *كوثر خالد احمد بغداد،العراق. جامعة بغداد، األمتحانية ،كلية الصيدلة،تدريسية وعضو اللجنة * بغداد،العراق. جامعة بغداد، كلية الصيدلة، تدريسي، ** بغداد،العراق. جامعة بغداد، كلية الصيدلة، تدريسية ورئيس وحدة ابن سينا للتعلم األلكتروني، *** بغداد،العراق. جامعة بغداد، عميد كلية الصيدلة، 1**** هالخالص ير اثرت جائحة كورونا على نظام التعليم بشكل أساسي من خالل التوسع في استخدام التكنولوجيا في البرامج التعليمية المختلفة. تمثل التأث التعليمية. فيما بعد األولي لجائحة كورونا في االنتقال الكامل والمفاجئ إلى التعليم عن بعد بغض النظر عن حالة االستعداد والجاهزية في المؤسسة مؤسسات أخرى ومنها مؤسسات توتدريجيًا ،بدأت العديد من المؤسسات التعليمية تعود إلى التعليم التقليدي الحضوري داخل الحرم الجامعي بينما تبن راسي الذي أجري في كلية الصيدلة بجامعة التعليم العالي في العراق نموذج التعليم المدمج. يقدم هذا التقرير جزًءا من استطالع تقييم نهاية الفصل الد . يهدف االستطالع إلى معالجة نقاط القوة والضعف المرتبطة بنموذج التعليم المدمج 20212020الدراسي الثاني من العام الدراسي بغداد للفصل تقييم نهاية الفصل الدراسي في صياغة تجربة أفضل وعلى وجه التحديد الجوانب المتعلقة بتقديم المحتوى االفتراضي للتعليم المدمج. تساهم نتائج للسنوات القادمة وتوجه عملية تعزيز التعليم عن بعد في التعليم الجامعي. .الطالب ، التعليم الصيدالني ، العراق ، التعليم المدمج ، رضا 19-التعليم اإللكتروني ، كوفيد : الكلمات المفتاحية introduction the use of technology in pharmacy education iraq prior to covid-19, the use of information and communication technologies (icts) in pharmacy education in iraq was largely limited. few institutions were implementing technologies like electronic course management (ecm), on-line sessions, electronic exams, video lectures, and others. however, these were sporadic attempts. during the academic year of 2019 -2020, and in response to covid-19 pandemic, all educational programs were suddenly moved to distance education over the period of few weeks (1). the sudden transition was a courageous yet challenging action step to continue the academic year requirement. however, the general lack of infrastructures and adequate training required for successful distance education forced the adoption of other education models for the academic year of 2020 – 2021. 1corresponding author e-mail: skathem@copharm.uobaghdad.edu.iq received: 12/10/2021 accepted: 23/ 12/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp269-277 iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report 270 the hybrid education experience for the academic year of 2020-2021 the ministry of higher education and scientific research (mohesr) decided that all universities and colleges will be adopting the hybrid education model for the academic year of 20202021. for medicine, dentistry, and pharmacy colleges and programs, it was determined that all lectures are to be delivered virtually. mid-term exams are to be given online for all courses, final exams on the other hand were determined to be conducted as on-campus paper exams for 60% of core classes. final exams for the remaining 40% core classes and non-core classes were determined to be administered on-line. the laboratory sections are to be delivered in a hybrid mode such that the lecture part and instructions for experiments are to be delivered virtually and students would come to perform the experiments in the lab on campus. this hybrid model of education allows maximum social distancing throughout the academic year by reducing the number of students present on-campus at any day. however, no further investments at infrastructures essential for distance education took place. in this format of hybrid education, distance learning represented a significant component of content delivery. as we reported earlier, distance education in iraq is a fairly new experience that was implanted without prior planning or preparation (1). therefor it was important that faculty receive appropriate training on various tools used in distance education. the training was available through the supervision and scientific evaluation apparatus in the mohesr, continuing education center at the university of baghdad, continuing education unit and ibn-sina unit for distance education at the university of baghdad college of pharmacy (uob copharm), and others. faculty members were highly encouraged to attend training courses and workshops on new tools in education and specifically distance education. it is very important to maintain the quality of education regardless of the education model implanted. as such, synchronous lectures were the main content delivery mode in the distance education component of the academic year. synchronous lectures were reported to be associated with a more positive learning experience compared to asynchronous delivery (2). however, all lectures were recorded and made available to students through the college youtube channel. this is to address the issue of poor internet service that faces some students and cause them to miss the synchronous lecture. end of semester evaluation student perceptions of the different components of the educational system is an integral part of “satisfaction with the educational program” which is a component of the “plan-do-check-act” cycle of quality assurance of education (3). end of semester evaluation should be done regularly and annually. results from the end of semester evaluation should be considered carefully to address areas of unsatisfaction without compromising the quality of the program. at uob copharm, end of semester evaluation has been regularly conducted. some instructors distribute an end of semester evaluation for the course they teach. instructors are encouraged but not required to distribute the end of semester evaluation. in the last two years, these evaluations have become very important due to the rapid changes in the education model enforced in response to covid-19 pandemic. over the course of 20 months, the students moved from attending lectures in lecture halls to full distance education and then partially back to being on campus in the hybrid education model. these rapid changes were enforced and implemented without sufficient preparations and necessary infrastructures. as such, these changes are expected to shift students’ satisfaction with the educational program. consequently, a central end of semester evaluation was conducted. the objective of the end of semester survey is to evaluate students’ satisfaction with the hybrid model in general and more specifically with the virtual component of content delivery. here we report the main findings of the end of semester evaluation and its future implications. methods the end of semester evaluation had quasi experiment design to conduct post-intervention assessment of new (hybrid) educational approach. the survey was developed using google form. the authors developed new survey items to evaluate the student perceptions toward the implementation of hybrid education model. the survey included measures evaluating students’ satisfaction with synchronous and asynchronous lectures for the different courses they take, students’ satisfaction with hybrid education in terms of scientific gain in distance education compared to traditional face-to-face education, hybrid mode of lab sessions, and in-class final exams following virtual delivery of course materials. additionally, the survey included a section comparing the distance education experience for the current academic year (2020 – 2021) (hybrid education model) compared to previous year (2019 – 2020) (mostly distance education). this last section is very important toward the success of information and technology implementation at the uob copharm where the college administration is actively working to expand the use of technology in different areas of the educational program. this section included measures of student perceptions of their skills in using google classroom and taking online exams, students’ satisfaction with online exam management by the college, technical support services provided, iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report 271 and the overall students’ satisfaction with distance education this year compared to last year. lastly, the survey included a question on students’ satisfaction with faculty understanding and support during the challenging times of covid-19 and a question on student perceptions of misconduct carried out by some students who took advantage of covid-19 pandemic to submit fake excuses and get permissions to skip assignments and quizzes. student recruitments the survey was distributed to the students via their university emails at the end of final exams for the spring 2021semester with two reminders every three days. the participation was optional. statistical analysis the analysis was conducted using the statistical package for the social sciences (spss, ibm, usa version 24). means, standard deviations (s.d.), and medians of participant responses were calculated. a 5-points likert scale (strongly disagree (1), disagree (2), neutral (3), agree (4), strongly agree(5)) was used for student responses. kruskal wallis test was used to compare means of student responses to different questions according to their year of the study. a multiple linear regression analysis was used to identify predictors of student perception of the education model implanted by the college. the multiple linear regression measured the association between 11 independent variables and the outcome variable (in general, our college successfully implemented distance education imposed due to covid-19). this survey was part of the end of the semester assessment. results and discussion a total of 548 student out of 1370 students responded to the survey giving rise to 40% response rate. responses from students had a median score of agree (4) for most measures used except for the following three measure: “in general, our college successfully implemented distance education imposed due to covid-19”, “in general, the faculty members had good understanding of students’ circumstances during covid-19 pandemic”, and “some students took advantages of the pandemic to not fulfill their academic commitments” which had a median score of neutral (3) (table 1). table 1. the descriptive characteristic of the student perceptions about the college teaching modes. measure n median mean std. deviation a although all the lectures were virtual, in-class final exam improved my scientific gain compared to online exams. 544 4 3.29 1.33 b having the practical part of the lab on campus improved my understanding even though the lecture part of the lab was virtual 544 4 3.35 1.27 c hybrid education model is better than distance education 543 4 3.53 1.27 d it would have been better to have the whole lab session on campus 545 4 3.57 1.32 e my scientific gain in distance education component is similar to in-class education 433 3 2.64 1.30 f in general, the experience of distance education this year is better than last year 432 4 3.45 1.12 g in general, the management of online exams this year is better than last year 432 4 3.50 1.04 h my skills in using google classroom this year is better than last year 432 4 4.11 0.78 i my skills in taking online exams this year is better than last year 432 4 4.03 0.81 j the technical support unit in the college was able to help resolve technical issues related to distance education 543 4 3.68 0.90 k the college administration worked hard to support the success of distance education 542 4 3.57 0.91 l i hope to continue using google classroom to distribute instructions and class materials after the resumption of full on-campus education 450 4 3.83 1.28 iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report 272 continued table 1. measure n median mean std. deviation m i will continue using university email to communicate with my instructors after the resumption of full oncampus education in the upcoming years 450 4 3.78 1.05 n in general, our college successfully implemented distance education imposed due to covid-19 432 3 3.38 1.00 o in general, the faculty members had good understanding of students’ circumstances during covid-19 pandemic 544 3 2.71 1.07 p some students took advantages of the pandemic to not fulfill their academic commitments 543 3 3.19 1.11 seven factors had significant (p-value < 0.05) positive association with the outcome variable according to the regression analysis (table 2). the seven significant positive factors included the students’ perceptions of the hybrid teaching compared to the in-class teaching, perceptions toward in-class exam, this year experience with virtual learning, the administration of the virtual exams, skills of using google classroom, technical help with the college team, and the college efforts to enhance the virtual learning (table 2). a more detailed presentation of the end of semester evaluation outcomes is presented below. table 2. multiple linear regression of factors associated with student perceptions toward college hybrid education approach. measure standardized coefficients (beta) pvalue my scientific gain in distance education is comparable to my gain in traditional face-to-face learning in the previous years 0.186 0.0001* although all the lectures were given virtually, the in-class exams enhanced my scientific knowledge compared to online exams 0.100 0.009* attending the practical side of the laboratory on campus helped me to understand the material better even though the lecture part was given online 0.064 0.127 hybrid education model in the practical course is better than distance education model -0.080 0.067 in general, virtual learning experience this year is better than last year 0.192 0.0001* in general, online exams administration and management is better this year compared to last year 0.171 0.0001* my skills to use google classroom this year is better than last year 0.102 0.044* my skills in taking and submitting online exams this year is better than last year -0.068 0.180 the electronic education unit team in the college was able to help me resolve technical issues experienced in distance education 0.127 0.001* the college administration worked hard to support the success of distance education experience 0.321 0.0001* in general, the faculty members had good understanding of students’ circumstances during covid-19 pandemic 0.007 0.853 *significant, p-value < 0.05. dependent variable: in general our college successfully achieved virtual learning due to covid-19. seven factors significantly (p-value < 0.05) positive associated with the outcome variable according to the regression analysis. iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report 273 students’ evaluation of the hybrid education experience the hybrid education model implemented for the theoretical part of the courses included online lectures and in-class exams. the in-class exams were important to ensure the integrity of the exam given the limited resources that hinder proper online exam proctoring. virtual delivery of the theoretical part was conducted in a synchronous and asynchronous mode. for the practical (lab) sessions, the lecture part was given electronically, and students performed the experiment on campus. student perceptions of the usefulness of synchronous lectures for their learning was trending toward neutral with an appreciable percentage of students expressing an attitude of disagree (figure 1a). student perceptions were trending toward an attitude of agree for the usefulness of video lectures for their learning (figure 1b-c). these outcomes are mainly driven by the poor internet services in iraq pertaining to non-optimal synchronous lecture experience. figure 1. student perceptions on the different modes of virtual content delivery used in the hybrid education model. a: synchronous lectures helped me understand the materials better, b: recorded video lectures that can be streamed on youtube is one of the advantages that came with distance education implementation, c: i prefer watching recorded video lecture over attending the synchronous lecture students’ responses on the different measures on their experience with hybrid education showed a median score of agree (4) (table 1 (a-d)). students perceived hybrid education to be better than e-learning for the lab sections of the courses where the majority of students agreed that attending the lab and performing experiments improved their learning (figure 2). however, students expressed their preference for traditional on-campus full lab section over the hybrid mode. the majority of students agreed that paper exams improved their learning outcomes compared to online exams (figure 2). while an appreciable percentage of students perceived distance education to be equally beneficial for their learning to traditional education, a good proportion of the students disagreed resulting in a median score of neutral (3) for that measure (table 1e). it is worth noting that student perceptions of their scientific gain in distance education compared to face-to-face education and the improvement of their scientific gain when giving in-class final exams had significant positive association with their perception of the college success in the distance education experience (table 2). these results are well expected giving the young age of experience for the faculty and the students with education models other than face-to-face education. iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report 274 figure 2. student perception of hybrid education model implemented during the academic year of 20202021. a: although all the lectures were virtual, in-class final exam improved my scientific gain compared to online exams. b: having the practical part of the lab on campus improved my understanding even though the lecture part of the lab was virtual, c: hybrid education model is better than distance education, d: it would have been better to have the whole lab session on campus. e: my scientific gain in distance education component is similar to in-class education. kruskal wallis test was used to compare means of student responses to different questions according to their year of the study. n: number of students responding to the survey. student perceptions of the distance education experience compared to previous year even though the academic year progressed in the hybrid model, the virtual component was still heavy compared to education prior to covid-19. while this was only the second academic year at which distance education is being implemented, it was not expected to be the last. this is because the covid-19 pandemic that enforced distance education is still ongoing. additionally, the administrations both at the university and college levels are determined on expanding the size of information and technology use in education as one mean of improving educational outcomes and institution performance (4). as such, it was important to learn about students experience with e-learning this year compared to previous year. the majority of students agreed that they had a better experience this year and that the management of online exams by the college was improved from last year where their responses showed a median score of agree on both measures (table 1f-g, figure 2a-b). this outcome can be driven by improved students’ skills in using the ecm system (google classroom) and in taking and submitting online exams. both of these measures showed median score of agree (4) where only 20% or less of students in any level disagreed (table 1(h-i), figure 3(c-d)). other factors that can contribute to students’ better experience is administration efforts and technical support provided by the e-learning unit at the college. all of these measures had a significant positive association with student perceptions of the college success in distance education implemented in response to covid-19 pandemic (table 2). students’ evaluation of the semester is significantly different across the different grades of the study program. the extent of experiencing the different models of education (face-to-face, hybrid, distance) was different for students in the different grades of the study program (figures 2-4). to elaborate, first and second year students did not experience traditional face-to-face education at the college level before as they were admitted into the program shortly before or during covid-19 pandemic. on the other hand, third-, fourth-, and fifth-year students experienced the traditional education model during the earlier year(s) in the program before they were forced to transition to distance education due to covid-19. these different experiences were reflected in student perceptions of the semester and the academic year. indeed, first and second year students’ responses showed the highest level of significant differences by the pairwise comparison iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report 275 test conducted for the different measures in the end of semester evaluation. responses from the first and second-year students showed higher ranked mean compared to students in other levels in questions on their willingness to continue using google classroom and university email for the remaining of their years in the study program (figure 4). figure 3. student perceptions on the distance education experience during the academic year of 2020 -2021 compared to that during the academic year 2019 – 2020. a: in general the experience of distance education this year is better than last year, b: in general the management of online exams this year is better than last year, c: my skills in using google classroom this year is better than last year, d: my skills in taking online exams this year is better than last year, e: the technical support unit in the college was able to help resolve technical issues related to distance education, f: the college administration worked hard to support the success of distance education. kruskal wallis test was used to compare means of student responses to different questions according to their year of the study. n: number of students responding to the survey. figure 4. student perceptions of the overall college performance in distance education and their intention to continue using ict tools following the complete resumption of on campus face-to-face education. a: in general our college was able to succeed in the distance education experienced implemented in response to covid19, b: i hope to continue using google classroom to receive notification, assignments and class materials even after fully going back to on campus face to face education, c: i will continue using university emails to communicate with my instructors after the resumption of on campus face-to-face education. kruskal wallis test was used to compare means of student responses to different questions according to their year of the study. n: number of students responding to the survey. iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report 276 on the other hand, responses from last year students showed the highest ranked mean, though not significantly different, to the question on student perceptions of technical team (ibn-sina unit for elearning) capabilities in providing required support (figure 3e). this possibly indicates a higher level of appreciation from the senior students to the efforts put by the college in transitioning from close-to-zero electronic communication to fully functioning electronic management system of communication and course delivery. first and second year students started their academic study with or after the establishment of formal electronic communication system. these students experienced distance education starting in their first year. video lectures and handouts were made available early in their study. as such they would have higher expectations for electronic services at the college. challenges in distance education implantation and future directions the overall rating of the college success in distance education showed a median score of neutral (3) (table 1). as mentioned earlier, students had a median score of agree for their willingness to continue using google classroom and university email for the remaining of their study program. this is a very good improvement from a study conducted at the beginning of distance education implantation in mid-2020 (5). the earlier study covered ten colleges of pharmacy in iraq including the uob copharm and reported a median score of disagree (2) for students’ intention to use electronic course management. even though the two reports are one year apart, the magnitude of improvement in the quality and extent of use of electronic course management (google classroom) is big. the earlier study reported students’ response shortly after the sudden transition to distance education where neither students nor faculty were trained enough for such transition. this current report comes after about two years of using google classroom as an electronic course management platform at uob copharm. these results reflect the importance of appropriate training for the implementation of a new educational tool. while the aforementioned outcomes about students’ intention to continue using ict in education are very promising, the actual implantation might be hindered by lack of essential resources. poor internet service, limited free internet service on campus for most institutions, lack of financial allocations required to provide essential exam monitoring services, limited number of technical support workers, and resistance of some faculty and students (anti-technology behavior) are only some examples of missing resources that challenge icts implantation in education in iraq. concluding remarks the level of icts implantation in education in iraq has been directly affected by covid-19 pandemic. given the limited infrastructure and resources available for proper icts implantation, the mohser is actively working to balance the quality of education and safety requirement enforced in response to the pandemic. hybrid education model was adopted in all universities and colleges including the uob copharm. virtual content delivery represented the main platform of the academic year of 2020 – 2021. the uob copharm has come a long distance in managing distance education which was well reflected in students’ response in the end of semester evaluation. the students expressed their appreciation for the administration effort and the technical support provided in virtual content delivery. an important outcome was students’ intention to continue using google classroom as the ecm platform and their university email in their education. the next step should be to continue using ecm and university emails after the full resumption of face-to-face education. moreover, virtual class meetings can be utilized to encourage international collaborations where educators from distant universities can be hosted to deliver content as part of the educational program curriculum. the academic year of 2021 -2022 was determined by the mohesr to be mainly in the traditional face-toface model for all core classes. this will put the college administration in a true challenge to continue using icts in education and at the same time might make their use more acceptable by the students. indeed, google classrooms are being used as the ecm at the uob copharm. the platform is mainly used to deliver handouts, assignment, notifications, and other course related materials. it is expected that such strategy will result in a better learning experience pertaining to higher level of student satisfaction with icts implantation for the academic year of 2021 – 2022. references 1. ahmed kk, salman ss, abbas wa, alkaisy sw, kathem sh. sudden transition of pharmacy education from traditional to distance learning in the era of covid-19: action steps of a leading pharmacy school in iraq. iraqi journal of pharmaceutical sciences 2020;29(2):271-8. 2. abriz s, mendzheritskaya j, stehle s. impact of synchronous and asynchronous settings of online teaching and learning in higher education on students’ learning experience during covid-19. front psychol. 2021;12(4544). 3. belash o, popov m, ryzhov n, ryaskov y, shaposhnikov s, shestopalov m. research on university education quality assurance: methodology and results of stakeholders’ satisfaction monitoring. procedia social and behavioral sciences. 2015;214:344-58. iraqi j pharm sci, vol.30(2) 2021 hybrid education experience: position report 277 4. basri ws, alandejani ja, almadani fm. ict adoption impact on students’ academic performance: evidence from saudi universities. education research international. 2018;2018:1240197. 5. al-jumaili aa, ahmed kk, al-jalehawi ak, alfatlawi bg, al-rekabi md, al-sawad os, et al. evaluating the use of informational technologies by students of healthcare colleges for academic purposes over a five-year period. education and information technologies. 2021;26:21. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ effect of chronic exposure of cadmium chloride in drinking water on structural and functional aspects of thyroid glandin matu iraqi j pharm sci, vol.19 (1) 2010 effect of cadmium chloride in thyroid gland 69 effect of chronic exposure of cadmium chloride in drinking water on structural and functional aspects of thyroid gland in mature male rabbits # samir h. cheyad* ,1 , kalisa k. khudier** and khtan a. al-mzain** * ministry of industry ,general commission for industrial research and development al-razi center for research and medical diagnostics. ** department of physiology , college of veterinary medicine ,university of baghdad , baghdad, iraq. abstract the effect of chronic exposure to two different levels of cadmium chloride (cdcl2) 30 ppb and 40 ppb in drinking water for 12 weeks on thyroid function of mature male rabbits was studied. eighteen mature male rabbits were randomly divided into three groups (each of six ) , control group (group i ): were offered ordinary tap water , and treated groups (ii and iii ) were offered tap water containing 30ppb and 40 ppb respectively for 12 weeks .serum concentration of thyroxin (t4 ) and triiodothyronine (t3) were measured every six weeks ,as an index of thyroid function , further more , section of thyroid gland were prepared for histological studies. the results showed that chronic exposure of male rabbits to two different levels of cdcl2 caused significant decrease (p<0.05) in serum t3 and t4 in both treated groups in compared with control group, in addition, there were histological changes in thyroid gland of treated groups manifested by hyperplasia with presence of large number of varying size of microfollicels and hypertrophic thyrocytes, small colloid with little secretion . key wards: cadmium, thyroid gland, thyroxin, triiodothyronin, thyrocyt الخالصة ى ( فً هاء جضء تالثلٍْ 03، 03اجشٌد ُزٍ الذساسح لوعشفح ذأثٍش الرعشض الوضهي لوسرٌٍْي هخرلفٍي هي كلْسٌذ الكادهٍْم ) اسًثا تالغا قسود عشْائٍا الى ثالز 21ذن اسرخذام 3اسثْع على ّظٍفح الغذج الذسقٍح فً ركْس االساًة الثالغح 21الششب ّلوذج ( ii group( اسرلود هاء عادي طٍلح فرشج الرجشتح ّهجوْعرً الوعالجح ) group iهجاهٍع هرساٌّح : هجوْعح السٍطشج ) ( اسرلود هاء الششب هضافا الٍَ iii group، جضء تالثالٍْى ( ّ ) 03ششب الوضاف الٍَ كلْسٌذ الكادهٍْم ترشكٍض )اسرلود هاء ال ّذشاي اٌْدّ t4اسثْعا ذن قٍاط ُشهًًْ الغذج الذسقٍح الثاٌشّكسٍي 21جضء تالثلٍْى ّلوذج 03كلْسٌذ الكادهٍْم ترشكٍض اخز الوقاطع الٌسٍجٍح للغذج الذسقٍح. اظِشخ الٌرائج اى ذعشض ركْس االساًة الوضهي كل سد اساتٍع فضال عي t3ثاٌشًٍّي ً فً هصل الذم ف t3 ّt4فً ذشكٍض ُشهًًْ لوسرٌٍْي هخرلفٍي هي كلْسٌذ الكادهٍْم فً هاء الششب قذ سثة اًخفاضا هعٌٌْا سٍجٍح للغذج الذسقٍح حذّز حالح فشط الٌسٍجهقاسًح هع هجوْعح السٍطشج كوا اظِشخ الفحْصاخ الٌهجاهٍع الوعالجح (hyperplasi ٍذوثلد تْجْد عذد كثٍش هي الخالٌا الذسقٍح ّتاحجام هخرلفح هحاطح تطثقح هي الخالٌا الطالئ )ح ) ــــhypertrophic thyrocyte ( فضال عي ّجْد ًقص ّاضح فً كوٍح الغشّاى )colloid ٌة هع قلح فً افشاصاذَ( داخل الجش . introduction cadmium is modern toxic metals (discovered in 1817) . its main use in electroplating because of its anticorrosive properties, it also used as color pigment of paints, plastics and as cathode material for nickel –cadmium batteries, cadmium is an important source of environment pollution (1) . hazardous waste disposal sites are large source of cd concentration found in soil and water . tobacco smoke is the main reason for cadmium accumulation in our body (2) ,on other hand , the major route of cadmium intake ( for non smoker ) is ingestion , this is largely due to the presence of cd ( 2-40 ppb ) in food staff of natural origin e.g. cereals beans , carrots , beverage, coffee and tea (3) ,or by the ingestion of contaminated food especially fish (4, 5) . the acute toxic effect of cd are generally restricted to the lung ,where as the effects following chronic cd exposure in human are multisystemic and include nephropathies emphysematous alteration in the lung , cardiovascular disease , and bone damage possibly ( osteomalacia and osteoperosis ) (6) . wealth of evidence suggested that heavy metals including cadmium exert profound toxic effects on the activities of a number of endocrine gland including thyroid gland (7,8) . # based on oral presentation in the seventh scientific conference of the college of pharmacy /university of baghdad held in 26-27 november 2008. 1corresponding author email : samir-cheyad@yahoo.com received :14/4/2009 accepted : 9/5/2010 mailto:samir-cheyad@yahoo.com iraqi j pharm sci, vol.19 (1) 2010 effect of cadmium chloride in thyroid gland 70 cadmium intoxication has been reported to reduce the thyroidal iodide uptake (7) and inhibits hepatic conversion of thyroxin (t4) to triiodothyronine (t3) in rats (9) and mice (10) . it has been previously reported that subcutaneous injection of cadmium chloride (cdcl2 ) (mg/kg b.w ) for ten weeks to male rabbits resulted in a significant reduction in serum t3 concentration and hepatic t4 production (11) . the direct relationship between heavy metals poisoning and thyroid dysfunction were studied in male rabbits by ghosh and battacharya ,1992 , within 24 hours of intramascular injection of cdcl2 (15mg/kg b.w) a significant increase in thyroid activity over the control with a concomitant rise in t3 titer were observed .. the toxic effect of cadmium on thyroid gland functions has been studied , yet its effect at above the permissible level and below the toxic one are questioned , to this aim the present study dedicated . materials and methods number of mature male rabbits 800 1000 g of local breed were acclimated to holding facilities for one week prior to commencement of dosing . animals in all stages of experiment were housed in clear plastic cages in conditioned room (22-25 c◦ ) with controlled lightening ,eighteen rabbits were randomly and equally divided into three groups and were treated for three months as fallow : group i, rabbits in this group were received tap water and served as a control group , the group ii were received 30 ppb cadmium chloride in drinking water , while the animals of group iii were received 40 ppb of cadmium chloride in drinking water . fasting blood samples were collected at zero, 6, and 12 th weeks of experiment via cardiac puncture technique , then serum was separated and frozen at – 20 c◦ for hormonal analysis ,tetraiodothyronint4 and triiodothyronin t3 were determined by using immuno assay detection (12) . for histological studies rabbits were killed and a portion of trachea with intact thyroid gland was dissected free of connective tissue and preserved in 10% formaline buffer solution till the preparation of histological sections . tissues were embedded in paraffin and several tissues sections were prepared, and stained with hematoxylineosin stain (13) .statistical analysis of data was done on the basis of two – way analysis of variance (anova) , using a significant level of p < 0.05 (14) . results the effect of the two different levels of cadmium chloride on mean values of serum t3 concentration of male rabbits are shown in table (1) , serum t3 concentration showed a significant decrease (p< 0.05 ) at the 6 th week of exposure to cadmium chloride in group iii and at the 12 th week of experiment in group ii as compared with control . in cadmium treated groups , in table(2) thyroxin serum concentration significantly decreased (p<0.05) compared to control , such decrement was observed at the 6 th –12 th week of experiment. within groups , the values tended to decrease significantly (p<0.05 ) with time (at 6 th -12 th week ) as compared with the pretreated period . the histological structure of thyroid gland of un treated rabbits was shown in figure (1) , the thyroid follicles containing colloid of uniform color , oval in shape , lined by cuboidal thyroidal follicular cells , figures 2,3 illustrated the histological changes in thyroid gland of rabbits following 30 and 40 ppb of cdcl2 ( group ii and group iii ),the figures showed a case of hyperplasia manifested by presence of thyroid microfollicels of varying size lined by high columnar thyroidal follicular cells (hypertrophic thyrocyte ) , small colloid with little secretion in follicular lumen ( figure 2) , while the figure 3 showed a large area of irregular and varying size microfollicels with hypertrophic thyrocyte with little colloid secretion . table 1 : serum triiodothyronine (t3) concentration (nmol/l) in rabbits treated with two different levels of cadmium chloride in drinking water. groups time ( weeks) i ii iii pretreatment 0 0.43± 0.01 a a 0.44±0.56 a a 0.44±0.01 a a d u r in g tr e a tm e n t 6 0.45±0.01 a a 0.43±0.01 a a 0.41±0.02 b b 12 0.44±0.01 a a 0.40±0.02 b b 0.40±0.02 b b values are expressed as mean ±se n = 6/group groups: i = control, ii = rabbits received 30 ppb of cadmium chloride in drinking water, iii = rabbits received 40 ppb of cadmium chloride in drinking water. capital letters denote between group differences, p< 0.05 vs. control. small letters denote within same group differences, p<0.05 vs. pretreated values. iraqi j pharm sci, vol.19 (1) 2010 effect of cadmium chloride in thyroid gland 71 table 2: serum tetraiodothyronine ( t4) concentration (nmol/l) in rabbits treated with two different levels of cadmium chloride in drinking water. groups time ( weeks) i ii iii pre treatment 0 58.17±0.7 a a 57.42±0.56 a a 57.12±0.42 a a d u r in g tr e a tm e n t 6 58.5±1.25 a a 51.0±2.4 b b 49.2±1.6 b b 12 58.35±1.3 a a 44.5±1.9 b c 41.7±0.4 b c values are expressed as mean ± se n = 6/ group groups: i = control, ii = rabbits received 30 ppb of of cadmium chloride in drinking water, iii = rabbits received 40 ppb of cadmium chloride in drinking water. capital letters denote between group differences, p< 0.05vs. control. small letters denote within group differences, p< 0.05 vs. pretreated values. figure 1:.section in thyroid gland of untreated rabbit (group i), note thyroid follicles with thin epithelial lining cells (thyrocyte) (arrow), filled within colloid secretion (c). h&e, 40x figure 2: section in thyroid gland from cadmium treated rabbit (group ii), note follicularcells of varying size, high hypertrophic thyrocytes (arrow), with a little area of micro colloid secretion.(c ) h &e. 40x figure 3 :section in thyroid gland from cadmium treated rabbit (group iii), showing large area of microfollicels of varying size with hypertrophic thyrocyte (arrow), and hardly little secretion in the lumen. (c) h&e. 40x discussion this study showed that exposure of rabbits to cadmium bring the animal to the case of hypothyroidism manifested by significant decrease in serum t3 and t4 concentration associated with biological changes . to the best of our knowledge , the effects of chronic exposure to cdcl2 at level 30 and 40 times above the permissive level on thyroid gland feature , has not been studied yet , however considerable body of evidence how exist attesting the involvement at heavy metals including cadmium in the toxicity of a number of endocrine glands including thyroid gland (8,9) . many studies suggested the mechanisms including affecting hepatic thyroxin metabolism through reduction in the activity of hepatic “ outer ring deiodinase ord” an c iraqi j pharm sci, vol.19 (1) 2010 effect of cadmium chloride in thyroid gland 72 enzyme which responsible for conversion of major circulating form of thyroid hormone (t4) to more biologically active form(t3) with disruption of t3 signaling leading to reduction in t4 conversion (15) . in addition cadmium may cause reduction of thyroidal i uptake by damages the structure and function of both follicular and parafollicular cells of thyroid (9,10) . long term exposure to cadmium may induce the activity of hepatic microsomal enzyme especially udp-gt (uridine diphosphate glucournyl tranferase ) (16) and phenol sulftransferase (17) resulting in rapid clearance of t3 and t4 , on other hand , accumulation of cadmium in mitochondria of thyroid follicular epithelial cells might disturb the oxidative phosphorylation of this organelle with subsequent loss of energy supply leading to inhibition in the synthesis and release of thyroid hormone (18) . the suspected selenium deficiency caused by cadmium treatment may lead to histological changes by activation fibrotic process in which inflammatory reaction and excess transforming growth factor b play a role in thyroid gland morphology . (19) gupta p and kar a (1999) study the relation between cadmium and selenium , vitamin e and thyroid dysfunction in chicken, in this study they suggested that cadmium decrease t3, probably by inhibiting hepatic 5monodeiodinase (5d-i)activity, which is a selenium dependent function. cadmium is known selenium antagonist while vitamin e facilitate selenium metabolism .vitamin e was shown to protect against cadmium toxicity and maintain 5d-i activity and t3 levels, while the experimenters concluded that the metal – induced inhibition in hepatic 5 d-i activity is mediated through lipid piroxidation my conclusion is that the cadmium inhibited 5d-i activity by decreasing selenium.while vitamin e does decrease lipid peroxidation it does this by facilitating selenium metabolism and selenium is the key metal in glutathione peroxidase which is a potent inhibitor of lipid peroxidation (9) accordingly we can speculate that selenium deficiency may occur in this study following cadmium exposure resulting in the damage of thyroid gland , decrease the concentration of both (t3 and t4) and inhibit monodeiodinase activity leading to state of hypothyroidism . acknowledgment the author is grateful to dr.barrh n. al-oqaily for his helps. references 1. flanoga i , tusek k , stengar m . mercury , selenium and cadmium in human autopsy from idrijaresidents and mercury mine workers . environ. res. 2000; 84(3): 211-8 . 2. iarc . iarc monographs on evaluation of carcinogenic risk of chemical to humans: beryllium, cadmium , mercury and exposure in the glass manufacturing industry ,vol.58world health organization , international agency for research on cancer , lyon , france 1993 ; pp: 119-146 , 210236 . 3. satarage s , haswell, e. and moore , m r . safe levels of cadmium intake to prevent renal toxicity . br. j. nutr. 2000 ; 84(6):791-802 . 4. hooth m j , deongelo , george a b , gaullord e t. subchronic sodium chloride exposure in drinking water result in concentration independent in rat thyroid follicular cell hyperplasia . toxicol. pathol. 2001 ; 17: 250-295 . 5. al-taai , m.s.. trace metals in water , sediments , fishes , and aquatic plants of shattalhilla . ph. d. thesis, college of science . university of babylon . 1999. 6. morimotol u k , kai k , okazaki y . uncoupling between bone formation and chronic resorption in overiectomized rats with cadmium exposure . toxicol.appl. phrmacol.2000 ; 164(3): 264-72 . 7. khalil badiei , pegah nikghadam . effect of cadmium on thyroid function in sheep . comp clin pathol .2009 ; 18: 255-259. 8. susan c , tlton ch, faran , m. effect of cadmium on reproductive axis of japanese medaka , comparative biochemistry and physiology part .2003 ; c136:265-267. 9. gupta p, kar a. cadmium induced thyroid dysfunction in chicken ; hepatic type i iodothyronine 5-d-i activity and role of lipid peroxidation . comp. biochm .physiolo. pharmacol. endocrinol.1999 ; 123(1): 39-44. 10. barbara p. structure and function of thyroid follicular cells in female rats chronically exposed to cadmium .bull.vet.inst.pulawy 2003; 47:157-163. 11. yoshida k , sugihira n , suzuki . effect of cadmium on t4 outer ring monodeiodination by rat liver . environment research1987; 40: 400405. 12. birsak h j , hotz a. the chemical and the thyroid j.nucl.med.1991; 18:761-778. 13. luna l g. manual of histological staining methods of the armed forces institute of pathology .1968 ; (3 rd ed) . iraqi j pharm sci, vol.19 (1) 2010 effect of cadmium chloride in thyroid gland 73 mcgrow hill book company . newyork . 14. snedecor g w, cochran w g. statistical methods,1980; 7 th ed . the lowa state university press, ames. 15. amma l l , wong c z , venstrom b , forrest d . distinct tissue specific roles for thyroid hormone receptors β and α – in regulation of type 1deiodinase expression . mol. endocrinol. 2001; 15(3) : 467-475. 16. wade m g , sophi p , kenneth w, edward y. thyroid toxicity due to subchronic exposure to a complex mixture of 16 organochlorines ,lead ,and cadmium . toxicological scienes. 2003; 67,207-18 . 17. ghosh n, bhattacharya s. thyrotoxicity of chlorides of cadmium and mercury in rabbits . biomed. environ. sci. 1992 ; 5(3) : 236-40 . 18. yoshizuko m , mori n , hamasaki k . cadmium toxicity in throid gland of pregnant rats. exp. mol. pathol. 1991; 55(1)97-104 . 19. ruze m , juna c, jose g. single and multiple selenium –zinc-iodine deficiencies affect rat thyroid metabolism and ultra structure .j. nutr.1999 ; 129:174-180. iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles doi: https://doi.org/10.31351/vol29iss1pp236-246 236 formulation and in-vitro evaluation of itraconazole floating microparticles taha a. basher*,1 and entidhar j. al-akkam* *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract itraconazole (itz) is an antifungal drug (bcsii) used for the treatment of local and systemic fungal infections. furthermore, itz used as an antifungal prophylaxis for immunocompromised patients. the objective of the study is to overcome the two problems of low and ph dependent solubility of itz by its preparation as floating microparticles. firstly, ph-dependent floating microparticles were prepared using oil in water solvent evaporation method, from which the best one (f7) selected as a best ph-dependent formula with composition of itz (200mg),ec (800mg), hpmc 15cps (200mg) and safflower oil (2ml) .then, f7 was compared with the selected relatively phindependent itz floating microparticles formula with composition of itz(200mg), a first coat of hpmc15cps (200mg), a second coat of ec (800mg), hpmc 15cps (200mg) and safflower oil (2ml) which prepared by a dual coating solvent evaporation method using a first coat which provides relatively ph-independent solubility, while the second coat applied as a bouncy producing agents. polyvinyl alcohol (pva) was used as a surfactant in both cases. the prepared floating microparticles were subjected to various evaluation parameters such as yield percent, drug loading and drug entrapment efficiency (ee), particle size analysis, invitro bouncy, drug release, fourier transforms infrared spectroscopy (ftir), differential scanning calorimetry (dsc) and x-ray diffractometry (xrd) studies. the selected relatively ph-independent formula (f16) has a particle size of 11.29 μm with a polydispersity index of 0.651, and the best ph-dependent formula (f7) has a particle size of 2.56 μm with pdi of 0.37. in vitro drug release per cent from the selected formula (f16) was 100 % at 10 hour (ph 1.2) while was 84% at 12 hour (ph1.2) for f7 (p > 0.05), 62 % at 12 hour (ph 3.7) for formula (f16) while was 22% at 12 hour (ph 3.7) for f7 (p < 0.05) , was 35 %at 12 hour (ph 6.8) from f16while, 7% at 12 hour (ph 6.8) from f7(p < 0.05). therefore itz release from the selected formula (f16)is significantly better(p < 0.05) and the bouncy per cent of selected formula (f16) was 95% that was significantly better than bouncy percent of f7 that was 86 %( p < 0.05) in formula 16. inf7, %dl and %ee were 97% and 89%, respectively, while in f16 both %dl and %ee were 96%. ftir results showed no change in the peaks of the itz microparticles functional group while xrd and dsc show change the physical state of itz which indicate conversion from crystalline to amorphous, which had better stability(f16). thus, dual-coated floating microparticles (f16) appeared to be a promising approach to improve solubility at different ph values and prolong the release of the itz within the stomach that may increase its oral bioavailability. keywords: itraconazole, hydroxypropylmethylcellulose 15cps, eudragits s100, solvent evaporation method, microparticles العائمة االتروكانازول لجسيمات تصييغ وتقييم خارج الجسم *عكاملاانتظار جاسم و 1*،طه عبد القادر بشير . العراق بغداد، بغداد، جامعة الصيدلة، كلية ،الصيدالنيات فرع* الخالصة كوقاية itzعالوة على ذلك ، يستخدم اتراكونازول هو دواء مضاد للفطريات يستخدم لعالج االلتهابات الفطرية الموضعية والجهازية. .مضادة للفطريات للمرضى الذين يعانون من نقص المناعة والذوبان في درجة الحموضة المعتمدة هما العامالن الرئيسيان المسؤوالن عن التوافر البيولوجي انخفاض القابلية للذوبان في الماء ة المنخفض عن طريق الفم. الهدف من الدراسة هو التغلب على هاتين المشكلتين من خالل صياغة االتراكونازول كجسيمات مجهرية عائمة مستقل الذوبان وإطالة أمد إطالق االتراكونازول والذي من الممكن ان يؤدي إلى زيادة التوافر الحيوي.نسبياً عن األس الهيدروجيني لزيادة قابلية 1corresponding author e-mail: babantaha497@yahoo.com received: 23/11/2019 accepted: 25/1 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp236-246 iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 237 نتج عنه نسبه اطالق ألنهصيغه كأفضل( 7) الصيغةأوالً ، تم إعداد الجسيمات الدقيقة العائمة المعتمدة على الرقم الهيدروجيني ،وتم اختيار المستقلة العائمة االتروكانازول جسيماتحضير ، ت (%86ساعه مقبول ) 12خالل العائمةساعه ونسبه الجسيمات 12( خالل %84دواء مقبول) كطبقة أولى توفر قابلية ذوبان مستقلة عن درجة hpmc15 cpsبطريقة تبخير مذيب الطالء المزدوج باستخدام الهيدروجينينسبيا عن االس كمثبت مساعد. pvaواستخدم العوم وزيت القرطم كعوامل توفر ecو hpmc15cpsنسبياً و ph)الحموضة) تلجسيماا على لمحملا جلعالا كمية، لمنتجةا يةولمايكرا تلجسيماا كمية جسيمات الدقيقة العائمة المحضرة التي تخضع لمعايير تقييم مختلفة ، مثلال ( ،ftir) الحمراء تحت باألشعة للطيف فورييه تحويل والدواء ، وتحرر( ، وتحليل حجم الجسيمات ، ، deeالدواء ) انحسار، وكفاءة يةولمايكرا (.xrd) السينية األشعة حيود و( dsc) التبايني المسح مسعر 0.651ميكرون مع مؤشر متعدد االختالفات يبلغ 11.29على حجم جسيم يبلغ (f16) تحتوي الصيغة المحددة المستقل نسبيًا عن األس الهيدروجيني كان (f16) نسبه تحرر الدواء من الصيغة المحددة .pdi 0.37 .مع 2.56لها حجم جسيم (f7) ، وأفضل صيغة تعتمد على األس الهيدروجيني ساعات 12٪ خالل f7 ،62(p> 0.05) من (ph1.2) ساعات 12٪ خالل 84( بينما كان 1.2ساعات )درجة الحموضة 10٪ خالل 100 ساعات 12٪ خالل 35، وكان f7(p <.05) نم (ph 3.7) ساعات 12٪ في 22بينما كان (f16) ( من الصيغة المحددة3.7)درجة الحموضة (ph 6.8) ، ساعة 12٪ في 7بينما (ph 6.8) f7 (p <.05) وبالتالي فإن تحرر ، itz من الصيغة المحددة أفضل بكثير (p <.05). بلغت نسبة .٪95كانت f16 وفيf7٪86الكريات المايكرويه الطافيه تغيير الحالة الفيزيائية لـ dsc و xrd ، بينما أظهرت itz الوظيفية للجزيئات الدقيقة من أي تغيير في موقف المجموعة ftir لم تظهر نتائج itz من البلورية إلى غير المتبلورة والتي كانت تتمتع بثبات أفضل مما قد يزيد من التوافر itzطالق وهكذا ، يبدو أن الجسيمات الدقيقة العائمة المزدوجة المغلفة هي طريقة واعدة لزيادة القابلية للذوبان وإطالة أمد إ البيولوجي عن طريق الفم. .طريقة تبخير المذيبات، الجسيمات الدقيقة , ,هايدروكسي بروبل مثيل سيليلوز ,اثيل سيليلوز : اتراكونازول ةالكلمات المفتاحي introduction oral drug administration is the most common route to get a systemic effect due to its numerous advantages like safety, noninvasive, painless, does not require assistance, more convenient for chronic drugs administration and the medicament not need to be sterile.(1)however, several factors can affect the oral bioavailability, including water solubility and ph-dependent solubility. hence, several techniques have been used to overcome these challenges include a reduction of the particle size that solves just water solubility problem(not the ph-dependent problem) such as micronisation or nanonisation, use of surfactants and conversion of crystalline to amorphous forms(2). oil in water solvent evaporation method is most straight forward method for microencapsulation, widely used in pharmaceutical industries and costeffective. this directly modified technique can be used to overcome many drugs physicochemical problems including both water solubility and phdependent solubility using specific coating materials (3). microparticles defined as solid particles with range of 1 and 1000 μm in diameter consisting of a core (drug) and coat layer prepared using one of the microencapsulation methods to overcome core (drug) related undesired physicochemical properties to obtain a more therapeutically effective drug(4). itraconazole is an active triazole, antifungal agent (c35h38cl2n8o4), which is active against a broad spectrum of fungal species including 𝘊𝘳𝘺𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴, 𝘊𝘢𝘯𝘥𝘪𝘥𝘢, 𝘈𝘴𝘱𝘦𝘳𝘨𝘪𝘭𝘭𝘶𝘴, 𝘉𝘭𝘢𝘴𝘵𝘰𝘮𝘺𝘤𝘦𝘴 and 𝘏𝘪𝘴𝘵𝘰𝘱𝘭𝘢𝘴𝘮𝘢 𝘤𝘢𝘱𝘴𝘶𝘭𝘢𝘵𝘶𝘮. (5, 6) itraconazole is (bcs ii) and has a pka of 3.7 (7). itz has the characteristic of ph-dependent solubility having the highest solubility at the acidic media (4μg/ml) compared to basic ph (1μg/ml). it is slightly soluble in alcohols and freely soluble in dichloromethane and practically insoluble in water (8), indicating that low aqueous solubility and phdependent solubility are the main reasons for low oral bioavailability. itraconazole is a weak basic drug with pka of (3.7), therefore at a low ph, of gastric juice, itz will be ionised therefore, the gastric acidity is essential for adequate dissolution, and its oral bioavailability increased when taken with food since gastric ph decrease after food intake(9).however, firstly the ph of the stomach is variable and impossible to remain below1.2 value, secondly there are cases that raise stomach ph value thus lead to decrease of itz solubility, and therefore bioavailability include fasting state, patients taking gastric acid-reducing agents such as protein pump inhibitors(ppi) and h2-blockers and furthermore patients with aids who complain from hypochlorhydria.(10). the objective of this study is to overcome both low water solubility and ph-dependent solubility of itraconazole by formulating it as floating microparticles and one with a relatively phindependent floating microparticles and compared between them thus to improve the solubility at variable ph values and prolong the release of the itz in stomach, which may lead to increase in itz bioavailability. materials and methods materials itraconazole (itz) powder (baji gaokang biotechnology co., ltd., china), hydroxy propylmethylcellulose (hpmc15cps)(shanghai ruizheng chemical tech co., ltd., china),ethylcellulose (ec)(bdh chemical ltd., england),extra virgin olive oil (evoo), rice bran oil (rbo), palm oil (po) and safflower oil (sfo) (furkan dogalurunler.tic.san.ltd, turkey). dichloromethane (dcm) (thomas baker chemicals pvt.ltd., india), ethanol, methanol and n-hexane (chem-labnv.,belgium). hydrochloric acid (hcl) (central drug house (p) ltd., india). eudragit s100 (ed s100) (athos chemicals, co., ltd., india). methods saturation solubility determination solubility studies for the pure drug achieved in 0.1n hcl (ph1.2), hcl buffer (ph 3.7) and phosphate buffer (ph 6.8) alone and with sls at a concentration of 2, 3 and 6% (w/v), respectively. iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 238 excess amount of pure itz was added to 10 ml of each solution in a test tube and agitated at 25±0.5°c in a rotary test tube shaker for 72hr. then, samples were filtered using 0.45µm millipore filters. each filtrate suitably was diluted with the respective solvent and analyzed by measuring the absorbance at the determined maximum wavelength (λmax) in each solvent usinguv-1800 uv-vis spectrophotometer (shimadzu scientific instruments inc., japan).the solubility (mg/ml) of itz was calculated (11). preparation of itz floating microparticles (phdependent) oil in water solvent evaporation method was used to prepare floating microparticles (phdependent) of itz. fifteen formulas prepared using different concentrations of polymers including; ec,hpmc 15cps and ed s100. different oils were also used such as; extravirigin olive oil (evoo), rice bran oil (rbo), palm oil (po) and safflower oil (sfo). polyvinyl alcohol (pva) was used as a surfactant, as shown in table (1). the vegetable oils were not used in formulas f1-f3. the used oils dissolved in a vehicle composed of (dcm) and ethanol at 1:1 ratio forming the oil phase to which the drug added. while the aqueous phase containing 0.75% pva as a surfactant. the oil phase was added to the aqueous phase drop wise using a bared syringe, under highspeed mechanical agitation of 1000rpm by utilizing magnetic stirrer for one hour at 25±1°c to allow the organic solvent to evaporate and get the microparticles that filtered and dried at room temp overnight. each formula was washed with 5 ml dcm to remove the excess oil if needed and dried again (12). table 1.composition of itz floating microparticles (ph-dependent) ingredient formula no. f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 f13 f14 f15 itz(mg) 200 200 200 200 200 200 200 200 200 300 200 200 200 200 200 ec(mg) 900 800 700 800 800 800 800 700 900 800 800 800 800 800 hpmc15 cps(mg) 100 200 300 200 200 200 200 300 100 200 200 200 200 200 200 eds100 (mg) 800 evoo (ml) 2 rbo (ml) 2 po(ml) 2 sfo(ml) 2 2 2 2 1 2 2 2 2 ethanol (ml) 20 20 20 20 20 20 20 20 20 20 20 20 20 20 methanol (ml) 20 dcm (ml) 20 20 20 20 20 20 20 20 20 20 20 20 20 20 20 dw(ml) 400 400 400 400 400 400 400 400 400 400 400 200 400 400 400 pva(%) 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 0.75 speed(rpm) 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 500 1000 iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 239 preparation of relatively ph-independent itz floating microparticles dual coating solvent evaporation method used for the development of the relatively phindependent itz floating microparticles. the first coat composed of hpmc 15 cps. it was applied by using oil in water solvent evaporation method then without filtration the second coat was an organic solution of hpmc15, ec and sfo in 1:1 of dcm: ethanol as a solvent(13) as represented in table 2. table2. composition of itz relatively phindependent floating microparticles ingredient f 16 itz (mg) 200 1st coat 2nd coat hpmc15 cps (mg 200 dcm (ml) 10 ethanol (ml) 10 ec (mg) 800 hpmc15 cps (mg) 200 sfo (ml) 2 ethanol (ml) 20 dcm (ml) 20 dw (ml) 400 speed (rpm) 1000 1000 characterization of the prepared floating microparticles percentage yield determination yield of the prepared microparticles calculated by dividing the dry weight of the prepared microparticles (practical weight) by the total weight of the drug and excipients used (theoretical weight) as stated in the following equation: 𝑌𝑖𝑒𝑙𝑑 (%) = (𝐴𝑐𝑡𝑢𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡/𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡) ×100 (1) particle size and polydispersity index (pdi) determination all of the prepared formulations were in the micro size that is bellow 212μm because all are sieved using sieve number 70. in addition , particle size and pdi of the prepared itz floating microparticles f7,f12, f14,f15 and f16 measured by dynamic light scattering (dls) using nano brook 90plus particle size analyzer (brookhaven instruments. the usa). measurements were performed in triplicate by measuring the intensity of light scattered by the molecules in the sample as a function of time at 90° scattering angle and constant temperature 25 °c. the pdi was determined, which measured the width of the size distribution of each formula; it is an index of spread or variation within the particle size (14). drug loading and entrapment efficiency (ee) determination 25 mg of each formula dissolved in 50 ml methanol. the solution filtered by 0.45 µm millipore filters and 1 ml was taken and diluted with methanol if required. the samples were analyzed for drug content spectrophotometricaly at 262 nm (14). ee (%) = (actual drug loading / theoretical drug loading) ×100 (2) drug loading (%) =(actual drug content / weight of powdered microsphere) ×100 (3) buoyancy per cent determination weight of the formula equivalent to 100mg itz was spread over the test media using dissolution apparatus (type ii) that filled with 900 ml of 0.1n hcl (ph 1.2, ph 3.7) and phosphate buffer (ph 6.8) separately in each case. the medium was agitated with a paddle rotating at 50 rpm for 12 h. both floating time (ft) and floating lag time were calculated. the floating and the settled portions of microspheres recovered separately were dried and weighed. all the bouncy studies were performed in duplicate (14) bouncy % = (wf / wf + ws)×100 (3) invitro release studies all formulations were subjected to in vitro release studies. the studies were carried out by usp apparatus ii (paddle method) at 50 rpm using 900 ml of 0.1n hcl (ph 1.2, ph 3.7) and phosphate buffer (ph 6.8). the temperature maintained at 37 ± 0.5 °c. at specific time intervals (every two hours), five ml aliquots were withdrawn and analyzed by uv spectrophotometer at the respective λmax after suitable dilution against suitable blank. the removed volume replaced with an equal volume of fresh solvent (9). all release studies performed in duplicate. (14) differential scanning calorimetry (dsc) the dsc of pure itz powder and selected formula (f16) was taken on (shimadzu dsc-60 plus, japan).ten milligrams sealed in the flatbottomed aluminum pan of the differential scanning calorimeter. data collection was achieved at a temperature range of 25–200°c and the heating rate was 5°c/min under nitrogen gas at a flow rate of 25 ml/min (15). fourier transform infrared spectroscopy (ftir) analysis the ftir spectra of pure itz and selected formula were recorded using ftir -7600, iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 240 australia spectrophotometer. powders were mixed with potassium bromide and compressed into disks using hydraulic press before scanning from 4000 to 400 cm-1 (16). x-ray powder diffractometry study the xrd patterns for pure itz and selected formula (f16) were analyzed using an xrd6000, shimadzu-japan. the sample chosen scanning was conducted over powder x-ray diffractometer at the axis of 2 thetas, with the continuous scan range of 5-80 degree. the operating voltage was 40 kv and current 30ma and scans step size of 0.050° (2θ) and scan step time of 60 seconds (17). statistical analysis to investigate the significance of the difference between the results of studied formulations, t-test used. the level of significance set at a  0.05, and (p <0.05) was considered to be statistically significant. results and discussion saturation solubility of pure itz the saturated solubility of itz were 0.1, 0.01 and 0.0044 in 0.1n hcl (ph 1.2), hcl buffer (ph3.7) and phosphate buffer for (ph 6.8), respectively as shown in fig (1). since sodium lauryl sulfate (sls) is a surfactant, so it used to increase the solubility of itz to fulfill sink condition. the saturated solubility of itz were 0.42, 0.37 and 0.36 at 0.1n hcl (ph1.2with2% sls), (ph3.7with3% sls) and phosphate buffer of ph 6.8 with 6% sls. figure 1. saturated solubility of pure itz at different ph. percentage yield the yield per cent of various floating microparticles formulas prepared depicted in figure (2).per cent yield does not reach 100% usually due to mechanical variables during preparation method and precipitation of the formula components on the tools used in the preparation. the percent yield was 58% in f2 were no vegetable oils used. however, the use of vegetable oils results in significant increase in yield per cent, f4 98%, f5 95%, and f6 91%, f7 86% were different vegetable oils used (p < .05). also, the higher sfo volume (f7 two ml versus one ml in f11) result in the higher per cent yield ( f7 92% and f11 88%) may be due to the higher oleic acid content when higher volume used. in f16, the lower yield was due to the longer stirring time that is three hours compared to one hour in f7 (different method was used). (18) similarly, the lower hpmc15 cps concentration results in a higher yield per cent, in f8 were 300mg of hpmc and 700 mg of ec the yield was 83% while in f9 were 100mg of hpmc and 900 mg of ec used a yield of 98%. this result may be due to increasing hpmc 15cps concentration result in a higher viscosity of the oil phase which results in flocculation and agglomeration of the prepared microparticles thus lower yield per cent. nepal et al. had found that the higher polymer concentration, the higher yield percent until an certain value after which the yield decreased (12). figure 2. percentage yield of itz floating microparticles. particle size analysis all the prepared formulations were in the micro size bellow 212μm because all are sieved using sieve number 70. however, f7, f12, f14, f15 and f16 particle size determined using, dynamic light scattering (dls) by nano brook 90plus particle size analyzer, and their particle size varied from 2.56µm to 6.086µm as shown in figure 3.the higher stirring rate; the smaller microparticles size due to less tendency of the formed microparticles to coalescence and aggregate (18,19). regarding the (f16), the particle size is bigger due to the higher drug-to polymer ratio and the longer stirring time (20). figure 3. particle size (μm) of itz floating microparticles iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 241 drug loading and entrapment efficiency (ee) determination the results of drug loading (dl) and drug entrapment efficiency (ee) have been shown in figure (4) and figure (5), respectively. inf4, f5, f6, f7, the %dl was 98, 98, 92 and 97%, respectively, and %ee was 98, 97, 84 and 89%, respectively while in f1, f2 and f3 the %dl was range from 50-79 and %ee range from 2046.using vegetable oils results in a significant increase in both drug loading and entrapment +efficiency (p < 0.05).vegetable oil with higher oleic acid content results in higher dl and ee (f4). similarly, these parameters increase upon using of lower concentration of hpmc15cps; in f8 were 300 mg of hpmc15cps used, %dl was 86, and ee was 71 while in f9 were 100mg of hpmc15cps used %dl was 99 and %ee was 98.the preparation technique dual coating solvent evaporation method also results in higher dl and ee (21). figure 4. drug loading of itz floating microparticles figure 5. entrapment efficiency of itz floating microparticles. buoyancy percent the floating lag time (flt) was zero for all formulas since floating has occurred immediately after the formula spread over the test media (23). the floating time (ft) was range from 3 %( f3) to 98 %( f4) as shown in fig (6).vegetable oils used were the key factor that provides bouncy; with higher oleic acid content of the vegetable oil; higher bouncy per cent obtained so in (f4) evoo used that had the highest oleic acid content among the used vegetable oil resulting highest bouncy per cent (94%). also, the concentration of hpmc15cps played an essential role in the determination of bouncy per cent with the lower the hpmc14cps concentration, the higher bouncy per cent (95% in f9). floating ability result from the low density of the prepared microparticles . similar results observed with floating microparticles of resperidone (12). figure 6. bouncy percent of itz floating microparticles in vitro drug release in vitro cumulative release per cent from the selected formula (f16) was 100 % at 10 hour (ph 1.2) while was 84% at 12 hour (ph1.2) from f7 (p> 0.05), 62 % at 12 hour (ph 3.7) from selected formula(f16) while was 22% at 12 hour (ph 3.7) from f7 (p < 0.05) , was 35 %at 12 hour (ph 6.8) while, 7% at 12 hour (ph 6.8) from f7(p < 0.05), therefore itz release from the selected formula is significantly better(p < 0.05)as shown in figures (7) and (8). these results indicated the efficiency of the dual coating solvent evaporation method and the ph-independent polymers used in solving the problem of the ph-dependent release of itraconazole. furthermore, since, itraconazole is bscii drug thus decreasing the particle size to micro-scale resulted in higher solubility this coordinated with noyes–whitney equation, in which the dissolution rate enhanced as the saturation solubility increased and the particle size decreased .also, relatively ph-independent solubility advantage resulted from the first coat, namely hpmc 15cps(13,22,23). another factor that may contribute to the fast release was the entrapment efficiency which had a direct effect on the drug release profile. as it increased, the release rate also increased (24). iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 242 figure 7. release profile for ph-dependent itz floating microparticles at various ph. figure 8. release profile for relatively phindependent itz floating microparticles at various ph. differential scanning calorimetry (dsc) the dsc was used to estimate the effect of excipients and conditions on the physical properties of the drug. pure itz exhibited a sharp endothermic melting peak at 170° (figure 9) indicating no change in its melting temperature and the drug had a crystalline nature with high purity. the dsc of the selected formula (f16) in figure 10 showed the complete disappearance of the melting peak of itz gave clear evidence of the transformation of the drug from crystalline to the amorphous state (25, 26). figure 9. dsc thermogram of pure itz at temperature range (20-250 °c) figure 10. dsc thermogram of selected formula (16) at temperature range (20-250 °c) fourier transforms infrared spectroscopy (ftir) the ftir spectrum of pure itz shown in figure 11. the main peaks of pure itz observed at wavenumbers,3466, 3130, 2965, 3067, 1698, 2822,1611, 1512 and 1453cm-1. it should be noted that all the characteristic bands of itz were detected in the spectrum of the physical mixture of drug, ec, hpmc15cps, as shown in figure 12. therefore, there is no interaction between the drug and polymers used. a sharp band at 1698 cm−1 in the pure drug spectrum was due to c= o, in the ftir spectrum of the selected formula (f16) all of the itz characteristic peaks are present as shown in figure 13 excepted that, the peak 1698 that shifted to 1745 this is because the fatty acids content of safflower oil has a characteristic peak at 1745 that shown in figure 14 (27) thus, in conclusion, the drug, polymers and safflower oil used in the formulation were compatible with each other.( 28,29). figure 11. ftir spectrum of pure itz. iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 243 figure 12. ftir spectrum of the physical mixture of drug, ec and hpmc 15cps. figure13.ftir spectrum of floating microparticles (selected formula f16) figure 14. ftir spectrum of safflower oil. (27) powder x-ray diffraction (pxrd) xrd test of the pure drug and the selected formula (f16) was done to detect the polymorphic structure of the drug before and after formulation. generally, the amorphous mater sate has higher physical stability. crystalline nature of pure itz easily detected through the numerous distinctive peaks as shown in figure 15 (27).the xrd of f7 and f16 showed disappearance of the characteristic peaks of pure itz as shown in figure 16 and 17 indicated that itz incorporated into the polymer matrix that was in the amorphous state (30). iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 244 figure 15. xrd of pure itz. figure 16.xrd of itz selected formula (f16) figure 17. xrd of itz best ph-dependent formula ( f7) conclusion dual coating solvent evaporation method was successfully used to produce relatively ph-independent itz floating microparticles by using the ph-independent water-soluble polymer(hpmc15 cps) that provided relatively ph-independent solubility of itz with a hydrophobic polymer (ec) and safflower oil that provided bouncy. different parameters, such as hpmc 15cps versus ec concentration, various vegetable oil including, safflower oil volume, stirring speed, oil to water ratio, co solvent type, drugto polymer ratio were investigated and optimized to produce a formula that provided both excellent bouncy for 12 hours with maximum drug release. ph-independent itz floating iraqi j pharm sci, vol.29(1) 2020 itraconazole floating microparticles 245 microparticles (f16) was significantly better than ph-dependent itz floating microparticles (f7) in both bouncy per cent at 12 hours and dissolution profile at all test media of different ph values (p < .05), therefore, dual coating solvent evaporation method is a promising method to improve ph-dependent and solubility problems of itz. references 1. tripathi kd. essentials of medical pharmacology. 6th edition. new delhi: jitendar p vij jaypee brothers medical publishers (p) ltd; 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mice received cinnamic acid 50 mg/kg in liquid paraffin orally by gavage. group iv: asthma model/ca 25 mg/kg; mice received 25 mg/kg in liquid paraffin orally by gavage. group v: asthma/ (50 mg/kg) cinnamic acid group; mice received cinnamic acid 50 mg/kg in liquid paraffin orally by gavage. the experiment continued for 14 days. on day 15, broncheo-alveolar lavage fluid, blood and lung tissue was collected. total cell count, tissue tnf-α, il-33, and serum ige increased considerably after sensitization to ovalbumin (ova). on the other hand, administration of cinnamic acid in 25mg and 50mg/kg has significantly decreased total wbc count, tissue tnfα, il -33, and serum ige results. these findings suggest that cinnamic acid has a protective effect against ova-induced allergic asthma in mice, possibly through its inhibitory activity on some proliferative modulating enzymes. keywords: allergic asthma, airway inflammation, ovalbumin ova, cinnamic acid (ca), chronic inflammation. التاثير الوقائي المحتمل لحمض السيناميك على نموذج الربو المستحدث في الفئران بوساطة بروتين البيض ** مناف هاشم زلزلة و 1*,حيدر خضر السَّفار ، بغداد، العراق. وزارة الصحة والبيئة * بغداد،العراق. جامعة بغداد، فرع االدوية والسموم ،كلية الصيدلة، ** الخالصة عالجية محدودية األدوية التقليدية تطوير تقنيات مزمن تلعب فيه الخاليا المناعية والهيكلية دوًرا. تقتضي الربو هو اضطراب تنفسي هذه في للربو. السيناميكمبتكرة لحمض المحتمل الوقائي التأثير من بالتحقق قمنا ، الناجم (ca) الدراسة الربو في عنعلى البيض البومين فئرا لكل مجموعة: 12( جراًما عشوائيًا وتم تقسيمها إلى خمس مجموعات 20-30) balb/c . تم اختيار ستين فئرا من الذكور من النوع انالفئر )الطبيعية(المجموعة تلقت \ pbs األولى: السيناميك(: )حمض التحكم الثالثة: المجموعة التحسسي(. )الربو الثانية: المجموعة السائل. البارافين مجم/كجم حمض السيناميك: 25مجم/كجم في البارافين السائل عن طريق الفم. المجموعة الرابعة: الربو التحسسي + 50الفئران حمض السيناميك اع الفئران تم + 25طاء التحسسي الربو الخامسة: المجموعة الفم. طريق عن السائل البارافين في السيناميك حمض من مج/كجم 50مجم/كجم سيناميك حمض الفئران تلقت السيناميك: لمدة 50حمض التجربة استمرت الفم. طريق عن السائل البارافين في اليوم 14مجم/كجم في يوًما. بشكل كبير بعد التحسس ige و il -33 و tnfα جمع سائل غسيل القصبات الهوائية والدم وأنسجة الرئة. ارتفعت مستويات الخامس عشر، تم مجم/كجم إلى انخفاض كبير في عدد كريات الدم 50مجم و 25. من ناحية أخرى ، أدى إعطاء حمض سيناميك (ova) بوساطة البومين البيض في الدم ، تشير هذه النتائج إلى أن حمض السيناميك له ige في نسيج الرئة ، ومستوىil -33 ، و tnfα ياتوانخفاض في مستو البيضاء ، . تأثير وقائي ضد الربو التحسسي في الفئران ، من خالل نشاطه المثبط او المعدل على االنزيمات التكاثرية ( ، االلتهاب المزمن.ca، حمض السيناميك ) ova، البومين البيض الكلمات المفتاحية: الربو التحسسي ، التهاب الُشعَب الهوائية introduction asthma is a chronic inflammatory respiratory illness characterized by wheezing, chest tightness, dyspnea, and cough, which are all symptoms of airway obstruction. they can occur on their own, in the early morning or late at night, following exposure to an allergen or physical activity (1,2). depending on where you reside, asthma has a broad variety of prevalence, mortality, and severity. although high-income countries have a higher incidence of asthma, asthma mortality is greatest in lowand middleincome countries. adults have greater prevalence of asthma-related mortality and healthcare consumption than children, despite the fact that children have a higher frequency and incidence of asthma (3). acute reversible airway obstruction, persistent airway hyperresponsiveness (ahr), and airway inflammation are all characteristics of allergic asthma in humans, which is described as a chronic inflammatory illness of the airways that affects the respiratory system (4,5). 1corresponding author e-mail: haydar77_k@yahoo.com received: 29/1 /2022 accepted:29 /4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp133-138 iraqi j pharm sci, vol.32(1) 2023 protective effect of cinnamic aid in asthma 134 structure alterations in the airway include subepithelial and airway wall fibrosis, goblet cell hyperplasia /metaplasia, smooth muscle thickening, and enhanced vascularity (6). 'airway modifications' or 'remodeling' are the consequence of repeated allergen exposure that causes recurring or continuing inflammation in the airways (7). chronic inflammation and structural alterations are hypothesized to contribute to asthma symptoms by having functional effects. airway remodeling, or structural changes in the airways associated with asthma, is a pathogenic aspect of chronic asthma that contributes to the disease's clinical presentations (8). t-helper 2 (th-2) cells release different pro-inflammatory cytokines that play essential roles in the development of allergic asthma, including a specific profile of interleukin (il)-4, il-5, and il-13, and this model replicates the clinical features of allergic asthma. furthermore, they can cause the creation of mucus, as well as the release of allergen-specific immunoglobulin (ige), chemokines, and eosinophils (9). hydroxycinnamic acids (hca) are a family of phytochemicals, or plant-derived chemicals, with a variety of health benefits, but they are not considered essential nutrients. this group of phenolic compounds is among the most often encountered in nature. phenolic substances are plant metabolites that are common in plantbased meals and drinks, according to research. they aid in defense against uv radiation and disease assaults in plants, and they also play a vital role in growth and development (10,11). since cinnamic acid is a natural chemical with an anti-inflammatory action as a regulator of particular enzyme activity, and stop cells from multiplying. the purpose of this study was to see whether cinnamic acid could protect against ova-induced allergic asthma. material and methods materials cinnamic acid and ova were purchased from sigma-aldrich chemie gmbh. diethyl ether supplied from roman pure chemistry, uk. paraffin oil supplied from applichem gmbh/germany. eliza (enzyme linked immunosorbent assay) kits for il-33, tnf-α, and ige were supplied by shanghai yehua biological technology co.ltd, china. experimental animal sixty adult albino male balb/c mice (weighing between 20 and 30 g) obtained from and kept in the animal house of the college of pharmacy/university of baghdad under specific pathogenfree conditions, provided with water and food ad libitum during a 12-hour light-dark cycle and kept during the study with regular room temperature (18 -21oc). animals were handled throughout this process in accordance with the helsinki declaration, which was approved by the university of baghdad's ethical committee for the use of animals in research. experimental protocol for allergic asthma model for preparation of allergic asthma model, ova is used which is the main protein found in egg white, which is not essentially immunogenic and therefore it must be systemically injected. multiple systemic allergen injections are often required in acute sensitization procedures. intraperitoneal administration of 1 ml of 10% ova on day 1, then exposed to an aerosol containing of (1% ova) for 14 successive days, 30 min /day, in an aerosol chamber built for this purpose from a portable nebulizer and a plastic food container as a nebulizing chamber (12,13). study design 60 mice were divided randomly into 5 groups 12 animals each: 1) group i: healthy control (phosphate buffer saline (pbs) / liquid paraffin). 2) group ii: asthma model (ova-challenged). 3) group iv: asthma model with cinnamic acid group: mice received (25 mg/kg) in liquid paraffin orally by gavage. 4) group v: asthma model with cinnamic acid group: mice received cinnamic acid (50 mg/kg) in liquid paraffin orally by gavage. 5) group iii: cinnamic acid control group: mice received cinnamic acid (50 mg/kg) in liquid paraffin orally by gavage. on day 15, mice were sacrificed; samples of blood were collected from the retro-orbital vein and centrifuged for 20 min at 3000g. subsequently, the serum was preserved at -20°c to determine ige levels. six mice from each group were assigned for bronchoalveolar lavage (balf), while the remaining six were sacrificed by cervical dislocation to retrieve lung tissue. bronchoalveolar lavage fluid (balf) collection following blood collection, six animals from each group were euthanized under deep anesthesia. to collect balf, the lungs were lavaged three times with 1 ml of normal saline. a total of around 2 ml of liquid was retained (14). centrifugation was used to separate the balf samples at 400 g for 7 min at 4o c, with the cellfree supernatant samples retained at 20 o c awaiting for experiments, ice is used to preserve the sediment for total wbc counting. total wbc counting balfs were collected and centrifuged for 7 min at 400 rpm/ min/ 4oc to separate entire cells into pellets. the pellet of entire cells was separated on ice after the supernatant was removed. the coulter cellular analysis system® was used to distinguish the wbc count on the same day. lung homogenate preparation phosphate buffer saline (pbs ph 7.4) at 4oc was used to wash the post-raval and right iraqi j pharm sci, vol.32(1) 2023 protective effect of cinnamic aid in asthma 135 inferior lobe tissue to eliminate any remaining blood and other debris. the tissue was then dried and weighed. for each 100 mg of lung tissue, 0.9 ml of cold pbs was added in an eppendorf tube. after that the eppendorf holding the tissue was placed in an icefilled beaker to keep it cool, the lung tissue was homogenized for 1 min at speed three on the homogenizer machine. the homogenate was centrifuged for 20 min at 4o c and 3000 rpm in a refrigerated centrifuge. the supernatant was extracted using a micropipette and kept at – 20oc until il33 and tnfα were analyzed. measurement of inflammatory cytokines levels il-33 and tnf-α levels were quantified using lung tissue homogenate supernatants, and serum levels of ige were determined using elisa. a microplate spectrophotometer was used to measure absorbance values at 450 nm (human, germany). standard curves were used to calculate the content of ige and inflammatory cytokines. histological sample preparation of lung tissue the middle and right superior lobes were selected for histopathological evaluation. lung tissue excised and fixed in neutral buffered formalin 10% (v/v), dried, immersed in paraffin, cut into 4-m slices, deparaffinized with xylene, and stained with hematoxylin and eosin (h&e) and periodic acid schiff (pas) (15). a light microscope (optika microscope, italy) was used to analyze tissue sections. on a scale of 0 to 4, the severity and the presence of inflammation and goblet cell hyperplasia in the lungs were evaluated (16). statistical analysis graphpad prism 7® was used to analyze the data. one-way analysis of variance (anova) was used to compare the means of the groups. all data were reported as mean and were considered significant when the p-value was less than 0.05. results effects of ca administration on wbc count in balf in the ova-challenged allergic asthma model; total wbc count in balf elevated considerably when compared to control group mice. in comparison with the ova-challenged allergic asthma model group, pretreatment with ca at a dose of 50 mg/ kg and 25 mg/kg resulted in a significant decrease in total wbc count in bal fluid. administration of ca alone had no impact on blood cell infiltration to bal fluid (figure 1). effect of cinnamic acid (ca) administration on il33 levels in lung tissue homogenate the levels of il-33 in ova-challenged model group were considerably higher than in the control group. when compared to the ovachallenged model group, pretreatment with ca at both doses had significant influence on il-33 levels. ca treatment in the non-sensitization animals didn't produce a substantial change in il33 level (figure 2a). effect of cinnamic acid (ca) administration on tnf-α level in lung tissue homogenate figure 2b shows that ova challenging revealed a significant increase in levels of tnf-α, a proinflammatory cytokine, compared to the control group. pretreatment with ca at a dose of 50 mg/kg and 25 mg/kg resulted in a significant decrease in tnfα level. on the other hand, ca treatment in the non-sensitization animals was resulted in nearcontrol levels of tnf-. effect of ca administration on serum ige levels compared to the control group, the data in figure 2d demonstrates a significant and substantial increase in serum ige levels of the ovachallenged group. when compared to the ovachallenged model group, pretreatment with ca at a dose of 50 mg/kg and 25 mg/kg resulted in a significant reduction in ige serum levels. finally, ca at a dose of 50mg/kg alone did not result in significant changes in serum ige levels when compared to the control group. histopathological assessment of lung tissue sections we carried out histological examination to illustrate the possible protective effect of ca against lung tissue remodeling in ova-challenged group as seen in (figure 3). the results show the significant difference between the ova challenged group and the control group; also, a significant difference is observed between the ova challenged / ca 50 mg group compared to the induction model (figure 3f). regarding the histological findings, we see the obvious protective effect of ca in the group treated with ca challenged with ova / 50 mg (figure 3d) compared to the control group (figure 3a) and the group challenged with ova (figure 3b). meanwhile, ca in a dose of (25 mg/kg) did not show much protection on a histological level as seen in (figure 3c). figure 1. effect of ca on total wbc count in balf for ova-induced allergic asthma in mice. (each value represents mean ± sd) * is significantly different compared with control group (p<0.05). # is significantly different compared with ovachallenged group (p<0.05). c o n t r o l o v a c h a l l e n g e d o v a c h a l l e n g e d + c a ( 2 5 m g / k g ) o v a c h a l l e n g e d + c a ( 5 0 m g / k g ) c a ( 5 0 m g / k g ) o n l y 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 5 0 0 t o ta l ce ll c o u n t c el l / cm 3 b a l f * * * ## iraqi j pharm sci, vol.32(1) 2023 protective effect of cinnamic aid in asthma 136 figure 2. effects of ca on the inflammatory contents in serum and lung tissue. il-33, tnf-α, and ige in serum and lung tissue for ova-induced allergic asthma in mice. (each value represents mean ± sd) * is significantly different compared with control group (p<0.05). # is significantly different compared with ova-challenged group (p<0.05). figure 3. effect of cinnamic acid on ova-induced pathological pulmonary changes. red arrow: hemorrhage or congestion. black arrow: alveolar sack & emphysema. a: normal lung (control); b: ova-challenged (model); c: ova+ca (25 mg/kg); d: ova+ca (50mg/kg); e: ca only (50mg/kg); f: histology scoring analysis. (each value represents mean ± sd) * is significantly different compared with control group (p<0.05). # is significantly different compared with ova-challenged group (p<0.05). c o n tr o l o v a c h a ll e n g e d o v a c h a ll e n g e d + c a ( 2 5 m g /k g ) o v a c h a ll e n g e d + c a ( 5 0 m g /k g ) c a ( 5 0 m g /k g ) o n ly 0 5 0 1 0 0 1 5 0 t n f  l u n g l e v e l ( n g / l ) * * * # # c o n tr o l o v a c h a ll e n g e d o v a c h a ll e n g e d + c a ( 2 5 m g /k g ) o v a c h a ll e n g e d + c a ( 5 0 m g /k g ) c a ( 5 0 m g /k g ) o n ly 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 i g e s e r u m l e v e l ( u g / m l ) * # # iraqi j pharm sci, vol.32(1) 2023 protective effect of cinnamic aid in asthma 137 discussion ovalbumin (ova) derived from chicken egg white is frequently used allergen that induces severe allergic lung inflammation in laboratory rodents (17). asthma problems have a complicated etiology. in asthma, cytokines play an important role in the immune system and inflammatory reactions. immunomodulatory properties may be found in a variety of natural compounds, such as influencing the expression of inflammatory cytokines and controlling inflammatory cell activity, according to evidence-based research of natural medicinal herbs in asthma treatment (18). in the present study, we found that ova induced an allergic reaction in the lungs of mice similar to allergic asthma compared to the control group manifested by elevated levels of total wbc in the bal fluid, high levels of il-33 and tnf-α in the homogenate of lung tissue, increased serum ige levels in the homogenate of tissue as shown in figure 1 and 2. pretreatment with ca at a dose of 50 mg / kg shows a significant alteration in results, showing a significant decrease in total wbc in balf, also a significant decrease in il-33 and tnf-α level in lung tissue homogenate, decreased serum ige levels. also, as shown in figures 1 and 2; ca in a dose of 25 mg/kg has a similar effect, but to a lesser extent. while ca in a dose of 50 mg/kg without ova challenging has near control values, suggesting that it only affects allergic parameter in disease state and in a dose-dependent matter. according to these observations, ca has immunomodulatory effects on total wbc, as well as il-33, tnf-, and ige concentrations. on a histological level, asthmatic individuals have aberrant alterations in the structure of the airway submucosa and epithelial tissue. pathologically, asthma is defined by increased th2 cytokine production, infiltration of inflammatory cells into epithelial tissue, metaplasia, and hyperplasia of goblet cells in epithelial tissue, smooth muscle hypertrophy, collagen deposition (collagen i, iii, and v, as well as tenascin c and fibronectin), vascular congestion, and mucous glands that are larger, resulting in narrowing of airways, increased mucous tissue remodeling is the term for these changes in airway anatomy (19,20). as we see in (fig 3 a, b, c, d, e and f) 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factors impacting hypertension awareness among hypertensive population: a quantitative study in a tertiary care hospital in malaysia ali haider mohammed*,1, bassam abdul rasool hassan**, azyyati mohd suhaimi***, ali blebil* , juman dujaili*and abdulrasool m. wayyes** *school of pharmacy, monash university malaysia, jalan lagoon selatan, 47500 bandar sunway, selangor, malaysia **department of pharmacy, al rafidain university college, baghdad, iraq **department of pharmacy practice, faculty of pharmacy, universiti teknologi mara, puncak alam campus, 42300 bandar puncak alam, selangor, malaysia abstract there are obstacles to high levels of hypertension awareness that are embedded in gender, income and lifestyle habits which need to be addressed. those obstacles could lead to high levels of undiagnosed and uncontrolled hypertension. this study aimed to explore the various factors which might affect hypertension awareness among a hypertensive population in a tertiary care hospital. an observational, cross-sectional study was conducted from november 2018 to march 2019. the study was conducted among hypertensive patients at a tertiary care hospital in selangor, malaysia. a validated and translated questionnaire was utilised as a data collection tool. descriptive and inferential statistical analysis was done using spss version 25. a thousand participants (female n=621, male n= 379) were recruited with mean age of 48± 11.09 years old. approximately half of the respondents (51.3%) were not informed by their doctor that they have hypertension. more than half of respondents (66.3%) were unaware of the normal range of systolic and diastolic blood pressure (bp). female gender, chinese race, urban resident, older adults, and tertiary education level are the most significant factors which play a main role in influencing the level of awareness among patients with hypertension. hypertension awareness needs to be addressed from a systemic point of view to solve the growing barriers through accessing the correct information about the disease. health care providers and authorities need to regulate the manner in which information on mortal diseases is presented to the public in order to reduce incidence of malpractice by also encouraging individuals to mostly consider information on public health which emanates from acknowledged authorities rather than going with popular and charismatic sentiments. keywords: hypertension, awareness, factors, malaysia دراسة : على الوعي بارتفاع ضغط الدم بين السكان المصابين بارتفاع ضغط الدمالمؤثرة العوامل مقطعية في مستشفى تخصصي في ماليزيا ، *بليبل ، علي ***ازيياتي محمد سحيمي ،**، بسام عبد الرسول حسن 1*،علي حيدر محمد ** عبد الرسول محمود ويس و* جمان دجيلي بندر صنواي ، سيالنجور ، ماليزيا 47500كلية الصيدلة ، جامعة موناش ماليزيا ، جاالن الجون سيالتان ، * العراق بغداد، ،الجامعة كلية الرافدين الصيدلة، قسم ** بندر بونشاك علم ، سيالنجور ، ماليزيا 42300جامعة مارا التكنولوجية ، بونشاك علم ، كلية الصيدلة ، ، الصيدلة السريريةقسم *** الخالصة هناك عقبات أمام المستويات المرتفعة من الوعي بارتفاع ضغط الدم المتضمنة في نوع الجنس والدخل وأسلوب الحياة والتي تحتاج إلى الية من ارتفاع ضغط الدم غير المشخص وغير المنضبط. هدفت هذه الدراسة إلى استكشاف معالجة. يمكن أن تؤدي هذه العقبات إلى مستويات ع يت دراسة العوامل المختلفة التي قد تؤثر على الوعي بارتفاع ضغط الدم بين السكان المصابين بارتفاع ضغط الدم في مستشفى الرعاية الثالثية. أجر . وقد أجريت الدراسة على مرضى ارتفاع ضغط الدم في مستشفى الرعاية الثالثية 2019رس إلى ما 2018رصدية مقطعية في الفترة من نوفمبر كأداة لجمع البيانات. تم إجراء التحليل اإلحصائي الوصفي واالستنتاجي باستخدام اإلصدار معتمد ومترجم في سيالنجور ، ماليزيا. تم استخدام استبيان عاًما. بينت 11.09± 48عمر ( بمتوسط 379، عدد الذكور = 621ألف مشارك )عدد اإلناث = . تم تطويعالتحليل األحصائيمن برنامج 25 ٪( لم يتم إخبارهم من قبل الطبيب بأن لديهم ارتفاع ضغط الدم. وأن أكثر من نصف المشاركين 51.3النتائج ان ما يقرب من نصف المشاركين ) ضغط الدم االنقباضي واالنبساطي. الجنس األنثوي ، والعرق الصيني ، والمقيمين في المدينة ، ٪( لم يكونوا على دراية بالمعدل الطبيعي ل66.3) الدم. يجب وكبار السن ، و التعليم العالي هي العوامل األكثر أهمية التي تلعب دوًرا رئيسيًا في التأثير على مستوى الوعي بين مرضى ارتفاع ضغط هة نظر نظامية لحل الحواجز المتزايدة من خالل الوصول إلى المعلومات الصحيحة حول المرض. يحتاج معالجة الوعي بارتفاع ضغط الدم من وج دوث سوء مقدمو الرعاية الصحية والسلطات إلى تنظيم الطريقة التي يتم بها تقديم المعلومات المتعلقة باألمراض المميتة للجمهور من أجل تقليل ح ًضا على التفكير في معظم األحيان في المعلومات المتعلقة بالصحة العامة التي تنبع من السلطات المعترف بها الممارسة من خالل تشجيع األفراد أي بدالً من التعامل مع الجمهور والمعتقدات والمشاعر الشعبية. ، ماليزياالمؤثرة عواملالوعي، الارتفاع ضغط الدم، الكلمات المفتاحية: 1corresponding author e-mail: alishanshool93@gmail.com received: 15/7/ 2021 accepted: 17/7 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp233-240 iraqi j pharm sci, vol.31(1) 2022 factors affecting hypertension awareness 234 introduction hypertension is one of the major risk factors to cardiovascular failure and mortality (1). the recent data indicated that 1 out of 5 patients has well-controlled blood pressure, and globally around 9 million deaths are because of hypertension (2). the level of hypertension in southeast asia (sea) countries is increasing dramatically without appropriate action. malaysia is one of sea countries where its population is also suffered from hypertension (2). according to a previous study, two out of every three malaysians have high blood pressure, affecting approximately ten million adults (3). hypertension can be averted or controlled by having healthy lifestyle habits, being aware of the risk factors and how to avoid them. therefore, one of the major methods in reducing hypertension is through raising hypertension awareness in both the young and adult population. this action would be across all ethnic barriers and status. there have been major advancements in public health education and increase the usage of social medias through which information can be disseminated; however, hypertension awareness in society is still under progression (4). nearly two thirds of adults indicate hypertension awareness, while the remaining of hypertensive population is unaware about the risk that might hypertension causes if left untreated. (3, 5). inadequate knowledge and awareness about blood pressure can lead to poor adherence to medications and, consequently, high rates of uncontrolled blood pressure ,therefore raising awareness remains a critical agenda in improving hypertension control and averting cardiovascular disease (2). in malaysia, the present healthcare system is paying more attention on therapeutic intervention rather than preventive care, with 70% of budget is allocated to pharmacological approach whereas 5% is to public health services (which include health promotion and prevention programmes) (6). a recent literature study which analysed the data from 20062015 of hypertensive population in malaysia, demonstrated that 85% of young adults (18-39 years old who were diagnosed with young-onset hypertension) are unaware of their diagnosis and less than 40% of them had controlled blood pressure (6). therefore, understanding the factors that probably influence the level of awareness towards hypertension is believed to be essential in the process of preventing and controlling hypertension (7). hence, this study aimed to explore the various factors which might affect hypertension awareness among a hypertensive population in a tertiary care hospital in malaysia. materials and methods study design, setting, and data collection. an observational, cross-sectional study was conducted from november 2018 to march 2019 through the application of an anonymous survey to patients who attended cardiology clinic at hospital kuala lumpur (hkl). a non-probabilistic sampling techniques was used to distribute questionnaire to participants who were willing voluntarily to participate in the study according to raosoft calculator, at least 385 participants must be recruited. the calculation was based on 50 % response distribution, 5 % margin of error and 95 % confidence interval. the online software foundation is based on widely utilized descriptive studies sample size estimation formula. setting the response distribution to 50% is the most conservative assumption. the assumption that the response rate is 50 % was based on the idea that both responses and response rates were completely unknown since only limited studies conducted in malaysia which were similar to the current study and the response rate was more than 50% (8). however, the research team have managed to recruit 1000 patients who were diagnosed as having hypertension by a specialist physician the inclusion criteria were 1) diagnosed as hypertensive by a physician;2) malaysian adult (age ≥18 years old); and 3) malaysian citizen whereas the exclusion criteria were 1) participants who were pregnant; 2) participants who were mentally incompetent.; and 3) participants who did not complete the questionnaire or refused to participate. participants were selected randomly from hypertensive patients who had their follow-up at cardiology clinic in hkl. participants started filling the questionnaires after taking their consent to participate in the study. the approximate time that each participant spent to compete the questionnaire was around 10-15 minutes. upon receiving the questionnaire from the participants, researcher checked and ensured that all items of the questionnaire were answered. study instrument the questionnaire for the survey was adapted from a previously validated literature study (8) and tailored to the local population to ensure its applicability among malaysian population. the original questionnaire was written in english; therefore, it was translated into bahasa malaysia using a forward and backward translation process. before being finalised, the accuracy and meaning of the translated versions, both forward and backward, were checked by bilingual malaysian expert then, any recommended amendments, wherever necessary, were discussed. it was pretested for content, design, readability, and comprehension on 20 individuals, and modification were made as necessary so that the questionnaire was simple to understand and easily to be answered by hypertensive population to give accurate responses. iraqi j pharm sci, vol.31(1) 2022 factors affecting hypertension awareness 235 the validity and reliability have been checked by conducting a pilot study on 30 hypertensive patients and the questionnaire was found to be reliable and valid in which cronbach alpha value was 0.89. prior to the application of the survey, sociodemographic information on the patients was collected: gender, age, race, marital status, education level, location and source of information about htn. a blood pressure measurement was carried out following the latest recommendations of american heart association by classifying participants whose blood pressure is >130/80 as htn. to evaluate the level of awareness, a score from 0 to 6 was awarded based on the 6 questions. a score of 0-2 was considered a poor level of awareness, a score between 3 and 4 was considered a fair level of awareness, and a score higher than 4 was considered a good level of awareness (9). data analysis the results obtained were recorded, scored, and tabulated into respective sections, with data described in the form of frequency and percentage, means and standard deviations as deemed on the nature of results and outcomes. the data was statistically analysed with spss version 25.0, and the significant level was determined when p is less than 0.05. association between independent variables (demographic outcomes) and dependent variables (level of awareness) was presented in the form of cross tabulations. multinomial logistic regression analysis was performed to predict the likelihood of association between awareness of the participants and their demographic variables. this test was used because dependent variable (level of awareness) has multiple categories (poor, fair, and high) in which the independent variables are participants’ gender, age, race, location, and education level. ethical approval the study was approved by the university technology mara (uitm) research ethics committee (uitm/rec/366/18), as well as the malaysian ministry of health medical research and ethics committee (nmrr-18-2591-43354). results demographic and professional characteristics of the study participants the mean age of participants is 48±11.09 where over half of the respondents (52.5%) were less than 50 years, with majority of the respondents (62.1%) were female. the malay race constituted the majority of the respondents (46.7%) followed by the chinese and indians. the vast majority (90.0%) of the respondents were from the urban areas. participants reported that healthcare professionals were the most source of information regarding hypertension. regarding marital status, majority of the respondents were married (55.6%) while minority was widowed (8.3%). about 39.2 % of the participants had secondary education level as shown in table 1. table 1. sociodemographic characteristics of hypertensive patients who responded to the survey (n = 1000) variables frequency (%) age ≤50 >50 522 (52.2%) 478 (47.8%) gender male female 379(37.9%) 621 (62.1%) race malay chinese indian others 467 (46.7%) 288 (28.8%) 198 (19.8%) 47(4.7%) location urban rural 900 (90.0%) 100 (10.0%) marital status single married divorced widowed 248 (24.8%) 556 (55.6%) 113 (11.3%) 83 (8.3%) education level illiterate primary school secondary school pre-university university 57 (5.7%) 122 (12.2%) 392 (39.2%) 246 (24.6%) 183 (18.3%) source of information about hypertension healthcare professional tv and radio newspaper and magazine leaflets school university/college friends family internet public health campaign 229 (22.9%) 179 (17.9%) 131 (13.1%) 66 (6.6%) 22 (2.2%) 21 (2.1%) 79 (7.9%) 94 (9.4%) 98 (9.8%) 81 (8.1%) hypertension awareness of the participants just over half of the respondents (51.3%) have been informed by a doctor or a health care professional that they have hypertension. the majority of the respondents (66.3%) were also told what their ideal blood pressure reading will be by either a doctor or health care practitioner. however, most of the respondents indicated that they did not receive sufficient information from their physician regarding the correct level of systolic (63.5%) and diastolic (69.6%) blood pressure (bp). the hypertension awareness among respondents is recorded in table 2. iraqi j pharm sci, vol.31(1) 2022 factors affecting hypertension awareness 236 table 2. awareness about hypertension among respondents (n=1000) no. questions frequency of positive answer, (n) percentage (%) frequency of wrong answer, n percentage (%) 1. have you ever been told by a doctor or health care provider that you have hypertension? 513 51.3% 487 48.7% 2. did your doctor or health care provider tell you what your personal blood pressure reading should be? 663 66.3% 337 33.7% 3. if told, what should your top number (systolic) be? 365 36.5% 635 63.5% 4. if told, what should your bottom number (diastolic) be? 304 30.4% 696 69.6% 5. has a doctor or health care provider ever told you that the top number is important to keep under control? 567 56.7% 432 43.2% 6. has a doctor or health care provider ever told you that the bottom number is important to keep under control? 569 56.9% 431 43.1% factors associated with the level of hypertension awareness the factors associated with the level of hypertension awareness were also recorded among the respondents. location, marital status and education level have displayed the most significant score among the respondents with a p-value of 0.0001. the least significant factor was gender which scored a p-value of 0.003. the factors associated with hypertension awareness are recorded in table 3. table 3. association between hypertension awareness and various factors factors *pearson chi-square value df p-value gender 5.43 2 0.003* age 13.42 2 0.0001* race 15.61 6 0.016* location 4.65 2 0.0001* marital status 5.90 6 0.0001* education level 1.51 8 0.0001* note: *pearson chi-square test, p<0.05. moreover, multinomial logistic regression test showed that chinese respondents have around 5 times higher awareness towards hypertension compared to malay and indian race (or 5.2, 95% ci 2.67-9.86, p=0.0001). it was also seen that both female and urban participants were having almost two times more likely to have awareness compared to their reference group (male and rural participants), respectively (or 2.7, 95% ci 1.525.31, p=0.001; or 2.4, 95% ci 1.03-4.64, p=0.042). independent predictors for hypertension awareness are recorded in table 4. table 4. independent predictors for hypertension awareness independent variables# or 95% ci for exp (b) *p (logistic regression) gender (female) 2.7 1.52-5.31 0.001 age (<50 years) 3.1 2.27-6.51 0.004 race(chinese) 5.2 2.67-9.86 0.0001 location (urban) 2.4 1.03-4.64 0.042 education level (university) 4.1 1.98-8.37 0.0001 * p<0.05; #dependent variables: level of awareness iraqi j pharm sci, vol.31(1) 2022 factors affecting hypertension awareness 237 discussion the current study has filled the gap and provided a clear insight about the factors that play essential role in affecting the level of awareness towards hypertension among hypertensive population in malaysia. hypertension awareness is higher among the age group above 50 years old than in younger participants. while hypertension affects all age groups based on lifestyle habits and genetics, it is more often taken seriously by the elderly rather than the younger population. it is typically observed that as one grows older, they are more likely to take their health matters seriously than when they were younger (10). this can be discussed in combination with the idea that the younger population has more physical capacity and energy to make lifestyle habits that lead to a healthier lifestyle, but according to the study of zhang & moran (2017), it is observed that theses habits are often driven by aesthetic factors rather than by health-centric motivations (5, 11). from the respondents, it is noted that there is a great difference in population between the females and males that are hypertensive. females constituted the majority of the hypertensive patients in the current study. this raises the question of whether gender is a risk factor for hypertension. a previous study elaborates on gender as a risk factor for hypertension. the study dismisses that women are more hypertensive than men, rather stating that women are less likely to be hypertensive with the proportion of 12% vs 27% (12). the study of reckelhoff (2018) also finds that women, though less hypertensive, are more aware of their hypertensive state than men with the proportion 32% vs 25%. this is consistent with another study which finds that women are faced with reproductive health issues monthly and therefore this prompts them to be more observant of their body changes and performance more than men (13). therefore, this brings to light that health care usage explains the difference in hypertension awareness based on gender in which women are more prone to hypertension than men. a possible explanation could be that the hormonal changes in females play an essential role in the mechanism of hypertension, which may lead to more disease prevalence in women than in men. additionally, pathophysiological studies of hypertension in women indicated that the shorter stature and the obligatory shorter arterial tree in women may have an impact in increasing heart rates and earlier reflected arterial pulse waves. hence, those factors may lead to the differences between males and females in systolic/diastolic blood pressure, pulse pressure amplification, and diastolic time (14). regular health care practice together with interaction with doctors and health care providers increases hypertension awareness. from the study, little information which patients have on hypertension was received from their consultation with either doctors or health care providers. a previous literature study reported that regular follow-ups of hypertensive patients to their doctor can help to increase their knowledge and awareness toward their disease (15). there are other media through which people can access information on hypertension (16). these include newspapers, social media, health care websites and even tv film and programmes. however, these media can only provide information to a certain extent (17). the retention of information on healthcare which is acquired from these mediums is often limited. there is often a very casual behaviour in the way in which information on hypertension would be disseminated. while the information is informative, most people do not pay full attention as they would consider hypertension is not a critical disease and easily curable (18). thus, healthcare professionals as well as media should play an important role in raising awareness about the potential risk of hypertension if left untreated. there is a rising trend on misrepresentation of information regarding hypertension in the media. according to the medical practice, there is a set of risk factors which are associated with hypertension (19). some of these factors include obesity, sedentary lifestyle, diets high in sodium and genetic factors. however, due to the wide access which people have to sharing information on the internet, there are overwhelming sources which disprove that the known risk factors of hypertension are valid (20). for example, there are overweight people who assert that they are healthy and not at risk of any disease. this is because many of young adults continue to have unhealthy lifestyles such as smoking or drinking alcohol without being aware about the risk that they bring to their health (21). therefore with this nature of influence in the media, hypertension awareness is impeded in young people, which are majority of the internet users (22). they end up acquiring information on hypertension which is less accurate and invalidates the known risk factors to hypertension. lower access to health care usage is another prevalent factor which affects hypertension awareness (23). it is noted that most people acquire their information on hypertension from doctors and health care providers. however, there are still areas where there is limited access to health care and there are also areas where health care is expensive. this excludes uninsured young adults, the less privilege and those with limited health insurance to have less access to the facilities from which they can gain information. therefore, the issue of access to these facilities translates in the low levels of awareness which is noted in the population of this study. according to fang et al (2019), when health care access and usage is high, it corresponds with the levels of awareness which the population has on hypertension, and their ability to receive preventive iraqi j pharm sci, vol.31(1) 2022 factors affecting hypertension awareness 238 care (24). however, this is disputed by the study of omboni (2019) which asserts that awareness often depends on the primary perspective which an individual adopts towards major health issues, which then places emphasis on behavioural and biological risk factors (25). ethnicity is also discussed as a factor which affects hypertension awareness. it is essential to note that despite certain genetic differences, hypertension does not discriminate based on ethnicity (26). however, it is often related to the typical lifestyle habits which the race perpetuates, such as their major physical habits and dietary preferences. however, this is refuted by the study of cuevas et al (2017) which asserts that the world is now a global village and major ethnic differences in lifestyle are now being erased. ethnicities are living in diversity with emphasis on equal access to information and facilities thus hypertension awareness among ethnicities would not be dependent on this factor (27, 28). there is limited awareness on the actual relevance of the systolic and diastolic numbers in one’s hypertension reading. in most cases, a patient would be notified of their reading, and given medical advice or even medication to lower the reading (29). therefore, in most cases, the patients are more concerns with seeing their reading decrease numerically without the awareness of the relevance of the figure. this reflects a shallow and unproductive method of approaching healthcare (30). this is consistent with the study of judy et al (2019) which indicates that most knowledge on public health tends to be shallow and number driven instead of addressing the biological and behavioural risk. patients would be oblivious of the biological risk which they face thus they would not consider awareness on the disease with much importance (31). this is reflected in the study of palatini et al (2018) which revealed that one of the primary reasons which caused patients to abruptly stop their hypertension medication is having a lower reading without awareness on the repercussions of their actions, which stem from limited awareness (32). hence, it is important for doctors to disseminate comprehensive information to their patients with diseases that place them at high risk of mortality. while some of the information may be too technical or scientific for patients to understand, there is need for this level of comprehensive awareness to avert incidences of patients acting out of this limited knowledge. urban location was indicated as a significant factor in hypertension awareness in the current study. this would suggest that residents of urban areas were at a more favourable position to have information on public health issues such as hypertension and access to facilities where they can get this knowledge (33). following this basis, it would be logical to conclude that hypertension levels in urban areas would translate to lower levels of hypertension in those areas. however, on the contrary, there are higher levels of hypertension in the urban areas than in the rural areas where there are significantly lower levels of awareness. this is rationalised in the study of qi et al (2018) which found that while awareness is significant, what is more necessary is to measure whether the individual is exposed to the biological and risk factors. in the case of hypertension, a typical rural lifestyle exempts them from the major risk factors of hypertension (34). due to subsistence farming the rural residents have more access to fresh food which has low levels of sodium. due to lesser machinery and appliances around the house (i.e., less vehicles and transportation), their level of physical activity is moderately high and consistent (35). therefore, these factors preclude them from being at the risk factors of hypertension, and awareness or lack of it would not be really significant in them becoming hypertensive. education level also proves to be a significant factor in hypertension awareness, with those who have reached university level in their study proving to be more knowledgeable about hypertension. therefore, this would emphasize the importance of education as a tool of enlightening the public on issues which affect their livelihood. furthermore, it would also reflect in having increased awareness of issues which affect the quality of life and an income to support their access to health care and information(36). overall, these factors have highlighted that the varying levels of awareness on public health matters can be traced to the inequalities which exist in society. healthcare then can tend to be more accessible to the privileged in society while those which are struggling to make ends meet can find themselves susceptible to diseases which can be avoided. limitations in terms of the limitations of the present study, it was conducted in a defined geographical area and did not represent whole malaysia so that findings should be generalized with caution. moreover, the questionnaire was made of close-ended questions which might have restricted the participants’ ability to explain the underlying reasons for certain outcomes. conclusion hypertension awareness remains a cause for concern in malaysia despite the increasing levels of public health and various mediums of disseminating information. various factors played a significant role in increasing or decreasing the level of awareness about hypertension. it is recommended to encourage individuals to mostly consider information on public health which emanates from acknowledged authorities rather than going with popular and charismatic sentiments. it is iraqi j pharm sci, vol.31(1) 2022 factors affecting hypertension awareness 239 also recommended to counter the inequalities to access to health care, by revising the policies which regulate access to health care facilities despite income and education levels. conflict of interest the authors declare that there is no conflict of 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cardiovascular risk associated with stage 1 hypertension defined by the 2017 acc/aha hypertension guideline. journal of the american college of cardiology. 2018;72(11):1201-10. 35. daştan i̇, erem a, çetinkaya v. urban and rural differences in hypertension risk factors in turkey. anatolian journal of cardiology. 2017;18(1):39. 36. patel l, alicandro g, la vecchia c. dietary approaches to stop hypertension (dash) diet and associated socio-economic inequalities in the uk. british journal of nutrition. 2020;124(10):1076-85. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 tnf-alpha gene polymorphisms doi: https://doi.org/10.31351/vol31iss2pp113-128 113 the effect of tnf-alpha gene polymorphisms at -376 g/a, -806 c/t, and -1031 t/c on the likelihood of becoming a non-responder to etanercept in a sample of iraqi rheumatoid arthritis patients. samer imad mohammed*.1 , munaf hashim zalzala** and faiq isho gorial*** *department of clinical pharmacy,college of pharmacy, university of baghdad, baghdad, iraq ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. *** college of medicine, university of baghdad, baghdad, iraq abstract tumor necrosis factor-alpha (tnf-α) antagonists’ therapy are expensive and has a non-responsive rate between 30% to 40% in rheumatoid arthritis patients. genetic variation plays a vital role in the responsiveness to this type of therapy.the aim of this study is to investigate if the presence of genetic polymorphism in the tnf-α gene promoter region at locations -376 g/a (rs1800750), -806 c/t (rs4248158), and -1031 t/c (rs1799964) affects rheumatoid arthritis patient's tendency to be a non-responder to etanercept. eighty ra patients on etanercept (etn) for at least six months were recruited from the rheumatology unit at baghdad teaching hospital. based on the european league against rheumatism response (eular) criteria, patients were divided into two groups: responders and non-responders. after polymerase chain reaction amplification of their dna, the amplified dna was sequenced by sanger method to determine the polymorphisms at the positions -376g/a, -806 c/t, and -1031t/c. the results of this study found that equally phi correlation and binary logistic regression analysis revealed a nonsignificant association for all genotypes in the three polymorphic sites with the tendency for being non-responder. moreover, there was no significant difference in tnf-α mean level or the change in disease activity score for 28 joints (das28) after six months of etanercept therapy between all genotypes for each polymorphic site. the present study concludes that there was no correlation between the polymorphisms in the tnf-α promoter region at -376g/a, -806 c/t, and -1031t/c with the tendency for being non-responder to etn. key words: rheumatoid arthritis, etanercept, genetic polymorphism, response. 376g/a ,806c/tأثير تعدد األشكال الجينية في عامل نخر الورم الفا بالمواقع ت على الميل لعدم االستجابة لأليتانرسبت في عينة من مرضى التهاب 1031t/c-و المفاصل الرثوي في العراق *** ايشو كولايرو فائق **مناف هاشم زلزلة ،1،*سامر عماد محمد * فرع الصيدلة السريرية، كلية الصيدلة، جامعة بغداد، بغداد، العراق. بغداد ، العراق بغداد، جامعة الصيدلة، كلية والسموم، دوية فرع اال** العراق بغداد، بغداد، جامعة الطب، كلية *** الخالصة هي عالجات باهظة الثمن ،وتصاحب استخدامها نسبة عالية من عدم استجابة قد تصل (tnf-α)العالجات المضادة لعامل نخر الورم .يلعب التباين الجيني دوًرا حيويًا في تحديد نسبة االستجابة لهذا النوع من العالج .٪ في مرضى التهاب المفاصل الرثوي40٪ إلى 30بين ما األ لتعدد وجود هناك كان إذا ما لمعرفة هو الدراسة الفاهدف الورم نخر عامل لجين المحفزة المنطقة في الجيني المواقع شكال 376 -في (rs1800750) g/a 806-و (rs4248158)c/t 1031-و (rs1799964)t/c تأثير في مرضى التهاب المفاصل الرثوي بالعراق وهل هناك تم تسجيل ثمانين مريًضا من مرض التهاب المفاصل الرثوي أن يكون غير مستجيب لعقار االيتانرسبت. في التباين الجيني على ميل المريض الهذ ة الرابطة والمستخدمين لعالج االيتانرسبت لمدة ستة أشهر على األقل من وحدة عالج المفاصل في مستشفى بغداد التعليمي. وبناًء على معايير استجاب ، تم تقسيم مرضى التهاب المفاصل الرثوي إلى مجموعتين: المستجيبين وغير المستجيبين. وبعد تضخيم (eular) األوروبية لمكافحة الروماتيزم تفاعل البلمرة المتسلسل ، تم اجراء تسلسل للحمض النووي المتضخم للمرضى المشاركين بأستخدام تقنية باستخدام تقنية الحمض النووي الخاص بهم .اهم نتائج الدراسة كانت كشف كل من معامل االرتباط1031t/c-و c/t 806-و g/a 376-لجيني في المواضع سانغر لتحديد تعدد األشكال ا االستجابة وتحليل االنحدار اللوجستي الثنائي عن عدم وجود ارتباط ذو داللة احصائية لجميع األنماط الجينية للمواقع الثالثة مع ميل المريض لعدم فاي معدل مستوى معامل نخر المرض لك لم يكن لتغيير االنماط الجينية ألي من المواقع الثالثة تأثير ذو داللة احصائية على مقدارلعقار االيتانرسبت.كذ مفصل خالل ستة اشهر من العالج. 28الفا في المريض وال على مقدارالتغيير الحاصل لنتيجة نشاط المرض لـ ط بين تعدد األشكال الجيني في المنطقة المحفزة لعامل نخر الورم الفا في المواقع ملخص هذه الدراسه اوضح انه لم يكن هناك ارتبا 376g/a 806-و c/t 1031-و t/c .مع قابلية مريض التهاب المفاصل الرثوي إلى عدم االستجابة لـأليتانرسبت , االستجابة.الكلمات المفتاحية : التهاب المفاصل الرثوي , االيتانرسبت , تعدد االشكال الجيني 1corresponding author e-mail: samer.jameel@copharm.uobaghdad.edu.iq received:2 /10 /2021 accepted:5 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp113-128 iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 114 introduction rheumatoid arthritis (ra) is a common chronic autoimmune joints disease. it is defined by progressive symmetric inflammation of the afflicted joints, which results in cartilage damage, bone erosion, and disability)1(. one influential proinflammatory cytokine that plays a critical role in controlling the inflammatory response and mediating ra pathogenesis is tumor necrosis factor-alpha (tnf-α) )2(. consequently, extensive research on tnf-α induced inflammatory processes has resulted in the conception of tnf-α antagonist medications to treat many inflammatory autoimmune diseases, including ra(3). they are produced from either a recombinant tnf-α receptor, like etanercept (etn), or a monoclonal antibody against tnf-α like infliximab and adalimumab(3). nowadays, tnf-α antagonists therapy has been widely used and is quite successful in treating ra(4). however ,they are prohibitively expensive, may induce some undesirable side effects, and not all ra patients respond well to these medications(5).in fact, between 30% to 40% of people with active ra do not respond successfully to tnf-α antagonists(6). the genetic disparity is a significant factor influencing tnf-α expression, and consequently, the response to tnf-α antagonists since the promoter region of the tnf-α gene is highly polymorphic (7). tnf-α antagonists are not all equally effective in all patients. as a result, being able to identify which tnf-α antagonist would be the most effective and should be taken initially would be tremendously beneficial in terms of reducing the time required to ensure an effective therapy, which has been repeatedly proved to be a significant factor in achieving long-lasting disease remission(8). etanercept , a recombinant human tnf-α receptor (p75) fusion protein that suppresses tnf-α competitively(9), is safe and effective in patients with active ra(10). as with other tnf-α antagonists, the clinical response to etn is variable, and one of the significant determinants of the response is genetic variability in the tnf-α gene(7). as a result, polymorphism testing aids in distinguishing patients with a high response rate from those with an insufficient response or even non-responsive (11). this information may be beneficial for some patients who concerned about their exposure to side effects or the associated high cost of biological therapy (11). several research articles published in the last few years have revealed conflicting outcomes about a possible association between tnf-α antagonists' response and polymorphisms in the tnf-α gene at several gene locations (12–14). due to the racial and ethnic variation in pharmacogenetics, which occurs as a result of the different in allele frequencies between populations, polymorphism analysis across populations is crucial for determining the genetic variables that may affect etn responsiveness. (15). earlier investigations did not examine the effect of single nucleotide polymorphisms (snps) in the tnf-α promoter region on the tendency to being non-responder to etn in iraqi ra patients; thus, the current study sought to determine whether the presence of snps in the promoter region of the tnfα gene at positions -376 g/a (rs1800750), -806 c/t (rs4248158) and -1031 t/c (rs1799964) may affect a patient's proclivity to be a non-responder. patients and methods this research article was a part of a large observational cross-sectional study conducted between october 12, 2020, and august 8, 2021. the study recruited a sample of eighty iraqi ra patients with established ra according to the revised 2010 american college of rheumatology (acr)/european league against rheumatism (eular) classification criteria for ra(16). the patients were recruited from the rheumatology unit of baghdad teaching hospital in baghdad, iraq. this unit provides services to a wide range of communities in iraq, including rural, urban, and inner-city districts from numerous governorates. the scientific and ethical committee of college of pharmacyuniversity of baghdad and rheumatology medical department at baghdad teaching hospital approved ethical permission with the number (recacpub-3102020b) on october 3, 2020. furthermore, written consent was obtained from all participants. patients selection ninety-seven patients with active ra received etn alone as a singular therapy during the study and met the inclusion criteria listed below. however, only eighty-six patients agreed to participate in this study, and only eighty completed the full requirements. the inclusion criteria: the patients must have been diagnosed with ra using the 2010 acr/eular ra classification criteria(16). also, patients with high disease activity according to disease activity score based on 28 joints and esr (das28-esr)(17) which is calculated as follows: das28 = 0.56 * sqrt(tender28) + 0.28 * sqrt(swollen28) + 0.70 * ln (esr) + 0.014 * patient global health. in order to be included, the das28-esr should be more than 5.1 at baseline. additionally, patients had to take etn subcutaneously and consistently for at least six months before enrollment, with no history of missed doses. iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 115 the exclusion criteria: • patient who has used etn for less than six months. • patients who use additional diseasemodifying anti-rheumatic drugs (dmards) with etn. • patients with co-existent other connective tissue diseases • patient with inadequate data like missed laboratory data or initial das28. patients classification: as presented in figure 1, the patients were divided into two groups according to eular response criteria(17), which are based on the clinical response as determined by (das28) (18) following at least six continuous months of etn treatment. when the das28 value after six months was reduced from a high value of ≥ 5.1 to a value less than 5.1 and with a change in das28 of greater than 0.6, the patient was classified as an etn responder. on the contrary, the patient has categorized as a nonresponder if the das28 value did not fall below 5.1 or if the change in the das28 was less than 0.6. accordingly, the patients are distributed regarding their responses into two groups. the first group (group a) contained forty-one ra patients who responded clinically to etn. the second group (group b) contained thirty-nine ra patients who failed to respond to etn. figure 1. flow diagram for the study participants. data collection demographic data (age, weight, disease duration, tender and swollen joints, and the visual analog scale (vas) for the patients and the evaluator) were obtained via direct patient interviews utilizing a patient information chart designed explicitly for this study. sample collection and preparation five milliliters of venous blood were obtained from each patient's forearm vein. then, two milliliters of blood were transformed into an ethylene diamine tetraacetic acid (edta) tube for dna extraction. at the same time, the remaining three milliliters of blood were put into a gel tube and centrifuged for ten minutes (4000 rpm). the remainder of the serum was collected in an eppendorf tube and stored at (-20 co) until all samples were obtained. tnf-α was then measured using the eliza approach. measurement of serum tumor necrosis factoralpha (tnf-α) the serum tnf-α level was determined using a cusabio elisa kit (wuhan, china; cat. no. csb-e04740h). this assay employs the quantitative sandwich enzyme immunoassay technique(19). dna extraction: the “promega reliapreptm blood gdna miniprep system” for genomic dna enables the straightforward purification of dna from blood samples. enzymatic amplification was performed using pcr and a hybrid thermal cycler. iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 116 quantitation of dna the "the quantus™ fluorometer" was used to determine the concentration of extracted dna and thus the sample's quality for downstream applications. (199 µl) diluted quantifluor dye was combined with 1 µl of dna, and then dna concentration readings were determined during a 5minutes incubation at room temperature. the primer: the tnf-α gene dna sequences were obtained from the ncbi genbank database. the primer premier 3 software was used to create pcr primers (table 1), with melting temperatures ranging from (58 to 62oc), primer lengths ranging from (18 to 23) nucleotides, and pcr amplicon lengths ranging from (800 to 1000) base pairs. table 1. the sequences, annealing temperature and size of the primers used in the study primer name sequence annealing temp. (°c) product size (bp) tnf-α_1-f 5`-tgtaaaacgacggccagtctcagagagcttcagggata-3` 60 966 tnf-α_1-r 5`-caggaaacagctatgaccgggacacacaagcatcaa-3` tnf-α_1-f: the forward primer. tnf-α_1-r: the reverse primer. primer preparation the forward and reverse primers used in this study were given in lyophilized form by the macrogen company. the primers were then dissolved in nuclease-free water to provide a stock solution with the highest concentration of (100 pmol/µl) that can be kept in the freezer at 20 °c. after that, a working solution for these primers was made by combining 10 µl of primer stock solution with 90 µl of nuclease-free water to yield a solution comprising (10 pmol /µl). primer optimization and pcr amplifications to determine the optimal annealing temperature for primers, the dna template was amplified using the identical primer pair (forward) (reverse) at annealing temperatures of 55, 58, 60, 63, and 65°c respectively. the best annealing temperature for the primer was 60°c as seen in figure 2. pcr amplifications were performed with 20μl volumes containing 10μl gotaq green master mix (2x); 1μl for each primer (10pmol); 6μl nuclease-free water and 2μl of template dna. pcr cycling was performed with pcr express (thermal cycler, biorad, usa) with the following temperature program: firstly, dna denaturation occurred at 94oc for 4 min followed by 30 cycles of denaturation at 94°c for only 30 sec; after that, annealing at 60°c for 30 sec; and extension at 72°c for 30 sec. a final extension incubation of 7 min at 72oc was included, followed by a 10 min incubation at 4oc to stop the reactions. figure 2. primer optimization at annealing temperatures of 55, 58, 60, 63, and 65°c. pcr products sequencing pcr product was sequenced by sanger method of sequencing using dna analyzer (abi3730xl) (macrogen corporation – korea). the results were obtained by email and analyzed with the use of geneious prime software (v 2021.1.1) (biomatters ltd., auckland, new zealand; www.geneious.com). iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 117 statistical analysis data were investigated using the spss for windows 26.0 software (spss, inc., chicago, il, usa) and graphpad prism version 7.04 (california, usa). continuous variables were stated as mean ± sem of the values. allele and genotypes are presented as percentages and frequencies. a probability that equals or less than 0.05 was considered significant. a shapiro–wilk test was used to test the normality of the results. the unpaired t-test was used for normally distributed data to determine if there is a significant difference in demographic characteristics and parameters between responding and nonresponding groups. a one-way analysis of variance (anova) test was used to analyze the difference between the means of more than two groups. then, a post-hoc analysis was used whenever a significant difference between three sample means was revealed by (anova). the chi-square test or fisher's exact test was used to test group differences of proportions. fisher's exact test was used if one of the expected values in a 2 x 2 comparison is < 5. phi correlation coefficient (phi) was used to measure the correlation between each genotype and the likelihood of being nonresponsive. the binary logistic regression analysis was used to estimate the relationship between tnfα level and the effect of genetic polymorphism on the likelihood of becoming a non-responder. results demographic data and clinical characteristics parameters of the study groups. table 2 summarizes the demographic characteristics of the study groups. in the current study, patients were matched. however, clinically, there was a significant difference in baseline das28 values between responder and non-responders (p. value <0.001). significant variations in das28 were also observed after six months of etn administration. in addition, after six months of etn medication, tnf-α levels were likewise shown to differ considerably between responders and nonresponders. table 2. demographic data and clinical characteristics parameters of the study groups. category responders group n=41 non-responders group n=39 p-value age(years) 49.46±10.54 51.18±11.95 0.497 a gender male n (%) 6 (14.6) 4 (10.3) 0.55 c female n (%) 35 (85.4) 35(89.7) 0.55 b weight(kg) 80.59±14.49 78.03 ±12.82 0.40 a disease duration (years) 10.10± 6.82 8.31±3.73 0.15 tnf-α (pg/ml) 78.63±34.1 113.35 ±54.54 0.001* a baseline das28 5.58± 0.343 6.06 ± 0.38 <0.001* a das28 after 6 months 3.34 ± 0.82 5.73 ± 0.54 <0.001* a results are reported as means ±sd or frequency (percentage). tnf-α: tumor necrosis factor-alpha. das28: disease activity score in 28 joints. a: independent 2 sample t-test. b: chi-square test. c: fisher-exact test dna concentration (µg/ml) the extracted dna concentration in all samples was found in a range of 20-30 µg/ml. pcr amplification result the amplification of the tnf-α genespecific region of human samples was presented in figure 3. the amplification of the tnf-α genespecific region of human samples was fractionated on 1.5% agarose gel electrophoresis stained with ethidium bromide (eth.br). figure 3. human samples' amplification of the tnf-α gene-specific region. the sample was fractionated on 1.5% agarose gel electrophoresis stained with eth.br. m: 100bp ladder marker. lanes 1-80 resemble 966bp pcr products. sanger sequences data analysis analysis of tnf-α ( -376 g/a) (rs1800750) snp figure 4 shows the analysis of (rs1800750) snp of the tnf-α gene using sanger sequencing. single "g" peak indicative of a (g) homozygous allele. the presence of the "g" and "a" peaks is indicative of the (g/a) heterozygous allele iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 118 figure 3. human samples' amplification of the tnf-α gene-specific region. the sample was fractionated on 1.5% agarose gel electrophoresis stained with eth.br. m: 100bp ladder marker. lanes 1-80 resemble 966bp pcr products. iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 119 figure 4 analysis of rs1800750 snp of tnf-α gene using sanger sequencing. analysis of tnf-α (806c/t) (rs4248158) snp figure 5 highlights the analysis of (rs4248158) snp of the tnf-α gene using sanger sequencing. single "c" peak indicative of a (c) homozygous allele. presence of the "c" and "t" peaks indicative of (c/t) heterozygous allele. figure 5. analysis of rs4248158 snp of tnf-α gene using sanger sequencing. analysis of tnf-α (-1031 t/c) (rs1799964) snp analysis of rs1799964 snp of tnf-α gene using sanger sequencing is presented in figure 6. single "t" peak indicative of a t homozygous allele. single "c" peak indicative of a ( c) homozygous allele. the presence of the "t" and "c" peaks is indicative of the (t/c) heterozygous allele. figure 6. analysis of rs1799964 snp of tnf gene using sanger sequencing. prevalence of genotypes polymorphism table 3 highlights the high proportions of gg genotypes of -376 g/a. the tt genotype was the most prevalent in more than half of the patients with -1031t/c. almost three-quarters of patients had the cc genotype for the -806 c/t variant. table 3 also shows the allele frequency for all the snps. iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 120 table 3. genotypes and alleles frequencies of tnf-α -1031 t/c, -806 c/t and -376 g/a gene polymorphisms in ra patients. n=80 patients genotypes -1031t/c genetic variant cc tc tt no. (%) 3(3.75) 22(27.5) 55(68.75) allele t c no. (%) 77(96.25) 25(31.25) -806 c/t genetic variant cc ct no. (%) 72(90) 8(10) allele c t no. (%) 80(100) 8(10) -376 g/a genetic variant ga gg no. (%) 4(5) 76(95) allele g a no. (%) 80(100) 4 (5) regarding the difference in alleles frequencies between the responders and non-responders, the results show no significant difference for all three polymorphic sites, as seen in table 4. additionally, the results of this study indicated that there was no significant difference in the distribution of all genotypes for all three sites in the tnf-α promoter region between responders and non-responders (table 4). table 4. difference in genotype frequencies of tnf-α -1031 t/c, -806 c/t and -376 g/a gene polymorphisms between responders and non-responders. genotypes responders group (n=41) no. (%) non-responders group (n=39) no. (%) p. value 1031 t/c cc 2 (4.9) 1 (2.6) 0.58 tc 12 (29.3) 10 (25.6) 0.71 tt 27 (65.9) 28 (71.8) 0.56 t 39 (95.1) 38(97.4) 0.97 c 14 (34.1) 11 (28.2) 0.40 -806 c/t cc 35 (85.4) 37 (94.9) 0.15 ct 6 (14.6) 2 (5.1) 0.15 c 41(100) 39(100) 1 t 6(14.6) 2(5.1) 0.17 -376 g/a ga 2 (4.9) 2 (5.1) 0.95 gg 39 (95.1) 37 (94.9) 0.95 g 41 (100) 39 (100) 1 a 2 (4.9) 2(5.1) 1 a chi-square test or fisher exact test was used to identify the statistical difference between the groups. association between genotypes and the likelihood of being non-responder table 5 shows the binary logistic regression analysis results, which are non significant for all genotypes of the three snps. this indicates that changing the genotype from the wild type to another polymorphic genotype cannot predict the tendency for being non-responder. table 5: binary logistic regression analysis of genotypes to predict the tendency of being non-responder for etn. parameter coefficient or p-value 95% ci. lower upper 1031 t/c 0.35 1.42 0.27 0.75 2.67 -806 c/t -0.88 0.41 0.10 0.14 1.21 -376 g/a 0.73 2.08 0.36 0.43 10.07 iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 121 similarly, by using phicorrelation all the demonstrated genotypes were either correlated positively or negatively with the tendency to be a non-responder, but none were statistically significant as seen in table 6. table 6. correlation between each genotype and the likelihood of being a non-responder. genotypes phi-coefficient p. value 1031 t/c cc -0.061 0.58 tc -0.041 0.71 tt 0.064 0.56 -806 c/t cc 0.189 0.09 ct -0.189 0.09 -376 g/a ga 0.006 0.95 gg -0.006 0.95 phi-correlation coefficient was used to find the correlation between each genotype and the likelihood of being a non-responder. the correlations between the genotypes and the difference in das28 over six months . as shown in table 7, most of the differences in das28 after six months of etn treatment were not statistically significant. table 7. association of the change in das28 over six months between different genotypes in -1031 t/c, 806 c/t and -376 g/a tnf-α genotypes polymorphism. genotypes -1031t/c genetic variant cc tc tt p. value δdas28 1.71±1.24 1.59±1.31 1.19±1.18 0.37 a -806 c/t genetic variant cc ct δdas28 1.22 ± 0.14 1.38 ± 0.54 0.72 b -376 g/a genetic variant ga gg δdas28 1.29 ± 0.13 0.73± 0.48 0.36 b results are reported as means ±sd, δdas28: the change in disease activity score of 28 joints over six months, a = one-way anova used to find the statistical difference, b = unpaired t-test used to find the statistical difference. tnf-α level in different genotypes after six months of continuous etn therapy, there was no significant difference in tnf α level between all genotypes of each snp, as reported in table 8. table 8. tnf-α level in different genotypes genotypes -1031t/c genetic variant cc tc tt p. value tnf-α 121.3 ±63.57 80.14±40.06 99.95±48.93 0.17 a -806 c/t genetic variant cc ct tnf-α 93.51 ± 5.67 111.1 ± 16.79 0.30 b -376 g/a genetic variant ga gg tnf-α 98.03± 15.86 95.35±5.61 0.91b results are reported as means ±sd, a = one-way anova used to find the statistical difference, b = unpaired ttest used to find the statistical difference. discussion etn has been proven to promote remission, reduce disease activity, and delay clinical and radiological disease progression in patients with ra. this has resulted in significant improvements in symptoms, function, and quality of life (20). as well known, the effectiveness of etn fluctuates considerably amongst ra patients, with around onethird of patients failed to clinically respond (21). as a result, early identification of ra patients who will not respond to tnf-α antagonists, including etn, enables a swift switch to alternative biological medications, thus increasing the patient's likelihood of promptly attaining treatment goals(22). studying the effect of genetic variation in response to etn is essential because, in contrast to other variables that can influence and modify the etn response, genetic determinants will remain consistent throughout a patient's lifetime (23). numerous snps were studied in the promoter regions of the tnf-α gene for their potential to alter the production of tnf-α or other cytokines, hence influencing the susceptibility or severity of ra(24,25). regarding the study's demographic characteristics, the results were equivalent in terms of the mean of age and duration of ra disease to those of another iraqi study that examined beliefs about medications among a sample of iraqi ra patients(26). in terms of iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 122 male-to-female ratio variation, the findings were comparable to other iraqi studies that confirm a high female-to-male ratio in ra illness(27–29). the results of this study identified three snps in the promoter region of the tnf-α gene in a sample of eighty ra patients treated with etn: -376 g/a, 806 c/t, and -1031 c/t. three earlier studies in iraq(27,30,31) investigated the association between a polymorphism in the promoter region of tnf-α and ra in a cohort of iraqi patients. however, no study has evaluated the relationship between these snps and etn responsiveness. furthermore, previous studies focused on only one or two snps, in contrast to the current study. many studies have examined the effect of snp combination in the tnf-α gene on the response to tnf-α antagonists (32). for instance, the snps -857c > t, -308g > a, 238g > a, and +489g > a in the tnf-α gene and their association to therapeutic efficacy were evaluated in retrospective study that involve 58 greek ra patients taking infliximab(33). similarly, an association study of three tnf-α related snps (-308g/a, -238g/a, and -857c/t) was conducted in poland and involve 280 ra patients of caucasian origin who treated with tnf-α inhibitors for at least 6 months (34). additionally, meta-analyses have been conducted to examine the relationship between a variety of snps and tnf-inhibitor responsiveness, including the 308 g/a polymorphism, the -857 c/t polymorphism, and the -238 g/a polymorphism (3,35).nonetheless, the current study was the only study that investigated the association between three snps -376 g/a, -806 c/t, and -1031 c/t on etn responsiveness. concerning the prevalence of -1031t/c genotypes, the current study found that more than 68 % of patients have the tt genotype. while the cc genotype had the lowest prevalence 3.75%. moreover, 97.5 %of patients possessed the t gene, while only 30% possessed the c allele. similarly, in the 1000 genomes database, the -1031 t/c promoter polymorphism has a reported minor c allele frequency of 22%. also, the results of this study are similar to those of saudi patients with diffuse large b-cell lymphoma(36), patients with behçet's disease in western algeria (37), and japanese patients with crohn's disease(38), all of whom had a high frequency of the tt genotype and a low frequency of the cc genotype. recent pieces of evidence have highlighted the associations between the -1031 t/c polymorphism and immune-mediated illnesses such as ra (38,39). it can thus be reasonably assumed that the polymorphism in this site may influence the response to etn. however, the current study found no statistically significant correlation between etn responsiveness and polymorphism in -1031t/c. no previous studies investigated the effect of-1031t/c on response to etn in ra patients nevertheless, the -1031 t/t genotype was predictive of a favorable response to tnf-α antagonists in chinese han ankylosing spondylitis patients(40). likewise, a case-control study which performed in spain and include 109 patients with psoriasis indicated that those with the tnf-α -1031 tt genotype responded better to infliximab(41). along with the small sample size, the current study's inconclusive results may be due to the use of etn in ra, whereas prior trials involved infliximab or adalimumab in diseases other than ra. moreover, ethnicity differences play a significant role in the pharmacogenetic investigations since allele frequencies in the populations studied may vary(42). regarding the prevalence of -806 c/t genotypes, the cc genotype was detected in 90% of ra patients enrolled in the current investigation. though, the t allele was present in only 11.25 % of patients, the c allele was present in all patients, and there was no statistically significant difference in the availability of these genotypes or alleles between the responders and non-responders. although the number of studies investigating the role of -806c/t in various conditions is limited, the results are consistent with those of a recent study examining the putative role of tnf-α gene polymorphisms in patients with south indian systemic lupus erythematosus (sle)(43). also, these values correlate favorably with results from a south german caucasian cohort research (44). furthermore, the findings are comparable to a casecontrol study conducted in taiwan that looked at the connections between snps in the promoter region of the tnf-α and susceptibility to nasopharyngeal carcinoma(45). the current study is the first to investigate the role of -806 c/t in ra patients' responsiveness to etn. the findings clearly demonstrated a non-significant connection between any genotype and a proclivity for non-response to etn. unfortunately, no comparable study exists to which the findings may be compared. nonetheless, the only comparable result was from a study in south indian systemic lupus erythematosus (sle) patients, which discovered no correlation between the 806c/t genotypes and the tendency to develop sle disease (43). this result may suggest that this polymorphic site plays a minor role in autoimmune disease and, consequently, in response to medications used to treat these diseases. in the case of -376 g/a genotypes, the current study discovered that 95 % of patients possessed the gg genotype while heterozygotes ga were found in 5% of the cases, and there were no homozygotes aa genotypes. likewise, the a allele was only found in iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 123 5% of ra patients; however, the dominant g allele existed in all patients. the findings are similar to those of a turkish study that examined the association of tnf-α (-376 g/a) polymorphisms in turkish tuberculosis patients(46). also, the result was parallel to an iranian study which examined -376 g/a tnf-α gene polymorphisms in iranian celiac patients to healthy controls(47). while various experimental studies which tried to address the relation between tnf-α polymorphisms and its expression, and the mechanisms controlling its expressions in many cell types and diseases highlights that the tnf -376g/a snps may have functional significance and may influence tnf-α gene expression levels(41,48,49); however, the tnf-α 376 g/a polymorphisms have received scant attention in ra patients, and their consequence on increased susceptibility to ra is uncertain (50–52). for instance, the polymorphism -376 g/a was not associated with an increased risk of ra in egyptian ra patients(53) or mexican patients(54). correspondingly, the study of kang et al. (55) found that -376g/a was not polymorphic in korean ra patients. the current study's finding failed to find a significant correlation between tnf-α (-376 g/a) polymorphisms and response to etn in iraqi ra patients. no earlier studies have examined the connection between the -376 g/a and the response to etn or even other tnf-α antagonists, this made the comparison with other studies in applicable. the most plausible reason for the result mentioned above, in addition to the study's small sample size, is that the polymorphism at -376 g/a was very marginally associated with ra(50–52) , despite its effect on tnf-α gene expression(41,48,49). the das28 score is the most often used outcome measure in investigating therapeutic response in ra (56). concerning the associations between various genotypes and das28 change after six months of continuous etn treatment, the current study could not confirm a significant change in das28 for all three sites. although he uses different response measures in his study, kang et al.(55) found a nonsignificant change in acr20 or acr70 linked with -1031t/c and -863c/a snps in a sample of korean ra patients. the discrepancy between the current study and other studies in the lack of a significant change in das28 might be related to the vulnerability of subjective outcome scores to heterogeneity, such as das28, are vulnerable to heterogeneity depending on the reporting clinician. moreover, it is generally established that measuring joint tenderness and patient perceptions of ra disease activity are two areas that can be challenging because there is no objective metric to validate clinical assessment(56). in addition to the differences in research design, sex ratios, sample size clinical outcomes assessed (das28esr vs. das28crp), anti-tnf medicine utilized, and concomitant use of disease-modifying antirheumatic therapies (dmards) with the tnf-α antagonist in some studies. regarding the difference in tnf levels between different genotypes, the current study results indicated that, polymorphism did not cause any significant difference in all three sites. although no prior research has explored the association between these variants and tnf-α levels in ra patients after taking etn therapy, one study analyzed the -1031 t/c polymorphism and tnf-α levels in patients with the acute coronary syndrome(57). the results indicated that tc carriers had significantly greater tnf-α levels than tt homozygotes(57). in contrast, another investigation discovered that carriers of the uncommon tnf-α 1031 c allele tended to have a more significant blood tnf-α level in chronic obstructive pulmonary disease patients(58). in the current study, the cc of carriers the -1031 t/c had the highest mean level of tnf-α but not significantly different from tt and tc carriers. also, tt carriers have a slightly higher but not statistically significant than tc carriers. regarding the -376 g/a and -806 c/t polymorphisms, the current investigation found that the polymorphic genotypes of these snps (-376 ga, -806 ct) had a slightly higher level of tnf, but the difference was not statistically significant. two investigations indicated that the a allele of the -376 g/a is associated with higher tnf-α production and transcription(59,60). however, this contrasts with musa et al., who discovered no correlation between the -376 g/a genotypes and tnf-α levels in egyptian patients with ra(53). on the other hand, katkam et al.(43) found no association between -806 c/t polymorphisms with the tnf-α level in a patient with sle. limitations the current study has several limitations. first, this study had a small sample size, which could be attributed to the small number of patients who used only etn and met all other inclusion criteria. second, because this study was conducted in a single center, caution is required when generalizing the findings to all iraqi rheumatoid arthritis patients. however, the chosen center treated patients from a variety of iraqi governorates. moreover, the study was conducted during the covid-19 era, and the overall number of patients included was limited due to repeated curfews that resulted in the loss of several patients as a result of their inability to attend the rheumatology units and obtained their medications. although the study excludes smokers and patients taking etn in combination with another dmard to rule out any response effect that could bias the iraqi j pharm sci, vol.31(2) 2022 tnf-alpha gene polymorphisms 124 results, one significant weakness is that the researchers focused exclusively on polymorphisms in the tnf-α promoter region, and ignored polymorphisms in other regions that could alter the response to etn. furthermore, because most of the patients were already on etn when they were enrolled, the baseline level of tnfwas not measured. the author relies on the difference in levels across genotypes to determine whether certain genotypes are associated with high levels of tnf-α, which may lead to non-responsiveness to etn. finally, the authors were unable to conduct a more powerful prospective cohort study by selecting only patients with specific genotypes and following them for six months due to the small number of patients who received only etn, insufficient financial resources, and the lengthy time frame required to recruit and conduct this type of study. conclusions this study has revealed that patients who do not respond to etn have a higher tnf-α level than respondents. sanger sequencing of the tnf-α gene promoter region revealed three polymorphic sites -1031 t/c, -806 c/t, and -376 g/a. the polymorphisms at these three locations did not affect the likelihood of iraqi ra patients being nonresponder. since the significance of these snps in ra patients' response to etn has not been established in the current study, and no previous study has inspected the effect of these snps in other populations. therefore, it is essential to examine this polymorphism in a larger 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influence of -308 tnf-α promoter polymorphism on the responsiveness to infliximab in patients with rheumatoid arthritis. scand j rheumatol (internet) 2004 (cited 2020 mar 18);33(4):228– 32. available from: https:// www. tandfonline. com/doi/abs/10.1080/03009740410005863 60. mugnier b, balandraud n, darque a, roudier c, roudier j, reviron d. polymorphism at position -308 of the tumor necrosis factor α gene influences outcome of infliximab therapy in rheumatoid arthritis. arthritis rheum (internet) 2003 (cited 2020 mar 18);48(7):1849–52. available from: https://onlinelibrary.wiley.com/doi/full/10.100 2/art.11168 this work is licensed under a creative commons attribution 4.0 international license http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ effect of adding cholesterol and different sugars to the nutrient medium on production the steroidal saponin " digitonin " from digitalis purpurea l iraqi j pharm sci, vol.19(1) 2010 in vitro digitonin production in digitalis plant 31 in vitro effect of cholesterol and different sugars on digitonin production in multiplied shoots of digitalis purpurea l. plant zainab j.awad *,1 * department of pharmacognosy ,college of pharmacy, university of baghdad ,baghdad, iraq. abstract production of the steroidal saponin digitonin in multiplied shoots of digitalis purpurea , (var. excelsior mixed) has been achieved in vitro by two experiments. in the experiment 1, shoot tips ( 1cm length ) explants from the sterilized seedlings were excised and cultured on ms medium ( murashige and skoog medium) supplemented with 0.5 mg/l tdz (thidiazuron) and cholesterol at the concentrations 0.0, 0.1, 0.3, 0.5, 1.0, 1.5, 2.0 or 4.0 mg/l. after 45 day, results showed that the treatment with 0.5 mg/l tdz and 2.0mg/l cholesterol had a positive effected on increasing the dry weight of multiplied shoots and their production of digitonin when compared with other treatments, where this treatment gave 2.98 g dry weight of multiplied shoots and digitonin at amount of 64.42 g/g dry weight, while the other studied characteristics of multiplied shoots (content the total chlorophyll, soluble sugars and starch.) also this treatment had appositive effected and was gave the following values:(3.51 mg/g fresh weight, 5.06% ,6.09% ) respectively. after that experiment 2 was carried out . the objective of this experiment was to increase the degree of digitonin production in multiplied shoots compared with the experiment 1.therfore in the experiment 2, we were selected supplements the best treatment of experiment 1(0.5mg /l tdz and 2.0mg/ l cholesterol ) and supplemented to the ms medium with the sugars glucose, fructose, sucrose or maltose at the concentrations 30,50,70 or 90 g/l for each sugar. after 45 days results showed that the treatment with 0.5 mg/l tdz, 2.0 mg/l cholesterol and 50g/l maltose was the best a compared with other treatments, where this treatment gave 4.52 g dry weight of multiplied shoots and digitonin at amount of 191.87 g/g dry weight . the content of multiplied shoots from the total chlorophyll, soluble sugars and starch also this treatment gave highest values and they are 4.97 g/g fresh weight , 5.91% and 8.30% respectively . key words: digitalis purpurea, cholesterol, sugars, digitonin . الخالصة digitalis purpuseaفٓ االفسع اىمخضاعفت ىىباث اىدٔضٕخاىس (digitonin)اوخاس اىسٕخسَئٕد اىصابُوٓ اىدٔضٕخُوٕه سم 1( حم اوضاشي خازس اىضسم اىحٓ بُاسطت حضسبخٕه فٓ اىخضسبت األَىّ، فُصيج اطساف االفسع بطُه excelsior mixed)ضسب ميغم/ىخس َاىنُىسخسَه باىخسامٕص 5.0بخسمٕص tdz)مساشٕل َسنُك( مضافاً اىًٕ msَشزعج عيّ َسظ مه اىبادزاث اىُمعقمت tdzميغم/ىخس 5.0ُٔماً أظٍسث اىىخائش بان اىمعاميت اىمخاىفت مه 00ميغم/ىخس، بعد 0.5اَ 0.5، 1.0، 1.5، 5.0، 5.0، 5.1، 5.5 ٓ عيّ شٔادة اىُشن اىضاف ىألفسع اىمخضاعفت َمحخُاٌا مه اىدٔضٕخُوٕه مقازوتً مع ميغم/ىخس مُىسخسَه ماوج ذاث حأرٕس أضاب 0.5مع مأنسَغم/غم َشن 20.00غم محخُٔت عيّ اىدٔضٕخُوٕه بمقداز 2..0اىمعامالث االخسِ، حٕذ بيغ اىُشن اىضاف ىألفسع اىمخضاعفت اىنيُزَفٕو اىنيٓ، اىسنسٔاث اىرائبت َاىىشاء، فقد ماوج صاف. اما اىصفاث االخسِ اىخٓ ُدزسج ىالفسع اىمخضاعفت ٌَٓ محخُاٌا مه %، 0.52ميغم/غم َشن طسْ , 0.01) -أفضو اىقٕم ٌَٓ : حٕذ أعطج ذاث حأرٕس إٔضابٓ عيّ ٌري اىصفاث أٔضاٌري اىمعاميت إوخاس اىدٔضٕخُوٕه فٓ األفسع %( َعيّ اىخُاىٓ. بعد ذىل أصسٔج اىخضسبت اىزاوٕت َاىٍدف مىٍا ٌُ إمناوٕت إحداد شٔادة أمزس فٓ .2.5 ميغم /ىخس 5.0اضافاث أفضو معاميت فٓ اىخضسبت األَىّ ٌَٓ msاىمخضاعفت مقازوت مع اىخضسبت االَىّ، ىرىل اضٕف اىّ َسظ tdz َ0.5 غم/ىخس ىنو 5.اَ 05، 05، 05ميغم/ىخس مُىسخسَه مع اىسنسٔاث سنسَش، ميُمُش، فسمخُش اَ ماىخُش َباىخسامٕص غم/ىخس 05ميغم/ىخس مُىسخسَه مع tdz ،0.5ميغم/ىخس 5.0ُٔماً، اظٍسث اىىخائش بان اىمعاميت اىمخاىفت مه 00عيّ حدي. بعد ُسنس 1.1.20غم محخُٔت عيّ مادة اىدٔضٕخُوٕه بمقداز 0.00ماىخُش ماوج ٌٓ األفضو، حٕذ بيغ اىُشن اىضاف ىألفسع اىمخضاعفت ألفسع اىمخضاعفت مه مادة اىنيُزفٕو، اىسنسٔاث اىرائبت َاىىشاء فقد أعطج ٌري اىمعاميت أضاً مأنسَغم/غم َشن صاف، أما محخُِ ا %( عيّ اىخُاىٓ.2.05%، 1..0ميغم/غم َشن طسْ، 0..0أفضو اىقٕم ٌَٓ: ) introduction digitalis purpurea ( fam: scrophularaceae ) is a biennial herb plant (1) . in the medicinal and pharmacy fields this plant has a great benefit because it contains the cardiac glycosides like" digitoxin" and steroidal saponin glycosides like" digitonin" both of these groups representing compounds of considerable importance to the pharmaceutical industry (2) . as yet, these compounds can not be synthesized economically by chemical or microbiological methods (3) . 1corresponding author email : merciful2003@ yahoo.com received : 24/3/2009 accepted : 9/1/2010 iraqi j pharm sci, vol.19(1) 2010 in vitro digitonin production in digitalis plant 32 in the field of pharmacy and medicine, the steroidal saponin digitonin had a great importance because it was used to determine the ratio of cholesterol in the blood, plasma and bile acids but the great importance and interest of this compound and some steroidal saponins like the diosgenin owing to their relation ship with some important substances as progesteron hormone, cortisone and vitamin d where the structures of these steroidal saponins have the same nucleus of these substances , then these steroidal saponins can be used as a precursors for the production of these substances . originally these substances were isolated from expensive and limit sources like adrenal cortex and bile acids of cattle (4 ,5) for this important many studies using modern technology like tissue culture technique ( in vitro) to increasing the production of steroidal saponins in some plants . where via the usage of this technology different explants could be cultured and inducted to different stage of growths as a source for these important medical compounds and in a high concentrations without being restricted to the environmental conditions as well as cleans of the pharmaco which could be obtained (6 ) .therefore the objective of our study using this technique to inducted the multiplication shoots of digitalis purpurea (var exlecior mixed) and increasing their production from the digitonin compound . material and method first :tissue culture of the plant digitalis purpurea; this work went through the following steps: 1. preparation of sterilized seedlings : that was, initiation a specific culture of seedlings free from any contamination which it will be a source for shoot tips and that was performed by: a. medium preparation : basic medium composition was that of ms (7) with 30g/l sucrose. the ph of this medium was adjusted to 5.5 then gelled with 8g/l difco bacto agar . ten milliliters of this medium was dispensed into 25x125 ml tubes. after capped with clear autoclavable lids, tubes were autoclaved at 121 0 c for 20 min . b. seeds germination : seeds of the plant digitalis purpurea ( var exlecior mixed) were surface sterilized in 70% ethyl alcohol for 30 second. this was followed with three sterilize distilled rinses ( 8) , after that the seeds were cultured on ms medium , and allowed to germinate by incubated in growth room at 25  2 c with artificial illumination given by fluorescent light of about 1600 lux at 16 hour / day ( 9) . incubation was for30 days and then the seedlings are ready to take the explants shoot tips which excised and cultured in step 2. 2. shoots multiplication and the production of steroidal saponin "digitonin": these processes were inducted by two experiments: experiment 1 this experiment was carried out to inducted multiplication the cultured shoot tips and their digitonin production and by the following steps: 1. preparation of the nutrient medium: basic medium ms was used supplemented with 30g/l sucrose plus 0.5 mg/l tdz (10) (for multiply the cultured shoot tip) and cholesterol at a concentrations of 0.0, 0.1, 0.3 , 0.5, 1.0, 1.5, 2.0 or 4.0mg/l ( for induct the production of digitonin ). the ph of the media were adjusted to 5.5 and gelled 8g/l difco bacto agar. twenty five milliliter medium was dispensed into 25x125 ml tubes. after capped with clear autoclavable, tubes were autoclaved at 121 0 c for 25 min . 2. cultured the shoot tip: excised the shoot tips ( 1cm length ) from the sterilized seedlings and one shoot tip in every tube was cultured, then allowed to multiplication by incubated in a growth room at the same condition of seeds germination ( 11) . experiment 2 this experiment was carried out after experiment 1 .the objective of this experiment was to increasing the degree of digitonin production in multiplied shoots compared with the experiment 1. therefore we were selected supplements the best treatment of experiment 1 (0.5 mg/l tdz and 2.0 mg/l cholesterol) and supplemented to ms medium with the sugars glucose, fructose, sucrose or maltose at a concentrations of 30,50,70 or 90 g/l for each sugar , the ph of these media and their gelled , distribution, autoclaving , cultured the shoot tip and condition the incubated in a growth room as in the experiment 1. second :analysis of the multiplied shoots . after 45 days in cultures, multiplied shoots are ready for studying the following characteristics: iraqi j pharm sci, vol.19(1) 2010 in vitro digitonin production in digitalis plant 33 1. total chlorophyll content:this characteristic was estimated according to goodwing method (12 ) . 2. dry weight estimation:the multiplied shoots were taken out of the tubs and washed in water to remove agar from them, after wiped with clean cloth ,the shoots were separated and differentiated on filter paper to be dried in an oven at 40c until they reached constant weight (13 ) , later, shoots were grinded to hard powder and kept in paper sacks in dry condition and dark place . 3. soluble sugars and starch content: these characteristics were estimated according to joslyn method (14 ) . 4. extraction of the steroidal saponin "digitonin" according to the procedure of brain et al (15 ) 5. hplc analysis of digitonin:-this analysis was carried out by using ( 16) c 18 – column and the mobile phase used was acetonitril: water (25:75). the flow rate was 1.5 ml/min .the measurements were carried out , using uv detector at 205 nm. statistical analyses in our study were made by using complete randomized design. the mean differences were tested utilizing lsd test. ( 17) results multiplication of cultured shoot tip and yielded the highest number of shoots in the experiment 1 and 2 could be inducted by supplemented 0.5 mg/l tdz to ms medium . in experiment 1, supplemented different concentrations of cholesterol to ms medium with o.5 mg/l tdz , showed positive effect on digitonin production in multiplied shoots and other studied characteristics for these shoots ( total chlorophyll content, soluble sugar, starch and dry weight ) as show in table (1). but a significant effect was observed with the concentration 2.0 mg/l cholesterol and 0.5 mg/l tdz. therefore we were selected this treatment and was used in the experiment 2 with different sugars . in this experiment , all concentrations of maltose sugar had signification effect on digitonin production, but the best concentration was 50 g/l which had more effect on the production of this compound and other studied characteristics of multiplied shoots as compared with other treatments of this experiment (table 2 ,3 ,4 and 5 ). table 1 : effect of different concentrations of cholesterol. with the presence of 0.5 mg/l tdz on the content of multiplied shoots of digitalis purpurea from the total chlorophyll , soluble sugars , starch , digitonin and on their dry weight. concentration mg/l total chlorophyll mg/g fresh weight soluble sugars (%) starch(%) digitonin g/g dry weight dry weight (g) 0.0 3.31 4.36 5.11 33.13 2.11 0.1 3.39 4.44 5.14 33.60 2.09 0.3 3.33 4.42 5.31 35.71 2.13 0.5 3.47 4.54 5.24 37.56 2.36 1.0 3.49 4.61 5.66 43.49 2.41 1.5 3.47 4.88 5.84 49.46 2.71 2.0 3.51 5.06 6.09 64.42 2.98 4.0 3.48 4.41 5.86 44.40 2.31 l.s.d.5% 0.05 0.21 0.17 6.19 0.23 table 2 : effect of different concentrations of sugars with the presence of 0.5mg/l tdz and 2mg/l cholesterol on total chlorophyll content of multiplied shoots of digitalis purpurea . total chlorophyll mg/g fresh weight. concentration g/l sugars maltose fructose glucose sucrose 4.69 3.18 3.41 3.51 30 4.97 3.85 3.51 3.90 50 4.31 3.84 3.56 3.96 70 3.60 3.61 3.15 3.42 90 0.23 l.s.d 5% iraqi j pharm sci, vol.19(1) 2010 in vitro digitonin production in digitalis plant 34 table 3 : effect of different concentration of sugars with the presence of 0.5 mg/l tdz and 2mg/l cholesterol on soluble sugars and starch content of multiplied shoots of digitalis purpurea . table 4 : effect of different concentrations of sugars with the presence of 0.5 mg/l tdz and 2mg/l cholesterol on digitonin content of multiplied shoots of digitalis purpurea . table 5 :effect of different concentrations of the sugars with the presence of 0.5 mg/l tdz and 2mg/l cholesterol on the dry weight of multiplied shoots of digitalis purpurea . dry weight (g) concentration g/l sugars maltose fructose glucose sucrose 3.14 2.28 2.42 2.96 30 4.52 2.91 3.61 3.18 50 4.03 2.71 3.72 3.51 70 3.90 2.51 2.31 2.40 90 0.25 l.s.d 5% discussion in the field of tissue culture, shoots cultures (multiplied shoots) of digitalis plant was considered one of the main sources to obtain some medicinal compounds like the cardiac and saponin glycosides (18) because the site of biosynthesis of these medicinal compounds is the shoots of this plant (19) for this reason , in the present study we were inducted multiplication the cultured shoot tip and formation the shoots cultures by supplemented the growth regulator tdz ( cytokinin – like ) to ms medium at a concentration 0.5 mg/l ( 10) , and because the strong effects of this growth regulator (20) , shoot cultures in our study were appeared as leaf masses consisted from a high number of small and dwarf shoots as shown in figure (1). soluble sugars (%) concentration g/l sugars maltose fructose glucose sucrose 5.69 4.59 4.78 5.06 30 5.91 4.55 4.81 5.31 50 5.49 4.38 4.20 5.54 70 4.44 3.33 3.08 4.01 90 0.30 l.s.d 5% starch(%) concentration g/l sugars maltose fructose glucose sucrose 7.91 5.74 5.79 6.09 30 8.30 5.82 5.87 6.81 50 7.30 5.50 5.55 6.76 70 6.12 4.49 5.01 5.65 90 0.41 l.s.d 5% digitonin g/g dry weight. concentration g/l sugars maltose fructose glucose sucrose 113.11 42.13 51.01 64.42 30 191.87 83.78 68.18 98.93 50 171.22 81.91 73.28 108.55 70 165.61 51.67 67.33 61.72 90 17.35 l.s.d 5% iraqi j pharm sci, vol.19(1) 2010 in vitro digitonin production in digitalis plant 35 figure 1 : multiplication shoot tip of digitalis purpurea (var. excelsior mixed) in the treatment 0.5 mg/l tdz and 2.0 mg/l cholesterol after 45 days. so it was difficult to distinguish the stem tips of these shoots then it was not possible to count their number and arrange the statistical analysis for them.total chlorophyll content, soluble sugars and starch of the shoot cultures was studied as an indicator to degree of the changes in quantity of digitonin in these shoots, where if the content of chlorophyll was high this mean the green plastids have high degree of differentiation and that reflected positively on the ability of green plastids to provide some necessary enzymes to produce these compounds (21, 22) . as for the soluble sugars and starch , these compounds are the main source for energy and carbon element which are necessary to formation the great carbon structure of steroidal compounds (23) . thus the rich places of chlorophyll , sugars and starch will be also rich in these medicinal compounds.in experiment 1, presence of the cholesterol with 0.5 mg/ tdz in ms medium had effected on increasing the internal level of digitonin inside the tissue of multiplied shoots especially in the concentration 2.0 mg/l , because this material was considered as a precursor to form the digitogenin ( aglycon part of digitonin ) (24) as shown in figure (2). also presence the cholesterol in ms medium was led to increasing the content of multiplied shoots from the total chlorophyll, soluble sugars and starch. this increasing may be due to analysis this precursor and supply of some elements for these shoots like the carbon element which is necessary to formation the chlorophyll , soluble sugars and starch (25) and reflected that positively on shoots production from the digitonin. all of these increases reflected positively on increasing the dry weight of these shoots ecpically in the concentration 2.0 mg/l . therefore we were selected this concentration with 0.5 mg/ l tdz and used in the experiment 2 with different sugars. in the experiment 2, the treatment 50 g /l maltose with 2.0 mg/ l cholesterol and 0.5 mg/l tdz significantly increased the digitonin , total chlorophyll, soluble sugars and starch in multiplied shoots and their dry weight as compared with other treatments of this experiment . the reason may be due, the maltose sugar is disaccharide and consist of two molecule of glucose . therefore presence of this sugar in ms medium was led to increasing the number of glucose molecules inside the tissue of these shoots and that reflected positively on different bioprocesses of these shoots like: 1increasing the supply of sugar part (glucose) which is necessary to building the digitonin where this compound consist from 2 molecules of glucose, 2 molecules of galctose and 1 molecule of xylose (25 ) . 2 – increasing the supply of energy which is necessary to growth and development of the shoots ( 23) , 3increasing the supply of carbon element which is necessary to building the great carbon structure of chlorophyll, different sugars, starch and digitonin (5,25 ) . iraqi j pharm sci, vol.19(1) 2010 in vitro digitonin production in digitalis plant 36 digitogenin figure 2 : the biosynthesis of steroidal sapogenin digitogenin (24) iraqi j pharm sci, vol.19(1) 2010 in vitro digitonin production in digitalis plant 37 references 1. tyler ,v.e, brod , l.r . and robbers , j.e.1988. pharmacognosy.9 th edition .k.m. varghese , com , bombay. 2. evans , f. j . and cowleg , p.s . 1972 .variation in cardenolides and sapogenins in digitalis purpurea during germination phytochemsitry 11.2729 – 2733 . 3. james , a.g.1999. physiology of plants and their cells : phytochemistry (saponins ).pergamon press inc .new york . 4. trease , g.e. and evans,w.c . 1983. pharmacognosy ( saponins , cardio – active drug and other steroids ) . 12 th edition . published by bailliere tindall a division of cassell ltd . 5. aua zeid , s.n.1986 . medicinal plants and herbales . dar al bihar . bayrute . 6. stafford, a. 1986. secondary metabolism in plant cell culture .(eds.).cambridge university press . cambridge , london , new rochelle , melbourme , sydney . 7. murashige, t. and skoog, f. 1962 .arevised medium for rapid growth and bioassay with tobacco tissue culture . physiol . plant .15:473 – 497 . 8. kubalakova , m., spitzova , i . and novak , f.j.1987 stability of lanatoside c content in the in vitro propagated digitalis lanata clones . biologia plant arum .29(1):7 – 9 . 9. hay, f.r. , robert , r.j. and coomber , s.a . 1997 . development of desiccation tolerance and longevity in seed detached capsules of foxgloves ( digitalis purpurea ) annals .botany . 79 : 419 – 427 . 10. zeinab , j.a. 2002 . the production of cardiac glycosides from the plant of zahrat al kishtiban ( digitalis purpurea l .) by using tissue culture technique .ph.d thesis agric.college .baghdad university .iraq . 11. coner, s.and reinhard , e.1986 . longterm cultivation of digitalis lanata clones propagated in vitro : cardenolide glycosides tent of the regenerated plants . plant medica . 7:478 – 481 . 12. goodwing , t.w .1976 . chemistry and biochemistry of plant pigments , 2nded academic press ,london , new york , sanfrencisco 13. -brugidou ,c. ,jacques ,m., cosson ,l., jarreau f.x. and ogerau , t.1988 . growth and digoxim content in digitalis lanata in controlled condition and natural environment .plant medical 262 – 265 . 14. joslyn , m.a. 1970 . method in food analysis , physical , chemical and instrumental methods of analysis ,2 nd .academic press , new york and london . 15. brain , g.l. , brain , k.r. 1976.influence of hormonal supplementation on steroid level during callus induction from seeds of trigonella foenum graecum . phytochemistry , 15: 1655 – 1660 . 16. barnes , j. , anderson , l.a . and phillipson , j.d. 2002 . herbal medicines , 2 nd ed , pharmaceutical press ,uk . 17. alrawi, k.m. and khalaf – allah, a.m. 1980 . design and analysis of agricultural experiments , mosul university . agric and forest college iraq . 18. abdul – mutalib , s.m . and mubashar , s.o. 1990 . the basic conception in plant cell , tissue and organ culture. university of baghdad , ministry of higher education and scientific research . iraq . 19. stuhlemmer , u., kreis, w., eisenbesis , m. and reinhard , e. 1993 . cardiac glyrosides in party submerged shoots of digitalis lanata .planta media ,59 : 539 – 545 . 20. carl , a.h. and jhon ,e.p.1993 . thidiazuron a potent cytokinin for woody plant culture .palnt cell tissue and organ culture .33:105 – 119 . 21. hagimori , m ., matsumoto , t. and obi , y.1982 .studies on the production of digitalis cardenolides by plant tissue culture .ii.effect of light and plant growth substances on digitoxin formation by undifferentiated cells and shoots – forming cultures of digitalis purpurea grown in light media .plant physiol .69 : 635 – 656 . 22. morales , c., cusido , r., palzon , j., bonfill , m.and pinol l.1999 . digitalis plants and tissue culture , improved conditions for cardinolide production .plant biology , 1:35. 23. alrawi, a.m. and sulieman ,r.r.1988 .metabolitei bioefficiencies .ministry of high education and scientific research ,baghdad university ,iraq . 24. margaret, l.v. and vickery , b.1981 .secondary plant metabolism . the macmillan press ltd. london and basingstoke . 25. mohamed, a.a.k.and younis , m.a.1991 .principles of plant physiology. v2 . ministry of higher education and scientific research .baghdad university . . college of agriculture .iraq . materials and methods iraqi j pharm sci , vol.18 (suppl.), 2009 effect of cox-2 selectivity on lipid profile 7 effect of cox-2 inhibitors selectivity on lipid profile in hyperlipidemic and normolipidemic type 2 diabetics * najwan k. fakree *,1 and shatha h. ali * department of clinical laboratory science , collage of pharmacy , university of baghdad, baghdad , iraq abstract development of nsaids based on inhibiting cyclooxygenase activity. however, the different physiological consequences arrised by appearance of new drugs with different selectivity to cox-2 enzyme upon their administration with their relevant affects on some cardiovascular risk factors. to study the potential effects of relatively diclofenac and highly specific celecoxib cox-2 inhibitors on lipid profile and serum c-reactive protein in type 2 diabetes, whom have hyperlipidemia to be compared by their effects with normolipidemic patients. a total number of 34 type 2 diabetics (14 normolipidemics and 20 hyperlipidemics) treated with either diclofenac 100mg/day or celecoxib 200mg/day for eight weeks. analysis of results indicated that diclofenac increased serum triglycerides (tg) whereas; celecoxib group exerted a significant reduction in total cholesterol (tc), triglycerides (tg) levels in hyperlipidemic patients. normolipidemic diabetics showed a significant elevation in serum total cholesterol, triglycerides and low density lipoprotein-cholesterol (ldl) with significant reduction in high density lipoprotein-cholesterol (hdl) in those treated with diclofenac , whilst those treated with celecoxib exhibited no modification of serum lipids. the results of the present study indicated that the net effect of treatment of hyperlipidemic type 2 diabetics by diclofenac was mostly qualitative as indicated by elevated tg/hdl ratio, to be a marker of atherogenicsmall dense ldl particles in diabetics, whereas celecoxib exerted no such effect in this group but produced a beneficial reduction in ldl/hdl ratio. meanwhile, diclofenac in normolipidemic diabetics exert a significant qualitative and quantitative modulation of their serum lipid components presented by net elevation in both ldl/hdl and tg/hdl ratios. as a conclusion the administration of relatively selective cox-2 inhibitors ( diclofenac ) to normolipidemic type 2 diabetics could adversely affect lipid metabolism by producing undesirable qualitative as well as , quantitative changes in serum lipid components, more than that observed in the hyperlipidemic diabetics. key words: diabetes, lipid profile, cox-2 selectivity لخالصة ا دزاست تبثُس ٍثبطبث اىسبَنيىاومسجُْس اىتبٍت واىْسبُت عيً ٍستىي اىدهىُ فٍ اىدً عْد ٍسضً اىسنسٌ ٍِ اىْىع ىغسض فٍ اىدً عْد هؤالء cاىبسوتُِ اىفعبه عيً تسمُص ومرىل دزاست تبثُس هرٓ االدوَت ذو اىتسمُص اىعبىٍ و اىطبُعٍ ىيدهىُ اىثبٍّ ٍيغٌ ٠١١ٍيغٌ َىٍُب ( او اىسُيُنىمسُب ) ٠١١ضب قد شبزمىا بهرٓ اىدزاست قد تٌ عالجهٌ اٍب ببىداَنيىفُْبك ) ٍسَ ۳٤اىَسضً . فبُ /اسببُع بعد تحيُو اىْتبئج وجد اُ اىداَنيىفُْبك َصَد ٍِ تسمُصثالثٍ اىغيسُسَد وَؤدٌ اىً شَبدة ّسبت) ثالثٍ اىغيسُسَد ٨ىَدة َىٍُب( تـــاىنىىستُسوه , ثالثٍ اىغيسُسَد وَؤدٌ اىً ّقصبُ فٍ ّسب مو ٍِمُصاتس اىدً بَُْب اىسُيُنىمسُب َقيو ٍِاىدهىُ عبىُت اىنثبفت( فٍ و اىتسمُص ذ. اٍب عْد ٍسضً اىسنس اىدهىُ عبىُت اىنثبفت( عْد ٍسضً اىسنس ذو اىتسمُص اىعبىٍ ىيدهىُ /) اىدهىُ واطئت اىنثبفت واىدهىُ واطئت اىنثبفت ٍع ّقصبُ فٍ شَبدة تسامُص مو ٍِ اىنىىستُسوه , ثالثٍ اىغيسُسَد اىً ٍ ىيدهىُ فبُ اىداَنيىفُْبك َؤدٌعاىطبُ اىدهىُ عبىُت /هىُ فٍ اىدً )ّسبت اىدهىُ واطئت اىنثبفتتسامُص اىدهىُ عبىُت اىنثبفت ومرىل َؤدٌ اىً شَبدة ٍيحىظت فٍ ّسب اىد بَُْب اىسُيُنىمسُب ال َؤثس بشنو ٍيحىظ عيً تسامُص اىدهىُ عْد ُت اىنثبفت( .اىدهىُ عبى /اىنثبفت( ومرىل )ّسبت ثالثٍ اىغيسُسَد فٍ اىدً c اىفعبه فٍ تسمُصاىبسوتُِ ٍيحىظ ُُ اىً ّقصبَبجُِ َؤدالومرىل وجد ٍِ خاله اىدزاست ببُ مال اىع هؤالء اىَسضً. قد بَنيىاومسجُْس( اىً ٍسضً اىسنس ذو اىتسمُص اىطبُعٍ ىيدهىُ بظ ّسبٍ الّصٌَ اىسواىرٌ َعتبس ٍث ).ىرىل فبُ اعطبء اىداَنيىفُْبك .اىتسمُص اىعبىٍ ىيدهىُ ٌوفٍ ٍستىي اىدهىُ فٍ اىدً امثس ٍَب هى ٍالحع عْد اىَسضً ذ ب وّىعُ ب َؤدٌ اىً شَبدة غُس ٍفضيت مَُ introduction diabetes mellitus (dm) is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. chronic hyperglycemia is associated with long – term failure of various organs, including small and large blood vessels (1) and peripheral neuropathy among the commonest complications in those patients.neuropathy accurse in 60-70% of diabetics, where excess sugar can injure the walls of the tinny blood vessels that nourish the nerves especially in the legs leading to tingling, numbness, burning or pain sensation in the fingers (2) . 1 corresponding author e-mail : najwan-aljalely@ yahoo.com received : 28/12/2009 accepted : 24/3/2009 iraqi j pharm sci , vol.18 (suppl.), 2009 effect of cox-2 selectivity on lipid profile ٨ treatment of diabetic neuropathy include primary prevention, management of early symptoms and relief of pain by drugs such as tricyclic antidepressant (tca), analgesics and anticonvulsant therapy. (3) meanwhile arthritis (joints inflammation) that causes pain and swelling due to damage in joints and connective tissues could be presented as osteoarthritis (oa) or rheumatic arthritis (ra) (4). osteoarthritis:-also known degenerative joint disease, this form of arthritis is usually limited affecting only the joints and is more painful later in the day. it’s thought to be caused by defect in cartilage that results in bone destruction. (5) rheumatic arthritis: can affect most of the joints of the body and is associated with permanent morning stiffness and swelling, it can involve internal organs such as heart and lunges. (5) because osteoarthritis and rheumatoid arthritis are so common so many people with these disorders could also have diabetes mellitus , diabetic patients also has specific musculoskeletal disorders that caused by `diabetes─ neuropathy ( nerve involvement ) , arthropathies (disorder of joints and connective tissues) and problems with skin , tendons and muscle (5) the most common therapy utilized to control arthritis pain in those patients are the non steroidal antiinflammatory drugs (nsaids) which permit their action by inhibiting cyclooxygenase enzymecox exists as two isoforms; cox1 which is housekeeping enzyme and mediates physiological responses like (cytoprotection of stomach, platelet aggregation, vascular homeostasis and kidney functions). while cox-2 is mainly expressed by cells that are involved in inflammation like macrophage and monocyte. (6) however, four categories of cox inhibitors have been proposed (7, 8) 1selective cox-1 inhibitors: these agents inhibit cox-1 activity without any measurable effect on cox-2 activity such as aspirin 2non selective cox inhibitors: these agents demonstrate no meaningful biologic or clinical differences in the inhibition of cox-1 versus cox-2 activity like ibuprofen, naproxen and indomethacin 3relaively selective cox-2 inhibitors: these drugs have analgesic and antiinflammatory at doses that cause inhibition of cox-2 butless inhibition of cox-1 such as diclofenac, meloxicam and nimesulide. 4highly selective cox-2 inhibitors: inhibit the cox-2 isoform but have no effect on cox-1 isoform like celecoxib and rofecoxib cox-2 inhibitors can adversely affect the cardiovascular system because they don’t inhibit cox-1 derived thromboxana2(txa2) which act as a vasoconstrictor and stimulates platelet aggregation. but prevent cox-2 derived prostacycline (pgi2) production which is a vasodilator with a potent inhibition of platelet aggregation, activation and adhesion of leukocytes and accumulation of cholesterol in vascular cells, (9, 10) . i.e. pgi2 act as endogenous antilipidemic agent thus the cardiovascular effect of txa2 would be expected to be exaggerated as the pgi2 was known to act as a general retrain to any recognized stimulus to platelet activation. deletion of the pgi2 receptor inositol phosphate (ip), like cox-2 inhibition elevates blood pressure and augment the pressure response to dietary sodium. meanwhile variation in other endogenous mediators such as no, could be expected to modulate the impact of cox-2 inhibition on cardiovascular function. thus, suppression of cox-2 dependent pgi2 formation can both augment the response to thrombotic and hypertensive stimuli and initiate and accelerate atherogenesis (10) the effect of selectivity to cox-2 enzymes on the various components of lipid profile were studied in both normoand hyperlipidemic( having total cholesterol ≥ 5.18mmol/l and/or triglycerides ≥ 1.69 mmol/l ) diabetes with arthritis. materials and methods this study was carried out at the national center for diabetes─ al mustansiria university, for the period from november / 2007 to july / 2008 the study included 34 patients with type 2 diabetes mellitus (14 normolipidemic and 20 hyperlipidemic) aging between ( 40 and56) years old with duration of diabetes from (3 to 6 ) years, having osteoarthritis or rheumatoid arthritis, under the supervision of a senior physicion of the center.all the patients have type 2 dm using aspirin therapy and maintained on oral hypoglycemic agents ( glibenclamide and / or metformine ) before and during the study period and not receiving insulin therapy . all pregnant , breast feeding women hypertensive , and patients with cardiovascular diseases were excluded, also patients with any history of gastrointestinal tract problem like peptic ulcer and duodenal ulcer,or having allergy to sulfa drugs and patients who used hypolipidemic drugs were excluded . all the patients received medical advice to keep their medications and diets under control throughout iraqi j pharm sci , vol.18 (suppl.), 2009 effect of cox-2 selectivity on lipid profile 9 the study. in addition to diabetic patients sixteen healthy subjects were included in the study as a control, with age and sex matching that of the patients .the selected subjects were categorized according to their therapy to be received as follows: group 1: included 20 hyperlipidemic diabetic patients(13 females and 7 males) with arthritis (13) of them were treated with celecoxib 200 mg/day taken as a single dose for 8 weeks, while the reminder (7) were treated with diclofenac 100mg/day after meals as a single dose for 8 weeks . group 2: included 14 normolipidemic diabetic patients (10 females and 4 males) with arthritis (7) of them treated with celecoxib 200 mg/day and (7) treated with diclofenac 100 mg/day after meal as a single dose for 8 weeks period. group 3: included sixteen apparently healthy subjects ( 9 females and 7 males) considered as the control group fasting venous blood sample were drawn from each patient at baseline and after 8 weeks of receiving either celecoxib or diclofenac therapy. then serum was separated and stored frozen for the performance the enzymatic assays of total cholesterol ( tc ) (11) , triglycerides ( tg ) (12) , and high density lipoprotein-cholesterol (hdl-c) (13) . low density lipoproteincholesterol (ldl-c) was determined according to freiwald equation (14) , plasma hs-crp was measured by votila method based on elisa technique (15) . results table (1) shows that diclofenac causes a significant increase in serum tg level (% change = +43.74) in hyperelipidemic diabetic patients (figure 2) without significant changes in serum tc, hdl-c, and ldl-c levels , while celecoxib produced significant decline in serum tc and tg (% changes are – 8.13, ─19.72) respectively (figures 1, 2) without significant changes in serum hdl-c and ldlc level. normolipidemic patients recieved diclofenac showed a significant increase in serum tc, tg and ldl-c with significant decline in serum hdl-c level ( % changes = + 12.51, + 21.34, + 27.21, 11.72) respectively (figures 1,2,4,3). while celecoxib produced non significant changes in serum tc, tg, hdl-c and ldl-c levels. table (2) shows that in hyperlipidemic diabetic patients diclofenac caused a significant increase in serum tg/hdl-c ratio % change +73.87 (figure 6) without significant difference in serum ldlc/hdl-c ratio. while, celecoxib showed a significant decline in serum ldl-c/hdl-c ratio ( % change -16.46 figure (5) without a significant effect on serum tg/hdl-c ratio .in normolipidemic patients diclofenac produce significant increase in both ldl-c/hdl-c and tg/hdl-c ratios (% changes =+46.79, 39.02 , respectively ( figures 5, 6 ) .wherase, celecoxib produce no significant changes in those ratios. table(1): effect of treatment with diclofenac or celecoxib on lipid profile in normolipidemic and hyperlipidemic type 2 diabetic patients low density lipoprotein (ldl) high density lipoprotein (hdl) triglyceride (tg) total cholesterol (tc) duration of therapy type of therapy group 2.50 ± 0.2 1.46± 0.05 1.22± 0.06 4.52± 0.22 control n=16 4.25± o.52*a 4.96 ±0.80*a 4.30±0.36*a 3.84±0.37*a 1.49 ± 0.09a 1.34±0.11a 1.48±0.10a 1.57±0.07a 2.05± 0.35*a 2.94± 0.54*b 1.94±0.24*a 1.56±0.15*b 6.68± 0.43*a 7.60 ± 0.68*a 6.66± 0.37*a 6.12±0.33*b baseline after 8 weeks baseline after 8 weeks diclofenac n= 7 celecoxib n= 13 hyperlipidemic n= 20 2.36±0.23a 3.00±0.36b 2.20±0.25a 2.86±0.52a 1.62±0.08a 1.43±0.11b 1.55±0.09a 1.68±0.11a 1.15±0.10a 1.40±0.13b 1.44±0.06*a 1.32±0.10a 4.51±0.20a 5.07±0.34b 4.42±0.26a 5.15±0.52a baseline after 8 weeks baseline after 8 weeks diclofenac n= 7 celecoxib n= 7 normolipidemc n= 14 data are presented as mean ± sem ( mmol / l) n = number of patients *p < 0.05 with respect to control group non identical superscript ( a , b ) within the same drug group represent significant difference , p < 0.05 iraqi j pharm sci , vol.18 (suppl.), 2009 effect of cox-2 selectivity on lipid profile ٠١ table (2): effect of treatment with diclofenac or celecoxib on lipid ratios (ldl/hdl) , (tg/hdl) in normolipidemic and hyperlipidemic type 2diabetics tg/hdl ldl/hdl duration of therapy type of therapy group 0.86± 0.06 1.72± 0.13 control n=16 1.41± 0.25*a 2.45± 0.61*b 1.47±0.28*a 1.03± 0.15a 2.95± 0.44*a 3.88± 0.80*a 3.04± 0.31*a 2.54± 0.27*b baseline after 8 weeks baseline after 8 weeks diclofenac n=7 celecoxib n=13 hyperlipidemic n= 20 0.71± 0.05a 0.99±0.09 b 0.94±0.05a 0.82 ±0.10a 1.51± 0.18a 2.21± 0.34b 1.46± 0.21a 1.77± 0.35a baseline after 8 weeks baseline after 8 weeks diclofenac n=7 celecoxib n=7 normolipidemic n= 14 data are presented as mean ± sem n = number of patients *p < 0.05 with respect to control group non identical superscript ( a , b ) within the same drug group represent significant difference , p < 0.05 figure (1): the % changes in serum total cholesterol concentration from baseline value after 8weeks treatment with diclofenac or celecoxib in hyperlipidemic and normolipidemic type 2 diabetic patients figure (2) :the % changes in serum triglycerides concentration frombaseline value after 8weeks treatment with diclofenac or celecoxib in hyperlipidemic and normolipidemic type 2 diabetic patients figure (3):the % changes in serum hdlcholesterol concentration from baseline valuet after 8weeks treatment with diclofenac or celecoxib in hyperlipidemic and normolipidemic type 2 diabetic patients figure (4) : -the % changes in serum ldl cholesterol concentration from baseline value after 8weeks treatment with diclofenac or celecoxibin hyperlipidemic and normolipidemic type 2 diabetic patients 14.26 12.51 -8.13 16.55 -10 -5 0 5 10 15 20 % c h a n g e f ro m b a s e li n e diclofenac hyperlipidemic diclofenac normolipidemic celecoxib hyperlipidemic celecoxib normolipidemic cholesterol 43.74 21.34 -19.72 -7.86 -20 -10 0 10 20 30 40 50 % c h a n g e f ro m b a s e lin e diclofenac hyperlipidemic diclofenac normolipidemic celecoxib hyperlipidemic celecoxib normolipidemic triglycerides 16.6 27.21 -10.58 29.68 -15 -10 -5 0 5 10 15 20 25 30 % c h a n g e f ro m b a s e lin e diclofenac hyperlipidemic diclofenac normolipidemic celecoxib hyperlipidemic celecoxib normolipidemic ldl -10.69 -11.72 5.86 8.16 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 % c h a n g e f ro m b a s e lin e diclofenac hyperlipidemic diclofenac normolipidemic celecoxib hyperlipidemic celecoxib normolipidemic hdl iraqi j pharm sci , vol.18 (suppl.), 2009 effect of cox-2 selectivity on lipid profile ٠٠ figure (5):-the % changes in serum ldl/hdl ratio concentration from baseline value after 8weeks treatment with diclofenac or celecoxib in hyperlipidemic and normolipidemic type 2 diabetic patients figure (6): % change in serum tg/hdl ratio concentration from baseline value after 8weeks treatment with diclofenac or celecoxib in hyperlipidemic and normolipidemic type 2 diabetic patients figure (7): the % change in serum hscrp concentration from baseline value after 8 weeks treatment with diclofenac or celecoxib in hyperlipidemic and normolipidemic type 2 diabetic patients discussion arthritis and diabetes are both common conditions that affects large population, the musculoskeletal system can be affected in diabetes , leading to several pathological conditions such as arthritis (5) in hyperlipidemic diabetics, the diclofenac produced a significant increase in serum triglycerides and exerts a qualitative changes in their serum lipid components ( increase tg/hdl ) .while hyperlipidemic diabetic patients treated with celecoxib showed a significant decline in serum total cholesterol level and producing a significant decline in ldl/hdl ratio (i.e. has beneficial quantitative effect on serum lipid ratio). these results agree with another study which reported that celecoxb decrease fat deposition in rats fed a highfat diet and that celecoxib could lower serum and hepatic lipids ( usually tg ) in rats by mechanism not related to cox-2 inhibition but by inhibition of fatty acid synthase expression which is a central enzyme in lipogenesis. (16) meanwhile other reports shown that cox-2 has anti –atherogenic activity so that rats given a selective cox-2 inhibitors (rofecoxib) had increased cardiovascular side effects not only due to the increase txa2 in circulation(as cox2 inhibitors don’t inhibit cox-1 derived thromboxana2) but also cox-2 deficiency resulted in accumulation of lipids in circulation ,and animals that given selective cox-2 inhibitors have higher serum cholesterol , tg, and hdl than animals not given cox-2 inhibitors (17). similarly other study applied on arthritis patients treated with celecoxib ( selective cox-2 inhibitors) and meloxicam ( non selective cox-2 inhibitors) had shown that celecoxib treated patients have higher serum cholesterol, tg and hdl than the control group, while those treated with meloxicam have higher serum cholesterol, tg but lower hdl than the control, indicating that selective cox2 inhibitors increased the cardiovascular risk markers in those patients (18) many reports study the effect of different types of nsaids on lipid profile had shown that nsaids ( among these drugs flufenamic acid , ibuprofen ,acetaminophen, indomethacin and acetysalicylic acid ) could lower serum cholesterol level in normocholesterolemic subjects because these drugs enhance ldl catabolism due to increased synthesis of mrna of ldl receptors proteins (19). also a study carried on animals had demonstrated that diclofenac therapy could lower serum lipids, oxidized ldl, serum antioxidant defenses and markers of oxidative stress in rats receiving diclofenac for 28 days (20) .we also studied the effect of celecoxib and diclofenac on hs-crp level which is a cut phase protein produced by the liver and rise significantly in response to 31.14 46.79 -16.46 21.55 -20 -10 0 10 20 30 40 50 % c h a n g e f ro m b a s e lin e diclofenac hyperlipidemic diclofenac normolipidemic celecoxib hyperlipidemic celecoxib normolipidemic ldl/hdl ratio 73.87 39.02 -29.66 -12.78 -40 -20 0 20 40 60 80 % c h a n g e f ro m b a s e lin e diclofenac hyperlipidemic diclofenac normolipidemic celecoxib hyperlipidemic celecoxib normolipidemic tg/hdl ratio -33.27 -31.1 -22.47 -21.08 -35 -30 -25 -20 -15 -10 -5 0 % c h a n g e f ro m b a s e li n e diclofenac hyperlipidemic diclofenac normolipidemic celecoxib hyperlipidemic celecoxib normolipidemic hs-crp iraqi j pharm sci , vol.18 (suppl.), 2009 effect of cox-2 selectivity on lipid profile ٠٠ injury, infection, and other inflammatory conditions and considered as a good marker of cardiovascular events (21) . results analysis had shown that both drugs lowered serum hs crp in both hyper and normolipidemic diabetics to similar degree with a greater effect produced by diclofenac. this agree with other study carried on humans and shown that when celecoxib given to patients with coronary artery disease for 2 weeks causes significant lowering in serum hscrp and oxidized ldl levels as compared with placebo (22) .another study involve diclofenac demonstrate that diclofenac administration to patients undergoing major urological surgery was associated with lower leukocyte count, crp concentration and temperature than the placebo group indicating that diclofenac may have antiinflammatory role in major surgery (23) . conclusion the administration of relatively selective cox-2 inhibiters (diclofenac) to normolipidemic type 2 diabetic could adversely effect lipid metabolism by producing qualitative and quantitative changes in serum lipid components more than that observed in hyperlipidemic patients. references 1. american diabetes association; diagnosis and classification of diabetes mellitus. diabetes care 2008. 31: s55 – s60. 2. dobretsorv m, romanovsky d and stimers: early diabetic neuropathy triggers and mechanism. world j gastroenterol 2007; 13(2): 175191. 3. keecia d king , pharma d, jocelyne d. jones, pharma d and jessica w arhen, pharma d. microvascular and macrovascular complications of diabetes mellitus. american journal of pharmaceutical education 2005 ; 69 (5) article 87 4. arthritis foundation . disease center avialable at : http://www. arthritis . org / conditions / disease center default asp ,2003 5. thomas pressely, md. arthritis and diabetes: acommon association. nfb 1992 , volume 7, number 4 6. jane a mitchell and timothy d warner . cyclooxygenase-2 pharmacology , physiology , biochemistry and relevance to nsaid therapy . british journal of pharmacology ( 1999 ) 128, 1121-1132 7. halis suleyman , berna demircan , yalein karagoz : anti-inflammatory and side effect of cyclooxegenas inhibitors. pharmacological reports 2007 , 59, 257 268 8. lipsky lp, abramson sb crofford l, et al . the classification of cyclooxygenase inhibitors ( editioral) rheumatol 1998 , 25 (12): 2298-303 9. burkhard hinz and kay brune. cyclooxygenase2-10 years later. journal of pharmacology and experimental therapeutics .february 2002, volum:300, issue 2, 367-375 10. tilo grosser , susanne fries and garret a . fitzgerald. biological basis for the cardiovascular consequences of cox-2 inhibitors therapeutic challenges and apportunities . j. clin. inves. (2006) 116 : 4-15 11. richmond w. preparation and proprieties of cholesterol oxidase from no─cardio sp. and its application to enzymatic assay of total cholesterol in serum clin chem. 1973, 19: 1350-51 12. fossati , p and prencipe , l.: serum triglyceride determined colorimetrically with an enzyme that produce h2o2 .clin chem. 1982;28(10): 2077-2080 13. burstein , m. ; scholinck , h.r. ; morfin. r-: early historical milestones in hdlcholesterol assay. j. lipid res. 1970 ; 11: 583-587 14. baron rb : lipid abnormalities , in current medical diagnosis and treatment , by : tierney lm; mcphee s j and papadakis ma (eds) , 43 rd ed , 2004 . mcgw─ hill book co, newyork , app 1191─1199. 15. votila m, rouslahti e and engvall e: two-site sandwich enzyme immunoassay with monoclonal antibodies to human alpha-fetoprotein. j immunol methods 1981; 42 (1): 11-15 16. suying lu and michael c. archer . celecoxib decreases fatty acid synthase expression via down regulation of c-june nterminal kinase . experimental biology and medicine 2007. 232-653 17. ajay narasimha, junji wateanable, james a. lin et al. anovel anti atherogenic role for cox-2 –potential mechanism for the cardiovascular side effects of cox-2 inhibitors . prostaglandins and other lipid mediators . augest 2007. volum 84, issue 1-2 pag 2433 18. ali m. hadi.: modulation of some cardiovascular risk markers induced by cyclooxygenase -2 inhibitors by vitamin e or low dose aspirin in rheumatoid arthritis or osteoarthritis patients. m.sc. thesis. clin. lab. sci. dept. collage of pharmacy / baghdad university. 2008: pp (114-115). iraqi j pharm sci , vol.18 (suppl.), 2009 effect of cox-2 selectivity on lipid profile ٠۳ 19. osama al rayyes; bo ahren and claes – henrik floren.: enhancment of low density lipoprotein catabolism by non steroid antiinflammatory drugs in cultured hpg2. europeon journal of pharmacology 1999; 372(3): 311-318. 20. emillio, c.; curcelli sergios; jose luiz, v.; b et al.: beneficial effects of diclofenac therapy on serum lipids, oxidized ldl, and antioxidant defenses in rats. life science 2008; 82: 892-898. 21. pepys, mb.; hirschfield, gm.: c-reactive protein: a critical update . j. clin. inves. 2003; 111(12): 1805-12. 22. rémy chenevard; david hürlimann; markus béchir; et al.: selective cox-2 inhibition improves endothelial function in coronary artery disease. circulation 2003; 107: 405-409. 23. a. m. mahdy; h.f. galley; m.a. abdelwahead.: differential modulation of interleukin-6 and interleukin-10 by diclofenac in patients undergoing major surgery. british journal of anaesthesia 2002, 88: 6797-802. iraqi j pharm sci, vol.22(1) 2013 active constituents in arctium lappa l. 18 phytochemical investigation for the main active constituents in arctium lappa l. cultivated in iraq dhuha a.al-shammaa *,1 , kawkab y. saour ** and zaid m. abdul-khalik *** * department of pharmacognosy, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. *** baghdad college of pharmacy, baghdad, iraq. abstract burdock ( arctium lappa), is among the most popular plants in traditional medicine and it is associated with several biological effects. literature survey revealed the presence of phenylpropanoid compounds .the most widespread are hydroxycinnamic acids ( mainly caffeic acid and chlorogenic acid) and lignans (mainly arctiin and arctigenin). this work will confirm the presence of these compounds in arctium lappa, cultivated in iraq, in both root and leaf samples. the dried plant samples were extracted by soxhlet with 80% methanol then separated the main constituents by thin layer chromatography (tlc) and high performance liquid chromatography (hplc). identification of the isolated compounds was carried out by uv, ir, and compared with reference standards using tlc, hplc and hptlc. keywords: actium lappa, hydroxycinnamic acid, lignan, phytochemical. دراسة كيميائية للمواد الفّعالة في نبات االرلطيون المستسرع في العراق ضحى عبذ الصاحة الشماع *،1 ، كوكة يعموب ساعور ** و زيذ محمذ عبذ الخالك *** * .قسى انعقاقٍش وانُباحاث انطبٍت ، كهٍت انصٍذنت ،جايعت بغذاد ، بغذاد، انعشاق ** .، كهٍت انصٍذنت ،جايعت بغذاد ، بغذاد، انعشاققسى انكًٍٍاء انصٍذالٍَت *** .بغذاد، انعشاق كهٍت بغذاد نهصٍذنت ، الخالصة . يٍ انُباحاث االكثش شعبٍت فً انطب انخقهٍذي وٌشحبط بانعذٌذ يٍ انقعانٍاث انبٍىنىجٍت, ٌعذ َباث االسقطٍىٌ وانًعشوف باسى انبهسكاء خصىصا حايض انكافٍك (االكثش اَخشاساهً حىايض هٍذسوكسً سٍُايكو . انسابقت وجىد يشكباث انفُم بشوباَىٌذ أظهشث انذساساث وهزا انعًم سىف ٌثبج وجىد هزِ انًشكباث فً َباث االسقطٍىٌ (.بصىسة سئٍسٍت األسكخٍٍ و األسكخٍجٍٍُ)وانهكُاٌ ( وحايض انكهىسوجٍُك وفصم ٪80انًٍثاَىلبىاسطت حى رنك باخز عٍُاث يٍ انُباث انجاف واسخخالصهاوقذ . و االوساق انًسخزسع فً انعشاق فً كم يٍ انجزوس ثى حى حشخٍص انًشكباث انًفصىنت باسخخذاو طٍف االشعت .عانٍت األداء انسائهتكشوياحىكشافٍا يع طبقت انشقٍقتال يشكباحها بكشوياحىكشافٍا باالضافت انى يقاسَخها يع انًىاد انقٍاسٍت باسخخذاو كشوياحىكشافٍا انطبقت انشقٍقت وكشوياحىكشافٍا فىق انبُفسجٍت واالشعت ححج انحًشاء . عانٍت االداء انسائهت . االرلطيون ، حامض هيذروكسي سيناميك ، ليغنان ، دراسة كيميائية :الكلمات المفتاحية introduction actium lappa (common name: burdock) is a flowering plant that belongs to the family asteraceae (compositae). with the advancement of different state-of-the-art analytical techniques, more active ingredients of actium lappa have been identified over the last decade (1) . the literature furnishes numerous data on their antiinflmmatory, hepatoprotective, antitumor, antimicrobial, antifungal, anti-aging and hypoglycemic effects (2) . the main active ingredients isolated from this herb are: caffeic acid (3, 4 – dihydroxy cinnamic acid); chlrogenic acid (1s,3r,4r,5r)-3-{[(2z)-3-(3,4 dihydroxyphenyl) prop -2 – enoyl] oxy }1, 4 ,5trihydroxycyclohexanecarboxylic acid; arctiin (3r,4r)-4-[(3,4-dimethoxyphenyl)methyl]-3 {[3-methoxy-4-[(2s, 3r, 4s, 5s, 6r) -3, 4, 5-trih ydroxy -6(hydroxymethyl) oxan -2-yl ] oxyphenyl ] methyl}oxolan-2-one; arctigenin (3r,4r)-4-[(3, 4-dimethoxyphenyl) methyl]-3 [(4hydroxy -3methoxy phenyl) methyl]-2tetrahydrofuranone (3) . dong wh. et al. ( 2006) (4) study the condition for extraction of arctiin from fruits of arctium lappa using supercritical fluid extraction, they found that optimal extraction conditions were: pressure 40 mpa, temperature 70 degrees c, using methanol as modifier carrier, while liu s. et al.(2005) (5) isolated and identified arctiin and arctigenin in leaves of burdock (arctium lappa l.) by polyamide column chromatography in combination with hplcesi/ms. 1 corresponding author e-mail:alduha_sun@yahoo.com. received: 18/7/2012 accepted:12/12/2012 http://www.botany.hawaii.edu/faculty/carr/aster.htm http://www.ncbi.nlm.nih.gov/pubmed?term=dong%20wh%5bauthor%5d&cauthor=true&cauthor_uid=17048565 http://www.ncbi.nlm.nih.gov/pubmed?term=liu%20s%5bauthor%5d&cauthor=true&cauthor_uid=15881114 iraqi j pharm sci, vol.22(1) 2013 active constituents in arctium lappa l. 19 ferracane r. et al. (2010) (6) were analyzed phytochemical compounds by liquid chromatography coupled to electro spray tandem mass spectrometry (lc/ms/ms) in negative mode. caffeic acid chlorogenic acid arctiin arctigenin experimental plant materials the plant sample was collected from the department of medicinal plants, college of agriculture, university of baghdad. identified by dr.saged auda mohammad , head of medicinal plant department. the plant leaves and roots were air dried in the shade at room temperature, coarsely powdered by mechanical grinder and weighed. chemicals all reagents and solvents used were analytical grade. standards used to identify the main active constituents (arctiin, arctigenin, caffeic acid and chlorogenic acid) were obtained from china (cheng du biopurify phytochemicals ltd.).uv was done in methanol. instruments electrical sensitive balance: sartorius/ germany, ultraviolet light : desaga heidelberg/ germany, rotatory evaporator: buchi rotatory evaporator, chiller: ultratemp 2000 , water bath: memmert/germany, hplc: water/irland, ftir: shimadzo ft-ir-84005 ir spectrometer, hptlc:eike reich/camag – laborator. extraction of plant 100 g of dried milled leaves and roots were extracted separately in a soxhlet apparatus with 80% methanol (3:1 solvent/plant ratio), for 6 hours. the extracts obtained were evaporated to dryness at 45 o c under vacuum using rotary evaporator (7) . general phytochemical screening by chemical tests the different parts of the plant have been screened for the occurrence of saponines, flavonoids, tannins and alkaloids by the following chemical reactions aferric chloride test for tannins. b froth test for saponin. cconc h2so4 with ammonia for flavonoids. dreagent for alkaloids (8) . isolation and identification mayer ' s of active constituents separation of the main active constituents from both leaves and roots of arctium lappa l. were carried out using preparative tlc: plc plates, 20x20cm and 1mm thickness of silica gel gf254 developed in solvent system (chcl3meoh 80:20) together with standard references http://www.ncbi.nlm.nih.gov/pubmed?term=ferracane%20r%5bauthor%5d&cauthor=true&cauthor_uid=19375261 http://upload.wikimedia.org/wikipedia/commons/c/c3/kaffees%c3%a4ure.svg iraqi j pharm sci, vol.22(1) 2013 active constituents in arctium lappa l. 20 the chromatogram was visualized by uv lamp (at 254 nm and 366 nm) (9) . identification of active constituents was done by: 1-matching with standards by tlc using the following mobile phases: s1: chloroform: aceton: formic acid (75,16.5, 8.5), s2: ethanol:acetic acid (85:15), s3: chloroform:methanol (80:20) (10) 2-high performance liquid chromatography (hplc): hplc analysis was done using c18 – column (150 × 4.6 mm) with specific conditions for each compound:  hplc conditions for arctiin, arctigenin and caffeic acid: the mobile phase is methanol: water (60:40) , flow rate: 0.5 ml/min at 280 nm (11) .  hplc conditions for chlorogenic acid: the mobile phase is acetonitrile: acetic acid: water (15: 0.5:85), flow rate 1ml/min at 320 nm (12) . 3fourier transform infrared spectroscopy (ftir) in kbr disk. 4-high performance thin layer chromatography (hptlc): the mobile phase is ethanol: acetic acid (85:15), the list of standards: arctiin std., arctigenin std., caffeic acid std., chlorogenic acid std., then leaf sample and root sample. results and discussion the preliminary phytochemical investigation revealed the presence of saponins, flavonoids, tannins in both parts of the plant, while the alkaloids are absent as shown in table( 1). table 1: the results of the general screening by chemical tests. identification for each isolated compound depend on : 1accurate measurement of rf values in tlc as shown in table 2 and figure 1 table 2: rf values of some plant constituents and their standards in different developing solvent systems in tlc. tlc rf (isolated compound) s1 s2 s3 tlc rf (standard) s1 s2 s3 compounds 0.66 0.67 0.72 0.67 0.68 0.71 arctiin 0.85 0.80 0.94 0.86 0.82 0.92 arctigenin 0.35 0.49 0.46 0.36 0.48 045 caffeic acid 0.03 0.04 0.03 0.02 0.03 0.04 chlorogenic acid figure 1 :thin layer chromatograms of both root and leaf sample with four references standard on silica gel gf254, developing in s1 solvent system and detection by uv light at 254 nm & 366 nm.[ 1:std. arctiin , 2: std.arctigenin , 3:std. caffeic acid , 4: std.chlorogenic, r: root extract, l: leaf extract.]. the plant sample plant part saponin alkaloid flavonoid tannin actium lappa l. root + + + leaf + + + iraqi j pharm sci, vol.22(1) 2013 active constituents in arctium lappa l. 21 2-high performance thin layer chromatography hplc hplc analysis can be carried out for qualitative analysis by comparison of retention times of analyzed samples and authentic standards at identical chromatographic conditions .the results are shown in figures (2, 3) and table 3. hplc of std. arctigenin hplc of std. arctin hplc of std. caffeic acid hplc of methanolic leaf extract hplc of methanolic root extract figure 2: hplc analysis of std. arctiin, std. arctigenin, std.caffeic acid and methanolic extract of leaf &root sample. iraqi j pharm sci, vol.22(1) 2013 active constituents in arctium lappa l. 22 hplc of std. chlorogenic acid. methanolic root extract methanolic leaf extract figure 3: hplc analysis of std. chlorogenic acid and methanolic extract of root & leaf sample. table 3: the retention time of each isolated compound with that of reference standards 3-ft-ir spectroscopy ir spectroscopy is most frequently used in phytochemical studies as a ' finge printing ' device, for comparing a natural with a synthetic reference standard .such comparisons are very important in the complete identification of many types of plant constituents as shown in table (4 ). rt of compounds in leaf extract rt of compounds in root extract rt of standard compound 5.074 5.231 5.063 arctiin 8.616 8.734 8.770 arctigenin 2.850 2.707 2.650 caffeic acid 9.541 9.818 9.889 chlorogenic acid iraqi j pharm sci, vol.22(1) 2013 active constituents in arctium lappa l. 23 table 4: ir absorption bands of isolated compounds ( in cm -1 ). 4-hptlc analysis hptlc was carried out for further identification of main active constituents present in methanolic extract of both leaves and root of actium lappa, by measuring the rf values and uv spectrum :the results obtained are shown in figure 4. all tracks at wavelengtth 254 nm arctiin std. arctigenin std. chlorogenic acid caffeic acid std. figure 4: hptlc analysis of methanolic extract with reference standards. aproximate positions of characteristic bands. compound 3470(oh), 1780(c=o),1594,1520(ar-h), 1080, 1030, 1020(β-glycopyranoside), 2930,2850, 1465, 1420,1262, 670. arctiin 3470(oh), 1770(c=o), 1610, 1520, 3030(ar-h), 1465, 1390 , 1270. arctigenin 3433, 3234, 2910, 1645, 1620, 1448, 1278 , 1217 , 1120, 974,576 caffeic acid 3421, 2929, 1697, 1635, 1456, 1398 ,1278,1182, 812. chlorogenic acid iraqi j pharm sci, vol.22(1) 2013 active constituents in arctium lappa l. 24 end rf values recorded for standards in hptlc which have small peak area, are excluded because these are due to impurities. all chromatographic data coincide with that reported and shown by the standards and so as the obtained uv & ftir results. this confirms the presence of arctiin, arctigenin, caffeic acid & chlorogenic acid in both leaves & root of arctium lappa. conclusion the iraqi plant , actium lappa, is a good source for many phenolic compounds with antioxidant activity. 80% methanol is the best solvent suitable for the extraction of the main active constituents from both root and leaf of the plant .the same constituents were obtained from both parts, however; the results showed that there was a slight different in the amount of each plant constituents. this study confirms the presence of chlorogenic acid, caffeic acid, arctiin and arctigenin in actium lappa plant cultivated in iraq. references 1. james a.duke; mary jo bogenschutzgodwin “hand book of medicinal herbs ” , second edition, 2002; by crc. 2. sheldonhendlerph.d.m.d.; david rorvik, “ pdr for nutritional supplements ” second edition, 2008(11): 156-159. 3. joanne barnes; linda a. anderson and j.d. phillipson, “ herbal medicines ” , 2007; sep. 4. dong wh; liu b., “study on condition for extraction of arctiin from fruits of arctium lappa using supercritical fluid extraction”, zhongguo zhong yao za zhi. 2006 (31): 1240-1276. 5. liu s, chen k; schliemann w; strack d,“ isolation and identification of arctiin and arctigenin in leaves of burdock (arctium lappa l.) by polyamide column chromatography in combination with hplcesi/ms”, phytochem anal. 2005(16): 8690. 6. ferracane r, graziani g, gallo m, fogliano v, ritieni a., “ metabolic profile of the bioactive compounds of burdock (arctium lappa) seeds, roots and leaves”, j pharm biomed anal. 2010(51): 399-404. 7. fabio bahls machado; rafael eidi yamamoto; karine zanoli.; “ evaluation of the antiproliferative activity of the leaves from arctium lappa by a bioassay-guided fractionation ” ,molecules, 2012(17): 18521859. 8. fabriciaspredes; analtg ruiz; joaoe carvalho; mary a foglio; “ antioxidant and invite anti proliferative activity of arctium lappa root extracts ” , complementary and alternative medicine , 2011(10): 11861472. 9. long-ze lin; james m harnly; “ identification of hydroxycinnamoylquinic acids of arnica flowers and burdock roots using a standardized lc-dad-esi/ms profiling method ” , journal of agricultural and food chemistry 2008(56): 1010510114. 10. liu s et al. “ isolation and identification of arctiin and arctigenin in leaves of burdock (arctium lappa l.) by polyamide column chromotography in combination with hplc-esi/ms ” , phytochemical anal 2005(16): 86-89. 11. xiao wang; fuwei li; qinglei sun; jingpeng yuan; ting jiang; chengchao zheng ; “ application of preparative highspeed counter-current chromatography for separation and purification of arctiin from fructus arctii”, journal of chromatography a 2005 (10): 247-251. 12. rosalia ferracane; giulia graziani; monica gallo; “ metabolic profile of the bioactive compounds of burdock (arctium lappa)seeds,root and leaves”, journal of pharmaceutical and biomedical analysis 2010(51): 399-404. http://www.amazon.com/s/ref=ntt_athr_dp_sr_2?_encoding=utf8&field-author=david%20rorvik&search-alias=books&sort=relevancerank http://www.ncbi.nlm.nih.gov/pubmed?term=dong%20wh%5bauthor%5d&cauthor=true&cauthor_uid=17048565 http://www.ncbi.nlm.nih.gov/pubmed?term=liu%20b%5bauthor%5d&cauthor=true&cauthor_uid=17048565 http://www.ncbi.nlm.nih.gov/pubmed/17048565 http://www.ncbi.nlm.nih.gov/pubmed?term=liu%20s%5bauthor%5d&cauthor=true&cauthor_uid=15881114 http://www.ncbi.nlm.nih.gov/pubmed?term=chen%20k%5bauthor%5d&cauthor=true&cauthor_uid=15881114 http://www.ncbi.nlm.nih.gov/pubmed?term=schliemann%20w%5bauthor%5d&cauthor=true&cauthor_uid=15881114 http://www.ncbi.nlm.nih.gov/pubmed?term=strack%20d%5bauthor%5d&cauthor=true&cauthor_uid=15881114 http://www.ncbi.nlm.nih.gov/pubmed/15881114 http://www.ncbi.nlm.nih.gov/pubmed?term=ferracane%20r%5bauthor%5d&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=graziani%20g%5bauthor%5d&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=gallo%20m%5bauthor%5d&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=fogliano%20v%5bauthor%5d&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=fogliano%20v%5bauthor%5d&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed?term=ritieni%20a%5bauthor%5d&cauthor=true&cauthor_uid=19375261 http://www.ncbi.nlm.nih.gov/pubmed/19375261 http://www.ncbi.nlm.nih.gov/pubmed/19375261 iraqi j pharm sci, vol.18(2) 2009 cosmetic products marketed in iraq 20 a study on cosmetic products marketed in iraq: microbiological aspect raghad a. razooki * ,1 , ebtihal n. saeed * and heyam hamza** * department of clinical laboratory science , collage of pharmacy , university of baghdad, baghdad , iraq ** microbiologist at national center for drug quality control and research centre, baghdad, iraq. abstract cosmetic products contain variable amounts of nutrients that support microbial growth. most contaminants in cosmetic products include bacteria such as staphylococcus, pseudomonas, klebsiella, achromobacter and alcaligenes. contaminated water is a likely source of organisms found in cosmetic products. products such as shampoo, hand and body lotion, facial cleanser, and liquid soaps were analyzed. in this study, out of 60 cosmetic products analyzed, 26.4% were found to be contaminated. most of the contamination was from bacteria and no fungal contamination was detected. the highest level of contamination occurred in shampoo.viable bacterial were not recovered from 100%, 92.8%, 91.6% and 89.2% of showed bath soaps, facial cleanser, hand and body lotion and shampoos, respectively. coliforms were recovered from one sample of shampoos. one isolate of shigella and pseudomonas aeruginosa was detected from two samples of shampoo. keywords: microbial contamination, cosmetic الخالصة ححُي مسخحضزاث انخجمٍم كمٍاث مخخهفت مه انمُاد انغذائٍت انخً حساعذ عهى ومُ انمهُثاث َحشمم ٌذي انمهُثاث اوُاع مه ٌعخبز انماء انمهُد مصذر . staphylococcus, pseudomonas, klebsiella, achromobacter, alcaligenesانبكخزٌا مثم حخعزض نٍا مسخحضزاث انخجمٍم مثم انشامبُاث، مسخحضزاث انسائهت الغزاض حجمٍهٍت اَ طبٍت، انمىظفاث انخً اٌضا آخز نهخهُد ان حقزٌبا اخذ حُانً سخُن مه ٌذي انمسخحضزاث َحم فحصٍا ماٌكزَبٍا َقذ اظٍزث انىخائج حمَانصابُن انسائم. فً ٌذي انذراست زي َاقهً ٌُ فطزي َان اعهى معذل نهخهُد َجذ فً انشامبُاث ٍنخهُد ٌُ بكخ% مه ٌذي انمسخحضزاث كاوج مهُثت َان معظم ا2..4 فً وُعٍه shigella َpseudomonasبكخزٌا ثمُجُدة فً احذ اوُاع انشامبُاث َاٌضا َجذ coliforms% َان بكخزٌا انـ 1..8 .(pathogenic micro organisms)ت ٍضً مه انبكخزٌا انمزآخزٌه مه انشامبُاث ٌَ introduction cosmetics are part of everyone's daily grooming routine. most cosmetic products are based on water/oil emulsion or oil/water emulsion and contain variable amounts of nutrients that support microbial growth. the raw materials used in cosmetic products may be grouped into categories (table 1). table 1 : raw material categories water acids, alkalis, salts oils, waxes, paraffin fatty acids, alcohol, esters surfactants, emulsifier talc, clay protein, starches, botanicals, gums and resin humectants colour and pigments preservatives, antioxidants and chelating agents fragrances, essential oils source: adapted from orth (1989). (1) microbial contamination in cosmetics, toiletries and personal care products is very common and has been of great concern to the industry for many years. bacteria, yeast and fungi are extremely diverse in their metabolic activities. the metabolic reaction of the microorganisms can cause health hazards because the metabolic products can be toxic, mutagenic.cosmetic products need protection against microbial spoilage, first of all in order to protect the consumer against potential dangers arising from pathogens and secondly to guarantee long-term stability (shelf life) of the formulae. preservatives play a vital role in product formulations. the selection of a germicidal is critical. in many cases, chemicals which are highly active against microbes also have similar effects against mammalian cells. therefore, a balance needs to be established with the preservatives of choice between killing organisms and not injuring the cells of consumer who uses the product. it is important to keep monitoring the cosmetic product for contamination because an increasing number of products are recalled each year , and the majority is contaminated with potential pathogenic micro organisms. (2) more knowledge on the reasons for contamination is needed. the aim of this study is to demonstrate the microbial content of unused cosmetic products at the point of sale. the cosmetic products were manufactured in iraq and were purchased from super market, saloons. 1 corresponding author e – mail : raghadrazooki@yahoo.com received : 14 / 10 / 2009 accepted : 22 / 12 /2009 iraqi j pharm sci, vol.18(2) 2009 cosmetic products marketed in iraq 21 microbiological contaminants of cosmetics the most frequent contaminants of cosmetic products include pseudomonas, klebsiella, achromobacier and alcaligenes (3) baird (4) surveyed 147 unused cosmetic products purchased in england. he recovered viable bacteria from 99 of the 147 products (table 2). gram-negative rods were isolated from 6.1% of the products. table 2 : gram-negative rods in cosmetics. contaminant product no. of organisms per ml/g pseudomonas aeruginosa lanolin hand cream 1.2x10 3 p. maltophilia mascara 7.0 x10 3 p. pseudoalkaligenes cleansing milk 3.1 x10 3 p. pseudoalkaligenes hair cream 1.9 x10 3 p. fluorescens hair oil 4.0 x10 3 p. putida cleansing jelly 2.5 x10 3 moraxella osloensis moisture cream 1.3 x10 3 enterobocter cloacas dental cream 2.3 x10 3 klebsiella aerogenes dental powder 3.4 x10 3 k. oxytoca dental powder erwina herbicola dental powder enterobocter cloacae dental powder source adapted from baird (1974). a number of surveys (1) reported the incidence of contaminants in unused cosmetic products (5) . the clinical and pharmaceutical significance of contamination for cosmetics has been reviewed by (6) . certain products, notably aqueous preparations were more susceptible to contamination than others. overall, these findings indicated that under the existing manufacturing conditions, some forms of contamination in the final product appeared to be inevitable. table (3) shows some potentially pathogenic bacteria isolated from cosmetic products and some of these organisms (7) are part of the normal human flora. table 3 : potentially pathogenic bacteria isolated from cosmetic preparations acinetobacter calcoaceticus escherichia coli providencia rettgeri citrobacter diversus hafnia alvei providencia stuartii citrobacter freundii klebsiella oxytoca pseudomonas cepacia clostridium spp. klebsiella pneumonia pseudomonas fluorescens enterobacter aerogenes morganella morganii serratia liquefaciens enterobacter agglomerans proteus mirabilis staphylococcus aureus enterobacter cloacea proteus vulgaris staphylococcus epidermidis enterobacter gergoviae enterobacter sakazakii source: adapted from norman (1984). hazards associated with microbial contamination infection from non-sterile products the roles of many organisms in cosmetic preparations were studied (8) by brunch (1972). he concluded that most are objectionable for application to damaged epithelium while others are opportunistic pathogens depending on the species, the site and the health of the recipient. contaminants in cosmetic products include bacteria such as staphylococcus, pseudomonas and other opportunistic bacteria. contamination of talc with closlridium tetani, infection of neonates with pseudomonas aeruginosa from contaminated cleansing solution and scalp infection from diluted stored shampoo leading to fatality in a granulocytopenia patient are some examples. the eye is particularty vulnerable to infection. the loss of eye sight after the use, during intraocular operation, of saline solution contaminated with pseudomonas aeruginosa (9) , and severe eye disorders caused iraqi j pharm sci, vol.18(2) 2009 cosmetic products marketed in iraq 22 by use of a cortisone ointment contaminated with the same organism are examples of the serious dangers posed by contaminated preparations. spoilage a spoiled product is one that has been rendered unfit for its intended use. mouldiness, colour change, fronting and packaging that bulge, leak or explode as a result of gas production are obvious effects of gross contamination and lead to chemical and physical changes in the products. discovery by the customer of a mould colony is not likely to encourage further purchase of the particular brand.recent reviews of the spoilage aspects of microbial contamination of medicines and cosmetics have been made by (6) . mixed flora introduced into the product by whatever means are often extremely versatile in their metabolic activity and can adapt to a broad range of environmental conditions. all cases of natural organic compounds are susceptible to degradation by one organism or another and synthetic material may also be attacked. products such as shampoos, which contain surfactants, are particularly susceptible to contamination by water-borne gram-negative bacteria, which may cause at the very minimum, a visible loss of lathering activity. active ingredients may also be rendered ineffective. visible effects contaminants may be seen as sediment, turbidity or pellicles in liquid products. on more solid preparations, coloured colonies may form. the appearance of bright yellow micrococcus colonies on a white cream is an alarming sight. this may result from use of natural ingredients such as dried egg, which can carry a large number of organisms, if improperly treated. contamination by pseudomonas spp., which is metabolically versatile, causes colour changes. this is due to alteration in product components as a result of direct consequence of metabolism or indirectly because of alteration of parameters such as ph or oxidation-reduction. the addition of organic material greatly increases the chances of growth and deposits or turbidity due to algae, mould, bacteria or yeast in a range of poorly preserved pharmacopoeia solutions. emulsions can become thin, separate, decolorize or change colour and become visibly heterogeneous owing to hydrolysis of the oil phase or change in ph of the aqueous phase. products such as shampoos are particularly susceptible to contamination by gram-negative water-borne bacteria (5) . slimy sediments, pellicles and discoloration may occur. degradation of actives constituents beveridge (10) reported on the inactivation of potent drugs and antimicrobial agents by a wide variety of microorganisms. penicillin can rapidly be destroyed by β-lactamases. other antibiotics, preservatives and disinfectants can be metabolized. atropine in eye drops can be destroyed by corynebacterium and pseudomonas spp (11) . and the transformation of hydrocortisone in the dermatological cream to a therapeurically different compound by a contaminant cladosporium herbarum. has been reported by (12) . methodology the objective of this study was to monitor the scenarios of iraqian market cosmetic products. in this study, various types of cosmetic products were purchased from supermarkets, saloons. these products were then subjected to microbiological tests to detect the presence of microorganisms at the point of purchase. aerobic plate count sterile materials and equipments were sterilized before use and aseptic techniques were used. the caps of the products were wiped with ethanol (70%).microbiological media were reconstituted and prepared from their dehydrated powder according to manufacturer instructions. by means of a syringe, or sterile spatula, one ml or one gram of the product was disintegrated in tryptic soy broth (9ml) according to b.p 2004 using a flask shaker and suitable serial dilations in tryptic soy broth were prepared. one-ml sample of each dilution was poured in a sterile petridish and then 15ml of sterile tryptic soy agar was poured on the samples, the plates were gently swirled in a round movement to allow a good mixing of the agar with the sample, then the plates were allowed to solidify on a leveled surface. triplicate plates for each sample were used and incubated at (35 0 c -37 0 c) for two days for bacteria. sabourand dextrose ager was used instead of tryptic soy agar-for the detection of fungi. the prepared plates were incubated at 25 0 c for 5 days. after incubation the number of colonies was counted by estimating the total count of the growing bacteria and fungi then the mean of three plates was calculated.a laboratory control count was performed using negative control blank (without product) and with positive control (contaminated product). more than two colonies on the negative control plate iraqi j pharm sci, vol.18(2) 2009 cosmetic products marketed in iraq 23 invalidated the test.the colonies were counted. colony counts exceeding 1000 were considered too high to count and the product further diluted. plates with colonies of 30-300 were selected. the microorganism content per milliliter or gram is the colony count multiplied by the appropriate dilution factor (10 or 100). samples samples used in this study included shampoos, liquid bath soaps, facial cleansers, hand and body lotions and moisturizers, which were purchased from supermarkets and saloons.detection for specific microorganisms such as escherichia coli, staphylococcus aureus, pseudomonas, and salmonella was performed following procedure under isolation and identification tests for specified microorganisms (b.p 2004) as shown in table (4). when results showed presence of any of these organisms, appropriate biochemical tests were performed. table 4 : isolation and identification tests for specified microorganisms (bp. 2004) organism enrichment primary test secondary test confirmation enterobacteriace ae lactose broth 35-37 0 c eeb-mossel 35-37 0 c for 24-48hr. vrbgla 3537 0 c growth of gram negatives e. coli as above macconkey broth 43-45 0 c for 18-24hr. macconkey agar 43-45 0 c for 18-24hr. indole at 43.544.5 0 c biochemical salmonella spp. as above for 524hr. tbbg broth 42-43 0 c 18-24hr. then subculture on: dca.xlda or bga for 35-37 0 c for 24-48hr. tsi agar 3537 0 c for 18-24 hr. biochemical serological p. aeruginosa saline peptone 35-37 0 c for 25hr casein digest broth 35-37 0 c for 24-48hr. cetrimide agar 35-37 0 c for 2448hr. oxidase test staph. aureus as for p.aeruginosa above as for p.aeruginosa above baird-parker 3537 0 c for 2448hr. coagulase, catalase, dnase tests eeb-m-mossel. enterobacteriaceae enrichment broth-mossel; vrbgla, violet red bile agar with glucose and lactose; tbbg, tetrathionate bile brilliant green broth; dca, deoxycholate citrate agar; xlda, xylose lysine deoxycholate agar; bga, brilliant green agar; tsi, triple sugar iron agar; dnase, deoxyribonuclease test. results and discussion of 60 products analysed for their total aerobic bacterial, coliforms and fungal counts, 26.4% were found to be contaminated table (5). most products were of bacterial contamination and no one were of yeast and mould. shampoos were more suceptible to contamination than other products presumably because they contain surfactants fig (1) and table (5).viable bacteria were not recovered from 100%, 92.8%, 91.6% and 89.2% of shower bath soaps, facial cleanser, hand and body lotion and shampoos, respectively. table (6) shows the microbial counts (c.f.u)/gm or ml) and types found in shampoos and body lotions ,only 2%, of shampoos were heavily contaminated by aerobic spore forming bacteria (bacilli) with more than 10 4 c.fu/gm or ml. while non of the others contained such a high number of bacteria. with regard to the medium range contamination levels; 5% of shampoos showed bacterial counts ranging from 10 2 to 10 3 c.f.u/gm or ml, compared to 2% of hand and body lotions which were contaminated to same level. coliforms were recovered from one sample of shampoos; staphylococcus were not recovered from any samples. one isolate of pseudomonas aeruginosa was also detected in a sample of shampoo. one isolate of shigella was also detected in a sample of shampoo. no fungal contamination was detected, as shown in table (6). the ph of all the tested samples was alkaline ph (8.2-9), which is well known to inhibit fungal contamination.bacterial contamination in unused cosmetic products is common because of the environment in which the products are manufactured, packed and the ingredients them selves. cosmetic ingredients are rich in nutrients and these provide organic substrates in the form of sugar, starch, protein, amino acid, organic acid, alcohol, amines, lipid and etc. for microbial growth. organisms such as pseudomonas putida possesses a mixed functions oxidase enzyme that enable them to utilizes substrates that many other organisms are unable to use. the ability of microorganisms to utilize substrates depends on their survival strategies. nutrients needed by organisms include nitrogen, sulphur, phosphorus and minerals. these materials, iraqi j pharm sci, vol.18(2) 2009 cosmetic products marketed in iraq 24 which are required for enzyme function and cellular osmoregulation are furnished as components of raw material or in water. water is a major ingredient in many cosmetic products and it has been the source of finished product contamination.malcom and woodroffe (3) reported that the most frequent contaminants of cosmetic products are pseudomonas, klebsiella, achromobacter and alcaligenes. they observed that these genera are common residents in water and contaminated water is likely source of the organisms found in contaminated cosmetic products. some microbes survive by forming endospores, biofilms, capsules, extracellular enzymes and by exhibiting acid tolerance. (14-18) our result is similar or like the report of okekre (19) , gram negative bacilli were seen in these studies, but unlike the report of hugo (20) .unlike the report of. (19), (20) , salmonella spp. was isolated in our study. and generally, microorganisms of interest in raw materials or cosmetic products grow best around neutral ph 7.0 and many yeasts and moulds are able to tolerate acid ph conditions. cosmetic ingredients supply nutrients for microbial growth. therefore, cosmetics should be produced in a perfectly clean hygienic environment. product premises, equipment, instruments, storage tanks and containers should accordingly be maintained in a high standard of cleanliness. in order to minimize contamination, cosmetic manufacturers should follow guidelines of good manufacturing practice (gmp). all starting materials should correspond to the agreed standards and be of consistently good quality (6) . ingredient listing is an important aspect of the labeling of cosmetic products. during the nances pharmaceutical control bureau cosmetics seminar 2002, one of the recpureinents discussed was labelling of cosmetic products (21) . table 5 : shows the microbial counts (c.f.u)/gm or ml) and types found in shampoos and body lotions. samples total no. of products contaminated products shampoo 15 10.8 hand and body lotion 15 8.4 shower bath soaps 15 zero facial cleaser 15 7.2 total 60 26.4 table 6 : microbial counts (c.f.u/ml or gm) and types found in shampoos and body lotions. shampoo counts types diagnoized 2% > 10 4 c.f.u/gm aerobic spore forming bacteria (bacilli) high level 5% 10 2 -10 3 c.f.u/gm aerobic spore forming bacteria (bacilli) medium level 1% 10 3 -10 4 c.f.u/gm 5.0x10 2 1.0x10 3 pseudomonas aeruginosa shigella spp. hand and body lotion 13x10 3 escherichia >10 4 c.f.u/gm 2% 10 2 -10 3 c.f.u/gm aerobic spore forming bacteria (bacilli) medium level 1% 10 3 -10 4 c.f.u/gm figure 1 : contamination based on product types. iraqi j pharm sci, vol.18(2) 2009 cosmetic products marketed in iraq 25 conclusion from this study it was found that bacterial contmination is more likely to occur than yeast and mould contamination. bacterial growth is favoured at neutral ph and most cosmetic products are at this range. microorganisms such as psudomonas, klebsiella, achromobacter and alcaligenes are the most frequently reported contaminants of cosmetic products. also, contamination is higher in shampoos than other products. this may be because they contain surfactants which are susceptible to contamination by water-borne gram-negative bacteria. cosmetic manufactures can prevent contamination by controlling raw materials, validating processes, instituting effective cleaning and sanitizing procedures, and training personnel. even low contamination does not necessarily mean that the manufacturers have followed the newly adopted eu regulation.there might be a possibility that the manufactuer used excessive preservatives in the product. a further study on preservatives will be carried out to detect the preservative level in cosmetic products marketed in iraq. references 1. orth, d s. handbook of cosmetic normax f e. the microbiology. (1989), marcel dekker. new york. 2. lundov, md and zacharian, c. recall of nicrobiologically contaminated cosmetics is eu from 2005 to may 2008. int. j. of cosmetic science, (2008), 30(6):471-474. 3. malcom, s and woodroffe, r. the susvial of bacteria in toiletries: inhibition and inactivation of vegetation microbes (skinnel, found hugo, weds). vol. 5 academic press, london. (1976), p.305314. 4. baird, r m. microbial contamination of cosmetic products. j. soc. cosmet. chem. (1974), 28: 17-20. 5. baird, r m. drugs and cosmetics. microbial biodeterioration (rose, a h ed.). academy press, london, (1981), p. 387-429. 6. russell, ad; hugo, wb; aylilfe, peter a. lambert. principles and practice of disinfection preservation and steriliaztion. academy pres london, (2004), p: 485487. 7. normam, fe. the cosmetic industry. scientific regulatory foundation. cosmetic science and tech. series, (1984). vol.2:21,301-320. 8. brunch, c w. possible modifications of usp microbial limits and tests. drug cosmet. ind., (1972), 110(6): 32-37, 116121. 9. ayliffe, g a j; barry, d r; lowbury. e j l; roper-hall, m j and walker, w m. postoperative infection with pseudomonas aeruginosa in an eye hospital. lancet, (1966), 1113-1117. 10. beveridge, e g. the microbial spoilage of pharmaceutical products. microbial aspects of deterioration of materials (lovelock, d w and / gilbert, r j eds.). academic press, london, (1975), p. 213235. 11. kedzia, w; lewon, j and wismienski, t. the breakdown of atripine by bacteria. j. pharma. phamacoal, (1961), 13: 614-619. 12. cox, p h and sewel, b s. the metabolism of steroids by cladosporium herbarum. j. soc. cosmet. chem., (1968), 19: 461467. 13. british phermacopoeaia. h. m. s. o, london appendix xvi ba 245, b. test for microbial contamination; (2004). 14. bos, ce, van doorne, hand derk, cf, microbiological stability tableto stored under tropical conditions inter. j. pharm.; (1989) 55:175-83. 15. barid rm and shooter ra. pseudomonas aerubine inflactions associated with the use of contaminated medicament. br medj. 16. myas ge and pasutto fm. microbiological contamin of cosmetics and toiletries. cem. j. pharm. sci. (1973), 19-23. 17. killings lo, ringerts o, silverton pe and ernenfell microbiological contamination of medical preparations. actapharm sci.(1966);219-22. 18. becks vanf lorenzoni: moisturizing creams and lotions distributed atropical developing country. jspp microbiol; (2001), 91: 922-928.n. pseudomonas aeruginosa out break in aneonatal intensium care unit. a possible link to contaminated hand lotion. amer jinf control; (1976); 2: 349-350. (1995), 23: 396-398. 19. okeke in and lomikanra a. bacteriobgied quality of skin 20. hugo pg, onyerwd: ao. microbial contamination and preservation capacity of sone brands of cosmetic creams. trop. j. of pharmaceutical research; (2003), 2: 229-234. 21. biro pengawalan farmaseutical kebangsaan. seminer on eu legislation for cosmetic products. 7 marcks(2002). kuala lumpar. iraqi j pharm sci, vol.31( 2 ) 2022 hepatic enzymes in major β-thalassemia doi: https://doi.org/10.31351/vol31iss2pp237-243 237 evaluation of hepatic enzymes in major β-thalassemic patients using deferasirox ahmed yahya ahmed dallal bashi* and fatimah haitham fathi**,1 *department of pharmacy, al-noor university college, mosul, iraq. **department of clinical laboratory science. college of pharmacy, university of mosul, mosul, iraq. abstract β-thalassemia major is a genetic disease that causes sever defect in normal hemoglobin synthesis. the patients with β-thalassemia major need periodic blood transfusions that can result in accumulation of body iron, so treatment with iron chelating agent is required. complications of this iron overload affecting many vital organs, including the liver. the aim of this work was to evaluate liver enzymes in β -thalassemia major patients with deferasirox versus without it. two groups of β-thalassemia major patients were involved in this study named group a; 40 β-thalassemia patients of blood transfusion dependent without deferasirox, group b; 40 β-thalassemia patients of blood transfusion dependent on deferasirox. in addition to group c, 40 normal subjects as a control group. samples of serum were obtained from all participants to be tested for alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase and ferritin. the biochemical data of the patients on blood transfusion without deferasirox showed significant increases in the mean serum levels of aminotransferases and ferritin in comparison with control. whereas the patients on blood transfusion with deferasirox exhibit significant increases in the means serum levels of alkaline phosphatase activity and ferritin in comparison with control. iron overload may cause liver injury, shown by significant increases of; alt and ast activities and elevated ferritin level in serum of transfusion dependent patients of β-thalassemia major. administration of deferasirox for βthalassemia major patients causes elevation of serum alp activity and ferritin level. key words: β-thalassemia, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, ferritin. الذين يستخدمون عقار من نوع بيتا الكبرى مرضى الثالسيميال الكبد يفةفحوصات وظ س الديفيراسيروك 1**،و فاطمة هيثم فتحي *احمد يحيى احمد دالل باشي قسم الصيدلة ، كلية النور الجامعة ، الموصل العراق * فرع العلوم المختبرية السريرية ، كلية الصيدلة ، جامعة الموصل ، موصل ، العراق ** الخالصة المتبررة إن األفراد المصيايين يعتمدنن لل لمليا ولل الدم . ن أالثالسييميا الببر ووع ييتا يخلل ف صصينيا الميموولويييتميز مرض ان المضييالتا الت صتعلب يالحديد الزا د ناارار الجاوبية . مما يؤدي ال زيادة الحديد ف الجسييم مما يسييتوجت اسييتعما دنا لحري الحديد الزا د السيييميا ووع ييتا ممن صمدف هذه الدراسيية لتلييم نئا ا الببد لد مر يي الث. للعالج يؤرران سييلبا لل العديد من األلضييا ف الجسييم نمنما الببد صشيمل الدراسية مجمولتان من مر ي الثالسييميا .نملاروتمم ما مر ي الثالسييميا ممن ي يسيتخدمون هذا العلار يسيتخدمون للار الديتيراسييرنك من يعتميدنن لل مريض م 40مريض ممن يعتميدنن لل النليل اليدنري لليدم، نالمجمولية أك صتبون من 40المجمولية أأ صتبون من . ووع ييتيا طتل من األصحا . صم جما لينا مصل 40ل مجمولة الضبط أج ن صتبون من إالنلل الدنري للدم نيتنانلون للار الديتيراسيرنك ، ياإل افة مصيل الدم. ائمر الدم لغرض قياس مسيتو فعالية إوزيم واقل أمين اييوين، نواقل أمين ايسيبارصي ، نالتوسيتاصيز اللالدي نمسيتو الحديدين ف كذلك وتا ح التحوصيا البيموييوية للمر ي الذين ي يسيتعملون للار الديتيراسييرنك زيادة معنوية ف معدي فعاليا إوزيما وواقل األمين ن اسييرنك ائمرنا زيادة الترصين ف مصيل الدم ملاروة يمجمولة الضيبط. يينما المر ي المعتمدين لل النلل الدنري للدم نيسيتعملون للار الديتير ود معنوية يمعد مسيتو فعالية التوسيتاصيز اللالدي نمسيتو الحديدين ف مصيل الدم ملاروة يمجمولة الضيبط. يسيتنتت من وتا ت هذه الدراسية نج سبارصي نكذلك زيادة الترصين ا حرايا ئاهرية ف نئا ا الببد نذلك يسبت زيادة مستويا فعالية إوزيم واقل أمين اييوين ن إوزيم واقل أمين اي نك ف مصيل الدم لد مر ي الثالسييميا الببر ووع ييتا المعتمدين لل النلل الدنري للدم. من جمة ا ر فلد اد اسيتعما للار الديتيراسيير سييتاصيز اللالدي نال زيادة فرصين مصييل لمر يي الثالسيييميا الببر ووع ييتا المعتمدين لل النلل الدنري للدم ال زيادة مسييتو فعالية إوزيم التو الدم. .بيتا ثالسيميا، إنزيم ناقل أمين االالنين، إنزيم ناقل أمين االسبارتيت، إنزيم الفوسفاتيز القاعدي، الفرتينالكلمات المفتاحية: introduction thalassemias are genetic disorders of hemoglobin synthesis where the production of normal hemoglobin is partly or completely suppressed. this suppression may be due to a defective synthesis of any globin chains. many forms thalassemia have been discovered and called by the defected globin chain 1corresponding author e-mail: ahmed.yahya50@alnoor.edu.iq received: 13/10 /2021 accepted: 26/1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp237-243 iraqi j pharm sci, vol.31(2) 2022 hepatic enzymes in major β-thalassemia 238 the common thalassemia of clinical interest are α and β-thalassemia(1). normally hemoglobin (hb) is made up of two α-globin and two β-globin chains (alpha2 beta2). β-thalassemia forms are hereditary diseases of blood in which reduced or absent βglobin chain production of hemoglobin (hb) tetramer (β+) or (β0) respectively, resulting in diminutive normal hb in red blood cells (rbc), diminutive production of rbc and then anemia(2) the formation of normal α-globin chains in β-thalassemia patients continues as normal, that result in accumulation of unmatched α-globin in the erythroid precursors. this increased free α-globin chains cannot synthesis tetramers of hb, instead precipitate in the bone marrow forming inclusion bodies that causes intramedullary destruction of erythroid precursors resulting in ineffective erythropoiesis characterized in all forms of βthalassemias(3). β-thalassemia major patients require repeated blood transfusion for normal life, patients on blood transfusion always have iron overload as a result to the periodic blood transfusion and ineffective erythropoiesis(4). the adverse effects of iron overload may be seen on certain vital organs like the liver, endocrine glands, heart, kidneys and lungs(5). iron over load can cause increased cellular accumulation of labile part of iron in certain parenchymal tissues, including the abovementioned organs causing iron toxicity. iron toxicity can cause production of reactive oxygen species (ros), such as free radicals where, it was proved that labile iron is the key mediator of the toxicity(6). deferasirox is the most recent drug used as an oral chelator that moves iron from stores by binding to the ferric atom (fe+3) of iron(7). the main metabolic pathway for this chelating agent is through glucuronidation and biliary excretion(7). the hepatic cells are the major tissues of iron storage so when a case of iron overload is present, it is regarded as the most important target for the therapy with deferasirox(8). the well-known complications that appear in thalassemic patients on deferasirox drug are the transient elevation of serum liver transaminases activities and serum creatinine(9). materials and methods patients and control selection: eighty β-thalassemic major patients all were dependent on blood transfusion with age ranged from 6-60 months who were presented to ibn-alatheer teaching hospital/ department of thalassemia in mosul city/ iraq were participated in this study since 1st of october 2011 to the 30th of march 2012. the enrolled participants were diagnosed as patients with β-thalassemia major according to hemoglobin variants using hemoglobin electrophoresis. those patients were classified into two groups: group a consisted of 40 patients depend on blood transfusion that were not received deferasirox as chelating therapy. their ages range from 6-60 months (with a mean of 21.12 months). group b consisted of 40 patients depend on blood transfusion that were treated with 10-30 mg/kg body weight of the chelating agent deferasirox daily. their ages range from 21-60 months (with a mean of 40.68 months). group c apparently healthy 40 normal subjects, with unknown diseases and not received any chronic therapy participate in this study. their ages range from 6-60 months (with a mean of 25.2 months). a written informed consent of the study details was obtained from each of the participant’s parents. the ethical approved on the study design and investigations was obtained from the local mosul medical college ethical committee. about 5 ml of venous blood was obtained from each child of the three studied groups in plain tubes, left 15 minutes at room temperature to form clot, followed by centrifugation to separate serum, that were then feezed deeply at -20 c°. the biochemical measurements were carried at the laboratory of ibn-alatheer teaching hospital in mosul / iraq.serum aminotransferases (ast and alt) activities were determined calorimetrically according to (wooton and freeman, 1982) by using a kit obtained from biomeriux company (france). serum alkaline phosphatase (alp) activity was determined by colorimetric reaction method, using a kit method supplied by (biolabo, france). serum ferritin was determined by an enzyme liked assay method(10), by using a kit obtained from biomerieux (france). statistical analysis the mean, standard deviation (sd), unpaired t-test, fisher freeman halton test, anova test were used as standard statistical analysis methods for analyzing the data of this work(11). the results were considered statistically significant when p≤0.05(12). results and discussion the comparison of serum alt activity in transfusion dependent β-thalassemia patients (group a) with controls (group c) showed a significant increase in the mean of serum alt activity in group a. while the comparison of transfusion dependent β-thalassemia patients using deferasirox (group b) with group c showed no significant increase in the mean of serum alt activity (tables 1 and 2 respectively). on comparing serum ast activity in group a with group c, there was a significant increase in group a, while in group b, there was no a significant difference in the mean of serum ast activity in iraqi j pharm sci, vol.31(2) 2022 hepatic enzymes in major β-thalassemia 239 comparison with that of group c (tables 1 and 2 respectively). table1. differences in serum alt, ast, alp and ferritin between group a and c. parameters mean ± sd group a group c alt activity (iu/l) 54.12±39.28 17.00±9.83 *p-value <0.001 ast activity (iu/l) 57.82±31.71 19.700±13.41 *p-value <0.001 alp activity (iu/l) 134.39±27.65 114.23±21.64 p-value > 0.05 (ns) ferritin (ng/ml) 1763.35±1285.44 43.6 ±18.85 *p-value <0.001 * significant difference between groups exists at p≤0.05. ns: not significant. anova (one away analysis of variance) test was used to compare the results of various parameters between thalassemic patients with the controls. table2. differences in serum alt, ast, alp and ferritin between group b and c. parameters mean ± sd group b group c alt activity (iu/l) 28.65±19.88 17±9.83 p-value > 0.05 (ns) ast activity (iu/l) 20.65±12.6 19.7±13.41 p-value > 0.05 (ns) alp activity (iu/l) 148.27±62.04 114.23±21.64 *p-value <0.001 ferritin(ng/ml) 2622.92±843.89 43.6 ±18.85 *p-value <0.001 * significant difference between groups exists at p≤0.05. ns: not significant. anova (one away analysis of variance) test was used to compare the results of various parameters between thalassemic patients with the controls. the mean of serum alp activity showed no significant increase in group a in comparison with group c, while in group b, there was a significant increase in the mean of serum alp activity (tables 1 and 2 respectively). the mean of serum ferritin showed a significant increase in both groups of patients (groups a and b) from that of group c (tables 1and 2 respectively). the results of this work showed significant differences in the serum of alt and ast activities between transfusion dependent β-thalassemia patients (group a) and controls (group c) (table1). iron-induced oxidative stress is known to be one of the most important factors causing liver cell injury in thalassemic patients (patpan et al.)(13), leading to increased liver enzyme activities(14). the results of this work are in accordance with that of another study (patpan et al.)(13) where, it was found that significant elevations in serum; ferritin, ast, alt activities in β-thalassemia major ( βtm) patients on blood transfusion group when compared with their controls. the results of this study also agree with other investigators (sonali et al.; setoodeh et al) (15,16). however, periodic blood transfusions is necessary as life-saving and improving the quality of life for the patients with βtm, even it causes excessive iron overload, that was regarded as an important clinical complication of the treatment (17). in the present study, the results of group b showed no significant increase in the mean of serum alt and ast activities in comparison to group c (table2). this can be attributed to the fact that deferasirox therapy reduces hepatocellular inflammation and improves liver functions that may be linked to the reduction in liver iron concentration and serum ferritin(18). in the present study, the results concerning the mean serum alt and ast activities that compared according to different age ranges for each of group a and group b patients, no significant changes were observed (table3 and 4 respectively). this may indicate that age did not adversely affect the liver functions. soliman et al. (2014)(19) reported that the variations in alt and ast activities in βtm patients who are under a regular treatment with deferoxamine were not correlated with the age of the patients. https://journals.sagepub.com/doi/full/10.1177/1179066018823534 https://journals.sagepub.com/doi/full/10.1177/1179066018823534 https://pubmed.ncbi.nlm.nih.gov/?term=setoodeh+s&cauthor_id=33520802 iraqi j pharm sci, vol.31(2) 2022 hepatic enzymes in major β-thalassemia 240 table3. effect of age on serum alt, ast, alp and ferritin in group a. parameters mean ± sd p-value < 2 years(n=20) ≥ 2 years(n=20) alt activity (iu/l) 54.80±40.04 53.45±39.54 p-value > 0.05 (ns) ast activity (iu/l) 56.2±28.86 59.45±35.01 p-value > 0.05 (ns) alp activity (iu/l) 114.8±16.03 153.99±22.46 *p-value <0.001 ferritin (ng/ml) 1212.1±427.73 2314.6±1602.31 *p-value <0.001 * significant difference between groups exists at p≤0.05. ns: not significant. anova (one away analysis of variance) test was used to compare the results of various parameters between thalassemic patients with the controls. table4. effect of age on serum alt, ast, alp and ferritin in group b. parameters mean ± sd p-value <3years (n=22) ≥ 3years (n=18) alt activity (iu/l) 29.72±20.66 27.33±19.41 p-value > 0.05 (ns) ast activity (iu/l) 24.13±14.4 16.38±8.58 p-value > 0.05 (ns) alp activity (iu/l) 136.44±40.59 162.74±79.95 *p-value <0.001 ferritin (ng/ml) 2450.09±839.09 2834.16±823.47 *p-value <0.05 * significant difference between groups exists at p≤0.05. ns: not significant. in the present study, the mean serum alt activity showed a significant increase according to increased ferritin ranges for group a only (table5 and 6 respectively). this result means that alt activity reflecting liver cell injuries due to toxic iron overload. table5. effect of ferritin levels on serum alt, ast and alp in group a. parameters mean ± sd p-value <1000 (n=9) 1000-2000 (n=22) >2000 (n=9) alt activity (iu/l) 33±17.05 56.09±37.93 70.44±51.42 *p-value <0.05 ast activity (iu/l) 49.33±18.1 54.68±30.57 74±41.45 p-value > 0.05 (ns) alp activity (iu/l) 116.58±25 131.84±19.76 158.45±32 *p-value <0.05 * significant difference between groups exists at p≤0.05. ns: not significant. table6. effect of ferritin levels on serum alt, ast and alp in group b. parameters mean ± sd p-value ≤ 2500 (n=20) >2500 (n=20) alt activity (iu/l) 24.45 ±16.37 32.85 ±22.5 p-value > 0.05 (ns) ast activity (iu/l) 18 ±11.81 23.3 ±13.11 p-value > 0.05 (ns) alp activity (iu/l) 152.15 ±41.67 144.4 ±78.31 p-value > 0.05 (ns) * significant difference between groups exists at p≤0.05. ns: not significant. iraqi j pharm sci, vol.31(2) 2022 hepatic enzymes in major β-thalassemia 241 the data of this work showed that the mean level of serum alp activity in group a is not significantly higher than in group c (table1) and this is in accordance with the results of younus and bashi, (2012)(20) who reported that the alkaline phosphatase activity was not significantly increased in the patients with βtm when compared with that of their control group. in the present study alp activity is significantly higher in group b in comparison to group c (table2). these results may be accounted to be due to that serum alp activity may originate from the liver, bone, intestine and placenta, in addition to the elevation of serum activity that result from hepatobiliary and nonhepatic (bone disease and childhood growth) causes of elevated serum alp activity(21). osteoporosis may result due to chronic request for blood cell production by over stimulation of the hematopoietic system that causes an increase in the number of osteoclasts and osteoblasts, leading to accelerated bone turnover and increase serum alp activity(22). bone demineralization which commonly occurs in β-thalassemia patients also causes an elevation in the serum alp activity. moreover, the results of the present study were in agreement with those of baldini et al. (2010)(23) where they studied the level of serum alkaline phosphatase activity in adult caucasian β-thalassemic who were on deferasirox therapy. the result of the present study showed a significant increase of serum alp activity according to age ranges for both groups a and b (table3 and 4 respectively) and for different serum ferritin ranges for group a only (table5 and 6 respectively). these results may be accounted due to iron overload itself, as it is known that it cause increase liver enzyme activities(14). in addition, the toxicity of iron on the bone lead to the bone demineralization and so increased serum alp activity. the results of the present study showed a significant increase in the mean level of serum ferritin (major physiological role is to store iron) in patient’s groups in comparison with the control group (table1 and 2 respectively) (iron overload is usually manifested by a high serum ferritin levels(5)). a significant increase in the mean level of serum ferritin also was seen in patient’s subgroups according to age (table3 and 4 respectively). there are many mechanisms to absorb or store or to transfer iron, but no mechanism to excrete iron outside the body. so it is important to find a way to get rid of excess iron that are accumulated due to periodic blood transfusion in βtm patients where it is found that every unit of blood contains about 200-250 mg of iron(24). another source of iron may exist in some βtm patients as more iron is absorbed from the diet as a response to ineffective erythropoiesis (25). the results of this study showed that the mean of serum ferritin levels in patients of group b was significantly higher than that of group a despite using deferasirox (table7). table7.differences in serum alt, ast, alp and ferritin between group a and b. parameters mean ± sd group a group b alt activity (iu∕ l) 54.1250±39.28 28.65±19.88 *p-value <0.05 ast activity (iu∕ l) 57.82±31.71 20.65±12.6 *p-value <0.001 alp activity (iu∕ l) 134.39±27.65 148.27±62.04 *p-value <0.001 ferritin (ng∕ ml) 1763.35±1285.14 2622.92±843.89 *p-value <0.01 * significant difference between groups exists at p≤0.05. ns: not significant. anova (one away analysis of variance) test was used to compare the results of various parameters between thalassemic patients with the controls this may be due to the short duration of administration of deferasirox which does not exceed 6 months or may be due to those patients in group b were older than that of group a (mean of age 21.12 and 40.68 months respectively. the results of the present work are in accordance with that of other investigators (26,27) where they reported, evidence of iron overload manifested as elevated serum ferritin levels in all patients with βtm whether were using or not using chelating agents. they proved that the serum ferritin level is in correlation with age. conclusion iron overload may cause liver injury. this is reflected by significant elevation of serum; alt and ast activities and 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15964 26. aboobacker f n, dixit g, lakshmi k m, korula,a, abraham a, george b, mathews v. outcome of iron reduction therapy in exthalassemics. plos one 2021; https:// doi. org/ 10.1371/ journal.pone.0238793 27. sobhani s, rahmani f, rahmani m, askari m, kompani f. serum ferritin levels and irregular use of iron chelators predict liver iron load in patients with major beta thalassemia: a crosssectional study. croat med 2019; 31;60(5):405413. doi: 10.3325/cmj.2019.60.405. this work is licensed under a creative commons attribution 4.0 international license. https://www.ncbi.nlm.nih.gov/pubmed/?term=turan%20s%5bauthor%5d&cauthor=true&cauthor_uid=21448327 https://www.ncbi.nlm.nih.gov/pubmed/?term=topcu%20b%5bauthor%5d&cauthor=true&cauthor_uid=21448327 https://www.ncbi.nlm.nih.gov/pubmed/?term=g%26%23x000f6%3bk%26%23x000e7%3be%20i%5bauthor%5d&cauthor=true&cauthor_uid=21448327 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3065317/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3065317/ 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https://pubmed.ncbi.nlm.nih.gov/?term=sobhani+s&cauthor_id=31686454 https://pubmed.ncbi.nlm.nih.gov/?term=rahmani+f&cauthor_id=31686454 https://pubmed.ncbi.nlm.nih.gov/?term=rahmani+m&cauthor_id=31686454 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 cht1 as a novel biomarker in nc doi: https://doi.org/10.31351/vol30iss1pp270-276 270 serum chitotriosidase level as a novel biomarker for therapeutic monitoring of nephropathic cystinosis among the iraqi children zainab a. al-kinani *,1 and shatha h. ali ** * ministry of health and environment, maysan , iraq. department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. abstract cystinosis is a rare autosomal recessive lysosomal storage disease with high morbidity and mortality. it is caused by mutations in the ctns gene that encodes the cystine transporter, cystinosin, which leads to lysosomal cystine accumulation. it is the major cause of inherited fanconi syndrome, and should be suspected in young children with failure to thrive and signs of renal proximal tubular damage. infants diagnosis can be missed since not all signs of renal fanconi syndrome are presented during the first months of life. elevated leukocytes cystine content is the cornerstone for the diagnosis of cystinosis. whereas, chitotriosidase (chit1 or chitinase-1) is primarily produced by activated macrophages. hence, inflammatory conditions including cystinosis could be associated with increased plasma chitotriosidase activity. this study is aimed to estimate serum chitotriosidase level, as a marker for screening as well as for therapeutic monitor for cystinosis disease, as the measurement of leukocyte cystine content is not available for iraqi children with cystinosis. the present study is a case-control study that included samples of 30 children with nephropathic cystinosis, compared to 25 healthy control children from those attending at the genetic rare diseases center / al-emamain al-kadhimain teaching hospital, baghdad-iraq. cystinotic children included in the study were those aged equal to less than 10 years, and diagnosed to have cystinosis according to clinical symptoms of the disease and eye examination; demonstrating corneal cystine crystals. blood specimens were obtained for separating white blood cell (wbc) for measuring their cystine content, within afew hours, by applying highperformance liquid chromatography (hplc). while aliquots of serum were utilized for analysis of chitotriosidase (elisa), creatinine and calcium concentrations. also, urine specimens were collected using urine cups from each participant for measuring glucose in the urine. the results reported that cystinotic patients had leukocyte-cystine content ranged between (0.5 to 6.2 nmol 1/2 cystine/mg protein), compared to age-matched healthy children which ranged between (0.07 to 0.32 nmol 1/2 cystine/mg protein). the patients also had abnormally higher levels of serum chitotriosidase ranged between (90 to 565 nmol/hr/ml) while for the control group ranged between (35 to 153 nmol/hr/ml). besides a significant associated with leukocyte-cystine content for cystinotic patients (r=0.957, p<0.001). estimation of serum chitotriosidase activity might aid in monitoring the therapeutic benefits of cysteamine therapy, as well as the prognosis of the disease when wbc cystine assessment is not available. keywords: cystinosis, chitotriosidase. لدى الكلوي يالسيستين لداء العالجي للرصد جديد حيوي كمؤشرالكايتوترايوسيديز في المصل مستوى العراقيين األطفال **و شذى حسين علي 1*،الكناني زينب احمد وزارة الصحة والبيئة ، ميسان ، العراق .* .الصيدلة، جامعة بغدادفرع العلوم المختبرية السريرية، كلية ** الخالصة المرض هو طفرات في جين لهذا الرئيسي والمراضة. السبب الوفيات معدل ارتفاع يسبب متنحي وراثي مرض هو داءالسيستيني؛ (ctns) .فانكوني لمتالزمة الرئيسي السبب إنهالمسؤول عن تشفير ناقل السيستين، السيستينوسين، يؤدي الى تراكم السيستين داخل الاليسوسوم الرضع، التشخيص تفويت يمكن. القريب الكلوي األنبوب تلف وعالمات النمو فشل من يعانون الذين الصغار األطفال عند به االشتباه الموروثة ويجب حجر هو البيضاء الدم خاليا في السيستين محتوى ارتفاع .الحياة من األولى األشهر خالل الكلوية فانكوني متالزمة عالمات جميع توجد ال ألنه تترافق أن يمكن وبالتالي، .النشطة بالعمال طريق عن أساسي بشكل إنتاجه يتم( 1-كيتينيز) زييدسيوايتوتراالك ألن نظًرا. التشخيص في ساساال إنزيم مستوى تقدير إلى الدراسة هذه تهدف. البالزما في كيتوتريوزيداز نشاط إنزيم زيادة مع السيستيني داء ذلك في بما االلتهابية الحاالت متوفر غير السيستين البيض الكريات محتوى قياس أن حيث ،يالسيستين داء لمرض عالجي ومراقب فحص كعالمة الدم في زييدسيوايتوتراالك السيستيني. بداء المصابين العراقيين لألطفال 1corresponding author e-mail: zainab.pharmacy2019@gamil.com received: 27/11/2020 accepted: 13/2 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp270-276 iraqi j pharm sci, vol.30(1) 2021 cht1 as a novel biomarker in nc 271 طفاًل 25 بـ مقارنة الكلوي، يالسيستين بداء مصابًا طفالً 30 من عينات ت والشواهد تضمنتحاالال دراسة عن عبارة الحالية الدراسة كان .العراق بغداد، التعليمي، الكاظمين يناإلمام مستشفى/ النادرة الوراثية األمراض مركز في يحضرون الذين أولئك من جيدة بصحة يتمتعون بداء إصابتهم تشخيص وتم أقل أو سنوات 10 أعمارهم تبلغ الذين أولئك هم الدراسة في المشمولين السيستيني مرض من يعانون الذين األطفال الدم خاليا لفصل الدم عينات على الحصول تم. السيستين في القرنية بلوراتالعين، ظهور وفحص للمرض السريرية لألعراض وفقًا السيستيني استخدمت . بينماhplc)) األداء عالية سائلة كروماتوجرافيا تطبيق طريق عن قليلة، ساعات غضون في السيستين، من محتواها لقياس البيضاء لقياس مشارك كل من البول أكواب باستخدام البول عينات جمع تم كما والكالسيوم.الكرياتينين تراكيزالكيتوتريوسيديز، لتحليل المصل من قسامات البول. في الجلوكوز نسبة بروتين(، ملغ/ سيستين 1/2 نانومول 6.2 إلى 0.5بين ) يتراوح البيض الكريات محتوى لديهم داء السيتيني كان مرضى أن بينت النتائج لدى كان. بروتين( ملغ/ سيستين 1/2 نانومول 0.32 إلى 0.07بين ) أعمارهم تراوحت والذين العمر مع المتطابقين األصحاء باألطفال مقارنة تراوحت مل( بينما/ ساعة/ نانومول 565الى 90بين ) تراوحت الدم في الكيتوتريوسيديز من طبيعي غير بشكل أعلى مستويات أيًضا المرضى داء السيستيني لمرضى السيستين البيض الكريات بمحتوى كبير ارتباط جانب إلى. مل(/ ساعة/ نانومول 153 إلى 35بين ) الضابطة المجموعة (r =0.957 > p 0.001.) يتوفر ال عندما بالمرض والتشخيص بالسيستامين للعالج العالجية الفائدة مراقبة فيالكايتوترايوسيديز في المصل نشاط تقدير يساعد قد في كريات الدم البيضاء. نيالسيستي تقييم المفتاحية: داء السيستيني،الكايتوترايوسيديز. الكلمات introduction cystinosis is a rare autosomal recessive lysosomal storage disease with high morbidity and mortality. it is caused by mutations in the ctns gene that encodes the cystine transporter, cystinosin, which leads to lysosomal cystine accumulation(1). three clinical forms of cystinosis can be distinguished depending on the age at presentation and the degree of disease severity. infantile nephropathic form: also known as renal fanconi syndrome, the most frequent and most severe form of the disease. patients are generally present before the age of 12 months with polyuria, polydipsia, and failure to thrive, caused by generalized proximal tubular damage(2). early symptoms of classical nephropathic cystinosis include renal tubular fanconi syndrome, rickets, impaired growth, hypothyroidism, and photophobia(3). however, cystine accumulation continued in non-renal organs, including the muscle, brain, bone marrow, liver, spleen, lymph nodes, cornea, conjunctiva, thyroid, pancreas, testes, and intestines(4). consequently, the clinical course of cystinosis changed from that of a largely renal disease to that of a multisystemic , including a distal vacuolar myopathy, decreased pulmonary function, swallowing impairment, deterioration of the central nervous system (cns), endocrinopathies, vascular calcifications, retinal damage, and other ophthalmic complications(5). definitive diagnosis is based upon a high index of suspicion because of the clinical presentation including: polyuria, thirst, failure to thrive, growth retardation, vomiting, periods of dehydration, constipation, developmental delay, and rickets in some patients(6). the patients are usually presented with hypokalemia, hypophosphatemia, metabolic acidosis, low serum uric acid, low carnitine, and, sometimes, hyponatremia. proteinuria can reach grams per day, and consists of low molecular proteins, albumin, and high molecular weight proteins(7)supported by slit lamp examination of the corneas showing crystals, which are generally present by 16 months of age (8). the detection of elevated intracellular cystine content is the cornerstone for the diagnosis. the methods for cystine determination differ depending on the cell type: mixed leukocyte preparation or polymorphonuclear (pmn) leukocytes(9). furthermore, several biochemical methods are currently used for cystine measurements, such as a cystine binding assay, amino acid chromatography or high performance liquid chromatography, making it difficult to compare the results of different laboratories(10) nationwide birth prevalence data concerning cystinosis are only reported in few populations, it is estimated at 1-9 per 1,000,000(11). however, the prevalence of cystinosis in iraq about is 163 patients according to data collected from the genetic rare diseases center/al-emamain alkadhimain teaching hospital, baghdad-iraq. the incidence of cystinosis is estimated as 1 in 100,000 –200,000 live births per year(12). cysteamine orally is the only specific targeted therapy available for managing cystinosis, as it act as cystine depleting therapy and mostly needs to be combined with cysteamine eye drops when corneal disease is involved(13). subjects and methods the present study is a case-control study included thirty child were diagnosed to have cystinosis from those attending at the genetic rare diseases center / al-emamain al-kadhimain teaching hospital, baghdad-iraq. this study was approved by the ethics committee of the college of pharmacy/university of baghdad. all participants were informed about the aim and the proposed benefits of the study before obtained their agreements. twenty-five age & sex matching apparently healthy individuals were included to serve as a control group. cystinotic children included in the study were those aged equal or less than 10 years, and diagnosed to have cystinosis according to clinical symptoms of iraqi j pharm sci, vol.30(1) 2021 cht1 as a novel biomarker in nc 272 disease and eye examination ; demonstrating corneal cystine crystals(14). sample collection and preparation from each participant ( patient and control subjects), venous blood sample were collected, but for cystinosis patients the blood venous blood samples were collected after at least 6 hours from taking treatment (cysteamine). six milliliters was withdrawn, about (2 ml) was transferred to a tube containing lithium heparin and stored at (2-8 oc, less than 24 hours) to be taken it to the laboratory (asco learning center in al-harthia city) for separation of white blood cell (wbc)(15). after that, the separated leukocytes were taken within less than 24 hours to be assessed at the ministry of science and technology / department of environment and water laboratories for measuring their cystine content by applying high performance liquid chromatography(16) (hplc system, sykamngermany). the other part (4 ml) of the blood sample was transferred to a plane tube for 30 minutes to clot and then centrifuged at (3000 rpm) for 5 minutes to obtain serum, which is used for measuring the level of creatinine(17)and calcium(18)(by taking precaution during blood sampling since the use of tourniquet might affect serum calcium level). the remaining aliquot of serum was kept in eppendorff tubes and frozen at (-20oc) for later analysis of serum chitotriosidase concentration using double sandwich elisa kit(19). glumerular filteration rate gfr was measured according to schwartz equation (20). also, urine specimens were collected using urine cups from each participant for measuring glucose in urine.notebly that the laboratory cystine assay procedures were blinded for the analyzer (regarding the sample was for a control or a patient) of the participants, i.e. samples were assayed in a random order. because, the lack of blinding could introduce bias into the assessment of subjective outcomes such as health-related quality of life and adverse events. additionally , it’s the first time to to report the measurement of wbccystine content for cystinotic patients in iraq. statistical analysis the analyses were conducted using the statistical package for the social science (spss, version 22, ibm, new york, usa). descriptive statistics (means, standards deviations, frequencies and percentages) of the participants (both patient and control group) were calculated. because the variables were not normally distributed (cystine & chitotriosidase), we used non-parametric tests including mann-whitney (between 2 groups) tests to measure the difference in multiple measures according to participating groups (patient vs control). spearman correlation was used to measure the relationships among different measures in patient group. a p-value of less than 0.05 was considered to be significant statistically. results patients and control group demographics and disease characteristics as shown in the table (1), the participating patients aged between 1.5 and 10 years (18 and 120 months) with a mean age of (65.20 ± 34.27) months about 5.4 years. the thirty cystinotic patients, included (17 male) and (13 female) representing 56.7% and 43.3% respectively. while, the control group had a comparable age to patients group with a mean age of (62.32 ± 35.45) months about 5.2 years and ranged between one and 10 years old (12 and 120 months). twenty-five control group included 13 male participants (52.0%) and 12 (48.0%) female. the patients group had lower weight, height, body mass index (table-1), gfr, and serum calcium (table-2) measures compared to the control group. on the other hand, the control children had significantly lower levels of serum creatinine, cystine, and chitotriosidase levels compared to the patients' group (figure-1). table 1. patient and control groups demographics n for patients=30, n for control=25, sd=standard deviations, m=male, f=female, bmi= body mass index, esrd=end stage renal disease characteristic patient group (mean± sd) patient group median control group (mean± sd) control group median age (months) 65.20 ± 34.27 66 62.32 ± 35.45 60 gender (m/f) (17/13) (13/12) weight (kg) 11.20 ± 3.00 10.5 22.28 ± 10.71 20 height (cm) 85.84 ± 11.88 85.5 108.56 ± 18.98 112 bmi kg/m2 14.68 ± 2.36 14.9 17.71 ± 2.96 17 age at diagnosis (months) 25.60 ± 20.88 18 disease duration (months) 39.53 ± 33.24 39 treatment duration (months) 19.93 ± 28.89 7 age at treatment started (months) 45.27 ± 31.04 36 age at reached esrd (month) 81.7 ± 28.26 81.5 iraqi j pharm sci, vol.30(1) 2021 cht1 as a novel biomarker in nc 273 table 2. studied biomarkers among patients and control gfr=glomerular filtration rate, normal s.cr range is 26 to 62 μmoles/liter, , normal cystine level (mixed leukocytes test) is less than 0.2 nm/mg protein, normal chitotriosidase level is < 78.5 nmol/hr/ml, normal s.calcium range is 2.2-2.6 mmol/l. the difference between patients and control groups in multiple demographics and disease measures in the table (3), there were non-significant differences between control and patients' groups in age (p-value > 0.05). there was a significant difference in mean rank (because most variables were not normally distributed, thus, mean ranked replaced the mean) for weight, bmi, serum creatinine, gfr, cystine, and chitotriosidase level of whole (30) cystinosis patients when compared with control (25) mean rank (p-value = 0.0001 < 0.05) as illustrated in figures (1). while serum calcium measures also showed significant differences between control and patient groups but with (p-value = 0.004 < 0.05) as shown in figure (1). table 3. the difference between patient and control groups. control group (mean rank) n=25 patient group (mean rank) n=30 p-value age (months) 27.18 28.68 .728 weight (kg) 39.02 18.82 .0001* bmi (kg/m2) 37.14 20.38 .0001* s. creatinine (µmole/l) 17.90 36.42 .0001* egfr (ml/min/1.73 m2) 40.98 17.18 .0001* s. calcium (mmol/l) 34.72 22.40 .004* wbc cystine level (nmol\mg) 13.00 40.50 .0001* s.chitotriosidase (nmol/hr/ml) 14.56 39.20 .0001* *significant (p-value <0.05) according to the mann-whitney test. figure 1.comparison between different parameters of control and patients *significant (p-value <0.05) difference between mean rank characteristic patient group (mean± sd) patient group median control group (mean± sd) control group median s. creatinine (µmol/l) 179.76 ± 177.53 99 38.86 ± 14.16 37 gfr (ml/min/1.73 m2) 44.09 ± 37.64 31.4 110.13 ± 12.16 109.5 wbc cystine(nmol/mg protein) 2.98 ±1.80 2.6 0.20 ± 0.06 0.18 s.chitotriosidase(nmol/hr/ml) 308.30 ± 134.79 293 115.96 ± 32.44 127 s. calcium (mmol/l) 1.80 ± 0.55 1.9 2.15 ± 0.20 2.1 iraqi j pharm sci, vol.30(1) 2021 cht1 as a novel biomarker in nc 274 correlations of cystine & chitotriosidase levels with each other and with other measures: as presented in table (4) both cystine and chitotriosidase had significant positive correlations with each other’s (r=0.957 p-value=0.000). meanwhile, these two parameters (cystine and chitotriosidase) also had significant positive correlations with serum creatinine level (r=0.895, r=0.927 p-value=0.000). on the other hand, cystine & chitotriosidase have significant negative correlations with serum calcium levels (r=-0.656, r= -0.612 p-value=0.000, respectively). furthermore, cystine & chitotriosidase correlates negatively with gfr (r=0.890, r= -0.917 p-value=0.000 respectively). also there is significant negative correlations between cystine level & bmi (r= -.426 p-value=0.019) as mentioned in table (4). table 4. spearman's correlations among patient demographics and disease characteristics ** correlation is significant at the 0.01 level (2-tailed) according to spearman's correlation. * correlation is significant at the 0.05 level (2-tailed). patient n=30 discussion as presented in table (1), the measured children height and weight for those aged (≤10 years) for the cystinotic patients, as the (mean ±sd) height was (85.84 ±11.88) cm and the (mean ±sd) weight (11.20 ±3.00) kg, compared to that of healthy children of the same age (108.56 ±18.98) cm and (22.28 ±10.71) kg, respectively. furthermore, the results also indicated that gfr and serum creatinine levels were statistically different between the patient and the control groups as in table (3). as the average glomerular filtration rate (gfr) of the patients was lower than that of the control group, while the average serum creatinine level was higher than of the control group owing to the progressive loss of the function of proximal tubular transporters in nephropathic cystinosis (nc) because of the accumulation of cystine, causing fanconi syndrome. the glomerular involvement progress with an increase in plasma creatinine level and a decrease in the (gfr) if no specified treatment is administered, which evolves to advanced chronic kidney disease (ckd) that it is likely because of interstitial precipitates of cystine crystals(21). the cystine wbc content of cystinosis patients was much higher than that of the control group. since the cystinosis disease is resulted by (ctns) gene mutations that encode the cystinosin (cystine transporter), which give rise to cystine accumulation in the lysosome. cystine levels increase up to 100-fold in affected individuals compared with control subjects(22). meanwhile, at diagnosis of cystinotic patients were observed the serum chitotriosidase activity was elevated significantly over the agematched healthy children and associated with leukocyte cystine level for patients. therefore, in patients with nephropathic cystinosis, chitotriosidase is considered a therapeutic monitor and a promised clinical biomarker. although the chitotriosidase enzyme is not specific for this disease, but it may aid in monitoring a patient’s response to therapy(23). chitotriosidase is associated with activated phagocytes; its expression and release are induced by lysosomal stress. thus, lysosomal stress can be present in cystinotic cells, as a result of cystine accumulation in cystinosis cells(24). meanwhile, serum calcium levels in cystinosis children were significantly lower than the control group despite that most of patients administered calcium supplements (table-3 or figure-1), this result is consistent with theodoropoulos et al. study , since the natural history of nephropathic cystinosis includes tubular reabsorption defects beginning in the 1st year of life in which proximal tubular cells fail to reabsorb parameters cystine chitotriosidase age correlation coefficient .385* .449* sig.(2 tailed) .036 .013 weight correlation coefficient .079 .200 sig. (2-tailed) .676 .289 bmi correlation coefficient -.426* -.281 sig. (2-tailed) .019 .132 s. creatinine correlation coefficient .895** .927** sig. (2-tailed) .000 .000 gfr correlation coefficient -.890** -.917** sig. (2-tailed) .000 .000 s. calcium correlation coefficient -.656** -.612** sig. (2-tailed) .000 .000 cystine correlation coefficient 1.000 .957** sig. (2-tailed) . .000 chitotriosidase correlation coefficient .957** 1.000 sig. (2-tailed) .000 . iraqi j pharm sci, vol.30(1) 2021 cht1 as a novel biomarker in nc 275 minerals, electrolytes, and other small molecules, having both hypercalciuria and hyperphosphaturia as risk factors for nephrocalcinosis and patients with renal tubular fanconi syndrome, excrete large amounts of calcium and phosphate(25) there was a significant correlation between the age of patients and leukocyte cystine level, as the age of the patient increase the level of cystine increase (table-4). additionally, the patient’s weight was increased with age thus requires the dose to be increased and it causes a burden to the patients which in turn influences the patients' compliance and in return the leukocyte cystine level. the adherence of patient to treatment is better in children ordinarily and head for fade in adolescents and adults(26). furthermore, the significant correlation between bmi and the leukocyte cystine levels due to the cystine crystals that precipitate throughout the body, including the bone leading to growth retardation(27). the accumulation of cystine inside the lysosome destroys different tissues at different rates, possibly due to promoting apoptosis. one of the earliest organs to be damaged are the kidney, due to its potent proteolysis resulting in great amounts of cystine in the lysosome or due to its relative incapability to originate new differentiation of parenchymal cells(28). thus, our results revealed a significant effect of the cystine level on renal function (manifested as lowered gfr and elevated serum creatinine level) as leukocyte cystine level raised leading to a deterioration of the renal function. meanwhile, the present study demonstrated a relationship between the elevation of wbc cystine level and lowering serum level of calcium (table-4), an interpretation of this result included cystineloaded cells was associated with mobilization intracellular ca2+ stores thus er stress may influence the intracellular ca2+ levels in cystinotic cells. the increased ca2+ influx following ctns knockdown may be a consequence of er stress(29). the elevation in serum chitotriosidase activity noticed in cystinosis patients may reflect a particular case of phagocyte activation concerned with cystine accumulation in the lysosome that causes excessive producing and/or releasing of the chitotriosidase. there is a linkage between cystine deposits and plasma chitotriosidase activity results from the noticing that the wbc cystine level and plasma chitotriosidase activity ran in parallel (the chitotriosidase level and wbc cystine level lowered concomitantly) (table-4) and were both influenced by the regimen of doses(30). conclusion in conclusion serum chitotriosidase activity estimation might aid in monitoring therapeutic benefit of cysteamine therapy, besides the prognosis of the disease when wbccystine assessment is not available, despite that chitotriosidase enzyme is not specific for this disease. however, it’s believed to be a useful clinical screening test and a promising therapeutic monitor since a remarkable link between cystine accumulation and plasma chitotriosidase activity comes from the observation that plasma chitotriosidase activity and leukocyte cystine content ran in parallel and were both affected by the dosing regimen. reference 1. gretz n. cystinosis in the federal republic of germany . coordination * and analysis of the data. 1985;8:8–10. 2. stokes mb, jernigan s, d’agati vd. infantile nephropathic cystinosis. kidney int [internet]. 2008;73(6):782–6. available from: http://dx.doi.org/10.1038/sj.ki.5002730 3. nesterova g, gahl w. nephropathic cystinosis: late complications of a multisystemic disease. pediatr nephrol. 2008;23(6):863–78. 4. gahl wa, kaiser-kupfer mi. complications of nephropathic cystinosis after renal failure. pediatr nephrol. 1987;1(3):260–8. 5. theodoropoulos ds, krasnewich d, kaiserkupfer mi, gahl wa. classic nephropathic cystinosis as an adult disease. jama. 1993;270(18):2200–4. 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http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 protective effects of cyanocobalamin against methotrexate nephrotoxicity doi: https://doi.org/10.31351/vol31iss2pp212-217 211 possible protective effects of two different doses of cyanocobalamin against methotrexate nephrotoxicity model in rats noor mohammed mohammed zakri*,1 and nada n al-shawi** *pharmacy section, alkarkh health directorate baghdad, moh, baghdad, iraq **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract nephrotoxicity is defined as rapid deterioration in kidney functions. it arises from direct exposure to drugs or their metabolites. methotrexate is a famous chemotherapeutic drug with anti-inflammatory and immunosuppressive properties. a high-dose methotrexate-induced renal dysfunction can be life threatening. cyanocobalamin, one of the forms of vitamin b12, acts as a coenzyme in the conversion of homocysteine to methionine in the cytosol, and the conversion of methylmalonyl-coa to succinyl-coa in the mitochondrion. the study was designed to investigate the possible protective role of intraperitoneal cyanocobalamin administered in 2 different doses, against rat model of nephrotoxicity induced by methotrexate. rats utilized in this study were randomized into 4 groups (ten rats per each group); group 1(control) rats intraperitoneally injected with 0.5ml normal saline once daily for 7 consecutive days. group 2rats intraperitoneally injected with 0.5ml normal saline once daily for 7 consecutive days; and at day 2, a single intraperitoneal dose of methotrexate (20mg/kg) is to be injected. group 3rats intraperitoneally injected with a 0.5mg/kg cyanocobalamin once daily for 7 consecutive days, and at day 2, a single intraperitoneal dose of methotrexate (20mg/kg). group 4rats intraperitoneally injected with a 2mg/kg cyanocobalamin once daily for 7 consecutive days, and at day 2, a single intraperitoneal dose of methotrexate (20mg/kg). co-administration of cyanocobalamin at doses cyanocobalamin 0.5mg/kg and 2mg/kg with methotrexate showed significant-reduction(p<0.05) in malondialdehyde, significant elevation (p<0.05)-in the glutathione level, significant upregulation in renal nrf2 expression and significant down regulation in renal keap1 expression each compared to corresponding levels in methotrexate-only treated group. in conclusion this study demonstrated that co-administration of cyanocobalamin at two different doses with mtx resulted in attenuation of its nephrotoxicity by the utilization of selected parameters. keywords: nephrotoxicity, methotrexate, cyanocobalamin, malondialdehyde, glutathione, nrf2, keap1. تجاه نموذج السمية الكلوية التأثيرات الوقائية المحتملة لجرعتين مختلفتين من السيانوكوباالمين في الجرذان ت يميثوتريكس لالمستحثة با ** ندى ناجي الشاوي و 1،*نور محمد محمد زكري العراق ، بغداد والبيئة، وزارة الصحة ، دائرة صحة بغداد الكرخ ، قسم الصيدلة * العراق فرع االدوية والسموم ،كلية الصيدلة، جامعة بغداد، بغداد، ** الخالصة تعرف السمية الكلوية بأنها تدهور سريع في وظائف الكلى. ينشأ من التعرض المباشر لألدوية أو مستقلباتها. الميثوتريكسيت هو دواء كون عالجي كيميائي مشهور له خصائص مضادة لاللتهابات ومثبطة للمناعة. ان الجرعات العالية من الميثوتركيست تسبب الخلل الكلوي وممكن ان ت ، بمثابة أنزيم مساعد في تحويل الهوموسيستين إلى ميثيونين في العصارة 12دة للحياة. يعمل السيانوكوباالمين ، وهو أحد أشكال فيتامين ب مهد مالونيل ميثيل وتحويل سكسينيل coa -الخلوية، المحتمل coa -إلى الوقائي الدور في للتحقيق الدراسة ُصممت الميتوكوندريون. في م باالمين داخل الصفاق الذي يتم تناوله في جرعتين مختلفتين ، تجاه نموذج الجرذان للسمية الكلوية التي يسببها الميثوتريكسات. تم تقسيللسيانوكو حقنت -مجموعات )عشرة فئران لكل مجموعة(. المجموعة األولى )مجموعة السيطرة( 4الجرذان المستخدمة في هذه الدراسة بصورة عشوائية إلى حقنت الجرذان بمحلول ملحي عادي -أيام متتالية. المجموعة الثانية 7مل داخل الصفاق مرة واحدة يومياً لمدة 0.5ذان بمحلول ملحي عادي الجر م/كغم(. ملغ 20أيام متتالية. وفي اليوم الثاني ، تم حقن جرعة واحدة داخل الصفاق من الميثوتريكسيت ) 7مل داخل الصفاق مرة واحدة يومياً لمدة 0.5 أيام متتالية ، وفى اليوم الثاني تم 7ملغم/كغم من السيانوكوباالمين داخل الصفاق مرة واحدة يومياً لمدة 0.5تم حقن الجرذان ب -المجموعة الثالثة ( تم حقن الجرذان داخل الصفاق بجرعة 20حقن جرعة واحدة من الميثوتريكسيت ملغم/كغم من 2ملغم/كغم( داخل الصفاق. المجموعة الرابعة: ملغم/كغم(. 20أيام متتالية ، وفي اليوم الثاني ، تم حقن جرعة واحدة داخل الصفاق من الميثوتريكسيت ) 7السيانوكوباالمين مرة واحدة يومياً لمدة ( في مستوى >0.05p) ملغم/كغم كل مع الميثوتريكسيت انخفاًضا معنويا 2ملغم/كغم و 0.5سبب االعطاء المشترك للسيانوكوباالمين بجرعات وانخفاض معنوي التنظيم في تعبير ال nrf2( في مستوى الجلوتاثيون، زيادة معنوية في التعبيرالكلوي >0.05pالمالونديالديهايد ، ارتفاًعا معنويا ) keap1 ه بان االستنتاج يمكن فقط. بالميثوتريكسيت المعالجة المجموعة في المقابلة بالمستويات مقارنة اإلعطاء الكلوي أن أوضحت الدراسة ذه المشترك للسيانوكوباالمين بجرعتين مختلفتين مع الميثوتريكسيت أدى إلى توهين السمية الكلوية عن طريق استخدام عوامل مختارة. nrf2 ،keap1ت ، سيانوكوباالمين ، مالونديالديهيد ، جلوتاثيون، يسمية كلوية ، ميثوتريكس: الكلمات المفتاحية 1corresponding author e-mail: noorpharmacist1984@yahoo.com received: 14/11 /2021 accepted: 16/1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp212-217 iraqi j pharm sci, vol.31(2) 2022 protective effects of cyanocobalamin against methotrexate nephrotoxicity 212 introduction nephrotoxicity can be defined as any renal damage caused directly or indirectly by drugs or their metabolites, with acute renal failure, tubulopathies, and glomerulopathies as prominent clinical presentations (1). methotrexate (mtx) is a famous chemotherapeutic drug with anti-inflammatory and immunosuppressive properties that is used to treat autoimmune disorders. despite its wide range of clinical use, mtx's efficacy is often limited by severe adverse effects, primarily nephrotoxicity and hepatotoxicity; it also has other side effects such as intestinal damage, myelosuppression (2-4). the pathogenesis of mtx nephrotoxicity involves several pathways, including oxidative stress (os) and inflammation (3). a characteristic hallmark of high dose-mtxinduced acute kidney injury (aki) is a rapid increase in serum creatinine after injection of high dosemethotrexate (hd-mtx) (5). two major pathways are thought to be involved in mtx-induced nephrotoxicity. the first is mtx-induced crystal nephropathy, which is caused by the precipitation of mtx and its metabolites within the renal tubules. the second mode is direct tubular toxicity; where, mtx can cause overproduction of the reactive oxygen species (ros) in the kidney (6). furthermore, researchers revealed that acidic urine can increase the risk of mtx-induced nephrotoxicity because mtx is poorly soluble at low ph, resulting in intratubular mtx crystallization and obstruction of urine flow (7). increased hydration, high-dose leucovorin, and glucarpidase (if needed) effectively lower serum mtx concentrations and protect cells from such drug, but these treatments must be started as soon as possible to -avoid further toxicity, -facilitate renal recovery, and -allow patients to resume hdmtx therapy once renal function has returned to normal (8). cyanocobalamin is the public name of the b12-active corrinoid (also known as cobalamin) with a cyanide ion (cn–) at the β-position of the cobalt atom (9). intracellular vitamin b12 is stored as methylcobalamin and deoxyadenosylcobalamin, two active coenzymes. methylcobalamin is a cofactor for cytoplasmic methionine synthase, which catalyzes homocysteine methylation to methionine. this transmethylation reaction also includes folate (vitamin b9), which is necessary for nucleic acid synthesis. the second active coenzyme (deoxyadenosylcobalamin) is a cofactor for methylmalonyl-coa mutase enzyme, which catalyzes the conversion of methylmalonyl-coa to succinyl-coa in the mitochondria. succinyl-coa then enters the krebs cycle where it is used to synthesize lipids and carbohydrates (10-12). vitamin b12 has several antioxidant properties including: (a) direct scavenging of reactive oxygen radicals (rors), mainly superoxide anion (o2 •–); (b) indirect activation of ros scavenging by restoration of reduced glutathione (gsh); (c) modulation of cytokine and growth factor (gf) production to offer protection from immune response-induced os; (d) lowering of homocysteine-induced os; and (e) reduction of os caused by advanced glycation end products (13). the nrf2 (nuclear factor-erythroid 2 related factor 2) is the key transcription factor that regulates the antioxidant response. under physiological condition, kelch-like ech-associated protein 1 (keap1) sequesters nrf2 in the cytosol. upon activation by ros, nrf2 dissociates from keap1, translocates into the nucleus, forms a dimer with a small musculoaponeurotic fibrosarcoma (maf) protein, binds to the antioxidant response element (are) and promote the transcription of several antioxidant and cytoprotective genes (14, 15). this study is designed to investigate the protective effect of cyanocobalamin (0.5mg/kg and 2mg/kg) each intraperitoneally (ip)-injected in combination on methotrexate (mtx)-induced nephrotoxicity in rats. materials and methods animals forty (40) white albino rats of both sexes, weighing 180-200g were used in this study. rats were obtained from the animal house of the college of pharmacy-university of baghdad; and maintained under controlled conditions of temperature, humidity and light/dark cycle. the animals were fed commercial pellets and tap water ad libitum. chemicals and kits methotrexate (mtx) vial (50mg/2ml) mylan, france; cyanocobalamin (vitamin b12 ampule 1mg/2ml) kontam, hong kong; rat gsh elisa kit mybiosource, usa; rat mda elisa kit mybiosource, usa. in addition, trizol reagent thermo fisher scientific (usa). gotaq® 1-step rt-qpcr system. promega, usa. pcr primers for nrf2, keap 1, and β–actin genes were synthesized and purchased from macrogen, korea. experimental design wistar rats utilized in this study were randomly-divided into 4 groups (10 rats each) and received their treatment by ip route as follows: group 1control/experimental healthy rats ip injected with 0.5ml normal saline (0.9% nacl) once daily for 7 consecutive days. group 2experimental healthy rats ip injected with 0.5ml normal saline (0.9% nacl) once daily for 7 consecutive days; and at day 2, a single dose of mtx (20mg/kg) is to be i.p injected (16). group 3experimental healthy rats i.p injected with a 0.5mg/kg cyanocobalamin (17) once daily for iraqi j pharm sci, vol.31(2) 2022 protective effects of cyanocobalamin against methotrexate nephrotoxicity 213 7 consecutive days, and at day 2, a single ip dose of mtx (20mg/kg) to be injected. group 4experimental healthy rats i.p injected with a 2mg/kg cyanocobalamin (17) once daily for 7 consecutive days, and at day 2, a single ip dose of mtx (20mg/kg) to be injected. the animals in each group were euthanized by anesthetic ether 24 hour after the end of the treatment. preparation of kidney tissue homogenate after the rat euthanized by anesthetic ether, the kidney was quickly excised, rinsed in ice-cold buffer phosphate saline ph 7.4 to remove excess thorough blood and weighed before homogenization, then minced tissue and homogenized with the aid of homogenizer after putting the tube in a beaker containing ice. after that the homogenate was then centrifuged for 20 min at 3000 rpm using cold centrifuge and the supernatant was utilized for the estimation of gsh, and mda levels. determination of mrna expression of renal nrf2 and keap 1 genes total rna was extracted from kidney tissues depending on the steps of trizol™ reagent protocol. quantus fluorometer was applied for the detection of the extracted rna concentration. the isolated rna was ready for use in cdna synthesis. one step rt-qpcr protocol used to investigate quantitatively mrna of the transcription factor nrf2 and keap 1. all real-time rt-pcr reactions were conducted in a total volume of 10 μl. the thermal profile used was at 37 °c for 15 min for rt. enzyme activation at 95 °c for 5min for initial activation denaturation, followed by 40 cycles of 95 °c for 20s denaturation, 56 °c for 20 s annealing and 72 °c for 20 s extension. as a reference gene, the βactin is used. after the pcr amplification, the δδ ct was used for calculating by subtraction of the β-actin ct from each sample ct, qrt-pcr primers nrf2, keap 1 and b-actin sequences (18, 19) were shown in table 1. statistical analysis data were analyzed as mean±standard error of the mean (sem); and performed by using the graphpad prism, version 8. the analysis of variance (anova) followed by a tukey's multiple comparisons test was done. data differences were considered significant at p<0.05. table 1. primers primer name sequence b actinf 5`-ccaccatgtacccaggcatt-3` b actinr 5`-acgcagctcagtaacagtcc-3` nrf2-f 5`-ttgtagatgaccatgagtcgc-3` nrf2-r 5`-tgtcctgctgtatgctgctt-3 keap1-f 5`-ggacggcaacactgattc-3` keap1-r 5`-tcgtctcgatctggctcata-3` results effect on reduced glutathione (gsh) level rats ip injected with mtx at day 2 at a dose of 20mg/kg (group 2) showed significant reduction (p<0.05) in the level of gsh compared to the corresponding level in control (group 1) rats. mean±sem of gsh levels were respectively 58.21± 4.254 and 77.85± 4.585. figure 1 furthermore, in figure 1 there were significant elevation (p<0.05) in gsh level in (group 3 and 4) rat treated with cyanocobalamin dose 0.5mg/kg and 2mg/kg for 1 week in combination with mtx each compared to corresponding level in (group 2) rats; where, mean±sem of gsh levels were respectively 76.57± 3.445, 78.05± 5.753 and 58.21± 4.254. 0 20 40 60 80 100 g s h c o n c e n tr a ti o n m m o l/ i control mtx 20 mg/kg mtx +cyanocobalamin 0.5mg/kg mtx +cyanocobalamin 2mg/kg a b b figure 1. bar chart showing levels of gsh in kidney tissue homogenate (mmol/l) in different experimental groups iraqi j pharm sci, vol.31(2) 2022 protective effects of cyanocobalamin against methotrexate nephrotoxicity 214 each value represents mean±standard error of means (sem) a l atter , is significantly different (p<0.05) compared to control group; b latter, is significantly different (p<0.05) compared to mtx group n=10. effect on malondialdehyde (mda) levels rats ip injected with mtx at day 2 at a dose of 20mg/kg (group 2) showed significant elevation (p<0.05) in the level of mda compared to that level in control (group 1) rats. the mean±sem of mda levels were respectively 2.882± 0.3345 and 1.838± 0.2085. figure 2 furthermore in figure 2 there were significant reduction (p<0.05) in mda level in (group 3 and 4) rat treated with cyanocobalamin dose 0.5mg/kg and 2mg/kg for 1 week each in combination with mtx at day 2 as compared to corresponding level in (group 2); where, mean±sem of mda levels were respectively 1.856± 0.08614, 1.881± 0.1498, and 2.882± 0.3345. 0 1 2 3 4 m d a c o n c e n tr a ti o n n m o l/ l control mtx 20 mg/kg mtx +cyanocobalamin 0.5mg/kg mtx +cyanocobalamin 2mg/kg a b b figure 2. bar chart showing levels of mda in kidney tissue homogenate (nmol/l) in different experimental groups each value represents mean±standard error of means (sem) .a latter, is significantly different (p<0.05) compared to control group; b latter, is significantly different (p<0.05) compared to mtx group. n=10. effect on nrf2 gene expression rats ip injected with mtx at day 2 at a dose of 20mg/kg (group 2) significantly (p<0.05) downregulated renal nrf2 mrna expression compared to control group (group 1); where, mean±sem of nrf2 level 0.3529±0.05385 and 1.982± 0.1495. figure 3.furthermore, figure 3 showed that there were significant (p<0.05) upregulation in renal nrf2 mrna expression in (group 3 and 4) rats treated with cyanocobalamin dose 0.5 mg/kg and 2mg/kg for 1 week in combination with mtx at day 2 ,respectively compared to rats treated with mtx (group 2); where, mean±sem of nrf2 level are 2.000±0.4949, 2.258±0.6545 and 0.3529±0.05385, respectively. 0 1 2 3 4 r e la ti v e m r n a e x p r e s s io n o f n r f2 /b -a c ti n control mtx 20 mg/kg mtx +cyanocobalamin 0.5mg/kg mtx +cyanocobalamin 2mg/kg a b b figure 3. bar chart showing levels of relative mrna expression of nrf2/b actin in kidney tissue in different experimental groups each value represents mean±standard error of means (sem) a latter,is significantly different (p<0.05) compared to control group; b latter, is significantly different (p<0.05) compared to mtx group. n=10. effect on keap1 gene expression rats ip injected with mtx at day 2 at a dose of 20mg/kg (group 2) upregulated renal keap1 mrna expression significantly (p<0.05) compared to that level in control (group 1) rats; where, mean±sem of keap 1 level 1.370±0.2115 and 0.7660± 0.0895. figure 4.furthermore, figure 4 showed that there were significant dawn-regulation (p<0.05) in renal keap1 mrna expression in (group 3 and 4) rats treated with cyanocobalamin dose 0.5 mg/kg and 2mg/kg for 1 week in combination with mtx at day 2, respectively compared to rats treated with mtx (group 2); where, mean±sem of keap1 level 0.6361±0.1280, 0.7690±0.04357 and 1.370±0.2115, respectively. 0.0 0.5 1.0 1.5 2.0 r e la ti v e m r n a e x p r e ss io n o f k e a p 1 /b -a c ti n control mtx 20 mg/kg mtx +cyanocobalamin 0.5mg/kg mtx +cyanocobalamin 2mg/kg a b b figure 4. bar chart showing levels of relative mrna expression of keap1/b actin in kidney tissue in different experimental groups iraqi j pharm sci, vol.31(2) 2022 protective effects of cyanocobalamin against methotrexate nephrotoxicity 215 each value represents mean ± standard error of means (sem) a latter, is significantly different (p<0.05) compared to control group; b latter, is significantly different (p<0.05) compared to mtx group. n=10. discussion methotrexate (mtx), a chemotherapeutic agent that is therapeutically-used for the treatment of various cancers. high doses of such drug can cause acute renal failure with an increase in the serum creatinine (scr) levels, uremia, and hematuria (20). one of the key mechanisms through which mtx can cause tissue damage is the os. since, it can increase ros production by suppressing homocysteine remethylation, depletion of the reduced form nicotinamide dinucleotide phosphate (nadph), stimulation of neutrophils, activation of nadph oxidase enzyme activity, and mitochondrial dysfunction; moreover, ros then can cause cell damage through oxidizing lipid and proteins, inactivating antioxidant enzymes, and causing dna damage, leading to a dysfunctional cellular protective response (18). furthermore, researchers reported that administration of high dose of mtx resulted in elevated mda level, -depletion of glutathione reservoirs, and -reduction in tissue antioxidant capacity (21). these ros negatively affect nephrons, resulting in structural and functional changes of glomeruli and renal tubules (22). the result of this study revealed that rats treated with 20mg/kg mtx ip, showed significant reduction in the level of gsh and significant elevation in the level of mda as compared to control group. additionally, mtx reduced the efficiency of the antioxidant enzyme defense mechanism, and making the cells more vulnerable to ros; furthermore, gsh is a sensitive marker of os and it plays an essential role in preserving the integrity of the cell; since such marker involved in several detoxification reactions in the organism and it is one of the most important non-enzymatic antioxidants (23). furthermore, ros-induced oxidation of polyunsaturated fatty acids (pufas) in biological systems resulting in the generation of lp products such as mda (24). although the endogenous antioxidant response system in human can strongly-regulate the level of ros and reduce correlated cellular damage, the exogenous antioxidants can play a significant role; since exogenous antioxidants were discovered to have a priming impact on the antioxidant response system; furthermore, both exogenous and endogenous antioxidant response systems work together to provide a more effective and efficient defense against harmful redox modulations (25). several molecular pathways play a remarkable role in kidney pathophysiology among them; kelch-like ech-associated protein1 (keap1)/ nuclear factor erythroid 2–related factor2 (nrf2). since, the keap1/nrf2 pathway is one of the most significant os cytoprotective pathways, and nrf2 plays a critical physiological role in protecting the kidney against a variety of illnesses (26). moreover, researchers mentioned that the increased in nrf2 expression was linked to lower os and enhanced antioxidant defenses (27). additionally, hassanein, e. h. m.et al (2018) reported that mtx significantly down-regulate renal nrf-2, while upregulate renal keap-1 (28). rats injected with a single dose 20mg/kg mtx downregulated renal nrf2 mrna expression and upregulated renal keap1 mrna expression significantly (p<0.05); while there were significant (p<0.05) -up-regulation in renal nrf2 mrna expression in rats treated with cyanocobalamin doses [0.5mg/kg and 2mg/kg] for 1 week in combination with mtx and -down-regulation in renal keap1 mrna expression in rats treated with cyanocobalamin doses [0.5 mg/kg and 2mg/kg] for 1 week in combination with mtx (figure 3 and 4). therefore, activation of nrf2/antioxidant signaling by cyanocobalamin can attenuate mtx-induced os, inflammation and kidney injury. researchers mentioned that the nrf2 signaling pathway played a vital role in the protection against cell injury induced by os and electrophiles; where, excessive production of ros and oxidative injury are associated with the pathogenesis of several diseases and disorders; and there is crosstalk between the nrf2/are signaling and other signaling pathways, including the nuclear factor kappa-lightchain-enhancer of activated b cells (nf-κb), which represented the main underlying mechanism by which nrf2 exerts its anti-inflammatory activities; furthermore, given the dual role of nrf2 activation in the prevention of os and inflammation, pharmacological activation of this signaling pathway could represent a powerful strategy for treating diseases associated with excessive release of ros and proinflammatory mediators (29). in agreement with the perception that the activation of the nrf2 pathway is coupled with antioxidant effects and was linked to the decreased levels of nfκb and inducible nitric oxide synthase (inos) expression in experimental rats, the nrf2 is important for the expression of antioxidant genes like superoxide dismutase (sod), catalase (cat) and heme oxygenase-1 (ho-1), researchers postulated that the improved efficacy of antioxidant nephroprotective defense may be explained by their ability to regulate the nrf2 signaling pathway positively (30). additionally it has been demonstrated that, mtx diminished nrf2/are/ho-1 signaling in the liver and kidney of rats (14). iraqi j pharm sci, vol.31(2) 2022 protective effects of cyanocobalamin against methotrexate nephrotoxicity 216 although exposure to moderate os can lead to nrf2 activation, the excessive and sustained ros generation can diminish nrf2 signaling in the kidney and liver of rats challenged with mtx; thus, the diminished nrf2/ho-1 pathway is a direct consequence of the sustained ros generation induced by mtx (31). conclusion according to the results obtained from this study, it could be concluded that the protective effect of cyanocobalamin at a dose of 0.5mg/kg and 2mg/kg were observed when co-administered with 20mg/kg mtx. notably, co-administration of cyanocobalamin at 2 different doses with 20mg/kg mtx resulted in attenuation of mtx induced nephrotoxicity. references 1. gabriel, t. montezuma, s. renato d. foresto. drug-induced 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menze, e. t., azab, s. s., & awad, a. s. involvement of nrf2/ho-1 antioxidant signaling and nf-κb inflammatory response in the potential protective effects of vincamine against methotrexate-induced nephrotoxicity in rats: cross talk between nephrotoxicity and neurotoxicity. archives of toxicology 2019, 5(93): 14171431. doi:10.1007/s00204-019-02429-2 31. younis, n.s.; elsewedy, h.s.; shehata, t.m.; mohamed, m.e. geraniol averts methotrexateinduced acute kidney injury via keap1/nrf2/ho-1 and mapk/ nf-κb pathways. curr. issues mol. biol. 2021; 43: 1741–1755. https:// doi. org/ 10. 3390 /cimb 43030123 this work is licensed under a creative commons attribution 4.0 international license. https://www.sciencedirect.com/science/journal/03043959 https://www.sciencedirect.com/science/journal/03043959/114/1 https://pubmed.ncbi.nlm.nih.gov/?term=abukhalil+mh&cauthor_id=31561418 https://pubmed.ncbi.nlm.nih.gov/?term=saghir+sam&cauthor_id=31561418 https://pubmed.ncbi.nlm.nih.gov/31561418/#affiliation-6 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 updates on covid-19 pandemic doi: https://doi.org/10.31351/vol30iss1pp56-65 56 genetic structure, transmission, clinical characteristics, diagnosis, treatment and prevention of coronavirus disease 2019 (covid-19): a review rawaa s. al-mayyahi*,1 and wa'il a. godaymi al-tumah** department of clinical laboratory science, college of pharmacy, university of basrah, basrah, iraq.  department of physics, college of science, university of basrah, basrah, iraq. abstract the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) caused a pandemic of coronavirus disease 2019 (covid-19) which represents a global public health crisis. based on recent published studies, this review discusses current evidence related to the transmission, clinical characteristics, diagnosis, management and prevention of covid-19. it is hoped that this review article will provide a benefit for the public to well understand and deal with this new virus, and give a reference for future researches. keywords: coronavirus, covid-19, sars-cov-2, pneumonia, respiratory infection. مقالة (covid-19) : 2019 كورونا فيروس مرض من والوقاية وعالج وتشخيص انتقال **الطعمه اللطيف عبد وائلو 1*،المياحي سالم رواء .السريرية ، كلية الصيدلة ، جامعة البصرة ، البصرة، العراق المختبرية العلومقسم * .قسم الفيزياء ، كلية العلوم ، جامعة البصرة ، البصرة ، العراق ** الخالصه 2019 كورونا فيروس مرض جائحة انتشار في تسبب( sars-cov-2) 2 الحادة التنفسية المتالزمة المستجد كورونا فيروس ان (covid-19 )ا المنشورة الدراسات على بناء . عالمية عامة صحية أزمة يمثل الذي هذا بانتقال المتعلقة الحالية األدلة المقالة هذه تناقش ، مؤخر معه والتعامل الجديد الفيروس هذا لفهم عامة فائدة المقالة توفرهذه أن المؤمل من .منه والوقاية والعالج والتشخيص السريرية والخصائص الفيروس .المستقبلية كمرجع للبحوث واستخدامها ، جيد ا التنفسي الجهاز عدوى ، رئوي التهاب ، cov-2 -سارس ، 19 كوفيد ، كورونا فيروسالكلمات المفتاحية : introduction since december 2019, a series of unexplainable pneumonia cases were identified in wuhan a central city in china (1–4). by january 2020, the results of deep sequencing data analysis from infected patient’s samples, confirmed that a novel coronavirus named severe acute respiratory syndrome-related coronavirus 2 (sarscov-2) is the causative agent for that observed mystery pneumonia (5). on february, 2020, the world health organization (who) named the disease caused by the sars-cov-2 as coronavirus disease-2019 (covid-19) and by march, 2020, the who announced the outbreak of covid-19 as a pandemic with confirmed cases in 114 countries. the number of infected patients exceeds 118,000, and the death toll exceeds 4000 (6). on april the 10th, 2020, there have been more than 1,500,000 reported cases and about 93,000 deaths in more than 130 countries (7). as of september 27, 2020, sars-cov2 has affected over 32.7 million reported cases and 991, 000 confirmed deaths (8). interestingly, the rate of new covid-19 cases has continued to increase and the rate of deaths has remained relatively stable. in october the 19th, more 42 million cases and 1.1 million deaths have been recorded around the world (figure 1) (9). the new type of coronavirus belongs to a large class of viruses called β-coronavirus. sars-cov-2 is similar to severe acute respiratory syndrome coronavirus (sars-cov) that caused the sarscov outbreak in 2002 and middle east respiratory syndrome coronavirus (mers-cov) that caused the mers-cov outbreak in 2012 (10–13). however, a novel coronavirus has different potential natural, intermediate and final hosts. these results in major problems for the prevention and management of viral infection. moreover, the new virus has high transmissibility and infectivity, and a low mortality rate compared with sars-cov and mers-cov (14). here, in this review we would like to display the genetic structure, route of transmission, diagnosis, clinical characteristics, source of infection and to discuss the foundational knowledge on the therapies that have been suggested for the treatment of covid-19. it is hoped that this review will provide researchers with an updated understanding of covid-19 and also to help follow-up research, prevention and treatment. 1corresponding author e-mail: rawaa.salim@uobasrah.edu.iq received: 11/11/ 2020 accepted:13 /2 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp56-65 iraqi j pharm sci, vol.30(1) 2021 updates on covid-19 pandemic 57 figure 1. number of covid-19 cases reported weekly by who region and global deaths (data as reported at on 19 october 2020) (9). genetic structure and pathogenic mechanism of sars-cov-2 coronaviruses are single-stranded ribonucleic acid viruses with a diameter of 80–120 nm. these viruses are found in humans and vertebrates, such as dogs, cats, chicken, cattle, pigs, and birds. coronavirus can infect the respiratory, intestinal, and central nervous systems in humans and other mammals (15,16). the genome sequence analysis showed that sars-cov-2 belongs to βcoronavirus family and it is similar to sars-like bat coronaviruses, this reveals that sars-cov-2 might originate from bats (14). interestingly, several studies have investigated that sars-cov-2 uses angiotensin-converting enzyme 2 (ace2) as an entry receptor (17–19). most of coronaviruses members use s proteins on their surface to recognize their corresponding receptors on host cells and gain entry into target cells and then cause infections (figure 2). the experimental results showed that sars-cov-2 binds to ace2 with ~10to 20fold higher affinity than sars-cov (20,21). however, the exact mechanism by which covid 19 cause infection in humans through binding of s-protein to ace2, a high human‐to‐human transmission rate, fast mutation and recombination and how sarscov-2 damage organs remain unclear, and therefore many studies are required. the current findings elucidate the rapid transmission ability of covid19 in humans compared with sars-cov, and the greater number of confirmed cases of sars-cov-2 compared with sars-cov disease. considering binding of sars-cov-2 with higher affinity with ace2 and soluble ace2 may be a possible candidate for the management of covid-19 (22). figure 2. viral entry mechanism of sars-cov-2 into the host cells. transmission of sars-cov-2 the natural hosts of sars-cov-2 are bats while the intermediate hosts are pangolins and snakes (23,24). virologic researchers found that sars-cov-2 is 96% similar to the whole-genome level of a bat coronavirus gene sequencing analysis (25). recently, it is considered that the essential source of sars-cov-2 infection is infected persons. epidemiologic results propose that droplets and close contact to patients with covid-19 are the most common mode of transmission. virologic studies suggest that sars-cov-2 is mainly transmitted from symptomatic people through coughing or sneezing. further, recent studies revealed that sars-cov-2 found in samples of stool, gastrointestinal tract, saliva and urine these cause an infection risk in sewage networks, sanitary settings, wastewater treatment plants, and the wider environment such as rivers and lakes (26,27). short exposures to asymptomatic contacts are less likely to cause transmission (27). aerosols (tiny droplets that remain suspended in the air) are another possible source of covid-19 infection in humans. also, contaminated surfaces are another possible mode of transmission (28,29). a case series of nine infected pregnant mother showed that there is a possibility of vertical transmission in the third trimester (30). in most case series, the pregnant women infection with covid-19 appeared in the third trimester of pregnancy and there were no maternal deaths and a favourable clinical course in the infants (30,31). however, researches on pregnant infected with covid-19 are insufficient; more studies are needed to verify the vertical transmission of sars-cov-2 from mother to neonates. the debate remains regarding sars-cov-2 transmissibility by indirect contact from inanimate surfaces. studies have shown higher levels of the virus on impermeable surfaces, such as plastic, stainless steel compared with permeable surfaces, such as cardboard (32). the previous studies have been shown that the coronavirus including the covid-19 can remain viable on different surfaces for a prolonged time under controlled laboratory iraqi j pharm sci, vol.30(1) 2021 updates on covid-19 pandemic 58 conditions (33). the epidemiological studies investigate that elderly people are most vulnerable to covid-19 (median age at death 75 years) (34). interestingly, the average age of hospitalized infected persons was 47 to 73 years, more men were admitted than women about (60% male) in most groups (35). approximately 2% to 5% of infected patients with covid-19 are younger than 18 years, with a median age of 11 years. a study of 1099 infected persons with covid-19 showed that the median incubation time was three days (range 0–24 days), and the median period from symptom start to death was 14 days (34,36). also, in a study of 52 critically ill individuals with covid-19 who were admitted to intensive care units, the mean age was 59.7 years, and the death rate was 61.5% by 28 days (37). based on current evidence and clinical experience, it was found that the death rate for those over 80 years is 5 times higher than the global average. eight out of 10 deaths reported in the united states were in adults over 65 years of age (38). also, it was reported that the percentage of death was increasing with age in united states (figure 3) (39). importantly, covid-19 may be asymptomatic or it may cause different symptoms that include fever, myalgia, with diarrhea, respiratory distress and life-threatening sepsis (40,41). interestingly, virologic studies investigate that sars-cov-2 can be transmitted by asymptomatic, presymptomatic, and symptomatic carriers (42). figure 3. percentage of death due to the coronavirus (covid-19) in united states, may 1–august 31, 2020 increase with age (39). clinical characteristics of sars-cov-2 the mean incubation period of sarscov-2 infection was (2-7) days; this is similar to the median incubation period of sars-cov which was (2–10) days (36,43,44). around 97.5% of infected persons who develop symptoms and signs will do so in 11.5 days of infection. the median time from symptom beginning to hospital admission is (3-9) days (44,45). the most common symptoms in hospitalized individuals are fever (90% of infected persons), dry cough (60%-86%), shortness of breath (53%-80%), fatigue (38%), vomiting or diarrhea (15%-39%), and myalgia (15%-44%) (5,46,47). for most infected persons with covid-19, 64% to 80% had olfactory and/or gustatory dysfunctions (48,49). furthermore, severe cases are susceptible to different complications, such as acute respiratory distress syndrome, coronary heart disease, hypertension, diabetes, cancer and other sever outcomes (50–53). also, it was observed that sarscov-2 can spread to other tissues organs and cause damage to kidney, liver, brain and ocular tissues (figure 4) (54,55). sars-cov-2 infection may be mild in children with a general case fatality rate less than 1%. the relationship between children and the infection of covid is unclear. the likely reasons may belong to their less robust immune responses and their lower rates of exposure to covid-19. despite most children cases are mild, about (<7%) of cases develop severe symptoms and need mechanical ventilation and therefore admitted to the hospital (42). figure 4. schematic figure displaying the potential complications of sars-cov-2 impacting organ systems. diagnosis of sars-cov-2 in general, the diagnosis of sars-cov-2 is made by using polymerase chain reaction testing. however, sars-cov-2 nucleic acid testing has low sensitivity and high specificity; therefore, falsenegative test results may occur. there are several factors contributing to false-negative test results such as specimen source, adequacy of the specimen collection method and time from exposure. it was observed that lower respiratory samples are more sensitive than upper respiratory samples. a research group in china collected 1070 specimens from 205 infected persons with sars-cov-2, they were observed that (93%) of positive rates of sars-cov2 pcr testing was in bronchoalveolar lavage fluid specimens, (72%) was in sputum, (63%) was in nasal swabs, and (32%) was in pharyngeal swabs. moreover, covid-19 can also be found in feces, but not in urine (56). saliva is another source of sars-cov-2 which required less personal protective equipment, fewer swabs, and more iraqi j pharm sci, vol.30(1) 2021 updates on covid-19 pandemic 59 validation (57). so, nasal swabs, laboratory, clinical and chest computed tomography (ct) scan can be also used in order to get a presumptive diagnosis (14,58). serological testing could help in the diagnosis of sars-cov-2 infection and also in the responses to novel vaccines. however, not all antibodies created in response to infection are neutralizing. therefore, the presence of antibodies may not produce immunity (59,60). a previous study succeeded to develop a rapid test (1 h) sarscov2 detection using sherlock technology (specific high-sensitivity enzymatic reporter unlocking) (61). also, a scientist group at peking university suggested developing a new method for rapid construction of the transcriptome sequencing library of sherry which is useful for rapid sequencing of covid-19. the transcriptome profiling by rna sequencing (rna-seq) is widely used to characterize cellular status. however, this method relies on second-strand complementary dna (cdna) synthesis to create initial material for library preparation (62). treatment of sars-cov-2 there are different classes of drugs being assessed and developed for the treatment of covid19. antiviral drugs have been reported as a promising treatment of a broad array of rna viruses. for example, remdesivir was initially developed for the management of ebola hemorrhagic fever. in vitro and in vivo studies suggest its activity against filoviridae, paramyxoviridae and coronaviridae such as merscov, sars-cov, and sars-cov-2 (5,63,64). holshue et al. found that remdesivir attained good results against sars-cov-2 (65). an in vitro study has shown the effectiveness of remdesivir in the control of covid-19 (66). although a large number of clinical trials have been performed, the efficacy of remdesivir remains uncertain. chloroquine and hydroxychloroquine have antiviral and immunomodulatory activities, they were clinically used against malaria and several viruses such as human immunodeficiency virus type 1 hepatitis b virus and herpes simplex virus type 1. chloroquine and hydroxychloroquine seam to prevent viral entry into cells and endocytosis of virus in vitro and may possess immunomodulatory effects in vivo (67,68). two previous studies among patients hospitalized for covid-19 showed no effect of hydroxychloroquine on the risk of intubation or mortality (69,70). one of these two previous cohort studies compared in-hospital mortality between 735 patients treated with hydroxychloroquine and azithromycin, 271 patients treated with hydroxychloroquine alone, 211 patients treated with azithromycin alone, and 221 patients without drugs. they found that there were no differences among the groups (69). however, both compounds may cause rare and serious adverse effects, most notably qt prolongation, hypoglycemia, neuropsychiatric effects, and retinopathy (69,71,72). the safety of chloroquine and hydroxychloroquine use during pregnancy was investigated (11). a review of 588 patients treated with chloroquine or hydroxychloroquine in pregnancy observed no overt child ocular toxicity (73). clinical trials still ongoing and must give more guidance. most antiviral agents that were previously used in sars-cov and mers-cov treatment are potential candidates to treat sars-cov-2. lopinavir-ritonavir is a protease inhibitor developed for the treatment of human immunodeficiency virus (hiv) (74). although lopinavir/ritonavir disrupts viral replication in vitro, a randomized, controlled, open-label trial of 199 hospitalized infected persons with severe covid19 shows no benefit when compared with standard care (75). ribavirin is rna-dependent rna polymerase inhibitors and uses against other novel coronaviruses. therefore, it is a candidate for the treatment of sars-cov-2 (76). however, its activity against sarscov in vitro was limited, needed high dose and combination therapy in order to inhibit viral replication. previous studies showed that patients received either intravenous or enteral administration while inhaled administration shows no benefit over enteral or intravenous administration (77,78). it was demonstrated that the high doses of ribavirin used in the sars-cov trials caused haemolytic anaemia in more than 60%of patients (78). the inconclusive efficacy results with ribavirin for other novel coronaviruses and its substantial toxicity propose that it has limited efficacy for covid-19 treatment. however, its clinical efficacy may be improved with combination therapy. ribavirin is also a known teratogen and contraindicated in pregnancy (79). sars-cov infections induced a disproportionate immune response. therefore, enhancing the body’s immunity is a possible candidate protocol for treatment of individuals with covid-19. interferon can induce both innate and adaptive immune responses resulting in inhibition of viral infection (11,80). corticosteroids have potent anti-inflammatory and immunomodulatory properties therefore; they are used to suppress lung inflammation, particularly in the advanced stages of the disease (81). low doses of corticosteroids caused downregulation of pro-inflammatory cytokine transcription resulting in preventing extended cytokine responses and increasing the resolution of pulmonary and systemic inflammation in pneumonia. moreover, corticosteroids can promote the dysregulated immune responses caused by sepsis (a potential complication of sars-cov-2) and may raise blood pressure in hypotensive patients (82–84). however, corticosteroids reduced viral clearance from the respiratory tract and blood and provoke viral replication. russell et al argued that the benefit of corticosteroids was greatest in patients with bacterial rather than viral infections (85). previous iraqi j pharm sci, vol.30(1) 2021 updates on covid-19 pandemic 60 studies have reported that there was no survival benefit (and possibility for harm) among patients with sars, mers-cov, and influenza receiving corticosteroids. however, preliminary results from the randomized evaluation of covid-19 therapy (recovery) trial, which showed that dexamethasone reduced mortality across sarscov-2 patients with severe respiratory complications. the mortality was reduced by onethird in 2104 patients with covid-19 receiving low doses of dexamethasone (6 mg daily, orally or intravenously) for up to 10 days and by one-fifth in patients taking oxygen only. the benefit was higher in patients who needed mechanical ventilation and in patients with symptoms for more than 7 days (81,86). furthermore, a previous cohort study in wuhan, china, showed that methylprednisolone treatment reduced risk of mortality (hazard ratio, 0.38 [95% ci, 0.20-0.72]) in 201 patients with covid-19 and acute respiratory distress syndrome (ards) (87). convalescent plasma from infected persons who have recovered from viral infections with high specific antibodies against sars-cov-2 is selected as an alternative treatment for covid19. it was first investigated during flu pandemic in 1918 and used to promote the survival rate of patients with sars, mers-cov, h1n1 and ebola, (88–91). indeed, convalescent plasma with neutralizing antibody was used in conjunction with continued methylprednisolone and antiviral treatment drugs to treat 5 critically ill sars-cov-2 patients with ards requiring mechanical ventilation. the findings showed improvement in clinical status among all participants (92). in randomized clinical trial of 103 infected persons with severe covid-19 observed there are no statistical differences in time to clinical improvement within 28 days across participants randomized to take plasma therapy vs standard treatment only (51.9% vs 43.1%) (93). however, consideration plasma-derived hyperimmune globulin and monoclonal antibodies against sars-cov-2 are another approaches deserve more consideration. prevention and vaccine development of covid19 sars-cov-2 is a potentially preventable infection. the epidemiology of the new disease around the world reveals the clear relationship between the intensity of public health action and the control of transmission (27,94–96). the rates of transmission can be reduced using different infection control measures such as the use of protective equipment, home quarantine after infection, physical distancing, travel restrictions and restricting mass gatherings (94,95,97). in addition, boosting the immune system is another way to prevent covid-19. sars-cov-2 may cause acute respiratory distress syndrome and sepsis, therefore, a moderate amount of vitamin c may be a way to ameliorate inflammation and vascular injury in covid-19 patients (98–100). moreover, the reduction in the amount of vitamin d and vitamin e in cattle may cause bovine coronavirus infection. therefore, suitable amount of vitamin d and vitamin e may increase resistance to sars-cov-2 (101). however, multiple interventions remain required until safe and potent treatments or vaccines become available. there are several approaches used to develop vaccines for sars-cov-2 such as nucleic acids (dna or rna), viral vectors, inactivated or live attenuated viruses and recombinant proteins or virus particles. however, the novel virus is an rna virus, therefore, rna-virus-related vaccines such as polio, measles, encephalitis b virus and influenza virus, represent the most promising alternatives. more than 120 vaccines are under development till now (102,103). however, who expected that a minimum of 12 to 18 months would be needed before widespread vaccine deployment. interestingly, on december the us food and drug administration (fda) endorsed pfizer/biontech’s and moderna as safe and effective covid-19 vaccines. both vaccines use messenger rna (mrna) technology, which includes instructions for human cells to make proteins that mimic part of the coronavirus. they target the crown-like spikes that found on the surface of the coronavirus (104). conclusions as of october 18, 2020, more than 40 million people worldwide had been infected with covid-19. therefore, covid-19 represents the greatest global public health emergency after the pandemic of spanish influenza in 1918. 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morgan r, smith j, wenham c. covid-19 vaccines and women's security. the lancet. 2020. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 fetuin-a, resistin serum level in iraqi multiple myeloma patients doi: https://doi.org/10.31351/vol29iss2pp80-87 80 estimation of beta two microglobulins, fetuin-a, resistin serum level in iraqi multiple myeloma patients ali s. salman*,1 , eman s.saleh* and mohammed s.abass** *department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq **ministry of health and environment, baghdad teaching hospital, medical city, baghdad, iraq abstract multiple myeloma is hematological disease produces many complications in the bone, kidney, neural and other complications. the study aims to measure serum biomolecules like fetuin-a and resistin and determined the possibility to use these biomarkers as disease predictor. blood samples were isolated from 58 patients and 24 sex and age-matched control, serum then isolated, and proper elisa kit then used to a determined level of β2 microglobulin, resistin, and fetuin-a. the result demonstrated significant increase in β2 microglobulin, fetuina and resistin in patients compare to control (1.347±0.714 vs. 0.913±0.253), p = 0.000, (14.003±10.352 vs. 9.259±4.264), p= 0.005, (1.967±3.595 vs. 0.604±0.622), p = 0.009, respectively. these differences give the possibility to use these biomolecules as a predictor in multiple myeloma. keywords: multiple myeloma,fetuin-a,resistin. مقارنة بين مستوى الفيتون أي والريزستين بين المرضى بمرض الورم النخاعي واالصحاء **محمد سليم عباسو * ، ايمان سعدي صالح 1*،علي صباح سلمان فرع العلوم المختبرية السريرية ، كلية الصيدلة ،جامعة بغداد ، بغداد ،العراق. * ، مستشفى بغداد التعليمي، مدينة الطب ، بغداد، العراق. وزارة الصحة والبيئة ** الخالصة الهدف من الدراسببة الورم النخاعي المتعدد هو احد امراض الدم الذي يسبببب معبباعفا في العال، ال لة، معبباعفا عصبببي و ر ، تحديد مسبببتو بعل الئايئا ااحيا ية في مصببب الدم التي هي فيتوين ي ،ريسببباتين وتحديد احتمالية اسبببتخدام هذم الئايئا كمتنب للمرض . ة شبببخح حبببحيب متطاب ين من حيس الئنم والعمر. بعدها عال المصببب وباسبببتخدام كتا ااايام احببب 24مريعبببا و 58عينا دم عالت من ماي روكلوبيولين في المرضبببى 2ماي روكلوبيولين، فيتوين ي، رياسبببتين. النتا ظ تهر زيادة محسبببوسبببة في مسبببتو بيتا 2لتحديد مسبببتو بيتا ، فيتوين ي اتهر زيادة محسبببببوسبببببة م ارنة بمئموعة p =0.000( قيمة 0.253±0.913ضبببببد 0.714±1.347م ارنة بمئموعة السبببببيطرة ±1.967، ريساتين اتهر زيادة محسوسة م ارنة بمئموعة السيطرة p=0.005( قيمة 4.264±9.259ضد 10.532±14.003السيطرة ، هذم النتا ظ تطينا احتمالية ااسببتفادة من هذم الئايئا ااحيا ية كمتنب لمرض الورم النخاعي p =0.000( قيمة 0.622±0.604ضببد 3.595 المتعدد. النخاعي ، الفيتون أي ، الريزستين .مرض الورم الكلمات المفتاحية: introduction multiple myeloma is a malignant disease characterized by a defect in plasma cells that result in a change in different organs including bone, kidney, and others (1). the disease starts as monoclonal gammopathy of undetermined significance (mgus) (2), which is developed to smoldering (asymptomatic) myeloma and finally becomes overt (symptomatic) myeloma (3). the bone disorder is the most common complication in the mm, the damage that occurs in the bone result from stimulation of osteoclast formation and activation that occurs in the area of the bone that is closed to myeloma cell. besides, to increase the bone resorption, there is a decrease in bone formation have been reported and this attributed to the suppression effect of myeloma cell on osteoblast cell and so inhibit bone formation (4). the first common cause of the renal disorder is due to light chain immunoglobulin (lci) (5), normally the monoclonal light chain reaches into proximal tubule after glomerular filtration that undergoes endocytosis in that cell by a specific scavenger receptor and then degrades in the lysosome (6). however, in mm patients the excess production of (lci) exceeds the catabolic capacity of the tubule to metabolize this protein within the tubular fluid result in accumulation of this protein with tamm-horsfall protein (loop of henle glycoprotein) and form cast that cause a tubular obstruction in the distal area and result in increased intraluminal pressure and decrees in glomerular filtration lead to renal function impairment (7). infection common among mm patients, one of the important reason is to reduce antibody response and abnormality in complement and granulocyte function, the infections represent one of the most important causes of death in those patients (8,9). 1corresponding author e-mail: alipharma2009@yahoo.com received: 5/1 /2020 accepted: 3/5/ 2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp80-87 iraqi j pharm sci, vol.29(2) 2020 fetuin-a, resistin serum level in iraqi multiple myeloma patients 81 beta2 microglobulin is a polypeptide presented in the serum its origin is the cell membrane of all nucleated cell in which found in tight junction with major histocompatibility complex 1(mhc1) (10), elevated its level reflected increase the intrinsic kinetic activity of tumor cell including dna and rna kinetic (11), therefore, it is important for staging, determining disease severity, response to chemotherapy and prognosis. fetuin-a is a multifunctional plasma protein secreted mainly from the liver to the bloodstream, fetuin-a consider as an inhibitor of insulin they act through inhibit tyrosine kinase insulin receptor and thereby participate in insulin resistance and metabolic syndrome and even type 2 d.m.(12,13). fetuin-a also has a role in calcium salt precipitation and calcification in the bone and teeth, also reported behaving anti adipokines action through antagonize adiponectin action and thereby affect the fatty acid uptake by adipocyte and precipitate atherosclerosis (12,14). fetuin-a is a negative acute phase reactant and therefore its level going to be decreased in inflammatory condition (12). one study showed the role of fetuin-a in the insulin resistance state as a result of increase free fatty acid in the circulation and they concluded ffa increase proinflammatory cytokine release from adipose tissue through enhancing expression of fetuin-a from the liver that in turn activated toll-like receptor 4 (tlr 4) in the adipose tissue to release these pro-inflammatory cytokines (14). the normal value of fetuin a 450-600 µg/ml and the difference in the level is genetically determined and the gender difference does not influence it's level (12). resistin is one of 114 amino acid adipocytokines that secreted primarily from macrophage and monocyte in human and from adipocyte in mice (15), play important role in regulating energy homeostasis and triglyceride storage and mobilization through not a well mechanism, resistin augmented type 2 dm by enhancing insulin resistance and enhancing the inflammation and its role in the obesity (16) a study showed increase resistin level with increasing obesity in the body (17-19), in the inflammatory process, resistin has shown to increase transcription event of several proinflammatory cytokines and thereby increase their levels, examples of these cytokines are interleukin-1 (il1), interleukin-6 (il-6), interleukin-12 (il-12), and tumor necrosis factor-α (tnf-α) (20-22). the study aimed to measure the level of fetuin-a and resistin in the serum of patients with multiple myeloma and determined the possibility to use these markers as a predictor of the disease. subject, materials, and methods the study was conducted in baghdad city in (baghdad hospital in the medical city and hematological center) from october 2018 to may 2019 were ( 58 ) patients diagnosed to have multiple myeloma and most of them regularly visit the hospital to receive chemotherapy, from the total number of the patients, (36) was male and (22) was female. the control subjects were randomly selected which were healthy, the control was age, sex, body mass index (bmi) matching to patients group. disposable syringe and needles used for blood collection, venous blood sample about six ml collected from patients and healthy volunteers in a plain tube, blood sample were centrifuge at 2000 rpm for 5 minutes to obtain serum. the serum was divide into an eppendorf tube and freeze in -20 co until all serum collected to measure the biomolecules by elisa technique using elisa kit. determination of serum level of β2 microglobulin serum β2microglobulin is determined through the use of a commercial kit from demediect by sandwich elisa method. in which highly purified anti-human-beta-2-microglobulin antibodies are bound to microwells. beta-2microglobulin, if present in diluted serum, plasma, or urine, binds to the respective antibody, washing of the microwells removes unspecific components, horseradish peroxidase (hrp) conjugated antihuman beta-2-microglobulin immunologically detects the bound patient beta-2microglobulin forming a conjugate/beta-2-microglobulin / antibody complex. washing of the microwells removes unbound conjugate. an enzyme substrate in the presence of bound conjugate hydrolyzes to form a blue color. the addition of an acid stops the reaction forming a yellow product. the intensity of this yellow color is measured photometrically at 450 nm. the amount of color is directly proportional to the concentration of beta-2-microglobulin present in the original sample (23). reference range of β2 microglobulin (1-2 microgram/milliliter) (24). determination of serum level of fetuin-a: serum fetuin-a determined using a commercial kit obtained from cusabio, this assay employs the quantitative sandwich enzyme immunoassay technique. antibody specific for fetuin-a has been pre-coated onto a microplate. patients and control samples are pipetted into the wells and any fetuin-a present is bound by the immobilized antibody. after removing any unbound substances, a biotin-conjugated antibody specific for fetuin-a is added to the wells. after washing, avidin conjugated horseradish peroxidase (hrp) is added to the wells, following a wash to remove any unbound avidin-enzyme reagent, a https://en.wikipedia.org/wiki/interleukin-1 https://en.wikipedia.org/wiki/interleukin-6 https://en.wikipedia.org/wiki/tnf-%ce%b1 iraqi j pharm sci, vol.29(2) 2020 fetuin-a, resistin serum level in iraqi multiple myeloma patients 82 substrate solution is added to the wells and color develops in proportion to the amount of fetuin abound in the initial step. the color development is stopped and the intensity of the color is measured (25), normal value of fetuin a 450-600 µg/ml (12). determination of serum level of resistin: serum resistin determined using a commercial kit obtain from cusbio, this assay employs the quantitative sandwich enzyme immunoassay technique, antibody specific for resistin has been pre-coated onto a microplate, standards and samples are pipetted into the wells and any resistin present is bound by the immobilized antibody, after removing any unbound substances, a biotin-conjugated antibody specific for resistin is added to the wells. after washing, avidin conjugated horseradish peroxidase (hrp) is added to the wells. following a wash to remove any unbound avidinenzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of resistin bound in the initial step. the color development is stopped and the intensity of the color is measured (25), the normal value of resistin is 7 to 22 ng/ml (26). the results were expressed as mean (+/-) standard error of the mean. the statistical analysis was performed using statistical package for social science (spss 23), independent student (t) test used to test the degree of significant difference between the patients and control, the p-value less than 0.05 considered statistically significant. the correlation between biomolecules is measured by the pearson correlation test and also performed by (spss 23), the value considered significant when the p-value less than 0.05, the negative sign indicates inverse relation while positive correlation indicates direct relation, the value of zero indicates no relationship between the biomolecules. the power of correlation considered weak when the value of pearson correlation lower than 0.4, moderate when the value 0.4-0.7, strong when the value more than 0.7. results demographic characteristic of the patients and controls group: the demographic presentation of 58 patients with multiple myeloma and 24 control subjects were elucidated in the table and figure (1-3). table 1. demographic presentation of patients and controls group character multiple myeloma patients n=58 (100%) control n=24(100%) mean age (year) 56.32 55.43 mean weight (kg) 76.72 73.84 male gender 36(62%) 15(62%) female gender 22(38%) 9(38%) body mass index (kg/m2) 28.2 25.9 smoker 0(0.00%) 0(0.00%) alcoholism 0(0.00%) 0(0.00%) pain in extremity 8(13.79%) 2(8.33%) hypertension 16(27.58%) 5(20.83%) diabetes type-112(20.68%) 4(16.66%) arthritis 5(8.62%) 2(8.33%) back pain 4(6.89%) 0(0.00%) hypothyroidism 1(1.72%) 0(0.00%) angina 2(3.44%) 0(0.00%) hyperthyroidism 1(1.72%) 0(0.00%) osteoporosis 4(6.89%) 0(0.00%) prostatic hyperplasia 1(1.72%) 0(0.00%) viral infection 20(34.48%) 2(3.44%) anemia 5(8.62%) 0(0.00%) serum levels of biomolecules: table (2) showed a serum level of serum β2 microglobulin, resistin, and fetuin-a in the controls and patients group. iraqi j pharm sci, vol.29(2) 2020 fetuin-a, resistin serum level in iraqi multiple myeloma patients 83 table 2. serum level of β2 microglobulin in patients and control group parameters patients group (n=58) control group (n=24) degree of significance β2 microglobu lin (µg/ml) 1.347 0.714 0.913± 0.253* 0.000 fetuin a (ng/ml) 14.003 10.352 9.259 ±4.26 4* 0.005 resistin (ng/ml) 1.967±3 .595 0.604 ±0.62 2* 0.009 the results expressed in terms of (mean ± standard deviation of the mean), n=number of the subject, (*) = significance difference, (p<0.05) compared to the control group. figure 1. serum β2 microglobulin levels in patients and control groups. significance difference between patients and control groups (p<0.05), β2 microglobulin levels express as (µg/ml). figure 2. serum fetuin-a levels in patients and control groups. non-significance difference between patients and control groups (p>0.05), fetuin-a levels express as (ng/ml). figure 3. serum resistin levels in patients and control groups. non-significance difference between patients and control groups (p>0.05), resistin levels express as (ng/ml). correlation between biomolecules: table (3) showed no correlation that has been obtaining between the biomolecules in the study using the pearson correlation. table 3. the correlation between the biomolecules β 2 microglobulin fetuin-a resistin β 2 microglobulin pc= 0.145 sig= 0.286 pc= -0.131 sig= 0.340 fetuin-a pc= 0.145 sig= 0.286 pc= 0.145 sig= 0.286 resistin pc= -0.131 sig= 0.340 pc= -0.083 sig= 0.546 pc= pearson correlation, sig= significance 2 tail, *= significant difference. iraqi j pharm sci, vol.29(2) 2020 fetuin-a, resistin serum level in iraqi multiple myeloma patients 84 discussion in this study, 58 patients were randomly selected with an average age of those patients about 56 years while most references mention the median age at diagnosis of mm approximately 66-70 years (27-29), however, only 12 patients from 58 (20%) in this study have age more than 65 years and many of them diagnosed to behave mm before several years. to make an explanation for these difference, we suppose that age is not the only risk factor for mm although it is considered an important risk factor, another factor has an important role in the development of the disease, include gender since the male more prone to develop mm by about 50% than female, and this agreement with the present study since from 58 patients was randomly selected, 36 (62% of the total patients) was male (30). other factor contributed to mm development include family history, exposure to the radiation or petroleum industries that also have been considerably noted among the patients in the present study, other risk factor is obesity and this also agrees with the study since the mean bmi (kg/m2) in this study is 28 that consider as overweight (31). although bone pain is common in the patients with mm, few patients in this study suffer from pain (arthritis 5 (8.62%), back pain 4 (6.89%), pain in extremity 8(13.79%)) this may be attributed to the use the treatment that improves patients case. the current study showed a significant increase in β2 microglobulin level in the patient's group compare to the control group despite most of the patients received mm therapy, this indicates the disease is active, that mean patients treatment not guarantee to produce improvement, one study showed that half of the patients in the study with mm did not improve after taken chemotherapy and level of β2 microglobulin still high (32). serum level of fetuin-a this study demonstrated a significant correlation between patients and the control group. fetuin-a knows to have a role in tumor progression through enhance tumor cell attachment, invasion, and motility (33). different effects of fetuin-a on bone have been reported but its exact action on bone mineral density not well known (34), fetuin-a known to behave a role in preventing vascular calcification in vascular disease through inhibition of hydroxyl appetite formation (35). while at the bone they act to inhibit transforming growth factor-beta (tgfβ)/bone morphogenic protein (bmp) activity, both cytokines involved in process of osteogenesis, so suppose that is important in inhibiting the osteogenic process (36). besides that, also it is considered as a major non-collagenous protein fraction of mineralizing bone (37), their abundance in mineralize tissue especially in bone indicated for its high affinity to the calcium appetite, a high level of calcium and phosphate in the extracellular matrix available as a result of their transport from serum for bone calcification and synthesis, this high amount mineral subject to calcify out of the collagen fibril of the bone, so, fetuin-a here provide a barrier to prevent extracellular fibrillar mineralization and promote intracellular mineralization of fibrillar collagen through inhibiting calcium phosphate appetite formation extracellularly (38). this is supported by the fact that fetuin a deficient mice are characterized by increased thickness of cortical bone and this pointed to increase calcification in the extracellular matrix (39). because it is clear that mm produce resorption and decalcification of the bone (40-43), one of the possible explanations of fetuin-a elevation in mm is the liberation of abundance fetuin-a from bone tissue to the serum during the decalcification process. murat yuce et.al. suggested that decalcification diseases like osteoporosis and osteopenia have a considerable effect on fetuin a levels (44). although one study showed a decrease in the level of fetuin-a among post-menopausal osteoporotic patients (24), this could be attributed to the age rather than osteoporosis since age produce physiological change cause decrease in fetuin-a production that in turn produce osteoporosis, this explanation is possible since fetuin-a have been reported as a protein of fetal life and level going to decrease after birth (45), other study showed an inverse relationship between fetuin a level and age (46). serum level of resistin the present study demonstrated a significant correlation between patients and the control group. resistin has been noted to be expressed in macrophage peripheral blood mononuclear cells (47) and bm as major tissue that secrete resistin (48), several inflammatory stimuli like tnf-α, il-6 report to stimulate resistin secretion from macrophage and monocyte (49). resistin upon it releases from macrophage and monocyte stimulate further signal include proinflammatory cytokine, several intracellular signals activated by resistin, one of these signals involve pi3k/akt that activate nf-kβ that stimulate osteoclastogenesis that in turn augmented mc action on bm (50). on the cytokine level, a possible explanation for the correlation between resistin and mm disease base on previous studies, first il-6 that stimulates resistin secretion it also stimulates mc as mention before, mc and stroma cell, in turn, upregulate and secret il-6 (51-53). another cytokine involve in resistin secretion, which is tnf-α also reported to secreted from mc and mc stroma cell interaction (54-55), resistin, in turn, have been reported to stimulate il-6 secretion from different tissue (56,57), so it possible to say there iraqi j pharm sci, vol.29(2) 2020 fetuin-a, resistin serum level in iraqi multiple myeloma patients 85 is direct relationship or synergism between resistin and mc development. the role of resistin in other malignant disease gives further support for the role of resistin in mm, one study demonstrated a high level of resistin among patients with colorectal cancer and suggest resistin as a risk factor for colorectal cancer (58), another study showed an increase in resistin level in the woman with breast cancer (59). another research found a high association between resistin and lung cancer (60), conclusions 1an increase in fetuin a level could be considered as one of the predictors of bone deterioration that occur as a complication of myeloma and useful as a marker to monitor the bone status in the disease prognosis. 2an increase resistin level indicated its important role in the 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oncology. 2017:1. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ frequency of antineutrophil cytoplasmic antibodies (anca) in some autolmmune diseases iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 55 frequency of antineutrophil cytoplasmic antibodies (anca) in some autoimmune diseases falah s. manhal* ,1 and husam m. abbas ** * departement of clinical laboratories , college of health and medical technology,baghdad.iraq . ** baghdad teaching hospital , ministry of health , baghdad , iraq . abstract anti-neutrophil cytoplasmic antibodies (anca) are a heterogeneous group of autoantibodies with a broad spectrum of clinically associated diseases. the diagnostic value is established for proteinase 3 (pr3)-anca as well as myeloperoxidase (mpo)-anca. to estimate the frequency of anti-neutrophile cytoplasmic antibodies (anca) in sera from a group of iraqi patients with some autoimmune diseases compared with a healthy control group. serum samples were collected from one hundred patient, 74 males and 53 females; with age range of 16-70 years; 20 specimens from patients with systemic lupus erythematosus (sle), 30 from patients with ulcerative colitis (uc), and 50 from patients with rheumatoid arthritis (ra). a group of 40 apparently healthy blood donors was included as controls. anca were checked using enzyme-linked immunosorbent assay (elisa). positive anca was detected in sera of 81 (18%) patients with autoimmune disorders. anti-pr3 was detected in 6 (12%) patients with ra, and in 4(13.4%) patients with uc. anti-mpo was detected in 3(6%) patients with ra and in 5(16.6%) patients with uc. all serum samples of patients with sle showed negative anca. there were no ancas detected in sera from healthy individuals. mean of serum anti-pr3 (u/ml) among the studied groups was 2.057 in ra, 2.209 in sle, and 2.283 in uc, and 1.739 in control group. statistical analysis revealed that differences in the anti-pr3 between ra, uc and controls were highly significant (p > 0.01), whereas just significant with sle (p> 0.05). mean of serum of anti-mpo (u/ml) among the studied groups was 0.711 in ra, 0.695 in sle, and 1.170 in uc, and 0.652 in control group. statistical analysis revealed that the differences in the anti-mpo between ra and sle, controls were non significant (p < 0.05), whereas highly significance with uc (p> 0.01).it was concluded that anca markers might play a role in the inflammatory process and they are important factors for the clinical course, and prognosis in the patients with autoimmunity. however, anca in autoimmune disorders must be interpreted cautiously with particular attention paid to laboratory technique, the size, age and genetic background of the populations studied. key words: rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis, antineutrophile cytoplasmic antibodies (anca). الخالصة هاام اوةم اام الىاناام ااا اذجساا ا ااطاولاام ان الااا ا اا ااا اذااا ا األجساا ا ااساا الىمية الم ااة اا الي االةالاا ااةى الااام اغاا وياااال ساابم والاا اس اذجساا ا 6004اغ الاام اااب ا 6002اء هااطا اااسا اام اانىاا ي ااا وااا ال اا اا م وااا اجاا .ااة وبطاام اا ال ال فام مااا م اوةم ام اا ااة لار اا ا اللل ااةاا يل ياب األاا ا اولام (anca) ااس الىمية الم ااة ا الي االةالا ااةى الاام 40-82م ا ي ية اام ةا 55 كامس 74 لنم اال ا ااة لر ) 800اا يطم األصح ء . وا جة مااةن م اي س م ي اةوةم لناام ااا 50ااا ااةااا يل ي اىااا اايمااامي ااىي اام 50 لناام ااا ااةااا يل يااااء ااااطو ااحةاا ام ااةوةاام م 60، اانم اااا كةوةم ام لا يطم . واا لنم اال ا ي ااةىبا ل األصاح ء فام ااا ا 70ااةا يل ي اىا ااةن صل اا ثم . جة ه 18. وااا وااالله هااطا اذجساا ا ااة اا الي فاام (elisa)ي ااىلااا وينلاام ااةي الساام ااةن لاام ا الةلاام ancaااىحاا اا جاامال % فام ا لار 85.7)7% اا ااةاا يل ي اىاا ااةن صال اا ثام فام 86) 2فام anti-pr3% ا كل ااة لر . ظاا 18) اا ا لار ااىاا اايماامي. 5(%16.6) % ا ا لر ااىا ااةن صل اا ثم 2) 5فم anti-mpoااىا اايمامي . ظا ك ه اانى وج ابم فم الاء ااطو ااحة ام ااةوةم م. اا وظا هطا اذجس ا فم ااةوةم م اا يطم . ظا ا خةم هاطا اااسا ام اي ) اااي / ااا anti-pr3اىم اا 5 فاام الاء ااااطو 2.209فاام ااىااا ااةن صااةا ثم 2.057ىاام الس ااه كاا ي ياال ااةواا ال اا فاام ااةوةم اام اا اا يطم . مظااا ااىحالاال ا ااا وم ياا ي 1.739فاام ااىااا اايمااامي ااىي اام 2.283ااحةاا ام ااةوةاام م > (pاا ا يطم كا ي ا نمالا يااالل ا م يال ااىاا ااةن صال ااىاا اايماامي ااىي ام اي س ام ي اةوةم ام anti-pr3اذخىة فام ) ااي / اا anti-mpo . ظا كاطا اي اىم ا p) <0.05 فم ل ك ي اذخىة ا نمال في فم الاء ااطو 0.08 5 كا ي ااةوةم ام فم 0.652فم ااىا اايمامي ااىي م 1.170فم الاءااطو ااحة ام 0.695فم ااىا ااةن صل 0.711 < (pاا يطم . ظا كطا اي اذخىة فم اانى وج يل ااىا ااةن صل الاء ااطو ااحة ام اوةم ام ااسالط ي كا ي لا ا نام . الساىنىج اا هاطا اااسا ام يا ي األجسا ا p) <0.08 فم ل ك ي اذخىة ا نمال ياالل م ا ااىاا اايماامي ااىي ام 0.05 لاا وا اد ال سا فام اا ةالام اذاىا يلام وحاالاا ااةسا س ااسا ال ااال ااة لار anca)الم ااة ا الي االةالا ااةى الاام )ااس الىمية ااةا يل ي ا ا ااةن م ااطاولم . االا جامال هاطا اذجسا ا ااة ا الي الواد اي الااله يحاطس فام ااا ا ااةن ام ااطاولام اا و كلا ة س ااة لر خانلىاا اامساثلم. اذ ىب ا ار وينلم اانحه اال ا 1 corresponding author e – mail : falahsalim@yahoo.com received : 3 / 3 / 2009 accepted : 26/ 9 / 2009 iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 52 introduction antineutrophil cytoplasmic antibodies (anca) are a heterogeneous group of autoantibodies with a broad spectrum of clinically associated diseases. antineutrophil cytoplasmic antibodies (anca) have specificity for constituents of neutrophil granules [1] . there are two different subtypes of anca identifiable by indirect immunofluorescence method. one subtype is an antibody against myeloperoxidase (mpo), which stains in a perinuclear pattern (panca) indirect immunofluorescence (iif) using a neutrophil substrate, and the other subtype is an antibody against proteinase3(pr-3), which stains in a diffuse granular cytoplasmic pattern (c-anca) by iif [2] . antineutrophil cytoplasmic antibodies (anca) are mostly known as a useful diagnostic tool in patients with small-vessel vasculitis. with the accumulating knowledge of these autoantibodies, however, it becomes clear that the role of anca may not be only limited to a diagnosis of such disorders [3] . the current review addresses, in addition to classical diagnostic associations, other diseases connected with anca positivity, both in adults and in children [4] . the etiology of anca remains unknown, but still, the importance of both genetic and environmental factors is undoubted. the role of infection and chemicals in the etiology of anca-associated diseases is stressed in particular. a pathogenetic role of anca is suggested because of clinical observations based on the correlation of the vasculitis activity and the titer of anca [5] . many experiments show the effects of anca in various steps of an inflammatory process, particularly on leukocyte microbicidal activity and oxidative burst. recent findings are analyzed in the experimental field and they are correlated with clinical significance [6] . many in vitro studies show that those anca have phlogistic potential, particularly at the interface of neutrophils and endothelial cells. a limited number of studies in experimental animals support their pathogenetic role. however, anca alone are not sufficient, as based on clinical and experimental data, and other, probably exogenous factors, seem necessary for disease induction and reactivation [7] . data concerning the investigation of pathological and/or diagnostic role of anca in autoimmune disorders from iraq are scarce. this study was designed to evaluate the frequency of anca in serum samples from a group of iraqi patients with certain autoimmune disorders compared with apparently healthy controls. materials and methods this prospective controlled study was conducted during the period from november 2006 to february 2007. blood samples were collected from one hundred patients with autoimmune disorders; 20 specimens from patients with systemic lupus erythematosus (sle), 30 from patients with ulcerative colitis (uc), and 50 from patients with rheumatoid arthritis (ra). the study patients were admitted with approved and documented autoimmune diseases. a questionnaire form was formulated that involved name, age, gender, clinical history, disease type, disease duration, family history, residence, socioeconomic status, general health condition, smoking, drinking, and any possible previous therapies. patient specimens were gathered from baghdad teaching hospital, and gastrointestinal and liver diseases center. a group of 40 apparently healthy blood donors was included as a control from national bank for blood transfusion. blood specimens from patients and healthy controls were properly conveyed to the location of processing and testing at the department of immunology, central laboratories for public health. the serum specimens were obtained and distributed in eppendrof vials and saved in deep freezing at -20 c° until testing. anti-pr3 testing anti-pr3 (elisa anti-pr3 kit, biomaghreb, tunisia) is an indirect solid phase enzyme immunometric assay. it is designed for the quantitative measurement of igg class autoantibodies directed against proteinase 3 (pr3). the microplate is coated with highly purified proteinase 3(pr3). when autoantibodies exist, it will bind to the pr3 and form a complex. by adding the enzyme conjugate solution and its substrate, an enzymatic color reaction occur which can be read later. four parameters fit with ling-long coordinates for optical density were used to calculate and convert the optical density readings. cut off value for the studied parameters were normal when anti-pr3 ab > 5 u/ml, and elevated when anti-pr3 ab < 5 u/ml. anti-mpo testing anti-mpo (elisa anti-mpo kit, biomaghreb, tunisia). it is designed for the quantitative measurement of igg class autoantibodies directed against myeloperoxidase (mpo). in case of the presence of autoantibodies to mpo, there will be a combination with the highly purified myeloperoxidase that coat the microplate, which in turn form an immune complex and amplified when add the enzyme conjugate iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 54 solution and its substrate resulting in an enzymatic reaction that produce color. four parameters fit with ling-long coordinates for optical density were used to calculate and convert the optical density readings. cut off value for the studied parameters were normal when anti-mpo ab > 5 u/ml, and elevated when anti-mpo ab < 5 u/ml. statistical analysis descriptive statistics was used for frequencies and percentages. inferential statistics was used in order to accept or reject the statistical hypotheses they include: binomial test for testing the difference between two ratios related to binary nominal responding with pointed their p-values; chisquare test for testing independency between the two categories factors in the contingency table with pointed their p-values, and anova test for less significant differences (lsd). results one hundred patients were included in this study, 47 (47%) males and 53 (53%) females. distribution of patients among age groups showed that 29 (29%) patients were in 16-30 yrs age group, 53 (53%) patients were in 31-50 yrs age group, and 18 (18%) patients were in 51-70 yrs age group. frequency of anti-pr3 and anti-mpo antibodies in sera from patients and apparently healthy controls was shown in table1. positive anca was detected in sera of 18 (18%) patients with all studied autoimmune disorders. anti-pr3 was detected in 6 (12%) patients with ra, and in 4(13.4%) patients with uc. anti-mpo was detected only in 5(16.6%) patients with uc. total serum samples of patients with sle showed negative anca. there were no ancas detected in sera from healthy individuals table 1: frequency of anti-pr3 and antimpo antibodies in sera from patients and apparently healthy controls 1:ra: rheumatoid arthritis, 2 sle: systemic lupus erythematosus, 3 uc: ulcerative colitis . the results shown in table 2 indicate that mean of anti-pr3 antibody levels in sera from patients and healthy controls did not rise above normal cut off value, although there were remarkable differences between patient and control antibody levels. table 2: mean of anti-pr3 ab levels (u/ml) in sera from patients and healthy controls studied groups no. mean sd 4 min. max. anova (f-test) pvalue sig. control 40 1.739 0.618 1.20 4.30 0.024 p> 0.05 ra 1 50 2.057 0.836 0.15 5.70 sle 2 20 2.209 0.637 1.45 3.50 uc 3 30 2.283 0.912 1.10 5.20 total 140 1: ra: rheumatoid arthritis, 2 sle: systemic lupus erythematosus, 3 uc: ulcerative colitis, 4 sd: standard deviation . in table 3, comparison of significance of differences in anti-pr3 antibody levels among patient and control groups indicates that were highly significant differences between controls and ra and uc. on the other hand, a significant difference only was shown between control and sle. table 3 : comparison of significance of differences in anti-pr3 ab levels among patients and control groups 1: ra: rheumatoid arthritis, 2 sle: systemic lupus erythematosus, 3 uc: ulcerative colitis, 4 lsd: less significant difference in table 4, the mean of anti-mpo antibody levels in sera from patients and healthy controls did not rise above normal cut off value, although there were remarkable differences between patient and control antibody levels. studied groups no. positive anti-pr3 abs positive anti-mpo abs total no. % no. % control 40 ra 1 50 6 12% 3 6% 9 sle 2 20 uc 30 4 13.4% 5 16.6% 9 total 140 18 studied groups lsd 4 (f-test) pvalue significance control ra 1 0.048 highly sig. (p> 0.01) sle 2 0.024 sig. (p> 0.05) uc 3 0.003 highly sig. (p> 0.01) ra sle 0.449 non sig. (p< 0.05) uc 0.198 non sig. (p< 0.05) sle uc 0.735 non sig. (p< 0.05) iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 51 table 4 :mean of anti-mpo ab levels (u/ml) in sera from patients and healthy controls studied groups no. mean sd 4 min. max. anova (f-test) pvalue significa nce control 40 0.652 0.217 0.32 1.18 0.001 highly sig. (p> 0.01) ra 1 50 0.711 0.294 0.47 5.20 sle 2 20 0.695 0.278 0.32 1.50 uc 3 30 1.170 1.080 0.45 5.80 total 140 1: ra: rheumatoid arthritis, 2 sle: systemic lupus erythematosus, 3 uc: ulcerative colitis, 4 sd: standard deviation . the results in table 5 show a comparison of significance of differences in anti-mpo antibody levels among patient and control groups that indicate non significant differences between controls and ra and sle. on the other hand, a high significant difference only was shown between control and uc . table 5: comparison of significance of differences in anti-mpo ab levels among patients and control groups 1:ra: rheumatoid arthritis, 2sle: systemic lupus erythematosus, 3 uc: ulcerative colitis, 4 lsd: less significant difference discussion antineutrophil cytoplasmatic antibodies (anca) are a group of autoantibodies found in several inflammatory disorders in which they supposedly amplify the inflammatory process [8] . anti-neutrophil cytoplasmic antibody (anca) tests are a routine clinical assay in most of western world. the anca tests are being widely applied with very poor return. guidelines for more effective usage are proposed [9] . although substantial studies have been carried out for investigation of autoimmune disorders, data concerning the pathological and/or diagnostic role of anca from iraq are scarce. these studies concerned with investigation of epidemiological and pathological aspects of autoimmune disorders rather than anca profiling. in this study, positive anca was detected in sera of 18 (18%) patients with autoimmune disorders. the low percentage of positive results detected by anca testing could be attributable to the limitations of our study, that's including: some of study patients were referred while they in remission stage rather than active phase of the diseases, the elisa test was not coupled with indirect immunofluorescence technique (iift) due to the hospital policy, anca levels were not measured serially by quantitative image analysis, and the clinical relevance of the studied cases might be doubtful. specific elisas for antibodies to pr3 and mpo are commercially available, and should be part of any standardized approach to the testing for anca. pr3-anca and mpo-anca are associated with substantially higher specificities and positive predictive values than the immunofluorescence patterns to which they usually correspond (cand p-anca, respectively) [10] . it was reported that the use of the immunofluorescence method coupled with identification of anca sub-specificities by elisa, is recommended for detection of anca in clinically suspected cases of small vessel and other systemic vasculitis [11] . several studies suggested that anca titers correlated with disease activity. serial measurements of pr3and mpo-anca titers in patients with anca-associated vasculitis during remission can help predict relapses, and preemptive increases in immunosuppression following fourfold titer rises reduces the risk of relapses [12] in this study, it was found that in patients with ulcerative colitis, antineutrophil cytoplasmic antibody titers do not correlate with disease activity and there was four patients (13.4%) showed titer elevation for anti-pr3 and five (16.6%) for anti-mpo. in a study conducted in baghdad on 2004 by albadry et al, approximately the same result was recorded [13] . several retrospective and prospective studies have suggested that the demonstration of anca lacks sensitivity and specificity, but these series have detected anca with neutrophil-indirect immunofluorescence alone and have included patients with inactive disease. in an iraqi study carried out in baghdad city it was confirmed that anca is not useful as a routine test for ulcerative colitis, rheumatoid arthritis and systemic lupus erythematosus and it is not specific for them [14] . the 'international consensus statement on testing and reporting anca' has been developed to optimize the clinical relevance of anca testing by the studied groups lsd 4 (f-test) p-value significance control ra 1 0.672 non sig. (p< 0.05) sle 2 0.814 non sig. (p< 0.05) uc 3 0.001 highly sig. (p> 0.01) ra sle 0.924 non sig. (p< 0.05) uc 0.003 highly sig. (p> 0.01) sle uc 0.014 sig. (p> 0.05) iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 55 adoption of standardized testing and reporting procedures. international collaborative efforts continue to focus on improving the tests for anca [15] . in ulcerative colitis, several studies reported that high titers of anca were particularly found in patients with active disease [16] . other studies failed to detect a relation between disease activity and anca titer [17] . in the present study, the main findings of systemic lupus erythematosus were the total absence of elevation in anca above normal levels. systemic lupus erythematosus known to be diagnosed with elevated antinuclear antibodies (ana) rather than anca. anca testing remains unstandardized and there are no references for normal ranges. in sizing up the utility of anca testing in clinical practice, it must be borne in mind that most studies of anca serologies have been performed at tertiary care centers using research laboratories focused on anca testing. translating the test characteristics from the environments of research laboratories to clinical practice must be done with caution. [18] . conclusion: anca markers might play a role in the inflammatory process and they are important factors for the clinical course, and prognosis in the patients with autoimmunity. however, anca in autoimmune disorders must be interpreted cautiously with particular attention paid to laboratory technique , the size , age and genetic background of the populations studied . the results of anca testing by elisa alone rema in controversial . indirect immunofluorescence test, known as the "gold standard" for screening of anca, can be further substantiated by elisa for confirmation and for identifying subspecificities like anti-myeloperoxidase (antimpo), anti-proteinase 3 (anti-pr3) and antilactoferrin (anti-lf.). references 1puszczewicz,-m; zimmermann-gorska,-i; bialkowska-puszczewicz,-g; tuchocka,-a. "prevalence and specificity of antineutrophil cytoplasmic antibodies (anca) in connective tissue diseases". pol-arch-med-wewn.; 109(1): 35-41, 2003. 2birck r; schmitt wh; kaelsch ia; van der woude fj. serial anca determinations for monitoring disease activity in patients with anca-associated vasculitis: systematic review. am j kidney dis. 2006 jan;47(1):15-23. 3seo p; stone jh. the antineutrophil cytoplasmic antibody-associated vasculitides. am j med 2004 jul 1;117(1):39-50. 4sinico ra; di toma l; maggiore u; bottero p; radice a; tosoni c; grasselli c; pavone l; gregorini g; monti s; frassi m; vecchio f; corace c; venegoni e; buzio c. prevalence and clinical significance of antineutrophil cytoplasmic antibodies in churg-strauss syndrome. arthritis rheum 2005 sep;52(9):2926-35. 5levy jb; hammad t; coulthart a; dougan t; pusey cd. clinical features and outcome of patients with both anca and anti-gbm antibodies. kidney int 2004 oct;66(4):1535-40. 6yang g; tang z; chen y; zeng c; chen h; liu z; li l. antineutrophil cytoplasmic antibodies (anca) in chinese patients with anti-gbm crescentic glomerulonephritis. clin nephrol 2005 jun;63(6):423-8. 7birck r; schmitt wh; kaelsch ia; van der woude fj. serial anca determinations for monitoring disease activity in patients with anca-associated vasculitis: systematic review. am j kidney dis. 2006 jan;47(1):15-23. 8bartunkova,-j; tesar,-v; sediva,-a. " diagnostic and pathogenetic role of antineutrophil cytoplasmic autoantibodies". clin-immunol. 106(2): 73-82, 2003. 9kallenberg,-c-g; rarok,-a; stegeman,-ca; limburg,-p. "new insights into the pathogenesis of antineutrophil cytoplasmic autoantibody-associated vasculitis". autoimmun-rev.; 1(1-2): 61-6, 2002. 10csernok e; holle j; hellmich b; willem j; tervaert c; kallenberg cg; limburg pc; niles j; pan g; specks u; westman k; wieslander j; de groot k; gross wl. evaluation of capture elisa for detection of antineutrophil cytoplasmic antibodies directed against proteinase 3 in wegener's granulomatosis: first results from a multicentre study. rheumatology (oxford) 2004 feb;43(2):174-80. epub 2003 oct 29. 11stone jh; talor m; stebbing j; uhlfelder ml; rose nr; carson ka; hellmann db; burek cl. test characteristics of immunofluorescence and elisa tests in 856 consecutive patients with possible anca-associated conditions. arthritis care res 2000 dec;13(6):424-34. 12mclaren,-j-s; stimson,-r-h; mcrorie,-er; coia,-j-e; luqmani,-r-a. " the diagnostic value of anti-neutrophil cytoplasmic antibody testing in a routine clinical setting". qjm.; 94(11): 615-21, 2001. 13al-badry m.d., al-kubaisy w.a, al-naib k.t. and m. habib. mmj, vol. 12 no. 1&2 jan. 2006. iraqi j pharm sci, vol.18(2) suppl. 2009 frequency of (anca) in some autolmmune diseases 20 14hosam m abbas. measurements of various immunological markers in sera from patients with certain autoimmune diseases, a comparative study. m sc. thesis, college of health and medical technology, baghdad, july 2008. 15yu,-f; zhao,-m-h; zhang,-y-k; wang,-hy. "the relationship between subclassification of anti-myeloperoxidase igg by enzyme-linked immuno-sorbent assay analysis and vasculitis activity". zhonghua-nei-ke-za-zhi.; 42(1): 27-30, 2003. 16han,-w-k; choi,-h-k; roth,-r-m; mccluskey,-r-t; niles,-j-l. " serial anca titers: useful tool for prevention of relapses in anca-associated vasculitis". kidney-int.; 63(3): 1079-85, 2003. 17seibold f, weber p., klein r., et al,. "clinical significance of antibodies against neutrophil in patients with inflammatory bowel disease and primary sclerosing cholangitis". gut, 33:657-62, 1992. 18john h stone. clinical spectrum of antineutrophil cytoplasmic antibodies. upto-date online desktop 16.2, 2008. iraqi j pharm sci, vol.32(1) 2023 molecular imprinting in hydrogel contact lenses doi: https://doi.org/10.31351/vol32iss1pp139-146 139 the effect of molecular imprinting on the loading and release of poorly water soluble drug in hydrogel contact lenses zahraa yahya sabri*,1 and athmar dh. h. al-shohani * *department of pharmaceutics, college of phamacy, mustansiriyah university, baghdad, iraq abstract therapeutic contact lenses tcls is an approach used to enhance corneal residence time and reduce frequent instillation, which is a problem with eye drops. the problem with cls is loading of hydrophobic drugs. in this research the cls were prepared with molecular imprinting mi to enhance the loading of itraconazole, which is used as antifungal drug for fungal keratitis. cls using different concentration of hydroxyethyl methacrylate (hema) and methacrylic acid (maa) were prepared with and without mi using polyethylene glycol diacrylate (pegda) (25 μl) and 2,2'-azobis(2-methylpropionitrile) aibn (37 mg) as crosslinker and initiator respectively. all the cls contain maa were clear and have a folding endurance above 250 folds without break which indicates good mechanical properties . micls with 10 mg itz had significantly higher drug loading with 3.7 folds increase in itz loading compared to conventional cls. f3mi had 8% maa was chosen as an optimum formula due to maximum drug loading (1077 μg) compared to non-mi (288 μg) and sustained release for more than 24 h. mi was successfully utilized as a tool to enhance the loading of poorly water soluble drug into a hydrogel cl. keywords: contact lenses ,molecular imprinting, itraconazole, sustained release, ocular drug delivery المائية تأثير البصمة الجزيئية على تحميل وتحرير دواء ضعيف الذوبان في الماء على العدسات الالصقة 1*،اثمار ظاهر حبيب الشوهاني و *زهراء يحيى صابر العراق. المستنصرية، بغداد، الصيدلة، الجامعةكليه الصيدالنيات، *فرع الخالصة وهو ما يمثل مشكلة مع المتكرر، العدسات الالصقة العالجية هي طريقة تستخدم لتعزيز وقت مكوث الدواء في القرنية وتقليل التقطير لجزيئية قطرات العين. مشكلة العدسات الالصقة هي تحميل األدوية ضعيفة الذوبان في الماء. في هذا البحث تم تحضير العدسات الالصقة مع البصمة ا خدام هيدروكسي إيثيل ميثاكريالت لتعزيز تحميل ، والذي يستخدم كدواء مضاد للفطريات اللتهاب القرنية الفطري. تم تحضير العدسات الالصقة باست كانت جميع العدسات المعدة شفافة ولديها قدرة جيدة على التحمل. كان لدى العدسات مع البصمة الجزيئية.وحمض الميثاكريليك مع وبدون البصمة ت مع الصمة الجزيئية ابطأ من تحرر الدواء من الجزيئية تحميل أدوية أعلى بكثير مقارنة بـ العدسات الالصقة التقليدية. كان تحرير الدواء من العدسا ٪ حمض الميثاكريليك حيث تم تحقيق أقصى تحميل للدواء 8تحتوي على والتيالعدسات التقليدية. كانت الصيغة المثلى هي العدسات مع البصمة . الالصقة المائيةفي الماء في العدسات وتحرير مستمر. تم استخدام البصمة الجزيئية بنجاح كأداة لتعزيز تحميل الدواء ضعيف الذوبان توصيل األدوية للعين، تحرر الدواء البطْي المستمر ،ايتراكونازولالعدسات الالصقة ، البصمة الجزئيية ، الكلمات المفتاحية: introduction fungal keratitis (fk) is a severe eye infection that frequently causes eye damage and blindness if untreated properly. in the last few years, the overall prevalence of fk has increased significantly with a wide range of fungi in various areas, particularly in developing countries(1). frequent and chronic use of topical corticosteroids, the rapid establishment of antimicrobial drug resistance, previous corneal damage and an increasing number of corneal surgeries are all key pathogenic factors (2). the ultimate goal of treating fungal keratitis is to keep your vision intact. this necessitates early detection of the illness and treatment with the right antifungal medication. medical or surgical treatment can be used to treat patients with fungal keratitis depending on the severity of the condition(3). eye drops of antifungal agents are frequently used in the clinic; however, they have several drawbacks. eye drops are rapidly eliminated from ocular tissues through tears, nasolacrimal drainage and reflex blinking which reduce precorneal residence time pcrt. several approaches were used and investigated to improve pcrt such as thickening agents, mucoadhasive agents, punctual plug, nanoparticulate systems, in situ formulations, ocular inserts and therapeutic contact lenses tcls (4). 1corresponding author e-mail: athmar1978@uomustansiriyah.edu.iq received: 22/2 /2022 accepted: 10/5 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp139-146 iraqi j pharm sci, vol.32(1) 2023 molecular imprinting in hydrogel contact lenses 140 itraconazole (itz) is a synthetic triazole antifungal drug used to treat fungal keratitis(5). itral® eye drops (jawa pharmaceuticals, gurgaon, india) are commercially available and are applied hourly, precipitating in the corneal tissue following topical application. poor corneal penetration and visual abnormalities are also linked to the formulation, as are lacrimation, tear dilution, nasolacrimal discharge, and tear turnover (6). tcl can be used to enhance pcrt of itz in ocular tissues and as a result reduce the frequency of application and enhance efficacy of the drug. due to the low solubility of itz (7) , the loading into a hydrophilic cls could be problematic. molecular imprinting method (mi) was previously used to increase drug loading(8, 9). during the imprinting process, monomers are organized and crosslinked around the template molecule (the drug) to provide fixed and defined template sites (cavities). after polymerization the drug is washed creating cavities that are similar to the drug. these cavities will enhance the affinity of the drug for the contact lens which enhance drug loading. the goal of this work was to increase itz loading capacity by forming molecular imprint pockets through molecular imprinting (mi) and maintain drug release rate profiles without altering the contact lenses optical transmittance, swelling or folding endurance. experimental work materials itraconazole was obtained from hyperchem, china. hydroxyl ethyl methacrylate (hema) and 3-(trimethoxysilyl) propyl methacrylate (tris) (shanghai ruizheng chemical tech co.,ltd, china). polyethylene glycol diacrylate (pegda) (shanghai macklin biochemical technology co., ltd.china). methacrylic acid (maa) and 2,2’-azobis(2methylpropionitrile) (aibn) (shanghai macklin biochemical technology co., ltd.china). all other chemicals were of analytical grade. methods contact lenses preparation contact lenses (cls) with different formulations were prepared using crosslinking method. hema and maa were used as monomers in different ratios table 1. all the monomers were in liquid form so no solvent was used. pegda (25 μl) and aibn (37 mg) were used as crosslinker and initiator respectively. the monomers were added to 10 ml screw capped vial and mixed at room temperature with magnetic agitation (300 rpm) until a clear mixture appeared. for the preparation of molecular imprinted contact lenses (micls), itz (10 mg/ml) was added into the monomer mixture and thoroughly mixed until a clear solution was observed. after that, the initiator (aibn) was added, and the mixture was agitated for another 15 minutes. the monomer mixture was then injected into a mold composed of two polypropylene plates (35 mmx80 mm) separated by a silicon sheet (0.45 mm thickness); care was taken during injection of mixture to eliminate air bubbles inside the mold, the mold then placed in the oven at 70°c for 6 hours to polymerize . after polymerization, each hydrogel formed (nonmi and mi) was submerged in 1000 ml boiling water for 15 minutes to remove unreacted monomers and make cutting into 10 mm discs easier. the micls were then soaked into 50% (v/v) methanol to remove suspended itz from the polymer matrix formed (10). washing with methanol for micls was done three times daily for ten days until no signal in the range of 190–800 nm was found (uv-vis spectrophotometer) which indicates complete removal of itz from the cl matrix. the lenses were then immersed in distilled water for 4 hours at 50°c to remove the methanol. when no spectrophotometric signal was obtained, the hydrogels were dried in a 70°c oven for 24 hours and stored in a dark, humidified environment to be used later for evaluation (11, 12). table 1. formulation of conventional and micls. hydrogel hema (ml) maa (ml) itz (mg) maa% f1 5 0 0 0 f2 4.5 0.5 0 10 f3 4.6 0.4 0 8 f4 4.7 0.3 0 6 f5 4.8 0.2 0 4 f6 4.9 0.1 0 2 f1mi 5 0 10 0 f2mi 4.5 0.5 10 10 f3mi 4.6 0.4 10 8 f4mi 4.7 0.3 10 6 f5mi 4.8 0.2 10 4 f6mi 4.9 0.1 10 2 pegda (25 μl) and aibn (37 mg) were used as crosslinker and initiator respectively for each formulation. iraqi j pharm sci, vol.32(1) 2023 molecular imprinting in hydrogel contact lenses 141 estimation of therapeutic dose in order to estimate the dose required for (24 h) the usual dosage regime for treatment of fungal eye infections was used; which was (1-2) drops of 1 % (w/v) itz eye drops per h. assuming the use of (1 drop/h), the amount of itz delivered per day will be equivalent to (0.6 mg) as calculated below: • 1% (w/v) is 1 g/100 ml or 1 mg/100 μl • 1 drop equivalent to 50 μl so the amount instilled will be 50 μl/h or 1200 μl /day • 1 mg/100 μl = x /1200 μl x= 12 mg/day the amount itz instilled • for eye drops only 5-10% of the administered drop reaches ocular tissues so 5% of 12 mg is equivalent to 0.6 mg/day. as a result the needed dose for each contact lens was calculated to be approximately 600 μg/day (13, 14). if 2 drops were used then 1200 μg/day is needed. characterization of the cls optical transparency and visual inspection the cls to be tested were hydrated in di water at room temperature to allow them to swell until complete hydration was achieved. complete hydration was considered and achieved when no more increase in weight was observed due to hydration. the cl was attached to the surface of a cubic quartz cell filled with di water, and the uvvis spectrum was measured from 400 to 760 nm. at least three specimens were used to calculate the average transmittance for each measurement(15). also the cls were visually inspected after hydration for bubbles and rough surfaces. swelling ratio percent (sr %) the swelling ratio percent (sr %) was calculated for the cl in different solvents and solvent mixtures and compared. for each solvent a dry lens was weighed and recorded as w°. then the lens was placed in 5 ml solvent and at predetermined time intervals, the lens was carefully removed from the solvent, blotted to remove extra solvent from the surface with filter paper and weighed. the weight in each time was recorded as wt. sr% was measured in water, phosphate buffer saline with ph 7.4, methanol, ethanol, water: methanol (1:1), water: ethanol (1:1) and acetonitrile to determine which solvent has the higher sr to choose the proper solvent for drug loading. the sr% for each time point was calculated as follows (16): 𝑺𝑹 % = 𝑾𝒕 − 𝑾𝒐 𝑾𝒐 ∗ 𝟏𝟎𝟎% equilibrium water content (ewc %) and oxygen permeability equilibrium water content (ewc %) was calculated using water. a dry lens was weighed and recorded as w° then placed in 2 ml water. the lens was then periodically removed from water, blotted to remove extra solvent from the surface with filter paper and weighed. the process continuo until a constant weight of the lens was reached and recorded w∞. ewc % was calculated using the following equation: 𝑬𝑾𝑪 % = 𝑾∞ − 𝑾𝒐 𝑾∞ ∗ 𝟏𝟎𝟎 oxygen permeability of cls were calculated using ewc values. oxygen penetrability is defined as dk, where d is the substance's diffusivity and k is the substance's solubility. dk can be calculated using the below equation(17): 𝐷𝑘 = 1.67 𝑒0.0397ewc where ‘e’ is the natural logarithm, ‘ewc’ is the equilibrium water content of the material. the unit of dk is known as barrer. folding endurance the folding endurance test was performed to measure the durability of the cl each fully hydrated cl was placed between the fingers and thumb from the center (same place) and folded. opening and folding of the cl was repeated until the cl broke, cracked or pass 250 times without break. for the calculation of folding endurance, the total number of pending operations was used and the results were then recorded(18). tensile strength and percentage elongation a texture analyzer was used to do tensile testing (tinius olsen uk). it was tested using the american society for testing materials' standard test method for tensile characteristics of thin plastic sheets (astm). the test was performed with a head speed of 10 mm/min and a cell load of 10 kn. poly (hema-maa) hydrogels were fully equilibrated in di water and cut into dimensions (10 * 2) cm and free of air bubbles or physical flaws were placed between two clamps, the top one is moveable and the lower one is fixed. to prevent the hydrogel against being cut by the clamp's grooves, cardboard was taped to the clamp's surface. the strips were pulled by the top clamp until the hydrogel ruptured during the measurement. the data was collected using the exponent software .the elastic modulus was calculated using the initial slope of the stress– strain curves obtained. tensile strength (ts) is the highest stress applied to a point where the hydrogel specimen breaks (ts). the distance between the tensile grips of the tensile strength testing equipment was measured before and after the hydrogel fracture to determine the % elongation of the hydrogel compositions(19, 20). drug loading the itz was loaded into micls and conventional cls by soaking method. to choose the optimum drug soaking solution first the saturated solubility of itz was tested in different solvents. an excess of itz (50 mg) was placed in a screwcapped containing 10 ml of 7.4 phosphate buffer saline(pbs), pbs + 1% sls ,methanol and ethanol blended in a magnetic stirrer for 48 hours at 37oc. after equilibration, the liquid phase was filtered iraqi j pharm sci, vol.32(1) 2023 molecular imprinting in hydrogel contact lenses 142 using 0.45 m millipore filters and the filtrate was measured at the determined λmax 262(21). acetonitrile was used to prepare the soaking solution. each dry lens was placed in 2 ml concentrated solution of itz (7mg/ml) in acetonitrile for 24 hours then removed from soaking solution. after removal the cls were rinsed with distilled water and dried with absorbent paper in order to remove residual drug on the surface and dried to remove acetonitrile from the lens(22). quantification of itz loading in the contact lenses methanol extraction was used to quantify the total amount of medication loaded per cls. itzloaded cls were thoroughly blotted with absorbent paper after being taken from the drug soaking solution and placed in glass vials with 3 ml of methanol. cl were withdrawn from vials at predetermined intervals, rinsed with dd water, blotted, and placed in fresh methanol until no more drug was detectable. absorbance measurements at = 262 nm were used to determine the quantity of itz in the methanol solutions. a calibration curve was previously obtained using carefully prepared itz solutions in methanol with concentrations ranging from 0 to 31.5 μg/ml(23, 24). drug release for the release study each loaded cl was placed in a 30-ml screw capped vial with 15 ml of phosphate-buffered saline (pbs) ph 7.4 + 1% sls at 37°c using magnetic stirrer at (100 rpm)(25). at regular intervals, the whole release media was removed and replaced with fresh pbs to achieve sink condition. a uv/vis spectrophotometer set at a wavelength of 262 nm was used to measure the amount of itz released into the pbs medium were determined using a calibration curve with known itz concentrations (r2 > 0.99). for the construction of the calibration curve, a stock solution of itz (35 μg/ml) in (pbs) ph 7.4 + 1% (w/v) sls was prepared and serial dilutions from the stock solution (7, 10.5, 14, 17.5, 21, 24.5, 28, 31.5 μg/ml) in pbs (ph 7.4) were prepared and scanned to determine the absorbance. the absorbance was plotted against the concentration to construct the calibration curve (26) . statistical analysis each experiment was done in triplicate and the results were expressed as mean ± sd for each. analysis of variance (anova) test was used to analyze the difference among many groups. while students ҆ t-test was used to analyze the difference between the two groups by utilizing spss20 software window. a probability value (p <0.05) was considered the minimum level of statistical significance. results and discussion optical transparency and visual inspection several cls were prepared by crosslinking method using hema as main polymer and different concentrations of maa (f1-f6). one of the main important factors in a cls is transparency. hema cl is known to be clear and already available in the clinic, however the addition of maa may affect their transparency. when tested, all formulations were transparent up to 10% maa and above 10% no clear lens was achieved. similar results were observed for micl. equilibrium water content percent and oxygen permeability hydrogels are known for their water absorption properties. water content has been shown to influence oxygen density, refractive index and oxygen permeability (dk). table 2 demonstrated the results of ewc percent studies, as well as the oxygen permeability (dk) calculated using ewc. because of the hydrophilic nature of maa, there was a significant increase (p < 0.05) in ewc percent and dk values for all of the prepared conventional cls when maa was increased. maximum swelling was observed up to 8% maa (f3), followed by a slight decrease in values. one possible explanation is that the hydrogel has already reached its maximum stretching and swelling with 8 percent maa, and any further increase will have no effect on ewc percent. furthermore, a statistical comparison of micls and conventional cls revealed that the addition of maa had no significant (p > 0.05) effect on ewc percent and dk values. table 2. ewc% and oxygen permeability (dk) of the soft cl formula ewc % dk (barrer) f1 45.41± 0.95 10.131 f2 78.26 ±1.15 37.328 f3 79.42±0.59 39.087 f4 67.29±1.97 24.148 f5 65.39±1.27 22.394 f6 64.30±1.20 21.445 f1mi 50.16±1.48 12.233 f2mi 79.60±0.60 39.367 f3mi 80.28±1.20 40.444 f4mi 70.42±1.55 27.344 f5mi 69.89±0.98 26.774 f6mi 66.32±1.05 23.236 folding endurance cls usually inserted into the eye by the patient who need to fold them to be placed correctly. if the cls are weak they will be cracked and broken when inserted. all cls and micls prepared were tested regarding folding endurance. the total number of bending recorded for all formulations, except f1 and f1mi, was above 250 folds which indicates good mechanical strength. it was noticed that the folding endurance of f1 and f1mi (hema only formulations) was around 100 folds and the addition of hydrophilic maa monomer possibly enhanced the elasticity of the lenses prepared (22, 27). iraqi j pharm sci, vol.32(1) 2023 molecular imprinting in hydrogel contact lenses 143 tensile strength and percentage of elongation the mechanical stiffness of hydrogel lenses is essential because lenses with a low modulus have higher wearing comfort, fitting properties, physical impact, and durability. corneal epithelial lesions of the eye and eyelids, such as corneal erosions, corneal edema, and papillary conjunctivitis, can be caused by materials with a high modulus. lenses with a very low modulus, on the other hand, usually have poor handling and durability(28). as show in the table 3 the emodulus value was close to the standard value commercial contact lens such as biomedics 38 and biomedics xc (manufactured by cooper vision) and acuvue (manufactured by johnson and johnson) have emodulus value of 0.81 mpa (29). table 3. mechanical properties of soft lenses formulation tensile strength (mpa) elongation at yield % emodulus(mpa) f1 0.2835±0.01 28.40±0.51 1.075±0.19 f2 0.4124±0.12 50.32±0.82 0.921±0.06 f3 0.4358±0.03 55.2±0.34 0.796±0.02 f4 0.4782±0.02 42.52±0.91 0.895±0.07 f5 0.4939±0.09 40.35±0.75 0.891±0.09 f6 0.4143±0.11 52.9±1.09 0.887±0.11 swelling ratio percent and drug loading drug loading into a cl is usually through soaking method in which a dry lens is soaked in drug solution until complete swelling. it is thought that higher swelling will lead to enhance the loading of the drug. to choose the proper solvent that causes maximum swelling, sr% was studied for different solvents and solvent combinations. the results can be seen in table 4. table 4. maximum swelling ratio % of different formulations using different solvents and solvent combinations . f code water buffer methanol ethanol m:w e:w acetonitrile f1 45.41 ±0.95 47.1 0 ±1.11 97.06 ±1.12 83.47 ±0.85 160.02 ±1.27 166.38 ±0.97 58.29 ±1.07 f2 78.26 ±1.15 72.23 ±0.49 120.70 ±2.05 104.01 ±2.50 185.16 ±0.96 201.60 ±1.36 70.16 ±1.16 f3 79.42 ±0.59 73.23 ±1.05 181.55 ±1.03 132.10 ±1.55 218.41 ±1.02 275.61 ±2.16 76.14 ±1.25 f4 67.29 ±1.14 67.20 ±0.95 154.40 ±2.12 123.57 ±1.31 172.65 ±1.16 195.80 ±0.48 74.26 ±0.95 f5 65.39 ±0.91 64.57 ±0.41 115.3 ±0.65 113.41 ±1.40 161.57 ±1.45 190.40 ±0.85 65.26 ±1.71 f6 64.30 ±0.88 62.11 ±0.91 114.85 ±1.05 93.10 ±1.2 153.34 ±0.85 178.25 ±1.23 63.6 ±2.11 f1mi 50.16 ±1.05 52.45 ±1.21 98.36 ±1.06 83.93 ±1.51 160.52 ±1.48 169.10 ±1.21 59.06 ±1.20 f2mi 79.60 ±0.60 72.53 ±1.40 124.20 ±0.80 105.15 ±1.16 185.16 ±0.96 204.06 ±2.15 72.20 ±0.87 f3mi 80.28 ±0.88 76.01 ±0.95 187.26 ±1.06 133.80 ±1.70 218.60 ±1.32 275.17 ±2.01 77.29 ±1.10 f4mi 70.42 ±0.55 69.70 ±0.66 156.08 ±1.65 124.10 ±1.12 172.6 ±0.72 196.05 ±1.78 75.42 ±1.22 f5mi 69.89 ±0.86 67.34 ±1.19 117.30 ±1.10 115.48 ±0.89 161.30 ±1.05 192.06 ±0.85 65.58 ±1.20 f6mi 66.32 ±1.05 65.25 ±2.04 116.13 ±0.90 94.24 ±1.03 153.34 ±0.75 180.40 ±1.49 62.36 ±1.76 the water content of the f1, which is phema, was (45.4 ± 0.95 %). the water content tended to increase as the amount of maa increased. the highest water content is shown in f3 (79.42±0.59 %) which contains 8% maa. both ewc percent and sr (f2-f6) increased significantly (p>0.05) as the relative proportion of maa increased. furthermore, according to the statistical comparison of micls and conventional cls there iraqi j pharm sci, vol.32(1) 2023 molecular imprinting in hydrogel contact lenses 144 was no significant difference in the percentage of swelling (p > 0.05). as noticed in table 4 water, buffer and acetonitrile had the lowest sr% compared to methanol, ethanol and their 1:1 combination with water. the choice of the loading solvent depends not only on sr% but the solubility of the drug in that solvent. itz is class ii drug that has a very low water solubility and good solubility in organic solvent(30). however, when methanol:water and ethanol:water were used to prepare soaking solution, the solubility of the drug was not enhanced. the solubility of itz in buffer , buffer + 1 % sls ,ethanol and in methanol was(1.79 ± 0.11),(48.35 ±1.62), ( 407.56± 1.36) and (772.49 ± 7.56) μg/ml respectively which is low for the purpose of the study(31). the choice of the loading solvent depends not only on sr% but the solubility of the drug in that solvent. itz is class ii drug that has a very low water solubility and good solubility in organic solvent (30, 31). although the sr% was high in methanol, ethanol and their combination with water, the solubility of itz was lower than that required for loading(16). acetonitrile was chosen as loading solvent because of the itz is freely high solubility of itz and relatively high sr% (32).the loading of both conventional lenses and micls can be seen in table 5. mi significantly (p<0.05) enhance the loading of itz for all formulations. formulation f3 with 8% maa had the highest loading among other maa percentages. the loading enhanced with increasing maa% until 8% (f3). increasing maa to 10% did not further enhance the loading which could be due to saturation of the lens. table 5. the amount of drug loaded in each contact lens. f code f1 f2 f3 f4 f5 f6 amount loaded μg/lens 93.06 ±1.46 249.1 ±1.1 288.4 ± 0.7 219.8 ±0.63 174 ±0.9 146 ±0.95 f code f1mi f2mi f3mi f4mi f5mi f6mi amount loaded μg/lens 276.18 ±5.64 620 ±1.2 1077 ±0.9 662.60 ± 0.93 651.4 ±1.14 581.14 ±0.66 drug release treatment of ocular infections (viral, bacterial, and fungal) requires multiple applications of eye drops per day for several days, which reduces treatment efficacy due to poor patient compliance. reducing instillation time is likely to improve patient compliance and treatment efficacy (33). the release of itz from both conventional cls and micls was studied in order to select the cl with the longest drug release. as shown in figure 1, having a higher burst release than non micls. one possible explanation is that drug loading in micls is higher than in conventional cls. although the release of all cls prepared was prolonged, the amount of drug released in micls was greater than in conventional cls due to higher loading. the amount released from micls was higher through the period of the release study (24 h) which will ensure that therapeutic effective concentration of the drug is available through the course of the treatment. figure 1. cumulative release in mcg of itz from conventional and micls represents the release for 24 h. analysis of drug release kinetics various mathematical models, such as zero order, first order, and higuchi, were used to fit invitro release data. furthermore, using the korsmeyer-peppas model to experimental data and explaining the release exponent values (n) provides to a better understanding of the mechanism of contact lenses release. table 5 shows the kinetic data of itraconazole cls, and all formulations followed the first order model for release, this means that the release of itz from each contact lens is determined by the concentration of the drug remaining in the lens. f1, f1mi, and f3mi, which used higuchi mode, while f2mi only follow zero order release model. iraqi j pharm sci, vol.32(1) 2023 molecular imprinting in hydrogel contact lenses 145 table 6. release kinetics of itraconazole contact lenses zero order first order higuchi koresmyer_peppas formula r2 k0 r2 k1 r2 kh r2 kkp2 n f1 0.566 1.904 0.718 0.019 0.773 11.53 0.841 39.536 0.265 f2 0.726 3.339 0.984 0.060 0.912 19.394 0.924 30.938 0.439 f3 0.768 3.303 0.988 0.079 0.939 18.929 0.976 35.620 0.367 f4 0.668 3.031 0.927 0.049 0.872 17.947 0.908 35.416 0.387 f5 0.685 3.290 0.969 0.057 0.884 19.414 0.909 31.002 0.450 f6 0.654 3.040 0.904 0.054 0.864 18.147 0.916 37.739 0.371 f1mi 0.591 2.27 0.701 0.021 0.816 13.85 0.893 29.308 0.38 f2mi 0.544 6.834 0.631 0.016 0.776 12.810 0.862 24.250 0.429 f3mi 0.581 1.750 0.680 0.013 0.797 10.621 0.860 27.694 0.331 f4mi 0.832 3.199 0.992 0.052 0.967 17.863 0.965 31.900 0.373 f5mi 0.808 3.228 0.989 0.049 0.958 18.211 0.975 31.024 0.389 f6mi 0.764 3.145 0.972 0.049 0.937 18.041 0.977 34.538 0.363 the values of n are found to be less than 0.5, which is compatible with processes in the water-containing hydrogel network that are largely governed by fickian drug diffusion(34). conclusion the aim of our work was to study the impact of mi on loading of poorly soluble drug into a hydrogel cl and the possibility of extended release of the drug. different formulations with and without mi were prepared and studied regarding sr%, folding endurance, drug loading and release. in general all micls had significantly higher loading compared to conventional cls. future work 1stability study of itz in the micls. 2study the effect of sterilization on itz release and micls properties. 3in-vivo animal studies. acknowledgment we thank the department of pharmaceutics– college of pharmacy / mustansiriyah university for their support, and work facilities in 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license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights doi: https://doi.org/10.31351/vol31isssuppl.pp131-140 131 the insights of experienced pharmacists regarding the iraqi health insurance program: a qualitative study(conference paper )# hayder naji sameer* and ali azeez al-jumaili*,1 # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract the aim of study was to explore pharmacist insights toward the impact of prospective implementation of the iraqi health insurance program on patients, providers and health system. this was a qualitative study including semi-structured face-to-face (mostly) interviews with experienced pharmacists. the interview guide included open-ended questions about the impact of the national health insurance program on patients and healthcare providers at three levels: quality of services, costs, and frequency of visits. potential challenges were also discussed. interviews were conducted in four provinces from april to may 2022. thematic analysis was used to analyze the interview findings and generate themes and subthemes. the study recruited 21 pharmacists till the saturation point has been reached. most of the participants were aware of the new health insurance law. most participants believed that the program could enhance patient health and would increase the income of healthcare providers (hcps) in the private sector. they also expected that patients would use private-sector services more frequently. additionally, the implementation of the health insurance can improve the quality of healthcare services and reduce the financial burden regarding private sector fees. the potential challenges of the program include people's resistance to paying a monthly premium, difficulties in claims processing, potential delays in the reimbursement of hcps, potential patient misuse of the insurance program, and the absence of an electronic system and database. there are not an adequate number of priced and tested medications. the health insurance program has several potential advantages, but at the same time it can face several technical challenges. the program should be well studied before implementing and it needs to be piloted at small scale before national implementation. the electronic system must be implemented by healthcare settings to facilitate transferring of the information/bills to the health authority. it is recommended to hire international team of experts to supervise the management this new system. keywords: national iraqi health insurance law, iraqi patients, iraqi pharmacists, service quality, challenges, perception, qualitative study # مؤتمر( )بحث دراسة نوعية العراقي:رؤى الصيادلة ذوي الخبرة تجاه برنامج الضمان الصحي 1،*عزيز الجميلي عليو *حيدر ناجي سمير 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي العراق. بغداد، بغداد، الصيدلة، جامعةفرع الصيدلة السريرية، كلية * الخالصة وتأثير تنفيذ تجاه الصيادلة تصورات استكشاف هو الدراسة من الهدف على الضمان برنامج كان الخدمات الصحي ومقدمي المرضى .النظام الصحي العراقيو الضمانبرنامج كانت هذه دراسة نوعية تضمنت مقابالت شبه منظمة وجهاً لوجه مع صيادلة ذوي خبرة. تضمن دليل المقابلة أسئلة مفتوحة حول تأثير ت الزيارات. كما تمت مناقشة التحديا عددالجديد على المرضى ومقدمي الرعاية الصحية على ثالثة مستويات: جودة الخدمات والتكلفة و الصحي عي لتحليل نتائج المقابلة وتوليد الموضوعات و. تم استخدام التحليل الموض2022 ايارإلى نيسانمن محافظاتالمحتملة. تم إجراء المقابالت في أربع والمواضيع الفرعية. الجديد. واتفقوا جميعًا على أن يالصح الضمان حتى الوصول إلى نقطة التشبع. كان معظم المشاركين على علم بقانون النياً صيد 2١الدراسة شملت القطاع الخاص. كما يتوقعون الصحية فيالقانون يمكن أن يعزز صحة المريض. يعتقد معظم المشاركين أن الخطة ستزيد من دخل مقدمي الرعاية م التأمين الصحي إلى تحسين جودة يمكن أن يؤدي تطبيق نظا ذلك، أن يستخدم المرضى خدمات القطاع الخاص أكثر من القطاع العام. باإلضافة إلى الشهرية،الناس لدفع األقساط معارضةخدمات الرعاية الصحية وتقليل العبء المالي المتعلق برسوم القطاع الخاص. تشمل التحديات المحتملة للخطة وعدم وجود ، الضمانوإساءة استخدام المريض لخطة الصحية، والتأخيرات المحتملة في السداد لمقدمي الرعاية المطالبات، والصعوبات في معالجة . والمفحوصةعدد كاٍف من األدوية المسعرة وعدم وجود نظام إلكتروني وقاعدة بيانات يمكن أن تواجه العديد من التحديات الفنية. يجب دراسة الخطة ولكنها في نفس الوقت المحتملة، الجديدة بالعديد من المزايا الصحي الضمانتتمتع خطة على نطاق صغير قبل التنفيذ الوطني. يجب أن يتم تنفيذ النظام اإللكتروني من قبل عيادات األطباء وال صيدليات جيدًا قبل تنفيذها ويجب تجريبها هيئة الصحة. الفواتير إلى الضمان الستعانة بفريق دولي من الخبراء ذوي الخبرة في إدارة خطة ا ب يوصى والمستشفيات لتسهيل نقل المعلومات/ لإلشراف على هذا النظام الجديد. نوعية. ، جودة الخدمة، التحديات، التصور، دراسةالصيادلة العراقيون العراقيون، الصحي، المرضى الضمانالكلمات المفتاحية: قانون 1corresponding author e-mail: ali.baraak@copharm.uobaghdad.edu.iq received: 15/7 /2022 accepted: 9/10 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp131-140 iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 132 introduction for more than three decades, the iraqi healthcare system has been experiencing several difficulties and challenges which have negative impacts on the quality of healthcare services (1). everyone in iraq is insured by the government’s health care system (2). every single health care facility that is part of the public sector is owned by the government and falls under the purview of the ministry of health (moh). every single health care provider that contributes to the public health system is an employee of the government. because private health insurance is not widely used in iraq, there are no reliable statistics available for the sector (3).the main current challenge for the public sector is inadequate funding to the moh as the budget allocated in 2019 was us $ 5.0 billion. in other words, the healthcare sector received just 4.5% of the state budget this proportion is low when compared to other middle eastern nations with similar budgets (1). the state company for marketing drug and medical appliances (kimadia) which is responsible for importing and distributing medicines, medical devices, and equipment for public healthcare settings in all 18 governorates of iraq. it was able to secure only 60% of the essential medicine list in 2019 because of the low budget(4). the iraqi population has risen exponentially over the last four decades, growing from a population of about 14.0 million in the 1980s to 40 million in 2020 (5). the directorate of population and manpower statistics in iraq indicated that the population census estimates in iraq for the year 2030 will reach 51,211,700 people(6).this large number afflicts burden on an already underdeveloped health sector and creates new challenges that need substantial solutions. health insurance is defined as an insurance system which covers medical expenses by a third party (7). a new iraqi health insurance law (ihil) has been approved in 2021. the beneficiaries (participants) should pay a monthly premium of 1.0% of the salary of almost all governmental employees, except those with high-ranking positions, who should pay 2.5% of their monthly income. this premium fee can cover the family members including jobless spouse, parents and children less than 21 years who are still studying. low-income people disabled and people with terminating diseases will have free coverage. emergency services in the public sector and preventive public health services such as vaccination and screening will still be provided for free by the moh (8). most of insured patients should pay co-insurance of 25% for prescriptions, lab tests, x-ray and medical dental services(8). they also should pay 10% of the physician visit fees. health insurance authority (hia) is governmental entity will regulate the new iraqi health insurance. hia will receives monthly bills from providers, premium from people and reimburses providers. each patient should register at a doctor in public or private sector and beneficiaries will have a health insurance card (8) (figure 1). the network can include healthcare providers from both the private and public sectors. current study was the first in-depth study investigating the pharmacist perceptions toward the prospective national health insurance. the pharmacists’ recommendations can help health officials to evaluate the potential barriers facing the national health insurance program. the study can explain in-depth the pharmacists’ concerns and requirements of successful implementation of the national health insurance. interview-based studies can provide in-depth information through follow-up questions which cannot be provided by surveybased studies. additionally, the interviews were interactive between the researcher (interviewer) and participants (interviewees) to have daily practice details about the topic. the aim of study was to explore pharmacists’ insights toward implementation and impact of new national health insurance program on patients, providers and health system. figure 1. the prospective iraqi health insurance program. iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 133 methods this qualitative study included face-to-face or phone semi-structured interviews with pharmacists working in public and/or private healthcare settings. the interview guide included open-ended questions covering the pharmacist perceptions about the impact on adopting the new health insurance program on patients and healthcare providers at three levels: quality of services, service cost and frequency of visits. potential challenges were also discussed. face-to-face interviews were conducted at public or private healthcare settings by one master student (pharmacist) in person or over the phone. the interviews were conducted from april through may 2022 in four provinces in iraq: dhi qar, baghdad, wasit and al-muthanna. most interviews were audio-recorded to transcribe every word. setting a total of twenty-one pharmacists have been interviewed by the researcher. he had met ten pharmacists in person (3 pharmacists at hospitals; five pharmacists at the pharmacy department; one pharmacist at a health centre in a rural area; and one pharmacist in the training and development center) and eleven pharmacists over the phone. thirteen of them agreed to be audio recorded, and the interviews lasted between 20 and 40 minutes. inclusion criteria all pharmacists must have at least five years of experience in the iraqi healthcare system, and all are registered with the syndicate of iraqi pharmacists (both public and private sectors). in other words, they should have completed their rotation and hospital residency at the public sector and had their own private pharmacy license. pharmacists’ recruitment two methods of sampling were used: purposive and snowballing. initially, the researcher used the purposive technique to target pharmacists in the public and private sectors in order to identify pharmacists with adequate experience in both public and private sectors. in addition, the researcher used a snowball strategy, which included asking participants about additional pharmacists who are interested to participate and meeting the inclusion criteria. in addition, several pharmacists who met the inclusion criteria refused to participate because of the excessive burden during work hours. before the interview, the participants got a guide for the interview and a brochure about the new ihil (figure 1) through whatsapp or telegram. we kept recruiting pharmacists until the saturation point was reached. before the interviews, interviewees gave their verbal permission, and their comments were made anonymous to protect their privacy. thirteen interviews were audio-recorded after obtaining the participants’ consent. to overcome any language barrier, the interviews were conducted using a combination of english and arabic languages. then, two bilingual authors translated all interview transcripts to english. ethical approval study proposal was approved by the central scientific committee of the university of baghdad college of pharmacy. before the interviews were conducted, verbal permission was acquired from the participants. participation was voluntary, and recording the interview was optional. to maintain participant anonymity, the interviews were unidentified. the participants received no reward. thematic analysis the qualitative data obtained from the interviews was analyzed using thematic analysis. the researchers gathered qualitative data from the participant responses to discover and develop themes. braun and clarke's (2006) six steps for thematic analysis were followed by the researchers (9). these steps include becoming familiar with the data (the comments), making initial codes, looking for themes, evaluating themes, defining and labeling themes, and writing the report. results interviews were conducted with 21 experienced pharmacists in four governorates, namely baghdad, dhi qar, almuthanna , and wasit. they were 16 men and five women. table 1 shows the demographic and professional information of the participants. iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 134 table 1. demographics characteristics of the participating pharmacists the code gender specialty degree workplace total years of experience ph 1 male chief senior pharmacist higher diploma dhi qar health department/training and development center 19 ph 2 male practitioner pharmacist bsc dhi qar health department / pharmacy department 8 ph 3 male practitioner pharmacist bsc dhi qar health department / al-haboubi teaching hospital 8 ph 4 male clinical pharmacist master wasit health department / fairuz hospital 10 ph 5 female practitioner pharmacist bsc department of pharmacy / department of health dhi qar 14 ph 6 male practitioner pharmacist bsc dhi qar health department/aljamiah health center 7 ph 7 male practitioner pharmacist bsc store manager in al muthanna province 14 ph 8 male practitioner pharmacist bsc dhi qar health department 8 ph 9 male practitioner pharmacist bsc dhi qar health department/mohammed al mousawi hospital 11 ph 10 male ` practitioner pharmacist bsc department of pharmacy / department of health dhi qar 21 ph 11 male practitioner pharmacist bsc dhi qar health department 6 ph 12 male practitioner pharmacist bsc dhi qar health department 9 ph 13 male master of clinical pharmacy master pharmacy department/ dhi qar health department 18 ph 14 female practitioner pharmacist bsc pharmacy department/ drug monitoring unit/ dhi qar health department 9 ph 15 female practitioner pharmacist bcs mohammed al mousawi hospital/ dhi qar 6 ph 16 male master of pharmaceutics master azizia hospital/ wasit 7 ph 17 male practitioner pharmacist bsc al-shuyoukh suq sector / dhi qar health department 10 ph18 female master's degree master al-amal hospital for cancer / baghdad 12 ph 19 male practitioner pharmacist bsc al-hussein teaching hospital/ dhi qar health department 7 ph 20 male practitioner pharmacist bsc dhi qar health department 8 ph 21 female practitioner pharmacist bsc dhi qar health department 11 iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 135 according to the participating pharmacists, the national health insurance program has several potential gains on patient clinical and economic outcomes, but at the same time it can face several technical challenges and requirements. the main themes and subthemes of the findings are summarized in table 2. opinions of the pharmacists participate in current study about the new iraqi health insurance law all pharmacists who were interviewed supported the implementation of the new iraqi health insurance program because it would subsidize medical services and reduce the financial burden on insured people: "i am supportive of this law because it allows patients who were not able to visit the private healthcare settings due to financial barriers. through this program, they will be able to follow up on their health condition"(ph-9). "health insurance is a good thing because when the patient visits hospitals or the private sector, the fees paid are low because the services are subsidized"(ph-14). the health insurance can enhance quality of medical services most pharmacists believed that the health insurance can enhance the quality of medical services in both the public and private sectors because there will be more competition among health care providers: " i'd like to add that competition between health care providers helps to make sure that provided services will be good"(ph-2). "as a result of competition between pharmacies, it is assumed that the level of services provided will improve, and the performance of workers in pharmacies will also improve. as for the public sector, the less workload on workers, the more time they will have to take care of patients in order to fully communicate information"(ph-18). some pharmacists suggested having financial penalties for low quality services. " healthcare providers should face financial penalties for short-term failures and low-quality services, so that the hospital bears the costs"(ph-8). the health insurance will increase the workload and providers' income in the private sector according to the perspectives of most pharmacists, the short waiting list and high quality of services in the private sector will make insured people more likely to go there. this will increase the income of hcps who have contracts with the health insurance authority. "patients go to the private sector more than the public sector, because the public sector is a collapsed sector, and the services are mostly unavailable, and you may need to wait, so the private sector is better in terms of speed and is supported at the same time" (ph-9). "the turnout of citizens participating in this program will be on the private sector because of the care, cleanliness, and easy access to medical services" (ph-18). "those who work in the private sector benefit more because they receive more patients, and this is accompanied by an increase in daily financial income" (ph-15). "the financial return will be better, especially in the private sector, because the number of patients for this sector is greater" (ph-12). the role of a family physician in organizing workflow within the new national iraqi health insurance program. the majority of pharmacists agreed that having family doctors involved in this project is important because they will have all patient's medical history and will be able to decide if patient needs to see a specialist doctor. this will reduce the workload on healthcare providers and give them time to keep track of important disease cases: "the family doctor is a sophisticated and necessary system to be applied for the purpose of knowing the history of the patient and their family as well. as you know, there are rare genetic diseases, and there is also sensitivity to certain drugs, and this information will be present with the family doctor, and he is also the decision-maker in referring patients to the specialized doctor"(ph-8). "it is better that the program includes a family doctor, because he knows all the health details of the patient and determines whether the patient needs to see the specialist doctor, thus reducing the time and effort for the specialist doctor"(ph-20). "i currently go to an ophthalmologist every six months because i have eye problem, and this is a result of late detection because we do not have a family doctor, and therefore early detection of diseases, including rare ones, is important so that we do not reach advanced stages"(ph-10). the clinical and economic effects of the new iraqi health insurance program on patients all the participating pharmacists expected the health program will reduce the financial burden on patients and this will reflect positively on their health: "it can reduce the financial burden on the patient because the services provided, such as operations and medicine, are subsidized"(ph-11). "every day i live with the suffering of patients because some medicines or doctors' prescriptions are expensive, and this law, if it is actually applied, will save them"(ph15). "the percentage of deduction from the employee’s salary is small, and therefore this monthly deduction will not affect them. therefore, when the employee is exposed to an emergency health situation in which they need a sum of money, the health insurance provides him with financial support"(ph -16). iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 136 "on health, the effect of this program is very significant and realistic. before implementation, citizen is not screened for health issues except in very necessary cases, but if the program is implemented, even in simple cases, he and his family will be seen by healthcare providers"(ph-9). potential challenges could face the implementation of the iraqi health insurance program the participants listed several potential challenges facing the implementation of the health insurance at national level. the potential challenges can be people resistance to pay monthly premium, inadequate expertise in management of financial bills, potential delay in the reimbursement of hcps, patient overuse of the insurance program and absence of electronic system and database. not adequate number of priced and quality tested medications according to seven pharmacists, one of the challenges that will face the implementation of this program is the testing and pricing of all medicines available in the public and private sectors, as there is currently a slowdown in the testing and pricing process. that is, after the program is put into place, there must be enough safe and effective medicines to go around. “will medicines in the iraqi market be tested and priced throughout iraq? because, to my knowledge, only one testing center is available, and it is impossible to quickly test medicines if only one is available"(ph-8). "when implementing this program, the medicines are supposed to be priced and tested, and this is a challenge for the state because most of the medicines in the pharmaceutical market are neither prices nor tested and entered the country through unofficial methods"(ph -11). people may not like the idea of monthly payment for the iraqi national health insurance nine pharmacists stated that getting people to accept this point related with health insurance is one of the main challenges and some people may even be against the implementation of this national program: "a large percentage of citizens will object to the program, and the reason for this is that they do not realize the importance of the health insurance law. this is considered the biggest challenge because this is expected at the beginning of the implementation of the program, but over time and when the benefits of its application are felt, the rejection will decrease"(ph-19). "employees, especially young people, refuse this thing [health insurance] because they do not complain about illness, so they must be educated that this law will greatly benefit them and their families"(ph-9). "citizens refuse to participate because they see that the state takes the money and does not provide services at the expected level"(ph-5). expected problems or delaying in the payment to healthcare providers most of the pharmacists revealed that there could be payment problems, which could affect the work of healthcare providers. "our work depends on the availability of medicine to provide the service. delaying payment to pharmacies will delay payment to the drug stores. therefore, payment must be made by the health insurance authority within a period of time not exceeding one month"(ph-2). one pharmacist was pessimistic and expected failure in the program after implementation. he thought that hcps may cancel the contract with the health insurance authority due to payment problems: "realistically, i do not think that there will be any development in the two sectors after the implementation of the program. it is possible that healthcare providers will withdraw from the program, and i believe that the patient will not benefit greatly from the service"(ph-13). no electronic health records are adopted currently seven pharmacists mentioned that the program cannot be carried out properly without the electronic database. "we do not have accurate statistics about the diseases. this issue will negatively affect the implementation of the program. as you know, most of patients have chronic diseases, so we need to study the topic carefully from an economic point of view "(ph-19). "we do not know how many people have high blood pressure, etc. i don't think we have an electronic system in iraq. do we have statistics about the number of healthcare settings in each region? i mean, are the settings sufficient to cover insured patients? for example, in nasiriyah, we do not have hospitals that can cover all residents "(ph-8). expecting difficulties in claims processin most pharmacists expected difficulties in management and processing of the insurance program claims. " we need qualified, experienced and trained staff people who can visit to institutions that have implemented this program, or attracting a team from abroad to supervise this system management"(ph-1). "the program must have a correct beginning, because poor programming means failure" (ph -4). potential patient misuse of the insurance program insured people may overuse the health insurance by increasing their unnecessary visits to healthcare providers because they are subsidized. "because the health services will be subsidized, insured individual can overuse of healthcare iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 137 services particularly in the private sector which puts additional strains on the fund. therefore, there are supposed to be programs to guide patients and set certain limits" (ph4). "it is also possible to give the health insurance card to other people, as long as the card is used by more than one person"(ph -3). pharmacists’ recommendation to hia for proper implementation of the health insurance program the participating pharmacist provided several recommendations toward a correct implementation of the health insurance including pilot adoption, using electronic system for claims’ processing, implementing electronic health records and hiring international team to help supervising the program management. pilot adoption before national implementation of the program is necessary some pharmacists recommended that the program should be piloted in certain regions or governorates. the pilot implementation can identify problems and obstacles and come up with solutions before the program is adopted at national level. "experiment with this law in a specific governorate to know the positives and negatives and the obstacles for the purpose of studying them, then the program will be gradually circulated to the country"(ph-11). "we must implement this program in specific governorates to monitor its progress, solicit the opinions of citizens in those governorates about the health insurance, determine how much they support the program and whether or not they enrolled into the health insurance "(ph-14). electronic system is pivotal for bill management all participants agreed that this program needs an electronic system. the electronic system is important for expediting work, keeping track of patients, and sending bills to the health insurance authority: "an electronic system is necessary because through it we can record and follow-up patients, and this is important. we also know the percentage of diseases for each disease through a database"(ph-8). electronic health records are essential to have clinical statistics about diseases the pharmacists recommended adoption electronic health records in all participating healthcare settings to have electronic medical data available on request. "there must be a successful and robust electronic system taken from countries that have previously used it because this system gives you information such as statistics for diseased cases, for example, statistics on diabetes, hypertension, and rare diseases. therefore, we will get good statistics, and here the system can be developed by directing a correct orientation, whether it is a private or public sector. "(ph2). "it is necessary to use an electronic system in all health institutions and to be linked with the health insurance authority for the purpose of smoothness and ease of work"(ph-7). it is important to hire an international team to help carried out the program correctly the majority of the pharmacists recommended recruiting international team of experts to help in supervising the program implementation and management. "it is necessary to bring in specialized teams that have experience from abroad to train the supervisors of this program in all the governorates"(ph-9). "we need a work team from abroad to supervise the staff of health insurance authority and train them on how to manage this program to be implemented successfully "(ph-17). "we need companies from abroad to implement the program and follow it, and it is better to apply it to an area for a year and wait for the results"(ph-13). discussion the goal of the iraqi health insurance program is to make sure that insured people have full health coverage and access to healthcare services(8). iraqi health officials confirmed that the law is supposed to go into effect this coming august 2022, but that it depends on the approval of the federal budget (10). all participating pharmacists supported the adoption of national health insurance as medical services would be subsidized, which could enhance patient accessibility to private healthcare services. the clinical and economic effects of the national health insurance on inured individuals according to the study findings, patient health is predicted to improve after implementing the health insurance because insured people would be able to access medical services at the private sector with subsidized fees. similarly, an american study compared the patient accessibility to healthcare services between two states (kentucky and arkansas) that expanded coverage by the affordable care act (aca) to low-income people and nonexpanded a state (texas). outcomes were evaluated after three years of the aca's coverage expansion using survey data obtained from low-income individuals in three states. by the end of 2016, there were more access to treatment, lower out-of-pocket costs, and more people who said they were in "excellent" health among the people gaining coverage in the states expanded the coverage of the aca (11). iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 138 some pharmacists believed that the poverty rate in iraq is increasing which hinders people accessibility to paid healthcare services and expected this health insurance can help insured people to get these services. their expectations are aligned with the collaborative assessment conducted by the ministry of planning, unicef, and the world bank which found that the spread of covid19 has had a significant impact on income and access to basic services for iraqi families. the proportion of children living in poverty in iraq has nearly doubled from one in five before covid-19 pandemic to two in five currently (12). it is worth mentioning that the proportion of the iraqi population that is at or below the poverty line reached 18.9% in 2016 (13).unfortunately, the iraqi health sector has been ignored by successive governments, and funding was inadequate over the last few years. this renders the preventive and curative health services and imposes financial burdens on low-income people. international health institutions say that more than 70% of treatment costs are paid for by the iraqi patient, even though the who says this number shouldn't be more than 30% (14). the effect of national health insurance on workload in the public and private sectors most of the pharmacists expected that insured patients would turn to private healthcare providers more frequently than the public sector and this would increase the workload on private providers. according to a systematic review of three studies from three countries about the effect of national health insurance on doctors in terms of workload and the level of services provided, most physicians work for long hours, up to 10 hours or more, and an average of ten minutes with each patient. consequently, it can lead to mental health problems for doctors, which can lower the quality of care they give to their patients. it can also lead to medication mistakes that cost money (15). on the other hand, a majority of pharmacists believe it is necessary to allocate family doctor for patients after implementation of program since they know all patient medical history and can determine whether they need to visit a specialist. this can reduce the workload on specialists and give them time to keep track of important disease cases. it is worth noting that the primary health care settings suffer from a shortage in family medicine physicians. the estimated number of qualified doctors in this field is 500, while the required number exceeds 1000 (1). the effect of the implementation of national health insurance on quality of services most of the participating pharmacists believed that under this program there will be competition between healthcare providers to provide better quality of services. in contrast, a study in nepal found problems in the level of services provided after pilot implementation of a health insurance program. those regions suffered from a scarcity of medicines as a result of the increased workload on providers and the participants waited a long time in hospitals(16). potential challenges facing the implementation of the national health insurance the pharmacists expected that the insurance implementation would face obstacles including rejection of people, lack of a database, lack of electronic system, misuse/overuse of the insurance, and inability to test and price medicines. a study in nigeria found extreme poverty, limited knowledge, little interest, superstitious beliefs, an inefficient payment method, a shortage of drugs, and inadequate supervision and monitoring are challenges facing the implementation of the national health insurance program (17). another study in four provinces in ghana found that the sustainability of the health insurance scheme is threatened by problems like a lack of and uneven distribution of medical facilities and health care professionals, rising costs, fraud and abuse, and not paying providers (18). electronic system in the national health insurance the electronic system was not implemented in the hospitals visited by the research, as most institutions in the government sector do not have an electronic system(3). the participating pharmacists in this study have made it clear that it is important during the implementation of this program to have an electronic system for the purpose of processing bills, following up on patients, and for the purpose of creating a database. a recent iraqi study found that inadequate documentation in most public hospitals which can negatively impact the monitoring of medication effectiveness and safety(19). study limitations the study had some limitations such as inequal participation in terms of gender which may be due to the concern of female pharmacists about being audio-recorded. additionally, some participants declined audio-recording. conclusion the implementation of the new iraqi health insurance program has many potential advantages, as this program is expected to have a positive clinical and economic impact on insured individuals because it will reduce the financial burdens resulting from out-of-pocket expenditures on medical services. it is expected that there will be an improvement in the quality of medical services due to competition between hcps which it will reflect iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 139 positively on insured individuals. hcps who work in the private sector and have contracts with hia will make more money because patients will visit them more often, which will be accompanied by an increase in workload for them. family physicians play an important role in this program at primary care settings as they will contribute to the follow-up of patients and decide whether cases require referral to hospitals or specialized centers, which reduces the workload on hcps. however, the program implementation needs several essential requirements which can threaten its successful adoption. the potential challenges can be people resistance to pay monthly premium, inadequate expertise in management of financial bills, potential delay in the reimbursement of hcps, patient overuse of the insurance program and absence of electronic system and database. the health insurance should be well thought out before being put into practice, and it should be tested on a small scale before implementing at national level. physician clinics, pharmacies, and hospitals must adopt an electronic system to submit information and bills to hia. it is also recommended to appoint an international team of experts to supervise the management of this new system. the quality of healthcare services provided by the insurance network of providers should be evaluated regularly. timely processing of the medical claims can help to have well timed payments to healthcare provider bills. table 2. main themes and subthemes of pharmacist insights regarding the national health insurance themes subthemes all pharmacists are supporters for the health insurance implementation ❖ subsidized fees (decrease out of pocket) ❖ decrease financial barriers to get services. the health insurance would increase the workload and providers' income in the private sector ❖ waiting time and quality of service ❖ increases patient visits to private providers ❖ family doctor can secure continuity of care ❖ more private provider visits, more income to them insurance can enhance quality of services. ❖ competition among health care providers ❖ financial penalties for low quality services. they expected several types of potential challenges facing the implementation of the national health insurance ❖ not adequate number of priced and tested medications ❖ people may not like the idea of monthly payment for health insurance. ❖ expected problems or delaying in the payment to healthcare providers. ❖ no electronic health records are adopted currently ❖ expecting difficulties in claims processing ❖ potential patient misuse of the insurance program. the clinical and economic effects of the new iraqi health insurance program on patients ❖ enhances accessibility ❖ enhances affordability several essential requirements are needed for successful implementation of the national health insurance. ❖ pilot adoption before national implementation. ❖ electronic system is pivotal for bill management ❖ electronic health records are essential to have clinical statistics about disease. acknowledgment we would like to thank dr. dhia jabbar kadhim, clinical pharmacy department at the university of baghdad college of pharmacy, for his help in selecting the study topic. we also appreciate the time and feedback of all the participating pharmacists. reference 1. alwan aa. health situation in iraq: challenges and priorities for action. baghdad: ministry of health, 2019. 2. amendments wi. official gazette of iraq public health law. 2020;(89):0–55. 3. al-jumaili aa. pharmacetuical country profile. 2020; (september) : 1–77.available from: https:// moh. gov. iq /upload /upfile/ ar/1375.pdf 4. al-jumaili aa, younus mm, kannan yja, nooruldeen ze, al-nuseirat a. pharmaceutical regulations in iraq: from medicine approval to postmarketing. east mediterr heal j. 2021;27(10):1007–15. 5. population, total iraq | data [internet]. [cited 2022 jul 10]. available from: https://data.worldbank.org/indicator/sp.pop.t otl?locations=iq 6. central statistical organization -iraq population estimates 2020 [internet]. [cited 2022 jul 11].available from: https:// www.cosit. gov.iq/ar /pop-main / manpower 7. marcinko de. dictionary of health insurance and managed care. springer publishing company; 2006. iraqi j pharm sci, vol.31(suppl.) 2022 iraqi health insurance -pharmacist insights 140 8. iraq health coverage law 2020. [cited 2022 apr 2]; available from: https://moj .gov.iq/ upload/pdf/4614.pdf. 9. creswell jw, clark vlp. designing and conducting mixed methods research. sage publications; 2017. 10. the ministry of health; iraqi news agency [internet]. [cited 2022 jul 12]. available from: https://www.ina.iq/154390--3-.html 11. sommers bd, maylone b, blendon rj, john orav e, epstein am. three-year impacts of the affordable care act: improved medical care and health among low-income adults. health aff. 2017;36(6):1119–28. 12. annual results 2021 | unicef iraq [internet]. [cited 2022 jul 10]. available from: https:// www. unicef. org /iraq /reports /annual results-2021 13. who emro | health system strengthening | priority areas | iraq site [internet]. [cited 2022 jul 13]. available from: http:// www. emro.who.int/iraq/priority-areas/healthsystem-strengthening.html 14. for the eastern mediterranean whoro. strengthening health financing systems in the eastern mediterranean region towards universal health coverage: health financing atlas 2018. 2019. 114 p. 15. sugiyatmi ta, hadi u, chalidyanto d, hafidz f, miftahussurur m. does the implementation of national health insurance affect the workload of a doctor and have an impact on service quality? a systematic literature review. j public health africa. 2019;10(s1):101–5. 16. paudel b. challenges for implementation of the health insurance program in nepal: implementer’s perspectives. oslometstorbyuniversitetet; 2021. 17. alawode go, adewole da. assessment of the design and implementation challenges of the national health insurance scheme in nigeria: a qualitative study among sub-national level actors, healthcare and insurance providers. bmc public health. 2021;21(1):1–12. 18. fusheini a, marnoch g, gray am. implementation challenges of the national health insurance scheme in selected districts in ghana: evidence from the field. int j public adm. 2017;40(5):416–26. 19. hiba laith 2022. hiba leith fahmi, ali azeez al-jumaili. understanding the experience of hospital pharmacists with the effectiveness, safety, adverse drug reaction reporting and interchangeability of biopharmaceutical medicines. iraqi j pharm sci, 2022;31(1):72– 86. this work is licensed under a creative commons attribution 4.0 international license http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 quinolone-resistance genes in salmonella enterica serotype typhi doi: https://doi.org/10.31351/vol31iss2pp218-222 218 occurrence of quinolone-resistance genes in ciprofloxacin-resistant salmonella enterica serotype typhi isolated form blood sample of patients with typhoid fever. jabbar s. hassan *,1, bushra j. al-tamimi** and fuad ghazi hassan *** *college of medicine, al-nahrain university, baghdad, iraq **college of medicine, university of babylon, babylon, iraq ***department of medical laboratories techniques,immunity, al-mustaqbal university college, babylon, iraq abstract salmonella is approved as a common foodborne pathogen, causing major health problems throughout the world particularly in low‐ and middle‐income countries. low-level fluoroquinolone resistance is conferred by both chromosomal and plasmid-encoded resistance, this research was carried out look into the occurrence rate of qnra,qnrb and qnrs genes in salmonella enterica serotype typhi cipr ofloxacin-resistant insulate from blood samples of patients with typhoid fever. fifteen salmonella enterica serotype typhi isolated previously from patients with typhoid fever were included in this study. all bacterial isolates were confirmed to have ciprofloxacin resistant by vitek 2 microbial identification system; after plasmid dna extraction; multiplex-pcr was done with primer sequences intended to plasmid-mediated quinolone-resistance genes which is qnra, qnrb, and qnrs. in this study; it was 21 qur genes amongst 15 isolates. the qnrs gene was the commonest (10/21, 47.6%) followed by qnra (6/21, 28.5%), whereas only 4 isolates were positive for qnrb (5/21, 23.8%). some isolates had more than one qnr genes. so, ciprofloxacin-resistant salmonella typhi can have more than one gene at the same time; and the most occurrence rate in regards to qnr gene in this study was qnrs compared to qnra and qnrb keywords: salmonella enterica serotype typhi , typhoid fever, fluoroquinolone, ciprofloxacin, qnr gene. وجود الجينات المقاومة للكوينولون في بكتريا السالمونيال المعوية المقاومة للسيبروفلوكساسين النمط للمرضى المصابين بالحمى التيفوئيديةالمصلي تايفي المعزولة من عينات الدم ***فؤاد غازي حسن و ** بشرى جبار التميمي حسان، 1*،جبار سلمان ، بغداد ، العراق كلية الطب جامعة النهرين فرع االحياء المجهرية * ، بابل ، العراق كلية الطب جامعة بابل فرع االحياء المجهرية** ، بابل ، العراق الطبية المختبرات تقنياتكلية المستقبل الجامعة قسم *** الخالصة تعتبر بكتريا السالمونيال أحد مسببات األمراض الشائعة المنقولة عن طريق األغذية ، والتي تسبب مشكلة صحية كبيرة في جميع أنحاء تحدث مقاومة الفلوروكينولون في هذه البكتريا عن طريق المقاومة المشفرة بالكروموسومات . العالم وخاصة في البلدان المنخفضة والمتوسطة الدخل ، أجريت هذه الدراسة للتحقق من معدل حدوث جينات في سالمونيال التيفوئيد المقاومة للسيبروفلوكساسين qnrsو qnrbو qnraوالبالزميد سابقا من مرضى تم استخدام خمسة عشر عزلة من السالمونيال التيفية التي تم عزلها. ئيدالمعزولة من عينات دم المرضى المصابين بحمى التيفو في هذه الدراسة تم التأكد من أن جميع العزالت البكتيرية لديها مقاومة للسيبروفلوكساسين بواسطة نظام التعرف الميكروبي . مصابين بحمى التيفوئيد vitek 2 .زميدي تم إجراء بعد استخراج الحمض النووي البالmultiplex-pcr باستخدام بادئات تم تصميمها من اجل ان تستهدف جينات كان جين . عزلة 15من بين qurجين 21في هذه الدراسة؛ كان هناك . qnrsو qnrbو qnraمقاومة الكينولون بوساطة البالزميد بما في ذلك qnrs يليه ( ٪47.6، 10/21)هو األكثر شيوًعاqnra (6/21 ،28.5٪ ) عزالت فقط موجبة لـ 4، بينما كانتqnrb (5/21 ،23.8٪ .) بعض وكان . المقاومة للسيبروفلوكساسين على أكثر من جين واحد في نفس الوقت هذة البكتريا لذلك يمكن أن تحتوي . qnrالعزالت لديها أكثر من جين qnrو qnraمقارنة بـ qnrsفي هذه الدراسة هو qnrأعلى معدل حدوث لجين السالمونيال التيفية ، حمى التيفوئيد ، الفلوروكينولون ، سيبروفلوكساسين : الكلمات المفتاحية introductions salmonella enterica serotypes is a gramnegative, rod shape, flagellated and aerobic bacteria; it is posing a great danger to human health, most notably in low‐ and middle‐income countries (1). for a long time, the first medication for salmonellosis infections included chloramphenicol and trimethoprim in addition to penicillin, but the increase in resistance to such treatments prompted most doctors and with the emergence of newer types of antibiotics to use new antibiotics particularly fluroquinolone groups (2). fluoroquinolone un-responsiveness are primarily caused by two mechanisms: chromosomally mediated mutations in topoisomerase's quinolone resistance determining regions (qrdr) and quinolone resistance determining region mutations; resistance genes belong to qnr groups plasmids mediated play a role in fluoroquinolone resistant (3). 1corresponding author e-mail: jabbarsalman30@yahoo.com received: 12/9 /2021 accepted:19 /1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp218-222 iraqi j pharm sci, vol.31(2) 2022 quinolone-resistance genes in salmonella enterica serotype typhi 219 low-level fluoroquinolone resistance is conferred by both chromosomal and plasmidencoded fluoroquinolone resistance (4). determinants belong to qnr have been found in a numeral of enterobacterial species from different parts of the world, including america, europe, and asia (5). so far, six variants (qnra1 to qnra6) have been discovered. quinolones produced by other plasmids qnrb (qnrb1 to qnrb5) and qnrs (qnrs1 and qnrs2) resistance determinants outlined in enterobacterial species (6). the current study aimed to investigate occurrence rate of qnra, qnrb and qnrs genes in ciprofloxacin-resistant salmonella typhi (s. typhi) insulate from blood samples of patients with typhoid fever. material and methods bacterial strain a retrospective study of archived isolates, including 15 salmonella enterica serotype typhi isolates which was previously recovered from blood samples of patients with typhoid fever were used in this study. resistance to ciprofloxacin for all bacterial isolates that included in the current study was detected by using of vitek 2 microbial identification system, the detection was carried out according to manufacturing company (biomerieux). the bacterial isolates were diagnosed and classified in medical microbiology department at the faculty of medicine, al -nahrain university plasmid dna extraction protocol salmonella enterica serotype typhi was harvested by using luria-bertani broth media, after centrifugation (8,000) rpm for two minutes; the supernatant was discarded and the pellet was collected. plasmid extraction of the salmonella enterica serotype typhi was performed as described by company instruction (wizard® plus minipreps dna purification system, promega) multiplex-pcr was carried out with primer sequences specific for plasmid-mediated quinoloneresistance genes (table 1), which is qnra, qnrb, and qnrs. the settings of the pcr as follows: after initial denaturation at 94°c for 7 min, the 35-cycle amplification profile consisting of 94°c for 30 s, 62°c for 30 s, and 72°c for 1 min using a cleaver scientific thermal cycler (tc 32/80-uk). the last elongation was place at 72°c for 10 minutes. for 1.5 hours, using 2% agarose at 7 v/cm (merckgermany) pcr product was identified. concomitantly, a molecular marker (1-kb dna ladder; bioneer) was used. after the gel was stained with ethidium bromide, dna bands were seen and photographed under uv light. table 1. primer nucleotide for identification of plasmid-mediated quinolone-resistance genes qnr genes nucleotide sequences (5' 3') products bp references qnra f gataaagtttttcagcaagagg atccagatcggcaaaggtta 593 7 r qnrb f gatcgtgaaagccagaaagg acgatgcctggtagttgtcc 469 8 r qnrs f tggaaacctacaatcatacatatcg ttagtcaggataaacaacaataccc 656 9 r results multiplex pcr was used to check for the presence of the plasmid-mediated quinolone resistance genes qnra, qnrb, and qnrs in 15 isolate of salmonella typhi that were resistant to ciprofloxacin; since amplicons product by pcr with the expected amplification product size qnra gene (593 bp), qnrb (469 bp) and qnrs (656 bp) respectively (figure 1). over 15 salmonella typhi isolate; 21 qur genes were detected; and the qnrs gene was the most common (10/21, 47.6%) followed by qnra (6/21, 28.5%), whereas only 4 isolates were positive for qnrb (5/21, 23.8%). some isolates had more than one qnr genes (table 2). iraqi j pharm sci, vol.31(2) 2022 quinolone-resistance genes in salmonella enterica serotype typhi 220 table 2. distribution of qnr genes through salmonella enterica serotype typhi isolates ciprofloxacin resistance. isolates qnrs qnra qnrb total genes 1 + 1 2 + 1 3 + + + 3 4 + 1 5 + + + 3 6 + 1 7 + 1 8 + + 1 9 + 1 10 + 1 11 + + 2 12 + 1 13 + 1 14 + 1 15 + 1 figure 1. gel electrophoresis of multiplex pcr products (2% agarose, 7 v/cm2,1.5hrs) for qnra, qnrb, qnrs of salmonella enterica serotype typhi positive isolates. lanes 7, 9, 10,11, 12 and 13 qnra gene (593 bp) positive isolates; lane 3,4,6,9,11,14: qnrb (469 bp) positive isolate; lanes 5,6,8,11,13 qnrs (656 bp) positive isolates. discussion previous studies have documented that salmonella disease in humans can vary from selflimited gastroenteritis usually connected with nontyphoidal salmonella (nts) to typhoid fever with obstacles such as a fatal intestinal perforation; this bacterium has a propensity to acquire resistance to multiple classes of antimicrobial agents, and eradication of infection by highly resistant salmonella typhi can be mostly difficult (10). this study tested the occurrence of qnr genes among ciprofloxacin resistance salmonella enterica serotype typhi isolated from the blood of patients with typhoid fever. out of 15 salmonella enterica serotype typhi isolates;( 21) qnr genes were detected. two isolates harbored the three qnr genes qnra, qnrb, qnrs whereas two isolates harbored two qnr genes in which one isolates harbored qnra and qnrs while the second isolates have qnra, qnrb. quinolone resistance is initiated mostly through chromosomal mutations. quinolone resistance caused by plasmids has been reported in numerous places of the world in the last few years. this type of resistance is caused by plasmidmediated qnra, qnrb, or qnrs genes (11). quinolone resistance at low levels has been linked to dna from transferrable plasmids. several investigations have found that qnr determinants are widely distributed among bacterial isolates all over the world. quinolones are antibacterial agents with a broad spectrum of action that are commonly utilized in both human and veterinary medicine. their widespread use has been linked to an increase in quinolone resistance. (11). hopkins et al., mentioned that qnr genes contributed to high-level ciprofloxacin resistance in salmonella species in chromosomal and plasmid mediated (12). there are many articles that reported an increase in non-susceptible bacterial strain to iraqi j pharm sci, vol.31(2) 2022 quinolone-resistance genes in salmonella enterica serotype typhi 221 fluroquinolone agents due to harboring the qnr genes may be contributed to using of such antimicrobials in food-producing animals (13,14). interestingly; in the current study; the qnrs gene was more prevalent (47.6%) than qnra and qnrb which were (28.5%) and (23.8%) respectively. qnrs gene can increased selective pressure on the drugs and subsequently contributes to resistance even in the absence of mutations and its it is more easily transmitted from animals to humans (15). the prevalence rate of qnr genes among gram negative bacteria varies depending on sample type and locational area; most studies reported that; the regional distribution of qnra genes is known to be wide (15). many enterobacteriaceae species have been found to have qnra-like determinants, and six variants have been identified in qnra and qnrb which is (qnra1 to qnra6) and (qnrb1 to qnrb6) while qnrs (qnrs1 and qnrs2), genes for plasmid mediated quinolone resistance (pmqr) have been found on many bacteria in addition to enterobacteriaceae such as pseudomonas species with varying in size and incompatibility specificity (16). cameron-veas et al. mentioned that 15% of salmonella enterica in brazil which have been isolated from pig harbored qnrb, and none was carrying qnra and qnrs (17). while in china lin d et al. reported that (66%) salmonella species carrying qnrs (18). it's worth noting that the transfer of resistance genes among enterobacteriaceae bacteria, such as quinolones genes, is a complicated process involving a variety of mechanisms, such as plasmidmediated resistance gene transfer and chromosomal alterations. types of clinical isolates, geographic location, and antibiotic usage rates in each country can all influence these pathways (19). many individuals in our country randomly use ciprofloxacin without following clinicians' prescriptions in self-medication. in addition, the use of this type of antibiotic in the treatment of animals could result in greater selective pressure on such groups of antibiotics, which could lead to resistance through the different types of qnr genes. conclusions this study reported that ciprofloxacinresistant salmonella enterica serotype typhi may harbor more than one gene at the same time; and the most prevalent qnr gene in this study was qnrs compared to qnra and qnrb. as far as we know, this is the first study in our country reported that results in salmonella enterica serotype typhi clinical isolates. acknowledgement we would like to express our gratitude to all patients who donate by samples that the search was completed. references 1. crump ja, sjölund-karlsson m, gordon ma, 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veterinary journal. 2018 apr 1; 234:36-42. 18. lin d, chen k, chan ew, chen s. increasing prevalence of ciprofloxacin-resistant foodborne salmonella strains harboring multiple pmqr elements but not target gene mutations. scientific reports. 2015 oct 5;5(1):1-8. 19. khalil zk. isolation and identification of different diarrheagenic (dec) escherichia coli pathotypes from children under five years old in baghdad. iraqi journal of community medicine. 2015;28(3):126-32. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ i r a q i j p h a r m s ci , v o l . 3 1 ( 1 ) 2 0 2 2 g l y co s yl a t i o n i n yo u n g i n f er t i l e ma l e doi: https://doi.org/10.31351/vol31iss1pp293-297 293 correlation between seminal fructosamine and glycosylation gap and some sex hormones in the young infertile male in mosul city moamin junaid salim* and muhammad a. alkataan*,1 * college of medicine, ninevah university ,ninevahm, iraq abstract infertility represents a growing health problem in mosul city and worldwide. infertility defined as a failure to induce pregnancy after unprotected sexual intercourse for more than 12 months. infertility in male is a multifactorial complex pathology that leads to different types of problems. this work try to explore the correlation between glycosylation gap and seminal fructosamine and another parameter in the young male patient in mosul city. the study included 50 subjects with age range 19-29 years with bmi 18-26 kg/m2, from october 2019 to july 2020. the infertility group include 25 patients newly diagnosed with infertility before starting any treatment; have no infection and no structural abnormality. the control group included 25 healthy subjects. hemoglobin a1c, serum fructosamine, serum and seminal testosterone, estradiol and testosterone: estradiol ratio.in addition to some plasma trace element as k, mg and zn also measured. there was a significant elevation in the glycosylation profile in the infertile male in compare to control (p<0.05). the results of this work showed that there was a significant elevation in glycosylation gap in the infertile group (p<0.01). testosterone and testosterone/ estradiol ratio significantly reduced in the infertile group in comparison to control group (p< 0.0004 and 0.0002 respectively). serum and seminal plasma testosterone/ estradiol ratio showed no significant changes between the two groups (p>0.05). in conclusion, there was a significant positive correlation seminal plasma fructosamine and glycosylation gap in infertile male group. keywords: seminal plasma, fructosamine, glycosylation وبعض الهرمونات الجنسية لدى الشاب المصاب المنوي و فجوة الجليكوزيل السائل االرتباط بين فركتوزامين بالعقم في مدينة الموصل 1*،القطان محمد عبد الغفور و *سالم مؤمن جنييد ، نينوى ، العراق .كلية الطب، جامعة نينوى* الخالصة ف العقم بأنه الفشل في إحداث الحمل بعد الجماع غير يمثل العقم مشكلة صحية متنامية في مدينة الموصل وفي جميع أنحاء العالم. يُعرَّ شهًرا. العقم عند الذكور هو أمراض معقد متعدد العوامل يؤدي إلى أنواع مختلفة من المشاكل. يحاول هذا العمل استكشاف 12المحمي ألكثر من في المرضى من الشاب في مدينة الموصل. اشتملت الدراسة كاليكوزيالشن والفركتوزامين المنوي وعوامل آخرى العالقة بين فجوة االرتباط بال تضم مجموعة .2020الى تموز 2019. للفترة من اكتوبر2كلغ/م 26-18عاًما بمؤشر كتلة الجسم 29-19شخًصا تتراوح أعمارهم بين 50على من األشخاص 25شذوذ هيكلي. ضمت المجموعة الضابطة مريًضا تم تشخيصهم حديثًا بالعقم قبل البدء في أي عالج ؛ ليس لديهم عدوى وال 25العقم البوتاسيوم في الدم اما الفركتوزامين ، والتستوستيرون ، استراديول والتستوستيرون / استراديول. كما تم قياس بعض العناصر مثل hba1cاألصح ا معامل في كبير ارتفاع هناك كان المنوي. السائل بالزما في الزنك و المغنيسيوم بمجموعة و مقارنة بالعقم المصابين في بالجليكوزيل الرتباط العقم .(p <0.05) السيطرة مجموعة في بالجليكوزيل االرتباط فجوة في كبير ارتفاع هناك التستوستيرون .(p <0.01) كان نسبة انخفضت أظهرت نسبة على التوالي. 0.0002و p <0.0004)) والتستوستيرون / استراديول بشكل كبير في مجموعة العقم مقارنة بمجموعة السيطرة التستوستيرون / استراديول في المصل والبالزما المنوية عدم وجود تغيرات معنوية بين المجموعتين. .جليكوزيل في مجموعة الذكور المصابين بالعقمالفي البالزما المنوية وفجوة االرتباط في الختام ، كان هناك ارتباط إيجابي معنوي بالفركتوزامين ، الفركتوزامين، كاليكوزيالشن. بالزما السائل المنوي: الكلمات المفتاحية introduction infertility represents a growing health problem in mosul city and worldwide. infertility defined as a failure to induce pregnancy after unprotected sexual intercourse for more than 12 months. infertility in male is a multifactorial complex pathology that leads to different types of problems(1). the seminal analysis is the main and the primary procedure for the diagnosis of the possible underlying cause of infertility(2). semen is testicular fluid consist of sperms (5%) and seminal plasma that produces by accessory sex glands (95%)(3,4).semen consists of proteins, lipid, inorganic ions, sugars and hormones that play a crucial role in the fertilization process(3,4). moh1977729@gmail.com :mail-corresponding author e1 received:13 /9/2021 accepted: 15/ 12/2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss1pp293-297 i r a q i j p h a r m s ci , v o l . 3 1 ( 1 ) 2 0 2 2 g l y co s yl a t i o n i n yo u n g i n f er t i l e ma l e 294 glycosylation process is a non-enzymatic binding of glucose or fructose to different types of proteins as haemoglobin and albumin(5). glycosylation leads to a significant impact on protein function that reflects as changes in the cellcell adhesion that affect sperm and oocyte cells due to changes in protein-carbohydrate interaction that guide specific cell surface recognition(6). fructosylation i.e. adding seminal plasma fructose to albuminone of the major glycoprotein in seminal plasma that presents due to high fructose level that inhibits sperm oocyte fusion due to conformational changes as described by olejnik et al and johnson et al (7,8). many works study the effect of changes in serum and seminal level of hormones on male fertility especially serum testosterone, estradiol and testosterone/ estradiol ratio that reflects as changes in spermatogenesis process(9). testosterone/ estradiol ratio guide the prepare spermatogenesis and maintain sperm viability before and after intercourse (10). serum and seminal plasma trace element as k, mg and zn also play a vital role in spermatogenesis(11). changes in serum glucose and fructose lead to a significant change in glycosylation process that reflected as elevate hba1c and serum fructosamine level in seminal plasma. the aim of this study: to explore the correlation between glycosylation gap and seminal fructosamine and another parameter in the young infertile male in mosul city. patients and methods the study included 50 subjects with age range 19-29 year with bmi 18-26 kg/ m2. the infertility group include 25 patients newly diagnosed by infertility specialist with infertility before starting any treatment; have no infection and no structural abnormality. the control group included 25 healthy subjects. this study carried out under ethical approval no. 45 that issued by ethical committee of ninevah college of medicine. all seminal plasma and serum samples collected from patients after follow the physician’s direction the assay was carryout in orkida-private laboratory in mosul. after abstinence for 2 to 4 days, semen samples collected by masturbation. seminal samples allowed to liquefy at room temperature for 30 minutes then centrifuged at 2500 g for 10 minutes supernatant collected according to the world health organization (1999) criteria(2). seminal plasma immediately separated and divided into 2 aliquots, then stored at -20°c until assayed. serum samples collection fasting venous blood was drawn. 2.5 ml of serum was collected in five eppendorf 0.5 ml tubes with 1 ml of the supernatant of seminal fluids. hba1c measured by chromatographic– spectrophotometric method (12). mean blood glucose (mbg); predicted hba1c and glycosylation gap (gg) calculated using equations(13). mbg =1.76*(hba1c) -3.67mmol/l p-hba1c = 0.017*fa + 1.61 gg = m-hba1c – p-hba1c serum and plasma of semen fructosamine by nbtspectrophotometric method (14). the concentrations of k, mg, and zn in serum and seminal plasma detected with electrolyte analyzer-psd (14) and the hormones assayed using multi-parametric immune analyzer mini vidas® automated immunoassay system by biomerix (france) (15). data will represent as mean ± sd and analyze using spss software. person s correlation use to show the correlation between the measured parameters. results the results of this work showed that there was a significant elevation in the glycosylation profile in the infertile male in compare to control (p<0.05). there was a significant elevation in glycosylation gap in the infertile group (p<0.001) table 1. table 1. glycosylation profile in young infertile male in compare to healthy controls parameter control infertile group p-value m-hba1c 5.16± 0.45 6.32±0.6 < 0.0001 s. fructosamine µmol/l 211±39 240±31 < 0.005 mbg 5.41±0.8 7.5±1.06 < 0.0001 p-hba1c 5.19±0.66 5.7±0.53 < 0.004 gg -0.026±0.01 0.63±0.13 < 0.0001 mbg=mean blood glucose, p-hba1c= predicted hba1c and gg= glycosylation gap serum (s.) estradiol showed a significant reduction in the infertile group (p<0.0001) while testosterone/ estradiol ratio showed a significant increase in comparison to the control group (p<0.03). serum testosterone showed no significant change between the control and infertile group (p<0.3). seminal plasma (se.) hormones showed testosterone and testosterone/ estradiol ratio significantly reduced in the infertile group in comparison to control group (p< 0.0004 and 0.0002 respectively). in contrast seminal plasma estradiol significantly elevated in the infertile group (p<0.05) table 2. i r a q i j p h a r m s ci , v o l . 3 1 ( 1 ) 2 0 2 2 g l y co s yl a t i o n i n yo u n g i n f er t i l e ma l e 295 table 2. serum and seminal plasma testosterone, estradiol and testosterone/ estradiol ratio in young infertile male in compare to healthy controls parameter control case p-value s. testosterone nmol/l 15.3± 2.12 14.3±5.13 0.3722 s. estradiol pmol/l 92.44±14.3 71±5.1 0.0001 s.t/e2 ratio 0.17±0.04 0.205±0.07 0.0349 se. testosterone nmol/l 4.62±0.75 3.78±0.82 0.0004 se. estradiol pmol/l 266±35 292±42 0.0214 se. t/e2 ratio 0.017±0.004 0.013±0.003 0.0002 serum testosterone/ estradiol ratio (s.t/e2) significantly increase in infertile group in comparison to control (p<0.05). seminal plasma testosterone/ estradiol ratio (se.t/e2) showed significant reduction in infertile group in comparison to control (p<0.001). in the control group, both serum and seminal plasma mg and zn significantly reduced in the infertile group (p<0.05, p<0.0001 respectively). serum k showed no significant change between both groups (p<0.91). seminal plasma mg and zn significantly reduced in the infertile group in comparison to control group (p<0.0001, p<0.0001 respectively). in contrast, seminal plasma k significantly elevated in the infertile group in comparison to control (p<0.0001) as shown in table 3. table 3. serum and seminal plasma potassium, magnesium and zinc levels in young infertile male in compare to healthy controls. seminal plasma/serum ratio showed significant elevation in k level (p<0.001) with no significant change seen in mg (p>0.05) while zn showed significant reduction (p<0.001) in infertile group when compare with control as shown in figure 1. figure 1. seminal plasma/serum levels of k, mg and zn. blue represent control group and red represent infertile male group.*=p<0.01. in both groups, there was a significant positive correlation between serum/seminal plasma fructosamine ratio and glycosylation gap (r= 0.81, p<0.001) figure 2. parameter control case p-value serum k meq/l 4.22± 0.64 4.24±0.62 0.9111 serum mg meq/l 2.1±0.3 1.93±0.28 0.0437 serum zn µg/dl 94.44±6.41 54.92±6.3 < 0.0001 se. k meq/l 10±1.31 23±3.45 < 0.0001 se. mg meq/l 11.12±1.01 8.74±1.46 < 0.0001 se. zn µg/dl 128.5±9.43 25.15±5 < 0.0001 i r a q i j p h a r m s ci , v o l . 3 1 ( 1 ) 2 0 2 2 g l y co s yl a t i o n i n yo u n g i n f er t i l e ma l e 296 figure 2. seminal plasma/ serum fructosamine (left) and glycosylation gap (right). blue represent control group and red represent infertile male group.*=p<0.01. discussion glycosylation of proteins play role in the male infertility that represent major health problem. seminal plasma proteins and other components determine the successfulness of fertilization process. in addition to the changes in seminal plasmahormones that guide spermatogenesis and sperm fit after ejaculation. in this work, glycosylation profile showed elevation in all parameters and this agree with results obtained by janiszewska & maria kratz(4), cheon et al(5), and kratz et al(17). testosterone and estradiol play vital role in spermatogenesis(10, 18, 19 )and this work study both serum and seminal plasma changes of these two hormones. many studies agree with the results obtained in this work that showed reduction in estradiol in the infertile group with significant elevation testosterone/ estradiol ratio (20, 21). seminal plasma testosterone and testosterone/ estradiol ratio significantly reduced in the infertile male and this agree with results obtained by chen et al22. seminal plasma estradiol elevated in the infertile male and this agree with chen et al. and collodel et al.22, 23. serum and seminal plasma testosterone/ estradiol ratio showed no significant changes between the two groups and this agree with results discussed by kratz et al 17. the results showed significant reduction in both serum and seminal mg and zn in the infertile male and this agree with both 24, 25, 26. seminal plasma k elevated in the infertile males and this also agree with gusani et al, 27. serum/seminal plasma fructosamine significantly elevated in infertile male janiszewska etal., and olejnik et al 4,7. to sum up, there was a significant positive correlation between glycosylation gap in blood seminal plasma fructosamine, some sex hormones and trace elements, which reflect that the elevation in glycosylation process may lead to increase susceptibility to develop infertility in young adult. recommendation this study recommended further study to the other components of seminal fluid with larger size sample. ethical approval this study carried out under ethical approval no. 45 that issued by ethical committee of ninevah college of medicine. acknowledgments our thanks for university ninevah college of medicine for the support of this work. references 1. deyhoul n, mohamaddoost t, hosseini m. infertility-related risk factors: a systematic review. int j women’s heal reprod sci. 2017;5(1):24-29. doi:10.15296/ijwhr.2017.05 2. barratt clr, björndahl l, de jonge cj, lamb dj, martini fo, mclachlan r, et al. the 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j clin diagnostic res. 2016;10(5):cc05-cc08. doi:10.7860/jcdr/2016/14393.7723 27. gusani ph, skandhan kp, valsa c, menta yd. sodium and potassium in normal and pathological seminal plasma. acta eur fertil. 1992;23(1):39-42. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis doi: https://doi.org/10.31351/vol29iss1pp12-32 12 nanotechnology-based topical drug delivery systems for management of dandruff and seborrheic dermatitis: an overview lena m. thomas*,1 and abeer h. khasraghi * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract dandruff and seborrheic dermatitis (sd) are common skin disorders affecting the scalp and extending to other body sites in case of sd. they are associated with pruritus and scaling, causing an esthetical disturbance in the population affected. treatment of such conditions involves using a variety of drugs for long terms, thus optimizing drug formulation is essential to improve therapeutic efficacy and patient compliance. conventional topical formulations like shampoos and creams have been widely used but their use is associated with disadvantages. to overcome such effects, novel topical nanotechnology-based formulations are currently under investigation. in the following article, we highlight recently published formulation approaches used to improve topical dandruff/sd therapy. keywords: dandruff, seborrheic dermatitis, topical therapy, vanotechnology لجلد الدهني: االتهاب القشرة الدهنية والتهاب المستخدمة في أنظمة توصيل األدوية الموضعية الجديدة نظرة عامة *عبير حسن خزعل و 1*،لينا مراد توماس الصيدالنيات ،كلية الصيدلة، جامعة بغداد، بغداد،العراق. فرع* الخالصة حالة التهاب قشرة الرأس والتهاب الجلد الدهني هي اضطرابات جلدية شائعة تصيب فروة الرأس ، وتمتد الى مواقع أخرى في الجسم في ادوية مختلفة استخدام ان عالج هذه الحاالت يتضمن تأثرين بالحالة.الجلد الدهني. ترتبط هذه الحاالت مع حكة وتقشر مما يسبب اضطرابا جماليا للم تم ل المريض.لتحسين الفعالية العالجية وامتثا ضروريقد تكون هناك حاجة إلى عالج طويل األجل ، وبالتالي فإن تحسين صياغة الدواء هو و للتغلب مساوْي.والكريمات على نطاق واسع ولكن استخدامها مرتبط باستخدام مجموعة متنوعة من المستحضرات الموضعية التقليدية مثل الشامبو اهج الصياغة التي تم نشرها في المقالة التالية ، نسلط الضوء على من .على هذه اآلثار ، يتم حاليًا البحث عن تركيبات جديدة تعتمد على تقنية النانو .هاب الجلد الدهنيالمستخدمة لتحسين عالج التهاب الجلد القشرة / والتومؤخًرا . قشرة الرأس ، التهاب الجلد الدهني ، العالج الموضعي ، تقنية النانو : الكلمات المفتاحية introduction seborrhoeic dermatitis (sd) is a recurrent, chronic inflammatory skin condition, which has pink to red greasy-looking skin with yellowish flaky scales, accompanied by itching. it affects areas rich in sebaceous glands, such as scalp, face, chest and intertriginous areas (1). dandruff is considered as a mild or initial form of seborrheic dermatitis and appears as white or gray flakes in scalp, accompanied by itching with no apparent inflammation, and is considered as an embarrassing disorder (2). there are many possible causes for dandruff/sd but most likely it is due to infection caused by malassezia fungus species . many factors are considered as possible contributors to the development of sd/dandruff which includes exogenous factors (e.g. humidity, heat and extended periods of sun exposure) and endogenous host factors (e.g. nutritional deficiency, stress, and immune response) (3). various topical treatment options are available such as antifungal agents, keratolytic agents and anti-inflammatory agents. nowadays, nanotechnology offers a revolutionary treatment for several skin diseases and proved to be safe and effective in the targeted delivery of many medicaments. this review article looks into some of the nanotechnology-based drug delivery systems with a focus on their potential role as nextgeneration carriers for medicaments used for topical therapy of dandruff/sd. 1corresponding author e-mail: lena.murad78@gmail.com received: 8/12/2019 accepted:1 /2 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp12-32 iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 13 topical pharmaceutical forms for the treatment of dandruff/seborrheic dermatitis conventional formulations many therapeutic agents are used for dandruff/sd and these are formulated in a variety of pharmaceutical preparations, including liquid preparations (solutions, shampoos, lotions, emulsions, hair oils), or semisolids preparations (ointments, creams, gels) so as to provide ease of application at multiple sites, along with maintaining effectiveness of the active agent. a summary of the main therapeutic agents used presently as different pharmaceutical formulations for management of dandruff/sd is represented in table 1. table 1.topical therapeutic agents used clinically for treatment of dandruff/sd * class drugs formulations available antifungals (azoles) ketoconazole 1, 2% shampoo 2% cream 2% foam 2% gel 2% emulsion bifonazole 1% shampoo 1% cream 1% gel climbazole 0.5, 1 and 2 % shampoo fluconazole 2% shampoo miconazole 2% shampoo 2% rinse clotrimazole 1% cream sertaconazole 2% cream 2% gel flutrimazole 1% shampoo 1% gel antifungals (hydroxy-pyridones) ciclopirox 1% shampoo 0.77% gel ciclopirox olamine 1%, 1.5% shampoo 1% cream piroctone olamine 1% shampoo antifungals (allylamines and benzylamines) terbinafine 1% solution 1% cream naftifine 1% gel butenafine 1% cream iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 14 continue table 1. topical therapeutic agents used clinically for treatment of dandruff/sd *. class drugs formulations available corticosteroids hydrocortisone 0.1% lotion 1% solution 1% liniment 1% cream 1% ointment desonide 0.05% lotion 0.05% cream 0.05% gel alclometasone diporpionate 0.05 % cream 0.05% ointment betamethasone valerate 0.12% foam 0.1% lotion 0.1% cream flucinolone acetonide 0.01% shampoo 0.01% solution clobetasole propionate 0.05 % shampoo clobetasole 17-butyrate 0.05% cream mometasone furoate 0.1% cream calcineurin inhibitors tacrolimus 0.03, 0.1% ointment 0.03 % cream pimecrolimus 1% cream keratolytic agents salicylic acid 2, 3, 4% shampoo salicylic acid /sulfur salicylic acid 2%, sulfur 2% shampoo salicylic acid 3%, sulfur 5% shampoo sodium sulfacetamide /sulfur 10% sodium sulfacetamide, 5% sulfur emollient foam tar containing products 2, 3, 4% solubilized coal tar extract shampoo 0.5, 1% whole coal tar shampoo 1, 5, 7% coal tar solution shampoo salicylic acid/coal tar 10% coal tar extract 2 or 3% salicylic acid shampoo azelaic acid 15% gel 15% foam 20% cream propylene glycol 15% solution 15% shampoo 30% liniment urea/bifonazole 40% urea and 1% bifonazole ointment miscellaneous agents selenium sulfide 1, 2.25, 2.5% shampoo zinc pyrithione 1, 2% shampoo iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 15 continue table 1. topical therapeutic agents used clinically for treatment of dandruff/sd *. class drugs formulations available lithium gluconate/succinate 8% lithium succinate ointment 8% lithium gluconate ointment or gel metronidazole 0.75, 1% gel benzoyl peroxide 2.5, 5 and 10% wash glycerin 10% lotion hyaluronic acid (sodium salt) 0.2% gel calcipotriol 50 µg/ gm cream 50 µg/ml solution tacalcitol 4 µg/ gm cream 4 µg/ gm ointment nicotinamide (vitamin b3) 4% cream *note: shampoos, foams, rinses and lotions are mostly used for treating dandruff/sd on the scalp; creams, emulsions and ointments are used to treat sd on face and body locations other than scalp; gels are used for scalp and non-scalp sd. treatment duration is usually for up to 4 weeks. novel nanotechnology-based formulations dandruff/sd patients require regular, longterm use of therapeutic agents, mostly used on daily bases. these are usually available as several conventional topical dosage forms. there is a strong need to develop innovative pharmaceutical formulations which are aesthetically and cosmetically more acceptable to the patient, and can be conveniently incorporated into a patient’s routine hairor skincare regimen to improve patient compliance. nanotechnology has emerged as an innovative drug delivery approach, allowing controlled , sustained and targeted drug delivery thus minimize undesirable drug side effects while maintaining or improving therapeutic efficacy (4). in the following sections, we highlight recently published work describing nanotechnology-based formulation approaches used to improve the efficacy of topically applied therapeutic agents used for dandruff/ sd management. table 2 summarizes research conducted with various nanotherapeutics as topical drug delivery systems used for dandruff/sd. table 2. summary of the most common nanocarriers for skin delivery of drugs used in dandruff/sd* nanocarrier dosage form drug loaded references microemulsion emulsion ketoconazole clotrimazole miconazole sertaconazole naftifine hcl salicylic acid lactic acid tacrolimus 7-11 11 15 16 22 23, 24 24 25, 27 iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 16 continue table 2. summary of the most common nanocarriers for skin delivery of drugs used in dandruff/sd nanocarrier dosage form drug loaded references microemulsion gel ketoconazole fluconazole sertaconazole butenafine hcl terbinafine hcl tacrolimus 10, 14 12, 13 17, 18 19, 20 21 26 nanoemulsion emulsion ketoconazole clotrimazole 34-38 39 nanoemulsion gel ketoconazole bifonazole terbinafine hcl 42 43 44-46 polymeric micelles dispersion clotrimazole fluconazole ketoconazole 49 49 50 liposome dispersion fluconazole miconazole ketoconazole sertaconazole terbinafine hcl ciclopirox olamine hydrocortisone betamethasone triamcinolone 55 56-58 36, 60-63 82 65, 66 68, 95 69, 71 69, 70 69 liposome gel ketoconazole terbinafine hcl hydrocortisone 64 67 71, 72 transferosome dispersion miconazole fluconazole clotrimazole terbinafine hcl hydrocortisone 76, 77 79 80 91 85 iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 17 continue table 2. summary of the most common nanocarriers for skin delivery of drugs used in dandruff/sd nanocarrier dosage form drug loaded references transferosome gel miconazole ketoconazole sertaconazole bifonazole sulphur and sa tacrolimus 78 81 82 83 84 86 ethosome dispersion fluconazole clotrimazole ketoconazole terbinafine hcl ciclopiroxolamine tacrolimus 55, 90 80 90 91-93 94, 95 96 ethosome gel terbinafine hcl 92, 93 niosome dispersion miconazole fluconazole ketoconazole ciclopirox olamine terbinafine hcl naftifine hcl benzoyl peroxide 100 101 102 103, 104 105 106 108 niosome gel ketoconazole naftifine hcl benzoyl peroxide 102 106 108 polymeric nanoparticles dispersion hydrocortisone betamethasone tacrolimus zinc pyrithione 113-115 116-119 120 121 iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 18 continue table 2. summary of the most common nanocarriers for skin delivery of drugs used in dandruff/sd nanocarrier dosage form drug loaded references solid lipid nanoparticles (sln) dispersion miconazole fluconazole ketoconazole clotrimazole hydrocortisone betamethasone-17-valerate clobetasol propionate 127-129 130 133 137 140 140 143 solid lipid nanoparticles (sln) gel bifonazole ketoconazole clotrimazole terbinafine hcl tacrolimus 132 134-136 135 138 148 nanostructured lipid carriers (nlc) dispersion miconazole fluconazole ketoconazole clotrimazole betamethasone dipropionate clobetasol propionate tacrolimus 128 131 133 137 142 144 146 nanostructured lipid carriers (nlc) ointment, gel ketoconazole clotrimazole terbinafine hcl betamethasone dipropionate clobetasol propionate tacrolimus 135 135 139 141 145 147, 148 metallic nanoparticles dispersions silver silver/ketoconazole sulphur selenium zinc oxide palladium 150-157 158, 159 161, 162 163 164 165 iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 19 1. microemulsions (mes) and microemulsion gels microemulsions are clear/transparent, thermodynamically stable dispersions of oil and water stabilized by emulsifiers, with droplet diameter usually within the range of 10 -100 nm (5). they have been widely studied to enhance the bioavailability of poorly soluble drugs, and represent an attractive option for enhanced dermal and transdermal administration of both hydrophilic and lipophilic drugs, as well as providing controlled or sustained drug release property (6). microemulsions have been used as carriers for antifungal drugs to ensure effective drug concentration levels in the skin after their dermal administration. several microemulsion formulations and microemulsion based gels of azole antifungals (ketoconazole (7-10), clotrimazole (11), fluconazole (12,13), miconazole (14, 15), sertaconazole (16-18)) and allylamine/benzylamine antifungals (butenafine (19,20) , terbinafine (21) and naftifine (22)) have been developed with a view to provide controlled drug release and to enhance the skin permeability with the potential efficacy for eradication of cutaneous fungal infections. a study showing the benefits of microemulsion-loaded hydrogel over conventional topical preparations is seen with butenafine hydrochloride microemulsion-loaded hydrogel. aerosol ot (surfactant), sorbitan monooleate (cosurfactant) and isopropyl palmitate (oil) were used in the preparation of microemulsion and carbopol 940 (1 %) was used as a gelling base for preparation of microemulsion-loaded hydrogel. the developed hydrogel has shown better ex vivo skin permeation and antifungal activity against candida albicans when compared to marketed cream. the greater drug penetration-enhancing activity of microemulsions may be attributed to the combined effects of both the lipophilic and hydrophilic domains of microemulsions while the greater antifungal activity may be due to enhanced permeation of microemulsion oil globules containing drug through the fungal cell wall (19). salicylic acid (sa) is a keratolytic agent with antimicrobial actions that have been used in topical products for the treatment of sd and dandruff. however, the topical use of sa is associated with burning sensation and irritancy. to minimize skin irritation and increase sa solubility, microemulsion loaded with sa was prepared and provided a better option for topical delivery with enhanced solubility in all the studied concentrations (23). in another study, a microemulsion composed of 12% salicylic acid and 4% lactic acid was prepared. this was composed of tween 80 as surfactant, propylene glycol as a co-surfactant, castor oil, ethyl alcohol and purified water. increasing the concentration of surfactant or co-surfactant, the microemulsion region becomes larger. such microemulsion could be a suitable vehicle for topical treatment of psoriasis, scaly patches, ichthyoses, dandruff, corns, calluses, and warts on the hands or feet (24). topical calcineurin inhibitors tacrolimus and pimecrolimus have shown safety and efficacy in the treatment of sd as an alternative to corticosteroids. tacrolimus is a lipophilic drug that is commercially formulated as a lipophilic ointment. a microemulsion-type colloidal carrier, as well as microemulsion based hydrogel of tacrolimus, were developed to improve the dermal availability of tacrolimus (25-27). 2. nanoemulsions (ne) and nanoemulgels nanoemulsions are biphasic dispersion of two immiscible liquids; an oily system dispersed in an aqueous system or an aqueous system dispersed in an oily system, stabilized by an amphiphilic surfactant. the droplet sizes in nanoemulsions are usually in the range of 100 400 nm. recently, the term nanoemulsions have been used specifically for systems having droplet diameter smaller than 250 nm that are in a metastable state compared with microemulsions (28). depending on constituents and relative distribution of the internal dispersed phase/phases and the external phase, nanoemulsions are termed as biphasic (o/w or w/o) or multiple nanoemulsions (w/o/w) (29). nanoemulsions offer several advantages for topical and transdermal delivery; they can be used to deliver both lipophilic and hydrophilic drugs to the skin or mucous membranes, have the capacity for site-specific drug targeting and delivery as well as their ability to increase the solubility and dispersion of drugs onto skin, thus will enhance skin permeation, extend the release of drugs and minimize their side effects by reducing the administrated dose. they are transparent/ translucent with a pleasant appearance that can be washed away easily after application and provide good skin hydration in cosmetic products (30, 31). on the other side, the disadvantage of these systems is their instability during storage and the fact that their preparation requires expensive, high energy input instruments as they require smaller amounts of surfactants compared to microemulsions (32, 33). antifungals are widely used in the treatment of sd. they are characterized by poor aqueous solubility and therefore have poor dispersibility in topical vehicles. formulation of antifungals as nanoemulsions enhances their solubility, and, subsequently, improves their subcutaneous absorption, and increases their efficacy for topical use. nanoemulsions of antifungal drugs for topical use were developed for ketoconazole (34-38) and clotrimazole (39). the use of topical nanoemulsions is limited due to their low viscosity and spreadability; such a problem is solved by the incorporation of gelling iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 20 agents to nanoemulsions and thus converting them to nanoemulgels (40). the latter can accommodate a higher amount of drugs due to their better solubilization capacity. moreover, because of their adhesion, nanoemulgels provide longer retention time and higher skin penetration along with the achievement of controlled drug release profile at the target site with fewer side effects (41). a variety of nanoemulgel formulations for the treatment of fungal infection incorporating ketoconazole (42), bifonazole (43) and terbinafine hydrochloride (44-45) have been formulated as a mean of more effective topical drug delivery system. a comparative assessment between terbinafine nanoemulgel for ex vivo drug permeation and in vivo antifungal activity compared to the marketed product, lamisil® emulgel was conducted. results showed that skin permeation and in vivo antifungal activity of terbinafine for candida infection from all the prepared nanoemulsion based gel formulae was improved significantly over the marketed emulgel (46). 3. polymeric micelles (pms) polymeric micelles are nanoscopic coreshell structures with diameters typically smaller than 100 nm, formed by self-aggregation of amphiphilic block copolymers dispersed in aqueous media, with the hydrophobic part of the polymer on the inside (core) and hydrophilic part on the outside (shell). pms have great potential as a drug delivery system as they increase the solubilization of poorly soluble molecules, provide sustained-release properties, and increase drug stability by the protection of encapsulated substances from degradation (47). despite their promising potential, significant problems have impeded the progress of pm and limited their applications as drug delivery systems, mainly due to lack of stability, limited polymers for use and lack of suitable methods for large-scale production. (48) researches have been conducted to utilize pms as drug delivery systems for different azole antifungal compounds using different copolymers. in one study, different azole antifungal compounds (clotrimazole, fluconazole, and econazole nitrate) were loaded in polymeric micelles with different copolymers. the best formulation was provided by the mpeg-dihexpla micelles loaded with econazole and incorporated with an efficiency of 98.3%. this micelle formulation showed significantly higher penetration than its commercial liposomal gel (pevaryl®) in both the porcine and human skins. the authors concluded that better skin delivery is due to the smaller size of formulation while the commercial formulation containing numerous penetration enhancers (49). another study reported that ketoconazole incorporated into methoxy poly (ethylene glycol)-b-poly (δvalerolactone) copolymeric micelles had 86-fold higher water-solubility than crude ketoconazole, and showed activity similar to crude drug with no skin irritation. in addition, the drug-loaded micelles demonstrated enhanced drug deposition in mice skin with no penetration through skin, as compared to marketed ketoconazole cream indicating selective skin delivery (50). 4. liposomes liposomes are colloidal spherical nanoparticle vesicles, composed of one or more lipid bilayers that can be produced from cholesterols, non-toxic surfactants, sphingolipids, glycolipids, long-chain fatty acids, and even membrane proteins. they have an aqueous core and can transport hydrophilic or hydrophobic drugs (51, 52). topical liposome formulations offer several advantages; they act as a solubilizing matrix for poorly soluble drugs, provide good skin penetration, associated with improved therapeutic efficacy and reduced side effects. they also act as a local depot that provides sustained drug release. however, the disadvantages of liposomes are associated with their low solubility, physical and chemical instabilities after long-term storage (53, 54). liposomes and liposomal gels have been used as a drug delivery system for a variety of antifungal drugs including fluconazole (55), miconazole (56-58), ketoconazole (59-64), terbinafine (6567) and ciclopirox olamine (68). liposomal dispersions and liposomal gels have also been developed for a variety of corticosteroids to increase their dermal delivery and hence, improve their topical bioavailability, reflected by improved therapeutic effect and reduced side effects. among the corticosteroids studied which have the potential for use in dandruff/sd are hydrocortisone, betamethasone, and triamcinolone (69-71). however, increased percutaneous penetration and efficacy combined with a decreased toxicity cannot be found for all steroids; the liposome characteristics can vary according to size, shape, surface charge and lipid composition (72). despite the improved therapeutic value of liposomes, it has become evident that classical liposomes remain confined to upper layers of the stratum corneum and fail to penetrate the skin layers deeply (73). to improve the elasticity of conventional liposomes, researchers have found and a new family of liposomal structures called transferosomes. 5. transferosomes transferosomes, also known as ‘deformable liposomes’ or ‘elastic liposomes’ are highly elastic vesicular systems, consisting of a complex lipid bilayer surrounding water-filled core. they differ from liposomes by the presence of edge activators (surfactants) in the lipid bilayer of vesicles; this will contribute to the deformability of the bilayers and provides transferosmes with better iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 21 skin penetration ability (74). transferosomes are used for topical or systemic administration of various hydrophilic and lipophilic drugs delivering them either into or through the skin; they have the ability for sustained release action with high efficiency. the main disadvantage of transferosomes is related to their chemical instability and cost of formulation (75). there have been numerous studies involving transferosomes and transferosomal gels as a drug delivery system for a variety of drugs useful for sd. antifungal drugs like miconazole (76-78), fluconazole (79), clotrimazole (80) ketoconazole (81), sertaconazole (8282) and bifonazole (83) were successfully encapsulated into transferosomes and transferosomal gels for topical delivery. in one study, miconazole transferosomes with a high encapsulation efficiency ranging from (67.98 ± 0.66%) to (91.47 ± 1.85%), with small particle sizes ranging from (63.5 ± 0.604 nm) to (84.5 ± 0.684 nm) were prepared. the optimized formulation of miconazole transfersomes was incorporated into a carbapol 934 gel base and showed higher antifungal activity than marketed product (daktarin® cream 2%), were the steady state flux after 24 h for miconazole transfersomal gel was 85.968 µg cm -2 h-1 as compared to a value of 72.488 µg cm -2 h-1 for daktarin®cream 2%. this could be attributed to the high deformability and flexibility of transfersomes, which allowed them to overcome skin barrier properties (78). sulfur and salicylic acid are effective for topical delivery in many skin-care products of many clinical conditions including sd due to their antiinflammatory and keratolytic activities. topical transferosomal gels of sulfur and salicylic acid were formulated and have shown an enhanced skin penetration compared with conventional gels (84). transferosomes have also been used for the delivery of anti-inflammatory agents such as hydrocortisone (85) and tacrolimus (86) with improved site-specificity and overall drug safety compared with traditional topical formulations, making such carrier a suitable one for the treatment of inflammatory skin disorders. a study reported the prepartion of tacrolimus transfersomes using different kinds of surfactants (sodium cholate, tween 80 and span 80). tween 80 was selected as the optimal carrier owing to the best deformability and the highest drug retentions. the optimized transferosomal formulations were further made into gel and in vitro drug release after 24 h of transferosomal gel and liposomal gel was 2.8 times and 2.3 times higher than the commercial ointment (protopic®). the optimized tacrolimus transferosomal gel displayed highest skin retentions compared with liposomal gel and commercial ointment. the amounts of tacrolimus in epidermis and dermis from transferosomal gel were 3.8 times and 4.2 times respectively as much as ointment, while liposomal gel was only 1.7 times and 1.4 times respectively as compared to ointment. in vivo therapy of mice atopic dermatitis, tacrolimus transferosomal gel took effect more quickly than liposomal gel and commercial ointment. thus transferosomes displayed superior performance and effective skin target for topical delivery of tacrolimus (86). 6. ethosomes ethosomes are a slight modification of liposomes. they are soft vesicles made of phospholipids, containing a high content of ethanol (20–45%) and water (87). compared to liposomes, skin penetration capacity of ethosomes is higher due to the capability of ethanol to cause disturbance of skin lipids, making this carrier system suitable for dermal and transdermal delivery of hydrophilic and lipophilic drugs. as with other lipid-based vesicular systems, stability is a major challenge for ethosomes (88, 89). ethosomes and ethosomal gels represent an efficient carrier for a variety of therapeutic agents used in the treatments of skin infection and inflammatory conditions, including sd. however, clinical studies are lacking but many researches have been conducted to prepare ethosomal formulations for a variety of antifungal agents including fluconazole (55), clotrimazole (80), ketoconazole 90), terbinafine (91-93) and ciclopirox olamine (94, 95). in one study, tacrolimus ethosomes were prepared and showed lower vesicle size and higher encapsulation efficiency as compared with traditional liposomes. in addition, tacrolimus ethosomes permeated to a greater degree than from commercial ointment (protopic®) suggesting the greater penetration ability to the deep strata of the skin for ethosomes (96) . 7. niosomes niosomes are vesicular nanocarriers similar to liposomes except that they are composed of mixtures of non-ionic surfactants, cholesterol and may contain small amounts of phospholipids (97). they can be used as carriers for hydrophilic or lipophilic drugs but are more popular than liposomes in the field of topical drug delivery due to their higher chemical stability because of using surfactant instead of phospholipids during their preparation, low production cost, high loading capacity and their ability to provide sustained drug release pattern (98, 99). in recent years, there have been much research on the use of niosomal dispersions and niosomal gels for the delivery of a variety of antifungal drugs such as miconazole (100), fluconazole (101), ketoconazole (102), terbinafine hydrochloride (103), naftifine hydrochloride (104) and ciclopirox olamine (105). in one study, ciclopirox olamine niosomes were prepared using span 60, iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 22 cholesterol, diacetyl phosphate. the obtained niosomes were in the size range of 170–280 nm, with entrapment efficiency 38–68%. a niosomal gel of the optimized batch was prepared by incorporating the niosomal dispersion in a 2% (w/w) carbopol 940 p. deposition of ciclopirox olamine into rat skin from niosomal dispersion and its gel was significantly higher than that of plain ciclopirox olamine solution and its marketed product. such findings suggest that niosomes are promising tools for cutaneous retention of ciclopirox olamine with expected reduction in the frequency of the application of the dosage form (106). benzoyl peroxide is widely used in the treatment of acne but has also been effective for the treatment of trunk and facial sd due to its antibacterial and keratolytic effects. 107(107) benzoyl peroxide loaded niosomes have been prepared to increase its solubility and was incorporated into gel by adding it to 1% carbopol 934 base to increase skin contact time to gain maximum benefits of the treatment. the prepared niosomal gel was advantageous because it controlled the release of the drug and enhanced its transdermal permeation. skin irritation studies conducted on mice showed that optimized niosomal gel formulation cause significant reduction in inflammation with very less irritation in comparison with plain benzoyl peroxide solution (108). 8. polymeric nanoparticles (pns) polymeric nanoparticles are solid colloidal particles with a diameter ranging from 1-1000 nm. they are made of non-biodegradable or biodegradable polymers (natural, semi-synthetic or synthetic) in which the active ingredient is dissolved, encapsulated, adsorbed or chemically attached. there are two types of nanoparticles depending on the preparation process: nanospheres and nanocapsules. nanospheres have a monolithic‑type structure (matrix) in which drugs are dispersed, encapsulated within the particles or adsorbed onto their surfaces, whereas nanocapsules have the drug confined in cavity of liquid core and surrounded by a polymeric membrane (109, 110). polymeric nanoparticles have been extensively studied as promising particulate carriers in the pharmaceutical and medical fields due to their subcellular size, potential to protect unstable active ingredients, ability to enhance the skin permeation poorly water-soluble lipophilic drugs, as well as their utility in providing controlledand sustaineddrug delivery (111). despite their proposed benefits, topically applied nanoparticles remain localized to proximal glands and hair follicles and are unable to deeply penetrate the stratum corneum; this makes their utility in obtaining prolonged skin retention and controlled release for the desired therapeutic effect debatable (112). many anti-inflammatory agents have been developed as pns, including hydrocortisone (113-115), betamethasone (116-119) and tacrolimus (120) with the aim of increased drug permeability through lipid membranes, long-term drug release potential as well as providing a safer approach for the treatment of dermatitis. zinc pyrithione (zpt), a widely used agent in anti-dandruff shampoos was prepared as nanoparticles with primary particle diameters in the range of 20-200 nm. it is expected that particles smaller than 25 nm in diameter would not be expected to significantly scatter light, and produce a clear anti-dandruff shampoo formulation, which exhibit a higher activity, be distributed more effectively on the scalp, and will require a less thickening agent in the shampoo formulation to ensure its stability against settling than the standard form of zpt (121). 9. lipid nanoparticles: solid lipid nanoparticles (slns) and nanostructured lipid carriers (nlcs) solid lipid nanoparticles (slns) are nanosized spherical structures composed of a coat of aqueous surfactant monolayer surrounding a high melting point lipid core that remain in a solid-state at the room as well as body temperature (122). slns can effectively encapsulate and solubilize lipophilic and hydrophilic drugs, but lipophilic drugs can be better delivered by solid-lipid nanoparticle (123). slns hold great promise to achieve controlled sitespecific drug delivery and increase in skin hydration. however, drawbacks associated with slns are uncontrolled drug expulsion from the carrier and limited drug loading capacity (124) . nanostructured lipid carriers (nlcs) are modified generations of slns consisting of a matrix composed of solid and liquid lipids, stabilized by an aqueous surfactant solution. the incorporation of liquid lipid causes structural imperfections of solid lipids to form a crystal lattice with many spaces. such arrangement increases spaces and allows for higher drug loading capacity (125). lipid nanoparticles (sln, nlc) have been reported as suitable carrier systems to control the penetration/permeation of highly lipophilic drugs and offer epidermal, follicular targeting, as well as controlled release of drugs, protecting them from degradation and enhancing their stability (126). slns and nlcs are among the nano-carriers that have conquered a better place in topical preparations and are applied either as an aqueous dispersion or incorporated in a suitable liquid or semi-solid preparations to provide an appropriate formulation for application upon the skin. they have been used to improve drug absorption by the skin for a variety of drug molecules intended for the topical treatment of multiple diseases. the development of slns/nlcs of antifungals might have a significant advantage for their clinical use. antifungals drugs such as miconazole nitrate (127-129), fluconazole (130, 131) , bifonazole (131, 132) , iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 23 ketoconazole (133-136) , clotrimazole (137) and terbinafine hydrochloride (138, 139) formulated as slns /nlcs upon incorporation into suitable semisolid preparations, have the potential to provide targeted and sustained drug release pattern, with reduction of fungal burden in the infected area. such findings could be exemplified with miconazole nitrate loaded sln (127). sln dispersions exhibited average size between 244 and 766 nm. all the dispersions had high entrapment efficiency ranging from 80% to 100%. miconazole nitrated-sln gel (2%) was prepared by incorporation with carbopol 940 gel base (0.3–1.0%), out of which 0.5% concentration showed good consistency. miconazole nitrated-sln gel produced significantly higher deposition of the drug in skin (57±0.65%) than marketed gel (30%±0.87) and this colloidal nanoparticulate gel, being sumbicron in size, enhances the drug penetration into the skin, remains localized for a longer period of time in skin as compared to conventional gel, thus enabling better drug targeting to the skin. incorporation of corticosteroids, such as hydrocortisone, betamethasone valerate and dipropionate (140-142), clobetasol propionate (143-145) into lipid nanoparticles enable such drugs to be deposited on skin with reduced systemic exposure and reduced local side-effects along with providing sustained release of drug in addition to more efficient penetration into skin layers than traditional formulations. tacrolimus, a calcineurin inhibitor, used in treatment of sd mainly due to its antiinflammatory effects, is not associated with the side effect profile of corticosteroids but topically is reported to have low penetration rate through the skin. a solid lipid nanoparticle (sln), nanostructured lipid carrier (nlc) and modified nano-lipid carrier formulations of tacrolimus were developed to overcome such a problem and subsequently improve its bioavailability (146148). 10. metallic nanoparticles (mns) recent advances in nanotechnology are the development of inorganic nanoparticles that remain stable for long periods and are useful for specific targeting and controlled release of carried drugs in the skin (149). a variety of metallic nanoparticles have been used in the treatment of a variety of skin diseases including sd. antidandruff shampoos have become popular in the treatment of dandruff using agents that combat the growth of the causative agent, malassezia furfur. recently, this yeast has developed resistance towards the commonly used antidandruff drugs, and as a result, it is necessary to develop a new class of novel antidandruff shampoos. silver nanoparticles (agnp) were developed for their bactericidal properties and used in the treatment of infectious diseases and have been used in several biomedical products, including wound or burn dressings (150). they have also been investigated as a potential fungistatic agent for various clinically relevant fungi including m. furfur involved in scalp related diseases such as dandruff. it is also reported that agnp may also have significant anti‐inflammatory effects (151, 152). the activity of silver nanoparticles depends on factors such as sensitivity to silver, the concentration of nanoparticles in the formulation, and their shape 153(153). silver nanoparticles can be synthesized from eco-friendly, cost-effective biological systems making them amenable to large-scale industrial production and are considered as cost-effective fungistatic agents in shampoo formulations for treating scalp problems, especially knowing that very small amount is required for producing desired antidandruff activity. there have been many reports using silver nanoparticles during the formulation of antidandruff shampoo with effective antifungal activity (154-157). a hybrid system of ketoconazole complexed with silver nanoparticles have been synthesized to enhance the activity against malassezia furfur. the anti-dandruff activity was highest with ketoconazole coated agnp when compared to ketoconazole and agnp individually (158, 159). there have been studies indicating that ag nps are toxic to the mammalian cells (160) therefore, sulfur nanoparticles were developed as a safer, more cost-effective alternative to silver nanoparticles as these are reported to possess broad-spectrum antimicrobial activity, as well as extensive antifungal activity against m. furfur, the main causative agent of dandruff (161, 162) . other metallic nanoparticles with potential anti-dandruff activity due to their antifungal activities against malassezia include selenium nanoparticles (senps) (163) with reported higher potency than the known anti-dandruff agent, selenium sulphide; zinc oxide nanoparticles (zno nps) (164) and palladium nanoparticles (pd nps) (165) reported to have antimicrobial as well as antidandruff activity. however, clinical research work is required before such metallic nanoparticles are introduced into anti-dandruff preparations. the potential benefits of the previously mentioned nanotechnology approaches over the conventional dosage forms and potential advantages of each nanotechnology formulation compared to the other nanotechnology techniques are summarized in table 3. iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 24 table 3. potential benefits of novel nanotechnology approaches over the conventional dosage forms and potential advantages of nanotechnology approach compared to the other nanotechnology approaches novel nanotechnology approach potential benefits of nanotechnology approach over conventional approach potential benefits of specified nanotechnology approach over other nanotechnology approaches references microemulsions (mes) and nanoemulsions (nes) emulsions are coarse dispersions with cloudy/ opaque semi-solid consistency whereas me and nes are clear/transparent colloidal dispersion with fluid consistency, suitable for delivering both lipophilic and hydrophilic drugs with higher stability, bioavailability and permeation than emulsions or conventional semisolids, with the capacity for site-specific drug targeting compared to other nanosystems, me and ne offer advantages in terms of simplicity and stability. 166 polymeric micelles solubilization of poorly soluble molecules, protection of encapsulated substances from degradation, thus enhanced stability and efficacy of encapsulated drugs as well as sustained and targeted delivery to desired site. limited benefit over other nanotechnology approaches due to lack of stability, low drug loading capacity, limited polymers for use and lack of suitable methods for large-scale production. 48 liposomes, transferosomes and ethosomes suitable carriers for both lipophilic and hydrophilic drugs, with better skin penetration, reduced side effects, improved therapeutic efficacy and stability of encapsulated drugs as well as ability to provide local drug depot, with sustained drug release action. improved localized as well as transdermal skin delivery of drugs 167 niosomes suitable carriers for both lipophilic and hydrophilic drugs, enhanced bioavailability, targeted delivery, and slow drug release. compared to lipid vesicles, niosomes are more stable, with higher drug loading capacity leading to reduction of dose, delayed clearance and ease of modification with lower production cost. 168 polymeric nanoparticles enhance lipophilic drug penetration through skin, with ability to protect unstable active ingredients and reduce skin irritation, with sustained drug release ability over prolonged periods of time. limited degree of enhancement in skin permeation and localization in the hair follicles; this may promote potential application of delivery of drugs to site of application during the treatment of dermatological conditions. 111 iraqi j pharm sci, vol.29(1) 2020 nanotechnology role in dandruff / seborrheic dermatitis 25 continue table 3.potential benefits of novel nanotechnology approaches over the conventional dosage forms and potential advantages of nanotechnology approach compared to the other nanotechnology approaches novel nanotechnology approach potential benefits of nanotechnology approach over conventional approach potential benefits of specified nanotechnology approach over other nanotechnology approaches references solid lipid nanoparticles (slns) and nanostructured lipid carriers (nlcs) effectively encapsulate and solubilize both lipophilic and hydrophilic drugs, with ability to improve penetration and follicular targeting, thus increases bioavailability. additionally, skin hydration effect is observed due to occlusive properties of lipid nanoparticles. higher stability and ability to protect chemically labile drugs against decomposition than lipid vesicles. nlc provide greater drug loading and better stability compared to sln. 126 metallic nanoparticles useful for controlled, localized and targeted drug 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http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32(1) 2023 colorimetric determination of salbutamol sulfate doi: https://doi.org/10.31351/vol32iss1pp45-52 45 colorimetric determination of salbutamol sulfate using spectrophotometry-continuous flow injection technique in bulk powder and pharmaceutical forms wasan a. al-uzri*,1, mariam jamal** and hind hadi* *department of chemistry, college of science, university of baghdad, baghdad, iraq **ministry of education, educational rusafa directorate ii abstract simple, precise and economic batch and flow injection analysis (fia)-spectrophotometric methods have been established for the determination of salbutamol sulfate (slb) in bulk powder and pharmaceutical forms. both methods based on diazotization coupling reaction of slb with another drug compound (sulfadimidine) as a safe and green diazotization agent in alkaline medium. at 444 nm, the maximum absorption of the orange azodye product was observed. a thorough investigation of all chemical and physical factors was conducted for batch and fia procedures to achieve high sensitivity. under the optimized experimental variables, slb obeys beer’s law in the concentration range of 0.25-4 and 10-100 μg/ml with limits of detection of 0.09 and 2.51 μg/ml for batch and fia procedures respectively. the high reproducibility of less than 1% (n=5) for both methods confirmed the applicability of these methods. using f and t tests, a statistical comparison of the recommended approaches with the standard spectrophotometric method revealed no significant differences in accuracy or precision. keywords: salbutamol, sulfadimidine, flow injection analysis, diazotization reaction. التقدير اللوني لكبريتات السالبيوتامول باستخدام تقنية الحقن الجرياني المستمر في االشكال النقية والصيدالنية * هند هاديو **، مريم جمال 1*،وسن االزري قسم الكيمياء، كلية العلوم، جامعة بغداد، العراق * وزارة التربية، مديرية تربية الرصافة الثانية ** الخالصة ( في slbتم وضع طرق بسيطة ودقيقة واقتصادية وهما طريقة الدفعة والحقن الجرياني المستمر، لتقدير دواء كبريتات السالبوتامول ) شف أزوتة الشكل النقي واالشكال الصيدالنية. اعتمدت كال الطريقتين على تفاعل االزوتة واالزدواج للدواء مع دواء اخر وهَو )السلفاديميدين( ككا االزو البرتقالية. تم التحقق من كل العوامل -نانومتر والتي عندها تم الحصول على اعلى امتصاصية لناتج صبغة 444ن في الوسط القاعدي. عند آم واء قانون دالكيميائية والفيزيائية لطريقتي الدفعة والحقن الجرياني للحصول على اعلى حساسية. وتحت الظروف التجريبية المثلٰى للمتغيرات اطاع ال مايكروغرام/مل لطريقتي الدفعة والحقن الجرياني على 2.51و 0.09مايكروغرام/مل وحدود كشف 10-100و 0.25-4بير عند مدى التراكيز اختباري ( لكال الطريقتين والذي اكدت امكانية تطبيق كال الطريقتين. وباستخدامn=5) %1التوالي. وكانت نسبة االنحراف القياسي النسبي اقل من ( تم عمل مقارنة احصائية للطريقتين المقترحتين مع الطريقة الطيفية القياسية والتي كشفت انه ال يوجد فرق واضح في الدقة والتوافقية t( وال )fال) لكال الطريقتين. التحليل بالحقن الجرياني ، تفاعل األزوتة. ،سلفاديميدين ،الكلمات المفتاحية : سالبيوتامول introduction salbutamol sulfate, also known as albuterol sulfate is chemically known as (rs)-1-(4hydroxy 3 hydroxy methyl-phenyl)-2-(tertbutylamino) ethanol sulfate, is beta 2 adrenergic receptor agonist bronchodilator drug have many uses such as treatment of patients with asthma (1). by reviewing the literature, it was found that there are many methods applied for estimation of slb in different samples such as liquid chromatographymass spectrometry (lc-ms) (2, 3), capillary electrophoresis (4), hplc (5, 6), spectrophotometry (79), flow injection analysis (10, 11), and voltammetry (12, 13). most of these methods are indirect or have drawbacks, such as reliance on expensive techniques, time demanding, and inappropriate for routine drug analysis (14-16) . as a result, it is essential to create a simple, quick, and low-cost approach for estimating slb in commercial dosage forms. spectrophotometric methods have been widely used in drug analysis; however, one of the most notable limitations of these approaches is the use of some hazardous chemical reagents for colorimetric reactions. as a result, replacement of these reagents with safer compounds like drugs is valuable option. 1corresponding author e-mail: wasanuzri@gmail.com received: 28/12 /2021 accepted: 28/2 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp45-52 iraqi j pharm sci, vol.32(1) 2023 colorimetric determination of salbutamol sulfate 46 fia has a higher sampling rate, uses fewer chemicals, has better precision, and is more versatile than batch approaches. because of the aforementioned benefits of fi, pharmaceutical analysis and quality control applications have seen a steady increase in interest. in the present work a drug compound was used as safe diazotization coupling reagent for estimation slb using direct batch and fia methods. sulfa drugs frequently contain multiple reactive amino compounds, one of which is sulfadimidine (sdm), which is utilized as a colorimetric reagent in this study to estimate slb. experimental instruments single beam spectrophotometer (shimadzu uv-visible 1240) was used for scan and absorbance measurements. the flow injection manifold composed mainly from 1 cm flow cell (50 μl), peristaltic pump type ismatec (ch–8152, switzerland) and injection valve (rheodyne, usa) used for injection micro liters of the sample. reagent solutions were continually pumped through flexible vinyl tubes while being mixed in a reaction coil (rc) consisting of teflon tubes (0.5 mm i.d.). with the use of a dual-channel manifold (figure 1); diazotized sulfadimidine (dsdm) was delivered through the first channel, while the second channel transported the nh4oh solution. through the second channel, through the injection valve, the slb drug solution was injected into the dsdm reagent stream and subsequently mixed with the ammonium solution inside the rc. the peristaltic pump was utilized for pumping the solutions and the azo-dye product's absorbance was measured at the manifold's end. figure 1. continuous fia manifold (dsdm, diazotized sulfadimidine; i.v, injection valve; p, pump; slb, salbutamol sulfate; r.c, reaction coil; d, detector; f.c, flow cell; w, waste). chemicals and reagents pharmaceutical grade slb and sdm (purity 99.9%) were supplied from iraq's state drug industries company (samara-iraq). butalin® tablets, labeled to contain 2 mg slb, manufactured by julphar industries-uae and purchased from local pharmacies. ammonia, sodium nitrite and hcl were purchased from bdh. solutions ● salbutamol sulfate solution (500 µg/ml): a standard solution of slb was prepared by dissolving 0.05 g of pure drug in 100 ml distilled water. ● diazotized sulfadimidine (0.01m): in 100 ml volumetric flask, 1ml of sdm standard solution (333 mg/ml) was transferred. after that a 3 ml of 1 m of hcl was added while keeping the temperature of the solution at 0-5 co in ice–bath. a weight of 0.069 g amount of nano2 was added with shaking for 5 min and then completed the volumetric flask with distilled water. ● hydrochloric acid (1m) and ammonium hydroxide (2m) aqueous solutions were laboratory prepared by simple dilution of concentrated hydrochloric acid (36.4% w/w) and ammonia solution (25% w/w) respectively and then standardized. solutions of marketed formulation after carefully weighing and grounding commercially available slb tablets, an amount of tablets’ powder equivalent to 0.05 g of pure drug was taken and dissolved in 50 ml of distilled water. the solution was then stirred vigorously for 15 minutes before being filtered into a 100 ml volumetric flask and diluted with distilled water. general procedure of batch method series volumes of standard solution of slb covers the range of concentrations of 0.25-4 μg/ml were transferred into 10 ml volumetric flasks. to each flask, 1.0 ml of dsdm (0.01m) and 4 ml of nh4oh (0.1m) solution were transferred then diluted with distilled water and mixed well. the reaction reaches to maximum intensity and stability after 10 min, and then the absorbance was measured at 444 nm (at 25 co) against reagent blank. the slb's amount was calculated using the calibration graph's regression equation. a concentration of 2 μg/ml slb was utilized in all batch optimization tests. general procedure of fia method a 150 μl of slb standard solution (range from 10-100 μg/ml) was injected through the injection valve utilizing a syringe. samples of slb were injected into 0.01m of dsdm stream. the resultant solution was mixed with 0.05 m nh4oh solution in the reaction coil. peristaltic pump propelled the solutions (flow rate of 2.87 ml/min) and the absorbance of the resulting orange product was monitored at 444 nm. a 50 μg/ml of slb was employed in the investigation of the fi system's optimum conditions. results and discussion the diazotization method is used for the determination of compound containing the primary aromatic amine group (17). most of these compounds are toxic, so it is preferable to use a pharmaceutical compound as a chromogenic reagent to make the iraqi j pharm sci, vol.32(1) 2023 colorimetric determination of salbutamol sulfate 47 methods less expensive and safer. in the present work, an orange dye produced from the coupling of slb with diazotized sdm (drug compound) was studied using batch and continuous fia methods. all the parameters that enhance the sensitivity of the reaction and consequently the assay of slb drug were studied. the influences of the chemical and physical parameters were carefully studied. absorption spectra and the mechanism of reaction the maximum value of absorption of azodye formed was estimated at 444 nm versus the blank in alkaline medium (figure 2). the supposed mechanism of the reaction was summarized in scheme 1. the aromatic amino group in sulfadimidine drug (the reagent in this study) is easily converted to diazonium salt during the diazotization reaction with nitrous acid. the diazonium salt then is coupling with phenolic drug (slb) in alkaline medium. using equimolar concentrations (4.2×10-4 m) of the drug and dsdm under the specified optimal conditions, the stoichiometry of the slb-sad dye was inspected by job's and molar ratio methods (18,19). a stoichiometry of 1:1 (slb: sad) was found with ε (molar absorptivity) value of 9.09×103 l/mol cm. figure 2. absorption spectra of (a) azo-dye product result from the reaction slb with dsdm/nh4oh measured versus the blank and (b) the blank versus distilled water. scheme 1. proposed reaction mechanism optimization conditions of batch method when dsdm is mixed with slb in the presence of ammonium hydroxide solution, an orange azo-dye product form very instantly. the azo-dye was found to absorb at 444 nm, fig. 2. the absorption of the orange dye was shown to be influenced by a number of factors, which were investigated consequently. the influence of altered volumes of 0.01m of dsdm (from 0.5 to 3 ml) and 0.1 m nh4oh (from 0.5 to 4 ml) were studied. maximum absorbance was obtained using 1 ml of 0.01m dsdm and 4 ml of 0.1 m ammonium hydroxide as shown in figure 3. iraqi j pharm sci, vol.32(1) 2023 colorimetric determination of salbutamol sulfate 48 figure 3. effect of the volumes of dsdm and nh4oh after 10 minutes from the start of the reaction, the product's absorbance was stable and remained constant for more than 30 minutes. the azo-dye product is formed and remains stable at room temperature, but as the temperature rises, the azodye decomposes; thus, room temperature (25 0c) is the optimal reaction temperature. also, different reagents addition orders were examined and it was found that the following order (dsdm+slb+nh4oh) was effective in achieving the desired findings (fig. 4) and was utilized in all subsequent tests. figure 4. order of addition (d=drug, b=base, r= diazotized reagent) optimization conditions of fia method manifold design the effects of all chemical and physical variables on the sensitivity estimation of slb using the fia approach were investigated. the fia manifold is the most essential parameter that must be optimized. to execute multiple reaction paths for slb drug with dsdm in alkaline medium, different types of manifold configuration (single and double channels manifolds) were studied. the two channels fi manifold provided the highest sampling frequency and maximal absorbance intensity. slb was injected into dsdm stream, which then combined with nh4oh using mixing coil as given in figure1. chemical variables the concentration effect of dsdm on the absorbance of dye was examined. into the stream of various concentrations of diazotized reagent (0.0050.05 m), a 150 μl of slb was injected. the results (fig. 5) indicated that a 0.01 m gave the best analytical signal and was chosen as optimum value. according to the previous studies, the reaction between slb and dsdm must carry out in an alkaline media; which could be because the phenolic drug (slb) is transformed to a more reactive phenoxide molecule. the influence of changing the concentration of nh4oh using a range of (0.01-0.1 m) was studied. the best results were obtained using 0.05 m of ammonium hydroxide (fig. 5) which was selected for the following experiments. figure 5. effect of concentration of reagent and base physical variables the flow rate has a great effect on the reaction sensitivity and sample frequency: therefore, this parameter was studied by using different rates ranged between 1.47-3.5 ml/min under the optimum conditions. when shown in figure 6a, the highest signal was attained at 2.87 ml/min, and then degreased gradually as the rate was increased due to increased dispersion. for that reason, a total flow rate 2.87 ml/min was selected as optimum rate. in the range of 25-100 cm, the influence of the mixing coil length was studied. a significant increase in absorbance (peak height) was detected with increasing the coil length up to 50 cm and then decreased (fig. 6b). this could be because of the fast coupling reaction between slb and dsdm, which eliminates the need to lengthen the reaction's steady time by increasing the rc length. because increasing the length of the rc creates increased dispersion and peak broadening, 50 cm was chosen for the following tests. the optimal injected volume of the sample was also tested using varied lengths of sample loop connected to the injection valve ranging from 75 to 200 µl, while keeping the other parameters constant. the results (fig. 6c) showed that 150 µl produced the highest intensity, thus that was chosen as the ideal volume. the flow system provided a sampling frequency of 39 samples h-1. iraqi j pharm sci, vol.32(1) 2023 colorimetric determination of salbutamol sulfate 49 figure 6. influence of (a) flow rate, (b) length of reaction coil, and (c) sample volume on intensity of azo-dye product. continued figure 6. summarized of optimum chemical and physical parameters table 1 lists all of the researched chemical and physical factors that may affect the azo-dye product's sensitivity for batch and fia techniques. figure 6. influence of (a) flow rate, (b) length of reaction coil, and (c) sample volume on intensity of azo-dye product. table 1. summary of studied parameters of batch and fia methods parameter studied range selected value batch fia concentration of dsdm (m) 5×10 -3-5×10-2 ---0.01 5×10-4-3×10-3 0.001 --- concentration of nh4oh (m) 0.01-0.1 ---0.05 0.005-0.05 0.04 --- injected sample volume (µl) 75-200 ---150 length of reaction coil (cm) 25-100 ---50 flow rate (ml/min) 1.47-3.5 ---2.87 methods validation calibration graphs at the optimized conditions (table 1), a linear calibration graphs were obtained for the analysis of slb drug utilizing batch and fia systems using a series of slb standard solutions. all analytical figures of merit were summarized in table 2 including linearity, repeatability, limit of detection (lod), and limit of quantification (loq). the slope, molar absorptivity, sandell’s sensitivity, and correlation coefficient for the calibration data were reported. batch and fia methods showed good linearity within the range 0.25-4 and 10-100 µg/ml of slb, respectively with correlation coefficients of at least 0.998. in comparison with the two techniques mentioned, fia is more convenient than the batch approach because of its greater linear range of calibration graph and high throughput samples (39 sample/h). the fia method is less sensitive than the batch approach, which could be owing to sample zone dispersion inside the carrier as well as reagent solutions arriving at the detector. accuracy, reproducibility and selectivity by analyzing three different concentrations of slb solutions using both approaches, the accuracy and reproducibility of the suggested procedures were assessed as relative error and relative standard deviation (% rsd), respectively (table 3). low relative error values (±3%) and good % rsd values (0.28-0.98%) point to the high accuracy and reproducibility of the present methods. iraqi j pharm sci, vol.32(1) 2023 colorimetric determination of salbutamol sulfate 50 table 2. analytical performance of proposed methods. variables batch method fia method regression equation y=0.2695x-0.0061 y=0.0039x+0.0723 linear range (µg/ml) 0.25-4 10-100 correlation coefficient, r 0.9981 0.9998 limit of detection (s/n = 3) (μg/ml) 0.09 2.51 limit of quantification (μg/ml) 0.29 8.36 molar absorptivity ε (l/mol cm) 9.09×104 1.32×103 sandell’s sensitivity, s (μg/cm2 ) 0.0037 0.2564 through-put (h-¹) 6 39 slope, b 0.2695 0.0039 intercept, a -0.0061 0.0723 sy/x 2.37×10 -2 3.17×10-3 sb 6.31×10 -3 4.34×10-4 sa 1.50×10 -2 2.66×10-3 table 3. accuracy and precision for batch and fia methods sample batch method fia method present conc. (µg/ml) found conc. (µg/ml) re (%) (rec.±rsd)% (n = 3) present conc. (µg/ml) found conc. (µg/ml) re (%) (rec.±rsd)% (n = 3) 1 1 1.03 3.00 103.00±0.76 25 24.90 -0.40 99.60±0.89 2 2 1.96 2.00 98.00±0.28 50 50.18 0.36 100.36±0.63 3 3 3.04 1.33 101.33±0.98 75 75.69 0.92 100.92±0.57 re. relative error, rec: recovery, rsd: relative standard deviation the interference of some additives that may be added to the drugs for manufacturing needs was evaluated in order to assess the selectivity and applicability of the recommended methods in routine analysis of slb in commercial tablets. to the 2 µg/ml of slb solution, fiftyfold of each interference (starch, lactose, polyvinylpyrolidone, and magnesium stearate) was added and evaluated separately utilizing batch and fia techniques. recovery ranged (97-101%) revealed that tablet additives had little to no effect, indicating that these methods could be used for slb quality control with high accuracy and precision. analysis of pharmaceutical formulations the two methods were utilized for the assay of slb in pharmaceutical formulations (tablets) and the results are given in table 3. the obtained results are in good agreement with the declared content. recovery studies for these tablets were used to evaluate the proposed procedures, and the findings showed that both approaches had outstanding recoveries values ranging from 95 to 98.8%. as demonstrated in tables 4, the findings of the two procedures were compared to the results produced by the official method (20) for the slb drug.the t and f-test values at 95% confidence limit (21) indicated that there are no considerable differences between batch, fia and standard methods for the assay of slb in dosage forms. iraqi j pharm sci, vol.32(1) 2023 colorimetric determination of salbutamol sulfate 51 table 4. application of the batch, fia and reference methods for the determination of slb in pharmaceutical dosage form. dosage form proposed methods standard method batch method nfia method conc.(µg/ml ) rec. (%)* rsd (%)* conc.(µg/ml) rec. (%)* rsd (%)* conc.(µg/ml) rec. (%)* rsd (%)* tak en found taken found taken found butalin® tablet (2mg ) 1 0.99 99.00 0.36 25 24.46 97.84 0.91 5 5.01 100.20 0.33 2 1.90 95.00 0.37 50 49.27 98.54 0.37 10 9.97 99.70 0.92 3 2.89 96.33 0.42 75 74.09 98.79 0.71 15 14.87 99.13 0.86 slb pure 100.78 100.29 99.98 t (4.303)** f (161.4)** 0.832 1.077 0.784 7.602 *for five determinations, conc., concentration; **theoretical value. conclusion the batch and continuous fia methods proposed for the analysis of slb in commercial dosage forms have the following characteristics: sensitivity, low cost, safe use of drug ingredient as reagent, and cover a wide variety of assays. the proposed methods were fully validated, with acceptable results for all of the method validation factors that were investigated. the fi process is suitable for analysis of slb in marketed formulations and is faster than batch methods due to its high sampling frequency (39 samples/h). there are several spectrophotometric and fia methods for determining slb, however many of them are insensitive, use hazardous reagents, or are quite expensive. experiments also showed that the results achieved using the two proposed methods are useful; therefore, these methods could be used as alternative methods for routine analysis of slb in different samples. references 1. reynolds, j.e.f. and martindale eds., (the extra pharmacopoeia), 30th ed., the pharmaceutical press: london; 2007; 1255. 2. zhang, d.; teng, y. and chen, k. determination of salbutamol in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry and its pharmacokinetic study. biomed. chromatogr. 2012; 26(10):1176-1182. 3. thuan, n. t. m. and linh, n. t. k. simultaneous determination of salbutamol and clenbuterol in human plasma using liquid chromatography coupled to tandem mass spectrometry. pharm sci asia. 2019;46 (2): 120-128. 4. bao, y.; yang, f. and yang, x. capillary electrophoresis coupled with electrochemiluminescence for the facile separation and determination of salbutamol and clenbuterol in urine. electroanalysis. 2012;24:7. 5. ibrahim, a n.; shimaa a f.; hala e z. and eglal a a. high-performance liquid chromatography method for simultaneous determination of guaifenesin, salbutamol sulfate and guaifenesin impurity (guaiacol). j chromatogr sci.2021, 59(5), 419-424. 6. selvadurai, m. and jayaraj k. high performance liquid chromatographic method development and its validation for salbutamol. british journal of pharmaceutical research. 2012, 2(4), 228-237. 7. abdoon, f. and yahyaa, s. validated spectrophotometric approach for determination of salbutamol sulfate in pure and pharmaceutical dosage forms using oxidative coupling reaction. journal of king saud university. 2020, 32(1), 709-715. 8. al-abachi, m. q. and abed, s. spectrophotometric determination of phenylephrine hydrochloride and salbutamol sulphate drugs in pharmaceutical preparations using diazotized metoclopramide hydrochloride. baghdad science journal, 2015, 12, 1. 9. al-enizzi, m. spectrophotometric method for assay of salbutamol in pharmaceutical formulations. basic sciences branch. 2015, 4, 12. 10. al abachi, m. q. and hadi, h. determination of salbutamol sulphate in pharmaceutical preparations using continuous/stopped flow injection method. international journal of research in pharmaceutical and biomedical sciences. 2012;3(3) :1189. 11. al-abachi, m. q. and subhi, s. flow injection spectrophotometric determination of salbutamol sulphate and pyridoxine hydrochloride using 2, 4dinitrophenylhydrazine. iraqi journal of scienc. 2013; 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16(3):60-67. 16. isabel, d. and moises k. flow-injection spectrophotometric determination of salbutamol with 4-aminoantipyrine. talanta. 2004; 64(5): 1233-1236. 17. bruice, p.y. organic chemistry. pearsons prentice. 2004. 18. job, p. advanced physiochemical experiments. 1964, 2nd ed., oliner and boyed, edinburgh, 54. 19. levie, r. principles of quantitative chemical analysis, the mc graw-hill companies, inc., singapore, 1997: 498. 20. british pharmacopaeia, h.m. stationary office, uk: london; 2019. 21. miller, j.c. and miller, j.n. statistics for analytical chemistry, ellis horwood: chichester, uk; 6th ed., 2010. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba doi: https://doi.org/10.31351/vol29iss2pp27-36 27 phytochemical investigation of aerial parts of iraqi cardaria draba alaa m. abd*,1 and enas j. kadhim** * ministry of health and environment, aldiwaniyah,iraq. **department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad,baghdad,iraq. abstract cardaria draba (l.) desv. (brassicaceae; syn. lepidum draba (l). commonly known as whitetop or hoary cress, is a perennial plant reproduces by seed and by horizontal creeping roots. brassicaceae or cruciferae family commonly known as the mustards family, contained flavonoids, alkaloids, saponins and a lot of dozens of glucosinolates. the aim of this research was to study chemical constituents of aerial parts of cardaria draba since no phytochemical investigation had been studied before in iraq for this plant. aerial parts of cardaria draba were defatted by maceration in hexane for 48 h. the defatted plant materials were extracted using soxhlet apparatus, with the aqueous methanol 90% as a solvent of extraction for 12 h, and fractionated with petroleum etherchloroform – ethyl acetateand n-butanol respectively. the n-butanol fraction was hydrolyzed with 10% hcl by reflex to break down the glycosideic linkage. flavonoids and phenolic acid compounds were isolated from hydrolyzed n-butanol fraction by preparative tlc to be then identified by hplc, tlc, ftir and melting point. the chromatographic and spectroscopic results showed the presence of luteolin, chlorogenic acid, caffeic acid, and resorcinol in aerial parts of c. draba. keywords: cardaria draba, flavonoids, phenolic acid, high-performance liquid chromatography (hplc) and fourier transform infrared spectroscopy (ftir). دراسة المحتوى الكيميائي لالجزاء الهوائية لنبات القانبري العراقي **كاظم جواد ايناس و 1*،االء ماجد عبد الرحيم وزارة الصحة والبيئة ، الديوانية ، العراق * .بية ، كلية الصيدلة ، جامعة بغداد، بغداد ، العراق فرع العقاقير والنباتات الط** الخالصة نبات القانبري العراقي من عائلة الخردليات وهو يحنوي على مركبات عديدة مثل الفالفونيييد القلويدات والصابونيات. يحنوي النبات ايضا على كالكوسيدات التي تحنوي على مركبات الكبريت. ازالة الدهون تم.يهدف البحث الحالي دراسِة المكونات الفعالة لالجزاء الهوائية لنبات القانبري, حيث لم تتم دراسته من قبل في العراق. ساعة في الهكسان. تمت عملية االستخالص لالجزاء المنزوعة الدهون في المحلول 48من االجزاء الهوائية للنبات بواسطة تنقيعها لمدة ساعة و باستخدام جهاز السكسوليت ثم تمت عملية تجزئة بعدة مذيبات عضوية شملت بالتتابع : 12لمدة %90المائي للميثانول %10خالت االثيل و البيوتانول االولي . تم كسر االصرة الكاليكوسيدية باستخدام حامض الهيدروكلوريك -كلوروفورم -لبتروليااليثرا preparative) . تم التحري عن محتوى المركبات الفينولية والفالفونويدية وعزلها بتقنية طبقة السليكا التحضيرية من الجزء المتحلل tlc) ف بمطياف الكشف تحت الحمراء والفصل الملون عالي الكفاءة ثم اجراء الكش(hplc) والتاكد من نقاوتها بتقنية الفصل الملون من نتائج الفصل الملون وكذلك نتائج تحليالت المطياف بينت . واختبار درجة االنصهار للمركبات المعزولة tlc)بطبقة السليكا الرقيقة ) البسيطة الريزورسينول , حامضي الكلوروجينيك والكافاييك في االجزاء الهوائية لنبات القنبري. ضوجود فالفونيد اللوتيولين واالحما . الحمراء تحت االشعة مطياف االداء، عالي السائل كرومرتوغرافيا ، الفالفونويدات والفينوالت ، القانبري نبات الكلمات المفتاحية : introduction cardaria draba (l.) desv. (brassicaceae; syn. lepidum draba (l). commonly known as whitetop or hoary cress, is a perennial plant reproduces by seed and by horizontal creeping roots (1). brassicaceae or cruciferae family commonly known as the mustards family, contained flavonoids, alkaloids, saponins and a lot of dozens of glucosinolates. they also have an enzymes called myrosinases which convert the glucosinolates into thiocyanates, isothiocyanate and nitriles which are toxic to many organisms, and so that help protector against herbivores. the plant oil content is mostly produced from the seeds of various species (2). the genus name arises from the greek word kardia (heart), which refers to the heart shaped fruit of c. draba. although not all the fruit in this genus are heart shaped. common names for c. draba are heart-podded, hoary cress, white-top, perennial peppergrass and in england it is known as hoary pepperwort, chalk weed and may be referred as whitlow pepperwort (devil's cabbage) (3). 1corresponding author e-mail: majidalaa622@gmail.com received: 20/10 /2019 accepted: 22/ 2/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp27-36 iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba 28 plants belonging to this genus are reported to have wide applications in folk medicines, as an anthelmintic, antiscorbutic, purgative and expectorant effects (4). the seeds have been used in treating flatulence and fish poison also used as a condiment. a decoction of the whole c. draba plant and seed is used as a diuretic in ethnomedicine in iran. it has been reported that the edible species in spain, rich in protein content higher than leaves of cabbage and spinach (5) .some flavonoids and phenols isolated from c. draba exhibited antihypertensive, anti-inflammatory, antimicrobial, antioxidant and antiradical activity. in iraq at aldiwaniya city, cardaria draba extract used to treat leishmaniasis (baghdad sore) topically. since there is no phytochemical investigation and separation of this naturally grown plant in iraq; the current research for flavonoids and phenolic compounds were investigated. c.draba contains alkaloids, saponins and flavonoids. in total of 16 compounds; isorhamnetin, quercetin and kaempferol were the most abundant flavonoids compounds while the most abundant phenolic compounds were sinapic acid, p-coumaric acid, caffeic acid and ellagic acid. phenolics are mostly produced in plants as secondary metabolites via the shikimic acid pathway (6-8). phenolic compounds are present in aerial parts of plant. plants have natural defense system against bacteria, insects, viruses and fungi, phenolic compounds consider an important part of this system and they can switch plant hormones. brassica species contain a wide and diverse range of polyphenols, namely the flavonoids and hydroxycinnamic acids, which serve as biochemical markers to differentiate members within different genera or even within the same species ( 9-11). chlorogenic and caffeic acids have antioxidant, anti-inflammatory, prevent diabetes, prevent premature aging, depigmentation, prevent neurodegenerative diseases, like parkinson’s disease, anti-hepatitis b virus activity also have been used to prevent sodium-selenite-induced cataract and reduce exercise-related fatigue (1223). luteolin has anti-oxidant activity, antiinflammatory, antimicrobial anticancer (24-29). resorcinol is an anesthetic found in throat lozenges also used as chemical intermediate for the synthesis of pharmaceuticals and other organic compounds (30-32). the dominant study objective is to investigate and isolate some flavonoids and phenols from n-butanol after hydrolysis fraction of c.draba grown in iraq since there were no previous studies concerning the iraqi species. experimental section plant material at the flowering stage, aerial parts of cardaria draba were collected from al hamza city of al-diwaniyah, iraq, in april 2018, identified at the iraq natural history research center and museum ,university of baghdad . extraction cardaria draba were air-dried for 3days. the aerial plant parts (75gm) were cut into small pieces, powdered and defatted by maceration in pure n-hexane for 48 hours, filtered through a whattman paper, shade dried, plant material was powdered then filled in the thimble and extracted with 700 ml of 90% methanol by a soxhlet extractor for 12 h. this extract was concentrated using rotary evaporator. after complete evaporation of the solvent, dry extract was weighted and dissolved in 100 ml water, partitioned with 100 ml (3times) petroleum ether, chloroform, ethylacetate and butanol. each fraction evaporated by rotary evaporator, each dry fraction weighted and revealed for preliminary test. the n-butanol fraction was hydrolyzed by reflex with 10% hcl, and then the hydrolyzed fraction was taken with n-butanol then dried for further investigation. screening of phytochemical components to identify the phytochemical derivatives in the methanolic extract, general phytochemical screening was performed according to literature (33). flavonoids test (shinoda test) few amount of the extract were dissolved in 1ml of 50% methanol then dried on steam bath. then a few drops of concentrated hydrochloric acid (hcl) were added followed by metallic magnesium. an orange or red color shows the presence of aglycone flavonoids. phenols test: this test was done by adding few drops of 1% fecl3 to few milligrams of aqueous methanol plant extracts. formation of dark greenish-blue color indicates the presence of phenols. alkaloids test by dragendorff's reagent the reagent is composed from two solutions, solution a and b solution a contains 60mg of bismuthsubnitrate in 0.2 ml hcl solution b contained 600 mg ki in 1ml distilled water. few drops from plant extract was added to the mixture of solutions a and b, should give brown precipitate that indicate presence of alkaloids. iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba 29 saponins test in a test tube few milligrams of extract were added to 5 ml of h2o. the solution was shaken strongly and observed for a stable persisting froth. few drops of olive oil were mixed with the frothing and shaken vigorously then it was observed for the formation of an emulsion. test for tannins in a test tube, few milligrams of the extract were boiled in 10 ml of water and filtered. a few drops of 0.1% ferric chloride (fecl3) were added and observed for a blueblack or brownish--green coloration. flavonoids and phenolic acid compounds isolation by preparative tlc from the hydrolyzed n-butanol fraction: flavonoids and phenolic compounds were isolated by preparative tlc from the hydrolyzed n-butanol fraction of c. draba. preparative silica gel gf254 plate of 20 cm×20 dimension with a layer thickness of 0.5cm.was reactivated by heating at 100oc for 15–20 min, then left to cool used for application. two mobile phase for nbutanol fraction after hydrolysis were used: first; (chloroform: methanol [90:10v/v]) and second mobile phase (chloroform: methanol: formic acid [87.5:10: 2.5]) placed in separated jars. the jars were lined with a filter papers closed tightly, and left for saturation. sample application was done by disolving0.5 g of the sample in absolute methanol and applied to the baseline of preparative tlc plate using capillary tubes. the isolated flavonoid and phenolic compounds from n-butanol after hydrolysis fraction of the aerial parts were recognized by hplc, tlc, ftir and melting point. by examination under uv light, the detection was done with wavelengths; 365& 254 nm. by analytical tlc, the purity of each band was checked until a single spot had been obtained in corresponding to reference standard. the pure compounds were scraped and extracted from adsorbed silica by methanol to be analyzed by hplc method. detection of isolated compounds by hplc analysis hplc analysis was achieved on pump system (knauer, bad homburg, germany), model 8300 hplc and an s-3210 photodiode-array detector (pda), with a water (150 × 4.6 mm i.d.) eclipse xdr-c18 column, using a binary solvent system: solvent a: dd h2o/ trifluoroacetic acid (97:3, v/v); and solvent b: acetonitrile. the following gradient program was used: 100–90% a from 0 to 10 min, 90– 30% a from 10 to 32 min, 30–0% a from 32 to 45 min at a flow rate of 1 ml/ min (34). standard solutions for hplc were: luteolin, resorcinol, caffeic acid and chlorogenic acid by dissolving 5 mg in 1 ml of hplc methanol. samples were firstly dried prepared for hplc analysis then dissolving them in hplc methanol and subjecting them to ultrasonication at (60% duty cycles for 25 min at 25°c then by centrifugation at 7500 rpm for 15 min). the pure upper layer of each sample was evaporated under vacuum. the residues were resuspended separately, in 1 ml of methanol hplc grade, standardizing using vortex mixer, and passing them through 2.5 µm disposable filter, then vortex mixer, and passing them through ( 2.5 µm disposable filter, finally stored at 3-5°c ). for hplc analysis; 0.02 ml of the sample was injected. detection of isolated compounds by ftir analysis: the isolated compounds were subjected to ftir analysis to detect the functional groups of these compounds. the following condition and apparatus were used shimadzu 3800 ft-ir/ japan in the pharmaceutical chemistry department at al mustansiriyah university / college of science. detection of isolated compounds by melting point using melting point apparatus stuart melting point /smp30 results phytochemical investigation for methanolic extract of c.draba table 1 showed the major active constituents present in the c.draba extract. table1. phytochemical analysis of aerial parts of cardaria draba extract the present study done for the cardaria draba showed the presence of medicinally active constituents. c. draba phytochemical active compounds were qualitatively analyzed and the results are presented in table 1.the positive and negative results, based on the presence or absence of color change. in this screening process, flavonoids, phenols, alkaloid and result phytochemical components + alkaloid + flavonoid + phenols + saponin tannin iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba 30 saponins gave positive (+) results and tannin offered negative (-) result. flavonoids and phenolic acid compounds isolation by preparative tlc from the hydrolyzed n-butanol fraction: tlc chromatogram for the hydrolyzed n-butanol fraction and non-hydrolyzed fraction showed in figure 1, which indicate the presence of chlorogenic acid, caffeic acid, luteolin and resorcinol in both fractions. figure 1-tlc chromatogram at 254nm(a), at 365nm(b) for n-butanol fraction before hydrolysis(left),and after hydrolysis(right); a:chlorogenic acid b: caffeic acid c: luteolin d :resorcinol, mobile phase: chcl3 :m2oh : formic acid (87.5 : 10: 2.5) for the preperative tlc, bright lines were investigateg which had been scrapted for isolated each compound,figure (2,3). figure .2. preparative tlc chromatogram of n-butanol hydrolyzed fraction at a: 254 and b:365, a : caffeic acid b: luteolin c : resorcinol , mobile phase chcl3 :meoh ( 90 :10) figure 3preparative tlc chromatogram of n-butanol hydrolyzed fraction at a: 254 & b:365, a :chlorogenic acid b:caffeic acid. c :luteolin, mobile phase chcl3 :meoh: formic acid ( 87.5 :10 :2.5) iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba 31 hplc chromatogram for the hydrolyzed nbutanol fraction hplc analysis were performed for the n butanol fraction (figure 4) and each isolated compound as shown in figures 5, 6, 7 and 8. figure 4. high performance liquid chromatogram (hplc) analytical of but. aft. hydrolysis fraction. figure.5. a: hplc of standard chlorogenic acid, b: hplc of isolated chlorogenic acid figure 6 .a : hplc of standard caffeic acid, b: hplc of isolated caffeic acid. iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba 32 figure 7 .a:hplc of standard luteolin b: hplc of isolated luteolin. figure 8. a: hplc of standard resorcinol b : hplc of isolated resorcinol tlc for the isolated compounds this was performed to insure the purity of the isolated compound which were isolated by scraping the isolate bands of the preparative tlc. figure 9. thinlayer chromatography for chlorogenic acid standard a and isolated chlorogenic acid b, on gf254 silica gel detection under uv light a: at 254 nm and b: 366 nm. figure10: thinlayer chromatography for caffeic acid standard a and isolated caffeic acid b, detected under uv light (a): at 254 nm and (b): at 366 nm. figure.11. thinlayer chromatography for ( a) : luteolin isolated a and standard luteolin b, (b) resorcinol standard a and isolated resorcinol (b) on gf254 silica gel revealing under 254 nm uv light ftir analysis for the isolated compounds the ftir spectral analysis of separated chlorogenic acid compound show peaks at 3600, 3400-3200, 1680, 1640, 1600, 1500, 1400, 1280. (figure12) iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba 33 figure12. ftir spectrum of isolated chlorogenic acid. ftir spectral analysis of isolated caffeic acid (compound show peaks at 3400, 32503200, 3000, 2900, 2800, 2500, 1640, 1620, 1600, 1530, 1440, 1290 (figure 13). figure13. ftir spectrum of isolated caffeic acid. ftir spectral analysis of isolated luteolin compound show peaks at 3400-3000, 2700, 2600,1650, 1600, 1550, 1500, 1500 ,1400 ,1300 ,1200 (figure 14). iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba 34 figure14. ftir spectrum of isolated luteolin. ftir spectral analysis of isolated resorcinol compound show peaks at3400-3000, 2880, 2600, 1600, 1400, 1370, 1290, 1100 (figure15). figure15. ftir spectrum of separated resorcinol. melting point ch1 compound melt at 205208 oc which match standard chlorogenic acid. ca 2 compound melt at 221-224 oc which match standard caffeic acid. l5 compound melt at 267-270 oc which match standard luteolin. r8 compound melt at 108-111 oc which match standard resorcinol. discussion natural products have at all times been a preferred choice of all as they play a great role in discovering new medicines. during extraction, solvents drawn-out into the solid plant material then solubilize compounds with similar polarity. through standard procedure plant's chemical constituent extraction and separation depend on selective solvents. flavonoids have an important role in the healthcare since they are a major class of natural compounds, broadly distributed in plants and numerous traditional medicine systems of the world. the preliminary phytochemical analysis confirmed the presence of alkaloids, phenols, and flavonoids. in (ir) spectral analysis, firstly iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of cardaria draba 35 the peak at 2947.33 cm−1 showed c-h stretching due to –ch 2, the peak at 3300.10 cm−1, a broad band is most probably the result of o-h stretching vibrations of phenol -oh group. the peak at 1697.41 cm−1 indicates the presence of( -c=o) carbonyl group. the peak at 1606.76 cm−1 showed the presence of -ch=ch group. the peak at1643& 1508.38 cm−1 revealed the presence of benzene ring. in addition to hplc, melting point and the above results approve that the isolated compound is chlorogenic acid. peak at 3100-3400, broadly band is utmost possibly the result of (o-h) stretching vibrations of phenol( oh) group, the peak at 1606.76 cm−1 showed the presence of( ch=ch )group. the peak at1643& 1508.38 cm−1 revealed the presence of benzene ring. in addition to hplc, melting point and the directly above results approve that the isolated compound is resorcinol. the importance of this study, is the first study which confirms the presence of chlorogenic acid, caffeic acid, luteolin and resorcinol in the iraqi species of c. draba. conclusion the results of this study exhibited the presence of phenols, i.e., chlorogenic, resorcinol and 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2017;1(10):370-5. 34. sharifi-rad j, hoseini-alfatemi sm, sharifirad m, da silva ja, rokni m, sharifi-rad m. evaluation of biological activity and phenolic compounds of cardaria draba (l.) extracts. j. biol. today's world. 2015;4(9):180-9 (modified). baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 occupational hazard of perchlorethylene doi: https://doi.org/10.31351/vol31iss2pp144-149 144 health hazards, hematological and biochemical alterations in drycleaning workers using perchloroethylene. bushra hassan marouf*,1 *department of pharmacology and toxicology, college of pharmacy, university of sulaimani, sulaimani, iraq abstract perchloroethylene (perc) is commonly used as a dry-cleaning solvent, it is attributed to many deleterious effects in the biological system. the study aimed to investigate the harmful effect associated with perc exposure among dry-cleaning workers. the study was carried out in the period between february 2019 to august 2019 and it was conducted on 58 adults in two groups. perc-exposed group; include thirty-two male drycleaning workers using exclusively perchloroethylene (perc) as a dry-cleaning solvent in sulaimani and erbil; northern-cities in iraq, and twenty-six healthy non-exposed subjects. history of perc exposure, use of personal protection equipment (ppe), safety measurement of the exposed group was recorded. blood sample was taken from each participant for measurement of hematological markers, liver and kidney function tests. the results showed that 28.1% of the workers were using ppe regularly while 71.9% of the workers were not. most of the workers 31(96.9%) were disposed of their waste products in improper way. non-significant differences were observed in hematological indices between perc-exposed group and non-exposed group. serum level of kidney and liver function markers of the dry-cleaning workers were higher with statistically significant difference compared with the non-exposed group. in conclusion, a non-significant alteration in hematological parameters, while significant changes in part of liver and kidney functions indices have been demonstrated. clinical observation indicated a harmful effect in perc-exposed group. keywords: occupational, perchloroethylene, personal protective equipment, dry cleaner المخاطر الصحية و التغيرات الدموية و البيوكيميائية في عمال الغسيل الجاف المستخدمين للبيركلوروأثيلين 1*،بشرى حسن معروف فرع االدوية والسموم، كلية الصيدلة، جامعة السليمانية، سليمانية، العراق * الخالصة للنظام بيركلوروأثيلين الضارة اآلثار من العديد الى يؤدي المحلول لهذا التعرض الجاف. للغسيل كمحلول شائعة بصورة يستخدم ت هذه البيولوجي. الهدف من الدراسة الحالية هو البحث عن التأثيرات الضارة المرتبطة بالتعرض لبيركلوروأثيلين بين عمال الغسيل الجاف. أجري من عمال التنظيف الجاف 32موعتين. المجموعة أالولى تشمل مجموعة التعرض لبيركلوروأثيلين وتحتوي على شخص في مج 58الدراسة على شخص سليم وغير معرض لمادة بيركلوروأثيلين. تم تسجيل 26المستخدمين للبيركلوروأثيلين كمحلول تنظيف. أما المجموعة الثانية فتحتوي على واستخدام معدات الحماية الشخصية للمجموعة المعرضة . و تم أخذ عينة الدم من كل مشارك لقياس المؤشرات مدة التعرض لمادة بيركلوروأثيلين ٪ من العمال 71.9٪ من العمال استخدموا معدات الوقاية الشخصية بانتظام في حين 28.1الدموية و اختبار وظائف الكبد والكلي. أظهرت النتائج أن ٪( تم التخلص من نفاياتهم بطريقة غير سليمة. اظهرت النتائج بان الفرق في المؤشرات 96.9)31لشخصية. معظم العمال لم يستخدموا معدات الوقاية ا و الدموية بكلتا المجموعتين غير ملحوظة. هناك فرق ملحوظ في مؤشرات الكبد والكلي في الدم بين المجموعتين حيث كانت نسبة مؤشرات الكبد وعة المعرضة أعلى عند مقارنته مع المجموعة غير المعرضة .الكلي في مصل الدم للمجم أستنتجت الدراسة ان هناك تغيير غير ملحوظ في المؤشرات الدموية مع وجود تغيير ملحوظ في جزء من مؤشرات وظائف الكبد والكلي. واشارت الدراسة السريرية الى وجود آثار ضارة في المجموعة المعرضة لبيركلوروأثيلين. ، غسيل الجاف معدات الحماية الشخصية، بيركلوروأثيلينالمهنية ، الكلمات المفتاحية : introduction perchloroethylene (perc), also known as tetrachloroethylene, is uncolored, volatile, nonflammable liquid and widely known for its use in dry cleaning of fabrics and clothes. perc is released into the environment from processes utilized in dry cleaning systems, by evaporation from dry cleaned clothing, from spills, and from waste products containing perc (1,2). acute exposure via inhalation may lead to irritation of the upper respiratory tract and the central nervous system. hepatic, renal, respiratory, hematological, ocular and dermal systems are target organs for perc toxicity in humans (3). additionally, the neurobehavioral function of healthy individuals is also influenced adversely as a result of chronic exposure to perc. chronic inhalation to perc is associated with headaches, loss of memory, motor neurobehavioral functioning, color vision impairment (4,5). 1corresponding author e-mail: bushra.marouf@univsul.edi.iq received: 24/9 /2021 accepted:15 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp144-149 iraqi j pharm sci, vol.31(2) 2022 occupational hazard of perchlorethylene 145 furthermore, united state environmental protection agency (us epa) declared perc as potential occupational carcinogen (6). metabolic pathways of perc are mainly cytochrome p450 pathway and glutathione conjugation pathway, the former one generates metabolite of triand dichloroacetate in liver, that are associated with hepatic toxicity and carcinogenicity. while the glutathione pathway is related to the formation of reactive metabolites targeted the kidney to induce renal toxicity and carcinogenicity (7). therefore, many studies elaborated the impact of perc exposure in various target tissues including liver, kidney and hematological tissues and highlighted the noncancerous toxicity of perc (8-11). furthermore, a toxicological review on perc has highlighted the scientific issues associated with deleterious effect of perc on the human health (12). a preliminary study conducted by habib et al (2018) has concluded that perc is a widelyspread cleaning solvent, and the exposure of perc among dry-cleaning workers is high. therefore, installation of local exhaust ventilation systems and monitoring devices of perc level is very important among dry-cleaning along with increase the awareness of workers about the harmful effects of perc and the significance of using personal protective equipment (ppe) while performing their job such as wearing appropriate chemical resistant gloves and clothing, safety goggles with a face shield for eye protection and respirator for respiratory protection (13). in some countries, manufacturers have begun using alternative cleaning methods that do not require the use of perc (14). however, in iraq especially in kurdistan region this solvent is the main dry-cleaning agent so far. therefore, the current study was designed to investigate the effect of perc on hematological markers, renal and liver function in the dry-cleaning workers, and to observe the awareness of the workers on the safety and occupational risks of this toxic substance in their workplaces. subjects and methods study population and data collection the study was designed as a crosssectional analysis, it was carried on 58 adult male subjects arranged in two groups, workers group (i.e. exposed to perc); includes thirty-two male drycleaning workers employed in 15 laundry shops that used exclusively perchloroethylene (perc) as a dry-cleaning solvent in sulaimani and erbil; northern-cities in iraq, and twenty-six healthy nonexposed subjects who were matched with the workers group in sex and age, subjects with different smoking habits have been excluded. the study was carried out in the period between february 2019 to august 2019. a written informed consent was obtained from each participant. the study protocol was approved by the ethical committee of the college of medicine, university of sulaimani (registration no#54 in 15.08.2017). a structured questionnaire, which was especially designed for the study, has been utilized and the responses of the participants were recorded by the researcher through face to face interview. the questionnaire was comprised of different sections to collect the information on the various aspects including demographic characteristics of the participants, exposure history to perc, level of awareness of the workers on the safety measurement and hazards of the perc, use of ppe during working hours, methods of disposal of perc waste byproducts; whether the management of perc waste is performed properly i.e through a specific management system or improperly through pouring them down the municipal sewer system. clinical and laboratory investigation a physical examination was performed by the researcher using a structured questionnaire to investigate the indoor-toxic effect of the perc on the workers including dizziness, headache, ataxia, eye irritation, respiratory tract effect and its dermatotoxicity. blood sample (10 ml) was taken from each subject. three milliliters of blood were collected in an edta tube and used for the determination of hematological markers including complete blood cells count using beckman coulter method (15). seven milliliters were collected in a plain tube for isolation of the serum by centrifugation at 5,000 rpm for 10 min. the serum was used for measurement of the markers of renal function (serum urea and creatinine) and liver function (total bilirubin, serum alanine aminotransferase (alt), aspartate aminotransferase (ast), alkaline phosphatase (alp) and lactate dehydrogenase (ldh)) spectrophotometrically using the clinical chemistry analyzer cobas c311 and ready-made kits (roche diagnostics gmbh, mannheim, germany) according to the manufacturer’s specifications. statistical analysis statistical analysis was performed using graphpad prism 5.0.1 softwares (graphpad software inc., la jolla, ca, usa). categorical data were analyzed with chi-square test. continuous variables were analyzed using independent sample t-test or the two-way anova and bonferroni’s post hoc test. a p-value of less than 0.05 was considered to be statistically significant. results the total number of the exposed drycleaning workers was 32 while of non-exposed subjects was 26. their mean age was (35.43±6.7) year for exposed group while (35.2±6.9) for nonexposed group. the occupational history of participants, ventilation status, vapor leakage and other characteristics of the workplace is summarized in table 1. the results reveal that most of the participants 20(62.5%) were with the duration of work of 5-10 years as a dry-cleaning worker. while iraqi j pharm sci, vol.31(2) 2022 occupational hazard of perchlorethylene 146 12(37.5%) were with more than 10-year duration. the other finding of the present study was relevant to the awareness of the workers on the safety and hazards of the perc. the results showed that 28.1% of the workers (n=9) were using the ppe such as chemical resistant gloves and clothing, goggles, apron and boots regularly while 71.9% of the workers (n=23) were not. almost all of the drycleaning laundry workers were disposed of perc hazardous waste products improperly 31(96.9%). simply they pouring them down the drain or into sanitary sewer system. the fact sheet on perc which provides information on health effects was available only for 13(40.6%) of the dry-cleaning laundry workers, while it was not available for 19(59.4%) of the them (p-value=0.081) (table 1). table 1. demographic characteristics of the drycleaning workers (n=32) and workplace condition. values are presented as percent or mean±sd; ppe: personal protective equipment. additionally, most of the workers had no education on occupational hazards 27(84.4%) and they had no a periodic health follow-up 31(96.9%). few numbers of the workers 2(6.3%) have taken a training course on the protection aspects of human exposure to perc in their workplace and nonremarkable number 1(3.1%) of dry-cleaning workplace has been visited by health authority (table 2). table 2. safety and health concerns of the drycleaning workers (n=32). values are presented as numbers and percentages. effect of perc vapor on hematological parameters is shown in table 3. non-significant differences were observed in hematological indices between the non-exposed group and perc-exposed group. parameter number (%) p-value age (year) 35.43± 6.7 duration of exposure to perc (year) < 5 years 0(0) 0.012 5-10 years 20(62.5) > 10 years 12(37.5) ventilation status in the workplace poor 5(15.6) 0.02 good 25(78.1) excellent 2(6.3) vapor leakage positive 6(18.8) 0.011 negative 26(81.2) protection facilities (ppe) available 9(28.1) 0.032 not available 23(71.9) waste disposal appropriately disposed 1(3.1) 0.01 inappropriately disposed 31(96.9) availability of fact sheets available 13(40.6) 0.081 not available 19(59.4) safety concern yes n(%) no n(%) education on workplace hazards 5(15.6) 27(84.4) periodic health follow-up 1(3.1) 31(96.9) reported cancer cases in workplace 0(0) 32(100) accidental death in workplace 2(6.3) 30(93.7) training in workplace 2(6.3) 30(93.7) follow-up by health authority 1(3.1) 31(96.9) iraqi j pharm sci, vol.31(2) 2022 occupational hazard of perchlorethylene 147 table 3. the hematological indices of both perc-non-exposed and perc-exposed group (n=58) values are presented as mean±s.d. n: number of participants; unpaired t-test was utilized to predict significance at p<0.05; hb: hemoglobin; hct: hematocrit; wbc: white blood cell. effects of exposure to perc vapor on the kidney and liver function markers of the drycleaning workers as shown in table 4. the results show significant higher serum urea, alt and alp level of the dry-cleaning workers group compared with non-exposed group (p-value 0.05). table 4. kidney and liver function markers of perc-non exposed and perc-exposed group (n=58) values are presented as mean±s.d; * significantly different compared with the non-exposed group values (p<0.05); alt: alanine aminotransferase; ast: aspartate aminotransferase; alp: alkaline phosphatase; ldh: lactate dehydrogenase the clinical observation of the perc exposed workers shows a profound clinical sign such as memory loss, dizziness, headache, irritation of the eye, respiratory tract irritation, ataxia and dermatological problems (table 5). table 5. percentage of certain clinical signs that appear frequently in perc-exposed dry cleaning workers (n=32) values are presented as numbers and percentages discussion perchloroethylene (perc), has been used commercially in many countries in dry cleaning, textile processing, and metal-cleaning operations (6). in dry-cleaning laundries, release of perc vapors into the environment and subsequent worker exposure to perc vapors is greatest during maintenance of dry-cleaning machine and disposal of waste products (16). although the present study has not measured the blood or urine concentration of perc or its metabolite in the exposed dry-cleaning workers, many studies have detected a significant measurable concentration of this substance in their biological fluids (9) which indicate that the drycleaning employees have occupational exposure to perc (10). further studies have shown that people exposed to perc either during work or residential proximity to perc dry cleaners have elevated levels of perc in blood, exhaled breath and urine (4,17). in the present study, the occupational observation of the laundry shops demonstrated inadequate safety profile that necessitate establishment of more educational and awareness program to overcome a potential risk of utilizing dry-cleaning solvent in these workplaces. azimi pirsaraei et al stated that the dry-cleaning workers are highly exposed to perc in their occupational settings, therefore protective measure should be considered to avoid the harmful effect of this toxic substance on the workers (3). the finding of the present study is inconclusive whether there is a correlation between using protective equipment and health status of the workers, however a recent study also emphasized on the proper use of personal protective equipment to avoid early dna damage in dry cleaners (11). in the present study the clinical status, hematological and biochemical parameters of the dry-cleaning workers in the laundry shops of parameters eerc-non exposed group (n=26) perc-exposed group (n=32) p-value hb (g/dl) 15.47±1.01 15.75±0.94 0.28 hct (%) 46.30±2.74 46.77±2.81 0.52 total wbc (x 109/l) 7.19±1.37 7.42±1.42 0.54 lymphocytes (x 109/l) 2.42±0.63 2.43±0.52 0.98 granulocytes (x 109/l) 4.54±0.90 4.52±1.15 0.93 platelets (x 109/l) 207.9±64.4 231.3±63.8 0.17 parameters perc-non exposed group (n=26) perc-exposed group (n=32) p-value s. urea (mg/dl) 26.2±3.2 30.9±6.7* 0.0017 s. creatinine (mg/dl) 0.83±0.15 0.84±0.18 0.76 s. bilirubin (mg/dl) 0.49±0.26 0.52±0.34 0.75 alt (u/l) 20.0±6.6 27.0±15.8* 0.04 ast (u/l) 22.3±6.7 23.9±9.6 0.46 alp (u/l) 67.0±23.2 87.0±40.4* 0.029 ldh (u/l) 154.9±31.0 161.3±28.3 0.42 clinical sign incidence n (%) memory loss 10(31.3) dizziness 9(28.1) headache 12(37.5) irritation of the eye 4(12.5) respiratory tract irritation 6(18.8) ataxia 3(9.4) dermatologic problems 4(12.5) iraqi j pharm sci, vol.31(2) 2022 occupational hazard of perchlorethylene 148 sulaimani and erbil cities were investigated. certain clinical signs in perc-exposed dry cleaning workers have been reported such as dizziness, headache, dermatological and respiratory tract effect. this clinical surveillance of the dry-cleaning workers in the present study is consistent with the other studies which have concluded that short-term inhalation of perc can lead to irritation of the nose and throat and depress the central nervous system (3). since exposure of perc at the workplaces primary occurs through inhalation and dermal contact where perchloroethylene is produced or used (18) and inhalation of perc can pose psycho-physiological effects include fatigue, anorexia, irritability, impaired memory, and confusion. direct skin contact with liquid perc may lead to erythema, papules and burns (19). furthermore, the findings of other study also demonstrated that the dry-cleaning workers exposed to perc are at risk of developing acute or chronic adverse health effects, depending on the concentrations of perc (13). other principle finding of the present study was a non-significant alteration in the hematological parameters in percexposed dry cleaning workers compare with the non-exposed group. meanwhile emara et al, has documented the hematotoxicity and immunotoxicity induced by perc in dry-cleaning workers (20). the liver and kidney are well-documented target organs for perc-induced toxicity. in a previously published animal study, a slight increase in weight of liver was noted after exposure to perc at different concentration which were coincided with very slight hypertrophy of centrilobular hepatocytes of the liver (21). our results are inconsistent with this previous report as the data of liver enzymes in the present study did not indicate a complete alteration in the liver function which can be explained by inadequate exposure of the workers to this dry-cleaning solvent due to non-commitment of the workers to the working hours in the studied laundry shops. other past study reported no effect of perc on liver and kidney function at the exposed level to dry-cleaning workers (9) which can support the finding of the present study. although the kidney has also been reported as a target organ for perc, previous reports stated that the effects of perc are typically noted at concentrations higher than those that induce liver effects after intermediate duration of exposure (22). the results of the present study demonstrated a significant alteration in serum urea level while nonsignificant changes in serum creatinine of the exposed group compare with the non-exposed participants. this finding is consistent with the previous study which had suggested a weak but dose-related effect of perc on the kidney of the dry-cleaning workers and it considered glutamine synthetase in urine as the most reliable biomarkers of renal changes in human exposed to low level of perc as it could indicate a specific effect of this solvent on the proximal tubules (9). recent study emphasized on urine sample as another non-invasive biomarker for detection and evaluation of perc in urine which might explain the potential deleterious effect of perc on renal system (23). for the present study, there are some limitations. first; biological level of perc has not been considered as a marker of the exposure. second; the environmental level of perc has not included as a studied parameter. however, the strength of the current study was that both clinical and biochemical parameters are included. conclusion a non-significant alteration in hematological parameters, while significant changes in some liver and kidney function indices have been demonstrated in the present study. additionally, inadequate use of protection facilities (i.e. personal protective equipment) has been reported among the workers that need awareness about hazards of perc and training on how to protect themselves from its deleterious effects. acknowledgment the author would like to thank the support of the college of pharmacy-university of sulaimani, and appreciates the help of all the drycleaning employees and the workers who participated in the study. ethical approval the study protocol was approved by the ethical committee of the college of medicine, university of sulaimani (registration no#54 in 15.08.2017). informed consent written informed consent was obtained from each participant. conflict of interest the author declares no conflict of interests for this article. funding source data this work is self-funded by the researcher. references 1. lynge e, tinnerberg h, rylander l, romundstad p, johansen k, lindbohm ml, et al. exposure to tetrachloroethylene in dry cleaning shops in the nordic countries. ann occup hyg. 2011;55(4):387-96., 2. usepa, “national emission standards for hazardous air pollutants (neshap) for perchloroethylene dry cleaning facilities,” final rule 71 fr 4274, july 2006. 3. azimi pirsaraei s, khavanin a, asilian h, soleimanian a. occupational exposure to perchloroethylene in dry-cleaning shops in tehran, iran. indust health. 2009;47(2):155159. iraqi j pharm sci, vol.31(2) 2022 occupational hazard of perchlorethylene 149 4. schreiber j, hudnell h, geller a, house d, aldous k, force m, et al. apartment residents' and day care workers' exposures to tetrachloroethylene and deficits in visual contrast sensitivity. environ health perspect. 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immunotoxicol. 2012; 10(3):311320. 22. atsdr (agency for toxic substances and disease registry). 1997. toxicological profile for tetrachloroethylene (perc) . atlanta , ga : u.s. department of health and human services. 23. modenese a, gioia t, chiesi a, abbacchini c, borsari l, ferrari d et al. evaluation of occupational exposure to perchlorethylene in a group of italian dry cleaners using noninvasive exposure indices. int j enviro res public health. 2019; 16, 2832. pii: e2832. doi: 10.3390/ijerph16162832. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 piroxicam liquid self-nano emulsion doi: https://doi.org/10.31351/vol29iss1pp174-183 174 formulation and characterization of piroxicam as self-nano emulsifying drug delivery system fahad f. salim*,1 and nawal a. rajab** *ministry of health and environment, baghdad, iraq **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract piroxicam (pir) is a nonsteroidal anti-inflammatory drug of oxicam category, used in gout, arthritis, as well as other inflammatory conditions (topically and orally). pir is practically insoluble in water, therefore the aim is preparing and evaluate piroxicam as liquid self-nanoemulsifying drug delivery system to enhance its dispersibility and stability. the dispersibilty and stability study have been conducted in oil, surfactant and cosurfactant for choosing the best materials to dissolve piroxicam. the pseudo ternary phase diagrams have been set at 1:1, 2:1, 3:1 as well as 4:1 ratio of surfactants and co-surfactants, also there are 4 formulations were prepared by using various concentrations of transcutol hp, cremophore el and triacetin oil. all the constructed prepared formulas have been assessed for in vitro drug dissolution, thermodynamic stability, polydispersity index, robustness to dilution, particle size distribution, drug content, and the dispersibility and emulsification time. from the presented research concluded that the self-nanoemulsifying drug delivery system is the convenient method for improving dispersibilty and stability of piroxicam. keywords: pseudo-ternary phase diagram, dissolution rate, snedds, piroxicam. اعداد وتقييم دواء البيروكسيكام كمستحلب سائل نانوي دقيق تلقائي التكوين **بنوال عيّاش رج و 1*،سالم فارس فهد ، بغداد ، العراق . وزارة الصحة والبيئة * عة بغداد، العراقمفرع الصيدالنيات، كلية الصيدلة، جا * الخالصة تثبيط طريق عن يعمل وهو ، أوكسيكام فئة إلى تنتمي التي الستيروئيدية االلتهابات الرثوية غير مضادات عن عبارة هوالبيروكسيكام سواء االلتهابية األمراض من وغيرها والنقرس المفاصل التهاب في المستخدمة لاللتهابات المضادة الفعالة األدوية هوأحد. البروستاجالندينصنع مستحلب إيصال نظام بشكل بيروكسيكام وتقييم إعداد هو الهدف فأن وبالتالي ، الماء في عمليا للذوبان قابل غير هو ، موضعيا عن طريق الفم أو لحل السواغات أفضل لتحديد مختلفة مركبات في الذوبان دراسة إجراء تم. واالستقرار الذوبان لتعزيز وتحسين سائل تلقائي التكوين نانوي ذاتي تحضير تم ، المشترك والسطح بالسطح الفاعل من 1: 4 و 1: 3 ، 1: 2 ، 1: 1 نسب في الثالثية الزائفة الطور مخططات تصميم تم. البيروكسيكام حجم لتوزيع المعدة المستحضرات جميع تقييم تم. , كريموفور و ترانسكيوتل األسيتامين ثالثي زيت من مختلفة تركيزات باستخدام تركيبات أربع الدواء تفكك وفي للتخفيف المتانة ، االستحالب ووقت التشتت ، الحراري الديناميكي االستقرار ، الدواء محتوى ، االنحراف تعدد مؤشر ، الجسيمات الدراسة نستخلص من. العادي الدواء مسحوق من (p <0.05) أعلى كان المحضرة المستحضرات جميع ومدى إطالق معدل أن وجد وقد. المختبري .للبيروكسيكام واالستقرار الذوبان قابلية لتحسين واعد نهج هو النانوي الذاتي الدواء إيصال نظام أن . ، البيروكسيكام sneddsالذوبان ، معدل ، الثالثية الزائفة المرحلة مخطط: المفتاحية الكلمات introduction there is poor water solubility in about fifty percent of new drug compounds, also the oral delivery of this type of drugs has indicated the presence of low bioavailability. there are alot of formulation plans like using solid dispersions, cyclodextrin inclusion complex, micronization, lipids, surfactants, permeation enhancers, salt formulation and nano-particles which are currently applied for overcoming such challenges (1). drug’s solubilizing in colloidal dispersion will enhance the availability and absorption of the drug (2). the physically stable formulations like emulsion preconcentrates, emulsions and the lipid solutions are widely applied to encapsulate the poorly soluble drugs (3). the (snedds) is defined as anhydrous forms thermodynamically stable and transparent or translucent non-ionized dispersion or nanoemulsion pre-concentrates. such systems are considered as anhydrous isotropic mix of co-surfactant, surfactant, oil and the drug, that spontaneously form o/w nanoemulsions when introduced into aqueous phase under conditions of gentle agitation, usually with globule size less than 200 nm. 1corresponding author e-mail: fahad85faris@yahoo.com received: 25/ 9 /2019 accepted:28 / 12/2019 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp174-183 iraqi j pharm sci, vol.29(1) 2020 piroxicam liquid self-nano emulsion 175 concerning the body, agitation that is needed to form nanoemulsions is made available via the gastrointestinal tract’s digestive motility (4). snedds works on improving the oral bioavailability related to drugs of poor water solubility via increasing colloidal dispersibilty and keeping the drug in dissolved form, in small oil globules, throughout its transit via gastrointestinal tract (2). piroxicam can be defined as nonsteroidal anti-inflammatory drug of the oxicam. the majority of nsaids actions are inhibiting the prostaglandin synthesis. piroxicam is effective anti-inflammatory drug used in gout, arthritis, in addition to the other inflammatory conditions (topically and orally) (5, 6). it is considered to be practically soluble in water, and soluble in anhydrous ethanol as well as methylene chloride. thus, piroxicam belong to class ii with low solubility and high permeability according to bcs parameters (7, 8), so this research aimed to formulate and evaluate piroxicam as liquid snedds for increasing its stability, wettability, solubilty in oil and colloidal dispersibilty to have convenient delivery of pir through oral cavity. materials and methods materials piroxicam,transcutol hp, cremophore el, and triacetin oil has been brought from hyperchem (china). methanol has been bought from sigma-aldrich (germany). hydrochloric acid has been bought from avantor performance materials ind. netherlands. methods solubility studies the saturation solubility test was made to select the best solvents for dissolving pir. pir’s solubility has been evaluated in various cosurfactants, oils and surfactants. excessive amounts of the pir powder was added for each vials consisting of (2ml) of chosen vehicle. following the process of sealing, mixture have been sonicated for five minutes, after that they have been subjected for shaking through the use of water bath shaker at a temperature of (25±1 oc) for forty-eight hours. after that, they have been subjected for centrifuging at 3000rpm for twenty minutes, followed by filtration through (0.45μm) membrane filter, and then subjected for dilution with methanol as solvent for dilution of oils, surfactant and co-surfactant, then assayed spectrophotometrically at their respective λ max for their drug contents. solubility results were calculated as mean ± sd (9, 10). construction of pseudo-ternary phase diagrams with regard to hydrophilic-lipophilic balance value as well as the results related to solubility studies, transcutol hp, cremophore el and triacetin oil have been selected as co-surfactant, surfactant and oil phase, respectively. through the use of water titration approach, the pseudo-ternary phase diagram has been constructed for determining snedds components’ ratios (11). surfactant as well as the co-surfactant (smix) have been subjected to mixing in various rations (1:1, 2:1, 3:1 and 4:1). slow titration of distilled water was added into the smix:oil mixture under gentle magnetic agitation until a stable, transparent system was created, observation of the transition from transparent to turbid point is noted down. the data gained was subjected to chemix software for construction of ternary plot (12, 13). preparing piroxicam liquid snedds series of liquid snedds formulas were prepared by using triacetin as oil, cremophore el as a surfactant in addition to using transcutol hp as a co-surfactant in ratios (1:1, 2:1, 3:1 and 4:1), also the oil:s mix ratio (2:8) keep constant as can be seen in (table 1). pir was dissolved in oil in screw-capped glass, after that it was mixed with other components at a concentration (10mg/0.4 ml) and heated in water bath for facilitating homogenization. vortex mixer was used for mixing the ingredients for 5 minutes to have uniform and clear mixture and again it was left to cool at room temperature, then the mixture was sonicated at room temperature for 10 minutes, finally the formulations were stored under visual inspection for minimum forty-eight hours and inspected for the presence of turbidity or phase separation before droplet size distribution study (1315). table 1. composition of piroxicam liquid snedds (% w/w) formula – code smix ratio oil: smix ratio triacetin oil % cremophore el % transcutal hp% snedds-1 1:1 2:8 20 40 40 snedds-2 2:1 2:8 20 53.33 26.66 snedds-3 3:1 2:8 20 60 20 snedds-4 4:1 2:8 20 64 16 iraqi j pharm sci, vol.29(1) 2020 piroxicam liquid self-nano emulsion 176 evaluations of the prepared piroxicam liquid snedds thermodynamic stability studies the prepared liquids sneddss formulations have been subjected for various thermo-dynamic stability tests (centrifugation, heating-cooling cycle and freeze-thaw cycle). centrifugation was performed at 3500rpm for thirty minutes and inspected for cracking, creaming, phase separation, as well as precipitation, and the stable formulations have been selected for heating-cooling cycle. there are 6 cycles between the refrigerator temperatures 4o c and 45o c with storage at each one of the temperatures for at least forty-eight hours, formulations, which are stable at these temperatures, were subjected to freeze-thaw cycle. there are threefreeze-thaw cycles between temperatures of (21 and +25o c) with storage at each temperatures for at least forty-eight hours. the prepared formulations, that were passed thermodynamic stress tests, have been provided for further studies (11). droplet size measurement and poly dispersity index (pdi) all the stable formulations subjected for mean droplet size and polydispersity index (pdi) test by applying 0.5ml of the liquid formula in 250 ml distilled water and gently mixed using magnetic stirrer at 25o c. droplet size and pdi were measured by using particle size analyzer instrument, a light scattering was monitored at a temperature of 25 oc at 90o angle (13, 16, 17). robustness to dilution the prepared formulas of snedds have been diluted to 50, 100, 1000 and 3000-fold with distilled water and 0.1n hcl in 2 separate glass vials. the resultant diluted nanoemulsion formulations have been subjected for shaking and after that visually observed following twenty-four hours for any form of phase separation, coalescence of droplets or drug precipitation (11, 13, 15). dispersibility tests and self-nano emulsification time the effectiveness related to dispersibility and self-nano emulsification time has been evaluated through the use of usp dissolution apparatus ii. each one of the snedds formulation in volume about (0.5ml) has been added to 500 milliliters of the distilled water kept at 37 ± 0.5 oc, at 50 rpm for gentle agitation. in vitro efficiency has been visually observed when obtaining transparent homogenous system and for determining time in minutes to reach complete nano-emulsification with the use of grading system (11, 13), as seen in table 2. table 2. grading system of in vitro performance of the snedds (dispersibility and self-nano emulsification time) grade time needed for nano-emulsion formation appearance a quickly forming (within one minute) nanoemulsion, have bluish or clear appearance b quickly forming (within one minute) a little less clear emulsion, having bluish white appearance c formed within 2min fine milky emulsion d slow to emulsify (longer than two min.). dull, greyish white emulsion that has a little oily appearance e slow to emulsify (> two min.). showing poor or minimum emulsification with large oil globules on the surface. determination of drug content snedds formulation’s drug content has been detected with the use of uv spectrophotometer assay, 0.4 milliliter (equal to 10 milligram pir) from each prepared formulation has been diluted to 100 milliliters with methanol and mixed thoroughly. the resultant solutions have been evaluated at estimated λ max (16, 18, 19). in vitro dissolution study the in vitro drug release regarding the pure pir powder and the prepared snedds formulations were assessed with the use of usp dissolution apparatus-ii (paddle type). paddle type using 0.1n hcl (900ml) as the dissolution media, at 37±0.5 oc and 50 rpm, with the use of dialysis bag approach (molecular cut off 12000 da) just for the formulas of snedds, they have been washed with deionized water for the purpose of getting rid of preservatives and then soaked in a dissolution medium (0.1n hcl) overnight to achieve equilibration state (20, 21). liquid snedds formula that contain pir equal to one dose was placed in a dialysis bag, and five milliliters of the dissolution medium has been withdrawn each ten minutes over sixty minutes (10, 20, 30, 40, 50 and 60) and replaced with fresh media (0.1n hcl) in each drawing. the dissolved drug’s amount was evaluated with the use of uv-spectrophotometer approach at its λ max (18, 19). selection of optimum piroxicam liquid selfnanoemulsion formula according to the evaluation tests (droplet size measurement and polydispersity index (pdi), in iraqi j pharm sci, vol.29(1) 2020 piroxicam liquid self-nano emulsion 177 vitro dissolution study and drug content) the best pir liquid snedds formula was selected. fourier transform infrared spectroscopy (ftir) infra-red spectra of piroxicam and selected liquid ssnedds formula were recorded by using kbr disc approach. the main goal of such study is determining the compatibility and the presence of interactions between components. powder sample was mixed with kbr and grind into fine powder, after that compressed to kbr disc. all kbr discs has been scanned over wave number region of 400cm-1 –4000cm-1 (22). field emission scanning electron microscope (fesem) morphology of the selected liquid snedds formula has been evaluated with the use of fesem. small amount of the nanoemulsion sample was assessed via fesem (mira 3 tescan, france fesem). the samples were examined at various magnifications which are ranged with instant data capture regarding the images onto personal computers (23). statistical analysis the results of the experiments were given as a mean of triplicate samples ± standard deviation and were analyzed according to the one way analysis of variance (anova) at the level of (p<0.05) to determine if the changes in the applied factors are statistically significant at level of (p ≤ 0.05) and nonsignificant at level of (p > 0.05). results and discussion determination of saturation solubility of piroxicam in different oils, surfactants and co surfactants pir solubility has been determined in various co-surfactants, surfactants and oils, amongst various oils that have been screened in (table 3), pir shows high solubility in triacetin oil. as can be seen in the (table 4), surfactant cremophore el and co-surfactant transcutol hp were shown a highest solubility for pir and therefore, they were chosen for the study . table 3. saturation solubility values of piroxicam in different oil types oil type solubility value (mg/ml) mean ±sd* oil type solubility value (mg/ml) mean ±sd* coconut oil 8.32 ± 0.066 triacetin oil 48.10 ± 0.057 sunflower oil 2.35 ± 0.11 corn oil 2.38 ± 0.14 peppermint oil 4.60 ± 0.047 paraffin oil 0.41 ± 0.067 castor oil 0.79 ± 0.089 linseed oil 1.15 ± 0.035 oleic acid oil 11.86 ± 0.093 avocado oil 1.21 ± 0.027 olive oil 2.06 ±0.032 almond oil 0.69 ± 0.041 sesame oil 2.33 ± o.o79 jojoba oil 0.64 ± 0.023 cinnamon oil 8.64 ± 0.051 aniseed oil 2.07 ± 0.072 table 4. saturation solubility of piroxicam in surfactant type and co-surfactant type surfactant type solubility value (mg/ml) mean ±sd* co-surfactant type solubility value (mg/ml) mean ±sd* span 20 2.018 ± 0.019 transcutol hp 30.9 ± 0.062 span 80 1.88 ± 0.089 peg 200 4.63 ± 0.048 tween 20 1.28 ± 0.13 peg 400 10.59 ± 0.12 tween 40 0.91 ± 0.086 peg 600 2.54 ± 0.023 tween 60 8.61 ± 0.11 ethylene glycol 0.61 ± 0.038 tween 80 6.17 ± 0.093 cremophore el 33.7 ± 0.054 *sd standard deviation from mean, n=3 pseudoternary phase diagram construction pseudo-ternary phase diagrams have been developed for identifying selfemulsifying regions and snedds formulations, pseudo-ternary phase diagram plot for various smix ratios (cremophore el: transcutol hp 1:1, 2:1, 3:1 and 4:1 was shown in figures 1. in the pseudo-ternary phase plot, the shaded area performs the area of nanoemulsions while unshaded area performs the area of the emulsion. the plot with a larger shaded area indicates the presence of perfect nano-emulsifying activity of formulated nanoemulsions and beneficial interaction among the smix, oil and aqueous phase (24). the oil:smix 1:9 and 2:8 ratios for all smix ratios remained as nanoemulsions even upon infinite water titration or dilution, this is possible as cremophore el with transcutol hp mixture hardly localized on surface regarding nanoemulsion droplets, decreasing interfacial free energy as well as offering mechanical barrier to coalescence causing automatic dispersion. as the ratio of the cremophore el increase showed best nanoemulsification characteristics indicates the presence iraqi j pharm sci, vol.29(1) 2020 piroxicam liquid self-nano emulsion 178 of perfect nano-emulsifying activity of formulated nanoemulsions (25). figure 1. the diagram plot for pseudo ternary phase for different s mix ratio (cremophore el: transcutol hp 1:1, 2:1, 3:1 and 4:1) preparation of piroxicam liquid snedds all the liquid pir snedd formulas that were visually noticed shows yellow and clear mixtures without phase separation or drug precipitation. assessment of the prepared liquid snedds thermodynamic stability studies all the prepared formulations of the pir snedds were passed the thermodynamic stability tests, since there was no indications of drug precipitation or phase separation at end of each cycle. this indicated that formulations are persistent against storage in extreme conditions. droplet size measurement and pdi droplet size measurement and pdi results are shown in (table 5). droplet size of nanoemulsion is very important factor in the selfemulsification performance since it affects the extent and rate of the drug release, in addition to its absorption (26). that in the small droplet size range, there will be high surface area provided for drug release and absorption (27). droplet size related to prepared snedds formulations have been (29.6 nm 112.7nm), as pdi approximately 0.3 apart from snedds-1 which has been 0.551, pdi lower than 0.3 indicates the optimum uniformity in droplet size distribution following dilution with water (28). table 5. droplet size measurements and poly dispersity index (pdi) of piroxicam liquid selfnano emulsifying systems drug delivery. f – code mean droplet size (in nm) polydispersity index (pdi) snedds1 112.7 00.551 snedds2 29.6 00.312 snedds3 66.7 00.379 snedds4 41.1 00.262 robustness to dilution dilution is caused by gastrointestinal fluids, also there is no possibility for properly corresponding amount of water for forming nanoemulsion with formulation, robustness to dilution has been carried out with excess of water and 0.1n hcl, also it has been stored for twenty-four hours. all the pir snedds formulations passed the test and visually observed as clear with no phase separation or precipitation. the capability of snedds formulation for keeping aqueous dilution has been outstanding. it was attributed to the high solubilizing characteristics of excipients, also the ability of forming stable nanoemulsion with small droplet size. this indicate that such formulations have been stable at the infinite water dilution and had high robustness to high dilution (29, 30). iraqi j pharm sci, vol.29(1) 2020 piroxicam liquid self-nano emulsion 179 dispersibility tests and self-nano emulsification time the emulsification studies have a high importance in estimating the self-emulsifying properties related to the prepared formulations. snedds must be rapidly and completely dispersed as soon as submitted to the aqueous dilution within mild agitation (31). the emulsification’s rate is significant important index to determine the emulsification’s effectiveness (32, 33). all prepared pir snedds formulations created nanoemulsion in not more than one minute with grade a and the difference in the self-emulsification times of various formulas in bulk liquid snedds has been extremely small and due to the fact that observation times have been fast (in seconds), it has been difficult to distinguish between the prepared formulas. drug content the drug content of all the prepared pir snedds has been more than 96% and there was no considerable difference between different formulations (p > 0.05), which meets the usp requirements and were within an acceptable range (90%-110%) (5), drug content percent of pir snedds illustrated in table 6. table 6. the drug content percent of piroxicam liquid self nanoemulsion (mean ±sd) n=3. f – code drug content % snedds-1 99.61 ± 0.078 snedds-2 96.82 ± 0.14 snedds-3 97.50 ± 0.084 snedds-4 98.24 ± 0.061 in vitro dissolution study the in vitro drug release profiles regarding f1 to f4 and pure pir have been assessed in 0.1 n hcl, 50 rpm and 37oc are shown in figure 2. dialysis membranes were used in this test since it is less susceptible to blockage and the size of the pores are very small (34). the prepared pir snedds formulas showed more than 92 % drug release at end of sixty minutes, yet f4 have higher release 97 %. since the drug is in dissolved state in snedds, the release will be faster, the faster release which is due to fine particle size and high surfactant mixture concentrations, that could simply emulsify oil for finer globule (35). figure 2. dissolution profile of pir snedds (f1, f2, f3 and f4) and pure piroxicam the release profile of pure pir is slower than prepared snedds formulas and reach 85% at the end of 60 min without dialysis membrane. all the prepared snedds formulas have no significant difference in extent and rate release profile as (p > 0.05), but have significant difference with extent and rate release profile of plain pir powder (p < 0.05). finally, the formulations of snedds led to spontaneous formation of nanoemulsion with small size of droplets, that allowed rapid rate of the drug release to aqueous phase, considerably faster in comparison to that of the pure drug powder. selection of optimum piroxicam liquid selfnanoemulsion liquid snedds formula f4 was selected as optimum pir liquid self-nanoemulsion due to the f4 has a higher drug content, higher in vitro release, small droplet size and lower value of pdi. fourier transform infrared spectroscopy ftir is an extremely powerful technique in discovering and evaluating chemical interactions between drug and any excipient and also to control chemical stability. the ftir spectra of the piroxicam and the selected formula f4 are shown in figure 3, 4 respectively. the main characteristic peak of pir ftir was the secondary amine n-h stretching appeared at 3392.17 cm-1 (36). iraqi j pharm sci, vol.29(1) 2020 piroxicam liquid self-nano emulsion 180 figure 3. ftir spectrum of pir. figure 4. ftir spectrum of selected pir snedds f4 formula it had been reported that pir has two interconvertible crystalline forms, the needle and cubic forms. the ir absorption peaks at 1634 cm-1 and 1629 cm-1 are assigned to stretching of amide carbonyl groups of the needle form and the cubic form of pir respectively, peak at 1527.35 cm-1 is because of stretching of the second amide band for both crystalline forms of pir. in this study, the peak at 1637.27 cm-1 was found in ir spectrum of pir, suggesting that the needle form of pir was used in the present study (37). the pir spectra also exhibited other characteristic peaks like, c=c stretching of aromatic ring at 1434.78 cm-1, c-n stretching at 1351.86 cm-1, c-o stretching at 1288.22 cm-1, s(=o)2 stretching at 1149.37cm-1, -so2‐n stretching at 1033.66 cm-1, aromatic ch bending at 825.38 cm-1, ortho-disubstituted phenyl at 771.39 cm-1 and c-s stretching at 669.18 cm-1, which indicate purity of drug (38-40). from the ftir results, it was found there was no changes in the peaks of pir spectrum to that of the spectra of the selected formula f4, the results indicate that no chemical interactions have been happened between the pir and excipients that utilized in the preparation. field emission scanning electron microscopic the liquid snedds f4 was observed under the (fesem), the results are illustrated in figure 5 .the obtained result indicated approximately spherical shape of the droplet. iraqi j pharm sci, vol.29(1) 2020 piroxicam liquid self-nano emulsion 181 figure 5. fesem image of pir selected snedds formula f4 conclusion from this study, a conclusion is made that snedds has provided important dosage form for oral water insoluble drug. snedds that was prepared from triacetin oil, cremophore el and transcutol hp was important approach for improving stability, wettability, 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http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 detection of epicatechin in tea leaves by tlc and hplc doi: https://doi.org/10.31351/vol31iss2pp304-312 304 detection of epicatechin in camellia sinensis leaves by thin layer chromatography and high performance liquid chromatography techniques ruaa mohammed ibrahim *,1 and zahraa suhail nassir** *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq. abstract the current study performed in order to detect and quantify epicatechin in two tea samples of camellia sinensis (black and green tea) by thin layer chromatography (tlc) and high performance liquid chromatography (hplc). extraction of epicatechin from black and green tea was done by using two different methods: maceration (cold extraction method) and decoction (hot extraction method) involved using three different solvents which are absolute ethanol, 50% aqueous ethanol and water for both extraction methods using room temperature and direct heat respectively. crude extracts of two tea samples that obtained from two methods were fractionated by using two solvents with different polarity (chloroform and ethyl acetate). qualitative and quantitative determinations of epicatechin in tea samples were investigated. epicatechin identification was made by utilizing preliminary chemical tests and tlc. this identification was also boosted by hplc and the quantity of epicatechin was determined in all ethyl acetate fractions of two tea samples. this research revealed the existence of epicatechin in black and green tea according to tlc and hplc. aqueous ethanol 50% was the best solvent for extraction of epicatechin from leaves of tea. quantitative estimation of epicatechin by hplc revealed that ethyl acetate fraction of dgtae contains the higher concentration of epicatechin than other analyzed fractions. conclusion, tea is an excellent source of catechins particularly epicatechin that possessed various pharmacological effects. keywords: black and green tea, tlc, hplc, epicatechin. بواسطة كروماتوغرافيا الطبقة الرقيقة الشايأوراق في epicatechin مادةالكشف عن والكروماتوجرافيا السائلة عالية األداء ** زهراء سهيل ناصر و 1*، رؤى محمد ابراهيم . العراقبغداد، ، كلية الصيدلة، جامعه بغداد، والنباتات الطبية فرع العقاقير * الخالصة كمية الكشف أجل من الحالية الدراسة أجريت بواسطة( واألخضر األسود الشاي) شايال من عينتين في epicatechinوقياس واألخضر األسود الشاي من epicatechin استخالص تم(. hplc) األداء عالية السائلة والكروماتوجرافيا( tlc) الرقيقة الطبقة كروماتوغرافيا على تشتمل مختلفة مذيبات ثالثة باستخدام( الساخن االستخالص طريقة) االغالء و( البارد االستخالص طريقة) النقع: مختلفتين طريقتين باستخدام الخام المستخلصات تجزئة تم. التوالي على المباشرة والحرارة الغرفة حرارة درجة باستخدام والماء المائي اإليثانول من ٪ 50 و ، المطلق اإليثانول التحديدات في التحقيق تم .(االثيل وخالت الكلوروفورم) مختلفة قطبية لهما مذيبين باستخدام طريقتين من عليهما الحصول تم الشاي من لعينتين تعزيز تم. tlc و األولية الكيميائية االختبارات استخدام خالل من epicatechin تحديد تم. الشاي في عينات epicatchin لمادة والكمية النوعية خالت جميع في epicatechin كمية تحديد ثم hplc بواسطة أيًضا التحديد هذا مادة وجود عن البحث هذا كشف . الشاي لعينتي اإلثيل بقات epicatechin لـ وفقًا واألخضر األسود الشاي في tlc و hplc .مادة الستخالص مذيب أفضل٪ 50 بنسبة المائي اإليثانول يعتبرepicatechin من أعلى تركيز على يحتوي dgtae من اإلثيل بقات خالت أن hplc بواسطة epicatechin لمادة الكمي التقدير أظهر. الشاي أوراق من . مختلفة دوائية تأثيرات تمتلك التي epicatechin وخاصة االكسدة لمضادات ممتاز مصدر هو الشاي ، الخالصة. تحليلها تم التي األخرى االجزاء والكروماتوجرافيا السائلة عالية األداء، كروماتوغرافيا الطبقة الرقيقةالشاي األسود و األخضر، حية :الكلمات المفتا introduction camellia sinensis (l.) is tea plant belongs to the family theaceae and implants in about thirty countries the whole world (1). it is originating in china and later distributed to japan and india, then to russia and europe (2). tea plants are woody shrubs of medium size with height reach to 1.8 meters (3). its leaves are pointed and oval at the tip with length 5-10 centimeters. its flowers are fragrant, white, of diameter 4 centimeters; contain 5 petals. the fruits are as three-angled capsule that contain three seeds (3) (figure 1). while its leaf buds and leaves are utilized to make tea. it has a good taste, attractive aroma, and health-reinforcing effects. these advantages made tea one of the most common beverages in the world. (4) 1corresponding author e-mail: received: / / accepted: / / iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp304-312 iraqi j pharm sci, vol.31(2) 2022 detection of epicatechin in tea leaves by tlc and hplc 305 there are three types of tea taken from camellia sinensis which are either not fermented like white and green tea, or partially fermented like oolong and red tea, and completely fermented like black tea, those compositions of those types are influenced by the process of fermentation(5). the most widely used is black and green tea that are obtained from the same species of plant (c. sinensis l.) but varying in their organoleptic taste, appearance, chemical contents and flavor attributed to their respective process of fermentation(2). the chemical composition of tea leaves includes alkaloids (theobromine, caffeine, theophylline), polyphenols (flavonoids and catechins), polysaccharides, volatile oils, amino acids, vitamins (like vitamin c), lipids, inorganic elements (like fluorine, manganese and aluminum), and other (2). figure 1. photo of camellia sinensis plant (5) camellia sinensis has been shown to possessed pharmacological and physiological activities such as anticancer (6), antioxidant (7), antiinflammatory (8), antibacterial (9), antidiabetic (10), hepatoprotective activity (11), and others. these activities are due to polyphenolic compounds mostly catechins and flavonoids that are considered as the major active components. recently catechins are the most significantly studied, including epicatechin (figure 2), catechin, epigallocatechin gallate and epicatechin gallat (12). therefore; the aim of this study was qualitative and quantitative determination of epicatechin in two tea samples of camellia sinensis (black and green tea). figure 2. chemical structure of epicatechin (1) material and method chemicals and reference standards the reference standard and chemicals that utilized in this work are listed with their purity percentage in table 1. table 1. chemicals and standard used with assay percentage chemicals assay percentage acetic acid glacial 99.8100.5 % acetone 99.9% chloroform 99.8% dioxan 98% epicatechin standard 99.9% ethanol 99.7-100% ethyl acetate 99% formic acid 88% methanol 99.5% n-butanol 99.7% toluene 99.5% instruments used for the conduction of the study the instruments that utilized in this work are summarized with their manufacture in table 2. table 2. instruments used and their manufacture instrument manufacturer electrical sensitive balance sartorius/ germany hplc system (shimadzu, 2010 cht) shimadzu/japan oven: memmert 854 buchi /germany rotatory evaporator: buchi rotatory evaporator attached to vacuum pump buchi/ germany ultraviolet light (desaga heidelberg) of 254 nm and 366 nm wave lengths desaga/germany plant materials dried green and black tea leaves obtained from the local market and identified by pharmacognosy and medical plants department in collage of pharmacy\ baghdad university and authenticated by prof. dr. sukaena abbas\ department of biology\ college of the science \university of baghdad. extraction of plant material aextraction by hot method (decoction) 15 gm of dried leaves from both tea samples were put in conical flask and extracted by direct heat (70 80 °c) for 20 mins, using three different solvents involved absolute ethanol, 50% aqueous ethanol and water (150ml of each) for extraction. (13) bextraction by cold method (maceration) 15 gm of dried leaves from each tea sample are placed in beaker and macerated for 5 days at room temperature in 3 solvents which are ethanol, 50% aqueous ethanol and water (150ml of each). after that, filtration was done for crude extracts obtained by both methods and concentrated by a rotary evaporator. the crude extracts were iraqi j pharm sci, vol.31(2) 2022 detection of epicatechin in tea leaves by tlc and hplc 306 hanging in water, then partitioning with chloroform (chcl3) and ethyl acetate by separating funnel in each solvent for 3 times to get their respective fractions rich with catechins and then concentrated (13) as shown in figure 3. preliminary phytochemical investigation ethyl acetate fractions were undergoing phytochemical analysis for flavonoids and phenolic acid. two chemical tests are utilized to detect their presence (14). phenolic acid test: in a test tube, few gm of ethyl acetate fraction were hanging in distilled water (1ml), then add 5% ferric chloride (few drops) and observed the color, a deep green to black coloration confirm the existence of phenolic acids. flavonoid test: few gm of ethyl acetate fraction were dissolved in 80% ethanol (20ml) and filtered, then take 1ml of the filtrate and dissolved in 1% potassium hydroxide (2ml) in a test tube, and observed the color. a yellow color confirms the existence of flavonoids. identification of epicatechin by tlc ethyl acetate fractions were analyzed by tlc for the presence of epicatechin, using plates of silica gel gf254 (20 x 10cm, 250 μm thickness); development occur in 20cm x 10cm double tank glass chamber presaturated for 30 minutes with different mobile phases as follow: (13,15) • s1: toluene: chloroform: acetone: formic acid (8: 4: 3: 3) • s2: toluene: dioxan: acetic acid (9.2: 4 :0.6) • s3: chloroform: ethyl acetate: formic acid (5: 4: 1) • s4: n-butanol: acetone: acetic acid (5: 5: 3) • s5: chloroform: acetone: formic acid (75:16.5:8.5) the detection was done by utilizing uv light at wavelength 254 nm and then calculated rf value. qualitative and quantitative estimations of epicatechin by hplc identification and quantitative determination of epicatechin in ethyl acetate fractions were made by using hplc (shimadzu, 2010 cht) with uv detector in which identification was performed by comparing the time of retention of analyzed fraction with that of reference standards at same chromatographic conditions with reversephase c18 column (targa) (250 × 4.6 mm, 5 𝜇) and the temperature of column was kept at 30∘c. mobile phase of hplc consist of solvent a prepared by dissolving 0.1ml of orthophosphoric acid in 0.9l of grade water of hplc and complete the volume to 1.0 l with water and then filtered by filter membrane (0.45 𝜇m) and degassed for 3 min in a sonicator, and solvent b which is acetonitrile. by gradient elution, mobile phase was developed at 0.01min 11% b; at 30 min 25% b; at 35-39 min 100% b; and at 40-50 min 11%b. the rate of flow of mobile phase was 1 ml/min and the volume of injection was 15 𝜇l. the wavelength of detection was 280 nm. (16) quantitative determination of epicatechin was performed by using calibration curve in which serial dilutions (10, 20, 30, and 40 ppm) from stock solution of epicatechin standard (10 ppm) was prepared. figure 3. systematic scheme for extraction of crude catechins (13) iraqi j pharm sci, vol.31(2) 2022 detection of epicatechin in tea leaves by tlc and hplc 307 results and discussion extraction of catechins in this work, the variation in yield percentages (%w/w) of ethyl acetate fraction of two tea samples was determined as shown in table 3. table 3. the yield percentages of ethyl acetate fractions of green and black tea. according to our work, the highest percentage of yield was obtained from maceration method of green tea (gt) which was (1.796) while for the decoction method of gt gave the highest yield which was (1.683), when we used 50% aqueous ethanol as solvent in two extraction method. and the best solvent for extraction of epicatechin is 50% aqueous ethanol. preliminary phytochemical investigation preliminary screening of phytochemicals confirmed that phenolic acid and flavonoids are present in ethyl acetate fractions of two tea samples. these chemical compounds may be in charge for different medicinal properties. identification of epicatechin by tlc the tlc results for all ethyl acetate fractions of two tea samples indicated the presence of epicatechin in which the color and rf value of epicatechin in analyzed fraction were identical to the epicatechin standard and identified as epicatechin, as shown in table 4 and figures 4-6. figure 4. tlc chromatogram of ethyl acetate fractions of green and black tea, st: epicatechin standard, 1: mgta, 2: mgte, 3: mgtae, 4: mbta, 5: mbte, 6: mbtae, 7: dgta, 8: dgte, 9: dgtae, 10: dbta, 11: dbte, 12: dbtae, using s1 as a solvent system. analyzed fraction percentage yield (%w/w) decoction green tea in ethanol (dgte) 1.005 decoction green tea in distilled water (dgta) 1.233 decoction green tea in aqueous: ethanol (1:1) (dgtae) 1.683 decoction black tea in ethanol (dbte) 0.718 decoction black tea in distilled water (dbta) 1.119 decoction black tea in aqueous: ethanol (1:1) (dbtae) 1.437 maceration green tea in ethanol (mgte) 1.147 maceration green tea in distilled water (mgta) 0.933 maceration green tea in aqueous: ethanol (1:1) (mgtae) 1.796 maceration black tea in ethanol (mbte) 0.932 maceration black tea in distilled water (mbta) 0.859 maceration black tea in aqueous: ethanol (1:1) (mbtae) 1.753 iraqi j pharm sci, vol.31(2) 2022 detection of epicatechin in tea leaves by tlc and hplc 308 figure 5. tlc chromatogram of ethyl acetate fractions of green and black tea, st: epicatechin standard, 1: mgta, 2: mgte, 3: mgtae, 4: mbta, 5: mbte, 6: mbtae, 7: dgta, 8: dgte, 9: dgtae, 10: dbta, 11: dbte, 12: dbtae, using s2 as a solvent system. figure 6. tlc chromatogram of ethyl acetate fractions of green and black tea, st: epicatechin standard, 1: mgta, 2: mgte, 3: mgtae, 4: mbta, 5: mbte, 6: mbtae, 7: dgta, 8: dgte, 9: dgtae, 10: dbta, 11: dbte, 12: dbtae, using s3 as a solvent system. the rf values of epicatechin in all ethyl acetate fractions with its standard in the best three mobile phases were calculated as shown in table 4. table 4. the rf values of epicatechin in all ethyl acetate fractions with epicatechin standard in best three mobile phases. mobile phase s1 s2 s3 st. 0.387 0.333 0.289 dgte 0.387 0.326 0.282 dgta 0.387 0.333 0.289 dgtea 0.380 0.319 0.275 dbte 0.373 0.326 0.282 dbta 0.373 0.319 0.275 dbtea 0.373 0.326 0.289 mgte 0.387 0.304 0.275 mgta 0.387 0.290 0.275 mgtea 0.380 0.304 0.268 mbte 0.373 0.312 0.275 mbta 0.373 0.319 0.275 mbtea 0.373 0.333 0.282 iraqi j pharm sci, vol.31(2) 2022 detection of epicatechin in tea leaves by tlc and hplc 309 qualitative and quantitative estimation of epicatechin by hplc in this study, identification and quantitative determination of epicatechin in all ethyl acetate fractions of two tea samples were made by hplc. the epicatechin peak in ethyl acetate fractions was detected by comparing the uv spectra and retention time (min) with that of standard at same conditions as given in figures 7-9. figure 7. hplc chromatogram for epicatechin standard figure 8. hplc chromatogram of ethyl acetate fraction of a: dga, b: mga, c: dgae, d: mgae, e: dge, f: mge. iraqi j pharm sci, vol.31(2) 2022 detection of epicatechin in tea leaves by tlc and hplc 310 figure 9. hplc chromatogram of ethyl acetate fraction of g: dba, h: mba, i: dbae, j: mbae, k: dbe, l: mbe . for quantitative determination, the curve of calibration was drawn using area under the curve (auc) vs. four levels of concentration of epicatechin standard. an equation of straight line was got from which the analyte concentration was determined in each ethyl acetate fraction as given in figure 10. the epicatechin was quantified in all ethyl acetate fractions and the results indicated that ethyl acetate fraction of dgtea showed the highest concentration of epicatechin (1104.34ppm) when compared to other analyzed fraction. the ethyl acetate fraction of mgta showed the smaller concentration of epicatechin (35.32ppm). the concentrations of epicatechin in ethyl acetate fractions of green and black tea are listed in table 5. iraqi j pharm sci, vol.31(2) 2022 detection of epicatechin in tea leaves by tlc and hplc 311 figure 10. hplc calibration curve of epicatechin table 5. the concentration of epicatechin in ethyl acetate fractions of green and black tea. conclusion camellia sinensis (tea) is a widely used beverage. tea contains several useful substances for human health. it is an excellent source of catechins (bioactive molecule) including epicatechin, catechin, epigallocatechin gallate, and epicatechin gallate. the 50% aqueous ethanol is better solvent for extraction of epicatechin from green and black tea than water and ethanol. the qualification of ethyl acetate fractions by tlc and hplc indicated that epicatechin was found in two tea samples. the hplc method is appropriate for the determination of epicatechin content in green and black tea samples. there was a difference in the concentration of epicatechin between the analyzed fractions but the content of epicatechin was higher in the ethyl acetate fraction of dgtae when compared with other fraction. it is suggested that the pharmacological activities of plant as an anticancer and antioxidant should be studied. references 1. graham hn. green tea composition, consumption, and polyphenol chemistry. preventive medicine, 1992; 21: 334-350. 2. sharangi ab .medicinal and therapeutic potentialities of tea (camellia sinensis l.) – a review. food research international, 2009; 42:529–535. 3. agarwal u, pathak d p, bhutani r, kapoor g, kant r. review on camellia sinensis : nature’s gift. international journal of pharmacognosy and phytochemical research, 2017; 9(8): 1119-1126. 4. narotzki b, reznick az, aizenbud d, levy y. green tea: a promising natural product in oral health. arch oral 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relationship between antimutagenic activity and major components of various teas. mutagenesis, 1996; 11(1): 37– 41. 10. shokrzadeh m, ebadi ag, mirshafiee ss, choudhary mi, effect of the aqueous green leaf extract of green tea on glucose level of rat, pakistan journal of biological sciences, 2006; 9(14): 27082711. 11. oyejide oo, olushola l, hepatoprotective and antioxidant properties of extract of camellia sinensis (black tea) in rats, african journal of biotechnology, 2005; 4(11):14321438. 12. oliveira rmmd. quantification of catechins and caffeine from green tea (camellia sinensis) infusions, extract, and ready-to-drink beverages. food science and technology (campinas), 2012; 32(1): 163-166. 13. row kh, jiny. recovery of catechin compounds from korean tea by solvent extraction. bioresource technology, 2006; 97: 790–793. 14. chang y, chamidha kumarihami h, kim h and song k. current status and prospect of molecular breeding in tea plant (camellia sinensis). the korean tea society, 2017; 23(1):74-84. 15. rashidinejad a, birch ej, hindmarsh j, everett dw. molecular interactions between green tea catechins and cheese fat studied by solid-state nuclear magnetic resonance spectroscopy. food chem. 2017; 215:228–234. 16. raju vss, nareshraju n, kannababu s, and gottumukkala v. determination of catechin and epicatechin content in chocolates by highperformance liquid chromatography. international scholarly research notices, 2014; 1:1-5. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 umbelliferone conjugates of nsaids doi: https://doi.org/10.31351/vol30iss1pp240-248 240 development, characterization and pharmacological investigation of umbelliferone conjugates of nsaids nija b.*,1, arun rasheed* and a kottaimuthu** *al shifa college of pharmacy, perinthalmanna, malappuram district, kerala, india **department of pharmacy, annamalai university, annamalai nagar-608 002, tamilnadu, india abstract the present investigation developed the ester prodrugs of non-steroidal anti-inflammatory drugs (nsaids), mefenamic acid(ma) and flurbiprofen(fbn) by conjugating with the natural antioxidant, 4-methyl umbelliferone that resulted in the formation of mefenamic acid-umbelliferone ester prodrug (mu) and flurbiprofen-umbelliferone ester prodrug(fu) .the principal objective of this study is the synthesis of the ester prodrugs of nsaids with the enhanced therapeutic activity and minimized side effects. prodrugs were synthesized by coupling method using n,n’dicyclohexylcarbodiimide/4-dimethylaminopyrimidine, and the resulted prodrugs were subjected to physical, chemical characterization, spectral characterization (ir, 1h nmr, 13c nmr and mass spectra), hydrolysis-kinetic study and pharmacological evaluation such as anti-inflammatory, ulcerogenecity as well as the effect of the nsaids in the central nervous system against degenerative mechanisms. the current study revealed that the umbelliferone conjugates of nsaids upon administration would release the parent drug by hydrolysis in the desired site with enhanced anti-inflammatory activity and reduction in the gastro intestinal toxicity. also, the synthesized pordrugs showed enhanced brain targeting efficiency with protective action against the degenerative processes. keywords: mefenamic acid, flurbiprofen, anti-inflammatory activity, prodrug, umbelliferone, gastrointestinal toxicity. introduction nsaids, well accepted for the therapeutic activities such as analgesic, anti–inflammatory and anti-pyretic activities based on the mechanism of cyclooxygenase(cox-1 and cox-2) enzyme inhibition and formation of prostaglandins (1) .the various pharmacological activities produced by the nsaids can be explained through different basic mechanisms such as nitric oxide system and transcriptional factors that showed direct relationship with cytokine expression which is having a significant role in the anti-inflammatory process (2). mefenamic acid, ma(2-(2, 3dimethylphenyl)amino benzoic acid) and flurbiprofen, fbn( 2 -(2 –fluorobiphenyl – 4 –yl ) propanoic acid) are nsaids widely used for the treatment of arthritic pain, inflammatory condition and dysmenorrhea etc (3). the major side effect produced by the nsaids is the gastric-duodenal ulceration due to the free carboxylic acid functional group in the structure also several mechanisms was put forward such as the inhibition of prostaglandin synthesis, irritant effect on the epithelial tissue, effect in the gastric-mucosal blood flow (4).the structure activity relationship of nsaids was proved that the free carboxylic acid functional group in the molecular structure of the nsaids is necessary for the binding with cox receptors to elicit the pharmacological action and the hydrolysis of the prodrugs produced the anti-inflammatory activity. so the novel strategies are appreciated for designing and developing the ester and amide compounds by derivatization of the – cooh functional group produced considerable therapeutic activity with the reduction in side effect due to the free carboxylic acid group in the structure (5).the modification of carboxylic acid functional group can improve the transport properties across blood brain barrier and provide enhanced distribution thus therapeutic activity (6). several studies have been conducted for the reduction of the side effects and improve the transport profile of nsaids. among that one of the most relevant approaches is prodrug based drug design. prodrug based scheme can overcome the limitations and side effects produced by the nsaids. the prodrugs based approaches such as carries linked and bioprecursor approaches have significant role in the drug research and development (7).the drug molecules after the development process may be failed in the required therapeutic activity because of the pharmacokinetic profile, transport profile and solubility etc and prodrug approach successfully overcome the above limitations (8).in the worldwide pharmaceutical market, about ten percentages of the drugs are considered as prodrugs that can effectively overcome the limitations of the nsaids. the gastric lesions produced by the long term use of nsaids are regulated by the release of reactive oxygen species. so this study expected to decrease the gastric ulceration by the synthesis of the prodrugs using the antioxidant conjugate (9). 1corresponding author e-mail: nija.b90@gmail.com received: 25/9/2020 accepted:12 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp240-248 mailto:nija.b90@gmail.com iraqi j pharm sci, vol.30(1) 2021 umbelliferone conjugates of nsaids 241 the nsaids-umbelliferone conjugates are developed with improved physical as well as chemical characteristics thus therapeutic profile. different nsaid derived compounds showed better activity, reduction in the side effect, transport profile and effective therapeutic activity (10). the natural antioxidant used in this study is the coumarin derivative, 4-methyl umbelliferone and studies showed that this natural compound has enough biological activities such as antioxidant, antiinflammatory, anti-diabetic, anti tumor and neuroprotective effect (11). this research work aim to develop the two ester prodrugs of nsaid, mefenamic acid and flurbiprofen, by conjugating with the antioxidant such as umbelliferone and identify the physical, chemical, hydrolytic test and pharmacological activities like anti-inflammatory activity, antiulcerogenic activity by using various methods expected to bring about improved characteristics. materials and methods materials and measurements the drug ma and fbn was obtained from tci chemicals (india)pvt.ltd., chennai, tamilnadu. the antioxidant 4-methyl umbelliferone was obtained from sigma aldrich chemicals pvt .ltd. mumbai. the ftir spectra of the compounds were recorded on ir spectrometer (bruker, software: opus), al shifa college of pharmacy, kerala. the elemental analysis by using elemental analyzer (thermo finnigan, italy, flash ea 1112 series) was done in sophisticated analytical instrumentation facility (saif), lucknow. 1h nmr (cryo-magnet spectrometer, bruker), (13) cnmr spectra (cryo-magnet spectrometer, bruker) and mass spectra (micromass q-tof micro) were performed in saif panjab university, chandigarh. the melting points of the prodrugs were recorded using melting point apparatus (sigma scientific products, tamilnadu), al shifa college of pharmacy, kerala. the absorbance was measured in the uv spectrophotometer (shimadzu, japan).determination of physicochemical properties and the pharmacological evaluations were carried out in department of pharmaceutical chemistry and department of pharmacology, al shifa college of pharmacy. the histo-pathological studies were carried out in department of pathology, kims and al shifa hospital, kerala. synthesis of nsaids -antioxidant prodrug: this research work developed the ester prodrug of nsaids, ma and fbn by conjugating with the antioxidant, 4-methyl umbelliferone thus mefenamic acid-umbellierone prodrug[mu] and flurbiprofen umbelliferone prodrug[fu]. to a stirred solution of 10 mmol of carboxylic acid in 15 ml of anhydrous dichloromethane (dcm) 110mg of 4dimethylaminopyrimidine (dmap) and 10mmol of 4-methyl umbelliferone was added. then 10mmol of n,n’dicyclohexylcarbodiimide (dcc) were added to the reaction mixture at 0-80c, which is then stirred for 5 minutes at 0-80c and 3hr at 20-250c.after completing the reaction, the precipitated urea was filtered off and the filtrate was evaporated. the residue was taken in 10ml dcm and washed with saturated sodium bicarbonate solution and then dried over magnesium sulphate. the solvent is removed by evaporation and the ester was purified by recrystallisation. before the recrystallisation the product was washed with alcohol to remove the excess of umbelliferone. if the product after synthesis was sticky, it was washed with petroleum ether two or three times. this procedure was used for the synthesis of both mu and fu and that was shown in the figure 1 and 2 (12). figure 1. scheme for the synthesis of ma-antioxidant prodrug iraqi j pharm sci, vol.30(1) 2021 umbelliferone conjugates of nsaids 242 figure 2. schematic representation of the synthesis of fbn-antioxidant prodrug physical and chemical characterization of the prodrugs the physical as well as chemical properties of the synthesized prodrugs were done by different methods such as solubility, determination of partition coefficient, melting point determination, thin layer chromatography, elemental and spectral analysis etc and the data obtained was compared with that of the parent nsaids, fbn and ma. the solubility of the prodrugs were evaluated in organic solvents such as chloroform, methanol, acetone, dimethylsulfoxide and the aqueous solvents such as water, sodium hydroxide (0.1n), hydrochloric acid (0.1n).this study is used to prove the hydrophilic and lipophilic nature of the drugs and prodrugs (13).thin layer chromatography was done to check the progression of the reaction and also confirm the purity of the synthesized compounds that was done on the pre coated silica g plates. the detection of the spots visually done by using uv chamber. the solvent system used here was ethyl acetate:hexane 1:2. the determination of melting point of the synthesized compounds and the results were compared with that of the reactants to confirm the formation of the product and also assure the purity of the synthesized compounds. the determination of partition coefficient aim to attain the knowledge of the lipophilic profile of the synthesized compounds and that was done by shake flask method in which n-octanol saturated with phosphate buffer (ph 7.4). the concentration of the drug and each produg was monitored by measuring the absence by uvvisible spectrometer (14). the quantification of the elements present in the synthesized compounds and the data was compared with that of the theoretical value was very much relevant and that determine the percentage of carbon, nitrogen, oxygen and hydrogen present in the synthesized ester prodrugs .the structure of the developed prodrugs were confirmed by ir spectra, 1h nmr spectra, 13c nmr and mass spectra. the spectral data of the prodrugs were compared with that of the standard nsaids, ma and fbn affirm the formation of the compounds. hydrolytic study simulated gastric fluid (sgf) and simulated intestinal fluid (sif) were used for the in vitro hydrolytic study having the ph of 1.2 and 7.4 respectively.10 mg of the prodrug was in 90ml of the sgf and sif and 15 ml of the solution was withdrawn and centrifuge and make up the volume with methanol up to 8 hours. after centrifugation 5ml of the supernatant was taken and monitored the free concentration of ma and fbn by uv-vis spectrometer at 288nm, 247 respectively. the rate of hydrolysis was calculated by k= (2.303/t) log [b/ (b − y)]: where k represents hydrolysis constant, t is the time in minutes, b is the initial concentration of prodrug, y is the amount of prodrug hydrolyzed and (b − y) is the amount of prodrug remaining (15). pharmacological evaluation the wistar albino rat was used for the study and all the animal experiments were conducted after obtaining the institutional ethical committee approval (reg.no:1195/po/re/s/08/cpcea), al shifa college of pharmacy, kerala. ant-inflamamtory activity the screening of anti-inflammatory activity was done by using the egg albumin induced inflammatory model. in this inflammation was induced by 0.1ml egg albumin in 1% normal saline and it was administered in to the sub plantar tissue of the right hind paw. the linear circumference of the injected paw was monitored before and after the administration of the agent by using vernier calipers (0.5, 1, 2, 3, 4 and 5 hours). inflammation is the iraqi j pharm sci, vol.30(1) 2021 umbelliferone conjugates of nsaids 243 difference in the paw circumferences between the control and other treated groups before and after the treatment of phlogistic agent (16). anti-ulcerogenic study gastro intestinal toxicity expressed as lesions produced by the drugs and prodrugs and the mucosal damage was examined by using of an electron microscope. the severity of the gastric toxicity was measured by the parameter mean ulcer index as per the procedure explained by arun rasheed et al., 2017 (17). activity in brain the distributed nsaids showed the activity in the brain can be evaluated by using behavioral test, antioxidant test and histopathology of the brain cortex valuation. the albino mice was used for the study and all the animal experiments were conducted after obtaining the institutional ethical committee approval (reg.no:1195/po/re/s/08/cpcea), al shifa college of pharmacy,kerala. the model used for pharmacological screening was aluminium chloride induced neurotoxicity model (18).the animals were divided in to six groups and each contain six animals. the group i received normal saline which acts as control, group ii received aluminium chloride (50mg/kg) that acts as a negative control, group iii,iv,v and vi received ma(3.08mg), fbn(1.92mg), fu(3.2mg) and mu(4.3mg) respectively. this chronic neurotoxicity model was conducted and evaluated for 90 days. behavioral tests open field tests the rats were placed in the open field apparatus and after placing the animals, allowed to move them without any disturbance for 5 minutes and number of head dips, line crossing and rearing were counted marble burying assay the marble burying test is a simple behavioral test conducted in rodents, especially rats and mice are exposed to glass marbles placed on thick bedding materials. thirty clean glass marbles were arranged evenly on the bedding. after 30 minutes exposure to the marbles, mice were removed and unburied marbles were counted. a marble was considered buried if its two‐third size was covered with saw dust and the total number of marbles buried was considered as an index of locomotion water maze test the rats were placed in the apparatus and escape latency was monitored. the apparatus consists of a large circular pool including a wooden material below (19). antioxidant parameters for the in vivo test of antioxidant parameters, after behavioral study the mice were sacrificed and brain tissue homogenate was prepared with normal saline and centrifuged. the supernatant was used for the tests. superoxide dismutase assay mixture contain 0.1ml supernatant,1.2 ml sodium pyrophosphate buffer (ph 8.3,0.025 m), 0.1ml phenazine methosulphate ,0.3ml nitroblue tetrazolium, 0.2 ml nadh (780μm) and make up the volume to 3ml by adding water.incubated at 300c for 90 s and 0.1 ml glacial acetic acid added, stirred with 4 ml n-butanol. it was allowed to stand for 10 min. the separated butanol layer having colour and it was measured by uvvisible spectrometer at 560nm. the sod activity was tabulated. catalase 0.1ml of the brain tissue homogenate was treated with 0.9 ml of phosphate buffer and 0.4 ml of hydrogen peroxide. after 60seconds 2 ml dichromate acetic acid mixture was added. the color developed and it was measured at the wavelength 620nm. the activity was expressed as units / g tissue)20(. histopathology of brain cortex histopathology of the brain cortex of the different treated groups were examined by hemotoxylin-eosin stain and monitored under electron microscope (21). statistical analysis statistical significance was done by anova and the values were expressed as mean ± sd. results and discussion physical and chemical characterization the two ester prodrugs of nsaids, fbn and ma were synthesized and physical as well as chemical characterization was done. the ester prodrugs of fbn and ma were synthesized by conjugating with the natural antioxidant 4-methyl umbelliferone by dcc/dmap coupling method and the two synthesized compounds were fu and mu. fu is 4methyl-2-oxo-2h-chromen-7-yl-2-(2-flouro-[1, 1’biphenyl]-4-yl) propanoate and mu is 4-methyl-2oxo-2h-chromen-7-yl-2-((2,3-dimethyl phenyl)amino)benzoate. the thin layer chromatography was used to determine the progression, formation and check the purity of the synthesized ester prodrugs. the single spot obtained from the thin layer chromatography and the rf values were 0.69 and 0.51 for fu and mu respectively. the molecular weight fu and mu was found to be 402 and 391 respectively. the remarkable difference in the melting point confirmed the completion of the synthesis of the compounds. the solubility studies proved the high solubility of the prodrugs in the organic solvents and that sowed the enhanced lipophilic behavior. iraqi j pharm sci, vol.30(1) 2021 umbelliferone conjugates of nsaids 244 also the enhancement of the lipophilic profile was proved by partition coefficient studies. the elemental analysis was done to find out the percentage of c, n, o and h present in the synthesized compounds and that is compared with that of the calculated values. the calculated and found values were comparable. the results of physical and chemical characterization were given in the table 1. table 1. physical and chemical characterization of fu and mu prodrug molecular weight colour melting point (0c) log p perce ntage yield rf value elemental analytical data calculated percentage found percentage fu [c25h19fo4] 402 pure white crystals 90-92 1.35 70 0.69 c 74.62 74.65 h 4.76 4.72 f 4.72 4.75 o 15.90 15.92 mu [c25h21no4] 391 yellowish white crystals 140-143 1.43 76 0.51 c 78.57 78.60 h 5.83 5.80 n 3.53 3.58 o 12.08 12.05 fu(4-methyl-2-oxo-2h-chromen-7-yl-2-(2-flouro[1,1’-biphenyl]-4-yl)propanoate):ftir(cm1,kbr):2,980(c– h),1,713(c=o,ester)and1,071(co,ester);1hnmr( cdcl3,): 7.56(m,j=7.75,3h)7.45(d,j=10.62,3h),7.37(m,j=5. 7,1h),7.23(dd,j=6.3,1h),7.06(d,j=2.15,1h), 7.01(d,j=8.65,1h), 6.26(s,1h), 4.028(q,j=7.1,1h), 2.415(d,j=7.1,3h),1.67(d,j=7.15,3h,); 1hnmr(d2o):7.56(m,j=7.75,3h)7.37(d,j=10.62,1 h),7.45(m,j=5.7,3h),7.23(dd,j=6.3,2h),7.06(d,j=2 .15,1h), 6.99(d,j=8.65,1h), 6.25(s,1h), 4.028(q,j=7.1,1h), 2.415(d,j=7.1,3h),1.67(d,j=7.15,3h,);mass(m/z):4 03;13cnmr(cdcl3,500mhz) 18.35(ch3),18.72(ch3),45.15 (ch, aliphatic), 110.29(ch, benzene),114.69(ch-coo, lactone), 115.25(c, benzene), 117.85(ch, benzene), 117.95(ch, benzene), 123.54(ch, benzene) , 125.38(c-c), 127.84(ch, benzene), 128.33(ch, benzene), 128.44(ch, benzene), 128.51(2c, ch benzene), 128.98(ch, benzene), 131.17(ch, benzene), 135.28(c, benzene), 140.68(c-c), 153.11(c-o, benzene), 154.17(ch, benzene), 158.85(c, benzene), 160.42(c-f), 160.83 (carboxyl, lactone -c=o), 171.9(carbonyl -c=o). mu (4-methyl-2-oxo-2h-chromen-7-yl-2-((2, 3dimethyl phenyl) amino) benzoate): ftir (cm-1 kbr): 3364 (n-h stretching), 2,980 (c–h), 1,718 (c=o ester), and 1,071 (c–o, ester); 1h nmr (cdcl3,δppm) 8.17(d,j=6.55,1h,), 7.67(d,j=8.6,1h,), 7.33(t,j=5.45,1h,),7.28(s,1h), 7.22(d,j=6.3,1h), 7.15(d,j=7.25,1h,), 7.12(t,j=7.75,1h,), 7.05(d,j=7.2,1h,), 6.77(d,j=10.06,1h,), 6.75(t,j=7.05,1h,), 6.29(1h), 2.46(s,3h), 2.32(s,3h), 2.18 (s,3h); 1 h nmr (d2o,δppm) 8.17(d,j=6.55,1h,), 7.67(d,j=8.6,1h,), 7.33(t,j=5.45,1h,),7.28(s,1h), 7.22(d,j=6.3,1h), 7.15(d,j=7.25,1h,), 7.12(t,j=7.75,1h,), 7.05(d,j=7.2,1h,), 6.77(d,j=10.06,1h,), 6.75(t,j=7.05,1h,), 6.29(1h), 2.46(s,3h), 2.32(s,3h), 2.18 (s,3h);mass(m/z):400; 13cnmr( cdcl3, 500 mhz)14.01(ch3),18.77(ch3),20.60(ch3),108.79( ch-coo, lactone) ,110.99 (c, benzene),113.38 (ch, benzene),114.54 (ch, benzene),,116.29 (c fused),117.89 (ch, benzene), 118.6 (ch, benzene),, 123.46 (ch, benzene), 125.4 (c-c), 126.05 (ch, benzene), 127.31(ch, benzene), 131.89(ch, benzene), 132.74 (c, benzene), 135.46 (ch, benzene), 138.12 (c, benzene), 138.38 (c, benzene), 150.64 (c, benzene), 151.98(c-o, benzene), 153.40(c-n), 154.32(c-fused), 160.58(carboxyl, -c=o), 166.69(carbonyl -c=o). in vitro hydrolysis study in vitro hydrolytic study of the synthesized ester prodrugs was conducted in the enzyme free simulated intestinal fluid (ph 7.4) and simulated gastric fluid (ph 1.2) and the results proved the enhanced stability of the prodrugs in gastric ph 1.2 and enhanced percentage hydrolysis of the synthesized ester prodrugs in the intestinal ph 7.4. the percentage hydrolysis of the synthesized ester prodrugs were graphically represented in the figure 2 and 3. the data showed that the synthesized ester prodrugs fu and mu showed the percentage of hydrolysis after 8 hrs in sgf was 29 and 29.50 respectively. the percentage hydrolysis of the fu and mu in sif was 76.50 and 75.32 respectively. this result established, the fact that the natural antioxidant conjugated prodrug showed considerable stability in sgf and considerable hydrolysis in sif. the kinetic study fu and mu follow first order kinetics that was understood from the correlation coefficient data with the half-life of 318 and 301 minutes respectively in sif. the results of pharmacokinetic study in sif were given in the table 2 and first order kinetic graph of the prodrugs in sif was given in figure 4. iraqi j pharm sci, vol.30(1) 2021 umbelliferone conjugates of nsaids 245 table 2. pharmacokinetic data in sif prodrug sif [correlation coefficient] first order kinetic data zero order first order rate constant half life(t1/2)[min] fu 0.9817 0.9928 0.00218 318 mu 0.9812 0.9956 0.00230 301 figure 3. percentage hydrolysis of the fu and mu in sgf figure 4. percentage hydrolysis of the fu and mu in sif anti inflammatory activity the drugs ma and fbn are good antiinflammatory agents but the synthesized umbelliferone conjugated prodrugs showed enhanced anti-inflammatory activity. the comparative study was shown graphically in figure 5.the data showed the enhanced activity from the six hours observations and the prodrugs showed the values 44.1 to 75.5, 46.0 to 78.4 in the case of mu and fu respectively. the activity profile of the prodrugs showed enhanced pharmacological activity. the statistical significance (p<0.05) was done by one-way anova and dunnet’s t test. figure 5. first order kinetic graph in sif ulcerogenic activity the side effect produced by the nsaid is the ulcer production in the gastric mucosa due to the free carboxylic acid functional group in the structure of nsaids. the ulcer formation in the different treated rodents was visually monitored and the parameter to access the ulcer formation is mean ulcer index. the photographs of the standard and test groups were given in the figure 6 and the mean ulcer index was graphically represented in the figure 7. figure 6. anti inflammatory activity figure 7. ulcer formation in different groups: i. ma, ii.fbn, iii. mu and iv. fu iraqi j pharm sci, vol.30(1) 2021 umbelliferone conjugates of nsaids 246 activity in brain pharmacological evaluation was done by monitoring the activity in the brain by behavioral studies, anti-oxidant test and microscopically examined the histopathology of the brain cortex and the obtained data analysis confirmed the protective nature of the nsaids against degenerated conditions in the brain. the neurotoxicity was induced by aluminum chloride (50 mg/kg) explained in the methodology. the behavioral tests assess the general behavior, memory, spatial learning, locomotor activity and anxiety etc. behavioral parameters were monitored by using the tests open field test, marble burying test and water maze test. the open field test explained graphically in figure 8. this behavioral evaluation proved that the number of head dipping, number of line crossing and rearing significantly (p<0.001) decrease in the neurotoxicity induced group ii compared to the normal saline treated control group. but the treatment of the animals with prodrugs showed remarkable increase in the number of head dipping, rearing and line crossing significantly (p<0.05 and p<0.01) compared to the fbn and mu treated groups. but the behavioral activity of the fbn and mu showed similarity to the group ii that may due to the limited distribution of nsaids in the brain. figure 8. ulcer index of the drugs and prodrugs the results of the marble burying study graphically represented in figure 9. the results indicated that the number of marbles buried by the group ii was significantly decreased (p<0.001) compared to that of the group i control. the nsaids treated groups showed similarity in the group ii significantly (p<0.001) and that proved the limited activity of the hydrophilic drugs in the brain. but the prodrug (fu and mu) treated groups showed significant (p<0.05 and p<0.01) increase in the number of marbles buried that proved the prodrug based synthesis provide the sufficient enhancement in the transport properties through the bbb and also the distribution, efficiency and protective effect. figure 9. open field test the water maze test evaluated the spatial learning and memory and the results were graphically represented in figure 10. the results showed that the negative control group has increased escape latency compared to the control group (p<0.01). the time taken to escape is decreased in the case of prodrug treated groups compared to that of nsaids treated groups. the time taken to escape by the fbn and ma treated groups is comparable with that of the negative control group with a significance of p<0. 001.the comparison in the data between the drug treated groups and prodrug treated groups revealed that after reaching the brain nsaid can perform an important role in the case of neuro-degenerative conditions. figure 10. marble burying test the evaluation of the antioxidant activity (sod and catalase) indicated the protective effect of the compounds in the brain. the amount of sod and catalase showed decreased in the negative control group compared with that of the normal control group (p<0.001). fu and mu treated groups showed significant increase (cp< 0.05, dp<0.01) in the sod and catalase activity when compared to that of the fbn and ma treated groups and the data was given in figure 11. this proved the protective effect of the nsaids in the central nervous system. iraqi j pharm sci, vol.30(1) 2021 umbelliferone conjugates of nsaids 247 figure 11. water maze test histopathology of mice brain were from the normal saline group, negative control group, ma,fbn,mu and fu treated groups and the results were analyzed. in the prodrug treated group, all the two different ester prodrugs showed normal cells of cortex without any spongiform cells, indicates the protective effect of synthesized natural compound conjugating ester prodrugs of ma and fbn that was shown in the figure 12. figure 12. antioxidant activity normal aluminium chloride fu mu figure 13. histopathology of brain cortex references 1. gunaydin c, bilge ss. effects of nonsteroidal anti-inflammatory drugs at the molecular level. eurasian j med. 2018; 50(2):116-121. 2. asanuma m, miyazaki i, ogawa n. neuroprotective effects of 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pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of convolvulus arvensis doi:https://doi.org/10.31351/vol29iss2pp62-69 62 phytochemical investigation of the aerial part of iraqi convolvulus arvensis mustafa h. alwan *,1 and maha n. hamad* **department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq abstract convolvulus arvensis is a species of bindweed that is rhizomatous and is in the morning glory family (convolvulaceae) native to europe and asia. the plant is naturally grown in iraq. the plant was reported to be used in traditional medicine from as early as 1730s. the aerial parts of convolvulus arvensis were macerated in 80% ethanol for 6 days. the concentrated extract was partitioned with n-hexane, chloroform, ethyl acetateand n-butanol successively. the n-hexane and ethyl acetate, fractions were examined for the presence of phytochemicals by thin layer chromatography and high performance liquid chromatography and its steroid and flavonoid contents were investigated. stigmasterol was isolated from n-hexane fraction and identified by liquid chromatography/mass spectroscopy. rutin was isolated from the ethyl acetate fraction and identified by liquid chromatography/mass spectroscopy. the aim is to examine the phytochemical constituents of the aerial parts of convolvulus arvensis, literature survey available so far revealed that there were no studies about the phytochemical investigation for convolvulus arvensis in iraq. different chromatographic techniques like thin layer chromatography and mass spectroscopy were used and the presence of stigmasterol and rutin in aerial parts of convolvulus arvensis was indicated. keywords: convolvulus arvensis, phytochemical analysis, steroids,flavonoids. دراسة كيمياويه للجزء الهوائي لنبات المديد العراقي *حمد و مها نوري 1*, علوان مصطفى حسن . لعراق، ا، بغداد فرع العقاقيروالنباتات الطبية،كليةالصيدلة،جامعة بغداد الخالصة يد نبات المديد هو نوع من انواع نباتات اللبالب وينتمي الى نباتات عائلة المجد الصباحي وينتشر غالبا في اسيا واوروبا. قد استخدم المد في الطب الشعبي منذ سبعينات القرن الماضي. لعالج امراض الجلد و داء الدمال ولعالج آالم المفاصل وعالج التورمات. من االيثانول المائي كمذيب لالستخالص %80من خالل الطريقة العامة لالستخراج للعالم )هاربورن( باستخدام تم استخالص النبات تخلص االيثانول بواسطة النقع . وقد تم اجراء الفحوصات الكيميائية االولية لنتائج االيض الثانوية المختلفة من خالل اختبارات كيميائية محددة على مس وجود ستيرويدات وفالفينويدات في نبات المديد . وكذلك تم الحصول على اربعة اجزاء مختلفة من المستخلص الخام والتي هي الخام وقد كشف عن وكذلك الروتين من جزء جزء الهكسان ، جزء الكلوروفورم ، جزء االثل استيت ، جزء بوتيل الكحول . تم عزل الستيكماستيرول من جزء الهكسان ل نقي باستخدام كروماتوغرافيا الطبقة الرقيقة التحضيري وقد استحدمت التقنيات الفيزيائية والكيميائية والتحليلية الطيفية لتحديد االثل استيت في شك فصِل تشخيِص وتركيبها الكيميائي ووزنه الجزيئي ، وتشمل : كروماتوغرافيا الطبقة الرقيقة ،والتحليل الطيفي الكتلي السائل .يهدُف البحُث لدراسِة و قبل .وتنقيِة بعِض المركباِت الكيميائية الموجودة في األجزاء الهوائية لنبات المديد، إذ لم تتم دراسة مكونات هذا النبات في العراق من نبات المديد،كروموتوغرافيا السائل عالي االداء، تحليل المواد الكيميائية النباتية،الستيرويدات، الفالفينويدات : الكلمات المفتاحية introduction convolvulus arvensis is a species of bindweed that is rhizomatous and is in the morning glory family (convolvulaceae) native to europe and asia(1). the genus convolvulus (convolvulaceae) has a cosmopolitan, though largely temperate distribution and comprises approximately 200 species worldwide. more than half of the species occur in the mediterranean region, macaronesia and western asia(2). the species convolvulus arvensis is a perennial plant that are woody at the base, with trailing or scrambling unarmed stems, petiolate leaves that are truncate or rounded at the base, flowers borne in axillary cymes, conspicuous peduncles that are generally shorter than the subtending bracts, and corollas that are blue, yellow or white (figure 1). (3) 1corresponding author e-mail: mustafa.7san@yahoo.com received: 25/9 /2019 accepted: 3/5 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp62-69 https://en.wikipedia.org/wiki/convolvulaceae https://en.wikipedia.org/wiki/europe https://en.wikipedia.org/wiki/convolvulaceae https://en.wikipedia.org/wiki/europe https://en.wikipedia.org/wiki/asia iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of convolvulus arvensis 63 figure 1. photo of convolvulus arvensis aerial parts of convolvulus arvensis was used as laxative, wound healing, anti-spasmodic anti-hemorrhagic, anti-angiogenetic and for the treatment of parasites and jaundice(4−6). in addition it was used as diuretic and in skin disorders such as anti-furunculosis, antidandruff and in spider bites(7). traditionally convolvulus arvensis was used as decoction in cough and flu, to treat the painful joints, inflammation and swelling(8), a purified water extract of leaves of bindweed is used to inhibit the growth of tumor cells, growth of blood vessels and enhance immune function(9). convolvulus arvensis reported to contain major compounds which are steroids, flavonoids, phenols, lipids and coumarins (10). campesterol, stigma-sterol and β-sitosterol were considered to be the most abundant steroid compounds in convolvulus arvensis, they exist in the aerial parts of the plant. campesterol has antiangiogenic activity and reduces cholesterol level (11), while stigma sterol has anti-fungal, potent antioxidant, hypoglycemic and thyroid inhibiting properties, laxative properties, reduce cholesterol level and anticancer (12,13), β-sitosterol has hypocholesterolemia activity, anti-diabetic effects, for benign prostatic hyperplasia (bph), and chemo preventive effect (14,15). quercetin, rutin and kaempferol are the most abundant flavonoids in the aerial parts of convolvulus arvensis, they have anti-oxidant, antiinflammatory and anti-cancer effects (16, 17).the most abundant phenols in convolvulus arvensis are chlorogenic acid, caffeic acid and ferulic acid which exist in the aerial parts of the plant. they possess anti-oxidant, anti-inflammatory anti-cancer effects and anti-diabetic effects (18-20). the aim: is to examine the phytochemical constituents of the aerial parts of convolvulus arvensis using different chromatographic (tlc, hptlc) and mass spectroscopy. literature survey available so far revealed that there were no studies about the phytochemical investigation for convolvulus arvensis in iraq. experimental section plant material the aerial parts of convolvulus arvensis were obtained from the farm of college of pharmacy/ university of baghdad. the plant was identified and authenticated by dr. khansaa rasheed/iraq natural history research center and museum /plant and environment department. extraction powdered plant material 400 grams were soaked in 1600ml ethanol (80%) with occasional shaking, at room temperature. after 2 days, the extract was filtered. the same process was repeated twice on the retained part. the filtrate (aqueous ethanol) from the three times was collected and evaporated to dryness under vacuum using rotary evaporator. a dark greenish residue was obtained. water 500ml was added to the residue and partitioned successively with n-hexane, chloroform, ethyl acetate, and n-butanol (3x500 ml) for each fraction. the first three fractions were dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. preliminary qualitative phytochemical analysis of crude extracts: 1.chemical tests the following tests were carried out on each extract which were obtained from the previously mentioned methods (21). test for flavonoids: few milligrams of each extract were placed in test tube suspended in few milliliters of ethanol and few drops of 5% alcoholic koh were added and noticed for the formation of yellow color which will disappear upon the addition of dilute hydrochloric acid. test for alkaloids: two tests were used for detection of alkaloids: a. dragendroff's reagent:dragendroff reagent (3drops) were added to 2 ml of ethanolic extract then observe of orange brown precipitate. b. mayer's reagent: mayer s reagent (4 drops) were added to 2 ml of ethanolic extract and observe of white creamy precipitate. test for phenols: 2 ml of the 5% ferric chloride was added to 2 ml of ethanolic extract the formation of green to deep blue indicates the presence of phenols. test for flavonoids: ethanolic koh was used for the detection of flavonoid which give yellow color. test for saponin about (2 gm) of the powdered sample from the leaves and seeds was boiled in (10 ml) of distilled water in a water bath and filtered. (5 ml) of the filtrate was mixed with (5 ml) of distilled water in a test tube and shaken vigorously. the formation of froth that persists for 15 minutes indicates the presence of saponins. iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of convolvulus arvensis 64 tests for steroids liebermann-burchard test: extract (3ml) was treated with chloroform, 5ml of acetic anhydride and drops of sulphuric acid was added. the formation of dark pink or red color indicates the presence of steroids. 2. analytical thin layer chromatography. ausing readymade plate aluminum coated tlc sheet g/uv254, 0.20 mm for n-hexane stationary phase used was silica gel, using liberman burchared spray reagent to detect the spot. the mobile phase was n-hexane: ethyl acetate (10: 4) (22). busing readymade plate aluminum coated tlc sheet g/uv254, 0.20 mm ethyl acetate fraction stationary phase used was silica gel, using uv lamp for detection of the spot. mobile phase: chloroform: methanol: formic acid (15: 4: 1) (23). 3. high performance thin layer chromatography analysis a-hexane fraction was analyzed also for its steroids contents utilizing hptlc (eike reich/camaglaboratory, switzerland, the chromatogram was developed in a mobile phase composed of ethyl acetate: hexane (50:50) examined at 254 nm wavelength. the number. of tracks were 3, injection volume was 100 µl, dosage speed 150 nl/s. the band length 8.0 mm. bethyl acetate fraction was analyzed also for its phytochemicals contents utilizing hptlc (eike reich/camag-laboratory, switzerland, the chromatogram was developed in a mobile phase mobile phase composed of ethyl acetate: formic acid: acetic acid: water (84: 4: 4: 10) examined at 254 nm wavelength. the number. of tracks were 16, injection volume was 100 µl, dosage speed 150 nl/s. the band length 8.0 mm. isolation of phytochemicals by preparative thin layer chromatography (tlc): a-isolation of steroids from hexane fraction using the same stationary and mobile phase in the analytical thin layer chromatography. bisolation of rutin from ethyl acetate fraction using the same stationary and mobile phase in the analytical thin layer chromatography. identification of isolated phytochemicals by lc/ms: the isolated flavonoid and steroid were recognized as rutin and stegmasterol respectively by liquid chromatography /mass spectrometry (lc/ms): analytical lc-ms was performed using a agillent 6410 qqq system under the following conditions: table 1. lc mass condition for flavonoid 1mobile phase , acetonitrile and water 2 flow rate 1 ml/min 3column size 0.19 mm 4c18 with 5μm particle size 5m/z range was 250 to 1000, 200k table 2. lc mass condition for steroids 1mobile phase, solvent a: 0.05% tfa in water, solvent b 0.05% tfa in methanol (ph 2.5). 2 c18 rp with 5μm particle size 3flow rate 1 ml/min 4column size 0.19 mm results and discussion 1preliminary phytochemical investigation like chemical tests were carried on the arial parts of convolvulus arvensis and showed the following results as shown in table 2. table 3: phytochemical analysis of the aerial parts of convolvulus arvensis extract. extract of c. arvensis phytochemical components + flavonoids + phenols + steroids alkaloids the table shows the presence of flavonoids, phenols and steroids. and the absence of alkaloids. 2analytical thin layer chromatography analysis for the hexane extract a-tlc test was carried on hexane fraction using a mobile phase of hexane: ethyl acetate (10: 4) and revealed the presence of steroid in the hexane fraction which was sprayed by liebermannburchard reagent and gave deep violet colour as compared with standard of stigmasterol as shown in (figure 2). figure 2 . thin layer chromotography for hexane fraction, a: beta-sitosterol (standard), b: hexane fraction, c: stigma-sterol (standard), mobile phase: hexane: ethyl acetate (10: 4). b-tlc for flavonoids was carried on ethyl acetate fraction of convolvulus arvensis using a mobile phase: chloroform: methanol: formic acid (15: 4: 1), the spot gave florescence under uv light 245 as compared with standard of rutin as shown in figure 3. iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of convolvulus arvensis 65 fig. 3. tlc for ethyl acetate fraction, a: ethyl acetate fraction, b: rutin (standard), mobile phase: chloroform: methanol: formic acid (15: 4: 1). 3hptlc analysis: ahptlc test was carried on hexane fraction of convolvulus arvensis using a mobile phase of hexane: ethyl acetate (50: 50) and revealed the presence of steroids as compared retardation factor (rf) with standard of stigma-sterol as shown in figure 4. figure 4. hptlc for hexane fraction, a: betasitosterol (standard), b: stigma-sterol, c: hexane fraction, mobile phase: hexane: ethyl acetate (50: 50). bhptlc was carried on ethyl acetate fraction using mobile phase: ethyl acetate: formic acid: acetic acid: water (84:4:4:10) and revealed the presence of rutin as compared the retardation factor (rf) with standard of rutin as shown in figures 5-7. figure 5. hptlc diagram for rutin standard figure6. hptlc diagram for isolated rutin iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of convolvulus arvensis 66 figure 7. hptlc for ethyl acetate fraction under uv 254 nm mobile phase: ethyl acetate: formic acid: acetic acid: water (84:4:4:10) isolation of phytochemicals by preparative thin layer chromotography a-preparative tlc test was performed on hexane fraction to isolate the steroid for further analysis using mobile phase: hexane: ethyl acetate (10: 4) as shown in (figure 8). figure 8 . preparative tlc for hexane fraction, a: beta-sitosterol (standard), b: stigma-sterol, c: hexane fration, mobile phase: hexane: ethyl acetate (10: 4). apreparative tlc test was performed on ethyl acetate fraction to isolate the rutin for farther analysis using mobile phase: chloroform: methanol: formic acid (15: 4: 1) as shown in (figure 9). fig. 9. preparative tlc for ethyl acetate fraction, a: ethyl acetate fraction, b: rutin (standard), mobile phase: chloroform: methanol: formic acid (15: 4: 1). iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of convolvulus arvensis 67 identification of isolated compounds by lc/ms analysis alc/ms test for steroid was performed to confirm the structure of the isolated steroids. the fragmentation pattern and molecular ion peak (412) indicated that the isolated steroid is stigmasterol as shown in (figure 10) . figure 10. lc/ms for stigma-sterol in hexane fraction fragmentation pattern was complained with that reported in the literature for stigmasterol from an earlier study shown in figure 11. figure 11: molecular ion and fragments of stigmasterol in hexane fraction (22). blc/ms for the isolated rutin confirmed the structure of the isolated rutin since fragmentation pattern and molecular ion peak (611) indicate that the isolated compound is rutin as shown in figure 12. iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of convolvulus arvensis 68 figure 12. lc/ms for rutin in ethyl acetate fractio. figure 13. mass spectrum of rutin in ethyl acetate fraction (23) conclusion the phytochemical investigation of the iraqi plant revealed the presence of stigma-sterol in hexane fraction and rutin in the ethyl acetate fraction of convolvulus arvensis aerial part of the plant. references 1. al-snafi, ali esmail: traditional uses of iraqi medicinal plants. iosr journal of pharmacy, 2018; 8: 32-96. 2. sa, fatima el zahra mahmoud abdallah, et al: the convolvulus species of the canary isles, the mediterranean region and the near and middle east. mededelingen van het botanisch museum en herbarium van de rijksuniversiteit te utrecht, 1967; 28: 3-288. 3. carine, mark a., robba, lavinia: taxonomy and evolution of the convolvulus sabatius complex (convolvulaceae). phytotaxa, 2010; 14: 1-21. 4. lakhdari, w., et al: ethnobotanical study of some plants used in traditional medicine in the region of oued righ (algerian sahara). 2016. 5. munz pa and keck dd: a california flora. university of california press, berkeley, ca 1959. 6. ali m, qadir mi, saleem m, janbaz kh, gul h, hussain l and ahmad b: hepatoprotective potential of convolvulus arvensis against paracetamol-induced hepatotoxicity. bangladesh j pharmacol 2013; 8: 300-304. 7. kaur, manbir; kalia, a. n. anticancer potential of the convolvulus arvensis: international journal, 2012; 1: 101-102. iraqi j pharm sci, vol.29(2) 2020 phytochemical investigation of convolvulus arvensis 69 8. meng xl, riordan nh, casciari jj, zhu y, zhong j and gonzález mj: effects of a high molecular mass convolvulus arvensis extract on tumor growth and angiogenesis. p r health sci j 2002; 21: 323-328. 9. sadeghi-aliabadi h, ghasemi n and kohi m: cytotoxic effect of convolvulus arvensis extracts on human cancerous cell line. research in pharmaceutical sciences 2008; 3: 31-34. 10. lee, jiwoo, et al: simultaneous determination three phytosterol compounds, campesterol, stigmasterol and daucosterol in artemisia apiacea by high performance liquid chromatography-diode array ultraviolet/visible detector. pharmacognosy magazine, 2015; 11: 297. 11. wang, gui-qi, et al: daucosterol inhibits colon cancer growth by inducing apoptosis, inhibiting cell migration and invasion and targeting caspase signalling pathway. bangladesh journal of pharmacology, 2016; 11: 395-401. 12. maalik, aneela, et al: pharmacological applications of quercetin and its derivatives: a short review. tropical journal of pharmaceutical research, 2014; 13: 15611566. 13. ali, huma, et al: isolation and evaluation of anticancer efficacy of stigmasterol in a mouse model of dmba-induced skin carcinoma. drug design, development and herapy, 2015; (9): 2793. 14. mahmood, talat, et al. complexation and antimicrobial activities of β sitosterol with trace metals. (cu (ii), co (ii), and fe (iii)). eur. acad. res, 2013; 1: 677-685. 15. saeidnia, soodabeh, et al: the story of betasitosterol-a eview. european journal of medicinal plants, 2014; 4: 590. 16. lin, xiang-qin; he, jian-bo; zha, zhenggen: simultaneous determination of quercetin and rutin at a multi-wall carbon-nanotube paste electrode by reversing differential pulse voltammetry. sensors and actuators b: chemical, 2006; 119: 608-614. 17. khare, chandrama p: ayurvedic pharmacopoeial plant drugs: expanded therapeutics. routledge, 2015. 18. aoudia, h., et al: phenolics, antioxidant and anti-inflammatory activities of melia azedarach extracts. ijarnp, 2013; 6: 19-29. 19. taher, mohammed a., et al: hypolipidemic effect of caffeic acid isolated from arctium lappa cultivated in iraq, in hyperlipidemic rat model. iraqi journal of pharmaceutical sciences 2015; 24: 18-24. 20. gohil, kashmira j.; kshirsagar, shashank b.; sahane, rajkumari s: ferulic acidcomprehensive pharmacology of important 21. bioflavonoid. int j pharm sci res, 2012; 3: 700-710. 22. h. wagner and s.bladt plant drug analysis, munich, march 1996 second edition. 23. tradit aj, altern c, sasidharan s, chen y, saravanan d, sundram km, et al: extraction, isolation and characterization of bioactive compounds from plants’ extracts institute for research in molecular medicine (inform), universiti sains malaysia, minden 11800, 2011; 8:1–10. 24. el-nabawy hi, ayyad dm, serag ms, abdelmogib m. phytochemical and biological evaluation of urospermum picroides. mansoura j chem. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 61 formulation of azithromycin suspension as an oral dosage form saba h. jaber * ,1 , zainab t.salih* and hiba m. salmo* *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract azithromycin is the drug of choice in the treatment of several bacterial infections, most often those causing middle ear infection, bronchitis, pneumonia, typhoid and sinusitis. it’s also effective against certain urinary tract infections and venereal diseases. this study was carried out to prepare an acceptable suspension either as dry physical mixture powder or granules to be reconstituted, through studying the effect of various type and concentration of suspending agent (xanthan gum, hydroxypropyl methylcellulose (hpmc), either alone or in combination) on the release profile of the drug. the best prepared suspension formulas (h& iii) were selected depending on the dissolution profile of each formulas and then compared with the reference suspensions (zithromax and azi-once).the viscosity, sedimentation volume, resuspendability and expiration date were evaluated for the chosen formulas (h&iii) and compared with references zithromax  and azi-once  .the result indicated that the chosen formula – h had better dissolution rate compared with references suspensions, also it was less viscous than them.while other chosen formula – iii had lower dissolution rate compared with zithromax  and higher dissolution rate than azi – once, also it was less viscous than both references.it was found that the dry physically mixed powder (formula – h) was more stable than the granular suspension (formula iii) since the expiration date for formula h and formula iii were 3.24 and 2.7 years respectively. key words: azithromycin, suspending agent, suspension. جحضير االزثرومايسين كمعلق فمىي صبا عبد الهادي جابر* ،1 سلمى*مىفق ، زينب ثابث صالح* و هبة *فزع انصٕذالوٕاث ، كهٕت انصٕذنت ، خامؼت بغذاد ، بغذاد ، انؼزاق الخالصة ا باالضافت انّ فؼانٕخً فٓ ػالج انؼذٔذ مه االسثزَمأظٕه ٌُ انذَاء انمفضم نؼالج انخٍاباث اندٍاس انخىفظٓ َحمّ انخٕفُئٕذ ٌذ امزاض انمظانك انبُنٕت َاالمزاض االوخمانٕت . حم اخزاء ٌذي انذراطت نخحضٕز مؼهك ثابج ) بشكم حبٕباث اَ مظحُق خاٌش نهحم ( مه بزَبم مثٕم طهٕهُس ( ػهّ طزػت حأثٕز اوُاع َحزاكٕش مخخهفت مه انمُاد انمؼهمت ) ساحثان كم ٌَأذرَكظٓدراطت االسثزَمأظٕه مه خالل اخخٕزث اػخمادا ػهّ مماروخٍا مغ انصٕغ االخزِ مه خالل لٕاص طزػت ححزر (iii & h )ححزر انذَاء . ان انصٕغ انخزكٕبٕت انمخخارة اص انفٕشٔائٕت نهصٕغ اَوض َ سثزَماكض ( . كذنك حمج دراطت انكثٕز مه انخُ –انذَاء ثم حمج كذنك مماروخٍا مغ انخزكٕبت انخدارٔت ) اسْ َحخمثم بدزٔان انمحهُل، حدم انمُاد انمخزطبت،اػادة انخداوض نهمحهُل بؼذ انزج َفؼانٕت انمُاد انحافظت (iii & h)انخزكٕبٕت انمخخارة خٓ اػطج طزػت ثزَماكض ( . َلذ اظٍزث انىخائح ان انصٕغ انخزكٕبٕت ان ساَوض َ -َمماروخٍا مغ انخزكٕباث انخدارٔت انمزخؼٕت ) اسْ مأظٕه انمحضز بشكم مظحُق خاٌش نهحم ٌُ وُػا ما َثزسححزر اػهّ مه انخزكٕبت انمزخؼٕت ٌٓ الم نشَخت. َكذنك َخذ ان مؼهك اال طىت نهحبٕباث . 2.7همظحُق َ طىت ن 3.2اكثز اطخمزارٔت مه انمؼهك انمحضز ػهّ شكم حبٕباث حٕث ان فخزة اوخٍاء انصالحٕت كاوج المفحاحية : أزثرومايسين ، مىاد معلقة ، معلق. الكلمات introduction suspension is preparation containing finely divided drug particle (suspensoid) distributed uniformly throughout a vehicle in which the drug exhibits a minimum degree of solubility (1) .some suspensions are available in ready liquid form that is, already distributed through a liquid vehicle with or without stabilizers and other additives. this preparation is termed as an “oral suspension “in the united state pharmacopeia (usp). other preparations are available as dry powders to be reconstituted when desired. this preparation is termed as “for oral suspension “in usp (1) .there are many physical and chemical consideration in the preparation and development of a suspension to satisfy its pharmaceutical requirements.some suspending agents are generally added to the dispersion medium in order that their structures help to maintain uniform dispersibility (2) or to prevent caking of the drug particles during shelf – life (3) . pharmaceutical suspension usually defined as a coarse dispersion (4) . 1 corresponding author email : sabahadee 77@yahoo.com received : 20/10/2011 accepted : 3/3/2012 mailto:77@yahoo.com iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 62 azithromycin is one of the worldś best selling antibiotics, and is derived from erythromycin. azithromycin is used to treat or prevent certain bacterial infections , most often those causing middle ear infection ,tonsillitis, throat infections, laryngitis, bronchitis, pneumonia, typhoid and sinusitis (5,6) .in recent year it has primarily been used to prevent bacterial infections in infants and those with weaker immune system.it is also effective against certain urinary tract infection and venereal diseases, such as non – gonococcal urethritis, chlamydia, gonorrhea and cervicitis (7,8) .unlike erythromycin, azithromycin is acid stable and can therefore be taken orally with no need of protection from gastric acids.it is readily absorbed, but its absorption is greater on an empty stomach (9) .azithromycin is practically insoluble in water, freely soluble in anhydrous ethanol and methylene chlorid (10) . materials and equipments materials azithromycn powder (supplied by kanawati medical products – syria) artificial flavor (raspberry flavor), xanthan gum , hydroxy propyl methyl cellulose (hpmc) , colloidal silicon dioxide, methyl paraben (m.p.) , propyl paraben (p.p.), disodium edta (supplied by samara drug industries (sdi) ), tribasic sodium phosphate dodecohydrate, sucrose, polyvinyl pyrrolidone (pvp) (supplied by bdh chemical ltd. pool, england),absolute ethanol (supplied by gcc gain land chemical company, u.k.) azi-once  powder for oral suspension (supplied by jam joom pharma). zithromax ® powder for oral suspension (supplied by pfizer italy). equipments miller hinton agar (supplied by rashmi – diagnostics, danglorc , india ), sartorious balance (sartorius ag. gottingen , germany ), ph 211 micro processor ph meter (hana, italy ), uv\vis –spectra photo meter – uv-9200 (sedico ltd .p.o.bo 20961, nicosia-1665 – cyprus), dissolution apparatus (erweka g.m.b.h. type dt6, w.germany), viscometer (ndj – 55 digital viscometer),oven (mem meter 854 schw bach, w. germany), oven (gallen kamp, bs size one, england), autoclave (webco f.g. bade of co laboratory equipment, hamburg, germany). method of preparation formulation of azithromycin suspension several formulas of azithromycin aqueous suspension were prepared, either as dry physical mixture powder for reconstitution or as granules for reconstitution. suspension prepared by physical mixing (as dry powder) table (1) shows different formulas of azithromycin suspension each was prepared as: ablend of 4.0 %( w/v) azithromycin was mixed with different excipients stated in the above table, in a dry amber bottle (11) . table 1 : different formulas of azithromycin powder prepared as physical mixture to be reconstituted as suspension (% w/v). materials (gm) formulas a b c d e f g h i j azithromycin 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 4.0 xanthan gum 0.3 0.5 0.3 0.5 0.3 0.5 0.5 0.5 hydroxy propyl methyl cellulose (hpmc) 0.3 0.5 0.5 0.5 0.3 0.3 0.3 0.3 colloidal silicon dioxide 0.5 0.1 the following substances have been added to each of the above formulas in the same quantities tribasic sodium phosphate dodecohydrate 0.8 methyl paraben (m.p) 0.18 propyl paraben (p.p) 0.03 disodium edta 0.1 artificial flavor (raspberry flavor) 0.49 sucrose 93 water for reconstitution 40 ml iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 63 suspension prepared as granular for reconstitution table (2) shows different formulas of azithromycin granules prepared by granulation method to be reconstituted as suspension as follows: a blend of 4.0 %( w/v) azithromycin was mixed with different excipients mentioned in the above table, followed by levigating with alcoholic pvp solution (0.5% w/v) as a granulating agent.the powder mixture passed through a sieve (300µm) to a petri dish for drying at 37°c before being transferred to an amber bottles (12,13) . the evaluation of prepared suspension and comparative studies of different variables affecting the selected formulas with marketed references (azi-once ® and zithromax ® suspension) were done. table 2 : different formulas of azithromycin prepared as granules to be reconstituted as suspension (% w/v). materials (gm) formulas i ii iii iv azithromycin 4.0 4.0 4.0 4.0 xanthan gum 0.3 0.5 0.3 0.5 hydroxry propyl methyl cellulose (hpmc) 0.5 0.3 0.3 0.5 the following substances have been added to each of the above formulas in the same quantities tribasic sodium phosphate dodecohydrate 0.8 methyl paraben 0.18 propyl paraben 0.03 disodium edta 0.1 artificial flavor (raspberry flavor) 0.49 sucrose 93 alcoholicpvp solution (0.5% w/v) 60 drops ( for granulation ) water for reconstitution 40 ml dissolution profile the dissolution rate of azithromycin drug from suspensions was studied using usp dissolution apparatus rotated at 50 r.p.m. the dissolution medium was 0.1n hcl (500 ml) maintained at 37°c, 5 ml sample of suspension was added to the medium. then a sample of dissolution medium was withdrawn at different time intervals (1, 2, 3, 4, 5, 7, 9, 12, 20, 30 minutes) through a pipette fitted with a filter paper. the same fresh dissolution medium was added to the jar each time to replace with drawn samples. each sample was suitably diluted and assayed spectrophotometrically at 210nm (10) for azithromycin content (14) . measurement of viscosity the viscosities were obtained at 37c with ndj – 55 digital rotational viscometer using spindle number 2 at 60 rpm. (15) . sedimentation volume measurement fifty ml of each suspension was diluted with distilled water to a volume of 100 ml in a stopper graduated cylinder. the suspensions were shaken vigorously to ensure uniformity, and then left undisturbed. the sedimentation volume was measured every 4 hours for a period of 48 hours (16) . re– suspendability of suspension the test consisted of manually shaking the cylinder after the sedimentation experiment was completed. based on the effort required to convert the sediment system to a homogenous suspension, the prepared product was rated as: resuspendable , resuspendable with difficulty or not resuspendable (17) . antimicrobial activity of suspension the antimicrobial activity of azithromycin suspensions were studied by the agar diffusion method (18, 19) . the basic of the diffusion test includes the diffusion of the material under test from a central reservoir into the surrounding agar which has been inculated with a sensitive organism. the growth of the organism is inhibited, and manifested as zone of inhibition.the relationship between the applied dose , kind of suspension and size of the inhibition zone in the basis of comparisons the gram +ve and the gram –ve bacteria which are sensitive to azithromycin were used . iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 64 gram + ve staphylococcus aureua . gram – ve escherichia coli. suspension tested azi – once ® . zithromax ® . formula h. formula iii. the test procedure steril miller – hinton agar media were poured in to flat bottom steril petri dish under aseptic technique and were allowed to harden , then incubated at 37°c for 24 hours after inoculated with the appropriate microorganism , all of these plates are divided into four sections as shown in figure (a) (14) .0.05ml (one drop) of each suspension formulas were inoculated in each sector of the agar media using a micropipette, each drop contains the equivalent of 2mg of azithromycin. figure (a) 1-represent azi – once ® suspension. 2-represent zithromax suspension. 3-represent formula h . 4represent formula iii . stability study the accelerated stability study was done in order to determine the expiration date of formula h and formula iii by placing the samples of both formulas in ovens at 40°c, 50°c and 60°c for 90 days.samples (dry physical mixture powder or granules) were taken and assayed for their drug content at a suitable time intervals using uv spectrophotometrical method for the two accepted formulas (h and iii) (20) . furthermore, physical stability (color, odor and ph) change for the samples were also examined . results and discussion xanthan gum was used as suspending agent because of its excellent suspending properties and also as an effective flocculating agent at relatively low concentration (21) . an increase in the concentration of xanthan gum (formula b) gave no substantial change in flocculation behavior, good dissolution properties and it produced a sediment layer that was easily redispersed upon shaking as shown in figure (1).on the other hand, hydroxy propyl methyl cellulose (hpmc) was utilized as suspending agent and binder as shown in figure (2). figure 1: effect of xanthan gum concentration on dissolution profile of azithromycin (formula a and b ) in 0.1 n hcl (ph=1.2) at 37 °c. figure 2: effect of hpmc concentration on the dissolution profile of azithromycin (formula cand d) in 0.1 n hcl (ph=1.2) at 37 °c. this polymer behave as a protective colloid by coating the solid hydrophobic particles with multimolecular layer, this will impart hydrophilic character to the solid and thus promote wetting (22) . but to a certain concentration, the dissolution of azithromycin decreased which is attributed to the fact that at high polymer concentration, the polymeric flocculating agent coat the whole surface of the suspended particles, so there is no free areas from adsorbate. these areas are necessary for cross-linking to be recure after the product was sheared (23) . the increase in the viscosity of the 4 2 3 1 iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 65 system may also have an effect on the dissolution rate of the drug (24) . furthermore, a combination of xanthan gum and hpmc (formula e , f, g and h ) resulted in a maximum enhancement in the dissolution of azithromycin , this could be due to the fact that their linear branched chain molecules form a gel – like net work within the system and become adsorbed on to the surface of the dispersed particles , thus holding them in a flocculated state ( bridging effect ) (20 ) as shown in figure (3) . formula – h produced a suspension with good dissolution as shown in figure (3). this formula (h) gave the most optimum physical stability and remarkable release profile, therefore it was chosen for extensive study and to be compared with reference suspensions.the addition of colloidal silicon dioxide as a dispersing agent (formula i&j) results in too viscous suspension as listed in table (3), with difficulty to redisperse upon shaking. this was a result of using a combination of three polymers (xanthan gum +hpmc and colloidal silicon dioxide) which leads to rheological synergism to be occurred due to stronger cross linking between these polymers (25) .these formulas (i &j) showed a substantial change in the dissolution behavior corresponding to the best formula –h and the releasing profile decreased by increasing the concentration of colloidal silicon dioxide as shown in figure (4) (26,27) . figure 3: effect of xanthan gum and hpmc on the dissolution profile of azithromycin ( formula e,f,g and h) in 0.1 n hcl (ph=1.2) at 37 °c. -formula h(0.5 % w/vxanthan gum + 0.3% w/v hpmc) -formula g (0.3% w/v xanthan gum + 0.3% w/v hpmc) -formula e (0.3% w/v xanthan gum + 0.5% w/v hpmc) -formula f (0.5% w/v xanthan gum + 0.5% w/v hpmc) table 3: the viscosities of the suspension formulas ( h , iii, i , j) and reference suspension, using spindle number 2 at 60 rpm. formulas viscosity (mpa.s) azionce ® 268 zithromax ® 240 formula – h 233 formula – iii 199.2 formula – i 450 formula – j 400 figure 4: effect of colloidal silicon dioxide concentration on the dissolution profile of azithromycin from the best formula – h ( formula i and j ) in 0.1 n hcl (ph=1.2) at 37 °c. finally, azithromycin suspension (as dry physical mixture powder) when prepared using 0.5%(w/v) xanthan gum as a single suspending agent together with 0.3%(w/v) hpmc as suspending agent and binder (formula –h) resulted in a suspension that showed high sedimentation volume (0.99) and was easily redispersed upon simple shaking.figure (5) shows the enhancement in the dissolution of azithromycin by the addition of a blend of different concentrations of xanthangum and hpmc (polymeric flocculation agent) to the formulas i, ii, iii and iv. these formulas were prepared as granules using alcoholic – pvp solution as a granulating agent (28) . the granules were found to be free flowing, not bulky, and of uniform size. the size of each granule was 300 µm according to the sieve number was used (no.50) (1,17) . formula iii was chosen since it gave good dissolution, stability although it produced sediment layer with simple shaking easily redispersable. figure (6) show the dissolution profile of azithromycin suspension for formulas h & iii compared with the reference zithromax ® and azi – once ® suspensions. iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 66 figure 5: effect of xanthan gum and hpmc concentration on the dissolution profile of azithromycin granules ( formula i,ii,iii and iv) in 0.1 n hcl (ph=1.2) at 37 °c. -formula i (0.3% w/v xanthan gum + 0.5% w/v hpmc) -formula ii (0.5% w/v xanthan gum + 0.3% w/v hpmc) -formula iii (0.3% w/v xanthan gum + 0.3% w/v hpmc) -formula iv (0.5% w/v xanthan gum + 0.5% w/v hpmc) figure 6: dissolution profile of azithromycin suspension for formulas h&iii compared with the reference zithromax ® & azi-once ® suspensions in 0.1 n hcl (ph=1.2) at 37°c . the results showed that azithromycin release from formula –h was higher than that form others ( azi-once and formula – iii ) but show approximately the same release as zithromax ® . formula – iii after reconstitution showed lower dissolution profile than formula h – and zithromax ® , this may be due to the granulation process, since pvp was used as a granulating agent which is water soluble binder and has good swelling and hydration capacity. these properties result in the high viscous region surrounding the drug particle (29,30) .on the other hand the viscosities of the products are represented in table (3). the results showed that the viscosity of azithromycin suspension was shear rate dependent and increased viscosity in the following order: azionce ® > zithromax ® > formula – h > formula – iii table (4,5) show the sedimentation volume and resuspendability after settling of the chosen azithromycin formulas (h,iii) and the reference suspensions after 48 hours undisturbing . the evaluation of azithromycin released as antibacterial agent was listed in table (6). table 4: sedimentation volume of azithromycin suspensions (formula h,iii,azi once and zithromax ® ) products f=hu / h0 zithromax ® 0.99 formula – h 0.99 formula – iii 0.9 azi – once 0.6 where hu is the ultimat height of the sediment as suspension settle ,h0 is the intial height the total suspension . table 5: resuspendability of azithromycin suspensions (formula h,iii,azionce and zithromax ® ) products resuspendaloility zithromax ® easily resuspendable formula – h easily resuspendable formula – iii easily resuspendable azi – once resuspendable with difficulty table 6: zone of inhibition by azithromycin in different formulas on the sensitive gram +ve and gram –ve organism (mm ). products gram +ve staph. aureus inhibition zone gram -ve e – coli inhibition zone zithromax ® 34 mm 33 mm formula – h 33 mm 28 mm formula – iii 34 mm 28 mm azi – once 32 mm 28 mm formula – h has less antimicrobial effect against gram positive microorganism compared to the references, while formula – iii has the same activity as references. formulas (iii and h) has the same antimicrobial activity against gram negative microorganism as azi – once ® but less than zithromax ®(31) .the accelerated stability study was applied on the chosen formula at higher temperatures(40°c,50°c and 60°c) to predict the expiration date of formulas h and iii . iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 67 the degradation of azithromycin in these formulas followed first order kinetics since straight lines,were obtained by plotting the logarithm of percent remaining of azithromycin versus time as shown in figures ( 7, 8) (32,33) . figure 7: degradation curve of dry physical mixture powder of azithromycin suspension ( formula – h ) at different temperature. figure 8: degradation curve of granular azithromycin suspension ( formula – iii ) at different temperature. table 7: degradation rate constant of azithromycin suspension dry powder (formula – h ) and granular suspension ( formula iii ) temperature k x 10 -3 (day -1 ) °c formula – h formula – iii 60 0.53 0.6 50 0.35 0.4 40 0.2 0.23 25 0.09 0.11 the degradation rate constants (k) at different temperatures were calculated from the slope of the straight lines and they were listed in table (7). arrhenius plots were then constructed and are shown in figures (9,10) for formula h and iii respectively.the linearity of the curve indicates their utility in predicting the rate of degradation at lower temperature. the rate constant at 25  c, obtained from those plot for dry physically mixed powder (formula – h ) and granular suspension ( formula iii) were equal to (0.09 x 10 -3 ) and (0.11 x 10 -3 )(day -1 ) respectively.since the degradation of the drug followed first order kinetics, the expiration date t 10% at 25  c could be calculated using the following equation. the expiration date for formula – h and formula – iii were 3.24 and 2.7 years respectively. figure 9: arrhenius plot for expiration date estimation of azithromycin suspension (formula – h ) at 25  c . figure 10: arrhenius plot for expiration date estimation of azithromycin suspension (formula – iii ) at 25  c references 1. ansels, pharmaceutical dosage forms and drug delivary system (8 th ed.) , 2005, chapter6,14, p. 187,386. 2. kawashima y. and iwamoto t. , preparation and characterization of a new controlled iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 68 release ibuprofen suspension for improving suspend ability , int .j. pharm . , 1991,75 (aug 30 ), 25-35. 3. martin, a. physical pharmacy (4 th ed.),lea and febiger, philadelphia ,london , 2000 ,chapter18,p.477. 4. remingtons, the science and practice of pharmacy (20 th ed. ) , 2000,chapter 22,p.317. 5. dagan r et al . , ((bacteriologic and clinical effeciency of amoxicillin /clavulanate vs. azithromycin in acute otitis media )) pediatric infections disease journal , february 2000;19(2):95-104. 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treki , m.s.; mahdy ,m.a. and mohammed , m.s. : effect of suspending agent on the dissolution and bioavailability of ampicillin . bull. pharm . sci. assuit university 1991; 14 ( 12 ) : 612 . 25. ciullo p.a,rheological properties of magnesium aluminum silicate /xanthan gum dispersions,j.soc.cosmet.1981,32,275– 285. 26. chang,s.h.,parrott ,e.l, relationship of dissolution rate in anionic polymeric solution to viscosity ,drug development and industrial pharmacy, 1991,17(2),p.201 – 213 . 27. sulayman h.t., formulation of naproxen as a suspension dosage form. thesis for m.s.c iraqi j pharm sci, vol.21(1) 2012 azithromycin suspension 69 degree , college of pharmacy , , university of baghdad, 2005. 28. shargel l. , applied biopharmaceutics and pharmacokinetics, (5 th ed.),2005, chapter 14, p.419. 29. shah m.b. and sheth b.b., effect of polymers on dissolution from drug suspension , .j.pharm .sci.1979,65(11),1618 – 1623. 30. martin dale , the extra pharmacopeia , 1999; 32 nd ed. , p 1122. 31. national committee for clinical laboratory standards. performance standards for antimicrobial disk susceptibility tests. eighth edition. approved standard nccls document m2 – a8 (isbn1 – 56238 – 485 – 6 ).nccls,940 west valley road, suite 1400, wayne ,pennsylvania 19087 – 1898 usa,2003. 32. hoogrheide , j.g. and wyka , b.e. : analytical profiles of drug substances . vol .п by fory , k.p., 1982 ; pp. (37 – 42 ) , ( 116 – 135 ). 33. walter lund : the pharmaceutical codex (12 th ed .) : principle and pratice of pharmaceutics . london, 1994; pp. (733 – 739) , 811 , 812 , 813 . iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 35 synthesis of new derivatives of ceftazidime as possible prodrugs shakir m. alwan *,1 and abdul-hafeedh h. abdul-wahab * * department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad,iraq. abstract five new ceftazidime derivatives were designed and synthesized in an attempt to improve the acid stability and may increase the spectrum of ceftazidime. the synthesized compounds included; schiff base of ceftazidime (compound 1), ceftazidime lysine amide schiff base (compound 2), ceftazidime lysine amide (compound 3), ceftazidime-di-lysine amide schiff base (compound 4) and ceftazidime-dilysine amide (compound 5). new ceftazidime derivatives were successfully prepared characterized and identified using spectral and elemental microanalysis (chns) analyses and the results comply with the calculated measurements. compounds 1 and 2 were subjected to a stability study in phosphate buffer (0.2m, ph 7.4) and in kcl/hcl buffer (0.2m, ph 1.2) at different time intervals (0 – 240 min) incubated at 37 °c. this revealed that both compounds in phosphate buffer (0.2m, ph 7.4) are significantly stable with t 1/2 of 18hrs and 24hrs respectively. however, compounds 1 and 2 are less stable in kcl/hcl buffer (0.2m, ph 1.2) with t1/2 of 3.48hrs and 3.13hrs respectively. the antibacterial evaluation of the new ceftazidime derivatives showed various degrees of antibacterial activities when compared with ceftazidime. the chemical modifications of ceftazidime showed slight effect on activities and most of compounds retained the antibacterial activities. compounds 2 and 4 afforded comparable antibacterial action. however, compounds 3 and 5 were equipotent with ceftazidime with respect to e.coli and staph. aureose. compound 4 has better activity than ceftazidime with respect to pseudomonas aeruginosa. schiff's base derivative of lysine (2, 6-bis(benzylideneamino) hexanoic acid) gave a reasonable antibacterial action towards escherichia coli and streptococcus spp; as compared with lysine which has no antibacterial activity. key words: ceftazidime, schiff bases, lysine. تخليق ودراست الفعاليت البكتيريت لمشتقاث جذيذة لعقار السفتازديم شاكر محمود علوان *،1 و عبذ الحفيظ حميذ عبذ الوهاب * * فزع انكًٍٍاء انظٍذالٍَت ،كهٍت انظٍذنت ،جايؼت بغذاد، بغذاد ، انؼزاق . الخالصة حى حظًٍى ٔححضٍز خًست يشخماث جذٌذة نؼمار انسفخاسٌذٌى ححٕي ػهى لٕاػذ شف ٔلٕاػذ شف نالسٍٍ ٔثُائً انالٌسٍٍ فً ج انًزكباث انًحضزة لٕاػذ شف نهؼمار يحأنت نشٌادة انفؼانٍت انبإٌنٕجٍت ٔححضٍز يشخماث حؼطى ػٍ طزٌك انفى. حضًُ -ثُائً االٌسٍٍ-( ٔسفخاسٌذٌى3( ٔسفخاسٌذٌى الٌسٍٍ اياٌذ )يزكب 2( ٔسفخاسٌذٌى الٌسٍٍ اياٌذ لاػذة شف )يزكب 1انسفخخاسٌذٌى)يزكب ٍ انخابؼت (. حضزث انًشخماث انجذٌذة بٕاسطت حفاػم يجًٕػت االي5ٍثُائً انالٌسٍٍ )يزكب –( ٔسفخاسٌذٌى 4لٕاػذ شف )يزكب ( .حضًٍ انبحث كذنك حفاػم انالٌسٍٍ يجًٕػخً 1نًجًٕػت االيٍُٕثٍاسٔل ٔانؼمار انًذكٕر يغ انبُشنذْاٌذ نخكٌٍٕ لٕاػذ شف )يزكب االيٍٍ االحادي يغ انبُشنذْاٌذ نخكٌٍٕ لٕاػذ شف نالٌسٍٍ ٔانذي حى ربطّ بًجًٕػت االيٍٍ انخابؼت نأليٍُٕثٍاسٔل بٕاسطت آطزة أياٌذ ( بٕاسطت ححهم 3(. كًا حى ححضٍز سفخاسدٌى انًزحبظ بانالٌسٍٍ )يزكب 2ٌٍ لٕاػذ شف نالٌسٍٍ انًزحبطت بانسفخاسدٌى )يزكب نخكٕ يجًٕػت لٕاػذ شف بٕاسطت حايض انٍٓذرٔكهٕرٌك. كًا حى ححضٍز سفخاسدٌى يزحبظ بثُائً انالٌسٍٍ انحأي ػهى لٕاػذ انشف بٕاسطت حايض 4. ٔبطزٌمت يشابٓت اجزٌج ػًهٍت ححهم نًجًٕػت لٕاػذ شف نًزكب (4بٕاسطت اطزة االياٌذ نخكٌٍٕ )يزكب ( .5انٍٓذرٔكهٕرٌك نخكٌٍٕ )يزكب حى ححضٍز انًزكباث بشكم َاجح ٔحى حشخٍض انخزكٍبّ انكًٍٍأٌت نهًزكباث انجذٌذة بٕاسطت انطزق انطٍفٍت ٔححهٍم انؼُاطز ٔدرجت فً يحانٍم انبفز 2ٔ 1فٍا انزلائك ٔجاءث انُخائج يطابمت نهخزكٍبت انًمخزحت. انًزكبٍٍ االَظٓار ٔانخحهٍم بٕاسطت كزٔياحٕكزا 254ٌ.و. بانخخابغ ٔانخً حخخهف ػٍ درجت ايخظاص انسفخاسٌذٌى) 222ٔ 222انفٕسفاث ٔانبٕحاسٍى اظٓزٔا درجت ايخظاص فً ٔكذنك يحهٕل كهٕرٌذ انذارئت (m, ph 7.4 0.27ٕو )( فً يحانٍم بفز فٕسفاث انظٕد2ٌٔ 1ٌ.و.(, درجت ثباث انًزكباٌ ) (. اظٓزث انذراست درجت ثباحٍت ٔاضحت ٔيؼمٕنت بانُسبت نهًزكبٍٍ اػالِ فً 0.2m ph 7.4انبٕحاسٍٕو ٔحايض انٍٓذرٔكهٕرٌك ) درجت 32ٔدرجت حزارة دلٍمت( 240-0يحهٕل انفٕسفاث ٔكاَج درجت ثباحٍت انًزكبٍٍ الم بانُسبت نهًحهٕل االخزنفخزاث سيٍُت ) يؤٌت.كًا حى حمٍٍى انفؼانٍت انًضادة نهبكخزٌا يغ إَاع يخخهفت يٍ انبكخزٌا كًا يٕضح فً انجذأل ٔكاَج انُخائج يُاسبت، حٍث اظٓزث يؼظى انًزكباث فؼانٍت يمبٕنت ٔجٍذة يمارَت يغ ػمار انسٍفخاسٌذو. ( فكاَج يخماربت يغ 5ٔ 3ت يغ ػمار انسفخاسٌذٌى ,ايا َخائج حمٍٍى فؼانٍت انًزكبٍٍ )( يخمارب4ٔ 2كاَج َخائج حمٍٍى انفؼانٍت نهًكزبٍٍ ) ( نذٌّ فؼانٍت يثهى ضذ بكخٍزٌا 4يزكب. ).aurosestaphylococcusٔبكخٍزٌا escherichia coliانسفخاسدٌى فًٍا ٌخض بكخزٌا pseudomonasaeruginosaٌسٍٍ ٔكاَج َخائجّ يمبٕنت ٔجٍذة ضذ بكخزٌا ( انذي ًٌثم لٕاػذ شف نال 2. ايا) انًزكب escherichia coli ٔstreptococcus pyogene ٍْٔذا افضم بكثٍز يٍ انالٌسٍٍ نٕحذِ انذي نٍس نّ فؼانٍّ ضذ ْذِ االَٕاع ي انبكخزٌا. . الاليسيه ، قواعذ شف، السفتازيذيم الكلماث المفتاحيت: 1 corresponding author e-mail:shakmawales@yahoo.co.uk received:28/1/2013 accepted:22/9/2013 iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 36 introduction chemical modifications of the c-3 carboxyl group and the c-7 amino group in the cephem nucleus produce various semisynthetic cephalosporins that have different antibacterial spectra, β-lactamase sensitivity/resistance and pharmacokinetic properties (1) . most of the known cephalosporins are prepared for parental administration, and still there is a great need for parental cephalosporin that has βlactamase-resistant and anti-pseudomonal activity (2) . oral activity conferred by the phenylglycyl substituent is attributed to increased acid stability of the βlactam ring, resulting from the presence of a protonated amino group on the c-7 acylamino portion of the molecules. carrier mediated transport of the dipeptide-like and zwitterion cephalosporin are important factors in achieving oral absorption (3) . a series of monofunctional and bifunctionalprodrugs of cefizoxime was synthesized and shown to have improved physicochemical properties and oral absorption (4) . ceftazidime is a semisynthetic, β-lactam antibiotic containing a pyridinium ring and used for parenteral administration. ceftazidime is stable to most β-lactamases produced by g (+) and g (-) organisms, and consequently is active against many ampicillin and cephalothin resistant strains, except methicillin resistant strains (5) . schiff bases have been shown to exhibit a wide range of biological activities including antimicrobial 6,7) , anti-inflammatory (8,9) , analgesic (9) , anti-tubercular (10-12) , antimycobecterial (13,14) , antioxidant (15) , antiviral (16) and enzyme inhibitors (17) . lysine based prodrugs are widely used for different purposes, such as targeting certain drugs to a specific site and improve pharmacokinetic properties. l-lysine linked with amphetamine was found useful for treatment of attention deficit hyperactivity disorder (18) . l-lysine was linked with aminoflavon through an amide bond in order to improve pharmacokinetic properties affording marked antitumor effect (19) . a new series of 8-aminoquinolons derivatives prepared by conjugation with amino acids, dipeptides and pseudo-dipeptide have been synthesized and showed promising cytotoxic, anti-leshmenial and broad spectrum of antifungal activities (20) . l-lysine linked to cefizoxime resulted in significant improvement in oral absorption (4) . a prodrug of cephalosporin (rwj-333441) was prepared by linking lysine, through an amide bond, resulted in an improvement in aqueous solubility that is useful for parenteral administration (21) . di/tripeptides and peptidelike drugs are absorbed across the brushborder membrane (bbm) by h + coupled peptide transporter (pept1), intracellular peptides and peptide-like drugs exit from cells across the basolateral membrane (blm) by an unidentified peptide transporter (22). in an attempt to produce new derivatives of ceftazidime that may have broader spectrum of activities, acid stable and could be used orally, a new series was designed and synthesized, as new schiff bases of ceftazidime and ceftazidime linked l-lysine schiff bases and ceftazidime-linked di-lysine schiff bases. ceftazidime is also designed to be linked with lysine and di-lysine through amide bonds and these derivatives may target the amino acid and dipeptide transport systems and may provide an approach of producing an oral ceftazidime preparation. experimental general melting points were determined (uncorrected) by using electrical melting point apparatus, electro-thermal 9300, usa. recording of the infrared spectra were performed in kbr disk using ftir spectrophotometer/shimadzu. elemental micro-analyses were performed by eurovector ea 3000a. uv spectra were recorded by uv spectrophotometer type analytikjenaspecord 40/ germany. the ascending tlc was run on silica gel f-254 (type 60) pre-coated aluminum sheets, for checking the purity of the products as well as monitoring the progress of the reaction. chemical synthesis five derivatives of ceftazidime (compounds 1-5) were designed and synthesized as schiff bases of ceftazidime, schiff bases of l-lysine linked to ceftazidime, l-lysine and di-lysine linked to ceftazidime that may improve pharmacokinetic properties and few could be applied for oral administration of ceftazidime and may broaden the antibacterial spectrum. benzaldehyde was used to prepare the schiff bases. synthesis of a new schiff base of ceftazidime sodium (compound 1) disodium mono(7-((z)-2-(2-((e)benzylideneamino)thiazol – 4 – yl) – 2-(2carboxylato propan – 2 – yl – oxyimino) -8oxo-3-(pyridinium – yl – methyl) – 5-thia-1azabicyclo(4.2.0) oct –ene – carboxylate).bicarbonate. this derivative was prepared by reaction of ceftazidime sodium and benzaldehyde in methanol at 60 °c (23) . iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 37 benzaldehyde (1.56 mmol, 0.18g) in methanol (10 ml) was added to ceftazidime (1.56 mmol, 1g) dissolved in methanol (100 ml) and to the above solution tea (1.56nmol, 0.48ml) was added. the mixture was refluxed for 6 hrs and was then acidified with (0.1n) hcl and filtered to remove the unreacted ceftazidime. the precipitate was washed with (2x30 ml) of distilled water and was dissolved in a solution of sodium bicarbonate solution (5%, 20ml) and dry acetone (60ml) was added and the solution was placed in a refrigerator to precipitate the product as yellowish-brown sodium salt of the schiff base of ceftazidime. the physical appearance, percent yield and rf values are listed on table 1. the ir characteristic bands (ѵ, cm -1 ) are recorded as; 3191 (n-h stretching of secondary amid), 1745 (carbonyl of β-lactam), 1647 (carbonyl stretching of carboxylate) and 1585 (c=n stretching). elemental microanalysis of compound 1 was listed on table 2. synthesis of schiff base of lysine, 2, 6-bis (benzylidene amino) hexanoic acid (2a) the two aliphatic amino groups of lysine were reacted with benzaldehyde to form schiff' bases (24) . schiff' bases were collected as solids and characterized by chemical and spectral analysis. lysine mono hydrochloride (26.3 mmol, 4.8g) was dissolved in distilled water (35ml) and naoh (26.3 mmol, 1g) was added and the mixture was cooled in an ice bath. benzaldehyde (52.6 mmol, 5.6 g) was added drop wise and the mixture was stirred for 2 hrs at room temperature. the obtained solid was excessively washed with distilled water and was recrystallized from methanol: ether (1:4) to afford a white needle-like crystals of the lysine schiff' bases (2a). the physical appearance, percent yield and rf values are listed on table (1). the ir characteristic bands (ѵ, cm -1 ); 3251 (o-h stretching of carboxylic acid), 3082, 3028 (ch aromatic stretching), 1703 (carbonyl group of carboxylic acid) and 1649 (c=n stretching). synthesis of new schiff bases of ceftazidimelysineamide disodium (compound 2) disodium mono (7-((z)-2-(2-(2,6-bis((e)benzylidene amino) hexaneamido)thiazol-4yl)-2-(2-carboxylato propan-2-yloximino)acetamido)-8-oxo-3-(pyridinum-1ylmethyl) -5-thia-1-azabicyclo(4.2.0)oct-2ene carboxylate) bicarbonate. the synthesis of compound (2) was achieved by reaction of compound 2a (lysine schiff base) and ceftazidime by the mixed anhydride method (25) , and as stated below. schiff base of lysine 2a (3.41mmol, 1.098gm) was suspended in (75 ml) of a mixture of dry acetone and dmf (1:2). to the above suspension, tea (3.41mmol, 0.48ml) was added and the mixture was placed in an ice bath at (-5 to -10c°). a solution of ethylchloroformate, ecf (3.41mmol 0.33ml) was added drop wise over a period of 10 min with continuous stirring, which was continued for further 30 min at room temerature. ceftazidime (3.41mmol, 2.171gm) was dissolved in distilled water (20ml) containing tea (3.41mmol, 048ml) and the solution was cooled to (0°c) and was added at once to the above mixture containing ecf with vigorous stirring for 4 hrs. the solvent was then evaporated and the resultant precipitate was washed with (0.1n) hcl. the suspension was filtered and the precipitate was collected and washed with ethanol (99.5%) to afford the hydrochloride salt. this was re-dissolved in a solution of sodium bicarbonate (25ml, 5%) and the ph of the solution was adjusted to 8. a cold dry acetone (60 ml) was added and the solution was placed in a refrigerator to precipitate compound 2 as sodium salt. the physical appearance, percent yield and rf values are listed on table (1). the ir characteristic bands (ѵ, cm -1 ); 3292 (n-h amide stretching), 1750 (carbonyl group of βlactam), 1660 (carbonyl group of carboxylate), 1620 (c=n stretching of imine) and 1573 (stretching of carbonyl of carboxylate anion). elemental microanalysis of compound 1 is listed on table 2. synthesis of ceftazidime-lysineamide disodium (compound 3) disodium mono (7-((z)-2-(2carboxylatopropan-2-yloxyimino) 2(2)-2,6diaminohexane amido) thiazol-4-yl) acetamido)-8-oxo-3-(pyridinuim-1-ylmethyl)5-thia-1-aza bicyclo (4,2,0)oct-2-ene-2carboxylate)bicarbonate. this compound was obtained by cleavage of the schiff' bases of compound 2 by reaction with hydrochloric acid solution at ph 2 incubated at 5-10˚c (26) , as shown in scheme (2). compound 2 was dissolved in distilled water (20ml), placed in an ice bath at (10°c) and a cold solution of hcl (1n, 10ml) was added drop wise to adjust the ph to 2 and the mixture was stirred for 4hrs. cold acetone (40 ml) was added to precipitate compound 3 and the precipitate was collected and washed with ethanol to afford a fine white powder of the hydrochloride salt. the ir characteristic bands (ѵ, cm -1 ); 3325, 3273 (n-h stretching iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 38 of primary aliphatic amine), 3197 n-h (stretching of secondary amide), 1758 (carbonyl group of β-lactam), 1665 (carbonyl group of carboxylate anion) and 1614 (n=c stretching of imine). elemental microanalysis of compound 3 is listed on table 2. synthesis of ceftazidime-dilysineamideschiff bases di sodium (compound 4) disodium mono (7-((z)-2-((2-(2-((e)benzylideneamino)-(hexanamido) hexanamido) thiazol-4-yl)-2-(carboxylate propan-2-yl oxyimino) acetamido)-8-oxo-3(pyridinum-1-ylmethyl)-5-azabicyclo (4,2,0)oct-2-ene-2-carboxylate) bicarbonate. the synthesis of this compound was achieved by reaction of compound 2a with compound 3 using the following procedure, as shown in scheme (2).schiff base of lysine 2a (3.41mmol, 1.098gm) was suspended in a mixture (75 ml) of dry acetone and dmf (1:2), and to the above mixture tea (3.41mmol, 0.48ml) was added and placed in an ice bath at (-5 to -10°c). a solution of ecf (3.41mmol 0.33ml) was added over a period of 10 min and the mixture was continuously stirred for further 30 min at room temperature. compound 3 (1.705mmol, 1.226gm) was dissolved in distilled water (20ml) containing tea (1.705mmol, 0.238ml) and was cooled to 0 °c and added at once to the solution of ecf-schiff base of lysine and the mixture was vigorously stirred for 4 hrs. the solvent was evaporated and the resultant precipitate was suspended with diluted hcl and was filtered and the precipitate was collected as the hydrochloride salt, which was washed with ethanol. the precipitate was dissolved in a solution of sodium bicarbonate (5%, 15ml) and the ph of the solution was adjusted to 8. a cold dry acetone (60 ml) was added and the mixture was placed in a refrigerator to precipitate compound 4, as sodium salt. the physical appearance, percent yield and rf value are listed on table (1). the ir characteristic bands (ѵ, cm -1 ); 3327 (n-h stretching vibration), 1758 (carbonyl group β-lactam), 1678 (carbonyl group stretching of carboxylate anion) and 1626 (c=n stretching of imine). elemental microanalysis of compound 3 is listed on table 2. synthesis of ceftazidime-di-lysineamide di sodium (compound 5) disodium mono(7-((z)-2-(2-(r-2,6-bis(2,6diaminohexanamido)-thiazol-4-yl)-2(carboxylate-propan-2-yloxyimino)acetamido)-8-oxo-3-(pyridinium-1yl-methyl)-5-thia-1-azabicyclo(4,2,0)oct-2carboxylate) bicarbonate. this derivative was prepared by reaction of compound 4 with hydrochloric acid solution at ph 2 and the mixture was incubated at 5-10 o c. compound 4 (1.08 mmol, 1.5gm) was dissolved in distilled water (20ml) and placed in an ice bath at 510°c. a cold solution of hydrochloric acid (1n, 10ml) was added drop wise and the ph was adjusted to 2. the mixture was stirred for 4 hrs and was filtered and the product was separated as the dihydrochloride salt in the filtrate. compound 5 as the dihydrochloride salt was neutralized by sodium bicarbonate (5%) and the ph of the solution was adjusted to 8. a cold dry acetone (60ml) was added to precipitate compound 5 as the sodium salt. the physical appearance, percent yield and rf value are listed on table (1). the ir characteristic bands (ѵ, cm -1 ); 3329, 3226 (nh stretching of primary aliphatic amine), 3194 (n-h stretching of amide), 1759 (carbonyl group of β-lactam) and 1622 (c=n stretching of imine). elemental microanalysis of compound 3 is listed on table 2. c h o methanol tea 12nahco3 reflux hco3. n+s n o h n o n oo s n nh2 o oh on+s n o h n o n o o s n n o oona+ na+ 5% scheme 1. synthesis of schiff base of ceftazidime sodium (compound 1). iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 39 o ho n n ecf,1dmf, acetone tea 2nahco3 hco3 . n+s n o h n o n oo oh s n h2n o o na+ na+ n+ s n o hn on o o os n h n o oo n n 5% hcl 5-10 c nahco312hco3. n+ s n o n h o n o o s n h n o o h2n nh2 +na-o +na-o 5% compound 1 compound 2 scheme 2. synthesis of compounds 2 and 3. iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 40 noh n n+ s n n h o n o o n s n h o h2n h2n o o -o ona+ na+ 1dmf, acetone 2nahco3 ecf, tea .hco3 hco3. hn o n o o n sh n o hn n h o n n o n n +na-o n s o o-na+ o n+ 5% hcl 5-10 c nahco3125% compound 4 compound 5 hco 3 . n+ s n o hn on o o os n h n o oo nhn h o h2n nh2 o h2n h2n na+ na+ scheme 3. synthesis of compounds 4 and 5. iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 41 noh n n + s n n h o n o o n s n h o h2n h2n o o -o ona + na+ 1dmf, acetone 2nahco3 ecf, tea .hco 3 hco 3. hn o n o o n sh n o hn n h o n n o n n + na o n s o o na + o n+ 5% scheme 4. synthesis of compound 4. results and discussion the ir characteristic bands shown in the spectra of compounds were used to identify and confirm their structures depending on the appearance and disappearance of certain chemical groups and consequently their bands (27, 28) . the ir spectra of the synthesized compounds showed characteristic bands of absorption, which were consistence with the proposed structures of the compounds. the ir spectrum of compound 1 (schiff base of ceftazidime) showed characteristic bands and are previously listed. the disappearance of the amine band of the aminothiazole moiety in the ir spectra gives a good indication for the formation of the newly synthesized amide bond (29) . the newly synthesized compounds (1,2 and 4) contain an extra imine bond with regard to the already existing imines, as shown clearly in their chemical structures. however, the presence of such number of imines resulted in very close bands in the ir spectra of these compounds. the ir characteristic bands for compound 2a (schiff base of lysine) showed imine group at 1649 cm -1 , which don’t exist in lysine and at the same time, disappearance of the primary amine group in the region 34003300 cm -1 . ceftazidime contain a primary aromatic amine of the aminothiazole moiety which appears at 3500-3400 cm -1 , while, compounds 3 and 5 contain primary aliphatic amine groups and their ir absorption bands appear at (3300-3200cm -1 ). the ir spectra of (c=o) of carboxylic acid of ceftazidime, which appeared at 1705 cm -1 , while the asymmetric vibration of (coo-) of these compounds appeared at (1647-1678 cm -1 ). elemental microanalyses were performed for the target compounds (1-5) to confirm their basic chemical formulas. the results were presented on table (2). the percent deviations of the observed to the calculated values were found to be acceptable and within the range of 0.4%. stability of compounds 1 and 2 in aqueous buffer solutions uv spectra of the aqueous solutions of the sodium salts of the parent and the new derivatives of ceftazidime were recorded on a double-beam uv-spectrophotometer and their λmax were determined. the λmax of ceftazidimepentahydrate is at 254nm, while λmax of ceftazidime schiff base (compound 1) is at 299 nm. λmax of ceftazidimelysine schiff base (compound 2) is at 277nm. under the experimental conditions (30), the behavior of the hydrolysis of compounds 1 and 2 follow pseudo-first order kinetic, since plot of log concentration vs. time resulted in a straight line and from the slope of this plot, the observed rate constant of hydrolysis was calculated. the degree of hydrolysis of compound 1(28 g ml) in cl cl buffer (0.2 ,p 1.2) and in phosphate buffer (0.2 , p 7.4) incubated at 37 c was studied at different time intervals (zero, 15, 30, 60,120 and 240 min).the half-life values were calculated from the pseudo-first order kinetic iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 42 law and found to be t½ 3.48hrs and 18.78hrs respectively. a similar procedure was conducted to study the stability of ceftazidime-lysine schiff base (compound 2) in kcl/hcl buffer (0.2m,ph 1.2) and in phosphate buffer (0.2m, ph 7.4) and the t½ values were calculated using the pseudo-first order kinetic law and the values equal to 3.13 hrs and 24hrs respectively. accordingly, the above studies have indicated significant increase in the values of t1/2 (18.78hrs) of compound 1 at slightly basic media (ph 7.4), which is much longer than the t1/2 of ceftazidime (2hr) (31) . however, the stability of compound 1 in kcl/hcl buffer (0.2m, ph 1.2) showed that thet1/2 was 3.48hrs compared with ceftazidime (2hr) (31) . compound 2 has a t1/2 value of 24hrs when incubated in phosphate buffer (0.2m, ph 7.4), which is a clear significant result that indicate a great stability of compound 2 at the slightly basic conditions (resembles the physiological ph). however, the stability of compound 2 in kcl/hcl buffer (0.2m, ph 1.2) was slightly better and reaching 3hrs, when compared with ceftazidime (31). table (1) physical parameters and percent yield of the synthesizedcompounds compound physical appearance yield % m.p. o c rf value 1 brown to yellow powder 70 165 decomposed 0.75 ( a ) 2a white powder 50 195-197 0. 69 (b ) 2 pale brown powder 68 210 decomposed 0.62 (b ) 3 white to pale yellow powder 52 266 decomposed 0.68 ( b ) 4 brown powder 58 225 decomposed 0.65 (a ) 5 pale yellow powder 50 284 decomposed 0.62 ( b ) table (2) elemental microanalysis of the synthesized compounds. elemental microanalysis % chemical formula compounds found calculated element molecular weight 48.57 48.65 c c30h26n6na2o10s2 740.09 1 3.46 3.54 h 11.24 11.35 n 8.88 8.66 s 53.36 53.97 c c43h42n8na2o11s2 956.95 2 4.57 4.42 h 11.84 11.71 n 6.92 6.70 s 44.79 44.61 c c29h34n8na2o11s2 780.74 3 4.50 4.39 h 14.10 14.35 n 8.48 8.21 s 59.48 59.64 c c69h74n12na2o13s2 1389.51 4 5.57 5.37 h 12.45 12.10 n 4.38 4.62 s 47.59 47.48 c c41h58n12na2o13s2 1037.08 5 5.49 5.64 h 16.55 16.21 n 6.37 6.18 s iraqi j pharm sci, vol.22(2) 2013 synthesis of new derivative of ceftazidime 43 antimicrobial activity assessment the newly synthesized derivatives were tested for their antimicrobial activity by discdiffusion method (32) against the following microorganisms: (a) gram-negative: escherichia coli and pseudomonas aeruginosa (b) gram-positive: streptococcus spp and staphylococcusaureus. type of media used: (nutrient medium), which contain 1g/l distilled water, peptone (5gm) and meat extract (3gm), and the ph was adjusted to 7.0. each of the synthesized compounds (30 g) was dissolved in dimethylsulphoxide to prepare the test solutions. ceftazidime (30 g) was used as the standard antibacterial drug. dimethylsulphoxide: water mixture (1:30) was used as the solvent. the results are shown on table (3). table (3) antimicrobial activity evaluation of the synthesized compounds. compounds s. pyogen. s. aureus. e. coli p. aeruginosa dmso ceftazidime + ++ ++ 1 + + ++ ++ 2 ++ ++ ++ ++ 3 + +++ ++ 4 ++ + +++ +++ 5 + ++ + 2a + + key to symbols: (-) = no inhibition, (14 mm) = +, (15-17 mm) = + + (more than 18 mm) = +++. the antimicrobial screening revealed that the newly synthesized compounds (2 and 4) showed reasonable antibacterial activities against g (+) strep. spp. in comparison with ceftazidime, which has no activity against this type of microorganism. compound 2 showed good activity against all 4 strains of bacteria used, as compared with ceftazidime. compound 4 showed good and reasonable activities against e. coli and p. aeruginosa comparable with ceftazidime. while, it showed good activity against strep. spp. and moderate activity against staph. aureus. compound 1 showed moderate activity against strep. spp. and staph spp. and good activity against e. coli and p. aeruginosa. compound 3 showed good and reasonable activity against e. coli and p. aeruginosa and moderately active towards staph. spp. however, compound 3 showed no activity against strep. spp. compound 5 showed good activity against e. coli and moderate activity against p. aeruginosa. generally, all the schiff bases of ceftazidime (compounds 1, 2 and 4) showed good and reasonable antimicrobial activity against the tested microorganisms especially g (+) bacteria. this increase in activity may be due to the incorporation of extra imine groups. the slight reduction in antibacterial activities of some of these derivatives (compounds 3 and 5) as compared with ceftazidime is observed, but these may still have potential to be used as therapeutic agents. compounds 3 and 5 that devoid of the extra imine groups and contain two primary amine groups have moderate to slight activities as compared to ceftazidime. compounds 2 and 4 showed broader antibacterial spectrum against both g (+) and g (-) bacteria. the schiff bases of lysine (2, 6-bis (benzylideneamino)) hexanoic acid (compound 2a) exhibit slight activities towards e. coli and strep. spp, as compared with lysine which has no antibacterial activity. however, compound 2a showed no antimicrobial activities against s. aureus or p. aeruginosa. references 1. roger g. 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spectrometric identification of organic compounds, john wiley and sons inc., (7 th ed.), new york, 2007; 72-108. 28. shriner, r.l., hermann, c.k.f., morrill, t.c., curtin, d.y. and fuson, r.c.: "infrared spectrometry". in, the systemic identification of organic compounds, (8th ed.), john wiley and sons inc, new york, 2004; 194-225. 29. hiroyuki, m., m. kasai, s. h., ken-ichi nishimura, s. nishizawa, kakeya, n., cephalosporin compounds: us patent, 5389625a, 1995; pub. no. wo91/06549. 30. buffer solutions, in: british pharmacopoeia, (5 th ed.), 2007;245-276. 31. paradis. d. et al; comparative study of pharmacokinetics and serum bactericidal activity of cefepenem, ceftazidime, ceftriaxone, imipenem and ciprofloxacin, antimicrobial agent chemotherapy. 1992;36(10) :2085-92. 32. finegold, s. m., martin, w. j., diagnostic microbiology, 6 th -ed., mosby london, 1982;450-458. iraqi j pharm sci, vol.31( 2 ) 2022 coenzyme q10 supplement for managing diabetic neuropathy doi: https://doi.org/10.31351/vol31iss2pp177-183 177 potential positive effects using coenzyme q10 supplement as adjuvant therapy to gabapentin for managing diabetic neuropathy rusul abdulkareem hadi *1, mohammed mahmood mohammed ** and isam noori salman *** *children welfare teaching hospital, medical city, ministry of health and environment, baghdad, iraq. ** department of clinical pharmacy, college of pharmacy, mustansiryah university, baghdad, iraq. *** consultant physician, national diabetes center for treatment and researches, mustansiryah university, baghdad, iraq. abstract the most prevalent chronic complication of diabetes mellitus is diabetic neuropathy. the pathogenesis of diabetic neuropathy is exacerbated by hyperglycemia-induced oxidative stress, which causes nerves to deteriorate in a programmed manner. many clinical trials depend on supplement in an attempt to improve neuropathy symptoms such as (pain & tingling) and patient quality of life, one of them is coenzyme q10 which is reported to have an antiinflammatory and antioxidant effects, and was totally nontoxic and non-reported side effects. this study aimed to evaluate using a coenzyme q10 supplement as an adjuvant therapy to gabapentin to improve the clinical symptoms of diabetic neuropathy in relation to its anti-inflammatory and antioxidant effects. this open-label interventional study involved 33 diabetic neuropathy patients divided into two groups: group (1) 16 patients were given 300 mg of gabapentin once a day at evening, plus group (2) 17 patients received 300 mg of gabapentin once a day in the evening plus coenzyme q10 200mg once daily. preand post-3 months of treatment, blood samples used to measure metabolic, antiinflammatory and antioxidant biomarkers (fasting blood glucose, glycated hemoglobin, tumor necrosis factor-α, iinterleukin-6 & superoxide dismutase) , as well as the michigan neuropathy screening instrument for assessment of clinical symptoms. after 3 months of coenzyme q10 use, the results showed that the group 2 produced a highly significant change in glycated hemoglobin & fasting blood glucose levels. meanwhile, there is no significant change in glycated hemoglobin & fasting blood glucose values in patients receiving just gabapentin. moreover, results showed highly significant differences in michigan neuropathy screening instrument, tumor necrosis factor-α, iinterleukin-6 & superoxide dismutase between the study groups at the completion of the research. finally, addition of coenzyme q10 to gabapentin for diabetic neuropathy patients result in improving the glycemic control & symptoms of the diabetic neuropathy, as well as decreasing effects of the inflammation in addition to oxidative stress after three months of treatment. keywords: diabetic neuropathy, coenzyme q10, gabapentin, neuropathy symptoms. كعالج coq10 لألكسدة لمكمل اإلنزيم المساعد ةالتأثيرات المحتملة المضادة لاللتهابات والمضاد للكابابينتين في مرضى اعتالل األعصاب السكري مساعد *** عصام نوري سلمان ** محمد محمود محمد 1،*رسل عبد الكريم هادي العراق ، بغداد، وزارة الصحة والبيئة ، مدينة الطب، مستشفى حماية األطفال التعليمي * العراق ، بغدادالجامعة المستنصرية/ ، كلية الصيدلة، فرع الصيدلة السريرية** العراق، بغداد، الجامعة المستنصرية ، المركز الوطني لعالج وبحوث السكري *** الخالصة: اعتالل األعصاب السكري هو أكثر المضاعفات المزمنة شيوًعا لمرض السكري. يؤدي اإلجهاد التأكسدي الناجم عن ارتفاع السكر في الدم إلى مما يساهم في أمراض االعتالل العصبي السكري. تعتمد العديد من التجارب السريرية على المكمالت في محاولة لتحسين لألعصاب ، هموت الخاليا المبرمج ه تأثيرات مضادة لاللتهابات لالذي ورد أن و coq10 المريض ،أحدها هو اإلنزيم المساعد أعراض االعتالل العصبي مثل )األلم والوخز( ونوعية حياة ن يلتحس مساعد لجابابنتينكعالج coq10ستخدام تهدف هذه الدراسة إلى تقييم ا. جانبيةالثار خالي من اآلسام وليس لديه اي تأثير ، و تماًما ومضادة لألكسدة لمعلومة التسميةاتداخلية ال دراسةشملت هذه ال .ألكسدةلمضاد الالمضاد لاللتهابات ووعالقتها بتأثير الدواء عتالل األعصاب السكري ال هاألعراض السريري مجم مرة واحدة يومياً في 300مريضاً تناولوا جابابنتين 16 (1)مريًضا يعانون من اعتالل األعصاب السكري تم تقسيمهم إلى مجموعتين. المجموعة 33 3مجم مرة واحدة يومياً. قبل وبعد coq10 200 مجم مرة واحدة يومياً في الليل باإلضافة إلى 300 مريضاً تناول جابابنتين 17 (2)الليل ، والمجموعة tnf-α و hba1c و (fbg ,ومضادات االلتهاب ومضادات األكسدة التمثيل الغذائي مؤشرات دم لقياسومصل ال أشهر من العالج ، استخدمت عينات أظهرت النتائج أن مجموعة co q10 استخدامأشهر من 3بعد السريرية.أعراض لتقييم شدة (mnsi) غانميشياختبار ، باإلضافة إلى sod )و il-6 و وفي الوقت نفسه ، ال يوجد تغيير كبير في مستويات co q10 أشهر من مكمالت 3بعد fbg و hba1c أنتجت تغيًرا مهًما للغاية في مستويات 2 hba1c & fbg عالية في معنويهذات داللة تاثيرعلى ذلك ، أظهرت النتائج عالوة وحده. لخدام جابابنتين في المرضى الذين عولجوا باست mnsi و tnf-α و il-6 و sod الدراسة نهاية في الدراسة مجموعات اخيرا", بين الجابابنتين coq10 مكملإضافة . العصبي إلى االعتالل مرضى في االلتهاب واإلجهاد التأكسدي بعد تاثيرفي نسبة السكر في الدم وأعراض االعتالل العصبي السكري ، باإلضافة إلى تقليل السيطرهالسكري يؤدي إلى تحسين .ثالثة أشهر من العالج . coq10 ,ل العصبيالكلمات المفتاحية: اعتالل األعصاب السكري ، الجابابنتين ، أعراض االعتال 1corresponding author e-mail: phd_pharm@yahoo.com received: 2/ 10/2021 accepted:29 /12 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp177-183 iraqi j pharm sci, vol.31(2) 2022 coenzyme q10 supplement for managing diabetic neuropathy 178 introduction diabetes mellitus (dm) would be a collection of metabolic disorders marked by the irregular metabolism of carbohydrate, fat, and protein; as a result to impaired insulin secretion or insulin action (1). according to the international diabetes federation (idf), there were roughly 425 million persons living with diabetes globally in 2017, with the number expected to rise to 629 million by 2045. (2). long-lasting hyperglycemia results in multi systemic problems of the eyes, nerves, kidneys, heart, and blood vessels. as a result of macroand micro-vessel disorders, diabetes causes high rates of morbidity and mortality (3). diabetic neuropathy, also known as peripheral nerve damage, is one of the most prevalent comorbidities of diabetes (4). it leads to an increased risk for significant functional limits and dangerous consequences, such as amputation of the legs (5). the pathologic process of diabetic neuropathy is characterized by three major changes: first, nuclear factor kappa b, activator protein 1, and mitogen-activated protein kinases are mostly activated by inflammation. second, polyol, hexosamine, protein kinase c, advanced glycosylation end-products, and glycolysis all seem to be involved in oxidative stress caused by hyperglycemia. third, the majority of reactive oxygen species are produced by mitochondrial malfunction. lipid peroxidation, protein modification, and nucleic acid damage are all caused by free radicals, which leads to axonal degeneration and segmental demyelination (6). several treatments have been assessed to minimize neuropathic deficits and enhance nerve function, which including oral anti diabetics, statins, anti-inflammatory, antioxidants, anticonvulsant, and other medications approved for neuropathy (7). coq10 is an endogenously produced molecule that serves as an electron carrier in the mitochondrial respiratory chain. coq10 would be an anti-oxidant that scavenges free radicals and inhibits lipid peroxidation, in addition to its particular role in the mitochondria (8). coq10 is shown to have therapeutic value in the treatment of diabetes and its related complications (9). serum coq10 levels in patients with diabetes are frequently low, which may be linked to subclinical diabetic cardiomyopathy that can be reversed with coq10 treatment (10). coq10 therapy increased serum coq10 values, enhanced endothelial function within brachial artery, which significantly reduced systolic and diastolic blood pressures, in addition to glycosylated hemoglobin levels (hba1c) (11). coq10 enhanced diabetic polyneuropathy nerve conduction measures and lowered oxidative stress while having minimal side effects; it decreases serum glucose levels by improving β-cells functions in addition to insulin requirements were lowered due to greater insulin sensitivity for patients with diabetes (12). these findings suggest that coq10 could be used as a supplement to treat peripheral neuropathy in people with type 2 diabetes in the future. patients and methods a prospective open-label randomizedcontrolled interventional trial designed to explore potential an anti-inflammatory & antioxidant effects of co q10 supplement as adjuvant therapy in diabetic neuropathy patients. the study was conducted from october 2017 to 31th july 2018. the study was performed on 40 iraqi patients of both genders (male & female) diagnosed with type 2 diabetic with neuropathy. all patients have been selected from the national diabetes center for treatment and research/ mustansiriya university, following approval of the scientific committee in the national diabetes center. seven patients excluded from the study due to incompliance and missed treatment dose (only 33 patients completed the study) patients were involved in the trial if they meet certain criteria: 1) adult patient 1870ـ years old. 2) mild-moderate cases diabetic neuropathy. but the following were the exclusion criteria: 1) pregnancy and breastfeeding. 2) severe case diabetic neuropathy. 3) patients with other comorbid diseases. 4) patients hypersensitive to any drug or supplement used in this study. over the course of 3 months, the employed patients were followed up on. they were divided into two groups based on the physician's treatment recommendations. (group 1): involved 16 patients treated with the conventional gabapentin capsules 300 mg as single daily dose at night. (group 2): included 17 patients treated with the conventional gabapentin capsules 300 mg as single daily dose at night plus coenzyme q10 capsules 200mg once daily for 3months. patient demographics, clinical symptoms, diabetes history, and diabetes test results (fbs, and hba1c) have all been documented. serum interleukin 6 (il6), tnf-alpha & sod were measured using elisa kits. clinical tests were carried out to determine the severity of symptoms of the dn; michigan neuropathy screening instrument (mnsi) (13). furthermore, specific quessionniare was used to assess the intensity of clinical symptoms pre and post-treatment (14). all patients were treated for 3 months, and blood samples were taken at the start of the trial (baseline values) and again three months later to assess any changes in the examined parameters. ethical consideration the scientific and ethical committee of mustansiriyah universitycollege of pharmacy gave their approval to this study. the national diabetes center for treatment and research came to an agreement. patient written agreement was iraqi j pharm sci, vol.31(2) 2022 coenzyme q10 supplement for managing diabetic neuropathy 179 obtained after a thorough description of the study's purpose, ensuring the accuracy of the data collected. statistical analysis the data were analyzed using the following software, microsoft excel, minitab v17, and spss v24. the results reported in this study were expressed as mean  sd. chi square test, paired ttest to comparing variables before and after treatment in the same group. the one-way analysis of variance (anova) was performed in order to determine and find differences among independent variables and to assess the degree of significance. probability values > (0.05) regarded not significant different, whereas p values < (0.05) were regarded significantly different, & p < (0.01) was considered highly significant different. results the table (1) summarizes the demographic information and baseline characteristics of the study participants. in this table, there were non-significant variations among patients allocated in each group regarding to age, gender, body mass index (bmi), family history, duration of diabetes, duration of neuropathy symptoms, smoking & alcohol drinking. table 1 .demographic characteristics of patients with diabetic neuropathy. demographic characters group 1 group 2 p-value© n (%) n )%( age (years) ≤ 60 7 )14) 8 16)) 0.942ns > 60 9 (18) 9 (18) gender male 8 (16) 5 (10) 0.426ns female 8 )16) 12 (24) bmi (kg/m2) < 18.5 0 (0) 0 (0) 0.087ns 17.5-25 4 (8) 0 (0) > 25 12 (24) 17 (34) family history yes 14 (28) 11 (22) 0.246 ns duration of neuropathy symptoms < 1 year 3 (6) 1 (2) 0.679 ns 1-5 years 9 (18) 12 (24) > 5 years 4 (8) 4 (8) duration of diabetes < 10 years 1 (2) 3 (6) 0.390 ns ≥ 10 years 15 (30) 14 (28) smoking yes 3 (6) 7 (14) 0.802 ns alcohol yes 0 (0) 0 (0) 1.000 ns the data is shown as (n): number of patients, and (%): percentage. ©chi square test (χ2) test for goodness of fit used to test counts between groups, ns: p value > 0.05 is considered no significant differences. as can be seen in table (2) and figure (1) the results demonstrated a significant difference (p<0.05) in michigan neuropathy screening instrument (mnsi) score between the two groups at the completion of the study. however, there was a highly significant improvement (p˂0.01) within each study group after 3 months of treatment. table 2. effect of treatment on mnsi in patients with diabetic neuropathy. group 1 group 2 a value-p mnsi score pre 11.25 ± 1.29 11.24 ± 1.09 ns0.972 post 8.81 ± 1.17 9.53 ± 0.72 * 0.045 b value-p 0.001** 0.001** data is presented in the form of mean ± sd, a independent t test used to test statistical differences between groups (horizontally), b paired t-test used to compare between pre and post within each group. ns: no significant differences (p≥0.05), (*) significant differences (p<0.05), ** highly significant differences (p<0.01). iraqi j pharm sci, vol.31(2) 2022 coenzyme q10 supplement for managing diabetic neuropathy 180 figure 1. effect of treatment on mnsi in patients with diabetic neuropathy. at the end of the trial, there were no significant variations in fbg and hba1c levels between the two groups (p≥0.05). however, patients received coenzyme q10 in addition to gabapentin group (2) demonstrated a significant and highly significant decrease in the fbg & hba1c levels, respectively. while no significant reductions were reported in group 1 for both fbg & hba1c levels at the finish of the study. table 3. effect of treatment on fbg & hba1c levels in patients with diabetic neuropathy. study groups group 1 group 2 a value-p fbg pre 225.94 ± 90.98 203.59 ± 64.87 ns0.426 post 188.69 ± 94.69 162.59 ± 53.79 ns0.344 b value-p ns 0.08 0.013* hba1 pre 9.09 ± 1.36 9.51 ± 1.17 ns0.360 post 8.73 ± 1.14 8.22 ± 0.79 ns0.129 bvalue -p ns 0.41 0.001** data presented as mean ± sd, a independent t test used to test statistical differences between groups (horizontally), b paired t-test used to compare between pre and post within each group. ns: no significant differences (p≥0.05), (*) significant differences (p<0.05), ** highly significant differences (p<0.01). figure 2. effect of treatment on fbg level in patients with diabetic neuropathy. figure 3.effect of treatment on hba1c level in patients with diabetic neuropathy. regarding to the anti-inflammatory and antioxidant effect of adding coenzyme q10 to gabapentinin treatment of diabetic neuropathy, tnf-α, il-6 and sod levels were measured. the findings of this research revealed that there are considerable variances (p<0.05) in tnf-α, il6ـ & sod levels at the end of the study between both groups. meanwhile, highly significant reductions were noticed in the levels of these three parameters after completing the study in respect to pretreatment levels, which are clearly shown in table (4) and figure (4, 5, & 6). figure 4.effect of treatment on tnf-α levels in patients with diabetic neuropathy. iraqi j pharm sci, vol.31(2) 2022 coenzyme q10 supplement for managing diabetic neuropathy 181 figure 5. effect of treatment on il-6 levels in patients with diabetic neuropathy. figure 6. effect of treatment on sod levels in patients with diabetic neuropathy. table 4. effect of treatment on tnf-α, il-6 & sod in patients with diabetic neuropathy. study groups group 1 group 2 a value-p tnf-α pre 22.26 ± 10.7 28.65 ± 10.4 0.092 ns post 10.20 ± 4.65 6.63 ± 2.42 * 0.012 b value-p ** 0.001 **0.001 il_6 pre 7.65 ± 5.04 6.93 ± 1.75 ns 0.594 post 3.05 ± 1.39 4.50 ± 1.37 * 0.002 b value -p 0.004** 0.001** sod pre 3.62 ± 1.79 4.49 ± 2.83 ns 0.295 post 1.10 ± 0.641 2.17 ± 1.63 *0.020 b value-p 0.001** 0.001** data presented as mean ± sd, a independent t test used to test statistical differences between groups (horizontally), b paired t-test used to compare between pre and post within each group. ns: no significant differences (p≥0.05), (*) significant differences (p<0.05), ** highly significant differences (p<0.01). discussion considering demographic and disease characteristic of the studied patients, the average age of the study's participants was 60.26 years. even though deals with a small sample of population, the mean of age is similar to that reported in other studies (15, 16). this is attributed to the fact that incidence of neuropathy increased with advanced age (17). the duration of time a person had diabetes was (10.43± 3.42) years old; long-term diabetics are predisposed to develop neuropathy due to prolonged exposure of peripheral nerves to hyperglycemia. the current evidence supports the involvement of glycemic management in the development and progression of dn. (18). within the present study significant (p<0.05) difference in mnsi score was noticed after 90 days of treatment between the groups. in this study, coq10 therapy provided early onset of clinically relevant pain reduction with only minimal and probably avoidable side effects. gabapentin mono therapy is a viable treatment option for people with neuropathic pain who have limited treatment options. it has advantages over currently available medications as a first-line treatment (19). these improvements could be due to gabapentin's one-of-a-kind action on calcium channels and consequently neurotransmitter release. gabapentin may be due to its anticonvulsants effects, acts predominantly on calcium channels & regulates the release of numerous neurotransmitters in the cns, which may result in gabapentin having a better influence (20). in a recent study, a clear link was shown between improved glycemic control and diabetes patients (21). many studies have revealed that coq10 has beneficial impacts on glucose homeostasis measures. schroeder et al., for example, demonstrated that coq10 as an antioxidant therapy may reduce glucose levels by protecting β cells from ros and glucose toxicity, as well as increasing insulin production (22). furthermore, coq10 reduces insulin resistance via modulating insulin as well as adiponectin receptors, along with glucose transporters, tyrosine kinase (tk), and phosphatidylinositol kinase (pi3 k), and also the redox system, soluble receptor of advanced glycated end products (srage), as well as adipocytokines (23). the current investigation found that the levels of il6ـ, tnf_α, and sod were significantly lowered iraqi j pharm sci, vol.31(2) 2022 coenzyme q10 supplement for managing diabetic neuropathy 182 compared to baseline. these effects of coq10 on cytokines are due to its anti-inflammatory properties. the exact mechanisms are unknown. however, coq10 has the ability to block the activation of the nf-kb signaling pathway as well as neutralize free radicals. (24(. in addition, coq10 has been shown to have anti-inflammatory properties through decrease the release of proinflammatory cytokines and cox-2 expression during inflammatory injury (25, 26) in the present study showed, elevation in sod enzyme activity at baseline, this suggested that diabetes individuals' superoxide radical generation may be increased as a result of oxidative stress caused by high glucose levels (27). oral coq10supplement did not result in an increased sod level, in fact it was decreased in both group rather than increased. this finding may be due to a number of reasons, the slightest variation in lipid peroxidation and antioxidant enzyme activity could be influenced by glucose management (27) & coq10, as an antioxidant and free-radical scavenger, may help to reduce oxidative stress in the central and peripheral neurological systems (28). tsai et al. found that supplementing with coq10 increased sod activity (29) and chio et al (30). conclusion addition of coq10 to gabapentin in diabetic neuropathy patients resulted in improving pain, physical activity and the glycemic control of patients, it is probably helpful in reducing severity and progression of neuropathy by reducing the impact of inflammation and oxidative stress. acknowledgment the authors would like to express their gratitude to mustansiriyah university / university of baghdad and all participants for contributing the study. references 1. gioacchini fm, albera r, re m, scarpa a, cassandro c, cassandro e. hyperglycemia and diabetes mellitus are related to vestibular organs dysfunction: truth or suggestion? a literature review. acta diabetol.2018dec;55(12):12011207. 2. n h cho, j e shaw, s karuranga, y huang, j d da rocha fernandes, a w ohlrogge, b malanda. idf diabetes atlas eighth edition 2017: global estimates of diabetes prevalence for 2017 and projections for 2045 diabres.2018.02.023. arambewela mh, somasundaram np, ranjan jayasekara hbp, kumbukage mp, jayasena pms, hemanthi 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yang yp, chiu th. a novel mechanism of coenzyme q10 protects against human endothelial cells from oxidative stress-induced injury by modulating no-related pathways. the journal of nutritional biochemistry. 2012 may 1; 23(5):458-68. 29. choi bs, song hs, kim hr, park tw, kim td, cho bj, kim cj, sim ss. effect of coenzyme q10 on cutaneous healing in skinincised mice. archives of pharmacal research. 2009 jun 1; 32(6):907-13. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ an investigation release and rheological properties of miconazole nitrate from emulgel iraqi j pharm sci, vol.18(2) 2009 miconazole nitrate emulgel 26 an investigation release and rheological properties of miconazole nitrate from emulgel lubna a.sabri *,1 , hala t. sulayman * and yehia i. khalil * * department of pharmaceutics ,college of pharmacy ,university of baghdad , baghdad , iraq abstract in this study miconazole nitrate was formulated as topically applied emulgel; different formulas were prepared using sodium carboxymethylcellulose (scmc) and carboxypolymethylene (carbomer 941) as gelling agents. the influence of type of gelling agent and concentration of both oil phase and emulsifying agent on drug release was studied and compared with commercially available miconazole nitrate cream (mecozalen ® ). the results of in vitro release showed that scmc emulgel bases gave better release than carbomer 941 bases and the release of drug increase from both bases as a function of increasing the concentration of emulisifying agent. the oil phase had retardation effect when its concentration increased. the formula which showed the highest drug release was chosen to evaluate its rheology and its stability .the rheological behavior of selected formula showed share-thinning flow indicating structural break down of intermolecular interaction between polymeric chains. moreover, the expiration date of the selected emulgel was found to be 1.7 year as well as their physical properties like, color, ph and consistency remains constant along storage time. keywords: emulgel, carbomer, miconazole nitrate, carboxymethyl cellulose. الخالصة ذٓذف ْذِ انذراسح انٗ صٛاغح َرزاخ انًٛكَٕاسٔل كًسرحهة جالذُٛٙ نالسرؼًال انجهذ٘، ٔ ذحضٛز ػذد يٍ انصٛغ تاسرخذاو كارتٕكسٙ يثٛم سٛهٛهٕس انصٕدٕٚو ٔانكارتٕيٛزكؼايم جالذُٛٛٙ.ٔكًا شًهد دراسح ذأثٛز َٕع ٔ ذزكٛش كم يٍ انؼٕايم انًساػذج ر انشٚرٙ ػهٗ سزػح ذحزر انذٔاء ٔ يقارَرٓا يغ انًسرحضز انرجار٘ )كزٚى يٛكٕسانٍٛ(. أشارخ َرائج ذحزر انذٔاء نالسرحالب ٔانطٕ ذزكٛش انؼٕايم صٛغ انكارتٕيٛز ٔأٌ سٚادج تاٌ أفضم سزػح ذحزر كاَد فٙ صٛغ كارتٕكسٙ يثٛم سٛهٛهٕس انصٕدٕٚو يقارَح يغ نٗ سٚادج سزػح انرحزر. أيا انطٕر انشٚرٙ فقذ كاٌ نّ ذأثٛز فٙ اػاقح انرحزر يغ سٚادج انًساػذج نالسرحالب فٙ كم يٍ انصٛغرٍٛ ادٖ ا ذزكٛشِ. نذنك اخرٛزخ انصٛغح انرٙ تُٛد اػهٗ سزػح ذحزر نذراسح ذذفقٓا ٔثثٕذٛرٓا.اضافح نذنك،فاٌ ذارٚخ انصالحٛح نهصٛغح انًخرارج نثثاذٛح انفٛشٚائٛح نهصٛغح انًخرارج يٍ حٛث انهٌٕ ٔاالص انٓٛذرٔجُٛٙ كاَد تحذٔد سُح ٔ سثؼح اشٓز يغ ػذو يالحظح أ٘ ذغٛز فٙ ا ٔانقٕاو طٛهح يذج انخشٌ. introduction dermal drug delivery offer many important advantages to overcome many problems associated with drug (1) . moreover this type of drug delivery is easy, painless and simple to terminate therapy if any adverse or undesired effects occur (2, 3) .skin delivery is an effective for targeting therapy for topical dermatological disorder as in antifungal agents (4) .emulsification as a physical process is widely used in pharmaceutical and cosmetic products for external use, since they are spread easily over effected area, mean while, the viscosity, appearance and degree of greasiness can be controlled by the formulator (5) . currently the great attention is devoted to the gels especially hydrogels formulation since they have an attractive appearance and develop pleasant cool feeling. they are easy to apply and remove (6) and generally provide faster drug release compared with ointment and cream (2) .eumlgels are emulsions either of oilin-water or water-inoil type, which are gelled by mixing with gelling agent. they have the advantages of both emulsions and gels. they have been recently used as vehicle to deliver various drugs to the skin (7, 8) .the validity of miconazole nitrate in treatment of fungal infections is well known. miconazole nitrate is used topically in management of fungal infections. clinical studies have shown that miconazole nitrate is effective for topically treatment of dermatophytoses, superficial mycoses, cutaneous candidiasis and mixed infections when applied twice daily (9) .the objective of this study is to formulate emulgel containing miconazole nitrate using two type of gelling agent, beside to that, determine the in vitro release of the drug from these bases, and investigation the influence of emulsifying agent, oil phase concentration on drug release, along with evaluation the rheological behavior of the prepared formulas. 1corresponding author e – mail : lubnahani_95@yahoo.com received : 21 / 1 / 2009 accepted : 24 / 5 /2009 iraqi j pharm sci, vol.18(2) 2009 miconazole nitrate emulgel 27 materials and methods materials miconazole nitrate powder kindly supplied by al safa factory, carboxypolymethylene ( carbomer 941) from ( goodrich, usa), sodium carboxymethylcellulose (scmc) from (bdh chemicals ltd, poole, england), sodium hydroxide (fluka ag, puriss.p.a, switzerland), tween 20, propylene glycol, span 20 from (merk-schuncherdt, germany), liquid paraffin (riedel-de haen ag seelze, hannover) , citric acid , methylparaben, propylparaben from (samara drug industries), all other reagents were of analytical grade. equipment sartorius balance (werke-gmbh, type 2842, germany), electrical mixer (janke and kunkel, rf16), water bath (memmert, germany) , ph-meter (hanna instruments ph 211 microprocessor, italy), usp dissolution apparatus, type ii (copley scientific tld, england), rotational viscometer (fungilab, spain) spectromemter ( specord 40, analytikjena, germany), hplc (waters, usa). methods preparation of carbomer and scmc gel fifty grams of the carbomer gel was prepared by dispersing one gram of carbomer powder in 50 ml purified water with aid of moderate speed stirrer (50 rpm), and then the ph was adjusted to 6-6.5 using 0.5n of sodium hydroxide (6,10) . while fifty grams of scmc gel was prepared by dispersing 2.5 grams of scmc powder in 50 ml of heated purified water, and the dispersion was cooled to room temperature and left overnight (5) . preparation of emulsion the general method was employed according to ansel h.c. et al (5) for preparation of an emulsion was as follows: the oil phase was prepared by dissolving 1.66 g of span 20 in liquid paraffin while the aqueous phase was prepared by dissolving 0.34 g of tween 20 in purified water. two grams of miconazole nitrate was dissolved in 2.5 g of ethanol, while 0.15 g of methylparaben and 0.05 g of propylparaben were dissolved in 5 gm of propylene glycol and both were mixed with aqueous phase. both the oily and aqueous phases were separately heated to 70-80 ° c. then, the oil phase was added to the aqueous phase with continuous stirring at 500 rpm until cooled to room temperature (5) . formulation of miconazole nitrate emulgel eight formulas of miconazole nitrate were prepared by dispersing the obtained emulsions with the gel in 1:1 ratio with gentle stirring until get homogenous emulgel as shown in table 1 table 1: different formulas of miconazole nitrate emulgel. (% w/w) f miconazole nitrate carbomer 941 scmc liquid paraffin span 20 tween 20 propylene glycol water observation ph 1 2 1 5 1.66 0.34 5 100 white viscous cream 6.5 2 2 2.5 5 1.66 0.34 5 100 white softy cream 6.2 3 2 1 5 3.32 0.68 5 100 white viscous cream 6.4 4 2 2.5 5 3.32 0.68 5 100 white softy cream 6.1 5 2 1 7.5 1.66 0.34 5 100 white viscous cream 6.5 6 2 2.5 7.5 1.66 0.34 5 100 white softy cream 6.1 7 2 1 7.5 3.32 0.68 5 100 white viscous cream 6.5 8 2 2.5 7.5 3.32 0.68 5 100 white softy cream 6.2 evaluation of miconazole nitrate emulgel drug released glassy beaker with 2.5 cm in diameter was filled with 3 g of each formula and mecolzen ® cream. the mouth of beaker was covered with a filter paper which was kept in place with rubber band and was inverted and immersed to about 0.5 cm of surface of citrate buffer ph 5.5 in a jar of dissolution test apparatus with stirring rate of 50 rpm. the study was carried out at 37±0.5 ° c.samples of 5 ml were withdrawn after (15, 30, 45, 60, 90, 120 and 180 minutes) through 0.45µm millipore filter paper and replaced with an equal volume of fresh buffer. the samples were then analyzed spectrophotomertrically at ג max 272 nm (11) . rheological studies rheograms were obtained at 37˚c using rotational viscometer. the prepared formulas were sheared with spindle r7 over the range of speed setting from 1 to 10 rpm with 30 seconds between each 2 successive speeds, and then in a descending order (11) . iraqi j pharm sci, vol.18(2) 2009 miconazole nitrate emulgel 28 physical properties the physical properties (color, odor, ph and appearance) of the selected formula (f4) was observed over the period of (4) months storage at (35 ° c) and at refrigerator temperature (4 ° c). the drug content was calculated using hplc method (12) to predicate the expiration date. the ph of emulgel was also measured by shaking up of one gram of emulgel with 100 ml of water. result and discussion formulation of miconazole nitrate emulgel one of the most challenges that appear in the formulation of drug as a topical preparation is the choice of the type of base used, so the choice of appropriate base type and concentration play an important role in the topical preparation design. the present study revealed that different formulas were succeeded in incorporation of miconazole nitrate 2% w/w in different bases as shown in table1. it was seen that using of carbomer 941 1% w/w as vehicle base for (formulas 1,3,5 and 7) gave white viscous cream compared with white soft cream resulted when sodium carboxymethylcellulose (scmc) used as vehicle base for (formulas 2,4,6 and 8). this effect may be attributed to the higher hygroscopicity of scmc compared with carbomer 941 1% w/w as vehicle base for (formulas 1,3,5 and 7) gave white viscous cream compared with white soft cream resulted when sodium carboxymethylcellulose (scmc) used as vehicle base for (formulas 2,4,6 and 8). this effect may be attributed to the higher hygroscopicity of scmc compared with carbomer (13) .meanwhile incorporation of emulsifying agent and liquid paraffin in different concentration for both types of formulas made of carbomer 941 and scmc gave marked effect on the consistency of the resulted base as a viscous or softy cream emulgel (14) , the later effect is well revealed when the viscosities of all formulas were determined as shown in table 2. table 2: viscosities (in poises) of miconazole nitrate emulgel at low and high rates of shear. formulas η max * η min ** formula 1 752.5 6306.46 formula 2 251.25 877.28 formula 3 873.67 8305.02 formula 4 494.63 2585 formula 5 986.32 6589.57 formula 6 615.9 3301.18 formula 7 517 3367.65 formula 8 410.39 2460.08 * viscosity at high rate of shear (10 rpm). * * viscosity at low rate of shear (1rpm). it was seen that both span 20 and tween20 increase the viscosity of formulas 1, 3 and 5 in which carbomer 941 used as abase, moreover the same effect was resulted in formulas 2, 4 and 6 when scmc used as a base. this effect is a consistent with the results obtained by wan l. et.al and ban n.b. et.al (14, 15) .on the other hand, increasing liquid paraffin content from 5 to 7.5 % w/w for formulas (5, 7) and formulas (6,8) at which carbomer 941 and scmc were used as a vehicle base, respectively, revealed a reduction in the viscosities at both low and high rate of shear, this result may be attributed to the ability of liquid paraffin to contribute in a formation of emulsion with water (15) , that make the utilization of span 20 and tween 20 as a surfactants is possible and then decrease the amounts of later surfactants in each previous later formula (16) . drug released effect of gel base type figure -1illustrates the effect of gelling agent type on release of miconazole nitrate from formulas 1 and 2, it was seen that two folds higher percent release of miconazole nitrate after 3 hours of release from emulgel when 2.5%w/w scmc used as a base in stead of 1% w/w carbomer. this result may be attributed to physical structure of the polymer network and shape of three dimension structure of the polymer, since the entrapment of miconazole nitrate within these structural network revealed high capability of 1% w/w carbomer941 compared with 2.5% w/w scmc. the same results were obtained when fluconazole was incorporated as a topical lipogel formulation using different type of cutina as gelling agent. they found that the kind of gelling agent have marked effect on release and flow properties of fluconazole (17) .in addition, the result may be also due to higher viscosity of the carbomer emulgel compared with scmc as shown in table -2-. figure 1: the effect of gel base type on the release profile of miconazole nitrate 2 % w/w at ph 5.5 and 37 ° c 0 10 20 30 40 50 60 0 30 60 90 120 150 180 210 time (min) % d ru g r e le a s e d f1(1%carbom er) f2(2.5%scmc) iraqi j pharm sci, vol.18(2) 2009 miconazole nitrate emulgel 29 effect of emulsifying agent concentration the effect of addition of span 20 and tween 20 as an emulisifying agent to produce emulgel structure is illustrate in figure -2. it was seen that increasing the concentration of emulsifying agent from 2% w/w to 4% w/w using carbomer 941(formula 1,3) and scmc (formula 2,4) as a gel base led to significant (p<0.05) increase in the amount of miconazole nitrate released in dissolution medium. this increasing was found to be 39% and 37.5% for both scmc and carbomer 941, respectively, compared with 27.5% and 15.8%. this effect may be referred to the ability of these emulsifying agents to lower the interfacial tension between oily and aqueous layer in the dispersion medium (18) , indicating an increasing the hydrophilicity of emulgel which in turn increase penetration of dissolution medium into the emulgel structure and then increasing the amount of miconazole nitrate released. this result was in consistent with that result obtained when increase the concentration of emulsifying agents from 1.5% to 2.5% in both carbopol and hydroxypropyl methyl cellulose emulgel base led to increase the release of chlorphenesin from topical emulgel (19) . figure 2: the effect of emulsifying agents concentration on release of miconazole nitrate 2 % w/w at ph 5.5 and 37 ° c. effect of oil phase concentration in an attempt to investigate the effect of oil phase concentration, when liquid paraffin was increased from 5% w/w to 7.5% w/w in formulas 5 and 6 showed significant (p<0.05) decrease in the amount of miconazole nitrate released from these bases, as shown in figure 3. this result may be explain according to the concept of escaping tendency of drugs (20) , it was supposed that increasing the thermodynamic activity which can be expressed in terms of relative solubility of drug lead to enhance the releasing of drugs from vehicle (21) .the same effect was obtained by elbary who proved that the increase liquid paraffin led to retardation of chloramphenicol release from its emulgel formulation (7) . on other hand, increase the emulsifying concentration with 7.5% w/w liquid paraffin as in formulas 7 and 8 showed significant (p<0.05) increase in release of miconazole nitrate as shown in figure 4.the treatment of obtained data with higuchi principle revealed that best fit mechanism of miconazole nitrate 2% w/w release from emulgel with linear relationship when the amount of drug released plotted with square root of time (22) as shown in figure 5.the later result of formula 4 was found to be pharmaceutically bioequivalent with the reference one mecozalen cream ® as shown in table 3. figure 3: the effect of paraffin concentration on the release of miconazole nitrate 2% w/w from carbomer 941 emulgel at ph 5.5 and 37 ° c. figure 4: the effect of paraffin concentration on the release of miconazole nitrate 2% w/w from scmc emulgel at ph 5.5 and 37 ° c. figure 5: the effect of emulsifying agent concentration on release of miconazle nitrate 2% w/w from emulgel. 0 10 20 30 40 50 60 0 30 60 90 120 150 180 210 time (min) % d ru g r el ea se d f3(4%ea in carbom er) f4(4%ea in scmc) f2(2%ea in scmc) f1(2%ea in carbom er) 0 10 20 30 40 50 60 0 30 60 90 120 150 180 210 time (min) % d ru g r el ea se d f1(2%e.a+5% paraffin) f3(4%e.a+5% paraffin) f5(2%e.a+7.5% paraffin) f7(4%e.a+7.5% paraffin) 0 10 20 30 40 50 60 0 30 60 90 120 150 180 210 time (min) % d ru g r el ea se d f2(2%e.a+5% paraffin) f4(4%e.a+5% paraffin) f6(2%e.a+7.5% paraffin) f8(4%e.a+7.5% paraffin) iraqi j pharm sci, vol.18(2) 2009 miconazole nitrate emulgel 30 table 3: the rate release constant (k) of miconazole nitrate from different emulgel bases. formulas k (mg. min -1/2 .ml -1 ) correlation coefficient (r) formula 1 0.0018 0.986 formula 2 0.0031 0.993 formula 3 0.004 0.986 formula 4 0.0038 0.992 formula 5 0.0012 0.998 formula 6 0.0015 0.999 formula 7 0.0014 0.998 formula 8 0.0022 0.992 mecozalen ® cream 0.0019 0.988 rheological properties of prepared gel in gel systems, consistency depends on the ratio of solid fraction, which produces structure, to liquid fraction. the difference in the kind of the gelling agents result changes in structure consistency (23) . the selected emulgel formula f4 demonstrates pseudoplastic flow with thixotropy (figures 6). the profiles showed that as share stress is increased, the normally arranged molecules align their long axes in direction of flow orientation reduce the internal resistance of material and hence decrease viscosity (24) .the presence of hysteresis loop indicated breakdown in structure occurred . figure 6: rheogram of formula 4 at 37 ° c ( scmc gel base). physical properties stability of prepared emulgel (effect of storage time) the selected miconazole nitrate emulgel, formula 4 was white creamy with homogenous appearance, no change in color and odor after 4 months of storage at refrigerate at 4 ° c and at 35 ° c. figure 7 show the degradation curve of miconazole nitrate of formula 4, from which the degradation rate constants was calculated from the slopes of straight lines. they was found to be 1.73 x 10 -4 and 1.64 x 10 -4 (day -1 ) after storage for 4 months at 4 ° c and 7 months at 35 ° c, respectively. arrhenius plot was constructed as shown in figure 8 and rate constant at 25 ° c was obtained. the expiration date of formula 4 was found 1.7 years ; with estimated ph value about 5.9. figure 7: degradation rate constant of formula 4 of miconazole nitrate 2% w/w at 35 ° c and 4 ° c. figure 8: arrhenius plot for expiration date estimation of miconazole nitrate emulgel (formula 4). conclusion miconazole nitrate can be formulated as emulgel with good release and consistency. emulsifying agent has pronounced effect on drug release from emulgel followed by the oil phase concentration and finally the type of gelling agent. the scmc based emulgel showed highest drug release when 5% w/w liquid paraffin and 4% w/w emulsifying agents were used and it is the formula of choice. the incorporation of gelling agent gives emulgel a proper consistency and exhibit shear-thinning behavior with thixotropy. 0 2 4 6 8 10 12 0 500 1000 1500 shear stress (dyne/cm 2 ) s h e a r ra te ( s e c -1 ) 1.984 1.988 1.992 1.996 2 0 2 4 6 8 months lo g % r e m a in in g 35° c 4° c -3.79 -3.78 -3.77 -3.76 -3.75 3.2 3.3 3.4 3.5 3.6 3.7 1/t x 10 -3 (k° ) l o g r a te c o n s ta n t (d a y 1 ) 3.35 iraqi j pharm sci, vol.18(2) 2009 miconazole nitrate emulgel 31 references: 1. bucks d.a., mcmaster j.r., maibach h.i. and guy r.h., bioavailability of topically administered steroids, j. invest. dermatology (1988), 91, 29-33. 2. ozguney s.i., karasulu y.h., kantarci g., transdermal delivery of diclofenac sodium through rat skin from various formulations, aaps pharm.sci.tech. 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(1962), 51, 802-804. 23. martin a., physical pharmacy, 4 th edition, b.i. wavery p.v.t.ltd, new delhi 1994 ,456-460. 24. yvonne t.f., khiany p., al-hanbali o., effect of carbopol and polyvinylpyrrolidone on mechanical rheological and release properties of bioadhesive polyethylene glycol gel, aasp pharm.sci.tech (2000), 1(3), article 24. comparative of different diagnostic methods iraqi j pharm sci, vol.19(1) 2010 helicobacter pylori 74 study the prevalence of helicobacter pylori infection by different diagnostic methods ibtihal n. saeed* ,1 and aseel i. ibrahim* * department of clinical laboratory science,college of pharmacy,university of baghdad, baghdad, iraq. abstract a total of 41 patients with gastro duodenal symptoms (show signs of inflammation with or without duodenal ulcer) . 21 males (51.2%) and 20 female (48.8%) with an average age 0f (20 – 80) years old under going gastrointestinal endoscopy at baghdad teaching hospital in internal disease clinical laboratory , between (february – june) 2009 . biopsies specimen of antrum , gastric fundus ,& duodenal bulb were examined by the following methods (rapid urease test , giemsa stain section to detect bacteria , & haematoxilin and eosin stained section for pathological study which are considered the gold standard methods , sera or plasma from these patients were tested by immunochromotography (icm),serological method for igg antibodies to h. pylori. history picture are( use of certain medication , tobacco , alcohol, and current infection are taken). the results showed that the percentage of prevalence (positive results)were (83%) by histopathological method while it gave only(73%) by serological method and(66%) by rapid urease test, and the prevalence in males was more than in females (44%), (39%)respectively ,and also the prevalence increase with age (40 – 60) 14 out of 15, most patients show gastritis and duodenal ulcer, 25 (60%) by endoscopy diagnosis and 7 (17%) show malignant cancer ,while 9(22%) without any symptoms. the sensitivity of urease test (82%) and specificity (88.1%) and by icm sensitivity (86%) and specificity (67%) comparing with gold standard methods 100% . the aim of this study is to compare the different diagnostic techniques of helicobacter pylori infection by using invasive methods (histological examination of gastric & duodenal biopsies stained by giemsa &haematoxilin & eosin methods , & rapid urease test which is considered the gold standard methods & non-invasive serological methods such as icm rapid test , all these tests provide information about the incidence and prevalence of h. pylori in population , diagnostic value for each test also the eradication of person. keywords: serology, helicobacter pylori , gastric ulcer , diagnosis الخالصة انًسببت نمشحت انًعذة ٔاالرُى عششي فً عٍُت عشٕائٍت يٍ helicobacter pylori ٌخضًٍ انبحذ دساست اَخشاس بكخشٌا انًشضى انزٌٍ ادخهٕا إنى شعبت انُاظٕس فً يسخشفى يذٌُت انطب انخعهًًٍ فً بغذاد ٔرنك باسخخذاو طشق حشخٍصٍت يخخهفت يُٓا ذاو صبغت انًٍٓاحٕكسٍهٍٍ ٔصبغت انكًضا ٔرنك نذساست انخغٍشاث انُسٍجٍت ٔفحص حٕاجذ انبكخشٌا فً انُسٍج , انفحص انُسٍجً باسخخ كزنك فحص انعٍُاث بطشٌمت انخحمك يٍ إَخاج إَضٌى انٍٕسٌاص باالعخًاد عهى ٔسظ انٍٕسٌا انسائم انحأي عهى دنٍم انفٍُٕل األحًش , ٔلذ ٔجذ اٌ َسبت icmباالعخًاد عهى انطشٌمت انسشٌعت iggٍش ٔنٕجً نألجساو انًضادة ٔكزنك بانطشٌمت انغٍش يباششة بانفحص انس . %93َسبٍا أعهى يٍ اإلَاد %::سُت ٔاٌ اإلصابت بانشجال 6>-;:االَخشاس ٔاإلصابت حضداد يع حمذو انعًش ٔخاصت بٍٍ األعًاس بًٍُا انًصابٍٍ بسشطاٌ %6>انًعذة ٔاالرُى عششي ْى حٕانً اٌ انخشخٍص األٔنً بانُاظٕس ٌبٍٍ اَّ انًشضى انًصابٍٍ بمشحت بانطشٌمت %19بًٍُا كاَج %39ٔاٌ اعهى َسبت نالَخشاس شخصج بطشٌمت انذساست انُسٍجٍت %71ٔانًشضى انعادٌٍٍ %88انًعذة بانذساست انُسٍجٍت ٔ %766عٍ طشٌك فحص اَضٌى انٍٕسٌاص , ايا كفاءة ٔحساسٍت ْزِ انطشق فخخشأح بٍٍ %>>انسٍشٔنٕجٍت ٔ .بانطشق انسٍشٔنٕجٍت ٔاالَضًٌٍت عهى انخٕانٍض 38%, %>3 introduction in 1983 warren and marshal (1) isolated a new curved gram negative bacillus from gastric mucosa of patients with active chronic gastritis , this bacteria was first named campylobacter pyloris then c. pylori and finally helicobacter pylori (2) , establishing an association between the bacteria , gastritis and peptic ulcer disease . h. pylori is the most important cause of chronic gastritis (3,4,5 ) , it is also the most important etiological factor responsible for duodenal ulcer (3,4,5) ,gastric ulcer (3,4,5) , and has an important role in the pathogenesis of gastric cancer (6,8 ) . h. pylori is also responsible for dyspeptic patients, and screening for h. pylori in those patients improve selectivity for gastroscopy (5) . the identified virulence factors of h. pylori include the flagella used for motility through the mucus , the urease activity used for neutralizing the acid from the stomach . the cytotoxin activity which vascuolize the epithelial cells (10,11) and this examined by histopathological study . since marshal and warren established the association between h. pylori , gastritis ,& peptic ulcer, a great number of diagnostic techniques have been developed (12) .the first rapid and simple test developed for the diagnosis of h. pylori infection was urease test based on the capacity of the organism to produce great quantities of this enzyme (13,14,15,16) . 1corresponding author email : ibtihal_noori@yahoo.com received : 15/2/2010 accepted : 26/5/2010 iraqi j pharm sci, vol.19(1) 2010 helicobacter pylori 75 the urease catalyzes the degradation of urea to ammonia and bicarbonate. this reaction produces an increase in the ph of the medium that can be detected by an acid – base indicator such as phenol red , that changes color from yellow to pink (15) .the velocity of the change of color depends on the urease concentration according to the numbers of bacteria present (17) .the great advantage of the urease test in the diagnosis of h. pylori is that the result can be obtained before the patient leaves the endoscopy room. the result were comparable in sensitivity and specificity with the histological and culture techniques and staining section by giemsa stain which are considered the gold standard methods (gastric biopsy is required to perform the test) (18,19) . mcnulty and wise (15) were the first ones to use this test to detect h. pylori infection . serological tests are useful in h. pylori infection because virtually all patients colonized with this organism under a local antibody response directed against antigens covering the surface and flagella of the organism and this antibody response detected in the serum (23,24,25) . also serological methods used to diagnose h. pylori in which no upper gastrointestinal endoscopy is required . maastrich 1996 working group cited by anon suggested that screening dyspeptic patient under 45 years of age for h. pylori might reduce the need of endoscopy (20,21) , but blood must be obtained to detect h. pylori antibodies (22) .h. pylori serology is alterative in comparison with other methods because it is simple , inexpensive , & less of a burden for the patient .several kits for detection h. pylori by serology have become commercially available since the discovery of h. pylori by warren in 1983 (1) , most of these kits are based on various antibody preparations and different techniques , this lead to an increase in the number of studies that have evaluated kit characteristics . different studies for comparison between kits to account for the different reference standards and designs used by various investigators (22,23,24,25) . serological diagnosis simplest and least expensive , non – invasive method for igg and or iga antibodies , latex agglutination methods are quick tests , useful for screening purposes .elisa based tests accurately quantities the amount of antibody (titer) present and are promising tool for assessing the efficacy of h. pylori eradication treatment (27) , also for rapid office – based serologic test , using immunochromotography (icm) , and the immunoblott for the diagnosis of h. pylori (26) . c13 / c 14 , urea breath test are reliable non – invasive methods for diagnosis of on going h. pylori infection (30,31) . material and methods samples : 41 gastric and duodenal biopsies from patients of the endoscopy department of baghdad teaching hospital – baghdad / iraq , were analyzed between (february – june) 2009 , at least two biopsies were taken from the antrum of each patient for histological study send to histopathological laboratory of the hospital stained by giemza method (luna 1968) (32) & haematoxilin & eosin method (modified m. of guyer ,1953 ) (33) by (gram wegurt) to study the histological change and detecting rod shaped h. pylori. phenol red rapid urease test a solution of urea 10% and solution of phenol red 1% were prepared for the working solution , 0.1ml of phenol red solution were mixed in 1 ml of the urea solution . the reagent is stable for two weeks of 4 – 8 °c each biopsy was embedded in 0.2 ml of the reagent and incubated at room temperature (22°c) for 1 min. serological diagnosis by (icm) immunochromotography method of (acon h. pylori one step –rapid test devise) serum / plasma is a sample test that utilized a combination of h. pylori antigen coated particles and anti – human igg to qualitatively and selectively detect h. pylori antibodies in serum or plasma in10 minutes after serum or plasma specimen is placed in the specimen well., it reads with h. pylori antigen coated particles in the test .the mixture migrate chromatographically along the length of the test strip and interacts with the immobilized anti – human igg , if the specimen contain h. pylori antibodies , a colored line appear in the test line region , indicating a positive result , if the specimen dose not contain h. pylori antibodies a colored line will not appear in this region , indicating a negative result comparing with positive control – test , the result should be read at 10 min.( acon lab. inc – 4/08 sorrento valley boulevard .san diego ,ca 9212,usa). personal information about past infection , treated use of certain medication , alcohol and tobacco , this result were analyzed according to age , sex ,race and another characteristics . calculation of sensitivity and specificity positive and negative predictive values were made using the following formula : iraqi j pharm sci, vol.19(1) 2010 helicobacter pylori 76 results a total of 41 patients were investigated, 21(51.2%) male and 20(48.8%) female with mean age 45 years old (range 20-80year), these patients under examination showed by endoscopy diagnosis that 14(34%) of them have gastric ulcer, 11(27%) duodenal ulcer, 7(17%) with gastric cancer and 9(22%) non ulcer dyspepsia. tables 1&2 show that the percentage of infection with helicobacter pylori or the prevalence of infection which studed by histopathological and giemsa staining section methods increase in males 18(44%) more than in females 16(39%) and also the percentage of infections increase with age between (40-60) years old 14(34%) out of 15(36.5%) patients, the percentage of infections more than in younger and older patients. table (3) shows the relation between the endoscopy diagnosis with positive and negative result of infection done by different diagnostic methods , in which histopathology and giemsa staining methods gives 34(83.0%) positive, 7(17%) negative, by serodaignosis (icm) test give 30(73%) positive, 11(27%) patients negative while by rapid urease test 27(66%) positive, 14(34%) patients negative. the positive value of serodignosis and urease test consist 88%,79% respectively from the true positive value by histopathological study (34+) patients.from these result the high prevalence of infections were obtained first by histopathological study then by serodiagnosis methods and later the lowest value by urease test. in all test used the prevalence over 75% considered high prevalence of infection in population (6) . also if we determined the positive value of diagnosis in relation with disease or endoscopy finding, histopathological study gives 90% positive in duodenal ulcer and 80% with gastric ulcer comparing with serodiagnosis (82%),(72%) and urease test (72%),(54%) irrespectively. table (5) show the sensitivity& specificity for each test depending on true positive, true negative, false positive, false negative values determined in table (4), comparing with a gold standard method of diagnosis and this gives the sensitivity & specificity of histopathology & giemsa staining methods 100% , by serodiagnosis (icm) method (86%), (67%) and by urease broth test (82%), (87%), means that the first method gives more accuracy result than others, with many disadvantage, and the other methods give less accuracy with many advantages discussed later in discussion. table 1 : show the prevalence of h. pylori infection in male & female. percentage of infection number of positive h. pylori percentage total number 44% 18 51.3% male (21) 39% 16 48.7% female (20) table 2 : show the prevalence of infection in different age groups. result (%) number of positive percentage (%) total number (41) age in years 14.6 6 21.95 9 20 – 30 12.5 9 29.2 12 31 – 40 17.0 7 17 7 41 – 50 17.0 7 19.5 8 51 – 60 7.3 3 7.3 3 61 – 70 4.8 2 4.8 2 71 80 iraqi j pharm sci, vol.19(1) 2010 helicobacter pylori 77 table 3 : show the percentage of positive and negative h. pylori infection diagnosed by urease , serological kit and endoscopy biopsies. sum. non ulcer dyspepsia no.(%) gastric cancer no. (%) duodenal ulcer no.(%) gastric ulcer no.(%) total number (41) 41(100%) 34(83%) 30(73%) 27(66%) 9 (22%) 6(14.63) 7(17%) 5 (12.19) 7 (17%) 7(17%) 4(9.75%) 5 (12.19) 11 (26 %) 10(24.4%) 9(21.95%) 8 (19.5%) 14 (34%) 11(26.8%) 10(24.4%) 9(21.95%) histopatholog giemsa stain sero.kit urease h. pylori (+) 7(17%) 11(27%) 14(34%) 3(7.3%) 2 (4.8% ) 4 (9.75 ) 0 3 (7.31%) 2 (4.87% ) 1(2.43%) 2 (4.87%) 3 (7.3%) 3(7.3%) 4 (27%) 5 (12.2% ) histopatholog giemsa stain sero. kit urease . h pylori (-) table 4 : number of true positive , true negative ,false positive ,false negative by different diagnostic methods. table 5 : values of different diagnostic methods . n.p.v. % p.p.v. % specificity % sensitivity % test 100 100 100 100 histopath. 100 100 100 100 giemsa stain 54 96 88 82 rapid urease test 45 94 67 86 rapid serodiagnosis (icm) if you know the prevalence of helicobacter pylori in your population you can make a judgment about the predictive value of a positive or negative test from the table(6). table 6 : predictive value of a test with 85% sensitivity and 79% specificity probability of disease with negative result(%) probability of disease with positive result(%) prevalence of disease 2 31 10 16 80 50 63 97 90 test true positive false positive true negative false negative histological changes 34 0 7 0 geimza stain 34 0 7 0 urease 27 1 7 6 icm (kit) serology 30 2 4 5 iraqi j pharm sci, vol.19(1) 2010 helicobacter pylori 78 discussion the aim of this study is to determine the prevalence of infection with h. pylori by different diagnostic methods in random population enter the endoscopy department for gastric and duodenal examination with or without symptoms of inflammation. from the 41 patients under investigations results in table (1) showed that prevalence of infection in males more than in females and these results agree with other studies done by nicholas et al (1992), in which they found that 47% males out of 82 patients, (44%) 0f them were infected by h. pylori (25) . table (2) showed that prevalence of infection increase with age between (40-60) years old agreed with nulty (1999) which found that the more likely age of infection in patients over 50 years old (42%) than in younger patients (21%) (22) , another group of liston r, et al (1996) cited by nulty(1999), found that (31.7%) of elderly patients with seropositive result had no evidence of active infection determined by endoscopy and urease test. older patients are more likely to have developed atrophic gastritis and h. pylori can not readily colonize this type of gastric mucosa ( 22) . it was recognized that prevalence of h. pylori infection increase with age in a symptomatic persons in developed countries and this tend to plateau at around the age of 60 years, related to socioeconomic status and ethnicity (5,23,25) .table(3)which showed high prevalence of infection given by histopathological study which are considered invasive gold standard methods (22,25,26) , (83%) of patients gave positive result ,while by serodiagnosis (icm) method (73%) and (65%) by urease test method which means that these methods gives lowest value of prevalence comparing with histopathological study and this because many factors were affecting on the value of the result such as for example. negative values in biopsy methods histopathology & giemsa staining section depend on patient that may be under treatment or past proven h. pylori infection treated with a course of antimicrobial drugs with proton pump inhibitors , that patient give negative and clearance of the disease in biopsy specimen but can give positive result with serodiagnosis, and so give false positive result and high prevalence than biopsies (23,25) . a negative value in urease test depend on non homogeneous distribution of the microorganism in the stomach and this situation is overcomed by use of several specimen from (3-5) for the same patient (34,35,36) so we minimize the specimen error and this explain the 13 patients which give negative result by this method, which lowering the percentage of infection comparing with other methods. -in serodiagnostic method the percentage of infection (73%) which gives positive result and 11(27%) patients comparing with histopathological study , 4 patients only were true negative and 5 patients were false negative and only 2 patients gave false positive result , this can be explained by:  patients who are in acute case of infection before an igg response has developed gave false negative serological result ,means that it may be positive result in biopsy method, also negative result may be due to patient not produce circulating antibody response which detectable only with complex type of antigen (preez-preez, et al. cited by nicholas(1992) (25) .  false positive result according to cross reaction antigen (3-9%) of patient have false positive result with h. pylori that might produce antibodies such as gastrospirillum hominis and this also depend on type of antigen used in test (23,25) or it may be that patient with past infection that gives false positive result with slowly return antibody, it may give positive test for over 6 months from clearance of the disease (25,26,34) . table (5)show the sensitivity & specificity of each test depend on the true positive ,true negative , false positive ,& false negative value in table (4), each positive value in histopathological study considered true positive value and each negative value considered true negative value (gold standard methods) and so sensitivity & specificity (100%) and this also agreed with other study which find that these methods gives sensitivity & specificity between (98-100%) (34) , and for serodiagnosis (86% ) , ( 67%) while urease test gives sensitivity & specificity (82%),(87%).the sensitivity & specificity of serological test reported by many workers varies from (76-96%) and half of the patients with false positive result were over 50 years age. other group found that elderly patient with positive serology had no evidence of active infection determined by endoscopy and urea breath test (14,22) , other workers for the same methods (icm) test find that the sensitivity is (96%) with(94%) specificity (26) which used this test to diagnose h. pylori iraqi j pharm sci, vol.19(1) 2010 helicobacter pylori 79 infections in thai children between(1.5-16 years old), other study compare different serological kits for h. pylori infection and also found that the sensitivity and specificity around (67%-86%) (22) , other outhers (25) find that sensitivity of commercial elisa kits had an accuracy between (89%95%).the sensitivity of urease test according to eugenia (34) is record to be (97%) by phenol red broth test with (100%) specificity also he mention that other authors have reported that urease test with specificity between (98%-100%) and sensitivity between(64%-98%),and this agreed by vaira (37,38) , thillainayagam (39) , mal.fertheiner (40) , mcnulty (15) , arvid, morris (42) , &westblom (14) reports specificities of (86%,98%,92%,100%) and sensitivities of (84%,92%,100%,88%) respectively, only hernandez reports a sensitivity of (72%) and specificity of (83%) for christensen's urea broth (43) .the presence of false negative and false positive result may be explained by the patchy distribution that h. pylori present in gastric mucosa ,especially in the body and fundus of the stomach, so the microorganism can be present in one biopsy and absent in another from the same patient (34, 44,45) . so the false negative value by this test were caused by the patchy distribution of the bacteria. conclusion it has been proposal that patient with dyspepsia could be screened for h. pylori status before it is recommended (25,22,23) and as h. pylori occurs in over (90%) of patient with chronic duodenal ulceration and up to (80%) of patient with chronic gastric ulceration (25,16,21) , it has been proposal that such an approach would help to reduce the need for endoscopy as well as cost, if such a policy were adopted only seropositive patient would undergo endoscopy and over 45 years of age or those taking anti inflammatory drugs, this would avoid up to (23%) of endoscopies. however ,further large prospective clinical studies are needed before such an approach can be accepted. also serological methods can be used for monitoring treatment and successful eradication of infection by detecting the fall in level of igg antibodies in serum after 3 months of treatment. the great advantage of the urease test in the diagnosis of h. pylori is that the result can be obtained before the patient leaves the endoscopy room, making clinical management easier. the studies suggest that urease result comparable in sensitivity and specificity with histological and culture techniques, being more economic and faster (12,34) , nevertheless an endoscopy is always necessary because a gastric biopsy is required to perform the test and also culture can be required for evaluating the 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32:467-9. 40. malfertheiner p, dominguez-munoz j, hekenmuller h, neubrand m, fisher h, sauer p. 1996. modified rapid urease test for detection of helicobacter pylori infection. eur. j. gastroenterol. heptol.;8:53-6. 41. arvid a, cook r, tabaqchali s, farthing m.1988. one minute endoscopy room test or campylobacter pylori. lancet;1:704. 42. morris a, mclntyre d,rose t,nicholson g.1986.rapid diagnosis of campylobacter pyloridis in infection .lancet;1:149. 43. hernandez f, rivera p, sigaran m, miranada j.1991. daignosis of helicobacter pylori: comparison of an urease test ,histological visualization of curved bacteria and culture.rev. inst. med. trop sao pablo; 33:80-2. 44. barthel j, everett d.1990. diagnosis of campylobacter pylori infections the"gold standard" and the alternatives. rev infect. dis.;12:5107-14. 45. azuma t, kato t, hirai m, ito s, kohli y.1996. diagnosis of helicobacter pylori inection. j. gastroenterol. hepatol; 11:662-9. iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel doi: https://doi.org/10.31351/vol30iss2pp196-207 196 formulation and in-vitro evaluation of carvedilol gastroretentive capsule as (superporous hydrogel) haider mohammed jihad *,1 and entidhar j. alakkam* *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract the carvedilol uses in cardiovascular disease has a major problem which is low solubility (class ii). the purpose of this study was to formulate and evaluate gastroretentive superporous hydrogel of carvedilol to improve its solubility and increase gastric residence time via utilization of various kinds and concentrations of hydrophilic polymers then incorporation of best prepared formula into capsules. sixteenth formulae of sph hybrid were prepared by gas blowing technique from the following materials; monomers (poly vinyl alcohol, and acrylamide), cross-linkers (methylene bisacrylamide, and glutaraldehyde), hybrid agent (chitosan), foaming agent (nahco3) and foam stabilizer (tween 80). different amounts or concentrations of these materials were utilized to investigate their effect on sph properties (density, porosity, floating, drug content, drug release, swelling time, and swelling ratio). the soaking procedure was utilized for loading of carvedilol into sph hybrid (6.25 mg/ 2.5 g sph). the choice of the best formula relied on the correlation of the resulted sph concerning to release profile of reference (carvedilol 6.25 mg tablet/ roche). the release of carvedilol was carried out by utilizing the dissolution apparatus (usp type ii). after analysis the results and application of similarity factor (f 2) equation, f8 was selected as the best formula then incorporated into capsule. the drug release data were applied to different mathematical kinetics and the results were shown to be fitted to higuchi model and the release mechanism was (fickian) diffusion. the overall results suggested that the prepared sph capsules for carvedilol are specific delivery to the stomach due to the increase in residence time and enhancement in solubility. key words: superporous hydrogel (sph), monomer, hybrid agent, cross-linker, and foaming agent. ) فائق المسامية كهالم مائي(في المعدة محتجزلول كبسول اليتصييغ وتقييم خارج الجسم للكارفيد *انتظار جاسم العكام و 1، * حيدر محمد جهاد بغداد ، العراق*فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، الخالصة .(يعتبر من الفئة الثانية من حيث التقسيم الصيدالني)لول لعالج امراض القلب هي قلة ذوبانيته يان اكثر مشكلة تواجه استخدام الكارفيد لول لتحسين قابلية الذوبان وزيادة يفي المعدة للكارفيد محتجزال فائق المساميةهالم المائي لل خارج الجسم الغرض من هذه الدراسة هو صياغة وتقييم .وقت البقاء في المعدة باستخدام انواع وتراكيز مختلفة من البوليمرات المحبة للماء ثم تحضير افضل صيغة على شكل كبسول )بولي فاينيل الكحول وأكريل المونومراتالمواد التالية؛ الهجين بتقنية االنتفاخ الغازي من فائق المسامية تم تحضير ستة عشر صيغة للهالم المائي ومثبت )بيكاربونات الصوديوم( ، العامل المولد للرغوة )كيتوسان( ، العامل الهجين)مثيلين بسأكريل أمايد و كلوتارالديهايد( المادة الرابطة أمايد( ، )الكثافة، المسامية، الطوفان، كمية فائق المسامية على خصائص الهالم المائي كمية أو تركيز هذه المواد اسة تأثير وقد تم در( . 80)توين الرغوة الهجين فائق المسامية الدواء، تحرر الدواء، وقت االنتفاخ ونسبة االنتفاخ(. لقد استخدمت طريقة التنقيع لتحميل الكافيدولول في الهالم المائي بشكل دمجهاتم وقد هي افضل صيغة يمكن اختيارها 8f ان الصيغةوجد )2f ( نتائج هذه الدراسة احصائيا وتطبيق معادلة العامل المشابهبعد تحليل صيغةكما ان بيانات تجربة تحرر الدواء تم تطبيقها على اكثر من صيغة رياضية وكانت النتيجة ان اقرب صيغة رياضية للبيانات هي كبسول. .االنتشار )فيكين(هيكوشي باإلضافة الى ان تحرر الدواء عن طريق دواء بشكل خاص الالبقاء لزيادة ذوبانية الكارفيديلول والهجين فائق المسامية ومما تقدم تظهر لنا النتيجة النهائية هي امكانية استخدام الهالم المائي .في المعدة ليتم امتصاصه هناك .، مونومر ، العامل الهجين ، مادة رابطة ، عامل مولد للرغوة فائق المساميةهالم مائي :الكلمات المفتاحية introduction the gastroretentive system can stay in the gastric region for many hours thereby prolonging the drug gastric residence time to get; better bioavailability, lower drug loss and enrich solubility for less soluble drugs in an environment with elevated ph (1). 1corresponding author e-mail: haider.mj80@yahoo.com received: 14/12/2020 accepted: 6/6 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp196-207 iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 196 several approaches to the gastroretentive drug delivery are being developed which involve: the floating system, the high-density sinking system, the muco-adhesive system, the magnetic system, the swellable, expandable, or unfoldable system, and superporous hydrogel (2). the sph is a system with 3dimensions of nonsoluble hydrophilic polymers that take up great amounts of water in a brief time due to the existence of interrelated microscopic holes. the used polymers are either natural like chitosan, alginate, gelatin, and carboxymethyl cellulose or synthetic like polyacrylamide, poly (acrylic acid), polyvinyl pyrrolidone (pvp), and polyvinyl alcohol (pva) (3). the hydrogel with hundreds of micrometers of pore size is considered as superporous hydrogel that varies from other forms of porous hydrogels like mesoporous and macroporous. the sph also has hundreds of time more surface area and smaller diffusion gap than traditional hydrogels due to its porous structure; these properties cause dried sph to swell very rapidly on contact with water to very large sizes (4). when sph is administered, the enlarged hydrogel can stay on in the stomach for an extended time and release the loaded drug as its volume are very large to be transferred across the pylorus sphincter. for use as the gastroretentive tool, sph not only has rapid swelling but also exhibits features such as biocompatibility, biodegradability, flexibility, high mechanical potency, great swelling potential, and acidic stability in the stomach (5). as example, sph hybrids of loratidine hydrochloride was formulated and characterized by dhingra et al (6). carvedilol is a weak base with a pka (7.8) and classified as class ii in the biopharmaceutical classification system (bcs). it favors solubilization in the stomach at low ph. however, it exhibits phdependent solubility therefore it undergoes formulation as gastroretentive superporous hydrogel (7, 8). carvedilol is 1-(9h-carbazol-4-yloxy)-3-[[2-(2methoxyphenoxy) ethyl] amino]-2-propanol with the chemical formula (c24h26n2o4) and has a chemical structure shown in figure (1). figure 1. chemical structure of carvedilol (9) it is a powder with white to off white color with melting point (m.p.) 114-115 ℃. it is practically insoluble in water, soluble in methylene chloride and methanol, sparingly soluble in ethanol and isopropyl alcohol, log p equal 4.19. this research is attempted to formulate and evaluate gastroretentive sph of carvedilol to improve its solubility and increase its residence time in the stomach via utilization of various kinds and concentrations of hydrophilic polymers and then incorporate the best-prepared formula into capsules. experimental work materials carvedilol (hangzhou-china), acrylamide (high media-india), methylene-bisacrylamide (high media-india), chitosan (alpha chemikaindia), tween 80 (alpha chemical-india), polyvinyl alcohol (me scientific-uk), sodium bicarbonate (riedel-germany), tetramethylethylenediamine, ammonium persulphate and glutaraldehyde (central drug house-india) preparation of super porous hydrogel hybrid of carvedilol the sph formation was performed by solution polymerization of monomers utilizing a gas blowing technique. the following constituents were applied sequentially to 10 ml beaker; acrylamide am 40% (1200 µl) as a monomer, polyvinyl alcohol pva (150 mg) as a monomer and hybrid agent, methyl bisacrylamide bis (450 µl) as a cross-linker, chitosan (400 µl) as a hybrid agent, tween 80 (0.2 ml) as a foam stabilizer, ammonium persulfate aps 20% (45 µl) and tetramethylehtylenediamine temed 20% (45 µl) as an initiator system. the admixture was shaken after adding each element. finally, nahco3 (100 mg) powder was added as a foaming agent. the polymerization was occurred after nahco3 addition and finished in a few minutes (table 1). the sph was removed from the beaker and washed with a blend of ethanol and water at a ratio of 3:1. then after, sph was dried for 72 hours at room temperature (10). the soaking procedure was utilized for loading of carvedilol. the amount of 0.1n hcl required for ideal swelling of sph was first determined thereafter, a solution of the drug (6.25 carvedilol/ 5 ml 0.1n hcl) was prepared. the sph was soaked in the prepared solution and left 20 minutes in the drug solution and let to dry overnight at room temperature (11). the effect of cross-linker concentration and type on the physical properties of sph hybrid (density, swelling, porosity, floating, drug content, and release) was studied as represented by f1-f7. each of f1-f5 was prepared with 450 µl of n, nmethylene-bisacrylamide (bis) as a crosslinker at 1, 1.5, 2, 2.5, and 3% w/v, respectively. while, each of f6 and f7 were prepared with 50 and 100 µl of glutaraldehyde (ga), respectively (table 1)。 in addition, to estimate the effect of chitosan (as a hybrid) concentration on sph features, f1, f8, f9, and f10 were prepared with different concentration iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 197 of chitosan; 1, 2, 3 and 4% w/v, respectively with keeping all other materials, and their amounts constant as demonstrated in the table (1). to investigate the effect of pva (as a monomer and hybrid agent) amount on the sph properties, formulas f1, f11, f12, and f13 were prepared with 150, 200, 250, and 300 mg pva, respectively (table 1). effect of foaming agent amount (nahco3) on properties of sph was also studied by f1, f14, f15, and f16 which were prepared with 100, 150, 200, and 250 mg of nahco3 with keeping other constituents constant as shown in table (1) characterization of the prepared sph drug content determination dry sph (about 2.5 g) containing 6.25 mg carvedilol was placed in a 100 ml volumetric flask and filled up to the mark with hcl buffer (ph 1.2). the resulting solution was filtered through whatman filter paper. then after, the absorbance was measured by utilizing uv spectrophotometer at the appropriate lambda max (λmax = 285) the amount of carvedilol in the tested formula was calculated (9, 12). density measurement for determination of density, solvent displacement process was used in which a dry sph was taken and weighed then immersed in a predetermined hexane volume in a graduated cylinder, sph volume was measured by increases in the volume of hexane. the density calculated by the following equation (13). density = wsph / vsph …… (1) where, wsph = weight of dried sph, vsph = volume of sph. swelling time determination this is an important feature of sph. the time of swelling of sph was determined by placing the hydrogel in a swelling media (water or 0.1n hcl) and the time for equilibrium swelling (constant weight) was recorded (14). swelling ratio measurement the completely dried sph was placed in excess of the swelling medium then removed from the media at a set time and weighed; the swelling ratio was measured by the following equation (14). qs = [(ws – wd) / wd] *100 …… (2) where, qs = swelling ratio, ws = weight of swollen sph, wd = weight of dried sph porosity measurement the dry hydrogel was immersed in the absolute ethanol overnight and weighed after excess ethanol on the surface was blotted; the porosity was calculated by following equation (15). porosity = (m2 – m1) / pv …… (3) where, m2 and m1 = weight of hydrogel after and before immersion in ethanol, respectively. p = absolute ethanol density, v = initial hydrogel volume. floating study the hydrogel was placed in a beaker containing 100 ml of 0.1n hcl, the time needed for rising of sph to the surface and floating was known as a floating lag time while, the total period time during which sph remained buoyant was known as a total floating time (16). in-vitro drug release the in-vitro carvedilol (6.25 mg) release test for sph hybrid (2.5 g) was performed by utilizing the dissolution apparatus (usp type ii) in 900 ml 0.1n hcl at 100 rpm speed of the paddle for 12 h at 37±0.5 ℃. a sample of dissolution medium (5 ml) was withdrawn at specified time intervals then; an equal volume of fresh medium solution was substituted. the absorbance of each sample was measured via uvspectrophotometer at the appropriate λmax of carvedilol. then after, the percent release of carvedilol was calculated (6). scanning electron microscopy (sem) analysis of sem was done to declare the morphology of dried sph. it was carried out to assure the porous structure produced during sph combination using the hummer sputter coater. samples were plated with gold carried with a jsm840 scanning electron microscope and collected the image using a digital capture card and digital scan generator (17). iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 198 table 1． formulas of sph. . formula component f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 f13 f14 f15 f16 am 40% (µl) 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 1200 pva (mg) 150 150 150 150 150 150 150 150 150 150 200 250 300 150 150 150 bis (µl) of 1% 450 450 450 450 450 450 450 450 450 450 1.5% 450 2% 450 2.5% 450 3% 450 ga (µl) 50 100 chitosan (µl) of 1% 400 400 400 400 400 400 400 400 400 400 400 400 400 2% 400 3% 400 4% 400 tween 80 (ml) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 aps 20% (µl) 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 temed 20% (µl) 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 45 nahco3 (mg) 100 100 100 100 100 100 100 100 100 100 100 100 100 150 200 250 total (g) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 199 statistical analysis results were expressed as mean ± sd for each. analysis of variance (anova) test was used to analyze the difference among many groups. while students ҆ t-test was used to analyze the difference between the two groups by utilizing spss20 software window. a probability value (p < 0.05) was considered the minimum level of statistical significance. best formula selection the choice of the best formula relied on the correlation of the resulted sph concerning to release profile of reference (carvedilol 6.25 mg tablet/ roche). the similarity factor (f 2) introduced by moore & flanner was utilized as a standard for determination of the best formula according to the following equation (4) where, n number of dissolution time points, r and t are the reference and test dissolution values at time t (10). fourier transform infrared (ftir) analysis the sph was desiccated and converted to a powdered form for ftir analysis to study the compatibility of the drug with polymers. the powdered specimen (2-3mg) was combined with kbr. the ftir spectrum was measured in the range 4000-600 cm-1 utilizing ftir spectrometer (18). capsule pre-formulation tests angle of repose measurement the angle of repose was measured by funnel method in which a funnel was held in a stand vertically at a specified height over a piece of paper put on the horizontal region, the bottom of the funnel was closed and sample powder was filled in the funnel, then the funnel was opened to release the powder to create a conical shape. conical diameter was measured in different directions and the conical height was measured by utilizing a scale. the angle of repose value was determined by the following equation: tan θ = h / r…… (5) where θ = angle of repose, h = conical height, r = conical radius the angle of repose values may be used as a powder flow guide as shown in table (2) (19). table 2. relationship between angle of repose and powder flow (20) angle of repose powder flow ˂ 25 excellent 25-30 very good 31-35 good 36-40 fair 41-45 passable 46-55 poor ˃ 56 very poor carr ҆ s index measurement carr ҆ s index provides a view about powder flow properties and can be calculated by measuring bulk and tapped densities. the bulk and tapped densities were calculated via simple filled cylinder tapping method. in this method, a graduated cylinder was filled with drug sample powder and the resulted volume (bulk volume) was measured. then after, the cylinder was tapped on a flat surface and the resulted volume changed to (tapped volume). both densities have been calculated by utilizing the following equations (21). dbulk = m/vb …. (6) dtapped = m/vp …. (7) where, m = mass powder, vb = bulk volume, dbulk = bulk density, vp= tapped volume, dtapped = tapped density % carr ҆ s index = [(dtapped – dbulk)/dtapped ] *100 ….(8) the correlation between index values and flow type is shown in table (3) table 3. relationship between carr ҆ s index and powder flow (20) carr ҆ s index (%) flow description ˂ 10 excellent 11-15 good 16-20 fair 21-25 passable 26-31 poor 32-39 very poor ˃ 40 very poor iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 200 evaluation of capsules weight variation test twenty capsules were taken randomly and weighed to calculate the average weight. then after, every capsule was weighed individually and weight of the empty capsule shell was subtracted from the gross weight. if the individual capsule weight in the range of (90-110%) of average weight, it passed the weight variation test (22). disintegration test this test was done by the disintegration apparatus; the water bath was filled to the mark with tap water and the temperature was adjusted to 37±0.5 ℃. the used beaker was filled with distilled water (600 ml) and suspended in the main bath, one capsule was placed in each of the six glass tubes and the disc may be used if the dosage form floats. the apparatus rotation was set at 28-32 cycles/minute and worked till all capsules were disintegrated or leaving the only small remnant of gelatin shell (soft mass with no firm core) on the mesh (23). drug content uniformity the content of five capsules was weighed accurately and the content of every capsule was poured in 100 ml volumetric flask and dissolved in 0.1n hcl (volume made up to the mark of flask). this solution was filtered through whatman filter paper, and then solution absorbance was measured at 285 nm by uv spectrophotometer to calculate drug content by using the equation of calibration curve (24). dissolution test the capsule rate of release was determined using the dissolution apparatus (basket type), in the beginning, the hard gelatin capsules were put in the basket, the apparatus was operated at 37±0.5c˚ and 100 rpm then the apparatus jar was filled with 900 ml of 0.1n hcl for each capsule. sample of 5 ml was withdrawn at 30 min interval for 4 h duration and replace with fresh buffer. the sample absorbance was measured using uv spectrophotometer at 285 nm after filtration via whatman filter paper to calculate the percent drug release using the equation of standard curve (25). mathematical models of drug release various mathematical models were employed to describe drug release kinetics from the sph loaded with active drug. however, the kinetics of carvedilol release from sph was concluded by finding the best fit of release data to zero order, first order, higuchi and korsmeyer-peppas plots (26). stability study (effect of temperature) the temperature effect on the selected formula (f8) of carvedilol was examined. this study was achieved by placing the capsules in ovens at various temperatures (40 and 60 c˚) for 7 weeks and samples were taken at certain intervals of time to calculate drug percent remaining (27). results and discussion carvedilol superporous hydrogel hybrid (sph) the mechanism for preparation of sph was polymerization of monomers in the presence of gas bubbles. the blowing technique utilized for the preparation of sph based on two processes polymerization and foaming, in the polymerization step the mixture of aps/temed redox pair will trigger radical polymerization then the monomers bond covalently to each other and form prolonged chain by crosslinking by bismethylacrylamide. while in the foaming process, the reaction of nahco3 with acidic content will generate carbon dioxide gas bubbles that stabilized by tween 80. the foam size was concluded by the amount of gas bubbles released that in turn was determined by the acid and nahco3 amount (10). effect of cross-linker concentration and type the effect of concentration and type of the cross-linker (bisacrylamide and glutaraldehyd) on the physical properties sph hybrid was represented by f1-f7 as shown in table (4). table 4. effect of cross-linker concentration and type on physical properties of carvidelol sph formula no. density g/3cm swelling time (min) swelling ratio porosity floating lag time (min) floating time (min) drug content (%) drug release (%) f1 1.2± 0.02 75 0.36 0.33± 0.025 70 61 99% 89% f2 1.23± 0.01 55 0.32 0.31± 0.015 70 75 106% 89% f3 1.24± 0.005 50 0.29 0.29± 0.05 75 72 93% 86% f4 1.27± 0.01 35 0.26 0.26± 0.01 80 70 108% 86% f5 1.28± 0.05 30 0.24 0.23± 0.025 _ 0 89% 80% f6 0.83± 0.05 75 0.46 0.15± 0.025 90 140 102% 96% f7 0.93± 0.05 55 0.41 0.1± 0.025 75 210 98% 95% iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 201 results showed that the increase in cross-linker concentration (both types) led to density increase because of diminishing of network space and less water entered the hydrogel. while, porosity decreased due to formation of strong bonds among the polymers tend to reduce interconnecting pores size. swelling ratio and time decreased due to the hydrogel took water molecules up by capillary force much faster than diffusion. however, there was drug release reduction because of reducing the porosity. since the polymers were compatible with carvedilol, the drug content was within the acceptable range. the floating property for bismethylacrylamide containing formulas was due presence of swellable polymer (chitosan) and effervescent agent (nahco3) except, (f5) because it exhibited the least swelling capacity. while formulas contain glutaraldehyde (f6 & f7) showed low densities (˂1 g/cm3) although the presence of swellable polymer and effervescent agent (17, 28). statistically the cross-linker effect was significant (p ˂ 0.05) on the density, swelling ratio, and drug release (29). effect of chitosan concentration the effect of chitosan concentration on the sph hybrid properties was represented by f1 (1%), f8 (2%), f9 (3%) and f10 (4%) as shown in table (5). table 5.effect of chitosan concentration on sph hybrid properties formula no. density g/3cm swelling time (min) swelling ratio porosity floating lag time (min) floating time (min) drug content (%) drug release (%) f1 1.2± 0.02 75 0.36 0.33± 0.025 70 61 99% 89% f8 1.21± 0.02 60 0.29 0.19± 0.02 84 160 86% 79% f9 1.24± 0.001 55 0.27 0.16± 0.001 55 80 102% 76% f10 1.28± 0.03 50 0.23 0.13± 0.02 55 80 87% 75% the results showed that increase in chitosan concentration resulted in porosity reduction and density increase (significant effect, p ˂ 0.05) due to prevent the bubbles from escaping from the solution mixture (increased viscosity) and accumulation at the periphery of the pore. as well as, there was a decrease swelling time and ratio since, the formed hydrogen bond between chitosan and other polymers decrease the ability to form hydrogen bond with water molecules thus limiting water absorption. there was a significant effect (p ˂ 0.05) on floating properties (a decrease in floating lag time and increase in floating time) because of increasing concentration of swelling polymer. the decrease in the drug release (significant effect, p ˂ 0.05) was due to decrease in the porosity. the drug content was in the acceptable range due to compatibility of chitosan with the active ingredient (28, 30). effect of pva amount the effect of pva amount on the sph hybrid properties was represented by f1, f11, f12 and f13 which were prepared with 150, 200, 250, and 300 mg pva, respectively. the results were shown in table (6). table 6. effect of pva amount on sph properties. formula no. density g/3cm swelling time (min) swelling ratio porosity floating lag time (min) floating time (min) drug content (%) drug release (%) f1 1.2± 0.02 75 0.36 0.33± 0.025 70 61 99% 89% f11 1.25± 0.04 70 0.35 0.2± 0.03 65 90 93% 80% f12 1.3± 0.01 65 0.32 015± 0.07 73 100 103% 77% f13 1.35± 0.03 55 0.3 0.12± 0.02 75 110 109% 75% the results showed that, the increase in pva amount led to; density increase due to cellulosic fibers presence in the structure of polymer, decrease in the porosity and swelling properties owing to the increase of solution viscosity during gelation, drug release reduction because of decreasing the porosity, a significant increase (p ˂ 0.05) in floating time due to the increased viscosity prevents the bubbles from escaping and because of compatibility of pva with iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 202 carvedilol, the drug content was within acceptable result (31, 32) . effect of foaming agent amount formulas; f1, f14-f16 were prepared with different amounts of foaming agent (nahco3) and evaluated to show the foaming agent amount effect on sph properties as demonstrated in table (7) . table 7. effect of foaming agent amount on sph properties. formula no. density g/3cm swelling time (min) swelling ratio porosity floating lag time (min) floating time (min) drug content (%) drug release (%) f1 1.2± 0.02 75 0.36 0.33± 0.025 70 61 99% 89% f14 1.18± 0.03 75 0.32 0.18± 0.02 70 110 85% 84% f15 1.15± 0.01 80 0.3 0.16± 0.04 60 130 98% 82% f16 1.1± 0.03 85 0.25 0.12± 0.02 55 150 95% 78% the results showed that increase in the foaming agent amount led to; decrease in porosity and density due to accumulation of generated carbon dioxide, slow swelling owing to the reduced porosity, since the foaming agent led to carbon dioxide generation which resulted to floating of sph. the reduced porosity led to decrease drug release while, drug content within acceptable range due to no interaction between carvedilol and nahco3 (33, 34). scanning electron microscopy (sem) of sph the scanning electron microscopic photographs of the selected formula (f8) at various magnification powers were shown in the figure (2). figure 2. scanning electron microscopic photographs of sph iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 203 the best formula by comparing the dissolution profile of formulas which exhibited significant differences in many properties (f2, f4, f5, f6 and f8) with that of reference (carvedilol 6.25 mg tablet/roche) to select best formula by similarity factor (f2) equation, f8 showed the higher value as shown in table (8) while, figure (3) showed the drug release comparison between selected formula (f8) and carvediol reference tablet. table 8. similarity factor values for selection of best formula figure 3. dissolution profile of carvidelol tablet/roche and selected sph (f8) fourier transform infrared (ftir) compatibility study to examine the compatibility of drug with polymers of sph, two samples were tested by ftir (pure carvedilol and dried sph) as shown in figures (4 and 5) and some variations were listed in table (9). figure 4. pure carvedilol ftir spectrum formula no. f2 value f2 29 f4 31 f5 32 f6 42 f8 56 iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 204 figure 5. dried sph hybrid ftir spectru table 9 variation in ftir analysis of carvedilol and sph hybrid (f8) no. assignment groups theoretical values carvedilol dried sph hybrid 1 aliphatic secondary nh stretch 3360-3310 3340.71 3338.95 2 secondary nh bond 1650-1550 1631.78 1630.54 4 aromatic secondary cn stretch 1350-1280 1346.31 1349.76 5 oh in plane bend 1350-1260 1303..88 1302.32 6 oh out of plane bend 720-590 621.08 597.59 7 aromatic ch in plane bend 1325-950 1211.3 1191.69 8 c-n stretch 1350-1000 1095.57 1322.19 9 c-o stretch 1150-1050 1118.71 1099.38 the results (figure 4 and 5) showed that, the major peaks of pure carvedilol and f8 were not affected and predominantly observed in the theoretical range, which indicated no interaction between the drug and the polymers. the change in figure (5) was in the near and mid ir region due to presence of (oh) group in the carvedilol and formula polymers (35, 36). results of capsule pre-formulation angle of repose and carr’s index the result values of angle of repose were in the range of excellent flow properties (ɵ = 15.622.3) of the pre-formulation mixture. as well as, all the resulted values of carr’s index were in the range of excellent, good, and fair flow properties (9%16%) (37). capsule evaluation no variation in weight of capsules since, results of weight variation were within acceptable limits (38). in addition, results of disintegration time were in the range 14-17 min. these results were passed the criteria of disintegration of capsules in the british pharmacopoeia (9). drug content uniformity results were within the range of 85-115% which indicated the uniform distribution and proper dose of active ingredient in the selected formula (capsule) (38). dissolution of carvedilol sph as capsule dissolution test was done for the selected formula (f8) after encapsulation and compared with that of reference tablet (carvedilol 6.25 mg tablet/roche). capsules of f8 gave the highest drug release within 4 hr. as shown in the figure (6). iraqi j pharm sci, vol.30(2) 2021 formulation and in-vitro evaluation of carvedilol capsule as superporous hydrogel 205 figure 6. drug release profile of carvedilol sph as capsule (f8) in comparison with carvedilol 6.25 mg tablet/roche mathematical model of drug release for determining the release mechanism of carvedilol capsule, table (10) showed the correlation coefficient for each model by using ms-excel software. table 10. mathematical models correlation coefficient values no. mathematical model correlation coefficient r2 1. zero order 0.9612 2. first order 0.5643 3. higuchi 0.9623 4. korsmeyerpeppas 0.9488 the results indicated that, dissolution release data were best fitted to the higuchi model due to the highest r2 value and the release mechanism was (fickian) diffusion. stability of carvedilol sph hybrid capsule the shelf life of carvedilol (f8) capsule was calculated by constructing the arrhenius plot as shown in figure (7). the rate constant of degradation k25 at 25˚c was found to be 1.58*10 -3 week-1 and the shelf life was 66.5 weeks since, carvedilol degradation followed first order kinetic (39). figure 7. arrhenius plot of carvedilol (f8) capsules conclusions based on the results gained, one can conclude the following: carvedilol sph is a good tool to improve the solubility (from the floating time, increase gastric residence time). the best formula of carvedilol sph was (f8) which composed of chitosan (2%), bis (1%), pva (150 mg) and nahco3 (100 mg). the release of carvedilol was best fitted to higuchi model with mechanism of fickian diffusion, and the estimated shelflife of carvedilol sph capsules was 66.5 weeks. references 1. gadge g, sabale v, khade a, mahajan u. current approaches on gastro retentive drug delivery system: an overview. international 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drug delivery systems, 10th ed. london: lippincott williams & wilkins; 2014. 39. sinko pj, singh y. martin's physical pharmacy and pharmaceutical sciences. 6th ed. philadelphia: lippincott williams & wilkins; 2011. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. doi: https://doi.org/10.31351/vol30iss2pp122-134 122 isolation and identification of phenolic compounds from dianthus orientalis wildly grown in iraq. khansaa hussein atiyah*, 1 and enas j kadhum* *ministry of higher education and scientific reserch, *department of pharmacognocy and medicinal plant, college of pharmacy, university of baghdad, baghdad ,iraq. abstract the plant dianthus orientalis that belongs to the caryphyllaceae family is one of the useful plants in iraq. its seeds are commonly used for toothache. this project provides the first comprehensive research done in iraq and the world to study the phytochemicals and the methods of extraction and isolation of active constituents from dianthus orientalis wildly grown in iraq. the plant was harvested from penjwin in al-sulaymaniyah city, iraq in september 2019.the whole plant were washed carefully, dried in shade area for two weeks, and milled in a mechanical grinder to a coarse powder. the plant was defatted by maceration with hexane for 7days and dried after that extracted by cold extraction methods using 80% methanol solvent for 9 days then fractionation with chloroform, ethyl acetate and n-butanol to separate the active constituents according to the change in polarities. the chloroform, ethyl acetate fractions were used for identification and isolation of phenolic compounds by tlc, ptlc, hplc and lc/mass, ftir. results of the phytochemical screening exposed the presence of, phenols in the plant extract. the phenolic compound (vanillic acid, coumaric acid, cinnamic acid, genistein, oleuropein) were separated and purified by ptlc. the isolated compounds were subjected to several chemical, chromatographic and spectral analytical techniques for their identification such as tlc, hplc, ftir and lc/mass. keywords: vanillic acide, coumaric acides, cinnamic acid, genistein, oleuropein, hplc, lc/mass. القرنفل البري الذي ينمو بصورة طبيعية في العراقعزل وتحديد المركبات الفينولية الموجودة في نبات *ايناس جواد كاظم و ،1خنساء حسين عطية* لعراق .،ابغداد ، جامعة بغداد ، كلية الصيدلة ،فرع العقاقير والنباتات الطبية * الخالصة ( من النباتات. المفيدة في العراق تستخدم بشكل caryphyllaceaeيعد نبات القرنفل المشرقي أو القرنفل البري الذي ينتمي إلى عائلة ) ستخالصها شائع كمسكن ألالم األسنان يعتبر هذا البحث أول بحث شامل في العراق وفي العالم لدراسة المواد الكيمياويه الموجودة في النبات وطرق ا وتم غسل وتجفيف النبات لمدة أسبوعان وطحنه 2019 وفصلها. تم جمع النبات من قضاء بنجوين في محافظة السليمانية في شهر أيلول من سنة %80عملية االستخالص بالطريقة الباردة طريقة نقع النبات في تمت ازالة الدهون بتنقيعها بالهكسان لمدة اسبوع وبعدها وتمبالمطحنة الميكانيكية الكلوروفورم، خالت االثيل والبيوتانول لفصل المركبات الفعالة اعتمادا ميثانول لمدة تسعة ايام. تمت عملية التجزئة باستخدام عدة مذيبات هي بالتتابع: ت مثل على االختالف في القطبية بين هذه المكونات. استخدمت طبقتي الكلوروفورم وخالت االثيل في التعرف وعزل المركبات الفينولية بعدة تقنيا رية وكروماتوغرافيا السائل عالية األداء وكروماتوغرافيا السائل وكانت نتيجة كروماتوغرافيا الطبقة الرقيقة وكروماتوغرافيا الطبقة التحضي حامض الكشوفات الكيميائية وجود مواد فينولية ومواد دابغة في المستخلص. وتم كشف وعزل المركبات الفينولية )حامض الفانلك وحامض السينامك و ا الطبقة الرقيقة وكروماتوغرافيا السائل عالية االداء،مطياف االشعه فوق البنفسجية, الكومارك والجنستين واالوليوروبين( بواسطة كروماتوغرافي كروماتوغرافيا السائل. كروماتوغرافيا السائل ، الكلمات المفتاحية: حامض الفانلك وحامض السينامك وحامض الكومارك والجنستين واالوليوروبين,كروماتوغرافيا السائل عالية االداء تلي. والطيف الك introduction dianthus l. is annual or perennial herb belongs to angiosperm’s family caryophyllaceae, subfamily caryophyllideae and tribe caryophyllideae(1). the family caryophyllaceae is well known for ornamental flowering plants(2). the unusual characteristic of the family is appearance of stable and endurable foam when parts of the plants are put into water and shaken. this behavior is due to the occurrence of high amount of saponins in the family(3). a number of other compounds such as fatty acid derivatives, benzenoids, phenyl propanoids, isoprenoids, and nitrogen containing compounds are also isolated from the plants belonging to the family(4,5-6). dianthus orentalis herbs suffruticous perennial, stems 25-50cm tall, sterile shoots absent, leaves pale green, linear, 1.5-5.5cm*0.5-3mm, flowers usually solitary. 1corresponding author e-mail: khansaa.sedo89@gmail.com received: 31/10/2020 accepted: 22/ 3/2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp122-134 iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. doi: https://doi.org/10.31351/vol30iss2pp122-134 123 epicalyx bracts ovate to ovate-oblong,4-10,covering 1l3-1l2 of calyx length .calyx pale green, conical, distinctly narrowed toward apex,1.7-2.4cm*2.53.5mm;petal limb pink,0.8-1.2cm long, fimbriate; petal claw 1.6-2.4cm long, exerted from calyx. habitats mountainsides and cliffs, rocky soil; elevation740-1650m, flowering in apr-may. occurance occasional and distribution in kurdistan iraq and iran(7) .taditional uses: dianthus orientalis seed used for tooth ache(8). dianthus caryophyllus is a very important species in caryophyllecea family it was used traditionally in the treatment of throat and gum infections, in the treatment of wounds, as cardio-tonic, diaphoretic, vermifuge and for the treatment of gastro-intestinal disorder. the plant traditionally used in china, japan and korea in the treatment of wounds and gastro-intestinal disorder and various other ailments(9-11).the chemical composition and the essential oil of the carnation flowers (dianthus caryophyllus) were studied. phytochemical tests showed that of dianthus caryophyllus contained triterpenes, alkaloids, coumarins and cyanogenic glycoside(12).the chemical composition and the essential oil of the carnation flowers (dianthus caryophyllus) was studied. twelve volatiles were identified by gas chromatography-mass spectrometry (gc-ms) as the main components of carnation flower oil. the major components were phenyl ethyl alcohol, eugenol, hexyl benzoate, hexenyl benzoate, benzyl benzoate, benzoin, nootkatone, benzyl salyicylate, m-cresyl phenyl acetate, hexadecanoic acid and eicosene(13).three flavonoids including apigenin-c-glycoside, kaempferol 3-o-β-d-glucopyranosyl-(1→2)-o-[α-lrhamnopyranosyl-(1→6)]-β-d-gluco-pyranoside and kaemp-ferol 3-o-[α-l-rhamnopyranosyl(1→6)]-β-d-glu-copyranoside(14-15),. two benzoic acid derivatives, protocatechuic acid and vanillic acid, flavonol glycoside peltatoside and flavone datiscetin were isolated from the plant(16). material and method collection of plant materials dianthus orientalis were obtained from penjwin in al-sulaymaniyah city, iraq in september 2019.the plant was identified and authenticated by dr.karzan aumar kadir /department of biology /college of sciences/ university of sulaimani the plant were washed thoroughly, dried under shade, and ground in a mechanical grinder to a coarse powder. equipment and chemical the instruments used were rotary evaporator (bȕchi rotavapor r-205, swiss), sonicator (branson sonifier, usa), highperformance liquid chromatography (hplc) (knuaer , germany). all chemicals and solvents used were of analytical grade and obtained from riedel-de haen, germany, except methanol, which is hplc grade was purchased from sigma-aldrich, germany. the standard vanillic acid, coumaric acid, were purchased from chengdu bio purify phytochemicals, china (purity >97). tlc aluminum plates pre-coated with silica gel (20 cm×20cm, 0.2 mm thick) used were obtained from machereynagel-germany. extraction the whole plant coarse powder 250gram was macerated with normal hexane for one week in conical flask 2000ml with shaking many times in the shade and then filter it, and then the organic layer was taken and dried in the shade the defatted powdered plant material was soaked in 1500ml methanol, with occasional shaking, at room temperature. after 3 days, the methanol soluble materials were filtered off and this method is repeated for three times (extraction will done in 9 days).the filtrate was evaporated to dryness under vacuum using rotary evaporator. a dark browngreenish residue was obtained. the residue twenty grams was suspended in 500ml water and partitioned successively with chloroform, ethyl acetate, and n-butanol (3x500ml) for each fraction. the first two fractions dried over anhydrous sodium sulfate, filtered, and evaporated to dryness. preliminary phytochemical investigation alkaloids, saponin, phenolic compounds investigation were carried out with: alkaloids test test for saponins tests for phenols: a: ferric chloride test, b: naoh test, isolation of phenolic compounds from the ethyl acetate fraction and chloroform fraction by preparative layer chromatography (plc): one gram of each fraction dissolve in 3 ml of methanol and applied on the number of plc plates as a semi concentrated solution in streak using a capillary tube on each plate, then the plate placed inside glass tank which contained the solvent system (chloroform: acetone :formic acid)(75:16:1). the band had been scrapped off, eluted with methanol and then filtered; the filtrate evaporated to dryness, the band that separated from ethyl acetate fraction was symbolized as e1. isolation from the chloroform fraction by preparative layer chromatography (plc): four bands were isolated from chloroform fraction utilizing the same procedure applied to the ethyl acetate fraction and using the same mobile phase (chloroform: acetone: formic acid) (75:16:1) the compounds were isolated from chloroform fraction were symbolized as c1, c2, c3, c4. https://doi.org/10.31351/vol30iss2pp122-134 iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 124 identification of the isolated phenolic derivatives from ethyl acetate and chloroform fraction of dianthus orientalis the compound that symbolized as e1, c1, c2, c3, c4 was identified by several methods including chemical, chromatographic, and spectral methods as: spraying with 5% ethanolic koh on tlc plate hplc analysis hplc technique (knuaer, germany) was applied for the detection of different constituents found in the ethyl acetate, chloroform fractions as flavonoids and phenolic acids, and for identification of the isolated compounds from dianthus orientalis.the mobile phase contains 1% aq. acetic acid solution (solvent a) and acetonitrile (solvent b), the flow rate was adjusted to 1 ml/min, the column was thermostatically controlled at 280 ˚c and the injection volume was kept at 20 μl. a gradient elution was performed by varying the proportion of solvent b to solvent a as shown in the table (1). the hplc chromatograms were detected using a photo diode array uv detector at three different wavelengths (272, 280 and 310 nm) flow rate 1ml|min(17). table 1. the gradient elution changing a and b proportion with time time mobile a % mobile b % 0 90 10 28 60 40 39 40 60 60 10 90 ftir identified chemical bands in molecules. ir spectra range of scanning was 4000-400 cm-1 lc/ms: analytical lc-ms was performed using agilente/system joined to an applied biosystems api 2000 mass spectrometer .mobile phase solvents acetonitrile and water a column of 0.19mm external diameter (75μm i.d.) and 200mm length was packed with thermo scientifice hypersil gold c18 with 5μm particle size. samples were run under the following conditions: m/z range was 250 to 10001, 200k resolution, and dynamic exclusion set to 1 with a limit of 90 seconds. result and discussion table 2. phytochemical screening of dianthus orientalis plant chemical test for phenols results a.ferric chloride test positive due to formation of dark brown color b.naoh positive due to formation of yellow color . 3.saponin test positive due to froth formation. 4.alkaloid test: dragendorff‘s reagent positive due to formation of orange-brown precipitate high-performance liquid chromatography (hplc) examination of ethyl acetate fraction and chloroform fraction figure 1. hplc chromatogram for ethyl acetate fraction iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 125 figure 2. hplc chromatogram for chloroform fraction. figure 3. tlc chromatogram for isolated cpd a: e1 and standard vanillic acid b: c4 and standard coumaric acid developed in the (chloroform acetone: formic acid) (75:16:1) solvent system, detect under uv light at 254. identification of e1 spraying with 5%ethanolic koh on tlc plate give yellow colored spot. hplc of isolated e1: the hplc chromatogram of standard vanillic acid and isolated cpd e1were shown in figure (4), spectrum of std vanillic acid and isolated cpd e1 and as shown in figures (5). ftir ir spectrum of isolated cpd e1 was showed in the figure (6) and interpretation of the bands in the table (3) lc/mass analytical lc-ms was performed using an agilent system joined to an applied biosystems api 2000 mass spectrometer. the lc-ms chromatogram of the isolated compounds e1 as in figure (7) . figure 4. hplc chromatogram of standard vanillic acid and isolated cpd e1. iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 126 figure 5. uv spectrum of standard vanillic acid and isolated cpde1. figure 6. ir spectrum of isolated cpde1 table3. interpretation of the ir bands for e1are shown below ir band of isolated cpde1 interpretation 3290 oh stretch vibrations band 2939 c-h asymmetric stretching 2828 c-h symmetric stretching 1663 c=o stretching 1448 c=c aromatic stretching 1266 in planec-h bending 1110 c-o-c stretching 809 out of plane c-h aromatic bending 679 out of plane c=c aromatic bending iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 127 figure 7. lc/ms chromatogram of isolated compound e1 all these data coincide with that reported for vanillic acid therefore compound e1 could be vanillic acid with mwt 168.1gram/mol. identification of c4 spraying with 5%ethanolic koh on tlc plate give yellow colored spot. hplc for isolated cpd c4: the hplc chromatogram of standard coumaric acid and isolated cpd c4 are shown in figure (8), spectrum of std coumaric acid and cpd c4 and as shown in figure (9). ftir: ir spectrum of isolated cpd c4 was showed in the figure (10) and interpretation of the bands in the table (4) iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 128 figure 8. hplc chromatograme of standard coumaric acid and isolatedcpd c4 figure 9. uv spectrum of standard coumaric acid and isolated cpd c4 ftir figure 10. ir spectrum of isolated cpdc4 iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 129 table 4. interpretation of the ir bands for c4are shown below. ir band of isolated cpdc4 interpretation 3307 oh stretch vibrations band 2972 c-h asymmetric stretching 2877 c-h symmetric stretching 2360 c=c stretching 1645 c=o stretching 1448 c=c aromatic stretching 1375 o-h bending 1085 c-o stretching 879 out of plane c-h aromatic bending 524 c-h stretching all these data coincide with that reported for coumaric acid therefore compound c4 could be coumaric acid. identification of c1 spraying with 5%ethanolic koh on tlc plate give yellow colored spot. hplc of isolated c1: the hplc chromatogram of standard oleuropien and isolatedcpd c1, spectrum of oleuropien standard and isolated cpd c1 were shown in the figures (11, 12) respectively. figure 11. hplc chromatograme of standard oleuropien and isolate cpdc1 figure 12. uv spectrum of standard oleuropien and isolate cpdc1 ftir: ir spectrum of isolated cpd c1 is showed in the figure (13) and interpretation of the bands in the table (5) iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 130 figure 13.ir spectrum of isolated cpdc1 table 5. interpretation of the ir bands for c1are shown below. ir band of isolated cpdc1 interpretation 3294 oh stretch vibrations band 2972 c-h asymmetric stretching 2875 c-h symmetric stretching 1683 c=o stretching 1492 c=c aromatic stretching 1379 o-h bending 1139 c-o stretching 1087 in plane c-h aromatic bending all these data coincide with that reported for oleuropien therefore compound c1 could be oleuropien. identification of c2 spraying with 5%ethanolic koh on tlc plate give yellow colored spot. hplc of isolated c2: the hplc chromatogram of standard genistein and isolated cpd c2 was shown in figure (14), spectrum of standard genistein and isolated cpd c2 was shown in figures (15). figure 14. hplc chromatogram of standard genistein and isolated cpd c2 iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 131 figure 15. uv spectrum of standard genistein and isolated cpdc2 ftir: ir spectrum of isolated cpd c2 was showed in the figure (16) and interpretation of the bands in the table (6). figure 16. ir spectrum of isolated cpdc2. table 6. interpretation of the ir bands for c2 were shown below. ir band of isolated cpdc2 interpretation 3204 oh stretch vibrations band 2941 c-h asymmetric stretching 2808 c-h symmetric stretching 2337 c=c aromatic stretching 1417 c=c aromatic stretching 1122 c-o stretching all these data coincide with that reported for genistein therefore compound c2 could be genisteine. identification of c3 spraying with 5%ethanolic koh give yellow colored spot. hplc of isolated c3: the hplc chromatogram of standard cinammic acid e and isolated cpdc3, spectrum of cinammic acid std and isolated cpd c3, were shown in figures (17, 18). . iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 132 figure 17. hplc chromatogram of standard cinammic acide and isolated cpd c3. figure 18. uv spectrum of cinammic acid std and isolated cpdc3 ftir: ir spectrum of isolated cpd c3 was showed in the figure (19) and interpretation of the bands in the table (7) figure 19.ir spectrum of isolated cpdc3 iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 133 table 7.interpretation of the ir bands for c3 were shown below. ir band of isolated cpdc3 interpretation 3385 oh stretch vibrations band 2970 c-h asymmetric stretching 2883 c-h symmetric stretching 1683 c=0 stretching 1446 c=c aromatic stretching 1389 o-h bending 1087 c-o stretching 1033 in plane c-h aromatic bending 879 out of plane c-h aromatic bending all these data coincide with that reported for cinammic acid therefore c3cpd could be cinammic acid conclusion the following points were pinched based on prior findings; 1. phytochemical screening of dianthus orintalis widely grown in iraq demonstrates the presence of various phytochemicals, which were separated from plant according to differences in their chemical nature. 2. the phenolic compounds: vanillic acid, coumaric acid, genistein, cinammic acid, and oleuropein were isolated from the plant. 3. isolated phenolic acids were identified by tlc, preparative tlc, hplc,ir lc/mass acknowledgements the authors are grateful to acknowledge the college of pharmacy/university of baghdad for providing the necessary facilities to carry out this study. references 1. bittrich, v., introduction to centrospermae. in: the families and genera of vascular plants, vol. ii, magnoliid, hamamelid and caryophyllid families, kubitzki, k., j.g. rohwer and v. bittrich (eds.). springerverlag, berlin, germany; 1993 pp. 13-19 2. holm l.g., plucknett d.l., pancho j.v., herberger j.p. the, honolulu: the world's worst weeds; university press of hawaii; 1977.pp. 111–114. 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sciences; 2017.vol. 9(6), 796-799. 9. chandra s, rawat ds, chandra d and rastogi j. nativity, phytochemistry, ethnobotany and pharmacology of dianthus caryophyllus. research journal of medicinal plant; 2016.10 (1): 1-9. 10. usher g. a dictionary of plants used by man. macmillan pub co 1974. 11. al-rawi a and chakravarty l. medicinal plants of iraq. 2nd ed., ministry of agriculture, baghdad; 1988: 93. 12. eltayeb ra. study of some chemical constituents of dianthus caryophyllus and elettaria cardamomum. thesis, university of khartoum; 2016. 13. el-ghorab ah, mahgoub mh and bekheta m. effect of some bioregulators on the chemical composition of essential oil and its antioxidant activity of egyptian carnation (dianthus caryophyllus l.). journal of essential oil bearing; 2006.9(3): 214-222. 14. galeotti f, barile e, lanzotti v, dolci m and curir p. quantification of major flavonoids in carnation tissues (dianthus caryophyllus) as a tool for cultivar discrimination. z naturforsch c; 2008. 63(3-4):161-168. iraqi j pharm sci, vol.30(2) 2021 dianthus orientalis l. 134 15. galeotti f, barile e, curir p, dolci m and lanzotti v. flavonoids from carnation (dianthus caryophyllus) and their antifungal activity. phytochemistry letters; 2008. 1: 44– 48. 16. curir p, dolci m, dolci p, lanzotti v and de cooman l. fungitoxic phenols from carnation (dianthus caryophyllus) effective against fusarium oxysporum f. sp. dianthi. phytochem anal; 2003.14(1):8-12. 17. seal, t. quantitative hplc analysis of phenolic acids, flavonoids and ascorbic acid in four different solvent extracts of two wild edible leaves, sonchusarvensis and oenanthelinearis of north-eastern region in india. journal of applied pharmaceutical science; 2016. 6(2), 157-166. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 hypolididemic activity of mistletoe leaves polysaccharides doi: https://doi.org/10.31351/vol32iss1pp75-83 75 lipid -lowering effect of polysaccharide (pectin) of viscum album l. plant in rats adzhiakhmetova s.l.* chervonnaya n.m.**. pozdnyakov d.i**,1 and oganesyan s.o.* department of organic chemistry, pyatigorsk medical and pharmaceutical institute, pyatigorsk. russia  department of pharmacology with course of clinical pharmacology, pyatigorsk medical and pharmaceutical institute, pyatigorsk. russia abstract disturbances of lipid metabolism is a predisposing factor of cardiovascular diseases. which are accompanied by high mortality rates of the population ischemic heart disease and ischemic stroke. plant polysaccharides can be promising remedies for correction of lipid imbalancein this regard. the aim of the study was to assess the hypolipidemic activity of polysaccharides isolated from the leaves of mistletoe. the test-object was the leaves of the white mistletoe (viscum album l.) growing on the apple (malus domestica borkh.). and the pear (pyrus communis l.). polysaccharides (water-soluble polysaccharides and pectin substances) were quantitatively determined by the gravimetric method followed by reprecipitation from ethanol. the qualitative composition was evaluated by thin layer chromatography. functional groups in polysaccharides were determined by titrimetric method. the hypolipidemic activity of the obtained polysaccharides was determined under oral administration at a dose of 100 mg / kg in wistar rats by the change of the concentration of total cholesterol. triglycerides. ldl and hdl cholesterol. the change of the content of cholesteryl ester transfer protein and niemann-pick c1-like1 protein was also determined. as a result. it was found that among the polysaccharides in the leaves of mistletoe. pectin substances are predominate. which belong to the group of low-esterified pectins. a significant content of free carboxyl groups in pectins from the leaves of mistletoe was also found. the study of hypolipidemic activity showed that. among the polysaccharides of mistletoe leaves. pectin substances exert a more pronounced effect on lipid metabolism. the use of which reduced the content of triglycerides. total cholesterol and ldl cholesterol with an increase in ldl cholesterol in rats. probably due to a decrease in the activity of cholesteryl ester transfer protein and niemannpick c1-like1 protein. at the same time. at the level of the trend. mistletoe pectins showed a higher level of activity than analogous compounds obtained from the leaves of host-plants. it should be noted that the severity of the pharmacological effect of mistletoe pectins did not depend on the host-plants. thus. based on the obtained results. it can be assumed that pectins obtained from the mistletoe leaves can be a potentially effective and safety hypolipidemic agent. keywords: plant polysaccharides. pectin substances. cholesterol-lowering activity. cept. npcl1. introduction white mistletoe (viscum album l.) is a semi-parasitic plant. as it is able to synthesize organic matter through photosynthesis. and receives moisture and minerals from the phloem of the affected plant the host with the help of a special vascular body. penetrating its roots deep under the bark(1-3). most often. viscum album parasitizes: populus nigra. pyrus communis. malus domestica; less often on carpinus betulus. quercus robur. juglans regia. robinia pseudoacacia; extremely rarely on juniperus communis. abies sibirica. pinus sylvestris(3-5). the leaves of hemiparasite white mistletoe are of significant interest for medicine and are diverse in chemical composition. the main biologically active substances of mistletoe leaves: flavonoids (quercetin. 3-methyl ester of quercetin. rhamnetin. isorhamnetin. rhamnazine. 3.7-dimethyl ester of quercetin). phenol carboxylic acids (caffeic and sinapic acids). carotenoids. organic acids. lectins. phenols and their derivatives. nitrogencontaining compounds and others(1-4). in ethnomedicine. mistletoe leaves are used for angina pectoris and chronic osteoarthritis treatment and as a hemostatic. astringent. painkiller. in scientific medicine. an aqueous extract in an experiment delays the growth of a cancerous tumor. inhibits the growth and development of metastases. is able to regulate metabolism. and nonspecifically increases the body's resistance(1). abroad. in the treatment of malignant neoplasms. mistletoe medication are widely used – iscador. abnoba viscum. iscucin. helixor. isorel. viscum compositum. which have high immunomodulatory and antitumor activity. 1corresponding author e-mail: pozdniackow.dmitry@yandex.ru received: 13/1 /2022 accepted: 6/4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp75-83 iraqi j pharm sci. vol.32(1) 2023 hypolididemic activity of mistletoe leaves polysaccharides 76 recent scientific studies have shown that iskador is an effective therapeutic agent for the treatment of chronic viral hepatitis (4,6,7). polysaccharides are vital plant metabolites and promising therapeutic agents. until recently. the study of the pharmacological activity of plant polysaccharides was very limited. but recently there has been significant progress in this area. so yin. et al.. 2019 established the presence of immunoregulatory properties in natural polysaccharides. which were expressed in an increase in the activity of macrophages(8). huang et al.. 2019. showed that plant polysaccharides have a pronounced antioxidant effect. moreover. the antioxidant properties of polysaccharides were realized through the effect on the enzymatic component of the endogenous antioxidant defense system superoxide dismutase and glutathione peroxidase(9). it was also found that polysaccharides isolated from plants prevent the effect of ionizing radiation on the body. while improving the immune status. hematopoietic function and preventing dna damage(10). thus. the high therapeutic potential of plant polysaccharides makes it relevant to study other aspects of the pharmacological activity of these compounds.in this regard. the aim of the study was investigate the polysaccharides in the leaves of the plant the semiparasite of mistletoe (viscum album l.). and the leaves of host plants the apple (malus domestica borkh.) and the pear (pyrus communis l.). materials and methods test objects the test object was the leaves of the white mistletoe (viscum album l.) growing on the apple (malus domestica borkh.). collected in the stavropol region. and the leaves of the white mistletoe (viscum album l.). growing on the pear (pyrus communis l .). collected on the territory of the belorechensky district of the krasnodar region. the raw material is the leaves of both plants. collected during the fruiting phase. the leaves of host-plants were also studied(1. 5). the studied samples of mistletoe and host plants were identified by specialists of the department of pharmacognosy and botany with the course of technology of phytopreparations (head of the department doctor of science (pharm.) konovalov d.a.) with the assignment of the number of the herbarium fund pmpig 17506 pmpig 17510. method of gravimetric separation of water-soluble polysaccharides (wsp) and pectin substances (ps). the quantitative determination of these fractions was determined gravimetrically(11-14). the removal of high molecular weight compounds was carried out by reprecipitation in ethyl alcohol and centrifugation (1000g. 15 min.) of the resulting precipitate (11. 12). to the investigation the qualitative monomeric composition of the isolated fractions. hydrolysis by sulfuric acid was carried out in 10 hours for wsp and in 48 hours for ps at t = 100°c. then. neutralization was carried out by barium carbonate solution using a universal indicator paper to ph = 7. filtered and evaporated in a water bath to a small residue(11, 13, 15, 16). the monosaccharide composition of the fractions was established by ascending thin layer chromatography using standard witness samples. the mobile phase was the solvent systems: pyridine-ethyl acetate-water (1: 2: 2) and n-butanolacetic acid-water (4: 1: 5). and the stationary phase was fn-7 paper (germany). aniline phthalate reagent was used as a developer(11, 16). quantitative analysis of functional groups in pectin substances the percentage of functional groups in pectin substances was determined by the titrimetric method (14, 17, 18, 19). to determine free carboxyl groups (kc) the test-samples of the ps near 1.0 g (accurately weighed) and was placed in conical flasks with a capacity of 300 ml. moistened with 95% ethyl alcohol (to avoid clumping). added 100 ml of distilled water. stirred and left night for complete dissolution of ps. then the resulting mixtures were titrated with a solution of sodium hydroxide (0.1 mol / l) until a red color appeared. which did not disappear within a minute. when 6 drops of hinton's indicator were added. the percentage of free carboxyl groups was calculated using the formula: кс. % = a/m0.45 where: a the volume of sodium hydroxide solution (0.1 mol / l) in ml used for titration; m is an amount of pectin substances. the percentage of methoxylated carboxyl groups (km.%) was calculated by the formula: km.% = b/m0.45 where: b the volume of sodium hydroxide solution (0.1 mol / l) in ml for the second titration; m is an amount of pectin substances. the total number of carboxyl groups (co) is equal to the sum of free and methoxylated carboxyl groups (in percent). co.% = kc.% + km.% the degree of methoxylation (esterification) of pectins was found as the ratio of the content of methoxylated carboxyl groups to the total amount of carboxyl groups (in percent): λ = km / ko ∙ 100% the percentage of methoxyl groups (ch3o) was calculated using the formula: сн3о.% = km(31/45) where: km the content of methoxylated carboxyl groups in pectin powder.%; 31 equivalent weight of ch3o groups; 45 is the equivalent weight of cooh groups. iraqi j pharm sci. vol.32(1) 2023 hypolididemic activity of mistletoe leaves polysaccharides 77 pharmacological study experimental animals the work was performed on 100 male wistar rats weighing 200-220 grams. 3 months old. the animals were obtained from the «rappolovo laboratory animal nursery» (russia. leningrad region) and during the experiment were kept under controlled conditions in the laboratory of living systems of the pyatigorsk medical and pharmaceutical institute. conditions of detention: ambient temperature 22 ± 20c. relative humidity 60 ± 5%. with a 12-hour change of the daily cycle. the rats were housed by 5 animals in macrolon cages on a granular hardwood bedding with free access to water and full diet feed. working with experimental animals was in accordance with generally accepted protocols of experimental ethics: directive 2010/63 / eu of the european parliament and of the council on the protection of animals used for scientific purposes. september 22. 2010 and arrive 2.0 guidelines. the local ethical committee (protocol # 21 dated 06.16.2020) approved the work. hypercholeterolemia model rats were set on paigen's high cholesterol diet (15% natural fat (sunflower oil). 1.25% cholesterol (panreac) and 0.5% cholic acid (panreac)) for 6 weeks. after the specified time the biomaterials were taken for research (20). study design in the course of the work. the following experimental groups (summary 10 groups) were distinguished (by 10 animals in each group): so sham-operated rats. nc negative control; pspc — a group of rats that received pectin from pear; psmd a group of rats that received pectin from a apple tree; psva/pc a group of rats that received pectin substances from mistletoe collected from pear; psva/md a group of rats that received pectin substances from mistletoe collected from apple tree; wsppc a group of rats that received soluble pear polysaccharides; wspmd a group of rats that received water soluble polysaccharides from apple tree; wspva/pc a group of rats that received watersoluble polysaccharides of mistletoe collected from common pear; wspva / md a group of rats that received water-soluble polysaccharides of mistletoe collected from apple tree. the test-compounds were administered at a dose of 100 mg / kg(21). per os. daily for 6 weeks in water solution form. after 6 weeks of administration the plasma lipoprotein profile and cholesteryl ester transfer protein (cept) concentration was determined. also. the small intestine was harvested from the rats to obtain the supernatant and assess the change of the niemannpick c1-like1 protein (npcl1) content was investigated. biomaterial sampling blood was collected from the abdominal part of the aorta into a citrate-filled syringe. then. whole blood was centrifuged at 1000 g for 10 min. to obtain a serum. in which the concentration of total cholesterol (tc). low density lipoprotein cholesterol (cldl). high density lipoprotein cholesterol (chdl). triglycerides (tg) and cept was determined. the small intestine was freed from the contents and washed in phosphate buffer solution pbs (ph = 7.4). the intestine were homogenized in a mechanical potter homogenizer in pbs (ph = 7.4) in a 1:7 ratio. the resulting homogenate was centrifuged at 10.000 g for 15 min to obtain a supernatant. in which the npcl1 content was determined. determination of tc content. the principle of the method is based on the spectrophotometric detection of the quinonimine dye. which is formed as a result of the oxidative azocoupling reaction of 4-aminoantipyrine with phenol. which occurs in the participation of hydrogen peroxide. which undergoes the oxidation of cholesterol to 4-cholesten-3-one. the color intensity of the reaction environ is proportional to the cholesterol content in the test material and is determined photometrically at a wavelength of 500 nm. determination of chdl content. the principle of the method is based on the fact that chylomicrons. very low density lipoproteins and low density lipoproteins are precipitated when phosphotungstic acid and mg 2+ ions are added to the test sample. after centrifugation (4000 g for 10 minutes). only chdl remains in the supernatant. the concentration of which is determined similarly to the concentration of tc. determination of cldl content. the principle of the method is based on the fact that after adding heparin to the test sample. ldl are deposited at their isoelectric point at ph 5.1. after centrifugation (4000 g for 10 minutes). cholesterol of chylomicrons. very low density lipoproteins and hdl remains in the supernatant. the cldl concentration is determined by the difference in total cholesterol and supernatant cholesterol. determination of tg content. the principle of the method is based on the detection of colored products of conjugaiuon reactions of lipase-mediated hydrolysis of fatty acids and oxidative azo coupling of 4aminoantipyrine and phenol with the formation of a quinonimine dye. which has an absorption maximum at 500 nm. iraqi j pharm sci. vol.32(1) 2023 hypolididemic activity of mistletoe leaves polysaccharides 78 determination of the concentration of cept and npcl1. the content of cept and npcl1 was determined by the method of enzyme-linked immunosorbent assay. assay kits were obtained from cloud clone (usa). the assay progress was in accordance with the kit manufacturer's instructions. the spectrophotometric signal was detected using an infinite f50 plate reader (tecan. austria). statistical analysis the results of experiment were statistically processed. the software package statistica 6.0 (statsoft. usa) was used in the work. statistical significant differences between the groups were determined by the anova method with the newman-keulse post-test. at a significance level of p <0.05. results isolation and investigation of polysaccharide complexes in mistletoe and host-plants leaves the quantitative determination of polysaccharide fractions was determined gravimetrically. table 1. qualitative and quantitative composition of polysaccharides isolated from leaves of white mistletoe. apple tree and pear note: mobility coefficients of standard samples in solvent systems: (1 pyridine ethyl acetate water (1: 2: 2)): 0.15 ± 0.01 (gal); 0.17 ± 0.01 (gal a); 0.27 ± 0.02 (ara) 0.38 ± 0.02 (rha); (2n-butanol acetic acid purified water (4: 1: 2)): 0.24 ± 0.02 (gal); 0.37 ± 0.02 (gal a); 0.45 ± 0.02 (ara); 0.56 ± 0.02 (rha); wsp-water soluble polysaccharides; ps pectin substances. after acid hydrolysis of the obtained fractions. it can be concluded that these fractions are characterized by a similar monomer composition. quantification of functional groups of pectin substances the study of the qualitative characteristicsof pectin substances and the determination of functional groups was of interest to substantiate the possibility of their use for medical purposes. table 2. content of functional groups in pectin substances isolated from leaves of white mistletoe. apple tree and pear test-objects functional group кс. % км. % ко. % -осн3. % (λ). % ps isolated from: leaves of mistletoe collected from apple tree 7.02 2.17 9.19 1.49 23.61 leaves of apple tree 9.63 4.10 13.73 2.82 29.86 leaves of mistletoe collected from pear 7.43 1.53 8.96 1.05 17.09 leaves of pear 8.64 3.83 12.47 2.64 30.69 testobject polysaccharides content% (n=6) monosaccharides and its mobility coefficients in thin layer chromatography (n=4) galactose (gаl) galacturonic acid (gal a) arabinose (ara) rhamnose (rha) solutions systems 1 2 1 2 1 2 1 2 leaves of mistletoe collected from apple tree wsp – 1.84±0.06 0.15± 0.01 0.25± 0.02 0.18± 0.01 0.35± 0.02 0.25± 0.02 0.45± 0.02 0.38± 0.02 0.57± 0.02 ps – 5.86±0.22 0.15± 0.01 0.26± 0.02 0.18± 0.01 0.35± 0.02 0.26± 0.02 0.44± 0.02 0.38± 0.02 0.55± 0.02 leaves of apple tree wsp – 2.08±0.06 0.14± 0.01 0.25± 0.02 0.18± 0.01 0.37± 0.02 0.27± 0.02 0.44± 0.02 0.37± 0.02 0.56± 0.02 ps – 7.86±0.36 0.15± 0.01 0.25± 0.01 0.17± 0.01 0.36± 0.02 0.26± 0.02 0.45± 0.02 0.39± 0.02 0.55± 0.02 leaves of mistletoe collected from pear wsp – 3.86±0.07 0.14± 0.01 0.23± 0.02 0.17± 0.01 0.36± 0.02 0.26± 0.02 0.44± 0.02 0.39± 0.02 0.55± 0.02 ps – 6.44±0.30 0.15± 0.01 0.24± 0.02 0.17± 0.01 0.35± 0.02 0.27± 0.02 0.43± 0.02 0.38± 0.02 0.56± 0.02 leaves of pear wsp – 5.27±0.08 0.14± 0.01 0.25± 0.02 0.16± 0.01 0.37± 0.02 0.25± 0.02 0.45± 0.02 0.39± 0.02 0.56± 0.02 ps – 12.02±0.37 0.14± 0.01 0.24± 0.02 0.17± 0.01 0.36± 0.02 0.26± 0.02 0.46± 0.02 0.40± 0.02 0.56± 0.02 iraqi j pharm sci. vol.32(1) 2023 hypolididemic activity of mistletoe leaves polysaccharides 79 the investigated pectin substances belong to the group of low esterified pectins (23.61%. 29.86%. 17.09%. 30.69%). because the degree of esterification of carboxyl groups is less than 50%. the significant content of free carboxyl groups (7.02%. 9.63%. 7.43%. 8.64%) indicates their rather high complexing ability and the possibility of using pectin substances as detoxicants. figure 1. a structural fragment of polygalacturonic acid. which is part of the studied pectin substances hypolipidemic activity of pectin substances and water-soluble polysaccharides obtained from mistletoe and host-plants leaves the block of pharmacological tests showed that in the nc group of rats under conditions of experimental hypercholesterolemia (table 3). an increase in the concentration of total cholesterol. cldl and tg was noted in relation to the so group of animals by 2.2 times (p <0.05); 4.1 times (p <0.05) and 1.6 times (p <0.05) respectively. with a decrease of chdl content by 54.5% (p <0.05). against the background of the administration of pectin substances obtained from the pear in relation to the nc group of rats. a decrease in the concentration of tc by 21.7% (p <0.05); cldl by 29.3% (p <0.05) and tg by 15.8% (p <0.05) was observed. at the same time. the content of chdl in animals that received pectin substances from pear increased by 40.0% (p <0.05) relative to the nc group of rats (table 3). against the administration of pectin substances from the apple tree. there was a decrease of the concentration of tc. cldl and tg in relation to the nc group of animals by 23.9% (p <0.05); 30.5% (p <0.05) and 15.8% (p <0.05). respectively. with an increase in chdl content by 30.0% (p <0.05). when animals were treated by pectin substances obtained from white mistletoe. which was collected from pear. a decrease in the concentration of tc. cldl and tg relative to the nc group of animals by 32.6% (p <0.05); 43.9% (p <0.05) and 21.1% (p <0.05) was noted. with an increase in chdl by 60% (p <0.05). in rats that were treated by pectin substances obtained from mistletoe. which was collected from a apple tree. the concentration of tc. cldl and tg was lower than that of the nc group of animals by 30.4% (p <0.05). 43. 9% (p <0.05) and 26.3% (p <0.05). respectively. while the content of chdl increased by 80% (p <0.05). it should be noted that the administration of water-soluble polysaccharides did not have a significant effect on the change in the lipid profile of blood serum in animals. table 3. influence of the test-substances on the change in the lipid profile of blood plasma in rats under conditions of experimental hypercholesterolemia group tc. мm/l ldl. мm/l hdl. мm/l тg.мm/l so 2.1±0.17 1±0.1 1.1±0.22 1.2±0.21 nc 4.6±0.25# 4.1±0.13# 0.5±0.19# 1.9±0.26# pspc 3.6±0.14* 2.9±0.24* 0.7±0.22* 1.6±0.17* psmd 3.5±0.13* 2.8±0.09* 0.7±0.24* 1.6±0.24* psva/pc 3.1±0.24* 2.3±0.25* 0.8±0.22* 1.5±0.14* psva/md 3.2±0.29* 2.3±0.18* 0.9±0.15* 1.4±0.29* wsppc 4.2±0.21 3.8±0.24 0.4±0.15 1.7±0.29 wspmd 4±0.28 3.5±0.11 0.5±0.23 1.9±0.16 wspva/pc 4.4±0.18 4.1±0.19 0.5±0.24 1.8±0.29 wspva/md 4.1±0.12 3.6±0.28 0.5±0.16 1.9±0.09 note: so sham-operated rats. nc negative control; pspc — a group of rats that received pectin from pear; psmd a group of rats that received pectin from a apple tree; psva/pc a group of rats that received pectin substances from mistletoe collected from pear; psva/md a group of rats that received pectin substances from mistletoe collected from apple tree; wsppc a group of rats that received soluble pear polysaccharides; wspmd a group of rats that received water soluble polysaccharides from apple tree; wspva/pc a group of rats that received water-soluble polysaccharides of mistletoe collected from common pear; wspva / md a group of rats that received water-soluble polysaccharides of mistletoe collected from apple tree. # statistically significant relative to the so group; * statistically significant relative to the nc group. iraqi j pharm sci. vol.32(1) 2023 hypolididemic activity of mistletoe leaves polysaccharides 80 also during the work it was found that the administration of pectin substances contributed to a decrease in the concentration of cept and npcl1. thus. in the nc group of rats. the content of cept and npcl1 was 2.6 times (p <0.05) and 2.9 times (p <0.05). respectively higher. than in so animals. against the background of the pspc. psmd. psva / pc and psva / md administration. a decrease in the concentration of cept in relation to nc in the group of animals by 28% (p <0.05); 23.4% (p <0.05); 38.6% (p <0.05) and 40.1% (p <0.05). respectively (fig. 2) and npcl1 (fig. 3) by 25.7% (p <0.05); 24.9% (p <0.05); 37.3% (p <0.05) and 40% (p <0.05). respectively was noted. at the same time. the content of cept and npcl1 in rats treated by water-soluble polysaccharides did not statistically significantly differ from that in the nc group of animals. figure 2. the effect of the test substances on the change in the concentration of cept in the blood of rats under conditions of experimental hypercholesterolemia. note: so sham-operated rats. nc negative control; pspc — a group of rats that received pectin from pear; psmd a group of rats that received pectin from a apple tree; psva/pc a group of rats that received pectin substances from mistletoe collected from pear; psva/md a group of rats that received pectin substances from mistletoe collected from apple tree; wsppc a group of rats that received soluble pear polysaccharides; wspmd a group of rats that received water soluble polysaccharides from apple tree; wspva/pc a group of rats that received water-soluble polysaccharides of mistletoe collected from common pear; wspva / md a group of rats that received water-soluble polysaccharides of mistletoe collected from apple tree. # statistically significant relative to the so group; * statistically significant relative to the nc group. figure 3. the influence of the test substances on the change in the concentration of npcl1 in the blood of rats under conditions of experimental hypercholesterolemia. note: so sham-operated rats, nc negative control; pspc — a group of rats that received pectin from pear; psmd a group of rats that received pectin from a apple tree; psva/pc a group of rats that received pectin substances from mistletoe collected from pear; psva/md a group of rats that received pectin substances from mistletoe collected from apple tree; wsppc a group of rats that received soluble pear polysaccharides; wspmd a group of rats that received water soluble polysaccharides from apple tree; wspva/pc a group of rats that received water-soluble polysaccharides of mistletoe collected from common pear; wspva / md a group of rats that received water-soluble polysaccharides of mistletoe collected from apple tree. # statistically significant relative to the so group; * statistically significant relative to the nc group. iraqi j pharm sci. vol.32(1) 2023 hypolididemic activity of mistletoe leaves polysaccharides 81 discussion lipid metabolism disorders leading to atherosclerosis are among the most common metabolic disorders and underlie the pathogenesis of a large number of diseases. the two leading non-infectious causes of mortality in the population (according to who) are ischemic heart disease and ischemic stroke. which are directly associated with progressive atherosclerosis and hyperlipidemia. in turn. hyperlipidemia is associated with an imbalance in the metabolism of cholesterol and triglycerides: in the lipid profile of blood serum. ldl cholesterol predominates. the concentration of triglycerides is increased. and the content of hdl is reduced. today. a wide range of medicines are used to treat mixed hypercholesterolemia. ranging from statins to newer pcsk9 inhibitors. the available medicines generally show excellent levels of efficacy and. when used rationally. can reach target blood cholesterol and triglyceride levels fairly quickly. however. despite the high efficiency. the use of statins is associated with the development of serious side effects. as pointed out by ward et al. 2019. deviation from the mode of use or dosing violation of drugs of the statin group is accompanied by the development of adverse reactions from the skeletal muscle (myopathy). liver (hepatotoxicity) and kidneys (myoglobin nephropathy) (22). pcsk9 inhibitors are a new group of highly selective cholesterol-lowering agents that can reduce ldl cholesterol levels by more than 70%. to date. the use of available agents of this group. alirocumab and evolocumab. is associated with an increased risk of developing upper respiratory tract infections (23) . a significant number of adverse reactions of the main cholesterol-lowering medicines makes urgent the search for alternative methods of lowering the concentration of cholesterol and triglycerides in the blood. one of such approaches may be the use of natural pectin substances. pectin’s are complex polysaccharides of the plant cell wall. localized in specific vacuoles. unevenly distributed. pectin’s play an important role in plant morphogenesis and physiology. and are also potentially effective therapeutic agents (24). li. et al. 2020 found that pectins isolated from abelmoschus esculentus prevent the development of fatigue by increasing the amount of glycogen. glucose and. accordingly. atp in skeletal muscle (25). the immunosuppressive and antiallergic properties of pectin’s. in particular galactan. arabinan. and apiogalacturonan. have also been investigated (26). there is information about the antibacterial activity of pectin’s. on multi-resistant strains (27). the hypolipidemic activity of some pectin’s also has been studied. a work by hu. et al.. 2019 showed that the use of pectin’s obtained from citrus fruits reduced the level of ldl cholesterol in c57bl / 6 mice (28). similar results for pectin substances were presented in a review by zhang et al.. 2021 (29). this study also established the hypocholesterolemic activity of pectin’s obtained from the leaves of the hemiparasite plant white mistletoe and host-plants pear and apple tree. at the same time. the use of pectin substances from mistletoe leaves contributed to a more pronounced (at the level of tendency) decrease of the concentration of tc. cldl and tg. as well as an increase of chdl. it should be noted that the administration of water-soluble polysaccharides to animals did not have a significant effect on the change in the lipid profile of blood plasma. analyzing the possible mechanisms of cholesterol and triglyceride reduction under the influence of the test pectin substances. it was suggested that these compounds can affect the function of cept and npcl1. cept is a hydrophobic glycoprotein that provides bi-directional transfer of cholesterol and triglycerides between plasma lipoproteins. the increased activity of cept leads to an increase in the transport of cholesterol and triglycerides from hdl to ldl. thereby increasing their amount and promoting atherogenesis (30). currently. several cept inhibitors are in 3 stage of clinical trials: torcetrapib. dalcetrapib. evacetrapib. and anacetrapib (31). in the course of the study. it was found that the administration of the test pectin substances contributed to a decrease in the concentration of cept. which contributed to the restoration of the balance of cldl / chdl. the transport protein npcl1 controls the absorption of cholesterol in the intestine and is a pharmacological target for some medicines. for example. ezetimibe(32). in the present study. it was shown that the studied pectin substances reduced the concentration of npcl1 in the small intestine. which can mediate impaired absorption of cholesterol and. accordingly. the hypocholesterolemic effect. also. one cannot exclude the possibility of the formation of difficult-to-absorb complexes of cholesterol with the studied pectin’s. since the latter have a sufficiently high adsorption capacity. iraqi j pharm sci. vol.32(1) 2023 hypolididemic activity of mistletoe leaves polysaccharides 82 conclusion this work showed no differences between the total content of polysaccharides in the leaves of the white mistletoe (viscum album l.) growing on the apple tree (malus domestica borkh.) and the leaves of the white mistletoe (viscum album l.) growing on the pear (pyrus communis l.). a study of the pharmacological activity of the isolated polysaccharides showed that pectin substances have a hypolipidemic effect. which is expressed in a decrease in the concentration of total cholesterol. ldl cholesterol and an increase in hdl cholesterol. as well as a decrease in triglycerides levels. at the tendency level. pectin substances obtained from white mistletoe had a more pronounced effect than analogous compounds isolated from carrier plants. at the same time. the ability of pectin substances to reduce the level of cholesterol and triglycerides in blood may be based on a decrease in the function of cept and npcl1. it should be noted that water-soluble polysaccharides did not exhibit cholesterol-lowering activity. references 1. plant resources of the ussr: 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kobayashi t. oikawa s. yamashita s. rakugi h. imai t. tanaka s. ohashi y. kuwabara m. ito h. ezetimibe lipid-lowering trial on prevention of atherosclerotic cardiovascular disease in 75 or older (ewtopia 75): a randomized. controlled trial. circulation. 2019;140(12):9921003. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.22(1) 2013 fatty acid pattern of silybum marianum and nigella sativa 25 comparative analysis of the fatty acid pattern of silybum marianum with nigella sativa by gas chromatography-mass spectrometry mohammed n. sabir *,1 and shwan k. rachid ** * department of pharmacognosy and pharmacy chemistry, school of pharmacy, university of sulaimani, sulaimaniyah, iraq. ** faculty of science and health, university of koya, koysanjaq, erbil, iraq. abstract many studied were conducted to evaluate the antihepatotoxic and antioxidant activities of silybum marianum and proved these actions. the naturally grown seed in iraqi-kurdistan region also were studied for its chemical contents and biological activities. vegetable oils occur in various plant parts mainly concentrated in the seeds. in this study comparison was made between the fatty acid patterns of two plant seeds, silybum marianum and nigella sativa. seed sample of silybum marianum and nigella sativa were exposed for extraction and isolation of the fatty acid contents using two different solvents (petroleum ether and n-hexane) at 60-80 o c using soxhlet apparatus and the oily extract were purified and analysed by gc-ms triplet system. result showed that extraction with petroleum ether yielded 23% and 24% of total weight of the seeds of silybum marianum and nigella sativa respectively and comparable results with n-hexane extract yielded 17% and 22% of total seed weight of silybum marianum and nigella sativa respectively showing significant differences in the fatty acids patterns of both plants under research. key words: fatty acids, gc-ms, silybum marianum, natural products, cardiovascular diseases. بواسطةدراسة تحهيهية مقاروة ألوماط األحماض اندهىية في وباتي انسهيماريه وانحبة انسوداء انفصم بانكروماتوغرافيا انغازي ذا انطيف انكتهي تقىية محمد ووزاد صابر *،1 شوان كمال رشيد و ** * . انعشاق،خايعح انسهًُاَُح ،كهُح انصُذنحفشع انعمالُش وكًُُاء انصُذنح ، ** . انعشاق،أستُم،كىَسُدك، خايعح كىَح ،كىنرٍ انعهىو وانصححاف الخالصة . فٍ هزا انشأٌ فاعهُرها ذى اثثاخ ولذ, نألكسذج كذواء فعال نحًاَح انكثذ و كًضاددساساخ نرمُُى فاعهُح انسهًُاسٍَ ال انعذَذ يٍ أخشَد وانفاعهُح انحُىَح وَحنهكشف عٍ يكىَاذها انكًُُا ألهُى كشدسراٌ انعشاقدساساخ أخشي ذى اخشاءها عهً تزوس انسهًُاسٍَ انُايُح طثُعُا فٍ . تصىسج يكثفح فٍ انثزوس ذرشكض وعادج يا ذرىاخذ فٍ أخضاء عذَذج يٍ انُثاخ انضَىخ انُثاذُح فكا هى يعهىو اٌ .نرهك انًكىَاخ أَثشاخ ) تاسخذاو َىعٍُ يٍ انًزَثاخ انكعىب و انحثح انسىداء وهًا ذٍُتزوس انُثاخلانذهُُح َىعُح األحًاضيماسَح دساسرُا هزِذًد فٍ دسخح يؤَح 80-60فٍ دسخح حشاسج حُث ذى اسرخالص وعضل األخًاض انذهُُح نًُارج يٍ تزوس انُثررٍُ (انثرشول و انهكساٌ انعادٌ . gc-msص تىاسطح خها هاٌلحمخو جانضَرٍ اخنًسرخهضذًد ذُمُحاتعذها و , تاٍسرخذاو خهاص انسىكسهُد نكم انثزوس ارجيٍ انضَىخ انثاترح َسثح انً وصٌ َى% 24و % 23أظهشخ انُرائح تأٌ عًهُح األسرخالص تاٍسرخذاو أَثشاخ انثرشول أعطد عهً %22و % 17 وانهرٍ تهغد يزَة انهكساٌ انعادٌوأكثش يٍ َسثها انًسرحصهح تىاسطح عهً انرىانٍ نهكعىب و انحثح انسىداءيٍ .انرراتع وهزا َُعكس عهً يحرىي األحًاض انذهُُح فٍ كهرا انُثررٍُ .األمراض انقهبية, انمىتوجات انطبيعية, سهيماريه, انطيف انكتهي انشامم-األستشرابية انغازية, األحماض اندهىية :انكهمات انمفتاحية introduction despite the advances and developments in synthetic chemistry contributing to the production of large number of drugs, natural products remain an attractive source for many new active metabolites (1,2,3) . these biologically active molecules have contributed in the last fifty years to pharmaceutical industries with unique drugs that exhibit potent pharmacological activities. natural products are known as robust sources of new drugs which are involved in production of 60% of the anticancers, and 75% of the antimicrobials (1,2) . prior to 2007 natural products accounted for approximately one-half of all licensed drugs in the world (3) . extensive studies on plant secondary metabolites revealed their potential biosynthetic capacity for production of biologically active molecules that are extensively used for the treatment of several acute and chronic disorders (4) . 1 corresponding author e-mail: hamashko@yahoo.com received: 10/6/2012 accepted: 7/1/2013 iraqi j pharm sci, vol.22(1) 2013 fatty acid pattern of silybum marianum and nigella sativa 26 research in epidemiological fields showed that free radical-induced oxidative damage of cell membranes, dna, and proteins played essential role in aging, degenerative diseases, such as cancer, atherosclerosis, and cataracts (5) . it has been confirmed that antioxidants metabolites extracted from plants, such as α-tocopherol, lascorbic acid, and β-carotene, have protective effects against the free radicals (6,7,) . in addition, the secondary metabolites, flavonoids showed wide range of therapeutic efficacy that contributed in management and supportive treatment of various diseases like cancer, liver damage, bacterial infections and diabetes (5) , for example; the silymarin which is composed mainly from flavonolignan silybins, is a component of the fruit and seed of the variegated milk thistle silybum marianum l. gaertn extract. this plant is a wild biennial herb which grows in many parts of the world including iraqi kurdistan-region (ikr) as an indigenous plant (8) . the fatty acid composition of silybum marianum seed oil was investigated by elmallah et al., 2003 showing oil rich in unsaturated fatty acids, including linoleic acid, oleic acid as well as saturated fatty acids including palmitic and stearic acids (33) . the seeds of nigella sativa linn. (ranunculaceae), which is commonly known as black seed, have been widely used in herbal medicine for the treatment and prevention of various diseases including diabetes, asthma and dyslipidaemia (11) . the seed contains both fixed and essential oils. researches show different pharmacological activities attributed to the crude extracts of the nigella sativa seeds namely antiinflammatory, analgesic, antimicrobial, antihepatotoxic and antinephrotoxicity (11,12) . the fixed oil pattern of nigella sativa have been analysed, eight fatty acids were identified in the extract including saturated and unsaturated fatty acids (lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid and eicosadienoic acid) (11) . the primary functions of fixed oils in the biologic system are to serve as energy storage (14) . the fixed oils are important products used pharmaceutically, industrially, and nutritionally (2, 3) . many drugs contain fixed oils as their principal constituents exemplified by omega-3 and omega-3,6,9, which are used for the treatment of variety of disorders including hypercholesterolemia (15) . vegetable oils occur in various plant parts, but they are mainly concentrated in the seeds (16) . dietary supplements in form of unsaturated fatty acids has shown the potential to reduce both the progression of cardiovascular diseases and related mortality including the sudden cardiac arrest, through lowering triglycerides levels in serum, acting as antithrombotic, decreasing the blood and plasma viscosity and showing improvement endothelial dysfunctions (17,18,19) . who data reveals that cardiovascular diseases (cvd) contribute to the highest mortality rates among population worldwide (20) . measurements have been taken to decrease the risks of cvd through changing the life style, dietary habits and exercise (21) , one of the important measurements that are employed in this manner is dietary supplements of food and medications rich in unsaturated fatty acids, these drugs are imported, putting burdens on the economy, meanwhile; they could be obtained from the available sources in the nature around us. silybum marianum seeds showed significant amounts of unsaturated fatty acids (table 2) making it a good source for these elements that could be obtained from the plant. materials and method soxhlet apparatus (pyrex), capacity 350ml. rotary evaporator (heidolf). all the solvents were of analytical grade. (gc-ms)(agilent 6890n gas chromatograph with a 5973 electron impact mass selective detector (agilent, waldbronn, germany) according to bode and ring (25,26) . the column temperature was kept at 130°c for 2.5 min and then increased to 240°c at 5°c/min. the mass selective detector was operated in scan mode, with a scanning mass range of m/z 40 to 500. sample preparation the wild silybum marianum plant was harvested during june 2011 from south east (industrial area) of al-sulaimaniah city, 5km far from the city centre. morphological features were used for characterization of the collected plant. nearly 600gm of the plant seeds were collected and stored under low temperature. in addition 1 kg of nigella sativa seeds were purchased (black seeds (kalonji) a1-quality, pakistan). according to the company's literature it was originated from western asia. two hundred grams of silybum marianum and nigella sativa seed samples were harvested dried under shade and kept frozen at 20 o c until use. the seeds were ground to coarse particles using iraqi j pharm sci, vol.22(1) 2013 fatty acid pattern of silybum marianum and nigella sativa 27 an electric mill. then the product was extracted using petroleum ether and n-hexane at 60-80 o c for six hours in a soxhlet apparatus (22,23) . the extract was then dried over anhydrous sodium sulphate, filtered and the solvent was removed from the filtrate by rotary evaporator under reduced pressure at 45 o c. the oily extract was subjected to three vacuum distillations in series to remove the debris (24) . the oil sample was stored in a vacuum desiccator for seventy two hours then centrifuged for ten minutes at 5000 rpm. finally, the samples were aliquoted into 1.0 ml portions and stored at -20°c. later, samples were analysed by gc-ms in triplicates. gc-ms spectrometry to 250 µl of each sample, equal volumes of methanol/toluene/sulfuric acid h2so4 (to separate the free fatty acids from the triglycerides) (50:50:2 v/v/v) mixture was added, and incubated overnight at 55°c. after cooling down, 400 µl of 0.5m sodium bicarbonate nh4hco3 solution was added. the mixture was centrifuged at 5000 rpm for 5 minutes at room temperature. 75 µl of the upper organic phase was mixed with 25 µl of the derivatization reagent, n-methyl-ntrimethylsilyltrifluoroacetamide (mstfa) (macherey-nagel, düren, germany) (to esterify the free fatty acids which ease the separation by gc) and incubated at 37°c for 30 minutes. 5 µl of each sample was analysed by gas chromatography coupled to mass spectrometry (gc-ms)(agilent 6890n gas chromatograph with a 5973 electron impact mass selective detector (agilent, waldbronn, germany), using a dimethyl(5% phenyl)-polysiloxane capillary column (agilent hp-5ms, 0.25 mm by 30 m by 0.25 µm) and helium as the carrier gas at a flow rate of 1 ml/min according to bode et al., (2006) and ring et al., (2006). samples were injected in split mode (split ratio, 10:1). the column temperature was kept at 130°c for 2.5 min and then increased to 240°c at 5°c/min. the mass selective detector was operated in scan mode, with a scanning mass range of m/z 40 to 500. fatty acids were identified using the national institute of standards and technology (nist) mass database and massbank database and based on mass spectral and retention index libraries for metabolomics and time of flight gas chromatography/gas chromatography. as control , fame mix reference (fatty acids essential standards) from (sigma-germany) was run under the same experimental condition. results and discussion in this work we have determined the fatty acid contents of wild silybum marianum, and compared to that of nigella sativa. fatty acids represent a chemically inert class of organic compounds that are easy to extract from biological material. normally, fatty acids are acids produced in cell after catabolism break down of fat. these compounds are hydrophobic and not water soluble. they are important part of a healthy diet and body requires them for organs and tissues and utilize them in many cellular activities (14) . fatty acid compounds found usually either in saturated or unsaturated state. saturated branched chain fatty acids are present as complex mixtures in numerous biological samples. branched chain fatty acids are usually saturated and the branch is normally a methylgroup. the common constituents of the lipids in plants are branched fatty acids, and they are rarely found in the integral lipid. unsaturated branched-chain fatty acids are normally found in marine animals, and branches other than methyl may be present in microbial lipids. the high-throughput gas chromatography/mass spectrometry (gc/ms) technology enables analysing a huge number of chemicals and biological samples. one of the important analyses of gc/ms data is compound identification. we characterized the fatty acid profiles of silybum marianum to show its difference from that of nigella sativa seeds. extraction solvent has great impact on fatty acid extraction yield (27) . petroleum ether and nhexane have low polarity index of 0.1, therefore in our work we aimed to use them as extracting solvents for the fatty acid content of the silybum marianum and nigella sativa seeds. as shown in table 1, the use of petroleum ether in extraction of 200 grams of each silybum marianum and nigella sativa seeds yielded 23.5% and 24% of the total weight respectively, while 17% and 22% of the seeds weight were yielded from silybum marianum and nigella sativa respectively by extraction using n-hexane (table-1). iraqi j pharm sci, vol.22(1) 2013 fatty acid pattern of silybum marianum and nigella sativa 28 table1: oil yields obtained from extraction of silybum marianum (l.) gaertn, and nigella sativa seeds using organic solvents petroleum ether and n-hexane the differences in lipid content between silybum marianum and nigella sativa by extraction with different organic solvents might resulted from genetic regulation of fatty acid biosynthesis machinery as well as differences in physiological and environmental effects. identification of the fatty acids contents was evaluated by comparison to spectra of highest similarity to the proposed substances in the nist chemistry webbook mass database library maintained by the national institute of standards and technology (nist) as a library of reference spectra and repetitive mass spectral data as query spectra (28,29,32) . the analysis of the plants seeds extract the fatty acids of variable chain length including both saturated and unsaturated structures (14:00, iso-15:0, 16:17, 16:00, 18:26,9, iso-17:0, 18:02, 18:01, and 18:00) were identified (figure 1). figure 1: diagram showing the relative amount of the fatty acid compound found in extracts of the silybum marianum and nigella sativa seeds using petroleum ether. the data was estimated by measuring the peak area of the gc analysis. the gas chromatogram of the extracts shows that the level of fatty acid content in silybum marianum extracts is significantly higher than the extracts of nigella sativa (figure 2). in addition, the use of petroleum ether in extraction of both seeds of silybum marianum & nigella sativa obtaining resulted in extraction of higher amount of fatty acids than extraction with n-hexane (table 1). this might be related to the differences in polarity of the solvents (23) . in comparison to the saturated fatty acid contents of the seeds extracts, low amounts of each branched (iso-15:0 and iso17:0) and unsaturated omega fatty acids were identified. although differences in the fatty acid content were observed, but the percentage of the compounds found in the plants extracts showed a high similarity (table 2). the only differences in fatty acid patterns is that of nigella sativa, in which no unsaturated omega fatty acid, 18:26,9 was investigated. the ratio of unsaturated fatty acids to the saturated in silybum marianum seed oil extract were compared and showed ratio of 1:54, which revealed significant higher content in the favour of the unsaturated omega fatty acids as shown in table 2. the percentage of unsaturated fatty acids was 60.67% comprises to the total fatty acid in the sample extract. while the percentage of omega fatty acids were 0.65% with pet-ether extract of the total unsaturated fatty acids. such a high ratio will make this plant a good candidate for production of omega fatty acids rich oil in the region. plant seeds extraction yield using organic solvents petroleum ether n-hexane silybum marianum (l.) gaertn 23% 17% nigella sativa 24% 22% iraqi j pharm sci, vol.22(1) 2013 fatty acid pattern of silybum marianum and nigella sativa 29 figure 2: gc-ms/ms spectroscopy analysis of the organic extracts a: silybum marianum seeds using petroleum ether and n-hexane, b: silybum marianum seeds using n-hexane, c: nigella sativa seeds extract using petroleum ether and n-hexane, d: nigella sativa seeds extract using n-hexane. the peaks 1= 14:00, 2= iso-15:0, 3= 16:17, 4= 16:00, 5= 18:26,9, 6= iso-17:0, 7= 18:02, 8= 18:01, and 9= 18:00 fatty acids). table 2: percentage of the fatty acid content of both silybum marianum & nigella sativa plant seed extracts using petroleum ether and n-hexane. no. fatty acid fatty acid ratio (%) sm-petet smnhex nspetet nsnhex 1 oh o 14:00 0.25 0.25 0.16 0.12 2 oh o iso-15:0 0.07 0.08 0.04 0.05 3 oh o 16:17 0.24 0.18 0.13 0.11 4 oh o 16:00 27.47 28.52 22.81 29.81 5 oh o 18:26,9 0.16 0.18 0 0 6 oh o iso-17:0 0.23 0.21 0.13 0.16 7 oh o 18:02 23.3 20.14 32.28 22.75 8 oh o 18:01 36.9 40 36.82 38.41 9 oh o 18:00 11.38 10.46 7.63 8.59 iraqi j pharm sci, vol.22(1) 2013 fatty acid pattern of silybum marianum and nigella sativa 30 conclusion the differences in fatty acid pattern can be used to differentiate between the two plants. however genetic analysis and transcriptomic studies are required to shed more light on the absence of the fatty acid in the extracts components. fatty acid (fa) profiles can be used as chemotaxonomic 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2009, 325: 161-165. 31. wen z., dumas t. e., schrieber s. j., et al., ―pharmacokinetics and metabolic profile of free, conjugated, and total silymarin flavonolignans in human plasma after oral administration of milk thistle extract‖, the am soc for pharmacol and exp ther, 2008, 36: 1: 36:65-72. 32. march r. e., ―quadrupole ion trap mass spectrometry: a view at the turn of the century‖, int j of mass spectrometry, 2000, 200: (1-3) 285-312. 33. el-mallah m. hassan, el-shami safinaz m., hassanein minar m. ―detailed studies on some lipids of silybum marianum(l.) seed oil‖, grasas y aceites, 2003, 54: 4, 397-402 http://webbook.nist.gov/ iraqi j pharm sci, vol.31( 2 ) 2022 12-hydroxyoctadecanoic acid organogels as a floating system doi: https://doi.org/10.31351/vol31iss2pp169-176 169 study the effect of 12-hydroxyoctadecanoic acid concentration on preparation and characterization of floating organogels using cinnarizin as modeling drug masar basim mohsin mohamed *,1, zainab saad qaddoori**and ghaidaa sulaiman hameed* * department of pharmaceutics , college of pharmacy , mustansiriyah university, baghdad, iraq **baghdad health directorate-al karkh, ministry of health and environments ,baghdad, iraq abstract this work targeted studying organogel as a potential floating system. organgel has an excellent viscoelastic properties, floating system posses a depot property. different formulations of 12hydroxyoctadecanoic acid (hoa) in sesame oil were gelled and selecting f1, f3 and f5 hoa organogels for various examinations: tabletop rheology, optical microscopy, and oscillatory rheology studies. also, the floating properties studies were conducted at in vitro and in-vivo levels. lastly, the in-vitro release study using cinnarizine (cn) was to investigate the organogel depot property. based on the results, the selected concentrations of hoa in sesame oil organogels showed temperature transitions from gel to sol higher than body temperature. these organogels scaffolds inner structures were a star-like shape. the formulation f5 hoa/so organogels were developing higher storage modulus values, which resulted from the amplitude sweep study. indeed, all the selected organogels were frequency sweep independent. the organogel’s in vitro floating properties were found positively proven our work’s aim and were buoyant for 24 hours as f5 hoa organogels remained for 12 hours in the rat’s stomach. the depot property showed the slow release of cn from f5 hoa/so organogel and not more than 65% w/w of cn released after 24 hours. keywords: organogel, floating, depot, (12-hydroxyoctadecanoic), sesame oil العائم الهالم العضوياوكتاديكانويك على تحضير وتوصيف ا هيدروكسي-12حمض دراسة تاثير للعقار نموذجسيناريزين كالباستخدام *غيداء سليمان حميد،**زينب سعد قدوري ،1،*باسم محسن محمد مسار العراق ، بغداد ، الجامعة المستنصرية ، كلية الصيدلة -فرع الصيدالنيات * العراق ، بغداد ، والبيئة وزارة الصحة ، الكرخ -دائرة صحة بغداد ** الخالصة استهدف هذا العمل دراسة الهالميات العضوية كنظام عائم يُعرف بالنظام اللزج المطاطي الممتاز والذي يمتلك خاصية المستودع العضوي. هيدروكسي أوكتاديكانويك في زيت السمسم واختيار ثالث صيغ لفحوصات مختلفة لعلم الريولوجيا المنضدية -12تم عمل تراكيز مختلفة من حمض ، كانت دراسات الخصائص العائمة على مستويات في المختبر وداخل الجسم. والفح ص المجهري البصري ودراسات الريولوجيا التذبذبية. أيًضا ، أظهرت أخيًرا ، كانت دراسة التحرر الدوائي في المختبر باستخدام دواء السيناريزين للتحقيق في خاصية المستودع العضوي. بناًء على النتائج هيدروكسي أوكتاديكانويك في هالميات زيت السمسم تحوالت درجة الحرارة من هالم إلى سائل او مائع أعلى من درجة -12غ المختارة من الصي هيدروكسي f5 (12حرارة الجسم. كانت الهياكل الداخلية للسقاالت العضوية حسب الفحص المجهري نجمية الشكل. كانت صيغ الهالميات العضوية نويك( ذات قيم معامالت تخزين أعلى ، والتي نتجت عن دراسة اكتساح السعة, اضافة الى كون جميع الهالمات العضوية المختارة كانت أوكتاديكا ملنا مستقلة عن اكتساح التردد في دراسة اكتساح التردد. اضافة تم التاكد من خصائص العوم في المختبر للهالميات العضوية متناسقة مع هدف ع ساعة. أظهرت 12هيدروكسي أوكتاديكانويك( من الهالميات العضوية في معدة الفئران لمدة 12) f5ساعة حيث بقيت 24نت مستمرة لمدة وكا ٪ وزن / 65هيدروكسي أوكتاديكانويك( والذي لم يتجاوز 12) f5خاصية المستودع التحرر البطيء لدواء السيناريزين من الهالميات العضوية ساعة. 24يدروكسي أوكتاديكانويك( بعد ه– 12وزن ) زيت السمسم ، هيدروكسي اوكتاديكانويك -12 ، المستودع ، العائم ، : الهالم العضوي الكلمات المفتاحية بالعربي introduction the major desirable route in drug delivery is the oral route for convenience intake and mastering the oral formulations. however, the following limitations like the low solubility of weakly basic drugs, the inconsistent absorption of some drugs, and the short stay in the stomach affect bioavailability. the gastroretentive systems overcome these limitations by helping keep the drug in the appropriate media of solubility throughout the gastrointestinal tract. gastroretentive systems are classified as swelling(1) , mucoadhesive(2), high density(3), and low-density systems(4); the floating system is a subdivision of low-density system(5). this current study focuses on the low molecular weight organogel to investigate organogel’s floating characteristics and depot property. two studies recently used the organogels of span 40, span 60, and stearic acid to investigate their gastric retention property, and the result showed instant buoyancy and long floating duration (6, 7). 1corresponding author e-mail: masarmohamed@uomustansiriyah.edu.iq received: 13/10 /2021 accepted: 21/12 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp169-176 iraqi j pharm sci, vol.31(2) 2022 12-hydroxyoctadecanoic acid organogels as a floating system 170 also, the organogel of hoa/ soybean oil delayed the release of the ibuprofen, and the drug release was indirectly related to the increase of hoa concentration in the organogel. the same outcome of the organogel slow release was shown even in the case of hydrophilic drugs the theophylline and ofloxacin (8, 9). to explore our aim, cinnarizine (cn) was used as a model drug for its solubility augmentation in the stomach environment and formulated with the organogel of low molecular weight gelator 12-hydroxyoctadecanoic acid (hoa) in sesame oil (so) to study the organogel's floating and depot properties. this organogel is filled in a hard gelatin capsule for patient intake as a suitable way to deliver the organogel. for the same gastroretintive approach, the hard gelatin capsule was used to load furosemide double-layer film for mucoadhesive and gastroretentive purposes(10). furthermore, the use of hard gelatin capsules to deliver organogel for the oral route was in many studies, such as the lecithin in sunflower oil organogel incorporated with metronidazole to obtain a controlled release(11). in addition, pereira et al was solubilized and loaded in hard gelatin capsules the hoa oragnogel for controlled and slowed release(12). material and methods materials hoa and cn were purchased from hangzhou hyper chemicals china and baoji guokang bio-technology– china, respectively. the so was obtained from the local market. methods preparation of organogel according to the following concentrations (1%, 3%, 5%, 7%, 10%, 13%, 15%, 18%, 20%) (w/w), the hoa was weighed out then completed the total weight to 1 gm with so in glass vials. these vials were placed in a water bath at 90℃ for 30 minutes, then let to cool overnight at room temperature. while for drug-loaded organogels, 25 mg of cn was weighed initially; then the hoa was added, followed by so to reach 1 gm of cn with organogel using the same preparation method. the flow of the organogel content upon inverting the vials correlated with the organogel formation as no flow means successful organogel formation. hoa/so organogels formulations were assigned and represented in table 1. phase transition (tabletop rheology) all the vials of the organogels incubation in a water bath at 90 ºc then the temperature of a water bath was reduced gradually to reach 32 ºc, where the average rate was 2°c/ 15 minutes. at the end of each 15 minutes, the vials were leaning 45° to check organogels status, whether solid or liquid. this phase represents the transition temperatures from liquid to solid for all organogel preparations followed by a reverse-phase by increasing the temperature (2°c/ 15 minutes) to build the transition temperatures from solid to liquid for all organogels. table 1. the compositions of the hoa organogels. fourmulation number cn (mg) hoa% (w/w) so % upto (w/w) f1 25 1 100 f2 25 3 100 f3 25 5 100 f4 25 7 100 f5 25 10 100 f6 25 13 100 f7 25 15 100 f8 25 18 100 f9 25 20 100 optical microscopy microscopic image preparation was by using an optical microscope and slides. the slides preparation was done by adding a drop of molten organogel on a glass slide while the vials of organogels were stilled set in the water bath at 90°c. a glass coverslip was placed on the top of the gel and flattened softly on the slide. after that, the slide was shifted into the microscope stage to examine and capture images using the software microcapture by the digital microscope camera mc500. the magnification of the microscope was x40. fourier transform infrared (ftir) ftir application for selected organogels was by using shimadzu ftir-8400s. the spectra recording were from 400 to 4000 cm-1, and the cell plate 201-77160-20 was for oils and krs-5 for kbr to test solid samples and organogels. oscillatory rheology studies rheological measurements were carried on anton par mcr302 rheometer using plate-plate configuration (pp25sn61895) for amplitude sweep test, and frequency sweep test at 25 ºc and the data evaluation was by rheoplus software. this study was carried out at the university of petra /pharmaceutical center (uppc). amplitude sweep the amplitude sweep test was applied to identify storage modulus (g'), loss modulus (g''), the linear viscoelastic region (lver), and the flow point for each preparation. the applied oscillatory strain range was set from 0% to 100% at angular frequency 10 rad s-1. frequency sweep the other oscillatory study was the frequency sweep; the chosen strain was within the range of lver values obtained from the amplitude sweep study for organogels where the angular frequency changed from 0.1 to 100 rad s-1. iraqi j pharm sci, vol.31(2) 2022 12-hydroxyoctadecanoic acid organogels as a floating system 171 investigation of the floating properties for organogels in-vitro floating study investigation of the floating parameters was done firstly with an in-vitro floating study for the organogel loaded in a capsule. a hot liquid organogel was poured into an empty hard gelatinous capsule’s body with the micropipette size 1 gram. the capsule was sealed carefully in a vertical position to be stored in a tube to ensure organogel stability. then, placing the capsule in a beaker filled with 200 ml of hcl solution ph 1.2, which was already prepared to be at 37°c with a constant stirring at 100 rpm. during this process, visual monitoring the gel status for 24 hours. in-vivo floating study this study followed the in-vitro floating to observe in-vivo floating property using five healthy adult female wistar rats weighing 200-210gm. according to the guidelines and approval of the ethical committee of research in the pharmacy college, mustansiriyah university for animal studies was this procedure. earlier, those rats reserved was for ten days in plastic cages under standard situations (12 hours of light and dark cycle, 24°c, 35-60% humidity) with free access to their nutrition and water. then, the rats were abstained from food for 24 hours before running the experiment still water-free access. methylene blue (0.1% w/w) was added to the selected organogels to discriminate the organogels from the stomach tissues. the ethanol addition was to keep the status of organogel as a liquid to make organogel intake by the rat possible, as ethanol was used in another study to liquefy organogel(13). provision one millilitre of the blue liquid preparation was to the rat with an oral gavages tube’s aid. these animals were anaesthetized by 50 mg/kg ketamine and 5 mg/kg xylazine intramuscular injection. then, a cut was made to the abdomen to investigate the floating preparations by the presence of the organogel in the rat’s stomach. photos for stomach were taken at 0 min before administration as a control, then at 1 hour, 2 hours, 6 hours, and 12 hours after giving the organogel. in-vitro release study in-vitro release study for cn loaded organogels capsules was accomplished using usp type ii apparatus (paddle type). the filled jar was to 900 ml of hcl ph 1.2 that adjusted at 37±0.5°c and 100 rpm. the capsule was positioned into the jars of the apparatus then according to the following time frame (0.083, 0.25, 0.5, 1, 3, 6, 9, 12, 15, 18, 21, and 24) hours; 5 ml was withdrawn from the release media then substituted with equal volumes of fresh medium. each sample filtration was by a millipore filter 0.45 μm papers and properly diluted if needed and measured by uv-visible spectrophotometer at 254nm (λ max of cn). each time point was representative for an average of 3 triplicates and transformed into a concentration using the following equation: y=0.0642x. this equation represents the calibration curve equation resulted from several dilutions of cn in an hcl ph 1.2. results and discussion preparation of organogel all hoa in so organogels were gelled at 25 0c and showed no flow after vial inversion, as shown in figure 1. the gelation concentrations were similar to another study using hoa in canola oil, diacylglycerol oil and unrefined sesame oil(14). furthermore, the addition of cn did not disturb the organogel formation. for the subsequent studies, three selected concentrations of organogels (f1, f3 and f5 organogels) were assigned to observe the differences among these organogels. figure 1.inverted vials of hoa in so at room temperature from left to right the organogel formulations (f1, f2, f3, f4, f5, f6, f7, f8, f9) iraqi j pharm sci, vol.31(2) 2022 12-hydroxyoctadecanoic acid organogels as a floating system 172 phase transition (tabletop rheology) this study was applied to find the temperature that could organogels transfer from one status to another. the selected organogels transition temperatures from sol-gel were from 60⁰c to 45⁰c, and the reverse transition temperatures of gel-sol within this range (47⁰c to 60⁰c) as shown in figure 2. these temperature transitions of all organogel indicated stable formulations in 37 °c the body temperature. our study’s gelation phase range was more stable and different than the hoa in light mineral oil organogel, which showed 20⁰c for the 2% w/w of the organogel and 70⁰c for the 10% w/w of the organogel(15). in conclusion, an increase in all selected organogels transitions temperature (sol-gel and gel-sol) as the organogel concentration increased and the transition temperatures were above 37⁰c, pointing to a solid status of organogel in the body temperature. optical microscopy this optical microscopy study was applied to probe the morphology of the scaffold that construct the organogels. the study showed, as presented in figure 3, “star-like aggregates”. this pattern showed a remarkable similarity to hoa organogels prepared in a different study in vegetable oil(16). it was noticed denser spherulites were obtained by increasing the concentration of hoa. in conclusion, the f5 organogel presented the most predictable formula that accomplished the aim of this work compared with the lower concentrations of the organogels as a more connecting scaffold with denser spherulites. figure 2. sol to gel and gel to sol transitions temperatures of selected hoa in so organogels by vial inversion method. figure 3. optical images of selected hoa formulations f1, f3 and f5 in so organogels as the images taken using x40 magnification and the magnification bar is 50 µm. fourier transform infrared (ftir) the hydrogen bonds between the functional groups, the carbonyl and the hydroxyl groups of hoa molecules were essential and proved to build the scaffold of organogels usually investigated by ftir. hence, the ftir has been executed; as shown in figure 4a, the carbonyl associated peaks for selected organogels showed peaks at 1745 cm-1 indicating the interactions between so and hoa molecules. this might indicate the high solubility of hoa in so. f5 showed a peak at 1698 cm-1 which is the exact position of the peak related to the carbonyl group of hoa representing the cyclic dimerization of hoa molecules that help in scaffold constitution(17, 18). this explicit appearance of carbonyl related peak at 1698 cm-1 results from the high content of the hoa.also, the peaks correlated to hydroxyl groups were almost disappearing in all selected organogels as this means hydrogen bonds between molecules, as shown in figure 4b.in conclusion, a righteous balance between the interand intra molecularinteraction represented by the gelator gelator interaction and gelatorsolvent interaction iraqi j pharm sci, vol.31(2) 2022 12-hydroxyoctadecanoic acid organogels as a floating system 173 figure 4. ftir spectra as the a shows the carbonyl group region, where b displays the whole spectrum. oscillatory rheology studies the dissemination of gels in stomach media reflects their weakness, which is not appropriate for our work that planned to keep the organogels intact to combat the stomach content and motion. thus, amplitude sweep was applied to test four parameters; firstly, the strength or the elasticity of the organogels via the g' (storage modulus represents the organogel strength or the elasticity), g'' (loose modulus represents the viscose status of the organogel). the third parameter is the lver (linear viscoelastic region signifies the persistence of the organogel elasticity by having an almost constant g' values) and the flow point (means g'=g'' and the begun of organogel destruction) as shown in figure 5 and table 2. the amplitude sweep figures were figures 5a, b and c, and it is clear that the g' and g'' values were augmented as the hoa concentration increased, whereas the lver and flow point values showed a reduction. these amplitude sweep study outcomes were similar to span 60 in so organogels. this effect might be because the increasing spherulite aggregates connections might diminish with gelator concentration augmentation(6). table 2 . the amplitude sweep parameters for hoa in so as each value represent the average of 3 values (n=3) ± sd. hoa/so formulation g' (pa) g'' (pa) lver (%) flow point (%) f1 6329±1954 1117± 443 0.18±0.07 2.4±0.17 f3 95683± 16925 19124± 3838 0.19±0.01 1.4±0.15 f5 442796± 148751 91376± 26634 0.08±0.01 0.96±0.057 g': represents storage modulus and its unit in pascal. g'': represents loose modulus and its unit in pascal. lver: represents the linear viscoelastic region on the g' curve and its unit % as in shear strain. the frequency sweep execution was to study the motion effect on the organogels, as was shown in figures 5 d, e and f. all the organogels were frequency-independent as g' and g'' were parallel, and g' curves were higher than g'' curves. this outcome might point to that the organogels kept their elasticity alongside different frequency values. this result was similar to the organogel of hoa when gelled in soybean oil and medium-chain triglycerides, which showed frequency not depending on organogels(19). to conclude, the increase in the hoa concentration in the organogel showed an increase in the g' values, reflecting the increase in the solid content. this result harmonized with the conclusion of the organogels image. iraqi j pharm sci, vol.31(2) 2022 12-hydroxyoctadecanoic acid organogels as a floating system 174 figure 5. rheology oscillatory figures, the a, b and c represent the amplitude sweep, and the figures d, e, and f represent the frequency sweep of the selected organogels of hoa in so. investigation of the floating properties for organogels in-vitro floating study after the organogel gelation in the capsules, the capsules were directly buoyant, and within minutes, the capsule’s shells dissolved in hcl media, and the solid organogel was buoyant. also, floating duration monitored all the selected hoa organogels and showed the same floating duration of 24 hours. similarly, oilentrapped calcium pectinate gel beads float for 24 hours(20). in a word, all selected organogels were floating for 24 hours. in-vivo floating study this study was executed to support the floating in-vitro outcomes of hoa organogels. photos were taken to show the gel formation and it’s residues for prolonged periods. the gels were still persistent at the four scarifying times and gradually degraded in the stomach within the time frame, as shown in figure 6. this result was like the results by aa aboelwafa et al for raft liquid grdds that persisted in the rat stomach for 8 hours(21). as a result, f5 organogel presented the floating and the persisting for 12 hours in the rat’s stomach, attaining this study’s aim. iraqi j pharm sci, vol.31(2) 2022 12-hydroxyoctadecanoic acid organogels as a floating system 175 figure 6: images showed sectioned rat’s stomach at different periods as the f5 organogel was stained with 0.1% w/v methylene blue. in-vitro release study the depot property was studied via an invitro release study using cn that was loaded and solubilized within the selected hoa organogels, as shown in figure 7. the cn release of f5 and f3 organogels, at 3 hours of the release study was not more than 31% w/w and 41% w/w respectively, and they were barely crossed the 65% w/w and 73% w/w respectively after 24 hours of the study. the f1 hoa oganogels released 69% w/w of cn at 3 hours of experiment, then cn was gradually released, reaching the 100% w/w of cn within the time frame of the release study. both f3 and f5 hoa organogels emulate in the slowing the cn release the best formulation of the floating tablets that contained carrageenan by nagarwal group (22). the f5 organogel presented the slowest cn release. this result might be due to the more connecting scaffold as shown in images and the strength presented by the higher values of g' as these might help capture the drugs within the scaffold of the organogel. in summary, the f5 was better in slowing the release of cn than other organogel concentrations. figure 7. the cn percentage release in stomach solutions ph 1.2 from selected concentrations of hoa/so organogels. conclusion f5 organogel presented the best values that accomplish the aim of this work compared with the lowest concentrations of the organogels as a more connecting scaffold as shown in microscopy study and higher g' that attributed to the more elastic or strong organogel as well as the slowest cn release for 24 hours. all the organogels were proven to be buoyant by in-vitro test for 24 hours, and the floating of f5 hoa/so was evident in the rat’s stomach for 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el-gazayerly, gastroretentive raft liquid delivery system as a new approach to release extension for carrier-mediated drug. drug delivery, 2018. 25(1): 1161-1174. 22. nagarwal, r.c., d.n. ridhurkar, and j.j.a.p. pandit, in vitro release kinetics and bioavailability of gastroretentive cinnarizine hydrochloride tablet. aaps pharmscitech, 2010. 11(1): 294-303. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ synthesis and charecterization of a new ligand schiff bases and its complexes co(ii),ni(ii), cu(ii), and zn(ii) ions iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 38 synthesis, characterization and antibacterial activities of ligand type n2o4 schiff base and its novel complexes with co(ii), ni(ii), cu(ii) and zn(ii) ions. ahmed t. numan * , manhel r. aziz *,1 and sinaa m. shaker ** *department of chemistry,college of education, ibn-al-haitham,university of baghdad,baghdad, iraq. ** microbiology, medical city, teaching laboratories, baghdad, iraq. abstract the reaction of 2-amino benzoic acid with 1,2-dichloroethane under reflux in methanol and koh as a base to gave the precursor [h4l]. the precursor under reflux and drops of ch3cooh which reacted with (2mole) from salicycaldehyde in methanol to gave a new type n2o4 ligand [h2l], this ligand was reacted with (mcl2) where [m= co (ii), ni(ii), cu(ii) and zn(ii)] in (1:1) ratio at reflux in methanol using koh as a base, to give complexes of the general formula [m(l)]. all compounds have been characterized by spectroscopic methods [ 1 h nmr ( just to the ligand), ftir, uv-vis, atomic absorption], melting point, conductivity, chloride content, as well as magnetic susceptibility measurements. from the above data, the proposed molecular structure of [co(l)], [ni(l)], [cu(l)] and[zn(l)] complexes adopting an octahedral about this metal ions. the synthesized ligand, along with their metal complexes were screened for their in vitro antibacterial activity against ten local strains of e. coli as gram-negative bacteria in addition to ten strains of salmonella typhi and to ten strains of acinetobacter baumannii and ten grampositive bacteria utilizing for locally strains of staphylococcus aureus, were tested also using the agar diffusion technique. keywords: schiff base , complexes . الخالصة تحا الصعاديد داي كلاووو أكااا -2,1ما أمينو حاام البنووكاك -2وذلك من مفاعلة [h2l] تضمن البحث تحضير الليكاند ومان الا ب تفاعاا الماادذ [h4l] ود هيدووكسايد العاودكوك كماعادذ اذ اعاات الصفاعاا الماادذ الو اايةاالوجااع يا الميااانوب و وجا عاات الالجا اذ ا ch3coohتح الصعاديد االوجااع يا الميااانوب ولاارا مان salicylaldehydeموب من 2م الو اية ( 1:1) ثم مفاعلة الليكاند ما دا الدنا ار الفلوكاة ا اصاداك الميااانوب و اااع للصفاعاا و نسابة (n2o2)نوع [h2l]الصفاعا الليكاند , حياث [m(l)]وتح الصعديد األوجاع , حيث تكون مدمدا جدكدذ ذوا العيغة الداماة كماعدذ وجود هيدووكسيد البوتا يوكو m= co(ii), ni(ii), cu(ii), and zn(ii) . شاع جمي المركبا الارق الايفية الصالية )االشادة تحا الحماراا واالشادة ياوق وطيا الارنين الناووي المغناطيسا و المرئية –البنفسجية 1 hnmr )الصحلياا الكما الادليك للدنا ار ما الصو ايلية و( )يماظ لليكاناد دوجااة االنعااباو والحسا ااية المغناطيسااية ماان نصااائا البحااث كااا التااكا الفرا اا الممصاار الموالوكااة الكبر ائيااة ومحصااوو الكلااوو و [co(l)], [ni(l)], [cu(l)], [zn(l)] . عتار دو الفدالية الباكولوجية الاوج الالية الحية لليكاناد ومدمداتاض ادثمان الساو ماان وعتاار عااوال salmonella typhiالاارو ماان اب أ و عتاار عااوال كبكصركااا ااالبة لعاابغة كااراك e. coliماان اب عااوال acinetobacter baumannii ماان اب عتاار عااوال وstaphylococcus aureus بكصركااا موجبااة لعاابغة كااراك حيااث جمياا ك األ ناف أالصبر أ صاداك تمنية الحيود. introduction schiff bases and their coordination compounds have played a great importance in medicine, industry and biochemistry. schiff bases are characterized by the (-n=ch-) (imine) group which imports in elucidating the mechanism of transamination rasemination reaction in biological (1,2) . during the past two decades, considerable attention has been paid to the chemistry of metal complexes containing nitrogen and other donor (3) . this may be attributed to their stability, biological activity (4) and potential application in many fields such as oxidation catalysis (5) and electrochemistry (6) . we have already drawn attention (7-11) to the strong relationship between metals or their complexes, and antibacterial (12-18) , and anticancer (19,20) activities. in 2009 a. thabit and co-workers reported the synthesis of a novel ligand type n2o2 and its complexes with co(ii), cu(ii), zn(ii), and cd(ii), which have been characterized by spectroscopy and elemental analysis (21) . this paper reports the synthesis and characterization of new derived from the reaction of α-amino carboxylic acid with 1,2dichloroethane in methanol, resulted (precursor)[h4l], which reacted with salicylaldehyde and its co(ii), ni(ii), cu(ii) and zn(ii) complexes. 1corresponding author email : manhelreaziz@yahoo.com received : 13/10/2009 accepted : 24/1/2010 iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 39 experimental chemicals used were analytical grades; metals were used as chloride salts. the complexes were determined by absorption technique, using shimadza (a.a) 680 g atomic absorption spectrophotometer. i.r data were recorded as (kbr) disc using a schimadza 4800 s ftir spectrophotometer in the range (4000-400) cm -1 which measured at the laboratories of ibn-sinaa company. 1 hnmr spectra were recorded in dmso-d6 using burcker 300 mhz spectrometer 1 which measured at amman jordan. (uv-vis.) spectra were obtained in (meoh) on a cecil, ce 2700 spectrophotometer in the range (200900) nm using quartz cell which measured at the college of ibnal-haithem. magnetic measurements were carried out on solid compounds using 6 bruker b.m. melting points were recorded on an electro thermal stuart apparatus and are not corrected. electrical conductivity measurements of the complexes were recorded at 25c for 10 -3 m solutions in (moh) as a solvent using a wissenchaftilich technique werksttaten, d1820 bweilheimi.f 42 conductivity meter which measured at the college of ibnal-haithem. the chloride contents for complexes was determined by potentiometric titration method on (686-titro processor-665), dosinat-metrom swiss, which measured at the laboratories of ibn-sinaa company. antibacterial screening was done at laboratories of medical city, baghdad using agar diffusion technique (22,23) . the ligand along with their metal complexes were screened for their in vitro antibacterial activity against gram negative bacteria (e. coli), gram-positive bacteria (staphylococcus aureus), salmonella typhi and acinetobacter baumannii bacterial strains. the ligand and their complexes have shown varied antibacterial activities against one or more bacterial strains and this activity enhanced on coordination / chelating. preparation of the ligand [h2l] the ligand was prepared by two steps: step (1): a solution of α-amino carboxylic acid (0.35 g, 2.25 mmole) in methanol (10ml) was added to it dichloroethane (0.2g, 2.23mmole) , (0.2 ml) with koh (0.18ml) as a base, the mixture was refluxed for 3 hours with stirring. then the mixture was allowed to cool at room temperature. the resulting a gray solid (precursor) [h4l] was obtained which filtered off and then washed with ethanol. yield (37%), (0.25g), mp (245-250 c˚ dec.). step (2): a solution of salicalyaldehyde (0.2 g, 1.66mmole), (0.18 ml) in methanol (5 ml) was added to the precursor solution [h4l] (0.25 g, 0.83 mmole), then (8) drops of ch3cooh was added slowly to the reaction mixture. the mixture was refluxed for (5 hours) with stirring. the resulting was orange solid of [h2l] as product was filtered of and then washed with ethanol. yield (42%), (0.18g), mp (220-226 c˚ dec.). preparation of the ligand [h2l] with metal ions (0.15 g, 2.94 mmole) of ligand solution in methanol (10ml), with koh as a base was added to a solution of (0.07 g, 2.94 mmole) cocl2.6h2o in methanol (10ml), the mixture was refluxed with stirring for (4 hours). the resulting was dark brown solid as product which was filtered of and then washed by water and re-crystallized with ethanol. the complexes [ni(l)], [cu(l)] and[zn(l)], were obtained in a similar method to that mentioned in the preparation of [co(h2l)2] complex described above, (table1) stated the quantity of starting materials and some physical properties of the prepared complexes. table 1 : the microanalysis results and some physical properties for the prepared ligand h2l and its complexes compounds formula colour m.p(c˚) yield% chloride metal m .wt h4l c16h16n2o4 gray 245-250 dec. 37 nil ---- 300.31 h2l c30h24n2o6 orange 220-226 dec. 42 nil ---- 508.52 [co(l)](1) c30h22n2coo6 dark brown 230 dec. 60 nil 10.42 (9.18) 565.44 [ni(l)](2) c30h22n2nio6 yellowish green 292 dec. 78 nil 10.38 (11.18) 565.20 [cu(l)](3) c30h22n2cuo6 dark green 320 dec. 52 nil 11.15 (11.37) 570.05 [zn(l)](4) c30h22n2zno6 light green 230 dec. 53 nil 11.43 (12.44) 571.90 iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 40 nh2 cooh + clch2ch2cl 2 koh methanol ref lux 3 hours 2-amin obenzoic acid dichloroethane h2n c o o ch2h2c o c o nh2 +2 ch oh o 2-hydroxybenzaldehyde ethane-1,2-diyl bis(2-aminobenzoate) n c o o ch2h2c o c o n ch hc hooh (z)-ethane-1,2-diyl bis(2-((z)-2-hydroxybenzylideneamino)benzoate) ref lux 5 hours methanol ch3cooh 8 drops + mcl2 n c o o ch2h2c o c o n ch hc oo m koh methanol ref lux 4 hours results and discussion synthesis of the ligand the ligand [h2l] was prepared according to the general method shown in scheme (1). the i.r spectral of the precursor [h4l] of the ligand [h2l], is shown in fig (1), the results were summarized in table (2) the figure exhibited band at (3329,3379) cm -1 which attributed to the stretching vibration of asymmetric and symmetric (n-h) for nh2 group, also the spectrum was showed bands at (1612)cm -1 , (1249) cm -1 and (1192) cm -1 attributed to υ (c=o) of ester group, υ (c-o) ester group and υ (c-o) phenolic respectively (21,24-27) , by comparing with the i.r spectrum of the ligand [h2l], fig (2), table (2) exhibited bands (3421) cm -1 , which can be attributed to υ (o-h) and (disappeared nh2 groups), also the spectrum showed bands at (1612)cm -1 , (1608) cm -1 , (1273) cm -1 and (1239) cm -1 attributed to υ (c=o) ester group, υ (c=n) imine group, υ (c-o) ester group and υ (c-o) phenolic respectively [21,24-27]. the (u.v-vis) spectra for precursor [h4l], fig (3), the results were summarized in table (3), the figure exhibits two high intense absorption peaks at (248) nm εmax (2532) molar -1 cm -1 , and (319) nm εmax (2484) molar -1 cm -1 , which assigned to (π→π * ), and (n→π * ) transition respectively [28,29], while the (u.v-vis) spectrum of the ligand [h2l] fig (4), (table3) exhibits three high intense absorption peaks at (250) nm εmax (2631) molar -1 cm -1 , (293) nm εmax (2660) molar -1 cm -1 , and (360) nm εmax (2581) molar -1 cm -1 , which assigned to (π→π * ), (π→π * ) and (n→π * ) transition respectively (28,29) . the 1 h nmr spectrum of the ligand (h2l), in dmso-d 6 , fig (5) shows proton of (o–h) group (ph–oh) which appears as a singlet peak signal at (10.2) ppm. the proton of (c–h) imine group appears as a singlet peak at (8.7) ppm. the multiples signals peaks at the range between (7-8)ppm, are due to aromatic hydrogen of carbon for the benzene ring which bonded with (c=o) carbonyl group, while the multiples signals peaks at the range between (6-7)ppm, are due to aromatic hydrogen of carbon for the benzene ring which bonded with (c=n) imine group, also the spectrum of the ligand appears a triplet peak at (4.7)ppm, which assigned to (-ch2-) methelene group, as soon as a singlet high peak at (2.5)pmm for the dmso-d 6 solvent [24,25,30,31]. where m= co(ii),ni(ii),cu(ii),and zn(ii) scheme 1 : the preparation of the ligand [h2l] and its complexes iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 41 table 2 : infrared spectral data(wave number ύ) cm -1 for the ligand h2l and its complexes figure 1: infrared spectrum of the precursor (h4l) figure 2 : infrared spectrum of the ligand (h2l) figure 3 : electronic spectrum of the precursor (h4l) compound υ(c=n) υ(c=o) ester υ(c-o) ester υ(c-o) phenolic υ(o-h) υ(m-o) phenolic υ(m-o) esteric υ(m-n) h4l ----1612 1249 1192 ---- --- ---- ---- h2l 1608 1612 1273 1239 3421 ---- -------- [co(l)](1) 1593 1612 1327 1300 ----505 461 416 [ni (l)](2) 1593 1612 1327 1300 -----536 440 430 [cu (l)](3) 1585 1609 1319 1288 -----540 468 428 [zn (l)](4) 1593 1611 1327 1300 ----516 459 445 iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 42 table 3 : electronic spectral data for the ligand h2l and its complexes c.t= charge transfer figure 4: electronic spectrum of the ligand (h2l) figure 5: 1 h nmr spectrum of the ligand (h2l) compound λ nm εmax molar -1 cm -1 assignment ratio molar conductivity s.cm 2 .mol -1 magnetic susceptibility b.m coordination h4l 248.9 319.7 2532 2484 π→π* n→π* ---- ----- ----- ------ h2l 250.0 293.6 360.8 2631 2660 2581 π→π* π→π* n→π* ---- ------ ----- ----- [co(l)](1) 248.5 295.6 414.0 629.8 2642 2531 1264 96 ligand field ligand field c.t 4 t1g(f)→ 4 t2g(f) neutral 18.1 3.87 (4.15) octahedral [ni(l)](2) 245.0 305.3 400.1 580.0 2597 1617 1317 62 ligand field ligand field c.t 3 a2(g)→ 3 t1g (p) neutral 14.9 2.83 (3.2) octahedral [cu(l)](3) 243.5 300.0 406.1 698.9 2608 2551 2636 52 ligand field ligand field c.t 2 e → 2 t2 neutral 12.46 1.7 (1.9) octahedral [zn(l)](4) 220.3 289.0 388.5 1483 584 371 ligand field ligand field c.t neutral 13.65 -----octahedral iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 43 synthesis of the complexes the reaction the of ligand [h2l] with co(ii), ni(ii), cu(ii) and zn(ii) was carried out in meoh. these complexes are stable in solution. the analytical and physical data, table (1) and spectral data, table (2) and table (3) are compatible with the suggested structure scheme (1). the i.r spectra of the complexes [co(l)](1), [ni(l)](2), [cu(l)](3), and [zn(l)](4). fig (6) and fig (7), respectively, the results were summarized in table (2), the figure exhibited at (1608) cm -1 in the free ligand spectrum which assigned to υ (c=n) imine group shifted to lower frequency and appeared at (1593) cm -1 , (1593) cm -1 , (1585) cm -1 and (1593) cm -1 for the complexes (1),(2),(3), and (4) respectively (25-28) . these bands were assigned the υ (c=n) stretches of reduced bond order, this can be attributed to the delocalization of metal-electron density into the ligand π-system (homo→lumo) [32,33]. (homo=highest occupied molecular orbital, lumo= lowest unoccupied molecular orbital). the phenolic (c-o) stretching vibration appeared at (1239) cm -1 in the free ligand was shifted to higher frequency and appeared at (1300) cm -1 , (1300) cm -1 , (1288) cm -1 , and (1300) cm -1 for the complexes (1), (2), (3) and (4) respectively, as well as ester group (c-o) stretching vibration appeared at (1273) cm -1 in the free ligand was shifted to higher frequency too, and appeared at (1327) cm -1 , (1327) cm -1 , (1319) cm -1 and (1327) cm -1 for the complexes (1), (2), (3) and (4) respectively, all that indicated a linkage between oxygen of phenolic group and oxygen of ester group and the metal (24,32,34) . the spectra showed the appearance of bands at (416) cm -1 , (430) cm -1 , (428) cm -1 and (445) cm -1 refer to υ (m-n) for complexes (1), (2), (3) and (4) respectively, these bands confirm the coordination of the nitrogen atom to the metal center, while the bands at [(505),(461)] cm -1 , [(536),(440)] cm -1 and [(540),(468)] cm -1 [(516),(459)] cm -1 assigned to υ (m-o) of complexes (1),(2),(3), and (4) respectively, theses bands indicating the phenolic and esteric oxygen in the ligand is involved the coordination with metal ions in complexes [3437]. the (u.v-vis) spectra for the complexes are shown in fig (8) and fig (9), table(3), complex [co(l)]: showed two high intense peak at (248) nm εmax (2642) molar -1 cm -1 and (295) nm εmax (2531) molar -1 cm -1 were assigned to the ligand field, while a medium intense peak at (414) nm εmax (1264) molar 1 cm -1 was assigned to the charge transfer (c.t), a weak broad peak at (629) nm εmax (96) molar -1 cm -1 was assigned to(d-d)electronic transition ( 4 t1g(f)→ 4 t2g(f)) suggesting octrahedral geometry (29) . complex[ni(l)]: showed two high intense absorption peaks at (245) nm εmax (2597) molar -1 cm -1 and (305) nm εmax (1617) molar -1 cm -1 are due to the ligand field, another high intense peak at (400) nm εmax (1317) molar -1 cm -1 was assigned to (c.t), while a weak broad peak at (580)nm εmax (62) molar -1 cm -1 was assigned to (d-d) electronic transition (3a2(g)→ 3 t1g(p)) suggesting octahedral geometry (29) . complex[cu(l)]: showed two high intense absorption peaks at (243) nm εmax (2608) molar -1 cm -1 and (300) nm εmax (2551) molar -1 cm -1 are due to the ligand field. a high intense absorption peak at (406) nm εmax (2636) molar -1 cm -1 was assigned to (c.t), while a weak broad peak at (698) nm εmax (52) molar -1 cm -1 was assigned to (d-d) electronic transition ( 2e →2t2 ) suggesting octahedral geometry (29) . complex[zn(l)]: showed two peaks at (220) nm εmax (1483) molar -1 cm -1 and (289) nm εmax (584) molar -1 cm -1 are due to the ligand field. while a weak broad peak at (388) nm εmax (371) molar -1 cm -1 was assigned to (c.t), the d 10 configuration of zn ii ion along with the data obtained confirms a octahedral structure around the ion (29) . the molar conductance of the complexes in methanol lie in the range(12.46-18.1 ohm -1 cm -2 mol -1 ), table(3), indicting their non-electrolyte having molar ratio of metal:ligand as 1:1 (38) . the magnetic moments for the complexes are shown in table (3) (39) . figure 6: infrared spectrum of the complex [co(l)](1) iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 44 figure 7: infrared spectrum of the complex [ni(l)](2) figure 8: electronic spectrum of the ligand [co(l)](1) figure 9: electronic spectrum of the ligand [ni(l)](2) biological activity the antibacterial activity of the synthesized ligand [h2l] and its complexes [co(l)](1), [ni(l)](2) , [cu(l)](3) , and [zn(l)](4) table (4, 5, 6 and7), fig(10 and 11) were tested utilizing the agar diffusion technique (40) . the organisms tested were staphylococcus aureus , e. collie , salmonella typhi , and acinetobacter baumannii. the agar media (muller-hinton agar) were inoculated with test organisms and a solution of the tested compound (100μg/ml) (41) , was placed separately in cups (6 mm diameter) in the agar medium. the inhibition zones were measured after 24 hours incubation at 35 c ˚ . separate studies were carried out with the solution alone of dmso and the showed no activity against any bacterial strains (41) . the results of these studies revealed that metals complexes showed an effective in the inhibition of acinetobacter baumannii, table (4). the ligand and (co +2 , ni +2 , cu +2 ,zn +2 ) complexes were showed an inhibition in some strains in each of staphylococcus, e. coli, and salmonella typhi, as shown in table (5, 6, and 7) . biological activity of the previous compounds in inhibition of bacterial growth could be attributed to one of the following mechanisms, the first mechanism is by the inhibition of the bacterial cell wall synthesis by bounding to the precursor of the cell wall (42) , second mechanism revealed that some antibodies have similar stereo structure to substrate (d-alanyl d-alanine). so it will act competitive inhibitions for the enzymes (transpeptidase and /or carboxpeptidase) which are the main enzymes catalyzed the end step in the biosynthesis of peptidoglycans of the bacterial iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 45 cell wall (43) . other mechanisms could contributed to the results found in the study which include the inhibition of biosynthesis of bacterial proteins by linking to the ribosoms by doing so, the ribosomes will not be in contact with trna, so the bacteria will not survive (44) . an other mechanisms were postulated that some antibodies inhibit the denovo synthesis of bacterial dna by splitting dna in dna-enzyme complexes by inhibition dna ligase (45) . table 4 : biological activity of acinetobacter baumannii bacteria of the ligand [h2l] and its complexes a= acinetobacter baumannii bacteria table 5 : biological activity of staphylococcus aureus bacteria of the ligand [h2l] and its complexes s= staphylococcus aureus bacteria table 6 : biological activity of e. coli bacteria of the ligand [h2l] and its complexes e= e. coli bacteria table 7 : biological activity of salmonella typhi of the ligand [h2l] and its complexes sal= salmonella typhi bacteria compound bacteria (zone of inhibition (diameter mm)) a1 a2 a3 a4 a5 a6 a7 a8 a9 a10 h2l 14 11 nil 14 16 14 15 12 13 16 [co(l)](1) 15 10 nil 15 14 15 12 nil 16 15 [ni(l)](2) 14 10 12 14 13 16 13 12 15 15 [cu(l)](3) 14 10 11 12 13 12 13 13 12 13 [zn(l)](4) 11 11 nil 14 13 14 14 12 15 16 compound bacteria (zone of inhibition (diameter mm)) s1 s2 s3 s4 s5 s6 s7 s8 s9 s10 h2l nil nil 16 nil nil nil nil nil nil nil [co(l)](1) nil nil 16 nil nil nil nil nil nil nil [ni(l)](2) nil nil 16 nil nil nil nil nil nil nil [cu(l)](3) 9 8 17 9 10 9 8 9 8 11 [zn(l)](4) nil nil 18 12 nil nil 12 nil nil nil compound bacteria (zone of inhibition (diameter mm)) e1 e2 e3 e4 e5 e6 e7 e8 e9 e10 h2l nil nil nil 12 12 10 nil 11 nil 10 [co(l)](1) 9 nil nil 10 7 10 nil nil nil 10 [ni(l)](2) 10 nil nil 11 8 12 nil 10 nil 8 [cu(l)](3) nil nil nil 12 8 12 nil 10 nil 10 [zn(l)](4) nil nil nil 8 8 8 nil 10 nil nil compound bacteria (zone of inhibition (diameter mm)) sal1 sal 2 sal 3 sal 4 sal 5 sal 6 sal 7 sal 8 sal 9 sal10 h2l nil 11 nil nil nil nil nil nil nil nil [co(l)](1) 10 12 9 nil nil nil nil nil nil 12 [ni(l)](2) nil nil 10 nil nil nil nil nil nil nil [cu(l)](3) nil nil nil nil nil nil 12 9 nil nil [zn(l)](4) 10 nil nil nil nil nil nil nil nil nil iraqi j pharm sci, vol.19(1) 2010 schiff base of aminobenzoic acid 46 figure 10: inhibition zones for acinetobacter baumannii utilizing agar diffusion technique figure 11: inhibition zones for staphylococcus aureus utilizing agar diffusion technique references 1. k. y. ,a.mayer , k.k.cheung , "synthesis of transition metal isocyanid complexes hydrogen bonding sits in peripheral location" , inorg, chem, acta , 1999 285:223-232. 2. a.s.shawali, n.m.s.harb, k.o.baghdad, "a study of tautomerism in diazonium coupling products of 4-hydroxycoumrin", 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(1991). 45. hopper d. c.: quinolones. in. g. l. mandell , j. e. bennett , r. dolin(ed.) mandell , douglas and bentell , s:" principles and practice infections disease". 5 th ed. churchill livingstone , philadelphia , (2002), 404-423. http://www.bactwise.edu/?bact330.html.(2002) iraqi j pharm sci, vol.32( 1 ) 2023 nanoparticle formulation design of piperine doi: https://doi.org/10.31351/vol32iss1pp14-30 14 an update on nanoparticle formulation design of piperine to improve its oral bioavailability: a review nindya kusumorini*, akhmad kharis nugroho**,1, suwijiyo pramono*** and ronny martien** *doctoral program in pharmaceutical science, faculty of pharmacy, universitas gadjah mada, yogyakarta 55281 indonesia **department of pharmaceutics, faculty of pharmacy, universitas gadjah mada, yogyakarta 55281 indonesia ***department of pharmaceutical biology, faculty of pharmacy, universitas gadjah mada, yogyakarta 55281indonesia abstract piperine, a crystalline alkaloid compound isolated from piper nigrum, piper longum, and other types of pipers, has many excellent pharmacological advantages for preventing and treating some specific diseases, such as analgesic, anti-inflammatory, hepatoprotective, antimetastatic, antithyroid, immunomodulatory, antitumor, rheumatoid arthritis, osteoarthritis, alzheimer's, and anticancer. however, its potential for clinical use through oral administration is hindered by water solubility and poor bioavailability. the low level of oral bioavailability is caused by low solubility in water and is photosensitive, susceptible to isomerization by uv light, which causes piperine concentration to decrease. a lot of nanoparticle formulation approaches have been applied to improve the poor oral bioavailability of piperine. oral nanoparticle formulation strategies have been successfully implemented in increasing the solubility and bioavailability of piperine within the body, such as the formulation of nanoparticles, nanosuspensions, liposomes, complexation using polymers, and micro/nano-emulsions. this nanoparticle formulation approach has been successful in increasing the solubility, permeability, and bioavailability of piperine effectively. in addition, this nanoparticle formulated piperine can deliver piperine in a targeted manner and increase the efficacy of piperine treatments, such as for alzheimer’s disease, epilepsy, rheumatoid arthritis, diabetes, breast cancer, colon cancer, and human brain cancer. keywords: piperine, bioavailability, solubility, nano-formulation, nanoparticle, nanoemulsion introduction piperine is a crystalline, yellow, odorless, and pungent alkaloid compound isolated from piper nigrum and piper longum. piperine is chemically known as (ee)-1-[5-(1,3-benzodioxol-5-yl)-1-oxo2,4-pentadienly]-piperidine, with the molecular formula of c17h19no3, a molecular weight of 285.34 g/mol, and melting point at 128-130oc (1–3). the piperine structure consists of three subunits, namely methylenedioxyphenyl ring, side chain with conjugated double bonds, and basic piperidine moiety attached through carbonyl amide linkage to side chain (4). piperine is unstable in acidic, alkaline, oxidizing , and sunlight environments. piperine can be degraded to trichostatin and cispiperyline under acidic conditions (hcl 2 m) and to piperanine, piperettine, and piperyline under sunlight (5). piperine contains four isomeric forms, namely trans-trans isomer (piperine), cis-trans isomer (isopiperine), cis-cis isomer (chavicine), and trans-cis isomer (isochavicine) (fig. 1). piperine contains four isomeric forms, namely trans-trans isomer (piperine), cis-trans isomer (isopiperine), cis-cis isomer (chavicine), and trans-cis isomer (isochavicine) (fig. 1). the isomerization of piperine compounds increases along with the increasing light intensity and exposure. transformation of piperine into chavicine isomers can be seen in long-term storage and spontaneously which causes a loss of spicy taste (6). the piperine ring influences the chemical stability and reactivity of piperine. this ring greatly influences steric-electronic properties, especially among the aromatic ring, carbonyl group, and alkene system (7,8). 1corresponding author e-mail: a.k.nugroho@ugm.ac.id received: 9/2 /2022 accepted: 10/5 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp14-30 iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 15 figure 1. structure of piperine and its isomers piperine can also be isolated from white and black pepper (piper nigrum) (9,10); root and fruit of piper longum (11,12); piper chaba (12,13); piper guineense fruit (14); and roots, stems, leaves, and fruit of piper sarmentosum (15). the alkaloid compound of piperine extracted from black and long pepper is commonly processed into traditional medicine in some asian countries. some medical products such as ayurveda, sidda, unani, and tibetan have used black pepper for treating cough, fever, sore throats, indigestion, insomnia, and heart disease (16–19). through the development of science and technology, many pharmacological benefits of piperine compounds have been successfully revealed. several studies show that piperine has therapeutic effects like antiallergic (20,21), antiinflammatory (22–25), rheumatoid arthritis (26), osteoarthritis (27), anticonvulsants, anti-epileptic, antidepressants, neurodegenerative disorders, and alzheimer's disease (24,28–32). piperine can also inhibit cell proliferation by reducing cell variability, increasing reactive oxygen species (ros), inducing cell shrinkage, fragmenting the dna by blocking cell cycle and caspase-3 activity (33), and effectively inhibiting cell proliferation and cell migration, inducing apoptotic processes in the her-2 gene that expresses breast cancer cells, inducing mmp-9, and reducing epithelial growth factor (egf) (34). piperine is safe to take orally at a 20 mg/kg bw (35). however, piperine is still not used clinically for disease prevention and treatment despite its broad pharmacological potential. due to its low solubility and bioavailability, piperine is poorly absorbed in the body. however, many scientists have designed the right formulation to solve this issue. recent strategies in oral nanoparticle formulated of piperine to increase their solubility and bioavailability are reviewed in detail. this review was conducted using several databases or computer-based electronic searches including pubmed, science direct, and google scholar to access all journals related to the nano-formulation of piperine. all major research papers must be published between 2017 – march 2022 with the keywords “piperine”, “formulation”, “nanoparticle”, “polymeric”, “nanocarrier”, “phospholipid”, and “nanoemulsion”. piperine physicochemistry piperine is the main alkaloid found in white pepper, black pepper, and long pepper, having a weak base (ph 8.0 – 8.5) with a pka of 12.22, and is poorly soluble in water with a solubility of 22.34 mg/l at 25oc (36). piperine has a melting point at 128-130oc, is soluble in ethanol (1g/15ml), methanol, petroleum ether (1g/1.7ml), and chloroform (1g/1.7ml) (3). piperine compounds are lipophilic with a log p value = 2.25 (36), which shows that they can be higher in octanol solvents than in water with a concentration ratio of 177.82:1. piperine is slightly soluble in water and has a low dissolution level, thus it is less effective for oral use. based on the results of piperine structure analysis, according to lipinski’s and veber’s rules, piperine compounds have good permeability in the membrane. the results of piperine permeability analysis are presented in the following section (37) (table 1). table 1. piperine structural permeability analysis (37). piperine structure analysis parameter results high permeability requirements log p 2.3 5 h-bonding donor 0 5 h-bonding acceptor 3 10 psa (polar surface area) 38.77 ao 140 ao number of hbonding donors & acceptors 3 12 number of rotating bonds 2 10 molecular weight 285.34 g/mol 500 biopharmaceutics properties of piperine based on the structural permeability analysis, piperine has good permeability across intestinal membranes. this is supported by some studies khajuria et al. (38) and suresh & srinivasan (39) which showed that piperine can be easily absorbed across the intestinal membrane barrier through passive diffusion and no metabolic changes observed during absorption. piperine is absorbed into the serosal fluid and intestinal tissue by 47-64% iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 16 and found to be transported predominantly to the duodenum (39–41). piperine can be rapidly absorbed into the intestine probably because the molecule is non-polar and lipophilic so that it can easily cross and get through the intestinal barriers. piperine does not experience biotransformation during intestinal absorption, 712% of absorbed piperine is found in the serosal fluid (39,40). the results of the intestinal examination showed that the highest concentrations in the stomach and small intestine could be reached within 6 hours after application, where less than 0.15% was detected in serum, kidney, and spleen from 30 minutes to 24 hours. the piperine’s membrane permeability analysis conducted in a previous study used caco-2-monolayer model, where the results obtained a permeability coefficient of 5.41 x 10-5 cm/s and 4.78x10-5 cm/s for basolateral-to-apical and apical-to-basolateral (42). piperine distribution begins after being absorbed into the intestine and then transported by serum albumin. piperine is bound to subdomain-1b of human serum albumin which is an important factor in the transport of piperine in the blood (43). based on a previous experiment conducted by suresh et al. (43) in rats that had been given a dose of 170 mg/kg orally, the maximum concentration of piperine was reached after 6 hours of application with 10.8% found in the tissues, and most of the piperine was found in the duodenum (8.8%). however, the result is in contrast to a study conducted by liu et al. (44), where the maximum concentration was obtained two hours after oral administration, with the highest concentration was in the liver. this difference may be due to the different forms of applied piperine, and other components may influence the distribution of piperine in the extraction process. formulation design developed to increase piperine’s oral bioavailability the oral method is ideally applied for drug administration because it makes the patients easily consume the medicine. oral administration can improve patients’ compliance, especially for longterm use compared to other consumption techniques (45). however, some latest chemical compounds being developed have low water solubility and low bioavailability, but high intraand inter-subject variability (46), and one of which is piperine. piperine has poor solubility, dissolution, and oral bioavailability (24% in rats) which are the main problems limiting its absorption (47). piperine is classified into the class ii biopharmaceutics classification system (bcs) based on its poor solubility in aqueous media and good permeability across the intestinal membrane barrier. many attempts and nanoparticle formulation designs have been developed to solve its low solubility and bioavailability. details of recent developments in the oral nano-formulation of piperine are discussed in the following sections: liposomes liposomes are spherical vesicles with an aqueous core and vesicle sizes from 30 nm to several micrometers, consisting of one or more phospholipid bilayer membranes or lamellae in which the polar head leads to the inner and outer aqueous phase of the vesicle (48). liposomes can deliver both hydrophilic and hydrophobic drugs. liposomes contain a lipid bilayer similar to the cell membrane. liposomes are made from a mixture of phospholipids, surfactants, and cholesterol. they function to increase the solubility of lipophilic and amphiphilic drugs, increase stability through encapsulation, reduce the toxicity of encapsulated drugs, are biodegradable, biocompatible, non-toxic, and non-immunogenic for systemic and nonsystemic administration (49). liposomes can increase the bioavailability of lipophilic drugs through oral administration by increasing cellular contacts and diffusion across epithelial and mucosal layers (50). the encapsulation of piperine into liposomes can increase the piperine’s dissolution and bioavailability levels. dutta & bhattacharjee (51) formulated piperine nanoliposomes for oral usage taken from soy phosphatidylcholine and tween 80 in a ratio of 1:1.2 and generated 78.6% entrapment efficiency, 29.75 ± 0.84 nm vesicle size, and resulted in 70% of piperine release for 8 hours in ph 6.4 buffer. imam et al. (52) formulated chitosan-coated piperine liposomes to improve the mucoadhesive properties of piperine liposomes. the liposomes were prepared through the thin-film dispersion method using a rotary evaporator and chitosan coating by electrostatic deposition. the chitosan functioned to increase the stability of piperine liposomes and protect them from chemical and enzymatic degradation in the digestive tract. the presence of chitosan on the liposomes’ surface showed an increase in stability and mucoadhesive properties by 2.8 times compared to those not coated with the chitosan. microemulsion, self-emulsifying drug delivery system (sedds), and self-nano emulsifying drug delivery system (snedds) microemulsions are single-phase isotropic mixtures consisting of thermodynamically stable and transparent oil, water, surfactant, and cosurfactant with an average droplet diameter of 10 to 140 nm (53,54). microemulsions are good candidates for oral drug consumption which is poorly soluble in water. they function to increase drug solubility, absorption rate, and bioavailability and reduce interand intra-individual variability in drug pharmacokinetics (55,56). microemulsions can improve the bioavailability of lipophilic drugs in several ways, such as protecting drugs from oxidation and enzymatic degradation and increasing iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 17 membrane permeability through lymphatic transport (57). lymphatic transport is the most contributing element to drug absorption in the intestine because it can protect against first-pass metabolism (58). the findings of a study by etman et al.(59) showed that piperine microemulsion for alzheimer’s therapy via oral route has succeeded in supporting and accelerating its delivery to the brain and producing better therapeutic results than pure piperine. piperine microemulsion has been successfully formulated with capryol 90 oil, surfactants tween 80 and cremophor rh40, and cosurfactant transcutol, which finally generates a particle size of lower than 150 nm with a negative zeta potential -30.36 mv. the size of the sedds particles lower than 500 nm enables an increasing absorption level of piperine through the lymphatic route (60). negatively charged nanoparticles can increase lymphatic uptake higher than positively and neutrally charged surfaces (61–63). a self-emulsifying drug delivery system (sedds) is an isotropic mixture of oils, surfactants, and cosurfactants that spontaneously forms an oilin-water emulsion during contact with gastrointestinal tract (git) fluids with mild agitation. the sedds system was then modified into self-micro emulsifying drug delivery systems (smedds) and self-nano emulsifying drug delivery systems (snedds) (64). sedds, smedds, and snedds formulations have some specific benefits, such as increasing the drug solubility and bioavailability, increasing drug absorption rate through the lymphatic pathway, decreasing the effects of first-pass metabolism, and increasing physical and chemical stability in long-term storage (65). the sedds, smedds, and snedds formulations can improve the bioavailability of lipophilic drugs through some mechanisms. they include lipid droplets built within self-emulsifying dispersions that can directly facilitate drug absorption and protect drugs from chemical and enzymatic degradation. they are localized in aqueous environments, influence the changes in gastrointestinal membrane permeability, and increase the drug absorption level via lymphatic pathways (66,67). the lipids in the git (gastrointestinal tract) stimulate the excretion process of bile salts and endogenous bile lipids which also supports lipid emulsification to form micelles and improves drug solubility in the git (65).surfactants can cause fluidization of intestinal membranes and opening of tight junctions which result in increased membrane permeability (68,69). the difference between sedds, smedds, and snedds systems can be viewed from oil and surfactant concentration. in this case, the sedds uses a surfactant with an hlb value lower than 12, while smedds and snedds use a surfactant with an hlb value higher than 12. kusumorini et al. (70) have formulated snedds piperine for oral administration using miglyol 812n oil, surfactant cremophor rh40, and cosurfactant peg400 with a ratio of 1:5, 6:2.9 w/w and piperine content of 2% w/w, with nanoemulsion size of 33.35 ± 1.97 nm and zeta potential of -22.87 ± 3.31 mv. the size of the particles is less than 200 nm. therefore, they can be easily absorbed by the intestinal epithelium and can prolong their living period which contributes to increased oral bioavailability. then, negatively charged drug carriers can also easily penetrate and cross the mucus barrier in the digestive tract, so that the drug can be smoothly consumed and enters the blood circulatory system (71). solid self-nano emulsifying drug delivery systems (s-snedds) the solid self-nano emulsifying drug delivery systems (s-snedds) are the changes of the snedds from liquid to dry powder supported by an inert adsorbent through a solidification process which can then be used to produce solid preparations, such as tablets, capsules, and pellets (72). s-snedds has several advantages, like increasing solubility and bioavailability, increasing stability, being easier to use for the scale-up process, to obtaining content uniformity, increasing patients’ compliance, and providing more accurate dosage (24,73). s-snedds, the solid form of liquid snedds, can form nanoemulsions within the droplets sized lower than 300 nm in aqueous media. it has the same solubility and bioavailability enhancement mechanism as the liquid snedds (74). piperine has been successfully formulated in the form of s-snedds using 9.39% glyceryl monolinoleic oil (gml), 17.38% poloxamer 188 surfactants, 9.39% transcutol cosurfactant hp, 56.33% adsorbent avicel ph-101, and 7.51% piperine using adsorption technique (75). s-snedds formulation results in a globule size of 73.56 ± 3.54 nm, a zeta potential of -28.12±2.54 mv, and bioavailability of 4.92 times higher than that of pure piperine dispersion. meanwhile, s-snedds piperine results in increased dissolution, bioavailability, hypertension effect, and better antioxidant and antibacterial activities than pure piperine. polymeric nanocarriers some polymeric nanocarriers such as dendrimers, polymer nanoparticles, micelles, nano gels, nanocapsules, and vesicles have recently been intensively used for drug delivery for their potential to modify the surface through chemical transformations to enhance drug loading and controlled release of specific ligands to reach specific sites where the drugs are designed (76,77). polymeric nanocarriers can increase the solubility of hydrophobic drug compounds, increase drugs bioavailability, pharmacokinetics and iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 18 biodistribution, minimize drug toxicity at the targeted sites, increase the stability of various therapeutic agents, and deliver drugs to the central nervous system by penetrating the blood-brain barrier (bbb) (76,78,79). ideally, polymeric nanocarriers should be biodegradable and non-toxic to minimize hypersensitivity reactions and provide good tissue compatibility (79). the polymeric nanocarrier systems are classified into nanocapsules and nanospheres. nanocapsules are vesicular systems in which drug molecules are surrounded by a membrane (encapsulated and trapped in lipids), while nanospheres are matrix systems in which drug molecules are dispersed across the particle surface (79). polymeric nanocarriers can increase the solubility and bioavailability of hydrophobic drugs in several specific ways, such as through diffusioncontrolled drug release where the polymer matrix does not have a membrane that acts as a barrier to diffusion. the drug release is controlled by the solvent, in which polymeric materials having a three-dimensional connective tissue structure can control the drug release by biodegradable polymer degradation through enzymatic decomposition. finally, the targeted drugs will be released at specific sites (80). therefore, polymeric nanocarriers play a crucial function in the piperine formulation for oral drug and piperine delivery in treating cancer. several formulations of piperine polymeric nanocarriers to increase piperine solubility are listed in table 2. the piperine nanocarrier formulation enhances the anti-cancer effect compared to pure ones, such as mcf-7 breast cancer, triple-negative breast cancer (tnbc) and mda-mb-468 cells, a549 cell lung cancer, hepg2 cell liver cancer, hela cervical cancer, and hs683cell brain cancer (81–86). the piperine nanocarrier formulation can increase solubility and bioavailability, extend drug circulation time, decrease toxic effects on normal cells, deliver the drugs to specific sites, penetrate the blood-brain barrier (bbb), and increase the piperine storage time. inorganic nanoparticle inorganic nanoparticles have more benefits than polymer and lipid ones. they have higher stability levels and are non-toxic and nonimmunogenic. there are inorganic carriers that have been used in drug delivery systems for drug therapy such as mesoporous silica, alumina, and zinc. mesoporous silica enforces higher encapsulation efficiency of the active ingredients, controlled structural properties, and biocompatibility (79). another example of an inorganic carrier for nanocarriers is hydroxyapatite. hydroxyapatite is a component of teeth and bone that exhibits good biocompatibility for delivering of prolonged-release drugs (87). piperine has been formulated with nanohydroxyapatite for anticancer therapy in vitro and performs a better anticancer effect on hct116 monolayer colon cancer cells (88). piperine nanohydroxyapatite produces spherical and amorphous nanoparticles with an average pore size of 9.7 ± 0.1 nm, drug loading of 16-22%, entrapment efficiency of 75-85%, and longer release at ph 7.4 (88). inorganic nanoparticles have increased the solubility and bioavailability of piperine through particle size reduction. they target the piperine at specific sites and release it controllably (89). solid lipid nanoparticle (sln) and nanostructured lipid carriers (nlcs) slns and nlcs are the two main types of lipid nanoparticles formulated by combining nontoxic lipid nanoparticles and excipients properties. slns and nlcs are promising drug carriers for delivering poorly water-soluble drugs (90,91). muchow et al. (92) stated, that sln is the first generation of lipid nanoparticles that structurally resembles an emulsion. slns are made of solid lipids at room and body temperatures stabilized by surface surfactants (93,94). meanwhile, nlcs are the second generation of lipid nanoparticles where generated lipid particles are a mixture of solid and liquid lipids and will be solid at a temperature of around 40oc (92). generally, the lipid excipients used to formulate slns and nlcs are biocompatible, biodegradable, and safe to use. slns and nlcs have some clinical advantages like increasing drug solubility and bioavailability, protecting drugs from chemical degradation, smoothly delivering drugs to specific sites, being easier to produce, and being stable during the storage period (95). bhalekar et al. (96) have developed an sln formulation of piperine for treating rheumatoid arthritis which could be consumed orally. the sln piperine formulation was prepared using the melt emulsification method with glyceryl monostearate and span 80 as the main ingredients. the sln piperine dispersion was successfully prepared with a drug and lipid ratio of 1:3 and a span concentration of 1% w/v. the results of the pharmacodynamic test showed that the piperine oral usage could provide a significant response compared to chloroquine suspension. chaudhari et al. (97) found a piperine formulation in the form of nlcs. nlcs were chosen because they could load the drugs maximally and had higher stability during the storage period than sln. piperine nlcs were made by solvent evaporation through high shear homogenization using solid lipid compritol 888 ato (50% w/w), liquid lipid squalene (25% w/w), piperine (20% w/w), span 80 (2 .5% w/w), and tween 80 (2.5% w/w). the piperine nlcs generated spherical particles, which were negatively charged, amorphous, resulting in higher drug release than pure piperine within 12 hours. zafar et al. (98) formulated the surface modification of nlcs with chitosan to improve the iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 19 therapeutic effect of piperine for diabetes therapy. the characterization of piperine nlcs coated with 0.2% w/v chitosan resulted in piperine encapsulated in lipid and amorphous form, within the size of 149.34 ± 4.54 nm. this could increase the piperine bioavailability ten times higher than pure piperine, and decrease the blood glucose three and half times better than that of pure piperine. quantum dots nanoparticle quantum dots (qds) are colloidal semiconductor nanocrystals that consist of atoms of groups ii-vi or iii-v of the periodic table and possess unique optical and fluorescent properties. the nanocrystal size of qds usually ranges from 2 to 10 nm and can be used for marking biological macromolecules, such as nucleosides and proteins (79). some of the commonly used qds include cadmium selenide (cdse), cadmium telluride (cdte), and indium arsenide (inas). there have been the latest developments for targeted piperine delivery in the formulation of qds nanoparticles. piperine is synthesized with copper oxide (cuo) qds and coated with hyaluronic acid (ha)/poly (lactic-co-glycolic acid) (plga) (99). piperine microspheres of cuoqds aim to deliver the piperine to the brain for treating epilepsy. piperine loaded in cuqds ha/plga shows enhanced neuroprotection and promoted astrocyte activation in epilepsyinduced mice in vivo. the particle size of cuoqds ha/plga is vital in delivering piperine through the bbb. smaller particle sizes can easily facilitate the drug delivery process into the systemic circulation. drug-phospholipids complexation phospholipids are the main components of mammalian cell membranes, are amphiphilic, and perform good solubility in water and oil. phospholipids are compatible with biological membranes and are hepatoprotective (100). they can increase the drug's bioavailability level with low water solubility and low membrane penetration, enhance drug absorption and release, protect against degradation in the gastrointestinal tract, reduce gastrointestinal side effects, and reduce the bitter taste of orally-consumed drugs (101). one of the benefits of phospholipids in drug delivery is their complexation with piperine compounds. piperine is complexed with hydrogenated soy phosphatidylcholine (hspc) with a molar ratio of 1:1.22 and exhibits complexation of the phospholipid group with the –c=o group of piperine through hydrogen bond interactions. the piperine/hspc complexation generates amorphous particles, increases solubility and sustained release of piperine, and increases piperine bioavailability 10.4 times compared to pure piperine (102). inclusion complexes inclusion complexes mean the formation of some complexes between the ligand as a complexing agent with a lipophilic cavity and a hydrophilic outer surface, which can interact with hydrophobic drug molecules (103). steric and thermodynamic factors function to form inclusion complexes of ligands and drug molecules. inclusion complexes of ligands and drug molecules may occur through non-covalent interactions such as hydrogen bonds, van der waals interactions, ion pairs, solvophobic effects, and/or hydrophobic interactions (104). the more hydrophobic the drug molecule, the more stable the inclusion complexes will be. applications of inclusion complexes in oral drug delivery include increasing oral solubility and bioavailability, increasing dissolution rate and rate, increasing drug stability at absorption sites, reducing drug-induced irritation, and masking unpleasant tastes (105). there are two types of formulations for forming inclusion complexes of ligands and drug compounds, namely binary and ternary inclusion complexes. binary inclusion complexes consist of complexing agents and drug compounds, while the ternary ones include complexing agents, drug compounds, and hydrophilic polymers increase drugs solubility (106,107). the binary inclusion complexes of piperine compound with βcyclodextrin with a molar ratio of 1:1 successfully increase solubility level, dissolution rate, and absorption rate of piperine in the intestine. the aromatic ring of piperine compounds interacts with the β -cyclodextrin cavity to form amorphous solids and work to increase the solubility level, dissolution rate, and bioavailability of piperine compounds (108,109). other forms of cyclodextrin, i.e α cyclodextrin and γ-cyclodextrin have also been successful in increasing the solubility and dissolution of piperine through hydrophobic interactions (110,111). the piperine inclusion complexes with ethylenediamine-β-cyclodextrin through hydrogen bonding at a molar ratio of 1:1 can completely increase the solubility and dissolution rate of the piperine. piperine compounds are present in the ethylenediamine-β-cyclodextrin cavity and build a relatively stable composite structure (112). the formation of ternary inclusion complexes assisted with hydrophobic polymers can increase the mixture of ligands and drug compounds. this can reduce ligands and the dose of the drug compounds (106,107). one of the polymers used for such complexes is hydroxypropyl methylcellulose (hpmc). the piperine ternary inclusion complexes with β-cyclodextrin and hpmc synergistically increase solubility and dissolution of piperine compared to the piperine binary inclusion complexes with β-cyclodextrin (106). other ligands that have been commonly used for inclusion complexes with piperine are cucurbiturils (113). they are macrocyclic oligomers that have the potential to increase drug solubility and bioavailability and are non-cytotoxic (114). the interaction between piperine and cucurbiturils occurs due to the interaction of dipole charges and iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 20 hydrogen interactions between the hydrogen atoms of methylene cucurbiturils and the aromatic part of the piperine molecules (113). the formation of these inclusion complexes can prevent the piperine’s isomerization reactions. table 2. summaries some types of nanoparticle formulation design of piperine. type form size (nm) composition method results reference liposom globular 243.4 ± 7.5 cholesterol, phospholipon® 90h, sodium cholate, chitosan thin film evaporation method increased solubility and permeability. showed anti breast cancer effect. (52) globular 29.75 ± 0.84 soya phosphatidylcholin e, tween 80 thin film evaporation method increased solubility, stability, and sustained release. (51) microemulsion spherical 161.50 ± 4.06 caproyl 90®, tween 80, cremophor rh 40, transcutol hp increased solubility and bioavailability. increased cognitive function in alzheimer’s disease. (59) sedds (selfemulsifying drug delivery system) snedds (selfnanoemulsifying drug delivery system) spherical 89.82 ± 2.16 ethyl oleate, tween 80, transcutol p increased solubility (5.90fold), bioavailability (5.2-fold), and permeability. (41) spherical 33.35 ± 1.97 miglyol 812n, cremophor rh40, peg400 increased solubility. (70) s-snedds spherical 73.56 ± 3.54 glyceryl monolinoleate (gml), poloxamer 188, transcutol hp, avicel ph-101 increased solubility (3.51fold), bioavailability (4.92-fold), and sustained release. (75) polymeric nanocarriers: albumin nanoparticle globular polymer 187.3 5.7 human serum albumin (hsa), ethanol, aqueous glutaraldehyde 8% (v/v) desolvation method, selfassembly method increased solubility and anticancer on mcf-7 cells. (81) micelle spherical 61.9 soluplus®, d-αtocopherol polyethylene glycol succinate (tpgs) thin-film hydration increased sustained release, solubility piperine, bioavailability (2.56-fold), physicochemical stability, cellular uptake, anticancer efficacy in a549 and hegg2 cells. (82) iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 21 continued table 2 . type form size (nm) composition method results reference polymer nanoparticle globular polymer 95 ± 10 poly (d, l, lactide-co-glycolic acid) (plga) ultrasonic atomization increased solubility, efficacy, and in vitro release (83) polymeric nanoparticle globular polymer 53 ± 1 methoxy poly (ethylene glycol)poly(lactic-coglycolic) (mpegplga), dichloromethane, lutrol® f68, sucrose single emulsion solvent extraction and thin-film hydration increased solubility and efficacy piperine inhibit breast cancer (tnbc) cells and mdamb-468 in breast cancer. (85) core-shell nanocarrier spherical dimethyl sulfate (dms), chitosan (c), pluronic f127, sodium dodecyl sulfate (sds), polyvinyl alcohol (pva), dichloromethane (dcm), aceton, brain cancer cell line hs683 nanoprecipit ation technique, ionic gelation increased sustained release and solubility. induction of apoptosis and cell cycle arrect in brain cancer cells. (86) nanoparticle globular polymer 130,20 ± 1,57 piperine, eudragit s100 (polymer), poloxamer 188 (surfactant) at 1:1:5 (w/w/w) solvent : ethanol nanoprecipit ation increased solubility, in vitro release, and bioavailability (2.7-fold). (115) starch nanoparticle globular polymer 88 ± 20 sago starch powder (natural biopolymer) in situ nanoprecipita tion increased solubility and stability. (116) nanosuspensio n spherical 172.5 hpmc (stabilizer), ethanol (solvent) (0.25% hpmc, 0.13% extract, rasio antisolventto-solvent 1:10) bottom-up approach increased solubility (5.13fold) and bioavailability (3.65-fold). (117) nanosponge nanoencapsulat ion system spherical β-cyclodextrin, diphenyl carbonate, ethanol, acetone, diclorometane microwaveassisted synthesis, solvent evaporation the ratio of -cd: dpc (1:10) resulted in the highest loading efficiency of piperine. (118) nanosponge nanoencapsulat ion system spherical β-cyclodextrin, diphenyl carbonate, ethanol solvent method increased loading efficiency and solubility. (119) core-shell nanoparticle spherical ĸ-carrageenan, zein, potassium chloride, coenzyme q10 antisolvent precipitation method, electrostatic deposition, ionic gelation increased photodegradation half-life (3.0fold), control release, chemical and physichochemica l stability. (120) iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 22 continued table 2 . type form size (nm) composition method results reference co encapculation microparticle spherical 922 zein, chitosan, pectin, coenzyme q10 ultrasoundassisted method, electrostatic deposition, solvent evaporation decreased chemical degradation. increased shelflive and physicochemical stability. (121) starch nanoparticle globular polymer 110 sago starch powder, anhydrous sodium sulfate (na2so4), propylene oxide, ethanol in situ nanoprecipita tion increased solubility and sustained release. (122) gelatin nanofiber globular polymer gelatin, glutaraldehyde, acetic acid electrospinni ng method improved controlled release profile. increased diffusion barrier and solubility. nanoencapsula tion spherical 68.2 gum rosin (polymer), acetone, oleic acid, pluronic-f emulsiondiffusion method increased sustained release and solubility. (123) inorganic nanoparticle spherical hydroxyapatite nanoparticle, gum arabic, folic acid hydrotherma l method, precipitation method increased solubility and inhibited monolayer hct116 colon cancer cell. (88) sln, nlc spherical 115,7 ± 6.26 compritol® 888 ato, squalene (liquid lipid), span 80, tween 80 solvent evaporation through high shear homogenizati on method increased solubility (2.0fold). (97) spherical 128,80 glyceryl monostearate, compritol 888 ato, precirol ato 5, tween 80, span 80 melt emulsificatio n increased solubility. (96) spherical 149.34 ± 4.54 chitosan 0.2% w/v increased piperine release (4.67-fold), bioadhesive, permeability (10.15-fold), bioavailability (3.76-fold), and antidiabetic. (98) qds spherical copper acetate dihydrate, 0.1 m naoh, ethanol, plga, ethyl acetate, poly (vinyl alcohol) (pva), hyaluronic acid precipitation method, emulsificatio n solvent evaporation method increased solubility and anticonvulsive efficiency. (99) iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 23 continued table 2 . type form size (nm) composition method results reference drugphospholipids complexation globular hydrogenated soy phosphatidyl choline (hspc), dichloromethane, n-hexane precipitation method increased bioavailability (10.40-fold) and half-life (20.55fold). (102) inclusion complexes cyclic hydroxypropyl beta cyclodextrin (hp β cd), d-αtocopherol polyethylene glycol 1000 succinate (tpgs) solvent evaporation method increased solubility (52.67fold) and dissolution (5.45fold). (124) cyclic hydroxy propyl methyl cellulose (hpmc), βcyclodextrin solvent evaporation and microwave irradiation methods increased stability, solubility and dissolution (4.43fold), (106) cyclic β-cyclodextrin freeze drying technique increased stability, bioaccessability, and antioxidant activity. (109) cyclic α-cyclodextrin, βcyclodextrin, γcyclodextrin physical mixture (pm) & ground mixture (gm) increased solubility. (110) cyclic mono(6ethylenediamine) β-cyclodextrin solvent evaporation method increased solubility (2fold). (112) cyclic cucurbit[n]uril increased solubility and stability. (113) the nanoparticle systems, especially in lipid carriers and biodegradable polymers, have been succesful in increasing the solubility, permeability, and bioavailability of piperine. the polymer nanoparticle formulation is very stable and can easily modify the surface; thus, drug release at specific sites can be managed well. biodegradable polymer nanoparticles can act as a drug depot that provides a sustainable supply of therapeutic compounds at the targeted sites (125). the biodegradable polymers (plga and pla) for nanoparticle production have been widely used for sustainable drug administration because they effectively deliver the drug to intracellular targeted sites (126). polymeric nanoparticle piperine using plga can also smoothly deliver the piperine to the targeted sites and increase its efficacy on hek-293 (human embryonic kidney cells) and mcf-7 (michigan cancer foundation-t and breast cancer cells) (83,84). lipid formulations can be easily absorbed by the body tissue after experiencing dissociation and release of the lipid system (127). the lipids in the gastrointestinal tract can also slow gastric emptying. therefore, drug lipids are stored longer in the small intestine, allowing better drug dissolution at the absorption sites to enhance absorption level. a high lipophilic drug with high solubility in triglycerides can be transported by the lymphatic system. in this way, the metabolism process in the liver can be bypassed, which can increase the drug bioavailability (127,128). lipid formulations can also increase drug bioavailability by inhibiting pglycoprotein efflux at the luminal membrane of epithelial cells in the colon, jejunum, and liver (129). although drug delivery on the nanoscale is very promising, nano-based formulations are still difficult to develop. for the pharmaceutical industry, drug formulation in nano form triggers complicated challenges because nanoformulations are easy to self-aggregate on long-term storage, affecting drug stability, variability, and efficacy (130– 132). other factors are related to the mechanism of solubility increase by nonionic surfactants, cosolvents of propylene glycol and polyethylene glycol, and the use of cyclodextrins. even though iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 24 nonionic surfactants, propylene glycol, polyethylene glycol, and cyclodextrin have shown to increase solubility, they can decrease permeability and lead to lower bioavailability levels despite high solubility (133–137). ingels et al. (138) found that the increase in solubility was disproportionate to drug absorption, and drug preparations cannot go through the epithelial barrier. although the solubility of the hydrobic drug was increased, it did not mean that there was an increase in its bioavailability. besides, increased solubility due to micellization did not necessarily increase the amount of drug available for passive diffusion across membranes and epithelial barriers in the gastrointestinal tract (git) (139). another challenge is related to scale-up and manufacturing processes. the nanoparticles are complex three-dimensional multicomponent products with a specific arrangement of components at the nanometer scale. therefore, they present typical challenges during the scale-up and manufacturing processes. nanoparticle formulation processes involving high-speed homogenization, sonication, organic solvents, cross-linking, milling, organic solvent evaporation, centrifugation, filtration, and lyophilization pose complex challenges during scale-up and manufacturing processes. different processing conditions during initial development on a small scale can affect chemical structure and conformation changes, denaturation, cross-linking, coagulation, and degradation of the active substances and excipients (140). for the scale-up process from small to industrial level, it is necessary to understand the characterization, testing, and release of nanoparticle products to identify critical parameters and analytical criteria; thus, the scale-up and manufacturing processes can result in high reproducibility and consistent products. identification of processing conditions involving polymer ratio, drug, target site, type of organic solvent, oil and water phase ratio, mixing temperature, pressure, ph of media, emulsifier, stabilizer, and cross-linker is also necessary before coming to the scale-up and manufacturing processes (141). conclusions the brief review shows that various formulation approaches have successfully increased the solubility and absolute bioavailability of piperine within the body. some approaches include solid dispersion, liposome, microemulsion, sedds, snedds, s-snedds, polymeric nanocarriers, inorganic nanoparticles, sln, nlc, qds, complexation with phospholipids, and inclusion complexes. however, most of the formulations do not consider the stability of piperine in dosage forms, especially for those in liquid preparations, because piperine compounds are very sensitive to light and can be easily degraded in liquid form. therefore, it is essential to further study the stability of piperine in the dosage form. some toxicity testing and demonstration of safety and efficacy should also be carried out to support the success of the piperine nano compounds formulation which later aims to increase its solubility and bioavailability. conflict of interest the authors have no conflict of interest. references 1. ezawa t, inoue y, tunvichien s, suzuki r, kanamoto i. changes in the physicochemical properties of piperine/β-cyclodextrin due to the formation of inclusion complexes. int j med chem. 2016;1-9. 2. gorgani l, mohammadi m, najafpour gd, nikzad m. piperine—the bioactive compound of black pepper: from isolation to medicinal formulations. comprehensive reviews in food science and food safety. 2017;16(1):124–40. 3. vasavirama k, upender m. piperine: a valuable alkaloid from piper species. int j pharm pharm sci. 2014;6(4):34–8. 4. chopra b, dhingra ak, kapoor rp, prasad dn. piperine and its various physicochemical and biological aspects: a review. open chemistry journal. 2016;3(1). 5. kotte scb, dubey pk, murali pm. identification and characterization of stress degradation products of piperine and profiling of a black pepper (piper nigrum l.) extract using lc/q-tof-dual esi-ms. anal methods. 2014;6(19):8022–9. 6. kozukue n, park m-s, choi s-h, lee s-u, ohnishi-kameyama m, levin ce, et al. kinetics of light-induced cis-trans isomerization of four piperines and their levels in ground black peppers as determined by hplc and lc/ms. j agric food chem. 2007;55(17):7131–9. 7. carosso s, miller mj. nitroso diels–alder (nda) reaction as an efficient tool for the functionalization of diene-containing natural products. org biomol chem. 2014;12(38):7445– 68. 8. wei k, li w, koike k, nikaido t. cobalt(ii)catalyzed intermolecular diels-alder reaction of piperine. org lett. 2005;7(14):2833–5. 9. kanaki n, dave m, padh h, rajani m. a rapid method for isolation of piperine from the fruits of piper nigrum linn. j nat med. 2008;62(3):281–3. 10. kusumorini n, nugroho ak, pramono s, martien r. development of new isolation and quantification method of piperine from white pepper seeds (piper nigrum l) using a validated hplc. indonesian j pharm. 2021;32(2):158–65. 11. mohapatra m, basak u. evaluation of piperine content from roots of piper longum linn., originated from different sources with iraqi j pharm sci, vol.32 (1) 2023 nanoparticle formulation design of piperine 25 comparison of zonal variation in odisha, india. international journal of pharma research & review. 2015;4:1–8. 12. rameshkumar kb, aravind apa, mathew pj. comparative phytochemical evaluation and antioxidant assay of piper longum l. and piper chaba hunter used in indian traditional systems of medicine. journal of herbs, spices & medicinal plants. 2011; 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licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 prevalence of ager gene polymorphism doi: https://doi.org/10.31351/vol31iss2pp202-210 202 prevalence of ager gene polymorphism in post menopause iraqi sample with osteoporosis and osteopenia in type 2 diabetes mellitus murooj g jameel *,1, zainab m. hashim **and mohammad h. al-osami*** *al-yarmok university college, diyala, iraq **department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq ***department of rheumatology, college of medicine, university of baghdad, baghdad, iraq abstract osteoporosis (op) is one of the most important metabolic disorders which is affected by interaction of genetic and environmental factors by almost 70% and 30% respectively. genetic components are identified to strongly effect bone mineral density, bone building and turnover, so they play central role in determining risk of op and fragility fractures. this case-control study consists of patient and control groups; group a: (70) postmenopausal women with op and osteopenia, group b: (20) control group. five milliliters of blood sample were divided into three tubes; one tube (1ml) contain gel for obtain serum to measure glucose level, the others tubes containing ethylene-diamine-tetra-acetic acid (edta), in second tube 2ml stored in deep freeze at (–40 co) until genomic analysis of dna for the performance of pcr genotyping of gene polymorphisms of rage, in third tube 2ml used to perform glycated hemoglobin % (hba1c) assays. hba1c and serum glucose levels is significantly increased in group a. frequency of c allele and cc genotype of rs1800625 were high significant in group a(p<0.001) than control. a allele and aa genotype of rs1800624 were high significant in group a(p<0.001) than control. homozygous 1800625 were (31%) and heterozygous 1800625(31%) compared to control homozygous 1800625 (5%) and heterozygous 1800625 (5%) respectively. homozygous 1800624 were (28.5%) and heterozygous 1800624(28.5%) in group a compared to control homozygous 1800624 (5%) and heterozygous 1800624 (20%) respectively. in conclusion, the cc genotype and c allele of rs1800625 snp and aa genotype and a allele of rs1800624 snp can be considered as indicators for op in post menopause iraqi women with type2 dm. keywords: osteoporosis, diabetes mellitus, rage gene polymorphisms وضعف في العينة العراقية بعد انقطاع الطمث مع هشاشة ager جين تقييم نسبه انتشار تعدد اشكال في مرضى السكري النوع الثانيالعظام * ** ومحمد هادي العصامي **زينب هاشم مجيد, 1،*مروج غازي جميل *كلية اليرموك الجامعة، ديالى، العراق *فرع العلوم المختبرية السريرية، كلية الصيدلة، جامعة بغداد، العراق مفاصل، كلية الطب، جامعة بغداد، العراق /**فرع الطب الباطني لخالصةا ٪ على التوالي. تم تحديد 30٪ و70هشاشه العظام من أهم االضطرابات األيضية التي تتأثر بتفاعل العوامل الوراثية والبيئية بنسبة تقارب بهشا اإلصابة مخاطر تحديد في مهًما دوًرا تلعب فهي لذلك العظام وبناء العظام، في المعادن كثافة على بقوة للتأثير الجينية الالمكونات عظام شة ( امرأة بعد سن اليأس مصابات بهشاشة العظام وضعف العظام، 70والكسور. تتكون هذه الدراسة من مجموعة المريض والمراقبة. المجموعة أ: ) ل مل يحتوي على هالم للحصول على مص 1( مجموعة تحكم. تم تقسيم خمسة مليلتر من عينة الدم إلى ثالثة أنابيب؛ أنبوب واحد 20المجموعة ب: ) مل المخزن في تجميد 2أسيتيك، في األنبوب الثاني تترا ديامين -لقياس مستوى الجلوكوز، واألنابيب األخرى التي تحتوي على حمض اإليثيلين ( للحمض النووي ألداء oc 40-عميق عند الجيني الثا rageلتعدد األشكال الجينية لـ pcr( ألجل التحليل مل إلجراء 2 لث، في األنبوب مستويات الجلوكوز ونسبه الهيموجلوبين السكري في الدم بشكل كبير في ارتفاعاضهرت النتائج (hba1c) .السكري فحوصات نسبة الهيموجلوبين سن اليأس في النساء بعد rs1800624و rs1800625تعدد األشكال rageالمجموعة )أ(مقارنه بمجموعه التحكم. تم اكتشاف ارتفاع معدل انتشار ٪( 31) 1800625٪( ومتغايرة الزيجوت 31متجانسة الزيجوت ) 1800625كانت . المصابات بهشاشة العظام في مرضى السكري النوع الثاني متجانسة الزيجوت 1800624٪( على التوالي. كانت 5كانت ) 1800625٪( ومتغايرة الزيجوت 5متماثلة الزيجوت ) 1800625مقارنة بالسيطرة ٪( على التوالي. في 20) 1800624٪( ومتغايرة الزيجوت 5) 1800624٪( مقارنة بالسيطرة 28.5) 1800624( ومتغايرة الزيجوت 28.5٪) سببًا لخطر اإلصابة بهشاشة العظام في النساء العراقيات بعد انقطاع الطمث المصابات rs1800624و rs1800625الختام، قد يكون تعدد األشكال ثاني بالسكري النوع ال rageتعدد األشكال الجيني ، مرض السكري النوع الثانيالكلمات المفتاحية: هشاشة العظام، 1corresponding author e-mail: mourogegazi@yahoo.com received: 6/ 11/ 2021 accepted:16 /1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp202-210 iraqi j pharm sci, vol.31(2) 2022 prevalence of ager gene polymorphism 203 introduction osteoporosis (op)is one of the most important metabolic disorders which is affected by interaction of genetic and environmental factors by almost 70% and 30% respectively (1). genetic components are identified to strongly effect bone mineral density (bmd), bone building and turnover, so they play a central role in determining risk of op and fragility fractures (2). diabetes mellitus (dm) is a common health problem worldwide counting about 1.2 million cases in iraq in 2015, type 2 dm (t2dm) represents about 90 to 95% of the overall diabetes types worldwide (3). leidig-bruckner et al. (2014) described prevalence of osteoporosis in women with t2dm was 21.9% and overall prevalence of low trauma fractures 5.7% (4). hyperglycemia can reduce the bone density through different pathways. toxic effects initiated by high serum glucose levels can directly reduction the function and number of osteoblasts (5). in fact, advanced glycation end products (ages) are dangerous heterogeneous compound of irreversible products resulting from non-enzymatic glycation. this reactions take place between the reactive carbonyl group of a reducing sugar, nucleic acids, lipids or proteins. the source of ages can be formed endogenously or exogenously under normal and pathological conditions (6). ages show a main role in the development of op, osteopenia and are associated with aging and hyperglycemia (7). a receptor advanced glycation end products also known as rage is type i cell surface receptor for ages belongs to the immunoglobulin "ig" superfamily and has been called as a pattern recognition receptor prr (8). the rager gene is located on the small arm of chromosome 6 "6p21.3" and the genomic sequence of this gene is polymorphic. this locus is associated in inflammatory and immune responses and is also the locus of main histocompatibility complex iii (mhc. iii). single nucleotide polymorphism "snp" is defined as a best common kind of polymorphism in which the change occurs in one nucleotide in sequence of dna, some previous studies show association between snps variation and op (9). therefore, the objective of this study was to evaluate the prevalence phenotype of rage and evaluate its relationship in a sample of iraqi post menopause with osteoporosis and osteopenia in type2dm. material and methods study design (case-control study) made over 4 months' period from march till june 2021 at medical city –baghdad teaching hospital rheumatology and rehabilitation consultation unit. this study consists of patient groups, this group were composed of (70) postmenopausal women with osteoporosis and osteopenia. in addition, to control group (20) as healthy women without osteoporosis or osteopenia. the age of subject is ≥ 45 years. disease female will be diagnosing as osteoporosis and osteopenia according to world health organization (who). the selection and diagnosis of patient will be doing under the supervision of rheumatologist physician in baghdad medical city. the current study protocol was approved by the local ethical committee of the college of pharmacy, baghdad university. exclusion criteria: • cushing’s syndrome, hyperparathyroidism, thyrotoxicosis. • gastrointestinal tract diseases: including ulcerative colitis, celiac disease and inflammatory bowel disease. • renal disease and epileptic • multiple myeloma, mastocytosis, lymphoma and leukemia. inherited disorders: including osteogenesis imperfecta, marfan´s syndrome, hemochromatosis. • rheumatic disorder such as rheumatoid arthritis and ankylosing spondylitis. • medication that can influence bone metabolism as steroid, vitamin d and hormone replacement therapy. after clinical examination, five ml of blood sample were collected from each subject in population study and divided into two parts: the first 3 ml of blood put in edta tubes for genotyping, and for perform glycated hemoglobin % (hba1c) assays, while the second part 2 ml was used for serum assessment of glucose tests. serum glucose level can be evaluated using a ready-made kit for this purpose, according to the method of barham and trindoer (1972) (10). glycated hemoglobin level was evaluated using a ready-made kit for this purpose according to abraham et al (1978) (11). determination of ager gene polymorphism genomic dna was isolated from blood sample according to the protocol of abio pure extraction. then genotyping depends on the analysis of promoter region of rage by utilizing pcr amplification and then sequencing according to method described by sanger and coulson 1975 (12). the primers used for amplify fragment of 797 bp of ager gene are: ager-f 5`tgtaaaacgacggccagtgaagaatggga agggagttatt-3` ager-r 5`caggaaacagctatgacaagagtccttcag gtactagag-3` the pcr amplification was carried out with 3 ng/µl of genomic dna, 2 µl of master mix, 10 µl of each primer, 7.5 µl nuclease free water. pcr cycles had been carried out as illustrated in (table 1). the pcr product then applied for gel electrophoresis and after that was sent for sanger sequencing using abi3730xl, automated dna sequences, by macrogen corporation – korea. the results were received and analyzed using genius software. iraqi j pharm sci, vol.31(2) 2022 prevalence of ager gene polymorphism 204 table 1. pcr amplification program statistical analysis the results were analyzed by statistical package for the social sciences (spss) (version 20.0) program and minitab (version18). one-way anova and t-test were used for the comparison of mean ± standard deviation (sd) of biochemical parameters of the patient and control groups and among the genotypes of rage polymorphism. alleles and genotypes frequency besides of odds ratios (or) and their 95% confidence intervals (ci) of the patients and control group were determined to utilize pearson’s chi-square test. the results of the analysis with a p value < 0.05 were considered significant and p<0.01 as highly significant. sequencing results insertion serum glucose and hba1c levels table 1 presents the mean ± sd of serum glucose and hba1c level obtained from 70 diabetic post menopause iraqi women with osteoporosis and osteopenia beside to 20 women as a control group. in group a, the serum glucose levels were significantly higher (146.13±12.79) compared to healthy control (84.56±10.25). also, hba1c levels were significantly higher compared (6.89±0.29) to healthy control (5.15±0.737). table 2. comparison between characteristic and biochemical parameters of the study groups p value < 0.05 considered significant and p value < 0.01 considered high significant genotypes and alleles frequency of rage after analysing pcr products of the ager polymorphism, 4 types of snp detected rs1800625 (-429t/c), rs1800624 (-374t/a), rs118122061 and rs143118560 as seen in figures (1,2&3). tables (3) lists the genotype and allele frequencies in % and the number of patients (no) having each genotype of the study groups. the distribution of genotypes in women with group a and control group was an agreement with hardy weinberg equilibrium. we detected a significant difference (p value <0.001) between the frequency of genotypes and alleles of rs1800625 (-429t/c), rs1800624 (-374t/a), in the patients group a compared with control group. the gene and allele frequencies for rs1800625 variant were tt 38%, tc 31%, cc 31%, (t53%, c47%) for group a, tt90%, tc 5%, cc 5% (t 92.5%, c 7.5%) for control healthy group. while the gene and allele frequencies for rs1800624 variant were tt 43%, ta28.5%, aa28.5%(t57%, a43%) for group a, tt75%, ta20%, aa5% (t85%, a15%) for control group. in other hand, there was non-significant difference (p-value <0.05) between the frequency of genotypes of ager gene variants rs118122061 and rs143118560 in the women with group a as compared to control group while allele of ager gene variant rs143118560 were significant (pvalue>0.05) and allele of ager gene variant rs118122061 non-significant. the gene and allele frequencies for rs11812261 variant were gg 79%, ga 21%, (g89%, a11%) for group a, gg85%, ga 15% (g 92.5%, a7.5%) for control group, while the gene and allele frequencies for rs143118560 variant was aa51%, at26%, tt23%(a64%, t36%)for group a, aa75%, at15%, tt10% (a82.5%, t17.5%) for control group. steps temperature °c time (m: s) m=minute, s=second cycle initial denaturation 95 05:00 1 denaturation 95 00:30 30 annealing 60 00:30 extension 72 00:30 final extension 72 07:00 hold 10 10:00 1 p value group b n=(20) group a n= (70) data 0.775 57.15 ±5.47 57.57 ± 6.68 age <0.0001 84.56±10.25 146.13±12.79 serum glucose(mg\dl) <0.001 5.15±0.737 6.89±0.29 hba1c(%) ethnicity 19(95%) 1(5%) 68(97%) 2(3%) arab kurd/turkman iraqi j pharm sci, vol.31(2) 2022 prevalence of ager gene polymorphism 205 table 3. comparison between genotypes and alleles frequency of rage polymorphism of the study entire group a p>0.05 is non-significant<0.05 significant and p<0.01 high significant. relationship between ager polymorphism and serum glucose and hba1c levels the results are given in table 3 shows the relationship between ager polymorphism and biochemical levels (glucose and hba1c) in group a. for rs1800625 snp, glucose level of cc genotype (147.27±14.94) were higher than tt (144.5±10.93) and tc (146.91±12.94) genotype respectively, but non-significant, also tc (146.91±12.94) were higher than tt (144.5±10.93) but still non-significant. while hba1c level of tt genotype (6.93±0.348) were higher than cc (6.91±0.222) and tc (6.84±0.29) respectively, but non-significant. for rs1800624snp, glucose level of tt genotype (150.83±12.26) were higher than aa (143.8±13.35) and ta (141.4±11.07); respectively but statistically non-significant, hba1c level of tt genotype (7.00±0.312) were higher than aa (6.88±0.338) and ta (6.93±0.302) respectively, but non-significant. for rs118122061snp, glucose level of gg (147.24±13.06) were higher than ga (142.07±11.27), but non-significant and hba1c level of gg genotype (6.9±0.26) were higher than ga (6.85±0.37), but also non-significant. for rs143118560snp, our data reported glucose level of aa (145.56±11.67) were higher than tt (143.5±13.55) and lower than at (147.56±12.61) respectively but also still non-significant and hba1c level of aa genotype (6.8±0.24) were lower than tt (6.92±0.33) (non-significant) and at (7.00±0.32) but significant. healthy control n= (20) group a n=(70) 95% ic or p value % n0. % no. genotypes snp 4.18-55.4 4.18-55.4 15.23 15.23 <0.001 90% 5% 5% 18 1 1 38% 31% 31% 26 22 22 tt tc cc rs1800625 (-429t/c) p value % no. % no. allele frequency 3.02-40.1 11 <0.001 92.5% 7.5% 37 3 53% 47% 74 66 t c 0.68-9.11 2.7-36.42 2.5 10 0.025 75% 20% 5% 15 4 1 43% 28.5% 28.5% 30 20 20 tt ta aa rs1800624 (-374t/a) p value % no. % no. allele frequency 1.16-15.48 4.25 0.001 85% 15% 34 6 57% 43% 80 60 t a 0.422-5.6 1.54 0.526 85% 15% 0 17 3 0 79% 21% 55 15 0 gg ga aa rs118122061 p value % no % no allele frequency 0.41-5.39 1.48 0.146 92.5% 7.5% 37 3 89% 11% 125 15 g a 0.68-9.1 0.9-12.13 2.5 3.33 0.167 75% 15% 10% 15 3 2 51% 26% 23% 36 18 16 aa at tt rs143118560 p value % no % no allele frequency 0.71-9.5 2.61 0.03 82.5% 17.5% 33 7 64% 36% 90 50 a t iraqi j pharm sci, vol.31(2) 2022 prevalence of ager gene polymorphism 206 figure 1. gel electrophoresis for pcr product of ager: the results of the amplification of ager specific gene region of human samples were fractionated on 1.5% agarose gel electrophoresis stained with eth.br. m: 100bp ladder marker. lanes 1-38 resemble 832bp pcr products. figure 2. gel electrophoresis for pcr product of ager: results of the amplification of ager specific gene region of human samples were fractionated on 1.5% agarose gel electrophoresis stained with eth.br. m: 100bp ladder marker. lanes 39-76 resemble 832bp pcr products iraqi j pharm sci, vol.31(2) 2022 prevalence of ager gene polymorphism 207 figure 3. gel electrophoresis for pcr product of ager: results of the amplification of ager specific gene region of human samples were fractionated on 1.5% agarose gel electrophoresis stained with eth.br. m: 100bp ladder marker. lanes 77-86 resemble 832bp pcr products table 4. comparison between biochemical parameters of the patients group a according to ager genotypes p value tc n0.22 tt no.26 p value cc no.22 tt no.26 parameter snp. rs1800625 0.488 146.91±12.94 144.5±10.93 0.462 147.27±14.94 144.5±10.93 glucose 0.342 6.84±0.29 6.93±0.348 0.803 6.91±0.222 6.93±0.348 hba1c p value cc no.22 tc n0.22 0.932 147.27±14.94 146.91±12.94 glucose 0.387 6.91±0.222 6.84±0.29 hba1c p value ta no.12 tt no.16 p value aa no.12 tt no.16 parameter snp. rs1800624 0.008 141.4±11.07 150.83±12.26 0.061 143.8±13.35 150.83±12.26 glucose 0.576 6.93±0.302 7.00±0.312 0.353 6.88±0.338 7.00±0.312 hba1c p value aa no.12 ta no.12 parameter 0.54 143.8±13.35 141.4±11.07 glucose 0.706 6.88±0.338 6.93±0.302 hba1c p value ga no.15 gg no.55 parameter snp. rs118122061 0.167 142.07±11.27 147.24±13.06 glucose 0.532 6.85±0.37 6.9±0.26 hba1c p value at no.18 aa no.36 p value tt no.16 aa no.36 parameter snp. rs143118560 0.566 147.56±12.61 145.56±11.67 0.579 143.5±13.55 145.56±11.67 glucose 0.024 7.00±0.32 6.8±0.24 0.216 6.92±0.33 6.8±0.24 hba1c p value tt no.16 at no.18 parameter 0.373 143.5±13.55 147.56±12.61 glucose 0.479 6.92±0.33 7.00±0.32 hba1c iraqi j pharm sci, vol.31(2) 2022 prevalence of ager gene polymorphism 208 discussion although several investigators have long addressed the question of how dm induces osteopenia and osteoporosis, the exact underlying mechanism is still unknown. many factors include food availability, absence of exercise, low sun exposure, gender, age, several diseases, inflammation, genetic polymorphism and ethnic group associated with development of osteoporosis. therefore, various investigated tried to know the etiology of osteoporosis or as a minimum identify its risk factor (13). the bmd levels in type 2dm patients were normal or high, several investigators reported a negative effect of type 2 dm on bmd. for instance, yaturu and colleagues found a significantly low bmd of hip in type 2 dm patients when compared to age-matched normal individuals (14). the current study shows a significant difference in serum levels of glucose when a comparison among group a (146.13±12.79) and control group (84.56±10.25), also significant difference found in levels of hba1c (%) in group a (6.89±0.29) when compared to control group (5.15±0.737), as shown in table 2 all this result was agreement with recent study (15) (16). it is commonly believed that hyperglycemia is a salient factor that has direct and indirect deleterious effects on osteoblast function, bone formation and also on proliferation and differentiation of mesenchymal stem cells (17). rage have a role in regulating bone homeostasis under physiological disorders and may implicated in several bone-associated diseases such as op and osteopenia however, the particular cell type that facilitates signaling of rage, and the downstream effects of rage activation on bone homeostasis and pathology, still unclear (18). this study is considered the first study that estimate the prevalence of the rage gene polymorphism in iraq. there are no studies in the neighboring countries that could be compared to the results and analyzing the impact of genes polymorphisms on the development of op in type 2 dm have been carried out; however, we compared the findings of this study to studies conducted on american, european, asia and african populations. several lines of evidence reported a role for rage and its ligands in stimulating osteoclastic activity and osteoclast maturation. in addition, hmgb1(high mobility group box 1)-rage signaling is concerned in recruitment of osteoclasts, osteoblasts, and blood vessels through bone formation. age-rage signaling may show an adverse role in osteoblast differentiation and function (19). several other studies have recommended that rage levels may actually be raised in op, osteopenia and dm with high levels of bone and cartilage turnover (20) (21). in the current study we reported the occurrence of polymorphisms in the promoter region of rage and identified a number of key polymorphisms. the results of our study showed that rage gene rs1800625 and rs1800624 polymorphisms were statistically different between group a compared to control group. rage gene rs118122061and rs143118560 polymorphisms were non-significant between group a compared to control, as exposed in table 3 and figures (1,2 and3). these data suggest that the polymorphisms involved in alterations in rage gene regulation that can influence on pathogenesis of dm post menopause women with op and osteopenia. prior studies that have noted the important role of race and ethnic on the epidemiology of osteoporosis (22). therefore, our results agree with ying several studies in the chinease population (23) (24) and disagree with raska et al study that found diabetes-specific parameters in addition to rage polymorphisms did not associate with bmd or fractures in t2dm postmenopausal women (25). the snps rs1800625 and rs1800624 was found to rise the transcription activity of ager in vitro. they involve an increase in rage expression, which might influence the pathogenesis of several inflammatory diseases and dm (9). result in table 4 shown biochemical parameters of the patient’s group according to rage genotypes, the women with rs 1800625 cc genotype had high mean ±sd of glucose levels (147.27±14.94) compared to tt (144.5±10.93) and tc (146.91±12.94) but still nonsignificant. also, the women with rs1800624 tt (150.83±12.26) genotype had high mean ±sd of glucose levels compared to aa (143.8±13.35) and ta (141.4±11.07), while women with rs118122061 gg (147.24±13.06) genotype had high mean ±sd of glucose levels than ga (142.07±11.27) genotype. in other hand women with rs143118560 at genotype had high mean ±sd (147.56±12.61) compared to tt (143.5±13.55) and aa (145.56±11.67) genotype. the women with rs 1800625tt genotype had high mean ±sd of hba1c levels (6.93±0.348) compared to cc (6.91±0.222) and tc (6.84±0.29). also the women with rs1800624 tt (7.00±0.312) genotype had high mean ±sd of hba1c levels compared to aa (6.88±0.338) and ta (6.93±0.302), while women with rs118122061 gg (6.9±0.26) genotype had high mean ±sd of hba1c levels compared to ga (6.85±0.37). in other hand women with rs143118560 aa (6.8±0.24) had lower mean ± sd compared to tt (6.92±0.33) (non-significant) and at (7.0±0.32) (significant), no statistical significance was found between glucose and hba1 levels with osteoporosis except in snp rs143118560 aa and at genotype of hba1c levels, and snp 1800624tt and ta genotype of glucose levels so absent clear effect of rage polymorphism on biochemical levels of glucose in women with osteoporosis, because glucose homeostasis affected by a variety of factors, glucose homeostasis is maintained by a complex neuro hormonal system, which modulates glucose uptake, glucose assembly, and exogenous glucose utilization following food ingestion (26) (27). there are several hormones iraqi j pharm sci, vol.31(2) 2022 prevalence of ager gene polymorphism 209 participate in glucose metabolism include insulin, glucagon, amylin, glucagon-like petide-1 (glp-1), epinephrine, cortisol, and growth hormone (gh). these hormones control glucose levels and act on several target tissues, involving muscle, liver, adipocyte (28). conclusion in the current study, a high prevalence of rage polymorphism rs1800625 and rs1800624 were detected in postmenopausal women with osteoporosis in type2dm. homozygous 1800625 were (31%) and heterozygous 1800625(31%) compared to control homozygous 1800625 were (5%) and heterozygous 1800625 were (5%) respectively. homozygous 1800624 were (28.5%) and heterozygous 1800624(28.5%) compared to control homozygous 1800624 were (5%) and heterozygous 1800624 were (20%) respectively. thus, the rs1800625 and rs1800624 polymorphism might be a causal risk allele for osteoporosis in post menopause iraqi women with type2 dm, also so it might be used as a biomarker. further research should be undertaken to confirm these results by suggesting more studies should be using a larger number of samples in different cities of the iraq. references 1. tamer a, gheita h, and nevin h. epidemiology and awareness of osteoporosis: a viewpoint from the middle east and north africa. int. j. clin. rheumatol. 2018; 13(3): 134-147. 2. rivadeneira f, mäkitie o. osteoporosis and bone mass disorders: from gene pathways to treatments. trends endocrinol metab.2016; 27:262–281. 3. 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characterization of aceclofenac nanosuspension (acns) for enhancement of percutaneous absorption using hydrogel dosage form aseel kadhem thamer*,1and ahmed najim abood** *basrah health directorate, al-fayhaa teaching hospital. ** department of pharmaceutics, college of pharmacy of basrah, basrah, iraq. abstract aceclofenac (ac) is an orally active phenyl acetic acid derivative, non-steroidal anti-inflammatory drug with exceptional anti-inflammatory, analgesic and antipyretic properties. it has low aqueous solubility, leading to slow dissolution and inadequate bioavailability(15%). the aim of the current study was to prepare and characterize ac-ns-based gel to enhance the dissolution rate and then percutaneous permeability. ns.s were prepared using solvent/antisovent precipitation method at different drug to polymer ratios (1:1, 1:2, and 1:3) using poly vinyl pyrrolidone (pvp-k25), hydroxy propyl methyl cellulose (hpmc-e5) and poloxamer® (388) as stabilizers alone and in combinations of two polymers (1:2 and 1:4 drug: polymer ratio). fifteen formulas of ac-ns.s were prepared and characterized for loading efficiency, particle size, polydispersity index and physical stability. the best formulas of ns were f11 (pvp k25, poloxamer® 338 and ac), and f15 (hpmc e5, poloxamer® 338 and ac) that gave the best results of physical stability and entrapment efficiency which were lyophilized to be characterized by ftir, dsc, p-xrd and sem. after that, the best prepared formula of ac-ns regarding the involved characterization methods was incorporated in gel dosage forms using (1% w/v carbopol®940). from this study, we conclude that the solubility and dissolution rate of ac were improved when the particle size was reduced to nano-scale as compared with pure drug. keywords: aceclofenac, nanosuspension, solvent/ antisolvent method, hydrogel. طبقات لوفيناك لزياده امتصاص الدواء فياسيكلعقارالتحضير والتقييم المختبري للمعلق النانوي ل الجلد باستخدام الهالم كشكل دوائي **احمد نجم عبودو 1*،اسيل كاظم ثامر مستشفى الفيحاء التعليمي . -دائرة صحة البصرة* . فرع الصيدالنيات،كلية الصيدلة،جامعة البصرة،البصرة،العراق** الخالصة دواء اسيكلوفيناك مشتق من حامض فينيل اسيتك . فعال عن طريق الفم وهو عقار مضاد لاللتهاب غير السترويدي ،مسكن لاللم وخافض ٪( ١٥ن في الماء مما يؤدي الى انحالل بطئ ونفاذيه منخفضة وعدم كفاية التوافر الحيوي)للحراره .يعاني االسكلوفيناك من انخفاض قابلية الذوبا جلد .تم .الهدف من الدراسة الحالية هو تحضير وتوصيف هالم مائي قائم على المعلقات النانوية لتعزيز معدل الذوبان ومن ثم النفاذيه عن طريق ال باستخدام نسب مختلفه من العقار والبوليمر )العقار: البوليمر( ترسيب باستخدام المذيب/مضاد المذيب(تحضير المعلقات النانوية باستخدام طريقة )ال ( كل على حدة او بمزجها معا. ( باستخدام )بولي فينيل بيروليدون،هيدروكسي بروبيل ميثيل سيليولوز و البولوكسيميرات١:٤، ۳:١، ۲:١، ١:١) ت النانوية وتميزت بكفاءة االنتاج والتحميل وتم اجراء االختبارات المختلفة عليها مثل حجم الجسيم والثبات تم تحضير خمسة عشر صيغة من المعلقا ، تحويل الفيزيائي ثم بعد ذلك جففت جزيئات المعلق النانوي واجريت عليها اختبارات اخرى مثل المجهر االلكتروني الماسح ، مسعر المسح التبايني لصنع الهالم المائي. ستنتج ٪١بنسبة ٩٤٠تحت الحمراء واختبار حيود المسحوق. ثم تم دمج المعلقات النانوية مع الكاربوبول فورييه الطيفي باالشعه . من نتائج التجربة انه تم تحسين الذوبانية والنفاذية لعقار االسكلوفيناك عندما تم تقليل حجم الجسيمات الى المستوى النانوي . المائي الكلمات المفتاحية: اسكلوفيناك ،المعلقات النانوية،طريقة الترسيب باستخدام المذيب/المذيب المضاد ، الهالم introduction the word ‘solubility’ is defined as a maximum quantity of solute that can be dissolved in a given amount of solvent. there are various techniques for solubility enhancement like particle size reduction, solid dispersion, use of surfactants, ph modification, complexation, hydrotrophy, use of co-solvent and others (1). when the particle becomes smaller, the surface area with volume ratio will be increases. the large surface area enables greater contact with the solvent which lead to elevate solubility. nanoparticles are particulate dispersions or solid particles with a size between 10-1000nm (2). 1corresponding author e-mail: kadhemaseel@gmail.com received: 4/1/2021 accepted: 1/3 /2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp86-98 iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 87 nanoparticles stabilization is crucial to ensure their effectiveness. the massive rise in surface area causes excessive surface energy, which is undesirable thermodynamically. the increase in surface energy accelerates particle agglomeration to reduce over-energy on the surface. agglomeration affects over all stability of the formulation of nanoparticles3. polymers like poloxamers® (338), hpmc (e5), pvp (k25) and others provide stabilization for nanoparticles by surface binding as well as through the steric bulk of their threedimensional structure (4). ac is a non-steroidal anti-inflammatory drug, it is a phenyl acetic acid derivative (figure-1), white crystalline solid, almost insoluble in water and soluble in ethanol (96%). after oral administration ac is absorbed and undergoes first pass hepatic metabolism (5). it is recommended for the short and long term therapy of the signs and symptoms of rheumatoid arthritis, osteoarthritis and ankylosing spondylitis (6). figure 1. chemical structure of aceclofenac (5) hydrogel is a water-swollen, cross-linked polymeric network formed by the simple reaction of one or more monomers (7). the main objective of this study is to modify ac particles to be prepared as ns using solvent/anti-solvent precipitation method and then incorporated in gel forming ns-based hydrogel type with improved dissolution rate and dermatological permeation. materials and methods materials aceclofenac powder (ac), poloxamer® (338) were purchased from lishui nanming chemical co., ltd (china). pvp®-k25, hpmc-e5 and carbopol® 940 are gifts from sama alfayhaa for pharmaceutical industries. all other materials are of analytical grades. methods characterization of aceclofenac powder determination of melting point melting point was determined by digital melting point apparatus. a few quantity of ac sample was taken and placed in a thin walled capillary tube which placed and heated in the device, when the sample start to melt, the melting point was recorded (8). spectrophotometric analysis ac powder (50mg) was weighed and dissolved in (50 ml) of each of ethanol, phosphate buffer (ph 7.4) and dw (in presence of ethanol 1% (v/v) as co-solvent) 9 to get stock solutions with concentrations (1mg/ml). from which, serial dilutions were made and inspected spectrophotometrically. the λmax values were determined and matched with literatures values (9). the absorbance values of resultant diluted solutions were measured by uv-visible spectrophotometer using the respective blank solvents (10). determination of saturation solubility excess amounts of ac powder were put in (25ml-conical flasks) containing (20ml) of each of the involved media as reported by maulvi et al. after that, the conical flasks were shaken with orbital shaking incubator at 37± 0.1°c for 24 hours, then kept in the incubator at 37°c for 24hours until equilibrium existed, the supernatant solution filtered through a 0.45μm filter paper and analyzed spectrophotometrically, three determinations were carried out (11). preparation of ac-nanosuspensions solvent/anti-solvent precipitation process was used to prepare ac-ns. (100mg) of ac powder was dissolved in 2ml of ethanol, then added by syringe dropping into (50ml) of d.w. containing fixed quantities of one stabilizer or combinedstabilizers at room temperature and subsequently stirred by using magnetic stirrer at (800rpm) for (1hour) at (40℃) to allow volatile solvents to evaporate (12). the compositions of the prepared formulas (1-15) were illustrated in table (1). evaluation of the prepared ac-ns.s measurement of particle size and polydispersity index (pdi) analysis of particle size was done using malvern mastersizer 2000 ms (worcestershire, great britain). average particle size and pdi of the prepared ns formulas were observed (the results of particle size was recorded as an average value depending on the device setting) to ensure that the particles are within nano-range size and pdi is acceptable (13). measurement of % entrapment efficiency (%ee) freshly prepared drug-loaded ns.s were centrifuged at 12500 rpm for 20 minutes. the concentration of drug in the supernatant was spectrophotometrically determined after filtration through 0.45μm filter paper at the estimated λmax. the ee of ac was calculated as follows (14). %ee= 𝑊𝑡 𝑖𝑛𝑡𝑖𝑎𝑙 𝑑𝑟𝑢𝑔−𝑊𝑡 𝑓𝑟𝑒𝑒 𝑑𝑟𝑢𝑔 𝑊𝑡 𝑖𝑛𝑡𝑖𝑎𝑙 𝑑𝑟𝑢𝑔 × %100 (1) iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 88 table 1. compositions of ac ns.s formulas using different stabilizers at different drug: stabilizer ratios with constant volume of injected organic solution (2 ml). formula code aceclofenac amount (mg) pvp k25 amount (mg) hpmc (e5) amount (mg) poloxamer (338) amount (mg) f1 100 100 f2 100 200 f3 100 300 f4 100 100 f5 100 200 f6 100 300 f7 100 100 f8 100 200 f9 100 300 f10 100 100 100 f11 100 200 200 f12 100 100 100 f13 100 200 200 physical stability assessment the prepared ns formulas were stored in a dark place at an ambient temperature for up to seven days. during this time, the particle size and visual appearance were monitored and determined (15). lyophilization of the prepared ns formulas for further characterization, the best formulas were lyophilized using freeze dryer (labconco usa). the powder yields were kept in a tight container at room temperature (16). characterization of the lyophilized powder scanning electron microscopy (sem) analysis the particle morphology of lyophilized powder and unprocessed drug were characterized by using sem. a small fraction was fixed on a doublesided conductive carbon tape and sputter-coated with 5 nm of a pt–pd alloy (17). compatibility studies differential scanning calorimetry (dsc) this test was used to examine the physical compatibility between ac, additives and methods conditions. the samples were precisely weighed and sealed hermetically with aluminum lid. the thermograms of lyophilized ac formulas and pure drug powder were recorded (18). fourier transform infrared spectroscopy (ftir) ftir scanning of kbr pellets containing powder samples of pure drug, lyophilized formulas in the wave number range 400-4000 cm-1 at a resolution of 4 cm-1 with speed of 2 mm/sec (19), powder x-ray diffraction (pxrd) using a diano x-ray diffractometer (usa) equipped with co-kα radiation (45 kv, 9 ma, scanned from 3 ° to 50 ° at 2 angles), samples from pure drug and lyophilized formulas powders were analyzed (20). preparation of ns-based hydrogel carbopol®940 was used as gelling agent in concentration of (1%) to prepare ns-based hydrogel from the selected ac-ns formula. the calculated amount of carbopol was dispersed in water using a magnetic stirrer with a speed of (1000 rpm) for (1 hour) until getting uniform dispersion, then a freshly prepared ac-ns formula (the selected one) was gradually added to the aqueous dispersion of carbopol. after that (1.6) ml glycerin was added as a viscosity modifier. finally, few drops of triethanolamine were added to initiate the hydrogel formation (21). the prepared hydrogel had been allowed to stand overnight to clear stuck air, then the prepared hydrogel formula was sealed in a tightly closed container at room temperature in a dark place for other tests (22). additionally, by the same procedure, plain hydrogel was prepared using ac pure form. evaluation of the prepared hydrogel measurement of ph the ph of the prepared hydrogel formula was determined using a ph meter at room temperature. this was done by fully placing the glass electrode in the gel system and recording ph (23). spreadability hydrogel formulas were found to be spreadable or not after (48 hour) of preparation, by measuring 2 g of gel spreading-diameter between two (10x10 cm) glass plates. determination of diameter after 1 minute of applying the weight. the spreadability can be calculated using the following equation: iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 89 s = m.l / t …….. (2) where: s is spreadability, m is the weight tied to the upper slide, l is the length of the glass slide and t is time consumed (24). rheological studies (viscosity analysis) they were done by drawell ndj-8s viscometer. the hydrogel was filled in a wide mouth container, so that the viscometer's spindle could be appropriately dipped without touching the bottom of jar. hydrogel samples were permitted to settle at constant temperature more than 30min. the viscosity was measured by increasing the speed of rotation starting from 0.3 rpm to 60 rpm. this done by using a spindle (number 4). the measurements were recorded at temperature of 25°c (25) . in vitro drug release study the ns-based hydrogel release analysis was performed by using assembly franze diffusion cell26 using a tube in which one end covered by dialysis membrane (8000-14000 dalton cut-off) that is soaked in a freshly prepared diffusion medium (phosphate buffer ph 7.4) over night. a specific amount of hydrogel equivalent to (100 mg) of ac was taken and spread on a semi-permeable dialysis membrane, the tube was partially submerged in a jar containing buffer. the temperature was held at 32± 0.5 °c stirred at 100 rpm. two milliliters-sample was taken at different time intervals and the drug concentration was spectrophotometrically determined at the determined λmax. against a suitable blank (27). the obtained data were fitted to different mathematical expressions to describe the kinetic and mechanism of aceclofenac release from the selected nanosuspension formulas with the help of ddsolver: an add-in program in microsoft excel (28). the kinetic models (zero order, first order, higuchi model and korsmeyer – peppas) were used29. the model that produced the highest correlation coefficient was selected as the best fitted model. stability studies (effect of temperature) for the assessment of the stability, the prepared hydrogel formula was kept at different temperature degrees: a refrigerator (4 °c), oven (40 °c) and room temperature (25 °c) for three months. samples were periodically withdrawn and tested for the physical appearance, homogeneity and viscosity (30). statistical analysis the results of particle size was recorded as an average value depending on the device setting, while others have been mostly provided as the mean value of triplicate readings ± standard deviation and have been assayed statistically using (anova) test for estimation if changes of involved variables have been statistically significant at level (p ≤ 0.05) and non-significant at level of (p>0.05). results and discussion determination of melting point it was found to be (156-158 °c) which is the same as reported reflecting the purity of the powder used in the study (31). spectrophotometric analysis the estimated λmax is 275nm that is agreed with the reported values (32). figures (2-a, 2-b and 2-c) showed the calibration curves in the involved media. a straight lines were obtained indicating that the calibration curves obeys beer-lambert law within the range of concentrations used (33). figure 2. calibration curves of ac in ethanol, phosphate buffer ph 7.4 and d.w. saturated solubility of ac powder the saturation solubility values of ac at different media were summarized in table (2). from which, we conclude that ac is practically insoluble iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 90 in water and soluble in ethanol34. the aqueous solubility will be increased by increasing ph due to weakly acidic properties of drug as in phosphate buffer medium ph 7.4 (35). table 2. solubility of ac at 37± 0.1 ℃ in different media (n=3) preparation of ac-ns in this study, fifteen formulas of ac-ns.s were prepared having particle size values within nanometer range as illustrated in tables (3 and 4) which are exposed for several characterization methods to get the best one. table 3. particle size, polydispersity index values of the prepared formulas using different types and amount of single stabilizer formula code average particle size (nm) pdi f1 270 0.3 f2 202 0.3 f3 164 0.3 f4 198 0.09 f5 271 0.07 f6 220 0.07 f7 98 0.3 f8 112 0.3 f9 85 0.3 table 4.particle size and pdi values for the prepared formulas (using combined stabilizers) characterization of the prepared ac-ns formulas particle size and pdi measurements the most important characterization parameters for the prepared ns.s are the average particle size and pdi which governs the physicochemical properties like saturation solubility, dissolution velocity and physical stability (36). the usual range of pdi obtained values are (0.10.25) which indicates narrow size distribution while pdi value more than 0.5 refer to very broad distribution (37). the pdi values of prepared formulas were ranged from (0.06-0.3). the best formulas (f11 and f15) have nano-sized particle with low pdi values (0.06 and 0.09) respectively as shown in table (4) which indicates good uniformity of nanoparticles. stabilizers such as hpmc, pvp and poloxamer were incorporated to prevent sedimentation, agglomeration and crystal formation (38). in addition to the safety and regulatory requirements, the choice of stabilizer is based on their ability to provide particle surface wetting and a barrier to prevent agglomeration of nanoparticles. formulas (f1-f3) give a good average particle size especially at drug: polymer ratio (1:3) with remarkable increase in average particle size, this may be due to role of pvp in inhibiting guest molecule crystallization and its excellent efficiency as agent for coating and high affinity of polymer to the nanoparticals surface (39). formulas f4-f6, also give a good average particle size with increase in size when changing the concentration of the polymer reaching to drug: polymer ratio (1:3). this is attributed to the fact, hpmc has both a hydrophobic alkyl chain and hydrophilic ohin its side chain. on the nano crystalline surface, the hydrophobic alkyl chain can be adsorbed, while the hydrophilic ohgroups are exposed to increase the wettability of the nano crystals (40). formulas f7-f9 had the best average size, at drug: polymer ratio 1:3, it gave (85 nm) size, this because of its high molecular weight and a greater polypropylene oxide (ppo) region of poloxamer. therefore, because of adsorption occurs via this hydrophobic ppo block, it has more 'anchoring capacity' to the nanoparticle surface (41). optimum stabilizer concentration is needed where the use of insufficient amount of stabilizer may not provide complete coverage of the drug surface, thus jeopardizing steric repulsion between particles (42). in our study, we see that as the concentration of stabilizer increases, the average particle size was decreased as seen in table (3) in which the formulas coded (f3 and f9) give smaller particle sizes (164 and 85nm) respectively at which highest amount of polymers use. according to the above result a smaller particle size (85nm) was seen when using poloxamer 338 in higher amount, since this polymer show reduction in average particle size because of the high molecular weight of polymer. smaller average particle size values were obtained for f11 about (43 nm) and f15 about (45 media saturation solubility (mg/ml) ± sd d.w 0.07 + 0.01 phosphate buffer 7.4 14.2 + 0.04 ethanol 40 + 0.02 formula code average particle size (nm) pdi f10 72 0.09 f11 43 0.06 f12 242 0.1 f13 237 0.07 f14 41 0.1 f15 45 0.09 iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 91 nm) when using combination of (pvp k25 + poloxamer 338) and (hpmc + poloxamer 338) respectively at ratio of (1 drug: 4 polymer) as shown in table (4). so these combinations showed a significant reduction in the average particle size (p< 0.05). also these formulas (f11 and f15) have pdi values (0.06 and 0.09) respectively which indicates a good polydispersity. these results may be obtained because of an excellent surface affinity of stabilizers toward drug molecules forming a large mechanical and thermodynamic barrier at the interface (43). the ratio of drug to polymer (1:4) looked to be optimum due to the effect of amount of polymer on the properties of the nanoparticles. the reason may be due to the fact, the viscous polymer solution has more difficulty to break up into smaller droplets at the same mixing input strength, leading to a rise in particle size (44). determination of %ee the %ee values of the prepared formulas were ranged from 86.3(f9) to 94.2 % (f15) as showed in table (5). there is no significant difference (p>0.05) between the prepared ac-ns.s which may give an impression of suitability of stabilizer mechanism (steric stabilization) especially when used in combination that give synergistic effect for stabilization of ac nanoparticles44. it is clear that the increase in stabilizer concentration can increase the %ee, but the study revealed that the concentration of stabilizers at ratio (1 drug: 4 stabilizer) was sufficient to give the optimized %ee. this may be due to the presence of optimum stabilizer type and optimum stabilizer concentration (45). table 5. ee values of the prepared formulas (n=3) formula code ee% ± sd f1 90.2 ± 0.06 f2 92.4 ± 0.03 f3 92.6 ± 0.04 f4 87.6 ± 0.01 f5 90 ± 0.1 f6 92.3 ± 0.11 f7 86.3 ± 0.1 f8 90.2 ± 0.08 f9 93.5 ± 0.06 f10 89.4 ± 0.005 f11 92.7 ± 0.01 f12 86.4 ± 0.01 f13 93.5 ± 0.17 f14 91.2 ± 0.02 f15 97.5 ± 0.05 physical stability of ac-ns the prepared formulas were tested for their physical stabilities for one weak at ambient temprature. formulas f11 and f15 were seemed to be clear after (1 weak) as seen in table(6). other formulas look to be precipitated due to increase particle size. the improved stability may be due to the increased thickness of layer of adsorbed polymer when poloxamer 338, hpmc e5 and pvp k25 were used in combinations, thus preventing the particles from aggregation and/or agglomeration by providing steric hindrance. ns stabilization can be done by electrostatic, steric, or electrostatic modulation (a combination of them) (46). table 6. physical stability study for f11 and f15 formulas measurement time f11 (particle size in nm) f15 (particle size in nm) initial time 43 45 2nd day 50 54 7th day 67 80.2 characterization of the lyophilized powder sem the morphological analysis and particle size values of (f11 and f15) in compare to ac row powder were performed by sem showing nanoparticles with a size of (43 and 45nm) as seen in figure (5). no aggregation of particles could be observed. a b iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 92 c figure 5. sem micrograph for pure ac powder at 36.23 kx magnification (a), lyophilized f11 at 78.00 kx magnification (b) and lyophilized f15 at 78.00 kx magnification (c). dsc dsc thermogram of pure ac powder {figure (6-a)} showed a sharp characteristic endothermic peak at (156.30 ºc) that is agreed with references. this gives an indication that the drug has crystalline nature with high purity. the dsc thermograms of the lyophilized formulas (figure (6-f11)) was showed shifting of the peak while figure (6-f15) showed loss of the melting endotherm which may be due to presence of small quantity of aceclofenac. changing in the peak was reflecting the changing in the crystalline state of aceclofenac to amorphous (47, 48). 50.00 100.00 150.00 200.00 temp [c] -20.00 -10.00 mw dsc 113 .23x100conset 153 .21x100cend set 108 .63x100csta rt 163 .75x100cend 128 .75x100cpea k -1.0 3x100j -11 4.78x100j/g hea t -12 .58x100m whei ght 50.00 100.00 150.00 200.00 temp [c] -10.00 -5.00 mw dsc 39.30x100conset 60.51x100cend set 29.92x100csta rt 70.32x100cend 51.05x100cpea k -21 4.11x100m j -35 .69x100j/g hea t -4.1 5x100m whei ght figure 6. dsc thermograms of pure ac powder, f11 and f15. iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 93 ftir spectra the characteristics absorption bands of ac were: 3318cm-1 (n–h stretching or o–h stretching), 3277 and 2936 cm-1 (c-h stretching) due to both aromatic and aliphatic stretching vibrations respectively in addition to 1771cm-1(–c=o stretching), 1585 and 1506cm-1 (–c=c stretching of aromatic), and some prominent bands as explained in figure (7). the spectrum of lyophilized powder showed the same characteristic peaks, albeit of smaller intensity as seen in figure (7). the results showed no major differences in the peaks of ac relative to the pure drug in the prepared formulas, suggesting the absence of any interaction between drug and polymers. a broad peak may be attributed to hydrogen bond between drug and polymer (49). figure 7. ftir spectra for pure ac powder, f11 and f15. f11 iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 94 pxrd pxrd assays were done for pure drug and the lyophilized formulas as showed in figure (8). the pxrd patterns of ac pure drug showed a sharp diffraction peaks indicating the crystalline nature of the drug as a consequence of different arrangement of the molecules in the crystal lattice. in the lyophilized formulas, lack of distinctive drug peaks or a significant reduction in the characteristic peaks indicating that ac was trapped in an amorphous or molecular shape within the mixture, this is agreed with the results obtained from the dsc assay (50). figure 8. pxrd graphs for ac powder, f11 and f15 preparation of hydrogel f15 was selected to be incorporated in hydrogel system. for comparison, blank hydrogel containing unprocessed ac powder was also prepared. characterization of ns based-hydrogel physical appearance visual inspection of the prepared ac-ns basedhydrogel revealing a good homogeneity, free of grittiness and no phase separation. the gel of f15 with carbopol concentration (1%) was appeared to be white in appearance. ph determination the ph values of the prepared ac hydrogels were (6.83 to 6.87) as seen in table (7). table 7.spread-ability and ph values of the prepared aceclofenac hydrogels (n=3) formula number spreadability (g.cm2/sec)± sd ph ± sd f15 14.3±0.01 6.83±0.02 blank 10.6±0.015 6.87±0.025 spread-ability measurement the prepared hydrogel was easily spreadable by applying little shear as seen in table (7). good spread-ability value is one of the essential properties for gel dosage form. spread-ability suggests gel propagation capability to a part of the skin. the therapeutic efficiency of gel determined according to spreading value (51, 52). determination of the viscosity the viscosity was measured at different shear rates and the data are represented in table (8). it can be seen in (figure-9) that the viscosity of hydrogel of f15 was ranged (180456-11306 cp), while for the blank hydrogel is (168435-10524cp). it was found that as the shear rate increased as the viscosity was decreased. therefore, it is a nonnewtonian flow behavior (53). the results showed that carbopol was a good gelling agent for preparation of hydrogels. iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 95 table 8.viscosity values for the prepared ac hydrogels speed (rpm) shear rate (1/sec.) gelled f15 viscosity (cp) blank hydrogel viscosity (cp) 1.5 0.32 178456 168435 2 0.41 140786 138576 2.5 0.49 99800 95723 3 0.6 83350 80836 6 1.32 60566 58243 12 2.48 48270 46387 30 6.4 24546 27534 60 13.16 13785 10524 determination of the viscosity the viscosity was measured at different shear rates and the data are represented in table (8). it can be seen in (figure-9) that the viscosity of hydrogel of f15 was ranged (180456-11306 cp), while for the blank hydrogel is (168435-10524cp). it was found that as the shear rate increased as the viscosity was decreased. therefore, it is a nonnewtonian flow behavior(53). the results showed that carbopol was a good gelling agent for preparation of hydrogels. figure 9.viscosity versus shear rate for gelled f15 and the blank hydrogel. in vitro drug release study in vitro drug release study was done in phosphate buffer (ph 7.4). figure (10) showed the release pattern of drug from the prepared hydrogels. at 10 hour period of the release assay, the cumulative percentages of drug release were in the order: gel of f15> plain hydrogel with 84.8% and 32.6% respectively. at 12 hr., the release of gel f15 reached to 100%, while plain hydrogel had the lowest release percentage, as we see there was a significant difference (p<0.05) between f15 hydrogel and plain hydrogel, this is due to improved dissolution rate and permeation due to small size of nanoparticles, i.e. the reduction in particle size leading to increased dissolution velocity. as drug particles are smaller, the corresponding surface area is greater, according to the noyes-whitney equation, so the nanocrystals have a significant elevation area. in addition, the diffusion distance decreases for very small particles. the increased surface area and the simultaneous decrease in diffusion distance could therefore significantly increase the velocity of dissolution of the substance (54). figure 10. the cumulative release of ac for gelled f15 and blank hydrogel the release of ac from hydrogel of f15 is best fit with higher correlation (r2) with zero order equation which show zero order release profile. while the blank hydrogel (r2 = 0.969) fitted with first order kinetics. it is worth mentioning that the diffusional exponent (n) obtained from korsmeyerpeppas can be utilized by calculating 60% release of the drug. when the liquid diffusion rate is slower than the relaxation rate of the polymeric chains, the diffusion is fickian or called case–i transport (n ≤ 0.5). when liquid diffusion rate and polymer relaxation rate are of the same order of magnitude, non-fickian diffusion or anomalous transport (0.5 < n < 1) is observed. when liquid diffusion rate and polymer relaxation rate are of the same time but diffusion rate more than relaxation rate are, super case – ii transport (n > 1.0) is observed. while the relaxation process is very slow compared with the diffusion, the case – ii transport occurs (zero order kinetic model) (29). the value of (n) for hydrogel of f15 (1%) was (0.822) and blank hydrogel was (0.873) that both of them were > 0.5 and < 1 which indicates that the drug release mechanism was non fickian, and suppose that aceclofenac hydrogel delivered their iraqi j pharm sci, vol.30(2) 2021 preparation and in vitro characterization of ac nanosuspension 96 active ingredients by non fickian diffusion (see table 9). table 9. in vitro release kinetics of ac hydrogel formulas formul a code zero orde r r2 first orde r r2 higuch i r2 kosmeyer -peppas (n) 15 0.974 0.943 0.917 0.822 blank 0.951 0.969 0.88 0.873 stability test the results of stability test were illustrated in table (10). the drug content was reduced at elevated temperature and this can be diminish by storage in refrigerator (55). table 10. stability tests of gelled f15 (n=3) stability paramet er 25 ℃ 40 ℃ 4 ℃ appearan ce homogen ous homogen ous homogen ous ph ± sd 6.83±0.02 6.57±0.05 6.62±0.03 drug content ± sd 97.5±0.05 1 96.43±0.0 15 97.5±0.01 conclusions ac-ns.s have been successfully prepared using various stabilizers in different stabilizer: drugs ratios (1:1, 2:1, 3:1 and 4:1). the data indicate that the process of solvent/anti-solvent precipitation is an efficient and a cost-effective method for preparing drug ns.s, and easily to implement for the manufacturing of drug nanoparticles. the results confirm that when ac particles are nano sized, both the solubility and dissolution are improved. ac-ns based hydrogel showed enhanced release rate of drug as compared with blank hydrogel. acknowledgments i would like to thank college of pharmacy of basrah and all the staff of the department of pharmaceutics, and especially assistant lecturer noor yousif. my deep thanks to sama al fayhaa pharmaceutical industry and pioneer 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perspective , j develop drugs 2013, 2:2 54. wei s, xie j, luo y, et al. hyaluronic acid based nanocrystals hydrogels for enhanced topical delivery of drug: a case study. carbohydr polym. 2018;202(july):64-71. 55. abdel-rashid rs, helal da, omar mm, el sisi am. nanogel loaded with surfactant based nanovesicles for enhanced ocular delivery of acetazolamide. int j nanomedicine. 2019;14:2973-2983. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 epigallocatechin gallate anticancer molecular mechanism doi: https://doi.org/10.31351/vol32iss1pp167-176 167 autophagy or apoptosis: anticancer molecular mechanism of epigallocatechin gallate with natural polyphenol effect on hepg2 cells viability rand r. hafidh*,1, zahraa q. ali**and ahmed s. abdulamir*** *department of microbiology, college of medicine, university of baghdad, baghdad, iraq. **department of anatomy, college of medicine, university of baghdad, baghdad, iraq. ***department of microbiology, college of medicine, al-nahrain university, baghdad, iraq. abstract the anticancer impact of epigallocatechin gallate (egcg) the highly active polyphenol of green tea was abundantly studied. however, the exact mechanism of its cytotoxicity is still under investigation. the current study was designed to investigate the effects of egcg on thirteen autophagyand/or apoptosis related genes in hepg2 cells. the apoptosis detection analyses such as flow cytometry and dual apoptosis assay were used on hepg2 cells. the genes expression profile of hepg2 cells was explored using the real-time quantitative-pcr. egcg increases g0/g1 cell cycle arrest and the real-time apoptosis markers proteins leading to stimulate apoptosis in 70% of the treated hepg2 cells. the up-regulation was recorded in two of autophagy inhibitory genes (fos-1, fos-2) and apoptosis inducer gene (ddit3). while the other ten genes expressed down-regulation after treatment. the down regulation genes involved the genes of mitochondrial autophagy marker proteins (bnip3, bnip3l, and nbr1), the autophagy regulator genes (birc5, mapk9), and the gene that implicated in protein biosynthesis and protein modification (itgb1). the genes that have pro-apoptotic function in cells (capns1, cflar, eif4g, and rb1) were also showed down-regulation after treatment. thus, the results demonstrated a potential effect of egcg to induce apoptosis rather than autophagy in the treated hepg2 cells that could play as good targeted therapy. keywords: egcg; apoptosis, autophagy, hepg2. cancer cell. الجزيئية المضادة للسرطان االلتهام الذاتي أو موت الخاليا المبرمج: اآللية epigallocatechin gallate لخاليا النمو و البقاء التأثير البوليفينولي الطبيعي على قابلية ذو hepg2 *** صاحب عبد األمير و أحمد **زهراء قاسم علي ،1*، رند رياض حافظ فرع األحياء المجهرية، كلية الطب، جامعة بغداد، بغداد، العراق. * التشريح، كلية الطب، جامعة بغداد، بغداد، العراق. فرع ** فرع األحياء المجهرية ، كلية الطب، جامعة النهرين، بغداد ،العراق. *** الخالصة والبوليفينول عالي النشاط للشاي األخضر باستفاضة. ومع epigallocatechin gallate (egcg)تمت دراسة التأثير المضاد للسرطان لـ على ثالثة عشر جينًا egcgذلك ، فإن اآللية الدقيقة لسميتها الخلوية ال تزال قيد التحقيق. األهداف: لذلك، فإن الدراسة الحالية مصممة للتحقيق في آثار خالي في المبرمج الخاليا موت أو / و الذاتي بااللتهام صلة اجراء hepg2ا ذات تم الطريقة: باستخدام . المبرمج الخاليا موت عن flowتحليالت cytometry و dual apoptosis assay خاليا علىhepg2 .تم الكشف عن مستويات الجينات باستخدام و rt-pcr الكمي. النتائج: يزيدegcg ٪ من 70مبرمج في الوقت الحقيقي مما يؤدي إلى تحريض موت الخاليا المبرمج في وبروتينات عالمات موت الخاليا ال g0 / g1من توقف دورة الخلية (. ddit3( وجين محفز لموت الخاليا المبرمج )fos-1 ،fos-2في اثنين من الجينات المثبطة لاللتهام الذاتي ) زيادة.المعالجة. تم تسجيل hepg2خاليا انخفاض األخرى العشرة الجينات أظهرت العالج. اتهامستويبينما المنخفضة بعد المستويات ذات الجينات الذاتي تتضمن االلتهام لبروتينات جينات ( ، والجين المتورط في التخليق الحيوي للبروتين birc5 ،mapk9( ، وجينات منظم االلتهام الذاتي ) nbr1و bnip3lو bnip3للميتوكوندريا ) و cflarو capns1في الخاليا ) pro-apoptotic(. كما أظهرت الجينات التي لها دور مسبق لموت الخاليا المبرمج itgb1وتعديل البروتين ) eif4g وrb1أظهرت النتائج تأثيًرا محتمالً لـ عليه( أيًضا انخفاض مستوياتها بعد العالج. الخالصة: و ،egcg ًللحث على موت الخاليا المبرمج بدال المعالجة والتي يمكن أن تكون هدفًا جيدًا للعالج. hepg2اتي في خاليا من االلتهام الذ الخاليا السرطانية. ،hepg2 ،االلتهام الذاتي ،موت الخاليا المبرمج ،epigallocatechin gallate ، egcgالكلمات المفتاحية: introduction epigallocatechin gallate (egcg) is a flavonoid that proved its therapeutic effect in cancer (1). it is the most active polyphenol in green tea, which has anti-inflammatory, antioxidant (2), and protective for cardiac muscle (3, 4). many in vivo and clinical studies (phase i / ii) have acclaimed that egcg is an efficient agent and quite safe in providing protection against cancer (5). the health benefits of green tea, particularly the positive role of egcg in stimulating autophagy in the treatment and prevention of liver cancer was proved (6). 1corresponding author e-mail: randriadh@comed.uobaghdad.edu.iq received: 23/2 /2022 accepted: 15/5 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp167-176 iraqi j pharm sci, vol.32( 1 ) 2023 epigallocatechin gallate anticancer molecular mechanism 168 moreover, egcg mediated cell death in glioblastoma cell line and cell line of the no small cell lung cancer by an induction to one of the apoptosis pathways (1, 7) . in cancer cells, apoptosis and autophagy play a critical role in the biological process of cancer (8). apoptosis (the programmed cell destruction) has a significant role in the treatment of cancer as it is a common target of many treatment strategies (9). autophagy has dual effect on tumorigenesis by suppressing the survival of cancer-cell and stimulating cell death. in addition, it accelerates tumorigenesis by inducing cancer-cell proliferation and tumor growth (10). natural products-prompted autophagy is mostly causing death of tumor cells when apoptosis is totally defective in cancer cells (11). the increment of apoptosis resistant has been proved among cancer cells together with a high rate of adverse events associated with chemotherapy/radiotherapy. highlight the needs to consider the activity of the natural products in regulating the cancer development events with focus on autophagy. the purpose of the current study is to explore the effect as anticancer agent on hepg2 cells by exploring certain genes that were not investigated before. it intended to investigate the role of egcg as a tumor-suppressor in treating or preventing hepatocarcinoma. the aim of this study was conducted by using dna analysis to explore the autophagyand apoptosis-related genes’ expression namely (fos-1, fos-2, ddit3, bnip3, bnip3l, birc5, mapk9, itgb1, capns1, cflar, eif4g, nbr1, and rb1). materials and methods the materials the following materials were purchased from sigma, germany: (-)-epigallocatechin gallate (egcg, minimum 95%, e4143-50mg), fetal bovine serum, dimethyl sulfoxide (dmso), and ribonuclease a. rpmi-1640 medium with l glutamine, amphotericin b, and penicillinstreptomycin were from (biowest, florida, usa). the proliferation assay of cell titer 96 aqueous one solution cell was from (promega, usa). propidium iodide was from (mp biomedicals, llc, iiikrick, france). apoptosis assay kit (dual), nucview™ 488 was from (biotium inc, usa). nucleic acid extraction kit gf-1 was from (vivantis technologies, malaysia). the iscript cdna synthesis kit was from (bio-rad, hercules, canada). rt-pcr master mixes were from (life technologies, usa). the compound preparation and cell culture egcg was dissolved in an absolute dmso giving a stock concentration of 50mg/ml and incubated at -20⁰c for subsequent analyses. the cell line: hepatocellular carcinoma cells (hepg2; atcc hb-8065), were cultivated in rpmi-1640 medium with 10% fetal bovine serum, 2.5 μg/ml of amphotericin b, and 50 u/ml penicillinstreptomycin at 37°c with 5% co2. cell cytotoxicity assay the cytotoxic effect of egcg on hepg2 cells was measured. freshly prepared egcg working concentrations of (10, 20, 40, 80, and 160 μg/ml) were diluted in 10% rpmi-1640 medium. hepg2 cells (1 × 105 cell/well) were treated with each concentration (200 μl/well) and incubated for 48 h in 5% co2 at 37°c. a negative control composed of dmso in 10% rpmi-1640 medium was used. later, a solution composed of 10% rpmi1640 medium (100 μl) and mts (20μl) was used to replace the wells’ contents. the absorbance at 490 nm, after 4 h was read by elisa reader (sunrise basic tecan, grödig, austria). the compound concentration that destroyed 50% of the cell line (ic50) was calculated using the following formula (12): cytotoxicity % = [1-(odt/odc)]* 100 in which (odt) represents the optical density of the tested compound and (odc) represents the optical density of the negative control. treatment of hepg2 cells based on egcg ic50 value obtained from the mts assay, duplicates of (1 × 105 hepg2 cell/well) was treated with10% rpmi-1640 medium with or without egcg ic50 for 48 h. both egcg-treated hepg2 cells and untreated cells were harvested and washed by 10% rpmi-1640 medium, after the end of the incubation time. the resulting cells were subjected to the following assays. analysis by the flow cytometry the treated and untreated cells were analyzed to explore the apoptosis level of cells and cell cycle arrest. the cells were fixed as (1:10 v/v) of ice-cold ethanol 70% with pbs. the fixed cells were incubated for 2h at −20°c and after that washed by pbs. staining dna solution (500 μl) in pbs was prepared, which composed of ribonuclease a (50 μl) as 1 mg/ml and propidium iodide (25 μl) as 1 mg/ml (13). the flow cytometry cyan adp apparatus was used for measurement (beckman coulter, usa). analysis was performed in triplicates of each sample and control (untreated cells) using the software summit (v4.3). a real-time detection of apoptosis a dual apoptosis assay kit was used along with sulforhodamine 101 (texas redtm) conjugated annexin v and caspase-3 substrate. this kit provides two real-time apoptosis events in intact cells, phosphatidylserine translocation and caspase3 activity. based on the instructions of the manufacturer, an aspiration of the wells’ contents was done to wash the treated and untreated cells with annexin v-binding buffer. later, the following materials were added to each well and incubated for 45 minutes: sulforhodamine 101-annexin v (5 μl), annexin-binding buffer (100 μl), and 0.2 mm iraqi j pharm sci, vol.32( 1 ) 2023 epigallocatechin gallate anticancer molecular mechanism 169 nucview tm 488 caspase-3-substrate (5 μl). fluorescence microscope (nikon eclopse 80 i) using fitc and texas-red filters, was used to observe the apoptotic events in the stained cells which were performed in triplicates. the apoptotic index was determined as apoptotic cells’ number as proportion of the total number of cells. the autophagy and apoptosis-related genes’ expressions rna extraction the expression of autophagyand apoptosis-related genes in hepatocarcinoma cells was investigated by extracting total rna from the collected cells, following instructions of gf-1 kit. rna concentration of (546 ng/ul) was measured by the spectrophotometer life science uv/vis (beckman coulter, usa). total rna concentration, quality, purity, and integrity were checked by the bioanalyzer agilent 2100 (agilent, usa). the isolated and checked total rna was stored at −80°c with an integrity number of (8.5). semi-quantitative real time-pcr the isolated rna from hepg2 cells (0.5 µg) was converted to cdna by the synthesis kit. following the manufacturer’s instructions, 5× reaction mix iscript (4 μl) was mixed with reverse transcriptase iscript (1 μl) and rna template (15 μl) in (20 μl/ reaction) as a final volume. by using thermo bath (finepcr, seoul, korea), the prepared mixture was incubated at 25°c for 5 min then at 42°c for 30 min. for 5 min, the temperature was raised to 85°c. the prepared cdna was kept at −80°c for further analysis. the targeted genes differential expression was detected using sybergreen rt-pcr master mixes on the detection system iq5 real-time pcr (bio-rad, usa) between treated and untreated hepg2 cells. the housekeeping gene β-actin (actb) was used for normalization with forward and reverse primers’ sequence (r: 5agcactgtgttggcgtacag-3, f: 5agagctacgagctgcctgac-3). the thirteen primers used to specifically amplify target gene cdna are shown in (table 1). the sequence of primers and the followed cycling regimen for the designed primers was carried out using primer3 and blast, nih, usa (the online basic local alignment search tool) at https:// blast. ncbi. nlm. nih. gov/blast.cgi. according to the specifications entered to the designing utilities and software and after in-lab verification of the pcr amplification, the thermal cycling conducted was as follows: one cycle at 95°c for 180sec followed with 40 cycles at 95°c for 60sec, and at 60°c for 70sec. quadruplicates of all samples were assayed. preparation of the negative controls (none-template controls) was done. the differential expression of the housekeeping gene actb along with the target genes was measured in folds according to delta-delta ct method, known as the e–∆∆ct method, where e stands for the value of pcr efficiency calculated out of the efficiency curve. in general, all runs of pcr turned out efficiency >90% with r2=0.99. a comparison between the expression levels of untreated controls with treated cells was performed. after low-efficacy wells expelled to be zero, pcr efficacy for all the used primers was measured. later, calculation of the fold changes in genes expression was carried out. table 1. gene symbols along with primer sequences applied in real-time pcr. gene name sequence avg. tm amp licon size ddit3 dna damage-inducible transcript 3 (human) f: 5′-gaacggctcaagcaggaaatc-3' r: 5′-ttcaccattcggtcaatcagag-3' 59.85 80 fos-1 fos proto-oncogene, ap-1 trancription factor subunit (human) f: 5′-cgtctccagtgccaacttca-3′ r: 5′-ggtccggactggtcgagat-3′ 60 233 fos-2 fos proto-oncogene, ap-1 trancription factor subunit (human) f: 5′cccggacttgcaccttactt-3' r: 5′actgatctgtctccgtctcct-3' 59.72 553 bnip3 bcl2 interacting protein 3 (human) f: 5′-ggagcctagtcaagcctgaga-3′ r: 5′-catccgttgatgtgcaatgcg-3′ 59 134 bnip3l bcl2 interacting protein 3 like (human) f: 5′-cctcgtcttccatccacaat-3′ r: 5′-gtccctgctggtatgcatct-3′ 58 211 iraqi j pharm sci, vol.32( 1 ) 2023 epigallocatechin gallate anticancer molecular mechanism 170 continued table 1. nbr1 nbr1 autophagy cargo receptor (human) f: 5′-attcaccccacagggatagc-3′ r: 5′-gcaggacgtggttagtgtca-3′ 59.9 865 birc5 baculoviral iap repeat containing 5 (human) f: 5′-tgacgaccccatagaggaaca-3′ r: 5′-cgcactttctccgcagtttc-3′ 60.1 186 mapk9 mitogen-activated protein kinase 9 (human) f: 5′-agtcatcctgggtatgggct-3′ r: 5′-aacctttcaccagctctccc-3′ 60 80 itgb1 integrin subunit beta 1 (human) f: 5′-gacgccgcgcggaaaa-3′ r: 5′-acatcgtgcagaagtaggca-3′ 60.3 209 capns1 cyclase associated actin cytoskeleton regulatory protein 1 (human) f: 5′-tgcggcgcagtgagtc-3′ r: 5′-ttcatgagttctgtggcgct-3′ 59.9 387 cflar casp8 and fadd like apoptosis regulator (human) f: 5′-gagtgccggctattggactt-3′ r: 5′-atagcaacatcccggcacaa-3′ 60.06 443 eif4g eukaryotic translation initiation factor 4 gamma 1 (human) f: 5′-gtggagattctaggcgaccg-3′ r: 5′-tcacgaggttcacaaggagc-3′ 59.9 435 rb1 retinoblastoma 1 (human) f: 5′-tttgtaacgggagtcgggag-3′ r: 5′-atcgaactgctgggttgtgt-3′ 59.7 648 analysis of data the data analysis was carried out using spss program version (16.0.0.2) and data were shown as (mean ± sd). the influence of the compound on cell growth inhibition was appraised by using 95% confidence intervals. linear regression index equations were used to calculate ic50 value. the student t-test was used to compare the mean of each cell cycle phase, in triplicates, of treated vs untreated cells in flow cytometric analysis. in real-time qpcr, the comparative ct method was used to build the used fold change calculation. the equation for comparative ct method which is also known as the 2-δδct method was as follows: δδct = δct treated – δct untreated. where: δct treated = ct treated – all mean treated and δct untreated = δct untreated – all mean untreated. less than 0.05 of p values were reflected as significant. results the cytotoxic effect of egcg in the current study hepg2 cell viability was affected by treatment with egcg in a dosedependent manner (figure 1). egcg concentration that destroyed 50% (ic50) of hepg2 cells was (10µg/ml). egcg effect on the selected genes’ expressions was studied using this concentration. figure 1. the mean percentages of egcg cytotoxic effect on hepg2 cells after treatment for (48h). the viability reduced in a dosedependent manner arrest in cell-cycle induced by egcg the treated hepg2 cells with egcg ic50 underwent a cell-cycle arrest in g0/g1 phase when compared to untreated one (figure 2). the treated cells were obstructed significantly in g0/g1 phase (68.35% ± 1.41) with (73.75% ± 2.21) in untreated cells, (p= 0.037). non-significant results were manifested when hepg2 cells treated with egcg after 48 h in comparison with untreated cells. in s phase the percentage was (7.10% ± 1.23) in treated cells with (8.82% ± 3.02) in untreated one, (p= 0.43). while, in apoptosis and g2/m phases the percentages were as follows: (16.40% ± 3.97) and (8.95% ± 3.96) comparing to untreated cells (9.62% ± 3.83) and (8.97% ± 1.71) with p value of (0.1) and (0.99), respectively. iraqi j pharm sci, vol.32( 1 ) 2023 epigallocatechin gallate anticancer molecular mechanism 171 figure 2. representative histogram of the flow cytometry analysis of hepg2 cells’ dna contents after 48 h, treatment with egcg ic50. treatment with egcg arrests cell-cycle in g0/g phase. the cells’ percentages with fractional dna content was detected by cyan adp apparatus and summit (version 4.3) program: s (r4), apoptotic cells (r2), g0/g1 (r3), and g2/m (r5) phases. the p value of less than 0.05 was considered as significant. revealing of the real-time apoptosis in intact hepg2 cells in order to detect the apoptosis induction by egcg treatment, the morphological changes of apoptosis in live-cells were investigated by fluorescence microscopy (figure 3). two important apoptosis markers were monitored by the kit. the monitored markers were the translocation of phosphatidylserine on the cell membrane which was identified by sulforhodamine 101-annexin v. in addition, caspase-3 activation was identified by nucviewtm 488 caspase-3 substrate activity within individual whole cells (14). the nucleus stained as a bright green after the cleavage of nucviewtm488 caspase-3 substrate by activated caspase-3 (figure 3c). besides, phosphatidylserine that had translocated from the inner to the outer leaflet of the plasma membrane, produced a red frame around the cell under a fluorescent microscope using a red filter (figure 3d). the morphological changes that could be detected after treatment like rounding of cells, detachment, and cell shrinkage reflected the apoptosis-related events (15). a comparison between the treated cells and untreated cells in the plates with negative control were done. after 48 h of treatment, egcg ic50 (20 µg/ml) stimulated apoptosis in 70% of hepg2 cells. figure 3. the morphological alterations of egcg treated hepg2 cells reveals apoptosis-related events. a) no morphological changes in the negative control hepg2 cell line were shown in the photomicrograph. b) the apoptosisrelated events were shown in the treated hepg2 cell line with egcg ic50 (20 µg/ml) after 48h. the cells viewed under a fluorescent microscope (nikon eclopse 80 i). the morphological alterations were manifested after 48h of treatment with egcg ic50. c) the photomicrograph of the treated hepg2 cell lines with egcg ic50 (20 µg/ml) for 48h and stained with caspases-3 substrate. caspase-3 activation was indicated by green nuclei in hepg2 cells. d) the photomicrograph of hepg2 cell lines treated with egcg ic50 (20 µg/ml) for 48h and stained with annexin-v substrate. the translocation of the phosphatidylserine from the inner to the outer leaflet of the cell membrane was indicated as red borders around hepg2 cells. e) the photomicrograph of the stained and merged hepg2 cell lines treated with egcg ic50 (20 µg/ml) for 48h. the apoptotic hepg2 cells were characterized by green nuclei surrounded by red frames. iraqi j pharm sci, vol.32( 1 ) 2023 epigallocatechin gallate anticancer molecular mechanism 172 upand down-regulated target genes after egcg treatment the profile of the gene expression of the hepg2 cells treated with ic50 of egcg for 48h was assessed by the semi-quantitative analysis of realtime qpcr. thirteen genes belong to different apoptosis or autophagy pathways that attributed to cancer progress in human normal cells were shown to be upor down-regulated after treatment (table 2). two of the fos member family were upregulated significantly, i.e., fos-1 (32.97 ± 9.24 folds) and fos-2 (10.31± 4.91 folds). the other gene that up-regulated in folds after treatment, was ddit3 (18.26 ± 4.51). the ten rest genes were down-regulated after treatment with egcg. downregulated genes was manifested with three of the mitochondrial autophagy marker proteins, bnip3 (0.48 ± 0.09 folds), bnip3l (0.42 ± 0.08 folds), and nbr1 (0.17 ± 0.002 folds). in addition, two autophagy regulator genes in cells were downregulated, birc5 (0.44 ± 0.07 folds) and mapk9 (0.43± 0.07 folds). moreover, genes related to metabolism/modification and biosynthesis process found to be affected by egcg treatment. two of these genes were downregulated, itgb1 (0.38 ± 0.02 folds) and eif4g (0.24 ± 0.01 folds). there were other three genes that have a role in apoptosis pathways downregulated after egcg treatment. they were capns1 (0.36 ± 0.02 folds), cflar (0.32 ± 0.02 folds), and rb1 (0.11 ± 0.001 folds). table 2. the expression levels of apoptosisand autophagy-related genes were shown in treated hepg2 cells after egcg treatment for 48h. discussion it has been proved that egcg low concentrations didn’t significantly alter hepg2 cells viability. in contrast, a significant reduction in the number of viable cells was detected with its high concentration treatment, thus egcg cytotoxicity is in a dose-dependent manner (6, 16). the results of this study prompt this fact as the egcg cytotoxic effect was in a dose-dependent manner, with high concentrations found to be cytotoxic to hepg2 cells after 48h. a study by ahn et al. has shown that the high level of egcg is associated with its cytotoxic effect. it is due to the expression of endoplasmic reticulum stress response proteins, such as atf3, gadd34, and chop (ddit3) (16). one of these endoplasmic reticulum stress response proteins; i.e. chop (ddit3) showed significant up-regulation after treatment with egcg in the current study. in 2019, guan et al. discovered the important role of chop as an inducer to autophagy and apoptosis by the endoplasmic reticulum stress (17). the role of the stress caused by a cytotoxic agent treatment was proved by a study in 2015. in which such treatment may induce damage of organelles and proteins in cancer cells. results in proapoptotic factors to be released due to a stress in mitochondria and endoplasmic reticulum and subsequent cancer cell death (11). phosphatidylserine (ps) exposure on the outer plasma membrane is considered as a unique characteristic of apoptotic cells that could be depended on annexinv staining of ps to distinguish apoptosis from other cell death pathways (18). this characteristic of ps externalization was investigated in this study to investigate the egcg efficacy on hepg2 cells. the results came to the fact that about 70% of the cells underwent apoptosis by checking the morphological changes in the stained intact hepg2 cells after treatment. the real-time apoptosis markers proteins investigation, particularly, caspase activation was strengthened by the g0/g1 cell cycle arrest that recorded by the flow cytometry analysis. these results were supported by a study which found that egcg increases caspase activity and the sub-g1 phase of the cell cycle leading to the stimulation of apoptosis in cancer cells (19). the effect of the autophagy related genes in cancer suppression or induction has been explored by many studies, in addition to their involvement in cancer development by different mechanisms (20, 21). based on the fact that the cytotoxicity effect of egcg on hepg2 cells is it mediated by autophagy or apoptosis, this study was designed. the data of this study recorded up-regulation of two of fos family members genes, namely, fos-1 and fos-2. these genes expressed as the activator protein-1 (ap-1) transcription factors in the host cells. these factors are participating in different aspects of cell survival or death, and cellular proliferation (22). hence, a study in 2010 found that hepg2 cells expressed gene name (fold change) up-regulated genes down-regulated genes fos-2 32.97 (fos gene family member) bnip3 0.48 ddit3 18.26 birc5 0.44 fos-1 10.31 (fos gene family member) mapk9 0.43 bnip3l 0.42 itgb1 0.38 capns1 0.36 cflar 0.32 eif4g 0.24 nbr1 0.17 rb1 0.11 iraqi j pharm sci, vol.32( 1 ) 2023 epigallocatechin gallate anticancer molecular mechanism 173 the ap-1 proteins c-jun and junb, but not c-fos, jund, and fra-1 can inhibit autophagy (23). bcl2 interacting protein 3 (bnip3) and bcl2 interacting protein 3like (bnip3l) are mitochondrial autophagy marker proteins (24). these two genes found to be down-regulated after egcg treatment to hepg2 cells. mitophagy is a mechanism which enhances cell protection in a healthy cell during mitochondrial damage (25). one of the mechanistic pathways that lead to a mitophagy is mediated by bnip3 and bnip3l proteins. each of these proteins provide joining of a mitochondrion to an autophagy apparatus via the interaction between receptor proteins like (nbr1), (26). nbr1 autophagy cargo receptor was downregulated after egcg incubation with hepg2 cells. it is a specific receptor for selective autophagosomal degradation of ubiquitinated targets and pexophagy (27, 28). autophagy is mediated by the formation of double-membrane vesicles known as autophagosomes that fuses with lysosomes. large cytoplasmic materials are selectively segregated and degraded in the lysosomes. substrate collection is mediated by ubiquitylation and enrollment of ubiquitin-binding autophagic receptors nbr1 that engulfed by autophagosome (21, 28). birc5 is verified as a straight autophagy regulator in cells (29). a study has come to a result that birc5 can be a potential therapeutic target for renal carcinoma cell lines by using a drug targets birc5 (30). the treated hepg2 cells with egcg displayed a down-regulation in birc5 gene when compared to untreated cells. mapk9 regulates autophagy gene expression that may stimulate autophagy by inducing the expression of certain autophagy related genes (31). data of this study found that mapk9 gene was significantly down-regulated which in turn may affect the mechanism of autophagic regulation. unlike a previous study done by ahn and his colleagues in 2009, they found that the expression of integrin subunit beta 1 gene (itgb1); a gene associated with protein modification and protein biosynthesis, was elevated after egcg treatment (16). the current study demonstrated downregulated effect of egcg on this gene after treatment. a recent study indicated that the treatment of breast cancer by a traditional chinese medicine targeting itgb1 could be a promising target to cease cancer progress and metastasis (32). this result may enhance our findings that egcg anticancer effect is associated with apoptosis induction rather than autophagy. translation initiation factors like eif4g have complex functions in cells (33). in 2010 a study carried by fan et al., proved the anti-cancer activity of the tested drug by disrupting the eif4e/eif4g association through binding to eif4e (34). studies display that the eif4e inhibition can lead to apoptosis-inducing and growth-inhibitory effects in cancer cells. hence, could induce the potential to use such molecular target for therapy (34, 35). these findings strengthen the current study results which found significant apoptosis induction by an inhibition to the selected genes, one of them eif4g. the calpain (capns1) is a family member of calcium activated cysteine proteases which implicates in both proor anti-apoptotic functions (36). capns1 depletion increases sensitivity to apoptosis (37) which support our results of capns1 gene down-regulation after treatment and increased the chance of apoptosis pathway for cytotoxic effect on treated cells. capns1 represents an encouraging target for cancer therapy since its action is tightly linked to tumor progress. it is required for autophagy and survival of cancer cells (37). thus, its downregulation affects negatively on cancer cells survival. the anti-apoptotic protein cflar (casp8 and fadd-like apoptosis regulator) has a significant attention as an anticancer therapeutic target in solid and haematological cancers where it is frequently overexpressed (38). cflar is a main apoptosisregulatory protein that inhibits necroptosis and autophagy (39). this gene was affected negatively by egcg, thus increased the chance to enhance apoptosis in treated hepg2 cells. additionally, the down-regulation to retinoblastoma 1 gene was in harmony with other findings who investigated its role in the apoptosis pathways. the retinoblastoma protein (prb) bounds to pro-apoptotic promoters that is transcriptionally active in the rb-dependent apoptotic pathways (40, 41). other study revealed the straight role for prb in the stimulation of apoptosis as response to genotoxic or oncogenic environment (42). the results of the current study agree with other findings which found that natural product treatment to cancer cells may induce signaling transduction but not in a sufficient way to induce autophagy (11). furthermore, a study on hepatocellular carcinoma in 2015 suggested that egcg prevents the development of cancer through cytocidal activity giving significant decrease in nfkappab and bcl-2 expression, and increased the expression of bax, p53, caspase-9, and caspase-3 with the release of cytochrome c (43). moreover, another study in 2018 has confirmed that egcg selectively targets hepatic de novo lipogenesis (dnl) pathway in hepg2 cells and thus inducing apoptosis by inhibiting certain key enzymes in this pathway, namely, atp citrate lyase, acetyl-coa carboxylase, and fatty acid synthase (fasn) (44). unlikely, other mechanism displayed by egcg cytotoxic effects on hepg2 cells was investigated by zhao et al., in 2017. they found that egcg inhibits alpha-fetal protein (afp) secretion and promotes the intracellular aggregation of afp, the protein that enhances tumor cell proliferation, which iraqi j pharm sci, vol.32( 1 ) 2023 epigallocatechin gallate anticancer molecular mechanism 174 induces autophagy to degrade afp and in turn inhibit the growth of hepg2 cells (6) . additional studies are critically in need to explore the exact pathway of cancer cell death after a natural product treatment. such studies need to focus on the regulation of multiple signaling pathways and the expression of molecules that are directly involved in autophagosome formation in cancer cells, particularly, hepg2 cells that could mediate the onset of autophagy and couldn’t be done by this study. conclusion with the increase incidence of apoptosisresistant cancer cells, the needs to investigate the cytotoxic effect of certain natural polyphenols products are increase. to indicate if their cytotoxicity is due to inducing the apoptosis or autophagy, the current study was conducted to investigate the effect of egcg on thirteen genes related to apoptosisand/or autophagy in cancer cells, particularly, hepg2 cells. some of these genes were investigated for the first time, i.e. (nbr1, itgb1, capns1, eif4g, and rb1). the flowcytometry analysis, dual apoptosis assay and qrtpcr all came to the conclusion that egcg as a natural polyphenol enhanced apoptosis-related genes but not autophagy-related genes and its mechanism in inducing the cytotoxicity is apoptosis. further studies are required to validate these results by using protein analysis for proteins involved in apoptosis or autophagy process. conflict of interests the authors have 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induces the apoptosis of hepatocellular carcinoma lm6 cells but not non-cancerous liver cells. int j mol med. 2015;35(1):117-24. 44. khiewkamrop p, phunsomboon p, richert l, pekthong d, srisawang p. epistructured catechins, egcg and ec facilitate apoptosis induction through targeting de novo lipogenesis pathway in hepg2 cells. cancer cell int. 2018;18:46. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 65 response surface methodology for development and optimization of theophylline pulmonary delivery system ali a. yas *,1 , yehia i. khalil * and mohamed s. mohamed ** * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmaceutics, fakulti farmasi, universiti teknologi mara, selangor, malaysia. abstract the aim of the present study was to develop theophylline (tp) inhalable sustained delivery system by preparing solid lipid microparticles using glyceryl behenate (gb) and poloxamer 188 (px) as a lipid carrier and a surfactant respectively. the method involves loading tp nanoparticles into the lipid using high shear homogenization – ultrasonication technique followed by lyophilization. the compositional variations and interactions were evaluated using response surface methodology, a box – behnken design of experiment (doe). the doe constructed using tp (x1), gb (x2) and px (x3) levels as independent factors. responses measured were the entrapment efficiency (% ee) (y1), mass median aerodynamic diameter (mmad, daer) (y2), zeta potential (zp, ξ) (y3), fine particles fraction (% fpf) (y4) and percentage of dissolution efficiency at 420 minutes (% de420) (y5). the optimized formula was characterized by differential scanning calorimetry (dsc), fourier transform infrared spectroscopy (ftir), scanning electron microscopy (sem) and x – ray powder diffraction (xrd) demonstrated that prolonged release physically due to the loaded tp exists mostly in its crystalline state . analysis of dissolution data of the optimized formula indicated that the best fitting is with higuchi model, whereas the mechanism of drug release pattern follows anomalous or non – fickian diffusion. key words: glyceryl behenate, solid lipid microparticles, theophylline nanoparticles مىهج الرد السطحً لحطىٌر وظام جحرٌر رئىي للثٍىفلٍٍه علً احمد ٌاس *،1 ، ٌحٍى اسماعٍل خلٍل * و محمد سالمة محمد *** * .فرع انصيذالَيبث ، كهيت انصيذنت ، جبيؼت بغذاد ، بغذاد ، انؼراق ** .فر انصيذالَيبث ، كهيت انصيذنت، انجبيؼت انخكُٕنٕجيت يبرا ، صيالَكٕر، يبنيزيب الخالصة انٓذف يٍ انذراصت ْٕ ححضير صيغت دٔائيت بطيئت انخحرر يٍ انثيٕفيهييٍ بشكم جضيًبث دُْيت يجٓريت يؼذة نالصخُشبق ببصخخذاو طريقت انخحضير حخضًٍ ححًيم انجضيًبث انُبَٕيت نهثيٕفهييٍ في انُبقم . صطحيكًثبج 188غهيضيريم بٓيُيج كُبقم دُْي ٔ انبٕالكضبيير حقييى انخغييراث انخركيبيت ٔ انخفبػهيت . ايٕاج فٕق صٕحيت يهيٓب حجفيف ببنخجًيذ – شذةانذُْي ببصخخذاو حقُيت يؼذنت حشًم حجبَش ػبني ال و انخجريبي حى حشيذِ ببػخًبد ػٕايم يضخقهت ٔ ْي يانخصى. انخجريبيبيُكيٍ –حصًيى بٕكش , ببصخخذاو االصخجببت انًُٓجيت انضطحيت يخٕصظ انقطر , (y1)ردٔد قيبصيت ٔ ْي كفبءة االَحببس ٔ( x3) 188 انبٕالكضبييرٔ ( x2) غهيضيريم بٓيُيج, (x1)انثيٕفيهييٍ بؼذ انًثبنيت نصيغت لثبج (. y5)انكفبءة انًئٕيت نالَحالل ٔ َضبت( y4)َضبت انجضيًبث انذقيقت , (y3)انزيخب االحخًبنيت , (y2)االيرٔدايُبييكي االنكخرَٔي ٔ حيٕد االشؼت انضيُيت نهًضحٕق يانًجٓرانًضح , رييت انطيفي نالشؼت ححج انحًراءٔححٕيم ف, حرارياجراء انًضح انخفبضهي ال ٔ نكٍ انيت انخحرر حخبغ اٌ انخحرر انطٕيم االيذ صببّ اٌ يؼظى انثيٕفيهييٍ انًحًم يٕجٕد بصيغت بهٕريت ٔ اٌ انخحرر يخبغ ًَٕرج ْيجٕحشي .غير فيكيبٌ .غلٍسٍرٌل بهٍىٍث ، الدهىن الصلبة المجهرٌة ، جسٍمات الثٍىفلٍٍه الىاوىٌة :الكلمات المفحاحٍة introduction pulmonary drug delivery in comparison to other routes of administration is most promising for local respiratory diseases (e.g., asthma, chronic obstructive pulmonary disease, tuberculosis, cystic fibrosis and cancer) as well as systemic diseases (e.g., thrombosis and diabetes) (1) . drug targeting via inhalation result a rapid drug deposition in a higher concentration at the site of action within lung tissues, thereby reducing the dose required and the side effects (2) . dry powder inhalers (dpis) are more preferable for delivering dry particulate drug to the respiratory tract, since they do not contain propellants as in the pressurized metered – dose 1 corresponding author e-mail: alistein76@yahoo.com received: 13/10/2012 accepted: 16/3/2013 iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 66 inhalers (pmdis), ensure drug stability and better patient compliance due to breath – actuated (3) . for an efficient conducting airways deposition, the particle size should be in the range of 2.5 – 6 µm, suitable shape, density and surface chemistry (4, 5) . sustained delivery inhalation therapy prolongs the duration of action and hence reduces dosing frequency (6) . although liposomes – based systems are biocompatible and tolerable, they are costly to be prepared and unstable during storage and nebulization (7) . polymeric nano – and micro – particles incorporated drug are prone to pulmonary toxicity, inefficient biodegradation, polymer accumulation and requirement of organic solvents for their production (8) . solid lipid microparticles which are solid lipid cores stabilized by surfactants; represent a unique sustained release inhalation therapy, prepared from biocompatible and biodegradable natural lipids with good tolerability and less respiratory toxicity (9) . nocturnal asthma is when asthma symptoms show exacerbation in the early hours of the morning and is associated with increased morbidity and lowered quality of life (10) . uniphyl ® 400 – 600 mg extended release tablets of tp, produced using contin ® chronopharmaceutical technology and administered once daily at evening for treating nocturnal asthma (11) . the inter – individual variability of tp absorption especially during night and the ubiquitous of cyclic nucleotide phosphodiesterases (pdes) enzymes that tp non – selectively inhibit results in a wide systemic side effects, necessitate pulmonary targeting preparation (12, 13) . according to the biopharmaceutics classification system (bcs); tp is a class i drug and in order to enhance loading and prolong its release from lipid microparticles, tp nanoparticles were prepared (14, 15) . this research focuses on the preparation, optimization and characterization of tp inhalable solid lipid microparticles. materials and methods materials pure theophylline (tp) and stearic acid (sa) were purchased from (sigma – aldrich / usa). glyceryl behenate (gb) (compritol ® 888 ato) was a gift from (gattefosse´ / france). poloxamer 188 (px) (lutrol ® f 68) was purchased from (basf – ludwigshafen / germany). all other chemicals / solvents used were of analytical grade. methods design of experiment box – behnken design is a class of second – order designs based on three – level incomplete factorial designs (16) . a design of three parts, each of two fully – leveled factors and a third factor set at zero level. the dots on the surface of a sphere are lying at on the middle of each edge of multidimentional cube and center point replicate (n = 3) was designed using (design – expert ® software version 8.0.7.1 / usa). this model is described by the following quadratic equation: y = t0 + t1x1 + t2x2 + t3x3 + t12x1x2 + t13x1x3 + t23x2x3 + t11x1 2 + t22x2 2 + t33x3 2 (eq. 1) where y is the measured response associated with each factor level combinations; t0 is the intercept and t1 to t33 are the regression coefficient computed from the observed value of y; x1, x2 and x3 are coded level of independent variables. the xixj (i = 1, 2 or 3 and j = 1, 2 or 3) and xi 2 (i = 1, 2 or 3) are interaction and quadratic terms, respectively. the independent factors selected were tp (x1), gb (x2) and px (x3). the dependent responses studied were the % ee (y1), mmad, daer (y2), zp, ξ (y3), % fpf (y4) and % de420 (y5). box – behnken design exhibit important advantages in comparison with other experimental designs: three factors are needed, and only twelve runs plus three replicates at the center point are required, costing less time and energy, each factor is studied and coded at three basic levels and it does not concern factors at extremely high or extremely low levels to avoid experiments in extreme conditions under which undesirable results might occur (17) . the composition of the box – behnken experimental design / factors levels are shown in (table 1). preparation of theophylline nanoparticles theophylline 250 mg and stearic acid 75 mg were dissolved in a mixture of dimethylformamide: ethanol, 45: 5 ml and then allowed to mix overnight. the solution was added to a beaker contained distilled water 50 ml via a microsyringe under sonication using a probe sonicator (vibra – cell tm / usa) at 35% amplitude – 20 second cycles for 6 minutes. the resulting nanosuspension was stored at – 80 ° c freezer (gfl ® / germany) and then lyophilized using a freeze dryer (labconco ® / usa). directly after nanosuspension preparation, sample of 2 ml was added to the quartz cell of the photon correlation spectroscopy ( malvern iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 67 zetasizer 1600 / uk) for zeta potential, mean particle size diameter and polydispersity index measurements. after lyophilization, the recovery was calculated using the following equation: recovery = wp wi x 100 (eq. 2) were wp – produced solid powder weight after lyophilization, wi – initial powder weight added of both tp and sa (18) . preparation of theophylline solid lipid microparticles the process parameters were optimized for the preparation of microparticles. theophylline nanoparticles (tp – nps) weight equivalent to tp (0.025, 0.050 or 0.100 gm) was added to a beaker containing molten gb (1.25, 2.50 or 5.00 gm) on a hot plate (favorit / malaysia) at 10 ° c above gb melting point. the mixture was then subjected to high shear homogenization using (x 120 cat / germany) at 15000 rpm for 3 minutes to allow dissolution / dispersion of tp. the still molten mass was then rapidly cooled at – 20 ° c freezer (hitachi / japan) for 2 days. the solidified mass was thoroughly grounded in a mortar and the obtained microparticles were dispersed in a 4 ° c – 25 ml aqueous phase of (1, 2 or 3 % w / v) px, homogenized at 15000 rpm for another 3 minutes and finally sonicated at 70 % amplitude – 20 second cycles for 12 minutes for cavitation forces to aid in further size reduction. the obtained suspension was stored at – 80 ° c freezer and then lyophilized to obtain water free microparticles (19) . entrapment / loading efficiency (% ee – y1) solid lipid microparticles weight approximately equivalent to tp 2 mg from each formula was washed on a filter with distilled water to remove the uncoated drug particles. then the washed microparticles were added to 100 ml of distilled water heated up to 10 º c above excipients melting point on a hotplate magnetic stirrer using (daihan hotplate stirrer / korea) and then stir at 1500 rpm for 5 minutes to extract tp. after being cooled to room temperature, the extract is filtered through 0.45 µm syringe filter and the content was determined spectrophotometrically at 274 nm against filtered extract from tp free solid lipid microparticles as a blank using spectrophotometer (hitachi / japan). the entrapment efficiency (ee) and the loading efficiency (le) were calculated by the following equations: table 1: box–behnken experimental design/factorslevels formulation tp (gm, x1) gb (gm, x2) px (% w / v, x3) f1 +1 0 +1 f2 -1 0 +1 f3 +1 0 -1 f4 0 +1 -1 f5 -1 +1 0 f6 +1 +1 0 f7 0 0 0 f8 0 +1 +1 f9 0 -1 -1 f10 +1 -1 0 f11 -1 0 -1 f12 0 0 0 f13 0 0 0 f14 -1 -1 0 f15 0 -1 +1 factor level (-) level (0) level (+) x1 0.025 0.050 0.100 x2 1.25 2.50 5.00 x3 1 2 3 ee = wf wi x 100 (eq. 3) where wf – tp weight in the finished microparticles and wi – tp weight initially added in the formulation. le = wtp wm x 100 (eq. 4) where wtp – tp weight in the microparticles and wm – microparticles weight (20) . mass median aerodynamic diameter (mmad, daer – y2) the solid lipid microparticles mass median aerodynamic diameter (mmad, daer) for iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 68 each prepared formula was calculated by the following equation: daer = d x 𝜌 𝜌1 (eq. 5) where daer – aerodynamic diameter, d – mass mean geometric diameter, ρ – particles density and ρ1 – reference density (1 gm / cm 3 ) (21) . the mean mass geometric diameter (d50) for each formula was measured by diluting a certain amount of solid lipid microparticles with distilled water, sonicated for 1 minute and then added to the sample dispersion unit of the laser diffractometer (malvern mastersizer 2000 / uk) (22) . the particles density (ρ) measurement was done by placing a certain amount of microparticles in a graduated cylinder, heated up to 10 ° c above excipients melting point and after being cooled to room temperature the final volume measured, according to the following equation: ρ = m v (eq. 6) where ρ – density of the solid lipid microparticles, m – weight of the solid lipid microparticles and v – volume of the molten lipid microparticles at room temperature (23) . zeta potential (zp, ζ – y3) after samples being diluted with distilled water and sonicated for 1 minute, the zeta (ξ) – potential is determined automatically by the photon correlation spectroscopy, by placing the solid lipid microparticles suspension in an electric field and measuring their mobility which is then related to the ξ at the interface, using the smoluchowski equation: ξ = ue η ε (eq. 7) where ξ – zeta potential (mv), ue – electrophoretic mobility (cm / sec), η – medium viscosity (centipoise) and ε – dielectric constant (24) . fine particles fraction (% fpf – y4) in order to evaluate the in vitro aerosolization efficiency of the solid lipid microparticles; andersen cascade impactor (aci) (graseby / usa) was used. the cascade impactor consists of a throat, a preseprator and eight stages of impaction plates (0 – 7) with cutoff aerodynamic diameters of 9, 5.8, 4.7, 3.3, 2.1, 1.1, 0.65 and 0.43 µm respectively. the impaction plates were precoated with a 2 % w / v of hydroxypropylmethylcellulose (4000 cps) gel in water to prevent particles bouncy and re – entrainment. a size ―2‖ hard gelatin capsules were filled with 20 mg powder from each prepared sample and aerosolized using rotahaler ® (cipla / india). the inhalation test was performed at an inhalation rate of 28.3 l / minute for 10 seconds. the fine particles fraction, which is total percentage deposited at stage 2 – 7 of the cascade impactor was used to evaluate aerosol performance using the following equation: fpf = fpd td x 100 (eq. 8) where fpf – fine particles fraction, fpd – fine particles dose (i.e., total weight of the solid lipid microparticles with size ≤ 5 µm) and td – total dose weight of the solid lipid microparticles delivered from the mouthpiece of the inhaler into the apparatus (25) . dissolution efficiency (% de420 – y5) the in vitro release was performed using franz diffusion cell system (permegear / usa). the receiver compartment was filled with 21 ml phosphate buffer ph 7.4, maintained at 37 ± 0.5 º c and stirred magnetically at 100 rpm. a cellulose acetate membrane (0.45 µm pore size and 2.5 cm 2 surface area) was inserted between the donor and the receptor compartments. a suitable aliquot of the solid lipid microparticles equivalent to tp 0.4 mg from each prepared sample was evenly spread on the cellulose acetate membrane that is pre – moistened with phosphate buffer ph 7.4 containing 0.1 % w / v tween 80 as a wetting agent and was occluded with a paraffin film. at time intervals of (5, 10, 15, 30, 60, 120, 180, 240, 300, 360 and 420 minutes), 3 ml aliquots of the receptor fluid were withdrawn, replaced by an equal volume of fresh medium and assayed spectrophotometrically against phosphate buffer ph 7.4 as a blank (26) . relying on correction for sampling, tp cumulated amount released q was calculated using the following equation: q = vs x cn − 1 n n =1 + vm x cn (eq. 9) where vs – volume of sample withdrawn, cn – 1 – drug concentration of the sample and vm – volume of the receptor medium (27) . the magnitude percentage of dissolution efficiency at 420 minutes (% de420) for each prepared sample was calculated from the area under the curve at time t and measured using the trapezoidal rule and expressed as a percentage of the area of the rectangle described by 100 % dissolution in the same time using the following equation (28) : % de420 = ( y x dt t 0 y 100 x t ) x 100 (eq. 10) the release data of tp formulas was fitted into various release kinetic models (29) (table 2). iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 69 table 2: mathematical models and dissolution mechanisms describing release kinetics. where % r – cumulative % tp released at time t, k – rate constant of the model and n – release exponent checkpoint analysis the checkpoint analysis was performed to confirm the reliability of the equations and plots in responses prediction. independent variables values were taken at three points / levels (0, 0.5, 0.5), (0.5, 0, 0.5) and (0.5, 0.5, 0). solid lipid microparticles formulations at these three checkpoints were prepared experimentally as shown in (table 3) and evaluated for the responses by analyses in triplicate to ensure reproducibility (30) . table 3: checkpoint formulas formulas x1 x2 x3 f16 0 -0.5 0.5 f17 0.5 0 -0.5 f18 -0.5 0.5 0 optimization of the solid lipid microparticles preparations in order to get the optimized formula (fo), the desirability function was run using design – expert ® version 8.0.7.1 software. the optimum formula was based on list criteria of maximum entrapment / loading efficiency, mass median aerodynamic diameter (mmad ≤ 5 µm), zeta potential (≥ ± 30 mv), maximum fine particles fraction and maximum cumulative percentage release in 420 minutes. differential scanning calorimetry thermal analyses were performed using differential scanning calorimeter (dsc – perkin elmer / usa). under nitrogen flow of 20 ml / minute, approximately 2 mg of pure tp, gb, sa, tp – nps, tp – gb physical mixture (1: 3) and fo was placed in a crimp – sealed aluminum pan and heated from 10 – 300 º c at a scanning rate of 10 º c / minute. an empty aluminum pan was used as a reference. fourier transform infrared spectroscopy fourier transform infrared spectroscopy was performed using (ftir – perkin elmer / usa) in order to characterize the possible interactions between the drug and the carrier in the solid state. samples of pure tp, gb, sa, tp – nps, tp – gb physical mixture and fo were prepared according to the kbr disc method. the scanning range was 4000 – 450 cm – 1 and the resolution was 4 cm – 1 . scanning electron microscopy the scanning electron microscopy analyses were performed using (fesem – fei / netherland). samples of pure tp, tp – nps and fo were mounted onto aluminum stubs and coated with a thin platinum layer. the scanning electron microscope was operated at an acceleration voltage of 5 kv and working distance of 7 – 10 mm. x – ray powder diffraction the powder x – ray diffraction patterns were recorded using (rigaku xrd / japan), under the following conditions: target cukα monochromatized radiation, voltage 40 kv and current of 20 ma at ambient temperature. the data of pure tp, gb, sa, tp – nps, tp – gb physical mixture and fo were collected in the continuous scan mode from 2 º – 40 º (2θ) at an angular increment of 0.02 º / second and count time of 1second / step. results and discussions theophylline nanoparticles characterization stearic acid was chosen as a stabilizer because it is naturally found in a small amount in the surfactant layer lying the lung epithelium, solid at room temperature and an amphiphilic surfactant that will act as an interface between tp – nps and water phase (31) . the tp – nps characterizations are shown in (table 4) and (figure 1). table 4: theophylline nanoparticles racterizations model mechanism equation zero – order concentration independent % r = k0 t (eq. 11) first – order concentration dependent % r = 100 (1 – e – k 1t) (eq. 12) higuchi diffusion % r = kh t 0.5 (eq. 13) hixson – crowell erosion % r = 100 (1 – (1 – khc t 4.6416 )) (eq. 14) korsmeyer – peppas diffusion % r = kkp t n (eq. 15) ζ (mv) d50 (nm) pi %recovery 41.205 ±1.453 722.040 ±6.677 0.558 ±0.031 75.361 ±16 iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 70 figure 1: tp – nps size distribution – size statistics report by intensity theophylline solid lipid microparticles preparation the rapid cooling leads to formation of solid solution (homogenous distribution) of drug in lipid matrix. drug release takes place by diffusion from the matrix and upon lipid degradation in vivo (figure 2) (32) . figure 2: three incorporation models of drugs in the solid lipid matrix table 5: observed responses entrapment / loading efficiency (% ee – y1) the entrapment efficiency (ee) varies from 34.762 % (f14) to 95.989 % (f3) for various factors level combinations (table 5). the independent factors affecting the ee were tp (x1) and px (x3) levels (p < 0.05, tables 6 – 7) and (figure 3). tp has a significant – positive effect on the ee. although tp hydrophilicity favors the external aqueous phase, its ees in some formulations were high, because tp localized at the interface (i.e., solid lipid microparticles surfaces) will induce outer surface saturation at higher concentrations and besides the internal phase viscosity (i.e., gb solidity due to its high melting point) will reduce tp leakage by reducing the external aqueous phase entrance into the lipid matrix (33, 34) . figure 3: response surface contour plot (up) and 3d plot (down) for tp and px effect on ee. formulation % ee (y1) mmad daer µm (y2) zp ξ mv (y3) % fpf ≤ 5 µm (y4) % de420 (y5) f1 75.964±5.463 4.849±0.456 -34.366±1.187 36.792±1.055 73.782±5.533 f2 47.596±6.458 4.902±0.227 -26.850±1.282 35.816±1.773 44.156±3.109 f3 95.989±4.146 6.168±0.083 -32.066±0.375 24.725±0.800 75.189±6.496 f4 67.017±7.147 6.514±0.149 -23.566±0.301 23.742±1.606 50.547±4.601 f5 51.578±4.542 6.104±0.229 -22.450±1.837 34.598±0.888 47.786±2.512 f6 85.263±3.917 6.225±0.196 -31.316±0.957 33.808±0.862 76.210±4.414 f7 56.428±5.931 6.232±0.209 -27.300±0.312 32.466±0.781 52.135±3.791 f8 41.578±3.605 5.278±0.478 -25.433±0.301 38.736±0.885 38.629±1.254 f9 78.247±5.174 6.456±0.106 -28.216±0.028 24.674±0.596 70.089±4.460 f10 83.789±6.955 6.099±0.064 -30.683±0.161 35.878±0.962 77.708±6.095 f11 58.754±5.777 6.354±0.205 -27.733±0.189 22.677±0.808 46.812±3.213 f12 55.357±5.896 6.181±0.208 -28.166±0.602 32.343±0.503 51.971±4.218 f13 55.714±5.786 6.283±0.211 -27.983±0.425 32.905±0.639 51.553±3.989 f14 34.762±4.301 6.256±0.213 -20.200±0.100 34.615±1.356 42.878±2.214 f15 39.368±3.568 5.115±0.224 -25.916±1.537 37.703±1.114 47.799±2.121 iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 71 table 6: statistical regression analysis table 7: analysis of variance (anova) *probability > f is the significance level and a value < 0.05 considered significant px has a significant – negative effect on the ee. tp becomes partly negatively charged at ph < 9 (i.e., neutral ph) and this result in the absence of attractive electrostatic interactions with px polyethylene oxide chains. in addition tp characteristic of ph independent solubility will precludes its hydrophobic interactions with the px hydrophobic cores and the px swelling upon hydration, all result in tp within the px layer release increment (35) . the effects on y1 can be explained by the following quadratic equation: y1 = 62.22 + 18.24x1 + 0.86x2 – 11.75x3 – 1.79x1x2 – 0.7x1x3 + 1.84x2x3 + 4.88x1 2 – 3.25x2 2 + 3.12x3 2 (eq. 16) there is an inverse relationship between the aqueous surfactant concentration and the sonication time but to a certain limit, because less energy will be required to achieve maximum dispersion of the produced solid lipid microparticles. any increase in px concentration will not aid in coverage, in contrary will lead to an increment in the micelles formation which has more negative effect on tp – solid lipid microparticles ee (36) . mass median aerodynamic diameter (mmad, daer – y2) the aerodynamic diameter is the diameter of a sphere of unit density, which reaches the same velocity in the air stream as a non – spherical particle of arbitrary density and expresses the mechanism of particle deposition in the iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 72 respiratory system (37) . the mass median aerodynamic diameter [mmad, daer] ranged from 4.849 µm (f1) to 6.514 µm (f4) (table 5) with the selected levels of variables. the most factors affecting the mmad are px (x3 and x3 2 ) and tp (x1 2 ) (p < 0.05, tables 6 – 7) and (figures 4 5). px has a significant – negative effect on the mmad. figure 4: response surface contour plot (up) and 3d plot (down) for tp and px effect on mmad generally, independent on the px molecular weight, the preserving of the mmad values is due to the px efficient coverage of the surfaces and sterically stabilizing microparticles by its long hydrophilic polyethylene oxide chains (poloxamer 188 average number of ethylene oxide ≈ 153 units) extend into solution and shield the particles surfaces preventing their agglomeration during the homogenization and sonication processes. also, these hydrophilic arms will potentially influence the shape of the resulting particles (38) . tp has a significant – negative effect on the mmad. this is attributed to the concentration effect of the non – incorporated tp molecules during lyophilization results in the formation of highly concentrated solutions of tp that its ionization will affect the zp and particles agglomeration. (39) . y2 can be described by the following quadratic equation: y2 = 6.24 – 0.024x1 + 0.035x2 – 0.66x3 + 0.061x1x2 + 0.026x1x3 + 0.034x2x3 – 0.17x1 2 + 0.11x2 2 – 0.49x3 2 (eq. 17) a part of the px covering layer of the solid lipid microparticles surfaces removed, a reduction in the repel force impart by px steric hindrance, enhance particles agglomeration through van der waals forces of attraction between particles and hence there will be an increase in the mean solid lipid microparticles size after freeze drying process (40) . figure 5: particle size distribution of the fo zeta potential (zp, ζ – y3) the zeta (ξ) – potential (zp) is the electrostatic potential at the boundary dividing the compact layer (charged solid surface – immobile conuterions) and the diffuse layer (liquid counterions – mobile counterions) (41) . the zp (> 30 or < 30 mv), indicates electrostatic repulsion among particles and good stability (42) . the zp values varied from – 34.366 mv (f1) to – 20.200 mv (f14) (table 5). tp (x1) and gb (x2 2 ) have statistical influential effects on the zp (p < 0.05, tables 6 – 7) and (figures 6 – 7). tp has a significant – negative effect on the zp. this is attributed to the free not incorporated tp that will be partly negatively charged at neutral ph and the increase in the zp will be in the negative direction (43) . figure 6: zeta potential distribution of the fo iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 73 figure 7: response surface contour plot (up) and 3d plot (down) for tp and gb effect on zp gb has a quadratic significant – positive effect on the zp. as gb content increased, there will be a reduction in the solid lipid microparticles ph together with zp increment in the positive direction due to the solid lipid microparticles surfaces tp protonation at low ph (ph < 4) (44) . both factors effects on y3 are shown in the quadratic equation below: y3 = 29.38 – 3.94x1 + 0.24x2 – 0.43x3 – 0.22x1x2 – 0.88x1x3 – 0.96x2x3 – 0.12x1 2 + 3.34x2 2 – 1.04x3 2 (eq. 18) the negative charge of the solid lipid microparticles core matrixes are involved in electrostatic interaction with the weakly basic tp molecules. thus, the solid lipid microparticles surfaces negative charge is the main contributor to the negative zp of the solid lipid microparticles (45) . the changes in the interfacial properties will have an influential effect on the zp and hence solid lipid microparticles size. this is due to the px layer covering the solid lipid microparticles surfaces will reduce the zp but will provide steric stability instead if solid lipid microparticles being efficiently covered with px (46) . fine particles fraction (% fpf – y4) andersen cascade impactor (aci) is a primary technique used for both development and testing of the inhaler products. size determination is based on the inertial impaction of aerosolized particles passing through decreasing nozzle apertures onto subsequent deposition stages each with a defined aerodynamic cut – off diameter (47) . the fine particles fraction (fpf) is lowest at 22.677 % (f11) and highest at 38.736 % (f8) (table 5). px (x3 and x3 2 ) and gb (x2 2 ) are the most factors affecting fpf (p < 0.05, tables 6 – 7) and (figure 8). simply by decreasing the cohesive forces between particles a lower aggregation tendency might gain and consequently lower surface energy was necessary to increase the flowability and finally the powder fpf. also; by reducing the particle density and tolerating an increase of the average particle size will enhance the aerosol efficiency. px has a significant – positive effect on the fpf. px percentage used has a special role concerning particles morphology, dispersibility and flowability. as px concentration increased, the fpf increased up to a certain limit that beyond highly charged particles produced (48) . figure 8: response surface contour plot (up) and 3d plot (down) for gb and px effect on fpf although gb has a quadratic significant – positive effect on the fpf, but it is a slight increment and only at higher gb concentration and that is shown from the surface truncated shape of the 3d plot for the response y4 range. effects of factors on response y4 explained by the following quadratic equation: iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 74 y4 = 32.37 + 0.33x1 – 0.35x2 + 6.69x3 – 0.64x1x2 – 0.34x1x3 + 0.56x2x3 + 0.37x1 2 + 1.99x2 2 – 3.08x3 2 (eq. 19) gb enables the production of low density aerodynamically inhalable particles and thus improving their respirable fraction by avoiding the natural clearance mechanism of the lungs (e.g., alveolar macrophage upake) due to the higher geometric diameter of the particles (49) . dissolution efficiency (%de420 – y5) the cumulative percentage of tp release at 420 minutes (% de420) varied from 38.629 % (f8) to 77.708 % (f10) (table 5).tp (x1) is the only factor affecting the % de420. tp has a significant – positive effect on the % de420 (p < 0.05, tables 6 – 7) and (figure 9). concerning tp effect only; tp release is biphasic, the first phase was a slight burst release due to the free non – incorporated tp on the solid lipid microparticles surfaces followed by prolong release due to the incorporated stable crystalline anhydrous tp (50) . figure 9: response surface contour plot (up) and 3d plot (down) for tp and gb effect on %de420 concerning gb non – significant – negative effect on the % de420 is due to the cold homogenization technique employed in the solid lipid microparticles production which results in a solid solution drug incorporation model and gb low crystallization degree. therefore, tp release rate will be prolonged for some formulations because the drug which is molecularly dispersed in the colloidal particles has a limited motion (51) . percentage tp released in 420 minutes can be described by the following quadratic equation: y5 = 55.23 + 15.15x1 – 3.17x2 – 4.3x3 – 0.04x1x2 + 1.56x1x3 + 1.35x2x3 + 4.15x1 2 + 1.77x2 2 – 0.64x3 2 (eq. 20) the reasonable explanation for the prolonged release is the three steps govern tp release from solid lipid microparticles; entrance of the dissolution medium into the solid lipid microparticles matrixes, dissolution of the dispersed tp particles / crystals and diffusion of the dissolved tp through the inert solid lipid microparticles matrixes (52) . checkpoint analysis three checkpoint formulations were prepared and evaluated for dependent responses (table 8). comparing the predicted and experimental values using student t – test, the differences were found to be insignificant (p > 0.05) indicate that the obtained mathematical equation is valid for predicting the dependent responses values. (eq. 21) was used in the percentage relative error calculation between the experimental and predicted values of each response: % relative error = 𝑷𝒓𝒆𝒅𝒊𝒄𝒕𝒆𝒅 𝑽𝒂𝒍𝒖𝒆−𝑬𝒙𝒑𝒆𝒓𝒊𝒎𝒆𝒏𝒕𝒂𝒍 𝑽𝒂𝒍𝒖𝒆 𝑷𝒓𝒆𝒅𝒊𝒄𝒕𝒆𝒅 𝑽𝒂𝒍𝒖𝒆 x 100 (eq. 21) optimization of the solid lpid microparticles preparations comprehensive search through desirability function revealed that the fo with 0.889 desirability has the composition of 0.1 gm tp, 1.27 gm gb and 3 % w / v px. by preparing the fo, the experimental responses are in good agreement with the predicted values (table 9). iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 75 table 8: checkpoint formulations comparing experiemntal / predicted values (n = 3) no. ex. pr. % re f16 % ee y1 60.187±3.074 56.740 0.060 mmad daer µm y2 6.025±0.435 6.634 0.091 zp ζ mv y3 -25.480±0.314 -23.399 0.088 % fpf y4 28.356±0.367 32.225 0.120 %de420 y5 67.344±3.773 61.142 0.101 f17 ex. pr. % re % ee y1 87.434±4.933 81.055 0.078 mmad daer µm y2 7.234±0.763 6.139 0.178 zp ζ mv y3 -35.132±1.145 -30.434 0.154 % fpf y4 17.315±0.310 15.727 0.100 %de420 y5 53.640±2.206 58.342 0.080 f18 ex. pr. % re % ee y1 54.345±2.629 45.132 0.204 mmad daer µm y2 6.431±0.173 5.985 0.074 zp ζ mv y3 -27.340±1.415 -24.156 0.131 % fpf y4 29.346±0.381 32.637 0.100 %de420 y5 38.343±1.146 46.635 0.177 table 9: optimized formula experiemntal / predicted values (n = 3) f o ex. pr. % re % ee y1 76.246±3.582 71.923 0.060 mmad daer µm y2 6.035±0.463 4.890 0.234 zp ζ mv y3 -29.734±2.509 -31.580 0.058 % fpf y4 35.819±1.343 38.731 0.075 %de420 y5 71.069±2.136 74.723 0.048 mechanism of dissolution the regression parameters obtained after fitting various release kinetic models to the in vitro dissolution data are listed in (table 10). the fit for various models investigated for drug release of the fo can be arranged in the following descending order: higuchi > korsemeyer – peppas > zero – order > hixson – crowell > first – order. the exact mechanism of release is non fickian or anomalous from the slope value of korsemeyer – peppas which is lie in the range of (0.450 < n < 0.890). the in vitro release profiles of tp, tp nps and fo are shown in (figure 10). the low release percentage of crude tp powder is due to the larger particle size in comparison with the tp – nps and fo. table 10: release kinetic parameters of the fo figure 10: in vitro release profiles of tp, tp – nps and fo differential scanning calorimetry (figure 11) shows the dsc of pure tp, gb, sa, tp – nps, tp – gb physical mixture and fo endothermic peak at 274 ° c, 77.41 ° c, 69.30 ° c, 64.83 – 272.20 ° c, 75.11 – 270.38 ° c and 76.18 ° c respectively. the endothermic peak corresponding to the melting point of tp is reduced to 272.20 ° c in the dsc thermogram of tp – nps and 270.38 ° c in the dsc thermogram of tp – gb physical mixture respectively, and this indicated that in tp – nps there is a lack of significant changes in tp crystalline state, whereas in the tp – gb physical mixture indicates tp saturation in the carrier. the disappearance of tp melting peak in the fo was noticed due to its percentage of (≈ 5 % w / w) so it is lowered, broadened and becomes dsc undetectable (53) . model slope intercept r 2 zero – order 0.179 12.510 0.916 first – order 0.003 0.933 0.578 higuchi 3.982 -0.952 0.990 hixson – crowell 0.006 2.013 0.633 korsmeyer – peppas 0.687 0.187 0.966 iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 76 figure 11: differential scanning calorimetry thermograms of tp (a), gb (b), sa (c), tp – nps (d), tp – gb physical mixture (e) and fo (f) fourier transform infrared spectroscopy as shown in (figure 12), the ftir spectrum of pure tp, gb, sa, tp – nps, tp – gb physical mixture and fo. the spectrum of tp shows characteristic peaks at 1710 cm -1 and 1665 cm -1 attributable to the (c = o stretching vibrations). the band at 1563 cm -1 is attributable to the (pyrimidine ring stretching vibration) (54) . the gb shows distinctive bands at 2916 cm -1 and 2849 cm -1 attributable to asymmetric and symmetric (aliphatic ch2 stretching vibrations). the band at 1737 cm -1 is attributable to the (ester c = o stretching vibration) (55) . the spectrum of sa resembles that of gb with little shift, i.e. the asymmetric and symmetric (aliphatic ch2 stretching vibrations) will be at 2920 cm -1 and 2851 cm -1 respectively, whereas the (ester c = o stretching vibration) will be at 1703 cm -1 . the spectrum of tp – nps still showed the sa 2920 cm -1 and 2851 cm -1 bands, whereas 1703 cm -1 disappear due to the tp 1710 cm -1 and 1665 cm -1 bands in the same region. the spectrum of tp – gb physical mixture still showed the gb bands at 2916 cm -1 , 2849 cm -1 and 1737 cm -1 , whereas tp 1710 cm -1 , 1665 cm -1 and 1563 cm -1 still shown with different intensity as a consequence of tp existence on the microparticles surfaces. the spectrum of fo shows gb bands at 2916 cm -1 , 2849 cm -1 and 1737 cm -1 , whereas tp band at 1563 cm -1 is the only shown due to the tp bands at 1710 cm -1 and 1665 cm -1 close proximity with respect to the gb 1737 cm -1 band. scanning electron microscopy the scanning electron microscope images for pure tp, tp – nps and fo are shown in (figure 13). both pure tp and tp – nps show a prismatic tp crystal habit, whereas the fo image shows irregular shape particles (56) . this irregularity is due to sonication done at 4 ° c by using an ice bath to prevent lipid melting by heat generated and so drug loading and homogeneity maintained. x – ray powder diffraction the xrd patterns of pure tp, gb, sa, tp – nps, tp – gb physical mixture and fo are shown in (figures 14). pure tp diffractogram has an important distinctive peak of high intensity at 12.63 ° (2θ), another two small and isolated peaks are present at 7.11 and 14.33 ° (2θ), whereas a group of small peaks covers the range 20 – 30 ° (2θ). gb has a high intensity peak at 21.22 ° (2θ) and a smaller one at 23.43 ° (2θ). sa has a high intensity peak at 2.22 ° (2θ) and two successive peaks at 21.5 ° (2θ) and 23.8 ° (2θ). tp – nps, tp – gb physical mixture and fo diffractograms still showed tp, sa and gb peaks but at reduced intensity. although tp melting peak disappearance in the fo corresponding dsc curve, tp isolated peaks in the above diffractograms confirms its crystalline state. iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 77 figure 12: fourier transform infrared images of tp (a), gb (b), sa (c), tp – nps (d), tp – gb physical mixture (e) and fo (f) figure 13: scanning electron microscopy images of tp ( a), tp – nps (b) and fo (c) iraqi j pharm sci, vol.22(1) 2013 theophylline pulmonary delivery system 78 figure 14: powder x – ray diffraction spectra of tp (a), gb (b), sa (c), tp – nps (d), tp – gb physical mixture (e) and fo (f) conclusions the selected variables main and interaction effects on the critical quality attributes of the inhalable solid lipid microparticles were determined by a box – behnken design of 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american association of pharmaceutical scientists journal 2009; 11(4): 771 – 777. http://www.sciencedirect.com/science/journal/13861425 http://www.sciencedirect.com/science/journal/13861425 http://www.sciencedirect.com/science/journal/13861425 iraqi j pharm sci, vol.18(2) .2009 genetic variations of echinococcus granulosus 61 genetic variations of echinococcus granulosus isolated from sheep and cows by using fingerprint dna method in iraq zaman a.a. ibrahim * ,1 , salwa s.muhsin ** and farhan a. risan *** * department of basic medical sciences, college of nursing, university of baghdad, baghdad, iraq, ** department of x ray, institute of medical technology, foundation of technical education, baghdad, iraq, *** department of medical pathological technology , college of health and medical technology ,baghdad , iraq. abstract the fingerprinting dna method which depends on the unique pattern in this study was employed to detect the hydatid cyst of echinococcus granulosus and to determine the genetic variation among their strains in different intermediate hosts (cows and sheep). the unique pattern represents the number of amplified bands and their molecular weights with specialized sequences to one sample which different from the other samples. five hydatitd cysts samples from cows and sheep were collected, genetic analysis for isolated dna was done using pcr technique and random amplified polymorphic dna reaction(rapd) depending on (4) random primers, and the results showed: 1-ability of opf-13 primer to find fingerprinting for dna of hydatid cysts collected from cows with (3) unique patterns and from sheep with (5) unique patterns. 2ability of opf-06 primer to found (4) unique patterns for cows and (2) unique patterns for sheep. 3-ability of opf-19 primer to find fingerprinting for dna of hydatid cysts collected from cows (5) unique patterns and (4) unique patterns for sheep. 4-opf-16 was unable to find the finger prints of dna isolated from cows and sheep keywords : hydatid cyst. opf primer .pcr. rapd. صة الخال لذ اسخخذهج في ُزٍ unique patternالخي حعخوذ على الٌسك الوخويض fingerprinting dnaاى طشيمت البظوت الْساثيت الذساست لخشخيض طفيلي األكيبط ألعذسيَ ّهعشفت االخخالفبث الْساثيت في هضبئفَ الْسطيت الوخخلفت "األغٌبم ّاألبمبس" . أر يوثل عذد الحضم الٌبحجت هع أّصاًِب الجضيئيت الخبطت ّألخخببع ألخبص ببلعيٌت ّالخي حويضُب عي بميت العيٌبث ليذ الذساست ّالخي الٌسك الوخويض كبًج حشخول على خوست عيٌبث لكل هي األكيبط العذسيَ ألوعضّلَ هي األغٌبم ّاألبمبس . ّبعذ ححليل العيٌبث ببسخخذام حمبًت الـ rapd ببدئبث عشْائيت. أظِشث الٌخبئج هب يلي 4ببالعخوبد على– ّبخٌسيك opf-6حن إيجبد بظوت ّساثيت للعيٌبث الخوست لذًب األكيبط العذسيَ الوعضّلت هي األغٌبم ّاألبمبس ببسخخذام الببدئ -١ للعيٌبث جويعب. unique patternهخويض ظوت ّساثيت لذًب عيٌبث األكيبط العذسيَ الوعضّلت هي األغٌبم ّبخٌسيك هخويض على الخْطل إلى إيجبد ب opf -13لذسة الببدئ -٢ ( أًسبق لألبمبس.۳بعذد خوست اًسبق ّ) على إيجبد بظوت ّساثيت لذًب عيٌبث األكيبط العذسيَ الوعضّلت هي الوضبئف الْسطيت األبمبس ّبٌسك opf -19لذسة الببدئ -٣ ( أًسبق لألغٌبم.4) ( ٥ّهخويض بعذد ) على إيجبد بظوت ّساثيت لذًب عيٌبث األكيبط العذسيَ الوعضّلت هي الوضبئف الْسطيت opf -16عذم لذسة الببدئ -٤ ّاألبمبس( الوسخخذهت في الذساست. األغٌبم ) introduction hydatidosis is a most serious zoonosis disease caused by larval stage hydatid cysts of tapeworms echinococcus granulosus which effect human and wide rang of livestock (1,2) and the ultimate growth of the cyst depends on location inside the host, so in some areas of the body they are unable to expand freely whereas in other most growth results in serious impairments to the function of vital structures or even in death (3) . these parasites live for long times in the hostile medium of the host and their struggle for life gave them various strategies that allow them to feed, reproduce and evade the immune system and immunosurveillance and promote chronic infection (4) .many methods were used for treatment of this disease by (surgically, chemotherapy, physically…etc), surgical remain the primary choice and method, others still ineffective or complementary) for controlling and inhibition of the disease distribution (5) , and many advanced laboratory methods were used for the diagnosis of this parasite in human and animals (6) . 1 corresponding author e – mail : zamanbrahim@yahoo.com received : 17 / 3 / 2009 accepted : 27/ 10 /2009 iraqi j pharm sci, vol.18(2) .2009 genetic variations of echinococcus granulosus 62 aim of the study the experimental studies at molecular level till now absent or hardly very rare about this parasite(adult and larval stage) in iraq , so this study was carried out using dna technique depending on polymerase chain reaction(pcr) method and strictly indicator random amplified polymorphic dna(rapd) to find out the fingerprinting which used to specify the sequences characterize the dna which isolated from cows and sheep because first of all ,the high sensitivity of this technique to determine the genetic variations between all animals hydatid cysts isolates, and secondly easy and perfect method to be used (7) . materials and methods. samples collection. genetic materials dna had been isolated from hydatid cysts specimens of cows and sheep which collected from different slaughterhouses in north, middle, and south of iraq during the period from june 2005 june 2006 design of experiments. the dna isolation was done as follow. one cm³ specimens from tissues germinal layer of hydatid cyst were isolate from cows and sheep & preserved into 70% ethanol with700µl of proteinase buffer was added {method was cited by (8) and minor modified by ( 9) .} the tissues had been cut by small and sharp scissor into small pieces, and then 35l of proteinase –k enzyme solution has been added and incubated for 24 hours at 55c in the incubator, ten µl rnaase enzyme solution has been added and incubated at 37c for 1hour, then the same volume of (chloroform, phenol, isomil alcohol) solution was added and mixed by vortex for 1 minute and centrifuged at 12000 rpm for 15 minutes. the upper layer pulled out and transferred to a clean, dry, tube, this process had been repeated many times till the middle white layer disappeared, then o.6 ml volume of isopropanol was added and then left at room temperature for 1hour and centrifuged at 1400 rpm for 10 minutes. the supernatants then removed and the precipitants washed with (70%, 95%) ethanol respectively, the tube left open to complete drying from ethanol, then the dna dissolved by 150 µl tris-edta (te) with ph 8 and incubated for 2 hours at 65 c to complete dissolving. then whole solution was kept at deep freeze (20c). after the dna characterization by measuring dna concentration and estimation of its purity ( 10, 11) using spectrophotometer with wave length 260nm, rapd was done according to (12) method which includes the following steps: 1the master mixture solution was prepared by mixing the following constituents in sterile plain tubes with following concentrations for each constituent: cows (5) samples sheep (5) samples final concentration volume of each sample constituents 424.8 354 17.7 distilled water 60 50 1 x 2.5 buffered solution x 10 12 10 200 µmole 0.5 dntps 08 40 pmol/r 2 primer 7.2 6 1.5µl 0.3 µl polymerase enzyme 2the constituents mixed well by vortex for 30 seconds and centrifuged for 30 seconds to precipitate all solution drops that suspended on tube wall. 3each tube which contained cows and sheep samples had been distributed in very sterile 0.5 ml eppendorf tube labeled with names of 5 hydatid cysts isolates of the study. 4two µl from each dna sample of both sheep and cows after performing several dilutions for the concentrated samples by d.w to reach the final required concentration starting the rapd reaction of dna chain which ranged between 10-50ngm / each 1 µl of dna sample and the final volume became 25µl (13) . 5the constituents were mixed and left for few minutes in centrifugation to collect the contents of reaction in the bottom of the tube, then 20-25µl of mineral oil to prevent the evaporation during the duplication process when the temperature reaches up to 94 c (14,15) . 6the tubes were transferred to thermocycler to start the duplication reaction according to the the following program:  one round for 2 minutes at 94 c for primary denaturation of dna strands .  forty duplicated rounds, each round for 2 minutes at 92 c to denatures the matrix ,1 minute at 36 c to bind the primers with dna matrix, and 2 minutes at 72 c for elongation of the bonded primers ,with last round for final elongation for 10 minutes at 72 c. iraqi j pharm sci, vol.18(2) .2009 genetic variations of echinococcus granulosus 63 7the tubes were removed from thermocycler, and volume of (23-24) µl from the contents was pulled from under the oil and mixed with 3 µl loaded solution. 8-agarose gel with concentration 1.2% was prepared and electrophoresis by well method was done on the samples with volumetric indicator formed from dna treated by (eco1) enzyme for 4-5 hours. 9the gel was examined after staining with ethidium bromide (1mg/ml) under ultra violet and black and white polaroid 667 photos were taken to the gel. 10the molecular weights of the duplicating segments were calculated depending on the bands positions with known molecular weights which produced from dna cutting enzyme (eco1) only that considered standard volumetric agents. four primers were tested. 1opf – 13 3' ggctgcagaa 5' 2-opf – 06 3' gggaattcgg 5' 3opf – 19 3' cctctagacc 5 4opf– 16 3' cgagtactgg 5' these primers were produced by (operon technologies alameda–a) to be used in final analysis of rapd experiments, and the results which appeared in the gel were converted into binary characters tables by putting number (1) if the band was found and number (0) if the band was not found and the molecular weights were calculated for the duplicated products in comparison with volumetric agent (eco1) only. in order to characterize each sample for the studied sample, we investigate the fingerprints or the duplicated bands and we determined the organized sequences characterization (14) . results and discussion: in iraq the studies about genetic variation between animals infected with hydatid cysts at molecular level almost very rare specially using fingerprint for identification. by using the four primers (opf 6, 13, 16, 19) the results showed their reactions in rapd technique that they were different in duplicated bands production and their molecular weights with each hydatid cyst dna samples taken from cows and sheep. the primer opf-13. this primer was able to find (3) types for unique patterns of dna which isolated from cows (figure 1a, table 1a) and ( 5 ) types for unique patterns of dna isolated from sheep (figure 1b, table 1b), and these results agree with the results conducted by (16) who they said that there are differences in sequences characterizes of duplicated bands and this primer gave a description for genetic material and genetic pattern of dna isolated from cows and sheep (17) . cows a no. of sample sheep b no. of sample figure 1: show the duplication production on agarose gel (1.2%) for hydatid cysts samples isolated from cows (a) and sheep (b) using opf-13 primer 3.5 hours and with 65 volts . m 1 2 3 4 5 m 1 2 3 4 5 iraqi j pharm sci, vol.18(2) .2009 genetic variations of echinococcus granulosus 64 table1: show the calculation method of sequences characterizes using primer opf13. a numbers of unique patterns (3) isolated from cows. (0) = duplicated bands not exist (1)=duplicated bands exit different colors means numbers of unique patterns. b number of unique patterns (5) for dna isolated from sheep. (0) = duplicated bands not exist (1) =duplicated bands exit different colors means numbers of unique patterns. the primer opf-6. this primer was able to found the fingerprint with (4) types of unique patterns for dna hydatid cysts which isolated from cows (figure 2a , table 2a) and ( 2) types for unique patterns of dna isolated from sheep ( figure 2b, table 2b)( 18) . figure 2 :show the duplication production on agarose gel (1.2%) for hydatid cysts samples isolated from cows (a) and sheep (b) using opf-6 primer for 3.5 hours and with 65volts ١ ٢ ٣ ٤ ٥ mw bp 8 8 1 1 1 ١011 8 8 1 1 1 ١٦11 8 1 1 1 1 ١١11 1 1 1 1 1 011 8 1 1 1 1 001 1 1 8 8 8 001 1 8 1 1 1 0٥1 1 1 8 8 8 0٣1 1 1 1 1 1 ٦01 1 1 1 1 1 ٦٣1 8 1 8 8 8 ٥٤1 1 8 8 8 8 ٤٢1 1 8 8 8 8 ٣01 1 1 1 1 1 ٣٦1 ١ ٢ ٣ ٤ ٥ mw bp 1 1 8 1 1 ١011 8 8 8 1 1 ١٦11 8 1 8 1 1 ١١11 1 1 1 1 1 011 1 8 1 8 1 001 1 1 8 8 8 001 1 1 1 8 8 0٥1 1 1 8 8 8 0٣1 8 1 1 1 1 ٦01 1 1 8 1 1 ٦٣1 8 1 1 8 8 ٥٤1 8 1 1 8 8 ٤٢1 8 1 1 8 8 ٣01 1 1 1 1 1 ٣٦1 cows a m 1 2 3 4 5 sheep b m 1 2 3 4 5 iraqi j pharm sci, vol.18(2) .2009 genetic variations of echinococcus granulosus 65 table2 : show the calculation method of sequences characterize using primer opf-6 a mw bp 1 2 3 4 5 1100 0 1 1 1 0 1000 1 0 0 1 0 990 1 1 1 1 1 800 1 0 0 0 0 770 1 1 1 1 1 650 1 1 1 1 1 450 1 1 1 1 1 numbers of unique patterns (4) for dna isolated from cows. (0) =duplicated bands not exist (1) =duplicated bands exist different colors means number of unique patterns. b mw bp 1 2 3 4 5 1100 0 0 1 1 0 1000 0 0 1 1 0 990 0 0 1 1 0 800 0 0 1 1 0 770 1 1 1 1 1 650 1 1 1 1 1 450 0 0 1 1 0 numbers of unique patterns (2) for dna isolated from sheep. (0)=duplicated bands not exist (1)= duplicated bands exist different colors means number of unique patterns. the primer opf -19 this primer showed (5) unique patterns for dna hydatid cysts samples of cows (figure 3a, table 3a), and (4) unique patterns for sheep dna hydatid cysts samples (figure 3b, table3b). it is clear from unique patterns number differences between the samples that the complementary loci for this primer which used in rapd indicators not evenly distributed between the samples (19) , and the appearance of unique pattern specific for cow's hydatid cysts dna represent fingerprint for these samples differentiated them from other samples by using this primer (20) . from this study, we could diagnosis the dna of sheep and cows hydatid cysts using rapd method with banding patterns and their molecular weights, so, by fingerprints the only three primers were able to diagnosis the genetic variations and differences between all hydatid cysts samples (21). no. of sample no. of sample figure 3: show the duplication production on agarose gel (1.2%) for hydatid cysts samples isolated from cows (a) and sheep (b) using opf-19 primer for 3.5 hours and with 65 volts . cows a sheep b m 1 2 3 4 5 m 1 2 3 4 5 iraqi j pharm sci, vol.18(2) .2009 genetic variations of echinococcus granulosus 66 table-3 show the calculation method of sequences characterizes using primer opf-19. a mw bp 1 2 3 4 5 1050 0 1 1 1 0 1000 0 1 1 1 1 980 8 1 1 8 8 860 8 8 1 1 8 750 1 1 1 1 1 630 0 1 1 8 1 numbers of unique patterns (5) for dna isolated from cows. (0) =duplicated bands not exist (1)= duplicated bands exist different colors means numbers of unique patterns. b mw bp 1 2 3 4 5 1050 0 1 0 0 0 1000 1 1 1 1 1 980 1 1 1 1 1 860 8 1 1 1 8 750 8 1 1 1 1 630 0 1 1 1 1 numbers of unique patterns (4) for dna isolated from sheep. (0) =duplicated bands not exist (1)= duplicated bands exist different colors means numbers of unique patterns. the primer opf-16 this primer had no ability to found hydatid cyst dna and gave them unique patterns by finger prints for cows and sheep, this primer failed to recognize the genetic variations by using rapd reaction and fingerprints method in the presence of hydatid cysts sample which had no broad genetic background and its high recognition ability failed to show a unique pattern of the studied samples (18) . acknowledgements sincerely, we must gift our deep thanks to all staff members in all baghdad–city slaughterhouses for their great help to obtain intact hydatid cyst with our best heart wishes for them. deep thanks to all members of department of medical bio-technology institute /university of baghdad for their great help. references 1. mcmanus, d.p.; zhang. w.; li, j. and bartley p.b. echinococcosis. lancet.2003.362: 1295 1304. 2. zhang,w.and mcmanus,d.p. recent advances in the immunology and diagnosis of echinococcosis . fems immunol . med .microbiol .2006. 47:2441. 3. john,d.t. and petri, w.a. markell and voge's.medical parasitoloy. 9 th ed ,saunders (elsevier).philadelphia.2006. pp. 224231. 4. rigano , r.;buttari,b .; profumo , e .; ortona ,e.; delunardo , f . ; margutti ,p .; mattei ,v .; teggi , a; sorice , m . and siracusano , a. echinococcus granulosus antigen b impairs human dendritic cell differentiation and polarizes immature dendritic cell maturation towards a th1 and th2 cell response . infect.immun.2007.75 (4):1667-1678. 5. ammann,r. and eckert,j. clinical diagnosis and treatment of echinococcosis in humans.in:echinococcus and hydatid disease(thompson,r.c.a.and lymbery,a.j.ed): cab international, wallingford, u.k. 1995. pp. 411-463. 6. mckerrow, j. parasitic disease. in: medical immunology. 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mmooddiiffiieedd vveerrssiioonn wwaass uusseedd ffoorr tthhee qquuaannttiittaattiivvee ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee ((aarrgg)) aanndd ggllyycciinnee (( ggllyy)) iinn aarrggiinniinnee aacceettyyll ssaalliiccyyllaattee –– ggllyycciinnee ccoommpplleexx .. aaccccoorrddiinngg ttoo tthhiiss mmeetthhoodd ttwwoo lliinneeaarr eeqquuaattiioonnss wweerree ssoollvveedd ttoo oobbttaaiinn tthhee aammoouunnttss ooff ((aarrgg)) aanndd ((ggllyy)).. tthhee ffiirrsstt eeqquuaattiioonn wwaass oobbttaaiinneedd bbyy ssppeeccttrroopphhoottoommeettiicc mmeeaassuurreemmeenntt ooff tthhee ttoottaall aabbssoorrbbaannccee ooff ((aarrgg)) aanndd ((ggllyy)) ccoolloorreedd ccoommpplleexx wwiitthh nniinnhhyyddrriinn .. tthhee sseeccoonndd eeqquuaattiioonn wwaass oobbttaaiinneedd bbyy mmeeaassuurriinngg tthhee ttoottaall aacciidd ccoonnssuummeedd bbyy ttoottaall aammiinnoo ggrroouuppss ooff ((aarrgg)) aanndd (( ggllyy)).. tthhee ttiittrraattiioonn wwaass ccaarrrriieedd oouutt iinn nnoonn- aaqquueeoouuss mmeeddiiaa uussiinngg ppeerrcchhlloorriicc aacciidd iinn ggllaacciiaall aacceettiicc aacciidd aass aa ttiittrraanntt.. tthhee ddeevveellooppeedd mmeetthhoodd iiss aaccccuurraattee,, pprreecciissee,, aanndd ffrreeee ffrroomm iinntteerrffeerreenncceess aanndd mmaayy pprroovviiddee aa uusseeffuull aapppprrooaacchh ttoo ccaalliibbrraattee iinn tthhee ddiirreecctt aannaallyyssiiss ooff ssoolliidd ssaammppllee aanndd iitt iiss ssuuiittaabbllee mmeetthhoodd ttoo bbee uusseedd aass aa qquuaalliittyy ccoonnttrrooll pprroocceedduurree.. kkeeyy wwoorrddss:: ggssaamm ,, aammiinnoo aacciidd aannaallyyssiiss .. الخالصةالخالصة بشمممه هلممٕه دسممخ خٌٕط وتممطن خوخميمميُ خومم مٍ وى ممٗخٌ gsamطفت خو يطسمميت خومطٌممت حممً خالممطن ح ممٗ ط يىمما ضط ممت خ مم لال لمميُ ٖوى لممٗن يىمما ٌمممطاوخيُ ل يخمميُ حممً اٌمم خو ط مم ت –خ ٌيّيمت خومم خلىت فمم حطليممـ خو ٍممم دسي ّيمم ُ دسيخ ط مم ه سطومم يليىيج ٖوممما ٖضط مممت خوخلممم ي فممم ٌ ممميد مٌمممط أ بخسمممخ خي مممطٌ خوىممم ِٗيت أ بخسمممخ خي ٌ ىمممٗن خوّّٕممم س ُ وى لمممٗن يىممما خوٍمطاومممت خ ٖذوممك بممم يممين خ سمميخط ه سطويلمميىك دسممي ٌممُ خوٍم مم بخ ممطفت ممطٌ ٬خوبيطلىٗس ممك لٍ ىممٗن ٌلمم وى لممٗن يىمما خوٍمطاوممت خو طِيممت ٖدسممخ الا خ سميخط ه سطويلميىك دسممي خوٍخ مطس بٗخسم ت خوٍمم ـ خومومٗ خ مط ٖح مم ط لٍيخمٓ ب ط ممت )ييمطس ٥٫٠خوٕيم سٖلىٗس كأ ُ خوخل ي أ خوخل ي خوٍبطشط دٖخ سيطي . خوّخط خوخ حً خوخٗصه خويٕط اوج يىا لَٗ ٔ ْ خو ط ت سط مت ٖخوّخط اقي ت ٖلطويمت ٌم ٌم ة . خوخ خلالث خوميٍيطٖ ت ٖم ح خطج يٕية iinnttrroodduuccttiioonn nnuummeerroouuss mmeetthhooddss ffoorr tthhee aannaallyyssiiss ooff aacceettyyllssaalliiccyylliicc aacciidd ccoouulldd bbee ffoouunndd iinn tthhee lliitteerraattuurree ((11--1100)) .. hhoowweevveerr,, tthheessee mmeetthhooddss aarree nnoott ssuuiittaabbllee ffoorr tthhee aannaallyyssiiss ooff aacceettyyllssaalliiccyylliicc aacciidd mmiixxttuurree wwiitthh aarrggiinniinnee aanndd ggllyycciinnee.. aarrggnniinnee aanndd ggllyycciinnee iinntteerrffeerree wwiitthh ddiirreecctt ttiittrriimmeettrriicc ddeetteerrmmiinnaattiioonn ooff aacceettyyllssaalliiccyylliicc aacciidd tthheerreeffoorree,, iitt wwaass iinntteennddeedd ttoo sseeppaarraattee aacceettyyllssaalliiccyylliicc aacciidd qquuaannttiittaattiivveellyy ffrroomm iittss ssaallttss bbeeffoorree iitt iiss ddeetteerrmmiinneedd bbyy ttiittrriimmeettrriicc mmeetthhoodd.. iitt wwaass iinntteennddeedd ttoo uussee ssppeeccttrroopphhoottoommeettrryy oorr ttiittrriimmeettrryy ffoorr ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee aanndd ggllyycciinnee .. tthheessee ttwwoo mmeetthhooddss aarree ssiimmppllee aanndd tthheerreeffoorree tthhee mmoosstt wwiiddeellyy uusseedd iinn rroouuttiinnee ddrruugg aanndd mmaannyy ccoommppoouunnddss aannaallyyssiiss aass ccoouulldd bbee sseeeenn ffrroomm tthhee llaatteesstt vveerrssiioonn ooff bbrriittiisshh ,, sswwiissss aanndd uunniitteedd ssttaattee pphhaarrmmaaccooppeeiiaass.. hhoowweevveerr,, ddiirreecctt aapppplliiccaattiioonn ooff tthheessee mmeetthhooddss ffoorr ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee aanndd ggllyycciinnee iinn aa mmiixxttuurree ccoonnttaaiinniinngg bbootthh ooff tthheemm iiss nnoott ppoossssiibbllee bbeeccaauussee tthheeyy iinntteerrffeerree wwiitthh eeaacchh ootthheerr.. aass aa rreemmeeddyy ffoorr tthhiiss ssiittuuaattiioonn iitt wwaass ddeecciiddeedd ttoo uussee tthhee ggssaamm wwhhiicchh hhaass wwiiddee aapppplliiccaattiioonnss ((1111--1177)) .. iitt iiss bbaasseedd oonn tthhee pprriinncciippllee ooff vvaarryyiinngg bbootthh ooff tthhee ssaammppllee mmoollaarr ccoonncceennttrraattiioonn aanndd pphh ooff ssoolluuttiioonn.. aaiimmeedd aatt tthhee vvaalliiddaattiioonn aanndd ssttaannddaarrddiizzaattiioonn ooff aannaallyyttiiccaall pprroocceedduurreess wwiitthh ddiirreecctt ssoolliidd ssaammppllee ccoonnttaaiinn ttwwoo ttyyppeess ooff aammiinnoo aacciiddss wwiitthhoouutt pprreevviioouuss iissoollaattiioonn.. mmaannyy mmeetthhooddss wweerree uusseedd ffoorr tthhee ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee aanndd ootthheerr aammiinnoo aacciidd iinnvvoollvveedd pprreevviioouuss iissoollaattiioonn lliikkee tthhee ccllaassssiiccaall aannaallyyttiiccaall tteecchhnniiqquuee uusseess aauuttoommaatteedd aammiinnoo aacciidd aannaallyyzzeerrss,, hhoowweevveerr tthheessee mmeetthhooddss rreeqquuiirree eexxppeennssiivvee ddeeddiiccaatteedd eeqquuiippmmeenntt dduuoo ttoo tthhee ppoosstt- ccoolluummnn ddeerriivvaattiizzaattiioonn ooff tthhee aammiinnoo aacciiddss ,, lloonngg aassssaayy ttiimmeess aanndd llaarrggee ssaammpplleess vvoolluummeess (( 1188 )) ..aallssoo tthhee ssttuuddiieess ooff ppiittcc aass tthhee ddeerriivvaattiizziinngg aaggeenntt,, ffoolllloowwiinngg tthhee ppiiccoo--ttaagg mmeetthhoodd uusseedd ffoorr tthhee ddeetteerrmmiinnaattiioonn ooff aarrggiinniinnee && ootthheerr aammiinnoo aacciidd ,, tthhoossee ssttuuddiieess rreeqquuiirree tthhee eessttaabblliisshhmmeenntt ooff ssttaannddaarrdd––aaddddeedd ccuurrvvee ((bbyy aapppplliiccaattiioonn ooff tthhee ssttaannddaarrdd aaddddiittiioonn mmeetthhoodd ))ttoo aavvooiidd pprrooppoorrttiioonnaall eerrrroorrss ((1199)) .. tthhee nnoonn--aaqquueeoouuss ttiittrraattiioonn wwaass uusseedd ffoorr ddeetteeccttiioonn ooff ffrreeee aammiinnoo ggrroouupp iinn aammiinnoo aacciidd ((2200)) ..tthhiiss mmeetthhoodd uusseedd ffoorr tthhee qquuaannttiittaattiivvee ddeetteerrmmiinnaattiioonn ooff mmaannyy cchheemmiiccaall ccoommppoouunnddss aanndd ddrruuggss iinn pphhaarrmmaacceeuuttiiccaall ffoorrmmss,, pprroovviiddiinngg pprreecciissee aanndd aaccccuurraattee rreessuullttss,, wwhhiicchh ccoouulldd bbee vveerriiffiieedd bbyy ssttaattiissttiiccaall mmeetthhooddss ((2211--2233)) .. aa ssppeeccttrroopphhoottoommeettrriicc mmeetthhoodd aarree wwiiddeellyy uusseedd ffoorr ddeetteerrmmiinnaattiioonn ooff aammiinnoo aacciidd bbaasseedd oonn tthhee rreeaaccttiioonn wwiitthh ccoolloorriinngg aaggeenntt aatt ddiiffffeerreenntt pphh ffoorrmmiinngg ccoolloorreedd ccoommpplleexx tthhaatt mmeeaassuurreedd aatt ssppeecciiffiicc wwaavveelleennggtthh ((2244--2277)) .. 1corresponding author email : aazzhhaarrmmjjkk@@yyaahhoooo..ccoomm received : 11/5/2010 accepted : 27/6/2010 mailto:azharmjk@yahoo.com iraqi j pharm sci, vol.19(2) 2010 mmooddiiffiiccaattiioonn ooff ggssaamm 32 eexxppeerriimmeennttaall mmaatteerriiaall aanndd mmeetthhooddss glacial acetic acid (99.8 %) was obtained from sigma. standard perchloric acid (0.1 ml) in glacial acetic acid was prepared by diluting (4.25 ml) of perchloric acid (72%) to (500 ml) with glacial acetic acid containing (10 ml) acetic anhydride. the prepared solution was standardized by titration with standard anhydrous sodium carbonate (0.106 gm) in glacial acetic acid (50 ml) the titration end point was determined potentiometrically with potentiometric titration equipped with glass – calomel combined electrode. the prepared standard was used in the titrimetric procedure for the determination of arginine and glycine in arginine acetylsalicylate–glycine complex. anhydrous sodium carbonate (99.5 %) , sodium hydroxide (0.5 m) and hydrochloric acid (0.5 m) were aldrich standards . arginine (98%, sigma) was standardized to determine the exact percentage of arginine by titration with standard hydrochloric acid (0.5 m). glycine (99%, fluka) was used after drying in an oven for 1hr at (105ºc). acetylsalicylic acid was purified by recrystilization from ethanol/ water mixture and standardized with standard (0.5 m) sodium hydroxide (10) . the determined percentage of acetylsalicylic acid was (99.4 %).the standards arginine , glycine and acetylsalicylic acid were used to prepare arginine acetylsalicylate – glycine complex . the later was used to test the validity of the proposed method. sodium acetate buffer (4m, ph 5.5) was prepared by dissolving sodium acetate trihydrate naoac.3h2o (bdh “analar “99.9) in 200 ml of deionized water. the solution was heated with stirring at (60ºc ) until a clear solution was obtained. glacial acetic acid (50 ml) was added and the volume was completed to (500 ml) with distilled water. the ph was adjusted by a drop wise addition of sodium hydroxide (4m) to ph (5.5). ninhydrin reagent was prepared as following : ninhydrine ( i gm) in 2 methoxy ethanol (25 ml) and stannous chloride sncl2.2h2o (0.07 g) with continuous stirring until completely dissolved (28) . sodium acetate buffer (8.3 ml) was added and the resulting solution was immediately transferred to dark glass reservoir bottle and a steam of nitrogen gas was babbled into the reagent solution for approximately (20 min). titration was made with potentiometric titrator (metrohm , switzerland ) titro process or equipped with(metrohm).dosimate and glass calomel combined electrodes. spectrophotometric measurement was performed on carry (100 conc) uvvisible spectrophotometer using (1 cm) cells. determination of acetyl salicylic acid in this complex (1gm) of arginine acetylsalicylate– glycine complex was weighed and dissolved in (25 ml) of distilled water. the salt was converted to free acetyl salicylic acid by acidification with hydrochloric acid (3 m) until ph (2.7) was reached. the acetyl salicylic acid was extracted by ether (3 × 20 ml). the ether portions were pooled together and ether was evaporated by a rotary evaporator under vacuum. the acetyl salicylic acid was collected to be determined quantitatively by back titration method or by direct titration method (5, 10) . the result obtained for acetyl salicylic acid was corrected for the presence of salicylic acid as an impurity during liberating and separating acetyl salicylic acid form the complex. salicylic acid was then determined in the liberated materials (29) . determination of arginine and glycine in this complex this was achieved by constructing and solving equations (2) and (4) using ninhydrin spectrophotometric and titrimetric method in non – aqueous media respectively. spectrophotometric method (36 mg) of the complex was precisely weighed and dissolved in (50 ml) distilled water to prepare a stock solution. an aliquot (1 ml) of this stock solution was diluted to (25 ml) with distilled water. an aliquot ( 1 ml ) of this solution was mixed with ( 1ml ) of ninhydrin reagent in a stoppered test tube , shacked and placed in a boiling water bath for ( 15 min ). ethanol (2.5 ml) and sodium acetate buffer (2.5 ml) were added to the mixture which was then cooled below (30 ºc ). the sample was shacked thoroughly for (30 second) and absorbance was measured at (570 nm) against a reagent blank using (1 cm).standard curves for arginine and glycine were constracted using (0.05 = 0.2 mm) standard solution for each. the values of the molar absorpitivity coefficient aa and ag in equation (2) were determined from slops of these calibration curves. titrimetric method in non – aqueous media (0.3 gm) of the complex was dissolved in glacial acetic acid (50 ml) in a dry flask. the glass electrode of the titroprocessor was immersed in the solution and the mixture was titrated against standard perchloric acid (reagent 1). a plot of ph against the titrant volume was constructed to obtain the titration end point. stability study identical aqueous solution (0.1 g/ 100 ml) of the complex was made. the stability was determined at different temperatures (25, iraqi j pharm sci, vol.19(2) 2010 mmooddiiffiiccaattiioonn ooff ggssaamm 33 40, 50, 60 & 70 ºc ) incubated in ovens for certain intervals. the decomposition of arginine acetylsalicylate – glycine complex was indicated by the release of acetylsalicylic acid , arginine and glycine as appears on tlc using a solvent system of ( n-propanol : 34% ammonia 7 :3 ) . the appearance of three spots on tlc plates for acetyl salicylic acid, arginine and glycine when the samples were incubated at (50, 60 & 70 ) ºc . while incubation at (25 and 40) ºc showed only one spot for arginine acetylsalicylate – glycine complex. effect of buffer glycine was used as a buffer to stabilize arginine acetylsalicylat complex. the concentration of glycine is (0.5 m) in respect to arginine (1m) to maintain the ph of the preparation at (4.7). rreessuulltt aanndd ddiissccuussssiioonn method of calculation the gsam is a traditional vector – matrix notation to construct and to solve a system of (n) linear equation in order to determine the concentration of a mixture of (n) substances. the mixture of compounds interferes with each other when measured by (n) different sensors that belong to any suitable analytical technique. according to gsam, the following system of two linear equations is required to determine arginine and glycine: aa11 == aa1111%%aa++ aa1122 %% gg aa22 == aa2211%%aa++ aa2222 %% gg where ; a1 and a2 are the total responses of two analytical sensors ( 1 and 2 ) to the percentage of arginine ( % a ) and glycine ( % g ) in the sample the factors a11 – a22 are absorptivity constant multiplied by the optical path length ( i.e of the sample ) .theory of gsam equations (1) gives precise result for %a and %g when the following two conditions are fulfilled: firstly there should be a large difference in magnitude between the ratio a11/a12 and the ratio a21/a22. secondly the precision is measuring a1 and a2 should be high. these requirements arise from the fact that the mathematical manipulation magnify the random error in measurement , so that large random error is produced in the calculated concentration of the measured substances ( i.e %a and %g ) (11,12) .according to the requirements stated in these two conditions, it was decided to construct one of the equations of the system by using spechtrophotometric technique, while the second equation is to be constructed using titrimetic method. ninhydrin was chosen as a color developing reagent in the spechtrophtometric procedure, as it is the most selective among other coloring agent for spechtrophotometric ddeetteerrmmiinnaattiioonn for amino acids (30-31) .the titrimetric method was used to obtain the second equation which could not be performed in aqueous medium because of the neutral behavior of salts in aqueous medium that prevent the occurrence of a clear titration end point . accordingly, a titrimetric method in nonaqueous media was adopted using glacial acetic acid as a solvent and a solution of perchloric acid in glacial acetic acid as a titrant. this method was used for quantitative determination of salt of amines with carboxylic acids (32-33) .the presence of an acidic salts in non-aqueous solvent behave as bases and therefore produce a clear titration end point when titrated with strong acid (34) .this experiment of procedure was intended to produce two linear equations in which the ratio a11/a12 and a21/a22 are very different in magnitudes. this was expected from the fact that arginine and glycine have nearly the same efficiency to produce colored complexes with ninhydrin reagent in spectrophotometric procedure (31) .while in the titrimetric procedure arginine molecules have two titratable amino groups in contrast to the glycine molecules which have one titratable group. the first equation which is obtained from the spectrophotometric procedure was derived starting from lambert – beer law as follows : … (2) equation (2) can be abbreviated as follows: a = a11 %a + a12 %g ……… (3) in equation (2) wasg gm of arginine acetyl salicylate – glycine complex sample was dissolved to prepare (vo ml) of stock solution. this stock solution was then subjected to serial dilutions (n) with f1,f2…fn dilution factors to reach the required final concentration. mw a and mw g are the molecular weight of arginine and glycine respectively. aa and ag are the molar absorpitivity constants of arginine and glycine respectively.the use of a ……… (1) زز.…… iraqi j pharm sci, vol.19(2) 2010 mmooddiiffiiccaattiioonn ooff ggssaamm 34 combination of titrimetry and spechtrophotometry satisfies the requirements stated in (2). the precision of the spectrophotometric method is moderate in general.while; the precision of the titrometric method is very high. therefore, the use of non aqueous titration method improves the overall precision of the results obtained from such combination of methods. the second equation, was obtained from titrimetric procedure as follows: …(4) equation (4) can be abbreviated as below: mpvp = a21%a + a22%g …….. (5) equation (4) v ml of glacial acetic acid containing v asg of the analyzed arginine acetyl salicylic-glycine complex was titrated with mp molar standard of perchloric acid, so that vp ml of the standard was required to reach the end point . equations (3) and (5) are mathematically compatible and can be solved linearly. the ratio a11/a12 and a21/a22 have large differences in their magnitudes since a11/a12 ≈ 2 while a 21/a 22 ≈ 1. development of titrimetric procedure for acetyl salicylic acid determination the complex sample solution must be acidified to liberate acetyl salicylic acid form its salt with arginine before extraction by ether. the optimum ph of the acidified aqueous solution was determined experimentally by applying the sample complex at different ph values of the extracted sample solution. the result of this study indicates that ph (2.7) is the optimum ph for acetyl salicylic acid extraction. at ph higher than (2.7) low result for acetyl salicylic acid were obtained due to incomplete liberation acetyl salicylic acid from its salt with arginine.the extraction step is vital in the development procedure because an attempt to perform direct titration with hydrochloric acid without performing apparent titration end point as could be seen from (figure 1). figure 1: titration curve of arginine acetylsalicylate -glycine solution with standard hydrochloric acid (0.5). analysis of arginine acetyl salicylate – glycine complex quantitative determination of acetyl salicylic acid. acetyl salicylic acid was determined quantitatively by hydrolysis and back titration method (29) . the result of the two methods was summarized in table (1). table 1: result of acetylsalicylic acid, arginine and glycine in arginine acetyl salicylate – glycine complex * hydrolysis and back titration method. * *direct titration method. quantitative determination of arginine and glycine in the complex the quantitative determination of arginine and glycine in the complex using a modified version of the gsam was achieved by a combination of colorimetric method ( to obtain the first equation ) and non-aqueous titration method ( to obtain the second equation).the colorimetric method using the colored complex of arginine and glycine with ninhydrin was applied once at different ph media and once by using different wavelengths; the results of these experiments were summarized in table (2) and (3) and in figure (2) and (3) respectively. table 2: molar absorptivity constant a* of arginine and glycine colored complex with ninhydrin at different ph values measured at 570nm *represent the molar absorptivity multiplied by the cell width c.v calculated percentage ( w/w ) ± sd expected percentage w/w item 0.4 % 0.6 % 49.6 ± 0.2 * 49.6 ± 0.3 * 50.0 acetylsalicylic acid 1.5 % 40.3 ± 0.6 40.0 arginin 2.0 % 9.7 ± 0.2 10.0 glycine ph of ninhydrin solution aa ag aa / ag 5.5 0.83 0.62 1.34 7.0 1.07 0.78 1.37 9.0 1.73 0.97 1.41 iraqi j pharm sci, vol.19(2) 2010 mmooddiiffiiccaattiioonn ooff ggssaamm 35 table 3: molar absorptivity constant a* of arginine and glycine colored co mp lex with ninhydrin at different wavelengths. ph of ninhydrin solution wavelength hs nm aa ag aa/ag 5.5 244 1.19 0.92 1.29 409 0.74 0.54 1.37 570 0.83 0.63 1.32 7.0 244 1.28 0.97 1.32 409 0.87 0.62 1.40 570 1.02 0.75 1.36 9.0 244 1.40 1.08 1.30 409 0.92 0.67 1.37 570 1.32 1.00 1.32 *represent the molar absorptivity multiplied by the cell width figure 2: standard curve of arginine and glycine colored complexes with ninhydrin at different ph values measured at 570 nm figure 3: uv-visible spectra of arginine and glycine colored complexes with ninhydrin at different ph values according to the non-aqueous titration method; the titration end point of the titration curve between standard perchloric acid with the complex was determined potentiometrically. a typical plot for the titration curve was exhibited in figure ( 4 ) iraqi j pharm sci, vol.19(2) 2010 mmooddiiffiiccaattiioonn ooff ggssaamm 36 figure 4: titration curve between standard perchloric acid (0.1n) and arginine acetylsalicylate –glycine complex. the result of stability study indicates the appearance of three spots on tlc at (50, 60, 79)ºc and only one spot at (25, 40) ºc as summarized in table (4). table 4: *rf values of the arginine acetylsalicylate – glycine complex at different temperatures for certain time intervals c intial 1 st 2 nd 4 th 25 0.42 0.42 0.42 0.41 40 0.42 0.42 0.42 0.41 50 0.42 0.43 0.46 0.55 60 0.42 0.44 0.45 0.57 70 0.42 0.45 0.55 0.56 *the solvent system is (n-propanol : 34% ammonia 7 :3 ) iinntteerrffeerreennccee as mentioned earlier the degradation product of arginine acetylsalicylate – glycine complex might interfere with the determination of arginine acetyl salicylic acid and glycine by the proposed method. the evacuation step in the acetylsalicylic acid analysis procedure ensures complete removal of acetic acid thus prevents its influence on the titration of acetyl salicylic acid with sodium hydroxide. beside, the determination of the amount of salicylic acid (20) and the subsequent correction eliminates its interference on the acetyl salicylic acid determination. the non – aqueous titration procedure is not affected by all the degradation procedure as this procedure is selective for basic groups such as the amino group. ccoonncclluussiioonn the proposed procedure is suitable to be used as a quality control procedure for the determination of arginine acetylsalicylate – glycine complex, as well as many formulas that contain essential and non essential amino acids (free or in combination)in different preparation like food supplement or other pharmaceutical preparation.this proposed procedure is simple, fast and accurate in compares with other procedures. rreeffrreenncceess 1. xie y., wang t. and goodeng r., xuexiao huaxue xuebao, 1980; 14(2) : 175 -179. 2. kiungel oh.,ensing k., hegge h., franke jp. and zeeuw d., j planer chromatogrmod tlc, 1933;6(2):112-116. 3. kmeted vj., 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f, anal bioanal chem, 2006; 386(4):1121-1136. 18. ignacio ferrandez-figares,luis cuadros rodriguez,antonio gonzalez-casado,j of chromatography b,2004;799:73-74. iraqi j pharm sci, vol.19(2) 2010 mmooddiiffiiccaattiioonn ooff ggssaamm 37 19. l.cuadrose rodriguez,l.gawiz gracia, e.m.amansa lopez,j.m.bosque sandra, trends anal chem.,2001(20):620. 20. n.r.meyckik, y.u.nikolaeva, i.p. ermakov , biochemistry (moscow), 2009; 74 (8): 933-937. 21. herida r.n. marowa,cristian c.c.lopes,simone g.cardoso ,lat.am. j. pharm., 2003;22(4):339-342. 22. yamamoto m,taguchi k,journal of the japanese association for petroleum technology,2000;65(5)469-476. 23. leigh m.clemow,w.roy jackson, fuel, 2002; 81(7):959-961. 24. ieggli, carine viana silva; cardoso, simone goncalves; belle, luziane potrich , journal of aoac international , 2005; september 1. 25. sheng yun li, yin zhe ren, feng lin zhao, chinese chemical letters, 2006; 17( 8): 1065-1068. 26. j. demeester, m. bracke, r. vochten, a. lauwers,j pharm sci,2006;67(5):729-730 . 27. d. kowalczuk, r. pietraś, j. baran, annales universitatisaari aecuriesklodowska lublin-polonia, 2007,vol. xx, n 2, 11:83-87. 28. british pharmacopoeia, the stationery offices, london, 2008:vol.4.a87. 29. jin x.,yaovw fenxi zazhi,1992; 12(3): 169-170 30. pin hs. organic chemistry,5th ed., mc graw-hill,inc,usa,1988:817,823. 31. holme j. and peck h., analytical biochemistry,2nd ed., longman group, uk, 1933;49:384-396. 32. mihajlovic r., anal chimicia acta, 1990; 229:287-290. 33. skoog ad.,west md. and holler jf. fundamentals of analytical chemistry, 5th ed., wb-saunders company, usa, 1988:249. 34. kontoyannis g., j pharm biomedical analysis, 1993; 13(1):73. iraqi j pharm sci, vol.32( 1 ) 2023 vitamin b combination and diabetic neuropathy doi: https://doi.org/10.31351/vol32iss1pp202-218 202 physicians’ perception and practice of prescribing vitamin b combination versus antiepileptic drugs for diabetic neuropathy: content validity, reliability and pilot study narmin s. essa*,1 and mohammed i. aladul* *department of clinical pharmacy, college of pharmacy ,university of mosul, nineveh, iraq abstract diabetic neuropathy (dn) is the most common complication of diabetes mellitus causing increased morbidity and mortality. although international guidelines did not include vitamins and dietary supplements in any line of management, these agents were prescribed by a significant number of physicians either as preventive or treatment of dn. this study aimed to develop a validated questionnaire that examines the physicians’ perception and practice towards prescribing vitamins or dietary supplements for dn treatment. the questionnaire was developed upon literature review via pubmed, medline, web of science, scopus, and google scholar. the questionnaire was revised upon experts’ views and opinions. the constructed questionnaire was validated by the means of content validity and internal consistency. finally, the developed questionnaire was piloted in a small representative sample of the original future aimed research target population, to test its applicability and feasibility. a total of 22 items questionnaire were developed in two domains (knowledge and perception domain and practice domain). content validity analysis results met a satisfactory level, in which the s-cvi/ua value was 0.86 for both clarity and relevance. the questionnaire also showed good reliability, in which the cronbach’s alphas was 0.805. in conclusion, this study showed the that constructed questionnaire had a good level of validity (content validity) and reliability that is able to cover different aspects of the current state of perception and practice among physicians regarding the management of dn. the preliminary results of the pilot study showed a good knowledge and perception of the respondents with dn. even in the absence of a local guideline, the respondents followed the international guidelines in choosing anti-neuropathic agents however, they tended to add vitamin b complex/b12 as an adjuvant in their management plan. keywords: diabetic neuropathy, neurotrophic b vitamins, physician, vitamins. عتالل وصف مزيج فيتامين ب مقابل األدوية المضادة للصرع الفي تهمتصور األطباء وممارس ب السكري: صحة المحتوى والموثوقية والدراسة التجريبية اعصاال *محمد ابراهيم العدولو 1*،نرمين سعيد عيسى ق ، العرا نينوىفرع الصيدلة السريرية، كلية الصيدلة، جامعة الموصل ، * خالصةال على الرغم من . والوفيات ضاعتالل األعصاب السكري هو أكثر المضاعفات شيوًعا لمرض السكري مما يؤدي إلى زيادة معدالت المر من خط أي في الغذائية والمكمالت الفيتامينات تتضمن لم الدولية اإلرشادية الدالئل العالج أن انه، خطوط الي اال هذه وصف الغذائية تم مكمالت م التحقق ثوير استبيان هدفت هذه الدراسة إلى تط. وقاية او عالج لمرضى اعتالل االعصاب السكريمن قبل عدد كبير من األطباء إما ك والفيتامينات تم تطوير االستبيان بناًء على .اعتالل االعصاب السكريمنه لفحص تصور األطباء وممارستهم تجاه وصف الفيتامينات أو المكمالت الغذائية لعالج الخبراء افكارلى تم تنقيح االستبيان بناء ع. google scholarو scopusو web of scienceو medlineو pubmedمراجعة األدبيات عبر أخيًرا، تم تجريب االستبيان المطور في عينة . تم التحقق من صحة االستبيان الذي تم إنشاؤه من خالل صحة المحتوى واالتساق الداخلي. وآرائهم المستقبل، المجتمع تمثلصغيرة في للبحث وجدواهوذلك المستهدف للتطبيق قابليته مجموعه .الختبار ما تطوير مجالي فق 22تم في استبيان رة والممارسة) قيمة (. اإلدراك كانت حيث مرٍض، مستوى المحتوى صدق تحليل نتائج والمالءمةل s-cvi / ua 0.86حققت أظهر . لوضوح كرونباخ ألفا كانت حيث جيدة، موثوقية أيًضا من . 0.805االستبيان جيد بمستوى يتمتع كان إنشاؤه تم الذي االستبيان أن الدراسة هذه أظهرت بعالج مرض الصالحية )صحة المحتوى( والموثوقية القادرة على تغطية الجوانب المختلفة للحالة الحالية لإلدراك والممارسة بين األطباء فيما يتعلق مرض اعتالل االعصاب ة التجريبية معرفة جيدة وتصور المستجيبين الذين يعانون من اعتالل االعصاب السكري. أظهرت النتائج األولية للدراس مرض االدوبة المناسبة لعالج الدولية في اختيار البروتوكوالتمحلية، اتبع المشاركون بروتوكوالت عالجية. حتى في حالة عدم وجود السكري عالجية. خطة الالكعامل مساعد في 12فيتامين ب المركب / ب اعتالل االعصاب السكري ومع ذلك، فإنهم يميلون إلى إضافة . ، طبيب ، فيتامينات لالعصاب مقويةالكلمات المفتاحية: اعتالل األعصاب السكري ، فيتامينات ب ال introduction diabetes mellitus is defined as a metabolic disorder caused by multifactorial aetiologies and presented by chronic hyperglycemia with defects in carbohydrate, fat, and protein metabolism resulted from either abnormal insulin secretion, insulin action or both (1). in 2021, the international diabetes federation stated that there are about 537 *corresponding author e-mail: nermeen.gul@hotmail.com. received: 6/4 /2022 accepted: 13/6 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp202-218 iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 203 million diabetes cases, and this number is projected to reach 643 million by 2030, and 783 million by 2045 (2). the rapid escalation in the number of cases will be accompanied by a direct increase in the prevalence of the chronic complications of the disease (3). diabetic neuropathy (dn) is the most common complication of diabetes mellitus (4) causing increased morbidity and mortality (5) furthermore causing a great burden on health care expenditure (6). dn is defined as a neurodegenerative disorder that affects the somatic and/ or autonomic peripheral nervous system in the setting of diabetes mellitus without other causes of neuropathy (7). despite its heavy burden on healthcare systems and the better understanding of the multifactorial pathogenesis of the disease, dn still underdiagnosed and undertreated (8). in the absence of evidence-based disease-modifying pharmacological agents, the mainstay of the management is based on glucose control, lifestyle changes, and pain management (9). despite the relatively high prevalence of dn in iraq (10), there is no local or regional guideline of dn management (11). neurotropic b vitamins (b1, b6 and b12) were well-known to have a crucial role in maintaining healthy nervous system (12). it was suggested that these vitamins may increase the availability and effectiveness of noradrenaline and 5-hydroxytryptamine in the descending inhibitory nociceptive system (13, 14) furthermore, in some countries they were classified as analegesic drugs (14).it was hypothesized that neurotropic b vitamins support directing the wallerian degeneration process (which is an active process that commenced after a nerve injury of degeneration of an axon) toward regeneration and remyelination (15), several clinical trials conducted to highlight the effect of administration of the neurotropic b vitamins together with different dose regimes (16 18), however the positive outcomes was limited to improvement in symptoms of pain numbness and paraesthesia with no improvement in neurophysiological parameters. thiamin (b1) had been shown to serve as a cofactor for three important enzymes that were involved in carbohydrate metabolism (19), in addition to have an important role in abolishing the metabolic dysregulations caused by high glucose level including polyol pathway, protein kinase c pathway, hexosamine pathway and increased advanced glycation end products which are the proposed mechanisms of dn pathogenesis (19, 20). benfotiamin which is a synthetic derivative of thiamin with better pharmacokinetic properties (21, 22) was shown to improve the neuropathy symptom score in a double-blind randomized placebocontrolled phase iii clinical study (bendip) (23). despite the fact that the association between vitamin b6 level and diabetes mellitus was well established (24), two clinical trials of pyridoxin administration in patients with dn were failed to improve subjective and objective parameters of the disease (25, 26). furthermore, concerns were raised about the safety profile of pyridoxin following several case reports about its possible toxic effects on nerves (27,28) which was proposed to be due to disruption of gammaaminobutyric acid (gaba) biosynthesis (29) which led several food safety authorities is several countries to decrease the allowed upper limit of pyridoxin dose (30,31). a long-term trial was showed a significant effect of metformin on b12 and methylmalonic acid (mma) level with concurrent worsening of neuropathy symptoms (32). several clinical trials concluded a positive effect of methylcobalamin (active form of vitamin b12) on the symptoms of dn with no effects on the nerve study parameters (33 35) until recently (2021) a prospective one-year, randomized double-blind, placebocontrolled trial, concluded positive effects of 1 mg methylcobalamin on in sural nerve conduction parameters along with michigan neuropathy screening instrument questionnaire (mnsiq), the level of pain, and the quality of life (36). although the latest international guidelines did not include vitamins and dietary supplements in any line of management (e.g., the national institute for health and care excellence national (nice) updated guideline in 2020 (37) and the american diabetes association (ada) guideline in 2021(38)). a recent study, that was conducted in saudi arabia, found that a significant number of physicians were prescribing vitamin b12 either as preventive or treatment to their patients with dn (55). the supplements have a heavy economic burden for patients in a developing country like iraq, which lacks national health insurance programs. in addition to the fact that the safety profile of some heavily prescribed vitamins is questionable (39). this study aimed to develop a validated questionnaire to assess physicians’ perception and practice towards the prescribing of vitamins and dietary supplements versus pharmacological agents for the management of dn. methods the study design involves three phases. in phase one, the construction of the first draft of the questionnaire from an interpretation of the literature on the research topic. in phase two, the content validity of the drafted questionnaire was tested to verify the applicability and appropriateness. finally, in phase three, the internal consistency of the questionnaires was piloted to ensure reliability and feasibility. phase one the literature review was conducted between the 1st of october 2021 and the 30th of december 2021 for published articles before the time of the research (no time limit was set to get as many as possible articles on the research topic). the search was only for full articles, written in english or arabic languages that are examining healthcare iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 204 professionals’ or physicians’ knowledge, attitude, perception, and practice toward neuropathic pain treatment or management. the exclusion criteria were; articles written in languages other than english or arabic, abstracts, articles examining patients’ perspectives rather than physicians. the literature review was conducted by the first author, a master student and the chief investigator, which were trained to search for relevant articles via the available databases and search engines including pubmed, medline, web of science, scopus, and google scholar. the medical subject headings (mesh) terms used in the search were supplied as a supplementary file. the number of results retrieved from the literature review through the above-mentioned databases was 86 researches of a variety of formats. following a screening of the retrieved researches and excluding irrelevant ones, the final number of researches chosen was 10 (figure 1). figure 1. workflow showing the process of literature review for constructing the first draft of the questionnaire based on the results of the literature review, the ten chosen articles were reviewed and used in the construction of the initial draft of the questionnaire. the questionnaire was constructed in english language since iraqi physicians are more familiar with medical terminology in english rather than arabic language. then the questionnaire was sent independently to two experts in kap (knowledge, attitude, and practice) tools at the university of baghdad and the university of duhok to verify the relevance and appropriateness of the questionnaires for the study topic. based on the experts’ views, the first draft was amended accordingly to improve the clarity and the structure of the questionnaire. this was followed by a second review process by another independent reviewer at the college of pharmacy, university of mosul to evaluate the questionnaire and eliminate redundancy and ambiguity, and to develop a final draft of the questionnaire. the questionnaire was developed to explore the physicians’ perception and practice towards the prescribing of vitamins and dietary supplements versus pharmacological agents for the management of dn. therefore, the items (questions) were allocated into two main domains (knowledge and perception domain and practice domain). the knowledge and perception domain aimed to closely examine the basic knowledge and the general perception of the physicians about dn (including the commonality of dn, its effect on patient’s iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 205 quality of life, mortality rate and the difficulty of treatment in addition to the perception about dietary supplements and vitamin b). the practice domain aimed to explore the physicians’ prescribing plans regarding anti-neuropathic agents and dietary supplements and indifferent cases. in addition to the factors that affect the choice of these agents. the time expected for completing the questionnaire (10 – 20 minutes) was enough for almost all of the respondents. content validity the six steps of the content validity procedure were conducted as follow: (i) content validity form development, (ii) assigning an expert review panel, (iii) distributing the content validation form to the expert (d) reviewing the domain and items (e) setting a score to each item (f) determining the content validity index (cvi) (40). in which the cvi uses item-cvi (i-cvi) and scale-cvi (s-cvi) to report content validity in questionnaire development (41). the i-cvi is determined by dividing the total number of experts by the number of experts who rated each item on a scale of 'very relevant' (score 1), 'relevant' (score 1), 'irrelevant' (score 0), and 'very irrelevant' (scoring 0) (42). the i-cvi value range of 0.8 or more indicates that the item is relevant, however, if the value is less than 0.8 and more than 0.69, this would suggest item revision, while a value of less than 0.7 indicates an item to be eliminated. the sum of all items with icvi equal to 1 divided by the total number of items for the s-cvi yielded the universal agreement (ua) among experts (s-cvi/ua). a content validity index of s-cvi/ua 0.8 indicates outstanding content validity (43). eight experts, including physicians and pharmacists from the university of mosul and ninevah university, as well as retired physicians, were recruited to judge the items using a scoring system ranging from 1 (the item is not relevant at all) to 4 (the item is highly relevant). if the items were scored lower than 3, in the comments, the experts were asked to offer a note or a suggestion to rephrase the item. reliability internal consistency is an indicator of a questionnaire's homogeneity (44). it reflects how close the questions (items) of a questionnaire are intercorrelated and hence measure the same concept. cronbach's alpha (α) value is used as a measure of internal consistency (45). the internal consistency reliability of the two domains (knowledge and perception domain, and practice domain) was tested using cronbach's alpha coefficient. forty-five participants were enrolled to complete the questionnaire, the sample size was satisfying the central limit theorem which assumed that the distributional assumption of the sample size of 30 or more to guarantee an equal mean between any sample and the target population (46). pilot study to test the feasibility and usability of the questionnaire, a pilot study was conducted. a total of 45 physicians in different specialties were recruited. a convenience purposive sampling from avicenna hospital and al-quds primary health centre in mosul, iraq. ethical approval was obtained from the medical research and ethics committee of the department of clinical pharmacy, college of pharmacy, university of mosul. ethical approval was also obtained from the collegiate committee for medical research ethics at the university of mosul (code: ccmre-pha-22-2) to conduct the study. the physicians were contacted to participate in this pilot via telephone and verbal consent was given to participate in the study. on the day of the interview, written consent was given by the participants. the researcher discussed the aims of the research and handed the paper survey questionnaire. the questionnaires were filled out in 15 minutes (±3 minutes). the face-to-face data collection started from the 20th to the 31st of january 2022. statistical analysis data obtained from the respondents (for content validity, reliability and pilot study) were entered to microsoft excel (2016) software to calculate the content validity. then the data was moved to the statistical package for the social sciences (spss) software version 24 to run the reliability test. descriptive analyses (percentages and frequencies) were carried out using spss software for the pilot study. results phase one the process of reviewing the literature via pubmed, medline, web of science, scopus, and google scholar databases resulted in 86 researches of a variety of formats (abstracts, posters, full articles, and books). these materials were screened by means of the titles and abstracts to remove duplicates and irrelevant articles. ten full-text articles were retrieved for further evaluation based on the inclusion and exclusion criteria (figure 1). the final number of researches chosen was 10 (table 1). iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 206 table 1. literature review articles author country aim sample size participants ' profession method hall et al. (2006) (47) uk to report drug treatment of neuropathic pain as managed by uk primary care physicians. 686 primary care centers. na utilization analysis aakash et al.(2008) (48) india to inquire about their prescribing preferences among the drug options that were provided, to treat painful diabetic neuropathy 89 physician survey possidente et al, (2009) (49) uk to evaluate provider practices for identification and treatment of painful diabetic peripheral neuropathy 357 physician, nurse, nurse practitioner, physician assistant, others, pharmacist survey benzon et al. (2013) (50) us to determine prescription patterns and whether these practices reflect current expert opinion. 474 physician survey mabrouk et al, (2013) (51) egypt to assess family physicians’ knowledge, attitude, and practice regarding dn. 60 physician survey lalonde et al. (2014) (52) canada to evaluate and identify the determinants of the kap of primary care physicians and pharmacists. 248 physician & pharmacist survey malik et al. (2017) (53) hong kong, malaysia, the philippines , taiwan, and thailand to examine the physician perceptions of painful dn and clinical practice behaviours in five countries in southeast asia. 100 physician survey provenzano et al, (2018) (54) us to assess the knowledge and practice of pcps in the management of chronic pain 300 primary care physicians survey aldossari et al, (2021) (55) saudi arabia to investigate local physicians’ knowledge and tendency to prescribe vitamin b12 or vitamin b complex for the treatment or prevention of diabetic peripheral neuropathy. 412 physician survey moon et al. (2021) (56) korea to analyze the pharmacological treatments for dn. na na utilization analysis the questionnaire was developed from the tools of the retrieved articles with modification to meet the aims of the developed questionnaire and to make it more suitable for the iraqi healthcare system, which differs massively from other healthcare systems where other articles were conducted. the questions were allocated into two main domains, namely, the knowledge and perception domain, including eight items, and the practice domain including 16 items. the first draft of the questionnaire was reviewed by two experts; these include deletion of two questions for redundancy and irrelevance. the experts also requested to rephrase three more questions to improve the clarity of the questions. on the second round of review, an expert suggested adding a “not sure” for questions with an answer of “yes” or “no” for closed-ended questions. the final draft consisted of 22 items (seven items in knowledge and perception domain and fifteen items in the practice domain) (table 2). iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 207 table 2. questionnaire’s domains knowledge and perception domain item 1 knowledge about dn response dn is a common complication of diabetic patients agree, neutral, disagree dn affects patient’s quality of life agree, neutral, disagree dn increases mortality rate agree, neutral, disagree dn is difficult to manage agree, neutral, disagree dn management plan is based on symptomatic relief agree, neutral, disagree dn can be reversed by strict blood glucose control agree, neutral, disagree dn incidence can be decreased by maintaining strict glucose control agree, neutral, disagree item 2 do you think that there is a definite treatment for dn? yes, no, not sure item 3 is there any local guideline for the management of diabetic neuropathy? yes, no, not sure item 4 knowledge about dietary supplements in dn vitamins and dietary supplements are approved by the fda agree, neutral, disagree vitamins and dietary supplements’ manufacturing undergoes quality control just like chemical medicines agree, neutral, disagree vitamins and dietary supplements are safe on long term use agree, neutral, disagree vitamins and dietary supplements have no side effects agree, neutral, disagree vitamins and dietary supplements have no interactions with other medications agree, neutral, disagree the cost of vitamins and dietary supplement worth the efficacy (outcome) provided. agree, neutral, disagree item 5 knowledge about vitamin b complex/b12 vitamin b complex/ b12 supplementation can prevent the development of dn agree, neutral, disagree vitamin b complex/ b12 supplementation can treat dn agree, neutral, disagree vitamin b complex/ b12 supplements are safe for long term use in treatment of dn agree, neutral, disagree item 6 what is the most common cause of dn? toxic effects of glucose, oxidative stress, vascular damage, vitamin b12 deficiency item 7 what is (are) the source of information does you follow in managing dn? information from senior colleagues, information from colleagues of different specialty, internet and social media reputable international guidelines, like american diabetes association (ada), nice, etc. textbooks or scientific journals, own practice practice domain response item 1 do you recommend/ prescribe a dietary supplement to your patients with dn yes no item 2 if you answered to the previous question with yes, how often do you recommend/ prescribe supplements to your patients with dn? always sometimes rarely iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 208 continued table (2) item 3 vitamins and dietary supplements in dn are usually prescribed by physicians as: treatment (monotherapy), adjuvant, tonic only, prophylaxis, satisfy the patient (placebo) item 4 the effectiveness of an agent is an important factor in choosing dn medication agree, neutral, disagree item 5 the safety of an agent is an important factor in choosing dn medication agree, neutral, disagree item 6 patient preference is an important factor in choosing dn medication agree, neutral, disagree item 7 drug drug interaction is an important factor in choosing dn medication agree, neutral, disagree item 8 cost of an agent is an important factor in choosing dn medication agree, neutral, disagree item 9 patient factors including co-morbidity are important factors in choosing dn medication agree, neutral, disagree item 10 medical advertisement of an agent is an important factor in choosing dn medication agree, neutral, disagree item 11 a 45 years old male diabetic patient (normal bmi and no comorbidities) is complaining of burning and tingling sensation pain in his feet especially at night, what is the drug of choice for this patient? amitriptyline, carbamazepine, duloxetine, gabapentin, pregabalin, vitamin b complex, vitamin b12 item 12 the extent of influences of international guidelines on your practice i never use them, i consult them occasionally, they are fundamental to my practice item 13 management plan of [mild dn] life style change (diet, exercise), strict control on blood sugar, supplements (vitamin b complex / vit b12, anticonvulsants or tca or snri, combination of (anticonvulsant+ tca and/or ssri+ supplement) item 14 management plan of [moderate dn] life style change (diet, exercise), strict control on blood sugar, supplements (vitamin b complex or vit b12,…etc), anticonvulsants or tca or snri, combination of (anticonvulsant+ tca and/or ssri+ supplement) item 15 management plan of [severe dn] life style change (diet, exercise), strict control on blood sugar, supplements (vitamin b complex or vit b12,…etc), anticonvulsants or tca or snri, combination of (anticonvulsant+ tca and/or ssri+ supplement) iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 209 phase two (content validity) to determine the content validity of the preliminary version of the questionnaire, an expert panel of eight healthcare professionals was recruited. the experts rated the items’ (questions’) clarity and relevance on a scale from (1 4) to calculate the i-cvi, s-cvi, and s-cvi/ua. tables 2 and 3 show the calculation of the validity indices for both clarity and relevance. the validity indices met satisfactory levels (tables 3, 4 and 5). table 3. calculations of the content validity for relevance it e m e x p e r t1 e x p e r t2 e x p e r t3 e x p e r t4 e x p e r t5 e x p e r t6 e x p e r t7 e x p e r t8 e x p e r t in a g r e e m e n t ic v i u a 1 4 4 4 4 4 4 4 4 8 1 1 2 4 4 4 3 4 4 4 4 8 1 1 3 4 4 4 3 4 3 3 4 8 1 1 4 4 4 4 4 4 4 4 4 8 1 1 5 4 4 4 4 2 4 3 3 7 0.875 0 6 3 4 4 4 3 4 4 4 8 1 1 7 4 4 4 4 3 4 4 4 8 1 1 8 4 4 3 4 3 4 4 4 8 1 1 9 4 4 4 4 4 4 3 4 8 1 1 10 4 4 4 3 3 4 3 4 8 1 1 11 4 4 4 4 4 4 4 4 8 1 1 12 4 4 4 4 4 4 4 4 8 1 1 13 4 4 4 4 4 4 4 4 8 1 1 14 4 4 4 4 3 4 4 4 8 1 1 15 4 4 4 4 4 4 1 3 7 0.875 0 16 4 4 4 3 3 4 3 4 8 1 1 17 4 4 4 4 4 4 3 1 7 0.875 0 18 4 4 4 4 4 4 3 4 8 1 1 19 4 4 4 4 4 4 4 4 8 1 1 20 4 4 4 4 4 4 4 4 8 1 1 21 4 4 4 4 4 4 4 4 8 1 1 22 4 4 4 4 4 4 4 4 8 1 1 table 4. calculations of the content validity for clarity it e m e x p e r t1 e x p e r t2 e x p e r t3 e x p e r t4 e x p e r t5 e x p e r t6 e x p e r t7 e x p e r t8 e x p e r t in a g r e e m e n t ic v i u a 1 4 3 4 4 4 4 4 2 7 0.875 0 2 4 4 4 3 4 4 4 4 8 1 1 3 4 4 4 3 4 3 4 4 8 1 1 4 4 4 4 4 4 3 4 3 8 1 1 5 3 4 4 4 4 3 4 4 8 1 1 6 4 4 4 4 4 4 4 4 8 1 1 7 4 4 4 4 4 3 4 4 8 1 1 8 4 4 4 4 4 4 4 4 8 1 1 9 4 4 4 3 4 4 3 4 8 1 1 10 4 4 4 4 4 4 4 4 8 1 1 11 4 2 4 4 4 4 4 4 7 0.875 0 12 4 4 4 4 2 4 4 4 7 0.875 0 13 4 4 4 4 3 4 4 4 8 1 1 14 4 3 4 4 4 4 4 4 8 1 1 15 4 3 4 4 4 4 4 4 8 1 1 iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 210 continued table4. 16 4 3 4 4 4 4 4 4 8 1 1 17 4 4 4 3 4 3 4 4 8 1 1 18 4 4 4 4 4 3 4 3 8 1 1 19 3 4 4 4 4 3 4 4 8 1 1 20 4 4 4 4 4 4 4 4 8 1 1 21 4 4 4 4 4 3 4 4 8 1 1 22 4 4 4 4 4 4 4 4 8 1 1 table 5. validity indices (clarity and relevance) validity index clarity relevance i-cvi 0.98 0.98 s-cvi 0.98 0.98 s-cvi/ua 0.86 0.86 phase three for the two domains, the calculated cronbach’s alpha coefficient for internal consistency was 0.804 which is in the range of accepted reliability. for the knowledge and perception domain, the cronbach’s coefficient was 0.726 while for the practice domain the coefficient was 0.823 suggesting a satisfactory result for reliability (table 6). table 6. calculated cronbach’s alpha coefficient for internal consistency of each domain knowledge and perception domain reliability statistics cronbach's alpha n of items 0.726 7 item-total statistics mean std. deviation corrected item-total correlation cronbach's alpha if item deleted item 1 17.31 2.076 0.226 0.743 item 2 2.53 0.625 0.539 0.669 item 3 2.33 0.739 0.389 0.706 item 4 12.36 3.009 0.556 0.665 item 5 6.22 1.795 0.58 0.659 item 6 2.20 0.968 0.519 0.674 item 7 0.69 0.468 0.28 0.731 practice domain reliability statistics cronbach's alpha n of items 0.823 15 item-total statistics mean sd. corrected item-total correlation cronbach's alpha if item deleted item 1 1.8 0.404 0.326 0.82 item 2 2.422 0.811 0.279 0.823 item 3 2.622 0.575 0.55 0.806 item 4 2.511 0.757 0.545 0.806 item 5 2.533 0.757 0.652 0.799 item 6 1.711 0.726 0.429 0.814 item 7 2.244 0.908 0.584 0.803 item 8 2.155 0.705 0.678 0.797 item 9 2.422 0.753 0.682 0.797 item 10 1.622 0.805 0.447 0.813 item 11 0.755 0.434 0.44 0.813 item 12 2.33 0.639 0.285 0.823 item 13 1.77 0.901 0.185 0.829 item 14 2.977 1.544 0.137 0.832 item 15 3.4 2.038 0.418 0.814 iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 211 pilot study a total of 45 physicians (56% male and 44% female) were recruited in piloting the final draft of the questionnaire. the age group of the majority (64%) of the participants was between 35-54 years. they were from different professional levels ranging from consultants (11%), specialists (51%), residents (22%), and general practitioners (16%). they were from different specialties (family medicine 29%, internal medicine 24%, general surgery 11%, and other specialties 36%). about half (49%) of the respondents were working in governmental hospitals while 36% were in primary care centers. only 42% of them were working in private clinics. more than half (56%) of the respondents have more than 10 years of experience in their field of specialty (table 7). table 7. demographics of the pilot study respondents variables n (%) gender male female 25 (56%) 20 (44%) age group 2534 years 3544 years 4554 years 55 years or more 7 (15.5%) 15 (33.3%) 14 (31.2%) 9 (20%) professional level consultants specialists residents general practitioner 5 (11%) 23 (51%) 10 (22%) 7 (16%) specialty family medicine internal medicine general surgery rheumatology cardio-surgery neurology orthopedic no specialty 13 (29%) 11 (24%) 5 (11.1%) 4 (9.5%) 3 (6.6%) 3 (6.6%) 3 (6.6%) 3 (6.6%) work setting primary care centers governmental hospitals tertiary care center academia retired 16 (36%) 22 (49%) 4 (9%) 1 (2%) 2 (4%) working in a private clinic yes no 19 (42%) 26 (58%) years of experience less than 5 years 5 10 years more than 10 years 6 (13%) 14 (31%) 25 (56%) knowledge and perception domain in the knowledge and perception domain, the majority of the respondents presented good knowledge of dn in terms of its commonality and effect on patients’ quality of life. while only about half of the respondents had good knowledge on the management plan’s mainstay and its difficulty along with the effect of dn on mortality rate. in the context of the effect of blood glucose control on dn, the majority of the respondents thought that controlling blood glucose could decrease the incidence of dn while only about one-quarter of the physicians thought that blood glucose control cannot reverse dn (table 8). regarding the knowledge of physicians towards vitamins and dietary supplements, about one half of the respondents thought that the dietary supplements are approved by the american food and drug administration (fda) and that they are quality controlled just like chemical medicines. while about one half of the respondents disagreed with the idea of vitamins and dietary supplements’ safety on long-term use, the lack of associated side effects and their lack of tendency to interact with other medications. the respondents were evenly split between supporters and opponents regarding the cost-benefit of vitamins and dietary supplements. about one-half of the respondents believed that vitamin b complex/ b12 has no role in the treatment of dn, nor in the prevention of dn. however, near half of the respondents believed that vitamin b complex/ b12 supplements are safe for long-term use in the treatment of dn. one-half of the respondents stated that no local (iraqi) guideline for the management of dn is available and about 34% of them were not sure if such guideline exists. similarly, about twothirds of the respondents thought that there was no iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 212 definite treatment for dn while about one-third were not sure about the availability of such treatment in the current time. more than one-half of the respondents believed that vascular damage, toxic effects of glucose, and vitamin b12 deficiency are the most common causes of dn. regarding the source of information for the management of dn, respondents rated textbooks and scientific journals, and international guidelines as the most important sources of knowledge (table 8). table 8. participants’ responses in knowledge and perception domain knowledge and perception domain agree neutral disagree dn knowledge dn is a common complication of diabetic patients 32 (71.1%) 13 (28.9%) 0 (0%) dn affects patient’s quality of life 42 (93.3%) 3 (6.7%) 0 (0%) dn increases mortality rate 20 (44.4%) 18 (40%) 7 (15.6%) dn is difficult to manage 28 (62.2%) 11 (24.4%) 6 (13.3%) dn management plan is based on symptomatic relief 28 (62.2%) 7 (15.6%) 10 (22.2%) dn can be reversed by strict blood glucose control 21 (46.7%) 9 (20%) 15 (33.3%) dn incidence can be decreased by maintaining strict glucose control 35 (77.8%) 6 (13.3%) 4 (8.9%) knowledge of dietary supplement in dn vitamins and dietary supplements are approved by the fda 22 (48.9%) 13 (28.9%) 10 (22.2%) vitamins and dietary supplements’ manufacturing undergoes quality control just like chemical medicines 19 (42.2%) 17 (37.8%) 9 (20%) vitamins and dietary supplements are safe on long term use 12 (26.7%) 15 (33.3%) 18 (40%) vitamins and dietary supplements have no side effects 8 (17.8%) 9 (20%) 28 (62.2%) vitamins and dietary supplements have no interactions with other medications 11 (24.4%) 10 (22.2%) 24 (53.3%) the cost of vitamins and dietary supplement worth the efficacy (outcome) provided. 14 (31.1%) 17 (37.8%) 14 (31.1%) knowledge of vitamin b supplements vitamin b complex/ b12 supplementation can prevent the development of dn 11 (24.4%) 10 (22.2%) 24 (53.3%) vitamin b complex/ b12 supplementation can treat dn 12 (26.7%) 15 (33.3%) 18 (40%) vitamin b complex/ b12 supplements are safe for long term use in treatment of dn 20 (44.4%) 15 (33.3%) 10 (22.2%) yes no not sure do you think that there is a definite treatment for dn? 3 (6.7%) 27 (60%) 15 (33.3%) is there any local guideline for the management of dn? 7 (15.6%) 22 (48.9%) 16 (35.6%) iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 213 continued table 8 . what is the most common cause of dn? (multiple response allowed) vitamin b12 deficiency 25 (55.6%) toxic effects of glucose 27 (60%) oxidative stress 15 (33.3%) vascular damage 31 (68.9%) what is (are) the source of information does you follow in managing dn? information from senior colleagues 11 (24.4%) information from colleagues of different specialty 11 (24.4%) internet and social media 7 (15.6%) reputable international guidelines, like american diabetes association (ada), nice, etc. 18 (40%) textbooks or scientific journals 26 (57.8%) own practice 16 (35.6%) practice domain the majority (80%) of respondents expressed their willingness to prescribe vitamins and/or dietary supplements to their patients with dn in a frequent manner. vitamin b complex was the most commonly mentioned dietary supplement when they were asked to give an example of dietary supplement that they recommend for their patients. and they recommend these dietary supplements (vitamin b complex) as adjuvant therapy. more than one-half of the respondents considered the effectiveness, safety, cost and patient factors as very important factors in choosing medications for the treatment of dn. more than two-thirds of the respondents chose one of the first line antineuropathic agents according to the latest guidelines for dn management, in which, gabapentin was the most chosen agents for the treatment of dn.the respondents’ opinions regarding the influence of international guidelines on their practice were in between consulting the guideline and utilizing the guideline. regarding their management plan of different degrees of dn, more than two-thirds of the respondents recommended the following lines of treatment: lifestyle change, strict control of blood sugar and dietary supplements (including vitamins) for the different severities of the disease. more than half of the respondents recommended an anticonvulsant as an option in managing moderate and severe dn, while about half of the respondents stated their recommendation of combining anticonvulsant, tricyclic antidepressant (tca), a dietary supplement with or without serotoninnorepinephrine reuptake inhibitor (snri) in managing severe dn (table 9). iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 214 table 9. participant’s responses in practice domain practice domain do you recommend/ prescribe a dietary supplement to your patients with dn yes 36 (80%) no 9 (20%) if you answered to the previous question with yes, how often do you recommend/ prescribe supplements to your patients with dn? always 28 (62.3%) sometimes 8 (17.8%) rarely 9 (20%) vitamins and dietary supplements in dn are usually prescribed by physicians as: treatment 7 (15.6%) adjuvant 26 (57.8%) tonic only 8 (17.8%) prophylaxis 3 (6.7%) satisfy the patient (placebo) 1 (2.2%) the effectiveness of an agent is an important factor in choosing dn medication agree 26 (57.8%) neutral 17 (37.8%) disagree 2 (4.4%) the safety of an agent is an important factor in choosing dn medication agree 27 (60%) neutral 11 (24.4%) disagree 7 (15.6%) patient preference is an important factor in choosing dn medication agree 9 (20%) neutral 16 (35.6%) disagree 20 (44.4%) drug drug interaction is an important factor in choosing dn medication agree 19 (42.2%) neutral 18 (40%) disagree 8 (17.8%) cost of an agent is an important factor in choosing dn medication agree 21 (46.7%) neutral 14 (31.1%) disagree 10 (22.2%) patient factors including co-morbidity are important factors in choosing dn medication agree 20 (44.4%) neutral 19 (42.2%) disagree 6 (13.4%) medical advertisement of an agent is an important factor in choosing dn medication agree 10 (22.2%) neutral 11 (24.4%) disagree 24 (53.4%) a 45 years old male diabetic patient (normal bmi and no comorbidities) amitriptyline 5 (11.1%) carbamazepine 2 (4.4%) duloxetine 3 (6.7%) is complaining of burning and tingling sensation pain in his feet especially at night, what is the drug of choice for this patient? gabapentin 18 (40%) pregabalin 7 (15.6%) vitamin b complex 7 (15.6%) vitamin b12 1 (2.2%) the extent of influences of international guidelines on your practice i never use them 4 (8.9%) i consult them occasionally 22 (48.9%) they are fundamental to my practice 19 (42.2%) iraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 215 continued table 9. management plan of [mild dn] life style change (diet, exercise) 37 (82.2%) strict control on blood sugar 29 (64.4%) supplements (vitamin b complex or vit b12,…etc) 28 (62.2%) anticonvulsants or tca or snri 9 (20%) combination of (anticonvulsant+ tca and/or ssri+ supplement) 1 (2.2%) management plan of [moderate dn] life style change (diet, exercise) 29 (64.4%) strict control on blood sugar 38 (84.4%) supplements (vitamin b complex or vit b12,…etc) 34 (75.6%) anticonvulsants or tca or snri 22 (48.9%) combination of (anticonvulsant+ tca and/or ssri+ supplement) 7 (15.6%) management plan of [severe dn] life style change (diet, exercise) 27 (60%) strict control on blood sugar 33 (73.3%) supplements (vitamin b complex or vit b12,…etc) 31 (68.9%) anticonvulsants or tca or snri 25 (55.6%) combination of (anticonvulsant+ tca and/or ssri+ supplement) 22 (48.9%) discussion the literature review process revealed no validated questionnaire that is targeted to explore physicians’ perception and practice towards the prescription of vitamins and other medicines in dn management. however, the review process found two articles that explored the utilization patterns of anti-neuropathic i.e (tca, snri, anticonvulsants, and opioids) in the uk and korea (47, 56). the prescribing preferences of healthcare professionals (physicians, pharmacists, and nurses) regarding antineuropathic medications, their knowledge, and beliefs about dn were examined in six researches (48-53). while provenzano et al., (2018) have investigated the practice of primary care physicians about the management of chronic pain (54). aldossari et al., (2021) had assessed the physicians’ knowledge and the tendency of prescribing vitamin b12 exclusively for the treatment or prevention of dn in primary care settings (55). our study illustrated the ability to produce a questionnaire with good psychometric properties on the physicians’ perception and practice toward prescribing vitamin b combination versus antiepileptic drugs in the management of dn along with the factors affecting their choice and the influence of the international guidelines to their current practice. the questionnaire of this study was developed by adapting items from the former-mentioned studies in addition to the self-constructed questions. the content validity was measured in this study since it is an essential step in questionnaire’s development. furthermore, it is an acceptable and important method to reflect the relevance and clarity of the instrument (57). the content validity indices ranged from 0.8 to 0.9 for both relevance and clarity which is satisfactory and reveals that the questions were relevant and representative of the intended aim of the developed tool (58). shi et al., (2012) indicated that i-cvis ≥ 0.78, s-cvi/ua, and s-cvi/ave ≥ 0.8 are considered excellent indices for relevance and clarity. the content validity indices of the constructed questionnaire of this study were within the above-mentioned ranges (59). the use of cronbach’s alpha in the multiple-item questionnaire was considered as a routine in knowledge, attitude, and practice studies (60), which is used to test and determine the internal consistency (reliability) of a questionnaire in applied research (61). nunnally and bernstein (1978) hypothesized that an alpha value of 0.70 and higher is an acceptable value in the early stages of developing a questionnaire (i.e., exploratory research) (62). on the other hand, george and mallery (2003) ranked alpha values according to acceptability into excellent ≥ 0.9, good ≥ 0.8, acceptable ≥ 0.7, questionable ≥ 0.6, poor ≥ 0.5, and unacceptable ≤ 0.5. the cronbach’s alpha coefficient for internal consistency of this study was 0.804 which is considered good (63). although the pilot study is time-consuming and leads to considerable data loss, it ensures the feasibility of the developed questionnaire, in other words, it detects any inappropriate and/or complicated questions that would ultimately end up by potential failure of the questionnaire. in our pilot study, the clarity of the questions was confirmed by face-toiraqi j pharm sci, vol.32(1) 2023 vitamin b combination and diabetic neuropathy 216 face short interviews with the respondents from different specialties (family medicine, internal medicine, general surgery, endocrinology, rheumatology, and neurology). as expected, the physicians showed a good perception of the commonality of dn, the impact of the disease on quality of life, and the role of glucose control in the etiology of dn and the management plan of the disease. diverse opinions were noticed regarding the process of manufacturing, fda approval, safety profile, and the cost-effectiveness of vitamins and dietary supplements. the lack of a local guideline was raised by the respondents as a reason for their different perspectives regarding the use of vitamins and medicines for the management of dn, in which their main source of information were reputable international guidelines like the american diabetes association (ada) and nice along with textbooks and scientific journals. however, physicians opted gabapentin, pregabalin, and duloxetine as their first pharmacological choice which was in line with the current international guidelines, the percentage of iraqi physicians who were adhered to international guidelines was higher than the percentage of the recent saudi study (55). although we expected that physicians would prioritize the efficacy and safety in their treatment choice, they also prioritized cost as an important factor in choosing the medication. interestingly, physicians are willing to prescribe vitamins and supplements for therapeutic purposes as an adjuvant which was consistent with the results of the saudi study (55) in which about half of the physicians rated the efficacy of vitamin b12 as moderate in both prevention and treatment of dn. regarding the management of mild, moderate, and severe dn, although the physicians adhered to the international guidelines that recommended lifestyle changes and strict blood glucose control as a primary intervention for dn. however, they unfollowed these guidelines by recommending vitamins and dietary supplements as an adjuvant treatment. this study has some limitations; first is that despite the fact that cronbach’s alpha is a commonly used test to determine the internal consistency in the literature it has limitations when used as a sole index for internal consistency. second, the small number of participants in the pilot study was the consequence of the fact that the target population (physicians) are limited in number and hard to reach, and that those who are participated in the pilot study would not be included in the main study (since this article is a part of a larger study and master degree dissertation); therefore, losing this number would affect the power of the main study. to the best of our knowledge, this study is the first to develop and validate a tool to explore physicians’ perceptions and practices regarding the use of vitamins and dietary supplements in the management of dn in different specialties. conclusion this study showed that the constructed questionnaire had good levels of validity (content validity) and reliability that is able to cover different aspects of the current state of perception and practice among physicians regarding the management of dn. the preliminary results of the pilot study showed a good knowledge and perception of the respondents with dn. even in the absence of a local guideline, the respondents followed the international guidelines in 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attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(1) 2012 total hip replacement home – care 39 assessment of patients’ knowledge toward total hip replacement home – care suad .j. mohamed * ,1 and abdullah .e. mecheser** *nursing college, university of baghdad ,baghdad, iraq. ** medical & health technical college,baghdad, iraq. abstract to assess the total hip replacement patients’ knowledge of home – care regarding pain management, medication therapy, wound care, mobility limitation and complications may occur in the post hip replacement surgery, and to assess relationship between some variables such as, age level of education, sex & marital status with homecare knowledge. a descriptive study was used to assess the hip-replacement patient home-knowledge, a purposive sampling of (60) hip-replaced –patients were selected from gazy alhariri hospital (central of surgical profession ) and alwasity hospital ( plastic surgery) , the questionnaire obtains two parts , part one, which included socio-demographical characteristics of the sample and part 2 , which included hip-replacement home-care knowledge , reliability and validity of the questionnaire were determined . data were collected through the use of the questionnaire, at the hip application of the interview techniques and review of the hip replacement surgery rehabilitation review literature. data were analyzed through descriptive and inferential data approach.the result of the study presented that the majority of the study sample were group at age (2535) years old, female married, housewife with (50%) of patients were at level of primary school education. they were lacking knowledge toward pain management at home and wound care , also discharged with very minimum information and rehabilitation training instruction toward practicing assistive walking and activity device or preventing potential complication such as infection and dislocation , besides the study revealed a significant relationship between totalhip replacement knowledge toward complication may be occurred and level of education ,which revealed that the educated hipreplaced patients can managing and preventing the complication may occurred better than those not educated.establishing a special rehabilitation unit in the ward to provide the patients with a rehabilitation program for training , also to provide them with a booklet or pamphlet illustrating all formations the patient may need at home. key words: total – hip replacement , patients’ knowledge of pain , patients’-knowledge of potential complication , patients' knowledge toward wound , patients’ knowledge of mobility. تقويم معرفت انمرظى نهعنايت انمنزنيت بعد عمهيت استبدال مفصم انورك *سعاد جاسم محمد ،1 **هللا عيادة مجيسر و عبد * وٍُت اٌخّزَض ،خاِعت بغذاد ، بغذاد ، اٌعزاق. ** وٍُت اٌخمُٕاث اٌصحُت واٌطبُت ، بغذاد ، اٌعزاق. الخالصة ٕاَت إٌّشٌُت بعذ عٍُّت اسخبذاي ِفصً اٌىرن اٌىاًِ ، فُّا َخعٍك باٌعٕاَت باألٌُ واٌعالج ُُ ِعٍىِاث اٌّزضً ٌٍعمُح اٌعاللت بُٓ بعض اٌّخغُزاث ِثً حمُُُ ووذٌه اٌذوائٍ واٌعٕاَت بدزذ اٌعٍُّت وححذَذاث اٌحزوت و اٌّضاعفاث اٌخٍ ححذد بعذ اٌعٍُّت ، . دراست وصفُت غُز احخّاٌُت ٌخمُُُ ِعٍىِاث ِزضً اسخبذاي ُت إٌّشٌٌٍعٕاَت واٌدٕس بّعٍىِاث اٌّزَض اٌعّز واٌّسخىي اٌثمافٍ ( ِزَض حُ إخزاء عٍُّت اسخبذاي ِفصً اٌىرن ٌهُ فٍ ِسخشفً اٌشهُذ غاسٌ 06)إٌّشٌُت وشٍّج اٌعُٕت ِفصً اٌىرن باٌعٕاَت حخضّٓ اداة االسخبُاْ ِٓ خشئُٓ:اٌحزَزٌ اٌّزوشٌ ٌٍدزاحاث اٌخخصصُت وِسخشفً اٌىاسطٍ ٌٍدزاحاث اٌخدٍُُّت ،و اٌدشء األوي: اٌّعٍىِاث اٌذَّىغزافُت ٌٍعُٕت. اٌدشء اٌثأٍ: ِعٍىِاث ِزضً اسخبذاي ِفصً اٌىرن اٌىاًِ ٌٍعٕاَت إٌّشٌُت. عض األدبُاث وحُ ححذَذ صذق األداة وخّع اٌبُأاث خالي اسخّارة اسخبُاْ عٓ طزَك اٌّمابٍت اٌّباشزة ِٓ لبً اٌباحذ ووذٌه ِزاخعت ب اٌخٍ حخص عٍُّاث اسخبذاي ِفصً اٌىرن وحأهٍُه، وحُ ححًٍُ اٌبُأاث ِٓ خالي اإلحصاء اٌخحٍٍٍُ واالسخذالي . سٕت، ٔساء ِخشوخاث ورباث بُىث . 52-52ِٓ اٌّدّىعت اٌعّزَت أظهزث اٌذراست أْ ِعظُ اٌّزضً اٌّشاروُٓ فٍ اٌعُٕت هُ %( ِٓ اٌّزضً واْ ِسخىاهُ اٌثمافٍ هى اٌذراست االبخذائُت فمظ ، اظهزث اٌذراست أَضا هٕاٌه ٔمص فٍ ِعٍىِاث اٌّزضً 26واْ) ٌّّارسه حأهٍه شفً بّعٍىِاث لٍٍُت خذا ٔحى اٌعٕاَت عٕذ حذود األٌُ فٍ إٌّشي ووذٌه اٌعٕاَت باٌدزذ، وَخُ خزوج اٌّزَض ِٓ اٌّسخ ٌخهاباث وعذَ حّىضع اٌّفصً فٍ ِىأت، ووذٌه اظهزث اٌحزوت وِٕع حذود ِضاعفاث خطزة واالاٌّشٍ وإرشاداث اسخعّاي ِسأذ اٌذراست عاللت لىَت بُٓ اٌّسخىي اٌثمافٍ ٌٍّزَض وِٕع حذود ِضاعفاث ٌّزضً اسخبذاي ِفصً اٌىرن اٌىاًِ. ّارسه إٌشاطاث ُز بزٔاِح حأهٍٍُ ٌٍّزَض ٌٍخذرَب عًٍ ِأوصً اٌباحثىْ أشاء وحذة حاهٍُُت ِخخصصت فٍ ٔفس اٌزدهت ٌخىف اٌّعٍىِاث اٌخاصت اٌخٍ حخعٍك واٌفعاٌُاث اٌُىُِت اٌخٍ َحخاخها اٌّزَض فٍ إٌّشي ووذٌه حىفُز وخُب َخضّٓ وً إٌشاطاث و . باٌخّارَٓ اٌحزوُت ٌُىىْ دًٌُ عًّ ٌه عٕذ اٌخزوج انكهماث انمفتاحيت : تبديم كامم انمفصم ، معرفت انمريط باألنم ، معرفت انمريط نمعاعفاث انمرض اندقيقت ، معرفت انمريط .بانجروح، معرفت انمريط بانحركت 1 corresponding author email : suad_jassim@yahoo.com received : 9/8/2011 accepted : 21/2/2012 iraqi j pharm sci, vol.21(1) 2012 total hip replacement home – care 40 introduction totalhip replacement provides significant relief of pain and improvement of function, it is the replacement of severely damaged hip with an artificial joint, indication for this surgery include arthritis, femoral neck fracture , expulsive trauma and problem resulting from congenital –hip disease (1 15 ) . hip replacement, have been an offer to patients with painful hip-joints for almost 50 years, millions of patients worldwide have benefited from this surgery and regained pain free, active lives (6,14) home as health setting, optimal management in the home occurs when a person can independently maintain a growth – promoting environment, the home is comfortable and safe, and a person perform self-care and hygiene tasks, interacts with others and engages in activities (9) .with maintaining femoral head component in acetabullar cup (2) .rehabilitation program and precautions necessary to protect newly implanted hip and to prevent problem called dislocation, these instructions and precautions given as sessions after surgery related to how to use a walker in walking and keeping a good alignment and balance in a usual time 2-4 weeks with cane and 4-6 weeks unassisted (2,12) .home care considerations include ongoing assessment of management , maintaining for infection , and prevention of deep vein thrombosis (dvt). not all patients will qualify for home nursing visit, the incision may be closed with metal staples ,which are removed at the surgeon's clinic, because of the high risk of dvt, prothrombin times will be determined weekly are commonly used to map the care of clients after total hip replacement because of the consistent postoperative needs of these population (4,8) . infection of all the surgical complication , is then most difficult to address .it can get into the hip joint by a variety of means ,may be transferred in top blood from skin at the time of operation or by the patients circulation (blood born contamination) , so in order to prevent infection the patient should takes a certain precautions, such as intra venous antibiotics (7) , an operations will be required to put things right , or may hip-joint opened and irrigated to remove infection after 6-weeks.outpatient physiotherapy treatment is tailored to patients need and speed recovery , usually about 6 7 physiotherapy sessions will be arranged over on 8 weeks period in participating walking on a regular basis and build-up a strength thigh and hip muscles (9) . methodology a descriptive design study was carried out to assess the patients knowledge toward hip replacement home-care knowledge a purposive (nonprobability) sampling of 60 patients on discharged from alhariri hospital and alwasity hospital a questionnaire, interview form was designed by investigator to measure the variables underlying the present study, which is consisted of two part: *. part one: demographical sheet, consisted of 6 items; as following age, gender, employment, marital status, level of education, and appliance aids used by patient.  part two: is composed of 4 sections, which are knowledge section to present the patients home care knowledge.  first section: patient knowledge of pain managment at home, which consisted of 5 items, scored as 3 for always, 2 for sometimes and 1 for never.  second section, wound care patient knowledge which consisted of 5 items, scored as 3 for always, 2 for sometimes and 1 for never.  third section: patients’ knowledge in mobility limitation and also scored 3, 2, 1 consisted of 8 items.  forth section: patients’ knowledge regarding potential complication, consisted of 4 items and also scored 3, 2, 1 for always, sometimes, and never respectively with 2 as cutting of point. data was collected by the researchers through the use of the designed questionnaire interview technique to patients on discharge the data collection was carried out from 1/october /2010 to 1/july/2011.the researcher took a long time to collect the data due to the rare cases and the surgery take place once a week.the validity of the questionnaire was determined for clarity, relevancy and adequacy. results distribution of the sample according to the occupation. iraqi j pharm sci, vol.21(1) 2012 total hip replacement home – care 41 table 1: demographical characteristics of the sample. % f item 33.3 21.7 10 25 10 20 13 6 15 6 25-35 35-45 46-55 56-65 66 or above age 100 60 total 46.7 53.3 28 32 male female gender 100 60 total 33.3 20 46.7 20 12 28 -employed -retired -housewife job 100 60 total 80 3.3 16.7 48 2 10 -married -single widowed marital status 100 60 total 50 33.3 16.7 30 20 10 -primary school -secondary school -college level of education 100 60 total f = frequency. this table indicated that the majority of the sample 33.3% were of (25.35) years age and employed,80% of them married, the level of education was primary school is 50% . table 2: distribution of patients' knowledge of pain management at home ms* never sometimes always item no. methods to reduce pain 2.6 0 20 40 taking bed-rest and maintaining alignment on-pain experience at operative site 1 2.9 9 35 16 practicing destruction and relaxation technique periodically 2 2.6 2 48 10 administrating medication when experience pain by care giver. 3 1.9 40 18 2 identify the action and doses of medication non-steroidal, anti-inflammatory and opioid drugs. 4 2.3 30 18 12 recognizing medication side-effect such as nausea, vomiting ,and constipation 5 *ms: main score this table showed the highest main score ( 2.9)on relaxation technique periodically and lowest main of score( 1.9) to identify the action and dose of medication that indicates lacking of knowledge related to treatment. iraqi j pharm sci, vol.21(1) 2012 total hip replacement home – care 42 table 3 : distribution of patients' knowledge toward wound management at home. ms never sometimes always item no. 2.16 20 10 30 keeping a wound incision clean and dry , until staple removed 1 1.5 30 6 14 changing wound dressing in a sterile technique 2 1.5 38 16 6 recognizing wound infection signs such as (redness, swelling, and drainage from incision) 3 2.2 12 26 22 fellow-up that wound suture is removed in fourteen days in out-patient clinic 4 3.8 2 3 28 taking antibiotic in time to avoid infection 5 this table showed highest main of score( 3.8) toward taking antibiotic in time while lowest main of score (1.5) to item recognizing wound infection . table 4: patients' knowledge of mobility-limitation. ms never sometimes always item no. 1.3 34 8 16 demonstrate safe use of assistive devices crutches ,walker ,and wheel chair 1 3.2 16 24 22 demonstrate, how to stand without flexing leg actually 2 2.2 18 12 30 recognizing the weight bearing limits especially on praying 3 2.1 12 28 20 recognizing avoidance of low seated chair and crossing legs in sitting 4 2.9 14 28 18 sleeping with abduction pillow , between legs to maintain alignment 5 1.9 18 34 10 participating in gradual activities and prescribed exercise regimen 6 1.9 36 10 10 accepting assistance in adl,(clothing, bathing himself) 7 1.7 26 24 10 demonstrating how to change position frequently 8 this table showed highest main of score( 2.9) in sleeping with abduction below between the legs while lowest main of score(1.3) to item demonstrating safe use of assistive devices crutches, walker and wheelchair. iraqi j pharm sci, vol.21(1) 2012 total hip replacement home – care 43 table 5 : distribution of patients' knowledge toward mobility limitation according to some demographical characteristics. sig. * df x2 total never sometimes always age no. 0.278 12 14.354 23 5 7 11 30-34 1 7 2 3 2 35-40 2 7 3 3 1 41-45 3 6 4 2 46-50 4 6 2 3 1 51-55 5 11 7 3 1 56-60 &above 6 60 19 23 18 total *d.f=degree of freedom this table indicated no significant relation between age group and patient knowledge at home toward activity and mobility limitation . table 6 : patients’ knowledge of potential complicationat home. ms never sometimes always item no. 1.6 48 6 6 identifies dislocation prosthesis signs; increase in pain, shortening of leg, inability to move leg. feeling of popping sensation in operative hip's site. 1 1.2 50 10 0 identifies deep-vein thrombosis signs; swelling calf pain. 2 1.6 32 16 12 identifies wound infection signs ,swelling ,pain, fever, purulent drainage from the wound incision 3 this table showed low main of score (1.6) to potential complication which indicating lacking of knowledge and information related to management of complication at home. table 7: the relation between the patients' knowledge of complication after hip-replacement at home and level of education . sig. df x2 total never sometimes always item no. s 4 2.75 29 9 13 7 primary school 1 s 4 2.74 14 3 5 6 secondary school 2 s 1 17 7 5 5 college 3 s 0.001 60 19 23 18 total this table reveals significant relation between level of education and patients' knowledge toward complication may occur after total hip-replacement at home , which indicating hipreplaced educated patients able to manage the complication that may occur iraqi j pharm sci, vol.21(1) 2012 total hip replacement home – care 44 discussion the current study showed that , the majority of the study sample were at age of 25-35 year,(table-1), this finding is not compatible with( smeltzer & bare) (1) who stated that total hipreplacement patients usually 60 years of age or older, and has unremitting pain or irreversibly damage hip joint . also the table revealed that 53.3% of the sample were female,46.7% they were housewife , married 80%,and 50% with primary level of education . concerning the patient-knowledge toward pain management at home, (table -2) the result of the study showed high mean score (2.9). toward practicing destruction and relaxation technique periodically and (2.6) on taking bedrest and maintaining alignment on experience pain, while low mean of score (1.9), identifying the action of a serious medication taken.these findings indicating a lack of knowledge of hip-replaced patients in managing pain at home, and also revealed that no discharge instruction was provided to them. it was stated that among patients undergoing hip replacement procedure ,pain is one of the five most undesirable complication .unrelieved pain can adversely affect the individual's ability to perform basic daily activities as well as increase of stay and rehabilitation (2 ,3) .one of the most important aspect to prevent postoperative infection is the knowledge of wound-care at home, (table-3) showed highest mian of score (3.8) on taking antibiotics in time, while lowest mian of score (1.5) for two serious item ,first for recognizing wound infection signs, second on changing dressing in a sterile technique , which also indicating lack of discharge knowledge and information given to patients at hospital.this result supported by( white) (7,13) , following a total hip replacement, the client is at increased risk for joint infection that may lead to the development of osteomylitis, also stated, the client will remain free from wound infection, as evidenced by normal temperature and white blood cells count (wbc), absence of purulent drainage from the wound and absence of redness or inflammation at the surgical site.practicing walking at home is the important aspect for hip-replacement patients as shown in (table 4) which indicating highest mean of score (3.2) to item of demonstrating, how to stand without flexing leg acutely, while lowest mean of score (1.3) to item of demonstrating safe use of assistive devices crutches, walkers and wheels chairs, this result revealed, that hip replacement patient discharged from hospital without any training programs or practicing the assistive devices provided to them for safety activity at home. this result supported by( micheals) (5, 11 ,15) who presented that many assistive devices have been developed to make activities easier and less stressful for joints and muscles, these devices will be helpful at home or work. also stated ( 6-7) physiotherapy sessions will be arranged over an 8 weeks period, the most important to do ,is how to walk on a regular basis and build up the strength in thigh and hip muscles , how to keep legs and knees apart, and avoid exercise flexion at the hip joint .the list of complication following total hip replacement was extensive ( table 6) showed that all mean of score for the three items of complication ,was(1.6)and(1.2) which indicating poor information and instruction were given related to complication may occurred at home ,that may keep them to be at high risk for these complication (8,10) supporting this result, who stated that home care consideration include ongoing assessment of pain management ,monitoring for infection and prevention of deep vein thrombosis. the patient should be instructed to obtain prophylactic antibiotics prior to any surgical procedures .no significant relation between the sample age group and patients knowledge toward mobility limitation at home (table 5) ,while significant relation between level of education and patients knowledge toward managing complication at home, which revealed that hip replacement educated patients can managing the complication at home (table7). conclusion the study presents that the majority of the sample were lacking knowledge related to pain management at home and wound care, also discharged with very minimum information and rehabilitation training instruction toward practicing assistive walking device and preventing potential complication such as infection and dislocation. recommendation 1. establishing a special rehabilitation unit at the ward to provide a program of training on assistive device instructed to patient apply to them before discharge to prevent complication of dislocation which is a very serious one. 2. provide the discharge patient with a pan phlet6 of all need instruction with figures illustrating to them all information they may need at home relating to activity and sleeping or medication 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"concerns over metal on metal hip implants" , the new york times, march (3), 2010 ;p. 655-661 12. huston,g. "medical device alert: all metal-on-metal (mom) hip replacements" ,medicines and healthcare , products regulatory agency. 22 april 2010. mda / 2010;p. 1132-37 13. stevan a," arthritis physical and occupational therapy " australian orthopedic association national joint replacement registry annual report. adelaide: aoa; 2008 ;(18),p. 431-438 . 14. barbara b , "the effect of pain on health related quality of life" , conference coverage, american academy of orthopedic surgeons (aaos), march 3, annual meeting of arthroplasty,2009;p. 753 -759. 15. jackson jl, "predictors of patient satisfaction with medical rehabilitation ",american journal of physical medicine& rehabilitation , 2003;(82) , 1123-1123 . http://www.mhra.gov.uk/publications/safetywarnings/medicaldevicealerts/con079157 http://www.mhra.gov.uk/publications/safetywarnings/medicaldevicealerts/con079157 http://www.medscape.com/viewarticle/588980 http://www.medscape.com/viewarticle/588980 acne vulgaris is a very common, chronic disorder, involving inflammation of the pilosebaceous units that can be varied in presentation and difficult to treat iraqi j pharm sci, vol.21(1) 2012 serratiopeptidase in acne 78 serratiopeptidase a hope in a rapid and better improvement of inflammatory acne vulgaris # ehab m. mikhael* ,1 and mahdi y. mohammed** * department of clinical pharmacy, college of pharmacy,university of baghdad,baghdad,iraq. **department of dermatology, abu-ghriab general hospital,baghdad,iraq. abstract acne vulgaris is a very common, chronic disorder, involving inflammation of the pilosebaceous units that can be varied in presentation and difficult to treat. inflammatory acne may yield both scarring and pigmentary changes so early and adequate therapy will, in all cases, decrease its severity and may entirely suppress this disease. serratiopeptidase has anti-inflammatory, anti-edemic and fibrinolytic activity and acts rapidly on localized inflammation. serratiopeptidase was added in aim to hasten acne resolution. during march to july 2010, a comparative study for a 50 healthy patient suffering from acne was divided into 2 groups: 1 st group treated by common acne modalities and the 2 nd one with same modalities plus serratiopeptidase. all patients were followed up in out patient clinic by a dermatologist after 1 st week of treatment and once weekly through the period of treatment. the results of this study showed that the effect of serratiopeptidase as adjuvant therapy for acne treatment result in a significant rapid improvement, this might be explained by serratiopeptidase ability in enhancing antibiotic efficacy and also increase the possibility of excellent improvement in acne appearance which may be due of it’s anti-inflammatory, anti-edemic. serratiopeptidase was found to create a good hope as additional therapy for complicated acne vulgaris and bring a rapid and better improvement in treating acne. key words: acne ,serratiopeptidase. زيم السيراتىتثتايديز أمل جديد تتحسن أسرع وأفضل لحة الشثاب الملتهةإن إيهاب مضر ميخائيل* ،1 و مهدي يحيى محمد** .* فشع انصٍذنح انسشٌشٌح ، كهٍح انصٍذنح ، جايعح تغذاد ، تغذاد ، انعشاق . ، انعشاق تغذاد يسرشفى اتٕ غشٌة انعاو ، ، **لسى انجهذٌح ألخالصة انشثاب يشض يُرشش ٔ يضيٍ ٌرضًٍ انرٓاب خالٌا انجهذ، نّ أشكال يخرهفح ٔيٍ انصعٕتح عالجّ. حثٕب انشثاب حة االنرٓاتٍح ذسثة أثاسا فً انٕجّ ٔذصثغاخ جهذٌح نزا فًٍ انضشٔسي عالجٓا تسشعح ٔتشكم يثانً نهرمهٍم يٍ حذج انًشض ٔإٌمافّ. ذًد إضافح إَضٌى رٓاب ٔانٕريح كًا اَّ ٌعًم يٕلعٍا نهرمهٍم يٍ االنرٓاتاخ.إَضٌى انسٍشاذٕتثراٌذٌض نّ خٕاص يضادج نالن ، دساسح يماسَح نخًسٌٕ يشٌضا 0202خالل انًذج يٍ آراس ٔحرى ذًٕص انسٍشاذٕتثراٌذٌض تٓذف اإلسشاع يٍ عالج حة انشثاب. ٔنى عٕنجد تاألدٌٔح انًعرادج نعالج حة انشثاب ٌعإٌَ يٍ دسجاخ يخرهفح يٍ حة انشثاب ذى ذمسًٍٓى إنى يجًٕعراٌ : انًجًٕعح األ جًٍع انًشضى ذًد يراتعرٓى تعٍادج خاسجٍح يٍ لثم ، ٔانًجًٕعح انثاٍَح عٕنجد تزاخ انعالجاخ يضافا نٓا إَضٌى انسٍشاذٕتثراٌذٌض. ٓشخ تاٌ ذأثٍش إَضٌى طثٍة جهذٌح يخرض تعذ األسثٕع األٔل يٍ انعالج ٔثى يشج أسثٕعٍا خالل فرشج انعالج.ْزِ انذساسح أظ انسٍشاذٕتثراٌذٌض كعالج يساعذ نحة انشثاب ْاو ٔيعُٕي السرجاتح أسشع نهعالج ٔلذ ٌفسش ْزا تماتهٍح اإلَضٌى نضٌادج فاعهٍح انًضاداخ نمذ ٔجذ إٌ اإلَضٌى ٔكزنك ٌضٌذ احرًانٍح انشفاء األفضم نحثٕب انشثاب تسثة ذأثٍشِ انًضاد نالنرٓاب ٔانٕريح. انحٌٍٕح انسٍشاذٕتثراٌذٌض لذ ٌخهك ايآل جٍذا كعالجا إضافٍا نعالج حة انشثاب انًسرعصً ، كًا اَّ ٌضًٍ عالجا أفضم ٔأسشع نعالج حة انشثاب. . سيراتىتثتايديزالكلمات المفتاحية : حة الشثاب ، introduction acne vulgaris is a very common, chronic disorder, involving inflammation of the pilosebaceous units that can be varied in presentation and difficult to treat. (1) four key factors have been identified in the etiology of acne: increased sebum production, follicular hyperkeratinization, colonization of the pilosebaceous unit with propionibacterium acnes and the production of inflammation ; (2) the inflammatory lesions vary from small papules with a red border to pustules to large, tender, fluctuant nodules . some of the large nodules were previously called cysts and the term nodulocystic has been used to describe severe cases of inflammatory acne. (3) inflammatory acne may yield both scarring and pigmentary changes (4) so early and effective treatment is essential to prevent and minimize the cosmetic disfigurement associated with acne scarring. # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : ehab_pharma84@yahoo.com received : 12/3/2011 accepted : 1/4/2012 iraqi j pharm sci, vol.21(1) 2012 serratiopeptidase in acne 79 adequate therapy will, in all cases, decrease its severity and may entirely suppress this disease. (1, 2) moderate or severe acne treated by use of topical therapy, plus antibiotics (5) , inflammatory lesions decrease and new lesions stop appearing within 2 to 6 weeks; (1) however, with the known therapy of acne patients should be counseled that an improvement may not be seen for at least a couple of months, (6) because antibiotic therapy can’t be truly evaluated until 6 weeks after starting. (1). general treatment guideline of moderate – severe acne according to global alliance algorithm (7) include antibiotic such as doxycycline which may play a therapeutic role in acne by reducing inflammation through anticollagenolytic, antimatrix-degrading metalloproteinase, and cytokine downregulating properties (8 ) plus topical retinoid with or without topical benzyl peroxide . late-onset and persistent adult acne are now more widely recognized and may necessitate consideration of an alternative approach to therapy. (2) serratiopeptidase (serratia) is a proteolytic enzyme (protease) produced by enterobacterium serratia sp. (9) some alternative medicine proponents claim that serratiopeptidase is beneficial for pain and inflammation (10) serratiopeptidase has antiinflammatory, anti-edemic and fibrinolytic activity and acts rapidly on localized inflammation. (11) . in this study we added serratiopeptidase to the standard acne treatment to limit the duration of active disease (hasten acne resolution) by early and effective treatment; therefore offers the possibility of minimizing both the physical and emotional scarring caused by acne. (2) methods during the period from march to july 2010, a comparative study for a 50 patients suffering from acne with varying degree of severity ranging from papulopustular acne to nodulocystic acne was divided into 2 groups: 1 st group 25 patients, (20 females and 5 males), age (18.36±2.464) years. in this group patients were received standard therapy according to guideline of global alliance algorithm: doxycycline capsule 100mg twice daily + retin a cream (isotretinoine 0.05%) twice daily plus panoxyl gel 5% ( benzyl peroxide) once daily, for 3 months . where as the 2 nd group include 25 patients, (20 females and 5 males), age (18.28±2.372) years. patients were given same therapy as in the 1 st group (for 3 month) plus danzen ® tablet (serratiopeptidase) 5 mg orally 3 times per day during the 1 st month of treatment only. all patients were followed up in out patient clinic by a dermatologist after 1 st week of treatment and once weekly for the 1 st month and then monthly in the next 2 months.clinical improvement based on: subside of inflammation, stop appearing of new lesions and finally acne subsides and starts remodeling with removal of residual hyperpigmentation. improvement more than 85% considered excellent, (65 – 85%) moderate, (50 – 65%) mild, while less than 50% considered as bad improvement. results a majority of patients showed significant (x2 = 4.0 , p = 0.05 ) mild – moderate improvement after 2 weeks in danzen treated group compared to control group; however there is non significant difference in this period between the 2 groups in term of excellent improvement. mean while, after 4 weeks, there is insignificant difference in the improvement of acne at different level between two groups, despite the significant difference ( x2 = 3.857 , p = 0.05 ) in excellent improvement between treated and control groups.after 4 weeks of treatment (danzen was stopped), recurrence reported in two patients who developed only mild improvement during the 1 st month.only one patient didn’t show any improvement ( resistant nodulocystic acne ) , another one patient suffer from git upset and erythematous papules in the body due to danzen use which led to stop danzen treatment just after 2 weeks despite his moderate improvement in acne appearance ( see table1 and 2 and figure 1 ). after 3 months ( at end of clinical trial ) despite non significant difference between treated and control group in number of acne patients that improved or showed excellent improvement , there is a reasonable percent of difference as shown in table 3 and 4 . http://en.wikipedia.org/wiki/protease http://en.wikipedia.org/wiki/enterobacteria http://en.wikipedia.org/wiki/alternative_medicine iraqi j pharm sci, vol.21(1) 2012 serratiopeptidase in acne 80 table 1: improvement in danzen treated group. number of weeks bad improvement no. of cases that show mild improvement no. of cases that show moderate improvement no. of cases that show excellent improvement 1 20 4 1 nill 2 9 12 3 1 3 5 4 8 8 4 2 3 5 15 8 3 1 3 18 12 2 1 1 20 table 2: improvement in control group. number of weeks bad improvement no. of cases that show mild improvement no. of cases that show moderate improvement no. of cases that show excellent improvement 1 23 2 0 0 2 18 5 2 0 3 11 8 4 2 4 6 8 5 6 8 5 5 2 13 12 4 3 2 16 table 3: percent of improvement in danzen treated group number of weeks % of patients that showed bad improvement % of patients that show mild improvement % of patients that show moderate improvement % of patients that show excellent improvement 1 80 16 4 0 2 36 4 12 4 3 20 16 32 32 4 8 12 20 60 8 12 4 18 72 12 8 4 4 80 table 4: percent of improvement in control group number of weeks % of patients that showed bad improvement % of patients that show mild improvement % of patients that show moderate improvement % of patients that show excellent improvement 1 92 8 0 0 2 72 20 8 0 3 44 32 16 8 4 24 32 20 24 8 20 20 8 42 12 16 12 8 64 figure 1: comparison between the numbers of improved patients of the 2 groups that treated with or without danzen 0 0 16 7 23 19 22 20 23 21 0 5 10 15 20 25 0 week 2weeks 4weeks 8 week 12weeks danzen control number of improved patients iraqi j pharm sci, vol.21(1) 2012 serratiopeptidase in acne 81 discussion the results of this study showed that the effect of serratiopeptidase as adjuvant therapy for acne treatment result in a significant rapid improvement , this might be explained by serratiopeptidase ability in enhancing antibiotic efficacy as was shown in other related studies to staphylococcus infections. (12) the addition of serratiopeptidase not only result in rapid improvement but also increase the possibility of excellent improvement in acne appearance which may be due of it’s anti-inflammatory, anti-edemic and fibrinolytic activities and it’s ability to act rapidly on localized inflammation. (11) in addition to that serratiopeptidase have synergistic effect with antibiotics. (13) after 12 months ( at end of clinical trial ) there is no significant difference between the 2 groups , this simply can be explained by the short period ( only one month ) usage of serratiopeptidase , however additional studies on large scale and for longer period of time are needed to confirm the benefit of addition of serratiopeptidase to acne treatment. conclusion serratiopeptidase was found to create a good hope as additional therapy for complicated acne vulgaris and bring a rapid and better improvement in treating acne; however, more studies on large scale and for longer period are needed to confirm this result. reference 1. arndt, kenneth a., hsu, jeffrey t.s: manual of dermatologic therapeutics. 7th edition: lippincott williams & wilkins; 2007. p. 4-19. 2. laura j savage and alison m layton: treating acne vulgaris: systemic, local and combination therapy. expert rev clin pharmacol. 2010;13(4):563-580 3. wolff, klaus , goldsmith, lowell a., et al. : fitzpatrick’s dermatology in general medicine , 7 th edition : mcgraw-hill ; 2008;p.690 – 703. 4. richard weller , j.a.a. hunter, j.a. savin, etal. : clinical dermatology , 4 th edition : blackwell publishing ; 2008; p. 164. 5. gollnick h, cunliffe w, berson d, dreno b, finlay a, leyden jj, et al. management of acne: a report from a global alliance to improve outcomes in acne. j am acad dermatol 2003; 49: s1–37. 6. british medical association and the royal pharmaceutical society of great britain. british national formulary. 57th ed. uk: bmj publishing group. 2009. p. 637. 7. thiboutot d, gollnick h, bettoli v, et al. new insights into the management of acne: an update from the global alliance to improve outcomes in acne group. j am acad dermatol 2009;60(5 suppl):s1– s50. copyright elsevier 2009. 8. skidmore r, kovach r, walker c, et al. effects of subantimicrobial-dose doxycycline in the treatment of moderate acne. arch dermatol 2003;139: 459†“ 464 . 9. nakahama k, yoshimura k, marumoto r, kikuchi m, lee is, hase t, matsubara h. : cloning and sequencing of serratia protease gene: nucleic acids res. 1986; 14 (14): 5843-5855. 10. kokate ck, purohit ap, gokhale sb. pharmacognosy (14th ed.). mumbai: nirali prakashan. : 2008; p. 12.11 11. mazzone a, catalani m, et al. : evaluation of serratia peptidase in acute or chronic inflammation of otorhinolaryngology pathology: a multicentre, double-blind, randomized trial versus placebo: j int med res. 1990 sep-oct;18(5):379-88. 12. mecikoglu m, saygi b, yildirim y, karadag-saygi e, ramadan ss, esemenli t.: the effect of proteolytic enzyme serratiopeptidase in the treatment of experimental implant-related infection: j bone joint surg am. 2006 jun;88(6):1208-14. 13. jyothi penta, kiran kumar jannu, radhika musthyala: antimicrobial studies of selected antibiotics and their combination with enzymes: international journal of pharmacy and pharmaceutical sciences issn-2010; 2, issue 3 :0975-1491. http://www.ncbi.nlm.nih.gov/pubmed?term=%22mazzone%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22catalani%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22mecikoglu%20m%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22saygi%20b%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22yildirim%20y%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22karadag-saygi%20e%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22ramadan%20ss%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22esemenli%20t%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22esemenli%20t%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed/16757752?log$=activity## http://www.ncbi.nlm.nih.gov/pubmed/16757752?log$=activity## iraqi j pharm sci, vol.19(2) 2010 59 anti-bacterial properties of melatonin against mycobacterium tuberculosis in vitro thamer m. jasim*, mustafa g.alabbassi** , suhad f. hatem almuqdadi* and jinan k. kamel*** * department of microbiology and biotechnology,al-mustansyria,university,college of pharmacy, baghdad, iraq. ** department of pharmacology and therapeutics, al-mustansyria university,college of pharmacy, baghdad, iraq. *** national reference laboratory abstract 57 isolates of mycobacterium tuberculosis and mycobacterium bovis were identified; they were isolated from different clinical sources which included sputum, bronchial wash, abscess, pleural fluid, gastric fluid, eye fluid, and csf, also urine and ear swab. this investigation was carried out on 198 patient attended national reference laboratory for t.b during september 2009. also the study declared that the ratio of separation of this bacterium from male was (67.6%) and it’s higher than the ratio of separation this bacterium from females which was (32.3%). the susceptibility of mycobacterium tuberculosis to melatonin was evaluated. many concentrations of melatonin were prepared to investigate it as antibacterial drug against multidrug resistant mycobacterium tuberculosis and mycobacterium bovis. suspension bacteria (10 -1 , 10 -3 and 10 -5 ) were cultured on lowensteinjensen media (lj) contains melatonin, while control media without this drug. six isolates were chosen according to their susceptibility patterns; they were resisting to rifampicin, streptomycin, isonicotinic –hydrazide and sensitive to ethambutol. in conclusion, these in vitro studies clearly demonstrate antibacterial effects of melatonin. among possible mechanisms, it is concluded that melatonin showed antibacterial effects against multidrug resistant t.b by reducing intracellular substrates. identifying the mode of action could be of great help in developing and researching new anti-bacterial drugs. key words: antibacterial, melatonin, mycobacterium tuberculosis. الخالصة يٍ انًصادس انسشٌشٌت انًخخهفت وانخً يىصبت يٍ انعصٍاث انذسٍَت انفطشٌت وانعصٍاث انذسٍَت انبقشٌت عضنت 75 شخٍصحى ح هزِ انذساست عهى َفزثويسحت األرٌ. ، انغسم انقصبً، انخشاس، سائم انضُب وانًعذة وانعٍٍ وانُخاعً كزنهك االدساس انقشعحضًُج %( 65.6)نزكىساٌ َسبت اصابت اوضحج انذساست أ .9009 شهش اٌهىلخالل خذسٌنه انًشصعً بش انىطًُساصع انًخخيشٌض 891 حشاكٍض يٍعذة انًٍالحىٍٍَ. حى اسخخذاو حى حقٍٍى حساسٍت انعصٍاث انخذسٍَت نعقاس %(:.9:) األَاد هً اعهى يٍ َسبت اصابت ج عذة صسع انًقاويت انًخعذدة نهًضاداث. راث m. bovisو m. tuberculosisضذ عصٍاث انخذسٌ هبكخشٌان انًٍالحىٍٍَ كًضاد 10 ) حخافٍف نهعانك انبكخٍشي -5 ,10 -3 ,10 -1 الٌحىي ًٍالحىٍٍَ بًٍُا وسظ انسٍطشة عقاس ان وانزي ٌحخىي عهى (lj) وسظ عهى ( يقاويت نهشٌفايبسٍٍ، انسخشبخىياٌسٍٍ، هزِ انعضالث حٍذ كاَج باألعخًاد عهى األًَاط انخحسسٍت عضالث اخخٍشث سخت. هزا انعقاس عهى انبكخشٌا نهًٍالحىٍٍَ انخزبٍطً نالٌزايبٍخىل. َسخُخش يٍ هزِ انذساست انخارٍشوحساست ، inhاٌضوٍَكىحُك هاٌذساصاٌذ بكخٍشي صذٌذ. فاق نًضاداَِ عهى داخم انخهٍت نهبكخشٌا وبزنهك ٌفخح بىاسطت حأرٍش وانًٍكاٍَكٍت انًحخًهت يخخبشٌا introduction although tuberculosis (tb) is a preventable and treatable disease, it causes 3 million deaths annually. the current situation of tb is unique, mainly due to two aspects: the association between tb and human immunodeficiency virus (hiv) infection, and the global spread of virulent strains resistant to key antituberculous drugs. there is a direct association between hiv infection and the reactivation of latent tb or the progression of tb following newly acquired infection (1) . melatonin originally identified as an effector for circadian rhythms, is now known to be a hormone involved in a vast range of homeostasis maintenance activities, for example seasonal timing, sexual development, the antioxidant defense system and immune response (2) . melatonin is synthesized from tryptophan within the serotonin pathway mainly in the pineal gland, and in a number of extrapineal organs such as retina, lens, bone marrow, intestine, skin and so on. to date, three mammalian melatonin receptors, gprotein coupled receptors mt1 and mt2, and a quinone reductase family receptor, mt3, have been identified (3) . melatonin is a highly studied endogenous molecule. this indolamine has a variety of beneficial effects within cells and organisms, including cell cycle regulation (4) . although there are a plethora of studies on melatonin, only a few of them relate to it in vitro antimicrobial activities (5–7) on to its effects in infectious diseases (8-10) . 1corresponding author email : received : 10/4/2010 accepted : 18/9/2010 iraqi j pharm sci, vol.19(2) 2010 60 new in vitro antimicrobial studies using melatonin suggested that it has limited antimicrobial properties (11, 12) while one study found that melatonin inhibited candida albicans at a concentration of 300 µg/ml (12) .in contrast to the in vitro studies, virtually all in vivo studies performed with melatonin in infectious disease models documented it as a successful therapy (9, 13) . melatonin is a highly versatile molecule. one example is its ability to limit the growth of a variety of tumor types. one of several proposed mechanisms to explain melatonin’s inhibitory actions on cancer growth is its ability to curtail the uptake of growth factors which promote cell proliferation (14) . linoleic acid (la), an essential omega-6 polyunsaturated fatty acid is a growth factor for a number of tumor types. via an action on the cell membrane, melatonin prevents the uptake of this fatty acid by cancer cells which reduces the activation of genes that promote cell proliferation (15) . similar actions of melatonin on the bacterial wall thus may restrict the survivability of bacteria. additionally, melatonin has a high metal binding capacity. melatonin binds iron (iii), copper and zinc thereby reducing their cytoplasmic availability. bacteria are strongly dependent on free metals, in particular, free iron for growth (16) . clearly, there are several potential mechanisms that may explain the possible antibacterial efficacy of melatonin. in the current study we tested the antimicrobial effects of melatonin against resistant strains of mycobacterium tuberculosis. materials and methods samples a total of 198 clinical samples from national reference laboratory for t.b, were included in this study. they were collected during november 2009. identification of these isolates as mycobacterium tuberculosis and mycobacterium bovis were based on biochemical properties (17) . the most samples were sputum; they are transfer to the laboratory in sterile container. three samples from each patient were taken to diagnose tuberculosis. susceptibility test was done to 13 isolates from 57 positive samples to tuberculosis according to routine work of national reference laboratory for tuberculosis. all samples treat with sodium hydroxide and hydrochloric acid to remove all microorganisms and epithelial cells (petroffs method). zeihl-neelsen stain was done for all specimens. culture the specimens the specimens was cultured on lowenstein-jensen media (lj) pouring in screw –capped tubes in final volume 6ml, these tubes put in oven at 80-85 c for 45 min. the media left for 24 hr to be insure that there is no contamination. the treated specimens will culturing by using pasture pipette, (0.4-0.2) ml from inoculme of specimen must be transferred to lj media and let the culture for 72 hr at 37 c in incubators( in slope position), then for 50 days at 37 c ( in vertical position). susceptibility test susceptibility test was done by using the proportional method (18) , four antibiotic solutions were used; rifampicin 40 µg/ml, streptomycin 4 µg/ml, ethambutol 2 µg/ml, isonicotinic hydrazide 0.2 µg/ml. mycobacterium tuberculosis suspension was prepared in many dilutions 10 -1 -10 -5, the primary dilution adjusted with macferland solution(3×10 8 ) cfu/ml. 100 µl from 10 -1 , 10 3 and 10 -5 dilution was cultured on media (lj) contain antibiotics and without antibiotics as control. after 6-8 weeks the result must be read, if the growth on media contains antibiotics more than control media this means resistant bacteria. while the growth consider sensitive when the growth less than control media, for this test we chose 4 isolates from mycobacterium tuberculosis and 2 isolates from mycobacterium bovis, according to their typing (catalase, nitrase test, niacin test and colony form). melatonin drug in this study, many concentration of melatonin dissolved in 96% ethanol (0.4, 0.2, 0.1, 0.05) mg/ml were prepared to investigate it as antibacterial drug against multi-drug resistant mycobacterium tuberculosis and mycobacterium bovis.suspension bacteria (10 1 , 10 -3 and 10 -5 ) were cultured on lj media contains melatonin, while control media without this drug. six isolates were chosen according to their susceptibility patterns, they were resist to rifampicin, streptomycin, isonicotinic –hydrazide and sensitive to ethambutol (19) . results table -1 showed us the number of mycobacterium tuberculosis isolated from many sources. it was found that 57 samples were positive samples. table-2 shows number and percentage of infection with m. tuberculosis and m. bovis according to the sex, it was found that 134(67.6%) of patients were males and 64(32.3%) were females with present of statistical significant. table-3 shows the percentage of infected with m. tuberculosis and m.bovis according to the age. the higher percentage rate (46.2%) was found in the group of (30-39 year) while the lowest iraqi j pharm sci, vol.19(2) 2010 61 percentage rate (6.7%) was found in the group (70≥ year) with present of statistical differences. table-3 shows the number and percentage of infection with mycobacterium spp. according to the sex, it was found that 134 (67.6%) of patients were males and 64(32.3%) were females with present of statistical significant differences. table-4 shows the effect of many concentration of melatonin on m. tuberculosis and m.bovis isolates. it was found that m. tuberculosis and m.bovis sensitized to the concentrations 0.05, 0.1, 0.2 and 0.4mg/ml. table-5 shows the susceptibility patterns of many dilution of m. tuberculosis and m. bovis to different concentration of melatonin. it was found that dilution of bacteria 10 -1 , 10 -3 and 10 -5 were sensitized to all concentrations of melatonin that were used. figure -1 shows the growth of m. tuberculosis and m bovis. resistant to rifampicin, streptomycin and inh and were sensitize to ethambutol.and melatonin. table 1 : number of m.tuberculosis and m.bovis isolated from many sources isolates source number of examined samples number of positive sample no. (%) sputum 145 55 96.5 bronchial wash 19 1 1.8 abscess 5 1 1.8 pleural fluid 13 gastric fluid 3 eye fluid 2 knee fluid 1 c.s.f 1 ear swabs 1 urine 8 total 198 57 table 2 : number and percentage of infection with m. tuberculosis and m. bovis according to the sex table 3 : percentage of infected with m. tuberculosis and m.bovis according to age. age groups number of examined samples number of positive sample no. (%) 10< year 6 0 0 10-19 year 16 6 *37.5 % 20-29 year 36 14 38.9 % 30-39 year 39 18 46.2 % 40-49 year 29 6 37.5 % 50-59 year 41 8 19.5 % 60-69 year 16 4 25 % 70≥ year 15 1 **6.7 % total 198 57 ** statistically significant difference table 4 : effect of many concentration of melatonin on mycobacterium spp. isolates code of isolates melatonin concentration (mg/ml) (0.4) (0.2) (0.1) (0.05) control s s s s r l2 s s s s r l3 s s s s r l4 s s s s r l5 * s s s s r l6 * s s s s r l =isolate, s =sensitive, r=resist, *= m. bovis table 5 : susceptibility patterns of many dilutions m.tuberculosis and m.bovis to many concentration of melatonin dilution of bacteria melatonin concentration mg/ml (0.4) (0.2) (0.1) (0.05) control 3×10 7 cfu/ml (10 -1 ) s s s s r 3×10 5 cfu/ml (10 -3 ) s s s s r 3×10 3 cfu/ml (10 -5 ) s s s s r sex of patients examined samples number of positive m. tuberculosis isolates number of positive m. bovis isolates no. % no. % no. % male 134 67.7 23 65.7 14 63.6 female 64 32.3 12 34.3 8 36.4 total 198 35 22 iraqi j pharm sci, vol.19(2) 2010 62 figure 1 : growth of m.tuberculosis resists to rifampicin,streptomycin, and inh and was sensitized to ethambutol and melatonin on a lowesten agar slant (from the left to the right). discussion the results of the present study indicate that melatonin has in vitro antimicrobial activity against strains of antibiotic-resistant mycobacterium tuberculosis. as melatonin is weakly soluble in water, investigators generally use ethanol to dissolve the indolamine. the antibacterial effects of melatonin could be a result of the metal binding capacity of the indolamine. normally, tissue fluids contain unsaturated iron-binding proteins including transferrin in plasma and lymph and lactoferrin in other secretions such as milk or mucous (20, 21) . these proteins ensure that the concentration of free iron in these fluids is virtually zero. this is essential for the normal bactericidal and bacteriostatic effects of plasma and extracellular fluids. if iron becomes freely available, the antibacterial effects of these fluids are lost. this can lead to rapid extracellular bacterial growth and a major increase in bacterial virulance. pathogenic bacteria have also ways of extracting essential iron from the low iron environment in vivo via siderophores such as enterochelin, which can remove iron from unsaturated transferrin or lactoferrin (22) . an intriguing aspect of this issue is that, because iron is absolutely essential for bacteria, it could make the development of bacterial resistance very difficult for any organism deprived of iron. of the concern being expressed over the increasing resistance to antibiotics now being encountered, particularly within hospitals, studies on the development of novel drugs against both iron transport and/or intrabacterial free iron availability should be undertaken. melatonin reportedly has a high metal binding capacity including iron. limson et al. (23) observed that melatonin and its precursors exhibited the ability to bind metals in situ. gulcin et al. (24) also noted that melatonin is an effective metal chelating agent. this feature of melatonin has been typically thought to be related to the antioxitant properties of the indole by making transition metals unavailable for the fenton reaction. however, in case of bacterial growth, an agent which binds free iron in the cytoplasm has great importance. as melatonin easily passes all biological barriers including bacterial cell wall, it may bind free iron in the cytoplasm and restrict bacterial growth via this mechanism. in the present study, melatonin was tested against resistant mycobacterium tuberculosis. in gram-negative bacteria, the cell envelope is composed largely of protein glycopeptide, lipopolysaccharide, and also, substantial amounts of lipid (25) . melatonin has been shown to limit the uptake of la and total fatty acids by human breast cancer cells. this feature may also work against an extremely fast dividing prokaryote. konar et al. (7) reported that melatonin, at the concentration of 1000 µg/ml, significantly reduced the lipid level of sacchoramyces cerevisae. furthermore in the same study, melatonin, at concentration of 300 µg /ml, was shown to be one of the most effective agents in reducing lipid levels of candida albicans. in organisms, melatonin can be administered via any of several routes, e.g. orally, sublingually, etc., and it is available either as an over-thecounter supplement or as a prescription drug (depending on the country). the molecule has a long shelf-life and is inexpensive relative to conventional drugs used to treat the variety of bacterial infections.melatonin is also very safe with few side effects and a very wide safety margin. the current in vitro studies should be expanded to investigate the efficacy of melatonin as an in vivo antibiotic. it has been previously shown that melatonin is beneficial in newborn humans suffering from septicemia (26) . those findings coupled with the data reported here suggest further investigations of the role of melatonin in reducing bacterial growth. references 1. abate g, hoffner s, stegard v, mqrner h. characterization of isoniazide-resistant starins of mycobacterium tuberculosis on the basis of phenotypic properties and mutations in katg. eur j clin microbiol infect dis , 2001; 20: 239-333. 2. mohd. a, yoshinao a, hajime f, tomoyuki m et al. serotonin and melatonin, neurohormones for homeostasis, as novel iraqi j pharm sci, vol.19(2) 2010 63 inhibitors of infections by the intracellular parasite chlamydia. journal of antimicrobial chemotherapy,2005;10:1-8. 3. chan as, lai fp, lo rk et al. melatonin mt1 and mt2 receptors stimulate c-jun n-terminal kinase via pertussis toxinsensitive and insensitive g proteins. cell signal, 2002; 14: 249–57. 4. reiter rj, gultekin f, flores lj et al. melatonin: potential utility for improving public health. kor hek, 2006; 5:131–158. 5. wang hx, liu f, ng tb. examination of pineal indoles and 6-methoxy-2 benzoxazolinone for antioxidant and antimicrobial effects. comp biochem physiol c toxicol pharmacol, 2001; 130:379–388. 6. ozturk ai, yilmaz o, kirbag s, arslan m. antimicrobial and biological effects of ipemphos and amphos on bacterial and yeast strains. cell biochem funct, 2000 ; 18 : 117–126. 7. robertson gt, doyle tb, du q, duncan l, mdluli ke, lynch as: a novel indole compound that inhibits pseudomonas aeruginosa growth by targeting mreb is a substrate for mexab-oprm. j bacteriol, 2007; 189: 6870–6881. 8. grandgirard d, leib sl. strategies to prevent neuronal damage in paediatric bacterial meningitis. curr opin pediatr, 2006; 18:112–118. 9. valero n, espina lm, mosquera j. melatonin decreases nitric oxide production , inducible nitric oxide synthase expression and lipid peroxidation induced by venezuelan encephalitis equine virus in neuroblastoma cell cultures.neurochem res , 2006 ; 31: 925– 932. 10. valero n, marinaespina l, bonilla e, mosquera j. melatonin decreases nitric oxide production and lipid peroxidation and increases interleukin-1 beta in the brain of mice infected by the venezuelan equine encephalomyelitis virus. j pineal res, 2007; 42:107–112. 11. wang hx, liu f, ng tb. examination of pineal indoles and 6-methoxy-2 benzoxazolinone for antioxidant and antimicrobial effects. comp biochem physiol c toxicol pharmacol, 2001; 130:379–388. 12. konar vv, yilmaz o, ozturk ai et al. antimicrobial and biological effects of bomphos and phomphos on bacterial and yeast cells. bio-organic chemistry, 2000; 28:214–225. 13. reynolds fd, dauchy r, blask d et al. the pineal gland hormone melatonin improves survival in a rat model of sepsis/ shock induced by zymosan a. surgery 2003; 134:474–479. 14. reiter rj. mechanisms of cancer inhibition by melatonin. j pineal res, 2004; 37:213–214. 15. blask de, dauchy rt, sauer la. putting cancer to sleep at night: the neuroendocrine / circadian melatonin signal. endocrine, 2005; 27:179–188. 16. ward cg, bullen jj, rogers hj. iron and infection: new developments and their implications. j trauma, 1996; 41:356– 364. 17. vestal al. in: procedure for the isolation and identification of mycobacteria. us department of health, education and welfare pub no. (cdc) 77 8230. atlanta, georgia: centers for disease control and prevention; 1977. p. 15-90. 18. guidelines for surveillance of drug resistance in tb 1997, who, iuatld. 19. canetti g, fox w, khomenko a, mahler ht, menon nk,mitchison da, et al. advances in techniques of testing mycobacterial drug sensitivity and the use of sensitivity tests in tuberculosis control programmes. bull world health organ, 1969; 41 : 21-43. 20. omer faruk, recaiogur1, ahmet korkmaz, abdullah kilic,russel j.reiter . melatonin as an antibiotic: new insights into the actions of this ubiquitous molecule. j.pineal res, 2008; 44:222–226. 21. bullen jj, rogers hj, spalding pb, ward cg. iron and infection: the heart of the matter. fems immunol med microbiol, 2005; 43:325–330. 22. bullen jj, rogers hj, spalding pb, ward cg. natural resistance, iron and infection: a challenge for clinical medicine. j med microbiol 2006; 55:251–258. 23. limson j, nyokong t, daya s. the interaction of melatonin and its precursors with aluminium, cadmium, copper, iron, lead, and zinc: an adsorptive voltammetric study. j pineal res, 1998; 24:15–21. 24. gulcin i,buyukokuroglu me, kufrevioglu oi.metal chelating and hydrogen peroxide scavenging effects of melatonin. j pineal res 2003; 34:278–281. 25. hebeler bh, chatterjee an, young fe. regulation of the bacterial cell wall: effect of antibiotics on lipid biosynthesis. antimicrob agents chemother 1973; 4:346–353. 26. gitto e, karbownik m, reiter rj et al. effects of melatonin treatment in septic newborns. pediatr res, 2001; 50:756–760. iraqi j pharm sci, vol.31( 2 ) isolation of catchin and epigallocatchin from iraqi r.coriaria doi: https://doi.org/10.31351/vol31iss2pp271-282 271 isolation of catchin and epigallocatchin from iraqi rhus coriaria by preparative high-performance liquid chromatography (phplc) nabaa m. ibrahem *,1, enas j. kadhim*, shihab hattab mutlag ** * department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract two compounds were isolated from the fruit part of rhus coriaria that grew wildly or cultivated in the north of iraq. the compounds were separated by preparative high-performance liquid chromatography and their structures were established based on detailed spectroscopic techniques like ftir and lc-.ms/ms. keywords: rhus coriaria, preparative hplc, lc-m/sms, ftir. phplc عزل كاتجين و ايبيكالوكاتجين من نبات السماك العراقي بواسطة ** و شهاب حطاب مطلك *ايناس جواد كاظم ، 1*، نبأ محمد ابراهيم العراق ، بغداد ، جامعه بغداد ، الصيدلة كلية فرع العقاقير والنباتات الطبية ، * العراق ، بغداد ، جامعه بغداد ، الصيدلة كلية ، االدوية والسموم فرع ** الخالصة وتم اخضاع (phplc) تم عزل مركبين من ثمار السماك العراقي الذي ينمو بشكل عشوائي / او مزروع بالقرب من القرى في شمال العراق بواسطة , جهاز" (hplc)اليه االداءالع المركبات المعزوله للعديد من التقنيات التحليليه والكيميائيه والكروماتو جرافيه و الطيفيه للتعرف عليها مثل كروماتوغرافيا السائله (lcmsms). , الكروماتوغرافيا السائله مع طيف الكتلة (ftir) الفوربيه" لتحويل طيف االشعه تحت الحمراء رافيا السائلة ( , الكروماتوغftir, طيف االشعه تحت الحمراء )) phplcالكلمات المفتاحية : نبات السماك , كروماتوغرافيا السائله عاليه االداء التحضيري) (. lc-ms-msمع مطياف الكتلة ) introduction medical plants have been used since ancient time for the treatment a variety of disorders, and in some countries, the use of medical plants is often associated with witchcraft and superstition, because people did not have the scientific insight to describe and predict the healing action of plants (1). local environmental purse derived from plants have play an important role in the provision of dietary and medical care for humans in many parts of the world. a very important factor buckle down to people’s interest in wild plants as food are times of famine or food scarcity, but on the other hand, eating wild products is becoming neat in our modern society (2). sumac is the common name of the rhus genus, which comprises 91 species names in the anacardiacea family, represented in iraq by one species namely r. coriaria l. which sprout wildly or cultivated in the north of iraq (3) .the name "sumac" comes from "summāq" which means "dark red" in arabic and syriac. r. coriaria has been used in spice blends and in traditional medicines for hundreds of years. the word "sumac" will be henceforth used to describe the spice product of rhus coriaria (4). rhus coriaria has long been used as a condiment spice, either alone or in combination with other spices. traditionally in iraq, it is used as a spice especially along with famous rich dishes like kabab and grilled meat as well as over salads that often accompany these dishes(3).the research efforts on sumac extracts to date show a promising potential for providing renewable bio products with the following reported bioactivities: antiinflammatory, antifungal, antifibrogenic, antimalarial, antimutagenic and antimicrobial .sumac had been used as traditional source of medication in different dietary cultures all over the world, the use of the plant in seasonings and flavoring agents has been the mainstay of indigenous remedies across the world (5). r. coriaria is used as a spice, and has been used in cooking for millennia. about two thousand years ago, the greek physician pedaniu dioscoride (40-90 a.d.) wrote in his voluminous "de materia medica" ("of medical matters") about the benefit properties of sumac, principally as a diuretic and anti-flatulent (6). traditionally ,sumac has been used in the treatment of diarrhea (7), hemorrhoids (8), ulcer (9), liver disease (10), animal bites, pain (11), diuresis, dysentery, hemorrhage, hematemesis, hemoptysis, ophthalmia, conjunctivitis, leucorrhea, and stomach tonic. (12). medical practitioners have also used sumac for cholesterol reduction (13), in the treatment of sore throat, and as an abortifacient (7). other reports also show its use in wound healing and as an antimicrobial (14). 1corresponding author e-mail: nabaaibrahim@copharm.uobaghdad.edu.iq received:23 /11 /2021 accepted: 1/2 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp271-282 iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 272 different parts of the plant have been used in various preparations in traditional system of medicine. powdered bark of sumac is effective for whitening the teeth. bark infusion is useful in early treatment of viral eye infections. bark is contused in water and put on the forehead for the first-aid treatment of epistaxis (15).sumac fruits powder are sprinkled on boiled egg and orally taken for the treatment of diarrhea (16). a decoction of sumac fruits is prepared and administered orally (150 cc) for the treatment of liver disease, diarrhea and urinary system disorders. decoction is taken three times per day until improvement occurs (7). the plant is globally distributed in temperate climate and tropical regions and can grow on marginal lands. the most common species of sumac is rhus coriaria which is grown commercially on global scale in mediterranean and middle east. this species had been cultivated for several years to produce a material of high quality for tanning. it is growing wild in the region extending from the canary island over the mediterranean coastline to iran and afghanistan. it is native to the mediterranean and the southeastern region of turkey (17). in iraq, sumac distributed mainly in north, north east and north west, in areas of an altitude more than 540m above sea level. r. coriaria sprout in mountain environments especially in rawandowz city. sulimania, which are the areas of most prevalent territories (18). more than two hundreds compounds have been specified from the r. coriaria and most of them are physiologically active. these chemical constituents can be set to various classes of the tannins (condensed and hydrolysable), conjugated phenolic acids, phenolic acids ,anthocyanins, , organic acids ,coumarins, flavonoids, terpenoids, xanthones, steroids, essential oils , and other groups of compounds have been detected (19) . so in this study, an investigation for the phytochemicals in the fruit of this plant grown at iraq north regions had been done using several hyphenated techniques include preparative hplc, high-performance liquid chromatography coupled with diode array detector (dad), and liquid chromatography and mass spectroscopy is adopted. preparative hplc are used to separate a specified amount of pure compounds and hplc-dad is used to confirm the purity of collecting fractions. the molecular structure was assigned by ftir and ms provides useful information about molecular masses and the structure of the isolated constituents of the crude methanolic plant extract. materials and methods plant material collection. rhus coriaria fruit were collected from the north of erbil in rawendos at april 2020, . after collection of plant, it sent to be identified and authenticated by specialist prof.( dr. sukaena abass), department of biology /college of sciences/ university of baghdad .the plant parts were cleaned thoroughly, dried under shade, and ground in a mechanical grinder to a fine powder. extraction method a-the air-dried powder of the fruit is weighted then defatted with n-hexane to get rid of chlorophyll and waxy material and then extracted in soxhlet with (1:10) aqueous methanol for 18hr then the extract is combined and dried by a rotary evaporator the dry residue is weighted and the yield of extraction is calculated. the dry fruit extract is suspended in water, then treated with various reagents to screen the type of phytochemicals present and then partitioned 2-3 times with solvent of various hydrophilicity such as n-hexane, chloroform, ethyl acetate and n-butanol. each fraction is dried and weighted and treated with screening tests to identify the phytochemicals present (20, 21). the ethyl acetate fraction is dried and weighted to identify the phytochemicals present and isolation different constituents using chromatographic techniques, preparative hptlc. identification of isolated compounds had been done by using different chromatographic and spectroscopic techniques like ftir and lc/ ms/ms. the preparative hplc specification and conditions table 1 represents the preparative hplc apparatus specification which consists of an autosampler, a pump, and a photodiode array detector coupled with an analytical workstation. table 1.preparative hplc apparatus specification component model or version company and origin 1 binary high pressure gradient pump p6.1l knuaer , germany 2 diode array detector dad 2.1l knuaer , germany 3 sample loop (20 µl) and injector d1357 knuaer , germany 4 analyses and system control software claritychrom , v 7.4.2.107 dataapex, czech republic 5 fraction collector foxy r1 teledyne isco, usa 6 mass spectrometry (lc/ms/ms) sciex-abi 3200 ms system sciex technologies, germany iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 273 the condition of hplc-dad analysis. the high-performance liquid chromatography was done by using stationary phase on acclaim c 18 column (5 μm particle size,250 x 4.6 mm), dionex ultimate 3000 liquid chromatograph. the mobile phase consist of 1% aqueous acetic acid solution (solvent a) and acetonitrile( solvent b) using gradient elution. the flow rate was kept to 0.7 ml/min, the column was thermostatically controlled at 2800c and the injection volume was adjusted at 20 μl. the gradient elution was changed from 10 % to 40% b in a linear manner for duration of 28 min, from 40 to 60 % b in 39 min, from 60 to 90 % b in 50 min. the mobile phase composition back to initial condition (solvent b: solvent a: 10: 90) in 55 min and pliable to run for another 10 min, before the injection of another sample. total time for analysis per sample was 65 min. the separation chromatograms were detected using a photo dad array ultraviolet detector at three different wavelengths (272, 280 and 310 nm) according to absorption maxima of analyzed compounds (22). able 2. the gradient elution time and flow rate of hplc analysis time mobile a concentration % mobile b concentration % flow rate ml/min 0 90 10 0.7 5 90 10 0.7 28 60 40 0.7 39 40 60 0.7 50 10 90 0.7 55 90 10 0.7 fraction collector factors peak recognition: slope 0.2 au/min , level 10 mau fraction size: 5 milliliters the detection of isolated compounds (compound i and ii) were performed by matching retention time and absorbance spectrum of the standards (epigallocatechin and catechin). the quantitative analysis of the detected components were calculated by serial concentrations of standard materials to build calibration curve between concentration and its equivalent peak area, the calculation was done according to the straight line equation y=ax +b, slop=y/x (yand b: b: y-intercept), where r2 regression factor. identification of compound by mass spectrometry apparatus and instrumentation conditions of -mass spectrometry (lc/ms) direct infusion scan were applied as follow: thems was performed using a sciex-abi 3200 ms system (sciex technologies, germany). the column jumper is used to connect liquid chromatography directly to the mass detector. using the auto sampling device, a sample volume of 20-μl was injected. solvent acontained millipore water with 5.0mm ammonium format/0.1%formic acid and solvent bconsisted of hplc-grade acetonitrile with 0.1% formic acid. the lc/ms was operated in the positive-ion mode with m/z scan range 50-900. the spectra of different peaks were studied in terms of lc-ms/ms chromatograms collected for 2 isolated fractions. the molecular masses of each product are calculated using a continuum. for molecular mass estimation, only molecular ion peaks were evaluated in each chromatogram. lc-ms/ms – spectra may be a valuable instrument to classify molecular mass components. the composition of a compound can be shown by the fragmentation patterns of different units. unknown compound fragmentation results have been compared to the already available data in the literature. acquisition method: \lcms-esi-pos. dam and all data had been shown in table (3) iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 274 table 3. time and flow rate of analyzed compounds step table: step total time(min) flow rate(µl/min) a (%) b (%) 0 0.00 1000 90 10 1 4 15 1000 90 10 2 20 1000 10 90 3 22 1000 10 90 4 5 25 1000 90 90 10 a= 5.0mm ammonium format/0.1%formic acid , b= acetonitrile/0.1formic acid , scan type: q1 ms (q1) range 50900 m+1, polarity: positive – esi, scan mode: profile . instrument specifications and details :software = analyst 1.6.4 version , pump agilent 1200 g1312a , column oven agilent 1200 g1316a , autosampler agilent 1200 g1367b , mass spectrometer api 3200 ab-sciex fourier transforms infrared (ft-ir) spectroscopy infra-red (ir) spectra of isolated compounds were recorded in shimadzu ftir spectrometer in the range of wave number 500 to 4000 cm–1 and operated in the transmittance mode. the mold and press of a pulver containing approximately 1 mg of substance were then used for preparing the thin disk under anhydrous conditions. results and discussion the results of the performed phplc analysis of ethyl acetate extract from the plant rhus coriaria fruits, confirmed the stable retention time of compound i and ii in the extract which were comparable to retention times of authenticated reference standards at identical chromatographic conditions as shown in figure (1). in addition to that, there is a matching between the uv spectra of the detected isolated compounds with corresponding standards uv spectra as shown in figures (2 and 3) which demonstrate a good fitness between the detected compounds and the standards. these results revealed that sumac plant contain epigallocatechin and catechin. the phytochemical constituents in plant extract are usually available in low quantity. for this reason a sensitive instrument was used in this study for detection and isolation. in this research, the phplc is a flexible, stable, and strongly enhanced column chromatography method, a technique commonly used for the separation of natural materials. preparatory high-pressure chromatography is an excellent purification technique and has been developed to ensure that important materials in the chemical, medicinal, biotechnological, and biochemical industries are insulated and identified (23) . gong and his colleagues have been isolated seven catechin compounds from fresh tea leaves using semi-preparative liquid chromatography technique ( 24) . figure 1. preparative hplc chromatogram for ethyl acetate fraction iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 275 figure2. uv spectrum of ethyl acetate fraction matched with standard uv spectrum of epigallocatechin. figure 3. uv spectrum of ethyl acetate fraction matched with standard uv spectrum of catechin. quantities of the studied compounds were evaluated by constructing a calibration curve using plotted peak area versus mass concentration of each standard using 5 different values of concentration. then applying the straight line equation of the standard curve as shown in figures (4 and 5) . the quantitative phplc analysis of the isolated compounds revealed that the catechin has higher concentration than epigallocatechin in fruit extract of rhus coriaria plant as shown in table (4). figure 4 . standard curves of epigallocatchin iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 276 figure 5 . standard curves of catchin table 4. quantitative analysis of ethyl acetate fraction mg/ml µg/ml (x) peak area (y) ethyl acetate fraction 0.1099 109.90 661.58 epigallocatechin 0.19911 1198.66 3166.77 catechin catechin and epigallocatechin are belonged to flavanols group of flavonoids. they have two chiral centers, one on c2 and other on c3. structurally, catechin has trans configuration and epigallocatechin has cis configuration with additional hydroxyl group (figure 6) ( 25). . catechin epigallocatechin figure 6 . chemical structure of catechin and epigallocatechin identification of isolated compound by the spectroscopic techniques identification of an isolated compound is usually achieved by a combination of several important spectroscopic techniques, such as ir and lc-ms/ms. the compound of interest can be identified by comparing its characteristic features with literature values. (no need to mention figure). ir – spectroscopy the fourier transforms infrared spectroscopy of compound i (table 5, figure 7 ) and compound ii (table 6 and figure 8) showed the appearance of characteristic peaks at 31973375cm1 due to phenolic oh stretching. band (h-bonded). the peaks 1610 cm-1,1519 cm-1 for compound i and 1604 cm-1, 1558 cm-1 attributed to the alkene stretching of aromatic ring . on the other hand, bending vibration band of phenyl o-h group is represent by 1350 cm-1 peak for compound i and 1365 cm-1 peak for compound ii. the appearance of peak at (1276-1284 cm-1 ) is related to the stretching of cyclic c-o. it has been observed that all chemical compounds show marked selective absorption in the ir spectrum of any molecule and are the fingerprints for structural confirmation. ir spectroscope technology is a very important tool for identifying several varieties of plant constituents in phytochemical studies. it also helps in the systemic elucidation of new compounds in plants (26). accordingly, isolated compounds were subjected to ir spectroscopy. from interpretation of the ir spectra, we concluded that the ir spectra of compound i & ii have been contrasted with that of authentic samples epigallocatechin and catechin respectively figure 7 & 8). iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 277 figure 7. ftir of compound 1(epigallocatechin) table 5. ftir interpretation of compounds i group frequency wave number in cm – 1 interpretation 32173348 phenolic oh str. band (h-bonded) 3005 aromatic c-h str. 2954 asymmetric ch2 str. 2850 symmetric ch2 str. 1610, 1519 ar. c=c str. bands 1458 bending vibration band of ch2 1350 bending vibration band of phenyl o-h 1284 cyclic c-o str. vibration band figure 8. ftir of compound 1i (catechin) iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 278 table 6. ftir interpretation of compounds ii. group frequency wave number in cm – 1 interpretation 31973375 phenolic oh str. band (h-bonded) 2916 a symmetric str. band of ch2 2850 symmetric str. band of ch2 1604, 1558 ar. c=c str. bands 1365 bending vibration band of phenyl-o-h 1276 cyclic c-o str. vibration band 1458 c-h bending 0f ch2 identification of isolated compound by lcms/ms spectroscopy identification of peaks of catechin and epigallocatechin isolated from rhus coriaria were carried out by comparing mass spectra of standard data and literature (27) . the mass spectrum of epigallocatechin was presented in( figure 9,10). the peak of protonated ion [м+h]+ occurred at m/z 307. while, the mass spectrum of catechin was presented in (figure 11,12) which show the m/z signal at 291 that represent a typical fragment ion detected in the mass spectrum of catechin (28,29). from the result,compound i according to ftir and lc-msms identical to epigallocatechin and compound ii according to ftir and lc-msms identical to catechin. lc-ms/ms is also a powerful analytical method that combines the physical separating capacity of fluid chromatography with the mass analysis capacities of mass spectrometry, providing a powerful instrument for analyzing complex mixes of analytical chemistry techniques. liquid chromatography will separate photo components based on the necessary time to appear at the end of appropriately packaged columns for individual components, while mass spectroscopy distinguishes each component based on the fragment mass that is produced when a beam of electrons bombarded the compound. lc-ms is used with high sensitivity and selectivity to analyze specific botanical extracts. it provides extensive information on structural elucidation of the substances generally and makes it easier to detect and identify possible chemical compounds in medicinal herbs, particularly when pure standards are not available. herbal medicinal drugs are being standardized and characterized as an area of scientific concern in the herbal drug industry. hplc is a good qualitative and quantitative technique, usually used for the evaluation of pharmaceutical because of its unique properties like high resolutions, high sensitivity (ppm repeatability, small sample size, moderate analysis condition, no need to vaporise the sample as in the gas chromatography, easy to fractionate the sample and purify , the use of hplc are increased day by day across the world(27) . figure 9.lc-msms of compound 1i +q1: 5.801 min from sample 6 (eg5) of nabaanabaa.wiff (turbo spray) max. 5.2e6 cps. 60 80 100 120 140 160 180 200 220 240 260 280 300 m/z, da 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% re l. in t. (% ) 306.7 130.0 101.9 75.8 82.5 151.8 59.1 114.2 219.4 302.8137.1 88.8 64.2 237.1 258.1161.9 182.661.1 103.3 139.4122.9 https://www.sciencedirect.com/science/article/pii/s2221169115002257#fig1 https://www.sciencedirect.com/science/article/pii/s2221169115002257#fig1 iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 279 figure 10. mass fragment of compound i epigallocatechin epigallocatechin = 306.27 g/mol m\z= 306.07 fragments : 1.289 2.164 3.122-125 figure 11. lc-msms of compound 1i iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 280 figure 12.mass fragment of compound ii catechin catechin =290.27 g/mol m\z= 291.0 fragments : 1180-181 2123 3256 4165.1 iraqi j pharm sci, vol.31(2) 2022 isolation of catchin and epigallocatchin from iraqi r.coriari 281 conclusion from the above study two flavonoids are isolated from rhus coriaria fruits (catechin and epigallocatechin) .phytochemical research is a scientific technique that is very important. this process identifies the main components found in the every plant element such as bark, leaves, stem, root and fruits. although nobody know how many different plants in the world today are used, but we aware that both herbal and western medicine are very valuable medicinal plants. references 1. bagetta g. herbal medicines: development and validation of plant-derived medicines for human health. clinical pharmacognosy. 2012; p.27 2. schunko, c, grasser, s., vogl, c.r.explaining the resurgent popularity of the wild: motivations for wild plant gathering in the biosphere reserve grosses walsertal, austria. journal of ethnobiology and ethnomedicine. 2015;11(55): 1–14. 3. thukaa zuhair abdul-jalil, rhus coriaria (sumac): a magical spice. herbes and spices book .2020 4. abu-reidah i m, jamous r m and ali-shtayeh m s: phytochemistry, pharmacological properties and industrial applications of rhus coriaria l. 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report doi: https://doi.org/10.31351/vol29iss2pp271-278 271 sudden transition of pharmacy education from traditional to distance learning in the era of covid-19: action steps of a leading pharmacy school in iraq kawther k. ahmed *, salema s. salman **, wafaa a. abbas***, shahad w. alkaisy ****and sarmed hashem kathem *****,1 * faculty and examination committee member, college of pharmacy, university of baghdad, baghdad, iraq. ** faculty and ibn sina unit for e-learning head, college of pharmacy, university of baghdad, baghdad, iraq. *** faculty and ibn sina unit for e-learning member, college of pharmacy, university of baghdad, baghdad, iraq. **** media unit head, college of pharmacy, university of baghdad, baghdad, iraq. ***** dean of the college of pharmacy, university of baghdad, baghdad, iraq. abstract education around the world has been negatively affected by the new coronavirus disease (covid-19) pandemic. many institutions had to adopt distance learning in compliance with the enforced safety measures. distance learning might work well for settings with stable internet connections, professional technical teams, and basic implementation of technology in education. in contrast, distance learning faces serious challenges in less fortunate settings with inferior infrastructure. this report aims to shed light on the immediate action steps taken at a leading pharmacy school in iraq to accommodate for the enforced changes in pharmacy education. the university of baghdad college of pharmacy went from less than minimal technology implementation to full distance learning in a remarkable time frame. pharmacy students were able to finish academic year requirements and move on with the program. final year students will graduate on time as competent pharmacists. keywords: e-learning, covid-19, online education, pharmaceutical education, : 19-االنتقال المفاجئ للتعليم الصيدلي من التعليم التقليدي إلى التعلم عن بعد في عصر كوفيد رائدة في العراقالصيدلة ال خطوات العمل لكلية و****و شهد وسام القيسي***، وفاء عبد االمير **سليمه سلطان سلمان، *كوثر خالد احمد 1*،****سرمد هاشم كاظم بغداد،العراق. جامعة بغداد، تدريسية وعضو اللجنة األمتحانية ،كلية الصيدلة،* .بغداد،العراق بغداد، جامعة الصيدلة، كلية تدريسية ورئيس وحدة ابن سينا للتعلم األلكتروني، ** .بغداد،العراق بغداد، جامعة الصيدلة، كلية تدريسية وعضو وحدة ابن سينا للتعلم األلكتروني،**** بغداد،العراق. جامعة بغداد، مسؤول وحدة اإلعالم، كلية الصيدلة، *** .بغداد،العراق بغداد، جامعة الصيدلة، كليةعميد **** الخالصه كان على العديد من المؤسسات االنتقال و( 19-كوفيد )د مستجتأثر التعليم في جميع أنحاء العالم سلبًا بجائحة فيروس كورونا ال تمتلك إنترنت مستقرة وفي االوساط التي تتوفر فيها خدمة التعلم عن بعد بشكل جيد يعمل . إلى التعلم عن بعد وفقًا لتدابير السالمة المطبقة في المقابل يواجه التعلم عن بعد تحديات خطيرة في . لتكنولوجيا في التعليما ات استخداماساسي فيها تطبيق تموي محترفة يةخبرات تقن صيدلة ال كليةيهدف هذا التقرير إلى تسليط الضوء على خطوات العمل الفوري التي تم اتخاذها في . لمتدنيةذات البنية التحتية ا ألوساطا تحولت كلية الصيدلة في جامعة بغداد من . ني بسبب جائحة كوروناتعليم الصيدالالرائدة في العراق الستيعاب التغييرات المفروضة في ال من إنهاء متطلبات الكليةطالب معه مكنقياسي ت التكنولوجيا إلى التعلم الكامل عن بعد في إطار زمنيإستخدام أقل من الحد األدنى من لممارسة مؤهلين كصيادلةفي الوقت المحدد من التخرج طالب السنة النهائية وتمكن البرنامج االكاديميسي والمضي قدًما في العام الدرا .المهنة ., العراقنيالصيدال التعليم ، التعليم عبر األنترنت،19-، كوفيدالكلمات المفتاحية : التعلم االلكتروني pharmacy education in the college of pharmacy educational institutions in iraq are centrally controlled and licensed through the ministry of higher education and scientific research (mohesr) and the university of baghdad college of pharmacy (uob copharm) is no exception. uob copharm offered a bachelor degree in pharmacy after the successful completion of a 5-year program and 172 units (215 credit hrs.). each academic year is divided into two 15-week semesters. each semester includes between 13 to 21 units/week (about 16 to 25 credit hrs./week). 1corresponding author e-mail: dean@copharm.uobaghdad.edu.iq received: 13/ 11/2020 accepted: 13/12/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp271-278 iraqi j pharm sci, vol.29(2) 2020 e -learning during covid-19 position report 272 the university of baghdad college of pharmacy (uob copharm) established in 1936, it is the first pharmacy school in iraq and the only one in the country till 1992. additionally, it is the first school to offer graduate studies (higher diploma, msc, and phd) in pharmacy in iraq. nowadays, a large percent of faculty members teaching in the different pharmacy schools in iraq are graduates of uob copharm at some point of their study program (bsc, msc, and phd). uob copharm had six divisions: pharmaceutical chemistry, pharmacology and toxicology, pharmaceutics, pharmacognosy, clinical and laboratory sciences, and clinical pharmacy. currently, uob copharm has a total of 91 faculty members, 1,128 undergraduate students and 113 first year postgraduate students across the three postgraduate programs (high diploma, msc, phd). before covid-19 pandemic, all pharmacy programs in iraq including uob copharm were face-to-face in-class programs across all classes and does not include any online courses (1). technology implementation at uob copharm before covid-19 era information and communication technology (ict) implementation in academia in iraq is limited (2) and uob copharm was no exception. at administer level, the importance of ict implementation and e-learning introduction was recognized. however, real life implementation was challenged by lack of infrastructure, a culture of technology rejection and common technology illiteracy among faculty and students (2,3). as part of the university of baghdad (uob) administrational attempts at introducing e-learning culture and practice, ibn sina unit for e-learning was established in each affiliated college in mid november 2018 to encourage e-learning, provide technological infrastructure and offer technical support. additionally, uob has an account through google’s g-suite for education and affiliated colleges and academic institutions were encouraged to implement various technologies in education. ibn sina unit at uob copharm conducted a number of workshops to introduce and train faculty members on using google classroom as an electronic course management (ecm) platform among other technical tools. despite these efforts, technologies like ecm, student response system, and even university email use were, in large, outcomes of sporadic personal efforts of technology-skilled instructors. even though official university emails were made available to faculty since 2018, only 20 out of 91 total faculty members have activated their emails and were using them before covid-19 era. official university emails were basically available for all students and faculty, however, students and faculty electronic communications were done mainly through social media platforms (1). additionally, ecm when implemented by the instructor, would be with students’ personal emails even if the instructors were using his/her official university email. before the establishment of ibn sina unit for e-learning, information technology services were administered through the “information technology unit” formerly known as “internet unit”. the it unit was providing basic support for faculty and students in accessing resources on the web and other basic services relevant to internet users. currently, the it unit is still operating and providing the same services in addition to other technical services. the unit also provides required support for ibn sina unit for elearning. education at uob copharm during covid19 period on march 17, 2020, the iraqi government announced a week long total curfew (4). curfew was extended for four weeks in an attempt to control the escalating covid19 cases. as such, mohesr announced moving all classes to virtual platforms to complete the academic year requirements with a modified academic calendar. universities and colleges were given the authorities to choose the educational platform, organize class schedules, and take control on monitoring the educational process to ensure education integrity. at uob copharm, distance learning was implemented using google classrooms where the university of baghdad had already registered for the g-suit for education. virtual classrooms were created by the instructors, some with their university emails and others with their personal emails. students had joined the class using class code that is initially conveyed to students through contacting class student representatives on their personal phone number. some instructors continued using social media for communicating with their students in an attempt to include all the class in the virtual classrooms. initially students did not accept the idea of e-learning and there was a bulk withdrawal from virtual classrooms. many reasons were behind this response. mainly students were experiencing sudden transition to a new platform of education in an unprepared setting. the college administration and ibn sina unit for e-learning took the lead on reassuring students and working with the faculty to iraqi j pharm sci, vol.29(2) 2020 e -learning during covid-19 position report 273 overcome the challenges experienced or anticipated by students in e-learning. this resulted in getting students back to the virtual classrooms to finish the first semester successfully. while this was a good start to the transition to e-learning, it came with its fair share of problems. with user (student) accounts outside the university domain, it was challenging to reach out to all students and ensure equality in educational material distribution. additionally, there were concerns about the privacy and integrity of the classrooms in terms of external users and assignments. the college students’ perspectives on e-learning were recorded as part of a survey conducted by ibn sina unit after the end of the first semester (april 2020). the survey was available to all undergraduate students across the five academic levels. the aim of the survey was to understand students’ perception and needs to prepare for the second semester. around 60% of all participating students agreed that e-learning was a good solution to avoid delaying their academic progress due to covid-19 pandemic (figure 1). in contrast, similar percent rejected the idea of continuing the use of e-learning as adjuvant to traditional learning after the pandemic ends (figure 1). however, more than 60% of students agreed that e-learning would be “good” if they had a better internet service (figure 1). the first semester concluded midapril and the second semester was to start on may 2nd. this two-week break was transformative thanks to the non-stopping efforts of the college administration and ibn sina unit for e-learning. action steps toward moving to full virtual education main action steps in preparation for the second semester can be categorized into six domains: establishing formal electronic communication system, virtual classes platforms organization, training and technical support to faculty and students, preparation for the final exams, supporting undergraduate students, and organizing class work for graduate students. below is an elaboration on the work done in each domain. establishing a formal electronic communication system within the college. this included: 1. creating formal university student emails for all undergraduate students (1,128 students). 2. activating formal university emails for all faculty. 3. creating official emails for all (113 students) first-year graduate students in preparation for coursework final exams for graduate students which was following a modified academic calendar. while these actions might be routine work for other institutions with established icts implantation, it took tremendous efforts to reach out to distant students and help them activate their university emails virtually. class schedules and virtual classes platforms organization. as mentioned earlier, the college of pharmacy has students in 5 levels, with 5-7 courses per semester in the undergraduate program. some courses had practical (laboratory) sections and the fifth (final) level had clinical sessions in public hospitals and public clinical laboratories. the laboratory and clinical practice parts were the most challenging to account for without impacting the students /graduates’ competences. the main action steps with regards to classes organization included: 1. creating 56 virtual classrooms for all undergraduate students in the 5-year bachelor of pharmacy program. these classes were all with official emails for both instructors and students. students were added to the classes to ensure all students are registered for required classes as opposed to leaving it to the students to register for their classes. this resulted in 100% student registration for electronic classes compared to varying percent in the first semester (figure 2). 2. class schedules included timetables for synchronous and asynchronous education. content delivery methods for the second semester included video lecture, powerpoint plus audio commentary, handouts in the format of powerpoint slides or pdf files, and live sessions (table 1). unlike the first semester, no lecture was to be delivered as handouts only. all instructors were providing asynchronous lectures as video (or powerpoint plus audio) and a typed handout in the format of pdf or powerpoint slides. the live sessions included discussions with the instructors over google meet, free conference call and zoom. 3. for classes with lab (practical) sessions, instructors were advised to do video demonstration of experiments. students were challenged with group and independent projects, assignments, and quizzes relevant to the experiment to ensure sound pharmacy education. 4. some pharmacy practice labs typically included patient-pharmacist consultation simulations with the instructors. for such labs, students submitted video recordings iraqi j pharm sci, vol.29(2) 2020 e -learning during covid-19 position report 274 of patient-pharmacist consultation simulations done at home with household members. instructors reviewed these videos and provided feedback. 5. clinical practice was replaced by virtual case presentations. cases were carefully selected to simulate previous years’ clinical practice experience. students were challenged with other cases, quizzes, and assignments to ensure best learning outcomes. 6. the college created a youtube channel where instructors would upload video lectures to enable easier lecture streaming by students due to poor internet services in iraq. the channel was organized by levels and instructors were trained for uploading lectures and importing links into the classrooms. the youtube channel was an innovative action step in response to students’ complains from the first semester about difficulties in streaming video lectures uploaded to google classrooms due to poor internet services. training and technical support to faculty and students. e-learning is a new experience for both faculty and students. mohser did provide virtual workshops and online resources for faculty to consult. similarly, the uob conducted many virtual workshops on distance education for affiliated institutions and faculty mainly through the uob central ibn sina unit for e-learning. however, both resources were open to faculty and instructors from different programs and not specifically tailored to pharmacy education. additionally, virtual workshops were attended by a massive number of faculty eager to learn about e-learning. to improve the quality of training experience, the administration and ibn sina unit for e-learning at uob copharm took the following action steps. 1. organization and administration of many workshops to train instructors on lecture recording. training covered different applications like powerpoint, flashback and other means of recording lectures. 2. organization and administration of workshops to introduce and train faculty on using google classroom features, taking attendance, engaging students into class materials, creating and managing assignments and quizzes and other topics relevant to e-learning. 3. preparation of educational and training materials for students on accessing their university account and related features using different electronic devices (smartphones, tablets, and computers). these training materials were in response to reports from students on difficulties in moving between personal and university accounts. 4. preparation of educational and training materials for students on using classroom features, submitting assignments, taking quizzes and other issues relevant to elearning. 5. instructors were advised on the possibility of using some classrooms in the college building for lecture video recording specially instructors who prefer using a white board during the lecture. 6. instructors and students were highly encouraged to seek technical help and ask questions in regard to using ecm, creating exams, answering exams, using live sessions applications and other issues relevant to e-learning. preparation for the final exams to conclude the academic year. with the increased incidence of covid-19 cases and to ensure safety of students and faculty, mohesr determined that final exams for all affiliated academic institutions will be administered on-line. exams were to be administered in a synchronous mode with specific timetables specified by the university/college. this was the first time in the history of pharmacy education in iraq that students take online final exams. while mohser issued regulations to ensure exam integrity and fair chance of students to participate, reality is that poor internet and, interrupted electricity service in iraq and unfamiliarity of students with online exams mandated immediate actions by uob copharm administration and ibn sina unit for e-learning to prepare for the worst. final exams were run and administered by the undergraduate exam committee and supported by ibn sina unit. main final exam preparations included: 1. creating classes dedicated to administering mock exams. mock exam classrooms intended to familiarize students with the exam process and types of expected questions. additionally, repeated mock exams helped reveal and resolve technical issues expected during real exams. three mock exams were administered for each level with varying students’ participation (figure 3a). 2. the exam was administered in 2-3 parts rather than one full part during the allocated time period. this was to avoid internet instability problems. to elaborate, students might lose internet connection during the exam. having the exam divided into more than one part ensures students iraqi j pharm sci, vol.29(2) 2020 e -learning during covid-19 position report 275 submission of at least partial work rather than losing the whole exam. additionally, in case of accidental reset of exam forms by the students before submission, the student will have to repeat that part only as opposed to reentering answers for the whole exam which would be time consuming and stressful for the student. 3. an emergency phone number was allocated for students to call if they experienced internet problems and lost all sorts of electronic communications with the exam committee during the exam. several students used that phone number and a member from the exam committee or ibn sina unit for e-learning was immediately available to work with them. 4. commencing virtual graduation project defense for final (fifth) year students. final year students have to submit independent graduation projects. students had already finished the practical part of the project before campus closure. instructors followed up with their students to finish project writing. a total of 181 students defended their graduation projects using synchronous video conference applications including google meet, zoom, and free conference call. supporting students and faculty through the new learning experience and stress associated with covid-19 pandemic. changes to educational programs usually starts with pilot trials and gradually introduced. following covid-19 pandemic, students, and faculty, were switched suddenly to virtual mode which caused stress and uncertainty for students. many students were questioning their capabilities and the system adequacy for e-learning. additionally, the ongoing covid-19 pandemic was another source of stress for students since some students were infected or had lost family member(s) due to covid-19. to ease out these unprecedented times for students in the uob copharm program, college administration, ibn sina unit for e-learning and all faculty had to be involved. 1. a separate classroom was created for each level in the undergraduate program to provide technical support and enable smooth communication between college administration and students. these classes were intended to be a hub for students to convey their concerns and problems directly to the dean office, the vice dean office for student affairs, and the ibn sina unit for e-learning. 2. many instructors continued communicating with students on social media applications to reassure students and relieve social isolation. 3. students reporting positive covid-19 themselves or their immediate family were constantly approached by instructors for reassurance. 4. as the sudden transition to distance education impacted faculty as well as students, a class dedicated to delivering technical support to faculty was created. additionally, faculty from the college were part of a free group chat application where they can informally exchange concerns, experience, and challenges during the new e-learning experience. organizing class work for graduate students. uob copharm caters the largest graduate pharmacy program in the country. it offers graduate studies in the six main disciplines of the college. the graduate program includes two course-work semesters for all first-year graduate students. after passing the first year of coursework, graduate students conduct research depending on the specific program (6 months for higher diploma students, one research year for msc students and two research years for phd students) and discipline they joined. there were a total of 113 graduate students in the first year across the three graduate levels in uob copharm for the academic year 2019-2020. the second semester for the graduate program started february 16, 2020. the first four weeks were in the traditional face-to-face delivery. after march 17, all classes were moved to online delivery. main action steps taken for the graduate program were similar to those taken for the undergraduate program: 1. virtual classrooms were created by the instructors and students joined with their personal emails. 2. a variety of content delivery methods were employed including synchronous and asynchronous education. live sessions were conducted using google meet, free conference call or zoom (table 1). 3. for final exams, official university emails were created for graduate students to ensure exam integrity. as stated earlier, three mock exams were conducted prior to final exams (figure 3b). exams were run by the graduate program exam committee and supported by ibn sina unit. 4. some graduate students had their thesis/dissertation defense scheduled between mid-march and mid-april where curfew was enforced. for those students, virtual thesis/dissertation defense were iraqi j pharm sci, vol.29(2) 2020 e -learning during covid-19 position report 276 held successfully as scheduled using goggle meet. outcomes of the unprecedented movement the experience of sudden transition from an all face-to-face program to an all online program can be viewed as a “full-size” pilot study where college administration, all faculty, and all students were exposed to pros and cons of applied icts and e-learning. faculty had to rethink their delivery methods for many courses, especially practical sections. they also had to rethink assessments and evaluations to harmonize the non-proctored elearning students. a range of delivery methods were used including mainly video lectures, slides plus audio commentary, handouts, external readings, and live session (table 1). in contrast to the first semester where some students left the virtual classrooms demonstrating their rejection for e-learning, no such case was recorded in the second semester. students enrollment in the virtual classrooms remained at 100% throughout the second semester where students had sensed the real willingness of all faculty and college administration to help them pass these challenging times. all the hard work and dedication of the college administration, ibn sina unit for elearning, and all the faculty at the uob copharm paid off at the end of the semester. for final exams, there was close to 100% participation of students in both the graduate and undergraduate levels (figure 3a & b). table 1.lecture format used in the graduate and undergraduate programs. lecture format undergraduate program graduate program (2nd semester) lecture/handout format first semester second semester phd msc h diploma live session 0 370 120 39 181 video 92 542 63 35 96 powerpoint + audio 122 359 82 64 141 powerpoint 76 358 110 40 131 pdf 85 508 63 48 135 figure 1. students’ perception about e-learning experience after the first semester and before starting the second semester. total number of students responding to the voluntary survey was 445 distributed across the five undergraduate levels of the program. the full questions wording on the survey was: elearning is good for the current pandemic status = do you think e-learning is good replacement for traditional learning in the current conditions (yes/no), e-learning with better internet service = what do you think about e-learning if the internet service was better? (good/ not good); continue using e-learning in hybrid education = do you like to continue using elearning as adjuvant to traditional face-to-face education after the pandemic ends? (yes/no). iraqi j pharm sci, vol.29(2) 2020 e -learning during covid-19 position report 277 figure 2. students participation in virtual classrooms by grade. first virtual semester: mid-march to mid-april; second semester: may to august 2020. in the first semester, it was left to the students to join the virtual class while in the second semester students were registered in google classrooms by ibn sina unit for e-learning using official students’ university accounts. each data point represents a single class (subject). figure 3. percent student participation in mock and final exams. three mock exams were organized for each of the 5 levels in the undergraduate program (a) and the 3 graduate programs (high diploma, master, phd), (b). mock exams were delivered in a separate virtual classroom for each level. final exams were delivered in a separate virtual classroom for each subject in each level for the undergraduate and graduate programs. iraqi j pharm sci, vol.29(2) 2020 e -learning during covid-19 position report 278 future directions the university of baghdad college of pharmacy is one of the lead pharmacy schools in iraq. it was and continues to be the pioneer in proposing new strategies for further pharmacy education in the country. the college administration is attentive to the need of implementing different icts into education to ensure graduates competences. while it was stressful and challenging to go from an all face-to-face program to all online program over couple weeks, this experience undeniably has positive future outlooks. the actions steps taken over the period from march through august represent the pillars for ict introduction to the uob copharm community including faculty, current and prospective students. for example, creating and activating all faculty and student university emails established professional electronic communications. the forced use of ecm exposed faculty and students to new organized platform for class materials that is accessible anywhere and anytime. additionally, students enjoyed the luxury of turning in their assignments and classwork while being miles away from campus and this is expected to help in future implantation of ecm. it is now in the plan of many faculty members to continue using this technology in case of resuming traditional faceto-face classes. this comes in accordance with the university of baghdad and the affiliated college of pharmacy strategic plan to increase the implementation of icts into education. the implementation of online courses as a permanent component of the uob copharm curriculum might not be feasible in the near future due to many regulatory and administrational considerations outside the scope of this report. however, it is still feasible to incorporate hybrid modules into existing courses. for example, flipped classroom methods can be applied where the didactic part of the course can be delivered as online video lectures. the in-class time would be then used for active learning and student engagement in group discussions. the next academic year (2020-2021, planned to start in december in the modified academic calendar) will mostly include sizable distance learning component in observance of covid19 pandemic. uob copharm administration and faculty have come across a long distance in optimizing pharmacy education for online delivery. the work is still ongoing to prepare for the next academic year. lessons learned from the 2019-2020 academic year will shape a better experience for faculty and students in the upcoming semester. acknowledgments the authors would like to thank dalia m. almufti from the it service for providing necessary information relevant to this report. references 1. al-jumaili, a. a., al-rekabi, m. d., alsawad, o. s., allela, o. q. b., carnahan, r., saaed, h., . . . sorofman, b.. exploring electronic communication modes between iraqi faculty and students of pharmacy schools using the technology acceptance model. american journal of pharmaceutical education, 2017. 81(5), 89. doi:10.5688/ajpe81589 2. alnuaimi, a. s.. iraq. in a. s. weber & s. hamlaoui (eds.), e-learning in the middle east and north africa (mena) region.2018, pp. 123-138. cham: springer international publishing. 3. ameen, n., willis, r., & abdullah, m. n. the use of e-learning by students in iraqi universities: potential and challenges paper presented at the 8th international visible conference on educational studies & applied linguistics.2017. 4. government-of-iraq (producer).. cabinet receives briefing on implementation of measures to contain covid-19. september 10, 2020. retrieved from https://gds.gov.iq/cabinet-receives-riefingon-implementation-of-measures-to-ontaincovid-19/ baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32(1) 2023 study lung protective effects of riboflavin and cyanocobalamin doi: https://doi.org/10.31351/vol32iss1pp100-106 100 study the lung-protective effects of riboflavin and cyanocobalamin against lung toxicity-induced by cyclophosphamide in rats waleed k. ghanim*,1, muhsin s. g. al-moziel*and hussein m. abood** * department of pharmacology and toxicology, college of pharmacy, university of basra, basra, iraq. ** electron microscope unit, college of pharmacy, university of basra, basra, iraq. abstract cyclophosphamide is a cytotoxic alkylating agent, it's used associated with different side effects including lung toxicity through oxidative damage. riboflavin and cyanocobalamin have lung-protective effects. this study was designed to evaluate the protective effects of both vitamins against lung toxicity induced by cyclophosphamide. seventy healthy adult albino rats of both sexes were divided into seven groups each group containing ten rats, all groups were treated for seven days then after (150 mg/kg) of cyclophosphamide was injected intraperitoneally at day seven, these groups were divided as follow; group one: intraperitoneally injected with (1ml/kg/day) of normal saline. group two: injected intraperitoneally with a single dose of cyclophosphamide (150 mg/kg). group three: administered orally riboflavin at a dose (10 mg/kg/day) and cyclophosphamide. group four: riboflavin administered orally at a dose (40 mg/kg/day) and cyclophosphamide. group five: riboflavin administered orally at a dose (0.1 mg/kg/day) and cyclophosphamide. group six: administered orally a mixture of riboflavin at a dose (10 mg/kg/day) and cyanocobalamin at a dose (0.1 mg/kg/day) and cyclophosphamide. group seven: administered orally a mixture of riboflavin at a dose (40 mg/kg/day) and cyanocobalamin at a dose (0.1 mg/kg/day) and cyclophosphamide. on day eight rats were sacrificed and serum was obtained for glutathione and the total antioxidant capacity measurement and lung extracted for the immunohistochemical study; both vitamins significantly (p<0.05) increased glutathione and the total antioxidant capacity and improve the immunohistochemical changes in comparison with the cyclophosphamide-treated group, these results indicate the protective effects of both vitamins against cyclophosphamide-induced lung toxicity. keywords: cyclophosphamide, vitamin b2, vitamin b12, lung toxicity دراسة التأثيرات الوقائية للريبوفالفين والسيانوكوباالمين ضد سمية الرئة التي يسببها سيكلوفوسفاميد في الجرذان **حسين محمود عبود و *محسن صغير المزيعل، 1، *وليد خالد غانم العراق ، البصرة ، جامعة البصرة ، كلية الصيدلة ، فرع االدوية والسموم * العراق ، البصرة، جامعة البصرة ، كلية الصيدلة ، وحدة المجهر االلكتروني** الخالصة عن الضرر مختلفة بما في ذلك تسمم الرئة الذي قد ينجم مرتبًطا بآثار جانبية ، يستخدم مؤلكل سام للخاليا عامل سيكلوفوسفاميد هو د تسمم الرئة الناجم التأكسدي. للريبوفالفين والسيانوكوباالمين تأثيرات واقية للرئة. صممت هذه الدراسة لتقييم التأثيرات الوقائية لكل من الفيتامينين ض ان ، وعولجت جميع جرذًا بالغًا سليًما من كال الجنسين إلى سبع مجموعات تحتوي كل مجموعة على عشرة فئر 70عن السيكلوفوسفاميد. تم تقسيم مجم / كجم( من سيكلوفوسفاميد داخل الصفاق في اليوم السابع ، تم تقسيم هذه المجموعات 150المجموعات لمدة سبعة أيام ثم بعد ذلك حقنت ب ) : تحقن داخل الصفاق مل / كغ / يوم( من محلول ملحي عادي. المجموعة الثانية 1على النحو التالي ؛ المجموعة األولى: الحقن داخل الصفاق ب ) ( سيكلوفوسفاميد من وحيدة ) 150بجرعة بجرعة الفم طريق عن الريبوفالفين تناول الثالثة: المجموعة كجم(. / يوم( 10مجم / كجم / مجم الخامسة: الريبوفالفين مغ / كغ / يوم( وسيكلوفوسفاميد. المجموعة 40وسيكلوفوسفاميد. المجموعة الرابعة: الريبوفالفين يؤخذ عن طريق الفم بجرعة ) مجم 10مجم / كجم / يوم( وسيكلوفوسفاميد. المجموعة السادسة: يعطى عن طريق الفم خليط من الريبوفالفين بجرعة ) 0.1عن طريق الفم بجرعة ) لفم خليط من الريبوفالفين مجم / كجم / يوم( وسيكلوفوسفاميد. المجموعة السابعة: يعطى عن طريق ا 0.1كجم / يوم( وسيانوكوباالمين بجرعة ) / مجم / كجم / يوم( وسيكلوفوسفاميد. في اليوم الثامن تم التضحية بالجرذان وتم الحصول 0.1مجم / كجم / يوم( وسيانوكوباالمين بجرعة ) 40بجرعة ) الكيميائية. كال الفيتامينان زادا بشكل على مصل الجلوتاثيون وقياس القدرة الكلية لمضادات األكسدة والرئة المستخرجة من أجل الدراسة المناعية ( المعالجة p <0.05ملحوظ المجموعة مع بالمقارنة الكيميائية المناعية التغيرات وتحسن الكلية لألكسدة المضادة والقدرة الجلوتاثيون من ) ئوي الناجم عن السيكلوفوسفاميد.بالسيكلوفوسفاميد ، وتشير هذه النتائج إلى التأثيرات الوقائية لكال الفيتامينات ضد التسمم الر ، تسمم الرئة 12، فيتامين ب 2الكلمات المفتاحية: سيكلوفوسفاميد ، فيتامين ب @yahoo.comph.wkg81 mail-corresponding author e1 received: 11/2 /2022 accepted: 19/4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp100-106 iraqi j pharm sci, vol.32(1) 2023 study lung protective effects of riboflavin and cyanocobalamin 101 introduction cyclophosphamide (cp) is a cytotoxic alkylating agent which is widely used to treat a wide range of malignant diseases including lymphoma, leukemia, breast cancer (1); however, its usage is associated with different side effects including lung toxicity, nephrotoxicity, hepatotoxicity (2). pulmonary toxicity induced by cp is believed to result from the active metabolite of cp which are phosphoramide mustard and acrolein that may induce oxidative damage to the alveolar tissues (3). riboflavin which is known also as vitamin b2 belongs to the group of vitamins that are soluble in water (4), that found in different types of food like cheese, egg, salmon, dairy products (5). riboflavin (vitamin b2) is considered an important precursor of two important nucleotides first one is flavin mononucleotide (fmn) and the second one is flavin adenine dinucleotide (fad) (6)these nucleotides have important roles in different redox reactions (6). it was found that riboflavin-deficit mice may show a reduction in fatty acid oxidation furthermore deficiency of vitamin b2 may cause anaemia, skin diseases, cardiomyopathy (7, 8). cyanocobalamin (vitamin b12) is a water-soluble vitamin (9) found in milk, fish, eggs, and meat (10). vitamin b12 (cyanocobalamin) is considered an important cofactor for the metabolism of homocysteine and methylmalonic acid (11), furthermore, its deficiency may be associated with numerous diseases like ischemic stroke, anaemia, disturbed vision (12, 13).the current study aims to evaluate the protective effects of riboflavin and cyanocobalamin against cyclophosphamide-induced pulmonary toxicity. materials and method experimental study seventy healthy adult albino rats of both sexes with a weight range from 200 to 230 gm were used in the present study, rats of both sexes equally distributed throughout the experimental groups; they were achieved from and kept under controlled temperature in the animal house of basrah university's college of pharmacy. the animals were fed commercial pellets and had free access to the water supply throughout the trial. drugs five hundred mg of cyclophosphamide vial was provided by baxter in the united states. amazing nutrition in the united states provided riboflavin capsules (400 mg). tq pharma, japan, provided the cyanocobalamin tablet (1 mg). study design the healthy experimental albino rats were divided randomly into seven groups each group consist of ten rats as follows: group one: rats in this group were received intraperitoneal (ip) injection of (1ml/kg/day) of normal saline (ns) for seven consecutive days; which represents the control group. group two: rats in this group were received intraperitoneal (ip) injection of a single dose of cp (150 mg/kg) (14) on day seven. group three: rats in this group were orally administered vitamin b2 at a dose (10 mg/kg/day) (15) for seven consecutive days and one intraperitoneal injection of cyclophosphamide at a dose (150 mg/kg) on day seven. group four: rats in this group were orally administered vitamin b2 at a dose (40 mg/kg/day) (15) for seven consecutive days and a single ip injection of cyclophosphamide at a dose (150 mg/kg), on day seven. group five: rats in this group were orally administered vitamin b12 at a dose (0.1 mg/kg/day) (15) for seven consecutive days and a single intraperitoneal injection of cyclophosphamide at a dose (150 mg/kg), on day seven. group six: rats in this group were orally administered a mixture of vitamin b2 at a dose (10 mg/kg/day) and vitamin b12 at a dose (0.1 mg/kg/day) for seven consecutive days and a single intraperitoneal injection of cp at a dose (150 mg/kg), on day seven. group seven: rats in this group were administered orally a mixture of vitamin b2 at a dose (40 mg/kg/day) and vitamin b12 at a dose (0.1 mg/kg/day) for seven consecutive days and single intraperitoneal injection of cp at a dose (150 mg/kg), on day seven.diethyl ether was used to euthanize rats 24 hours after the end of the management period (i.e., on day 8). intracardiac puncture yielded 5 ±1 ml of blood, which was collected in special tubes containing gel and clot activator to yield serum for glutathione and serum total antioxidant capacity (tac) measurement, lungs were extracted and washed with phosphatebuffered saline and then stored in 10% formalin solution for immunohistochemical study. immunohistochemical study the terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (tunel) assay is used to determine apoptosis in lung tissue (16). statistical analysis statistical analysis was performed by spss version 25 for windows software. data were expressed as mean ± sd. a one-way analysis of variance was used to examine the statistical significance of the differences between the groups (anova). p-values less than 0.05 were considered statistically significant differences. results table 1 showed that rats intraperitoneally injected with cp at day seven at a dose of 150mg/kg (group two) resulted in a significant decrease (p<0.05) in the serum level of glutathione in comparison with the relevant level in control (group one). mean ± sd of the levels of glutathione in the serum for (group two and group one) was found to be respectively, 33.4 ± 0.516 and 87.6 ± 0.516. moreover, (table 1) illustrated that there was a significant increase (p<0.05) in serum glutathione iraqi j pharm sci, vol.32(1) 2023 study lung protective effects of riboflavin and cyanocobalamin 102 level in groups treated with vitamin b2 (10mg/kg/day and 40 mg/kg/day) each for one week prior to cp (ip 150mg/kg) (groups three, and four respectively), vitamin b12 (0.1mg/kg/day for one week prior to cp (ip 150mg/kg) (group five), a mixture of vitamin b2 (10mg/kg/day) with vitamin b12 (0.1mg/kg/day) prior to cp (ip 150mg/kg) (group six) and vitamin b2 (40mg/kg/day) with vitamin b12 (0.1mg/kg/day) (group seven) for one week prior to ip injection of 150mg/kg of cp compared to the relevant serum level to (group two) rats ip injected with cp (150mg/kg). mean ± sd of serum glutathione levels for groups (three, four, five, six, seven and two) were respectively, 47.4 ± 0.516, 50.9 ± 0.737, 54.1 ± 0.316, 61.3 ± 0.483, 71.5 ± 0.527, and 33.4 ± 0.516. table 1. effects of vitamin b2 and vitamin b12 on serum total antioxidant capacity level group/treatment mean tac mmole/l ±sd group one (control)/ injected ip with 1ml/kg/day ns 1.42± 0.0186a group two/cp ip 150mg/kg 0.05 ± 0.003b group three/ vitamin b2 (10mg/kg/day) prior to cp (ip 150mg/kg) 0.56 ± 0.008c group four/ vitamin b2 (40mg/kg/day) prior to cp (ip 150mg/kg) 0.86 ± 0.03d group five/ vitamin b12 (0.1mg/kg/day) prior to cp (ip 150mg/kg) 1.03 ± 0.008e group six/ vitamin b2 (10 mg/kg/day) plus vitamin b12 (0.1mg/kg/day) prior to cp (ip 150mg/kg) 1.16 ± 0.024f group seven/ vitamin b2 (40mg/kg/day) plus vitamin b12 (0.1mg/kg/day) prior to cp (ip 150mg/kg) 1.27 ± 0.017g each value represents mean ± standard deviation (sd). values expressed in small letters (a, b, c, d, e, f, and g) are significantly different (p<0.05). number of animals in each group=10 immunohistochemistry (tunel assay) of rats' lung tissue a section of rats’ lung tissue of (group one) shows normal control lung tissue showing part of lung alveolar shows normal architecture cells ( no apoptosis, greencolored cells) as shown in figure (1a). immunohistochemistryical changes in rats’ lung intraperitoneally injected with a single dose of cp (150mg/kg) (group two) revealed damages included inflammatory cells are found with nuclear fragmentation (red arrow) and pyknosis (black arrow) with a thickness of the alveolar wall characterized by massive apoptosis as shown in the figure (1b).section of rats’ lung orally administered of (10 mg/kg/day, 40mg/kg/day) of vitamin b2 (group three and group four; respectively) for seven consecutive days before ip injection of (150mg/kg) of cp and orally administered of ( 0.1 mg/kg) of vitamin b12 group five) for seven days before ip injection of (150mg/kg) of cp at day seven showed fewer histopathological changes such as inflammatory cell (black arrow) karyorrhexis (red arrow) and destruction of alveolar-like emphysema with infiltration inflammatory cells (red arrow). as shown in figures (1c, 1d, and 1e) respectively. in addition to that combination of oral administration vitamin b2 (10, 40mg/kg) with vitamin b12 (0.1mg/kg) for seven consecutive days before ip injection of (150mg/kg) of cp at day seven (group six and group seven) respectively show improvement in lung tissue compared with other treated groups that have noted normal architecture alveolar wall (black arrow). as shown in figures (1f, and 1g) respectively. iraqi j pharm sci, vol.32(1) 2023 study lung protective effects of riboflavin and cyanocobalamin 103 immunohistochemical study of lung figure 1. light micrograph of immunohistochemical changes in lung tissue of rats are presented on the plate. a) the normal control lung tissue showing part of lung alveolar shows normal architecture cells (black arrow). b) lung tissue of rats treated with 150 mg/kg of cyclophosphamide has revealed damages included inflammatory cells are found with nuclear fragmentation (red arrow) and pyknosis (black arrow) with a thickness of the alveolar wall. c) lung tissue treated with 10 mg/kg of vit. b2 shows fewer histopathological changes such as inflammatory cell found (black arrow) karyorrhexis (red arrow). d) lung tissue treated with 40 mg/kg of vit. b2 shows destruction of alveolar-like emphysema (black arrow) with infiltration inflammatory cells (red arrow). e) lung tissue treated with 0.1gm/kg of vit. b12 shows fewer infiltration cells (red arrow) with a normal alveolar wall (black arrow). f) lung tissue treated with 0.1gm/kg of vit. b12 combination with 10 mg/kg of vit. b2 shows improvement in lung tissue compared with other treated groups that have noted normal architecture alveolar wall respectively (black arrow). g) lung tissue of rat treated with a combination of vit b12 0.1g/kg and vit b2 40mg/kg shows normal lung tissue (black arrow). tunnel stain 40x. iraqi j pharm sci, vol.32(1) 2023 study lung protective effects of riboflavin and cyanocobalamin 104 discussion in the present study, rats were treated with a single dose of cp (150 mg/kg) group two produce a significant reduction in a glutathione level (p< 0.05) compared to group one (control group) this attributed to the biotransformation of cp since, cyclophosphamide is prodrug required metabolic bioactivation by cytochrome p450 (cyp-450) enzyme system producing 4hydoxycyclophosphamide which undergo ringopening to aldophosphamide which decompose spontaneously to phosphoramide mustard (which is responsible for antitumor activity) and acrolein, both metabolites( phosphoramide and acrolein) will induce oxidative damage and free radicals formation, furthermore acrolein will form adduct with glutathione leading to decrease in glutathione level, glutathione considered as the most important intracellular non protein thiol donor group, playing important functions in detoxification of reactive oxygen species, acting as cofactor for other enzymes and regulate dna synthesis, so cyclophosphamide may result in reduction of glutathione level (17). furthermore, pre-treatment with vitamin b2 (10 mg/kg/day, and 40 mg/kg/day) group three and four respectively; and pre-treatment with (0.1mg/kg) of vitamin b12 group five and pretreatment with the combination of both vitamins group six and seven before cp injection produce a significant increase in glutathione level this attributed to the role of vitamin b2 as being the source for two important coenzymes which are flavin mononucleotide and flavin adenine dinucleotide (18) which have an important role in the oxidation-reduction reaction, also for the activity of superoxide dismutase and catalase (19), furthermore for conversion of oxidized form glutathione(gssg) to reduced one (gsh) (15, 20). whereas vitamin b12 plays an important role in the maintenance of glutathione level by direct-acting as a superoxide scavenger (21), furthermore vitamin b12 might have a protecting role against (lowgrade) inflammation-induced oxidative stress by affecting or modulating the expression of growth factors and inflammatory cytokines (22). rats treated with cp (150 mg/kg) group two produce a significant reduction in tac level (p< 0.05) compared to group one (control group) this effect could be explained by damaging effects produced by cp as a result of its metabolism and oxidative stress which induce a reduction in tac (23). furthermore, pre-treatment with vitamin b2 (10 mg/kg/day, and 40 mg/kg/day) group three and four respectively; and pre-treatment with (0.1mg/kg) of vitamin b12 group five and pretreatment with the mixture of both vitamins group six and seven respectively; before cp injection produce a significant increase in tac levels this could be explained by their ability to maintain antioxidant enzyme levels including superoxide dismutase, glutathione level in which vitamin b2 plays an important role (24); furthermore the role of vitamin b12 as a scavenger for free radicals (21, 25) and its role in modulating the level of the inflammatory cytokines (25). in the current study, rats injected with a single dose of cp (150 mg/kg) group two shows inflammatory cells that found with nuclear fragmentation and pyknosis with a thickness of the alveolar wall which confirms the effects of cp when compared with group one, however rats pre-treated with different doses of vitamin b2 groups three and four and a fixed dose of vitamin b12 group five prior to cp shows fewer histopathological changes such as inflammatory cell, karyorrhexis and destruction of alveolar-like emphysema when compared with group two furthermore; rats pre-treated with combinations of different doses of vitamin b2 and fixed-dose of vitamin b12 groups six and seven prior to cp injection shows improvement in lung tissue compared with other treated groups that have noted normal architecture alveolar wall this attributed to the effects of both vitamin b2 and vitamin b12 as the antioxidant activity and their ability to increase the level of glutathione which consist with the finding of bashandy sa et al and moshiri m et al which founds that vitamin b2 and vitamin b12 have an important role in the maintenance glutathione level and conversion of glutathione from oxidized form to reduced form (26, 27). conclusion cyclophosphamide has serious lung toxicity side effects, so to reduce this effect, pretreatment with a combination of vitamin b2 and vitamin b12 may have a beneficial role to reduce this toxic effect. acknowledgment the authors gratefully thank the college of pharmacy, university of basrah, for supporting the present work. ethical clearance in iraq, the research ethical committee oversees scientific research with ethical approval from the ministries of the environment, health, higher education, and scientific research conflict of interest 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attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage doi: https://doi.org/10.31351/vol29iss1pp260-267 260 effects of vitamin d3 on methotrexateinduced jejunum damage in rats farah k. abdul-wahab *, 1 and nada n. al-shawi * *department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract both methotrexate and vitamin d3 are used in combination for the treatment of various diseases. the aim of this study is to highlight the effect of vitamin d3 on methotrexate-induced jejunum damage using biochemical and histopathological studies. seven groups of both sexes of rats were selected and treated as follows: (group i and group ii) : control 1,control 2 (i.p normal saline) daily for 14 and 21 days respectively ; (group iii and group iv) :vitamin d3 groups (500 iu/rat/day) orally for 14 and 21 days, respectively;(gro up v): a single dose of methotrexate 20mg/kg, i.p injected for 4 days;(group vi):vitamin d3 (500 iu/rat/day) single for 14 days and methotrexate (20 mg/kg i.p) injected only at day 10;.(group vii) vitamin d3 (500 iu/rat/day) orally for 21 days and methotrexate (20 mg/kg i.p) injected only at day 17; then the jejunum was removed and used for measuring malondialdehyde (mda) content, total antioxidant capacity (taoc) level; in addition histopathological study of jejunum tissue. administration of vitamin d3 for 21 days and a single dose of methotrexate at day 17 resulted in non-significant difference (p>0.05) in mda; while significant reduction (p<0.05) in the taoc level in jejunum tissue; furthermore , sever villi damage ,crypts abscess, epithelial atrophy , mixed inflammatory cells infiltrate and goblet cells depletion were observed in comparison with methotrexate group. so the study demonstrates that vitamin d3 plays a synergistic role with methotrexate therefore the combined use of vitamin d3 and methotrexate may be used as a strategy to overcome dose limitations and side effects when use for the treatment of cancer, rheumatoid arthritis and psoriasis. key words: jejunum damage, methotrexate, oxidative stress, rats, vitamin d3. عه المَثوحرٍكسَج فٌ الجرذان علي حلف الصائم الىاجم 3 دحأثَر فَخامَه فرح قَس عبدالوهاب ،*1 و ودى واجٌ الشاوً * .*فشع االدٌٔح ٔانسًٕو ، كهٍح انصٍذنح، خايعح تغذاد، تغذاد،انعشاق الخالصة. انضٕءعهى ْٕذسهٍظ انذساسح ْزِ يٍ أيشاض يرعذدج.انٓذف نعلج ًٌكٍ اسرخذايًٓا يعا 3د ٔفٍرايٍٍ انًٍثٕذشٌكسٍد يٍ لا ك اٌ دشراٌان يٍ يدايٍع اخرٍاسسثع ذى ٔانُسٍدٍح.، انحٌٍٕح انكًٍٍائٍح انذساساخ تاسرخذاو انًٍثٕذشٌكسٍد عٍ انُاخى انصائى ذهف عهى 3د ذؤثٍشفٍرايٍٍ صسلد داخم انصفاق 2انسٍطشج ٔيدًٕعح 1يدًٕعح انسٍطشج : ( )انًدًٕعح االٔنى ٔانثاٍَح انُحٕانرانً: عهى ٔاعطٍد اندُسٍٍ يٍ كل دٔنٍح /خشر/ٌٕو( ٔحذج 555 ) 3د فٍرايٍٍ )انًدًٕعح انثانثح ٔانشاتعح(: يدايٍع ، انرٕانً عهى ًٌٕيا 21ٔ 14نًذج )يحهٕل انًهح انطثٍعً( ٌٕيًٍا أٌاو؛4كغى ٔصٌ اندسى داخم انصفاق( نًذج يهغى/ 25ٔاحذج ) خشعح )انًدًٕعح انخايسح(:انًٍثٕذشٌكسٍد ؛ انرٕانً ًٌٕياعهى 21ٔ 14نًذج فًٌٕا يهغى/كغى( انزي25) ٔانًٍثٕذشٌكسٍد ًٌٕيا 14نًذج ٌٕيًٍا ٔاحذج يشج] ( ٌٕو خشر/ دٔنٍح / ٔحذج555 3د [ ()انًدًٕعح انسادسح(:فٍرايٍٍ ًٌٕيا 21نًذج انفى طشٌك دٔنٍح / خشر/ ٌٕو( عٍ ٔحذج 555 ) 3د فٍرايٍٍ داخم انصفاق؛)انًدًٕعح انساتعح(: 15انٍٕو فً فمظ صسق ٔانسعح انًانَٕذاي انذٌٓاٌذ، نمٍاط ٔاسرخذايّ انصائى إصانح ذًد ؛ثى داخم انصفاق 11انٍٕو فً فمظ حمٍ يهغى / كغى( انزي 25ٔانًٍثٕذشٌكسٍد ) يٍ ٔاحذج ٔخشعح ًٌٕيا 21( نًذج 3دفٍرايٍٍ ) إعطاء يٍ انصائى. َرح يشضٍح َسٍدٍحألَسدح فضل عٍ دساسح نألكسذج انًضادج انكهٍح نـهسعح انكهٍح انًضادج نألكسذج اَخفاضا يعٌُٕا كاٌ ( تًٍُاp>0.05انًانَٕذاي انذٌٓاٌذ ) َسثح فً غٍش يعُٕي اَخفاًضا 11انٍٕو فً انًٍثٕذشٌكسٍد (p<0.05ٔذهف ) تًدًٕعح يماسَح انكؤط خلٌا َٔضٕب االنرٓاتٍح انًخرهطحانخلٌا ٔذسهم ٔضًٕسانطلئٍح االلثٍح فً ٔخشاج انضغة فً شذٌذ ٔانًٍثٕذشٌكسٍد3د ًٌكٍ اسرخذايٓا يعا فٍرايٍٍ نزنك ، انًٍثٕذشٌكسٍد يع دًٔسايرآصًسا ٌهعة 3د فٍرايٍٍ أٌ انذساسح ذٕضح ، . نزنكانًٍثٕذشٌكسٍد ٔانرٓاب انًفاصم انشٔياذٌٕذي ٔانصذفٍح. اندشعح ٔاَثاس انداَثٍح عُذ علج انسشطاٌ حذٔد عهى نهرغهة كإسرشاذٍدٍح . 3 فَخامَه د,الجرذان ,االكسدة ,المَثوحرٍكسَج ,الكلماث المفخاحَت : حلف الصائم 1 corresponding author e-mail: farah77kais@yahoo.com. received: 20/ 12 /2019 accepted:8 /2 /2020 iraqi journal of pharmaceutical science iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 261 introduction methotrexate (mtx), a folic acid antagonist, inhibits the enzyme dihydrofolate reductase (dhfr) and it is utilized as an antineoplastic and anti-rheumatoid agent (1) . the effects of mtx are not specific in action against tumour cells but also it can damage normal rapidly proliferating cells, namely intestinal epithelial cells (2) .the jejunal damage induced by mtx may be due to several factors including oxidative stress (os), inflammation and apoptosis .therefore, it is imperative to combine it with another drug, which could decrease its toxicity and increase its efficacy. it has been reported that different in vitro and in vivo studies have shown that os and dysregulation of antioxidant enzymes can play an important role in mtx-induced jejunal damage (3, 4) . moreover, researchers reported that there is no specific treatment for mtx-induced jejunum damage once it has occurred (2, 5) . vitamin d, a fat-soluble vitamin obtainable from the diet as well as produced in the skin from sunlight. vitamin d as a precursor of a potent steroid hormone, undergoes two-step metabolism in the liver and kidney to synthesize a biologically active form, calcitriol, which binds to the vitamin d receptor (vdr) (6, 7) that found in most cells, resulting in wide spread actions of vitamin d3 on most physiologic and pathologic processes. the primary sites of action of such vitamin are the intestine, bone, and kidneys; thus, the biologic functions of vitamin d3 are multiple and include its classic role in bone and mineral metabolism, and other non-classic actions, including cell proliferation, immunomodulation and cell differentiation (8) .vitamin d3 implicated in the pathophysiology of immune-mediated diseases including multiple sclerosis (ms) and inflammatory bowel disease (ibd) and it insufficiency has been linked to higher rates of cancers including colorectal, prostate and breast cancers (9,10) . objective: this study aims to study the effect of vitamin d3 at two different administration durations against mtx-induced jejunal damage in rats using biochemical and histopathological studies. materials and methods experimental animals forty nine (49) adult albino rats of both sexes, weighing 150-250gm were randomly allocated into seven groups (7 animals each) were used in this study. rats were obtained from and maintained in the animal house of the college of pharmacy, university of baghdad under conditions of controlled temperature. the animals were fed commercial pellets and tap water ad libitum throughout the experiment period. the study was approved by the scientificand the ethical committees of the college of pharmacy/ university of baghdad. chemicals and drugs methotrexate vial [(50mg/2ml vial) milan, italy]; vitamin d3 [(10,000 drops), diabase, italy]; malondialdehyde (mda) elisa kit (elabscience biotechnology, china); total anti-oxidant capacity kit (elabscience biotechnology, china). experimental protocol experimental rats used in this study were randomly divided into seven groups (7 animals /group) as follows: group i: control 1 [(i.p normal saline (0.9% nacl)] daily for 14, this group served as negative control; group ii: control 2 (i.p 0.9% nacl) daily for 21 days this group served as negative control; group iii: vitamin d3 group (500 iu/rat/day) (11) orally for 14; group iv: vitamin d3 group (500 iu/rat/day) (11) orally for 21 days; group v: methotrexate (mtx) single dose (20mg/kg, i.p) for 4 days, this group served as positive control (12) ; group vi: vitamin d3 orally (500 iu/rat/day) single for 14 days and methotrexate (20 mg/kg i.p), which injected only at day 10; group vii: vitamin d3 (500 iu/rat/day) orally for 21 days and methotrexate (20 mg/kg i.p), which injected only at day 17. at the end of the experiment, all rats were anesthetized by diethyl ether and sacrificed by cervical dislocation. then, the jejunum tissue samples were taken for the determination of mda contents, total antioxidant capacity (taoc), and for histopathological examination. determination of malondialdehyde (mda) contents contents of mda in the jejunum tissue homogenate were quantitatively estimated using mda kit based on elisa method (#e-el-0060; elabscience).the content of mda in jejunum tissue homogenate samples can be calculated by comparing the od of the samples with the standard curve. level of mda is expressed as ng/ml (13) . determination of total antioxidant capacity (taoc) level total antioxidant capacity(taoc)level was measured according to the manufacturers’ protocol using colorimetric method by the utilization of the total antioxidant capacity assay kit (#e-bc-k136; elabscience).many antioxidants in the body can reduce fe +3 to fe +2 , which in turn can form stable complex with phenanthroline substance. the taoc level in jejunum tissue samples can be calculated by measuring the absorbance at 520 nm (14) . histopathological examination the jejunum tissue samples were fixed in 10% formalin, dehydrated in graded ethanol, and https://en.wikipedia.org/wiki/jejunum iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 262 embedded in paraffin. sections of jejunal tissue were cut with a microtome set at a thickness of 5 μm, mounted on clear glass slides, and stained with haematoxylin and eosin (h&e). sections were examined and photographed using light microscopy (micros) and evaluated by the specialist pathologist (15) . an overall score for the severity of the jejunal damage was assessed in stained jejunum tissue sections as follows: avilli damage (shortening and fusion); bcrypt damage; c-epithelial atrophy; dinflammatory cells infiltrate in the lamina propria; and egoblet cell depletion as 0, none; 1, mild; 2, moderate; 3, severe (16) . statistical analysis data were expressed as the mean ± standard error of the mean (sem). the statistical significance of the differences among various groups was determined by one-way analysis of variance (anоva) followed by least significant difference (lsd) test by spss statistics version 25. differences were considered statistically significant for p-value less than 0.05. results figure 1 showed that there were nonsignificant differences (p>0.05) in the content of mda in jejunum tissue homogenate among group i (control 1), group ii (control 2), group iii (vitamin d3 for 14 days) and group iv (vitamin d3 for 21 days ); while the content of mda in the jejunum tissue homogenate in group of rats ip injected with group v (mtx) was significantly elevated(p<0.05) compared to groupi (control 1, group ii (control 2), group iii (vitamin d3 for 14 days) and group iv (vitamin d3 for 21 days ) furthermore, although the content of mda was reduced in group vi (mtx+ vitamin d3 for 14 days) and group vii (mtx+ vitamin d3 for 21 days) but still non-significantly different when each compared to mtx-treated rats (group v) (p>0.05). figure 1. effects of various treatments on malondialdehyde (mda)contents in the jejunum tissue homogenate of rats. each value represents mean ± standard error of means (sem). values expressed in small letters (a, and b) are significantly different (p<0.05). figure 2 showed that there were non-significant differences (p>0.05) in the taoc level in jejunum tissue homogenate among group i (control 1), group ii (control 2), group iii (vitamin d3) and group iv (vitamin d3); furthermore, figure 2 also showed that the level of taoc in jejunum tissue homogenate of rats ip injected with mtx (group v) was significantly reduced (p<0.05) compared to group i (control1), group ii (control 2), group iii (vitamin d3) and group iv (vitamin d3); additionally, there were significant reduction (p<0.05) in the taoc level in jejunum tissue sample in group of rats treated with mtx+vit d3 (group vi) and mtx+vit d3 (group vii) compared to mtx-treated rats (group v) (p<0.05). figure 2. effects of various treatments on total antioxidant capacity (taoc) levels in the jejunum tissue of rats. each value represents mean ± standard error of means (sem). values expressed in small letters (a, b, c, and d) are significantly different (p<0.05). histopathological examination of rats' jejunum tissue for histopathological study in the jejunum tissue of control 1 (group i), and control 2 (group ii) of rats, there were normal appearance of villi and crypts, with no epithelial atrophy,but a mild– moderate degree of mixed inflammatory cells infiltrate were seen within lamina propria; additionally, no goblet cell depletion was observed in figure 3 (a and b). furthermore, in group of rats orally-administered vitamin d3 for 14 days (group iii), there wasa mild-villous and crypts damage, -epithelial atrophy, and –mixed inflammatory cells infiltrate were seen in the lamina propria; furthermore, mild goblet cells depletion was also observed (figure 3-c). while in group of rats orally-administered vitamin d3 for 21 days (group iv), there are focal widening ,and shorting of villi, mild crypt damage (fusion of crypt) and a mild mixed inflammatory cells infiltrate in the lamina propria and mild goblet cells depletion were observed in (figure 3-d). concerning section of jejunum tissue of methotrexate-treated rats (group v), there were moderate villous (v)and crypts (c)damage which was represented by shortening, widening and fusion of villi with v/c ratio 2/1; moreover, moderate epithelial atrophy , moderate inflammation is seen in lamina propria and a moderate degree of goblet cells depletion. (figure 3-e ). iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 263 in group vi of rats-treated with methotrexate+ vitamin d3 (for 14 days), figure 3-f showed a severe damage that represented by shorting, widening and fusion of villi (villous atrophy),and crypt damage (abscess); with villous to crypts ratio 1/1; furthermore, moderate epithelial cells atrophy, a heavy mixed inflammatory cells infilterate , and moderate goblet cells depletion was also observed. moreover, in group vii of rats treated with methotrexate+ vitamin d3 (for 21 days), figure 3g showed that there were severe damages that represented by loss, widening and fusion of villi with v/c ratio 1/1, severe crypt damage (the crypt epithelium is atrophied and few cells are sloughed out in the lumen of the crypt(apoptosis); additionally, severe epithelial cells atrophy , heavy mixed inflammatory cells infiltrate were seen in the lamina propria and sever goblet cells depletion. figure 3. sections of the jejunum tissue of various rats groups (haematoxylin and eosin (h&e) x 10).a and b) group i and group ii: normal appearance of villi (v) and crypts (c), no epithelial atrophy, with a mild to moderate degree of mixed inflammatory cells infiltrate is seen in lamina propria, and no goblet cell depletion. c) group iii: a mild villous, crypts damage, mild epithelial atrophy and goblet cells depletion compared to the area pointed by the large arrow which shows goblet cells. d) group iv: focal widening ( )and shorting of villi, a mild crypt damage, a mild mixed inflammatory cells infiltrate in the lamina propria and the tips of villi showed mild goblet cell depletion compared to the sides of the villi where by goblet cells are still present. e) group v: moderate villous and crypts damage represented by shortening, widening and fusion of villi with v/c ratio 2/1, moderate epithelial atrophy and moderate inflammation were seen in lamina propria and moderate goblet cells depletion. f) group vi: severe damage that represented by shorting, widening and fusion of villi (villous atrophy) with v/c ratio 1/1, crypt abscess, moderate epithelial cells atrophy, a heavy mixed inflammatory cells infiltrate is also seen in lamina propria, and goblet cells depletion. g) group vii: severe damage represented by loss, widening and fusion of villi with v/c ratio 1/1, crypt abscess, severe epithelial atrophy, heavy mixed inflammatory cells infiltrate is seen in the lamina propria and sever goblet cells depletion. yellow arrows indicate villi damage, white arrows indicate crypt abscess, and blue arrow indicates goblet cells and black arrows indicate goblet cells depletion. a b d e f g c iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 264 discussion the intestinal epithelium is the largest surface area of the body in contact with the external environment (17) . concerning mtx, such chemotherapeutic drug belongs to the antimetabolite class of medication; where, it structurally resembles folic acid, and it competitively inhibited dihydrofolate reductase (dhfr) enzyme (18, 19) . researchers described that the therapeutic use of mtx has been limited by its impact on the rapidly-dividing cells of crypts through the inhibition of the epithelial cell proliferation and induced apoptosis in the small intestinal crypts (20) ; moreover, multiple factors may have role in mtx-induced small intestine damage such as the dose and the treatment duration of such drug, type of disease, in addition to apoptotic factors (2) . additionally, several mechanisms have been hypothesized to underline small intestine injury induced by mtx such as oxidativeand nitrosative stress, up-regulation of inflammatory mediators that may promote apoptosis which in turn may augment inflammation and tissue injury of intestinal tissue which play important role in the pathogenesis of small intestine damage induce by mtx in rat (21, 22) . regarding the oxidative stress marker malondialdehyde (mda), which is an end product of lipid peroxidation an events in the cells are recognized as an indicator of oxidative stress (23) . figure 1 showed that there were non-significant differences (p>0.05) in the content of mda in jejunum tissue homogenate among group i (control 1), group ii (control 2), group iii (vitamin d3) and group iv (vitamin d3); while , in group of rats ip injected with mtx 20mg/kg (group v) caused elevation in mda contents accompanied by the reduction of taoc level in rats' jejunum tissue homogenate compared to control1 (group i), control 2 (group ii), vitamin d3-orally administered rats for 14 days (group iii) and vitamin d3-orally administered rats for 21 (group iv), this comes in line with previous animal and clinical studies, which demonstrated that os and lipid peroxidation are hallmarks of mtx-induced jejunum damage (24-26) ; furthermore, authors reported that the cell damage can be initiated by the reaction of free radicals with biological macromolecules producing lipid peroxides with the depletion of first-line antioxidant enzyme systems, including reduced glutathione (gsh) (3, 27) .although there were reduction in mda contents in rats' jejunum homogenate that treated with mtx with vitamin d3 orally-administered for 14 days (group vi) and mtx with vitamin d3 orallyadministered for 21 days (group vii) but still nonsignificantly different when each compared to mtx-treated rats (group v). on the other hand, results of this study showed that the taoc level was significantly reduced in groups vi and vii rats each compared to groups v rats as shown in figure 2. for histopathological study in the jejunum tissue of control 1 (group i), and control 2 (group ii) of rat treated with normal saline for 14 and 21 days, respectively, there were normal appearance of villi and crypts, with no epithelial atrophy, but a mild– moderate degree of mixed inflammatory cells infiltrate were seen within lamina propria; additionally, no goblet cell depletion was observed in figure 3 (a and b). furthermore, in group of rats orally-administered vitamin d3 for 14 days (group iii), there was a mild -villous and crypts damage, -epithelial atrophy, and –mixed inflammatory cells infiltrate were seen in the lamina propria; furthermore, goblet cells depletion was also observed [figure 3-c]. while in group of rats orally-administered vitamin d3 for 21 days (group iv), there are focal widening, and shorting of villi, mild crypt damage (fusion of crypt) and a mild mixed inflammatory cells infiltrate in the lamina propria were observed in (figure 3-d). the suggestion concerning the effect of vitamin d3 may be due to its pro-oxidant effect that caused an increase in ros, which in turn may decrease gsh level; thus os can consequently be resulted in cells. in addition, this study showed that there were histopathological changes in the jejunum tissue of rat treated with mtx for 4 days which characterized by moderate villous damage that represented by shortening, widening and fusion of villi and crypts damage with v/c ratio 2/1, moderate epithelial atrophy and moderate inflammation were also seen in the lamina propria; furthermore, a moderate degree of goblet cells depletion is seen compared to group i, group ii, group iii, and group iv rats; and these changes are consistent with that observed in previous studies (11, 28) .authors mentioned that mtx act as a prooxidant that may cause depletion of the tetrahydrofolate, suppressed dna synthesis, inhibited epithelial proliferation, and induced apoptosis in the small intestinal crypt (4) . in addition, the release of free radicals by moderate infiltrate inflammatory cells in lamina propria may have a role in jejunum pathogenesis and this comes in line with previous study (29) . iraqi j pharm sci, vol.29(1) 2020 vitamin d3 on methotrexate induced jejunum damage 265 moreover, many authors described that the oxidative damage induced by mtx in jejunum tissue can be prevented by different vitamins and natural plant extracts that may act as cytoprotectors to protect normal cells of the jejunum from mtx damage (2, 5) ; but, results of the current study showed that histopathological jejunum sections of rats administered vitamin d3 for 14 days and mtx at day 10 (group vi) (figure 3-f); where severe damage that represented by loss, widening and fusion of villi, crypt abscess, severe epithelial cells atrophy, a heavy mixed inflammatory cells infiltrate were seen in the lamina propria and sever goblet cells depletion (figure 3-g).flanagan l, et al (2016) reported that vitamin d3 can synergistically act with mtx; where, vitamin d3 can induce apoptosis which may be due to the action of such vitamin as pro-oxidant and caused an induction of reactive oxygen species (ros) by altering redox state of cell (30) . in addition, abu el maaty ma and wölfl s (2017) described that vitamin d3 can enhance the activity of mtx by increasing intracellular availability (31) . additionally, zhao r et al in 2013 demonstrated that the intestinal absorption of mtx and folate is mostly mediated by the proton-coupled folate transporter (pcft), which mainly expressed in the proximal part of the small intestine at the apical brush-border of enterocytes' membrane; and vitamin d3 can increase expression of intestinal pcft and an enhancement of cellular folate uptake (32) . moreover, the potential mechanism for increasing intestinal absorption of mtx can be brought about via simultaneous treatment of mtx with vitamin d3; in other word, vitamin d3 can affect the bioavailability of mtx (33) ; additionally, vitamin d3-mtx interaction was reported to be through transporters; where, such vitamin can alter the pharmacokinetics and pharmacodynamics of mtx that can result in variability of efficacy and involved in the mtx disposition in the body and in the regulation of intracellular metabolism in targets cells (31, 34) . thus, beneficial effects may be brought about when vitamin d3 and mtx used together for the treatment of different diseases such as cancer, inflammatory bowel disease, rheumatoid arthritis (ra), and psoriasis by decreasing the dose of mtx so decrease side effects and increase efficiency; but, adverse effects instead of beneficial effects were observed in this study. conclusion the present study demonstrates that vitamin d3 plays a synergistic role with methotrexate by causing histopathological damage in jejunum tissue of rats; where, vitamin d3 can act as pro-oxidant and/or it can enhance the entrance of mtx inside the cell; and, adverse effects resulted from combination of vitamin d3 and mtx are observed in this study. acknowledgments this article was abstracted from the phd thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the authors gratefully thank family, friends in the college of pharmacy of baghdad university, dr. ban a. abdul majeed, working at 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pharmacological roles. curr opin pharmacol. 2013;13 (6):875-80. 33. eloranta jj, zaïr zm, hiller c, häusler s, stieger b, kullak-ublick ga. vitamin d3 and its nuclear receptor increase the expression and activity of the human proton-coupled folate transporter. mol pharmacol. 2009;76 (5):106271. 34. inoue k, yuasa h. molecular basis for pharmacokinetics and pharmacodynamics of methotrexate inrheumatoid arthritis therapy . drug metab pharmacokinet. 2014; 29 (1): 1219. creative commons attribution 4.0 is licensed under a bijpsbaghdad iraqi journal pharmaceutical sciences by d.university of baghda -copyrights© 2015 college of pharmacy .international license https://www.ncbi.nlm.nih.gov/pubmed/24383099 https://www.ncbi.nlm.nih.gov/pubmed/19666701 https://www.ncbi.nlm.nih.gov/pubmed/?term=inoue%20k%5bauthor%5d&cauthor=true&cauthor_uid=24284432 https://www.ncbi.nlm.nih.gov/pubmed/?term=yuasa%20h%5bauthor%5d&cauthor=true&cauthor_uid=24284432 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https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.jstage.jst.go.jp/article/dmpk/advpub/0/advpub_dmpk-13-rv-119/_article/-char/ja/ https://www.ncbi.nlm.nih.gov/pubmed/24284432 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 indole based n-acyl hydrazones with antibacterial activity doi: https://doi.org/10.31351/vol29iss1pp207-2015 207 synthesis, characterization and antibacterial activity evaluation of new indole-based derivatives ahmed t. sulaiman* and susan w. sarsam*, 1 *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract indole is widely distributed heterocycle found in natural biologically active molecules, drugs, and other substances. starting from indole-3-propionic acid (ipa) a metabolite produced by gut’s bacteria, new series of nacyl hydrazones (4a-g) was synthesized. these n-acyl hydrazones were prepared by the reaction of 3-(1h-indol3-yl) propane hydrazide and aldehyde in the presence of glacial acetic acid as a catalyst. 1hnmr and ft-ir analyses were used to identify the synthesized compounds. in vitro study was performed to evaluate the antibacterial activity of the synthesized compounds against six different types of microorganisms by using well diffusion method. all the tested n-acyl hydrazones (4a-g) displayed moderate activity against the gram-negative e.coli which was comparable to amoxicillin, except compound (4e), which showed high activity. also, selective moderate activities against other gram-negative bacteria were shown by compounds (4a, 4c, 4e, 4f and 4g), while, compounds (4b) and (4d) exhibited intermediate activity against gram-positive b.subtilis. all the synthesized compounds exhibited selective lower antibacterial activity compared to ciprofloxacin. additionally, no activity was exhibited by any of the examined compounds against the gram-positive s. aureus. keywords: n-acyl hydrazone, indole-3-propionic acid, antibacterial. تحضير وتشخيص وتقييم الفعالية المضادة للبكتريا لمشتقات اندول جديدة 1*،و سوزان وديع سرسم *أحمد طالل سليمان *فرع الكيمياء الصيدالنية، كلية الصيدلة، جامعة بغداد، بغداد، العراق . الخالصة الطبيعية واألدوية. في هذا البحث تم تحضير سلسلة يعتبر اإلندول من اوسع الحلقات الغير متجانسة إنتشاراً في المركبات البايولوجية . هذه المركبات حضرت عن طريق بروبيونيك -3 مض اإلندولاتحتوي على مجموعة االسيل مشتقة من ح( 4a-g)زونات جديدة من الهيدرو بروبيونك مع عدة الديهايدات بوجود حامض الخليك الثلجي كعامل مساعد. تم تشخيص جميع -3 -الهايدرازايد المشتق من حامض اإلندول مفاعلة أُجريت دراسة مختبرية لتقييم المركبات المحضرة .مطياف األشعة تحت الحمراء وجهاز الرنين النووي المغناطيسي المركبات المحضرة عن طريق ( أظهرت فعالية متوسطة ضد 4a-gبكتيرية لستة انواع مختلفة من البكتريا.جميع المركبات المحضرة )كمضادات ,4a, 4c, 4eحيث كان له فعالية عالية. كما أظهرت المركبات ) (4e)والتي كانت مماثلة لألموكسيسلين،عدا المركب اإلشريكية القولونيةبكتريا 4f, 4g) فعاليات متوسطة ضد االنوا( 4ع البكترية االخرى السالبة لصبغة الغرام. بينما المركباتb, 4d ) كان لها فعالية متوسطة ضد البكتريا سين. إضافة العصوية الرقيقة الموجبة لصبغة الغرام. جميع المركبات المحضرة كان لها فعالية منتقاة ضد البكتريا أقل عند مقارنتها بالسيبروفلوكسا الذهبية الموجبة لصبغة الغرام. المكورات العنقوديةمن المركبات فعالية ضد بكتريا إلى انه لم تظهر اٌي بروبيونيك، مضاد بكتيري. -3-مض اإلندولاوزونات المرتبطة بمجموعة االسيل، حراكلمات مفتاحية: الهيد introduction indole (benzo[b]pyrrole) is widely distributed heterocycle found in natural biologically active molecules, drugs, and other substances. in the biological system, several indole-based biomolecules occur with different effects such as serotonin (5-hydroxytryptamine) a neurotransmitter, melatonin the sleep hormone, tryptamine and related amino acid tryptophan. also, natural alkaloids with an important pharmacological activity contain indole base had been isolated, among them; vinblastine and vincristine (vinca alkaloids) with anti-cancer activity (1). also, many synthetic drugs carrying indole pharmacophore are now available such as sunitinib an anticancer drug, delavirdine an antiviral clinically used for hiv, indomethacin, etodolac nsaids, and many other drugs with various pharmacological activities (2). indole propionic acid (ipa) is a type of plant auxin (plant hormone) which is involved in plenty of developmental ways through the growth period of the plant (3). in human, ipa is detected in serum and cerebrospinal fluid (4), it originates from clostridium sporogenes an intestinal flora, its presence in plasma depends on this type of bacteria (5), and recently it was discovered other types of gut bacteria are capable of the production of ipa, which are peptostreptococcus anaerobius and three strains of clostridium cadaveris (6). 1corresponding author e-mail: sarsam14@yahoo.com received:28 / 9 /2019 accepted: 14/ 1/ 2020 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp207-2015 iraqi j pharm sci, vol.29(1) 2020 indole based n-acyl hydrazones with antibacterial activity 208 these types of bacteria produce ipa through deamination of tryptophan by bacterial enzyme tryptophan deaminase (7). ipa has antioxidant activity and acts as a free radical scavenger which completely protects nerve cells against the beta-amyloid’s toxic effect, so it perhaps applied in the treatment of alzheimer’s disease (8, 9). also, ipa is considered a ligand for pregnane x receptor (pxr), and together regulates mucosal homeostasis and integrity of git by decreasing tumour necrosis factor-alpha and increases junctional protein-coding mrnas (10, 11). recently, it was observed that ipa has antituberculosis activity in vitro and in vivo by a mechanism which involves inhibition of bacterial tryptophan biosynthesis through a negative feedback mechanism (12, 13). hydrazones constitute a significant group of organic compounds contain (-nh-n=c-) moiety. they are synthesized by the reaction of hydrazine derivatives with carbonyl-containing compound (aldehyde or ketone) (14). hydrazones have received considerable interest by many medicinal chemistry researchers owing to the ease of their synthesis as well as the exhibition of wide range of pharmacological effects. they have been notified to have antibacterial, analgesic, antianticonvulsant, antiplatelet, anti-tubercular, inflammatory and antitumor activities (15). n-acyl hydrazone (nah) is considered a privileged structure, which resembles the smallest essential subunit, shared in many drugs or leadcompounds, capable of interacting with a single receptor or more than one class of receptors. also, due to its ease of production, the stability to hydrolysis, changing h-bonding (donating ↔ accepting) and altering conformation which results in different molecular property with diverse pharmacological activities (16). several drugs containing nah pharmacophore are now available for clinical use such as dantrolene the only drug approved for malignant hyperthermia treatment (17), nitrofurantoin, an antibacterial and carbazochrome which has been used for the treatment of capillary and parenchymal haemorrhage (18). the aim of the present investigation was to synthesize new indole derivatives containing n-acyl hydrazone starting from indole-3-propionic acid then to evaluate their antibacterial activity. materials and methods starting material 3-indole propionic acid (ipa) and aldehydes were purchased from himedia (india), hydrazine hydrate from merck (germany), methanol from thomas baker (india) and ethanol from scharlau (spain). the progress of reactions as well as the purity of newly synthesized compounds was checked out by thin-layer chromatography (tlc) by using silica gel gf (type 60) pre-coated aluminium sheet from merck (germany). uv-254 lamp was used to visualize the spots, and the elution system used was (ethyl acetate: toluene: methanol (2:2:1)). stuart smp3 melting points device was used to measure the melting points and were incorrected. infrared spectra were made at the college of pharmacy/ university of baghdad using ft-ir (ir affinity-1) spectrometer (shimadzu, japan). 1hnmr spectra were obtained using bruker (400 mhz) device and dmso-d6 as a solvent and were performed at the central instrumental laboratory/ college of science/ university of tehran / iran. all the synthesized compounds were identified and their structures were determined by ftir and 1hnmr spectral analyses (19) synthesis of ester (methyl 3-(1h-indol-3-yl) propionate) (2) (20) ten gm (0.052mole) of indole propionic acid (1) was dissolved in (50 ml) of methanol and chilled to 0 oc using an ice bath. then 3 ml of concentrated h2so4 was added to the cold solution drop by drop with vigorous stirring, and then the temperature of the solution was raised gradually to 70 co and started the reflux. the reaction was monitored by tlc, after 4 hours of reflux the reaction completed and the solution was poured into a crushed ice containing beaker, a precipitate was formed, and then the solution was neutralized with 5% (w/v) nahco3. the final precipitate was obtained by filtration and recrystallized from (methanol/water) to yield off-white fine crystals. yield 94%; m.p. 75-76 oc; rf = 0.86; ir (v, cm-1): 3390: (n-h) str. of indole. 3086: ar (c-h) str., 2951, 2916: (c-h) asym. str. of ch3 & ch2, 2897, 2854: (c-h) sym. str. of ch3& ch2, 1716: (c=o) str. of ester, 1165: (c-o-c) str.; 1hnmr (δ, ppm) : 2.66 (2h, t, -ch2-ch2-cooch3), 2.95 (2h, t, ch2-ch2-cooch3 ), 3.58 (3h, s, -cooch3), 6.96,7.05 (2h,2t, ar-h), 7.10 (1h, s ,ar-h), 7.32,7.50 (2h,2 d, ar-h), 10.79 (1h,s, indole n-h). synthesis of acid hydrazide (3-(1h-indol-3-yl) propanehydrazide) (3) (21) 5gm (0.024 mole) of compound (2) was dissolved by heating and stirring in 30 ml ethanol, then (20ml, 0.41 mole) of hydrazine hydrate was added gradually to the hot solution with continuous agitation and started the reflux. after 5 hours, tlc showed hydrazide spot only, the reaction was stopped and left to cool, then poured into a beaker contains (50 ml) of distilled water. excess ethanol was evaporated and the formed precipitate was obtained by filtration, dried, rinsed with ether then recrystallized from water to obtain white needle shape crystals. yield =88%; m.p 143 oc; rf = 0.48; ir (v, cm-1): 3309, 3278: (n-h) str. of indole and hydrazide, 3082: ar. (c-h) str., 2897, 2854: (c-h) str. (asym.& sym.) of ch2, 1666: (c=o) str. of amide, 1508: ar. (c=c) str.; 1h nmr (δ, ppm): 2.38 (2h, t, -ch2ch2-conhnh2), 2.90 (2h, t, -ch2-ch2conhnh2 ), 4.16 (2h, s, -nh-nh2), 6.96, 7.04 iraqi j pharm sci, vol.29(1) 2020 indole based n-acyl hydrazones with antibacterial activity 209 (2h,2 t, ar-h), 7.08 (1h, s ,ar-h), 7.31, 7.50 (2h, 2d, ar-h), 8.99 (1h,s, -nh-nh2), 10.75 (1h,s, indole n-h) (22). synthesis of n-acyl hydrazones compounds (4a-g) (23). equimolar (1mmole) of acid hydrazide (3) and appropriate aldehyde were dissolved in suitable solvent (ethanol or water) and reacted, using glacial acetic acid as a catalyst then refluxed for 1-2 hour. ethanol was used as a solvent except for the reaction with vanillin where water was used instead (24). when the reaction was finished, the residue was filtered and dried. the quantities of each reactant are listed in table 1. table 1. quantity of reactants that equal to 1 mmole. compound quantity acid hydrazide (3) 203.1 mg p-oh benzaldehyde 122.1 mg p-cl benzaldehyde 140.5 mg p-br benzaldehyde 185 mg benzaldehyde 0.1 ml p-no2 benzaldehyde 151.1mg p-n(ch3)2 benzaldehyde 149.2 mg vanillin 152.1 mg n'-(4-hydroxybenzylidene)-3-(1h-indol-3-yl) propanehydrazide (4a) white powder, yield 73%; m.p. 208-209 oc; rf = 0.7; ir (v, cm-1) : 3429: (o-h) str. of phenol, 3302, 3186 : (n-h) str. of indole and hydrazone, 3059: ar (c-h) str., 2900, 2862: (c-h) str.(asym.& sym.) of ch2, 1624 : ( c=o) str. of amide, 1604: (c=n) str., 1566: ar. (c=c) str.; 1hnmr (δ, ppm): 2.53 (2h, t, -ch2-ch2-co-nh), 2.97 (2h, t, -ch2-ch2-co-nh-), 6.79 (2h, t, ar h ), 6.96, 7.05 (2h,2 t, ar-h), 7.12 (1h, d ,ar-h), 7.32 (1h, d, ar-h), 7.46 (2h, dd, ar-h), 7.55 (1h, d, ar-h), 7.87, 8.02 (1h, 2s, -n=ch-), 9.83, 9.87 (1h, s, -oh) , 10.77 (1h,s, indole n-h), 11.07, 11.16 (1h,2s, -nh-n=c-). n'-(4-chlorobenzylidene)-3-(1h-indol-3-yl) propanehydrazide (4b) white grain-like crystals, yield 69%; m.p. 197-198 oc; rf = 0.77; ir (v, cm-1 ): 3383, 3170: (n-h) str. of indole and hydrazone, 3055: ar. (c-h) str., 2958, 2843: (c-h) str. (asym. & sym.) of ch2, 1654: (c=o) str. of amide, 1612: (c=n) str., 1558: ar. (c=c) str.; 1hnmr (δ, ppm): 2.58 (2h, t, -ch2ch2-co-nh-), 3.00 (2h, t, -ch2-ch2-co-nh-), 6.97, 7.06 (2h,2t, ar-h), 7.13 (1h,d,ar-h), 7.32 (1h, d, ar-h), 7.46 (2h, dd, ar-h), 7.55 (1h,d, arh), 7.66 (2h,dd, ar-h), 7.96, 8.13 (1h, 2s, -n=ch), 10.78 (1h,s, indole n-h), 11.35, 11.45 (1h,2s, nh-n=c-). n'-(4-bromobenzylidene)-3-(1h-indol-3-yl) propanehydrazide (4c) white fine crystals, yield 72%; m.p. 175176 oc; rf = 0.78; ir (v, cm-1 ): 3433, 3174 (n-h) str. of indole and hydrazone, 3062: ar. (c-h) str., 2970, 2862 : (c-h) str. (asym.& sym.) of ch2 , 1654 : (c=o) str. of amide, 1608: (c=n)str., 1558: ar. (c=c) str.; 1hnmr (δ, ppm): 2.57 (2h, t, -ch2ch2-co-nh-), 2.97 (2h, t, -ch2-ch2-co-nh-), 6.97, 7.06 (2h,2 t, ar-h), 7.12 (1h,d ,ar-h), 7.32 (1h, d, ar-h), 7.54 (2h,dd, ar-h), 7.56 (1h,d, arh), 7.61 (2h,d, ar-h), 7.94, 8.11 (1h, 2s, -n=ch-), 10.77 (1h,s, indole n-h), 11.33,11.44 (1h,2s, -nhn=c-). n'benzylidene3-(1h-indol-3-yl) propanehydrazide (4d) white fine needle-like crystals, yield 63%; m.p. 173-174 oc; rf = 0.74; ir (v, cm-1): 3294, 3182 (n-h) str. of indole and hydrazone, 3020: ar (c-h) str., 2900, 2846: (c-h) str. of ch2 (asym. & sym.), 1620: (c=o) str. of amide, 1600: (c=n) str., 1566: ar. (c=c) str. 1hnmr (δ, ppm): 2.57 (2h, t, -ch2ch2-co-nh-), 3.00 (2h, t, -ch2-ch2-co-nh-), 6.97, 7.06 (2h,2t, ar-h), 7.13 (1h,d ,ar-h), 7.32 (1h, d, ar-h), 7.37-7.45(3h, m, ar-h), 7.55 (1h,d, ar-h), 7.64 (2h,dd, ar-h), 7.98, 8.14 (1h, 2s, n=ch-), 10.78 (1h,s, indole n-h), 11.28, 11.38 (1h,2s, -nh-n=c-). 3-(1h-indol-3-yl)-n'-(4-nitrobenzylidene) propanehydrazide (4e) yellow fine crystals, yield 98%; m.p. 238239 oc; rf = 0.76; ir (v, cm-1 ): 3387, 3190 ( n-h) str. of indole and hydrazone, 3055: ar (c-h) str., 2954, 2835: (c-h) str. of ch2 (asym & sym.), 1678 : (c=o)str. of amide, 1581: (c=n) str., 1512: ar. (c=c) str. & asym (no2) str., 1334: (no2) sym. str.; 1hnmr (δ, ppm): 2.61 (2h, t, -ch2-ch2-co-nh), 3.02 (2h, t, -ch2-ch2-co-nh-), 6.97, 7.05 (2h, 2t, ar-h), 7.13 (1h,d ,ar-h), 7.32, 7.55 (2h,2d, arh), 7.89 (2h,dd, ar-h), 8.06, 8.22 (1h, 2s, -n=ch), 8.26 (2h,dd, ar-h), 10.77 (1h,s, indole n-h), 11.57, 11.67 (1h,2s, -nh-n=c-). n'-(4-(dimethylamino) benzylidene)-3-(1h-indol3-yl) propanehydrazide (4f) white fluffy crystals, yield 83.8%; m.p. 206-207 oc; rf = 0.74; ir (v, cm-1): 3163 ( n-h) str. of indole and hydrazone overlap, 3043: ar (c-h) str., 2974, 2908, 2850: (c-h) str. of ch3 & ch2 (asym. & sym.), 1643 : (c=o) str. of amide, 1597: (c=n) str., 1527 : ar.(c=c) str.; 1hnmr (δ, ppm): 2.52 (2h, t, -ch2-ch2-co-nh-), 2.93, 2.95 (6h,2s, -n(ch3)2 ), 2.97 (2h, t, -ch2-ch2-co-nh-), 6.71 (2h, t, ar-h), 6.97, 7.05 (2h,2t, ar-h), 7.12 (1h,d ,ar-h), 7.32 (1h, d, ar-h), 7.45 (2h,dd, ar-h), 7.55 (1h,dd, ar-h), 7.84,7.98 (1h, 2s, -n=ch-), 10.77 (1h,s, indole n-h), 10.98, 11.06 (1h, 2s, -nh-n=c). iraqi j pharm sci, vol.29(1) 2020 indole based n-acyl hydrazones with antibacterial activity 210 n’-(4-hydroxy-3-methoxybenzylidene)-3-(1hindol-3-yl) propanehydrazide (4g) white powder, yield 65%; m.p. 160-165 oc; rf = 0.68; ir (v, cm-1 ): 3406 : (o-h) str. of phenol, 3383, 3217 ( n-h) str. of indole and hydrazone, 3032: ar. (c-h) str., 2939, 2846: (c-h) str. of ch2 (asym. & sym.), 1635 : (c=o) str. of amide, 1581 (c=n) str., 1516 ar.(c=c) str., 1265 : ar (c-o-c) str.; 1hnmr (δ, ppm): 2.54 (2h, t, ch2-ch2-co-nh-), 2.98 (2h, t, -ch2-ch2-conh-), 3.75, 3.80 (3h, s, -och3), 6.79 (1h, dd, arh), 6.94 (1h, t, ar-h), 7.00 (1h,s, ar-h), 7.06 (1h,t, ar-h), 7.13 (1h,d, ar-h), 7.21 (1h, d, ar-h), 7.32, 7.55 (2h, 2d, ar-h), 7.86, 8.01 (1h,2s, -n=ch-), 9.45, 9.49 (1h, s, -oh) , 10.77 (1h,s, indole n-h), 11.11, 11.18 (1h, 2s, -nh-n=c-) (25,26). antibacterial activity the antibacterial activity was tested by well diffusion method, by using bacterial suspension obtained from mcfarland turbidity standard (number 0.5). mueller-hinton agar mha plates were inoculated by this suspension. in each agar plate of examined bacteria, four wells were made, and 80μl from the solution of the synthesized compound was added to them. the plates were incubated at 37 °c for 24 hours, and then the antibacterial activity was evaluated by measuring the diameter of the inhibition zone around the well in (mm) (27). four types of gram-negative bacteria (escherichia coli, pseudomonas aeruginosa, klebsiella pneumonia and proteus mirabilis) and two types of gram-positive bacteria (staphylococcus aureus and bacillus subtilis) were tested in vitro for antibacterial activity. all types of bacteria have already been isolated and identified in the college of the medicine/ university of alnahrain. ciprofloxacin and amoxicillin were used as standards for antibacterial activity. the concentrations of standers were 1mg/ml in dimethyl sulfoxide (dmso). results and discussion chemistry the pathway for the synthesis of the targeted n-acyl hydrazones (4a-g) is depicted in scheme 1. compound 4a 4b 4c 4d 4e 4f 4g r1 oh cl br h no2 n(ch3)2 oh r2 h h h h h h och3 scheme 1. general synthetic pathway of the titled compounds iraqi j pharm sci, vol.29(1) 2020 indole based n-acyl hydrazones with antibacterial activity 211 the synthetic pathway started by the preparation of methyl ester derivative (2) of ipa by fischer esterification method using h2so4 as a catalyst. the acid protonates oxygen of the carbonyl group and makes carbon more electrophile, subjected to nucleophilic attack by the lone pair of electrons of alcohol. the nucleophilic attack breaks carbonyl group and forms the tetrahedral intermediate oxonium ion. at this point, the excess of alcohol will deprotonate the tetrahedral intermediate oxonium ion to form neutral molecule. then, transfer of the proton to hydroxyl group which is eliminated as water and the carbonyl bond is regenerated (28) (scheme 2).this method showed considerable yield with ease of production. . scheme 2. mechanism of carboxylic acid esterification (fischer esterification) (28) hydrazine hydrate (n2h4.h2o) was used to prepare the acid hydrazide (3) from compound (2). the reaction is base catalyzed ester aminolysis. the mechanism involves two hydrazine molecules either occurred in single step (concert pathway) in which aminolysis occurs through carbonyl attack by amine associate with proton abstraction from the amine, while departure of leaving group associated with proton transfer from ammonium ion, this process happen in one step. the second possibility occurs through multi steps pathway in which the first hydrazine molecule attacks the electrophilic carbonyl group to form tetrahedral intermediate and the second hydrazine molecule will transfer the proton to methoxy group resulting in dissociation of the methoxy group(29-32) (scheme 3). iraqi j pharm sci, vol.29(1) 2020 indole based n-acyl hydrazones with antibacterial activity 212 scheme 3. mechanism of hydrazide formation from ester (ester aminolysis) (29, 30) subsequently, the final n-acyl hydrazones (4a-g) were synthesized by condensing 3-(1h-indol3-yl) propanehydrazide compound (3) with the corresponding aldehyde in acidic media as a catalyst. in this reaction, one water molecule had been eliminated to form a carbon-nitrogen double bond (imine or schiff base) (33) (scheme 4). the target compounds (4a-g) were obtained in good to excellent yields (63-98%). scheme 4. mechanism of imine formation (schiff base) (34) iraqi j pharm sci, vol.29(1) 2020 indole based n-acyl hydrazones with antibacterial activity 213 the ir spectra demonstrated shifting of the absorption band of c=o from 1716 cm-1 in ester to 1666 in hydrazide (amide formation), in addition, two bands appeared at 3309, 3278 attributed to primary amine n-h stretching overlapping with nh stretching of indole. the infrared spectra of the synthesized n-acyl hydrazones (4a-g) had a characteristic absorption band at 1581-1612 cm-1 of carbon-nitrogen double bond (c=n) stretching of imine. additionally, compounds (4a-g) had recognized bands at 1620-1678 cm-1, attributed to the carbonyl group (c=o) stretching of amide. the 1hnmr spectra of the ester, acid hydrazide and nahs were consistent with the assigned structures. two sets of two separated singlets were displayed in the 1hnmr of n-acyl hydrazones (4a-g), attributed to both the –n=ch and -conhprotons. these protons appeared as two singlets resonating in the regions (7.84 – 8.22) and (10.98 – 11.67) ppm, respectively. the presence of these paired signals can be explained on the basis of the existence of hydrazones as e/z geometric isomers around the c=n double bonds as well as the cis/trans amide conformers (35, 36). antibacterial activity table 2 shows the activity of the synthesized compounds (nahs) against the selected bacteria at a concentration of 1mg/ml. table 2. in vitro antibacterial activity of the synthesized n-acyl hydrazones at 1mg/ml . zone of inhibition (mm) compounds p. mirabilis k. pneumoniae e. coli p. aeruginosa b. subtilis s. aureus 12 14 14 4a 12 11 4b 14 12 4c 11 11 4d 14 18 4e 13 12 4f 12 12 4g 10 15 38 35 amoxicillin 45 18 30 50 29 52 ciprofloxacin dmso (-) = no activity, (zone of inhibition between 5-10 mm) = slightly active, (zone of inhibition between 10-15 mm) = moderately active, (zone of inhibition more than 15 mm) = highly active (37). the data illustrated in the table 2 reveals that all the synthesized tested compounds showed moderate activity against e.coli comparable to that of amoxicillin, except compound 4e, which showed high activity. also, compounds 4a and 4e showed moderate activity against klebsiella pneumonia, which was greater than amoxicillin. in addition, moderate activity was viewed by compounds 4a and 4f against proteus mirabilis. also, moderate antipseudomonas activity was seen with compounds 4c and 4g, which was lower compared to amoxicillin. all the tested compounds demonstrated various antibacterial activities against selected bacteria, which were lower compared to ciprofloxacin. generally, all the tested compounds had no activity against the selected gram-positive bacteria, except compounds 4b and 4d, which showed moderate activity against bacillus subtilis. the using of local isolates and not the control strains (as american type collection culture strains atcc), may be the cause of low activity of the synthesized compounds due to mutation and resistance (38). conclusion new n-acyl hydrazone derivatives containing indole base were successfully synthesized and the structures of newly synthesized compounds were characterized using ir and hnmr spectral analyses methods. the synthesized compounds had moderate activity against escherichia coli and selective activity against other types of bacteria. antituberculosis activity evaluation of the synthesized compounds and using the control strains of bacteria instead of local isolates, are recommended for a future study. acknowledgement the authors are grateful to the college of pharmacy/ university of baghdad for all the facilities to perform the research. references 1. mekky h, al-sabahi j, abdel-kreem mf. potentiating biosynthesis of the anticancer alkaloids vincristine and vinblastine in callus cultures of catharanthus 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37. dabholkar vv,gavande rp.synthesis and antimicrobial activities of novel 1, 4benzothiazine derivatives. arab. j. chem. 2016; 9:225-229. 38. landecker h. antibiotic resistance and the biology of history. body & society. 2016;22(4):19-52. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ enhancement of of atorvastatin tablet dissolution using iraqi j pharm sci, vol.19(1) 2010 atorvastatin dissolution 82 enhancement of atorvastatin tablet dissolution using acid medium kahtan j.hasson* ,1 * department of pharmaceutical chemistry ,college of pharmacy,al-mustansyria,university.baghdad .iraq . abstract in this study some generic commercial products of atorvastatin tablets were evaluated by dissolution test in acid medium by comparing with that of parent drug lipitor of pfizer company. some of solubilizing agents were studied in formulation of atorvastatin tablet including; surface active agent and peg 6000 .the most effective factor was the use of peg6000 in formulation of atorvastatin tablet which improved the dissolution and the results of dissolution profile of formulated tablet in this work was bioequivalent to that of lipitor .the quantitative analysis of this work was performed by using reversed phase liquid chromatography and a proper mixture of mobile phase which give a retention time for atorvastatin about 6 minutes . خالصة عهي حثوب االذوزفاسراذيٍ انًُرجح يٍ قثم عدج يعايم دوائيح قي انوسط انحايضي ساء فحص االَحالنيح في هرا انثحث ذى أج وفي ()حثوب نثيروزسكح فايززوانًُرجح يٍ انشسكح األو وهي شذيٍ اتهيح االَحالل نحثوب االذوزفاسرا ساء دزاسح يقازَح يع قوقد ذى أج ذعرثس يساعدج نهروتاٌ يثم صوديوو نوزيم سهفيد يواد تإضافحذجازب إجساءعهي , كًا اٌ هرا انثحث قد احروىيضي نحاانوسط ا خ في دحيث ساعاالذوزفاسراذيٍ صيغح حثوب عهي وقد كاَد انًادج االخيسج ذاخ ذاثيس فعال 0666ويادج توني اثيم غاليكول َوع اٌ االصهي ) نثيروز( ًسرحضسان اني ءيكافي انًسرحضس انًصُع في هرا انثحث وأصثح اَحالنيح االذوزفاسراذيٍ ذحسيٍ دزجح تاسرعًال انطوز انعكسي نهكسوياذوغسافيا انسائهح وتاسرعًال خهيط يُاسة نهطثقح انًرحسكح أجسيدفي هرا انثحث قد انرحهيالخ انكًيح دقيقح ذقسيثا . 0نيعطي وقد انفصم introduction atorvastatin calcium is a synthetic lipidlowering agent. it is an inhibitor of 3-hydroxy3-methylglutaryl-coenzyme a (hmg-coa) reductase. this enzyme catalyzes the conversion of hmg-coa to mevalonate, an early and rate-limiting step in cholesterol biosynthesis.the absolute bioavailability of atorvastatin is approximately 14%. the low systemic availability is attributed to hepatic first-pass metabolism or mucosa gastrointestinal presystemic clearance (1) . the plasma concentrations of atorvastatin and other related compounds which are metabolized by cytochrome p450 isoenzyme cyp3a4 were increased when these drugs are taken with repeated use of grape fruit juice due to inhibition of this enzyme which is located in the gastro intestinal tract (2) . atorvastatin calcium is practically insoluble in aquouse medium below ph4 (3) . this physical property of insolubility in water, obviously, will lead to low dissolution in stomach medium. the first study of best dissolution formula which held by pfizer co. was carried on gastric medium ,and the use of alkalizing agent (calcium carbonate) and the surfactant ( sodium lauryl sulphate ) were recommended in formula of atorvastatin tablet (4) .several studies were reported in attempt to enhance the dissolution of atorvastatin in its solid dosage forms; complexation of atorvastatin by cyclodexetrin significantly enhanced its dissolution, but it is rather tedious procedure (5) . atorvastatin calcium was prepared as a redispersible dry powder for emulsion which was formulated by using dextran as a carrier and poloxamer 188 as a surfactant, its dissolution showed a 2.33 fold increase compared to the pure atorvastatin calcium powder in vitro gastrointestinal medium (6) in this present study, lipitor tablet( 20 mg) , the patent drug formulation of atorvastatin calcium tablet is tested for dissolution in acid medium and the result was about 92 % dissolved atorvastatin to the labeled amount. different generic products of atorvstatin tablet (commercial products) are evaluated by determination of their dissolution profiles in acidic medium .in addition,some experimental formulations of atorvastatin calcium were made in this work and the using of sodium lauryl sulfate in different concentrations and peg 6000 have been evaluated by their influences on the rate of dissolutions .determinations of the dissoluted amounts of atorvastatin in acid medium are carried in this work by using a reversed phase hplc method, prescribed below. several previous methods of analysis have been used for quantitative determination of atorvastatin in tablet including spectrophotometry (7) , and hplc method for separation of atorvastatin and amlodipine in pharmaceutical formulation (8) . 1corresponding author email : kahtan48@yahoo.com received : 9/11/2009 accepted : 1/6/2010 http://www.rxlist.com/script/main/art.asp?articlekey=2710 iraqi j pharm sci, vol.19(1) 2010 atorvastatin dissolution 83 experimental work materials atorvastatin calcium crystalline powder ( supplied by cadila healthcare limited company ,india ), sodium lauryl sulphate ( shouguang pharm. ind.co.ltd,china ), poly ethylene glycol 6000 ( sasol germany gmbh. ), potassium dihydrogen phosphate (reagent grade).acetonitrile and methanol (hplc grade). apparatus uv.vis.spectrophotometer , specord 40, analytikjena.hplc; knauer, dual piston pump, uv detector and computerized recorder,tablet dissolution tester usp procedure 6 tablets of each product were tested, one tablet in each of six vessels of the dissolution apparatus of paddle system and 50 rpm ,using 900ml of 0.1m hcl as a medium .the filtered samples were taken at intervals; 10,15,20,25,30,35,40,45 minutes and subjected to hplc analysis to determine the percent of atorvastatin calcium dissolved in medium. method of analysis an hplc method for the analysis of atorvastatin calcium was carried by using the following chromatographic conditions. column: ods, 25 cm x 4.6 mm mobile phase: acetonitrile: 0.01m potassium dihydrogen phosphate solution (40:60), adjusted to ph 4.0.with phosphoric acid. detection: by u.v at 240 nm. flow rate: 1.0 ml/minutes.atorvastatin peak appeared in 6 minutes and the analysis accomplished in 8 minutes (figure 1) figure 1: hplc chromatogram of atorvastatin tablet in which the peak of atorvastatin appeared in 6 minutes and the peaks of the excipients are eluted at the first minute which indicated that there is no interference with the assay of atorvastatin . the accuracy and precision of this hplc method were proved since the relationship of different concentrations of atorvastatin with their peak area ratio have shown a straight line with correlation coefficient of value 0.999. figure 2: the standard curve of atorvastatin dilutions with percent recovery peak area using the reversed-phase hplc method. dissolution profile three commercial products of atorvastatin tablet were tested for dissolution in acid medium and compared with that of patent product (lipitor). most of the generic formulas of atorvastatin tablet showed low bioequivalence to lipitor, (see figure 3 ) figure 3 :dissolution profiles of some commercial products of atorvastatin tablets; where product a ( avas20,micro lab.india) was bioequivalent to lipitor tablet ,however the product b (atorvatin ,alfaris co.syria) and product c (atorvast 20,avanzor,syria) showed low dissolution . relation between conc.of std atorvastatin and their % recovery of peak areas 0 10 20 30 40 50 60 70 80 90 1 2 3 4 5 6 7 8 mg/100 of std atorvastatin % r e c o v e r y r e l a t e d t o t h e p e a k a r e a s r= 0.9998 dissolution profiles of atorvastatin tablets of different company compared to lipitor tablet 0 10 20 30 40 50 60 70 80 90 100 10 15 20 30 45 m inutes % o f a t o r v a s t a t in r e le a s e d lipitor tab. product a product b product c iraqi j pharm sci, vol.19(1) 2010 atorvastatin dissolution 84 in my attempt to achieve good atorvastatin tablet formulation ,different experiments were carried in this work considering the acceptable physical properties ;hardness,friability and disintegration time in addition to the main requirement; the high dissolution .the first experimental product is termed as (product d) which contained atorvastatin calcium,calcium carbonate ,microcrystalline cellulose ,lactose,colloidal silicone dioxide ,pvd,magnesium stearate and talc .the dissolution profile of this first formula of product d was too low comparing to lipitor tablet ( figure 4 ) figur4: dissolution profile of product d (the first experimental batch in this work) which showed very low dissolution. therefore, product d formula was subjected to improvement and several experiments were made to evaluate the addition of solubilzing agents 1. effect of surfactant in addition to the ingredients of atorvastatin tablet mentioned above, different concentrations of sodium lauryl sulphate( sls) were added in an attempt to increase the dissolution of atorvastatin . figure (5) shows that there is significant increase in dissolution of atorvastatin tablet but it is lower than 80% and there is no significant differences between the added concentrations of 10,20and 40 mg of sls in enhancement of dissolution . figure 5: effect of sls on dissolution of atorvastatin tablet in product d. 2. effect of poly ethylene glycol (peg 6000) as it is demonstrated in step 1 the minimum effective concentration of sls was 10 mg per tablet ,in addition to this substance ,peg6000 ( finely sieved powder) was incorporated in different concentrations( 10 and 20mg ) with the active constituent of atorvastatin tablet formulations and the dissolutions profile for each formula was tested ( see figure -6) . the attempts to increase the concentration of peg6000 more than 20mg per tablet were failed because the other physical properties of tablet (hardness and friability) were affected. figure 6 : effect of peg 6000 on dissolution of atorvastatin tablet in product d effect of sls in atorvastatin tablet on its dissolution 0 10 20 30 40 50 60 1 2 3 4 5 6 7 8 minutes % o f d i s s o l v e d a t o r v a s t a t i n no sls 5mg/tab.sls 10mg/tab.sls 20mg/tab.sls 40mg/tab.slsdissolution profile of product d ( atorvastatin tablet) first experimental batch 0 10 20 30 40 50 60 70 80 90 100 10 15 20 30 45 minutes % o f d i s s o l v e d a t o r v a s t a t i n i n a c i d m e d i u m lipitor tab. prod.d effe ct of diffe re nt conce ntrations of peg 6000 on dissolution 0 20 40 60 80 100 5 10 15 20 25 30 35 45 minutes % o f d i s s o l u t i o n without peg 10 mg peg 20 mg peg iraqi j pharm sci, vol.19(1) 2010 atorvastatin dissolution 85 conclusion this study indicated that the formulation of atorvastatin 20mg tablet with the presence of sls (10 mg) and peg6000 (20 mg) per tablet has enhanced the dissolution of atorvastatin to a value equivalent to that of parent product (lipitor). references 1. martindale, drug reference, 2007. pharmaceutical, press, london. 2. effects of grape fruit juice on pharmacokinetics of atorvastatin and ravastatin in japanese , ichiro fukazawa ,et al , british journal of clinical pharmacology, v57, 2004, page 448-455. 3. clark`s analysis of drugs and poisons, 3rd edit., pharm. press london ,atorvastatin monograph . 4. u.s.patent issued on april 18, 2006. 5. solubility and stability enhancement of atorvastatin by cyclodextrin complexation, by china reddy et al, j.pharm.sci.and technol.,may 2009, 63; 217-225. 6. preparation, characterization and in vitro intestinal absorption of a dry emulsion formulation containing atorvastatin calcium ,by yong-mei yin,et al, drug delivery ,vol.16,jan 2009,page 30-36 . 7. simultaneous spectrophotometric determination of atorvastatin and amlodipine besylate in tablet, khan mr and jain d. indian j.pharm.sci.2006; 68; 546-458. 8. stability indicating rp-hplc estimation of atorvastatin and amlodipine besylate in pharmaceutical formulations , da shahi et al ,indian journal of pharmaceutical sciences ,2008,70.page 754-760 . iraqi j pharm sci, vol.29(1) 2020 immuno-protective effect of nigella sativa seed oil doi: https://doi.org/10.31351/vol29iss1pp253-259 253 evaluation of the possible immuno-protective effect of nigella sativa seed oil on cyclophosphamideinduced myelosuppression in mice shahad a. hussein*1 and sarmed h. kathem** * ministry of health and environment, medical city health directorate, baghdad, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad ,iraq. abstract myelosuppression is one of the serious adverse effects of cancer chemotherapy that lead to life threatening febrile neutropenia and considered a limiting factor for successful therapy. cyclophosphamide a widely used anticancer drugs, induces severe bone marrow suppression by damaging hematopoietic stem cells. as cancer incidence expands globally, the demand for an effective myeloprotective therapy during cancer treatment is also increasing. nigella sativa seed oil, a well-known plant extract that widely used for various health conditions. this study aims to evaluate the myeloprotective activity of nigella sativa seed oil in cyclophosphamide-induced myelosuppression mice model. myelosuppression induced by single intraperitoneal injection of cyclophosphamide (200 mg/kg). animals were divided into 4 groups each with 6 mice. first group served as negative control group received only normal saline. a second group served as experimental myelosuppression model group achieved by cyclophosphamide. additional 2 groups were mice received nigella sativa seed oil (1ml/kg/day) or (2ml/kg/day) orally for 6 consecutive days starting day 1 with cyclophosphamide on day 3. blood were collected from retro–orbital area on day 7 for total and differential leukocytes counts. compared to model group, it is reported that nigella sativa seed oil (1ml/kg) significantly (p<0.05) increases total leukocytes count (1000±73 versus 700±36 cell/μl) and bone marrow cells viability (50.17±5.36 versus 9.5±0.76%). furthermore, increasing nigella sativa seed oil dose to (2ml/kg) resulted in further improvement, where we reported a significant (p<0.05) increase in total leukocytes count (1267±352 versus 700±36 cell/μl) and bone marrow cells viability (67.67±5.49 versus 9.5±0.76%). the study concluded that nigella sativa seed oil has a promising strong myeloprotective and immunomodulatory effects against cyclophosphamide-induced myelosuppression. keywords: cyclophosphamide, nigella sativa seed oil, total leukocytes count, bone marrow cells viability. العظم المحفز بواسطة عقار تقييم الوقاية المناعية المحتملة لزيت بذور حبة البركة على تثبيط نخاع في الفئرانالسايكلوفوسفامايد **هاشم كاظم سرمد و 1*،شهد عمار حسين العراق الطب، بغداد،مدينة والبيئة، دائرةوزارة الصحة * كلية الصيدلة، جامعة بغداد، العراق والسموم، االدوية فرع** الخالصة التأثيرات الجانبية للعالج الكيميائي والذي يؤدي الى نقص الخاليا المتعادلة المهدد للحياة ويعتبر يعتبر تثبيط نخاع العظم واحداً من ابرز واحداً من اكثر االدوية المضادة للسرطان استخداماً والذي ويعتبر عقار سايكلوفوسفامايد ذلك من اهم العوامل المحددة لنجاح هذا النوع من العالج. نسبة حدوث السرطان اصبحت واسعة على الصعيد للدم. إنبدوره يحفز تثبيط نخاع العظم الحاد وذلك بواسطة تحطيم الخاليا الجذعية المكونة إن زيت بذور حبة البركة هو العالج الكيميائي اصبح في تزايد ايضاً.العالمي لذلك فأن الطلب على العالجات التي تحمي نخاع العظم خالل فترة تهدف هذه الدراسة الى تقييم فعالية زيت بذور حبة البركة في حماية نخاع العظم على مستخلص معروف وكثير االستخدام في حاالت صحية كثيرة تم تقسيم الحيوانات ملغم/كغم( .200ن عقار السايكلوفوسفامايد بجرعة )تم تحفيز تثبيط نخاع العظم بواسطة حقن جرعة منفردة م نموذج الفئران. الى اربعة مجاميع تحتوي كل مجموعة على ستة فئران. اعتبرت المجموعة االولى مجموعة السيطرة وحقنت الفئران بمحلول ملحي عادي. السايكلوفوسفامايد. وفي المجموعتين االضافيتين اعطيت الفئران زيت المجموعة الثانية اعتبرت نموذج تثبيط نخاع العظم وحقنت بعقار مل/كغم/يوم( عن طريق الفم لمدة ستة ايام متتابعة تبدأ من اليوم االول وحقنت الفئران بعقار السايكلوفوسفامايد 2مل/كغم/يوم( او )1بذور حبة البركة ) بالمقارنة مع الدم من منطقة مدار محجر العين لغرض احصاء عدد خاليا الدم البيضاء. في اليوم الثالث بجرعة منفردة. في اليوم السابع تم جمع مقارنة 73±1000مل/كغم تزيد من عدد خاليا الدم البيضاء بصورة معنوية )1مجموعة النموذج, اظهرت النتائج انه زيت بذور حبة البركة بجرعة باألضافة الى ذلك فأن زيادة (.%0.76±9.5مقارنة مع 5.36±50.17يا نقي العظم )خلية/مايكروليتر( وتزيد من قابلية نمو خال 36±700مع بالمقارنة مع 352±1267مل/كغم( ادى الى زيادة اضافية في عدد خاليا الدم البيضاء بصورة معنوية )2جرعة زيت بذور حبة البركة الى ) ويمكن االستنتاج بأنه زيت بذور حبة البركة (.%0.76±9.5بالمقارنة مع 5.49±67.67خلية/مايكروليتر( ونمو في خاليا نقي العظم ) 700±36 عقار السايكلوفوسفامايد.تثبيط نخاع العظم المحفز بواسطة يمكن ان يكون لديه تأثير قوي في حماية نخاع العظم وتأثيرات مناعية ضد .نخاع العظم خاليا ، حيوية عدد خاليا الدم البيضاء السايكلوفوسفامايد ، زيت بذور حبة البركة ، : الكلمات المفتاحية 1corresponding author e-mail: shahad.amaar.89@gmail.com received: 28/11/2019 accepted: 1/ 2/2020 iraqi journal of pharmaceutical scienc https://doi.org/10.31351/vol29iss1pp253-259 iraqi j pharm sci, vol.29(1) 2020 immuno-protective effect of nigella sativa seed oil 254 introduction bone marrow is the major site that responsible for hematopoiesis by hematopoietic stem cells (hsc) which are continuously proliferating cells that can be differentiated to form different mature blood cells that include red blood cells, white blood cells (granulocytes, monocytes and lymphocytes) and platelets (1). white blood cells (leukocytes) are major part of the immune system that protect the body and defend against pathogens and fight infections. normal level of total leukocyte count in adult human is (4000 – 10000 cells/mm3) (2), while in mice is (2000-10000 cells/mm3) (3). myelosuppresion is the most common and popular adverse effect of several chemotherapy which are the most effective therapy for cancer (4). the myelotoxicity caused by cytotoxic drugs lead to dose reductions of these drugs and delays in therapy, these can compromise the outcomes of chemotherapy and reduces overall survival (5,6). cyclophosphamide (cp) is one of the oldest and the most successful anticancer drugs, it acts as alkylating agent that belong to oxazaphosporines group (7). the initial clinical trials of cyclophosphamide for cancer treatment were performed in 1958, and in 1959 approved by the fda as a cytotoxic agent. in fact, it is well established that cyclophosphamide is a prodrug metabolize to generate active alkylating metabolites that include 4-hydroxy cyclophosphamide, aldophosphamide mustard , which can interfere with dna synthesis in rapidly dividing cells and can lead to apoptosis. cyclophosphamide has toxic effects on different organs that include bone marrow, heart, gonadal and bladder. the main cyclophosphamide toxicity is on bone marrow and bladder (8), the cyclophosphamide myelotoxicity as a result of lack specificity in anti-tumour activity. when killing tumour cells, it also cause serious damage on normal cells, for instance, hematopoietic stem cells which are rapidly dividing cells in the bone marrow, this leads to decrease the ability of these cells to proliferate and differentiate with reduction in the formation of different blood cells. myelosuppresion can lead to anaemia, leukopenia, and thrombocytopenia. leukopenia is a life-threatening condition that lead to febrile neutropenia combined with bacterial and fungal infections (9). different plants extract found to have a protective effect against a variety of toxicity that induced by chemotherapy (10). nigella sativa is an annual herb belonging to the ranunculaceae family. it is also known as (black cumin), the seed have been used as a seasoning spice and food additive in the middle east and mediterranean areas (11). nigella sativa seeds contain proteins, saponins, alkaloids, fixed oil, and essential oil. the biological effects of nigella sativa are attributed to the various characterized constituents. thymoquinone (tq), the main bioactive constituent of the essential oil may be responsible for major therapeutic effects of nigella sativa (12) . in addition to the seed, its cold pressed oil is utilized as natural dietary supplements and therapeutic agents to support the immune system, treat asthma, allergic rhinitis, diabetes, gastrointestinal disturbance and other conditions in the european union and other developed countries (13). nigella sativa seed oil (ng oil) used to evaluate its immune-protective and immunomodulatory effect on myelosuppresion induced by cyclophosphamide. the aim of this study is to evaluate the immune-protective and immunomodulatory effect of nigella sativa seed oil on cyclophosphamideinduced myelosuppression in mice. materials and methods animals forty two adults’ albino male mice weighing (20-25) gram were brought from and maintained in the animal house of college of pharmacy/university of baghdad under normal conditions of temperature, humidity and light/dark cycle. the animals were fed commercial pellets and tap water ad libitum throughout the experimental period. the study was approved by the scientific and ethical-committees of the college of pharmacy/university of baghdad. drugs cyclophosphamide as monohydrate (1000mg vial) was purchased from (baxter health care ltd, germany). nigella sativa seed oil 100% pure was purchased from toroslar company (turkey), which was prepared by cold pressing from fresh nigella sativa seeds and packaged in amber glass bottle to protect it from sunlight exposure. experimental protocol mice were randomly allocated into four groups, each containing 6 mice as follow: group i: mice were received a single intraperitoneal injection of 0.15 ml 0.9 % normal saline at day 3. this group served as a negative control. group ii: mice were received a single dose of intraperitoneal cyclophosphamide (200mg/kg body weight) on day 3. this group served as experimental model (14). group iii: mice were administered a dose of nigella sativa seed oil (1ml/kg body weight/day) orally by oral gavage for 6 consecutive days starting day 1, with a single dose of intraperitoneal cyclophosphamide (200mg/kg body weight) on day 3. group iv: mice were orally administered nigella sativa seed oil at a dose of (2ml/kg body weight/day) by oral gavage for 6 consecutive days starting day 1 , with a single dose of intraperitoneal cyclophosphamide (200mg/kg body weight) on day 3. iraqi j pharm sci, vol.29(1) 2020 immuno-protective effect of nigella sativa seed oil 255 in groups (iii and iv) animals, each dose of nigella sativa seed oil was administered once daily for 6 consecutive days starting day 1, and on day 3, they received a single dose of cyclophosphamide (200mg/kg body weight) by ip injection. twentyfour hours after the end of the treatment duration (day 7), the animals were euthanized by diethyl ether (bdh chemicals, england) and cervical dislocation (15). samples collection 1blood collection after animals have been euthanized on day 7, 0.5 ml of blood was drawn from retro–orbital area of mice eyes and collected in edta tube. samples were then prepared for the analysis of total leukocyte count (16). 2bone marrow extraction. after mice euthanized, femur bone was extracted and used to extract bone marrow cells by flushing the marrow cavity with phosphate buffered saline (pbs) ph 7.4 and collected as cell suspension, that was used immediately for bone marrow cell viability test (17,18). analysis estimation of total leukocytes count in blood: total white blood cells counts were performed on an automated hematology analyzer (xp 300, sysmex / japan) using direct detection method (19). bone marrow cells viability test this test was used to determine the number of viable bone marrow cells present in a cell suspension to the total cell count (20). equal parts of 0.04% trypan blue solution and bone marrow cell suspension were mixed to form a mixture that utilized for counting of the viable cells (clear) and nonviable cells (blue) immediately (within 3-5 minute after mixing) using neubaur hemocytometer under light microscopy. viable bone marrow cells calculated as percentage of total cells. calculation performed by the following equation: viable cells (%) = [number of viable cells / total number of cells] * 100 statistical analysis the numeric data presented in the study expressed as mean±standard error of the mean (se). all statistical analyses were carried out using the statistical package of social science (spss) software version 25. intergroup comparisons were made using nonparametric tests (kruskalwallis h test, mann-whitney u test). differences were considered significant at p<0.05 (21). results effect of two doses of nigella sativa seed oil on total leukocyte count in cyclophosphamide induced myelosuppression. the data presented in (table 1) and (figure 1) showed that administration of a single dose of intraperitoneal cyclophosphamide (200mg/kg) to mice on day 3 (group ii, experimental model) resulted in the significant (p<0.05) reduction of total leukocyte count (700 ± 36) compared to the negative control animals (group i, 5800 ± 520), that lead to the severe suppression of bone marrow function. however, in group iii mice, which administrated nigella sativa seed oil at a dose of (1ml/kg/day) 3 days prior to a single intraperitoneal dose of cyclophosphamide (200mg/kg) on day 3 resulted in significant attenuation of the cyclophosphamide myelosuppressing effect (table1) (figure 1), the total leukocyte count in those animals (group iii) increased (1000 ± 73) significantly (p<0.05) compared to animals that received cyclophosphamide only (group ii, experimental model). furthermore, increasing the dose of nigella sativa seed oil to 2ml/kg/day administered 3 days before cyclophosphamide treatment resulted in further improvement of bone marrow function (table1) (figure 1). data showed that animals received (2 ml/kg/day) of nigella sativa seed oil (group iv) resulted in significant rise (p<0.05) in total leukocyte count (1267 ± 352) compared to the experimental model group (group ii). effect of two doses of nigella sativa seed oil on bone marrow cells viability in cyclophosphamideinduced myelosuppression. the data presented in table 1 and figure 2 showed that administration of single intraperitoneal dose of cyclophosphamide (200mg/kg) causes aggressive suppression of bone marrow function manifested as highly significant reduction (p<0.05) in bone marrow cells viability (9.5 ± 0.76) in the experimental model (group ii) compared to the negative control animals ( 92 ± 0.58) (group i). however, in group iii mice received nigella sativa seed oil at dose of (1ml/kg/day) 3 days prior to (200mg/kg) cyclophosphamide therapy resulted in significant (p< 0.05) increase in bone marrow cell viability (50.17 ± 5.36) compared to model group (group ii) (table 1) (figure 2). furthermore, increasing the dose of nigella sativa seed oil to (2ml/kg/day) administered 3 days before cyclophosphamide treatment (group iv) resulted in further increase in bone marrow cells viability (67.67 ± 5.49) and the results appear a significant increase in the viability of bone marrow cells (p<0.05) (group iv) compared to the experimental model group (group ii) (table 1) (figure2). iraqi j pharm sci, vol.29(1) 2020 immuno-protective effect of nigella sativa seed oil 256 table 1. effects of nigella sativa seed oil on total leukocytes count and bone marrow cells viability in cyclophosphamide-induced myelosuppresion in mice. groups type of treatment total leukocyte count (cell/μl) (mean ±s.e.m) bone marrow cell viability (%) (mean ±s.e.m) group i (negative control) normal saline 5800 ± 520 * 92 ± 0.58 * group ii (experimental model) cyclophosphamide (200mg/kg) 700 ± 36 9.5 ± 0.76 group iii cyclophosphamide (200mg/kg) + nigella sativa seed oil (1ml/kg) 1000 ± 73 * 50.17 ± 5.36 ⃰ group iv cyclophosphamide (200mg/kg) + nigella sativa seed oil (2ml/kg) 1267 ± 352 * 67.67 ± 5.49 ⃰ data are expressed as mean ± standard error of means (sem). * significantly different (p<0.05) with respect to the experimental model group. figure 1. bar chart showing total leukocytes count in various experimental mice groups. * significantly different (p<0.05) with respect to the model group. figure 2.bar chart showing bone marrow cells viability test in various experimental mice groups. * significantly different (p<0.05) with respect to the model group. discussion myelosuppression is commonly seen with chemotherapy through interfering with normal cell production in bone marrow leading to myelotoxicity that manifested as a reduction in total white blood cells causing a serious life threatening leukopenia combined with secondary infections (22), so the clinical outcome of treatments with chemotherapy is severely limited, owing to their toxicity to normal tissues. therefore, there is a necessity to discover adjuvant therapy which may be used in conjunction with anticancer drugs to reduce their associated toxic adverse effect and improve the efficacy of the therapy(23). the main dose-limiting toxicities for cyclophosphamide are febrile neutropenia combined with secondary infections, often result in dose reductions, therapy delays and lowers overall survival (24). the current study evaluates the immunuo protective effect of nigella sativa seed oil on cyclophosphamide-induced myelosuppression. the results obtained from this study revealed a beneficial modulating effect on periphral total leukocytes count and bone marrow cells viability (table 1). the present study revealed that cyclophosphamide induced myelosuppression, which was evident by the high reduction of total leukocytes count and bone marrow cells viability in animals treated with cyclophosphamide only compared to the negative control animals (table 1) (figure 1 and 2) that resulted in aggressive suppression of bone marrow function. the most obviously change in white blood cells count due to their short life cycles; the results are in agreement with studies of other researchers, who observed the myelosuppressive effect of cyclophosphamide administration to animals manifested as reduction of the total leukocyte count after 3-4 days from last cyclophosphamide dosing (25, 14). this experimental iraqi j pharm sci, vol.29(1) 2020 immuno-protective effect of nigella sativa seed oil 257 model of cyclophosphamideinduced myelosuppresion is a well validated and widely used animal model to study the effects of chemotherapyinduced myelosuppresion in terms of pathogenesis and therapy (18, 26). this study was in line with study of others in cyclophosphamide induced bone marrow suppression by decreasing bone marrow cells numbers in animals treated with cyclophosphamide (27). data obtained from this study revealed an interesting dose-dependent myeloprotective and immunostimulatory effects of ng oil on bone marrow function. this beneficial effect obtained from the reported increase in total leukocyte count and bone marrow cell viability (table 1), which imply an improvement in bone marrow function after chemotherapy-induced myeosuppression. this is an important result and may open the way for a therapeutic application of ng oil. this is the first study that investigates the effect of ng oil on bone marrow function in chemotherapy-induced myelosuppresion and there are little studies in this regard. however, ng oil used in other studied showed immunostimulatory actions in experiments with different settings. a study showed increase in the total white blood cells, lymphocyte count and neutrophil count in mice treated with ng oil (28). in addition, other study showed that ng oil has an immunostimulatory effect on trypanosoma brucei infected animals and revealed a significant rise in total white blood cells of infected animals (29). another study revealed the immune-potentiation effect of ng oil in immunocompromised animals by stimulation of macrophage phagocytic activity either directly or via lymphocytes activation (30). furthermore, the immuno-protective effect of ng oil was observed in gamma-irradiation induced immunusuppression in animals, which considerably normalized leukopenia and produced significant regeneration in spleen and thymus lymphoid follicles (31). the study showed the effect of thymoquinone, the major constituent of ng oil, resulted in increase of the total count of white blood cells in healthy animals which are received specific dose from thymoquinone (32), specific concentration of thymoquinone may regulate self-renewal and immunomodulatory potential of mice bone marrow mesenchymal stem cells in vitro (33) . another study revealed the treatment of animals with ng extract revealed a significant enhancement in the bone marrow cellularity, total leukocyte count and spleen weight compared to normal control animals (34). all the above mentioned studies and results are consistent with the results obtained from this study although each study has its own experimental settings. on the other hand, some studies showed results in contrast to the results of the current study. in this regard, a study in which type 2 diabetes mellitus patients who received ng oil for 40 days, total leukocyte count remain statistically unchanged (35). additional study observed a decrease in leukocyte count after chronic treatment of healthy animals with ng oil for 12 weeks (36) . the difference in the results of these two studies may be attributed to the longer duration of treatment with ng oil if compared with current study. nigella sativa seed oil 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breast cancer doi: https://doi.org/10.31351/vol29iss2pp99-106 99 assessment of some hematological parameters in iraqi women with different breast cancer stages zahra m. ali* and shatha h. ali* and forat y. mohsen** *department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq **ministry of health and environment, medical city, oncology teaching hospital, baghdad, iraq abstract breast cancer (bc) is the most commonly diagnosed cancer in women. the metabolism of iron is closely regulated by hepcidin which exerts its action by interacting with a ferroportin. the aim of the present study was to assess the alterations in the levels of some serum biomarkers that have a role in iron homeostasis (hepcidin and ferroportin) in addition to hematological parameters (hemoglobin, leukocyte and platelets count) in different stages of bc. this study included 66 women with bc. the patients were categorized as follows : group 1 includes :22 patients with stage i disease ,group 2 includes: 22 patients with stage ii disease ,and group 3 include: 22 patients with stage iii disease .group 4 includes :22 apparently healthy women as control. data analysis revealed a significant elevation of serum hepcidin levels of patients groups 1, 2, and 3 (437.2±26.4, 501.4±31.8 and 558.5±21.3 pg/ml respectively) ,vs (179.4±19.8 pg/ml) of control, with steady elevations from stage i to iii . furthermore, serum ferroportin levels were significantly lowered in groups 1 and 3 compared to control (0.589±0.107 and 0.733±0.1 vs 1.37±0.28 ng/ml respectively).while blood hemoglobin level of group 3 were lower (11.96±0.18 vs 12.7±0.13g/dl ) compared with controls . blood leukocyte count of patients (all groups) (7.39±0.28 ,8.93±0.48,9.86±0.52 (^103/µl) respectively) were markedly increased compared to controls (6.06±0.23), while mean platelet count for patients in group 2&3 were significantly increased compared to controls (313.9±19.3,309.2±25.3 vs 233.3±9.1 respectively). in conclusion, hepcidin, ferroportin and hematological markers including hemoglobin, wbc count and platelets count are altered in women with bc compared to healthy control. the changes occur mostly in accordance with disease stages . key words: breast cancer, hepcidin , ferroportin. العراقيات المصابات بمراحل مختلفة من سرطان الثدي تقييم بعض المعلمات الدموية لدى المريضات **فرات يحيى محسن و *شذى حسين علي، 1*,زهراء محمد علي . العراق،بغداد ، جامعة بغداد ، كلية الصيدلة ،فرع العلوم المختبرية السريرية * . العراق،بغداد ، مستشفى االورام التعليمي ، دائرة مدينة الطب ، وزارة الصحة والبيئة ** الخالصة يعمل بالتنسيق مع الذي بواسطة بروتين الهبسدين بدقة ان ايض الحديد منظم.سرطان الثدي هو اكثر سرطان مشخص لدى النساء الفروبورتين. ( باالضافة الى قياس الهبسدين والفروبورتين ) التي لها دور في تنظيم معدالت الحديد بروتينات في مصل الدم فائدة قياستهدف هذه الدراسة الى تقييم وسائل تساعد في دراسة تقدم سرطان الثدي .كالعوامل الدموية )الهيموغلوبين وكريات الدم البيضاء والصفائح الدموية( بالمرحلة )مريضة 22:المجموعة االولى شملت حسب مراحل المرضمصابة بسرطان الثدي ,تم تقسيم المريضات انثى 66شملت هذه الدراسة بالمرحلة الثالثة من )مريضة 22,المجموعة الثالثة شملت (بالمرحلة الثانية من المرض)مريضة 22,المجموعة الثانية شملت (االولى من المرض . كمجموعة مقارنة تبدو بصحة جيدة انثى 22.المجموعة الرابعة شملت (المرض 21.3±558.5 و 31.8±501.4و 26.4±437.2) 3و2و1ت الهبسدين لدى كل مجاميع المرضى في معدال تحليل النتائج اظهرت ارتفاع مهم (pg/ml لمجموعة المقارنة وكانت هناك زيادة تتناسب مع تقدم المرض من المرحلة االولى الى المرحلة الثالثه . 19.8±179.4بالتتابع مقابل ) روبورتين لدى مرضى المرحلة االولى والثالثة بالمقارنة مع معدالت مجموعة االصحاءكان هناك نقصان مهم في معدالت الف باالضافة الى ذلك 0.18±11.96بالتتابع( .بينما اظهرت معدالت الهيموغلوبين للمجموعة الثالثة نقصان مهم 0.28±1.37مقابل 0.1±0.733و 0.589±0.107 0.28±7.39هي 3و2و1كان متوسط معدالت كريات الدم البيضاء لمجاميع المرضى .بالمقارنة مع مجموعة االصحاء 0.13g/dl±12.7مقابل مرتفعة ارتفاعا مهما مقارنة لمجموعة 3و2بينما كانت متوسطات معدالت الصفيحات الدموية للمجاميع بالتتابع 0.52±9.86و 0.48±8.93و بالتتابع . 9.1±233.3مقابل 25.3±309.2و 19.3±313.9واالصحاء ) الهبسيدين والفروبورتين والعوامل الدموية )الهيموغلوبين واعداد كريات الدم البيضاء واعداد الصفائح الدموية( اظهرت نتج ان يمكن ان نستوبذلك انحرافات في النساء المصابات بسرطان الثدي بالمقارنة مع مجموعة االصحاء .معظم هذه االنحرافات كانت متناسقة مع مرحلة المرض. . الفروبورتين،الهبسدين ،: سرطان الثدي الكلمات المفتاحية 1corresponding author e-mail: obaydizahra@yahoo.com received: 21/10 /2019 accepted: 20/5 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp99-106 iraqi j pharm sci, vol.29(2) 2020 serum hepcidin and ferroportin in breast cance 100 introduction breast cancer (bc) is the most commonly diagnosed cancer in women, it is estimated to affect (24.2%) of women worldwide. in iraq, the number of new cases in 2018 for both sexes, and for all age groups was 5141 which represent 20.3% of all new cases for all cancer types (25320), while the number of prevalent cases of all cancer types over 5 years for all ages was 54809 cases (1). breast carcinomas is the most common type of bc although different types of sarcomas and lymphomas also can be encountered (2). chronic inflammation is a key participant in cancer development and progression (3). the mechanism by which chronic inflammation is related to bc prognosis is not fully clear .however , complex processes through which chronic inflammation may promote carcinogenesis such as polarization of m2 tumor-associated macrophages via inflammatory cytokines with the subsequent production of tumor growth factors or promotion of angiogenesis (4,5).in addition, several prognostic factors are correlated with inflammatory status such as body fatness, physical activity, and cardiovascular comorbidity, which may affect tumor prognosis through alternate mechanisms. meanwhile, the presence of undetected cancer cells may result in inflammation, thus inflammation may not only contributor to tumor promotion, but also result of these cancer cells (6). the american society of clinical oncology (asco) recommends the use of tumor markers in prevention, screening, treatment and surveillance of bc. the most common clinically applied tumor markers for breast cancer are ca 15-3, ca 27.29, carcmoembryonic antigen (cea), estrogen receptor (er), progesterone receptor (pr), human epidermal growth factor receptor 2 (her2), urokinase plasminogen activator (upa), plasminogen activator inhibitor 1 (pai-1) and multiparameter assays for gene expression (7) . the metabolism of iron is closely regulated by hepcidin which is liver-derived peptide. it exerts its action by interacting with a ferroportin (fpn), a transmembrane protein implicated in iron efflux from the body iron stores (8).binding of hepcidin with fpn exerts a negative effect on erythropoiesis by inducing internalization and then subsequently destruction of fpn (8,9). increased erythropoiesis suppresses hepcidin production, while iron loading and inflammation induce its production. blood loss, anemia, increased erythropoietin or hypoxia stimulate bone marrow erythropoiesis and then reduce hepcidin (10,11) . increase in serum hepcidin has been reported increasingly from diverse clinical states, (neoplastic diseases, inflammation, and sepsis) (10,12,13). sideropenic anemia is frequently reported in cancer patients, (14) that is mediated by the inflammatory cytokines produced by cancer cells and by macrophages infiltrating the cancer tissue (15). these cytokines stimulate the hepatic production of hepcidin, which in turn inhibit iron absorption in the duodenum and promote iron sequestration by macrophages limiting its recycling (16). moreover, the inflammatory cytokines decrease fpn expression in duodenal enterocytes blocking the transport of iron from enterocytes to transferrin (16). il-10 also induces an increase in serum ferritin and soluble transferrin receptor (stfr) levels causing anemia (17) . the role of peripheral wbc in predicting incident bc remains to be determined in spite of the established role of peripheral wbcs in bc prognosis (18-19). many studies showed the lack of association between total white blood cell count (twbcc) and bc risk (20-21) ; yet, twbcc serves as a significant predictor of certain types of bc and for other types of cancer like lung (20) ,gastric (22) ,and colorectal cancers ( 23). furthermore, platelets have important roles in various physiological and pathophysiological processes, including inflammation, immunity, angiogenesis, wound healing, and cancer progression (24-25). platelet dysfunction and thrombotic disorders are important manifestations of cancer progression (24-26). thrombocytosis is associated with poor cancer prognosis, suggesting a potential role for platelets in the pathogenesis of the disease (27-28). the aim of the present study was to assess the alterations in the levels of some serum biomarkers that have a role in iron homeostasis (hepcidin and ferroportin) in addition to hematological parameters (hemoglobin, leukocyte and platelets count) in different stages of bc. material and methods this study was conducted at the oncology teaching hospital / baghdad medical city from october/2018 to february/2019 . subjects enrolled in the study were 66 women that were recently diagnosed as having bc at different stages of the disease; and 22 apparently healthy women of comparable age to serve as control group. the age range of the subjects was 30-70 years. specialist oncologist performed diagnosis and staging of bc according to breast cancer staging, nccn guidelines version 4.2017 (29); a specialized pathologist using the who grading system, (30) performed the histological confirmation and grading. bc patients at stage iv of the disease is characterized by metastasis to other organs, and those at stage 0 having either ductal carcinoma in situ (dcis) or lobular carcinoma in situ (lcis) of disease, were excluded from the study. other excluded patients were those whom were already undergoing chemo-, radio-, or hormonal therapy; or those with autoimmune diseases or hemoglobinpatheis, except iron deficiency anemia. iraqi j pharm sci, vol.29(2) 2020 serum hepcidin and ferroportin in breast cance 101 subjects enrolled in the study were grouped as: group 1 including 22 bc women at stage i of the disease, group 2 including 22 bc women at stage ii of the disease, group 3 including 22 bc women at stage iii of the disease and group 4 including 22 apparently healthy women serving as control group. blood specimens were obtained from all participants. serum levels of hepcidin and fpn were estimated quantitatively by sandwich type of enzyme linked immune-sorbent assay (elisa), (31) using kits purchased by r&d® (usa) for hepcidin and mybiosource® (usa) for ferroportin. measurements of total white blood cell count (32), platelet cell count (33) were performed utilizing whole blood samples based on dynahelix flow technology. estimation of blood hemoglobin was performed utilizing colorimetric surfactant method (34). statistical analysis analysis of data was carried out using the available statistical package of spss-23 (statistical packages for social sciencesversion 23). data were presented in simple measures of mean, standard error, and range. student t-test was used for testing the significant difference between two groups, whereas anova among more than two groups by lsd post hoc multiple comparisons of data. person’s correlation was calculated for studied quantitative variables. statistical significance was considered whenever the p value was less than 0.05. results age range for group 1 was 32-67 years (51±2.3), for group 2 was 34-68 years, (47.63±1.87), and for group 3 was 32-65 year (45.5±1.75), while age range for control group was 31-60 years (44.3±1.5). table-1 summarized the descriptive date of bc patients and healthy control regarding age, body weight, height, and body mass index (bmi). there was no significant difference between total patients group and healthy control group regarding age, body weight, and height (p value 0.064,0.502 ,0.064 respectively) and significant difference regarding bmi (p value 0.041). table1. descriptive data of total patients and control groups parameter bc patients (n=66) healthy control (n=22) p value age (years) 47.9±1.14 44.3±1.5 0.064 body weight (kg) 74.07±1.15 72.04±2.7 0.502 height (cm) 159.8±0.81 163.04±2.05 0.064 bmi (kg/m2) 28.8±0.3 26.96±0.82 0.041* data are expressed as mean± se , bc: breast cancer, bmi: body mass index,* significant difference from control group (p˂0.05) table 2 presents the data of studied groups regarding age, body weight, height and body mass index table2.descriptive data of studied groups. group parameter group 1 group 2 group 3 group 4 age (years) 51.5±2.3 46.2±1.66 45.9±1.79 44.3±1.5 body weight (kg) 73.3±1.76 72.4±1.96 76.4±2.24 72.04±2.75 height (cm) 160.1±1.42 157.9±1.46 161.6±1.26 163.04±2.05 bmi(kg/m2) 28.4±0.45 28.9±0.41 29.1±0.7 26.9±0.82 data are expressed as mean± se , bmi: body mass index data of table-3 describe the differences between patients and control groups regarding family history, a number of smoker subjects, presence of diabetes mellitus , presence of hypertension . sixteen of bc patients were presented with one or more first degree relative with bc in contrast to two of female control, all healthy control females were selected as none of them having diabetes mellitus nor hypertension and all of them were nonsmoker, while five of bc patients were diabetic and eight of them were hypertensive. table 3 . medical history of studied groups parameter bc patients n=66 healthy control n=22 family history 16 2 smoker ** 2 none dm 5 none hypertension*** 8 none bc: breast cancer, n=number, dm: diabetes mellitus, iraqi j pharm sci, vol.29(2) 2020 serum hepcidin and ferroportin in breast cance 102 table 4 describes the differences among studied groups of patients regarding hormonal receptors state [estrogen receptor (er), progesterone receptor (pr), and human epidermal growth factor 2 receptor (her-2)], and histological classification of bc as invasive ductal carcinoma (idc) was the predominant type followed by ductal carcinoma in situ (dcis) with idc , then invasive lobular carcinoma (ilc) with idc and finally ilc . table4. basic criteria of studied patients in different stages of breast cancer group parameter stage i (n=22) % stage ii (n=22) % stage iii (n=22) % total (n=66) % er positive 10 45.4 16 72.7 17 77.2 43 65.1 pr positive 12 54.5 16 72.7 16 72.7 44 66.6 her-2 positive 9 40.9 10 45.4 9 40.9 28 42.4 idc 16 72.7 15 68.1 14 63.6 45 68.1 dcis&idc 5 22.7 6 27.2 4 18.1 15 22.7 ilc 0 0 1 4.5 0 0 1 1.5 ilc&idc 1 4.5 0 0 4 18.1 5 7.2 er: estrogen receptor, pr : progesterone receptor, her-2:human epidermal growth factor receptor, idc :invasive ductal carcinoma, dcis: ductal carcinoma in situ, ilc :invasive lobular carcinoma. as presented in table-5, there were significant elevations in serum hepcidin level, blood wbc count and blood platelet count of total bc patients as compared to control group, while there was significant decrease in blood hemoglobin level of total patients as compared to control and no significant difference between these groups regarding a serum ferroportin level. table5. blood and serum levels of the studied parameters among total patients and control groups group parameter total patients (n 66) control group (n 22) p value s. hepcidin pg/ml 499.09±16.4 179.4±19.8 0.000* s.ferroportin ng/ml 0.794±0.11 1.37±0.28 0.07 b.hemoglobin gm/dl 12.1±0.16 12.7±0.13 0.009* b.wbc count (ˆ103/µl) 8.73±0.28 6.06±0.23 0.000* b. platelet count (ˆ103/µl) 301.8±12.4 233.3±9.16 0.000* data are expressed as mean ±se, wbc: white blood cell ,b: blood ,s: serum table6. blood and serum levels of the studied parameters among the different study groups group parameter group 1 (n=22) group 2 (n=22) group 3 (n=22) group 4 (control) (n=22) p value s. hepcidin pg/ml 437.2± 26.4 501.4± 31.8 558.5± 21.3 179.4± 19.8 0.000* s.ferroportin ng/ml 0.589± 0.107 1.06± 0.31 0.733 ± 0.102 1.37± 0.28 0.07 b.hemoglobin gm/dl 12.2± 0.28 12.4± 0.37 11.9± 0.18 12.7± 0.13 0.17 b . wbc count ^103/µl) 7.39± 0.28 8.93± 0.48 9.86± 0.52 6.06± 0.23 0.000* b.platelet count(^103/µl) 282.2± 20.09 313.9± 19.3 309.2 ±25.3 233.3 ±9.16 0.016* data are expressed as mean ±se, wbc: white blood cell , b: blood ,s: serum figure-1 illustrates that serum hepcidin levels of patient groups are significantly higher than that of controls through different stages from i-iii. hepcidin levels tends to increase as the disease progresses; as presented by significantly higher levels in stage iii compared to that in stage i. serum levels of fpn of stage i and iii were significantly lower than that of control group with no significant difference between patient groups (figure-2). blood hemoglobin levels of stage iii was significantly lower than that of control group with no significant difference between patients groups (figure-3). iraqi j pharm sci, vol.29(2) 2020 serum hepcidin and ferroportin in breast cance 103 figure1. mean value of serum hepcidin among studied groups *= significantly different from control group (p˂0.05) figure 3. mean value of blood hemoglobin among studied groups *= significantly different from control group (p˂0.05) correlation studies were done between all studied parameters among all groups including total patients, group 1, group 2, group 3, and group 4 and the significant results (that indicated positive or negative correlation) are tabulated in table-5. serum hepcidin levels were significantly correlated with serum fpn levels in bc patients (total patient r=0.407, stage ii r=-0.678, stage iii r=-0.433). more advanced stages of bc (stage iii) were presented with more significant correlation between fpn levels and blood platelet count (r=-0.438). figure2. mean value of serum ferroportin among studied groups *= significantly different from control group (p˂0.05). table 5. correlation study among studied groups x-axis y-axis r-value p value group s.hepcidin s.ferroportin -0.678 0.001 stage ii s.hepcidin s.ferroportin -0.433 0.044 stage iii s.hepcidin s.ferroportin -0.407 0.001 total patient s.ferroportin b.platelet -0.438 0.041 stage iii s: serum, b: blood discussion globally, more than 95% of bcs diagnosed as adenocarcinomas (35) , while idc is the most common form of invasive breast cancer (accounting for 55% of incidence upon diagnosis) (36) ,and ilc constitutes only 5%–15% of invasive breast carcinoma (37) .the results of this study agree with these results ,as 68.1% of the patients having idc ,and only 1.5% of patients having ilc as mentioned in table-2. the important role of iron in dna synthesis could lead to cell cycle arrest after iron depletion, (38) whereas chronic sub toxic levels feeding of iron to cancer cells transform them into a the more aggressive phenotype which is prone to metastasis (39). hepcidin induces fpn degradation, thus, interfering with iron efflux and cause iron sequestration in tumor cells. moreover, hepcidin blocks iron export from cells such as macrophages and enterocytes (40). serum levels of hepcidin are strictly controlled by different stimuli; iron status is the prime controller of hepcidin expression under basal conditions (41); yet, other factors can also control liver hepcidin expression, such as hormones, growth factors and heparins (42-44). in this study, serum levels of hepcidin in all patients groups are significantly higher than that of the control group and the elevation was coordinated with the stage of the disease (p value 0.000, 0.000, 0.000 respectively) with significant elevation of stage iii as compared to stage i (p value 0.001), while there was no significant difference between levels of stage i 1&ii, stage ii&iii (p value 0.076 iraqi j pharm sci, vol.29(2) 2020 serum hepcidin and ferroportin in breast cance 104 ,0.115 respectively). while, serum fpn levels are significantly lower in patients with stage i and iii than in the control group (p value 0.015 , 0.047 respectively). serum fpn levels in patients with stage ii was also lower than that of the control group but the difference was statistically non-significant (p value 0.325),and there were no significant differences between stages i&ii and stages i&iii and stages ii&iii (p value 0.142 ,0.65 ,0.307 respectively ). similar findings were reported in previous studies. orlandi et al (45) reported that patients with bc and benign breast lesions had significantly higher hepcidin levels than healthy control group. similarly, ciniselli et al (46) found that when compared with patients with benign breast diseases, patients with bc had significantly higher hepcidin levels, also bc patients were reported to have higher serum hepcidin that is associated with lower levels of fpn when compared to healthy controls (45,47) . patients with stage iii showed significantly lower blood hemoglobin levels compared to that of the control group (p value 0.032) and nonsignificant decrease of stage i and stage ii as compared to control group (p value 0.138 ,0.325 respectively ) ,and there were non-significant differences among patients groups (stages i&ii ,stages i&iii ,and stages ii&iii ) (p value 0.613 ,0.498 ,0.238 respectively ) . these changes occur in accordance with the high hepcidin and low fpn levels. twelve out of twenty two of bc patients at stage iii have iron deficiency anemia at time of bc diagnosis. the number of anemic bc patients at stage i and stage ii was much lower than that of patients at advance stage (stage iii) (4/22 and 5/22) respectively; these findings point the important role of hepcidin in iron metabolism. hepcidin overexpression is not only associated with various hematological malignancies and some solid organs tumors such as breast, prostate cancers, but it also participate in the development of anemia in those patients (10,45,48) . in this study, there was a steady increase in twbcc throughout disease stages ,and there were significant elevations in all patients groups (stage i ,ii ,and iii ) as compared to control group (p value 0.022 ,0.000 ,0,000 respectively ) , even that the increase is still within normal range of blood wbc count [410(ˆ103/µl)] (49).there were significant elevations of stage ii and iii than that of stage i (p value 0.008 ,0.000 respectively ) ,and no-significant elevation of stage iii than that of stage ii (p value 0.103). elevated wbc count has been shown to be associated with increased risks of the lung (19), gastric (22), and colorectal (23) cancers. leukocytosis is associated with thrombocytosis which is negative prognostic indicator in patients with cancer (50-51).thrombocytosis is associated with a poor cancer prognosis, suggesting a potential role for platelets in the pathogenesis of the disease (28) . in this study , platelet count of all patients group still within normal values. there were significant negative correlations between serum hepcidin levels and serum ferroportin levels of total patients, stage ii patients group and stage iii patient group, and these results may be related to negative regulatory role of hepcidin on fpn by induction of the later degradation (8,9) as these groups showed significant elevation of serum hepcidin level with decrease in fpn levels as compared to control. also previous studies reported elevation of serum hepcidin in association with decreased fpn levels as compared to healthy control (45 ,52). conclusion hepcidin, ferroportin and hematological markers including hemoglobin, wbc count and platelets count are altered in women with bc compared to healthy control. the changes 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survival in patients with metastatic renal cell carcinoma. cancer. 2006; 107: 1793800. 52. pogribny ip. ferroportin and hepcidin: a new hope in diagnosis, prognosis, and therapy for breast cancer. breast cancer res 2010; 12:314. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 nanocapsules loaded with orange peel extract doi: https://doi.org/10.31351/vol32iss1pp194-201 194 chitosan(prunus avium) gum nanocapsules loaded with orange peel extract ***karabet and francois* , hoda habbal *, **,1idee -tahani al *department of food science, faculty of agriculture, damascus university, syria ** department of food technology, general commission for scientific agricultural research, syria. *** department of chemistry, faculty of science, damascus university, syria. abstract the orange peel extract (oe) is an additive material that has been used widely as a natural antioxidant source and bioactive compound in the pharmaceutical and food sectors. the poor stability and degradation of this extract were considered problems in the industry. new technologies have been introduced recently to prevent this degradation such as encapsulation. in this work, chitosan (cs) and prunus avium gum (pg) were proposed as promising materials for the encapsulation of oe via the ionic gelation method. the effect of different ratios of cs: pg and cs: oe on encapsulation efficiency (ee %) of oe and the capsule size was investigated. the ee% of cs-pg ranged from 60.63 to 87.06 % with a size range of 40 to 95nm according to atomic force microscope (afm) images. the formulation with the highest ee% was chosen to be characterized by ftir, scanning electron microscope (sem), and in vitro release study. the ftir spectra confirmed the cross-linking between the nh3+ group in cs and the negative functional group (-coo-) in pg. according to sem micrographs, the capsules showed a spongy porous structure. the in vitro release study indicated that the release of oe from the cs-pg matrix in the acidic and neutral medium was 55.15 and 52.67% respectively after incubation for 240 minutes. this study found that cs-pg can be used as an effective wall matrix for encapsulating oe and delivering it into the gastrointestinal system. keywords: orange peel extract, encapsulation, chitosan, prunus avium gum. النانوية المحملة بمستخلص قشور البرتقال (prunus avium)صمغ -كبسوالت الكيتوزان *** فرانسوا قره بت و *هدى حبّال ، 1*,**, تهاني العايدي .سورية، جامعة دمشق، كلية الزراعة ، قسم علوم االغذية * سورية ، الهيئة العامة للبحوث العلمية الزراعية ، قسم تكنولوجيا األغذية** سورية. ، جامعة دمشق، كلية العلوم، قسم الكيمياء *** الخالصة والمركبات الفعالة حيوياً ( مادة مضافة تم استعالها على نطاق واسع كمصدر طبيعي لمضادات األكسدةoeمستخلص قشر البرتقال )يعد مشكلة في الصناعة. أدخلت تقنيات جديدة تعد . في المجاالت الصيدالنية والغذائية لمنع -كالكبسلة -مؤخًراعدم الثباتية وتدهور هذا المستخلص وفق ( oeكمادة جدارية للكبسوالت النانومترية لمستخلص قشور البرتقال ) prunus avium التدهور.هدف البحث إلى استعمال الكيتوزان وصمغ دُرس تأثير نسب إضافة مختلفة من الكيتوزان: الصمغ ونسب المستخلص : الكيتوزان في كفاءة الكبسلة، وحجم الكبسوالت طريقة الهالم األيوني. نانومتر. تم اختيار المعاملة التي حققت أعلى كفاءة كبسلة 95و 40كبسوالت بين % وأبعاد ال87.06و 60.63الناتجة. تراوحت كفاءة الكبسلة بين للكبسوالت حدوث ftirأكدت أطياف الـ .( والقدرة على التحرر في الزجاج. semوالمجهر اإللكتروني الماسح ) ftirووصفت باستعمال تقنية في الصمغ. ظهرت الكبسوالت ببنى coo−)− (ومجموعات الهيدروكسيل في الكيتوزان )-nh3+ (تجاذب كهربائي وارتباط بين مجموعات األمين ي لقشور مسامية وفقاً لصور المجهر اإللكتروني الماسح. أوضحت دراسة القدرة على التحرر في الزجاج بأن النسبة المئوية لتحرر المستخلص الفينول على التوالي بعد التحضين لمدة 52.67و 55.15البرتقال في األوساط الحامضية والقلوية وصل إلى دقيقة. أظهرت الدراسة بأنه يمكن %240 .جدارية فعالة لتغليف مستخلص قشور البرتقال وادكم prunus aviumاستعمال الكيتوزان وصمغ prunus aviumالكلمات المفتاحية: مستخلص قشور البرتقال، الكبسلة، الكيتوزان، صمغ introduction the processing industries of fruits and vegetables generate a huge amount of by-products, such as peels, seeds, pulp, and certain other remnants that are discarded (1). scientists are interested in these wastes because they are sources of high-added value (2). orange peels, which remain after juice extraction, are an important source of bioactive compounds like phenols, pigments, and essential oil. these components, which have biological properties such as antioxidant, antibacterial, and anti-inflammatory, can be employed in the pharmaceutical and food industries (3). despite their importance, these compounds have limited bioavailability and low stability under processing conditions. the majority of phenolic compounds found in food are esters, polymers, or glycosides, and cannot be absorbed in this form (4). encapsulation in biodegradable polymeric nanoparticles is one approach to circumventing these constraints (5). hussein et al (6) reported that the nano-capsulation process increased the thermal stability of rosemary essential oil. tavakoli et al (7) claimed that the encapsulation of the olive leaf extract rich in polyphenols masks its bitter taste and increases the nutritional value and stability of the extract in yogurt. 1corresponding author e-mail: tahane.alidee@yahoo.com received: 2/2 /2022 accepted: 7/6 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp194-201 iraqi j pharm sci, vol.32(1) 2023 nanocapsules loaded with orange peel extract 195 several methods for forming nanocapsules have been developed like emulsification, coacervation, ionic gelation, electrospinning, etc (8). the ionic gelation approach was widely employed in this field due to the use of non-toxic and highly biocompatible polymers. this method is based on the interaction between oppositely charged polymers (9) like chitosan and gums. chitosan (cs) is a naturally occurring cationic polysaccharide composed of glucosamine and n-acetyl-glucosamine produced by the alkaline deacetylation of crustacean chitin (10). the amine groups of chitosan are ionized in acidic conditions. plant gums are the high molecular hydrophilic polysaccharides produced from different parts of the plant (e.g. plant cell walls, tree exudates, seeds, tuber/roots) (11). prunus avium gum (pg) is a rosaceae tree that exudates gum (12). it is a by-product of the cherry tree in syria as the result of the gummosis phenomenon. the physicochemical and functional properties of prunus gums exudate showed promising properties for usage in a variety of sectors. ( 13, 14, 15), like nanoencapsulation of bioactive compounds. gums can react with cationic polymers during ionic gelation by their negative functional groups (16). salarbashi et al (17), produced prunus armeniaca gum nanoparticles loaded with curcumin and achieve high encapsulation efficiency (86.1%). a new nanoparticle delivery system for the encapsulation of quercetin was also created utilizing a mixture of almond gum (amygdalus communis l.) and shellac as biopolymers (18). several studies indicated that the preparation parameters such as polymer concentration, chitosan–gum relative ratio, and the concentration of bioactive compounds can affect the encapsulation efficiency and particle sizes (19, 20, 21). the aim of this study is to prepare and evaluate oe-loaded chitosan nanocapsules using prunus avium gum (pg) as an ionic crosslinker. the impacts of the ratios of cs: gum and cs: oe, on encapsulation efficiency and capsule size were examined. furthermore, the capsules with the highest encapsulation efficiency were characterized using a scanning electron microscope (sem), and fourier transforms infrared (ftir) and their release was investigated in acidic and neutral mediums. materials and methods materials the peels of sweet orange (citrus sinensis) were obtained -in 2018from local juice shops in damascus, syria. a sample of cherry tree gum exudates (pg) was taken from the trunks and branches of cherry trees (prunus avium) in suwayda governorate, southern syria. chitosan (cs) low molecular weight (batch #stbh2613). all of the high-purity reagents and solvents utilized in this work were purchased from sigma-aldrich. preparation of orange peels extract (oe) the peels of valencia sweet orange (citrus sinensis l.) were washed and dried at 40°c for 48 hours. the orange peel extract was prepared according to the optimal conditions studied previously. grounded and dried orange peels were mixed with ethanol (50%) at the ratio of 1:10 (w/v) and put in a water bath at 36 °c for 2 hours and then centrifuged at 4500 rpm for 6 minutes. the supernatant was collected, filtrated through 0.45μm membranes, and kept in glass containers at −18 °c for further processing. determination of total phenolic compounds total phenolic compounds of oe were estimated using a folin–ciocalteu method. the extract samples (0.5 ml) were mixed with 2.5 ml of folin-ciocalteau reagent (0.2 n). after 5 minutes 2.0 ml of sodium carbonate (75 g/l) were added, and the mixture incubated at room temperature for 2 hours. the absorbance of the reaction was measured at 760 nm. gallic acid at concentrations ranging from 25 to 250 mg/l was used in the preparation of a standard curve (22) purification of pg gum the gum was purified using the modified method reported by bhushette et al (23). the samples were powdered in an electric grinder (starmix) and sieved (500μm sieve) to obtain a uniform particle size. the gum solution (5 %w/v) was heated to 50°c for 3 h, filtered to remove impurities, and concentrated twice at 50°c. the cherry gum exudate was precipitated with absolute ethanol (exudate solution: ethanol, 1:3 v/v). the precipitated gum was separated by centrifugation at 4500 rpm for 3 minutes, dried in the oven at 50°c for 48 hours to achieve a consistent weight, and then stored in an airtight container for future use (figure1.). figure 1. a and b: the sample of (prunus avium) gum exudates, c: gum precipitated with ethanol, d: purified gum after separated by centrifugation . ( a b c d iraqi j pharm sci, vol.32(1) 2023 nanocapsules loaded with orange peel extract 196 preparation of cs-pg nanocapsule nanocapsules loaded with oe were produced according to the method described by akinluwade et al (24) with some modifications. cs (0.5%) was dissolved in an acetic acid solution )1 %( then, oe was added to cs with different oe: cs ratios (0.5:1, 1:1, and 1.5:1 v/v) and stirred at 1300 rpm for 1 hour using a magnetic stirrer. after that, and during stirring the pg (2%w/v) was added to the cs-oe solution by drop-wise method at different pg: cs volume ratios (1:1, 2:1, and 3:1 v/v), and the mixture was stirred at room temperature for 2 hours. the formed capsules were separated by centrifuging at 13,000 rpm for 30 minutes at 4°c, and the supernatant was used to determine the encapsulation efficiency. encapsulation efficiency (ee) the free phenolic compounds were determined in the supernatant after the separation of capsules by the folin-ciocalteau method which was described by ebrahimzadeh et al (22) using a spectrophotometer at 765 nm and the ee% was calculated according to the following equation (1): ee% = total amount of phenols−free phenols total amount of phenols ×100 (1) capsules sizes the size, the particle size distribution, and surface morphology were examined using atomic force microscopy measurements (afm, nanosurf easyscan2, pgitzerland: tapping mode (tap190 alg), nanosensors™, neuchatel, switzerland). characterizations of nanocapsules the freeze-dried nanocapsules with the maximum ee% were characterized (alpha12ldplus-germany) by scanning electron microscopy (sem), fourier transform infrared spectroscopy (ftir), and in vitro release profile. scanning electron microscopy (sem) sem (sem, xmu ii-vega) was used to study the morphology of prepared nanocapsules using a secondary electron detector at an acceleration voltage of 20 kv. in vitro release study in vitro release study was conducted using a modified method of yadav et al (25). the release of oe from the capsules was performed in phosphate buffer solution (0.2 m, ph=7.2) and hcl (0.1n, ph=1.2). the nanocapsules (0.03g) were placed in an eppendorf tube with 1 ml of the release solution and incubated at 37°c in a shaking water bath for 240 min. at regular intervals (0,15, 30, 60, 90, 120, 150, 180, 210, and 240 minutes), the sample was withdrawn and centrifuged at 3500 rpm for 10 minutes to separate the released oe from simulated fluids. then, the quantity of releasing oe was measured spectrophotometrically using the folin– ciocalteu method as described by ebrahimzadeh et al (22). infrared spectrum analysis ft-ir spectra for cs, pg, and cs-pg capsules were carried out using an ftir-4200 jasco spectrometer in the wavelength range of 4000–400 cm-1 at a resolution of 4 cm−1 using the kbr disc method . statistical analysis the results of experiments were expressed as a mean± standard deviation of three replications of the experiment. analysis of variance (anova) two-way as a statistical model was performed, followed by fisher's least significant difference (lsd) test to evaluate the significant difference between treatments at the 5% level using genstat software 12. results and discussion encapsulation efficiency (ee %) the ee % of oe ranged from 60.63 to 87.06% for all formed nanocapsules with different ratios of cs: pg and cs: oe (table 1). the results of ee% were lower than those reported by mahmoud et al (26), the difference in results could be attributed to the differences in methods of preparation nanocapsule and the difference in the physicochemical properties of wall materials. mahdavi et al (27) reported that the type of coating material and the ratio of core material to coating material significantly impact the ee%. table 1. ee% of oe -loaded nanocapsules created via different treatments. cs: oe ratio cs: pg ratio 1:1 1:2 1:3 1:0.5 87.06±0.39a nf nf 1:1 72.47±0.78bc 73.85±0.36b 79.62±0.79ab 1:1.5 60.63±5.84d 65.03±5.3cd 76.05±1.67b *mean values ±sd values. different letters indicate a significant difference (p<0.05) between the results. nf: not formed . iraqi j pharm sci, vol.32(1) 2023 nanocapsules loaded with orange peel extract 197 according to the statistical analysis in table 2, the oe ratio had a significant influence on the ee% (p<0.05). our results showed that as the ratio of oe increased the ee % decreased, this may be explained by the fact that when the ratio of oe increased the solution density increased, and the cross-linking between the matrix and oe was lost. this finding was in agreement with a previous study for preparing viola odorata linn. extract microcapsules (28). on the other hand, the statistical analysis revealed that the ee% was dependent on the ratio of cs: pg, and the maximum ee% was achieved at the high ratio of cs: pg. several previous studies have found that increasing the ratio of encapsulating material improves ee% (19, 16). this increase in the ee% with an increasing cs: pg ratio could be explained by insufficient encapsulation materials to form a strong structural matrix and a protective layer at a low ratio of cs: pg. while a denser and stronger wall layer with more active sites is formed around nanocapsules at the higher ratios of cs: pg, leading to a higher ee % (29). particle morphology and size distribution afm imaging is an -efficient approach for providing surface morphology as well as more precise size and size distribution (30). the results in table 2 and afm images confirmed that the capsules of cs-pg prepared via the ionic gelation method were in nanometre dimensions (<100 nm). the afm microscopy in figure. 2 showed that the produced nanocapsules had a uniform spherical shape. one of the most important aspects of particulate system formulation is the particle size distribution (31). the histograms (figure 2, c) revealed the capsules created by cs and pg are with the same mean size. figure 2. 2 × 2μm2 afm images (a: 2dand b: 3d images) and capsules size distribution (c) of capsules entrapping oe: (1:1 of cs-pg and 1:0.5 of cs: oe ) the results in table 2 showed that the capsules produced in this study have a mean size in the range of 40 to 90 nm. the results also showed when the cs: pg ratios were, 1:2 and 1:3 and cs: oe ratio was 1:0.5 no capsules were formed. this result may be due to the viscosity of pg at high ratios resulting in a limited reduction in the functional groups (32) and limiting the interaction between pg hydroxyl groups and cs amine group table 2. size of cs-pg capsules loaded with oe (nm). cs: oe ratio cs: pg ratio 1:1 1:2 1:3 1:0.5 95±7.07d nf nf 1:1 70±1.36c 40±1.71d 41±1.41 d 1:1.5 60±3.4bc 60±1.42 a 52.5±3.53 b * mean ± sd values of triplicate samples. different letters in the results indicate a significant difference (p<0.05).nf: not formed. as shown in table 3 the individual variables and their interactions had a significant effect on capsule size (p<0.05). a significant decrease in size was observed when the pg concentration was increased. this is due to a stronger interaction between the negative charge of the pg and the positive charge of the cs, which is reflected in the small size (33). these results are consistent with those reported by vahidmoghadam et al (34).on the other hand, the size of the nanocapsules was impacted significantly by the ratio of cs: oe added in the preparation, and as the cs: oe ratio increased the size of the capsules increased. rajabi et al (19) claimed that the nanocapsule’s sizes were increased when the core increased because of a rise in the surface of the carriers as a result of their increased load, resulting in a thicker layer of complexes chitosan-gum around them. based on the findings of akdeniz et al (35), when core material ratios increase, the coating material is insufficient to encapsulate it, resulting in the coalescence of capsules and larger particle size. a previous study had found similar results (36). table 3. analysis of variance of cs-pg capsules sizes. source of variation d.f m.s. f gum ratio 2 417.3333 <.001 phenolic extract ratio* 1 675 0.005 gum×phenolic extract 2 217 <.001 *the ratio of phenolic extract (1:0.5) which did not form any capsules was excluded from the statistical analysis of cs-cg nanocapsules. iraqi j pharm sci, vol.32(1) 2023 nanocapsules loaded with orange peel extract 198 nanocapsules structure figure 3 illustrates the sem microscopy of the nanocapsules with the highest ee% (1:1 cs-pg and 1:0.5 cs: oe). the nanocapsule particles seemed to be in an irregular form with noticeably sharp edges (figure. 3). also, the porous structure was observed in freeze-dried powder. tavares et al (37) reported similar results for encapsulation of garlic extract. kuck et al (38) observed the irregular structure and a broken glass shape for freeze-drying particles of grape skin phenolic extract. the spongy porosity structure of the particles is a result of the freezedrying process and the development of ice crystals in the substance (39). figure 3. sem images of capsules entrapping oe at different magnification scales; a:200μm, b: 20 μm, and c: 5 μm. the release profiles of oe from nanocapsules encapsulated structures should not only preserve the bioactive compounds entrapped in them, but they should also be able to release them so that they may perform their biological functions (40). the release profile for the best formulation, based on the highest ee%, was studied in simulated gastric fluid (ph =1.2) and in simulated intestinal fluid (ph= 7.2). it is nutritionally preferable for the capsules to release slowly in simulated gastric media and quickly in simulated intestinal media (41). in acidic conditions, the solubility of cs increases and the polymer matrix swells, resulting in an increase in the target material's release rate (19). as shown in figure 4, phenolic compounds were released from capsules during the incubation periods, and 55.15% of oe was released from the cs-pg matrix after 240 min. at ph> 6.5 the porosity in the structure of nanoparticles observes due to the swell in the gum chain, resulting in the release of bioactive compounds (16). as illustrated in figure 4, oe release in neutral media was significantly lower than in acidic media, with oe release reaching 52.67 after 4 hours. the time required to release 50% of the encapsulated oe (t50%) is about 207.51 and 236.01 min under acidic and neutral media respectively. the amount of phenolic extract released in the literature is higher than that reported in this study; rajabi et al (19) reported that about 80% and 70% of the saffron extract was released from chitosan-arabic gum nanocapsule after 240 minutes in acidic and neutral media, respectively. this difference can be explained by differences in the chemical compositions of the two environments, as well as differences in cs solubility. on the other hand, the chemical structure of saffron extract and oe extract differs in terms of molecular weight and size of molecules, as well as negative charge potential, resulting in differing release behavior of the two substances. our results indicate that the nanocapsules created by cs-pg could be used as an efficient strategy for retaining oe bioactive components and delivering them safely into the gastrointestinal tract. figure 4. in vitro cumulative release of oe from cs-pg nanocapsules in various ph media. fourier transform infrared analyses (ftir) the interactions between the wall components (chitosan and gum) and between wall components and extract within the nanocapsules were studied using ftir measurements (figure. 5). the cs spectra showed peaks at 3362.28 (oh and nh2 stretch), 2880.17 (ch stretch), 1652.7 (c꞊o iraqi j pharm sci, vol.32(1) 2023 nanocapsules loaded with orange peel extract 199 stretching in the amide i), 1423.21 (c-n stretch in amine i), and 1068.37 cm−1 (coc stretch). pg powder show peaks at 3320.82 (o-h stretching), 2924.52 (ch stretching), 1617.98, 1429.96 (stretching vibration of carboxyl), and 1039.77 cm−1 (stretching of the c-o bond). an increase in the wavenumber of the hydroxyl group from 3362.28 in cs to 3399.89 cm−1 in nanocapsules, indicated the improvement of hydrogen bonding between oe and cs (37). the peak of amide i in cs and the peak of carbonyl in pg shifted from 1617.98 and 1652.7 cm-1 to 1621.84 cm-1. also, the peak at 1429.96 and 1423.21 cm-1 in pg and cs shifted to 1449.24 cm-1 in nanocapsules. these changes indicate the complex formation via electrostatic interaction between the negative functional groups of the gum and the positive ones of chitosan within the nanoparticles (19, 43). figure 5. ftir spectra: (a) cs, pg, and their nanocapsules conclusion in this work, oe-loaded cs-pg nanocapsules were successfully prepared via the ionic gelation method. the ratio of cs: pg and cs: oe significantly affects both the capsules size and ee%. the highest ee% (87.06%) with nanometre dimensions (95 nm) was obtained when the cs: pg and cs: oe were added at the ratios 1:1 and 1:0.5 respectively. the release experiments demonstrated that the produced nanocapsules can release oe over time (55.15 and 52.67 % were released after 240 min in acidic and neutral media respectively). according to sem images, the nanocapsules had a spongy porous structure. the findings revealed that cs-pg may be utilized as wall materials for bioactive compound delivery in food sectors and that pg could be used as a natural and safe polymer. future research will focus on a study the effect of fortification of various food products with oe encapsulated with cs-pg on their quality parameters. acknowledgments we are grateful to the general commission for scientific agricultural research. especial thanks to the department of food science -faculty of agriculture, physics department, and chemistry department faculty of science at damascus university. references 1. suleria, h. a., barrow, c. j., and dunshea, f. r.. screening and characterization of 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http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 pathogenesis of obesity-related glomerulopathy doi : https://doi.org/10.31351/vol30iss1pp22-28 22 the pathological mechanisms of obesity-related glomerulopathy: a review article ali a. kasim*,1 and ahmed h. ataimish** *department of clinical laboratory sciences, college of pharmacy, university of baghdad; baghdad-iraq ** department of pharmacology and toxicology, college of pharmacy, university of baghdad; baghdad-iraq abstract the rising prevalence of obesity-related glomerulopathy (org) occurs in concordance with the rising prevalence of obesity worldwide. clinically org is manifested by slowly progressing microalbuminuria that may develop to clinically evident proteinuria. pathological characteristics of org include; glomerular hypertrophy in the presence or absence of focal segmental glomerulosclerosis (fsgs). org can develop into clinically overt chronic renal insufficiency or even end-stage kidney disease. this article reviews the most important mechanisms involved in the development of org; that are related to alteration of renal hemodynamics, stimulation of reninangiotensin-aldosterone system (raas), impairment of insulin sensitivity, ectopic lipid deposition, adipose tissue cytokine disorder and local renal micro-inflammation. keywords: obesity-related glomerulopathy, renin-angiotensin-aldosterone system, insulin resistance االليات المرضية العتالل الكبيبات البولية المرتبط بالسمنة : مقال مراجعة علي عبد الحسين قاسم *،1 و احمد حامد اطيمش** * فرع العلوم المختبرية السريرية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . جامعة بغداد ، بغداد ، العراق . فرع العلوم المختبرية السريرية ، كلية الصيدلة ،** الخالصة سريريًا يتجلى .العالميحدث بالتوافق مع انتشار السمنة في جميع أنحاء بالسمنةالمرتبط البولية إن االنتشار المتزايد العتالل الكبيبات تشمل . سريريًا ظاهرةبروتينية بيلة التي قد تتطور إلى والتقدم ئةبحدوث البيلة األلبومينية الدقيقة بطي بالسمنةالمرتبط البولية اعتالل الكبيبات يمكن و. تصلب كبيبات مقطعي بؤري بانعدام وجودأو البولية بوجود تضخم الكبيبات بالسمنةالمرتبط البولية عتالل الكبيباتالخصائص المرضية ال .لمرض الكلىالنهائية رحلة سريريًا أو حتى الم واضحإلى قصور كلوي مزمن هذا االعتالل أن يتطور -وتحفيز نظام الرينين الطبيعية،ديناميكا الدم الكلوية غير وتشمل؛ اعتالل الكبيبات البولية المرتبط بالسمنة هذه المقالة تستعرض أهم اآلليات لتطور اخيرا و نسيج الدهنيال سايتوكينات، واضطراب خارج النسيج الدهني، وضعف حساسية األنسولين ، وترسب الدهون ألدوستيرون -أنجيوتنسين .االلتهاب الدقيق الموضعي الكلوي ،اضطرابات التمثيل الغذائي للدهون، مقاومة األنسولين ؛ألدوستيرون -أنجيوتنسين -نظام رينين ،المرتبط بالسمنةالكبيبات البولية اعتالل : الكلمات المفتاحية االلتهاب introduction obesity represents a global public health problem. according to the world health organization (who) estimations in 2016, the overweight population worldwide accounted for approximately 1.9 billion adults, of which approximately 650 million are obese (1). obesity is not just over nutrition, but it is closely related to many diseases. pathologically, org is usually manifested by glomerular hypertrophy, with focal segmental glomerulosclerosis (fsgs), occurring in obese individuals (2). org usually has an insidious onset, manifested by slowly progressing microalbuminuria or clinically evident proteinuria, with or without impairment of renal function, and a small number of patients have microscopic hematuria or nephrotic syndrome (3). the prevalence of obesity-related glomerulopathy (org) increases in parallel with the increasing prevalence of obesity (3). the incidence of org is not well documented due to the variation in renal biopsy policy between different countries, and because org can occur without overt signs or symptoms (4). keeping in mind that in obese patients with diabetes mellitus, it cannot be determined whether diabetes or obesity is the principal cause of proteinuria. in a large-scale retrospective study evaluating kidney biopsies, kambham et al. has recorded a tenfold increase in the prevalence of org over 15 years (5). 1corresponding author e-mail: ali.qasem@copharm.uobaghdad.edu.iq received: 27/ 9 /2020 accepted: 14/ 11/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp22-28 iraqi j pharm sci, vol.30(1) 2021 pathogenesis of obesity-related glomerulopathy 23 obesity is involved as an independent risk factor in the development of chronic kidney disease (ckd) (6), thus, org has attracted increasing attention. this article discusses the pathogenesis of org. pathogenesis of org several mechanisms may contribute to the development of org; and these mechanisms are mostly interconnected: alteration of renal hemodynamics obesity alters the renal blood flow; both the glomerular filtration rate (gfr) and the filtration fraction increases in obese individuals regardless of blood pressure (7). the elevated gfr and filtration fraction increases protein concentration in the postglomerular peritubular capillaries with the subsequent increase in oncotic pressure within these capillaries, and hence, increases sodium reabsorption from the proximal tubules (7). moreover, increased activity of the reninangiotensin-aldosterone system (raas) is reported in obese individuals (8-10); an effect that is mediated by increased synthesis of renin and renin precursors by adipose tissue (8). angiotensin ii promotes luminal na+-h+ exchange and basolateral na+-k+ atpase action, activating the epithelial sodium channel (enac), with the subsequent increase in sodium reabsorption (11). sodium reabsorption diminishes solute transfer to macula densa leading to inactivation of the tubuloglomerular feedback and dilation of the glomerular afferent arterioles, i.e. increasing gfr (12). in addition to its effect on proximal tubular sodium reabsorption, angiotensin ii has direct vasoconstrictor effect on the glomerular arterioles. the vasoconstrictor effect of angiotensin ii on the efferent arteriole is greater than the afferent arteriole, increasing the gfr. in addition, other dietary factors such as high-salt diet and highprotein diet may increase gfr in obese patients (13). finally, visceral adiposity imposes physical pressure on the visceral organs including the kidneys, with the consequence of elevated intrarenal pressure that compresses the loop of henle and peritubular capillaries reducing the flow of glomerular filtrate through the renal tubules promoting sodium reabsorption by them(14, 15). long-term high perfusion and high filtration increase the pressure within the glomerular capillaries, and thus, endothelial cells, epithelial cells and mesangial cells damage, which further leads to proteinuria, glomerular hypertrophy, segmental sclerosis, and interstitial fibrosis. this can be mediated by a variety of transmitters, including angiotensin ii, angiotensin receptor (atr), transforming growth factor beta (tgf-beta), tgf-β receptor and phospholipase d (16). renin-angiotensin-aldosterone system activation as discussed earlier raas is overactivated in obese individuals (8-10), and beside the aforementioned effects of this system on renal perfusion, it participates in renal endothelial cell dysfunction and proteinuria, increased inflammation and tissue fibrosis. these detrimental effects are mediated by several mediators such as matrix metalloproteases, cyclooxygenase 2 (cox-2), endothelial nitric oxide synthase (enos), reactive oxygen species (ros), and many cytokines (17-22). insulin resistance several hypotheses were proposed to explain the link between obesity and insulin resistance, such as inflammation, mitochondrial dysfunction, lipotoxicity and most importantly hyperinsulinemia. these entire hypotheses are centered on interrupting of insulin signaling (23, 24). in insulin resistance, the body secretes compensatively elevated levels of insulin. hyperinsulinemia has been reported to promote the synthesis of growth factors including insulin-like growth factor-1 (igf-1) and igf-2 and transforming growth factor-β1 (tgf-β1), which hasten extracellular matrix deposition aiding in glomerular hypertrophy and fibrosis (25, 26). moreover, hyperinsulinemia increases renal tubular reabsorption of uric acid via glut9 transporter (27). also, hyperinsulinemia stimulates hepatic lipoprotein synthesis resulting in hyperlipidemia with the subsequent increase in the need for nadph that is met by the de novo purine nucleotide synthesis, speeding uric acid production (28). hyperuricemia contributes to renal inflammation (29, 30), vascular endothelial dysfunction (31, 32), fibrosis (33), glomerulosclerosis (34, 35) and proteinuria (36, 37). binding of insulin to its receptor on the podocytes is essential to regulate morphological adaptation of podocytes in response to changes in capillary pressure and gfr after meal (38). accumulation of non-esterified fatty acids (nefa) in podocytes in obese individuals impairs insulin signaling and induces apoptosis. the remaining podocytes become hypertrophic to compensate for the destroyed ones (39, 40). renal gluconeogenesis is activated in the context of insulin resistance. in response to the renal hemodynamic and metabolic changes in obesity, the proximal tubules become hypertrophic (41); an effect mediated by the activation of mammalian target of rapamycin complex 1 (mtorc1) in the proximal tubules cells. insulin activation mtorc1, promote lipid synthesis, angiogenesis, protein synthesis, cellular growth (42). chen et al. has reported that the homeostatic model assessment of insulin resistance (homa-ir) index, the most commonly used measure of insulin resistance, to be significantly correlated with the prevalence of org and with proteinuria; and suggested the screening for this index as predictive marker for renal damage in obese individuals (43). iraqi j pharm sci, vol.30(1) 2021 pathogenesis of obesity-related glomerulopathy 24 ectopic lipid deposition ectopic lipid deposition within mesangial cells results in foam cell formation and glomerular hypertrophy. mesangial cells are exposed lipoproteins as no basement membrane separates them from the glomerular endothelium (40). endothelial dysfunction results in lipoprotein outflow to mesangial cells; beside, the phagocytic functions of mesangial cells that make them engulf various lipid particles. lipoproteins enter mesangial cells via binding to the low-density lipoprotein (ldl) receptors, while, long-chain fatty acids enter via scavenger receptors (44). lipoprotein lipase hydrolyzes lipoproteins releasing triacylglycerols (45). ldl receptor feedback, which important in preventing cellular cholesterol accumulation, is disrupted by the micro-inflammatory status in obesity, causing unrestricted lipid buildup (46). the deposited lipids in mesangial cells result in the formation of foam cells and loss of contractile function, leading to reduced structural integrity of glomerular arterioles and glomerular hypertrophy (40). lipid deposition in podocytes and proximal tubular cells due to the impairment of insulin signaling is discussed above. adipose tissue cytokines disorder the function of adipose tissue is not limited to lipid storage and energy supply; it is considered as an endocrine organ that secretes many cytokines (adipokines) involved in regulation many biological functions and implicated in the pathogenesis of several organ specific diseases, including renal diseases (47). the adipo-renal axis is important for normal renal functions along with the response of the kidney to injury. obesity is associated with dysregulated synthesis and release of a number of adipokines (48). many of these adipokines have been reported to disrupt renal cells’ functions in vitro, which might mediate org (49, 50). adipokines whether those produced by the peripheral adipose tissue or those produced by the renal adipose tissue contribute to org in obese patients. leptin and adiponectin have both noninflammatory and inflammatory roles in this regard. the roles of pro-inflammatory adipokines will be discussed separately with the role of microinflammation in org. leptin is mainly produced by white adipose tissue, and acts to regulate energy-related metabolism. obese individuals are in a state of hyperleptinemia and leptin resistance that are shown to be independently associated with insulin resistance (51). both indirect and direct actions of leptin contributes to the development of org in obese individuals. binding of leptin to its functional brain receptor (ob-rb) activates the sympathetic nervous system, increasing blood pressure, renal blood flow and gfr (52, 53). while, binding of leptin with glomerular leptin receptor (ob-ra), increases expression of glomerular transforming growth factor-β1 (tgf-β1), leading to an increase in the synthesis of type iv collagen in extracellular matrix, promoting fibrosis and glomerulosclerosis (54). furthermore, leptin has significant proinflammatory actions; it regulates cells involved in both innate and adaptive immune responses, including monocytes/macrophages and t-cells (55). leptin enhances macrophage infiltration to the kidneys (56), and central t-cell production along with peripheral shift toward the pro-inflammatory t helper-1 (th1) adaptive immune responses (57). meanwhile, leptin enhances t-cell survival and promotes production of pro-inflammatory cytokines (58). in addition, leptin structurally and functionally resembles pro-inflammatory cytokines, such as interleukin-6 (il-6) (55). finally, it binds to creactive protein (crp) and may modulate its activity (59). crp is an inflammatory mediator involved in the initiation and progression of atherosclerosis and renal disease (60). adiponectin is an adipokines with protective properties; it has anti-inflammatory, antiatherogenic and insulin sensitization effects (61). adiponectin levels has been reported to be lower in overweight and obese individuals compared to normal weight individuals, and levels are negatively correlated with increased visceral fat (62, 63). adiponectin helps to maintain structural integrity of podocytes (64). kim et al. showed that binding of adiponectin to its intrarenal receptor (adipor1), improves oxidative stress status and inhibits podocytes apoptosis by ameliorating the intracellular pathways associated with lipid deposition and endothelial dysfunction (64). moreover, adiponectin is suggested to have significant anti-inflammatory effects by the suppression of tumor necrosis factor-α (tnf-α) production with the subsequent prevention of nuclear factor-κb (nf-κb) activation (65). adiponectin also inhibits the expression of vascular cell adhesion molecule 1 (vcam-1) and intercellular adhesion molecule 1 (icam-1) (66), hence decreasing monocyte adhesion to endothelial cells as well as macrophage-induced cytokine production (67). moreover, adiponectin is inversely correlated with crp expression in human adipose tissue (68). the role of micro-inflammation obesity has been considered a state of chronic low-grade inflammation (69). adiposity induces an inflammatory microenvironment in the kidneys. adipose tissue of obese individuals is highly infiltrated by macrophages (70), and it has been estimated that macrophages are roughly accounting for 40% of the total cells within adipose tissue of obese individuals (71). adipose tissue macrophages contribute to key regulatory physiological functions such as tissue remodeling iraqi j pharm sci, vol.30(1) 2021 pathogenesis of obesity-related glomerulopathy 25 (72), and insulin sensitivity (73). macrophages secrete both anti‐ and pro‐inflammatory cytokines (74). anti‐ inflammatory cytokines secreted by adipose tissue macrophages such as il-4 and il-10 conserve insulin sensitivity by neutralizing inflammatory responses (73). macrophages also secrete proinflammatory cytokines such as tnf-α, il-1β, il-6, il-8, il-12 crp, monocyte chemoattractant protein-1 (mcp‐1), and plasminogen activator inhibitor-1 (pai-1) in response to inflammatory stimuli. with progressive obesity and adipocytes hypertrophy, adipose tissue macrophages secrete chemoattractants, such as mcp‐1 that recruits more macrophages to renal adipose tissue. proinflammatory cytokines promote a microenvironment of chronic low-grade inflammation and insulin resistance in the kidneys (75). macrophages and adipocytes communicate with each other via different mediators. for example, fatty acids released from adipocytes stimulate macrophages for the secretion of tnf-α which increases il-6 secretion by adipocytes. both tnf-α and il-6 are pro-inflammatory cytokines that amplify inflammation in the kidneys as well as in the adipose tissues (76). moreover, tnf-α plays an important role in the development of renal fibrosis (77). it was found that the expressions of tnf-α and its receptor is enhanced in renal biopsy samples collected from org patients, referring to a potential role of tnf-α in the pathogenesis of org (78). systemically, il-6 is mainly 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commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 chronic kidney disease doi: https://doi.org/10.31351/vol32iss1pp59-66 59 clinical significance of osteoprotegerin, vitamin d, obestatin and some biochemical variables in kidney failure patients hazhar m. balaky*,1, akam jasim mustafa** and parween abdulsamad ismail*** *mergasor technical institute, erbil polytechnic university, erbil, kurdistan region, iraq. **department of chemistry, faculty of science, soran university, kurdistan region. iraq. ***department of chemistry, college of education, university of salahaddin, iraq abstract chronic kidney disease (ckd) is described as an abnormality of renal function existing for a long period of time. because the initial stages of ckd can be asymptomatic, its early diagnosis is difficult. the earlier stage of ckd can lead to several complications, such as anemia and bone mineral metabolism disorders. the study was conducted to assess the effect of the chronic renal disease stage on osteoprotegerin, 1,25 dihydroxyvitamin d, obestatin levels and some biochemical parameters in patients who have not undertaken dialysis therapy. in this case-control study, fifty-five patients with kidney failure and forty healthy people were examined. circulating concentrations of osteoprotegerin, 1,25 dihydroxyvitamin d and obestatin were estimated by elisa technique, serum urea, uric acid, total protein and total calcium were estimated using spectrophotometry technique. the serum concentration of osteoprotegerin, obestatin, and renal function markers (urea, creatinine and uric acid) in patient groups were higher (328.3±41.68 pg/ml; 15.52±4.28 pg/ml; 183.2±10.35 mg/dl; 8.88±0.54 mg/dl; 6.57±0.22 mg/dl) respectively in comparison with the control group (172.6±55.48 pg/ml; 11.64±3.26 pg/ml; 33.45±1.08 mg/dl; 0.95±0.03 mg/dl; 4.35±0.22 mg/dl) respectively. vitamin d, total calcium and total protein level in the patient group were lower (24.49±2.53 ng/ml; 8.06±0.18 mg/dl; 6.49±0.12 g/dl), respectively, as compared to controls (57.11±12.39 ng/ml; 10.15±0.23 mg/dl; 6.82±0.10 g/dl) respectively. the current study reveals that circulating levels of osteoprotegerin and obestatin are remarkably linked with renal failure; the current data identify a high prevalence deficiency and insufficiency of 1,25 dihydroxyvitamin d in patients with moderate and severe chronic nephropathy. keywords: osteoprotegerin, 1,25 dihydroxyvitamin d, obestatin, kidney failure. introduction chronic kidney disease (ckd) is one of the leading causes of death and disability. ckd is associated with a wide range of life-threatening diseases (1). it is a disease condition associated with premature mortality, increased healthcare expenditures, and decreased quality of life. the terminal stage of the disease, end-stage renal disease (esrd), necessitates dialysis or kidney transplantation (2). osteoprotegerin (opg) is a glycoprotein that plays an important regulatory role in the skeletal, vascular, and immune systems. it has been shown that opg predicts chronic kidney disease (ckd). pg is increased in patients with nondiabetic and diabetic chronic kidney disease (ckd), where it predicts deterioration of kidney function, vascular events, and cardiovascular and all-cause mortality. consistent with it, it has been recently reported that elevated opg is associated with increased 5and 10-year risk of rapid renal decline, renal disease hospitalization, and/or deaths in elderly women (3). vitamin d deficiency is very frequent in ckd, affecting more than 80% of patients in predialysis. vitamin d insufficiency arises at an early stage of the disease and tends to worsen with the progressive loss of renal function (4). moreover, decline in glomerular filtration in ckd patients causes vitamin d deficiency, derangements in calcium and phosphate homeostasis, and secondary hyperparathyroidism resulting in bone destruction and vascular calcification (5). however, the role of native vitamin d supplementation (ergocalciferol, cholecalciferol or calcifediol) remains unclear in chronic kidney disease (ckd), particularly in the pre-dialytic phase (4). obestatin, a peptide hormone, might be participated in the loss of appetite in endstage renal disease patients. the obestatin hormone is encoded by the same gene as ghrelin and has the opposite effects compared with ghrelin; it blocks nourishment and causes weight gain, whereas ghrelin promotes nourishment. the balance between concentration of obestatin and ghrelin in final-stage nephropathic patients influences balance of energy and appetite, which involves malnourishment in final-stage nephropathic patients. thus, obestatin is a nourishment parameter identifying the fattening of the body and insulin resistance (6). urea is the primary metabolite derived from dietary protein and tissue protein turnover. it is freely filtered at the glomerulus but not secreted, and it is reabsorbed by the renal tubules. in addition, as urine flow rates decrease, more urea is reabsorbed. blood urea nitrogen (bun) measures the nitrogen component of serum urea levels are inversely 1corresponding author e-mail: hazharbalaky86@yahoo.com received: 27/12 /2021 accepted:6 /4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp59-66 iraqi j pharm sci, vol.32( 1 ) 2023 chronic kidney disease 60 correlated with the decline of kidney function and are also affected by extrarenal factors such as protein intake, gastrointestinal bleeding, catabolic states, malnutrition, heart failure, dehydration, use of glucocorticoids, and hepatic urea synthesis (7). the role of uric acid in chronic kidney disease (ckd) has been a controversial topic for decades. studies supporting the role of uric acid as an important mediator of ckd are extensive. experimentally raising uric acid could cause low-grade kidney damage and accelerate established ckd (8). creatinine is the most commonly used marker to measure gfr. gfr is estimated from measurements of creatinine concentrations in blood, using various prediction equations. in adults, the chronic kidney disease epidemiology collaboration (ckd-epi) and the modification of diet in renal disease (mdrd) study equations are the most widely used (9). several pieces of research have demonstrated that nourishment factors are essential in averting the development of persistent nephropathy. according to prior research, a very low protein food source as a section of nourishment treatment has serviceable impacts in decelerating the development of chronic kidney disease (10). nevertheless, restricted research has investigated the effect of continuous protein consumption and its crucial sources on the beginning of chronic kidney disease, comprising the protein of both plant and animal sources. in an investigation, it was recorded that a higher intake of animal protein (red meat) contributes to elevated chronic kidney disease health risk, and replacing animal protein with plant protein diminished the health risk of chronic kidney disease (11). methods study population and design ninety-five participants with age range (29-67) years, have been subjected to the present study, and these selected participants were divided into two groups. group (1) includes subjects with chronic renal failure (n=55). group (2) consisted of apparently healthy individuals (n=40). patients were clinically diagnosed with kidney failure. the samples were collected from ashty hospital in soran city. patients were assessed by full medical history to exclude any existing systemic disease. the two groups were matched in age and gender. collection of blood samples five to six ml of venous blood were taken from each participant, and collected in gold-top serum separator tubes (sst), to get the serum, the samples were centrifuged at (3000 rpm) for fifteen minutes. the obtained serum samples were transferred immediately to pre-labelled and coded eppendorf tubes. these samples were frozen at – 20°c for subsequent analysis. biochemical assays circulating concentrations of osteoprotegerin (opg), 1,25dihydroxycholecalciferol and obestatin in serum samples were estimated by sandwich enzyme-linked immunosorbent assay (elisa) technique using biovision (usa) research-purpose kits. the concentration of urea, creatinine, uric acid, total protein and total calcium were estimated using the enzymatic colorimetric method using biolabo kits (france). statistical analysis statistical data analysis was performed using spss version 21 and graphpad prism version 8 computer programs. comparing the study parameter means between patients and control groups was performed by using unpaired t-test (man-whitney u) test. bar graphs and statistical test results were expressed as mean±se. results and discussion as shown in table 1, osteoprotegerin, urea, creatinine, and uric acid levels were significantly higher in renal failure patients (328.3±41.68pg/ml; 183.2±10.35 mg/dl; 8.88±0.54 mg/dl; 6.57±0.22 mg/dl) respectively. similarly, the levels of obestatin were higher in kidney failure patients (15.52±4.28 pg/ml), but the variation was not statistically remarkable. the levels of vitamin d, total proteins and total calcium were significantly lower in kidney failure patients (24.49±2.53 ng/ml; 6.49±0.12; g/dl 8.06±0.18 mg/dl) respectively than that of the control group (57.11±12.39 ng/ml; 6.82±0.10 g/dl; 10.15±0.23 mg/dl) respectively. table 1. comparison between biochemical variable levels in kf and control group parameters controls patients p-value osteoprotegerin (pg/ml) 172.6±55.48 328.3±41.68* 0.0001 obestatin (pg/ml) 11.64±3.26 15.52±4.28 0.241 vitamin d (ng/ml) 57.11±12.39 24.49±2.53* 0.030 urea (mg/dl) 33.45±1.08 183.2±10.35** <0.0001 creatinine (mg/dl) 0.95±0.03 8.88±0.54** <0.0001 uric acid (mg/dl) 4.35±0.22 6.57±0.22** <0.0001 total proteins (g/dl) 6.82±0.10 6.49±0.12* 0.0503 total calcium (mg/dl) 10.15±0.23 8.06±0.18** <0.0001 value of * indicates significant, ** indicates highly significant iraqi j pharm sci, vol.32( 1 ) 2023 chronic kidney disease 61 serum level of opg table (1) and figure (1) showed significant elevation (p=0.0001) in circulating concentration of opg in patients (328.3±41.68 pg/ml) as compared to controls (172.6±55.48 pg/ml). figure 1. serum level of opg possible explanations for increased serum concentration of osteoprotegerin (opg) with ckd may include decreased clearance of opg or increased inflammatory activity. inflammatory mediators are known to promote the production of opg. as renal failure is associated with increased inflammatory activity, likely, inflammatory responses may partly explain increased serum opg in ckd. several mechanisms underlying opg increase in ckd might include low-grade inflammation, fibroblast growth factor 23 (fgf-23) elevation, and kidney function. inflammation might elevate opg levels as several proinflammatory cytokines, such as tnf-α, regulate opg production in vascular smooth muscle cells (12). secondly, increased concentrations of fibroblast growth factor 23 found in persistent nephropathic patients might also promote osteoprotegerin expression (13). thirdly, given that renal secretion is assumed to control the clearance of osteoprotegerin, the reservation of osteoprotegerin attributed to kidney destruction might supply another section to explain the mechanisms whereby osteoprotegerin levels are elevated in chronic kidney disease (14). as for inflammation, it has been revealed that osteoprotegerin promoted the expression of endothelial cells of cell surface proteins mediating the interaction between cells. it elevated white blood cell adhesion to the single layer of squamous endothelial cells, as for scarring of the tissue. it has been revealed that osteoprotegerin could begin transforming growth factor beta 1 -dependent alters in stromal cells of the vascular wall, promoting multiplication, fibrosis and inflammation. furthermore, osteoprotegerin expression is elevated in response to transforming growth factor beta 1, which could contribute to a vicious cycle that causes the self-elicitation of both osteoprotegerin and transforming growth factor beta 1 (3). osteoprotegerin transportation remarkably elevated the gene expression of interleukin six and transforming growth factor beta 1, as well as the quantity of protein cysteine nitrosylation, which all participated in the progression of renal damage (15). in this process, not only transforming growth factor beta 1 but also various cytokines and growth factors stimulate the abnormal aggregation of collagen protein, fibronectin glycoprotein, and other constituents accountable for kidney scarring (16). consequently, our finding supposes that osteoprotegerin has the possibility to directly instigate renal damage (17). serum levels of obestatin table (1) and figure (2) showed a nonsignificant increase (0.241) in the circulating level of obestatin in patients (15.52±4.28 ng/ml) as compared to controls (11.64±3.26 ng/ml). figure 2. serum level of obestatin obestatin, originally identified as an anorectic peptide, plays a vital role in several pathological activities besides physiological activities, such as substance and generation of energy from the metabolic pathway, motility of gastrointestinal tract, pancreatic secretion, cell multiplication, memory and sleep, etc. (18). obestatin-arginine vasopressin (avp) contributes in the regulation of metabolic pathway of body water. the secretion of arginine vasopressin (avp) in basic status is elevated after the management of obestatin antiserum; the secretion of arginine vasopressin is additionally elevated after water impoverishment. earlier investigations have examined that the release of obestatin is abnormally elevated in patients with persistent heart failure and nephropathy. compared to other groups, the current study found that patients with complicated renal failure have remarkably increased concentrations of obestatin (19,20). circulating level of 1,25-dihydroxycholecalciferol the results in table (1) and figure (3) showed that there was a remarkable decline p=0.030) in circulating concentration of vitamin d in patients (24.49±2.53 ng/ml) as compared to controls (57.11 ± 12.39 ng/ml). iraqi j pharm sci, vol.32( 1 ) 2023 chronic kidney disease 62 figure 3. serum level of vitamin d. our results accompany previous findings proposing that 1,25 dihydroxyvitamin d insufficiency is vigorously linked with significant advancement in chronic kidney diseases among adults. among 14,679 united states adult participants, the mean circulating concentration of 1,25dihydroxycholecalciferol was lower in patients with stage four-five persistent nephropathy in comparison with normal renal function (21). likewise, another research estimated the circulating concentration of 1,25-dihydroxy cholecalciferol in patients with persistent nephropathy. the mean serum concentration of 1,25dihydroxycholecalciferol was lower in patients with stages three-four (22). furthermore, participants with a serum concentration of 1,25dihydroxycholecalciferol less than 15 ng/ml had a 2fold greater occurrence of final stage nephropathy than those with more than 15 ng/ml during a longlasting, follow-up (23). additionally, few researches revealed that low 1,25 dihydroxyvitamin d concentrations were independently linked with albumin-urea in chronic kidney diseases and t1dm (24,25). the present result illustrates that 1,25 dihydroxyvitamin d mild decrease / greater decrease is a persistent state in patients with chronic kidney diseases, mainly those with an estimated glomerular filtration rate of less than 15 ml/min/1.73 m2. various mechanisms might describe the deficiency of 1,25dihydroxycholecalciferol in persistent nephropathic patients. first, most patients with chronic kidney diseases have limited protein sources besides caloric consumption, so 1,25 dihydroxyvitamin d is fairly few. second, many chronic kidney disease patients have restricted environmental activities with declined sunlight exposure (26). finally, a considerable lack of urinary 1,25 dihydroxyvitamin d metabolites takes place in patients with urinary protein-creatinine ratio ≥1.0 g protein/g creatinine. investigators reported an interrelationship between parathyroid hormone, 1,25dihydroxycholecalciferol and persistent nephropathy (27). in chronic kidney diseases, the kidney 1α-hydroxylase expression is blocked to remunerate for phosphate reservation, which results in an elevated expression of 24‐hydroxylase for the breakdown of 1,25-dihydroxycholecalciferol. the concentration of 1,25 dihydroxyvitamin d in dialysis patients is lower than in healthy individuals (28). renal malfunctioning, usually found among patients with renal diseases, is involved in the generation of 1,25-dihydroxycholecalciferol insufficiency that contributes to lowering serum calcium levels and secondary hyperparathyroidism, which results in secondary osteoporosis (29). it has been recorded that 1,25 dihydroxyvitamin d insufficiency in renal disorders is elevated in severe stages of chronic kidney disease (28). serum level of kidney function markers table (1) and figure (4) revealed a significant (p<0.0001) increase in serum level of urea, creatinine and uric acid in patients (183.2±10.35 mg/dl), (8.88±0.54 mg/dl) and (6.57±0.22 mg/dl) respectively as compared to controls (33.45±1.08 mg/dl), (0.95±0.03 mg/dl) and (04.35±0.22 mg/dl), respectively. figure 4. serum levels of (a) urea, (b) creatinine (c) uric acid. iraqi j pharm sci, vol.32( 1 ) 2023 chronic kidney disease 63 the current research revealed that higher blood urea nitrogen concentrations were linked with negative kidney consequences unconstrained of estimated glomerular filtration rate in patients with moderate to severe kidney damage. previous cohort study recorded that both blood urea nitrogen and serum osmolality were unconstrained health risks for persistent nephropathy in patients with conserved renal action (30). urea formation directly corresponds to dietary protein consumption, and curtailment of daily protein consumption contributes to decrease urea formation. various research concerning the potency of daily protein curtailment on renal disease development have been performed; however, findings have been incompatible. the wide-ranging irregular trial, the improvement of diet in kidney disorder study, revealed that a diet with very lowprotein provided with oxoacids contributes to the reduction of the degree of development of chronic kidney disease compared with a diet that contains a limiting amount of high protein foods, but this was not statistically remarkable (7). serum creatinine descriptive of glomerular filtration rate has acquired its restriction since a decline in glomerular filtration only contributes to an imperceptible elevate in serum creatinine since its tubular release elevated; so, an imperceptible elevate in serum creatinine does not definitely means that glomerular filtration rate is normal, but a serum creatinine elevate more than 2 mg/dl result in saturation of excretion begins to consider glomerular filtration rate (31). the current study supports that hyperuricemia is associated with a greater decline in renal function and a higher risk of progressing to kidney failure. the influences of hyperuricemia on renal function decline and the risk of kidney failure are more significant in patients without proteinuria than those with proteinuria (32). uric acid is the final oxidation product of purine metabolism and is renally excreted. therefore, elevated serum uric acid levels are seen in patients with reduced glomerular filtration rate (gfr). however, it has been proposed that uric acid itself plays a causal role in the pathophysiology of chronic kidney disease and possibly in acute kidney injury. a review of the literature demonstrates uric acidrelated cellular changes that contribute to renal disease. studies performed on rats have demonstrated that there are fundamental changes in the renal vasculature in the presence of hyperuricemia. it was found that uric acid decreased the expression of e-cadherin in epithelial cells resulting in a loss of cell-to-cell contact in the renal tubular cells of rats. without cell-to-cell contact, epithelial cells are unable to coordinate efforts to secrete substances needed to increase renal blood flow, such as nitric oxide (33). in addition, a recent study utilizing immortalized proximal tubular epithelial cells from normal adult human male kidneys has demonstrated that increased uric acid levels cause napdh-dependent oxidative changes that promote apoptosis (34). this finding shed light on the connection between hyperuricemia and tubulointerstitial renal damage. serum level of total protein table (1) & figure (5) revealed a nonsignificant decrease (p=0.0503) in serum level of total protein in patients (6.49±0.12 mg/dl) as compared to controls (6.82±0.10 mg/dl). figure 5. serum level of total protein it has been reported that serum total protein concentrations are strongly associated with estimated glomerular filtration rate decline, rapid egfr decline and incident ckd. these findings were independent of albuminuria, self-reported health, biomarkers of inflammation, as well as demographic and clinical risk factors for kidney disease progression (35). serum total protein concentration is a well-known predictor of mortality in patients with ckd. only a few studies have examined serum albumin as a risk factor for the development of esrd (36,37). few studies have examined the associations of serum albumin with kidney function decline. however, in one study of eight inflammatory markers, including serum albumin, serum albumin was the only laboratory measure associated with kidney function decline. few studies have examined the associations of serum albumin with kidney function decline. however, in one study of eight inflammatory markers, including serum albumin, serum albumin was the only laboratory measure associated with kidney function decline (38). the underlying mechanisms for these associations are unclear. to our knowledge, there is no direct physiological basis for low serum albumin to cause the development of ckd. serum albumin levels may be reduced for several reasons: liver damage and disease may decrease production; they may decrease as a negative acute phase reactant or in response to inflammation; and substantial and sustained albuminuria can lead to lower serum concentrations. uncommonly low serum albumin can also reflect malnutrition, but the only malnourished state that consistently produces lower iraqi j pharm sci, vol.32( 1 ) 2023 chronic kidney disease 64 serum albumin is kwashiorkor, a rare disease in the developing world (39). previous studies hypothesized the strong relationship between lower serum protein and kidney function decline in their research must be multi-factorial, representing general ill health. lower protein concentrations may reflect much broader abnormalities in inflammation, thrombosis and microvascular function processes than can be captured by the markers available in this cohort. lower serum protein and kidney function decline may also be related to multiple factors that are currently poorly understood. these findings are consistent with the results from our previous study of hiv-infected women and other studies of serum protein in esrd (35). in a 2011 retrospective cohort analysis of 1800 men and women from the uk, wagner et al. found that serum albumin levels were an essential component of a multivariable risk model for mortality in patients with esrd (36). with some types of kidney disease, protein may be lost in the urine (proteinuria). with peritoneal dialysis, some protein crosses the peritoneal membrane and exits the body in the effluent dialysate (the solution drained from the peritoneal cavity). this loss increases in a person with peritonitis, an infection of the peritoneum. serum level of total calcium table (1) and figure (6) revealed a remarkable reduction (p<0.0001) in circulating concentration of total calcium in patients (8.06±0.18 mg/dl) as compared to controls (10.15±0.23 mg/dl). figure 6. serum level of total calcium throughout nephropathy, the renal may no more filter out additional phosphorus and eliminate it from the body. with time, phosphorus may elevate in the blood. ca and po-34 in the body interact in opposite directions: as blood ca levels rise, po-34 levels fall. with renal disorder development, elevated phosphorus concentrations may cause low circulating calcium concentration by accumulating onto the osseous matter and other tissues. disturbances in the metabolic pathway of calcium metabolism disorders are characteristic features of chronic kidney disease (40). in various investigations, the circulating concentration of phosphorus was confirmed as a principal health risk for heart failure and all-cause death in kidney failure patients. in an american group of 35114 patients undertaken hemodialysis with an average follow-up of one year, among the patients with excessive remaining kidney urea authorization, there was an increased mortality health risk in patients with an elevated circulating concentration of phosphorus and decreased serum calcium (41). conclusion the study showed disturbances in the circulating concentration of osteoprotegerin, obestatin, and 1,25 dihydroxyvitamin d in patients with kidney failure. the serum osteoprotegerin level and obestatin were considered contributors to kidney failure. these findings indicated that opg, a bone-modulating protein, might have a role in developing kf patients. decreased concentrations levels of 1,25 dihydroxyvitamin d are frequently observed in patients suffering from chronic kidney failure; these serve as a principal element in the progression of kf. more investigations are needed to explain the importance of these findings in the etiopathogenesis of kidney failure. acknowledgement we would like to express our gratitude to the technical and support staff of the dialysis center ashty hospital in soran city for providing specimens. conflict of interest the authors declare no conflict of interest during this study. references 1. hasan m, sutradhar i, gupta rd, sarker m. prevalence of chronic kidney disease in south asia: a systematic review. bmc nephrology. 2018;19(1):1-2. 2. chukwuonye ii, ohagwu ka, adelowo oo, chuku a, obi ec, onwuchekwa u, anyabolu en, oviasu e. prevalence and predictors of chronic kidney disease in a semiurban community in lagos. international journal of nephrology. 2019 ; 2. 3. bernardi s, toffoli b, bossi f, 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american society of nephrology, 21(2), pp.223-230 40. yu g, cheng j, jiang y, li h, li x, chen j. serum phosphorus and calcium levels and kidney disease progression in immunoglobulin a nephropathy. clinical kidney journal. 2021 jan 25. 41. wang m, obi y, streja e, rhee cm, lau wl, chen j, hao c, hamano t, kovesdy cp, kalantar-zadeh k. association of parameters of mineral bone disorder with mortality in patients on hemodialysis according to level of residual kidney function. clinical journal of the american society of nephrology. 2017;12(7):1118-27 this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ the survival of different fungal spores during tabletting iraqi j pharm sci, vol.19(1) 2010 survival of fungal spores during tabletting 1 the survival of different fungal spores during tabletting raghad a.al-shikli *,1 , alaa a.abdul-rasool ** and mustafa m. al-hiti * * department of clinical laboratory science,college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the survival of dried spores of a.flavus, penicillia spp., and cladosporia spp.inoculated into multivitamins and folic acid tablets were examined at different compression pressures.survival of fungal spores decreased with increasing compression pressure. the level of survival at particular pressures was shown to depend on the size of the contaminating fungal spores.the lethal effect of tabletting was attributed to shearing forces upon the contaminating spores generated by interparticulate movement. this hypothesis was supported by the dependence of survival upon spore size. key words: fungal spores, microbial contamination. الخالصة لقد تن تصٌيع ًوعيي هني ألقرنصألا أللدئألةينل أللصنيد ًيل ئانض انفهل أللمولنع ئهلووعنل أللميوفهيٌنفا ئتنن اننفع منمنل ألًنوأل هني ألسبفرجلس كنائسبوريل ئبوصكيزيي -أللمطصيفا ئاض بٌيسيليل ٕ ٔٓ, ٤ ئتن كبسهف بطصيقل أللكبس أللوبفشنص تتنض ون و غصألم/سبور ٓٔ ي ى زينفا أللغنن ؤ تنااى أللنا ًقصننفى انض عنندا أللسنبورألا ئألى هتندع أللبقننف تتنض ونن ؤ هتنيي يتووند علننا النن أللبننو هخولمنل ئرند تبنني . انهٍ أللمصونيِل َكفًنْض interparticulateًُِسَب لنا رَنّ أللقنوألِا علنا بوي نفِا أللوَلوينَ ئلّنَد بتصكنِل tablettingأللولوث.أللوأميص أللقفتل ع علا الِن أللبوي ِل. هدعوهل هي ربل عووفِا أللبقف ِ introduction microbiologically contaminated tablets may cause disease and under humid storage conditions lead to visible deterioration of the tablet.various workers have examined the effect of compaction process upon natural contaminants present in the granulate.in all cases, compression of granules to significant reduction in the levels of microbial contamination. (13) tabletting machines exert some antimicrobial effect . the shearing forces and localized heat involved in pressing tablets is sufficient to destroy many mould spores and vegetative organisms, although bacillus spores appear to survive. reduction (67-93)% in total viable counts of dry blended product has been reported to be produced by tabletting (1) increasing compression pressures and tabletting speed enhance the anti microbial effect (4-8.) .fassihi and parker (9) granulated lactose powder using a granulating fluid containing a. niger spores .they demonstrated a linear relationship between the log survival and the applied pressure (28-271 mn/m2). such linearity was not observed by yanagita et al. (10) for rhodotorula glutinins, e.coil and bacillus subtilis contaminated crystalline cellulose.plumpton (11) found that the levels of survival within four formulations were similar for a given organism, yet patterns emerged according to the mechanism of compaction of the material.survival following compaction in lactose and emdex , which compact by a process of fracture, was inversely related to pressure over the entire range tested , whilst for sta-rx and potassium chloride such relationship was only exhibited up to compaction pressure of (194 mn/m2). at low compaction pressure (0-39 mn/m2)there was a little inactivation of the microorganisms in any of the four formulations. also plumpton (11) found that levels of survival were inversely related to the size of the organisms for any given pressure, survival being greatest for b.megaterium spores (cell dameter approximately 1.5µm), intermediary for a.niger spores (4µdiameter) and least for s.cerevisiae (mean cell diameter approximately 8 µm). similar trends in survival according to cell size were suggested from the work of yanagiitu et al. (10). these workers examined the survival during compaction of vegetative cells of e.coli and r.glutinis and spores of b.subtillis within an avicel/skin milk formulation. b.subitillis spores were found to be highly resistant to tabletting. where as vegetative cellsof r.glutinis were much more sensitive to tabletting than those of e.coil. blair (7) found that extent of kill on tablet compression was great for the larger organism, e.cloacase, on studying separate batches of lactose monohydrate , maize starch and cellulose 1 corresponding author email : raghadrazook@yahoo.com received : 28 / 12 / 2008 accepted : 31 / 5 / 2009 iraqi j pharm sci, vol.19(1) 2010 survival of fungal spores during tabletting 2 microcrystalline contaminated with staphylococus aureus or enterbacter cloacae and tabletted at various compression force,indicating that size is important and cell rupture by shear,rather than heat,is the mechanism of kill.in this study,we examined the effect of tabletting on survival of dried spores of a.flavus,penicillia spp. and cladosporia spp. inoculated into the multi vitamines and folic acid tabletsduring compression with two levels (10 2 and 10 4 )spore/gram at three compression pressure and one conpression pressure (10 2 and10 4 ) spore/gram respectively. materials, instruments and methods chemicals acetonitrile ,acetone, ammonium hydroxide ,anhydrous sodium sultate , benzene, chloroform, glacial acetic acid ,hydrous disodium hydrogen phosphate (na2hpo4.12h2o), , methanol, potassium hydroxide, potassium chloride, sulfuric acid, sodium hydroxide, sodum 1-hexane sulfonate, sodium perchlorate, sodium chloride, and tween 80 (polysorbate 80) suppied by bdy england. monobasic potassium phosphate from fluka-swizerland. heexan supplied by merch-w. germany. microcrystalline cellulose (avicel ph 101, avicel ph 301), folic acid, maize starch, vitamin b1 (thiamin mononitrite), vitamin b2 (riboflavin),vitamin b6 (pyridoxine hcl), methionine, talc and magnesium stearate supplied by fmc coproation and kindly supplied from (sammara drug industires sdi. iraq). microorganisms aspergillus flavus , penicillia spp. and cladospria cladosporoids were obtained from college of agriculture ,university of baghdad. culatures were stored on sabouared ager slants following incubation at 25°c for 5 days. fresh cultures were prepared every 4 weeks . culture media sabouraud dextrose agar. rosebengal agar macconkey broth solution used for dilutions and preparation of spore suspension relative humidity of the prepared solutions . extraction solvent of aflatoxin. relative humidity containers . tablet formulations prepation of dried spore powder 0.1 ml aliquots of 7days cultures of a.flavus, cladosporia and penicillia were inoculated onto the surfaces of predied sabourad dextrose agar plates, incubated at 25°c for 5days. after this time spores were clearly visible on all the plates. three ml of sterile water containing 0.1%tween 80 (as a dispersin agent) were add to each plate . the spores were then dislodged by using glass spreader. the spore suspensions obtained were then stirred by using a vortex mixer for one minute. the spore suspension were then filtered through a sterile cotton wool in order to get rid of the hypha. the filtrates were then harvested by centrifugation at (10,000xy for10 min ). the supernatant liquids were then decanted and the residues were resuspended in 20 ml of sterile distilled water and washing were repeated three times. the number of spores of the resultant sopre suspensions was determined by viable count technique, spore suspensions were adjusted to obtain 2.16x10 6 spore/ml for a.flavus, 1.68x10 6 spore/ml for penicillia spp. and 1.98x10 6 spore/ml for c. cladsporoids. multivitamin tablets and folic acid tablets were prepared using the formulas listed in tables (1) and (2), respectively . the following excipient were used avicel ph 301 as a direct compression excipient, starch (5% w/w)as a disintegrant, magnesium stearate (0.5%)and stearic acid (2% w/w) as lubricants, were mixed with the active ingredients and compressed directly using a single punch tabletting machine with 7-mm flat –faced punches. table 1 : the formula of the prepared folic acid tablet ingredient amount / tab folic acid 1mg avicel ph 301 118.6 mg maize starch 6.5 mg mg.stearte 2.6 mg talc 1.3 mg total 130 mg table 2 : the formula of the prepared multivitamin tablet ingredient amount/tab thiamin mononitrite 1.5 mg riboflavin u.s.p 20 mg pyridoxine hcl u.s.p 2.0 mg methionine 2.0 mg avicel ph 301 105 mg maize starch 6.5 mg talc u.s.p 6 mg stearic acid ( powdered ) 3.0 mg mg.stearte ( powdered ) 2.0 mg total 130 mg iraqi j pharm sci, vol.19(1) 2010 survival of fungal spores during tabletting 3 preparation of contaminated tablets for preparation of 500 contaminated tablets a(0.3 ml, 30µl) of a.flavus .(0.4 ml 40µl) of penicillia spp and ( 0.3 ml, 30 µl) of c. cladosporiods were transferred to a sterile mortars and placed in the incubator until completely evaporation of water .dried spores were scraped off and were included in direct compression formulation by dry mixing to 102 get spore / gram and 104 spore / gram for each of a.flavus, penicillia spp. and c. cladosporids respectively. ingredients including dried microorganisms spores weighed and lightly mixed in a glass morter by the method of geometric dilution technique for 20 min. preliminary experiments had established that this method gives a uniform distribution of the microorganisms within the formulation , screen in the lubricant (magnesium stearte or stearic acid) and mixed for an additional 5 minutes. quantities each of 130mg were accurately weighed and poured into 7-mm diameter die. determination of viable number of spores in the prepared tablets viable number of spores in prepared tablets was determined immediately after their production at different compression forces and after storage up to 8 weeks. eight tablets (total wt.=1gm) were disintegrated in trypic soy broth (9ml) according to b.p 98 using a flask and suitable serial dilution in tryptic soy broth were prepared. oneml sample of each dilution was poured in a sterile petridish and then 15 ml of molten dextrose agar was added to the plate. the sample and molten sabouraud dextrose agar were mixed together in forward and backward movement and swirled movement. the plates were allowed to solidify on a leveled surface. the plates were incubated at 35°c for 2-5 days. survivals as colony forming units were estimated as the mean of triplicate determinations and expressed as a percentage relative to an uncompressed control sample of the contaminated formulations. physical properties of tablets the result of physical properties of tablets are shown in table (3). tablet weight and thickness, friability, hardness and disintegration time were measured. table 3 : physical ,chemical and microbiological ( control ) evaluation of folic acid and multivitamin tablet tablet evaluation multivitamin folic acid weight ( 7min ) 130 mg 130 mg c.pressure 148.3mn/m 3 wt.uniformity 0.9% 0.9% hardness (kp) 6.6  0.4 6.5  0.6 thickness ( mm ) 2.85 2.65 friability % 0.2 0.2 disintegration time 2.3 min 2 min assay b6 pyridoxine % 133.76% b1 thiamin % 148.2% folic acid % 905 microbiological quality one day after preparation less than 10 * cfu/gm after storage for 8 week at 35 c 75 % rh less than 10 * cfu/gm after storage for 8 week at 35 c 85 % rh less than 10 *cfu/gm after storage for 8 week at 35 c 95 % rh less than 10 * cfu/gm interrelation of compression pressure and survival of a. flavus, penicillia and cladosporia spores in multivitamin and folic acid tablets multivitamin formulation were contaminated with a.flavus spores or cladosporium spores or penicillia spores using 102 and 104 spore/gm. tablets were prepared at a variety of compression pressure (137.9, 144.8, and 148.3) mn/m2 and the number of survival was determined immediately upon ejection of tablets from the die. the number of survival was plotted as logarithmic function of compression pressure. results and discussion tablet evaluation folic acid and multivitamin tablets were prepared as previously mentioned (tables 1 and 2, respectively). the prepared folic acid and multivitamin tablets were evaluated physically, chemically and microbiologically. the results areshown in table 3. interrelation of compaction pressure and survival of a. flavus, penicillia and cladosporia spores in multivitamin and folic acid tablets the results of the effect of compression force upon percent survival are shown in figure(1) and table (4a)for a contamination level of 102 spore/gm using three compression pressures of (137.9, 144.8 and 148.3 mn/m2) to get tablets with optimum physical properties. the results (fig. 1) and (table 4a) in which a 10 2 was used indicate that a loss of iraqi j pharm sci, vol.19(1) 2010 survival of fungal spores during tabletting 4 50.0, 49.0, and 94.0 percent in viability of the spores was obtained at pressure of 148.3 mn/m 2 for a.flavus spores , penicillia spp. spores, and c.cladosporoids spores, respectively. the data also show that the survival at a contamination level of 102 spore/gm was inversely proportional to the compression pressure i.e, increasing pressure from (137.9 to 148.3)mn/m 2 caused a decrease in survival from 60 to 50.0 percent for a.flavus spores, from 50.0 to 41.0 percent for penicillia spp . spores and from 10.0 to 6.0 percent for c.cladospooids. that reduction is statistically significant (p=0.05). in general a higher reduction in viability was observed for cladosporia than of penicillia and a.flavus, size of the spores were in order of(3-6)µm in diameter, (4-6)µm in diameter, 3-7(-11)x2-4(5) µm in diameter )for a.flavus, penicillia, and c.cladosporoids, respectively. on the other hand, figure (2) and table (4b) show the effect of compression pressure upon percent of survival using 104 spore/gm contamination level and (148.3mn/m2) compression pressure to to obtain optimum physical properties of the tablet. the results indicate that the pressure applied caused a significant reduction in percent of survival (p=0.05)as 50.0 ,37.0 and 16.0 percent for a.flavus, penicillia, and c.cladosporoids ,as shown in figure (2). (4b).these results are well support the hypothesis that an inverse relationship is existed between spore size and compression pressure. chesworth et al. (1) attributed the lethal effects of compression to a combination of two events, generation of heat and shearing between particles causing mechanical damage to the cells.fassihi et al. (12) regarded localized heating within the formulation as being the critical parameter associated with inactivation of a.niger spores during compression of lactose granules .they proposed that during compression process, the pressures applied between the upper and lower punches were exerted only upon those particles directly in contact with the punch surface and that stresses on the remaining particles were through inter particulate contact . such a phenomenon would result in very high pressures being exerted over small areas of inter particulate contact .this might cause the existence of localized (hot spots) within the tablets and bring about death of the microorganisms by heat alone. such high temperatures have been shown to result in the melting of some materials during compression (13-18). if such a mechanism were applicable in our systems one would expect the levels of survival for different organisms to reflect their heat sensitivity rather than cell size. this was not the case, results suggested therefore that shearing forces(size) are contributory to the observed lethal effects of compression rather than heat per sec.if pressure alone was responsible for the observed inactivation of microbial spores then once again no differences would have been expected between organism types. conversely, larger sized microorganisms would be more likely to be subjected to shearing forces within a compact than smaller sized ones, supporting the hypothesis that inactivation is brought about by a direct physical trauma. figure 1 : effect of compression pressure upon the survival of ( 10 2 spore/gram ) different fungal spores in multivitamin tablet l.s.d = 3.7 figure 2 : survival of 10 4 asp flavus penicillia and cladosporia spores at constant pressure in multivitamin tablet iraqi j pharm sci, vol.19(1) 2010 survival of fungal spores during tabletting 5 table 4: effect of pressure on survival of different fungal spores expressed as a % in multivitamin and folic acid tablets using 10 2 spore /gm and using 10 2 spore/gm contamination levels 4a 4b conclusion the results emphasize on the existence of relationship between survival of fungal spores and the pressure used during tableting. survival of fungal spores decreased with increasing compression pressure and level of survival at particular pressure depends upon the size of the spore. the lethal effect of tabletting was attributed to shearing forces upon the contaminating spores. references 1. chesworth, k.a.c.; sincletr, a.; atertten , r.j; and hayer,w.p. micro bios lettus 1977,4:41. 2. morris,s.jproceedings of thye guild of hospital oharmacists, hondon, ,1981 10:63. 3. ayorinde, j.o. odeku, o.a. and tida, o.a. the survival of b.subtilis spore in dicacium phosphate, hactose and starch and ther binary mixtures during tableting. pharmaceutical technology, 2005, 29 (12):56-67 4. plumpton e.j.;gilbert,p.; and fell, j.t the survival of microgranisms during tableting int.j. pharm. 1986a ,30:241-246; 5. plumpton e.j.;jilbert,p.;and fell j.t.effect of special distribution on contaminant microorganisms with in tablet formulations on subsequent in in activation through compaction int.j.pharm.1986b,30:237-240; 6. fassihi,a.r;and parker,m.s.inimicable effects of compaction speed on microorganisms in powder systems with dis-similar compaction mechanisms j.pharm.sci,1987, 76:466-470. 7. blair t.c. ; buckton ,g. ;and bloomfield ,s.f; baird, r;leak r.e .;and leech , r (ods). microbial quality assurance in pharmaceuticals , cosmetics and toiletries .ellis.horwood,chichest :1991,pp:104-161. 8. odeku,o.a,awe,o.o.,popoola,b.odenigi, m.a.and itida,o.a.compreccssion and me mechanical properties of tablet formulations containing corn,sweet potato and cocoyam starches.phar.tech ,2005 29(4):82-90. 9. fassihi,a.r.;and parker,m.s.journal of applied acteriology,43meetings xvii;1977a 10. yanagtta,t.;miki,t.;sakat,t.;and itorkoshi ,chemical and pharmaceutical bulletin, 1987 ,26:185-190. 11. plumptone.j.studies upon the survival of various microorganisms in solid dosage forms,ph.d.thesis, university of manchester;1982 12. fassihi,a.r.;and parker, m.s.the influence of water activity and oxygen tension upon the survival of aspergillus and pencillia species and tablets .int .biodeterior . bull , 1977,13 :75-80. 13. bowden,f.p.and tabor , d . (friction and lubrication of solids) , vol. 2 , clarendon press,oxford;1964 . 14. jayasinghe ,s.s;pllpei,n.and harwood ,c.f.materals science and engineering ,5 ,287 ;1969,1970 15. odeka,o.aand itiolao a.evaluation of khaya gum as abinder in paracetol tablet formulation on pharm.pharmacol commum, 1998, 4:183 -188. 16. espinasse.v,mperria cornet.j, amartceat, gervais. p.high pressure in activation of dried microorganisms. drug developed and industrial pharmacy, 2002,81ssae: 329-337. 17. if eynwa f.obuekwe and florence eichie , actapolonae pharmaceutica-drug research , ;2006 ,6 (2) :121-125. 18. obuekwae,i.f.,ogbimi a . o. , pakistan j. sc. ind. res. 2002,45:341. pressure (mn/m 2 ) % mean no. of spore / gm for 10 2 spore/gm contamination level a.flavus penicillin spp. c. cladosporoids 137.9 60.0 50.0 10.0 144.8 56.0 45.7 7.7 148.3 50.0 41.0 6.0 pressure (mn/m 2 ) % mean no. of spore / gm for 10 2 spore/gm contamination level a.flavus penicillin spp. c. cladosporoids 148.3 50.0 37.0 16.0 iraqi j pharm sci, vol.28(1) 2019 guggulsterone and nutritional steatohepatitis doi: https://doi.org/10.31351/vol28iss1pp17-23 17 possible amelioration of the severity of nutritional steatohepatitis by guggulsterone in mice sara a. nafeer*,1 and munaf h. zalzala* department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract non-alcoholic fatty liver disease (nafld) has become one of the most common chronic liver diseases worldwide, which is characterized by steatosis, inflammation, and fibrosis. the aim of this designed study is to evaluate the ability of guggulsterone to prevent high fat diet induced steatohepatitis in mice. five groups of male mice were selected and treated as the following: group i, mice had free access to standard commercial diet and considered as control group, group ii, mice were fed a specially formulated high-fat diet for 12 weeks to induce non-alcoholic liver disease, while groups iii, iv and v the mice were administered high fat diet containing guggulsterone at 500, 1000 and 2000 ppm concentration respectively for 12 weeks. maintaining mice on fat rich diet only resulted in inducing the metabolic and histological changes related to nafld. while the treatment with guggulsterone significantly improves the evaluated markers. these results demonstrate guggulsterone may be useful in preventing the development of steatohepatitis. keywords: guggulsterone, steatohepatitis. الفئرانلستيرون في والتقليل من شدة التهاب الكبد الدهني التغذوي باستخدام الكيك ةامكاني *مناف هاشم زلزله و 1*،ساره أمين نفير فرع االدوية والسموم ،كلية الصيدلة،جامعة بغداد،بغداد،العراق.* الخالصة أصبح مرض الكبد الدهني واحد من أكثر أمراض الكبد المزمنة شيوعا حول العالم, الذي يتميز بالتشحم مصاحبا له االلتهاب والتليف في الكبد. الهدف من هذه الدراسة هو تقييم قدرة المركب الكيكولستيرون على الوقاية من التهاب الكبد الدهني المستحث باستخدام خمس مجاميع من الفئران الذكور تم اتخاذها ومعالجتها كالتالي: المجموعه االولى الفئران فيها لدهون على الفئران. حمية غذائية عالية ا غذيت على نظام غذائي اعتيادي واعتبرت كمجموعه السيطره, المجموعه الثانية تم تغذية الفئران على نظام غذائي عالي الدهون فقط ة الثالثة والرابعة والخامسة تم تغذيه الفئران فيها بنظام غذائي عالي الدهون اسبوع الحداث مرض الكبد الدهني, اما المجموع 12لمدة . ابقاء الفئران على حمية عالية الدهون ادى على التواليجزء من المليون 2000, 1000, 500ويحتوي على الكيكولستيرون وبتراكيز ة قد يكون المقاسة.بالنتيجالمعاييرلستيرون ادى الى تحسن الى احداث تغيرات ايضية ونسيجية متعلقه بالمرض .اما العالج بالكيكو .استخدام الكيكولستيرون مفيد في منع تطور التهاب الكبد الدهني . التهاب الكبد الدهني ، الكلمات المفتاحيه: كيكولستيرون introduction non-alcoholic fatty liver disease (nafld) has become the most common chronic liver disease worldwide; it is a multi-factorial disorder with the contribution of a variety of environmental and genetic factors(1). the progression of nafld was introduced originally as a disease of two consecutive hits which suggest the accumulation of fat in the hepatocytes sensitize the liver hepatocytes to a second metabolic insult associated with oxidative stress and proinflammatory cytokines (tnf-α, il-6, il-8,il-1β) release, that result in cellular damage by inflammation, steatonecrosis, subsequently fibrosis(2). the primary mechanism underlying hepatocyte dysfunction that leads to disease progression is lipotoxicity. lipotoxic injury occurs due to excessive accumulation of lipids especially free fatty acid (ffa) lead to oxidative stress through the generation of bioactive lipotoxic metabolites and reactive oxygen species (ros), proinflammatory cytokines expression upregulation, and βoxidation inhibition(3)(4). guggulsterone [4,17(20)-pregnadiene-3,16-dione] is the active ingredient obtained from oleogum resin, known as gum guggul of commiphora mukul plant; it has been used for a long time to treat many disease states such as hyperlipidemia, atherosclerosis, arthritis, obesity, and other inflammatory disorders in ayurvedic medicine. guggulsterone is an farnesoid x receptor (fxr) antagonist, guggulsterone inhibits fxr induction of ileum bile acid-binding protein ibabp, bile salt export pump bsep and the small heterodimer partner (shp) expression (5). guggulsterone also found to be able to inhibit nuclear factor-kappa b (nf-κb) activation by suppressing the activity of nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor alpha (ikba) kinase in hepatic stellate cells (hscs) thus guggulsterone act as an antifibrotic agent(6). 1corresponding author e-mail: kriyuzero@yahoo.com received: 21/8/2018 accepted: 24/9/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp17-23 iraqi j pharm sci, vol.28(1) 2018 guggulsterone and nutritional steatohepatitis 18 materials and methods experimental protocol thirty-five white male mice of six weeks’ old were provided by the animal house of the college of pharmacy/ university of baghdad. their weights ranged between 20-30 gm. the animals were housed in well-ventilated plastic cages, and were maintained under conditions of relatively controlled temperature, humidity, and 12 hrs. light / dark cycle. with a free access to a standard commercial diet that was purchased from the local market and tap water ad libitum. the animals randomly were divided into five groups of 7 animals in each group, as the following: group i: mice utilized in this group were fed standard commercial diet and considered to be the control group. group ii: mice utilized in this group were fed a specially formulated high-fat diet (hfd) for 12 weeks to induce nonalcoholic liver disease. group iii: mice utilized in this group were fed a high-fat diet that contains guggulsterone at a concentration of 500 ppm for 12 weeks. group iv: mice utilized in this group were fed a high-fat diet that contains guggulsterone at a concentration of 1000 ppm for 12 weeks. group v: mice utilized in this group were fed a high-fat diet that contains guggulsterone at a concentration of 2000 ppm for 12 weeks. the animal’s weight of all groups was measured routinely once weekly and at zero time. the animals of each group at the end of the experiment after overnight fasting were anesthetized using diethyl ether, and blood was collected from the heart by cardiac puncture, then the animals were sacrificed and the liver were obtained and weighed for calculating liver index (by dividing liver weight in mg by the last body weight in gram was recorded), then stored for analysis. samples preparation and analysis collected blood from the anesthetized animal was placed in a plain eppendorf tube and then centrifuged at 3000 (rpm) for 15 minutes. the supernatant was collected and used in the estimation of liver enzymes activity, total bilirubin (tb), tumor necrosis factoralpha (tnfα), and lipid profile, by using ready-made kits mice specific for each parameter. histological examination various sections of liver tissues obtained from mice utilized in this study were histologically examined according to the junqueira et al. 1995method (7). liver specimens were collected from the tissue and kept in 10% formalin solution ph 7.4 and then undergo a serial of dehydration in different grades of alcohol. cleaning of tissues was done by means of xylol, and then the tissues were embedded in paraffin wax. the blocks were cut by microtome into 5µm slices, and then washed in a water bath and left in an oven for dewaxing. then, the blocks sectioned and stained with hematoxylin and eosin (h&e) stain. light microscopic examinations were performed, and photomicrographs were taken, this was done in a blind fashion by a senior pathologist. statistical analysis all the comparisons between groups were conducted by using student’s t-test. a level of p < 0.05 was considered statistically significant. all data are represented as a (mean ± standard deviation) for the continuous variables. results effects of guggulsterone on liver index in figure-1, the liver index (measured by dividing the liver weight by the last record of the total body weight) of high-fat diet only mice diet showed significant increase in comparison with the control group (p<0.05). treatment with guggulsterone at 2000 ppm caused a significant decrease in liver index (p<0.05) in comparison with hfd groups, while the treatment with guggulsterone of (500 and 1000) ppm concentration caused nonsignificant difference when compared to hfd groups(p>0.05). figure( 1) effects of guggulsterone on the liver index of mice models with high-fat diet induced nafld, * is a significantly different compared to the control group (p<0.05). # is significantly different compared to the hfd group (p<0.05). effects of guggulsterone on the serum lipid profile in figure 2, mice fed with high-fat diet showed a significant increase in cholesterol level in comparison with the control (p<0.05). treatment with guggulsterone at (500, 1000 and 2000) ppm showed a significant decrease in cholesterol level compared to the hfd group (p<0.05). in figure 3, the treatment with guggulsterone showed nonsignificant (p>0.05) effect on tg level in comparison with hfd. https://doi.org/10.31351/vol28iss1pp17-23 iraqi j pharm sci, vol.28(1) 2018 guggulsterone and nutritional steatohepatitis 19 in figure 4, treatment with guggulsterone at (500 and 1000) ppm showed non-significant (p>0.05) difference in ldl level in comparison with hfd while treatment with guggulsterone at (2000) ppm showed significant decrease in ldl level in comparison with hfd(p<0.05). figure (2) effects of guggulsterone on cholesterol level of mice models with high-fat diet induced nafld. *is a significantly different compared to the control group (p<0.05). #is significantly different compared to the hfd group (p<0.05). figure( 3) effects of guggulsterone on tg level of mice models with high-fat diet induced nafld. figure 4. effects of guggulsterone on the ldl level of mice models with high-fat diet induced nafld. *is a significantly different compared to the control group (p<0.05). #is significantly different compared with the hfd group (p<0.05). effects of guggulsterone on the serum levels of liver enzymes activity in figure 5, the treatment at (1000 and 2000) ppm concentration of guggulsterone resulted in a significant decrease in ast level compared to the hfd group (p<0.05). there was a marked increase in ast level in hfd group compared to control group, as the control group showed a higher standard deviation value, the increase in ast level appeared to be statistically non-significant. in figure 6, only the treatment with concentration of (2000) ppm of guggulsterone caused a significant decrease (p<0.05) in alt level compared to hfd group. there was a marked increase in ast level in hfd group compared to control group, as the control group showed a higher standard deviation value, the increase in ast level appeared to be statistically non-significant. in figure 7, high-fat diet only group showed a significant increase in serum alkaline phosphatase (alp) level compared to control group (p<0.05), the administration of guggulsterone at (1000 and 2000) ppm caused a significant decrease in alp serum level compared to hfd group (p<0.05). figure (5) effects of guggulsterone on serum ast level of mice models with high-fat diet induced nafld. # is significantly different compared to hfd group (p<0.05). https://doi.org/10.31351/vol28iss1pp17-23 iraqi j pharm sci, vol.28(1) 2018 guggulsterone and nutritional steatohepatitis 20 figure (6) effects of guggulsterone on serum alt level of mice models with high-fat diet induced nafld. # is significantly different compared with hfd group (p<0.05). figure( 7) effects of guggulsterone on serum alp level of mice models with high-fat diet induced nafld. * is a significantly different compared to control group (p<0.05). # is significantly different compared to hfd group (p <0.05). effects of guggulsterone on the serum levels of total bilirubin (tb) in figure 8, there is a significant increase in the level of tb in mice maintained on high-fat diet compared to the control group (p<0.05). while all guggulsterone treated groups showed a significant decrease in the levels of serum tb in comparison with hfd group (p<0.05). effects of guggulsterone on the serum levels of creatinine kinase (ck) in figure 9, mice fed with high-fat diet showed a significant increase in ck level compared to the control group (p<0.05). while groups treated with (1000 and 2000) ppm concentration of guggulsterone showed a significant decrease in the levels of ck compared to hfd group (p<0.05). effects of guggulsterone on the serum tnf-α level in figure 10, mice fed high-fat diet only showed a significant increase in the level of serum tnf-α compared to the control group (p<0.05). the treatment with 2000 ppm guggulsterone showed a significant decrease in the levels of tnf-α (p<0.05) compared to the hfd group, while the treatment with 500 & 1000 ppm concentration of guggulsterone result in non-significant effect in comparison with hfd group. figure (8) effects of guggulsterone on serum tb level of mice models with high-fat diet induced nafld. * is a significantly different compared to the control group (p<0.05). # is significantly different compared to the hfd group (p<0.05). figure (9) effects of guggulsterone on serum ck level of mice models with high-fat diet induced nafld. * is a significantly different compared to the control group (p<0.05). # is significantly different compared to the hfd group (p<0.05). figure (10) effects of guggulsterone on serum tnf-α level of mice models with high-fat diet induced nafld. * is a significantly different compared to the control group (p<0.05). # is significantly different compared to the hfd group (p<0.05). https://doi.org/10.31351/vol28iss1pp17-23 iraqi j pharm sci, vol.28(1) 2018 guggulsterone and nutritional steatohepatitis 21 effects of guggulsterone on the liver tissue histology blinded histological assessment was performed on hematoxylin and eosin stained hepatic tissue to detect the presences of steatosis, ballooning degeneration and inflammation (figure 11), there was no evidence of steatosis, ballooning degeneration or inflammation observed in control group. the h and e stained hfd liver tissue section showed the presences of steatosis in the form of pronounced micro-vesicular fatty change (small, clear, optically empty lipid vacuoles), and macrovesicular fatty change in which the size of the vacuoles increases pushing the nucleus to the periphery of the cell, giving the cell an empty ringlike appearance, that with the development of inflammation and ballooning degeneration. the h and e stained liver tissue section of guggulsterone treated group at 500 ppm showed mild steatosis in the form of small lipid droplets, while the treatment with guggulsterone at 1000 and 2000 ppm reduced the liver tissue to look like that of the control group with no sign of steatosis, ballooning degeneration or inflammation. figure (11) histological assessment of nash activity score (steatosis, inflammation and ballooning degeneration). representative h and e stained sections from; control (a), hfd (b), gs 500ppm (c), gs 1000ppm (d) and gs 2000ppm (b), mouse models.a variable size of clear, optically empty,unstained fat vacuoles microvesicular (thick arrow) and macrovesicular (thin arrow) fatty changes. a b c d e https://doi.org/10.31351/vol28iss1pp17-23 iraqi j pharm sci, vol.28(1) 2018 guggulsterone and nutritional steatohepatitis 22 discussion non-alcoholic fatty liver disease (nafld) has turned to be one of the most common chronic liver disease worldwide; it is a multifactorial disorder with the contribution of a variety of environmental and genetic factors(1). these results came in tune with the previous investigations that guggulsterone suppressed lipid accumulation in a dose-dependent manner(8), via inhibiting farnesoid x receptor (fxr) that consider as a major coordinators of adipocyte gene expression and differentiation, that result eventually in reduced liver index(9)(5). liver has a primary role in controlling plasma levels of ldl cholesterol because most of the ldl receptors are located in the liver. guggullipid extracts have been used widely as a lipidlowering agent; it has been well documented for its hypolipidemic activity(10). guggulsterone inhibits lipoproteins formation and lower the rate of intestinal fat and cholesterol absorption that result in an increase in the rate of fecal excretion of bile acids and cholesterol. guggulsterone lipid lowering effect is due to farnesoid x receptor (fxr) antagonistic activity, studies showed it inhibit expression of fxr agonist-induced genes and bsep expression induction(11)(12). the results from the present study have further proved the efficacy of the treatment with purified guggulsterone in lowering the serum total cholesterol and serum ldl levels compared to mice fed with high fat diet only. meanwhile, the treatment with guggulsterone resulted in a not significant minor decrease in serum tg levels compared with hfd group, such outcome might be attributed to differences in animal model and the time period in which this study was carried out, since some studies showed that the treatment with guggulsterone in fischer rats lowered serum triglycerides and concomitantly increased serum hdl levels(13)(14). liver enzymes mainly the alanine aminotransferase (alt), aspartate aminotransferase (ast) and alkaline phosphatase (alp) considered as liver toxicity markers, the increment in their level that is due to increased ros generation(15)(16). this study data showed a reduction in the level of alanine aminotransferase (alt), aspartate aminotransferase (ast) and alkaline phosphatase (alp) in guggulsterone treated groups remarkably with the higher concentration, compared to the hfd-treated group, thus consequently alleviated the damage in the liver caused by hfd. this is due to the antioxidant activity of guggulsterone, which reported to be hepatoprotective agent(17). guggulsterone enhance the transcription of bile salt export pump that lead to reduced hepatic lipid content, also due to guggulsterone own activity as antioxidant, guggulsterone reduce the need for the bilirubin that act as free radical scavenger this results in reduced oxidative stress (18)(19) eventually reduced production of bilirubin, as noted in the presented results that serum tb is significantly lower in guggulsterone treated groups compared with the hfd group that showed a markedly elevated tb level. the results showed that the level of ck elevated remarkably in the group that maintained on hfd only. studies showed that the higher serum level of ck is associated with higher body fat mass, and consider as biochemical evidence of metabolic abnormality and the presence of nafld(20)(21), guggulsterone treatment resulted in lowering the level of ck, the higher the concentration result in the lower level of ck, this can be attributed to the hypolipidemic ability of guggulsterone that resulted in lower body visceral fat(19)(18).tnf-α is used as a predictor of fibrosis in patients with steatohepatitis(22).the elevation in tnf-α is related to aberrant production of cytokines by adipose macrophages and kupffer cells(23)through the activation of tlr4 in macrophages that initiates downstream signaling pathways including nuclear factor-kappa b (nf-κb) complex that play an important role in acute and chronic inflammatory conditions in response to liver damage caused by hfd(24)(25). the treatment with guggulsterone as shown in the results had significantly decreased the elevated serum level of both tnf-α compared to the hfd group. this reduction in cytokines level is related to the antiinflammatory effect of guggulsterone, as it has the ability to inhibit the nf-κb signaling pathways as it had been shown in previous studies(26)(27). the histological assessment of liver tissue in this study showed that the hfd group clearly developed an early sign of fibrotic nash with marked hepatosteatosis and ballooning degeneration. while the guggulsterone treated groups had showed a healthy like liver tissue especially with higher concentration. the result supports the role of guggulsterone as hepatoprotective and antifibrotic agent(6). conclusion the results obtained from this study demonstrate that guggulsterone protected the treated mice against high-fat diet induced nafld changes, and the (1000 and 2000) ppm concentration of guggulsterone effectively provided the highest level of protection to the liver against high-fat diet induced nafld. therefore, guggulsterone may be useful in preventing the development of steatohepatitis. reference 1. clark jm. the epidemiology of nonalcoholic fatty liver disease in adults. j clin gastroenterol. 2006;40(suppl. 1):5–10. 2. james cpdofw. steatohepatitis: a tale of two ‘“hits.”’ 1998; 3. neuschwander-tetri ba. hepatic lipotoxicity and the pathogenesis 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derivative gg-52 inhibits nf kappa b signaling in gastric epithelial cells and ameliorates ethanol-induced gastric mucosal lesions in mice. ajp. 2013;(12):193–203. 27. cheon jh, kim js, kim jm, et al. plant sterol guggulsteroneinhibits nuclear factork b signaling in intestinal epithelial cells by blocking i k b kinase and ameliorates acute murine colitis. 2006;12(12):1152–61. https://doi.org/10.31351/vol28iss1pp17-23 phytochemical and antimicrobial study of some flavonoids present in the fruits of two ammi l iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 48 phytochemical study of some flavonoids present in the fruits of two ammi l. species wildly grown in iraq thukaa z. abdul-jalil * , kawkab saour **,1 and abdulmutaliba. nasser *** * department of pharmacognosy , college of pharmacy , university of baghdad , baghdad , iraq . ** department of pharmaceutical chemistry, college of pharmacy,university of baghdad , baghdad, iraq . *** department of pharmacognosy , baghdad college of pharmacy , baghdad , iraq. abstract ammi species belong to the family umbellifereae that provide a host of bioactive compounds (mainly coumarins and flavonoids) of important biological activities, like prevention and treatment of heart and vascular disease and some types of cancer. literature survey revealed that there was no study concerning ammi flavonoids in iraq. ammi majus and ammi visnaga, which are wildly grown in iraq, were chosen for this study. this study concerned with extraction, identification, isolation, and purification of some biologically important flavonols quercetin and kaempferol from the fruits of ammi majus and ammi visnaga. extraction of these flavonols was carried out using 85% methanol and 90% ethanol. identification of these flavonols quercetin and kaempferol was done using thin layer chromatography (tlc) where different solvent systems had been tried. ultra violet (uv) light and iodine vapor where used for detection. this identification was further augmented by using high performance liquid chromatography (hplc) and then these flavonols were isolated and purified. the most suitable extraction, isolation and purification procedures of flavonols were fully described in this study. the identification of isolated flavonols (quercetin & kaempferol) was carried out using melting point (m.p.), thin layer chromatography (tlc), and infrared spectroscopy (ir).this study confirms the presence of quercetin and kaempferol in ammi majus & an ammi visnaga fruit, the percentage of quercetin was higher in ammi visnaga than ammi majus, while the percentage of kaempferol was higher in ammi majus than ammi visnaga. key words : ammi majus , ammi visnaga , quercetin , kaempferol . الخــالصـة ( و هى ٌححىي على الكثٍز من المزكبات )منها الكىمارٌن والفالفٍنىٌداات( اات umbellifereaeجنس الخلة من العائلة المظلٍة ) لفعالٍدة احيٍائٍددة الحددً ج ددحخاي للىلاٌددة وعددالل امددزاا الملددض والادزاٌٍن ونعدد اظددىاع ال ددز اظاتا وظظددز لعدداي وجددى را ددات يددى ا ( .ammi majus lالفالفٍنىٌاات فً ظبات الخلة فً العزاق ,لذلك اصبح من احهمٍة را ة نع اصناف الخلة ومنها الخلة الادٍااظً ) ( الحددً جنمددى نزٌددل فددً العددزاق افددً هددذس الارا ددة جدد ا ددحخالع وكادد وف دد وجنمٍددة نعدد .ammi visnaga lamوالخلددة البلدداي ) ( ammi visnaga(والندىع )ammi majusالفالفٍنىٌاات المهمة من النايٍة احيٍائٍدة وهمدا الكىر دحٍن والكدامبفٍزو مدن لمدارالنىع ) %ا جد الكاد عدن ۹٠% ومدن لد المدذٌض العمدىي اٌثداظى نن دبة ٥٨يٍث جد اح دحخالع نا دحخااي المدذٌض العمدىي مٍثداظى نن دبة الفالفٍنىٌاات )الكىر حٍن والكامبفٍزو ( نا حخااي جمنٍة كزوماجىغزافٍا الابمة الزلٍمدة نا دحخااي مدذٌبات مخحلفدة كى دٍل ظالد والكاد ا اح اء العددالً ال ددائلة ونعدداها جمددث عملٍددة الف دد عنهددا نا ددحخااي احفددعة فددىق البنف دداٍة ونخددار احٌى ٌن وكددذلك جمنٍددة كزوماجىغزافٍدد والحنمٍةا كمدا جد فدً هدذس الارا دة ا حٍدار الازٌمدة المنا دبة لال دحخالع والف د والحنمٍدة وفدزيها نازٌمدة محكاملدة ا وكدذلك ا دحخامث هدا والحدً فدملث ر رجدة احظ دهار مامىعة من الحمنٍات للححمٍك من ظىعٍة المزكبات المف ىلة )كىر حٍن و الكامبفٍزو ( و رجة ظماوج للمزكبددات المف ددىلة وكزوماجىغزافٍددا الابمددة الزلٍمددة وكددذلك ماٍدداف احفددعة جحددث الحمددزاء االبحددث هددذس الارا ددة وجددى الفالفٍنىٌدداات ا ددة ك كمٍددة س الارذ)الكىر ددحٍن والكددامبفٍزو ( فددً لمددار الخلددة الاددٍااظً و الخلددة البلدداي الحددً جنمددى نزٌددا فددً العددزاق ا كمددا هددزت هدد الكىر حٍن كاظث كثز فً الخلة البلاي من الخلة الاٍااظً ما الكامبفٍزو فأك كمٍحه كثز فً الخلة الاٍااظً من الخلة البلايا introduction ammi is a genus of 3-6 species of flowering plants in the apiaceae family; they are native in southern europe, northen africa and south west asia .(1,2) ammi majus and ammi visnaga are one of the most important medicinal plant species in the world ( figure 1 , figure 2 ). (3) in iraq ammi majus usually found in fields and gardens and by the side of channels, often as weed of cultivation. it's collected from kut, baghdad, hawija and many other areas. while ammi visnaga are widely distributed in erbil, mousl, baghdad, sulaimania and kirkuk in north of iraq .(4) 1corresponding author email : dr.ksaour@yahoo.com received : 13/10/2009 accepted : 13/2 / 2010 mailto:dr.ksaour@yahoo.com iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 49 figure (1) photography of ammi majus figure (2) photography of ammi visnaga epidemiological data, clinical investigations, and animal studies provide strong evidence that the main active constituents of this genus are: coumarin and their derivatives, flavonoids, volatile oil and fixed oil .(5) all the flavonoids described in ammi species can be classified into flavonols (quercetin , kaempferol , isorhamnetine ) and flavones (apigenin , luteolin , chrysoeriol ) these types of flavonoid have been show to be powerful antioxidant and free radical scavengers. (68) among these flavonoids : quercetin which is a flavonol type of flavonoids that has been shown to help prevent the development of a variety of condition related to inflammation and free radical damage, including arthritis, allergies, macular degeneration, heart disease, gout, and various forms of cancer .(9,10) the other flavonol found in these plants is the kaempferol, which is a strong antioxidant and help to prevent oxidative damage of our cells, lipids and deoxyribonucleic acid (dna). it seems to prevent arteriosclerosis by inhibiting the oxidation of low density lipoprotein and the formation of platelets in the blood. studies have also confirmed that kaempferol acts a chemopreventive agent which means that it inhibits the formation of cancer cell (9,11) materials and methods a. plant materials the plant materials (dried ripe fruits) of ammi majus l. (apiaceae) was collected during the months of march and april from local fields about 2 km south of kut. while the fruits of ammi visnaga (apiaceae) were collected from the botany garden in the college of pharmacy, uuniversity of baghdad. (pharmacognosy department). both of them were identified by the department of pharmacognosy, college of pharmacy, university of baghdad and authenticated by national iraqi herbarium, botany directorate at abu – ghraib. a 50 gm of powdered fruits of ammi majus and ammi visnaga were packed in a thimble of soxhlet extractors. 500 ml of petroleum ether (b.p 40 – 60 c 0 ) was used in a soxhlet extractor for three hours to get rid of lipids and fat. (9) the defatted powdered fruits after drying over night were extracted with 500 ml of 85% methanol for 12 hours by use of soxhlet apparatus for flavonoids extraction as free and glycosides. (6,7) the methanolic extract was then filtered and then a portion had been taken and kept aside for further work. the remaining portion of filtrate was evaporated under reduced pressure at a temperature not exceeding 40 °c (f1).the methanolic extract (f1) residue was weighted and subjected for identification and purification procedures. to continue the extraction process, the previously extracted plant materials (fruits) of both ammi majus and ammi visnaga were dried at room temperature, and then were extracted again with 500 ml of 90 % ethanol for 12 hours by use of reflux apparatus for extracting the remaining flavonoids. (6,7) . the contents of the flask were filtered while hot and the ethanolic extract was allowed to cool at room temperature. a portion of ethanolic extract had been taken and kept aside for further work. the remaining portion of ethanolic extract was concentrated under reduced pressure to dryness (f2) and the dried extract (f2) was weighted and subjected for identification and purification procedures.later on, equal volumes of methanolic extract and ethanolic extract of both ammi majus and ammi visnaga were mixed together to give methanolic ethanolic extract which also concentrated under reduced pressure to dryness (f3). the dry extract (f3) was weighted and subjected for identification and purification procedures.figure (3 and 4) show schematic procedure for the extraction method. iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 50 50 gm of powdered fruits of ammi majus soxhelt with petroleum ether (b.p 40 – 60 c 0 ) for 3 hours filtrate marc defatted fruits no flavonoid appear after test soxhelt with 85 % methanol for 12 hours marc (fruits) filtrate reflux with 90 % ethanol for 12 hours evaporation to dryness (f1) filter hot filtrate marc evaporation to dryness (f2) figure 3 : schematic procedure for the extraction method of flavonoids from ammi majus fruits. 50 gm of powdered fruits of ammi visnaga soxhelt with petroleum ether (b.p 40 -60 c 0 ) for 3 hours filtrate marc defatted fruits no flavonoid appear after test soxhelt with 85% methanol for 12 hours marc (fruits) filtrate reflux with 90 % ethanol for 12 hours evaporation to dryness (f1) filter hot filtrate marc evaporation to dryness (f2) figure 4 : schematic procedure for the extraction method of flavonoids from ammi visnaga fruits. iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 51 b. identification of flavonoid identification of f1,f2,f3 were carried by thin layer chromatography (tlc) using a ready made aluminum plates of silica gel gf254, two different detection methods, first by using uv light wave length 254 nm and 366 nm, second by using iodine vapor in the jar,in comparison with three different solvent systems s1,s2,s3. standard flavonoids: quercetin (fluka-austia) kaempferol (sigma-aldrich,usa) different developing solvent systems that were: (12-15) s1= chloroform:aceton:formic acid(75: 16.5 : 8.5 ) s2 = chloroform: methanol (90:10) s3 = toluene: chloroform: aceton (40: 25: 35) c. isolation and purification of quercetin and kaempferol after locating of quercetin and kaempferol of the extract in comparison with standards, preparative thin layer chromatograghy was done to isolate and purify them. the portion of mixture methanolicethanolic extract (f3) was used to obtain the final product by applying it as a concentrated solution in arrow of spots using capillary tube and the standard sample was applied in one side of the plate. the mobile phase used was s1 = chloroform : aceton : formic acid ( 75 : 16.5 : 8.5 ) the separated compound appear as a band identified using u.v light detection method.the band corresponding to the standard was scrapped out and collected in a beaker and eluted with gentle heating and filtered. then the filtrate was evaporated to dryness under reduced pressure to give yellow precipitate. the precipitate then recrystallized using hot ethanol and maintained for tlc and measuring melting point and infra red spectrum. d. qualitative and quantitative estimation of flavonoid using hplc technique qualitative and quantitative estimations of quercetin and kaempferol were done by using knauer/germany high performance liquid chromatography (hplc) in which identifications were made by comparism of retention time obtained at identical chromatographic conditions of analyzed samples and authentic standards.the hplc conditions are listed in the following table (1): table 1 : hplc conditions (16) sample mobile phase column flow rate detection quercetin acetonitrile: methanol : glacial acetic acid (70:30: 0.1) c18 5 mm x 150 mm 0.5 ml / min uv. detector at λ 306 nm kaempferol methanol : water ( 7.5 : 92.5 ) c18 ods 1.5 ml / min uv. detector at λ 308 nm results and discussion three extraction portions were obtained from the experimental work in which methanolic extract (f1), ethanolic extract (f2) and the third extract portion, which is a mixture of methanolic – ethanolic extract (f3). results showed that the third extract portion was the best, because the amount of both extract and flavonoid were higher than the two other extract portions. as shown in (table 2). table 2 : percentages of extract and flovonoids (quercetin, and kaempferol) in the fruits of ammi majus and ammi visnaga. plant ammi majus ammi visnaga extraction portions f1 f2 f3 f1 f2 f3 percentage of extract 8.5 4.8 10.0 9.2 6.0 11.2 percentage of quercetin 0.019 traces 0.036 0.020 traces 0.042 percentage of kaempferol 0.025 traces 0.045 0.018 traces 0.035 iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 52 identification of flavonoids by tlc tlc of the extracts (f1,f2,f3) obtained from dried ripe fruits of ammi majus and ammi visnaga , confirms the presence of quercetin and kaempferol in all extraction portions in comparison with standards. as represented in table (3) and figure (5) . table 3 : showed the rf values of flavonoid (quercetin and kaempferol) and their standards in different developing solvent systems in tlc. solvent system s1 s2 s3 rf value of standard quercetin 0.4 0.45 0.78 rf value of quercetin in ammi majus 0.38 0.43 0.76 rf value of quercetin in ammi visnaga 0.39 0.44 0.77 rf value of standard kaempferol 0.52 0.6 0.85 rf value of kaempferol in ammi majus 0.51 0.61 0.84 rf value of kaempferol in ammi visnaga 0.49 0.59 0.86 mee q k mee mee q k mee a.v. a.m. a.v. a.m. ( 1 ) ( 2 ) mee q k mee a.v. a.m. ( 3 ) figure 5: tlc of fruits extracts of ammi majus and ammi visnaga obtained by extraction method using silica gel gf254 as adsorbent and (s1)as a mobile phase. detection by uv-light at (1) 254 nm , (2) 366 nm , (3) iodine vapor. ( a.m: ammi majus , k : kaempferol standard, mee: methanolic – ethanolic extract, a.v: ammi visnaga, q: quercetin standard ) iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 53 isolation and quantitative determination of quercetin and kaempferol by preparative tlc from the investigation of the fruits extracts (fractions) of ammi majus and ammi visnaga on tlc plates, it was found that quercetin present in both fruits extracts but higher in ammi visnaga while kaempferol also present in both fruits extracts but higher in ammi majus. the percentage of both quercetin and kaempferol were obtained by weighing of the isolated compounds as shown in table (4) table 4 : percentages of quercetin and kaempferol present in the fruits of ammi majus and ammi visnaga. plant fruits (total) ammi majus ammi visnaga quercetin 0.036 % 0.042 % kaempferol 0.045 % 0.035 % characterization of the isolated kaempferol and quercetin tlc both isolated compounds appeared as a single spot having the same color and rf value as that of reference standards. measuring melting points the isolated compounds were identified to be quercetin and kaempferol from their sharp melting point. since one of these compound showed a melting point of 314 – 316 c 0 compared to quercetin melting point 316 c 0. the other compound showed a melting point of 275 – 276 c 0 compared to melting point 276 -278 c 0 for standard kaempferol. ir. the ir spectra of each isolated quercetin and kaempferol were recorded as kbr disc using ir spectra photometer buck scientific model 500, gave identical results were compared with authentic standard samples; which confirm that our isolated compounds are quercetin and kaempferol, (17) as shown in figures (6-7). figure 6 : ir spectrum of isolated kaempferol o oh oh oh ho o kampferol iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 54 figure 7 : ir spectrum of isolated quercetin hplc analysis generally, the percentage of quercetin and kaempferol is higher in methanolic – ethanolic extract than in methanolic extract and the methanolic extract contains higher amount of quercetin and kaempferol than ethanolic extract. in addition, ammi majus had shown different percentages of quercetin and kaempferol than ammi visnaga. since the percentage of quercetin in ammi majus was lower than the percentage of quercetin in ammi visnaga in all extracts. while the percentage of kaempferol in all ammi majus extracts was higher than the percentage of kaempferol in ammi visnaga extracts.the result indicates that the hplc method was efficient for qualitative identification and quantitative determination of quercetin and kaempferol. as show in table (5) and figures (8-13). table 5 : percentage of flavonols in ammi majus and ammi visnaga. extraction solvents percentage of the quercetin in the plant fruits. percentage of the kaempferol in the plant fruits. methanolic extract of ammi majus (f1) 0.026 0.037 methanolic extract of ammi visnaga (f1) 0.033 0.025 ethanolic extract of ammi majus (f2) 0.011 0.018 ethanolic extract of ammi visnaga (f2) 0.015 0.012 methanolic – ethanolic extract of ammi majus (f3) 0.045 0.052 methanolic – ethanolic extract of ammi visnaga (f3) 0.050 0.043 o oh oh oh oh ho o quercetin iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 55 figure 8 :hplc analysis of kaempferol standard figure 9 : hplc analysis of methanolic-ethanolic extract of ammi majus figure 10 : hplc analysis of methanolic-ethanolic extract of ammi visnaga iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 56 figure 11: hplc analysis of quercetin standard figure 12 : hplc analysis of methanolic-ethanolic extract of ammi majus figure 13 : hplc analysis of methanolic-ethanolic extract of ammi visnaga iraqi j pharm sci, vol.19(1) 2010 detection of flavonoids in ammi l. species 57 conclusions phytochemical investigation of ammi majus and ammi visnaga fruits, grown in iraq revealed the presence of important group of medicinal natural products belong to flavonoid derivatives. quercetin and kaempferol were isolated and identified in ammi majus and ammi visnaga fruits by using simple and reproducible tlc and hplc method. the flavonoid, quercetin and kaempferol are found in the fruits of amm majus and ammi visnaga, were quercetin present in large quantities in the fruits of ammi visnaga than that of ammi majus, while kaempferol present in the fruits of ammi majus in large quantities than in ammi visnaga. references 1. w.h.o.: who monographs on selected medicinal plants. 2007; 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( واحد من اهم الترايتيربينويد خماسية الحلقهoleanolic acid الذي تم استخالصه من أوراق الزيتون بطريقتين مختلفتين معروفتين ،) الن التحديد مع الطريقتين كلتا في األولينوليك حمض وجود إثبات تم وقد القاعدية، الحمضية والطريقة الحمضي المائي التحلل طريقة وعي وهما لسرطان الثدي بقيمة amj13نشًطا ضد خط خاليا بالطريقة االولى مستخلص كان ال ،(hplcوالكمي باستخدام الفصل اللوني السائلة عالية األداء) ic50 ميكروغرام / مل. 0.8936تبلغ amj13 الخاليا خط ، األداء عالية السائلة اللوني الفصل ، األساسية الحمضية الطريقة ، الحمضي المائي التحلل طريقة األولينوليك، حمض: المفتاحية الكلمات introduction olea europaea (olive), family oleaceae, is an evergreen small tree(1); native to the mediterranean region, regarded as a significant crop owing to the remarkable nutritional and therapeutic oil effects(2). steroids, terpenoids, reducing sugar flavonoids and tannins were detected in olive tree leaves(3). oleuropein(phenolic compound) and oleanolic acid(triterpene) are the most abundant key components phytochemicals in olive leaves(4,5). leaves and fruits content of oleanolic acid differ among varieties, olive fruit maturation leads to acid content drastically decreases by 70-80%(6). oleanolic acid is a secondary metabolite that belongs to an oleanane type pentacyclic triterpenic acid(7), widely distributed in the plant kingdom in greater than 1620 plant species; particular in oleaceae family, as do in spices, edible crops and medicinal plants; clove, apple, olive, and lanata shrub are included(8-10). chemically oleanolic acid is 3β-hydroxyolean-12en-28-oic acid(3), naturally, oleanolic acid present in free form (aglycone of many saponins) and as glycoside derived from the mevalonic acid pathway(9,11,12). oleanolic acid along with its natural and synthetic derivatives attracts much attention because of its diverse pharmacological effects as hepatoprotective, anti-osteoporosis, antiinflammatory, neuroprotective, hypoglycemic, antitumor and other effects(13). oleanolic acid demonstrated proved anticancer effect versus lung, gallbladder, pancreatic cancers, leukemia, and osteosarcoma(14). an exhaustive literature survey revealed that quantitative estimation of oleanolic acid from this plant has been recruited by different methods recently,(15) however, this study articulates the frame to view the first report with regard to extract, isolate, identify oleanolic acid using tlc and hplc, to estimate its quantity using hplc, and evaluate its cytotoxic effect on breast cancer (amj13) cell line. 1corresponding author e-mail: zakaa.abd@copharm.uobaghdad.edu.iq received: 16/10 /2021 accepted:26 /1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp244-250 https://en.wikipedia.org/wiki/triterpenoid iraqi j pharm sci, vol.31(2) 2022 determination of. oleanolic acid and its cytotoxic effect 245 materials and method plant materials and extraction of oleanolic acid olea europaea leaves were collected in december 2020 from baghdad, the identification was done by the college of pharmacy/ university of baghdad while authentication by prof. dr sukaena abbas\ department of biology\ college of the science \university of baghdad. the leaves were washed with water, and then shade dried at room temperature for three weeks. 40 gm of milled leaves were placed in thimble and extracted with 400 ml methanol for 3 hours using soxhelt apparatus. the solvent was evaporated in a rotary evaporator till dryness to give crude methanolic extract 8.22 gm(16, 17). extraction of oleanolic acid two methods were used for extraction method 1: acidic hydrolysis of crude methanolic extract four grams of methanolic extract was reflexed using 100 ml of 5% hcl for 2 hours. crude hydrolysis products were extracted with 100ml ethyl acetate two times. ethyl acetate extract was concentrated in rotary evaporator to give 1.543 gm and the percentage yield was 3.86%. ethyl acetate extract was stored for further examination(18, 19). method 2: basic –acidic extraction method four grams of methanolic extract was reconstituted with an appropriate amount of water and dissolve in the water bath. to about 30 ml of aqueous extract, add10% koh dropwise till basification (ph about 9). centrifuge to remove precipitate, take supernatant, and add 20% hcl drop by drop to get an acidic medium (ph about 4) and the precipitate is formed. centrifuge to get precipitate which contains oleanolic acid and related compounds(20). identification of oleanolic acid by chromatographic techniques two simple and accurate chromatographic techniques were developed for the determination of oleanolic acid in iraqi olea europaea leaves: qualitative identification by thin layer chromatography an analytical tlc was performed for oleanolic acid identification in which silica gel tlc (gf 254) plate was used as a stationary phase, toluene: methanol (18:2) was the solvent system, standard solution of oleanolic acid (1 mg/ml), solution of ethyl acetate fraction and extracted oleanolic acid were applied on tlc plate. after development, detection the chromatogram was done by spraying 5% ethanolic h2so4 and heated in the oven. oleanolic acid was observed as a purple spot and rf value was calculated(21). qualitative and quantitative identification by rphplc the oleanolic acid isolated from olea europaea leaves was analyzed using sykmn germane hplc system. the detection was performed at 215 nm, rpc18-ods column (250cm x 4.6mm,) was used and the column was maintained at room temperature, the mobile phase was composed of acetonitrile: water (85:15 v/v), the flow rate was 1ml/min and the isocratic mode of elution was used. the volume of the injected standard and sample was 0.1 ml(22, 23). evaluation of bioactivity of the ethyl acetate fraction using mtt cytotoxicity assay to determine the cytotoxic activity of ethyl acetate fraction; human breast cancer cell (amj13) was obtained from the iraq biotech cell bank unit and preserved in rpmi-1640 (capricorn, germany), supplemented with 10% fetal bovine (capricorn, germany), 100 unit/ml penicillin, and 100 µg/ml streptomycin. cells were passaged using trypsinedta (capricorn, germany), reseeded at 50% confluence twice per week, and incubated at 37 °c. the mtt cell viability assay was performed on 96well plates, cell lines were seeded at 1 × 104cells/well, 24 hrs later or when the confluent monolayer was done, cells were treated with the tested compound (serial concentrations of ethyl acetate fraction). after 72 hrs of treatment, the viability of the cell was measured by removing the medium or supernatant, mtt solution was added to each well (and incubating the cells for 1.5 hrs. at 37 °c; the mtt test is a sensitive colorimetric and quantitative test, reflects cell viability, proliferation, and toxicity and is based on the ability of the mitochondrial nadph-dependent oxidoreductase enzymes to reduce the soluble yellow mtt (3-[4,5dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) to insoluble purple formazan crystals, the darker the solution the greater number of viable cells(24). after eliminating the mtt solution, the remaining crystals in the wells were dissolved by adding 130 µl of dmso (dimethyl sulphoxide) followed by incubation at 37 °c for 15 min with shaking(25). the absorbance was measured on a microplate reader at 492 nm (test wavelength); the assay was carried on in triplicate. the inhibitory rate of cell growth (the percentage of cytotoxicity) was calculated according to the following equation(26): % cytotoxicity = 100 – [(a 492 in extract treated cell / a 492 in extract untreated cell) (100)] statistical analysis the obtained data were statically analyzed using an unpaired t-test with graphpad prism (27). the values were presented as the mean ± sd of triplicate measurements (28). results and discussion extraction is the crucial primary step in the analysis of medicinal plants to get the desired chemical components for further separation and identification. olea europaea leaves are regarded to be economical and appreciable source of oleanolic acid; therefore, different extraction methods iraqi j pharm sci, vol.31(2) 2022 determination of. oleanolic acid and its cytotoxic effect 246 (conventional and non-conventional) were utilized for extraction and separation of oleanolic acid from olea europaea leaves but for the first time the acidic hydrolysis method and basic acidic method were applied for assessing oleanolic acid presence. methanol was used as extracting solvent because oleanolic acid is freely soluble in alcoholic solvents with optimum solubility in methanol(17, 29-31). tlc method development analytical tlc (figure 1) revealed the presence of oleanolic acid in ethyl acetate fraction (method 1) and an oleanolic acid fraction (method 2) since one of the separated spots in these fractions has similar color and rf value to that of oleanolic acid standard (rf for oleanolic acid in toluene: methanol (18:2) is 0.43. hplc identification and quantification of oleanolic acid hplc was chosen as a qualitative and quantitative method for oleanolic acid determination as it is a simple, rapid, efficient, and comprehensive method for triterpenic acids separation in plant extracts(31) . the retention time for the oleanolic acid standard was 9.69 min (figure 2, a). the chromatogram for ethyl acetate and extracted oleanolic acid fractions (figure 2, b and 2, c) demonstrated the presence of distinct peaks at 9.70 and 9. 79 min which reflects the presence of oleanolic acid respectively in both examined fractions figure 1. tlc chromatogram of (st: oleanolic acid standard, m1: ethyl acetate fraction by method 1, m2: extracted oleanolic acid by method 2) after derivatization with 5 % h2so4 reagent and observation at daylight. . figure 2. hplc chromatogram of a: standard oleanolic acid, b: ethyl acetate fraction (method 1), c: extracted oleanolic acid fraction (method 2) iraqi j pharm sci, vol.31(2) 2022 determination of. oleanolic acid and its cytotoxic effect 247 concerning quantitative determination of oleanolic acid in the desired fractions serial dilutions of standard oleanolic acid in methanol were prepared (10, 20,30, and 40 ppm). a plot of area vs concentration of oleanolic acid standard shows a linear fit (figure 3). this calibration chart was subsequently used for the quantification of oleanolic acid in the analyzed fractions. figure 3. hplc calibration of oleanolic acid standard oleanolic acid amount in the method 2 fraction is lower than its amount in the ethyl acetate fraction; illustrated in table 1, as the area under the curve and concentration of both fractions prove. this may be attributed to the fact that the oleanolic acid in olea europeae leaves may be present in bound forms (monodesmosidic and didesmosidic type of glycoside) and acidic hydrolysis of glycosides release aglycone from glycone(s)(19, 32, 33). in olea ferruginea leaves oleanolic acid glycosides had been isolated(34). table 1. auc and concentration of ethyl acetate and extracted oleanolic acid fractions fraction auc (mau.s) concentration (ppm) ethyl acetate (method 1) 6111.985 70.414 extracted oleanolic acid (method 2) 1866.144 21.4 cytotoxic test the cytotoxic test was performed to evaluate the toxicity of ethyl acetate fraction of olea europaea leaves on human tumor cell line; breast ductal carcinoma amj13 cells by the mtt test. as shown in figure 4, ethyl acetate fraction revealed a cytotoxic activity against the amj13 cell line with a minimum cytotoxic effect at 6.25 µg/ml and a maximum cytotoxic effect at 200 µg/ml (table 2). this effect is concentration dependent. figure 4. effect of ethyl acetate fraction over amj13 cell growth. the results are expressed as % of cytotoxicity. table 2. % cytotoxicity of ethyl acetate fraction at various concentration concentration 200 µg/ml 100 µg/ml 50 µg/ml 25 µg/ml 12.5 µg/ml 6.25 µg/ml % cytotoxicity 73.40 71.70 69.00 64.30 59.20 36.60 the (ic50) concentrations inducing 50% cell growth inhibition for examined fraction was 0.8936 μg/ml (figure 5). morphology of amj13 cell before treatment, and after treatment with ethyl acetate fraction is seen in figure 6. figure 5. ic50 of ethyl acetate fraction on amj13 cell line iraqi j pharm sci, vol.31(2) 2022 determination of. oleanolic acid and its cytotoxic effect 248 figure 6. morphology of amj13 cell a: before treatment, b: after treatment previous studies demonstrated the anti-proliferative and pro-apoptotic effect of oleanolic acid against breast cancer mcf-7, mda-mb-231 cells line (35,36). oleanolic acid inhibits the cell cycle at various phases and promote apoptosis in the cancer cells via alteration in the expression of the tumor cell cycle regulatory proteins differently (37). conclusion to the best of our knowledge, this is the first study on the extracted oleanolic acid from olea europaea leaves by acidic hydrolysis and basic acidic methods with hplc quantification and evaluation of cytotoxic activity against new human breast 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membrane sensors for the selective determination of metoclopramide hydrochloride and their applications to pharmaceutical analysis abdul-muhsin a. al-haideri*, najwa i. abdulla* ,1 and ibtihaj k. malih* *chemistry department, college of education ibn al-haitham, university of baghdad, baghdad, iraq. abstract metoclopramide (mcp) ion selective electrodes based on metoclopramide-phosphotungstic acid (mcp-pt) ion pair complex in pvc matrix membrane were constructed. the plasticizers used were tri-butyl phosphate (tbp), di-octyl phenyl phosphonate (dopp), di-butyl phthalate (dbph), dioctyl phthalate (dop), di-butyl phosphate (dbp), bis 2-ethyl hexyl phosphate (behp). the sensors based on tbp, dopp, dbph and dop display a fast, stable and linear response with slopes 59.9, 57.7, 57.4, 55.3 mv/decade respectively at ph ranged 2-6. the linear concentration range between 1.0×10 -5 – 1.0×10 -2 m with detection limit 3.0×10 -6 and 4.0×10 -6 m for electrodes using tbp, dopp and dbph while electrode using dop shows a linear concentration range between 3.0×10 -5 – 1.0×10 -2 m and detection limit 6×10 -6 m. the selectivity coefficient values were calculated for mcp with respect to different inorganic cations, sugars and amino acids using the mcp depending on tbp plasticizer which is also used for the determination of mcp in pure solutions and in pharmaceutical preparations by direct, standard addition methods and as an indicator electrode in potentiometric titration with phosphotungstic acid (pt) giving satisfactory results. key words: metoclopramide-hcl membrane sensors , metoclopramide determination , pharmaceutical analysis , pvc membrane sensors غشائية بىليمرية انتقائية لتعيين ميتىكلىبرامايد هيدروكلىرايد وتطبيقاتها في أقطاب التحليل الدوائي عبد هللا* اسحاق، نجىي الحيدري* حميدعبد ال عبد المحسن ،1 * مالح كاظم و إبتهاج .*قسم انكُمُاء ، كهُح انرزتُح اته انهُثم ، خامعح تغذاد ، تغذاد، انعزاق الخالصة (mcp-ptذىاوند انذراسح ذسضُز اقطاب اورقائُح غشائُح تىنُمزَح سائهح نهمُرىكهىتزاماَذ تاسرعمال انزوج االَىوٍ ) ( كىسُظ نها مع انمىاد انمهذوح:pvcكمادج مرسسسح فٍ مادج كهىرَذ مرعذد انفىُم ) tri-butyl phosphate (tbp), di-octyl phenyl phosphonate (dopp), di-butyl phthalate (dbph), di-octyl phthalate (dop), di-butyl phosphate (dbp), bis 2-ethyl hexyl phosphate (behp). انمعرمــذج عهً انمــىاد انمهذوــــح األقطاب، ان األقطابدراســـــح خـــىاص ومىاصفـــــاخ هذي أظهزخ dop, dbph, dopp, tbp 55.3، 55.4، 55.5، 55.5اسرداتح سزَعح ومسرقزج وخطُح تاوسذاراخ قذرها أعطد mv/decade 1ما ذزاوذ مذي انرزاكُز انخطُح تُه فُ 6 – 2عهً انرىانٍ عىذ دانح زامضُح ذزاوزد11 -5 111 -2 مىالرٌ 311وزــذ ذسسس -6 ،3 11 -6 ،411 -6 أعطًعهً انرىانٍ تُىما dbph, dopp, tbpانمعرمــذج عهً نألقطابمىالرٌ 311اسرداتح خطُح نهرزكُز ذزاوزد تُه dopانقطة انمعرمذ عهً -5 111 -2 611مىالرٌ وزذ ذسسس -6 الرٌ. زسثد مى قُم معامم االورقائُح نقطة انمُرىكهىتزاماَذ تىخىد اَىواخ مىخثح وسكزَاخ وزىامض امُىُح مخرهفح تاسرعمال انقطة انمعرمذ عهً . ذم اخرثار انقطة انمذكىر فٍ ذعُُه ذزاكُز انمُرىكهىتزاماَذ فٍ مسانُم وقُح مسضزج مخرثزَاً ومسانُم دوائُح تاسرعمال tbpانمهذن زَقح انمثاشزج وطزَقح االضافاخ انقُاسُح فضال عه اسرعمال انقطة كذنُم فٍ انرسسُر انمدهادٌ ضذ مسهىل انط phosphotungstic acid (pt) .معطُاً ورائح خُذج ية الغشائ األقطابغشائية للميتىكلىبرامايد هيدروكلىرايد، تقدير الميتىكلىبرامايد، التحليل الدوائي، أقطاب: مفتاحيهالكلمات ال الدوائية. introduction metoclopramide hydrochloride 4amino-5-chloro-n-[(2-diethyl amino) ethyl]-2methoxy benzamide hydrochloride, mcp is an antiemetic procaine derivative which is currently used in gastrointestinal (gi) diagnostics. it is the active ingredient of many pharmaceutical formulations concerned with the treatment of (gi) disorders due to the elective character of its action in various digestive manifestations in medical practice such as, nausea, meteorism, vomiting, epigastric discomfort and to increase the gastrointestinal motility. this drug act on the muscles within the wall of the upper intestinal tract causing them to contract and to move food and fluids along. it also crosses from the blood stream into brain cells and may cause significant side effects e.g., head ache, dizziness, fatigue, dry mouth, rash. (1-3) 1 corresponding author email : najwa_issac@yahoo.com received : 12/10/2011 accepted : 13/3/2012 iraqi j pharm sci, vol.21(1) 2012 polymeric membrane sensors and pharmaceutical analysis 71 several analytical procedures have been reported for the quantitative determination of metochlopramide in dosage forms or in biological fluids. among these are fluorimetric, (4) spectrophotometric, (5-8) voltammetric, (9) gas chromatography (gc), (10) high performance liquid chromatography (hplc), (11,12) flameless atomic absorption spectrophotometry (13) and membrane sensors. (14,15) in recent years, the development and application of new ion selective electrodes (ises) have played an important role in pharmaceutical and biological analysis. (16-18) the great challenge in this field is the combination of simplicity, low cost, relatively fast response, reasonably selectivity, low detection limit, high accuracy and applicability to colored and turbid solutions. this work deals with the construction of membrane sensors that insure the determination of mcp based on the mcp ion-pair with phosphotungstic acid embedded in a pvc polymer matrix plasticized with different plasticizers. the performance characteristics of the proposed electrodes (e.g., slope, detection limit, concentration range, response time, ph effect, and selectivity) were studied and the determination of mcp in pharmaceutical formulations has also been investigated. experimental part equipment all potentiometric measurements were carried at 25c ± 1c in a constant stirring with a philips pw9421 (england) digital ph/mv meter using the following cell assembly: ag, agcl internal filling solution  membrane  test solution  saturated calomel electrode. a philips pw 9418 ph meter with an orion 91-02 combined glass electrode (switzerland) were used for ph adjustment. reagents all chemicals used were of analytical grade and were used without further purification. metoclopramide hydrochloride was provided by the state company of drugs and medical supplies industry (sdi, samara, iraq), phosphotungstic acid (pt), tetrahydrofuran (thf) and pvc for ises by bdh, dioctyl phthalate (dop), dibutyl phosphate (dbp), dibutyl phthalate (dbph), dioctyl phenyl phosphonate (dopp), tributyl phosphate (tbp) and bis 2-ethyl hexyl phosphate (behp) by fluka. pharmaceutical formulations were obtained from the sdi, iraq (meclodin tablets and oral pediatric drops); zmc hamborg gmbh, germany (placeela injection ampoules); the arab company for drugs industry, jordan (clopram syrup). preparation of the ion association complex the mcp-pt ion association was prepared by mixing 40 ml of an aqueous solution of metoclopramide hydrochloride (1 mmol) with 40 ml solution containing an equimolar amount of phosphotungstic acid. the resultant precipitate was filtered, washed thoroughly with double distilled water then dried at room temperature. this ion association complex was used as electro-active material in the proposed sensors. electrode preparation ion association complex (40 mg) was mixed with a plasticizer (360 mg) and pvc (170 mg). the mixture was dissolved in thf (7 ml). the solution was poured into a glass ring (3.5 cm in diameter) standing on a leveled glass plate, covered with a filter paper and the solvent was allowed to evaporate slowly at room temperature. (19) a transparent membrane was obtained, from which a disc of about 8mm in diameter was cut out and glued to the smoothened end of a 3 cm long pvc tube by means of a pvc-thf solution. the other end of the tube was connected to a glass tube which was ¾ filled with 10 -2 m solution of mcp hydrochloride as the internal reference solution in which the ag/agcl reference electrode was dipped. the constructed electrode was conditioned by soaking in 10 -2 m standard solution of drug for at least 2 hours before measurements. electrode calibration standard metoclopramide hydrochloride solutions (20 ml of 1.0×10 -1 1.0×10 -6 m) were transferred into a 50 ml beaker and the sensor with the reference electrode were immersed in the solution. the measured electromotive force (e.m.f.) values were plotted as a function of a logarithm of the drug concentrations. the electrode was periodically recalibrated over a period of nine weeks. selectivity of the electrode selectivity coefficients pot ba k , were evaluated by the separate solution method (20) according to the following equation: log pot ba k , = (eb – ea)/s+(1-za/zb) log aa where ea , eb ; za , zb and aa , ab are the potentials, charge numbers and activities of the primary a and interfering b ions respectively at aa = ab . potentiometric determination of mcp in pure samples and pharmaceutical formulations pure sample: aqueous solutions of mcp hydrochloride equivalent to 1.0×10 -2 , 1.0×10 -3 m were prepared. tablets: ten tablets (meclodin, 5 mg per tablet) were finely iraqi j pharm sci, vol.21(1) 2012 polymeric membrane sensors and pharmaceutical analysis 72 powdered, well mixed and an accurate weight required to prepare 10 -3 m mcp hydrochloride solution was dissolved in a minimum amount of double distilled water, filtered into a 25 ml volumetric flask and diluted to the mark. injection ampoules (placeela), oral pediatric drops (meclodin) and syrup (clopram): an accurate volumes (12 ml, 7.5 ml, 30 ml) of each formulation respectively were quantitatively and separately transferred into 100 ml volumetric flasks and the volumes were completed to the mark with double distilled water to get solutions of 10 -3 m mcp hydrochloride. as pure and pharmaceutical samples: a 20 ml aliquot of the drug solution was potentiometrically measured as described and the potential reading was compared with the calibration plot. alternatively the standard addition method was applied by measuring the potentials of the drug test solution before and after the addition of small increments (0.2 ml) of a standard solution (10 -1 m) of mcp hydrochloride. the change in the electrode potential (e) at constant temperature of 25c was recorded and used to calculate the concentration of the drug. (21) a potentiometric titration was also applied when 10 ml aliquot of the drug test solution were diluted to 100 ml with double distilled water. the resultant solution was titrated with (10 -2 and 10 -3 m) standard solution of pt using mcp membrane electrode as the sensor. results and discussion membrane composition and electrode response metoclopramide hydrochloride phosphotungstate is a stable water insoluble ion pair complex but readily soluble in organic solvents such as tetrahydrofuran. the complex was dispersed in a pvc membrane as an active material with the following plasticizers: tributyl phosphate (tbp), dioctyl phenyl phosphonate (dopp), dibutyl phthalate (dbph), dioctyl phthalate (dop), dibutyl phosphate (dbp) and bis(2-ethyl-hexyl phosphate) (behp). the performance characteristics of the proposed sensors based on data collected over a period of nine weeks were evaluated according to iuapc recommendations. (22) the results were summarized in table 1. a typical calibration graph of mcp sensors is shown in figure1. it is well known that the nature of the plasticizer significantly influence the sensitivity, linearity and life time of the polymeric membrane sensors. the plasticizers insured the mobility of the membrane constituents within the membrane phase by lowering the viscosity of the polymer matrix, set the dielectric constant of the membrane phase and provide a suitable mechanical properties of the membrane. (23,24) nonnernstian slopes were obtained with sensors depending on dbp and behp. their slopes were (40.7 and 29.0) mv/decade with correlation coefficients of 0.9982 and 0.9979 respectively. the linear range of concentration were 1.0×10 -4 1.0×10 -2 m and 6.0×10 -4 5.0×10 -2 m with detection limits 4.0×10 -5 m and 2.0×10 -4 m respectively. the poor results for sensors using dbp and behp can be attributed to the low solubility or low distribution of mcp-pt ion pair in these plasticizers that have high viscosity (112.89 and 272.54) cst. on the other hand the membranes based on dopp, dbph and dop gave a near nernstian slopes of (57.7, 57.4 and 55.3) mv/decade with correlation coefficients of 0.9996, 0.9996 and 0.9986 respectively. the linear range of these membranes were 1.0×10 -5 1.0×10 -2 m for membranes using dopp and dbph, and 3.0×10 -5 1.0×10 -2 m for dop with detection limits of 4.0×10 -6 m, 4.0×10 -6 m and 6.0×10 -6 m respectively. a nernstian slope of 59.9 mv/decade with correlation coefficient of 0.9998 was obtained for membrane based on tbp and a linear concentration range 1.0×10 -5 1.0×10 -2 m and detection limit 3.0×10 -6 m. the stability of electrodes was monitored continuously by measuring the potential drift and evaluate it for a period of 15 days. the standard deviations of the potential drift obtained for these 15 days were ± 1, ± 1, ± 1, ± 4, ± 9 and ± 12 mv for membranes no. i, ii, iii, iv, v and vi respectively. figure 1: calibration curves of metoclopramide hydrochloride electrode -1 00 -5 0 0 50 100 150 200 250 1.0e-7 1.0e-6 1.0e-5 1.0e-4 1.0e-3 1.0e-2 1.0e-1 1.0e+0 e (m v ) concentration of mcp (m) tbp dopp dbph dop iraqi j pharm sci, vol.21(1) 2012 polymeric membrane sensors and pharmaceutical analysis 73 table 1: parameters of metoclopramide hydrochloride selective electrodes parameters electrode number i ii iii iv v vi plasticizer tbp dopp dbph dop dbp behp slope mv/decade 59.9 57.7 57.4 55.3 40.7 29.0 correlation coefficient 0.9998 0.9996 0.9996 0.9986 0.9982 0.9979 linear range (m) 1.0×10 -5 – 1.0×10 -2 1.0×10 -5 – 1.0×10 -2 1.0×10 -5 – 1.0×10 -2 3.0×10 -5 – 1.0×10 -2 1.0×10 -4 – 1.0×10 -2 6.0×10 -4 – 5.0×10 -2 detection limit (m) 3.0×10 -6 4.0×10 -6 4.0×10 -6 6.0×10 -6 4.0×10 -5 2.0×10 -4 response time 1.0×10 -1 – 1.0×10 -5 5 – 9 5 – 15 5 – 15 7 – 12 30 – 39 60 – 75 life time (day) 52 52 50 64 28 4 potential drift (mv/day) 1 1 1 4 9 12 soaking effect membranes that are freshly prepared must be soaked in their standard solutions before using to form a thin surface gel layer for ion exchange process to occur. (25) soaking require different times depending on the diffusion and equilibration at the electrode-test solution interface. a fast equilibrium establishment is a condition of a fast potential response. (26) it was observed that continuous soaking of the mcp electrodes in 1.0×10 -2 m solution of mcp hydrochloride at room temperature negatively affects their response to mcp due to leaching out of the membrane components to the external solution. the optimal conditioning time for the proposed mcp membranes was found to be about 2-3 hours. life time the life time of the sensors was studied by recalibrating the sensors periodically (every three days) over a period of nine weeks. the electrode depending on dop was used successfully for 64 days and the electrodes depending on dopp, tbp and dbph were used for 52 and 50 days without significant changes in the electrode parameters. however, the life times of the electrodes depending on dbp and behp were 28 and 4 days due to leaching out of the membrane incompatible components. response time the response time of an electrode is evaluated by measuring the average time required to achieve a potential within ±1 mv of the final steady state potential, upon successive immersion of a series of interested ions, each having a ten-folds difference in concentration. it is notable that the experimental conditions like the stirring or flow rate, the ionic concentration and composition of the test solution, any previous usage or preconditioning of the electrode, and the testing temperature have an effort on the experimental response time of a sensor. (27) the response times for the mcp proposed electrodes at different concentrations of mcp solutions ranging from 1.0×10 -5 to 1.0×10 -1 m were calculated and listed in table 1. a plot of the electrode response with time for electrode no.i is shown in figure 2. the response time for all electrodes ranged (5-7) sec. for 1.0×10 -1 m solutions and (9-15) sec. for 1.0×10 -5 m solutions except the electrodes depending on dbp and behp, where they showed longer response time (39 – 30) sec. and (75 – 60) sec. for 10 -5 – 10 -1 m respectively which could be attributed to the high viscosity of the plasticizers used which slow down the mobility of the ion pair within the membrane and cause the equilibrium process to take longer time. figure 2. the response time of tbp electrode using 10 -5 m metoclopramide hydrochloride ph-effect the influence of the ph on the response of mcp membrane sensors was checked using 10 -2 m and 10 -3 m mcp hydrochloride solutions and recording the e.m.f. at different ph values. the ph was adjusted by the addition of very small volumes of 0.1 m hydrochloric acid or sodium hydroxide solutions. as can be seen in figure 3, the electrode potential is practically iraqi j pharm sci, vol.21(1) 2012 polymeric membrane sensors and pharmaceutical analysis 74 independent of the ph range 2.0 – 6.0 for membrane depending on tbp. the considerable decrease of the potential observed at ph values higher than 6.0 is due to the mcp base was precipitated and this leads to the decrease in the concentration of the protonated species and lowers e.m.f. readings. this can be viewed from the consideration that the dissociation constant of mcp hydrochloride, pka is 9 so, at ph values higher than 6 (decrease in the concentration of h + ) the dissociation of mcp hydrochloride will increase and more precipitate will form in the presence of sodium ions which leads to a decrease in the concentration of the protonated species. at ph below 2.0 a sharp increase of the e.m.f. was noticed indicating that the membrane sensor responds to hydrogen ions (14) . figure 3:ph effect on electrode response using electrode based on tbp, 0.001m, 0.01m metoclopramide hydrochloride table 2 illustrates the working ph range for all the constructed mcp-pt the membrane sensors.the mcp electrode depending on tbp was selected for carrying out the remaining studies. it showed a good, fast and stable response over a period of 52 days with a wide linear range and low detection limit. table 2: working ph range for metoclopramide electrodes electrode no. plasticizer ph range i tbp 2.0 – 6.0 ii dopp 3.9 – 6.4 iii dbph 3.0 – 6.0 iv dop 3.2 – 5.5 v dbp 6.0 – 9.2 vi behp 5.0 – 8.0 selectivity measurements one of the important characteristic of any selective sensor is its response to the primary ion (mcp) in the presence of other ions (j) present in solutions which is expressed in term of the potentiometric selectivity coefficient pot j zmcp k  , . it is well known that the selectivity of ion-pair association based membrane sensors depends on the selectivity of the ion exchange process at the membranetest solution interface, the mobility of the respective ions within the membrane and the hydrophobic interactions between the primary ion and the membrane. the influence of some inorganic cations, sugars and amino acids on the mcp electrode was determined by the separate solution method. figure 4 illustrates a typical plot for the selectivity of mcp electrode to lactose. as can be seen from table 3, none of the investigated species was found to interfere as shown by the very small values of pot j zmcp k  , which means that the mcp-pt ion pair used in the sensor was completely dissociated in the organic phase of the membrane and reflects the high selectivity of the mcp-pt electrode towards mcp. the inorganic cations do not interfere owing to the differences in ionic size and consequently their mobility and permeability as compared with those of mcp + . the selectivity in the case of sugars and amino acids is attributed to the differences in the polarity and lipophilic nature of these molecules relative to mcp. table 3: selectivity coefficient values of various interfering compound for metoclopramide electrode i interfering ion concentrations of metoclopramide hydrochloride 10 -1 m 10 -2 m 10 -3 m 10 -4 m 10 -5 m 10 -6 m ka,b ka,b ka,b ka,b ka,b ka,b k + 4.0×10 -1 4.2×10 -1 2.6×10 -4 4.3×10 -3 1.6×10 -2 6.0×10 -2 nh4 + 7.7×10 -3 7.7×10 -3 8.0×10 -2 7.9×10 -1 1.9×10 -1 1.6×10 -1 cu 2+ 6.3×10 -5 5.8×10 -5 2.0×10 -4 5.4×10 -4 1.9×10 -3 1.2×10 -2 glucose 2.8×10 -4 9.3×10 -4 8.9×10 -3 8.9×10 -2 6.4×10 -1 6.3×10 -1 lactose 2.4×10 -4 6.7×10 -4 6.3×10 -3 5.8×10 -2 5.8×10 -1 8.7×10 -1 sucrose 1.2×10 -4 6.0×10 -4 3.7×10 -3 6.6×10 -2 3.6×10 -1 1.1×10 -2 alanin 1.5×10 -3 4.3×10 -3 5.4×10 -2 5.1×10 -1 1.7×10 -1 1.5×10 -1 glycine 2.3×10 -3 4.5×10 -3 8.0×10 -3 3.6×10 -1 2.1×10 -1 1.3×10 -1 iraqi j pharm sci, vol.21(1) 2012 polymeric membrane sensors and pharmaceutical analysis 75 figure 4: selectivity of electrode using tbp to lactose interfering by seperate solution method analytical application the usefulness of metoclopramide electrodes in the determination of mcp hydrochloride in pure solutions 10 -2 , 10 -3 m and in some pharmaceutical preparations was examined by direct, standard addition, multiple standard addition and potentiometric titration. the statistical analysis of the analytical results illustrated in table 4 encouraged us to use the proposed electrode for the determination of mcp hydrochloride in some pharmaceutical preparations (tablets, ampoules, drops and syrup) the results are listed in table 5 presented by recovery %r, relative standard deviation %rsd and relative error %re. the relative recovery was calculated for 5 additions of 0.1 m mcp hydrochloride solutions. table 4: metoclopramide hydrochloride pure samples analysis by potentiometric techniques using electrode i* sample (m) measurements by potentiometric methods direct s.a. m.s.a. titration 1×10 -2 1.001×10 -2 1.005×10 -2 0.992×10 -2 0.995×10 -2 %rsd 1.409 1.404 0.565 0.353 %rc 100.1 100.5 99.2 99.5 %re 0.1 0.5 -0.8 -0.5 1×10 -3 1.002×10 -3 1. 003×10 -3 0.991×10 -3 0.993×10 -3 %rsd 1.408 1.407 0.635 0.504 %rc 100.2 100.3 99.1 99.3 %re 0.2 0.2 -0.9 -0.7 *each value represents an average of 3 measurements. table 5: determination of metoclopramide hydrochloride in some pharmaceutical formulations by potentiometric methods using electrode i* pharmaceutical formulation sample (m) direct s.a. m.s.a. tablets 1.000 10 -3 1.002×10 -3 1. 018×10 -3 1. 003×10 -3 %rsd 1.411 1.386 1.407 %rc 100.2 101.8 100.3 %re 0.1 1.8 0.3 injection ampoules 1.000 10 -3 0.997×10 -3 0.995×10 -3 1. 001×10 -3 %rsd 1.418 2.125 1.400 %rc 99.7 99.5 100.1 %re -0.3 -0.5 +0.1 oral pediatric drops 1.000 10 -3 0.992×10 -3 0.991×10 -3 1. 002×10 -3 %rsd 1.420 0.630 1.272 %rc 99.2 99.1 100.2 %re -0.8 -0.9 0.2 syrup 1.000 10 -3 1. 003×10 -3 1. 001×10 -3 0.990×10 -3 %rsd 2.100 0.700 1.331 %rc 100.3 100.1 99.0 %re 0.3 0.1 -1.0 *each value represents an average of 3 measurements. iraqi j pharm sci, vol.21(1) 2012 polymeric membrane sensors and pharmaceutical analysis 76 a typical plot of antilog e/s versus the volume of the standard mcp together with the concentration of pure 10 -3 m of mcp is shown in figure 5. gran’s plot paper with 10% volume correction was used. for potentiometric titration of mcp hydrochloride, a typical titration plot of 10 -3 m mcp with 10 3 m pt is shown in figure 6. the values of standard deviation and recovery given in table 4 and 5 prove that the electrode depending on tbp plasticizers is very successful for the determination of mcp either in pure or pharmaceutical formulations. the good agreement between data obtained by the proposed sensor and the standard pharmacopial method (28) and the spectrophotometric method (8) confirming the applicability of the sensor for the analysis of controlled drugs. figure 5: plot of antilog (e/s) vs. the 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(conference paper )# hasan raid fadhil*, ali azeez al-jumaili*,1 and nizar abdulateef al ani ** # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq **rheumatology unit, department of medicine, college of medicine, university of baghdad, baghdad, iraq abstract the study objective was to conduct a pharmacoeconomic cost-effective analysis between infliximab reference (remicade) and its biosimilar (remsima) in patients with rheumatoid arthritis (ra) in two iraqi hospitals. this is a retrospective study (natural trial) ) that involved a prospective data collection phase as well in which data were collected from patients' medical records and face-to-face interviews from december 2021 to april 2022. the study included 57 patients who were categorized into two groups according to the type of infliximab they received. 27 patients received reference infliximab (remicade) and 30 patients received biosimilar infliximab (remsima). the two groups had comparable demographic and baseline disease parameters, with a mean age of 49.6 years and a bmi of 30.0. the vast majority of participants were women (82.5%) with a low level of formal education (65%). overall, both infliximab biopharmaceuticals had good effectiveness to reduce ra disease activity (cdai) and improve patient quality of life. they both had comparable adverse reactions including uti, fatigue, and headache. there was no significant difference (p-value >0.05) in disease activity between the two groups according to ra clinical disease activity index (cdai) score across all three-time series: before biological therapy, 14 weeks post-therapy, and 30 weeks post-therapy. in 2019, remicade was slightly linked with better quality of life, but costlier ($41,896 per qaly) than remsima. it was not clear whether the reference biologic (remicade) or its biosimilar (remsima) was more costeffective. in 2021, remicade was more cost-effective compared to remsima because remicade was less expensive and relatively more effective according to its measurement by cdai and eq-5d-5l scores. registering and purchasing both reference infliximab and its biosimilar is a good idea to keep the competition in price and maintain infliximab procurement for iraqi ra patients. keywords: cost-effectiveness, rheumatoid arthritis, infliximab, biosimilar, quality of life, iraq. ( remicade)الرميكيد المرجعي (infliximab) دواء االنفلكسيمابلـدراسة الجدوى االقتصادية ) بحث مؤتمر (# ( في مرضى التهاب المفاصل الرثويremsima) الرمسيما الحيوي بالبديل مقارنةً ** عبد اللطيف العاني و نزار 1،*علي عزيز الجميلي ،1حسن رائد فاضل 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي * العراق. بغداد، بغداد، جامعة الصيدلة،كلية السريرية، فرع الصيدلة العراق بغداد، بغداد، جامعة الطب، كلية الطب، قسم الروماتيزم، وحدة أمراض ** الخالصة )الريميكيد( وبديله الحيوي )الرمسيما( في مرضى المرجعي دواء االنفلكسيماب بيناالقتصادية جدوى دراسة المن هذه الدراسة هو اجراء الهدف . التهاب المفاصل الرثوي في المستشفيات العراقية المرجعي ابتم اجراء هذا التحليل لالقتصاد الدوائي في مستشفيتين تعليمية حكومية في بغداد العراق التي تحتوي على مراكز إلعطاء دواء االنفلكسيم كانون االول وبديله الحيوي لمرضى التهاب المفاصل الرثوي. تم جمع البيانات من السجالت الطبية للمريض والمقابالت الشخصية مع المرضى من شهر .٢٠٢٢الى شهر نيسان سنة ٢٠٢١سنة مريضا يعانون من التهاب المفاصل الرثوي. تم تصنيف المرضى الى مجموعتين وفقا لنوع االنفلكسيماب الي تلقوه خالل ٥٧شملت الدراسة ٣٠ .. وتلقى ٢٧اسبوع المرجعي)الريميكيد( االنفلكسيماب تلقى الحيوي ٣٠مريضا البديل مرضية ال)مريض صفات المجموعتين لدى كان رمسيما(. %( مع ٨٢.٥. كانت الغالبية العظمى من المشاركين من النساء )٣٠عاما ومعدل كتلة جسم يبلغ ٤٩.٦وديموغرافية واساسية متشابهة، بمتوسط عمر يبلغ جدا لتقليل نشاط مرض التهاب %(. بشكل عام كان كال من االنفلكسيماب المرجعي وبديل٦٥مستوى منخفض من التعليم الرسمي ) ه الحيوي فعالية جيدة والصداع. لم المفاصل الرثوي وتحسين نوعية حياة المريض. كان للدوائين اعراض جانبية غير مرغوبة مماثلة بما في ذلك التهاب المسالك البولية والتعب عند جميع نقاط القياس الثالثة .. قبل العالج البايولوجي (cdai) لسريرييكن هناك فرق كبير في نشاط المرض بين المجموعتين وفقا لمؤشر نشاط المرض ا ٤١،٨٩٦ولكنه اكثر كلفة جدا )ويوفر جودة حياة افضل كان الريميكيد اكثر فعالية قليال ٢٠١٩في عام .اسبوعا بعد العالج ٣٠اسبوعا بعد العالج و ١٤و لم يكن من الواضح ما اذا كان الدواء البيولوجي المرجعي )الريميكيد( او البديل الحيوي )الرمسيما( اكثر لذلك .( مقارنة بالرمسيما qalyدوالرا لكل اقل كان الريميكيد الن بالرمسيما مقارنة تصادية( )افضل جدوى اقكان الريميكيد اكثر فعاليه من حيث التكلفة ٢٠٢١فعالية من حيث التكلفة. في عام . حيث الفعالية وفقا لنتائج بقليل من تكلفة واكثر 1corresponding author e-mail: ali.baraak@copharm.uobaghdad.edu.iq received:15 /7 /2022 accepted:6 /10 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp100-110 iraqi j pharm sci, vol.31( suppl. ) 2022 cost-effectiveness analysis of infliximab 101 (. كان تسجيل وشراء كل من النوعين لدواء االنفلكسيماب له فكرة جيدة eq-5d-5lالحياة ) ( وجودةcdaiمؤشر نشاط المرض السريري ) للحفاظ على المنافسة في السعر والحفاظ على توفير عالج االنفلكسيماب لمرضى التهاب المفاصل الرثوي . جودة الحياة، الحيويالبديل ، دواء االنفلكسيماب، رثويالتهاب المفاصل ال, اقتصاديات الدواء المفتاحية:الكلمات introduction rheumatoid arthritis (ra) is a lifelong, systemic autoimmune inflammatory disease that can cause severe joint inflammation and ultimately joint damage (1). bone and cartilage deterioration and erosions are hallmarks of rheumatoid arthritis (ra), which is characterized by persistent inflammation of cartilaginous diarthrodial joints(2). pain, stiffness, and inflammation of peripheral joints are the primary symptoms of ra(3). it has a negative impact on patients’ quality of life and impairs their ability to carry out daily tasks (4). the global prevalence of ra is 0.46% (5). the prevalence of ra in iraq was 1% in 1978 and the incidence was 3% in 2011 (6,7). this disease is associated with considerable health and economic costs (8). the objective of treating ra is to diminish impairments in physical function and quality of life by obtaining remission or low disease activity (9). conventional synthetic disease-modifying antirheumatic drugs (csdmards) like methotrexate (mtx) are suggested as first-line therapy for ra (9). the use of biologic dmards (bdmards), such as a tumor necrosis factor inhibitor (tnfi), is advised by the european league against rheumatism (eular) for patients who do not respond adequately to conventional dmards (10). infliximab is a monoclonal antibody that inhibits tnfalpha and is used to treat rheumatoid arthritis and other autoimmune diseases (11). the development of targeted biological therapy for rheumatoid arthritis (ra) is one of the major achievements of contemporary medicine. this is especially true for rheumatoid arthritis (ra), (12). due to the complexity of biological medicine research and manufacturing processes, the prices of these medicines are quite expensive, and they create a significant strain on healthcare systems. furthermore, the lack of patient access to biological medicines has been a growing problem in several countries (13,14). numerous reference biological medicines are reaching patent expiration, prompting the development of so-called 'biosimilar' pharmaceuticals. a biosimilar is described as "a biotherapeutic product that is equivalent in quality, safety, and effectiveness to an existing licensed reference biotherapeutic product" (15). the introduction of biosimilars has resulted in price competition and a significant drop in the net prices of biological therapy (11,16). in various countries, biosimilar infliximab (remsima) has been approved for use in all indications approved for reference infliximab (remicade) including rheumatoid arthritis (17). remicade is manufactured and marketed by janssen biotech while remsima is developed by celltrion healthcare and commercialized by hikma pharmaceuticals. the common infliximab adrs include fatigue, rash, back pain, headache, nausea, infections (such as urinary tract infection and respiratory tract infection), infusion-related reaction, and dyspepsia (23). the state company for marketing drugs and medical appliance (kimadia), formed in 1964, is the organization in charge of procuring and distributing medications, medical appliances, and equipment for public health care settings (public hospitals and primary healthcare settings) throughout iraq (18). one of the main causes for the recent lack of important medications in iraq has been insufficient funding granted to kimadia (18). in order for biosimilars to be successfully included in treatment regimens in iraq, healthcare professionals and government decision-makers must possess an extensive understanding of biosimilars (19). due to the rising usage of infliximab and its biosimilar in iraq as a routine treatment for moderate-to-severe rheumatoid arthritis, their costeffectiveness must be evaluated. such a pharmacoeconomics study can provide more information and feedback to the ministry of health (moh) officials to assist them to approve and procure biopharmaceutical medicines in a way that saves money and enhances the patient clinical outcome. this is the first pharmacoeconomics study in iraq measuring the cost-effectiveness of reference biological medicine (infliximab) compared to its biosimilar. the study objective was to conduct a pharmacoeconomics study (cost-effective analysis) between reference infliximab reference (remicade) and its biosimilar (remsima) in patients with rheumatoid arthritis in iraqi hospitals. patients and method study design this is a retrospective multicenter study (natural trial) that involved a prospective data collection phase as well. the study was conducted at two large teaching governmental hospitals in baghdad, iraq (baghdad teaching hospital and alyarmouk teaching hospital), which normally provide infliximab to ra iraqi patients. data were collected from patients’ medical records and face-toface interviews with the patients from december 2021 to april 2022. patients and settings this study recruited a convenient sample of 57 adult outpatients who were diagnosed according to the iraqi medical practice which usually follows the american college of iraqi j pharm sci, vol.31( suppl. ) 2022 cost-effectiveness analysis of infliximab 102 rheumatology/european league against rheumatism (acr/eular) 2010 criteria (20). those ra patients who received their infliximab doses at baghdad teaching hospital or al-yarmouk teaching hospital: 30 patients received remsima and 27 patients received remicade. thus, the inclusion criteria were ra patients who received infliximab for at least 14 weeks and had a medical record with infliximab follow-up data. the exclusion criteria were: 1) patients with other autoimmune diseases; 2) cognitive impairment that prevents them from understanding or completing the questionnaires and data collections forms; 3) missing data from their medical records regarding disease activity scores from previous visits, and 4) patients who developed immunogenicity to infliximab and switched to another medication. the infliximab intravenous dose for the treatment of moderate to severe ra was 3-5 mg/kg and given as an induction regimen at 0, 2, and 6 weeks, followed by a maintenance regimen of 3-5 mg/kg every 8 weeks. according to the data that is currently available, the clinical response is often attained within three months after therapy initiation (21,22). the dose and type of infliximab given to the patients were determined by rheumatologists. each rheumatologist is responsible for a small number of patients. data collection data were collected from medical files using a previously prepared data collection form. additionally, demographic and clinical data were also collected directly from the patient. at the time of approaching the patient, the interview, demographic information (age, gender, weight, height, disease duration, smoking, type of occupation, and prior biological medicines used for ra) was collected. the frequency of drug adverse reactions (adrs) was evaluated by asking the patients to report any adrs that they suffered after adding infliximab to the treatment regimen. before each dose, the attending physician assessed the infliximab efficacy using the rheumatoid arthritis clinical disease activity index (cdai) and noted it in the patient's medical records. the cdai score ranged from 0 to 76. the cdai was calculated using the following formula: cdai = tjc + sjc + pdga + edga. the tjc is the tender joint count of 28 joints (0-28). the sjc is the swollen joint count of 28 joints (0-28) the pdga is the patient disease global assessment of disease activity on a visual analog scale (vas)(0–10). the edga is the evaluator/physician disease global assessment of disease activity on a visual analog scale (0–10) (23). remission per cdai is defined as a score < 2.8; low disease activity is when the cdai score equals 2.8– 10; moderate disease activity is with a score of 10– 22; high disease activity is when the score > 22 (24). to measure the clinical response in term of cdai score (cdai improvement), the following formula was used (cdai improvement = cdai week 0 – cdai week x). the eq-5d-5l was utilized to assess the patient's quality of life (qol) after receiving approval from the european euroqol group's foundation. a validated arabic version of the eq5d-5l was used to conduct in-person interviews with ra patients and assess their qol at two points. the first point is before starting infliximab treatment which is measured retrospectively (recall qol) and the second point is at present (visit) time. the eq5d-5l is a qol tool that has been implemented across a variety of illness areas. it consists of the five dimensions: mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. each dimension has five possible responses (no problems, mild problems, moderate problems, severe problems, and extreme problems). answers are displayed as a single-digit number between 1 and 5 for each dimension, indicating the chosen severity level (25). by using the eq-5d-5l index value set for the general population of zimbabwe, we were able to convert the health status reported in the eq5d-5l questionnaire into a utility value (26). the researchers hypothesized that among the nine nations for which a value index is available, zimbabwe's living conditions are more comparable to those of iraq. the utility is a single numerical value, often between 0 and 1. that reflects the individual's health-related qol at a given moment (27). the utility value is used to calculate qualityadjusted life years (qaly) which is used as an outcome measure in our cost-effectiveness analysis. qaly was calculated using the following formula (qaly= (utility before treatment – utility after treatment) * duration of time spent in health state). the costs of the reference infliximab (remicade) and its biosimilar (remsima) for the years (2019 & 2021) were obtained from the kimadia website (28). after data collection, the direct cost of infliximab was determined. the costeffectiveness analysis was conducted from a payer (iraqi ministry of health) perspective. the incremental cost-effectiveness ratio (icer) was calculated by dividing the difference in total costs (incremental cost) of two medications by the difference in the chosen measure of health outcome (incremental effect) between the two medications using this equation (𝑰𝑪𝑬𝑹 = (𝑪𝒐𝒔𝒕 𝑹𝒆𝒎𝒊𝒄𝒂𝒅𝒆 − 𝑪𝒐𝒔𝒕 𝑹𝒆𝒎𝒔𝒊𝒎𝒂)/ (𝑶𝒖𝒕𝒄𝒐𝒎𝒆 𝑹𝒆𝒎𝒊𝒄𝒂𝒅𝒆 − 𝑶𝒖𝒕𝒄𝒐𝒎𝒆 𝑹𝒆𝒎𝒔𝒊𝒎𝒂)) (29). the icer result is the 'extra cost per extra unit of health effect'. in this study, the icer was calculated by two health outcomes. the first one is cdai improvement after 14 weeks of therapy initiation, and the second one is quality-adjusted life years (qaly). iraqi j pharm sci, vol.31( suppl. ) 2022 cost-effectiveness analysis of infliximab 103 the ethical approval of the study proposal was obtained from the university of baghdad college of pharmacy and the two participating hospitals. also, verbal approval was obtained from the patients before the face-to-face interview. statistical analysis descriptive statistics (means, standard deviation, frequencies, and percentages) were conducted for all study items. data were analyzed using statistical package for the social sciences (spss) software version 25. since most variables were not normally distributed, we used nonparametric tests to measure the difference between the two groups and when multiple measures of each group. the mann-whitney test was used to measure the differences in cdai, utility, and qol measures between the two infliximab groups. pearson chisquare was used to measure the association between the infliximab adverse reactions and the type of infliximab used. friedman's test and pairwise comparisons were used to measure the difference across the three measures of cdai (baseline, 14 weeks, and 30 weeks post-treatment) within each group. the wilcoxon signed ranks test was used to measure the difference in the utility, and qol domains preand post-treatment within each group. a p-value of less than 0.05 was considered statistically significant. results the study sample was 57 patients with rheumatoid arthritis (ra). the patients were categorized by the rheumatologists into two groups according to the type of infliximab they received for more than 14 weeks: 27 patients received reference infliximab (remicade) and 30 patients received biosimilar infliximab (remsima). the two groups had comparable demographic and baseline disease parameters, with a mean age of 49.6 years and a bmi of 30.0. the vast majority of participants were women (82.5%) with a low level of formal education (65%) (table 1). table 1. the characteristics of the participating rheumatoid arthritis patients parameter all (n=57) mean (std. dev) remicade (n=27) mean (std. dev) remsima (n=30) mean (std. dev) age (years) 49.58 (10.25) 49.59 (11.29) 49.57 (9.42) weight (kg) 79.14 (16.93) 81.63 (16.05) 76.90 (17.65) height (cm) 162.12 (8.78) 163.63 (6.93) 160.77 (10.10) bmi * 29.99 (5.47) 30.38 (5.05) 29.65 (5.88) total dose (mg) 308.70 (78.56) 296.30 (80.77) 320.00 (76.11) dose (mg/kg) 3.99 (0.99) 3.71 (1.07) 4.24 (0.84) n (%) n (%) n (%) gender • male • female 10.0 (17.50) 47.0 (82.50) 6.0 (22.20) 21.0 (77.80) 4.0 (13.30) 26.0 (86.70) smokers 6.0 (10.55) 1.0 (3.70) 5.0 (16.70) occupation • sedentary • physical 51.0 (89.50) 6.0 (10.50) 24.0 (88.90) 3.0 (11.10) 27.0 (90.00) 3.0 (10.00) education level • illiterate or primary • secondary • graduated 37.0 (64.90) 13.0 (22.80) 7.0 (12.30) 15.0 (55.60) 7.0 (25.90) 5.0 (18.50) 22.0 (73.30) 6.0 (20.00) 2.0 (6.70) *bmi = body mass index the adverse drug reactions with the highest prevalence were fatigue (26.3%), nausea (22.80%), headache (17.50%), and urinary tract infection (uti) (29.90%) (table 2). the remsima-containing treatment regimen was associated with a significantly higher incidence of nausea (76.9%) compared to the remicade-containing treatment regimen group (23.2%). on the other hand, there was no significant association (p-value ˃ 0.05) between the type of infliximab-containing regimen (reference vs biosimilar) regarding the incidence of other main three adverse reactions (headache, fatigue, and uti) (table 2). iraqi j pharm sci, vol.31( suppl. ) 2022 cost-effectiveness analysis of infliximab 104 table 2. the adverse reactions of infliximab-containing treatment regimen in the participating ra patients. adverse reaction all (n=57) (%) remicade (n=27) (%) remsima (n=30) (%) pvalue nausea 13.0 (22.80) 3.0 (11.10) 10.0 (33.30) .046* headache 10.0 (17.50) 3.0 (11.10) 7.0 (23.30) .304 fatigue • mild • moderate severe 11.0 (19.30) 4.0 (7.00) 4.0 (14.80) 1.0 (3.70) 7.0 (23.30) 3.0 (10.00) .241 urinary tract infection • mild • moderate severe 3.0 (5.30) 14.0 (24.60) 1.0 (3.70) 6.0 (22.20) 2.0 (6.70) 8.0 (26.70) .576 allergic reaction 8.0 (14.00) 4.0 (14.80) 4.0 (13.30) na rash 3.0 (5.30) 1.0 (3.70) 2.0 (6.70) na dyspepsia 3.0 (5.30) 1.0 (3.70) 2.0 (6.70) na respiratory tract infection 2.0 (3.50) 0.0 (0.00) 2.0 (6.70) na *significant (p-value <0.05) according to pearson chi-square. na=not applicable due to the small sample. the majority of patients (71.9%) were new to biological disease-modifying antirheumatic drugs (dmards), with comparable percentages between the remicade (74.1%) and remsima (70.0%) groups. other rheumatoid arthritis drugs were also used as concurrent treatments, such as methotrexate and non-steroidal anti-inflammatory drugs (nsaids). more than half of the ra patients were taking methotrexate concomitantly with infliximab (59.30% of the remicade group and 63.30% of the remsima group) (table 3). table 3. the medication history of the participating ra patients other ra medicines all, n=57 (%) remicade, n=27 (%) remsima, n=30 (%) methotrexate use 35.0 (61.40) 16.0 (59.30) 19.0 (63.30) nsaid* drug use 27.0 (47.40) 11.0 (40.70) 16.0 (53.30) corticosteroids drug use 22.0 (38.60) 9.0 (33.30) 13.0 (43.30) infliximab treatment duration • patients reached 14 weeks • patients reached 30 weeks 57.0 (100.00) 41.0 (71.90) 27.0 (100.00) 24.0 (88.89) 30.0 (100.00) 17.0 (56.67) previous biological therapy • no previous biological therapy • etanercept • adalimumab • rituximab 41.0 (71.90) 16.0 (28.10) 4.0 (7.00) 2.0 (3.50) 20.0 (74.10) 7.0 (25.90) 3.0 (11.10) 2.0 (7.40) 21.0 (70.00) 9.0 (30.00) 1.0 (3.30) 0.0 (0.00) other medications • leflunomide • pregabalin • ssz* • hcq* 5.0 (8.80) 2.0 (3.50) 2.0 (3.50) 8.0 (14.00) 2.0 (7.40) 2.0 (7.40) 0.0 (0.00) 4.0 (14.81) 3.0 (10.00) 0.0 (0.00) 2.0 (3.50) 4.0 (13.33) *nsaids=non-steroidal anti-inflammatory drugs. ssz =sulfasalazine. hcq =hydroxychloroquine. there was no significant difference (pvalue > .05) in disease activity between the two groups according to ra clinical disease activity index (cdai) score across all three-time series: before biological therapy , 14 weeks post-therapy, and 30 weeks post-therapy. the differences between the two groups in terms of the improvements in cdai score were not statistically significant (pvalue >0.05) after 14 weeks of infliximab therapy initiation. iraqi j pharm sci, vol.31( suppl. ) 2022 cost-effectiveness analysis of infliximab 105 there were no significant differences (pvalue > 0.05) in the quality-of-life dimensions and utility of patients between the treatment groups before starting infliximab therapy (table 4). but after at least 14 weeks of infliximab therapy, the two groups had significant differences according to two qol measures (pain and anxiety/depression), utility, and duration of disease. in other words, the utility in the remicade group was significantly (p-value <0.05) higher than that of the remsima group after treatment (table 4). additionally, the two negative dimensions of eq-5d-5l qol (pain/discomfort and anxiety/depression) were significantly (p-value <0.05) lower in the remicade group than that in the remsima group (table 4) table 4. the difference in the quality-of-life dimensions and utility measures between the groups before treatment and at least 14 weeks after treatment. before treatment after ≥14 weeks of treatment. qol/utility measures infliximab type n mean rank p-value mean rank p-value. mobility limitation remicade 27 29.22 .916 27.22 .425 remsima 30 28.80 30.60 self-care limitation remicade 27 25.15 .073 25.76 .128 remsima 30 32.47 31.92 usual activities limitation remicade 27 27.07 .358 25.63 .134 remsima 30 30.73 32.03 pain/ discomfort remicade 27 29.07 .970 23.33 .011* remsima 30 28.93 34.10 anxiety/ depression remicade 27 27.74 .564 23.70 .017* remsima 30 30.13 33.77 utility remicade 27 30.96 .369 34.02 .030* remsima 30 27.23 24.48 *significant (p-value <0.05) according to mann-whitney test. after 14 weeks and 30 weeks of receiving remsima or remicade therapy, the cdai scores have significantly improved (p-value <0.05) compared to the baseline (before treatment). however, the cdai score did not improve significantly (p-value >0.05) between 14 weeks and 30 weeks post-remsima or remicade therapy (table 5a&b) (table 6 a&b)). table 5-a. the difference in cdai scores across three measures before (baseline) and after remsima treatment (14 weeks and 30 weeks post-therapy). remsima -null hypothesis test p-value the distributions of cdai 0 time, cdai 14 weeks and cdai 30 weeks are the same. related-samples friedman's two-way analysis of variance by ranks .001* *significant (p-value <0.05) according to friedman's test table 5-b. pairwise comparisons sample 1-sample 2 test statistic std. error std. test statistic p-value adj. pvalue cdai 30 weeks cdai 14 weeks .056 .33 .16 .868 1.000 cdai 30 weeks cdai 0 time 1.11 .33 3.33 .001 .003** cdai 14 weeks cdai 0 time 1.05 .33 3.16 .002 .005** ** significant (p-value <0.05) according to pairwise comparisons test. iraqi j pharm sci, vol.31( suppl. ) 2022 cost-effectiveness analysis of infliximab 106 table 6-a. the difference in cdai scores across three measures before (baseline) and after remicade treatment (14 weeks and 30 weeks post-therapy). remicade-null hypothesis test p-value the distributions of cdai 0 time, cdai 14 weeks and cdai 30 weeks are the same. related-samples friedman's two-way analysis of variance by ranks .000* *significant (p-value <0.05) according to friedman's test table 6-b. pairwise comparisons sample 1-sample 2 test statistic std. error std. test statistic pvalue adj. pvalue cdai 30 weeks cdai 14 weeks .10 .28 .36 .718 1.000 cdai 30 weeks cdai 0 time 1.14 .28 3.96 .000 .000* cdai 14 weeks cdai 0 time 1.04 .28 3.60 .000 .001* *significant (p-value <0.05) according to pairwise comparisons the ra patients had a high level of limitations (4.27 to 4.4 out of 5) in their physical activities (mobility, self-care, usual activities) in addition to a high level of pain/discomfort and anxiety/depression (3.9 and 4.53 out of 5) before remsima therapy (table 7). before remicade therapy, the ra patients had a high level of limitations (3.85 to 4.37 out of 5) in their physical activities (mobility, self-care, usual activities), in addition to a high level of pain/discomfort and anxiety/depression (3.78 and 4.56 out of 5) (table 7). these qol dimensions, utility, and vas scores have been significantly improved (p-value <0.05) after at least 14 weeks of receiving remsima and remicade (tables 7). table 7. the measures of utility and qol dimensions of the ra patients before and after remsima or remicade use. qol dimensions remsima remicade n mean std. deviatio n n mean std. deviation mobility limitation before treatment 30 4.27 .90 27 4.37 .62 self-care limitation before treatment 30 4.37 .89 27 3.85 1.23 usual activities limitation before treatment 30 4.40 .89 27 4.26 .81 pain/discomfort before treatment 30 4.53 .62 27 4.56 .57 anxiety/depression before treatment 30 3.90 1.39 27 3.78 1.28 utility before treatment 30 .140 .226 27 .189 .19 mobility limitation after treatment 30 2.70 1.055 27 2.41 1.24 self-care limitation after treatment 30 2.37 1.47 27 1.74 1.05 usual activities limitation after treatment 30 2.93 1.33 27 2.41 1.21 pain/discomfort after treatment 30 3.03 .85 27 2.37 1.21 anxiety/depression after treatment 30 2.83 1.28 27 2.04 1.25 utility after treatment 30 .576 .216 27 .683 .181 according to the listed prices in 2019, remicade yielded slightly higher qalys and slightly higher disease activity (cdai) improvement, but with a higher cost. the incremental cost-effectiveness ratio (icer) for remicade was $41,896 to gain one qaly per patient and $405 to reduce one cdai for one patient (per 14 weeks). after reducing the cost of remicade in 2021, remicade was dominant/more cost-effective compared to remsima because it yielded slightly higher qalys and slightly higher (cdai) improvement with lower cost (table 10) (table 11). iraqi j pharm sci, vol.31( suppl. ) 2022 cost-effectiveness analysis of infliximab 107 table 10. cost and benefit of infliximab. cost and benefit of infliximab cost/outcome remicade remsima difference between remicade and remsima cost in 2019 in usd ($) • one vial cost (direct cost) • total cost per patient per first 14 weeks • total cost per patient per 1 year $ 405 $ 3,645 $ 10,935 $ 315 $ 2,835 $ 8,505 $ 90 $ 810 $ 2,430 cost in 2021 in usd ($) • one vial cost (direct cost) • total cost per patient per first 14 weeks • total cost per patient per 1 year $ 148 $ 1,332 $ 3,996 $ 200 $ 1,800 $ 5,400 $ -52 $ -468 $ -1404 mean cdai improvement in the first 14 weeks 11.1 9.1 2 mean qaly gained in 1 year (utility improvement) 0.494 0.436 0.058 no. of visits in: • first 14 weeks • 1 year 3 9 3 9 table 11. the incremental cost-effectiveness ratio (icer) of remicade to remsima. incremental cost-effectiveness ratio cost per one cdai unit in 14 weeks cost per one qaly icer 2019 $ 405 $ 41,896 discussion the majority of participants were illiterate women with an overweight bmi. the ra incidence is higher in women than in men due to sex hormones’ role in which estrogens can boost specific immune responses (30). a higher bmi was connected with a higher incidence of rheumatoid arthritis (31). in this study, the most common adverse drug reactions (adrs) of the infliximab-containing regimen were urinary tract infection (uti), fatigue, nausea, and headache. the incidence of adrs was comparable in both groups except for nausea, which occurred significantly higher in the remsima group. uti was the most common adr (29.9%) in the participating patients. an american study concluded that infliximab and its biosimilar have a similar safety profile (32). infliximab seems to have the highest proportion of adverse drug reactions (23%) reported to the iraqi pharmacovigilance center (iqphvc) compared to other biological therapy. (33). nearly all participants (50 out of 57) used concurrent medications with infliximab therapy, including conventional synthetic disease-modifying antirheumatic drugs (csdmards) such as methotrexate (mtx), leflunomide, hydroxychloroquine (hcq), and sulfasalazine (ssz). josef s smolen et al., (2020) mentioned that biological disease-modifying antirheumatic drugs (bdmard) should be combined with csdmard for ra patients to increase their efficacy (10). according to registry data, about 30% of patients receiving biological therapy do not receive a concomitant dmard (34). in our study, the majority of ra patients used mtx as a concurrent medication with infliximab therapy. mtx remains the pivotal drug in the ra therapeutics regimen (10). additionally, nsaids were used occasionally as analgesic and anti-inflammatory for a short period for pain management. the majority of the participating patients were naïve to biological therapy before receiving infliximab, but some of them had received one or two different biological therapy before switching to infliximab. this may be due to a lack of response or availability issues. a previous study mentioned that if one biologic dmard has failed, adding another biologic dmard to the treatment regimen should be considered (10). iraqi j pharm sci, vol.31( suppl. ) 2022 cost-effectiveness analysis of infliximab 108 after 14 weeks of therapy, the disease activity (as measured by the cdai score) decreased significantly from baseline and remained relatively constant thereafter in both groups. although there was a higher cdai score improvement in the remicade group compared to the remsima group, this improvement was not statistically significant. this result was comparable with the findings of earlier swedish and multinational studies (35,36). the eq-5d-5l questionnaire was used to evaluate qol (utility) in order to compare the two groups and conduct a cost-effectiveness analysis. because of its uniformity, patient acceptance, and well-established utility, the eq-5d-5l was selected as the utility outcome (35,36). all the participating ra patients (in both groups) had a very low qol before starting infliximab treatment. it is well known that ra has a significant influence on qol and functional performance, with qol being significantly worse compared to the general population (37). the use of biological infliximab for 14 weeks resulted in a significant improvement in the qol of patients with ra. then qol remained relatively stable for both groups thereafter. a previous study found an enhancement in qol after receiving infliximab (35). remicade was related to a somewhat greater increase in qol than remsima, although the difference between the two groups was not statistically significant. we considered the direct infliximab costs in both 2019 and 2021 since the only cost available at the beginning of the study was 2019 then at the end of the study, the costs changed in 2021. according to the 2019 price list obtained from the kimadia website, the total cost of infliximab per patient for 14 weeks (3 doses) was $ 3,645 for remicade and $ 2,835 for remsima. and the oneyear total cost (9 doses) was ($ 10,935) for remicade and ($ 8,505) for remsima. the remicade yielded the icer of $ 405 per one unit reduction of cdai in 14 weeks and $ 41,896 per qaly. in 2019, remicade had a higher cost and slightly higher improvement in cdai and qol compared to remsima. in other words, remicade was more effective but more expensive. because the iraqi ministry of health (moh) did not have a specific willingness to pay per qaly, we could not decide which one was more cost-effective. however, if we can assume that willingness to pay is three times the gross domestic product (gdp) per person, iraqi moh's willingness to pay can be about $15,000 per qaly (38). in this scenario, remicade was not cost-effective in 2019 compared to remsima. in 2021, there was a reduction in the costs of both remicade and remsima according to the price list obtained from the kimadia website. the total cost of infliximab per patient for 14 weeks (3 doses) was $ 1,332 for remicade and $ 1,800 for remsima. the one-year total cost (9 doses) was ($ 3,996) for remicade and ($ 5400) for remsima. in 2021, the remicade was dominant (more costeffective) because it had a lower price and slightly higher improvement in cdai and qalys. the study had some limitations. the study covered two centers in one province (baghdad) and recruited a relatively small sample size with a follow-up period of 30 weeks. conclusion in 2019, remicade was slightly more effective and provide a better qol, but was costlier ($41,896 per qaly) compared to remsima. because the iraqi moh did not have a specific willingness to pay per qaly, it was not clear whether the reference biologic (remicade) or its biosimilar (remsima) was more cost-effective in patients with ra. however, if we can assume that willingness to pay is three times the gdp per person, iraqi moh's willingness to pay can be about $15,000 per qaly. in this scenario, remicade was not cost-effective in 2019 compared to remsima. in 2021, remicade was more cost-effective compared to remsima because remicade was less expensive and relatively more effective according to cdai and eq-5d-5l scores. overall, both infliximab biopharmaceuticals had good effectiveness in reducing ra disease activity (cdai) and improving patient qol. they both had comparable adverse reactions, including uti, fatigue, and headache. registering and purchasing both reference infliximab and its biosimilar was a good idea to keep the competition in price and maintain infliximab procurement for ra patients. acknowledgment the authors would like to thank the medical staff and administrations of both rheumatology units at baghdad hospital and al-yarmouk hospital for their collaborations. references 1. smolen js, aletaha d, barton a, burmester gr, emery p, firestein gs, et al. rheumatoid arthritis. nat rev dis prim. 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[cited 2022 jul 12]. available from: https://www.worldbank.org/en/home. this work is licensed under a creative commons attribution 4.0 international license http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(suppl.) 2022 exploring iraqi herbalists knowledge and practice doi: https://doi.org/10.31351/vol31isssuppl.pp178-187 178 a cross-sectional survey of iraqi herbalist practicing in the middle euphrates area with a recognition of their knowledge, practice and attitude(conference paper )# suhad s. humadi*,1, saif m. hassan*, salam w. ahjel * # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of pharmacy , al-zahrawi university college , karbala ,iraq. abstract high percentage of the iraqi population profoundly rely on folk medicine to meet their health needs which makes their safety questionable. this study aims to evaluate iraqi herbalists' knowledge and practice to ensure the public's safety. this study was conducted in a cross-sectional design between october 2021 and march 2022, using a pretested questionnaire administered to iraqi herbalists practicing in middle euphrates area. through face-to-face meetings, participants completed a multicomponent questionnaire comprising 15 items in four sections. the data obtained were analyzed using a statistical package for social sciences; chi-square was used to correlate some variables, and p-values of <0.05 were considered significant. a total of 54 male herbalists from five iraqi provinces joined this survey, the majority practicing in kabala. most herbalists had 1020 years of experience, and more than 50% had a university degree. data showed that 72.2% of herbalists identify their herbal items using their own experience, and 35.2% use this experience as their sole source of knowledge. only 35.1% use herbal books in conjugation with their experiences, and a few (1.9%( use multiple sources of information. herbalists with more years of experience (79.6%( evaluate patient conditions properly, follow up (40.7%(, and refer patients to their physicians when needed (42.6%(. although fifty percent of herbalists educate their patients regarding the storage condition of remedies, most of them refrain from writing the complete ingredients on their final product regardless of their experience. results also showed that most herbalists do not have a record tracking adverse reaction. most iraqi herbalists lack the proper system for prescribing and dispensing their remedies and adequately identifying the sold herbs. the study showed a variation in practice among herbalists using approaches based mainly on their experience. keywords: herbalism, herbalists, herbal remedies, practice survey, clients دراسة المسح المقطعي للعشابين العراقيين في منطقة الفرات االوسط والتعرف على مهاراتهم المعرفية #مؤتمر( )بحث والسلوكية سهاد سامي حمادي*،1، سيف حسن* وسالم عاجل* 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي العراق. الصيدلة، كربالء، الجامعة، قسمكلية الزهراوي * الخالصة سالمتهم في محل تساؤل عن مدى اهلية الصحية مما يجعل احتياجاتهم تعتمد نسبة عالية من سكان العراق اعتمادا كبيرا على الطب الشعبي لتلبية تشرين نفي الفترة ما بيتصميم مقطعي بأجريت هذه الدراسة هذه الدراسة لتقييم معرفة واداء العشابين لضمان سالمة الجمهور. هنا تأتي أهمية نالعشابين. وم هذا االستبيان اعطاءتم .في أربعة أقسام مبوبا سؤاال خمسة عشرمن مؤلف فقرات,متعدد الشامل و باستخدام استبيان , ٢٠٢٢ شهر آذارالى ٢٠٢١ االول تم باإلضافة الى ذلك .spssالنظام االحصائي باستخدام المستحصلة احصائيا تحليل البيانات األوسط ومن ثمفي منطقة الفرات ارسي الطب العشبي لمم نتائج االستبيان استحصلت من وذات داللة احصائية . مهمة ٠,٠5 البالغة اقل من القيمة االحتماليةاستخدام مربع كاي لربط بعض المتغيرات، واعتبرت نية تتراوح عشابا في خمس محافظات عراقية مختلفة و أظهرت النتائج فيها ان العشابين هم من الذكور واغلبهم يمارسون العمل في كربالء وخبرتهم المه 5٤ بالمئة من العشابين يستدلون على اعشابهم ٧٢.٢كما واظهرت البيانات ان ية.على شهادة جامع لينحاصبالمئة منهم 5٠أكثر من عاما علما ان ٢٠و ١٠بين بالمئة يعتمدون على هذه الخبرة كمنبع اساسي للمعرفة. ٣5.٢الطبية من خالل الخبرة المهنية الذاتية دون االعتماد على المصادر العلمية الموثوقة حيث ان بالمئة ١.٩تخدمون المراجع العلمية الخاصة باألعشاب باإلضافة الى الخبرة المهنية في ممارساتهم التطبيقية ويس بالمئة ٣5.١ واثبتت هذه الدراسة ايضا ان ( يقومون بالتقييم الصحيح لحاالت ٪ ٧٩,٦فقط هم الذين يستخدمون المصادر المختلفة للمعرفة. ومن الجدير بالذكر أن العشابين ذوي أعوام الخبرة الطويلة ) ( من العشابين يقومون بإحالة الحاالت المستعصية لألطباء االختصاص. ٤٢.٦٪ونسبة ) متابعة تطور أوضاعهم الصحية ( يقومون ب ٪ ٤٠,٧ المرضى وان ) واس العشبية المواد خزن بطرق يتعلق فيما زبائنهم بتثقيف يقومون العشابين من بالمئة خمسين أن من الرغم على انه ايضا الدراسة تخدامها واوضحت أظهرت النتائج أيضا أن معظم سنوات الخبرة و النهائي بغض النظر عن العشبي منتجهم يمتنعون عن كتابة المكونات الكاملة على الصحيح، اال ان معظمهم المعطاة. لألعراض الجانبية للمنتجات العشبية خاص ليس لديهم سجل تتبع العشابين 1corresponding author e-mail: suhadsami04@yahoo.com received: 14/5/2022 accepted: 4/ 12/2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp178-187 iraqi j pharm sci, vol.31(suppl.) 2022 exploring iraqi herbalists knowledge and practice 179 صحيح المباعة بشكل األعشابوتحديد عالجاتهم العشبيةالعراقيين إلى النظام المناسب لوصف وصرف العشابين أغلب يستخلص من هذه الدراسة افتقار العشابين في ممارساتهم التطبيقية والمبنية بشكل اساسي على الخبرة الذاتية المكتسبةفي الممارسة بين واضحا تباينا ايضا الحالية . أظهرت الدراسةودقيق زبائن ، العشابين ، استبيان شامل ، الطب الشعبي الكلمات المفتاحية: introduction herbalism is a folk medicine practice using plants and plant extracts to treat ailments (1) .the use of herbal medicine maintains to grow globally, with an estimated 80% of the population relying on these products to treat different health disorders in various national healthcare settings (2-5).in the mediterranean region, consumers highly utilize herbal medicine, with many studies stating high percentages (6) .in the gulf region, 43.2-76.0% of the population claims to use herbal medicine regularly (7) .studies in iraq show a 71.6-76.4% use of herbal products among patients (8-10) .the widespread use of herbal products may be due to cultural acceptability, accessibility, or cost affordability (11-13) . historically, herbalists have been the source of supplying herbal remedies. several ethnobotanical studies have been performed in iraq to investigate medicinal plants used by local herbalists in managing different diseases (1417) .however, limited studies were conducted to evaluate herbalists' practice and knowledge in this field (16) .researchers observed that most traditional herbalists practice their healthcare services illegally and are not officially identified by the authorities in many countries (18,19) .the incorrect use of traditional practices can lead to severe consequences; thus, an investigation needs to determine the efficacy and safety of such practices. our analysis is the first survey performed in southern iraq to investigate herbalists' knowledge and practices. the primary aim of this study is to evaluate iraqi herbalists' knowledge and practice to ensure their client's safety, performed through a crosssectional structured interviewer-administered questionnaire using bloom's cut-off categories to measure the knowledge and practice scores (20-22) .questions were chosen after reviewing studies in this field and modified to serve the aim of this study (16,32) .the secondary aim is to provide the baseline data for researchers for further investigations regarding herbalist practice, thus generating a proper model for herbalist practice. materials and methods study design the study is a cross-sectional questionbased survey of herbalists practicing in the iraqi southern area. the survey took place between october 2021 to march 2022. the chosen area for this study is the middle euphrates area, denoted by five governorates: karbala, al-najaf, babylon (hilla), al-muthanna, and al-qadisiya. this area is recognized for its cultural and religious contributions that nourished herbalist knowledge , especially during religious rituals. for the two governorates, al-muthanna and al-qadisiya, we chose the biggest cities represented by al-samawah and aldiwaniyah, respectively. data collection data collection took place in the selected herbalist’s shops through face-to-face interviews with the herbalist. each interview lasted about 2030 minutes, and consent was implied upon agreement to complete the questionnaire. ethical approval for the study was achieved through the ethical board at al zahrawi university college (azuc ref no.174/09/2021). the sample size was estimated according to the following formula n=(z1-α( 2×p(1−p(/d2 where n=number to sample. z1-α is a standard normal variation (at p<0.05 = 1.96 (, p is the excepted proportion of herbalists in the population depending on previous studies set to 50% as no available data in the literature for the actual proportion of herbalists or registered herbalists shop (23,24,32) d is the maximum tolerable error for the prevalence estimate (e.g., ±0.05). questionnaire the form used in this study was created following a complete revision of relevant literature and modified to fit the aim of this study (6,14,16). it mainly contained closed-ended questions of 15 items and was divided into four parts. the questions were formulated to detect the system followed by herbalists prescribing and dispensing herbal remedies to their patients and the source of information used in their practice. part a consisted of five questions about demographic data (gender, age, level of education, years of experience, and the location of the herbalist's shop). part b of the questionnaire consisted of 2 questions to identify the sources of used herbs and identity recognition of these plants. part c of the questionnaire had 7-questions to detect the herbalist's practices concerning herbal remedies. it includes evaluating the patient's chronic disease and allergies before dispensing remedies, advising the patient on the safe use and storage conditions for the product, listing all ingredients on the final product administered, reporting any adverse effects, referring to their physician or pharmacist whenever necessary, and monitoring the progress of the given treatment. part d of the survey addressed the herbalists' knowledge used in their job with questions of multiple choices about their source of information. they were advised to choose from the https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4020364/#b4 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4020364/#b4 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4020364/#b4 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4020364/#b4 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4020364/#b4 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4020364/#b4 iraqi j pharm sci, vol.31(suppl.) 2022 exploring iraqi herbalists knowledge and practice 180 three to four answer options listed in the form and to add other sources if not mentioned. bloom's cut-off categories were used to measure the knowledge and practice scores. for the knowledge score component, two questions with multiple choice options were asked, identifying the source of information used to assess patient condition and identifying herbs given to clients. several answer statement options were used, a positive statement was given one point, whereas a negative statement was given zero. total points were added and calculated to measure the total score value. the original bloom’s cut-off points represented as 80.0%–100.0%, 60.0%–79.0%, and <59.0% were modified from the kap study conducted on thalassemia among indonesian youth (21) they were applied to categorize kp (knowledge and practice( into three levels. the scores for knowledge varied from 0 to 7 points and were grouped into three levels as follows: high knowledge level: 6 to 7 scores; moderate level: 4-5 scores; and low level: 0 to 3. for the practice component, a total score based on the answers to seven questions on a fivepoint likert scale (5=always; 4=usually; 3=often; 2=rarely,1= never (was calculated. the total points given corresponded to practice were 35. a practice score from 28 to 35 indicated good practice, whereas a score from 21 to 27 indicated fair practice. furthermore, a score of less than 20 indicated poor practice (21,23) the inclusion criteria were adults aged 20 years or older practicing traditional medicine in a herbalist shop, and the exclusion criteria were the incomplete form and younger ages. the questionnaire form was validated regarding content rationality by three specialists in the pharmacognosy field and translated to arabic with the aid of 2 native speakers with english linguistic specialty and a ph.d. in pharmaceutical science. the translation process involved several steps (25): • preparation of the original form by authors who had different fields of experience • forward translation to the target language by a native speaker with a ph.d. in english linguistics • back translation with a different translator to investigate any discrepancies • harmonization to prevent any deviations of meaning • cognitive debriefing, and testing on two herbalists to ensure clearance and understandability, then the final approval of the form one of the obstacles faced in the pilot study was the translation of adverbs of frequencies used to determine herbalist practice utilizing the 5-point likert rating scale (always, usually, often, rarely, never). this problem was overcome using percent numbers to determine the difference between the used adverbs and to ease the herbalist's understanding of the meaning of these words. statistical analysis statistical software for social sciences was used to analyze the studied data (spss version 26, chicago us ). age, education, and experience proportions were estimated using descriptive statistics. the chi-square test was utilized to estimate the statistical significance of differences in proportion types between the groups, and a p-value of less than 0.05 was considered significant. results a sum of 85 herbalists was approached; there wasn’t official statistical data available on the number of registered herbalists' shops in the studied area and this was also noted in other studies (32), only 54 responded to the survey giving a response rate of 63.5 %. most herbalists did not agree to release information due to competition and safety issues that brought this participation rate. a total of 54 male herbalists from five different iraqi provinces participated in this study. interestingly, womenfolk medicine was not noticed or observed during this survey. for the score of kp (knowledge and practice), it was noted that the majority of herbalists (94.4%) had poor knowledge, and 35.1% had poor practice as shown in tables 2 and 3. table 2. distribution of knowledge scores using bloom's cut-off categories. level n (54) percentage high knowledge level (6-7) zero zero moderate level knowledge (4-5) 3 5.5 % poor level knowledge (0-3) 51 94.4% the minimum scores point zero, maximum score point 7 table 3. distribution of practice scores using bloom's cut-off categories level n (54) percentage good practice (2835) 18 33.3% fair practice (22-27) 17 31.4% poor practice (0-21) 19 35.1% minimum scores point 11, maximum score points 35 data showed that most herbalists (38.9%) were recorded in karbala, followed by babylon province (29.6%) and other provinces with the lowest percentage. nearly half were young adults (48.1%)and middle-aged (42.6%). amusingly, it was found that more than half of the respondents (57.5 % collectively graduated and postgraduate (have university degrees as their level of education, and only 11.1 % were https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4020364/#b4 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4020364/#b4 iraqi j pharm sci, vol.31(suppl.) 2022 exploring iraqi herbalists knowledge and practice 181 illiterate. data showed that less than half of the participants (42.6%) have more than ten years of experience and about one-third (35.2%) have more than 20 years of experience. a comprehensive demographic data of the study respondents is demonstrated in table 4. table 4 . demographic characteristics of the study population variants type n % chisquire pvalue herbalist residence babylon 16 29.6 19.519 0.001 al-qadisiya 6 11.1 al-muthanna 5 9.3 karbala 21 38.9 najaf 6 11.1 total 54 100.0 herbalist age/year 20-39 26 48.1 14.333 0.001 40-59 23 42.6 60-79 5 9.3 total 54 100.0 herbalist education degree illiteracy 6 11.1 38.407 0.000 primary 5 9.3 secondary 12 22.2 university 28 51.9 post-graduate 3 5.6 total 54 100.0 herbalist experience/year <5 4 7.4 17.852 0.000 5-10 8 14.8 10-20 23 42.6 >20 19 35.2 *(p<0.05) part b of the survey addresses the herbalist's plant source and identification method; data demonstrated that 81.5 % of herbs were imported, while only 18.5 % came from the local market. the method used for the identification process depended mainly on the herbalist's experience (72.2%), and only a minority (14.8%) of herbalists bought their herbs from authenticated places. statistical analysis showed a significant difference (p<0.05) between herbalists’ years of experience and the method of herbal identification. results are demonstrated in figure 1 iraqi j pharm sci, vol.31(suppl.) 2022 exploring iraqi herbalists knowledge and practice 182 figure 1 herbalist’s method of plant identification and years of experience. part c of the survey discussed the herbalist's practice in dispensing their remedies to patients. results showed that more than threequarters of herbalists (79.6%) constantly assess patients and follow up (40.7%) with their clients on the progress of given treatment. only a minority of herbalists never evaluate patient conditions nor monitor their treatment, with a low percentage of 7.4% and 13.0 %, respectively. our findings showed a significant correlation (p<0.05) between both patient disease assessment & follow-ups and herbalists' years of experience. it was shown that herbalists rarely (29.6 %( or never (25.9%) keep a record of side effects that occur with remedies given, but most of them (42.6%) direct their clients to their health care providers when such side effects occur. there was a significant difference (p < 0.05) correlating years of experience of herbalists who always refer their patients in case of remedy's side effects. other observations from the analysis showed a comparable proportion of herbalists write expiration on their given products (40.7%) compared to those who never (29.6%) write expiration. for the consultation on storage conditions, about half of the respondents (50.0%) always advised patients about the proper storage conditions of the given remedy. a further note about herbalist practice was writing the complete ingredients on the final remedy package. data showed that 40.7% of them never, and 14.8% rarely put this information on the product given, as compared to 22.2% who always do so; it was also found that there was a significant correlation to years of experience as it was a part of their practice. the detailed information analyzing practice is shown in table 5 table 5 .herbalist’s practice and years of experience type <5 5-10 10-20 >20 total chisquare pvalue n % n % n % n % n % patient disease assessment always 4 9.3% 6 14.0% 16 37.2% 17 39.5% 43 79.6% 9.64 0.00 never 0 0.0% 1 25.0% 1 25.0% 2 50.0% 4 7.4% often 0 0.0% 0 0.0% 2 100.0% 0 0.0% 2 3.7% rarely 0 0.0% 0 0.0% 2 100.0% 0 0.0% 2 3.7% usually, 0 0.0% 1 33.3% 2 66.7% 0 0.0% 3 5.6% herbalist follows up and monitor always 4 18.2% 3 13.6% 7 31.8% 8 36.4% 22 40.7% 21.93 0.00 never 0 0.0% 1 14.3% 3 42.9% 3 42.9% 7 13.0% often 0 0.0% 2 22.2% 5 55.6% 2 22.2% 9 16.7% rarely 0 0.0% 1 9.1% 8 72.7% 2 18.2% 11 20.4% usually 0 0.0% 1 20.0% 0 0.0% 4 80.0% 5 9.3% iraqi j pharm sci, vol.31(suppl.) 2022 exploring iraqi herbalists knowledge and practice 183 continued table 5. herbalist records any side effects from the preparation type <5 5-10 10-20 >20 total chisquare pvalue n % n % n always 2 14.3% 4 28.6% 4 28.6% 4 28.6% 14 25.9% 23.22 0.12 never 0 0.0% 1 7.1% 8 57.1% 5 35.7% 14 25.9% often 1 11.1% 1 11.1% 4 44.4% 3 33.3% 9 16.7% rarely 1 6.3% 2 12.5% 6 37.5% 7 43.8% 16 29.6% usually 0 0.0% 0 0.0% 1 100.0% 0 0.0% 1 1.9% herbalist refers patient with side effect to physician or pharmacist always 2 8.7% 3 13.0% 10 43.5% 8 34.8% 23 42.6% 9.00 0.00 never 1 7.7% 0 0.0% 6 46.2% 6 46.2% 13 24.1% often 1 14.3% 2 28.6% 2 28.6% 2 28.6% 7 13.0% rarely 0 0.0% 0 0.0% 2 66.7% 1 33.3% 3 5.6% usually 0 0.0% 3 37.5% 3 37.5% 2 25.0% 8 14.8% herbalists put expirations on the product sold always 3 13.6% 5 22.7% 7 31.8% 7 31.8% 22 40.7% 32.85 0.00 never 0 0.0% 0 0.0% 7 43.8% 9 56.3% 16 29.6% often 0 0.0% 0 0.0% 3 100.0% 0 0.0% 3 5.6% rarely 0 0.0% 1 16.7% 4 66.7% 1 16.7% 6 11.1% usually 1 14.3% 2 28.6% 2 28.6% 2 28.6% 7 13.0% herbalist gives information to clients about the storage condition always 2 7.4% 5 18.5% 10 37.0% 10 37.0% 27 50.0% 15.92 0.002 never 0 0.0% 0 0.0% 4 40.0% 6 60.0% 10 18.5% often 1 12.5% 0 0.0% 5 62.5% 2 25.0% 8 14.8% rarely 0 0.0% 0 0.0% 2 66.7% 1 33.3% 3 5.6% usually 1 16.7% 3 50.0% 2 33.3% 0 0.0% 6 11.1% herbalist records information of all ingredients on the package sold always 3 25.0% 2 16.7% 3 25.0% 4 33.3% 12 22.2% 16.926 0.003 never 0 0.0% 0 0.0% 11 50.0% 11 50.0% 22 40.7% often 0 0.0% 2 28.6% 4 57.1% 1 14.3% 7 13.0% rarely 1 12.5% 2 25.0% 3 37.5% 2 25.0% 8 14.8% usually 0 0.0% 2 40.0% 2 40.0% 1 20.0% 5 9.3% *(p<0.05) the last part of the survey addresses the herbalist's knowledge and source of information. it was found that most herbalists (35.2%) rely on their gained experience as their sole source of information needed in their practice, compared to (31.5 %) who use herbal books as an additional source of information. the herbalist who uses multiple sources represented a low percentage of 1.9%. the obtained data also showed that herbalists with higher years of experience tend to use fewer sources of information and depend on their acquired knowledge. (table6) iraqi j pharm sci, vol.31(suppl.) 2022 exploring iraqi herbalists knowledge and practice 184 table 6 .herbalists information source and years of experience years of experience <5 5-10 10-20 >20 total chisquir e pvalue n % n % n % n % n mean ±sd % knowledge answers: herbalist information source experience 0 0.00% 1 12.50% 1 0 43.50% 8 42.10% 19 4.75± 35.20 35.20% 19.66 0.186 experience + internet 1 25.00% 1 12.50% 2 8.70% 1 5.30% 5 1.25± 9.30 9.30% herbal medicine book + experience 1 25.00% 4 50.00% 8 34.80% 4 21.10% 17 4.25± 31.50 31.50% herbal medicine books 1 25.00% 0 0.00% 3 13.00% 4 21.10% 8 2± 14.80 14.80% herbal medicine book + experience + internet 1 25.00% 1 12.50% 0 0.00% 2 10.50% 4 1± 7.40 7.40% herbal medicine book + experience + study 0 0.00% 1 12.50% 0 0.00% 0 0.00% 1 0.25± 1.90 1.90% *(p<0.05) discussion there have been continuing arguments about the likelihood of regulating practitioners of herbal and traditional medicine globally since they met vital criteria that included risk to the public through poor practice (26-28). the world health organization (who) recommends that countries take action to regulate traditional and complementary medicine practices and practitioners. according to who reports, no more than 25 of the 191 member nations have a federal policy on traditional herbal medicine, and only 64 countries control herbal practices (16,26). in light of these facts, it is crucial to detect the current herbalist practices to reveal the steps needed to design a general system for herbalist practice. the study revealed several important observations regarding herbalist practice and knowledge. the first observation noted was the method used to identify the herbal products dispensed in herbalist shops. this is an essential initial step in managing the patient condition on the one hand and their safety on the other hand. depending on experience rather than references is a part of the concern, being a source of error since many species may look the same and necessitate using references and documentation for complete identification. our findings matched the observation noted by nedhal a. al – douri study in iraq, performed between 2009 and 2011 (16), highlighted this vital step in patient care. mislabeling of herbal products and its impact on patient safety has been extensively studied (36,37). therefore, it is important to impose regulations on imported herbal products and to be sold only in licensed and registered stores under the supervision of the ministry of health. these products should be fully labeled with the correct botanical name in addition to their local name in the market to avoid any mislabelling in dispensing herbs to patients. for the practice part, our findings showed that most herbalists perform patient assessments before dispensing their remedies, and most follow up with their clients on the treatment progress. the above approach was noted with more experienced herbalists. this result showed that herbalists acquire their knowledge mainly through their actual practice or with what we call "trial and error" in patient management; accordingly, herbalists with fewer years of experience practice differently and impose higher risks on their patients. this finding was also confirmed by other studies performed in other countries (32-34). the inconsistency in practice among herbalists is another concern in patient safety, which necessitates regulating the practice with general standards. another helpful note extracted from our data showed that herbalists only sometimes track records of adverse effects from the herbal remedies given. this recording is crucial to the safety of patients and essential in generalizing the pool of data for herbs (12,29,30) as most of our data regarding adverse effects generally depend on the feedback and prognosis from patients. iraqi j pharm sci, vol.31(suppl.) 2022 exploring iraqi herbalists knowledge and practice 185 interestingly, most herbalists refer clients to their physicians in case of unwanted side effects which is a part of a collaboration between some herbalists and health care providers. this practice was also noted in other studies (32,35). a further note about herbalist practice was to refrain from writing the complete ingredients on the final remedy package. herbalists believe such information may impact their success, being their profession's secret. this finding brings us to a significant safety issue for the patients, especially in case of toxicity, allergy, and possible drug interaction with an unknown product, as most herbalists use a combination of herbs and rarely prescribe a single herb entity (31) the study also revealed that herbalists use their acquired experience as their primary source of information; a similar observation was noted by reem a. issa's study, which was performed in jordan in 2017 (32). this practice can impose some risk on clients as information is not up to date through references and education programs (3234). herbs can produce a broad scale of unwanted or adverse reactions, some of which can trigger severe damage, life-threatening conditions, and even death. various and undeniable cases of poisoning have been stated in the literature (38-40). limitations of the study study participants' recruitment was based on their interest in the survey; thus, an unequal number of herbalists were joining from different provinces. herbalists' rejection to participate in the study was due to illegal practice, limited time, fear of releasing information about their practice, and competition issues. another limitation was the difficulty in obtaining the official number of registered herbalists' shops. moreover, no studies have been performed to estimate the proportion of herbalists in the population. further studies are required to investigate herbalist practice in other parts of iraq and recruit more significant numbers to generate national data. conclusion the study showed that iraqi herbalists practicing in the studied area use different approaches in prescribing and dispensing their remedies to clients and identifying their herbal plants. herbalists with higher experience tend to practice differently than those less trained. this variation in practice may impose health risks on the public which necessitates creating a standard of practice followed by all herbalists. the regulation should start from the initial step of importing herbal products down to the final step of dispensing these products to patients. recommendations it is recommended to schedule continuous education programs for updated information regarding herbal product consumption, safety, and effectiveness for herbalists to ensure safety. the ministry of health can enforce this as a program needed to renew their license periodically. it is essential to set a system for the correct identification and verification of the different medicinal plants and their various species and to train herbalists on this system. it is also recommended to have frequent follow-ups and visits scheduled by the ministry of health to check on different herbalist shops and their practice, focusing on the importance of writing the complete list of ingredients, storage, and expiration on the package of herbal remedies. moreover, there is a need for an advanced system for reporting unwanted side effects and reactions from different sold herbal products to create a database on the safe use of such products and their possible adverse reactions. conflict of interest in this study, there was no conflict of interest references 1. si-hua gao, shu-feng zhou, zhi-ling yu, hou-qi chen, shuo-feng zhang, min-ke tang, jian-ning sun, and kam-ming ko; 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53: 2–3 39. cosyns j. p., jadoul m., squifflet j. p., wese f. x, van ypersele de strihou c. ;urothelial lesions in chinese-herb nephropathy. american journal kidney. 1999; 33 :1011–1017 40. ernst e . toxic heavy metals and undeclared drugs in asian herbal medicines. trends pharmacology sciences. 2002, 23 136–139 this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 flaxseed effects on menopause associated symptoms and biochemical parameters doi: https://doi.org/10.31351/vol32iss1pp257-265 257 effects of flaxseed (linum usitatissimum) on postmenopausal symptoms and its clinical parameters kawa dizaye*, dereen adeeb alchalabi*and bushra hassan marouf**,1 *department of basic sciences, college of medicine, hawler medical university, erbil, iraq iraq department of pharmacology and toxicology, college of pharmacy, university of sulaimani, sulaimani,** abstract menopause is the irreversible and permanent cessation of menstruation after one year of amenorrhea. common symptoms include vaginal dryness, hot flushes, night sweating, and bone pain. traditionally linum usitatissimum (flaxseeds) has been used for the treatment of postmenopausal symptoms. however, the effect of this medicinal herb on the satiety hormone; leptin and body weight in menopausal women has not been elucidated well. this study was designed to evaluate the effect of flaxseeds on serum estrogen, progesterone, leptin, and malondialdehyde, body mass index and blood pressure in healthy women. moreover, the flaxseed effect on climacteric symptoms was also investigated. the study was an open-label interventional clinical trial. it was a sixweeks (short duration) oral administration of 1000 mg flaxseed powder twice daily by menopausal women. serum levels of estrogen, progesterone, and leptin hormones as well as total plasma malondialdehyde were determined pre-and-post flaxseed intervention. clinical symptoms and bothersome complaints of postmenopausal women such as hot flushes, vaginal dryness, bone pain, and night sweating were also evaluated. furthermore, blood pressure elements and body mass index were also measured. the results showed that linum usitatissimum seed powder significantly reduced vaginal dryness, hot flushes, bone pain, and night sweating in menopausal women. the same dose of flaxseed had no significant effects on serum estrogen and progesterone. however, it significantly decreased serum malondialdehyde and increased serum leptin. flaxseed had a non-significant effect on body mass index and blood pressure. in conclusion linum usitatissimum seed had significant effect in relieving vasomotor symptoms and increasing serum levels of leptin in menopausal women. keywords: climacteric, estrogen, progesterone, leptin, linum usitatissimum, malondialdehyde, menopause, progesterone. ( على أعراض سن اليأس والمعايير السريرية في linum usitatissimumالكتان ) آثار بذور النساء بعد سن اليأس 1**،بشرى حسن معروف و *، ديرين عادل الجلبي *كاوه دزه يي ، العراق اربيل قسم العلوم األساسية، كلية الطب، جامعة هولير الطبية، * ، العراق سليمانية كلية الصيدلة، جامعة السليمانية، والسموم، دوية فرع اال** الخالصة االعراض .من دون دورة شهريةسنة كاملة ويتم التشخيص بذلك بعد مرور دائمة انتهاء دورات الحيض بصورة مرحلة هو سن اليأس ق الليليالمهبل، هبات الحرارة، جفاف الشائعة لهذه المرحلة هي واآلالم العضام. تم تقليديا استخدام بذور الكتان لعالج اعراض المصحوبة التعرُّ على الهورمون الشبع الليبتن و مؤشر كتلة الجسم. لذلك تم تصميم هذا البحث لتقييم تأثير بذور الكتان على ابالسن اليأس. ولكن لم يتم توضيح تأثيره نساء نسبة هورمون األستروجين و البروجستيرون و الليبتن و عامل أألكسدة مالونديالديهايد و مؤشر كتلة الجسم و الضغط الدم في مجموعة من ال ملغم من مسحوق البذور لدى المشاركين 1000بحث على شكل تداخلي قبل و بعد التناول المادة. تضمنت الدراسة تناول في السن اليأس. تم تصميم ال . تم تحليل نسبة الدم لهورمون األستروجين و البروجستيرون و الليبتن وكذلك نسبة مالونديالهايد قبل و بعد أسابيعفي الدراسة مرتين باليوم لمدة ستة ق الليليالمهبل، جفاف وتم تقييم األعراض السريرية و المعاناة المزعجة لهذه الحالة منها الهبات الحرارة، تناول البذور. واآلالم العضام و التعرُّ أظهرت النتيجة بأن بذور الكتان خفضت شدة التعرق الليلي في النساء المشاركات في البحث. حيث لم كذلك ضغط الدم وقياس مؤشر كتلة الجسم. ر كبير جد تأثير كبير على نسبة األستروجين و البروجستيرون و لكن انخفضت نسبة مالونديالديهايد و ارتفعت نسبة الليبتن في الدم. لم يحدث تغييو عراض بأن لبذور الكتان تأثير كبير وواضح على تقليل وارتياح من األ من ذلك نستنتج في مؤشر كتلة الجسم وضغط الدم لدى المشاركات في البحث. الحركية الوعائية وتخفيض عامل األكسدة و ارتفاع نسبة هورمون الشبع الليبتن في الدم عند المرأة في السن اليأس. ، مالونديألديهايد الكلمات المفتاحية: سن اليأس، األستروجين، البروجسترون، الليبتن introduction menopause is a naturally occurring phenomenon that is not related to a pathological condition. it is characterized by hormonal changes, permanent and irreversible cessation of menstruation. a woman who has experienced twelve consecutive months of amenorrhea is confirmed to be diagnosed as menopause. the typical age for menopause to occur in most women is 45-52 years (1,2). 1corresponding author e-mail: bushra.marouf@univsul.edu.iq received: 21/ 4/2022 accepted: 30/7 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp257-265 iraqi j pharm sci, vol.32( 1 ) 2023 flaxseed effects on menopause associated symptoms and biochemical parameters 258 the majority of women experience complicated symptoms, such as hot flushes, night sweats “i.e. vasomotor symptoms”, disturbance of sleep and sexual dysfunction, mood change, weight gain, bone pain, and cognitive impairment. furthermore, menopause has an impact on other body systems such as genitourinary, cardiovascular, and psychogenic (3). menopause-associated symptoms such as vulvovaginal atrophy, atrophic vaginitis, or urogenital atrophy which are recently named as genitourinary syndrome of menopause usually last five to seven years although they may persist longer. these health consequences of menopause are thought to be caused by the lack of estrogens during menopause (4,5). typically, the primary treatment for menopausal symptoms is hormonal therapy. however, due to the adverse effects associated with the use of hormones, most women cannot use this approach (6). herbal products enriched with estrogen are frequently used in relieving menopause symptoms. phytoestrogenic herbs elevate the level of estrogen in menopause women and can attenuate the frequency of vasomotor symptoms via escalation of the estrogen level and regulating the hormonal balance (7,8). phytoestrogens can be found in grains, vegetables, and fruits of many medicinal plants. flaxseed (linum usitatissimum); is regarded as a well-known and enriched source of phytoestrogens, it is a member of the genus linum in the family linaceae. linum usitatissimum oil has been traditionally used for the treatment of menopause symptoms. it has a unique nutrient profile that contains omega-3 fatty acids, lignans, α-linolenic acid, protein, and fiber (9). hypo estrogenic state during menopause age is proven to enhance metabolic dysfunction thus leading to higher body mass (10). a preclinical study proposed the anti-obesity effect of flaxseed in an animal model of metabolic syndrome via a mechanism in which flaxseed is capable of removing leptin resistance then enhance lipolysis and fatty acid oxidation and inhibiting lipogenesis (11). leptin is a protein mainly synthesized in adipose tissue, and its production is associated with total body fat (12), it acts as a satiety hormone by acting on the hypothalamic centers, and suppressing appetite and food intake, thereby regulating body weight homeostasis (13,14). in the previous studies, various levels of leptin have been recorded in postmenopausal women when compared with premenopausal women, some have increased (15), equal (16), or decreased leptin levels (17), these discrepancies may be associated with concomitant changes in body mass index (bmi), body composition, and insulin sensitivity along with declining endogenous estradiol levels (14). the effect of this medicinal herb on the satiety hormone; leptin and body weight in menopausal women has not been elucidated well. based on many clinical and pre-clinical studies, this plant has also been suggested to has an ameliorative effect on postmenopausal symptoms (18,19). one of the speculated mechanisms is through the modulation of oxidative stress. a decrease in estrogen production in menopause women, obesity and aging causing an imbalance in oxidative stress (20–22). malondealdehyde as a marker of oxidative stress is elevated in post menopause compared to their premenopausal peer, it can be used as a marker of cardiovascular risk (23). although advanced work on the beneficial action of flaxseed have been conducted however, the flaxseed antioxidant effect on improving clinical symptoms and oxidative stress in menopause women remain to be elucidated and there is no enough research to support a recommendation for its use. therefore, this study was designed to evaluate the potential effects of flaxseeds on serum estrogen, progesterone, the satiety hormone leptin, and malondialdehyde and to investigate the climacteric symptoms in postmenopausal women using flaxseed powder for six weeks. subjects and methods study design and setting the study was an open-label interventional clinical trial; it was conducted at the outpatient’s unit in maternity teaching hospital in erbil/kurdistan region. the duration of the study was eight months from july 2020 to february 2021 including patient recruitment, follow up and data analysis. it was carried out under the declaration of helsinki and its amendments to the ethical guidelines for human studies and the currently adopted regulations of the iraqi ministry of health. approval of this study was obtained before its onset from the ethical committee of hawler medical university. all the women included were informed about the nature and scope of the study and written informed consent was obtained from each before participation in the study. eligible women diagnosed as menopausal by senior gynecologists were selected. a total of 48 patients were screened for eligibility. forty patients were eligible for inclusion. twenty-three patients completed the six-weeks study, seventeen patients were dropped out from the study (figure 1). the study was a six-week oral administration of 1000mg flaxseed powder twice daily, the dose was selected based on the previous studies with modification (24-26) (figure 1). flaxseed powder was prepared by grinding the seeds, then a capsule dosage form was prepared from the milled powder, it was given with full instruction. the duration of the study was eight months including patient enrollment, intervention, follow up and laboratory investigations. weight (in kilograms) and height (in centimeters) were measured while iraqi j pharm sci, vol.32( 1 ) 2023 flaxseed effects on menopause associated symptoms and biochemical parameters 259 participants were wearing light clothing and no shoes. blood pressures (bp) were recorded in a sitting position with a standard mercury sphygmomanometer after minutes of rest. all the participants have been instructed to eat flaxseed-free diet. figure 1. flow chart of the study design. bmi; body mass index, mda; malondialdehyde. inclusion criteria the included participants in this study were the postmenopausal women (last menstrual bleeding was at least 1 year before) who were diagnosed by a gynecologist, who had not taken any hormonal therapy, supplements, vitamins, phytoestrogens six months before the recruitment. women between the ages range of 47 to 59 years were included in the study. all the participants have been informed to eat flaxseed-free diet. exclusion criteria patients who underwent oophorectomy, hysterectomy, or patients with chronic disease such as hypertension, diabetes mellitus, thyroid disease (hypothyroidism / hyperthyroidism) or cardiovascular disease, cancer, and women who received hormone replacement therapy were excluded. blood sample five milliliters of blood samples were drawn from each patient in the morning. blood samples have been taken before (first day) and after the treatment (last day after six weeks) to determine estrogen, progesterone, leptin hormones, and malondialdehyde (mda) levels in the postmenopausal women. venous blood samples were drawn into plain tubes without anticoagulants. the blood was allowed for 30 minutes to clot and after centrifugation for 5 minutes at 3000 rpm; the serum was collected in a plain tube and kept frozen for analysis. outcome measures serum levels of estrogen, progesterone, and leptin hormones were determined pre-and posttreatment (i.e. at day 1 and at day 42) using an enzyme-linked immunosorbent assay (elisa) kit. additionally, antioxidant status was observed, and particularly the concentration of total plasma mda was determined pre-and post-treatment. clinical symptoms and bothersome complaints of post menopause women such as bone pain, vaginal dryness, night sweat and hot flushes were also evaluated pre and post-flaxseed intervention. bone pain was measured by using visual analogue scale (vas). while hot flushes and night sweat were measured based on women own self-reports, the women were provided with a diary and instructed to record the time when hot flushes and night sweat occur with its severity rating mild, moderate and severe (27). furthermore, blood pressure elements and bmi were also measured twice. to monitor patient adherence to the study protocol and appearance of possible adverse effects, weekly telephone calls were done to all the participants. statistical analysis all data were expressed as mean ± standard error of means (m ± sem) and statistical analysis was carried out using statistically available software (spss, version 24). a chi-square test for the association was used to compare proportions. the student's t-test was used to compare two means. a p-value of less than 0.05 was considered statistically significant. iraqi j pharm sci, vol.31(2) 2022 flaxseed effects on menopause associated symptoms and biochemical parameters 260 results 1-the basic characteristics of the participants has been summarized in table (1). table1. basic characteristics of the participants n=23. 2-effects of linum usitatissimum on serum estrogen and progesterone level in post-menopause women. after six weeks of daily administration of linum usitatissimum powder for women presenting with menopausal symptoms, flaxseed has no significant effect on estrogen and progesterone level as shown in table(2). table2. effect of linum usitatissimum powder on serum estrogen and progesterone levels in postmenopause women (n=23). non-significant p-value is ≥ 0.05 3-the effect of linum usitatissimum on leptin and malondialdehyde in women presented with postmenopausal symptoms. daily administration of 1000 mg linum usitatissimum powder for six weeks significantly increased serum leptin level and significantly decreased serum malondialdehyde as showed in the table(3). table3. effect of daily administration of linum usitatissimum powder on serum leptin and malondialdehyde level in post-menopause women for six weeks (n=23). parameters pre-flaxseed intervention (mean±se) post-flaxseed intervention (mean±se) p. value leptin (ng/ml) 53.13 ± 7.16 73.84 ± 6.42 0.004 ** malondialdehyde(nmol/ml) 77.43 ± 15.30 40.80 ± 6.44 0.007 ** **indicates significant p-value is <0.01 4-effect of linum usitatissimum on body mass index in postmenopausal women.flaxseed powder had no significant effect on the body mass index (bmi) after six weeks of daily administration in women presenting with menopausal symptoms in table(4). table4. the effect of linum usitatissimum on body mass index in women presented with post-menopausal symptoms. non-significant p-value is ≥ 0.05 5-effect of linum usitatissimum on diastolic blood pressure, systolic blood pressure, mean blood pressure, and heart rate in post-menopausal women. six weeks of daily administration of flaxseeds powder showed no significant effects on diastolic blood pressure, systolic blood pressure, mean blood pressure, and heart rate table(5) in post-menopausal women. parameters flaxseed treatedparticipants n=23 age (year) ± sd 57.13±2.89 body mass index (kg/m2) ± sd 27.72±0.36 duration of menopause period (month) ± sd 6.25 ±1.1 estrogen (pg./ml) 39.48±16.71 progesterone (ng/ml) 0.33±0.03 parameters preflaxseed intervention (mean±se) post-flaxseed intervention (mean±se) p. value estrogen (pg/ml) 39.48 ± 16.71 33.32 ± 13.62 0.408 progesterone(ng/ml) 0.33 ± 0.03 0.35 ± 0.03 0.577 parameters preflaxseed intervention (mean±se) post-flaxseed intervention (mean±se) p. value body mass index(kg/m2) 27.72 ± 0.36 27.75 ± 0.38 0.668 iraqi j pharm sci, vol.31(2) 2022 flaxseed effects on menopause associated symptoms and biochemical parameters 261 table5. effect of linum usitatissimum on diastolic blood pressure, systolic blood pressure, mean blood pressure, and heart rate before and after treatment (n=23). parameters preflaxseed intervention (mean±se) post-flaxseed intervention (mean±se) p. value diastolic blood pressure (mmhg) 67.82 ± 1.21 69.69 ± 1.09 0.257 systolic blood pressure (mmhg) 120.73 ± 2.31 121.52 ± 2.23 0.350 mean blood pressure (mmhg) 85.39 ± 1.47 86 ± 1.30 0.320 heart rate (b/min) 71.34 ± 1.38 71.08 ± 0.87 0.871 non-significant p-value is ≥ 0.05 6-the effect of linum usitatissimum on vaginal dryness in postmenopausal women. daily administration of linum usitatissimum powder for six weeks significantly reduced vaginal dryness (pvalue = 0.001) in women presented with postmenopausal symptoms as showed in figure (2). figure 2. effect of linum usitatissimum on vaginal dryness in postmenopausal women (n=23). ***indicates significant p-value is ≤ 0.001 7-the effect of linum usitatissimum on hot flush in post-menopausal women.after six weeks of daily administration, linum usitatissimum powder significantly reduced the frequency of hot flushes (p-value = 0.001) in post-menopausal women presented with a history of a hot flush (figure 3). figure 3. the effect of linum usitatissimum on symptoms of a hot flush in post-menopausal women (n=23). **indicates significant p-value is ≤ 0.01 and ***indicates significant p-value is ≤ 0.001. 8-the effect of linum usitatissimum on bone pain in post-menopausal women using visual analogue scale (vas). linum usitatissimum significantly reduced bone pain measured by visual analogue scale (vas) in menopausal women who presented with a history of bone pain after six weeks of daily use of flaxseed powder as shown in table(5). table5. effect of linum usitatissimum on bone pain in post-menopause women (n=23). **indicates significant p-value is ≤ 0.01 9-the effect of linum usitatissimum on night sweating in post-menopausal women. daily ingestion of flaxseed for six weeks significantly reduced the symptoms of the night sweating in women who presented with menopausal symptoms as showed in table(6). table6. effect of linum usitatissimum powder on night sweating in post-menopausal women (n=23). symptoms severe moderate mild p.value (n) % (n) % (n) % 0.001*** night sweating measured by women’s own self-reports) pre-flaxseed intervention (8) 34.8 (9) 39.1 (6) 39.1 post-flaxseed intervention (1) 4.3 (2) 8.7 0 ***indicates significant p-value is ≤ 0.001 symptoms severe moderate mild p-value (n) % (n) % (n) % 0.005** bone pain (measured by vas) pre-flaxseed intervention (4) 17.4 (12) 52.2 (7) 30.4 post-flaxseed intervention (1) 4.3 (5) 21.7 (9) 39.1 iraqi j pharm sci, vol.31(2) 2022 flaxseed effects on menopause associated symptoms and biochemical parameters 262 discussion more than two-thirds of the women in the menopause period have some symptoms such as hot flush, night sweating, vaginal dryness, and sleep disturbances are due in part “not entirely” to estrogen depletion (28). these symptoms are frequent and may be severe that can cause considerable emotional distress. many treatment approaches exist to alleviate these bothersome symptoms, including hormone replacement therapy, non-hormone drug treatment, and plant-based therapies such as soybean, lentils as a source of isoflavones, and flaxseed, vegetables, grains, fruits as lignans phytoestrogen sources (29). safety concerns regarding hormone replacement therapy have developed an interest in the use of phytoestrogens in the management of complaints associated with menopause age. flaxseed extract is the main origin of lignan phytoestrogen named secoisolariciresinol diglucoside. after ingestion and in the colon, it is converted into enterodiol and enterolactone; the active mammalian lignans (30). in the previous studies when postmenopausal women used a flaxseed supplement diet, their menopausal symptoms were decreased and their quality of life has improved (24). the health benefits of flax meal are due to its alphalinolenic acid (31), fiber (32), and lignan (33). the high omega-3 and protein content also make flaxseed meals unique and superior to other fiber supplements and food ingredients (26). the mammalian lignans are believed to work by binding to estrogen receptors on cell membranes. they show low estrogenic activity or anti-estrogenic activity with potentially pharmacological advantages (34). in the current study, intake of one gm flaxseed for six weeks showed no significant effect on total serum estrogen and serum progesterone. however, in a clinical study, menopause women consuming 10g ground flaxseed daily for 7 weeks significantly reduced serum level of 17beta-estradiol and estrone sulfate and increasing serum prolactin concentrations (35). additionally, in another clinical intervention, haggans et al showed that 10g flax consumption daily for 7 weeks significantly increases the 2hydroxy estrone/16-α hydroxy estrone ratio in urine and the 2-hydroxy estrone excretion levels were higher of 10 g/day flax group compare to the control group, this difference in the result may be mostly due to the dosage of flaxseed and the duration of therapy (36). effects on the other endogenous sex hormones were not statistically significant this finding suggested that flaxseed affects certain circulating sex hormone levels in favor of prevention of breast cancer since a high ratio of 2-hydroxylation to 16α hydroxylation pathway estrogen metabolites have been associated with reduced breast cancer risk (37). in this study, after six weeks of flaxseed intake, flaxseed increased the serum level of leptin in postmenopause women, when compared with pretreatment serum level. this rise in serum leptin levels in post-menopausal women could be related to flaxseed high content of omega-33 fatty acid and α-linolenic acid which increases leptin expression (26,38). mccullough et al reported that consumption of flaxseed significantly increased plasma and adipose levels of α-linolenic acid and consequently leptin levels were elevated in animals (rabbits) taking a diet supplemented with 10 % flaxseed (10gm flaxseed /100gm regular rabbit diet in that study. changes in leptin expression by flaxseed supplementation are inversely correlated with the risk of atherosclerosis and effects on lipid metabolism through reducing total cholesterol, ldl, and increasing hdl levels (38). a systematic review and meta‐analysis of randomized controlled trials on the effect of flaxseed supplementation on circulating adiponectin and leptin concentration in adults reported that flaxseed supplementation had no significant effect on adiponectin and leptin levels in adults (39). although in the current study consumption of 1000 mg of flaxseed powder has no significant effect on bmi. however, a meta-analysis that included 45 randomized placebo-controlled trials showed a significant reduction in body weight, bmi, and waist circumference following long-term supplementation of flaxseed i.e. more than 12 weeks, in doses ≥30 g/day durations, this controversial finding might be related to the smaller quantity and shorter duration of flaxseed consumed in the present study. flaxseed consumption decreases bmi due to the presence of dietary fiber, as flaxseed is a rich source of dietary fiber both soluble as well as insoluble fibers. dietary fibers from flaxseed were found to have a direct relation to health in particular in body weight regulation through diminished nutrient absorption (40,41). additionally, a recent in vivo study conducted by luo et al. has elucidated the mechanism of the ant obesity effect of flaxseed polysaccharide via inducing satiety due to removal of leptin resistance and enhancing lipid metabolism through the ampactivated protein kinase (ampk) signaling pathway and suppression of lipogenesis (11). the menopause transition period is accompanied by dysregulation of lipid metabolism and production of many adipocytokines, proinflammatory cytokines, and reactive oxygen species which cause lipid peroxidation (42). the current study aimed to attenuate the deleterious effect of oxidative consequences of this metabolic alteration by flaxseed. administration of flaxseed in this study to women presented with symptoms of menopause showed a statistically significant reduction in malondialdehyde serum level. the result is iraqi j pharm sci, vol.31(2) 2022 flaxseed effects on menopause associated symptoms and biochemical parameters 263 consistent with the finding of other studies that demonstrated the antioxidant potential of flaxseed and its phenolic components (43–45). it was found that the phenolic compounds (lignans, phenolic acid, flavonoids, phenylpropanoids, and tannins) of flaxseeds are excellent in preventing the excess of free radicals and avoiding their pathological effects. phenolic compounds exert their antioxidant effect by acting as reducing agents, hydrogen donors, singlet oxygen quenchers, or metal chelators (45). furthermore, the reduction in malondialdehyde serum level could be achieved through the flaxseedrich content of the α-linolenic acid, because αlinolenic acid can be stored in adipose tissue, and some of its beneficial actions may be due to its effects on the adipose tissue. consumption of 1000 mg flaxseed for six weeks showed a non-significant change in mean blood pressure, systolic blood pressure, and diastolic blood pressure in normotensive post-menopause women. however, in a coronary heart disease high-risk population consuming a diet with alpha-linolenic acid there was a decrease in diastolic blood pressure, and increase serum triacylglycerol concentration (46). additionally, in patients with hypertension flaxseed consumption reduces blood pressure by altering circulating oxylipins “derived from polyunsaturated fatty acids regulate vascular tone” via an α-linolenic acid-induced inhibition of soluble epoxide hydrolase (47). wilkinson et al., (2005) measured the diet-related change in blood pressure and total peripheral resistance responses to a diet high in flaxseeds, they recorded a non-significant change in systolic and diastolic blood pressure (48). however, in patients with peripheral arterial disease intake of 30g of milled flaxseed per day for six months resulted in significant decreases in both systolic and diastolic blood pressure (49). whereas this result disagreed with the meta-analysis carried out by khalesi et al (2015) who recorded an improvement in diastolic blood pressure after consuming whole flaxseed for twelve weeks’ duration (50). daily consumption of 1000 mg flaxseed for six weeks in women presenting with menopausal symptoms showed a significant reduction in frequency and duration of menopausal symptoms such as hot flashes and night sweat, this effect was not consistent with the clinical studies conducted on postmenopausal women in which the participants had been allocated in different groups for consuming flaxseed, the results of those studies showed that consumption of a flaxseed-rich diet is not more effective than a placebo in alleviating the climacteric symptoms of postmenopausal women (51,19). in the current study, all the post-menopausal women had less dry vagina when they have taken 1000 mg of flaxseed powder for six weeks. a similar result was observed in postmenopausal women who have taken 1 g of milled flaxseed daily for 24 weeks 19. this is because flaxseed is rich in phytoestrogen and omega 3, that combat vaginal dryness. in the current study, flaxseeds showed a significant reduction in bone pain in post-menopausal women. this was in agreement with the study of griel et al (2007) who reported that the α-linolenic acid and omega-3 fat found in flaxseed promotes bone health by preventing excessive bone turnover (52) . conclusion menopause is characterized by significant bothersome clinical symptoms, endocrine and metabolic changes. 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fibers lower cholesterol and increase fecal fat excretion, but magnitude of effect depend on food type. nutr metab. 2012;9(1):8. doi:10.1186/1743-7075-9-8 41. ibrügger s, kristensen m, mikkelsen ms, astrup a. flaxseed dietary fiber supplements for suppression of appetite and food intake. appetite. 2012;58(2):490-495. doi:10.1016/j.appet.2011.12.024 this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( 2 ) 2022 cyp2c19 polymorphisms in ischemic stroke patients doi: https://doi.org/10.31351/vol31iss2pp251-259 251 correlation between cyp2c19 polymorphisms and recurrent risk in patients with ischemic stroke treated with clopidogrel in kurdistan region-iraq hind s. jardaq*, 1 and mohammed y. jamal** * department of clinical pharmacy, college of pharmacy, mosul university, mosul, iraq. ** department of clinical pharmacy, college of pharmacy, university of baghdad , baghdad, iraq. abstract clopidogrel is a prodrug that must be transformed into an active metabolite by hepatic cytochrome p450 (cyp) isoenzymes in order to be active in preventing platelet clotting. polymorphisms of the cyp2c19 gene can cause a reduction or complete loss of cyp2c19 enzyme activity resulting in inhibiting clopidogrel metabolism, effectiveness and increment of stroke recurrence risk in ischemic stroke patients. this study aims to investigate the correlation between genetic polymorphisms in cyp2c19*2 and*3 and recurrent risk in patients with ischemic stroke taking clopidogrel 75mg in kurdistan region–iraq. this retrospective case-control study was carried out at kurdistan, erbil, medicina medical center, and rizgary general hospital from january 2021 to august 2021. the blood sample was taken from the participants and tested for genotyping. the collection of retrospective data was done by reviewing patients' medical files in the rizgary hospital and patients’ electronic records in the neurology clinic of medicina medical center. sixty patients participated, (34) were male and (26) were female, with age range (38-96) years, diagnosed with ischemic stroke in not more than two years and on 75 mg clopidogrel maintenance dose. genotyping analysis showed that 61.7 % were homozygotes for wild allele *1, 26.7% (*1/*2) and 6.7 % (*1/*3) heterozygotes genotype. the homozygotes for mutant alleles cyp2c19*2,*3 were 3.3 % (*2/*2) and 1.7 % (*3/*3) respectively. the (*2/*3) was not detected in the study population. results revealed a significant correlation between the risk of stroke recurrence with the existence of variant allele cyp2c19 *2, and angiotensin converting enzyme inhibitors/angiotensin receptor blockers (aceis/arbs) usage (p = 0.024, p = 0.039, p = 0.24 respectively). on the other hand, there was no significant relationship between the risk of stroke recurrence and carrying the variant allele cyp2c19 *3 (p = 1.000). in conclusion, survivors of ischemic stroke treated with clopidogrel who carry cyp2c19*2 allele had a higher risk of recurrent stroke as it is associated with reduced metabolic activity of cyp2c19 enzyme leading to reduction of clopidogrel metabolism and bioavailability. keywords: cyp2c19, gene polymorphism, clopidogrel, stroke. االصابة بجلطة ثانية في المرضى الذين يعانون من السكتة الدماغية و خطورة cyp2c19 صيغالعالقة بين تعدد ويتناولون عقار كلوبيدوجرل في إقليم كردستان العراق **محمد ياوز جمال و 1*،هند سالم جردق العراق. ، الموصل ، جامعة الموصل ، كلية الصيدلة ، فرع الصيدلة السريرية * العراق. بغداد ، بغداد، جامعة ، كلية الصيدلة ، فرع الصيدلة السريرية ** الخالصة السيتوكروم متوازنة إنزيمات بواسطة نشط مستقلب إلى تحويله يجب أولي دواء هو الصفائح cyp p450الكلوبيوجريل تجلط لمنع أو فقده تماًما مما يؤدي إلى تثبيط استقالب الكلوبيدوجريل cyp2c19في تقليل نشاط إنزيم cyp2c19الدموية. يمكن أن يتسبب تعدد أشكال الجين من العالقة بين تعدد األشكال الجينية ، تقليل فعاليته وزيادة خطرتكرار االصابة بالسكتة الدماغية للمصابين بها الول مرة. تهدف الدراسة إلى التحقق مجم في ٧٥و خطر االصابة بجلطة ثانية في مرضى السكتة الدماغية الذين يتناولون عقار كلوبيدوجريل cyp2c19 *3و cyp2c19 * 2في إلى ٢٠٢١كاري العام من يناير إقليم كردستان العراق. أجريت هذه الدراسة بأثر رجعي في كردستان ، أربيل ،مركز ميديسينا الطبي ومستشفى رز اخذت البيانات من السجالت الطبية للمرضى في المستشفى تم أخذ عينات الدم من المشاركين واختبارها من أجل التنميط الجيني. ٢٠٢١أغسطس نساء, تتراوح اعمارهم ( من ال٢٦( من الرجال و ) ٣٤االعصاب .شارك ستون مريضا ، ) والسجالت الطبية اإللكترونية للمرضى من عيادة طب ملغ.أظهر تحليل التنميط ٧٥( سنة. تم تشخيص هؤالء المرضى بسكتة دماغية لمدة ال تزيد عن عامين ، مع تناول عقار كلوبيدوجريل ٩٦-٣٨بين ) (، بينما ٣/ * ١٪ )* ٦.٧( و ٢/ * ١٪ )* ٢٦.٧، متغايرة الزيجوت مقسمة إلى ١٪ كانت متجانسة الزيجوت لألليل الطبيعي * ٦١.٧الجيني أن ( ٣/ * ٢(. لم يتم الكشف عن )* ٣/ * ٣٪ )* ١.٧( و ٢/ * ٢٪ )* ٣.٣،موزعةالى cyp2c19*2,*3 متجانسة الزيجوت لألليالت الطافرة التمثيل الغذائي ، انخفاض نشاط cyp2c19 * 2ثانية مع حمل األليل المتغير دماغية بسكتة في مجتمع الدراسة. وجدت عالقة بين خطر االصابة على التوالي(. من ناحية أخرى ، لم تكن هناك عالقة ٠.٠٢٤= p، ٠.٠٣٩= p، ٠.٠٢٤=p) aceis / arbsواستخدام cyp2c19إلنزيم ن خطراإلصابة بالسكتة الدماغية المتكررة كا=p).١( cyp2c19 *3ذات داللة إحصائية بين خطر االصابة بجلطة ثانية وحمل األليل المتغير ويحملون الكلوبيدوجريل يتناولون الذين الدماغية الجلطة مرضى لدى إلنزيم cyp2c19 * 2اعلى األيضي النشاط بانخفاض مرتبطة ألنها cyp2c19 .مما يؤدي إلى تقليل فعالية عقار كلوبيدوجريل ، السكتة الدماغية ، تعدد األشكال الجيني ، كلوبيدوجريل cyp2c19 الكلمات المفتاحية: 1corresponding author e-mail: hind.gardaq@yahoo.com received: 8/10 /2021 accepted: 30/1 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp251-259 iraqi j pharm sci, vol.31(2) 2022 cyp2c19 polymorphisms in ischemic stroke patients 252 introduction stroke is a leading cause of longterm disability and death globally (1). ischemic stroke is defined as a brain, spinal cord, or even retinal infarction (2) produced by a blood vessel obstruction due to thrombosis or embolism, resulting in impaired blood supply to the part of the brain fed by this vessel and loss of neurological function (3) nearly half of ischemic stroke or transient ischemic attack (tia) patients are at high risk of recurrent stroke within a few months of the initial episode (4). in the united states, one in every six people will have a stroke throughout their lifetime. more than 13.7 million people suffer a stroke each year, and 5.8 million people die as a result. over 80 million people have lived through a stroke (1). ischemic stroke accounts for roughly 70% of all strokes, with hemorrhagic stroke accounting for the remaining 30%. (5). cardio-embolism and large artery atherosclerosis are the most common known causes of ischemic stroke (6). clopidogrel, a p2y12 receptor inhibitor, exhibits higher efficacy, with no added risk of adverse effects, to aspirin in reducing the risk of ischemic stroke, myocardial infarction (mi) and vascular mortality when clopidogrel is added to aspirin (7,8). when compared to antiplatelet monotherapy, dual antiplatelet therapy (duat), a combination of aspirin plus clopidogrel is more effective in reducing the risk of ischemic vascular events (9). clopidogrel is an inert prodrug that must be activated in the liver through a complicated biochemical process. about half of the dose of clopidogrel is absorbed from the intestine after oral intake (10). the carboxylesterase enzyme hydrolyzes over 85% of the absorbed dose of clopidogrel, turning it into an inactive metabolite. the remaining 15% of the dose is transformed into the active metabolite 2-oxoclopidogrel active metabolite in a two-step bio-activation process by several cytochromes p450 enzymes (11-13). as a result, only 2% of the clopidogrel dose is converted into an active metabolite and enters the systemic circulation (14). cyp2c19 is responsible for 44.9 percent of clopidogrel conversion to 2oxoclopidogrel and roughly 20% of active thiol metabolite synthesis from 2-oxoclopidogrel. as a result, cyp2c19 is required for both clopidogrel activation steps (15). pharmacogenetics is the field that studies how genetic variation affects individuals' response to drugs. this inter-individual variation can range from inadequate therapeutic efficacy to serious, potentially life-threatening adverse drug reactions (16). the cyp2c19 gene is located within a cluster of cytochrome p450 genes on chromosome 10q24 containing 8 introns and 9 exons(15). the cyp2c19 gene was expressed in the liver to synthesize a catalytically active enzyme cyp2c19 that is a primary enzyme involved in the conversion of the antiplatelet clopidogrel to the active 2-oxo metabolite (17). currently, there is around 25 different type of cyp2c19 mutations, with cyp2c19*2 and cyp2c19*3 being the most prevalent mutant alleles (11). the frequency of these mutations varies depending on trace or ancestral origin, with asians having a higher frequency than caucasians and african americans (2). the two cyp2c19 mutant alleles cyp2c19*2 and *3 are the key loss of function (lof) alleles that can cause a reduction or complete loss of cyp2c19 enzyme activity resulting in inhibiting clopidogrel metabolism (11). individuals can be classified into three phenotypes based on the number of lof alleles they carry: non-carriers or extensive metabolizers (ems; *1/*1), patients with one lof allele or intermediate metabolizers (ims; *1/*2 and *1/*3), and patients with two lof alleles or poor metabolizers (pms; *2/*2, *2/*3, and *3/*3) (18). reduced development of the active clopidogrel metabolite and reduced clopidogrel effectiveness was observed in people who have one or two nonfunctional cyp2c19 alleles. the presence of a nonfunctional cyp2c19 allele has been linked to an increased risk of cardiovascular events in numerous studies (2). this study aims to assess the correlation between genetic polymorphisms in cyp2c19*2 and*3 and recurrent risk in patients with ischemic stroke on daily 75 mg clopidogrel tablets in kurdistan region–iraq. methods patients and study design from january 2021 to august 2021, an observational mixed retrospective and prospective case-control study was conducted in erbil, kurdistan, in two medical centers: the first is the neurology clinic in medicina medical center, and the second is the neurology ward in rizgary hospital, one of erbil's largest hospitals, in collaboration with a neurologist who served as project supervisor. sixty patients of convenient sample were chosen after meeting the particular inclusion and exclusion criteria. this study's sample size was comparable to that of alhazzani in 2017, a study conducted in a neighboring country, saudi arabia which included fifty patients divided equally into two groups: clopidogrel responders and clopidogrel nonresponders (15). these patients were diagnosed with ischemic stroke for at least two years, taking a 75 mg clopidogrel daily maintenance dose. patients' blood samples were gathered and examined for genotyping by polymerase chain reaction (pcr) searching for the presence of cyp2c19*2 and *3 polymorphisms ; patients were divided into two groups based on the presence of stroke recurrence: 30 patients with recurrent stroke (case group) and 30 patients without recurrent stroke (control group). the distribution inside each group is dependent on the existence of polymorphisms (cyp2c19*2 and *3 loss of iraqi j pharm sci, vol.31(2) 2022 cyp2c19 polymorphisms in ischemic stroke patients 253 function) according to pcr test and arranged into those with mutant genes and those without the mutant gene. the collection of data was done by reviewing the medical files in the hospital and from patients' electronic medical records in the neurology clinic. the demographic factors of patients were chosen according to the risk factors stated in the american heart association/american stroke association (aha/asa) guidelines for the prevention of stroke in patients with ischemic stroke or transient ischemic attacks such as age, gender, race, and family history, weight, diabetes mellitus, hypertension, hyperlipidemia (high ldlcholesterol levels), ischemic heart disease (ihd) and current smoking.the use of cardio-protective medications (statins, angiotensin-converting enzyme inhibitors/angiotensin receptor antagonist [acei/arb], beta-blocker, calcium antagonist, and aspirin) might play an important role in secondary stroke prevention (19), therefore they also were registered with the risk factors in a patient information sheet to be correlated with recurrence of the stroke. as well, the clopidogrel drug brand is selected as one of the patient demographic factors that can affect the risk of ischemic stroke recurrence based on that modifying the drug salt formulation could change its physicochemical properties, thereby affecting clinical efficacy and safety (20). in addition, the quality of the clopidogrel drug brand is may be different from that of generic one as it can be affected by the level of impurities present in many copies of the original drug (21). inclusion criteria patients who are diagnosed with ischemic stroke and taking 75 mg clopidogrel as a daily maintenance dose were included in the study. exclusion criteria 1. patients who are taking drugs interacting with clopidogrel for example tricyclic antidepressants (tcas), antipsychotics, warfarin, and glycoprotein iia/iiib antagonists. 2. patients with a history of alcohol or drug abuse. 3. patients with clotting or other blood disorders. 4. patients with liver and kidney diseases. instruments and procedure molecular analysis two milliliters of venous blood were collected from each participant in a 3 ml ethylene diamine tetra acetic acid evacuated tube (edta). samples were stored at -20º before sending for genomic isolation. genomic dna isolation from white blood cells was done by using the solid-phase dna extraction method with a kit provided by promega (reliaprep tm blood gdna miniprep system). the quality of extracted dna was checked using ethidium bromide stained 0.5% agarose gel electrophoresis and visualized by uv light. polymerase chain reaction and dna sequencing the cyp2c19*2 and *3 polymorphisms were detected by the tetra arms (amplification refractory mutation system) polymerase chain reaction method which uses the thermal cycler (biometra tadvanced 384 g, 230 v) and four primers (forward and reverse outer primers, forward and reverse inner primers) for amplification. this method is of acceptable specificity because the inner primers designed to be allele-specific. the amplified products of each reaction were separated on 1.5% agarose gel. automated sanger sequencing was performed to confirm the different allelic variants of cyp2c19*2 and cyp2c19*3. sequencing of the purified products was conducted with the sequencing kit according to the instructions of the manufacturer (3700 the bigdye® terminator v3.1 cycle sequencing kit, applied biosystems, usa). the sequencing was performed on abi prism 3700 genetic analyzer from applied biosystems. the sequencing results were analyzed by finch program sequencing analysis software. statistical analysis continuous data were expressed as a mean (sd) based on a normal distribution, whereas categorical data were described as numbers and percentages. for comparing nominal categorical variables that are patient-related factors, chisquared test and fisher exact test (fisher test was utilized for 2*2 cells) were used. fisher exact test was used to calculate phenotype and genotype statistics. p-values are of or less than 0.05 were considered statistically significant. statistical package for the social sciences version (spss) 23.0 was used to do all calculations. results characteristics of the patients sixty patients of 34 males and 26 females, with ages ranging from 38 to 96 years were involved in the study. the proportions of diabetes, smokers and hyperlipidemia were higher in the recurrent stroke group whereas the proportions of hypertension, ischemic heart diseases (ihds), family history and clopidogrel drug brand are nearly equal in both groups. however ace/arbs, betablockers and statins usage percentages are higher in the non-recurrent group. only the cyp2c19*2 was associated with the incidence of stroke recurrence (p=0.024), but not the cyp2c19*3 loss of function (p=1.000). the gel photographs of cyp2c19 polymorphisms are depicted in figure 1. among the rest of variables, there was also a significant correlation between the usage of aces/arbs and stroke recurrence (p=0.024). iraqi j pharm sci, vol.31(2) 2022 cyp2c19 polymorphisms in ischemic stroke patients 254 tableerror! no text of specified style in document.1. demographic and clinical characteristics of the study sample ihd = ischemic heart disease, aceis/arbs =angiotensin-converting-enzymes-inhibitors, ccbs =calcium channel-blockers, anti-dms = anti-diabetics, lof = loss of function. *significant difference using fisher exact test ≤ 0.05 level a b figure1. polymerase chain reaction and tetra arms pattern of (a):cyp2c19*2 (681 g>a) polymorphism and (b):cyp2c19*3 (636 g>a) polymorphism. arms=amplificationrefractory -mutation-system. pcr = polymerase chain reaction. wt. wild type, bpbase pair. genotype distribution in the study population (n = 60), the frequency of cyp2c19 genotypes is summarized in table 2. more than half of the participants (61.7%) are homozygotes for the wild type allele (*1/*1); 26.7 % are heterozygotes for the cyp2c19*2 allele (1*/2*); 6.7 % are heterozygotes for the cyp2c19*3 allele (*1/*3); 3.3 % homozygotes for the ( *2 / *2) ; 1.7 % are homozygotes for cyp2c19*3 (*3/*3) as shown in figure 2. in the sample population, the (*2/*3) genotype was not observed. based on these frequencies of genotypes, p-value non-recurrent group(n=30),% recurrent group(n=30),% total(n=60),% variable 0.602 gender 18 (60.0) 16 (53.3) 34(56.7) male 12 (40.0) 14 (46.7) 26(43.3) female 0.100 age, yrs. 5 (16.7) 2 (6.7) 7(11.7) <50 7 (23.3) 7 (23.3) 14(23.3) 50-59 14 (46.7) 9 (30.0) 23(38.3) 60-69 4 (13.3) 12 (40.0) 16(26.7) >70 0.176 81 ± 11.883 77 ±11.305 weight, kg (mean ± sd) 0.739 25 (83.0) 24 (80.0) 49(81.7) hypertension 0.426 10 (33.3) 13 (43.3) 23 (38.3) diabetes 1.000 4 (13.3) 3 (10.0) 7 (11.7) ihd 0.417 21 (70.0) 18 (60.0) 39 (56.0) hyperlipidemia 0.095 3 (10.0) 8 (26.7) 11 (18.3) smoking current therapy 0.024 25 (83.3) 17 (56.7) 42 (70.0) aceis/arbs 0.472 6 (20.0) 3 (10.0) 9 (15.0) beta-blockers 0.795 16 (53.3) 17 (56.7) 33 (55.0) ccbs 0.152 24 (80.0) 19 (63.3) 43 (71.7) statins 0.592 10 (33.3) 12 (40.0) 22 (36.7) anti-dms 0.706 3 (10.0) 5 (16.7) 8 (13.3) aspirin 0.739 5 (16.7) 6 (20.0) 11 (18.3) stroke family history 1.000 2 (6.7) 3 (10.0) 5 (8.3) clopidogrel drug brand 0.024 5 (16.7) 13 (43.3) 18 (30.0) cyp2c19*2 lof 1.000 3 (10.0) 2 (6.7) 5 (8.3) cyp2c19*3 lof iraqi j pharm sci, vol.31(2) 2022 cyp2c19 polymorphisms in ischemic stroke patients 255 cyp2c19*1,*2,*3 allele frequencies were 75.83%, 16.7%, and 5%, respectively. table 2. genotype distribution of cyp2c19. figure2. genotype distribution cyp2c19 polymorphism and risk of stroke recurrence poor metabolizers (pms) were found in two cases in the recurrent ischemic stroke group and one case in the non-recurrence group. thirteen intermediate metabolizers (ims) patients experienced a recurrent stroke, while seven patients had no recurrent stroke. the phenotypic distribution in both groups was not significant (p=0.176). the detailed data is portrayed in table 3.the distribution of cyp2c19*2 and cyp2c19*3 genotype in the two groups is shown in table 4, with the recurrent group accounting for the majority of the heterozygote instances (12) for the cyp2c19*2 allele. the statistical significance of this distribution was determined to be 0.039. there was one case with the (*3/*3) genotype and one case among four cases with the (1*/3*) genotype in the recurrent is group. this distribution of cyp2c19*3 genotype and phenotype was not statistically significant (p=0.617). table 3. genotype and phenotype association with recurrent ischemic stroke. phenotype genotype recurrent group(n=30),% non-recurrent group (n=30),% p-value ems gg 15(50) 22(73.3) 0.176 ims ga 13(43.3) 7(23.3) pms aa 2(6.7) 1(3.3) ems = extensive metabolizers, ims = intermediate metabolizers, pms = poor metabolizers. *significant difference using fisher exact test ˂0.05 level. table4.cyp2c19*2 and *3 genotype and phenotype association with recurrent ischemicstroke. p-value nonrecurrent group (n=30),% recurrent group(n=30),% genotype phenotype cyp2c19*2 0.039 25(83.3) 17(56.7) gg(*1/*1) ems 4(13.3) 12(40) ga(*1/*2) ims 1(3.3) 1(3.3) aa(*2/*2) pms cyp2c19*3 0.612 27(90) 28(93.3) gg(*1/*1) ems 3(10) 1(3.3) ga(*1/*3) ims 0(0) 1(3.3) aa(*3/*3) pms *significant difference using fisher exact test ˂0.05 level. cyp2c19 genotype frequency % cyp2c19*1 (*1/*1) 37 61.7 cyp2c19*2 (*1/*2) (*2/*2) 16 2 26.7 3.3 cyp2c19*3 (*1/*3) (*3/*3) 4 1 6.7 1.7 total 60 100 iraqi j pharm sci, vol.31(2) 2022 cyp2c19 polymorphisms in ischemic stroke patients 256 discussion genetic variants in cyp2c19, an enzyme essential for clopidogrel bio-activation, have been linked to lower clopidogrel antiplatelet efficacy and therefore to a higher risk of recurrence of ischemic stroke. the impact of the pharmacogenetics of the cyp2c19 polymorphisms cyp2c19*2 and cyp2c19*3 on the risk of stroke recurrence in ischemic stroke patients treated with clopidogrel for secondary prevention is reported in this study. patients with cyp2c19*2 loss of function variant allele had a greater rate of stroke recurrence than those who did not have the mutant gene. cyp2c19*1,*2,*3 allele frequencies were 75.83%, 16.7%, and 5%, respectively, while extensive metabolizers (ems), intermediate metabolizers ims, and poor metabolizers pms allele frequencies were 61.7%, 20%, and 5%, respectively. the distribution of the cyp2c19*2 allele (16.7%) was found to be similar to an earlier iraqi study which was done in 2015, on 221 people of iraqi nationality and arabic ethnicity who were not relatives with a cyp2c19*2 allele frequency of (15.2%) in addition to several studies included other middle eastern populations, such as jordanians (16%), lebanese (13.4%), and palestinians (15.5%),(2225). the cyp2c19*3 allele frequency in this study was (5%) which falls within the frequency range for asian populations (3.36 % 11.66%) which is reviewed in a meta-analysis study that included 78 original study articles (26). the frequency of cyp2c19*3 allele in the sample population of this study was 5%, which differs from prior data in another study in 2018, duhok city, in the kurdistan region that was done by mohammad and al-allawi in 2018 on 201 iraqi patients on clopidogrel undergoing percutaneous coronary intervention which didn’t document any case to carry this allele (27). however, the number of patients carrying the heterozygote genotype (*1/*3) and homozygote genotype (*3/*3) in the both groups was small to compare, therefore we cannot depend considerably on them and we need a larger sample. the data of the current study reveal that possessing the variant allele cyp2c19*2 (p=0.024) but not cyp2c19*3 (p=1.000) is associated with a potential risk for recurrent stroke and that the risk is linked with the ims and pms of cyp2c19*2 carriers (p=0.039). similar findings were obtained in a study of patients treated with percutaneous coronary intervention (pci) and dual antiplatelet therapy (dapt), which indicated a significant link between main adverse cardiovascular events )mace( and cyp2c19*2, but not cyp2c19*3 (25). in contrast to the present study's findings, a study in melbourne, australia, found that cyp2c19*17 carriers had a higher risk of ischemia events (p=0.04), but cyp2c19*2 and cyp2c19*3 carriers had no significant association with the outcomes including ischemic events (28). the explanation for the difference between these conflicting results and the findings of the present study , regarding the relation of cyp2c19*2 and *3 polymorphisms with the recurrent risk of stroke, is may be due to the difference in the allelic frequency between populations since it is highest in asian populations compared to others(26), as a result the low frequency of cyp2c19*2 and *3 in populations of australia in the last study was not sufficient to make a strong correlation with the ischemic events. with regards to the significant relation of cyp2c19*17 with ischemic outcome, although this variant is associated with enzyme hyper-functioning leading to hemorrhagic events(29), this relation can be explained in two findings present in a review article made by jiang et al, 2015, first one is that pharmacogenomics of antiplatelet intervention study (papi) founds that clopidogrel levels were similar in participants carrying the cyp2c19*17 allele and corresponding peers holding the cyp2c19*1 allele, suggesting that the cyp2c19*17 mutation has a minor impact on clopidogrel pharmacokinetics. secondly, a recent pharmacogenetics study identified a linkage between the cyp2c19*17 allele and cyp2c19*4, an lof mutation, which suggest that the high metabolic capacity of cyp2c19*17 carriers is altered if the cyp2c19*4b haplotype is also present in these individuals(10). in conclusion, the ischemic outcomes may be resulted from another factor or mechanism particularly there was no association between cyp2c19*17 polymorphism and platelets activity as mentioned earlier. many additional non-genetic factors may contribute to an increased risk of stroke recurrence such as gender and race. stroke prevalence is higher in black men than among other races(30). all patients in that study were white without significant association with sex (p=0.602). hypertension, ischemia, endothelial dysfunction, and pressure overload all promote local tissue ace production, which can lead to long-term structural alterations such as myocardial and vascular remodeling. ace inhibitors are thought to work by lowering blood pressure as a result of vasodilation and salt depletion as well as other benefits mediated by distinct vascular protective actions that lead to atherosclerosis regression or prevention(31). however, the study findings revealed a significant relationship with acei/arb medication therapy (p=0.024) and there was no significant association between recurrence risk and hypertension (p=0.739). these results were consistent with the findings of another prospective study in china 2019, done on 289 patients with ischemic stroke treated with clopidogrel for prevention where the iraqi j pharm sci, vol.31(2) 2022 cyp2c19 polymorphisms in ischemic stroke patients 257 correlation of drug therapy during follow-up (acei/arb) was significant with risk of stroke recurrence (p=0.006) and contrast with hypertension which was an independent risk factor of recurrence risk (p=0.04)(32), this can be due to that most of the hypertensive patients in the current study were of age below 60 years old, they had either hypertension alone or with one other risk factor, mostly hyperlipidemia without other comorbidities or they had controlled blood pressure. the majority of stroke prevention data in diabetic individuals comes from primary prevention rather than secondary prevention, glycemic management has been proven to lower the risk of microvascular problems, but not stroke(30). the correlation between stroke recurrence risk and diabetes in this study was not significant (p=0.426), this result was compatible with the other study results in china in patients undergoing stent plantation for cerebral artery stenosis (33). in opposite, the correlation with diabetes was significant (p=0.009) in a study that was done in spain on 209 tia patients among and ischemic heart disease patients managed with stenting and dual antiplatelet follow-up for ischemic events(34). brand clopidogrel is approved for clinical use as hydrogen sulfate (bisulfate) salt whereas generic clopidogrel is designed with different salt formulations(35). different salt forms of any active component can vary markedly in physicochemical properties, such as solubility, hygroscopicity, stability and flowability, in addition to the presence of different levels of impurities between many copies of drug, can affect the biological activity of drug (20,21). as a result, the efficacy of clopidogrel may vary with the use of brand and generic clopidogrel affecting the risk of stroke recurrence. however, the current study findings revealed that there is no difference in effect between patients using clopidogrel (p=1.000), similar to a retrospective study in usa, resulted that generic clopidogrel is as effective as the brand clopidogrel (p=0.77) for the inhibition of platelet aggregation(36),the same results also were found in other study in ontario, canada, on patients hospitalized for acute coronary syndrome including those had stroke or transient ischemic attack (tia) taking branded and generic clopidogrel where there was no significant difference between clinical outcomes of death or re-hospitalization with the type of clopidogrel used (p=0.605) for ischemic stroke or tia patients (37) . conclusion 1. this study provides certain evidence on the genetic effect of cyp2c19*2 on the risk of stroke recurrence in intermediate and poor metabolizers of clopidogrel drug in ischemic stroke patients since this variant can decrease the activity of cyp2c19 enzyme leading to decrease or even inhibit clopidogrel metabolism resulting in lowering the clopidogrel active metabolite level. as a result the antiplatelet action of clopidogrel is diminished. 2. this study affirmed that the ace/arbs drugs play a protective role in ischemic stroke patients since they are contributing to the prevention of ischemic stroke recurrence. recommendation the cyp2c19 gene polymorphisms of patients were discovered to be effective in guiding therapeutic customized antiplatelet therapy. modifying antiplatelet medication treatment based on cyp2c19 genotype such as increasing drug dosage, mixing drugs, or trying with new antiplatelet agents could be possible options to decrease clopidogrel resistance. limitations this study has a number of drawbacks. particularly, the study time length was insufficient in considering the recurrence of stroke, as well the limited sample size of the study. references 1. phipps ms, cronin ca. management of acute ischemic stroke. bmj. 2020;368. 2. cavallari lh, momary km. pharmacogenetics in cardiovascular diseases. pharmacogenomics. 2019. 133–179 p. 3. catanese l, tarsia j, 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geudelin b, et al. mortality and adverse events with brand and generic clopidogrel in the us food and drug administration adverse event reporting system. eur hear j cardiovasc pharmacother. 2019;5(4):210–5. 35. westphal es, aladeen t, vanini d, rainka m, mccadden k, gengo fm, et al. generic clopidogrel: has substitution for brand name plavix® been effective? j pharm pract. 2021; 36. ko dt, krumholz hm, tu j v., austin pc, stukel ta, koh m, et al. clinical outcomes of plavix and generic clopidogrel for patients hospitalized with an acute coronary syndrome. circ cardiovasc qual outcomes. 2018;11(3):1– 9. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(2) 2021 covid 19 and hypertension doi: https://doi.org/10.31351/vol30iss2pp23-30 23 exacerbation of covid 19 in hypertensive patients （ a review ） noor h. naser*,1 and ammar abdul aziz alibeg* * department of pharmaceutical chemistry, faculty of pharmacy, university of kufa. abstract since its discovery in december 2019, corona virus was outbreak worldwide with a very rapid rate, so it described by world health organization (who) as a pandemic. it associated with severe acute respiratory distress syndrome, and can invade the cells through angiotensin converting enzyme 2 (ace 2) receptor which play an important role as regulator for blood pressure. hypertension is a potential risk factor for sever acute respiratory syndrome covid-19, and associated with high mortality rate as shown in many epidemiological studies. a lot of published article reviews, retrospective and meta-analysis regard the association between covid-19 and hypertension. moreover, specific antihypertensive medications that infected patients were receiving are not known; only data about renin-angiotensin-aldosterone system (raas) are available. objective: to summarize the most updated data of covid-19 in hypertensive patients. keywords: covid-19, hypertension, angiotensin converting enzyme 2 (ace2) الدم ضغط ارتفاع مرضى في covid 19 تفاقم *و عمار عبد العزيز علي بيك 1*،نور هاتف ناصر .العراق ،النجف،جامعة الكوفة ،كلية الصيدلة ،فرع الكيمياء الصيدالنية* الخالصة العالمية الصحة منظمة وصفته لذلك ، للغاية سريع بمعدل العالم أنحاء جميع في كورونا فيروس انتشر ، 2019 ديسمبر في اكتشافه منذ ace) 2 لألنجيوتنسين المحول اإلنزيم مستقبل خالل من الخاليا إلى يدخل أن ويمكن ، الشديدة الحادة التنفسية الضائقة بمتالزمة يرتبط. جائحة بأنه الوخيمة الحادة التنفسي الجهاز لمتالزمة المحتملة الخطر عوامل أحد هو الدم ضغط ارتفاع ويعتبر .الدم لضغط كمنظم مهًما دوًرا يلعب والذي (2 covid-19 ، الخافضة المحددة األدوية فإن ، ذلك على عالوة. الوبائية الدراسات من العديد في موضح هو كما الوفيات معدل بارتفاع ويرتبط .(raas) األلدوستيرون-أنجيوتنسين-الرينين نظام حول بيانات فقط حيث تتوفر ؛ معروفة غير المصابون المرضى يتلقاها كان التي للضغط . ace2)) 2االنزيم المحول لالنجيوتنسين،ارتفاع ضغط الدم ،covid-19الكلمات المفتاحية: introduction coronavirus is a novel corona virus (ncov), or called corona virus disease covid-19 as it appear firstly in 2019 in wuhan, china, then it is worldwide outbreak (1,2). this virus belong to a family of positive single stranded rna (+ssrna) with a diameter ranging from (60-140 nm), which can be classified as α, β, δ, and γ (3,4). the covid19 is a type of β genus, which consist of outer capsule, from which a mushroom-like protein spike was projected and give the crown like shape for the virus, these structures facilitate the virus entry into host cell (5,6). figure 1 represents the structure of covid-19.it is highly infectious disease that attack the respiratory system and cause sever acute respiratory distress syndrome. due to the high genome similarity with sars-cov, (86.9%), so it is called (sars-cov-2) (7). figure 1. structure of corona virus (8) 1corresponding author e-mail: noorh.naser@uokufa.edu.iq received:21 /12/2020 accepted:23 / 5/2021 published online first: 2021-12-09 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp23-30 iraqi j pharm sci, vol.30(2) 2021 covid 19 and hypertension 24 since its discovery in december 2019, the virus has transmitted at an extremely high rate around the world and was caused confusion in all joints of life, including the health and economic aspects (9). pathogenesis of the disease even though much is identified about the mortality of the disease, much less is understood about the pathophysiology of the virus. in addition the anti-inflammatory mechanisms of the virus are unclear, and much of the events can be obtained depending on the previous studies on sars-cov (10). according to infected cells, covid-19 can be categorized into three different phases which are associated with different three manifestations (11). stage 1: asymptomatic stage occurs within one to two days after the virus was inhaled, which was attached to the epithelial cells in the nasal cavity and initiate replicating through ace2 receptor, which considered the main receptor for both sars-cov2 and sars-cov (12, 13). in this stage the virus targeted the ciliary cells (14), and this hypothesis require some clarification because single-cell rna indicates low levels of ace2 expression in the conduction of airway cells and no evidence predilection for the cell type (15). stage 2: upper airway stage in this stage, the virus proliferates and migrates down the respiratory tract along the conducting airways, which alert more intensive innate immune reaction. at this time the disease became clinically apparent, and the virus sarscov-2, should be discharged in nasal swabs and sputum secretion (16). at this level cxcl10 (an interferon responsive gene that has an excellent signal to annoyed ratio in the alveolar type ii cell responsive to both sars-cov and influenza). evaluating the innate immune response of the patient enhance forecasts of the disease and lead to rapid monitoring (17). the disease may be moderate in about 80% of the infected patients, and predominantly confined to the upper and conductive airways and by ending this stage, the disease can develop into the third stage (pneumonia). the patients with conserved symptomatic therapy may be monitored at home (18). stage 3: hypoxia, ground-glass infiltration only 20% of the infected patients with covid-19 will progress to stage 3, which associated with pulmonary infiltration, and some of these will be associated with very severe illness(19). this stage associated with maximum production of pro-inflammatory and anti-inflammatory cytokines as interleukin-2 (il2), interleukin-7 (il7), interleukin-10 (il10), and tumor necrotic factors-α (tnfα) (20). at this stage the virus hits the gas exchange system of the lung and penetrate type ii alveolar cells, the virus replicates and divided rapidly within these cells, and generate huge number of viral particles, then the cell induce apoptosis and perish. the outcome is possibly self-replicating pulmonary toxins, as the viral particles that generated infected type ii cells in adjacent unit (21). the pathological outcome of the virus is a wide spread alveolar injury with fibrin-rich hyaline membrane and a scare of multinucleated giant cells. the intensive scaring and fibrosis may progress to abnormal lung tissue repair (22). therefore an aggressive and innate immune response and epithelial regeneration will be required for rehabilitation. so, the patients with depleted immune response may permit the viral disseminate to the lung gas exchange units very readily (23). renin-angiotensin-aldosterone system (raas) renin-angiotensin-aldosterone system is one of the most important hormonal mechanisms that regulates the body hemodynamic, through regulation of blood pressure, fluid volume, and na+k+ balance. therefore any disturbance in one of the system biomolecules lead to alteration in the body hemostasis and blood pressure developing (24). renin is a hormone synthesized in the kidney and release to the circulation in response to hypotension and low intra-tubular sodium level, this hormone responsible for conversion of angiotensinogen to angiotensin i (ang i), which cleaved by angiotensin converting enzyme (ace) to give angiotensin ii (25). aldosterone is another biomolecule that affecting the body homeostasis, which play an important role in the reabsorption of sodium ions, potassium excretion, and water retention from the distal nephrons of the kidney, by that modulate the extracellular space volume and blood pressure (26). angiotensin converting enzyme (ace) is a membrane bounded glycoprotein, which play an important role in the blood pressure homeostasis, through negative modulation of renin-angiotensin aldosterone system (raas) converting of angiotensin i into angiotensin ii, which is potent vasoconstrictor that associated with elevation in blood pressure (27). due to the vital role of these biomolecules in regulation of body homeostasis, therefore most patients with elevating blood pressure, and cardiovascular diseases are treated with angiotensin receptor blockers (arbs), and angiotensin converting enzyme inhibitors (ace-i) (28), a major concern was raised about the safety and/ or persistent beneficial effects of these drugs in sarscovid-19 infected patients (29). it was suggested that the binding of sars-cov-2 with ace2 in hypertensive patients is an important factor for covid-19 exacerbation and associated with increased mortality (30,31,32). as hypertension and cardiovascular disease are important risk factors for severity and mortality in covid-19 infected patients and considered as targets that must be iraqi j pharm sci, vol.30(2) 2021 covid 19 and hypertension 25 intensively addressed in the management of covid-19 (33). physiological role of angiotensin converting enzyme2 (ace2) angiotensin converting enzyme-2, (ace2) is a trans-membrane glycoprotein (monocarboxypeptidase), which is a homologue of ace, which responsible for conversion of ang. ii into its protective metabolites, ang1-7 (34). in addition it converts the angiotensin 1 into ang1-9, which are then converted by ace and ace2 into ang1-7 (35,36). by these mechanisms ace2 can suppress the effect of raas and reduce vasoconstriction and cardiac remodeling(37). angiotensin converting enzyme2 (ace2) as entry receptor for covid-19 the recent researches reported that sarscov2 can interact and block the ace2, therefore they may represent a therapeutic option for covid19 (38,39). the entry of the virus into the human cells occur through binding of the viral spikes with the rbd (receptor binding domain) of ace2 (angiotensin converting enzyme2) (40), which is highly expressed in the type ii alveolar cells and lymphocytes (41), and can be expressed in the blood vessels (42), kidney (43), and gastrointestinal tract especially (esophagus, stomach, colon (44), ileum, and rectum) (45). this lead to internalization of ace2 , after the binding and endocytosis of covid 19, the ace2 will reduce, and subsequent increase in atii accumulation (46). the spike protein then undergo proteolytic cleavage by trans-membrane protease serine 2 (tmprss2) enzyme (47), which allows fusion to the cell (48). such binding determines viral entry and cell injury, which is directly proportional to ace2 expression (49,50). figure 2. role of ace2 in the entry of covid 19 into the host cells (51). the association between hypertension and covid 19 the association between hypertension and covid 19 was concern of many studies (52,53). since the pathogenesis of covid 19 interact with ace2 receptors a one of the components of reninangiotensin system (ras), which is the key of blood pressure regulation (54). ace2 is the receptor that mediates the invasion of covid 19 into the cells by a spike (s) glycoprotein-ace2 binding pathway (55). after infection, the ace2 level was reduced due to the binding with the spike protein of covid 19, resulting in an imbalance between ace1 and ace2 (56). the renin-angiotensin iialdosterone axis was recognized as a key regulator of blood pressure in the hypertensive patient, with angiotensin ii regulated through ace (57). thus, due to imbalance between ace1 and ace2 result from viral infection, the hypertensive patients tend to appear more serious organs injury (58). in hypertensive covid 19 patients more sever clinical types of mortality were observed, leading to a suggestion that covid 19 associated the clinical outcome of covid 19 (59). f. xei et al study, found that 31% of hypertensive patients also have other forms of cardiovascular diseases, associated with increased risk of death in covid 19 patients (60). accordingly, preexisting hypertension, rather than cardiovascular disease was considered the underlying cause of increased susceptibility to rapid progression of the disease, and more sever covid 19 infection (61,62). wu et al. (63) and zhou et al.(64) had found hypertension to have a hazard ratio of 1.70 and 3.05 for death in 201 and 191 patients with covid-19, respectively. there is an important question, whether angiotensin converting enzyme-inhibitors (ace-i), and angiotensin receptor blockers (arb), which are a group of important antihypertensive agents medications, have favorable impact on the patients infected with sars-cov-2, or associated with deleterious effect (65), because it was found that ace-i, and arb induced over expression of ace2 receptors, which are facilitating the entry of the virus to the host cell, and propagation of the cell injury. raas activation plays a major pathogenic role in hypertension through hemodynamic actions and cytokines and intracellular signaling pathways, which lead to adverse cellular effect result in systemic damage. many hypothesis have been raising about which is more beneficial or should withdrawing the medications? although the number of fatal covid-19 positive patients treated with ace-is was more than twice the number of those treated with arbs, it cannot absolutely conclude the risks or benefits of using such medications, due to iraqi j pharm sci, vol.30(2) 2021 covid 19 and hypertension 26 the association of other factors as age, environment, and impact of unidentified comorbidities on outcome with the covid-19 patients (66,67). ace2 alleviates the vasoconstriction, and pro-fibrotic effect of angiotensin-ii through its degradation and by counteracting its action through formation of ang 1-7. the high expression of ace2 in the cardiovascular system, type ii alveolar cells, and enterocytes, demonstrates its essential role in the cardiovascular and immune systems. sanchis gomar et al study, suggested that the usage of arbs may be a better treatment option for hypertensive patients with covid-9 patients at higher risk of sever forms of the disease due to the equal efficacy, but much more fewer side effects than that of aceis(68,69). g. chao et. el. (70) and v. muthiah (71) had found that abrupt withdrawal of ace-i and arbs, in high risk patients, such as those with heart failure or myocardial infarction may result in clinical instability and adverse health outcomes. so, these studies suggested that the raas inhibitors should be continued in patients in otherwise stable conditions who are at risk for, being evaluated for, or with covid-19 (72,73). figure 3. effect of sars-cov-2 on physiological action of renin-angiotensin-aldosteronesystem. 1. reduction of ace2 during corona virus infection 2. increase ace2 and ang (1-7) after ace-is and arbs administration (74). ace: angiotensin converting enzyme, ace2: angiotensin converting enzyme2, arb: angiotensin ii receptor blockers, at1-r: angiotensin ii receptor type i, mas r: mas receptor, no: nitric oxide, olp: oligopepidase and neprilysin (in blood circulation). conclusion the entry of sars-cov-2 into the cells occur through protein binding domain (pbd) of the ace2 receptor, which 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angiotensin-converting enzyme inhibitors/angiotensin receptor blockers on onset and severity of severe acute respiratory syndrome coronavirus 2 infection. j am heart assoc. 2020;9:e016219. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31( suppl. ) 2022 pharmacokinetics of fluoxetine doi: https://doi.org/10.31351/vol31isssuppl.pp153-161 153 effect of food on the pharmacokinetics of fluoxetine in healthy male adult volunteers(conference paper )# duaa jaafar jaber al-tamimi*,1, khalid kadhem al-kinani**, salam shanta taher** and ahmed abbas hussein** # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of pharmacy, al-nisour university college, baghdad, iraq. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. ***baghdad college of medical sciences, baghdad, iraq. abstract fluoxetine (fx) is an antidepressant drug administered only orally in humans. despite the wide use of fx, until now, there is only limited number of literature concerning the pharmacokinetics (pk) of fx and the effect of food on its pk. the objective of this investigation was to study the pk of fx in arabic healthy male adult volunteers under fasting and fed conditions. the study was designed as a single dose , two periods. for both conditions; fx 20 mg capsules (prozac®, eli lilly, canada) were administered to 41 volunteers. blood samples were drown from each volunteer immediately before dosing (zero time) and then after different time intervals after dosing (one hr. interval for the first 12 hrs., then 12 hrs. interval from 12-72 hrs. and finally 24 hrs. interval from 72-144 hrs.). in fasting study, fasting was overnight for 12 hrs. while, the fed study was conducted after 90 days wash-out period following the completion of the fasting study. the same subjects who received fx in the fasting study were administered the drug directly after a fatty breakfast. the concentrations of fx were assessed in the plasma samples obtained from each volunteer by rapid, accurate, precise, sensitive, and specific method. the pk parameters were calculated according to standard methods using non-compartmental data analyses using kinetica software. the mean ± sd values of cmax, tmax, auc0-t, auc0-∞, kel, t0.5, mrt, cl/f and vd/f of fluoxetine 20 mg capsules under fasting conditions were 11.5 ± 3.0 ng/ ml, 5.4 ± 1.6 hr, 408.9 ± 177.1 ng.hr/ml, 453.8 ±192.2 ng.hr/ml, 0.022 ± 0.005 hr-1, 33.5 ± 7.4 hr, 51.1 ± 11.2 hr, 58.3 ± 33.0 l/h and 2845 ± 1807 l, respectively. the corresponding values of these parameters for fluoxetine 20 mg capsules under fed conditions were 10.9 ± 3.3 ng/ ml, 6.7 ± 1.9 hr, 329.1 ± 181.0 ng.hr/ml, 417.6 ±192.2 ng.hr/ml, 0.022 ± 0.008 hr-1, 35.5 ± 11.6 hr, 51.9 ± 10.8 hr, 58.8 ± 24.2 l/h and 2482.4 ± 1371 l respectively. the current investigation demonstrated no statistical differences in the fx pharmacokinetic parameters cmax, auc0–t, auc0–∞, kel, t1/2, mrt, cl/f, and vd/f after fasting compared to the fed conditions, whereas there was statistically significant elongation in the tmax values after food intake. therefore, this study concludes the absence of food effect on the pk of fx (except tmax) in the arabic population and confirms the method of administration mentioned in the product information but also concludes high interindividual variation in fx exposure (auc), which suggest that therapeutic drug monitoring (tdm) might be advisable when feasible. keywords: fluoxetine, pharmacokinetics, food effect, arabic volunteers. #) بحث مؤتمر ( تأثير الغذاء على الحرائك الدوائية للفلوكستين في المتطوعين البالغين األصحاء *** عباس حسين واحمد** سالم شنته طاهر ،**خالد كاظم الكناني ،1*،دعاء جعفر جابر التميمي 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي العراق. بغداد، ، ةالجامعكلية النسور الصيدلة، قسم * العراق بغداد، بغداد، جامعة الصيدلة، كلية الصيدالنيات، فرع ** العراق. بغداد، الجامعة، كلية بغداد للعلوم الطبية الصيدلة، قسم *** الخالصة ، حتى اآلن ، ال يوجد fx البشر. على الرغم من االستخدام الواسع لـدواء مضاد لالكتئاب يتم تناوله عن طريق الفم فقط في (fx) الفلوكستين الخاصة به. كان الهدف من هذه الدراسة هو دراسة حركية الدواء وتأثير الطعام على fx لـ (pk) سوى عدد محدود من األدبيات المتعلقة بحركية الدواء ف الصيام والتغذية. صممت الدراسة كجرعة وحيدة على فترتين. لكال الظرفين تم في متطوعين بالغين أصحاء عرب في ظل ظرو fx لـ حركية الدواء متطوًعا. تم سحب عينات الدم من كل متطوع مباشرة قبل تناول الجرعات )صفر مرة( ثم بعد 41إلى (prozac®, eli lilly, canadaإعطاء كبسوالت) ساعة(. 144-72ساعة. فترة من 24ساعة ، وأخيراً 72إلى 12ساعة ، من 12ساعة ، ثم 12فترات زمنية مختلفة بعد الجرعات )فترة ساعة واحدة ألول يوم من فترة الغسل بعد االنتهاء من دراسة الصيام. 90ساعة. بينما أجريت دراسة التغذية بعد 12في دراسة الصيام ، كان الصيام بين عشية وضحاها لمدة في عينات البالزما التي تم fx الصيام تم إعطاؤهم الدواء مباشرة بعد وجبة إفطار دسمة. تم تقييم تركيزات في دراسة fx نفس األشخاص الذين تلقوا وفقًا للطرق القياسية باستخدام تحليالت البيانات الحركية الدوائية وحساسة ومحددة. تم حساب معلمات الحصول عليها من كل متطوع بطريقة سريعة ودقيقة و t0.5 و kel و ∞-auc0 و auc0-t و tmax و cmax لـ االنحراف المعياري ± كانت قيم المعدل kinetica خدام برنامجغير المقسمة باست mrt و cl / f و vd / f 177.1± 408.9ساعة ، 1.6± 5.4نانوغرام / مل ، 3.0± 11.5مجم تحت ظروف الصيام 20لكبسوالت فلوكستين 58.3ساعة ، 11.2± 51.1ساعة ، 7.4± 33.5، 1-ساعة 0.005± 0.022نانوغرام ساعة / مل ، 192.2± 453.8نانوغرام / ساعة / مل ، . لتر ، على التوالى 1807± 2845لتر / ساعة ، ±33.0 1corresponding author e-mail: duaa.jaafaraltamimi@yahoo.com received: 23/5 /2022 accepted: 23/10 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp153-161 iraqi j pharm sci, vol.31( suppl. ) 2022 pharmacokinetics of fluoxetine 154 181.0± 329.1ساعة ، 1.9± 6.7نانوغرام / مل ، 3.3± 10.9مجم تحت ظروف التغذية 20كانت القيم المقابلة لهذه المعلمات لكبسوالت فلوكستين 58.8ساعة ، 10.8± 51.9ساعة ، 11.6± 35.5، 1 -ساعة 0.008± 0.022نانوغرام / ساعة / مل ، 192.2± 417.6نانوغرام / ساعة / مل ، لتر على التوالي. أظهرت الدراسة الحالية عدم وجود فروق ذات داللة إحصائية في معامالت الحرائك الدوائية 1371± 2482.4لتر / ساعة و ±24.2 مقارنة بظروف التغذية ، بينما كان هناك إحصائيًا بعد الصيام vd / f و cl / f و mrt و t1 / 2 و kel و auc0 و auc0 t و fx cmax لـ في tmax باستثناء fx لـ الحركية الدوائية بعد تناول الطعام. لذلك ، خلصت هذه الدراسة إلى عدم وجود تأثير غذائي على tmax زيادة كبيرة في قيم fx (auc)ج أيًضا تباينًا كبيًرا بين األفراد في التعرض لكمية دواء الـاالشخاص العرب وتؤكد طريقة االعطاء المذكورة في معلومات المنتج ولكنها تستنت .عندما يكون ذلك ممكنًا (tdm)، مما يشير إلى أن قد يكون من المستحسن تطبيق المراقبة الدوائية حركية الدواء ، تأثير الغذاء ، متطوعون عرب الكلمات المفتاحية: فلوكستين ، introduction fluoxetine is indicated in adults for the symptomatic relief of various types of depression involving major depressive disorder, obsessivecompulsive disorder and some eating disorders (bulimia nervosa). moreover, fx should only be used in combination with psychological therapy when prescribed to children or young persons with moderate to severe major depressive disorder. the usual initial dosage is 20 mg, administered once daily in the morning. if the expected clinical improvement does not take place after a trial period of several weeks, a gradual dose increase is recommended and should not exceed a maximum of 60 mg daily (1, 2). chemically, fluoxetine base (fx) is unrelated to tricyclic, tetracyclic or other available antidepressant agents. fluoxetine is the first highly specific phenylpropylamine derivative belonging to the selective serotonin reuptake inhibitors (ssri) class (1). it facilitates serotoninergic neurotransmission through potent and selective inhibition of presynaptic serotonin reuptake. fluoxetine gained approval by usfda in 1987. the molecular formula of fx is c17h18f3no, and its molecular weight is 309.33 g/mol while that of fluoxetine hydrochloride is c17h19clf3no, and its molecular weight is 345.79 gm/mol. fluoxetine base is very lipophilic with logp equal to 4.05. each 20 mg prozac® capsule contains 22.36 mg of fx hydrochloride which equivalent to a 20 mg fx-free base (1, 2). oral fx is well absorbed from gastrointestinal tract (git) with bioavailability (ba) of less than 90% due to hepatic first-pass metabolism. peak plasma levels (cmax) of 15-55 ng/ml were achieved after 6 to 8 hours of a single 40 mg dose. the cmax of the fx 20 mg capsule was 11.754 ng/ml. the fx capsule is bioequivalent to the oral solution; however, the fx tablet demonstrates lower ba than the capsule formulation making the capsule slightly more effective than the tablet dosage form. fluoxetine exhibits a nonlinear pk profile; therefore, it should be used with caution in patients with hepatic dysfunction. the age does not affect the pk of fx. besides, the drug exhibits a better tolerability profile than tricyclic antidepressants making fx particularly appropriate for elderly patients with depression. moreover, the pk of fx is not influenced by renal impairment or obesity (3-5). fluoxetine is extensively metabolized by demethylation in the liver to the active metabolite norfluoxetine and other unidentified metabolites. the fx’s terminal elimination half-life (t1/2) is 4 to 6 days, and for its active metabolite, norfluoxetine is 4 to 16 days (3) . the pharmacological activity of norfluoxetine is similar to the parent drug fx, and it contributes to the long duration of action of fx. this unique property makes the drug provide an antidepressant effect by less frequent administration (once weekly) than the usual daily doses. the apparent volume of distribution (vd) of fx and its metabolite are high and vary between 20-42 l/kg. the total body clearance (cl) of fx is reported to be 0.576 l/hr/kg. fluoxetine is highly plasma protein bound (about 94%), allowing fx and its active metabolite (norfluoxetine) to be distributed to the brain. the fx is mainly eliminated in the urine. about 60% of an oral fx dose is excreted in urine within 35 days, and about 12% of the dose is excreted in the feces within 28 days (3-5). fluoxetine (as the hydrochloride salt) is administered in oral dosage forms only and is available as 20 mg tablets, 20 mg capsules, delayedrelease capsules (hard-shelled and soft gel), and as a solution containing 20 mg/5 ml. the tablet is presented as a quick-release, delayed-release, chewable, or orally dissolving tablet (odt), which breaks down in saliva. the chewable and odt dosage forms can be helpful for patients having trouble or difficulty swallowing (3-5). other dosage forms are under investigation, such as transdermal (6), fast dissolving film (7), and other drug delivery systems (8, 9). knowledge of drugs pk is essential to obtaining safe and effective drug products. besides, the administration of medications with food may significantly impact the rate and/or extent of drugs' absorption and, consequently, their ba. therefore, several pk/ba/be investigations were conducted to elucidate the pk characteristics of different drugs and dosage forms in the arabic population. among these investigations are studies that have been published recently for antibacterial drugs such as azithromycin (10), cefixime (11), levofloxacin (12), cefuroxime (13), fluconazole tablet and capsule (14, 15); immunosuppressant drugs as cyclosporine (16); drugs indicated for benign prostatic hyperplasia like doxazocin (17); drugs indicated for ménière’s disease primarily betahistine (18) and antihypertensive agents iraqi j pharm sci, vol.31(suppl.) 2022 pharmacokinetics of fluoxetine 155 such as amlodipine, valsartan, hydrochlorothiazide (19). the current study was conducted to demonstrate the pk of fx in arab healthy male adult volunteers under fasting and fed conditions to see whether there is a necessity to change the dosage regimen and method of administration of fx in the arabic population. materials and methods ethical commitments a study protocol containing all the details of this research involving the informed consent form was issued by the principal investigator according to the international council for harmonization (ich) guidelines for good clinical practice (gcp) and the recent version of the helsinki declaration (20-22). the protocol was reviewed and approved by the clinical investigator and the institutional review board/ethical committee (irb/ec) before conducting the research. any alterations and/or amendments in any section of the protocol suggested by the clinical investigator and/or the irb/ec which may have a remarkable impact on the study conduct were made by the principal investigator, followed by further approval by the clinical investigator and the irb/ec; unless if the suggested alterations or amendments were regarded minor according to the principal investigator’s decision such as logistical or administrative issues. informed consent each volunteer, together with two witnesses in addition to the clinical investigator, personally signed two original copies of the consent form, one copy was delivered to the volunteer, and the other copy was saved in the research file as part of the source documents. according to the study protocol and based on the principal and/or clinical investigator’s decision, any volunteer’s participation was terminated at any time during the research according to certain criteria. these criteria include the following: violation and/or bad compliance of the volunteer with the protocol, and in the case continued participation may impact the volunteer’s health such as illness, and abnormal vital signs were detected. moreover, any volunteer was allowed to withdraw from the study due to any personal reason without undue delay. volunteers forty six arabic adult male healthy volunteers were screened in the study based on the following inclusion criteria: ages between 18-50 years; body mass index of 18-30 kg/m2; nonsmokers or light smokers (consume less than 10 cigarettes per day); non-vegetarians, no previous allergic responses to fx and related compounds, no hospitalization, blood donation or participation in any clinical trials such as ba/be/pk two months before the current study; the volunteers had normal physical and clinical examinations including electrocardiograms (ecg), vital signs (systolic/diastolic blood pressure, pulse and temperature), absence of cardiovascular, pulmonary, renal, hepatic, gastrointestinal, hematological, endocrinal, immunological, dermatological, neurological and psychiatric diseases; no evidence of poor motivation, antagonistic personality, emotional or intellectual problems that may impact the validity of the volunteer’s consent to participate in the study or limit the ability to comply with requirements of the study protocol. moreover, the volunteers had normal clinical laboratory tests involving complete hematological and biochemical profiles, routine urinalysis, negative virologic tests (hiv, hepatitis b and c) and no alcohol and drug abuse. clinical procedures and dose administered the study was designed as a single dose , two periods, in which the same subject were given the drug under fasting and fed study separated for 90 days wash out interval between dosing. in the fasting study, the volunteers were admitted to the clinical site the evening before fx administration day at about 6:00 p.m., and then a standard dinner was served at 8:00 p.m. the volunteers were confined to the clinical site for 24 hours post drug administration. after overnight fasting of 12 hours (at 8:00 a.m.), the volunteers were given a single dose of the investigational drug prozac® capsules (eli lilly, canada) containing 20 mg fx with 240 ml of potable water. in the fed study, fx capsules were administered to the same volunteers who received the drug in the fasting study after a washout period of 90 days after ending the fasting study by applying the same clinical procedures mentioned above, except fx capsules, were administered to the volunteers directly after serving a fatty breakfast. food and drink were not allowed until 4 hours after dosing (other than water, which was allowed 2 hours after drug intake). standard lunch and dinner were served after 4 and 8 hours of fx administration, respectively. beverages containing caffeine were banned 12 hours before dosing and up to 24 hours post-dosing. grapefruit-containing beverages were prohibited 48 hours pre-dosing and until the end of both the fasting and fed studies. the volunteers were allowed to sit or walk around, but they were forbidden from strenuous activity. besides, the volunteers were not allowed to lay or sleep during the first 4 hours of fx intake. approximately 5 ml blood samples were drawn from each volunteer to determine fx concentration in plasma. each blood sample was placed into an evacuated heparinized glass tube through an indwelling cannula inserted in the forearm vein. the sampling time table was as follow: 0 (immediately before fx administration), then at 1, 2, 3, 4, 5, 6, 7, iraqi j pharm sci, vol.31(suppl.) 2022 pharmacokinetics of fluoxetine 156 8, 9, 10, 12, 24, 36, 48, 60, 72, 96, 120 and finally at 144 hours after dosing. the blood samples were centrifuged at 3500 rpm for 10 min and the separated plasma was transferred directly into two plastic tubes in approximately equal aliquots and stored frozen at -25±5 ◦c pending measuring fx levels in the plasma. in-house procedure proved that the storage temperature (-20±5 ◦c) was adequate for saving fx stable for two months. the actual clock time for each blood sample was recorded. directly after each blood sample withdrawal, normal saline (0.5 ml) containing 20 units of heparin per ml was injected into the cannula to push the residual blood and prevent blood clotting. about 0.2 ml of blood was discarded from the cannula before each blood sample withdrawal to eliminate any residual blood in the cannula. twenty blood samples were sampled from each volunteer. the whole blood volume withdrawn from each volunteer for the fasting study was approximately 120 ml, including the blood obtained for screening and clinical laboratory testing at the last blood sample taken at 144 hours post-dosing before the volunteer’s discharged from the fasting study and the blood taken before one day of admission to the fed study. the same blood volume was withdrawn from each volunteer for the fed study. since the volume of blood (120 ml) sampled during six days for each of the fasting and the fed studies and the washout interval between both studies is about three months, thus there is no physiological concern regarding the total volume of blood taken from each volunteer. a confidential system for tube labeling was applied based on in-house standard operating procedure (sop) referring to the drug name (fx), the volunteer’s identification number, and a number indicating the time of each blood/plasma sample withdrawal. safety evaluation the entire clinical phase of the fasting and fed studies, including volunteer admission, drug administration, blood sampling and safety evaluation of fx, was accomplished by a qualified medical crew and under the supervision of the clinical investigator. also, the qa personnel monitored all clinical procedures to assure adherence to the study protocol. the vital signs (blood pressure, pulse, and temperature) were recorded for each volunteer almost one hour before fx administration and then at 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, 72, 96, 120 and ultimately upon volunteer’s discharge at 144 hours after dosing. moreover, laboratory tests for hematology, biochemistry, and routine urinalysis were achieved at the end of both the fasting and the fed studies. the clinical investigator, together with the clinical team, handled and recorded any adverse events (ae), adverse drug reactions (adr), and serious adverse effects (sae). in addition to that, several safety measures were taken into consideration, such as the availability of clinical facilities to handle any urgent case beyond the capability of the clinical site. fluoxetine determination the concentrations of fx were assessed in the plasma samples obtained from each volunteer by a previously described analytical method (23) following fda bioanalytical method validation guidance (24, 25). the method was proved to be rapid, accurate, precise, sensitive, and specific to determine fx in plasma. the linearity of the method was established for fx concentrations ranges of 0.05-100 ng/ml with a lower limit of quantification of 0.05 ng/ml and a lower limit of detection of 0.03 ng/ml (23). a standard calibration curve involving a blank matrix was generated for each bioanalytical run to assess fx concentrations in the unknown authentic plasma samples. no determination was done by extrapolation below or above fx concentrations ranges of 0.05-100 ng/ml established in the standard calibration curve. pharmacokinetic computations kinetica software was used for pharmacokinetic (pk) calculations and statistical analysis, including anova tests and descriptive statistics. microsoft excel was applied for data plotting. from each volunteer’s fx plasma concentration-time data, the pk parameters were calculated according to standard methods using noncompartmental data analyses (26, 27). the following pk parameters were computed for each volunteer: cmax = maximum or peak blood concentration. tmax = time at which cmax occurs. both cmax and tmax were obtained directly from the concentration-time profile of each volunteer. kel = terminal elimination rate constant estimated by linear regression of at least three points of log-plasma concentrations versus time in the last terminal elimination phase. t1/2 = terminal elimination half-life calculated from 0.693/k. auc0-t = area under concentration-time curve measured by trapezoidal rule from time zero to the last time (tlast) of blood sampling. clast = last measurable fx concentration at tlast that meets or above the lower limit of quantification (0.05 ng/ml). auct-∞ = extrapolated area under plasma concentration-time curve from tlast to infinity calculated from clast/kel. this area is also termed aucextrapolated, aucresidual or auctail. auc0-∞ = area under the concentration versus time curve from t0 to t∞ equal to auc0-t + auct-∞. the % extrapolated auc was calculated as 100 x (auct-∞/auc0-∞). mrt = mean residence time equivalent to aumc/auc; aumc is the area under the moment curve. cl/f = total body clearance measured from fx dose (20 mg) divided by auc0-∞ of each volunteer. f is the absolute bioavailability. vd/f = apparent volume of distribution of fx calculated from cl/kel of each volunteer. iraqi j pharm sci, vol.31(suppl.) 2022 pharmacokinetics of fluoxetine 157 results and discussion volunteers due to the relatively slow absorption rate (tmax range from 2-9 hours) and long t1/2 of fx, which was reported to be about two days (3-5, 2830), this study was designed to sample the drug in blood frequently and for 144 hours after fx administration to obtain a reliable and clear description of the pk behavior of the drug in the body including the absorption and disposition phases. forty-six volunteers were screened for the study; two of them were medically not eligible to be enrolled in this research, one volunteer did not come to admission the day before fx administration at the fasting study, one volunteer withdrew during the fasting study, and another withdrew during the fed study due to personal causes; thus, the remaining 41 volunteers completed both the fasting and the fed studies. clinical procedures the current trial was executed according to a study protocol based on ich guidelines for good clinical practice (20, 21), the declaration of helsinki (22), fda guidance on bioanalytical method validation (24, 25), and the standard methods for pk data analysis (26, 27). no violation of the study protocol was reported throughout all study phases. the usual initial therapeutic dose of the fx capsule is 20 mg, administered to patients once daily (1-5). if no clear clinical improvement occurs after a trial period of several weeks, it is recommended to escalate the dose gradually, provided that it should not be more than 60 mg daily (1-5). therefore, in the current research, the dose strength (20 mg fx capsule) was decided to be evaluated to balance fx blood concentrations for reliable determination of the pk parameters of the drug on the one hand and to expose the volunteers to a safe therapeutic dose on the other hand. fluoxetine pharmacokinetics figure 1 shows fx plasma concentrationtime profiles after fasting and fed conditions (each point is represented as a mean±sd). while figure 2 and figure 3 show a direct comparison in mean fx plasma concentrations versus time curves between fasting and fed studies. as can be seen in figure 1, fx demonstrates clear interindividual variabilities in its plasma concentrations at all data points of the absorption and disposition phases for fasting and fed conditions. fluoxetine reveals a slow absorption phase, followed by relatively slow distribution and then a long terminal elimination phase under both fasting and fed states. the absorption rate of fx seems to be delayed when the fx capsule was given directly after a fatty breakfast (fed study) compared to the absorption rate seen when the drug was administered on an empty stomach. figure 1. plasma concentration-time profiles of fluoxetine after administering fluoxetine 20 mg capsules to healthy adult arabic subjects under fasting (a) and fed (b) conditions (each point is mean±sd , n=41 for each data points). iraqi j pharm sci, vol.31(suppl.) 2022 pharmacokinetics of fluoxetine 158 figure 2 .comparison of mean plasma concentration-time profiles (t=0-24 hr) of fluoxetine after administering fluoxetine 20 mg capsules to healthy adult arabic subjects under fasting and fed states. figure 3. comparison of mean plasma concentration-time profiles (t=0-144 hr) of fluoxetine after administering fluoxetine 20 mg capsules to healthy adult arabic subjects under fasting and fed states. the pk of fx resulting from the fasting and the fed studies are also enlisted in the given table below. the mean ± sd values of cmax, tmax, auc0-t, auc0-∞, kel, t0.5, mrt, cl/f and vd/f of fluoxetine 20 mg capsules under fasting conditions were 11.5 ± 3.0 ng/ ml, 5.4 ± 1.6 hr, 408.9 ± 177.1 ng.hr/ml, 453.8 ±192.2 ng.hr/ml, 0.022 ± 0.005 hr-1, 33.5 ± 7.4 hr, 51.1 ± 11.2 hr, 58.3 ± 33.0 l/h and 2845 ± 1807 l, respectively. the corresponding values of these parameters for fluoxetine 20 mg capsules under fed conditions were 10.9 ± 3.3 ng/ ml, 6.7 ± 1.9 hr, 329.1 ± 181.0 ng.hr/ml, 417.6 ±192.2 ng.hr/ml, 0.022 ± 0.008 hr-1, 35.5 ± 11.6 hr, 51.9 ± 10.8 hr, 58.8 ± 24.2 l/h and 2482.4 ± 1371 l respectively. visual inspection of table 1 indicated that fx exhibit low interindividual differences in both fasting and fed conditions (cv% about 30%) for the pk parameters cmax, kel, tmax, t1/2, and mrt (table 1). higher intersubject variabilities (cv% ranges 42-64%) were found for the parameters auc0–t and auc0–∞, cl/f, and vd/f. the same findings were reported in other literature (3-5, 28-30). the current research demonstrated that the computed % extrapolated auc had a small contribution to the total auc (a mean = 5.7 ng.hr/ml, a range of 1.3-13.8 ng.hr/ml) in the fasting study and (a mean = 6.2 ng.hr/ml, range of 1.6-14.9 ng.hr/ml) in the fed study. these results indicated that 144 hours of blood sampling after fx administration and a lower limit of quantification of fx in plasma (0.05 ng/ml) were quite enough for a complete description of the concentrations versus time profiles of the drug and reliable determination of all pk characteristics of fx including the absorption and the terminal elimination phases for both fasting and fed studies. thus, due to the high interindividual differences in the total drug exposure (auc) after fasting and fed states as shown in the table given later which may lead to high interindividual differences in the extent of drug effectiveness, the current investigation suggests that fxs therapeutic drug monitoring (tdm) can be useful in clinical practice to obtain safe and effective plasma concentrations. the tdm of antidepressant agents has been declared as an important tool for demonstrating therapeutic and economic advantages. recently, efforts have emphasized establishing more effective and safer regimens for antidepressants like fx by considering the individual pk properties of the patients. accordingly, tdm of fx has been recommended for problems solving such as therapeutic failure and/or toxicity, dose titration, special populations (like children, pregnant women and elderly patients), a certain situation such as the high potential for clinically relevant drug interactions and pharmacokinetically important and special circumstances (31-37). statistical analysis applying anova tests to compare the pk parameters of fx obtained after the fasting study in comparison to the corresponding parameters obtained after the fed study as shown in the table given below.the p values obtained from anova tests for the pk parameters after fasting against the corresponding parameters obtained after the fed condition were 0.42853, 0.0020779, 0.69204, 0.68997, 0.99092, 0.39977 and 0.73251 for the parameters cmax, tmax, auc0–t, auc0–∞, kel, iraqi j pharm sci, vol.31(suppl.) 2022 pharmacokinetics of fluoxetine 159 t1/2, and mrt, respectively. the results demonstrated no statistical difference (p < 0.05) for all the pk parameters cmax, auc0–t, auc0–∞, kel, t1/2, mrt, cl/f and vd/f except the parameter tmax which showed significant difference (p > 0.05) for the fasting versus the fed states due to delay in the absorption rate of fx after food intake. thus, fx onset of action delay would be expected if fx capsules are taken with food. table1. pharmacokinetic parameters of fluoxetine 20 mg capsules under fasting and fed conditions fasting stat cmax (ng/ml) tmax (hr) auc0-t (ng.hr/ml) auc0-∞ (ng.hr/ml) kel (hr-1) t0.5 (hr) mrt (hr) cl/f (l/h) vd/f (l) mean 11.5 5.4* 408.9 453.8 0.022 33.5 51.1 58.3 2845 ±sd 3.0 1.6 177.1 192.2 0.005 7.4 11.2 33.0 1807 %cv 26 30 43 42 25 22 22 57 64 min 5.3 3 118.0 122.7 0.014 17.8 31.5 24.8 886 max 16.4 10 775.6 807.5 0.039 49.4 72.7 163 7601 * median = 5 fed stat cmax (ng/ml) tmax (hr) auc0-t (ng.hr/ml) auc0-∞ (ng.hr/ml) kel (hr-1) t0.5 (hr) mrt (hr) cl/f (l/h) vd/f (l) mean 10.9 6.7* 329.1 417.6 0.022 35.5 51.9 58.8 2482.4 ±sd 3.3 1.9 181.0 192.2 0.008 11.6 10.8 24.2 1371 %cv 30 28 55 46 36 33 21 48 55 min 3.5 2 94.9 107.9 0.012 17.3 35.6 23.6 820 max 15.8 12 787.9 848.1 0.040 57.5 74.8 135.9 6630 * median = 7 safety and tolerability for both fasting and fed studies, fx 20 mg capsules were tolerated well by all volunteers, and no ae, adr, and sae were noticed during each study period of 144 hours. besides, the clinical examinations, including vital signs and laboratory tests, were not significantly altered after fx dosing compared to the baseline. conclusions this article demonstrated the pk parameters of the therapeutic dose of fx 20 mg capsules administered to the arabic population. no, statistically significant differences after the fasting versus the fed conditions were found in the primary and secondary pk parameters including cmax, auc0–t, auc0–∞, kel, t1/2, mrt, cl/f and vd/f. however, the absorption rate was slower, and consequently, the tmax was significantly longer after food intake. thus, food intake with fx capsules may influence the onset rather than the intensity of the effect of fx. interestingly, it was 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of psychotropic drugs. pharmacopsychiatry. 2006;39(4):121-127. this work is licensed under a creative commons attribution 4.0 international license https://www.ncbi.nlm.nih.gov/pubmed/?term=zaid%20an%5bauthor%5d&cauthor=true&cauthor_uid=17176627 https://www.ncbi.nlm.nih.gov/pubmed/?term=bowirrat%20a%5bauthor%5d&cauthor=true&cauthor_uid=17176627 https://www.ncbi.nlm.nih.gov/pubmed/?term=kort%20jj%5bauthor%5d&cauthor=true&cauthor_uid=17176627 https://www.ncbi.nlm.nih.gov/pubmed/?term=oscar-berman%20m%5bauthor%5d&cauthor=true&cauthor_uid=17176627 https://www.ncbi.nlm.nih.gov/pubmed/?term=oscar-berman%20m%5bauthor%5d&cauthor=true&cauthor_uid=17176627 https://www.ncbi.nlm.nih.gov/pubmed/17176627 https://www.ncbi.nlm.nih.gov/pubmed/17176627 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.19(2) 2010 lithotripsy of urinary stone by carum copticum seeds 38 lithotripsy of different urinary tract stones by using seeds of carum copticum ahmed g. sabar* ,1 * department of community health ,college of health and medical technology, baghdad,iraq . abstract it has been a well-known practice to use seeds and the essential oil of carum copticum as a strongly antiseptic , antispasmodic , aromatic , bitter , diaphoretic , digestive , diuretic , expectorant and tonic. also used for cure influenza, asthma, and rheumatoid arthritis. to our knowledge it will be the first time to use the seeds of this herb as a urinary tract stone lithotripsy.this research aimed to the use of these seeds as a lithotripsian against different types of urinary stones and determine the efficiency of these preparation against which types of stone.a liquid solution was prepared from dissolving the seeds powder in cow milk and then concentration this preparation was done by boiling at 100 ° c to reduce the volume of solution to the half.the treatment was given via oral administration for successive 9 days before breakfast. 350 patients with urinary stone of different type took part in this research. all patients were subjected to ultrasonography and intravenous pyelography examinations to localized the position and detect diameter of stone. the above examination and also biochemical tests for diagnosis of stones ingredients were repeated after the administration of treatment and excretion of stone fragments in urine. the results were so promising especially against pure caoxalate stone. key words: carum copticum; lithotripsy; ca-oxalate stone; mixed stone الخالصة اسزخذِذ ثذٚر ٔجبد إٌخٛح )وّْٛ اٌٍّٛوٟ( ٚوذٌه اٌش٠ٛد اٌط١برح اٌّسزخٍصخ ِٓ اٌجذٚر وّٛاد ِضبدح ٌٍّغص ,ِذرح, ِسبعذح ٌٍٙضُ, ِمشعخ , ٚاسزعٍّذ ا٠ضب ٌعالج اٌجزد ٚاٌسعبي ٚاٌزثٛ ٚاالسٙبي ٚاٌزٙبة اٌّفبصً اٌزِٚبرشِٟ. ٚاسزخذِٕب ثذٚر ٘ذا ٝ اٌّجبرٞ اٌج١ٌٛخ حست ِعٍِٛبرٕب الٚي ِزح فٟ ٘ذا اٌّجبي . ٠ٙذف ٘ذا اٌجحث اٌٝ اسزخذاَ ثذٚر ٔجبد إٌجبد وعالج ٌزفز١ذ حص إٌخٛح إٌٙذ٠خ وعالج ٌزفز١ذ حصٝ اٌّجبرٞ اٌج١ٌٛخ ثبٔٛاعٙب اٌّخزٍفخ, ٚرحذ٠ذ اٌفعب١ٌخ االوجز رجبٖ اٞ ٔٛع ِٓ أٛاع اٌحصٝ . رُ درجخ ِئ٠ٛخ الخزشاي اٌى١ّخ اٌٝ 011اَ ح١ٍت االثمبر ٚرُ رزو١ش إٌم١ع ثغ١ٍبٔٗ اٌٝ رحض١ز ٔم١ع ِٓ ِسحٛق ثذٚر إٌجبد ثبسزخذ ِز٠ضب ِّٓ ٠عبْٔٛ ِٓ ٚجٛد 051ا٠بَ ِززب١ٌخ لجً االفطبر .شبرن فٟ ٘ذٖ اٌذراسخ 9إٌصف . اعطٟ اٌخ١ٍظ عٓ طز٠ك اٌفُ ٌّذح فٛق اٌصٛر١خ( ,ٚاالشعخ اٌٍّٛٔخ ٌزحذ٠ذ ِٛلع ٚحجُ اٌحصٝ , ثُ اٌحصٝ فٟ اٌّجزٜ اٌجٌٟٛ اخضعٛا اٌٝ فحٛصبد اٌسٛٔبر )اٌّٛجبد اجزٞ ٌىبفخ ع١ٕبد اٌجحث فحص االدرار اٌعبَ .اع١ذد ٘ذٖ اٌفحٛصبد ثعذ اسزخذاَ اٌعالج وذٌه اجز٠ذ اٌفحٛصبد اٌجب٠ٛو١ّ١ب٠ٚخ الج فعبي ثىفبءح عب١ٌخ رجبٖ حصبح اٚوشالد عٍٝ اٌحص١بد اٌّزفززخ ٚإٌبسٌخ فٟ االدرار ٌزشخ١ص ِىٛٔبرٙب ح١ث رج١ٓ اْ ٘ذا اٌع اٌجٛربس١َٛ إٌم١خ ٚثذرجخ الً ٌٍحصٛاد ِٓ االٔٛاع االخزٜ . introduction renal stones (nephrolithiasis) are concretion composed of crystalline components and organic matrix [1]. although the symptomatic presentations may be similar ;the disorder is heterogeneous as to composition and etiology.today, most urinary stones in patients in most countries are renal stones. about 1-4% of the population is believed to have kidney stones every year in usa and europe. about 2-5% of population in asia, 815% in europe and north america and 20% in saudia arabia develop kidney stone in their lifetime [2, 3, and 6] . renal stones tend to recur, and the rate of recurrence is about 75% during 20 years environmental and genetic factors [4-5] . a specific diagnosis for every patient with kidney stones, may give very important information about the stone-formation mechanism and the pharmaceutical manner to prevent recurrent stone formation [5-6] .carum copticum with herbarium number 2930303-1 is a plant in umbelliferae family with a white flower and small, brownish seeds. this plant is commonly grows in india,iran,egypt and europe [7] .the seeds and especially the essential oil are strongly antiseptic, antispasmodic, diuretic, and used in the treatment of so many diseases.the seeds contains about 4-6% essential oil, of which 4555% is thymol [8] , while the essential oil in the dried fruits (2.5-5% ) is dominated by thymol (35-60%) [9] .from south india carum copticum fruits, almost pure thymol has been isolated (98%) , but the leaf oil was found to be composed of monoterpenoids and sesquiterpenoids: 43%cadinene, 11% longifolene, 5% thymol, 3% camphor and others [9] . the effective components of this 1corresponding author email : ahmedalqaicy@yahoo.com , ahmed@hotmail.com received : 16/11/2009 accepted : 1/8/2010 mailto:ahmedalqaicy@yahoo.com mailto:ahmed@hotmail.com iraqi j pharm sci, vol.19(2) 2010 lithotripsy of urinary stone by carum copticum seeds 39 plant , responsible for the observed bronchodialatory effect [6] ,despite the availability of modern medication the propensity towards the traditional medications is growing through out the word [7,8] which needs scientific investigation for evaluating the therapeutic effects and their mechanism of action. indeed no acute toxicity data were available for carum copticum ,although animal studies of putative beneficial effect of this plants seeds involved the use of a dose of 500mg/kg body weight in mice and rat without morbidity or mortality ascribable to the herb, suggesting that the oral ld50 of carum copticum (fruit/seeds) is likely to be higher than that figure [9] . the present study was carried out to determine the role of carum copticum seeds as a herbal medication for treatment urinary tract stones. materials and methods the study of effects of carum copticum seeds as a lithotripsian agent was carried out in 2008 on (350) patients diagnosed by specialist physician in private clinic from different sites of iraq (baghdad,diala,mousol) and the experimental part was undertaken during the period (2001-2008). materials seeds of carum copticum (other latin name : trachyspermum ammi ), were collected from local market in baghdad, identified under expert guidance ,taxonomy was performed by national herbarium of iraq at 1997,depended on ayurvedic pharmacopoeia of india (api) [10] , and kew herbarium [11] , liquid milk, sugar. the dose normally recommended in traditional ayurvedic use is 3-6 gm / day , presumably being 3 gm once or twice a day [12] . also this dose was documented in arabic antique manuscript (tathkarat daood al-antaky) [13] . methods before started giving the preparation we obtained the written consent of the patients who included in the study; (15gm) of seeds were ground to a very fine powder.(total dose for each patients taken with in 9 days) ;(5gm) of grounded seeds were boiled with 150 ml of liquid cow milk and 25 gm sugar, until half of the volume was obtained; the preparation was kept cool [13] ;this preparation divided to ( 3 ) equal doses, each patients was given single dose a day before breakfast for a period of 3 days ; this procedure was repeated for remainder (10gm) of grounded seeds ; ultrasonography (u /s) and intravenous pyelography (ivp), were performed pre-, and post treatment to be sure of curing urinary tract from any stone; urinary tract stones were of different sizes ranging from 5mm and 1.2 cm and they were seen in the kidney (renal stone) at upper pole calyx, mid renal part and lower pole calyx, also they were seen in the ureter (uretral stones) , and finally they were seen inside urinary bladder (vesical stones) . in addition general urine examination (gue) was done for all patients ; qualitative analysis of stone / fragments passed after herbal treatment, a procedure described by hodgkinson [14] , was employed to figure out the chemical constituent of urinary stone results the present study included (350) patients with different urinary tract stones were treated with liquid extract of carum copticum seeds. table 1 : chemical constituent of stones patients age range(years) % no. of patients type of stone (20-45) 48,57 170 pure ca-oxalate 20-50)) 28.57 100 (ca-oxalate/uric acid) mixed stone (22-50) 22,86 80 ca-oxalate/) hydroxyapatite) mixed stone 100% 350 total table 2 : lithotripsy events table 3 : duration of lithotripsy % no. of lithotripsed stone no. of patients type of stone 100 170 170 pure ca-oxalate 53 53 100 (ca-oxalate./ uric acid) mixed stone 31.25 25 80 ( ca-oxalate./ hydroxyapatite) mixed stone 350 total time (days) type of stone 2-7 pure ca-oxalate 7-12 ca-oxa./uric acid))mixed stone. 7-15 ca-oxalate./hydroxyapatite) mixed stone. iraqi j pharm sci, vol.19(2) 2010 lithotripsy of urinary stone by carum copticum seeds 40 table 4 : response the types of stones to the treatment with carum copticum * h.s: highly significant discussion the results of this study including the investigation of the efficiency of carum copticum seeds (liquid solution), locally prepared on urinary stone among (350) patients.the results present in table (1) showed that pure caoxalate consist the larger percentage among types of stones. since a (170) patients out of (350) patients (48.57%) have a pure ca-oxalate urinary stone.results of table (1) revealed that (28.57), (22.86) of patients got mixed stone (caoxalate\uric acid) and (ca-oxalate \hydroxyapatite) respectively.the results cleared out in table (2) indicated that the local preparation of carum copticum seeds have a good affectivity on ca-oxalate stones , since a hundred percent of this type of urinary stone had been lithotripsed , comparing to (53%) for mixed (ca-oxalate\uric acid) and (31.25%) for mixed (ca-oxalate\hydroxyapatite) . recently in india had successfully purified an anticalcifying protein from the seeds of carum copticum using oxalate depletion assay and deciphered its inhibitory activity against ca-oxalate crystals growth .the antilithiatic potential of carum copticum was confirmed by its ability to maintain renal functioning, reduce renal injury and decrease crystal excretion in urine and retention in renal tissue [15] .it was obvious from the results presented in table (3) that pure ca-oxalate required the minimum duration to complete lithotripsy. while mixed stone of both types required a longer time to complete lithotripsy. the response of types of stone was indicated in table (4) which showed that pure ca-oxalate was the most affected types of stones by the treatment with carum copticum seeds compared with other two types of urinary stones mainly (caoxalate\uric acid) mixed stone and (caoxalate\hydroxyapatite) mixed stone. on the other hand type two of stone (ca-oxalate\uric acid) mixed stone was affected by treatment with carum copticum seeds more than the mixed stone (ca-oxalate\hydroxyapatite). our results confirmed the antilithiatic properties of carum copticum seeds that authorized by many researchers [16-19] . biostatistical analysis (binomial-test and z-test) confirmed these results. it could be argued for that the effect of herb extract (crude extraction) is depending totally on chemical structure of urinary tract stones, also it is clear from the results presented in table (4) that ca-oxalate whenever existed as one of the constituent of urinary tract stones it will provoke or stimulate the action of herbs seeds extract, in other word ca-oxalate is decisive component of urinary tract stone that encourage lithotripsy whenever carum copticum seeds extract are available.in this view, mixed stones which are either (ca-oxalate\uric acid) or (caoxalate\hydroxyapatite) showed different response to the treatment with carum copticum seeds extraction and that in our view is corresponding to the amount of ca-oxalate present in the mixture .in general, the lithotripsic effects of carum copticum seeds against urinary tract stones were mentioned by arabic scientist sheikh dawood antaki (about 1008 a. h) [13] . conclusion thus, the present study suggests the potential of carum copticum seeds in lithotripsy of urinary stone, especially calcium oxalate and forms the basis for the development of antilithiatic drug interventions against urolithiasis. acknowledgment the author is grateful to dr. nabeel fadel , dr. nasseer abed agha, and dr. akram mohd al-mahdawy, for help in the diagnosis of the caseas. thank also to dr. zuhair nouman al-ani a professor of pharmacogenetic for his scientific advices. references 1. pak cyc. kidney stones. williams textbook of endocrinology 1992; 1519-36. 2. pak cyc.kidney stones. lancet 1998; 351: 1797-801. 3. kamoun a, daudon m, abdelmoula j, hamzaoui m,chouachi b, houissa t, zghal a, ben ammar s, belkahia c, lakhoua r. urolithiasis in tunisian children. pediater nephrol 1999; 13(9):920-5. 4. gault mh, chafe l. relationship of frequency, age, sex, stone weight and composition in 15 624 stones. j urol 2000; 64 (2): 302-7. 5. kourambas j, aslan p, the cl, mathias bj, preminger m. role of stone analysis in metabolic evaluation and medical treatment of c.s (p-value) between each pairs of types ca-oxa.×mixed(1) ca-oxa.×mixed(2) mixed(1)×mixed(2) response (%) type of stone 0.003 h.s 0.000 h.s 0.000 *h.s 170 (100%) pure ca-oxalate. 53 (53%) ca-oxalate./uric acid mixed stone. (1) 25 (31.25%) caoxa./hydroxyapatite mixed stone. (2) iraqi j pharm sci, vol.19(2) 2010 lithotripsy of urinary stone by carum copticum seeds 41 nephrolithiasis. j endocrinol 2001; 15 (2): 181-6. 6. thomas se, stapleton fb. leave no (stone) untreated; understanding the genetic bases of calcium-containing urinary stones in childhood. adv pediatr 2000; 47: 199221. 7. seyed hassan hejazian; mohd hossein ,and mohd dashti,.(antinociceptive effect of carum copticum extract in mice using formalin test).,world appl.sci.j. 2008,3(2):215-219 8. chopra. r. n., nayar. s. l. and chopra. i. c. glossary of indian medicinal plants (including the supplement). council of scientific and industrial research, new delhi. 1986. 9. versita,warsaw. fumigant toxicity of essential oil from carum copticum against indian meal moth,journal of plant protection research,dec. 2008, 48(4);14274345. 10. ayurvedic pharmacopoeia of india,a jamoda.government of india.part 1980; vol.i,first edition:2-3. 11. bull.of miscellaneous information(royal garden,kew), 1931 ; 1931(6); 299 344. 12. the ayurvedic pharmacopoeia of india,appendix.,government of india.part i,volume ii,first edition: 1999; 189-93. 13. shaikh dawood antaki. tadhkirat uli al albab wal jamia lil ajab alujab. medieval medical book. 10 th century, product code : 1568. 14. hodgkinson a. a combined qualitative and quantitative procedure for the chemical analysis of urinary tract calculi. j.clin.path. 1971, 24,147-151. 15. tan zeer kaur , rakesh, et al., in vivo efficacy of trachyspermum ammi anticalcifying protein in urolithiatic rat model. jour. of ethnopharmacology .2009, 126(3) 459-462. 16. a k pathak, n nainwal et al., pharmacological activity of trachyspermum ammi : a review. jour.of pharmacy research, 2010; 3(4); 895899. 17. gurinder j and daljit s.a., bioactive potential of anethum graveolens,foeniculum vulgare and trachyspermum ammi belonging to the family umbelliferae-current status. jor. of medicinal plant research 2010; 4(2), 87-94. 18. shazia s. ,mir a.k, mushtaq a. and muhammed z., indigenous knowledge of folk medicines by the women of district chakwal, pakistan . jor.ethnobotanical leaflets 2006; 10: 243-253. 19. muhammed h.,sumera a.,mir ajab k, ethnopharmacology, indigenous collection and preservative technique of some frequently used medicinal plants of utror and gabral,district swat,pakistan. afric.jor.of tradition. ,complem.,and altenet. med. 2006 ; 3(2) , 57-73. iraqi j pharm sci, vol.28(1) 2019 rebamipide ocular inserts doi: https://doi.org/10.31351/vol28iss1pp24-36 24 preparation and evaluation of extended release ocular inserts of rebamipide for local effect using casting technique zainab a. radhi*,1 and mowafaq m. ghareeb* * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the aim of this study is to prepare and evaluate an extended release rebamipide ocular insert using hydroxypropyl methylcellulose as the main components of this drug delivery system for dry eye syndrome management, to have the advantage of both rebamipide inducing mucin secretion and hydroxypropyl methylcellulose lubricating properties. solvent casting technique was used to prepare the inserts; the amino acid l-arginine was used to solubilize the drug, hydroxypropyl methylcellulose grades (hpmc e5 and k15m) and poly ethylene glycol 200 were used as excipients. the inserts were evaluated for their physical and mechanical properties, moisture loss% and absorption %, surface ph, and in-vitro drug release. the use of l-arginine exhibited an enhancement of rebamipide solubility in both deionized water and phosphate buffer (ph 7.4) by approximately 274 and 2.8 folds, respectively. the formulae showed uniform weight, thickness and drug content except for formula (f1) that composed mostly of hpmc e5: hpmc k15m at ratio of 1: 0.17 and had no plasticizer (poly ethylene glycol 200) in its composition; it showed haziness in its appearance and brittleness of the insert. while formula (f3) which contained mainly hpmc e5: hpmc k15m at ratio of 1: 0.4 with poly ethylene glycol 200 showed good physical and mechanical properties thus was selected for in vitro release and was compared to the marketed brand mucosta® ophthalmic suspension unit dose 2%w/v; f3 showed significant larger t50% and t80% (p-values 0.034 and 0.015) compared to those of the reference marketed brand and similarity factor value was (f2 = 37.27). the results of this study showed that rebamipide ocular inserts have good potential for futuristic rebamipide extended release ocular delivery system. keywords: rebamipide, l-arginine, hydroxypropyl methylcellulose, ocular insert, casting technique تحرير الريباميبايد للتأثير الموضعي باستخدام تقنية الصب تحضير و تقييم الصق عيني الطالة **و موفق محمد غريب 1*،زينب عبد المحسن راضي .بغداد،العراق جامعة بغداد، ،كلية الصيدلة، الصيدالنياتفرع * الخالصة الهدف من هذه الدراسة هو تحضير وتقييم الصق عيني الطالة تحرير الريباميبايد باستخدام هايدروكسي بروبيل مثيل سولولوز كمكونات ان اص ورئيسية في نظام توصيل دوائي لعالج متالزمة العين الجافة ، وذلك لالستفادة من كل من خواص الريباميبايد الذي يحفز إفراز الميوسين وخ -مض األميني لتم استخدام الحو تقنية الصب باستخدام المذيب لتحضير اللواصق العينية ؛ تروكسي بروبيل مثيل سولولوز المرطبة. استخدمالهايد تم تقييم خصائص كسواغ. 200( والبولي إيثيلين جاليكول k15mو e5الهايدروكسي بروبيل مثيل سولولوز ) . كما تم استخدامأرجينين إلذابة الدواء هر ظاللواصق الفيزيائية والميكانيكية ، ونسبة فقدان وامتصاص الرطوبة ، و االس الهيدروجيني لسطح الالصق ، وتحرير الدواء في المختبر. أ 274حو ( بن7.4أرجينين زيادة في ذوبانية الريباميبايد في كل من الماء منزوع األيونات ومحلول الفوسفات المنظم)الرقم الهيدروجيني -استخدام ل ( التي تتكون في الغالب من الهايدروكسي بروبيل f1مرة ، على التوالي. أظهرت الصيغ وزنًا وُسمًكا ومحتوىًا متجانًسا باستثناء الصيغة ) 2.8و في أظهرت ضبابية حيث ( في تركيبها ؛ 200ال تحتوي على ملدن )بولي إيثيلين جاليكول ( و1:0.17ة )بنسب (e5: k15m) مثيل سولولوز (e5: k15m)الهايدروكسي بروبيل مثيل سولولوز ( التي تحتوي بشكل أساسي علىf3في حين أن الصيغة ) مظهرها و هشاشة في اللواصق . ر الختبار تحرير الدواء في المختب أظهرت خصائص فيزيائية وميكانيكية جيدة وبالتالي تم اختيارها 200مع بولي إيثيلين جاليكول ( 1:0.4ة )بنسب و 0،034القيمة االحتمالية ) t80٪و t50٪نسبة أكبر من f3وزن/حجم. أظهر ٪2المعلق العيني ميوكوستا تمت مقارنتها بالعالمة التجارية و (. وأظهرت نتائج هذه الدراسة أن الالصق العيني = 37،27f2 ( مقارنة بتلك الخاصة بالعالمة التجارية المسوقة وقيمة عامل التشابه )0،015 .للريباميبايد لديه امكانات مستقبلية جيدة كنظام توصيل دوائي للعين يعمل على اطالة تحرير الدواء ., الالصق العيني, تقنية الصب هايدروكسي بروبيل مثيل سولولوز ارجنين, -الكلمات المفتاحية: ريباميبايد, ل introduction dry eye syndrome is a multifactorial disease that affects the tears and ocular surface, and lead to various symptoms as visual disturbance, discomfort, gritty feeling and burning sensation; it may also cause tear film instability and possible defects of ocular surface and inflammation. (1) this disease may occur due to the decreased ability of lacrimal functional unit (lacrimal glands, ocular surface, and lacrimal drainage pathways) to produce sufficient amount of tear film components which results in a poor quality or/and low quantity of tear film (2,3,4,1) . dry eye management includes patient’s education, dietary and environmental changes, artificial tears, punctal plugs, topical and systemic medications; in addition to surgical intervention which can be a possible option. the choice of a suitable management is based on the etiology and severity of the syndrome (4,1,5) . 1corresponding author: zainabradhi@yahoo.com received: 31/8/2018 accepted: 14/10/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp24-36 iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 25 rebamipide is a quinolone compound, its molecular formula is (c19h15cln2o4) and it has a molecular weight (370.79 g/mole), the structure is illustrated in figure (1). it is classified as class iv according to the biopharmaceutical classification system (bcs).(6) rebamipide acts pharmacologically by increasing mucus production and secretion, improving wound healing, and it has exhibited cytoprotective and anti-inflammatory properties. (1,7) the compound was first introduced, in 1990, for gastric ulcer treatment; then investigating rebamipide ability to induce mucin secretion from ocular cells, has led to its approval for dry eye treatment in 2011 and it was marketed, in 2012, as (mucosta® ophthalmic suspension unit dose 2%) in japan. (8,9) figure (1 ) chemical structure of rebamipide (9) ocular inserts are solid or semisolid sterile preparations that can be prepared with various shapes and sizes and should be suitable for ocular application; the inserts are usually placed into the cul-de-sac and less often in the upper fornix or on the cornea. the most important advantage is to increase the dosage form’s contact time with the conjunctiva and it varies from few hours to days; when compared to solution which has contact time no longer than fifteen minutes (10), the inserts showed a great enhancement in residence time. (11,12) artificial tears and lubricants are the firstline management for dry eye syndrome because they are obtainable, non-invasive, and have minimum side-effects; they usually contain hydrophilic polymers in their composition. (4) lubricating inserts were developed to substitute the artificial tear drops in order to overcome the frequent dosing problems of conventional eye drop. these inserts are generally composed of water soluble polymers as cellulose derivatives (for example hyroxypropyl cellulose and hydroxylpropyl methyl cellulose) and may also contain plasticizers. the plasticizers used in these inserts should be soluble in the lacrimal fluid as polyethylene glycol, glycerin, and propylene glycol; they may exist in the ocular inserts in a range from (1-40% by weight). (13) the objective of this study is to prepare and evaluate an extended release rebamipide ocular insert using hydroxypropyl methylcellulose as the main components of this drug delivery system for dry eye syndrome management, to have the advantage of both rebamipide inducing mucin secretion and hydroxypropyl methylcellulose lubricating properties. solvent casting technique was used to prepare the ocular inserts. materials and methods materials rebamipide and hydroxypropyl methyl cellulose (hpmc) k15m were purchased from shijiazhuang aopharm medical technology co. ltd. (china). hpmc e5 and potassium dihydrogen phosphate monobasic (kh2po4) were obtained from himedia laboratories pvt. ltd. (india). l-arginine was obtained from central drug house ltd. (india). polyethylene glycol (peg 200) was supplied by sinopharm chemical reagent co. ltd. (china). sodium hydroxide was provided by techno pharmchem (india). potassium chloride was supplied by merck group (germany). absolute ethanol was obtained from panreac chemicals (spain). methanol was provided by thomas baker chemicals (india). dichloromethane was supplied by gainland chemical company (uk). methods characterization of rebamipide determination of melting point rebamipide melting point of was determined by using differential scanning calorimetry (dsc), a sample of three mg was heated from 25ºc to 350ºc at a rate of 10ºc/min in a sealed aluminum pan. the peak shown in the thermogram represents the drug’s melting point. determination of λ max stock solutions of rebamipide with a concentration of (0.2mg/ml) were prepared by dissolving 20 mg of the drug in up to 100 ml of phosphate buffer (ph 7.4), methanol and ethanol respectively. from these stock solutions, diluted solutions with concentrations of (0.009 mg/ml) for rebamipide in phosphate buffer and (0.008mg/ml) for rebamipide in both ethanol and methanol were prepared, and then uv spectrophotometer was used to determine rebamipide λmax at 200-400 nm. (14) construction of calibration curve rebamipide calibration curve was constructed in three vehicles phosphate buffer (ph 7.4), methanol and ethanol by using diluted solutions of the drug with different concentrations (1, 2, 3, 4, 5, 6, 7, 8 and 9 µg/ml) prepared from a (0.2 mg/ml) stock solution. samples were analyzed using uv-spectrophotometer at rebamipide λmax. the recorded absorbance of each sample was plotted against its respective concentration. fourier transform infrared spectroscopy (ftir) rebamipide ftir spectrum was obtained by using pressed-disk technique. a small amount of drug was ground with kbr powder then the mixture was pressed into a disk. the ftir spectroscopy was used to investigate the prepared disk at wave lengths range of 4000-400 cm-1 (15) . iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 26 determination of rebamipide solubility the solubility of rebamipide was determined in different solvents (deionized water, methanol, ethanol, and phosphate buffer (ph 7.4)). an excess amount of the drug was dissolved in 10 ml of solvent and shaken for 72 h at room temperature. the mixtures were centrifuged at 4000 rpm for ten minutes and then filtered with filter syringe (0.45 μm).(16,17) the filtrate was analyzed using uvvisible spectrophotometer at rebamipide λmax, and then the concentration of rebamipide saturated solution was calculated.(18) the solubilization effect of amino acid on rebamipide was determined by adding an equimolar amount of l-arginine in both deionized water and phosphate buffer (ph 7.4); the equimolar ratio of rebamipide to l-arginine is (1: 0.47). preparation of rebamipide inserts calculations of rebamipide insert size each insert has a surface area of 40 mm2 that contains 2mg of rebamipide. the surface area of the inserts and their number in each batch was calculated by dividing the petri-dish surface area over the insert’s surface area as follow: surface area of petri-dish = 56 cm2 surface area of the rectangular insert = 58= 40 mm2 =0.4 cm2 number of inserts/ batch = 56/0.4 = 140 insert total amount of rebamipide per each batch = 140  2= 280mg total amount of l-arginine per each batch = 140  0.95= 133 mg preparation of rebamipide solution for casting to prepare homogenous liquid of the drug for casting, different solvent were used include deionized water, methanol, and ethanol. also, trial to prepare rebamipide solution using equimolar quantity of amino acid (l-arginine) in water was studied. rebamipide was solubilized by using an equimolar amount of the amino acid l-arginine. a calculated amount of l-arginine (133 mg per batch) was dissolved in 5 ml of deionized water, and then an equimolar amount of rebamipide (280 mg per batch) was added with continuous stirring using a magnetic stirrer for five minutes. formulation of rebamipide inserts four formulas were prepared using solvent casting technique. they were simply prepared by mixing the components to form a matrix film, table (1). the polymer hpmc with two grades (e5 and k15m) were prepared as aqueous solutions with the concentrations (10%, and 2% w/v) respectively. homogenous polymer mixture was measured with a specified weight for each formula as stated in table (1); it was poured into the rebamipide/larginine solution, then peg200 (35% of polymer weight) was added as a plasticizer with continuous stirring by magnetic stirrer for two hours, then the mixture was set aside for twelve hours to ensure air bubbles removal. the resultant solution was casted into a flat surface polystyrene petri-dish and left to dry at room temperature until complete solvent evaporation. (13, 19, 20) a dry film was obtained and cut into rectangular inserts (5 mm x 8 mm), packed in aluminum foil and stored at room temperature. table( 1) composition of formulas of matrix rebamipide ocular inserts evaluation of rebamipide ocular inserts: physical appearance physical appearance of the insert’s color, transparency and texture was observed and inspected visually (21) . weight uniformity five inserts were selected randomly from each batch (5mm x 8mm), they were weighed individually using a digital balance; the mean weight and standard deviation were calculated.(20) batches with a variation of more than ±5% were rejected.(22) thickness measurements five inserts were selected randomly from each batch; the thickness of each insert was measured at five positions using a digital vernier caliper.(23) the mean thickness and standard deviation were calculated. batches with a variation of more than ±5% were rejected.(22) content uniformity ten inserts from each batch were selected randomly. each insert was dissolved in 50 ml of phosphate buffer (ph 7.4), and then the solution was filtered through 0.45µ filter syringe. one milliliter of the filtrate was diluted up to ten milliliters; the drug content was measured by analyzing the diluted solution with uv-visible spectrophotometer.(24,25) the mean and standard deviation were calculated. the dosage uniformity meets the requirements stated in the usp when the first ten units have an acceptance value less than or equal to 15% (26) . formula rebamipide (mg) l-arginine (mg) hpmc e5 (mg) hpmc k15m (mg) peg 200 (%) f1 2 0.95 12 2 0 f2 2 0.95 12 2 35% w/w of polymer weight f3 2 0.95 10 4 f4 2 0.95 14 0 iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 27 folding endurance folding endurance is done to evaluate the flexibility of the inserts; it is the number of folds that can be achieved without breaking the insert, when repeated folding at the same position is done. folding endurance of three inserts of (5 × 16 mm) of each formulation was carried out; the mean value and the standard deviation were recorded.(24,27) moisture loss% moisture loss% was determined by placing pre-weighed inserts in a desiccator containing silica beads for 72 hr. at room temperature, then the inserts were weighed again and the mean moisture loss% was calculated using the equation(27): moisture loss% = 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡−𝑓𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 ∗ 100 moisture absorption% three inserts of the selected formulas were weighed and placed in a desiccator that contains a saturated solution of potassium chloride, that maintains relative humidity of about 84%(28), for 72 hr. at room temperature; then the inserts were reweighed and the moisture absorption% was calculated using the equation(27): moisture absorption% = 𝐹𝑖𝑛𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡−𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 ∗ 100 surface ph surface ph of the inserts was determined by allowing the insert to swell for thirty minutes with 5 ml of de-ionized water in a glass vial, and then the electrode of a ph-meter was placed as close as possible to the insert surface and the ph was recorded. the process was done in triplicate.(19) in-vitro drug release study this study was performed using a modified diffusion cell shown in figure (2), which contains a receptor and a donor chambers; the receptor chamber was filled with 65 ml of phosphate buffer (ph 7.4) as the dissolution medium which was maintained at 34 ± 1º c (19,29), and the medium was stirred at 50 rpm using a magnetic stirrer. the insert of the selected formula was placed on a dialysis tubing membrane (mw 1200014000 dalton) which was mounted between the two chambers. a sample with a 2 ml volume was withdrawn with a syringe at specified time intervals (0.25, 0.5, 1, 2, 3, 4, 5, 6,7,8,9,10, 11 and 12 hrs.) which was replaced by a fresh medium to preserve the sink conditions. the sample was filtered by a filter syringe (0.45 μm), and then the filtrate was diluted and analyzed using uvvisible spectrophotometer at rebamipide λmax. the cumulative drug release % of rebamipide was calculated.(30) four drops of mucosta® ophthalmic suspension 2%w/v were used as the reference brand; these four drops are equivalent to 2 mg, the amount of the rebamipide in the ocular insert. t50% and t80% (hr.) that denote for the time required for 50% and 80% of the drug to be released, respectively, were used to compare the rebamipide release from the selected formula with its release from marketed mucosta® ophthalmic suspension unit dose 2%w/v, and these parameters were tested statistically using two-tailed student’s t-test; similarity factor (f2) was also used to compare the drug release profiles; when two curves are identical (f2 = 100), the fda considers two release curves to be similar when f2 ≥ 50. this factor is calculated by the equation: (31) 𝑓2 = 50 × 𝑙𝑜𝑔 {[1 + 1 𝑛 ∑ (𝑅𝑡 − 𝑇𝑡 ) 2𝑛 𝑡=𝑖 ] −0.5 × 100} where n is the number of time points, rt and tt are the drug% released values at time t from the two curves being compared. figure (2) modified diffusion cell used in the invitro drug release study compatibility study this study was done to identify any possible interaction between rebamipide and the polymers used in the inserts preparation. fourier transform infrared spectroscopy (ftir) in addition to the ftir spectrum of the pure rebamipide, the spectra of the pure polymer hpmc e5 and the prepared medicated insert of formula f3 were attained to detect the compatibility of the drug within the formulation. the procedure discussed previously in characterization of rebamipide section. differential scanning calorimetry (dsc) to confirm the compatibility of rebamipide with the polymers, the dsc of the polymer hpmc e5, hpmc k15m and the prepared medicated insert f3 were studied according to procedure mentioned previously in the determination of melting point section, along with the dsc of the pure rebamipide. iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 28 statistical analysis the experimental data were conducted in triplicates unless otherwise stated, and expressed as means and their standard deviations (mean± sd). one-way analysis of variance (anova) and two tailed student’s t-test were used to analyze the results of the study and asses the statistically significant differences in comparing means; pvalues considered: significant at a level of (p ≤ 0.05) and not significant at a level of (p > 0.05). results and discussion characterization of rebamipide determination of melting point dsc graph showed a peak at 307ºc figure (3), which is close to the values stated in the references. (16,17) 100.00 200.00 300.00 400.00 temp [c] -10.00 -5.00 0.00 5.00 mw dsc 307.06x100c figure (3) dsc thermogram of pure rebamipide powder determination of λ max the diluted solution of (0.009 mg/ml) rebamipide in phosphate buffer (ph 7.4) and (0.008mg/ml) rebamipide in methanol and ethanol were scanned by uv-visible spectrophotometer at 200400 nm. the obtained spectrum of rebamipide in phosphate buffer (ph 7.4) had a λmax at 227nm and in agreement with the value reported in reference.(14) while the spectra of both rebamipide in methanol and ethanol had the same λmax at 229nm. construction of calibration curve the calibration curve of rebamipide was constructed by plotting the absorbance of the diluted solutions of rebamipide in phosphate buffer (ph 7.4), methanol and ethanol against its respective concentration as shown in figure (4), figure (5) and figure (6), respectively. straight lines with high coefficients of determination, (r2 =0.999), (r2 =0.9955) and (r2 =0.9948) respectively, were obtained; those indicate that the calibration curves of rebamipide obey beer’s law. figure (4) calibration curve of rebamipide in phosphate buffer (ph 7.4) figure (5) calibration curve of rebamipide in methanol iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 29 figure (6) calibration curve of rebamipide in ethanol fourier transform infrared spectroscopy (ftir) the ftir spectrum of pure rebamipide figure (7) was compared to the reference ftir spectrum of rebamipide reported in japanese pharmacopoeia.(7) the characteristic peaks of ftir spectrum of pure rebamipide are illustrated in figure (7), at wave numbers (in cm-1): 685 for out of plane n-h wagging of amide (800-666), 758 for out of plane n-h wagging of lactam (800-700), 872, 843, and 832 for out of plane c-h bending bands of aromatic ring (900-675), 1235, 1183, and 1092 for in-plane c-h bending of aromatic ring (1300-1000), 1273 for c-o stretching of carboxylic acid (13201210), 1420 for c-c stretching of aromatic ring (15001400), 1539 for n-h bending of amide band ii (1650-1515), 1642 for c=o stretching of amide band i (1680-1630), 1725 for c=o stretching of carboxylic acid (1730-1700), 3060 for c-h stretching of aromatic ring (3100-3000), 3088 for oh stretching of carboxylic acid (3300-2500), 3271 for n-h stretching of amide (3300-3060). (15) these interpretations indicated the drug purity. figure (7) ftir spectrum of pure rebamipide powder. determination of rebamipide solubility the solubility of rebamipide was determined in different solvents at room temperature as shown in table (2). table (2) solubility of rebamipide in several solvents solvent solubility (mg/ml) deionized water 0.045± 0.011 methanol 1.41± 0.076 ethanol 0.83± 0.038 phosphate buffer (ph 7.4) 3.88± 0.25 these results confirmed the poor solubility of rebamipide in both aqueous and organic solvents and they are in agreement with the references. (7, 17, 16) the poor solubility can be explained by the high lipophilic nature of the drug. (32) rebamipide solubility in phosphate buffer (ph 7.4) was slightly higher than its solubility in deionized water and the other organic solvents used; this result can be explained by acidic properties of the drug (pka = 3.38), thus its solubility is ph dependent. (32) finding a way to solubilize the drug is essential in this study in order to form a homogenous solution suitable for casting, and the solvent should be highly evaporative to obtain a dry film with minimal residual solvent, thus from the results and the literature survey it was concluded that it is not possible to solubilize the drug with solvent or solvent system. after the failure of the trials to solubilize the rebamipide with the solvents, thus enhancing rebamipide solubility by using amino acid was considered and an excess of equimolar amounts of rebamipide and l-arginine (1:0.47) were added in both deionized water and phosphate buffer (ph 7.4) as shown in table (3). (33, 34) iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 30 table (3) solubility of rebamipide in presence of l-arginine solvent solubility (mg/ml) deionized water 12.33 ± 1.136 phosphate buffer (ph 7.4) 11.01 ± 1.911 the addition of l-arginine enhanced the solubility of rebamipide in both deionized water and phosphate buffer (ph 7.4) by approximately 274 and 2.8 folds respectively. this can be explained by the formation of rebamipide arginine salt.(34) preparation of rebamipide inserts preparation of rebamipide solution for casting in solvent casting technique, the drug is preferred to be in solution to ensure its even distribution throughout the casted film, and since the drug is intended for ophthalmic use then it should have a particle size less than ten µm, to be an acceptable ophthalmic preparations and avoid irritation of the eye.(35) rebamipide’s poor solubility in many solvents, makes it difficult to formulate; thus l-arginine was added to solubilize the drug and to prepare a homogenous solution as discussed in previous section. formulation of rebamipide inserts hpmc e5 was used in all four formulae as a film forming polymer (primary polymer), while hpmc k15m which is a higher molecular weight grade of hpmc was used in (f1-f3) as a secondary polymer in order to optimize the rebamipide ocular inserts properties and retard the drug release, peg 200 was used as a plasticizer in (f2-f4). the inserts dimensions are shown in figure (8). figure (8) photograph showing the rebamipide ocular insert’s dimensions in millimeters (5mm x 8mm) evaluation of rebamipide ocular inserts physical appearance the physical appearances of the inserts are shown in figure (9), while the detailed texture, transparency and color of each formulation are stated in table (4). formula f1 had a hazy appearance and a rough texture compared to the other formulas, which points out the major effect of the plasticizer in the inserts appearance. figure (9) photograph showing the physical appearance of the prepared rebamipide ocular inserts iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 31 table (4) physical appearance description of rebamipide inserts formula texture transparency color f1 rough hazy colorless f2 smooth clear colorless f3 smooth clear colorless f4 smooth clear colorless all the inserts of formulae (f2-f4) had good physical appearances, clear, colorless and smooth. while f1 which lacks peg200 in its composition had dramatic change in its physical appearance; the ocular inserts were rough and hazy, because the plasticizer can affect the polymer properties as its optical clarity which might be the reason for the haziness; (36) peg200 may associate with enhancement of rebamipide solubility in the film according to the reference (16), hence its absence may result in precipitating out of the drug when the solvent evaporated and gave the rough texture to the film. weight uniformity the mean weights of the samples of all formulae were ranged between (16.23±1.39 mg – 20.30±0.92 mg) as shown in table (5). thickness measurement the mean thickness of all formulae ranged between (0.31±0.030 mm0.40±0.012 mm) as showed in table (5). drug content uniformity the prepared inserts of all formulae had drug content means ranged between (1.84±0.14 mg 2.16±0.17 mg); the results are presented in table (5). according to usp dosage uniformity criteria the acceptable range would be (1.7mg -2.3 mg).(26) the inserts of formulae (f2-f4) had good uniformity parameters and the results were within the acceptable criteria; except for formula f1 that had variation of more than 5% from its weight and thickness means and it also showed an effect on content uniformity; although the drug content mean was within the acceptable range but some of the samples had drug content out of the acceptable range which are apparent from its standard deviation value. this suggests that the process of casting and evaporation did not result in a uniform film; the absence of the plasticizer in f1 composition may affect its pour-ability. folding endurance the results of folding endurance are shown in table (5). f1 was very brittle due to the absence of the plasticizer in its composition, which ensures the importance of the plasticizer presence in formulating the inserts. the plasticizer role can be explained by its improvement of the flexibility of the polymers by lowering their glass transition temperature degree (tg). the small plasticizer molecules occupy the intermolecular spaces among the polymer chains leading to the reduction of the secondary forces between them that lead to the reduction of the required energy for polymer molecular motion and hence, the polymer flexibility is enhanced.(36) on the other hand, f4 which composed of only one grade of hpmc e5 with lower molecular weight had significantly lower folding endurance compared to f2 (p-value 0.004) and compared to f3 (p-value <0.001); while f3 that had hpmc e5: k15m ratio of (1: 0.4) showed significant (p-value <0.001) increase in folding endurance compared to f2 which had hpmc e5: k15m ratio of (1: 0.17) table (5) physical parameters of rebamipide inserts moisture loss% moisture loss% values of all formulas are shown in table (6), the values are within the range of (3.31±2.17%6.76±1.81%). the plasticizer and polymer ratio showed no effect on moisture loss% on the ocular inserts. in f4 which had only hpmc e5 in its polymer composition showed significantly higher moisture loss% compared to formulae f2 and f3 with (p-values 0.028 and 0.014) respectively, this indicates that the presence of higher viscosity grade of hpmc k15m in the composition of these formulae hindered the water evaporation and kept the moisture within the insert, and since water is a natural plasticizer then the ocular insert flexibility will be maintained. (36) moisture absorption% the results of moisture absorption% of all formulas are shown in table (6), the values ranged between (16.43± 2.75 % 22.68± 6.53 %). there was a significant difference (p-value 0.036) between f1 and f2, which indicates that the plasticizer formula weight (mg) thickness (mm) content (mg) folding endurance f1 16.23±1.39 0.31±0.030 2.16±0.17 very brittle f2 19.63±0.58 0.39±0.006 1.95±0.11 77.67±6.314 f3 19.47±0.53 0.38±0.007 1.86±0.06 288.00±15.18 f4 20.30±0.92 0.40±0.012 1.84±0.14 57.00±11.37 iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 32 addition in f2 has increased the moisture absorption%. this increment may be due to the hygroscopic nature of the polymer peg which under certain values of relative humidity, has the ability to absorb water from the surrounding air and experiences the phenomenon of deliquescence.(37) there were no significant differences in moisture absorption% of the formulas in which the hpmc ratio or grade had been changed, thus these factors have no effect on the moisture absorption % of inserts. surface ph surface ph of the inserts are shown in table (6), and found to be between (7.52±0.05 6.93 ±0.04); this range of values would not change the ph value of the tears(38), since tears have ph value equal to 7.4 with some buffering capacity. table (6)rebamipide insert moisture loss%, absorption% and surface ph evaluation formula moisture loss% moisture absorption% surface ph f1 3.89±1.94 16.43±2.75 7.52±0.05 f2 3.74±2.25 22.68±6.53 7.22±0.07 f3 3.31±2.17 17.79±5.12 6.99±0.06 f4 6.76±1.81 17.30±1.82 6.93±0.04 from the results of the evaluation of the physical and mechanical properties of rebamipide ocular inserts, f3 showed the best results and was selected for further evaluation. in-vitro release study the in-vitro drug release profiles of selected formula f3 and the reference are shown in figure (10), and their t50% and t80% (hr.) are listed in table (7). formula f3 which contains hpmc e5: k15m (1:0.4) and peg200 (35%w/w of total polymer weight) showed significantly larger t50% and t80% compared to the mucosta® ophthalmic suspension (p-values 0.034 and 0.015) respectively; the similarity factor between then was (f2 =37.27). these results indicate that the prepared rebamipide ocular insert (f3) release profile was different from that of the reference and had noticeable drug release retarding effect. this result supports the objective of this study to extend the drug release from the insert compared to available marketed dosage form. figure (10) cumulative% release profile of rebamipide in phosphate buffer (ph 7.4) at 34ºc from f3 and reference table (7) drug release parameters from the insert f3 and mucosta® suspension (4drops) formula t50% (hr.) t80% (hr.) f3 2.30 5.53 reference mucosta® 0.90 1.87 compatibility studies fourier transform infra-red spectroscopy (ftir) the ftir spectra of the pure rebamipide figure (7), polymer hpmc e5 and formula f3 are shown in figure (11) and figure (12) respectively. the characteristic peaks of ftir spectrum of rebamipide are illustrated in figure (12), at wave numbers (in cm-1): 1115 for in-plane c-h bending of aromatic ring (1300-1000), 1316 for c-o stretching of carboxylic acid (1320-1210), 1412 for c-c stretching of aromatic ring (15001400), 1644 c=o stretching of amide band i (1680-1630) ; this indicate that there is no interaction between rebamipide and the excipients. iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 33 figure (11) ftir spectrum of pure hpmc e5 powder figure (12) ftir spectrum of the selected rebamipide ocular insert f3 differential scanning calorimetry (dsc) dsc thermogram image of the pure rebamipide was shown in figure (3); dsc images of polymers used in the selected formula hpmc e5 and hpmc k15m; and for the selected formula f3 shown in figure (13), figure (14) and figure (15) respectively. the dsc thermogram of selected formula (f3) of rebamipide ocular insert did not show a noticeable peak; this may be due to the dilution effect of mixing small amount of rebamipide within the formula with higher proportions of polymers and other excipient. 100.00 200.00 temp [c] 2.00 3.00 4.00 5.00 mw dsc 55.64x100c figure (13) dsc thermogram of hpmc e5 iraqi j pharm sci, vol.28(1) 2018 rebamipide ocular inserts 34 100.00 200.00 temp [c] 0.00 1.00 2.00 3.00 mw dsc 79.39x100c figure (14) dsc thermogram of hpmc k15m 100.00 200.00 300.00 temp [c] -4.00 -2.00 0.00 2.00 4.00 mw dsc figure (15) dsc thermogram of selected rebamipide ocular insert f3 conclusion based on the results obtained from the study, it was concluded that rebamipide ocular inserts have good potential for futuristic rebamipide extended release ocular delivery system for dry eye treatment. few points are noticed from the experimental work that should be considered during rebamipide insert formulation; the enhancement of rebamipide solubility by adding l-arginine to the formulation, the importance of using plasticizer in the insert composition to improve physical and mechanical characteristics of the insert, the incorporation of higher molecular weight polymer (hpmc k15m) lead to improving the inserts flexibility and extended the drug release. references 1. milner ms, beckman ka, luchs ji, allen qb, awedeh rm, berdahl j, et al. dysfunctional tear syndrome : dry eye disease and associated tear film disorders new strategies for diagnosis and treatment. curr opin ophthalmol. 2017;28(1):3–44. 2. gipson ik. the ocular surface: the challenge to enable and protect vision. invest ophthalmol vis sci. 2007;48(10):4390–8. 3. melorose j, perroy r, careas s. gray’s anatomy. 41st ed. standring s, editor. elsevier limited; 2016. 4. phadatare sp, momin m, nighojkar p, askarkar s, singh kk. a comprehensive review on dry eye disease : diagnosis , medical management , recent developments , and future challenges. adv pharm [internet]. 2015;2015(2):1–12. available from: http://dx.doi.org/10.1155/2015/704946 5. zhang x, vimalin jm, qu 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http://linkinghub.elsevier.com/retrieve/pii/s002 2354916303719 38. pawar p, khurana g, arora s. ocular insert for sustained delivery of gatifloxacin sesquihydrate: preparation and evaluations. int j pharm investig [internet]. 2012;2(2):70. available from: http://www.jpionline.org/text.asp?2012/2/2/70/ 100040 iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd doi: https://doi.org/10.31351/vol27iss2pp32-41 32 belief about medications among sample of iraqi patients with inflammatory bowel disease nisreen j. jebur *,1, dheyaa j. kadhim* and nawal. m. firhan ** *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** gastroenterology and hepatology teaching hospital, medical city, baghdad, iraq. abstract inflammatory bowel disease includes both crohn’s disease and ulcerative colitis, is a chronic, progressive relapsing disease of gastrointestinal tract that require long-term treatment or maintenance therapy. taking patient’s beliefs about the prescribed medication in consideration had been shown to be an important factor that affects compliance of the patient in whom having positive beliefs is a prerequisite for better compliance. the aim of the current study was to investigate and assess beliefs about medicines among a sample of iraqi patients with inflammatory bowel disease and to determine possible association between these beliefs and some patient-specific factors. this study is a cross-sectional study carried out on 150 already diagnosed inflammatory bowel disease patients who attended the gastroenterology and hepatology teaching hospital/medical city/baghdad. the mean age of the patients was (31.7 ± 11.4 years).the number of ulcerative colitis patients was 74, while the number of crohn’s disease patients was 76. belief about medicines was measured using the arabic version of the beliefs about medicines questionnaire. the majority of the patients (58%) had strong beliefs in the necessity of treatment (specific-necessity score greater than specific-concern). for all patients (ulcerative colitis and crohn’s disease together), there was a significant inverse correlation between male gender and specific concern score. number of infliximab doses directly correlated with specific necessity score and inversely correlated with specific concern score. future studies should investigate how interventional approaches addressing these predictors may lead to improve beliefs about medicines among inflammatory bowel disease patients and their impact on disease control. key words: inflammatory bowel disease, beliefs about medicines, beliefs about medicines questionnaire, crohn’s disease, ulcerative colitis. داء االمعاء االلتهابي العراقيينالمعتقدات عن األدوية لدى مرضى **مهدي فرحانو نوال *، ضياء كاظم جبار 1*، جمعه جبر نسرين بغداد،بغداد،العراق.،كلية الصيدلة،جامعة السريريةالصيدلة فرع * مستشفى أمراض الجهاز الهضمي والكبد ، مدينة الطب ، بغداد ، العراق. ** الخالصة ان مرض التهاب االمعاء والذي يضم مرضي التهاب القولون التقرحي ومرض كرون هو مرض مزمن متفاقم ومنتكس يصيب األخذ بعين االعتبار ان معتقدات المريض حول الدواء الموصوفمع االمعاء ويحتاج الى العالج بصورة مستمرة وعلى مدى طويل. العالج ب واحدة من أهم العوامل التي تؤثر على االلتزام بالعالج ، حيث ان المعتقدات اإليجابية حول األدوية هي شرط أساسي لاللتزام تعد اط وتحديد االرتبالتهاب االمعاء مرضى لدىل األدوية الموصوف للمريض . الهدف من الدراسة الحالية هو فحص وتقييم المعتقدات حو مريض تم تشخيصهم 150بعض العوامل الخاصة بالمريض. هذه الدراسة هي دراسة مستعرضة أجريت على والمحتمل بين هذا االعتقاد مريضا اما عدد 74كان . عدد مرضى التهاب القولون التقرحيسنة( 11.4± 31.7العمر )متوسط التهاب االمعاء مرضسابقا ب دى المعتقدات عن االدوية. وكان ل . تم تقييم المعتقدات عن االدوية باستخدام النسخة العربية من استبيانمريضا 76ى كرون فكان ضمر ( معتقدات قوية في ضرورة العالج )نسبة ضرورة العالج كانت أكبر من نسبة القلق من االثار الجانبية والمستقبلية %58غالبية المرضى ) بالنسبة للمرضى جميعهم )مرضى التهاب القولون التقرحي ومرض كرون( وجد هناك ارتباط معنوي عكسي بين جنس الذكور (.لألدوية دواء انفليكسيماب فقد ارتبط طرديا مع معيار الحاجة الخاصة وعكسيا مع معيار القلق الخاص. الخاص. اما عدد جرع قوبين معيار القل داء ى لدى مرضيجب ان تبحث الدراسات المستقبلية كيف يمكن للتداخالت المهتمة بهذه العوامل ان تحسن من المعتقدات حول االدوية االمعاء االلتهابي وتأثير ذلك على السيطرة على المرض. مرض كرون, التهاب القولون التقرحي. ،استبيان المعتقدات حول االدوية ، المعتقدات حول االدوية ، داء االمعاء االلتهابي الكلمات المفتاحية : 1corresponding author: e-mail:asmrnn@yahoo.com received: 4/ 8 / 2018 accepted: 22/ 9 / 2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp24-31 https://doi.org/10.31351/vol27iss2pp24-31 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 33 introduction inflammatory bowel disease (ibd) includes both crohn’s disease (cd) and ulcerative colitis (uc), is a chronic, progressive and relapsing disease of gastrointestinal tract, that is characterized by epithelial injury and intestinal inflammation (1-4). the exact etiology of ibd is still not fully understood, however, it is clear that many etiologic factors, including genetic susceptibility, the gut flora, and environmental exposures, are important contributors to the ibd (5). in general, symptoms may be similar in both patients with uc or cd (6). symptoms vary from mild to severe during relapse which may decrease or disappear during remission (7). amino salicylates drugs are among the most commonly pharmacological therapy used for both inducing and maintaining of remission in patients with mild-moderate ibd (6). corticosteroids are used mainly as “rescue” therapy for patients experiencing flare of the disease (8). immunosuppresants (e.g., azathioprine) are used to maintain remission in both forms of ibd (9). several biological agents are currently available (e.g., infliximab) to treat ibd patients (10). the patients usually weigh the benefits of drugs against its potential risks (11). accordingly, two medication beliefs categories were proposed by horne et al, i.e., necessities and concerns. patients can have concerns about the possible unwanted harmful long-term effects as well as dependence on their medication (12) whereas necessity beliefs are related to the expected beneficial effects of a drug on health (13). accordingly, taking patient’s beliefs about the prescribed medication in consideration had been shown to an important factor that affects compliance of the patient where having positive beliefs is a prerequisite for better compliance (14-16). to measure beliefs about medicines, the beliefs about medicines questionnaire (bmq) was developed by horne et al (17). the aim of the current study was to investigate and assess beliefs about medicines among a sample of iraqi patients with inflammatory bowel disease and to determine possible association between this belief and some patient-specific factors. patients and method patients the current cross-sectional study was carried out on 150 already diagnosed ibd patients who attended the gastroenterology and hepatology teaching hospital/medical city/baghdad during september 2017 till january 2018. the mean age of the patients was (31.7 ± 11.4 years). the number of uc patients was 74 (49.3%) while the number of cd patients was 76 (50.7%). inclusion criteria the inclusion criteria for the current study were: 1-ibd patients who are aged 18 years or more of either sex. 2-disease duration of 6 months or more. 3-patients are taking at least one drug on regular base. 4-patients have not changed their drugs on the last three month. 5-patients should be able to communicate effectively and willing to participate in the study. exclusion criteria the exclusion criteria for this study were: 1-patient who had speech, hearing, or cognitive deficits that would affect their understanding of the questions. 2-patient being on treatment for any psychological or neurological diseases. 3-if they were receiving no drug. 4-patients providing conflicting or incomplete information also excluded. method the questionnaires in the current study, belief about medicines was measured using the arabic version (18) of the beliefs about medicines questionnaire (bmq) developed by horne et al (17) (figure-1). the bmq includes two parts; general and specific. the specific part evaluates patients’ beliefs about medications given for a specific disease and in turn, it contain two subparts; specific necessity of prescribed drugs for a particular disease (5 statements) and specific concern about the potential adverse unwanted consequences of drugs (5 statements). the general part of the bmq is about beliefs about medicines in general and also contains two subparts, the general overuse part about the way in which drugs are prescribed by the doctors (4 statements) and the general harm part which evaluates the degree to which patients perceive drugs as harmful. patients answered each question in subparts using a 5-point likert scale, where: 1 = strongly disagree, 2 = disagree, 3 = uncertain, 4 = agree, and 5 = strongly agree. thus, points of each question are summed to give the score. higher specific necessity scores indicate stronger personal need for the drugs. higher specific concerns scores indicate stronger concerns about the adverse effects of the drugs. higher scores on the general harm part indicate more negative views about drugs iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 34 as a whole while higher scores on the general overuse part indicate more negative views about the way in which drugs are prescribed and that they are overused by doctors (18). figure 1.the beliefs about medicines questionnaire (bmq) (17). study design the pilot study a pilot study was carried out on ten ibd patients to test and optimize the arabic words of the questionnaires used in the current study, the data obtained from this study was not included in the major study. strongly disagree disagree uncertain agree strongly agree specific necessity 1-my life would be impossible without medicine 2-without medicine i'll be very ill 3-my health , at present depend on my medicine 4-my medicine protected me from becoming worse 5-my health in the future depends on my medicine specific concern 6-i sometimes worry about the long term effect of my medicine 7-having to take medicine scares me 8-i sometimes worry about becoming too dependent on my medicine 9-my medicine disrupt my life 10-my medicines are mystery to me general-harm 11-people who take medicines should stop their treatment for a while every now and again 12-most medicines are addictive 13-medicines do more harm than good 14-all medicines are poison general-overuse 15-natural remedies are safer than medicines 16-doctors use too many medicines 17-doctors place too much trust on medicines 18-if doctors had more time with their patients they would prescribe fewer medicines iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 35 administration of question ِ naires the data related to the study were collected by the researcher . when the patients arrived to the hospital to receive their programmed doses of biological agent, they were asked if they accept to participate in the study, a complete explanation to the questions in the questionnaire was done and each patient spent about 5 minutes to fill the research questionnaire completely . statistical analysis. discrete variables presented using their number and percentage, chi square test used to analyze the discrete variable. two samples ttest used to analyze the differences in means between two groups (if both follow normal distribution with no significant outlier). binary logistic regression analysis was used to calculate the odd ratio (or) and their 95% confidence intervals. linear regression analysis performed to assess the relationship between different variables, if one or both of them follow normal distribution person regression used but if both did not follow normal distribution spearman correlation will used. negative sign indicate inverse relationship, but positive sign represent direct relationship. statistical package for the social sciences (spss) version 20.0.0 (chicago, il), medcalc statistical software version 14.8.1 (medcalc software bvba, ostend, belgium; 2014), and graphpad prism 7.0 software package were used to make the statistical analysis, p value considered when appropriate to be significant if less than 0.05. results smoking was significantly higher in patients with cd compared to the uc. also the frequency of rural was significantly higher in patients with uc compared to cd, and the rest of the variables did not show a significant difference between cd and uc as illustrated in table 1. table 1. comparison of socio-demographic between both types of ibd variables ulcerative colitis crohn's disease p-value number 74 76 age (years), mean ± sd 32.2 ± 10.9 31.2 ± 12.0 a0.602 <20 years 9 (12.2%) 12 (15.8%) b0.877 20 – 29 years 26 (35.1%) 28 (36.8%) 30 – 39 years 23 (31.1%) 18 (23.7%) 40 – 49 years 9 (12.2%) 10 (13.2%) 50 – 60 years 7 (9.5%) 8 (10.5%) gender, no. (%) female 34 (45.9%) 30 (39.5%) b0.509 male 40 (54.1%) 46 (60.5%) social status, no. (%) single 29 (39.2%) 42 (55.3%) c0.052 married 45 (60.8%) 34 (44.7%) education level, no. (%) illiterate 1 (1.4%) 1 (1.3%) d0.902 primary 16 (21.6%) 14 (18.4%) secondary 28 (37.8%) 27 (35.5%) college 29 (39.2%) 34 (44.7%) residence, no. (%) urban 58 (78.4%) 71 (93.4%) b0.008 rural 16 (21.6%) 5 (6.6%) smoking, no. (%) 3 (4.1%) 11 (14.5%) b0.028 drinker, no. (%) 0 (0%) 1 (1.3%) c1.0 a: independent t-test, b: chi-square test, c: fisher exact test, d: fisher-freeman-halton exact test the duration of disease was significantly longer in patients with uc compared to cd, the frequency of patients with active disease was significantly higher in patients with uc, the frequency of patients had surgical treatment was higher in patients with cd, and the frequency of patients treated in out-patients setting was higher in the cd as illustrated in table 2. the total score with the sub-scores of patients believes for all patients are shown in table 3 as well as figure 1. iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 36 table 2. comparison of disease haracteristics between both types of ibd the total score with the sub-scores of patients believes for all patients are shown in table 3 as well as figure 1. table 3. patients believes scores for all patients variables value patients believes specific necessity score, mean ± sd (range) 19.23 ± 5.21 (5 -25) specific concern score, mean ± sd (range) 14.95 ± 6.60 (5 25) general harm score, mean ± sd (range) 9.90 ± 3.35 (4 20) general overuse score, mean ± sd (range) 10.22 ± 3.30 (4 18) total score, mean ± sd (range) 54.30 ± 9.61 (31 -80) figure 1. belief about medications for all patients regarding the components of bmq, all the components are not statistically different between cd and uc, as illustrated in table 4. table 4. patients believes scores for each disease variables ulcerative colitis crohn's disease p-value number 74 76 patients believes specific necessity score, mean ± sd 19.58 ± 4.87 18.89 ± 5.53 a0.421 specific concern score, mean ± sd 15.30 ± 6.63 14.61 ± 6.59 a0.523 general harm score, mean ± sd 9.81 ± 3.06 9.99 ± 3.62 a0.748 general overuse score, mean ± sd 9.69 ± 3.10 10.74 ± 3.43 a0.052 total score, mean ± sd 54.38 ± 10.14 54.22 ± 9.13 a0.922 : chi square testbtest, -: independent ta variables ulcerative colitis crohn's disease p-value disease duration (years), mean ± sd 5.88 ± 4.66 4.16 ± 3.98 a0.017 disease activity, no. (%) remission 43 (58.1%) 60 (78.9%) b0.006 active 31 (41.9%) 16 (21.1%) surgical treatment, no. (%) 6 (8.1%) 19 (25.0%) b0.006 the number of chronic drugs, no. (%) single medication 31 (41.9%) 32 (42.1%) b0.979 multiple medications 43 (58.1%) 44 (57.9%) the number of chronic diseases, no. (%) no disease 71 (95.9%) 71 (93.4%) c0.609 single disease 3 (4.1%) 3 (3.9%) multiple disease 0 (0.0%) 2 (2.6%) admission, no. (%) in-patients 17 (23.0%) 5 (6.6%) b0.005 out-patients 57 (77.0%) 71 (93.4%) doses of infliximab, mean ± sd 7.15 ± 6.53 7.95 ± 6.39 a 0.450 a: independent t-test, b: chi-square test, c: fisher-freeman-halton exact test iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 37 the majority of the patients (58%) had strong beliefs in the necessity of treatment (scores bmq specific-necessity greater than score bmq specific-concern). however, (22%) of the patients reported strong concerns about the treatment (scores bmq specific-concern greater than score bmq specific-necessity).the remaining of the patients (20%), have equal scores for bmq specific-necessity and specificconcern scores (table 5) suggests that they have an equal agreement on both concept of the subpart where they share the same score. there was significant inverse correlation between gender (male vs. female) and specific concern score, while significant direct correlation was present between smoking and general overuse score, number of infliximab doses directly correlated with specific necessity score and inversely correlated with specific concern score, number of drugs used directly correlated with both specific concern and general harm scores for all patients as illustrated in table 6. table 5. bmq necessity-concern differential table 6. linear regression analysis between bmq components and other variables for all patients variables specific necessity specific concern general harm generaloveruse r p-value r pvalue r pvalue r pvalue gender 0.127 0.121 -0.198 0.015 -0.058 0.479 0.082 0.317 age 0.075 0.362 0.051 0.536 -0.052 0.525 -0.020 0.812 disease duration 0.065 0.428 -0.041 0.619 -0.092 0.263 -0.066 0.419 disease (cd vs. uc) -0.066 0.421 -0.053 0.523 0.026 0.748 0.159 0.052 disease activity -0.047 0.568 0.093 0.258 -0.010 0.904 -0.080 0.330 social status (married) 0.089 0.279 0.021 0.801 -0.016 0.842 0.003 0.976 education level -0.091 0.266 -0.018 0.823 -0.103 0.210 -0.080 0.332 residence(rural) -0.011 0.896 -0.043 0.597 -0.051 0.533 0.014 0.866 smoking 0.003 0.969 -0.140 0.087 -0.107 0.192 0.201 0.013 surgical treatment -0.103 0.210 0.091 0.270 -0.083 0.312 0.052 0.530 number of drugs used -0.053 0.521 0.199 0.014 0.218 0.007 0.107 0.194 number of chronic disease -0.053 0.518 0.094 0.254 0.067 0.415 0.046 0.574 place of management (out-patients) -0.094 0.253 -0.009 0.912 -0.075 0.364 -0.041 0.618 doses of infliximab 0.188 0.021 -0.214 0.008 -0.146 0.075 0.015 0.855 the number of drugs used was directly correlated with specific concern score also number of chronic diseases directly correlated with general overuse score in uc patients, as illustrated in table 7. necessity – concern differential n percentage necessity > concern 87 58.0 concern > necessity 33 22.0 necessity = concern 30 20.0 iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 38 table 7. linear regression analysis between bmq components and other variables for uc patients variables specific necessity specific concern general harm generaloveruse r p-value r p-value r pvalue r pvalue gender 0.105 0.372 -0.172 0.142 -0.039 0.738 0.101 0.393 age 0.086 0.466 -0.085 0.474 -0.044 0.708 0.031 0.791 disease duration 0.105 0.374 -0.053 0.654 -0.148 0.209 0.008 0.948 disease activity -0.176 0.134 0.120 0.310 -0.001 0.992 -0.039 0.743 social status (married) -0.012 0.917 0.028 0.814 0.050 0.671 0.072 0.543 education level -0.029 0.804 -0.019 0.875 -0.055 0.641 -0.085 0.469 residence (rural) -0.151 0.198 0.046 0.697 0.022 0.853 0.096 0.418 smoking 0.004 0.975 -0.176 0.134 -0.077 0.513 -0.046 0.697 surgical treatment -0.025 0.829 -0.081 0.492 0.035 0.769 -0.083 0.484 number of drugs used 0.116 0.327 0.255 0.028 0.019 0.874 0.086 0.467 number of chronic disease 0.160 0.174 0.095 0.422 -0.010 0.934 0.243 0.037 place of management (out-patients) -0.087 0.460 -0.044 0.712 -0.203 0.083 -0.034 0.772 doses of infliximab 0.166 0.158 -0.131 0.267 -0.017 0.885 -0.007 0.952 residence (rural) inversely correlated with specific concern score, smoking was directly correlated with general overuse score, number of drugs used directly correlated with general harm score, number of infliximab doses inversely correlated with both specific concern and general harm scores in cd patients as illustrated in table 8. table 8. linear regression analysis between bmq components and other variables for cd patients variables specific necessity specific concern general harm generaloveruse r pvalue r pvalue r p-value r pvalue gender 0.156 0.178 -0.217 0.059 -0.078 0.504 0.048 0.679 age 0.062 0.597 0.168 0.146 -0.057 0.627 -0.049 0.676 disease duration 0.003 0.980 -0.052 0.657 -0.034 0.772 -0.083 0.477 disease activity 0.051 0.662 0.041 0.725 -0.007 0.952 -0.055 0.638 social status (married) 0.157 0.176 -0.002 0.984 -0.063 0.589 -0.008 0.944 education level -0.139 0.230 -0.012 0.915 -0.147 0.206 -0.094 0.420 residence(rural) 0.150 0.196 -0.227 0.048 -0.147 0.206 -0.011 0.927 smoking 0.021 0.854 -0.118 0.310 -0.134 0.249 0.295 0.010 surgical treatment -0.127 0.273 0.225 0.051 -0.159 0.171 0.071 0.540 number of drugs used -0.166 0.152 0.162 0.161 0.352 0.002 0.111 0.341 number of chronic disease -0.144 0.213 0.108 0.355 0.100 0.389 -0.064 0.580 place of management (out-patients) -0.082 0.479 0.081 0.485 0.073 0.531 -0.161 0.165 doses of infliximab 0.217 0.060 -0.293 0.010 -0.259 0.024 0.016 0.888 iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 39 discussion inflammatory bowel diseases, are chronic gastrointestinal conditions characterized by alternating episodes of symptom exacerbation and periods of general well-being. as in the majority of other chronic illnesses, lifelong therapy is required (19). it is a worldwide health-care problem with a continually increasing incidence (20). regarding socio-demographic characteristics of patients involved in the current study, smoking was significantly higher in patients with cd (14.5%) compared to the uc (4.1%). approximate to this result was found in study by (c. de bie et al., 2015) where more cd patients were active smokers compared to uc (40% vs. 17%) (21). smoking was found to increase the risk of developing cd but not uc (22). in addition, the frequency of rural uc patients (21.6%) was significantly higher compared to rural cd patients (6.6%) and this is in agreement with study by (alireza t. s et al. 2013) where frequency of rural patients was higher in uc (13.6%) than cd (8.3%) (23). the rest of the socio-demographic variables did not show a significant difference between cd and uc as illustrated in table 1. with respect to disease characteristics, the duration of disease was significantly longer in patients with uc (5.88 ± 4.66 years) compared to cd (4.16 ± 3.98 years) which approximate the results reported by (i˙ lhami y. et. al., 2009) study in turkey where uc duration was longer than cd (24). this may be due to that it takes longer to diagnose cd than uc (25). the frequency of patients with active disease (relapsed) was significantly higher in patients with uc than in cd patients (41.9% vs. 21.1%) respectively. this result came opposite to that of (weigert et. al., 2010) in which the frequency of ptients with active disease was (38.7%) for uc and (64%) for cd patients (26). the frequency of patients had surgical treatment was higher in patients with cd 25% compared with 8.1% for uc patients which is consistent with the result of other studies (ozin et.al. 2009), that found surgical interventions were more common in cd (25). in addition, frequency of patients treated in out-patients setting was higher in cd patients which means that patients with cd may undergo less relapses or any other causes that lead to hospitalization and then cd patients usually treated as outpatients more than uc patients. regarding the components of bmq, all the components are not statistically different between cd and uc. the majority of the patients (58%) had strong beliefs in the necessity of their ibd treatment ( scores bmq specific necessity greater than score bmq specific-concern). this result came approximate to another study (horne et al 2008) which state that (48%) of ibd patients were (high necessity, low concerns) (27). in its simplest form, where necessity beliefs outweigh concerns, the patient is likely to be adherent. in the present study (22%) of the patients reported strong concerns about the treatment (scores bmq specific-concern greater than score bmq specific-necessity). this group of patients was worried about the adverse effect of their prescribed ibd medications. these concerns about long-term effects of using ibd medications should be assessed and reduced by healthcare providers to improve medication intake. the current study showed that there was a significant inverse correlation between gender (male vs. female) and specific concern score that mean male patients have less specific concern about ibd medications than female patients. other studies did not show any significant association between gender and bmq sub scores (28, 29). in the present study, significant direct correlation was present between smoking and general overuse score. smoking has seldom been studied to determine the association with bmq sub scores (over use). also, results of current study showed that the number of infliximab doses directly correlated with specific necessity score and inversely correlated with specific concern score (as number of infliximab doses increase the specific necessity score will increase and specific concern score will decrease). this result may be due to that biological agents are associated with a normalization in quality of life (qol) for ibd patients (30), and this will increase the belief of patients about the specific necessity of infliximab. the number of drugs used directly correlated with both specific concern and general harm scores for all patients (table 6) , this result similar to the result of ( konstantina tsianou 2016) , where the number of drugs used correlated positively with the general harm sub score (29). a possible explanation of this result is that patients using high number of drugs surely will have high concerns about these drugs. in the current study, the number of chronic diseases directly correlated with general overuse score in uc patients (table-7); however, other study found that the number of chronic conditions did not correlate with any of the bmq subs score (28). it seems that accumulation of diseases and/or medicines, has a positive impact on concern (31). also the present study found that residence in rural areas in patients with cd inversely correlated with specific concern score that mean that patients who live in rural areas have less iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 40 concern about their medication than patients who live in urban areas. a possible explanation could be lack of adequate education and/or lack of information to assist a greater understanding of medicines and their effects (31). conclusions the majority (58%) of iraqi ibd patients sample had strong beliefs in the necessity of their ibd treatment where the medication-necessity score was greater than medication-concern score with male patients had less specific concern about ibd medications than female patients. references 1. walker aw, sanderson jd, churcher c, et al. high-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease. bmc microbiology. 2011; 11(1):1-12. 2. shimshoni e, yablecovitch d, baram l,et al . ecm remodelling in ibd: innocent bystander or partner in crime? the emerging role of extracellular molecular events in sustaining intestinal inflammation. gut. 2014; 21: gutjnl-2014:367-372. 3. morgan xc, tickle tl, sokol h, et al. dysfunction of the intestinal microbiom:gutjnl e in inflammatory bowel disease and treatment. genome biology. 2012; 13(9):1-18. 4. neurath mf. cytokines in inflammatory bowel disease. nature reviews immunology. 2014; 14(5):1-14. 5. schaefer js, attumi t, opekun ar, et al. microrna signatures differentiate crohn’s disease from ulcerative colitis. bmc immunology. 2015; 16(1):1-13. 6. marie a. chisholmburns. pharmaco therapy principles and practice. 4th edition 2016. p: 307-320 . 7. bernstein cn, fried m, krabshuis jh, et al. world gastroenterology organization practice guidelines for the diagnosis and management of ibd in 2010. inflammatory bowel diseases. 2010; 16(1):112-124. 8. waljee ak, wiitala wl, govani s, et al. corticosteroid use and complications in a us inflammatory bowel disease cohort. plos one. 2016; 11(6):1-14. 9. bernstein cn. treatment of ibd: where we are and where we are going. the american journal of gastroenterology. 2015; 110(1):113. 10. danese s, vuitton l and peyrin-biroulet l. biologic agents for ibd: practical insights. nature reviews gastroenterology & hepatology. 2015; 12(9):1-9. 11. clarris s, sunitha c, william t, et al. the role of insight into and beliefs about medicines of hypertensive patients. cardiovasc j afr. 2007 nov-dec; 18(6): 353–357. 12. horne r, weinman j. self-regulation and self-management in asthma: exploring the role of illness perceptions and treatment beliefs in explaining nonadherence to preventer medication. psychol health. 1999; 14:1–24. 13. sieta t, joost c, rosalie v, et al. medication beliefs, treatment complexity, and non-adherence to different drug classes in patients with type 2 diabetes. journal of psychosomatic research. 2014; 76: 134– 138. 14. rob h, sarah c, rhian p, et al. understanding patients’ adherence-related beliefs about medicines prescribed for long-term conditions: a meta-analytic review of the necessity-concerns framework. plos one. 2013; 8 (12): 1-24. 15. mohamed m, salmiah m, mohd d. beliefs and adherence to medicines among malaysian malay type 2 diabetes. international journal of current research. 2014; 6 (02):5026-5033. 16. james e, john d. diabetic patients’ medication underuse, illness outcomes, and beliefs about antihyperglycemic and antihypertensive treatments. diabetes care. 2009; 32(1): 19-24. 17. horne r, weinman j, hankins m. the beliefs about medicines questionnaire: the development and evaluation of a new method for assessing the cognitive representation of medication. psychol health. 1999; 14: 1–24. 18. raniah m, waleed m, adham s , et al. beliefs about medicines and self-reported adherence among patients with chronic illness: a study in palestine. journal of family medicine and primary care. 2014; 3 (3): 224-229. 19. d’inca r, bertomoro p, mazzocco k,et al. risk factors for non‐adherence to medication in inflammatory bowel disease patients. alimentary pharmacology & therapeutics. 2008; 27(2):166-72. 20. zhang yz, li yy. inflammatory bowel disease: pathogenesis. world journal of gastroenterology: wjg. 2014; 20(1):91-99. 21. de bie c, ballet v, hendriks n,et al. smoking behaviour and knowledge of the health effects of smoking in patients with inflammatory bowel disease. alimentary pharmacology & therapeutics. 2015; 42(1112):1294-302. https://www.ncbi.nlm.nih.gov/pubmed/?term=shiri%20c%5bauthor%5d&cauthor=true&cauthor_uid=18092108 https://www.ncbi.nlm.nih.gov/pubmed/?term=srinivas%20sc%5bauthor%5d&cauthor=true&cauthor_uid=18092108 https://www.ncbi.nlm.nih.gov/pubmed/?term=futter%20wt%5bauthor%5d&cauthor=true&cauthor_uid=18092108 iraqi j pharm sci, vol.27(2) 2018 belief about medications in ibd 41 22. rosenfeld g, bressler b. the truth about cigarette smoking and the risk of inflammatory bowel disease. the american journal of gastroenterology .2012; 107:1407 – 1408. 23. taghavi sa, safarpour ar, hosseini sv,et al. epidemiology of inflammatory bowel diseases (ibd) in iran: a review of 740 patients in fars province, southern iran. ann colorectal res. 2013; 1(1):17-22. 24. yüksel i, başar ö, ataseven h, et al. mucocutaneous manifestations in inflammatory bowel disease. inflammatory bowel diseases. 2008; 15(4):546-50. 25. ozin y, kilic mz, and nadir i, et al.clinical features of ulcerative colitis and crohn’s disease in turkey. j gastrointestin liver dis. 2009; 18(2):157-62. 26. weigert j, obermeier f, neumeier m,et al. circulating levels of chemerin and adiponectin are higher in ulcerative colitis and chemerin is elevated in crohn's disease. inflammatory bowel diseases.2010; 16(4):630-7. 27. horne r, parham r, driscoll r,et al. patients' attitudes to medicines and adherence to maintenance treatment in inflammatory bowel disease. inflammatory bowel diseases. 2008; 15(6):837-44. 28. tsianos ev, tsianou k, tsipouras mg, et al. beliefs about medicines questionnaire (bmq) in inflammatory bowel disease patients in greece. ιs this useful?. european journal for person centered healthcare. 2016; 4(1):187-95. 29. tsianou k. preliminary analysis of drug adherence of patient suffering with ulcerative colitis. (diploma thesis) 2016. 30. rocchi a, benchimol ei, bernstein cn,et al. inflammatory bowel disease: a canadian burden of illness review. canadian journal of gastroenterology and hepatology. 2012; 26(11):811-7. 31. komninis id, micheli k, roumeliotaki t,et al. adaptation and validation of the beliefs about medicines questionnaire (bmq) in primary care patients in greece. european journal for person centered healthcare. 2013; 1(1):224-31. iraqi j pharm sci, vol.31(2) 2022 adverse events following the covid-19 vaccination doi: https://doi.org/10.31351/vol31isssuppl.pp168-177 168 knowledge, perception, and reporting practices of healthcare providers about adverse events following the covid-19 vaccination in iraq(conference paper )# farah hatem nsaif * and basma zuheir al-metwali**,1 # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 * ministry of health and environment, department of health in baghdad rusafa , ibn al-balady hospital ,baghdad, iraq ** department of clinical pharmacy , college of pharmacy,university of baghdad/baghdad, iraq. abstract routine vaccination activities, such as detection, reporting, and management of adverse events following immunization (aefis), are generally handled by healthcare providers (hcps). safe vaccines against severe acute respiratory syndrome coronavirus (sars-cov-2) were introduced to control the coronavirus disease-19 (covid-19) pandemic. the study aimed to assess the knowledge, perceptions, and practice of hcps in iraq about reporting adverse events following covid-19 vaccination, and their association with sociodemographic variables. the study was a cross-sectional study that was carried out between august and september 2021 at the covid-19 vaccination centers in iraq. this study used an online and paper-based questionnaire, which was distributed among hcps (physicians and pharmacists) in covid-19 vaccination centers. a total of 117 pharmacists and physicians responded to the survey. two-thirds of respondents were pharmacists. the majority of the respondents (49.6%) had fair knowledge levels on aefis. the perception of 43% of the participants was very good, whereas the perception of 28%, 23%, and 6% of the participants was fair, good, and poor, respectively. the reporting practice of hcps was inadequate in 53% of respondents. the number of pharmacists who had good knowledge of aefis was significantly higher than that of the physicians. the age group (30-39) years of hcps was significantly associated with more positive perception towards aefis. the number of pharmacists that had good perception was significantly higher than that of the physicians. furthermore, hcps aged 30 to 39 years had significantly higher reporting practices than other age groups. the study highlighted that the hcps working at the covid-19 vaccination centers have low knowledge of aefis. on the other hand, hcps had more positive perception towards reporting aefis. education programs and reference materials are needed to increase their awareness about aefis. keywords: adverse events following immunization, covid-19, healthcare providers, knowledge, perception, reporting practice. معرفة وفهم وممارسا ت مقدمي الرعاية الصحية في االبالغ عن األثار الجانبية بعد التطعيم بلقاح #) بحث مؤتمر ( في العراق 19 -كوفيد ** بسمة زهير المتولي و 1*، فرح حاتم نصيف 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي .العراق، بغداد ، الرصافة، مستشفى ابن البلد، دائرة صحة بغداد والبيئة، ة الصحةروزا* .العراق ، بغداد ، جامعة بغداد ، كلية الصيدلة فرع الصيلة السريرية، ** الخالصة مثل الكشف عن اآلثار الجانبية بعد التطعيم واإلبالغ عنها ومعالجتها. يتولى مقدمو الرعاية الصحية عموًما أنشطة التطعيم الروتينية ، . تهدف الدراسة إلى تقييم معرفة وتصورات وممارسات مقدمي الرعاية 19-تم إدخال لقاحات آمنة ضد فايرس كورونا للسيطرة على جائحة كوفيد بلقا التطعيم بعد الجانبية األثار عن اإلبالغ حول العراق في كوفيدالصحية الرعاية 19ح لمقدمي الديموغرافية الخصائص تأثير لتقييم وكذلك ، . كانت الدراسة عبارة عن دراسة مقطعية 19-الصحية على معرفتهم وتصوراتهم وممارساتهم في اإلبالغ عن األثار الجانبية بعد التطعيم بلقاح كوفيد نا في العراق. استخدمت هذه الدراسة استبيانًا عبر اإلنترنت واستبيانًا ورقيًا ، تم في مراكز التلقيح ضد فيروس كورو 2021أجريت بين اب و ايلول صيدليًا وطبيبًا ردودهم. كان ثلثا المستجيبين من الصيادلة. غالبية المستجيبين 117توزيعه بين األطباء والصيادلة في تلك المراكز. قدم ما مجموعه ٪ و 28٪ من المشاركين تصور جيدًا جدًا ، بينما كان تصور 43ثار الجانبية بعد التطعيم. كان لدى ٪( لديهم مستويات معرفة مقبولة باأل49.6) ٪ من المستجيبين. كان عدد 53٪ مقبوال وجيدًا وضعيفًا على التوالي. كانت ممارسة اإلبالغ عن مقدمي الرعاية الصحية غير مناسبة في 6٪ و 23 ( سنة لمقدمي الرعاية الصحية 39-30تأثير الضار لالثار الجانبية أعلى بكثير من األطباء. ارتبطت الفئة العمرية )الصيادلة الذين لديهم معرفة جيدة بال ء. عالوة بشكل كبير مع التصورات تجاه اآلثار الجانبية الضارة بعد التطعيم. كان عدد الصيادلة الذين لديهم تصور جيد أعلى بكثير من عدد األطبا عاًما ممارسات إبالغ أعلى بكثير من الفئات العمرية األخرى. سلطت 39و 30قدمي الرعاية الصحية الذين تتراوح أعمارهم بين على ذلك ، كان لم بعد الجانبية األثار عن منخفضة معرفة لديهم كورونا فيروس ضد التطعيم مراكز في العاملين الصحية الرعاية مقدمي أن على الضوء الدراسة ية أخرى ، كان لدى مقدمي الرعاية الصحية تصور أكثر إيجابية تجاه اإلبالغ عن اآلثار الجانبية بعد التطعيم. هناك حاجة إلى برامج التطعيم. من ناح .تعليمية ومواد مرجعية لزيادة وعيهم حول اآلثار الجانبية بعد التطعيم .ممارسات ،فهم ،معرفة ،اية الصحيةمقدمو الرع ،-كوفيد، الكلمات المفتاحية: األعراض الجانبية بعد التطعيم 1corresponding author e-mail: basma.naji@copharm.uobaghdad.edu.iq received: 11/7/2022 accepted: 20/11 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp168-177 iraqi j pharm sci, vol.31(suppl.) 2022 adverse events following the covid-19 vaccination introduction adverse events following immunization (aefis) is defined as "any untoward medical occurrence which follows immunization and which does not necessarily have a causal relationship with the use of the vaccine. the adverse event may be any unfavourable or unintended sign, abnormal laboratory finding, symptom or disease."(1). to improve vaccination safety, there is a need to increase the reporting of aefis in order to identify problems and take appropriate corrective action (2,3). to ensure that any safety signals can be detected rapidly and responded to in an appropriate and timely manner, aefis reporting from health facilities may need to be more frequent than usual reporting.(4) reports should be done by using a standardized aefi reporting form(5). as most of the infected countries, iraq has reported its first confirmed case of sars-cov-2 infection in 24th february / 2020 (6). high rates of successful vaccinations may aid in overcoming the global health threat posed by the covid-19 pandemic )7(. regarding vaccine status in iraq, a total of 4,908,532,010 individuals had received the vaccine by september 12, 2022 (8). although adverse events from the different covid-19 vaccines may vary, those commonly reported include pain at the injection site, fatigue, ache of muscles, chills, and fever (9). these side effects normally subside within a few days and are a sign that the immune system is functioning properly(10). if an individual receives the vaccine and experience a negative reaction ,s/he should be informed about the possible adverse events to seek medical attention(11). the covid-19 vaccine is a new vaccine(12). when a new vaccine is introduced, adverse events associated with this new vaccine should be reported(13). all aefis, both major and minor, encountered should be reported(14). this is done by using the standard covid19 aefi reporting form(15). in iraq, there are numerous routes for receiving and documenting aefi reports that were established within the system which has evolved with time. the major sources are through the designated safety personals at the vaccination centers that are informed and then fill and submit the paper-based or online reporting aefi form to document the report. in addition to that, the online self-assessment form was designed to be filled by the vaccines themselves and can be accessed through the whatsapp number of the iraq pharmacovigilance center (ipvc)(16) .the iphvc has an official reporting form for the covid-19 vaccine. routine immunization activities, such as detection, reporting, and management of aefis are generally performed by hcps in hospitals and primary health care centers. they must have good knowledge of aefis and its management in order to effectively carry out these obligations(17). it is noteworthy that there is a study of vaccine safety conducted using the iphvc database in the iraqi ministry of health from 2014 till the end of 2018. the results of this study have shown that the number of reports of aefi reports had increased during 2017 and 2018 where they were 1080 (51%) and 684 (32%), respectively, which may be related to increased awareness of hcps compared with previous years(18). the published studies in iraq were more focused on the medicines adverse drug reactions and were not specific to vaccines. despite the pharmacists having a positive attitude, they lacked of adequate knowledge and reporting practice to adverse drug reactions (19,20). it is very important to assess these aspects of knowledge, attitude, and practice of aefis, particularly those concerning the covid-19 vaccine being a new agent introduced to the market. the aims of the current study were to assess the knowledge, perceptions, and practice of hcps (physicians and pharmacists) about reporting adverse events following covid-19 vaccination in iraqi vaccination centers, and their association with sociodemographic variables. subjects and methods this was a cross-sectional study carried out between 16th august until 16th september/ 2021 in covid-19 vaccination centers in iraq. the study population included a convenient sample healthcare providers (physicians and pharmacists) involved in covid-19 vaccination who gave consent to participate in the study. the exclusion criteria were healthcare providers (physicians, and pharmacists) who were not involved in covid-19 vaccination, those who did not give consent to participate in the study and those who provided incomplete information during the completion of the questionnaire.a paper-based, self-administered questionnaire in english language was used for data collection. the study was conducted in primary health care institutions and hospitals that were concerned with covid-19 vaccination in baghdad. these centers were al-qanat health care center, al-mustansirya health care center and al-kindy teaching hospital. because of the low response rate due to the workload, an online questionnaire through a google form was used for this study in addition to the paper-based questionnaire and distributed through specialized medical groups on social media throughout the study period. the questionnaire was adopted from two previous studies with some modifications made to be suitable for covid-19 vaccines (17,21). the questionnaire was reviewed and approved by members of the ethical and scientific committee at the college of pharmacy/university of baghdad. no validation of the questionnaire was performed. iraqi j pharm sci, vol.31(suppl.) 2022 adverse events following the covid-19 vaccination 170 there were four sections to the questionnaire. the first section consisted of five questions, which were used to collect the data about socio-demographic characteristics. in the second section, there were six questions, which were used to collect data on their aefis knowledge. section three comprised eight questions, which were used to assess the respondents' aefis perceptions. the last section included eight questions about the practices of reporting aefis among the respondents. some of the questions in this section were not given a score since they were designed to evaluate the information rather than the respondents' reporting practices. the questionnaire was first pretested on ten hcps to ensure the clarity of the questions. then it was distributed to other hcps. this study was approved by the ethical and scientific committee at the college of pharmacy/university of baghdad. the participants were informed about the research objectives and confidentiality of their responses. the statistical analyses were performed using microsoft excel and statistical package for social sciences (spss) (version 23.1). the study population's characteristics were summarized using descriptive statistics. frequencies and percentages were used to represent categorical variables. the knowledge score was determined by allocating a score of 1 to each correct response and a score of zero for each incorrect response. to calculate the knowledge score, all correct responses were summed, divided by all possible correct responses, and multiplied by 100. a knowledge score of more than 50% was considered as good knowledge on aefi; whereas a score of 40-50% was considered fair knowledge and a score of less than 40% was considered as poor knowledge.(17) for the determination of perception, 'yes' responses were considered as positive perception and were given a score of 1 whereas 'no' answers were considered as a negative perception and were given a score of 0. participants who scored less than 2 were considered to have a poor perception, those more scoring 2-5 to have a fair perception, those scoring 5-7 to have a good perception, and those who scored 7 or to have very good perception.(17,21) for reporting practice, each correct response represented an adequate practice and was scored one, whereas incorrect response represented an inadequate practice and was scored zero. a cumulative score of 50% or more was considered as good reporting practice, whereas poor reporting practice was considered when the cumulative score was less than 50%. (17) the effect of demographic characteristics on knowledge, perception, and reporting practice was calculated using the chi-square test or fisher's exact test if needed. fisher's exact test was employed when more than 20% of cells have anticipated frequencies of less than 5. the p value of less than 0.05 was considered to be significant. results demographic characteristics of the study population a total of 117 pharmacists and physicians provided their responses, where 28 responses were paper-based and 89 responses were online. all responses obtained were complete and 9 hcps did not consent to participate in the paper-based study. the largest age group was 30–39 years (55.6%). approximately, two-thirds of respondents (67.52%) were pharmacists. the demographic characteristics are summarized in table 1. table 1. demographic characteristics of the study population. variables frequency (n) percentage (%) age (years) <30 44 37.6 30-39 65 55.6 40-59 8 6.8 profession physicians 38 32.48 pharmacists 79 67.52 place of work primary health care centre 70 59.83 hospital 47 40.17 gender male 32 27.35 female 85 72.65 degree/ qualification bachelor 102 87.18 post graduate 15 12.82 knowledge of healthcare providers of aefis: knowledge was assessed by asking questions about the causes, and features of aefis. the majority of the respondents 50% had fair knowledge levels on aefi while 40% had poor and 10% had good knowledge (figure 1). iraqi j pharm sci, vol.31(suppl.) 2022 adverse events following the covid-19 vaccination 171 figure 1. knowledge scores of healthcare providers. good knowledge: score of > 50%, fair knowledge: score of 40-50% and poor knowledge: score of < 40%. the majority of the respondents (74%) knew that vaccine-product related reaction is one of the aefis causes, while only 25% of them knew that immunization error-related reaction is considered as a cause of it and 19% knew that immunization anxiety-related reaction is also classified as a form of aefis. about three quarters of the respondents knew that the methods of reporting adverse events that following the covid-19 vaccines is by filling aefi reporting form, but also 6% of the respondents considered talking to colleague as a method of reporting. also 65% of the respondents did not know about the covid-19 vaccine reporting form. when asked participants about adverse events of covid19 vaccine that should be reported, 62.4% of the respondents knew that all of adverse events should be reported and only 37.6% of them did not know that. the results also demonstrated that only 14% of the participants knew that the investigation of aefis should be started within 24 hours (table 2). table 2. knowledge of study participants about aefis of covid-19 vaccine. perceptions of healthcare providers about aefis: forty-three percent of the participants had very good perception while 28%, 23%, and 6% had fair, good, and poor perception, respectively (figure 2). figure 1. perceptions of aefis among healthcare providers most of the respondents (91.5%) who reported an aefi believed that reporting aefis does not involve personal consequences or punishment. also 76.9% of the respondents felt that inadequate monitoring of adverse events can lead to a decline in immunization coverage. furthermore, 92.3% agreed that improving aefis observation can aid in public trust in the immunization program. moreover, 97.3% of respondents wanted to learn more about how to diagnose, report, investigate, and manage aefis (table 3). questions frequency(n) percentage aefis causes: (multiple answers possible): vaccine-product related reaction 86 74 vaccine-quality defect-related reaction 26 22 immunization error-related reaction 29 25 immunization anxiety-related reaction 22 19 coincidental reaction 26 22 methods of covid-19 vaccine aefi reporting: filling of aefi form 85 73 reporting via social media group 15 13 email/online 10 9 talking to colleague 7 6 aefi can be caused by reconstituted vaccines stored longer than normal, vaccine reaction, inappropriate route of administration, vaccines stored beyond the expiry date, or contaminated vaccines. yes 48 41 no 69 59 do you know about the covid-19 vaccine reporting form? yes 41 35 no 76 65 the investigation of an aefi should start within 24 hours: yes 17 14 no 100 86 iraqi j pharm sci, vol.31(suppl.) 2022 adverse events following the covid-19 vaccination 172 table 2. perceptions of study participants about aefis. *‘yes’ answers signify positive perception whereas ‘no’ answers signify negative perception. reporting practices of healthcare providers on aefis overall, the reporting practice of hcps was inadequate in 53% of respondents while it was adequate in 47% of them (figure 3). more than half of the respondents (57.3%) stated that they encountered aefis. the most commonly encountered aefi with covid19 vaccine was pain and swelling at the injected site (83%), fever (76.1%), fatigue (61.2%), and headache (55.2%). about 50% of the hcps indicated that they routinely report the aefis that they encounter and most of them (62%) reported within 24 hours of detecting aefi. the methods used for aefi reporting by the respondents were filling aefi forms (67.5%) and via other methods (32.5%). approximately half of the respondents (49.6%) reported that they have seen an aefi reporting and investigation form and the rest indicated that they have not. in addition, more than half of the respondents (56.4%) indicated that they didn’t have aefi reference guidelines in their facilities and 86.3% of the respondents stated that they routinely tell the patients about the side effects of a vaccine. on the other hand, 49.6% of hcps stated that they did no routinely report the aefis that they encounter. about 32.7% of the participants who did not report aefis stated that they didn’t know how and where to report it. only 8.6% of them felt that it was not related to immunization. the results of the reporting practice are shown in table 4. figure 2. level of reporting practice of health care providers toward aefi. adequate reporting practice: cumulative score of ≥ 50%, inadequate reporting practice: cumulative score of < 50%. yes* no* variables frequency (n) percentage frequency (n) percentage i believe that reporting an aefi cannot lead to personal consequences/ punishment 107 91.50 10 8.50 i believe that reporting an aefi will not make me feel guilty about having caused harm to a person taking the vaccine 106 90.60 11 9.40 i believe that healthcare providers are willing to report an aefi even when they are not confident about the diagnosis 82 70.10 35 29.90 i believe that poor monitoring of adverse events can cause a reduction in immunization coverage. 90 76.90 27 23.10 i believe that the process of reporting an aefi is not long and boring? 93 79.50 24 20.50 i believe that if adverse events are reported, something will be done about it. 103 88 14 12 i believe that enhancing the observation of aefi can help build public trust in the immunization program. 108 92.30 9 7.70 i desire to learn more about how to diagnose, report, investigate and manage aefi 114 97.40 3 2.60 iraqi j pharm sci, vol.31(suppl.) 2022 adverse events following the covid-19 vaccination 173 table 3. reporting practice of aefi of covid-19 vaccine by study participants association of socio-demographic characteristics and aefi knowledge, perception and practice respondents’ profession was found to significantly affect knowledge on aefi (table 5). the percentage of pharmacists who had good knowledge of aefis was significantly higher than that of the physicians. in contrast, respondents' age group and profession were significantly associated with perception towards aefis (table 6). the age group (30-39) had significantly higher perceptions than other groups. in addition, age group was significantly associated with reporting practice where the age group (30-39) had significantly higher reporting practice than other age groups (table 7). table 5. association between aefi knowledge and study participant characteristics knowledge aefi classification variables poor fair good p-value age (years) <30 19 17 8 0.154 30-39 25 37 3 40-59 3 4 1 profession physicians 14 24 0 0.018* pharmacists 33 34 12 place of work primary health care centre 20 21 6 0.615 hospital 27 37 6 gender male 15 15 2 0.536 female 32 43 10 degree/ qualification bachelor 43 50 9 0.075 diploma 0 0 0 master 3 3 2 phd 1 0 1 board certificate 0 5 0 *significant at (p-value < 0.05) according to chi-square tests. table 6. association between study participants' characteristics and perception of aefi frequency (n) percentage frequency (n) percentage have you encountered a covid-19 aefi in your practice? 67 57.3 50 42.7 do you routinely report an aefi you encountered? 59 50.4 58 49.6 have you ever seen an aefi reporting and investigation form? 58 49.6 59 50.4 do you have aefi reference guidelines materials at your workstation? 51 43.6 66 56.4 do you routinely tell the patient about the side effect of a vaccine? 101 86.3 16 13.7 perception of aefi classification variables poor fair good very good p-value age (years)# <30 5 11 6 22 0.008* 30-39 0 21 19 25 40-59 2 1 2 3 profession physicians 1 8 4 25 0.005* pharmacists 6 25 23 25 place of work primary health care centre 3 13 12 18 0.776 hospital 4 20 14 32 iraqi j pharm sci, vol.31(suppl.) 2022 adverse events following the covid-19 vaccination 174 continued table 6. *significant at (p-value < 0.05) according to chi-square tests/ fisher exact test. # fisher exact test. table 7. association between study participants' characteristics and reporting practices on aefis reporting practices on aefis classification variables inadequate adequate p-value age (years) <30 31 13 0.008* 30-39 29 36 40-59 2 6 profession physicians 20 18 0.957 pharmacists 42 37 place of work primary health care centre 25 21 0.857 hospital 37 33 gender male 15 17 0.461 female 47 38 degree/ qualification bachelor 55 47 0.938 diploma 0 0 master 4 4 phd 1 1 board certificate 2 3 *significant at (p-value < 0.05) according to chi-square tests. discussion to our knowledge, this is the first study in iraq concerning aefis with covid-19 vaccines among pharmacists and physicians working in vaccination centers. the quality and safety of immunization services, as well as the monitoring of aefi, are influenced by hcps' knowledge, perceptions, and reporting practices on aefi. furthermore, it promotes public confidence in vaccinations, resulting in increased vaccine coverage and a reduction in the burden of infectious diseases as more people are vaccinated(22). this is especially important in the case of the covid-19 pandemic which has had a wide spread impact on health, including significant mortality among older adults and those with pre-existing health conditions, as well as global economic repercussions caused by physical distancing measures(23). based on the scores obtained in this study, knowledge about aefis was inadequate. a small percentage of the study participants had good knowledge, with the vast majority having fair to poor knowledge. this low level of knowledge of study participants may be due to their inadequate knowledge about the causes of aefis, the presence of a covid-19 aefis reporting form, and the time within which investigations of aefis should be started. this is similar to the findings of an albanian study in which majority of the respondents had poor knowledge levels on aefi and only 7.8% of hcps have good knowledge (24). most of the study respondents knew that reporting of covid-19 vaccine adverse events is done by filling a form. this is similar to a study done in lagos, nigeria when the participants were asked about aefi reporting forms were 92.7% of them knew about filling the form as a method of reporting (25). however, a small percent of the study participants believed that reporting can occur via social media groups which indicate the need to educate them that reporting is done only via the official form of covid-19 vaccine according to this study, the majority of the respondents did not know about the covid-19 vaccine reporting form, which can be considered perception of aefi classification variables poor fair good very good p-value gender male 2 10 7 13 0.974 female 5 23 20 37 degree/ qualification bachelor 6 25 25 46 0.227 diploma 0 0 0 0 master 1 4 1 2 phd 0 0 1 1 board certificate 0 4 0 1 iraqi j pharm sci, vol.31(suppl.) 2022 adverse events following the covid-19 vaccination 175 among the reasons that lead to a decrease in reporting adverse events of the vaccine(26). also, most of the respondents were unaware that the investigation of aefis should be started within 24 hours. on the contrary high percentage of respondents (62.3%) who were recruited in a similar study in ghana showed good awareness(21). the investigation of aefis must start nearly to the time an event occurs, especially for serious as well as unexpected aefis to assess the causality of aefi and also managing of it in hospitals(3). in addition, a previous study conducted in baghdad has shown that iraqi pharmacists had insufficient knowledge about the iraqi pharmacovigilance system(27). knowledge regarding pharmacovigilance is very important for hcps to report aefi to the pharmacovigilance center and have a positive perception towards reporting the perception of aefis reporting was very good for the majority of the study participants. the majority of respondents believed that reporting aefis does not involve personal consequences and will not make them feel responsible for causing harm to people receiving the vaccine. additionally, a good perception level was obtained from the respondents who believed that most of the hcps were willing to report aefis even if they were not confident about the diagnosis and that poor monitoring may cause a reduction in immunization coverage. a positive and encouraging finding from the current study is that most of the health workers were conscious that reporting aefis can help build trust in immunization programs and most of them also desire to learn more about how to diagnose report and manage aefis. this will be essential to immunization managers, especially at the health center level, to offer aefi training opportunities. these findings, however, differ from the low perception levels on aefis recorded by nurses in nairobi, kenya(22). among the reasons for the low of perception level is the fear of being blamed and personal consequences after reporting. in contrast to the study in ghana which showed that about threequarters of study participants believed that reporting an aefi can lead to such personal consequences(21). the profession and age were the only factors that significantly affected the perception of aefis. pharmacists' perception was more positive than the perception of the physicians. however, it is worth noting that fewer physicians participated in the present study compared to pharmacists. the good perception was also affected by the age (the age group 30-39) years. this may suggest that most of the study participants were within this group of age and they have more practice in their work than other groups. this is in agreement with similar to study on aefi among nurses in kenya were respondents aged 30–39 years were three times more likely to have a good perception towards aefi surveillance(22). the majority of the respondents' reporting practice toward the aefis was inadequate. this suggests that although hcps had good perception about aefis, they did not have as much good practice of reporting aefis. in contrast, in the study of nigeria, the reporting practices were appropriate in (86.8%) respondents(17). this under reporting practice may be due to that they didn’t have aefi reference guidelines materials at their workstation. the participants in the current study that didn't report aefis stated the main reasons behind this were that they were not aware of how and where to report it, reporting form was not available, and they were busy and had no time as a result of their work pressure the most commonly used method for reporting was through filling the aefi form. similar findings have been reported in australia where all nurses were familiar with paper reporting procedures to the local department of health and also described their workplace reporting processes, such as having the report forms on hand and/or an existing protocol for reporting adverse events(28). good practices such as routinely telling the patient about the side effects of the vaccine were observed among the majority of the respondents. however, this study results also indicated that age was the only socio-demographic characteristic that significantly influenced the reporting practice of aefis because age reflects years of experience in the practice. gender, profession, and place of work did not have a significant influence on aefi reporting practice. the major limitation of the study was the small and convenient sample size and the short time for data collection. another limitation was that physicians were not well represented, as compared to pharmacists. conclusion a low level of knowledge was observed among hcps towards aefis, so it is important to raise awareness and improve the knowledge of all hcps through regular training programs. on the other hand, an adequate perception level was observed in approximately two-thirds of the study participants. however, the reporting practice of hcps was inadequate in more than half of the participants. because the covid-19 vaccines are newly introduced to the market, healthcare providers who provide immunizations require extra education about their role in vaccine safety and reporting adverse events. therefore, there is a need to increase the knowledge of pharmacists and physicians about aefis and the importance of their reporting. this can be achieved by providing opportunities like training and 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commonly used antibiotic for anaerobic bacterial infections, protozoal infections, and microaerophilic bacterial infections by interacting with dna and thereby inhibiting the protein synthesis. metronidazole is administered orally, intravenously, or topically and has a half-life of 6.5 ± 2.9 hours. a number of studies have recently been conducted on the selective substitution of hydrogen with deuterium, which increases the bond strength thereby increasing the biological half-life and, consequently, the drug's metabolic stability. in an attempt to check whether the deuterated metronidazole possessed similar pharmacological activity as that of metronidazole, deuterated metronidazole was synthesised using deuterium oxide in the presence of benzoic acid under nitrogen atmosphere. the synthesised deuterated metronidazole was characterised by ir, 1hnmr and mass spectroscopy and were tested for antibacterial, antifungal, and anti-tubercular activities. the metronidazole and its deuterated compound showed equipotent antifungal activity and aerobic antibacterial activity. also, when compared with the non-deuterated compound, deuterated metronidazole exhibited better anaerobic antibacterial and anti-tubercular activity. keywords: deuterated metronidazole, antibacterial activity, antifungal activity, anti-tb activity. introduction the development of newer molecules is a time-consuming and expensive since only one out of 1, 00, 000 molecules become a potent lead. the reason for the low success rate is due to metabolic instability, poor absorption, oral bioavailability, and toxicity of the compounds. to overcome these challenges, medicinal chemists all over the world are working on various strategies such as bioisosteres, hybridisation techniques, scaffold hopping etc. one such recent technique is deuteration of molecules, which has gained interest in developing novel chemical entities with enhanced desired physicochemical properties (1-7). deuterium is a naturally occurring non-radioactive hydrogen isotope that was reported in 1932. hydrogen has a mass of 1.008 atomic mass units (amu) and includes one electron and one proton, but deuterium additionally contains a neutron and has a mass of 2.014 amu. deuterium has a natural abundance of around 1 part in 6400, or 0.015 percent, allowing huge amounts of deuterium to be extracted as heavy water (d2o) with extremely high isotopic purity. d2o can then be used as a direct or indirect source of deuterium in a variety of chemical reagents and building blocks for deuterated drug manufacturing. deuterium and hydrogen atoms are freely interchangeable in synthetic processes and deuteration leads to the replacement of deuterium for hydrogen in a molecule, although the carbond bond is known to be stronger than the carbon-h bond due to lower zero-point energy, the kinetic isotope effect may occur as a result of the change in bond strength. when one or more hydrogen atoms are replaced with deuterium, the ratio can be as high as 6to 10-fold. this process might have an impact on interactions between deuterated molecules and drug-metabolizing enzyme systems. selective deuterium substitution preserves the pharmacologic profile of physiologically active compounds while also positively modifying their metabolic fate in some situations. deuterium substitution can theoretically increase the safety, effectiveness, and/or tolerance of a medicinal drug (8-14). deutetrabenazine was the first deuterated pharmaceutical molecule approved by the fda for the treatment of chorea, "an involuntary movement disorder” linked to tardive dyskinesia and huntington's disease (figure 1) (15-18). the primary reason for d exchange is to enhance their metabolism and absorption characteristics, resulting in differentiated pharmaceuticals with enhanced safety and/or efficacy. deuteration of bioactive chemicals is gaining popularity, with numerous deuteriumlabelled entities now in clinical studies. as a result, scientists are developing new synthetic technologies for deuterated compounds in order to accommodate the increase in demand. many deuterated compounds will not exhibit a beneficial 1corresponding author e-mail: drmkkathir@gmail.com received: 26/ 4 / 2022 accepted:29 / 6/2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31iss2pp297-303 iraqi j pharm sci, vol.31(2) 2022 evaluation of deuterated analogues of metronidazole 298 and substantial exchange as compared to the precursor; yet, exhibiting a spectacular final product. despite various obstacles, researchers are continuing to look at the possibility of replacing ch with c-d to increase the active ingredient's halflife while simultaneously improving pharmacokinetics and toxicological characteristics. recent papers focused on the deuteration-based synthesis of a new structural scaffold by converting the ch3 group of metronidazole to the cd3 group (1922). metronidazole [2-(2-methyl-5nitroimidazol-1-yl) ethanol] is a synthetic antibiotic derived from azomycin that was first discovered in streptomyces spp. cultures in the 1950s. actinobacteria, such as streptomyces eurocidicus and nocardia mesenterica, as well as proteobacteria (pseudomonas fluorescens), produce azomycin. metronidazole is metabolised in the human liver to produce more polar metabolites. the gut microbiota can also modify metronidazole, resulting in reduced metabolites including hydroxyethyl oxamic acid and acetamide. further literatures indicates that one of the metabolites like acetamide is the responsible for the cytotoxicity. hence if the metronidazole is deuterated thereby the half-life will be increased and could result in reduction of cytotoxicity. the metabolism of metronidazole is explained in figure 2 (23-27). figure 2. metronidazole metabolism in humans metronidazole is now used to treat bacteria, including clostridia infections, fusobacteria, and rosacea, as well as oral and dental infections, septicemia, endocarditis, joint and bone infections, gynaecological infections, and respiratory tract infections. even though it is widely used, metronidazole has been associated with neurotoxicity, genotoxicity, painful urination, cystitis, and pelvic pain due to long-term therapy (figure 2). tamoxifen, which is used to treat breast cancer causes genotoxicity but its deuterated derivative is found to be less genotoxic. similarly, genotoxicity caused by metronidazole can be overcome by its deuterated analogue. also, the halflife of metronidazole which is found to be 6.5 ± 2.9 hours can be increased in deuterated analogue due to increase in bond strength. in continuation to our ongoing work on the development of antimicrobial agents, we herein for the first time hitherto unreported deuterated analogues of metronidazole. the aim of the study is to convert the ch3 group of metronidazole to the cd3 group by deuterium exchange and to determine the physicochemical properties with their evaluation for anti-microbial activity (28-31). methods and materials in silico studies the physicochemical characteristics of metronidazole and deuterated metronidazole were investigated using molinspiration (www.molinspiration.com) and swiss adme. the logp parameter is used to determine whether or not the cell membrane is permeable. drug absorption, bioavailability, drug-receptor interactions, iraqi j pharm sci, vol.31(2) 2022 evaluation of deuterated analogues of metronidazole 299 metabolism, and toxicity are all influenced by the hydrophilic or lipophilic properties of drug molecules. total polar surface area (tpsa) is a good predictor of drug transport qualities such as intestinal absorption, bioavailability, and bloodbrain barrier penetration because it is directly connected to a molecule's hydrogen bonding potential. a score of -1 to -4 on the log s distribution is excellent for improved medication absorption and distribution in the body. the number of rotatable bonds is used to evaluate the molecular flexibility and the molecule with more rotatable bonds is more flexible, and its binding affinity with its binding pocket is better. drug similarity data (lipinski's rule of five) may be used to compare the molecule's characteristics and structural aspects to recognized medications. the bioactivity score for deuterated metronidazole and metronidazole like gpcr ligand, nuclear receptor ligand, a kinase inhibitor, and ion channel modulator is calculated using the molinspiration drug-likeness score (www.molinspiration.com) (3235). synthesis of deuterated metronidazole in a nitrogen environment, metronidazole (0.0342g 0.2 mmol), benzoic acid (0.0049g, 0.04 mmol), and 1ml of d2o were refluxed for 48 hours at 120 o c. the progress of the reaction was monitored by tlc. on completion, the mixture was neutralised with a saturated nahco3 solution and extracted three times with ethyl acetate (3ml). the separated organic layers were combined, and anhydrous sodium sulphate were added to dry the organic layer. the organic layer was then distilled under reduced pressure to obtain crude product which were purified using 99.9% ethanol (36). scheme 1. synthesis of deuterated metronidazole biological evaluation the biological tests were carried out at the central research laboratory at maratha mandal in belgaum. test microorganisms the test microorganisms that are considered for the study are gram-positive (e. faecalis, staph aureus, and fusobacterium nucleatum), gram-negative (e. coli, pseudomonas, porphyromonas gingivalis, and prevotella intermedia), and mycobacterium tuberculosis strain h37rv. candida albicans and a. niger were also used to evaluate the antifungal activity of the newly synthesized deuterated compound. antifungal and aerobic antibacterial activity aerobic antibacterial and antifungal activities were carried out according to the protocols described in the literature (37). anaerobic antibacterial activity anaerobic antibacterial activity was performed as per the procedures cited in the literature (38). antitubercular activity anti-tubercular activity was carried out according to the protocols outlined in the literature (39-41). results and discussion pharmacokinetics profile of metronidazole and its deuterated analogue an orally active drug, according to lipinski's rule of five, should contain no more than 5 hydrogen bond donors (oh and nh groups), not more than 10 hydrogen bond acceptors (mostly n and o), molecular weight under 500 g/mol, and a partition coefficient log p of less than 5. metronidazole and deuterated metronidazole results were found to be compatible with lipinski's 'rule of five,' with a molecular weight of 171.16 and 174.13 respectively. both metronidazole and its deuterated compound showed no violations according to lipinski’s rule and the results are as stated in table1. bioactivity score for metronidazole and its deuterated analogue molinspiration also predicted the bioactivity scores for metronidazole and its deuterated compound for drug targets, which are provided in table 1. a molecule with a bioactivity score of greater than 0.00 is most likely to have significant biological activity, whereas values between -0.50 and 0.00 are considered moderately active, and scores less than -0.50 are considered inactive. both metronidazole and its deuterated molecule had equal bioactivity scores for gpcr ligand, kinase inhibitor, nuclear receptor ligand, iraqi j pharm sci, vol.31(2) 2022 evaluation of deuterated analogues of metronidazole 300 ion channel modulator, enzyme inhibitor, and protease inhibitor. according to the data, metronidazole and deuterated metronidazole have a modest interaction with an enzyme receptor inhibitor. table 1. in silico studies for metronidazole and its deuterated analogue logp – partition coefficient, tpsatopological polar surface area, mwmolecular weight, nrotb-number of rotatable bonds, non no. of hydrogen bond acceptor, nohnh – no. of hydrogen bond donor. synthesis of deuterated metronidazole deuterated metronidazole was synthesised from commercially available metronidazole as starting material using d2o at nitrogen atmosphere in the presence of benzoic acid. the synthesised deuterated metronidazole was obtained as a light brown solid with 90% yield; mp:166-1680c; and rf value of 0.6 (toluene: chloroform: methanol (3.0:2.0:0.6 v/v)).ir (kbr, vmax) cm -1: 1523: asymm. (no2) str., 1368: symm. (no2) str., 2856: (cd3) str., 3123: (ch2) str. of alkyl group, 3420: (oh) str. of alcohol. 1h nmr (500 mhz,dmso-d6) 8.05 (s, 1h, imidazole), 7.45 (s, 1h, oh), 4.73 (t, 2h, o-ch2, 4.63 (t, 2h, n-ch2). deuterated metronidazole showed an m/z of 174. aerobic antibacterial activity the mass differences associated with the replacement of hydrogen by deuterium in a molecule are likely to have a major influence on the physical and chemical properties of the molecule. the results for the aerobic antibacterial activity of deuterated metronidazole and metronidazole against two grampositive bacteria (staph aureus and e. faecalis) and two gram-negative bacteria (pseudomonas and e. coli) are shown in table 2. from the results, it is clear that deuterated metronidazole exhibited better aerobic antibacterial activity against e. faecalis, with a mic of 25μg/ml when compared with metronidazole 50μg/ml. the mic values for metronidazole and its deuterated analogue showed similar values of 25μg/ml against staph aureus, and 50μg/ml against e. coli. bu in the case of pseudomonas, deuterated metronidazole had a mic of 50μg/ml, compared with 25μg/ml of metronidazole. anaerobic antibacterial activity the anaerobic antibacterial activity of deuterated metronidazole and metronidazole against two gram-negative bacteria (porphyromonas gingivalis and prevotella intermedia) and one gram-positive bacteria (fusobacterium nucleatum) are represented in table 2. deuterated metronidazole had a mic of 0.8μg/ml in thioglycolate broth for anaerobic antibacterial activity in fusobacterium nucleatum, and 3.12μg/ml in porphyromonas gingivalis compared to 1.6μg/ml and 6.25μg/ml of metronidazole respectively. also, in prevotella intermedia, deuterated metronidazole showed a better mic of 3.12μg/ml, compared with 6.25μg/ml of metronidazole. from the results, it is clear that deuterated metronidazole exhibited stronger anaerobic antibacterial activity against gram-positive bacteria compared to gram-negative bacteria. deuterated metronidazole has superior antibacterial activity against anaerobic bacteria. this is due to the absence of electron transport proteins in aerobic cells with negative redox potential. as a result, the drug is only effective against bacteria with anaerobic metabolisms, even if it is effective against some microaerophiles like h. pylori. pharmacokinetics parameters metronidazole deuterated metronidazole logp -0.47 -0.47 tpsa 83.88 83.88 natoms 12 12 non 4 4 nohnh 1 1 n violations 0 0 nrotb 3 3 mw 171.16 174.13 bioactivity score gpcr ligand -1.09 -1.09 ion channel modulator -0.87 -0.87 kinase receptor inhibitor -0.59 -0.59 nuclear receptor ligand -1.74 -1.74 protease receptor inhibitor -1.68 -1.68 enzyme receptor inhibitor -0.32 -0.32 iraqi j pharm sci, vol.31(2) 2022 evaluation of deuterated analogues of metronidazole 301 antifungal activity the mic values for antifungal activity of deuterated metronidazole and metronidazole are shown in table 2. when tested for antifungal activity in brain heart infusion broth, both deuterated and its parent compound exhibited mic values of 25μg/ml against candida and 100μg/ml against a. niger. in comparison to a. niger, both the compounds showed stronger antifungal activity against candida. antitubercular activity the compounds were screened against m. tuberculosis h37rv using the microplate alamar blue assay (maba). table 2 shows that deuterated compound, with a mic of 1.6μg/ml, has superior anti-tb action than metronidazole, which has a mic of 3.12μg/ml. deuterated metronidazole's increased activity could be due to a primary isotope effect at the target site, in which the rupture of c-d bonds is directly involved, or the deuterated molecule's increased stability could play a role in the bacteria's inability to metabolise deuterated compound as easily as the protio form. when the antitubercular activity of deuterated metronidazole was compared to that of the standard drugs isoniazid and ethambutol, this showed similar activity with a mic of 1.6μg /ml. in addition, when compared to the standard medication pyrazinamide, which has a mic of 3.125μg /ml, deuterated showed significantly greater antitubercular activity. table 2. antimicrobial activity of metronidazole and deuterated metronidazole conclusion to combat drug resistance, enhance pharmacokinetic profile and reduce cytoxicity of metronidazole, deuterated metronidazole was developed. the physicochemical parameters of metronidazole and its deuterated derivative were assessed using molinspiration and swiss adme, and both exhibited identical physicochemical properties and bioactivity scores. the antibacterial activity (aerobic and anaerobic) of metronidazole and its deuterated derivative against gram-positive, gram-negative bacteria, and fungus, as well as the m. tuberculosis h37rv bacterium, was investigated. deuterated metronidazole had a minimum inhibitory concentration (mic) of 0.8 μg/ml in fusobacterium nucleatum, compared to 1.6μg/ml for metronidazole, and a mic of 1.6μg/ml in porphyromonas gingivalis, compared to 3.12μg/ml for metronidazole. in prevotella intermedia, deuterated compound had a mic of 3.12μg/ml, whereas metronidazole had a mic of 6.25μg/ml. deuterated metronidazole's higher activity might be owing to a primary isotope effect at the target site, in which c-d bond breaking is directly involved. because c-d bonds are more stable than c-h bonds, the molecule's enhanced stability may affect the rate of deuterated metronidazole metabolism. similarly, when it comes to anti-tb activity, the deuterated compound, which has a mic of 1.6μg/ml, is superior to that of metronidazole, which has a mic of 3.12μg/ml. deuterated derivatives were also shown to have nearly equivalent activity to metronidazole against gram-positive and gram-negative aerobic bacterial strains, as well as fungus. according to the findings, deuterated metronidazole is a good starting point for rational antibacterial activity design. in addition, pharmacokinetic and pharmacodynamic s. no microorganism minimum inhibitory concentration (μg/ml) deuterated metronidazole metronidazole standard drug aerobic antibacterial activity ciprofloxacin 1 pseudomonas 50 25 <4 2 e. coli 50 50 2 3 e. faecalis 25 50 2 4 staph aureus 25 25 2 anaerobic antibacterial activity moxifloxacin 1 fusobacterium nucleatum 0.8 1.6 <0.125 2 porphyromonas gingivalis 1.6 3.12 <0.125 3 prevotella intermedia 3.12 6.25 <0.125 antifungal activity fluconazole 5 candida 25 25 16 6 a. niger 100 100 8 anti-tubercular activity isoniazid pyrazinamide 7 m. tuberculosis h37rv 1.6 3.12 1.6 3.125 iraqi j pharm sci, vol.31(2) 2022 evaluation of deuterated analogues of metronidazole 302 investigations must be conducted to demonstrate the deuterated compound's activity. despite the study and development of various deuterated medications, their efficacy, safety, and a complete understanding of the deuterated drugs' specific processes remain unsolved and difficult. 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2007. 39. lourenco mc, de souza mv, pinheiro ac, ferreira md, gonçalves rs, nogueira tc, peralta ma. evaluation of anti-tubercular activity of nicotinic and isoniazid analogues. arkivoc. 2007; 15:181-91. 40. munna s, basha sc, reddy pr, pramod n, kumar yp, basha gm. antitubercular activity of actiniopteris radiata linn. phytochemical analysis. 2014; 10:12. 41. franzblau sg, witzig rs, mclaughlin jc, torres p, madico g, hernandez a, degnan mt, cook mb, quenzer vk, ferguson rm, gilman rh. rapid, low-technology mic determination with clinical mycobacterium tuberculosis isolates by using the microplate alamar blue assay. journal of clinical microbiology. 1998;36(2):362-6. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ abstract iraqi j pharm sci, vol.19(2) 2010 tinidazole bioadhesive vaginal gels 64 in vitro evaluation of tinidazole bioadhesive vaginal gels zainab t.salih* , 1 *department of pharmaceutics, college of pharmacy, university of baghdad,baghdad, iraq. abstract many trials were made to prepare tinidazole 2% as bioadhesive vaginal gels using different gel bases including hydroxypropyl methyl cellulose (3 and 4% w/w), methylcellulose (3 and 4%w/w) and carboxymethylcellulose (2 and 3% w/w) .swelling index of the polymers,ph , viscosity , bioadhesive force , and in-vitro drug release to the simulating vaginal fluid (s.v.f.) were investigated for all the prepared bioadhesive gels . the mechanism of drug release from the gel bases was also investigated. the results revealed that c mc 3% gave the highest viscosity and bioadhesive strength with the lowest release rate while lowest viscosity and bioadhesive force was obtained with hpmc gel base with the highest release rate. the mechanism of drug release was affected by the type of gel base. fickian diffusion was obtained with all gel bases. key words: tinidazole, bioadhesive polymers, gels الخالصة بروبيم يثيم % حضر عهً شكم هالو يهبهي يخاطي االنتصاق باستخداو قىاعد هالييت يختهفت تشًم هيدروكسي2تيُدازول تى قياش االش انهيدروجيُي وانهسوجت وقىة ااالَتفاخ نها. كًسيهيهىز وانكاربىكسي يثيم سيهيهىزوانتي تى قياش َسبت ,انًثيمسيهيهىز .وقد اظهرث 4.2نهالييت انًحضرة في انىسظ انًشابه نهًحيظ انًهبهي باش هيدروجيُي تصاق وتحرر اندواء يٍ انتراكيب ااالن اعطً اعهً نسوجت واعهً انتصاق هيهىزكاربىكسي يثيم سي %3وأٌ ,انُتائج اٌ تحرر اندواء يتاثر بُىع وتركيس انبىنيًر انًستخدو واعهً َسبت اَتفاخ وابطا تحرر. ايا انهايدروكسي بروبيم يثيم سيهيهىز فقد اعطً اعهً تحرر نهدواء ونى يعطي اي َسبت اَتفاخ في انتركيس انًستخدو . introduction the vaginal route of administration has many advantages compared to other routes (1). the vaginal epithelium is permeable to a wide range of drugs like hormones, peptides, proteins and antimycotics agents that need to reside at the site of infection for a prolonged period to be more effective. the vagina provides a promising site of systemic drug delivery because of its large surface area and its rich blood supply. in addition , a prolonged contact of a delivery system with the vaginal mucosa may be achieved more easily than at other route of drug absorption site like rectum or intestinal mucosa (2). . tinidazole ( tnd ) is {1[ 2(ethylsulphonyl) ethyl ] – 2 – methyl – 5 nitroimidazole} has been proved to be very effective for the therapy of amoebiasis , trichomonas , lambliasis and anaerobic infections (3) .the drug is well tolerated and widely used in clinical practice in the form of i.v infusion , tablets and vaginal pessaries (4). the efficacy of tnd in the treatment of trichomonas vaginalis has been well documented (5) . tnd is similar to metronidazole intravaginal gel can be used to treat bacterial vaginosis (6,7), it is effective , safe , easy to apply and has fewer side effect compared to oral dosage forms of this medication (8,9) patients tolerate gels better than vaginal inserts or ointments. (10) this study was conducted to formulate tnd in a suitable mucoadhesive vaginal gel through studying different variables affecting the physicochemical properties and the in vitro release of the drug from these preparations. materials and methods materials tinidazole ( tnd ) was supplied by sigma chemical co., methylcellulose( mc) and carboxymethylcellulose(cmc)from(bdh limite ( hpmc) from ( shin-etsu chemical co.– germany) . all other materials used were of analytical grade. methods preparation of simulating vaginal fluid (s.v.f) simulating vaginal fluid (s.v.f ) was prepared by dissolving 3.51gm of sodium chloride ,1.4gm of potassium hydroxide ,0.22gm of calcium hydroxide , 0.018gm of bovine serum albumin, 2 gm of lactic acid , 1gm of acetic acid ,0.16gm of glycerol ,0.4gm of urea and 5gm of glucose in 1 liter distilled water then the ph was adjusted at 4.2 (11). 1corresponding author email : zainabthabit@yahoo.com received : 3/5/2010 accepted : 18/9/2010 iraqi j pharm sci, vol.19(2) 2010 tinidazole bioadhesive vaginal gels 65 preparation of tnd vaginal aqueous gel bases the tested aqueous gel bases were prepared by dispersing the polymers powder of 3 and 4%(w/w) mc ,2 and 3%(w/w) cmc and 3 and 4%(w/w) hpmc in cold freshly distilled water with the aid of a high speed stirrer , until a solutions were complete (12) .the products were refrigerated for 1hour before use to obtain a suitable consistency .tnd gels were prepared by dispersing 2% drug in different gel bases as shown in table(1). table 1:composition of tnd bioadhesive vaginal gel in vitro dissolution of tnd from gel bases the release study was carried out in a usp apparatus type і at 100 rpm ,a basket of 2.5 cm in diameter was enclosed with a multifold filter paper (dialysis cell) in order to be filled with 1gm of each base containing 2%w/w tnd. after connecting to a stirrer motor , the dialysis cell was immersed to about 1 cm of its surface in 500 ml of simulating vaginal fluid ph4.2 at 37 ◦ c for 5 hours .samples were withdrawn after 0.5,1,2,3,4 and 5 hours and replaced with an equal volume of the same fluid solution . the samples were analyzed for their drug content at its λmax 320nm (13, 14). . the effect of polymer type on the release of tnd from the gel bases the effect of mc, cmc and hpmc bases on the release of tnd from gel bases was studied. the effect of polymer concentration on the release of t nd from the gel bases the effect of 3 and 4% w/w mc , 2 and 3% w/w cmc and 3 and 4% w/w hpmc on the release of tnd from gel bases were studied. measurement of the swelling index of dry polymer in s.v.f the swelling index is the volume in ml occupied by 1 gm of polymer after it has swollen in aqueous liquid (s.v.f used in the study) for four hours. the process is carried out by placing 1gm of dry polymer in 25ml graduated cylinder , moisten it with 1ml (ethanol 96%) and with an addition of 25 ml s.v.f shaking vigorously every 10 minute for one hour ,then allowing to stand for three hours . after1.5houre from beginning the test any volume of liquid retained in the layer of the polymer and any particles of polymer floated at the surface of the liquid were released (15) .the swelling was measured according to this equation: (final volumeinitial volume) swelling = initial volume mechanism of tnd release from gel bases to understand the mechanism of the drug release , the release profiles from different gel bases, the % fraction drug released verses time profiles were plotted using linear fitted equations proposed by koresmeyer peppas ( 16 ) f = mt/mo = ktⁿ at time t (1) ln f = ln k + n ln t (2) the equations express the fraction (f) release of drug from gel where mt is the amount released at time t ,mo is the initial amount of drug ,k is the rate constant and n is the releasing exponent value indicative the mechanism of drug release( if n is 0.5 or less (fickian ) holds the release, while when n greater than 0.5 this indicate nonfickian (anomalous) releasing mechanism), n value could be result from the slope obtained by the drawing of ln. fraction released verses ln. time. viscosity measurement a brook field digital viscometer with a suitable sample adaptor was used to measure the viscosity in centipoises of the bioadhesive prepared gel. (17) determination of ph the ph of the bioadhesive gels were determined by digital ph meter .one gram of gel was dissolved in 25 ml of dislilled water and the electrode dipped into gel formulation for 30 minute..the measurements of ph of each system were replicated three times (18) iraqi j pharm sci, vol.19(2) 2010 tinidazole bioadhesive vaginal gels 66 determination of mucoadhesive force the mucoadhesive potential of each system was determined by measuring the force required to detach the gel from sheep vaginal mucosal tissue by using a modified chemical balance. asection of vaginal mucosa was cut from the sheeps vaginal cavity and instantly fixed with mucosal side out onto each glass vial using a rubber band. the vials with vaginal mucosa were stored at 37 ◦ c for 5 minutes .then next vial with a section of mucosa was connected to the balance in inverted position, while first vial was placed on a height adjustable pan. fixed amount of sample of each gel system were placed onto the vaginal mucosa of first vial. then the height of second vial was adjusted so that mucosal surfaces of both vials come in intimate contact .five minutes contact time was given to ensure intimate contact between tissues and the sample. then weight was kept rising in the pan until vials get detached. the bioadhesive forces as the detachment stress in dyne/cm 2 , was determined from the minimal weights that detached the tissues from the surface of each gel using the following equation (19): detachment stress(dyne/cm 2 )= m  g/a where, m= weight required for detachment of two vials in grams. g= acceleration due to gravity [980cm/s 2 ]. a=area of tissue exposed. the vaginal mucosa was changed for each measurement .measurements were repeated three times for each of the gel system. results and discussion in order to fortify the adhesion of administered drug onto the mucosal surface, tnd was formulated in a suitable mucoadhesive polymers , mc, cmc and hpmc bases to take a full advantageous of the contact time , the drug can be dispersed in the gel giving a concentration that is higher than corresponding to the solubility of the drug (20) ,as well as ,it can be considered as a way for sustaining the release of the drugs from gel dosage form . mc, cmc and hpmc were used as a gel base because of there safety use for vaginal application as well as there compatibility with tnd, with other vaginal secretion. the effect of polymer type and concentration on the releasing of tnd from the gel bases figures 1, 2 and 3 showed the amount of tnd released from gel bases in s.v.f medium .it was found that the amount of tnd released from gel bases were significantly influenced (p<0.05) by the type of the base and the concentration the polymer used. (21) in general it was found that highest amount of tnd was released from cmc gel base and decreased with an increasing in polymer concentration. (22) ,also the drug release rate was decreased with the increase in the polymer concentration. (23) estimation of swelling capacity the swelling index (capacity) for 1gm dry mc and cmc are 50% and 80% respectively. hpmc is soluble and nonswollen polymer at the concentration used . the high swelling % of cmc in s.v.f indicates that the swelling of this polymer takes place at a slower rate. the straight lines obtained from plotting of t/v ( t: time in hour , v: volume of aqueous s.v.f absorbed per volume of dry polymer) against time t in hour as shown in figures (4and 5) were utilized to calculate the kinetic of swelling including initial rate of swelling (rceiprocal of the intercept ) and swelling equilibrium( reciprocal of slope) (24) , as shown in table 2 table 2 : the initial rate of swelling (reciprocal of intercept) and swelling equilibrium (reciprocal of slope) correlation coefficient r initial rate of swelling (ml of fluid solution /hr.ml of dry polymer ) swelling to equilibrium size(ml of fluid/ml of dry polymer ) polymer type 0.99 74.626 1.797 mc 3% 0.9885 12.987 1.496 mc 4% 0.9944 11.454 1.951 cmc 2% 0.995 14.619 1.500 cmc 3% iraqi j pharm sci, vol.19(2) 2010 tinidazole bioadhesive vaginal gels 67 figure 1 : the effect of mc concentration on the release profile of tnd at ph 4.2 and 37  c figure 2: the effect of cmc on the release profile of tnd at ph 4.2 and 37  c figure 3 : the effect of hpmc on the release profile of tnd at ph 4.2 and 37  c figure 4 : the swelling mechanism of 3 & 4% w/w mc in simulating vaginal fluid ph 4.2 and 37  c figure 5 : the swelling mechanism of 2 & 3% w/w c mc in simulating vaginal fluid ph 4.2 and 37  c releasing mechanism of tnd from gel bases it was found that formulation with higher swelling index retard the release of drugs more than those with lower swelling indices , this swelling also depends on the ph of the medium and the presence of electrolytes , where the s.v.f ph 4.2 has a lot of electrolytes content such as : sodium chloride , potassium hydroxide , calcium hydroxide (25). this swelling is important since its significantly decreases the releasing rate of the drug and increases in the amount of the drug release after five hours ,as shown in table-3.the reason behind this increasing in the amount released due to erosion of the gel as a result of severe hydration or relaxation of the polymer , so interchain intermolecular force will no longer be able to resist any external forces ,once gel erodes ,it breaks up into smaller and smaller particles ,more surfaces will be exposed to the fresh swelling medium figure-3the amount released of tnd to s.v.f 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 100 200 300 400 time(minute) f f r a c ti o n r e le a s e d 2% w/w cmc 3% w/w cmc figure-4-the amount released of tnd to s.v.f 0 0.05 0.1 0.15 0.2 0.25 0.3 0 100 200 300 400 time(minute) f f r a c ti o n r e le a s e d 3% w/w hpmc 4% w/w hpmc figure-2the effect of mc cocentration on the release profile of tnd atph 4.2 and 37 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0 100 200 300 400 time(minute) f f r a c ti o n r e le a s e d 3% w/w mc 4% w/w mc 0 0.5 1 1.5 2 2.5 3 3.5 1 2 3 4 5 time(hr) t /v ( h r /m l f lu id s o lu t io n / m l c m c ) 3% cmc 2% cmc iraqi j pharm sci, vol.19(2) 2010 tinidazole bioadhesive vaginal gels 68 and hence more drug will be released leads to an increasing in the amount of the drug released from cmc gel base more than other swollen mc gel base after five hours (18) .as a result , cmc gel bases hold a fikian release mechanism. table 3 :the release rate, amount released and the releasing mechanism of the tnd from gel bases viscosity measurment viscosity is an important parameter for characterizing the gel as it affects the spreadibility ,extrudability and release of drug (26) .cmc gel base show the highest viscosity, and as the polymer concentration increase ,the viscosity will be increased as shown in table 4. on the other hand , minimum level of viscosity was seen with f5 and f6 explain the rapid release of drug from this gel system and this due to non swelling property of hpmc polymer at these concentration . table 4:the viscosity and ph of the prepared tnd bioadhesive vaginal gel determination of ph the ph of gels were found to be within the range of 5-6.2 which is within the limit of semisolid specification and at this ph the gel will be non irritant to vagina .(12). the physical appearance of the gel was evaluated with naked eye . the gel was smooth without any lumps and of uniform color ,no color change /liquification /separation were observed . determination of mucoadhesive force scheme 1 indicates the vaginal bioadhesive properties of the prepared gels in sheep vagina,and the resultes showed that all vaginal bioadhesive strengths were found in the following order f2,f4>f3>f1>f6>f5 indicating that (f2)and (f4) showed the highest bioadhesive properties. scheme 1: bioadhesive strength of tnd bioadhesive vaginal gels conclusion results of this study confirm that, the physical properties of the prepared gel were affected by the type and concentration of the polymer used in the preparation. the more sustained preparation with highest bioadhesive force and highest viscosity was obtained by the use of 3% cmc as a gel base.in addition to that the mechanism of drug release from tinidazole 3%cmcgel base was diffusion mixed with erosion. references 1. richardson ji illum l the vaginal route of peptide & protein drug delivery .adv. drug delivery rev 1992 ; 8: 341-366 . 2. claudia, kv, constantiae, irene h. , andreas, sb . development and in vitro evaluation of a mucoadhesive vaginal delivery system for progesterone. j. controlled release 2001; 77: 323-332. mechanism of tnd release amount release (mg/5hr) (mean±sd,n=3) k % minⁿ releasing rate n exponent release value polymer type and concentration fickian 7.05±0.14 5.3974 0.41 mc 3% fickian 4.466±0.13 3.5744 0.49 mc 4% fickian ±0.15 7 4.1594 0.509 cmc 2% fickian 4.35±0.12 3.582 0.52 cmc 3% fickian 5.2±0.03 6.1472 0.29 hpmc 3% fickian 4.58±0.11 3.1864 0.5 hpmc 4% formula no. viscosity (cps) ph f1 1450 6.02 f2 3240 5.2 f3 1700 5.03 f4 6800 5.17 f5 14.5 5.02 f6 21.6 5.2 iraqi j pharm sci, vol.19(2) 2010 tinidazole bioadhesive vaginal gels 69 3. trac jw. webster lt. drugs used in the chemotherapy of protozoal infection .in:hardman, j.,g., limbird,l. ,e.,the pharmacological basis of therapeutics 9 th ed. mc graw –hill,new york 1996 ; 995-998 4. martindale. the complete drug reference, 32 ed., pharmaceutical press 1999; 594: 1099, 1541. 5. bnf, british national formulary, british medical association and royal pharmaceutical society of great britain 2004; 47 ed.: 287, u.k. 6. milani,m., barcellona,e., agnello,a., efficacy of the combination of 2g oral tinidazole and acidic buffering vaginal gel in comparison with vaginal clindamycin alone in bacterial vaginosis :a randomized ,investigator -blinded, controlled trial .euro .j. of obst. and gyne 2003 ;109: 67-71. 7. livengood, . g. h. mc gregor , ., j.,a., soper, d., e, newton, e., thomason, j.,l., 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κ-carrageenan matrices. aaps pharmscitech. 2004; 5(2): article 25. 25. omidian, h, park, k., swelling agents and devices in oral drug delivery ,j.drug .del.sci.tech. 2008; 18(2): 83-93. 26. sanap, g.s., smita, t., tarannum, s. and sangita, g. formulation and evaluation of mucoadhesive beads of glipizide by 2 3 factorial design .jou. of phar. res. 2009; 2(5): 934938. iraqi j pharm sci, vol.28(2) 2019 5 fluorouracil conjugate with pyrrolidine dithiocarbamate 23 -https://doi.org/10.31351/vol28iss2pp17 doi: 17 synthesis, characterization and preliminary cytotoxic activity study of new 5 fluorouracil conjugate with pyrrolidine dithiocarbamate as a mutual anticancer prodrug ikram k. shihab*,1 and mohammed h. mohammed** * ministry of health and environment directorate-rasafa, imam ali hospital, baghdad-iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq abstract 5-fluorouracil (5-fu)s one of the commonly used chemotherapy drugs in anticancer therapy; unfortunately treatment with 5-fu solely has many drawbacks low lipophilicity, low permeability, low molecular weight, and its relatively poor plasma protein binding; also a brief half-life, therefore frequent administration is required to maintain the optimal therapeutic plasma level which in addition to its poor selectivity, drug resistance and limited penetration to cancer cells; leads to increased incidence of side-effects to healthy cells/tissues and low response rates. in order to minimize these drawbacks; 5-fu was chemically conjugated with pyrrolidine dithiocarbamate (pdtc) in a mutual prodrug moiety (s-(9h-purin-6-yl) 3-((pyrrolidine-1carbonothioyl)thio)propanethioate) "compound [iv]" with (chloroacetic acid) and (chloroethanol) being the linkers ;synthesized prodrug and intermediates were characterized and identified using ftir ,1h nmr and all the results shown good agreements with the proposed chemical structures of the synthesized compounds. ; in-vitro preliminary cytotoxicity study was conducted for compound [iv] and 5-fu on cal 51 and b16v cell lines ,results showed enhanced cytotoxic effects for [iv] over 5-fu. keywords: 5-fluoruracil, prodrug, pdtc, cytotoxicity. مع فرورويوراسيل -5لــل مستحدث لمقترن السرطان ضد للفعالية اولي وتقييم وتشخيص تحضير السرطان ضد مزدوجة فعالية ذو دوائي كمقدم ثايوكارباميت ثنائي بايروليدين **و محمد حسن محمد 1،*اكرام خليل شهاب . العراق ، بغداد ،علي االمام مستشفى ،الرصافة بغداد صحة دائرة،والبيئة الصحة وزارة * .العراق ، بغداد ،الصيدالنية/ الكيمياء فرع ،بغداد جامعة ، الصيدلة كلية** الخالصة -5 استعمال ان المؤسف من , السرطان مرض ضد المستعملة العالجات في االستعمال الشائعة االدوية من فلورويوراسيل-5 االرتباط على يفةالضع قابليته و الصغير الجزيئي ه,وزن المنخفضة ,نفوذيته للدهون الفته انخفاض تشمل: عديده مساوء فيه منفردا فلورويوراسيل السمية نتقائيتها سوء الى باالضافة الذي االمر فعال, بالزمي تركيز الدامة جرعاته تكرار يتطلب مما القصير النصف عمر وكذلك البالزما ببروتينات عيةالطبي واالنسجة الخاليا عند الجانبية االعراض حدوث نسب زيادة الى ,يؤدي السرطانية الخاليا الى المحدودة ونفاذيته له الدوائية والمقاومة ةفعالي ذو دوائي كمقدم ثايوكارباميت( ثنائي )بايرولدين مع فلورويوراسيل-5 اقران تم المساوئ هذه تحجيم الجل له؛ االستجابة معدالت وانخفاض -1) واستعمل 4 رقم مركب لمسمىا thio)propanethioatecarbonothioyl 1 yl)3((pyrrolidine 6 purin (s(9h( ) يدعى مزدوجة لحمراءا تحت االشعة طيف بواسطة الوسطية والمركبات النهائي المركب تشخيص ؛تم بينهما روابط ك ايثانول(-كلورو-2و) الخليك( حامض-كلورو و المصنع لعقارل الخلوية للسمية اولية مختبرية دراسة اجريت ؛ المقترحة الكيميائية للهيكلية مطابقة النتائج واظهرت المغناطيسي النووي الرنين و لخلويةا السمية تحسن اظهرت ,الدراسة للفأر( الميالنومي الجلد )سرطان و البشري( الثدي )سرطان : السرطانية الخطوط على فلورويوراسيل-5 فلورويوراسيل.-5 بال مقارنة 4 للعقار خلوية. سميّة و ثايوكارباميت ثنائي بايروليدين دوائي, مقدم فلورويوراسيل,-5المفتاحية: الكلمات introduction most anticancer chemotherapies activate nuclearfactor kappa b( nf-κb), so does gamma irradiation, activation of nf-κb leads to resistance to apoptosis induced by chemotherapy or irradiation, a state of antiapoptosis take place which eventually leads to resistance to anticancer treatment, here comes the role of nf-κb blockers as chemopreventive agents which if administered in combination with chemotherapeutic agents or gamma irradiation give synergism(1). pdtc a novel stable dithiocarbamate with antioxidant (2,3) and potent nf-κb inhibitory effect . (4–6) where nf-κb inhibition is independent of its antioxidant property (7). 5-fluorouracil (5-fu) the fluorinated analogue of uracil, one of the commonly used chemotherapy drugs in anticancer therapy, for a variety of human malignancies breast cancer, head, and neck cancer (8), skin cancer,(9) colorectal and other gastrointestinal cancers (10) . also ovarian and liver cancers (11) . treatment with 5-fu by itself has many 1corresponding author e-mail: ik20shihab@gmail.com received: 3 /1/2019 accepted: 20 / 3/ 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp17-23 iraqi j pharm sci, vol.28(2) 2019 5 fluorouracil conjugate with pyrrolidine dithiocarbamate 18 drawbacks; high water solubility, low permeability, low molecular weight, and its relatively poor plasma protein binding; also a brief half-life of about 10– 20 min requires frequent administration to maintain the optimal therapeutic plasma level (12–14) .properties like poor selectivity, drug resistance and limited penetration to cancer cells; not only lead to low response rates but also have potentially serious side-effects to healthy cells/tissues. among 5-fu's known side effects are myelosuppression, mucositis, nausea, emesis hand-foot-syndrome,(15,16) neurotoxic effects (cerebellar syndrome and encephalopathy) (17). prodrug strategies may be the most advanced research area that enables the safe delivery of cytotoxic drugs to neoplasm tissues and activate it specifically by over-expressed enzymes at the cancer microenvironment leading to the controllable drug release in target sites, researches also showed that there are higher concentrations of esterase in cancerous cells than in normal ones and rational design of ester prodrugs is the increased bioavailability of drugs with high polarity making ester drugs good targets for prodrug research (18,19) . when used in combination, pdtc act as an enhancer of 5-fluorouracil-induced growth inhibition of colon carcinoma cells (19). this work aims to synthesize a novel mutual prodrug of pdtc and 5-fu linked by an ester linkage and evaluates the synthesized compound for in vitro cytotoxic activity. materials and methods materials 5-fluorouracil (5-fu) was purchased from baoji guokang biotechnology/china, all the chemicals and solvents used in synthesis were of analytical grade and used without further purification. reactions' progress and the purity of compounds were monitored and confirmed by thinlayer chromatography (tlc), using silica gel gf254 (type 60) pre-coated aluminium sheets, merck (germany) exposed to uv-254nm light, the chromatograms were eluted with ethyl acetate/methanol (1:1) for [i] and [iii], ethyl acetate/n-hexane (4:1)for [iv]; melting points were determined using stuart smp30 melting point apparatus with one-end sealed capillary method, and are uncorrected. fourier-transform infrared spectroscopy (ftir), were recorded using specac quest atr (diamond)-uk on (iraffinity-1) spectrophotometer, shimadzu, japan at baghdad university/college of pharmacy. 1h nmr was recorded on (nmready-60 spectrometer by nanalysis corp. 60 mhz, canada) at central laboratory service /college of education for pure sciences / ibn al-haithem /university of baghdad. the chemical shift was expressed as (δ=ppm) and coupling constants in hz, methanol-d6 or dmso-d6 were used as solvents. methods of chemical synthesis synthesis of (pyrrolidine-1-carbodithioate triethylammonium) [i](20) in a 250 ml beaker a stirred solution of pyrrolidine (pyr) (5 g, 0.07 mole) and triethylamine (tea)(9.7 ml, 0.07 mole)in 70 ml dry ethanol was cooled on an ice bath to -5° c, carbon disulfide (cs2) (5 ml,0.084mole) was added from a dropping funnel slowly (over a period of 30 mins) stirring was maintained for additional 1h at room temperature after cs2 dropping has stopped then the precipitate filtered and recrystallized from ethanol/ether (1:2) to give compound i as light beige powder the. the % yield: 87% m.p.=130 °c (d) , rf= 0.8. ir( υ= cm 1 ):2300-2966 broad n-h of tert. amine, , 2966 c-h str. of ch3 2858,2704 c-h str. of ch2,1454 ch2 scissor vibration,1373,1323 c=s str. of dithiocarbamate;999 and 937 c-s str. 1hnmr(60 mhz, methanol-d6, δ= ppm):0.91.35(9h,m,ch3(tea's)); 1.5-2 (6h,m,ch2( pyr 's)); 2.8-3.24(4h,m,ch2(pyr's)); 3.43-4.15 (4h,m,ch2(tea's)). synthesis of 2-hydroxyethyl pyrrolidine-1carbodithioat e[ii] (21) to a stirred solution of compound [i] (2g,0.008mole) dissolved in 41 ml acetone, chloroethanol (0.504ml,0.008 mole) was added, and the reaction mixture was stirred for 18 hrs. at ambient temperature (30°c) after which time filtered in the freezer and the precipitate was washed with cold acetone dropped on the filter paper and the filtrate was devoted from its solvent and the resulting oil re-dissolved in chloroform, extracted with brine sol. and the organic layer was dried with mgso4 and used directly in the next reaction as the product was a volatile oil . physical properties: oil coffee brown in color with greenish hue ir (υ= cm1):3600-3200 broad (o-h) str.,2970,2870 c-h str. of ch2; 1435,1222,1161 c=s str. of dithiocarbamate; 999 c-s str. synthesis of 2-(5-fluoro-2,4-dioxo-3,4dihydropyrimidin-1(2h)-yl)acetic acid( 5-fu acetic acid)compound [iii] (22) in a 100 ml flat bottom quick-fit boiling flask mixed 5-fu (2g , 0.0153 mole) with 15ml freshly prepared 10% koh solution mixing continued at room temperature until 5-fu completely dissolved where chloroacetic acid (2.18g , 0.0153mole) dissolved in 5 ml d.w was added and stirring continued for 30 mins (at room temperature) thereafter the ph of the mixture was adjusted to ph10 by the addition of 10% koh and the reaction was put under reflux for 2 hr. then cooled to room temperature, acidified with concentrated (35-38%) hcl to ph=5 and left to precipitate at 4°c (for 2hr) slowly, the precipitate got filtered away and the filtrate was acidified once again with concentrated hcl to ph = 2.2 ,cooled at 4°c to slowly give the iraqi j pharm sci, vol.28(2) 2019 5 fluorouracil conjugate with pyrrolidine dithiocarbamate 19 product, recrystallized by dissolving in (5%) nahco3 and precipitation by concentrated hcl addition to give compound iv as white transparent short needle-like crystals. percent % yield= 62%, m.p. =275-276°c (ref=276-277°c). (23);rf=0,7. ir( υ= cm-1 ): 3200-2800 (broad) coo−h stretching (h bonded) ; 3186 (n-h)str.of imide; 3055c (c-h)str ; 2970,2846 asym. and sym. c−h stretching of (ch2);1735,1685,1662 are (c=o)of cooh overlapped with (c=o) str in pyrimidine ring, 1141 (c-f). 1hnmr (60 mhz,dmso-d6,δ= ppm): 4.31( 1h,s,ch2) ; 7.99(1h,d,ch); (11.79,s,n-h);11.87 (1h,s,c=ooh). synthesis of ((pyrrolidine-1carbonothioyl)thio)methyl 2-(5-fluoro-2,4-dioxo3,4-dihydropyrimidin-1(2h)-yl)acetate compound[iv](22) to a stirred solution of compound [iii] (1g, 0.0053 mole) in 30 ml dry acetone cooled on an ice bath, dicyclohexyl carbodiimide (dcc) (1.23g, 0.006 mole) was added and stirring continued for 30 min. then reaction cooled and compound [ii] (0.5g, 0.0026 mole )was added gradually, the reaction mixture was left to stir in cold condition under temp= -10 to -20°c (average of -15°c) for three consecutive days and on the fourth day stopped, filtered ,dried completely from solvent then redissolved in ethyl acetate and cooled for 2hrs then filtered again and washed successively with: 0.1n hcl (50ml x3), dw(50ml x3), nahco3 5% (50 ml x3) and finally brine sol. (50ml x2), the organic layer was dried with anhydrous mgso4 and concentrated under reduced pressure to give a reddish-dark brown oil that was triturated with petroleum ether and recrystallized with ethyl acetate/n-hexane (1:3) to give compound iv as light brown powder .percent yield yield=52% , m.p.=128 oc (d) ,rf=0.75. ir( υ= cm -1 ):3263(n-h) str. of imide;2931,2850asym. & sym (c-h) str of ch2 ; 1701 (c=o) of ester;1728,1658 (c=o)str. of uracil; 1338,1157 (c=s) of thiocarbamate.1hnmr (60 mhz, dmso-d6,δ= ppm): 1.5-2.15 (4h,m,ch2 (pyr)); 3.4-4(2h,m,ch2); 4-4.6 (4h,m,ch2) two signals combined ; 8.06 (1h,m,c-h); 11.8 (1h,s,nh). cytotoxicity study a preliminary in vitro cytotoxicity assay (cell viability assay on cancer cell line) for the compounds ([iv], and 5-fu(as a standard) has been carried out at the iraqi centre for cancer and medical genetic research (iccmgr). both cal51 (human breast cancer) and b16v (mouse melanoma) cell lines were maintained using rosswell park memorial institute (rpmi)-1640 media supplemented with 20% fetal calf serum and seeded on micro-titration (96well plates at a concentration of 1×104 cells/well) and incubated at 37 oc until the cells became confluent monolayer and various concentrations of tested compounds ([iv] and 5-fu) were added in (12.5,25,50, and100 mcg/ml) prepared by serial two-fold dilutions using maintenance media from stock solution of test sample in triplicate form of each concentration. the negative control wells contained only the cells with culture media, then the 96-well cell culture plate incubated at 37ᵒ c in an incubator supplemented with 5% co2 gas for 72 hrs. (24)the cytotoxic activity of compounds was evaluated by crystal violet assay (25), the optical density of each well was measured by using elisa (enzyme linked immuno sorbent assay) reader at a transmitting wavelength on 492 nm. the inhibition rate of cell growth (the inhibition rate %(ir%)) was calculated as (a˗b)/a×100, where a is the optical density of untreated wells (control conc.=zero), and b is the optical density of treated wells (26,27) .data of in vitro cytotoxicity were subjected to unpaired t-test to compare the ir% mean values and standard error of the mean (sem) were calculated , using microsoft excel; also a non-linear regression analysis to obtain dose-response curves and ic50values (using aat bioquest quest graph). results and discussion chemistry compound [i] was prepared by reacting carbon disulfide with pyrrolidine in the presence of (tea) as a base, the reaction is exothermic so was carried out over an ice bath .(28) then the reaction of comp. [i] with 2-chloroethanol using acetone as a solvent took overnight to give comp [ii] that was characterized by the appearance of broad o-h stretching vib. between 3600-3200 and disappearance of broad nh str vib.of tea at 23002966 ,as shown in scheme 1 shows path of synthesis of [i] &[ii] scheme 1.synthesis of compound [i] and [ii] to synthesize compound [iii] , n1 of 5-fu was subjected to alkylation with chloro acetic acid using koh as a base to capture the chloride (scheme 2),[iii] structure was confirmed by appearance of 1735 cm-1 which is (c=o) stretching of carboxyl in ftir along with o-h proton signal at δ=11.87 in 1hnmr. (scheme 2) coupling of compounds [ii] and [iii] was through the catalysis of dcc(29), it took 3 days and continuous cooling to give the final product [iv] ftr showed disappearance of the broad o – h str. of compound [iii] and the appearance of c= s str. of thiocarbamate at 1338 and 1157 cm-1 .alao o – h signal in proton nmr was not observed with appearance of new proton signals for the pdtc indicating ester formation (scheme 2). iraqi j pharm sci, vol.28(2) 2019 5 fluorouracil conjugate with pyrrolidine dithiocarbamate 20 scheme 2 .synthesis of compounds [iii] and [iv] cytotoxicity study results are represented in tables 1 and 2, the inhibition rate percentage (ir %), and in figures 1 and 2 as histograms of ir% versus concentration; the estimated ic50 values for 5-fu and [iv] are illustrated in table 3. figures 3 and 4 exhibit visual comparison between cancer cells before and after treatment with compound [iv]. table 1. cytotoxic effect (cell viability assay) on cal-51 cell line of 5-fu & compound [iv] by crystal violet assay method (25 ). cell line concentration cal51 12.5 mcg/ml 25 mcg/ml 50 mcg/ml 100 mcg/ml 5-fu i.r.% 35.9 34.56 36.35 37.4 pvalue (compared to control) 05-8.474e s 05-1.229e s 11-6.605e s 09-1.40022e s [iv] i.r.% 16.1357 4.68567 19.4429 60.139 pvalue (compared to control) 0.0647192 ns 0.61836 ns 0.03287 s 05-3.4937e s p-value (compared to 5-fu) 0.03375 s 0.01857 s 0.05167 s 0.003274 s (p<0.05) considered significant, s=statistically significant, ns= non significant. table 2. cytotoxic effect of 5-fu and compound [iv] on b16v cell line by crystal violet assay method(25) cell line concentration b16v 12.5mcg/m l 25mcg/ml 50mcg/ml 100 mcg/ml 5-fu i.r.% 14.999 23.45011.32270.630 pvalue (compared to control) 0.001549 s 0.0399 s 0.4786 ns 0.00311 s cpd [iv] i.r.% 7.771956 50.60804 27.25844 40.91145 pvalue (compared to control) 0.75201 ns 0.005268 s 0.060048 ns 0.011975 s fu)-5 to (compared value-p 0.76965 ns 0.000355 s 0.0666 s 05-7.85e s (p<0.05) considered significant, s=statistically significant, ns= non significant, (negative values indicate increased proliferation) iraqi j pharm sci, vol.28(2) 2019 5 fluorouracil conjugate with pyrrolidine dithiocarbamate 21 figure 1. inhibition rate %(%ir) versus different concentrations of compound [iv] and 5fu, on cal-51 cell lines at 72 hrs. figure 2. inhibition rate% (%ir) versus different concentrations of compound [iv] and 5fu, on b16v cell lines at 72 hrs. (negative values indicate increased proliferation). table 3. ic50 values in mcg/ml for 5-fu and comp. [iv]. cal51 b16v 5-fu 50.914 13.503 [iv] 56.108 10.502 as illustrated in table 1 and figure 1 on cal-51, the percent inhibition rate of 5-fu showed moderate but consistent cytotoxicity whereas compound iv gave significantly higher cytotoxicity (60%) at conc. =100 mcg/ml. b16v cells resisted 5-fu as the i.r% flipped to negative most of 5-fu's concentrations, the opposite case is seen for compound iv that showed moderate but superior activity when compared to 5-fu's on the same cell line. compound iv gave significant ir % at conc.= 25and 100mcg/ml see table 2 and figure 2 . compound iv has superiority over 5-fu considering the ic-50 (table 3). since most of the prodrug's weight is not 5fu (100mcg of prodrug contains 36 mcg 5-fu) even though the comparisons are not entirely fair for compound iv regarding the 5-fu quantity provided, the drug made a good impression of enhanced cytotoxic effect ,this enhancement could be explained by the increased lipophilicity and eventually improved cellular penetration , also pdtc's nf-κb inhibiting properties may aid in decreasing esistance to 5-fu by stimulating apoptosis (1) . added cytotoxic effects are probably due to the direct anticancer action of pyrrolidine dithiocarbamate , through its antioxidant, antiproliferative and nf-κb inhibiting nature ; (4– 6)this dual effect of the prodrug is mostly pronounced when it was found killing b16v cells that were resisted by 5-fu alone, figure 2 . figure 3. cells of cal-51; a: effect of control. b: effect of 100mcg/ml of compound [iv] at 72 hrs. figure 4. cells of b16v ; a: effect of control. b: effect of 100mcg/ml of compound [iv] at 72 hrs. conclusion the procedure for the synthesis of the target compound was achieved successfully; characterization of the structures was done by ftir spectroscopy and 1h nmr for the final and intermediate compounds, purity confirmed by rf values and melting points. preliminary anticancer activity of the final product [iv] shows that it gave considerable cytotoxic activity against two types of cancer cells, and an improvement in cytotoxicity compared to the parent drug 5-fu. acknowledgments we are grateful for the facilities provided by college of pharmacy –dept. of pharmaceutical chemistry – university of baghdad, and the experimental therapy dept., iraqi center for cancer and medical genetic research, mustansiriyah university. iraqi j pharm sci, vol.28(2) 2019 5 fluorouracil conjugate with pyrrolidine dithiocarbamate 22 references 1. bharti ac, aggarwal bb. nuclear factor-kappa b and cancer: its role in prevention and therapy. biochem pharmacol 2002;64(5–6):883–8. 2. zhu b-z, carr ac, frei b. pyrrolidine dithiocarbamate is a potent antioxidant against hypochlorous acid-induced protein damage. febs lett [internet]. 2002; 4;532(1–2):80–4. 3. ivan alm, campanini mz, martinez rm, ferreira vs, steffen vs, vicentini ftmc, et al. pyrrolidine dithiocarbamate inhibits uvbinduced skin inflammation and oxidative stress in hairless mice and exhibits antioxidant activity in vitro. j photochem photobiol b biol. 2014; 138:124–33. 4. parodi fe, mao d, ennis tl, bartoli ma, thompson rw. suppression of experimental abdominal aortic aneurysms in mice by treatment with pyrrolidine dithiocarbamate, an antioxidant inhibitor of nuclear factor-κb. j vasc surg. 2005;41(3):479–89. 5. schreck r, meier b, männel dn, dröge w, baeuerle pa. dithiocarbamates as potent inhibitors of nuclear factor kappa b activation in intact cells. j exp med . 1992;175(5):1181– 94. 6. cuzzocrea s, chatterjee pk, mazzon e, dugo l, serraino i, britti d, et al. pyrrolidine dithiocarbamate attenuates the development of acute and chronic inflammation. br j pharmacol. 2002;135(2):496–510. 7. woods js, ellis me, dieguez-acuña fj, corral j. activation of nf-kappab in normal rat kidney epithelial (nrk52e) cells is mediated via a redox-insensitive, calcium-dependent pathway. toxicol appl pharmacol. 1999;154(3):219–27. 8. naghizadeh s, hassanzadeh nemati n, hassani najafabadi a, niknejad h, khani mm. controlled release of fluorouracil (5-fu) from chitosan-co-poly(ethylene glycol)/ poly(glycerol sebacate)-co-poly(ethylene glycol)-coated iron oxide. int j polym mater polym biomater. 2017;4037:1–9. 9. petaccia m, condello m, giansanti l, la bella a, leonelli f, meschini s, et al. erratum: inclusion of new 5-fluorouracil amphiphilic derivatives in liposome formulation for cancer treatment (med. chem. commun. 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coupling reagents. chem soc rev. 2009;38(2):606–31. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(1) 2012 anti-inflammatory effect of ammi majus alcoholic extract 82 dose dependent anti-inflammatory effect of ammi majus alcoholic extract in rat: chronic study shihab h. mutlag *,1 * department of pharmacology and toxicology,college of pharmacy,university of baghdad,baghdad,iraq. abstract during treatment of inflammatory diseases, many conventional therapies (non-steroidal antiinflammatory drugs) used to relief pain and inflammation. chronic use of the intended drugs is frequently associated with serious side effect, which may lead to discontinuation of treatment . the efficacy and doseresponse effect of ammi majus extraxt (2 , 4, 8 , 16, and 32 mg/rat) were assessed using formalin to induce paw edema in rats as a model of chronic inflammation respectively. in this study, 42 rats were used and allocated into 7 groups each containing 6 rats, representing control (distilled water) , standard (piroxicam ) and test extract ( 2 , 4 , 8 , 16 and 32 mg/rat of ammi majus alcoholic extract ). the test extract and control were given orally before induction of inflammation paw edema was measured by using vernier caliper after 7 days for chronic inflammation. the result indicated that ammi majus alcoholic extract significantly lower paw edema (p<0.05) compared to standard and control, while the dose 16mg/rat also lower the paw edema compared with other test groups but less compared with the dose 32mg/rat. in conclusion, ammi majus alcoholic extract possess anti-inflammatory activity in animals model of chronic inflammation and the effect increased with increasing the dose. key words: ammi majus alcoholic extract, chronic inflammation , dose-dependent . العالقة بيه الجرعة والتأثير للفعالية المضادة لاللتهاب للمستخلص الكحىلي للخلة الشيطاوية في الجرذان : دراسة مسمىة شهاب حطاب مطلك* ،1 ح تغذاد، تغذاد ، انعشاق.ظايع ،كهٛح انصٛذنح فشع األدٔٚح ٔانسًٕو ،* الخالصة انًسرًش ذمهم االنرٓاب نكٍ االسرعًال أٔ األنىانرٙ ذسكٍ انًسرعًهحُْانك انكصٛش يٍ االدّٔٚ االنرٓاتٛح األيشاضفٙ عالض ادٖ انٗ انثحس لطع انعالض انسثة انز٘ إنٗظاَثّٛ لذ ذؤد٘ تآشاسٚكٌٕ يصحٕتا انًضيُحاالدّٔٚ خصٕصا عُذ يشضٗ االنرٓاتاخ ِنٓز سخص شًُٓا . إنٗ إضافحانرٙ نٓا يفعٕل انعالظاخ انًصُعح ٔأشاس ظاَثّٛ الم انُثاذٛح انطثٛحعٍ عالظاخ تذٚهّ يرًصهّ تانًسرخهصاخ نك يعشفح انٓذف يٍ ْزِ انذساسح ْٕ ذمٛٛى فعانّٛ انخهّ انشٛطاَّٛ فٙ انًُارض انرعشٚثّٛ نالنرٓاتاخ انًضيُح فٙ انحٕٛاَاخ انًخرثشّٚ ٔكز ظشر يخرثش٘ ٔلسًد انٗ سثعح يعايٛع فٙ كم 24 انذساسحال.اسرعًم فٙ ْزِ أٔ انعشعحفًٛا ارا كاَد فعانٛح انعالض ذضداد تضٚادج يعًٕعح سد ظشراٌ نرمٛٛى ذؤشٛش انخهّ انشٛطاَّٛ فٙ كم ًَٕر ض يٍ انًُارض حٛس ذًد دساسح انرؤشٛش انًضاد نالنرٓاب نعشع يخرهفّ ٔكزنك ذى اسرخذاو يادج انثاٚشٔكسٛكاو ٔانًاء انًمطش عٍ طشٚك انفى نهعشر يهغى24يهغى 61ٔيهغى ،8يهغى ،2يهغى ،4نهًسرخهص % فٕسيانٍٛ ذحد ظهذ كم ظشر عهٗ 4. ذى اسرحذاز االنرٓاب فٙ ًَارض االنرٓاب انًضيُح تحمٍ كًُارض لٛاط ٔسٛطشِ اشُاء انرعشتّ لٛاط انسًك.فٙ ْزِ أداجاٚاو يٍ اسرحذاز االنرٓاب ٔذى لٛاط َسثح ذؤشٛش انعالض تطشٚمح 7يُح تعذ انرٕانٙ . ذى ذمٛٛى االنرٓاب انًض يهغى نهخهّ انشٛطاَّٛ فعانّٛ راخ فشق يعُٕ٘ فٙ ذمهٛم انٕريّ انُاذعّ يٍ حمٍ انفٕسيانٍٛ فٙ ًَٕرض 24انعشعّ أظٓشخ انذساسح يٕاص٘ نرؤشٛش االدٔاخ انمٛاسّٛ انًسرعًهح ٔانًرًصهّ تًضاداخ االنرٓاتاخ غٛش االنرٓاتاخ انًضيُّ عهٗ طٕل فرشج انرمٛٛى ٔترؤشٛش 24يهغى فٙ ذصثٛظ االنرٓاتاخ انًضيُّ ترؤشٛش الم يماسَح تانعشعح 61انسرشٔٚذّٚ )تاٚشٔكسٛكاو( ٔاضٓشخ انُرائط اٚضا ذؤشٛش نهعشعّ ائط انذساسّ تؤٌ ذؤشٛش انخهّ انشٛطاَّٛ اضٓش َرائط ظٛذِ فٙ ًَارض ًٔٚكٍ االسرُراض يٍ َر يهغى حٛس اصداد انرؤشٛش تضٚادج انعشعح. فعٕل انعالض ٚضداد تضٚادج انعشعح.ٔاٌ ييهغى 24االنرٓاب انًضيُّ ٔخصٕصا فٙ انعشعّ .الكلمات المفتاحية : المستخلص الكحىلي للخلة الشيطاوية ، االلتهاب المسمه ، تأثير زيادة الجرعة introduction inflammation is an important physiological reaction which occurs in response to a wide variety of injurious (bacterial infection or physical trauma) ultimately aiming to perform the dual function of limiting damage and promoting tissue repair (1) . it requires the participation of various cell types expressing and reacting to diverse mediator along a very precise sequence (2) . the inflammatory response is often initiated by the activation of resident macrophage through pattern-recognition receptors: this triggers the sequential release of pro-inflammatory mediators such as eicosanoids, cytokines, chemokines and protease which derive leukocyte recruitment and activation (3) . 1 corresponding author email : shihab_hattab@yahoo.com received : 22/10/2011 accepted : 1/4/2012 mailto:shihab_hattab@yahoo.com iraqi j pharm sci, vol.21(1) 2012 anti-inflammatory effect of ammi majus alcoholic extract 83 chronic inflammation is a process of prolonged duration (weeks or months to years) in which active inflammation, tissue injury and healing proceed simultaneously (4) . it is characterized by ainfiltration with mononuclear cells including macrophage, lymphocytes and plasma cells b-tissue destruction, largely induced by the product of inflammatory cells crepair, involving new vessel proliferation (angiogenesis) and fibrosis (5) .resolution of inflammation (antiinflammatory response) is an active process controlled by endogenous mediators that suppress pro-inflammatory gene expression and cell trafficking, induce inflammatory cell apoptosis and phagocytosis. an optional balance between proand anti-inflammatory response is required to prevent the highly detrimental effect of extensive, prolonged or unregulated inflammation (3) .ammi majus is ancient herbal remedy used in the treatment of various therapeutic conditions , asthma and angina (6) , an infusion is used to calm the digestive system, while the decoction of the seed, taken after intercourse appears able to prevent implantation of fertilized ovum in the uterus (7) and the seed contain furancoumarins which stimulate pigment production in skin expose to bright light (6,7) .ammi majus contain linear furancoumarins (xanthotoxin , bergabten , imperatorin and isioimpiellin) which inhibt human liver cyp450 (8,9) simple coumarins induce number of enzymes like aldehyde reductase , glutathione stransferase(gst), and nad(p)h quinone oxidoredctase in the liver that are possible for detoxification of alfatoxin b1 (10) and also contain flavoniods (quercetin and keampferol) which has antioxidant and anti-tumor activity (11,12) the present study was designed to evaluate the efficacy and dose response effect of ammi majus alcoholic extract in experimental animal model of chronic inflammation. materials and methods the present study was carried out on 42 rats of both sexes weighing 250, selected from the animal house of the college of pharmacy, university of baghdad. the animals were maintained on normal temperature, humidity and light/dark cycle. they fed standard rat pellet diet and had free access to water until the night of the day of investigation.the animals were allocated into seven groups 6 animals each as follows . the control group was administered with 2ml/kg distilled water orally by gastric gavage tube , the standard group was treated with 5mg/kg piroxicam intraperitoneally while the test groups was treated with either 2, 4, 8, 16 or 32 mg/rat orally respectively. chronic inflammation was induced by injection of 0.1 ml of 2% formalin into sub planter area of the right hind paw of the rat (13) . all treatment were administered 30 minutes prior to formalin injection and continued for seven consecutive days. the increase in paw thickness was measured by vernier caliper method (14) before and seven days after induction of inflammation.. statistical analysis all data were expressed as mean ±sem. comparisons between treated groups were performed by anova and students t-test to evaluate the statistical difference. the p value < 0.05 was considered significant. results the anti-inflammatory effect of ammi majus alcoholic extract on chronic inflammatory model was illustrated in( table 1 )and (figure 1). treatment with piroxicam significantly reduce formalin induce paw thickness (p<0.05) compared to control . ammi majus alcoholic extract 16mg /rat and 32mg/rat showed significant reduction in paw thickness (p<0.05) compared to control, while ammi majus alcoholic extract 2mg, 4mg and 8mg/rat showed non-significant reduction in paw thickness( p>0.05) compared to control .both piroxicam and ammi majus alcoholic extract 16mg/rat produced comparable effect on formalin-induced chronic inflammation while ammi majus alcoholic extract 32mg/rat showed significant difference to that produced by control , standard and all other treated groups (2mg, 4mg, 8mg and 16mg/rat ) which produced the greatest effect with increase the dose of ammi majus alcoholic extract . figure 1: mean increase in paw thickness during experimentally-induced chronic inflammation of the study groups. iraqi j pharm sci, vol.21(1) 2012 anti-inflammatory effect of ammi majus alcoholic extract 84 table 1: effect of different doses of ammi majus alcoholic extract on formalin-induce chronic inflammation in rats. % of inhibition mean increase in paw thickness(mm) after 7 days treatment groups 2.96 ± 0.37 control ( 2ml/kg d.w) n=6 16.11 1.63± 0.28 *a piroxicam 5mg/kg n=6 0 2.86 ± 0.12 b extract 2mg/rat n=6 0.23 2.9 ± 0.14 b extract 4 mg/rat n=6 0.94 2.85 ± 0.19 b extract 8 mg/rat n=6 12.79 2.06 ± 0.25 *c extract 16 mg/rat n=6 24.88 1.2 ±0.24 *d extract 32 mg/rat n=6 data are presented as mean ± sem; n=number of animals; *p<0.05 with respect baseline value ; values with non-identical superscript (a,b,c and d) among different groups are considered significantly different (p<0.05). discussion the inflammatory process is invariably characterized by a production of prostaglandins, leukotrienes , histamine , bradykinin , platelet-activating factor (paf) and by a release of chemicals from tissues and migrating inflammatory cells (15) . the initial phase of inflammation (edema,0-1 hour) which is not inhibited by nsaid like indomethacin or aspirin , has been attributed to the release of histamine , 5-hydroxytryptamine and bradykinin (16) , followed by a late phase (1-6 hours) mainly sustained by prostaglandin release and more recently has been attributed to the induction of inducible cyclooxygenase (cox-2) in the tissue (17) . inflammatory events are initiated, enhanced, or coordinate by the action of various chemical mediators such as mast cells, platelets, and leukocytes are responsible for the release of inflammatory mediators and play an important role in the development of inflammation. these mediators include cytokines and chemokines , which promote inflammation and further function to amplify the response (18) , low molecular weight lipids derived from arachidonic acid (aa) , gases like nitric oxide (no) and carbon monoxide , reactive oxygen species (ros) and nucleotides (19) , small peptides such as kinins, complement and clotting system and finally amines such as histamine and 5-ht.chronic inflammation begins 2-4 days after the onset of the acute response and can last for weeks to months or years due to the persistence of the initiating stimulus, interference of the normal healing process, repeated bouts of acute inflammation or low-grade smoldering due to continued production of immune response mediators (4) . the histological characteristics of chronic inflammation are the increase presence of macrophage and increased numbers of fibroblasts and other tissue matrix cells, such as osteoclasts and chondrocytes, via cytokine and growth factor-induced proliferation. fibroblast is associated with secretion of both collagen and collagenase leading to fibrosis and reactive tissue remodeling. phagocytic cell can also contribute directly to tissue injury through the release of proteolytic enzymes and free radicals (20) .the effect of the of ammi majus alcoholic extract on formalin-induced paw edema, as chronic inflammatory models was assessed by vernier caliper method. ammi majus alcoholic (32mg/rat) significantly reduced paw thickness (p<0.05) and the level of inhibition was found to be higher than standard drug utilized in the study as shown in (figure 1) and (table 1) while ammi majus alcoholic extract (16mg/rat) show less effect in paw thickness compared with piroxicam. ammi majus alcoholic extract (2mg , 4 mg and 8mg/rat) showed no effect on paw thickness compared to other test groups .the antiinflammatory effect of ammi majus alcoholic extract may be explained by the many active constituents of the extract : 1(quercetin), which is the most common flavoniod that scavenger both reactive oxygen species (ros) and reactive nitrogen species (rns). consequently the flavonoid might be used to reduce both oxidative stress i.e an imbalance between the production of and protection against reactive species, and the inflammation. moreover , querectin can also inhibit tf-kb activation, thereby directly reducing the cytokine production via this transcription factor (21) . both these capacities of the intended flavonoid may contribute to the counteracting effect of quercetin on the lipopolysacchride(lps) -induced tumor necrosis factor alpha(tnfα) . 2(kaempferol) suppressed nuclear factor –kappab(nfkappab) activating and expression of its target genes cyclooxygenase-2 inducible nitric oxide synthase , monocyte chemoattractant protein-1 , and regulate upon activation , and normal tcell expressed and secreted in aged rat kidney. furthermore , kaempferol suppressed the increase of the pro-inflammatory nf-kappab iraqi j pharm sci, vol.21(1) 2012 anti-inflammatory effect of ammi majus alcoholic extract 85 cascade through modulation of nuclear factorinducing kinase (nk)/kappab kinase(ikk) and mitogen-activated protein kinases (mapks) in aged rat kidney (22) . 3(coumarines) like bergabten showed significant antiinflammatory and analgesic activity; however xanthotoxin only have anti-inflammatory activity and isoimperatorin only analgesic effect . the anti-inflammatory and analgesic constituents seem to be related to the peripheral inhibition of inflammatory substance and to their effect on the central nervous system (23) . imperatorin and isoimperatorin showed dual inhibitory activity due to their significant effect on 5lipoxygenase and showed comparable inhibition on cycloxygenase1( cox1) and cycloxygenase2( cox2), when compared to indomethacin and nimesulide . only imperatorin caused a significant reduction of nitric oxide (no)generation (24) . also psoralen , xanthotoxin have shown cox-2/5-lo dual inhibitory activity (25) . oxypeucedanin , imperatorin and isoimpertorin these compound have been reported to exhibit pharmacological effect such as inhibition of lipopolysaccharideinduced prostaglandin e2 (26) , inhibition of il1b-induced cyclooxygenase-2 (cox2) (27) and inhibitory effects on the gaba degradative enzyme , gaba transaminase (28) . in conclusion , ammi majus alcoholic extract in a dose dependant pattern was effective in decreasing chronic inflammatory reaction in experimental model, where the antiinflammatory activity of ammi majus alcoholic extract increase up to 32mg/rat and the effect increased with increasing the dose. references 1. nathan c. points of control in inflammation. nature 2002; 420:846-852. 2. gouwy m, struyf s, proost p, van damme j, synergy in cytokine and chemokine network amplifies the inflammatory response. cytokine growth factor rev 2005; 16:561-580. 3. lawrence t, willoughby da, gilroy dw. an ti-inflammatory lipid mediators and insights into the resolution of inflammation. nat rev immunol 2002; 2:787-795. 4. whicher j , chambers r, mechanisms in chronic inflammation . immunol today 1984;5:3-4. 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–arch.pharm res. 2006;29:617-623. 26. ban hs, lim ss , suzuki k, jung sh , lee s, lee ys, shin kh and ohuchi k. inhibitory effect of furocoumarins isolated from the roots of angelica dahuricae on prostaglandin e2 production . plant medica 2003;69:408-412. 27. lin ch, chang cw, wang cc, chang ms and yang ll. byakangelicol, isolated from angelica dahuricae, inhibit both activity and induction of cyclooxygenase 2 in human pulmonary epithelial cells, journal of pharmacy and pharmacology 2002 ;54:1271-1278. 28. kim dk, lim jp, yang jh, eom do, eun js and leem kh, acetylcholinesterase inhibitor from the roots of angelica dahuricae . archives of pharmacia research 2002;25:856-859. http://www.ncbi.nlm.nih.gov/pubmed?term=%22park%20mj%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22lee%20ek%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22heo%20hs%22%5bauthor%5d iraqi j pharm sci, vol.32( 1 ) 2023 helicobacter pylori infections and moxifloxacin-triple therapy doi: https://doi.org/10.31351/vol32iss1pp107-114 107 treatment of helicobacter pylori infections using moxifloxacin-triple therapy compared to standard triple and quadruple therapies ahmed mansur kadhim *,1, mohammed mahmood mohammed**, hussein muhammad hassan abdul-hussein *** and yasir sabeeh abdulridha *** * ministry of health and environment, wassit health directory, iraq ** department of clinical pharmacy, college of pharmacy, al-mustansiriya university, baghdad, iraq *** ministry of health and environment, al-zahraa teaching hospital, wassit province, iraq **** ministry of health and environment, imamaen kdhimaen medical city, karkh health directory, iraq abstract helicobacter pylori (h. pylori) is one of the most common infectious human pathogens. h. pylori could induce inflammation, that causes illnesses and disorders of upper gastrointestinal which including peptic ulcer diseases, dyspepsia, gastroesophageal reflux disease and gastric mucosa-associated lymphoid tissue (malt) lymphoma. it is important to use a better tolerated and greatly effective eradication regimen. this study aimed to evaluate the efficacy, safety, tolerability of prescribing moxifloxacin-based triple therapy compared to that of using bismuth-based quadruple therapy and clarithromycin-based triple therapy in treatment of h. pylori infection, and the patients abo blood group phenotypes as an interrelated disease affecter. in this study, 75 newly diagnosed adult patients with h. pylori infection were included and completed the study. they were allocated into three groups with three different treatment regimens for h. pylori eradications; group a (25 patients) received oral standard clarithromycin-based triple therapy for 14 days. group b (25 patients) received oral bismuth based-quadruple therapy for 10 days. group c (25 patients) received oral moxifloxacin-based triple therapy for 14 days. the results reported in this study indicated a significant higher eradication rate of group b and group c (84% and 80%, respectively) of patients with h. pylori infections compared to that of group a (52%). the incidence of adverse effects was appeared as 64%, 72% and 24% of patients in group a, b and c respectively. the use of moxifloxacin triple regimen for h. pylori eradication, present with eradication efficacy parallel to that of quadruple regimen which was significantly higher compared to that of clarithromycin triple regimen. in this study, the eradication rates of triple clarithromycin regimen, quadruple regimen and triple moxifloxacin treatment regimen were low in h. pylori infected patients whom carrying blood group o phenotype compared to those having other blood groups phenotype. however, no statistically significant differences were yielded in eradication rates of all treatment regimens in regards to the type of blood groups phenotype. also, moxifloxacin triple therapy is more tolerable and does not increase the incidence of overall adverse effects compared to other regimens used in this study. keywords: h. pylori, moxifloxacin, clarithromycin, triple therapy, quadruple therapy. الموكسيفلوكساسين الثالثي مقارنة بالعالجات القياسية الثالثية البوابية باستخدامعالج عدوى البكتريا والرباعية *** ياسر صبيح عبد الرضا و ***حسين محمد حسن عبد الحسين ،** محمد محمود محمد ،1*، احمد منصور كاظم ، دائرة صحة واسط، العراق وزارة الصحة والبئية * الصيدلة السريرية، كلية الصيدلة، الجامعة المستنصرية، بغداد، العراق فرع ** ، مستشفى الزهراء التعليمي، دائرة صحة واسط، العراقوزارة الصحة والبئية *** العراق الكرخ، ، مدينة اإلمامين الكاظميين الطبية، دائرة صحةوزارة الصحة والبئية *** الخالصة البوابية االلتهاب الذي يسبب ملويةة شيوًعا. يمكن أن تسبب الالبوابية هي واحدة من أكثر مسببات األمراض البشرية المعديالملوية البكتريا المعدي المريئي واألنسجة سترجاعأمراًضا واضطرابات في الجهاز الهضمي العلوي بما في ذلك أمراض القرحة الهضمية وعسر الهضم ومرض اال للغاية. هدفت هذه الدراسة إلى يمكن تحمله وفعال جيدمن المهم استخدام نظام استئصال (. .(maltاللمفاوية المرتبطة بالغشاء المخاطي المعدي ) على البزموت والعالج معتمد على الموكسيفلوكساسين مقارنةً باستخدام العالج الرباعي ال معتمد وصف العالج الثالثي ال مالئمةتقييم فعالية وسالمة و . في هذه الدراسة مرضالب كعامل مرتبط مرضىلل aboعلى كالريثروميسين في عالج عدوى الملوية البوابية ، وأنماط فصيلة الدم معتمد الثالثي ال ة عالج مريًضا بالغًا تم تشخيصهم حديثًا بعدوى الملوية البوابية واستكملوا الدراسة. تم تقسيمهم إلى ثالث مجموعات مع ثالثة أنظم 75، تم تضمين يوًما. تلقت المجموعة 14اسيًا يعتمد على الكالريثروميسين لمدة مريًضا( عالًجا ثالثيًا قي 25البوابية ؛ تلقت المجموعة أ ) ملويةمختلفة الستئصال ال الموكسيفلوكساسين مريًضا( عالًجا ثالثيًا يعتمد على 25ج )أيام. تلقت المجموعة 10مريًضا( عالًجا رباعيًا يعتمد على البزموت الفموي لمدة 25ب ) ٪ 80و ٪84) ج هي والمجموعة بمعدل استئصال أعلى للمجموعة ان دراسة إلى في هذه ال مثبتةيوًما. أشارت النتائج ال 14عن طريق الفم لمدة ٪ 64الضائرة بنسبة . ظهرت نسبة حدوث التأثيرات (52٪ ). أ ة البوابية مقارنةً بالمجموعةملوي على التوالي( من المرضى المصابين بالبكتيريا ال ة البوابية ملويعلى التوالي. إن استخدام نظام موكسيفلوكساسين الثالثي الستئصال ال٪ من المرضى في المجموعة )أ( و )ب( و )ج( 24٪ و 72و 1corresponding author e-mail: ahmed.abd2200p@copharm.uobaghdad.edu.iq received: 7/11 /2021 accepted: 19/4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp107-114 iraqi j pharm sci, vol.32( 1 ) 2023 helicobacter pylori infections and moxifloxacin-triple therapy 108 الدراسة ، كانت كالريثروميسين. في هذه ظهر فاعلية استئصال موازية لتلك الخاصة بالنظام الرباعي الذي كان أعلى بكثير مقارنة بالنظام الثالثيا .نظام الكالريثروميسين الثالثي والنظام الرباعي ونظام العالج الثالثي موكسيفلوكساسين منخفًضا في المرضى ل ستئصالمعدالت اال مقارنة بأولئك الذين لديهم النمط الظاهري لفصيلة الدم oة البوابية الذين يحملون النمط الظاهري لفصيلة الدم ملويةالمصابين بالبكتيريا ال دم. األخرى. ومع ذلك ، لم تسجل فروق ذات داللة إحصائية في معدالت االستئصال لجميع نظم العالج فيما يتعلق بنوع النمط الظاهري لفصائل ال ثي موكسيفلوكساسين أكثر قابلية للتحمل وال يزيد من حدوث اآلثار الضارة الكلية مقارنة باألنظمة األخرى المستخدمة في هذه أيًضا ، يعد العالج الثال الدراسة. . ة البوابية ، موكسيفلوكساسين ، كالريثروميسين ، العالج الثالثي ، العالج الرباعيملويالكلمات المفتاحية: ال introduction helicobacter pylori is one of the most common infectious human pathogens, and accounts for high risk of morbidity and suffering (1). worldwide, h. pylori infects about fifty percent of populations and it highly associated with duodenal ulcers (du) and benign gastric ulcers (gu) (2). the incidence is more in developing countries compared with developed countries (3). h. pylori infection is usually transmitted via feco-oral or oro-oral routes, in addition to gastro-gastric route (2). h. pylori is one of the most important causes of upper gastrointestinal illnesses, including dyspepsia, peptic ulcer diseases (pud), gastroesophageal reflux disease (grd) and gastric mucosa-associated lymphoid tissue (malt) lymphoma (4). according to the american college of gastroenterology (acg 2017), h. pylori infection testing can be done for patients with all diseases mentioned above (5), and because the high prevalence and serious health burden of such infection, it is necessary to use a highly effective and well tolerated eradication regimens (3). different therapeutic regimens used for eradication of peptic ulcer infection includes antisecretory medications; proton pump inhibitors (ppis), h2-receptor antagonists (h2ras) and other medications (6). anti-infective drugs for h. pylori eradications are included in many regimens as; 1) clarithromycin-based triple drugs regimens, for 14 days. howover, due to the high clarithromycin resistances reported in north america, its use as first-line treatment option is diclined recently (5, 7); 2) anothor drug regimen is a quadruple regimen; which include bismuth-based quadruple drug regimens and non-bismuth based quadruple drug regimens.the main advantage of this regimen is no clarithromycin resistance and minimal effect of metronidazole resistance which overcomes by extended duration of 10–14 days. other drug regimens also used for treatment of h. pylori infection include hybrid regimen , and sequential regimens (5,8,9). based on available results of meta-analysis and clinical trials studies, moxifloxacin-based triple therapy is safe and effective and shows better outcome parameters compared to the standard clarithromycin-triple therapy in either first-line or second-line therapies in the treatment of patients with h. pylori infections (10-12). this study aimed to evaluate the efficacy, safety, tolerability of prescribing moxifloxacin-based triple therapy compared to that of using bismuth-based quadruple therapy and clarithromycin-based triple therapy in the treatment of h. pylori infection, and the patients abo blood group phenotypes as an interrelated disease affecters i.e. eradication rate of moxifloxacin-based triple therapy compared to other standard h. pylori eradication therapies and their relations to abo blood group phenotypes. patients and methods the current study was a prospective randomized-controlled interventional open-label clinical trial, performed in a single health center. this study was conducted on iraqi patients, who attended the gastrointestinal endoscopy unit of alzahraa teaching hospital/ wassit province from october 2016 to september 2017 and screened with suspected h. pylori infection. the patients were selected by a gastroenterologist physician and assigned as having a positive endoscopic examination of h. pylori infection (with clinical indications for h. pylori treatment and presented with positive stool antigen test). however, patients with h. pylori negative test, patients who previously received eradication therapy for h. pylori infection, patients with poor compliance, patients with advanced gastric cancer or other malignancy or comorbid disease, in addition to pregnant or lactating women were excluded from the study. eligible patients were allocated randomly into three groups and the treatments were divided as follows: group a (25 patients; 13 male and 12 female) received oral standard conventional triple therapy. group b (25 patients; 11male and 14 female) received oral bismuth quadruple therapy. group c (25 patients; 14 male and 11 female) received oral moxifloxacin-based triple therapy. dempgraphic data and clinical symptoms were collected through direct interview with the patient. eligible patients were allocated randomly into three groups and the treatments were divided as follows: group a received oral standard conventional triple therapy (esomeprazole tab. 40 mg twice daily (b.i.d), amoxicillin tab. (1000 mg b.i.d), clarithromycin tab. (500 mg b.i.d)) for fourteen days. group b received oral bismuth quadruple therapy (esomeprazole 40 mg b.i.d, a capsule containing three agents (pylera®); metronidazole 125mg, bismuth subcitrate potassium 140mg, and tetracycline 125mg, were taken four times daily (q.i.d)) for ten days. group c received oral moxifloxacin-based triple therapy (moxifloxacin iraqi j pharm sci, vol.32( 1 ) 2023 helicobacter pylori infections and moxifloxacin-triple therapy 109 tab. 400 mg once daily, amoxicillin tab. (1000 mg b.i.d.) and esomeprazole 40 mg b.i.d) for fourteen days. six weeks after completion of treatment, clinical outcomes were evaluated by the following: 1. h. pylori eradication was checked by using stool antigen test. patients with negative stool antigen test were classified as h. pylori free (13), while those who presents with positive stool antigen test were considered as h. pylori infected. furthermore, a clinical examination was done for each patient to assess their condition and response to therapy. 2. adverse drug reactions associated with the three h. pylori eradication regimens.biopsy samples: because h. pylori does not evenly distribute throughout the gastric mucosa, three to four gastric antral and two body mucosal biopsy specimens were taken from every patient in the endoscopy unit before starting the study as a gold standard diagnostic tool (14). for all gi mucosal biopsies that were used for histopathological diagnosis, 10% buffered formalin was used as a fixative agent. two experienced histopathologists reviewed samples and they were blinded to the endoscopic findings. stool sample: stool specimens were collected from each patient before starting the study (as a diagnostic tool) and 6 weeks after the study ends (to assess the eradication regimen activity) , and the stool antigen test was performed according to the principle of h. pylori rapid antigen test (15). the h. pylori antigen rapid test device (feces) used from abon biopharma, china.blood samples: blood samples were drawn and collected immediately after endoscopy from all patient groups. h. pylori test was performed based on the principle of h. pylori antibody rapid test device (serum/plasma) (16). the anti h. pylori igg antibody rapid device used from abon biopharma, china. and anti abo and anti-d monoclonal kit used from spinreact, spain. statistical analysis: data were analyzed by using statistical package for social sciences (spss) (student version 23, mcgraw hill company 2015). continuous variables (expressed as mean ± sd), and categorical variables (expressed as number (n) and percentage (%)) like demographic data, eradication rate, eradication rate according to abo blood group phenotypes, and incidence of adverse effects. demographic data (age, bmi, gender, family history and duration of symptoms) were converted to categorical data and analyze statistically by chi square test, in addition to eradication rate and incidence of adverse effects among the three groups. fisher’s exact test used to analyze abo blood group phenotypes distribution, and eradication rate with their relations to abo blood group phenotypes. 𝑃 value less than 0.05 was considered to be statistically significant. results this study was conducted on 119 iraqi patients, who attended the gastrointestinal endoscopy unit, screened for suspected h. pylori infection, from them, 88 patients were h. pylori positive and the other 31 patients were h. pylori negative who were excluded, only 75 adult patients were with h. pylori infection whom completed the study by per protocol analysis.the results of this study presents with no significant differences among the three groups regarding to age (p value = 0.553), bmi (p value = 0.806) and gender (p value = 0.688). moreover, blood group phenotype, a, b, ab and o represented by 24%, 30.7%, 12% and 33.3% of all patients, respectively. regarding to each blood group phenotype, no significant differences were reported among the three groups (p value = 0.326). finally, no significant association between family history of dyspepsia and type of treatment used in this study (p value = 0.820), as seen in table 1. table 1. demographic distribution and disease characteristics variables study groups p value group a group b group c age (years) mean±sd (range) 38.6±11.1 (20-65) 38.9±14.4 (20-63) 36.8±9.6 (22-60) 0.553 a bmi (kg/m2) mean±sd (range) 25.7±3.7 (19-32) 25.5±3.6 (19-31) 26.1±4.5 (19-35) 0.806 a no (%) no (%) no (%) gender male 13 (52) 11 (44) 14 (56) 0.688 a female 12(48) 14(56) 11(44) abo blood group a 5 (20) 5 (20) 8 (32) 0.326 b b 4 (16) 12 (48) 7 (28) ab 5 (20) 2 (8) 2 (8) o 11 (44) 6 (24) 8 (32) family history +ve 6 (24) 7 (28) 8 (32) 0.820 a -ve 19(76) 18(72) 17(68) duration of symptoms <1 year 13 (52) 6 (24) 13 (52) 0.069 a ≥ 1 year 12 (48) 19 (76) 12 (48) bmi = body mass index a fisher’s exact test used for abo blood groups to examine the degree of significance. b pearson chi square test used for other demographic and disease characteristics to examine the degree of significance. p >0.05 are not significantly different iraqi j pharm sci, vol.32( 1 ) 2023 helicobacter pylori infections and moxifloxacin-triple therapy 110 the prevalence of h. pylori infection in this study that determined by stool antigen test, histology, and antibody test shows that 88 (73.95%) of enrolled patients were h. pylori positive (however, 75 patients only completed the study) while 31 (26.05%) of the patients were h. pylori negative. table-2 shows that the use of quadruple regimen (pylera®) eradicated 84% of patients with h. pylori infection, triple moxifloxacin regimen eradicated 80% of patients with h. pylori infection, while triple clarithromycin regimen eradicated only 52% of patients with h. pylori infection. the best eradication rate was achieved by quadruple regimen and triple moxifloxacin regimen which were significantly different to that achieved by triple clarithromycin regimen (p value = 0.023) table 2. eradication rate of moxifloxacin-based triple therapy compared to other standard h. pylori eradication therapies significant difference among different groups (p>0.05). data analyzed by pearson chi-square test. group a patients received oral standard clarithromycin-based conventional triple therapy group b patients received oral bismuth quadruple therapy group c patients received oral moxifloxacin-based triple therapy table-3 shows that there was no statistically significant association between type of drug regimens and blood group phenotypes of patients; p value is > 0.05 in all conditions with lower percent of eradication rate of o blood group phenotype compared to others for the three study groups. table 3. eradication rate of moxifloxacin-based triple therapy compared to other standard h. pylori eradication therapies and their relations to abo blood group phenotypes blood group a b ab o p value study groups eradication rate n (%) n (%) n (%) n (%) group a 4 (80) 2 (50) 3 (60) 4 (36.4) 0.530 group b 4 (80) 11 (91.7) 2 (100) 4 (66.7) 0.574 group c 6 (75) 6 (85.7) 2 (100) 6 (75) 1.000 p value 0.939 0.253 0.482 0.411 data presented as n= number and (%) = percentage the data were analyzed by fisher’s exact test to examine the degree of significance. p >0.05 are not significantly different the overall adverse effects of drug regimens appeared during the treatment were documented to determine the tolerability of drug regimens. low incidence of taste disturbance (bitter taste), diarrhea and gastric upset were observed in some patients. all adverse effects were mild to moderate and there was no sever adverse effect which necessitates cessation of the treatment. table-4 shows that overall adverse effects appeared on 72%, 64% and 24% of patients used quadruple pylera®, triple clarithromycin and triple moxifloxacin respectively. the incidence of adverse effects related to triple moxifloxacin used was significantly different from that achieved in quadruple pylera® and triple clarithromycin using patients (p value = 0.001). table 4. the incidence of adverse effects of moxifloxacin-based triple therapy compared to other standard h. pylori eradication therapies. study group adverse effects p value yes no n (%) n (%) group a 16 (64) 9 (36) 0.001(a) group b 18 (72) 7 (28) group c 6 (24) 19 (76) data presented as n= number and (%) = percentage the data were analyzed by chi square test to examine the degree of significance. (a) (p<0.01) high significant difference. study groups patients number n eradication rate n (%) group a 25 13 (52) group b 25 21 (84) group c 25 20 (80) p value --0.023* iraqi j pharm sci, vol.32( 1 ) 2023 helicobacter pylori infections and moxifloxacin-triple therapy 111 discussion in the present study, the prevalence of h. pylori infections is 73.95% were h. pylori positive. hooi et al reported the highest prevalence in africa (79.1%), and asia (54.7%). in contrast, h. pylori prevalence is lowest in northern america (37.1%), while in western asia; iran (59.0%), saudi arabia (65.9%), turkey (77.2%) (17). two studies conducted in iraq showed that (78%) and (68.97%) of adults respectively were infected with h. pylori (18, 19). the current study explored the eradication rate of first line standard clarithromycin tripleregimen was (52%) and thus the eradication failure (resistance) was (48%). the result of this study is consistent with several studies; malfertheiner et al founded that 55% of patients were eradicated in the standard clarithromycin therapy (20) and makhlough et al recorded eradication rate 70% achieved with clarithromycin triple therapy as a first-line regimen (21). in iraq, studies by abbas et.al and ali et.al recorded that per protocol eradication rate 57.89% and 57.8 % respectively, achieved with a first-line therapy of clarithromycin based-triple regimen (19,22). therefore, in cases where the h. pylori resistance to clarithromycin drug regimen is higher than 20%, it recommended that treatments include clarithromycin should be avoided in the eradication of h. pylori (23). so, because of high prevalence of resistant rate of conventional clarithromycin triple regimen we must use another type of treatment in the area of high clarithromycin resistant such as bismuth based-quadruple regimen and moxifloxacin triple regimens and considered as first-line treatment (7,24). in the present study, the eradication rate of the first line quadruple therapy using three in one capsule (pylera)®; plus esomeprazole was (84%) found significantly higher than standard clarithromycin based triple therapy (p value <0.023), this result was in agreement with other studies; scalese et al which has shown that 87% of patients got eradication of h. pylori after treatment with quadruple therapy by using (pylera®) capsule given with ppi (25). the major effect of bismuth is to add an additional 30%–40% to the success with resistant infections (26). although metronidazole resistance is high worldwide, but it does not interfere with the therapeutic effects of bismuth, tetracycline and metronidazole combination due to metronidazole synergism with bismuth (20, 27). the duration of quadruple therapy (pylera)® is prepared for a ten days duration, if extending the duration to 14 days, would not increase the therapeutic effectiveness (28). this differs to what has been suggested for standard triple therapies with 14 days duration (29). the eradication rate of moxifloxacin-based triple therapy in the current study, equal to (80 %). this result was in consistent with data reported that per protocol eradication rate was (84.8%) by using triple regimen consist of moxifloxacin, amoxicillin and esomeprazole (30). other studies showed that the moxifloxacin-based triple therapy eradication rate was found to be over (90 %) by per protocol analysis (23). a study compared moxifloxacin based-triple regimen for 10 days, and bismuth based-quadruple regimen for 14 days resulted with eradication rates of 82.6%, and 90.5% respectively (11). consequently, moxifloxacin-based triple therapies could be safe and could be suggested in clinical practice and showed higher rates of eradication compared to the standard triple therapy in the treatment of h. pylori infection and well tolerated with a good compliance and few adverse effects in comparing with the standard triple therapy (12, 23, 31). it is obvious in this study that the eradication rates of triple clarithromycin regimen, quadruple regimen and triple moxifloxacin treatment regimen were low in h. pylori infected patients whom carrying blood group o phenotype (36.4%, 66.7% and 75% respectively) compared to those having other blood groups phenotype. however, no statistically significant differences were yielded in the eradication rates of all treatment regimens in regards to the type of blood groups phenotype, as shown in table-3. the blood group o individuals express higher inflammatory responses to h. pylori, demonstrating significant association between positive caga h. pylori strain and the development of peptic ulcers among patients belonging to the blood group o (32). it was demonstrated that bacterial load in patients with positive cag a was greater than in patients with a negative cag a, both in the corpus and antrum (33). a significant reduction in the eradication rate after h. pylori treatment was associated with high antral density of h. pylori (34). moreover, blood group o phenotype possibly has higher antibiotic resistance compared with other blood groups phenotypes (35, 36) . in total, these may provide a possible explanation to the low response of blood group o patients to h. pylori treatment by standard clarithromycin triple therapy, and for both quadruple drug therapy and moxifloxacin triple therapy. the incidence of overall adverse effects in this study showed statistically highly significant differences among the three study groups with more frequency for group b, bismuth quadruple therapy, and most of the adverse effects were mild to moderate in suffered patients. these results come in consistent with other studies which showed that 60% and 51% of patients respectively had medication adverse effects due to the treatment with clarithromycin triple therapy (20, 37). a study by delchier et al reported that in patients treated for 10 days with quadruple pylera®, iraqi j pharm sci, vol.32( 1 ) 2023 helicobacter pylori infections and moxifloxacin-triple therapy 112 67.3% of patients reported adverse events (38). other study done by liou et al found the frequency of adverse events was 47% in patients treated with 14day clarithromycin triple therapy and 67% in patients treated with 10-day bismuth quadruple therapy (39). quadruple regimen needs more frequent use of drugs that may affect patient's adherence and its cost-effectiveness is an important issue, so bismuth-quadruple therapy still a challenge for both the physician and the patient (40). finally, the reported adverse effects were mild, self-limited, and had no influence on the patient’s tolerability to medications. conclusions both 14 days moxifloxacin triple regimen and ten days quadruple regimen showed higher eradication effectiveness and symptoms improvement compared with standard clarithromycin triple regimen. moreover, moxifloxacin triple therapy is more tolerable and does not increase the incidence of overall adverse effects compared to other regimens. 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https://doi.org/10.31351/vol32iss1pp92-99 92 the ameliorating effect of oral paquinimod administration against imiquimod induced psoriasis-like inflammation in mice raghad abdulsalam khaleel*,1 and munaf hashim zalzala** *department of pharmacology, college of medicine, university of al-iraqia, baghdad, iraq. *department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract psoriasis is a chronic, inflammatory condition that primarily affects the skin, hair, and joints and is associated with significant humanistic and economic consequences. psoriasis was induced in mice in this work using an imiquimod 5% cream, an immune response modifier that can cause psoriasis-like skin inflammation when given orally. paquinimod is prepared as a suspension and has been orally given to mice before imiquimod application.in this study, albino mice were allocated into five groups and treated as follows: the control group received only daily application of cream based on shaved back (62.5mg/2cm) with a daily oral dose of vehicle for 14 consecutive days with the oral vehicle. imiquimod group received a daily oral dose of vehicle one hour before imiquimod 5%application on shaved back (62.5mg/2cm) for 14 consecutive days.the paquinimod-treated group received different daily oral doses of paquinimod one hour before imiquimod 5% application on shaved back (62.5mg/2cm) for 14 consecutive days. dexamethasone -treated group received a daily oral dose of dexamethasone oral solution (2mg/5ml) one hour before imiquimod 5% application on shaved back (62.5mg/2cm) for 14 consecutive days. paquinimod only group received a daily oral dose of paquinimod for 14 consecutive days. the current study found that the administration of paquinimod suspension resulted in a significant decline in tnf-α,il-23, il17 level, reduced psoriasis area and severity index, spleen index, skin thickness, and gene expression of tnf-α, nf-kb, il-1b, il-17in the (paquinimod suspension+imiquimod) group substantially more than that in the (vehicle suspension+imiquimod) groups. in conclusion, paquinimod has a strong ameliorating effect as compared with dexamethasone against imiquimod-induced psoriasis-like inflammation in mice. keywords: psoriasis, paquinimod, imiquimod 5%, tnf-α, il-23. تأثير االستخدام الفموي لعالج الباكوينيمود لتخفيف الصدفية الناتجة عن االستخدام الموضعي لاليميكويمود في الفئران **مناف هاشم زلزلةو 1*,رغد عبد السالم خليل . ، العراقبغداد ، العراقية الجامعة ، الطب ، كليةاالدويةفرع * العراق ، بغداد، بغداد جامعة، الصيدلة كلية ، والسموم رع االدوية ف ** الخالصة .تم استحداث .إنسانية واقتصادية كبيرة مشاكل الصدفية هي حالة التهابية مزمنة تؤثر بشكل أساسي على الجلد والشعر والمفاصل وترتبط ب التي تكون اشبه بالتهاب الجلد الذي يتميز باالختناق واالحمرار االرتشاحي ( في الفئران %5الصدفية عن طريق االستخدام الموضعي لاليميكويمود) الباكوينيمود كمعلق فموي وتم اعطاؤه كمعلق فموي للفئران رتحضي تم في هذه الدراسة. ةوتسلل الخاليا االلتهابية والخاليا العدلة والخاليا الليمفاوي :المجموعة االولى تم تقسيم الفئران البيضاء إلى خمس مجموعات وتم عالجها على النحو التالي .قبل ساعةمن وضع الكريم الموضعي لاليميكويمود يوم ,المجموعة الثانية تلقت 14ة الشعر من منطقة الظهرلمدة سم( على الجلد بعد ازال2ملغم/62.5تلقت جرعة موضعيةلحامل الكريم بشكل يومي) يومي بشكل لاليميكويمود الموضعي الكريم لحامل الموضعية اليومية الجرعة تلقيها من ساعة قبل الباكوينيمود حامل من يومية فموية ) جرعة الثالثة تلقت جرع يومية مختلفة من عالج الباكوينيمود بشكل يوم,المجموعة14سم( على الجلد بعد ازالة الشعر من منطقة الظهرلمدة 2ملغم/62.5 سم( على الجلد بعد 2ملغم/62.5) فموي قبل ساعة ساعة من تلقيها الجرعة اليومية الموضعية لحامل الكريم الموضعي لاليميكويمود بشكل يومي الظهرلمدة منطقة من الشعر مخت14ازالة يومية جرع تلقت الرابعة لمدة يوم,المجموعة الباكوينيمود عالج من فموي بشكل بشكل 14لفة يوم بشكل مل قبل ساعة من تلقيها الجرعة الموضعية لاليميكويمد 5ملغم/2متتالي,المجموعة الخامسة تلقت جرعة يومية فموية من محلول الديكساميثازون تائج هذه الدراسة اظهرت ان المعلق الفموي للباكوينيمود قلل الى نيوم.14سم( على الجلد بعد ازالة الشعر من منطقة الظهرلمدة 2ملغم/62.5يومي) و il-1b و nf-kb و tnf-α والتعبير الجيني لـ il17 ومستوى il-23 و tnf-α حد كبير مقياس مساحة منطقة الصدفية ومؤشر شدتها il-17 وفي ي.للباكوينيمود مقارنة بالمجموعة المعالجة بحامل المعلق الفموفي المجموعة المعالجة بالمعلق الفموي ومؤشر الطحال وسمك الجلد و في الفئران. نهاية الدراسة تم االستنتاج من انه الباكوينيمود له تاثير اقوى من الديكساميثازون في تقليل الصدفية المستحدثة بواسطة االيميكويمود tnf-α، il-23، il17 ، باكوينيمود، االيميكويمود ، الصدفية الكلمات المفتاحية : 1corresponding author e-mail: raghad_salam@yahoo.com received: 15/2 /2022 accepted: 19/ 4 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp92-99 iraqi j pharm sci, vol.32(1) 2023 oral paquinimod administration against imiquimod induced psoriasis 93 introduction psoriasis is a chronic, inflammatory condition that primarily affects the skin, hair, and joints and is associated with significant humanistic and economic consequences. it occurs in about 1% to 3% worldwide, with a higher prevalence in western countries(1) . the precise cause of psoriasis is still unclear. psoriasis is closely related to genetics, but environmental exposure plays an essential role in disease development(2). this immune-mediated disease entails a dysregulated interplay between immune cells and keratinocytes. while the pathogenesis is not fully understood, intense research has been ongoing to explore the underlying mechanisms involved. the topical application of imiquimod (imq), a ligand of tolllike receptors (tlr) 7 and 8, has been reported to induce psoriasis-like dermatitis with many hallmarks of human psoriasis, such as the formation of micro abscesses, skin thickening, hyperkeratosis, acanthosis, scaling, and erythema(3). paquinimod is an immunomodulatory agent, in vivo, paquinimod reduces splenic counts of ly6chicd115 inflammatory monocytes and cd11b+cd11c+ dendritic cells in a mouse model of experimental autoimmune encephalomyelitis(3). franse´n pettersson n, et al. (2018) has been reported that the immunomodulatory quinoline-3carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis(4).in 2015 martin stenström also reported that paquinimod reduces skin fibrosis in tight skin 1 mice, an experimental model of systemic sclerosis(5). jong-uk lee (2021) approved that the inhibitory effect of paquinimod on a murine model of neutrophilic asthma induced by ovalbumin with complete freund’s adjuvant(6). tahvili s, et al (2018) has been reported that paquinimod prevents the development of diabetes in the non-obese diabetic (nod) mouse(7). in this work, paquinimod was prepared as a suspension and given orally to mice before psoriasis induction via imiquimod. in view of the above considerations, this study was conducted to examine the ameliorating effect of oral paquinimod administration against imiquimod induced psoriasislike inflammation in mice and to compare this effect with the dexamethasone administered orally pointing to its ability to cause significant reduction in pasi score and gene expression of tnf-α, nfkb, il-1b, il-17. methodology chemicals and kits the chemicals that were used in this work include formalin (merk chemicals, germany), diethyl ether (bdh chemicals, india), xanthan as a suspending agent (samara drug industry iraq ( , imiquimod cream (meda germany) , paquinimod (sigma aldrich usa ) , dexamethasone syrup (samara drug industry iraq), and dulbecco’s phosphate-buffered saline (euro cyclone s.p.a, italy). the kits used in this study include (interleukin-23, interleukin-17, tumor necrosis factor alfa) all of them from (bioassay technology laboratory, china) also rna purification kit (genezoltm trirna pure kit, general, india), gdna remover and cdna synthesis super mix (transgen, china) and green qpcr supermix (transgen, china). methods animal treatment forty (40) balb/c albino mice (male, 8wk) (22-40 gm wt.) they were obtained from and maintained in the animal house at the college of pharmacy/university of baghdad under conditions of controlled temperature, humidity and light periodicity (12-hour light/dark cycle). they were fed commercial pellets and tap water ad libitum throughout the experimental period with ethical and animal care approval. preparation of paquinimod suspension: paquinimod 7.5mg was triturated in the mortar with 5ml of glycerin until a smooth paste is formed. the smooth paste was then diluted gradually by adding distilled water with continuous mixing until a homogenous mixture is formed. the mixture is transferred to the graduated cylinder. the preservative (methylparaben 0.09gm, propyl paraben 0.015gm) is dissolved in 10ml distilled water and added to the mixture in the cylinder. the volume is completed to 100ml. final concentration of paquinimod is 7.5 mg/100ml dose of dexamethasone human dose (mg / kg) = animal does (mg / kg) × (animal km / human km) as the km factor for each species is constant, the km ratio is used to simplify calculations(8) . km ratio = 0.162 human dose (mg / kg) = animal does (mg / kg) × 0.081 human dose of dexamethasone = 18 mg /day 18 mg / 60 kg (reference body weight for human) = 0.3 mg /kg animal does (mg / kg) = 0.3 (mg/kg) / 0.081 animal does (mg / kg) = 3.7 (mg/kg) experimental design this study was started in february; 2021 to september; 2021.approximately forty (40) mice have shaved their back area using a traditional manual eraser (trimming not zero shaving), and all of the mice were weighed at zero time and ear thickness of the right ear was measured using vernier every two days for 14 days consecutively. the oral experiment includes 5 groups of mice (each group includes 8 mice), as the following: iraqi j pharm sci, vol.32(1) 2023 oral paquinimod administration against imiquimod induced psoriasis 94 the control group were received only daily application of cream based on shaved back (62.5mg/2cm) and right ear (5mg) with a daily oral dose of vehicle for 14 consecutive days with the oral vehicle. imiquimod group in which mice have received a daily oral dose of vehicle one hour before imiquimod 5%application on shaved back (62.5mg/2cm) and right ear (5mg) for 14 consecutive days. paquinimod -treated groups in which mice were received different daily oral doses of paquinimod one hour before imiquimod 5% application on shaved back (62.5mg/2cm) and right ear (5mg) for 14 consecutive days. dexamethasone -treated group in which mice have received a daily oral dose of dexamethasone oral solution (2mg/5ml) one hour before imiquimod 5% application on shaved back (62.5mg/2cm) and right ear (5mg) for 14 consecutive days. paquinimod only group in which mice have received a daily oral dose of paquinimod for 14 consecutive days. to determine the severity of inflammation of the back skin, an objective the scoring system was developed based on the clinical pasi score. erythema, scaling, and thickening were independently scored on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. a higher pasi score was associated with poorer quality of life and poorer satisfaction with a skin condition. furthermore, an improvement in pasi was associated with an improvement in conventional pasi score for general bodily pain for mice (9). measurements of spleen index all mice have been weighed and when mice have been sacrificed, the spleen index is calculated by dividing the spleen weight of the mice in mg by body weight in gm (10). measurements of skin thickness at the end of the experiment, the skin thickness of the back area has been measured in mm via digital vernier caliper and comparing measured thickness between groups (11). assessment of inflammation approximately 1 to 1.5 ml of blood was collected from a retro-orbital vein placed in an eppendorf tube and centrifuged at 3500 rpm for 15 minutes to obtain serum, which was separated and stored at -20 c until the day of analysis. serum was utilized for the estimation of serum il-23 and il-17 levels by elisa kits according to the manufacturer's protocols (12). tissue homogenate skin tissue from the back area was rinsed in pbs (ph7.4) to thoroughly remove excess blood and weighed before homogenization. the tissue was homogenized in pbs (ph 7.4) by homogenizer on ice, and thawed at 2-8 ° c or frozen at -20 ° c. finally the homogenized tissue was centrifuged at 2000-3000rpm for approximately 20 minutes (13). polymerase chain reaction procedure total rnas were extracted using trizol from (genezoltm trirna pure kit, geneiad, india). reverse transcriptase pcr was performed following standard procedures. the primer pairs of the expected products were shown in table 1. all primers were purchased from applied bio system (13), and are designed according to online tool of integrated dna technology (14) table 1. gene name and primer sequences for products. gene id primer sequence (forward/reverse) gapdh (housekeeping) forward:5-ctttgtcaagctcatttcctgg-3 reverse: 5-tcttgctcagtgtccttgc-3 tnf-alpha forward: 5-tagcccacgtcgtagcaaac-3 reverse: 5-acaaggtacaacccatcggc-3 nf-κb forward: 5-aagacaaggagcaggacatg-3 reverse: 5-agcaacatcttcacatccc-3 il-1b forward: 5-acggaccccaaaagatgaag-3 reverse: 5-ttctccacagccacaatgag-3 il-17 forward: 5-tccagaatgtgaaggtcaacc-3 reverse: 5-tatcagggtcttcattgcgg-3 iraqi j pharm sci, vol.32(1) 2023 oral paquinimod administration against imiquimod induced psoriasis 95 histopathological examination the skin of the back area and the right ear was removed from mice. then it was washed with normal saline solution for the preparation tissue to histopathological examination. after washing tissues were fixed with formaldehyde (10% of formaldehyde in water). the thin section slide for tissues was stained with hematoxylin and eosin stain and mounted with the protective coverslip (15). statistical analysis data were expressed as mean values, the mean± standard error of the mean (sem). where one-way anova analysis was used for testing the significant difference between the means of groups. differences were considered statistically significant when the p-value was less than 0.05. results oral effect of paquinimod on the pasi score the pasi score was significantly increased in the model animal treated by imiquimod in comparison with the control group. the antiinflammatory effect of paquinimod was successfully decreased the pasi score compared to model animals. dexamethasone suspension administered orally significantly decrease the pasi score compared to imq treated group (figure 1). figure 1.the area and severity index of psoriasis (pasi) after oral administration of paquinimod in imiquimod-induced psoriasis in mice. each value represents mean ± standard deviation (sd). * significantly different (p<0.05) concerning the control group. # significant compared to the vehicle susp + imq group (p<0.05). effect of paquinimod on the skin thickness figure 2 showed that the increase in skin thickness in the model group was highly significant compared to the control group. the treatment with dexamethasone and paquinimod produce a significant decrease in the skin thickness is compared to the model group. the skin thickness in the (paquinimod susp only) group remains normal as in the control group. effect of oral administration of paquinimod on spleen index in imiquimod-induced psoriasis in mice. figure 3 revealed the spleen index for mice in the imq-treated group was significantly elevated compared to the control group. the spleen index in the paquinimod sus + imq group remains approximately normal compared to the spleen index in the control group, while in the dexamethasonetreated group the measured spleen index decreases significantly compared to the model group (vehicle susp+imq) group. figure 2. effect of oral administration of paquinimod on skin thickness in imiquimodinduced psoriasis in mice. each value represents mean ± standard deviation (sd).* significantly difference concerning the control group.# significant difference in comparison to the imq treated group. figure 3. spleen index after oral administration of paquinimod in imiquimod-induced psoriasis in mice. each value represents mean ± standard deviation (sd).* significantly different (p<0.05) concerning the negative control group. # significant compared to the vehicle susp + imq group. effect of oral administration of paquinimod on serum il-23, il-17, and tnf- levels in imiquimod-induced psoriasis in mice. figure 4 a & b showed that there was significant elevation (p<0.05%) in serum levels of il23 and il-17 in the imq-treated group compared to the normal control group, as well as the treatment with dexamethasone showed significance in reduction serum il23 (p<0.05) compared to the model group. also, the oral administration of paquinimod was significantly decreased the il-23 and il-17 serum levels in comparison with (vehicle susp + imq) group (p<0.05). serum il23 and il-17 levels significantly decreased by the effect of oral paquinimod suspension in comparison with the normal control group (p<0.05). figure 4 c revealed a significant elevation in tissue tnf-α level (p<0.05) in the group (vehicle susp + imq) group as compared to the normal control group, there was iraqi j pharm sci, vol.32(1) 2023 oral paquinimod administration against imiquimod induced psoriasis 96 also a significant reduction in tissue tnf-α level (p<0.05) in the group (paquinimod susp + imq) group (vehicle susp+ imq) group, besides there was a significant reduction (p<0.05) in tissue tnfα level in the group (dexamethasone sol+ imq) group as compared to the group (vehicle susp + imq) group. in the group (paquinimod susp only) group there was a significant reduction (p<0.05) in tissue tnf-α level compared to the group (vehicle susp + imq) group. figure 4. effect of oral administration of paquinimod on (a: serum il-23 level, b: il-17 level, and c: tnf alfa level) in imiquimodinduced psoriasis in mice. each value represents mean ± standard deviation (sd). * significantly different (p<0.05) concerning the negative control group. # significant compared to the vehicle susp + imq group effect of oral administration of paquinimod on gene expression of il-1b, tnf-α, nf-b, and il17 in imiquimod-induced psoriasis-like inflammation in mice. figure 5a displayed that gene expression for il-1b in mice treated with imq was highly significantly expressed. the anti-inflammatory effect for paquinimod revealed a significant reduction for il-1b’s expression is compared to the model group. on the other hand, the relative level of il-1b mrna in skin tissue for mice treated with dexamethasone or paquinimod alone showed significant inhibition compared to control mice. in figure 5b, the model mice treated with imq have expressed the level for tnf-α in the skin in a highly significant increase than control mice. the gene expression of tnf-α for (paq susp + imq) group was significantly reduced compared to the mrna level for tnf-α in (vehicle susp+imq) group. on other hand, gene expression of tnfα for mice of (dexamethasone sol+imq) and (paq point only) groups were showing significant differences compared to gene expression of tnf-α for mice of (control) group. figure 5c manifested that the oral administration of paquinimod produces a significant reduction in mrna level for nf-κb in the skin and inhibits the inflammatory effect for imiquimod. dexamethasone has a clear anti-inflammatory effect on nf-κb level in comparison to the model group. the gene expression for il-17 in the back skin for the model group was elevated higher than that in a normal animal. paquinimod successfully reduced the gene expression for il-17 in comparison to model mice. dexamethasone produces a paquinimod like an effect on the il-17 level. (figure 5d). iraqi j pharm sci, vol.32(1) 2023 oral paquinimod administration against imiquimod induced psoriasis 97 figure 5. effect of oral administration of paquinimod on gene expression of il-1b, tnf-α, nf-κb, and il-17 in imiquimod-induced psoriasis-like inflammation in mice. each value represents mean ± standard deviation (sd). * significantly different (p<0.05) concerning the negative control group. -# significant compared to the vehicle susp+ imq group. effect of oral administration of paquinimod on histological examination in imiquimod-induced psoriasis in mice. in the sections of the skin of the back area of the normal control group, the histological sections showed a normal appearance as in figure 6 a. while in the skin of the section of the back area (vehicle oint + imq), the histological section showed marked hyperkeratosis and acanthosis (5 cells) of the epidermis with heavy inflammatory cells in the upper dermis as in figure 6 b. also, paquinimod pretreatment produce a section of back area skin that showed the normal-looking appearance of skin tissues as in figure 6 c. in the dexamethasone suspension pretreated mice, the section of the back area showed a thin epidermis layer, there is still hyperkeratosis with mild acanthosis and a rete ridge with mild inflammatory cells as in figure 6 d. in the group (paquinimod oint only) group, the histological sections back area showed a normal-looking appearance as in figure 6 e. iraqi j pharm sci, vol.32(1) 2023 oral paquinimod administration against imiquimod induced psoriasis 98 figure 6. effect of paquinimod on imq-induced pathological changes in psoriasis on the skin of the back area. staining: hematoxylin and eosin (h&e).black arrows: sign of the thickness of the epidermal layer of the right ear section. a: control group; b: vehicle susp+imq group; c: paq susp+imq group; d: dexamethasone suspension+imq group; e: paq susp only group; f:histology scoring analysis. each value represents mean ± sd. * significantly different compared to the control group (p<0.05). # significantly different compared with the vehicle+imq group (p<0.05). discussion psoriasis is a chronic, inflammatory, immune-mediated disease of the epidermis with systemic involvement. psoriatic lesions appear as itchy, reddish raised plaques covered with silvery scales(16). in this study, imiquimod has been used to induce psoriasis-like skin inflammation by topical application on the back area for 14 consecutive days which have been led to skin inflammation as monitored by increased skin thickness when measured at sacrificing time, and histopathological examination also confirmed that skin of the back area was inflamed, acanthosis, hyperkeratosis, and elongation of the rete-like ridge as shown in the model group. the quinoline-3-carboxamides (q compounds) are small molecule compounds that have exhibited beneficial effects in several mouse models of inflammatory disease. in this study, paquinimod is prepared as a suspension and has been orally given to mice before imiquimod application. importantly, the present study showed that paquinimod ameliorated the psoriasis like inflammation induced by imq, evidenced by the significant decrease in the serum levels of tnf-α,il-17,il-23 , coupled with a significant decrease in pasi score ,skin thickness and spleen index.greater than that serum il-17, il-23 level was significantly reduced in (paquinimod susp+imq) group as compared with the control group which indicates that paquinimod suspension act as an antiinflammatory agent by decreasing the serum level of these interleukins. il-23, mainly produced by dendritic cells, macrophages, and keratinocytes acts on numerous target cells via either an il-17dependent or an il-17-independent mechanism. in the first, il-23 stimulates th17 cells via il-23r and induces the release of molecules such as il-17 or il22(16). these, by binding their cognate receptors il17r or il-22r, eventually activate the “effector cells” keratinocytes, b cells, osteoclast precursors, macrophages, and fls. alternatively, the same subset of target cells can be directly challenged by the il-23 in an il-17-independent manner(16). the overall effect of the activation of the il-23 pathway consists of the recruitment of inflammatory cells within the inflamed tissue. the pro-psoriatic activity of il-23 has been suggested by the occurrence of psoriatic-like skin lesions in a mouse model of il23 administration intradermal(16). il-23 can indeed act both independently of il-17 by promoting the epidermal hyperplasia and activating the keratinocyte proliferation via the increase of keratin 16 (k16) expression, and in association with il-17 by enhancing dermal acanthosis, neutrophil recruitment and infiltration of il-22 and il-17producing cells into the lesional skin (17). skinresident cells such as keratinocytes, fibroblasts, and endothelial cells, which express the receptor machinery for responding to il-17 and il-22 stimulation, react by up-regulating the expression of pro-inflammatory cytokines, chemokines (e.g., cxcl1 and ccl20), and anti-microbial peptides (such as ll-37 or β-defensins)(16). overall, il-17 stimulation can increase keratinocytes proliferation, neo-angiogenesis, recruitment, and activation of mast cells, neutrophils, and macrophages, while reducing the expression of adhesion molecules thus favoring the disruption of the skin barrier(16). findings regarding gene expression of il-1b, tnfα, nf-b, and il-17 showed that in mice treated with imq was highly significant expressed. these results are consistent with the previous researches that reported the potent disease-inhibitory effects in experimental models of autoimmune diseases(16).a study by martin stenström(2016) revealed that paquinimod reduces skin fibrosis in tight skin 1 mice, an experimental model of systemic sclerosis they found that paquinimod reduces skin fibrosis in an experimental model of ssc, and this effect correlates with local and systemic effects on the immune system(4).another study by nina franse´n pettersson(2018) showed that the immunomodulatory quinoline-3carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis(18).the antiinflammatory effect for paquinimod revealed a significant reduction in the expression of paquinimod treated group in comparison to the model group, indicating the strong antiinflammatory effect of paquinimod against psoriasis induced by imiquimod and this can be proved by histopathological examination of mice skin section which has been revealed that paquinimod reduced skin inflammation, scaling, thickness, parakeratosis and epidermal infiltration that have been induced by imiquimod. iraqi j pharm sci, vol.32(1) 2023 oral paquinimod administration against imiquimod induced psoriasis 99 conclusion in conclusion, the present study revealed that the treatment of mice with paquinimod have ameliorative effects, that are comparable to those of dexamethasone, against imiquimodinduced psoriasis through reduction in tnfα il17, il-23 level, reducing (pasi, skin thickness, spleen index) and a significant reduction in the gene expression with paquinimod being more effective than dexamethasone .all the findings indicated the strong ameliorative effect of paquinimod and it is a promising intervention for psoriasis treatment, which may explain its anti-inflammatory activity. acknowledgment the data of this article were abstracted from the ph.d. thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the authors are extremely grateful to the college of pharmacy/university of baghdad for supporting this work. references 1. abdulridha sh, kadhim dj, abdul razzak sa. assessment of quality of life in a sample of iraqi patients with psoriasis. iraqi j pharm sci. 2020;29(2):161–8. 2. kamiya k, kishimoto m, sugai j, komine m, ohtsuki m. risk factors for the development of psoriasis. int j mol sci. 2019;20(18):4347. 3. jabeen m, boisgard as, danoy a, kholti n el, salvi jp, boulieu r, et al. advanced characterization of imiquimod‐induced psoriasis‐like mouse model. pharmaceutics. 2020;12(9):1–18. 4. pettersson nf, deronic a, nilsson j, hannibal td, hansen l, schmidt-christensen a, et al. the immunomodulatory quinoline-3carboxamide paquinimod reverses established fibrosis in a novel mouse model for liver fibrosis. plos one. 2018;13(9):1–14. 5. stenström m, nyhlén hc, törngren m, liberg d, sparre b, tuvesson h, et al. paquinimod reduces skin fibrosis in tight skin 1 mice, an experimental model of systemic sclerosis. j dermatol sci. 2016;83(1):52–9. 6. lee ju-, park js, jun ja, kim mk, chang hs, baek dg, et al. erratum: inhibitory effect of paquinimod on a murine model of neutrophilic asthma induced by ovalbumin with complete freund’s adjuvant .canadian respiratory journal. 2021:9 (8896108) . 7. tahvili s, törngren m, holmberg d, leanderson t, ivars f. paquinimod prevents development of diabetes in the non-obese diabetic (nod) mouse. plos one. 2018;13(5):1–17. 8. nair ab, jacob s. a simple practice guide for dose conversion between animals and human. j basic clin pharm. 2016;7(2):27-31. 9. id mb, nerviani a, afflitto gg, pitzalis c. role of the il-23 / il-17 axis in psoriasis and psoriatic arthritis : the clinical importance of its divergence in skin and joints. 2018. 10. wang s, zhu l, xu y, qin z, xu a. salvianolic acid b ameliorates psoriatic changes in imiquimod-induced psoriasis on balb/c mice by inhibiting inflammatory and keratin markers via altering phosphatidylinositol-3kinase/protein kinase b signaling pathway. korean j physiol pharmacol. 2020;24(3):213221. 11. zhang s, liu x, mei l, wang h, fang f. epigallocatechin-3-gallate (egcg) inhibits imiquimod-induced psoriasis-like inflammation in balb / c mice. bmc complement altern med. 2016;16(1):1–11. 12. nakajima k, kanda t, takaishi m, shiga t, miyoshi k, nakajima h, et al. distinct roles of il-23 and il-17 in the development of psoriasis-like lesions in a mouse model. j immunol. 2011;186(7):4481–9. 13. coo luna lg. preparation of tissue. man histol. 1968;(april):1–36. 14. atawodi se, atawodi jc, dzikwi aa. polymerase chain reaction: theory, practice, and application: a review. sahel medical journal. 2011; 13. 15. zainab h. ahmed, munaf h. zalzala, manal a. ibrahim . guggulsterone suppresses ovalbumininduced inflammation in rat asthmatic model. indian journal of forensic medicine & toxicology.2021; 15(1). 16. sahi fm, masood a, danawar na, mekaiel a, malik bh. association between psoriasis and depression: a traditional review. cureus. 2020;12(8):8–13. 17. id mb, nerviani a, afflitto gg, pitzalis c. role of the il-23 / il-17 axis in psoriasis and psoriatic arthritis : the clinical importance of its divergence in skin and joints. 2018; 18. m. stenström, et al., paquinimod reduces skin fibrosis in tight skin 1 mice, an experimental model of systemic sclerosis, j dermatol sci 2016. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 evaluation of new nsubstituted phthalimide derivatives doi: https://doi.org/10.31351/vol29iss1pp247-252 247 synthesis and preliminary biological activity evaluation of new n substituted phthalimide derivatives halah a. sahib*,1 and mohammed h. mohammed* *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract a new series of bases of schiff (h2-h4) derived from phthalic anhydrideweresynthesized. these schiff bases were prepared by the reaction of different amines (tyrosine methyl ester, phenylalanine methyl ester, and isoniazid) with the phthalimide derived aldehyde with the aid of glacial acetic acid or triethylamine ascatalysts. all the synthesized compounds were characterized by (ft-ir and 1hnmr) analyses and were in vitro evaluated for their antimicrobial activity against six various kinds of microorganisms. all the synthesized compounds had been screened for their antimicrobial activity against two gram-positive bacteria “staph. aureus, and bacillus subtilis”, two gram-negative bacteria “escherichia coli, and klebsiella pneumoniae”, and two fungi species “candida tropicalis and candida albicans” using concentrations of 62.5, 125 and 250 µg\mlof derivative in dimethyl sulfoxide(dmso). all the synthesized compounds showed no activity at all against gram-positive bacteria, for gram-negative bacteria and fungi they showed moderate or no activity except compound h1revealedhigh antifungal activityagainstcandida tropicalisat concentrations 125 and 250 µg\ ml. keywords: schiff base, phthalic anhydride, antimicrobial. لمركب النتروجينناتجة من تعويض جزيئي على موقع ذره دوائية جديدةتصنيع وتقييم اولي لمركبات الفثاالمايد *و محمد حسن محمد 1،*هالة عبد الصاحب .*فرع الكيمياء الصيدالنية، كلية الصيدلة، جامعة بغداد، بغداد، العراق الخالصة قواعد بواسطة تفاعل األمينات المختلفة هذه ال. تم تحضير المستمدة من أنهيدريد الفثاليك )4h-2h(شيف تم تصنيع سلسلة جديدة من قواعد راي إيثيل أمين )تيروزين ميثيل إستر ، فينيل أالنين ميثيل إستر ، وإيزونيازيد( مع ألدهيد المشتت من الفثاليميد بمساعدة حمض األسيتيك الجليدي أو ت واستعمال مطياف االشعة تحت الحمراء ومطيافالرنين النووي المغناطيسي جميع المركبات المركبة بواسطة تحليالت ) تشخصتكعامل مساعد. ة ( وتم تقييمها في المختبر لنشاطها المضاد للميكروبات ضد ستة أنواع مختلفة من الكائنات الحية الدقيقة. تم فحص جميع المركبات المركبللبروتون الجرام )المكورات العنقودية الذهبية ، والعصية الرقيقة( ، واثنان من البكتيريا لصبغة لنشاطها المضاد للميكروبات ضد اثنين من البكتيريا إيجابية و 62.5( باستخدام تركيزات المبيضات المكورنة والمبيضات( ، ونوعان من الفطريات )اإلشريكية القولونية ، والكلبسيلة الرئويةسالبة الجرام ) لم تظهر جميع المركبات المركبة أي نشاط على اإلطالق ضد .ثنائي مثيل السلفوكسايد المذيبميكروغرام / مل من مشتق في 250و 125 كشف 1h لجرام والفطريات باستثناء أن المركبلصبغة اسلبية ضد بكتريا أو بال نشاط الجرام ، فقد أظهرت نشاًطا معتدً لصبغة الالبكتيرياإيجابية .ميكروغرام / مل 250و 125رية بتركيزات عن نشاط مضاد للفطريات مرتفع ضد المبيضات المدا .المكروبات، مضاد فثالك انهايدرايد، قواعد شيفكلمات مفتاحية: introduction the development of multidrug microbial resistance is the main challenge that modern scientists have so far been facing in the recent decades. the fact that many pathogenic microorganisms taking charge of numerous human and animal diseases have caused resistance mechanisms to the conventional therapies have encouraged hard work investigations in the fields of natural and synthetic chemistry, to discover new drug classes having many efficient therapeutic profiles(1). phthalimide derivatives have a structural core (–co-n(r)co-) and an imide ring which confer a biological activity to them. phthalimides have wide range uses that include as antiinflammatory agents(2)&(3), antidiabetic(4), antioxidant(5), anticonvulsant(6), hiv-1 reverse transcriptase inhibitor(7), as protective agent(8), as an antimicrobial agent using schiff base principle(9), (10)&(11).the structural feature of the five-membered ring shows they are hydrophobic and this enhances their passage across the biological membranes. 1corresponding author e-mail: halah_sahib@yahoo.com received:8 /10/2019 accepted: 1/2 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp247-252 iraqi j pharm sci, vol.29(1) 2020 248 as a result, the phthalimide subunit has been designed as a hybrid with other molecules to give either synergistic, additive or new biological activity(11,12) .on another hand, it was found that the schiff bases are of great benefit for the synthesis of various bioactive medicinal compounds from the primary amine. they possess antimalarial, anticancer, antimicrobial, antioxidant, anticonvulsant and anti-inflammatory characters(13). schiff bases are the compounds own imine or azomethine (–c=n–) functional group. the nitrogen atom of azomethine may participate in the hydrogen bonding with the active sites of cell components and intervene with normal cell functions(14). amino acids schiff base is of high interest in the research world. numerous scientists developed new strategies due to the special bioactivity of this design. zahraa salim et al(15), have studied the anti-acid phosphatase activity of amino acid schiff base, while yan zhang et al(16), have investigated the increased herring sperm dna intercalating of tryptophan vanillin schiff base. saroji kumar et al(17), in two separate pieces of research work, have proved the beta-lactamase activity of tryptophan and phenylalanine amino acids schiff bases with substituted benzaldehyde. some researchers have linked phthalimide moiety with amino acid either directly proving antimicrobial activity(10)or using a spacer, having no schiff base structure, with antiviral activity as a result(18). similarly, isoniazid schiff bases had been of wide attention in medicinal chemistry. many researchers condensed isoniazid with various aldehydes to produce schiff bases with enhanced anti-tubercular and antimicrobial activities, for example, s. syed tajudeen et al(19), produced schiff base complexes of isoniazid with significantly enhanced antibacterial activity against microbial strains. as well as joseph n. yonge et al(20), prepare disoniazid’s schiff bases by the reaction of isoniazid with pyridine carboxaldehyde which showed good activity agains tbacteria compared to isoniazid alone. no research work linked isoniazid with phthalimide moiety through schiff base has been reported. experiment materials and methods starting material phthalic anhydride and aldehydes were purchased from (riedel de has̈en,germany), tyrosine methyl ester, phenylalanine methyl ester and 4aminobenzaldehyde from (hyperchem,china), isoniazid from (judex,england), acetic acid)bdh,china) ethanol& methanol ) biosolve,netherland), dmso&ch2cl2 (romil,uk) and dmf)thomas baker, india). thin-layer chromatography (tlc) was used to follow up the reaction and to check the purity of synthesized compounds, by using silica gel gf (type 60); pre-coated aluminum sheet from (merck germany), uv-254 lamp was used to visualize the spots, and the elution system used was (methanol: ethyl acetate: n-hexane (0.5: 2: 3)). stuart smp3 melting points apparatus was used to measure the melting points, and were uncorrected. ir spectra were made using ft-ir (ir affinity-1) spectrometer, shimadzu, japan at the university of baghdad college of pharmacy. the 1h-nmr spectra were performed at the college of education for pure sciences (ibn al-haitham), university of baghdad. instrument model: nmready-60 spectrometer, 60 hz by n analysis corp., canada. 1hnmr spectra were obtained on bruker model ultra shield 400 mhz spectrophotometer dmso-d6 used as a solvent for samples measurement; it was performed at central instrumental lab, school of chemistry, college of science, the university of tehran, iran. chemical synthesis synthesis of 4-(1,3-dioxoisoindolin-2yl)benzaldehyde(h1) compoundh1was synthesized by direct mixing equimolar quantities of “phthalic anhydride” with “4-amino benzaldehyde” (16.5 mmol) followed by refluxing in acetic acid 60 ml for 4 hours. after cooling, solid particles were separated out then cold water was added to the resultant mixture and was filtered using buchner, washed with water several times; and dried over silica gel under vacuum. the precipitate was collected by filtration and recrystallized from ethanol to obtain a bright yellow powder(21). yield68%; m.p.202-204oc;rf= 0.8;ir(v, cm-1): 3012: ar.(c-h) str., 2754: ald. (c-h) str.,1782, 1697: asym. &sym.str. of imide (c=o),1720: aldehyde (c=o) str.,1600-1465: ar.(c=c) str.,1080, 717: aromatic in plane& out of plane (c-h) bend.;1hnmr(δ,ppm):10.06 (1h,s, hco), 7.7-8.07 (8h, m, ar-h). synthesis of methyl 2-((4-(1,3-dioxoisoindolin-2yl) benzylidene) amino)-3-(4-hydroxyphenyl) propanoate (h2) a mixture of “l-tyrosine methyl ester” (0.5 g, 2.6 mmol) in 100 ml dichloromethane, compound h1 (0.643 g, 2.55 mmol), and anhydrous magnesium sulfate (0.75 g, 6.25 mmol) was refluxed for 6 hours), schiff base formation is indicated by conversion of solution into a yellow-color. the solution allowed cooling at room temperature, the precipitated particles (magnesium sulfate) were discarded by filtration. the filtrate solvent was evaporated to obtain a yellow-colored product and was recrystallized from ethanol(22). yield =40%; m.p209-210oc; rf = 0.58; ir(v,cm1):3394: phenolic (o-h) str.,3024: ar(c-h) str., 2970& 2873: asym. & sym. aliphatic (ch3) str., 2935 & 2854: asym. & sym. aliphatic (ch2) str.,1782 &1701: asym. & sym. of imide (c=o) iraqi j pharm sci, vol.29(1) 2020 249 str.,1720:(c=o) str. of ester, 1639: (n=c) schiff base str.,16001450: ar-(c=c) str., 1080 & 721: in and out of plane (c-h) bend.; 1h nmr(δ,ppm):9.18 (1h,s,phenolic oh),7.52-8.14 (8h, m, arh),8.16(1h,s,ch=n),6.62 (2h, d)& 6.67 (2h,d) arh of tyrosine part,4.2 (1h, t, ch),3.63 (3h, s, och3),2.88 & 3.14 (2h, 2d, ch2). synthesis of methyl 2-((4-(1,3-dioxoisoindolin-2yl) benzylidene) amino)-3-phenylpropanoate (h3). an ethanolic solution of 2-phenyl alanine methyl ester hydrochloride (2 g, 9.27 mmol, after stirring for half an hour with triethylamine), was added to hot dry ethanolic solution of compound h1 (1.9 g, 7.56 mmol), the mixture refluxed in water bath for 7 hours, a yellow-colored solution indicates schiff base formation. the mixture was concentrated then allowed to cool. the precipitated schiff base was filtered, washed with ethanol several times, and dried in the oven at 60 oc. the solid product was then stored under vacuum(23). yield70%;m.p.207-209 oc;rf= 0.41;ir(v,cm1):3055:ar(c-h) str., 2966 & 2889: asym. & sym. aliphatic (ch3) str., 2943 & 2873: asym. & sym. aliphatic (ch2) str., 1782 &1701: asym. & sym. of imide (c=o) str., 1728: (c=o) str. of ester,1639: (n=c) schiff base str., 16001450: ar-(c=c) str., 1080-721: in and out of plane (c-h) bend.; 1hnmr(δ,ppm):8.14 (1h, s, ch=n), 7.177.90 (13h, m, ar-h), 4.2 (1h, t, ch), 3.6 (3h, s, och3), 3.02& 3.14 (2h, 2d, ch2) synthesis of n'-(4-(1,3-dioxoisoindolin-2-yl) benzylidene) isonicotinohydrazide (h4) a methanolic solution of isoniazid (1.37 g, 10mmol) was added gradually to a hot methanolic solution of compound h1(1.66 g, 10 mmol, after stirring with 2 drops of glacial acetic acid), after few minutes solid particles appeared and refluxing was continued for 1 hour. the pale yellowish solid separated was filtered, washed several times with methanol,dried and stored over silica gel in a desiccated jar. it was recrystallized from dmf/ methanol to give a shiny yellow powder(20). yield 81%; m.p. 344-346oc; rf = 0.25; ir (v, cm-1): 3282: sec.amide (n-h) str., 3059: ar-(c-h) str., 1789 &1701: asym. & sym. of imide (c=o) str., 1666: (c=o) amide str., 1604: imine (c=n) str., 16041512: ar-(c=c) str., 1083 & 717cm-1 in and out of plane (c-h) bend.; 1hnmr (δ, ppm): 12.16 (1h, s,nhco), 7.48-8.80 (12h, m, ar-h),8.5 (1h, s, ch=n). antimicrobial activity the antimicrobial activity was screened using well diffusion methods. two types of gramnegative bacteria “klebsiella pneumoniaand escherichia coli” and two types of gram-positive bacteria “staphylococcus aureus and bacillus subtilis” were used for testing thein-vitro antibacterial activity, and two fungi species “candida tropicalis and candida albicans” for testing the in-vitro antifungal activity isolated from a patient in nahrain teaching hospital, baghdad, iraq. cefotaxime and miconazole used as standards against antibacterial and antifungal respectively, and dmso as a solvent.it was done in “the university of baghdad, college of education for pure sciences ibn al-haitham, central service laboratory”. results and discussion chemistry the pathway for the synthesis of the targeted schiff bases(h2-h4) is depicted in scheme 1. scheme 1. general synthetic pathway of the titled compounds. the synthetic pathway started by the preparation of aldehyde of phthalimide compound (h1) through imide bond formation using glacial acetic acid as solvent and catalyst. this method showed good yield with ease of production. the schiff baseswereprepared by reaction of compound (h1) with different amines (tyrosine methyl ester, phenylalanine methyl ester and isoniazid with the addition of few drops of “glacial acetic acid or triethylamine”. the reaction involves elimination of one water molecule in order to form a carbonnitrogen double bond (imine or schiff base). all the synthesized compounds were characterized and their structures were confirmed by “ftir and 1hnmr” spectral analyses. the ir spectra for the parent nucleus (h1)demonstrated characteristic two absorption bands of asymmetrical& symmetrical imide (c=o) stretching displayed at (1782&1697) cm-1. in iraqi j pharm sci, vol.29(1) 2020 250 addition, 1720 and 2754 cm-1 are accounted for (c=o)&(ch) stretching of aldehyde respectively. for compounds (h2) and (h3), the disappearance of the characteristic aldehyde band and appearance of the band at 1639 cm-1 is a good indication for schiff base formation i.e. linkage has occurred between the phthalimide core and (tyrosine or phenylalanine methyl ester moiety). compound )h2( has an obvious band at 3394 cm-1 is attributed to phenolic oh group stretching. while compound (h4) shows three important bands one is at 3282 cm-1 for secondary nh stretching and the second is at 1666 for amidic carbonyl stretching, and 1604 cm-1 for imine band stretching. the 1hnmr spectra of the synthesized compounds were consistent with the assigned structures. compound(h1) showed characteristic signals recorded at δ=10.06due to cho of the aldehyde, besides, the aromatic protons displayed at their expected region as a multiplate at δ=7.78.07 ppm. compound (h2) displayed ch=n peak as a singlet at δ=8.16, while compounds (h3) imine ch=n peak appeared at δ=8.14 ppm.a peak of singlet at δ=9.18 ppm is due to the phenolic group in compound (h2). two doublet peak in the region of δ=3.01 ppm is attributed to the ch2 group of the amino acid part for (h2) and 3.08 is due to (ch2) of (h3). finally, a sharp singlet peak integrated for three protons at δ=3.6 ppm is due to the och3 group of both compounds. compound (h4) showed characteristic peaks at δ=12.1 ppm, attributed to nhco, also singlet peak at δ= 8.5 ppm is due to ch=n. antibacterial activity tables1 and 2 show the antimicrobial activity of the synthesized compounds at concentrations of (62.5, 125 and/or 250 µg\ml). table 1. antibacterial activity of the tested compounds table 2. antifungal activity of the tested compounds (-) = no activityslightly active (zone of inhibition between 5-10 mm), moderately active (zone of inhibition between 10-20 mm), highly active (zone of inhibition more than 20 mm).(24) compound name conc. µg/ml s. aureus b. subtilis e. coli k. pneumoniae zone of inhibition(mm) h1 62.5 11 125 12 250 12 h2 62.5 11 10 125 12 10 250 h3 62.5 125 250 10 h4 62.5 125 250 10 12 cefotaxime 100 49 43 51 25 dmso compound name conc. µg/ml candida tropicalis candida albicans zone of inhibition(mm) h1 62.5 18 125 24 250 22 h2 62.5 14 125 15 250 h3 62.5 14 125 250 h4 62.5 12 125 13 250 miconazole 100 15 23 dmso iraqi j pharm sci, vol.29(1) 2020 251 the data illustrated in tables1 and 2 reveals that all the synthesized compounds had been screened for their antimicrobial activity against two gram-positive bacteria “staph. aureus, and bacillus subtilis”, two gram-negative bacteria “escherichia coli, and klebsiella pneumoniae”, and two fungi species “candida tropicalis and candida albicans” using concentrations of 62.5, 125 and 250 µg\ml of derivatives in dmso. for antibacterial activity:moderate activity against g-negative “escherichia coli, and klebsiella pneumoniae”appeared at concentrations(62.5 and 125 µg\ml) and250 µg\ml for compounds (h2)and (h4), respectively. compounds (h1andh3) were moderately active against only e. coli at concentrations (62.5, 125 and 250 µg\ml) and250 µg\ml, respectively. however, all of them exhibited no activity against gram-positive “staph. aureus, and bacillus subtilis”. the zero mm inhibition zone at concentration250µg\ml while containing inhibitory zone at lower zones at concentrations62.5 and 125 µg\ml for compound(h2) may be due to at high antimicrobial concentration a small number of bacterial resistant mutants can provide the protection to others by producing signaling molecule to turn on the drug effluxpumps which are transport proteins involved in the extrusion of toxic and antibiotic substrates from within cells into the external environment of bacteria, enhancing the survival capacity of the overall population. (25) on the other hand, the antifungal activity against candida tropicalis and candida albicans showed that compound (h3) has only moderate activity against candida albicans at a concentration of 62.5 µg\ml. compounds (h2andh4) were only moderately active against candida tropicalisat concentrations 62.5 and 125µg\ml. finally, the best activity among all the derivatives was obtained from compound (h1) which proved high activity against candida tropicalis at concentrations (125, 250 µg\ml). conclusion new schiff bases derivatives containing phthalimide core were successfully synthesized and their structures were characterized by “ir and 1hnmr spectral” methods. all the synthesized compounds showed no activity at all against grampositive bacteria, for gram-negative bacteria and fungi they showed moderate or no activity except compound (h1) revealed high antifungal activity against candida tropicalisat concentrations 125 and 250 µg\ml. acknowledgment we are grateful to the “college of pharmacy/ university of baghdad” for all the efforts in performing the research. references 1. tsemeugne j, fondjo es, tamokou j, rohand t, et al. activity of a novel trisazo dye from 3-amino-4h-thieno [ 3, 4-c ][ 1 ] benzopyran4-one. int j med chem. 2018;2(18):1–8. 2. bach d, liu j, kyung w, et al. bioorganic & medicinal chemistry synthesis and biological activity of new phthalimides as potential antiinflammatory agents. bioorg med chem [internet]. 2017;25(13):3396–3405. available from: http: // dx .doi .org /10. 1016/j.bmc.2017.04.027 3. 3. lima lm, castro p, machado al, et al. synthesis and anti-inflammatory activity of phthalimide derivatives, designed as new thalidomide analogues. bioorg med chem. 2002;10(9):3067–3073. 4. jawed ham, mohammed mh, ismeal sh, et al. synthesis, characterization and alpha glucosidase inhibition activity of new phthalimide derivatives. iraqi j pharm sci. 2018;27(1):100–108. 5. nayab ps. new phthalimide ‐ appended schiff bases : studies of dna binding, molecular docking, and antioxidant activities. luminescence. 2016;32(5):1–10. 6. abdel-hafez aa. synthesis and anticonvulsant evaluation of n-substituted isoindolinedione derivatives. arch pharm res. 2014;27(5):495–501. 7. vajragupta o, boonchoong p. new leads of hiv1 integrase inhibitors new leads of hiv-1 integrase inhibitors. mahidol univ j pharm sci. 2016;29(3–4):1–10. 8. behalo ms. facile synthesis of novel amino acids derivatives as potential antibacterial agents using sustainable materials. j chinese chem soc. 2017;64(10):1–9. 9. al-azzawi am, shawqi m, razzak a. synthesis, characterization and antibacterial activity of several new schiff bases linked to phthalimide moiety. kerbala j pharm sci number. 2011;2:124–133. 10. maheswara cuma, jayakar rb, srinivasan r. international journal of pharma and bio sciences synthesis and antimicrobial activity of α n-phthilimido and acetimido derivatives from amino acids and anhydrides. int j pharma bio sci. 2010;1(4):81–86. 11. lamie pf, philoppes jn, el-gendy ao, et al. design, synthesis and evaluation of novel phthalimide derivatives as in vitro antimicrobial, anti-oxidant and antiinflammatory agents. molecules. 2015;20(9):16620–16642. 12. bosquesi pl, regina t, melo f, et al. antiinflammatory drug design using a molecular hybridization approach. pharmaceuticals. 2011;4(11):1450–1474. iraqi j pharm sci, vol.29(1) 2020 252 13. muhi-eldeen za, al-kaissi e, suaifan gary, et al.synthesis and biological screening of aminoacetylenic tetrahydrophthalimide analogues as novel cyclooxygenase. 2017;9(2):160-165. 14. kajal a, bala s, kamboj s, sharma n, saini v. schiff bases : a versatile pharmacophore. j catal. 2013;2(13);1–14. 15. al-garawi zsm, tomi ihr, al-daraji alihr. synthesis and characterization of new amino acid-schiff bases and studies their effects on the activity of acp, pap and npa enzymes ( in vitro ). j chem. 2012;9(2):962–269. 16. zhang yan, wang x, ding l. interaction between tryptophan-vanillin schiff base and herring sperm dna. j serb chem soc. 2010;75(9):1191–1201. 17. sah sk, rasool u, shanthi b, et al. antibacterial potential of l-tryptophan on extended-spectrum betalactamase producing escherichia coli strains : a pathological promising approach. asian j pharm clin res. 2018;11(7): 368-374. 18. al-masoudi na, abood e, al-maliki zt,et al. amino acid derivatives. part 6. synthesis, in vitro antiviral activity and molecular docking study of new n α -amino acid derivatives conjugated spacer phthalimide backbone. med chem res. 2016;25(11):2578–2588. 19. tajudeen ss, kannappan g. indian journal of advances in chemical science schiff base – copper ( ii ) complexes : synthesis, spectral studies and anti-tubercular and antimicrobial activity. indian j adv chem sci. 2016;4(1):40– 48. 20. yong jn, mainsah en, ntum sj, et al. synthesis, characterization and antibacterial studies of some isoniazid-derived schiff bases. international research journal of pure and applied chemistry. 2016; 12(1): 1-8. 21. lima lm, castro p, machado al, et al. synthesis and anti-inflammatory activity of phthalimide derivatives, designed as new thalidomide analogues. bioorg med chem. 2002;10:3067–3073. 22. albert j, crespo m, granell j, rodríguez j, et al. cyclopalladation of schiff bases from methyl esters of α-amino acids. unexpected activation of the o-me bond with formation of a bianionic tridentate metallacycle. organometallics. 2010;29(1):214–225. 23. yorur-goreci c, demir z, altas n. green synthesis of new amino acid schiff bases and their biological activities. turkish chem soc. 2016;3(3):15–26. 24. dabholkar v v, gavande rp. synthesis and antimicrobial activities of novel 1, 4benzothiazine derivatives. arab j chem [internet]. 2016;9(s1):s225–s229. available from: http://dx.doi.org/10.1016/j.arabjc.2011.03.009. 25. li j, xie s, ahmed s, et al. antimicrobial activity and resistance: influencing factors. front. pharmacol. 2017; 13(8):1-11. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ dedication iraqi j pharm sci, vol.21(1) 2012 oxidative stress in prostate cancer 56 changes in serum levels of tumor necrosis factor–alpha and antioxidant status in different stages of malignant prostate cancer patients in iraq ebtehal s. mohammed * , wafa f. al-taie * , intesar t. numan ** and saad a. hussain **,1 * department of chemistry, ibn al-haitham college of education, university of baghdad,baghdad,iraq ** department of pharmacology and toxicology, college of pharmacy, university of baghdad,baghdad,iraq abstract chronic inflammation can induce proliferative events and posttranslational dna modifications in prostate tissue through oxidative stress. the present study was designed to evaluate the changes in serum levels of tnf-α, malomdialdehyde (mda) and total antioxidant status (tas) patients with different stages of malignant prostatic cancer (pca) and benign prostatic hyperplasia (bph). one hundred males (age range of 58-72 years) with different stages of malignant pca were recruited from the radiotherapy and nuclear medicine teaching hospital in baghdad during the period from september 2010 to april 2011. the patients were categorized according to the 4 disease stages (i, ii, iii, and iv); 25 patients with benign prostatic hyperplasia (bph) and 25 normal healthy subjects were considered as comparator groups. blood samples were taken from all subjects for analysis of tnf-α tas and mda levels. the results showed significant differences between the four stages of pca patients in all parameters; however, highly significant difference was observed in stage iv compared to control and bph patients. in conclusion, tnf-α and total antioxidant status could be utilized for marking the advanced stages of malignant pca. key words: malignant prostate cancer, inflammation, tnf-α, oxidative stress األكسذة فٍ مصم انذو نذي مرضً سرطان وحانت مضاداث tnf.αانتغييراث فٍ مستىي انبروستاث انخبيث انعراقييه بمختهف مراحهه و سعذ عبذ انرحمه حسيه** انطائٍ*، اوتصار طارق وعمان** فاضممحمذ * ، وفاء صبرٌ إبتهال .خايعت بغذاد ،بغذاد ،انعزاق انٓيثى ، ابٍ* قسى انكيًياء ، كهيت انخزبيت .ٔانسًٕو ،كهيت انصيذنت ، خايعت بغذاد ،بغذاد ،انعزاق ٔيتاألد** فزع الخالصة انًشيُت دٔر فعال في حطٕر ًَٕ ٔحخصص األٔراو انسزطاَيت يٍ خالل سيادة حكٕيٍ إَٔاع يخخهفت يٍ اندذٔر نالنخٓاباث األكسذةانَٕذايانذيٓايذ، انحانت انكهيت نًضاداث انفا، انً-عايم انخُخز انسزطاَيِ انذراست نخقييى انخغيزاث في يسخٕٖ ذانحزة. حى حصًيى ْ بيٍ أاضح اانُخائح اخخالف أظٓزثخالل انًزاحم انًزضيت انًخخهفت نًزضٗ سزطاٌ انبزٔسخاث انخبيث. األكسذةٔيعايم حانت حا في يسخٕٖ انًزحهت انزابعت يٍ انًزض فزقا يعُٕيا ٔاض أظٓزثحال أيتنًزضٗ سزطاٌ انبزٔسخاث ٔعهٗ األربعتانًزاحم اسخخذاو ْذِ انًعاييز بإيكاَيت االسخُخاجانًعاييز انخي حًج دراسخٓا يقارَت بًداييع انسيطزة ٔيداييع حُسح انبزٔسخاث انحًيذ. ٔيًكٍ كعٕايم يضافت في ححذيذ يسخٕٖ حطٕر انًزض ٔيخابعت عالخّ. رط االكسذة .، ف tnf.αانكهماث انمفتاحيت : سرطان انبروستاث انخبيث ،االنتهاب ، introduction chronic inflammation can induce proliferative events and posttranslational dna modifications in prostate tissue through oxidative stress. in fact, repeated tissue damage and oxidative stress related to this event may provoke a compensatory cellular proliferation with the risk of hyper-plastic growth or neoplastic modifications (1) . it is well accepted that regions of prostatic inflammation can generate free radicals and many reactive species of oxygen. in particular, macrophages and neutrophil infiltrations provide a source of free radicals that can induce hyper-plastic or precancerous transformations through the oxidative stress to the tissue and dna (2) . cytokines can contribute to cancer development in several ways. tnf-α has been shown to enhance the formation of reactive oxygen species (ros) by inflammatory cells, and thereby increase the risk for dna damage and inhibition of dna repair in tumor cells (3) ; other mechanisms include direct stimulation of cell growth, induction of angiogenesis, and recruitment of inflammatory neutrophils (4) . 1 corresponding author email : saad_alzaidi@yahoo.com received : 8/10/2011 accepted : 28/2/2012 iraqi j pharm sci, vol.21(1) 2012 oxidative stress in prostate cancer 57 in addition, an imbalance of proand antiinflammatory cytokines, caused by point mutations or polymorphisms, may prevent the normal self-limiting nature of the immune response, leading to prolonged inflammation with chronic exposure to cytotoxic mediators (5) . in prostate cancer (pca), many studies have related serum levels of tnf-α to the development of the disease, associating the serum levels of tnf-α, the disease development and presence of metastasis (6) . the expression and action of tnf-α and its receptors have been reported in several tumors such as esophageal (7) , follicular thyroid (8) , skin (9) , ovarian (10) and breast cancers (11) . while increased ros generation has traditionally been associated with tissue injury or dna damage, new and exciting information points to an essential role for its increased generation in several cellular processes associated with neoplastic transformation and aberrant growth and proliferation (12) . thus, excessive production of ros or inadequacy in a normal cell’s antioxidant defense system (or both) can cause the cell to experience ros oxidative stress and the increased may play a broader role in cellular processes associated with initiation and development of many cancers including pca. the present study was designed to identify the expected correlation between tnf-alpha levels and oxidative stress markers in different stages of malignant pca and bph patients and reveal their possible importance as future diagnostic markers for stage classification. patients and methods after screening 132 patients, a prospective case control study was carried on 100 patients with malignant prostate cancer (pca) at the radiotherapy and nuclear medicine teaching hospital in baghdad. their age range was 58-72 years, and all of them are newly diagnosed with the disease. after careful clinical, biochemical and histopathological evaluation by oncology specialists, they were classified into four groups according to the clinical stage of the disease (stages i, ii, iii and iv) and each class include 25 patients. other 25 patients, with the same age ranges, diagnosed for benign prostatic hyperplasia, were included and served as comparator with bph. twenty five healthy subjects, with the same age ranges, were selected and served as control group for comparison of the studied parameters, at the department of urology/baquba teaching hospital. from all subjects, blood samples were collected by vein puncture. tnf-α enzyme linked immunosorbent assay (elisa) was utilized depending on a technique called quantitative sandwich immunoassay (13) . total antioxidant status (tas) was determined by using a kit supplied by randox (14) . malondialdehyde (mda) was determined by using a kit supplied by northwest (15) . the data were statistically evaluated using unpaired student's t-test and multiple-way anova; p values less than 0.05 were considered statistically significant. results table 1 indicated that tnf-α levels in all stages of malignant prostate patients were significantly higher compared to control (485.88%, 1152.04%, 2469.73%, and 3640.16%, respectively, p<0.05), and (126.67%, 384.39%, 894.19% and 1347% respectively, p<0.05) compared to bph patients; significant differences were observed between the four stages of pca patients; however, highly significant difference was shown in stage iv of malignant pca patients compared to control and bph patients (p<0.05) (figure 1). table 1: serum levels of tnf-α in healthy subjects, bph patients and patients with different stages of malignant prostate cancer. patients groups serum tnf-α pg/ml healthy subjects 28.61±8.9 a bph patients 73.95±16.3 b stage i p. cancer 167.62±38.5 c stage ii p. cancer 358.21±96.5 d stage iii p. cancer 735.2±126.0 e stage iv p. cancer 1070.1±250.6 f values represent mean ±sd; values with nonidentical superscripts (a,b,c,d,e,f) indicated significant differences between groups (p<0.05). figure 1: % differences in tnf-α in pca patients with different stages of the disease compared to bph patients and controls. iraqi j pharm sci, vol.21(1) 2012 oxidative stress in prostate cancer 58 table 2: serum levels of mda, total antioxidant status (tas) and the oxidative stress index (osi) in healthy subjects, bph patients and patients with different stages of malignant prostate cancer. patients groups serum mda (μmol/l) serum tas (mmol/l) osi healthy subjects 2.44±0.6 a 1.84±0.2 a 0.14±0.04 a bph patients 6.21±1.7 b 1.49±0.1 b 0.41±0.13 b stage i pca 10.49±0.8 c 1.28±0.1 c 0.82±0.1 c stage ii pca 13.61±2.6 d 1.15±0.1 d 1.19±0.24 d stage iii pca 17.49±2.4 e 1.09±0.2 e 1.68±0.5 e stage iv pca 20.64±3.1 f 0.81±0.25 f 2.93±1.4 f values represent mean ±sd; values with non-identical superscripts (a,b,c,d,e,f) indicated significant differences between groups (p<0.05). table 2 clearly demonstrated that mda levels in all stages of pca patients were significantly higher compared to control (329.92%, 457.79%, 616.8%, and 745.9% respectively), and 68.92%, 119.16%, 181.64%, and 232.37%, respectively compared to bph patients, and significant differences were observed between the four stages of pca; however, highly significant difference was observed in stage iv of malignant pca patients compared to control and bph patients (p<0.05) (figure 2). table 2 also indicated that tas levels in all stages were significantly lower compared to control (30.43%, -37.5%, -40.76%, and -55.98%, respectively) and -13.5%, -22.3%, -26.35% and 45.27%, respectively compared to bph patients, and there is significant difference reported between the four stages of pca; however, lower significant difference was observed in stage iv of malignant pca patients compared to control and bph patients (p<0.05) (figure 3). figure 2: % differences in serum mda levels in pca patients with different stages of the disease compared to bph patients and controls. moreover, oxidative stress index (osi) values in all stages were significantly higher compared to control (485.71%, 750%, 1100%, and 1992.86%, respectively), and 100%, 190.24%, 309.76%, and 614-63%, respectively compared to bph patients; significant differences were observed between the four stages of pca; however, highly significant difference was observed in stage iv of malignant pca patients compared to control and bph patients (p<0.05) (figure 4). figure 3: % differences in serum tas levels in pca patients with different stages of the disease compared to bph patients and controls. figure 4: % differences in osi value in pca patients with different stages of the disease compared to bph patients and controls. iraqi j pharm sci, vol.21(1) 2012 oxidative stress in prostate cancer 59 discussion chronic inflammation orchestrates a tumor supporting microenvironment that is an indispensable participant in the neoplastic process. in the present study, we reported significantly elevated serum levels of tnf-α in patients with pca compared with control and bph, and there was a strong association between the serum levels of the tnf-α, disease stage and the presence of metastatic disease (table 1, figure 1). these results were compatible with those observed by others (11,16) . this clearly indicates that the role of tnf-α has been linked to all steps involved in tumorigensis, including cellular transformation , promotion, survival, proliferation, invasion, angiogenesis, and metastasis. prostate cancer is mostly a disease of elderly men. the progressive inherent or acquired changes in cellular metabolism occurring with aging may play an important role in the development of this disease. reactive oxygen species (ros) generated either endogenously or from external sources, due to decrease in intracellular ros scavenging system plays a vital role in regulating several biological phenomena (12) . the involvement of oxidative stress as an early event in pca development was suggested by miyake et al. (17) who showed that androgen suppression is capable of decreasing oxidative stress. polybarchou et al. reported that over production of h2o2 plays a major role in androgen-independent cell proliferation and migration of lncap cells (18) . thus in the present study, we have demonstrated the status of lipid peroxides and antioxidants in plasma of pca patients in comparison with normal and bph subjects; this result indicates that mda in stage iv of pca patients was significantly higher compared to other groups (table 2, figure 2), which means overproduction of free radicals by the inflammatory processes of pca cause potential oxidative injury to erythrocytes and erythrocyte membranes and damage their antioxidant defense systems. similar reports of higher mda levels in pca were observed by others (19,20) . in contrast to our finding, no significant change in lipid peroxidation in patients with pca was reported by dogruabba soglus (21) . the data presented in table 2 also showed that tas was significantly lower in patients with carcinoma of prostate when compared to controls and bph; however, when tas levels in all stages were compared significantly lower values observed in stage iv (figure 3); the lower tas levels may be due to increased turnover of tas for preventing oxidative damage in those patients. this finding was compatible with those reported by ealon et al. (22) . under certain conditions the increases in oxidants and decrease in antioxidants cannot be prevented and the oxidative/antioxidative balance shifts towards the oxidative status, accordingly, table 2 and figure 4 clearly showed significant elevation in osi values in stage iv pca patients compared to other groups. in conclusion, tnf-α and total antioxidant status could be utilized for marking the advanced stages of malignant pca. acknowledgment authors thank university of baghdad for support, and the staff of nuclear medicine teaching hospital in baghdad and baquba teaching hospital for their assistance. references 1. palapattu gs, sutcliffe s, bastian pj, platz ea, et al. prostate carcinogenesis and inflammation: emerging insights. carcinogenesis 2005; 26:1170-1181. 2. sciarra a, di silverio f, salciccia s, autran gomez am, et al. inflammation and chronic prostatic diseases: evidence for a link? eur urol 2007; 52:964-972. 3. de miguel mp, roguela m, bethencourt fr, et al. 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280(49):4042840435. 19. ayding a, sara a, zorica f, soyal a, et al. oxidative stress and antiioxidant status in non-metastatic pca and bph. clin biochem 2006; 39:176-179. 20. surapaneni km, venkata gr. lipid peroxidation and antioxidant status in patients with carcinoma of prostate. indian j physiol pharmacol 2006; 50(4):350-354. 21. dagru-abba soglu sj, aykac-toker g, kocak t, unluer e, uysal m. antioxidant enzyme activities and lipid peroxides in the plasma of patients with bph or pca are not predictive. j cancer res clin oncol 1999; 125(7):402-404. 22. ealon jw. calatases and peroxidases and glutathione and hydrogen peroxide: mysteries of the bestiary. j lab clin med 1991; 18:3-4. http://www.ncbi.nlm.nih.gov/pubmed?term=%22jentzsch%20am%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22bachmann%20h%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22f%c3%bcrst%20p%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22biesalski%20hk%22%5bauthor%5d iraqi j pharm sci, vol.32( 1 ) 2023 slow release organogel doi: https://doi.org/10.31351/vol32iss1pp31-39 31 formulation and assessment of delayed/slow-release diclofenac sodium edible organogel utilizing low molecular weight organogelators zahraa yahya aziz*, masar basim mohsin mohamed *,1 and marwa hazim jasim* * department of pharmaceutics, college of pharmacy, university of al-mustansiriyah, baghdad, iraq abstract organogel as a delayed and slow drug release dosage form was prepared and characterized various gelators, including 12hsa (12-hydroxystearic acid), span 60. span 40 were used; castor oil (co) and anise oil (ao) were also used as a liquid phase. to achieve the aim of this work, diclofenac sodium (ds) was used as a model drug. organogels specifications were characterized by estimating thermal attitude using tabletop rheology and differential scanning calorimetry (dsc). the organogel strength study was characterized by applying oscillatory rheology tests the amplitude sweep and the frequency sweep. the morphology of the organogel was observed using the optical microscope. co and ao binding capacity was also measured. the transition temperatures for all organogels were reversible. imaging demonstrated the existence of spherulites aggregates in organogels prepared using 12hsa and span 40 in co and ao. however, span 60 containing organogels in both oils existed as fibers aggregates. furthermore, 20 wt% 12hsa organogels exhibited viscoelastic characteristics with a linear frequency-independent elastic modulus (or g’ and g’’). the results revealed that the hpmc (hydroxyl propyl methyl cellulose) capsule containing the organogel resisted the dissolution in the acidic media for two hours. moreover, organogels slowed the release of ds for 24 hours in an alkaline medium. finally, all the selected organogel in co exhibited a high oil binding capacity. keywords: 12hsa, span 60 , span 40, sodium diclofenac, slow release-organogel. سبان ، هيدروكسي ستيارك اسد 12تصميم وتقييم الهالم العضوي البطئ والمتأخر التحرر باستخدام كعناصر مكونة للهالم ذات وزن جزيئي منخفض في زيوت صالحة لألكل باستخدام 40وسبان 60 دواء الدكلوفيناك صوديوم *مروة حازم جاسم و * ، مسار باسم محسن محمد1*، زهراء يحيى عزيز بغداد، العراق ، فرع الصيدالنيات، الجامعة المستنصرية، كلية الصيدلة* الخالصة هيدروكسي ستيارك اسد 12تم استخدام .الهالم العضوي كنظام لتقدير أهليته على تأخير وابطاء تحرر الدواء في االثنى عشريتقييم لتحقيق اهداف هذا العمل، تم استعمال .مكونة للهالم وزيت الخروع وزيت اليانسون كطور سائل للهالم العضوي كعناصر 40 وسبان 60سبان ، .تم تحديد مواصفات الهالم العضوي بتقييم سلوكه الحراري باستخدام علم السيالن المنضدي والمسح التفاضلي المسعر .ديومدواء الدكلوفيناك صو تم التحقق من شكل الهالم العضوي باستخدام .ايضا تم استخدام اختبارات علم السيالن التذبذبي التي تتضمن اختبار مدى السعة واختبار مدى التواتر قد بينت النتائج أن كبسولة هيدروكسي بروبيل مثيل سيليولوز .اضافة الى ذلك تم ايضاح قدرة ربط زيت الخروع وزيت اليانسون .صريالمجهر الب التصوير بالمجهر اظهر تجمعات .درجات الحراة االنتقالية للهالمات العضوية كانت قابلة لالنعكاس .قاومت التحلل بالوسط الحامضي لمدة ساعتين 60اما بالنسبة للهالمات العضوية الحاوية سبان .في زيت الخروع 40هيدروكسي ستيارك اسد وسبان 12للهالمات العضوية المحتوية على كروية % وزن 20اضافة الى ذلك، فقد امتلكت بعض الهالمات العضوية صفات لزوجة مطاطية كما تبين في .فقد اظهرت تجمعات خيطية في كال الزيتين .ساعة بالوسط القاعدي 24الهالمات العضوية ابطأت تحرر الدواء لمدة .ستيارك اسد في كال الزيتين كان غير معتمد على التردد روكسيهيد 12من .من ناحية اخرى، كل الهالمات العضوية المختارة بزيت الخروع اظهرت قوة ربط مرتفعة بالزيت هالم عضوي -تحرر بطئ -صوديوم دكلوفيناك -40سبان -60سبان -هيدروكسي ستيارك اسد 12الكلمات المفتاحية: introduction organogels have obtained the interest of many researchers for their simple and inexpensive preparation. the organogel is neither non-crystalline nor glassy, consisting of an organic phase that is liquid engaged in a three-dimension tangled network of small molecular weight gelators (such as 12hsa, span 60, and span 40) or polymeric gelators (such as poloxamer and carbopol). physical or chemical interactions occur amongst gelators, leading to fibrous self-assembled that engage with each other to cause the structure of the three dimensionalnetwork(1). commonly seen gelators are sorbitan monostearate,lecithin and cholesteryl, 12hsa, anthraquinone derivates, and sterol. organogel usages are various, including pharmaceuticals, chemistry, biotechnologies, cosmetics, and food technology(2). numerous organogel formulations were prepared to deliver the drugs by various administration routes. in a previous study, 12hsa has been used to prepare organogels with soyabean oil to deliver ibuprofen. the release rates showed a reduction in the organogels release rate with the rise in the gelator quantity(3). 1corresponding author e-mail: zahraayahyaaziz@yahoo.com received: 28/11 / 2021 accepted: 22/2 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp31-39 iraqi j pharm sci, vol.32( 1 ) 2023 slow release organogel 32 cyclosporine a offered good effectiveness when taken orally in organogel of span 60(4). span 40 was formulated with various oils such as mustard oil and groundnut oil as organogels solid content was 20 wt% and used for the controlled delivery of metronidazole(5). organogel was also formulated to achieve gastroretentive characteristics using span 60, span 40, and stearic acid as gelators with olive and sesame oil to obtain a floating gastric system(6, 7). this study aimed to investigate the organogel for the slow and delayed-release using the gelators (12hydroxystearic acid (12hsa), span60, span 40) with oils (castor oil (co) and anise oil (ao)). the drug model was diclofenac sodium (ds) as the delayed-release in the duodenum representing the site of absorption could be acquired using an hpmc acid-resistant enteric capsule to overcome ds gastric irritation. to our knowledge, the selected gelators and oils in this work were not formulated previously together except span 40, which was priorly gelled with co and loaded with metronidazole to achieve controlled drug delivery for topical use(8). materials and methods materials ds was a kind gift from safa pharma. 12hsa(purity 90%), span 60, and span 40 were purchased from hangzhou hyper chemicals china and sinopharm chemical reagent co., ltd, respectively. also, co and ao were bought from rs spain and hemani herbal, respectively. methods formulation of organogel in the beginning, the drug unloaded organogel was prepared by weighing out the particular quantity of each (span 40, span 60, and 12hsa) in the vials, then completed to 1 gm with co and ao for each gelator as in the following concentrations (0.5, 1, 3, 5, 7, 10, 13, 15, 17, 20) wt% respectively (as clarified in table 1). the vials incubation in the water bath was for 40 min at 85°c until a clear solution was obtained to confirm the solubility of gelators in oils. the vials were then brought out from the water bath and allowed to cool at room temperature (these gelators mechanisms of action are upon cooling make aggregates of fibers that constitute the three-dimension scaffold). after a period, an overturn to the vials was to examine the formulation of the organogel. a solid organogel is obtained when there is no flow in the preparation, but if flow occurs upon overturning, the result will be liquid formulation. finally, ds-loaded organogel was formulated as the above procedure. the 75mg of ds was weighed with the chosen quantity of the gelators and oil added to complete the weight to 1 gm, then solubilization of the contents in oils occurred at 85 ºc (9). table 1 . organogel contents concentrations oil concentration (wt%) gelator concentration (wt%) 99.5 0.5 99 1 97 3 95 5 93 7 90 10 87 13 85 15 83 17 80 20 tabletop rheology organogel vials were incubated in the water bath at 85 ºc. the reduction in the temperature then was allowed to be 2 ºc every 15 minutes until 32 ºc reached. the vials were tilted 45°c at the termination of each 15 minutes to check whether the organogel formulation was sol or gel. the transition temperatures from sol to gel for all organogels were revealed at this stage of the work. this portion of the study was pursued by adverse phase via raising the heating proportion (2°c/ 15 minutes) to 85°c for all organogels. the transition temperatures from gel to sol will be recorded. for each organogel, this procedure was performed in triplicate (10, 11). the tabletop rheology test was done using the water bath by applying different temperatures from 85 ºc to 32 ºc as rheology in this test means the transfer of organogel status from sol to gel. differential scanning colorimetry (dsc) thermal analysis of the organogel was performed with a setaram dsc evo 131 differential scanning calorimeter (china). a weighing 10 mg of organogel and the gelators was in an aluminum pan and tightly sealed. the organogel was heated from room temperature to 110°c at 10°c/min (12). optical microscopy the morphology of the organogels was examined by utilizing an optical microscope and slides to pick up the microscopic image. a drop of melting organogel withdrawn by micropipette, directly after taking the vial out of the water bath, added on the glass slide. the liquid organogel was pressed softly using a glass coverslip placed over the drop for 15 minutes at 85ºc. then the slide was transmitted into the microscope stage after cooling. utilizing the software micro and the digital microscope camera mc500 to capture the images as the magnification was x40(13). iraqi j pharm sci, vol.32( 1 ) 2023 slow release organogel 33 oscillatory rheology study the rheology was carried out utilizing anton par mcr 302 rheometer using plate-plate configuration (pp 25/ sn 61895). all measurements were in triplicate at 37 ºc, and the acquired data estimation was by rheoplus software(10). these tests were carried out at the university of petra/pharmaceutical center, amman, jordan, for ds-loaded organogel. the prepared organogel as shown in the organogel formulation section was scooped and placed between the two plates of (pp 25/ sn 61895). amplitude sweep the amplitude sweep test was performed to determine storage modulus (g'), loss modulus (g''), flow point for each formulation, and the linear viscoelastic region (lver). this study executed the oscillatory strain range from 0% to 100% at angular frequency 10 rad s-1. frequency sweep the second oscillatory test that followed the amplitude sweep was the frequency sweep. picking the applied strain on the organogel was from the range of lver data of the amplitude sweep test for each organogel (0.01-0.08 %). then, different speeds were applied to the organogel sample via the angular frequency that transformed from 0.1 to 100 rad s-1. in-vitro release study the ds organogel-loaded hpmc capsule was first subjected to in vitro release in hcl solution ph 1.2 for 2 hours to examine the resistance of these capsules to the acidic media. then, ds organogels release was for 24 hours to test the organogel slow release in sodium phosphate buffer (ph 6.8 containing 0.1 % w/v sodium lauryl sulfate) using usp apparatus the paddle type; where their jars were filled till 900 ml of hcl solution or alkaline media, that adjusted at 37±0.5°c and 75 rpm. the hpmc capsule that loaded with the organogel was put into the jars of the dissolution apparatus then the samples were withdrawn as the following time setting (0,1, 0,5, 1, and 2) hours for acidic media. subsequently, the release of organogel in the alkaline media and the sample withdrawing was within this time frame (0.083, 0.25, 0.5, 1, 3, 6, 9, 12, 15, 18, 21, and 24 hours)(7, 14). a withdrawal of 10 ml each time was replaced with a similar quantity of the media. after that, the withdrawn samples were filtered by utilizing a millipore filter paper membrane (0.45 μm). after filtration, these samples were diluted and measured by a uv-visible spectrophotometer at its λmax in a phosphate buffer solution of ph 6.8 and 0.1 n hcl solution of ph 1.2 were 276 nm and 280 nm, respectively. their calibration curve equations were y = 32.618x with a regression coefficient value r2 = 0.9941 for ph 6.8 and y = 18.848x +0.0185 with a regression coefficient value r2 = 0.997 for ph 1.2. this study was carried out three times. oil binding capacity a certain amount of organogel was centrifuged to determine the oil binding capacity (obc). one gm of organogel was prepared and left at room temperature for 24 hours. after that, the tube containing the organogel was centrifuged for 15 minutes at 6,000 rpm. a whatman filter paper was weighed, and then the vial was inverted over it and stayed for 5 minutes to absorb the free liquid oil dripping from the organogel. after that, the filter paper was weighed to calculate the mass of expressed oil. obc is determined by this equation: oil binding capacity (%) = (1 mass of expressed oil 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑠𝑎𝑚𝑝𝑙𝑒 𝑚𝑎𝑠𝑠 ) × 100 obc was performed by calculating the average of 3 times of this study±sd(15). this test reflects the organogels capacity for oil holding. statistical analysis statistical test the standard deviation was executed for release results using one way anova with the aid of spss version 16.0 . results and discussion organogel formulation the organogel formulations in different gelator concentrations and oils lead to assessing the minimum gelation concentration for any gelator. hence, the minimum gelation concentration (mgc) is essential to evaluate the least concentration of the gelators that gels oils, which, in turn, reflects the organogel strength(16). as shown in figure (1 a and, b), the formulations of 12hsa organogels at room temperature were gelled in ao, and co and the mgc were 3 and 7 wt%, respectively. in addition, figures (1 c and d) show the span 60 organogels, and the mgc was 10 wt% for the ao, and co. last, the span 40 organogels were shown in figure 1 e and f, and their mgc were 7 and 20 wt% for ao, and co. the diversities in the solubility of gelators in oils cause variations in the mgc of different gelators (17). at the elevated temperature (85°c), the gelators were soluble in oils, but they produced varied gelation concentrations when they were left to cool. this variation was imputed to the selfaggregation of the gelator when cooled, which was, at lower gelators concentration, not adequate to make a gel or the 3d structure. in conclusion, 12hsa and span 40 gave varied gelation concentrations in ao, and co while span 60 showed a similar gelation concentration. iraqi j pharm sci, vol.32( 1 ) 2023 slow release organogel 34 for the subsequent studies, the chosen organogels within the range of the selected concentrations were 20 wt% of each gelator. since previous studies about organogels showed that the highest gelator concentration led to the slowest drug release, this represents the core of our aim. the attribution of the organogels to the slow release was due to the fiber density, which is a result of the aggregation's gelator molecules. this was shown in 12hsa/ pg (propylene glycol) organogel in which the highest concentration, the 14 wt% slowed the release of the n4-myristoyl gemcitabine in tumor ph media(18). furthermore, the 20 wt% of span 40 and 60 in sesame oil organogels slowed the release of cinnarizine in stomach media(7). figure 1. organogels of 12hsa in ao and co as in a and b, organogels of span 60 in ao and co as in c and d while in e and f are span 40 in ao and co organogel. the concentrations of gelators were from right to left (0.5, 1, 3, 5, 7, 10, 13, 15, 17, and 20) wt%. tabletop rheology tabletop rheology is an uncomplicated and suitable process that describes the phase transitions from gel to sol and from sol to gel(7). table (2) represents the temperatures at which the phase is transmitted from gel to sol and vice versa. the selected organogel showed the transition temperature from gel to sol (tgel-sol) higher than body temperature, which indicated thermal stability. this thermal stability was ascertained via the reverse phase represented by the transition temperature from sol to gel (tsol-gel). previous work utilizing span 60 and span 40 in sesame oil has also shown convergent results(7). all selected oraganogels needed a temperature higher than 37°c to solidify except the span 40 in co as shown in table (2) (tsol-gel) ºc. table 2. organogels phase transition temperatures figure 2. dsc characterization of the organogels. 12hsa pure powder, 20 wt% of 12hsa in co and ao organogels (a), span 60 pure powder and the 20 wt% span 60 in co and ao organogels (b), span 40 pure powder, and 20 wt% of span 40 in co and ao organogels (c). gelator 20 wt% oil (tgelsol) ºc (tsol-gel) ºc 12hsa co 58 48 ao 68 54 span 60 co 56 42 ao 54 40 span 40 co 64 34 ao 64 40 iraqi j pharm sci, vol.32( 1 ) 2023 slow release organogel 35 differential scanning colorimetry (dsc) dsc defines a precise thermal analysis to specify the organogel transition temperature (11, 19). this is essential in our work as the aim is to have an intact organogel inside the body to achieve the slow release of the drug. all the selected organogel’s thermograms and the corresponding gelator powder were displayed in figure (2 a, b, and c). in the case of 12hsa, the temperature at which it was melted at 80 ºc and a minor peak was noticed at 60 ºc which might be caused by the impurities of 12hsa (12hsa purity 90%), an outcome that also was noticed in the previous research with 12hsa gel in toluene(20). while for the organogels that contain co and ao with 12hsa, the transition temperatures were 63 ºc. and 73 ºc, respectively. regarding span 60, the transition temperature was 59 ºc. however, the conversion temperature peak for the organogel of co did not appear in the thermogram. this led to the conclusion that the fibers which form the organogel were amorphous. ao with span 60 organogel thermogram might also point to amorphous formation as this kind of figure is attributed to phase separation of the oil from gelators as both were amorphous and had different glass transition temperatures (21). the thermogram of span 40 shows the peak of conversion at 59 ºc. both organogels of co and ao with span 40 did not produce peaks which can also be explained as that amorphization in the organogel fibers occurred. organogel amorphization can also be attributed to oil composition and this was also shown in a previous study of span 40 in co organogel(8). the 12hsa in both oils showed higher thermal transition than the span 40 and 60 organogels. most of the transition temperatures were closed to the transition temperature of tabletop rheology results for all organogels. optical microscopy study optical microscopy was performed to study the morphology of the entangled network of the organogel with different gelators as this network is responsible for forming the 3-dimensional structure that held the oils and evidence of organogel formation. this study shows that different gelators give different microscopic shapes. as shown in figure (3), the images revealed that all 12hsa organogels in ao and co exhibited spherulite aggregates, and this outcome resembles a previous study that utilizes 12hsa with canola oil(22). the spherulites appeared in the microscopic images of span 40 organogels formulated in ao and co, similar to this result showed in former research that employed span 40 with sunflower oil(23). whereas the scaffold span 60 organogels in oils ao and co present as fibers aggregates, another study of organogel prepared from span 60 and soya-bean oil showed the same fibers(24). different scaffold morphology could be attributed to the difference in the solubility of the gelators in oils that upon cooling led to the different molecular hierarchy assembly(25). in conclusion, all images showed scaffolds; however, different underlying scaffold structures. 12hsa/ao 12hsa/co span 60/ao span 60/co span 40/ao span 40/co figure 3. microscopic images of organogels containing 12hsa, span 60 and span 40 in ao and co, all images were captured using objective x40 and scaled against 50µm. iraqi j pharm sci, vol.32( 1 ) 2023 slow release organogel 36 oscillatory rheology studies rheology studies include amplitude sweep and frequency sweep, which are essential tests to investigate the strength of the organogel that is required to resist the motion of the gastrointestinal tract (26). amplitude sweep in this study, the first portion examined ds-loaded organogels utilizing the amplitude sweep test for the selected organogel to determine storage modulus (g'), loose modulus (g''), lver, and the flow point. the solid phase was represented by g', which reveals the organogels elasticity, whereas g'' indicates the liquid phase. the amplitude sweep test was performed by putting the organogel to face a growing strain from zero to 100%. if the gel reveals good strength, the result of g' is higher than g'' in parallel curves, and constant values of g' and g'' until attaining a value of strain that the gel cannot keep the same g' value and begins weakening as before this point is the lver. after that, with the continued rising strain, the gel attains a breaking point. at this point, the values of g' and g'' are the same, defined as the flow point. all the organogel parameters are illustrated in figure (4) and table (3). the values of g' of all organogels in this experiment appeared larger than g''. this relation was also shown in the rheological study of bis-ureabased organogels in different primary alcohols (27). the 12hsa in ao presented the highest g' value, and the lowest g' was detected by span 60 in ao organogel. the lver of 20 wt% 12hsa in both oils were approximate. the span 60 and 40 organogels appeared the same trend of the lver as co organogels were higher than ao organogels. the flow point of 20 wt% 12hsa in co organogel was the highest value compared with other organogels, whereas the lowest percentage was 20 wt% span 60 in ao. these different points for different organogels might pertain to different transient bonds which connect fibers that are responsible for the organogels structure(28). to sum up, 12hsa organogels were the best to show the viscoelastic properties via presenting high g', lver and, flow points at 37°c as this indicates the best resistance to the gastrointestinal motility. table 3. amplitude sweep parameters g', g'', lver, and flow points for the six selected organogels as each value is an average and standard deviation of 3 values. the study was set at a strain from 0% to 100%, angular frequency 10 rad.s-1, and temperature at 37°c. gelators oil g' (pa) g'' (pa) lver (%) flow point (%) 20 wt% 12hsa co 439000 46.8 0.04475 20.3 ao 3230000 349 0.04335 6 20 wt% span 60 co 25100 2.66 0.0458 5 ao 17545 1.9384 0.0293 0.65 20 wt% span 40 co 111000 117.15 0.175 4 ao 74008 8.901185 0.0221 6.5 figure 4.the amplitude sweep test for 12hsa in co and ao organogels, span 60 in co and ao organogels and last span 40 in co and ao organogels. iraqi j pharm sci, vol.32( 1 ) 2023 slow release organogel 37 frequency sweep this study was carried out to examine the ability of organogel to stay solid at different speeds or motions. the strong organogel usually shows non-intersecting g' and g'' curves. as shown in figure (5) the 20 wt% 12hsa in both oils have a similar pattern and were frequency-independent at high rate frequencies; nevertheless, at low frequencies, the organogels were highly affected by the low rate of frequency as the curves of g' and g'' intersected. the span 60 organogels in co and ao showed the same pattern; but the g' and g'' values were constant alongside the whole range of frequencies, indicating elastic organogels (7). the last organogels, span 40 in both oils, showed almost similar attitudes as stable curves within the selected range of frequencies just the span 40 in ao curves of g' and g'' were close at low frequency, this result is similar to a previous study in which organogelator molecules of amino-acid type was used(29).in summary, span 60 and 40 in co and ao showed constant curves (g’ and g’’) at different frequencies, indicating frequency-independent organogels, while 12hsa organogels in both oils were impacted at low frequencies. figure 5. frequency sweep test as the left column represents organogels of 12hsa, span 60 and span 40 in co while the right column shows the same sequential organogels in ao. the angular frequency was set from 0 to 100 rad s-1, and the temperature was 37°c. in-vitro release study the in vitro release was executed to explore the organogels as formulations that can slow the release of ds. firstly, the ultraviolet reads for the samples withdrawn from the acidic media revealed that ds did not release from the hpmc capsule in this media, which supports the concept that the enteric hpmc capsule resists dissolution in the acidic media (30). thus, the in-vitro release study for organogels was in small intestinal ph 6.8 for 24 hours to check the depot characteristic of organogels and the effect of different gelators on the ds release. with all the release examinations, a release for control formulation consisted of 75 mg ds mixed with co and ao individually to understand the release of ds from the organogels as oily formulations are well-known as dosage forms with the depot property. figure (6a) shows the release profiles of ds in co of different organogels. as observed, 12hsa in co shows the most depot release in this study; span 40 and 60 came sequentially. statistically, the release profiles showed significant variation (p ≤ 0.05) in the release percent amongst the three gelators in co organogel. figure (6b) shows the release profiles of ds in ao, which revealed that the organogel of 20 wt% 12hsa, span 60 and span 40, their release profile was close to each other within the frame time experiment. the control of ds in co and ao showed a higher difference (p ≤0.05) in the ds release profile than the organogels, which is attributed to the organogel scaffold rather than oil property.in a nutshell, the 12hsa, span 60, and span 40 gave different release patterns in oils due to the different solubility of these gelators. a sodium alginate gel in another study was used as a carrier to sustain ds release. it showed a closed pattern to our organogel 12hsa/co, as 73 wt% ds after 12 hours was released a promising matrix to slow the ds release (14). the organogels 12hsa in co was the closest to the aim of the current study via showing the slowest release, which was supported by the strength outcome of the amplitude sweep test presenting the best values of g' and flow point. iraqi j pharm sci, vol.32( 1 ) 2023 slow release organogel 38 figure 6. in-vitro ds release in ph 6.8 phosphate buffer solution at 37°c as a and b represent 12hsa, span 60, and span 40 in co and ao, respectively. each release curve is an average of triplicate ± standard deviation. oil binding capacity (obc) the binding capacity for oils to any organogel reflects the scaffold’s strength that constructs the organogel and held the oil; hence, this test was carried out (31). table (4) reveals the obc results as all the selected organogel in co showed a high binding capacity. at the same time, the organogels in ao produce a noticeable low obc compared with the co organogels and this was supported by dsc results of span 60. these findings support the in vitro release as the organogels in co; specifically, the 12hsa organogel showed slower ds release and the higher obc, which means that these co organogels were more capable of holding the ds and oil. table 4.obc of 20 wt% 12hsa, span 60 and span 40 in co and ao 20 wt% gelators /oil obc of co (%) ±sd obc of ao (%) ±sd 12 hsa 99.9±0.06 89.61±0.1 span 60 95.6±0.1 72.61±0.17 span 40 99.3±0.05 58.61±0.28 conclusion the target of this study was to fabricate organogel with the delayed and slow release of ds. this was achieved with the organogels formulated in co compared to those organogels prepared in ao. those organogels showed a slower release profile and presented good viscoelastic properties: higher g' and flow points. furthermore, they were frequency independent in most applied frequency ranges. also, they showed strong gelators binding toward co. acknowledgment the authors appreciate the sustenance and the support from mustansiriyah university, college of pharmacy, for working and searching in the college’s laboratories. references 1. shapiro yejpips. structure and dynamics of hydrogels and organogels: an nmr spectroscopy approach. prog polym sci. 2011;36(9):1184-253. 2. esposito cl, kirilov p, roullin vgjjocr. organogels, promising drug delivery systems: an update of state-of-the-art and recent applications. jcr. 2018;271:1-20. 3. mujawar nk, ghatage sl, yeligar vcjijop, chemical, sciences b. organogel: factors and its importance. ijpcbs. 2014;4(3):758-73. 4. singh vk, anis a, al sesame -zahrani s, pradhan dk, pal kjijes. ftir, electrochemical 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4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions doi: https://doi.org/10.31351/vol29iss2pp17-26 17 analysis of docetaxel adverse drug reactions: a retrospective study based on iraqi pharmacovigilance center database ahmed m. hameed*,1 , dheyaa j. kadhim* and manal m. younus** * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** iraqi pharmacovigilance center, directorate of technical affairs, ministry of health and environment ,baghdad, iraq. abstract docetaxel is a chemotherapeutic agent approved for management of various cancers, but the occurrence of significant adverse drug reactions may affect the drug use and overall effectiveness in clinical practice. the purpose of the current study was to measure the distribution of adverse drug reactions of docetaxel reported in iraq and to assess the causality, severity, seriousness, preventability, expectedness and outcome of these adverse reactions. a retrospective study conducted on individual case safety reports from the iraqi pharmacovigilance center / ministry of health. the study included 118 individual case safety report containing 236 adverse drug reactions. most of the adverse drug reactions were related to “skin and subcutaneous tissue disorders” (26.7%), followed by “respiratory, thoracic and mediastinal disorders” (20.8%), “gastrointestinal disorders” (17.4%) and “general disorders and administration site conditions” (10.6%). the majority of these reactions with possible causality (68.6%), severity level 3 according to hartwig’s severity assessment (55.5%), expected (80.5%), possibly preventable (93.2%), and serious (80.5%). in addition, the most common outcome of adverse drug reactions was recovered / resolved (46.19%). keywords: docetaxel, pharmacovigilance, adverse drug reactions, iraqi pharmacovigilance center, anti-cancers. مركز بيانات قاعدة إلى تستند استرجاعية دراسة: للدوسيتاكسيل الضارة الدوائية التفاعالت تحليل العراقي الدوائية اليقظة ** و منال محمد يونس *ضياء جبار كاظم 1*,احمد ماجد حميد .فرع الصيدلة السريرية ،كلية الصيدلة، جامعة بغداد، بغداد، العراق* .العراقبغداد ،، والبيئة المركز العراقي لليقظة الدوائية، دائرة االمور الفنية، وزارة الصحة ** الخالصة تفاعالت بحدوث تتأثر أن يمكن السريرية الممارسة في فعاليته لكن ، السرطانات من كثيرة ألنواع ومعتمد فعال عالج دوسيتاكسيل هو وشدتها السببية وتقييم العراق في عنها الُمبلغ للدوسيتاكسيل الضارة الدوائية التفاعالت توزيع قياس هو الحالية الدراسة من الهدف كان. ضارة دوائية اليقظة الدوائي مركز من الفردية الحاالت سالمة تقارير على أجريت رجعي بأثر هذه الدراسة هي دراسة. ونتائجها وتوقعها منها والوقاية وخطورتها دوائيا ضاًرا. تفاعاًل 236 على يحتوي الحاالت لسالمة فرديًا تقريًرا 118 على الدراسة اشتملت. الصحة وزارة/ العراقي والمنصف والصدر التنفسي الجهاز اضطرابات تليها ،٪( 26.7) الجلد تحت األنسجة واضطرابات بالجلد الضارة الدوائية التفاعالت معظم ارتبطت الضارة الدوائية التفاعالت غالبية٪(. 10.6) اإلعطاء موقع وحاالت العامة واالضطرابات٪( 17.4) الهضمي الجهاز واضطرابات ،٪( 20.8) 80.5) وخطيرة ،٪( 93.2) منها الوقاية يمكن وربما ،٪( 80.5) ومتوقعة ،٪( 75.4) معتدلة وشدة ،٪( 68.6) محتملة كانت ذات سببية هذه .(%46.19)حلها او التعافي منها هي الضارة للتفاعالتاالكثر شيوعا جةالنتي كانت ذلك إلى باإلضافة٪(. . السرطان مضادات ، العراقي الدوائية اليقظة مركز ، الضارة الدوائية التفاعالت ، الدوائية اليقظة دوسيتاكسيل ،: المفتاحية الكلمات introduction according to the world health organization (who), “pharmacovigilance (pv) is defined as the science and activities relating to the detection, assessment, understanding and prevention of adverse effects or any other drug-related problem”(1). pharmacovigilance is a combination of communication systems, registries, and databases in a complex structure for a patient using a drug, pharmacovigilance represent an essential tool for the early identification of risk signals faced by the patient due to drug usage(2). the main steps in the pharmacovigilance process is the identification and reporting spontaneously the (adrs) which happened throughout the treatment, the process of pharmacovigilance should be applied carefully and continuously so that it can achieve its target which is the optimum safety of drug and in order to achieve this target the entire health care professionals should participate in the pharmacovigilance process also patients should be involved in the process through continuous patient education(3). 1corresponding author e-mail: ph.ahmed4747@gmail.com received: 21/12 /2019 accepted: 15/2 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp17-26 iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 18 as defined by who, the adr is “any noxious or unintended response to a drug, which occurs at doses normally used in man for prophylaxis, diagnosis or therapy of disease, or for modification of physiological function”(4). adrs constitute a burden on the health care system because adrs are a major cause of morbidity, hospital admission, increasing health care cost, and even increasing mortality rates(5). the increase in the cost represent a huge burden on the health care system, health care facilities may spend 20% of their budget to deal with the complications encountered due to drug usage in some countries(6). cancer represent a major cause of mortality and morbidity with a yearly mortality rate of 12% worldwide(7). chemotherapeutic regimens are complex approaches for the treatment of cancer and patients with cancer are generally more prone to adverse drug reactions due to their decreased immune system function compared to normal individuals or patients of other disease areas(8). docetaxel is an important antimicrotubule agent from the family of taxane’s. it is a paclitaxel semisynthetic derivative of but it is more potent, derived from extracts of the leaves of the european yew tree (taxus baccata), was discovered in the 1980’s(9). docetaxel is highly effective when used as single therapy or in combination with other anticancer drugs for a number of cancers including breast cancer in different stages, head and neck cancers, gastric cancer, non-small cell lung cancer and androgen-independent metastatic prostate cancer(10). treatment regimens that contain docetaxel produce better outcomes in the metastatic, adjuvant, and neoadjuvant settings(11). according to the summary of product characteristics (smpc) by european medicines agency (ema), “the most commonly reported adverse reactions of docetaxel alone are: neutropenia, anemia, alopecia, nausea, vomiting, stomatitis, diarrhea and asthenia. the severity of adverse events of docetaxel may be increased when docetaxel is given in combination with other chemotherapeutic agents”(12). adrs associated with docetaxel may cause stopping of treatment process, interruption of treatment process or may cause the dose of the drug being used to be decreased and thus affecting the treatment process. in some cases if the adrs are not effectively treated the encountered risk may outweighs the potential benefit of docetaxel but results from clinical trials showed that when adrs are managed effectively the quality of life and survival are greatly improved by docetaxel(13). the aim of the present study was to demonstrate the distribution of docetaxel adrs reported to the pharmacovigilance center and to analyze the causality, severity, seriousness, preventability, expectedness and outcome of these adrs. subjects and method a retrospective study conducted on individual case safety reports (icsr) obtained from the iraqi pharmacovigilance center / ministry of health. the reports were collected via vigiflow – iraq. vigiflow is provided by uppsala monitoring center (umc) which is a who collaborating center for adverse drug reactions from many national centers. the study included 118 individual case safety report containing 236 adverse drug reaction which were analyzed for demographic distribution, adr classification, causality, severity, expectedness, preventability and seriousness. adverse drug reactions were classified by the system organ classification (soc) (groups of adverse reaction pertaining to the same systemorgan), and also classified according to the preferred term (pt) (principal terms used for describing drug adverse reactions) according to the medical dictionary for drug regulatory affairs (meddra)(14). the adrs in pt was sorted in tables according to their soc distribution to show count and percentage of the reported adrs. causality was assessed using who-umc criteria and were categorized into certain, probable, possible, unlikely, unclassified, and unclassifiable (table 1) (15). iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 19 table 1. the who-umc criteria for causality assessment (15). causality term assessment criteria certain • event or laboratory test abnormality, with plausible time relationship to drug intake • cannot be explained by disease or other drugs • response to withdrawal plausible (pathologically, pharmacologically) • event definitive pharmacologically or phenomenologically (i.e. an objective and specific medical disorder or a recognized pharmacological phenomenon) • rechallenge satisfactory, if necessary probable/ likely • event or laboratory test abnormality, with reasonable time relationship to drug intake • unlikely to be attributed to disease or other drugs • response to withdrawal clinically reasonable • rechallenge not required possible • event or laboratory test abnormality, with reasonable time relationship to drug intake • could also be explained by disease or other drugs • information on drug withdrawal may be lacking or unclear unlikely • event or laboratory test abnormality, with a time to drug intake that makes a relationship improbable (but not impossible) • disease or other drugs provide plausible explanations conditional/ unclassified • event or laboratory test abnormality • more data for proper assessment needed, or • additional data under examination unassessable/ unclassifiable • report suggesting an adverse reaction • cannot be judged because information is insufficient or contradictory • data cannot be supplemented or verified to assess the severity of the adverse events, the modified hartwig and seigel criteria have been used (table 2) (16). table 2. hartwig’s severity assessment (16). level of severity the criteria level 1 an adr occurred but required no change in the treatment with the suspected drug level 2 the adr required that treatment with the suspected drug be held, discontinued, or otherwise changed. no antidote or other treatment requirement was required. no increase in length of stay (los) level 3 the adr required that treatment with the suspected drug be held, discontinued, or otherwise changed. and/ or an antidote or other treatment was required. no increase in los level 4 any level 3 adr which increases length of stay by at least 1 day. or the adr was the reason for the admission level 5 any level 4 adr which requires intensive medical care level 6 the adverse reaction caused permanent harm to the patient level 7 the adverse reaction either directly or indirectly led to the death of the patient 2 expectedness analysis was based on the summary of product characteristics (smpc) for each drug which is approved during the marketing authorization, each reported adr was reviewed to check if it is included in the smpc or not, if the adr is included in the smpc then the adr is considered an expected adr, if the reported adr was not mentioned in the smpc then the adr is considered unexpected (17). the preventability assessment was based on the modified schumock and thornton criteria where the adrs were either preventable or non-preventable. if there was missing data making it not possible to answer all questions very clearly then the adr is possibly preventable (table 3)(18, 19). iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 20 table 3. schumock and thornton preventability assessment criteria(18,19). question yes no 1 was there a history of allergy or previous reaction to the drug? 2 was the drug involved inappropriate for patient's clinical condition? 3 was the dose, route, or frequency of administration inappropriate for the patient's age, weight or disease? 4 was there any required therapeutic drug monitoring, or other laboratory tests not performed? 5 was a drug interaction involved in the adr? 6 was poor compliance involved in the adr? 7 was a toxic serum concentration or a laboratory? monitoring test documented? seriousness analysis was based on the protocols followed by the national center of pharmacovigilance and applied by the center staff, seriousness was assessed according to the icsr paper (table 4). table 4. seriousness assessment in the individual case safety report (20). the outcome for each icsr was recorded and categorized into one of following categories by the who: fetal, not recovered/not resolved, recovered / resolved, recovered / recovered with sequelae, recovering /resolving and unknown in case of missing data in this field of the report. ethical approval the study had been approved by the scientific committee of the university of baghdad/ college of pharmacy and the iraqi ministry of health/ department of research and development before it was conducted statistical analysis analysis of data was carried out using microsoftexcel. data were presented in simple measures of frequency and percentage. results age group analysis showed that adults were the major group (87.29%). regarding gender distribution, the majority of the reports was for females (78.81%). pharmacists were the most common reporters (74.58%). regarding the province of reports, it was not available in (22.03%) of the reports and the highest number of reports were from najaf (18.64%) (table 5). do you consider the reaction to be serious? yes no if yes, please tick () to indicate why the reaction is considered to be serious: the patient died due to the reaction involved or prolonged inpatient hospitalization life threatening involved persistent or significant disability or incapacity congenital anomaly medically significant, please give details: iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 21 table 5. age group, gender, reporter and province distribution of icsrs. age group icsr number (%) adult 103 (87.29%) elderly 10 (8.47%) n/a 3 (2.54%) infant 2 (1.69%) gender icsr number (%) female 93 (78.81%) male 19 (16.10%) n/a 6 (5.08%) reporter icsr number (%) pharmacist 88 (74.58%) n/a 23 (19.49%) physician 5 (4.24%) other health professional 2 (1.69%) province icsr number (%) n/a 26 (22.03%) al-najaf 22 (18.64%) nineveh 20 (16.95%) baghdad 13 (11.02%) karbala 9 (7.63%) al-basra 8 (6.78%) al-anbar 6 (5.08%) babylon 6 (5.08%) salah al-din 4 (3.39%) kirkuk 3 (2.54%) diyala 1 (0.85%) characteristics of icsr icsr number (%) docetaxel as a single agent 100 (84.7%) combination with cyclophosphamide and doxorubicin 7 (5.9%) combination with trastuzumab 5 (4.2%) combination with cisplatin 4 (3.2%) combination with gemcitabine 1 (0.8%) combination with 5-flurouracil 1 (0.8%) n/a: not available, icsr: individual case safety report according to soc, the adrs distribution for docetaxel showed that “skin and subcutaneous tissue disorders” (26.7%), “respiratory, thoracic and mediastinal disorders” (20.8%), “gastrointestinal disorders” (17.4%) and “general disorders and administration site conditions” (10.6%) were the most frequently reported adrs. docetaxel adverse drug reactions are listed in (table 6) in the preferred term grouped by soc. iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 22 table 6. adrs in preferred term grouped by system organ classification for docetaxel adrs skin and subcutaneous tissue disorders 63 (26.7%) pruritus 13 (5.5%) dermatitis exfoliative 10 (4.2%) nail discoloration 10 (4.2%) skin exfoliation 7 (3.0%) skin discoloration 6 (2.5%) erythema 5 (2.1%) skin burning sensation 4 (1.7%) skin ulcer 2 (0.8%) hyperhidrosis 1 (0.4%) rash 1 (0.4%) rash generalized 1 (0.4%) rash papular 1 (0.4%) skin depigmentation 1 (0.4%) swelling face 1 (0.4%) respiratory, thoracic and mediastinal disorders 49 (20.8%) dyspnea 22 (9.3%) interstitial lung disease 13 (5.5%) cough 3 (1.3%) hyperventilation 3 (1.3%) choking 2 (0.8%) epistaxis 2 (0.8%) respiratory disorder 2 (0.8%) choking sensation 1 (0.4%) oropharyngeal pain 1 (0.4%) gastrointestinal disorders 41 (17.4%) nausea 19 (8.1%) vomiting 11 (4.7%) diarrhea 4 (1.7%) abdominal pain 2 (0.8%) abdominal discomfort 1 (0.4%) abdominal pain upper 1 (0.4%) aphthous ulcer 1 (0.4%) epigastric discomfort 1 (0.4%) mouth ulceration 1 (0.4%) general disorders and administration site conditions 25 (10.6%) pyrexia 6 (2.5%) swelling 4 (1.7%) administration site extravasation 2 (0.8%) chest discomfort 2 (0.8%) influenza like illness 2 (0.8%) administration site rash 1 (0.4%) chest pain 1 (0.4%) chills 1 (0.4%) iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 23 table 6 continued . adrs in preferred term grouped by system organ classification for docetaxel adrs extravasation 1 (0.4%) fatigue 1 (0.4%) feeling cold 1 (0.4%) injection site pain 1 (0.4%) instillation site erythema 1 (0.4%) edema peripheral 1 (0.4%) immune system disorders 15 (6.4%) hypersensitivity 12 (5.1%) drug hypersensitivity 2 (0.8%) immune system disorder 1 (0.4%) musculoskeletal and connective tissue disorders 11 (4.7%) arthralgia 5 (2.1%) myalgia 3 (1.3%) back pain 1 (0.4%) bone pain 1 (0.4%) muscular weakness 1 (0.4%) nervous system disorders 8 (3.4%) headache 3 (1.3%) hypoesthesia 2 (0.8%) burning sensation 1 (0.4%) demyelination 1 (0.4%) dizziness 1 (0.4%) infections and infestations 6 (2.5%) candida infection 1 (0.4%) oral candidiasis 1 (0.4%) oral fungal infection 1 (0.4%) q fever 1 (0.4%) skin infection 1 (0.4%) wound infection bacterial 1 (0.4%) cardiac disorders 3 (1.3%) tachycardia 3 (1.3%) metabolism and nutrition disorders 3 (1.3%) decreased appetite 2 (0.8%) fluid retention 1 (0.4%) blood and lymphatic system disorders 2 (0.8%) anemia 1 (0.4%) neutropenia 1 (0.4%) eye disorders 2 (0.8%) lacrimation increased 2 (0.8%) injury, poisoning and procedural complications 2 (0.8%) burn esophageal 1 (0.4%) ligament rupture 1 (0.4%) vascular disorders 2 (0.8%) flushing 1 (0.4%) iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 24 table 6 continued. adrs in preferred term grouped by system organ classification for docetaxel adrs hypotension 1 (0.4%) ear and labyrinth disorders 1 (0.4%) tinnitus 1 (0.4%) investigations 1 (0.4%) respiratory rate decreased 1 (0.4%) neoplasms benign, malignant and unspecified (including cysts and polyps) 1 (0.4%) cancer pain 1 (0.4%) reproductive system and breast disorders 1 (0.4%) menstruation irregular 1 (0.4%) grand total 236 (100.0%) causality assessment showed that most of the adrs was in the “possible” category with 68.6% followed by 24.2% for probable, 3.4% for unclassified, 2.5% for unlikely and 1.3% for certain category. severity analysis showed that level 3 was the major category with 55.5% of adrs falling in this category followed by 19.9% for level 4, 12.3% for level 1, 8.1% for level 2, 3.8% for level 5 and 0.4% for level 7. expectedness analysis showed that 80.5% of the adrs associated with docetaxel was expected adrs while 19.5% of the adrs were unexpected. preventability analysis showed that 93.2% of the adrs were in the possibly preventable category, 3.8% were non-preventable and 3% were recorded as preventable. the outcome of the adrs associated with docetaxel were mainly in the recovered / resolved category with 46.2%, followed by 15.3% (15.25%) in the not recovered / not resolved / ongoing category, 6.4 (6.36) % were in the recovering / resolving category, 0.9 (0.85)% in the recovered / resolved with sequelae category, 0.4 (0.42)% of the adrs were recorded as fatal and 30.9 (30.93)% of the adrs were missing the data regarding the outcome of the reaction. the seriousness analysis of the adrs with docetaxel showed that 80.5% of the adrs were serious while 14.8% of the adrs were non-serious, 4.7% of the adrs were missing some data to assess seriousness. table 7. causality, severity, expectedness, preventability, seriousness and outcome of adrs reported for docetaxel causality number of adrs (%) certain 3 (1.3%) probable 57 (24.2%) possible 162 (68.6%) unclassified 8 (3.4%) unlikely 6 (2.5%) severity level 1 29 (12.3%) level 2 19 (8.1%) level 3 131 (55.5%) level 4 47 (19.9%) level 5 9 (3.8%) level 7 1 (0.4%) expectedness expected 190 (80.5%) unexpected 46 (19.5%) preventability non-preventable 9 (3.8%) possibly preventable 220 (93.2%) preventable 7 (3.0%) outcome fatal 1 (0.42%) not recovered / not resolved / ongoing 36 (15.25%) recovered / resolved 109 (46.19%) recovered / resolved with sequelae 2 (0.85%) recovering / resolving 15 (6.36%) unknown 73 (30.93%) seriousness no 35 (14.8%) unknown 11 (4.7%) yes 190 (80.5%) iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 25 discussion the current study showed that most of the adrs were among the adult age group (87.29%) (table-5), the gender distribution showed more adrs in the female patient group than in the male patient group this may be attributed to the greater use of docetaxel in the treatment of breast cancer which lead to the development of more adrs for this medication in the adult and female gender. the results showed that the reporters of the adrs were mostly pharmacists which indicate that the pharmacovigilance responsibility in the healthcare facilities are more held by pharmacists. regarding the province of reporting, al-najaf and nineveh showed the most icsrs reported across iraq despite the unstable security situation in nineveh and this result is consistent with result from another pharmacovigilance study in iraq that showed that nineveh was the major contributor to the icsrs reporting(21), although docetaxel was a single medication in around 85% of cases, the presence of concomitant anti-cancer medications in less than 20% of the total cases affected the level of final certainty. the most commonly observed adrs in the current study were in the “skin and subcutaneous tissue disorders” (26.7%), followed by “respiratory, thoracic and mediastinal disorders” (20.8%), “gastrointestinal disorders” (17.4%) and “general disorders and administration site conditions” (10.6%) (table 6) and the most common adrs were nausea, vomiting, dyspnea, skin disorders, nail discoloration and hypersensitivity respectively which is consistent with the most commonly occurring adrs according the summary of product characteristics by the ema but the hematological adrs like neutropenia and anemia were under observed in the study with (0.4%) for each (table 6) compared to the summary of product characteristics (12), according to a report published by the iraqi pharmacovigilance center there was an increase in cutaneous and respiratory adverse reactions of docetaxel few years ago that required a regulatory action at that time (22). adverse drug reactions causality assessment showed that most of the adrs were in the possible category followed by probable and very few in certain category that’s because for an adr to be considered as certain, many criteria must be met (time sequence, disease or other drug causality ruled out, dechallenge and rechallenge)(23) and these criteria specially the rechallenge is rarely experienced with adrs occurring after an anticancer medication, rechallenge may not be needed for a certain classification in a small number of situations such as when a cytotoxic drug extravasates and causes tissue damage which is the case with the 3 certain adrs reported in the study, but for probable category rechallenge is not required so more adrs fall in this category. for possible category, the adr is suspected to be caused by other drugs or can be caused by the disease condition being treated. for severity assessment of adrs, level 3 severity accounts for (55.5%) of the adrs as shown in (table 7) indicating that the majority of the adrs required antidote or other medication for the adrs with no increase in hospitalization time which takes place in the level 4 category which represent 19.9% of the adrs. both level 3and 4 constitute the moderate category of the severity assessment(17). there was one adr that result in death of the patient and the reaction was (bacterial infectious disorders) with possible causality. the preventability analysis showed that most of the adrs were possibly preventable due to missing data that lead to the conclusion weather it was definitely preventable or non-preventable. the outcome for docetaxel adrs was mostly recovered / resolved adrs with (46.19%) while data regarding adrs outcome were missing in (30.93%) (table 7). a study for the presentation and management of docetaxel related adrs was conducted in canada stated that most of the common treatment related toxicities are resolved either between cycles of the drug or by treatment discontinuation(11). seriousness analysis showed that 80.5% of adrs were serious which may reflect the idea that non-serious adrs were underestimated by the health care providers and was not reported accordingly and the focus was directed on reporting serious adrs to the national pharmacovigilance center. the current study is not without limitations. the main limitation was the incompleteness of the reports reported by the health facilities to the pharmacovigilance center as the reporter is mainly focusing on the adrs but less focus on the patient medical history, concomitant medications and patient follow up and these data are necessary to establish the causality, preventability and other parameters more accurately. conclusions and recommendations docetaxel has a wide range of side effects profile affecting mainly the skin, respiratory and gastrointestinal systems with most of these side effects being expected, serious and moderately severe. more focus should be directed towards side effects for proper management and prevention also there should be a continuous awareness regarding the importance of pharmacovigilance in all health care facilities and the employees responsible in these facilities should be professionally trained so that the reported data be accurate and can be analyzed properly for better health outcome. also patients should be educated about the possible side effects for their medications and how to report in case these side effects occur. iraqi j pharm sci, vol.29(2) 2020 analysis of docetaxel adverse drug reactions 26 references 1. who. 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maysaa a. abdul khaleq* ,1 , abdulkareem h. abd** and maysaa a. dhahi*** * department of pharmacotheraputics,college of pharmacy, almustansiryh university,baghdad,iraq. ** department of pharmacology, college of medicin, alnahrain university, baghdad, iraq. *** department of microbiology, college of medicin, al-nahrain university, baghdad, iraq. abstract seventy five e. coli isolates were collected from urine of patients with urinary tract infections in al-kadhimia and al-yarmook teaching hospitals in baghdad for a period between 22/11/2009 to 15/3/2010, from these samples twenty five isolates were selected according to their pattern of the highest resistance as these showing multi-drug resistances and tested to specify their minimum inhibitory concentration for (meropenem, gentamicin and amikacin), meropenem was found having the lowest mic comparing with others. this study also includes in vitro effects of various combinations of three types of antimicrobials (meropenem, gentamicin and amikacin) against twenty five e. coli isolates.among combinations the combination of meropenem with the other types of antimicrobials showed high synergistic effect when 1/4+1/4 mic for each antimicrobial were used. while combinations of amikacin with gentamicin in some isolates showed additive effect when 1/2+1/2 mic for each antimicrobial were used. the plasmid profile for the twenty five e. coli isolates were studied using pure yeild ™ plasmid miniprep systemcat.# a1220 – promegausa. in order to determined the presence of plasmid for antimicrobials resistance. الخالصة جَعج خَست ٗسبعُ٘ عضىت ٍِ االشٞشٝشٞا اىق٘ىّ٘ٞت ٍِ ادساس ٍشضٚ اىَجاسٛ اىب٘ىٞت اىزِٝ ساجع٘ا ٍسخشفٚ اىناظَٞت عيٚ ٍا ٌٍْٖٗ حٌ اخخٞاس خَست ٗعششُٗ عضىت اعخَادا ۲۰۱۰⁄ ۳ ⁄۱۱اىٚ ۲۲⁄۱۱⁄۲۰۰۰ٗاىٞشٍ٘ك اىخعيَٜٞ فٜ بغذاد ىيفخشٓ ٍِ اىجْخَاٝسِٞ ،ىيَضاداث ) اىَٞشٗبٌْٞ (mic)اداث اىجشثٍ٘ٞت ثٌ حذدث اىخشامٞض اىَثبطت اىذّٞا بذحٔ ٍِ ٍقاٍٗت عاىٞت ٗ ٍخعذدة ىيَضٴ ا قو حشمٞض ٍقاسّت باىَضاداث ٴ مثش فاعيٞت ٗ رىل بخثبٞطٔ َّ٘ اىبنخشٝا باٴ ظٖشث اىْخائج باُ ٍضاد اىَٞشٗبٌْٞ ٕ٘ االٴ ٗقذ ا )ٗاالٍٞناسِٞ ٗقذ e. coli ((in vitroححاد اىَضاداث اىحٞ٘ٝت ضذ خَست ٗعششُٗ عضىت ٍِ ثٞش اٴ حضَْج ٕزٓ اىذساست اسخقصاء حا.خشٙٴ اال ٝشٞش اىٚ حاثٞش حاصسٛ عاىٜ عْذ اسخعَاه )ُ اححاد اىَٞشٗبٌْٞ ٍع بقٞت اىَضاداث اىحٞ٘ٝت)اىجْخَاٝسِٞ ٗاالٍٞناسِٞ ٴ ظٖشث اىْخائج اٴ ا الٍٞناسِٞ ٍع اىجْخاٍاٝسِٞ فٜ بعض اىعضالث ٝشٞش اىٚ حاثٞش بَْٞا اححاد ا .ىنو ٍضاد حٞ٘ٛ mic)سبع اىخشمٞض اىَثبط االدّٚ ) ىَْط اىبالصٍٞذٛ ٴ دساست ا ىذساست اٝضا ٴ شَيج ا .ىنو ٍضاد حٞ٘ٛ mic)اضافٜ فقط عْذ اسخعَاه ّصف اىخشمٞض اىَثبط االدّٚ ) ُ اىعضالث ٴ ظٖشث اىْخائج باٴ ٗقذ ا miniprepسخخذاً عذة ىعضه اىبالصٍٞذ ب٘اسطت ّظاً ٴ با e. coliىخَست ٗعششُٗ عضىت ٍِ بنخشٝا .ىَضاداث اىحٞ٘ٝتٴ ( حاٍئ ىبالصٍٞذ ىَقاٍٗت ا٬،۳۲،۳۳،۱۳) introduction urinary tract infections (utis) are one of the most common bacterial infections in humans both in the community and hospital setting (1) . escherichia coli have been documented to be the most important pathogen associated with symptomatic urinary tract infections (2) .plasmid dna molecule is separate from, and can replicate independently of, the chromosomal dna (3) . in this study we use combination of meropenem (which is a broad spectrum antimicrobial agent with more activity against gram-negative bacilli and less activity against gram-positive cocci than is imipenem) (4) , with aminoglycosides which are polar compound with more activity against aerobic gram-negative bacilli and little activity against an aerobic bacteria and use with other antimicrobial agent against gram positive bacteria (5) . # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : maysaa_ali82a@yahoo.com received : 12/3/2011 accepted : 10/5/2011 iraqi j pharm sci, vol.20(2) 2011 combinations of antimicrobials against resistant e. coli 67 material and methods the e. coli identification depended on morphological, biochemical testes in addition to api 20e system. susceptibility of isolates to seventeenth antimicrobials was tested using disk diffusion assay according to modified kirby–bauer method (6) . meropenem, nitrfurantoin , amikacin and imepenem were to be the most effective antimicrobials, while the other antimicrobials were less effective. minimum inhibitory concentration (mic) was determined using tubes dilution method (7) . the combination of antimicrobials weather it’s synergistic, additives, antagonistic, or indifference depending on the fractional inhibitory concentration (fic) was determine as follow: (≤0.5) synergism, (0.5–<1) additive, (1–<4) indifference,(≥4) antagonism, and calculated using the following equation (8) . mic for antibiotic in combination fic = ———————————————–– mic for antibiotic alone plasmid dna isolated using pure yeild ™ plasmid miniprep system, according to the manufacture manual. then the extracted plasmid dna was loaded in 0.8% agarose gel stained with ethidium bromide and electrophoresis for 60 minutes at 2v/cm using 1x tbe buffer. then agarose gel was visualized using uv-transluminator. result and discussion colonies of e. coli had marked as a flat smooth and pink in color as a result of lactose fermentation in the media on macconky agar, while on blood agar it gave small pink convex colonies surrounded by zone of βhaemolysis. in microscopic examination it showed as small single bacilli non spore forming with red color (gram –negative bacteria), it occurred separately and singly, but often they are accumulated in groups. the result of biochemical tests for most of e. coli showed its ability to catalase production and lactose fermentation while it gave a negative result in oxidase, urease and simmon citrate tests. further identification of the isolates was done by using api 20e system, as in figure (1). e. coli (4) figure 1: identification of e. coli by api20e system antimicrobial sensitivity test 1-qualitative method (disc diffusion test) in this study we found that antimicrobials sensitivity among e. coli isolates varied according to the nature of antimicrobials. the percentage of resistant isolates to each antimicrobial is shown in figure (2).standard disc diffusion assay was used to detect the sensitivity of pathogenic bacteria and results obtained were compared with those of clinical and laboratory standard institute (9) .the results of the current study (figure 2) revealed that most of e. coli isolates resist the βlactam antimicrobials (like ampicillin and amoxicillin) (10) .noted the high resistance rates of gram positive and gram negative species to penicillins and some of cephalosporins. increasing of bacterial resistance rates to this group of antimicrobials may be a result of either production of β lactamase enzyme that had the ability to destroy the βlactam ring in these antimicrobials (11, 12) . also it may be due to minimizing the interaction of antimicrobials with target site (penicillin binding proteins) (13) .augamentin ( amoxicillin + clavulanic acid) had more activity than other penicillin due to its presence of clavulanic acid, which inhibit βlactamase enzyme, and increase the spectrum of amoxicillin against grampositive iraqi j pharm sci, vol.20(2) 2011 combinations of antimicrobials against resistant e. coli 68 and gramnegative bacteria (14) . many research illustrated the higher activity of imipenem and meropenem (related to carbapenems group) against grampositive and gramnegative bacteria (15) .regarding aminoglycoside group, amikacin was more active than gentamicin on the current e. coli isolates, many researches showed that the increasing resistance against aminoglycoside group was due to production of the modified enzymes and losing outer membrane pores, which are responsible of permeability of surface cell layer to antimicrobials (16) . the current results (figure 2) was in agreement with that of shevelev et al. (2002) (17) who found in a study that the resistance percentage of the isolates to amikacin was (0%) , while the resistant rate to gentamicin was (48.6%). the results also was in agreement with bashir et al.( 2008) (18) who found in a study in pakistan that the resistance percentage of the isolates to gentamicin was (49%) . resistant to tobramycin was (40.7%) and this result was near that found by pape et al. (2004) (19) who found that the resistant percentage of e. coli to tobramycin was (30%).many studies were illustrated the activity of naldixic acid, and most of quinolones antimicrobials against wide range of bacteria that were in a good agreement with the currently result. for example the resistant rate to ciprofloxacin was (40.7%) this result was comparable to the result of shamm et al.(2001) (20) found in a study that the resistant percentage of e. coli to ciprofloxacin was (39%). resistance to pipracillin was (85.5%), this result was in agreement with that of bujdakova et al.(1998) (21) who found that (86%) of e. coli isolates resistant to pipracillin , and this may be due to the ability of e. coli to develop resistance to these antimicrobials through the production of β-lactamase enzyme which break the β-lactam ring of pipracillin. resistance to nitrofurantoin was (2.6%), this result was in agreement with akyar (2008) (22) who found that the resistant rate of e. coli against nitrofurantoin was (3%).resistance to trimethoprim/ sulfamethoxazole (sxt) was (43.4%), this result may be attributed to the wide use of (sxt) as empirical therapy for urinary tract infection, however this result was in agreement with gupta; hooton and stamm (2001) (23) who found that the resistance to (sxt) among e. coli isolates from patient with utis has increased, with a prevalence of resistance which is reported 30 to 50 percent . figure 2: percentage of resistant e. coli isolates to antimicrobial tob: tobramycin; cn: gentamicin; sxt: triomethoprime and sulfamethoxazole; cip: ciprofloxacin; na: naldixic acid; ctx: cefotaxime; ipm: imipenim;am: ampicillin; cl: cephalexin; cro:ceftriaxone;amc:amoxicillin and clavulonicacid; f:nitrofurantoin; azm:azithromycin;prl:pipracillin;mpm: meropenem; ax:amoxicillin ak:amikacin 2quantitative method (minimum inhibitory concentration) (mic) table 1 showed that mic of meropenem ranged from (0.003-12.5μg/ml) this result was in agreement with marie et al. (24) who found in his study that e. coli was moderately susceptible to meropenem at mic (8μg/ml) .the results of this study also showed that the mic of gentamicin ranged from (12.5 to 480 μg/ml), this result was in agreement with jakobsem et al. (25) who found in his study that the mic of gentamicin distributed from (8-› 512 μg/ml)..on the other hand mic of iraqi j pharm sci, vol.20(2) 2011 combinations of antimicrobials against resistant e. coli 69 amikacin ranged from (0.3-2.5μg/ml), this result was in agreement with shrivastava and chaudhary (26) whose found that the mic of amikacin in e. coli was (2μg/ml).while celine et al. (27) who found in his study that the mic of amikacin in e. coli ranged from (1 to16 μg/ml). table 1: mic value for three antimicrobials (µg/ml) tested against e. coli isolates e. coli isolates meropenem µg/ml gentamicin µg/ml amikacin µg/ml mic mbc mic mbc mic mbc a1 0.12 0.125 300 300 2.5 5 a2 1.25 12.5 200 300 1.25 2.5 a3 0.12 1.25 300 300 1.25 2.5 a4 1.25 1.25 300 480 0.6 1.25 a6 0.12 1.25 480 480 0.3 0.6 a7 1.25 1.25 300 300 2.5 5 a10 12.5 12.5 480 480 0.6 1.25 a11 0.003 0.003 12.5 12.5 1.25 2.5 a13 0.12 0.12 300 300 2.5 5 a24 0.12 1.2 300 300 1.25 2.5 a28 0.03 0.03 200 200 1.25 2.5 a32 12.5 12.5 480 480 0.6 1.25 a35 1.25 1.25 200 300 0.6 1.25 a37 0.12 1.25 480 480 0.3 0.6 a41 12.5 12.5 100 200 1.25 2.5 a42 12.5 12.5 100 200 1.25 2.5 a43 1.25 1.25 200 300 0.6 1.25 a44 0.06 0.06 200 300 2.5 5 a45 1.25 12.5 200 300 1.25 2.5 a47 0.12 1.25 480 480 0.3 0.6 a51 12.5 12.5 100 200 1.25 2.5 a55 0.06 0.03 200 300 2.5 5 a57 0.12 1.25 480 480 0.3 0.6 a58 0.12 0.12 300 300 2.5 5 a67 0.12 0.125 300 300 2.5 5 lsd value 4.945 * 5.418 * 137.95 * 118.38 * 0.830 * 1.651 * * (p<0.05), lsd: least significant difference 3antimicrobials combination the result in table2 shows that the synergistic effect noticed from combination of meropenem with gentamicin on isolate no. (1, 2, 3, 4, 6, 7, 10, 13, 24, 28, 35, 37, 41, 42, 43, 44, 45, 47, 51, 55, 57, 58, 67), this result similar to that shown by richared et al. (28) found that aminoglycoside synergized with βlactams antimicrobials against e. coli isolates, because of the latter action on cell wall synthesis, which enhance diffusion of the aminoglycoside into the bacterium. while isolate no.(32) show the additive effect with combination of meropenem with gentamicin, and that may be due to their resistance to gentamicin (mic 480) and to meropenem (mic 12.5). table3 shows another synergistic effect resulted from combination of meropenem with amikacin when its effect tested on isolate no. (1, 2, 3, 4, 7, 10, 13, 24, 28, 37, 41,42,43, 44,45,47, 51, 55, 57, 58, 67) this result was in agreement with and piroska et al. (29) whose found that there is synergistic effect result from combination of meropenem with amikacin against e. coli isolates . while isolates no. (6, 32, 35) showed no effect toward combination of meropenem with amikacin.on the other hand combination of amikacin with gentamicin ( table 4 showed additive effect when tested on isolates no. (1, 2) but other isolates show no effect. iraqi j pharm sci, vol.20(2) 2011 combinations of antimicrobials against resistant e. coli 70 table2: results of combination of meropenem with gentamicin (1/4+1/4mic) result fic mic of gentamicin after combination (ug/ml) mic of gentamicin before combination (ug/ml) mic of meropenem after combination (ug/ml) mic of meropenem before combination (ug/ml) e. coli isolates syn 0.5 75 300 0.03 0.12 a1 syn 0.5 50 200 0.31 1.25 a2 syn 0.5 75 300 0.03 0.12 a3 syn 0.5 75 300 0.31 1.25 a4 syn 0.5 120 480 0.03 0.12 a6 syn 0.5 75 300 0.31 1.25 a7 syn 0.5 120 480 3.125 12.5 a10 syn 0.5 75 300 0.03 0.12 a13 syn 0.5 50 200 0.007 0.03 a28 syn 0.5 75 300 0.03 0.12 a24 syn 0.5 50 200 0.31 1.25 a35 syn 0.5 120 480 0.03 0.12 a37 syn 0.5 25 100 3.12 12.5 a41 syn 0.5 25 100 3.12 12.5 a42 syn 0.5 50 200 0.31 1.25 a43 syn 0.5 50 200 0.015 0.06 a44 syn 0.5 50 200 0.31 1.25 a45 syn 0.5 120 480 0.03 0.12 a47 syn 0.5 25 100 3.12 12.5 a51 syn 0.5 50 200 0.01 0.06 a55 syn 0.5 120 480 0.03 0.12 a57 syn 0.5 75 300 0.03 0.12 a58 syn 0.5 75 300 0.03 0.12 a67 --122.23 * 213.56 * 4.234 * 5.030 * lsd value * (p<0.05); lsd: least significant difference; syn: synergism; fic: fractional inhibitory concentration table 3: results of combination of meropenem with amikacin (1/4+1/4 mic): result fic mic of amikacin after combination (ug/ml) mic of amikacin before combination (ug/ml) mic of meropenem after combination (ug/ml) mic of meropenem before combination (ug/ml) e. coli isolates syn 0.5 0.62 2.5 0.03 0.12 a1 syn 0.5 0.31 1.25 0.31 1.25 a2 syn 0.5 0.31 1.25 0.03 0.12 a3 syn 0.5 0.15 0.6 0.31 1.25 a4 syn 0.5 0.62 2.5 0.31 1.25 a7 syn 0.5 0.15 0.6 3.12 12.5 a10 syn 0.5 0.62 2.5 0.03 0.12 a13 syn 0.5 0.31 1.25 0.03 0.12 a24 syn 0.5 0.31 1.25 0.007 0.03 a28 syn 0.5 0.07 0.3 0.03 0.12 a37 syn 0.5 0.31 1.25 3.12 12.5 a41 syn 0.5 0.31 1.25 3.12 12.5 a42 syn 0.5 0.15 0.6 0.31 1.25 a43 syn 0.5 0.62 2.5 0.01 0.06 a44 syn 0.5 0.31 1.25 0.31 1.25 a45 syn 0.5 0.07 0.3 0.03 0.12 a47 syn 0.5 0.31 1.25 3.12 12.5 a51 syn 0.5 0.62 2.5 0.01 0.06 a55 syn 0.5 0.07 0.3 0.03 0.12 a57 syn 0.5 0.62 2.5 0.03 0.12 a58 syn 0.5 0.62 2.5 0.03 0.12 a67 --122.23 * 213.56 * 4.234 * 5.030 * lsd value * (p<0.05); lsd: least significant difference; syn: synergism; fic: fractional inhibitory concentrations iraqi j pharm sci, vol.20(2) 2011 combinations of antimicrobials against resistant e. coli 71 table 4: antimicrobials combination (1/2+1/2 mic for each antimicrobials) e. coli isolates antimicrobials combination mic of first antimicroal alone (µg/ml) mic of first antimicrobial in combination (µg/ml) mic of second antimicrobial alone (µg/ml) mic of second antimicrobial in combination (µg/ml) fic results a32 mpm+cn 12. 5 6. 25 480 240 1 add a1 ak+cn 2. 5 1. 25 300 150 1 add a2 ak+cn 1.25 0. 625 200 100 1 add add: addition; fic: fractional inhibitory concentration mpm: meropenem; cn: gentamicin; ak: amikacin. extraction of plasmid dna the result of figures (3and 4) indicate that each of the isolates (a6 , a37)containing two bands of plasmid dna with approximate molecular weight (2000 and 1900) bp comparing with molecular weight marker. also, isolates no.(a32, a57) containing one plasmid dna with approximate molecular weight (2000) bp when comparing with molecular weight marker.there are many studies referred to the isolation of antimicrobial resistance plasmid from e. coli. joseph et al. (2001) (30) found in their study that e. coli isolates contain plasmid coding for resistance of aminoglycoside antimicrobials, including gentamicin and tobramycin. also, march galimand et al(2003) (31) found in their study that e. coli isolated from patient suffering from urinary tract infection contain plasmid coding high level of resistance to aminoglycoside. piddock (1999) (32) found in his study that e. coli contain plasmid coding for resistance of flouroquinolone .sisson et al. (2002) (33) found in their study that resistance to nitrofurantoin may be chromosomal or plasmid mediated. minch chau phuc nguyen et al. (34) found in their study that the plasmid gene that confers resistance to azithromycin had recently emerged in non multidrug resistant e. coli; philippon; arlet and jacoby (2002) (35) found in their study that e. coli contains plasmid coding for resistance of ampicillin. in the other hand, other e. coli isolates that show no plasmid may be due to carrying plasmids with low copy number. figure 3: plasmid profile of e. coli strains lane (a6, a37, a57, a32): plasmid dna extracted from e. coli strains; m.w: molecular weight marker of lambda dna digested with ecori+hindiii . electrophoresis was carried in 0.8% agarose gel at (2v/cm) for 30 min. 5148-4973bp 4268bp 3530bp 2027bp 1904bp 1584bp 1375bp 947bp 831bp 564bp chromosome plasmids iraqi j pharm sci, vol.20(2) 2011 combinations of antimicrobials against resistant e. coli 72 figure 4: plasmid profile of e. coli strains isolated from utis patients lane (a6, a37, a57, a32): plasmid dna extracted from e. coli strains; m.w: molecular weight marker of lambda dna digested with ecori+hindiii . electrophoresis was carried in 0.8% agarose gel at (2v/cm) for 60 min. reference 1. david, s.; 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a.; arlet, b.; jacoby, ga. plasmid –determined ampctype βlactamases. antimicrobial agent chemotherapy; 2002; 46:1-11. iraqi j pharm sci, vol.28(2) 2019 burn treatment by umbilical cord serum doi: https://doi.org/10.31351/vol28iss2pp165-173 165 use of human umbilical cord serum to treat animal skin burns intesar j. alramahi*, hanan j. kassab**,1, maysoon a. merdaw***, alaa a. alasadi****, sawsan al mousauy****, rownak ahmed ****, israa ismaeel**** and dorees al-sultani****. *ibn sina drug research center (director), ministry of industry and minerals, baghdad, iraq. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq ***department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad, iraq. ****al-razi center for research and diagnostic kits, general commission for research and industrial, department, ministry of industry and minerals, baghdad, iraq abstract the objective of the study was to test the hypothesis that human umbilical cord blood crude serum applied topically may promote an early healing for animal models with burn injury. human umbilical cord serum hucs was collected and screened for transmitted diseases such as hepatitis b, hepatitis c and hiv. the hucs was subjected to microbial testing to demonstrate the presence or absence of viable contaminating microorganisms. mice (no=45) and rabbits (n=16) were scalded by boiling water then treated with hucs in comparison to untreated animals. another group of rabbits (n=24) were subjected to hot water (70°c) and 1n naoh and treated with hucs in comparison to cetrimide 0.5% cream treated group and control group (without treatment). topical application of hucs promoted the healing process; complete healing was seen after 10 days in the mice group and 7 days in the rabbit group. cetrimide group applied to the second rabbit group showed slow healing and needed 10 days for hair regrowth. control group in both mice and the two rabbits’ group, showed very slow response and the burn area diameter remained the same for over 10 days, and no hair regrowth was observed after 10 days. in conclusion the results of the current study indicated that hucs is a promising therapy for healing of burns caused by boiling water alone or by alkali with hot water. more clinical trials are needed to explore the long-term effects after ucs use and incorporation of hucs in suitable dosage form. keywords: hucs, burn injury, cetrimide, animal models. استعمال مصل الحبل السري البشري لعالج حروق جلد الحيوان ، ****، سوسن الموسوي****، االء االسدي***ميسون عبد الزهرة مرداو ،1،**حنان جالل كساب، *انتصار الرماحي ****س السلطانييدور و ****، اسراء إسماعيل****رونق احمد سينا لألبحاث الدوائية، وزارة الصناعة والمعادن، بغداد، العراق مركز ابن* الصيدالنيات، كلية الصيدلة، جامعة بغداد، بغداد، العراق فرع** العلوم المختبرية السريرية، كلية الصيدلة، جامعة بغداد، بغداد، العراقفرع *** للبحث والتطوير الصناعي، وزارة الصناعة والمعادن، بغداد، العراقمركز الرازي للبحوث والعدد التشخصية، الهيأة العليا **** الخالصة الغاية من البحث هو إلثبات فرضية ان المصل المستخلص من دم الحبل السري لإلنسان قد يعجل الشفاء عند مسحه على جلد الحيوان بعد التأكد من خلوه من االمراض االنتقالية كالتهاب الكبد الفيروسي نوع )ب( و)ج( ومرض نقص hucsالمحروق. تم جمع مصل الحبل السري ( واالرانب 54المناعة المكتسب واخضع للتجارب الميكروبية المختبرية للتأكد من خلوه من البكتيريا والعفن المرضية. تم حرق جلد الفئران )عدد صل الحبل السري ومقارنتهم بالذي لم يتلق عالج. ومجموعة أخرى من االرانب تم حرقها بواسطة ماء ( بالماء المغلي وعالج البعض بم61)عدد ( بالمقارنة مع مجموعة لم تخضع cetrimide( وثم عالجها بمصل الحبل السري او بالستراميد )1n naoh)س( والصودا الكاوية °07حار ) أيام(، اما مجموعة االرانب التي 0أيام( واالرانب )خالل 67امل في مجموعة الفئران )خالل للعالج. ان مصل الحبل السري عجل الشفاء والتئام ك أيام لنمو الوبر من جديد. الحيوانات التي لم تتلق أي 67تم عالجها بالستراميد تحسنت ولكن ابطأ من المجموعة التي عولجت بالمصل واحتاجت لنوعي الحرق، أظهرت تحسن بطيء جدا وبقي مساحة الحرق كبيرة، ولم ينم وبرها حتى بعد عالج سواء كانت الفئران واالرانب التي تعرضت مايام. نستنتج ان مصل الحبل السري وسيلة فعالة لتعجيل الشفاء للحروق الناتجة عن الماء المغلي او الماء الحار والصودا الكاوية. ويلز67مرور . بعد استخدام المصل ودراسة طرق تحضيره كمستحضر صيدالني دراسة سريرية مستفيضة لمعرفة االثار الناتجة الكلمات المفتاحية: مصل الحبل السري، حروق جلد الحيوان، الستراميد. *corresponding author e-mail : hanan70k@gmail.com received: 22/2/2019 accepted: 7/9/2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp165-173 mailto:hanan70k@gmail.com iraqi j pharm sci, vol.28(2) 2019 burn treatment by umbilical cord serum 166 introduction burn is a thermal injury caused by contact to a physical, chemical or electrical source (1) and degree of the burn depends on the temperature of the skin, temperature of heat source and the duration of exposure (2). burn injury represents a cellular stress in the skin. burns are associated with inflammation which exacerbate pain and impair wound healing, and infection due to damage of the external epidermal layer which disrupts the innate immune system and increases susceptibility to bacterial infection (3). one of the most important complications of burn healing is infection, especially burnt inpatients which suffer from multi-drug resistance (4). burns can result in fibro proliferative scarring, skin contractures, or chronic wounds that take weeks or months to heal. burn injuries are highly individualized owing to wound-specific differences such as burn depth and surface area, in addition to patient-specific factors including genetics, immune competency, and age (5). other extrinsic complications such as microbial infection can complicate wound healing, resulting in prolonged inflammation and delayed reepithelialization, with high risk of hypertrophic scarring and infection which can lead to the development of a pronounced immune response, accompanied by sepsis or septic shock, which results in hypotension and impaired perfusion of end organs, including the skin – all processes that delay wound healing (3,6,7). burns are classified into three main grades; first grade (epidermal layer), second grade superficial (superficial dermal), deep second grade (mid dermal) and the third grade (deep dermal and full dermal thickness), the third grade burns are painless, with dry appearance, with no bleeding, this type of burn is hard to heal (1,8). management of burn include antibacterial administration (topical and oral), and moisture retention dressing and /or graft replacement in third grade burns (9). sometimes antibiotics alone fail to eradicate the microorganisms causing infection, therefore the use of unconventional treatment is needed (10). human umbilical cord serum hucs normally contains two arteries and a single vein, all embedded in wharton’s jelly, which is a collagenous matrix within the umbilical cord that protects the umbilical vessels and contains an abundance of hyaluronic acid (ha)(11) . the possibility of hucs topical application to wounds, including burns, can provide a cheap and accessible wound care for many of the neediest patients in the world (12,13). like peripheral blood serum, hucs contains hyaluronic acid (14) and many growth factors and stem cells (15). therefore, it can support healing, by providing the skin basic elements for regeneration that are lacking in the conventional medications (16). human umbilical cord blood serum was proposed to improve morbidity and mortality (13). since 2003 the number of studies using stem cell from different origin (adipose tissue, bone marrow, and umbilical cord). have increased, either applied topically as a collagen-chitosan scaffold or as biodegradable poly (β-amino esters) nanoparticles or via intradermal injection (17-20). but the number of studies using umbilical cord blood for skin injuries remains minor; liu et al. injected ucb intravenously into burnt mice, while dai et al. isolated mononuclear cells from mesenchymal stem cells of human umbilical cord blood and transplanted into injured nude mice by tail vein injection (21,22). there are several advantages which the cord blood serum can offer than other blood-derived preparations; mainly, a large volume of serum can be gained from the umbilical vein at one time, to overcome the need for sampling of the peripheral blood from each patient and avoid repeated blood collection from patients. furthermore, avoid the difficulty of obtaining blood from patients with infectious diseases or with abnormal materials in blood dyscrasia, also it is considered as medical waste (23,24). burn healing in third grade burns by human umbilical cord serum (hucs) was investigated in this study. in the current study, acute burn injury was inflicted, and the subcutaneous layer (full thickness) was compromised by boiling water or by hot water (70°c) with 1n naoh in which, infection is the major associated complication. materials and methods umbilical cord collection fresh human umbilical cord blood was collected from donor pregnant women at the time of delivery from al-karkh hospital (after receiving their consent) and screened for transmitted diseases such as hepatitis b, hepatitis c and hiv. the cords and placenta were collected immediately after delivery and the blood was drained by gravity from the placenta and strained by hands and collected in sterile containers. the blood was left to clot, and the serum was isolated by centrifuging at 2000 rpm for 10 min and stored at -20°c in vials each containing 1ml (25). sterility test the hucs was subjected to microbial testing. all sampling was carried out under aseptic conditions in a laminar flow cabinet, and hucs was streaked on a nutrient agar plate (n/a) then incubated for 24 hours at 37° c to demonstrate the presence or absence of extraneous viable contaminating microorganisms. iraqi j pharm sci, vol.28(2) 2019 burn treatment by umbilical cord serum 167 müeller-hinton agar (mha) is more commonly used for the routine susceptibility testing of nonfastidious (26).well diffusion assay was done in mha using a sterile cork borer to punch holes on an agar plate; using the swab with the test organism to streak the mha plate for a lawn of growth. the selected microorganisms included the common skin and mucosal contaminants staphylococcus epidermidis (atcc 700562) and staphylococcus aureus (atcc 6538), respectively; the pathogenic gram-negative pseudomonas aeruginosa (atcc 15442); and candida albicans (atcc 10231), a yeast commonly isolated from umbilical cord blood. after the streaking is complete, the plate was allowed to dry for 5 minutes. the hucs was placed in a well to study the antimicrobial effect. then the inoculated müeller-hinton agar plates were placed in an incubator for 24 hours at 37° c. animals mice weighing 18-23g (10-12-wk. old) and albino rabbits weighing 2-2.5kg (6 m. old) from the laboratory animal house of al-razi center in accordance with the institutional animal care and use committee. the animals were kept in the animal house at a temperature of about 25°c, in which the sun was the light source, and they were provided with standard diet and drinking water ad libitum. the animals were held tight and their back hair was shaved. thermal burns were induced on the animals by two methods, first; wet thermal source (scalds), this was performed on the mice and rabbits, the shaved area was subjected to boiling water (100ºc) for 5 sec., the burn diameter for mice was about 1cm in diameter, while the first rabbit group burns were about 7-8 cm in diameter (27). while the rabbit second group which were subjected to both scalding by water 70°c for 5sec., and chemical burning by applying 2-3 drops of (1 n naoh) on 4 circles points on each rabbit’s back of about 1 cm2; all the animals showed 3rd degree burns. this procedure was sufficient to induce burns of third grade (24). mice were chosen due larger population possibility (27) .rabbits were chosen because of larger size and will not be affected by the burn area size (28). methods in the initial experiments hucs was taken out of the freezer on the day of experiment and thawed to room temperature and applied few drops to sterile gauze. the mice and rabbits’ wet thermal burns were dressed daily with hucs impregnated sterile gauze. digital photographs were taken on day 3, 7, 10 and 20 for macroscopic evaluation to measure the wound surface and wound contracture and compared with control (animals without treatment). while, the hucs was swabbed on rabbits back (burnt by wet thermal and chemical burns daily). the treatment method was divided into three groups:  group a this group received hucs treatment.  group b which received (cetrimide 0.5% cream) celavex® sdi a mild antiseptic daily.  group c which didn’t receive any treatment as a control. statistical analysis the statistical analysis systemsas (2012) program was used to detect the effect of difference groups in study parameters. least significant difference –lsd test or t-test was used to significant compare between means (0.05 and 0.01 probability) in this study (29). results and discussion microbiological assessment showed after incubation under testing conditions, no growth was seen on nutrient agar streaked with the hucs (figure 1a, 1b) which means that hucs did not support microbial growth, and no inhibition zone was seen (figure 1c, 1d) against the tested microbes. however, satisfactory results, only indicated that, no contaminating microorganism had been found in the sample examined under the conditions of the test, also cord serum had bacteriostatic effects due to antimicrobial peptides (30,31). iraqi j pharm sci, vol.28(2) 2019 burn treatment by umbilical cord serum 168 the efficacy of hucs in the healing of skin burns was evaluated by the clinical evidence of injured area decrease in diameter, inflammation reduction, and hair regrowth after treatment. the results may be related to growth factor supplementation, and angiogenesis (32).the burnt area was proportional to the animals’ surface area. results in table 1 and figure 2 showed the burn healing process when the hucs was applied topically to mice, in 3 days the burn area started to contract and fibrin clot was formed, and after 10 days remission was complete, and the hair started to grow again. the accelerated healing process by hucs was evident, and mice back hair grew tremendously faster after hucs application in comparison with control group. although experiments using mice are more convenient, but the mouse model has many disadvantages, mouse have a thinner epidermis and dermis compared to humans, the mouse hair cycle is usually three weeks, where as human hair cycles can last several years (33). also, mice wound healing process involves contraction while human healing process involves re-epithelialization and granulation (34). table 1. comparative mice groups at different stages of treatment with control subjected to scalding duration of treatment with hucs (day) treated group untreated control group t-test (p-value) 0 no of animals (n=25) (n=20) burn area % 23.86034 ± 0.382082 23.86034 ± 0.382082 1.035 ns (0.548) 3 no of animals (n=17) (85%) death % 15% died (n=3) burn area % 15.97429 ± 2.172889 18.81667±2.016667 4.932 ns (0.092) 7 no of animals (n=15) death % 11.7% died (n=2) overall (25%) burn area % 6.034755 ± 0.289341 11.20269 ± 2.434742 3.834 * (0.0372) 10 no of animals (n=10) death % 33.3% died (n=5) overall (50%) burn area % 3.125701 ± 0.52276 6.357143±0.071429 1.638 * (0.0252) 20 no of animals (n=7) death % 30% died (n=3) overall (65%) burn area % 0.721154 ± 0.240385 3.911765±1.088235 1.085 * (0.349) * (p<0.05), ns: non-significant. a b c d figure 1.microbiological assessement of the hucs on nutrient agar (a, b) and müeller-hinton agar (c, d) iraqi j pharm sci, vol.28(2) 2019 burn treatment by umbilical cord serum 169 day after treatment treatment group control group 0 3 7 10 20 figure 2. the difference in healing process between the mice treated with hucs in comparison with control without treatment iraqi j pharm sci, vol.28(2) 2019 burn treatment by umbilical cord serum 170 rabbits were used as a second model; the results were almost the same as mice. the rabbits were subjected to boiling water (figure 3, and table 2), the burn area of the group treated with hucs showed accelerated healing and wound coverage with complete remission in 3 weeks in comparison with the untreated group. table 2. comparative burn area (%) in rabbit groups subjected to scalding by boiling water ** (p < 0.01), ns: non-significant, (n = 8) rabbit group subjected to boiling water scalds day treatment group control 0 7 10 20 figure 3. the difference in healing process between the scalded rabbit group treated with hucs in comparison with control without treatment while the second group of rabbits which were subjected to both hot water (70°c) and 1 n naoh burns (group a) which received the hucs as shown in the figure 3, showed the fastest healing, decrease in diameter of the burn, reduced inflammation around the burn area, formation of fibrin clot at day 3 and hair regrowth after 7 days . group b which received cetrimide showed slow healing , after 7 days the wound formed a fibrin clot and the inflammation area around the duration of treatment with hucs (day) treated group burn area % untreated control group burn area % t-test (p-value) 0 22.91667±2.172889 22.91667±2.172889 3.784 ns (0.688) 3 11.15177± 0.037422 19.21856±0.01221 1.0953 ** (0.0001) 7 7.414966± 0.748299 17.84059±0.697734 3.029 ** (0.0001) 10 3.846154± 0.675676 11.86331± 0.636694 3.261 ** (0.0001) 20 0.500992± 0.054563 7.450054± 0.713212 1.369 ** (0.0001) iraqi j pharm sci, vol.28(2) 2019 burn treatment by umbilical cord serum 171 burn decreased and needed 10 days for hair regrowth. group c showed very slow response and burn area diameter remained the same for over 10 days, the inflammation was at its peak at day 3, fibrin clot formed slowly, and no hair regrowth was obvious after 10 days. table 3. comparative burn area (%) in rabbit groups subjected to hot water (70°c) and chemical burns within naoh duration of treatment with hucs (day) group a (hucs applied) burn area % group b (cream 0.5%) burn area % group c control burn area % lsd (p-value) 0 48.4308±5.039109 48.4308±5.039109 48.4308±5.039109 4.711 ns (0.794) 3 26.07895±2.453303 33.73737±2.626263 37.69737±0.197368 2.176 * (0.0377) 7 22.04199± 1.046244 26.02026±1.252463 25.15527±0.796291 2.017 * (0.046) 10 15.57353± 2.073529 20.51948±2.337662 22.22413±2.134844 4.703 * (0.0359) 20 8.465608± 1.058201 12.41176±3.588235 14.53595±1.464052 3.066 ** (0.0027) * (p<0.05), ** (p<0.01), ns: non-significant, lsd least significant difference (n = 8). hot water (70°c) and 1 n naoh day group a (hucs) treated group b (cetrimide) treated group c control (without treatment) 3 7 10 20 figure 4. the difference in healing process between rabbit group subjected to both hot water (70°) and 1 n naoh burns treated with hucs in comparison with those treated with 0.5% cream and control without treatment iraqi j pharm sci, vol.28(2) 2019 burn treatment by umbilical cord serum 172 the use of hucs topically accelerated the burn healing without any side effects or any sign of injury microbial contamination. wharton's jelly are reported to possesses biological properties (collagen 1, hyaluronic acid, laminin, and fibronectin) that stimulate cellular responses important for soft tissue healing and promote scar-less wound healing (11). the level of satisfaction with the hucs treatment was very high, for all animals tested, with improvement of their skin injury as early as day 3, reaching full satisfaction at endpoint of 3 weeks of the experiment. the hucs had been shown to have anti-inflammatory effects which promote angiogenesis and enhance healing. therefore; hucs was recommended for burnt human. conclusion in conclusion, this work confirmed that hucs is a promising therapy for the healing of burns by alkali substance and/hot or boiling water. the area exposed to boiling water or hot water with alkali decreased in size and the burn improved after 10 days of treatment. the extent of area of burn injury rather than its duration was the main factor determining the early response and repair process. the hucs need to be prepared by well-equipped and trained laboratory staff. preparation steps include efficacy and microbiology controls for a reproducible quality of the final product. more clinical trials are needed to further explore the longterm effects with the hucs and ultimately provide safer and more effective therapies for future clinical applications. acknowledgments the authors gratefully thank the al-razi center for research and diagnostic kits/ general commission for research and industrial department/ ministry of industry and minerals for helping in carrying out this research to a fruitful outcome. references 1. ja g, vb a, eh o, 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stromal cells in a rat model of stroke. basic exp res. 2009;87(3):350–9. 33. mccoll d, cartlidge b, connolly p. real-time monitoring of moisture levels in wound dressings in vitro: an experimental study. int j surg. 2007;5(5):316–22. 34. gurtner gc, wong vw, sorkin m, et al. surgical approaches to create murine models of human wound healing. j biomed biotechnol. 2011;2011(article id 969618):8. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ the role of chloroquine phosphate on acute phase reactant proteins in patient with knee osteoarthritis iraqi j pharm sci , vol.18 (1) ,2009 chloroquine and knee osteoarthritis 56 the role of chloroquine phosphate on acute phase reactant proteins in patients with knee osteoarthritis # eman s. saleh *, 1 , kismet m.turki ** and mohammed h.al-osami *** *department of clinical laboratory sciences,college of pharmacy,university of baghdad,baghdad , iraq. ** college of medicine , university of baghdad , baghdad , iraq . ***department of rheumatology and rehabilitation . medical baghdad city ,baghdad , iraq . abstract the acute phase response is a major pathophysiologic phenomenon that accompanies inflammation whether acute or chronic. complement (c3 and c4) and c reactive protein (crp) are positive acute phase proteins (+ ve apps ). their production takes place in hepatocyte and the blood concentration of these parameters are increased in osteoarthritis (oa). chloroquine (cq) is a diprotic weak base traditionally used to treat malaria. recently the phosphate salt of cq is used to decrease this type of (+ve apps) . in this study, patients who suffered from knee osteoarthritis (koa) are treated with oral dosage form of chloroquine phosphate (cqp) for one month, twice daily. our results demonstrate that cqp improves the patient status by decreasing complement and c-reactive protein in blood. key words: chloroquine , knee osteoarthritis , acute phase proteins , complement , c-reactive protein. الخالصة نذٖ انًشضٗ انزيٍ يعإٌَ crp, c4, c3نقذ ذًد دساعح دٔس عقاس فٕعفاخ انكهٕساكٕيٍ عهٗ يغرٕٖ انثشٔذيُاخ انرفاعهيح 22اَثٗ ٔ 45يشيضا ) 44ركش( ٔ 25أَثٗ , 35شخصا يٍ االصحاء ) 55يٍ انفصال انعظًي نهشكثح.أجشيد ذجشتح عشٕائيح عهٗ ( ذى ذشخيصٓى يٍ قثم انطثية 2552ُح انطة انعيادج االعرشاسيح يٍ) شٓش كإٌَ انثاَٗ انٗ ايهٕل ركش( في يغرشفٗ يذي االخرصاصي حغة طشيقح انكهيّ األيشيكيّ نًثحث انشثيّ حيث ذى ذحشيض األعشاض تٕاعطح يؤشش ) كهيكشيٍ ٔنٕسَظ ( نهفصال ٔ c3يع يغرٕٖ كم يٍ أنًرًًيٍ c -د يغرٕٖ انثشٔذيٍ انرفاعهيانعظًي ، أخزخ صٕس شعاعيّ أياييّ خهفيّ نًفصم انشكثّ ٔحذ c4( يشذيٍ تانيٕو ٔنًذج شٓش. عحثد عيُح يٍ دو االصحاء ٔانًشضٗ 255. ٔصف نٓى حثٕب فٕعفاخ انكهٕساكٕيٍ ترشكيض )يهغى نرفاعهيح في انًصم. نٕحظ اَخفاض أالً ثى عيُح يٍ دو انًشضٗ انزيٍ اعرعًهٕا ْزا انعقاس تعذ شٓش نغشض قياط َغة انثشٔذيُاخ ا يغرٕياخ ْزِ انعٕايم تصٕسج ٔاضحح ٔيعُٕيح عُذ اعرعًال انعالج حيث صيادذٓا ذهعة دٔسا يًٓا في ذغشيع ذآكم انغضاسيف ٔذكٕيٍ .يحانُرٕءاخ انعظً introduction: oa is the most common disease in the world and a major cause of pain and disability which usually develops in distal inter phalangeal ( dip )joints of finger, the weight bearing joints of leg and the movable portion of spine (1) . it is associated with a breakdown of cartilage in any joint in the body (2) . pathologically, oa was defined as a gradual loss of articular cartilage combined with thickening of subchondral bone/ bony out growth (osteophyte) at joint margins with mild to chronic non specific synovial inflammation (3) . cq is 4 -aminoquinoline approved for treatment and prophylaxis of malaria. recently cqp is used by some authors in the oa as disease modifying anti –rheumatic drug ( dmard) (4) , claimed to cause lowering of blood level of proinflammatory mediators (5) . our study shows the effect of cqp on the serum concentration of crp, c3 and c4 in patients with koa.c-reactive protein is a laboratory marker that is important in the assessment of inflammation, serve as a predictor and indicator of response to therapy and overall outcome in various disorders (6) . the major function of crp is to bind phosphocholine, this is by permitting recognition of foreign pathogens and phospholipid constituents of damaged cells (7) , so the activation of complement system and / or binding to phagocytic cells will take place , and to initiate elimination of targeted cells by interaction with both humoral and cellular affecter system of inflammation , as aresult crp is a component of innate immune response (8) ,and useful in early detection of low grade inflammation (9) . # based on oral presentation in the seventh scientific conference of the college of pharmacy /university of baghdad held in 26-27 november 2008 . 1 corresponding author e-mail : www.faith_1960@yahoo.com received : 31/12/2008 accepted : 28/3/2009 http://www.faith_1960@yahoo.com/ iraqi j pharm sci , vol.18 (1) ,2009 chloroquine and knee osteoarthritis 57 c3 and c4 serve proinflammatory roles, including chemotaxis, plasma protein exudation at the site of inflammation and opsonization of infectious agents and damaged cells (10) .c3 is beta 2 protein 180 kda , cleave by c3 convertase into c3a and c3b. c3b reacts with factor b to produce more c3 convertase and activate c5, so the serum level of c3 is increased in acute inflammation (11) . c4 is beta -1 protein, 210 kda cleave by c1s to produce c4a and c4b, c4b interact with c2b to activate classical pathway c3 convertase. (12) patients and method fifty healthy people (30 female and 20 male) as control and seventy four patients (45 female and 29 male) are selected randomly from the out patient clinic in baghdad teaching hospital / medical city / baghdad from january to september 2008 with inflammatory koa diagnosed according to american college of rheumatology (13) . all patients were assessed by kellegren and lawrence grading criteria for radiographic severity of knee osteoarthritis with different signs and symptoms such as joint pain, stiffness, bony enlargement, bony tenderness and crepitus (14) . their ages are ranged from ( 45 to 78 ) years with mean value ± standard error of mean ( 55.07 ± 6.18).chloroquine phosphate tablet (medoquine 250mg/ medochem company is equivalent to 150mg cq base), was prescribed by rheumatologist as twice daily after meal for one month,because cqp is 4-aminoquinoline derivative( cq+phosphate) ,it absorbed completely and rapidely from git ,its mean absorption half –life is four hours with a lag time slightly more than 30 minutes (15,16) ,and its duration of action is extended from sixteen to forty five days (17) .the serum analysis of all patients and control was done in the general health laboratory center / baghdad, before using this drug and one month later in order to estimate the level of c3, c4 and crp.c3 and c4 are determined by radial immune diffusion (11) while crp is assessed turbiditmetrically by antigen antibody reaction technique (17) . results were analysed by statistical package for social science (spss). results the presented data in this study showed the comparisons between level of c3,c4 in microgram per milliliters (μg/ml) and crp in milligram per liter(mg/ l) in healthy and patients before the treatment as well as after one month of using chloroquine phosphate are depicted in table (1),figures(1,2,3).there were a significant decrease in c3,c4 and crp in male,female and total patients compared to their level at baseline and control. table (1): the serum level of c3,c4 and crp before using cqp at baseline and after one month of the treatment. control baseline p value control, baseline after one month p value pre-,posttreatment c3 (μg/ml) t m f 950.1±21.1 877.3±17.3 987.4±20.2 1794.4±34.2 1788.2±48.8 1798.4±47.16 s p>0.01 1504.5±31.1 1503.4±40.9 1505.3±41.7 s p<0.05 c4 (μg/ml) t m f 320.23±12.2 318.1±15.7 307.2±13.4 396.08±14.6 367.7±20.2 414.3±19.9 s p>0.01 325.03±12.7 288.03±17.1 323.09±17.9 s p<0.05 crp (mg/l) t m f 1.08±0.1 1.1±0.12 2.09±0.17 4.3±0.3 3.8±0.6 4.63±0.4 s p>0.01 2.02±0.2 1.8±0.37 2.1±0.23 s p<0.05 sem: standard error of mean. significant p value : (p<0.05),(p>0.01). iraqi j pharm sci , vol.18 (1) ,2009 chloroquine and knee osteoarthritis 58 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 total male female control pre-treatment post-treatment figure (1): the level of serum c-reactive protein at baseline (pre-treatment), after one month of using chloroquine phosphate (post-treatment) and control. 0 200 400 600 800 1000 1200 1400 1600 1800 2000 total male female control pre-treatment post-treatment figure (2): the level of serum complement three at baseline and one month later of using chloroquine phosphate. 0 50 100 150 200 250 300 350 400 450 total male female control pre-treatment post-treatment figure (3): the level of serum complement four at baseline and one month later of using chloroquine phosphate. iraqi j pharm sci , vol.18 (1) ,2009 chloroquine and knee osteoarthritis 59 discussion c3, c4 and crp are components of +ve apps , their production are increased by hepatocyte (18) . the elevation of these proteins are detected in patient with oa which is due to releasing of inflammatory molecules (cytokines) (19) . jawad et al in 2004 (4) demonstrated that the using of cqp as dmard for three months in patients with koa, lead to decrease the serum crp level. in our study the presented data shows a significant decrease in this parameter after one month of using cqp (p<0.05), table (1), figure (1), so our result is in agreement with all explanation and findings. chloroquine phosphate decreases serum level of crp, c3 and c4 depending on it's ability to enter lysosomes and all acidic compartments of the cells (lysosomotropic effect) (20) . it interferes with intracellular processing, receptor recycling (21) and the secretion of proteins which lead to decrease the production of cytokines and other inflammatory mediators decreases lymphocyte proliferation as an immune effect (22) . non lysosomotropic effect of cqp includes the inhibition of phospholipase, antagonization of prostaglandin stabilization of lysosomal membrane in synoviocytes (23,24,25) . in 2006 , numman et al used silymarin to treat patients with koa instead of cqp (26) . silymarin is a plant (mixture of flavolignans),isolated from the ripe seeds of silybum marianum (milk thistle), it proved to have effective inhibitory effects on cyclooxygenase and 5lipooxygenase in vitro and experimental animals (27) . their results showed that this drug when prescribed for two months in koa, it decreases the serum level of c3 and c4 significantly. our study shows a significant decrease in c3 and c4 at the end of trial ( p<0.05) table (1), figure (2) and (3). all findings, trials in addition to the mode of action of cqp are supported the results. conclusion: crp, c3 and c4 are decreased after using cqp for one month in patients with koa. recommendation further study is needed to asses other parameters in serum and synovial fluid. references 1. morehead k , sack ke . osteoarthritis : what therapies for this disease of many causes ? postgraduate med . 2003;114, 5. 2. brandt kd .osteoarthritis. in: kasper dl(ed) .harrison , s principles of-internalmedicine,6 th . ed ,mcgraw-hill companies,2005;2036-45. 3. berenbaum f. osteoarthritis: a. epidemiology, pathology and pathogenesis . in: klippl jh, crofford lj, stone jh, wegand cm (eds). primer on rheumatic disease 12 th ed. atlanta, georgia. arthritis foundation, 2001; p.285-89. 4. jawad hm, salman s ,mohammed l. the effect of chloroquine phosphate as a disease modifying agent in osteoarthritis . ph d thesis submitted to university of baghdad/college of medicine and the committee of postgraduate studies in clinical pharmacology, 2004. 5. jelab a, jawad hm, salman s, mohammed l: the effect of chloroquine phosphate on pro-inflammatory interleukins in osteoarthritis. msc thesis submitted to university of baghdad/college of medicine and the committee of postgraduate studies in clinical pharmacology,2007. 6. baddour vt , bradley jd . clinical assessment and significance of inflammation in knee osteoarthritis. curr rheumatol rep,1999;1:59-63. 7. volanakis je. acute phase protein in rheumatic disease. in: koopman wj (ed.).arthritis and allied condition. a textbook of rheumatology. williams wilkins, baltimore 1997; p.505. 8. hoffman ja, kafatos fc, janeway ca, ezekowitz ra.phylogenetic perspective in innate immunity.science,1999;248,1313. 9. visser m, bouter lm,mcquillan gm,et al. elevated creactive protein levels in over weight and obese adult .jama,1999;282, 2131-35. 10. molenaar et,voskuyl ae,et al. complement activation in patients with rheumatoid arthritis mediated in part by c-reactive protein. arthritis rheum, 2001;44, 997. 11. feldkamp cs. immunochemical techniques. in: kaplan la, pesce aj, kazmierczak sc (eds.) , fourth edition. textbook of clinical chemistry , mosby usa,2003, p.227-45. 12. kyle ra. classification and diagnosis of monoclonal gamma pathies. in: rose nr, friedman h and fahey jl (eds.). manual of clinical laboratory immunology (3 rd ed.). washington dc; american society of microbiology 1986; p.152. 13. recommandation for the medical management of osteoarthritis of the hip and knee.update. american college of iraqi j pharm sci , vol.18 (1) ,2009 chloroquine and knee osteoarthritis 60 rheumatology, subcommittee on osteoarthritis guidelines .arthritis rheum,2000;43,p.1905. 14. kellgren hj, lawrence js. radiologic assessment of osteoarthritis. ann rheum dis,1957;16, 494-501. 15. smith dg, aronson jk. drug for arthritis. in: grahame-smith dg(ed.), textbook of clinical pharmacology and drug therapy ,3 rd . ed ,oxford university press.2002;p.355-56, 509-10. 16. furst de. pharmacokinetics of hydroxychloroquine and chloroquine during treatment of rheumatic diseases. lupus,1996; 5, 11. 17. black s, kushner i, samols d. serum creactive protein. j biol. chem.,2004; 279, 478. 18. morley jj, kushner i. serum c-reactive protein level in diseases. ann ny acad. sci. ,1982; 389, 406. 19. gabay c, kushner i. acute phase protein and other systemic responses to inflammation. n engl. j med.,1999; 340, 448. 20. petri m. hydroxychloroquine use in baltimore lupus cohort: effect on lipid ,glucose and thrombosis.lupus,1996; 5, 16. 21. rynes ri: antimalarial drug . in: kelley wn , harris ed jr, ruddy s, sledge cb (eds.) .textbook of rheumatology, (5 th ed.) . wb saunders. philadelphia ,1997; p.59-1. 22. karres i, kremer jp, dietle i, steckholzer u, jochum m, ertel w. chloroquine inhibits proinflammatory cytokines release into human whole blood. am j physiol/ regul integ comp physiol, 1998; 274, 1058-64. 23. fox r. antimalarial drug. possible mechanism of action in autoimmune disease and prospects for drug development. lupus, 1996; 5, 4. 24. wallace dj. anti-malarial therapies in: wallace dj, hahu bh (eds.). dubois lupus erythematosus ( 5 th ed.) williams wilkins, baltimore, 1997;p. 1117. 25. stuhimeier km. mepacrine inhibits matrix metallo proteinase-1 (mmp-1) and (mmp-9) activation in human fibroblastlike synoviocyte. j rheumatol, 2003; 30, 2330. 26. numan it, hussain sa, al-ani ta. evaluation of the clinical use of silymarin in knee osteoarthritis: application of dual inhibition concept of cycloxygenase and 5-lipoxygnase, a ph d thesis submitted to university of baghdad/college of pharmacy and the committee of postgraduate studies in pharmacology, 2006. 27. pepping j. milk thistle: silybum marianum. am j health syst pharm,1999;56,1195-97. iraqi j pharm sci, vol.32( 1 ) 2023 sub-chronic effect of different doses of diclofenac sodium doi: https://doi.org/10.31351/vol32iss1pp227-236 227 sub-chronic effect of different doses of diclofenac sodium on female reproductive system in rats bedoor a. salim*, muhsin s.ghalib** and ausama ayob jaccob**,2 *ministry of health and environments , maysan health directorate, baby and maternity hospital, maysan, iraq. **department of pharmacology and toxicology, college of pharmacy, university of basra, basra, iraq. abstract non-steroidal anti-inflammatory drugs (nsaids) are widely utilized drugs in today's world. these medications are well-known for their anti-inflammatory and analgesic properties. the goal of this study was to suggest and explain the subchronic effects of low and high doses of diclofenac on female reproductive system in rats. a total of 24 female rats were divided into 4 groups, six rats in each. the first group was given distilled water as a control for 35 days, the second and third groups were given diclofenac (1 mg/kg) and (5 mg/kg) for 35 days respectively. the fourth group was given a combination of diclofenac and mefenamic acid for 35 days. hormonal, biochemical, and hematological tests were performed. low dose diclofenac showed no significant change regarding luteinizing hormone (lh), progesterone, prolactin, and glutathione, but an increase in follicle stimulating hormone ( fsh), and decrease in prostaglandin e2 pge2 and estrogen compared to control group were documented. in contrast, high dose diclofenac alone or combined with mefenamic acid showed significant impact on female reproductive system documented by biochemical and histopathological evaluations. at hematological levels diclofenac decrease red blood cells (rbc), hemoglobin concentration (hgb), and platelet account but no change in the total white blood cells (wbc) were found. sub-chronic use of diclofenac sodium (ds) alone or in combination with mefenamic acid have a deleterious impact on the female reproductive system, oxidative stress and hematological parameters. keywords: nsaids, diclofenac sodium, female reproductive toxicity. ناث أل الجهاز التناسلي على الصوديوم ديكلوفيناك من مختلفة لجرعات التأثير دون المزمن الجرذان ** يعقوبايوب أسامة و **غالب محسن صغير ,*عباس سالم بدور العراق. ، ميسان ، مستشفى الطفل والوالدة ، وزارة الصحة, دائرة صحة ميسان * .العراق البصرة، جامعةالبصرة، كليةالصيدلة، فرع االدوية والسموم، ** الخالصة المضادة لاللتهابات والمسكنات. تستخدم مضادات االلتهاب غير الستيرويدية على نطاق واسع في عالم اليوم. تشتهر هذه األدوية بخصائصها نثى في الفئران. كان الهدف من هذه الدراسة هو اقتراح وشرح التأثيرات دون المزمنة للجرعات المنخفضة والعالية من ديكلوفيناك على الجهاز التناسلي لأل ألولى أعطيت الماء المقطر كعنصر تحكم ، المجموعة مجموعات ، ستة فئران في كل مجموعة. المجموعة ا 4أنثى من الجرذان إلى 24تم تقسيم إجمالي مجم / كجم( على التوالي. المجموعة الرابعة أعطيت مزيج من ديكلوفيناك وحمض الميفيناميك لمدة 5مجم / كجم( و ) 1الثانية والثالثة أعطيت ديكلوفيناك ) ، lh، اك أي تغيير معنوي فيما يتعلق بـ. لم تظهر الجرعات المنخفضة من ديكلوفينيوما. تم إجراء االختبارات الهرمونية والكيميائية الحيوية والدموية 35 هرمون االستروجين مقارنة بمجموعة البروستاكالندين و وانخفاض في ، fshالبروجسترون ، البروالكتين ، والجلوتاثيون ، ولكن تم توثيق زيادة في يكلوفيناك بمفردها أو مع حمض الميفيناميك تأثيًرا كبيًرا على الجهاز التناسلي لألنثى موثقًا من خالل التحكم. في المقابل ، أظهرت الجرعات العالية من د ، وحساب الصفائح الدموية الهيموغلوبينالتقييمات البيوكيميائية والتشخيص المرضي . في مستويات الدم ، يقلل ديكلوفيناك من عدد كرات الدم الحمراء ، له تأثير ضار على حمض الميفينامك وحده أو باالشتراك مع لدايكلوفيناك االستخدام شبه المزمن . wbcلى أي تغيير في إجمالي ولكن لم يتم العثور ع .الجهاز التناسلي األنثوي ، واإلجهاد التأكسدي والمعايير الدموية . لألنثى التناسلية السمية ، الصوديوم ديكلوفيناك ، الستيرويدية غير االلتهاب مضادات: المفتاحية الكلمات introduction nonsteroidal anti-inflammatory drugs (nsaids) are a family of pharmaceuticals authorized by the fda for the treatment of different diseases as antipyretic, anti-inflammatory, and analgesic (1). they are indicated for muscular discomfort, dysmenorrhea, arthritic diseases, gout, and as opioid-sparing medications in some acute traumatic diseases (2,4). at the mechanistic levels, nsaids inhibit the production of prostaglandins by blocking the cyclooxygenase (cox) enzymes. cox enzymes are divided into two types: cox-1 and cox-2. the first one is constitutively responsible for the creation of prostaglandins necessary for organ function, gastric protection, platelet aggregation, and vasoconstriction. the cox-2 isoform is inducible and found in the kidneys and vascular endothelium, and it is only created when the body is inflamed (5). 1corresponding author e-mail: ausama.jaccob@uobasrah.edu.iq received: 14/4 / 2022 accepted: 26/ 6 / 2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp227-236 iraqi j pharm sci, vol.31(2) 2022 sub-chronic effect of different doses of diclofenac sodium 228 prostaglandins are made from the polyunsaturated phospholipid (arachidonic acid) by a series of multistep enzymatic processes, such enzymes are targeted by a variety of medications, the most common of which being (nsaids). it appears that chronic use of nsaids might have a negative impact on bodily tissues including renal dysfunction, blood pressure, hepatic damage, platelet inhibition, gastrointestinal, cardiovascular and reproductive system as recently studied organs(6,7). diclofenac sodium (ds) is one of the most commonly prescribed analgesic. it is a phenylacetic acid derivative that has been used as human medicine for many years to treat rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, and primary nocturnal enuresis (8). in the short term, it might potentially be used to treat acute musculoskeletal injuries and dysmenorrhea (9). the availability of ds as an over-the-counter medication may lead to its misuse, resulting in gastrointestinal, liver, renal, and neurological consequences(10–12). furthermore, it can be seen that the hematological and biochemical indicators of rats may be significantly affected by ds. as a consequence, it can be inferred that ds, at various dosages and over different periods, may have negative impacts on critical animal tissues, resulting in hematological disorders, hepatic and renal impairments (13). it was found that ds produce histological changes in the male rat's testicles, reduce sperms count, motility, viability, and significantly reduce testosterone level(10). little data are available regarding the effect of nsaids on the female reproductive system (frs), however, ovulation, whether primary or secondary may be affected. normally, prostaglandins play an essential role in the ovulation process. when luteinizing hormone (lh) surges and progesterone levels increase before the onset of ovulation, prostaglandins help to facilitate ovulation by digesting the collagen surrounding the ovarian follicle and also by stimulating smooth muscle contraction. however, the exact mechanism by which prostaglandins accomplish these steps is still unclear and required further research to declare it(14).nsaids can cause reversible infertility in women due to the suppression of cyclooxygenase enzymes and prevent formations of prostaglandin. since nsaids are widely used and abused in women during their reproductive years, it has been proven that these women will have "luteinized unruptured ovarian follicles"(15). fortunately, natural ovulation was reported to be retained after these agents were stopped(16). cölçimen n et al. investigated the morphological and prenatal follicle number after low dose (1mg/kg) ds administration, no significant differences were observed in pregnant rats compared to the remaining groups(17). due to the limited knowledge are available on the effect of nsaids on the female reproductive system, the present work was designed to evaluate the possible effect of subchronic administration of different doses of ds alone or in combination with mefenamic acid on the female reproductive system in rats. materials and methods materials diclofenac sodium obtained from alfayhaa pharmaceutical company in al-basrah city, iraq, mefenamic acid (ponstan forte) tablet as 500 mg active ingredient was purchased from (pfizer manufacturing deutschland gmbh, betriebsstatte freiburg, germany). all hormonal analysis kits were purchased from abbott co. (usa), while pge2 and gsh elisa kits, were obtained from shanghai yl biotech co. (china). animals this study employed twenty-four mature female albino rats weighing between 150 and 250 g. they purchased from the animal holding unit of the university of basrah, the college of veterinary medicine, iraq. the rats were maintained in plastic cages with a temperature of 25–30 °c and a 12-hour light/12-hour dark photoperiodicity. they were fed a standard pelletized diet and had free access to water daily, and were weighed before the beginning of the work as the starting point, then every 7days, and at the end of the experiment. after two weeks of acclimatization to the new laboratory environment, the rats were enrolled in the current study. the study followed the national institute of health guidelines for the use of laboratory animals, and ethical approval was received in october/2021 from the pharmacy college/university of basrah/ethics committee 3/5/293. experimental design twenty-four female rats were chosen randomly and divided into control and three (3) treated groups, with six animals in each group. the control group received only distilled water (as a vehicle of diclofenac sodium) orally administered daily to be compared and exclude vehicle, stress, and environmental effects. group 1 rats received 1mg/kg (9) of ds gavaged daily. group2 rats treated with a high dose of ds (5mg/kg) (18) were gavaged in a daily manner. group3 rats received a combination of both ds (5mg/kg) and mefenamic (20mg/kg) via the oral route. all control and treated groups administered their scheduled doses for a period of 35 days as sub-chronic study model. the rats were weighed every 7 days, and the doses changed accordingly. after the last day of medication treatment, 5 ml of blood from each rat was withdrawn via posterior vena cave with a needle syringe while they were anesthetized with chloroform. then the blood was distributed into two tubes one of them for hematological parameters evaluation, while the remaining were allowed to clot iraqi j pharm sci, vol.31(2) 2022 sub-chronic effect of different doses of diclofenac sodium 229 and centrifuged at 10000rpm for 20 minutes, and the resulting serum was extracted, frozen, to be used later for biochemical and hormonal analysis. the ovaries of the rats were excised and fixed in formalin for histopathological investigation. hormonal assay serum levels of lh, fsh, progesterone, estradiol, and prolactin were measured using prepared kits of chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of these hormones following the manufacturer's procedure while utilizing a diagnostic automated laboratory analyzer (abbott architect i4000, usa). biochemical assay serum glutathione and pge2 were determined using an elisa (enzyme-linked immune sorbent assay) based on the biotin double antibody sandwich technology according to the manufacturer's instructions. hematological parameters the anticoagulated blood was used to calculate the hematological parameters. red blood cells (rbc) count, hemoglobin (hb) concentration, packed cell volume (pcv), total white blood cells (wbc) count, monocytes count, lymphocytes count, granulocytes count, platelets count, mean corpuscular volume (mcv), mean hemoglobin concentration (mchc), and mean corpuscular hemoglobin (mch) were all determined using automated hematology analyzer nano 3 (genolabtek, windsor, canada). histopathological examination ovaries of each rat were sliced into tiny pieces, cleaned in normal saline, and stored in 10% formaldehyde. a sample of tissue was dried and buried in paraffin before being sliced into 3–4 mm slices and mounted on a thin glass slide. staining was done with hematoxylin and eosin (h&e), and then a comprehensive histopathological investigation was performed under supervision of professional pathologist. statistical analysis one-way anova analysis was used among groups. then turkey’s post-hoc analysis test was utilized for further assessment of data to obtain clear p values between groups. data were expressed as mean± sem with p<0.05 significance. graphpad prism software (version 6.0). results administration of ds for 35 days in group 1 and group2 decreased body weight gain compared to the control group as shown in figure (1). group3 rats received co-treatment of ds and ma as a combination to evaluate the possible sub-chronic unwanted effect of these compounds. no changes in body weights were documented when correlating statistically between changes in body weights and periods of time (35 days) p=0.0687. compared to the control group, changes in body weight in group3 were different and no weight gain was documented, nevertheless, there is a significant increase in body weight in the remaining groups. figure 1. changes in rats body weights with respect to days of the experiments. no significant changes in body weights during time were documented in in group 3 p>0.05. * represents significant difference in comparison to control p<0.05. results are presented as mean ± sem in this study, measurement of serum pge2as prognostic biomarker is important point to explain and correlate the relationship between nsaids use and female reproductive hormone effect. figure (2) clearly proves this idea where a significant reduction in serum pge2 level in group1 (treated with low dose ds), group2 (treated with a high dose of ds) p=0.0051 and group3 (treated with combination) p=0.0006 compared to the control group. figure 2. impact of sub-chronic use of low and high pharmacological doses of diclofenac sodium alone (group1, 2) and in combination with mefenamic acid (group3) on serum concentration of prostaglandin e2 (pge2) in rats (n=24). values are expressed as mean± sem. *represent significant difference p<0.05 among groups. serum concentrations of fsh were significantly elevated in all treated groups as shown iraqi j pharm sci, vol.31(2) 2022 sub-chronic effect of different doses of diclofenac sodium 230 in figure 3a in comparison to the control group. rats ingroup1 showed the highest significant value compared to other treated and control groups. administration of pharmacological doses of ds in combination with mefenamic acid significantly increase serum lh value in group3 as shown in figure 3b. groups 1 and 2 are well-matched with the control group and no significant differences were documented. figure 3. impact of sub-chronic use of low and high pharmacological doses of diclofenac sodium alone (group1, 2) and in combination with mefenamic acid (group3) on a: serum concentration of (fsh), b: serum concentration of (lh) in rats (n=24). values are expressed as mean± sem. *represent significant difference p<0.05 among groups. ** represent significant difference p<0.01 among groups. serum concentrations of estrogen were significantly reduced in all treated groups as shown in figure 4a compared to normal control group. oral administration of ds is associated negatively with serum estradiol concentrations. in contrast, administration of a high pharmacological dose of ds in (group2) and in combination with mefenamic acid (group3) significantly increases serum progesterone value compared to the normal control group and group1 as seen in figure 4b. groups 1 associated with a non-significant (p>0.05) compared to control. figure 4. impact of sub-chronic use of low and high pharmacological doses of diclofenac sodium alone (group1, 2) and in combination with mefenamic acid (group3) on a: serum concentration of estradiol, b: serum concentration of progesterone in rats (n=24). values are expressed as mean± ser. *represent significant difference p<0.05 among groups. iraqi j pharm sci, vol.31(2) 2022 sub-chronic effect of different doses of diclofenac sodium 231 serum prolactin levels were evaluated in this work to obtain a complete explanation about the effect of nsaids on the female reproductive system. there are no significant differences were observed between groups 1, 2, and control groups, all of them are well-matched as seen in figure 5. a significant reduction in serum prolactin level was detected in group3 compared to the remaining group. figure 5. impact of sub-chronic use of low and high pharmacological doses of diclofenac sodium alone (group1, 2) and in combination with mefenamic acid (group3) on serum concentration of prolactin in rats (n=24). values are expressed as mean± ser. *represent significant difference p<0.05 among groups. figure 6 summarizes serum concentrations of glutathione in control and all studied groups. serum glutathione concentration significantly decreased in group 2(rats exposed to a high pharmacological dose of ds) compared to the control group. no significant differences were documented between groups1, 3, and control groups, all of them have comparable glutathione concentrations. figure 6. impact of sub-chronic use of low and high pharmacological doses of diclofenac sodium alone (group1, 2) and in combination with mefenamic acid (group3) on serum concentration of glutathione levels in rats(n=24). values are expressed as mean± sem. *represent significant difference p<0.05 among groups. the microscopic finding of ovaries of control rats showed normal primordial follicles, primary and secondary follicles, graafian's follicles, and normal parenchymal cells (figure 7a). in contrast to the treatment groups showing numerous follicles with thick and dark dyed granulosa and theca layers, graafian follicles with pyknotic oocyte, multi follicles ovary, corpus luteum (cl), and asterisk fibrotic (figure 7b,c and d). figure 7. light micrographs of the ovary section stained with h&e x20, a) representative image showed normal follicle growth, primary and secondary (thick arrowhead), oocyte with healthy nucleus (thin arrow), graafian and corpus luteum with healthy appearance (thick arrow). b) representative image showed numerous follicles with thick and dark dyed granulosa and theca and layers(black arrow), graafian follicles with pyknotic oocyte(thick arrowhead), multi follicles ovary (thin arrow), corpus luteum (cl); asterisk, fibrotic(red arrow).c) representative image showed numerous follicles(thin arrow), thick arrow, graafian follicles, oocyte, nucleus with healthy appearance, and graafian follicles with pyknotic nucleus(whit arrow).d) representative image showed (white arrow) graafian follicles with healthy appearance, thick and dark dyed granulosa and theca and layers(blue arrow), corpus luteum with asterisk, fibrotic(red arrow). iraqi j pharm sci, vol.31(2) 2022 sub-chronic effect of different doses of diclofenac sodium 232 blood was collected form rats and characteristic complete blood count of all groups are summarized in table (1). the concentrations of wbc were comparable in all treated and control groups. the finding of differential cell count showed that the percent of lymphocyte, and mid% were significantly reduced in group3 and group1 respectively compared to control. no significant differences were documented in granulocyte concentration in all treated and control groups. the red blood cell count and hemoglobin values were negatively associated with nsaids administration in all treated groups compared to control. data on mcv, mch, and mchc give us an idea about the amount and average concentration of hemoglobin in rbc. significant reduction in their values was detected in groups 1 and 2 and 3 in comparison to that in control group. results of the present work indicate a significant reduction in platelet count in all groups that received ds alone or in combination with mefenamic acid compared to the control group. table 1. impact of sub-chronic use of low and high pharmacological doses of diclofenac sodium alone (group1, 2) and in combination with mefenamic acid (group3) on complete blood count in rats (n=24). values are expressed as mean± sem. *represent significant difference p<0.05 compared to control. parameter control mean± ser group1 mean± ser group2 mean± ser group3 mean± ser p-value wbc (10^3/ul) 10.8± 1.45 11.29 ± 2.35 13.8 ± 1.52 12.25 ± 1.46 p<0.05 lym% 78.3±2.19 71.95± 4.65 72.65±3.53 60.37± 3.81* p>0.05 gran% 13.46 ±2 12.86 ± 2.57 16.5 ±3.01 17.23± 2.158 p<0.05 mid% 8.23 ±0.53 5.18 ± 2.15* 10.83 ± 1.02 7.4± 0.86 p>0.05 rbc(10^6/ul) 6.93± 0.15 5.16 ± 0.17* 5.43± 0.22* 6.22 ± 0.09* p>0.05 hgb( g/dl) 13.22±0.2 11.08 ± 0.24* 11.08± 0.57* 11.5± 0.15* p>0.05 hct % 38.76 ± 1.1 34.06 ± 0.71 34.4± 1.96 36.26 ± 0.27 p<0.05 mcv(fl) 65.75±1.75 59.72± 0.41* 59.25± 1.6* 59.52±0.53* p>0.05 mch(pg) 20.81 ±0.97 21.55± 0.54 19.81 ± 0.4 18.25±0.2* p>0.05 mchc(g/dl) 33.3±0.31 32.5± 0.1 33.18 ± 0.07 33.55±0.1 p<0.05 rdw-cv % 13.18±0.27 15.01± 0.66* 14.46 ± 0.44 13.85± 0.32 p<0.05 rdw-sd(fl) 34.38±1.17 39.81± 1.46* 34.38 ±1.08 32.15± 1 p<0.05 plt(10^3/ul) 567.7±20.28 421.5±23.42* 466.7±19.96* 399.3 ± 20.23* p>0.05 mpv(fl) 8.75±0.522 7.06 ± 0.25* 7.08 ± 0.13* 6.96 ±0.16 * p>0.05 pdw(fl) 8.75±0.52 8.08 ± 0.46 8.35± 0.31 8.13 ± 0.27 p<0.05 pct% 0.3618±0.007 0.29 ± 0.01 0.46 ± 0.05 0.519±0.05 * p>0.05 p-lcr% 11.41±1.6 8.9± 1.45 8.5± 0.6 8.18 ±0.99 p<0.05 p-lcc10^9/l) 62.5±8.58* 34.67±3.49 59± 7.96 58.83 ± 7.687 p>0.05 discussion (frs) maybe affected by different toxicants that may in turn induce direct or indirect adverse outcomes. certain chemicals digested and bio-transformed locally into toxic free radicals, whereas others cause toxic damage through hormonal regulation (19).ds is one of the most commonly used nsaids among women of reproductive age for the management of a range of women's illnesses, including dysmenorrhea and menorrhagia(20). chronic use and abuse of nsaids may lead to an unexpected effect on frs, from this the current work was intended to explain how the sub-chronic use of ds affected the female reproductive system in rats. an experiment in this study has shown administration of ds resulted in a reduced body weight gain in group1 (received low dose of ds) and group 2 (received a high dose of ds) as compared to the control group. decrease in food intake, gastric discomfort associated with nsaids causes stomach and intestinal lesions as the most prominent adverse effects are the principal reasons for our results. our findings are seen to be consistent with different studies, elliott sn et al showed that use of ds in dose (5 mg/kg), bodyweight growth was significantly reduced and alabi qk et al found the iraqi j pharm sci, vol.31(2) 2022 sub-chronic effect of different doses of diclofenac sodium 233 treatment of the rats with ds resulted in a reduction in body weight. (18,21). on other hand, other articles demonstrated a positive correlation between nsaids use and an increase in body weight. this may be explained by edema and impairment of renal function occurring in ds abuse (22). in the current study, low and high doses of ds (group1 and 2) alone or when combined with mefenamic acid (group3) diminished serum pge2 concentration significantly compared to the control group. mechanistically, this reduction can be explained due to inhibition of pge2 synthesis both centrally and peripherally by competitive reversible inhibition of cox enzymes (23). these findings align with nakatsugi s et al. who found that nsaids have the ability to inhibit the pge2 production through cox-2 from exogenous and endogenous arachidonic acid (24). furthermore, abdelhalem ms et al found that ds was a powerful inhibitor of pge2 biosynthesis in the brain (25). measurement of serum prostaglandins in this experiment is important to correlate and further interpret our finding regarding the impact of nsaids abuse on frs. oral administration of ds alone and in combination with ma resulted in a significant elevation in serum fsh levels in comparison to the control group, the highest significant value seen in group 1. on the other hand, significant elevation in serum lh level in group 3, compared to the remaining groups. measurement of serum menotropins levels in relation to other female hormones are interesting because the impact of negative and positive feedback mechanisms of all these hormones cascade is considered the key to discussing our results. unfortunately few articles have been carried out on this field, however, these hormonal changes are similar to that found by. ji k et al found a significant increase in transcription of fshr and lhr, genes after exposure to nsaids (26). in contrast, some studies showed treatment with nsaids causes a decrease in both serum lh and fsh levels (7), or non-significant changes in the serum levels of fsh and lh (27,28).the cystic appearance of the treatment animals' ovaries compared to the normal control group confirm these results. these histopathological changes was due to the failure of ovulation of a certain number of graafian follicles that had been converted into luteinized unruptured follicles (29). inhibition of pg synthesis in the preovulatory follicles by nsaids hampered the completion of the ovulatory process, resulting in follicular rupture failure and thus cyst formation(30).our findings matched those of tomioka rb et al, who reported an increase in luf syndrome in juvenile idiopathic arthritis ( jia) patients on nsaids (15). also jesam c et al who showed non-steroidal antiinflammatory medicines (meloxicam or rofecoxib) are known to have comparable effects on ovulation when used systemically over several days. these medications caused luteinized un-ruptured follicles (luf), or dysfunctional, delayed ovulation (31). evaluation of serum estrogen along with serum progesterone levels in this study are important to fully summarized the effect of ds on complete blood hormones of frs. serum estrogen levels were significantly reduced in all treated groups compared to the control group. estrogen is a hormone that plays a major role in female reproduction. it is responsible for the ovulatory follicle's development, activating the pre-ovulatory surge of gonadotropins in the middle of the menstrual period, changing the condition of cervical mucus to make it easier for sperm to travel, and preparing the uterine endometrial lining for implantation (32). pge2 increases estrogen production by increasing the activity of aromatase, the enzyme that converts androgens to estrogens (33). these results are in agreement with wangwa ek et al found that nsaids (piroxicam) has been shown to have detrimental effects on female fertility by lowering levels of estrogen, progesterone, and gonadotropin, also hudson ag et al found that serum estradiol levels in nsaid users are considerably lower than in non-users (7,34). regarding progesterone levels assay, we found the opposite results compared to estrogens values. serum progesterone levels were significantly elevated in both groups 2 and 3 compared to the control group while the group1 showed a non-significant effect because of limited articles talking about this project. however, drug molecules that inhibit pg synthesis could adversely affect progesterone value and consequently female hormones. these findings are similar to that of amiridis gs et al who reported that meloxicam was used at various periods after insemination to reduce pgf2a release, raise luteal progesterone levels, and prevent early embryonic death (35). on other hand, these results disagree with previous studies that found nsaids cause a decrease in serum progesterone levels like dzięcioł m et al showed single dose administration of meloxicam cause a significant drop in progesterone levels, also amina s et al showed that usage of mefenamic acid cause the progesterone levels to drop with dose equal to 0,5 mg/kg 1 mg/kg, 1,5 mg/kg, and 2 mg/kg(36,37). to complete the view about the effect of nsaids on frs, serum prolactin levels were measured. a non-significant change in the serum prolactin levels in treated groups compared to the control group were obtained except in group 3 which showed a significant reduction in serum prolactin level compared to the control group. one reason for that was inhibition of prostaglandins (pgs) biosynthesis in the central nervous system (cns), pgs may stimulate prolactin release in the hypothalamus (25). the current result comes in tune with previous studies that showed ds causes a rapid and sustained decrease in prolactin plasma levels in healthy volunteers (38). in contrast, our finding was iraqi j pharm sci, vol.31(2) 2022 sub-chronic effect of different doses of diclofenac sodium 234 challenged by adeyemi wj et al demonstrated ds treatment cause an increase in the level of prolactin in male rats (39). glutathione is the main abundant thiol antioxidant in the body. it's a natural antioxidant found in both male and female gametes, in varying quantities. it has been proven that it is involved in the fertilization process and early embryo development. it protects eggs from oxidative stress during folliculogenesis, and as a result, egg quality is highly dependent on it (40). excessive reactive species generation, such as reactive oxygen species (ros), has been linked to diclofenac metabolism in vivo, resulting in oxidative stress, genomic damage, and cell death via apoptosis (41). in this study, there is a significant decrease in serum glutathione concentration in group 2 treated with high dose ds compared to the control group. these findings are consistent with the results that reflect high doses ds administration to rats lower glutathione concentrations significantly compared to control groups (10,42). finally, the considerable changes in hematological markers in diclofenac sodium-treated rats might indicate toxicity. the overall wbc count did not change significantly in any of the treatment groups during this study. this is in agreement with previous studies (43). although abd-el megid s et al showed that nsaids causes a significant decrease in wbc count (44). the considerable drop in white blood cell counts found in the treatment groups might be attributed to the participants' reduced feed consumption, as noted during the experiment. this is in line with previous research, which found that reduced feed intake had a significant influence on the hematopoietic system, resulting in lower numbers of white blood cells, platelets, and reticulocytes (45).the significant decrease in rbc and hgb levels in all treatment groups when compared to the control group might indicate druginduced toxicity, which is characterized by excessive red blood cell destruction and anemia (46). it might also be attributed to erythrocyte loss from gastrointestinal hemorrhage. when the body loses a significant amount of blood. the results of this investigation are consistent with those of orinya oa et al (13), and abdel-rahman on et al (47). significant reductions in mcv, mch, and mchc values were detected in all treated groups compared to the control group. similar outcomes were observed in previous studies (48). significant reduction in platelet count in all treated groups compared to control group, it's reasonable that the lower platelet count in this study might be due to ds's inhibitory influence on platelet synthesis, lowering platelet aggregating strength. this is in agreement with earlier studies (13,49). in conclusion, according to the present finding, we can assume that sub-chronic use of ds alone or in combination with ma has a deleterious impact on the female reproductive system through disruption of hormonal concentrations and regulations, shifting the balance toward oxidative stress by reduction of glutathione concentrations, and ovarian 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between asthma, obesity and leptin level in iraqi asthmatic patients and the main risk factors that are associated with leptin level # hiba a. hasan* ,1 *department of clinical laboratory science,college of pharmacy, al-mustansiryah university,baghdad,iraq. abstract this study tries to clear the correlation and association between asthma, obesity and leptin levels. also it will work to indicate the main risk factors which play role in the elevation of leptin level within asthmatic patients. this is a case control study conducted on (38) asthmatic patients and (20) healthy control who were closely similar by age, gender and bmi. the main statistical tests used were student t test, linear regression test and correlation test. significance was set at p < 0.05. sampling method used for this study was convenience sampling method. the main results of this study show a significant association and positive correlation between age (old age ≥ 40 years old), female gender, bmi (overweight and obese) and steroid utilization with leptin elevation in iraqi asthmatic patients since p values < 0.05. this study concludes that there is a correlation between obesity, age, sex and utilization of steroid with leptin level and they were the main risk factors which play role in the mechanism of elevation of leptin in iraqi asthmatic patients. key words: leptin, asthma, bmi. الخالصة ا االساسَ٘ الخٖ حلعب دّس الخطْسةّ هسخْٓ اللبخ٘ي ّ ححذٗذ عْاهل السوٌتب٘ي الشبْ ّ العالقت إٗجادعلٔ الذساستحِذف ُزٍ ( هي 02) بالشبْ ّ ا هصاب ا هشٗض (83علٔ ) الذساستحشخول ُزٍ فٖ اسحفاع هسخْٓ اللبخ٘ي عٌذ الوشضٔ الوصاب٘ي بالشبْ . , tُْ اخخباس الذساستالوسخخذم فٖ ُزٍ اإلحصائٖالخحل٘ل الجسن. الوخقاسب٘ي بكل هي العوش ّ الجٌس ّ كخلَ األصحاء األشخاص طشٗقَ االخخ٘اس بْاسطتطشٗقَ جوع العٌ٘اث كاًج هعٌْٗت. الضٗادةحعخبش >p 2,20كاًج إرا العالقت.اخخباس الخطٖ ّ اخخباس االسحذاد كخلَ الجسن (, اإلًاد,02اّ ٗسإّب٘ي العوش )اكبش هي >p 2,20 هعٌْٗت عالقتُٖ الذساستالوشٗح. كاًج الٌخائج االساسَ٘ لِزٍ ٌُاك باى الذساستّ اسخِالك السخ٘شّٗذ هع هسخْٓ اللبخ٘ي عٌذ الوشضٔ العشاق٘٘ي الوصاب٘ي بالشبْ.حسخٌخج ُزٍ البذٗي()الْصى الضائذ ّ عْاهل الوشضٔ الوصاب٘ي بالشبْ ّ حعخبش هي هسخْٓ اللبخ٘ي عٌذ صٗادةاسخِالك السخ٘شّٗذ ّ الجٌس, العوش, السوٌت,ب٘ي عالقت بالشبْ.ُشهْى اللبخ٘ي عٌذ الوشضٔ العشاق٘٘ي الوصاب٘ي صٗادةفٖ ه٘كاً٘كَ٘ ا االساسَ٘ الخٖ حلعب دّس الخطْسة introduction leptin is a 16 kda (kilo dalton) nonglycosylated circulating hormone, secreted mainly by the adipose tissue and its mrna is mostly synthesized in mature adipocytes, although it is also found in other tissues (1) . it's major function is the regulation of fat mass by decreasing food intake (it decreases the content of neuropeptide y at the hypothalamus) and increasing resting energy expenditure (2) .therefore it is often considered as an antiobesity hormone, though its primary function seems to be mediating adaptation to fasting (1). there is scientific evidence showing that leptin is involved in the initiation of puberty, pregnancy, breast-feeding, immune system regulation, inflammatory processes, respiratory function, sex hormones, growth hormone, and thyroid hormones, in addition to bone tissue metabolism, septic processes, hematopoiesis, cachexia, and others (35) . in case of asthma leptin exerts its action through the leptin receptor (ob-r) by activating both phosphatidylinositol-3-oh kinase and mitogen-activated protein kinase signaling pathways. so leptin function as a regulatory link between the endocrine and immune systems and might represent a bridge between inflammation and tissue repair. it also contributes to regulation of the maturation of fetal lung cells and to homeostasis of the endothelium (68) .leptin is increased during allergic reactions in the airways and might play a role in the relationship between obesity and asthma (9) . the specific role of the leptin/leptin receptor pathway in the bronchial epithelium from asthmatic patients is still largely unexplored (6) . in obese asthmatic patients, there is increasing evidence for an important role played by the epithelium in orchestrating the inflammatory responses and in producing a chronic wound scenario involving tissue injury and aberrant repair leading to airway remodeling (6) . # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1corresponding author email : hibaalichemist@yahoo.com received : 19/3/2011 accepted : 20/9/2011 iraqi j pharm sci, vol.20(2) 2011 leptin in iraqi asthmatic patients 97 airway remodeling has been related to the severity of asthma, and consistently, major tissue changes have been observed in patients with asthma (10) . asthma and obesity are both important current public health problems; but the underlying mechanisms have not yet been established (11). for these reasons this presented study tries to clear the relation between asthma, obesity and leptin levels. also it will work to indicate the main risk factors which play a role in the elevation of leptin level within asthmatic patients. materials and methods subjects this is a case control study conducted on (38) asthmatic patients and 20 healthy control who were closely similar by age, gender and bmi but the only difference that the control were not suffering from asthma. those asthmatic patients were admitted to alkhadhimeia teaching hospital and alyarmook teaching hospital between 23 th november 2010 to 30 th december 2010. clinical data that related with patients age, gender, bmi and waist to hip ratio were collected from patients' files by specific data sheet form designed for this study. methods blood samples were drawn between 9:00 am and 12:00 pm. all specimens were centrifuged at 4000 rpm within 2 hour of collection and serum stored at -20°c until analysis. after preparation of serum, the levels of leptin were measured with (eliza) method using drg (drg instruments gmbh\ germany) kit. all of the patients who were involved in this study fulfilled the inclusion criteria. the inclusion criteria include adult asthmatic patient ≥ 18 years old, male or female and were admitted for treatment of asthma. the exclusion criteria include smokers, alcoholism and chronic obstructive pulmonary disease (copd).the following anthropometric measurements were obtained: weight, height, bmi (body mass index), and waist and hip circumferences. weight was measured after calibration the scale before each weight measurement. height was obtained with an aluminum cursor stadiometer graduated in millimeters. the subject was barefoot, with heels, back, and head in contact with the stadiometer in horizontal plane. body mass index (bmi) had been estimated from person's weight & height, it was calculated by dividing weight (in kilograms) by height (in square meters). bmi≤18.5 (underweight) bmi=18.5-24.9(normal weight) bmi=25-29.9(over weight) bmi≥ 30(obese) (12) waist and hip circumferences (wc and hc, respectively) were measured with a tape measure to the nearest 0.5 cm. the waist-tohip ratio (whr) was calculated by dividing waist measurement (in centimeters) by hip measurement (in centimeters). the cutoff points of risk for whr were ≥0.8 for women and ≥1.0 for men; wc cut-off points were ≥102 cm for men and ≥88 cm for women (3) . statistical analysis since the type of data collected were continues type, therefore the statistical tests used were linear correlation test and linear regression test the main reasons for selecting these two tests are the data were normally distributed and this linear correlation test will help us to detect the type of correlation depending on (r) (r value range +1 to –1) whether it is positive or negative correlation but these results will mean nothing only when p value < 0.05 (significant result). while student t-test was used to find if there is a differences between the measured leptin values and the control values, significance was set at p < 0.05. sampling method used for this study was convenience sampling method which is a type of non probability sampling which involves the sample being drawn from that part of the population which is close to hand. that is, a sample population selected because it is readily available, convenient and within inclusion criteria. it may be through meeting the person (13) . the results were expressed as mean ± standard deviations (sds). results clinical characteristics of asthmatic patients are summarized in table 1. the mean values for age, weight, height, whr and bmi did not show any difference between the groups of asthmatic patients and healthy control since all p values > 0.05. leptin level for the total asthmatic patients is significantly higher than the healthy control , also leptin level for female asthmatic patients is significantly higher than healthy control since p < 0.05. but leptin for male asthmatic patients show no significant differences than control one. we notice also leptin level for female higher than for male. while in table 2 the results show that the leptin level in postmenopausal asthmatic women is significantly higher than that which was found in premenopausal asthmatic women since( p =0.041). risk factors that are associated with elevation of leptin levels in asthmatic patients will be shown in the table 3. according to the results which are shown in table 3 gender, age, bmi and cortisone iraqi j pharm sci, vol.20(2) 2011 leptin in iraqi asthmatic patients 98 treatment are the main risk factors that play a role in increasing leptin level in asthmatic patients. also as shown in the results the female gender is highly associated and correlated with increases in leptin level than male gender, the age above or equal to 40 years is strongly associated and correlated with elevation of leptin within asthmatic patients. while in case of bmi according to the results, obesity is highly correlated and associated with elevation of leptin level in asthmatic patients followed by overweight. normal weight and underweight both show a very weak correlation with leptin level. according to linear regression test, steroids treatment (prednisolone or dexamethasone) which have been received by 34 asthmatic patients and only 4 asthmatic patients did not receive it show a significant and strong positive correlation with elevation of leptin level among asthmatic patients. so according to the results of this study it had been shown that obesity and receiving cortisone treatments are the two parameters highly associated and play a role with elevation of leptin level among iraqi asthmatic patients. table1: asthmatic patients and healthy control variables variables asthmatic control p value * demographic data n 38 20 gender, female/male 22/12 10/10 utilization of steroid, yes/no 34/4 age (years) 52.7±14.4 50.4±12.7 0.274 weight (kg) 75.94± 21.53 73.7±19.83 0.649 high (cm) 158.92±9.78 159.77±9.48 0.359 waist (wc) (cm) 107.29±19.74 106.73±17.45 0.802 waist-hip ratio (whr) 0.97±0.14 0.95±0.13 0.074 bmi (kg/ m 2 ) 30.48±8.89 28.79±6.92 0.182 leptin total (ng/ml) female (ng/ml) male (ng/ml) 40.52±25.49 48.18±24.80 24.10±11.34 27.33±7.20 27.93±6.64 22.46±6.41 0.031 0.037 0.083 *student t test is used. table2: difference between leptin level of post menopausal women with pre menopausal variables of asthmatic patients n leptin level p value * leptin menopausal status post menopausal status 15 47.29±13.09 0.041 pre menopausal status 7 29.73±17.52 *student t test is used. table3: association and correlation of asthmatic patients variables with leptin levels variables of asthmatic patients p value * correlation (r) gender 0.044 female (p = 0.001) male (p= 0.117) 2.015 0.873 age (years) 0.029 ≥ 40 (p= 0.031) < 40 (p= 0.573) 1.127 0.941 bmi 0.001 underweight (p= 0.114) normal (p= 0.281) overweight (p= 0.006) obesity (p= 0.000) 0.116 0.629 1.924 3.052 utilization of steroid 0.000 3.004 *linear regression test iraqi j pharm sci, vol.20(2) 2011 leptin in iraqi asthmatic patients 99 discussion the aim of this study was to investigate serum leptin levels and its relationship with the main risk factors which play role in it's elevation within iraqi asthmatic patients. the results of student t -test show that there is a significant increase in leptin level among asthmatic patients than healthy control. also serum leptin levels of asthmatic patients and especially of asthmatic female were higher than those of healthy controls in spite of no difference in age, weight, high, waist-hip ratio and bmi levels. the lack of difference in leptin levels between asthmatic and healthy males could be a result either of a true modifying effect of sex on leptin or a small sample size to detect a difference (14) .the results also show that leptin level in female is higher than in male. this can be explained by the effect of sex hormones on leptin expression , as testosterone can inhibit and ovarian sex steroids can increase the expression of leptin (11,15) .serum leptin levels are relatively low in prepubertal ages, showing a gradual rise in both sexes before the onset of puberty, followed by a significant increase during puberty in girls and a decrease in boys (16,17) . this finding was in agreement with previous observations for higher serum leptin concentration in women than in men (15) .also according to the results of our study it had been shown that the leptin level among postmenopausal women is higher than that found in premenopausal women, the reason may be due to that in postmenopausal woman who already obese the incidence of asthma attack is higher than the non obese woman because the concentration of storage endogenous estrogen in obese woman is higher than non obese one and this is what leads to high incidence of asthma attack. so both factors postmenopausal and obesity are the main two factors which play role with incidence of asthma attack (18) .this result was in agreement with this mentioned by sood et al (15) for higher serum leptin concentrations association with current asthma in women and this association may be stronger in postmenopausal women. findings of our study revealed that the age play role in the elevation of leptin level since it shows a significant association (p < 0.05). the explanation for that in aging people there are several factors that will start to increase too like immunological factors and leptin that presuppose to hyperleptinemia. but the age factor will play a risk role within leptin level elevation when the patients are overweight women therefore age alone as an isolated factor is not so risky factor without bmi and gender (15) . these findings are similar to those reported in other studies, which showed an increase of leptin with aging (19, 20) and an inverse to those found in other literatures which showed decrease of plasma leptin with aging (21) .also our results show that bmi specifically obese and overweight is a strong risk factor which plays a role in the elevation of leptin level among asthmatic patients. this point also was mentioned by mai and his colleague (11) that bmi (overweight or obese) is one of the main risk factor which play a role in the elevation of leptin level within asthmatic patients (6) .also sood and his colleague (15) indicated a high association of bmi in females than males because of high production of hormones in female than in male which lead to this point (9) . moreover bmi and gender (specifically old women) considered as a main risk factors for elevation of leptin among asthmatic patients (3) . since these two points i.e., bmi and gender (old women) are already found in our study so this can explain the strong association between bmi factor and leptin level. these observations have led some to conclude that leptin may be important in explaining the link between obesity and asthma, particularly in women.our results is clear that utilization of steroids which had been used by the asthmatic patients also is considered as a one of the risk factor that play a role in the elevation of leptin level. since the use of steroid by asthmatic patients is considered as one of the main factors which play a role in the elevation of leptin level (11) . the main explanation for this is that the use of dexamethasone by patients suffering from problems with their pulmonary system will leads to elevation of leptin level. the cortisone treatment will mainly effects on adipoinsular and hypothalamic-pituitary-adrenal (hpa) axis in preterm in those patients. this adipoinsular mainly responsible for regulation of energy and activity and since leptin is responsible for such things so this will leads to increases in leptin level. so administrated steroid will lead to elevation in leptin level through its' direct effect on adipoinsular (22) . it has been assessed that synthetic dexamethasone stimulates both leptin synthesis in preadipocytes and adipocytes and increases leptin secretion and leptin receptor mrna expression in choriocarcinoma cells (6, 23). conclusion the results of our study suggest that asthma and obesity have a strong relation to leptin level. the risk factors which play a role in elevation of leptin level in iraqi asthmatic patients are gender, age, bmi and utilization of steroids. to be more accurate [old women x iraqi j pharm sci, vol.20(2) 2011 leptin in iraqi asthmatic patients 100 bmi (overweight or obese) x utilization of steroid] were considered as the main risk factors which cause elevation of leptin level within iraqi asthmatic patients.also our study proves that leptin level among post menopausal women is significantly higher than that of pre menopausal ones. therefore these results clear the main factors that will cause the elevation of leptin within asthmatic patients and the relation between asthma, obesity and leptin. so this can help the iraqi physicians and clinicians how leptin can play role within incidence and increases of asthma severity, and also it clear that the detection of leptin level is very important in order to control its level. in order to reduce or prevent the incidence of asthma attack. acknowledgment the author likes to show appreciation to prof. dr. hedef d. el-yasin\dep. of physiological chemistry\college of medicine\baghdad university. dr. hydar noori\al-khadhimeia teaching hospital, ms. intisar sh. ali\ al-yarmook teaching hospital for technical assistance. references 1. hegyi, k., fülöp, k.a., kovács, k.j., falus, a., & tóth, s.,high leptin level is accompanied with decreased long leptin receptor transcript in histamine deficient transgenic mice. immunology letters. 2004;92: 193-197. 2. alema´n, m. r., santolaria, f., batista, n., de la vega, m.j., gonza´lez-reimers e., milena, a., llanos, m., & go´mezsirvent, j.l., leptin role in advanced lung acncer . a mediator of the acute phase response or a marker of the status of nutrition.cytokine .2002;19(1): 21-26. 3. mendoza-nunez, v. m., garcı´asa´nchez, a., sa´nchez-rodrı´guez, m., galva´n-duarte, r.e., & fonseca-yerena, m. e.overweight, waist circumference, age, gender, and insulin resistance as risk factors for hyperleptinemia. obesty research .2002;10(4): 235-259. 4. soares mj, piers ls, o’dea k, collier gr. plasma leptin concentrations, basal metabolic rates and respiratory quotients in young and older adults. int j obes relat metab disord. 2000; 24:1592–9. 5. corsonello a, buemi m, artemisia a, giorgianni g, mauro vn, corica f. plasma leptin concentrations in relation to sick euthyroid syndrome in elderly patients with nonthyroidal illnesses. gerontology. 2000;46:64–70. 6. bruno, a., pace, e., chanez, p., gras, d., vachier, i., chiappara, g., laguardia, m., gerbino, s., profita, m., & giomarkai, m. leptin and leptin receptor expression in asthma. the journal of allergy and clinical immunology,2009; 124, 230-237. 7. bergen, h.t., cherlet tc, manuel p, scott je. identification of leptin receptors in lung and isolated fetal type ii cells. am j respir cell mol biol. 2002;27:71–77. 8. wolk r, deb a, caplice nm, somers vk. leptin receptor and functional effects of leptin in human endothelial progenitor cells. atherosclerosis. 2005;183:131–139. 9. shore sa, schwartzman im, mellema ms, flynt l, imrich a, johnston ra. effect of leptin on allergic airway responses in mice. j allergy clin immunol. 2005;115:103–109 10. james al and wenzel s. clinical relevance of airway remodelling in airway diseases. eur respir j. 2007;30:134–155 11. mai, x. m., chen, y., & krewski, d.,does leptin play a role in obesity asthma relatioship.pediatr allergy immunol .2009;20: 207-212. 12. mei,grummer-strwn,l.m.,pietroblli ,a.,gouding,m.i.,&dietz,w.h.validity of body mass index compared with other body composition screening index for assessment of body fatness in children &adolescent. am. j. clin. nut. 2002 ;7: 597-599. 13. hinton, p. r., brownlow, c., mcmurrary, i., & cozens, b. spss explained,2 nd edition, routledge/ taylor and francis group, london, 2004;31-44. 14. . guler, n., kirerleri, e., ones, u., tamay, z., salmayenli, n., & darendeliler, f. leptin: does it have any role in childhood asthma? j allergy clin immunol 2004;114: 254-259. 15. sood, a., ford, e.s., & camargo, c.a., association between leptin and asthma in adults.thorax 2006; 61: 300-305. 16. blum wf, englaro p, hanitsch s, juul a, hertel nt, mu¨ ller j, et al. plasma leptin levels in healthy children and adolescents: dependence on body mass index, body fat mass, gender, pubertal stage and testosterone.j clin endocrinol metab 1997;82:2904-10. 17. garcia-mayor rv, andrade ma, rios m, lage m, dieguez c, casanueva ff. serum leptin levels in normal children: relationship to age, gender, body mass index, pituitary-gonadal hormones, and pubertal stage. j clin endocrinol metab 1997;82:2849-55. iraqi j pharm sci, vol.20(2) 2011 leptin in iraqi asthmatic patients 101 18. barr, r.g., wentowski,c. c. grodstein, f.,somers,s.,c.,stampfer m.j.,schwartz j.,speizer,f.,e., carlos a. camargoc.,a., jr.prospective study of postmenopausal hormone use and newly diagnosed asthma and chronic obstructive pulmonary disease.arch intern med.2004;164:379-386. 19. van den saffele jk, goemaere s, debacquer d, kaufman jm. serum leptin levels in healthy ageing men: are decreased serum testosterone and increased adiposity in elderly men the consequence of leptin deficiency? clin endocrinol. 1999;51:81–8. 20. monroe mb, schiller bc, seals dr, jones pp. relation of leptin and insulin to adiposity-associated elevations in sympathetic activity with age in humans. int j obes relat metab disord. 2000;24:1183–7. 21. ostlund re, yang jw, klein s, gingerich r. relation between plasma leptin concentration and body fat, gender, diet, age and metabolic covariates. j clin endocrinol metab. 1996;81:3909–13. 22. ng, p. c., lam, c.w.k., lee, c.h., fok, t.f., chan, his., ma, k.c., & wong, e. changes in serum leptin concentration after corticosteroid treatment in preterm infants. acta paediatrica,2002; 91, 684690. 23. . lee mj, wang y, ricci mr, sullivan s, russell cd, fried sk. acute and chronic regulation of leptin synthesis, storage, and secretion by insulin and dexamethasone in human adipose tissue. am j physiol endocrinol metab. 2007;292:e858–e864. iraqi j pharm sci, vol.29(2) 2020 osteoporosis in post kidney transplantation doi: https://doi.org/10.31351/vol29iss2pp1-7 1 evaluation the risk factors that are associated with osteoporosis in post kidney transplantation in a sample of iraqi patients angham a. hasan *,1, munaf h. abd alrazak ** and hassan m. abbas al-temimi *** * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq *** ministry of health and environment , baghdad, iraq. abstract renal transplantation is a principal treatment option for end-stage kidney failure. bone loss and fracture are serious complication of kidney transplantation, associated with morbidity and mortality. the pathogenesis of post transplantation bone loss is multifactorial and complex. there are changes in the normal bone remodeling system which will lead to more accelerated osteoporotic changes compared to normal individuals. the current work aimed to investigate the incidence of osteoporosis in post kidney transplant patients when compared to the general population. and study the relationship between post kidney transplant immunosuppression therapy and osteoporosis and determine some biochemical changes. also to evaluate the bone mass and the possible correlation between demographic data and the development of osteoporosis. a case control study, conducted in the kidney transplant center – medical city complex during the period (from october 2018 till april 2019), seventy-five kidneys transplant patients were participated in the present study including (23 females &52 males). apparently healthy seventy-five subjects were selected to participate as a normal group for comparison (control) including (35 females and 40 males). all participants were examined for their bone density using dexa scan (t – score) and those with cut– point ≤2.5 were diagnosed as having osteoporosis (lumbar and hip bones were examined). the prevalence of osteoporosis and osteopenia was significantly higher in transplant patients compared to control for lumbar and hip bone (for lumbar bones: 33.3% vs. 2.7% for hip bones: 60% vs. 14.7%). tscore was significantly lower in the transplant patients compared to control for both lumber (-1.9±0.8 vs. 1.1±0.7) and hip bones (-2.3±0.9 vs. -1.3±0.8).in logistic regression analysis; only gender and bmi were the predictors of osteoporosis for lumbar bone, while; the bmi and serum calcium were the predictors of osteoporosis for hip bones. in conclusion , osteoporosis in post-renal transplant patients is high when compared to general population, only corticosteroids significantly increase risk of osteoporosis, biochemical marker serum level in post kidney transplant patients are significantly different when compared with the general population but did not increase risk of osteoporosis and body mass index is a risk factor for both lumbar and hip bones osteoporosis ,while gender and serum calcium are risk factors for osteoporosis in lumbar and hip ;respectively. keywords: osteoporosis, renal transplant, t – score, immunosuppressant drugs. عوامل الخطورة مع هشاشة العظام في عينة من المرضى العراقيين الذين يخضعون عالقة تقييم لعمليات زرع الكلى ***و حسن محمد عباس **، مناف هاشم عبد الرزاق 1*،انغام احمد حسن داد ، العراق فرع الصيدلة السريرية ، كلية الصيدلة ، جامعة بغداد ، بغ* فرع االدوية والسموم ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق.** وزارة الصحة والبيئة ، بغداد ، العراق *** الخالصة عملية زرع الكلى تعتبر العالج الرئيسي لمرضى المراحل االخيرة للفشل الكلوي. هشاشة وتكسر العظم تعتبر من المضاعفات الخطيرة تعددة ة زرع الكلى وتكون مرتبطة مع تفاقم الحاله وزيادة خطر الوفاة للمريض .أن عملية تطور فقدان العظم ما بعد زرع الكلى تكون معقده و ملعملي قارنة العوامل .حيث ان بعد عملية ألزرع تحدث تغييرات في ألنظام ألطبيعي ألعادة تصميم العظم وهذا يؤدي الى التسريع أكثر في هشاشة العظم م هذه الدراسة صممت للتحقق في حدوث هشاشة العظم في مرضى زرع الكلى مقارنة باالشخاص االصحاء و لمعرفة العالقة باألشخاص العاديين. في مصل مرضى زرع المحتملة بين نوع العالج المناعي بعد زرع الكلى وهشاشة العظام. ولدراسة التغيرات الكيميائية الحيوية في بعض المؤشرات وإليجاد العالقة المحتملة بين البيانات الديموغرافية وتأثيرها على هشاشة العظام. ملعظاكتلة التقييم وايضا الكلى ( ، 2019إلى أبريل 2018مجمع مدينة الطب وللمده)من أكتوبر -تم إجراء عملية مراقبة لحالة الحاالت في مركز زراعة الكلى ( 65 15العمر )من الذكور(. كان مدى 52من االناث و 23لية زرع الكلى في هذه الدراسة ، بما في ذلك )وشارك خمسة وسبعون مريًضا في عم من الذكور(. 40و من االناث 35سنة. تم اختيار خمسة وسبعون من االشخاص االصحاء للمشاركة كمجموعة طبيعية للمقارنة بما في ذلك ) 1corresponding author e-mail: pharmacistangham@yahoo.com received: 28/11 / 2019 accepted:15 / 2/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp1-7 iraqi j pharm sci, vol.29(2) 2020 osteoporosis in post kidney transplantation 2 (. تم فحص جميع المرضى لمعرفة كثافة العظام باستخدام فحص كثافة العظام )درجة تي(، وتم تشخيص 65 15العمر )وكان مدى ن بهشاشة العظام )تم فحص عظام الفقرات القطنية والورك (، باإلضافة إلى ذلك تم قياس نسبة هرمون على أنهم مصابو 2.5 -≥المصابين بنقطة مدة الزرع كان متوسط دي،الزالل، الفوسفاتيز القلوية،الفوسفات، والكالسيوم في مصل االشخاص المشاركين في الدراسة.الغدة خلف الدرقية، فبتامين ٪ يستخدم السيكلوسبورين و 34,7٪ يستخدمون التكروليموس ، 50,7٪( ، في حين أن 98,7لميكوفينوالت )سنوات، تلقى غالبية المرضى ا 4 ٪ يستخدمون سيروليمس.وجميع المرضى كانوا يستخدمون الكورتيكوستيرويد النسبة المئوية لمعدل انتشار كانت أعلى بكثير في مرضى زرع 9,3 ٪ 33,3٪بالنسبة لعظام الورك: 14,7٪ مقابل 60عظام الفقرات القطنية وعظم الورك )لعظام العمود الفقري: االكلى مقارنةً باالشخاص العاديين في ( وعظام 0,7± 1,1-مقابل 0.8± 1,9-أقل بكثير في مرضى الزرع مقارنةً بالتحكم لكل من الفقرات القطنية )تي ٪(. كانت درجة 2.7مقابل -كان هناك عالقة عكسية كبيرة بين مؤشر كتلة الجسم ودرجة تي في االشخاص العاديين)درجة تي = (.0,8± 1,3-مقابل 0,9± 2,3-الورك ) وكان هناك ارتباط معنوي ،( لعظام الفقرات القطنية0,401( ، في حين أن هذه العالقة تصبح مباشرة وهامة لمرضى الدراسة)درجة تي 0,285 الجنس وجد ان ليل االنحدار اللوجستي وبتح ( بالنسبة لعظم الورك.0,232-فقط)درجة تي = مباشر بين درجة تي والجنس في مجموعات الدراسة لعظام ومؤشر كتلة الجسم كانت عوامل تنبئ بهشاشة عظام الفقرات القطنية، في حين كان مؤشر كتلة الجسم والكالسيوم عوامل تنبئ بهشاشة العظام عقارالكورتكوستيريد عامل لعظام لدى مرضى زرع الكلى أعلى مقارنة مع االشخاص األصحاء،نسبة هشاشة ا ان من هذه الدراسةنستنتج الورك. في مصل مرضى زرع الكلى يختلف بشكل كبير عن االشخاص االصحاء خطوره وبشكل كبير لهشاشة العظام، مستوى المؤشرات الكيميائيه الحيويه عامل الجسم هي عامل خطورة لهشاشة عظم الفقرات القطنيه و يعتبرمؤشر كتلة الجسم مؤشر كتلة لكنها ال تعتبر عواما خطورة لهشاشة العظام، هشاشة العظام في عظام الفقرات العنقيه وعظم الورك كما يعتبر الجنس و مستوى الكالسيوم في المصل عوامل خطورة لهشاشة عظام خطورة ل الفقرات القطنية والورك؛على التوالي . االدوية المثيطة للمناعة ،فحص كثافة العظام )درجة تي(هشاشة العظام ، عملية زرع الكلى، الكلمات المفتاحية: introduction the primary function of kidney is to maintain stable internal equilibrium by eliminating excess water, electrolytes, and other byproducts, through the formation of ultrafiltrate from the plasma by the filtration action of glomerulus system, which can serve as a system for reabsorption or section of other materials and byproduct(1). the main stay of treatment of end stage renal failure (esrf) is either dialysis (temporary solution), or kidney transplant. transplant result in better outcome in comparison with dialysis (2). however, transplant had its drawbacks, including low availability of kidney from donors, use of chronic immunosuppressant medications and others factors; these factors together with superiors outcome compared to dialysis lead to the development of criteria for selective candidates for the operation to include as many as possible patients to benefit from transplantation (3). in order to prevent graft loss that caused by immune reaction, several protocol develop, these protocols will prevent acute rejection. currently reduction of side effects that caused by these protocols become as important as their role in reducing the incidence of acute rejection. in the present time, intensive immunosuppression therapies in the early stages of the transplant become a paramount, followed by maintenance protocol to reduce the risk of rejection (4). however, many of these drugs have side effects that will result in more deterioration of bone density and osteoporosis. osteoporosisa disease that show reduction in bone mass, micro architectural disruption, and enhanced skeletal fragility, with subsequent low bone strength and high rates of fracture (5). after renal transplantation, there are changes in the normal bone remodeling system which will lead to more accelerated osteoporotic changes compared to normal individuals, while for transplantation-related bone loss results from both an increase in the rate of resorption and a decrease in the rate of bone formation(6) .the current work aimed to investigate the incidence of osteoporosis in post kidney transplant patients when compared to the general population, and study the relationship between post kidney transplant immunosuppression therapy and osteoporosis and determine some biochemical changes. also to evaluate the bone mass by using dual x-ray absorptiometry (dexa) and the possible correlation between demographic data and the development of osteoporosis . subjects and method study design a case control study applied in kidney transplant center – medical city complex during the period from october 2018till april 2019. patients and controlled subjects seventy-five kidney transplant patients were participated in the present study including (23 females & 52 males). the age range was (15 65) years. apparently seventy five healthy subjects were selected to participate as a normal group for comparison (control) including (35 females & 40 males). the age range of these subjects was (15 65). the follow up of kidney transplant patients was made by specialist's surgeon. inclusion criteria 1. patient age range (15 -65) years. 2. more than 6 months’ post kidney transplant operation exclusion criteria 1. endocrine diseases. 2. inflammatory diseases. 3. gastrointestinal diseases. 4. lung disease. 5. patients using drug that effect calcium level. iraqi j pharm sci, vol.29(2) 2020 osteoporosis in post kidney transplantation 3 bone density assessment dexa scan was used for the assessment of bone density, with t – score ≤ 2.5 to define osteoporosis and between – 1.0 to – 2.5 to define osteopenia (7). laboratory procedure a 5 ml venous blood sample from each participant was collected and then sent for laboratory analysis in the medical city complex campus, serum (calcium, phosphorous (po4), alkaline phosphatise (alp), vitamin d3, parathyroid hormone(pth), and albumin) measurement were recorded. statistical analysis for the assessment of continuous variables, independent t – test was used, while for categorical variables chi square testused, ordinal logistic regression analysis used to examine the risk of osteoporosis (in which the order of category from lowest to highest was normal bone, osteopenia, and osteoporosis). all analysis carried out using spss version 22.0.0 (chicago, il) and graphpad prism version 8.2 ( san diego, california usa), p value considered when appropriate to be significant if less than 0.05 results the study included 150 participants, mean age of patients was not significantly different in the study group compared to control(40.9±12.2 vs. 38.4±11.5 years, respectively), with age range from 15 – 65 years,the commonest age group for both study and control group was between 40 – 49 years (26.7% vs. 28.0%, respectively).bmi was significantly lower in the study group compared to control (25.2±3.8 vs. 27.0±7.5 kg/m2, respectively).there was no significant difference in gender between study and control groups .with male to female ratio (2.26:1 vs.1. 14:1, respectively), as illustrated in table.1. table 1. assessment of demographic, clinical, and laboratory data. variables control study p-value number 75 75 age (years), mean ± sd 38.4±11.5 40.9±12.2 0.191 # bmi (kg/m2), mean ± sd 27.0±7.5 25.2±3.8 0.068 # gender, n (%) 0.065 # female 35 (46.7%) 23 (30.7%) male 40 (53.3%) 52 (69.3%) transplant duration (years), median (iqr) 4.0 (2.0 – 7.0) treatment, n (%) mmf 74 (98.7%) cyclosporine 26 (34.7%) sirolimus 7 (9.3%) tacrolimus 38 (50.7%) s.ca (mg/dl), mean ± sd 9.0±0.4 9.5±0.7 <0.001# s.po4(mg/dl), mean ± sd 4.1±0.6 3.3±0.6 <0.001 # s.alp(u/l), mean ± sd 65.6±17.7 101.2±35.8 <0.001 # s.vitamind3( ng/ml), mean ± sd 22.5±15.0 22.9±13.4 0.871 * s.pth(pg/ml), mean ± sd 39.4±22.8 82.6±66.9 <0.001# s. albumin(mg/d), mean ± sd 3.8±0.6 3.6±0.3 0.002# * significant difference indicates p-value <0.05, compared to control # non-significant difference indicates p-value ≥0.05 mmf: mycophenolatemofetile s.alp: serum alkaline phosphatase, s.ca: serum calcium, s.pth: serum parathyroid hormone, s.po4: serum phosphorus. the prevalence of osteoporosis and osteopenia was significantly higher in transplant patients compared to control for bone lumbar and hip bones (figure 1 and 2), also; t-score was significantly lower in the transplant patients compared to control for both lumbar and hip bones ;as illustrated in table 2. iraqi j pharm sci, vol.29(2) 2020 osteoporosis in post kidney transplantation 4 table 2. assessment of bone status. variables control study p-value number 75 75 t – score, mean ± sd spine bone -1.1±0.7 -1.9±0.8 <0.001 hip bone -1.3±0.8 -2.3±0.9 <0.001 lumbar bone, n (%) <0.001 normal 39 (52.0%) 14 (18.7%) osteopenia 34 (45.3%) 36 (48.0%) osteoporosis 2 (2.7%) 25 (33.3%) hip bone, n (%) <0.001 normal 43 (57.3%) 7 (9.3%) osteopenia 21 (28.0%) 23 (30.7%) osteoporosis 11 (14.7%) 45 (60.0%) figure 1. osteoporosis status for spinal (lumbar) bones . *** highly significant difference (p-value < 0.001). **** very highly significant difference (p-value < 0.0001) figure.2. osteoporosis status for hip bone *** highly significant difference (p-value < 0.001) **** very highly significant difference (p-value < 0.0001) only gender and bmi were the predictors of osteoporosis for lumbar bone, as illustrated in table 3, while the bmi and serum calcium were the predictors of osteoporosis for hip bone, as illustrated in table 4 . table 3. ordinal regression analysis of the predictor of osteoporosis for lumbar bone in the study group β or 95%ci p-value age 0.020 1.020 0.984-1.057 0.284 gender (female) -1.107 3.026 1.118-8.188 0.029 * bmi 0.163 0.850 0.748-0.966 0.013 * transplant duration 0.047 1.048 0.961-1.143 0.286 mmf cyclosporine 0.252 1.286 0.516-3.208 0.589 sirolimus 0.035 1.036 0.249-4.317 0.961 tacrolimus -0.162 0.850 0.362-2.0 0.710 s.po4 0.141 1.151 0.562-2.358 0.700 s.alp -0.003 0.997 0.986-1.008 0.591 s.vitamin d3 0.011 1.011 0.977-1.047 0.522 s.pth 0.001 1.001 0.995-1.008 0.644 s.albumin -0.023 0.977 0.275-3.475 0.971 s.calcium -0.315 0.730 0.379-1.406 0.346 β: ordinal logistic regression analysis,or: odd ratio, ci: confidence interval it cannot be calculated for mmf *significant relationship between variables (p-value<0.05) iraqi j pharm sci, vol.29(2) 2020 osteoporosis in post kidney transplantation 5 table 4.ordinal regression analysis of the predictor of osteoporosis for hip bone in the study group. β or 95%ci p-value age 0.017 1.017 0.979-1.056 0.377 gender (female) 0.933 2.543 0.951-6.802 0.063 bmi -0.139 0.870 0.760-0.996 0.043 * transplant duration -0.007 0.933 0.917-1.076 0.872 mmf cyclosporine 0.279 1.321 0.511-3.419 0.566 sirolimus -0.646 0.524 0.098-2.807 0.451 tacrolimus 0.023 1.023 0.414-2.527 0.961 s.po4 -0.444 0.642 0.292-1.409 0.269 s.alp 0.002 1.002 0.989-1.014 0.790 s.vitamin d3 0.020 1.020 0.982-1.061 0.308 s.pth 0.002 1.002 0.995-1.009 0.542 s.albumin 0.417 1.517 0.384-5.995 0.552 s.calcium -0.665 0.514 0.265-0.997 0.049 * β: ordinal logistic regression analysis, or: odd ratio, ci: confidence interval it cannot be calculated for mmf *significant relationship between variables (p-value<0.05) discussion disturbances in bone metabolism are common complications that affect patients after successful renal transplantation. the usual method for assessing bmd by dxa scan in which osteoporosis defined as t score ≤ 2.5 standard deviation (one standard deviation represent the average of young adult) (8). bmd in patients who have undergone renal transplantation has been reported to decrease by a mean of 5.5% to 19.5% during the first 6 months but only 2.6% to 8.2% between months 6 and 12 after surgery (9). in the present study, mean age ±sd of transplant patients were 40.9 ± 12.2 years, with 73.3% of them distributed between 30 – 59 years, this result is close to coco et al; study which included 59 kidney transplant patients with mean age of 45.5 ± 13 years (10), also in agreement with walsh et al; study which included 93 transplant patients with mean age±sd for treatment was (46.1 ± 12.77 years) and for control (46.1 ± 12.93 years) (11).in this study patients were younger than patients included in the smerud et al; study with mean age 51.4 ± 13.8 years for all the 129 transplant patients (12), also lower than other study with mean age of 50.7 ± 15.5 years for 49 control transplant patients (13). in the present study, mean t – score for lumbar-1.9±0.8 ,while for hip bone it was -2.3±0.9 in study group .in marcén et al study, for the lumber bones (l2-l4) t-score, 1.88±0.99, and for femoral neck was 1.52±0.88, which is close to present findings (14). in mazzaferro study, t – score was 1.290 ± 1.286 which higher than current study (15). in durieux et al study, t – score was – 2 ± 1.3 at the lumbar spine and 1.9 ± 1.2 at the femoral neck which is close to current study (16). in the current study, the prevalence of osteoporosis, osteopenia, and normal bone for spinal bone were 33.3% ,48.0% ,and 18.7% ;while for hip bone it was 60.0% ,30.7% ,and 9.3% . in gregorini et al, a retrospective cohort study, 60.3% of the patients had normal bone, while osteopenia and osteoporosis were present in 24.6% and 15.1%, respectively (17), which is in disagreement with the current study since osteoporosis in the current study is higher. in marcén et al study, 20% had normal bmd in the lumbar spine; 52.5% had osteopenia and 27.5% had osteoporosis. while for femoral neck, 35.0% had normal bmd; 50.0% had osteopenia; and 15.0%, osteoporosis (which mean lower osteoporosis rate when compared to the present result) 14. in durieux et al; studyis agree with current study for the lumbar spine 37% had osteoporosis, and 44% had osteopenia, but disagree for the femoral neck 37% had osteoporosis and 40% had osteopenia 16. in marcen et al study, 41.9% had osteopenia and 14% had osteoporosis (18). the high rate of osteoporosis that observed in this study can be explained by the long duration of transplantation that can will more progressive bone disease, also all patient received corticosteroids (cs) and for extended period of time, since cs is known to cause bone loss by its inhibitory effect on osteoblast cells, and activation of osteoclastic activity, reduction of ca absorption from the git, enhance renal ca excretion, and increased section of pth(19). in the present study, 34.7% of the patients received cyclosporine(csa) (as part of combination therapy), also there is no significant relationship between cya with osteoporosis, (with or, 95%ci = 1.286 ,0.516-3.208; for lumbar bone and = 1.321 , iraqi j pharm sci, vol.29(2) 2020 osteoporosis in post kidney transplantation 6 0.511-3.419 for hip bones(, which in agreement withmartín-fernández(20). this can be explained by the lack of effect of csa on bone, or since the decrease in bmd in transplant recipients is difficult to evaluate because csa is usually administered together with glucocorticoids(20). tacrolimus was received in the present study by 50.7% patients (as part of combination therapy), also there was no significant relationship between tacrolimus and osteoporosis, (with or, 95%ci =0.850, 0.362-2.0; for lumbar bone and, =1.023, 0.414-2.527 ;for hip bones).fk506-based regimens may benefit the skeleton by lowering steroid exposure in transplant recipients. in a small group of kidney-transplant recipients followed for 1year, goffin et al. (21); noted that those who received tacrolimus had a small net increase in bone density compared to a loss observed in those who received cyclosporine. sirolimus might impair bone formation by interfering with the proliferation and differentiation of osteoblasts and might contribute to the impairment of osteoclast-mediated bone resorption(20). in the present study, only 9.3% of patients received sirolimus, there was no correlation between the use of sirolimus and osteoporosis(with or, 95%ci =1.036,.249-4.317; for lumbar bone and, =0.524,0.098-2.807 ;for hip bones), it is a more novel immunosuppressive agent that produces lower effects on bone(22). in the current study, 98.7% of patient received mycophenolatemofetil (mmf) (as a part of combination therapy), and there was no significant relationship between (mmf) and osteoporosis. mycophenolatemofetil has no influence on bone formation and mass in clinical observations (23). also, the logistic regression analysis revealed that the bmi (or, 95%ci = 0.850 ,0.7480.966) and gender (or, 95%ci = 3.026 , 1.1188.188) were risk factors that are associated with osteoporosis risk for lumbar bone.while for hip bone, logistic regression analysis for bmi (or, 95%ci = 0.870 ,0.760-0.996), and serum calcium (or, 95%ci = 0.514 ,0.265-0.997) were risk factors that are associated with osteoporosis in hip bone. these results study disagree with the results of another study, where no significant relationship was observed between osteoporosis and gender and body mass index (24). also differs from another study, where no significant relationship was observed between osteoporosis and serum levels of calcium (25) but it agrees with the same study, regarding nonsignificant relationship between osteoporosis and serum phosphorous and serum parathyroid hormone level. osteoporosis after kidney transplantation is multifactorial, while pathophysiologic mechanisms responsible for this condition is not completely elucidated. pre-transplantation risk factors include duration of dialysis, high or low parathyroid hormone (pth) levels and preexisting bone disease. post-transplantation risk factors associated with bone loss and/or fractures are deceased kidney donor, immunosuppressive regimen choice (glucocorticoids, calcineurin inhibitors), and time since transplantation, hypophosphatemia and graft dysfunction. additional risk factors such as postmenopausal status for women and presence of diabetes have been considered as possible culprits, in adjunction to the classical osteoporosis risk factors such as age and female gender(26). conclusion osteoporosis in post-renal transplant patients is high when compared to general population, t-score was significantly lower in the transplant patients when compared to control for both lumbar and hip bone, immunosuppressant therapy (mycophenolate mofetile, tacrolimus, cyclosporine and sirolimus) did not increase the risk of osteoporosis, but corticosteroids significantly increase risk of osteoporosis, biochemical marker serum level in post kidney transplant patients are significantly different when compared with general population but did not increase risk of osteoporosis. body mass index is a risk factor for both lumbar and hip bones osteoporosis, while gender and serum calcium are risk factors for osteoporosis in lumbar and hip; respectively. conflict of interest there are no conflicts of interest. references 1. bailey ma, shirley dg, unwin rj. renal physiology. in: johnson rj, feehally j, floege j. comprehensive clinical nephrology. 5th ed. philadelphia: elsevier saunders; 2015. p. 1427. 2. 2.gabriel co, helen b, and john l.r.. impact of cadaveric renal transplantation on survival in patients listed for transplantation. j am socnephrol .2005; 16 (6) 1859-1865. 3. fritsche l, vanrenterghem y, nordal kp, grinyo jm, moreso f, budde k, et al. practice variations in the evaluation of adult candidates for cadaveric kidney transplantation: a survey of the european transplant centers. transplantation. 2000;70(10):1492-7. 4. wiseman ac, cooper je. prophylaxis and treatment of kidney transplant rejection. in: johnson rj, feehally j, floege j, editors. comprehensive clinical nephrology. 5 th ed. philadelphia: elsevier saunders; 2015. p. 117687. 5. anne k, lucile a, tamsen b, steven n. b, scott d. b, david r. g. nih consensus development panel on osteoporosis prevention diagnosis, and therapy. osteoporosis prevention, diagnosis, and therapy. jama.2001; 285: 785–95. iraqi j pharm sci, vol.29(2) 2020 osteoporosis in post kidney transplantation 7 6. stein e, shane e.osteoporosis in organ transplant patients. in: marcus r, feldman d, dempster dw, luckey m, cauley ja. osteoporosis (4th ed). san diego: academic press; 2013. p. 1349-74. 7. cummings sr, bates d, black dm. clinical use of bone densitometry :scientifi c review. jama2002; 288: 1889–97. 8. rachner td, khosla s, hofbauerlc.osteoporosis: now and the future. the lancet 2011;377(9773):1276-87. 9. lindsay r, silverman s, cooper c et al. risk of new vertebral fracture in the year following a fracture. j am med ass 2001; 285: 320–323 10. coco m, glicklich d, faugere mc, burris l, bognar i, durkin p, et al. prevention of bone loss in renal transplant recipients: a prospective, randomized trial of intravenous pamidronate. journal of the american society of nephrology 2003;14(10):2669-76. 11. walsh sb, altmann p, pattison j, wilkie m, yaqoob mm, dudley c, et al. effect of pamidronate on bone loss after kidney transplantation: a randomized trial. american journal of kidney diseases2009;53(5):856-65. 12. smerud kt, dolgos s, olsen ic, asberg a, sagedal s, reisaeter av, et al. a 1-year randomized, double-blind, placebo-controlled study of intravenous ibandronate on bone loss following renal transplantation. american journal of transplantation 2012;12(12):331625. 13. .torregrosa jv, fuster d, gentil ma, marcen r, guirado l, zarraga s, et al. open-label trial: effect of weekly risedronate immediately after transplantation in kidney recipients. transplantation 2010;89(12):1476-81. 14. marcén r, caballero c, uriol o, fernández a, villafruela jj, pascual j, et al. prevalence of osteoporosis, osteopenia, and vertebral fractures in long-term renal transplant recipients. transplantation proceedings 2007;39(7):2256-8. 15. mazzaferro s, diacinti d, proietti e, barresi g, baldinelli m, pisani d, et al. morphometric xray absorptiometry in the assessment of vertebral fractures in renal transplant patients. nephrology, dialysis, transplantation 2006;21(2):466-71. 16. durieux s, mercadal l, orcel p, dao h, rioux c, bernard m, et al. bone mineral density and fracture prevalence in long-term kidney graft recipients. transplantation 2002;74(4):496500. 17. gregorini m, sileno g, pattonieri ef, corradetti v, abelli m, ticozzelli e, et al. understanding bone damage after kidney transplantation: a retrospective monocentric cross sectional analysis. transplantation proceedings 2017;49(4):650-7. 18. marcen r, caballero c, pascual j, teruel jl, tenorio m, ocana j, et al. lumbar bone mineral density in renal transplant patients on neoral and tacrolimus: a four-year prospective study. transplantation 2006;81(6):826-31. 19. rubin mr, bilezikian jp: clinical review 151: the role of parathyroid hormone in the pathogenesis of glucocorticoid-induced osteoporosis: a re-examination of the evidence. j clinendocrinolmetab 2002; 87: 4033-4041 20. martin-fernandez m, rubert m, montero m, de la piedra c. effects of cyclosporine, tacrolimus, and rapamycin on osteoblasts. transplantation proceedings 2017;49(9):221924. 21. goffin e, devogelaer j-p, lalaoui a, et al: tacrolimus and low-dose steroid immunosuppression preserves bone mass after renal transplantation. transplint2002;15:73-80. 22. campistol jm, holt dw, epstein s. bone metabolism inrenal transplant patients treated with cyclosporine or sirolimus. transplint .2005; 18:1028 -35. 23. lei s, xu-biao x, long-k. p, shao-j. y, yat. p. mechanism and treatment strategy of osteoporosis after transplantation. international journal of endocrinology 2015; p 10. 24. ahmadpoor p, reisi s, makhdoomi k, ghafari a, sepehrvand n, rahimi e. osteoporosis and related risk factors in renal transplant recipients. transplproc2009; 41:2820–2822. 25. esra o. h, hussein y. s.prevalence and risk factors of osteoporosis in kidney transplant recipients: dual-energy x-ray absorptiometry scan study.medical journal of babylon. 2018; 15: 267-270. 26. evangelia d, konstantinosl ,theodoros e, vassilios l. osteoporosis after renal transplantation. international urology and nephrology. 2015;47:503–511. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.19(1) 2010 design and synthesis of new nsaid 6 design and synthesis of new non-steroidal anti-inflammatory agents with expected selectivity toward cyclooxygenase-2 inhibition nadeem a. abdul razzak *,1 , samera.f.hussan * and ahlam j. qaseer * * department of pharmaceutical chemistry , college of pharmacy,university of baghdad ,baghdad, iraq. abstract this study includes design and synthesis of new non-steroidal anti-inflammatory agents (nsaids) with expected cyclooxygenase-2 (cox-2) selective inhibition to achieve better activity and low gastric side effects. two series of compounds have been designed and synthesized as potential nsaids,these are: salicylamide derivatives (compounds 3,4,5 ) and diflunisal derivatives (compounds 10&11). in vivo acute anti-inflammatory effect of one of the synthesized agents (compound 3) was evaluated in the rat using egg-white induced paw edema model of inflammation. preliminary pharmacological study revealed that compound 3 exhibited less anti-inflammatory effect compared to that of aspirin after 120 and 210 minutes, which encourage the continuation of the search to demonstrate or identify the preliminary pharmacological activity for the synthesized compounds and to identify their selectivity toward cox-2 isoenzymes. keywords:nonsteriodal anti-inflammatory drugs(nsaid),aspirin derivatives ,diflunisal, cyclooxygenase -1(cox-1),cyclooxygenase-2(cox-2) الخالصة حصًيى وحخهيق يشكباث جذيذة غيش سخيشويذيت يضادة نالنخهاباث راث فعانيت يخىقعت كًزبطاث نالَضيى انذساستحخضًٍ نهحصىل عهى فعانيت افضم واعشاض جاَبييت اقم. حى حخهيق يجًىعخيٍ يٍ انًشكباث وهي:يشخقاث (cox-2) 2 سايكهىاوكسجُيض أجشيج دساست انخقييى انذوائي األوني نهفعانيت انًضادة (.00و01انًشكباٌ يشخقاث انذيفهىَيسال )و(,54,4انًشكباث انسهسيم أيايذ ) ( بطشيقت اسخحذاد وريت ححج جهذ يذ انجشر باسخخذاو صالل ,نالنخهاباث غيش انشرىيت نىاحذ يٍ انًشكباث انًخهقت انجذيذة )انًشكب ألسبشيٍ بعذ يٍ ااقم أظهش حأريشاً يضاداً نالنخهاباث قذ ,األونيت إٌ انًشكب انبيىنىجيتانفعانيت انبيط )األنبىييٍ(..أشاسث انُخائج انخقييى انذوائي األوني نبقيت انًشكباث انًخهقت وكزنك يعشفت دسجت اَخقائها نًعشفت انبحذ عهى إكًال يشجعدقيقت، يًا 201و 021 .ضانًزبط إلَضيى انسايكهىأوكسجُي introduction inflammation defined as a complex series of tissue changes that result in pain and fever (1) ; or it is a normal, protective response to tissue injury caused by physical trauma, noxious, chemical, or microbiologic agents. inflammation is the body's effect to inactivate or destroy invading organisms, remove irritation, and set the stage of tissue repair (2) . inflammation can be divided into three phases; acute, chronic and immune response (3). there are two cyclooxygenase (cox) enzymes, cox-1 and cox-2. cox-1 is a constitutive enzyme, involved in tissue homeostasis; while cox-2 is induced in inflammatory cells and produces the prostanoid mediators of inflammation. also cox-3 has recently been described (4) . although cox-1 and cox-2 have similar structures, there are slight differences that affect the drug binding and lead to different actions (5) . both enzymes have a long narrow channel into which arachidonic acid enters and be converted into pgs, with cox-2 has an additional side pocket. selective cox-2 inhibitors have chemical structure with rigid side extension that binds in this side pocket . .nsaids have three major pharmacological desirable actions, all of which result mainly from the inhibition of cox-2 in inflammatory cells and the resultant decrease in prostanoid synthesis; these are: anti-inflammatory action,antipyretic effect and analgesic effect. we have two types of nsaid these are traditional nsaid and selective nsaid (5) .traditional nsaids (tnsaids) are mainly carboxylic acid containing compounds that are either aromatics or aliphatics. they include different chemical classes with different physical properties (6) .they are frequently prescribed for muscloskeletal complications and are often taken without prescription for minor aches and pains. there are new many different nsaids on the market and non of these is ideal in controlling or modifying the sign and symptoms of inflammation (7) . 1corresponding author email : nadeem_mmr@yahoo.com received : 16/6/2009 accepted : 1/11/2009 iraqi j pharm sci, vol.19(1) 2010 design and synthesis of new nsaid 7 for example salicylic acid derivatives e.g. aspirin, (acetyl salicylic acid) has been in use as a pharmaceutical agent for over 100 years (8) . aspirin is unique among cox-inhibitors because it covalently modifies the protein of enzymes and irreversibly inhibits them (9) . aspirin transfers its acetyl group to serine-530 (ser530), which prevents proper binding of arachidonic acid in the cox active site .no other residues on either cox-1 or cox-2 are acetylated (10,11,12) .aspirin suffers from the side effects associated with local gastrointestinal irritation; therefore a number of attempts or modifications have been carried out to overcome this problem such as salt formation, e.g. calcium acetyl salicylate ,buffered aspirin (magnesium or aluminum salts) and such as nuclear modification such as diflunisal (13) .diflunisal ,5-(2',4'-difluorophenyl) salicylic acid, is not converted to salicylic acid in vivo. it is largely devoid of antipyretic effect, perhaps because of poor penetration into the cns. it is 3-4 times more potent than aspirin (6) .the higher potency of diflunisal may be resulted from the existence of two vanderwaals binding sites (aromatic rings) and the ortho fluoro atom which promotes non-coplanarity between the two aromatic rings which is required for effective binding to the cox enzyme. in addition, the presence of para-fluoro atom will retard metabolism, by preventing oxidation of the para-position, hence increase duration of action (13) .cox-2 selective inhibitors, or coxibs, were developed in an attempt to inhibit prostacyclin synthesis by cox-2 isoenzyme induced at the site of inflammation without affecting the action of the constitutively active cox-1 isoenzyme found in the gastrointestinal tract, kidney, and platelets (3) . preferentially selective cox-2 inhibitors for example : meloxicam which is a novel nsaid acting by preferential inhibition of cox-2 (14) . it has a selectivity towards cox-2 up to 100 fold over cox-1 depending on the test system (15) .. an isosteric functional groups to 2-amino-5-methyl thiazole moiety in meloxicam are investigated as a possible bioisosteric analogues (16) . highly specific cox-2 inhibitors since the loss of selectivity is a potential problem with large dosage of nsaids that, preferentially inhibit cox-2; thus new agents that are more specific towards cox-2 even at large dose were synthesized, these agents include:celecoxib,roficoxib,valdicoxib,etoricoxib, and imricoxb. experimentals a.chemistry materials: 2-aminobenzothiazole,2aminopyridine&2-aminopyrimidine (bdh,england ), acetyl salicylic acid(judex england),dcci, diflunisal(jordon),ethanol 95%and 99%,all solvents were of analar type and used without further purification general procedure melting points(uncorrected) were determined by capillary method on thomas hoover apparatus (england)and ir spectra were recorded on model 500 scientific ir spectrophotometry ,buck company(usa) in pharmacy collage ,baghdad university.ascending thin layer chromatography (tlc) was run on dc-kartan si alumina 0.2 mm to check the purity and progress of reaction. the chn analysis was done using an exeter ce-440 elemental microanalyzer (germany). the analysis was carried out at micro analytical center, faculty of science-cairo university.the identification of compounds was done using iodine vapor and the chromatograms were eluted by two solvent systems: a:thf : diethyl ether : cyclohexane (40:40:20) (17) b:methanol : acetic acid : diethyl ether : benzene (1:18:60:20) (18) method of preparation of aspirin anhydride aspirin (5.0gm, 27.77mmole) was dissolved in methylene chloride (75ml), and dicyclohexyl carbodiimide (dcci) (2.86gm, 23.84mmole) was added. the reaction mixture was continuously stirred at room temperature for 3.5 hours. a white precipitate of dicyclohexylurea (dcu) was formed, then removed by filtration. the filtrate was evaporated under vacuum and an oily product was formed to yield compound (1) (18) . synthesis of n-(2-pyridyl)-acetyl salicylamide compound 1: (4.0gm, 11.69mmole), 2aminopyridine (1.1gm, 11.69mmole), zinc dust (catalytic amount, 0.01gm), glacial acetic acid (1.12ml, 19.64mmole) and dioxane (32ml) were placed in a flask, equipped with reflux condenser. the reaction mixture was refluxed gently for 90 minutes, the solvent was evaporated under vacuum, the residue was dissolved in the minimum volume of ethyl acetate, washed with nahco3 (10%, 3x), hcl (1n, 3x), and distilled water (3x), dried using anhydrous magnesium sulfate. the filtrate was evaporated under vacuum to give a crude product 2. the recrystilization was carried out using ethyl acetate-petroleum ether (60-80 o c) iraqi j pharm sci, vol.19(1) 2010 design and synthesis of new nsaid 8 mixture, a white crystalline product was obtained compound(2) (19, 20) . compound (2) :melting point (115-117),yield (41.14 white powder),ir in kbr disk :3390 n-h stretching vibration of secondary amide,1768 c=0 stretching vibration of acetate ester ,1604-1577&1532 c=o stretching vibration of aromatic and n-h bending (amide ii)is also included in this region. synthesis of n-(2-pyridyl)-salicylamide compound 2 (0.5gm, 1.953mmole) was dissolved in a minimum volume of absolute ethanol 99%: tetra hydro furan(thf) (3:1) mixture. the solution was cooled to 18 o c, and then sodium hydroxide solution (2n, 1.162ml, 2.325mmol) was added drop wise, with continuous stirring over a period of 30 minutes. stirring was continued at 18 o c for additional three hours. the reaction mixture was neutralelized with equivalent quantity of hcl, excess of cold water was added to precipitate the phenolic compound, which was then filtered and dried to give compound (3) (20). the same method of synthesis has been done for the other salicylamide derivatives but with two different heterocycles (2-amino benzothaiazole) and (2-amino pyrimidine) to yield the final compounds (4)&(5). compound (3):melting point (218220),yield(51% white crystals),rf value (0.75a&0.79b) ;ir in kbr disk :3000-3500 broad o-h stretching vibration of phenol,3238 n-h stretching vibration of secondary amide , 1675 c=o stretching vibration of secondary amide (amide i), 1611-1604, 1541-1537 c=c stretching vibration of aromatic and n-h bending vibration of secondary amide,1234c-o stretching vibration of phenol (figure 1) . chn analysis found c 66.71, h 4.63,n 12.74. chn calculated c 67.28,h 4.67,n 13.08 compound (4):melting point 290 decomposed ,yield (47% white crystals ). compound (5):melting point (191193),yield(56%white crystals). ir in kbr disk for both compounds :30003500 broad band stretching vibration of phenol , 3330 &3301 n-h stretching vibration of secondary amide ,1678&1623 c=o stretching vibration of secondary amide (amide i),1245&1224 c-o stretching vibration of phenol. figure 1 : ir spectrum of compound( 3) in kbr disk. synthesis of 5-(2 ' ,4 ' -difuorophenyl)acetylsalicylic acid, compound (6) in a 250ml boiling flask, equipped with reflux condenser, diflunisal (5.0gm, 20mmol) and acetic acid anhydride (15ml, 159mmole) were placed and 3 drops of concentrated sulfuric acid were added drop wise. the reaction mixture was refluxed gently for 1 hour, then allowed to cool with occasional stirring. ice-water was then added until a precipitate was formed, which was filtered using pump, washed with cold distilled water several times, the crude product was collected. the recrystalization was carried out using ethanol 95% to give compound( 6) (19) . compound (6): melting point (172-174),yield (89% white crystals ) ir values in kbr disk 3000-2676 o-h stretching vibration (hbonded ) of carboxylic acid ,1768 c=o stretching vibration of acetate ester ,1700 stretching vibration of carboxylic acid ,1319 o-h bending vibration of carboxylic acid ,1274 c-o stretching vibration of carboxylic acid. synthesis of 5-(2 ' ,4 ' -difluorophenyl)-acetyl salicylic acid anhydride compound(7): compound 6 (5.0gm, 17.1mmole) was dissolved in thf (30ml), then dcci (1.75gm, 8.55mmole) was added. the reaction mixture was continuously stirred at room temperature for 4 hours. a iraqi j pharm sci, vol.19(1) 2010 design and synthesis of new nsaid 9 white precipitate of dcu was formed which was then removed by filtration. the solvent was evaporated under vacuum and a solid product of compound (7) was obtained (18) . compound (7): melting point (149-150),yield (80% white powder ) ir value in kbr disk :1815&1743 c=o stretching vibration of anhydride (asymmetric &symmetric bands),1277&1173 c-(c=o)-o(c=o)-c stretching vibration of anhydride. synthesis of 5-(2,4-difluorophenyl)-n-(2pyridyl) acetyl salicylamide compound(8) compound 5 (2.5gm, 4.4mmole), 2aminopyridine (0.418gm, 4.4mmole), zinc dust (0.004gm), glacial acetic acid (0.425ml) and dioxane (25ml) were placed in roundbottom flask, equipped with reflux condenser. the reaction was carried out as in the synthsis of compound (2). the recrystilization was carried out using ethyl acetate-petroleum ether (60-80 o c) mixture, a white crystalline product was obtained( (compound 8) (19, 20) . compound (8): melting point (180-181),yield (42.2 white powder) . the same method of preparation has been done with 2-amino benzothiazole to yield compound (9) compound (9):melting point (161164),yield(39.5% white crystals) . ir values in kbr disk for compounds (8&9): 3335&3333 n-h stretching vibration of secondary amide ,1774&1753 c=o stretching vibration of acetate ester ,1696&1686 c=o stretching vibration of secondary amide (amide i). synthesis of 5-(2,4-difluorophenyl)-n-(2pyridyl) salicylamide, compound (10) to a cooled solution(18 o c) of compound (3) (0.4gm, 1.085mmole) in absolute ethanol 99%: thf (3:1) mixture, sodium hydroxide (2n, 0.651ml, 1.302mmol) was added drop wise, with continuous stirring over a period of 30 minutes. then the reaction mixture was worked up as described previously in preparation of compound (3).the same method of preparation has been done for diflunisal anhydride with (2-amino benzothiazole) to yield the final product compound (11). compound (10):melting point (230-232),yield (48,%of white powder),rf value (0.53 a&0.69b),ir in kbr disk: 3376-3227 o-h stretching vibration of phenol, 3304 n-h stretching vibration of secondary amide,1660 c=o stretching vibration of secondary amide (amide i), 1598 and 1530 c=c stretching vibration of aromatic and n-h bending of secondary amide (amide ii), 1262 c-o stretching vibration of phenol ( figure 2). chn analysis found :c 64.93,h 3.80, n 8.33 chn calculated : c 66.26,h 3.68, n 8.59. figure 2 : ir spectrum of compound 10 in kbr disk. compound (11): melting point (251253),yield 42% faint yellow crystals).ir value in kbr disk :3450-3100 o-h stretching vibration of phenol and n-h stretching vibration of amide is buried within this band ,1677 c=o stretching vibration of secondary amide (amide i),1604-1534 c=c stretching vibration of aromatic and bending vibration of secondary amide (amide ii). b.pharmacology acute anti-inflammatory activity of one of the chemically synthesized compounds, compound( 3) was evaluated in vivo using eggwhite to induce paw edema in rats. the decrease in paw thickness is the basis of screening the newly synthesized compound for its anti-inflammatory activity.eighteen albino rats of either sex, weighing 300 ± 10 gm supplied by the animal house of the college of iraqi j pharm sci, vol.19(1) 2010 design and synthesis of new nsaid 10 pharmacy, university of baghdad were used in this study. animals were kept under standardized conditions (12 light-12 dark cycle) for 7 days for acclimatization; and were fed commercial chaw and had provided with water. rats were brought 1 hour before performing the experiment to the laboratory, and were allocated into 3 groups (each of 6 rats) as follows: a/ six rats served as control; they received drug vehicle (0.5ml propylene glycol in water 50% v/v). b/ six rats received aspirin as a reference substance in a dose of (100mg/kg, i.p.) in propylene glycol (21) .c/ six rats received compound 3 suspended in propylene glycol (118.8mg/kg, i.p.) (22) . the anti-inflammatory activity of the tested compound was studied using egg-white induced edema model. acute inflammation was produced by a subcutaneous injection of 0.05ml of undiluted fresh eggwhite into the planter side of the left hind of the rats; 30 minutes after intraperitoneal injection of the drug or the control. the paw thickness was measured by vernea at eight time intervals (0, 30, 60, 90, 120, 150, 180 and 210 minutes) after the drug administration. statistical analysis students t-test was used to make comparisons with respect to baseline, while comparisons between different groups at specified time was done using analysis of variance (anova). p values less than 0.05 were considered significant. result and conclusion the designed compounds have been synthesized successfully as shown in scheme (1) & (2) and their structures were confirmed, using elemental microanalysis (chn), infrared spectroscopy (ir spectra) and their purity was confirmed by their physical data (melting points and rf values).the conversion of carboxylic acid group of aspirin and diflunisal to corboxamide group by conjugating the selected moiety of heterocyclic compound may produce new non-steroidal anti-inflammatory agents with expected selectivity toward cox2 inhibition and hence less gastric irritation. preliminary pharmacological evaluation has been done for one of the designed compounds (compound 3) and it has been found that this compound exhibit slightly less potent antiinflammatory effect than aspirin as shown in (figure 3 and table 1). scheme (1): synthesis of compounds (3,4 ,5). h3c o oh o o dcc h3c o o o h3c o o o o nh2n h3c o o o n n h methylen chloride aspirin 1 reflux 2 (2) hcl (1) naoh oh o n n h 3 n sh2n reflux h3c o o o n s n h (2) hcl (1) naoh oh o n s n h 4 n nh2n reflux h3c o o o n n n h (2) hcl (1) naoh oh o n n n h 5 2 dcc iraqi j pharm sci, vol.19(1) 2010 design and synthesis of new nsaid 11 4 4.2 4.4 4.6 4.8 5 5.2 5.4 5.6 5.8 6 6.2 0 120 210 time (min) pa w th ic kn es s (m m ) propylene glycol aspirin compound 35 a a a a b b a b b scheme (2): synthesis of compounds (10 and 11). figure 3: effect of vehicle (propylene glycol), aspirin and compound 3 on paw edema in rat after 120 and 210 minutes of egg-white injection. results are expressed as mean ± sem (n=6/group). time zero is the time of egg-white injection. non-identical superscripts (a,b) among different groups represent significant difference (p<0.05). acetic anhydride reflux diflunisal oh ho of f ho of f o h3c o thf o of f o h3c o dcc of f o h3c o o h nf f o h3c o r-nh2 reflux r (1) naoh (2) hcl o h nf f oh r n 6 7 n n s 22 rcompound no. 10 11 compound no. r 8 9 n s p a w t h ic k n e ss ( m m ) dcc acetic anhydride rnh2 (1) naoh (2)hcl compound no. compound no. iraqi j pharm sci, vol.19(1) 2010 design and synthesis of new nsaid 11 table 1:effect of control (propylene glycol), reference drug (aspirin) and tested compound (compound 3) on egg-white induced paw edema in rats. time (min) paw thickness (mm) control (n=6) aspirin (n=6) compound 3 (n=6) 0 5.45 ± 0.12 a 5.39 ± 0.11 a 5.54 ± 0.12 a 30 6.15 ± 0.15 5.85 ± 0.13 5.79 ± 0.09 * 60 6.41 ± 0.07 * 5.64 ± 0.14 * 5.85 ± 0.11 * 90 6.56 ± 0.11 * 5.48 ± 0.12 * 5.68 ± 0.09 * 120 5.85 ± 0.12 * a 5.18 ± 0.12 * b 5.43 ± 0.07 * b 150 5.41 ± 0.09 * 4.95 ± 0.15 * 5.21 ± 0.11 * 180 5.30 ± 0.12 * 4.70 ± 0.11 * 4.98 ± 0.09 * 210 5.25 ± 0.07 * a 4.58 ± 0.06 * b 4.75 ± 0.13 * b  data are expressed as mean ± sem.  n= number of animals.  control, reference drug and tested compound were given 30 minutes before the injection of eggwhite.  time (0) is the time of injection of egg-white (induction of paw edema).  *p<0.05 with respect to time (0).  non-identical superscripts (a, b) among different groups represent significant difference (p<0.05). references 1. guyton, a.c. and hall, j.e. (eds.): resistance of body to infection: leuckcytes, graneulocytes, the monocytemacrophase system and inflammation. in: textbook of medical physiology (10 th ed.). harcourt asia pteltd, philadelphia, 2000; pp. 397. 2. harvey, r.a. and champe, p.c. (eds.): anti-inflammatory drugs and autocoids. in: lippincott’s illustrated review pharmacology (3 rd ed.), lippincott williams and wilkins, philadelphia, 2006; pp. 495-498. 3. waghner, w.; katzung, b.g. and furst, d.e.: nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, non-opioid analgesic and drugs used in gout. in: basic and clinical pharmacology (9 th ed.). katzung, b.g (ed). mcgraw hill, new york, 2004; pp. 573-576. 4. rang, h.p., dale, m.m. and ritter, j.m. 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pharmacol. 2004; 36(3): 148-150. abstract: iraqi j pharm sci, vol.20(1) 2011 hba1c and foot ulceration 19 measurements of hba1c for patients with diabetes mellitus and foot ulceration mohammed a.taher *,1 , mayada m. moustafa * , aqeel s. mahmood ** *department of clinical laboratory sciences,college of pharmacy,university of baghdad,baghdad, iraq. **medical city , baghdad teaching hospital, baghdad, iraq. abstract people with diabetes can develop different foot problems. in the blood stream glucose reacts with hemoglobin to make a glycosylated hemoglobin molecule called hemoglobin a1c or hba1c, the more glucose in the blood the more hemoglobin a1c will be present in the blood. the hbalc test is currently one of the best ways to check diabetes to be under control. the aim of study is to compare between the blood investigations which includes the fasting blood sugar and hbalc (glycosylated hemoglobin), and to evaluate the benefit of hbalc (measurement for diabetic patients with foot ulcer, to be a good indicator for controlling blood glucose). sixty patients with type2 diabetes mellitus from the outpatient clinic of baghdad teaching hospital, medical city over the period from nov. 2006 to nov. 2008, were included in the study. follow up was done only to 30 patients with diabetic foot ulcer. twenty (66.66%) were males and 10(33.33%) were females their age range from (23-75) years (mean age of 52years), and 21 normal subjects as control. a (glycohemoglobin hbal-test/fast lon-exchange resin separation method) kit was used. the data finding that there is a greater association between hbalc level and foot ulceration healing. there is a relationship between the age of the patients and the hbalc level. the patients who used (glibenclamide+metformin) have the lower range of hbalc, while those who use (metformin) have the higher level of hbalc. hbalc (glycosylated hemoglobin) is most accurate test to determine actual reading over the past 2-3 months, and to evaluating the risk of glycemic damage to the tissues. so, we recommend the hbalc testing, but it can't be used to monitor day-to-day blood glucose concentration because it's not influenced by fluctuation in blood concentration. key words: diabetic foot ulcer, hbalc الخالصة ّىٓ اْ تتطٛر ٌد٠ُٙ اٌحاٌة اٌّزظي١ة اٌيٝ اابيابة بتمز يا ليٟ اٌميدَ ج ليٟ ِميزٜ اٌيدَ ش٠ يا ّاٌّصاب١ٓ بداء اٌسىزٞ ِٓ اٌ ٚوٍّيا وأيك و١ّية اٌىٍٛويٛس hba1cاٚ a1cاٌىٍٛوٛس تٍتصك با١ٌّٙٛغٍٛب١ٓ ٌتى٠ٛٓ وال٠ىٛس١ال٠تد ١ّ٘ٛغٍٛب١ٓ ٚتدعٝ ١ّ٘ٛغٍيٛب١ٓ ٚا يد ِيٓ hba1cٚ يد ليٟ ااٚٔية ااة١يزن بياْ ااسيتاأة باةتتيار ِسيتٜٛ اوثز ج ٌمد a1cلٟ اٌدَ اوثز وٍّا وأك و١ّة ا١ٌّٙٛغٍٛب١ٓ اٌغا٠ة ِٓ ٘ذٖ اٌدراسة ٌٍّمارٔة بي١ٓ اٌطيزق اٌّتتاية ٚاٌّاتّيد ع١ٍٙيا ليٟ ٞ اٌٛالع تحك اٌس١طزن جالعً اٌطزق ٌٍتحمك ِٓ ِزض اٌسىز ٌسيىزٞ ليٟ اٌية اٌصي١اَ ِٚمارٔتيٗ باةتتيار ِسيتٜٛ اٌس١طزن عٍٝ ٔستة اٌسيىز ٌيدٜ اٌّزظيٝ اٌسيىزٞ ِيٓ إٌيٛن اٌثيأٟ خ ِثيً اةتتيار ا 30شخص ِصاب١ٓ بّزض اٌسىزٞ ِٓ إٌيٛن اٌثيأٟ ٚاسيتّز اٌدراسية عٍيٝ 60اٌىال٠ىٛس١ال٠تد ١ّ٘ٛغٍٛب١ٓ ج ا ز٠ك اٌدراسة عٍٝ %( ج 33.33) 10( ٚاأيا 66.66%) 20سٕة ٚواْ عيد اٌيذوٛر 75-23شخص ُِٕٙ ِصاب بتمز ا لٟ اٌمدَ ٚبّادي عّزٞ ِٓ شخص س١ٍُ غ١يز ِصياب بيداء اٌسيىزٞ ٌٚي١ه ٌد٠يٗ تمز يا )ِمّٛعية سي١طزن( ٚليد ٚ يدٔا ٔت١مية ٘يذٖ اٌدراسية بياْ 21بـ وّا استا١ٓ ج وّيا ٚ يدٔا ٕ٘يان عاللية بي١ٓ عّيز ٚشيفاء تمز يا اٌميدَ hba1cتف١يد بياْ ٕ٘يان عاللية وت١يزن بي١ٓ ِسيتٜٛ إٌتائج اٌّستحصيً ع١ٍٙيا ٛس١ال٠تد ١ّ٘ٛغٍٛب١ٓ لٟ اٌدَ خ ويذٌه ٚ يدٔا ِيٓ ةيالي ٘يذٖ اٌدراسية بياْ اٌّزظيٝ اٌّاياٌم١ٓ بّمّٛعية اا ٠ٚية اٌّز٠ط ٚٔستة اٌىال٠ى اٌّخفعيية ٌٍسييىز )١ِتفييٛر١ِٓ ي و١ٍت١ٕىالِا٠ييد( لييد أخفييط ِسييتٜٛ اٌىال٠ىٛسيي١ال٠تد ١ّ٘ٛغٍييٛب١ٓ اوثييز ِييٓ ِمّٛعيية اٌّزظييٝ اٌييذ٠ٓ ٠ىٛس١ال٠تد ١ّ٘ٛغٍٛب١ٓ اعٍٝ ج تستٕتج اٌدراسة باْ اٌىال٠ىٛس١ال٠تد ١ّ٘ٛغٍٛب١ٓ ٘يٛ استاٍّٛا )ا١ٌّتفٛر١ِٓ( لمط ١ث واْ ِستٜٛ اٌىال اشٙز سابمة ٌٚتمد٠ز اٌخطٛرن اٌتٟ ٠تازض ٌٙا اٌّصياب ج ٌيذٌه ٔٛبيٟ باسيتاّاي 3 – 2ابح اةتتار ٌتا١١ٓ اٌم١ّة اٌحم١م١ة ٌٍسىز بّدن جّة اٌسىز اٌّتغ١زن ١ِٛ٠ااط١ٕا ل١اةتتار اٌىال٠ىٛس١ال٠تد ١ّ٘ٛغٍٛب١ٓ تٝ ٚاْ واْ ا٠ introduction people with diabetes can develop different foot problems [1,2] . foot problems most often occur when there is neuropathy, poor blood flow , or changes in the shape of feet toes [2-4] , ulcers occur most often on the ball of the foot or in the bottom of the big toe. neglecting ulcers can result in infections, which in turn may lead to loss of a limb [5] . diabetic patients are at higher risk and they show 2 4 times more likely to have heart disease or suffer a stroke than people without diabetes [5-7] . in the blood stream glucose reacts to the hemoglobin to make a hemoglobin molecule called hemoglobin a1c (hba1c), the more glucose in the blood , the more hemoglobin a1c will be formed in the blood [6,7] . about 90% of hemoglobin is hemoglobin a [7] . 1corresponding author email : mayada_aga@yahoo.com received : 9/5/2010 accepted : 3/1/2011 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://www.medicinenet.com/script/main/art.asp?articlekey=3690 http://www.medicinenet.com/script/main/art.asp?articlekey=3690 iraqi j pharm sci, vol.20(1) 2011 hba1c and foot ulceration 20 although one chemical component accounts for 92% of hemoglobin a, approximately 8% of hemoglobin a is made up of minor components that are chemically slightly different. these minor components include hemoglobin a1c, a1b, a1a1, and a1a2. hemoglobin a1c is a minor component of hemoglobin to which glucose is bound. thus it's referred to as glycosylated or glucosylated hemoglobin [8-10] .the hba1c test is currently one of the best ways to check diabetes is under control. the hba1c level changes slowly, over 10 week, so it can be used as a ((quality control)) test [11-13] . hemoglobin a1c was first separated from other forms of hemoglobin by huisman and meyering in 1958 using a chromatographic column. [12] it was first characterized as a glycoprotein by bookchin and gallop in 1968. [13] its increase in diabetes was first described in 1969 by samuel rahbar and coworkers the reactions leading to its formation were characterized by bunn and his co-workers in 1975. [14, 15] the use of hemoglobin a1c for monitoring the degree of control of glucose metabolism in diabetic patients was proposed in 1976 by anthony cerami, ronald koenig and coworkers [16] . in the normal 120-day life span of the red blood cell, glucose molecules react with hemoglobin, forming glycosylated hemoglobin. in individuals with poorly controlled diabetes, the quantities of these glycosylated hemoglobins are much higher than in healthy people [16, 17] .once a hemoglobin molecule is glycosylated, it remains that way. a buildup of glycosylated hemoglobin within the red cell therefore reflects the average level of glucose to which the cell has been exposed during its life cycle [18, 19] . measuring glycosylated hemoglobin assesses the effectiveness of therapy by monitoring long-term serum glucose regulation. the hba1c level is proportional to average blood glucose concentration over the previous four weeks to three months [20] . some researchers state that the major proportion of its value is related to a rather shorter period of two to four weeks. [7] . the 2010 american diabetes association standards of medical care in diabetes added the a1c ≥ 6.5% as another criterion for the diagnosis of the diabetes [21] . there were numbers of laboratories techniques used to measure glycosylated hemoglobin [22] :  high-performance liquid chromatography (hplc).  immunoassay. patients and methods patients with type 2 diabetes mellitus with foot ulcers seen in the outpatient clinic of baghdad teaching hospital, medical city over the period from nov. 2006 to nov. 2008. a total number of 30 patients with diabetic foot ulcer and 21 normal subjects.. the age of patients range from 23-75 years (mean age 52), as shown in table (1) and occupation of the patients, in table (2). diagnosis of the presence of foot ulcers was made by a specialist physician through physical examination and xray examination. for every case, the following had been done, 1. patient medical history recorded. 2. full physical examination, a complete blood picture and renal function test. 3. lab. investigations: a. fasting blood sugar: was measured in serum obtained for all subjects blood by a commercial kit obtained from biomaghreb, using the enzymatic method (23) . b. hbalc (glycosylated hemoglobin): method of measurement was followed according to the instructions mentioned in glycohemoglobin hba1-test kit which obtained from wiesbaden-germany using blood specimens and edta as anticoagulant (24) . 4. request form was given to all patients which include the details of age, sex, occupation, symptoms, site of ulcers, table (3). 5. all 30 patients with diabetic foot ulcer were put on therapy with oral hypoglycemic drugs for 3months (we measured their fasting blood sugar and hbalc before and after the therapy). these drugs, metformin [500mg, 2/day] glibenclamide [5mg, 2/day] and combination of glibenclamide 5mg (2/day)+ metformin 500mg (2/day) (25) , table (5). 6. the relationship of patients age and (hbalc and fbs) were determined, table (7). 7. a control groups: the control group was represented by 21 apparently healthy persons and their fbs (fasting blood sugar) and hbalc were measured, table(4). 8. data were expressed as mean ± standard deviation and differences between means were analyzed by paired student's t-test. p value less than 0.05 were considered significantly different. http://www.medicinenet.com/script/main/art.asp?articlekey=3608 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/chromatography http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/glycoprotein http://en.wikipedia.org/wiki/hba1c#cite_note-pmid4874776-2 http://en.wikipedia.org/wiki/samuel_rahbar http://en.wikipedia.org/wiki/hba1c#cite_note-pmid1201013-4 http://en.wikipedia.org/wiki/anthony_cerami http://en.wikipedia.org/wiki/anthony_cerami http://en.wikipedia.org/wiki/hba1c#cite_note-pmid934240-5 http://en.wikipedia.org/wiki/red_blood_cell http://en.wikipedia.org/wiki/red_blood_cell http://en.wikipedia.org/wiki/glycation http://en.wikipedia.org/wiki/diabetes http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/biological_life_cycle http://en.wikipedia.org/wiki/biological_life_cycle http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/hba1c#cite_note-6 http://en.wikipedia.org/wiki/american_diabetes_association http://en.wikipedia.org/wiki/hba1c#cite_note-6 http://en.wikipedia.org/wiki/hba1c#cite_note-1 http://en.wikipedia.org/wiki/high-performance_liquid_chromatography http://en.wikipedia.org/wiki/immunoassay iraqi j pharm sci, vol.20(1) 2011 hba1c and foot ulceration 21 results the percentages of diabetic foot ulcer were greater over 70 years old and less under 30 years old, table (1). while table (2), shows the more effected diabetic patients with foot ulcer were laborers and employees and less were students. the biggest sites of foot ulcer were on the bottom of the big toe and less at the ball of the foot, table (3).table (4), shows significant difference between fbs & hba1c for diabetic foot ulcer patients and control group. in table (5), patients who used metformin or glibenclamide and the combination of them (glibenclamide + metformin) the mean±value hbalc decreased significantly after treatment p<0.05. fbsof patients who used metformin or glibenclamide and combination of them (glibenclamid +metformine) the mean± value of fbs decreased significantly compared with the mean value before treatment ,and table (6), shows patients who used combination of (glibenclamide+metformin) have higher percentage (53.84%) of healing than patients treated with glibenclamide 30.0% or metformin 28.57% .table (7), shows the highest level of hbalc and fbs over 70 years old patients with foot ulcer and the lowest level of hba1c and fbs under 30 years old. fig.(1) show the significant +ve correlation between age and hba1c% r=0.82, p< 0.01. fig. (2): show that age of the patients is proportional to fbs were r=0.8, p< 0.01. for people without diabetes mellitus, the normal range for the hba1c≤6.2%. (70%) of patients whom have hba1c level less than (9.79%) did not complain from any complications. and (30%) of patients with high hba1c (9.79-11.07%) shows that they suffered from some of the following complications: a. retinopathy (impairment of vision, exudation and retinal hemorrhage). b. neuropathy (pain, numbness, and loss of sensation). c. nephropathy (pleural effusions, ascites, subcutaneous oedema in legs, high blood urea levels, high serum creatinine levels and albuminuria). d. gastropathy . table 1 : age distribution n = 30 table 2 : the occupation of the patients occupation percent farmer 15 % housewife 24.5 % laborers and employees 32.5 % student 3.5 % retired 24.5 % total 100 % n = 30 table 3 : the site of foot ulcers distribution. site number of patients percent ball of the foot 12 40% bottom of the big toe 18 60% total 30 100% n = 30 table 4: fasting blood sugar and glycosylated hemoglobin in control subjects and diabetic foot ulcer. before starting treatment. control (n=21) diabetic foot ulcer (n=30) fbs (mg/dl) 83 ± 5.04 a 243.83 ± 7.10 b hba1c % 3.41 ± 0.17 a 9.25 ± 0.21 b data are expressed as mean ± sd. n=number of patients. non-identical superscripts (a,b) represent significant difference, p<0.001). age group percentage of diabetic foot ulcer less than 30 5.5 % 30-39 8.5 % 40-49 7.5 % 50-59 24.5 % 60-69 21.5 % 70-75 32.5% total 100 % iraqi j pharm sci, vol.20(1) 2011 hba1c and foot ulceration 22 table 5 : hba1c and fbs levels for patients with diabetic foot ulcer after and before treatment hba1c % fbs before after before after metformin (n=7) 9.06 ± 0.2 a 6.64 ± 0.08 a* 238.85 ± 8.08 a 138.98±5.01 a* glibenclamide (n=10) 9.64 ± 0.32 a 6.65 ± 0.06 b* 248.08 ± 7.10 a 134.00±5.54 b* metformin + glibenclamide (n=13) 9.05 ± 0.12 a 6.31 ± 0.07 c* 244.56 ± 6.13 a 124.6 ± 3.94 c* data are expressed as mean ± sd. n=number of patients. *p<0.05 with respect to pre-treatment value. non-identical superscripts (a,b,c) represent significant difference among groups, p<0.05). table 6 : percentage of healing after treatment for diabetic foot ulcer patients. treatment patient no. dose healing cases percentage of healing metformin 7 500mg (2/day) 2 28.57% glibenclamide 10 5mg (2/day) 3 30.0% glibenclamide + metformin 13 5mg (2/day) + 500mg (2/day) 7 53.84% table 7 : distribution of mean hba1c and fbs among the patients with foot ulcer. age group hba1c% fbs (mg/dl) <30 8.18 ± 0.12 205 ± 5.25 30-39 8.56 ± 0.13 218.66 ± 6.67 40-49 9.79 ± 0.29 275.5 ± 13.07 50-59 10.99 ± 0.29 345.75 ± 18.46 60-69 11.01 ± 0.27 344.4 ± 19.39 >70 11.07 ± 0.64 354.5 ± 41.07 figure 1 : the relationship between hba1c and age. figure 2: the relationship between fbs and age. discussion and conclusion the present study reflecting the recommended tests and examinations, to assess the diabetic care (26,27) . diabetes is most commonly associated with many micro and macrovascular abnormalities. one of these serious complications is the foot ulcer development in patients with poor glucose level control. hba1c is the a useful indicator of how well the blood glucose level has been controlled in the recent past and may be used to monitor the effects of diet,excrcise and drug therapy on blood glucose in diabetic patients ..most patients in this study (60%) have ulcer in bottom of the big toe, while 40% have ulcer 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 70 80 h b a 1 c % age (year) "r=0.82, p<0.01" 0 50 100 150 200 250 300 350 400 450 500 0 20 40 60 80 f b s ( m g /d l ) age (year) "r=0.8, p<0.01" iraqi j pharm sci, vol.20(1) 2011 hba1c and foot ulceration 23 in ball of foot. this may be due to the low rate of blood circulation in these two sites, table (3) (16) . the groups of the patients were treated with either metformin, glibenclamide or a combination of (glibenclamide + metformin) showed no significant difference among them in respect to hba1c and fbs before treatment. the mean value of the hbalc and fbs after treatment in all groups decreased significantly p<0.05, table (5). however, combination therapy (glibenclamide+metformin) showed high percentage of healing than other patients who were on the metformin or glibenclamide alone. so this combination was the best treatment to control the hba1c thus controlling the glucose level table(6), hence faster healing of foot ulcers. in this study we found that the older patients have higher hbalc and fbs levels. that means older patients were with less compliance than younger patients, table (7). fig. (1), fig. (2). the higher hba1c level was detected in ages higher than 70 years old, table (1) (28) , indicating bad glycemic control. more percent of the patients were those with occupation as laborers and employees. this may be due to their hard work with defective circulation due to their diabetes, table (2). in july,2009, an international expert committee published a report that made the case for using the hemoglobin alc assay to diagnose type2 diabetics (29) . moreover as of january 2010, the american diabetes association included alc as an appropriate diagnostic test (21) . it has been reported that high hba1c levels increase the development and progression of eye, kidney and nerve complications in diabetes mellitus poor glucose control also increases the risk of short-term complications of surgery such as poor wound healing (30-32) . in our study patients with high hba1c levels (9.79-11.07%) suffered from eye, nerve or kidney complications, while patients with hba1c level less than (9.79%) did not suffer from complications. since hba1c is not influenced by daily fluctuations in blood glucose concentration. we recommended that people with diabetes should keep their hba1c level less than (6.63%) by following diet and drug instructions, diabetes out of control could result in complications .patients with diabetes mellitus should make hba1c test every three months to determine whether their blood glucose have reached the target level of the control. patients who have glucose level under good control may be able to wait longer between the blood tests, but it's recommended to be checked at least 2 times a year (33) . references 1. edward j.b , jessie h.a , victoria , c.etal , prediction of diabetic foot ulcer occurrence using commonly available clinical information . diabetes care . 2006 ; vol. 29 , no. 6 . 2. abbott ca, carrington al, ashe h, et al. the north-west diabetes foot care study incidence of, and risk factors, new diabetic foot ulceration in a community-based patient cohort. diabet med. 2002; 19:377384. 3. boulton aj. , kirsner rs , vileikytel ; clinical practice : neuropathic diabetic foot ulcers.n engl j . med.2004 ; 351 : 48 – 55 . 4. nieto fj. jrib arren c, gross md, comstock gw. cuher rg. uric acid and serum antioxidant capacity. areaction to atherosclerosis. atherosclerosic 2000, 148, 131-9. 5. baker jf, krishnan e, chen l, schumacher hr. serum uric acid and cardiovascular diseas: recent development, and where do the leave us. amj med 2005; 118:816-826. 6. dehghan a, van hoek m, sijbrands ej, hofman a, witteman jc. high serum uric acid as a novel risk factor for type 2 diabetes mellitus. diabetes care 2008 feb; 31(2):361-2. 7. kanellis j, kang dh. uric acid as a mediator of endothelial dysfunction, inflammation, and vascular disease. semin nephrol 2005, 25:39-42. 8. waring ws, mcknight ja, webb dj, maxwell sr. uric acid restores endothelial function in patients with type 1 diabetes and regular smokers. diabetes 2006; 55:3127-3132. 9. american diabetes association. standards of medical care in diabetes-2008. diabetes care. 2008; 31:s12-s54. 10. cowie cc, rust kf, byrd-holt dd, eberhardt ms, flegal km, engelgau mm, saydah sh, williams de, geiss ls, geiss ls, gregg ew. prevalence of diabetes and impaired fasting glucose in adults in the u.s. population: nhanes 1999-2002. diabetes care. 2006; 29(6): 1263-1268. 11. smith nl, barzilay ji, shaffer d, savage pj, heckert sr, kuller lh, kronmal ra, resnick he, psaty bm. fasting and 2-hour postchallenge serum glucose measures and risk of incident cardiovascular events in the elderly: the cardrovascular health study. archives of internal medicine. 2002; 162:209-216. 12. meigs jb, nathan dm, d'agostino rb sr, wilson pw; framingham offspring study. fasting and post challenge glycemia and cardiovascular disease risk: the iraqi j pharm sci, vol.20(1) 2011 hba1c and foot ulceration 24 framingham offspring study. diabetes care 2002; 10 1845-1850. 13. coutinho m, gerstcin hc, wang y, yusuf s. the relationship between glucose and incident cardiovascular events. a metaregression analysis of published data from 20 studies of 95, 783. individuals followed for 12.4 years. diabetes care. 1999; 22:233-240. 14. expert committee on the dignosis and classification of diabetes mellitus report of the expert committee on the diagnosis and classification of diabetes millitus. diabetes care. 1997; 20: 1183-1197. 15. acton kj, burrows nr, geiss ls, thompson t. diabetes prevalence among america, indians and alaska natives and over all population-united state, 19942002. morbidity and mortality weekly report, 2003; 52 (30); 702-704. 16. saydah sh, geiss ls, tierney e, benjamin sm, engelgan m, brancati f. review of the performance of methods to identify diabetes cases among vital statistics, administrative, and survey data. annals of epidemiology. 2004; 14(7):507-516. 17. gu k, cowie cc, harris mi. mortality in adults with and without diabetes in a national cohort of the u.s. population, 1971-1993, diabetes care. 1998; 21: 11381145. 18. hu fb, stampfer mj, solomon cg, liu s, willett wc, speizer fe, nathan dm, manson je. the impact of diabetes mellitus on mortality from all causes and coronary heart disease in women: 20 years of followup. archives of internal medicine: 2001; 161: 1717-1723. 19. tsai c, hayes c, taylor gw. glycemic control of type 2 diabetes and sever periodontal disease in the u.s adult population community. dentistry and oral epidemiology, 2002; 36(3): 182-192. 20. larsen ml, horder m, mogensen ef. 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"studies of unusual hemoglobin in patients with diabetes mellitus". biochem. biophys. res. commun.1969; 36(5):838-43. 23. dingeon b.ann bio clin. 1975;33:3. 24. nuttall, fd, diabetes care 1998;21,14751480 25. ellsworth, witt, mosby's medical drug reference. 2003 26. reynolds tm, smellie ws, tomey pj; glycated haemoglabin (hbaic) montoring. bmj. 2006 sep 16; 333(7568): 586-8. 27. beach kw; a theoretical model to predict the behavior of glycosylated hemoglobin levels. j theor biol. 1979 dec; 81(3): 54761. 28. muller is,de grauw wj, van gerwen wh, bartelink ml,van den hoogen hj,rutten ge: foot ulceration and lower limb amputation in type 2 diabetic patients in dutch primary health care.diabetes care 2002; 25,570-574 29. the international expert committee. international expert committee report on the role of the a1c assay in the diagnosis and diabetes. care,2009; 32:1327-1334 30. ollerton. rl, playle r, ahmed k, dunstan fw, luzio sd, owens dr. day-to-day variability of fasting plasma glucose in newly diagnosed type2 diabetic subjects. diabetes care, 1999; 22:394-398. 31. selvin e, crainiceanu cm, brancati fl, coresh j., short-term-varibility in measures of glycemia and implication for the classification of diabetes. arch. intern. med. 2007 jul 23;167(14): 1545-51. 32. champe,p. (ed) ''biochemistry''.4 th edition 2008;3:25-39. 33. samann a, tajiyeve o. prevalence of the diabetic foot syndrome at the primary care level in germany. diabet med. 2008; 25(5):557-63. effects of ficus carica l iraqi j pharm sci , vol.18 (suppl. ), 2009 preventive effect of pentoxyfilline 39 preventive effects of different doses of pentoxyfilline against ccl4induced liver toxicity in rats jameel i. abd al-zahra * , dawser k. ismael ** and nada n. al-shawi **,1 * al-elwiya hospital for pediatrics, baghdad, iraq. ** department of pharmacology and toxicology,college of pharmacy,university of baghdad,baghdad,iraq. abstract the liver protective effects of pentoxifylline were studied through pre-treatment of rats with various intraperitoneal (ip) doses (25, 50 and 100mg/kg/day) 14 days before induction of liver toxicity by carbon tetrachloride (ccl4). the parameters of oxidative stress, malondialdehyde (mda) and reduced glutathione (gsh) were measured in liver homogenate in addition to histopathological examinations. analysis of data revealed significant amelioration of oxidative stress in groups of animals pre-treated with different doses of pentoxifylline (ptx) compared to group of animals intoxicated by ccl4 as evidenced by lowering mda contents and elevation of gsh levels in liver tissue homogenate but the levels still significantly different compared to controls. additionally, increasing doses of ptx produce a dose-dependent improvement in liver tissue damage induced by ccl4, as evident histologically by the stained liver sections. in conclusion, these findings suggest that, the hepatoprotective effects of pentoxifylline are dose dependent. key words: pentoxifylline, ccl4, liver toxicity. الخالصة اةخمملررع غممدرل مملريي مماناةزرعممترم( رةلزنممد ر055،ر25،ر52)رزممامرعتدممما رعممترةليفتاينممي ي يتلريدرةلتمميحيارةلائمم يممتممدراسة مم رت ةألالم رةلا ةم ربهم ا رفما رئيم ررحيجرتمدرابا ب ع ري اسيمرةل باة ط رسرةلتنمدرةل يميررئيلرأ تحمةثريازر01رف رةلزاذة رةلص ق ماةرب ضام ف رةلمرراسة م رسنميزي رب حمعرع م يرعمترسنميذرةل يممرةلمصم رتحم ررفم رسنميذرةل يممرmda , gshرةألينم رعخلرتاةييم ةسا م رعمترخم رمرماارتحنمترظم ارفم رعدم ييارفما رةألينمم رأظهارتح يلرةلفت ذرةلمزهارةلضا رلمت بد رةلمتغياةترةلح ة رفيه.ر ةضرة رعنمتاي ترةألالم رةلا ةم رب ما ررةلمصم ب رزماعم ع سسم رب لم رسنميذرةل يممرفمرgshمأست م مرعنمتاىررmdaعنتاىررعدفايرف ،ريمم رأظهماترةلفتم ذرةلتميحيارةلائم رل يفتاينمي ي يترعمترخم رعفدمهرحمممثرررعات رعترعنتاي ته رف رعزماع رةلنيطا رةألينم رب ي لممىرةلزماذة .رمعمترخم رةلفتم ذرةلتم رتمدرةلحصما رر ةلتيحياةترةلت يم رمةأللته بيم رةلتم ر مييته رةلزاعم رةلنم ع رلابم ع ري اسيممرةل ابما رف رةل يم.ع يه ،ريم فف رةض تفت درباراارع ئ رمحي ربيترةلزاع رةلمنتامع رعترةليفتايني ي يترمفد ليتهرةلائ ي رامرةلتنمدر ر introduction carbon tetrachloride (ccl4) is a vehicle for organic compounds, formerly used as fire extinguisher, dry cleaner; however, its use for these purposes has now been abandoned because safer alternatives are available, but it is still used in fumigation of grain and insecticides (1) . it has been suggested that, free radical-mediated damage to the hepatocytes play an important role in the development of liver toxicity with ccl4 (2) . the injury seems to be mediated by a reactive metabolites trichloromethyl free radical (·ccl3) formed by homolytic cleavage of ccl4 or by an even the more reactive species, tri-chloromethyl peroxy free radical (cl3coo · ), formed by the reaction of · ccl3 with o2 (3) . the reaction of the above mentioned free radicals with lipids and proteins causes the peroxidation of polyenoic lipids of the endoplasmic reticulum and the generation of secondary free radicals derived from these lipids (a chain reaction) (4) . destructive lipid peroxidation leads to the breakdown of membrane structure and function. furthermore, ccl4 causes damage to the mitochondria and this consequently lead to decrease atp synthesis and the hepatocytes accumulate large droplets of triglycerides in its cytosol as a result of membrane damage (5) . pentoxifylline (ptx) is a synthetic dimethyl xanthine derivative designated chemically as 3, 7dimethyl-l-(5-oxohexyl) xanthine (c13h18n4o3) (6) . it is nonselective phosphodiesterase enzyme (pde) inhibitor with rheologic property, 1 corresponding author e-mail : nada alshawi @ yahoo.com received : 24/12/2008 accepted : 6/6/2009 iraqi j pharm sci , vol.18 (suppl. ), 2009 preventive effect of pentoxyfilline 40 as it inhibits erythrocytes pde which results in an increase in erythrocytes cyclic adenosine monophosphate (c-amp) activity, which in turn allows the erythrocytes membrane to maintain its integrity, with increasing red blood cell flexibility, promoting enhancement in tissue perfusion and improvement in regional microcirculation (7) , thus, the drug is used in patients with venous leg ulcers or peripheral vascular disease to improve blood flow (8) . in addition, ptx reduces plasma fibrinogen concentration and increases the fibrinolytic activity, thus decreasing blood viscosity (9) . apart from its effect on blood cell rheology, ptx has, in addition, anti-inflammatory properties through inhibition of cytokine production. it inhibits lipopolysaccharide-induced production of tumor necrosis factor alpha (tnf-α) by monocytes and t-cells as well as interleukin two (il 2)-induced adherence of leukocytes (10, 11) . moreover, the anti-inflammatory properties of pentoxifylline are probably related to its ability to suppress oxygen radical production and scavenge reactive oxygen species (ros) in vitro from activated neutrophils (12) or in human erythrocyte membrane (13) . in addition, pentoxifylline and its metabolites modulate polymorphnuclear (pmn)adherence, superoxide production, deregulation, and migration altered by mononuclear leukocyteproduced inflammatory cytokines (14) .this study was designed to evaluate the possible hepatoprotective effect of different doses of pentoxifylline against ccl4-induced liver toxicity in rats. methods sixty white albino male rats weighing 200-250g were used in this study; they were obtained from and maintained in the animal house of the college of pharmacy, university of baghdad under conditions of controlled temperature. animals were fed commercial pellet and tap water ad libitum. animal groups are treated as follows: group i – ten rats received single daily dose of ip injection of 2 ml /kg /day of d.w for 14days. the animals were killed by anesthetic ether on the day 15.the group served as control. group iiten rats received single ip injection of pentoxifylline 100 mg/kg/day alone for 14 days to examine the effect of pentoxifylline on the liver function. the animals were killed by anesthetic ether on the day 15. group iii – ten rats received single daily dose of ip injection of 2 ml /kg /day of d.w. for 14 days. at the day 15, the animals received single dose of ccl4 (99%) (2 ml of a mixture of 1:1 v/ v in a corn oil /kg /day) orally by gavages tube to induce liver damage in rats. the animals were killed by anesthetic ether 24 hr after ccl4 administration (15) . the group served as positive control of hepatotoxicity. group ivthirty rats were utilized to study the possible protective effects of different doses of pentoxifylline against ccl4-induced liver damage and allocated as follows:  ten rats received single daily dose of ip injection of pentoxifylline 25 mg/kg/day started 14 days prior treatment with ccl4. the animals were killed by anesthetic ether on the day 16.  ten rats received single daily dose of ip injection of pentoxifylline 50 mg / kg / day started 14 days prior to treatment with ccl4. the animals were killed by anesthetic ether on the day 16.  ten rats received single daily dose of ip injection of pentoxifylline (100 mg/kg/day) started 14 days prior to treatment with ccl4. the animals were killed by anesthetic ether on the day 16. tissue homogenate was prepared by standard procedure (16) and the levels of malondialdehyde (mda) (17) and reduced glutathione (gsh) (18) were analyzed in liver tissue homogenate. small pieces of hepatic tissues were prepared for histopathological examination according to standard procedure and evaluated by ordinary microscope after staining with hematoxyline and eosin (19) . statistical analysis of data was performed utilizing student's t-test and anova. 95% confidence of data was considered for significance. results the data presented in (table 1) showed that, ccl4 produces a highly significant increase in mda contents of liver tissue homogenate compared to control group. (p > 0.05); while it produces a significant decrease in the level of gsh in rat's liver homogenate compared to control group. (p < 0.05). single i.p. injection of 100mg.kg -1 pentoxifylline given to rats for 14 days, showed a nonsignificant difference (p < 0.05) on either the contents of lipid peroxidation product (mda) in rat's liver homogenate or on the level of reduced glutathione (gsh) compared to control group as shown in (table 1). pretreatment of rats with 25mg.kg -1 .day -1 , 50mg.kg -1 .day -1 and 100mg.kg -1 .day -1 pentoxifylline ip 14-days before orallyadministered ccl4, resulted in 29.63%, 50.5% and 68.16% decline in hepatic mda contents iraqi j pharm sci , vol.18 (suppl. ), 2009 preventive effect of pentoxyfilline 41 compared to ccl4-treated animals, respectively (p>0.05); but still significantly higher (167.29%, 88% and 20.91% respectively) compared to control group (p > 0.05). (table1). moreover, table 1, showed a significant difference between different doses of pentoxifylline on mda contents of rat's liver homogenate (p > 0.05). pre-treatment of rats with single i.p. injection of 25mg.kg -1 , 50mg.kg -1 and 100mg.kg -1 pentoxifylline for 14 days before orally-administered ccl4, resulted in significant increase in hepatic gsh levels 106%, 183.98% and 344.13% ,respectively (p > 0.05); but still significantly lower 65.74%, 53.39% and 27.11% respectively, compared to the control group (p > 0.05). (table 1). concerning the effect of different doses of pentoxifylline on hepatic gsh levels, the results showed that, there were significant differences between 25mg.kg 1 .day -1 , 50mg.kg -1 .day -1 and 100mg.kg -1 .day 1 pentoxifylline. (p > 0.05). (table 1).the histological examination of liver sections from each animal treated with ccl4 showed wide areas of severe ballooning degeneration, necrosis of hepatocytes, severe cholestasis especially around central vein (zone 3) with severe inflammatory cells reaction and severe steatosis (figure 2 iandii) as compare with normal architect liver in control group (figure 1).increasing doses of pentoxifylline, produces a dose-dependent improvement in the degree of fatty degeneration in the scattered hepatocytes, previously induced by ccl4 administration, associated with the attenuation of the degree of congestion and hemorrhage. the extent of the inflammatory cell infiltration was decreased as seen in (figures 3, 4 and 5). sections of the rat's liver treated with 100mg.kg -1 .day -1 pentoxifylline alone showed normal looking appearance of livers with mild congestion of blood vessels and mild dilatation of sinusoid in continuation with central vein (figure 6). table (1)the effects of pre-treatment with different doses of pentoxifylline and 100mg.kg -1 pentoxifylline alone on (mda) contents and reduced glutathione (gsh) levels in rat's liver homogenate compared to ccl4 and control groups. -each value represents mean ± sd. -values with non-identical superscripts (a, b, c, d and e) within each parameter are considered significantly different (p >0.05). -n = number of animals. -values with non-identical superscripts (a, b and c) within each parameter of pre-treated groups with different doses of ptx for 14 days are considered significantly different (p >0.05). group i : control group. group ii : animals treated with 100mg.kg -1 .day -1 ptx alone. group iii : ccl4-treated group. group iv-a: animals pre-treated with 25mg.kg -1 .day -1 ptx prior to ccl4 intoxication. group iv-b: animals pre-treated with 50mg.kg -1 .day -1 ptx prior to ccl4 intoxication. group iv-c: animals pre-treated with 100mg.kg -1 .day -1 ptx prior to ccl4 intoxication. n mda nmol/g tissue gsh µg/g tissue group i 10 108.912 ± 10.19 a 12.76 ± 2.488 a group ii 10 117.024 ± 15.78 a 11.878 ± 2.96 a group iii 10 413.686 ± 7.317 b 2.094 ± 0.634 b group iv-a 10 291.116 ± 12.65 c a 4.371 ± 0.559 c a group iv-b 10 204.76 ± 18.086 d b 5.947 ± 0.466 d b group iv-c 10 131.68 ± 9.05 e c 9.3002 ± 1.186 e c iraqi j pharm sci , vol.18 (suppl. ), 2009 preventive effect of pentoxyfilline 42 figure(1):section showing normal rat's liver magnification: (10*10) ; staining: haematoxylline and eosin. i ii figure (2): sections showing morphological alteration of liver from ccl4-treated rats -a: necrotic damage. -b: ballooning degeneration. -c: cholestasis. -d: steatosis .e: inflammatory cell infiltration. magnification: i (10*10), ii (10*20); staining: haematoxylline and eosin. figure( 3): section of rat's liver showing the effect of pre-treatment with 25mg.kg -1 .day 1 ptx for 14 days prior to ccl4. -a: necrotic damage. -b: steatosis. -c: inflammatory cell infiltration -d: normal hepatocyte. magnification: (10*10); staining: haematoxylline and eosin. figure (4): section of rat's liver showing the effect of pre-treatment with 50mg.kg 1 .day 1 ptx for 14 days prior to ccl4. -a: necrotic damage. -b: inflammatory cell infilteration -c: kupffer cell no hyperplasia. -d: normal hepatocyte. magnification: (10*10); staining: haematoxylline and eosin. iraqi j pharm sci , vol.18 (suppl. ), 2009 preventive effect of pentoxyfilline 43 figure (5): section of rat's liver showing the effect of pre-treatment with 100mg.kg 1 .day 1 ptx for 14 days prior to ccl4. -a: mild steatosis -b: mild sinusoid dilatation. -c: normal hepatocyte. magnification: (10*10); staining: haematoxylline and eosin. figure (6): section of rat's liver treated with 100mg.kg -1 .day -1 ptx alone for 14 days. -a: normal hepatocyte -b: congestion. -c: mild sinusoid dilatation. magnification: (10*10); staining: haematoxylline and eosin. discussion carbon tetrachloride (ccl4) exerts its toxicity through its metabolites, including the free radicals (·ccl3) and (cl3coo · ), both of which injure the hepatocytes by causing lipid peroxidation and binding covalently to cell systems associated with the formation of protein-lipid cross linkage, and among these also the binding of ( ·ccl3) adducts with dna proteins and lipids (20) . the most serio consequence of free radical-induced liver injury is lipid peroxidation, and it has been found that, free radical can cause oxidative damage to cellular proteins and alter cellular function (21) .the biochemical and histological evidences presented in this study clearly demonstrated the state of oxidative stressinduced hepatic tissue damage by ccl4 treatment, manifested by the elevation of mda contents in liver tissue homogenate, which is associated with sever depletion of gsh levels as shown in table 1. these results are consistent with those observed by others (22) . the present work showed that, treatment of rats with different doses of ptx i.p., 14 days before orally-administrated ccl4, resulted in a significant decrease in the mda contents in liver tissue homogenate (table 1). many in vivo and in vitro studies concerning the antioxidant activity of ptx are conflicting. reports relating to its antioxidant property are associated with leukocyte-driven radicals by inhibiting phagocytosis and superoxide production in vitro from activated neutrophils and monocytes (23) . pentoxifylline may act as agonist for both adenosine receptors, a1 and a2; in addition to its property for inhibiting phosphodiesterase enzymes both effects leading to inhibition polymorphneuclear cells response (24) . the reduction of the activated neutrophils by ptx might be important, because increased oxidative stress is a feature of the ccl4-induced liver injury and significant protection was obtained with the use of antioxidants (25) . noyan, t. et al in 2006 (26) reported that, pre-treatment of mice with 50mg.kg -1 ptx for 7 days prior to ccl4 the present work decreases mda contents in liver tissue homogenate. the results of our work showed a similar data to that reported by previous authors (26) . moreover, ptx has been shown to inhibit superoxide anion production by kupffer cells in rat liver preservation and transplantation models (27) . the reduction of hepatic lipid peroxidation induced by ccl4 after treatment with different doses of ptx may be associated the inhibition of phosphodiesterase enzyme activity and increase of camp and cgmp levels (28) . in the contrary, our results are inconsistent with the results of the in vitro studies done by oytun, p. et al in 1999 (29) ; and by horvath, b. et al in 2002 (30) , where 50mg.kg -1 ptx neither preventing oxidative damage after short-term hepatic ischemia / reperfusion, nor have significant antioxidant capacity at therapeutic concentration against phenazine methosulphate, a free radical-generation agent, respectively. the present work showed that, pre-treatment of rats with different doses of iraqi j pharm sci , vol.18 (suppl. ), 2009 preventive effect of pentoxyfilline 44 ptx (25mg.kg -1 .day -1 , 50mg.kg -1 .day -1 and 100mg.kg -1 .day -1 ) for 14 days prior to orallyadministrated ccl4, significantly increases gsh levels in liver tissue homogenate (106%, 183.98% and 344.13%), respectively compared to ccl4-treated animals (table 3-1). glutathione has powerful antioxidant properties through direct and indirect action. the main action of gsh is direct effect through the reduction of hydroperoxides, a reaction that is catalyzed by the enzyme glutathione peroxidase, while the indirect effect is through the reduction of other antioxidants like recycling vitamins (c and e) (31) . these reactions run nonenzymatically and depend on the presence of reducing agents (34) . pentoxifylline has been shown to increase the activities of glutathione peroxidase (gsh-px) and catalase enzymes, which found to be decreased in serum of mice intoxicated by ccl4 (26) .pentoxifylline has largely been used in peripheral venous and cerebral circulatory disorders characterized by defective regional microcirculation (32) . it increases red blood cell flexibility, reduces blood viscosity, and decreases platelets aggregation, thereby improving microcirculation (33) and supporting the tissue with more oxygen, which in turn has been showed to potentiate the activity of gsh (21) . the effect of pentoxifylline in elevating gsh levels observed in the present study (table 1) may be attributed to the vasodilator effect of pentoxifylline through the release of no * (34) and prostacycline (35) , in addition to its properties for improvement the circulation and increased tissue oxygenation (36) , which in turn as similarly observed in previous report (37) may potentiate gsh activity for decreasing lipid peroxidation caused by ccl3 * and ccl3oo * , a free radicals of ccl4 and preventing the covalent binding of ccl3oo * radical but not ccl3 * to cellular macromolecules (21) . the vasodilator effect of ptx is clearly evident histologically in section of rat's liver pre-treated with 100mg.kg -1 .day -1 prior to ccl4 and in livers of animals treated with 100mg.kg -1 .day -1 ptx alone, figures (5 and 6), in which ptx promotes dilatation of sinusoids in continuation with central vein and congestion of blood vessels. the decline in the severity of degeneration and necrosis in rats pre-treated with 25mg.kg -1 .day -1 and 50mg.kg -1 .day -1 pentoxifylline and the disappearance of the degeneration and necrosis seen in rats pre-treated with 100mg.kg -1 .day -1 pentoxifylline may be due to its inhibitory effects on lipid peroxidation as evidence by decrease in mda content and the ability for preserving gsh levels in liver tissue homogenate (table 3-1). the data are consistent with others (26) . the inflammatory features observed by histological examination of rat's liver pre-treated with different doses of pentoxifylline 14 days prior to ccl4 treatment was decreased from wide dispersed inflammatory cells to very few or no hyperplasia of kupffer cells with few polymorphneuclear cells. (figures 3, 4 and 5), this indicates that, pentoxifylline may have anti-inflammatory effects against ccl4-indued hepatic damage and the degrees of inflammation were gradually decreased upon increasing the dose of pentoxifylline. the data are comparable with the results obtained by the work of abdel-salam and his colleagues, in 2003 (38) where pentoxifylline was given at doses comparable to those used in man for the treatment of circulatory disorder (36 or 72mg.kg -1 ) before the inflammatory agent carrageenan, significantly reduces carrageenan-induced paw edema response. similarly, pentoxifylline was shown to suppress the activation of kupffer cells and thereby decreasing liver injury after transplantation (39) . rockey et al in 1997 (40) , suggested that, acute ccl4 intoxication can results in a state of necro-inflammation through enhanced production of cytokines that could activate inducible nitric oxide synthase in the kupffer cells. nitric oxide (no * ) and superoxide anion (o2 *) can form peroxynitrites with the subsequent release of highly reactive sulfhydryl oxidation and lipid peroxidation (41) . pentoxifylline has a property for suppression the inducible nitric oxide synthase at mrna levels (42) ; and this may be one of the reasons that ptx attenuates liver damage and necro-inflammation. moreover, pentoxifylline may inhibit the generation of mrna for tnf-α in vitro and decrease the in vivo production of tnf-α in human and in experimental animals (43) . these inhibitory effects of ptx on tnf-α could be one of the reasons for its attenuating effects against ccl4induced liver injury and this supported by histological examination observed in the present work. 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(1) it is characterized by chronic anovulation and hyperandrogenism. (2). insulin resistance and associated hyperinsulinemia also have been recognized as important pathogenic factors in determining the majority of pcos women particularly when obesity is present . (3) most but not all women with pcos have hyperinsulinemia with insulin resistance (4) .the association between hyperinsulinemic insulin resistance and pcos well recognized and play an import role in the development of pcos (5) .hyperinsulinemia has been shown to reduce sex hormone binding globuline (shbg) synthesis in liver (6) and insulin has a direct effect on ovarian steroidogenesis in theca cell. (7) metformin is the oldest and still most insulin sensitizer world wide in the treatment of type2 diabetes mellitus and pcos-associated with insulin resistance. it is a biguanide derivative and considered as an insulin sensitizer since it lowers glucose levels without increasing insulin secretion . (8) 1corresponding author email : mohammed_taher34@yahoo.com received : 16/1/2010 accepted 26/5/2010 iraqi j pharm sci, vol.19(2) 2010 polycystic ovary syndrome and silymarin 12 silymarin is an active polyphenolic flavenoid extracted from fruits(seeds) of medicinal plant silybum marianum (milk thistle), extracts were standardized to contain 70-80% silymarin complex which comprised mainly of three major flavolignans , silybinin silychristin and silydianin of which silybinin is the most biological active. silymarin is considered to be very safe and there are only few reports on its adverse effects,mainly a mild laxative effect has been observed in occasional instances and there are no known contraindications or side effects reported during its regular use. (9) according to the multiple pharmacological actions of silymarin, silybinin have been clinically evaluated in diabetics for their therapeutics value reduces the lipoperoxidation of cell membrane and insulin resistance significantly, decreasing endogenous insulin overproduction and the need for exogenous insulin administration. (10) so this study was designed to evaluate the efficacy of silymarin as insulin sensitizer improving an ovulation rate by treatment of pcos and consequently its effect on hormonal and biochemical profile of the patients and comparing it with a classical one, metformin. materials and methods patients this study was conducted into baghdad city ,in al-elwia maternity teaching hospital from 12/2006-6/2007.the study groups included 80 women selected randomly, 60 patients with pcos aged (19-39) years with a mean age ( 27.5) years and 20 healthy control women aged (21-32) years with mean age (24) years.the diagnosis of pcos was made by the gynaecologists depending on ultrasound examination ,clinical features and laboratory tests according to diagnosis criteria of ( rotterdam 2003) (11) . table-1 shows that the clinical presentations of patients in present study like those reported in other studies of polycystic ovary syndrome in that it is a heterogeneous disorder investigations included : serum fasting glucose levels, fasting insulin levels, serum testosterone, serum progesterone and serum leutinizing hormone (lh).all patients participitated in this study were diagnosed having pcos and were non-diabetic, not hypertensive, not pregnant , and not taking any medications that affect the reproductive or metabolic functions with 90 days of study. the patients were followed weekly regularly under gynecologist supervision during the period of treatment.the women were grouped into 4 groups as follow: group 1: included 20 pcos patients ,with bmi (31.22±1.138 kg/m2),and age (19-31) years. they received sylimarin tablets (750mg/day) in 3 divided doses after meals for 3 months. group 2: included 20 patients with bmi 30.84±1.23kg/m2) and age (20-35) years. the treatment was including metformin tablets 1500mg/day in 3 divided doses (500mg after meals for 3 moths. group 3: included 20 patients with bmi 32.83±1.37 kg/m2), age (22-39) years. the treatment was consisting of combination of 2 drugs (sylimarin 750 mg/day ) and metformin ( 1500 mg /day) in 3 divided doses for 3 months. group 4: included 20 healthy women with bmi 28.4±1.01kg/m2) ,age (21-32) years and these women were with regular cycle (21-32 days) who were taken from outside of the hospital and selected as controls. sample collection venous blood sample withdrew after overnight fasting ( at least 12 hours of fasting ) from pcos women and the control group . for the subjects with regular cycles ,the samples were taken at 3-5 days after the cycle for determination of serum lh and the sample for progesterone were taken at 21 days of the cycle .the other patients with irregular cycle ,the samples were taken randomly. the samples were taken from the patients before starting and after one month of treatment . biochemical analysis determination of serum glucose and insulin levels fasting serum glucose and insulin levels were measured by commercial kit obtained from randox using enzymatic method (12,13) . determination of homeostasis model assessment of insulin resistance (homa-ir) , homa ir was calculated using the following formula (14) : homa-ir=fasting glucose (mmol/l)× fasting insulin (pmol/ml)/22.5. insulin resistance patients were defined as having homa>2.7. determination of serum testosterone (15) and lh levels (16) : serum testosterone and lh levels were determined by radioimmunoassay(ria) method using a kit provided by sigma-aldrich. determination of serum progesterone & ovulation rate serum progesterone levels were determined using kit obtained from sigma-aldrich , using (ria) method, and the ovulation rate was determined according to mid-luteal phase progesterone level that was equal to or more than 16nmol/l (5ng/ml) . (17) determination of body mass index(bmi) bmi was calculated using standard formula : bmi= weight (kg)/high (m2). iraqi j pharm sci, vol.19(2) 2010 polycystic ovary syndrome and silymarin 13 obese patients were defined as having mbi > 27kg/m2 (18). ultrasound study transvaginal ultrasound study scan is performed for each patient at about day 12 of the cycle in order to to confirm follicular changes that appear through biochemical and hormonal changes , also it was repeated for each patient who had serum progesterone levels higher than or equal to 16nmol/l in order to confirm improvement of fertility and response of patients to treatment and follow up follicular development . (19) diagnosis hyperandrogenism based on criteria of androgen excess society (aes 2006), which recommended the following diagnostic criteria for pcos hyperandrogenemia. (20) 1. hyperandrogenism ( hirsutism and /or hyperandrogenemia ) 2. ovarian dysfunction (oligo-anovulation and /or pcos). 3. exclusion of related disorders such as hyperprolactenemia and congenital adrenal hyperplasia. hirsutism based on ferriman-gallwey score , evaluates nine body sites including the face, chest , areolae , linea alba , upper back, lower back, buttocks, inner thighs and external genetalia . (21) infertility inability of any couple to conceive a child within a 12 months period of unprotected coitus ( sexual intercourse) . (22) statical analysis student t-test was used to examine the quantitative differences in the mean parameters. the results are expressed as mean±sd and the p-values <0.05were considered statically significant. results table-1 shows that 43.3% of the patients were with hirsutism and 36.6% with acne .most patients were obese 68.3% and 31.6% were lean.the percentage of infertility among the patients were 31% and only 7% were with regular cycle while the percentage of amenorrhea and oligomenorrhea were 19% and 34% respectively.the percentage of insulin resistance was 78.3%, morover the androgenemia feature was the highest (85%).table 2 shows a significant elevation ( p<0.05) in mean serum insulin levels (pmol/l) of base line levels in the three study groups compared with control group and it declined significantly (p<0.05) after 1 st ,2 nd and 3 rd month of treatment in all groups of patients. there is significant increment (p<0.05) in mean serum glucose levels (mmol/l) of base line levels in three groups compared with control group and it declined significantly (p<0.05) after 1 st ,2 nd , and 3 rd month of treatment in all groups of patients except in 1 st month of first group , it was non-significant (p>0.05).also the same table illustrated significant increment (p<0.05) in mean homa-ir of baseline level in 3 pcos groups compared with control and it declined significantly (p<0.05) after 1 st ,2 nd , and 3 rd month of treatment in all groups of patients. there was significant increment (p<0.05) in mean serum testosterone levels (nmol/l) of base line levels in 3 groups compared with control group and it declined significantly (p<0.05)after 1 st ,2 nd and 3 rd month of treatment in all groups of patients. mean serum progesterone levels (nmol/l) of baseline levels in three groups decreased significantly (p<0.05) compared with control group and elevated significantly (p<0.05) after 1 st ,2 nd , and 3 rd month of treatment in all groups of patients except 1 st first group , it was non-significant (p>0.05).this table also demonstrate significant increase(p<0.05) in mean serum lh levels (u/l) of base line levels compared with the control group and it declined significantly (p<0.05) after 1 st .2 nd . and 3 rd month of treatment in all groups of patients except in 1 st month of group 1 and 2 ,it was non-significant (p>0.05).table-3 illustrated that, the percentage of increment in mean serum progesterone levels (nmol/l) was 4.28 % ,8.72 and 12.22 in group1 ,also 4.324%,8.42% and 15.9% in group2 and 4.179% ,8.79% and 17.51 in group 3 after 1 st ,2 nd .and 3 rd month of treatment for each group respectively. the numbers of women who had ovulated were 4,5 and 10 in group 1,2,3 respectively. table 1: demographic data of 60 women with polycystic ovary syndrome. feature no. of patients (% ) hirsutism 26(43.3) acne 22(36.6) obesity 41(68.3) lean 19(31.6) infertility 31(51.6) amenorrhea 19(31.6) oligomenorrhea 34(56.6) regular cycle 7(11.6) insulin resistance 47(78.3) hyperandrogenemia 51(85) iraqi j pharm sci, vol.19(2) 2010 polycystic ovary syndrome and silymarin 14 table 2: effect of metformin and /or silymarin on insulin ,glucose ,homa-ir ratio, total testosterone and progesterone in women with pcos. g r o u p s analytes control base line after 1 m after 2m after 3m 1 insulin(pmol/l) 57.5±0.359 92.18±4.73 89.35±0.35* 85.65±4.28* 81.44±3.66* glucose(mg/dl) 5.1±0.17 5.29± 0.29a 5.01± 0.192ns 4.88±0.128* 4.73±0.128* homa 2.13±0.015 3.11± 0.244a 2.865±0.233* 2.673±0.178* 2.02±0.178* testosterone(nmol/l) 1.45±0.03 4.59± 0,223a 4.427±0.242* 4.242±0.303* 3.396±0,318 progesterone(nmol/l) 17.15±0,02 12.84±0,612a 13.39±0.682ns 13.96±0.804* 14.41±0.942* lh(u/l) 5.2±0.365 9.38± 0.317a 9.18± 0.284ns 9±0.245* 8.71±0,376* 2 insulin(pmol/l) 57.5±0.359 83.7±4.49a 82.1±3.468 80.8±3.01* 74.5±4.73* glucose(mg/dl) 5.1±0.17 5.35± 0.362a 4.49± 0.209* 4.63±0.35* 4.25±0.229* homa 2.13±0.015 2.68± 0.226a 2.59± 0.212* 2.39±0.199* 2.02±0.178* testosterone(nmol/l) 1.45±0.03 4.07± 0.199a 3.938±0.213* 3.765±0.185 3.9±0.167* progesterone(nmol/l) 17.15±0,02 12.95±0.967a 13.517±0.941* 14.04±1.01* 15.01±1.33* lh(u/l) 5.2±0.365 111.13±0.87a 10.56±0.839ns 10.10±0.644* 9.52±0.741* 3 insulin(pmol/l) 57.5±0.359 106±6.6 94.05±4.26* 84.16±4.50* 77.22±3.09 glucose(mg/dl) 5.1±0.17 5.12±0.301a 4.58±0.330* 4.27±0.369* 3.87±0.22* homa 2.13±0.015 3.55±0.172a 2.75±0.144* 2.298±0.245* 1.91±0.135* testosterone(nmol/l) 1.45±0.03 4.59±0.942a 4.18±0.176* 4.06±0.159* 3.9±0.167* progesterone(nmol/l) 17.15±0,02 13.88±0.875a 14.46±0.792* 15.10±0.673* 16.31±0.916* lh(u/l) 5.2±0.365 10.08±0.510a 9.33±0.480* 8.89±4.22* 8.54±0.515* values are mean±sd , a p< 0.05 for comparison with control group ,*p<0.05 for comparison with baseline , ns non-significant p>0.05 table 3: comparison among mean % of increament in progesterone levels (nmol/l) and number of women who had ovulated during the study in all groups of pcos patients. discussion the percentage of patients with hirsutism and acne was 43.3% and 36% respectively (table-1) and this finding was consistence with other study performed in diagnosis of pcos.cutaneous manifestations of hyperandrogenism in pcos include hirsutism,acne or combination , and male –pattern hair loss ( androgenic alopecia); whereas acanthosis nigrigans is a cutaneous marker of hyperinsulinemia . (23) the study demonstrated that percentage of obese patients was 68.6% while it was 31.6% for lean , this is common in pcos and it is in line with other studies which demonstrated that 40-60% of women with pcos are obese (bmi>27 kg/m2). (24,25) the present study showed that (51.6%) of the patients were infertile , 31.6% with amenorrhea, 56% with oligomenorrhea ,11.6% with regular cycle,78.3% with insulin resistance and 85% with hyperandrogenemia ,these results are in agreement partly with other results which demonstrate the presence of infertility by (55-75%) , amenorrhea (26-15%) ,oligomenorrhea (50-90%) regular cycle (22%) and hirsutism (60-90%) in women with pcos . (24,26) the high levels of androgens lead to chronic anovulation , menstrual disturbances and hirsutism. pcos patients typically have elevated lh levels and lh:fsh ratios. (27) because hyperandrogenism leads to abnormal folliculogenesis and endome % of 1 st month % of 2 nd month % of 3 rd month no.of women ovulated group1 (sylimarin) 4.28 8.72 12.22 4 group2 ( metformin) 4.324 8.42 15.9 5 group 3 (combination) 4.179 8.79 17.51 10 iraqi j pharm sci, vol.19(2) 2010 polycystic ovary syndrome and silymarin 15 trial development . (28,29) hyperandrogenemia is a key feature of the syndrome; but it is not always linked to hyperandrogenic symptoms such as acne or hirsutism; indeed ,ethnic groups such as asian shown insulin resistance and associated hyperinsulinemia are also now recognized as important pathogenic factors in determining hyperandrogenism in the majority of pcos women ,particularly when obesity is present . (30) the present study illustrated a significant (p<0.05) increase in serum insulin and glucose baseline levels and homa-ir index baseline value compared with control group, the results were compatible with those observed by laure c.,et al. (31) , as characteristic features of women with pcos.during three months of treatment with metformin and /or silymarin a significant (p<0.05) reduction in these parameters in all groups was observed except the effect of silymarin on glucose levels in the first month, was non-significant (p>0.05) , as shown in table (2). metformin leads to increase glucose utilization, decrease hepatic glucose production, imcrease insulin receptor binding and insulin receptor tyrosin kinase activity, but it has adverse effect on gastrointestinal tract and liver function (32,33) , while silymarin, represents a new possibility in the treatment of pcos, the underlying mechanistic links for this effects may be due to different possible mechanismas; silymarin increases, normalized and stimulated pancreatic activity of antioxidant and free radical quenching enzymes, ( glutathion peroxidase,superoxide dismutase and catalase). (34.35) silymarin may produce its effect on glucose and insulin levels by another mechanism through blockage of tnf-α where that serum tnf-α concentration have been high in normalweight pcos women and even higher levels in obese women with pcos. (36) when combination of silymarine and metformin were used , a powerful synergism effect occurred and led to best results as illustrated in third group , because each drug act by different pharmacological mechanism and different receptor sites which means that they may not compete one with each other to get same response, so that pronounced reduction in glucose , insulin and homa-ir values was occurred.the present study demonstrated a significant (p<0.05) increase in baseline testosterone levels compared with control group ,this result was compatible with other studies which demonstrate that serum concentration of testosterone and androstenedion are elevated in women with pcos ( the mean concentration are 50%-150% higher than controls). (37) during the 3 months of treatment with metformin and /or sylimarin, a significant reduction(p<0.05)from baseline of testosterone was observed ( table -2 ).these results partly in agreement with velazqwz et al. concerning metformin effect who reported that in an uncontrolled study, treatment with metformin for 8 weeks results in reduction of serum free testosterone in 29 non-diabetic women with pcos , mostly overweight. (38) most studies on this subject suggest that insulin lowering agents may affect the entire spectrum of endocrine, metabolic , and reproductive abnormalities in pcos patients.however not all studies have assessed the effects of metformin in hyperandrogenic women have confirmed these findings. interestingly, where insulin levels were reduced by treatment , serum androgens were lowered as well. (39) in an uncontrolled trial that assessed 26 obese women with pcos before and after treatment with 1500 mg metformin /day for 8 weeks , a reduction in insulin concentrations and in serum free testosterone were reported and shbg increased by 23% (38).the combination of silymarin and metformin resulted in a more remarkable reduction in testosterone levels than group 1 and 2 , this may be contributed to additive effect of these two drugs.it has been reported in this study significant decrease in baseline progesterone levels compared with control group, this result was compatible partly with other study. (40) treatment with metformin and /or silymarin for 3 months , demonstrated a significant increament (p<0.05) in serum progesterone levels in all groups , except in first month of group 1 ,it was non-significant (p>0.05) (table 2 ). the improvement in ovulation rate ( as assessed by measurement of mid-luteal phase progesterone level (>5ng/ml or >16nmol/l) was evaluated according to the percent of increment in baseline progesterone levels and number women who had ovulated , ( table 3) which reflect that third group showed highest percentage of increment in progesterone levels and number of women who had ovulated. however , other researchers found a significant enhancement in luteal progesterone levels in pcos women treated with metformin and they suggested that insulin resistance and hyperinulinemia may be responsible for low progesterone levels during luteal phase in pcos (41) ; therefore the luteal progesterone levels may be enhanced in pcos by decreasing insulin levels with metformin. it had been reported that an improvement in menstrual pattern or ovulation with only modest improvement in insulin resistance and hyperinsulinemia is sufficient to promote preovulatory follicular maturation. (42) silymarin was not different entirely from metformin concerning its effect on ovulation rate and progesterone levels as a result of their effect on insulin resistance and hyperinsulinemia .there is a significant negative correlation between insulin and proiraqi j pharm sci, vol.19(2) 2010 polycystic ovary syndrome and silymarin 16 gesterone, and between progesterone and lh concentration. (41) therefore it is probaple that effect of silymarin on progesterone levels were consequences of its effect on insulin resistance and hyprinsulinemia. there was remarkable response to combination treatment because each drug act by its own mechanism and higher increment in progesterone and ovulation rate exerted by each drug alone may be enhanced by their combination . the base line lh levels in this work increases significantly (p<0.05) compared with control group, and it was compatible with other studies which demonstrate that women with pcos , have 55-75% of a high lh to fsh ratio due to increased levels of lh than low levels of fsh. (43) elevated serum concentrations of lh are common in all reported series of women with pcos. (44) typically , pcos associated with increased lh and androgens but with normal or low serum concentrations of fsh.most investigations have also documented an increased lh pulse amplitude and frequency as characteristic feature of pcos. (45) during three months of treatment with metformin /and or silymarin a significant reduction (p<0.05) in serum lh levels were observed in all groups except in first month of group 1 and 2 , it was non-significant (p>0.05), (table 2 ). the reduction of plasma levels of lh are not a primary event in the reduction of hyperandrogenism induced by metformin because many studies have reported a reduction in plasma androgens but not concomitant reduction in lh, indicating that in these cases the reduction of steroid synthesis cannot be secondary to reduced stimulation of lh also. it is possible that spontaneous or induced ovulation or reduction in androgens may lead to a secondary reduction in lh.therefore androgens returned to pretreatment levels when metformin was suspended and that rise preceded the rise in lh, sustaining the hypothesis that a primary disorder of androgen hypersecretion is the cause of lh hypersecretion. (46) the effect of silymarin can be explained in the same manner, although its action on insulin levels are more pronounced when compared with metformin in current study, however there is a positive correlation between hyperinsulinemia and lh levels (41) , the possible effect of silymarin on lh though its action on hyperinsulinemia and insulin resistance , indeed improvement in hyperinsulinemia may lead to decrease response of lh to gnrh. as expected from above mechanism of each drug , a highest reduction in lh levels were observed when combination used ,( table2 ) which indicates that each drug may improve the other. references 1. vincenzo de leo,antonio ia marca and felice petraglia.insulin-lowering agents in the management of polycystic ovary syndrome.endocrine reviews.2003;24(5):633667. 2. acien p,quereda f,matalin p,et al.insulin,androgen& obesity in woman with polycystic ovary syndrome:a heterogenous group of disorder.fertil steril.1999;7:32-40. 3. poretsky l,cataldo na,rosenwaks 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protection against oxidative stress-induced insulin resistance in rat l6 muscle cells by micro molar concentrations of alpha-lipoic acid.diabetes . 2001;50:404-410. 36. gonzalez f, thusu k, abdel-rahman e, et al. elevated serum levels of tumor necrosis factor in normal –eight women with polycystic ovary syndrome.metabolism. 1999; 48:437-441. 37. conway gs , honour jw,jacobs hs. heterogenety of polycystic ovary syndrome: clinical , endocrine and ultrasound feaures in 556 patients.clin endocrinol (oxf) 1989;30:459-470. 38. valezquezz em, mendoza s, hamer t, et al. metformin therapy in pcos reduces hyperinsulinemia , insulin resistancd, hyperandrogenemia , and systolic blood pressure , while facilitating normal menses and pregnancy.metabolism. 1996;43:647-654. 39. acbay o, gundogdu s. can metformin resuce insulin reistance in polycystic ovary syndrome? fertil steril.1996;65:946-949. 40. velazqquez e,acosta a, mendoza sg. menstrual cylicity after metformin therapy in pcos.obsest 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management of polycystic ovary syndrome .2003;24(5):633-667. single daily dose of aminoglycosides in comparison with multiple daily dose in iraq iraqi j pharm sci , vol.18 (1) , 2009 single daily dose of aminoglycoside 21 the safety profile of single daily dose of aminoglycosides in comparison with multiple daily dose amanj a. baker * , ibrahim a. majeed** ,1 and dawser k. ismail*** * department of pharmacology , college of pharmacy , university of hawler , iraq . ** department of clinical pharmacy, college of pharmacy, university of baghdad , baghdad ,iraq *** department of pharmacology and toxicology ,college of pharmacy,university of baghdad,baghdad,iraq abstract to overcome the problems which associated with the standard multiple daily doses (mdd) of aminoglycosides (ags) like high incidence of toxicity(nephrotoxicity, ototoxicity)(5-25%) and high cost, an alternative approach was developed which was single daily dose (sdd).this new regimen was designed to maximize bacterial killing by optimizing the peak concentration/minimum inhibitory concentration(mic)ratio and to reduce the potential for toxicity. the study includes 75 patients selected randomly, 50 of them received sdd regimen of age range of 17-79 years and the remaining received mdd regimen of age range of 13-71 years. the study was designed to evaluate the safety of sdd regimen in comparison with mdd regimen. all the patients in sdd group received a constant dose of 5-mg/kg/day of gentamicin and 20mg/kg/day of amikacin with a drug administration interval based on estimated creatinine clearance(clcr): if ≥60 ml//min every 24 hours (q24h), 5940 ml/ min every 36hours and 3930 ml/min every 48 hours.the calculated dose was diluted with 0.9% normal saline or 5% dextrose to 50-100 ml and given as intravenous infusion over 30-60 minutes. in sdd group , the mean length of therapy was 6.4±1.73 days .gentamicin accounted for 96% of the aminoglycoside use, and the majority(58%) of patients received the drug every 24 hours.the 36and -48 hours intervals were used for 34 and 8% of the population, respectively.while in mdd group , the mean length of therapy was 5.0±0.91 days. gentamicin accounted for all (100%) of aminoglycoside use, and all of the patients received the drug every 8 hours. no clinically apparent ototoxicity and nephrotoxicity were observed in the patients in the sdd group, in contrast to the patients in mdd group, in whom 4 patients (16%) were developed nephrotoxicity and 1 patient (4%) was developed ototoxicity. the obtained results indicate that sdd regimen was safer through decreasing the incidence of both nephrotoxicity and ototoxicity.for statistical analysis, anova test was used with p<0.01.each mean was expressed as mean±sem(standard error of mean). key words: aminoglycosides, single daily dose, nephrotoxicity and ototoxicity. الخالصة مو اجل حجاوز النشاكل النصاحبت للجرع القياسيت النخعددة مو دواء االمثبوكالكوساادد سبابا البانيت العاليات كخلال الالان واال ه ن الجدداد مانل لسداادة القاسليات %( والالل العاليت . اسخحدثج دراست سددلت فا اساخادان ىماان الجراات اليوميات الواحادة نااا اليماا53 -3) ماردط 53القاحلت للباخردا او غردق زدادة ىببت قينت الخركيس / اقل حركيس مثبػ واالقالل مو االاراض الجاىبيات . حشانل الدراسات الان دد النخبقا كااه ( سايت اساخلنوا الادواء سيماان الجراات الواحادة العا 57 – 75ماردط كااىوا سااناار ) 35ثل اخخيارنل اشوائياً ظانيمل ( سيت اساخلنوا الادواء سيماان مخعادد الجارع . ماننج الدراسات لخقيايل ساالمت ىماان الجراات الواحادة سالنقارىات 57 – 71سنعدل االانار ) ملغال 55ملغال /كغال /داون ماو ماادة جيخنادبايو و 3سيمان مخعدد الجرع جنيع النرظن فا ىماان الجراات الواحادة اساخلنوا جراات ثاسخات مال / دقيقات دع ان 45/دون مو مادة ام كيبيو مع فامل اخا العاال ااخنااداً الان وظاوك الاردااحيييو الناناو ا ا كااه اقال ماو /كغل سااات . الجراات النبانوحت خ اج سنحلاول 24مال / دقيقات كال 1517سااات و 14مل / دقيقات كال 25 – 37ساات 52الدواء كل دقيقات فا 45 – 15مل واا او غردق اليعوك الورددي خالل 755 – 35% الن 3دكبخروز % او محلول ال 5.7النلح ال بيع % ماو النرظان والغالبيات مايمل 74اداان حال اا ااء الجيخامادبايو الان 7.51 ± 4.2ىمان الجرات الواحدة كاه معادل غاول فخارة العاال %( مااو النرظاان سااالخوال . سيينااا فاا ىمااان 4%( ) 12) ساااات اسااخادن لاا 24و 14ساااات فواماال الاا 52%( اسااخلنو الاادواء كاال 34) الدوجااد . ساااااث 4%( اسااخلنوا جيخامادباايو كاال 755ادااان جنيااع النرظاان ) 5 77 ± 3مخعاادد الجاارع كاااه معاادل غااول فخاارة العااال مخعادد الجارع حيا ظمور سردري ف حبانل اال ه والالان اياد النرظان النباخادميو ليماان الجراات اليوميات الواحادة . سييناا فا ىماان %( .اليخائج حؤكد سااه ىماان الجراات الواحادة كااه اكثار اماىااً ماو خاالل 2%( كلن وحالت حلل حبنل اال ه ) 74حاالث حلل ) 2ظمرث جدددة مو الخلل ف اال ه والالن .حقليل الحاالث ال 1 corresponding author : e-mail : ibrahimalbayati54@yahoo.com received : 11/6/2008 accepted : 28 / 12/2008 iraqi j pharm sci , vol.18 (1) , 2009 single daily dose of aminoglycoside 22 introduction clinical experience over the past 50 years has shown that multiple daily dosing strategy to be both labor and lab. intensive and the correct multiple daily dosing of aminoglycoside often requires pharmacokinetics expertise, close monitoring of drug serum levels and renal function (1) .currently many centers are adopting the sdd regimen as the standard / preferred dosing method and by the year 2000, about 80% of the hospitals worldwide use sdd regimen. (2) . the rationales of using aminoglycosides as sdd were due to their concentration dependent killing (3) ,significant post – antibiotic effect (pae) (4) , avoidance of the adaptive post – exposure resistance (5) ,and aminoglycosides' uptake into renal tubule cells and inner ear is a saturable process (6) .the toxicities of aminoglycosides include nephrotoxicity, ototoxicity (vestibular and auditory). approximately 8% to 26% of patients who receive aminoglycoside for more than several days develop mild renal impairment which is reversible because the proximal tubular cells have the capacity to regenerate (7) .aminoglycosides are poly cationic in nature binding to the anionic site on the endothelial cells of the glomerulus leading to reduction in the glomerular filtration rate. (8) they are almost exclusively filtered by the glomerulus and excreted unchanged. filtered aminoglycosides undergo proximal tubular reabsorption by binding to anionic phospholipids in the brush boarder, followed by endocytosis and sequestration in lysosomes of the s1ands2 segments of the proximal tubule . (9) the earliest lesion observed following clinically relevant doses of aminoglycosides is an increase in the size and number of lysosomes. (10) .these lysosomes contain myeloid bodies, which are electrondense lamellar structures containing undergrated phospholipids .the renal phospholipidosis produced by the aminoglycosides is thought to occur through their inhibition of lysosomal hydrolyases,such as sphingomyelinase and phospholipases (11) ,as a result the lysosomes become progressively distended until they rupture, releasing lysosomal enzymes and high concentration of aminoglycosides into the cytoplasm .the released lysosomal contents can interact with various membranes and organelles that trigger cell death (12) . aminoglycosides also inhibit various atpase including na + -k + atpase, adenylate cyclase; alter the function of mitochondria and ribosome. (13) aminoglycosides induce irreversible ototoxicity (vestibular and auditory) in about 2 to 25% of the patients (14) . the precise mechanism of hair cell destruction in both forms of ototoxicity is unclear,but it has been suggested that aminoglycosides interfere with active transport system essential for the maintenance of the ionic balance of the endolymph (15) . this would lead to alteration in the normal concentration of ions in the labyrinthine fluid with impairment of electronic activity and nerve conduction .eventually, the electrolyte changes, or perhaps the drugs themselves damage the hair cell irreversibly. several factors have been associated with a higher incidence of ototoxicity including duration of therapy (>8 days), cumulative dose, total daily dose, trough serum drug concentration, concurrent diuretic therapy, underlying disease state, previous exposure to aminoglycoside therapy , age, specific aminoglycosides (16) . sdd regimen was suitable for all patients requiring aminoglycoside therapy except those having great changes in aminoglycoside pharmacokinetics like pregnant patients, children etc.....the drug dosage in sdd regimen was by fixing the dose of the drug with changing the interval of drug administration according to the estimated clcr (6) and this consist of giving a constant dose of 5mg/kg/day for gentamicin and tobramycin, 20mg /kg/day for amikacin and 15 mg/kg/day for streptomycin (17) . the interval of drug administration will depend on the estimated clcr calculated by using cockcroft equation (17) .the dose is calculated based on actual body weight (abw) unless the patient is ≥20 % more than the ibw .for obese patients, a dosing weight (dw) should be calculated. monitoring of renal toxicity was done through measurement of basal and every 2-3 days of serum creatinine concentration (s.cr) (6) .the standard measurement of peak and trough level is not required due to the high peak level and drug –free interval obtained with sdd regimen. in patients with adequate renal function (clcr >60ml/min), the trough aminoglycoside level would be near zero (<< 1mg/l) so, the initial dosing interval would be maintained and no further drug level determination was necessary as long as clcr remains unchanged (18) . in general, monitoring of the sdd regimen can be achieved by taking a single random blood sample through 6-14 hours. after starting of the infusion and the level will be evaluated on a nomogram .even this single random drug concentration may no longer be necessary to be measured for the following classes of patients (17) :-patients receiving sdd every 24 hours, patients without concurrently administered nephrotoxic iraqi j pharm sci , vol.18 (1) , 2009 single daily dose of aminoglycoside 23 agents ,patients without exposure to contrast media, patients not in the intensive care unit, patients less than 60 years age. a baseline and weekly audiometry are recommended for patients who require greater than 2 weeks of therapy (`7) . materials and methods this comparative study was done in alkirkuk general hospital in kirkuk city on (75) patients admitted to surgery, medicine and gynecology wards under medical supervision by the specialist physician in each ward. the patients were selected randomly and they were classified in to two groups, sdd group (50 patients, age 17-79 years) and mdd group (25 patients, age 13-70 years). the patients that were eligible for the study include all the patients requiring aminoglycoside therapy except those with great change in ag pharmacokinetics. the patients in sdd group were received aminoglycoside antibiotic (whether gentamicin or amikacin) in a dose depending on their actual body weight (abw) unless their abw is >20% above their ideal body weight (ibw) .for these patients, a dosing weight (dw) , which is based on the ibw plus 40% of the estimated adipose tissue mass, was calculated for dosage determination. the interval of drug administration is based on the calculated creatinine clearance (clcr) according the cockroft equation, which is equal to: clcr (male) =ibw (kg) (140-age) /72[s.cr mg/dl] clcr (female) = 0.85 ibw (kg) (140age) /72 [s.cr mg/dl] where: ibw is ideal body weight. therefore, if clcr is ≥ 60 ml/ min, the interval of drug administration would be 24 hours, clcr is 59-40 ml/ min; the interval of drug administration would be 36 hours, clcr is 39-30 ml / min; the interval of drug administration would be 48 hours, clcr is < 30 ml/ min, aminoglycosides would not be recommended and other alternative antibiotics should be used. culture and sensitivity test was done before starting therapy using modified kirbybauer method to know the reason of failure (if occur) whether due to drug resistance or due to the regimen itself. the following laboratory tests and parameters were monitored for all patients who enrolled in the study including: basal and every 2-3 days measurement of serum creatinine concentration, white blood cell count(wbc) and general urine examination (g.u.e)(for those having uti) , basal and daily recording of body temperature.monitoring of nephrotoxicity was done through basal and periodic measurement of s.cr, while ototoxicity was monitored through baseline audiometry and documentation of auditory function, and daily monitoring of any changes in hearing status under the supervision of specialized physician. results and discussion age was considered an important factor in aminoglycosides toxicities since with increasing age there was a decrease in renal function and subsequent reduction in aminoglycosides excretion and hence there accumulation. table(1) showed percent of patients in respect to their age distribution. table (1): percent of patients in respect to their age distribution. group ≤40 years 4150 years 5160 years 6170 years >70 years sdd 40% 18% 26% 12% 4% mdd 52% 20% 20% 8% 0% figure (1) showed that in the sdd group, majority (58%) of patients received the drug(gentamicin and amikacin) every 24 hours.the 36and -48 hours intervals were used for 34 and 8% of the population, respectively. while all the patients in mdd group were received therapy every 8 hours, renal function is an important criteria in aminoglycosides therapy since it determines the dose and interval of drug administration because they are mainly renally excreted (19) .renal function status was reflected by normal serum [s.cr] and creatinine clearance (20) . the normal range of serum creatinine was 0.7-1.2 mg/dl (70-150 µmol/l) and of creatinine clearance was 60-125ml/min (21). figure (1) : percent of patients (sdd( group in respect of intreval of drug administration 0 10 20 30 40 50 60 70 every 24 hrs. every 36 hrs. every 48 hrs. interval of drug adm inistration % o f p a t ie n t s sdd group iraqi j pharm sci , vol.18 (1) , 2009 single daily dose of aminoglycoside 24 table(2): the mean pre and post treatment serum creatinine concentration [s.cr] for sdd and mdd group. each value represents mean±sem, values with non identical superscripts (a,b,c) are considered significantly different (p<0.01) . table (2) showed that the pre treatment renal function in both sdd and mdd group was normal (reflected by the normal [s.cr]) while the post treatment was abnormal in mdd group (reflected by the elevated [s.cr]) which indicate the occurrence of nephrotoxicity.statistical analysis (p>0.01) revealed that there was no significant difference between the mean pre and post treatment [s.cr] in sdd group while there was a significant difference (p<0.01) in mdd group. this indicates the superiority of sdd regimen over mdd regimen since the incidence of nephrotoxicity with mdd regimen was higher and occur in a wide number of patients (5-25%) as many studies reported (22) duration of therapy (>8 days) was one of the most important determinant risk factor in aminoglycosides toxicity (23) and they receive therapy for various duration ranging from ≤3 days to > 14 days, figure (2). figure(2) : percent of patient in respect to their length of therapy the mean length of therapy in sdd group was 6.4±1.73 days while 5.0±0.91 days in mdd group. the results of statistical analysis indicated that there was no significant difference (p>0.01) in the mean length of therapy between the sdd and mdd group.24% of the patients in sdd group received therapy for ≥ 8 days compared to only 8% in group indicating the safety of sdd regimen since no toxicity was observed in spite of higher percent of patients who received longer duration of therapy due to the safety profile of sdd regimen (24) figure (2) . ninety six percent of the patients in sdd group received gentamicin and 24% received amikacin, while all the patients in the mdd group received gentamicin only. the total number of the patients who received gentamicin and amikacin in sdd group were > 50 because most of the patients were first treated empirically with gentamicin then converted to amikacin according to the results of culture and sensitivity test which revealed gentamicin resistant bacteria and amikacin sensitive bacteria. the mean dose of gentamicin was 282.9±6.8 {range, 212.5 438.4} mg in sdd group and 235.2±4.8 {range, 120240} mg in mdd group, while for amikacin it was1182±71.3 . {range, 914 1680} mg. the results of statistical analysis showed that their was a significant difference (p<0.01) in the mean dose of gentamicin between sdd and mdd group. this proves the safety of sdd regimen because no incidence of toxicity despite the use of higher dose, in contrast to development of toxicity in mdd group (25) . all patients who are given the sdd and mdd regimen either had improved or complete resolution of their infections except two patients given mdd regimen had failures. concerning the toxicity (nephrotoxicity and ototoxicity) of both sdd and mdd regimen, no toxicity was observed in any patients received sdd regimen while 20% of the patients who received mdd regimen developed toxicity, figure( 3 ). figure(3) : percent of patients mdd group in respect of drug toxicity occurance groups pretreatment mean [s.cr](mg/dl) post treatment mean serum [s.cr] (mg/dl) sddgroup 1.0±0.09 a 0.97±0.09 a mdd group 1.15±0.32 b 1.7±0.33 c 0 5 10 15 2 0 2 5 3 0 3 5 4 0 4 5 5 0 <=3 4 .0 5 .0 6 .0 7 .0 8 .0 10 .0 11.0 14 .0 >14 length of therapy(days) % o f p a ti e n ts mdd sdd 4 16 80 ototoxicity nephrotoxicity no toxicity iraqi j pharm sci , vol.18 (1) , 2009 single daily dose of aminoglycoside 25 nephrotoxicity was defined as an increase in s.cr. of ≥0.5 mg/dl above the baseline value. patients were not evaluated for nephrotoxicity if they had been treated with aminoglycoside in the previous week, treatment with aminoglycoside was resumed within one week of stopping treatment, hemodailysis was started within 48 hour after the start of therapy and if the patient met the criteria for nephrotoxicity within the first 24 hour of therapy since in that case the decline in renal function is unlikely to be the result of the aminoglycoside treatment (26) . nephrotoxicity was not detected in any of the patients who received sdd regimen. this agree with other study which also indicate no nephrotoxicity with sdd regimen (27) , but david p. nicolau study showed that sdd regimen may be associated with small percent (1.2%) of nephrotoxicity (6) . sixten percent of the patients in mdd group met the criteria of nephrotoxicity in our study, figure (3). figure (4) show the age distribution of the patients in whom toxicity occurred. all the patients who developed nephrotoxicity in mdd group were male and 75% were of age of ≤ 40 years with mean age of 35±9.5 years and mean dose 210±20.0 mg. this agree with other studies that revealed 5-25% of nephrotoxicity with mdd regimen (28,29) .the mean length of therapy in those who developed nephrotoxicity was 9.3±2.9 since duration of therapy was of the most important determinant factor in nephrotoxicity (30) , figure(4) : percent of patient mdd group in whom toxicity occur in respect to age distribution figure(5) . no significant difference in the mean age and dose was observed between the patients in mdd group who developed nephrotoxicity and those who did not, but there was a significant difference ((p<0.01) in the mean length of therapy, 5 days compared to 9.3 days, indicating the importance of duration of therapy on toxicity. ototoxicity, in the form of vestibular manifestation, was not observed in any of the patients in sdd group as in benjman m. limson et al study which also revealed no ototoxicity with sdd regimen (31) while other study revealed only 0.14% ototoxicity (6) . four percent of the patients in mdd group developed ototoxicity with age >50 years old, and duration of therapy of >14 days as bates, d.e. study indicate a high percent of ototoxicity with mdd regimen ranging from 5-25% (32) . in spite of the presence of greater risk factors for aminoglycosides toxicity in sdd group including :  elderly patients(>70 years) were found in sdd group but not in mdd group;  the percentage of patients who received therapy for ≥8 days were greater in sdd group;  administration of amikacin in sdd group for a median length of therapy of 13 days(range 3 to 42 days) which is greater than the recommended duration of therapy for amikacin(i.e., 7 to 10 days);  greater median dose of gentamicin in sdd group;  use of sdd regimen in diabetic and hypertensive patients with impaired renal function; figure(5) : percent of patients mdd group in whom nephrotoxicity occur in respect of length of therapy with no appearance of toxicity in sdd regimen indicates it’s safety over mdd regimen. this safety was due to the new pharmacodynamic data of aminoglycosides which is the saturability of aminoglycosides accumulation in renal tubular cells and inner nephrotoxicity 0 10 20 30 40 50 60 4.0-5.0 6.0-7.0 >14 length of therapy(days) % o f p a ti e n ts 0 20 40 60 80 100 120 <40 51-60 age (year) % o f p a ti e n ts nephrotoxicity(control) ototoxicity(control) iraqi j pharm sci , vol.18 (1) , 2009 single daily dose of aminoglycoside 26 ear at low concentration (threshold concentration) and the time above this threshold concentration was the determinant of toxicity (6, 33). the threshold for toxicity was corresponds to a plasma concentration of 2 mg/l. the once-daily regimen produces a threefold higher plasma concentration, which enhance efficacy that otherwise might be compromised due to the prolonged sub-mic concentration later in the dosing interval compared with every-8-hour regimen. oncedaily regimen provides a 12-hour period during which plasma concentration are below the threshold for toxicity, thereby minimizing the toxicity that otherwise might result from the early high plasma concentration. the every-8-hour regimen, in contrast, provides only a brief period (< 3 hours) during which plasma concentrations are below the threshold for toxicity (33,34) , (figure 5). conclusion on the basis of the obtained results, one can conclude that sdd regimen appears to be safer than the conventional mdd regimen through reduction in the incidence of nephrotoxicity and ototoxicity. references 1. .marik p.e., havlik i., monteagudo f.s.e., lipman j. pharmacokinetics of amikacin in critically ill adult and pediatric patients: comparison of onceversus twice-daily dosing regimens. j. antimicrob. chemother. 1991; 27:81-89. 2. freeman c.d., nicolau d.p., belliveau p.p., and nightingale c.h. once-daily dosing of aminoglycosides: review and recommendations for clinical practice. j. of antimicrob. chemother. 1997; 39(6):677-686. 3. henry f. chamber .aminoglycosides .in: laurence l.brunton. goodman and gilman’s the pharmacological basis of therapeutics.11 th edition. new york: mcgraw-hill, 2006; 1155-1171. 4. prins jm, koopmans rp, büller hr, kuijper ej, speelman p. easier monitoring of aminoglycoside therapy with once-daily dosing schedules. eur. j. clin. microbiol. infect. dis. 1995 ; 14(6):531–535. 5. karlowsky j.a., zhanel g.g., and davidson r.j. et al. once-daily aminoglycoside dosing assessed by mic reversion time with pseudomonas aerugenose. antimicrob. agents chemother. 1994; 38:1165-1168. 6. david p. nicolau, collin d. freeman, paul p. belliveau, charles h. nightingale, jack w. ross, and richard quintilian. experience with a once-daily aminoglycoside program administered to 2,184 adult patients. antimicrob. agents and chemother., 1995; 39:650–655. 7. smith, c.r., lipsky, j.j., laskin, o.l., et al .double-blind comparison of the nephrotoxicity and auditory of gentamicin and tobramycin .new engl. j. med., 1980; 302:1106-1109. 8. gilbert d.n. once-daily aminoglycoside therapy. antimicrob. agents chemother., 1991; 35:399-405. 9. cojocel c: aminoglycoside nephrotoxicity in sipes ig. mcqueen c.a., gandofil a.j (eds)\: comprehensive toxicology. oxford, england: elsevier. 1997; 7: 495-524. 10. de bore me. renal injury due to environmental toxins, drugs and contrast agents. in: berl. t. bonventre jv (eds): atlas of dis. of the kidney, philadelphia: current med., 1999; 11.2-11.14. 11. verpooten g.a., talkens p.m., bennett w.m., aminoglycosides and vancomycin. in: de broe m.e., porter g.a., bennett a.m., verpooteng.a. (eds). clinical nephrotoxicants, renal injury from drugs and chemicals. the netherlands: kluwer, 1998; 105-120. 12. humes, h.d., sastrasinh, m., and weinberg, j.m. calcium is a competitive inhibitor of gentamicin-renal membrane binding interactions, and dietary calcium supplementation protects against gentamicin nephrotoxicity. j. clin. invest., 1984; 73:134-147. 13. queener, s.f., luft, f.c., and hamal, f.g. effect of gentamicin treatment on adenylate cyclase and na+, k+ – atpase activities in renal tissues of rats . antimicrob. agents chemother., 1983; 24:815-818. 14. carol s., candace s., and dina k. once – daily aminoglycoside therapy: potential ototoxicity, antimicrob. agents and chemoth., 1996; 40:2209-2211. 15. neu, h.c., and bendush, c.l. ototoxicity of tobramycin: a clinical overview. j.infec. dis., 1976; 134:s206-s218. 16. zaske, d.e. aminoglycosides.in: w. evans, j.j.schentag, and w.j.jusko (ed.).applied pharmacokinetics; principles of therapeutic drug monitoring, 3ed edition. applied therapeutics, spokane, wash., 1991; 14.1-14.48. 17. preston s.l., briceland l.l. single daily dosing of aminoglycosides. pharmacotherapy, 1995; 15:297-316. 18. begg ej, barclay ml, duffull sb. a suggested approach to once-daily iraqi j pharm sci , vol.18 (1) , 2009 single daily dose of aminoglycoside 27 aminoglycoside dosing. br. j. clin. pharmacol.1995; 39: 605-609. 19. jones r.n. method preferences and test accuracy of antimicrobial susceptibility testing: update from the college of american pathologists microbiology surveys program. arch. pathol. lab. med. 2001; 125:1285. 20. a.f. smith, d.j. beckett, f.w. walker and p.w.h. rae. lecture notes and clinical biochemistry, 6th edition. the black well sciences, logo, u.k. 1998; 4:51-68. 21. murray l., ian w., tom t., and chee k. cheung. oxford handbook of clinical medicine, 7th edition. new york, oxford press, 2007; 292. 22. prins jm, weverling gj, van ketel rj, speelman p. circadian variations in serum levels and the renal toxicity of aminoglycosides in patients. clin. pharmacol. ther. 1997; 62:106-11. 23. c. bartal, a. danon, f. schlaeffer, k. reisenberg, m. alkan, r. smoliakov, a. sidi, and y. amlog. pharmaceutical dosing of aminoglycosides: a controlled trial. am. j. med., 2003; 114(3): 194-198. 24. demczar dj, nafziger an, bertino js. pharmacokinetics of gentamicin at traditional versus high doses: implications for once-daily aminoglycoside dosing. antimicrob. agents chemother. 1997; 41:1115-1119. 25. 25. bertino js jr, booker la, franck pa, jenkins pl, franck kr, nafziger an. incidence of and significant risk factors for aminoglycoside-associated nephrotoxicity in patients dosed by using individualized pharmacokinetic monitoring. j infect dis., 1993; 167(1):173–179. 26. de vries, p. j., r. p. verkooyen, p. leguit, and h. a. verbrugh. prospective randomized study of once-daily versus thrice-daily netilmicin regimens in patients with intra abdominal infections. eur. j. clin. microbiol. infect. dis. 1990; 9:161–168. 27. jan m. prins, gerrt j. weverling, koen d. blok, ruud j. van ketel. and peter speelman. validation and nephrotoxicity of a simplified once-daily aminoglycoside dosing schedule and guidelines for monitoring therapy. j. antimicrob. agents and chemother. 1996;40: 2494-2499. 28. lerner, a.m., reyes, m.p., cone, l.a., et al. randomized, controlled trial of the comparative efficacy, auditory toxicity, and nephrotoxicity of tobramycin and netilmicin. lancet, 1983; 1:1123-1126. 29. the international antimicrobial therapy cooperative group of the european organization for research and treatment of cancer. efficacy and toxicity of single daily doses of amikacin and ceftriaxone versus multiple daily doses of amikacin and ceftazidime for infection in patients with cancer and granulocytopenia. am. inter. med., 1993; 119: 584-593. 30. zaske, d.e. aminoglycosides.in: w. evans, j.j.schentag, and w.j.jusko (ed.).applied pharmacokinetics; principles of therapeutic drug monitoring, 3ed edition. applied therapeutics, spokane, wash., 1991; 14.1-14.48. 31. benjman m. limson, vicente x. genato, and genaro yusi. a randomized multicenter study of the single daily dose regimen vs. thee multiple daily dose regimen of netilmicin in the treatment of systemic infection. j. microb. infec. dis. 1989; 18:47-52. 32. bates, d.e. aminoglycoside ototoxicity. drugs today. 2003; 39:277-285. 33. henry f. chamber .aminoglycosides .in: laurence l.brunton. goodman and gilman’s the pharmacological basis of therapeutics.11 th edition. new york: mcgraw-hill, 2006; 1155-1171. 34. gilbert d.n. aminoglycosides. in: mandell gl, bennett je, dolin r, eds. douglas and bennett's. principles and practice of infectious diseases. new york: churchill livingston, 1995; 279-301. iraqi j pharm sci, vol.29(1) 2020 nebivolol-loaded supersaturable self-nanoemulsion 225-doi.org/10.31351/vol29iss1pp216https:// :doi 216 comparison between conventional and supersaturable self-nanoemulsion loaded with nebivolol: preparation and in-vitro/ex-vivo evaluation lubna a. sabri*1 and ahmed a. hussein* * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract nebivolol (nbh) is a third-generation β1-blocker with high selectivity and vasodilation activity. nevertheless, nebivolol exhibits low oral bioavailability, which may adversely affect its efficacy. recently, supersaturable self-nanoemulsion (su-sne) is an advanced sne approach that can address low bioavailability. the study aims to prepare nebivolol-loaded su-sne by reduction the amount of the prepared conventional sne to half. besides, an appropriate polymer type and concentration to prevent nbh precipitation upon oral administration have investigated. a conventional self-nanoemulsion (formula a) was prepared by dissolving nbh in 500 mg vehicle mixture of imwitor®988: cremophor-el: propylene glycol. then, eight su-sne formulas with the absence or presence of four different polymers were prepared and evaluated. in-vitro precipitation assay was performed to assess the precipitation inhibition capacity of polymers. the ex-vivo permeation through rat intestinal mucosa was also conducted for determination of permeability parameters. results revealed that (susna formula sas1) containing 5% soluplus could effectively retard the nebivolol precipitation. there was no statistical difference between formula a and sas1; both maintained a higher apparent nbh concentration for approximately 240 min in 0.1n hcl. the permeation rate of conventional (formula a) and soluplus-based susne (formula sas1) was significantly improved, and the permeation enhancement ratio was found 2.7 and 3.2, respectively, as compared with non-formulated nbh. consequently, it is concluded that developing soluplusbased nebivolol sne is a promising alternative approach. it can enhance nebivolol stability and permeability with half the amount of conventional sne components. keyword: imwitor988, permeation enhancement, supersaturable self-nanoemulsion, soluplus. ل: تحضيره وتقييمه لوالذاتي التكون التقليدي والمشبع المحمل بالنيبيفو النانوي مقارنة بين المستحلب المختبر و خارج الجسم الحيداخل *و احمد عباس حسين 1*،لبنى عبد الكريم صبري .العراق بغداد، ، بغداد جامعة ، الصيدلة كلية ، ياتنالصيدال * فرع الخالصة توافر له ينتمي عقار النيبيفولول الى الجيل الثالث من حاصرات بيتا مع انتقائية عالية وتأثير موسع لألوعية الدموية. ومع ذلك ، فإن كنهج( su-sne) مفرط االستيعاب ذاتي التكون استحدث المستحلب ، األخيرة اآلونة في حيوي فموي منخفض ، مما قد يؤثر سلبا على فعاليته. عن بالنيبولول محملة su-sne تحضير إلى الدراسة تهدف هذه الحيوي المنخفض. التوافر لمعالجة sne)) للمستحلب الذاتي التكون التقليدي متقدم بعد تناوله نيبيفولولترسب لمنع المناسب البوليمر ايجاد نوع وتركيز ، ذلك إلى باإلضافة. النصف إلى المحضرة التقليدية sne كمية تقليل طريق يكول. بعد ذلك ، تم تطويروكريموفور وبروبلينكال 988مغلم من مزيج االمويتر 500تم تحضير الصيغة التقليدية باذابة النيبيفولول في لقد .فمويا في المختبر لتقييم قدرة البوليمرات على أربعة بوليمرات مختلفة. ثم تم إجراء اختبار الترسيب بغياب او وجود su-sneصيغ من نوع ثمان وتقييم التقليدي sneخالل امعاءالفئران للمقارنة بين معلق النيبيفولول و من تثبيط الترسيب. عالوة على ذلك ، تم إجراء دراسة خارج الجسم الحي للنفاذية تختلف ال والتي ٪ سوليبلس يمكن أن تؤخر ترسب النيبيفولول بشكل فعال 5المحددة. وكشفت النتائج أن الصيغة الحاوية على su-sneوصيغة لى اضافة ألمول من حامض الهيدروكلوريك. با 0.1دقيقة في 240دة لم عالي للنيبيفولولتركيز على حافظا كالهما ألن. a الصيغة عن إحصائيا التقليدية والصيغة الحاوية على السوليبلس ، ووجدت ان نسبة تعزيز النفاذية حوالي sneاختالف كبير في معدل النفاذ بين وجود يالحظ، لم كذل ا و نهجيعدتالي ، يمكن أن نستنتج أن تحضير النيبيفولول المعزز بالسوليبلس ،على التوالي، بالمقارنة مع النيبيفولول الغير مصاغ. وبال 3.2و 2.7 .المكونات المطلوبة للمستحلب الذاتي التكون التقليدي كمية نصف مع والنفاذية النيبولول استقرار التركيزعزيزي ا, يمكن انواعد بديال فائق التشبع ، سويبلس. ذاتي التكون النانوي، تعزيز النفاذية،مستحلب 988امويتر : المفتاحية الكلمات introduction nebivolol (nbh) is a third-generation β1blocker with high selectivity. it also has vasodilator and oxidative stress reduction effect through promoting endothelium nitric oxide release (1). it has been prescribed for the management of hypertension and heart failure, particularly preferred for elderly patients and those with other co-morbid conditions like diabetes and peripheral vascular disease (2). nevertheless, nebivolol is proposed based on the biopharmaceutical classification system as a class ii drug. 1corresponding author e-mail: lubnasabri14@gmail.com received: 2/11/2019 accepted: 25/1 /2020 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol29iss1pp216-225 mailto:lubnasabri14@gmail.com iraqi j pharm sci, vol.29(1) 2020 nebivolol-loaded supersaturable self-nanoemulsion 217 nebivolol is poorly soluble with extensive hepatic metabolism leading to variable and low oral bioavailability about 12%, which may adversely affect its efficacy (3). therefore, different approaches have been adopted to improve solubility and hence, its oral bioavailability through formulating as nanofiber (4), β-cyclodextrin complex (5), solid dispersion (6) and co-crystal (3). lipid-based drug delivery system (lbdds) is another promising formulation approach. todays, selfnano-emulsifying drug delivery system (snedds) is considered as one of the most attractive lbdds that could address the absorption issue in oral drug delivery (7). snedds refers to a preconcentrate or anhydrous nanoemulsion. it is typically composed of a drug candidate dissolved in a uniform, transparent mixture of oil(s), surfactant(s) and co-surfactant(s). upon contacting with gastrointestinal fluids, snedds can rapidly form in-situ a stable o/w nanoemulsion. peristalsis aids nanoemulsion formation. the nano-size droplets provide a large surface area for drug releasing and absorption (8). however, snedds application has limited by a high amount of surfactants which leads to side effects, such as mucosal irritation (9). meanwhile, reducing surfactant quantity may result in supersaturated state upon dilution. in other words, it causes insufficiently drug dispersing. consequently, drug precipitation may occur and ultimately leading to a decrease in drug absorption. therefore, the concept of "spring and parachute" is emerged. the term spring used to describe the generation of the supersaturation to increase the free drug concentration with high chemical potential gradient, which acts as a driving force for enhancing luminal absorption. supersaturable selfnanoemulsion (su-sne) is one of the spring formulations, which produces supersaturation in git. on other hands, parachute refers to the preservation and prolongation of the supersaturation state without drug precipitation (10). it is well known that the two primary steps for drug precipitation are firstly nucleation and then crystal growth. these steps can be avoided by using various polymers or amphiphilic copolymers. so they are named as precipitation inhibitor excipients. they can thermodynamically or kinetically inhibit precipitation for an extended period, adequate for drug absorption (11). so, this study aimed to prepare nebivolol supersaturable sne to have physio-chemical characterisations similar to conventional sne with reducing the total quantity of sne vehicle. the impact of different type and concentration of polymers on drug precipitation was also studied to select optimised one. moreover, the intestinal permeability parameters of the optimised polymerbased supersaturable formula, crude nebivolol suspension and conventional sne were investigated for comparison. materials and methods materials imwitor® 988 was kindly gifted by (ioi oleochemical, gmbh, germany). the other following materials were purchased from; nebivolol hydrochloride (baoji guokang bio-technology co., china), cremophor®el (international laboratory, usa), propylene glycol (evans medical ltd, liverpool, england), hpmc-k100, pvp-k30 (hyperchem, chain), soluplus (basf, germany) and peg6000 (himedia laboratories pvt ltd, india). all other reagents utilised were of analytical grade. methods preparation of conventional and supersaturated nebivolol loaded self-nanoemulsion the data obtained from a preliminary study had used to select efficient sne vehicle for nbh. among various tested oils, surfactants and cosurfactants, the mixture of imwitor-988: cremophorel: propylene glycol (pg) had excellent miscibility. it also exhibited a satisfying monophasic region in the pseudo ternary phase diagram. it found that a combination of 10% w/w imwitor, 45% w/w cremophor and 45% w/w pg had proper sne properties, physical stability and nebivolol-loading capacity of 18.1 ± 1.33 mg/ml. so, it coded as (formula a) and considered as conventional sne. while supersaturable self-nanoemulsion coded as (formula sa) was developed by reducing the amount of conventional sne vehicle to half. both formulas a and sa were prepared by adding nebivolol to a homogenous vehicle. the mixture was vortexed and then, sonicated until a clear liquid obtained. on other hands, hydroxypropyl cellulose (hpmc k100), polyethylene glycol (peg 6000), polyvinylpyrrolidone (pvp k30) and soluplus were selected. the effect of different selected polymer on formula sa performance was studied. so, each polymer powder was mixed with the formula (sa) and then, vigorously vortexed to obtain a clear liquid or uniform suspension (12). table 1 illustrates the contents of the prepared sne formulations. all prepared formulations were kept at room temperature until further evaluation. iraqi j pharm sci, vol.29(1) 2020 nebivolol-loaded supersaturable self-nanoemulsion 218 table 1. nebivolol-loaded supersaturable liquid self-nanoemulsion formulations. formulation composition (mg) a sa sah sag sap sas1 sas2 sas3 sas4 nebivolol * 5 5 5 5 5 5 5 5 5 imwitor-988 50 25 25 25 25 25 25 25 25 cremophor-el 225 112.5 112.5 112.5 112.5 112.5 112.5 112.5 112.5 propylene glycol 225 112.5 112.5 112.5 112.5 112.5 112.5 112.5 112.5 hpmc-k100 12.5 peg 6000 12.5 pvp k30 12.5 soluplus 12.5 2.5 6.5 25 total weight (mg) 505.5 255.5 268 268 268 268 258 262 280.5 nebivolol* 5mg equivalent to 5.5 mg nebivolol hydrochloride added to each formula. characterization of supersaturable-sne formulations droplet size distribution and polydispersibility index measurements each of the prepared formulations was dispersed in distilled water with (1:100) dilution and agitated by a magnetic stirrer for 5 minutes. the mean droplets size of the nanoemulsion resulted were determined using laser particle size analyser (brookhaven zetaplus, holtsville, usa). determination of self-nanoemulsifying time for supersaturable formulations the self-emulsification time was assessed by adding each formula, that represents a single dose of nebivolol to 100 ml of 0.1n hcl in a glass beaker on a magnetic stirrer, which stirred gently at 50 rotations per minutes (rpm). the time needed for complete homogenisation of dispersion was evaluated visually by the formation of clear or slight bluish liquid appearance (13). in-vitro precipitation test under non-sink condition the in-vitro precipitation test was carried out in order to evaluate the impact of the different type and concentration of polymers on maintaining a supersaturation state as a function of time (14). four doses of the conventional and supersaturable-sne were added to 100 ml of 0.1n hcl to ensure supersaturated state in tested media. the stirring speed was adjusted at 100 (rpm), and the temperature was 37 oc. samples of 3 ml were withdrawn from the test medium at 10, 15, 30, 45, 60, 120, 180 and 240 min without volume replenishment. then, the withdrawn samples were filtrated with a filter syringe of 0.22 μm (15). the filtrated samples were suitably diluted. their nebivolol content was determined using uvspectrometry (uv1100 model, emc-lab, germany). the apparent nebivolol concentrationtime profile was finally constructed. morphological visualization by transmission electron microscope (tem) transmission electron microscope (zeiss libra 120 plus carl zeiss nts, germany) was used to investigate the morphology of the sne formulas (a and sas). before analysis, each formula was diluted with distilled water (1:10 v/v). then, a few drops of the prepared dispersion were applied to a carbon-coated copper grid to form a thin liquid film, and the excess of dispersion was blotted with the aid of filter paper. the film was negatively stained with phosphotungstic acid (2% w/v) solution for 5 min and left to dry under room temperature (16). the stained sample was viewed, and the photographs were taken at suitable magnification power ex-vivo permeability study non-everted rat gut sac method, as described by ruan et al. (17) with mild modification, was carried out to study ex-vivo permeation of the prepared nbh loaded-sne (formula a) and supersaturable-sen (formula sas1) to be compared with pure nbh powder. five male sprague–dawley rats, their weight approximately 180–220 g, were supplied by the animal house in the college of pharmacy-university of baghdad. the search ethics committee iraqi j pharm sci, vol.29(1) 2020 nebivolol-loaded supersaturable self-nanoemulsion 219 approved the procedure followed in this experiment. all animals had received humane care, which complied with the guideline for the care and use of laboratory animals published by the us national institutes of health (nih publication no 85–23, revised 1996). the rats were only permitted to drink water with no food given overnight before conducting the study. each rat was sacrificed by firstly anaesthetised by inhalation of diethyl ether before cervical dislocation for the experiment. the entire small intestine was detached after making midline abdominal incision of 4–5 cm, and then an equal length of the jejunum segments was cut. immediately, the segments were cleaned with icecold normal saline solution (sodium chloride 0.9% w/v) utilising a syringe equipped with a blunt needle. after tying one end of each segment with silk thread, the intestinal sac was filled with one dose of nbh-loaded of the selected formulations (a, sas1 and pure nbh) that diluted to 1 ml with phosphate buffer. then, the sac was carefully ligated to the paddle of the dissolution apparatus ii (pharmatest, germany) after tightly closing of both sac sides. each intestinal sac was immersed in a 500 ml of the permeation media (phosphate buffer saline ph 7.4) at 37°c in a dissolution apparatus which operated at 100 rpm and continuously gassed with oxygen (approximately~ 20 bubbles/ minute) (18) . at predetermined time intervals, a sample of 5 ml was collected and directly replenished with the same volume of fresh medium. the collected samples were filtered through 0.22 μm millipore syringe filter and assayed for its nebivolol content. in this study, the cross-sectional area of the intestinal sac (s) was equal to 11.775 cm2, which calculated by applying (equation 1). taking the length of the sac (h) was 15 cm and assuming that it has a cylindrical shape with an inner radius (r) was 0.125 cm. s = 2π r h (equation 1) the apparent permeability coefficients (papp) were calculated using (equations 2): papp = (d q/dt) / (s * co) (equation 2) where (d q/dt)/s is the drug flux into the acceptor solution. the steady-state rate (flux) can be achieved by plotting the cumulative amount of drug permeated across the intestinal membrane versus time. by applying linear regression analysis of the data. the slope of the linear part of the graph would identify, which represents the flux. while co is the initial drug concentration in the mucosal side (19). the permeation enhancement by formulation was calculated by dividing the permeation rate at steady state (flux) of the selected formulas on plain nbh suspension. the cumulative nbh diffused (q120min) into acceptor jar after 120 minutes was also calculated. the extrapolation of the linear steadystate line to the time axis represented the lag time. compatibility study the drug-excipient compatibility was determined by fourier transform infrared (ftir) spectroscopy (shimadzu, japan) to identify pure powder and detect any possibility of interaction or complexation between drug and excipients. the ftir spectra of the pure nbh and su-sne formula (sas1) were determined by placing directly without any previous sample preparation onto the crystal and scanned over the range between 4000 400 cm-1 wavenumber at a resolution of 8 cm. statistical analysis the experiments results were given as a mean of triplicate samples ± standard deviation (sd). by employing one-way analysis of variance (anova) followed by post hoc analysis can detect the differences between the data of interest. the result was significantly considered different when the probability level (p) was less than 0.05 using microsoft excel 2007 and ibm statistical package for the social sciences (spss), version 23. results and discussion preparation of conventional and supersaturated nebivolol loaded self-nanoemulsion in order to achieve our aim, the amount of each sne components of the optimum formula (a) was reduced to half. thus, the saturated formula (sa) may be prone to the risk of drug precipitation after dilution with an aqueous solution. therefore, different hydrophilic polymers as a precipitation inhibitor were added to formula (sa) to protect the supersaturated state and stabilise the colloidal dispersion. the prepared formulations appeared as clear mixture except for hpmc k100, and pvp k30loaded formulas. they appeared as opaque homogeneous suspension, which may be attributed to the inability of hpmc and pvp to dissolve in the sne components. however, they may be useful once added to the aqueous medium to suppress precipitation. characterization of supersaturable-sne formulations droplet size distribution and polydispersibility index measurements the average droplet size of the prepared formulations was listed in (table 2). as shown from the table that the average droplet size of formula (sa) and (a) was the same. since both formulas (a) and (sa) composed of low drug proportion compared to vehicle proportion (1:100) and (1:50), respectively. as a result, they maintained their selfnano-emulsifying capacity after dispersion into aqueous media. the same result was also observed by ke et al. (20) furthermore, supersaturable formulations containing peg-6000 or pvp-k30 also exhibited an average droplet size that approximated similar to iraqi j pharm sci, vol.29(1) 2020 nebivolol-loaded supersaturable self-nanoemulsion 220 conventional sne formula. while, both hpmc and soluplus-incorporated sne had average size significantly (p < 0.05) higher than formula (sa). that could happen when the droplet surface coated by the polymer. also, it found that an increased amount of incorporated soluplus caused a slight increase in average size. such results indicated that the added polymers were either adsorb and/ or incorporate onto the surface of the droplets (21). on another hand, all formulations had low pdi of < 0.5, represented a homogeneity of the size distribution that could be further confirmed by tem. determination of self-nanoemulsifying time for supersaturable formulations the emulsification efficiency of the supersaturable sne formulations was shown in (table 2). it had appeared that time taken to form a clear or slight bluish dispersion upon dilution was less than two minutes for all prepared formulations. it was worth noting that the addition of a small amount of different polymers to the formula (sa) did not significantly change in the emulsification time, that ranged between 34 and 73 seconds. in the same concern, lee d r et al. found that supersaturable formulas containing kollidon va64 or pvp were instantly emulsified upon contact with the aqueous medium (12). however, the employment of 5% hpmc k100 (formula sah) or 10% soluplus (in formula sas4) as precipitation inhibitor would increase the emulsification time to 73 and 65 seconds, respectively. that might due to increased viscosity of the system, and thus delay spreading of the formulas in the aqueous medium (11). table 2.characterization of supersaturable formulation and comparison to conventional liquid selfnanoemulsion formula. formula code droplet size (nm) ± sd pdi ± sd emulsification time (sec) ± sd a 19.6 ± 0.4 0.213 ± 0.025 24 ± 1.02 sa 20.04 ± 2.03 0.228 ± 0.013 37 ± 2.5 sah (5%) 278.91 ± 0.36 0.319 ± 0.015 73 ± 1.5 sag (5%) 18.65 ± 0.78 0.169 ± 0.058 34 ± 3.1 sap (5%) 23.47 ± 2.57 0.343 ± 0.024 52 ± 2.5 sas1 (5%) 55.28 ± 1.22 0.294 ± 0.005 36 ± 1.2 sas2 (1%) 46.14 ± 1.4 0.348 ±0.005 53 ± 1.5 sas3 (2.5) 57.62 ± 4.7 0.344 ± 0.01 35 ± 1.5 sas4 (10%) 61.4 ±1.28 0.251± 0.013 65 ± 3.5 in-vitro precipitation test under non-sink condition the non-sink condition helps to achieve the required degree of supersaturation and hence, ensure the susceptibility of the drug to precipitate when subject to aqueous media (14). the in-vitro precipitation evaluation for formulations in the present and absence of precipitation inhibitor revealed in (figure 1). it can be seen from the apparent nbh concentration-time curves that the formula (sa) showed decrease concentration of nbh to about 66.5 % at 2 hours compared to conventional sne formula (a), which maintained nbh concentration above 90% along the experimental period. that indicated the loss of solubilising capacity of sne vehicle and hence, resulted in nbh precipitation to nearly its saturation solubility after 6 hours when diluted with 0.1n hcl. in contrast, the addition of hydrophilic polymer to supersaturable formula (sa) showed a significant difference (p > 0.05) in apparent nbh concentration-time profile. the results of delay precipitation came in agreement with that reported for other drugs by researchers who explained them based on precipitation inhibitor′s chemical and physical nature (11, 22). figure 1. the apparent concentration-time profile of nebivolol-loaded conventional and polymer-based supersaturable sne formulations in 0.1n hcl. iraqi j pharm sci, vol.29(1) 2020 nebivolol-loaded supersaturable self-nanoemulsion 221 statistically, it was found that the addition of peg, pvp or soluplus revealed a significant difference (p<0.05) from formula sa, which not contain any polymer. meanwhile, hpmc addition showed no significant difference (p >0.05) from formula sa. among different polymers, soluplus 5% (formula sas1) showed a comparable apparent concentration to the conventional sne, formula a profile with no statistically significant difference (p > 0.05) between them. such results could explain that the interaction at the crystal surface can slow nucleation and crystal growth leading to hampering precipitation kinetics. furthermore, soluplus may prevent aggregation and/or destruction of the drug-loaded oil droplet in a gastric fluid as reported by song et al. through incorporation ability of a hydrophobic portion of soluplus into the drug-loaded droplets and formation of a more condensed structure. besides that, its polyethyleneglycol group would sterically maintain colloidal dispersion (23). on the other hand, a different amount (1, 2.5, 5 and 10% w/w) of soluplus-based supesaturable formulations was prepared to assess the effect of the amount of soluplus on supersaturated condition. results exhibited in (figure 2) shown that the presence of 5% soluplus appeared sufficiently useful for delay crystallisation. subsequently, the formula (sas1) was selected as an optimum su-sne for further characterisation. figure 2. apparent concentration-time profile of supersaturable nebivolol-loaded sna with a different amount of soluplus in 0.1n hcl. morphological visualization by transmission electron microscope (tem) photographs depicted in (figure 3) revealed that conventional sne (formula a), and supersaturable-sne that containing 5% soluplus polymer (sas1) consisted of regular spherical, smooth surface globules with the size below 100 nm. the nanodroplets appeared as uniform dark, while the surroundings were found to be bright. there was no aggregation or precipitation. microscopic observation showed that both tested systems exhibited similar rounded shape morphology, suggesting that suitable self-nano-emulsifying ingredients can effectively generate nbh-loaded nanoemulsion. figure 2. transmission electron micrographs of conventional sne (formula a; left side) and supersaturable-sne containing soluplus (formula sas; right side). iraqi j pharm sci, vol.29(1) 2020 nebivolol-loaded supersaturable self-nanoemulsion 222 ex-vivo permeability study the amount of nebivolol permeated through intestinal mucosa was demonstrated in (figure 4). the steady-state flux was calculated from the slope of penetration profiles; the permeation rate and cumulative amount permeated from conventional nbh-loaded sne and soluplus-based supersaturable sne were found to be significantly higher (p < 0.05) than plain nbh suspension. the diffusion parameters were summarized in (table 3). figure 4.the cumulative amount of nbh permeated through rat intestinal sac from nbhloaded conventional, supersaturable sne and plain suspension. table 3. the diffusion parameters of permeability study for nebivolol from plain suspension, conventional and supersaturable sne. formula code cumulative amount diffused at 120 min (q120 minµg) flux (dq/dt.s) (µg/min.cm2) permeability coefficient (papp*10 -5 cm/min) lag time (min) pure nbh 965.6 ± 4.8 0.71 ± 0.02 14.31 ± 0.31 6.5 a 2895.9 ± 5.6 1.98 ± 0.01 39.6 ± 0.14 2.5 sas 3210.14± 15.4 2.37 ± 0.07 47.5 ± 0.04 2.3 from the above (table 3), it deduced that the conventional and supersaturable sne showed permeation enhancement ratio of 2.7 and 3.2, respectively, as compared with non-formulated nbh. moreover, it was found that after 120 min, about 57.92± 0.11 % and 64.2 ± 0.31 % of the initial amount of nebivolol were permeated from formula a and sas1, respectively, compared to only 19.31± 0.1 % from nbh suspension. it is noteworthy that the supersaturable formula (sas) provided high luminal concentrations of the drug in molecular form, thus resulting in a higher flux across the intestinal membrane. however, there was no statistical difference in permeation rate with (p > 0.05) between formulas a and sas1. the obtained data came in agreement with previous studies, concerned with the impact of selfnanoemulsion formulation on intestinal permeation enhancing (24). they ascribed a significant permeation improvement by self-nanoemulsion formulations could be attributed to presenting of the drug in a wholly solubilised form at its absorption sites. additionally, the nanometric size of droplets of resultant emulsion provides a large interfacial surface area for penetration. besides that, the employment of permeability enhancer components in the sne formulation might improve the intestinal permeation by disturbing the intestinal membrane integrity and increasing its fluidity (25). in this study, cremophor-el and pg were used as a surfactant and co-surfactant, respectively. both are known for their permeation enhancing properties (26, 27). also, soluplus has reported for its action as permeation enhancer of insoluble drug and inhibitor of the efflux pump function (28). compatibility study nbh displays characteristic peaks at 3390, 3190.26, 1072.42 cm-1 correspondings to o-h stretching, n-h stretching and c-n stretching of a secondary amine, respectively as illustrated in (figure 5). the other principal intense peaks are related to n-h bending at 1489.05 cm-1, aromatic ch in-plane bending at 1211.3 cm-1 and alcoholic co stretching observed at 1101 cm-1. other peaks are 1620.21 and 1543.05 cm-1 for aromatic c=c stretching. finally, peak at 1141.86 cm-1 is related to c-o-c stretching of cyclic ether (3, 5). meanwhile, the ftir spectrum of su-sne formula (sas1) showed fewer peaks of the drug. the peaks were overlapping in the fingerprint region, which indicated trapping of nbh inside the sne components. moreover, there was a broad peak of o-h at wavenumber ranges (3600–3200 cm–1) which hidden nbh characteristic peaks at 3390 and 3190.26 cm-1. that can be ascribed by hydrogen bond formation between nbh and sne components, that reflected nbh solubilisation in the formula. also, the principal peaks of nbh at 1624.06 and 1543 cm-1 for aromatic c=c, as well as, 1211.3 cm1 maintained in their known position with a slight shift of n-h bending peak to 1492.9 cm. moreover, no new peaks were noticed, which indicated that ftir spectroscopy validated the compatibility between nbh and oil, surfactant, co-surfactant excipients of formula sas1. iraqi j pharm sci, vol.29(1) 2020 nebivolol-loaded supersaturable self-nanoemulsion 223 figure 5.the ftir spectrum of (a) pure nebivolol powder, and (b) the soluplus-based supersaturable selfnanoemulsion (sas1) formula. conclusion from this study, it is deduced that successful supersaturable self-nanoemulsion (susne) could be prepared by adding amphiphilic polymer, 5% w/w soluplus. the optimum su-sne (formula sas1) composed of nebivolol therapeutic dose, which dissolved in 262.5mg mixture of imwitor 988, cremophore el, propylene glycol and soluplus. it emulsified rapidly upon dilution within 36±1.2 sec to yield nano-size emulsion as confirmed by tem. the formula sas1 could maintain nebivolol supersaturation without precipitation in 0.1n hcl. that indicated by high apparent nebivolol concentration more than 90% over 240 min, which was similar to conventional self-nanoemulsion. moreover, 5% soluplus-based supersaturable selfnanoemulsion enhanced nebivolol intestinal permeability by 3.2 folds compared to pure powder. so the nebivolol supersaturable self-nanoemulsion can be considered as an alternative approach to conventional self-nanoemulsion. acknowledgement the authors are thankful to assistant lecturer ammar a. fadhel for his technical helping in conducting the ex-vivo study. the authors are also grateful to the ministry of science and technology for their cooperative to accomplish this study. references 1. fongemie j, felix-getzik e. a review of nebivolol pharmacology and clinical evidence. drugs, 2015;75:1349.–1371. 2. toblli je, digennaro f, giani jf, dominici fp. nebivolol: impact on cardiac and endothelial 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nimisha roy, mehul agrawal, shubhangi chaudhary, vipin tirkey, mishra n. review article on permeation enhancers: a major breakthrough in drug delivery technology. international journal of pharmaceutical science research 2017; 8(3): 1001-1011. 28. zi p, zhang c, ju c, su z, bao y, gao j, et al. solubility and bioavailability enhancement study of lopinavir solid dispersion matrixed with a polymeric surfactant-soluplus. european journal of pharmaceutical science 2019;134:233-245 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.28(2) 2019 itraconazole nanosuspension doi: https://doi.org/10.31351/vol28iss2pp124-133 124 formulation and characterization of itraconazole as nanosuspension dosage form for enhancement of solubility asmaa m. rashid*,1 and shaimaa n. abd-alhammid* department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract itraconazole (itz) is a triazole antifungal agent given orally for the treatment of oropharyngeal and vulvovaginal candidiasis, for systemic infections including aspergillosis, candidiasis, and for the prophylaxis of fungal infections in immunocompromised patients. the objective of the study was to formulate a nanosuspension for a practically water-insoluble itz to increase aqueous solubility and improve its dissolution and oral bioavailability. itz nanosuspension was produced by a solvent-antisolvent nanoprecipitation method in the presence of different stabilizers (poloxamer-188 and hpmce5) at different ratios with the drug alone or combination with surfactants (tween 80 and sls). the results exhibit that the particle size values of all prepared itraconazole formulations were in the nano size range. the best formula (f6) has a particle size of 42 nm and zeta potential of 21.86 mv. in vitro cumulative release percent from the nanosuspension was 88 % at 30 min and from lyophilized nanoparticles was 98.2% when compared to the pure drug 13.5%. additionally, the effects of different parameters on the prepared formulas were investigated. several characterization methods were done for the optimized nanoparticles prepared by lyophilization technique, such as ftir, dsc, xrd and sem, which showed smooth uniform particles within the nano size. ftir shows no change in the position of the itz nanosuspension functional group; xrd and dsc show no significant change in the crystallinity of lyophilized nanoparticles. thus, nanosuspension appears to be a promising approach to increase the water solubility and the dissolution rate of itz. keywords: itraconazole, nanoprecipitation method, nanosuspension وتقييم عقار االتروكانازول كمعلق نانوي لزيادة الذوبانية تصييغ وتوصيف *و شيماء نزار عبد الحميد 1*،اسماء محمد رشيد بغداد،العراق. جامعة بغداد، ،كلية الصيدلة، الصيدالنيات فرع * الخالصة البلعومي وفطريات المبيضات المهبلية وداء -من االدوية المضادة لاللتهابات الفطرية كداء المبيضات الفموي االتراكونازولعقار .الرشاشيات وللوقاية من الفطريات المرافقة اللتهابات نقص المناعة كل ة ذوبانه من اجل الحصول على شكشكل دوائي فموي لتحسين قابلي لالتراكونازولالهدف من هذه الدراسة هو تحضيير وتقييم معلق نانوي . صيدالني فموي مستقر كمعلق نانوي تم تحضيره باستخدام طريقة الترسيب بالمذيب ومضاد المذيب باستعمال مثبتات مختلفة مثل ) بوليكسمر االتراكونازول مع دراسة تأثير عوامل مختلفة (tween 80,sls ) وبتراكيز مختلفة لوحدها او مع مثبت مساعد مثل e 5, هايدروكسي بروبيل مثيل سيليلوز 811 . على التحضير ( 6كانت ضمن حجم النانو لجميع التركيبات وكانت افضل صيغة رقم) االتراكونازولاظهرت النتائج ان الحجم الحبيبي للجسيمات لعقار .8:4:4بوبنس (18( ومثبت مساعد )تووين 811-( ملفولت باستخدام مثبت مستقر)بوليكسمر48.16-سطحي ) جهد( نانومترو24لها حجم حبيبي ) للمعلق النانوي الجاف مقارنة بالعقار االصلي %81للصيغة السائلة و 08(في الدقيقة %11وتراوحت النسبة المئوية النحباس الدواء ) , ftir المجفف النانويلتقييمات مختلفة لغرض فحص الحالة البلورية للمسحوق مختارة لنفس المدة .خضعت الصيغة ال %80.5 والذي يبلغ dsc , xrd,sem وكانت النتائج تشير الى كونها جزيئات ذات حجم نانوي وذات سطح ناعم مما يشير الى ان تقنية تصنيع المعلق النانوي .ذوبان عالية وانتشار سريع قابلية ذو اتراكونازول دة والفعالة لصياغةبالترسيب بالمذيب من الطرق الواع االتراكونازوللعقار . الترسيب بالمذيب،معلق نانوي, ، اتراكونازول :الكلمات المفتاحية introduction low bioavailability is one of the serious problems correlated with poorly soluble drugs. foremost struggles have been ended for the development of customized drug carriers to overcome the disappointing invivo fates of the drug. hence, there is a growing necessity for a unique strategy that can tackle the formulation associated problems related to the delivery of hydrophobic drugs in order to improve their clinical efficacy and optimize their therapy concerning pharmacoeconomics. various solubilization techniques have been used for the improvement of solubility and drug dissolution rates including a reduction of the particle size , by micronisation or 1corresponding author: e-mail: asmaamohammad68@gmail.com received: 27 / 5 /2019 accepted: 18/8 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp124-133 iraqi j pharm sci, vol.28(2) 2019 itraconazole nanosuspension 125 nanonisation to increase the surface area, use of surfactants, cyclodextrin complexation, pro-drug formation, conversion of crystalline to amorphous forms, polymeric conjugates and solid dispersion(1) nanosuspension has shown to be a more cost-effective and technically simpler method. it can be defined as the dispersion of particles in the nanometer size range, where stabilization is accomplished by the addition of surfactants, stabilizer or a combination of both (2,3). nanosuspension consists of a poorly watersoluble drug without any matrix material suspended in dispersion resulting in the formulation having high dissolution velocity and increased saturation solubility (4). it can be applied to several administration routes such as oral, parenteral, pulmonary, ophthalmic, and nasal routes (5). itz is an active triazole, antifungal agent(c35h38cl2n8o4), which is active against a broad spectrum of fungal species including cryptococcus, candida, aspergillus, blastomyces and histoplasma capsulatum. it inhibits lanosterol 1,4demethylase, the enzyme that converts lanosterol to ergosterol (6,7). itz is belonging to class ii as classified by the biopharmaceutical classification system (bcs), soluble in lipids and has a pka of 3.7 (8). itz has the characteristic of ph-dependent solubility having the highest solubility at the acidic media (4μg/ml) compared to basic ph (1μg/ml). it is slightly soluble in alcohols and freely soluble in dichloromethane and practically insoluble in water(9), indicating that poor aqueous solubility is the main reason for lower plasma concentrations. itz at a low ph, in gastric juice, will be ionized and therefore the gastric acidity is essential for adequate dissolution and its oral bioavailability was increased when taken with food (7,10). in nanoprecipitation method, the drug is dissolved in a suitable solvent. this solution is mixed with a miscible antisolvent system in the presence of surfactants. rapid addition of drug solution into the antisolvent leads to the sudden supersaturation of drug in the mixed solution forming ultrafine drug solids. precipitation method involves two phases nuclei formation and crystal growth (10). the objective of this study is to optimize and characterize the formulations prepared by the nanoprecipitation method for the preparation of itz nanosuspension. materials and methods materials itz powder was purchased from baji gaokang biotechnology co., ltd., china. tween 80 was purchased from scharlau s. l spain. poloxamer 188 and hpmc e5 were provided by hangzhou hyper chemicals limited, zhejiang, china. dialysis membrane70 provided by himedia (mumbai, india). all other chemicals and solvents were of analytical reagent grade. methods saturation solubility determination solubility studies for the pure drug were achieved in distilled water, 0.1n hcl( ph 1.2) and phosphate buffer ph 6.8. in each case, the excess amount of sample added to 10 ml of solvent and agitated at 25°c in a rotary test tube shaker for 72 hrs. after equilibration, samples were filtered using 0.45 µm millipore filters, suitably diluted with the respective solution, and analyzed by measuring the absorbance at the determined wavelength of max. absorbance (256) nm, when this solution was scanned in the uv range from 200800nm using uv-visible spectrophotometer, in 0.1 n hcl and (263)nm in methanol, d.w, and phosphate buffer ph 6.8 to determine the amount of the drug dissolved (11,32). preparation of itraconazole nanosuspension by precipitation method nanosuspension precipitation method is used to prepare nanosuspension of itz using different concentrations of polymer and surfactant. in brief, 50 mg of itz was dissolved in an organic solvent ( 5 ml of methanol). the aqueous solution containing the selected stabilizers p-188 and hpmc-e5 at different ratios (1:1), (1:2) and (1:4) drug to stabilizers, or in combination with co-surfactant (tween 80 and sls), which acts as the antisolvent system. this was followed by the addition of the organic solution into stabilizer/surfactant aqueous solution at a rate (1ml /min) by the help of syringe, under high-speed mechanical agitation of 3000 rpm using, homo disperser for 30 min at 25±1°c to allow the organic solvent to evaporate and get the desired nanosuspension (13) . the batches were prepared according to the formulation design (table 1). iraqi j pharm sci, vol.28(2) 2019 itraconazole nanosuspension 126 table 1. composition of itraconazole nanosuspension formulation. f: formula, itz: itraconazole, p-188: poloxamer 188, sls: sodium lauryl sulphate ,hpmce5 :hydroxypropyl -methylcellulose, rpm: revolution per min. characterization of the prepared nanosuspension: particle size and polydispersity index (pdi) particle size analysis and pdi of the prepared itz nanosuspension were measured by dynamic light scattering (dls) using nano brook 90plus particle size analyzer (brookhaven instruments. usa). measurements were performed in triplicate by measuring the intensity of light scattered by the molecules in the sample as a function of time at 90° scattering angle and constant temperature 25 °c. the pdi was determined which measured the width of the size distribution of each formula, it is an index of spread or variation within the particle size (5,14). determination of entrapment efficiency (%dee) ten ml of freshly prepared nanosuspension was centrifuged at 6000 rpm for 20 minutes using ultracentrifuge. the supernatant solution was filtered and separated. one ml of this filtrate was diluted with 10ml water, and the absorbance at maximum λ max was measured by uv spectrophotometer using water as blank. the amount of unincorporated drug in the formulations was measured, and the entrapment efficiency was calculated by subtracting the quantity of free drug in the supernatant from the initial amount of drug taken (eq.1). the outcomes were analyzed in triplicate(15,20). 𝐷𝐸𝐸% = (𝑡𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑡ℎ𝑒 𝑓𝑜𝑟𝑚𝑢𝑙𝑎−𝑓𝑟𝑒𝑒 𝑑𝑟𝑢𝑔) 𝑡𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 𝑖𝑛 𝑓𝑜𝑟𝑚𝑢𝑙𝑎 ×100 (1) determination of zeta potential the zeta potential of the selected formula of nanosuspension was measured by using ‘nano brook 90plus-zeta seizer (brookhaven instruments usa). sample of the selected formula was placed in the electrophoretic cell and measured three times at 25 ±1° c, and the average values were calculated. zeta potential gives information about the surface charge properties and furthers the long-term physical stability of the nanosuspension. to obtain an electrostatically stabilized nanosuspension, a minimum zeta potential of ± 30mv is required. in the case of a combination of electrostatic and steric stabilization, a minimum zeta potential of ± 20mv is desirable (16,25). scanning electron microscopy (sem) the morphology of pure drug and the selected itz nanosuspension (f6) were examined by scanning electron microscope (vega3tuscan czech republic) operated with a secondary detector at different acceleration voltage and at different magnification .the morphology of pure drug was done by direct deposition of powder on double-sided carbon tape and coated with gold at 1k, 2k, 5k and 500x magnification. while for liquid sample f6, it was prepared by the droplet evaporation technique. a droplet of liquid was deposited on double-sided carbon tape and dried at room temperature for the evaporation of water and then coated with gold (17,18). lyophilization of selected itz nanosuspension lyophilization of the selected formula was accomplished using christ (alpha 1-4 ld plus) to recollect nanoparticles in a dried-powder state from the nanosuspension and to complete characterization of nanosuspension and show the effect of lyophilization on nanoparticles size and solubility. the selected formula was used containing 2% w/w mannitol ,as the cryoprotectant, was frozen in a refrigerator at -70 °c for 24 h. then the sample was lyophilized using vacuum freeze dryer at a controlled temperature of -58 °c and the pump operating at the pressure of 150 militorr over the range of 48–72 hr. the obtained powder was used for further studies (5,13). characterization of itz lyophilized nanosuspension differential scanning calorimetry (dsc) the dsc of pure itz powder and lyophilized itz nanoparticles formulation were taken on (shimadzu dsc-60 plus, japan). ten milligrams were sealed in the flat-bottomed aluminium pan of the differential scanning calorimeter. data collection was achieved at a temperature range of 0–200°c and the heating rate formula no. f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 itz(mg) 50 50 50 50 50 50 50 50 50 50 50 p-188(mg) 50 100 200 400 100 100 200 200 hpmce5(mg) 50 100 200 tween80(ml) 0.1 sls (mg) 50 methanol(ml) 5 5 5 5 5 5 5 5 5 5 5 dw(ml) 20 20 20 20 20 20 20 20 20 20 20 speed(rpm) 3000 3000 3000 3000 3000 3000 1500 500 3000 3000 3000 iraqi j pharm sci, vol.28(2) 2019 itraconazole nanosuspension 127 was 5°c/min under nitrogen gas at a flow rate of 25 ml/min (19). fourier transform infrared spectroscopy (ftir) the ftir spectra of bulk itz, and lyophilized itz nanoparticles were recorded using ftir -7600, australia spectrophotometer. powders were mixed with potassium bromide and compressed into disks using hydraulic press before scanning from 4000 to 400 cm-1 (19). x-ray powder diffractometry studies the xrd patterns for bulk itz and lyophilized itz nanoparticles were analyzed using an xrd-6000, shimadzu-japan. the freeze-dried samples scanning was conducted over powder x-ray diffractometer at the axis of 2 thetas ,with the continuous scan range of 5-80 degree. the operating voltage was 40 kv, and current 30ma and scan step size of 0.050° (2θ) and scan step time of 60 seconds (19). in -vitro dissolution studies the in-vitro dissolution test was estimated using (paddle assembly) type ii dissolution test apparatus. accurately weighed bulk drug and the selected nanosuspension formula (f6) and the lyophilized formula (equivalent to 50mg nanoparticles)were determined under sink conditions using himedia dialysis membrane 70 (mwco 12 kd). briefly, 20ml of optimized formulations of itz nanosuspension was placed in the pretreated dialysis bag soaked in dissolution media overnight before use; and fitted with a paddle then dispersed in 900ml of 0.1 n hcl (ph 1.2) kept at 37± 0.5 °c at a rotation speed of 100 rpm. sink condition was maintained throughout the study. an aliquot of 5ml samples was withdrawn from the receiver compartment at predetermined time intervals (5, 10 , 15 ,20 ,30 , 45, 60 ,90 ,and 120 min) respectively and refilled with the equivalent volume of fresh dissolution medium to maintain the constant volume. then, samples were filtered and the amount of drug determined spectrophotometrically on uv spectrophotometer at the determined wave length of max. absorbance in 0.1n hcl at 257 nm wavelength. the experiment repeated in triplicate for each formulation (5,16). results and discussion saturation solubility of pure itz and freeze-dried nanoparticles the solubility of pure itz and itznanosuspension ( f6) (itz: p-188: tween80 1:2:2) that was selected for lyophilization; was carried out in purified water, ph 1.2 hcl buffer and phosphate buffer ph 6.8. the saturation solubility of the lyophilized itz nanosuspension was increased significantly(p< 0.05) over the pure drug in all vehicles used. this increment due to the decrease in particle size and increasing the surface area as illustrated in figure 1 (20). particle size analysis and polydispersity index measurement the mean particle size of various batches of nanosuspension prepared is depicted in table 2. all the prepared formulations were in the nano size. the mean particle size varied from 42nm – 685.2 nm. the stabilizer p-188 and tween 80 resulted in smaller particle size indicating effective stabilization of prepared nanosuspension which agrees with that obtained by kalvakuntla who formulate aprepitant nano-suspension(27). the pdi of each formula was determined which is varied from 0.005-0.336. the entrapment efficiency of itz-nanosuspension the results of entrapment efficiency have been shown in table 2. the values of drug entrapment efficiency of f3, f6, f7 was high compared to other formulations in figure 2 which may be attributed to the presence of optimum concentrations of stabilizers (p-188) and the presence of surfactant ( tween 80). it is clear that increasing the stabilizer concentration, and the addition of surfactant increased the drug entrapment efficiency. the low values of drug entrapment efficiency point out the relatively low affinity of the drug with the polymer matrix. the concentration of stabilizer used is the most effective factor in entrapment efficiency, and this agrees with that obtained by preeti singh. et al. who formulates satranidazole nanosuspension and by patil et al. who formulates spry dried chitosan nanoparticles containing doxorubicin (21,22). effect of stabilizer concentration on the particle size and polydispersity index figure 3 shows the effect of stabilizer concentration on particle size and pdi by using four different ratios of polymers 1:1,1:2, 1:4 and 1:8 ( f1, f2, f3 and f4 for p-188) and( f9,f10,f11 for hpmc e5). the formulation showed pdi in the range of (0.005-0.336), and this low value will indicate good stability of the nanosuspension. the results showed that the particle size decrease with the increasing of concentration of polymer. mean particle size of formula f1 was 531.2 nm compared with f4 that had mean particles size 53 nm for p-188 and for hpmc e5 ,f9 was 241.6 nm compare to f11 that had mean particles size 90 nm. the choice of suitable stabilizers and its concentration are the most important factors to control the size and stability of the nanosuspension during nanoprecipitation methods (23). iraqi j pharm sci, vol.28(2) 2019 itraconazole nanosuspension 128 table 2.physicochemical characterization of itraconazole nanosuspension formula no. stabilizer drug:stabilizer co-stabilizer stirring speed (rpm) particle size(nm) polydispersity index %ee f1 p-188 1:1 3000 532 ±0.0 0.269 ±0.0 96.4±0.2 f2 p-188 1:2 3000 196.2±0.0 0.336 ±0.0 98.1±0.2 f3 p-188 1:4 3000 131.8±0.0 0.005 ±0.0 99.98±0.3 f4 p-188 1:8 3000 53±0.0 0.041 ±0.0 99.2±0.32 f5 p-188:sls 1:2:2 3000 540.1±0.0 0.321 ±0.0 93.41±0.5 f6 p-188: tween80 1:2:2 3000 42±0.0 0.086 ±0.0 99.99±0.11 f7 p-188 1:4 1500 424±0.0 0.005 ±0.0 99. 4±0.05 f8 p-188 1:4 500 685.2±0.0 0.005 ±0.0 97.82±0.53 f9 hpmce5 1:1 3000 241.6±0.0 0.260 ±0.0 81.94±0.38 effect of stirring speed on the particle size and polydispersity index three different speeds 3000, 1500 and 500 rpm were used to prepare three formulas( f3 ,f7 and f8) to show the effect of stirring speed on particle size as shown in figure 4. in this study the optimum speed for formulas have a drug to stabilizer ratio 1:4 was found to be 3000 rpm that produces mean particle size 131.8nm. pdi of these formulations was 0.005 an increasing stirring speed would result in lower particle size. as a result, high shear stress was necessary to break down particles to the submicron range (24). influence of stabilizer type on particle size and pdi two types of stabilizer were used to prepare nanosuspension , hpmc e5 and p-188 at the (1:1,1:2,1:4) ratio. as shown in figure 5 , the mean particle size obtained is (241.6,95,90 nm) for hpmc e5 , while for p-188 stabilizer the mean particle size obtained is (531.2,196.2,131.8nm) for the same ratios. comparing results, hpmc e5 stabilized nanosuspension , demonstrated the lowest particle sizes than p-188. hpmc e5 has a good affinity toward the drug particles, and thereby, it can provide an active steric barrier against particles growth (26). zeta potential the zeta potential for the selected formulation of itz nanosuspension was 21.86 mv, as shown in figure 6. the charge was negative due to adsorbed tween 80 and p-188 on the drug particles; the importance of zeta potential that it reflects the degree of repulsion between adjacent, similarly charged particles in the dispersion. the obtained value for selected formulation indicates that stable nanosuspension value can be related to the stability of colloidal dispersions. zeta potential is an important parameter for the prediction of the stability of nanosuspension (24). in vitro drug release the release of itz from the nanosuspension of selected formulation and the lyophilized nanoparticles was higher than the release profile of the pure drug in 120 min as shown in figure 7. the %cdr of the selected lyophilized nanoparticles was 98% in 30min and 88% for itz-nanosuspension formula in 0.1n hcl media as compared to 13.5% of the pure drug in the same media indicating the poor drug solubility and thereby dissolution (25). so there is significant differences (p<0.05) in the release of the pure drug and selected formula ,which coordinated with noyes–whitney equation, in which the dissolution rat is enhanced as the saturation solubility increased and the particle size decreased(23) . other factor that may contributing to the fast release is the entrapment efficiency which has a direct effect on the drug release profile. as it increased, the release rate also increased (26). scanning electron microscope (sem) the sem of pure itz is presented in figure 8 ,showed agglomerates of irregular, rough surface, large shape of itz particles while the images of the sem of the selected formula of the nanosuspension (f6) is represented in figure 9 , showed smooth uniform particles within the nano size which could be assigned to the presence of stabilizer that coated particles, the surfactant which was stabilized the particles could be adsorbed to the surface of the crystals by hydrophobic interaction. the sem images confirmed that an increase in particle size was observed after freeze-drying, but it was still in the submicron level and lower in size in comparison with the pure drug, but not below 100 nm (16,27). differential scanning calorimetry (dsc) the dsc was used to estimate the effect of excipients and conditions on the physical properties of drug. the dsc of pure itz powder, (figure10 ) depicted a sharp endothermic peak at 166.19 °c indicating no change in its melting temperature and the drug has a crystalline nature with high purity. for lyophilized powder, (figure11), the slightly lower melting temperature, at 165.59ºc might be due to small size of itz crystals resulted from surrounding of the stabilizer to drug crystals and small particle size (5.13). iraqi j pharm sci, vol.28(2) 2019 itraconazole nanosuspension 129 powder x-ray diffraction analysis (pxrd): to affirm the crystallinity of itz, x-ray diffraction was performed for pure itz and lyophilized nanoparticles. the x-ray diffraction pattern of itz exhibited characteristic sharp peaks (figure12) at 14.48°, 17.51°, and 20.37° that indicate the crystalline state of itz (20).some of characteristic peaks appeared related to p-188 at 19.10, broader one between 22°, and 23° and a characteristic peaks at 9.7° appeared for mannitol (30). the diffractogram of lyophilized nanoparticles exhibited the diffraction peaks at 9.630, 13.550, 19.370, 20. 290 21.830, 23.110, 24.560, 36.580, 37.790 and 44.010 which arise due to the overlapping contribution of excipients and drug. (20) however, slight decrease in intensity of peaks was observed with and the absence of two characteristic peaks in the xrd pattern of selected lyophilized nanoparticles compared to the pure itz as seen in figure 13 may explain a reduction in the size of itz crystal and nanoparticles was not affected significantly by the freeze-drying process (5,16). ft-ir analysis the ftir spectra of pure itz is shown in figure14.the main peaks of pure itz were observed at wave numbers, 3466, 3130, 2965, 3067, 1698, 2822,1611, 1512 and 1453 cm-1.the absorption bands between 2800 cm1 and 3200 cm-1 were vibration stretching of c-h for both alkane and aromatic c-h. the band located at 3466 cm−1 was assigned to the stretching vibration of ch of furan ring group and 733cm-1due to c-cl stretching in itz molecule and those at 3130 and 3067 cm−1 resulted from stretching vibration of the amino group. a sharp band at 1698 cm−1 is due to c= o, and the bands at 1611 and 1454 cm−1 are assigned to stretching vibration of (c= c) and (c-n )bonds, respectively. it should be noted that all the characteristic bands of itz were detected in the spectrum of the lyophilized nanoparticles as shown in figure 15, indicated that the drug and the stabilizer and other excipients used in the formulation are compatible with each other.( 20,32). conclusion nanoprecipitation method was successfully used to produce stable itz nanosuspension by using the proper stabilizer (p188) and surfactant (tween 80). different parameters, such as stabilizer type, concentration, stirring speed, were investigated and optimized to produce the smallest drug nanoparticles. the dissolution rate of the prepared itz nanosuspension and lyophilized nanoparticle were significantly enhanced as compared with the pure itz powder. figure 1. saturated solubility of pure itz and selected lyophilized formula figure 2. the entrapment efficiency of itz nanosuspension figure 3. effect of stabilizer concentration on the particle size iraqi j pharm sci, vol.28(2) 2019 itraconazole nanosuspension 130 figure 4. effect of stirring speed on particle size figure 5.effect of stabilizer type on particle size figure 6. zeta potential of the selected formula (f6) itz-ns figure 7. release profile for pure drug, nanosuspension formulation(f6) and itz lyophilized nanoparticles (ln) figure 8. sem of pure itz figure 9. sem of itzselected formula (f6) iraqi j pharm sci, vol.28(2) 2019 itraconazole nanosuspension 131 figure 10. dsc thermograms of pure itz figure 11. dsc thermograms of itz lyophilized powder of selected formula (f6). figure 12. xrd of pure itz figure 13. xrd of itz lyophilized powder of selected formula ( f6) figure 14. ftir of itraconazole figure15. ftir of itz lyophilized powder of selected formula (f6) figure 16. particle size distribution of the selected formula (f6) references 1. bagavatula h, lankalapalli s, tenneti vv, beeraka nm, bulusu bt. comparative studies on solubility and dissolution enhancement of different 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tt,serajuddinat.characterizatin of solid dispersion of itraconazole prepared by solubilization in concentrated aqueous solutions of weak organic acids and drying. pharmaceutical research. 2016 jun 1;33(6):1456-7 32. vyas pr, patel kj. enhancement of solubility of itraconazole using novel liquisolid technique. pharma science monitor. 2015 apr 1;6(2). 269-286. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(suppl.) 2022 antibiotic self-medication doi: https://doi.org/10.31351/vol31isssuppl.pp9-17 9 self-medication towards antibiotic use among nonmedical university staff (conference paper )# haydar f. al-tukmagi*, harith kh. al-qazaz**, sadeel a. shanshal**,1 and muhanad m. al-kaisey* # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *baghdad college of medical sciences, baghdad, iraq. ** department of clinical pharmacy, college of pharmacy, university of mosul, mosul, iraq abstract the use of antibiotics without prescription (self-medication) is growing globally and is associated with increased bacterial resistance, ineffective treatment and adverse reactions. this study aimed at assessing the practice of antibiotic self-medication in the iraqi population. a cross-sectional study design was adopted in this work. the sample was comprised of 303 staff members from the non-medical colleges in iraq. an online questionnaire was distributed between the 29th of june to the 14th of september 2021 to collect data including socio-demographic characteristics and questions about antibiotic self-medication. most of the participants had a university degree and a moderate monthly income. the majority (88%) have practiced self-medication at least once before. a “simple” condition and convenience were the main motivators behind self-medication, which was mainly used for sore throat, fever and cough. own experience was the most reported determining factor for selecting an antibiotic, and community pharmacies were the main source for obtaining the antibiotics. about 40% of the participants admitted to switching the antibiotic or changing its dose during the treatment course. self-medication with antibiotics is a major issue in our community and measures have to be taken to reduce its impact on public health through the development of bacterial resistance. keywords: self-medication, antibiotics, bacterial resistance. التدريس المضادات الحيوية بين أعضاء هيئة باستخدامالتطبيب الذاتي في الكليات غير الطبية #مؤتمر ( ) بحث * مهند مروان القيسي و 1,** عبدالمنعم شنشل سديل ، **حارث خالد القزاز، *التكمجيحيدر فخري 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي .كلية بغداد للعلوم الطبية، بغداد، العراق* فرع الصيدلة السريرية، كلية الصيدلة، جامعة الموصل ، الموصل ، العراق ** الخالصة غير الفعال الاستخدام المضادات الحيوية بدون وصفة طبية )التطبيب الذاتي( يزداد عالميًا ويرتبط ذلك بزيادة المقاومة البكتيرية لها، والعالج نإ طعية في هذا العمل, حيث والتفاعالت الضارة. تهدف هذه الدراسة إلى تقييم مشكلة التداوي الذاتي بالمضادات الحيوية لدى سكان العراق. تم اعتماد دراسة مق لجمع البيانات 2021سبتمبر 14يونيو و 29منتسب في الكليات غير الطبية داخل العراق. وقد تم توزيع االستبيان عبر اإلنترنت بين 303تكونت العينة من لمشاركين حاصلين على شهادة جامعية ودخل شهري بما في ذلك الخصائص االجتماعية العامة وأسئلة حول التطبيب الذاتي بالمضادات الحيوية. كان معظم ا ٪( مارسوا التطبيب الذاتي على االقل مرة واحدة من قبل. الحالة كانت "بسيطة" وسهولة الحصول على المضادات الحيوية هي 88معتدل. الغالبية الكبرى ) لحلق والحمى والسعال. الخبرة الشخصية كانت هي العامل األكثر شيوًعا الدوافع الرئيسية وراء التطبيب الذاتي ، حيث كان يستخدم بشكل أساسي في التهاب ا ٪ من المشاركين قد ذكروا 40الختيار المضادات الحيوية ، وكانت الصيدليات المجتمعية هي المصدر الرئيسي للحصول على المضادات الحيوية. حوالي التدابير لعالج. التطبيب الذاتي بالمضادات الحيوية هو قضية رئيسية في مجتمعنا ويجب اتخاذ انهم قاموا بتبديل المضاد الحيوي أو تغيير جرعته أثناء فترة ا .للحد من تأثيره على الصحة العامة من خالل تطوير المقاومة البكتيرية ةالالزم التطبيب الذاتي، المضادات الحيوية، المقاومة البكتيرية : المفتاحية الكلمات introduction self-care is any practice adopted by an individual for the purpose of maintaining own health and protecting from diseases, and one element of self-care is self-medication (1). the world health organization (who) defines self-medication as “the use of drugs to treat self-diagnosed disorders or symptoms, or the intermittent or continued use of a prescribed drug for chronic or recurrent disease or symptoms” (2). self-medication is growing to be a major public health problem and cultural, political and economic factors have contributed to triggering a global increase in this practice (3). differences in the attributes of the health care systems between the developed world and the developing world such as t the access to care, guidelines of compensation and the policies for dispensing drugs result in varied prevalence of self-medication between these two worlds (4). in the developing countries, infections continue to be a very common cause of death, and that is why antibiotics are very important therapies (5). they are within the most commonly sold medications globally (6). self-medication with antibiotics may be viewed as irrationally using the antibiotics (7). the consumption of antibiotics without a physician prescription has been mounting recently and has contributed significantly to antimicrobial resistance (8). 1corresponding author e-mail: sadeelshanshal@uomosul.edu.iq received: 16/5 /2022 accepted: 26/ 7/2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp9-17 iraqi j pharm sci, vol.31(suppl.) 2022 antibiotic self-medication 10 antibiotics resistance is becoming a global health catastrophe due to continuous overuse and misuse of antibiotics throughout the world, the main reason behind this disaster is attributed to selfmedication (8)(9). this irrational use is also associated with various health hazards that could result from mistakes in the diagnosis, the dose, the route of administration or even the formulation. risks of adverse effects and drug interactions can also contribute to the health hazards (10). the extensive inappropriate use of antibiotics combined with unawareness of the individuals about the proper use of the antimicrobials, their adverse effects or correct doses are possible causes of misdiagnosis, antibiotic resistance increased morbidity (8). for all these reasons, many countries have laws to prohibit selling antimicrobial drugs without prescription to avoid the evolution of super bacteria resistant to all known antimicrobials (11). unfortunately, this is not the case in all countries, where such drugs can be purchased relatively easy without prescription. a study conducted by nusair et al in jordan showed that the practice of antibiotics self-medication was still high in 2021 and this should urge the authorities to find an approach to limit antibiotics dispensing to prescriptions only (12). another recent study in turkey revealed that selfmedication with antibiotics continues to be a major health concern in the society that requires educational campaigns in order to warn about the consequences of inappropriate use of antibiotics (13). in iraq, laws do exist that prohibit over-the-counter sale of antibiotics, but they are not enforced, and it has been reported that these drugs can be purchased directly from the pharmacy (14). little is known about the self-medication of antibiotics among the staff of non-medical colleges in iraqi universities, therefore; this study aims to shed light on this important topic in order to reduce the chance of fueling antimicrobial resistance in our society. material and methods a cross-sectional study design was used in this study by conducting a web-based survey among the staff of non-medical colleges in iraqi universities (baghdad and mosul universities as model). the participants were recruited during the period from 29th of june to 14th of september 2021. a convenience sampling with snow-balling technique was used to invite the participants. the inclusion criteria were being a staff member in baghdad or mosul universities. the teaching staffs in medical colleges were excluded from this study. the survey was conducted in arabic language to be comprehensible and easily understood by the university staffs. the original english version was adapted from previously published works (15–17). face validation was conducted through an expert panel to ensure the cultural adaptation within the arabic version. it was designed by google forms, and the survey link was sent three times to a variety of social media closed groups (like whatsapp, viber, and messenger); at the beginning of the study on 29th of june, on 25th of july and on 11th of september. settings of the google form survey were set to “limit to 1 response” in order to ensure a one response being received from each participant without duplication. the front page in the survey included information about the aims of the study, a confirmation that the participation was completely voluntary, and that all data would only be used for the research purpose. the first part of the survey was used to collect data about the socio-demographic characters of the participants (age, gender, university, residence, education, and monthly income). part two consisted of questions about self-medication by using antibiotics; the first two questions asked the respondents whether they knew antibiotics and if they had used antibiotics previously. the remaining questions were regarding antibiotic self-medication asking about the frequency of the practice, the main driving force and complaint behind self-medicating, factors affecting the choice of the antibiotics, the source of the drugs, whether the instructions were being checked or not, the possibility of changing the antibiotic or its dose and the reasons behind stopping the treatment. all the data were processed and analyzed by using microsoft excel 365 and statistical package for social sciences version 26 (ibm spss statistics for windows, ibm corp., armonk, ny, usa). descriptive statistics (frequency and percentage) were used to describe demographic characters. pie and clustered column charts were used to illustrate the responses of the participants. before conducting the study, the protocol of the work was authorized by the scientific committees in baghdad college of medical sciences and the department of clinical pharmacy at the college of pharmacy / university of mosul. the participants were asked to put a tick in the google form survey to confirm that their participation was voluntary and to inform them that their data would only be used for research purposes. results a total of 303 participants were recruited in this study with age ranged between 19 and 70 years and the mean (sd) of the age was 41.47 (10.55) years. the other demographic characteristics of the study participants are illustrated in table 1. iraqi j pharm sci, vol.31(suppl.) 2022 antibiotic self-medication 11 table 1. demographic characteristics of the study sample variable n (%) gender male female 147 (48.5) 156 (51.5) university mosul baghdad 141 (46.5) 162 (53.5) residence urban rural 286 (94.4) 17 (5.6) education secondary university post graduated 49 (16.2) 113 (37.3) 141 (46.5) monthly income limited (< 500,000 iqd) moderate (500,000 – 1,000,000 iqd) high (> 1,000,000 iqd) 63 (20.8) 205 (67.7) 35 (11.6) almost all participants (96%) declared that they knew a group of drugs called antibiotics, and about 95% of the studied population mentioned that they had used antibiotics in the last two months (figure 1). out of the 303 participants in this study, 265 (88%) said that they have practiced self-medication with antibiotics before. when they were asked about the frequency of self-medication with antibiotics in the past year, 133 (43.9%) participants said that they rarely self-medicated with these drugs compared to 29 (9.6%) and 24 (7.9%) who admitted to selfmedication usually and always respectively. figure 1. a. the of percentage participants having an antibiotics knowledge b. the percentage of participants who have used antibiotics in the previous two months having a condition that was seen as simple by the participant was the main driving factor behind seeking self-medication with antibiotics. figure 2 summarizes these factors. figure 2. reasons for self-medication with antibiotics iraqi j pharm sci, vol.31(suppl.) 2022 antibiotic self-medication 12 regarding the complaint behind using antibiotics without prescription, sore throat came first, followed by fever and cough while skin wounds and vomiting came last (figure 3a). own experience was the major determining factor when choosing antibiotic for self-medication followed by recommendation from a pharmacist or having leftovers from a previous prescription (figure 3b). when the participants were asked about the considerations behind selecting a particular antibiotic for selfmedication, the majority chose the type, indications and the brand of the drug as their answers. side effects and price were not very important (figure 3c). community pharmacies were by far the most important source for the antibiotics used by participants for self-medication. other sources existed but were much less significant (figure 3d). figure 3. a. complaint for self-medication with antibiotics b. antibiotic selection basis c. considerations when selecting antibiotics d. source of antibiotics iraqi j pharm sci, vol.31(suppl.) 2022 antibiotic self-medication 13 when asking the participants if they ever check the instruction of antibiotics, 160 (52.8%) mentioned that they always read it, while 92 (30.4%) answered they sometimes check it and only 51 (16.8%) said that they never do that. the pharmacist was the principal source of the information regarding the appropriate antibiotics’ doses, followed by reading the leaflets and own experience. doctors, family or friend and internet came after (figure 4). figure 4. source of dosage instructions. during the course of treatment, approximately 60% of the participants admitted to never switching the antibiotic used while only 4% said that they always change their medication. concerning the dose used in self-medication, about 40% of the non-medical staff said they sometimes change the dose deliberately, in contrast to 172 (56.8%) participants who assured they never change the dose by themselves. improvement of the medical condition was a major reason behind changing the dose of the antibiotic whereas worsening of the condition was a minor one according to the respondents (figure 5). figure 5. reasons for dosage adjustment regarding concern about taking a counterfeit product, about one third of the respondents said that they are always very much concerned about this issue compared to one half who are never concerned about having a counterfeit antibiotic. the participants divided approximately equally into either keeping the same product brand or changing it throughout the treatment course. figure 6 shows the responses of non-medical staff participants about the reasons for stopping treatment with the antibiotics; disappearance of symptoms and completing the course were the main reasons. iraqi j pharm sci, vol.31(suppl.) 2022 antibiotic self-medication 14 figure 6. when to stop antibiotic more than two-thirds of the participants (72.9%) have suffered from some type of adverse reaction to the antibiotics during usage. the main response when this happened was to stop the antibiotic followed equally by consulting a pharmacist or a family or friend. doing nothing and switching to another antibiotic were also recorded by some participants (figure 7). figure 7. response to side effects more than half of the participants (58.4%) think that self-medication with antibiotics is an acceptable practice. the remaining participants were distributed between thinking that either it is a good (19.8%) or non-acceptable (21.8%) practice. at the same time, 114 (37.5%) respondents assured that self-medication cannot treat common infections and only 83 (27.4%) said that it may be able to treat these infections in contrast to 106 (35%) who were confident that they will benefit from selfmedication. discussion self-medication with antibiotics reflects individuals’ wish to take the responsibility of treating some health problems. there are many pros and cons associated with self-medication; reducing healthcare costs and the momentum on doctors to devote themselves for more urgent cases are among the advantages. on the other hand, inappropriate usage of antibiotics could lead to worsening of the condition as well as the development of antibiotic resistance (18–20). the mean age of the respondents in the current study was 41.47 years which was similar to the age of participants in another study conducted in sri lanka (21). but different from studies that were conducted in bangladesh and india as the bangladeshi and indian participants were younger (22,23). this is because the target population was different between the studies. more than three quarters of the participants in this study have graduated from universities (37.3% university educated and 46.5% post graduated), and this is comparable to the education of the participants in a saudi study (24). ninety-six percent of the participants in the current study reported that they knew antibiotics as a drug group; this percentage was higher than that found by ateshim et al (25) in eretria. the majority of the participants in this study (88%) have practiced self-medication with antibiotics, this is comparable with other studies that were conducted in sudan (26) and india (27). however, such percentage is much higher than results of other studies conducted worldwide (15,25,28–30). this variation may be attributed to the presence of more rigorous laws governing the use of antibiotics or to iraqi j pharm sci, vol.31(suppl.) 2022 antibiotic self-medication 15 differences in economic characteristics in targeted populations. forty-three percent of the participants in this study said they rarely used antibiotics last year, while in eretria, a similar ratio of respondents self-medicated with antibiotics once or twice in the 12 months prior to the study by ateshim et al (25). being sick with a simple condition was the main reason behind antibiotics self-medication in the current study. this agreed with the findings of seam et al (22), zawahir et al (21), and ateshim et al (25), but was in contrast to the findings of a study conducted in afghanistan as cost and lack of time came first (31). the low income of the afghani population could be blamed for having cost come first as a driving force for self-medication. in our study, sore throat was the first indication for self-medicating with antibiotics and this was in agreement with the other studies (15,17,27,32–34). there were studies, however, which disagreed with our results; in an afghani study (31), cough was the main complaint for which antibiotics were used, in a bangladeshi study (22) fever was the major reason behind antibiotics self-medications, in eritrea (25) the antibiotics were mainly self-prescribed for skin wounds and in sri lanka (21), common cold and cough came first. although the indications for antibiotic self-medication seem different between countries, simple upper respiratory tract problems appear in the majority of the studies (15,17,21,27,31–35) regarding the factors affecting choosing antibiotics, participants in this study stated that own experience was one of the main factors affecting their choices, this agreed with other studies (17,22). the finding of this study concerning the basis of selecting a particular antibiotic were the type beside the indications which came in accordance with another study (31). this may give a clue that the public are educating themselves about drugs by reading the leaflets or asking the pharmacist, but such education should not be regarded as a good practice if it leads to self-medication. when asking the respondents about the source for the antibiotics community pharmacies came as the major source in accordance with other studies (21,22,31,36–38). a flaw in the laws governing the sale of antibiotics or defects in enforcing them is probably the reason why the public can obtain antibiotics from the pharmacy without a prescription. only 51 (16.8%) participants said that they never check the instructions of antibiotics before usage. the same percentage was recorded in afghanistan as 45 (16%) participants admitted that they never read the package (31). leaflets are put there for a reason, to inform the patients of the uses, dosage and potential side effects of the drug (39) and ignoring them may increase the already existing risk of selfmedication. when asking participants about the source of their information; the pharmacist was the main source in our study similar to another study (31), while previous experience came first in a study conducted in jordan (15). in this study, highest percent of the respondents (60%) assured that they never switch antibiotic they use and this is consistent with the findings of a nepali and indian studies (17,38). about 57% of the participants in this study assured that they never deliberately change the dose of antibiotics by themselves, the respondents who changed the dose of antibiotics admitted that the improvement in clinical condition was the main cause to adjust dose without prescription. this ratio was comparable to that reported by haque et al (11). switching antibiotics or changing their dosage during treatment without consulting a specialist add to the problems of self-medication such as mistreatment, bacterial resistance and adverse effects (40). about half the study sample expressed no concern at all in buying counterfeit antibiotics. although this practice may be attributed to the lower cost of such products, the actual cost may increase as the use of counterfeit drugs is associated with treatment failure and the development of resistance (41) disappearance of symptoms was the main reason why the respondents stopped self-medication with antibiotics in the current study followed by completing the course just like other studies (25,31,38). however, this disagrees with the findings of rajendran et al (17) who reported the completion of the course as the main reason. although it is widely known that a course of antibiotics should be completed even after the cessation of symptoms to avoid the development of resistance, recent reports suggest that this practice is not supported by solid evidence (42). in this study, about 73% of the participants said that they may have experienced some side effects due to the use of antibiotics. in contrast, pant et al (38) found that about 80% of the participants did not express any side effects. for those who suffered from adverse reactions, the majority from the two studies stopped the antibiotics course when this happened. regarding the opinion of the participants in this study about the practice of self-medication, 58.4% of them considered self-medication as an acceptable practice, and this result was less than other studies (17,38). the fact that the percentage in the current study was lower than other studies should not be used to lessen the negative impact of self-medication practice in our community. this is because the percentage is still too high (more than half of the population) and will increase to about 78% if the percentage of those believing that self-medication is a “good” practice was added. more than one third of the participants were confident that antibiotic self-medication will treat common infections. this ratio agrees with the findings of pant et al (38). one third of the population should not be regarded as insignificant since these people can affect the opinion and practice of their iraqi j pharm sci, vol.31(suppl.) 2022 antibiotic self-medication 16 family members and friends, expanding the problems of self-medication. limitations of the study the current study had some limitations. the cross-sectional design of the study measures each variable only once as both exposure and outcome are assessed at the same time and any causal relationship will need further assessment. the use of an online tool may also be viewed as a limitation since it confined the questionnaire to close-ended questions and eliminated the interviewer who could obtain more realistic responses from the participants. conclusions the findings of this study confirm that selfmedication with 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a systematic review. cureus. 2018 apr;10(4):e2428–e2428. 41. ajibola o, omisakin oa, eze aa, omoleke sa. self-medication with antibiotics, attitude and knowledge of antibiotic resistance among community residents and undergraduate students in northwest nigeria. diseases. 2018 apr;6(2):32. 42. langford bj, morris am. is it time to stop counselling patients to “finish the course of antibiotics”? can pharm j. 2017 oct;150(6):349–50. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin doi: https://doi.org/10.31351/vol30iss1pp258-269 258 the role of ginkgo biloba extract as monotherapy in improving the outcomes of patients with metabolic syndrome: a pilot comparative study with metformin tavga a. aziz*,1 *department of pharmacology and toxicology, college of pharmacy, university of sulaimani, kurdistan, iraq abstract the present study evaluates the effects of ginkgo biloba extract as monotherapy on the glycemic status, insulin resistance (ir), body mass index (bmi), and visceral adiposity index (vai), in addition to the inflammatory markers, oxidative status and leptin level in patients with metabolic syndrome in comparison with metformin. the study is a randomized, double-blind pilot study conducted during the period may to september, 2020. fifty patients were recruited in the study and they were allocated into two groups (25 per each group): ginkgo biloba and metformin groups, they received (120 mg ginkgo biloba extract/ capsule) and (500 mg metformin/ capsule) respectively; orally as a single dose for 90 days. blood samples were taken at zero time and after 90 days and utilized for analysis of blood glucose, hba1c, insulin and leptin levels, lipid profile, taos, hscrp, tnfα and il-6. additionally, hematological markers, liver and kidney function tests also measured. body mass index, waist circumference (wc), ir, and vai were determined at baseline and after 90 days of gkb extract and metformin treatment. ginkgo biloba significantly decreased ir (4.3±2 vs 8±6.4), (p=0.047), bmi (30.7±2.7 vs 31.5±2.2) (p<0.048), vai (183.7±101 vs 245.7±104.5), (p=0.036) and leptin level (4976±1803 vs 7317 ± 2807), (p=0.037) compared with baseline value. however, no significant decrease was observed on hba1c and insulin level. gkb also significantly decreased il-6 level (19.8±19 vs 28±22), (p=0.018) and tnfα level (130.6±33.7 vs 182.8±36.6), (p<0.001) with significant increase in hdl level (41.3±11.6 vs 30.7±4.8), (p=0.01) and taoc (52.8±27 vs 37±19.5), (p=0.01) compared to the baseline values. metformin led to a significant decrease (12.9±6 vs 27 ±19 µiu/ml) in insulin level (p= 0.032) and ir (6.7±5 vs 12.8±9), (p=0.039), with significant increase in hdl level (49.21±4.4 vs 38.08±3.8), (p=0.001) compared with the pre-treatment value. the use of 120mg gkb as monotherapy was effective in improving the outcomes of metabolic syndrome suggesting it as a good candidate to be used in the clinical setting with a larger sample size and for a longer period of time. keywords: ginkgo biloba, metabolic syndrome, glycemic status, visceral adiposity index, inflammatory markers. لمرضى المتالزمة االيضية: دراسة اولية و مقارنة مع احادي في تحسين النتائج كدواء ر مستخلص الجينكوبيلوبادو دواء الميتفورمين 1*،عزيز احمد ةفكتا العراق ،كردستان ،السليمانية جامعة ،الصيدلة كلية ، والسموم االدوية فرع* الخالصة لقد صممت هذه الدراسة لتقييم تاثير مستخلص الجينكوبيلوبا على مقاومة االنسولين و نسبة مؤشر كتلة الجسم ومحيط الخصر ومؤشر االيضية.السمنة الحشوية و مستوى االلتهابات وواالكسدة ومستوى الدهون لمرضى المتالزمة تم اعتماد طريقة الدراسة السريرية العشوائية المزدوجة. اجريت على خمسين مريض تم تشخيصهم بمرض المتالزمة االيضية حيث تم تقسيمهم ملغم من مستخلص 120عشوائيا الى مجموعتين )خمسة وعشرون مريضا لكل مجموعة( المجموعة االولى تم اعطائهم كبسولة تحتوي على ملغم من الميتفورمين, كل االدوية اعطيت عن طريق الفم كجرعة واحدة لمدة تسعين يوما في كلتا 500يلوبا والمجموعة الثانية اعطيت الجينكوب االضافة الحالتين. خالل فترة الدراسة تم تقييم كل من مؤشر كتلة الجسم ومحيط الخصر. تم قياس مستوى السكر و مستوى السكر المتراكم في الدم ب ى ى مستوى االنسولين ومؤشر مقاومة االنسولين و مستويات الدهون ومؤشر السمنة الحشوية ومؤشرات االلتهابات و مستوى اللبتين باالضافة الال ء.قياس مستوى القدرة المضادة لالكسدة وايضا تم تقييم سالمة المستخلص خالل تأثيره على وظائف كل من الكبد والكلى قبل وبعد اعطاء الدوا ظهرت النتائج بان مستخلص الجينكوبيلوبا تسبب في انخفاض ملحوظا في مستويات كل من مقاومة االنسولين و مؤشر كتلة الجسم ومؤشر السمنة ا أ الحشوية ومؤشرات االلتهابات كما وبينت عن ارتفاع ملحوظ في القدرة المضادة لالكسدة ومستوى الكولسترول الحميد نسبة الى نتائج وقت بد راسة.اسة. ومن الجدير بالذكر ان مستخلص الجينكوبيلوبا لم يؤثر بشكل سلبي على كل من الدم ووظائف الكبد والكلى بعد تسعين يوم من بدأ الدالدر كمكمل مستخلص الجينكوبيلوبا كدواء احادي اظهر فعالية جيدة في تحسين وضع مرضى المتالزمة االيضية مما يرجح اقتراحه استنتجت الدراسة ان غذائي للسيطرة على مرض المتالزمة االيضية. . مستوى االكسدة ،ومؤشر السمنة الحشوية ،مؤشرات االلتهابات ،مؤشر الجهد السكري ،المتالزمة االيضية،الكلمات المفتاحية: جينكوبيلوبا 1corresponding author e-mail: tavga.aziz@univsul.edu.iq received: 21/11/2020 accepted: 21/1 /2021 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp258-269 iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin 259 introduction metabolic syndrome (mets) is a debatable clinical body characterized by metabolic disturbances. the etiology of the disease contributes to several factors upon which genetic and environmental factors have a critical role in the disposition of the disease(1). there are some risk factors such as obesity, insulin resistance, dyslipidemia, and hypertension that contribute to the pathogenesis of the disease. improper management of mets may end up with cardiovascular events and type 2 diabetes (2, 3). obesity is considered pandemic and the prevalence has increased pronouncedly (4). large bodies of evidence proved the relation between body weight, hyperlipidemia, (5) metabolic syndrome (6) and type2 diabetes(4). in addition to the pivotal role of obesity in the development of insulin resistance (4, 7). weight reduction is the first nonpharmacological strategy in the management of mets, however people are not willing to adhere with a restricted low calories diet for a long period of time (1, 8, 9). additionally, some inflammatory markers are known to increase in patients with mets (10). however, the relationship between inflammation and metabolic syndrome are not clear. one of the theories that explain this link could be the fact that obese peoples with mets have high level of adipose tissue that contribute in the release of proinflammatory cytokines directly into the circulatory system (11) . the increase in the levels of the inflammatory markers do not resemble that of acute or chronic inflammation since it is not associated with infection or serious tissue damage, it is a low grade of inflammation mainly known as meta-inflammation( 12) . the multifactorial etiologies of mets render the management of the disease to be difficult because we need more than one medication to control the symptoms of the disease like hyperglycemia, hypertension, and dyslipidemia with truncal obesity (1, 13). polypharmacy is usually associated with increasing incidence of non-adherence, adverse reactions and drug interactions (14, 15). therefore, the search for new medication that possesses more than one mechanism to control the disease is of value. medicinal plants have a long history in the management of various metabolic illnesses (16), ginkgo biloba l. leaf extract (gkb extract), is among the nutraceuticals that gained great attention by the researchers because of its numerous biologically active constituents that may modify insulin action and/or production (17, 18). the plant is known for its antioxidant (19), anti-inflammatory (20) and hypolipedemic activity (21, 22). furthermore, ginkgo biloba proved to be effective in improving glycemic status and insulin sensitivity (23, 24). recently gkb was shown to be effective as add-on therapy with metformin in patients with t2dm and mets (25) , suggesting it as a good candidate to be tested as monotherapy in these diseases. accordingly, the present study was designed to evaluate the effect of gkb alone on the outcome of metabolic syndrome. patients, materials and methods patients the study protocol was approved by the ethical committee of the college of medicine/university of sulaimani (certificate no 507/1024), and has been approved in iranian registry of clinical trials with registration reference irct20200803048285n1. the study was carried out in accordance with the principles of the declaration of helsinki as revised in 2000 (a set of ethical principles regarding human experimentation developed for the medical community by the world medical association (26) . written informed consent was obtained from each participant prior to enrollment in this pilot study. materials ginkgo biloba extract, as a standardized powder (egb761), was obtained from apollo healthcare resources, singapore; metformin (met) tablets (500 mg, merck sante ҆ s.a.s., france) were obtained from the verified and licensed pharmacy. methods study design and patient treatment the study was a randomized, doubleblinded pilot study conducted between may to september 2020 at the center of diabetes and endocrine glands, directory of health/ sulaimani city. the patients were recruited from public hospitals and private clinics according to the selection criteria. according to the inclusion criteria, patients of both sexes with the age range of 25-65 years, diagnosed by a specialist physician as having metabolic syndrome depending on the guideline of metabolic syndrome (27). the exclusion criteria included pregnancy, ischemic heart disease, cardiac arrhythmias, glucose-6-phosphate dehydrogenase (g6pd) deficiency, bleeding disorders, seizures, and known hypersensitivity to any component of the trial drugs (gkb extract and metformin). moreover, patients on supplements that contain multivitamins and polyphenols were also excluded. sixty patients were originally screened for eligibility; only 50 were eligible and randomized into two groups (25 patients each) as follows: metformin (500 mg tablet) group and gkb group received gkb extract (120 mg/capsule) as a single dose for 90 days. the gkb capsules were prepared in the laboratory of pharmaceutics, college of pharmacy, university of sulaimani. patient followup was performed on monthly base to ensure patient compliance. iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin 260 unfortunately, only 39 patients; 19 patients from the first group and 20 patients from the second group were completed the study and included in the final data analysis (figure 1). figure 1. flowchart shows the screening, recruitment and randomization of patients. anthropometric outcomes anthropometric measures were evaluated at baseline and after 90 days at the end of the treatment. height and weight were measured by an electronic scale and a wall-mounted stadiometer. waist circumference was measured by a tape measure. each measurement was taken twice, and the average values were recorded. in addition, the body mass index (bmi) was calculated according to the following formula: bmi = weight (kg)/height2 (m2) (28). visceral adiposity index (vai) was also measured. the vai is an empirical mathematical model, which is gender specific and based on simple anthropometric (bmi and wc) and functional parameters (tg and hdl-c), which is an indicator of body fat distribution and function. calculation of vai was according to the formula given by amato et al (29) . the formula is a linear equation derived by extrapolation from the relationship between bmi and wc in a healthy normal/overweight population. distribution mode of adipose tissue was corrected for tg and hdl-c levels to determine the vai as follows: female vai = (wc/36.58 + (1.89 × bmi)) × (tg/0.81) × (1.52/hdl) male vai = (wc/39.68 + (1.88 × bmi)) × (tg/1.03) × (1.31/hdl) where wc is expressed in cm, bmi in kg/m2, tg in mmol/l, and hdl in mmol/l. biochemical and hematological tests patients were informed to be fasted for exactly 12 h, then about 10 ml blood was taken from each patient at zero time (before starting treatment) and after 90 days’ treatment by vein puncture. from which, about 2.0 ml was drawn in edta containing tubes and utilized for analysis of hematological markers fasting blood glucose (fbg) 30, and hba1c 31 using colorimetric methods (roche-cobas c 311, roche diagnostics gmbh, mannheim, germany). insulin resistance calculated using homa-ir (32). the remained 8.0 ml was drawn in plain tubes and left to clot, then centrifuged at 3000 rpm for 20 min to obtain serum. the serum was stored at –20oc unless analyzed immediately. the serum insulin content was measured using an immunoassay method 33 (roche-cobas e 411; hoffman-la roche ltd.). the serum was used for analysis of leptin 34, total lipid profile 35 using colorimetric methods (roche-cobas c 311, roche diagnostics gmbh, mannheim, germany). tnfα and il6 were measured using human tnfα and human il6 elisa kit respectively by a colorimetric method 36, 37 (chromate, awareness technology, inc., palm city, fl, usa) and hscrp measured using the elisa kit 38. the serum was also used for the analysis of total antioxidant capacity using total antioxidant capacity (t-aoc) assay kit by a colorimetric method 39 (visible spectrophotometer 721, china). liver (40) and kidney functions test (41, 42) and hematological markers (43) were determined calorimetrically using ready-made kits (randox, london, uk) according to the manufacturer’s instructions. statistical analysis the statistical analysis was performed using the graphpad prism 5.1 software (graphpad software, inc., la jolla, ca, usa). descriptive statistics was utilized to compare the patient’s characteristics between the two groups. paired t-test was utilized to evaluate the difference between the pretreatment mean and post treatment mean of the same group. unpaired t-test was utilized to evaluate the differences between post treatment mean of the different groups. two-way analysis of variance, supported by bonferroni’s post hoc analysis, and analysis of covariance were used to determine the difference between the mean of independent samples at p-value < 0.05. results the baseline data of the mets patients were shown in table 1. there were no statistically significant differences (p>0.05) in all parameters between the metformin and gkb groups including the age, gender, weight, wc, bmi and hba1c. after 90 days of treatment, fbg levels of both gkb treated group and metformin treated group were non-significantly decreased (125±17 mg/dl vs 126 ±30) and (135±11 mg/dl vs 141 ±17) respectively, (p>0.05) compared with baseline values (figure 2 a). regarding the effects on insulin, 90 days’ treatment with gkb extract non-significantly decreased (20.6±17 µiu/ml vs 36.7 ±28) level (p= 0.07) compared with baseline values, while treatment with metformin led to a significant decrease (12.9±6µ iu/ml vs 27 ±19) in insulin levels compared with baseline values (p= 0.032) (figure 2 b). concerning the level of hba1c, gkb treated iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin 261 group produced a non-significant decrease (7%±0.8% vs 7.2%±1%) compared to the baseline value (p=0.6) with no significant change produced by metformin (figure 2 c). regarding insulin resistance, there was a significant decrease after 90 days of treatment with each of gkb extract and metformin compared with the pre-treatment value (4.3±2 vs 8±6.4), (6.7±5 vs 12.8±9), (p=0.047) and (p=0.039) respectively (figure 2 d). m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 5 0 1 0 0 1 5 0 2 0 0 f a s ti n g b lo o d g lu c o s e ( m g /d l) a m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 2 0 4 0 6 0 8 0 s e ru m i n s u li n ( u u /m l) * b m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 2 4 6 8 1 0 h b a 1 c ( % ) c m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 1 0 2 0 3 0 in s u li n r e s is ta n c e a a ,b a , c a * * d figure 2: effect of gingko biloba extract (gkb) on the serum level of (a) fasting glucose (b) insulin (c) hba1c (d) insulin resistance of patients with metabolic syndrome. values were presented as mean ± s.d; * significantly different compared with baseline values (paired t-test, p<0.05). values with non-identical letters (a, b, c) were significantly different among each other (anova, p<0.05). table 1. baseline characteristics of the randomly allocated metabolic syndrome patients parameters metformin n=19 gkb n=20 p value age (yr) 44.15±9.5 45.7±7.8 0.25 male (%) 4 (21%) 5 (25%) 0.42 weight (kg) 79.46± 13.46 80.7±17 0.18 bmi (kg/m2) 32 ±7 31.5±2.2 0.56 wc (cm) 97.6±10.2 102.2±5.5 0.23 hba1c (%) 7±0.95 7.2±1 0.68 p-value consider significant if p<0.05. iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin 262 figure 3 a clearly shows that the use of 120mg gkb extract, as a single oral dose for 90 days, significantly decreased bmi (30.7±2.7 vs 31.5±2.2) (p<0.048) compared with baseline values, while administration of metformin resulted in a nonsignificant decrease (32.4±6.9 vs 32.8±7) in bmi compared with baseline values. moreover, gkbtreated patients showed a non-significant decrease in waist circumference values (99.6±5.5 vs 102.2±5.5) (p=0.06) compared with baseline values, while metformin did not significantly affect this parameter after 90 days (figure 3 b). regarding the effect on visceral adiposity index (vai), (figure 3 c) shows that in gkb treated group, there was a significant decrease (183.7±101vs 245.7±104.5) in vai after 90 days of treatment compared with the baseline value (p=0.036); meanwhile, a non-significant decrease (158±77.8 vs 195.6±145.5) in vai was observed after 90 days of treatment with metformin (p=0.26) compared with the pre-treatment value. gkb also resulted in a significant decrease (4976±1803 vs 7317 ± 2807) in the level of leptin after 90 days’ treatment (p=0.037); compared with the pretreatment value with no significant change (6169±2414 vs 7176 ± 2812) observed with the use of metformin (p=0.22) (figure 3 d). m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 1 0 2 0 3 0 4 0 5 0 b m i (k g /m 2 ) * a m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 5 0 1 0 0 1 5 0 w a is t c ir c u m fe re n c e ( c m ) b m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 1 0 0 2 0 0 3 0 0 4 0 0 v is c e ra l a d ip o s it y i n d e x * c m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 5 0 0 0 1 0 0 0 0 1 5 0 0 0 s e ru m l e p ti n ( n g /m l) * d figure 3. effect of gingko biloba extract (gkb) on the serum level of (a) bmi (b) waist circumference (c) vai and (d) leptin of patients with metabolic syndrome. values were presented as mean ± s.d; * significantly different compared with baseline values (paired t-test, p<0.05). after 90 days of treatment total cholesterol, triglyceride and serum ldl were non-significantly changed with each of gkb extract and metformin compared with the pre-treatment value (figure 4 a, b and c). while serum hdl levels was significantly increased after 90 days of treatment with each of gkb extract (41.3±11.6 vs 30.7±4.8), (p=0.01) and metformin (49.21±4.4 vs 38.08±3.8), (p=0.001) compared with the pre-treatment value (figure 4d). m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 1 0 0 2 0 0 3 0 0 s e ru m t o ta l c h o le s te ro l (m g /d l) a m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 1 0 0 2 0 0 3 0 0 s e ru m t ri g ly c e ri d e s ( m g /d l) b iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin 263 m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 5 0 1 0 0 1 5 0 2 0 0 s e ru m l d l ( m g /d l) c m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 2 0 4 0 6 0 8 0 s e ru m h d l ( m g /d l) * * d figure 4. effect of gingko biloba extract (gkb) on the serum level of (a) total cholesterol (b) triglycerides (c) ldl and (d) hdl of patients with metabolic syndrome. values were presented as mean ± s.d; * significantly different compared with baseline values (paired t-test, p<0.05). regarding tnfα there was a significant decrease after 90 days of treatment with gkb extract (130.6±33.7 vs 182.8±36.6), (p<0.001) compared with the pre-treatment value and (130.6±33.7 vs 181.3±74), (p<0.05) compared with metformin treated group (figure 5 a). while the use of 500 mg metformin produced no significant change compared with the baseline value. ninety days’ treatment with gkb extract also produced a significant decrease in the level of il6 (19.8±19 vs 28±22), (p=0.018) compared with the pre-treatment value with non-significant decrease produced by group treated with metformin (figure 5 b). moreover, hs-crp was non-significantly decreased in gkb treated group and non-significant increase produced by metformin treated group (figure 5 c). serum taoc was significantly increased after 90 days of treatment with gkb extract (52.8±27 vs 37±19.5), (p=0.01); compared with the pretreatment value. m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 1 0 0 2 0 0 3 0 0 s e ru m t n f - ( p g /m l) * a a a b a m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 2 0 4 0 6 0 s e ru m i l -6 ( p g /m l) * b m e t f o r m in 0 m e t f o r m in 9 0 g k b 0 g k b 9 0 0 5 1 0 1 5 s e ru m h s c r p ( m g /l ) c figure 5. effect of gingko biloba extract (gkb) on the serum level of (a) tnf-α (b) il6 (c) hs-crp of patients with metabolic syndrome. values were presented as mean ± s.d; * significantly different compared with baseline values (paired t-test, p<0.05).values with non-identical letters (a,b) were significantly different among each other (anova, p<0.05). iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin 264 metformin also increased the level of taoc however it was statistically not significant (figure 6). all the biochemical analyses regarding the liver functions indicated no alteration of values after 90 days of gkb treatment (table 2), except serum alp where significant decreases were reported in each of gkb and metformin treated groups compared with baseline values (p=0.02) and (p<0.001) respectively. for the kidney function tests; serum creatinine levels were significantly elevated compared with baseline values in the metformin-treated group (p=0.014) but still within the normal range, with no significant change in blood urea level in both treated groups (table 3). regarding the hematological markers; (table 4) reveals significant increase (p=0.038) in hb concentration in the gkb-treated patients after 90 days compared with baseline values. meanwhile, platelet count significantly decreased in gkb treated group (p=0.014) and metformin treated group (p=0.011). m e tf o r m in 0 m e tf o r m in 9 0 g k b 0 g k b 9 0 0 2 0 4 0 6 0 8 0 1 0 0 s e r u m t a o c ( u /m l) * figure 6. effect of gingko biloba extract (gkb) on the serum level of taoc of patients with metabolic syndrome. values were presented as mean ± s.d; * significantly different compared with baseline values (paired t-test, p<0.05). table 2. effect of gingko biloba extract (gkb) on the liver function markers of patients with metabolic syndrome. parameters metformin (n=19) gkb (n=20) baseline after 90 days baseline after 90 days serum ast (u/l) a20.21±7.25 a19.12±4.82 a21.23±7.1 a21.39±5.4 serum alt (u/l) a17.11±8.2 a15.7±5.2 a24.57±13.5 a23.15±8.8 serum alp (u/l) a75.4±25.5 a* 64.46±20 a86.7±24.8 a*75.8±21.9 values were presented as mean±s.d; n: number of patients; * significantly different compared with baseline values (paired t-test, p<0.05); values with different superscripts (a,b) within each parameter were significantly different (anova, p<0.05). table 3. effect of gingko biloba extract (gkb) on the renal function markers of patients with metabolic syndrome. parameters metformin (n=19) gkb (n=20) baseline after 90 days baseline after 90 days serum urea (mg/dl) a25±6.5 a6±8.4. 25 a27.44±7.7 a29.56±7.7 serum creatinin (mg/dl) a0.65±0.12 a*0.75±0.12 a0.64±0.17 a0.7±0.17 values were presented as mean±s.d; n: number of patients; * significantly different compared with baseline values (paired t-test, p<0.05); values with different superscripts (a,b) within each parameter were significantly different (anova, p<0.05). table 4. effect of gingko biloba extract (gkb) on the hematological markers of patients with metabolic syndrome. parameters metformin (n=19) gkb (n=20) baseline after 90 days baseline after 90 days hb (g/dl) 12.9±1.2 13.4±1.5 13.3±1.2 13.9±1.3* hct (%) 37.9±3.8 39.4±4.6 38.9±4.6 40.8±4.1 rbc countx106 (cells/μl) 4.4±0.5 4.6±0.5 4.7±0.58 4.9±0.4 wbccount x103 cells/μl 8.5±2.3 8.1±1.6 8.1±1.8 7.6±1.7 platelets count x109 cells/l 257±50 223±57* 237±43 201±40* values were presented as mean±s.d; n: number of patients; * significantly different compared with baseline values (paired t-test, p<0.05). iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin 265 discussion the first step in the treatment of metabolic syndrome is modification of life style via avoiding sedentary life style and this includes decreasing food intake and increasing physical activity, however unfortunately only a few patients are willing to modify their life style without the aid of medications. in the present study, gkb for the first time has been used as a monotherapy in patients with mets; and it was effective in modifying and improving some of the components of mets such as insulin resistance, bmi, vai and hdl in addition to the inflammatory markers and the antioxidant status. similarly, many studies have shown the effectiveness of gkb in improving insulin sensitivity (23, 44, 45). the glycemic status have been improved by the use of gkb in the current study however, the change was statistically not significant which could be attributed to the small dose and/or the short period of treatment, additionally, the exact effect of the plant cannot judge on such a small sample size. moreover, it was effectively enhanced insulin sensitivity and this finding was in tune with other studies (44, 24). obesity secondary to high fat food produces alterations in the regulation of peripheral metabolism and food intake, subsequently decreasing insulin sensitivity, enhancing weight gain, and other metabolic disorders (46, 47). in many countries, obesity is considered as a major health problem and the primary goal is how to decrease the prevalence of obesity (48), and in spite of the efforts for minimizing obesity, still non-pharmacological interventions are inadequate to achieve satisfaction (9, 49, 4), and because of the high risk associated with obesity in developing insulin resistance (5); additional medications are required to accomplish the necessity in the treatment of obesity. nutraceutical recognized from traditional medicinal plants may represent a good choice for the development of new medications targeting obesity. in the current study, gkb extract successfully decreased bmi and wc; and this effect could be attributed to the terpenoid component of the plant that have the ability to inhibit the activity of pancreatic lipase (pl), which may in part give some clues about the reduction of body fat mass. moreover, gkb was found to significantly inhibit pl (50), which may attribute to the reduction of bmi in the current study. while metformin did not affect bmi and wc which could be due to the small dose used in the study and relatively short period of treatment (51). gkb was effective in decreasing vai and this finding was consistent with other study which demonstrated that prolonged treatment with gkb stimulated a noticeable visceral adiposity loss, improvement of insulin sensitivity via stimulation of insulin signaling cascade in gastrocnemius muscle (52). another parameter that screened in this study is leptin; leptin is increasingly being involved in the etiology of metabolic disorders. it is one of the vital hormones expressed by the adipose tissue, and it has a critical role in regulating food consumption and energy production through its action on the hypothalamic nucleus. it has been reported that leptin shares the same signaling pathway with insulin.(53, 54) mutation in leptin or its receptor results in improper leptin signaling and eventually increases food consumption and attenuates energy liberation in humans and experimental animals in spite of obesity (55). in the present study, gkb significantly decreased the serum levels of leptin and the exact mechanism of such finding is unclear. however, the previously reported decrease in visceral adiposity (18), could be of value in explaining the changes in serum leptin. additionally, gkb might exert a positive antiinflammatory effect on the hypothalamus, with a consequent reduction of the levels of orexigenic peptide and/or increasing anorexigenic peptide levels, which may promote weight loss via suppressing the appetite (24). in the present study, gkb as monotherapy in patients with mets was effective in modifying and improving some of the components of mets such as hdl and metainflammation. many studies have shown the effectiveness of gkb in improving glycemic status (23, 44, 45), and ameliorating the inflammatory response (56, 24). recently, the role of the oxidative stress and the pro-inflammatory mediators have been proved in the pathogenesis of insulin resistance; hence, attenuating the process of the inflammatory response is one of the therapeutic strategies to prevent the development and progression of insulin resistance (57). the antiinflammatory effect of gkb demonstrated through attenuating the inflammatory markers; the study showed a pronounced decrease in the level of tnfα which demonstrates a significant change with the baseline value and with the group treated with metformin. gkb also produced a significant decrease in the serum level of il6 compared to the baseline value. this anti-inflammatory effect could be attributed to the modulating effect on the expression of many inflammatory mediators and the ability of the plant to downregulate nitric oxide level and prostaglandin e2 formation along with decreasing proinflammatory cytokines and upregulating nf-kb factor (24, 58, 59). iraqi j pharm sci, vol.30(1) 2021 ginkgo biloba extract and metformin 266 the relationship between oxidative stress and inflammation is greatly documented (60, 61). evidence from many studies proved the role of reactive species in the pathology of many chronic inflammatory diseases (62, 63). among the inflammatory cytokines that modulate the inflammatory response is tnf-α which has a pivotal role in the generation of reactive species and enhance the expression of other inflammatory cytokines. targeting tnf-α may participate in the downregulation of the inflammatory responses (64). the current study revealed a potent antioxidant capacity exhibited by gkb; many mechanisms are proposed for this effect such as chelation of transition metals, scavenging of reactive oxygen species and enhancing the production of antioxidant molecules (65). furthermore, gkb has been reported to attenuate oxidative damage in previous studies (19’ 65). moreover, the plant exerted no deleterious effect on the liver, kidney and the hematological markers. taking all these findings together suggests gkb as a good candidate to be tested on a larger sample size of mets patients and for a longer period of time to explore the exact beneficial effect of the plant in this respect. limitations the major limitations of this study are the small sample size and the relatively short duration of treatment. therefore, future studies are warranted to determine the long-term effect of gkb extract by following up a larger study population. conclusion the use of 120mg gkb as monotherapy was effective in improving the outcomes of metabolic syndrome through decreasing insulin resistance, bmi, vai, and leptin levels, and it was also effective in decreasing the inflammatory markers and increasing the antioxidant capacities in comparison with metformin suggesting it as a good candidate to be used in the clinical setting with a larger sample size and for a longer period of time. acknowledgments the author appreciates the college of pharmacy, university of sulaimani, and the center of diabetic and endocrine glands for their support and for providing the facilities to complete this project. disclosure the author reports no conflicts of interest in this work. references 1. srikanthan k, feyh a, visweshwar h, shapiro 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http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions doi: https://doi.org/10.31351/vol29iss1pp41-54 41 formulation and investigation of lacidipine as a nanoemulsions rajaa a. dahash*,1 and nawal a. rajab** *ministry of health and environment, baghdad **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract lacidipine (lcdp) is a calcium-channel blocker with low aqueous solubility and bioavailability. nanoemulsion (ne) is one of the popular methods that has been used to solve the solubility problems of many drugs. lcdp was formulated as a ne utilizing triacetin as an oil phase, tween 80 and tween 60 as surfactants and ethanol as a co-surfactant. nine formulas were prepared, and different tests performed to ensure the stability of the nes, such as thermodynamic stability, particle size, polydispersity index, zeta potential, dilution test, conductivity test, drug content, viscosity and in-vitro drug release. results of characterization showed that lcdp ne (f-5) using triacetin, tween80 , ethanol and ddw in a ratio of (10:60:30) was selected as the best formula, since it has excellent thermodynamic stability with a particle size of 13.42, low pdi 0.234 , zeta potential (14.5mv), good dilution without drug precipitation , efficient electrical conductivity 0.241ms/cm , higher percent of drug content (99.14%) with acceptable viscosity, and complete release of the drug after (30 min.) with significantly higher (p<0.05) dissolution rate in comparison with pure drug powder. the selected formula (f-5) subjected to further investigations as drug and excipient compatibility study by fourier transform infrared spectroscopy (ftir) and high performance liquid chromatography (hplc) and atomic force microscope (afm) . the outcomes of the (ftir) explain that the distinctive peaks for lcdp were displayed the same functional group's band with very slight shifting, which suggests the presence of hydrogen bonding. this indicates that there was no interaction between lcdp and other ne components, while the hplc demonstrated that was no change in retention time and no extra peaks reported. therefore, these excipients were found to be compatible with lcdp. in conclusion, the ne was found to be an efficient method to enhance the solubility and dissolution rate of drugs that have poor water solubility (lipophilic drugs). keyword: lacidipine, , triacetin, tween 80, tween60 , nanoemulsion . صياغة وتوصيف الالسيدبين كمستحلب نانوي ** نوال عياش رجب و *رجاء عباس دهش العراق ،بغدادوزارة الصحة والبيئة، * جامعة بغداد، العراق الصيدلة، الصيدالنيات، كليةفرع ** الخالصة عقبة رئيسية تقيد استخدامها في اصبحتلعديد من المركبات الصيدالنية الفعالة لديها مشاكل في الذوبان حتى اآلن، والتي ان ا منخفض جدا. ان عقارالالسيدبين هو مانع لقنوات الكالسيوم له ذوبان مائي وتوافر حيوي. المستحضرات الصيدالنية المستحلب النانوي هو واحد من االنظمة الشائعة وبان وذابة لمثل هذه الجزيئات القليلة الذج هي صيغ دوائية ان الصيغ الصيدالنية الدهنية 80، توين كزيتباستخدام ثالثي األسيتامين لقد تم تصييغ الالسيدبين كمستحلب نانوي. التي تم استخدامها لحل مشاكل الذوبان في العديد من األدوية تم تحضير تسعة تركيبات سائلة وأجريت لها اختبارات مختلفة لضمان ثبات المستحلب .واإليثانول كمضاد للشد السطحي كخافض للسطح 60وتوين تقدير ،اختبار الموصلية الكهربائيةالنانوي مثل االستقرار الديناميكي الحراري قياس حجم القطيرات، مؤشر التشتت، جهد زيتا، اختبار التخفيف، والتي تحتويلمستحلب الالسيدبين 5لقد أظهرت نتائج التوصيف أن التركيبة رقم . في المختبر ومستوى تحرر العقار توى اللزوجةمس محتوى العقار، oil: smix : ddw جهد زيتا0.234)مؤشر التشتت ) نانومتر، (13.42) لها حجم قطيرةصيغة كأفضل (10:60:30) بنسب ، (mv 14.5 (%99.14) ءمن محتوى الدوا عالية، نسبة مئوية (سم /مللي 0.241)، عدم ترسب الدواء عند اجراء اختبار التخفيف، الموصلية الكهربائية الفعالة ( ( قد f-5)الصيغة المفضلة .عند المقارنة مع مسحوق الدواء النقي( >p 0.05) واعلى بكثير دقيقة 30وتحرر العقار الكامل بعد لزوجة مقبولة مع المجس و ((hplc( وftirبواسطة الفحص باألشعة تحت الحمراء ) المكونات االخرىدراسة التوافق مع مثلخضعت لمزيد من االختبارات النانوي والمكونات األخرى. لمستحلباأن القمم المميزة للـعقار لم تتأثر وهذا يشير إلى وجود توافق بين (ftir) ، حيث يوضحااللكتروني الماسح أنه لم يكن هناك تغيير في وقت االستبقاء وليس hplcبينما أثبت فحص .العقار ومكونات الصيغة المفضلة عدم وجود تفاعل بينمما يؤكد على (f5) المحددة للصيغة .المجس االلكتروني الماسح هناك أي قمم إضافية. لذلك تم التأكد من توافق العقار مع مكونات المستحلب النانوي االخرى من النتائج التي تم الحصول عليها تبين ان المحلول النانوي وسيلة فعالة لزيادة قابلية الذوبان .تجميع دون صغير الكروية الجسيمات ان حجم اثبت لكثير من االدوية. لعقار )الالسيديبين( وبهذا يمكن اعتباره طريقة جيدة لتحسين الذوبانية المائية .مستحلب نانوي ،60توين ،80توين ،ترايستين ،: الالسيدبين الكلمات المفتاحية : 1corresponding author e-mail: fatima.yousef2022@gmail.com accepted: 16/ 9 / 2020 received: 6 / 5 /2019 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp41-54 iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 42 introduction oral delivery of drugs is regarded as the optimal route to achieve therapeutic and prophylactic effects against various diseases, especially chronic conditions. it may have poor bioavailability as a hurdle, leading to challenges for pharmaceutical manufacturers to design delivery system(s) that can provide improved pharmacokinetic profiles and hence therapeutic responses (1,2). the term nanoemulsion (ne) refers to a thermodynamically stable isotropically clear dispersion of two immiscible liquids, such as oil and water, stabilized by an interfacial film of surfactant molecules. their size varies from 10 to 1000 nm. (3,4). lacidipine (lcdp) is a dihydropyridine calcium-channel blocker developed for oral administration .it used in the treatment of hypertension and atherosclerosis and possessed an antioxidant effect (5). chemical name of lcdp is (e)-4-[2-[3-(1,1dimethylethoxy) -3-oxo-1-propenyl] phenyl] 1,4-dihydro -2,6-dimethyl -3,5-pyridine dicarboxylic acid diethyl ester, the molecular weight (455.5) and pka 2.5 , log p (octanol/water) is 5.51,the powder is a white to pale yellow powder, melt at 178°c (6,7) , and the chemical structure is shown in figure(1) (8) . figure 1. the chemical structure of lcdp (8) lcdp poorly absorbed from the gastrointestinal tract after oral doses and undergoes extensive first-pass metabolism the bioavailability has been reported to be 2 to 10%. the rate limiting step for drug absorption in this class is dissolution (9). this study aims to prepare lcdp as a nanoemulsion to improve its dissolution rate. materials and methods materials lcdp was obtained from baoji guokang bio-technology co., ltd, china, triacetin was purchased from hyper-chem ltd co, china, tween 20 obtained from himedia chemicals, india, tween 60 was obtained from avonchem, england, tween 80 was purchased from riedel-de-haen, germany, ethanol was purchased from sigma-aldrich, germany, hydrochloric acid was purchased from thomas baker, india . all other chemicals were of analytical grade. methods characterization of lcdp differential scanning calorimetry analysis (dsc) the dsc study was performed for the pure drug to evaluate the thermotropic properties and thermal behavior of the lcdp. approximate weighed samples (about 3 mg) were put in sealed aluminium pans and warmed at a scanning rate of 10°c/min (11). saturation solubility study of lcdp saturated solubility of lcdp was estimated in various oils, surfactants, cosurfactants and dissolution media. the measurement of solubility was done as follows: the excess amount of lcdp was added to (5 ml) of each selected individual oils, surfactants and co-surfactants contained in stoppered vials separately, then shaken utilizing a water bath shaker at 25±1°c for 72 hours to prepare a saturated solution. after accomplishing the equilibrium, the mixtures were centrifuged at 3000rpm for 15min, followed by filtration through a 0.45micrometer millipore filter. samples were suitably diluted with ethanol and analyzed by uv/vis spectrophotometer at λ max of lcdp. the measurements were done in triplicate (12,13 ). construction of pseudo-ternary phase diagrams the aqueous titration method was utilized to construct the pseudo-ternary phase diagram. based on the solubility studies, triacetin was selected as an oil phase, tween 80 and tween 60 were selected as surfactant and ethanol were selected as a co-surfactant, and deionized water (ddw) used as an aqueous phase. the oil: surfactant:co-surfactant (smix) mixed at different ratios ranging from (1:9 to 9:1). smix ratios was 1:3, 1:2, 1:1, 2:1and 3:1 for smix (tween 80/ethanol) and 1:2, 1:1, 2:1 and 3:1 for smix tween 60/ethanol (14,15) . preparation of lcdp nanoemulsion different o/w ne formulations (table 1) were prepared using the smix and oil concentrations according to pseudo-ternary phase diagrams; primary lcdp emulsion was prepared via dissolving (2 mg) of the drug in the selected oil. the magnetic stirrer used to ensure complete mixing then the selected smix added slowly in a fixed ratio till the clear solution was obtained. iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 43 ddw added dropwise to the clear solution with the continuous stirring at (~500 rpm ) at room temperature until the formation of a clear emulsion. after that, the prepared emulsions were ultrasonicated via utilizing a 20 khz sonicator for 10 min (16,17). table 1. composition of lcdp nanoemulsion ne-f triacetin %w/w surfactant cosurfactant smix ratio smix % w/w ddw %w/w f-1 10 tween 80 ethanol 1:3 60 30 f-2 10 tween 80 ethanol 1:2 60 30 f-3 10 tween 80 ethanol 1:1 60 30 f-4 10 tween 80 ethanol 2:1 60 30 f-5 10 tween 80 ethanol 3:1 60 30 f-6 10 tween 60 ethanol 1:2 60 30 f-7 10 tween 60 ethanol 1:1 60 30 f-8 10 tween 60 ethanol 2:1 60 30 f-9 10 tween 60 ethanol 3:1 60 30 lcdp nanoemulsion characterization visual transparency optical observation for ne formulas was intent utilizing good light source for transparency and flow ability (18). thermodynamic study i. centrifugation study: in this study, formulas were centrifuged at 5000 rpm for 30 min and then checked for instability such as phase separation. the formulations that did not show any signs of instability were chosen for heatingcooling cycle (19). ii. heating-cooling cycles test: stability of ne depends on the variation of temperature was studied by heating-cooling cycle. formulations subjected to six cycles between refrigerator temperature 5 °c and at 50 °c storage at each temperature for not less than 48 hours (20). iii. freezing–thawing test: this test was done by exposing the formulations for two different temperatures which are (-21°c) and (25°c) using refrigerator and the time for each temperature not less than 24 hours (21). droplet size measurement the droplet size of ne was established by analyzing the fluctuations in light scattering due to the brownian motion of the particle utilizing dynamic light scattering technique (zetasizer nano zs, malvern, uk) (22). polydispersity index measurement (pdi) the estimation of the (pdi) gives information about the uniformity of droplet size within the formulated ne. the lower pdi value (near zero) indicates a monodisperse droplet population (23). zeta potential measurement (ʓ – potential) the droplet charge (zeta potential) of the nes was determined to utilize dynamic light scattering technique, zeta potential was believed to be sufficient for ensuring the physical stability of nes (24). dilution test the aqueous dilution test was performed, one ml of each ne formulas (f1f9) diluted to 50 ml,100 ml and 500 ml with distilled water at 25ºc with constant stirring at 50 rpm and observed visually for turbidity, clarity and phase separation (25). conductance measurement the o/w nes are highly conducting since the water is the external phase, whereas w/o nes are not conducting as they have water in the internal phase (26). drug content estimation accurately 10 ml of each ne formula which contains (2 mg) was diluted with ethanol to the sign (100 ml) in a volumetric flask and subjected to the centrifugation for 15 minutes (3000 rpm), then filtered using 0.45 μm filter syringe and suitably diluted. determination of the contents of lcdp nes via utilizing uv/vis spectrophotometer at the selected λ max (27,28). viscosity measurement determination of viscosities determines whether the system is o/w or w/o emulsion (29). iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 44 in vitro drug dissolution study the in vitro release of lcdp loaded ne occurs using usp dissolution apparatus type–ii. dialysis bag (molecular cut off 12000da) was utilized. ten ml of each formula which contain 2mg of lcdp was put in the bag, and this bag was immersed in 500 ml of dissolution medium. the rotation speed was 50 rpm, and the dissolution medium was 0.1n hcl with 1% tween 20 at 37 ± 0.5 °c (30). samples (5 ml) were withdrawn at a regular time intervals (5,10,15, 20,30, 40,50 and 60 min) from the dissolution medium and the samples then filtered by using through a 0.45 μm filter syringe and were analyzed by uv/vis spectrophotometer at the λ max. of the drug (31). selection of the optimum formula the choice of the optimum formula was accomplished, and this achieved according to the globule size analysis, pdi, zeta potential measurements, electrical conductivity, viscosity, drug content and in vitro release studies. evaluation of the selected lcdp optimum formula drug and excipient compatibility study by ftir to demonstrate any possible interaction between the drug and the utilized excipients in the selected formula. samples were mixed with potassium bromide and pressed in the form of a disc; ftir spectroscopy analyzed the disc from 4000-400 cm-1 (32) . validation of the hlpc method hplc method used in the investigation and to determine the possible interactions between oil, drug and other excipients. a waters hplc system used which was equipped with a spa-20a detector. the system was controlled through breez software. the mobile phase which consisted of acetonitrile: water (65:35%v/v) with a flow rate of 1ml/min at ambient temperature and the injection volume was 10 µl. the detective wave length was set at 239 nm. the mobile phase was filtered through (0.45µm) in millipore solvent filtration apparatus before use (33). atomic force microscopy (afm) study the afm is capable of scanning the surfaces in controlled environmental conditions and can measure the particle size of nanoparticles accurately. afm confirmed the size and surface morphology of lcdp nanoparticles after drying of the selected formulations. droplets of optimized formulas were deposited on freshly cleaved mica and dried 15 minutes in the oven by the droplet evaporation technique. particle size, 3ddimension graph, and a histogram of particle size distribution were obtained (34). results and discussion differential scanning calorimetry analysis (dsc) pure lcdp powder showed a characteristic endothermic peak at (185.80 °c °c), such sharp endothermic peak signifies that drug used was in a pure crystalline state and it was near the reported one (35), as shown in figure 2 . figure 2. differential scanning calorimetry thermogram of lcdp. saturation solubility study of lcdp as demonstrated in the table (2), higher solubility of lcdp was in triacetin while the lower solubility was in liquid paraffin. to ensure the drug is solubilized form, triacetin was utilized in the formulations, no precipitation of drug will occur since lipophilic drugs can be easily present in a solubilized form (36,37). regarding surfactants, tween 80 and tween 60 were chosen as a surfactant to obtain a one-phase clear solution (38). ethanol was found to have a higher solubilizing capacity for lcdp (39). iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 45 table 2. saturation solubility study of lcdp sd solubility(mg/ml) oil ±0.65 32.139 coconut oil ±0.27 15.224 corn oil ±0.32 4.165 grape seed oil ±0.48 6.175 lavender oil ±0.89 1.281 liquid paraffin ±0.95 58.126 oleic acid ±0.65 16.366 olive oil ±0.85 6.156 sunflower oil ±0.78 63.137 triacetin surfactant ±0.53 18.221 propylene glycol ±0.78 41.156 tween 80 ±0.85 39.187 tween 60 ±0.89 35.043 tween 20 co-surfactant ± 0.72 65.164 ethanol ±0.96 52.189 methanol ±0.26 46.232 peg200 ±0.87 38.562 peg400 construction of pseudo-ternary phase diagrams figures (3 and4) showed the pseudoternary phase diagram for the o/w nes using triacetin as an oil phase, tween 80 and tween60 as a surfactant and ethanol as a co-surfactant. figure 3.pseudo-ternary phase diagram o/w emulsion diagram using triacetin, tween 80: ethanol in different ratios. iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 46 figure 4. triangular co-ordinate o/w emulsion diagram using triacetin, tween60: ethanol in different ratios. lcdp ne characterization thermodynamic study nes are thermodynamically stable systems, formed of particular concentrations of oil, smix and ddw with no phase separation and no cracking or creaming. small droplet size prevents any flocculation, enabling the system to remain dispersed with no separation (40), as shown in table (3). table 3. results of thermodynamic stability studies for lcdp nanoemulsions. droplet size table (4) showed the results of droplet size measurement. the results illustrated that when the concentration of surfactant increased the particles size reduced since this high surfactant concentration decreases surface tension and stabilizes newly developed surfaces during homogenization and production of smaller particles (41). figures 5 and 6 show the droplet size measurement of the lcdp nes formulas 1-9. table 4. particle size measurement for lcdp nanoemulsion. freezethawing cycles heatingcooling cycles centrifugation test f-code pass pass pass ne-1 pass pass pass ne-2 pass pass pass ne-3 pass pass pass ne-4 pass pass pass ne-5 pass pass pass ne-6 pass pass pass ne-7 pass pass pass ne-8 pass pass pass ne-9 mean particle size (nm) fcode mean particle size (nm) fcode 304.3 f-6 345.7 f-1 220.6 f-7 258.8 f-2 15.05 f-8 205.5 f-3 13.47 f-9 13.42 f-4 13.42 f-5 iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 47 figure 5 . particle size distribution of lacidipine nes formulas (1, 2, 3,4,5 and 6) respectively figure 6. particle size distribution of lcdp nes formulas (7, 8 and 9) respectively. polydispersity index measurement (pdi) pdi refers to the quality of the dispersion; this index represents uniformity and homogeneity of the particles in the nes , as revealed in table (5) (35). table 5.the polydispersity index of lcdp nanoemulsions zeta potential measurement (ʓ – potential) zeta potential governs the degree of repulsion between adjacent, similarly charged, dispersed particles. when the nonionic surfactants adsorb onto the nanoscale droplets, they lowering the zeta potentials and preserve stability (42). table (6) and figures 7 and 8 show the values of the zeta potential of formulas 1-9 table6. zeta potential of lcdp nanoemulsions f-code pdi f-code pdi f-1 0.581 f-6 0.261 f-2 0.269 f-7 0.362 f-3 0.370 f-8 0.410 f-4 0.381 f-9 0.186 f-5 0.234 fcode zeta potential (mv) fcode zeta potential (mv) f-1 -3.84 f-6 -3.04 f-2 -5.4 f-7 -10.8 f-3 -9.62 f-8 -11.8 f-4 -6.45 f-9 -3.84 f-5 -14.5 iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 48 figure 7. zeta potential values of lacidipine nes formulas (1,2,3 and 4) respectively. figure 8. zeta potential values of lcdp nanoemulsions formulas(5,6,7,8and 9) respectively . dilution test dilution test confirmed the high physical stability of the lcdp ne under dilution with water. in less than 1 minute, all ne formulas (f1-f9) showed clear and fine bluish ne indicating o/w type, proved that they are maintaining the nanosized character and could be diluted in gi fluids without drug precipitation (43). conductance measurement electrical conductivity had a potent relation with the type or nature of the external phase of ne. higher values for electrical conductivity indicate higher conductivity of water (44). the results in a table (7) showed higher conductivity values. table 7. conductivity measurement of lcdp nanoemulsions. fcode σ(ms/cm) f-code σ(ms/cm) f-1 0.267 f-6 0.123 f-2 0.143 f-7 0.156 f-3 0.154 f-8 0.0691 f-4 0.176 f-9 0.105 f-5 0.241 drug content estimation all nes formulas agreed with the requirements of the british pharmacopeia range (95%105%) as shown in table (8), which indicated that high content uniformity and revealed the adequacy of the preparation method (45). iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 49 table 8.drug content of lcdp formulations (mean ±sd, n=3). fcode % drug content sd ne-1 99.14 ±0.81 ne-2 96.29 ±0.21 ne-3 98.03 ±0.56 ne-4 96.15 ±0.32 ne-5 99.14 ±0.68 ne-6 99.26 ±0.42 ne-7 99.16 ±0.26 ne-8 96.03 ±0.97 ne-9 97.48 ±0.46 measurementviscosity from figure (9), it was demonstrated as the concentration of the surfactant increased, the viscosity increased this may be due to entrapping of the water molecules in crosslinking surfactants chains and also highest surfactant concentration would make the dispersion medium more rigid, as well as formulas that contain tween 60 has a higher viscosity than that contain tween 80 since tween 60 has a higher molecular weight than tween 80 (46,47) . the results also showed that the viscosity decreased as the rotation speed increased (shear rate) indicating the pseudoplastic (shear thinning liquids) flow of the preparation (48). figure 9. viscosities of lcdp nanoemulsions in vitro drug dissolution study the release of the drug from all nes formulations was found nearly 100% at the end of 60 min.; higher the dissolution, faster the absorption, and hence quicker and higher the drug action can be obtained by smaller the particle size of a drug in the dosage forms (49). figure (10) demonstrated that the release of lcdp from the formulas that contain tween 80 as a surfactant was higher than that contain tween 60 which could be explained by the smaller droplet size of formulas containing tween 80 as compared to that formula which contains tween 60 leading to a higher rate of dissolution. the higher hlb value of tween 80, which is 15 enhanced the continuous distribution and solubilization of the incorporated lipophilic drug within the system (50,51). figure 10.a comparative dissolution profile of lacidipine nes (f2, f-6 and pure lcdp) in 500ml of 0.1 n hcl (with 1% tween 20) dissolution medium at 37 °c selection of the optimum formula after studying the characterization of prepared lcdp nes (f1-f9), it was found that (f-5) is selected as the best formula that is characterized by a low particle size (13.42), low polydispersity index (pdi) (0.234) , zeta potential (-14.5), good spreadability on the filter paper, efficient electrical conductivity (0.241 ms/cm) , good ph value (5.9), good percent of light transmittance (99.10), accepted viscosity , higher drug content percent (99.14) and higher dissolution rate. the optimized formula would be subjected to further studies. evaluation of the selected lacidipine optimum formula drug and excipient compatibility study by ftir ftir is an extremely powerful technique in discovering and evaluating any possible chemical interaction between lcdp and any excipient during nes preparation. iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 50 ftir spectra of pure lcdp powder showed characteristic peaks which are: 3348.78 cm−1 due to (n−h) stretching vibration, 3109.65–2976.59 cm−1 corresponding to (=c−h) stretching, 2930.31 cm−1 due to aliphatic (c−h) stretching, 1674.87 cm−1 for ester (c=o) stretching, 1495.53 cm−1 and 1451.17 cm−1 due to aromatic −c=c stretching, 1372.10 cm−1 corresponding to aliphatic c−h bending, 745.35 cm−1 for disubstiuted ortho benzene stretching and 982.55 cm−1 for c−n stretching. the ftir spectrum of pure lcdp and selected formula (f-5) displayed the same functional groups band with very slight shifting, which suggests the presence of hydrogen bonding (52,53). figures 11 and 12 showed the ftir spectra of the lcdp and the selected formula (f-5) respectively. figure11.the ftir spectrum of lcdp. figure 12.ftir spectrum of the selected formula ( f-5 ) . validation of the hlpc method the chromatograms of pure drug lcdp and lcdp in the selected formula (f-5) there was no change in retention time, and no extra peaks reported . therefore , these excipients were found to be compatible with lcdp (54) . figures (13 and 14) showed the chromatograms of lcdp, triacetin, tween80 ethanol and f5 respectively. figure 13. chromatograms of pure lcdp and triacetin, respectively. iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 51 continue figure 13. chromatograms of pure lacidipine and triacetin, respectively. figure 14. chromatograms of tween80 and f-5 respectively atomic force microscopy (afm) study afm is capable of scanning and measures the properties and characteristic of the surfaces. with the high accuracy of the afm, it is possible to determine the dimensions of nanoparticles with high reliability. the morphological analysis and particle size of formula f5 performed by afm were close to spherical in shape and smooth surface (55,56), as shown in figure 15. iraqi j pharm sci, vol.29(1) 2020 lacidipine nanoemulsions 52 figure 15 the afm image of lcdp ne (f5) where the scanning area is 2µm*2µm conclusion after discussing the previously obtained data, it is easy to deduce the following points: 1. all the ne formulas prepared with triacetin as an oil phase, tween 80 and tween 60 as a 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aldosterone and calcification doi: https://doi.org/10.31351/vol28iss1pp53-63 53 serum aldosterone level in patients with diabetic nephropathy in relation to vascular calcification balqies h.saleh *,1, shatha h.ali * and khalid i. allehibi** * department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. ** consultant endocrinologist,specialized center for endocrinology and diabetes, , baghdad, iraq. abstract diabetic nephropathy(dn) is a complex disease manifested by persistence microalbuminuria occurring due to the interaction between hemodynamic and metabolic pathway that activates the local renin-angiotensin-aldosterone system resulting in a decline in renal functions. this study aimed to quantify the associations between serum aldosterone concentration and fetuina as a marker of calcification in type 2 diabetic patients with and without microalbuminuria from one side, and study the possible relationship between aldosterone and fetuin-a with glycemic indices, serum electrolyte, renal function and microalbuminuria and body mass index from the other side. a case-control study involved eighty-six adult subjects classified into three groups after testing urine microalbumin including thirty-two diabetics type 2 patients with positive microalbuminuria and twenty-eight diabetics type 2 patients with negative microalbuminuria and 26 healthy subjects during their visit to al kindy specialized center for endocrinology and diabetes / baghdad. those patients were compared to control group of 26 apparently healthy subjects, fasting blood samples was obtained from each of them in one occasion only to measure: fasting serum glucose, electrolyte, aldosterone, fetuin-a, urea, and creatinine. in addition to glycoheamoglobin, glomerular filtration rate and body mass index. despite the presence of microalbuminuria in thirty-two of the studied diabetics, there was no positive correlation between aldosterone and fetuina, besides that no significant variations in serum aldosterone ,glomerular filtration rate(gfr) values, while both groups showed a significant increase in fasting serum glucose and glycaoheamoglobin ,significant decrease in serum sodium and chloride in comparison with the control group , significant increase was detected in serum fetuin-a mean values in microalbuminuric diabetics. whereas, negative microalbuminuric diabetics measures expressed a positive correlation between both serum sodium and chloride levels and fetuin -a. the conclusion of this study diabetic patient are prone to vascular calcification (vc) might be due to increase in aldosterone level or due to diabetic itself from this study we can conclude microalbuminuria can occur without a decline in renal function or a change in estimated gfr ,no definite correlation occur between aldosterone and fetuina, fetuina mean values are higher in diabetic patient with microalbuminuria compared to diabetic patients without microalbuminuria and control group and this referred to uncontrolled diabetes ,aldosterone show a correlation with weight and body mass index while fetuina does not show such correlation. in general, electrolyte disturbances (hypernatremia) is more obvious in this study , and its occurrence is due to diabetic (osmotic diuresis) or drugs, while sodium retention which is a sign of aldosterone increment does not occur. hypochloremia that occur in this study is due to chloride and it is in parallel with sodium level. keywords: aldosterone, fetuin a, vascular calcification, type 2 diabetes mellitus, glomerular filtration rate, diabetic nephropathy (dn). مستويات االلدوستيرون في مصل الدم في مرضى السكري الذين يعانون من اعتالل الكلى السكري وعالقتة .بتكلس االوعية الدموية **خاالد ابراهيم اللهيبيو *شذى حسين علي˓1،*ام صالحشبلقيس ه بغداد،بغداد،العراق.فرع العلوم المختبرية السريرية،كلية الصيدلة،جامعة * ، بغداد ، العراق.المركز التخصصي المراض الغدد الصماء والسكري،استشاري بامراض الغدد الصماء والسكري.** الخالصة دوستيرون أللاإعتالل الكلى السكري هو مرض معقد يحدث بسبب التفاعل بين مسار الدورة الدموية والتمثيل الغذائي الذي ينشط نظام الرينين أنجيوتنسين .المحلي مما يؤدي إلى انخفاض في وظائف الكلى 1corresponding author e-mail: b_h_s86@yahoo.com received: 23/8/2018 accepted: 11/11/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp53-63 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.28(1) 2019 aldosterone and calcification 53 ز االلدوستيرون في المصل والفتوين أ كعالمة للتكلس عند مرضى السكري النوع الثاني الذين يالعالقة بين تركتقيم هدفت هذه الدراسة إلى يطرحون زالل دقيق في االدرار من جهة ودراسة العالقة الممكنة بين الاللدوستيرون والفتوين أ مع موشر السكري واالمالح في المصل يطرحون او ال فحص وظائف الكلى والزالل الدقيق في االدرار ومؤشر كتلة الجسم من جهة اخرى . و اثنان يتضمنون , ى ثالث مجاميع بعد فحص الزالل الدقيق في االدراربالغا يصنفون ال شخصا ثمانونالتحكم : ستة و -تضمنت دراسة حالة يس لديهم بول ل من النوع الثاني بالسكري من النوع الثاني لديهم بول زاللي دقيق و ثمانية وعشرون مريًضا مصابًا بالسكري بامصا مريضا وثالثون مقارنة هؤالء تمتمراض الغدد الصماء والسكري / بغداد. الز ألكندي التخصصي لمرك زيارتهم, أثناء دقيق وستة وعشرون شخصا اصحاء زاللي مرة ل مالمرضى بمجموعة السيطرة المكونة من ستة وعشرين شخًصا يبدو أنهم أصحاء ، تم جمع عينات الدم الصيامي و الحصول عليها من كل واحد منه ىأ ، اليوريا والكرياتينين. باإلضافة إلى الهيموجلوبين الغليكوزيالتي وترشيح الكل-ن ، فيتوين ، األلدوستيرواالمالحلقياس: جلوكوز المصل ، واحدة فقط الكبيبي. للدراسىىىىة ، لم تكن هنات اختالفات كبيرة في على الرغم من وجود البول الزاللي الدقيق في اثنين وثالثين من مرضىىىىى السىىىىكري الذين خضىىىىعوا و الهيموكلوبين الصىىىىيامي السىىىىكرقيم بينما كال المجموعتين اظهرت تغير كبير في، و معدل ترشىىىىيح الكلى الكبيبي المصىىىىل في معدل ألدوسىىىىتيرون أ -لفتوينيم لمتوسىىىط الق فيزيادة كبيرة ولكن تم اكتشىىىا الكاليكوزيالتي وانحفاض كبير للصىىىوديوم والكلورايد في المصىىىل مقارنة بمجموعة السىىىيطرة ين لديهم زالل دقيق بالمقارنة مع الذين ليس لديهم زالل في االدرار و مجموعة السىىىيطرة وهذا يشىىىير الى السىىىكر الغير المسىىىيطر لمجموعة المرضىىىى الذ على حد سىىىىىىىواء.مع الفتوين أ الصىىىىىىىوديوم والكلوريد بينفي حين أعربت مجموعة المرضىىىىىىىى الذين ليس لديهم بول زاللي ارتبااات إيجابية . عليهة او عن بتكلس االوعية الدموية ربما لزيادة االلدوسىىتيرونبالسىىكري من النوع الثاني عرضىىة لةصىىابة ينالمصىىاب المرضىىىهذه الدراسىىة االسىىتنتام من من هذه الدراسىىىة نسىىىتطيع ان نسىىىتنتا ان االدرار الزاللي الدقيق يمكن ان يحصىىىل بدون انخفاض بوظائف الكلية او تغير بمعدل , اريق مرض السىىىكري أ اعلى في مرضىىى السىىكري الذين لديهم ا ادرار -أ ,معدل قيم الفتوين -الكبيبي كما ان التوجد هنالك عالقة واضىىحة بين االلدوسىىتيرون والفتوين الترشىىيح هر تيرون يظمجموعة السيطرة وهذ يشير الى السكري الغير مسيطر عليه كما ان االلدوسالذين ليس لديهم ادرار زاللي دقيق و زاللي دقيق بالمقارنة مع أ لم يظهر مثل هذه عالقة , بصورة عامة اختالل االمالح )انخفاض الصوديوم(واضح في هذه الدراسة -عالقة مع الوزن ومؤشر كتلة الجسم بينما الفتوين االلدوسىىىتيرون لم يحدث. ىوالذي حدث نتيجة لمرض السىىىكري )االدرار التناضىىىحي( او االدوية بينما انحباس الصىىىوديوم الذي هو عالمة الرتفاع مسىىىتو الصوديوم. ىانخفاض في الكلورايد الذي حصل في هذه الدراسة النة الكلورايد متوازي مع مستو .أعتالل الكلية السكري،معدل الترشيح الكبيبي، السكري النوع الثاني ، , تكلس االوعية أ – الفتوين ،االلدوستيرون الكلمات المفتاحية: introduction diabetic nephropathy (dn) is one of the microvascular complications that develops in about 30% of patients with type1 diabetes mellitus and about 40% in those with type2 diabetes mellitus (t2dm) (1). it’s characterized by albuminuria, irreversible decrease in glomerular filtration rate (gfr) and arterial hypertension(2). microalbuminuria is an earlier sign of general vascular dysfunction and nowadays is considered a predictor of worse outcomes for both kidney and heart (3). hence, patients with t2dm should be screened for microalbuminuria from the date of diagnosis (4). with diagnostic reference standard of 30 to 300 mg of albumin in a 24-hour urine sample (5). persons with type 1 or 2 diabetes and microalbuminuria should continue to be tested for albuminuria annually to observe disease progression and response to therapy (4). essential causes in the pathogenesis of diabetic nephropathy are metabolic and hemodynamic pathways (6). heamodynamic pathway had been reported to include activation of local renin angiotensin aldosterone system (raas) in proximal tubular epithelial cells, mesangial cells, and podocyte (7). with consequences of reactive oxygen species(ros) generation, inflammation, over expression of transforming growth factor-β (tgf-β), and deregulations of different vascular growth factors such as the vascular endothelial growth factor-a (vegf-a)(8). activating local (raas) results in increased angiotensin ii (ang ii), in addition to action adrenoctincorticotrpic hormone ( acth ), and potassium are three principal factors that adjusted aldosterone secretion (9,10). classical effects of aldosterone are to promote sodium retention and potassium loss by the kidney, although it exerts similar but lesser effects on the colon, sweat, and salivary glands (11). aldosterone/mineralocorticoid receptor (mr) system plays an important role in cardiovascular and renal diseases, particularly in the presence of excessive salt intake. in individuals with metabolic syndrome, adipocyte-derived aldosterone-releasing factors cause inappropriate release of aldosterone in the adrenal glands during salt loading, resulting in the development of salt-induced hypertension, cardiac and renal damage (12). aldosterone also plays a definitive role on systemic and vascular insulin resistance, as the vascular insulin resistance is considered an early cause to vascular damage. accordingly, aldosterone impairs insulin receptor (ir) signaling by changing in the phosphatidylinositol 3-kinase (pi3k) / nitric oxide (no) pathway and by inducing oxidative stress and crosstalk between the ir and the insulin-like growth factor-1 receptor (igf-1r) leading to proliferation, oxidative stress and inflammation. meanwhile, aldosterone exerts negative effects on structural and functional integrity of the pancreatic β-cell by encouraging inflammatory and oxidative stress conditions, which lead to decreased insulin release and actions, including actions in the vasculature (13). aldosterone is a new and axial factor that causes vascular calcification (vc) (14). also, it has a detrimental effect in the vasculature (enhances iraqi j pharm sci, vol.28(1) 2019 aldosterone and calcification 54 vascular oxidative stress), promote vessel inflammation and apoptosis (15). one of hemodynamic pathway factors is aldosterone hormone, aldosterone can promote vascular change and calcification in patients with diabetes through several mechanisms (16). a wide range of biomarkers has been studied for identification of type 2 diabetes patients at microvascular and macrovascular risk. fetuin-a is a novel biomarker that is used with metabolic complication to understand the causes that lead to microvascular or macrovascular changes that occur in diabetic nephropathy(17) .fetuin-a: is a 60 kda (kilo dalton) glycoprotein produced exclusively by the liver and secreted into serum in relatively high concentrations in humans(18). fetuin-a is known to inhibit ectopic calcium deposition and protect from vascular calcification (19). epidemiological studies suggested that higher serum fetuin-a levels are associated with insulin resistance (ir), metabolic syndrome (ms) and type2dm (20). subjects and methods the study was conducted on patients with type2 diabetes mellitus in al kindy specialized center for endocrinology and diabetes. for the period from november/2017 to february 2018. a total number of 60 diabetic patients (30 males and 30 females) were included in this study, 32 patients were positive for microalbuminuria and 28 were negative for microalbuminuria ,and their age ranged between (40) and (65) with a mean ±sd of (53.28±7.20 years). patients were selected after excluding those on insulin therapy, hypertensive patients, or those with thyroid disorder or other endocrinopathies, or those with active liver diseases, pregnant females and smokers. those patients were compared to 26 apparently healthy subjects (13 males&13 females). the study was approved by the local research ethics committee and all subjects were signed on a written informed consent to participate in this study. testing for microalbuminuria was performed by using (combina 13) urine test strip (21). purchased by human diagnostic worldwide, germany .while, fasting glucose, creatinine and urea were measured by cobas c 311 roch analyzer (22-24) glycoheamoglobin(hba1c) was measured by spectrophotometer apel pd-303(25) .serum electrolytes (sodium, potassium, and chloride )were estimated based on the potentiometric difference between the sample (electrodes) and reference standard, using fuji dri-chem nx 500 electrolyte analyzer(26). specific competitive binding elisa kit for human aldosterone, purchased by demiditic, germany (27). serum fetuin a levels were measured by utilizing quantitative sandwich elisa kit from cusabio, shanghai /china(28). glomerular filtration rate determination : 1. the cockcroft-gault formula ccr={((140age) x weight)/(72 scr)} x 0.85 if female , where ccr is expressed in milliliters per minute, age in years, weight in kilograms, and serum creatinine (scr) in milligrams per deciliter(29). 2. modification of diet in renal disease equation (the 4-variable mdrd study equation): gfr = 175 x (standardized scr)1.154 x (age)-0.203x (0.742 if female) x (1.210 if african american) gfr is expressed in ml/min/1.73 m2 cr is serum creatinine expressed in mg/dl, and age is expressed in years (30). statistical analysis was performed using the spss statistical package for social sciences (version 20.0 for windows, spss, chicago, il and usa). data are presented as means ± sd. significance was set at p < 0.05. cases and controls were compared using either the t-test for independent samples. the pearson coefficient test was used to test the relation between studied parameters. results subject’s characteristics are listed below in table-1. iraqi j pharm sci, vol.28(1) 2019 aldosterone and calcification 55 table (1) subject characteristics group p value parameters patient (n=60) control (n=26) mean sd mean sd age (years) * gender (m/f) 53.28 30:30 7.20 47.88 13:13 6.35 0.001 0.999 weight (kg) * 77.81 18.61 70.77 7.11 0.013 bmi (kg/m2) * 29.46 6.18 26.32 2.14 0.001 duration of dm (years) 7.29 5.20 . . fsg (mmol/l) * 12.20 4.64 5.12 .63 0.005 hba1c (%) * 8.24 1.84 4.62 .88 0.005 creatinine (µmol/l) 71.57 37.56 71.38 8.25 0.972 urea (mmol/l) 4.21 1.13 3.87 .93 0.179 gfr mdrd (ml/min1.73m2) 105.26 42.35 94.42 9.47 0.064 gfr crock ( ml/min) 117.98 45.39 106.35 10.45 0.065 albuminuria( mg) * 34.00 46.07 .00 .00 0.005 sodium (meq/l) 119.5 15.0 140.7 3.9 0.005 potassium(meq/l) 3.9 .5 4.1 .1 0.199 chloride(meq/l) 82.5 11.0 99.6 4.3 0.005 sbp (mmhg) * 13.43 1.80 12.15 .54 0.005 dbp( mmhg )* 8.47 1.19 8.08 .39 0.026 aldosterone (pg/ml) 145.63 68.51 116.03 134.14 0.179 fetuin-a 628.76 829.14 254.83 151.00 0.001 *= significantly different from control, body mass index (bmi), fast serum glucose (fsg), glycohemoglobin (hba1c),glomerular filtration rate (gfr).systolic blood pressure(sbp), diastolic blood pressure (dbp) as shown in (figure -1), the mean values of fasting serum glucose (fsg) and glyco hemoglobin (hba1c) of group a (diabetic patients with positive +ve microalbuminuria) and group b (diabetic patients with negative –ve microalbuminuria) were significantly higher than group c(control). iraqi j pharm sci, vol.28(1) 2019 aldosterone and calcification 56 figure ( 1) mean values of fasting serum glucose and hba1c among groups. furthermore, mean values of gfr (cockcroftgault formula) for groups a and b were not significantly different from that of group c (the control), although these values were elevated as compared to that of group c .as shown in (figure -2). figure (2) mean of gfr (ml/min) (cockcroftgault formula) in: group a (diabetic positive microalbuminuria). group b (diabetic negative microalbuminuria) . group c (control). mean values of serum creatinine level for groups a and b were not significantly different from that of group c (the control) figure (3) mean values of serum creatinine level in: group a =diabetic positive microalbuminuria. group b =diabetic negative microalbuminuria. group c =control. data analysis of the estimated serum values of aldosterone among studied groups indicate a non significant difference between diabetics (with and without microalbuminuria) and the control subjects (figure-4) . figure (4) mean values of serum aldosterone levels iraqi j pharm sci, vol.28(1) 2019 aldosterone and calcification 57 as illustrated in figure-5 serum fetuin-a levels were significantly elevated in group a (diabetic positive microalbuminuria) when compared to group b (diabetic negative microalbuminuria) as well as when compared to group c (control). figure (5) mean values of serum fetuin-a levels figure (6) significant correlation between bmi and gfr (crock-gault) in group a whereas, in group b (diabetic negative microalbuminuria) pearson’s correlation coefficient values of 0.388 and p value 0.041 indicating significant correlation between weight and aldosterone levels (figure-7). figure (7) significant correlation between weight and aldosterone in group b. as shown in (figure-8) pearson’s correlation coefficient values of 0.390 and p value 0.040 indicating significant correlation between serum chloride (cl) and fetuin-a levels in diabetics without microalbuminuria. figure (8) significant correlation between chloride and fetuin-a in group b. whereas, pearson’s correlation coefficient values of 0.431and p value 0.022 indicating significant correlation between serum sodium and fetuin-a in non-albuminuric diabetics, as presented in (figure-9). iraqi j pharm sci, vol.28(1) 2019 aldosterone and calcification 58 figure (9) significant correlation between sodium and fetuin-a in group b. discussion the level of glycemic control seems to be the strongest factor influencing transition from normoalbuminuria to microalbuminuria(31). glycemic control plays as vital role in diabetic microvascular complications. it was shown that for each 1% reduction in updated mean of hba1c there is a 37% reduction in microvascular complication risks (32). the fsg in diabetic patients mean value was (12.20±4.64 m mol/l) and hba1c mean was (8.24±1.84 %) which is significantly higher than the control group (mean fsg 5.12±0.63 m mol/l, hba1c mean 4.62±0.88%), as summarized in (figure-1). there was no positive correlation between (fsg, hba1c) and microalbuminuria, in contrast to a study by chen et al (31). considering renal function assessment; the mean values of serum creatinine and estimated gfr were within normal range for both groups (group a diabetics with positive microalbuminuria and group b diabetics with negative microalbuminuria as shown in (figure-2) and (figure-3). estimated gfr and serum creatinine depend on the renal hemodynamics, systemic blood pressure, urinary findings, and susceptibility to therapeutic intervention. on the basis of these findings, it is concluded that microalbuminuria may not be associated with abnormal creatinine or creatinine clearance. (33) among the important parameters that had been shown to directly affect renal haemodynamics and alter the afferent/efferent balance, is bmi which could result in glomerular hypertension , hyperfiltration and ultimately, renal injury(34). the present study shows a positive correlation between (bmi, weight) and estimated gfr (crock-gault) in diabetic patient with positive microalbuminuria as in (figure-6). studies had explained the assessment of gfr in relation to body dimensions, however, it is important to be aware that renal function equations are subjected to bmi-associated bias, for example: kwakernaak et al compared between cockcroft–gault and modification of diet in renal disease (mdrd) in healthy subject, and found that the effect of bmi on overestimation of gfr was the largest for cockcroft– gault and less for mdrd (35). the renal haemodynamic profile in overweight and obesity, and in subjects with a central body fat distribution, may be affected by other factors which are sodium intake and volume homeostasis, as well as long-term susceptibility to renal damage. (36, 37). interestingly, in young normotensive subjects, overweight is associated with a rise in filtration fraction (ff) in response to high salt intake, whereas in lean subjects gfr increases without a rise in ff. in overweight subjects, moreover,a high salt intake is associated with a larger increase in the extra cellular volume (ecv) than in lean subjects, supporting the impact of subtle changes in renal haemodynamics on volume homeostasis(38), as being presented in this study with a positive correlation between body weight and serum aldosterone (figure-7) in group b diabetic patients.the long-term consequences of this unfavorable renal haemodynamic profile, elicited by the combination of overweight and excess sodium intake may well contribute to the development of salt-sensitive hypertension and renal damage later in life(39). diabetic patients are more prone to develop electrolyte disturbances because of disease state itself and associated disruption of blood glucose homeostasis. the use of antidiabetic drugs also leads to the development of electrolyte disturbances(40).in the present study, the mean values of serum na+ level in both groups of diabetic patients were significantly reduced compared to control group (table-1) . similar findings were observed by alaka das (41). there were individual variations in sodium level in both groups of diabetic patients some of them were within normal range, but others were under normal range .hyponatremia is the most common electrolyte abnormality in clinical practice and is associated with increased morbidity and mortality and even small decreases of serum sodium are associated with increased probability for adverse outcomes (42). drugs represent a common cause of hyponatremia in individuals with diabetes (42). such as the first generation sulphonylureas (tolbutamide and chlorpropamide) (43), (44). other drugs of common utilizations include nsaids, angiotensin converting iraqi j pharm sci, vol.28(1) 2019 aldosterone and calcification 59 enzyme inhibitors, rosiglitazone or even amlodipine. patients with central nervous system disorders, pulmonary disorders including lung infections, and malignancies may exhibit hyponatremia due to the syndrome of inappropriate antidiuretics. patients with diabetic nephropathy and chronic renal failure are very prone to the development of hyponatremia due to decreased water excretion (42). furthermore, potassium levels in both groups of diabetic patients were near to normal range , with no significant change in serum k+ level had been observed between the diabetic group and control group. similar findings were reported by alaka das (41). although there were elevated serum aldosterone levels in microalbuminuric diabetics but the wide variation of these values when analyzed statistically were not of enough significance when compared with mean value of diabetic negative microalbuminuria and control group (figure4) which are similar to the result obtained by hollenberg et al (45). meanwhile, a positive correlation between weight and serum aldosterone level was observed in group b patients (figure-7), similarly tuck et al. demonstrated that weight loss is accompanied by reductions in pra (plasma renin activity) and aldosterone, irrespective of sodium intake, and this affects the decline in bp in obese patients (46). interestingly, high levels of pra, ace, aldosterone, and insulin with sodium retention and potassium loss were found in patients with visceral obesity, but all of these tended to disappear upon weight reduction and were not found in patients with peripheral obesity (47). another study demonstrated that the pac (plasma aldosterone concentration) is positively correlated with the amount of visceral adipose tissue and is inversely correlated with insulin sensitivity, independent of the pra level. this suggested that a fat derivedsubstance contributes to aldosterone excess in patients with visceral obesity (48). fetuin -a had been reported to have a role in causing vascular calcification in diabetic patient, fetuin-a gives a picture if the patient has calcium deposition in vascular (calcification occurs or not), it is a multifunctional glycoprotein predominantly secreted by the liver and mainly involved in promoting insulin resistance(49). accumulating experimental and epidemiological studies reported that it was associated with a spectrum of cardiometabolic disorders, such as metabolic syndrome (50), nonalcoholic fatty liver disease (51), t2dm (52), and cardiovascular diseases (cvd) (53).a study by lv x et al showed a positive association between serum fetuin-a levels and albuminuria in patients with metabolic syndrome or t2dm (54), however, no such correlation had been detected in this study. furthermore, the mean value of serum fetuina in diabetics with positive microalbuminuria was higher than that in diabetics with negative microalbuminuria and control group (figure-5) and this indicated to uncontrolled dm . in this study, we evaluated the associations of some parameters related to microvascular diseases in patients with t2dm and early diabetic nephropathy as the degree of albumin excretion, renal function (gfr and serum creatinine). all of these parameters showed no significant correlation with fetuin a, nor to correlate with some clinical and metabolic parameters as bmi, blood pressure which are similar to the study by ayman ramadan et al (55). however, it had been found that fetuin-a levels seem to be associated with prevalent macrovascular disease (as coronary artery disease, stroke and peripheral artery disease) in t2dm, and fetuin-a serum levels are not associated with microvascular complications (24 h urinary albumin excretion) in patients with early diabetic nephropathy (56) which almost in agreement with our study in some extent in that fetuin-a levels do not correlate with metabolic parameters in t2dm patients with prevalent late complications. on the other hand, serum fetuin -a showed a positive correlation with serum chloride in group b as shown in (figure-8) and this is obvious that chloride is most commonly associated with proportionate changes in sodium concentration so therefore, the concentration of cl usually parallels to that of sodium and may correlate with fetuin a in same mechanism that control serum level of sodium through (raas activation, higher insulin concentration) causing sodium retention with a parallel increase in chloride level(57). similarly, a positive correlation between serum sodium level and fetuina is present as shown in (figure-9). in diabetic patient, there are 2 mechanisms that cause sodium retention, first one is the activation of (raas), whereas the secon, t2dm, have a high circulating insulin level but lack its functional role in regulating glucose level,and insulin thought to have a role in increasing sodium level by its antidiuretic effect. high concentration of fetuin a in patients with type 2 diabetes is associated with increasing insulin level; these two mechanism may counteract the osmotic diuresis that occur due to hyperglycemia (58). according to ther result of this study, it is concluded that in diabetic patient with microalbuminuria it may not necessary to have high serum level of aldosterone besides that aldosterone hormone is not the only factor that cause calcification. calcification may occur due to diabetes itself .microalbumiuria in diabetics may not associate with the decline in renal function or a change in estimated iraqi j pharm sci, vol.28(1) 2019 aldosterone and calcification 60 gfr. in this study diabetic patients with microalbuminuria are characterized with high level of (fsg &hba1c) in comparison with control group and this increment may have a relationship with the increment of fetuin a . in the current study, metabolic parameter like weight and body mass index showed a correlation with aldosterone level but lack this relation with fetuina. acknowledgements i would like to thank all 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pharm sci, vol.19(2) 2010 stability of cfm and cfz with ca against βlactamase 42 evaluation of stability of cefamandol and ceftazidime with clavulanic acid against extended spectrum βlactamase siham s. shaokat * and hamoudi a. hameed* ,1 * ministry of industry and minerals, food and drugs sector, baghdad, iraq. abstract the aim of this study is to evaluate in-vitro activity of cefamandol (cfm) and ceftazidime (cfz), in combination with clavulanic acid (ca) against ten complicated multiresistant uropathogenic e.coli .one hundred clinical strains were isolated from patients with chronic urinary tract infections (utis), these isolates were identified by the api identification systems. the antimicrobial susceptibility tests were determined by kirby-bauer method, all of them were sensitive to imipenem (imp). ten strains were chosen for the present study, they were resistant to ampicillin (amp), amoxicillin (amo), carbenicillin (cb), ticarcillin (tic), azlocillin (azl), amoxicillin\ potassium clavulanate {augmentin(amc)}, (amo\ca), ticarcillin\ potassium clavulanate {timentin} (tic\ca) ,cefazolin (cfo) ,cefaloridin (cfr), cefamandol, (cfm),cefoxitin, ceftazidime (cfz), cefixime (cxm), cefoperazone( cfp) and aztreonam (atm), also resistant to other antibiotics, tetracycline(tc),cloramphenicol(cm),gentamycin(g),amikacin (amk), ciprofloxacin (cip) and trimethoprim. 50% of the isolates were resistant to nalidixic acid and rifampicin. the minimum inhibitory concentrations of cefamandol and ceftazidime were determined, by tube method. transfer of plasmids were done by direct conjugation test to sensitive standard e.coli ,cell free βlactamases were prepared and detected by macro-iodometric method. the activity of each cell free ß– lactamases extract against cfm and cfz were determined by disks diffusion method (microbiological masuda method) .excellent activities were obtained against these strains when cfm and cfz, combined with ca, therefore complete zones of inhibition were obtained indicated the prevalence of extended spectrum βlactamases in e.coli. the stability of cfm and cfz in the presence of ca were useful in the treatment of chronic urinary tract infections caused by multiresistant βlactamase (esbl) producer e.coli. key words: extended spectrum βlactamases, imipenem, aztreonam, ceftazidime. الخالصة -e( باضافة حايض انكالفجىالَك ججاِ ) cfz( وانسٍفحازدٌى )cfmٌهذف انبحث انى دراسة فعانٍة انسٍفايُذول ) coli اٌ ( انًقاوية نًجًىعة يضادات انبٍحاالكحاو يعسونة يٍ يرضى ٌعاَىٌ يٍ انحهاب انًجاري انبىنٍة انًسيٍ وقذ بٍُث انذراسة واٌ ثباجٍة كم يٍ انسٍفاياَذول وانسٍفحازدٌى انفعانٍة انًثهى نكم يٍ انسٍفاياَذول وانسٍفحازدٌى جكىٌ بىجىد حايض انكالفجىالَك ( انًقاوية نًضادات e-coliبىجىد انحايض جشٍر انى ايكاٍَة اسحخذايهًا فً يعانجة انحهاب انًجاري انبىنٍة انُاججة عٍ ) ل رراق انحقٍٍى وانحشخٍ انحً اسحخذيث فً انبحث . انبٍحاالكحاو يٍ خال introduction clavulanic acid is a βlactam; structurally it differs from pnicillins in two respects, the replacement of sulfur in the penicillin thiazolidine ring with oxygen in the clavam oxazolidine ring and the absence of the side chain at position 6. clavulanic acid a naturally occurring clavam isolated from streptomyces clavuligerus has poor antibacterial activity but exerts a potent and irreversible inhibitory effect on βlactamases especially penicillinase by blocking the active sites of these enzymes and is strongly synergistic with most of the βlactamines in vitro (1) . due to this combination, amoxicillin is protected from degradation and its spectrum is therefore extended to include bacteria normally resistant to amoxicillin and other β lactam antibiotics (2) . in the case of βlactam resistant bacteria a bacterial enzyme, β lactamase, cleaves the βlactam ring and renders the antibiotic inactive. β-lactamases are a large and diverse group of enzymes in which four clinically relevant classes are known (3) . β-lactamase continues to be the leading cause of resistance to βlactam antibiotics among gram-negative bacteria. in recent years there has been an increased incidence and prevalence of extendedspectrum β-lactamases (esbls), enzymes that hydrolyze and cause resistance to oxyiminocephalosporins and aztreonam.the majority of esbls are derived from the widespread broad spectrum βlactamases tem-1 and shv-1. 1corresponding author email : hamodiabas@yahoo.com received : 11/6/2009 accepted : 3/8/2010 iraqi j pharm sci, vol.19(2) 2010 stability of cfm and cfz with ca against βlactamase 43 esbls have become widespread throughout the world and are now found in a significant percentage of e.coli and klebsiella pneumoniae strains in certain countries (4, 5, 6, 7) . there are also new families of esbls, including the cefotaximase (ctx-m) and oxatype enzymes, ceftazidimase, as well as novel unrelated βlactamases (8, 9, 10). the stability of different cephalosporins to the most important βlactamases was assessed and many clinical studies have shown that up to 75% of the βlactamases responsible for β lactam resistance in g-negative bacteria were r-plasmid mediated (11) . recently, new fourth generation cephalo-sporins, such as cefepime, cefpirome, cefoselis, cefditoren, cefozopran (12) , were introduced into antibacterial chemotherapy and their activities were compared with other β-lactams such as ceftazidime,imipenem and carbapenem, against..p.aeruginosa, enterobacteriaceae (e.coli, klebsiella pneumoniae) and gpositive bacteria. in addition several drug combinations have been produced which contain both a βlactam antibiotic and a β lactamase inhibitor; the inhibitor has high affinity for βlactamase, irreversibly binds to it, and thereby preserves the activity of the β lactam. currently, four penicillin inhibitor combinations are in clinical use: ampicillinsalbactam (unasyn), amoxicillinclavulanate (augmentin), ticarcillin –clavulanate (timentin) and pipracillintazobactam (zosyn) (12) . urinary tract infections (utis) are cause a significant health problem and e.coli has been reported to be the primary pathogen in approximately 80% of cases. e.coli, express structures called adhesins fimbriae or pili that help them bind to specific tissue (13) . the aims of the study are: 1. to know the prevalence of extended spectrum βlactamase (esbl) in multi drug resistant (mdr) strains of e.coli isolated from complicated urinary tract infections. 2. to evaluate the following combinations: cefamandol/clavulanateand ceftazidime / clavulanate for their in vitro antimicrobial activity against complicated urinary tract infections caused by esbls βlactamases. materials and methods standard strains with plasmid – mediated beta – laectamases were used: 1-e.coli k12 (tem-1 type βlactamase with isoelectric point 5.4) confer plasmid(r 111) and e.cloacae p99 ). 2-e.coli k12 (shv-1 type βlactamase pitton (type ii) i.p 7.7 (10).3-e.coli k12 600 rif and e.coli k12 600 nal sensitive to antibiotics (10) . 4-clinical isolates of e.coli. 5-pure enzyme of med labs. 6e.coli atcc 25922 provided by medical city identification of e.coli. a total of 100 strains of e.coli were selected and identified by api 20 e . system (biomerieux vitek, inc) (14) . antibiotic susceptibility test (disk diffusion method (10) the resistance pattern for antibiotics were determined by kirby/bauer diffusion assay on mueller – hinton agar (20ml / plate) the inoculum was 10 4 – 10 5 cfu / ml, of 6 hours cultures at 37ºc for 24 hours.the antibiotics used were as follow: amoxicillin (amo) 30 µg, augmentin(amc) (amo 20µg + ca10µg),(tic),azlocillin 100µg, timentin (tim) (75µgtic+ca 10 µg), cefaloridin(cfr) 30µg ,cefamandol (cfm) 30µg and ceftazidime (cfz) 30µg, cefixime(cxm) 30μg, ceftriaxone (ctr) 30µg, cefoperazon (cfp) 30µg ,aztreonam (atm) 30µg rifampicin (rif) 30µg, nalidixic acid (nal)30µg, ciprofloxacin(cip) 10mcg, amikacin (amik)10µg (tc)30µg, chloramphenicol (cm)30µg, gentamicin (gm)30µg, and cotrimoxazole (trimethoprime 2.5 µg + sulfamethaxazole 22.5 µg) (tm). minimum inhibitory concentrations (mics) mics were determined by dilutions of different concentrations of cfm, cfz, alone and in the presence of clavulanic acid (ca). according to the method recommended by the national committee for microbiology laboratory standards (france) powders of β -lactam antibiotics were obtained from (russell and beecham). (15) transfer of genetic information by direct conjugation method conjugal transfer of 3gc resistant esbl producing strains was done at 35°c -37°c in liquid medium {brain heart infusion (b.h)} or in solid media {trypticase soya agar (t.s.a) or mueller – hinton (m.h)} using e. coli k12 600 rif and e.coli k12 600 nal as recipient. equal volumes (1 ml) of culture of the donor and the recipient strain (108-109 cfu/ml) grown with agitation in tryptic soya broth were mixed and incubated statically for 18 hours at 35°c. transconjugants were selected on m.h agar containing 64-µg/ml nalidixic acid to inhibit the growth of donor and 2.5 µg/mlcfz to inhibit the growth of recipient strain (11) . iraqi j pharm sci, vol.19(2) 2010 stability of cfm and cfz with ca against βlactamase 44 phenotypic confirmatory disc diffusion test (pcddt) for esbl (18) ten µl of ca solution was added to discs of cfz and cxm one hour before culture, these were applied to the surface of a muller hinton agar, seeded with a suspension of 10 4 10 5 /cfu of bacteria under test.. an increase in zone diameter for either antimicrobial agent tested in combination with ca versus its zone when tested alone was observed. for cfz an increase in zone diameter of> 5mm and for cxm > 3 mm was considered as an esbl producer. extraction of ß– lactamase cell free beta –lactamases were prepared from strains known to be good producers of the desired enzymes, (ß–lactamases, type tem-1 and shv-1, r-plasmid mediated enzymes) and ß–-lactamase from e.cloacae p99 ( cephalospornase) as references. crud enzymes also prepared from test isolates of e.coli (10) . detection of ß– lactamase by macro iodometric method. this test is based on the reaction of the (oic) acid of penicillin with iodine. ß– lactamasehydrolyze penicillin to penicilloic acid, which in turn react with iodine, the presence of ß– lactamase in a test system was shown by decolorization of starch-iodine complex, observed in 1-18hours at 4°c (16). assessment of stability of ß– lactams to cellfree ß– lactamases (17) the surface of muller hinton agar was seeded with a suspension of sensitive indicator e.coli atcc. four discs containing ß– lactams under test were placed near filter papers discs; each of them was impregnated with 30μl of the extract enzymatic. the plates were incubated at 37°c for 18hours, the ß– lactamase activity was observed like half moon zone of inhibition. masuda microbiological method (17) ten clinical isolates were screened for ß– lactamase inhibitors using 10μl ca in combination with 30 μl of cfz or cfm. sensitivity discs containing cfz or cfm and a filter disc incorporated with10μl enzyme and 10μl (ca) as potassium clavulanate were placed on agar plate on which a bacterial suspension of sensitive e.coli atcc (standard) was spread the inoculum was 10 4 – 10 5 cfu / ml, of 6 hours cultures at 35 c 0 37c 0 for 24 hours. unchangeable inhibition zones demonstrate stability of the antibiotic to the enzyme. results and discussion extended –spectrum ß– lactamases ( esbls) are derivatives of enzymes such as shv-1 and tem-1 that have undergo site specific mutation that enable them to hydrolyze , and thus inactivate , oxyimino – cephalosprins such as, cefotaxime and ceftazidim. (19) . all clinically important reactions of ß– lactamase inhibitors , such as tazobactam , sulbactam, and calvulanic acid , involve ß– lactam ring cleavage during acylation of an active site . although other clavams produced in nature may possess antibacterial and antifungal properties, clavulanic acid is the only one known clavam with potent ß– lactamase inhibitory activity owing in part to its 3r ,5r stereochemistry , it is a potent inhibitor of ß– lactamase enzymes produced by many strains of staphylococcus aureus , e.coli , klebsiella , proteus , sheglla , pseudomonas , and haemophilus influenzae (20,21,) . 100% of the isolates were found to be resistant to amp, amo,cb,tic, azl, cfr,cfo, tc,cm and tm , 10% were resistant to cfm cxm, cfz,cfp,ctr , atm tim, and amc.also resistant to g,amk,cip and tm , 50% of the isolates were resistant to nal and rif as shown in table 1. esbl was detected in 10 isolates by pcddt the zone of inhibition increased in presence of ca. for cfz >10mm, and for cfm and cxm>5mm, potentiation of the inhibiton zone of 3gc in the presence of ca was observed.. indicated esbl production in ten strains; the diameters zone of inhibition for amc and tim were range from 0 5mm while the normal diameters zones of inhibition were for amc 14-21mm and for tim is 13mm. the critical normal mics for tim and amc were (4-16) and (128) respectively. the mics were studied for ten clinical isolates of e.coli in comparison with standard resistant strains, the range of mics for cfm was 512 2048 μg/ml and for cfz 32-64 μg/ml, while for non esbl producer it ranged from 0.02-8 µg/ml. after the addition of ca eight-fold reduction or more in mics (table 2 ) . these results was in iraqi j pharm sci, vol.19(2) 2010 stability of cfm and cfz with ca against βlactamase 45 agreement with the investigation of chaudhary ,u, aggarwal-r (17) indicated esbl producers. all the isolates were sensitive to imp, but among the non ß– lactam antibiotics cip and amk were most effective drugs 90 strains were sensitive. resistance to cfz was transferred to recipient e. coli k12 c600 rif or e. coli k12 c600 nal strains, along with resistance to other ß– lactam antibiotics, esbl production is coded by genes on conjugation plasmids which are easily transmitted among different members of enterobacteriaceae, all esbls have serine at their active sites. the results of detection of ß– lactamases by iodometric method were positive for 10 strains comparing with standard negative and positive ß– lactamases r111 (tem-1) and e.coli esbl producer. the inhibition of betalactamase production by ca has been demonstrated with many strains of bacteria, this effect potentiates the action of many beta lactams, such as amp, amo, cb and azl. many clinical reports of combination of amo with ca have been encouraging, in urinary tract infections due to ß– lactamase-producing organisms type tem and shv, whilst amo alone had no effect, the addition of ca (as salt) dramatically change the half moon inhibition zone to complete inhibition zone (3,4,11) . figure 1 indicate the activity of ß– lactamase extracts against ß-lactams antibiotics, figure 2 indicate the antibiotic – enzyme interaction by the highly sensitive double disks technique, demonstrated their hydrolysis, however ß-lactamase of e.cloacae not effected by amc and inhibited by azl and hydrolyzed all cephalosporins. ( 5,6,20) . table 1: sensitivity tests of ten strains determined by disk diffusion test . abbreviation's : amo: amoxicillin amc:amoxiclave; cb: carbenicillin;azl: azlocillin; tim: timentin; cxt cefoxitin ; cfm: cefamandol; cfz: ceftazidime; cxm:cefixime,ctr:ceftriaxone, cfp :cefoperazon; amk: amikacin, cip: ciprofloxacin , rif: rifampicin; nal: nalidixic acid; the diameters of zone of inhibition for ampicillin, amoxicillin, carbenicillin,ticarcillin, azlocillin,cefazolin(cfo), cefaloridin(cfr),cefoperazone(cfp) aztreonam(atm) , tetracycline ; chloramphenicol; trimethoprim and gentamicin were zero. all of them sensitive to imipenem (imp) 17-23mm and cefoxitin 15-22mm .normal zone for amc: 14-21mm; tim:13mm; cfz,ctr,ctx: 15-21mm; cfm:15-22mm. amk:25-32mm; rif :19-32mm;cip:30-40mm. table 2: minimum inhibitory concentrations of ten uropathogenic e.coli comparing with standard strains . no. of isolate e.coli mics mcg/ml imp cfm cfz cfm+ca cfz+ca 1,2,3 2 512 64 2 1 4,5,6 1 1024 64 0.5 2 7,8,9,10 4 2048 32 0.25 0.5 e.coli (453)* shv-1 (7.7) (19) 16 16 0.05 0.25 0.25 e.coli (r111) tem-1 (5.4)* ) 16 32 0.02 0.25 0.25 e.cloacae(p99** (8.3)* 64 512 64 32 64 e.coli (esbl) 4 128 64 0.25 2 * isoelectric points. ** cephalosporinase. normal values of mics : cfm s<8 mcg\ml r >32mcg\ml cfz s<4 mcg\ml r >16mcg\ml no of isolates diameters of zone of inhibition / mm amo amc tic tim cxt cfm cfz cxm ctr amk cip rif nal 1 0 4 0 5.5 16 2 3 10 11 10 12 19 0 2 0 4.5 0 7 16 3 3 12 10 11 10 0 18 3 0 3.5 0 8 18 2 5 8 5 14 12 19 0 4 0 4 0 8.5 17 0 4 4 13 12 11 10 0 5 0 5 0 9 17 6 14 15 12 12 11 19 0 6 0 5.5 0 9 19 10 14 15 13 12 18 0 0 7 0 6 0 7 20 11 11 15 12 18 20 0 18 8 0 6 0 6.5 20 5 10 13 10 18 24 0 19 9 0 6 0 9.5 20 5 11 12 10 15 22 19 19 10 0 7.5 0 9 17 7.5 13 10 10 12 10 19 18 e.coli (esbl) 0 0 0 5 17 7 13 12 10 12 18 11 17 e.coli atcc 25922 21 21 13 13 22 22 21 21 21 32 40 32 33 iraqi j pharm sci, vol.19(2) 2010 stability of cfm and cfz with ca against βlactamase 46 ceftriaxone azthreonam ceftazidime cefixime imipenem cefaloridin azlocillin carbenicillin figure 1: activity of ß– lactamase against ßlactams antibiotics figure 2: antibiotic –enzyme interaction ,by the highly sensitive double disks technique (24) demonstrated a : hydrolysis of ceftazidime and cefalmandol by β-iactamases producing e.coli b: inhibition by clavulanic acid (ca) . conclusions the ten clinical isolates in this study were very resistant to amc, tim,cfm ,cfz,cxm,cfp, ctr and atm, but sensitive to imipenem comparing with standard tem-1 and shv-1 (plasmidic penicillinases) and e.clocae p99 ) (chromosomic cephalosporinase) indicating the prevalence of extended-spectrum β-lactamases (esbls) enzymes that hydrolyze and cause resistance to oxyimino-cephalosporins and aztreonam. our study shows presence of esbl producer e.coli in ten clinical isolates. the routine antimicrobial sensitivity test may fail to detect esbl, mediated resistance against 3gc and detection of esbl production should be carried out as a routine in diagnostic laboratories by pcddt as it is a simple and cost effective test, the combination with clavulanic acid bringing the susceptibility back, confirms the esbls. esbls have become widespread throughout the world and are now found in a significant percentage of e.coli and klebsiella pneumoniae strains in certain countries, (6, 7, 9, 10) . the increasing emergence of cephalosporins resistant e.coli has leaded to concern about the use of various combination therapies. references 1. jensen, -s-e; 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22(2): 75-80 19. matthew kalp et al. why extended – spectrum ß– lactamases shv-2 and shv5 are ' hypersuceptible' to mechanism based inhibitors, biochemistry , 2009, 48 (41) , 9912-9920 20. mattew kalp et al . efficient inhibition of class a and class d ß– lactamases by michaelis complexes , journal of biological chemistry , 2007.28(30) . 21. mary l. raber , micheal f. freeman and criag a.twonsend , journal of biological chemistry , 2009, 284 (1). 22. siham-ss. marc o. sirot-djoly-b. and cluzel –r spread of shv-1 betalactamases in e.coli isolated from fecal samples in africa.1987. antimicro. agent and chemother.vol 31 (6)943-945. 23. -ma, -y; li, -j-y; yao, -l; zhang, -l; hu,-c-q; jin,-s-h zhonghua-yi-xueza-zhi. antimicrobial resistance of escherichia coli isolates collected from inpatients and outpatients] 2003 25; 83(12): 1046-8. a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci , vol.17 (2) , 2008 single dose in oral surgery 41 single dose antibiotic prophylaxis in outpatient oral surgery comparative study fadia y. alhamdani 1,* and faaiz y.alhamdani ** * department of clinical pharmacy, college of pharmacy, university of baghdad. , baghdad , iraq. ** department of oral and maxillofacial surgery ,university of almusransiriyah,college of dentistry. abstract it is clear that correct application of antibiotic prophylaxis can reduce the incidence of infection resulting from the bacterial inoculation in a variety of clinical situations; it cannot prevent all infections any more than it can eliminate all established infections. optimum antibiotic prophylaxis depends on: rational selection of the drug(s), adequate concentrations of the drug in the tissues that are at risk, and attention to timing of administration. moreover, the risk of infection in some situations does not outweigh the risks which attend the administration of even the safest antibiotic drug. the aim of this study was to compare between 2 prophylactic protocols in out patients undergoing oral surgical procedures. thirty patients, selected from the attendants of oral surgery clinic in al-karamah dental center, were subjected to different oral surgical procedures under local anesthesia. these patients were given single dose antibiotic prophylaxis in 2 groups; 1st group were 15 patients given 1 million i.u. of procaine penicillin i.m. 30 minutes before oral surgery, 2nd group were 15 patients given 600mg clindamycin orally 1 hours before oral surgery. the maximum time for all procedures was 2 hours. there was no difference between procaine penicillin (1 million i.u.), and clindamycin (600mg), regimens concerning post operative infection in out patient’s oral surgical procedures. key words: antibiotic prophylaxis, outpatient oral surgery ةصالخلا ـةضو ا ن م أ ئوووو اأن أ ةضو ي ا تأد لـ ـضي ءا عإ حووحص ا قي طتلا أأ حضوولا ن تاطاا أ وأل عإ أ ضنقضن أ ـةض تا لص أ حضلا ن أ قءضضت ي أ ت رسي تاطاا أ ت و أ قءضضتع ب قب ووتا تاطاا أ ة ن أ ت ت ووي أ ضتو ي . تاطالتخالا لك عنمي ال حيحصلأ قـيبطتلا نإ .ةعونتملا ةــيريرسلا تالاحلا نم ددــع ببسإ أ ضنقضون ع ىبئص حضل تاطاص أ وـ ـض ح ئ ا أ ةضوع لض ت حأ أأ قءضضت ي ل ءتا ضنضا أ ضلحت تاطاا أ حضلا ن أ ويوات بلل . تاطالتخالا لك عنمي ال حيحصلأ قـيبطتلا نإ .ةعونتملا ةــيريرسلا تالاحلا نم ددــع ببسإ أ تضضسي أ أ حضض ل أ تنحض حلح ل أ لنأ لأ ضتلضل أ ء بض حلحوو ل )أ ئ ا أ ةضوع( بض أ ت ض أ لتا ب حضلو أ قءضضتع لأ اضق ط حضوـضي تت ضضت ض تاطالتخالا لك عنمي ال حيحصلأ قـيبطتلا نإ .ةعونتملا ةــيريرسلا تالاحلا نم ددــع ببس ن م أ ئ ا لمو ع تاطات ا ضحقض را طضأ تاطاا ن تاطالتخالا لك عنمي ال حيحصلأ قـيبطتلا نإ .ةعونتملا ةــيريرسلا تالاحلا نم ددــع ببس ن م أ ئ ا أ ةضوع . تاطاا أ ست ت ب الت تاطال ات بل أ حضلا ن أ قءضضت ي ع تاطا ا . تاطالتخالا لك عنمي ال حيحصلأ قـيبطتلا نإ .ةعونتملا ةــيريرسلا تالاحلا نم ددــع ببسإ وتد ماط أ تلأضي مو أ ح لاوي بضا بتلرولو ضا ليضضا لـ ـضضا ح تلأ أ ا ا لئلوإ ضتأحلن نعلات أ ئ اأ يتأطضي بض أ لض اأن أ ستأطضي نب أ ىضت إ )رةي أ ضلت ت أ وللض( .ر أحضض ل تلتوإ تاطات ئ تاطاا ـقووص أ تأيلضا أ ا ا للللإ ـ أ ستأطي بض تاطاتلل أ ءتأ تاطاي أ ضلــووض نب أ ىضت إ طضم ر تاطالتخالا لك عنمي ال حيحصلأ قـيبطتلا نإ .ةعونتملا ةــيريرسلا تالاحلا نم ددــع ببسحئ لوو ضتأحلن يتأطضي تاطاضتو ي رةي أ ضلت ت أ وللض . مي م أ تلأ إ ر تاطالتخالا لك عنمي ال حيحصلأ قـيبطتلا نإ .ةعونتملا ةــيريرسلا تالاحلا نم ددــع ببس ن رم يت ي تاطاوتات تاطاا حو ل أ قتلل ا بت حضا لمض تاطاحضوإ لطتت ضي لر تاطالتخالا لك عنمي ال حيحصلأ قـيبطتلا نإ .ةعونتملا ةــيريرسلا تالاحلا نم ددــع ببس ن رم ئحض ) ح س و ي تاطات ئ ( وو ضس لم 15 تاطاحل ا ات ن أ و ) ح س و ي أ ا اضي لرئ 600 تاطات ئ ( ل ح ل أ ءوحتت تاطا ضا 15أ ىل أ أ ءواي تاطاا أ وـوي أ لم ايتأم أ ضتأحلن أ ستأطضي ض ض إ.ل اي أ تضضسي أاا ءا مت ت عع أحضلر بضا أ س و ضضا تاطاا ا طضي ت طتلا أ حضلاو ن أ قءضضت ووي بلت أ ضتأحص أ ستأطض أ و وع بلاوأ ا . introduction the use of antimicrobial agents to prevent infection is effective in many circumstances, and it is limited to specific, well-accepted indication to avoid excess cost, toxicity, and antimicrobial resistance.(1) preoperative topical, oral, and intravenous antimicrobial prophylaxis has been important in decreasing the incidence of surgical site infection.(2,3) the time taken for an antibiotic to reach an effective concentration in any particular tissue reflects its pharmacokinetic profile and the route of administration. (4) administration of prophylaxis more than three hours after the start of the operation significantly reduces its effectiveness. for maximum effect, it should be given just before or after the start of the operation. (5) preoperative antimicrobial surgical prophylaxis is recommended for operative procedures that have a high rate of postoperative wound infection, when foreign materials must be implanted, or when the wound infection rate is low but the development of a wound infection results in a disastrous events. (2,3,6) 1 corresponding author : e-mail : samanmerseds@yahoo.com received : 16/7/2008 accepted : 22/11/2008 42 infection of the incised skin or soft tissues is a common but potentially avoidable complication of any surgical procedure. some bacterial contamination of a surgical site is inevitable, either from the patient's own bacterial flora or from the environment. (7) in procedures that require the insertion of implants or prosthetic devices, the term surgical site infection is used to encompass the surgical wound and the implant. surgical site infection also encompasses infections involving the body cavity (e.g. a. subphrenic abscess ), bones, joints, meninges and other tissues involved in the operation. (8) prophylactic administration of antibiotics inhibits growth of contaminating bacteria and their adherence to prosthetic implants, thus reducing the risk of infection. (9) the goals of prophylactic administration of antibiotics to surgical patients are to: reduce the incidence of surgical site infection, use antibiotics in a manner that is supported by evidence of effectiveness, minimize the effect of antibiotics on the patient’s normal bacterial flora, minimize adverse effects and cause minimal change to the patient’s host defenses.(2) it is important to emphasize that surgical antibiotic prophylaxis is an adjunct to, not a substitute for, good surgical technique. antibiotic prophylaxis should be regarded as one component of an effective policy for the control of hospital-acquired infection. (10,11) the american college of surgeons classified wound surgery into 4 categories: clean, clean-contaminated, contaminated and dirty wound, according to this classification trans-oral wound is considered clean contaminated, that is, class ii, these wounds should receive protection if (a) the patient has depressed host defenses. (b) a prosthetic device is being inserted. (c) the sequel of an infection is serious; and (d) some aspect of the procedure, such as increased duration or decreased local blood supply, makes infection more likely. (8,11) prophylactic antimicrobial agents should be administered not more than 30 to 60 minutes before surgery.(8-9) exceptions to this rule are cesarean procedures, colonic and urologic procedures. therapeutic concentrations of antimicrobial agents in tissue should be present throughout the period that the wound is open. the duration of antimicrobial prophylaxis for the majority of procedures is controversial; however, experts recommend at most one or two postoperative doses. (2,3) the antibiotics chosen for prophylaxis can be those used for active treatment of infection. however, the chosen antibiotics must reflect local, disease-specific information about the common pathogens and their antimicrobial susceptibility.(12) procaine penicillin is one of the semi-synthetic penicillin obtained by alterations in the prosthetic group differ from the naturally occurring product (penicillin g ) in three dimensions: their resistance to acid makes oral administration possible, they may be resistant to the action of penicillinase and their spectrum of antimicrobial activity is usually broadened for many streptococcal infections. (14) it is bactericidal, act by interfering with bacterial cell wall synthesis.(10) clindamycin is a bacteriostatic act by interfering with protein synthesis of bacteria. it is active against gram positive cocci, including streptococci and penicillinresistant staphylococci, and also against many anaerobes, especially b. fragilis( 15). subjects and methods after a thorough history taking, clinical, and radiographic examination, thirty patients attending al-karamah dental center were selected to participate in this study. these patients are mostly from the residents of the neighborhood, which is a relatively a low socioeconomic level. none of patients had medical history or active infectious process. all patients in this study are not allergic to penicillins. these patients were subjected to oral surgical procedures under local anesthesia maximally 2 hours the surgical procedures involved bone and soft tissue and these includes: removal of impacted lower 3rd molar, apicectomy for upper central and lateral incisors. patients were classified into two groups according to the antimicrobial agent: 1. 1st group were 15 patients given single i.m. doses of 1 million i.u. procaine penicillin 30 minutes before oral surgery. 2. 2nd group were 15 patients given 600mg clindamycin orally 1 hour before surgery. number of female patients included in our study was 17, while the number of male patients was 13. patients were classified into 3 groups. group one (10-19) nine patients, group two (20-29) thirteen patients and group 3 (3039) eight patients. surgical procedures included in this study were: removal of impacted lower rt 3rd molar (11 cases), removal of impacted lower lt 3rd molar (8 cases), removal of impacted of upper rt 3rd molar (1 case), apicectomy for upper rt central incisor (5 cases) apicectomy for upper lt central incisor (4 cases) apicectomy for upper rt lateral incisor (1 case).meticulous handling of the tissues, avoidance of unnecessary surgical trauma and copious irrigation of the wound before closure to remove foreign bodies and debris, leaving 43 no potential foci for bacterial infections were of crucial importance in our measures. patients were examined 48 hours post-operatively to investigate the presence of any local and general signs of post operative infection these signs are: increased pain or tenderness, post operative swelling at the site of surgery, enlarged, tender regional lymph node and fever. the same investigated parameters were also examined 7th day after surgery, for suture removal. results characterization of patients according to age, gender and type of oral operation is given in figures 1, 2, and 3. no postoperative infections were recorded in the two groups, and no postoperative complications in the two groups. figure (1) no. of patients according to gender figure (2) : no. of patients according to age group figure( 3) no. of patients according to surgical procedures discussion although some studies found that antibiotic prophylaxis in some oral surgical procedures is controversial (12,16,17).its generally agreed that when antibiotic prophylaxis is decided, the antibiotic must be present in the systemic circulation at a high level at the time of surgery and is usually given as one dose (17,18,19). in spite of the fact that preoperative antibiotic prophylaxis is an established practice (17, 20), there is no consistent protocol for the method or duration of drug administration in oral surgical procedures, (21) and this is true for iraqi dental surgical centers. although it is agreed that procedures entailing entry into the oropharynx or esophagus, need antibiotic coverage of aerobic cocci is indicated. prophylaxis has been shown to reduce the incidence of severe wound infection by approximately 50 percent. (22). our choice for procaine penicillin depends on two factors 1. most of oral infections caused by penicillin sensitive bacteria (23) 2. the use of penicillin is an established clinical practice in advanced surgical centers (22,23), on the other hand some of the studies select clindamycin for antimicrobial prophylaxis in oral surgery, clindamycin is occasionally chosen for orthopedic surgical prophylaxis, where it has an excellent activity against staphylococcus spp. and bacteroides fragilis, but have no activity against enteric microorganism.(22,24). also it has good reputation for tissue penetration, with almost the same effectiveness of penicillin against anaerobes. (13) the minimum inhibitory concentration (mic) of clindamycin is achieved within the first 2-3 dose intervals. thus, stable drug concentration is then maintained for greater than 6 hours 0 2 4 6 8 10 12 14 16 18 males females 0 2 4 6 8 10 12 14 group 1 group 2 group 3 0 2 4 6 8 10 12 impaction lower rt 3rd molar impaction lower lt 3rd molar impaction upper rt 3rd molar apicectomy upper rt centeral incisor apicectomy upper lt central incisor apicectomy upper rt lateral incisor 44 after the last dose. (13) in our selected sample; female patients were more than the males, this may be explained by the fact that females are more interested in oral hygiene. we have noticed that the number of patients in the age group (20-29) is higher than other age groups; this could be attributed to the fact that the problems of impacted 3rd molar or its complications are usually experienced in this age group. no post operative infections were recorded in our sample, for all patient groups (no difference between parenteral and oral route of administration). we conclude that there is no difference in surgical prophylaxis between procaine penicillin (1 million i.u.), and clindamycin 600mg concerning post operative infection in out patient’s oral surgical procedures, and this may be explained by the fact that both antibiotics used in this study covered both pathogens that are mostly involved in oral infections. this conclusion shown in figure (4) which represents surgical removal of impacted lower 3rd molar (group 2)and figure (5) which represents apicectomy for upper central incisor intraoperatively (group 1), figure (6) postoperatively for the same case, while figures 7,8 and 9 represent apicectomy for lower central incisor, preoperative, intraoperative and postoperative respectively (group 2). figure (4): apicectomy with periapical dental cyst enucleation for upper central incisor (intra operative picture) the patient has been given clindamycine 600 mg 1 hr. preoperatively (group 2) figure (5): surgical removal of impacted lower 3rd molar (intra operative picture) the patient has been given 1 million i.u. procaine penicillin 30 minute pre operatively (group 1) figure (6) : postoperative picture (3rd postoperative day)for the site of operation (postoperative oedema subsided, no signs of infection noticed) figure(7): 21 years old female with extra oral sinus due to infected cyst associated with necrotic lower central incisor (pre operative picture), (group 2) 45 figure (8): inta operative picture after the removal of the infected cyst. this patient has been given 600 mg clindamycin 1 hr. pre operatively figure (9): extra oral picture after one month of the operation shows the process of healing of the extra oral sinus references 1. mums ca, playfair jhl, roitt im, wakelin d and williams sr. medical microbiology 1998; 2nd edn, pp 32−35 mosby, st louis . 2. nooyen smh, overbeek bp, brutel driviere a, storm aj, langemeyer jjm. prospective randomised comparison of single-dose versus multiple-dose cefuroxime for prophylaxis in coronary artery bypass grafting. eur j clin microbiol infect dis 1994;13:1033-1037 . 3. centers for disease control and prevention. staphylococcus aureus with reduced susceptibility to vancomycin— united states, 1997 [published erratum appears in mmwr morb mortal wkly rep 1997;46:851]. mmwr morb mortal wkly rep 1997;46:765-766 . 4. southorn pa, plevak dj, wright aj, wils on wr. adverse effects of vancomycin administered in the perioperative period. mayo clin proc 1986;61:721-724 5. sekhar ch, narayanan v and baig mf. role of antimicrobials in third molar surgery: prospective, double blind, randomized, placebo-controlled clinical study. br j oral maxillofac surg 2001; 39: 134−137. 6. centers for disease control and prevention. reduced susceptibility of staphylococcus aureus to vancomycin— japan, 1996. mmwr morb mortal wkly rep 1997;46:624-626 . 7. emmerson am, enstone je, griffin m, kelsey mc, smyth et. the second national prevalence survey of infection in hospitals – overview of the results. j hosp infect 1996; 32: 175-90. 8. tornqvist io, holm se, cars o. pharmacodynamic effects of subinhibitory antibiotic concentrations. scand j infect dis 1990; 74: 94-101. 9. cars o, odenholt-tornqvist i. the postantibiotic sub-mic effect in vitro and in vivo. j antimicrob chemother 1993; 31: 159-66. 10. moss f, mcnicol mw, mcswiggan da, miller dl. survey of antibiotic prescribing in a district general hospital. i. pattern of use.lancet 1981;2:349-52. 11. goldmann da, weinstein ra, wenzel rp, tablan oc, duma rj, gaynes rp, et al. strategies to prevent and control the emergence and spread of antimicrobialresistant microorganisms in hospitals. a challenge to hospital leadership. jama 1996; 275: 234-40. 12. heit jm, farhood vw, edwards rc; survey of antibiotic prophylaxis for intraoral orthognathic surgery j.oral and maxillofac. surg.1991 apr;49(4):340-2. 13. mcgowan je. cost and benefit of perioperative antimicrobial prophylaxis: methods for economic analysis. revinfect dis 1991; 13: 879-89. 14. martin c. antimicrobial prophylaxis in surgery: general concepts and clinical guidelines. french study group on antimicrobial prophylaxis in surgery, french society of anesthesia and intensive care. infect control hosp. epidemiol, 1994; 15: 463-71. 15. page cp, bohnen jm, fletcher jr, mcmanus at, solomkin js, wittmann dh. antimicrobial prophylaxis for surgical wounds. guidelines for clinical care. arch surg 1993; 128: 79-88. 46 16. m. v. martin1, a. n. kanatas2 and p. hardy3 antibiotic prophylaxis and third molar surgery british dental journal (2005); 198, 327-330 . 17. peterson l j,booth df; efficacy of antibiotic prophylaxis in intraoral orthognathic surgery; j. oral surgery 1976 dec;34(12):1088-91. 18. shapiro m. prophylaxis in otolaryngologic surgery and neurosurgery: a critical review. rev infect dis 1991; 13(suppl 10):s858-68. 19. page cp, bohnen jm, fletcher jr, mcmanus at, solomkin js, wittmann dh. antimicrobial prophylaxis for surgical wounds. guidelines for clinical care. arch surg1993;128:79-88. 20. mcdonald m, grabsch e, marshall c, forbes a. single-versus multiple-dose antimicrobial prophylaxis for major surgery: a systematic review. aust n z j surg 1998;68: 388-96. 21. worrall sf. antibiotic prescribing in third molar surgery. br j oral maxillofac surg 1998; 36: 74−76. 22. david h. perrott, orthognathic and reconstructive surgery 1997, manual of oral and maxillofac. surg. massachusetts general hospital, 3rd edition: 273. 23. larry j. peterson: principles of antibiotic therapy 1994; oral and maxillofacial infections 3rd edition ; chapter 5 : 166, 186-190 24. burke jf. the effective period of preventive antibiotic action in experimental incisions and dermal lesions. surgery 1961;50:161-168. iraqi j pharm sci, vol.19(2) 2010 protein profiles in children with kala-azar 70 urine protein sds-page reveals different profiles in iraqi children with kala-azar yassir mk al-mulla hummadi* ,1 * department of pharmaco-therapeutics, college of pharmacy, al-mustansiriyah university, baghdad, iraq. abstract urine proteomics have been an area of interest and recently in kala-azar as an alternative sample type for serum or plasma. because of simplicity, noninvasiveness of collection and simpler matrix. many studies had detected an increased protein excretion in the urine of patients with active kala-azar due to renal involvement particularly by an immunological related mechanism(s). this study have demonstrated the presence of three different protein profiles in iraqi children (patients: including 60 children aged 4-60 months) with defined kala-azar using the conventional sds-page on urine samples. urine protein profile in kala-azar patients revealed three groups of banding patterns: group1(33.4)% of the patients show the pattern of 5 bands with a mw. ranged (512.861-158.489), groups-2, 3 were (55, 11.6) % of the patients showing 2 banding proteins with a mw. ranged (512.861-199.526) , (199.526-181.97) respectively. these findings may be correlated with other epidemiological studies that revealed the presence of different clinical presentations like fever, spleenomegally, hepatomegally, thrombocytopaenia, and different response to leishmania therapy. furthermore, the presence of different protein patterns may also be related to the chronicity of infection and the degree of renal involvement. the presence of a similar protein band between group-1 and 2 may be of diagnostic purpose and further studies on expanded number of patients are required to identify that kind of protein or other urine protein profiles. key words: protein band, protein pattern, sodium dodecyl sulphate-poly acrylamide gel electrophoresis (sds-page), urine proteome, visceral leishmaniasis. الخالصة ٔيؤخشاً فٙ داء انهشًبَٛبث االحشبئّٛ كًُٕرج بذٚم نعُٛبث انًصِم أَٔ اْخًبو ةحشكم انبشٔحُٛبث فٙ عُٛبث انبٕل دائش انبالصيب. بسبب انبسبطِت فٙ انحصٕل عهّٛ ٔسٕٓنت خًعّ بخدُب انٕسبئم انببضعّ كبالدٔاث اندبسحّ اضبفّ انٗ بسبطت يبدحّ ٍِ يخضاٚذ فٙ بِٕل انًشضٗ ا ٍْ انِذساسبِث طشَذ بشٔحٛ نًصببٍٛ ببنهشًبَٛب االحشبئّٛ فٙ انفخشِ انُشٛطت نهًشض االسبسّٛ. إكخشفْج انعذٚذ ِي ٍِ يخخهفِت فٙ ًَبرج بٕل ِ٘ خصٕصبً بًب ٚخعهق ببٜنِٛت انًُبعّٛ. كشفج ْزِ انذساسِت ٔخَٕد ثالد يدبيٛع اًَبط بشٔحٛ بسبب انخبثشانكهٕ ٍِ )انًشضٗ انًشخصٍٛ ببنهشًبَٛب االحشبئّٛ: شٓش( ٔرنك ببسخخذاو حقُٛت انخشحٛم انكٓشببئٙ ٓٙ-ٗطفم بعًِش ٓٙاألطفبِل انعشاقٛٛ ( % يٍ انًشضٗ اظٓشث خًس حضو ٗ.ٖٖ) ٔانخقهٛذ٘ )ْالو يخعذد االكشٚم ايبٚذ( عهٗ عُٛبِث انبِٕل. ٔقذ كبَج كًب ٚهٙ: يدًٕعت انًشضٗ اظٓشث ( % يٍ ٙ.ٔٔ، َ٘٘كبَْج ) ٖٔ ٕ( دانخٌٕ، يدًٕعّ 23ٗ.2٘ٔ-2ٙٔ.ٕٔ٘بشٔحُّٛٛ َيع ٔصٌ خضٚئٙ حَشأَذ بٍٛ ) ٍِ نكم يًُٓب َيع ٔصٌ خضٚئٙ حَشأَذ بٍٛ ) ( دانخٌٕ ٔ عهٗ انخٕانٙ. 2ٔ.31ٔ-ٕٙ٘.33ٔ، ) (ٕٙ٘.33ٔ-2ٙٔ.ٕٔ٘حضيخٍٛ يٍ انبشٔحٛ ًَّٗ ى انكبذ حضخ ’ْزِ انُخبئحِ قَْذ حُشْحبَظُ ببنِذساسبِث انٕببئّٛ األخشٖ انسببقّ ٔانخٙ َكشفْج ٔخٕد انعٕاسِض انسشٚشِٚت انًخخهفِت يثم انُح ًَ ٍِ انًخخهفِت نَُشبَّ ب أٚضبً ٔانطحبل، قهت انصفٛحبث، ٔاسخدبببّث يخخهفّ نعالجِ انهشًبَٛب االحشبئّٛ. عالٔة عهٗ رنك، حضٕس أًَبِط انبشٔحٛ . كًب ِ٘ ٍِ يًبثهِت بٍٛ انًدًٕعِت إٌُيخََعهّقت ببصيبٌ انًشض ٔدسخِت انخذّخِم انكهٕ ٍْ قَْذ حَُكَٕبٌ ٕٔ ٔٔخٕد حضيت بشٔحٛ بًكبٌ انفبئذةِي ِٙ يًب ٚسخذعٙ اخشاء ِدساسبِث ٍِ أَٔ كشف أخشٖنهغشِض انخشخٛص ٍْ انبشٔحٛ ٍْ انًشضٗ نخًَٛٛض رنك انُٕعِ ِي أًَبطٔعهٗ عذِد يّٕسعِ ِي يٍ انبشٔحُِٛبث فٙ عُٛبث انبِٕل. أخشٖ introduction visceral leishmaniasis and cutaneous leishmaniasis caused by leishmania donovani and leishmania tropica or leishmania major respectively are known endemic diseases in iraq.visceral leishmaniasis had been prevalent in iraq for the last 2-3 decades causing serious problems (1) . it represents an important factor in infant's mortality in this country (2) .diagnosis of visceral leishmaniasis generally based on clinical presentation and microscopical demonstration of the parasite in smears, aspirates of bone marrow or culture of these aspirates on certain culturing media. however, the techniques are painful, hazardous and need skilled personnel and equipped hospitals some times difficult to be obtained (3) . since visceral leishmaniasis is occurring in places of poor socioeconomic conditions where health services are poorly developed (4) , the diagnosis in this case is difficult to be obtained using these techniques (3) . however, presence of leishmania donovani soluble antigen, corresponding antibody and component of the complement in the serum of patient with active kala-azar has been demonstrated in a number of studies (1) , al-bashir (5) . 1corresponding author email : yassir.almullahummadi@ymail.com received : 25/5/2010 accepted : 29/9/2010 iraqi j pharm sci, vol.19(2) 2010 protein profiles in children with kala-azar 71 on the other hand, the detection of leishmania donovani parasite antigen in the urine samples of patient with active kala-azar has been demonstrated in a number of studies by immunoblotting (6) ; enzyme linked immunosorbent assay (elisa) (7) ; latex agglutination test (lat) (8) and direct agglutination test (dat) (8, 9) .furthermore, urine samples are more easily to be collected especially from young children, as well as facilitating field studies (9) .therefore, this study aims to demonstrate the presence of variations in protein profiles in a number of iraqi patients with defined visceral leishmaniasis using the conventional sodium dodecyl sulphate-poly acrylamide gel electrophoresis (sds-page) in urine samples. patients and methods this study was divided into two groups: 1. patients: including 60 children aged 4-60 months, admitted to paediatric ward of alkadhimiyah teaching hospital-al-nahrain university from december 2007febriwary 2009. all were proven cases of kala-azar by: clinical manifestation of fever, hepato-spleenomegally, the demonstration of the parasite the amastigotes from indirect smear of bone marrow and serological diagnosis by indirect immunofluorescence antibody test ifat. 2. control: consist from 10 healthy children all were seronegative. urine samples 24 hours urine specimens were collected from each child in the control or the diseased group. urine samples from all children were concentrated 100 times using amicon apparatus (amicon corporation davers, ma) and kept at -20 o c till use to avoid bacterial contamination as described by baqir et al. (2002) (6) ; al-bashir and ali (2003) (8) .total protein for each sample was measured according to the method of lowery et al. (1951) (10) . the protein electrophoresis was dependent on the conventional polyacrylamid gel electrophoresis page as described by fehrnstrom and morberg (1977) (11) . urine protein profile urine protein electrophoresis was performed as described by fehrnstrom and morberg (1977) (11) , using coomaise brilliant blue staining.migration distance of the calibration proteins were measured after staining the protein bands. the relative mobility (rm) was measured too according to the following equation: the calibration curve was constructed by plotting rms versus log. molecular weights for calibration proteins fig.(1). the molecular weight of proteins of interest was determined from the position of its rm value on the calibration curve. figure 1: standard curve for approximate estimation of molecular weights of various proteins inurine samples. results conventional polyacrylamide gel electrophoresis has been used to differentiate between protein patterns in serum of normal and patients with kala-azar. coomassie brilliant blue stain has been used to visualize the band separated on the gel (12) . urine of the control group has a specific protein pattern with one protein bands fig.(2). control group (1) group (2) group (3) figure 2 : urine protein profiles in normal and kala-azar children. the relative mobility value (rm) was 0.48 with a molecular weight 199.525.urine protein profile in kala-azar patients revealed three banding patterns ranged from (2-5) protein iraqi j pharm sci, vol.19(2) 2010 protein profiles in children with kala-azar 72 bands. the (rm) values were ranged from (0.22-0.52). see fig.(2). the first protein pattern demonstrates five bands. the rm values were ranged from (0.22-0.52), with molecular weights ranged from (512.861 158.489) ×10 3 dalton respectively. four out of these bands were shown to be abnormal from the control these are bands (1, 2, 3 and 5) as shown in table (1). this band picture was found in 20 patients (33.4 %) table (2). the second protein pattern shows two bands the rm values were ranged from (0.22-0.48), with a molecular weights (512.861 and 199.526) ×10 3 dalton respectively. one of these two bands (band-1) was shown to be different from the control as shown in table (1). this profile was found in 33 patients (55 %) table (2).the third protein shows two bands the rm values were ranged from (0.48-0.50), with a molecular weights (199.526 and 181.970) ×10 3 dalton respectively. one of these two bands (band-2) was shown to be different from the control as shown in table (1). this profile was found in 7 patients (11.66 %) table (2). table 1 : rm values and molecular weight for each band in different urine protein profile groups. band no. control rm control mw×1 3 dalton g1rm g1mw×10 3 dalton g2 rm g2mw×10 3 dalton g3rm g3mw×1 3 dalton 1 0.48 199.526 0.22 512.861 0.22 512.861 0.48 199.526 2 0.32 316.227 0.48 199.526 0.50 181.970 3 0.37 236.026 4 0.48 199.526 5 0.52 158.489 rm: relative mobility, mw: molecular weight in daltons. table 2 : different urine protein profiles with the percentage of patients in each group. group total no. bands no. abnormal bands no. patients % patient 1 5 4 20 33.4 2 2 1 33 55 3 2 1 7 11.6 discussion the detection and identification of trace amounts of proteins in complex samples is a major challenge in biomarkers discovery and validation (13, 14) . samples of interest for detection of protein biomarkers are typically serum or plasma. however, with the rapidly growing interest in human urine proteome (15, 16) , because of simplicity and noninvasiveness of collection, making urine an alternative sample type for many diagnostic tests (17) .furthermore, urine contains a relatively small number of proteins typically present at low concentrations and thus simpler matrix for detecting proteins as compared to serum (17) . some human diseases, excess proteins are found in the urine as can occur in patients with compromised kidney function. as a result, many of the proteins normally present in blood will also be excreted in to the urine. this condition is known as proteinuria, is often observed in acute inflammation, acute urinary tract infection, amyloidosis, diabetic nephropathy, kidney failure, multiple myeloma, nephrotic syndrome and severe yeast infection (18, 19 ) . kidney involvement has been demonstrated in patients with kalaazar with significant proteinuria (20) .the detection of leishmania donovani soluble antigen and antibody (igm and igg) has also been shown in urine samples of kala-azar patients (21, 22).abnormal renal functions particularly, glomerular filtration rate, urinary concentration, acidification that may be correlated to an immunological mechanisms and the evidence of renal proximal tubular damage have been demonstrated recently (23, 24) . lima et al. (2009) (24) have also detected the presence of different kinds of proteins in the urine of patients with kala-azar particularly, albumin, igg, β2-microglobulin, α1 , α2 , β and γ-globulins. all these studies had confirmed renal involvement during active kala-azar disease. however, to the best of our knowledge, no study till now tried to demonstrate the types of protein patterns or profiles that may be observed in patients with the active disease. therefore, in this study we tried to investigate-using urine samplesthe presence of different protein profiles in a number of defined children with kala-azar in iraqi j pharm sci, vol.19(2) 2010 protein profiles in children with kala-azar 73 iraq using the conventional sds-page.the results of this study revealed the presence of one protein band in the control healthy children and the presence of three different protein patterns groups in the defined children with kala-azar. as shown in the results, patients involved in this study were divided into three groups according to their urine protein profile (see the above results).groups 2 and 3 showed only 2 banding proteins in 55 and 11.6 % of the studied patients, while group 1 showed 5 different banding proteins in 33% of the studied patients. these differences in the number of bands for each banding pattern group may be attributable to the chronicity of infection (24) and to the difference in the degree of renal tubular dysfunction with differences in the types and amounts of proteins excreted in the urine (23, 24) .furthermore, clinical and epidemiological studies in iraq showed a large differences in the clinical picture of the disease in terms of fever, hepatomegally, spleenomegally (2, 25, 26), anaemia, thrombocytopaenia and response to therapy (sodium stibogluconate) (26) . similar findings have been reported in other endemic areas with kala-azar like sudan (27), india (28) and iran (29). this may explain the different protein patterns among different groups in this 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http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22lima%20verde%20em%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/pubmed?term=%22zijlstra%20ee%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22ali%20ms%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22el-hassan%20am%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22el-toum%20ia%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22satti%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22ghalib%20hw%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22sondorp%20e%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22winkler%20a%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract javascript:al_get(this,%20'jour',%20'trans%20r%20soc%20trop%20med%20hyg.'); javascript:al_get(this,%20'jour',%20'trans%20r%20soc%20trop%20med%20hyg.'); javascript:al_get(this,%20'jour',%20'trans%20r%20soc%20trop%20med%20hyg.'); iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge doi:https://doi.org/10.31351/vol27iss2pp66-76 66 studying the effect of variables on acyclovir microsponge noor y. fareed *,1 and hanan j. kassab** *department of pharmaceutics, college of pharmacy, university of basra, basra, iraq.  department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the aim of the present investigation was to develop a microsponge delivery system of acyclovir to control its release when applied topically thereby reducing dosing frequency and enhancing patient compliance. the microsponge was produced by oil in oil emulsion solvent diffusion method. the effect of different formulation and process variables such as internal phase volume, polymer type, drug polymer ratio, stirring speed and stirring duration on microsponge production yield, loading efficiency, particle size, and in vitro drug release was evaluated. the results showed that the microsponge f2 prepared from eudragit rs polymer had optimum physical properties regarding loading efficiency of 99.71±0.7 % and production yield which was 85%. also, f2 showed 66% drug release through 8 hours. accordingly, the oil in oil emulsion solvent diffusion method is an effective technique to formulate microsponge with maximum production yields and loading efficiency for acyclovir. keywords: microsponge, acyclovir, controlled topical release, oil in oil emulsion solvent diffusion method. ةدراسة تاثير المتغيرات على اسفنجيات االسايكلوفر المايكروي **و حنان جالل كساب 1* ، نور يوسف فريد . العراق، البصرة ،جامعة البصرة، كليه الصيدلة ، فرع الصيدالنيات* .العراق، بغداد ،بغداد جامعة ،الصيدلة كليه ، فرع الصيدالنيات* الخالصة كاسفنجيات مايكرويه للتحكم بتحرر العقار عند وضعه على الغرض من الدراسة المقدمة هو تصنيع وتقييم عقار االسايكلوفير الجلد لغرض تقليل عدد الجرع اليومية وزيادة امتثال المريض للعالج. حضرت االسفنجيات المايكرويه بواسطة طريقة شبه المستحلب رعة الخلط نسبة الدواء إلى البوليمر، سالمتضمن انتشار المذيب إلى المكون الخارجي الزيتي ثم اختبر تأثير متغيرات طريقة التحضير ك ،وحجم المذيب في الطور الداخلي للمستحلب . كما تم تقيم االسفنجيات بالنسبة الى حجم الجسيمات ، نوع البوليمر ، ، مدة الخلط ( أبدت أفضل الخصائص ٢خصائص السطح وتحرر الدواء خارج الجسم. أثبتت النتائج ان التركيبة) ، كفائة التحميل ، الحصيلة اإلنتاجية . كما أن التركيبة % ٨٥وقابلية التحميل التي كانت، % ٧١،99 ± ٧،٠كالقابلية اإلنتاجية بمقدار، الفيزيائية لالسفنجيات المايكروية ساعات . من خالل نتائج الدراسة تبين نجاح طريقة شبه المستحلب المتضمن انتشار ٨خالل % ٦٦( أبدت تحرر للدواء خارج الجسم ٢) المذيب إلى المكون الخارجي الزيتي لتحضير أسفنجيات االسايكلوفير المايكروية بقدرة إنتاجية و كفاءة تحميل عالية. ، االسايكلوفير ، التحكم بتحرير العقار ، طريقة شبه المستحلب المتضمن انتشار المذيب. اسفنجيات:الكلمات المفتاحية introduction microsponges are polymeric porous microspheres used for controlled delivery of medications (1) .this technology has been used for topical formulations and recently for oral administration. microsponges are biologically safe and are believed to contribute towards reducing side effects, improved stability, increased elegance and enhanced formulation flexibility(2). even though microsponges are very small in size; they are not able to permeate the skin and are not absorbed systemically; therefore, they are considered drug delivery systems for topical use. microsponges accumulate in the tiny nooks and crannies of the skin, and release slowly the entrapped drug as the skin requires(3). microsponges ms can be incorporated into a wide variable of pharmaceutical dosage form, like tablets(4), capsules(5), powders, creams(6), topical emulsions (7), gels (8–10) , etc. acyclovir acv was the first antiviral to be approved for the treatment of viral infections such as herpes simplex, varicella zoster virus and epstein barr infections(11). acv is hydrophilic in nature having water solubility of 2.5mg/ml at 37° c. the chemical structure of acv is shown in figure 1. 1corresponding author e-mail: nalbassam@ymail.com received: 8 / 8 / 2018 accepted: 3 / 10 / 2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp66-76 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge 67 topical delivery of acv is considered to be attractive choice for viral infection of the skin, as targeted therapy is enabled, circulating drug levels and risk of renal insufficiency will be reduced(12). poor penetration across the skin decreases the therapeutic value of topical acv marketed creams (13). high potency formulation and five applications per day are required to achieve the desired antiviral effect, which may cause skin irritation and decrease patient compliance. the aim of the present study was to develop acv microsponges intended for controlled topical delivery of acv. the o/o emulsion solvent diffusion method was adopted to prepare acv microsponge due to low solubility of acv in the external oil phase; the drug loss was minimized leading to an increase production yield and drug loading efficiency. different formulation variables (internal phase volume, polymer type, drug to polymer ratio) and processing variables (stirring speed and stirring duration) were evaluated to maximize the encapsulation efficiency and optimize release characteristics. figure 1. the chemical structure of acyclovir materials and methods materials acyclovir acv was purchased from hangzhou hyper chemicals limited, china. eudragit rs 100 and eudragit rl 100 were from evonik rӧhm gmbh, germany. light liquid paraffin, acetone, n-hexane, magnesium stearate, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium hydroxide, and cellulose nitrate filter with molecular weight cut off 8000-14000. methods all acv microsponge formulas were prepared by oil in oil quasi-emulsion solvent diffusion method. the internal phase was prepared by dissolving the polymer into acetone by sonication using sonorex (bandelin, berlin, germany) to obtain a clear polymeric solution. the drug and mg-stearate were added to the prepared polymeric solution. the internal phase is added drop wise at a rate of 1 ml per min to 100ml of liquid paraffin with stirring at (500, 1000and 1500 rpm) by using mechanical overhead stirrer (rzr 2041 heidolph tm, schwalbach, germany) for (30, 60 and 120 min) at which the organic solvent could evaporate, and the solidified ms precipitated. the ms were collected by filtration using of büchner filtration device and was washed six times using nhexane and left to dry over night at room temperature. the composition of various ms formulations is given in table 1. table 1 .composition of acv loaded microsponges formula code polymer type drug / polymer ratio solvent volume (ml) stirring rate (rpm) stirring time (min.) f1 ers 1:1 5 500 60 f2 ers 1:1 7.5 500 60 f3 ers 1:1 10 500 60 f4 ers 1:1 7.5 500 30 f5 ers 1:1 7.5 500 120 f6 ers 1:1 7.5 1000 60 f7 ers 1:1 7.5 1500 60 f8 ers 2:1 7.5 500 60 f9 ers 1:2 7.5 500 60 f10 erl 1:1 7.5 500 60 f11 ers: erl 1:1 7.5 500 60 *all microsponges were prepared by using 0.5 g of polymer and 100 ml of liquid paraffin. *magnesium stearate was added as a percentage weight relative to the acetone volume iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge 68 characterization of microsponges formulation determination of production yield the production yield of the microsponge was obtained from the weight ratio of filtered and dried microsponge amount of each formulation to total solid material amount in the dispersed phase (drug, polymer and mg stearate) as given in the following equation: py(%)= 𝑷𝒓𝒂𝒄𝒕𝒊𝒄𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒕𝒉𝒆 𝑴𝑺 𝐓𝐡𝐞𝐨𝐫𝐞𝐭𝐢𝐜𝐚𝐥 𝐰𝐞𝐢𝐠𝐡𝐭 (𝐝𝐫𝐮𝐠,𝐩𝐨𝐥𝐲𝐦𝐞𝐫 𝐚𝐧𝐝 𝐌𝐠 𝐬𝐭𝐞𝐚𝐫𝐚𝐭𝐞) × 𝟏𝟎𝟎% determination of loading efficiency le the drug content was determined in the prepared microsponge by crushing the ms using a porcelain mortar. accurately weighed 100 mg of this powder was extracted in 100 ml of naoh. the solution was sonicated for 15 min and left for 24 hours. the solution then was filtered and diluted (if necessary, a sample of 1ml was withdrawn from this solution, diluted to 100 ml with naoh) and assayed spectrophotometrically at 264 nm (cecil spectrophotometer ce7200;uk) to determine the acv content. the loading efficiency was determined according to the following equation: 𝑳𝑬(%) = 𝑨𝒄𝒕𝒖𝒂𝒍 𝒅𝒓𝒖𝒈 𝒄𝒐𝒏𝒕𝒆𝒏𝒕 𝒐𝒇 𝑴𝑺 𝑻𝒉𝒆𝒐𝒓𝒆𝒕𝒊𝒄𝒂𝒍 𝒅𝒓𝒖𝒈 𝒄𝒐𝒏𝒕𝒆𝒏𝒕 𝒐𝒇 𝑴𝑺 × 𝟏𝟎𝟎% determination of particles diameter the optical microscope (olympus, japan) was equipped with graduated eye piece and was used to estimate the average diameter of acv microsponges, under (10 x) magnification power, in which, 100 ms particles of each batch were examined. the average particle diameter was determined according to the following equation: d av = ∑ 𝒏𝒅 ∑ 𝒏 where: dav is the average diameter of particles (μm), n is the number of particles per group, and d is the middle value (μm). scanning electron microscope (sem) study the surface topology and particle morphology of the prepared microsponge was studied using carl zeiss supra 55vp fesem, germany. the samples were first loaded on sample stub using a double side carbon tape and sprinkle the powder on it then tight all stubs on the specimen’s holder after simple blowing to remove non-adherent particles. the prepared samples were loaded on sem via air-lock door which depends on low voltage to avoid charging. solid state characterization of microsponge dsc, xrd and ftir were employed to characterize the physical state of the drug in the prepared microsponge and to confirm the absence of interaction within formulation. the pure drug (acv), polymer (eudragit rs), mgstearate, the physical mixture of all formula components and avc ms, were investigated individually using each of these techniques. differential scanning calorimetry (dsc) thermal analysis was carried out using dsc-600, shimadzu, japan by loading 5 mg of the powder into a sealed aluminum pan, which was compared with a sealed blank aluminum crucible as reference. the temperature was raised from 25°c to 300°c in a nitrogen atmosphere at the rate of 10°c per minute. the flow rate of nitrogen was 50 ml per minute. x-ray diffractometry (xrd) an xrd pattern was determined using x-ray diffractometer-6000, shimadzu, japan. the results were recorded over a range of 5–50° (2θ). the operating conditions were: voltage 40 kv, currente30 ma, scanning speed 8 deg /min. fourier transform infrared spectroscopy (ftir) the ir spectrum was recorded using ftir 600 spectrometer, uk. the sample was crushed with potassium bromide with a porcelain mortar and pestle. the mixture was then compacted to form a translucent pellet with the aid of a mechanical die press to obtain kbr discs. these discs were scanned in frequency range of 4000-400 cm-1. in vitro release studies drug release rate from acv microsponge was carried out through dialysis bag technique using usp dissolution test apparatus-ii. the dissolution media consisted of 900 ml phosphate buffer having a ph of 7.4. accurately, weighed samples of acv microsponge equivalent to 50 mg of the drug was suspended in 5 ml phosphate buffer (ph 7.4) in a dialysis bag and the rotation speed was adjusted at 50 rpm at 37°c. at prescheduled time intervals (30min, 60min, 120min, 180min, 240min, 300min, 360min, 420min, and 480min) 10 ml of the media were withdrawn and replaced by an equal volume of dissolution medium to maintain a constant volume. the samples were filtered through a (0.45 μm, millipore) filter, suitably diluted (if necessary ,1 ml of filtrate diluted to 10 ml with phosphate buffer solution) and analyzed at 252 nm using a double-beam cecil spectrophotometer ce 7200; uk. these experiments were conducted in triplicate. kinetic modeling of acv release the data obtained from the in vitro release studies were fitted to different iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge 69 mathematical expressions to describe the kinetic and mechanism of acv release from the microsponge selected formula with the aid of ddsolver an microsoft excel add-in (14). the kinetic models used were zero order kinetic , first order kinetic , higuchi model and korsmeyer peppas (15). the model with the highest correlation coefficient was the best fitted model. statistical analysis results of each experiment was reported as a mean ± standard deviation and were analyzed according to the one-way analysis of variance (anova) test, using microsoft excel program 2010. differences were statistically significant at p < 0.05. results and discussion in this study. two types of eudragit polymers (eudragit rs and eudragit rl) were used as a matrix forming agent for acv microsponges. these polymers are known for their release retarding properties (16). the internal phase solvent used in this experiment was acetone. the compatibility of acetone with liquid paraffin system is attributed to the partial polarity of acetone (17). the oil in oil emulsion diffusion method successfully produced microsponge particle having a uniform spherical shape with high production yield and excellent loading efficiency owning to the low drug solubility in the external phase. the rationalization for mg-stearate use, is the ability of metal stearate to form a protective shell surrounding the sponge particle and of great benefit in decreasing the coalescence and aggregation (18). the microsponge production yield and loading efficiency is summarized in table 2 table 2. characterization of acyclovir microsponges characterization of f1-f3 revealed that the ms were formed when the volume of inner phase ranged from 5 to 10 ml. the particle size significantly (p < 0.05) decreased with increasing the inner phase volume, because the lower inner phase viscosity led to breaking of the emulsion into smaller droplets forming microsponges with small particle size (19). the loading efficiency was found to decrease at larger internal phase volumes because the reduction in the viscosity of the internal phase enhance drug escape to the periphery(20). accordingly; 7.5 ml of acetone were found to produce microsponge of acceptable properties in term of loading , yield and particle size. stirring duration was found to have significant effect on microsponge formation and 60 minutes was found to be most effective, while 30 minutes of stirring was insufficient for complete solvent diffusion and resulted in a lower yield. stirring for 120 minutes did not significantly affect the microsponge particle size or loading efficiency (p > 0.05). the effect of different polymers and polymer combination on acv microsponge was studied in f2, f10 and f11 prepared from eudragit rs, eudragit rl, and eudragit rs eudragit rl at 1:1 drug to polymer. particle size was found to be insignificantly affected with changing polymer type (p > 0.05) as the viscosity of the employed eudragit polymers is similar resulting in comparable internal phase viscosity and similar viscosity difference between the internal and external phase (21) . the loading efficiency was significantly lower (p<0.05) with eudragit rl microsponges because higher content of the ammonium group facilities the diffusion of some of the entrapped drug to the surrounding medium during formation of the microsponges(22). increasing the ratio of the drug to the polymer resulted in particle size significant reduction (p<0.05), since the amount of the polymer available per each microsponge was relatively lower than microsponge prepared at lower drug to polymer ratio (23). also, the loading formula code production yield (%) loading efficiency (%) particle diameter (um) f1 67 98.77±0.3 47.23±0.75 f2 85 99.71±0.7 32.36±0.85 f3 88 88.59±0.36 26.57±0.51 f4 75.7 98.72±0.54 39.05±0.73 f5 87 99.28±0.2 30.64±0.5 f6 57 99.35±0.2 21.06±0.3 f7 45 97.31±0.5 16.84±0.4 f8 73 96.65±0.47 28.86±0.31 f9 85 94.05±0.23 51.13±0.32 f10 82 90.84±0.28 31.65±1.65 f11 83 98.12±0.35 30.23±1.45 iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge 70 efficiency was significantly reduced (p<0.05) as lower amount of polymer to the drug produced lower viscosity enhancing drug escape to external media thereby reducing loading efficiency and drug entrapment in the ms(24). increasing the stirring speed reduced the production yield, due to adherence of the polymer to the walls of the glass container as a result of the vigorous turbulence created in the external phase (25). also, the particle size was significantly decreased (p<0.05) as the stirring speed increased, since high mechanical shear applied, resulting in a rapid splitting of the formed droplets, allowing less chance of coalescing into bigger droplets(26). the most suitable stirring speed was found to be 500 rpm. scanning electron microscope (sem) sem analysis of the selected microsponge formula is shown in figure (2). ms were spherical in shape, having uniform size distribution with rough surface contains pores and crakes resulted from diffusion of the solvent. figure 2. sem of microsponge f2 [a] 1.5kx magnification [b] 1.0kx magnification solid state characterization of microsponge according to dsc analysis illustrated in figure 3, acv displayed a sharp characteristic endothermic peak at 259°c corresponding to the melting point of the drug in the crystalline form, while eudragit rs thermogram showed one transition only at 65°c which represents the glass transition temperature of the polymer in the amorphous state. the drug peak was obvious in the physical mixture which excludes in-compatibility. the disappearance of the melting point peak of acyclovir from the selected microsponge formula thermogram is explained by the uniform dispersion of the drug into the polymeric matrices (27) . 100.00 200.00 300.00 temp [c] -10.00 0.00 mw dsc 259.48x100c 176.83x100c 100.00 200.00 300.00 temp [c] -3.00 -2.00 -1.00 0.00 mw dsc figure 3. dsc thermograms of [a]pure acv [b] eudragit rs100 a b a b iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge 71 100.00 200.00 300.00 temp [c] -10.00 -8.00 -6.00 -4.00 mw dsc 115.50x100c 176.50x100c 257.39x100c 100.00 200.00 300.00 temp [c] -1.00 0.00 1.00 2.00 mw dsc 176.33x100c 92.40x100c continued figure 3. eudragit rs100 [c] physical mixture [d] microsponge f2 xrd diagrams are shown in figure 4a,4b eudragit rs100 showed typical diffraction form of amorphous materials, whereas the pure drug showed the diffractogram pattern of a crystalline material at 2θ of 7.0154 °, 29.1349° and 26.0690°. the physical mixture and the selected microsponge formula showed a diffraction pattern in which acv characteristic crystalline diffraction peaks present at 2θ are in the same position of the pure sample but with reduced intensity due to the presence of the polymer(28) . figure 4. a. xrd of pure acv [a]eudragit rs100 [b] c d a b iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge 72 figure 4b. xrd of physical mixture [c] microsponge f2 [d]. the ftir absorption bands of pure acv were noticed at 3519 cm-1 (o-h stretching), 3471 cm-1 and 3440 cm-1 (n-h stretching), (2962 cm-1 , 2927 cm-1) (aliphatic c-h stretching a symmetric), 2854cm-1, 2871 cm-1 (aliphatic c h stretching symmetric), 1485cm-1 (aliphatic c -h deformation), 1716cm-1 (c = o stretching) and 1049. cm-1 ( co stretching), while for eudragit rs100, asymmetric c h stretching bands appeared at 2993 and 2954 cm-1, while the band at 2850 cm-1 represent symmetric c-h stretching, the carbonyl group c=o of an ester exhibited a stretching vibration at 1732cm-1, as seen in figure 5. the characteristic absorption bands of the drug were retained in the physical mixture and the selected microsponge formula with no new peaks appearance which confirms chemical computability between the formulation component and the absence of any chemical interaction. c d iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge 73 figure (5): the ftir spectrum of [a]pure acv [b] eudragit rs100 [c] physical mixture [d] microsponge f2 study of acv release profiles from the microsponge in vitro release study was performed to investigate the effect of different variable on drug release from the microsponges. the effect of changing drug polymer ratio on drug release was illustrated in figure (6). the best retardation is obtained as the amount of polymer to the drug is increased since increased particle size decreases the effective surface area and increasing the path length traveled by the drug molecule (29). besides , at higher drug ratio , more drug would present near to and on the surface of the microsponge which is in direct contact with the dissolution media accounting for the rapid release(30). the release profile is also affected by varying polymer type as shown in figure 7. acv release rate increased from 66 % with f2 (ers) to 80.22 % for f11(ers: erl) and 92.99 % for f10 (erl) for a duration of 8 hours. eudragit rs was the least permeable of the three formulas and the most capable of sustaining drug release. this is explained by different structural properties of the two polymers in term of percentage of the quaternary ammonium group present which is (10%) in eudragit rl 100 while eudragit rs 100 contains only (5%). thus, eudragit rl 100 is more permeable and release the drug at a faster rate than eudragit rs(21). the effect of stirring speed on drug release was evaluated by comparing the release behavior from f2, f6 and f7 prepared at stirring speeds of 500, 1000 and 1500 rpm as provided in figure 8. drug release was faster for f7 than f6 and f2 since it has lower particle size resulted from high stirring speed which led to an increase surface area exposed to the dissolution medium and subsequently release rates are faster.(31) . figure 6. effect of drug: polymer ratio on % drug release from f2, f8 and f 9. a b c d iraqi j pharm sci, vol.27(2) 2018 acyclovir microsponge 74 figure 7. effect of polymer type on % drug release from f2, f10 and f 11. kinetic analysis of acv release data from f2 microsponge the release of acv from microsponge f2 obeys zero order release as their r2 values gave the higher result, the release depends on the ms pore size and release of avc from the pore rather than the concentration of avc inside the ms. the mechanism of drug release is non fickian diffusion supercase ιι as the release exponent " n " value of f2 microsponges is more than 1. supercase ιι release behavior meaning that the release depends on polymer relaxation rather than water diffusion inside the microsponge as shown in table 3. figure 8. effect of stirring speed on % drug release from f2, f6 and f7. table 3 . kinetic analysis of acv release data from f2 microsponge f o r m u la mathematical model for drug release kinetics korsmeyer-peppas zero order first order higuchi hixsoncrowell f2 k0 r2 k1 r2 kh r2 khc r2 r2 kkp n 0.116 0.9386 0.001 0.8965 2.064 0.7570 0.000 0.9115 0.9452 0.047 1.153 conclusion it can be concluded from the results obtained in this study that the oil in oil emulsion solvent diffusion method is an effective technique to formulate microsponge based delivery system of acyclovir with maximum production yields and drug loading efficiency. the acv microsponge formulated with the eudragit rs at drug: polymer ratio of 1:1 is the best formulation among all the prepared batches in terms of capacity of extending drug release behind 8 hours thereby decreasing the number of 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evaluation of eudragit microspheres containing acetazolamide. international journal of pharmaceutics. 2004;269:131– 140. nitric oxide, peroxynitrite and malondialdehyde levels iraqi j pharm sci, vol.21(1) 2012 markers for nitrosative/oxidative stress and sle 87 nitric oxide, peroxynitrite and malondialdehyde levels as markers for nitrosative/oxidative stress in iraqi patients with systemic lupus erythematosus shaimaa m. mohammed* ,1 , inaam a. amin* and zeina z. sabri* *department of clinical laboratory sciences,college of pharmacy,university of baghdad,baghdad, iraq. abstract systemic lupus erythematosus is an autoimmune disease of unknown aetiology affecting multiple organ system. reactive nitrogen and oxygen species are claimed to play a role in this disease. however, the potential of nitrosative/oxidative stress to elicit an autoimmune, response remain till now largely unexplored in humans. this study was done to investigate the status and contribution of nitrosative/oxidative stress in iraqi patients for systemic lupus erythematosus. blood samples from 19 patients with systemic lupus erythematosus and 19 age-and sexmatched apparently healthy controls were evaluated for serum levels of nitrosative/oxidative stress markers including nitric oxide, peroxynitrite and malondialdehyde. nitric oxide levels were measured by spectrophetometric method depending on griss method, while peroxynitrite levels were measured by spectrophetometric method based on peroxynitrite mediated nitration of phenol. malondialdehyde levels were measured by the thiobarbitoric acid method. serum nitric oxide levels were significantly elevated in sle patients (mean + se 263.58 + 35.42 mol/l) as compared with healthy control (162.48 +10.42 mol/l). peroxynitrite levels were also significantly elevated in a disease group (mean + se 7.23 +0.92 mol/l) as compared to healthy control (4.47 + 0.38 mol/l). on the other hand, malondialdehyde levels were slightly elevated in sle patient (mean + se 4.53 + 0.22 nmol/ml) as compared to control group (4.32 + 0.58nmol/ml). the study findings support an association between nitrosative/oxidative stress and sle through elevated level of no, peroxynitrite and mda in the serum of sle patients. key words: nitric oxide, peroxynitrite, sle. قٍاس مستىٌاث أوكسٍد الىتزٌك والبٍزوكسً واٌتزاث والمالىن داي الدهاٌد كعالماث لفزط ألجهاسيالىتزجت واألكسدة عىد المزضى العزاقٍٍه المصابٍه بداء الذئب اإلحمزاري شٍماء مىذر محمد* ،1 احمد أمٍه * و سٌىت سهٍز صبزي* أوعام، و١ٍت اٌص١ذٌت ، جاِعت بغذاد ، بغذاد ، اٌعشاق * فشع اٌعٍَٛ اٌّخخبش٠ت اٌغش٠ش٠ت ، الخالصة داء اٌزئب اإلحّشاسٞ اٌجٙاصٞ ٘ٛ ِشض ِٕاعٟ ِغبباحٗ غ١ش ِعشٚفت ٠ٚص١ب اٌعذ٠ذ ِٓ أجٙضة اٌجغُ. ِغبباث حفاعالث ٚاظحت ٌحذ ا٢ْ باإلٔغاْ. ٟ غ١شإٌخشجت ٚاألوغذة ٠ُظٓ أْ حٍعب دٚساً فٟ ٘زا اٌّشض، ٌىٓ غش٠مت اعخثاسحٙا ٌٍجٙاص إٌّاعٟ اٌزاح ٘زٖ اٌذساعت أُجش٠ج ٌّعشفت حاٌت ِغبباث إٌخشجت ٚاألوغذة فٟ اٌّشظٝ اٌعشال١١ٓ اٌّصاب١ٓ بذاء اٌزئب اإلحّشاسٞ اٌجٙاصٞ. ع١ٕاث ١ىٛٔٛا ِٓ األصحاء ظا٘ش٠اً اٌّخّاث١ٍٓ باٌعّش ٚاٌجٕظ ٌ 91ِش٠عاً بذاء اٌزئب اإلحّشاسٞ اٌجٙاصٞ ٚوزٌه ِٓ 91اٌذَ ُعحبج ِٓ ِجّٛعت اٌغ١طشة ِٓ األصحاء، ٚرٌه ٌخم١١ُ حشو١ض عالِاث ِغبباث فشغ إٌخشجت ٚاألوغذة ٚاٌخٟ حشًّ أٚوغ١ذ إٌخش٠ه ٚاٌب١شٚوغٟ ٌمذ حُ ل١اط حشو١ض أٚوغ١ذ إٌخش٠ه باعخعّاي غش٠ك وشط باعخخذاَ ِم١اط اٌعٛء اٌط١فٟ ٚوزا حُ ل١اط اٞ اٌذ٘ا٠ذ. ٔا٠خشا٠ج ٚاٌّاٌْٛ د ٔا٠خشا٠ج بطش٠مت ِم١اط اٌعٛء اٌط١فٟ اٌّعخّذة عٍٝ ٔخشجت اٌف١ٕٛي باٌب١شٚوغٟ ٔا٠خشا٠ج، أِا اٌّاٌْٛ داٞ اٌذ٘ا٠ذ فمذ حُ اٌب١شٚوغٟ ل١اعٗ بطش٠مت حاِط اٌثا٠ٛباسبج١ٛسن. إْ حشو١ض أٚوغ١ذ إٌخش٠ه فٟ ِصً اٌذَ ٌّشظٝ داء اٌزئب االحّشاسٞ اٌجٙاصٞ لذ واْ ِا٠ىشِٚٛي/ٌخش( ِماسٔتً بّجّٛعت اٌغ١طشة ِٓ األصحاء 6.58. + 62..87)ِع١اس اٌخطأ( +عذي ِشحفعاً اسحفاعاً ِع٠ٕٛاً )اٌّ + .١ِ3.8ىشِٚٛي/ٌخش(. حشو١ض اٌب١شٚوغٟ ١ٔخشا٠ج أ٠عاً واْ ِشحفعاً اسحفاعاً ِع٠ٕٛاً عٕذ ِجّٛعت اٌّشظٝ ) 94.58 + 978.52) ِا٠ىشِٚٛي/ٌخش(. ِٓ ٔاح١ت أخشٜ، حشو١ض اٌّاٌْٛ 2..4 + 5.53ِا٠ىشِٚٛي/ٌخش( ِماسٔتً بّجّٛعت اٌغ١طشة ِٓ األصحاء ) 4.18 ٔأِٛٛي/ًِ( ِماسٔتً بّجّٛعت 4.88 + .5.6داٞ اٌذ٘ا٠ذ واْ ِشحفعاً اسحفاعاً غف١فاً عٕذ ِشظٝ داء اٌزئب االحّشاسٞ اٌجٙاصٞ ) جت ٚاألوغذة ِٚشض داء اٌزئب االحّشاسٞ ٔأِٛٛي/ِٛي(. ٔخائج اٌذساعت ب١ٕج أْ ٕ٘ان عاللت ب١ٓ فشغ إٌخش 4.62 + 8..5اٌغ١طشة ) اٌجٙاصٞ ِٓ خالي اسحفاع ِغخٜٛ أٚوغ١ذ إٌخش٠ه ٚاٌب١شٚوغٟ ٔا٠خشا٠ج ٚاٌّاٌْٛ داٞ اٌذ٘ا٠ذ فٟ ِصً اٌذَ ٌذٜ اٌّصاب١ٓ باٌّشض. .ألجهاسي داء الذئب اإلحمزاري، البٍزوكسً واٌتزاث، أوكسٍد الىتزٌكالكلماث المفتاحٍت : 1 corresponding author email : shaimaa_m77@yahoo.com received : 8/1/2012 accepted : 1/4/2012 iraqi j pharm sci, vol.21(1) 2012 markers for nitrosative/oxidative stress and sle 88 introduction systemic lupus erythematosus (sle) is an autoimmune disease of unknown aetiology affecting multiple organ system (1) . the most remarkable feature of sle is autoantibody production, a function of the acquired immune response. however, an inappropriately active and sustained innate immune response is implicated in both the initiation and the pathogenic consequences of the autoantibody production in sle (2) . an important part of the innate immune response is the production of reactive nitrogen species (rns) and reactive oxygen species (ros) (3) .reactive nitrogen species include nitric oxide (no) and peroxynitrite (onoo ), while reactive oxygen species include superoxide (so) and hydrogene peroxide (h2o2). nitric oxide is a biological messenger mediating many important physiological functions but also pathological process. it plays a vital role in host defense and immunity by modulating inflammatory processes (4) .it's synthesized from l-arginine by both a constitutive no synthase (cnos) & inducible no synthase (inos) (5) .the effect of no production on the cellular processes largely depends on its concentration and the local presence of other free radicals. lower concentrations of no have direct effects on processes e.g. proliferation and cell survival, while high concentrations have indirect effect through both nitrosative stress by modifying proteins and oxidative stress by influencing the cytoplasmic redox balance through generation of onoo following its reaction with so (6) . peroxynitrate can oxidize lipids such as those found in ldl or arachidonic acid (7). peroxynitrate can also act as peroxide substrate for peroxidoses such as those found in cyclooxygenase (8) and finally onoo can nitrate dna (9) . nitric oxide dependent tissue injury has been implicated in a variety of rheumatic diseases, including sle and rheumatoid arthritis (ra), and recent evidence suggests that no contributes to t cell dysfunction in these autoimmune disease (10) . in murine model of sle, no production has shown to be increased with the progression of the disease and lead to glomerular, joint and dermal pathology (2) . on the other hand, pharmacological inhibition of inos in these models significantly reduced both no and ros production (11) . these findings suggest that inos activity and its products may contribute to the inflammatory lesions in sle (3) . like rns, reactive oxygen species could play a significant role in a pathogenesis of sle, in that, excessive generation of ros (i.e.) super oxide anion (o2 ) and/or hydroxyl radical ( oh) have the potential to initiate damage to lipids, proteins and dna (12,13) . lipid peroxidation (lp), an oxidative degeneration of poly unsaturated fatty acids leads to the formation of highly reactive aldehydes such as malondialdehyde (mda) which can bind covalently to proteins resulting in their structural modifications and affecting biological function (14) . it was reported that high level of mda in sle patients indicates that ros damage might play a role in sle (15) .the potential for nitrosative/oxidative stress to elicite an autoimmune response or to contribute to sle pathogenesis remains largely unexplored in humans. this study was undertaken to investigate the status of nitrosative/oxidative stress in patients with sle. patients and methods nineteen patients with sle (17 females, 2 males) age range (19-45) years who were attending the rheumatology consultation clinic of baghdad teaching hospital, and 19 apparently healthy controls (17 females, 2 males) age range (21-46) years were included in the study after obtaining their informed consent. sle was diagnosed on the basis of the revised criteria of the american college of rheumatology (acr) (16) . exclusion criteria were pregnancy, the presence of active infection and the presence of cancer, since all can affect serum no level. ten ml blood samples were collected from all patients by vein puncture; 2 ml of each sample were transferred to edta (ethylene diamine tetraacetate) tube for erythrocyte sedimentation rate (esr) determination according to the westergreen method. the rest 8 ml were transferred to 10 ml sterile plane tube, allowed to clot for 30 min at room temperature and centrifuged at 3000 g for 5 min to obtain serum. serum aliquots were divided into three 1ml eppendroff's tubes for mda, nitric oxide and peroxynitrite measurment. estimation of biochemical analysis determination of serum mda level malondialdehyde level was estimated as described by hunter et al. (17) . to 0.5 ml of serum was added 0.5 ml of 35% trichloroacetic acid (tca). after vortex-mixing, 0.5ml tris/hcl buffer (50m m; ph 7.4) was added followed by further mixing and incubation at room temperature for 10 min. one ml of 0.75% thiobarbituric acid (tba) in 2m na2so4 was added and then the mixture was heated at 100 o c for 45 min. after cooling, 1ml of 70% tca was added, the mixture was iraqi j pharm sci, vol.21(1) 2012 markers for nitrosative/oxidative stress and sle 89 vortexed and then centrifuged at 950 xg for 10 min. the absorbance of the supernatant was determined at 530 nm.total tba-reactive material were expressed as mda, using a molar extinction coefficient for mda of 1.5×10 5 cm -1 m -1 . determination of no level is done by 2 steps: 1. deprotinization step: deprotinization of serum sample is done by addition of 6mg of zinc sulphate powder to 400l of serum (15 gm/l) followed by vortex and centrifugation, clear supernatant is taken and kept frozen at -18 o c until nitric oxide estimation. 2. serum no measurement step: measurement of serum no was performed according to the method of miranda et al. (2001). deprotinized sample from step 1 was thawed at room temperature, and 70l of supernatant was applied to a microtiter plate well, 70l vanadium chloride (8mg/ml) was added to each well for reduction of nitrate to nitrite and this was followed by addition of the griss reagents [35l sulfanilamide (2%) and 35l n-(lnaphthyl) ethylendiamine dihydrochloride (nedd) (0.1%)]. after 30 min, incubation at 37 o c, absorbance was read at 540 nm using elisa reader. concentration of no in serum samples were determined from linear standard curve established by 0-200 mol/l sodium nitrite (18) . determination of peroxynitrite level serum peroxynitrite level was determined according to the method described by beckman et al. (19) , cited by van uffelen et al. (20) . in which the peroxynitrite mediated nitration of phenol was measured spectrophotometrically at 412 nm. in brief 100l of serum was placed in glass test tube, to which 5mm phenol in 5m sodium phosphate buffer ph 7.4 was added to a final volume of 2 ml, the resulting solution after mixing is then incubated for 2 hours and then 15 l of 0.1 m naoh was added and the absorbance is read at 412 nm. statistical analysis data were translated into a computerized database structure. an expert statistical advice was sought for statistical analysis using spss version 12 computer software. data in this study was presented as mean + standard error (mean + se). student's ttest was used to compare the group means. a pvalue <0.05 was considered to be statistically significant. results table (1) shows the demographic characteristics of the subjects. there was no significant difference between the control and sle patients regarding gender, age, weight and body mass index (bmi). serum analysis showed significantly elevated levels of no in the 19 patients with sle mean + se (263.58 + 35.42 mol/l) compared with controls (162.48+10.42mol/l) pvalue <0.05 serum level of peroxynitrite also were significantly elevated in the 19 sle patients mean +se(7.23+0.92mol/l) as compared with the controls (4.47+0.38mol/l), pvalue <0.05. lipid peroxidation measured as serum mda levels were higher in patients with sle mean + se (4.53 + 0.22 nmol/ml) as compared to healthy controls (4.35 + 0.58 nmol/ml). yet, it failed to reach a level of significance. erythrocyte sedimentation rate levels were significantly higher in sle patients (71.00 + 7.05 mm/hr) as compared with the control group (14.21 + 0.45mm/hr) p<0.05. table (2) shows the level of no, onoo , mda and esr in serum of patients with sle and healthy controls. table 1: demographic data of the studied groups characteristic sle controls number 19 19 gender f/m 17/2 17/2 age 29 + 2.42 31 + 2.62 weight 67.63 + 2.82 70.37 + 2.71 bmi 25.34 + 0.99 24.82 + 0.96 values were expressed as mean + sd. table 2 : nitric oxide, peroxynitrite and malondialdehyde and erythrocyte sedimentation rates in serum of patients with systemic lupus erythematosus and healthy controls sle patients healthy controls p-value no (mol/l) 263.58+35.42 162.48+10.42 0.017* onoo (mol/l) 7.23+0.92 4.47+0.38 0.025* mda (nmol/ml) 4.53+0.22 4.35+0.58 0.774 esr (mm/hr) 71.00+7.05 14.21+1.57 0.000* no: nitric oxide, onoo : peroxynitrite, mda: malondialdehyde, esr: erythrocyte sedimentation rates values were expressed as mean + sd. * p<0.05, students' t-test. iraqi j pharm sci, vol.21(1) 2012 markers for nitrosative/oxidative stress and sle 90 discussion systemic lupus erythematosus is a puzzling disease due to its multifactorial etiology including genetic, hormonal and environmental triggers, the molecular mechanisms underlying this systemic autoimmune response remain largely unknown (15) . in recent years, free radical mediated reactions have implicated considerable attention as the potential mechanism in the pathogenesis of sle (21) . studies using animal models of sle also suggested an association between nitrosative/oxidative stress and autoimmunity (2,22,23) . however, relevance of nitrosative / oxidative stress in the pathogenesis and progress of sle in human is not fully understood. our results present in this study show significantly elevated levels of no as compared to healthy controls; this came in accordance with previous studies demonstrating higher level of no in active sle patients (3,24,25) , also considerable evidence supports that no production correlate with disease activity and damage in sle (3) . on the other hand, this study also demonstrated significantly elevated level of onoo in sle groups as compared to healthy controls. it is also came in accordance with previous studies which suggest that overproduction of inos and increased production of onoo may contribute to glomerular and vascular injury in sle and other autoimmune diseases (24,26,27) .thus these rns could play an important role in the pathogenesis of sle, in that the potential of no in disease pathogenesis could lie largely to the extent of its production and generation of superoxide radical (o2 ), leading to the formation of peroxynitrite which is a potent nitrating and oxidizing agent (5) . peroxynitrite can react with tyrosin residues forming nitrotyrosine forming a neoepitopes on nucleophilic domain of self antigens (21,28) .in addition onoo mediated modifications of endogens proteins and dna may enhance their immunogenicity leading to a break in the immune tolerance (21,29,30) .other important mechanism by which no can play a central role in the pathogenesis of sle is through its ability to regulate t-cell function (10) . nitric oxide under physiological condition has been shown to regulate t-cell function, but overproduction of no may contribute to t-cell dysfunction and result in no-dependent tissue injury (31,32) .on the other hand and concerning lipid peroxidation, this study shows slightly elevated level of mda in serum of sle as compared to healthy controls which came in agreement with earlier reports and confirming the presence of increased oxidative stress in sle (33-36) .malondialdehyde is the most abundant aldehyde resulting from lipid peroxidation and high level of it indicates that ros damage might play a role in sle (36,37) .these ros can cause cross linking of proteins or could cause oxidative inactivation of certain enzymes causing functional impairment of cells and libration of cytoplasmic proteases (38) . they can also induce damages in dna which results in new antigenic determinants and stimulation of anti dna antibody formation and autoimmunity (39,40) . conclusion the study findings support an association between nitrosative/oxidative stress and sle through elevated level of no, peroxynitrite and mda in the serum of sle patients which might have a role in the disease pathogenesis and progression, however, such suggestion need future studies to confirm it. references 1. c.y. ho, c.k. wong, e.k. li, l.s. tam and c.w.k. lam. elevated plasma concentration of nitric oxide, soluble thrombomodulin and soluble vascular cell adhesion molecule-1 in patients with systemic lupus erythematosus. rheumatology. 2003; 42: 117-122. 2. jim c. oates. the biology of reactive intermediates in systemic lupus erythematosus. autoimmunity, 2010, 43(1): 56-63. 3. jim c. oates, stephanie r. shaftman, sally e. self and gary s. gilkeson, association of serum nitrate and nitrite levels with longitudinal assessments of disease activity and damage in systemic lupus erythematosus and lupus nephritis, arthritis rheum. 2008 january; 58(1): 263-272. 4. farrel aj, blake dr.. nitric oxide, ann rheum dis. 1996; 55: 7-20. 5. griffith ow, stuehr dj. nitric oxide synthase properties and catalytic mechanism. ann rev physiol, 1995; 57: 707-36. 6. chung ht, pae ho, choi bm, billiar tr, kim ym. nitric oxide as 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uffelen be, van der zee j, de kostes bm, van stereninck j, elferink jg. intracellular but not extracellular conversion of nitroxyl anion into nitric oxide leads to stimulation of human neutrophil migration, biochem j, 1998; 330: 719-722. 21. kurien bt, hensley k, bachmann m, scofield rh. oxidatively modified autoantigens in autoimmune disease. free radic biol med., 2006; 41: 549-556. 22. khan mf, wu x, ansari ga. anti malondialdehyde antibodies in mrl +/+ mice treated with trichloroethene and dichloroacetyl chloride: possible role of lipid peroxidation in autoimmunity. toxicol appl pharmacol,2001;170: 88-92. 23. wang g, wang j, ma h, khan mf. increased nitration and carbonylation of protein in mrl+1+mice exposed tp trichloroethene: potential role of protein oxidation in autoimmunity. toxical appl pharmacol, 2009; 237: 188-195. 24. belmont h.m., levartousky d., goel a., amin a., giorno r., rediske j., skovron m.l., abramson s.b. increased nitric oxide production accompanied by the upregulation of inducible nitric oxide synthase in vascular endothelium from patients with systemic lupus erythematosus. arithritis and rheumatism, 1997; 40(10): 1810-1816. 25. oates jc, christensen ef, reilly cm, self se, gilkeson gs. prospective measure of serum 3-nitrotyrosine levels in systemic lupus erythematosus: correlation with disease activity. proc assoc am physicians, 1999; 111(6): 611-621. 26. morgan pe, sturgess ad, davies mj. evidence for chronically elevated serum protein oxidation in systemic lupus erythematosus patients. free radical res, 2009; 43: 117-127. 27. nagy g, clark jm, buzas ei, gorman cl, cope ap. nitric oxide, chronic inflammation and autoimmunity. immunol lett, 2007; 111: 1-5. 28. khan mf, wu x., kaphalia bs, boor pj, ansari ga. nitrotyrosine formation in iraqi j pharm sci, vol.21(1) 2012 markers for nitrosative/oxidative stress and sle 92 splenic toxicity of aniline, toxicology, 2003; 194: 95-102. 29. habib s, moinuddin, ali r. peroxtnitritemodified dna: a better antigen for systemic lupus erythematosus anti-dna autoantibodies. biotech appl biochem, 2006; 43: 65-70. 30. dixit k, ali r. role of nitric oxide modified dna in the etiopathogenesis of systemic lupus erythematosus. lupus, 2004; 13(2): 95-100. 31. nagy g, konez a, philips pe, perl a. mitochondrial signal transduction abnormalities in systemic lupus erythematosus. curr immunol rev, 2005; 1: 61-67. 32. perl a, fernandez dr, telarico t, doherty e, francis l, phillis pg. t-cell and b-cell signaling biomarkers and treatement targets in lupus. curr opin rheumatol, 2009; 21: 454-464. 33. seyithan taysi, mustafa gul, refik ali sari, fatih akcay and nuri bakan. serum oxidant/antioxidant status of patients with systemic lupus erythematosus. clin chem lab med, 2002; 40(7): 684-688. 34. mohan ik, das un. oxidant stress antioxidants and essential fatty acids in systemic lupus erythematosus. prostaglandius leukot essent fatty acids, 1997; 56: 193-8. 35. turi s, nemeth i, torkos a, saghy l, varga i, matkovics b, et al. oxidative stress and antioxidant defense mechanism in golmerular disease, free radic biol med, 1997; 22: 161-8. 36. kurien bt, scofield rh. free radical mediated peroxidative damage in systemic lupus erythematosus. life sci, 2003; 73(13): 1655. 37. ahsan h, ali a, ali r. oxygen free radicals and systemic autoimmunity. clin exp immunol, 2003; 131(3): 398. 38. bauer v, bauer f. reactive oxygen species as mediators of tissue protection and injury. gen physiol biophys, 1999; 18: 7. 39. du j, gebicki jk. dna degradation and protein peroxidation in cells exposed to hydroxyl free radicals. redox rep, 2002; 7(5): 329. 40. sies h. impaired endothelial and smooth muscle cell function in oxidative stress. exp physiol, 1997; 82: 291. iraqi j pharm sci , vol.17 (2) ,2008 frusemide oral liquid dosage forms 1 a study on the stability of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion fatima j. jawad *,1 * department of pharmaceutics, college of pharmacy, university of baghdad. , baghdad , iraq abstract the present study aim at preparing frusemide in liquid form suitable for oral use. this is achieved through preparing different liquid forms of frusemide. the frusemide liquid is prepared in the following forms: oral solution, syrup and elixir with intensity of 1, 0.4 and 0.8% weight /volume respectively and in combination with potassium carbonate, polysorbate 80, alcohol and phosphate buffer solution of ph8 to dissolve the frusemide in the above mentioned forms. the different forms of the prepared medicine have been stored in glass bottles that can provide protection against light and at 40, 50, 600c for four months. besides the ph has been checked to decide the period of validity. the results show that the expiration date of frusemide have lasted for 1.8, 1.07 and 1.22 years respectively for the oral solution, the syrup and the elixir. the suspensions of frusemide are formulated in combination with the following: polyvinyl pyrolidine, xanthan gum, the combination of (xanthan gum and sodium carboxymethyl cellulose), the combination of (xanthan: methyl cellulose) and chitosan. the formulas which give suitable release of the drug are chosen for assessment according to the following considerations: the rat of sedimentation and apparent zero order degradation constant at 250c. in conclusion, it is found the best formula is that which includes poly vinylpyrolidine, tween20, glycerol, sorbitol, cocoa syrup and parabens at ph7. the fluidity of this chosen formula is psendoplastic type and its validity has lasted for about three years. the emulsion of frusemide is also prepared extemporaneously by using the commercial frusemide tablets in combination with acacia and olive oil. this should be consumed within 45 days of the date of production. key word: frusemide, elixir, suspension, emulsion. ةصالخلا لَيلتلايئل تتللدثهت ةسفتتنِبتمعيِيتاا هُ ثنةتيتموتوريتل يثبٌت نوما تلاس ةيتلا ةساتتلا نِتت . رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا فدهت % ُ%ه/ رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا فسلتبااتلامبةاتتملتوةئ ِىة%تلايِلة ثِلتُلايِاةت ِئ ة%ت0.8ت ت0.4 ت1لا هُ ثنةتيتونلاِيتتنِبتُسهلحتُلا ثهت عِلت .8ل%تلايلئستتئالتلتُلا لِيتُملاِيتلا ِ 80 وةهتلايُل تلانلدهت ةس ةاٌتلانوما تت تا ل تتلا هُ ثنةتيتتةتلاس ةيتلانلوِئلتلبًر 8 ليُ ت ئزتتميِتتتانيلت ئ بتت سَهتملتبيأتلا تلاَثيئُزثيةت60 ت50 ت40تةتايةىةت%زةزثتتمدة لتاادِ ت يئزة%ت رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا فهلئلت ت يتتتةتلانلاِيتلا نِبت1.22ت ت1.07ت ت1.8لنَه%تلايمةسرت اهتميلتلر رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا فثة%تلا هُ ثنةتيتوةىظت امليتيتلا مهلتلاةميثتتاا ر رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا فثت. مويدوصلاو ناثنازلا غمص) لتل يثلتمباعة%تلا هُ ثنةتيتملت ِاةت ةتهُايتوُتلنوتلاةلىاةهتُمصلا ت)لنوتلاةلىاةهتُلا ِ تِلت ُلاعهلحتُلا ثهت ةامبةات. لامهلوثتتلامةتلبيظتللهئتمية تتاايُل تللتلومثةئٍةتاامعثثلتموت ماثفت اثاِ%( ُمصلا ت)لنوتلاةلىاةه :لاناثفت اثاِ% (ُلا ثمِ ةه. تميِتتتُايتُزيت ةهتل رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا ف وتلهوثيتتٍةت25وريتاثة ت هبتتلامه تت مويدوصلاو ناثنازلا غمص) ة ظتلام تانهليتتلا هتلاتةٍهتتتلالهوثتت يئزتت رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا فهلئلتلالت .7تُواث هُيتُ ِئ مِيتُسهلحتلا ةوةُتُلايهل ثية%تبييتل تٍثيئُزثيةت20لامةتوةىظتللمِبتباات ِاةتتثيفت ة هُايتوتُلِتوت رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا فدهتم ملاتتلا هُ نةتيتلىثةضت ُس فتزهتةهتٍلًتلامهوثيتتلانومةئلت اِتت ثيُ ر مث ةتلمةتلر رضح .ةيومفلا ةلئاسلا لاكشالا فلتخمب هعينصت لالخ نم دياميسورفلل لوبقم يومف لئاس ريضحت ةساردلا فثةلَةتوةىظت ليُ ت مويدوصلاو ناثنازلا غمص) رنت يِل%. .45موتلاهلوتلا هُ ثنةتيتلامسةئتتتملتلا نوتلابه ةتُ%تظتلاةتمِهتبااتلهتتملتليلُاٌتوريت تتِلتموتللدثًه introduction frusemide which belongs to the group of loop diuretic is very effective indraining all kinds of oedemas (of cardiac, hepatic or renal origin), in mild or moderate hypertension or used in greater doses in acute and chronic renal failure, oliguria. (1) commerrically available as tablets (20, 40, 80 and 500mg) and injection (10 and 20 mg/ml) and frusemide oral solution which mentioned in usp. (2) many studies concered frusemide to prepare it in defferent pharmaceutical dosage forms as frusemide containing rectal suppositories to increase the drug liberation with the use of non-ionic surfactants (solutol of hs 15, cremophor rh60 and montanox 60df).(3) frusemide adhesive micro-spheres in hard gelatin capsules, frusemide granules with dika fat with maize starch and microcapsules of frusemide with acrycoat e30 acrylic polymer were prepared and evaluated in man resulting in sustained release. (4) to the patients who have difficulty in swallowing the oral liquid dosage forms (syrup, elixir, suspension and emulsion) and rectal are offen supplied. 1 corresponding author : e-mail : thepharmacycollege16@yahoo.com received : 14/4/2008 accepted : 9 / 7/2008 iraqi j pharm sci , vol.17 (2) ,2008 frusemide oral liquid dosage forms 2 frusemide is week organic acid such as barbiturates and the sulfonamide. its solubility in water is increased as the ph is increased by addition of a base. therefore syrups which are sweet viscous oral solutions can be prepared as well as elixir which is sweet hydroalcoholic solution containing flavoring materials. (5) also frusemide as insoluble materials can be prepared in liquid media by means of appropriates suspending agents or mix with oil which dispersed as small globules in water in presence of emulsifying agent to form emulsion. (6) in hospitals because the absence of commercial liquid dosage forms solutions and suspensions of frusemide are prepared extemporaneously from injections and tablets respectively may be susceptible to sedimentation of insoluble frusemide, chemically degraded by gastric acid and impractical in case of injection due to many ampoules required.(7) the aim of this study is to prepare frusemide in different liquid dosage forms (syrup, elixir, suspension and emulsion) because these forms are not commercially available. then test its stability and compatibility to decide on an appropriate formulation and assigne an expire date. materials, instruments and methods frusemide (usp), xanthan gum, cherry flavor and sorbitol supplied by sdi, iraq; sodium carboxy methylcellulose, methylcellulose, methyl and propyl paraben from hopkin and williams ltd, england; tween20 and 80 (merck-schanchard germany); ethanol gcc gainland chemicals company, u.k.; polyvinyl pyrrolidin (pvp) and potassium carbonate from bdh chemical ltd. pool, england. sodium saccharin and aspartam (bdh limited pool, england); sodium hydroxide (flukaag); disodium hydrogen phosphate and potassium dihydrogen orthophosphate (atlas chemie, wgermany); frusemide 80 mg tablets (hoechst marion roussel) and date of production is 9-2007. sartorius balance ag gottingen, bl210s, ce, germany; ph meter, orchidis labrotaries, france and hanna instruments type, france; dissolution apparatus type ii, dis 6000, copley scientific, nottingham, u.k.; uv. visible spectrophotometer, gitra 5,gbc scientific equipment, u.s.a.; oven 50, 400c memmert 854 schwabach, w. germany; oven 600c gallenkamp, b5 size one, england. experimental formulas i, ii and iii which are summarized in table (1) were prepared according to the following methods. table (1): different formulas of frusemide prepared as solution, syrup and elixir (i, ii and iii respectively) matarials formulas i ii iii frusemide (gm) 1.0 0.4 0.8 potassium carbonate (gm) 0.5 -- tween 20 (ml) --5 tween 80 (ml) -6 - sodium carboxy methyl cellulose (1%w/v) -5 ml - aspartam (gm) -0.054 - sodium saccharin (gm) --0.08 glycerol (ml) 30 -- sorbitol 70% w/w (ml) -32.4 10 alcohol 95% (ml) -10 40 citric acid (gm) 0.1 cherry flavor (gm) 0.05 phosphate buffer 8 (ml) qs -100 100 purified water (ml) qs 100 -- frusemide oral solution (formula i) 0.5 gm potassium carbonate was dissolved in 45 ml purified water. then frusemide 1gm and citric acid 0.1gm were added with stirring. glycerine 30 ml was measured by graduated cylinder and added to previous mixture. before the volume was completed to 100ml, the cherry flavor was added. finally the ph was adjused by using ph-meter. (6) frusemide free sugar based syrup (formulaii) 0.4 gm of frusemide was mixted with 6m1 tween80 and ethanol 10ml with stirring. aspartame and citric acid were added to previous mixture. iraqi j pharm sci , vol.17 (2) ,2008 frusemide oral liquid dosage forms 3 sorbitol 32.4ml and 5ml dispersion of sodium carboxy methyl cellulose (1% w/v) were measured in graduated cylinder and added to resulting product with stirring. after the product was filtered by cotton the cherry flavor was added. the volume was completed to 100ml by phosphate buffer 8. finally the ph was adjusted by using ph-meter.(8) frusemide elixir (formula iii) 0.8gm of frusemide was dissolved in 10ml sorbitol plus 40ml ethanol. then citric acid 0.1 gm and sodium saccharin 0.08gm were mixed in 20 ml purified water puls 5 ml tween 20. then the aqueous solution was added to the alcoholic solution to maintain the highest possible alcoholic strength at all times so that the minimal separation occurs when the two solutions were completely mixed, the cherry flavor was added. then the volume was completed to 100ml by phosphate buffer 8. the elixir was permitted to stand for a few hours to ensure saturation of alcoholic solvent. the product was filtered by using talc as filter aid to prevent cloudy appearance. finally the ph was adjusted by using ph-meter. (6) phosphate buffer ph8 was prepared by mixing 50ml of a solution of 0.2 m potassium dihydrogen orthophosphate with 46.8ml of 0.2m sodium hyproxide then diluted to 200ml with water.(2) formulation of frusemide suspension table (2) shows 6 formulas of frusemide suspension prepared by the following method: frusemide, methyl plus propylparaben, sorbitol and glycerol were levigated in the mortar with tween20 and part of prepared dispersions of suspending agents (pvp, xanthan gum, sodium carboxy methyl cellulose, methylcellulose and chitosan) in different concentration as summarized in table (2). the remaining amounts of the dispersions were added in divided portions to the mixture. the mortar was rinsed several times with purified water and the rinsed volume of dispersion was added to cylinder, cocoa syrup was added before the volume was completed to 100ml by adding purified water. (5)comparison studies of formulas a, b, c, d and e the following parameters were used to compare the prepared formulas a, b, c, d and e. table (2): different formulas of frusemide prepared as suspension (a, b, c, d and e). and emulsion (f). matarials formulas a b c d e f frusemide (gm) 2.5 2.5 2.5 2.5 2.5 frusemide tablet (80mg) 5 pvp (gm) 10 xanthan gum (gm) 0.5 0.5 0.5 sodium carboxy methyl cellulose (gm) 0.5 methyl cellulose (gm) 0.25 chitosan (gm) 1.5 acacia (gm) 6 olive oil (ml) 18 tween 20 (ml) 1 glycerol (ml) 10 sorbitol (ml) 5 cocoa syrup (ml) 20 methyl + propyl paraben (gm) 0.18 +0.03 purified water qs(ml) 100 90 dissolution rate measurement the dissolution medium was 900ml of phosphate buffer 6.8. the temperature of study was 370c and the rotating velocity was 100 rev. min-1. 5 ml of each formulas a, b, c, d and e was transferred to the jar bottom using a syringe. at appropriate intervals samples of 5ml were taken from the jar and analyzed for total content of frusemide by uvspectrophotometer. detection was done at 330nm. 5ml of fresh phosphate buffer was added to the jar with each time intervals to keep the volume constant.(10) sedimentation volume 100 ml of each formulas (a, b, c, d and e) was transferred to the stoppered graduated cylinder. the suspension were shaken vigorously to ensure uniformly then left undistributed. the sedimentation volume was measured at selected time intervals during storage without agitation for a period of 8weeks and was calculated in terms of the ratio of ultimate settled height (vu) to the original height (vo).(11) iraqi j pharm sci , vol.17 (2) ,2008 frusemide oral liquid dosage forms 4 extemporaneous preparation of frusemide emulsion (formula f) acacia was triturated in mortar to be in powder form. 12ml water was added to get primary emulsion. 18ml olive oil was added drop by drop with continuous trituration in same direction until clickuing sound was heard. spread frusemid powder from grinded (5) tablets of 80 mg strength. the primary emulsion was diluted to 90ml by purified water. the contents of formula f are showed in table (2) as well as the dissolution rates of formula f were measured as described previously.(8) stability study 2ml samples of formulas i, ii and iii were stored in closed tubes at 40, 50 and 60 0c measurement on: 0, 7, 15, 30, 60, 90, and 120 day.sample of 100ml suspension was inspected for change in color, odor, ph and precipitant. analysis for remaining frusemide was carried by diluting 2ml of sample with distilled water to 200ml. the 5 ml of resultant solution was taken and completed to 50 ml with 0.1 n naoh. the absorbance of later solution was detected by a uv-method at 271 nm.(9) the accelerated stability test were also carried out on the suspensions showing the heighest sedimentation volume which were formulas a and d. the suspension of each formula was centrifuged to get supernatant solution. 1 ml samples of the resultant solution were stored in closed tubes at 40, 50 and 60 0c measurement on: 0,7, 15, 30, 60, 90 and 120 day. samples of 100ml suspension were inspected for change in color, odor, ph and precipitant. analysis of remaining frusemide was carried out by diluting 1ml of supernatant solutions with phosphate buffer 6.8 to 25 ml. the absorbance of latter solutions were determined by a spectrophotometer at 330 nm.(9,12) the shelf life calculated from the initial concentration [a0] and the apparent zero-order rate of degradation (k0) accordings to the following equations. (13) 0 0 %10 0 ][10.0 ]lub[ k a t ilityofrusemideskxk   the stability of extemporaneous frusemide emulsion was done as those of suspension which mentioned previously. rheogram rheogram was obtained for the selected formula at 370c with brook field dv-ii+pro viscometer which read shear stress versus shear rate. results and discussion oral frusemide solution was claimed to produce agreater diuretic with congestive heart failure than tablet so formula i prepared as oral solution containing potassium carbonate which added to increase ph up to 7. the effect of ph on solubility is critical in the formulation of liquid dosage forms. the solubility of frusemide (pka=3.9) is often ph dependent. furthermore, the ph control is at least as important to fully control the crystallize habit and the stat of agglomeration to ensure quality, efficacy and safety of the drug.(10,14) also frusemide prepared as syrup(formula ii) which containing tween 80 to increase solubility of frusemide. alcohol is present in formula ii to serve as a solvent and preservative. while benzoates and parabens was excluded from this formula (ph8) because they are ineffective as preservative in alkaline solutions which frusemide freely soluble in it. sorbitol is compatible with alcohol as much as 10 percent (v/v) before crystallization is observed. (8, 15) formula iii having a high alcoholic content (elixir) contains saccharin which is required only in small amount rather than sucrose which is only slightly soluble in alcohol and required greater quantities for equivelant sweetness. thisتformulaتisتselfتpreservingتandتdon’tتrequireتtheت addition of antimicrobial agent because it contains more than 10-12% of alcohol. the carboxymethylcellulose, a derived gum function as viscosity builder agent.(6) the presence of glycerine and sorbitol in formula i, ii and iii contributes to solvent effect, assists in the dissolution of the solute and enhance the stability of the preparation. however, the presence of these materials also adds to the viscosity. (6) the effectiveness of cherry flavour in masking the taste of frusemide is enhanced by presence of weak acid (citric acid). (8) the accelerated studies applied on formulas i, ii and iii at higher temperatures (40, 50, and 600c) were employed to predict the expiration date of these formula using uv-spectrophotometer. the degradation of frusemide in these formulas shows first order kinetics since straight lines were obtained by plotting the logarithm of percent remaining of frusemide versus time as shown in figures (1,2 and 3) according equation (1). log c= log co – ----------1 in which co is the initial concentration; c is the remaining undecomposed concentration at time t; and k is the first order rate constant and –k/2.303 is the slope of the line from which the value of the rate constant is obtained. (12) 303.2 k t iraqi j pharm sci , vol.17 (2) ,2008 frusemide oral liquid dosage forms 5 1.984 1.986 1.988 1.99 1.992 1.994 1.996 1.998 2 2.002 0 1 2 3 4 5 time (months) lo g % r em ai ni ng 40 c 50 c 60 c figure (1): degradation curve of frusemide oral solution (formula i) at 40, 50, 60 0c 1.984 1.986 1.988 1.99 1.992 1.994 1.996 1.998 2 2.002 0 1 2 3 4 5 time (months) lo g % r em ai ni ng 40 c 50 c 60 c figure (2): degradation curve of frusemide syrup (formula ii) at 40, 50, 60 0c 1.955 1.96 1.965 1.97 1.975 1.98 1.985 1.99 1.995 2 2.005 0 1 2 3 4 5 time (month) lo g% r em in in g 40 50 60 figure (3): degradation curve of frusemide elixir (formula iii) at 40, 50, 60 0c table (3) summarized the degradation rate constant of formulas i, ii and iii. arrhenious plots were constructed to predict the degradation rate constant of frusemide at 250c as shown in figures (4, 5 and 6). the results indicate no significant differences (p>0.05) between k25 0 c for formulas (i, ii and iii). the expiration data of frusemide was calculated according to first order reaction equation: c k t 025 104.0%10  -----------------2 table (3): degradation rate constants of frusemide in formulas i, ii, iii at 40, 50 and 60 0c. formulas k40(x10 -3) month-1 k50(x10-3) month-1 k60(x10-3) month-1 i 5.9 7.1 8.0 ii 10.7 26 35 iii 10.1 15 20 0 1 2 3 4 5 6 7 8 9 2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 3.35 3.4 1/tx 10 3 (kelvin -1) kx 1 0 3 ( m o un th -1 ) figure (4): arrhenious plot for estimation of the expiration date of formula i at 25 0c. 0 5 10 15 20 25 30 35 2.9 3 3.1 3.2 3.3 3.4 1/tx103(kelvin-1) kx 10 -3 (m o nt h -1 ) figure (5): arrhenious plot for estimation of the expiration date of formula ii at 25 0c iraqi j pharm sci , vol.17 (2) ,2008 frusemide oral liquid dosage forms 6 0 2 4 6 8 10 12 14 16 18 20 2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 3.35 3.4 1/tx10 3(kelvin-1) kx 10 3 ( m o nt h -1 ) f igure (6): arrhenions plot for estimation of the expiration date of formula iii at 25 0c the expiration dates were found to be equal to 1.8, 1.07 and 1.22 years for formulas i, ii and iii respectively as shown in table (4). table (4): degradation rate constants at 250c and the corresponding expiration dates of the prepared formulas. formulas no. k25(x10 -3) month-1 t 10% (years) i 4.8 1.8 ii 8.09 1.07 iii 7.0 1.22 figures (7 and 8) show the dissolution rate profile of frusemide for formulas a, b, c, d, e and f. the results showed that frusemide amounts released increases in the following order: f<e<c<d<b<a. the differences was significant (p<0.05). formula a showed the highest dissolution rate for a short periods of time (10 minutes), this could be due to the wetting effect of water soluble polymer (10%pvp) solution with intrinsic rapid dissolution properties especially in the practice of the presence solubilizer glycerol and sugar alcohol such as mannitol and mixture thereof . optionally a surfactant such as tween 20 may be added to facillate wettability within formulation. (16) figure (9) shows the sediment volume of formulas a, b, c, d and e during 8 weeks after 48 hours undisturbed. 0 20 40 60 80 100 120 0 10 20 30 40 50 60 time min % d is so lv ed o f fr us em id e a b c pol y. (b) pol y. (b)figure (7) : dissolution rate profile of frusemide formula (a, b and c) 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 time (min) % d is so lv ed o f f ru se m id e d e f figure (8) : dissolution rate profile of frusomide formulas (d, e and f) 0 0.2 0.4 0.6 0.8 1 1.2 0 2 4 6 8 10 time (week) s ed m en ta tio n vo lu m e a b c d e figure (9): sedimentation volume of formulas (a, b, c, d and e) the sediment volume measured according to the ratio of the final height of the sediment after settling (vu) to the initial height of the total product (v0). vo vu f  -------(3) iraqi j pharm sci , vol.17 (2) ,2008 frusemide oral liquid dosage forms 7 the results show that the sediment volume increases in the following order: e<b<c<d<a the sedimentation volume depends on the types and concentrations of suspending, wetting and viscosity enhancing agents used. as a result, a high sediment volume observed in formula a could be a result of the increase in the viscosity of the pvp dispersion in alkaline media which may be caused by the expansion resulting from electrostatic repulsion between negatively charged carboxylate group. (17, 18) in formula d the increase in viscosity due to strong cross-linking between the nonionic gum (methylcellulose) and anionic gum (xanthan gum).(19) the presence of glycerol, sorbitol and cocoa syrup adds to the viscosity of suspension especially cocoa syrup (chocolate syrup) which is a suspension of cocoa powder in vehicle containing liquid glucose, glycerine, vanillin and sucrose. it is effective because of its high viscosity and enhance the palatability by coating the tongue and thus it tends to inhibit diffusion of the frusemide to the taste buds. (8) the resultant solutions from centrifuging of formulas a and d suspension was with no reservoir of frusemide to replace that depleted so frusemide degradation in them follows the first-order expression as in equation (4). ][ ][ ck dt cd  -------(4) in which c is the concentration of frusemide remaining undecomposed at time and k is known as a first-order rate constant. when the concentration [c] is rended constant as in the case of a suspension, the equation (5) is applied. okck ][ ------(5) in which k0 is apparent zero order rate constant, [c] is the solubility of frusemide at 250c which equal 0.086 gm/100ml at ph 7 and k is first order rate constant at 250c the first order rate constant for frusemide degradation in supernatant centrifuged solution of formula (a and d) were calculated from slopes of straight lines which result from plotting log% remaining of frusemide in these solutions versus time at elevated temperatures (40, 50 and 60 0c) as shown in figure (10). then by plotting the log of these rate constants versus reciprocal of the absolute temperature. first order rate constant k25 0 c was obtained by extrapolating the straight line in arrhenious plot as shown in figure (11). k0 = k25 0 c x [ frusemide in solution] as in equation(5) k0 = (8.07 x 10 -2 month-1) x (0.086 g/100ml) k0 = 6.94 x 10 -5 g/ml. month-1 then, the expiration date of frusemide suspension formula (a and d) which showed the best release and sedimentation volume than other formulas was calculated according to apparent-zero order reaction equation o 0 k ][10.0 %10 a t  ----------(6) 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 time (month) lo g % r em ai ni ng 40 50 60 figure (10): degradation curve of centrifuged frusemide solution (formula a and d) at 40, 50, 60 0c 0 2 4 6 8 10 12 14 16 2.9 3 3.1 3.2 3.3 3.4 1/tx103(kelvin-1) k x 10 -3 ( m o nt h -1 ) figure (11): arrhenenius plot of centrifuged frusemide solution (formula aandd) the expiration dates were found to equal 3 years for both formula a and d. the shelf life of extemporaneously frusemide prepared emulsion (formula f) was calculated from the initial concentration and the apparent zero-order rate of degradation (k0) as the steps followed by estimation of expiration data of formulas a and d frusemide iraqi j pharm sci , vol.17 (2) ,2008 frusemide oral liquid dosage forms 8 suspensions. the shelf life of formula f was 45 days. acacia used in preparation of formula f because it is preferred in the formulation of the most extemporaneous prepared products. as well as the experimental studies in animal and clinical studies in humans showed that acacia gum has a urea lowering effects so this preparation is useful for patients suffering from hypertension with chronic renal failure. (20) formula a was selected to study the rheologyتasتshownتinتfigureت(12)تbecauseتofتit’sت highest release, sediment volume and shelf life than other formulas. the rheogram of formula a has a high viscosity at low shear stress while at higher shearing stress it has low viscosisty due to the flow behavior of pvp at concentration above 5% witch is typical pseudoplastic and thixotropic characteristics.(18) therefore formula a which containing pvp, tween20, glycerol, sorbitol, cocoa syrup and parabens considered a well formulated suspension because it was physically and chemically stable due to pvp is compatible with many drugs such as frusemide, paracetamol, salicylic acid and testosterone. (16) 0 100 200 300 400 500 600 700 800 0 200 400 600 800 1000 1200 shear stress (dyne cm -1 ) s he ar r at e (s ec . -1 ) f igure (12): rheogram of the selected frusemide suspension formula (a) references: 1martindale, “theتextraتpharmacopoeia” 31st ed., royal pharmaceutical society; 1996; p 871-874. 2british pharmacopeia vol.2; 1993; p 784. 3szilvia, b., geza, r.j., eszter, d., george, f. and istvan, in vivo and in vitro study in rats of rectal suppositories containing frusemide, european jaurnal of pharmaceutics and biopharmaceutics, may 2002, 53, issue 3, p 311-315. 4akiyama, y., nagahara, n. e., kitano, m., iwasa, s., yamamoto, i., azuma, j., evaluation of oral mucoadhesive microspheres in man on the basis of the pharmacokinetics of frusemide, j-pharm-pharmacol; 1998; 50(2); 159-66. 5aulton, m.e., pharmaceutics: the science of dosage form design, 2nd ed., 2002, pp 242, 9193. 6ansel, h.c., allen, jr.l., popovich, n.g., pharmaceutical dosage forms and drug delivery system, 2005, seventh ed., pp 338-342, 360-365. 7wood,d.j.,extemporaneous formulationsproblems and solutions, j-paediatric and perinatal drug therapy, 1997; 1:25-29. 8lewis, w.d., sprowls american pharmacy,an introduction to pharmaceutical techniques and dosage forms, 7th ed., 1974, 76-88, 209-23. 9u.s.pharmacopeia , by authority of the united states pharmacopeial convention , inc. 2004 , p 844,845 . 10bauer, b., couteau, a.,monjanel, f., effects of the physical characteristics of frusemide on its release from generic tablets, stp pharma practiques, 2002, 12(2), 76-84. 11florence, a.t., attwood, a., physicohochemical principles of pharmacy, 2nd ed., 1988, 257, 264265. 12martin, a., kinetics, physical pharmacy, physical chemical principles in the pharmaceutical science, lea and febiger philadelphia. london 4th ed., 1993-286. 13 qasem, j. g, bummer, p.m and digenis, g. a, kinetics of pactitaxel, pharm scitech, 4(2), 2003,21. 14cohen, n., golik, a., effect of frusemide oral solution versus frusemide tablet, j-miner electrolyte metab, 1996; 22(4); pp 248-52. 15shihad, a.r., mustafa, r.m., effect of polyethylene glycol, sodium lauryl sulfat and polysorbate80 on the solubility of frusemide, international journal of pharmaceutics, 1979, 4, p 13-20. 16ghebre, s., isac, j., solid pharmaceutical dispersion, united state patents, oncology out sourcing partner in development, 2004, (13), p 15. 17eczacilik, b., studies on furazolidone suspension formulation, acta pharmaceutic turcica, 1992. 13, 97-103. 18kassem, m. a., matha, a. g., rheologic studies on dispersion of polyvinylpyrrolidone, pharmaceutical acta helvetiae, 1970, 18-26. 19walker, c. v., wells, j. i., rheological synergism between ionic-nonionic gums, international journal of pharmaceutics, 1982, 11, p 309-322. 20chesney, r. m, patters, a., acacia gum in chronic renal failure; j-therapy, 2006, 3(2), p 183-185. iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel doi: https://doi.org/10.31351/vol28iss1pp64-74 64 preparation and characterization of etodolac as a topical nanosponges hydrogel maysam m. abbas*,1 and nawal a. rajab* *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract nanosponges (ns) of etodolac (eto) was prepared using the emulsion solvent diffusion method. etodolac took as a model drug because of its short half-life, low water solubility; ns is non-irritating, non-mutagenic, nonallergenic and non-toxic suitable to bind to poorlysoluble drugs within the matrix and improve their bioavailability .the effects of drug: polymer ratio, the effect of level concentration of internal phase and stirring time and other variables that effect on the physical characteristics of ns were investigated and characterized.the selected formula was lyophilized then incorporated into hydrogel ; which also evaluated .the results show that the formulation that contain drug:polyvinyl alcohol: ethylcellulose in ratio 1:3:2 is the best with smallest particle size 40.2±0.098nm with polydispersibility0.005 and in vitro release 97.6%±0.11, , eto ns carbopol hydrogel produced a significant(p<0.05) improvement of the in vitro release than pure eto hydrogel. keywords: etodolac, nanosponges, hydrogel. موضعي مائي كمستحلب نانوية كإسفنجيات االيتودوالك دواء ميوتقي تحضير **رجب عياش نوال و 1*،عباس محمد ميسم العراق بغداد،بغداد، جامعة ، الصيدلة كلية الصيدالنيات، فرع* الخالصة بأشكال تحرير و الذوبان لتعزيز وذلك الذوبان قليل االيتودوالك دواء من نانوية اسفنجيات من هالم صياغة تقديم الدراسة من الهدف من المذيبات انتشار بطريقة االسفنجيات حضرت الهضمي الجهاز على الجانبية االثار لتقليل بديلة كوسيلة الجلد طريق عن استخدامه و موضعية تقيم تم و المائي الهالم الي االسفنجيات اضفنا وبعدها و التحميل كفاءة لإلسفنجيات الفزيائية الخصائص تأثيرات عليها اختبرت ثم مستحلب شبه مختبريا االسفنجي المائي الهالم تحرر تفوق النتائج أظهرت و مختبريا، المائي الهالم . مائي هالم النانوية، اإلسفنجيات االيتودوالك،: المفتاحية الكلمات introduction the most significant challenge with topical drug delivery is the barrier nature of the skin that restricts the entry of most drugs, to increase the therapeutic effectiveness of the existing drug molecules is by formulating them using novel nanocarrier systems and incorporating them into topical preparations (1). nanosponges are a novel class of nanoparticles, exhibiting promising potential in controlled drug delivery, especially topical formulations. bezawada et al., define nanosponges(ns) as biocompatible porous nanoparticles formulate as nano-sized colloidal carriers having the shape of tiny sponges in nature; with a size of about a virus and the average diameter (250 nm -1μm) which will fill with a variety of materials. different methods of preparation of ns; ultrasound assisted synthesis ,emulsion solvent diffusion method, solvent method and hyper cross-linked cyclodextrins (2); pervious work on ns ; like using lemongrass oil a volatile oil extracted from the leaves of cymbopogon citratus incorporated in an ec ns revealed that the spongy structure with minute pores and the sustained integrity of the ns structure when incorporated in the hydrogel showed no skin irritation and increasing bioavailability (2). etodolac (eto) is a pyranocarboxylic acid that falls in the (nsaids) category. eto is a weakly acidic drug that belongs to the biopharmaceutical classification system (bcs) class ii type (practically water insoluble). the chemical name is [(±) (1, 8 -diethyl-1, 3,4, 9-tetrahydropyrano[3, 4b] indole 1-acetic acid] with a molecular formula of c17h21no3, and molecular weight is 287.36g/mol, fig. (1) demonstrated chemical structure of eto. (3) 1corresponding author: e-mail: maya_vv56@yahoo.com received:8 / 9/2018 accepted: 14/11/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp64-74 iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 65 figure (1) chemical structure of etodolac(3) materials and methods materials etodolac powder supplied by hangzhou hyper chemicals limited, china, ethyl cellulose polymers powder n-type obtained from hangzhou hyper chemicals limited, china,, gcc analytical reagent, uk supplied dichloromethane. all other materials used in this study were of analytical grade. methods preparation of etodolac nanosponges eto ns was prepared by emulsion solvent diffusion method table (1). the internal organic phase consisted of different amounts of eto and ethyl cellulose; all dissolved in dichloromethane (dcm) or acetone (internal or disperse phase); while pva dissolved in 100 ml water (external or aqueous continuous phase). the disperse phase was added very slowly and continuously with the help of syringe into the aqueous phase then we put the formula under mechanical agitation for 2 hours at room temperature (4, 5). table (1) composition of eto ns. formula no. ingredient f1 f2 f3 f4 f5 f6 f7 f8 etodolac (gm) 4 2 1 1 1 1 1 1 pva (gm) 3 3 3 2 3 2 3 4 ec (gm) 2 2 2 2 2 2 3 2 dichloromethane (ml) 20 20 20 20 _ 20 20 20 acetone(ml) _ _ _ _ 20 _ _ _ distilled water(ml) 100 100 100 100 100 100 100 100 stirring speed (rpm) 1000 1000 1000 1000 1000 2500 1000 1000 methods evaluation of the prepared nanosponges particle size and size distribution particle size and polydispersity index determination done by using the nano brook 90plus particle size analyzer; which is a dynamic light scattering, works by measuring the intensity of light scattered by the molecules in the sample as a function of time (6). the in-vitro dissolution profile of nanosponges in-vitro drug release studies with a pretreated dialysis bag (schuchardt dialysis membrane mwco 12,000 da) cut off. an aqueous dispersion of ns (10ml), is placed in dissolution test usp apparatus type ii. that fixed on 100 rpm and 37 ±0.5°c, eto loaded ns evaluated by immersing it in 900 ml 7.4ph phosphate buffer (7,8) . zeta potential measurement (zp) zp for selected formula determined by the nano brook 90plus zeta sizer (brookhaveninstruments usa) (9). freeze drying of the selected etodolac nanosponges the formed ns sonicated for 10 minutes to avoid the presence of aggregates. then centrifuged at 3000rpm for15min at room temperature to separate the uncomplexed drug as a residue below the colloidal supernatant. after that we filtrate by using filter paper to remove any undissolved particles (10). the colloidal supernatants were freeze-dried to obtain eto loaded ns (11). drug entrapment efficiency of nanosponges lyophilized powder for the drug entrapment efficiency tests, 10mg of the ns powder was dissolved in 10ml phosphate buffer solution 7.4, in a volumetric flask then placed in bath sonicator to break the complex the free drug remained in supernatant while entrapped drug retained in the ns, the measurements was carried out in triplicate and standard deviation values was taken (12). saturation solubility of lyophilized powder performed for the selected formulation by dissolving the excess amount of lyophilized powder in 10 ml of phosphate buffer ph 7.4. then the mixture was shaken for 48 hours at 25°c then filtered; and analyzed. (13) the dissolution profile of lyophilized powder in-vitro drug release studies were carried out using usp type ii dissolution apparatus (50 rpm, 37 ±0.5ºc); 1 gm of eto loaded nanosponges lyophilized powder put in pretreated dialysis bag (cut off 12,000 da). the dissolution media contain 900 ml of phosphate buffer ph7.4; at each fixed time intervals samples were taken and suitably diluted and analyzed; in triplicate (n = 3). (14) fourier transform infrared spectroscopic analysis (ftir) ftir helps to check out the compatibility between the drug and the excipients.the ftir spectra of pure eto and lyophilized powder of the selected formula were obtained using (ftir-8300 shimadzu, japan)(15). iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 66 x-ray powder diffraction (xrpd) powder x-ray diffraction can be used to emphasize the crystalline nature of the substances in the solid state, the diffractograms of eto pure powders and lyophilized powders of the selected formulations analysis using shimadzu xrd-6000 powder x-ray diffractometer (16). scanning electron microscope (sem) sem can be used to study morphology, and surface topography of the prepared ns and the difference in morphology state of the pure drug and the product by using a vega/tescan scanning electron microscope (17). preparation of nanosponges loaded hydrogel the gel-forming polymer (carbopol 934) 1 gm was soaked in 100 ml water for 2 hrs. then dispersed by agitation using a magnetic stirrer to get a uniform dispersion. the dispersion was allowed to stand for 15 minutes so that all the entrained air expelled. methylparaben (preservative); dissolved in a sufficient quantity of water pre-warmed to 40°c and incorporated. then add triethanolamine (2% v/v) drop by drop with continuous mixing to neutralize the ph of polymer aqueous solution. finally ethanolic solutions of lyophilized eto ns and second formula of pure eto powder incorporated in the polymer aqueous solution (18) . the composition of eto nanosponge loaded in carbopol 934 hydrogel in the table (2). table (2) physical properties of the prepared hydrogel ingredients (% w/w) f1 f2 etodolac ns eq. to 2 gm etodolac 12 etodolac (gm) 2 carbopol 934(gm) 1 1 triethanolamine 2.0 2.0 ethanol (95% v/v) 20 20 deionized water q.s 100 100 methyl paraben(mg) 100 100 determination of viscosity rheology of prepared ns loaded hydrogel formulation was studied by myr rotational (cup and bop) digital viscometer with spindle no. r7, at 2200 rpm, at room temperature; (n=3) (19). determination of etodolac content in the hydrogel formula eto content in the hydrogel was determined by taking specific quantity of the prepared gel which is equivalent to 10 mg of etodolac and transferred to 100 ml volumetric flask containing phosphate buffer (ph 7.4). the mixture then allowed to sonicate for 5 minutes followed by filtration then the filtrate was analyzed at λ max of eto (20). in-vitro dissolution test of etodolac nanosponges loaded hydrogel the in-vitro release of eto from ns loaded hydrogel and eto hydrogel formulas performed by using dissolution apparatus-ii (paddle type). hydrogel (2 gm) was performed using dialysis bag method with pretreated (dialysis membrane cut off 12,000 da). the dissolution test performed ; with 900 ml dissolution media phosphate buffer ph 7.4, at 37 ± 0.5 °c with stirring speed of 100 rpm. samples filtrated, diluted and analyzed; usually the drug release experiments were conducted in triplicate (n = 3); the statistic mean value of three readings was taken. result and discussion this study presents a new approach for the modification of polymeric nanoparticles as well as a new system for enhancing dissolution and release of poorly soluble drugs. particle size analysis and polydispersity index measurement the effect of different parameters on the particle size and polydispersity index was studied using eight different formulations. the mean particle size (effective diameter) for formulations varied in the range from 46.9±0.358 nm to 1043.35±10.2 nm. the particle size and pdi for different formulations of different parameters is showing in the table (3). table (3) the particle size, pdi of different formulations formula no. particle size ±se* pdi % drug release (% ± se) f1 526.5± 0.125 0.352 59%± 8.767 f2 224.3 ± 90.7 0.005 66%± 8.022 f3 46.9± 6.5 0.005 97.6%± 12.201 f4 172.8± 0.169 0.005 89 %± 11.603 f5 354.68± 0.8 0.329 75%± 10.242 f6 111.5± 9.2 0.246 45 %± 5.998 f7 711.6± 90.7 0.005 50% ± 17.224 f8 1043.35± 10.2 0.352 45%± 8.870 *se standard error, n=3 variables affecting formulations effect of the drug to polymer ratio the formula 1 and 2 as seen in the figure 2 and figure 3; revealed that as the amount of drug increase; the particle size increase and invitro drug release reduced significantly (p< 0.05) ; bohrey et al. observe the same result ; and explained it by the fact that a more considerable amount of drug results iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 67 in a more viscous organic phase (dispersed phase), making complex the mutual dispersion of the aspects and forming bigger nanoparticles (22). while in formula 3 and 7 figure 2 and 4 ; amount of ethyl cellulose polymer increased from 2 gm to 3 gm ;this cause an increase in particle size and significantly (p< 0.05) decrease in the amount of drug released; the same result was seen with kılıçarslan et al., they prepared verapamil hcl loaded microspheres and discuss the effect of the drug/polymer ratio ; as the amount of polymer in the formulation increased, the drug release decreased; due to rising in thickness of polymer wall of ns particles will affect the diffusion path thus the drug not be quickly released in to the dissolution medium (23). figure (2) the effect of the drug to polymer ratio on the particle size of etodolac loaded nanosponge figure (3) dissolution profile of formulas f1, f2 in pbs of ph 7.4. figure (4) dissolution profile of formulas f3, f7 in pbs of ph 7.4. effect of pva concentration the presence of pva molecules stabilizes the emulsion nano droplets and prevents them from aggregation with one another. the pva molecules must cover the organic/aqueous interfacial area of all the droplets. hence a specific amount of pva molecules is required to achieve small size of nanoparticles. we can notice from table (3) and fig. 5 and fig. 6 that the most uniform size distribution was obtained from 3% pva in f3; as the amount of pva increased f8 or decreased f4 from 3 % w/v; this cause insignificant effect (p > 0.5) on in -vitro drug release and particle size. as the concentration of pva increased as in f8 this cause the micro size particle appearance instead of nanoparticles due to the increased viscosity of the aqueous phase causing reduction in the net shear stress available for droplet break down so the pva concentration stays in the continuous phase. in this case pva does not play a role, either in the emulsification or in the stabilization of the sponges. the same result was noticed with zweers et al. and feng et al. (24, 25) . while in formula 4 as the amount of pva decreased from specific level; the particle size decreased but cause the fragility of the formed ns during the process of dispersion. which was noticed by a significant decrease in invitro drug release for f4. the particle size of ns seems to be dependent on the pva concentration in continuous phase; the same result was reported by kemala et al. (26). figure (5) pva concentration effect on eto loaded nanosponge figure (6) dissolution profile of formulas f3, f4 and f8 in pbs of ph 7.4. iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 68 effect of type of internal phase solvent organic solvents have essential role in the preparation of ns. beside dissolving the drug and polymer; organic solvents ensure the initial thermodynamic equilibrium with the aqueous phase. also, during development the organic solvent diffused into the external phase and form ns; and this diffusion and consequently elimination of organic solvent mainly depend on its boiling point (27). formula 3 and 5 from table 3 and fig.7; demonstrate this effect of change the type of organic phase causing increase in mean particle size with significantly (p< 0.05) decreasing invitro drug release. because they depend on the physical properties of the organic solvent used; the solubility of dcm in water is low, but vapor pressure is very high. therefore, dcm rapidly diffused into water and evaporated out resulting in fast precipitation of ns droplets (containing polymer with drug) without giving much time for drug molecules to partition into aqueous phase and aggregation. while acetone have higher boiling point (b.p. 56 °c) than dcm (b.p. 39 °c) so the evaporation is slower; therefore the in vitro release of acetone is low (28,29). figure (7) dissolution profile of formulas f3, f5 pure drug in pbs of ph 7.4. effect of stirring speed formula 4 and 6 from table 3 and fig.8; show the impact of mixing speed on ns formulations; increasing the stirring speed from 1000 to 2500 rpm cause insignificant (p > 0.5) decreasing of in-vitro drug release with decreasing of mean particle size; this explained by srinivas et al.; at higher stirring rate decrease the mean particle size and the production yield was reduced because the polymer adhered to paddle due to high turbulence created within the external (1). figure (8) dissolution profile of formulas f4, f6 in pbs of ph 7.4. zeta potential measurement (zp) high zp value improve stability of the dispersion and will resist aggregation while colloids dispersion with low zeta potentials tends to coagulate or flocculate for the selected formulation of eto loaded ns was-30.8 mv, the charge was negative due to the surface negative charge of pva hydroxyl group that anchored on the surface of the ns ; the same result noticed with salah et al., (30) the ns adequately stabilized by pva nonionic surfactant that appeared to be the most suitable surfactant in reducing aggregation between nanoparticles the same result was reported by lakshmi et al. (31). saturation solubility of freeze-drying nanosponges the best formula for lyophilization; the batch f3 gave use the small particle size and lowest polydispersity index with best dissolution profile; table 1,3 and figure 3,4. the quantity f3 gives rise to fluffy mass powder showing highly porous structure; with a white cotton-like, these spongy materials was the same result that reported by singireddy et al. (32). the saturation solubility of the lyophilized powder was increased significantly (p < 0.5) from the pure drug solubility; it expands to 12± 0.7folds in ph 7.4; the same result reported by rao et al. (33). the dissolution profile of lyophilized powder the lyophilized powder of ns selected formula (f3) shown in fig. 9. the release of eto from the ns was higher than the release profile of pure drug within 90 min. the percent of cumulative drug release of lyophilized f3 was more than 90% in less than 30 min as compared to less than 20% and 25% of the pure drug in the same in phosphate buffer ph 7.4 media. factors that are contributing to a fast release attributed to the reduction in particle size causing increase in the surface area and consequently enhanced the contact between particles and dissolution medium the same result seen with torne et al. (34) . also, the plastic designs of the prepared ns structure and hydrophobic nature of ec, drug particles near the surface of the ns matrix could be initially diffused into the surrounding medium, iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 69 resulting in the increase in the pores and thus facilitating further drug release (21). figure (9) in -vitro drug release profile of eto loaded ns lyophilized powder in pbs ph 7.4 drug entrapment efficiency the results show that each10 mg of lyophilized powder of ns of formulas f3 contain 97.45 % of eto. fourier transform infrared spectroscopic analysis (ftir) the ftir spectrum of pure etodolac and lyophilized powder given in figure (10) a, b. the results showed that the characteristic peak of eto was c=o stretching vibration of cooh group in1743.33cm−1 is present in all the spectrums indicating that there is no chemical interaction between the drug other excipients (35). figure (10) a.ftir spectrum of pure etodolac figure (10) b . ftir spectrum of eto loaded ns lyophilized powder scanning electron microscope (sem) it can be seen that the raw drug particles have a rough surface with large particle size fig (11); while the sem of f3 lyophilized powder fig (12) showed finely spherical, smooth, and porous due to diffusion of dcm from the surface penjuri et. al. reported the same result (36). iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 70 µm)(11) sem of raw drug magnification 500x; size of particles (100 urefig figure (12) sem of f3; at 498x,500x,1.00kx and 2.00 kx magnification iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 71 powder x-ray diffraction analysis (pxrd) pxrd patterns of eto as a pure powder showed sharp diffraction peaks in the fig. (13) indicates the crystalline nature of the pure etodolac. however, these characteristic peaks disappeared in the pattern of lyophilized powder as seen in fig. (14) producing a diffused pattern of very lowintensity peaks and shifting to a lower degree with complete absence of and diffraction peak, this mean eto in the lyophilized powder is in an amorphous state; the same was reported by rao et al (37) . figure (13) pxrd of pure drug. figure (14) pxrd of selected formula f3 physical properties of the prepared hydrogel the physical appearance showed that systems gelled and pale to white non-transparent, with smooth, homogeneous constancy. this result agrees with that of research on miconazole nitrate loaded nanosponge gel (18). the result of ph for eto loaded ns hydrogel is 5.9±0.01; while the ph of eto hydrogel was 6.7±0.01; which lies in the normal ph range of the surface; indicating the suitability of the formulations for application on the skin (38). the viscosity of nanosponges hydrogel carbopol 934 hydrogel showed approximate viscosity between (32,000 -134,150) cp for eto loaded ns hydrogel while eto hydrogel showed viscosity between (45,000 140,546) cp; figure (15). the viscosity value of eto loaded ns hydrogel is excellent because it provides easy removal of preparation from the container and smooth application of formulations on the infected surface; there is a significant difference (p<0.05) between formulations.(39) figure (15) the rheogram of nanosponges hydrogel and pure etodolac hydrogel iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 72 determination of etodolac loaded nanosponges content in the hydrogel formulas the eto ns contents in hydrogel are 97.5% ± 0. while in eto hydrogel contents are 90.1% ± 0.44 from this we can conclude that eto uniformly distributed in both hydrogel formulations. in -vitro drug release study from the release profile fig. (16); eto ns loaded hydrogel has produced a significant improvement in the dissolution rate which is significantly higher (p<0.05) than that of pure eto hydrogel. in -vitro release of eto nanosponge from hydrogel showed fast and complete statement in comparison to pure eto hydrogel, the same result seen with; aldawsari et al.; they prepared a topical hydrogel of lemongrass-loaded ns; the higher porosity of the larger particles would allow for the leakage of the eto to the hydrogel during preparation, resulting in faster release rates (21) . figure (16) dissolution profile of eto from nanosponges hydrogel formula and pure eto hydrogel in pbs (ph 7.4) the eto loaded ns lyophilized powder and in the liquid state showed the same cumulative % of drug release profile more than 97.67% release of eto, within the same time; 90 minutes whereas the eto ns loaded hydrogel released more than 86% of eto in the same period fig. (12).this delayed in the release of eto ns loaded in hydrogel because of embedding of eto ns into the hydrogel base which effectively controlled the release of eto, so the release was a combination of the version of the drug from ns matrix carriers and subsequent diffusion through the microchannel structures of the carbopol hydrogel base song et al. reported the same result (40). conclusion emulsion solvent diffusion method was used because of its simplicity and reproducibility. also, this method is seem to be promising for the preparation of eto ns; it can achieve that ns carbopol hydrogel was greatly enhanced dissolution and release of etodolac when compared to pure etodolac hydrogel. references 1. sriniwas p, sreeja k. formulation and evaluation of voriconazole loaded nanosponges for oral and topical delivery. international journal of drug delivery research 2013; 5(1): 55-69. 2. bezawada s, charanjitha , reddy v. m, naveena , gupta v r m . nanosponge –a concise review for emerging 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solvents on nanoparticle formation and surfactants on release behavior invitro using costunolide as model anticancer agent. international journal of pharmacy and pharmaceutical sciences int j pharm pharm sci, 2014 ; 6 ( 4) : 638-645. 30. salah s, mahmoud a a, kamel a o. r e s e a r c h article : etodolac transdermal cubosomes for the treatment of rheumatoid arthritis: ex vivo permeation and in vivo pharmacokinetic studies drug delivery journal , 2017; 24(1): 846–856 31. lekshm u m d, poovi g, reddy p n. in vitro observation of repaglinide engineered polymeric nanoparticles. digest journal of nanomaterials and biostructures, 2012; 7 (1), 1 – 18. 32. singireddya a, subramaniana s. cyclodextrin nanosponges to enhance the dissolution profile of quercetin by inclusion complex formation journal particulate science and technology an international journal, 2014; 34 (3) : 341-346. 33. rao m r p, chaudhari j, trotta f , caldera f. research article investigation of cyclodextrinbased nanosponges for solubility and bioavailability enhancement of rilpivirine . american association of pharmaceutical scientists pharm sci tech, 2018 ;19(5):23582369 iraqi j pharm sci, vol.28(1) 2019 etodolac topical nanosponges hydrogel 74 34. torne s, darandale s, vavia p , trotta f , cavalli r . research article: cyclodextrin-based nanosponges: effective nanocarrier for tamoxifen delivery. pharmaceutical development and technology, 2013; 18(3): 619–625 35. ruhidas b, naskar d, banerjee s , karan s , chatterjee t k .evaluation of gum katira as a model sustained release adjuvant in the preparation of etodolac loaded microsphere . indian j. of pharmaceutical education and research, 2016; 50 ( 1) : 146-158 36. penjur s c b, ravouru n, damineni s, sailakshmi b n s , poreddy s r. formulation and evaluation of lansoprazole loaded nanosponges. turk j pharm sci, 2016; 13(3) : 304-310. 37. rao m , bajaj a , khole i , munjapara g, trotta f . original article: in vitro and in vivo evaluation of b-cyclodextrin-based nanosponges of telmisartan. journal of inclusion phenomena and macrocyclic chemistry , 2013; 77 (1–4 ) :135–145 38. el-kased r f, amer r i, attia d , elmazar m m . honey-based hydrogel: in vitro and comparative in vivo evaluation for burn wound healing. scientific reports j. .2017;(7):211 39. drais h k. design and evaluation of nystatin gel-based emulsion for oromucosal fungal infection. world j.of pharmacy and pharmac. scien., 2016; 5 (6 ): 297-307. 40. song s h , lee k m, kang j b , lee s g , kang n j , choi y w. improved skin delivery of voriconazole with a nanostructured lipid carrier-based hydrogel formulation. chem. pharm. bull., 2014; 62(8) : 793–798. a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci , vol.17 (2) , 2008 interleukins and abortion 74 effects of interleukin-2 (il-2) and interleukin-6 (il-6) in recurrent spontaneous abortion (rsa). dhamraa w. ahmed*,1 *collage of medical and health technology , baghdad , iraq abstract recurrent spontaneous abortion (rsa) is the most painful experience for couples expecting a child. this study aimed to determine the relevance of il-2 and il-6 in recurrent spontaneous abortion (rsa). serum samples were collected from 90 women attend al kadhmiya teaching hospital in baghdad. 60 women (first group) had recurrent abortion the women were negative for rubella virus, herpes simplex virus and toxplasma gondii. and they were negative from bacterial infection eg. niesseria gonorrhea and chlamydia trachomatis. the histopathological tests for fetus abnormalities were negative in this group, and 30 women (second group) with successful pregnancy (normal delivery). all samples were analyzed for il-2 and il-6 by commercially available enzyme-linked immunosorbent assay (elisa) kits. the data showed highly significant increase in the serum level of il-2 in group 1 compared with group 2 (p<0.001). however, il-6 showed highly significant increase level in group 2 compared with group 1. in addition, there was no significant correlation between these two markers in studied groups. the data of this study strengthen the possibility that high level of il-2 and low level of il-6 may explain the role of type-1 cytokines in the pathogenicity of recurrent spontaneous abortion. key words: interleukin 2, interleukin 6 and recurrent spontaneous abortion (rsa) ةصالخلا رأايلباجعإيلالليليلايس لذيوعيلاساعذسيلاايي يذليبيعجيلا ن يعيلاللا يعيذلي طاعسيلا دعي . نع يرحتلل ةساردلا هذه تيرجأ .لافطألا باجنإ يف نيبغارلا نيجوزلا ةايح يف ةميلألا براجتلا نم لركيملايلااذلا ياقسإلايدعي يذلي بالليلباجعإيلالليليلايس لذيبيني يأكييحأا يديس يوعميوعيوايادسيعيوعيطحعايل نققعي افيوحس دفي6ين2 ننذيلرطسلاا يعي لا عليي يلاسأقييليلاياياد يلانافياسا يديس ي عطكيوعيطحعايدعطاليوعيلب كعةيلالليليلايس لذينلاميليا يينلع كيققامعيوعي ين نلايي herpes simplex virus لاسسعاقلي لاإا ,نذيلنحيrubella virusلباعا ياديلنحيلاإع يلاايعطي ي)لاإييللا( ينلا مويارعي niesseria gonorrhea ,ين لاايدابي اعاسكعيا سلرعيلايح يلاحيم gondii toxoplasma لايجااعلي chlamydia trachomatisين لااي نع يرحتلل ةساردلا هذه تيرجأ .لافطألا باجنإ يف نيبغارلا نيجوزلا ةايح يف ةميلألا براجتلا نمع سكيلااذلا يلاسحياي يوعيدابين ا نيي امعليذلي نع يرحتلل ةساردلا هذه تيرجأ .لافطألا باجنإ يف نيبغارلا نيجوزلا ةايح يف ةميلألا براجتلا نم س يملايلاياياد .لاياياد يلاتعطي ي عطكي ( اقسإلايدعيelizaوعيعمعا يديس ي عطكيوعيطحعايننأعيبيقكعياعاذجي يأي . ييويلاأيسعليذإعكيانلرج يلراي لي) يذليوعاييلاياياد يلانافيوجعذط يووي2 , نع يرحتلل ةساردلا هذه تيرجأ .لافطألا باجنإ يف نيبغارلا نيجوزلا ةايح يف ةميلألا براجتلا نملكلليلاسسعيويذلنييوأسار يدعاي يذليوحسارعليلرطسلاا يعي6يني2 يالرطسلااا يذليوعاييلاياياد يلاتعطي يوليدأ يوجعذط يوويلاياياد ي6ن لااي عطكيوأارليلرطسلاا يعي , (p< 0.001لاياياد يلاتعطي يي) لاناف .اعبنعذ ي افي ااياييياكليلاسسعيويلذي عإيوأساايايعيملرعيلاالاييعيذليلاياعويويإيايلااذلا ي .طسعيويملايلااذلا ي نديكي .6ينلطلدعةيلرطسلاا يعي2( ذلي نع يرحتلل ةساردلا هذه تيرجأ .لافطألا باجنإ يف نيبغارلا نيجوزلا ةايح يف ةميلألا براجتلا نموللني يلباجعإيلاسقجعيليلايس لذيذليبعرليلذيدع يلرطسلاا يعي1لبسيعاي ي ننذ)لاحعرسا عرعي introduction recurrent spontaneous abortion (rsa) is one of the important complications in pregnancy. half of recurrent miscarriages loss is multifactorial, can be explained by genetic, hormonal,anatomical, metabolic abnormalities infections or autoimmune mechanisms and an be divided into embryological driven causes (mainly due to abnormal embryonic karyotypes) and maternally driven causes which affect the endometrium and/or placental development (1). known causes of maternal defects include coagulation disorders, autoimmune defects, endocrine disorders and endometrial defects (2). mammalian pregnancy is thought to be a state of immunological tolerance. the mechanisms underlying this phenomenon are still poorly understood, successful mammalian pregnancy depends upon tolerance of a genetically incompatible fetus by the maternal immune system. when tolerance is not achieved pregnancies fail (3) . 1 corresponding author : e-mail : dhamora@yahoo.com received : 16/7/2008 accepted : 16/12/2008 iraqi j pharm sci , vol.17 (2) , 2008 interleukins and abortion 75 immunological rejection of the fetus due to recognition of paternal antigens by the maternal immune system, resulting in abnormal immune cells and cytokine production, is postulated to be one cause of unexplained pregnancy loss (4). cytokines have traditionally been divided into families dependent upon the immune cell of origin and the immunological effects that they bring about. cd4+ t-helper cells are the major immune cells involved in cytokine production, and these can be divided into functional subsets based on their cytokine production, t helper 1 (th1) cells produce interferon gamma (ifng), il-2 and tumor necrosis factor beta (tnfb) are the main effectors of cell mediated immune response (5) t-helper 2 (th2) cells produce il-4, il-5, il-6 and il-10, which are the main effectors of antibody-mediated humoral responses (6). local mechanisms may play an important role in evading immune attack because maternal alloreactive lymphocytes are not systemically depleted. the specialized fetal tissue in contact with maternal uterine tissue might contribute to tolerance by several mechanisms, such as depleting tryptophan,(7) by inactivating natural killer cells through hla-g expression,(8) or by provoking apoptosis of activated maternal lymphocytes (9). incomplete tolerance might therefore result in disturbed pregnancy such as spontaneous abortion and pre-eclampsia. further, th1/th2 cytokine balance has been seen as a very important mechanism determining the survival of the fetus in the maternal uterus. recent evidence suggests that maternal tolerance is established at the feto-maternal interface, by factors deriving from the decidualized endometrium and from the trophoblast itself, is maintained throughout gestation in physiological pregnancy (10). cytokines released at the feto-maternal interface have been proposed to play an important role in regulating embryo survival controlling not only the maternal immune response but also angiogenesis and vascular remodeling (10; 11). th-1 cytokines are considered to be detrimental to pregnancy, via direct embryo toxic activity, or via damage to the placental trophoblast, or possibly by activating cells that are deleterious to the conceptus, whereas th-2 cytokines may directly or indirectly contribute to the success of pregnancy by down regulating potential th-1 reactivity (12; 13). protect the fetus and placenta from being rejected and to aid in the maintenance of normal pregnancy. in humans an important role for the t-helper 2 immune response has also been reported during normal pregnancy (14; 15). methods : studied group this study included ninety (90) women from the obstetrics and gynecology department of alkadhmiya teaching hospital in baghdad. patients' ages ranged between (18-36) years withيaيmeanيofي(30.1ي−ي27.5)يyear.يtheيpatientsي were divided into two groups: group 1: sixty (60) women were admitted to the hospital for recurrent spontaneous abortion (3-6 numbers of abortions) for evacuation. group 2: thirty (30) women with successful pregnancy (normal delivery) as control group. sample collection from each women included in the study blood samples were collected to obtain the serum. procedure * enzyme linked immunosorbent assay (elisa) for the detection of il-2, il-6 in serum: il-2, il-6: elisa test kits provided by (mabtech australia pty ltd). product cod: (3460-ia-6) il-6, (3430-ia-6) il-2. estimation of il-2, il-6 level in serum or plasma by elisa method. this method has two immunological steps. in the first step, the cytokine is captured by monoclonal antibody bound to the wells of a micro titer plate. in the second step a monoclonal antibody linked to a biotinylated monoclonal antibody is add together with streptavidine-peroxidase conjugate. the solid phase antibody-antigen complex and in turn, binds the conjugate. after incubation, the wells are washed and the antigen complex bound to the well detected by addition of a chromogenic substrate. the intensity of the color developed is directly related to the specific monoclonal antibodies concentration of the sample (16). statistical analysis the student test (t-test) analysis program was used to calculate the values, mean, and standard error were all used in the analysis and the relationship between the indicators was measured qualitatively by using the correlation coefficient iraqi j pharm sci , vol.17 (2) , 2008 interleukins and abortion 76 (r).values of p<0.05 were considered as statistically significant (17). results the expression of il-2 and il-6 was detected by elisa technique. table (1) shows the mean value of concentration (pg/ml) of il2 in sera of studied group which show highly significant (p<0.01) increased expression of il-2 in aborted women compared with control. table (2) shows the mean value of concentration (pg/ml) of il-6 in sera of studied group which show highly significant (p<0.01) increased expression of il-6 in aborted women compared with control. in addition, the study failed to found a significant correlation (p>0.05) between il-2 and il-6 in aborted women and control groups, as shown in table(3) . table (1): the mean value of concentration (pg/ml) of il-2 in sera of studied group. table (2): the mean value of concentration (pg/ml) of il-6 among studied group. table (3): pearson correlation (r) between il-2 and il-6 levels in serum of studied group pearson correlation il-2 il-6 abortion r 0.532 p-value >0.05 sig. ns control r 0.421 p-value >0.05 sig. ns ns= non significant difference discussion the level of il-2 increased in women with abortion in comparison with that successful pregnancy as shown in table (1). evidence supporting this result showed that the administration of one of the th-1 cytokines like interferonγي(ifnγ),يtumorيnecrosisي factorαي(tnf-α)يorيinterleukin-2 (il-2) to normal pregnant mice causes abortion (18). during a normal pregnancy, allogenic fetal tissues are exposed to the maternal immune system. rejection reactions normally develop after allogenic recognition following the principle of transplantation immunology (19). although the immune system is functional in the uterus and the embryo expresses paternal major histocompatibility complex (mhc) molecules, the conceptus nevertheless escapes the deleterious effect of maternal rejection. it may be due to local tolerance or even perhaps immune suppression after interactions between fetal antigens and the maternal immune system(19). t cells must change from a resting to an activated state during immune responses and lead to de-novo synthesis of interleukin il2 and expression of il-2 receptors {il-2rs} (20). interaction of il-2 and its receptors triggers cellular proliferation, culminating in the emergence of effectors t cells that are required for the full expression of immune responses. (21) spontaneous abortions in humans have been shown to be associated with increased production of interleukin (il)-2 and ifn by peripheral blood mononuclear cells (pbmc) and with decreased production of il10, as compared to normal pregnancy (22). studies by hill and colleagues have shown that studied group n mean± std. error comparison of significant p-value sig. control 20 97.45± 0.90 0.00 highly sig. (p<0.01) aborted women 20 259.32± 82.6 total 50 studied group n mean± std. error comparison of significant p-value sig. control 20 375.30±73.3 0.00 highly sig. (p<0.01) aborted women 20 82.41±0.86 total 50 iraqi j pharm sci , vol.17 (2) , 2008 interleukins and abortion 77 trophoblast antigens activate the pbmc of women with a history of unexplained recurrent spontaneous abortion (rsa) to produce the embryotoxic cytokines ifn and tnf-ß(22,23). interleukin-2, tumor necrosis factor, and interferon are deleterious and used to stimulate the apoptosis of human primary villous trophoblast cells. in addition this study reveals in table 2 that the expression of il-6 proteins in circulation of women with successful pregnancy was significantly higher (p<0.001) than that of women with abortion. further studies showed that there is a greater increase in il-6 secretion during pregnant compared to not pregnant state that may be detected by elisa of endometrial biopsy samples (24). human trophoblasts express il-6 receptor and produce il-6, which induces the production of hcg in an autocrine manner, suggesting a role of il-6 in early implantation and its continuation in early pregnancy(25). il-6 may play a role in physiological mechanisms involved in uterine contractions and the propagation of labour. thus, increased concentrations of il-6 may reflect a systemic reaction in the mother, leading to labour and delivery (26). this study showed that il-6 concentrations were lower in women with rsa than in those undergoing normal delivery. considering that il-6 is a th2-type cytokine and that normal pregnancy appears to be a th2-biased condition. these cytokines encourage vigorous antibody production, and are therefore commonly associated with strong antibody responses that are important in combating infections with extracellular organisms. il-6 may also induce prostaglandin synthesis by intrauterine tissues; thus, it seems to play a physiological role in labour development. high levels of il-6 have been detected in pregnant women at term and in preterm at labour (27). such human studies do not clearly indicate a cause-andconsequence between increased il-6 concentrations and events associated with labour, studies in monkeys have shown that an increase in il-6 concentrations precedes uterine contractions that may play a role in physiological mechanisms involved in uterine contractions and the propagation of labour (28). the il-6 found in the serum may originate from the trophoblast (29). thus the study by makhseed and colleagues, demonstrated that il-6 concentrations were lower in women with rsa than in those undergoing normal delivery, considering that il-6 is a th2-type cytokine and that normal pregnancy appears to be a th2-biased condition (13). th1 and th2 cells are mutually inhibitory to each other when th1 reactivity is high, th2 reactivity is usually low and vice versa (30). the current study shows in table (3) no significant correlation (p>0.05) between il-2 and il-6 in women with recurrent spontaneous abortion (rsa) and successful pregnancy. this un relation might be associated with different role of these two cytokine during pregnancy thus we suggested further study focusing on the role of il-2 and il-6 in pregnancy and rsa in placental tissue. in conclusion, il-2 might play a pathological role in pregnancy in contrast il-6 might play a role in successful pregnancy. references 1. aplin, j. maternal influences on placental development. semin. cell.dev. biol. 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(1989) th1 and th2 cells: different patterns of lymphokine secretion lead to different functional properties. annu. rev. immunol., 7, 145–173 iraqi journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 antiplatelts activity of vitamin e 25 antiplatelts activity of vitamin e in relation to dose and duration of therapy zaid o. ibrahim , shatha h. ali *,1 * department of clinical laboratory sciences, college of pharmacy , university of baghdad , baghdad , iraq abstract vitamin e, having the well known antioxidant activity through scavenging free radicals و it occurs in several isomeric forms , these isomers have relatively different functions . one of these actions is related to its ability to inhibit platelets aggregation and hence thrombosis. the present study included a total number of apparently healthy 62 males . 11of them served as standard group , treated with 100 mg aspirin /day for more than one month . another 31 subjects were randomly grouped into 5 groups that received different daily doses of α – tocopherol : 400 iu , 800 iu and 1200 iu for 2-6 months.the remainder ( 20 ) subjects served as a control group ( received no therapy ) . platelets function was assessed based on measuring bleeding time and slide platelets aggregation time ( spat ) meanwhile , thiobarbituric acid reactive substances (tbars) were measured as a marker for oxidative stress. the results showed that the commercially available vitamin e preparations (α tocopherol ) could exert anti-coagulant effect ,such effect is more dependant on duration of therapy , rather than dose related action .in addition to it’s antioxidant effect, which seems to be significantly correlated to it’s antiplatelets effect ( r=0.994 ,p<0.05).hence , long term administration of high doses of vitamin e could be effective in decreasing the incidence of thrombosis ,which in turn depends on platelets function. such effect might not affect bleeding time obviously , but it could reduce chances for platelets recruitment , which might represent an additional advantageous action for vitamin e over other antioxidants. key words : vitamin e , α tocopherol , antiplatelets . ةصالخلا ت انتتلن لا لوأول هتينتص فالت ن فادس ن ل داض اتهتيص فبواور فيأن فاتي . ت صاخ فورعم أ ن ات ف ن ي ر نرعمن وصاخ صخيتض فاهتبذ فبورعم أانر دخ له ت ههتة وووتةي لهتبذ . ت صت عأأخ تتختل ات بن فهذنيتة ف ل ع هدأ تبنات ح فتخأ ت فسأ له ل لوا ف انتتن فبارتر تنا أل فان ت 62فبتيتخ وأي ع فهيأن ف ل ع .ألاه صاخ ف ر ت – خههت دأ و ه رن فس ةن فا لوتلنو أتهتوض فذت 31 لبال ع لنت وفا ن ديأ ل خ أ و ت .وأل أرسنل 100 تصتخأع . لت تات فس أع و فست لخ أ وت ن وفن ع لنت ( وفا لهتبذ أأ وت ن 1200 و800 و400أ د انأوض وأل لهتبذ ) فاستردن تف ر ت ار أل فتخترصل لوا ف لرتر فل عتهتوف لا فال .أل فتات فذي يتة فاهتخأع فهتي ذوتفن فهذنيتة ف ل ع ليخ انت ل ن فهرل و ف ا فادي فبهذنيتة ف ل ع فبتواو فبا فساع وتا فا انت لست فا فاتذتفب لو وص ات eتتلا فيتع تر نو رد د فنخ ف رن صن ت فتتدس ا. ن أة فهتتةد ت فه ل فات اأ ات ت ب فوأ ب ل فذنتتلن صلب عتي ت ل ل فذت أ د انأوض ار عاي ت عي و أت نأ ة فبا فابن أوب ف ي .و ت صا فتت نأ انات عخ و عوتا ه رن و وأل – دخأ فبا ل ن تتاأ ر فوال ديأ ل رأخت ص تفوأف فاو تن ل صا فذنتتلن .فافا ت أهتوض انتتلن لا فذت أ انأوض عاي ت عو ت أت نأ لذن ل داض أربنخ تتاتفن صيت تهيأ ة ف ي ل داض – ديأ ل داس خ أ –دخنأن سخنت وفا ن عب أت نأصت فبا اوتفن فهذنيتة ف ل ع وصا فتت نأ صعوت ت عاي ت عي ت أت نأ وتانت فتذلنخ انتتلن لا فبا لنأخ ل فا فالت ن فادس ن . introduction vitamin e has been known as an essential nutrient to maintain normal reproduction since 1922 (1).the large scale studies have shown an inverse correlation between its high dietary intake and the incidence of coronary heart diseases ( 2,3 ) . however,these studies did not provide sufficient evidences , that vitamin e administration can prevent cardiovascular events , nor the subsequent cardiac deaths(4 ). such confliction could be attributed to the fact that , clinical studies usually utilizes the commercially available vitamin e preparations , which almost contain αtocopherol alone , whereas vitamin e in food occurs in several other forms.thus ,the absence of other tocopherols – than α-tocopherol –in the pharmaceutical preparations ( utilized in the previously mentioned studies ) may account for such unexplained results ( 5,6 ). naturally vitamin e occurs as a family of eight members , four of them are referred to as tocopherols and the other four members are known as trienols . both tocopherols and tocotrienols are subdivided into 4 types : alpha (α ), beta ( β ) , gamma ( γ) and delta(δ) , according to their substitutions on the molecule(7),as shown in figure (1) and table 1 . 1 corresponding author : e-mail : dr_ shathahali @ yahoo.com received : 23/9/2007 accepted : 11/6/2008 iraqi j.pharm.sci., vol.17 (1) ,2008 antiplatelts activity of vitamin e 26 table 1 different types of vitamin e r 1 r 2 r 3 alpha c h3 ch3 ch3 beta ch3 h ch3 gamma h ch3 ch3 delta h h ch3 r = substituted groups in the general structure of tocotrienols general structure of tocopherole general structure of tocotrienols figure 1 the most abundant forms of vitamin e in nature are alpha and gamma – tocopherols ( 8 ) .hence , some members of vitamin e was shown to exert specific functions , that may not be found in others ( 9 ) .considering vitamin e action , it can act as scavenger of free radicals , thereby it can provide protection from free radicals – produced damage ( 10 ) .also , it had been reported that vitamin e could exert an antiinflammatory effects through inhibiting lipooxygenase action , thus inhibiting leucotrienes release ( a powerful mediator of inflammation ) ( 11) .meanwhile , vitamin e can decrease the cyclooxygenase cascade in leukocytes which interferes with inflammatory process (12) .some observations by chan and leith ( 1981 ) and gilber et al ( 1983 ), demonstrated that vitamin e enhances the release of prostacyclin – a potent vasodilator and inhibitor of platelets aggregation – in a dose-dependent manner. this study was designed to evaluate the antiplatelets action of α –tocopherol in the commercially available vitamin e preparations in iraqi market , in relation to dose and duration of therapy . subjects and methods : the study included 62 male subjects with age ranged between 32 and 55 years old (45 ±4.2) . the contributing subjects were selected to have no past history of cardiovascular disease or thrombotic disorder, from those attended a private clinic for inferlility at alsadoon street /baghdad , under supervision of a senior physician for the period julynovember 2005 . twenty of them served as a control group ( received no therapy ) .another group of 11 subjects were treated with a daily dose of 100 mg aspirin for more than one month ( 1-3 months ) . the remainder ( 31 subjects ) were subdivided into 5 groups to be treated with vitamin e (α-tocopherol ) as follows : group a : included 6 subjects treated with 400 iu /day for less than 5 months (2-4 months ). group b : included 7 subjects treated with 400 iu / day for more than 5 month (5-6 months) . group c : included 6 subjects treated with 800 iu/ day for less than 5 months (2-4 months ). group d : included 6 subjects treated with 800 iu / day for more than 5 months (5-6 months). group e : included 6 subjects treated with 1200 iu / day for less than 5 months (2-4 months) . the treatment in all groups did not exceed six months . blood specimens were obtained by venipuncture , to perform the platelets assessments anticoagulant(edta-k2) was added ,whereas those aliquots used to assess serum tbars were obtained by centrifugating blood specimens after clotting . platelet function was evaluated by measuring slide platelet aggregation time spat tm , based on measuring time required by platelets to aggregate on a slide in the presence of a potent soluble aggregating agent ( 30 micromol propylgallate) , purchased from analytical control system acs inc (15) . bleeding time was measured for each subject at the end of treatment period according to tvy method ( 16). oxidative stress was assessed by measuring thiobarbituric acid reactive substances tbars in serum according to beuge and auest method(1978). r2 ho ch3 ch3 ch3 ch3 r2 o r3 general structure of tocopherols r2 ho ch3 ch3 ch3 ch3 r2 o r3 general structure of tocotrienols figure -1 r2 ho ch3 ch3 ch3 ch3 r2 o r3 general structure of tocopherols r2 ho ch3 ch3 ch3 ch3 r2 o r3 general structure of tocotrienols figure -1 iraqi j.pharm.sci., vol.17 (1) ,2008 antiplatelts activity of vitamin e 27 results was expressed as mean ± sd , student t-test ( unpaired ) were used considering p values less than 0.05 to be significant (18). results effects of α –tocopherol therapy on bleeding time : bleeding time values showed no significant change in subjects treated with daily dose of 400 iu of α –tocopherol ( group a), even in those continued therapy for more than five months ( group b) figure (2) . whereas , the effect of a dose of 800 iu /day was time– dependent , as shown by results of the group treated for more than 5 months ( group d).higher doses ( 1200 iu/day) of α – tocopherol for the same period ( less than 5 month) also failed to produce significant change in bleeding time values ( group e) . while , the standard therapy with antiplatelet agent(aspirin ) produced a significant elevation . figure 2 . effects of alpha – tocopherol on bleeding time group a : included subjects treated with 400 iu /day less than 5 months (2-4 months ) . group b : included subjects treated with 400 iu / day more than 5 month ( 5-6 months ) . group c : included subjects treated with 800 iu/ day less than 5 months (2-4 months ) . group d : included subjects treated with 800 iu / day more than 5 months ( 5-6 months ) . group e : included subjects treated with 1200 iu / day less than 5 months (2-4 months). aspirin group : included subjects treated with 100 mg / day (1-3months) . * = significantly different from control ( < 0.05 ) effects of α –tocopherol therapy on spat values : the results of spat test for the studied groups are illustrated in figure (3) . significant alterations in spat values were observed in those subjected to therapy that continued more than 5 months , by either doses : 400 or 800 iu α –tocopherol /day – i.e. groups band d , respectively . figure 3 . effects of alpha –tocopherol on slide platelet aggregation test ( spat) values group a : included subjects treated with 400 iu /day less than 5 months (2-4 months ) . group b : included subjects treated with 400 iu / day more than 5 month ( 5-6 months ) . group c : included subjects treated with 800 iu/ day less than 5 months (2-4 months ) . group d : included subjects treated with 800 iu / day more than 5 months ( 5-6 months ) . group e : included subjects treated with 1200 iu / day less than 5 months (2-4 months). aspirin group : included subjects treated with 100 mg / day (1-3months) . *=significantly different from control(p< 0.05) ** =significantly different from control ( p< 0.01) . effects of α –tocopherol on serum tbars levels : figure (4) summarizes the changes in serum tbars levels in response to tested therapy regimens .a significant reduction was detected in groups treated with aspirin and those given α –tocopherol in doses of 400 or 800 iu/day for more than five months. ** * * * iraqi j.pharm.sci., vol.17 (1) ,2008 antiplatelts activity of vitamin e 28 figure 4 . serum thiobarbituric acid reactive substances ( tbars) concentration in various groups group a : included subjects treated with 400 iu /day less than 5 months (2-4 months ) . group b : included subjects treated with 400 iu / day more than 5 month ( 5-6 months ) . group c : included subjects treated with 800 iu/ day less than 5 months (2-4 months ) . group d : included subjects treated with 800 iu / day more than 5 months ( 5-6 months ) . group e : included subjects treated with 1200 iu / day less than 5 months (2-4 months ) aspirin group : included subjects treated with 100 mg / day (1-3months) . * = significantly different from control ( p < 0.05) . discussion one of the reported actions for vitamin e is the anti-coagulant and antiplatelets activity. such effect is mainly presented by the gamma ( γ ) form of tocopherols , as prolongation of bleeding time and abnormal platelet aggregability results after ingesting high doses of vitamin e for long period of time (19,20). in vitro , alpha ( α ) , gamma ( γ ) and delta (δ) – tocopherols have similar effects on human platelet aggregation and a combination of these tocopherols has a synergestic platelets inhibitory effect over the α –tocopherol alone ( 21,22 ). one of the proposed mechanisms for vitamin e antiplatelet activity could be through its interference with vitamin k activity with subsequent disturbance of the cascade of reactions for clot formation ,that predispose to thrombosis . however, such interruption depends on the isomer of vitamin e used , dose and period of administration(23) . another proposed mechanism to explain the platelet antiaggregability effect of vitamin e is related to no bioactivity (24,25) . decreased bioavailability of no is a characteristic feature in patients with coronary artery disease and impaired platelet no production which predicts acute coronary syndrome ( 26 ) . platelets -derived no has been found to inhibit platelets aggregation and to reduce recuiment to grow to thrombus (27) . incorporation of α – tocopherol might increase no producion in platelets by its free radicals scavenging activity and by preventing no quenching by peroxyl radicals ( 28,29 ). the results of the present study shows that α –tocopherol when administered alone could exert significant modifications in platelets function as presented by changes in spat values ( figure -3 ).such effect seems to be related to duration of therapy rather than to dose administered . although , such effect was less obvious in bleeding time values ( figure -2-) .however , the concomitant changes in serum tbars in the studied groups (figure 4 )could strongly suggest a relationship to exist between antioxidant activity of vitamin e with it’s antiplatelets activity (30) , indicated by a significant correlation between tbars and spat values (r=0.994,p<0.05) .although some reported that antiplatelets activity of vitamin e is independent on it’s antioxidant effect (31).the lowering effect of tbars by vitamin e may represent an index for delivering vitamin e to membrane structures of different cells including the platelets, which is reflected by a decrease in platelets aggregability upon longer time of exposure to these doses of α –tocopherol , through increasing the amount of α –tocopherol inside body with possible participitation of it’s antioxidant activity to affect spat values. aspirin administration for more than one month could lower tbars levels ( figure -3-) through increasing the apoferritin level , whose duty is to quench free iron in plasma , since free iron catalyses free radicals generation through fenton‘s reaction ( 32,33). long-term ingestion of α –tocopherol (more than 5 month) is needed to exert it’s antiplatelts activity which may be explained on the bases of its pharmacokinetic behavior , because it is stored initially in adipose tissues before its action appears in circulation(34,35). thus to get greater benefit from vitamin e administration , it may be preferable to take other forms of tocopherols ( i.e. γ–tocopherol ) with a pharmacokinetic behavior that does not require to build up a concentration after accumulation in adipose tissues (36,37 ). however , similar studies including larger number of subjects and longer duration of therapy could provide more clear picture about such effects of different isomeric forms of tocopherols. in conclusion , * * iraqi j.pharm.sci., vol.17 (1) ,2008 antiplatelts activity of vitamin e 29 vitamin e administration can produce significant effects in those patients with high risk of thrombus formation to be preferred over other antioxidanats like vitamin c . acknowledgment this study was abstracted from a high diploma dissertation in clinical lab. sciences references 1evans h. m. and bishop k.s. :on the existence of a hitherto unrecognized dietary factor essential for reproduction . science 1922,56 : 650-651. 2rimm e.b.,stampfer m. j.,ascherio a., et al :vitamin e consumption and the risk of coronary heart disease in men .n engl j med 1993; 328 : 14501456 . 3stampfer m.j.,hennekens c. h.,manson j.e.,et al :vitamin e consumption and the risk of coronary disease in women .n engl j med 1993; 328 : 1444-1449. 4yusuf s., dangenais g.,pogue j., et al : the heart outcomes prevention evaluation study investigators . n engl j med 2000; 342:154-160. 5wolf g. : γ tocopherol : an efficient protector of lipids against nitricinitiated peroxidative damage . nutr.rev 1997; 55 : 376 – 378. 6ohrvall m.,sundlof g.and vessby b. :gamma but not alpha tocopherol levels in serum are reduced in coronary heart disease patients .j 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the arachidonic acid cascade in platelets . prostagl leukot med 1986 ;21 :177-185 . 21mabile l.,bruckdovfer k. r., rico-evans c.: moderate supplementation with natural alpha – tocopherol decreases platelet aggregation and low-density lipoprotein oxidation .atherosclerosis 1999 ; 147 :177-185. 22liu m.,wallmen a ., olsson-mortlock c. et al :mixed tocopherols inhibit platelet aggregation in humans; potential mechanisms .am j clin nutr 2003, 77 (33) :700 –706. 23freedman j.e.and keaney j.f. : vitamin e inhibition of platelet aggregatrion is independent of antioxidant activity .j nutr 2001 ; 131 : 3745-37775. 24 li d.,saldeen t., romeo f., et al : different isoforms of tocopherols enhance nitric oxide synthase phosphorylation and inhibit human platelet aggregation and lipid perxidation ; implications in therapy with vitamin e. j cardiovasc pharmacol ther 2001 ; 6 : 155161 . 25 freedman j.e.,sauter r., battinelli e.m., et al : deficient platelet –derived nitic oxide and enhanced hemostasis in mice iraqi j.pharm.sci., vol.17 (1) ,2008 antiplatelts activity of vitamin e 30 lacking the nos iii gene . circ res 1999 ; 84 : 1416-1421 . 26 freedman j.e., ting b., hankin b.,et al : impaired platelet production of nitric oxide predicts presence of acute coronary syndromes. circulation 1998; 98 : 14811486. 27 freedman j.e., loscalzo j.,barnared m.d., et al : nitic oxide released from activated platelet recruitment . j. clin. invest. 1997, 100 : 350-356. 28huie r. e. and padmaja s. : the reaction of no with superoxide .free radic res commun 1993; 18 : 195199 29rubbo h.,radi r.,trujillo m., et al : nitric oxide regulation of superoxide and peroxynitritedependent lipid peroxidation; formation of novel nitrogen – containing oxidized lipid derivatives .j biol chem 1994; 269 : 26606-26075 . 30muller m. and sorrell t.c. : oxidative stress and the metabolism of arachidonic acid in stimulated human platelets ; role of hydroxyl radical .prostagl. 1997; 54 : 493 509 . 31-jane e freedman andjohn f keaney jr : vitamin e inhibition of plalelets aggregation is independent of antioxidant activity . j nut 2002;131:374s -377s. 32-buczynski a., wachowicz b., kedziorakornatowska k. et al : changes in antioxidant enzymes activities . aggregability and malondialdehyde concentration in blood platelets from patients with coronary heart disease. atheroscler 1993; 100 : 223-228. 33-okuma m.,steiner m., baldini g., et al :studies on lipid peroxidation in platelets ii ; effect of aggregating agents and platelets antibody . lab.clin. med 1971;77:728-742 . 34brigelius-flohe r. and traber m. g. : vitamin e ; function and metabolism. faseb j 1999 ; 13 : 1145-1155. 35mehta j., li d. and mehta j . l. : vitamins c and e prolong time to arterial thrombosis in rats . j nutr 1999 ; 129 : 109-112. 36 li d., suldeen t., romeo f., et al : relative effects of alpha and gamma tocopherol on low density lipoprotein oxidation and superoxide dismutase nitric oxide synthase activity and protein expression in rats . j cardiovas pharmcol ther 1999 ; 4 : 219 226 . 37unchern s,laohruangpanya n, phumala n, et al : the effects of vitamin e on platelets activity in beta thalassaemia patients . br j haem 2003;123(4) :738744. comparative evaluation of using intranasal desmopressin, parenteral diclofenac or their combination in the management of acute iraqi j.pharm.sci., vol.16 (2) ,2007 effect of silibinin on iop ٣٤ effect of silibinin in lowering the intraocular pressure in normotensive rabbits: interaction with pilocarpine and cyclopentolate saad a. hussain *,1 , haider m. mohammed* , munaf h. abdulrazzaq* , ahmed t. numan* *department of pharmacology and toxicology, college of pharmacy, university of baghdad , baghdad , iraq. abstract previous data indicated the effectiveness of silibinin as intraocular pressure (iop) lowering agent. the present study was performed to evaluate the interaction of silibinin with pilocarpine or cyclopentolate in lowering iop in normotensive rabbits. the effects of topically instilled silibinin hemisuccinate solution (0.75%) alone or adjunctly combined with 2% pilocarpine or 1% cyclopentolate on the iop of normotensive rabbits were evaluated using indentation tonometry. the results showed that 0.75% solution of silibinin was found more potent than pilocarpine (2% drops) in lowering iop of normotensive rabbits, while their combination results in longer duration of action. moreover, the elevated iop values produced by cyclopentolate (1%drops) were decreased by silibinin, while prior instillation of cyclopentolate did not interfere with the iop-lowering effect of silibinin. in conclusion silibinin lowers iop in normotensive rabbits more than pilocarpine, and their combination elongates the duration of the iop-lowering effect. this might be due to interference with aqueous humor formation as a possible mechanism. in addition, cyclopentolate did not significantly alter the effect of silibinin on iop. key words: silibinin, iop, pilocarpine, cyclopentolate الخالصة لقد ثبت في دراسة سابقة فعالیة السیلیبینین في خفض الضغط الداخلي لعین االرنب، تم تصمیم ھذه الدراسة لتقییم التداخل بین موضعیا في العین. تمت دراسة تأثیر االستخدام الموضعي في العین السیلیبینین والبایلوكاربین أو السایكلوبنتولیت عند استخدامھم % سایكلوبنتولیت على خفض الضغط الداخلي ١% قطرة بایلوكاربین أو ٢% سلیبینین لوحده أو بوضعھ قبل أو بعد ٠.٧٥لمحلول % سیلیبینین في خفض ضغط ٠.٧٥. أظھرت النتائج بأن تأثیر indentation tonometryلعین االرانب الطبیعیة باستخدام تقنیة % بایلوكاربین وأن استخدامھما معا یؤدي الى اطالة مدى مثل ھذا التأثیر. كما ثبت بأن للسیلیبینین القدرة ٢العین الداخلي أكبر من ین عند استخدام %، بینما لم یرتفع ضغط الع١على تقلیل االرتفاع الحاصل في ضغط العین الداخلي الذي أحدثتھ مادة السایكلوبنتولیت االخیر قبل السیلیبینین. وعلیھ یمكن ان نستنتج بأن السیلیبینین افضل من البایلوكاربین في خفض ضغط العین الداخلي لعین االرانب یة وان استخدامھما معا یطیل مدة ھذا التأثیر، ویمكن أن یعزى ھذا التأثیر الى التداخل مع عملیة تكوین ماء العین الداخلي كمیكانیك اضافة الى ان الساكلوبنتولیت لم یؤثر على فعالیة السیلیبینین في خفض الضغط الداخلي للعین.عمل للسیلیبینین. introduction two mechanisms underlay the effectiveness of various drugs implicated in the management of elevated intraocular pressure (iop), the reduction in aqueous humor (ah) inflow and enhancement of ah outflow (1). reduction of aqueous inflow was observed due to the use of ß-adrenoceptor blockers (2), carbonic anhydrase inhibitors (3) and others including forskolin (4). however, reduction of ah inflow involves either vascular (5, 6) or ionic mechanisms (7). in a recent study, we reported on a decrease in iop of normotensive rabbits after ocular instillation of silybinin solution (8). it has been suggested that a direct pharmacological effect on trabecular pathway to be likely involved in pilocarpine-induced fall in iop (9). the role of camp in ah regulation is very well explained (10, 11). it inhibits ah inflow by blocking ion transport across ciliary epithelium (12). inhibition of na+-k+ -2cl¯ by camp decreases the uptake of cl¯ by ciliary epithelium (13), and the increase in camp level, stimulated by ß2 -adrenergic receptor activation in the trabecular meshwork cells, suggested to be responsible for the increased outflow facility after application of ß2-agonists (14). silibinin hemisuccinate, a powerful antioxidant flavonoid (15), inhibits campphosphodiestrase enzyme, even more potent than theophylline or papaverine (16), an effect that can be utilized for the interference with ah formation and iop regulation. the present study was designed to examine the interaction between silibinin and pilocarpine or cyclopentolate in lowering iop of normotensive rabbits, and to provide preliminary evidence for the mechanism of this effect. 1 corresponding author : e-mail : saad_alzaidi@yahoo.com received : 15/7/2007 accepted : 30/12/2007 iraqi j.pharm.sci., vol.16 (2) ,2007 effect of silibinin on iop ٣٥ -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 0 0.5 1 1.5 2 2.5 3 tim e (hours) % i o p r e d u c ti o n 0.75% silybinin hemisuccinat e 2% pilocarpine hcl 1% cyclopent olat e * * * * * * * * * materials and methods thirty-five new zealand white rabbits weighing 1.5-2.5 kg were used in this study. animals were kept in the animal house of the college of pharmacy, university of baghdad, under standardized conditions (12 hrs lightdark cycles at room temperature), and were fed a standard diet (quality control laboratories, moh, iraq) and water ad libitum. drugs treatment silibinin hemisuccinate in pure form was a gift from tolbiac srl, argentina, and all other compounds used during the study were supplied by the department of pharmacology and toxicology, college of pharmacy, university of baghdad. silybinin hemisuccinate was dissolved in arachis oil and used freshly prepared (0.75%) solution. pilocarpine (2%) and cyclopentolate (1%) were used as commercial eye drop formulas (alcon pharmaceuticals ltd, cham, switzerland). the animals were allocated into 7 groups (5 rabbits each) for studying the effect of topically instilled silybinin hemisuccinate alone, pilocarpine alone, cyclopentolate alone, the effect of pilocarpine or cyclopentolate instilled 30 min prior to silybinin and the effect of prior instillation of silybinin 30 min before pilocarpine or cyclopentolate. measurement of iop indentation tonometry, using schiotz tonometer, was utilized in this study for measuring iop before and after instillation of drugs or drug vehicle (arachis oil). thirty minutes before starting drug instillation, the cornea was anesthetized with 0.5% tetracaine hcl (chauvin pharmaceuticals ltd, surry, england) and baseline iop was measured using schiotz tonometer. after instillation of 1 drop of silibinin hemisuccinate (0.75%), pilocarpine (2%) and cyclopentolate (1%), measurement of iop was performed every 30 min for 3 hr (17). after each measurement the eyes were washed with normal saline and the instrument was cleaned with diethyl ether. all experiments were conducted in a masked manner, and were performed during a fixed time of the day (from 10:00 am to 3:00 pm) to exclude the effect of circadian changes in iop. for assessment of the pre-instillation effect of pilocarpine or cyclopentolate, baseline iop was recorded before instillation of these drugs to both eyes, and then 1 drop of freshly prepared silibinin hemisuccinate oily solution was applied after 30 min to both eyes. the iop was measured every 30 min for 3 hr. the same later approach was followed to evaluate the effect of pre-instillation of silibinin on that produced by pilocarpine and cyclopentolate. all results were expressed as mean ± s.e. comparisons with baseline were made using student’s paired t-test, while a single-factor analysis of variance (anova) for repeated measurements was used to evaluate differences between groups. p values less than 0.05 were considered significantly different. results effects of 0.75% silibinin, 2% pilocarpine and 1% cyclopentolate in normotensive rabbits, instillation of 0.75% silibinin decreases iop significantly for 2.5 hr, and remains significantly different with respect to baseline value at all measured time points (figure 1). the maximal decrease in iop was achieved after 1 hr of instillation (38.56 %) compared to baseline (p<0.05). pilocarpine eye drops (2%) significantly reduces iop after 30 min of instillation (21.5%, p<0.05), decreased gradually to nonsignificant value (0.46%, p>0.05) at the end of 3 hr compared to baseline (figure 1). instillation of 1 drop cyclopentolate (1%) significantly elevates iop, reaching maximum value after 0.5 hr (34.76 %); then gradually decreases with time (4.24 %) after 2.5 hr of instillation. figure 1: effect of 0.75% silibinin hemisuccinate, 2% pilocarpine hcl and 1% cyclopentolate hcl on iop in normotensive rabbits; results are presented as mean of percent changes ± sem; n=10 eyes for each group; * p < 0.05 with respect to baseline; † p < 0.05 with respect to 0.75% silibinin alone. iraqi j.pharm.sci., vol.16 (2) ,2007 effect of silibinin on iop ٣٦ effects of pre-treatment with 2% pilocarpine and 1% cyclopentolate the iop-lowering effect of pilocarpine was enhanced when silibinin instilled 30 min latter, 26.87% compared to 19.0% produced by pilocarpine alone (p<0.05). the reduction in iop observed in this procedure remains significantly high (40.88%, 49.5%, 43.34%, and 26.87%) compared to that produced by pilocarpine alone (9.23%,4.16%,1.81% and 0.46%) for the time intervals 1.0, 1.5, 2.0, and 2.5 hr respectively (p<0.05) (figure 2). moreover, this level reduction in iop was significantly different compared to that produced by silibinin alone along the entire period beyond the addition of silibinin. when cyclopentolate instilled 30 min prior to silibinin, the latter still have the ability to reverse the elevation of iop produced by cyclopentolate (from 34.76% to 17.3%) after 30 min of instillation (p<0.05); these changes are not significantly different at the time intervals 1.0, 1.5, and 2.0 hr after instillation of silibinin, p>0.05 (figure 3). figure 2: effect of preand post treatment with 0.75% silibinin hemisuccinate on iop lowering effect of 2% pilocarpine in normotensive rabbits; results are presented as mean of percent changes ± sem; n=10 eyes for each group; * p < 0.05 with respect to baseline; † p < 0.05 with respect to 2% pilocarpine alone. figure 3: effect of preand post treatment with 0.75% silibinin hemisuccinate on iop rising effect of 1% cyclopentolate in normotensive rabbits; results are presented as mean of percent changes ± sem. n=10 eyes for each group; * p < 0.05 with respect to baseline; † p < 0.05 with respect to 1% cyclopentolate alone; pre-treatment with silibinin hemisuccinate did not significantly differ from silibinin hemisuccinate alone p > 0.05. effects of pre-treatment with silibinin pre-treatment with silibinin, 30 min before instillation of pilocarpine resulted in additive effect on the iop lowering activity of pilocarpine (37.5%), which was significantly different, compared to the effect of pilocarpine alone (21.5%). this reduction in iop remains significantly different with respect to that produced by pilocarpine after 1.0, 1.5 and 2.0 hr. however, the effect of pre-treatment with silibinin did not significantly differ compared to that produced by silybinin alone during all time intervals (p>0.05) (figure 1). even when cyclopentolate instilled 30 minutes latter, pretreatment with silibinin results in highly significant reduction in iop (31.47% p<0.05), and remains significantly high 1.0, 1.5, and 2.0 hr after instillation of cyclopentolate (-20.08%, -8.92%, -4.12% respectively) compared with the rise in iop (28.36%, 18.29%, 10.18% and 10.18%) produced by cyclopentolate alone (p<0.05). cyclopentolate appeared to slightly reverse the action of silibinin when instilled 30 min latter; however, such effect did not significantly differ from that produced by silibinin alone (p>0.05). -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 0.5 1 1.5 2 2.5 3 time (hour) % i o p r e d u c ti o n 2% pilocarpine alone pre-treatment with 0.75% silybinin hemisuccinate post-treatment with 0.75% silybinin hemisuccinate * * * * * * * * * * * * * * -45 -35 -25 -15 -5 5 15 25 35 0.5 1 1.5 2 2.5 3 tim e (hour) % c h a n g e i n i o p 1% cyclopent olat e alone p ost -t reat ment wit h 0.75% silybinin hemisuccinat e p re-t reat ment wit h 0.75% silybinin hemisuccinat e 0.75% silybinin hemisuccinat e alone * * * * * * * * * * * * * iraqi j.pharm.sci., vol.16 (2) ,2007 effect of silibinin on iop ٣٧ discussion it has been reported previously in our laboratory that corneal instillation of silibinin lowers iop in normotensive rabbits in a dose dependent manner. it also delays iop recovery rate after i.v infusion of 20% sodium chloride solution (8). although inhibition of campphosphodiesterase is proposed as a suspected mechanism for the action of silybinin on iop (18), interference with the cholinergic influence in this respect was evaluated. in the present study, the effect of silibinin on iop was higher than that produced by pilocarpine, and their combination results in an additive effect. muscarinic agonists, including pilocarpine, lower iop through enhancing ah outflow due to contraction of the iris sphincter (19). according to the reported mechanisms of action of silibinin, targeting ah formation and interference with ion transport can be suggested as possible mechanisms (20). consequently, the mechanisms through which pilocarpine and silibinin produce their effects can be utilized for explaining the additive effect reported when both of them are used at the same time. to confirm the idea that silibinin lowers iop through a mechanism not related to the cholinergic system, the interaction of silibinin with anticholinergic agent like cyclopentolate was evaluated. although there is no practical evidence on elevation of iop in rabbits due to instillation of cyclopentolate, the results reported in this study demonstrated such effect, which can be attributed to the abnormal sensitivity of the locally bred strain of rabbits to the effect of cyclopentolate. in the present work, the rise in iop produced by instillation of cyclopentolate was effectively reversed by silibinin; meanwhile, the iop lowering effect of silibinin was not affected by postinstillation of cyclopentolate. based on these data, one can postulate that silibinin interferes with iop regulation through reduction of ah inflow. taken together with the data obtained in previous study (8) about the effect of silibinin on iop recovery rate and its contralateral effect, one can suggest the interference with ah inflow as a mechanism involved in pilocarpine-silibinin and cyclopentolatesilibinin interactions. the iop-lowering effect of cholinomimetics is mediated via the activation of the inositol triphosphate (ip3) pathway that linked to m3-receptors in the ciliary and iris-sphincter muscles (21). silibinin, on the other hand, strongly inhibits camp phosphodiesterase with consequent elevation of camp levels (16). the later mediates many biological effects including the inhibition of ion transport by na+-k+2cl– co-transporter across ciliary epithelium and trabecular meshwork (13, 22), which is similar to that produced by activation of the ip3 pathway initiated by pilocarpine. regulation of cell volume is very important phenomenon that involved during exposure to hypoor hypertonic environment, and na+ k+2cl – co-transporter is the system responsible for such regulation (23, 7, 24). infusion of hypertonic sodium chloride solution resulted in shrinkage of ciliary epithelium, and to retain the original volume, ion transporters should be activated to transport na+ and cl– across ciliary epithelium (25). inhibition of this co-transporter by camp delayed osmotic recovery rate, an effect reported after silibinin administration. in conclusion, corneal instillation of silibinin lowers iop in normotensive rabbits, probably through a mechanism not related to the interference with cholinergic influence on iop. acknowledgment the authors gratefully thank college of pharmacy, university of baghdad for supporting the project and tolbiac srl, argentina for providing pure silibinin powder as a gift. references 1. moses r a. intraocular pressure. moses ra, hart w. (eds.), adler’s physiology of the eye. clinical application, 8th edition, the c.v. mosby, santa louis, 1987; 223. 2. robinson jc, kaufman pl. dosedependent suppression of aqueous humor formation by timolol in the cynomolgus monkey. j glaucoma 1993; 2: 251. 3. shahidullah m, william m, wilson s, et al. effects of ion transport and channelblocking drugs on aqueous humor formation in isolated bovine eye. invest ophthalmol vis sci 2003; 44: 1185-1191. 4. caprioli j, sears m, bausher l. forskolin lowers intraocular pressure by reducing aqueous inflow. invest ophthalmol vis sci 1984; 25: 268. 5. bill a, stjernschantz j. cholinergic vasoconstrictor effects in the rabbit eye: vasomotor effects of pentobarbital anesthesia. acta physiol scand 1980; 108: 419. 6. millar jc, carr rd, humphries rg, et al. drug effects on intraocular pressure and vascular flow in the bovine perfused eye using radiolabeled microspheres. j ocul pharmacol ther 1995; 11: 11. 7. do cw, to ch. chloride secretion by bovine ciliary epithelium: a model of aqueous humor formation. invest ophthalmol vis sci 2000; 41: 1853-1860. 8. mohammed hm, abdulrazzaq mh, iraqi j.pharm.sci., vol.16 (2) ,2007 effect of silibinin on iop ٣٨ hussain sa. effects of silibinin hemisuccinate on the intraocular pressure in normotensive rabbits. saudi med j 2007, 28(9): 1397-1401 9. erickson ke, schroeder a. direct effects of muscarinic agents on the outflow pathways in human eyes. invest ophthalmol vis sci 2000; 41: 1743-1748. 10. do cw, kong cw, to ch. camp inhibits transepithelial chloride secretion across bovine ciliary body epithelium. invest ophthalmol vis sci 2004; 45: 3638-3643. 11. stamer wd, roberts bc, epstein dl. hydraulic pressure stimulates adenosine 3΄, 5΄-cyclic monophosphate accumulation in endothelial cells from schlemm’s canal. invest ophthalmol vis sci 1999; 40(9): 1983-1988. 12. neufeld ah, sears ml. cyclic-amp in ocular tissues of the rabbit, monkey, and human. invest ophthalmol vis sci 1974; 13: 475. 13. crook rb, takahashi k, mead a, et al. the role of na-k-2cl cotransport in blood-to-aqueous chloride fluxes across rabbit ciliary epithelium. invest ophthalmol vis sci 2000; 41: 2574-2583. 14. lu y, li m, shen y. the effects of epinephrine and adrenergic antagonists on adenosine 3΄, 5΄monophosphate level of bovine trabecular cells in vitro. zhonghua yan ke za zhi 1998; 34 (2): 124-126. 15. altorjay i, dalmi l, sari b, et al. the effect of silibinin (legalon) on the free radical scavenger mechanisms of human erythrocytes in vitro. acta physiol hung 1992; 80: 375-380. 16. koch hp, bachner j, loffler e. silymarin: potent inhibitor of cyclic amp phosphodiesterase. methods find exp clin pharmacol 1985; 7(8): 409-413. 17. vareilles p, lotti vj. effects of timolol on aqueous humor dynamics in the rabbit. ophthalmol res 1981; 13: 72-79. 18. berets a, anton r, cazenave jp. the effects of flavonoids on cyclonucleotide phosphodiesterase, cody v, middleton e, harborne jb. (eds.), plant flavonoids in biology and medicine: biochemical, pharmacological and structure-activity relationships. alan r. liss inc. new york, 1986; 281-296. 19. wiederholt m, bielka s, schweig f, lütjen-drecoll e. regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye. exp eye res 1995; 61: 223. 20. ruckstuhl m, landry y. inhibition of lung campand cgmpphosphodiesterases by flavonoids and other chromone-like compounds. biochem pharmacol 1981; 30: 697-702. 21. berridge m. inositol triphosphate and calcium signaling. nature 1993; 361: 315325. 22. o'donnell me, martinez a, sun d. endothelial na-k-cl cotransport regulation by tonicity and hormones: phosphorylation of cotransport protein. am j physiol cell physiol 1995; 269: 1513-1523. 23. civan mm, peterson-yantorno k, sanchez-torres j, coca-prados m. potential contribution of epithelial na+ channel to net secretion of aqueous humor. j exp zool 1997; 279: 498-503. 24. crook rb, von brauchitsch dk, polansky jr. potassium transport in nonpigmented epithelial cells of ocular ciliary body: inhibition of a na+, k+, clcotransporter by protein kinase c. j cell physiol 1992; 153: 214-220. 25. civan m.m, coca-prados m, petersonyantorno k. pathways signaling the regulatory volume decrease of cultured nonpigmented ciliary epithelial cells. invest ophthalmol vis sci 1994; 35: 2876-2886. iraqi j pharm sci, vol.28(2) 2019 beta-thalassemia major 8 -https://doi.org/10.31351/vol28iss2pp1 doi: 1 clinical complications of beta-thalassemia major ali j. shawkat *,1 and ahmed h. jwaid ** * ministry of health and environment,baghdad,iraq. **department of pharmacology, college of pharmacy, university of baghdad, baghdad, iraq. abstract beta thalassemia syndrome (β-tm) syndrome is a group of hereditary blood disorders that are mainly characterized by reduction or absence of β-globin chain synthesis. without iron chelation therapy (ict) the regular blood transfusion would increase the iron stores to several times. endocrine glands are vulnerable to iron overload causing endocrine dysfunction. iron deposition within the parathyroid gland causes hypoparathyroidism particularly after ten years of age. pancreatic islets are very susceptible to oxidative damage due to iron overload; their high divalent metal expression makes them highly susceptible to iron-catalyzing oxidative stress. the pathogenicity of osteopathy in β-tm is multifactorial comprising environmental (diet and lifestyle), iatrogenic (medicines), genetic and acquired factors (expansion of bone marrow, hemochromatosis, deficiency of growth hormone, hepatitis and hypogonadism). the increase in blood transfusion and rbcs break down in addition to iron accumulation and deposition are the main factors causing splenomegaly. liver disease is one of the major complications affecting patients with β‐tm.liver damage is multifactorial with iron overload is considered the main causative factor, as well as hepatitis c (hcv) and hepatitis b (hbv) infections which are acquired on recurrent blood transfusions. the free radicals of deposited iron overcome the cellular antioxidant mechanisms resulting in peroxidative cellular injury. as a result, iron overload is the leading cause of left ventricular cardiomyopathy development. keywords: beta-thalassemia major, iron overload, endocrinopathies المضاعفات الناتجة عن مرض الثالسيميا الكبرى ** احمد حامد جويد و 1، *علي جالل شوكت .العراقبغداد ، ،والبيئة وزارة الصحة* .العراق،بغداد ،جامعة بغداد ،كلية الصيدلة ،والسموم فرع االدوية** الخالصة . بيتا-لوبين.أو انعدام توليف سلسلة غ اضطرابات الدم الوراثية التي تتميز بشبل رئيسي بقلةهي مجموعة من نوع بيتا-الثالسييميا الببر الزائد للحديد للفرط( فإن نقل الدم المنتظم من شييهنأ أن يزيد من مزازن الحديد عدة مرات. الغدد الصييماض عرضيية ict) المببل للحديدبدون العالج ن وتاصية بعد عشر سنوات مهرمون الباراثايرويد في قلةالدرقية يسيب جن رسي الحديد داتل الغدة مما يسيب اتتال واائف الغدد الصيماض. ت على الزالياتبافؤالمعدني الثنائي ال وجود التعبيرالزائد للحديد ؛ إن الفرطالبنبرياس معرضية بشيدة لرضيرار التهكسيدية بسييب تاليا بيتا في العمر. تي تشييمل ال و متعدد العواملالثالسيييميا الببر يبون مؤكسييد المحف ز للحديد. التسييب في مرا هشيياشيية العظام في يجعلهم أكثر عرضيية لهجهاد ال ، موص هرمون الن، العوامل الوراثية والمبتسييييبة )توسييييل نزاع العظم ، الصييييباق الدمو ، نقاالدويةالعوامل البيئية )النظام الغذائي ونمط الحياة( ، كرات الدم الحمراض باإلضيافة إلى تراكم الحديد وترسبأ من العوامل اإلضافية لتخزم زيادة تحطم . يعد نقل الدم و(لغدد التناسيليةالتهاب الببد ونقص ا ومن العوامل متعدد الثالسيييميا الببر حيي يبونالببد أحد المخيياعفات الرئيسييية التي تؤثر على المرضييى الذين يعانون من اعتال الطحا ، يعد تبتسييي ( التي (b نوع ( والتهابات الببدc) نوع التهاب الببد باالضيييافة الىالمسيييب الرئيسيييي لذل ، ويعتبرالحديد فرطزيادة ه العوامل هواهم هذ مؤكسد حطم تعلى آليات مخادات األكسدة الزلوية مما يؤد إلى الناتجة عن فرط الحديد في عمليات نقل الدم المتبررة. تتغل الجذور الحرة للحديد .عتال عخلة القل ال . نتيجة لذل ، يعتبر الحمل الزائد للحديد هو السب الرئيسيلزاليا القل ،الغدد الصماء . الزائد للحديد الفرط ، نوع بيتا-الثالسيميا الكبرىالكلمات المفتاحية: introduction beta thalassemia major is a group of hereditary blood disorders that are mainly characterized by reduction or absence of β-globin chain synthesis, resulting in a reduction of hemoglobin in red blood cells (rbcs), decreased production of rbcs and consequently anemia (1,2). thalassemia disease was first described by thomas b. cooley in 1925(2,3). the β globin synthesis is normally regulated by two β genes; one on each copy of chromosome 11(4). thalassemia is an autosomal recessive genetic condition, caused by point mutation within or near β-gene. such mutations result in either absence of the β globin (β0 thalassemia) or reduction in synthesis of the β globin(β+-thalassemia)(5). β-thalassemia is clinically classified into three major types: 1) thalassemia minor (β-thalassemia carrier/trait): this type is heterozygous with a mild clinical phenotype since one normal copy of the beta globin chain is present (e.g. β+/β, β0/β). 2) β-thalassemia intermedia: a heterogeneous genetic mutation that still allows for some β-globin chain formation (β+/β0, β+/β+). patients develop symptoms later than in thalassemia major most often between 2-6 years of age with heterogeneous clinical findings that are similar but milder than β-thalassemia major (β-tm). 1corresponding author: e-mail: alijalal0135@gmail.com received: 15/1 /2019 accepted: 3/7 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp1-8 mailto:alijalal0135@gmail.com iraqi j pharm sci, vol.28(2) 2019 beta-thalassemia major 2 although patients are capable of surviving without regular transfusion, development and growth are retarded. 3-) β-thalassemia major (cooley's anemia): refers to a severe phenotype which occurs when patients are homozygous or compounds heterozygous for β chain mutation (severe β+/β+, β+/β0,β0/β0), patients commonly present with symptoms within the first two years of life (3,11,14). the reduction or absence of the βglobin chains results in a relative excess of α-globin chains, which when unpaired are unstable and precipitate in the erythroid precursors in the bone marrow, consequently there is ineffective erythropoiesis and the mature cells that reach the circulation have a shortened lifespan(6). the cornerstone management for patients with β-tm is based on lifelong transfusion and iron chelation. the aims of transfusion are to correct anemia and suppress ineffective erythropoiesis(7). the ironloading rate from the blood transfusion in transfusiondependent thalassemias (tdt) is about ten times the rate in non-transfusion-dependent thalassemias (ntdt); a unit processed from 420ml of donor blood contains approximately 200mg of iron. in β-tm the equivalent of 100-200 ml of blood per kg body weight per year is transfused, this means iron accumulation in mg is equivalent to 116-232mg/kg/ year, or 0.32-0.64 mg/kg/day. without iron chelation therapy (ict) the regular blood transfusion would increase the iron stores to several times the normal levels (11,26).iron overload results in severe complications including cardiac disease, osteopathy and endocrine complications (11, 26). the non-transferrin-bound iron (ntbi) and labile plasma iron (lpi) circulate in plasma and are deposited in susceptible cells in which ntbi rather than using transferrin receptor, enters cells through the voltagedependent ca+2 channel (figure 1). myocardial iron overload causes death from cardiomyopathy and heart failure in the second decade of life, pituitary damage causes growth retardation, hypogonadism and delayed puberty, endocrine complications include diabetes, hypoparathyroidism and hypothyroidism, and liver disease ending with cirrhosis and hepatocellular carcinoma(8,9). figure 1. pathologic mechanisms and consequences of iron overload (10). clinical complications of β-thalassemia major growth retardation endocrine glands are susceptible to excess iron causing endocrine dysfunction (significantly hypogonadotropic hypogonadism hh) which is a common complication in β-tm that requires recognition and treatment. patients with the β-tm present with a delay in growth and puberty and reduction of the average height. growth failure pathogenesis is multifactorial: including iron overload, chronic anemia and hypoxia, zinc and folic acid deficiency, chronic liver disease, intensive use of chelating agents, endocrinopathies and growth hormone-insulin like growth factor-1(ghigf-1) axis dysregulation(11). normal growth of children is disrupted by anemia, iron overload and ineffective erythropoiesis. however, maintenance of hb above 9 with adequate chelation therapy makes β-tm patients growing as normal as their nonthalassemic peers(11).zinc is a trace element that has an important role in the synthesis of fat, enzymes, immune system, and rbcs' survival and its deficiency causes membrane fragility(12). despite zinc deficiency and hyperzincuria due to chelation therapy in β-tm patients, there is no significant difference between those with or without growth impairment regarding zinc concentrations in the body(13). folic acid deficiency results in complications such as anorexia, growth failure and git disorders besides megaloblastic anemia. folic acid deficiency is more severe among β-tm patients; however, microcytosis of thalassemia may mask the hematological characteristics of folic acid deficiency. folic acid in a dose of 1mg/day should iraqi j pharm sci, vol.28(2) 2019 beta-thalassemia major 3 be given to thalassemic patients with deficiency states(14).intensive chelation therapy, mainly deferoxamine (dfo) when given as early as 2-5 years may have deleterious effects on growth, it has been found that dfo inhibits cell proliferation, dna synthesis, mineral deposition, and collagen formation; all of which result in shortening of the spinal height and consequent truncal shortening(11,15). hypogonadism hypogonadism is the most common reported endocrinopathy; caused by iron deposition in the pituitary gland, gonads or both. it has been shown that gonadotropes require much more iron than other anterior pituitary cells, and are most affected by iron-overload resulting in declined levels of lh (luteinizing hormone) and fsh (follicular stimulating hormone)(16).the direct effect of ntbi on testes and ovaries is still unknown; however, ovaries reserve is still preserved in most females with thalassemia even if they were amenorrheic since they can still ovulate after hormonal treatment. males testes on histological examination from autopsies revealed testicular interstitial fibrosis and hyperpigmentation of undifferentiated seminiferous tubules with a diminished number of leydig cells(17).anterior pituitary gland iron deposition occurs in the first decade of life, but the clinical manifestations appear only around puberty in which firstly only a diminished gonadotropin reserve and intact gonadotropin pulse is demonstrated, but, later the reserve of the gonadotropin is dramatically diminishing that may result in irreversible damage of the hypothalamic-pituitary-gonadal (hpg) axis(16). hypothyroidism thyroid dysfunction frequently occurs in β-tm but its prevalence and severity depend on the age of the studied populations, ferritin levels, duration, frequency of blood transfusion, and dose and type of chelating agent used(18). t3 (triiodothyronine) and t4 (thyroxine) levels are regulated strictly by hypothalamic-pituitary-thyroid axis. thus, hypothyroidism may be central or primary depending on the iron deposition in hypothalamus, pituitary or thyroid gland. thyroid hormones affect many systems including cardiovascular, nervous, reproductive, and digestive systems. thyroid function should be monitored in all patients with β-tm as they are considered hypertransfused, and in those who receive suboptimal chelation therapy. patients with an elevated tsh (thyroid stimulating hormone) level should be followed up yearly, and a proper chelation therapy can prevent and reverse iron overloadinduced hypothyroidism, l-thyroxin should be instituted in hypothyroid β-tm patients with moderate to severe iron overload(19). hypoparathyroidism the secretion of the parathyroid hormone (pth) takes place by the parathyroid gland and functions in calcium homeostasis together with vitamin d and calcitonin, pth maintains calcium levels within normal range by facilitating its absorption from the gastrointestinal tract, or by phosphorous excretion and calcium reabsorption from the kidney; or by bone resorption. pth also plays a role in the conversion of vitamin d to its active form (1, 25 dihydroxycholecalciferol) in the kidneys(20).patients with β-tm undergoing frequent transfusions, which causes iron deposition within the parathyroid gland with a resultant hypoparathyroidism particularly after ten years of age; as a consequence of that low pth and vitamin d ensue. hypoparathyroidism is also a leading cause of hypocalcemia, and several neurological complications may evolve such as tetany, seizures, and paresthesia(21,22).patients with β-tm require a yearly screening for hypoparathyroidism to estimate hypocalcemia associated problems. thalassemia patients in their second decade of life require supplementation with calcium and vitamin d to prevent complications such as neurological complications and fractures, and to maintain normal bone growth(23). diabetes mellitus (dm) dm constitutes 20-30% of endocrine complications worldwide in patients with β-tm(24). pancreatic islets are very susceptible to oxidative damage due to iron overload; their high divalent metal expression makes them highly susceptible to iron-catalyzing oxidative stress. another proposed mechanism of glucose abnormalities in β-tm is autoimmunity against antigens of the β-cells, in which iron deposition within these cells provides an environment that triggers an autoimmune reaction against β-cells and consequently destroying them. zinc deficiency is a common problem in β-tm, and it has been shown that zinc deficiency results in exacerbation of inability of the pancreas to secrete sufficient amount of insulin in response to glucose load in β-tm patients, also iron deposition within parenchymal tissues such as the pancreas induces inflammation, recent studies demonstrated elevated levels of circulating il-1α (interleukin-1alpha), tnf-α (tumor necrosis factor alpha) and il-6 (interleukin -6) in β-tm patients which explains the progressive and gradual deterioration of these cells(25).according to the uk and international thalassemia management guidelines recommend regular screening by annual oral glucose tolerance test (oggt) around puberty or 10 years when a family history is presented. this allows for a proper treatment of hyperglycemia or intensification of iron chelation therapy which would improve glycemic control or prevents future diabetes development(26).patients with β-tm are aimed at preventing, detecting and managing diabetes-related iraqi j pharm sci, vol.28(2) 2019 beta-thalassemia major 4 complications comprising macrovascular complications (cardiovascular, cerebrovascular and peripheral vascular diseases), and microvascular complications (diabetic retinopathy, nephropathy, and neuropathy)(26).the use of hba1c for assessing glycemic control in thalassemia patients with hemoglobinopathies is less accurate; hence an alternate method for assessment that is not affected by abnormal hemoglobin variants is the use of fructosamine levels measurement which depends on serum protein glycosylation(25). bone diseases osteopenia and osteoporosis (op) affect about 40-50% of patients with β-tm and are responsible for a vast majority of morbidity in this population with an increased risk for pathological fractures. the pathogenicity of osteopathy in β-tm is multifactorial comprising environmental (diet and lifestyle), iatrogenic (medicines), genetic and acquired factors (expansion of bone marrow, hemochromatosis, deficiency of gh or igf-1, hepatitis and hypogonadism(27). the pathogenesis of osteopathy is summarized by the following points: 1role of receptor activator of nuclear factor-κb, receptor activator of nuclear factor-κb ligand, and osteoprotegerin (rank/rankl/opg) system: physiologically two distinct cell types are in charge of the maintenance and renewal of the bone: the osteoblasts which are responsible for bone formation and the osteoclasts which are responsible for bone resorption and remodeling. in β-tm, the "aging" process of the bone begins as early as in childhood due to the gradual development of an imbalance between enhanced osteoclastic resorption and insufficient osteoblastic formation(28). the essential pathway that links osteoclast-mediated bone resorption with osteoblast-mediated bone formation consists of a paracrine system that comprises receptor activator of nuclear factor-κb ligand (rankl), its receptor activator of nuclear factor-κb (rank), and a soluble protein osteoprotegerin (opg). rankl is produced by osteoblasts and their precursors, binds to the rank receptor, promoting osteoclast differentiation and proliferation. opg functions as a decoy receptor to block the action of rankl. this system provides a balance between bone formation and resorption and through which a wide variety of biological mediators like (hormones, cytokines, and growth factors) affect bone homeostasis(29). it has been found that rankl which enhances osteoclastic function is elevated in β-tm patients, while opg and opg/rankl ratio were reduced, associated with low bone mineral density (bmd) this gives evidence that opg/rankl system plays a vital role in the pathogenesis of osteoporosis in β-tm, moreover cytokines levels (interleukin-1α, tnf-α, and interleukin-6) were elevated in β-tm in which these inflammatory cytokines inhibit growth plate proliferation and differentiation, increase apoptosis and result in reduction of matrix synthesis. circulating il-1α concentrations were found to correlate with rankl serum levels indicating an association between these cytokines and altered bone turnover in β-tm patients(28,30). 2genetic factors: the genetic factors play a significant role in the pathogenesis of osteopenia and osteoporosis in β-tm accounting for 70% of the variance in (bmd). polymorphisms of several genes affecting bmd have been identified most important are collagen type i a1 (colia1), transforming growth factor-beta 1 (tgfβ1) and vitamin d receptor (vdr). in a study of thalassemia male patients with (colia1) gene polymorphism associated with more severe osteoporosis of the spine and the hip and there was a failure of improvement of spinal op using bisphosphonate therapy. tgfβ1 is the most available growth factor in human bones, and it is produced by osteoblasts has the main function of inhibiting osteoclasts proliferation and activity and stimulates proliferation and differentiation of pre-osteoblasts. polymorphisms of this gene have been studied and play a role in lowered bmd and susceptibility to osteoporotic spine fractures. polymorphism of vdr gene have found to be associated with short stature and lowered lumbar spine and femoral neck bmd in thalassemia patients(30). 3-bone marrow expansion: as mentioned above that ineffective erythropoiesis results in erythroid hyperplasia and marrow expansion secondary to extramedullary hematopoiesis(31). this results in expansion of the medulla, thinning of cortical bone and resorption of cancellous bone causing a generalized loss of bmd(30). 4-endocrine complications: hypogonadism: in transfusion-dependent thalassemia hypogonadism results primarily from pituitary failure and to a lower extent of gonadal failure(16). hypogonadism is associated with lower bmd in β-tm(32). generally, low estrogen and progesterone concentrations result in a decrease in the inhibition of osteoclast activity and bone formation. low testosterone levels result in the reduction of its direct stimulatory effect on the proliferation and differentiation of osteoblasts(33). gh-igf dysfunction: gh and igf have an anabolic effect on which is very important during adolescence for the acquisition of bone mass and during adult life for maintaining skeletal architecture(30). defective gh-igf axis causes a decrease in osteoblast proliferation and bone matrix and increases in osteoclasts activation and subsequent bone loss(33). hypoparathyroidism: vitamin d and calcium deficiency increase bone turnover by stimulating pth production; consequently increased pth stimulates osteoclastogenesis and subsequent bone resorption to maintain normal circulating calcium concentrations. hypothyroidism: chondrogenesis and bone mineralization are regulated by t3; t3 iraqi j pharm sci, vol.28(2) 2019 beta-thalassemia major 5 augments osteocalcin and collagen synthesis, increase proliferation and differentiation of osteoblasts. in adult life t3 is an anabolic hormone that is necessary for growth to stimulate peak bone mass accrual. nevertheless, it regulates bmd and bone turnover; i.e., hypothyroidism is a leading cause for growth retardation and bone deformation(30). diabetes mellitus: insulin stimulate osteoblasts proliferation and maintains endochondral bone growth, insulin deficiency is a known risk factor for the development of osteoporosis(30). 5-iron overload and deferoxamine (dfo) toxicity: deposition of iron in the bones impairs osteoid maturation and inhibits mineralization locally, this occurs by a mechanism that includes incorporation of iron into calcium hydroxyapatite crystals which consequently affects their growth(34). dfo is responsible for bone growth and metabolism disorders. it exerts a direct effect by interfering with bone growth and by altering bone metabolism due to chelation of trace metals. dfo exerts deleterious effects on osteoblasts through inhibition of dna synthesis, osteoblasts proliferation, differentiation of osteoblastic precursors, and in patients receiving high doses it enhances osteoblasts apoptosis(35). 6-reduced physical activity: patients with β-tm have a reduced physical activity either due to the complication of the disease or overprotection of the parents who do not encourage their children to make any muscular activity; in either case, this results in increased osteoclastic function and/or reduced osteoblastic activity after which bone destruction ensues(33,34) . thalassemia facies the majority of craniofacial manifestations in β-tm are due to hemolytic anemia, hypoxia and ineffective erythropoiesis with a resultant bone marrow hyperplasia and expansion. these include frontal bossing, larger cheekbones, depressed nasal bridge, and protruding maxilla. craniofacial changes give a distinctive "chipmunk" – like appearance(36,37) (figure 2). figure 2.the facial appearance of a child with β‐ tm(1). splenomegaly the spleen is the major organ of hb catabolism of the old rbcs. in β‐tm, there is an increased rate of hemopoiesis to compensate for anemia, causing an increase in production of abnormal rbcs and consequent clearance, as well as other changes such as extramedullary hemopoiesis resulting in splenomegaly. the increase in blood transfusion and rbcs break down in addition to iron accumulation and deposition are additional factors for splenomegaly(38). hypersplenism is problematic in patients who are inadequately transfused and results in leucopenia, thrombocytopenia, and exacerbation of anemia(39). splenectomy is indicated for patients with: increased blood requirement (200-220 ml/kg/year) which prevents adequate control with icts, symptoms of upper quadrant pain or feel of early satiety and/or massive splenomegaly with a danger of rupturing(9,40). although splenectomy relieves anemia and decreases the frequency of blood transfusion; it is associated with risks such as infections because the spleen is a major site of antibodies production, and thrombotic risk due to increased thrombin generation and decreased proteins c and s(40,41) liver disease the primary site of iron storage is in the liver. furthermore, it is the only site of transferrin and ferritin synthesis(42). liver disease is one of the major complications affecting patients with β‐ tm.liver damage is multifactorial with iron overload is considered the main causative factor, as well as hepatitis c (hcv) and hepatitis b (hbv) infections which are acquired on recurrent blood transfusions(43). normally iron is stored proteinbound in the liver; which in cases of iron overload free iron is very toxic and catalyzes the production of free radical with a resultant lipid peroxidation causing hepatotoxicity(42). iron overload provokes malignant transformation in the liver by accelerating fibrosis and ultimate cirrhosis formation. this ccurs by activating stellate cells and by the profibrogenic effect of lipid peroxidation. hepatocellular carcinoma (hcc) is caused by iron overload and chronic viral hepatitis; which was not a common complication of β‐tm in the past since patients were used to die at a younger age because of heart failure(44). acute or chronic injury to the liver would result in elevated concentrations of alanine transaminase (alt) and aspartate transaminase (ast)(45). alt serum level is considered an indicator of the necroinflammatory process of the liver(46). to assess the extension of liver cell damage a sensitive indicator of liver function is used namely serum bilirubin; hyperbilirubinemia in β‐tm could be pre-hepatic (hemolytic), hepatic or post-hepatic (obstructive)(47). the incidence of gallbladder stones (cholelithiasis) and hepatic duct stones (choledocholithiasis) is 30-80% in patients with β‐ iraqi j pharm sci, vol.28(2) 2019 beta-thalassemia major 6 tm. approximately two-thirds of β‐tm patients have multiple calcified-bilirubin stones by age 15. thalassemia patients have gallbladder dysmotility with delayed small bowel transit and autonomic dysfunction; resulting in gallstone formation. 30% of gallstones are due to hemolysis, and about 30% due to cholestasis, chronic liver disease, ileac disease or resection, and 40% of cases are idiopathic(48). patients who undergo splenectomy should have their gallbladder evaluated for gallstones before surgery, especially if the patients have symptoms suggestive of biliary tract disease; at which time concomitant cholecystectomy should be performed(9). cardiac disease cardiomyopathy and arrhythmia in β-tm are the most important complications caused by iron overload accounting for 71% of deaths globally(49). patients with β‐tm undergo chronic hemolysis that causes anemia; if left untreated leads to increased cardiac output, which ultimately results in left ventricular (lv) dilatation and hypertrophy ending with high rate heart failure (hf). on the other hand in iron overload, excess iron (ntbi) after saturation of reticulocyte system, deposits in cardiomyocytes through t and l-type calcium channels. the free radicals of deposited iron overcome the cellular antioxidant mechanisms resulting in peroxidative cellular injury. as a result, iron overload is the leading cause of lv cardiomyopathy development. arrhythmias including atrial fibrillation, supraventricular tachycardia, ventricular tachycardia, and ventricular arrhythmia; is increased in parallel with increased cardiac siderosis(50,51). neither serum ferritin nor liver iron concentration (lic) gives a reliable measure of cardiac iron concentrations, even modern measures such as tissue doppler imaging has a poor correlation with cardiac iron. endomyocardial biopsy is an invasive and unreliable as well for measuring cardiac iron due to the small samples which cause sampling error(52). an alternative non-invasive technique for measuring cardiac siderosis is by using mri-t2*(52). mri-t2* also detects early ventricular dysfunction and can be used to monitor cardiac iron during ict. in β‐tm normal myocardial t2* (t2*˃ 20 ms) is associated with normal lv and rv ejection fraction (ef). on the other hand, lower myocardial t2* (t2*˂20) is associated with lv and rv dysfunction, and an improvement in t2* results indicate improvement in lv and rf ejection fraction(49,52,53). complications associated with iron chelation therapy (ict) deferoxamine (dfo) has a negative impact on thalassemia patients' quality of life (qol) as the infusions can be very troublesome, painful and time consuming with restrictive activity(54,55). compliance with dfo and its rigorous requirements of daily subcutaneous infusion still a limiting factor for treatment success. dfo chelates iron only during the time infused, hence, poor compliance with dfo results in gaps in chelation coverage, leading to an increase in lpi levels which cause further tissue damage(56). many side effects are associated with dfo treatment including local skin reaction,growth retardation,visual disturbances and hearing loss, and infections are negatively impact thalassemia patients. although the oral deferasirox (dfx) iron chelation is more favorable, it is associated with nausea, vomiting, abdominal pain,rash, and acute renal failure, in addition to its high cost that may complicate the disease(9,57). conclusion the iron-loading rate from the blood transfusion in transfusion-dependent thalassemias (tdt) is about ten times the rate in non-transfusiondependent thalassemias (ntdt). iron overload causes multiple organs damage including; liver, endocrine glands, bones, and heart.better managing of iron overload and prevention of complications should be considered to prevent morbidity and mortality in those patients. references 1. cossio mlt, giesen lf, araya g, et al. hoffbrands essential haematology. 7th ed. hoffbrand av, moss pah, editors. vol. xxxiii, wiley blackwell. john wiley & sons ltd.; 2016. 73-85 p. 2. marengo-rowe aj. the thalassemias and related 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clinics of north america. 2010;24(6):1109–30. 56. porter jb, evangeli m, el-beshlawy a. the challenges of adherence and persistence with iron chelation therapy. international journal of hematology. 2011;94(5):453–60. 57. marsella m, borgna-pignatti c. transfusional iron overload and iron chelation therapy in thalassemia major and sickle cell disease. hematology/oncology clinics of north america. 2014;28(4):703–27. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics doi: https://doi.org/10.31351/vol27iss2pp42-54 42 impact of osteocalcin level on vascular calcification in type 2 diabetics in relation to fibroblast growth factor-23(fgf-23) inas a. ali*, 1 and shatha h. ali** * ministry of health and environment, karbala health directorate, karbala, iraq ** department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq abstract the present study aimed to assess the potential impact of serum concentration of undercarboxylated osteocalcin (the active form of osteocalcin) and fibroblast growth factor-23 on the incidence of cardiovascular diseases in type 2 diabetics with carotid artery calcification and the possible association with metabolic changes in relation to glucose and minerals homeostasis. this study included 52 men with carotid artery calcification type 2 diabetes mellitus. these patients were categorized; as follows: group a includes 30 patients who had cardiovascular disease and group b includes 22 patients who had no cardiovascular disease. these groups were compared with 25 apparently healthy control (group c). it has been shown that fasting serum glucose, hba1c, homeostatic model assessment of insulin resistance and undercarboxylated osteocalcin values were significantly different in group a and b as compared with control. also, undercarboxylated osteocalcin was negatively correlated with fasting serum glucose and hba1c in group a and b. furthermore, mean serum fibroblast growth factor level was significantly different among the three studied groups, with highest levels in group a. in type 2 diabetic patients with normal kidney function and carotid artery calcification, fibroblast growth factor-23 is associated with cardiovascular disease while undercarboxylated osteocalcin does not. keywords: diabetes mellitus type 2, vascular calcification, undercarboxylated osteocalcin, fibroblast growth factor-23 تأثير مستوى االوستيوكالسين على تكلس االوعية الدموية في مرضى السكري من النوع (23-)اف جي اف 23الثاني والمتعلق بعامل نمو االرومة الليفية **شذى حسين عليو 1،* ايناس عبد المحسن علي وزارة الصحة والبيئة، دائرة صحة كربالء، كربالء، العراق. * برية السريرية،كلية الصيدلة،جامعة بغداد،بغداد،العراق.علوم المختفرع ال** الخالصة الى تقييم التأثير المحتمل لتركيز المصل من االوستيوكالسن غير الكاربوكسيلي )الشكل الفعال ة تهدف هذه الدراس 2رضى السكري من النوع على االصابة بامراض القلب واالوعية الدموية في م23-لالوستيوكالسين( وعامل نمو االرومة الليفية والمصابين بتكلس الشريان السباتي وامكانية االرتباط مع التغيرات االيضية فيما يتعلق بتوازن الكلوكوز والمعادن. تم تصنيف هؤالء المرضى .يمع تكلس الشريان السبات مصابا بمرض السكري من النوع الثاني شملت هذه الدراسة اثنين وخمسين رجال لتالي: المجموعة أ تضم ثالثين مريضا والذين يعانون من أمراض القلب واألوعية الدموية والمجموعة ب تشمل اثنين وعشرين على النحو ا أمراض القلب واألوعية الدموية. تمت مقارنة هذه المجموعات مع اشخاص على مايبدو بصحة جيدة عانون منال ي نمريضا والذي من االصحاء. )المجموعة ج( شملت خمسة وعشرين شخصا ن قيم تقييم نموذج التوازن لمقاومة االنسولين واالوستيوكالسي ،الهيموغلوبين السكري جلوكوزالمصل الصيامي، قيملقد لوحظ أن ير ايضا االوستيوكالسين غ غير الكاربوكسيلي كانت مختلفة بشكل معنوي في المجموعة أ و ب مقارنة مع مجموعة السيطرة)االصحاء(. لي كان مرتبطا سلبيا مع جلوكوز المصل الصيامي والهيموغلوبين السكري في المجموعة أ و ب . وعالوة على ذلك ، كان الكاربوكسي بشكل ملحوظ بين المجموعات الثالث المدروسة ، مع أعلى المستويات مختلفًا المصل في 23 مستوى عامل نمو األرومة الليفيةمتوسط في المجموعة أ. و االرومه كلى طبيعية لوحظ ان عامل نم لسكري من النوع الثاني والذين يعانون من تكلس الشريان السباتي ويتمتعون بوظيفةا في مرضى يرتبط مع أمراض القلب واألوعية الدموية في حين االوستيوكالسين الغير كاربوكسيلي ال يرتبط . 23-الليفية . 23 ، عامل نمو االرومة الليفيةغير الكاربوكسيلي األوعية الدموية ، أوستيوكالسين ، تكلس 2: مرض السكري النوع المفتاحية الكلمات 1corresponding author e-mail: enas.abdalmuhsen@gmail.com received: 21/8/2018 accepted: 29/9/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp42-54 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics 43 introduction diabetes mellitus(dm), notably, type 2 diabetes mellitus (t2dm) is a common chronic metabolic disease (1). in the last two centuries; pathogenesis, epidemiology, prevention, and treatment of dm have been well established (2). additionally, both types of diabetes (1&2) are associated with a higher risk of bone fractures (3). in t2dm, bone mineral density (bmd) is equivalent or increased according to a metaanalysis (4), but the risk of fracture is increased despite this bmd increment (5). the paradoxical increment in fracture rate in patients with t2dm with bmd increment might result from an increased falling rate (6). in addition, a higher risk of fracture in t2dm is thought to be attributed to the decreased bone formation and decreased bone quality (7). hyperglycemia can reduce the bone density through different pathways. toxic effects caused by high serum glucose levels can directly decrease the function and number of osteoblasts (8). generally, osteoclastogenesis is enhanced in dm as shown in human studies (9). meanwhile, vascular calcification, as osteogenesis, involves multiple interactions of different cells that produce the matrix vesicles with subsequent mineralization (10). the most important direct mechanism that combines the bone loss with the vascular calcification is an increased release of calcium and phosphate from the bone, in the form of calcium-phosphate complexes, by an enhancement of osteoclast-induced resorptive process. subsequently, these mineral ions of calcium and phosphate and/or complex may be localized to the vascular sites and form a nidus for future mineralization or cause locally increased calcium and/or phosphate levels that induce precipitation of calcium-phosphate complexes in the artery (11). osteocalcin (oc), a biomarker for the bone formation, has been well documented to be lower in t2dm patients than in non t2dm controls. hence, suggesting that glucose levels would affect oc levels in t2dm patients, so apparently, the formation of bone is suppressed if compared with non t2dm controls (12). the oc can regulate energy metabolism through the enhancement of pancreatic β-cells to secrete insulin (13). furthermore, insulin signaling in the osteoblasts may regulate the hemostasis of whole-body glucose via control of oc activation (14).on the other hand, it has been found that decreased oc levels are associated with insulin resistance and t2dm reflecting the fact of oc ability to modulate the risk of cardiovascular disease (15). while, fibroblast growth factor-23 (fgf-23) is a hormone predominately expressed in the osteocytes and has a role in the mineral homeostasis through induction of hyperphosphaturia, inhibition of calcitriol synthesis and inhibition of parathyroid hormone (pth) secretion (16). it is well demonstrated that the higher levels of fgf-23 are associated with an increased risk of arterial stiffness and atherosclerosis even in patients with no renal impairment (17). fgf-23 is considered as an inhibitor factor of bone mineralization (18), and because, it can regulate the phosphate metabolism, it may be a good marker of the time-averaged hyperphosphatemia, and subsequently, the fgf-23 is found to be as a marker of the vascular calcification (19). the goal of this study was to assess the potential impact of serum concentration of undercarboxylated osteocalcin (ucoc) (the active form of oc) and fgf-23 on the incidence of cardiovascular diseases in type 2 diabetics with carotid artery calcification and the possible association with metabolic changes in relation to glucose and minerals homeostasis. subjects and methods a case-controlled study was carried out at al-hassan specialized center for diabetes and endocrine diseases / imam hussain medical city in karbala (from october/2017 to march/2018). a total number of one hundred diabetic male patients were selected from the outpatient clinic under the supervision of an endocrinologist. diabetes was diagnosed according to the american diabetes association criteria (20). after excluding those with bone-related disorders :osteomalacia, fracture, tumors, paget's disease, multiple myeloma, malignant hypercalcemia, hyperthyroidism and hypothyroidism, hyperparathyroidism and hypoparathyroidism ,or those on medication(drugs affect vitamin k status like warfarin, ketoconazole and vitamin k), bisphosphonate, glucocorticoid,1,25(oh)2-d3, heparin and anticonvulsant. after a doppler ultrasonography for all of the diabetic patients (one hundred) looking for carotid artery calcification. only 52 out of these patients, who were recognized to have a carotid artery calcification, were included in our study. the patients were arranged into two groups according to the presence of cardiovascular disease like ischemic heart disease [based on electrocardiograph (ecg) results] (21) and/or hypertension [depending on the history, blood pressure measurement and ecg changes ] (22,23); as follows: iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics 44  group a: thirty diabetic patients with calcification of the carotid artery who had cardiovascular disease.  group b: twentytwo diabetic patients with calcification of the carotid artery who had no cardiovascular disease. these groups were compared with apparently healthy control cases (group c); included twenty-five healthy subjects. after an overnight fasting, a blood sample was withdrawn to perform the required investigations. the study was authorized by the local research ethics committee and all subjects were provided with a written informed consent to participate in this study. serum glucose (24), urea (25), calcium (26), phosphate (27) and alkaline phosphatase (alp) (28) were analyzed using specific kits purchased by spinreact (spain). while, glycosylated hemoglobin (hba1c) was measured by automated fluorescent immunoassay system (afias) kit (29) purchased by afias, boditech med incorporated(korea).whereas, fgf-23 (30) and ucoc (31) were estimated using specific enzyme-linked immunosorbent assay(elisa) sandwich method kits, supplied by cusabio(china) using elisa plate reader beckman coulter(austria). insulin (32), pth (33) and thyroid stimulating hormone (tsh) (34) were measured by specific electrochemiluminescence immunoassay (eclia) kits supplied by cobas, roche diagnostics (gmbh, switzerland) using cobas e411analyzer roche, hitachi(switzerland). while serum creatinine (35) was determined using a specific kit purchased by shenzhen mindray bio-medical electronics co., ltd. (china). glomerular filtration rate (gfr) was predicted to assess kidney function by the estimation of creatinine clearance (ccr) based on serum creatinine (scr) concentration in the adult male by using cockcroft and gault formula (36) : ccr= {((l 40–age(yrs)) x weight (kg) ) / ( 72 x scr (mg/dl))}x 0.85 (if female) while the homeostatic model assessment of insulin resistance (homa-ir) is used to estimate insulin resistance from fasting glucose and fasting insulin, by applying the following formula (37) : homa-ir ={fasting insulin(μiu/ ml) * fasting glucose(mg/dl)} / 405 statistical analysis of data is presented as means ± sd. significance was set at p <0.05. cases and controls were compared employing either the t-test for independent samples or the pearson coefficient, for normally distributed variables. results the subjects' characteristics are illustrated in table-1. table 1. subjects characteristics variable group n mean± sd p-value age (year) a 30 58.40±7.81 0.001** b 22 50.41±6.98 c 25 46.84±7.60 bmi ( kg/m2) a 30 29.11±3.56 0.57 b 22 28.51±5.15 c 25 30.10±6.77 systolic blood pressure (mmhg) a 30 134.83±14.88 0.001** b 22 121.59±10.40 c 25 119.32±8.16 diastolic blood pressure (mmhg) a 30 83.50±18.62 0.01** b 22 77.50±6.50 c 25 72.08±6.57 duration of diabetes (year) a 30 7.83±7.35 0.001** b 22 6.50±6.23 c 25 0.00±0.00 iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics 45 continued table 1. variable group n mean± sd p-value b.urea(mg/dl) a 30 32.20±6.59 0.02* b 22 29.41±7.15 c 25 27.64±3.78 s.creatinin(mg/dl) a 30 0.86±0.19 0.27 b 22 0.79±0.12 c 25 0.81±0.11 gfra (ml/min) a 30 113.65±29.42 0.04* b 22 133.07±38.87 c 25 137.89±40.51 pth(pg/ml) a 30 41.37±14.17 0.09 b 22 41.76±17.48 c 25 49.37±11.53 tsh(nmol/l) a 30 1.85±1.14 0.88 b 22 1.83±0.95 c 25 1.97±1.02 a = gfr based on cockcroft gault equation. * = significant difference (p≤0.05). **: highly significant difference (p≤0.01). n: number of subjects, bmi: body mass index, gfr: glomerular filtration rate, pth: parathyroid hormone, tsh: thyroid stimulating hormone. date illustrated in table -2, showed that fasting serum glucose (fsg), hba1c and homa -ir values were significantly elevated in diabetic patients with cardiovascular disease ( group a ) and those without cardiovascular disease ( group b) as compared with the control (group c) (p<0.01), but there were no significant differences between diabetic patients groups (p>0.05). while fasting serum insulin mean values were not significantly different among the three studied groups (p>0.05). table 2. descriptive statistics of glycemic parameters and significant comparative studies between studied groups **: highly significant difference (p≤0.01). n: number of subjects, hba1c: glycosylated hemoglobin, fsg: fasting serum glucose, homa-ir: homeostasis model assessment of insulin resistance variable group n mean± sd p-value fsg(mg/dl) a 30 192.90±57.25 0.001** b 22 186.53±38.81 c 25 98.72±10.06 hba1c a 30 8.37±2.27 0.0001** b 22 8.59±2.09 c 25 5.44±1.23 insulin (μiu/ ml) a 30 14.08±10.68 0.32 b 22 12.51±7.01 c 25 10.76±4.35 homa-ir a 30 7.11±7.08 0.004** b 22 5.65±3.41 c 25 2.63±1.14 iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics 46 as presented in the table-3, the mean serum calcium, phosphate, calcium×phosphate (ca×pi), as well as alp values were not significantly different among the studied groups (p>0.05). table3. descriptive statistics of serum calcium, phosphorus, (calcium*phosphate) product and alkaline phosphatase among groups. n: number of subjects, s.ca: serum calcium, s.pi: serum inorganic phosphate, ca×pi: calcium×phosphate, alp: alkaline phosphatase. as illustrated in figure-1, the mean of serum ucoc level was significantly lowered in diabetic patients with cardiovascular disease (group a) and diabetic patients without cardiovascular disease (group b) when compared with control group (p<0.01), while there was no significant variation between diabetic patients groups (p>0.05). figure1. serum undercarboxylated osteocalcin levels in diabetics and control groups. *=significantly different from control a correlation study was revealed that ucoc was negatively correlated with fsg and hba1c in the diabetic patient with cardiovascular disease group (p<0.01) [figure2&3] and diabetic patients without cardiovascular disease group (p<0.05) [figure4&5]. serum human fgf-23 level was significantly elevated in diabetic patients with cardiovascular disease (group a) as well as with diabetic patients without cardiovascular disease (group b) as compared to the control group (p<0.01). besides a significantly higher mean value of fgf-23 in diabetic patients with cardiovascular disease (group a) over that of diabetic patients without cardiovascular disease (group b) (p<0.01)[figure -6]. figure 2. correlation between undercarboxylated osteocalcin (ucoc) (ng/ml) and fasting serum glucose (fsg) (mg/dl) in diabetic patients with cardiovascular disease group. variable group n mean± sd p-value s.ca(mg/dl) a 30 9.27±0.64 0.63 b 22 9.41±0.59 c 25 9.25±0.60 s.pi (mg/dl) a 30 3.68±0.64 0.76 b 22 3.80±0.63 c 25 3.70±0.45 ca×pi (mg/dl)2 a 30 34.34±7.30 0.66 b 22 35.87±7.34 c 25 34.28±5.26 alp( u/l) a 30 81.63±27.83 0.17 b 22 101.91±65.10 c 25 83.84±20.91 iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics 47 figure 3. correlation between undercarboxylated osteocalcin (ucoc) (ng/ml) and hba1c in diabetic patients with cardio vascular disease group figure 4. correlation between undercar boxylated osteocalcin (ucoc) (ng/ml) and fasting serum glucose (fsg) (mg/dl) in diabetic patients without cardiovascular disease group. figure 5. correlation between undercarboxylated osteocalcin (ucoc) (ng/ml) and hba1c in diabetic patients without cardiovascular disease group. figure 6. serum fibroblast growth factor -23 in diabetics and control groups. *=significantly different from control. ∆=significantly different from group b discussion it has been observed that patients with t2dm are associated with high insulin resistance and may have apparently normal or elevated insulin levels, the higher levels of serum glucose in those patients would lead to even higher insulin levels with the normal function of their β-cells (20). as agreed with other results, insulin levels appear within the normal range. therefore, there was no significant difference between diabetic patient groups and control group. the association between insulin resistance and cardiovascular disease occur through its relation to hypertension, dyslipidemia, atherosclerosis, and hypercoagulability (38). despite the non-significant higher value of homa-ir in diabetic patients with cardiovascular disease as compared with diabetic patients without cardiovascular disease group, this higher value may be responsible for the linkage between group (a) and the presence of cardiovascular disease. as mentioned previously, insulin resistance as assessed by homa-ir, was significantly higher in a diabetic patient with and without cardiovascular disease as compared with the control group. in patients with t2dm, the insulin resistance leads to increment in serum glucose levels; consequently, the prolonged hyperglycemia will reduce the bone formation by the osteoblasts(39). this will result in a decrement in the production of ucoc and oc that can cause a further increment in the insulin resistance via reduction of adiponectin formation in the adipocytes (40). iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics 48 based on these findings, both the high glucose and the high insulin levels can be included in the list of factors inducing vascular calcifications (41). in this study, an interesting observation on the association between the insulin resistance and the vascular calcifications was provided as agreed with a previous study (41) since homa-ir mean value was significantly higher in diabetic patients groups with vascular calcification when compared with control group. in the present study, the mean serum calcium level was not significantly different among different studied groups (p>0.05) as shown in the table-3. however, even though serum calcium was within the normal reference range, may play a role in developing vascular calcification. this result occurs in accordance with other studies that showed higher levels of the circulating calcium, even within the normal range, were found to be linked with the thickening of the carotid plaque which represents an initial marker of cardiovascular diseases (42). furthermore, as illustrated in the table-3, the mean serum phosphate level showed no significant difference among the studied groups (p>0.05). however, it was suggested that phosphate, even within the normal reference range, acts as a contributing factor in the vascular calcification. this is consistent with foley et al. who observed in multivariate models that the phosphate levels were significantly associated with the calcium level of the coronary artery and the higher levels of serum phosphate, even if it is within the normal range, may form a risk factor in the process of coronary artery atherosclerosis in the healthy young group (43) additionally, the current study showed that the values of (ca×pi) were not significantly different among the studied groups (p>0.05) as shown in the table-3. as previously mentioned, the arterial calcifications are linked with an increased (ca×pi) product. serum phosphate may also act directly to increase the vascular calcification, especially when the (ca×pi) product levels are high as noticed in patients with chronic renal disease (44) or in subjects without chronic renal disease (implied to as a dystrophic calcification). an ectopic calcification is mainly called a dystrophic calcification if it is associated with a normal systemic mineral balance. commonly, these areas show evidence of changes and/or necrosis in the tissues. dystrophic mineralization is usually noticed in the soft tissues because of disease, injury, and aging (45). additionally, the (ca×pi) products have a positive relationship with the risk of cardiovascular disease in subjects free of chronic renal disease and cardiovascular disease in the community (46). in addition, the data presented in table3 showed that serum alp was not significantly different when compared neither with control group nor within the patient’s groups (p>0.05). however, diabetic patients without cardiovascular disease group showed an elevation of mean serum alp concentration level above the normal reference range while diabetic patients with cardiovascular disease group showed a high-normal level. this result is consistent with a maxwell et al. study who had observed an elevated alp level in diabetic patients (47). since, mean fsg was significantly higher in the group with elevated alp, indicating a relation between the severity of diabetes and diabetic bone disorder. furthermore, an increment in the alp levels is linked to the extensive vascular calcification, resulting in premature atherosclerosis and cardiovascular disease. alp induces vascular calcification through its action on inorganic pyrophosphate (ppi) which is regarded as a powerful inhibitor for the passive calcium phosphate deposition. the biochemical actions of ppi are the prevention of the calcium and phosphate aggregation, and hydroxyapatite crystal growth. in vivo study, it was reported that the ppi is hydrolyzed into phosphate by the serum alp. consequently, an increment in the alp activity can promote an imbalance between phosphate and ppi, inducing an ectopic calcification (48). so, in this study, the elevated level of alp could be considered as one of the contributing factors in the vascular calcification. however, these results lack the significant difference between the different studied groups. statistically, the lack of significance can be attributed to the sample size of this study in comparison with the other previous studies. many studies had appraised that as the active form of oc, ucoc has an effect on the glucose and lipid metabolism (40). serum oc was observed to be reduced in patients with hyperglycemia, dm, obesity, insulin resistance, and metabolic syndrome (49). as consistent with these studies, the mean of serum ucoc level was significantly reduced in diabetic patients groups (a and b) when compared with control group (p<0.01) as illustrated in figure-1 the reduction of serum ucoc level in patients with t2dm can be explained by the following causes: iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics 49 1. presence of insulin receptors on the osteoblasts, hence, reduction of insulin secretion and insulin resistance in dm can affect the function of the osteoblasts(50). also, insulin stimulates the decarboxylation of oc indirectly through osteoclast activation(13). so, in t2dm, decreased inulin sensitivity can decrease the decarboxylation of oc and ucoc levels. 2. the serum level of vitamin d is significantly lower in patients with t2dm; nevertheless, it can organize the processes of the transcription and translation of the oc gene (51). 3. hyperglycemia prevents the osteoblast function and may have a direct toxic effect on the osteoblast (8). 4. it was reported that the reduction of oc was associated with the suppressed level of the pth in dm and a low level of vitamin d could explain this relation (52) because vitamin d stimulates oc production by the osteoblast (53). decreased plasma oc levels were demonstrated to be linked with higher incidence of the pathological cardiovascular events, such as arterial and valvular calcification, more carotid intima-media thickness, and carotid atherosclerosis (54). the ectopic cardiovascular calcification is considered as one of the pathological vascular alterations that lead to the development of the cardiovascular disease (55). on the contrary to a study by guo et al. (56), the current study did not show significant variations in the mean ucoc level between diabetic patient groups (p>0.05). however, the mean level of ucoc in diabetic patients with cardiovascular disease was lower than the mean of diabetic patients without cardiovascular disease. despite this difference, statistically, it was of no significance as presented in figure-1. guo et al. supposed a possible association between the serum levels of ucoc with the severity of a vascular complication in t2dm, showing the relationship between ucoc and the cardiovascular diseases, as well as, providing a clinical evidence that serum ucoc level is reduced in individuals with vascular complications of t2dm, and there was an inverse relationship between ucoc and t2dm in chinese men involving ucoc as a future therapeutic target to treat the vascular complications in t2dm (56). the possible mechanisms that can explain the relationship between the oc and the risk of cardiovascular disease are still unclear. it has been shown that oc can enhance the proliferation of pancreatic β-cell, secretion of insulin, and release of adiponectin, thus increasing the insulin sensitivity. therefore, the lower levels of oc may be combined with more resistance of insulin or t2dm and lower levels of adiponectin, thereby, promoting more cardiovascular damage. however, it has been demonstrated that the lower levels of oc may directly influence the cardiovascular risk (54). so, a partial explanation of these conflicting data can be done by the gender and ethnic differences that present among the individuals. moreover, the status of the vitamin d and vitamin k, which may influence the circulating levels of oc and ucoc, are actually not taken into account in the available and many of the cited clinical studies. the vitamin k is considered as a cofactor needed for the carboxylation process of oc. hence, the dietary factors can have an essential role in oc synthesis. however, the current study did not investigate the effect of diet on the serum oc or ucoc. moreover, different risk factors for the cardiovascular disease like hypertension and dyslipidemia had existed in this and other studies, so the potential effects of these associated diseases with the corresponding therapeutic agents may influence the results of the current study. as observed in this study from the figures (2-5), serum ucoc level was significantly and negatively correlated with fsg and hba1c in diabetic patient with and without cardiovascular disease , that was consistent with previous studies (57,58) revealing that the ucoc acts as a hormone that promotes responsiveness to the insulin and glucose tolerance which was consistent with the results of fulzele et al. study (14). as observed in this study (figure-6), serum fgf-23 level was significantly elevated in diabetic patients with cardiovascular disease (group a) as well as in diabetic patients without cardiovascular disease (group b) as compared to the control (p<0.01).the increased levels in t2dm could be attributed to: firstly: there is a strong relation between the fgf-23 and the bmd in t2dm patients. the serum level of fgf-23 may give an idea about the number of osteocytes and a higher bmd can be found in t2dm as agreed with reyes-garcia et al. study (59). iraqi j pharm sci, vol.27(2) 2018 osteocalcin and vascular calcification in diabetics 50 secondly: the circulating fgf-23 levels are elevated as the kidney function decreases as revealed by larsson et al. (60). fgf-23 is a powerful biomarker of early chronic renal disease and may reflect the incident kidney disease in diabetics (61). thirdly: the insulin resistance had an independent association with fgf-23 levels. an increment in homa-ir levels is associated with an increment in fgf-23 levels among subjects with normal kidney function (62). because a patient with increased insulin resistance may reabsorb more phosphate, thus, a higher level of fgf-23 is reached in order to eliminate the phosphate (63). moreover, the fgf-23 has the ability to conserve the calcium despite suppressed hormonal synthesis of vitamin d that induced by fgf-23 itself. so fgf-23 have a role in the development of vascular calcifications (64). the increment in the circulating fgf-23 levels, which was also noticed in the diabetic animal models, may escalate the existing endothelial dysfunction and induce a vascular calcification, subsequently, resulting in atherosclerosis in dm patients (65). as reported in the figure (6), there was a significant elevation in fgf-23 level in diabetic patients with cardiovascular disease group when compared with diabetic patients without cardiovascular disease group (p<0.01). this difference was related to the role of fgf-23 in the development of cardiovascular disease. previous studies demonstrated a strong doseresponse link between the increased fgf-23 levels and the future cardiovascular disease and death in both patients with chronic renal disease (66) and with no chronic renal disease (19). in addition, the higher fgf-23 level was associated with increased arterial stiffness as demonstrated in patients with t2dm (59) and even in the general population(67). furthermore, voigt et al. study reported that the fgf-23 was detected in the calcified carotid atherosclerotic lesion from individuals with normal renal function (68). on the other hand, in vivo study showed that fgf-23 effect on the blood vessels is indirect because fgf-23 can suppress the production of vitamin d hormone which is an important factor in the regulation of the endothelial function (69) and proliferation of the cardiomyocyte (70). the reduction in 1,25(oh)2-d3 can elevate the angiotensin ii production through an increment in the renin expression, which leads to cardiac hypertrophy and hypertension (71). moreover, fgf-23 may enhance endothelial dysfunction through direct interference with the nitric oxide-induced vasodilation (72). an important limitation of this study is the lack of generalizability of the results to both sexes and they are only limited to elderly men. further studies about the same issue can be conducted on both sexes. also, another limitation is the sample size of this study was relatively small. therefore, we premise that the results of this study could be a base for future studies in larger groups of subjects. in conclusion, the present study suggested that the higher serum fgf-23 level was associated with carotid artery calcification and cardiovascular disease (ischemic heart disease and/or hypertension) in t2dm patients with normal kidney function. while lowered serum ucoc level was associated with carotid artery calcification but does not associate with cardiovascular disease. even so, by connecting the dots, both elevated fgf-23 and reduced ucoc levels are related to altered bone metabolism in relation to abnormal glucose homeostasis which would contribute to vascular calcification, and consequent cardiovascular complications in t2dm patients. since ucoc was inversely correlated with fsg and hba1c, confirming the role of ucoc in glucose metabolism and thereby cardiovascular risk. for this reason, further studies are needed with a big sample size to investigate the role of ucoc in the development of cardiovascular disease in diabetics. finally, serum ucoc and fgf-23 may supply a reliable marker for carotid artery calcification in t2dm patients with normal kidney function. for this reason, more investigations are 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journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 sertraline hcl microspheres 64 preparation and characterization of biodegradable microspheres containing sertraline hydrochloride laith h. samein* , ahmed a. hussein*, 1, alaa a. abdulrasool*, jabar a. faraj** * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. ** university of kentucky college of pharmacy, lexington, ky, usa. abstract four batches of sertraline hcl microspheres were prepared using a poly (d-l-lactide-coglycolide) (plga) polymer ( mw. 9, 27, 30 and 83 kda) as a delivery system. the microspheres were prepared by a dispersion/solvent extraction-evaporation method and characterized for drug loading by uv, particle size by laser diffractometry and surface morphology by scanning electron microscopy (sem). the in vitro sertraline hcl release was studied. spherical microspheres with a mean diameter of 21 to 26 µm loaded with 24.6 – 38.2% were produced. the in vitro drug release was shown to be depend on polymer molecular weight and also on the drug loading. differential scanning calorimetry (dsc) was employed to investigate the physical state of sertraline hcl inside the microspheres and stability and polymer interaction study were performed in solution. key words: sertraline hcl , microspheres , plga polymer ةصالخلا ران ددعتم م د غهن اتي ب رةا sertraline hcl) دي غي اي سن هجمن كغن ن غي ة ا ) غ ب ت ( نكةم و لةد ا . سن هجمن ران 83 , 30 , 27 , 9( ) ا جا ) plga يي ا س س ا) عتم ص هس يمغن ,اا ل اي رغا : هغ ت هغح ا د عش وتي ي م ينلكجغ ,رج سن /دشن ا وقترة . ب ع sem)( ,ا رسح كش د عش هكب دة هجمن و ستناق )laser diffractometryد عش تمةا ارت غا ) %ح قتج .38.2-24.6 ا ه دنكي mµ 26-21 ت ن كغن ن غي ة ا سة جك . ن اجمن را اي د حشن غح هكب تلن ا ت ن سة جك ا ح ممن قل يته د ل ا جا ) ي غهن ا س ا د ل هغ هغح ا . ( ح عتم م تال دي ة اغا ةا كغن ن غي ة ا ب سح سن هجمن , س ا ب ع dsc) كين وعتان ا لةدح ي غهن ب سح ه د . introduction sertraline hcl is the second most potent inhibitor of serotonin reuptake and the second most selective blocker of serotonin over noradrenaline uptake. it has been approved in 1997 in france, and is currently widely prescribed in europe and the united states(1). it has been also used for the treatment of depression, obsessive-compulsive disorder (ocd), depression relapse and social phobia(2,3). it is the only selective serotonin reuptake inhibitor (ssri) that binds to dopamine transporters(4). sertraline hcl exhibits linear pharmacokinetics(5). after single doses between 50 and 200 mg, t1/2 is similar for single dose and steady-state conditions(6). the elimination rate constant is higher in young males than in females or subjects 65 years old or older(7). the hepatic metabolism is the most important pathway, with only 0.2% of an oral dose being excreted unchanged in the urine(8). the ndemethylation is the main metabolic step in the biotransformation of sertraline(9). drug absorption from the git is slow, but complete with maximum plasma concentrations (cmax) attained within 6-8 hr and compared to other ssris, a relevant portion of oral sertraline is excreted in the feces (~50%) (10). increasing evidence from randomized controlled trials of ssris show their efficacy in treating pediatric depression. the number of prescriptions for sertraline hcl use in pediatric populations has exploded recently with figures ranging from 600,000 children and adolescents(11 13). 1 corresponding author : e-mail : ahmed_sura@yahoo.com received : 30 /4/2008 accepted : 28/6/2008 iraqi j.pharm.sci., vol.17 (1) ,2008 sertraline hcl microspheres 65 the oral administration of this drug to children is hard to control compared to adults. in addition, to frequent administration, chances of drug misuse following oral administration are high. this necessitates administration of the drug via a different route. therefore, there is a strong need for a non-oral controlled delivery dosage form for this drug. this paper investigates the feasibility of formulating sertraline hcl into biodegradable microspheres using plga polymer to be used as injectable dosage form. in addition, drug stability and drug-polymer interactions were studied. finally, in vitro release efficacy from the formulations was also assessed. experimental materials poly(d,l-lactic-co-glycolic acid) (plga); resomer 502h (9 kd), 503 h (27 kd), 503 (30 kd), plga 50:50 and 6535dl (83 kd) plga 65:35 ;were supplied by boehringer ingelheim (ingelheim, germany). polyvinyl alcohol (m.wt. 30000-70000; pva) and sertraline hcl were supplied by sigma (st. louis, mo, usa). all other chemicals were obtained commercially as analytical grade reagents. preparation of microspheres four batches of sertraline hcl microspheres (ms) were formulated with polymers at increasing molecular weight. the microspheres were prepared by dispersing the homogenous suspension of polymer and drug into a 0.35% pva solution continuous phase followed by solvent extraction / evaporation /dilution as already described(14). in detail sertraline hcl was sieved through a 150 mesh sieve and amount corresponding to 25, 40 and 45% loading of the sieved powder was dispersed in methylene chloride and properly sonicated. the polymer in the proportion of 12% was then added to the resulting suspension and after its complete dissolution the suspension was slowly injected into phosphate buffer saline ( ph 8.9) containing 0.35% poly vinyl alcohol (pva) and mixed at 900 rpm. at 4°c. the microspheres hardening and complete evaporation of the solvent were accomplished increasing slowly the temperature up to 20°c in on hour. at the end of the process the microspheres have been recovered by filtration through a 0.65µm harvesting filter and freeze-dried overnight .table 1 outlines the preparation parameters for sertraline hcl microspheres. the s/o/w method is represented in figure 1. table 1 preparation parameters and particle size of sertraline hcl ms. particle characterization: particle size distribution the prepared particles were sized by laser diffractometry using a malvern 2600 laser sizer (malvern 2600 particle sizer, malvern, uk). the average particle size was expressed as the volume mean diameter vmd in microns (µ ). surface morphology surface morphology was examined by scanning electron microscopy (sem) (hitachi model s800 japan) after palladium/gold coating of the microspheres sample on an aluminum stub. drug content 10 mg of microspheres were dissolved in dimethyl sulfoxide (dmso). the sertraline hcl was extracted, since polymer and drug were soluble in dmso. in detail, triplicate samples of 10 mg of the microspheres were quantitatively transferred to 12 ml glass test tube. the microspheres was solubilized in 2 ml polymer m.w. (kda) method ph of cp target load % w/w drug content % w/w encapsulation efficiency (%) particle size (μ) 503h 27 s/o/w 8.8 45 38.2 84.9 19.6 502h 9 s/o/w 8.8 25 24.6 98.4 21.0 503 30 s/o/w 8.8 40 34.3 85.8 26.0 6535dl 83 s/o/w 8.8 40 34.5 86.3 24.0 iraqi j.pharm.sci., vol.17 (1) ,2008 sertraline hcl microspheres 66 of dmso, then 10 ml of 0.1m acetate buffer ph 5 was added and the tubes were agitated by a wrist action shaker for 1 hr. the sample were centrifuged at 3000 rpm and the aqueous layer was analyzed spectrophotomery at 273 nm. absorbance measurements were made at a selected wavelength (λ max = 273 nm).absorbance measured values were fitted against a calibration curve based on a lambert–beer law(15). mg plga + 3600 mg ch2cl2 sertraline hcl 150 mg + ch2cl2 polymer conc. 12% w/w 150, 240 or 270 mg of sertraline hcl in 2000 mg ch2cl2 were mixed and stirred for 5 min. [dispersed phase (dp)] mixture was mixed with 50 ml phosphate buffer saline ( ph 8.9) containing 0.35% pva and mixed at 900 rpm at 4°c the microspheres hardening and complete evaporation of the solvent were accomplished increasing slowly the temperature up to 20°c in on hour. microspheres were collected on filter paper and washed for 3 times with distilled water microspheres were freeze-dried over night figure 1: preparation of plga sertraline hydrochloride microspheres using dispersion/solvent extraction-evaporation method. iraqi j.pharm.sci., vol.17 (1) ,2008 sertraline hcl microspheres 67 dsc analysis sertraline hcl thermotropic behavior inside the microspheres was investigated by a dsc 2920, de differential scanning calorimeter. samples of sertraline loaded microspheres and blank microspheres were scanned at 5 °c/min heating rate in the range – 10°c to 300°c. in addition, dsc scans were run on the drug, polymers and physical mixtures of the drug with polymers used in the preparation of microspheres. further measurements were carried out on drug powder after suspension and sonication for 20 seconds in dichloromethane, then evaporation of dichloromethane and on the drug, postsieving, to detect any structural modification due to the preparation process. all the samples were freeze-dried dried over night before the analysis(16). drug-polymer interaction drug-polymer interaction studies were carried out in solutions containing lactic acid, glycolic acid and with mixtures of lactic acid and glycolic acid ( 50:50 la : ga) at 37°c. sampling was performed periodically (5, 10, 20, 30, 40 and 50 days) followed by uv analysis at 273 nm. all analysis were performed in duplicate(17). in vitro drug release study long-term (48 days) in vitro drug release was carried out in 0.1m acetate buffer, ph 5 at 37 c. the ph of this buffer is close to that of an acidic microenvironment that form within the plga matrix(18). briefly, 10 mg of microspheres were suspended in 10 ml of the buffer. at each time point(1, 3 , 8, 10, 15, 20, 27, , 34, 41, and 48 days) 1 ml of supernatant was withdrawn from each tube after centrifugation (2min, 3000 rpm) and an equivalent volume of fresh buffer was then added to replace the amount collected. analyses were carried out using uv spectrophotometry at 273 nm on triplicate or duplicate samples. results and discussion preparation of microspheres preparation of sertraline hcl loaded microspheres was accomplished by the s/o/w method already described in the experimental section. the reason for choosing such procedure is the low solubility shown by the drug into most of the solvents commonly used in microsphere formulation. various preparation conditions and materials were investigated in order to obtain the best results concerning loading and drug release. microspheres morphology and size distribution and in vitro release behavior to test the feasibility of sertraline formulation. the results shown in table 1 reveal the remarkable encapsulation efficiencies (84.9% 98.4%). a critical step at this point was the complete drug dispersion that is fundamental to have a uniform distribution of the drug inside the microspheres and higher encapsulation efficiency. plga polymers were employed with increasing molecular weight (9-83 kda.) and different glycolic/lactic ratio (50:50 and 35:65) in order to investigate the effect of these parameters on the release behavior of such formulations. the best batches resulted plga based preparations and especially microspheres with plga resomer 503h and 502h polymers showed the best results in term of encapsulation efficiency and drug content. microspheres characterization sem analysis on sertraline hcl microspheres showed that the microspheres were successfully fabricated with a spherical shape, a certain fragility and relatively low porosity (figure 2). the average particle size was approximately 22µm which is suitable for intramuscular or subcutaneous injections(19). (a) (b) ( c ) (d) figure 2: sems of sertraline hydrochloride loaded microspheres. 503 (a), 502h (b), 6535dl (c) , 503h (d) drug content and encapsulation efficiency dispersion /solvent extraction-evaporation method has been used succefully in the incorporation of hydrophobic drugs with good yield value loading percentage(20). loading 68 efficiencies ranged from 84.9-98.4 as illustrated in table 1. yield value are function of the efficiency of preparation method and values up to 70% were accepted(21). sertraline hcl , being a water insoluble molecule is better dispersed in organic solvent then emulsified in aqueous solution of the surfactant where minimum amount of the drug would be in the aqueous continous phase(22). the loading efficiency of 502h microspheres was the highest among other batches(table 1). this result may be due to its lowest target load (25%), since a higher target load of bioactive material is likely to decrease the entrapment efficiency of drug in plga(23-25). the drug content ranged from 24.6-38.2 % . drug-polymer interaction there was no detectable decrease in sertraline hcl concentration in 0.1m acetate buffer, ph 5.0 for the entire duration of study (50 days) at temperatures 37 °c. there is no significant change in drug levels when incubated with solutions of lactic acid, glycolic acid and a 50:50 mixture corresponding to the molar amount that would be obtained on complete hydrolysis of the plga polymers , 502h and 503h. the dsc analysis the dsc analysis confirmed a high drug-polymer affinity. the comparison of thermal profiles of drug, polymer, physical mixture and drug loaded microspheres revealed that the drug was present as a dispersion in the polymeric matrix for all the microsphere batches as demonstrated by the lack of sertraline hcl melting peaks (figure 3 a-d). differences in glass transition temperature (tg) between drug loaded microspheres and raw polymer suggest that the drug has a plasticizing effect on the internal structure of the polymer(26). the drop in the tg was greater for microspheres prepared from high molecular weight polymers. tg values of all the systems studied are shown in table 2. table 2 : the tg of the sertraline bpowder, microsphere and the physical mixture of sertralin with polymer. figure 3a : dsc scan of sertaline hcl, sertaline hcl-502h polymer physical mixture, 502h polymer and sertaline hcl 502h microspheres. figure 3b : dsc scan of sertaline hcl, sertaline hcl-503h polymer physical mixture, 503h polymer and sertaline hcl 50 microspheres. rg503h tg (°c) rg502h tg (°c) sertraline503h phys. mix. 43.09 sertraline502h phys. mix. 34.90 pure polymer 45.60 pure polymer 33.62 503h ms 37.79 502h ms 32.27 rg503 tg (°c) 6535dl tg (°c) sertraline-503 phys. mix. 46.25 sertraline6535 dl phys. mix. 43.01 pure polymer 47.05 pure polymer 46.25 503 ms 30.15 6535 dl ms 33.80 69 figure 3c : dsc scan of sertaline hcl, sertaline hcl-503 polymer physical mixture, 503 polymer and sertaline hcl microspheres. figure 3d : dsc scan of sertaline hcl, sertaline hcl-6535dl polymer physical mixture, 6535dl polymer and sertaline hcl 6535dl microspheres. in vitro drug release a pathway for sertraline hcl release was provided by microsphere degradation where water-soluble degradation products (i.e. monomers and oligomers ) leave the microspheres matrix for the surrounding aqueous medium. since oligomers are close to the surface they can leach out faster than that located deeper within the matrix, carboxylic acid oligomers trapped within the matrix autocatalyze further ester bond hydrolysis, resulting in the increasing rate of mass loss(27). four batches of microspheres were subjected to long-term in vitro release (48 days) at 37°c in 0.1m acetate buffer, ph 5.0. the data in figure (4) showed complete sertraline hcl release from 503h and 502h microspheres throughout 35 days with no significant variation between them(p< 0.01). on the other hand, 503 and 6535dl microspheres gave total drug release about 82% and 59% respectively within 35 days. the high drug release from 503h and 502h microspheres can be attributed to the highest loading percent of the drug for 503h microspheres and to the low molecular weight for 502h polymer, and these two effects may fasten the hydrolysis of microspheres(28,29). in paired comparison ( 503h vs 503 ), where the overwhelming majority of structure are chemically identical, and the difference between them is whether the polymer end groups are a carboxylic function (503h) or a long-chain fatty ester(503), the more hydrophilic polymer , the greater amount of drug bound . in a similar study, release of bone morphogenetic protein-2 from hydrophilic plga microspheres was higher than that from hydrophobic one(30). the slow release of sertraline hcl from dl6535 microspheres might be due to the slow hydration and degradation of the high molecular weight polymer(31). this result was expected and similar results reported by researchers(32-34). figure 4: in 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2 (3) article 17.) 31yoo jy., kim jm., seo ks., jeong yk., lee hb., and khang g. characterization of degradation behavior for plga in various ph conditionby simple liquid chromatography method. bio-medical materials and engineering. 2005; 15: 279– 288). 32lima km., rodrigues jm. . poly-dllactide-co-glycolide microspheres as controlled release antigen delivery system. braz j med bio res, 1999; 32(2): 171180). 33pekarek kj, dyrud mj, ferrer k, jong ys, mathiowitz e. in vitro and vivo degradation of double-walled polymer microspheres. journal of controlled release. 1996; 40: 169-178. 34burton kw, shameem m, thanoo bc , deluca pp. extended release peptide system through the use of plga microsphere combinations. j. biomater. sci. polymer edn. 2000; 11: 715-729. http://www.springerlink.com/content/105282/?p=549cd2d3e00c4f3fa9211ef57e6be6ea&pi=0 http://www.springerlink.com/content/r327j687q818/?p=549cd2d3e00c4f3fa9211ef57e6be6ea&pi=0 http://www.ingentaconnect.com/content/klu/pham;jsessionid=19sqvu55xe59s.alexandra the effect of atenolol on iraqi j pharm sci , vol.18 (1) ,2009 atenolol and cpk-mb levels 16 the effect of atenolol on ck-mb levels in hypertensive patients # inaam a. amin *,1 * department of clinical laboratory sciences,college of pharmacy,university of baghdad,baghdad , iraq abstract atenolol is one of beta-adrenergic receptor blocking agent. it is widely used for the treatment of hypertension as a selective antihypertensive drug. but long term usage of atenolol may cause one of the cardiovascular diseases like myocardial infarction. to prove the relationship between atenolol and cardiovascular disease, measurement of creatinekinase-mb as a diagnostic indicator in early and long term usage of this drug by hypertensive patients is recommended. a comparative study was conducted in al-yarmouk teaching hospital–emergency departmenton 30 hypertensive patients using atenolol. they were divided into (2) groups a and b according to the duration of the drug usage. group a(15) patients with a mean age (56+6) years. they used atenolol for a period of (1-5) years. group balso (15) patients with mean age (60+6) years. they used atenolol for (6-20) years. both groups were with nearly the same number of males and females. all subjects of the study groups were screened to exclude evidence of hyper or hypothyroidism, diabetes and chronic renal failure. venous blood samples were taken in first 8 hours after onset symptoms of cardiac attack from each patients and the levels of creatine kinase-mb were estimated and compared between the (2) groups. there is a significant correlation between levels of serum creatine kinase-mb of group a and group b (p<0.05). atenolol causes increased level of serum ck-mb and this increase was directly proportional to the duration of the drug usage. ck-mb is one of cardiac markers that released from heart muscle when it is damaged as a result of myocardial infraction. so, atenolol has a significant correlation with development of myocardial diseases. key words: hypertension , atenolol and side effects , creatine kinase-mb , atenolol antihypertensive. : الخالصة التُنىرمُن هى واحد مدن موعى دأل ايةوَدأل التدٍ علعدغ دً ق دت مظدت، وه ُتدخد وهدى َظدتع ف مداغ واطدت ادٍ ملخلودأل ارع دخ ظغػ ال ف ولهذا اإن هذا الل،خر ق َصنف ك واء اختُخرٌ لت، ُغ ارع خ ظغػ ال ف ولان اطتع اف هذا الل،خر دً مد ي غىَدغ قد َظد من امزاض الوهخس ال، ٍ الى خئٍ. وإلث خه اللوقأل ُن ،خر التُندىرمُن وممدزاض الوهدخس ال، دٍ الى دخئٍ ىاطدسأل قُدخص ح وث واح منشَم الازَخعُن كخَنُض ف ب كاخشدف عل ُ دٍ دً العد ي ال،صدُز وال لُد مطدتع اف هدذا الل،دخر لد ي مزظدً ارع دخ ظدغػ الد فد ولهدذا ( مزَعددخي َلددخنىن مددن ارع ددخ ظددغػ الدد ف ٠٣ ددً -قظددم السددىار -ظتمدد ً الُزمددىت التل ُعددٍمجزَددد ةراطددأل م،خرنددأل حددخمه اددٍ م وَظددتلع ىن عُنددىرمُن كلددوت وعددم ع،ظددُم هددلمء العزظددً ألددً موعددى تُن م وب( وا،ددخي ألددً غددىل اتددزي اطددتلعخلهم لل،ددخر التُنددىرمُن. ( طدنأل ١-٥طدتلع ىا التُندىرمُن ل تدزي سمنُدأل عزاوحدد دُن ( طدنأل وا٥+١٥( مزَعدخيد ملد ل م عدخرهم ٥١العوعى أل م( وعتألف مدن ( طدنأل. ٠٣-٥( طنأل اطدتلع ىا التُندىرمُن لعد ي عزاوحدد دُن ٥+٥٣( مزَعخيد مل ل م عخرهم ٥١والعوعى أل ب( وعتألف مَعخي من ز مصدخ ُن دأمزاض الغد ي ال رقُدأل كو العوعى تُن ع،زَ خي ععم ن ض الل ة من كو الونظُند وكغ العزظً العمدعىلُن هدذا ال راطدأل قُد وم اء الظازٌ مو وش الا ُأل العشمن. عم مخذ ُنخه من ةف كدغ مدزَط خدول الظدخ خه اليعخنُدأل ايولدً لد رهدىر م دزاض ايسمدأل َنُض ف ب ادٍ ال، ُأل ل،ُخص مظتىي منشَم الازَخعُن كخَنُض ف ب وععدد م،خرندأل النتدخئي دُن العوعدى تُن. كدخن ملد ل ايندشَم كزَدخعُن كدخ . ومدن خدول النتدخئي التدٍ عدم اللصدىل ُهدخ >p ٣٠٣١ مصىل ةف العوعى أل ب( م،خرندألي خلعوعى دأل م( خلُدأل و علنىَدأل واظدلأل ع ُن من ،خر التُنىرمُن َلةٌ ألدً ارع دخ وسَدخةي مظدتىي ايندشَم كزَدخعُن كدخَنُض ف ب وهدذا الشَدخةي عتنخطد غزةَدخي مدت غدىل ال تدزي مطتلعخل التُنىرمُن و عدخ من هدذا ايندشَم ملشدز حُدىٌ خدخض يمدزاض الوهدخس ال، دٍ الى دخئٍد لدذا ادإن ،دخر التُندىرمُن لد الشمنُأل وقأل ملنىَأل ل وث ممزاض الوهخس ال، ٍ الى خئٍ وخصىصخي احتمخء اللع أل ال، ُأل. introduction: atenolol is one of beta blockers acts by blocking beta receptors that are found in various parts of the body, and prevents the action of nor-adrenaline and adrenaline (1) . atenolol is rapidly absorbed from the gut. blood level reached a peak concentration in (23) hours (2) . metabolism of atenolol is minimal and almost the total absorbed drug (85-100)% is cleared via excretion in the urine in an unaltered manner (3) . although atenolol is the drug of choice in different cardiovascular diseases as angina pectoris, hypertension, arrhythmias and in prevention of heart attack (4) . the prolong use of this drug as antihypertensive may show different side effect which may develop to symptoms of cardiovascular disease. creatine kinase-mb is one of the isoenzymes of creatine kinase which is mostly found in the heart. i measured creatine kinase-mb as an important biological marker, when it appears in abnormal level>10u/l in serum. # based on oral presentation in the seventh scientific conference of the college of pharmacy /university of baghdad held in 26-27 november 2008 1 corresponding author e-mail : inaam1960 @yahoo.com received : 31/12/2008 accepted : 31/3/2009 iraqi j pharm sci , vol.18 (1) , 2009 atenolol and cpk-mb levels 16 this means that there is a myocardial injury. ck-mb shows increases above normal in a person's blood test about four to six hours after the start of a heart attack. it reaches its peak level in about 18 hours and returns to normal in 24 to 36 hours (5) . ck-mb is both a sensitive and specific marker for mycocardial infarction, most commonly used to confirm the existence of heart muscle damage. materials and method: this comparative study was done in the emergency department in al-yarmouk teaching hospital on (30) hypertensive patients (48-68) years who received atenolol tablet 100mg as antihypertensive drug for a duration of (1-20) years. the patients were divided into (2) groups according to the duration of drug use: group a: consists of (15) patients with a mean age (56+6), they used atenolol for a period of (1-5) years. group b: consists of also (15) patients with a mean age (60+6), they used atenolol for a period of (6-20) years. venous blood samples were obtained from each patient of both groups for measuring the level of ck-mb. the method used for measuring ck-mb is immunoinhibition assay (randox) in which an antibody is incorporated in the ck reagent. this antibody will bind to and inhibit the activity of the m subunit of ck-mb. this means that only the activity of the b subunit in serum is measured (6,7) . the sample is serum, heparinized or edta plasma. haemolysis interferes with the assay. reagents are a mixture of ck-mb buffer/glucose (imidazole buffer, glucose, mg-acetate and edta) with enzymes/coenzymes/substrate/antibody (adp, amp, diadenosine pentaphosphate, nadp, hk, g-6-pdh, n-acetylcysteine, creatine phosphate and antibody to ck-m). a patient sample is added to the reagent mixture read the absorbance directly at 340nm (a1), the second reading is after five minutes exactly (a2). a = a2 – a1 a multiplied by 1651 (kit factor) gives the concentration of ck-mb in u/l. this procedure is done at room temperature 25 o c. results: after collection and categorization of data from the (30) patients included in the study, statistical analysis was done [table 1 and fig.1] which revealed the following: 1. the correlation between atenolol duration 1-5 years and ck-mb (u/l) in patients included in the study ( y = 2.4336x+2.5759, r 2 = 0.236, r=0.486, p=0.066 (not significant)). 2. the correlation between atenolol duration 6-20 years and ck-mb (u/l) in patients included in the study (y= -0.3751x+24.188 , r2= 0.277, r= -0.166, p= 0.553 (not significant)). 3. the correlation between atenolol duration (years) and ck-mb (u/l) in total 30 patients included in the study (y=0.9507x+9.3164, r 2 =0.1757, r=0.419, p=0.021 (significant direct correlation) as shown in fig. (2). table(1) : the ck mb(u/l) concentration duration of use of atenolol in hypertensive included in the study figure(1) : correlation between time of atenolol usage and serum ck-mb atenolol duration of use (years) 1-5 years 6-20 years c k m b ( u /l ) mean 10.12 20.59 sd 8.58 8.60 minimum 1.6 7.1 maximum 31.3 36.9 ck-mb (u/l) 0 5 10 15 20 25 30 1-5 years 6-20 years iraqi j pharm sci , vol.18 (1) ,2009 atenolol and cpk-mb levels 16 figure(2) : the correlation between the duration of atenolol treatment (years) and ck-mb (u/l) in total 30 patients included in the study discussion: atenolol is widely used all over the world for the treatment of hypertension. it is an efficient antihypertensive but it has many side effects which sometimes they might be serious. enzymology is a diagnostic indicator for cardiovascular disease in hypertensive patients with atenolol treatment (8) . ck-mb, the primary indicator used to diagnose a heart attack because it exists in the highest amount in the heart helps in converting creatine to creatinine, a reaction that is necessary for metabolism and energy production. so, the level of ck-mb determines the effectiveness of antihypertension drug which provides a diagnostic clinical evidence (8) . rise in the level of this enzyme (ck-mb) has been reported in hypertension with myocardial infarction patients (9,10) . enzymes always have been identified as a specific and sensitive markers of both clinical and subclinical myocardial injury (11) . therefore biological marker like ck-mb to quantify myocardial injury has been widely used in clinical practice. in cardiac muscle they are tightly bound to the contractile apparatus and therefore plasma concentrations is extremely low. with acute myocardial injury, there is release of ck-mb into the serum, the extent of the elevation in serum depends on the severity of the myocardial injury. and the entry of this enzyme in circulation depends upon the rate of passive diffusion of the enzyme from infarct myocardium cells (12) . one of the most reliable and commonly tested cardiac enzyme is ckmb which released specifically from injured heart muscle (13) .increased serum levels of ckmb in hypertensive patients taking atenolol is directly proportional to the duration of the atenolol usage. long exposure of cardiac muscle to atenolol leads to escape of ck-mb to circulation. the mechanism by which atenolol causes myocardial injury is not yet known and this may be due to cardiac muscle which becomes fatigue with prolonged exposure to atenolol causing it unable to contract efficiently and ending with failure (14) . conclusion: atenolol should be used selectively and in acute urgent cases for different cardiac diseases. for hypertensive patients of long term usage checking should be followed continuously to make sure if any symptoms of cardiac injury appears and in such a case terminates using atenolol and other antihypertension drug should be described. references 1fisherman w.h., atenolol and timolol two new systemic b-adrenceptor antagonist, n. england j.med., 1992; 306(24), 1456-1462. 2fitzgerald jd, ruffin r., smedstad kg, roberts r., et al., studies on the pharmacokinetics and pharmacodynamics of atenolol in man, eur.j.clin.pharmacol., 2003; 13: 81-89. 3williams da, lemke tl, foye wo., foye's principles of medicinal chemistry, hagerstown, md: lippincott williams & wilkins, 2002. 4rosendorff c., black hr, cannon cp, et al., treatment of hypertension in the prevention and management of ischemic heart disease: a scientific statement from the american heart association council for high blood pressure research and the councils on clinical cardiology and epidemiology and prevention, circulation, 2007; 115: 2761-88. 5book-wu, a., editor. cardiac markers, washington, dc: american association of clinical chemistry (aacc) press, 1998. 6szasz, g., and e.w. busch, paper presented at 3 rd eur.congr.clin.chem., brighton/england, june 1979; 3-8 (abstract). 7wurburg, u., et al., clin.chem., 1976; 54: 357. 8m.m. kamble, s.m. vaidya, effect of antihypertensive drugs on cardiac enzymes in hypertension with myocardial infarction in niddnm, indian journal of clinical biochemistry, 2002; 17(2) 60-63. iraqi j pharm sci , vol.18 (1) ,2009 atenolol and cpk-mb levels 16 9stein w., med.weit, 1985; 36, 572. 10yi-chun, z., yi-zhum, z., heidi, s., peter, g., and thomas, u., substrate metabolism, hormone interaction and angiotensin converting enzyme inhibitors in left ventricular hypertrophy diabetes, 1996, 45 (suppl-1); 559-565. 11adams, j., abendschein, d., jaffe, a., biochemical markers of myocardial injury= 1s mb creatine kinase, the choice for the 1990s? circulation, 1993; 88, 750763. 12burton e.s., robert r., and kenneth b.i., considerations in the use of biological markers of ischemic injury, circ.res., 1976, 38 (suppl-1); i-99-i-106. 13european heart journal 2004, 25(14): 1187-1196; doi: 10. 1016/j.ehj.2004.04.026 copyright © 2004 by the european society of cardiology, role and importance of biochemical markers in clinical cardiology. 14fitzgerald jb, the biological and clinical effect of atenolol (tenormin) a cardioselective beta antagonist. in goldberg me ed. pharmacological and biological properties of drug substances, vol.2, american pharmaceutical association, washington, 1999; 98-147. clinical evaluation of melatonin alone and in combination with pizotifen in the prophylaxis of migraine iraqi j.pharm.sci., vol.16 (1) ,2007 benfotiamine in cardiotoxicity 14 protective effect of benfotiamine against doxorubicin-induced cardiotoxicity in rabbits. munaf h. abdulrazzaq* ,1 * department of pharmacology and toxicology, collage of pharmacy, university of baghdad, baghdad, iraq. abstract : the protective effect of benfotiamine against doxorubicin-induced cardiotoxicity was evaluated in rabbits. pretreatment of rabbits with 70mg/kg benfotiamine orally 7 days before induction of cardiotoxicity with i.v 15mg/kg doxorubicin. injection resulted in significant reduction of the activities of lactate dehydrogenase and creatine phosphokinase enzyme in the serum compared to doxorubicin treated animals; benfotiamine also improves the histological changes produced by doxorubicin in the cardiac muscle compared to control. in conclusion, benfotiamine when used concomitantly with doxorubicin protects the myocardium against the cardiotoxicity induced by this cytotoxic drug. الخالصة ة أٌ ْذِ اندراسة قد جى جصًًٛٓا نحقٕٚى انحأثٛز انٕقاايٙ انًتحًام نًااال انوُيٕجٛاايٍٛ لاد انحها اناذ٘ جداووّ يااال اندٔندٕرٔعٛداٍٛ اٙ اه ل انقهب ٙ األراَب يٍ خالل سٚاال ٕايم انحاندد ٔجتيٛش انًٕت انًوزيج نهخهٛة ٔيٍ خالل ٕايم اخزٖ . أظٓزت انُحاايج أٌ ع ااام يااا أاٖ يهاغ/نغى اٍ يزٚال انٕرٚاد(5أٚاو قوم جز اة اندٔندٕرٔعٛداٍٛ ) 0يهغ/نغى يٍ خالل انيى( نألراَب ٔنًدل 07انوُيٕجٛايٍٛ ٔعجز ة ) يقارَاة عًداحٕٚاجٓا ُاد األراَاب اناذٍٚ اساحهًٕا جز اة اندٔندٕرٔعٛداٍٛ ldh ٔcpkعنٗ اَخياض يعُإ٘ عًداحٕٚات عانٛاة أَشًٚاٙ وُيٕجٛايٍٛ ٙ جتدٍٛ انحه انتاصم ٙ َدٛج انع هة انقهوٛة ندٖ يجًٕ ة األراَب انحٙ اسحهًث جز اة انوُيٕجٛاايٍٛ قوام قط. نذنك أسٓى ان جز ااة اندٔندٕرٔعٛدااٍٛ يقارَااة عحهااك انحااٙ اسااحهًث جز ااة اندٔندٕرٔعٛدااٍٛ قااط. ٔأخٛاازام َدااحُحج أٌ اسااحعًال ياااال انوُيٕجٛااايٍٛ قواام انكا ٛة نُدٛج هة انقهب يٍ انحه انذ٘ ٚدووّ قار اندٔندٕرٔعٛدٍٛ. اندٔندٕرٔعٛدٍٛ قد ٕٚ ز انتًاٚة introduction : the anthracycline antibiotic, doxorubicin, is an important antineoplastic agent that show high efficacy against various types of malignancies (1) . it produces cardiotoxicity as a specific adverse effect mostly attributed due to extensive formation of reactive oxygen species (ros) that associated with many events related to nucleic acid metabolism and induction of the immune system (1,2) . recently, apoptosis of cardiomyocytes has been suggested as a common mechanism of acute and chronic loss of myocytes (3) , and doxorubicin was reported as one of the most potent inducers of apoptosis in many cell lines of many types (4,5) . it has been suggested also that phosphokinase c (pkc) is one of the intracellular signaling mediator for the action of tnf-α, which is responsible for cytotoxicity and apoptosis in many cell types (6) . benfotiamine, the lipid soluble prodrug of thiamine, is converted after administration to the biologically active thiamine pyrophosphate (tpp) (7) , and due to its relatively higher rate of oral absorption and greatest intracellular access (8, 9) ; it can replace the conventional thiamine wherever indicted. it inhibits diacylglycerol-protein kinase c pathway and nfkβ activity through activating the transketolase, one of the pentose phosphate pathway enzymes (10) . this work was designed to evaluate the protective effect of orally administered benfotiamine against doxorubicininduced cardiotoxicity in rabbits. materials and methods : adult rabbits (locally bred white strain weighing 1-1.5kg) of both sexes, maintained in the animal house of the collage of pharmacy, university of baghdad, were used for this study. animals were allowed for a standard pellet diet and tap water ad libitum. the animals were allocated into three groups and treated as follow: group 1 includes six rabbits treated with normal saline only, served as negative controls. group 2, includes six rabbits given normal saline orally for seven days before induction of cardiotoxicity with i.v. injection of 15mg/kg doxorubicin, and served as positive controls. group 3 includes six rabbits, pretreated with benfotiamine (70mg/kg) orally for 7 days before induction of cardiotoxicity with i.v injection of 15mg/kg doxorubicin. the animals were sacrificed 48 hr after administration of doxorubicin by an overdose of thiopental (100mg/kg). blood samples were collected for the preparation of serum and estimating serum enzymes activities of lactate dehydrogenase (ldh) (11) , creatine phosphokinase (cpk) (12) and glutamic oxaloacetic transaminase (got) (13) . histological sections of the myocardial tissues were prepared for evaluating the histopathological changes with ordinary microscope after fixing hearts in 10% formalin, processed and embedded in paraffin. 3μm. thick sections were cut on glass slides and stained with hematoxylin and eosin (h&e). the data were presented as mean + s.e. the significance of differences between mean values 1 corresponding auther : e-mail: munafzalzala@yahoo.com received : 1/8/2006 accepted : 4/3/2007 mailto:munafzalzala@yahoo.com iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 15 0 50 100 150 200 250 300 350 control doxorubicin doxorubicin + benfotiamine l d h l e v e l u / l 0 20 40 60 80 100 120 control doxorubicin doxorubicin + benfotiamine c p k l e v e l u / l v was evaluated utilizing unpaired student's t-test. p alues less than 0.05 were considered significantly different. results : figure (1) indicated that serum level of ldh activity was significantly increased in doxorubicin treated rabbits compared to controls (p<0.05), while pretreatment with benfotiamine significantly reduces serum ldh activity compared to doxorubicin treated group. in figure (2), treatment with doxorubicin significantly elevated serum level of cpk activity compared to controls, and pretreatment of rabbits with benfotiamine before administration of doxorubicin resulted in cpk activity value that was significantly lower compared to doxorubicin treated animals and comparable to that of controls. there are no significant changes in serum levels of got activity in doxorubicin treated group and that group which pretreated with benfotiamine before doxorubicin (figure 3). histological examination of tissue section from the heart muscle clearly showed that mild focal edema, focal cellular injury, vaculation of cardiomyocytes and disoriented nuclei were observed in doxorubicin treated group (figure 5). pretreatment with benfotiamine before doxorubicin administration was protects cardiac tissue against doxorubicininduced damage that shows normal heart tissue with no significant degenerative changes when compared with normal heart tissue slide (figure 4 & 6). * figure (1) : effect of pre-treatment with benfotiamine on the serum level of lactate dehydrogenase activity in rabbits with cardiac toxicity induced with doxorubicin. *p<0.05 compared with the control. * figure (2) : effect of pre-treatment with benfotiamine on the serum level of creatine phosphate kinase (cpk) activity in rabbits with cardiac toxicity induced with doxorubicin. * p<0.05 compared with the control. figure (3) : effect of pre-treatment with benfotiamine on the serum level of glutamate-oxaloacetate aminotransferase (got) activity in rabbits with cardiac toxicity induced with doxorubicin. 0 2 4 6 8 10 12 14 control doxorubicin doxorubicin + benfotiamine g o t l e v e l u / l iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 16 figure (4) : section showing normal rabbits myocardial tissue . magnification : 20x, staining: haematoxylline & eosin. figure (5) : section showing morphological alteration of heart from doxorubicin-treated rabbits. black arrow represents the vaculation of cardiomyocytes with focal edema. magnification: 20x, staining: hematoxylene & eosin. figure (6) : section showing the protective effect of benfotiamine (70mg/kg) against cardiotoxic effect of doxorubicin. there are no significant changes. magnification: 20x, staining hematoxylene & eosin. discussion : the major adverse effect associated with doxorubicin use is the high incidence of cardiomyopathy and heart failure (14) . several reports suggested that doxorubicin-induced apoptosis plays an important role in its cytotoxicity, which is linked to formation of reactive oxygen species (ros) derived from redox activation of doxorubicin (15, 16) ; and many other studies have focused on doxorubicin-induced apoptosis signaling mechanism (17, 16) . in the present study, the cardiotoxicity of doxorubicin was clearly demonstrated as a significant elevation in serum activity of ldh and cpk in doxorubicintreated rabbits compared to control (figures 1and 2). while benfotiamine pretreated rabbits showed a significant reduction in ldh and cpk activity when compared to doxorubicin treated group. (p < 0.05). during the state of oxidative stress induced by doxorubicin treatment, cytotoxicity was mediated through the expression of different cytokines, especially tnf-α (18) ; and benfotiamine, used in this study to protect the myocardium against toxicity by doxorubicin, was found to inhibit excessive release of tnf-α through a mechanism related to the inhibition of pkc enzyme that involved in the generation of nfkβ the important signaling molecule in tnf-α release pathway (19, 20) . modulation of tnf-α expression constitutes an attractive therapeutic choice in the alleviation of doxorubicin-induced apoptotic cardiotoxicity. substances like benfotiamine, iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 17 which inhibits expression of tnf-α during the oxidative stress states, may have potential therapeutic value in this respect. accordingly, the reported cardioprotective effect for benfotiamine in this study against doxorubicin-induced cardiotoxicity can be explained. histological examination strongly supported this idea revealing the ability of benfotiamine in ameliorating the toxic effects of doxorubicin on the cardiomyocytes (figures 5 & 6). the histological features appeared in the heart of doxorubicin-treated rabbits clearly showed that the apoptotic cellular damage was more prevail than the necrotic one, mostly due to the absence of inflammatory cells and swelling of myocytes which consider the critical feature of necrosis (figure 5). therefore, benfotiamine may protect the heart against cytotoxic effect of doxorubicin by interfere with the process of toxicity through its ability to inhibit the apoptosis. in conclusion, benfotiamine protect the myocardium against doxorubicin-induced toxicity through a mechanism not related to antioxidant properties. acknowledgment i would like to express grateful thanks to dr. ahmed d. al-eesa for help in histological study and valuable comments. references : 1. gianni l, myers c. the role of free radical formation in the cardiotoxicity of anthracycline. muggia f m, green m d, speyer jl. (eds.) cancer treatment and the heart, john hopkins university press, baltimore; 1992: 9-46. 2. dorr rt. cytoprotective agents for anthracyclines. semin oncol 1996; 23(suppl.8): 23-34. 3. saraste a, pulkki k, henriksen k, kallajoki m, parvinen m, voipio-pulkki lm. apoptosis in human acute myocardial infarction. circulation 1997; 95: 320-323. 4. ling y h, priebe w, perez-soler r. apoptosis induced by anthracycline antibiotics in p388 parent and multidrug-resistant cells. cancer res 1993; 53: 1845-1852. 5. zhang j, clark jr, herman eh, ferrans v j. doxorubicin-induced apoptosis in spontaneously hypertensive rats: differential effects in heart, kidney and intestine, and inhibition by icrf-187. j mol cell cardiol 1996; 28: 1931-1943. 6. chang, q, tepperman bl. the role of protein kinase c isozymes in tnf-α-induced cytotoxicity to a rat intestinal epithelial cell line. am j physiol gastrointest liver physiol 2001; 280: g572-g583. 7. singleton ck, martin pr. molecular mechanisms of thiamine utilization. current molecular medicine. 2001; 1(2): 197-207. 8. hilbig r, rahmann h. comparative autoradiographic investigations on the tissue distribution of benfotiamine versus thiamin in mice. arzneimittelforshung 1998; 48(5): 461468. 9. gleiter ch, schreeb kh, freudenthaler s. comparative bioavailability of two vitamin b1 preparations: benfotiamine and thiamin mononitrate. in: gries fa, federlin k. (eds). benfotiamine in the therapy of polyneuropathy. new york, georg thieme verlog, 1998:29-33. 10. hammes h, du x, edelstein d, taguchi t, matsumura t, ju q. et al. benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. nature medicine 2003; 9(3): 294300. 11. howell bf, coll b. clin chem 1979; 25:269. 12. fleisher g. automated method for the determination of serum cpk activity. j clin path 1967; 13:233. 13. reitman s, frankle s. in vitro determination of transaminase activity in serum. am j clin path 1975; 28: 56-60. 14. buzder au, morcus c, smith tl, blumenschein gr. early and delayed clinical cardiotoxicity of doxorubicin. cancer 1985; 55: 2761-2765. 15. sawyer db, fukazawa r, arstall ma, kelly ra. daunorubicin-induced apoptosis in rat cardiac myocytes is inhibited by dexrazoxane. circ res 1999; 84: 257-265. 16. nakamura t, ueda y, juan y, katsuda s, takahashi h, koh e. fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats. circulation 2000; 102: 572. 17. wang l, markovich r, chen jw, wang ph. regulation of cardiomyocyte apoptotic signaling by insulin-like growth factor i. circ res 1998; 83: 516-522. 18. meldrum dr, cleveland jc, caun bs, meng x, harken ah. increased myocardial tnf-α in a crystalloid perfused model of cardiac ischemic-reperfusion injury. ann thorac surg 1998; 65: 439-443. 19. moscat j, diaz-meco mt, rennert p. nf-kb activation by protein kinase c isoforms and bcell function. embo reports 2003; 4(1): 3136. 20. barnes pj, karin m. nuclear factor-kb a pivotal transcription factor in chronic inflammatory diseases. new england journal of medicine 1997; 336(15): 1066-1071. 21. beltramo e, berrone e, buttiglieri s, porta m. thiamin and benfotiamine prevent increased apoptosis in endothelial cells and pericytes cultured in high glucose. diabet metab res rev 2004; 20(4): 330-336. effects of enalapril vs losartan on uric acid concentrations in patients with metabolic syndrome iraqi j pharm sci, vol.20(2) 2011 losartan versus enalapril on serum uric acid levels 75 effects of losartan versus enalapril on serum uric acid levels in hypertensive patients with metabolic syndrome # zeina a. a. al-thanoon* ,1 and isam h. mahmood** * department of pharmacology, college of pharmacy, university of mosul, mosul, iraq. ** department of pharmacology, college of medicine, university of mosul, mosul, iraq. abstract to investigate the effects of losartan and enalapril on serum uric acid in hypertensive patients with metabolic syndrome, one hundred and twenty six newly diagnosed mild hypertensive patients, having markers of metabolic syndrome included in the study. the patients were divided into two groups. group 1 (60 patients) was given losartan (50 mg/ day) and group 2 (66 patients) enalapril (20 mg/ day) for a duration of 2 months. a control group of seventy apparently healthy individuals were included. metabolic syndrome was diagnosed according to diagnostic criteria of metabolic syndrome related to the american national cholesterol education program-adult treatment panel iii. serum uric acid levels were measured before and after drug administration. the results revealed a significant higher levels of uric acid were found in the hypertensive patients as compared with control group and a significant drop of uric acid was noted after treatment with losartan but not with enalapril. in conclusions: this study demonstrates significantly higher serum uric acid concentrations in hypertensive patients having markers of metabolic syndrome. losartan but not enalapril therapy produced a significant fall in the serum uric acid level. losartan can be useful therapeutic agent to control blood pressure and to reduce serum uric acid level in hypertensive patients having markers of metabolic syndrome and hyperuricaemia. key words: hypertension, metabolic syndrome, uric acid, losartan, enalapril. الخالصة عهً يسخىي انحايِط انبىنٍ فٍ يصم انذو نذي يزظً ارحفاع ظغػ انذوِّ اإلَاالبزَمنخَحّزٌ حأثُزاِث عقارٌ انهىسارحاٌ و ونذَهى عالياث انعانٍ يٍ انُىع انخفُف بانعغػ إصابخهى حذَثا يزَعا شخصىا 1ٕٔعهً وانًخالسيت األَعُِت، أخزَج هذِ انذراست . أعطُج انًدًىعت األونً عقار انهىسارحاٌ يج يدًىعت انًزظً إنً يدًىعخاٌ حسب انعالج انًعطً نهى. قس انًخالسيت األَعُتِ يٍ سهًُا شخصا 0ٓ خخُارإحى يهغ َىيُا، .أسخغزقج فخزة انعالج يذة شهزٍَ. ٕٓيهغ َىيُا، وانًدًىعت انثاَُت عقار اإلَاالبزَم 0ٓ بزَايح انىغٍُ نخعهُى ان يعاَُزيدًىعت انعبػ. شخصج انًخالسيت األَعُت حسب انًخطىعٍُ )َبذوٌ أصحاء( غبُعٍ انعغػ نُكىَىا نكم يٍ يدًىعت انًزظً) قبم وبعذ انعالج( ويدًىعت انعبػ. أظهزث انحايط انبىنٍيسخىي قُاس انكىنسخزول األيزَكٍ. حى ٍِ فٍ يصم انذو نذي يزظً ارحفاع ظغػ انذوَّ بانًقارَت يع يدًىعت انعبػانَُخائِح ارحفاعا يعُىَا يهحىظا فٍ يسخىي انحايِط انبىن ٍِ بعذ انًعاندت بعقار انهىسارحاٌ نكٍ نََُس َيع عقار األَاالبزَم. فٍ االسخُخاخاث: أظهزث واَخ فاظا يعُىَا فٍ يسخىي انحايِط انبىن ٍِ فٍ يصم انذو نذي يزظً إرحفاع ظغػ انذوَّ وانذٍَ نذَهى هذِ انذراسِت أٌ هُاك ارحفاعا يعُىَا يهحىظا فٍ يسخىي انحايِط انبىن يهحىظ فٍ يسخىي اَخفاض يعُىٌإنً يِت األَعُِت. أدي انعالج بعقار انهىسارحاٌ نكٍ نََُس انعالَج بعقار األَاالبزَمعالياُث انًخالس َُْطَزة عهً ظغِػ انذّو ونخَخفُط يسخىي انحايِط ٌَ انهىسارحاٌ عالّخاً يفُذاً نهَس ٌْ ََُكى ٍُ أَ ٍِ فٍ يصم انذو. ًَُك انبىنٍ فٍ انحايِط انبىن ٍ يزظً إرحفاع ظغػ انذوِّ وانذٍَ نذَهى عالياُث انًخالسيِت األَعُِت وفزغ انحايط انبىنٍ فٍ انذو.يصم انذو ف introduction some investigators have suggested that uric acid plays a causal role in the development of cardiovascular disease (1) whereas others have concluded that uric acid merely reflects other concomitant risk factors, such as hypertension, insulin resistance, obesity, or lipid abnormality (2) . elevated serum uric acid concentrations are also found in healthy offspring of parents with coronary heart disease, indicating a possible causal relationship (3) . krishnan et al (4) demonstrating that hyperuricemia increases the risk of developing hypertension by approximately 80%, independent of baseline blood pressure measurements, renal function, serum lipid levels, body mass index, proteinuria, alcohol use, and age. johnson et al (5) reported that elevated uric acid level was observed in 40% to 60% of hypertensive subjects; similarly, hypertension was observed in 50% to 65% of subjects with gout. johnson et al (6) reported that hyperuricemia was observed in 25% of treated hypertensive subjects, 50% of those without treatment, and 75% to 100% of those with malignant hypertension or renal dysfunction. # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : zeinaalkazaz@yahoo.com received : 7/3/2011 accepted : 19/7/2011 iraqi j pharm sci, vol.20(2) 2011 losartan versus enalapril on serum uric acid levels 76 serum uric acid (sua) levels are often increased in subjects with ms. however, none of the proposed sets of diagnostic criteria include sua levels in the definition of ms (7,8) . in 2001, the national cholesterol education program adult treatment panel iii (ncep atp iii) published the most widely used set of diagnostic criteria. these criteria include elevated plasma triglyceride (trg) levels (≥150 mg/dl[1.69 mmol/l]), decreased levels of high-density lipoprotein cholesterol (hdl-c) (<40 mg/dl[1.04 mmol/l] in men and <50 mg/dl[1.29 mmol/l] in women), elevated blood pressure (bp)(≥130/85 mm hg), increased fasting plasma glucose levels (≥ 110 mg/dl [6.1 mmol/l]), and abdominal obesity (waist circumference >102 cm in men and >88 cm in women) (9) . it is possible that the increased cardiovascular disease risk associated with the ms is partially attributed to elevated circulating sua concentration (7,8) . large epidemiologic studies demonstrated that the prevalence of ms showed a graded increase according to sua levels (10-12) . moreover, sua concentration was positively correlated with blood pressure (bp), body mass index, levels of fasting plasma glucose, triglycerides, highsensitivity c-reactive protein, and inversely correlated with high density lipoprotein cholesterol levels (hdl-c) (8) . many drugs have hypouricaemic properties, in addition to their main therapeutic effects. the oral weight loss agent sibutramine decreases serum uric acid in obese patients by 20% to 25% (13) . similarly, in patients with type 2 diabetes and hyperuricemia, the insulin sensitizing agent troglitazone lowers serum uric acid by 20% to 25% (14) . ramipril was found to increase the excretion of uric acid in a number of hypertensive patients (15) . the present study was conducted to investigate the effects of losartan compared with enalapril on uric acid levels in hypertensive patient having markers of metabolic syndrome. patients and methods one hundred and twenty six hypertensive patients having markers of metabolic syndrome participated in this study. they were divided into two groups according to the type of the drug taken. group 1 was given losartan (angizar 50mg, micro pharmaceutical industries, co. ltd., india) in doses of 50mg daily. they are 28 males and 32 females, with a mean age of 56.68±6.32 years. group 2 received enalapril (enalapril 20mg, asia pharmaceutical industries, co. ltd., alepposyria) in doses of 20 mg once daily. they are 30 males and 36 females with mean ages of 52.80±7.23 years. another 70 healthy, non obese, normotensive individuals, age and gender matching with study patients, participated in the study as a control group. they were 34 males and 36 females, with mean age of 53.51±6.66 years. this open 2-month, controlled, comparative clinical trial was conducted on hypertensive patients having markers of metabolic syndrome who were seen at ibn-sina teaching hospital in mosul, iraq. the study protocol was approved by the ethics committees of the college of medicine and mosul health administration. non-diabetic patients with mild hypertension (stage 1: systolic 140 159 mmhg and diastolic 90 99 mmhg) (16) , who met the diagnostic criteria of metabolic syndrome according to the american national cholesterol education program-adult treatment panel iii (ncepatp iii) (9) were included in this study. those with hepatic or renal diseases, pregnancy and lactation, hypertensive patients on antihypertensive therapy, hypersensitive patients on losartan or enalapril, gouty patients and inflammatory diseases such as rheumatoid arthritis were excluded. markers of metabolic syndrome including, waist circumference, blood pressure, serum glucose concentration, triglycerides, and hdl-cholesterol were determined before and at the end of study period. the presence of 3 or more of such markers indicates metabolic syndrome state. blood pressure was measured by standard mercury sphygmomanometer. goal bp after treatment was less than 140/ 90 mmhg. serum glucose concentration, total cholesterol, triglycerides, and hdl-cholesterol were measured by using special kits. ldlcholesterol was calculated from friedewald equation (17) . serum uric acid concentration was measured at baseline and after 2 months therapy with losartan or enalapril by enzymatic method using a kit supplied by biolabo laboratories (france). statistical methods standard statistical methods were used to determine the mean and standard deviation (sd). paired student t-test was used to compare the results between before and after drug therapy. unpaired t-test was used to compare the results of cases before and after losartan or enalapril therapy with control and to compare the results between losartan and enalapril treated groups.. the statistical results were considered significant at p=0.05 or less. iraqi j pharm sci, vol.20(2) 2011 losartan versus enalapril on serum uric acid levels 77 results baseline measurement of waist circumference, body mass index (bmi) , blood pressure, serum glucose concentrations and lipid profile of the patient's groups showed a significant elevation as compared with the control group, while hdl-cholesterol showed lowered values as compared with the controls ( p<0.001) (table.1). baseline uric acid levels were 306.69±67.72 µmol/l for losartan group and 302.94±56.86 µmol/l for enalapril group which showed a significant elevation (p<0.001) as compared with the control (284.95±76.52 µmol/l) (table.1 and table.3) respectively. comparison of uric acid levels before and after 2 months of therapy by each drug showed a significant reduction in losartan group (p<0.001) (table .2) but not in enalapril group (p=0.123) (table .4). comparison of uric acid levels between losartan group and enalapril group showed a significant reduction in the losartan group (p<0.001) as compared with the enalapril group (table .5). table 1: comparison of data between control and losartan group ( before and after therapy). parameters mean  sd control (n=70) before (n=60) after (n=60) bmi (kg/m 2 ) 22.2  1.8 33.46  2.08*** 30.95  1.8*** waist circum. (cm) 83.95  6.2 106.79  8.53*** 104.08  8.3*** sbp (mm hg) 127.05  6.93 143.60  7.72*** 136.82  8.4*** dbp (mm hg) 79.24  4.91 92.18  6.21*** 83.92  6.3*** fsg (mmol/l) 4.75  0.9 6.6  0.4*** 5.12  0.7*** total-c (mmol/l) 4.45  0.63 5.28  0.74*** 4.65  0.8*** tg (mmol/l) 1.48  0.6 1.67  0.37 1.23  0.5* hdl-c (mmol/l) 1.60  0.28 1.32  0.32*** 1.54  0.4*** ldl-c (mmol/l) 2.20  0.70 3.20  0.67*** 2.84  0.8*** uric acid ( mol/l) 284.95  76.52 306.69  67.72*** 275.92  61.63*** * significant difference from control at p<0.05, ** at p<0.01 and *** at p<0.001 using unpaired t-test. table 2 : comparison of the effects of losartan before and after therapy . parameters mean  sd before (n=60) after (n=60) p-value bmi (kg/m 2 ) 33.46  2.08 30.95  1.8*** <0.001 waist circum. (cm) 106.79  8.53 104.08  8.3*** <0.001 sbp (mm hg) 143.60  7.72 136.82  8.4*** <0.001 dbp (mm hg) 92.18  6.21 83.92  6.3*** <0.001 fsg (mmol/l) 6.6  0.4 5.12  0.7*** <0.001 total-c (mmol/l) 5.28  0.74 4.65  0.8 0.135(ns) tg (mmol/l) 1.67  0.37 1.23  0.5 0.240(ns) hdl-c (mmol/l) 1.32  0.32 1.54  0.4*** <0.001 ldl-c (mmol/l) 3.20  0.67 2.84  0.8 0.098(ns) uric acid ( mol/l) 306.69  67.72 275.92  61.63*** <0.001 ***significant difference at p<0.001 using paired t-test. ns= not significant. iraqi j pharm sci, vol.20(2) 2011 losartan versus enalapril on serum uric acid levels 78 table 3:comparison of data between control and enalapril group ( before and after therapy). parameters mean  sd control (n=70) before (n=66) after (n=66) bmi (kg/m 2 ) 22.2  1.8 32.79  1.9*** 30.6  2.18*** waist circum. (cm) 83.95  6.2 103.44  8.87*** 100.8  8.53*** sbp (mm hg) 127.05  6.93 145.78  5.39*** 136.95  7.58*** dbp (mm hg) 79.24  4.91 91.44  6.15*** 86.07  5.0*** fsg (mmol/l) 4.75  0.9 6.55  0.38*** 5.35  0.66*** total-c (mmol/l) 4.45  0.63 5. 40  0.93*** 5.42  0.76*** tg (mmol/l) 1.48  0.6 1.36  0.60 1.2  0.51* hdl-c (mmol/l) 1.60  0.28 1.40  0.3*** 1.57  0.32*** ldl-c (mmol/l) 2.20  0.70 3.26  0.72*** 3.27  0.99*** uric acid ( mol/l) 284.95  76.52 302.94  56.86*** 289.99  50.28*** * significant difference from control at p<0.05, ** at p<0.01 and *** at p<0.001 using unpaired t-test table 4 : comparison of the effects of enalapril before and after therapy . parameters mean  sd before (n=60) after (n=60) p-value bmi (kg/m 2 ) 32.79  1.9 30.6  2.18*** <0.001 waist circum. (cm) 103.44  8.87 100.8  8.53*** <0.001 sbp (mm hg) 145.78  5.39 136.95  7.58*** <0.001 dbp (mm hg) 91.44  6.15 86.07  5.0*** <0.001 fsg (mmol/l) 6.55  0.38 5.35  0.66*** <0.001 total-c (mmol/l) 5. 40  0.93 5.42  0.76 0.205(ns) tg (mmol/l) 1.36  0.60 1.2  0.51 0.193(ns) hdl-c (mmol/l) 1.40  0.3 1.57  0.32 0.178(ns) ldl-c (mmol/l) 3.26  0.72 3.27  0.99 0.716(ns) uric acid ( mol/l) 302.94  56.86 289.99  50.28 0.132(ns) ***significant difference at p<0.001 using paired t-test. ns= not significant. table 5: comparison of data after losartan and enalapril therapy. parameters mean  sd losartan (n=60) enalapril (n=66) p-value bmi (kg/m 2 ) 30.95  1.8 30.6  2.18 0.026 (ns) waist circum. (cm) 104.08  8.3 100.8  8.53 0.605(ns) sbp (mm hg) 136.82  8.4 136.95  7.58 0.134 (ns) dbp (mm hg) 83.92  6.3 86.07  5.0* 0.05 fsg (mmol/l) 5.12  0.7 5.35  0.66* 0.05 total-c (mmol/l) 4.65  0.8 5.42  0.76 0.120(ns) tg (mmol/l) 1.23  0.5 1.2  0.51 0.321(ns) hdl-c (mmol/l) 1.54  0.4 1.57  0.32 0.062(ns) ldl-c (mmol/l) 2.84  0.8 3.27  0.99 0.126(ns) uric acid ( mol/l) 275.92  61.63 289.99  50.28*** <0.001 * significant difference at p<0.05 and *** at p<0.001. ns= not significant. iraqi j pharm sci, vol.20(2) 2011 losartan versus enalapril on serum uric acid levels 79 discussion the present study demonstrates significantly higher uric acid levels in subjects with metabolic syndrome as compared with the control group. these results are in consistent with the results obtained from many articles which also demonstrate increased levels of uric acid in patients with metabolic syndrome (8, 12, 18) . several mechanisms were attributed to the increase of ua levels in ms. one of these mechanisms is related to insulin resistance, which is accompanied by ms. proximal tubular reabsorption of ua occurs by an active transport mechanism closely linked to or identical with the tubular reabsorption of sodium. insulin can enhance renal tubular sodium reabsorption in humans. furthermore, renal excretion of ua is reduced in situations with increased renal tubular reabsorption of sodium . this relationship suggests an altered tubular sodium handling and uric acid metabolism which is constituent with hyperinsulinemia. insulin resistance being the possible pathophysiological link (19) . another mechanism for the increased sua levels in ms is that ms is associated with increased oxidative stress (20) . because uric acid is considered to be an effective antioxidant. the elevated sua levels encountered in individuals with ms may reflect a compensatory mechanism counteracting the increased oxidative stress associated with the ms (21) .in the present study, only losartan causes a significant reduction of serum uric acid concentrations in patients with metabolic syndrome after 2 months of therapy. these results indicate that losartan have uricosuric effects. many studies have demonstrated that the uricosuric effect of losartan was due to the parent compound and not to the active metabolite exp 3174 and that this effect is independent of angiotensin ii receptor blockade and is due to unique biochemical properties of losartan (22-24) . the hypouricemic effect of losartan may be due to that losartan target the urate anion exchange and diminish urate reabsorption in the proximal convoluted tubule; as a result, the urate excretion fraction is increased by 13%-30% and increases renal uric acid excretion (25) . this aspect of losartan therapy might have therapeutic advantages by reducing the risk of elevated uric acid in patient with ms, since elevated serum uric acid levels in patient with ms is regarded as a risk factor for the development of cv diseases (26) and may ameliorate hyperuricemia induced by other drugs. it was reported that the risk of death due to ischemic heart disease increased by 77% (men), and by 30% (women) when serum uric acid levels where in the highest quartile (>416 µmol/l) compared with the lowest quartile (<321 µmol/l) (27) . data obtained from the present study showed that enalapril produce non significant effects on uric acid concentration in patients with metabolic syndrome. data from the literature demonstrates different results. no effect was reported by tikkanen et al., (28) , rise in sua levels reported by de rosa et al (29) , and and others demonstrates sua lowering effect (8, 30) . in conclusion: this study demonstrates significantly higher serum uric acid levels in hypertensive patients having markers of metabolic syndrome. losartan therapy but not enalapril therapy produced a significant fall in serum uric acid levels. losartan can be a useful therapeutic agent to reduce serum uric acid level in hypertensive patients having markers of metabolic syndrome and hyperuricaemia. references 1. torun m, yardim s, simsek b, burgaz s. serum uric acid levels in cardiovascular diseases. j clin pharm ther 1998; 23: 2529. 2. tatli e, aktoz m, buyuklu m, altun a. the relationship between coronary artery disease and uric acid levels in young patients with acute myocardial infarction. cardiol j 2008; 15: 21-25. 3. ronora f, targher c, zenere mb, saggiani f, cacciatori v, tosi f, travia d, zenti mc, branzi p, santi i, muggeo m. relationship of uric acid concentration to cardiovascular risk factors in young men. role of obesity and central fat distribution. int j obest relat met disord 1996; 20: 975-980. 4. krishnan e, kwoh ck, schumacher hr, kuller l. hyperuricemia and incidence of hypertension among men without metabolic syndrome. hypertension 2007; 49: 298-303. 5. johnson rj, feig di, herrera acosta j, kanog dh. resurrection of uric acid as a causal risk factor in essential hypertension.hypertension2005;45:18-20. 6. johnson rj, segal ms, srinivas t. essential hypertension, progressive renal disease, and uric acid: a pathogenetic link? j am soc nephrol 2005; 16:19091919. 7. hoieggen a, alderman mh, kjeldsen se, julius s, devereux rb, defaire u, et al. the impact of serum uric acid on cardiovascular outcomes in the life study. kidney int 2004; 65: 1041-1049. 8. tsouli sg, liberropoulos en, mikhailidis dp, athyros vg, elisaf ms. elevated serum uric acid levels in metabolic iraqi j pharm sci, vol.20(2) 2011 losartan versus enalapril on serum uric acid levels 80 syndrome: an active componenet or an innocent bystander. metab clin and exper 2006; 55: 1293-1301. 9. national cholesterol education programadult treatment panel iii. executive summary of the third report (ncep-atp iii) (2001). 10. conen d, wietlisbach v, bovet p, shamlaye c, riesen w, paccaud e, et al. prevalence of hyperuricemia and relation of serum uric acid with cardiovascular risk factors in a developing country. bmc public health 2004; 4: 9-12. 11. yoo tw, sung kc, shin hs, kim bj, kim bs, kang jh, et al. relationship between serum uric acid concentration and insulin resistance and metabolic syndrome. circulation 2005; 69: 928-933. 12. lee je, kim yg, choi yh, huh w, kim dj, oh hy. serum uric acid is associated with microalbuminuria in prehypertension. hypertension 2006; 47: 962-967. 13. mcmahon fg, fujioka k, singh bn, mendel cm, rowe e, rolston k, et al. efficacy and safety of sibutramine in obese white and african american patients with hypertension: a 1-year, double-blind, placebo-controlled, multicenter trial. arch intern med 2000; 160: 2185-2191. 14. iwatani m, wasada t, katsumori k, watanabe-takahashi c, kamatani n, iwamoto y. troglitazone decreases serum uric acid concentrations in type ii diabetic patients and non diabetics. diabetologia 2000; 43: 814-815. 15. labeeuw m, pozet n, zech py, hadjaissa a, finaxz de villaine j, luville m. influence of acute administration of ramipril on the excretion of uric acid. arch mal coeur vaiss 1987; 80: 870-874. 16. the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (express). national heart, lung, and blood institute (jnc-7). nih publication 2003, no.03-5233: 25-32. 17. friedewald wt, levy ri, fredrickson ds. estimation of the concentration of ldl-c in plasma without use of preparative ultracentrifuge. clin chem 1972; 18: 499502. 18. schmidt mi, duncan bb, watson rl, sharrett ar, brancati fl, heiss g. a metabolic syndrome in whites and african-americans. the atherosclerosis risk in communities baseline study. diabetes care1996; 19:414-418. 19. cappuccio fp, strazzullo p, farinaro e, trevisan m. uric acid metabolism and tubular sodium handling. results from a population-based study. jama 1993; 270; 354-359. 20. evans ji, maddux ba, goldfine id. the moleculer basis for oxidative stress induced insulin resistances . antioxid redox signal2005; 7:1040-1052. 21. nieto fj, iribarren c, gross md, comstock gw, cutler rg. uric acid and serum antioxidant capacity: a reaction to atherosclerosis?atherosclerosis 2000; 148:131-139. 22. liberopoulos en, christides d, elisaf m. comparative effects of losartan and irbesartan on serum uric acid in hypertensive patients with hyperuricemia and gout. j hypertens 2002; 20:347-351. 23. alderman m, aiyer kj. uric acid: role in cardiovascular disease and effects of losartan. curr med res opin 2004; 20:369-379. 24. rayner bl, trinder ya, baines d, isaacs s, opie lh. effect of losartan versus candesartan on uric acid, renal function, and fibrinogen in patients with hypertension and hyperuricemia associated with diuretics. am j hypertens2006; 19:208-213. 25. john db, mike ac. serum uric acidlowering therapies: where are we heading in management of hyperuricaemia and the potential role of uricase. curr rheum rep 2004; 6:240-247. 26. puddu pe, lanti m, menotti a, mancini m, zanchetti a, cirillo m, et al. serum uric acid for short-term prediction of cardiovascular disease incidence in the gubbio population study. gubbio study research group. acta cardiol 2001; 56:243-251. 27. fang j, alderman mh. serum uric acid and cardiovascular mortality. jama 2000; 283: 2404-2410. 28. tikkanen i, omvik p, jensen ha. comparison of the angiotensin ii antagonist losartan with the angiotensin converting enzyme inhibitor enalapril in patients with essential hypertension. j hypertens1995; 13:1343-1351. 29. de rosa m, cardace p, rossi m, baiano a, de cristofaro a. comparative effects of chronic ace inhibition and at1 receptor blocker losartan on cardiac hypertrophy and renal function in hypertensive patients. j hum hypertens 2002; 16:133140. 30. reyes aj. cardiovascular drugs and serum uric acid. cardiovasc drugs ther 2003; 17:397414. http://www.ncbi.nlm.nih.gov/pubmed/8732701?ordinalpos=3&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/8732701?ordinalpos=3&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/8732701?ordinalpos=3&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/8732701?ordinalpos=3&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/10580179?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/10580179?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/10580179?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum 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http://www.ncbi.nlm.nih.gov/pubmed/15025846?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22rayner%20bl%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22trinder%20ya%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22baines%20d%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22isaacs%20s%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22isaacs%20s%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22opie%20lh%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'am%20j%20hypertens.'); javascript:al_get(this,%20'jour',%20'am%20j%20hypertens.'); http://www.ncbi.nlm.nih.gov/pubmed/11573830?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/11573830?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/11573830?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/11573830?ordinalpos=2&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_defaultreportpanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22tikkanen%20i%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22omvik%20p%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=%22jensen%20ha%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_discoverypanel.pubmed_rvabstractplus javascript:al_get(this,%20'jour',%20'j%20hypertens.'); javascript:al_get(this,%20'jour',%20'j%20hypertens.'); hand hygiene has been considered to be the most important tool iraqi j pharm sci , vol.18 (1) , 2009 microorganisms from hospital soaps 28 isolation of some microorganisms from bar soaps and liquid soaps in hospital environments sarmad m.h. zeiny *,1 * department of microbiology, college of medicine, university of baghdad, baghdad , iraq abstract this study was designed to determine the colonization of the in-use hand washing soaps in hospital settings. it is a comparative cross-sectional research in a surgical specialties and baghdad teaching hospital in baghdad, iraq. swabs from surfaces of bar soaps and from liquid soaps via their applicator tips; at the sinks of toilets of hospital staff and working rooms of the wards were taken in january 2008. conventional microbiologic methods were used for culture of the swabs and identification of the isolates. colonization was detected 60% and 15.9% in bars and liquid forms respectively. and this lead to the conclusion that bar soaps could be colonized with microorganisms excessively. liquid hand washing soaps are more appropriate in hospital environments. proper using conditions of the hand washing items should be defined in health care settings. keywords: bar soap, liquid soap, pseudomonas aeruginosa, nosocomial infections. الخالصة حيت إنٗ انًزيض. حهٕد انيذ بانبكخزيا يؼخبز يٍ أْى انطزق الَخقال انؼذٖٔ بيٍ انًزضٗ أٔ يٍ انؼايهيٍ في يجال انزػايت انص ْذفج ْذِ انذراست نخحذيذ بؼض اإلحياء انًجٓزيت انًسخؼًزة َظافت انيذ حؼخبز أْى أداة في انسيطزة ػهٗ حاالث انؼذٖٔ انًسخشفٕيّ. صًًج ْذِ انذراست ػهٗ شكم يقارَت يقطؼيت ٔحى جًغ يسحاث يٍ أسطح انصابٌٕ ػهٗ إَٔاع انصابٌٕ انًسخخذو في انًسخشفياث. أظٓزث هب ٔيٍ فخحاث جٓاس اإلػطاء نهصابٌٕ انسائم في يسخشفٗ بغذاد انخؼهيًي ٔ انجزاحاث انخخصصيت في بغذاد, انؼزاق.انص % نهصابٌٕ انسائم. ْذِ انُخائج حؤكذ أٌ اسخؼًال انصابٌٕ 1..9% بيًُا كاَج 06انُخائج أٌ َسبت حهٕد انصابٌٕ انصهب كاَج بٌٕ انصهب.انسائم بانًسخشفياث اَسب يٍ انصا introduction hand carriage of bacteria is an important route of transmission of infection between patients or from the health care worker to the patient. 1-6 hand hygiene has been considered to be the most important tool in nosocomial infections control. failure to perform appropriate hand hygiene is supposed to be the leading cause of nosocomial infections and the spread of multiresistant microorganisms, and has been recognized as a significant contributor to outbreaks. the microbial flora of the skin of hands consists of resident and transient microorganisms. the resident microorganisms survive and multiply on the skin. the transient microorganisms represent recent contaminants of the hands acquired from colonized or infected patients/clients or contaminated environment or equipment. transient microorganisms are not consistently isolated from most persons. in contrast to the resident microorganisms, the transient microorganisms found on the hands of health care personnel are more frequently implicated as the source of nosocomial infections. the most common transient microorganisms include gram negative coliforms and staphylococcus aureus. hand washing with plain soap is effective in removing most transient microrganisms. 7-9 the mechanical action of washing and rinsing removes most of the transient microorganism present. 1012 .health care workers wash their hands in two ways: (a) the social hand wash, which is the cleaning of hands with plain, non-medicated bar or liquid soap and water for removal of dirt, soil, and various organic substances; (b) the hygienic or antiseptic hand wash, which is the cleaning of hands with antimicrobial or medicated soap and water. most antimicrobial soaps contain a single active agent and are usually available as liquid preparations. appropriate hand washing results in a reduced incidence of both nosocomial and community infections. 13 much studies have been written and debated regarding the use of bar versus liquid skin cleansers in relation to infection control. 7,14-22 in this study, the aim was to detect and compare bacterial contamination of soap bars and liquid soaps. 1 corresponding author e-mail : smzeiny2002@yahoo.com received : 9/11/2008 accepted : 22 / 2/2009 iraqi j pharm sci , vol.18 (1) , 2009 microorganisms from hospital soaps 29 materials and methods setting surgical specialties and baghdad teaching hospital in baghdad, at the middle of iraq. materials in january 2008, 50 swabs from surfaces of bar soaps at the sinks of toilets and working rooms of the wards were collected; 44 swabs were collected from tips of the applicators of liquid soaps containers approximately at the same hospital points. swabs were collected from wet surfaces of bars and tip of the containers of liquid soaps. soap bars were plain soaps (duru, turkey). liquid soaps (johnson, turkey) included formaldehyde (in trace amount as antiseptic agent according to label information). despite of the liquid soaps in this settings were called antibacterial by the manufacturer; formaldehyde that was included in the liquid soaps considered as preservative rather than antibacterial effect. microbiology collected swabs were dipped into tubes containing 1 ml sterile normal saline (0.9%). samples were brought to the microbiology laboratory without delay. tubes were shaked and ten microliter of substance was inoculated on blood agar and eosin-methylen-blue (emb) agar incubated at 37 0 c for 22-24h. sabouraud`s agar media are enforced with chloramphenicol (16 µg/ml) to inhibit the growth of contaminating bacteria; incubated at 30c˚, for 30 days were used to rule out fungi . unfortunately anaerobic laboratory conditions could not be accomplished during this study due to the shortage in laboratory facilities. yielded microorganisms were identified by conventional microbiological methods and by using api 20e, api 20ne & api strep (biomerieux, usa). statistics t-tests were used for calculating significance of difference of colonization rates between bar soaps and liquid soaps and also comparing the frequency of yielding microorganisms. results among 50 swabs of bar soaps, 30 (60%) swabs were found colonized. a total of 44 microorganisms were isolated. numbers of isolate are shown in figure 1. pseudomonas aeruginosa (41%) was the most frequent isolated bacteria followed by escherichia coli (13.6%) and acinetobacter baumanii (11.4%).from liquid soaps, 6 microorganisms were detected at only 7 tips (15.9%) of the total 44 containers. this includes 4 (66.6%) p. aeruginosa, one (16.6%) proteus penneri and one (16.6%) flavimonas oryzihabitans. comparison of the rates of bacterial colonization between bar soaps and liquid soaps are shown in figure 2 and 3.bar soaps were found more colonized than the liquid soaps significantly (p<0.05). p. aeruginosa was the most frequent isolate in both two group whereas isolation rate was significantly higher (p<0.05) in bar soaps but not in the liquid soaps (p>0.05) as statistically. iraqi j pharm sci , vol.18 (1) , 2009 microorganisms from hospital soaps 30 figure 1. distribution of bacterial agents colonizing on the soaps figure 2. bacterial colonization rates of bar soaps figure 3. bacterial colonization rates of liquid soaps 4 18 6 5 2 2 1 1 1 1 1 1 1 1 0 2 4 6 8 10 12 14 16 18 numbers p.aeruginosa e.coli a.baumanii e.aerogenes e.cloaca s.aureus p.penneri c.idolegenens f.brevis candida diphtheroid f.oryzihabitans a g e n t s distrution of bacterial agents colonizing on the soaps bar soap liguid soap bacterila grow th, 60% no grow th, 40% bacterila growth no growth bacterila grow th, 16% no grow th, 84% bacterila growth no growth iraqi j pharm sci , vol.18 (1) , 2009 microorganisms from hospital soaps 31 discussion the most common hand-cleaning agents are bar soap and liquid soap in disposable plastic containers. when in use, bar soaps are frequently misused because they are typically stored in contact with moisture and remain moist for long periods of time. it is usually kept in a container, on or next to a wash basin. more often than not, it resides in surface water. the resulting jelly mass is unsightly, difficult to use effectively. this supplies an environment which provides the perfect opportunity for bacteria and organisms to grow. most bars of soap in communal areas are used by a number of different people. this means that one bar of soap can be in direct contact with skin bacteria from more than one person, and may harbour live pathogenic bacteria. 22 cross infection can and does occur under these circumstances. 23 when using a bar of soap, the cdc (centre for disease control) recommends placement on a drainable rack between uses. 6 soap racks that promote drainage of all water from the bar should be installed. in addition, there should be easy access to replacements when soap is lost, dropped, melted, or consumed. small soap bars were also recommended that can be changed and used in preference to larger bars that are more likely to melt or become colonized with bacteria. 24 liquid soap on the other hand is much better to use. liquid soap is dispensed straight from a plastic container. it has not been exposed to skin bacteria or other contaminants. as a result, cross contamination is not likely to occur, providing a more cleaning and more hygienic alternative. 23 mcbride et al reported that bar soaps were found to have higher bacterial cultures after use than liquid soaps. 23 in another study, kabara and brady obtained samples from bar and liquid soaps from 26 public bathrooms which were investigated. liquid soaps were found to be negative for bacteria, while 100% of the 84 samples obtained from bar soaps yielded positive cultures. 15 in an epidemiological study, the researchers isolated several strains of pseudomonas from 45 of 353 environmental samples used by multiple providers (13%) and found that the 5 most common strains were frequently found on patients. they also affirmed that the hands are a major vehicle for the transfer of pseudomonas bacteria and implicated bar soap in its spread. 14 other groups of researchers have found that bacteria survive on soap bars in continuous use in public lavatories, even when cultured 48 hours following their last use. 15,22 the role of the soap dish in infection control has also been studied. despite of cdc recommendations most health care settings like our hospital are using soap dishes instead of drainable racks. jarvis et al showed that supplies used for hand washing can be contaminated with gramnegative organisms if they are not completely dried. swabs were collected from soap dishes on 6 wards and from a bacteriology laboratory on 4 consecutive days. the sludge of the dish was found to be colonized with predominantly gram-negative bacteria. this colonization persisted, even when medicated iodophor bar soap was used. 25 in our study, dishes were found wet, and surfaces of soaps were generally covered by squashy mass and bars were found heavily contaminated (%88). this study revealed quite lower contamination rate in liquid soaps compared with bar soaps, although they didn’t include suggested antibacterial agents for hand antisepsis such as triclorasan or chlorhexidine. however, liquid soaps would be expected to be sterile. so, there should be problems with the handling. honestly, in this study any strict procedures had not been followed in the wards for the how often liquid dispensers should be cleaned, disinfected or exchanged. after the results were obtained, procedures were described for handling and usage of liquid soaps and dispensers immediately. in conclusion, correct use of hand washing materials is more important choosing kind of soaps. hypothesis of transferring microorganisms to healthcare workers’ hands via contaminated soap bars have not confirmed, antibacterial or not, liquid soaps seem more suitable alternative for hygienic hand washing. proper handling of liquid soap should be implemented wherever they are used in the hospital. compliance of the hand washing is more important than the kind of the soap. references 1. larson e. a causal link between hand washing and risk of infection? examination of the evidence. infect control hosp epidemiol 1988; 9:28-36. 2. larson e, mayur k, laughon ba. influence of two handwashing frequencies on reduction in colonizing flora with three handwashing products used by health care personnel. am j infect control 1989; 17:83-88. 3. nystrom b. impact of handwashing on mortality in intensive care: examination of the evidence. infect control hosp epidemiol 1994;15:435-436. 4. gruendemann bj, larson el. antisepsis in current practice. in: rutala wa, ed. iraqi j pharm sci , vol.18 (1) , 2009 microorganisms from hospital soaps 32 disinfection, sterilization and antisepsis in health care. washington, dc: association for professionals in infection control and epidemiology, inc and polyscience publications, inc, 1998:183-195. 5. larson el. hand washing and skin preparation for invasive procedures. in: olmsted rn, ed. apic infection control and applied epidemiology principles and practice. st. louis: mosby, 1996:chapter 19. 6. boyce jm, pittet d. guideline for hand hygiene in health-care settings. morbidity and mortality weekly report 2002; 5:1– 48. 7. larson el, apic guidelines committee. apic guideline for hand washing and hand antisepsis in health care settings. am j infect control 1995; 23:251-269. 8. reybrouck g. handwashing and hand disinfection. j hosp infect 1986; 8:23. 9. bettin k, clabots c, mathie p et al. effectiveness of liquid soap vs chlorhexidine gluconate for the removal of clostridium difficile from bare hands and gloved hands. infect control hosp epidemiol 1994; 15:697-702. 10. gould d, chamberlain a. gram-negative bacteria. the challenge of preventing cross-infection in hospital wards: a review of the literature. j clin nurs 1994; 3:330345. 11. gould d. the significance of hand-drying in the prevention of infection. nurs times 1994; 90:33-35 12. noskin ga, stosor v, cooper i et al. recovery of vancomycin-resistant enterococci on fingertips and environmental surfaces. infect control hosp epidemiol 1995;16:577-581 13. kampf g, kramer a. epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs. clin microbiol rev 2004; 17:863-893. 14. bruun ds, mcgarrlty gj, blakemore ws, coniell ll. epidemiology of pseudomonas aeruginosa infections: determination of pyocin typing. j clin microbial 1976; 3:264-271. 15. kabara jj, brady mb. contamination of bar soaps under 'in use' conditions. j environ pathol toxicol oncol 1984: 5:114 16. kabara j. bar soap and liquid soap. j am med assoc 1985; 253:1560-1561. 17. skewes s. skin care rituals that do more harm than good. am j nurs 1996; 96:3335. 18. gilmore ds, aeilts gd, aeilts ba. bruce sk, jlmenez em, schick dg, et al. effects of bathing pseudomonas and klebsiella colonization in patients with spinal cord injuries. j clin microbial 1981; 14:404-407. 19. heinze je. bar soap and liquid soap. j am med assoc 1985; 251:3222-3223. 20. heinze je. the safety of bar soap. j environ pathol toxicol oncol 1986; 6:5964. 21. heinze je, yackovich f. washing with contaminated bar soap is unlikely to transfer bacteria. epidemiol infect 1988; 101:135-142. 22. larson el, eke p1, wilder mp, laughon be. quantity of soap as a variable in hand washing. infect control 1987; 8:371-375 23. mcbride me. microbial flora of in-use soap products. appl environ microbiol 1984; 48: 338-341. 24. nix dh. wound care: factors to consider when selecting skin cleansing products. j wocn 2000; 27:260-268. 25. jarvis jd, wynne cd, enright l, williams lid. handwashing and antiseptic-containing soaps in hospital. j clin pathol 1979; 32:73. microsoft word bagpha05114 _4_ iraqi j. parma. sci., vol.14, 2005 31 -------------------------------------------------------------------------------------------------------------- magnesium, zinc, and copper in serum, erythrocyte, urine and dialyzate fluids of haemodialysis patients sami, n. najem.** department of biochemistry .college of medicine al-anbar university. (to whom correspondence should be addressed) ** department of biology, college of science, al-anbar university abstract: copper (cu) zinc (zn) and magnesium (mg) in serum, rbc, urine and dialyzate fluids were studied in 39 patients, who have been undergoing chronic haemodialysis treatment. they were divided in to polyuric , oliguric and anuric depending on their urinary output. elevated serum and rbc mg was observed before dialysis, while decreased serum and rbc level was noticed except serum mg of polyuric patients. before dialysis elevated serum and rbc zn were observed. while after dialysis these parameters were increased. normal rbc cu value before dialysis was observed. while low serum cu was noticed. after dialysis serum cu showed raised value, while rbc level decreased in oliguric and increased in polyuric patients. zn / cu ratio found to be high in those patients. all these results were discussed in relation to urine content and also to the dialyzate fluid. key words: trace elements, haemodialysis, renal failure :الخـالصـة لنحاس في مصل وكریات الدم الحمر وإدرار ومحلول الغسیل الناتج ل�دى تس�عة قیست تراكیز المغنیسیوم والخارصین وا رظھ, اإلدرارغزیري وقلیلي وعدیمي المطروحة إلىحسب كمیة اإلدرار واللذین قسمواوثالثین مریضا عولجوا باإلنفاذ الدموي غزی�ري ع�دا مص�ل ,بع�دھا هانخف�ض تركی�ز الدم الحمر قب�ل المعالج�ة ف�ي ح�ین مصل وكریاتارتفاع في تركیز المغنیسیوم في وبقي تركیز النحاس طبیعیا في كریات الدم الحمر ,المعالجةارتفع تركیز الزنك في المصل وكریات الدم الحمر قبل وبعد ,اإلدرار قلیل�ي كری�ات ال�دم الحم�ر ف�ي مرض�ى انخف�ض ف�ي وارتفع بع�د المعالج�ة بینم�ا ,المصلفي هقبل المعالجة في حین انخفض تركیز نوقش�ت جمی�ع النت�ائج عل�ى .المرض�ى في جمی�ع وارتفعت نسبة الزنك إلى النحاس ,اإلدرارمرضى غزیري وارتفع لدى ,اإلدرار .العناصرضوء محتوى اإلدرار ومحلول الغسیل من ھذه iraqi j. parma. sci., vol.14, 2005 32 -------------------------------------------------------------------------------------------------------------- introduction: in most dialysis units, little attention was given to the trace element contents of renal dialysis fluids or to the possibility of contamination of such fluids with trace metals .a number of considerations suggest that trace element disturbances might occur in dialyzed patients, must at least in part be ascribed to the dialysis treatments itself during which these constituents may either be transferred to or removed from the patients (1). isolated cases of acute copper and zinc toxicity have occurred in haemodialysis patients as results of contamination of dialysis equipments or fluids with these metals (2). other reported hypozincemia in haemodialysis patients (3). previous study reported elevated silicon and aluminum in those patients (4) . the present study was intended to clarify levels of serum zinc (zn), copper (cu), and magnesium (mg), according to the patient’s urine output, erythrocytes zn, cu, and mg and comparing them with control group. also studying levels of these elements in the patient urine and the dialyzate. patients and methods: subjects: thirty-nine patients (25 males and 14 females), aged between 18 and 70 years, treated with haemodialysis (h.d), 17 were polyuric, 14 oliguric and 8 anuric, they were maintained with four hour session of (h.d). twenty-five normal individuals (15 males and 10 females) aged between 1864 years served as control group protocol: ten ml of venous blood was collected from each of the patients and control group. . serum was aspirated and kept in deep freeze (at –20 co) until the time of assay. serum zn, cu, and mg were measured using an atomic absorption spectroscopy (pu9100 x philips) by direct aspiration of the sample after being diluted with deionized water (1-10) for zn and cu, and (1-100) for mg (5) from the residual erythrocyte .cu, zn and mg were determined according to whitehouse et–al (6) urine cu, zn and mg were determined following the same methods as in serum. determination of these elements in the dialyzate performed in the same method with out dilution. the statistical comparison were made using t-test the probability less than 0.05 were considered significant. results: serum as shown in table 1. mg increased (p<0.05) in all patients decreased after treatments (p<0.01) in unuric patients, while it increased in olig and polyuric patients (p<0.01). table 1. serum mg, cu, zn and zn /cu before and after haemodialysis. before dialysis cases serum mg++ mean +sd mg/100ml serum cu++ mean +sd µmol/l serum zn++ mean +sd µmol/l s. zn++/s.cu++ mean +sd control n=25 1.6 +0.04 20.72 +3.22 14.3 +4.7 0.67 +0.20 anuric haemodialysis n=8 2.3** +0.6 19.68*** +9.87 27.64*** +6.79 1.42** +0.67 oliguric haemodialysis n=17 2** +0.02 17.98*** +13.67 23.12*** +8.48 1.29** +0.33 polyuric haemodialysis n=14 2.4** +0.6 16.54*** +7.78 24.12*** +5.77 1.45** +0.74 after dialysis anuric haemodialysis n=8 1.78* +0.3 39.86*** +29.83 38.08*** +9.87 0.95** +0.91 oliguric haemodialysis n=17 2.3* +0.6 23*** +6.77 24.13*** +8.61 1.04** +0.87 polyuric haemodialysis n=14 2.9*** +0.5 14.47*** +3.89 35.61*** +15.98 2.46** +1.52 iraqi j. parma. sci., vol.14, 2005 33 -------------------------------------------------------------------------------------------------------------- cu decreased significantlly (p<0.001) in all patients befor treatment, and increased (p<0.001) in unuric and oliguric patient but it decreased (p<0.05) in polyuric patients after treatments. zn found to be increased (p<0.001) in all patients befor and after treatment compaired with control group .zn/cu ratio increased (p<0.001) in all patients and after treatment it decreased (p<0.05) in oliguric and unuric patients, while it increased (p<0.05) in polyuric patients rbcs as shown in table 2. mg increased (p<0.05) in patint group before dialysis while decreased (p<0.01) in oliguric and unuric patients and in polyuric patients after treatment. (p<0.05). table 2.erythrocytes mg ,cu, zn and zn/cu before and after haemodialysis . before dialysis cases ery. mg++ mean +sd mg/100ml ery. zn++ mean +sd µmol/l ery. cu++ mean +sd µmol/l ery. zn++ /cu++ mean +sd control n=25 4.5 +0.51 1412 +230 119.4 +0 11.8 +7.2 anuric haemodialysis n=8 5.3** +0.28 1836*** +154 81.14*** +15.3 22.6*** +13.2 oliguric haemodialysis n=17 6.2** +0.38 1898*** +165 118* +40.3 16*** +9.5 polyuric haemodialysis n=14 5.8** +0.29 1433*** +199 111.2*** +30.5 12.8*** +5.4 after dialysis anuric haemodialysis n=8 5.6* +0.37 1965* +240 81.3 +15.5 24.16** +13.5 oliguric haemodialysis n=17 5.8* +0.25 1895* +157 114** +35.4 16.6* +12.6 polyuric haemodialysis n=14 3.3** +0.18 1543* +160 119.4** +45.9 12.9* +5.2 cu decreased (p<0.001) in unuric patients befor dialysis it increased (p0.05) after dialysis in polyuric patients. zn increased (p<0.001) in all patients befor dialysis and it increased (p<0.01) in all patients after dialysis. zn/cu ratio increased (p<0.001) in all patient befor dialysis, while increased in unuric (p<0.01) in olig and poly uric patients (p<0.001) after dialysis. urine table 3 showed that cu, mg and zn increased (p<0.001) in olig and polyuric compared with control group. table 3. mg, cu and zn levels in patients urine. cu ++  mol/day mean  sd zn ++  mol/day mean  sd mg ++ mmol/day mean  sd control n=25 3.51.2 15.67.2 0.30.1 polyuric patients 58.430.5 35.5615.9 4.90.95 oliguric patients 15.52.5 2912.3 183.977.5 iraqi j. parma. sci., vol.14, 2005 34 -------------------------------------------------------------------------------------------------------------- dialyzate solution dialyzate solution seems to be contained cu, zn and mg before treatment, the higher amount of cu found in unuric and polyuric patient after dialysis, while zn increased in un uric and oliguric , mg increased in polyuric patients only ( table4). table 4. mg, cu and zn levels in the dialyzate . cu ++  mol/l meansd zn ++  mol/l meansd mg ++ mmol/l meansd use solution for treatment 3.70.01 2.70.01 2.50.02 solution of unuric patients 20.05 1.10.02 0.30.01 solution of oliguric patients 0.60.02 10.02 0.40.03 solution of polyuric patients 1.30.2 0.90.25 0.430.1 discussion: there are many sources of disturbances in metal ions during chronic renal failure, some of them are impairment of kidney to execrete most trace elements, restricted protein intake, and dialysis technique itself which aggravates the condition by transferring to or removing trace element from them (7, 8) . the results showed variable increase in serum and rbc mg but still in the upper normal range (table 1, 2). this is in agreement with other studies (3,4). the increase in mg level may be due to the long use of phosphate binder medication or drinking water containing high mg concentration. the lowest value of mg appear in the oliguric patients could be due to the amount of mg excreted in their urine (table3). after dialysis serum and rbc mg levels were increased in oliguric and polyuric patients this could be explained in view of transferring mg from the dialyzate solution (table 4) which showed that the dialyzing solution of polyuric patients containing a high amount of mg comparing with the dialyzate from oliguric and anuric patients this also reported by other workers (3, 9). moreover, there have been reports on the deleterious effect of dialysis on the free radicals and lipid peroxidation that added to the suffering of those patients (10). this oxidative stress, particularly of the superoxid dismutase, directly affects some trace elements, particularly cu. in this study, we found normal erethrocytic cu (table 2), which may be due to intercellular binding with protein, and low serum cu before dialysis (table 1).or due to high urine cu (table 3). the lowest cu value found in serum of polyuric patients and the highest value found in their urine, while the increase in serum cu during dialysis could be explained by increase in the absorption of cu during dialysis session (3) {high level of cu was found in the dialyzate ( table4)}. or the redistribution of cu, which associated with cereluloplasmine (carrier protein). serum zn showed high values before dialysis (table 1). which confirms other reports (11, 12) while disagreed with others (3, 9). this might be due to low urinary excretion as shown in table (3). the lowest value of urinary zn found in oliguric patients while the highest was in polyuric patients, which reflects the variation in serum and rbc zn (tables 1, 2). zinc was not effectively lost through dialysis thus elevated serum zn was observed after treatments (table 1). also most serum zn is protein bound (13) it will not be in equilibrium with zn ion in the dialyzate. zn may cross from the dialyzate to the plasma even when total zn concentration in the latter is higher than the dialyzate concentration this finding reflects the highest amount of zn in the dialyzate of anuric patients table (4). this may be due to accumulation of this ion in patients serum and the use of a huge amount of contaminated water through dialysis. zn/cu ratio was found to be high (table 1). which has been suggested predisposing factor in cardiovascular diseases (14, 15) . along with other cardiovascular risk factors found in that patient (16) . they might be under high risk of cardiovascular diseases. so the correction of this ratio may affect positively in decreasing risk factors of cardiovascular diseases. iraqi j. parma. sci., vol.14, 2005 35 -------------------------------------------------------------------------------------------------------------- references: 1halsted, g. a. and smith, jr. j. c. plasma zinc in health and disease.lancet , 1970, 1 ,322 2petrie , j . j .and row , p . g. dialysis anemia caused by subacute zinc toxicity .lancet 1977, 1,1178 . 3mahler ,d.j . ;walsh , j. r. and haynie , g . d. magnesiume, zinc and copper in dialysis patients .amer.j. clin. path, 1971, 56,17-23. 4atia ,m . m .;al-hamdany, m .a.; hassan, m .j. and hammad , e . f. disturbance in serum silicon level in hemodialysis patients in ramadi.tanta medical journal, 1999, 27(1), 213-217. 5whiteside , p .j. pye-unicam atomic absorption data book,2nd ed.,pye-unicam ltd.england, 1976, 6whitehouse,r.c., prasad,a.s., rabban,p.i. and cossack,z.t. zinc in plasma,neutrophils,lymphocytes and erythrocytes as determind by flameless atomic absorption spectrophotometry. clin.chem, 1982, 28 (3), 475. 7de haese ,p .c . and de bore , m . e . tarce elements in dialysis fluid . nephron .dial . transpl, 1996, 11 (2),92-97. 8krachlet ,m ., wilnsberg ,g. and irgolic , k .j.,trace element status of hemodialysed patients. biol . trace.elem .res, 1997, 58 (3),209-221. 9al-timimi,d.j.:al-shamma,g.a.;al-shaarbaf,h.;al-ghabban,s.s. and sultan,t.r. serum zinc,copper and magnesium in patients with chronic renal failure and dialysis.j.fac. med. baghdad, 1988, 30 (3),259-264. 10 biasioli ,s. schiavon, r. and petrosion,l. free radicals and oxidation stress challenge dialysis patients . asaid journal, 1997,43 (5) ,766-772. 11bogden, j.d., oleski ,j.m.:weiner,b.:smith,l.g.: and najem,g.r. elevated plasma zinc concentration in renal dialysis patients. amer.j. clin. nut, 1980,33 (4) ,1088-1095. 12krachler,m.: wirnsberger,g. and irgolic, k.j. trace element status of hemodialysis patients.biol.trace elem.res. (united state) , 1997, 58 (3) 209-221. 13bogden,j.d. blood zinc in health and disease. in: zinc in the environment. part ii: health effects , new york : wiley interscience, 1980, 137-169. 14atia ,m. m. serum zinc ,copper , total and hdl-cholesterol after acute myocardial infarction .j.fac.med. baghdad, 1996, (38) 3, 262-265. 15al-timimi ,d.j, al-shamma,g., aziz,a.a. changes in serum ,zinc , copper and magnesium concentration after myocardial infarction. and other heart disease.iraqi j. pharm.sci, 1983, 2(1): 43-52. 16atia, m.m. ,al-jumaili, t.i. , al-rawi , k.a. and najem ,s.n. lipid profil abnormalities in patients with renal faliure undergoing haemodialysis in al ramadi general hospital (iraq). in press, 2001. الخـلاصـة: cases serum mg++ mean +sd mg/100ml serum cu++ before dialysis cases a comparative biochemical study of iraqi j pharm sci, vol.19(2) 2010 study proteins profile with β–thalassemia 19 a comparative biochemical study of proteins profile in iraqi children and adolescent with β–thalassemia ali m. malik* , emad m. malik**, nawal mj al-shammaa*** and zeinab m. al-rubaei*** *central children hospital,ministry of health,baghdad,iraq . **departement of pharmaceutical chemistry,risafa directurate,ministry of health,baghdad,iraq . *** department of chemistry,college of education , university of baghdad ,baghdad,iraq . abstract the aim of the present research is to study different protein fractions in sera of children and adolescent with β –thalassemia major and minor and to compare the results with that of healthy control.one hundred fifty children and adolescents were enrolled in this study,including 50 patients with βthalassemia major , 50 patients with βthalassemia minor as pathological control group and another apparently 50 healthy individuals as a control group. the age of all studied groups ranged from (4-18)years.total protein, albumin and immunoglobulins were estimated in sera of all subjects. a significant decrease was found in the total protein and albumin levels in sera of both major and minor thalassemic patients compared to normal groups. a significant increase in immunoglobulin levels (igg, igm and iga) was found in the sera of major and minor β-thalassemia patients compared to control.different protein parts in sera of all subjects were detected using cellulose acetate electrophoresis.the results revealed significant reduction in βglobulin fractions in βthalassemia major patients compared to the normal and pathological control groups. significant elevations in γ globulins fractions were observed in both major βthalassemia and minor βthalassemia as compared to normal control group. as a conclusion the alteration in some protein parts occurred which is more obvious in major thalassemia patients compared to the normal and pathological control groups. key words: protein parts , elecrtrophoresis , β-thalassemia. الخالصة شخصنامم الصنراف نل العن،اخ س نم ننه منابا مصنا ا من، لن، دم ال ان، اس ن الم ن نظ 051جمعت نمادج الذم من 51ال انن، اس ن الم ن ننظ الخ نني خ الننود اج ن،خا طمةمنجننة نن ، م، نن ة س خالشنذدذس خنم نننه مننابا مصنا ا منن، لنن، دم البنذ من ال انر دفا نة مخ ني ( ناة.01-4شخصا م اسصااء طمةمنجة ، ط ع ة.ت ،اخح اجماف المةام ع المذفخ ة ) الصننراف ننل العنن،اخ خملافن بننا مننع المةمنجننة اجنااء ال نن،خت ننل م، ننم لنن، دم ال انن، اس نن الم ن ننظ نننن ننا منن اسشننخا اشنافم تا ق اس طل من ال ن،خت الي نل خاسل ننم خاسم انط ن نل انام نل مصنل طنل من المةنام ع الم، ن ة خالضنا ة. الضا ة. انط ن نل انام )الا ائج الم خجند نلصاه معاني ل م نى ال ،خت الي ل خاسل نم منع يدناد خا ناة نل م ن نى طنل من اسم igm خ igg خ( iga ل طل م مةام ع الم، م المذفخ ة ملافنة منع المةمنجنة الضنا ة ال ع نة. تنا دفا نة اجنااء ال ن،خت نل مصنل م، م ل، الذم ال ان، اس ن الم ن نظ خ المةمنجنة الضنا ة ال ع نة لا نة ال ،ه نل اليب، نائل خ اشنافم الا نائج النم جنذم طاما ن مةنام ع الم، نم ا مع يداد خا اة ل ط ن نل -ال ا اما هااك نلص خا ح ل ط ن نل –ل ط ن نل خجند نلص الشذدذ خالخ ة. تا اس ا اج اه ال ر ، ل اجااء ال ،خت معاندة خناصة ل م، م ل، دم ال ا،اس الم ن نظ الشنذدذ ملافننة الم ن ظ الخ ي خالمةمنجة الضا ة ال ع ة .مع م، م ل، دم ال ا،اس introduction thalassemia is the name of a group of geneticlly (inherited), blood disorders , all of which involve under production of haemoglobin , and partial or complete failure of synthesis a specific type of globin chain.the defect may affect the α, γ and δ chain or may affect some combination of the β, γ and δ chains in the same patient, but never α and β chain together, unmatched globins could precipitate and damage rbc membranes causing their destruction while still in the marrow [1,2] .beta (β)thalassemia manifest clinically has three major groups: 1-βthalassemia major, 2βthalassemia intermedia and 3-βthalassemia minor (trait) [2] .β -thalassemia major occurs at a high gene frequency throughout the mediterranean populations, the middle east, india and southern china through thailand populations [3] .the prevalence of β– thalassemia in iraq have not taken much intention in previous studies in spite of the large population affected by this haematological disease.proteins are substances that made up of smaller building blocks called amino acids [3] , which are an important constituents of all cells and tissues. human serum contains more than 125 well identified 1corresponding author email : elaf95@yahoo.com received : 11/1/2010 accepted : 2/6/2010 iraqi j pharm sci, vol.19(2) 2010 study proteins profile with β–thalassemia 20 proteins. so there are many different kind of proteins in the body with many different functions,for the example:enzymes, some hormones, hemoglobin, immunoglobulin (antibodies) [4] . the major sites of synthesis of plasma proteins are the liver and the immune system [5] . total protein level depend on the balance between their synthesis and their catabolism or loss from body . a test for total serum protein measures total amount of protein in blood serum as the amounts of albumin and globulins [6] . albumin has a single polypeptide chain of (580) amino acids. it is a very stable protein with a high net negative charge at the physiological ph. it has a molecular weight of (66)kda . albumin molecule could serve as hormones and various metabolites as well as drugs and antibiotics carrier. albumin also functions in the maintenance of proper osmotic pressure [7] .the immunoglobulins which are antibodies, are a heterogeneous group of plasma proteins produced by b lymphocytes . .these proteins are important in preventing and fighting infections. elevation in the serum levels of immunoglobulin are seen in infectious diseases and thalassemia [8] .many studies have been carried out to evaluate changes of the immune system in thalassemia patients, considering the humoral and cellular immune system; but no consistent defect in white cells or immune functions had been documented [8] .the aim of the present research is to study different protein fractions in sera of children and adolescent with β -thalassemia major and minor and to compare the results with that of healthy control. materials and methods selection of subjects and blood sampling six ml of venous blood sample was obtained from 150 children and adolescent attending ibn al-baladi hospital . 50 patients were with β -thalassemia major, 50 patients were with βthalassemia minor (as pathological control group) and 50 apparently healthy individuals as control group.the age of all studied groups were ranging from (4-18) years.the blood samples were transferred into plain tubes, allowed to stand for 15 minutes at room temperature then centrifuged at 3500 rpm for (10) minutes. the resulting serum was separated and frozen at (-20 0 c) till used for the estimation of total ptotein (tp), albumin, igm, iga, igg and performing electrophoresis for sera. determination of total protein(tp) the concentration of total proteins was determined according to the colorimetric method described by gornall a. [9-10] the peptide bonds of proteins react with cu 2+ in alkaline solution to form a colored complex in which the absorbance at 550 nm was proportional to the concentration of total protein in the specimen. the biuret reagent contains sodium potassium tartrate to complex cupric ions and maintains their solubility in alkaline solution . determination of albumin albumin concentration in serum was measured using manual procedure, teco diagnostics kit.serum albumin binds selectively to the dye bromcresol green at the ph 4.2. the absorbances of the resulting albumin-dye complex, was read at 630 nm, was proportional to the albumin concentration [9] . determination of igm, iga and igg immunoglobulins (igm, iga and igg) were determined by using immunoturbidimetric method [11] .immunoglobulins (igm, iga and igg) form a complex with antibodies in solution. the absorbance of the complex could be measured spectrophotometrically at 340 nm. immunoglobulin concentrations for each sample was estimated by the equation obtained from a comparable standard curve for each type . serum protein electrophoresis electrophoresis is referring to the transport of electrically charged particles in an electric field,that can be utilized for the characterization of their components after a comparison to references.to perform an electrophoretic separation requires the support material (cellulose acetate) which was made with buffer previously placed in the electrode chamber then sample (serum) was applied to the support, and electrophoresis was performed by conducting to electric power for 40 min, using a constant voltage (150 v). the support was then removed from the electrophoresis unit stained with 1% penceau s. after washing out excess dye, the support media was dried then the electrograms were scanned [9] . statistical analysis data presented as means and standard deviation. study of t-test (p) was used to compare the significance of the difference in the mean values of any groups( p ≤ 0.05) were considered statistically significant. the overall predictive values for the results in all studied groups were performed according to biostatistics by daniel in 1987 [12] . iraqi j pharm sci, vol.19(2) 2010 study proteins profile with β–thalassemia 21 results and discussion table (1) summarizes the results of estimated levels of serum total protein (tp) ,albumin ,globulin and (a/g) ratio expressed as (mean ± sd) in sera of normal control,thalassemia minor and βthalassemia major . table 1 : total protein (tp), albumin,globulin and (a/g) ratio levels in sera of βthalassemia major, minor thalassemia patients and normal control . groups tp gm/dl (no.=50) albumin gm/dl (no.=50) globulin gm/dl (no.=50) no.=50 (a/g)ratio t-test thalassemia major 5.46± 0.54* 3.83± 0.54* 1.626± 0.011* 2.35 * p≤ 0.05 (thalassemia minor) 6.88 ± 0.66* 4.55± 0.49* 2.335± 0.150* 1.94 * p≤ 0.05 control 7.99 ± 0.59 5.21 ± 0.48 2.775 ± 0.402 1.88 p≤ 0.05 p significant difference from control values. *: significant difference between minor and major thalassemia patients. the (mean ± sd) level of tp in sera of control group, and βthalassemia patients (minor &major) in gm /dl were (7.99 ±0.59), (6.88 ± 0.66) and (5.46 ± 0.54), respectively. the results showed a significant decrease of tp in βthalassemia major and minor comparing with control. also a significant decrease in tp of βthalassemia major compared to minor was found . these results are in agreement with one study who claimed that the decrease in serum total protein is due to secondarily decreased synthesis of protein by the liver [13] .the results of serum albumin measurements revealed that the mean values of albumin in the three studied groups in gm/dl were (5.21 ± 0.48) , (4.55 ± 0.49) and (3.83 ± 0.54) , respectively.the low levels of albumin may roughly be balanced by arise in immunoglobulin levels . this is quite common combination;where most of individual proteins , other than albumin , make relatively small contribution to total protein because of quite large percentage change in the concentration of one of them may not be detected as change in total protein so only low albumin levels are of clinical importance [14] .the (a/g)ratio of βthalassemia patients (major &minor) were 2.35 and 1.94 respectively decreased significantly to 1.88 in the control group. a ratio much less than one can give clues about problems in the body [15,16]. the serum levels of igg, igm and iga in sera of control,minor and βthalassemia major patients are shown in table (2). furthermore,the results showed high levels of igg, igm and iga in sera of thalassemia patients compared to control. also a significant higher increase in immunoglobulins in sera of major thalassemic patients was noticed compared to thalassemia minor patients. table 2 : serum immunoglobulin levels (iga, igm and igg) of control, pathological control and βthalassemia major patients. groups igg mg/dl (no.=50) igm mg/dl (no.=50) iga mg/dl (no.=50) t-test thalassemia major patients 1519 ± 37.60* 220 ± 6.55* 241.6 ± 7.48* p≤ 0.05 thalassemia minor patients 1182 ± 18.41* 148.68 ± 4.13* 202 ± 11.12* p≤ 0.05 control 1004 ± 19.00 120.6 ± 3.04 168.6 ± 5.05 p≤ 0.05 p significant difference from control values. * significant difference between minor and major thalassemia patients such observations can be attributed to many factors. for instance repeated blood transfusion in βthalassemia patients would result in a continuous exposure to various antigens and might lead to increased levels of serum immunoglobulins.repeated infections also stimulate the immune system and may result in increased immunoglobulin levels [17,18] . iron overload was suggested by some investigators as an important contributing factor in altering the immune parameters in thalassemia patients that results in increased iraqi j pharm sci, vol.19(2) 2010 study proteins profile with β–thalassemia 22 migration of t helper cells to the gut and lymph nodes and this causes an increase in serum immunoglobulin levels in thalassemia patients [19,20] .figure (1) showed sera electrophoresis pattern of thalassemia major ,minor and control groups. abnormal pattern of serum protein in sera of patients groups is obvious by the decrease shown in the albumin band and change in the pattern of β and γ globulins which include main proteins specially those of immunoglobulins.the decrease in albumin levels of patient groups could be due to decrease rate of synthesis ,or to an increase in catabolism rates which occurred in many diseases [14] .meanwhile results revealed a mild reduction in β globulin that may be due to the presence of transferrin (the iron bound protein)in this band,which is considered as to be the major component of the β-globulin fraction and appears as a distinct band on high resolution serum protein electrophoresis [22] . also figure (1a) of electrophoresis showed a highly significant increase in γglobulins in major βthalassemia major patients compared to normal control, while figure (1b) showed a mild increase in γ globulins in pathological control compared to normal control.the increase in γ-globulins related to hemoglobinopathy diseases [9] . a a c c b figure 1 : serum protein electrophoresis pattern (c control) a(left):normal control and major β-thalassemia patients b-(right):normal control and pathologicalcontrol (minor) references 1. noguchi,c.,butterwoth,j.,karawajew,l.,k uppers,r.,favaloro, f. ,and jacobsohn , d. : hematologica , 2004,89,1281-1283. 2. hope r., longmore j., mc maunus s., and wood a.: "xford hand book of clinical medicine"15 th ed.oxford university press,2001,22-25. 3. cappolini n., cohena., and proter j.:"guidelines for the clinical monagement of thalassemia". thalassemia international federaton,2000,1-29, 79-92. 4. lissaure t., and colayden g.: "illustration text book of paeidiat rics",2 nd ed. , mosby international lmt.,2001,305. 5. kanieco j., harvey j., and bruss. m. : "clinical biochemistry of domestic animals " 5 th ed. , academic press. ,1990 , 120-226. 6. michael l., janet l., and edward p.: "clinical chemistry", 4 th ed. lippincott williams & wilkins,2002,172. 7. nelson d. , cox m. : "lehninger principles of biochemistry" 3 rd ed. ,worth publishers , 2000 , 2, 877. 8. ahmad a., susan j., reza a., soheila a., nima j.,mehrank.:evalusion of serum levels of immunoglobulins in thalassemia patients,british journal of haematology , 2005,214: 220-223. 9. bishop m.,pody e.,schoff l.:"clinical chemistryprocedures , correlations ",5 th ed., lippincott williams&wilkins,2005,105106, 194212. 10. gornall a.:biolabo regents ,biolabo. manufacturer : biolabo sa, ref. 8216,maizy,france. + albumin alfa-1 fraction alfa-2 fraction beta fraction gamma fraction iraqi j pharm sci, vol.19(2) 2010 study proteins profile with β–thalassemia 23 11. shivananda nayak b.: "manipal manual of clinical biochemistry ", 3 rd ed., medical publishers ltd., new delhi,2007,96-100. 12. dainel w.: "biostatestics: a foundation for analysis in the health science", 4 th ed.,1987, 127-139. 13. ismaail h.: sialic acid and other biochemical parameters level in βthalassemia major child patients. m. sc.thesis college of science.almustansiriyah university,2001.. 14. zilva p.m., and philip d. m.: "clinical chemistry in diagnosis and treatment'' 6 th ed.,2006,159-160. 15. international myloma foundation : the scientific and medical communties, 2008, 818, 4487-7455. 16. 16-dambro, goldman:cecil textbook 0f medicine,1 st ed,2000,300-309. 17. henk, s.,leo,m. and anneke ,b.: allontibodies after blood transfusions in a nonhematologic allommunized patients, transfusion,2006,46(4),630-635. 18. weatherland d.,clegg j.:"the thalassemia syndromes", 4 th ed.,london: black well scientific publications,2000,120124. 19. weatherland d., clegg j.,higgs d., wood w.:the hemoglobinopathies. in: the metabolic basis of inherited disease. 8 th ed. mc graw-hill,2000,4000– 4656. 20. ranjna c.: "practical clinical biochemistry ", 3 rd ed.,new delhi,medical publishers,2003,600-605. iraqi j pharm sci , vol.18 (suppl.), 2009 silymarin in acute inflammation 14 dose-dependent anti-inflammatory effect of silymarin in experimental animal model of acute inflammation kasim m. juma'a * , saad a. hussain **,1 , intesar t. numan ** and assad h. siqar ** * department of pharmacy, baquba general hospital, diyala, iraq ** department of pharmacology and toxicology, college of pharmacy,university of baghdad,baghdad,iraq abstract silymarin, a flavolignans from seeds of „milk thistle‟ “silybum marianum” has been widely used from ancient times because of its excellent hepatoprotective action. it has been used clinically to treat liver disorders including acute and chronic viral hepatitis, toxin/drug-induced hepatitis and cirrhosis and alcoholic liver disease. the efficacy and dose-response effect of silymarin (125, 250 and 500 mg/kg) were assessed using egg albumin-induced paw edema in rats as a model of acute inflammation. in this model, 56 rats were used and allocated into 7 subgroups each containing 8 rats. all treatments were given intraperitonealy 30 minutes before induction of inflammation by egg albumin and then the increase in paw edema was measured 1h, 2h and 3h after induction of inflammation by using the vernier caliper. the results indicated that silymarin, at doses range used, significantly lowered paw edema (p<0.05) an effect comparable to that produced by the reference drugs, acetyl salicylic acid, meloxicam and dexamethazone. paw edema suppressive effect of silymarin 250 and 500 mg/kg was comparable and both of them were significantly different from that of silymarin 125 mg/kg (p<0.05). therefore, silymarin exert an important anti-inflammatory activity in animal model of acute inflammation, which was significantly increased as the dose increased up to 250 mg/kg. key words: silymarin, acute inflammation, dose-response الخالصة نمذ اسزخذو األَسبٌ ويُز انمذو يبدح انسهًُبسٍَ انًسزخهصخ يٍ ثزوس َجبد انكعىة ثسجت رأثُشهب فٍ حًبَخ انكجذ. ونمذ اسزخذيذ سشَشَب فٍ انىلذ انحبظش نًعبندخ أيشاض انكجذ انحبدح وانًضيُخ وانُبشئخ عٍ انفُشوسبد أو انزعشض نهسًىو وانكحىل. رى فٍ هزِ ًَىرج األنزهبة انحبد يهغى/كغى( فٍ ١٢٢، ٢١٢، ٥٢١انزأثُش انًعبد نألنزهبة ندشع يخزهفخ يٍ يبدح انسهًُبسٍَ ) انذساسخ رمُُى خشراٌ. رى ٨يدًىعبد كم يدًىعخ ظًذ ٧أثُط لسًذ انً خشرا ١٦انًسزحذس ثىاسطخ األنجىيٍُ فٍ لذو اندشراٌ. رى اسزخذاو ثضسق صالل انجُط فٍ دلُمخ يٍ اسزحذاس األنزهبة ۳٢ك انضسق ثبنجشَزىٌ لجم َغشعالج اندشراٌ ثدشع يخزهفخ يٍ انسهًُبسٍَ عٍ لذو اندشراٌ، ورى لُبط يسزىي ركىٍَ انىريخ ثعذ سبعخ واحذح، سبعزٍُ وثالس سبعبد ثعذ اسزحذاس انىريخ ثبسزخذاو انفُشَُخ. أظهشد ثبنًشكجبد انمُبسُخ انًسزخذيخ نهًمبسَخ )حبيط أسُزُم مبسَخ ثأٌ نهسهًُبسٍَ فعبنُخ يعبدح نألنزهبة رعزًذ عهً اندشعخ ي انُزبئح بة سبنُسُهُك، دَكسبيُثبصوٌ وانًُهىكسُكبو(. ويٍ خالل انُزبئح انزٍ رى انزىصم أنُهب ًَكٍ األسزُزبج ثأٌ نهسهًُبسٍَ رأثُش يعبد نألنزه .يهغى/كغى. ٢١٢فٍ انًُبرج انًخزجشَخ نألنزهبثبد انحبدح وانزٌ َضداد ثضَبدح اندشعخ حزً introduction inflammation is an important physiological reaction, which occurs in response to a wide variety of injurious agents (bacterial infection or physical trauma) ultimately aiming to perform the dual function of limiting damage and promoting tissue repair (1). it requires the participation of various cell types expressing and reacting to diverse mediators along a very precise sequence (2). the inflammatory response is often initiated by the activation of resident macrophages through pattern-recognition receptors; this triggers the sequential release of proinflammatory mediators such as eicosanoids, cytokines, chemokines, and protease, which drive leukocyte recruitment and activation (3). resolution of inflammation (anti-inflammatory response) is an active process controlled by endogenous mediators that suppress proinflammatory gene expression and cell trafficking, induce inflammatory cell apoptosis and phagocytosis. an optional balance between proand anti-inflammatory responses is required to prevent the highly detrimental effect of extensive, prolonged or unregulated inflammation (3). silymarin, the seed extract of milk thistle (silybum marianum), is an ancient herbal remedy used to treat a range of liver and gallbladder disorders, including hepatitis, cirrhosis, and as a hepatoprotectant against poisoning from wild mushroom, alcohol, chemical, and environmental toxins (4). milk thistle is one of the best-studied medicinal plants for the treatment of liver disease (4 -7). 1 corresponding author e-mail : saad_alzaidi@yahoo.com received : 13/1/2009 accepted : 4/5/2009 mailto:saad_alzaidi@yahoo.com iraqi j pharm sci , vol.18 (suppl.), 2009 silymarin in acute inflammation 15 most of these effects have been attributed to direct and/or indirect antioxidant capacity of silymarin, such as being a scavenger of ros, phenylglyoxylic ketyl radicals and a chain breaking antioxidant (8). the present study was designed to evaluate the efficacy and dose response effect of silymarin in experimental animal model of acute inflammation. materials and methods silymarin powder used in the present was obtained as standardized pure extract from luna company (egypt) and was dissolved in 98% dimethyl sulfoxide solution to produce stock solution with concentration of 250 mg/ml, from which different concentrations were obtained by dilution. dimethyl sulfoxide and diethyl ether solutions were obtained from merck company (germany), dexamethazone (american regent, inc. usa), acetyl salicylic acid (sanofi, france) and hand caliper (germany). in the present study, 56 sprague dawley rats of both sexes (180-220 g) were used and allocated into 7 subgroups, each containing 8 rats. these groups represent control, standard and test groups. silymarin was tested for its ability to suppress acute inflammation using fresh egg albumin-induced edema in rats as a model according to the technique established by winter et al (9) . rats were fasted overnight, and deprived of water during the experiment to ensure uniform hydration and to minimize variability in edematous response (10) . the rats were separated into 7 groups (8 rats each); control group treated with dimethyl sulfoxide 2 ml/kg; the three standard groups treated with acetyl salicylic acid 100 mg/kg, meloxicam 10 mg/kg and dexamethazone 1 mg/kg respectively; while the three test groups treated with silymarin (125, 250 and 500 mg/kg, respectively). all drugs were administered intraperitonealy and 30 minutes post-treatment, inflammation was induced by injecting 0.1 ml of fresh egg albumin (phlogistic agent) (11,12) into the sub planter surface of the right hind paw. the increase in paw edema, as a result of inflammation, was measured using vernier caliper before and 1 hr, 2 hr and 3 hr after induction of inflammation. the difference in paw thickness before and after induction of inflammation was calculated and presented as mean increase in paw thickness (mm). the ability of anti-inflammatory drugs to suppress paw inflammation was expressed as percentage of inhibition of paw edema (13) . all results were expressed as mean ± sem. the significance of difference between the control and treated groups were determined using one-way analysis of variance (anova), followed by student's t-test. p-values < 0.05 were considered significant. results the anti-inflammatory effect of silymarin on acute inflammatory model was shown in table 1 and figure 1. silymarin (125, 250 and 500 mg/kg), acetyl salicylic acid, meloxicam and dexamethazone significantly reduced egg albumin-induced paw edema (p<0.05) compared with control group after 1 hr, 2 hr and 3r h from induction of inflammation. there is significant difference between silymarin (125 mg/kg) group and all other treatment groups (p<0.05) along three hours of assessment, while no significant difference exists between silymarin doses 250 and 500 mg/kg, and with dexamethazone and meloxicam groups at the second and third hour of assessment. silymarin (250 and 500 mg/kg) produced an effect which is significantly different from that of acetyl salicylic acid at the second and third hour of assessment (p<0.05) (except for silymarin 500 mg/kg group which produces non significant effect at the second hour).the dose-response effect of silymarin on acute inflammation was illustrated in figure 1. the suppressive effect of silymarin on paw edema was significantly increased (p<0.05) as the dose doubled from 125 to 250 mg/kg. however, further increase in the dose up to 500 mg/kg did not show significant increase in the anti-inflammatory activity (except after the first hour, where silymarin 500 mg/kg significantly differs from silymarin 250 mg/kg). 2.0 2.2 2.4 2.6 2.8 3.0 log doses of silymarin 20 24 28 32 36 40 p e rc e n ta g e o f in h ib it io n after 1 hr. after 2 hrs. after 3 hrs. figure (1): dose-response effect of different doses of silymarin on egg albumin-induced acute inflammation in rats. iraqi j pharm sci , vol.18 (suppl.), 2009 silymarin in acute inflammation 16 table 1: effect of different doses of silymarin on egg albumin-induced acute inflammation in rats. % of inhibition mean increase in paw thickness (mm) treatment groups 3 h 2 h 1 h 3 h 2 h 1 h _ _ _ 1.76 ± 0.06 2.29 ± 0.05 3.06 ± 0.06 dimethyl sulfoxide 2 ml/kg 40 41 37 1.05 ± 0.04* a 1.35 ± 0.04* a 1.91 ± 0.06* a acetyl salicylic acid 100mg/kg 35 35 35 1.15 ± 0.05* a 1.49 ± 0.05* b 1.99 ± 0.05* a meloxicam 10mg/kg 37 40 42 1.10 ± 0.05* a 1.37 ± 0.06* a 1.78 ± 0.06* b dexamethazone 1mg/kg 22 23 27 1.38 ± 0.06* b 1.76 ± 0.05* c 2.24 ± 0.06* c silymarin 125 mg/kg 29 34 33 1.25 ± 0.06* c 1.51 ± 0.06* b 2.05 ± 0.07* a silymarin 250 mg/kg 31 37 39 1.21 ± 0.04* c 1.45±0.04* b 1.85 ± 0.05* b silymarin 500 mg/kg data were expressed as mean ± sem; number of animals = 8 in each group; *p<0.05 with respect to control group; values with non-identical superscripts (a, b, c) among different groups are considered significantly different (p<0.05). discussion the inflammatory response is a physiological characteristic of vascularized tissues (14) . exudation, which is a consequence of increased vascular permeability, is considered as a major feature of acute inflammation (15) .egg albumin-induced paw edema in rats is an in vivo model of inflammation used to screen agents for antiinflammatory effect (16) . the characteristic swelling of the paw is due to edema formation. inhibition of increased vascular permeability and hence the attendant edema modulate the extent and magnitude of the inflammatory reaction. the paw edema that induced by injection of egg albumin is peaked after 1 h and then progressively decreased with time. many chemical mediators like histamine, 5ht, kinins and prostanoids mediate acute inflammation induced by phlogistic agents including egg albumin (17) . in accordance with marsha-lyn et al (18) , inflammation occurs through three distinct phases: an initial phase mediated by histamine and 5-ht (up to 2 hour); an intermediate phase involving the activity of bradykinin and a third (late) phase with prostanoid synthesis by cox (19) . the anti-inflammatory activity of silymarin extract was evaluated by egg albumin-induced paw edema using vernier caliper method and the results were shown in table 1 and figure 1. three different doses (125, 250 and 500 mg/kg) of silymarin were evaluated for the aim of finding dose-response relationship; the results clearly indicated the significant antiinflammatory activity of this flavonoid within the dose range utilized, compared to the standard anti-inflammatory agents used in this respect. it efficiently suppressed early, intermediate and late phases of acute inflammation as illustrated in table(1) . however, the effect of silymarin on early and intermediate phases was better than that on the late phase as shown in figure (1). the antiinflammatory effect of silymarin, especially at the doses 250 and 500 mg/kg was comparable to that of standard drugs and there was no significant difference between them. additionally, silymarin shows a dosedependent effect up to 250 mg/kg, and further increase of the dose was not associated with further increase in activity. the in vivo antiinflammatory activity of silymarin was tested in different experimental models of inflammation, and the results suggested that an important anti-inflammatory action was achieved by inhibition of neutrophils migration into the inflamed site which lead to the release of ros, rns and proteolytic enzymes resulting in microvascular endothelial injury, increase endothelial barrier permeability and edema (20) . silymarin at doses range 25, 50 and 100 mg/kg, when administered orally, significantly reduced papaya latex-induced paw inflammation. however, it was not effective against carrageenan-induced inflammation; it also reduced experimentallyinduced ear edema in mice (36.84%) but the reduction was statistically not significant (21) . silymarin has many biological activities as antioxidant, anti-inflammatory, cytoprotective and anticancer effects, it scavenges free radicals, increases cellular gsh content and induces superoxide dismutase (sod) activity and causes a significant reduction in lipid peroxidation and consequently protecting and stabilizing cell membranes (22, 23) . the cell membrane stabilizing effect of silymarin on iraqi j pharm sci , vol.18 (suppl.), 2009 silymarin in acute inflammation 17 mast cell (20) may explain its effect on the initial phase of acute inflammation model mediated by histamine and 5-ht. it may suppress mast cell degranulation and inhibits the release of these mediators that initiate early phase of paw inflammation. silymarin blocked tnf-α-induced activation of nf-κb which regulates the expression of various genes involved in inflammation, cytoprotection and carcinogenesis in a dose and time dependent manner (24) , and it is completely blocked the mrna expression of il-1β and cox-2 in lipopolysaccharide (lps) stimulated raw 264.7 cells (25) . additionally, the strong inhibition of leukotriene b4 (ltb4) formation by silybinin, which is the major component extracted from milk thistle and considered to be most biologically active in terms of its antioxidant and anti-inflammatory properties (26) , was confirmed in experiments with phagocytic cells isolated from human liver, with a significant inhibition of 5-lox achieved in vivo (27) . inhibition of pg synthesis and cox expression by silymarin may explain its effect on the third phase of inflammatory reaction. it is now clear that silymarin suppresses early, intermediate and late phase of acute inflammatory model through interference with the initiation and propagation phases of acute inflammation. however, there is limited data about the role of silymarin in the resolution phase of inflammation, which is recently considered as an important target for drugs used in inflammatory diseases. leukocytes migration contributes to initiation and propagation of acute inflammation which was significantly inhibited by silymarin. propagation phase also mediated by a diverse number of inflammatory mediators whose release, activity and genetic expression was significantly inhibited by silymarin (25) . in comparison with silymarin, nsaids inhibit propagation phase of inflammation through inhibition of pg synthesis, but they have negative effect on the resolution phase of inflammation (except acetyl salicylic acid) through inhibition of 15d-pgj2 synthesis, which is important pre-resolving antiinflammatory mediator (28) . on the other hand, steroids are considered as a gold standard of anti-inflammatory drugs where they inhibit all phases of acute inflammation. they inhibit initiation and propagation phases through suppression of leukocytes migration and inflammatory mediators genetic expression while enhance resolution phase of inflammation through augmentation of macrophages capacity for phagocytosis of apoptotic cells (29) . in conclusion, silymarin in a dose dependent 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pharmacy ,college of pharmacy, university of baghdad,baghdad,iraq. abstract the use of antibiotics (ab) in surgery focused in either treating established infection or to prevent suspected post-operative infection. inappropriate use of antibiotic for treatment of patients with common infections is a major problem worldwide, with great implications with regards to cost of treatment and development of resistance to the antimicrobial agent. moreover, antibiotics may often be dispensed without a clear clinical indication. this study was conducted to estimate the medication errors in using antibiotic for surgery patients which may effect their wound healing. a 260 patients with clean-contaminated and contaminated surgery were included from two teaching hospitals, 160 patient from medical city hospital and 100 from al-kadhimiya hospital, 86% were female and 32% were male, their age range was 40 +/15. the study shows that there are medication errors related to different causes: firstly, medical team error which include the nurse (70.9%) and the physician which include 1) delay in patient follow up after operation(5.9%) , 2) changing the ab without doing culture and sensitivity test (48.8%), and incomplete prescription order(13.1%). second: ordering error which include: 1) the absent of original source of ab (44.5%), 2) error in availability of the chosen ab (74.8%), and third: error related to the patient itself include 1) socioeconomic situation (14.5%), 2) educational state (54.3%), finally error related to increase cost in dispensing more than one ab needed (80.1%), although the healing was (63.6 %), delay in response (25%) and complicated wound infection (5%), significant results were arrange nurse error and poor drug availability. in conclusion: medication errors are still common problem in our hospitals, which are mostly related to medical team and the pharmacists should give more effort to avoid these errors. key wards: acquired error, antibiotic, surgery patients. الخالصة نًثثرح أٔ نًُؼٓا انًرٕقغ تؼذ انؼًهٛح. انًضاداخ انحٕٛٚح فٙ اندشاحح ٚرشكض أيا فٙ يؼاندح األخرالطاخ انثكرٛشٚح ا اسرخذاو انًضاداخ انحٕٛٚح تشكم غٛش يالئى نؼالج انًشضٗ يغ االخرالطاخ انثكرٛشٚح انشائؼح يٍ أْى انًشاكم انًُرششج ػانًٛا", يغ اسرخذاو , انًضاداخ انحٕٛٚح غانثا" انؼايم رٔ انًؼاداج انًٛكشٔتٙ. ػالٔج ػهٗ رنك إنٗكهفح انًؼاندح ٔذطٕس انًقأيح إنَٗرائح كثٛشج َسثح انرٙ اندشاحح نًشضٗانًضاداخ انحٕٛٚح اسرخذاوْزِ انذساسح أخشٚد نرقٛٛى األخطاء انطثٛح فٙ ٔاضحح. سشٚشّٚياذصشف تذٌٔ دالنح يٍ أثٍُٛ يٍ انًسرشفٛاخ يهٕثح ٔخشاحح يهٕثح ضًُٕا-يٍ سدْح اندشاحح يغ خشاحح َظٛفح يشٚض 022. خشٔحٓىقذ ذؤثش ػهٗ شفاء % 20% كإَا َساء 62ٔ, يشٚض يٍ يسرشفٗ انكاظًٛح 022يشٚض يٍ يسرشفٗ يذُٚح انطة ٔ 022انرؼهًٛٛح, ذضًُد انذساسح أسثاب يخرهفح: أٔال", خطأ انفشٚق انطثٙ انز٘ إنٗأظٓشخ أخطاء طثٛح ذؼٕد ْزِ انذساسح.01 -+/02سخال, يذٖ أػًاسْى كاٌ انًضاداخ انحٕٛٚح تذٌٔ ( ذغٛٛش0 %(, 1.7) ذأخش فٙ يراتؼح انًشٚض تؼذ انؼًهٛح (0 ٔذضًٍ %( ٔانطثٛة92.7ذضًٍ انًًشضح ) انًصذس ( غٛاب 0 ٔٚرضًٍ طهثٙ : خطأثاَٛا"%(, 02.0يرطهثاخ انٕصفح ) اكرًال( ػذو 2%(, ٔ 06.6) انحساسٛح ٔانضسع اخرثاس انًشٚض َفسّ إنٗخطأ ٚؼٕد %(, ثانثا": 90.6%(, ٔ خطأ فٙ ذٕفش انًضاد انحٕٛ٘ انًخراس )00.1) األصهٙ نهًضاداخ انحٕٛٚح فٙ صشف صٚادج انركهفح إنٗخطأ ٚؼٕد %(, ٔأخٛشا" 10.2%(, انحانح انرؼهًٛٛح )00.1) االقرصادٚح االخرًاػٛح( انحانح 0ٔٚرضًٍ ذهٕز خشٔذ يؼقذ %( 01ٔ) االسرداتح%(, ذأخش فٙ 22.2اء كاٌ )نهشف االسرداتح %(, نك62.0ٍيضاد حٕٛ٘ يطهٕب ) ألكثش يٍ ٔانصٛادنح أسثاب انكادس انطثٙ إنٗ: األخطاء انطثٛح ذثقٗ يٍ انًشاكم انشائؼح فٙ يسرشفٛاذُا ٔانرٙ غانثا" يا ذؼٕد االسرُراجفٙ %(.1) انزٍٚ ٚدة أٌ ٚثزنٕا خٕٓد أكثش نردُة ْزِ األخطاء. introduction postoperative wound infection is still one of the most frequent complications observed in surgery patients. currently, surgical antibiotic prophylaxis (sap) accounts for over 30% of antibiotic prescriptions in general hospitals. however, in surrey centers, it can be as high as 95%. (1,2) therefore, monitoring sap is critical in ensuring appropriate use of antimicrobial agents in this setting. this helps to increase the effectiveness of sap and minimize the consequences of its misuse, such as the risk of developing antibiotic resistance, adverse events and a higher cost to the institution. the choice of antimicrobial agent, the timing of administration and the duration of prophylaxis are factors that can affect the appropriate use of sap. (3,4) in a surgery centre, the appropriateness of sap can be affected by the level of surgical activity, the number of surgical specialities and medical teams working in the same unit. these factors predispose to high variability in sap practices, leading to antimicrobial misuse. (2) # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1corresponding author email : fadiaalhamdani@ymail.com received :2/4/2011 accepted :3/10/2011 iraqi j pharm sci, vol.20(2) 2011 medication error in using ab 103 antibiotic usage is one sign of hospital care and cost inclusion that has received much attention over the past decade. since the misuse of antibiotics has been well documented, (5,6) improved practices for prescribing antibiotics have been suggested as a major goal of quality assurance and cost inclusion (7,8) . in practice, however, individual practitioners and hospitals have worked together to formulate programs that aim to have measure, change, and often improve practices for prescribing antibiotics (9,10) . antibiotics are high on the list of drugs used for self-medication, (11,12) and physicians may sometimes prescribe antibiotics without a clear clinical indication. (13,14) the aim of the present study was designed to evaluate the prevalence of medication errors of using ab after surgery, and to estimate the causes behind these error. patients and method this study was carried out at two teaching hospitals (medical city and alkadhimiya hospital) during 3 months. we recorded prospective information from the case sheet of patients after surgical operation. the clean-contaminated and contaminated surgery was included in this study. the demographic data of 260 patients (160 patients from medical city hospital and 100 from alkadhimiya hospital), include their age (40 +/15), and gender (86% were female and 32% were male). the acquired medication errors was classified as medical team error, ordering errors, error related to the patient itself, and error related to the cost. medication errors data were collected and analyzed descriptively. result and discussion 1types of medication errors table (1) shows errors obtained from both hospitals which are classified into four groups with their percentage. the nurse error included the incomplete administration of the night dose representing (70.9%), while the physician errors include (5.9%) delay in patient follow up after operation, (48.8%) changing the ab without doing culture and sensitivity test, and (13.1%) incomplete prescription order. ordering errors include absent of original source of ab (44.5%), nonavailability of the ab necessary for each surgery (74.8%). patient errors include (54.3%) poor knowledge about the ab and (14.5%) of patient compliance concerning proper medication use to prevent any complications. cost error includes (80.1%) the use of more than one ab needed for each surgery.the institute of medicine (iom) defines medical errors as the failure to complete a planned action as intended or the use of a wrong plan to achieve an aim (15) .the study showed the higher percentage of medication errors were related to cost error which was most common cause. table 1 : type of medication error in both hospitals. (%) percentage of error type of medication error 70.9 5.9 48.8 13.1 medical team error nurse error physician error adelay in follow up bchanging ab c incomplete prescription order 44.5 74.8 ordering error aabsent of original source of ab. berror in availability of ab 14.5 54.3 error related to the patient asocioeconomic situation beducational state 80.1 cost the iom report estimates that medical errors cost the nation approximately $37.6 billion each year; about $17 billion of those costs are associated with preventable errors. about half of the expenditures for preventable medical errors are for direct health care cost, medical team error (nurse error), and prescribing error (availability of ab). (16,17) with many hospital processes, medication delivery is a highly complex, multi-faceted operation involving multiple people and numerous steps. the medication delivery process consists of five basic stages: prescribing ordering, order transcription, dispensing, administering and monitoring. within each of these stages there are multiple actions, each presenting potential for error. (18) on the other hand, professional nurse is the practitioner most often associated with the responsibility of medication administration. an essential part of every nurse’s training is committing to memory and practice the ―five rights‖ checklist: right drug, right dose, right route, right time, and right patient (19,20) .the nurse may deliver the ―right drug‖ based on the prescribed order, but if the dosage is incorrect, the pharmacist and the nurse missed the opportunity to correct the error. a multidisciplinary approach is necessary to improve medication administration (21) .also, a primary factor contributing to medication errors within the drug ordering system is due to lack of iraqi j pharm sci, vol.20(2) 2011 medication error in using ab 104 prescriber information regarding drug therapies. errors regarding the choice of drug or dosage have been found to be the most likely type to cause injury. following errors associated with the prescribing/ordering process, medication administration errors are the second most frequent type (22,23) . 2comparison between medication errors in two iraqi hospitals in table (2) we notice that there was significant differences concerning errors related to the nurse (66.5% versus 75.3%) and the availability of ab (78.4% versus 71.1%) between alkadimiya hospital and medical city. in the above comparison we did not include patients error because the same type of population found in both hospitals. numerous factors in the health care system contribute to medication safety and errors. some of these factors can be attributed directly to provider organizations, while others can be attributed to the medication-use system itself. in many cases, multiple factors are involved related to professional practice, health care products, procedures, and systems, including prescribing; order communication; product labeling, packaging, and nomenclature; compounding; dispensing; distribution; administration; education; monitoring; and use. according to a variety of sources, the root cause of medical errors is due to the complexity of todays healthcare system. (24) the iom emphasized that most medical errors are systems related and not attributable to individual negligence or misconduct. the key to reducing medical errors is to focus on improving the systems of delivering care and not to blame individuals. health care professionals are simply human and, like everyone else, they make mistakes (25) . medication errors reported to the fda may stem from poor communication, misinterpreted handwriting, drug name confusion, lack of employee knowledge, and lack of patient understanding about a drug's directions. "but it's important to recognize that such errors are due to multiple factors in a complex medical system," says paul seligman, m.d., director of the fda's office of pharmacoepidemiology and statistical science. "in most cases, medication errors can't be blamed on a single person." (26,27) so our results could be part of these medication errors reported regarding the complex medical system in iraqi hospitals. to avoid these medications misuse, the pharmacists should give information and education to the patients until they understand the role of medications in their health. besides, educating the pharmacists to increase their roles in community sitting, also avoiding medication errors requires vigilance and the use of appropriate technology to help ensure proper procedures are followed. (28,29) computerized physician order entry reduces errors by identifying and alerting physicians to patient allergies or drug interactions, eliminating poorly handwritten prescriptions, and giving decision support regarding standardized dosing regimens. (30,31) moreover we recorded the percentage of healing after surgery regarding more than one ab needed.the complete healing was (63.6 %), delay of response (25 %), and complicated wound infection (5 %), and this is because in such types of clean contaminated and contaminated surgery, the most commonly used ab are a combination of cephalosporins, aminoglycoside, and metronidazole (32,33) to cover the most common infecting organism suspected to cause surgical site infection. (34,35) we concluded that lack of knowledge about drugs and lack of employee knowledge is one of the most common causes of medication errors. a systematic plan for routine and ongoing education for nurses and other clinicians who administer medications should be developed and implemented, in addition , effective role of pharmacist in community and with medical team is most warranted. table 2 : comparison between percentage of medication error in two iraqi hospitals. chi square (p-value) medical city hospital al-kadhimiya hospital 0.0005 * 75.3% nurse error 66.5% 0.003 7.5 % physician error 4.3% 0.005 51.9% changing ab 45.6% 1.00 45% absent of original source of ab 44% 0.0005 * 71.1% availability of ab 78.4 % 0.586 15.8% incomplete prescription order 10.4% 0.002 83% cost 77.3% * represent significant differences with p<0.005 references 1. amarasingham r, plantinga l, dienerwest m, gaskin dj, powe nr. clinical information technologies and inpatient outcomes: a multiple hospital study. 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ondansetron hcl nanoparticles for transdermal delivery amjed h. noor*,1 and mowafaq m. ghareeb** * ministry of health and environment, babylon health directorate, babylon, iraq. **department of pharmaceutics, college of pharmacy, baghdad university, baghdad, iraq abstract ondansetron hcl (ond) is a potent antiemetic drug used for control of nausea and vomiting associated with cancer chemotherapy. it exhibits only 60 – 70 % of oral bioavailability due to first pass metabolism and has a relative short half-life of 3-5 hours. poor bioavailability not only leads to the frequent dosing but also shows very poor patient adherence. hence, in the present study an approach has been made to develop ond nanoparticles using eudragit® rs100 and eudragit® rl100 polymer to control release of ond for transdermal delivery and to improve patient compliance. six formulas of ond nanoparticles were prepared using nanoprecipitation technique. the particles sizes and zeta potential were measured using zeta-plus analyzer. the particle morphology was also studied using scanning electron microscopy (sem). the in-vitro release of the drug from the nanoparticles was carried out in phosphate buffer saline ph 7.4. the particle size of the prepared nps were in nano size which ranged from (95.34 to 275.84 nm) with positive zeta potential. the drug entrapment efficiency was varied with the drug polymer ratio from 41.87% to 78.45%. the sem showed uniform shape and regularly distributed particle sizes. the in-vitro drug release study exhibited the sustained release of ond with burst release. the cumulative percentage released after 12 hr. were between were 77.89 and 96.01%. also the transdermal permeation study show that nanoparticles permeate more efficiently than aqueous solution of the drug through the skin by approximately two fold. ond nanoparticles were prepared successfully using nanoprecipitation method. the controlled drug release aimed for transdermal drug delivery could be obtained by using eudragit rs100 and eudragit rl100 polymers which can reduce dosing frequency, decrease side effects and improve patient compliance. keywords: ondansetron hcl, nanoprecipitation method, eudragit rs100, eudragit rl100, sem. تحضير وتقيم جسيمات نانوية لالوندانسترون هيدروكلورايد ال عطائها عن طريق الجلد **و موفق محمد غريب 1،*أمجد حسين نور .العراق بابل، دائرة صحة بابل,والبيئة، الصحة وزارة* .،العراق بغداد ، بغداد جامعة ، الصيدلة كلية ، الصيدالنيات فرع** الخالصة رتتتتي دواا فعتتتال ميتتتاد لل تتتتطا ستتتنلدث للستتتتيءرة ملتتت الغاليتتتاي وال تتتتطا ال تتتر ب بتتتتالع ond))االوندانستتتنروي ريدروكليرا تتتد ٪ متتتتت النتتتتيافر الحيتتتتيف متتتتت ضر تتتتا الاتتتتل بستتتتبل الن اليتتتتل الغتتتت ائط لل تتتترور ا ول ولتتتت م تتتتر 70 – 60ف تتتت الكي يتتتتائط للستتتترضاي. عتتتتر تتتا تتتر يتتتد ا تتتعيا ا 5-3نصتتتص رصتتتير نستتتبيا متتتت ستتتامات. النتتتيافر الحيتتتيف اليتتتعيص ال تتتردف ف تتت رلتتت اليرمتتتات ال نكتتتررة ولكتتتت ي ج لنءتتتي ر جستتتي ات ناني تتتة منغاريتتتة الصتتتغر متتتت االوندانستتتنروي ال يدروكليرا تتتدجتتتد ا لل تتتر ل. وبالنتتتالط ، فتتتط رتتت إ الدراستتتة ، تتتل ر بتتتاع ن تتت للنحكل فط حر ر الدواا مبر اليلد لنحسيت امنالال ال ر ل للع . rl100و rs100ث اال يدراجيت اسنلداب س حيتتتتاث اليت ئتتتتات تتتتل رمتتتتداد ستتتتنة صتتتتين متتتتت اليستتتتي ات الغاني تتتتة ل وندانستتتتنروي باستتتتنلداث غيتتتتة النرستتتتيل الغتتتتانيف. تتتتل ريتتتتا (. و تتتل semوري تتتة ج تتتد ز نتتتا باستتتنلداث ج تتتاز محلتتتل ز نتتتا بلتتتس. ك تتتا تتتت دراستتتة اتتتكل اليستتتي ات باستتتنلداث مي تتتر ال ستتت ا لكنرونتتتط ) .7.4 ذو س ريدروجيغط اجراا م لية حر ر الدواا فط ال لنبر مت اليسي ات الغاني ة باسنلداث محليل بار ملحط مت الايساات نتتتانيمنر( متتت ري تتتة ج تتتد ز نتتتا ميجتتتل. 275.84رلتتت 95.34ي حيتتتل اليستتتي ات الغاني تتتة بحيتتتل الغتتتاني النتتتط راوحتتتت بتتتيت )الغنتتتائجا كتتتا ٪. و ظ تتتترت صتتتتير مي تتتتر ال ستتتت 78.45 -٪ 41.87كتتتت لا با غتتتتت كاتتتتااة ح يتتتتل التتتتدواا متتتت ادتتتتن نستتتتبة البتتتتيلي ر متتتت التتتتدواا متتتتت ظ تتتتترت دراستتتتتة ذوبتتتتتاي التتتتتدواا فتتتتتط ال لنبتتتتتر حر تتتتترا منياصتتتتت زمتتتتتة بانن تتتتتاث. االلكنرونتتتتتط ااتتتتتكال و حيتتتتتاث اليستتتتتي ات ميحتتتتتدإ و مي ستتتامة متتتا بتتتيت 12و كانتتتت الغستتتبة النراك يتتتة ال نحتتتررة بعتتتد لألوندانستتتينروي ريدروكليرا تتتد متتت حر تتتر ستتتر للتتتدواا فتتتط بدا تتتة االدنبتتتار كتتتت لا ظ تتتترت دراستتتتة االدنتتتتراق مبتتتتر اليلتتتتد ي اليستتتتي ات الغاني تتتتة نللتتتتل بكاتتتتااة اكالتتتتر متتتتت ال حلتتتتيل ال تتتتائط ٪. 96.01٪ و 77.89 تتتتل حيتتتتير اليستتتتي ات الغاني تتتتة ل وندانستتتتنروي ريدروكليرا تتتتد بغيتتتتا باستتتتنلداث ضر تتتتة النرستتتتيل تتتتعايت.للتتتتدواا مبتتتتر اليلتتتتد بحتتتتيالط والتتت ف تتتد رلتتت ستتتليل التتتدواا مبتتتر اليلتتتد باستتتنلداث بتتتيلي رات اال يدراجيتتتت الغتتتانيف. كتتتت الحصتتتيل ملتتت حر تتتر التتتدواا ال ستتتيءر مليتتت rs100 وrl100 والنط كت ي لل مت يا ر اليرمات و ليل اآلثار اليانبية و حسيت امنالال ال ر ل للع. .االلكترونيالمجهر المسح , rs100,rl00الكلمات المفتاحية: أوندانسترون هيدروكلورايد, طريقة الترسيب النانوي, االيودراجيت 1corresponding author e-mail: amjadnoor29@yahoo.com received: 22/12 /2019 accepted: 3/5 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp70-79 mailto:amjadnoor29@yahoo.com iraqi j pharm sci, vol.29(2) 2020 transdermal ondansetron nanoparticles 71 introduction nanoparticles (nps) offer a number of advantages for dermal drug delivery, including improved drug solubility and stability, adjustable surface properties, increased surface adhesion, drug targeting, controlled drug release and increased drug penetration and permeation through the skin and mucus membrane(1). nanoparticle surface charge has a significant effect on adhesion and penetration of nanoparticles through the skin and mucus membrane. the skin is negatively charged under normal physiological conditions and positively charged nanoparticles may adhere to it. cationic aminoeudragit nanoparticles penetrated deeper into the skin in comparison to negatively charged nanoparticles. this is attributed to lack of electrostatic interaction with negatively charged nanoparticles that impaired access to the outermost skin layer(2, 3). the most common form of drug delivery is the oral route; this route of administration has notable advantages and also have significant drawbacks like first pass metabolism, drug degradation in gastrointestinal tract due to enzymes, ph etc. to overcome these difficulties a novel drug delivery system was developed. in recent years it has been shown that the skin is a suitable route for drug delivery to the systemic circulation(4). the skin has been an essential route for drug delivery when topical, local, or systemic effects are preferred. however, skin constitutes an excellent barrier and presents difficulties for the transdermal delivery of therapeutic agents, since limited drugs possess the features necessary to penetrate throughout the stratum corneum in adequate amounts to reach a therapeutic concentration in the blood(5). in order to enhance drug transdermal absorption, various strategies have been considered, developed, and patented. development in physical permeationenhancement technologies has led to renewed interest in transdermal drug delivery. some of these novel technologies include iontophoresis, electroporation, ultrasound, microneedles to open up the skin, and more recently the use of transdermal nanocarriers (6). transdermal drug delivery system (tdds) includes all topically administered drug preparations intended to deliver the active ingredients into the circulation(7). tdds can improve the therapeutic efficacy and safety of drugs by more precise spatial and temporal employment of the drug within the body thereby decreasing both the size and number of doses and also improving its effectiveness with optimal dose concentrations. appropriate drug choice and an effective drug delivery system play an essential role in achieving optimal therapeutic results(8). ondansetron hcl a 5ht3 antagonist is a potent antiemetic drug used for control of nausea and vomiting associated with cancer chemotherapy (figure 1). it exhibits only 60 – 70 % of oral bioavailability due to first pass metabolism and has a relative short half-life of 3-5 hr(9). figure 1. chemical structure of ondansetron hcl(10) the objective of this study is to formulate and evaluate of ond nanoparticles for transdermal drug delivery and to improve patient compliance. materials and methods materials ondansetron hcl (gift from pioneer co. for pharmaceutical industries) polyvinyl alcohol (pva), eudragit rs 100 (rhom pharma, germany) and eudragit rl 100 (rhom pharma, germany), ethanol (thomas baker chemical, mumbai, india). all other chemicals used were of analytical grade. methods preparation of ond nanoparticles six formulas of ond nanoparticles were prepared using solvent/ antisolvent precipitation technique (nanoprecipitation method). a certain amount of pure drug of ond and polymer was completely dissolved in water miscible solvent (ethanol). the obtained drug-polymer solution was then injected at speed of 0.5 ml / min(11) using syringe infusion pump into the water containing stabilizer (1% pva) with continuous stirring of 1000 rpm. precipitation of solid drug particles occurred immediately upon mixing. the precipitated nanoparticles are sonicated for 5 min using probe sonicator. the organic solvent was then evaporated under room temperature and then lyophilized using freeze drying system (copley, uk) to obtain the nanoparticles powder. the composition and variable condition of preparation of different formulas of nanoparticles are listed in table (1) (12). iraqi j pharm sci, vol.29(2) 2020 transdermal ondansetron nanoparticles 72 table 1. composition of ond nanoparticles code no. ond (mg) eudragit rl 100(mg) eudragit rs 100 (mg) pva % d:p ratio f1 8 8 1 1:1 f2 8 16 1 1:2 f3 8 24 1 1:3 f4 8 8 1 1:1 f5 8 16 1 1:2 f6 8 24 1 1:3 characterization of ond nanoparticles particle size analysis particle size distribution, mean diameters, and polydispersity index of nanoparticles were determined by dynamic light scattering (dls) techniques using particle size analyzer (zetaplus, brookhaven, usa) at a scattering angle of 90° at room temperature. for each sample, measurements were achieved in triplicate(13). zeta potential it is a physical property in suspension. it is defined as the difference between the bulk solution (dispersing medium) and the surface of the hydrodynamic shear (slipping plane). it can be used to optimize the nanoparticle formulation for long time stability. it was measured by zeta-plus analyzer (zetaplus, brookhaven, usa) (14). measurements were performed in triplicate. surface morphology the morphological examination of the nanoparticles was performed using scanning electron microscopy (sem; tescan, uk)(15, 16). entrapment efficiency (ee): weighed samples of drug-loaded nanoparticles (10 mg) were dissolved in 10 ml of methanol under sonication for 2 hr. the samples were filtered through a membrane filter and analyzed spectrophotometrically at λmax 310 nm using a uv/vis spectrophotometer (emc lab, germany). the entrapment efficiency was determined using the following equation(15); % 𝑬𝑬 = 𝒎𝒂𝒔𝒔 𝒐𝒇 𝒅𝒓𝒖𝒈 𝒊𝒏 𝒏𝒂𝒏𝒐𝒑𝒂𝒓𝒕𝒊𝒄𝒍𝒆𝒔 𝒎𝒂𝒔𝒔 𝒐𝒇 𝒅𝒓𝒖𝒈 𝒖𝒔𝒆𝒅 𝒊𝒏 𝒑𝒓𝒆𝒑𝒂𝒓𝒂𝒕𝒊𝒐𝒏 × 𝟏𝟎𝟎 the measurements were performed in triplicate and values were the mean ± sd. in vitro drug release studies four milliliter of nanodispersion (8mg drug) were placed in dialysis bags (8000-14000 d), which were sealed and placed in 500 ml dissolution medium (phosphate buffer ph 7.4 containing 0.25 % brij-35). drug release study was carried out employing the usp type ii dissolution apparatus (pharma test, germany) at 37 °c± 0.5 and 50 rpm for 20 hr. at each time interval of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 20 hr. aliquots 5 ml of sample was collected and replaced with fresh buffers. the collected samples were analyzed spectrophotometrically at λmax 310 nm (17). the measurements were performed in triplicate and values were the mean ± sd. in-vitro skin permeation study the abdominal skin of adult male wistar rats weighing 250 ± 10 g obtained from animal house of college of pharmacy/ university of baghdad were used for in-vitro permeation study of nanoparticles. the rat skin was fixed between the donor and receptor compartment with the stratum corneum facing upper side of the inverted glass tube in beaker (modified diffusion cell). to maintain sink conditions 100 ml phosphate buffer ph 7.4 containing 0.25% (w/v) brij-35 were added in receptor chamber. the temperature was maintained at 37 ± 1°c. receptor media was continuously stirred with magnetic stirrer at 50 rpm, in a way that the rat skin surface just flushes the diffusion fluid. the formulation (4 ml) was gently placed in a donor compartment. at time interval of 1, 2, 3, 4, 5, 6, 7, 8 and 12 hr aliquots of 2 ml sample were withdrawn from the receptor compartment and replaced as soon as possible with the same volume of receptor fluid. the samples were analyzed for drug content using uv spectrophotometer at λmax 310 nm. each experiment was performed in triplicate. the cumulative amount of drug permeated (q) at different time intervals and various parameters like steady-state flux (jss), lag time (tl) and apparent permeation coefficient (papp) were calculated (18). the measurements were performed in triplicate and values were the mean ± sd. iraqi j pharm sci, vol.29(2) 2020 transdermal ondansetron nanoparticles 73 compatibility study fourier transform infrared spectroscopy (ftir) before the development of formulation, ftir spectra of physical mixtures of ond with polymers were compared with the standard ftir spectrum of the pure drug (ond) were performed using ftir spectroscopy (alpha ii, bruker, germany). spectra were recorded between 400 and 4000 cm−1 range(19). thermal analysis: differential scanning calorimetry (dsc) dsc analysis was performed using thermal analysis instrument (std q600 v20.9 build 20, usa). the samples (pure drug, physical mixture and selected formula) were weight (4 mg) in a sealed aluminum pans and heated at a rate of 10 °c/ min in a 30 to 350 °c temperature under nitrogen flow of 40 ml/min(20). powder x-ray diffractometric (pxrd) study the powder x-ray diffraction configuration of the achieved sample of the ond was determined to confirm the crystalline nature of the drug. the study was confirmed using powder xray diffractometery (xrd-6000, shimadzu, japan 220v/50hz); the operating voltage and current were 40 (kv) and 30 (ma) respectively. samples were scanned at 2θ from 0-80° for qualitative studies and the scanning rate was 4°/min(21). statistical analysis the outcomes of the experimental work are demonstrated as a mean of triplicate models ± sd and were examined in relation to the one-way analysis of variance (anova) to determine if the changes in the applied factors are statistically significant at level of (p < 0.05) and nonsignificant at level of (p > 0.05). results and discussion ondansetron hcl loaded nanoparticles were prepared by nano-precipitation method without using toxic harmful organic solvents. additionally, this method has an advantage of single step, no need of high shear/ stirring rate or high temperature. this technique is suitable for compounds that are soluble in ethanol or acetone. two grades of eudragit polymer, (eudragit rs 100 and eudragit rl 100) were used. although both show ph-independent drug release properties, they differ in their water permeability. eudragit rs100 has very low water permeability, while eudragit rl100 has high water permeability(22). additionally, the ability of eudragit polymers to form nanodispersion with smaller particle size, positive surface charge (due to the quaternary ammonium groups on the polymer surface) excellent stability, and lacking of irritant effect are advantageous. eudragit® rl 100 has great water permeability, due to the higher quaternary ammonium group content than eudragit rs100 which allowed more water penetration and, consequently, more drug wetting and release(23). the effect of drug: polymer ratio exhibited a wide effect on particle size and distribution (p < 0.05). all the formulas confirmed a small mean particle size. the mean particle size varied from 95.34 to 275.84 nm with polydispersity index ranging from 0.271 to 0.367 (table 2), the results showed that increasing the concentration of the dissolved polymer leading to increase the viscosity of organic phase and reduced the stirring efficiency resulted in the formation of the bigger emulsion droplets which lead to give larger particle size. the same results were recorded by meltem c. et al(24). type of polymer had no significant effect on particle size (p> 0.05) for all formulation. both types of polymer gave nanoparticles with practically same particle size range, these outcomes were in agreement with aisha, et al. (25). all formulations with eudragit showed a positive zeta potential due to present of quaternary ammonium group (figure 6) with values ranging from +15.72 to +31.69 mv (table 2). table 2. mean particle size, pdi, zeta potential and entrapment efficiency of ond nanoparticles formulas code particle size* (nm) pdi* zeta potential* (mv) entrapment efficiency* f1 95.340±13.24 0.271± 0.012 +15.72± 0.67 48.93± 0.63 f2 136.69±21.67 0.278± 0.020 +23.98± 1.44 73.76± 0.77 f3 246.43±24.21 0.267± 0.021 +19.19± 1.37 78.45± 2.13 f4 111.66±18.45 0.367± 0.035 +18.01± 1.81 41.87± 1.54 f5 145.670±9.56 0.312± 0.041 +23.93± 1.26 65.12± 1.64 f6 275.84±27.13 0.253± 0.035 +31.69± 1.13 71.72± 1.32 *average ± standard deviation (n=3) poly vinyl alcohol (pva) is a water soluble, synthetic polymer, used in this preparation assists dual purposes; firstly , it acts as a non-ionic surfactant and prevents the particle growth. secondly, it maintains the viscosity of the preparation required for improve stability of nanoparticles. iraqi j pharm sci, vol.29(2) 2020 transdermal ondansetron nanoparticles 74 the entrapment efficiency of the drug was ranged from 41.87% to 78.45% for the prepared formulations. the results showed that the entrapment efficiency of the prepared nanoparticles was improved by increasing the ratio of polymer (p < 0.05). it has been displayed that increase in polymer ratio in organic phase improves drug entrapment due to increase in viscosity of organic phase which enables the diffusional resistance of drug molecules from organic phase to aqueous phase, leading to entrapping greater quantity of drugs in the nps(3). in vitro drug release profile of the prepared nps using dialysis membrane at beginning showed a quick release characteristic of ond unrelated to the processing conditions. the release curve exhibited that initially fast release up to 30 min and then controlled release was achieved. rapid release at the beginning may due to free, unencapsulated and adsorbed drug on the surface of the nps. drug release was slow from rs 100 compared to rl 100 nanosuspension and this may be due to the greater aqueous permeability of eudragit rl100 polymer. the release rate was correlated to the ratio of drug and polymer. the in vitro drug release profile of the formulations (f2, f3, f5 and f6) were 85.02 %, 77.89 % , 96.01% and 82.69 %, respectively for 12 hr. hazender and dortunc also detected unlike drug release profiles when eudragit rl 100 was used in place of eudragit rs 100(26). generally, all the prepared nanoparticle formulas exhibited a sustained release and burst effect that could be detected (figure 2). it suggests that percent drug release is dependent on the type of polymer used. on the basis of particles size, encapsulation efficiency and release profile, batches f2 and f5 were chosen to complete other study to select the optimized batch for the preparation of nanoparticles. figure 2. dissolution profile of the prepared ond nanoparticles (f2,f3,f5 and f6) in pbs (ph 7.4). the in vitro permeation study of the formulation f2 and f5 in comparing with the aqueous drug solution using rat skin show a significant improvement (p < 0.05) in the penetration of ond (figure 3). the flux (jss) values for ond nanoparticles (f2 and f5) were 177.93and 163.12µg/ cm2.hr respectively, and for aqueous drug solution was 80.44µg/ cm2 hr. polymeric ond nanoparticles permeation was found to be higher than that for aqueous drug solution. the higher flux and permeation values of nanoparticles suggest that it might be able to cross the skin easily as compared with the aqueous drug solution. the permeation profiles of ond nanoparticles (f2 and f5) and aqueous drug solution are shown in figure 3. the permeated parameters such as steady state flux rate, lag time and apparent permeability coefficient (papp) are given in (table 3). the total flux of nanoparticles was approximately two times higher than that of aqueous drug solution(27). the permeation study indicating that f2 gave the highest drug penetration with lowest lag time (p > 0.05) in comparison with f5 and aqueous solution of the drug, so, it was chosen as the selected formula. table 3. permeation parameters of ondansetron hcl formulation flux* (jss) (µg/ cm2. hr) permeability coefficient* (p) (cm/ hr) lag time* (t l) (hr) f2 177.93± 5.32 8.9 * 10-2± 0.003 0.47± 0.021 f5 163.12± 4.67 8.1* 10-2± 0.0024 0.76± 0.063 aqueous solution 80.44± 4.12 4* 10-2± 0.0013 1.16± 0.14 *average ± standard deviation (n=3) figure 3. permeation profiles of ond through rat skin from f2, f5 and aqueous drug solution sem (figure 4) photograph of the selected formula (f2) exposed that ond loaded nps have uniform shape and regularly distributed particle size which are correlated with the results obtained by zeta plus analyzer. iraqi j pharm sci, vol.29(2) 2020 transdermal ondansetron nanoparticles 75 figure 4. sem of selected ond nanoparticles (f2) the thermal analysis is an important method to decide any likely interaction between the drug and excipients used. two endotherms peaks were achieved with ond at 202.53°c for drug melting and 111.46 °c (28) which corresponds to the dehydration process in ond, since it is a dihydrate (figure 5). the relatively decreased intensity of the endothermic peaks (figure 6) in physical mixture may be due to dilution effect with small shift in melting point of 3.16 °c indicating that there is no interaction between the drug and the polymer, the same result was recorded by pattnaik s. et al.(29). in dsc thermogram of ond loaded nanoparticles (figure 7), endothermic melting peak of drug at 202.53°c was completely disappeared, which indicate ond entrapment, presence of ond as molecularly dispersed form, and reduction in drug crystallinity in the nanoparticles matrix due to the solvent evaporation process, the same outcome were recorded by kharb v. et al (21). figure 5. dsc of ond pure drug figure 6.dsc of physical mixture (ond and eudragit rs 100) iraqi j pharm sci, vol.29(2) 2020 transdermal ondansetron nanoparticles 76 figure 7. dsc of ond nanoparticles x-ray diffractogram of ond (figure 8-a) shows sharp high intensity peaks at diffraction angles 2ө of 6°, 11.96°, 16.42°, 18.2°, 24°, 25.52° and 30.1° indicating that the drug is crystalline. xrd diffraction pattern of drugpolymer physical mixture (figure 8-b) shows several characteristic sharp peaks of ond with reduced intensity which can be attributed to mixing process. this proved that ond was still present in crystalline form in the physical mixture and no drug polymer interaction occurred. xrd analysis of ond-loaded nanoparticles did not show any characteristic peaks of ond at its particular diffraction angle and the absence of peaks suggested the absence of crystallinity i.e. amorphous form in ond nanoparticles (figure 8-c). figure 8. xrpd of (a) ons, (b) physical mixture of ond and eudragit rs100 and (c) selected formula (f2) of ondnps iraqi j pharm sci, vol.29(2) 2020 transdermal ondansetron nanoparticles 77 fourier transform infrared spectroscopy (ftir) the ir spectra of the pure drug exhibits spectra at 3398.67 cm-1 (oh stretching), 1632.5 cm-1 (c=o stretching) and at 754.54 cm-1 ( c-h bending). the spectra of physical mixtures of the drug with polymers shows simple shifting in position and intensity of characteristic peeks specially for oh stretching of ond-pva physical mixture which is due to h-bond formation (table 4), these outcomes indicating the compatibility of the drug with the polymers used in the formulation of nanoparticles (figures 911). table 4 . characteristic peak of pure drug and physical mixture no. type of peak pure drug (cm-1) ond-eudragit rs physical mixture (cm-1) ondpva physical mixture (cm-1) 1 oh stretching 3398.67 3400.64 3373.36 2 c=o stretching 1632.5 1633.48 1632.32 3 c-n stretching 1080.77 1082.34 1081.56 4 c=n stretching 1453.36 1453.97 1452.49 5 ch bending 754.54 755.56 755.44 figure 9. ftir spectra of ond pure drug figure 10. ftir spectra of (ond and pva) physical mixture iraqi j pharm sci, vol.29(2) 2020 transdermal ondansetron nanoparticles 78 figure 11. ftir spectra of (ond and eudragit rs 100) physical mixture conclusion ondansetron hcl nanoparticles were prepared successfully using nanoprecipitation method. drug: polymer ratio of the system were important to obtain nanoparticles with desired size. the encapsulation efficiencies were acceptable for all nanoparticles obtained. the release profile of the drug from nanoparticles were dependent on the type and concentration of the used polymers and the transdermal permeation study show that nanoparticles permeate efficiently than aqueous solution of the drug through the skin by approximately two fold. the controlled drug release of ond aimed for transdermal drug delivery could be obtained by using eudragit rs100 and eudragit rl100 polymers which can reduce dosing frequency, decrease side effects and improve patient compliance. the prepared ond 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under hypochlorhydria. asian journal of pharmaceutics (ajp): free full text articles from asian j pharm. 2016;10(3). 29. pattnaik s, swain k, mallick s, lin z. effect of casting solvent on crystallinity of ondansetron in transdermal films. international journal of pharmaceutics. 2011;406(1-2):106-10. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.28(2) 2019 fibromyalgia syndrome in women related with age and obesity doi: https://doi.org/10.31351/vol28iss2pp65-72 65 the assessment of some serum biomarkers of fibromyalgia syndrome in pre and postmenopausal women in relation to obesity eman s. saleh*,1 * department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq abstract the objective of this study is to evaluate the level of parathyroid hormone (pth), co enzyme q (co q) and total antioxidant status (tas) in serum’s women with fibromyalgia syndrome firstly, then to assess the influence of age and obesity on these biomarkers. this study was performed at rheumatology and rehabilitation consultation unit in baghdad teaching hospital. blood sampling from (59) women with fibromyalgia syndrome (fms) and (30) control be taken. the mean age of fms group was (42.22±15.34) years and for control was (40.7±18.22 years) and body mass index (bmi) for patients and control were (31.83 ± 5.05 and 30.09± 4.87) correspondingly. those participants were subdivided into four different groups according to menopausal status; fms before menopause were (30.14±10.58 and for control were 29.74± 12.21) years, post menopause fms age were (53.2 ± 16.18 and control were 47.8 ±12.51) years and bmi to measure serum (pth and co q) by enzyme linked immune sorbent assay (elisa) and (tas) by spectrophotometry. results analyzed by spss version 24, independent samples t-test and pearson’s correlation, p values < 0.05 were considered significant. the results record increment of serum’s pth levels significantly, decrement of co q (p value<0.05) and nonsignificant decreased tas (p value> 0.05) in fms comparing with control group. considering the age; pth serum level showed insignificant increase and decrease of (co q & tas) (p value >0.05& p value< 0.05) respectively in post menopause comparing with pre menopause. according to bmi; serum pth level record a significant raise in obese group (p value <0.003), reduction of serum co q significantly (p value< 0.05) and insignificantly for tas (p value> 0.05). the mean serum level of pth before menopause correlate significantly with tas (p value= 0.034) also with co q of both fms and control groups when bmi>30 (p value= 0.029 and p value= 0.043) respectively. in post menopause of both (fms and control) pth correlate positively (p value=0.05). independent ttest for pre and post menopause fms showed significance variance in tas only (p value=0.004). independent ttest for obese and nonobese fms showed significant variance in pth and tas (p value=0.032 and p value= 0.0001). in conclusion the obesity and menopause play an imperative role in the etiology of fms relative with serum’s biomarkers (pth, co q and tas). keywords: obesity, menopause, fibromyalgia syndrome, parathyroid hormone, total antioxidant status, co enzyme q. تقدير بعض المؤشرات الحيويه في مصل النساء قبل وبعد سن اليأس في متالزمة الم الليف العضلي وعالقتها بالسمنه 1*،ايمان سعدي صالح فرع العلوم المختبرية السريرية،كلية الصيدلة،جامعة بغداد،بغداد،العراق. * الخالصة مستوى هرمون الجار الدرقي ،االنزيم المشارك ف وحالة مضادات التأكسد الكليه في النساء العراقيات المصابات وضعت الدراسة لتقييم لتابعه ابمتالزمة الم الليف العضلي اوال ولبيان تاثيرالعمر ومؤشر كتلة الجسم عليهم. تمت الدراسة في شعبة امراض الروماتيزم والتأهيل الطبي المنحرف ±السيطره. معدل العمر ( نموذج من مجموعة 03( اضافة الى )95ب نموذج من دم المريضات البالغ عددهم) لمستشفى مدينة الطب بسح تقسيم المرضى واالصحاء الى اربعة مجاميع تم سنه على التوالي. 42.44±23.4و 49.02± 24.44القياسي للمريضات وغير المريضات هو ( سنه وبعد سن اليأس ) 44.44± 45.42و 43.92±03.42المريضات واالصحاء قبل سن اليأس) حسب العمر وكتلة الجسم ,معدل اعمار ( سنه تم قياس الهرمون واالنزيم في المصل بطريقة مقايسة الممتز المناعي المرتبط باالنزيم ) األليزا( بينما 44.94±24.2و 90.4±41.42 مقايسة مضادات االكسده بطريقة التعيين اللوني. 1corresponding author e-mail:dr.emansaadi@yahoo.com received: 10/ 3 /2019 accepted: 11/5 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp65-72 iraqi j pharm sci, vol.28(2) 2019 fibromyalgia syndrome in women related with age and obesity 66 ندع ظهرت النتائج زياده معنويه في مستوى الهرمون الجار الدرقي بالمصل ونقصان معنوي للألنزيم والمعنوي لمضادات األكسده.بالنسبه للعمر ين بمقارنة مجموعتي )المريضات مع السيطره(. لوحظ زياده غير معنويه للهرمون و قله لألنزيم, نقصان معنوي لمضادات األكسده عند المقارنه تختالف غير القبل و بعد سن اليأس. بينت النتائج هناك زياده معنويه عند النساء البدينات المصابات بالمتالزمه عند مقارنتها بمجموعة السيطره لكن ا ملموس في مجموعتي البدينات وغير البدينات. وأيضا سجلت حالة مضادات األكسده قله معنوية في مجموعة البدينات فقط. في مصل (p value= 0.034)وضحت النتائج هناك عالقه معكوسه معنويه بين مستوى الهرمون الجار الدرقي وحالة مضادات األكسدة الكلية باألعتماد على مؤشر كتلة الجسم الكثر من (p value= 0.029 and p value= 0.043)واألصحاء وايضا مع األنزيم ف مجموعتي المريضات في قياس الهرمون بين المجموعتين اعاله بعد سن اليأس. (p value=0.05). ايضا هناك عالقه معنويه حرجه 03 عالقه وجدت فقط. للمريضات االكسده لمضادات معنويه عالقه وجود ينت ( سنه ب99وبعد 29عالقة التباين بأتختبار تي لما )قبل واالنزيم للمريضات البدينات وغير البدينات اضافة الى وجود تباين واضح بالهرمون ومضادات االكسده لكال المجموعتين الهرمون بين معكوسه (p value=0.032 and p value= 0.0001) األنزيم المشارك ف. ، حالو مضادات التأكسد الكلية،الهرمون الجار الدرقي،متالزمة ألتهاب الليف العضلي ،سن اليأس، المفتاحيه: السمنهالكلمات introduction fibromyalgia syndrome (fms) is a chronic disorder which has been defined by a history of widespread pain and the presence of marked tenderness to palpation at standard anatomically defined tender points (1). fibromyalgia (fm) was recognized as a true syndrome with the publication of the american college of rheumatology (acr) classification criteria in 1990, which were updated in 2010 (1, 2). the parathyroid glands maintain the serum levels of calcium (ca) and phosphorus (p). calcium plays a dynamic role in metabolic processes (3). there is a large symptom overlap between hyperparathyroidism (hp) and fm so the subjects experience “fatigue, musculoskeletal pain, headache, cognitive dysfunction, and mood disturbance” (4). coenzyme q ( co q ) or q10 is a small fat-soluble, vitamin-like substance located in mitochondria that transfers reducing equivalents from complexes i and ii to complex iii of the respiratory chain. some chronic disease conditions are also thought to either reduce the biosynthesis and/or increase the demand for co q in the body, but there are no definite data to support these claims (5, 6). reactive oxygen species (ros) is produced in metabolic and physiological processes. under certain conditions, the increase in oxidants or decreased in antioxidants that cannot be prevented, and the disorders related to oxidative stress would develop. antioxidant molecules prevent or inhibit these harmful reactions. the antioxidant system consist of enzyme and nonenzyme anti-oxidants. antioxidants prevent free radical induced tissue damage by preventing the formation of radicals, scavenging them, or by promoting their decomposition (7-9). the definition of tas is the sum total of endogenous and food derived antioxidants of the extra cellular fluid of an individual (10). aim of study to estimate serum biochemical markers {parathyroid hormone (pth), co q and total antioxidant status (tas)} in women with fms and evaluate the effect of age and obesity on these biomarkers. subjects, materials and methods the work was started in september/ 2014 to january/ 2015 at baghdad teaching hospital / rheumatology and rehabilitation consultation unit, by the assortment of women with fms which was accomplished under observation of rheumatologist according to american college of rheumatology (acr) criteria (11). in this study, blood sample were withdrawn from (59) females with fms and (30) control subjects without fms in order to obtain the serum after centrifugation of the clotted specimens. all applicants were classified consistent with their menopausal status before 45 years refer to pre menopause and after 55 years be in post menopause as shown in (figure 1). note: each subject must follow above condition. the mean age of fms before menopause were (30.14±10.58, control were 29.74± 12.21) years; the post menopause fms were (53.2 ± 16.18 and control were 47.8 ±12.51) years. determination of (bmi) was by dividing body weight in kilograms over the height in meters square {bmi = weight (kg) / height (m2)} (12) (figure 2). the assessment of serum pth (demeditec diagnostics gmbh/germany) and co enzyme q (cusabio biotech co., ltd, /china) were done by enzyme linked immune sorbent assay (elisa); this assay employs the competitive inhibition enzyme immunoassay technique. the colorimetric method randox kit used to assess total antioxidant status (tas) (13-15). inclusion and exclusion criteria for subjects selected to participate in the study was shown in table 1. iraqi j pharm sci, vol.28(2) 2019 fibromyalgia syndrome in women related with age and obesity 67 table 1. the selection criteria for fms and control women inclusion criteria exclusion criteria the age range of subjects 20-60 years. the mean age (m) ±standard deviation of mean (sd) of the patients was (42.22± 15.34) years and the control group was (40.7± 18.22) years as well as complete blood picture and erythrocyte sedimentation rate (esr) tests were within normal range all women had any endocrinopathiy, autoimmune, inflammatory, rheumatologic disorders, chronic and systemic disease in addition the pregnant and lactating subjects. figure 1 refers to distribution of subjects shared this work classified according to their ages less than 45 years as premenopause and more than 55 years as post menopause while figure 2 expressed the classification of both fms& control along with their bmi as normal bmi if less than 25 and obese bmi more than 30. figure1. demographic distribution of volunteers (fms and control) according to age in this study. figure2.demographic distribution of (fms and control) women according to bmi in this study. the statistical analysis was done by using spss (version 24) for values of measurement of serum biomarkers levels which expressed as mean± sd. also independent ttest sample were used to complete the analysis and pearson’s correlation between studied biomarkers in different groups. p value is significant < 0.05. the results this trial showed a significantly (p value < 0.01) increased serum level of pth in fms when compared with control, while the significant decrease of co q (p value < 0.01) and insignificant (p value > 0.01) decline in tas for all subjects as shown in (figures 3,4 and 5, respectively). the mean age ± sd of both fms and control were (42.22±15.34) and (40.7±18.22) years and bmi were (31.83±5.05) and (30.09 ±4.87) kg/m2 respectively. figure 3. the mean serum pth level (pg/ml) in fms and control groups (p value<0.01) figure 4. the mean serum co q level (ng/ml) in fms and control (p value <0.01) figure 5.the mean serum tas level (mmol/l) in both fms and control groups (p value > 0.05) subject no. =89 fms no. = 59 42.22± 15.34 y post menopause= 41 age>55 years pre menopause=18 age< 45 years control no. = 30 40.7± 18.22y pre menopause=25 age< 45 years post menopause= 5 age> 55 years subject no. =89 fms no. = 59 bmi= 31.83± 5.05 fms with bmi ˃30 kg/m2 no.= 39 fms with bmi˂25 kg/m2 no.=20 control no.=30 bmi= 30.09± 4.87 bmi ˂25 kg/m2 no.= 25 bmi˃30 kg/m2 no.=5 iraqi j pharm sci, vol.28(2) 2019 fibromyalgia syndrome in women related with age and obesity 68 above groups were discrete into four subdivisions. the first two groups demonstrate the levels of serum biomarkers in fms and control as post menopause while reminder two groups in pre menopause, these information are listed in table 2. the significant increasing of serum level of pth in both age groups with fms (p value< 0.005) , significant decreasing of serum co q level (p value< 0.005 ) and significant decrease in tas in post menopause group but insignificant in pre menopause groups when compared with control (p value< 0.05 and p value>0.01 respectively) table 2. the serum level of biochemical markers in both fms patients and control distributed according to their ages represented by (mean± sd). age > 55 years fms patient no. =41 control no.=5 serum pth: pg/ml coq: ng/ml tas: mmol/l pth:pg/ml coq:ng/ml tas:mmol/l mean± sd 52.15±16.26** 2.36± 0.68** 1.025± 0.06* 30.20±12.51 5.51±2.07 1.39±0.15 age <45 years fms patient no.=18 control no.=25 serum pth:pg/ml coq:ng/ml tas:mmol/l pth:pg/ml coq:ng/ml tas:mmol/l mean± sd 49.07±24.29** 2.38±0.72** 1.12±0.16 29.74±14.21 5.24±2.63 1.43±0.2 table 3 shows the effect of obesity on serum biomarkers levels in normal and obese (fms and control) groups. the results appear that the value of pth in serum was increased significantly and highly significant (p value<0.01, p value <0.005) in both normal and obese respectively when compared with control groups. the serum level of co q shows high significant decreasing in normal and obese fms groups, insignificant decreased for tas in comparison with control group (p value< 0.005& p value>0.05). table 3.the serum level of biochemical markers (mean± sd) in fms and control distributed according to their bmi. bmi <25 fms patient no.= 20 control no. =25 serum pth:pg/ml coq:ng/ml tas:mmol/l pth:pg/ml coq:ng/ml tas:mmol/l mean± sd 35.42±12.04* 2.20±0.85** 1.36± 0.1 27.80±12.89 4.12±0.4 1.72±0.13 bmi >30 fms patient no.= 39 control no.=5 serum pth:pg/ml coq:ng/ml tas:mmol/l pth:pg/ml coq:ng/ml tas:mmol/l mean± sd 52.57±22.65** 2.4±0.68** 1.04±0.09 30.22±14.11 5.51±2.7 1.35±0.14 the mean serum level of pth before menopause correlate significantly with tas (p value= 0.034) also with co q of both fms and control groups when bmi>30 (p value= 0.029 and p value= 0.043) respectively. in post menopause of both (fms and control) pth correlate positively (p value=0.05). independent ttest for pre and post menopause fms showed significance variance in tas only (p value=0.004). independent ttest for obese and nonobese fms showed significant variance in pth and tas (p value=0.032 and p value= 0.0001). discussion fibromyalgia syndrome (fms) is classified as a first-order syndrome or a symptom complex with unknown or unclear etiology, heterogeneous pathogenesis and a defined phenotype; second order syndromes (sequences) are defined by unknown etiologies, homogeneous pathogenesis and defined phenotypes; third-order syndromes are defined by homogeneous etiologies, unknown or insignificant pathogenesis and defined phenotypes types (16). iraqi j pharm sci, vol.28(2) 2019 fibromyalgia syndrome in women related with age and obesity 69 the results show hyperparathyroidism of fms group if comparing with control so it is in concomitant with the result of armagan et al (17) measured serum pth, calcium, phosphorus and active vitamin d showing in significant increase of pth. another study done by shaheen et al through approved the deficiency of vitamin d in fibromyalgia (18). ferrari r et al studied the rate of prevalence related with primary hyperparathyroidism in widespread and localized musculoskeletal pain were 6.4, 5.5, and 6.1 %, respectively (19). previous studies (20, 21) show fms have a predisposition to osteopenia. measurement co q, in this study showed deceasing in serum level of fms so this clarify the mitochondrial dysfunction of muscle & neural cell which is a part of electron transport chain (etc), affect the synthesis of atp pools and finally lead to widespread pain (22). study on osteoarthritis demonstrated the same results (23). in us (24); co q supplements were set as foods not as drug. about tas and oxidative stress: this study illustrate that the level of tas was decreased insignificantly because of presence of reactive oxygen species (ros) in excess result in cell damage and lipid peroxidation (25). chung et al study match with current study in relation with oxidative stress and fms (26).turkish authors compare between fms& obstructive sleep apnea syndrome in assessing anti-oxidant enzymes (catalase, superoxide dismutase and glutathione peroxidase) were significantly lowered at the same time malondialdehyde was increased so this finding like the present study’s result (27). the role of oxidants/antioxidants, mitochondrial dysfunction, and autophagy in fibromyalgia was published recently (28). the effect of age on serum biochemical markers showed that the variation between pre and postmenopausal groups were unclear insignificant but when compare with control group demonstrate significant differences this finding belongs pth similar to recent study done by guncha k and gagan d that showed significant correlation of age with serum pth in both pre-menopausal and postmenopausal women (29) because an impairment of ovarian function in postmenopausal women alter the metabolism of ca and therefore reduce bone mass (30). borgia et al. demonstrate the role of hyperparathyroidism in musculoskeletal pain of fibromyalgia (31). in correspondence of present study with cammozi et al and safi et al observations that parathyroid hormone was significantly low in postmenopausal women (32,33). eastell et al and khosla et al in different studies described the progress of age lead to increase turnover of bone due to raise the blood level of parathyroid hormone and lower estrogen (34, 35). the role of estrogen in inhibiting interleukin six was decreased with age so lead to prolongation of osteoclast’s life span (36). sainaghi et al demonstrated the relation between low levels of vitamin d in different rheumatic diseases with higher production of pth (37). recent turkish study bone mass and bone turnover measurement for premenopausal women with fms and degenerative disc disease control patients; the authors demonstrated that presence of depression, general pain and other clinical findings in fms created women to be susceptible to osteoporosis (38). oxidative stress (os) and co q play a role in neuromusculoskeletal conditions such as fm so the providing of antioxidants kept tissues faraway from unpleasant effects of os (39-42). in egyptian study by soliman et al showed that the level of (os) increased with decreased of antioxidant capacity significantly these findings in concomitant with the present study in premenopausal age (43). another spanish study determined total anti-oxidant capacity in 82 postmenopausal fm women and compared with 25 apparent healthy as control their results showed significantly decreased tas when compared with controls( zinc, uric acid, ferritin, iron, catalase, sod, gpx ) as a result of enhancement of os in fm because of elevation of protein peroxidation and oxidative dna damage significantly and generated advanced glycation endproducts (ages) so this modified proteins became more resist to be digest, as well as excite cytokines, adhesion molecules, and growth factors expression through nfκb stimulant(44). niklowitz et al mentioned the blood level of co q was decreased in old german people so the redox status be shift to oxidized direction (45). the impact of obesity on current fms study and comparison between obese and non obese, looked through elevation of pth increased significantly at the same time decreased of tas serum level. this syndrome is one of musculoskeletal disorder with obesity agree with old study by christensen et al in their demonstrating the combination of obesity with certain rheumatologic conditions specifically knee osteoarthritis (46). other studies showed the effects of obesity on clinical examination of fms by measuring tenderness, symptoms, quality of life (47, 48). okifuji et al assessed catecholamines, cortisol, c-reactive protein and interleukin-6 as neuroendocrine indices as well as measuring some clinical manifestation such as symptoms, treadmill testing and indices of sleep in thirty eight fms patients (49).there are different mechanism to explain the association of fibromyalgia and obesity in accordance to endocrine dysfunction; one of them is impairment of growth hormone and insulin growth factor-1 (igf-1) secretion this finding illustrated in fm patients by bennett et al. and in obese subjects by maccario et al.(50-52). authors in 2008 published their results that demonstrate bone mineral density and serum level of osteocalcin and igf-1 which decreased significantly and insignificant decrease calcium, phosphorus, vitamin d3 and pth in premenopausal mailto:rferrari@shaw.ca http://www.ncbi.nlm.nih.gov/pubmed/?term=sainaghi%20pp%5bauthor%5d&cauthor=true&cauthor_uid=22019806 iraqi j pharm sci, vol.28(2) 2019 fibromyalgia syndrome in women related with age and obesity 70 fms with mean bmi 25.63± 3.14 kg/m2 (16). in spain; aparicio et al. observed the effect of obesity and over weight on pain, fatigue, stiffness and physical functioning and play a role in progress of these fm symptoms (53). the association between pain of fm and obesity showed in old study due to central sensitization (54, 55). kim et al mentioned that the greater bmi had greater fibromyalgia-related symptoms through their study on wide range of bmi by classify the patients into four groups (nonobese, overweight, moderately obese and severely obese) (56). jameel m. g. et al demonstrated the effect of obesity on serum total calcium, myeloperoxidase and vitamin d3 in iraqi fms patients in addition to fetuin –a and thyroid function test in two different publishing studies (57, 58). in the longitudinal analyses demonstrate an association between weight loss of obese middle-aged patients with localized weightbearing joints pain by studying c reactive protein, interleukins, tumor necrosis factor-α and interferonγ(59). brazilian authors studied the effect of overweight and obesity on serum levels of some adipokines in fm middle aged women (60). turkish study include 124 fms women classified into normal bmi, overweight and obese to assess pain, tender point count, disease activity, anxiety and depression in ages between 1855 years (61). cross sectional study in turkey was done to evaluate the effects of obesity and over weight on fms severity in relation to general health and psychological status (62). conclusion: the study clarify the influence of progressing age in general but chiefly after menopausal age when compared with age less than 45 years as well as obesity on serum level of pth, co q and tas in iraqi fms women through enhancing the oxidative stress and tissue damage as result directly or indirect accelerating pathophysiology, etiology and severity. thus fms women must recommended anti-oxidant supplements, vitamin d and minerals especially calcium, as well as advise them to improve lifestyle in order to decrease the undesirable effects of fms. references 1. anil kumar jain, bruce m. carruthers: fibromyalgia syndrome: a clinical case definition 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https://www.ncbi.nlm.nih.gov/pubmed/?term=fischer%20a%5bauthor%5d&cauthor=true&cauthor_uid=27257350 https://www.ncbi.nlm.nih.gov/pubmed/?term=palussen%20m%5bauthor%5d&cauthor=true&cauthor_uid=27257350 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4865593/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc4865593/ http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.19(2) 2010 gravimetric estimation of caffeine 48 gravimetric estimation of caffeine in different commercial kinds of tea found in the iraqi market maha n. hamad* ,1 and dhuha a. abdul-hussain* *department of pharmacognosy, college of pharmacy, university of baghdad, baghdad,iraq . abstract caffeine (1,3,7-trimethylxanthine), which is the most widely consumed stimulant in the world, had been isolated and estimated gravimetrically in fifteen different commercial kinds of tea found in the iraqi market.the kinds of tea were chosen according to their differences in the degree of fermentation and the method of processing i.e. black , gray and green . the isolated caffeine was identified by melting point, sublimation, tlc, chemical tests, uv , ir , hplc and chno analysis. key words: caffeine, purine, tea. الخالصة م, ذم فصهه وذعٍٍه كمٍره تطشٌقح ثالثً مثٍم صاوثٍه( انزي ٌعرثش مه أكثش انمىاد انمىثهح إسرعماالً فً انعان – 7, 3, 1كافاٌٍه ) وصوٍح فً خمسح عشش وىعاً مخرهفاً مه انشاي انمىجىد فً األسىاق انمحهٍح. ذم أخرٍاس ومارج مه انشاي عهى دسجاخ مخرهفح مه انرخمٍش انزوتان ,. ذم ذشخٍص انكافاٌٍه انمعضول تطشق مخرهفح مىها قٍاط دسجح اي االسىد وانشصاصً واالخضش و طشق مخرهفح نهرحضٍش وكشوماذىغشافٍا انطثقح انشقٍقح , وفحىصاخ كٍمٍاوٌح , وطٍف األشعح فىق انثىفسجٍح واألشعح ذحد انحمشاء انرسامً , .وانكشوماذىغشافٍا انسائهح راخ انكفاءج انعانٍح وحساب وسة عىاصش انكشتىن ,انهٍذسوجٍه,انىٍرشوجٍه واالوكسجٍه introduction thea or tea consists of the prepared leaves and leaf buds of camellia sinensis (formerly known as thea sinensis) of the theaceae family. there are three main commercial types of tea: green , oolong (gray) and black, depending on the method of processing. the leaves may be fermented or left unfermented. fermented teas are referred to black tea, unfermented teas as green tea and partially fermented teas as oolong tea.black tea is prepared by heaping the fresh leaves until fermentation has begun, then they are rapidly dried artificially with heat, while green tea is prepared by rapidly drying the freshly picked leaves in copper pans over a mild artificial heat, or the leaves are often rolled in the palm of the hand as they dry. gray tea is partially fermented by heaping then they are dried on artificial heat. tea contains caffeine (theine) and small amounts of adenine, theobromine, theophylline, gallotannic acid and volatile oil. 1,2 caffeine (1,3,7-trimethylxanthine), the molecular formula of which is c8h10n4o2 , is the most widely consumed stimulant in the world can be considered to be constructed from the purine ring system, which is important biologically, being found in nucleic acids and nucleotide and in few organisms as alkaloids. 3 caffeine was first discovered in tea in 1827, and was named theine , and later it was found in mate , coffee and various other plants and the term theine was then dropped. 4 purines are considered pseudo alkaloids since they are not derived from an amino acid precursor. 5 n n n n h nh h n n n h n n n o o o h3c-n ch3 ch3 o purine xanthine caffeine 1corresponding author email : manoha_1957@yahoo.com received : 28/4/2010 accepted : 8/8/2010 iraqi j pharm sci, vol.19(2) 2010 gravimetric estimation of caffeine 49 caffeine acts as a cns stimulant , mild diuretic, it increases the heart rate and blood pressure and stimulate gastric secretions. it also acts as a natural pesticide that paralyze and kills certain insects feeding on the plants. 6,7 caffeine with uv can kill some kinds of algae and there are evidences that it enhances mutations in bacteria and viruses and also induce chromosome damage. 8,9,10,11 caffeine is an ingredient of several dozen proprietary products, for the most part, these combination with acetyl salicylic acid, ascorbic acid, codeine, paracetamol, and other analgesics and antipyretics.caffeine is found in a number of beverages ingested by people in addition to tea as coffee, and to some extent cocoa. other , less commonly used sources of caffeine include the plants guarana, and yerba mate' which are sometimes used in the preparation of teas, and recently ,energy drinks. tea leaves contain 1-4% caffeine, while coffee 1-2% yet a cup of brewed coffee contains about 100-150 mg caffeine while a cup of tea contains 60-75 mg. caffeine is also a common ingredient of soft drinks such as cola. soft drinks typically contain about 10-50 mg of caffeine per serving . the range of caffeine contents in various beverages is shown table 1 table 1 : range of caffeine in various beverages approximate caffeine content of various beverages range of mgs of caffeine coffee (5oz. cup) 40-170 soft drinks (12 oz. can) 10-50 black tea (one tea bag) 25-110 oolong tea (one tea bag) 12-55 green tea (one tea bag) 8-30 decaffeinized tea(one tea bag) 1-4 energy drinks ( 12 0z. can) 75-90 in this paper we have estimated caffeine gravimetrically in fifteen different kind of tea found in the market black, gray and green tea . materials and method  samples of tea were chosen randomly to represent black, gray and green tea in the form of tea bags or unpacked form.  all reagents are anhydrous solvents were of analar type and generally used as received from the commercial suppliers.  silica gel used in the form or ready made aluminum plates of silica gel gf254 , merck co.  uv was run in methanol , ir was run in kbr disk.  hplc was done using knauer/ germany hplc.  standard caffeine is from evans medical ltd , liverpool. isolation of caffeine 25 gm of tea were boiled with 200ml of water for fifteen minutes in a covered beaker . the extract was filtered through muslin and the mark was re boiled with 120ml of water for five minutes, filtered and the mark over the muslin was washed with 70ml of boiling water, the muslin was then squeezed till exhaustion. the combined extracts were cooled and mixed with 4gm of sodium carbonate with stirring, then transferred to a separatery funnel and partitioned with three successive portions of methylene chloride each of 50ml (i.e. 3x50ml), each time the funnel was inverted back and forth ten times. the methylene chloride layers were combined together and dried over anhydrous sodium sulfate , filtered and evaporated to dryness under vacuum. the obtained caffeine was re crystallized from boiling ethanol, filtered and weighed. the percentages of caffeine was calculated as w/w. the extraction procedure is shown in diagram(1). diagram 1 : isolation of caffeine from tea iraqi j pharm sci, vol.19(2) 2010 gravimetric estimation of caffeine 50 two samples of each kind were used and the average weights of the isolated caffeine were taken for calculation of the percentages and comparison. identification of caffeine the isolated caffeine was identified by several methods :  it was identified by measuring its melting point and compare it with standard caffeine and also using a mixed sample from isolated caffeine and the standard . 12 the results are shown in table (2). table 2 : melting points of isolated and standard caffeine melting point sample 236.7°c isolated caffeine 237.3°c standard caffeine 237°c mixed isolated and standard caffeine then caffeine was identified by two chemical tests : 1murexide test : isolated caffeine gave a purple color. 2isolated caffeine was treated with hydrogen peroxide and 2% hcl. after evaporation to dryness , a bright red color was obtained. the color turn purple upon addition of drops of 5% ammonia. 13 also caffeine was identified by sublimation and this process was achieved by introducing a small quantity of caffeine in a porsalen dishand covered with a watch glass , the porsalen dish was subjected to heat while the watch glass was covered with a plastic sack containing ice .upon heating caffeine started to sublime and condense on the lower surface of the watch glass. caffeine was also identified by tlc using silica gel gf254 plates developed in three different mobile phases and comparing the rf values of isolated caffeine with standard using single and mixed spots , and detection was done under uv254nm. 14,15,16,17 mobile phases used are: mobile phase i : ethyl acetate : acetic acid 95:5 . mobile phase ii : chloroform : ethyl acetate : formic acid 5: 4 : 1 mobile phase iii : petroleum ether : methylene chloride : ethyl acetate 1: 1 : 2. the result of tlc are shown in table (3) table 3 : rf values of isolated and standard caffeine standard caffeine rf values isolated caffeine rf value mobile phase 0.257 0.226 i 0.516 0.490 ii 0.110 0.110 iii  the uv absorption spectrum exhibits a pair of absorption bands peaking at (209) and (272)nm with a shoulder between them 18 . the uv spectrum of the isolated caffeine is shown in fig.(1) figure 1 uv spectrum of the isolated caffeine also caffeine was identified by ir 19 and the spectrum is shown in fig. (2) figure 2 : ir spectrum of the isolated caffeine iraqi j pharm sci, vol.19(2) 2010 gravimetric estimation of caffeine 51 also caffeine was identified by chno analysis ,whereby the percentage of each element measured and compared with the calculated one.the results are shown in table 4. table 4 : chno analysis of caffeine found percentages calculated percentages element 49.877 49.484 carbon 5.199 5.155 hydrogen 29.109 28.866 nitrogen 16.709 16.495 oxygen caffeine was finally identified by hplc 18 using c18 5x150mm column and a mobile phase composed of methanol/water 90:10 with flow rate of 0.8ml/minute and detection with uv detector at 275nm. the retention time of the isolated caffeine was compared with that of the standard. the results are shown in figures 3 and 4. figure 3 : hplc of isolated caffeine figure 4 : hplc of standard results and discussion the average weights and percentages of the caffeine isolated from each kind of tea are shown in table (5) and fig. (5). the idea of this method of isolation of caffeine is to extract the water soluble materials in the tea leaves in a hot water . (the solubility of caffeine in water is 22 mg/ml at 25 o c , 180 mg/ml at 80 o c and 760 mg/ml at 100 o c ) . the caffeine is extracted from the water after cooling with dichloromethane (140 mg/ml) than in water (22 mg/ml), it readily dissolves in the dichloromethane.however, the tannins are slightly soluble in dichloromethane but upon addition of sodium carbonate to the extract the tannins will be converted to phenolic anions (since phenols are acidic enough to be converted to phenolic salts i.e. deprotenation of oh group ) upon addition of sodium carbonate). the phenolic salts thus formed are not soluble in dichloromethane , soluble in water, as shown below: aroh + na + 2co3 -2 → arona+ + na+ hco3 tannins soluble tannins salts in water, dichloromethane soluble in water insoluble in dichloromethane iraqi j pharm sci, vol.19(2) 2010 gravimetric estimation of caffeine 52 table 5 :the percentage of caffeine in different kinds of tea % of caffeine weight of caffeine in 25 gm of tea tea brand no 1.924 1.40 1.288 1.240 1.176 0.952 0.800 0.720 0.360 0.440 1. 364 1.308 0.884 0.520 0.480 0.481 0.351 0.322 0.310 0.294 0.238 0.2 0.180 0.090 0.110 0.341 0.327 0.221 0.130 0.120 al-otoor (black) al-rabeea (black) mahmood (black) ahmad (black ) al-wazza (black ) al-tuffaha (black ) ahmad (black , tea bags ) ration tea ( black ) lipton (black , tea bags ) al-okozay ( gray , tea bags ) al-attar ( green , tea bags ) lipton (green , tea bags ) ahmad (green , tea bags ) green tea (un packed) alokozay ( green , tea bags ) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 figure 5 : percentages of caffeine in different kinds of tea caffeine being a xanthine derivative was first identified by the murexide test. the core of the reaction is apparently that caffeine through a number of steps , is oxidized into the intermediate that ultimately condenses to murexoin. the ammonium salt of murexoin is the principal contributor to the purple color of the final solution. 20 the quantitative differences obtained in different kind of tea is probably due to the method of processing of each kind since caffeine sublimes with out decomposition upon exposure to heat, therefore it should be expected that caffeine could be lost during fermentation and processing. also the differences of caffeine quantity and consequently the percentage may be due to different time of harvesting of leaves of the plant. acknowledgement we are deeply greatfull to mr. dhulfiqar a. abid ( msc.) and mrs. sahar m. shakir ( msc. ) and mr. zaid m. abdul-khalik for running the uv , ir , and hplc spectrum. references 1. tyler v.e. , brady l.e., "pharmacognosy" 9 th edition, lea and febiger philadelphia 1988. 2. trease g.e. and evans w.c., "pharmacognosy" 15 th edition , wb saunders co. ltd london 2002. 3. dey p.m. , harborne j.b. , "plant biochemistry" academic press 1997. 4. weinberg ba., bealer bk., "the world of caffeine" routledge, newyork and london 2001. 0 0.5 1 1.5 2 2.5 % p e rc e n ta g e o f ca ff e in iraqi j pharm sci, vol.19(2) 2010 gravimetric estimation of caffeine 53 5. cordell g.a., "introduction to alkaloids" john wiley and sons , inc canada 1981, 6. 6. jinno, d. , "comperhensive medical chemistry" pergamon press 1996. 7. eaton k., "caffeine could be healpful" las vegas review journal , 2010, 23. 8. srivastava b.s., kumar h.d., singh h.n. , "the effect of caffeine and light on killing of the blue-green algae anabaena doliolum by uv radiation" archives of microbiology 1971, 78(2), 139-144. 9. siderpoulos a.s., shankel d.m., "mechanism of caffeine enhancement of mutations induced by sub lethal uv dosages" j. bacteriology 1968, 96(1), 198204. 10. lytle c.d., "the effect of caffeine on the survival of uv-irradiated herpes simplex type 1 virus" j. general virology 1974, 24, 381-383. 11. cremer c., cremer t., and gray j. w., "introduction of chromosome damage by uv light and caffeine" cytometry 1982, 2(5), 287-290. 12. the merck index , 8 th edition merck and co. inc. rahway, nj, usa 1996. 13. cave' a., "pharmacognosy , phytochemistry, medicinal plants" 3 rd edition , intercept ltd, england 1995, 882883. 14. fenske m., "caffeine determination in human saliva and urine by tlc anduv absorption densitometry" chromatographia 2007, 65(3-4) , 233-238. 15. pavlik j.w., "the detection of caffeine in commercial products" j. of chemical education 1973, 50(2), 134. 16. franciszek b., sabina a., urszula h., "densitometric determination of caffeine in tea beverages after tlc e-separation" chemia analityczna, 2006, 51(4), 603-611. 17. stahl, e., "thin layer chromatography" springer-verlag berlin. heidlberg n.y. 1969. 18. pavlova v., petrovsk s., "simultaneous determination of amphetamine, methamphetamine, and caffeine in seized tablets and hplc" acta chromatographica, 2007, 18, 157-164. 19. cook d., regnier z.r., "the infrared spectra of caffeine salts" canadian journal of chemistry, 1967, 45, 2895-2897. 20. pedersen o., "pharmaceutical chemical analysis : methods for identification and limit tests" 2006, 86-88. ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 fo rmulation of rifampicin sus pension 1 formulation of rifampicin suspension lubna a. sabri*1 , alaa a. abdul-rasool** and muayad a. shehab** * de partm ent of pha rm a ce utic s, colleg e of p ha rm a cy, univ ersity of b agh da d bag hda d-iraq * *de pa rtm en t o f ph arm ace utics, co lle ge o f pha rm acy , un iv ersity of bag hd ad ,bag hd ad-ira q abstrac t rifa mpicin is the drug of c hoice in treatment of tube rc ulosis. als o, it is e ffec tive in trea tme nt of various bacterial infe ctions.this study was c arried out to pre pare a s ta ble sus pension for rifampicin through prepa ra tion of diffe re nt formula s of rifampicin aqueous sus pension either a s rea dy to use or as gra nular powder to be reconstituted.the selec te d formula (a) was e valua te d and c ompared with comme rc ia l brand of rifampicin (rifac tine ®) as a re fe re nce through meas uring their diss olution rates and othe r physica l prope rties.the res ults indicated tha t the selec te d formula had be tter dis solution ra te compared with the referenc e sus pension and the rheogram showed that the se le cted formula was les s vis cous tha n the re fe rence one.als o, it wa s found that the gra nular rifa mpicin was more stable than the ready to use suspe nsion, sinc e the e xpira tion date of granula r rifampicin was 2.6 yea rs , while the expiration date of re ady to use s uspens ion was 1.8 yea rs . k ey words :rifampicin , suspending a ge nt ,powder for rec ons titution , aque ous s uspe ns ion الخالصة مختلفة رن الرئوي وااللتهابات البكتيرية ال عالج التد ي ن هو الدواء المفضل ف سي ريفامبي راء هذه الدراسة . إن ال تم إج ق جـاهز عـل شـكل م ا ب ـم مبيسـين أ ق الريفا معـل ة ل ختلـف ة م ركيبـي ر صـيغ ت من خالل تحضي معلق ثابت للريفامبيسين ر لتحضي وق جاهز للتعل كمسح و مال أ ركيبية المختارة . يقلالستع مستحضر المرجع ) أ(أن الصيغة الت مع ال مقارنة رت للتقييم وال اختي ن( خرى من) الريفاكتي ص الفيزيائية األ وا رر و الخ رعة التح رة . خالل قياس س مختا كيبية ال غة التر أوضحت النتائج أن الصي ر عيين التدفق أظه و ت رعة تحرر مقارنة مع المستحضر المرجع جة من أعطت افضل س أن الصيغة المختارة هي اقل لزو مرجع حيث . المستحضر ال علق الجاهز لالستعمال ٬ من الم ستقرارية حبيبي هو اكثر ا مبيسين ال معلق الريفا ن كذلك وجد أ و ي هي معلق الحبيب عول ال مف هاء مدة انتهاء مف) ٦,٢(أن مدة انت عمال هيسنة بينما علق الجاهز لالست ول الم . سنة) ۸,١(ع in troduction an oral p harma ceutica l susp ens ion ha s lo ng be en one of the mo st fav ora ble dos age fo rms fo r ped ia tric patients o r pa tients una ble to to le ra te s olid dos age fo rms (1). the re a re ma ny phys ic al and che mical co nside ra tions in the p re paration and de velop ment o f a s us pension to s atisfy its pharmace utical requireme nts. so me suspe nd ing age nts are ge nerally add ed to the disp ersion me dium in o rd er that their structure s he lp to mainta in unifo rm disp ersibility (2) o r to preve nt c aking o f the d rug p article s d uring shelf-life (3). rifampicin is bactericidal agent aga inst wide ra nge o f microorga nism (4). it is o ne of the ve ry s lightly s oluble drugs, thus is suitab le fo r s uspension dosa ge form. b ut rifa mpicin is poo rly wetted with water d ue to its hyd ro phobic na ture . el-ba ry et a l. s tudied the wettab ility of rifampic in p owde r using diffe re nt c onc entrations of v ario us s urfa ctants and po lyhydroxy co mpound s .the we ttab ility of rifampic in was found to be d irec tly p rop ortional to surfac ta nt hlb a nd c onc entration (5). in this s tudy, rifampicin is formula te d as an a que ous s usp ension either a s re ady to use or a s dry po wde r and the s elec te d fo rmula is co mpa re d with the re fe re nc e suspe ns io n. experimenta l ma terials and eq uipmen ts rifampicin powd er, po lyso rb ate 80 , ra spberry flav or, xantha n gum, guar gum , methylpa ra be n, prop ylparab en (s upp lied b y sama ra drug indus tries (sdi)). 1corresp ond in g author ema il lub naha ni_95@yahoo .comlub naha ni_95@yahoo .com rec eived 26-1 1-200 5 accepted 24-5-2 006 mailto:lubnahani_95@yahoo.com ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 fo rmulation of rifampicin sus pension 2 rifa ctine® susp ens ion ( supplied by me dical union p ha rma eutica l, egypt) sod ium sa cc harin, sorbitol 70%, so dium citra te (supplied by ibnse ina drug re se arch cente r, baghd ad). citric ac id monohydrate (al-rahma pha rmace utic al co., j ordan). disod ium ed etate, so dium me ta bisulp hite , hyd ro chloric acid, co lloida l silic one d io xide (ae ro sil), methylc ellulo se , s odium ca rb oxymethylc ellulo se (bdh chemica ls ltd , poo le , england). spe ctro photomete r (pye -unicom-sp -8 -1 00 mod el 2 92mk, engla nd ). disso lutio n app aratus (erewka g.m.b.h. type dt6, w.germany). ph mete r (orchidis la boratories, france). visco meter (cole-pa rme r, rotationa l visc omete r u.s.a). ove ns (memme rt 85 4 schwaba ch, w.ge rmany). method o f pr ep ara tion for mulatio n of rifampicin sus pe nsion : sev eral formula s of rifa mpicin aqueo us suspe ns io n were prep ared, either as read y-to us e aqueous suspe ns io n o r dry p owde r fo r re co nstitution. ready-to-use aqueous rifampicin su spension different fo rmula s of rifa mpicin susp ens ion were p re pared us ing d ifferent s usp ending agents a s shows in tab le (1), e ach fo rmula w as prepa re d as follows : rifa mpicin, me thylpa ra ben a nd propylp arabe n were lev igated in a mo rtar with the pre pa re d d is persion of the s usp ending agent. the mixture wa s tritura te d with a p es tle until a s mooth p as te was fo rme d. with co ntinuous tritura ting, the pa ste was diluted with the rema ining amount o f the disp ersion of suspe nd ing agent the n tra nsfe rred to grad uated c ylinder. the re quired amo unt o f so dium s ac cha rin and d is odium ed etate o r so dium metabis ulphite we re d is solve d in a sma ll portio n of dis tille d water a nd ad ded to the gra dua te d cylinder. f inally sorbitol, glyc erol and ras pbe rry flav or were add ed fo llowe d by ad ding s ufficient distilled wa te r to ma ke up to vo lume. the suspe ns io n wa s shake n thoroughly a nd the p h wa s ad justed to 5 with few drop s of 5m s od ium c itrate. ta ble ( 1 ) diffe re nt formula s of ready-to use rifampicin s uspe ns io n (% w/v) material fo rmula a b c d e f g r ifa mpicin 2 2 2 2 2 2 2 agar x y methy lc el lu lo se x xantha n gu m x y x scmc x x p olysorba te 80 x x x x x x x disodiu m edetate 0.1 0.1 0.1 0.1 0 .1 sodium me tabisulph ite 0 .1 5 0.15 glyc erol 5 5 5 5 so rbito l 7 0 % 5 5 5 5 sodium saccharin 0 .2 me thylp araben 0 .18 p ropy lp araben 0 .03 r aspbe rry flav or 0 .05 fina l v olume 100 ml ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 fo rmulation of rifampicin sus pension 3 powder fo r reconstitu tion diffe re nt s us pending agents we re us ed to prepa re rifa mpicin s usp ensions as p owde r form re ad y fo r re co nstitutio n are shown in ta ble (2 ). eac h was prep ared by tritura ting rifa mpicin pow der with the s elected c ompo nents. the pow der mixture was p as sed through a siev e (1 50 μm) b efore b eing tra nsferred to a mber glas s b ottle s (6).the re was an e xce ptio n for fo rmula i, in whic h a po wder b le nds wa s moistened with (0 .5 % po lyvinylp yrrolidone in alco hol). the da mp mass wa s then pas se d thro ugh the siev e (10 00 μm) and the gra nule s were drie d at 3 7 ˚c. the d ried gra nule s were re siev ed through the sa me siev e before be ing transferred to ambe r gla ss b ottles . the ea se of re co nstitution a nd s ta bility were e va luated to se le ct the pro pe r formula, whic h will b e subjecte d to further stud y. table ( 2 ) diffe re nt formulas o f rifa mpicin powde r to be re constitute d as sus pe nsion. ( % w/v) com para tive studies of the s ele cte d form ula with rifactine® suspen sio n: the s elected formulas (a a nd i) we re comp ared with the reference rifactine ® utilizing the following pa ra meters: dis so lu tion pr ofile the diss olutio n ra te o f rifamp ic in sus pe ns ions wa s stud ie d us ing usp d is so lution app aratus . the d is so lution me dium was 0 .1 n hcl (900 ml), 5 ml s amp le o f susp ens ion was add ed. the n a sample o f diss olution med ium was withdrawn at different time interva ls (2, 5,10,15 ,3 0 and 4 5 minutes ) thro ugh a p ip ette fitte d with a filter pa pe r. fres h d is so lution medium was a dde d to the ja r eac h time to re place withd ra wn sa mples. eac h sa mple was suitab ly d iluted a nd as sa yed spe ctro photometric ally a t 47 5 nm for rifa mpicin co ntent. me as ure ment of rheo gra m rhe ogra ms were ob ta ined a t 37 ˚c using cole-parme r ro ta tio nal visco meter. sed ime ntatio n volume mea sur eme nt fifty ml o f ea ch s usp ension was d ilute d with dis tille d water to a vo lume of 100 ml in a stopp ered gra dua te d cylind er. the susp ens ions were sha ke n vigorously to e nsure unifo rmity, then left und is turb ed . the se dimenta tion volume was me asure d ev ery 4 ho urs for p erio d of 4 8 hours (7). res usp end ability of sus pen sion the tes t co ns is te d of manua lly shaking the cylind er a fter the se dimenta tion e xp erime nt was c omplete d. ba se d on the e ffort req uire d to convert the s edime nted system to a homo ge nous susp ens io n, the prepa re d product was ra ted as : re sus pe nda ble, res us pendab le with difficulty or no t re susp end able. stab ility study: the acc elerated s ta bility stud y was d one in orde r to de termine the e xp iratio n date of fo rmula a a nd i b y plac ing the sa mples o f bo th fo rmula s in ove ns a t 35 ̊ c, 45 ˚c a nd 5 5 ̊ c for 120 d ays . sa mples we re taken a nd a ss aye d fo r drug conte nt a t s uita ble time interva ls (0, 15,30 ,6 0,90,12 0 d ays) us ing uv spe ctro photometric method a t 4 75 nm. result an d disc ussion the nonio nic surfac ta nt (po lyso rb ate 80) was inc orporated in the formulatio n of rifa mpicin suspe ns io n a s a w etting a gent to increas e diss olutio n rate of the drug (5). in add ition, xanthan gum was a ls o use d a s a sus pe nding age nt be cause of its e xc elle nt sus pe nding pro pe rtie s and also a s an e ffec tive floc culating agent a t re la tive ly lo w conce ntra tion(8). an inc re as e in the conce ntra tion of xanthan gum (as in fo rmula mate rial fo rmula i ii iii iv rifampic in 2 2 2 2 gua r gu m x y 2y scmc x ae rosil x x p olys orbate 80 x s ucrose 2 0 s odium sa cc harin 0.08 metylparabe n 0.18 propylparabe n 0.03 disod ium e deta te 0 .1 s odium citra te 0.06 citric ac id 0.03 raspberry fla vor 0.05 ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 fo rmulation of rifampicin sus pension 4 b) gave no s ub stantial cha nge in flo cc ulatio n be hav io r a nd a visc ous susp ens io n wa s obta ined which pour with d iffic ulty. on the other ha nd, sod ium ca rb oxymethylc ellulo se (scmc) was us ed a s in fo rmula d. it ga ve good diss olutio n prope rties a nd it prod uce d se diment la ye r that was e as ily redisp erse d by s haking. furthermore, a c ombina tion o f xa ntha n gum and scmc re sulted in to o visc ous suspe ns io n, which po ured with d ifficulty. methylc ellulo se utilized in formula e produce d a s us pe nsion with low d is so lution ra te a s shown in figure (1 ). fina lly, a ga r was utilized as thic kening age nt and to c ontrol flocc ulatio n. good res ults were ac hieve d with fo rmula f but ha rd c ake wa s fo rme d with formula g. fo rmula a ga ve the mos t o ptimum physica l stability a nd re markab le re le as e profile, therefore it w as chose n fo r exte ns iv e stud y a nd to be co mpared with re fe re nc e suspe ns io n. on the o ther hand, rifa mp ic in s us pe ns io n (as dry powde r) when prepa re d using guar g um as a single suspending age nt ( formula ii) resulted in a suspe nsio n tha t sho wed low se dimenta tion volume (0.2) but was easily re disperse d. the addition of a erosil to formula iii and iv re sulted in eas ily re dispersed suspens ions with high sed iment v olume (0.8 and 0 .9 for iii a nd iv, respectively). be ing fine ly divided , aerosil a ggre gates to form thre e dimensional network toge ther with its ability to absorb large a mount o f wate r, hence it p revents ca king (6). in ad dition sodium ca rboxymethylcellulose was used as a suspe nd ing age nt in c omb inatio n with po lyso rbate 80 to enhance the d isso lution (formula i). this for mula was prepared as granule s using a lcoho lic pvp so lution as a granula ting age nt. the granules were found to be fre e flowing and not bulky . also , rifamp ic in granule s were found to be good in app eara nce and their pa rticles we re uniform in size. fo rmula i was c hos en s ince it gave goo d stability although it prod uce d sed ime nt layer with ea sily re dispe ra bility by s ha king. figure (2) s hows the d is so lution rate of rifa mpicin suspe ns io n for formulas a and i comp ared with the reference rifac tine sus pe ns ion. the results s ho wed tha t rifamp ic in re le ase d from formula a wa s higher tha n that from o thers. fo rmula i after re constitution sho wed the lowes t diss olutio n ra te and this may be due to granulation proc ess , s ince pvp was used a s granulating age nt which is water solub le binder a nd has go od swe lling and hyd ra tion cap acity (9). the se p ro pertie s re sult in high visco us re gion surrounding the drug partic le . rhe ograms o f the prod uc ts a re rep re sente d in figure (3). the grap h showed that the vis co sity of rifampicin sus pe nsions wa s s he ar ra te dep end ent a nd increa sed in the fo llowing order: formula a < rifa ctine < formula i the re sults illus tra te d that the p re pa red fo rmula s (a a nd i) e xhib ited ps eudop la stic flow prope rties due to the s us pending agents use d, whic h were xanthan gum and sod ium carbo xymethylce llulo se . 0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 35 40 45 50 55 60 time (min) % r if a m p ic in r e le a s e d 50 55 60 65 70 75 80 85 90 95 100 0 5 10 15 20 25 30 35 40 45 50 55 60 time (min) % d is s o lv e d o f r if a m p ic in rifactine formula a formula i fig ure (1) : dis so lution ra te profile o f rifa mp ic in s us pe nsion (formula e) in 0 .1 n hcl a t 37 º figure (2) : dis so lution rate profile of rifa mpicin suspe ns io n (formulas a and i) a nd rifac tine in 0 .1 n hcl at 37 ˚c. ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 fo rmulation of rifampicin sus pension 5 . tab le s (3 and 4) show the s edimentatio n volume a nd res usp endab ility after s ettling of rifampic in s us pensions, res pe ctiv ely. the d ata indica te d that fo rmula a, which wa s prepa re d with xa ntha n gum, ha d s ed ime ntation volume eq ual to 2. this res ult wa s a ttrib uted to the ne two rk o f flo cs formed in the s us pension , which wa s s o loo se and fluffy tha t ca n b e exte nde d thro ughout extrave hicle. the sa me re sult was rep orte d by j awad (10). table ( 3 ) se dime ntatio n volume o f rifa mpicin suspe ns io ns (fo rmulas a, i and rifactine ) ta ble ( 4 ) re suspe ndability of rifampicin suspe ns io ns (fo rmu la s a,i and r ifac tine ) products res us pe ndability rifa ctine ea sily resuspended f ormula a no sedimenta tion fo rmula i ea sily resuspended rifa ctine ea sily resuspended in the s ta bility stud y of rifa mpicin sus pe ns ion and granular rifamp ic in figures (4 and 5) sho wed tha t the de grada tion o f rifamp ic in for fo rmula s a a nd i res pe ctiv ely, whic h fo llows firs t orde r kinetics s ince straight line s we re obtaine d by plotting the lo garithm o f perce nt re maining o f rifamp ic in v ersus time . pro duc ts se dime nta tion v olume f = hu / ho f rifac tine 45 /50 0.9 fo rmula a 1 00/50 2 formula i 20 /50 0.4 0 25 50 75 100 125 150 175 200 225 0 50 100 150 200 250 shear stress (dyne/cm 2 ) s h e a r ra te ( s e c . -1 ) rifactine formula a formula i 1.93 1.94 1.95 1.96 1.97 1.98 1.99 2 2.01 0 30 60 90 120 150 time (days) l o g % r if a m p ic in r e m a in in g 35 c 45 c 55 c o o o figure (3) : rheo gram at 3 7˚c o f rifampic in s us pe nsions fo rmu la s and rifa ctine . whe re : hu is u ltimate he ig ht of the se dime nt a s s us pe nsion settle ho is the initial he ight of the to ta l suspe ns io n. figure (4) :deg radatio n curve o f re ady-touse rifa mpicin s uspe ns ion (formula a) a t diffe re nt te mpe ra tures . ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 fo rmulation of rifampicin sus pension 6 the d egra dation rate co nstants (k) at d ifferent te mpe ra ture s were ca lc ulated fro m the s lo pe s of the s traight line s and the y were listed in ta ble (5). ta ble ( 5 ) de grada tion ra te cons ta nts of rifa mpicin sus pe nsions rea dy -to-use (for mula a) a nd gra nular sus pe nsion (for mula i). arrhe nius plots were then c ons tructed and are shown in figures (6 and 7 ) fo r fo rmula i and a, res pec tive ly. the linearity o f the c urve s indica te s their utility in pred ic ting the rate of de grada tion a t lowe r tempe ra tures . the rate co ns ta nts at 25 ˚c, obta ined fro m those plots fo r read y-to -use (formula a) a nd granular sus pe ns io n (formula i) were e qual to 0.15 3 x 10-3 a nd 0 .1 09 x 10 -3 (d ays-1) re spe ctiv ely. since the degra dation o f the d rug fo llowe d first order kinetics , the e xpiratio n da te t1 0% at 2 5˚c could be c alcula te d us ing the fo llowing equatio n: 0.10 5 t1 0% = k25˚c te mpe ra tu re (˚c) k x1 0-3 ( da y-1) for mula a for mula i 55 1 .206 0 .822 45 0 .619 0 .429 35 0 .318 0 .224 25 0 .153 0 .109 1.95 1.96 1.97 1.98 1.99 2 2.01 0 30 60 90 120 150 time (days) l o g % r if a m p ic in r e m a in in g 35 c 45 c 55 c o o o 0.1 1 10 3 3.1 3.2 3.3 3.4 (1/t) x 1000 (kelvin-1) k x 1 0 -3 ( d a y -1 ) 0.1 1 10 3 3.1 3.2 3.3 3.4 (1/t) x 1000 (kelvin-1) k x 1 0 -3 ( d ay -1 ) figure (5 ): deg ra dation c urve of gra nula r rifa mpic in s us pe nsion (fo rmula i) at diffe re nt te mpe ra tures figure (6) :arrhe nius p lo t fo r e xpira tion date e stima tion o f rifa mpicin suspe ns io n formula i a t 25˚c. fig ure (7) : arrhe nius plot for expiration da te e stima tion o f rifampicin suspe ns io n fo rmu la a at 25˚c. ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 fo rmulation of rifampicin sus pension 7 the e xpiratio n d ates fo r formula a and fo rmula i were 1.8 and 2.6 ye ars re spe ctively, indica ting tha t rifampicin is mo re s ta ble whe n it is p re pared as granula ted p owde r for re co nstitution, as a ls o indicate d b y p atankar and bajaj (11) referen ces 1. ans el h.c., allen l.v. and p opo vich, n.g., pha rmace utic al dos age fo rms a nd d rug de live ry s yste m, 17 th e d., 1 999 ,c hap ter 13 , p. 347 -3 64. 2. kawa shima y. and iwa moto t., prep ara tio n and charac te riza tion of a new c ontrolle d re le as e ib uprofen s us pension for improving . suspe nd ability, int. j. pharm., 199 1, 7 5 (aug 30), 2 5-35. 3. zatz j.l., sc hnitze r l. and sa rp otdar p ., flocc ulation of sulfame ra zine s us pension b y a ca tionic po lymer. j. p harm. sc i., 1 979 , 68, 149 1-149 4. 4. physician des k reference, c omp act d is k, 200 1. 5. el-ba ry a.a., elmone m h.a. a nd naim o., fo rmula tion a nd bioa vailability of rifa mpicin suspe ns io n, egyp t j. p harm. sc i., 199 2, 3 3 (5 -6 ), 997 -10 12 . 6. el – khes hen s.a. , ba dawi s .s . and bada wi a.a., optimization of rec ons titutab le sus pe ns ion of rifa mpicin using 24 fac to rial des ign , drug de v .ind .pha rm.,19 96 , 22(7),6 23 -63 0 . 7. lac hman l., lieb erman h.a. and kanig j.l., the theory and p rac tic e o f indus trial pha rma cy,3 rd ed., 1 986 ,c hap ter 16 a nd 18 , p. 480 -4 88, 535 . 8. f elmeister a., kuc htya k g., kozio l s. and felmeis te r c., po lymer induced flocc ulation of pha rma ce utical suspe ns io ns , j. pharm. sc i., 197 3, 62 (12), 1972 -1 973 . 9. aulto n-m.e, pha rma ceutic s: the sc ie nce of d osa ge form de sign, 19 88,chapte r 15 and 1 8, p.264 , 26 9-278 , 31 8-320 . 10. j awa d f .j ., a co mbine d formulation of dilo xanid e furo ate and metronida zo le b enzoa te as a s usp ension, thes is , co llege of pharmac y, baghda d unive rs ity, 20 00. 11. p atankar, l. and bajaj, a., f ormulatio n of rifa mpicin and iso niazid gra nules for re constitution, indian j . pharm. sc i, 199 4 ,56 (5 ), 1 69 -17 3. iraqi j pharm sci, vol.21(1) 2012 antibacterial and phytochemical study of salvia officinali 93 antibacterial and phytochemical study of iraqi salvia officinalis leave extracts lana y. muttalib* and alaadin m. naqishbandi* ,1 *department of pharmacognosy, college of pharmacy, hawler medical university, erbil, iraq. abstract sage (salvia officinalis), belong to labiatae family is indigenous to iraq and other mediterranean areas but now cultivated worldwide, principally for its use as culinary herb. in the present study preliminary screening for the important phytochemical natural product groups indicated the presence of flavonoid, saponin, hyrolysable and condensed tannin groups. the antibacterial activity of two concentrations 10 mg/ml and 100 mg/ml of chloroform and hydroalcoholic extracts from salvia officinalis leaves was evaluated against four strains of gram negative bacteria (escherichia coli, pseudomonas arigenossa, klebsiella pneumonia, and proteus spp) and two strains of gram positive bacteria (staphylococcus aureus, bacillus cerus) using agar well diffusion method. chloroform extract 100mg/ml was found active against two types of bacteria, staphylococcus aureus with mic of 90 mg/ml and proteus spp with mic of 80 mg/ml. bioassay guided separation using tlc led to the separation of 6 constituents from the active extract, one of them was identified as thujone. key words: salvia officinalis, antibacterial, tlc, thujone. دراسة الفعالية التثبيطية للبكتيريا والمكونات الكيميائية لمستخلصات أوراق الميرمية التي تنمو في العراق عالء الذين محمذ النقشبنذي* ،1 و النا يوسف مطلب* *فشع انعمالُش وانُباتاث انطبُت ، كهُت انصُذنت ، جايعت هىنُش انطبُت ، اسبُم ، انعشاق الخالصة وانتٍ تًُىا بصىسة طبُعُت فٍ انعشاق و يُاطك labiataeانً انعائهت انشفىَت (salvia officinalis)انًُشيُت تُتًٍ اخشي فٍ انششق األوسظ و تزسع فٍ يُاطك يختهفت يٍ انعانى بانذسجت انشئُسُت كعشبت نألكم. انبحث تًهُذٌ نُىاتج انكًُُائُت ( . و لذ أختبشث flavonoid, saponin, hyrolysable and condensed tanninتج ) انطبُعُت فٍ انُباث ادث انً كشف انُىا c1 10mg/ml , c2 ( بتشكُزٍَ )ethanol 75% ( و يزَب انكحىنٍ )chloroformيستخهصاث انًزَب انعضىٌ ) 100mg/mlَ ٍىع انصبغت انسانبت ) ( نغشض يعشفت انفعانُت انتثبُطُت نهبكتُشَا يفابم أسبعت سالالث يٍ انبكتُشَا ي escherichia coli, pseudomonas arigenossa, klebsiella pneumonia ، proteus spp وَىعٍُ يٍ انصبغت انًىجبت ) (staphylococcus aureus, bacillus cerus( باستخذاو تمُُت )agar well diffusion ٌاظهشث انُتائج باٌ انًزَب انعضى .) 100mg/ml تثبُطُت يمابم سالنتٍُ يٍ انبكتُشَا، رو فعانُتstaphylococcus aureus انحذ األدًَ نهتشكُز انًثبظ )و ب mic)90 mg/ml و proteus spp ب( انحذ األدًَ نهتشكُز انًثبظ mic) 80 mg/ml الحما تى فصم و يطابمت انًكىَاث. ( و لذ تى فصم ستت tlc) تمُُت كشويىتىغشافُا انصفائح انشلُمتان باستخذاوانشئُسُت نًستخهص انفعال ألوساق َباث انًُشيُت . thujone بأَها إحذاهًايكىَاث سئُسُت وتى تشخُص ، الميرمية ، الفعالية التثبيطية للبكتيريا tlcالكلمات المفتاحية : introduction sage (in kurdish and arabic, meramea) derived from (salvia officinalis) belong to labiatae family, indigenous to iraq and other mediterranean areas but now cultivated worldwide, principally for its use as culinary herb. salvia is a perennial herbaceous to shrubby herb growing up to 50cm in height ( 1, 2) . sage is used as therapeutic agent and in food, beverages, spirits, and cosmetics and in the production of relaxing and curative herbal tea (3) . sage reportedly has antibacterial, fungistatic, virustatic, astringent, secretion-stimulating, and perspiration-inhibiting effects. phenolic acids (e.g., salvin and salvin monomethyl ether) isolated from sage have antimicrobial activities, especially against staphylococcus aureus (4) .the volatile oil of sage consists of about 5% of αand βthujone together with cineole, borneol and other constituents (2, 3) . non volatile components of the leaf include diterpenes, phenolic glycosides based on caffeic acid, phydroxybenzoic acid and tannins (3-8%) both hydrolysable and condensed tannins (2) . thus, the present study is aimed to carry out preliminary screening for the important phytochemical natural product groups and to investigate and establish the antibacterial activity of iraqi salvia officinalis leave extracts and finally bioassay guided separation and identification of the main active constituents in the active extract (s). 1corresponding author email : alaadinmn@hotmail.com received : 12/5/2011 accepted : 3/4/2012 mailto:alaadinmn@hotmail.com iraqi j pharm sci, vol.21(1) 2012 antibacterial and phytochemical study of salvia officinali 94 materials and methods plant material salvia officinalis leaves were collected during september 2009 in erbil and authenticated by the department of pharmacognosy, college of pharmacy-hawler medical university. phytochemical screening fifty g of dried powdered salvia officinalis leaves were extracted using 75% ethanol for the phytochemical investigation using ultra sonic bath for 1hr at 40 0 c (5) . preliminary qualitative tests were carried out on the plant extracts to detect the most important natural product groups (6) . alkaloid test: hydroalcoholic extract of sage leaves (75% ethanol) was treated with dilute sodium hydroxide (5% naoh) solution; extraction is then carried out with chloroform. the concentrated organic liquid is then shaken with 5% hcl and allowed to separate. the aqueous extract was used for detection of alkaloidal compounds (2) . few drops of dragendorff reagent were added to 1ml of extract. the appearance of a reddish-brown to orange precipitate indicated the presence of alkaloid (2, 7, and 8) . flavonoid test: 2 ml of hydroalcoholic extract of sage leaves were tested for the presence of flavonoid glycoside by the addition of few drops of naoh solution. the formation of intense yellow color which turn to colorless on addition of few drops of dilute acid solution indicated the presence of flavonoids (9) . anthraquinone test: the tested sage extract was boiled with 1ml of dilute hcl in a test tube over a pre-heated water bath for 5 minute. the contents were cooled and extracted with chloroform, the chloroform layer was separated and ammonia solution was added. the appearance of a rose-pink color in ammonia layer is indicated the presence of anthraquinone glycoside (9) . cardioactive glycoside test: the dried sage extract was dissolved in chloroform, and then evaporated to dryness. dissolve the residue in (0.4ml) glacial acetic acid with few drops of fecl3, concentrated sulphuric acid (h2so4) was added along the side of the test tube to settle at the bottom. the reddish brown color changing to bluish green color appears at the junction of the two reagents within 2-5 min. spreading slowly into the acetic acid layer indicated the presence of cardioactive glycoside (9) . saponin test: a volume of 2ml of hydroalcoholic sage extract were shaken with 1ml of water. the formation of semipermanent foam (15 min) indicated the presence of saponin natural products (6) . tannin test: a few drops of ferric chloride reagent 1% were added to 1ml of sage extract. the appearance of blue color indicated the presence of hydrolysable tannins and appearance of green color indicated the presence of condensed tannins (2, 6) . antibacterial evaluation extraction: plant material was collected, dried in air for seven days, and all plants were powdered with mechanical grinder. 50gm of dried powdered plant material was extracted separately with 1000 ml of chloroform using ultra sonic bath for one hour at 40 0 c (5) . the extracts were obtained by filtration through buckner funnel, and evaporated to dryness by rotary evaporator yielding chloroform extract (5.4 g). the residual plant materials were dried then re-extracted using 75% ethanol using ultra sonic bath for one hour at 40 0 c (5) . the obtained extracted from filtration by buckner funnel evaporated to dryness by rotary evaporator yielding ethanol extract (9.2 g). plant extract preparation: the plant extracts were evaluated separately at two different concentrations 10mg and 100mg. the chloroform extracts and ethanol extracts were dissolved separately in 1ml of 20% tween 80 and 10% dimethyl sulfoxide (dmso) respectively. tested microorganism: bacteria which were used in the process of investigation are obtained from biology department, science college, salahaddin university. bacterial strains include two gram positive bacteria (staphylococcus aureus, bacillus cerus) and four gram negative bacteria (escherichia coli, psedomonus arigenossa, klepssila spp and proteus spp). the bacterial samples are frozen at -4 0 c in cooled incubator, later reactivated before it’s used. method of antibacterial evaluation: the antibacterial activity of the two types of plant extracts were tested against six strains of bacteria using agarwell diffusion method (10, 11) . from the frozen bacteria inoculation was done into nutrient agar media, and incubated at 37 o c for 24 hr.the grown bacteria were suspended in a normal saline solution (0.85% sodium chloride w/v) to a turbidity of 0.5 mc farland standards (108 cfu/ml).the prepared bacterial suspension was used to inoculate into muller-hinton agar plate with a sterile nontoxic cotton swab on a wooden applicator. four wells were done by a sterile cork borer of 5mm in diameter in each plate.100 µl of dilution of plant extracts in 10% dmso for ethanol extract and 20% tween 80 for iraqi j pharm sci, vol.21(1) 2012 antibacterial and phytochemical study of salvia officinali 95 chloroform extract to give final concentration of 1mg and 10mg of each plant extract was added in each well, 10% dmso and 20% tween 80 were used as negative control and streptomycin antibiotic used as positive control in concentration of (100mg/ml) added in a well in each plate. plates are incubated at 37ºc for 24 hr. determination of minimum inhibitory concentration (mic): mic values for biolog ically active extracts against staphylococcus aureus and proteus spp. were determined by agar well diffusion method (10, 11) . separation and identification of the main active constituents thin layer chromatography (tlc) technique was applied for the separation and identification of active constituents using silica gel gf 254 nm, layer thickness 0.2 mm; 20x20 cm aluminium cards (fluka, switzerland) (7, 12) . different solvent systems were tried as a mobile phase [toluen: ethyl acetate (93:7), toluen: ethyl acetate: formic acid (5:4:1), ethyl acetate: formic acid: glacial acetic acid: water (10:1.1:1.1:2.6), benzene: pyridine: formic acid (7.2:1.8:1)] (13, 14) .fifty mg of dried chloroform extract was dissolved in 1 ml chloroform and 1µlof thujone reference substance (chromadex, usa) was dissolved in 1ml toluene, 10 µl of both solutions were applied to tlc plate and developed in different mobile phase systems. the plate was dried at room temperature. the tlc plate was sprayed with anisealdehyde sulphuric acid reagent after the development process. the plate was heated to 100–110°c for 5–10 min. and visualized under visible light (13) . a number of constituents were separated, and their rf values were calculated, the procedure was triplicated and mean values were taken, table (1). table 1: rf value and spot color of separated constituent (s3) separated constituent rf value spot color mobile phase (1) mobile phase (2) mobile phase (3) mobile phase(4) s3 0.5 0.31 0.13 0.43 violet standard thujone 0.52 0.34 0.15 0.44 violet mobile phase (1): toluen: ethyl acetate (93:7) mobile phase (2): toluen: ethyl acetate: formic acid (5:4:1) mobile phase (3): ethyl acetate: formic acid: glacial acetic acid: water (10:1.1:1.1:2.6) mobile phase (4): benzene: pyridine: formic acid (7.2:1.8:1) results sage leaves chloroform extract shows activity against two strain of bacteria staphylococcus aureus and protues spp. at 100mg\ml concentration with inhibition zone diameter (20±0.265 and 10±0.2) respectively with corresponding mic value 90 mg\ml against staphylococcus aures and 80 mg\ml against proteus spp, table (2). the phytochemical screening tests showed positive results for flavonoid, saponin hydrolysable and condensed tannin, while the other natural product groups including alkaloid, anthraquinone, and cadioactive were found absent. the results of separation by tlc revealed the presence of a number of constituents in chloroform extract, one of the constituent (s3) was found to be with identical color and rf value with that of thujone reference substance,figure (1). figure 1: tlc chromatogram of sage leaves, mobile phase [toluen: ethyl acetate (93:7)]; chloroform extract (ce), thujone reference substance (r). iraqi j pharm sci, vol.21(1) 2012 antibacterial and phytochemical study of salvia officinali 96 table 2: antibacterial activity for sage leaves chloroform extract, c1 (10mg/ml) and c2 (100mg/ml). no. microorganism inhibition zone diameter (mm), mic value ( mg/ml) chloroform extracts ethanol extracts c1 c2 c1 c2 1staphylococcus aureus --20±0.265, (90)* ---- 2pseudomonas aeroginosa -------- 3 escherichia coli -------- 4klebsila pneumonae -------- 5bacillus cerus -------- 6proteus spp. --10±0.2, (80)* ---- *all readings were repeated three times, the mean of triplicates ± sd values were taken. discussion sample preparation is the crucial first step in the analysis of herbs, because it is necessary to extract the desired chemical components from the herbal materials for further separation and characterization. thus, the development of modern sample preparation techniques with significant advantages over conventional methods analysis of medicinal plants is likely to play an important role in the overall effort of ensuring and providing high quality herbal products to consumers worldwide (15) . the plant expresses antibacterial activity against two strains of bacteria. from the results, the chloroform extract was active while hydroalcoholic extract was inactive. the activity of the plant as showed in the literature review may be due to phenolic acids (example, salvin and salvin monomethyl ether) previously isolated from sage, especially against staphylococcus aureus (4) . the obtained results were in agreement with the finding of zhi-he and hiroyuki, (1996) (16) who were found that sage leaves extract at 0.2% concentration was gave strong activity against staphylococcus aureus, in the same time rogério et al, (2004) (17) found that the essential oils of salvia officinalis was inhibited 83.3% of proteus spp. while gislene et al, (2000) (18) were found that the collected sage in brazil was inactive against staphylococcus aureus and proteus spp. literature reviews on salvia officinalis revealed that there was few phytochemical studies conducted on the iraqi species of sage, and due to the antibacterial activity of its chloroform extract and from phytochemical screening which was found to contain four groups of phytochemical natural product groups, it was tried to separate and identify the main active constituents in the active extract by using tlc. the proposed tlc method was regarded as suitable for quality control of herbal products containing salvia officinalis and for its extracts. the method is simple, sensitive, and specific, and both the standards and the samples can be analyzed simultaneously, without the requirement for sophisticated equipment (13) . separation of the main active constituents in chloroform extract by the tlc revealed the presence of six constituents figure (1). one of the constituents (s3) had the same rf value and color as thujone the reference compound. chemotaxonomic markers are sometimes used as indicators of botanical identity; thujone is the marker for salvia officinalis (19) . miladinović and miladinović (2000) (20) found that the main component of the salvia officinalis essential oils was thujone (24.88%) and camphor (16.3%). the carriers of antimicrobial activity of the sage oil were found to be thujone and camphor (19, 21) . further study using advanced spectroscopical methods and gas-liquid chromatography is recommended for more confirmation of the identity of (s3) constituents and to identify the other separated constituents in the chloroform extract. references 1. banso, a. phytochemical and antibacterial investigation of bark extracts of acacia nilotica. j. med. plants res., 2009; 3: 082085. 2. evans w.c. trease and evans pharmacognosy. 2000, elseveir, uk. 3. davut karaasian; and mensure őzgűven. determination of qualitative and quantitative of sage (salvia officinalis l.) essential oils. pakistan journal of biological science, 2001;4(1): 41-43. 4. british herbal manufacturers association (b.h.m.a). british herbal pharmacopoeia, 1983; isbn 0-903032-07-4. iraqi j pharm sci, vol.21(1) 2012 antibacterial and phytochemical study of salvia officinali 97 5. bartram, t. encyclopedia of herbal medicine, 1st ed; 1995, grace publishers: bournemouth. 6. ashutush kar : pharmacognosy and pharmacobiotechnology. 2003, new age international, k.k.gutpa: new delhi. 7. harborn, j.b. in: phytochemistery methods. 1984, new york wiely. 8. seema e. isolation and identification of cyaniding pigment from allium cepa l. and its application as antioxidant. m.sc. thesis. 2008, university of basra. 9. alupuli a.; calinescu i.; and lavric v. ultrasonic vs. microwave extraction intensification of active principles from medicinal plants. aidic conference series, 09, 2009; 1-8doi: 103303/ acos0909001. 10. park, y.k.; ikegaki, m.; and contado, j. l. comparison of flavonoid aglycone content of apis mellifera propolis from various region of barazil. arquivor de biologiae techno, 1997; 40: 97 -106. 11. trukoglu, a.; duru, m. e. and mercan, n. antioxidant and antimicrobial activity of russula delica fr; an edible wild mushroom. eurasian j. of anal. chem., 2007; 1:54-67. 12. ming-weil, z.; bao-jiang, g.; rui-fenl, z.; jian-weil, c.; zhenchengl, w.; zhi-hongl, x.; yanl, z. and xiao-junl, t. separation, purification and identification of antioxidant compositions in black rice. agri. sci. in china. 2006; 5:431-440. 13. durōn, r.r.; l.c. almaguer; a. de j. garza-juárez; ma. de la luz salazar cavazos; and n. waksmande-torres. development and validation of thin-layer chromatographic methods for quality control of herbal products. acta chromatographica, 2009; 21: 203-215. 14. jancsák, g.; and i. máthē. parallel determination of rosmarinic and cafeic acid by tlc-densitometry. chromatographia, 1997; 46:5/6. 15. carmen w. huie. a review of modern sample-preparation techniques for the extraction and analysis of medicinal plants. anal bioanal. chem. 2002; 373:23–30. 16. zhi-he liu and hiroyuki nakano. antibacterial activity of spice extracts against food related bacteria. j. fac. appl. biol. sci. 1996; 35:181-190. 17. rogério santos pereiraa; tânia cristina sumitaa; marcos roberto furlanb; antonio olavo; cardoso jorgec; and mariko uenod. antibacterial activity of essential oils on microorganisms isolated from urinary tract infection. rev saude publica, 2004; 38(2). 18. gislene g. f. nascimento; juliana locatelli; paulo c. freitas; giuliana l. silva: antibacterial activity of plant extracts and phytochemicals on antibiotic resistant bacteria. brazilian journal of microbiology, 2000; 31:247-256. 19. wagner h.; s. bladt; and e.m. zgainski. plant drug analysis: a thin layer chromatography atlas. 1984, springer, berlin, germany. 20. miladinović, d. and miladinović, lj. antimicrobial activity of essential oil of sage leaves from serbia. facta universities, physics, chemistery and technology series2 2000; (2): 97-100. 21. dragan t. veliĉković; milena t. nikolova; stephanie v. ivancheva, jelena b. stojanov; and vlada b. veljkovi. extraction of flavonoids from garden (salvia officinalis l.) and glutinous (salvia glutinosa l.) sage by ultrasonic and classical maceration. j. serb. chem. soc., 2007; 72 (1): 73–80. iraqi j pharm sci , vol.18 (suppl.), 2009 benfotiamine in liver injury 74 study of the protective effects of benfotiamine against ccl4-induced hepatotoxicity in rats tavga a. aziz * , zheen a. ahmed ** , kasim m. juma'a *** munaf h. abdulrazzaq **** and saad a. hussain ****,1 * department of pharmacology, college of pharmacy, university of sulaimaniya, kurdistan, iraq ** department of pharmacology, college of medicine, university of sulaimaniya, kurdistan, iraq *** department of pharmacy, baquba general hospital, diyala, iraq **** department of pharmacology and toxicology, college of pharmacy,university of baghdad, baghdad, iraq abstract liver is considered as the first target for the toxic effects of toxins and other xenobiotics, and this can be attributed to its role as a site which receive all absorbed xenobiotics from the gastrointestinal tract and its role as a major site for biotransformation of xenobiotics. the present study was designed to evaluate the possible hepatoprotective effect of benfotiamine against ccl4-induced hepatotoxicity in rats. the study was conducted on 48 male albino rats; the animals were allocated into 8 groups (6 rats in each group) and treated as follow: 4 groups treated with oral doses of either normal saline, benfotiamine (100 mg/kg), thiamine (100 mg/kg), n-acetylcystein (400 mg/kg) only without induction of hepatic damage. the other 4 groups were treated as indicated previously with induction of hepatic damage with ccl4; at the end of treatment period, rats were scarified, blood samples obtained and livers excised for the assessment of the oxidative stress parameters (mda and gsh), cholesterol and triglycerides levels. additionally, serum levels of total bilirubin, albumin, total protein and the activities of alt, ast and alp enzymes were evaluated before and after treatment with benfotiamine. tissue sections were prepared for evaluation of histopathological changes. the results indicated that benfotiamine has the ability to protect hepatic tissue against the toxicity induced by ccl4, revealed through reduction of serum levels of tsb and liver enzymes, decrease in the hepatic tissue mda levels and elevation of gsh there. histological evaluation of tissue sections prepared for this purpose confirmed the previous finding. in conclusion, benfotiamine is capable to protect liver tissue against ccl4-induced toxicity in rats more than thiamine. key words: benfotiamine, ccl4, hepatotoxicity الخالصة ٚعحبر انكبد انٓدف األٔل نهحأذٛر انطاو نهًركبات انًخحهفة نكَّٕ انًٕقع األٔل انذ٘ ٚطحهى شًٛعع انًركبعات انحعٙ ٚعحى ايحااععٓا عٍ طرٚععا اناُععاه انٓ ععًٛة اكععا ة نكَٕععّ يركععس انحلععٕ ت األٚ ععٛة نٓععذِ انًركبععاتس جععى جاععًٛى ْععذِ اندعاضععة نحاٛععٛى انلًاٚععة انًحٕقعععة نًععا ه شعراا جبعٛح ثٛعد جعى جاطعًٛٓا انعٗ 7٤ٛايٍٛ كد انحطًى انًطحلدخ بربا ٙ كهٕعٚد انكربٌٕ ٙ انصعرااٌس جشرٚعث اندعاضعة هعٗ انبُفٕج يلهععٕل يهلععٙ ٕاثععد يععٍ انًركبععات انحانٛععة نبصععرع ععٍ طرٚععا انفععى يُٓععا شععرااٌ نكععم يصًٕ ععةال جععى عع ز جعبعععة٦ذًاَٛععة يصًٕ ععات يهغى/كغعىس جيعا انًصًٕ عات األعبععة األفعرٖ حًعث 7١١يهغى/كغعى، جضعٛحٛم ضطعحٍٛٛ ٠١١ٍٛ يهغى/كغعى، ذٛعاي ٠١١يحٕازٌ، بُفٕجٛايٍٛ يعانصحٓا بُفص انطرٚاة انًذكٕعه آَفا يع اضحلداخ جهف كبد٘ بٕاضطة عبا ٙ كهٕعٚد انكربٌٕ، ٔ ٙ َٓاٚة حره انع ز جى قحم انلٕٛاَعات ًانَٕعدا٘ اندٚٓاٚعدال، انكٕنٛطعحرٔل ٔانمعلٕو عٙ نانحأكطعد انكهٕجعاذٌٕٛ ٔا نهلإل هٗ جكبا ْا ٔ ُٛات يٍ يٓعاس جعى قٛعاش يععاٚٛر عرط ال عٙ ياعم ast ،alt ،alpف عة َطٛس انكبد باألكا ة انٗ قٛاش يطحٕٖ يا ه انافراء، انبعرٔجٍٛ ٔ عانٛعة األَسًٚعات انكبدٚعة س جى جل ٛر يااطع َطٛصٛة نهكبعد ٔيحابععة انحغٛٛعرات فرٖاندو ٔيااعَة يطحٕٖ ْذِ انًعاٚٛر قبم ٔبعد انع ز بانبُفٕجٛايٍٛ ٔانًركبات األ انلاعهة بٕاضطة انًصٓرس جذبحث َحائس اندعاضة يادعه انبُفٕجٛايٍٛ هٗ ثًاٚة َطٛس انكبد كد انحطًى انًطحلدخ بٕاضعطة عبعا ٙ كهٕعٚعد انٗ ففح يطحٕٚات انكٕنٛطحرٔل ٔانمعلٕو انكربٌٕ يٍ ف ل ففح يطحٕٚات يا ه انافراء ٔ عانٛة األَسًٚات انكبدٚة ٙ اندو، جكا ة ٔانًانَٕدا٘ اندٚٓاٚد ٙ َطٛس انكبد يع ع ع يطحٕٖ انكهٕجاذٌٕٛ ُْاك، كًا جكدت َحائس انفلص انُطٛصٙ يااكر آَفعاس ًٚكعٍ األضعحُحاز بعأٌ رااٌ اكرر يٍ انرٛايٍٛس نهبُفٕجٛايٍٛ انًادعه هٗ ٔقاٚة َطٛس انكبد كد ان رع انًطحلدخ بٕاضطة عبا ٙ كهٕعٚد انكربٌٕ ٙ انص introduction the liver plays a central role in carbohydrate, protein and fat metabolism and allows the detoxification of various xenobiotics. additionally, it regulates the synthesis and secretion of bile. (1) toxic injury occurs in the liver more often than other organs, because all ingested substances that are absorbed are first presented to the liver and that the liver is responsible for the metabolism and elimination of many substances. (1) many xenobiotics such as acetaminophen, ccl4 ,and yellow phosphorus produce liver damage in a predictable and dose-dependent manner; the most frequent mechanism of hepatocellular injury involves production of injurious metabolites by the cytochrome p450 system. (2,3) preventive care can significantly reduce the progression of liver disease. (4) 1 corresponding author e-mail : saad_alzaidi@yahoo.com received : 3/1/2009 accepted : 6/6/2009 mailto:saad_alzaidi@yahoo.com iraqi j pharm sci , vol.18 (suppl.), 2009 benfotiamine in liver injury 7٤ one of the drugs that used for this purpose is n-acetylcysteine (nac) which has been used clinically for the treatment of acetaminophen poisoning (5) . oral supplementation with (nac) provides an alternate means of boosting intracellular glutathione via elevated intracellular cysteine, and this can scavenge peroxynitrite and hydroxyl radicals as well as convert hydrogen peroxide to water. (6) benfotiamine (s-benzoyl thiamine-o-monophosphate) has been shown to reduce the formation of advanced glycation end products (age) byactivating transketolase. (7, 8) benfotiamine has been noted to possess clinical efficacy in the treatment of diabetic cardiomyopathy, (9) diabetic nephropathy (10) and diabetic neuropathy. (11, 12) moreover, benfotiamine has been shown to reduce the oxidative stress through a mechanism unrelated to age formation. (13) activation of akt (protein kinase b) has been demonstrated to stimulate enos activity, increase the bioavailability of no and reduce the generation of ros. (14) benfotiamine has been reported to improve the function of endothelium by activating akt and subsequently stimulating enos and inhibiting the generation of ros. (15,16,17) benfotiamine is thought to act by at least three different mechanisms. first, activation of the hexosamine pathway with subsequent decrease in the accumulation of deleterious glucose metabolites seems to be involved, second, normalization of pkc activity along with prevention of nuclear factor kappa b (nf-κb) activation has been found in retinas, and third, correction of imbalances in the polyol pathway by decreasing aldose reductase activity, sorbitol concentrations and intracellular glucose levels seems to play a role. (7) absorption of benfotiamine was better than thiamine itself, and levels of thiamine and thiamine pyrophosphate (tpp) remain higher for longer period of time. (18) absorption of thiamine in the form of benfotiamine is found to be five times greater than the absorption of the conventional thiamine supplements, and because of greatest intracellular access of benfotiamine, its tissue availability and effects are more impressive, especially in the brain and muscles. (19) the present study was designed to evaluate the possible protective effect of benfotiamine against ccl4-induced hepatotoxicity in rats. materials and methods forty eight sprague-dawley rats of both sexes weighing 180-220 g were obtained from the animal house in the college of pharmacy, university of baghdad and used in the study. the animals were housed in the animal house of collage of pharmacy, university of sulaimaniya under conditions of controlled temperature and allowed free access to water and food. the study protocol was approved by the committee for medical research, college of medicine, university of sulaimaniya. the animals were allocated into eight groups, each contain 6 rats and treated as follow: group i, received single oral daily dose of normal saline for 7 days. the group served as control; group ii received single oral daily dose of normal saline for 7 days, at day 8 the animals received single dose of ccl4 (2 ml of a mixture of 1:1 v/v in a corn oil /kg/day) orally by oral needle to induce liver damage. (20) the animals were sacrificed 24 hr after ccl4 administration; (21) group iii received single oral daily dose of thiamine (70 mg/kg/day) for 7 days; group iv received single daily oral dose of thiamine (70 mg/kg/day) started 7 days prior to treatment with ccl4 at day 7; group v received single oral daily dose of benfotiamine (70 mg/kg/day) for 7 days; group vi received single daily oral dose of benfotiamine (70 mg/kg/day) started 7 days prior to treatment with ccl4 at day 7; group vii received single oral daily dose of n-acetylcysteine (400 mg/kg/day) for 7 days; group viii received single daily oral dose of n-acetylcysteine (400 mg/kg/day) started 7 days prior to treatment with ccl4 at day 7. all animals were sacrificed on the day 8. after killing the animals by over dose of thiopental (panpharma s.a. france) (100mg/kg), livers were obtained and utilized for preparation of tissue homogenate. the organ was quickly existed and placed in a chilled phosphate buffer solution (ph 7.4), blotted with filter paper, and 10% homogenate was prepared in the same solution utilizing metal head tissue homogenizer at 4 o c. all preparations were frozen (-18 0 c) unless worked immediately. after killing the animals, blood was collected by intracardiac puncture. the clot was dispersed with glass rod and then centrifuged for 15-20 minutes at 2000 rpm and the supernatant was used for the estimation of alt and ast, (22) alp, (23) albumin and total protein (24) as parameters of liver function tests and total serum bilirubin (25) as excretory function test. samples of liver tissue homogenates were used for determination the malondialdehyde (mda), (26) reduced glutathione (gsh), (27) cholesterols and triglycerides levels. (28,29) tissue sections were prepared for histological examination according to the method of bauer, (15) using paraffin sections technique. the significance of differences between the mean values was calculated using unpaired student's t -test. piraqi j pharm sci , vol.18 (suppl.), 2009 benfotiamine in liver injury 74 values less than 0.05 were considered significant for all data shown in the study. results table 1 showed that serum activities of alanine-aminotransferase (alt) and aspartate aminotransferase (ast) were significantly elevated in ccl4-intoxicated animals (847.5% and 2381.6% respectively) compared to control group (p>0.05). pre-treatment of rats with single oral doses of thiamine, benfotiamine or n-acetylcysteine for 7 days prior to intoxication with ccl4 showed a marked decline in the serum alt and ast activities (36.6%, 32.8% and 88.6% respectively for alt and 39.75%, 77.97% and 94.2% respectively for ast) compared to ccl4-treated animals (p>0.05). serum alkaline phosphatase (alp) activity was significantly elevated in ccl4-treated animals (53.4%) compared to control group (p>0.05, table 1), and pre-treatment with single oral doses of thiamine, benfotiamine or n-acetylcysteine for 7 days, prior to induction of liver toxicity with ccl4, showed a marked decline in the level of serum alp activity (17%, 37.9% and 32.2% respectively) compared to ccl4-treated animals. table 1. effects of benfotiamine on serum levels of alt, ast and alp activities in experimental animal model of ccl4-induced liver toxicity. type of treatment serum alt u/l serum ast u/l serum alp u/l saline treated only 8.0 ± 1.06 6.0 ± 0.6 79.3 ± 18.3 saline + ccl4 75.8 ± 11.5 * a 148.9 ± 17.5 * a 121.7 ±26.6* a thiamine only 10.2 ± 1.5 b 6.5 ± 0.71 b 90.5 ± 19.1* b thiamine + ccl4 48.0 ± 9.5 * c 89.7 ± 16.1 * c 101.0 ±20.2* b benfotiamine only 9.6 ± 1.2 b 7.1 ± 0.9 b 80.9 ± 8.1 c benfotiamine + ccl4 50.9 ± 8.2 * c 32.8 ± 6.1 * d 75.5 ± 10.6 c n-acetylcysteine only 9.0 ± 1.2 b 7.2 ± 0.9 e 72.0 ± 9.0 c n-acetylcysteine + ccl4 8.6 ± 1.4 b 8.6 ± 1.2 * e 82.5 ± 10.2 c each value represents mean ± sd; * significantly different compared to saline only treated group (p<0.05); values with non-identical superscripts (a,b,c,d,e) are considered significantly different within the same parameter (p<0.05). table 2 showed that animals intoxicated with ccl4 presented with a highly significant increase in mda (95.6%) and decrease in gsh (72.2%) contents of liver tissue homogenate compared to control (saline treated) group (p>0.05, table 2). pre-treatment of rats orally with single doses of thiamine, benfotiamine or n-acetylcysteine for 7-days before induction of hepatotoxicity with orally administered ccl4 resulted in 32%, 31.1% and 51% decrease in hepatic mda contents respectively, and significant increase in gsh (340%, 237% and 246% respectively) compared to ccl4treated only animals (p>0.05, table 2). table 2. effect of benfotiamine on liver tissue malondialdehyde (mda) and reduced glutathione (gsh) levels in experimental animal model of ccl4-induced liver toxicity type of treatment liver tissue gsh µmol/g tissue liver tissue mda nmol/g tissue saline 27.0 ± 6.75 230.0 ± 46.0 saline + ccl4 7.5 ± 0.81* a 450.0 ± 48.0* a thiamine 21.0 ± 4.2 b 246.0 ± 36.0 b thiamine + ccl4 33.0 ± 6.0* c 306.0 ± 14.0* c benfotiamine 25.5 ± 5.2 b 218.0 ± 35.0 d benfotiamine + ccl4 25.3 ± 2.6 b 310.0 ± 22.0* c n-acetylcysteine 31.2 ± 4.5 c 221.0 ± 26.0 d n-acetylcysteine + ccl4 26.0 ± 5.3 b 220.0 ± 18.0 d each value represents mean ± sd; * significantly different compared to saline only treated group (p<0.05); values with non-identical superscripts (a,b,c,d) are considered significantly different within the same parameter (p<0.05). iraqi j pharm sci , vol.18 (suppl.), 2009 benfotiamine in liver injury 0١ in table 3, total serum bilirubin (tsb) levels were significantly elevated in ccl4treated animals (85.7%) compared to control group (p>0.05, table 3), and pre-treatment with single oral doses of thiamine, benfotiamine or n-acetylcysteine for 7 days, prior to induction of liver toxicity with ccl4, showed a marked decline in the level of serum tsb (33.8%, 41.5% and 30.7% respectively) compared to ccl4-treated animals. serum albumin and total protein were shown to be significantly decreased in ccl4-treated animals (31.8%) compared to control group (p<0.05), while total protein levels showed a non significant differences in ccl4-treated animals (1.3%) compared to control group (table 3). pre-treatment of rats with single oral doses of thiamine, benfotiamine or n-acetylcysteine for 7 days, prior to intoxication with ccl4, showed increase in the level of serum albumin (45.4%, 36.36% and 13.6% respectively) compared to ccl4-treated animals (p>0.05, table 3), while total protein levels were not significantly changed. however, rats treated with single oral dose of thiamine or benfotiamine for 7 days showed nonsignificant differences on both serum albumin and total protein (p < 0.05) compared to control group. meanwhile, single oral dose of n-acetylcysteine for 7 days showed a significant increase in both serum albumin and total protein levels (p > 0.05) compared to control group (table 3; figure 4).serum triglyceride and cholesterol levels in liver tissue homogenate were shown to be significantly increased in ccl4-intoxicated animals (333% and 212.5% respectively) compared to control group (p> 0.05, table 3). however, pre-treatment of rats with single oral doses of thiamine, benfotiamine or nacetylcysteine for 7 days, prior to induction of hepatotoxicity with ccl4, showed a marked decline in the levels of triglycerides (20%, 57% and 58% respectively) and cholesterol(24.5%, 44.9% and 42.2% respectively) compared to ccl4-treated animals (p>0.05, table 3 ). histological examination of liver tissues of rats exposed to toxic dose of ccl4 showed a marked hepatic damage revealed as zonal necrosis, extensive diffuse vacuolar degeneration of the hepatocytes, together with ballooning and fatty changes, dilatation and congestion of the central vein; the later showed focal hemorrhage with variable degree of inflammatory cell reaction were seen also (figure 1). treatment of rats with benfotiamine, thiamine or n-acetylcysteine before induction of toxicity clearly demonstrated protective effects against ccl4induced toxicity, where benfotiamine showed the greatest level of protection compared to that produced by n-acetylcysteine and thiamine respectively (figures 2, 3 and 4). table 3. effect of benfotiamine on serum levels of total serum bilirubin, albumin, total protein and liver tissue triglycerides and cholesterol in experimental animal model of ccl4-induced liver toxicity type of treatment total serum bilirubin mg/dl serum albumin mg/dl liver tissue triglycerides µmol/g tissue liver tissue cholesterol µmol/g tissue serum total protein mg/dl saline treated only 0.35 ± 0.01 2.9 ± 0.08 12.0 ± 2.4 8.8 ± 0.3 6.17 ± 0.4 saline + ccl4 0.65 ± 0.14* a 2.2 ± 0.1* a 52.0 ± 7.2 * a 18.7 ± 2.1* a 6.3 ± 0.6 a thiamine only 0.31 ± 0.02 b 3.2 ± 0.2 * b 11.5 ± 2.1 b 9.3 ± 1.8 b 5.9 ± 0.38 a thiamine + ccl4 0.43 ± 0.1* c 3.2 ± 0.1* b 41.5 ± 3.6 * a 14.1 ± 3.6* c 5.8 ± 0.3 a benfotiamine only 0.29 ± 0.03 b 2.9 ± 0.1 b 10.2 ± 1.3 b 8.5 ± 0.8 b 5.3 ± 0.7 a benfotiamine + ccl4 0.38 ± 0.04 d 3.0 ± 0.1 b 22.3 ± 3.4 * c 10.3 ± 0.9 * d 5.9 ± 0.5 a n-acetylcysteine only 0.39±0.02 d 4.5 ± 0.4 * c 9.3 ± 1.6 b 8.3 ± 0.6 b 7.2 ± 1.2* b n-acetylcysteine + ccl4 0.45 ± 0.02* c 2.5 ± 0.2 b 21.8 ± 2.6 * c 10.8 ± 0.5 * d 6.0 ± 1.1 a each value represents mean ± sd; * significantly different compared to saline only treated group (p<0.05); values with non-identical superscripts (a,b,c,d) are considered significantly different within the same parameter (p<0.05). iraqi j pharm sci , vol.18 (suppl.), 2009 benfotiamine in liver injury 0٠ figure 1. section from liver tissue after ccl4 intoxication. (x400, h and e stain) figure 2. section of liver tissue after intoxication with ccl4 and protection with orally administered n-acetylcysteine. (x400; h and e stain) figure 3. section from liver tissue after ccl4 intoxication and protection with orally administered thiamine (x 10; h and e stain) figure 4. section of liver tissue after intoxication with ccl4 and protection with orally administered benfotiamine. (x400; h and e stain) discussion in animals acutely exposed to ccl4 orally, the liver appears to be the primary target organ. (21) numerous studies showed that metabolism of (ccl4) is required for its toxicity; (30) it is known to be rapidly transformed by cytochrome p450-2e1 of the hepatocyte endoplasmic reticulum to ccl3 • which is converted into ccloo • in the presence of oxygen. (31) peroxidative degradation of membrane lipids of endoplasmic reticulum rich in polyunsaturated fatty acids leads to the formation of lipid peroxides, these in turn give products like mda that cause damage to the membrane (32) and alter cellular function. (33) the reduction in gsh is due to consumption for conjugation of metabolites, and then redox potential of the tissue will be impaired. (34) gsh depletion also results in lipid peroxidation and impaired antioxidant enzyme activities. (35) the data presented in this study clearly demonstrated the state of oxidative stress induced in hepatic tissues by ccl4 treatment manifested by elevation of mda content in tissue homogenate, which is associated with depletion of gsh content, these results are compatible with those obtained by others. (36) administration of benfotiamine results in increased intracellular thiamine diphosphate levels, a cofactor of transketolase enzyme; activation of this enzyme by thiamine may reduce superoxide overproduction through directing advanced glycation and lipoxidation end products substrates to the pentose phosphate pathway. (7,8) the data presented in this work showed a significant decline in mda level and increase in gsh level in animals treated with benfotiamine and thiamine prior to ccl4 administration, this might be attributed to the up-regulation of transketolase but could be a positive side effect of benfotiamine, which show an intrinsic antioxidative activity by itself. (37) it is important to mention that the effect of benfotiamine was much better than thiamine concerning gsh levels. lipid peroxidation and oxidative stress were attenuated by nac administration to the rats prior to ccl4, which may be due to enhancement of intracellular gsh biosynthesis. lipid peroxidation has been proposed to disrupt cellular membrane, resulting in loss of membrane integrity, (38) and may lead to leakage of alt and ast and increasing their activities in the plasma. the plasma transaminases (alt and ast) are known to be increased significantly in rats after exposure to toxic doses of ccl4. (39) the data presented in table 1, clearly support the iraqi j pharm sci , vol.18 (suppl.), 2009 benfotiamine in liver injury 05 above explanation and seems to be consistent with those obtained by others. (40) the sharp elevation in serum activities of the enzymes that localized in bile ducts is a useful biochemical index for bile duct damage, particularly alkaline phosphatase (alp). in the present work, serum activity of alkaline phosphatase that is present in the lining membrane of the hepatocytes was also increased in the ccl4-treated rats compared to normal control animals, and these results are consistent with that reported by other investigators. (41) the major finding of the present study is that treatment of animals with benfotiamine prior to ccl4 elicits beneficial effects on the structure and functions of the liver; regarding the enzymatic activities, there is a significant decrease in ast level compared with that intoxicated with ccl4, the reduction was three times more than that obtained through the use of thiamine prior to ccl4 administration, but, still the protection was less than that observed in the animal group treated with nac prior to ccl4. the data also revealed attenuation of alt activity induced by ccl4 due to the use of benfotiamine, which was approximately the same as that noted with thiamine; however, the protection was much less than that observed due to the use of nac. concerning serum alp, the serum activity level of this enzyme almost normalized by benfotiamine, again the protection was better than thiamine even it precede that of nac. the data presented in this study regarding the effect of nac was consistent with those observed by others. (38) the suggested mechanisms behind these effects of benfotiamine may be due to indirect action of the lipophylic pro-drug through decreasing lipid peroxidation, which is the main factor affecting membrane permeability and integrity, and consequently preventing further leakage of cytosolic enzymes; (38) another possible explanation may be related to its negative action on protein kinase c activities. 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produces h2o2. clin chem 1982; 28(10): 2077-2080. 30. martinez m, mourelle m, muriel p. protective effect of colchicine on acute liver damage induced by ccl4. role of cytochrome p-450. j appl toxicol 1995; 15: 49-55. 31. poyer jl, mccay pb, lai ek, et al. confirmation of assignment of the trichloromethyl radical spin adduct detected by spin trapping 13c-carbon tetrachloride metabolism in vitro and in vivo. biochem biophys res commun 1980; 94: 1154–1160. 32. cotran rs, kumar v, robbins sl. cell injury and cellular death. in: robbin’s pathologic basis of disease, 5th edition, prism book pvt. ltd., 1994: 379-430. 33. johnston de, kroening c. mechanism of early ccl4-toxicity in cultured rat hepatocytes. pharmacol toxicol 1998; 39: 231-239. 34. percival m. anti-oxidants. clin nutr 1998; 10: 1-4. 35. husain k, morris c, whitworth c, et al. 4-methylthiobenzoin acid protection against cisplatin nephrotoxicity: antioxidant system. fundam appl toxicol 1996; 32(2): 278-284. 36. sotelo-felix ji, martinez-fong d, de latorre p. protective effect of carnosol on ccl4 –induced acute liver damage in rats. eur j gastroenterol hepatol 2002; 14(9): 1001-1006. 37. schmid u, schupp n, heidland a, et al. new approaches for the treatment of genomic damage in end stage renal disease. j ren nutr 2008; 18(1): 127-33. 38. recknagel ro, glende ea, dolak ja, et al. mechanisms of carbon tetrachloride toxicity. pharm ther 1989; 43:139-154. 39. agarwal ak, mehendale hm. potentiation of ccl4 hepatotoxicity and lethality by chlordecone in female rats. toxicology 1983; 26: 231-241. 40. gole mk, dasgupta s. role of plant metabolites in toxic liver injury. asia pac j clin nutr 2002; 11(1): 45-50. 41. ulicna o, greksak m, vancova o, et al. hepatoprotective effect of rooibos tea (aspalathus iinearis) on ccl4-induced liver damage in rats. physiol res 2003; 52: 461-466. iraqi j pharm sci , vol.18 (suppl.), 2009 benfotiamine in liver injury 07 ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 37 effect of temperature on the stability and release profile of ibuprofen microcapsules issraa r. abed alrahman *1 *de pa rtme nt o f pharm aceu tic s, college of pha rm acy, baghda d un iversity, bag hdad iraq. abstract the s tability and releasing profile of 2:1 core: wa ll ratio ibuprofen mic roc apsules prepa red by aqueous coa cerva tion (gelatin and ac acia polymers coa t) and an orga nic coace rvation methods (ethyl c ellulose a nd sodium alginate polymers c oat) in weight equivalent to 300mg drug, we re studied using different storage tempe ra tures 40°c, 50°c ,60°c and refrigerator tempe ra ture 4°c in an ope ned a nd closed containe r for three months (re leas ing profile) and four months (s tability s tudy).it wa s found that, the se ibuprofen mic rocapsules we re stable with e xpira tion da tes of 4.1 a nd 3.1 yea rs for aqueous and an organic method res pec tive ly.aqueous prepa red ibuprofe n microcapsules were found more stable than those microcaps ules prepa red by organic method with activation ene rgy (ea) 4804.8 c al/mol a nd 5033.6 ca l/mol of a drug respec tive ly.the releasing perce ntage of ibuprofen for all microcapsules prepa red by both me thods was decreased as the storage te mperatures increased, exce pt for mic rocapsules prepared by a que ous me thod, whic h were found to be the same at 25-40°c as the standard one which stored at 25°c temperature, on the other hand , as the te mperature decrease d to 4°c (refrige ra tor ) of a n open and c losed conta iner ,the amount of drug de te cted in microparticles is increased. these differe nces in the amount of drug re leased may be re ferred to the c hange in physica l prope rties in polymer coa ts or in the amount of drug de te cted in a whole microca psules. keyword: ibuprofe n mic rocapsules , storage tempe ra ture, stability. الخالصة كبسوالت رة ل مت دراسة تأثير درجات الحرا رة مختلفة ت را ي درجات ح وفين ف اليبوبر م٬ ° ٥۰,م °٦۰(ا حرارة ) م° ٤۰ عها في درجة رى بوض ق (م°٤واخ غل واألخر م وح على ثباتية وطريقة تحرر مادة )في وعائين مفت ن المائية ) ۱ :۲ (األيبوبروفين من النسبة ريقتي جالتين والصمغ (للكبسوالت المجهرية المحضرة بالط غلفة بمادتي ال الم وية )العربي وم (والطريقة العض عادل ) لسليلوز األثيل والجينيت الصودي ن لمدة ۳۰۰بوزن ي وبروفي ثالثة ملغم من مادة األيب واء راسة ثباتية الد ر لد ربعة اشه وا واء رة .اشهر لدراسة تحرر الد ريه المحض كبسوالت المجه سبة من ال كان ثباتية تلك الن مدى صالحية ك واضحا مع ن ريقتي منهمابالط ريقتين المائية و العضوية على التوالي ۱.۳ و ۱.٤ل ولقد وجد أن .سنوات للط مائية أكثر ثباتية بسبب وجود المادة المصلبة للغالف هايد(الكبسوالت المجهرية المغلفة بالطريقة ال مالدي زيد من ) الفور التي ت جات رة منخفضة كان ذلك واضحا أيضا من خالل قلة كفاءة التغليف وقابلية تحمله للحرارة العالية أو الرطوبة مع در را ح واء زيئات الد حفيز ج ؤدي .طاقة ت ن تلك الكبسوالت عند زيادة درجات حرارة الخزن ت من جانب أخر وجد أن تحرر الدواء م رات الفزياوية التي تحدث في الم غي ود ذلك الى الت كس بانخفاض درجات الحرارة ويع ى الع ادة الى قلة في سرعة التحرر وعل ن من الكبسوالت المجهرية المخزونة عند وبروفي وفين والمشابهة لتحرر األيب كبسوالت المجهرية لأليبوبر م °۲٥المغلفة لل ها في نفس تلك الدرجة زن مائية ال تتغير ,عند خ ريقة ال مجهرية المحضرة بالط كبسوالت ال كما لوحظ تحرر الدواء من ال . م°٤۰۲٥ مقارنة للنسبة األصلية عند الدرجة 1corresp ond in g author ema il issraap har m @yah oo.com rece ived 3-5-2006 accepted 1 3-12-20 06 ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 38 in troduction: microenca psula tio n technique prov id es a go od tool to protec t the drug from environmenta l factors (te mperature , humid ity, light, o xygen) by imp ro ving its s ta bility thro ugh e nc lo sing of s mall drug pa rticles of liquid s, s olids or gas es by a n inta ct s he ll(1,2). acc elerated s ta bility stud y at e le va te d te mpe ra ture s is an impo rta nt stud y whic h indica te s the effec t of te mpe rature o n che mical stab ility of microc aps ules as we ll a s the ir physica l p rop erties (3) . che mic ally b y pred ic ting the ra te of drug d eco mpos ition a fter exp erime ntally ev aluating the v elocity co nsta nt o f a pa rticular re ac tion, the amount of a ctive drug pres ent at any time and expiration d ate c ould b e de te rmine d the s pe cific manor in whic h the ra te of rea ctio n va ries with the c onc entration of the reac ta nts (3,4). the arrhenius eq uation reve als the e ffec t of te mpe ra ture o n an obs erve d rate co ns ta nt b y affe cting the mo le cular motio n thro ugh de te rmining the arrhenius a ctiv atio n e ne rgy (ea) , whic h is the minimum kinetic e ne rgy that a molec ule must p oss es s in order to undergo rea ctio n (4) . the physica l e ffe ct of te mpe ra ture , o n microca psule s by a ltering the glas s transitio n te mpe ra ture (tg) of mic ros pheres polymers co at ; is a na rrow te mpe ra ture ra nge o ver which mic roc ap sule s polymer rev ersibly change from phase to phas e w hich oc curs in amo rp hous polymers and in a morpho us c hains pa rtly c rystalline polymers, for exa mple tg of pure glyco lide -l-lac tide (plga) polymer of microca psule wa ll was found to be 49 oc at which the rmo -rev ersible ge la tion in which the lo ng and fle xible p olymer c hains tend to bec ome entangle d and attra ct e ac h o ther by sec ondary side cha in forces , this phys ic al cha nges in microc ap sules p olyme r cause an alte ring in the diffus ion o f the d rug fro m mic ro cap sule s wall by affe cting the d riving fo rc es of p hysico-chemica l po te ntia l grad ie nt and transpo rt parame ters(5,6). on the other hand , protein microc ap sules stored at 4oc o ve r the c ourse of 2 8 d ays produced physica l c hange s in shap e or inte grity of microca ps ules that a ffec te d the re le asing behavior of these s pheres (7). aim of the work to stud y the effe ct of different storage te mpe ra ture s o n the s ta bility (kinetic study) and re le asing profile of 2:1 ib uprofen mic ro cap sule s prepa re d b y aqueous (gelatin and a ca cia co at) and organic metho ds (sod ium alginate a nd e thylcellulos e polyme r c oat)(8). ma terials and methods ibup ro fe n powd er sdi, iraq. gelatin granula ted (fluka a-g, ch-94 70 buchs, switzerla nd). ethylcellulose , chlo roform, methanol a nd etha nol 96%, (bdh che mic al ltd, england). phe no phtha line s olutio n, calcium chlorid e, sod ium a lginate (judex la boratory regents , sud bury, midd le s, engla nd). s odium hydroxide , po ta ss ium dihyd ro gen phos pha te (e-me rk, da rmstadt, we st germany). formalde hyde so lution (ho pkin a nd eilliams ess ex, england ). aca cia, is oprop ano l (ried el de haen ag, s eelze hannov er, ge rma ny). pre paration of ib upr ofen microc aps ules ib uprofen microcapsules were prep ared freshly using a queous metho d (co mplex coacerva tion phase separa tion) a nd o rganic metho d as shown in sc he me 1 and 2 respec tive ly (8): ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 39 ge la tin (2%) w/w ib uprofen + acac ia (2%) w/w 40 0c 40 0c mix with s tirring at 25 0 r.p.m adjust to ph 4 se t a s ide for 50 minutes with stirring (4 00c) add 10 ml forma ldehyde (5%) with s tirring for 10 minute s co ol d own to 5 0c filte r resuspend ed in isop ropa nol filte r dry (roo m te mp.) sche me -1 pre pa ra tion o f ibuprofe n microcaps ules by aque ous me tho d. ibuprofen + na -a lginate (2%) ethylc ellulose in c hloroform (20 %) 40 0c roo m temp erature mix dropp ing into 1% calcium c hloride so lution dry in o ven a t 6 00c sche me -2 pre pa ration of ibuprofe n microca ps ules by org anic me thod. ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 40 ass ay o f micr oca psules content 0 .4 50gm of 2: 1 core : wa ll ibup ro fe n microca psule s p re pared by a que ous and orga nic metho d (eq . to 207 mg (6 9%) a nd 26 1 mg (87%) ib uprofen rec ep tive ly) was extrac te d in 5 0ml o f methano l, 0.4ml o f pheno phthaline so lution a s indicato r was a dde d, the n titra te d against 0.1m s odium hydro xide until a re d co lo r was o btaine d, b la nk titration was carrie d out, and a mount of d rug co ntent was determine d ,eac h 1 ml of 0.1m naoh is equiv alent to 20.63 mg of c13h18o 2 (9,10). deter minatio n of micr ocapsules pr operties microenca psulatio n yield and encapsula tion efficiency of 2:1 co re : wall ra tio ib uprofen mic ro ca psules prepared b y aq ueous and organic metho d we re es tima te d us ing the fo llowing e xp ress io ns . actual wt. of mic ro cap sule s ga ined mic ro enc ap sula tion yie ld = x 10 0 (%) the oretic al wt. of mic roc ap sule s ac tual drug lo ad ing enc ap sulation e fficienc y = x 10 0 (%) the oretic al drug load ing effec t of tempera ture on s tability of ibupro fe n microca psu les : the e ffec t of different storage te mpe ra ture s on the d egra dation rate of the selec ted formula 2: 1 c ore: wa ll ra tio ibup ro fe n mic ro cap sule s prepa re d by a que ous and o rganic me thod wa s stud ie d. the stud y wa s d one by incub ated the prepa re d mic ro ca psule s in a n amb er co lo re d glas s c ontaine rs at diffe re nt te mpe ra ture s (6 0oc, 50 oc, 40oc) a nd 4oc of refrigera tor in an o pened a nd clos ed c onta iners fo r 4 months. samples of mic ro ca psule s of both prep aratio n metho ds (aqueous and o rganic ) in we ight of 652 mg and 517 mg re spe ctively eq uivalent to 300 mg ib up rofe n we re taken at des ired time intervals (e very month) and a ss aye d fo r the co ntent o f drug ac cording to the p ro ced ure mentioned b efore fo r stab ility s tudy. the e ffec t of te mper atur e on the diss olutio n be havio r of ibu pro fe n mic ro cap sules : in vitro drug relea se from 2:1 co re : wall ratio ib uprofen mic ro cap sule s sa mples using b as ket method in we ight equiv alent to 300 mg d rug of both prep aratio n metho ds wa s stud ie d us ing u. s. p d is so lution a ppa ra tus with 900 ml of ph6.8 pho sphate buffer at 37 oc  1 and stirring s pe ed 15 0 r.p.m. at a pprop riate time inte rv als 5ml s amp le wa s withdrawn, filte re d, and me as ured sp ec trop hotome trica lly at λmax 264 nm. this as sa y w as re pea te d for three months , ino rd er, to study the effe ct o f diffe re nt storage temperature (4 0oc, 50 oc, 60oc), 4oc ope ned co ntaine r a nd clos ed one ) o n the re le ase profile of ibup ro fe n fro m s elected fo rmula o f bo th method s. results a nd disc ussion micr oc aps ules c onten ts microencap sula tion yie ld a nd enca psula tion efficiency of 2 :1 co re : wall ra tio ib uprofen mic ro cap sule s prep ared b y aq ue ous and organic me thod we re ca lc ulated a s sho wn in ta ble 1. tt aa bbllee (( 11)) mmiicc rroo cc aa ppss uullee ss ccoo nnttee nn ttss oo ff 22 :: 11 ccoo rree ttoo wwaa llll rraa tt iioo iibb uupp rr oo ffee nn mmiicc rroo cc aa ppss uu llee ss pprree ppaa rree dd bbyy aaqquuee oo uuss aa nndd oo rr gg aa nn iicc mmee tthh oo dd.. (2 :1) co re : wall ra tio d rug amo unt (gm) coa t (gm) m icro e nca ps ulation yie ld pe rce nt yie ld d rug loa ding (mg ) encapsul atin efficien cy (%) gelatin aca cia ethylcellu lose n a algina te t heoretical (gm ) actual (gm ) theoretical (m g) actual (mg) aq ueo us ly prepared ra tio 8 2 2 12 8 6 7 300 207 69 orga nicly prepared ra tio 8 2 2 12 1 1.7 97.5 300 261 87 ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 41 effe ct o f elevated temp era tu re o n stability of ib upr ofen micr oca ps ule s f igures (1) and (2 ) sho w the c hange in the lo g % rema ining o f ibup ro fe n versus time at different eleva ted te mperature 40oc, 50 oc, 60oc.the obtaine d profile s were linear fo r bo th 2: 1 c ore :wall ra tio ibup ro fe n mic ro cap sule s prepa re d b y a que ous a nd o rganic metho d indica ting that ibuprofen de grad atio n fo llowe d first ord er kinetics. the s lo pe o f this line arity wa s de te rmine d and the ca lc ulated rate co ns ta nts are s umma rize d in ta bles 2 a nd 3 for b oth metho ds , resp ec tive ly. arrhe nius plots are shown in figure s (3 ) and (4 ) .the ra te c ons tants (k) at 2 5oc o btained from the plot were 2.13 x 10-3 mo nth-1 a nd 2.8 1 x 10-3 mo nth-1 for aqueo us and orga nic metho d re spe ctively. -3 -2.5 -2 -1.5 -1 -0.5 0 2.9 3 3.1 3.2 3.3 3.4 3.5 (1/t)x10-3 l o g k 1.982 1.986 1.99 1.994 1.998 2.002 0 1 2 3 4 time (month ) lo g % re m a in in g 40 c 50 c 60 c 1.977 1.982 1.987 1.992 1.997 2.002 0 1 2 3 4 5 time (month ) l o g % re m a in in g 40 c 50 c 60 c -3 -2.5 -2 -1.5 -1 -0.5 0 2.9 3 3.1 3.2 3.3 3.4 (1/t) *10-3 lo g ( k) figure (1): acce le ra te d bre akdown of ibupro fe n in 2 :1 co re : wall ratio mic roc apsules at diffe re nt e le va te d te mpe ra ture s us ing aqueo us formula figure (3): arrhe nius plot for es timatio n s he lf life o f ib uprofe n microca psule s us ing 2:1 c ore : wall ratio pre pare d by (aque ous me thod) figure (4 ): arrhe nius plot for e stima tion she lf life o f ibuprofe n micro ca psule s using 2 :1 c ore : wall ra tio pre pare d by (organic me tho d) figure (2): acce le rate d bre akdown o f ibuprofe n in 2:1 co re : wall rat io mic roc aps ules at diffe re nt e le va te d te mpe ratures using orga nic fo rmula ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 42 ttaa bbllee ((22)):: ddee gg rraa ddaa tt iioo nn rraa ttee ccoo nn ss ttaa nn ttss (( kk)) oo ff iibbuu pprr oo ffee nn mmiicc rroo ccaa pp ss uu llee ss uuss iinngg 22 :: 11 ccoo rree :: wwaa llll rraa tt iioo aa tt ddiiffff ee rree nntt ttee mmppee rr aa tt uurree ss (( aaqquuee oo uu ss mmee tt hhoo dd )) ta ble (3): deg radatio n rate constants (k) of ibupro fe n mic rocaps ule s using 2 :1 co re : wall ra tio at diffe re nt te mpe ratures (orga nic me thod). te mpe rature k ( mont h-1) 40oc 4.67 x 1 0-3 50oc 5.77 x 1 0-3 60oc 7.59 x 1 0-3 sinc e the de grada tion o f the d rug follow ed first order kine tic (stra ight line ), the expiration d ate co uld be c alcula te d by the following e qua tion: t10% = c25 o k 0.104 (4). it was eq ua l to 4 .1 years (aque ous ) and 3.1 ye ars (orga nic). while the arrhenius a ctiv atio n e ne rgy (ea) w as c alcula te d fro m the slop e of arrhe nius plots .it wa s fo und to be 480 4.8 ca l/mol and 5 03 3.6 cal/mol res pe ctiv ely. the ab ove res ults indica te d tha t, the microsp heric partic le s pre pa re d from gelatin and aca cia co at (aq ueo us me thod) were more stab le be ca use of p re se nce of c ro ss linking agent (formalde hyde) (11), as we ll as , ac tiv atio n energy was les s than tha t of orga nic metho d (12), which is in c ons istent with the res ults obta ined by s iv akumar-p a w ho found that the stab ility o f ibup ro fe n lipo so pmes increas ed b y us ing a cros slinking a gent(13). the other manifes ta tio ns o f ge ne ra l ap pea ra nc e like color o f microca ps ules we re not c hange d afte r 4 mo nths for b oth formulas . effe ct of tempe ra tur e on the diss olutio n be havio r of ibu pro fe n mic ro cap sules : figures 5 and 6 ind ic ate tha t no la rgely difference wa s ap pe ared in the releas ing profile of a ll s tore d ib uprofen mic roc ap sule s of bo th metho ds at 60 oc, 5 0oc, 40oc), 4oc in an ope ned and c lose d co ntainer (refrigerator) after one mo nth in co mpariso n with s tand ard 2:1 core: wall ratio ib up rofe n mic ro sp heres prepa re d by aq ue ous and o rganic method stored at ro om temperature(14) ,while a fter sec ond and third mo nths , the re le asing behavior o f microca ps ules for bo th me thods was c le arly c hange d a s shown in figure s 7, 8 and 9,10 for aq ueo us and o rganic metho d, re spe ctively. te mpe rature k (month-1) 40 oc 3.96 x 10 -3 50 oc 4.75 x 10 -3 60 oc 6.45 x 10 -3 0 20 40 60 80 100 120 15 30 4 5 60 7 5 90 1 05 s td 600c 500c 400c 40c c lose % d ru g r e le a se d 40c op en figure (5 ): the re le asing be havior of 2 :1 core : wall rat io ibuprofe n microc aps ules pre pare d by aqueo us me tho d store d at diffe re nt te mpe ratures afte r one month us ing 6.8ph o f phosphate buffe r fig ure (6): the re le as ing be havior of 2 :1 core : wa ll ratio ibuprofe n micro ca psule s pre pa re d by organic me thod store d at diffe re nt te mpe rature s after one mo nt h using 6 .8 ph of phos phate buffe r. ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 43 f igures 7 a nd 8, illus tra te the re le as ing be hav io r of 2: 1 co re : wall ratio ibup ro fe n microca psule s prepa re d by aqueo us metho d stored a t 40 oc in which the re le ase of the d rug was the same as the s tand ard one . the sa me re sult wa s obta ined by ca ro l-as, he found that the releas e of tryptophan and theo phylline fro m gel mic roc ap sules stored at 25oc and 40oc re maine d uncha nged(15). on the other hand, microc aps ules s tore d at 50oc and 60oc provide d lo wer relea se o f drug due to re mova l of s olve nt, de nse r pe riphery (v is cous bounda ry) of microc aps ules (16), b ut when the te mperature of storage d ec re ase d to 4oc of refrige ra to r of an ope n a nd close conta iner, the releas e of ibuprofen increa sed bec ause of physica lly changes in the integrity of microc aps ules s ha pes whic h be came more spheric al with s moo th s urfa ce that pro vided la rger s urfa ce area with p orous s urfa ce a fter comp le tion of s ta bility stud y(17,18). also , microca psule s stored in a n o pen conta iner pro vide d highe r relea se of ib uprofen than c lo sed o ne d ue to pres enc e of humidity in the refrigerator environment that mad e the gelatin swe ll fas te r in the d is solution med ia with ra pid diffusion of the drug from the te xture of the gel(10). the results o btained from figure 9 a nd 1 0 sho wed the d elay in re le as ing p rofile of ib uprofen from micro ca psule s pre pa re d by organic metho d when the tempe ra ture of storage inc re as ed from 40 oc to 6 0oc d ue to te nde nc y o f microca ps ules to s tic king which was reve rs ible afte r co oling to roo m te mpe ra ture . the slightly dec re ase d of disso lutio n rate and the s ticking phe no mena could b e exp la ined by c ha nge in the po lymer film d ue to its low gla ss tra ns ition temp erature, which could be a dditio na lly red uce d by disso lv ed ib uprofen mo le cules in a dditio n, storage a t higher tempe ra ture a llowe d e thyl cellulos e film to b ec ome more dense d ue to curing effect as it was re po rted for eudragit l10 0-5 5 po lymer(19). in s pite of increas ing the re le as e of ib uprofen from micro ca psule s pre pa re d by organic me thod when s to re d a t re frigerator te mpe ra ture in co mparison with standard 2: 1 core: wall ratio s tored at ro om tempe ra ture , the re le ase o f drug fro m mic roc ap sules s tored in an op en c onta iner a t 4 oc s howe d lo wer re le ase of d rug at firs t 15 -3 0 minutes b ec aus e of some amorphous ibuprofen in the s olid d is persion proba bly re acte d with the p olymer by c atalytic action o f water vap or d uring storage(20). 0 20 40 60 80 100 120 15 30 45 60 75 90 105 st d 60 0 c 50 0 c 40 0 c 4 0 c ope n 4 0 c cl ose 0 20 40 60 80 100 120 15 30 45 60 75 90 105 st d 60 0 c 500 c 40 0 c 4 0 c o p en 4 0 c clo se % d ru g r e le a se d fig ure (7): dissolutio n be havior o f ibuprofe n mic roc apsules pre pare d by a que ous me thod store d at diffe re nt te mpe ratures us ing pho sphate buffe r ph 6.8 a fte r 2 nd mo nth of storage. fig ure (8): dissolution be hav io r of ibuprofe n microc aps ules pre pare d by aque ous me thod s to re d a t diffe re nt te mpe ratures after third mo nt h o f stora ge us ing p hos pha te buffe r ph 6 .8 ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 44 conclusion 2:1 c ore : wa ll ratio ib uprofen mic roc ap sule s prepa re d by b oth aqueo us a nd orga nic metho d were sta ble with s helf life o f 4.1 a nd 3.1 ye ars. all physica l changes a ffec te d the re le as ing be hav io r of this ratio o ccurred at d ifferent storage tempe ra ture were re vers ib le o n le av ing thes e formulas at 25oc in tightly c lo se d conta iner. 2:1 core: wall ratio ib uprofen microc ap sules prepa re d by a que ous method can b e stored at a te mpe ra ture 2 5-40oc without altering its stability o r the releas e profile . refe renc es 1. uddin, m.s., ha wlader, m.n., zhu ,h.j ., mic roencapsula tion o f ascorbic a cid; e ffec t of process va ria bles on product charac te ris tics journal microe ncaps ul., 200 1; 18, p .1 99209 . 2. berto lini, a.c., siani, a.c., grosso ,c.r., stability o f mono terpenes e ncaps ulated in g um ara bic by sp ra y drying, j-agric-foo d-che m., 200 1;49,780-5. 3. ismat a. hamid, industrial p harmac y, 197 5; 2 01 -204. 4. alfre d ma rtin, the p hysica l p ha rma cy,; 4th editio n; 1993,313 -3 14 . 5. we n, i., ki mbe rly, w.a., ki ne tic and ther modyna mic mo deling of the formation of polyme ric mic rosp heres using solve nt extraction /evapo ra tion ,jo urna l of co ntrolle d re lease, 1995; 3 7, 187 -198. 6. yo shio ka,s.,aso ,y.,kojima,s., drug re lease fro m poly dllactide mic rosp heres controlled by ga ma – irradiation ,journal of controlled release , 1995 ; 37 , 263-267. 7. ma gee ,g.a., ha lb ert,g.w., wil mott, n., effec t of process variables o n the in v itro degra da tion of p ro te in microspheres , journal of c ontrolled re lease ,19 95;37 ,1 1-19. 8. iss raa, r.a., p repa ra tion of ib up rofe n mic ro ca psules as a s us ta ined re le ase s olid dosage form, thesis , colle ge of p harmac y, unive rs ity of baghda d ,2000. 9. european pharmacopoeia [disk], ib uprofen, france , 1998. 10. britis h pharmac opoeia, vo l. 1 and 2, 199 3; p .305,3 01. 11. chowd hury, s.r., mukherje e, p. k., mic roencapsula tion of ib uprofen by c omple x coace rvatio n tec hnique, east-p harm, 19 95; 38[mar], 137-138 . 12. nguyen, m.t., he ndrick, m., mo del studies o n the s tability of folic acid a nd 5methyl tetra hyd ro fo lic a cid de grada tio n during thermal trea tme nt in co mbina tion with high h ydros tatic pressure , j-agric –fo odche m, 20 03; 21[may], 51 , 33 52-7. 13. s ivakumar, p.a., mythily , s., liposome ib uprofen co njuc ate a nd the rele ase cha racteristics o f ibuprofen, indian-drugs, 199 4;31[dec], 568573. 14. el-bary, a.a.,el-naba rawi, m.a., moha med ,m.i., prop io nic acid deriv atives fro m their d osage forms , drug-de r-ind-pha rm. 199 8; 2 4, 439 -45. figure (9 ): dissolution be hav io r of ibuprofe n micro ca psule s pre pare d by organic me thod store d at diffe re nt te mpe ratures afte r 2 nd month of s to rage using 6.8 ph p hos phate buffe r. figure (10): dissolut io n be havior o f ibuprofe n micro ca psule s pre pare d by organic me thod store d a t diffe re nt te mpe ra tures afte r 3rd mo nth of s to rage using 6 .8 phos phate buffe r. ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 ac ce lerated stability of ib upr ofen micr oc aps ules 45 15. charala mbos , g.v.,da vid, g.d., carol,a.s ., zero-order rele ase from b ip ha sic polyme r hydrogels, jo urna l of co ntrolle d re lease ,1995;34, 185-192 . 16. we n, i.l., kmbe rly, w.a., predic tion of peptide-lo ade d plga mic rosp he res p repa re d by solve nt e xtra ction/evap oratio n metho d ,j ournal o f co ntro lled release , 19 95;37 , 1 99214. 17. rajes h, r.d., ra je sh, h.p., fac to rial effe ct of p ro cess paramete rs o n pharmace utical characte ristics and s tability stud y o f p lga microca ps ules co ntaining wa te r-so luble drug, plga microsheres , 2 004 ;1 -1 4. 18. davidson, i.g., la gne r,e.j .,re lease mec hanism of ins ulin e ncaps ulated in trehalose es te r de riva tive microcapsules delivere d v ia inhalation ,int.-jpharm., 20 03; 26[mar],25 4, 211 -22. 19. we ib , g., kno ch,a., micro encapsula tio n of ibup ro fe n by a co acervation process us ing eud ra git l1 00-55 as an e nteric polyme r, drug develop ment and ind ustria l pha rmacy, 19 93; 19, 2751-2764. 20. ma koto,o.,mika,o.,hygroscooic sta bility and d isso lution p rope rties of spray-d ried s olid dispe rsion o f furo se mid e with e ud ra git, journal of pharmac eutica l s ciences ,19 93; 82,32 -3 8. comparative evaluation of using intranasal desmopressin, parenteral diclofenac or their combination in the management of acute iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 93 effect of silibinin in lowering the intraocular pressure in normotensive rabbits: interaction with betaxolol saad a. hussain * ,1 , haider m. mohammed* , ahmed h. jwaied * , munaf h. abdulrazzaq* *department of pharmacology and toxicology, college of pharmacy, university of baghdad , baghdad , iraq. abstract previous reports demonstrated the effectiveness of silibinin hemisuccinate as a potential intraocular pressure-lowering agent. the exact mechanism by which silibinin exerted this effect has not yet been documented, but might suggested to interfere with aqueous humor formation. the present study was designed to evaluate the comparative efficacy of silibinin as iop lowering agent to that of betaxolol in normotensive rabbits, and the interaction of silibinin with betaxolol as a way for investigating the possible mechanism of action of silibinin in this respect. the effects of instillation of 0.75% silibinin solution and 0.5% betaxolol eye drops in the eyes of normotensive rabbits were evaluated using indentation tonometry. the results showed that 0.75% solution of silibinin was more potent than betaxolol (0.5%) in lowering iop in normotensive rabbits. furthermore, the effect of preand postinstillation of silibinin-betaxolol combination showed a characteristic antagonistic feature. in conclusion, silibinin appears to be more potent than betaxolol in lowering iop in normotensive rabbits; the preand post-instillation of silibinin provide experimental evidence for the possible antagonistic effect of betaxolol with the iop-lowering effect of silibinin. key words: silibinin, betaxolol, camp, pde-inhibitors الخالصة ثبج يٍ دساست سابقت فعانٍت يادة انسٍهٍبٍٍٍُ فً خفض انضغظ انذاخهً نهعٍٍ، اال اٌ يٍكاٍَكٍت يثم ْزا انخأثٍش نى حعشف بعذ عهى انشغى ال حذاخهٓا يع حكٌٍٕ انسائم انذاخهً نهعٍٍ. حى حصًٍى ْزِ انذساست نًقاسَت فعانٍت انسٍهٍبٍٍٍُ فً خفض انضغظ انذاخهً نهعٍٍ يٍ احخً ة يقاسَت بًادة انبٍخاكسٕنٕل فً االساَب، ٔكزنك دساست انخذاخم بٍٍ انبٍخاكسٕنٕل ٔانسٍهٍبٍٍٍُ عُذ اسخخذايًٓا يعا كٕسٍهت نخكٌٍٕ فكش % بٍخاكسٕنٕل قطشاث فً 5.0% سٍهٍبٍٍٍُ ٔ 0..5ت عًم انسٍهٍبٍٍٍُ. حًج دساست حأثٍش االسخخذاو انًٕضعً نًحهٕل حٕل يٍكاٍَكٍ . أظٓشث انُخائح فعانٍت انسٍهٍبٍٍٍُ فً خفض indentation tonometryعٌٍٕ أساَب بٍضاء راث ضغظ داخهً طبٍعً باسخخذاو حقٍُت نزي ٌحذثّ انبٍخاكسٕنٕل، كًا اٌ اسخخذاو انسٍهٍبٍٍٍُ قبم ٔبعذ انبٍخاكسٕنٕل اظٓش حضادا انضغظ انذاخهً نهعٍٍ بشكم افضم يٍ رنك ا ٔاضحا فً انخأثٍش. ًٌكٍ االسخُخاج بأٌ االسخخذاو انًٕضعً نهسٍهٍبٍٍٍُ فً انعٍٍ ٌؤدي انى خفض انضغظ انذاخهً نهعٍٍ اكثش يٍ ٍ اٌ ٌعطً فكشة أٔنٍت عٍ يٍكاٍَكٍت عًم ْزا انًشكب.انبٍخاكسٕنٕل، كًا اٌ انخذاخم انسهبً يع انبٍخاكسٕنٕل ًٌك introduction although glaucoma is no longer defined as elevated intraocular pressure (iop) but rather a condition comprises characteristic optic nerve head and visual filed abnormalities (1) , lowering iop is still the major strategy in slowing down glaucomatous damage to the inner structures of the eye and visual filed (2) . all current treatment strategies are designed to reduce iop by reducing the rate of aqueous humor (ah) formation and/or enhance its drainage out of the eye (3) . the ciliary epithelium has α2and 2-adrenergic receptors. stimulation of α-receptors or inhibition of 2-receptors was thought to reduce ah formation (4) . topical instillation of epinephrine decreases the rate of ah formation, an effect thought to be mediated by -receptor induced increase in camp in the ciliary epithelium (5) . the participation of camp in this effect has been supported by finding that activators of adenylcyclase (cholera toxin and forskolin) decrease ah formation and hence iop in experimental animals and human (6) . targeting of this cl transport system is thought to be the newer proposed mechanism for the lowering of iop by the oldest antiglaucomatus drug, timolol (7) . these findings support the major involvement of increased rather than decreased camp as a second messenger mechanism in the control of ah formation in normal physiology, as well as in pathological conditions. interestingly, the action of -blockers in the reduction of ah formation is now suggested to involve campindependent mechanism (7) . furthermore, timolol was shown to reduce epinephrineinduced increase in uveoscleral outflow when the two drugs applied concurrently (8) . inhibition of phosphodiesterase (pde) by flavonoids has been previously described (9,10) ; silib 1 corresponding author : e-mail : saad_alzaidi@yahoo.com received : 15/7/2007 accepted : 30/12/2007 iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 05 powerful antioxidant flavonoid (11) has been shown to reduce iop in normotensive rabbits when used alone in different concentrations, with greater effect achieved with 0.75% dose (12) . the site of action did not exactly pointed but the drug shown to delay iop recovery rate after i.v. infusion of 20% nacl solution. this largely suggests an interference with ah inflow mechanism. interestingly, silybinin was shown to inhibit campphosphodiesterase enzyme more potent than theophylline or papaverine (13) . this study was conducted to evaluate the possible interaction of silibinin with the iop-lowering effect of the -adrenergic blocker betaxolol in normotensive rabbits. materials and methods twenty new zealand white rabbits weighing 1.5-2.5 kg were used in this study, and treated according to the ethics of animal experiments approved by the university of baghdad. animals were kept in the animal house of the college of pharmacy, university of baghdad, under standardized conditions (12 hrs light-dark cycles at room temperature), and were fed standard diet and given water ad libitum. silibinin hemisuccinate in pure form was a gift from tolbiac s.r.l. (argentina) and all other chemicals were supplied by the department of pharmacology and toxicology, college of pharmacy, university of baghdad. silibinin hemisuccinate was dissolved in arachis oil (as a vehicle) and used as freshly prepared (0.75%) solution. betaxolol 0.5% drops (alcon, cham, switzerland) were used as commercial eye drop formula. rabbits were allocated into four groups (5 animals each) for studying the effect of topically instilled silibinin hemisuccinate, betaxolol, the effect of topical silibinin instilled 30 min prior to betaxolol, and the effect of topical betaxolol instilled 30 min prior to instillation of silibinin . measurement of iop: indentation tonometry using schiotz tonometer was utilized in this study for measuring iop before and after application of drugs or vehicle. thirty min before starting any application, the cornea was anesthetized with 0.5% tetracaine, and baseline iop was measured using schiotz tonometer. after topical instillation of 1 drop of silibinin (0.75%) and betaxolol (0.5%), measurement of iop was performed every 30 min for 3 hr (14) . after each measurement, eyes were washed with normal saline and the instrument was cleaned with diethyl ether. all experiments were conducted by trained subject who is completely unaware about the type of treatment followed, and performed during a fixed time of the day (from 10:00 am to 3:00 pm) to exclude the effect of circadian changes in iop.for studying the pre-instillation effect of betaxolol, baseline iop was recorded after the instillation of betaxolol, and then one drop of silibinin solution was instilled after 30 min into both eyes. iop was measured every 30 min for 3 hr. the same later approach was followed to evaluate the effect of preinstillation of silibinin on that produced by betaxolol. results were presented as a mean value of iop  sd. comparisons with baseline were made using student’s paired t-test, while a single-factor analysis of variance (anova) was used to test the statistical differences between groups. p values less than 0.05 were considered significant. results effects of 0.75% silibinin and 1% betaxolol: in normotensive rabbits, ocular instillation of 0.75% silibinin decreases iop for 2.5 hr compared to baseline value and remains significantly different at all measured time points (table1). the maximum decrease in iop was achieved after 1 hr of instillation of silibinin (38.56%) compared to baseline value (p<0.05). application of 1 drop betaxolol (0.5%) eye drop resulted in significant decrease in iop, with maximum reduction achieved after 1hr (22.08%); then decreased with time and became (2.05%) after 3.0 hr, which found non-significantly different compared to baseline (table1). effects of pre-treatment with 0.75% silibinin corneal instillation of 0.5% betaxolol eye drops produced highly significant reduction in iop when preceded (30 min) by instillation of 0.75% silibinin (38.35%-3.9%, p<0.05) compared to the effect produced by betaxolol alone (22.08%-6.61%, p<0.05) (table 1, figure 1). the higher magnitude of reduction in iop was achieved during the first 30 min of instillation of 0.5% betaxolol to silibininpretreated eyes (38.35%, p<0.05) compared to 10.78% produced by betaxolol alone. the reduction in iop continued to be significantly high during the next 30 min interval (27.76%, p<0.05) compared to 22.08% produced by 0.5% betaxolol alone. however, the reduction in iop after this period seems to be nonsignificant (17.71%, 10.52%, 3.94%, p>0.05) compared to 19.49%, 20.87% and 6.61% produced by 0.5% betaxolol alone at the intervals 1.5, 2.0 and 2.5 hr (p>0.05). the reduction in iop was significantly different from that produced by 0.75% silibinin alone for the intervals 0.5, 1.0 and 1.5 hr (p<0.05), while found non-significant for the rest of time (p>0.05) (table, figure 1). iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 04 effects post-treatment with silibinin the results presented in table 1 and figure 1 showed that the ocular hypotensive effect of 0.5% betaxolol was slightly changed following the addition of silibinin, and during the first 30 min of application, 0.75% silibinin resulted in 13.61% reduction in iop compared to 22.08% produced by betaxolol alone (p>0.05). although the iop was reduced following the next 30 min (26.52%), it was found comparable to that produced by betaxolol alone (p>0.05). table 1. effects of instillation of silibinin 0.75% and betaxolol 0.5% on the intraocular pressure (iop) in normotensive rabbits. intraocular pressure (mmhg) group baseline 0.5hr 1.0 hr 1.5 hr 2.0 hr 2.5 hr 3.0 hr silibinin 0.75% 28.6 ± 1.9 22.1 ± 0.6* a 17.5 ± 2.8* a 19.1 ± 3.7* a 25.3 ± 4.0* a 27.5 ± 2.9* a 28.6 ± 1.9 a betaxolol 0.5% 31.5 ± 2.3 28.1 ± 3.1* b 24.5 ± 3.9* b 25.4 ± 3.1* b 24.9 ± 2.3* a 29.4 ± 2.4* a 30.8 ± 2.0 a silibinin pre-treatment 31.7 ± 2.5 24.6 ± 2.2* c 19.7 ± 2.3* a 22.9 ± 2.7* a 26.1 ± 2.2* a 28.4 ± 2.5* a 30.5 ± 2.4 a silibinin post-treatment 31.7 ± 2.8 28.1 ± 3.4* b 27.2 ± 3.6* b 23.1 ± 2.6* b 24.7 ± 3.1* a 29.2 ± 2.6* a 31.5 ± 2.7 a values are expressed as mean ± sd; number of eyes in each group = 10; * p< 0.05 with respect to baseline value; values with non-identical superscripts (a,b,c) among different groups are considered significantly different (p<0.05). figure 1. effects of preand posttreatment with 0.75% silibinin hemisuccinate on iop-lowering effect of 0.5% betaxolol in normotensive rabbits; results are presented as mean of % reduction; number of eyes in each group = 10; *p<0.05 with respect to 0.75% silibinin hemisuccinate alone. 45 40 35 30 25 20 15 10 5 0 0.5 1 1.5 2 2.5 3 time (hours) % i o p re d u c ti o n 0.75% silibinin hemisuccinate 0.5% betaxolol hcl pre-treatment with silibinin hemisuccinate post-treatment with silibinin hemisuccinate * * iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 04 discussion in the present study, topical instillation of silybinin was shown to strongly lower iop in normotensive rabbits, an effect that appears significantly greater than that produced by 0.5% betaxolol (table1). betaxolol binds to βadrenergic receptors of the ciliary processes with high affinity (15) . since agonists to all βadrenergic receptors (β1, β2, and β3) stimulate adenylcyclase via interaction with gs-protein to increase camp production, betaxolol was thought to lower iop by reducing the intracellular concentrations of camp (16) . however, it has long been unclear whether the putative reduction in camp itself causes the reduction in iop, an observation reported by liu et al (1981) who demonstrated that dtimolol, another β-blocker might be as effective as l-timolol in decreasing aqueous flow (17) , despite stereospecificity of the βadrenergic receptors for the l-isomers (18) . meanwhile, if betaxolol reduces aqueous humor formation by blocking β-adrenergicmediated increase of camp production, one would expect camp itself to increase inflow. however, camp certainly does not markedly increase aqueous inflow. accordingly, caprioli et al (1984) reported a decrease in inflow following administration of forskolin, which stimulates endogenous production of camp (6) . however, the foregoing considerations do not preclude the possibility that betaxolol reduces secretion of aqueous humor exclusively through its action as a nonselective β-adrenergic antagonist, but have raised doubts about that hypothesis. a conflicting result have recently been reported by mclaughlin et al (2001) who demonstrated that application of camp did not reverse timolol’s effects; and that timolol and levobunolol produced camp-independent inhibition of the regulatory volume increase (rvi) in ciliary cells and increased intracellular ca 2+ and ph; they suggested that inhibition of cl /hco3 exchange mediates timolol’s inhibition of aqueous humor formation as an alternative mechanism for the reduction of aqueous inflow and then iop (7) . the iop lowering effect of silibinin is thought to occur via reduction of ah formation, and the site of action has been postulated to be the ciliary epithelium; this is based on previous data reported in our laboratory that revealed delayed recovery time following intravenous infusion of 20% nacl and a profound contralateral effect on untreated eyes (12) . the present study demonstrated that when compared with betaxolol, silibinin was found more effective in lowering iop. it appears that neither prenor post-instillation of each one of them improves significantly the iop-lowering effect produced by any one of them alone. preinstillation effect of silibinin appears to completely abolish that of betaxolol; however, the higher magnitude of reduction in iop already produced by pre-instillation of silibinin (figure 1) might be due to the action of silibinin alone. these effects are very interesting in that the potent action of silibinin might mask that of betaxolol especially when given 30 min before, and this might explain the predominance of silibinin action over that of betaxolol (figure 1). however, silibinin did not augment the effect of betaxolol when administered latter suggesting interference with its action by previous instillation of betaxolol. this conclusion can be accepted pharmacodynamically since -blockers are known to initiate a decrease in camp levels required for the action silibinin (as pdeinhibitor) and the only effect shown in figure 1 might be attributed to betaxolol alone.the mechanism through which betaxolol reduced iop is now difficult to correlate with its inhibitory effect on camp. isoproterenol, which stimulate camp production, has been reported to increase aqueous humor inflow (19) . in contrast, forskolin, which also stimulate camp formation, has been found to reduce inflow (20) , and isoproterenol itself has reported to reduce iop in water-loaded rabbits (21) . since silibinin has been proved to inhibit pde (9) and to produce higher magnitude of reduction in iop (37.84%) compared to that of betaxolol (22.0%), one could suggest that increasing intracellular camp could be the major event through which silibinin reduces iop. involvement of camp as a target in the events of lowering iop was clear in many studies that involve application of forskolin (an adenylcyclase activator) via either topical or systemic route (22) . forskolin reduced net aqueous humor inflow in rabbits, and increased ciliary blood flow through activation of adenylcyclase in ciliary epithelium; this action was not blocked by timolol (6) . this is quite important to explain why betaxolol blocks the effect of silibinin (a pde-inhibitor). β-blockers did not block the action of forskolin because the latter was still capable to stimulate synthesis of camp, while silibinin requires the presence of already synthesized camp to elongate its half-life. for this reason blockade of camp synthesis by betaxolol diminish the activity of silibinin. cyclic amp was found to inhibit transepithelial cl secretion across bovine ciliary epithelium by uncoupling the intracellular gap junction (23) and to inhibit other important regulator of iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 09 aqueous humor dynamics, the na + -k + -2cl cotransporter (24) . inhibition of this cotransporter has found to be associated with higher degree of reduction in iop. interestingly, this profile of activity is similar to that produced by 0.75% silybinin (38.5%) in the present study. although the effect of silibinin on the ocular phosphodiesterase (pde) has not been studied, a study on beef heart pde revealed that silibinin was more potent as pde-inhibitor than theophylline and papaverine in this regard (13) . from these findings one can suggest that the strong ocular hypotensive effect produced by silibinin might be attributed to the inhibition of pde, and the resultant accumulation of camp inhibits na + k + -2cl cotransporter in ciliary epithelium as well as in trabecular meshwork cells. both effects on inflow and outflow of aqueous humor dynamics could be the possible mechanisms through which silibinin produces this effect, and became a new drug candidate for the reduction of elevated iop.in conclusion, the results obtained in this study provide experimental evidence that silibinin is more potent in reducing iop than betaxolol through a mechanism that might be related to increase camp levels. acknowledgment the authors thank the college of pharmacy, university of baghdad for supporting the project, and tolbiac srl, argentina for providing pure standardized silibinin hemisuccinate as a gift. references 1. anderson dr. glaucoma: the damage caused by pressure. am j ophthalmol 1989; 108: 485. 2. leske mc, heijl a, hussein m, bengtsson b, hyman l, komaroff e. early manifest glaucoma trial group: factors for glaucoma progression and the effect of treatment: the early manifest glaucoma trial. arch ophthalmol 2003; 121: 48-56. 3. moses r a. intraocular pressure. in: moses ra, hart w. (eds.), adler’s physiology of the eye: clinical application, 8th edition, the c.v. mosby, santa louis, 1987; 22. 4. andrew s, titcomb l. glaucoma in: walker r, edwards c.(eds), clinical pharmacy and therapeutics, (2 nd ed.),churchill livingstone, london. 1999; 807-821. 5. neufeld ah, sears ml. cyclic-amp in ocular tissues of the rabbit, monkey, and human. invest ophthalmol vis sci 1974; 13: 475. 6. caprioli j, sears m, bausher l. forskolin lowers intraocular pressure by reducing aqueous inflow. invest ophthalmol vis sci 1984; 25: 268. 7. mclaughlin wc, peart d, purves dr, carre ad, peterson-yantorno k, mitchell hc, et al. timolol may inhibit aqueous humor secretion by campindependent action on ciliary epithelial cells. am j physiol cell physiol 2001; 281: 865-875. 8. schenker ji, yablonski me, podos sm, linder l. fluorophotometric study of epinephrine and timolol in human subjects. arch ophthalmol 1981; 99: 1212. 9. ruckstuhl m, landry y. inhibition of lung cyclic ampand cyclic gmp phosphodiesterases by flavonoids and other chromone-like compounds. biochem pharmacol 1981; 30: 697-702. 10. beretz a, anton r, cazenave jp. the effects of flavonoids on cyclic nucleotide phosphodiesterases. in: cody v, middleton e, harborne jb. (eds.), plant flavonoids in biology and medicine: biochemical, pharmacological and structure-activity relationships, alan r. liss inc, new york, 1986; 281-296. 11. altorjay i, dalmi l, sari b, imre s, balla g. the effect of silibinin (legalon) on the free radical scavenger mechanisms of human erythrocytes in vitro. acta physiologica hungarica 1992; 80: 375-380. 12. mohammed hm, abdulrazzaq mh, hussain sa. effects of silibinin hemisuccinate on the intraocular pressure in normotensive rabbits. saudi med j 2007, 28(9): 1397-1401 13. koch hp, bachner j, loffler e. silymarin: potent inhibitor of cyclic amp phosphodiesterase. methods find exp clin pharmacol 1985; 7(8): 409413. 14. vareilles p, lotti vj. effects of timolol on aqueous humor dynamics in the rabbit. ophthalmol res 1981; 13: 72-79. 15. 15.vareilles p, silverstone d, plazonnet b, ledouarec jc, sears ml, stone ca. comparison of the effects of timolol and other adrenergic agents on intraocular pressure in the rabbit. invest ophthalmol vis sci 1977; 16: 987-996. 16. hoffman bb, lefkowitz rl, taylor p. neurotransmission: the autonomic and somatic motor nervous system. in: hardman jg, limbird le, molinoff pb, ruddon rw, gilman ag. (eds.), goodman and gilman’s: the iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 00 pharmacological basis of therapeutics, (9th ed.), mcgraw-hill, new york, 2001; 105-139. 17. liu hp. chion cy. continuous stimulation and instant display of aqueous humor dynamics with a microspectrometer and a sensitive drop counter. exp eye res 1981; 32: 583-592. 18. lefkowitz r.j, mukherjee c, coverstone m, caron mg. steriospecific [ 3 h] (-) alprenolol binding site, β-adrenergic receptors and adenylate cyclase. biochem biophys res commun 1974; 60: 703-709. 19. brubaker, r.f.: clinical measurements of aqueous dynamics: implications for addressing glaucoma. in: civan mm. (ed.), the eye’s aqueous humor: from secretion to glaucoma, academic press, san diego, 1998; 233-284. 20. tian b, geiger b, epstein dl, kaufman pl. cytoskeleton involvement in the regulation of aqueous humor outflow. invest ophthalmol vis sci 2000; 41: 619-623. 21. gieser sc, juzych m, robin al, schwartz gf. clinical pharmacology of adrenergic drugs. in: ritch r, shields mb, krupin t. (eds.), the glaucoma, (2nd ed.), mosby, st. louis, 1996; 14251448. 22. lee py, podos sm, mittag t, severin c. effect of topically applied forskolin on aqueous humor dynamics in cynomolgus monkey. invest ophthalmol vis sci 1984; 25: 1206-1209. 23. gabelt bt, kaufman pl. prostaglandin f2 alpha increases uveoscleral outflow in the cynomolgus monkey. exp eye res 1989; 49(3): 389-402. 24. okada y, hazama a. volume regulatory ion channels in epithelial cells. news physiol sci 1989; 4: 238-242. iraqi j pharm sci, vol.31( suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala doi: https://doi.org/10.31351/vol31isssuppl.pp62-74 62 phytochemical screening of petroleum ether fractions by gc/ms and isolation of lupeol from two different parts of iraqi leucaena leucocephala. (conference paper )# azal satar al-baaj *,1 and thukaa z. abdul-jalil1* # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq. abstract this work is considered the first study for the components of the iraqi leucaena leucocephala plant, where the different phytochemical compounds that present in the aerial parts were identified by using the gas chromatography/mass spectrometry technique (gc/ms). the type of the components and their concentration will differ according to the part of the plant used and the method of extraction (hot and cold). this study made a comparison in lupeol concentration that was identified and isolated from petroleum ether fractions of leucaena leucocephala by using gas chromatography/mass spectrometry (gc/ms), high-performance thin-layer chromatography (hptlc), and preparative high-performance liquid chromatography (p-hplc). the plant leaves and stems were collected in september, dried under shade, and powdered (separately), then extracted by two extraction methods: hot soxhlet and cold maceration method using 85% ethanol, then the result crude extract was fractionation with petroleum ether by using a separator funnel. the results of gc/ms, hptlc, and phplc indicated that the leaves contain a higher concentration of lupeol than the stems and the cold maceration method is more efficient than the hot soxhlet extraction method. lupeol has many pharmacological activities applied in alternative medicine such as anti-inflammatory, antimicrobial, anti-arthritic, anticancer, antidiabetic, and antioxidant activities with wide future applications. keywords: leucaena leucocephala, lupeol, gas chromatography/mass spectrometry (gc/ms), high-performance thin-layer chromatography (hptlc), and preparative high-performance liquid chromatography (p-hplc). الفحص الكيميائي النباتي الجزاء االثير البترولي بواسطة تقنيات كروماتوغرافيا الغاز / قياس الطيف ) بحث مؤتمر (# وعزل مادة اللوبيول من جزأين مختلفين من نبات الليوسينا العراقي gc/msالكتلي *زهير عبد الجليل و ذكاء 1*،أزل ستار ألبعاج 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي جامعة بغداد ، بغداد ، العراق . الصيدلة، كلية الطبية، فرع العقاقير والنباتات * الخالصة ) شجرة الرصاص أالبيض ( المزروع في ألعراق ) ألتابع هذا العمل ، وألول مرة ، دليل على وجود مادة اللوبيول في نبات الليوسينا اجراء مقارنة في الى عائلة البقوليات( من خالل تحديد المركبات الكيمونباتية في االجزاء العليا من النبات ) االوراق و السيقان (. في هذه الدراسة تم كروماتوغرافيا الغاز /قياس الطيف ستخلصة من نبات الليوسينا باستخدام تقنياتتركيز مادة اللوبيول المشخص والمعزول من أجزاء البتروليم ايثر الم تم جمع أوراق و سيقان . phplc ، و الفصل اللوني السائل العالي االداء hptlc ، الفصل اللوني للطبقة الرقيقة عالية االداءgc/ms الكتلي ، بعدها تم االستخالص بإستخدام طريقتين: طريقة االستخالص منفصل عن االخر( النبات في شهر سبتمبر ، و جففت تحت الظل، ثم طحنه )كل جزء ٪ من االيثانول، بعدها تم تجزئة ٨٥الساخن باستخدام جهاز السوكسلت و طريقة االستخالص البارد باستخدام طريقة النقع ، و كال الطريقتين باستخدام .ام قمع الفصلالمستخلص النباتي الخام مع البتروليم أيثر بأستخد الى ان االوراق تحتوي على تركيز اعلى من اللوبيول من السيقان و أن طريقة (phplc ,hptlc ,gc/ms) أشارت نتائج التقنيات المستخدمة البدي الطب في المطبقة الدوائية أالنشطة من العديد اللوبيول يمتلك الساخن. االستخالص طريقة من كفاءة أكثر البارد فاعليته االستخالص مثل ل .كمضاد لاللتهابات، مضاد للميكروبات، أللتهاب المفاصل، مضاد للسرطان، مضاد للسكر، ومضاد لالكسدة مع تطبيقات مستقبلية واسعة الكروماتوجرافيا السائلة عالية ، كروماتوغرافيا الطبقة الرقيقة عالية األداء ، ، كروماتوجرافيا الغاز / قياس الطيف الكتلي وبيولل ،: ليوسينا مفتاحية كلمات . األداء التحضيري introduction from the beginning of life on earth, there was an association between humans, animals, and plants in which it supplied some of the needs that are important for life continence such as oxygen, food, and medicine for treating their diseases. over time and with many tries , humans learned how to use herbal materials in their life in a beneficial way. aft er thousands of years, the traditional medicine systems were used all over the world, the ayurvedic and unani of the indian subcontinent, the chinese and tibetan of other parts of asia, the native americans of north america, the amazonian of south america, and several local systems within africa. (1) azal.satar1200m@copharm.uobaghdad.edu.iq :mail-corresponding author e1 received:18 /5 / 2022 accepted:5 /9 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp62-74 iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 63 leucaena belongs to the fabaceae family and mimosoideae’s subfamily which contain approximately 50 species of shrubs and trees. l. leucocephala has high nutritious value and a lot of uses around the world such as human food, firewood, timber, green manure, and shade; so, it is known as “a miracle tree’’. (2) l. leucocephala is used in many countries by a human who eats the young leaves, flowers, and young pods in soups such as in indonesia, central america, and thailand. (3), (4) l. leucocephala tree proved to have a high medicinal value as studies revealed that it contains various active chemical compounds such as flavonoids, cardiac glycosides, tannins, phylobatanins, alkaloid, saponins, ester, and ketone. so, it has a lot of pharmacological activities such as antimicrobial, anthelmintic, antibacterial, anti-proliferative, antidiabetic, anticancer, diuretic, anti-inflammatory, antioxidant, antitumor, antihistaminic, anti-androgenic, and hepatoprotective properties. (2) this study articulates the frame to view the first report with regarded phytochemical screening by gc/ms, determination and isolation of lupeol from petroleum ether fractions of two different parts of iraqi leucaena leucocephala by hptlc and phplc respectively. figure1. photos of flowers, leaves, fruits, seeds, and stems of the iraqi leucaena leucocephala plant (27) . materials and methods plant material collection and authentication in september 2021, the plant was collected from one of the farms located in al_ diwaniyah city. the plant parts (leaves and stems) were isolated, cleaned, and dried for a week under the shade until they dried completely. the leaves and stems were separately ground to be ready for extraction. the plant has been authenticated by dr. zainab abed aoun ali, ph.d. in plant taxonomy / department of life sciences / college of science / the university of baghdad. preparation of petroleum ether extracts. the dry powdered leaves and stems were extracted by two extraction methods: 1. soxhlet (hot method): 100 grams of each powdered part was extracted with 1000 ml of 85% ethanol for 21 hours, and the result fractions were concentrated by a rotary evaporator and then fractionation two times with 200 ml of petroleum ether in a separatory funnel, the results p.e fractions were analyzed by using gc/ms, hptlc, and phplc. 2. maceration (cold method): 100 grams of each powdered part was macerated with 1000 ml of 85% ethanol (two times, each time for two days), and the result fractions were concentrated by a rotary evaporator and then fractionation two times with 250 ml of petroleum ether in a separatory funnel, the results p.e fractions were analyzed by using gc/ms, hptlc, and phplc. iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 64 figure 2. extraction, fractionation & identification scheme of lupeol from iraqi leucaena leucocephala. leaves stems evaporate by using a rotary evaporator extraction with 85% ethanol hot method by soxhlet cold method by maceration crude ethanolic extracts of leaves and stems (separately) that result from the hot and cold extraction methods result of four fractions of petroleum ether crude extracts: 1st: from leaves by hot extraction method 2nd: from leaves by cold extraction method 3rd: from stems by hot extraction method 4th: from stems by cold extraction method all the resultant organic layers were concentrated by using a rotary evaporator to get crude extracts gc/mass hplc hplc hplc p-tlc hplc tlc hplc drying under shade, pulverized into powder in separatory funnel, all ethanolic extracts are fractionation with water and petroleum ether (100-250ml) *2 evaporate petroleum ether layers aqueous layer p_hplc hplc gc/ms hplc hptlc hplc iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 65 preliminary identification and isolation of lupeol in petroleum ether extracts of iraqi leucaena leucocephala gas chromatography/mass spectrometry (gc/ms) analysis for petroleum ether fractions. four fractions of petroleum ether were analyzed by using gc/ms chromatography which is found in ibn al-bittar center / ministry of science and technology / baghdad / iraq. lupeol in p.e fractions of leucaena leucocephala was identified by matching the mass spectra with libraries spectra. the quantity of lupeol is calculated as a percentage of the relative peak area. table (1) shows the gc/ms conditions that were used in the analysis of the p.e fractions. table 1. gc/ms conditions. (4) instrument agilent (7820a) usa gc mass spectrometer analytical column agilent hp-5ms ultra (30 m length x 250 µm diameter x 0.25 µm inside diameter) injection volume 1µl pressure 11.933 psi temperature gc inlet line temperature: 250 ˚c aux heaters temperature: 310 ˚c carrier gas he 99.99% injector temperature 250 ˚c scan range: m/z 25-1000 injection type split less oven program temperature ramp 1 60˚c hold to 3 min. ramp 2 60˚c to180 ˚c 7 ˚c/min. ramp 3 180˚c to 300˚c 8 ˚c/min ramp 4 300˚c hold to 3 min. qualitative estimation of lupeol by using highperformance thin layer chromatography (hptlc(. all p.e extract fractions of leaves and stems that were prepared by different extraction methods were qualitatively identified by using the hptlc which is found in baghdad college of medical sciences/ baghdad/ iraq. table (2) shows the hptlc conditions that were used in the analysis of the p.e fractions. table 2. hptlc conditions. (5) instrument eike-reich/camag-laborator/ switzerland and operated by win cats software using a tungsten lamp stationary phase tlc plates silica gel 60 f254 pre-coated layer (20 cm x 10 cm), thickness 0.2mm band length 8 mm standards lupeol samples four p.e fractions of leaves and stems from two extraction methods for iraqi leucaena leucocephala solubility methanol application volume 3 μl development chamber camag, adc 2chamber (20x 10) chamber saturation time 5 minutes development mode ascending mode distance run 75 mm slit dimensions 4.00 x 0.30 mm scanning speed 20 mm/s measurement mode absorbance mobile phase and detection lupeol toluene: ethyl acetate: chloroform (5:1:4). (26) uv 225 nm and 5% h2so4 spray iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 66 qualitative and quantitative identification of proposed lupeol by preparative high-performance liquid chromatography (phplc). lupeol was identified and quantified from petroleum ether fractions of leucaena leucocephala by using preparative high-performance liquid chromatography (phplc) which is found in the ministry of science and technology/ department of water and environment/ baghdad/ iraq. table (3) shows the phplc conditions that were used in the analysis of the p.e fractions. table 3. phplc conditions. (6) instrument cyknm high-performance liquid chromatography column mediterranea c18 (5 µm 15 x 2.12 cm) mobile phase acetonitrile (a) and water (b) gradients 0-1 min 3% a; 10-45 min 3-21% a; 45-60 min 21-40% a. (25) samples petroleum ether fractions (leaves and stems) standard lupeol standard column temperature room temperature application volume 100 μl for identification, 1 ml for isolation injection concentration 1mg /1ml for each sample detection wavelength uv detector at λ 210 nm results and discussion this work is considered the first attempt to recognize the biologically active constituents of leucaena leucocephala in iraq. the weights of the result crude ethanolic extracts were (cold 16.78, hot 14,53) grams, after fractionation with p.e, the weight of the p.e fractions were (leaves, cold 2.89 / leaves, hot 1.85 / stems, cold 1.9 / stems, hot 0.4) grams. the results of gc/ms, hptlc, and phplc indicate that there are the same compounds that are found in the leaves and stem but in different concentrations. these compounds are belonging to various chemical classes, these are; fatty acids, volatile oils, phytosterols, triterpene, diterpene, fatty alcohols, vitamin e isomers, alkanes, and alkenes. gas chromatography/mass spectrometry (gc/ms) analysis for petroleum ether fractions. after analyzing the petroleum ether extracts of leucaena leucocephala by gc/ms, it was found that they contain many active compounds, which are summarized in the following figures and tables. figure 3. gc-ms analysis of petroleum ether leaves extract of leucaena leucocephala by hot extraction method. iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 67 table 4. gc-ms analysis of petroleum ether leaves extract of leucaena leucocephala by hot extraction method. no. chemical class compound rt (min.) molecular formula similarity index references 1 lactams caprolactam 12.039 c6h11no 98 (7) 2 natural monoterpenoid phenol thymol 12.776 c10h14o 94 (8) 3 macrocyclic organosiloxane cyclohexasiloxane dodecamethyl 13.018 c12h36o6si6 94 (9) 4 macrocyclic organosiloxane cycloheptasiloxane, tetradecamethyl 16.187 c14h42o7si7 93 (10) 5 fatty acid methyl ester methyl tetradecanoate 21.255 c15h30o2 99 (11) 6 fatty acid methyl ester. hexadecanoic acid, methyl ester 25.240 c17h34o2 99 (11) 7 long-chain fatty acid ethyl ester hexadecanoic acid, ethyl ester 27.099 c18h36o2 98 (12 8 fatty acid methyl ester 9,12-octadecadienoic acid (z, z)-, methyl ester (linoleic acid) 28.344 c19h34o2 98 (13) 9 essential fatty acid 9,12,15-octadecatrienoic acid, (z, z, z) 28.488 c18h30o2 99 (9) 10 fatty acid methyl ester 9,12,15-octadecatrienoic acid, methyl ester, (z, z, z) 28.488 c19h32o2 99 (14) 11 diterpenoid phytol 28.657 c20h40o 99 (2) 12 fatty acid methyl ester methyl stearate 28.964 c19h38o2 99 (15) 13 long-chain fatty acid ethyl ester linoleic acid ethyl ester 29.531 c20h36o2 91 (12) 14 fatty acid ethyl ester 9,12,15-octadecatrienoic acid ethyl ester, (z,z,z) 29.649 c20h34o2 95 (15) 15 diester hexanedioic acid, bis(2ethylhexyl) ester 33.399 c22h42o4 95 (16) 16 fatty acid methyl ester docosanoic acid, methyl ester 35.636 c23h46o2 97 (17) 17 triterpene squalene 39.790 c30h50 97 (14) 18 steroid stigmast-4-en-3-one 40.671 c29h48o 94 (11) 19 lipids gamma-tocopherol 43.214 c28h48o2 99 (18) figure 4. gc-ms analysis of petroleum ether leaves extract of leucaena leucocephala by cold extraction method. https://pubchem.ncbi.nlm.nih.gov/#query=c6h11no https://pubchem.ncbi.nlm.nih.gov/#query=c6h11no https://pubchem.ncbi.nlm.nih.gov/#query=c6h11no https://pubchem.ncbi.nlm.nih.gov/#query=c6h11no https://pubchem.ncbi.nlm.nih.gov/#query=c6h11no https://en.wikipedia.org/wiki/monoterpene https://en.wikipedia.org/wiki/phenols https://pubchem.ncbi.nlm.nih.gov/#query=c12h36o6si6 https://pubchem.ncbi.nlm.nih.gov/#query=c12h36o6si6 https://pubchem.ncbi.nlm.nih.gov/#query=c12h36o6si6 https://pubchem.ncbi.nlm.nih.gov/#query=c12h36o6si6 https://pubchem.ncbi.nlm.nih.gov/#query=c12h36o6si6 https://pubchem.ncbi.nlm.nih.gov/#query=c12h36o6si6 https://pubchem.ncbi.nlm.nih.gov/#query=c12h36o6si6 https://pubchem.ncbi.nlm.nih.gov/#query=c12h36o6si6 https://pubchem.ncbi.nlm.nih.gov/#query=c14h42o7si7 https://pubchem.ncbi.nlm.nih.gov/#query=c14h42o7si7 https://pubchem.ncbi.nlm.nih.gov/#query=c14h42o7si7 https://pubchem.ncbi.nlm.nih.gov/#query=c14h42o7si7 https://pubchem.ncbi.nlm.nih.gov/#query=c14h42o7si7 https://pubchem.ncbi.nlm.nih.gov/#query=c14h42o7si7 https://pubchem.ncbi.nlm.nih.gov/#query=c14h42o7si7 https://pubchem.ncbi.nlm.nih.gov/#query=c14h42o7si7 https://pubchem.ncbi.nlm.nih.gov/compound/31284 https://pubchem.ncbi.nlm.nih.gov/#query=c15h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c15h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c15h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c15h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c15h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c15h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12-octadecadienoic%20acid%20(z%2cz)-%2c%20methyl%20ester%22%5bcompletesynonym%5d%20and%205284421%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12-octadecadienoic%20acid%20(z%2cz)-%2c%20methyl%20ester%22%5bcompletesynonym%5d%20and%205284421%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c19h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h34o2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12%2c15-octadecatrienoic%20acid%2c%20(z%2cz%2cz)-%22%5bcompletesynonym%5d%20and%205280934%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12%2c15-octadecatrienoic%20acid%2c%20(z%2cz%2cz)-%22%5bcompletesynonym%5d%20and%205280934%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c18h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h30o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h30o2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12%2c15-octadecatrienoic%20acid%2c%20methyl%20ester%2c%20(z%2cz%2cz)-%22%5bcompletesynonym%5d%20and%205319706%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12%2c15-octadecatrienoic%20acid%2c%20methyl%20ester%2c%20(z%2cz%2cz)-%22%5bcompletesynonym%5d%20and%205319706%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12%2c15-octadecatrienoic%20acid%2c%20methyl%20ester%2c%20(z%2cz%2cz)-%22%5bcompletesynonym%5d%20and%205319706%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c19h32o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h32o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h32o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h32o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h32o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h32o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c19h38o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h38o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h38o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h38o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h38o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h38o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h36o2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12%2c15-octadecatrienoic%20acid%20ethyl%20ester%22%5bcompletesynonym%5d%20and%206371716%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229%2c12%2c15-octadecatrienoic%20acid%20ethyl%20ester%22%5bcompletesynonym%5d%20and%206371716%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22hexanedioic%20acid%2c%20bis(2-ethylhexyl)%20ester%22%5bcompletesynonym%5d%20and%207641%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22hexanedioic%20acid%2c%20bis(2-ethylhexyl)%20ester%22%5bcompletesynonym%5d%20and%207641%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c30h50 https://pubchem.ncbi.nlm.nih.gov/#query=c30h50 https://pubchem.ncbi.nlm.nih.gov/#query=c30h50 https://pubchem.ncbi.nlm.nih.gov/#query=c30h50 https://pubchem.ncbi.nlm.nih.gov/#query=c29h48o https://pubchem.ncbi.nlm.nih.gov/#query=c29h48o https://pubchem.ncbi.nlm.nih.gov/#query=c29h48o https://pubchem.ncbi.nlm.nih.gov/#query=c29h48o https://pubchem.ncbi.nlm.nih.gov/#query=c29h48o https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22(%2b%2f-)-gamma-tocopherol%22%5bcompletesynonym%5d%20and%2014986%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c28h48o2 https://pubchem.ncbi.nlm.nih.gov/#query=c28h48o2 https://pubchem.ncbi.nlm.nih.gov/#query=c28h48o2 https://pubchem.ncbi.nlm.nih.gov/#query=c28h48o2 https://pubchem.ncbi.nlm.nih.gov/#query=c28h48o2 https://pubchem.ncbi.nlm.nih.gov/#query=c28h48o2 iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 68 table 5. gc-ms analysis of petroleum ether leaves extract of leucaena leucocephala by cold extraction method. no . chemical class compound rt (min.) molecular formula similarity index references 1 caprolactams caprolactam 12.013 c6h11no 98 (8) 2 natural monoterpenoid phenol thymol 12.763 c10h14o 94 (9) 3 macrocyclic organosiloxane cyclohexasiloxane, dodecamethyl 13.018 c12h36o6si6 93 (10) 4 macrocyclic organosiloxane cycloheptasiloxane, tetradecamethyl 16.174 c14h42o7si7 93 (11) 5 fatty acid methyl ester methyl tetradecanoate 21.255 c15h30o2 99 (12) 6 fatty acid methyl ester hexadecanoic acid, methyl ester 25.220 c17h34o2 89 (12) 7 long-chain fatty acid ethyl ester hexadecanoic acid, ethyl ester 26.492 c18h36o2 98 (13) 8 fatty acid methyl ester 9,12-octadecadienoic acid (z, z)-, methyl ester 28.318 c19h34o2 98 (2) 9 fatty acid 9,12,15-octadecatrienoic acid, (z, z, z) 28.612 c18h30o2 91 (2) 10 diterpenoid phytol 28.612 c20h40o 99 (2) 11 fatty acid methyl ester methyl stearate 28.944 c19h38o2 99 (16) 12 triterpene squalene 39.803 c30h50 99 (15) 13 phytosterols beta-sitosterol 39.953 c29h50o 96 (14) 14 pentacyclic triterpenoid lupeol 42.327 c30h50o 96 (14) 15 very long-chain primary fatty alcohol 1-heptacosanol 44.414 c27h56o 98 (20) 16 fat soluble tocopherols vitamin e 44.669 c29h50o2 95 (14) figure 5. gc-ms analysis of petroleum ether stems extract of leucaena leucocephala by hot extraction method. iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 69 table 6. gc-ms analysis of petroleum ether stems extract of leucaena leucocephala by hot extraction method. figure 6. gc-ms analysis of petroleum ether stems extract of leucaena leucocephala by cold extraction method. table 7. gc-ms analysis of petroleum ether stems extract of leucaena leucocephala by cold extraction method. no . chemical class compound rt (min.) molecular formula similarity index references 1 fatty acid methyl ester hexadecanoic acid, methyl ester 23.604 c17h34o2 99 (11) 2 fatty acid ethyl ester hexadecanoic acid, ethyl ester 24.445 c18h36o2 98 (12) 3 fatty acid methyl ester 9-octadecenoic acid (z)-, methyl ester 25.864 c19h36o2 99 (21) 4 diterpenoid phytol 26.100 c20h40o 93 (2) 5 diester hexanedioic acid, bis(2-ethylhexyl) ester 29.211 c22h42o4 89 (16) 6 diterpenoid alcohol trans-geranylgeraniol 33.276 c20h34o 90 (22) gc-ms chromatogram of the petroleum extracts of leucaena leucocephala shows many peaks indicating the presence of different compounds. some of these compounds are considered as a major and others are minor. lupeol is one of the detected compounds in p.e extracts, was identified by matching its mass spectra with libraries spectra the quantity of lupeol is calculated as a percentage of the relative peak area.. qualitative identification by hptlc hptlc is consider an advanced forms of tlc, it is very efficient for qualitative and quantitative analysis. automated application of sample is more precise than manual application and that will prevent the difference in volume of application, so no . chemical class compound rt (min.) molecular formula similarity index references 1 fatty acid methyl ester hexadecanoic acid, methyl ester 23.594 c17h34o2 96 (11) 2 phthalate ester that (diester) dibutyl phthalate 24.464 c16h22o4 93 (19) 3 fatty acid 10-octadecenoic acid methyl ester 25.854 c19h36o2 99 (14) 4 enol 2-methyl-z,z-3,13octadecadienol 28.161 c19h36o 93 (20) 5 phytosterols beta -sitosterol 30.308 c29h50o 99 (13) https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c18h36o2 https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229-octadecenoic%20acid%20(z)-%2c%20methyl%20ester%22%5bcompletesynonym%5d%20and%205364509%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229-octadecenoic%20acid%20(z)-%2c%20methyl%20ester%22%5bcompletesynonym%5d%20and%205364509%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%229-octadecenoic%20acid%20(z)-%2c%20methyl%20ester%22%5bcompletesynonym%5d%20and%205364509%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://pubchem.ncbi.nlm.nih.gov/#query=c20h40o https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22hexanedioic%20acid%2c%20bis(2-ethylhexyl)%20ester%22%5bcompletesynonym%5d%20and%207641%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22hexanedioic%20acid%2c%20bis(2-ethylhexyl)%20ester%22%5bcompletesynonym%5d%20and%207641%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22hexanedioic%20acid%2c%20bis(2-ethylhexyl)%20ester%22%5bcompletesynonym%5d%20and%207641%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://pubchem.ncbi.nlm.nih.gov/#query=c22h42o4 https://en.wikipedia.org/wiki/alcohol_(chemistry) https://en.wikipedia.org/wiki/alcohol_(chemistry) https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22all-trans-geranylgeraniol%22%5bcompletesynonym%5d%20and%205281365%5bstandardizedcid%5d https://www.ncbi.nlm.nih.gov/pcsubstance/?term=%22all-trans-geranylgeraniol%22%5bcompletesynonym%5d%20and%205281365%5bstandardizedcid%5d https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o https://pubchem.ncbi.nlm.nih.gov/#query=c20h34o https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c17h34o2 https://pubchem.ncbi.nlm.nih.gov/#query=c16h22o4 https://pubchem.ncbi.nlm.nih.gov/#query=c16h22o4 https://pubchem.ncbi.nlm.nih.gov/#query=c16h22o4 https://pubchem.ncbi.nlm.nih.gov/#query=c16h22o4 https://pubchem.ncbi.nlm.nih.gov/#query=c16h22o4 https://pubchem.ncbi.nlm.nih.gov/#query=c16h22o4 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o2 https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o https://pubchem.ncbi.nlm.nih.gov/#query=c19h36o https://pubchem.ncbi.nlm.nih.gov/#query=c29h50o https://pubchem.ncbi.nlm.nih.gov/#query=c29h50o https://pubchem.ncbi.nlm.nih.gov/#query=c29h50o https://pubchem.ncbi.nlm.nih.gov/#query=c29h50o https://pubchem.ncbi.nlm.nih.gov/#query=c29h50o iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 70 that will decrease the differences in development of spots through the plate that may occur. it’s flexible enough for one hptlc system to analyze different samples. use of stationary and mobile phase is depending on the number of samples being analyzed (23) . two parts of leucaena leucocephala and two extraction methods were selected to make a comparison between them based on the percentage yield obtained from each method. the presence of lupeol in petroleum ether fractions was obtained. qualitative identification was made by comparison of the maximum retardation factor (max rf) and uv spectrum of lupeol in petroleum ether fractions with its corresponding lupeol standard. figure 7. hptlc chromatogram of lupeol standard. figure 8. hptlc chromatogram of lupeol in petroleum ether fractions of leaves and stems of leucaena leucocephala by hot and cold extraction methods. (a)leaves, hot extraction method (b)leaves, cold extraction method (c)stems, hot extraction method (d)stems, cold extraction method. iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 71 identification and isolation of proposed lupeol by phplc. identification and isolation of lupeol from petroleum ether fractions of the plant was done by using phplc which is considered as the most common method for purification in pharmaceutical industries. (24) phplc chromatogram showed many peaks which represent many different compounds according to their retention time, one of them is lupeol at retention time of (30.08, 30.00, 30.07, 29.94), are the major peaks which were identified by comparison with standard lupeol at retention time of (30.11). the major peaks were collected by fractions collector after monitoring it according to the time (time from beginning of the appearance to disappearance of the peak), then the sample obtained from phplc was dried over anhydrous sodium sulfate, weighted and subjected to different identification methods. figure 9. phplc chromatogram of petroleum ether fraction of leaves that extracted by cold method. figure 10. phplc chromatogram of petroleum ether fraction of leaves that extracted by hot method. figure 11. phplc chromatogram of petroleum ether fraction of stems that extracted by cold method. iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 72 figure 12. phplc chromatogram of petroleum ether fraction of stems that extracted by hot method. figure 13. phplc chromatogram of standard lupeol. according to linear least square regression equation, quantitative determination was carried out by using a calibration curve for the isolated lupeol with a correlation factor equals’ 0.9998278 and peak areas of the new detected compound in fraction of petroleum ether was used to determine the concentration as shown in table 8 and figure 14. figure 14. calibration curve of lupeol standard. iraqi j pharm sci, vol.31(suppl.) 2022 phytochemical screening of two different parts of iraqi leucaena leucocephala 73 table 8. amount of lupeol in p.e extracted fractions. fraction auc (mv.s) amount (ppm) p.e, leaves, cold extraction method 8963.08 589.787 p.e, leaves, hot extraction method 6589.11 277.548 p.e, stems, cold extraction method 5789.25 370.918 p.e, stems, hot extraction method 3658.99 296.946 phplc revealed that leaves contain higher amount of lupeol than stems, and the cold extraction method give best results than hot one, so it will be preferred in the extraction of leucaena leucocephala. conclusions the identification methods that applied on these fractions elucidate that leaves and stems of leucaena leucocephala have many phytochemicals that belongs to different chemical classes (fatty acids, volatile oils, phytosterols, triterpene, diterpene, fatty alcohols, vitamin e isomers, alkanes and alkenes). these phytochemicals differ in their quantity and types from leaves to stems, and also differ according to the method of extraction (hot or cold method). phytochemicals were detected in higher amount in the leaves that extracted by cold maceration method. also, the findings of the study show that hplc method can be adopted for the determination of concentration of lupeol in various extract petroleum ether fractions from various plant parts and isolation with shorter run time and good efficiency. so, it is advisable to consume the leaves of leucaena leucocephala for extraction and isolation of these compounds. acknowledgement thanks to the university of baghdad, college of pharmacy, members of the pharmacognosy and medicinal plants branch for providing necessary facilities and assistance. references 1. mamedov n. medicinal plants studies: history, challenges and prospective. med aromat plants. 2012;01(08). 2. zayed mz, samling b. phytochemical constituents of the leaves of leucaena leucocephala from malaysia. int j pharm pharm sci. 2016;8(12):174–9. 3. brewbaker jl, plucknett dl, gonzales v. varietal variation and yield trials of leucaena leucocephala (koa haole) in hawaii. res bull hawaii agric exp stn. 1972;(166):29pp. 4. isbilen o, volkan e. allium willeanum holmboe exerts anticancer activities on metastatic breast cancer cells mcf-7 and mdamb-231. heliyon. 2021;7(8):e07730. 5. rao kvb, nidhi h, dipankar d, garima d, kumar g, karthik l. research article phytochemical profile,. 2015;31(42):235–41. 6. nowak j, kiss ak, wambebe c, katuura e, kuźma ł. phytochemical analysis of polyphenols in leaf extract from vernonia amygdalina delile plant growing in uganda. appl sci. 2022;12(2). 7. thi hd, d’hooghe m. an update on the synthesis and reactivity of spiro-fused β-lactams. arkivoc. 2019;2018(6):314–47. 8. nagoor meeran mf, javed h, taee h al, azimullah s, ojha sk. pharmacological properties and molecular mechanisms of thymol: prospects for its therapeutic potential and pharmaceutical development. front pharmacol. 2017; 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2020;12(4):850– 6. 26. rao kvb, nidhi h, dipankar d, garima d, kumar g, karthik l. research article phytochemical profile,. 2015;31(42):235–41. 27. pandey vc, kumar a. leucaena leucocephala: an underutilized plant for pulp and paper production. genet resour crop evol. 2013;60(3):1165–71. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ comparative evaluation of using intranasal desmopressin, parenteral diclofenac or their combination in the management of acute iraqi j.pharm.sci., vol.16 (2) ,2007 desmopressin in renal colic 1 comparative evaluation of using intranasal desmopressin, parenteral diclofenac or their combination in the management of acute renal colic pain in iraqi patients ibrahim a. majeed* 1 *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract there is a suggestion that an antidiuretic hormone-induced decrease in diuresis might contribute to the rapid relief of the acute pain in renal colic. this study was designed to evaluate the efficacy of desmopressin nasal spray compared with diclofenac given intramuscularly in patients with acute renal colic. the study included 75 patients randomized into three different groups; group a received desmopressin (40 μg, nasal spray), group b diclofenac (75 mg) intramuscularly and group c, both desmopressin and diclofenac. pain was assessed using a visual analogue scale (a 10-cm horizontal scale ranging from `no pain' to `unbearable pain') at baseline, 10, 20 and 30 min after administering the treatments. on admission, the pain level was the same in all three groups. at 10 min the pain decreased in all groups to a level that was not significantly different. at 20 min groups b and c had similar mean pain levels (5.8), whereas in group a it was 5.7. at 30 min, groups b and c scored 3.0 and 2.5 respectively, and group a 6.1. all three treatments were equally effective at 10 and 20 min but at 30 min there was a stabilization/slight increase in pain level in group a. in conclusion, these results indicate that desmopressin may be used to treat renal colic either alone or combined, increasing the analgesic effect of other drugs like diclofenac key words: renal colic, intranasal desmopressin, diclofenac الخالصة : لشالو اللوا الحوبد حوٍ صوىل ال.لُو ا هوري الدزا و ًَىخد مقتسذ علً أن هىزمىن المضبد للتدزز المحث لىقصه اإلدزاز والرٌ قد َوىعص للو موسَ بصوىزل 57صممت لتقُُا كفبءل مبدل دَسمىبسَسُه عه طسَق العضول للمسىوً المصوببُه بحصوىل ال.لُو ا الدزا و ا توى علوً مُ.سوكوسا شذذ انوو ا المدمىعو )ة( داَ.لىحُىوب 04تا تقسُمها الً ثالث مدبمُع المدمىع )أ( ا وتلمت دَسمىبسَسوُه عشىائُ 14ملغا عه طسَق العضل ا والمدمىع )ج( ا تلمت كال الدوائُه دَسمىبسَسُه و داَ.لىحُىوب انلوا قوُا بىا ول القُوبض العُىوٍ ) 57 دقُق بعد تىبول العالج ا عىد الدخىل مستىي 04 04 14لمدزج مه ن الً غُس محتمل انلا ( عىد خظ الشسوع -انحقٍ ا للقُبض 04دقبئق قل حٍ كل المدبمُع الً المستىي الرٌ لا َ.ه هىوب حوسم معىوىٌ عىود الدقُقو 14انلا كبن متسبوٌ حٍ خمُع المدبمُع عىد 7ا0دقُق مدمىع ) ة ج ( كبوت القُم 04 عىد 5ا7بُىمب مدمىع )أ( كبن 7ا5ة ج ( لهب متى ظ مستىي انلا مدمىع ) دقُقو كوبن هىوب 04دقُقو ول.وه حوٍ 04 14 كل العالخب الثالثو كبووت متسوبوَ التو ثُس حوٍ 1ا1علً التىالً ولمدمىع )أ( 7ا0 لً وقصبن ىئُل حٍ مستىي انلا عىد لمدمىع )أ( ا حوٍ ان وتىتبج تلول الىتوبئح اوىوحت ان دَسمىبسَسوُه قود َسوتعمل لعوالج ا تقسازاً ع المغص ال.لىٌ مىفسداً أو مسكببً لصَبدل الت ثُس المس.ه لالدوَ انخسي مثل داَ.لىحُىب ا سَسُه داَ.لىحُىب : مغص كلىٌ داخل تدىَ انو دَسمىب مفتاح الكلمات introduction renal colic is caused by an increase in pelvi-ureteric pressure secondary to an obstruction of the urinary tract. this increase in pressure causes a prostaglandins-mediated increase in renal blood flow and a subsequent increase in diuresis which, in turn, further increases intrapelvic pressure. modulation of adh is probably one of the most important mechanisms leading to an increased diuresis (1, 2) and one of the roles of prostaglandins (pgs) seems to be blocking the action of antidiuretic hormone (adh) by interfering with camp mediated signal transmission (3) . nsaids (inhibitors of pg synthesis) have long been used as effective agents in the treatment of renal colic. they block other pg-induced effects, such as afferent arteriolar vasodilatation, which causes an increase in diuresis and consequently raises pelvic pressure. they also reduce local oedema and inflammation, and inhibit the stimulation of ureteric smooth muscle, which is responsible for increased peristalsis and subsequently increased ureteric pressure. there is a suggestion that an adh-induced decrease in diuresis might 1 corresponding author : e-mail : ibrahimalbayati54@yahoo.com received : 23/9/2006 accepted : 8/7/2007 mailto:ibrahimalbayati54@yahoo.com iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 0 contribute to the rapid relief of the pain of renal colic (4) . desmopressin (1-desamino-8-dargininevasopressin) is a synthetic structural analogue of adh. compared with adh, it has a greater antidiuretic effect, a longer duration of action and reduced vasopressor activity. these properties make it a first-line drug for replacement therapy in central diabetes insipidus (5) and a very effective agent in the treatment of nocturnal enuresis (6) .the marked antidiuretic effect of desmopressin is probably responsible for its efficacy in the treatment of renal colic (1) . peripherally, it has been shown that desmopressin suppresses the spontaneous contractions of circular smooth muscle fibers in the renal pelvis of rabbits (7) . the same effect could be possible in humans. some authors reported the role of desmopressin in stimulating the secretion of b-endorphins by the hypothalamus (8-11) , which could explain a possible additional central analgesic effect of the drug. to assess the efficacy of intranasal desmopressin in relieving the acute pain of renal colic caused by urolithiasis, we compared the analgesic efficacy of this drug with of one of the most widely used nsaids in renal colic, diclofenac. we also compared desmopressin alone with desmopressin plus diclofenac. the study was enhanced by using the recently marketed intranasal spray form of desmopressin patients and methods this prospective, randomized trial was conducted between may and june 2005 in the emergency department, al-nasirya general hospital, and included 75 patients (45 men and 30 women, mean age 40.3 ± 3.4 years) admitted to the emergency service with renal colic caused by lithiasis and who had previously received no treatment. the patients were randomly assigned to three groups; group a received desmopressin 40 µg intranasally, group b diclofenac 75 mg intramuscularly and group c, both desmopressin and diclofenac. a detailed history was taken and the patients examined. the time of onset and duration of the pain and associated symptoms were recorded, with the number and dates of former episodes, the elimination of calculus and eventual previous documentation of stones by imaging. vital signs and positive findings of the routine physical examination were evaluated and recorded. patients with evidence of high blood pressure, coronary disease, rhinitis, influenza, anticoagulant therapy, and peptic ulcer, renal or liver failure were excluded from the study, as were any pregnant women. a visual analogue scale was used to assess the intensity of pain; this consisted of a 10-cm horizontal scale ranging from `no pain' to `unbearable pain', with values recorded to the nearest millimeter. the pain was assessed on admission and at 10, 20 and 30 min after therapy was administered. in all patients a plain x-ray of the urinary system was taken and any adverse reactions were recorded. the results, presented as mean + sd, were assessed statistically by comparative statistics (one-way anova). results after the random assignment, each group includes 25 patients. the mean duration of pain was 15.07 h, with slight differences among the three groups (14.5, 19.8 and 12.7 in groups a, b and c, respectively). the mean number of previous episodes was 1.3 (1.5, 1.32 and 1.09, in a, b and c, respectively). there were no significant differences in age, blood pressure, radial pulse, or axillary temperature, or in the laboratory values, i.e. for factors related to urinary osmolarity. the intensity of pain at presentation was similar in all groups (.table 1). after 10 min the pain scores were also similar, but at 20 min groups b and c had the same score, whereas group a had a higher score (5.3), and at 30 min, the scores were lower in groups b and c than in group a. in table (2) there were significant differences in pain score with time from baseline in all groups (p<0.01). scores at 0, 10 and 20 min between groups were similar, but after 20 min the pain scores were lower in groups b and c. after 30 min, the differences between a and b, and between a and c, were significant (p<0.01). although the differences between b and c were not significantly different, the score was lowest in group c. in group a, there were significant differences between the first pain assessment and those at 10 and 20 min, but not after 30 min (i.e. pain increased after having diminished at 10 and 20 min). iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 0 table 1. the changes in mean pain score in the three groups with time after administration of therapy. values were expressed asmean + sd, number of patients= 25 in all groups, values with nonidentical superscripts (a, b, c, d) were considered significantly different p < 0.05. table 2. number of responders to treatment and their percentage after administration of drugs. number of responders to treatment response time (min) desmopressin 40µg diclofenac 75 mg combinatio n 10 9 (36%) 21 (84%) 22 (88%) 20 8 (32%) 2 (8%) 3 (12%) 30 1 (4%) 2 (8%) 0 (0%) total 18 (72%) 25 (100%) 25 (100%) number of patients in each group = 25. discussion treating renal colic with intranasal desmopressin 40 μg induced prompt pain relief, with significant decreases in pain scores after only 10 min. this effect was maintained at 20 min and then decreased slightly, in contrast to a former study (1) in which the effect lasted longer (thus reflecting the progressive intranasal absorption of desmopressin, with a plasma peak that can occur up to 4 h after administration, indicating a relatively slow absorption through the nasal mucosa) (10) . within group a (as in group c), there were apparently two subgroups of patients with marked differences in their response to therapy (table 2). thus, although the mean response to therapy after 10 and 20 min was similar in the three groups, the response of individual patients showed that groups a and c had a greater proportion of patients with a marked decrease in their pain scores. thus there seem to be two populations of individuals who will or will not respond to desmopressin. this was reported previously in two different studies (1, 2) , in which 44% and 54% of patients, respectively, had complete pain relief. however, the underlying mechanism(s) are unknown; some authors suggest that it could be caused by individual variation in the intranasal absorption of desmopressin (10) . explaining these findings may detect factors that could be used to identify those patients in whom intranasal desmopressin may be more effective. as in a previous study (1) , the administration of an nsaid with desmopressin was very effective in relieving pain, although the desmopressin was given before the nsaid and not simultaneously. nsaid action is more effective in the presence of higher plasma levels of adh (4) . in group c, none of the patients remain not responding to treatment after 20 min, which suggests that an nsaid with desmopressin may potentate each drug's analgesic effect, with no significant increase in adverse reactions. the mechanisms of action of nsaids and desmopressin were mentioned previousely (8-12) . the antidiuretic effect of desmopressin is more intense than that induced by pg inhibition, but it is not caused by a decrease in renal blood flow. the antidiuretic action of nsaids may in effect be nephrotoxic, by decreasing renal blood flow and the gfr (through an increase in preglomerular resistance) in an already obstructed, dysfunctional kidney. this functional compromise is not clinically detectable, as pg inhibitors act selectively on the obstructed kidney, leaving the contralateral organ unscathed and allowing serum creatinine levels to remain within normal limits (13) .the ease of administration of desmopressin, its low cost, good tolerability and lack of clinically relevant side effects make it safe. studies using desmopressin therapy for up to 3 years have shown no toxic reactions or significant changes in laboratory values. thus the results of the present study suggest that desmopressin intranasal spray may be a useful addition to the therapy for renal colic, either alone or combined with nsaids. it is a safe drug which is easy to administer and apparently effective in treating renal colic. other issues which need to be explored include the optimum dosage, method of use (i.e. in an ambulatory setting), whether there is a reduction in the need for diagnostic or pain score response time (min) desmopressin 40µg diclofenac 75 mg combination 0 7.5±1.2 a 7.7±2.0 a 7.65±1.5 a 10 5.7±0.9 b 5.8±1.1 b 5.8±1.0 b 20 5.3±0.8 b 3.8±0.6 c 3.7±0.7 c 30 6.1±0.9 b 3.0±0.6 c 2.5±0.3 d iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 0 therapeutic interventions and whether it reduces the rate of hospital admissions. that there seem to be some patients who do not respond or respond only minimally to desmopressin needs further clarification; characteristics should be identified in this group which might explain their lack of response. in conclusion, these results indicate that desmopressin may be used to treat renal colic either alone or combined, increasing the analgesic effect of other drugs like diclofenac references 1. el-sherif ae, salem m, yahia h, al-sharkawy wa, al-sayrafi m. treatment of renal colic by desmopressin intranasal spray and diclofenac sodium. j urol 1995; 153: 1395-1398. 2. constantinides c, kapralos v, manousakas t, mitropoulos d, alamanis c, dimopoulos c. management of renal colic with intranasal desmopressin spray. acta urol belg 1998; 66: 1-3. 3. marumo f, edelman is. effect of ca++ and prostaglandin e1 on vasopressin activation of renal adenyl cyclase. j clin invest 1971; 50: 1613-1620. 4. grenabo l, aurell m, delin k, holmlund d, sjodin jg. antidiuretic hormone levels and the effect of indomethacin on ureteral colic. j urol 1983; 129: 941-943. 5. robinson ag. ddavp in the treatment of central diabetes insipidus. new engl j med 1976; 294: 507-511. 6. miller k, goldberg s, atkin b. nocturnal enuresis: experience with long-term use of intranasally administered desmopressin. j pediat 1989; 114: 723-726. 7. kimoto y, constantinou ce. effects of (1-desamino-8-arginine) vasopressin and papaverine on rabbit renal pelvis. eur j pharmacol 1990; 175: 359-362. 8. patchev vk, rackeâ k, almedida of. adrenalectomy and experimental hypercorticalism modulate the basal, corticotropinreleasing-hormone and arginine-vasopressinstimulated release of hypothalamic beta-endorphin. neuroendocrinology 1991; 54: 111-117. 9. kjaer a. vasopressin as a neuroendocrine regulator of anterior pituitary hormone secretion. acta endocr 1993; 129: 489496. 10. seif sm, zenser tv, clarochi ff, davis bb, robinson ag. ddavp (1-desamino8-d-arginine-vasopressine) treatment of central diabetes insipidus ± mechanism of prolonged antidiuresis. j clin endocr metab 1978; 46: 381-388. 11. kapcala lp, weng cf, juang hh. protein kinase c activators stimulate b-endorphin secretion from hypothalamic cells. brain res bull 1992; 29: 553-557. 12. kjaer a, knigge u, bach fw, warberg j. permissive, mediating and potentiating effects of vasopressin in the acth and beta-endorphin response to histamine and restraint stress. neuroendocrinology 1993; 58: 588-596. 13. perlmutter a, miller l, trimble la, marion dn, vaughan ed , felsen d. toradol, an nsaid used for renal colic, decreases renal perfusion and ureteral pressure in a canine model of unilateral ureteral obstruction. j urol 1993; 149;926-930. rosiglitazone , metformin or both for treatment of polycystic ovary syndrome iraqi j pharm sci , vol.17 (2) , 2008 rosiglitazone , metformin in polycystic ovary syndrome 80 rosiglitazone , metformin or both for treatment of polycystic ovary syndrome mohammed a.taher*,1 , waleed r. sulaiman* , hillal y. al-khairi** * department of clinical laboratory sciences,college of pharmacy,university of baghdad,baghdad, iraq. ** ministry of health-alnauman hospital,baghdad, iraq. abstract this study was designed to show the advantages of using the combination of metformin and rosiglitazone over using each drug alone in treatment of women with polycystic ovary syndrome (pcos).forty four women with pcos were classified into 3 groups , group 1 received rosiglitazone (4mg/day) for 3 months , group ιι received metformin ( 1500 mg/day)for three months and groupιιι received the combination ( rosiglitazone 4mg/day + metformin 1500 mg/day) for the same period of treatment . the blood samples were drawn before treatment and after 3 months of treatment . the fasting serum glucose , insulin , progesterone , testosterone , leutinizing hormone were measured before and after treatment. the reduction of serum insulin , glucose ,homostasis model assessment of insuline resistance ( homa-ir) , lh and testosterone levels were greater in the group received the combination of rosiglitazone with metformin than that those taken each one alone. testosterone levels decreased significantly (p<0.05) from baseline level 1±0.04ng/ml to 0.073±0.32ng/ml after treatment with combination.the rate of ovulation is 29.4%,36.4% , 62.5% in rosiglitazone , metformin and combination of both, respectively.the combination of rosiglitazone with metformin has more beneficial effect on ovulation rate. key words: polycystic ovary syndrome, rosiglitazone, metformin, ovulation rate . ةصالخلا لك دلت سو ىددي در ت د ع رل سٌردلا لم ن دد ٌ عت د عفلم ٌلع ممص سٌردلا هذ م ل م د د جل ددململى مدٌ ع و ددن ت ٌرل ءه لل . تمب تمرع ده ملمو مدٌ ع و ددن ت ٌرل ءه لل جد دص مو فع ل ددله ةعرجب نوزاتيلكيزورلا تلوانت ىلوالا ةعومجملا : ت ةعرجب نوزاتيلكيزورلا تلوانت ىلوالا ةعومجملا : تو لا دد دعمو ددلل و ً ل دي 4 ةعرجب نوزاتيلكيزورلا تلوانت ىلوالا ةعومجملا : ت ٍ دعملى : دد دعمو ء دص ً ل دي د عفلم ٌلع م مو مو فع ل 4 مو فع ل ددله ةعرجب نوزاتيلكيزورلا تلوانت ىلوالا ةعومجملا : ت ةعرجب نوزاتيلكيزورلا تلوانت ىلوالا ةعومجملا : تو لا ل دد دعمو ددلددو ةل ً ل دي لم ن د عفلم ٌلع م مو 1500 دد ٌ عت م مو مو فع ل د له در د .لدلبد دلس سدني نذ در د مرل ةعرجب نوزاتيلكيزورلا تلوانت ىلوالا ةعومجملا : ت ةعرجب نوزاتيلكيزورلا تلوانت ىلوالا ةعومجملا : تو لا در د. دٌي م 1500 دد ٌ عت م مو جٌعفلى دلمعهعع , ءلجعد , دن ك جٌج , داعت ع دمعً م دٌجٌعسٌ هل مدعلل هد م لم ن د عفلم ٌلع دد ٌ عت دل م ةلدو لل هذ .هدل جٌعذ دٌجٌعسٌ فةذ ر عفل م جٌعذ دة ح مرل در د ملدد هل . م د عفلم ٌلع دد ٌ عت همٌل دل رل ممص دٌ ً ل لم ن 62,5 , 36,4, 29,4 هللي لجنو دةلمم و ممص لٌلد دن ت م د عفلم ٌلع دد ٌ عت دت ًل ةعرجب نوزاتيلكيزورلا تلوانت ىلوالا ةعومجملا : ت لل ب ممص لجنو لٌلد دن ت. introduction polycystic ovary syndrome (pcos) is the most common abnormality in women during reproductive age, it is a hetrogenous disorder of uncertain etiology (1). it is characterized by chronic anovulation and hyperandrogenism (2) affecting approximately 5-10 % of reproductive age women. the association between hyperinsulinemic insulin resistance and pcos is well recognizes and may play an important pathogenic role in development of pcos (3). obese and lean women with pcos manifest insulin resistance independent on body weight, although obesity is an additive factor which aggravates insulin resistance (4). there is some data to suggest that insulin enhances the effect of lh on preovulatory ovarian follicle arrest (5). it is possible that hyperinsulinemia due to insulin resistance drives the lh affect on ovarian theca cells to cause androgen excess which are intrinsically programmed to produce more androgens (6) . excess androgens are known to interfere with the process of follicular maturation , thus inhibiting ovulation and producing more arrested follicles. it has been postulated that in pcos ovaries there is an increased rsistance to all insulin functions , except for steroidogenic effect and the ultimate result is excess androgen production even with normal insulin level(7). metformin is abiguanide hypoglycemic agent that is approved for the management of type ιι diabetes (8). its main mechanism of action is the reduction of hepatic glucose production ( hnhibition of gluconeogenesis ). it also increases insulin mediated glucose utilization in peripheral tissue and decreases intestinal absorbtion of glucose(9). 1 corresponding author : e-mail : mohammed_taher43@yahoo.com received : 19/9/2007 accepted : 30/12/2008 iraqi j pharm sci , vol.17 (2) , 2008 rosiglitazone , metformin in polycystic ovary syndrome 81 several authors (10,11) have demonstrated the additional benefits of using metformin such as these related to menstrual cycle regulation and induction of ovulation, protection from pregnancy losses ,improvement of cardiovascular risk factors ,moreover metformin markedly increases both spontenous ovulation rate and clomiphene-induced ovulation rate for obese women with pcos (12) . many studies have shown improvements in ovulatory function , development of normal menses , and restoring of fertility (13). in spite all of these benefits , many workers reported that metformin effect may be to some extent transient and some cellular adaptation may occur during more prolonged therapy (14). rosiglitazone was approved in (1999) by food and drug administration (fda) as an oral antidiabetic agent for the management of type ιι dibetes as montherapy and in combination with oral hypoglycemic agents (15). rosiglitazone increases insulin sensitivity without stimulating insulin secretion, its mode of action is by binding and activation of the nuclear peroxisome proliferators activator gamma (ppar-γ) which is found in key target tissues for insulin action as adipose tissue, skeletal muscle and liver. activation of (ppar-γ) regulates the transcription of insulin –responsive gens involved in control of glucose and fatty acid metabolism (9,11) . therefore this study was designed to show whether combination of ( rosiglitazone and metformin ) has advantages over using each drug alone in the treatment of women with pcos. methods and materials this study was conducted at baghdad city in al-elwia maternity teaching hospital from october-2004 till june-2005.the study groups included 60 raqi women, (44) case with pcos aged 17-40 years with a mean of age 27.3±5.07 years , and 16 normal control subjects aged 18-38 years with a mean of age 27.1±6 years. the patients included in this study were diagnosed with pcos were nondiabetic, non-hypertensive and nonpregnant.the patients were under gynecologist supervision during period of treatment. the diagnosis of pcos was made by gynecologist depending on ultrasound examination, clinical features and laboratory tests (hormonal assay). the patients involved in this study were on normal diet.they were divided randomly into 3 groups: 1. groiup 1 included 17 patients (bmi 28.8±3.9 kg/m2), age 29.7±6.4 years. the patients received rosiglitazone 4mg daily in two divided doses (2mg) in the morning and (2mg) in the evening after meals for 3 months. 2. -group ιι included (11) patients (bmi 29.1±5.2kg/m2), age 24.9± years). the patients received metformin 1500mg daily in three divided doses (500mg after each meal ) for three months. 3. -group ιιι included (16) patients (bmi 34.2±6 kg/m2) , age 26.5±4 years. the patients received a combination of the two drugs ( rosiglitazone 4mg/day + metformin 1500mg/day ) for three months. 4. -control group included (16) normal women (bmi 30±4.8 kg/m2), age 27.1±6 years. sample collection: eight millilitrs (8ml) of venous blood samples used in this study were drawn from pcos patients.the first sample was collected before treatment as a baseline level, and after three months of treatment with insulin sensitizing agents.fasting blood samples were used for the measurement of glucose , insulin, hormones (lh, testosterone and progesterone). blood samples were left at room temperature for 30 minutes for clotting, centrifugated and then serum was separated and collected in small aliquots(0.5ml) and stored at (-20 c) until biochemical and hormonal analysis was performed. the serum was used for measurement of fasting blood sugar,insulin ,testosterone, lh and progesterone levels. biochemical and hormonal assay: fasting insulin levels were determined using a commercial kit obtained from randox, using radioimmunoassay (ria) method (17,18). serum testosterone levels were determined using a commercial kit obtained from immunotech, based also on (ria) (19). by using of a kit from immunotech, the radioimmunoassay of progesterone is compitiotion assay (20). serum lh determined using kit from immunotech, by the immunoradiometric assay (irma) which is sandwich type assay (21).fasting serum glucose was measured by a commercial kit obtained from biomghreb, using the enzymatic method(22). diagnosis of infertility depends on that inability of any couple to conceive a child within a 12 months period of unprotected coitus ( sexual intercourse) (23). body mass index (bm i) was calculated using the standard formula : bmi=weight (kg)/hight (m2). obses patients were defined as having bmh> 27 kg/m2(24,25). iraqi j pharm sci , vol.17 (2) , 2008 rosiglitazone , metformin in polycystic ovary syndrome 82 homeostasis model assessment of insulin rsistance (homa_ir) was calculated using the following formula: homa-ir= basal glucose (mmol/l).basal insulin(μiu/ml) 22.5 insulin resistance patients were defined as having homa>2.7 (26). the drugs used in this study were: rosiglitazone 4mg tablets purchased from (sunpharma)indiaand metformin 500mg tablets purchased from (mb and c) -syria statistical analysis 1-the results were expressed as man±sd mean. 2student t-test was used to examine the difference in the mean of parameters tested. 3p-value of less than (0.05) was considered significant. results the most patients in this study were with oligomenorrhea (68.1%) and (22.7%) of the patients were infertile. the hirsutism was obvious symptom in (63.6%) of the patients ( table 1). the combination of metformin and rosiglitazone reduced the levels of serum insulin, glucose , homa-ir, lh and testosterone which are more than that produced by rosiglitazone or metformion alone (tables 2 , 3 and 4 )(p<0.05).the testosterone was significantly decreased (p<0.05) only after treatment with combination compatred to the baseline levels. the ovulation rates were 29.4%, 36.4%, 62.5% in rosiglitazone, metformin and combination of both , respectively (table 5). table(1): demographic data of 44 patients with pcos. character number of patients (%) obese 31(70.4%) lean 13(29.5%) amenorrhea 10(22.7%) oligomenorrhea 30(68.1%) regular cycle 4(9%) infertility 10(22.7%) hirsutism 28(63.6%) table (2): effect of treatment of rosiglitazone(4mg/day) on levels of insulin, glucose, homair,lh and testosterone in group1. variables control levels (n=16) baseline levels (before treatment) (n=17) after treatment (n=17)(%) insulin (μu/ml) 8.4±1.7 15.6±5.5a 10.4±3.6(33.5) glucose (mg/100ml) 82±4.9 89.8±7 84±5.8(6.4) homa-ir 1.7±0.43 3.4±1.4a 2.1±0.9(38.2) lh (mμ/ml) 4.5±0.15 11.6±3.4a 10.6±3.1a(8.6) testosterone(ng/ml) 0.34±0.01 0.94±0.44a 0.71±0.43a(24.4) values are means±sd n= no. of women. %= percentage of change compared with base line levels. a p<0.05 for comparison with control group. homa= homeostasis model assessment of insulin resistance. lh= leutinizing hormone. iraqi j pharm sci , vol.17 (2) , 2008 rosiglitazone , metformin in polycystic ovary syndrome 83 table (3) : effect of treatment with metformin(1500mg/day) on levels of insulin , glucose , homa-ir,lh and tstosterone in group ιι. variables control levels (n=16) baseline levels (before treatment ) (n=11) after treatment (n=11)(%) insulin (μu/ml) 8.4±1.7 14.5±4.3a 10.3±2.8(28.9) glucose (mg/100ml) 82±4.9 84.2±4.2 82±5.1(2.6) homa-ir 1.7±0.43 2.9±0.9a 2±0.5b(13) lh (mμ/ml) 4.50.15± 12.4±4.7a 11.5±3.9a(7.2) testosterone(ng/ml) 0.34±0.01 0.91±0.51a 0.71±0.37a(21.9) values are means ±sd. n=no. of women. %=percentage of change compared with baseline level. a p<0.05 for comparison with control group. table (4): effect of treatment with the combination of metformin(1500 mg /d) and rosiglitazone (4mg/d) on the levels of insulin , glucose , homa-ir,lh and testosterone in group ιι. variables control levels (n=16) baseline levels (before treatment) (n=16) after treatment (n=16)(%) insulin (μu/ml) 8.4±1.7 21.2±8a 12.5±4.8(41) glucose (mg/100ml) 82±4.9 90.3±8.3a 84.1±6.5(6.8) homa-ir 1.7±0.43 5±1.5a 2.6±0.8(48) lh (mμ/ml) 4.50.15± 11.9±2.8a 10.2±2.3a(14.2) testosteroe (ng/ml) 0.34±0.01 1±0.46a 0.73±0.32a,b(27) values are means ±sd. n= no. of women . %=percentage of change compared with baseline level. a p<0.05 for comparison with control group. b p<0.05 for comparison with base line alone . table (5): ovulation rate in pcos patients for treatment with insulin sensitizing agents. groups secondary outcome group 1 (n=17) group ιι (n=11) group ιιι (n=16) total ovulation 5(29.4) 4(36.4%) 10(62.5)%111 19 no ovulation 12(70.6%) 7(63.6%) 6(37.5%) 25 total 17 11 16 44 n=no. of women group 1: women treated with rosiglitazone 4mg/d alone. grouop ιι: women treated with metformin 1500mg/d alone. iraqi j pharm sci , vol.17 (2) , 2008 rosiglitazone , metformin in polycystic ovary syndrome 84 group ιιι : women treated with combination of roziglitazone 4mg/d and metformin 1500mg/d. iraqi j pharm sci , vol.17 (2) , 2008 rosiglitazone , metformin in polycystic ovary syndrome 85 discussion in this study , the administration of insulin sensitizing agents rosiglitazone and metformin alone or in combination for three months showed non-significant reduction (p>0.05) in serum glucose levels , serum insulin levels nor homa _ir index. the efficacy and percentage of improvement was seen to be more obvious in combination group than with either drug alone (table 2,3 and 4) . roziglitazone showed more improvement than metformin . however, in present study , rosiglitazone and metformin treatment improved insulin resistance because there was an improvement in fasting insulin and fasting glucose levels, similar results were reported by other studies (27,28). all groups of patients who received rosiglitazone and metformin alone or in combination showed a slight ( non-significant) decline in lh levels when compared with baseline levels .lack of change in lh levels also reported by many researchers(28,29). the effect of rosiglitazone and metformin combination for three months was associated with significant decline in testosterone levels (p<0.05) ( table 4). the study also shows a greater decrease in insulin and homa-ir index leading to more decrease in testosterone level. these results are in agreement with studies showed a reduction in serum androgen levels after the reduction of insulin levels by insulin sensitizing agents, and these effect were independent in body weight (28,30,29). in general, the favorable effect of insulin sensitizing agents on hyperandrogenemia in pcos may be due to reduced pituitary secretion of lh, reduced ovarian androgen secretion, and increased levels of sex hormone binding globulin (shbg)(31).the adminstrastion of rosiglitazone or metformin alone or both of them for three months demonstrated an improvement in ovulation rate assessed by measurement of mid-luteal phase progesterone level in group ιιι more than group ιι and ι ( table 5) . this may be due to the synergestic effect of two drugs which lead to decrease the testosterone significantly (p<0.05). the percentage of ovulation was (62.5%0, (36.4%) and (29.4%) in groups ιιι , ιι , ι respectively. several studies investigated effect of metformin on menstrual cyclicity, and a significant improvement in the frequency of menstrual cycles has been reported , with an incrase in the percentage of ovulatory cycles as assessed by mid-luteal phase progesterone (32,27). k.j meenanakumaari et al (2004) found a significant negative correlation between insulin and progesterone , and between progesterone and lh concentration in pcos women treated with metformin and suggested that insulin resistance / hyperinsulinemia may be responsible for low progesterone levels during the luteal phase in pcos. the luteal progesterone may be enhanced in pcos by decreasing insulin levels with metformin (33). richardo azziz et al (2001) studied the effect of rosiglitazone on menstrual cyclicity and ovulation in pcos women. azziz reported an increase in the mean rate of ovulation in doserelated fasion and he expected an improvement in the menstrual cycle after the improvement in ovulation (34). nicholas a. cataldo et al (2006) showed a favorable effect of rosiglitazone on menstrual pattern or ovulation independent of rosiglitazone dose, furthermore they have found that ovulation occur in association with only modest change in insulin rsistance and insulinemia and claimed either that a small metabolic improvement is sufficient to promote preovulatory follicular maturation or that rosiglitazone exerts its effect indepently of insulin (35). similar results were reported by didem dereli et al ( 2005)(36). in conclusion it is preferable to use a combination of rosiglitazone and metformin in infertile pcos women as it has more potent effect in the improvement of ovulatiobn rate. the combination is also more beneficial to alleviate the hyperandrogenemioa in women with pcos. acknowledgment we would like to thank dr.mohammed g.chabuk ( gynecologist) for his help in preparing and diagnosis of patients in al-elwia maternity teaching hospital. also we are grateful to assisstant prof.dr. abdulwahab al-shekli in college of pharmacy ( universty of baghdad ) for his expertise in laboratory work. refrences 1. adam balen,kathy michelmore.what is polycystic ovary syndrome? are national views important. hum reprod. 2002;17(9):2219-2227. 2. acien p, quereda f, matalin p, et al. insulin , androgen and obesity in women with and without polycystic ovary syndrome : a heterogenous group of disorders . fertil steril.1999;7:32-40. 3. andrea dunaif.insulin resistance and the polycystic ovary syndrome: mechanism and implications for pathogenesis . endocrine reviews. 1997;18(16):774-800. 4. dunaif a, segal kr , futterweit w. et al . 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polycystic ovary syndrome. new england journal of medicine, 1996;335:617-623. 31. vincenzo de leo . antonio la marac, felice ppetrag;ia . insulin lowering agents in the management of polycystic ovary syndrome. iraqi j pharm sci , vol.17 (2) , 2008 rosiglitazone , metformin in polycystic ovary syndrome 87 endocrinol reviews ,2003 ; 24(5) :633-667. 32. velazquez e, acosta a, mendoza sg. menstrual cyclicity after metformin therapy in polycystic ovary syndrome. obstet gynecol. 1997;90:392-395. 33. meenanakumaari kj, agarwal s, krishna a, et al. effect of metformin treatment on luteal phase progesterone concentration in polycystic ovary syndrome. brazelian j medical and /biological research. 2004:1637-1644. 34. aziz r, ehrmann d, lergo rs et al . troglitazone improves ovulation and hirsutism in polycystic ovary syndrome multicenter, double blind, placebocontrolled trial. j clin endocrinol metab 2002;86:1626-1632. 35. nicolas a. cataldo , fahim abbasi , tracy l. metabolic and ovarian effects of rosiglitazone treatment for 12 weeks in insulin resistance women with polycystic ovary syndrome . hum reprod. 2006;21(1) : 109-120. 36. dereli d, dereli t, bayraktar f, et al. endocrinol and metabolic effects of rosiglitazone in non-obese women with polycystic ovary syndrome . endocrinol journal. 2005;52(3):299-308. iraqi j pharm sci, vol.28(2) 2019 prevalence of ugt1a1*93 and abcc5 doi: https://doi.org/10.31351/vol28iss2pp24-29 24 prevalence of ugt1a1*93 and abcc5 polymorphisms in cancer patients receiving irinotecan-based chemotherapy at al-najaf al-ashraf ahmed f. al-talkani*, 1 and sarmed h. kathem ** * department of clinical pharmacy, college of pharmacy, al-ameed university, karbala, iraq. ** department of pharmacology, college of pharmacy, university of baghdad, baghdad, iraq. abstract irinotecan (cpt-11) is a semisynthetic derivative of the antineoplastic agent camptothecin used in a wide range as an anti-cancer agent in many solid tumors because of its cytotoxic effect through the interaction with the topoisomerase i enzyme. the major limiting factors for irinotecan treatment are its association with potentially life-threatening toxicities including neutropenia and acute or delayed-type diarrhea, results from distinct interindividual and interethnic variability due to gene polymorphism. this is a cross sectional pharmacogentics study was conducted on 25 cancer patients to estimate the prevalence of ugt1a1*93 and abcc5 allele single nucleotide polymorphism (snp) in iraqi cancer patients treated with irinotecan-based therapy at middle euphrates cancer center. four drops of venous blood was drawn for each patient and was applied onto the fta classic card to perform a genotyping assay for the 2 snps. after dna isolation and purification, real time pcr was performed to detect the snps of each gene. results of this study showed the prevalence of one allele variant (heterozygous mutation) of ugt1a1*93 was 64% compared to 36% of patients were wild type to this snp. no patient (0%) could be detected with homozygous polymorphism of the ugt1a1*93. for the abcc5 polymorphism, results revealed that 32% of patients have one polymorphic allele (heterozygous), while 28% of them have two polymorphic alleles (homozygous mutation). wild type abcc5 gene constitutes 40% of patients. as a conclusion, high prevalence of ugt1a1*93 and abcc5 polymorphic alleles were detected in patients at middle euphrates cancer center which may explain the high toxicity features associated with irinotecan therapy. keywords: irinotecan, ugt1a1*93, abcc5, polymorphism. لدى مرضى السرطان abcc5و ugt1a1*93 تقيم نسبة انتشار الطفرة الوراثية في الموروثتين الذين يتعاطون عالج االيرينوتكان في النجف االشرف ** سرمد هاشم الخطيب و 1،*احمد فالح الطالقاني .العراق ،كربالء ،العميدجامعة ،كلية الصيدلةفرع الصيدلة السريرية، * .العراق ،بغداد ،جامعة بغداد ،كلية الصيدلة والسموم، فرع االدوية** الخالصة عالج االيرينوتيكان هو مشتق م الكامتقوييشتي الشتتة عتناعا و الست يسقفدم فا عالج العديد م االواام الصلتة و للد لهداتى علع من . م اعظم االستتتتتام المعراللة الستتتتقفدامى هو اتتتتنوا اعراح جانتية مصتتتتا تة لى مدال نهصتتتتا عدد العدال الدموية 1ايزوميريز -انزيم ال توبو نية بي يباالاتافة الع االستتنال بنوعية ل ال اد و الم(جالل القا يقعقهد ان اتنواوها المقتاي لدم المراتتع يعقمد علع ا قالفا فا المواويا ال و dnaو م يم اسقفدامنا السقفالص ال ftaالطرا دم م االوادة الطرفية و واعنا فا بطاالا ال 4س ب المراتع او بي الم قمعا . الكقشاف وجود الطفرا فا المواويا ال ينية. pcrدها بدء الدواا ال رااية فا ال بع الطفرة الواايية المقغايرة ل الضتتتتتتاهرال فا االليال الوا دل ها الستتتتتتتائدة مهاانقة بال الطفرة الواايية المقمايلة ل الضتتتتتتتاهرال فا ايلا اظنر النقائج م المراتتتع ي ملون %32م المراتتتع ي ملون افرا واايية مقغايرة و %23مهابال ان . بالugt1a1*93المواويةل فيما يفص المواوية و ugt1a1*93ان النستة العالية التشاا الطفرا المواايية فا المواويقي االستقنقاج.abcc5افرا واايية مقمايلة فيما يفص ال مواوية abcc5 لدم المراع الد تفسر النستة العالية لضنوا االعراح ال انتية المصا تة لعالج االيرينوتيكان. . ugt1a1*9 ،abcc5 اختالف في الموروثات الجينية، الكلمات المفتاحية : introduction toxicity and efficacy as a result of the administered drugs is the field of interest for any health care providers (hcp) during the patients follow up. there is an evidence suggesting that the effectiveness and toxicity of any drug may be affected, at least in part, by a genetic polymorphism 1corresponding author: e-mail: skathem@copharm.uobaghdad.edu.iq received: 20/1 /2019 accepted: 2/3 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp24-29 iraqi j pharm sci, vol.28(2) 2019 prevalence of ugt1a1*93 and abcc5 25 (changes in the genetic code that found in more than 1% of the human population) responsible for drug metabolism or drug targets(1). pharmacogenetics is the filed that interest in the study of how genetic variation affects individuals’ response to drugs(1,2). this inter-individual variation can range from inadequate therapeutic efficacy to serious, potentially life-threatening adverse drug reactions.(2,3) the clinical use of this field aims to reduce the potential for adverse drug reactions and maximize drug response by matching the best available drug or dose to an individual’s genomic profile(4–6). irinotecan (cpt-11) is a semisynthetic derivative of the antineoplastic agent camptothecin. is water-soluble and derives its name from the camptotheca tree where it was first isolated. (7) irinotecan is a pro-drug, and it activated through a hydrolysis reaction catalyzed by two isoforms of carboxylesterases enzyme (ces1 and 2) in vivo to active metabolite 7-ethyl-10-hydroxycamptothecin (sn-38) through cleavage of the ester-bond at c10(8,9). its active metabolite sn-38 is 100–1000 times more potent than its parent drug. sn-38 is actively transported into the hepatic cells by the organic anion transporting polypeptide (oatp) 1b1 transporter (i.e., slco1b1). then it is further conjugated and detoxified in the hepatic cells by udp-glucuronosyltransferase (ugt) 1a1 and 1a9 enzyme and extrahepatic by ugt1a7 enzyme to yield its h-glucuronide, sn-38 g.(10) sn-38 and sn38g are substrates for protein pumps, responsible for a unidirectional compound efflux from hepatocytes into bile and urine [atp binding cassette (abc) transporters] to be eliminated. (11) irinotecan used in a wide range as an anticancer agent in many solid tumors because of its cytotoxic effect through the interaction with the topoisomerase i enzyme.(12) it is approved worldwide for first-line therapy in combination with 5-fluorouracil and leucovorin for patients with metastatic colorectal cancer and as a single agent for second-line therapy of fluorouracil refractory metastatic colorectal cancer(13). in addition, irinotecan-based therapy is effective in the lung, pancreatic, esophageal and ovarian cancers. the major limiting factors for irinotecan treatment are its association with potentially life-threatening toxicities include neutropenia and acute or delayedtype diarrhea, with distinct interindividual and interethnic variability as a result of the unique pharmacogenetic profile of each patient and/or ethnicity.(14) screen patients for dna variations prior to selecting irinotecan therapy or dose can guide the decision-making process in terms of individually tailored irinotecan dose adjustments with subsequent toxicity risk reduction while maintaining therapeutic benefits.(15). in june 2005, the fda changed the irinotecan package insert for ugt1a1*28 pharmacogenetics testing in patients, recommending reduced irinotecan doses in homozygous ugt1a1*28 carriers, without specifying the extent of reduction(16). this irinotecan label revision based on genetic studies have been established that patients who are ugt1a1*28/*28 allele carrier are at the highest risk of developing severe toxicity of irinotecan(14). recently, and after identifying the irinotecan pharmacology in many studies, a series of genes has investigated for their possible contribution to the variability in irinotecan disposition and adverse effects.(17,18) other polymorphisms in metabolizing enzymes (ugt1a1, ugt1a7, ugt1a9, and ces) and transporters [atp-binding cassette (abc) and slco1b1] genes extensively studied in relation to pharmacodynamics and pharmacokinetics of irinotecan(11,19–21). ugt1a1*93 (rs569189,3156c>t) and abcc5 (rs562, 1243t>c) show a significant relation with irinotecan-induced neutropenia and diarrhea respectively in many recent studies(22-24). in this study, we will determine the prevalence of these polymorphisms in middle euphrates cancer center patient who received irinotecan as a preliminary study to demonstrate their relation with irinotecan toxicity in future work. patients and methods patient selection and study design twenty-five cancer patients at middle euphrates cancer center were selected to participate in the current cross-sectional study and were all on irinotecan monotherapy or irinotecanbased regimen therapy. four drops of venous blood were drawn for each patient and applied onto fta classic card to perform a genotyping assay for the 2 genes. toxicity features were graded according to the national cancer institute (md, usa) – common terminology criteria for adverse events (ctcae) version 5.0(25). ethical approval was obtained from the department of clinical pharmacy at the college of the pharmacy and from the center management. singed informed consent from all the patients were also obtained. a total of 25 patients were included in the study. the age distribution of the patients was 16% with an age of (20-39 years), 68% with an age of (40 -59 years) and the remaining 16% with an age of ≥60 years. males included in the study were (72%) and females (28%). the higher percentage of the population in this study were from al-najaf governorates (44%), while the rest distributed from the neighboring governorates. the majority of the patient were arab (96%). the demographic data and the patient characteristics are illustrated in table 1. iraqi j pharm sci, vol.28(2) 2019 prevalence of ugt1a1*93 and abcc5 26 table 1. demographic data and patient characteristics variable category number percent age group 20-39 y 4 16.0% 40-59 y 17 68.0% ≥ 60 y 4 16.0% sex male 18 72.0% female 7 28.0% province babil 3 12.0% baghdad 1 4.0% diwania 3 12.0% muthanna 3 12.0% najaf 11 44.0% ninava 1 4.0% thiqar 1 4.0% wasit 2 8.0% ethnicity arab 24 96.0% kurd/ turkmen 1 4.0% genotyping genomic dna was isolated from venous blood according to the protocol of reliaprep™ blood gdna miniprep system (promega, usa). from national center of biotechnology information (ncbi) database rs562 and rs569189 information was obtained, and specific primers designed in order to make specific assay for allelic detection using amplification refractory mutation system–polymerase chain (arm system –pcr). primer sequence listed in table 2. real time pcr was performed in a 10µl total reaction mixture containing 1 µl (10-30 ng) of dna template, 0.5µl (10 µm) of each primer, 5µl of gotaq qpcr master mix (promega, usa). after several trails for optimization, pcr conditions consisted of an initial melting step of 10 minutes at 95°c (initial denaturation); followed by 40 cycles of 10 sec. at 95°c (denaturation), 10 sec. at 60°c (annealing) and 72 c for 20 sec. (extension). melting curves were constructed by increasing the temperature from 70°c to 95°c (0.3 c/sec). table 2. primers sequences and annealing temperature gene polymorphism primer name sequences annealing temperature abcc5 rs562-inner-f 5`-cacgacatgcaacgctgaccattccat-3` 60oc rs562-inner-r 5`-aggtgggcgtggtcactgctgtcataag-3` rs562-outer-f 5`-cccttgcaaccaaccagctttgctacca-3` rs562-outer-r 5`-ccgcagtcgtcgcacagtctctctctct-3` ugt1a1*93 rs569189-inner-f 5`-gacatttctggacacaccctgggcaat-3` rs569189-inner-r 5`-ccagtactgggccttttcatccaaggaag-3` rs569189-outer-f 5`-ccgtcccataacccttgtctgcacagtt-3` rs569189-outer-r 5`-ccaccacagctggaaatgtgctgagtct3` statistical analysis statistical package for social sciences version 24 (spss v24) was used to analyze data. continuous variables presented as means with standard deviation and discrete variables presented as numbers and percentages. results clinical characteristics of patients thirty two percent of pateints have positive family history for the occurance of cancer distributed as 16% for their father and 16% for their siblings. non-pharmacological treatment for both radiotherapy plus surgery constitue (36%). iraqi j pharm sci, vol.28(2) 2019 prevalence of ugt1a1*93 and abcc5 27 irinotecan dose modification (dose reduction) was done for 12%. the most common toxicity associated with irinotecan included diarrhea (grade 3 and 4) (54.2%), vomiting (16.7%), severe neutropenia toxicity (grade 3 and 4) 20.8%, and toxic alopecia (25%). the clinical characteristics of the patients are shown in table 3. table 3. clinical characteristics of patients. variable category number percent positive family history of malignancy  father 4 16.0%  mother 0 0.0%  sibling 4 16.0% adjuvant non-pharmacological treatment  none 8 32.0%  surgery 8 32.0%  radiotherapy 0 0.0%  both 9 36.0% adose modified  yes 3 12.0%  no 22 88.0% bsever toxicity features  alopecia 6 25.0%  neutropenia 5 20.8%  diarrhea 13 54.2%  vomiting 4 16.7% a: irinotecan dose reduction (25% -35%) b: grade 3 and 4 toxicity prevalence of ugt1a1*93 and abcc5 single nucleotide polymorphism: genotyping assay of ugt1a1*93 snp revealed that patient have one allele polymorphism (heterozygous mutation) constitute 64% compared to 36% of patient were wild type for this snp. no patient has homozygous mutation could be detected in the studied population (table 4). concerning abcc5 genotyping, results showed that the one allele variant (heterozygous mutation) prevalence calculated as 32%, while the homozygous mutation 28%. patients carried wild type abcc5 gene comprised 40% (table 4). table 4. prevalence of studied mutation in patients gene allele number percent ugt1a1*93 t/t 9 36.0% t/c 16 64.0% c/c 0 0.0% abcc5 c/c 10 40.0% c/t 8 32.0% t/t 7 28.0% discussion up to our knowledge, this study is considered the first study that estimate the prevalence of the ugt1a1*93 and abcc5 polymorphisms in iraq. there are no other studies in the neighboring countries that could be compared to the results reported in this study; however we compared the findings of this study to studies conducted on american, european and african populations. it was found that the heterozygous variant of the ugt1a1*93 detected in (64%) of the study population, while there was no homozygous variant could be detected. innocenti et al. study which was conducted in the usa on african american patients in which no homozygous polymorphism was detected (21). other studies reported the prevalence of homozygous variant in american whites (13%), france (7%) and united kingdom (7%)(21,22,26). another study measured variant alleles frequency in european, asians, and africans reported a prevalence of 26%, 8% and 36% respectively (17). the reasons for the recorded variation between the present study and other studies are not clear, but could be attributed to ethnicity-related variations, and/or due to low sample size of the studied population(26,28). many patients participated in this study were complaining from several severe toxicity features including diarrhrea, vomiting, neutropenia, and alopecia (table 3). these adverse effected reported in the study could be attributed to the presence of polymorphism in genes sequences that are iraqi j pharm sci, vol.28(2) 2019 prevalence of ugt1a1*93 and abcc5 28 responsible for the metabolism of irinotecan (27). this finding was consistent with li et al. and crona et al studies, who postulated that a polymorphism of ugt1a1*93 was a strong predictor of irinotecan induced neutropenia, that is associated with higher sn-38 auc and lower absolute neutrophils3 counts (anc) nadirs. (11,29) the abcc5 transporter function was unclear until recently, in which it was found that abcc5 transporter plays a role in the transport of cyclic nucleotides and platinum-based and nucleosidebased analog used in anticancer treatment (e.g. irinotecan and its active metabolite sn-38).(18) among abcc5 gene was detect that (40%) of studied patients carried a wild type allele, the homozygous mutation represent (32%) and (28%) of them with a homozygous mutation. di martino et al. study which was conducted in italy metastatic colorectal cancer patients showed that the polymorphic allele (t) frequency was (51%) while the (c) allele detected in (48%) of the patient, with homozygous mutation (5/26) and (14/26) of the patient a heterozygous carrier(23). another study measured variant alleles frequency in asians breast cancer patient reported that (t) allele carried by (60%) of the patient and only (40%) of them was carried (c) allele. the genotyping assay through lal et al. was showed that (17%) of the patient with homozygous mutation and (43%) with heterozygous mutation.(30) this high polymorphic allele frequency incidence in iraqi patient could be attribute the high sever gi toxicity among them (table 3). this finding was consistent with chen et al., who assume that a polymorphism of abcc5 was a strong predictor of irinotecan induced diarrhea. (18) conclusion a high prevalence of ugt1a1*93 and abcc5 polymorphic alleles were detected in patients at middle euphrates cancer center. further studies should be conducted in multicenter all around iraq to evaluate the effects of such gene variants on irinotecan associated toxicity features and to maximize the treatment efficacy. acknowledgement special thanks to middle euphrates cancer centers employees. we are appreciative to dr. ali j. hekmt for his help in accomplishing the research. references 1. osanlou o, pirmohamed m, daly ak. pharmacogenetics of adverse drug reactions. advances in pharmacology. 2018; 1st ed. 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diseases. 2003;31(1):98–101. 29. crona dj, ramirez j, qiao w, et al. clinical validity of new genetic biomarkers of irinotecan neutropenia: an independent replication study. pharmacogenomics journal. 2016;16(1):54–9. 30. lal s, sutiman n, ooi ll, et al. pharmacogenetics of abcb5 , abcc5 and rlip76 and doxorubicin pharmacokinetics in asian breast cancer patients. pharmacogenomics journal 2016;17(4):337– 43. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ development of modified release nicotine tablet formulation for treatment of ulcerative colitis iraqi j pharm sci, vol.19(2) 2010 modified release nicotine tablet 75 development of modified release nicotine tablet formulation for treatment of ulcerative colitis marwan y. al-hurr *,1 * department of pharmaceutics, college of pharmacy, university of baghdad,baghdad,iraq. abstract one of the therapeutic effects of nicotine is used as a protective against developing ulcerative colitis . ulcerative colitis is an inflammatory disease of the bowel affecting the superficial lining mucosa in the rectum and large intestine. in this study nicotine tablets were formulated as modified release tablets targeted to the colon. all formulas were studied for drug release , effect of diluent, retardant concentration, avicel grade,and compression force, the selected formula was then further studied for drug release in 3 different ph ( coated tablets) .the kinetic study revealed acceptable shelf life . finally the selected formula was given to 6 patients in a pre-liminary clinical study which showed that nicotine can stabilize mild to moderate ulcerative colitis attacks. key words: ulcerative colitis, nicotine, modified release , colon delivery. الخالصة التهاب القىلىن التقزحي احذ التهاتاخ الجهاس الهضمي التي تصية الطثقح المخاطيح الظطحيح للمظتقيم و االمؼاء الغليظه هذا الثحث تم تحضيز مضغىطاخ الىيكىتيه محىرج التحزر مىجه الى في ويؼتثز الىيكىتيه ػامل حمايح ضذ تطىر هذا المزض. القىلىن. وتم دراطح تحزرالذواء و تأثيز وىع المىاد المضافح غيز الفؼالح و تزكيش المىاد المثثطه للتحزر للصيغ المحضزج, وتأثيز قىج ر الىيكىتيه في اوطاط مختلفح االص الهيذروجيىي و وتم أجزاء دراطح اوطغ للصيغه المختارج مه حيث تحز الكثض ػلى تحزر الىكىتيه. مزضى للصيغح المختارج حيث أظهزخ 6مه حيث ثثاتها في درجاخ حزارج مختلفح. وتم ايضا اجزاء دراطح طزيزيه اوليح ػلى .الذراطح ان الىيكىتيه يمىغ تطىر المزض و يشيذ مه اطتقزاريح هذا المزض في الزاحل الثظيطح و المؼتذلح الشذج introduction colonic drug delivery has gained increased importance not just for the delivery of the drugs for the treatment of local disease associated with the colon like crohns disease , ulcerative colitis and irritable bowel syndrome..etc., but also for the potential systemic delivery of proteins and therapeutic peptides. the large intestine, though difficult to reach by preoral delivery is still deemed to the delivery of agents to cure the local disease of the colon (1,2) . colonic delivery formulations are in general may be designed to provide either the burst release or for sustained/ prolonged release once reaching the colon (3) . the proper selection of a formulation approach depends upon several important factors (4) which are : the pathology and pattern of the disease , the physicochemical and biopharmatical properties of the drug and finally the desired release profile of the active ingredient. the most common physiological factors considered in the design of delayed release colonic formulations is ph gradient of the gastrointestinal tract (5,6) .delayed release formulations such as single unit or multiparticulate system for colon targeting , nanoparticulate system, microspheres, pelletsand beads, coating with ph sensitive polymers, embedding in matrices and bioadhesive systems (7) can be considered for the design of colon deliveryformulations. a wide array of polymers has been employed as drug release retarding agents each of which presents a different approach to matrix concept. plastic matrix system , due to their chemical inertness and drug embedding ability , have been widely used for sustaining the release of drugs. plastic polymers e.g. ethylcellulose and acrylates, which are capable of forming insoluble or skeleton matrices, have been widely used for controlled release of drugs due to their inertness and drug embedding ability . acrylate polymers are widely used as tabletcoating and as retardents for drug release in sustained release formulations (8) . ulcerative colitis (u.c) is an inflammatory disease of the bowel affecting the superficial lining mucosa in rectum and large intestine. the disease typically starts from the rectum and continues through the large bowel sparing the deeper layers of the intestinal wall (9) . a variety of antiinflammatory, immunosuppressive, and biological agents have been used to induce and / or maintain remission in uc . sulfasalazin, olsalazine, balsalazide, oral and rectal mesalamine and topical corticosteroids are the standard first line therapies for uc.patients who fail to respond to these agents are usually treated with oral corticosteroids. 1corresponding author email : m@marwanpharm.com , mngateway2000@yahoo.com received : 1/ 9/ 2010 accepted : 11/12/2010 mailto:m@marwanpharm.com mailto:mngateway2000@yahoo.com iraqi j pharm sci, vol.19(2) 2010 modified release nicotine tablet 76 there is a need for additional first line treatments in patients with uc and for alternatives to corticosteroid therapy in refractory patients. nicotine may be such an agent for which epidemiologic studies have shown that smoking protects against the development of uc and controlled clinical trials have demonstrated that transdermal nicotine is efficacious for active uc (10-15) . nicotine is a drug obtained from the plant nicotina tabacum. it’s a weak base. its available as a colorless to pale yellow oily liquid with an unpleasant tobacco-like odour and burning taste (16) . its most preferred that absorption of nicotine occurs predominantly in the colon. so post-gastric delayed release composition, the composition will pass through the small intestine in about 4-8hr and will resides in the colon for about 48hr. further more nicotine is absorbed more slowly in the colon than in the small intestine, therefore, nicotine delivered for absorption predominantly in the colon will be absorbed more slowly over a sustained period and will give rise to more uniform plasma concentration. by predominant absorption from the colon we mean to include preferably 80-90% of the total dose of nicotine (17) .the present study is to develop modified release of 20mg nicotine tablets as a targeted delivery system in the colon with pre-liminary clinical study. eudragit rs pm as a retardant polymer which is responsible for the sustained release behavior of the drug was used for preparation of the core matrix and the selected formula was targeted to release in the colon by using enteric coating ( a mixture of eudragit l and s ). materials and method nicotine (sigma) gifted from pharmacognosy department. college of pharmacy/ university of baghdad, sodium hydroxide (fluka chemie ag. buchs/scheiz), dibutylphthalate ( usb, b. brussels,belgium), hydrochloric acid , iospropanol, and orthophosphoric acid (riedal de haen ag seelze hanover), polyvinylpyrrolidone ( pvp k30 ) , acetone, potassium dihydrogen phosphate, ethanol 99% ( bdh chemicals, ltd, liverpool, england )microcrystalline celluloseavicel ® ph101, ph302, ph200 (fmc corperation, pennsylvania, usa), eudragit ® l100, s100, rs pm – rhöm pharma gmbh weiterstadt, germany),trisodium phosphate, talc (hopkins and williams ltd. england), coloring agent ( deep orange lakes) zinc stearate (barlocher, gmbh, germany), disodium hydrogen phosphate, mannitol, starch ( merk, germany). table (1) summarizes 8 formulas to prepare modified release nicotine tablets by wet granulation method with alcohol. a known weight of the granules were mixed with a speicified amount of zn stearate ( 1%) in a well closed container and compressed into tablets using 9mm punches ( tablet machine single punch – korch, type eko, erweka gmbh, kr offenbanch/ germany ). table 1 : different formulas of nicotine prepared as modified release tablets. constituents formulas ( mg) 1 2 3 4 5 6 7 8 nicotine 20 20 20 20 20 20 20 20 pvp (10%) 20 20 20 20 20 20 20 20 avicel ® ph302 40 40 40 40 40 40 eudragit ® rs pm 20 20 40 60 20 20 20 20 starch 100 100 100 100 100 100 100 zinc stearate 1% 1% 1% 1% 1% 1% 1% 1% mannitol 100 avicel ® ph101 40 avicel ® ph200 40 compression force 4kg 4kg 4kg 4kg 4kg 4kg 6kg 8kg total weight of final tablet 200 200 200 200 200 200 200 200 evaluation of the prepared tablets the following parameters were used to compare the prepared formulas to obtain the final selected formula. 1. effect of diluents type on the percent released of nicotine. formula 1 and 2 were used to study the effect of two different diluents ( starch andmannitol ) on drug release. 2. effect of eudragit rs pm concentration. formula 1,3 and 4 were utilized to study the effect of different concentrations (10% , 20% and 30% respectively) of eudragit rs pm on the drug release. iraqi j pharm sci, vol.19(2) 2010 modified release nicotine tablet 77 3. effect of avicel grade. formulas 1,5 and6 which contain avecil ph 302, ph101,and ph200 respectively were used to study the effect of different grades of avecil as channeling agent on the drug release. 4. effect of compression force on nicotine release. formulas 1, 7and 8 were used to study the effect of changing the compression force 4kg, 6kg and 8kg respectively on the drug release. drug release : ( usp dissolutuion apparatus type ii, coply scientific ltd, england) the medium used : ph 6.8 phosphate buffer 750ml, apparatus ii , rotation 75 rpm, with a sampling time: 1,2,3,4 and 6hr. the amount of nicotine dissolved was determined spectrophotometecally at λmax. 260nm of filtered samples.( uv visible spectrophotometer, carrywin uv. varian, australia). the samples were diluted with dissolution medium if necessary and compare with a standard solution having a known concentration of nicotine in the same medium. assay: hplc analytical method the chromatographic separation was achieved on a c-18 colum with uv detection at 260nm the hplc system comprised a (waters 1500 series hplc pump( usa) , waters 2487 dual λ absorbance detecter, water breeze soft ware.) was operated at ambient temperature and used citrate buffer: methanol (85: 15 % v/v ) with an appearent ph 2.4 as the mobile phase. the flow rate was maintained at 0.7 ml / min. and the retention time 6.94 min. (18) . preparation of coating formula the coating solution was prepared according to the rhom pharma recommendations (the manufacturer) as follows: formula : eudragit * 6gm isoprpanol 115.7gm acetone 77.1gm dibutylphthalate 1.2gm talc 3.25gm magnesium stearate 0.25gm color 0.25gm titanium oxide 1.55gm semithicone q.s. *mixture of eudragit l 100 and s 100 in a ratio 1:2 the final coating solution formula prepared was 205.3gm. procedure the formula was prepared by mixing the solvents together with the plasticizer ( dibutylphthalate) in a high shear mixer mlw type lr10 ( veb ml w prufgerate-werk, medingen/ stizfrtal/ germany). eudragit mixture was added slowly at room temperature , the powder was thoroughly wetted and care was taken to ensure that nothing settled at the bottom or formed lumps . mixing lasted for at least 30mins, until the solution was clear, the fillers were added step by step . calculation of the amount of lacquer required ( 19) a specific thickness of coating is required based on the purpose of the coating and the amount needed depending on the surface area of the cores which may be calculated from the following equation assuming that the tablets are cylindrical in shape: s.a= л ( d.h + ½ d 2 ) where d is the diameter (mm) h is the height (mm) s.a is the surface area (mm 2 ) the nicotine tablets had a diameter of 9mm and a surface area approximately equals to 240mm 2 . since 3-5mg lacquer / cm 2 of tablet cores required to produce a core resistant to acidic environment ( enteric coated tablets). so multiplying the surface area of the tablet core by the amount required and dividing it by the weight of tablet, the quantity of the lacquer to be applied as a percentage will be obtained. the amount to be applied ( % dry lacquer substace)= s.a( mm 2 ) / w ( mg) x ( mg/ mm 2 ) = 240 / 200 x 5 = 6% tablet coating the selected formula was coated by dipping method . each tablet was held by forcipes and dipped in the coating lacquer in and out 15-20 times , the coat was dried by a stream of warm air between each dip .( 20) dissolution study of the coated tablets ( 21) the dissolution rate of the selected formula for nicotine ( coated tablets) was determined using usp apparatus at 37+ 0.5 o c with paddle and the rotation speed was set at 75rpm in order to simulate the ph change of iraqi j pharm sci, vol.19(2) 2010 modified release nicotine tablet 78 the git , ph change dissolution procedure was applied as follows: 2hr. testing in 0.1n hcl solution followed by testing for one hour in phosphate buffer ph 4 obtained by adding 195ml 0.2m tribasic sodium phosphate solution during which samples were withdrawn at specified times and replaced immediately by fresh medium. then the medium was changed to ph 6.8 by adding 55ml 0.2 m tribasic sodium phosphate adjusted by 2n naoh or 2n hcl if required . samples were withdrawn at different time intervals and analyzed spectrophotometrically at 260nm. kinetic study effect of temperature: the effect of temperature on the degradation of the selected formula of nicotine modified release tablets was studied . the study was done by storing 90 tablets in ovens (mermert ul 80 ( rostfrei, schwach, germany) )at different temperatures 50 o c, 60 o c and 70 o c. samples were taken at specified time intervals and analyzed for nicotine. since the degradation of the drug follows 1 st order kinetics, the expiration date t10% at 25 o c could be calculated using the following equation : t10% = 0.105/ k 25 o c pre-liminary clinical study before giving the preparation we obtained a written consent of the patients who were included in this study. the modified release nicotine tablets of the selected formula was given to 6 patients suffering from mild to moderate ulcerative colitis (high mucus secretions, irritable bowl syndrome, mild to moderate bleeding and gases). all patients were put on 20mg single dose of nicotine for 2 weeks. the patients were evaluated clinically(physical examination and endoscopy ) before and during treatment ( physical examination ) under the supervion of dr. mumtaz k. hanna at his private clinic. results and discussion effect of diluent’s type on nicotine release although diluents are normally thought to be inert ingredients they can significantly affect the biopharmaceutical, chemical and physical properties of the final tablets .(22) formula 1and 2 which contain maize starch and mannitol as diluents, it was seen that starch gave the best drug release compared with mannitolas shown in fig. 1 .this behavior may be attributed to the swellability property of starch when compared with mannitol which the release is due to water solubility. (23) figure 1: effect of diluent type on the release of nicotine at ph 6.8 and 37 o c effect of eudragit concentration: eudragit rs pm can be in corperated in a percent of 10-30% (w/w) by weight to provide suitable granules and matrix tablets. the amout of eudragit rs pm to be added , depends upon the solubility characteristics of the drugs and the rate required (20). formulas 1 , 3 and 4 which contain 10% , 20% and 30 % w/w of eudragit ( as a retardant) respectively were evaluated . formula 1 gave the best modified release of nicotine when compared with the requirements of drug release to the colon (17). the results from dissolution profiles of formulas 1 ,3 and 4 indicates that the retardant content affects the release of nicotine from the tablet, this result is in a consistent with the results obtained when eudragit rspm polymer was used as a retardant material for diclofenac sodium and indomethacin tablets. (24) it appears that the amount of retardant needed is 10 % as shown in fig. 2 .this is in agreement with the reported data which indicated that the retardation effect on the release of drug is dependent on the amount of eudragit included (25) figure 2 : effect of eudragit rs pm concentration on the percent released of nicotine at ph 6.8 and 37 o c. 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 6 time ( hr.) % d r u g r e le a s e d starch mannitol 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 6 time ( hr.) % d r u g r e le a s e d 10% eudragit rspm 20% eudragit srpm 30% eudragit rspm iraqi j pharm sci, vol.19(2) 2010 modified release nicotine tablet 79 effect of avicel grade avicel is microcrystalline cellulose , it is partially depolymerized cellulose prepared by treating alphacellulose obtained as a pulp fibrous plant material with acids. the grade of avicel depends on its normal loss on drying , bulk density and degree of polymerization values. (26 ) the results showed that formula 1 in which avicel ph 302 present, gave the best dissolution profiles while the difference in the release that occurred in the other formulas 5 and 6 which contain avicel ph 101 and ph 200, respectively. is due to the difference in the porosity , surface area, particle size and density of avicel as stated (27 ) as shown in fig. 3 figure 3 : effect of avicel grade on the percent released of nicotine at ph6.8 and 37 o c. effect of compression force compression forces affect the hardness of a tablet and its thickness at a constant die fill as additional compression force is applied , the hardness values increase and the thickness decrease and the decrease as shown in fig.4 ( 19) , thereby the dissolution of the drug decrease in surface area and increase in hardness. this may be related to the low porosity resulted from the increase in compression force. (8) therefore, formula 1 was selected because it gave the best drug release profile which complies with absorption properties of nicotine from the colon and it was further investigated for enteric coating, kinetic study and preliminary clinical study (17). figure 4 : effect of compression force on percent released of nicotine at ph 6.8 and 37 0 c. coating formula the tablets showed good appearance with no signs of craking or splitting or peeling. induction of hydrophobic materials and inert fillers ( mg stearate , talc , titanium oxide , aluma lakes with an orange color ) these fillers facilitate processing of the lacquer by reducing its stickness, help to smooth the permeability to water and decrease the tackiness of the drying lacquer. in addition they reduce the permeability of the film as long as the mechanical strength is maintained thereby enhancing the enteric properties of the film ( 28,29 ). formula 1 was coated to target the drug to the colon. the coated tablets showed no drug release in 0.1 n hcl for 2hr. period of the test and the release of the drug increased rapidly when the ph changed to 6.8 as shown in fig 5. figure 5 : the cumulative percent of enteric coated nicotine tablets release at different ph-media at 37 o c. kinetic study effect of temperature the stability of the coated modified release tablets were studied at different exaggerated temperatures ( 50 o c , 60 o c and 70 o c ) for 3 months. fig 6 shows the change in the log percentage remaining of nicotine versus time at different temperatures. the obtained profiles were linear , indicating that nicotine degradation follows 1 st order kinetics. the slopes of these lines were determined and the calculated rate constants (k) are summarized in fig (6). arrhenius plot was then constructed as shown in fig 7. the linearty of the curve indicates its utility in predicting the rate of degradation at lower temperatures. since the degradation of the drug followed 1 st order kinetics the expiration date can be calculated at 25 o c for nicotine coated matrix tablets and it was 52 month. 0 20 40 60 80 100 0 1 2 3 4 6 time ( hr.) % d r u g r e l e a s e d avicel ph302 avicel ph 101 avicel ph 200 0 20 40 60 80 100 1 2 3 4 5 6 7 8 9 10 time (hr) % d ru g r e le a s e d the release profile of enteric coated tablets in ph solutions ( ph 1.2, 4, 6.8) ph1.2 ph 4 ph 6.8 iraqi j pharm sci, vol.19(2) 2010 modified release nicotine tablet 80 figure 6 : percent remaining of drug versus time. fig7: arrhinous plot for expiration date estimation of nicotine tablet (formula 1). clinical study nicotine was given to 6 non smoking patients ( 5 males and 1 female ) with an age range of 2765 yr. the patients took 20mg once a day for 10 days suffering from mild to moderate ulcerative colitis. the out come of this preliminary study indicates that 67% ( 4 out of 6 patient ) were responsive to nicotine therapy ( relief of bleeding and most sign and symptoms) although all patients suffered from adverse effect reaction towards nicotine therapy because the patient were non smokers (lightheadedness or dizziness, nausea, headaches, central nervous system stimulation and tachycardia). (30) further studies in the future should be done including in vivo nicotine blood concentration to optimize the dose. references 1. davis ss. overcoming barriers to the oral administrationof peptide drugs. trends pharm sci 1990;11:353-5. 2. vandenmooter g, kinget r. oral colonspecific drug delivery: a review. drug delivery 1995;2:81-93. 3. pinhasi a, gomberg m, avramoff a. us200467030442004. 4. brahma ns. modified – release solid formulations for colonic delivery. recent pat drug deliv formul 2007;1:53-63. 5. evans df, pye g, bramley r,clark ag, dyson tj, hardcastle jd. measurement of gastrointestinal ph profiles in normal ambulant human subjects. gut 1988; 29:1035-41. 6. nugent sg, kumar d, rampton ds, evans df. intestinal luminal ph in inflammatory bowel disease: possible determinants and implications for therapy with aminosalicylatesand other drugs. gut 2001;48:571-7. 7. das s, deshmukh r, jha ak. role of natural polymers in the development of multipartculate systems for colon drug 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nicotine in pharmaceutical formulation. j. chromatogr. a. 1996: 749: 81-85. 19. rohm pharma information sheets , rohm pharma gmbh, weiterstadt; info. eudragit l100-55 a/e , eudragit s100 4/e , 1/s-7e 2006. 20. lachman l., lieberman h.a., kanig j.a; the theory and practice of industrial pharmacy 3 rd ed. leo and febiger . chap. : 1986;12:372. 21. usp 2007 compact disk. 22. banker g., baley p., baley g.; tablet formulation and design, liberman and lachman (eds) pharmaceutical dosage form tablets, vol.1 , marcel deckker, new york 1982; 61-108. 23. laako r., eerkainen s.;effect of core components on indomethacin release from film-coated granules; int.j. pharm. :1991; 67(1):79-88. 24. al-hurr m.y. ; formulation andclinical study of diclofenac sodium and indomethacin as an oral modified release tablets, thesis for m.sc. degree, college of pharmacy, university of baghdad, 2003. 25. khanfar m.s., salem m.s. najib n.m., pillai g.k, dissolution behavior of sustained release formulation of indomethacin with eudragit rs acta. pharm. hung 1997; 67(6):235-239. 26. handbook of pharmaceutical excipients 6 th ed. washington dc., american pharmaceutical association 2009. 27. al-ebady m.n. ; design and in vitro evaluation of prednisolone tablets as a potential colon delivery system, thesis for m.sc. degree, college of pharmacy, university of baghdad, 2006. 28. chatfield h.w,; science of surface coating, van nostrand, newyork 1962. 29. kassab h.j., formulation and clinical study of sulphasalazine tablets, m.sc. thesis college of pharmacy, university of baghdad, 1999. 30. sandborn wj, tremaine wj, offerd kp, transdermal nicotine for mildly to moderately active ulcerative colitis . a randomized double – blind , placebo controlled trial . ann intern med :1997; 126: 364 -71. iraqi j pharm sci, vol.20(2) 2011 prevalence of microorganisms in h1n1 patients 81 the prevalence of microorganisms in h1n1 patients compared to seasonal influenza in a sample of iraqi patients # mohammed f. almarjani * ,1 , saba s. khazaal * , thana m. zayer * , yasir a. atahia ** and kadhim a. kadhim *** *department of biology , college of science , almustansiriya university,baghdad , iraq. ** college of al-kindy medicine , university of baghdad, baghdad , iraq. *** college of pharmacy / almustansiriya university, baghdad , iraq. abstract this study provides valuable information on secondary microbial infections in h1n1 patients compared to seasonal influenza in iraqi patients. nasopharynx swabs were collected from (12 ) patients infected with seasonal influenza (11 from baghdad and 1 patient from south of iraq) ,and ( 22 ) samples from patients with 2009 h1n1 ( 20 from baghdad and 2 from south of iraq). the results show that the patients infected with 2009 h1n1 virus were younger than healthy subjects and those infected with seasonal influenza. and the difference reached to the level of significance (p< 0.01) compared with healthy subjects.two cases infected with 2009 h1n1 virus (9.1%) were from south of the iraq and remaining 20 cases were from baghdad . polymicrobial isolates from nasopharynx swab were observed in patients infected with 2009 h1n1 virus. polybacterial infections (2-7 microorganisms) and fungal infection were reported in 21 out of 22 patients (95.5%) and 5 out of 22 (22.7 %) respectively.the predominant isolated microorganisms were streptococcus pyogenes , staphylococcus aureus and streptococcus pneumoniae were found in 95.2 % , 95.2 % and 90.5 % respectively .the results also show that seven microorganisms were isolated from 10 (47.6 %) patients infected with 2009 h1n1 , no microorganism was isolated from patients infected with seasonal influenza or healthy persons. key words: seasonal influenza , 2009 h1n1, nasopharynx swabs الخالصة ( h1n1فبٌشط بفبٌشط أوفيىوضا اىخىبصٌش ) هذفج اىذساست اىحبىٍت اىى معشفت اىمسبببث اىمبٌنشوبٍت اىثبوىٌت عىذ اىمصببٍه ر حم خمع ومبرج مه اىعشاقٍٍه ، أ( عىذ اىمشضى seasonal influenzaومقبسوخهب بخيل اىمعضوىت مه اىمصببٍه ببألوفيىوضا اىمىسمٍت ) مصبة بفبٌشط 11 ، و مه بغذاد و مصبة واحذ مه خىىة اىعشاق( 22مصبة ببألوفيىوضا اىمىسمٍت ) 21األوف واىحىدشة مه h1n1 (12 1مه بغذاد و )أظهشث اىىخبئح ان اىمصببٍه بفبٌشط .مه خىىة اىعشاقh1n1 هم مه اىفئبث اىعمشٌت األقو مقبسوت حصو اىى أمثش مه h1n1، وان هىبك أصبببث مبٌنشوبٍت مخعذدة عىذ اىمصببٍه بفبٌشط بٍه ببألوفيىوضا اىمىسمٍت واألصحبء ببىمصب و streptococcus pyogenes ان األوىاع اىبنخٍشٌت اىسبئذة هًوىع مبٌنشوبً واحذ ) بنخٍشي وفطشي( ، و staphylococcus aureus و % 2,51بىسبت عضه streptococcus pneumoniae مزىل أظهشث %,225بىسبت عضه. بٍىمب ىم حعضه مه اىمصببٍه ببألوفيىوضا اىمىسمٍت واألصحبء. h1n1أوىاع بنخٍشٌت مه عشش مشضى مصببٍه بفبٌشط 7اىىخبئح عضه introduction in late march and early april 2009, an outbreak of h1n1 influenza a virus infection was detected in mexico, with subsequent cases observed in many other countries including the united states (1) . on june , 2009, the world health organization raised its pandemic alert level to the highest level, phase 6, indicating widespread community transmission on at least two continents (2,3). between 1958 and 2005, 37 cases of swine influenza among civilians were reported . six cases (17 percent) resulted in death. forty-four percent of infected individuals had known exposure to pigs. cases were reported in the united states, former czechoslovakia, the netherlands, russia, switzerland, and hong kong (4) .influenza virus is present in respiratory secretions of infected persons. as a result, influenza virus can be transmitted through sneezing and coughing via large-particle droplets . transmission via contact with surfaces that have been contaminated with respiratory droplets or by aerosolized smallparticle droplets may also occur. in addition to respiratory secretions, certain other bodily fluids (eg, diarrheal stool) should also be considered potentially infectious (5) . # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : marjani20012001@yahoo.com received : 26/3/2011 accepted: 19/7/2011 iraqi j pharm sci, vol.20(2) 2011 prevalence of microorganisms in h1n1 patients 82 influenza predisposes individuals to developing bacterial community-acquired pneumonia .during each of the influenza pandemic of the 20 th century , secondary bacterial pneumonia was a frequent cause of illness and death and streptococcus pneumoniae was reported as the most common etiology .these findings also apply to seasonal influenza (6) .the aim of this work was to study the prevalence of microorganisms in h1n1 compared to seasonal influenza in nasopharyngeal swabs of iraqi patients. materials and methods 1. sampling: nasopharyngeal swabs were collected from (11 ) patients infected with seasonal influenza from baghdad and (1) patient from south of iraq ,and ( 20 ) samples from patients with 2009 h1n1 from baghdad and (2) from south of iraq during the period 1/1/2009 to 15/2/2010 . 2. bacterial isolates : samples were streaked onto blood , chocolate and mrs agar , under aerobic and anaerobic conditions , the plates were incubated at 37c for 24-72 h. the isolates were subjected to the microscopic and biochemical tests for the diagnosis as mentioned in ( 7 ,8 ) . results table 1. shows that the patients infected with 2009 h1n1 virus were younger than healthy subjects and those infected with seasonal influenza. and the difference reached to the level of significance (p< 0.01) compared with healthy subjects .two cases infected with 2009 h1n1 virus (9.1%) were from south of iraq and the remaining 20 cases were from baghdad . polymicrobial isolates from nasopharyngeal swabs were observed in patients infected with 2009 h1n1 virus.polybacterial infections (2-7 microorganisms) and fungal infection were reported in 21 out of 22 patients (95.5%) and 5 out of 22 (22.7 %) respectively ( table 2).the predominant isolated microorganisms were streptococcus pyogenes , staphylococcus aureus and streptococcus pneumoniae wich was found in 95.2 % , 95.2 % and 90.5 % respectively, while actinomycetes are isolated from 47.6% (10 out 21) ( table 2).table 3 shows that seven microorganisms were isolated from 10 (47.6 %) patients infected with 2009 h1n1 ,no microorganism was isolated from patients infected with seasonal influenza or healthy subjects. table 1: the charactereristics of the study . healthy subjects (n=11) patients with seasonal influenza patients with 2009 h1n1 gender : (male:femal) 7:4 6:6 11:11 age(year): median mean ± sd 38 40.9 ± 13.7 32.5 36.3±15.4 30 27.4±12.6 * residency: baghdad south of iraq 11 11 1 20 2 * p< 0.01 compared with healthy subjects. table 2 : the frequency (%) of microorganisms isolated from nasopharynx swabs. microorganisms frequency streptococcus pyogenes 20(95.2) staphylococcus aureus 20(95.2) streptococcus pneumoniae 19(90.5) streptococcus mitis 12(57.1) haemophillus influenzae 15(71.4) actinomycetes 10(47.6) candida albicans 5(23.8) table 3: distribution of 2009 h1n1 cases according to the number of microorganisms. no. of microorganisms frequency (%) of patients 0 1(4.5) 1 0(0) 2 1(4.5) 3 0(0) 4 6(27.3) 5 1(4.5) 6 3(13.6) 7 10(45.5) http://en.wikipedia.org/wiki/probiotic#cite_note-vanderhoof-6 iraqi j pharm sci, vol.20(2) 2011 prevalence of microorganisms in h1n1 patients 83 discussion in april 2009, a novel influenza virus (2009 influenza a [h1n1] virus) was first reported in humans . this was followed by case series of patients with severe 2009 influenza a (h1n1) viral infection (3). this report provides valuable information on secondary bacterial infections in h1n1 patients compared to seasonal influenza in iraqi patients. the rate of infection in the united states has been highest among individuals ≤24 years of age (9) .tsigrelis etal (10) describe two patients with early onset secondary bacterial pneumonia due to s. aureus that occurred as a complication of 2009 h1n1 viral infection. the emergence of pneumonia caused by community-associated methicillin-resistant s. aureus (ca-mrsa) during the 2003–2004 and subsequent influenza seasons has further altered the microbiological spectrum of secondary bacterial pneumonia in the setting of influenza (11,12). during the 1968–1969 influenza pandemic, there was a large increase in the number of patients presenting to a hospital in atlanta, georgia, us, in january 1969 with acute respiratory infections which led to the admission of all patients with clinical or radiographic evidence of pneumonia. pneumococcus was the most common aetiology but cases of pneumonia related to s. aureus increased, in relative terms, more that due to any other organism, from 6% of cases during the previous influenza season to 19 % during the pandemic. in contrast, 61% of community-acquired pneumonia (cap) cases were caused by pneumococcus during the previous influenza season compared to 48 % during the pandemic [10] . although secondary bacterial pneumonia due to staphylococcus aureus was a major cause of death in patients with influenza during prior pandemics but it has not been well characterized during the study of morens et al . (13). in a review of 37 cases of human infections with swine influenza virus reported between 1958 and 2005, six cases (17 percent) resulted in death, all of which were due to pneumonia. influenza virus was the only pathogen identified from the lungs in four patients; in one individual, streptococcus viridans, neisseria spp, and klebsiella spp were also identified in addition to influenza virus (14 , 15). conclusions the patients infected with 2009 h1n1 virus were younger than healthy subjects and those infected with seasonal influenza. and the difference reached to the level of significance (p< 0.01) compared with healthy subjects . references 1. centers for disease control and prevention (cdc). intensive-care patients with severe novel influenza a (h1n1) virus infection—michigan, . mmwr morb. mortal. wkly. rep. 2009;58:749– 52. 2. centers for disease control and prevention (cdc). hospitalized patients with novel influenza a (h1n1) virus infection—california, april–may, 2009. mmwr morb .mortal. wkly. rep . 2009;58:536–41. 3. centers for disease control and prevention (cdc). swine influenza a (h1n1) infection in two children— southern california, march–april 2009. mmwr. morb. mortal. wkly. rep. 2009;58:400–2. 4. myers, k.p. ; olsen, c.w. and gray, g.c. cases of swine influenza in humans: a review of the literature. clin. infect. dis. 2007; 44:1084. 5. united states centers for disease control and prevention. interim guidance on infection control measures for 2009 h1n1 influenza in healthcare settings, including protection of healthcare personnel. http://www.cdc.gov/h1n1flu/guidelines. 6. united states centers for disease control and prevention. pregnant women and novel influenza a (h1n1): considerations for clinicians.http://www.cdc.gov/h1n1flu/cli nician_pregnant.htm (2009). 7. greenwood, d.; slack, r.c. and peutherer, j.f. medical microbiology. (sixteenth ed.). churchill livingston ,2002. 8. carr, f.j.; chill, d. and maida, n. the lactic acid bacteria: a literature survey. critical rev. in microbiol., 2002 ; 28(24). 9. belshe, rb. implications of the emergence of a novel h1 influenza virus. n engl j med 2009; 360:2667. 10. tsigrelis ,c; mohammad ,m; fraimow ,h.s; dellinger ,m;archesani,r.p and reboli,a.c. secondary bacterial pneumonia due to staphylococcus aureus complicating 2009 influenza a (h1n1) viral infection. infection . 2010, 38:237– 239. 11. hageman, j.c.; uyeki, t.m.; francis, j.s., et al. severe community-acquired pneumonia due to staphylococcus aureus, 2003–2004 influenza season. emerg infect dis. 2006;12:894–9. 12. centers for disease control and prevention (cdc). severe methicillinhttp://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=4 http://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=4 http://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=4 http://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=4 http://www.cdc.gov/h1n1flu/guidelines iraqi j pharm sci, vol.20(2) 2011 prevalence of microorganisms in h1n1 patients 84 resistant staphylococcus aureus community-acquired pneumonia associated with influenza—louisiana and georgia, december 2006–january 2007. mmwr morb. mortal. wkly. rep. 2007;56:325–9. 13. morens dm, taubenberger jk, fauci as. predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness. j. infect. dis. 2008; 198: 962–70. 14. wentworth, de, thompson, bl, xu, x, et al. an influenza a (h1n1) virus, closely related to swine influenza virus, responsible for a fatal case of human influenza. j. virol 1994; 68:2051. 15. blyth, cc, iredell, jr, dwyer, de. rapidtest sensitivity for novel swine-origin influenza a (h1n1) virus in humans. n engl j med 2009; 361:2493. http://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=85 http://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=85 http://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=85 http://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=85 http://www.uptodate.com/home/content/abstract.do?topickey=pulm_inf%2f18836&refnum=85 iraqi j pharm sci, vol.28(1) 2019 mentha extract in mucositis doi: https://doi.org/10.31351/vol28iss1pp37-43 37 the protective effect of mentha spicata ethanolic extract on irinotecaninduced mucositis in mice. aliaa a. s. abdul jabbar* and sarmed h. kathem *, 1 *pharmacology and toxicology department, college of pharmacy, university of baghdad, baghdad, iraq abstract irinotecan induced-mucositis is an inflammatory event of the intestine caused by an increase in the concentration of active metabolite 7-ethyl-10-hydroxycamptothecin (sn-38) in the intestine. irinotecan must first be converted by a carboxylesterase to the active metabolite (sn-38), which is subsequently glucuronidated by the hepatic enzyme to sn38g. the sn-38g is deconjugated in the intestine to sn-38 via β-glucuronidase produced by the intestinal bacterial flora, which accounts for sn-38 delayed intestinal mucositis of irinotecan. the present study was designed to evaluate the protective effect of mentha ethanolic extract on irinotecan-induced mucositis, intestinal mucositis induced by i.p injection of irinotecan (75mg/kg/day) for 4 days. mentha ethanolic extract orally administered to mice for 7 days starting one day before irinotecan dose. results showed that mentha ethanolic extract significantly decreased both jejunal tissue il-1β (3.47±1.23 vs. 6.5±0.36 ng/ml), and fecal βglucuronidase activity (79.78± 10.7 vs. 120.6± 8.3 u) compared to the model control group, also histopathological sections showed improvements in mucositis features in the mentha ethanolic extract treated animals. as a conclusion, mentha ethanolic extract has a protective effect on irinotecan-induced mucositis. keywords: intestinal mucositis, irinotecan, mentha ethanolic extract, il-1β, fecal β-glucuronidase. مستخلص الكحولي ألوراق النعناع المدبب ضد االلتهاب المعوي الناتج من للالتأثيرالوقائي االيرينوتيكان في الفئران علياء عبد الستار عبد الجبار* و سرمد هاشم كاظم*، 1 ،العراق . جامعة بغداد،كلية الصيدلة ،فرع االدوية والسموم * الخالصه في االمعاء, االيرينوتيكان يجب (sn-38) االلتهاب المعوي المتسبب بواسطه االيرينوتيكان نتيجه لزياده تركيزااليض الفعال لاليرينوتيكان والذي سوف يقترن بصوره متسلسله بواسطه انزيم الكبد (sn-38) الى دواء فعال (carboxylesterase) بواسطه انزيم الان يكون متحوال اوال الذي ينتج من البكتريا الموجوده بصوره طبيعيه في االمعاء والتي (β-glucuronidase) والذي سوف يزال االقتران بواسطه انزيم (sn38g) الى والمسؤوله عن االلتهاب المعوي المتاخر لاليرينوتيكان. لدراسه تاثير مستخلص النعناع للحمايه من االلتهاب (sn-38) تودي الى زياده تركيز مليغرام 75المعوي المتحفز بواسطه االيرينوتيكان. يحفز االلتهاب المعوي لدى الفئران بواسطه حقن االيرينوتيكان في التجويف البروتوني بجرعه ايام تبدا قبل يوم واحد من جرعه االيرينوتيكان. نتيجه الدراسه 7ايام . يعطى مستخلص االيثانولي للنعناع فمويا للفئران لمده 4احد ولمده في اليوم الو في فضالت الفئران ( β-glucuronidase) للنسيج المعوي وقلل من فعاليه انزيم (il-1β) شوهد ان مستخلص االيثانول للنعناع قلل نسبيا من مقارنه مع المجموعه المعالجه لاليرينوتيكان فقط. ولقد شوهد في مقاطع التشريح النسيجي تحسن في خصائص االلتهاب المعوي في الحيوانات واسطه دواء تج باالمعالجه بواسطه المستخلص االيثانولي للنعناع . ولالستنتاج, مستخلص االيثانول للنعناع لديه تاثير للحمايه من االلتهاب المعوي الن .االيرينوتيكان االيرينوتيكان، النعناع المدبب، االلتهاب المعوي الكلمات المفتاحية : introduction intestinal mucositis is damage occurs to the intestinal mucosal membrane, one of the most harmful side effects of chemotherapy and radiotherapy treatment. mucositis presents with symptoms as intestinal and oral pain, vomiting, diarrhea, sore mouth and destruction of the gastrointestinal epithelium that contributes to poor absorption of nutrients and, consequently, weight loss (1). the development of mucositis can be divided into five phases, the first phase, known as the initialization phase, occurs after chemotherapy administration resulting in cell injury. this lesion might be a result of direct dna damage or caused via generation of reactive oxygen species (ros) (2). the second phase is a primary damage response that dna strand breaks and reactive oxygen species (ros) activate a series of interacting biological events by the activation of various transcription factors (3). the third phase is signal amplification, the presence of pro-inflammatory cytokines induces heightened tissue damage leading to a vicious circle in the signal amplification keep on increasing cytokines and oxidative stress levels then resulting in more extreme tissue damage and apoptosis and the epithelium initiates to lose integrity (2). 1corresponding author e-mail: skathem@copharm.uobaghdad.edu.iq received: 17 /9/2018 accepted:14 /10/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp37-43 iraqi j pharm sci, vol.28(1) 2019 mentha extract in mu cositis 38 the fourth phase is ulceration phase, clinical appearances of mucositis become apparent as mucosal integrity is lost and formed painful lesions (2). the final healing phase occurs after the ending of cancer therapy. irinotecan (cpt-11) is a topoisomerase i inhibitor. it's the hydrochloride salt of a semisynthetic derivative of camptothecin with higher water solubility than camptothecin. irinotecan was first approved for the treatment of metastatic colorectal cancer (crc) in the united states in 1996 (4). irinotecan interacts with cellular topo i–dna complexes and has s-phase-specific cytotoxicity. at greater concentrations of irinotecan non-s-phase cells can also be killed. the mechanism of non-s-phase cell killing appears to be related to transcriptionally induced dna damage and through apoptosis mechanism (5). irinotecan must first be converted by a carboxylesterase (ces) to the active metabolite 7-ethyl-10-hydroxycamptothecin (sn-38). the sn-38 is the major metabolite believed to be responsible for irinotecan’s biologic effects which including, efficacy and toxicity. the (sn38) is detoxified chiefly by uridine diphosphate glucuronosyltransferases (ugts) specifically the hepatic ugt1a1 and ugt1a9, in the liver to form inactive sn-38 glucuronide (sn-38g). common unwanted adverse effects of irinotecan are bone marrow toxicity leading to abnormal blood counts, in specific leukopenia, and mucositis, which leads to diarrhea (6). sn-38g is deconjugated to sn-38 via βglucuronidase produced by the intestinal bacterial flora which accounts for the late sn-38 double peaks in the plasma, and responsible for the delayed intestinal toxicity of irinotecan (7). cpt-11 also increases nuclear factor-κb (nf-κb) and cytokines (tnf-α, il-1β, il-6, and kc levels) and myeloperoxidase activity in intestinal tissue which involved in the pathogenesis of mucositis (8). mentha spicata synonym mentha viridis, among the family of lamiaceae (labiatae). used as fresh or dried leaves or powder, as a seasoning and flavouring herb or traditionally as an herbal. the mentha is also commonly used as an antimicrobial agent and as a preservative in food (9). the polar extracts of mentha leaves are characterized mostly by a high content of phenolic compounds, such as rosmarinic acid, luteolin and apigenin derivatives (10). some of these components have been revealed to have antioxidant properties, so, the mentha spicata could also be considered an antioxidant source (11). more interestingly, the anti-inflammatory properties of mentha extract rich in phenolic compounds have been confirmed in vivo in rats (12). in term of biological uses, mentha acts as antispasmodics and anti-platelets and insecticides (13). also, mentha is considered as herbal medicine in folklore remedies for treating of colds and flu and respiratory tract problems, gastralgia, hemorrhoids, and the stomachache. the goal of this study to evaluate the protective effect of mentha spicata ethanolic extract on irinotecan-induced intestinal mucositis in mice. materials and methods chemicals and kits the chemicals that were used in this work includes, diethyl ether (bdh chemicals, england), formalin (merck chemicals, germany), irinotecan vial (kocak pharma, turkey), mice interleukin-1-β for enzyme-linked immune sorbent assay (elisa) (shanghai, china), 4nitrophenyl-b-d-glucuronide substrate for enzyme (sigma, usa), dimethyl sulfoxide (merck chemical, germany), ethanol solution (merck chemical, germany). animal selection twenty four albino female mice (8 weeks) weighing between (25-38 gm) were selected randomly and maintained in the animal house at the college of pharmacy/university of baghdad. animals were maintained under controlled conditions of temperature, humidity and light/dark cycle. they were fed standard rodent pellet diet and have free access to water. plant collection fresh plant of mentha spicata collected from different areas of baghdad/al-resafa at october, the plant was identified for the authentication purpose by (dr. sucaina at the department of biology/college of science/university of baghdad). the leaves of the mentha were used and dried at room temperature, then crushed by mortar and pistol to be extracted by maceration method. extraction of the plant one hundred fifty gram of air-dried leaves of mentha plant was crushed in mortar and pistol to be extracted by maceration with 1500 ml of ethanol solvent in a closed container at room temperature for three day, then the mentha ethanolic extract filtered using a whitman filter paper and evaporated under reduced pressure by rotary evaporator at 42 c to concentrate and dry the extract (14). after that, the collected amount was weighted. preparation of stock solution for ethanolic extract fraction a stock solution of mentha spicata ethanolic extract was freshly prepared by dissolving 960 mg of ethanolic extract in 10 ml of 2% of dimethyl sulfoxide (dmso) to obtain a concentration (9.6mg/0.1ml) according to mentha ethanolic extract dose for mice 320 mg/kg/day (15). experimental protocol the animals used in this study divided into four groups each with 6 mice as follows: iraqi j pharm sci, vol.28(1) 2019 mentha extract in mu cositis 39 group i: six mice received a single oral dose of 0.1ml of normal saline for 7 successive days starting day 0. this group served as a blank control group. group ii: six mice received a single dose of 0.1ml intraperitoneal irinotecan (75mg/kg) for 4 successive days starting day1. this group served as a model control group (16). group iii: six mice received a single dose of 0.1ml intraperitoneal irinotecan (75mg/kg) for 4 successive days starting day 1 with 0.1ml of 2% dmso by oral gavage for 7 successive days starting day 0. group iv: six mice received 0.1ml single orally dose of mentha ethanolic extract (320mg/kg/day) for 7 successive days starting day 0 with an intraperitoneally single dose 0.1ml of irinotecan (75mg/ kg) for 4 successive days starting day 1 (17). animal received mentha ethanolic extract 0.1 ml by oral gavage in the morning at 8 am before 1/2 hour of irinotecan injection. the extracts treatment started one day before irinotecan administration (day 0) and continued to the two days after the last irinotecan dose was administered, a total of 7 days. preparation of jejunum tissue homogenate animals have been euthanized at (day 7) under light diethyl ether anesthesia. abdomen is opened by a midline incision and two horizontal incisions then the jejunum of each animal utilized in this study is quickly dissected, placed in chilled phosphate buffer saline solution (pbs) at (ph7-7.2) at 4cº to remove excess blood, then the tissue dried with filter paper and weighed then was minced to small pieces; where, 1 g of jejunum was put in tube containing 9 ml of (0.01m) phosphate buffer saline solution. the resulting homogenate was centrifuged at 5000 rpm for 10 minutes at 4cº then the supernatant is separated using micropipette and store at -20cº until the day of analysis. the jejunum tissue homogenate utilized for the estimation of interleukin one beta (il-1β) level. fecal β-glucuronidase assay the activity of intestinal bacterial βglucuronidase in mice feces was measured by the method described by shiau and chang (18). briefly, fresh fecal pellets were collected in the morning before and after irinotecan administration in (day 0 and day 5), then weighed and mixed with pbs (0.01 m, ph 7.5) at a (wt/wt) ratio of (1:100). after softening for 20 min, the fecal pellets were homogenized. then 0.05 ml of 4-nitrophenyl β-dglucuronide (0.01 m, sigma, usa) was added to 1 ml diluted fecal homogenate to produce the following reaction:  4-nitrophenyl β-d-glucuronide + βglucuronidase → 4-nitrophenol + βglucuronide all reaction solutions were incubated at 37°c for 60 min and then centrifuged at 3000g for 10 min. the absorbance of each supernatant solution was measured at 405 nm in a spectrophotometer (aplepd303, japan). the concentration of 4-nitrophenol in each sample was calculated by reference to a standard curve at the same time and conditions. the activity of fecal β-glucuronidase was measured as one unit of an enzyme that cleaves 1 nmol of 4nitrophenol per hour at 37°c under saturated substrate concentrations. histopathological examination of the mice jejunum at the end of the experiment period, all the animals were sacrificed, 5-cm ring from the proximal area (close to the duodenojejunal flexure) of each harvested jejunum was carefully removed, washed in a normal saline solution and fixed in 10% formalin solution. jejunum tissues were prepared for histological examination according to the method of junqueira lc. et al. (19), using paraffin sections technique. statistical analysis the experiment design used for these studies was rationalized complete block design (rcbd). the results were presented as means ± standard deviation (sd). one way analysis of variance (anova) followed by dunnett method. the differences between the means are considered significant at the 5% confidence level. results effects of mentha ethanolic extract on intestinal tissue il-1β level mice that orally treated with normal saline (group i) and mice that orally treated with mentha ethanolic extract and cpt-11 (group iv) showed a significant reduction (p<0.05) in the tissue level of il-1β compared with model control group (group ii), as shown in table 1. table (1) effects of mentha ethanolic extract on jejunum il-1β level in mice. treatment groups n=6 type of treatment il-1β (ng/ml) mean ± sd i normal saline 0* ii irinotecan 6.5±0.36 iii irinotecan +dmso 6.9±0.32 iv mentha extract +irinotecan 3.47±1.23* (): significant difference with respect to the model control group (p0.05). n: number of animals. effects of mentha ethanolic extract on fecal βglucuronidase enzyme activity as shown in table 2, on the day (0), the mean for mentha ethanol extract group showed nonsignificant changes in β-glucuronidase activity (p>0.05) compared with model control (group ιi). iraqi j pharm sci, vol.28(1) 2019 mentha extract in mu cositis 40 on the day (5), mice treated with mentha ethanolic extract and cpt-11 (group iv) showed a significant reduction in fecal β-glucuronidase activity (p<0.05) compared with model control (group ιi), as shown in table 2. table( 2) comparison of the intestinal β-glucuronidase activity in different groups. treatment groups n=6 type of treatment day 0 β-glucuronidase activity (u) mean ± sd day 5 β-glucuronidase activity (u) mean ± sd i normal saline 83.9 ± 12.2 84.45± 11.5* ii irinotecan 74.32± 13.6 120.6± 8.3 iii irinotecan +dmso 78.69±15.7 111.25± 11.2 iv mentha extract +irinotecan 81.19± 13.3 79.78± 10.7* notes: one unit of enzyme activity is the amount of enzyme that will cleave (1 nmol) of the colorimetric substrate per hour at 37°c under saturated substrate concentrations. (): significant difference with respect to the model control group (p 0.05). n: number of animals. histopathological examination of mice jejunum sections histopathological examination of group i (normal saline group) showed the normal structure of intestinal villi, no inflammatory cell infiltration and without dysplastic cells change (figure 1). figure (1) cross section is showing normal mice jejunum in group ι. magnification (100x), staining: haematoxyline and eosin. red arrow: sign to normal intestinal villi. histopathological examination of group ii (irinotecan group) showed atrophy of intestinal villi with heavy chronic inflammatory cells infiltration and moderate dysplastic cell change (figure 2). figure (2) cross sections showing abnormal mice jejunum in group ιi. magnification: (100 x and 400 x); staining: haematoxyline & eosin. a: villi atrophy. b: chronic inflammatory cells infiltration. c: dysplastic cell change iraqi j pharm sci, vol.28(1) 2019 mentha extract in mu cositis 41 histopathological examination of group iii (cpt11+ dmso) showed atrophy of intestinal villi with heavy chronic inflammatory cells infiltration with mild to moderate dysplastic cell change (figure 3). figure (3) cross sections showing mice jejunum in group iii. magnification: (100x, 400x), staining: haematoxyline & eosin. a: villi atrophy. b: chronic inflammatory cells infiltration. c: dysplastic cell change. histopathological examination of group iv (mentha ethanolic extract +cpt-11) showed shortening of intestinal villi with chronic inflammatory cell infiltration but less than the model control group. sections showed no dysplastic changes (figure 4). figure (4) cross sections showing mice jejunum in group iv. magnification: (100 x and 400 x), staining: haematoxylin & eosin a: marked shortening of intestinal villi. b: chronic inflammatory cell infiltration. discussion mucositis is the painful inflammation and ulceration of mucous membranes lining the digestive tract, as a result of chemotherapy and radiotherapy treatment for cancer (20). cytotoxic chemotherapy can cause functional and structural changes in the gastrointestinal tract (git). common gastrointestinal symptoms following chemotherapy include heartburn, abdominal pain, diarrhea and constipation, bloating and nausea. these symptoms arise as the result of the damage caused by chemotherapy agents (1). the pathogenesis of irinotecan-induced intestinal toxicity may involve oxidative stress; irinotecan enhanced production of reactive oxygen species (ros) and reactive nitrogen species, which induce cell death. the ros can also activate the transcription nuclear factor kappa-lightchain-enhancer of activated b cells (nf.κb) (2). the study has shown significant increases in the expression of pro-inflammatory cytokines, with key offenders tnf, il-1β, il-4, il-6 (2). microbiota can influence the efficacy and toxicity of drugs by regulating the levels of metabolic enzymes and transporters. inactivated metabolite of irinotecan, sn-38g, is reactivated to sn-38 by bacterial βglucuronidase in the intestinal tract (21). in the present study, histological examination of the jejunum sections from mice received irinotecan showed atrophy of intestinal villi with heavy chronic inflammatory cells infiltration, few crypt glands with moderate dysplastic cell change. these changes indicated that irinotecan-induced intestinal mucositis as shown in (figure 2). this result confirmed by other study reported that irinotecan causes severe jejunum and colonic damage like villous atrophy, crypt hypoplasia, increased apoptosis, dilation, and excessive mucous secretion. increased levels of cell apoptosis combined with the histopathological changes in both the jejunum and colon and the changes in goblet cell numbers may iraqi j pharm sci, vol.28(1) 2019 mentha extract in mu cositis 42 cause changes in absorption rates, possibly leading to diarrhea (22). the present study showed a significant increase in the il-1β in mice jejunum that confirmed irinotecan-induced intestinal mucositis mediated by inflammation. other study confirmed that il-1β production plays a crucial role in mucositis (23). importantly, this study showed that irinotecan significantly increased fecal β-glucuronidase activity. the structure of the intestinal microbial community and increase β-glucuronidase activity by irinotecan. these changes increase irinotecan toxicity as more sn-38 (the active form of the drug responsible for toxicity) will be liberated (24). histological examination of the jejunum section from animal received irinotecan with mentha ethanolic extract showed improvements in histopathological finding compared with models control group for intestinal toxicity induced by irinotecan. these improvements include mild shortening of intestinal villi, with mild chronic inflammatory cells infiltration, no dysplastic cell change compared with the model control group. the successful reduction in intestinal toxicity induced by irinotecan in mice treated with mentha ethanolic extract showed in the jejunal histopathological sections confirmed by reduction in tissue il-1β level. the polar extract of mentha leaves is characterized by a high content of phenolic compounds, such as rosmarinic acid, luteolin and apigenin derivatives (10). more interestingly, the antiinflammatory properties of mentha extract rich with phenolic compounds have been confirmed in vivo in rats (12). in addition, mentha ethanolic extract significantly decreased fecal β-glucuronidase activity (table 2). the lower β-glucuronidase activity by mentha extract leads to the lower liberation of sn-38, which ultimately decreased intestinal mucositis. the resulted improvements in irinotecan-induced mucositis obtained from mentha extract may be explained by first, the novel direct inhibitory effect of the extract on β-glucuronidase activity that reduced mucositis as confirmed by this study (12). second, the indirect effect of the extract on mucositis due to mentha anti-inflammatory effect as confirmed in this study by reduction of the il-1β level (25,26). conclusion data obtained from the presented study suggested that mentha ethanolic extract has antiinflammation activity and anti-β-glucuronidase activity, by the decreased intestinal level of il-1β and fecal β-glucuronidase activity, respectively. so, the ethanolic mentha extract has protective effects against irinotecan-induced mucositis. references 1. stringer am, gibson rj, bowen jm, logan rm, yeoh as-j, keefe dmk. chemotherapyinduced mucositis: the role of gastrointestinal microflora and mucins in the luminal environment. j support oncol [internet]. 2007;5(6):259–67. 2. araújo rs, de barros alb. intestinal mucositis induced by chemotherapy: an overview. j mol pharm org process res [internet]. 2015;03(03):18–9. 3. sultani m, stringer am, bowen jm, gibson rj. anti-inflammatory cytokines: important immunoregulatory factors contributing to chemotherapy-induced gastrointestinal 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baghdad, iraq. ***head of iraqi pharmacovigilance center, directorate of technical affairs, ministry of health, iraq. abstract immunization is one of the most cost-effective and successful public health applications. the results of immunization are difficult to see as the incidence of disease occurrence is low while adverse effects following the immunization are noticeable, particularly if the vaccine was given to apparently healthy person. there are high safety expectations of population regarding the vaccines, therefore they are more prone to hesitancy regarding presence of even small risk of adverse events which may lead to loss of public trust to the vaccination programs. vaccine safety monitoring is needed as they are now administered to the general population and also available to special categories such as pregnant women and patients with different diseases whom not subjected to clinical trials as well as the incorrect administration rout and the presence of rare or delayed onset adverse events make the presence of surveillance system necessary. the aim of the current study was to measure the distribution, percentage, and frequency of adverse reactions related to vaccines administration in iraq and to assess the causality, severity, seriousness of these adverse reactions. this study is a retrospective descriptive study for surveillance of vaccine safety conducted using iraqi pharmacovigilance centre database from 2014 till the end of 2018. this study conducted in period from 1st september 2018 till february 2019 and was in corporation with iraqi pharmacovigilance center in iraqi ministry of health. 2116 adverse events were included and outcomes, severity, seriousness, and causality of adverse events were assessed. majority of adverse events following immunization cases (90.97%) were mild, non-serious (94.47%) and recovered (94.23%). most reports were for general disorders and administration site conditions and the majority were for elevated body temperature and injection site reactions. keywords: vaccine, pharmacovigilance, adverse events following immunization, iraqi pharmacovigilance center, immunization. االستناد لقاعدة ة باثر رجعي بشدة و جدية االثار الجانبية بعد التطعيم في العراق : دراس ، ةيتقييم السبب انات مركز اليقظة الدوائي في العراقيب ***و منال محمد يونس **كاظم ضياء جبار ، 1،*أحمد كاظم عبد كلية الصيدلة ، جامعة الكفيل ، النجف، العراق. * فرع الصيدلة السريرية ،كلية الصيدلة، جامعة بغداد، بغداد، العراق.** مدير المركز العراقي لليقظة الدوائية، دائرة االمور الفنية، وزارة الصحة، العراق. *** الخالصة نسبة . من الصعوبة مالحظة نتائج عمليات التلقيح و ذلك النخفاض فعاليةً من حيث الكلفةيعد التطعيم من اكثر التطبيقات الصحية لقاحات تعطى الشخاص المعظم في حين ان االعراض الجانبية التي قد تحصل نتيجة اللقاحات تكون واضحة للعيان , خاصة و ان ضااالمرحدوث نتشار افي حمالت التطعيم و بالتالي قد تزيد من امكانية تهتخاذ اقصى درجات السالمة و الحذر اهمية لضمان عدم فقدان المجتمع ثقاسليمين . ان الجانبية معروفة اثناء عمل التجارب االولية لكن بعض االثار ال يمكن معرفتها اال بعد فترة اللقاح اعراضض غير المرغوبة بها . قد تكون المراا تكون االعراض الجانبية نادرة او قدو الذين قد يعانون من امراض مختلفة للحوامل او اعطائها , كاعطاء القاح بطريقة غير صحيحة من الزمن ان الهدف من الدراسة نا تاتي اهمية متابعه اللقاحات من حيث االعراض الجانبية التي قد تسببها . من ه بعد فترة زمنية و تكون متأخرة وتحدث . يعد البحث جديتهاشدتها و مدى سببية االعراض الجانبية للقاحات معرفةمعرفة توزيع و نسب حدوث هذه االعراض الجانبية و الحالية هو لغاية نهاية 4102للتقارير الموجودة و التي هي من سنة يقظة الدوائيلالمركز العراقي ال بيانات قاعدة علىبحثا وصفيا باثر رجعي باالعتماد عرض جانبي و تجميع المعلومات حول 4002تجميع , 4102لغاية فبراير 4102في الفترة من اول من سبتمبرسنة , تم خالل البحث 4102 ( و غير % 21.29بشدة بسيطة ). تبين النتائج ان معظم االعراض الجانبية كانت تحديد سببيتهاو ية االعراض الجانبعواقب و, وخطورةشدة , . معظم االعراض الجانبية كانت تعود تحت تصنيف ( من الحاالت %22.49( و قد تعافت االعراض الجانبية في ) % 22.29جدية بنسبة ) تفاعالت الحاالت التي تصيب مناطق اعطاء و زرق اللقاحات و التي معظمها تعود الرتفاع درجة حرارة الجسم و الاالضطرابات العامة و لة في مناطق التلقيح في الجسم .الحاص . التطعيم ، المركز العراقي لليقظة الدوائية ،االثار الجانبية بعد التلقيح، اليقظة الدوائية ،اللقاحات الكلمات المفتاحية: 1corresponding author e-mail : ahmad.kadhum.pharm@gmail.com received: 29/ 5 /2019 accepted:18 /8 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp142-150 mailto:ahmad.kadhum.pharm@gmail.com iraqi j pharm sci, vol.28(2) 2019 adverse event following immunization 143 introduction vaccine term is of broad meaning which refers to the biological product that enhances the immunity to prevent or sometimes treat diseases (1). based on the way of formulation and preparation, there are many types of vaccines given by different ways of administration. monovalent vaccines are those with a single strain of antigen such as measles vaccine while polyvalent vaccines are those that contain more than one strain or serotype of the antigen such as of oral poliovirus vaccine (opv). on other hand, combination vaccines are produced by combining more than one antigen in the single formula so the vaccine could protect from several diseases in the same time as well as reducing the need for multiple injections or administration (2). live attenuated vaccines are obtained through weakened organism that causes the disease to reduce their ability to be pathogenic, this kind of vaccine provides an immune response that mimics what happens when the infection that is caused by organisms such as opv (3). inactivated vaccines are found using physical or chemical treatment so microorganisms get killed like inactivated polio vaccine (ipv) (4). toxoid vaccines are another type of vaccines in which the toxin of microorganisms isolated, purified then inactivated such as tetanus toxoid (5). subunit vaccines, like the inactivated vaccine, contain no live organism, but this type uses an antigenic portion of the microorganism instead of the whole killed microorganism such as hepatitis b vaccine which use the surface antigen (hbsag) (4). adverse events following immunization (aefi) is defined as “any untoward medical occurrence which follows immunization and which does not necessarily have a causal relationship with the usage of the vaccine.” the adverse event (ae) may be any unfavourable or unintended sign, abnormal laboratory finding, symptom or disease(6). there are five known causes of aefi: a. vaccine productrelated reaction, b. vaccine quality defect-related reaction, c. immunization error-related reaction, d. immunization anxiety-related reaction, e. coincidental event (2,7). the most common systems that are widely used worldwide for data collection of adverse reactions is the spontaneous reporting system (srs) in national pharmacovigilance centres which is recommended by world health organization (who) (9,10). pharmacovigilance (phv) according to who is defined as “the science and activities relating to the detection, assessment, understanding, and prevention of adverse effects or any other possible drug-related problems.” the purpose of phv is to improve medication safety and health care, it also acts as an indicator for clinical care standards(2,10). expanded program on immunization (epi) was established in 1985 in iraq which support the immunization services , it has strength points as it has good surveillance system and integration between authorities as well as coordination with who (10). vaccine safety monitoring is needed as they now are administered to the general population and also available to special categories such as pregnant women as well as incorrect administration rout and presence of rare or delayed onset adverse events make the presence of surveillance system necessary (2). the aim of the current study was to measure the distribution, percentage, and frequency of adverse reactions related to vaccines administration in iraq and to assess the causality, severity, seriousness of these adverse reactions. subject and methods this study is a retrospective descriptive study for monitoring of vaccine safety conducted using the iraqi pharmacovigilance database. ethical approval was acquired from the iraqi ministry of health as well as college of pharmacy/university of baghdad prior to conducting the study along with the approval of the iraqi pharmacovigilance center to order an account for the access to the vigiflow. the data of reports has been extracted from vigiflow which is a management system for individual case safety report (icsr) by uppsala monitoring center (umc) and vigibase which is the who international icsr database (11). the study conducted between 1st of september 2018 rill february 2019. the data were reported from 2014 untill the end of the 2018 in corporation with iraqi pharmacovigilance center in ministry of health. there were 2141 adverse events for 1221 reports; some reports were excluded from the study based on the duplication of the reports and presence of identification number (id) as well as missing of vital data for the assessment of the report. id is a unique number is given for each report for facilitating their organization and tracking when needed. after exclusion, the included reports were 1209, which contains 2116 reported adverse events regarding vaccines. the data of adverse events collected and classified. inclusion criteria included any report with available and valid data for the assessment of the case while some cases were excluded in the correlations as they have missing data. adverse event following immunization were classified according to system organ class (soc) which is the major category that aefi belongs to , according to the medical dictionary for drug regulatory affairs (meddra) (11). in order to assess the severity of the adverse events, the modified hartwig and seigel criteria have been used (12) as illustrated in table 1. iraqi j pharm sci, vol.28(2) 2019 adverse event following immunization 144 table 1.the modified hartwig and seigel severity assessment scale for adverse drug reactions (12) mild level 1 an adr occurred but required no change in treatment with the suspected drug level 2 the adr required that treatment with the suspected drug be held, discontinued, or otherwise changed. no antidote or other treatment requirement was required. no increase in the (los) moderate level 3 the adr required that treatment with the suspected drug be held, discontinued, or otherwise changed. and/or an antidote or other treatment requirement was required. no increase in los level 4 any level 3 adr which increase los by at least 1 day. or the adr was the reason for admission severe level 5 any level 4 adr which requires intensive medical care level 6 the adr caused permanent harm to the patient level 7 the adr either directly or indirectly led to the death of the patient adr: adverse drug reactions; los: length of stay. the outcomes of the aefi were collected as they founded in icsr of aefi. the outcomes that the reporter would choose from according to who include: fatal, not recovered/not resolved, recovered / resolved , recovered / recovered with sequelae, recovering / resolving and unknown.the seriousness assessed according to six criteria that were adopted by who, same seriousness criteria are used in the iraqi icsr (13) and in vigiflow reports (11) form as is shown in table 2. table 2. seriousness assessment in the individual case safety report (13) the revised who classification (second edition) was used for assessment of causality of aefi which provided as a guideline for assessing the icsr for causality (14) as shown in table 3: table 3. world health organization causality assessment algorithm for adverse event following immunization (14) adequate information available adequate information not available consistent with causal association to immunization indeterminate inconsistent with causal association to immunization unclassifiable a1 vaccine product related reaction ( as per publishe d literature ) a2 vaccine quality defectrelated reaction a3 immuni zation error related reaction a4 immunizati on anxiety related reaction b1 temporal relation is consistence but there is insufficient definitive evidence for vaccine causing event (may be new vaccine linked event) b2 reviewing factors result in conflicting trends of consistency and inconsistency with casual association to immunization c coincidental underlying or emerging condition(s) , or conditions caused by exposure to something other than vaccine specify additional information required for classification : do you consider the reaction to be serious? yes no if yes, please tick () to indicate why the reaction is considered to be serious: the patient died due to the reaction involved or prolonged inpatient hospitalization life threatening involved persistent or significant disability or incapacity congenital anomaly medically significant, please give details: iraqi j pharm sci, vol.28(2) 2019 adverse event following immunization 145 statistical analysis the demographic parameters regarding the distribution of age, gender of patients as well as the type of vaccines and the year of the reports measured to show the distribution pattern of the reports using descriptive statistic using microsoft excel 2016. the relations were tested using chisquare ,calculated by statistical package for the social sciences (spss) version 24.0. results the reports of adverse events included in the study were 2116 from 2014 reports until the end of 2018 in which a number of reports were 77, 62, 213, 1080 and 684 for the years 2014, 2015, 2016, 2017 and 2018 respectively. the most age group of which adverse events (aes) reported was infants by 1865 aes (88.14%), while aes for adults were 166 (7.84%), children 35 (1.65%), neonates 34 (1.61%) and adolescent 14 (0.66%). two reports did not specify the age group of which they reported for as it demonstrated by table 4 . table 4 . demographic distribution of the adverse events following immunization year aefi count (%) 2014 77 (4%) 2015 62 (3%) 2016 213 (10%) 2017 1080 (51%) 2018 684 (32%) total 2116 (100%) gender aefi count (% ) male 1012 (47.83%) female 932 ( 44.05%) n/a 172 (8.13%) total 2116 (100%) age group aefi count (%) infant 1865 (88.14%) adult 166 (7.84%) child 35 (1.65%) neonate 34 (1.61%) adolescent 14 (0.66%) n/a 2 (0.09%) total 2116 (100%) n/a :not available it was found that hexavalent vaccines had the most number of reports by 977 (46.17%) followed by pentavalent ii 473 (22.35%). the third-most reports were for pentavalent i vaccine 233 (11.01%) as shown below in table 5. table 5. types of vaccines associated with adverse event following immunization . hexavalent : (diphtheria, tetanus, acellular pertussis– hepatitis b -inactivated poliohaemophilus b ) vaccine, pentavalent i : (diphtheria, tetanus, acellular pertussis inactivated polio-haemophilus b, ) vaccine , pentavalent ii : (diphtheria, tetanus whole cell pertussis -hepatitis b-haemophilus b) vaccine , bcg: bacillus calmetteguérin vaccine, mmr: measles ,mumps and rubella vaccine, opv: oral polio vaccine the most aefi was related to general disorders and administer site conditions (57.75%), followed by skin and subcutaneous tissue disorders (29.11%). in addition, 8.65% of the reports were for immune system disorder as shown in table 6. vaccines administered aefi count (%) hexavalent 977 (46.17%) pentavalent ii 473 (22.35%) pentavalent i 233 (11.01%) tetanus 168 (7.94%) bcg 103 (4.87%) mmr 51 (2.41%) measles 40 (1.89%) hepatitis b 22 (1.04%) influenza 14 (0.66%) pneumococcal 13 (0.61%) rota virus vaccine 10 (0.47%) opv 6 (0.28%) hepatitis a 2 (0.09%) meningococcal 2 (0.09%) tick-borne encephalitis 1 (0.05%) pneumococcal & hexavalent 1 (0.05%) grand total 2116 (100%) iraqi j pharm sci, vol.28(2) 2019 adverse event following immunization 146 table 6. adverse event following immunization classified by system organ class soc: system organ class. regarding the severity assessment of aefi, it was found that 90.97% of the reported aes were mild, while 2.5% were of moderate severity, 0.14% were severe while the severeity class of 6.38% of the reports couldn’t be specified as shown in table 7. table 7. severity types of adverse event following immunization severity count % mild 1925 90.97% moderate 53 2.50% severe 3 0.14% unknown 135 6.38% total 2116 100.00% outcomes of aefi were collected according to the final result that seen by the reporter regarding the aes where 94.23% of the aes were resolved, 2.79% of aes were with unknown outcome while not available data was found in 2.27% of reports as shown in table 8. table 8.adverse event following immunization outcomes n/a: not available, aefi: adverse event following immunization the majority of cases were not serious cases (94.47%) while 1.47% of the reports were seen to increase hospitalization and 1.13% of total reports were life-threatening as shown in table 9. regarding the six aefi reported as led to disability (table 9), two were for bcg due to lymphadenopathy, two for hexavalent vaccine due to injection site swelling, one for measles for rash and one for polio vaccine due to weakness of the injected limb. the first five cases recovered while the polio ae were reported as not recovered. 25% of the life-threatening conditions were reported to measles vaccine and they were due to anaphylaxis and anaphylactoid responses. soc count of soc % general disorders and administration site conditions 1222 57.75% skin and subcutaneous tissue disorders 616 29.11% immune system disorders 183 8.65% infections and infestations 30 1.42% blood and lymphatic system disorders 29 1.37% respiratory, thoracic and mediastinal disorders 9 0.43% gastrointestinal disorders 9 0.43% nervous system disorders 6 0.28% vascular disorders 5 0.24% metabolism and nutrition disorders 2 0.09% musculoskeletal and connective tissue disorders 2 0.09% cardiac disorders 1 0.05% congenital, familial and genetic disorders 1 0.05% injury, poisoning, and procedural complications 1 0.05% grand total 2116 100.00% the outcome in reports of aefi count % recovered/resolved 1994 94.23% unknown 59 2.79% n/a 48 2.27% not recovered/not resolved 7 0.33% recovering/resolving 5 0.24% fatal 2 0.09% recovered/resolved with sequelae 1 0.05% total 2116 100% iraqi j pharm sci, vol.28(2) 2019 adverse event following immunization 147 table 9. seriousness of adverse event following immunization the two fatal cases were reported for bcg vaccine. relation of gender with outcomes, severity and seriousness was tested and it showed no statistically significant relation of gender with each of these three parameters as shown in table 10 table 10. relation of the outcome , severity and seriousness of adverse event following immunization with gender parameter male female total p. value outcomes no. % no. % no. recovered 952 95.97% 886 97.15% 1838 0.159 n.s others* 40 4.03% 26 2.85% 66 total 992 100.00% 912 100.00% 1904 severity no. % no. % no. mild 913 96.72% 860 97.40% 1773 0.656 n.s moderate 29 3.07% 22 2.49% 51 severe 2 0.21% 1 0.11% 3 total 944 100.00% 883 100.00% 1827 seriousness no. % no. % no. yes 947 94.89% 887 96% 1834 0.246 n.s no 51 5.11% 37 4% 88 total 998 100% 924 100% 1922 *others : unknown, not recovered/not resolved, recovering/resolving, fatal, recovered/resolved with sequelae , ns : not significant , (p<0.05) regarding the relation of age group with each of outcome, severity and seriousness, there is a significant relationship between age group with each of these parameters. recovery outcome was significantly lower in adolescent group compared to other age groups. regarding severity, aefis of mild severity was significantly lower in child age group compared to other age groups (p-value <0.0001). with respect to seriousness, serious aefis was significantly higher in child age group compared to other age groups (p-value <0.0001) as shown in table 11. table 11. outcome , severity and seriousness of adverse events following immunization in respect to age group parameter age groups p-value neonate infant child adolescent adult total outcomes no. % no. % no. % no. % no. % no. recovered 29 90.6% 1761 96.6% 32 96.97% 8 72.7% 162 97.6% 1992 <0.0001 others* 3 9.4% 63 3.4% 1 3.03% 3 27.3% 4 2.4% 74 total 32 100% 1824 100% 33 100% 11 100% 166 100% 2066 severity no. % no. % no. % no. % no. % no. mild 23 92.% 1711 97.44% 25 80.65% 8 100% 156 98.11% 1923 <0.0001 moderate 2 8.% 43 2.45% 6 19.35% 0 0.00% 2 1.26% 53 severe 0 0.% 2 0.11% 0 0.00% 0 0.00% 1 0.63% 3 total 25 100% 1756 100% 31 100% 8 100% 159 100% 1979 seriousness no. % no. % no. % no. % no. % no. yes 8 23.53% 66 3.58% 10 30.3% 2 14.29% 7 4.22% 93 <0.0001 no 26 76.47% 1779 96.42% 23 69.7% 12 85.71% 159 95.78% 1999 total 34 100% 1845 100% 33 100% 14 100% 166 100% 2092 *others : unknown, not recovered/not resolved, recovering/resolving, fatal, recovered/resolved with sequelae seriousness count % not serious 1999 94.47% increase hospitalization 31 1.47% life threat 24 1.13% unknown 24 1.13% medically significant … 30 1.42% disability 6 0.28% iraqi j pharm sci, vol.28(2) 2019 adverse event following immunization 148 the results of causality assessment are shown in table 12 where the majority (95.98%) of aefis were of a1 classification which almost was for body temperature conditions and local adverse eventst. table 12. the causality assessment of adverseevent following immunization the majority of the causality classified as vaccine product related reaction ( a1) , which count for 95.98% of the total 2116 aefi , folowed by 1.18% for b1 and b2 with 1.09% . six cases reported and classified as a4 and they were mostly a psychological events . some reportes could not be classified in one of the categories due to unavailability or mising of some data in the reports therefore classified as “unclassified”. discussion in order to identify and assess adverse reactions and events of drugs, the pharmacovigilance depends on collection of information regarding the post-marketing events in post-marketing or approval phase of medications by a system that relays on spontaneous reporting system (srs) in order to detect any problems with the vaccines, vaccination program or emergence of new adverse events that not reported in the trials (15). the results of the current study revealed that the percent of aefi reports is increased during 2017 and 2018 compared with previous years which may be related to increased awareness of health care workers. on its establishment, the iraqi pharmacovigilance center (iphvc) has adopted the mission of sensitizing and raising the awareness of the importance of reporting adrs in ensuring patient safety through face-to-face communication with all health care workers in iraq, where the need to create a reporting culture will be an intense challenge (16). infants were of highest reporting aes and it may be owed to the fact that they are the most targeted group regarding the immunization programs. regarding the type of vaccines associated with aes (table 5), in the current study the hexavalent had 46.17% followed by pentavalent ii with 22.35% . in a study regarding vaccine surveillance in oman showed that pentavalent ii vaccine was third most vaccine that aes reported for and they mostly were minor local reaction (17). in studies comparing combination versus separated administered vaccines, the reported adverse event indicate that combining antigens usually does not increase adverse effects-in fact, it can lead to an overall reduction in adverse events (18). the combination vaccines development increase the compliance and rates of vaccination coverage (19) while , the disadvantage combination vaccines is that it may not always be clear which component is responsible for a particular adverse event (20). as is shown in table 6, general disorder and administration site conditions followed by skin and subcutaneous tissue disorders both had the highest reported percent (57.75% and 29.11% respectively), in which the most reported aefi were for body temperature conditions and different type of injection site reactions such as redness, swelling, and pain. in one study in switzerland from 1991 – 2001, it was also reported that the majority of aes were for general disorder and administration site conditions (47%) (21). in the united states during 11 years of aefi surveillance the most reported aes were fever and injection site reaction (about one quarter for each) (22). regarding the severity assessment of aefi (table 7), the majority of the aefi cases (90.97%) were mild and it was mentioned in the supplementary information of vaccine safety , that the majority of vaccine reactions are “common”, mild, settle without treatment, and have no long-term consequences (23). regarding the outcomes of the aefi (table 8), the majority of cases were recovered with 94.23% percent,while with respect to seriousness (table 9) the majority of aefi cases were nonserious (94.47%) . in one study in 2016 in switzerland regarding the aefi summery, it was found that 55% of the reports were not serious adverse events (24). in this study two cases were reported as fatal, both were reported for bcg vaccine as disseminated bacillus calmette-guerin infection but there was no sufficient evidence to accuse the vaccine product. in fact, fatal dissemination of bcg infection is very rare to occur (up to 0.000159%) and almost in severe immunocompromised individuals (25) and the estimated rate of severe reaction occurrence with bcg is 1 in 1000 to 1 in 50.000 given doses (2) . among life threatening conditions, 25% of reports belonged to measles vaccine due to anaphylaxis and anaphylactoid responses in the results of this study and it is known that measles vaccine causes anaphylaxis in 1 per 100,000-million administered doses (26). regarding causality assessment of aefi (table 12), the majority (95.98%) of aefis were of a1 classification which almost was for body temperature conditions and local adverse events. in causality count % a1 2031 95.98% b1 25 1.18% b2 23 1.09% a3 16 0.76% unclassified 15 0.71% a4 6 0.28% total 2116 100% iraqi j pharm sci, vol.28(2) 2019 adverse event following immunization 149 switzerland surveillance of aefi from 1991 – 2001, it was found that 21.3% of the reported aes were of very likely or certain causal relationship of which 86.6% related to local reactions (21). once vaccines are induce immune response by the vaccine antigen interaction with immune system which is essential in developing the immunity against the diseases, manifestation of mild adverse reaction is common such as redness, swelling at injection site. fever as well is considered as inflammatory response associated with the immune response (14). b1 classification as shown in table 13, was account for 1.18% of the aefi. in b1 the temporal relation is established but the evidence to cause the vaccine is insufficient. it actually may be a new vaccine event and details of this events must retained. with time, if similar reports recorded so it may aid in suggestion a new potential causal relation (14) . if there is a conflicting trends between inconsistency and consistency with causality present, the aefi will have b2 classification, despite of adequate information but it can not clearly classify the ae (14). in this study it was found that 1.09% of the aefi were classified as b2. in study performed in india regarding the causality assessment of aefi it was found that b1 and b2 classifications accounted for 1% and 7% respectively among 1037 reports (27). the a3 causality was seen in mostly for bcg vaccine, which counted for 75% for all a3 cases. it was reported and known that bcg vaccine cause a local reaction in 90-95% of patients, incorrect site of injection also leads to local reaction and abscess if bcg given by sc route, the incorrect injection site that lead to abscess for example ,was classified as a3 causality which refers to immunization error (2). in a study conducted in india for aefi, immunization error related was count for 13% of the reports and classified as a3 in one indian study (27). six cases (0.28%) were classified as a4 causality as they were mostly a psychological event. upon administering of tetanus–diphtheria vaccine in 1998 in jordan, 122 individual admitted to the hospital as they felt chest tightness, feeling faint, and dizziness and this accident was reported to be associated with mass psychogenic illness (28). adolescents, especially if immunized in mass clinical settings, are more prone to have anxiety related vasovagal reactions resulting in fainting (29). in a study conducted in india for aefi, it was found that 17% of reports related to a4 classification (anxiety related) (27). the current study is not without limitations, lack of some information in the reporting of adverse events due to absence of understanding or careless filling the data makes the assessment of severity and seriousness of the cases difficult as well as difficult or impossibility of tracking the machines that associated with the adverse events. conclusion and recommendations in general, the administered vaccines in iraq were safe and the majority of them were mild and nonserious adverse events. further investigation is required regarding the causality for unclassified category, some adverse events that reported and classified as b1 recommended to be tracked as they might be new adverse events of vaccines. it may be of benefit to share these results with the media in order to increase the trust of the public regarding the immunization programs. references 1. stanley a.,vaccine fact book. phrma,usa,2013;1-102. 2. molly mort, adele baleta, frank destefano,et al. ,vaccine safety basics learning manual, who press , switzerland , 201, 1-207. 3. maurice r. ,robert e, eugene b., et al., live , attenuated mumps virus vaccine , the new england journal of medicine, 1967,319;252 available from: http:// dx. doi. org /10. 1016 /s0140 -6736(18)31132 4. rie s. kallerup , camilla foged, subunit vaccine delivery, advances in delivery science and technology chapter 7 , springer ,2015 ,15-29 . available from: http: // link .springer .com /10.1007/978-1-4939-1417-3 5. palihawadana p, peiris s, amarasinghe a, et al. immunization handbook , third edition , epidemiology unit ministry of health ,sri lanka,2012,1-295 , available from: http://www.epid.gov.lk/web/images/stories/im munization_guide_2012.pdf 6. council for international organizations of medical sciences & who, definition and application of terms for vaccine pharmacovigilance: report of cioms/who working group on vaccine pharmacovigilance, cioms publications, geneva,2012 , 1-195 7. s. olsson , the role of the who programme on international drug monitoring in coordinating worldwide drug safety efforts, drug safety,august 1998, 19(1) ,1-10. 8. adverse drug event monitoring/pharmacovigilance guideline , food, medicine and healthcare administration and control authority,third edition , ethiopia, 2014, 1-37 9. n arthur , a bentsi-enchill , m r couper , et al .,the importance of pharmacovigilance, who publications,uk , 2002,1-52;1–38. available from: http:// doi .wiley .com /10 .1002 /0470853093 10. who emro,| expanded programme on immunization (epi) programmes , iraq [internet]. cited 2019 apr 4. available from: http: // www . emro .who . int / irq /programmes / expanded-programme-onhttp://www.emro.who.int/ iraqi j pharm sci, vol.28(2) 2019 adverse event following immunization 150 immunization-epi.html 11. vigiflow user guide, uppsala monitoring centre, sweden ,2014 ,1-120 12. steven c. hartwig , siegel j., philip j, preventability and severity assessment in reporting adverse drug reactions, american journal of hospital pharmacy,49:2229-32, october 1992. 13. maytham hadi , guidelines for the iraqi pharmacovigilance system ( iphvc ), ministery of health , 2012;1–34. 14. causality assessment of an adverse event following immunization (aefi): user manual for the revised who classification,second edition, who, switzerland , 2018. 1–62 p. available from: http://www.who.int/vaccine_safety/publication s/aefi_manual.pdf?ua=1 15. lei j, ram m, gidudu jf, et al., use of a new global indicator for vaccine safety surveillance and trends in adverse events following immunization reporting 2000 – 2015,vaccine ,elsevier, (36) 1577-1582 . available from: https://doi.org/10.1016/j.vaccine.2018.02.012 16. manal yonus , iraq aleap to safety ,uppsala report ,sweden, 2011;(january),52,1-24 17. awaidy s al, bawikar s, prakash kp, et al. ,. surveillance of adverse events following immunization : 10 years ’ experience in oman, eastern mediterranean health journal 2010;16(5),474-480 18. halsey na, combination vaccines: defining and addressing current safety concerns. clin infect dis , 33:312–8 [cited 2019 may 10];. available from: http://www.ncbi.nlm.nih.gov/pubmed/1170976 5 19. decker m, principles of pediatric combination vaccines and practical issues related to use in clinical practice. pediatr infect dis j , 2001, 20(11 suppl):10-8 [cited 2019 may 10];. available from: http:// www. ncbi .nlm .nih. gov /pubmed/11704718 20. elliman d, bedford h. safety and efficacy of combination vaccines. bmj . 2003 326(7397):995–6 . available from: http: // www . ncbi.nlm.nih.gov/pubmed/12742896 21. schumacher z, bourquin c, heininger u, surveillance for adverse events following immunization (aefi) in switzerland—1991– 2001. vaccine ,elsvier, 2010 ,28(24):4059–64. available from: https:// www .sciencedirect .com /science/article/pii/s0264410x10004950 22. surveillance for safety after immunization: vaccine adverse event reporting system (vaers) , cdc,united states, 1991-2001 [internet]. [cited 2018 sep 13]. available from: https://www.cdc.gov/mmwr/preview/mmwrh tml/ss5201a1.htm 23. english o. supplementary information on vaccine safety part 2 : background rates of adverse events following immunization, who, 2000,1-112 24. swissmedic pharmacovigilance unit , summary of adverse events following immunization reported in switzerland during 2016. 2016;1–9. 25. world health organization, bcg vaccine. who position paper. wkly epidemiol rec. 2004 jan 23;79(4):27-38. available from: http: //www .ncbi. nlm .nih .gov /pubmed /14768305 26. duclos p, ward bj. measles vaccines. drug saf , 1998 ,19(6):435–54. available from: http: //www .ncbi.nlm. nih.gov /pubmed /9880088 27. singh ak, wagner al, joshi j, et al. expert review of vaccines causality assessment of serious and severe adverse events following immunization in india : a 4-year practical experience. expert rev vaccines , 2018;00(00):1–8. available from: https://doi.org/10.1080 /14760584. 2018. 1484285 28. s. kharabsheh, haidar al-otoum, j.clements, et al ., mass psychogenic illness following tetanus-diphtheria toxoid vaccination in jordan, bulletin of the world health organization ,2001, 79,764-770. available from: https: //www .ncbi .nlm .nih .gov /pubmed /11545334 29. cdc,syncope after vaccination , cdc,united states, january 20052007, 2008 / 57 (17) ; 457 460 web site cited 2019 may 10. available from: https: //www .cdc. gov /mmwr /preview /mmwrhtml /mm5717a2.htm. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://www.ncbi.nlm.nih.gov/pubmed/14768305 https://doi.org/10.1080%20/14760584 https://www.ncbi.nlm.nih.gov/pubmed/11545334 https://www.ncbi.nlm.nih.gov/pubmed/11545334 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 naproxen liquid suppository 31 formulation and in vitro evaluation of in-situ gelling liquid suppositories for naproxen noor n. al-wiswasi ** , eman b.h. al-khedairy* , 1 * department of pharmaceutics, college of pharmacy, university of baghdad , baghdad ,iraq ** abi-ghraib general hospital, ministry of health , baghdad ,iraq abstract in-situ gelation is a process of gel formation at the site of application, in which a drug product formulation that exists as a liquid has been transformed into a gel upon contact with body fluids. as a drug delivery agent, the in-situ gel has an advantage of providing sustained release of the drug agent. in-situ gelling liquid suppositories using poloxamer 188 (26-30% w/w) as a suppository base with 10% w/w naproxen were prepared, the gelation temperature of these preparations were measured and they were all above the physiological temperature. additives such as polyvinylpyrrolidin "pvp" ,hydroxylpropylmethylcellulose "hpmc", sodium alginate and sodium chloride were used in concentration ranging from (0.25-1%w/w) to modulate the gelation temperature and gel strength .the best preparation was obtained through using a combination of poloxamer 188, sodium alginate, naproxen and distilled water (29,0.5,10and 60.5 % w/w respectively)with gelation temperature of 33.6ºc±0.2 and gel strength of 28±2 seconds. the release of drug from this preparation was sustained for about 12 hours and it was faster than conventional solid suppository (proxen® 500) and oral tablets (naproxen®500) using dialysis tubing method. key words: naproxen, in-situ gelation, liquid suppository, poloxamer188 ةصالخلا رلإض ءئاإمعثافيإمع متمإعء يثاذلإمعتلم مإمعإإا عإومإيف مإمهلثل ايإتثذ اإرضحاسإمع يثاذلإ لوةإلا ةإلمع نإ اثافيإمعإإا عإضاتإت الرإيمإلفم ةإمع يلإإلياإوفم تإا إمعل ءئاإر ماإتياضتإضءإإتالسإمعتلمسإ لوةإىفاةإمحيت .ع عهإتلإتاذئلإ %ل ل /ل ل( م اةدإملالئاإعثاذئلإ٣٠-٢٦)١٨٨معثاايئةإمعيا ءاإمع ف لئاإمعثافيإمعإإا عإإعءاا للميئاإ الثل ايإمعافعفميايلإ %ل ل /ل لإإياإمعاا للميئاإلضاتإلئااإةسواإ لمسدإمعثافيإمعإإا عإعم إمع يثاذلمبإلوتإم ماإوف إةسواإ١٠معثاايئةإيمإ لمسدإمع يلإمعجلافعفوئا .لعثدئئلإةسواإ لمسدإللفدإمعثافيإمعإإا عإعم إمعثاايئةإتلإرلثيتمعإيفمةإر اوئاإياةإ فعمإوائةإ %ل ل/ل ل( ٠,١ -٢٥,٠ االلعتللإدإائتسلميمإ لل ئةإيائةإلءئءف دإمع ائةإمعبفةافعإلمءفساتإمعبفةافعإ ثلممئلتثلمللإ ئاإ) ٠ ,٥إدإ٢٩دإمع ائةإمعبفةافعإدإمءفساتإمعبفةافعإلإياسإيلحلإ)١٨٨.لرلإموذةإييثاذلإتلإمعابفيإضءئرإياإيلحإمعافعفميايلإ إيا ئا .لعلتإميملبإ٢ ±٢٨عإإل لفدإº ٢,٠ ± ٣٣, ٦ % ل ل /ل لإ اعثلال%إ( تسواإ لمسدإتافيإمعإإا عإ٥,٦٠إدإإإ١٠ , معتسملاإمعياسوئاإعءثالسإلمعثمإمولاةإإ ولثيتمعإلاادإمع ئلإمعدلا مإ ةلإتالسإمعتلمسإياإا مإمع يثاذلإمالإىفاةإمحيتإ تدإ لاضاإتللاااإم اإلر رإمللاإياإمعثاايئةإمعبءااإمعثلءئتااإلمعاافيإمعج فاا .١٢ introduction naproxen is a non steroidal antiinflammatory drug used for painful and inflammatory rheumatic arthritis, osteoarthritis, non articular rheumatism, in acute injury, migraine and tension headache, postoperative pain and pain associated with gynecological procedures.(1) its main adverse effects are gastrointestinal, of which peptic ulcer, with or without bleeding is the most sever effect. in addition esophageal ulceration may rise due to incorrect consumption. therefore rectal delivery has been explored as a potential method of avoiding gastric irritation that may occur when this drug is administered orally. (2) the ideal suppository would be easy to administer without any pain during insertion and would remain at the administered site avoiding the first-pass effect in the liver and the gastrointestinal tract. several problems are associated with the solid suppositories such as giving the felling of alien, discomfort and refusal to the patient with the possibility of lowering patient compliance. furthermore, solid suppositories might reach the end of the colon allowing the carried drug to undergo the first-pass effect. (3) to solve these problems, there have been several attempts to develop suppositories which exist as liquid at room temperature but gels at physiological one so they are easy to administer to the anus. (4, 5) suitable gel strength with suitable bioadhesive force is required in liquid suppositories so as not to leak out from the anus after administration and not to reach the end of the colon (6, 7) 1 corresponding author : e-mail : emanalkhedairy@yahoo.com received : 3 /3 / 2008 accepted : 11 /6 / 2008 iraqi j.pharm.sci., vol.17 (1) ,2008 naproxen liquid suppository 32 poloxamer series is a group of non ionic surface active compounds of polyoxyethylenepolyoxypropylene-polyoxyethylene block copolymer .(8) it is the most common base of liquid suppositories in which their solutions were known to exhibit the phenomenon of reverse thermal gelation; remaining as a solution at low temperature and gelling when the temperature increases. by modulating the gelation temperature of different poloxamer solutions, liquid base can be formulated which form a gel in the rectum at body temperature with suitable gel strength (4, 9, 10) furthermore, poloxamers were reported not to cause any damage on mucosal membrane. (11) this study was carried out to prepare an acceptable in-situ gelling liquid suppository for naproxen through studying different variables affecting the physicochemical properties and the in vitro release of the drug from this preparation. materials and instruments materials naproxen, hydroxypropylmethylcellulose (hpmc) supplied by samara drug industry (sdi), poloxamer 188( basf, united pharmaceuticals, jordan), polyvinylpyrrolidone (pvp), disodium hydrogen phosphate , potassium dihydrogen phosphate (bdh chemicals, ltd, liverpool, england ) , sodium alginate (hopkins and williams, ltd, england), sodium chloride ( evans medical , ltd, liverpool, england) dialysis tubing 36/32 with clips (medicell international ltd, liverpool, england), proxen®500 suppositories (grünenthal pharma ag, suisse), naprox®500 tablets (medical bahri, syria) instruments hot plate with magnetic stirrer (ika®weke, copley,mbh and co.kg, d-7921, germany), manually modified gardener cantype mobilometer , sartorius balance (werkegmbh, type 2842, germany),ph-meter (hanna instruments ph 211 microprocessor, italy), usp dissolution apparatus, type ii (copley scientific ltd, england), uv.visible spectrophotometer (carrywin uv.varian,100i spectrum, australia), water bath (mammert, germany) method preparation of liquid suppository naproxen and various amounts of excipient, except the liquid suppository base "poloxamer 188", were completely dispersed in the specified amount of distilled water with continuous agitation at room temperature and cooled down to 4°c. poloxamer 188 was then slowly added to the solution with continuous agitation. the liquid suppository was left at 4°c until a clear solution was obtained. (4) several preparations were prepared according to the factors that affect the physicochemical properties of the resultant liquid suppositories in order to get an acceptable formula. evaluation of in-situ gelling liquid suppositories: 1. gelation temperature: a 20-ml transparent vial containing a magnetic bar and 10 g of the liquid suppository was placed in a low-temperature thermostat water bath.a digital themosensor connected to a thermositor was immersed in the liquid suppository. the liquid suppository was heated at a constant stirring "100 (rpm)".when the magnetic bar stopped moving due to gelation, the temperature displayed on the thermistor was determined as the gelation temperature. (4) only those liquid suppository preparations that pass the gelation temperature test were subjected to the next tests. 2. gel strength: the gel strength of the liquid suppositories was measured using manually modified gardener can type mobilometer (figure 1 ).(12) a 50 g of the liquid suppository was transferred to a 100 ml graduated cylinder, and the cylinder was kept at 36.5°c for 30 minute in a water bath.a "35 g" weight of an appropriate size was then placed on the surface of the liquid suppository in the cylinder, and the time in second(s) required for this disc to move 5 cm down through the gelled suppository was measured and taken as an arbitrary index of gel strength. (13) only those liquid suppository preparations that pass the gel strength test were subjected to the next test. figure 1.(11): gel-strength measuring device (a): weight, (b): device, (c): mess cylinder , (d): liquid iraqi j.pharm.sci., vol.17 (1) ,2008 naproxen liquid suppository 33 3. dissolution test: to perform the in vitro release test for liquid suppositories a semipermeable membrane "dialysis tubing 36/32"(14) of 7 cm in length with clips (15) was used. a 5 g weight of the liquid suppository containing 500 mg naproxen was inserted into the semipermeable membrane tubing and both sides of the tube were closed with the clips to prevent leakage. the semipermeable membrane tube was then placed in a dissolution tester. drug release test was performed at 36.5°c using the usp dissolution apparatus type ii at 100 rpm with 500 ml sorensen's phosphate buffer ph 6.8 as a dissolution medium.at one hour intervals, 5 ml of the dissolution medium was sampled and filtered. the volume inside the jar was kept constant by addition of an equal volume"5 ml" of the same buffer solution. (6, 9) the filtrate was analyzed spectrophotome trically for naproxen content at 331 nm. factors affecting the physicochemical properties of the in-situ gelling liquid suppositories 1. effect of poloxamer 188 concentration different concentration of poloxamer 188 ranging from 26-30%w/w (4) were used to prepare a plain liquid suppository, and the effect of poloxamer 188 concentration on the gelation temperature and gel strength was studied. 2. effect of addition of naproxen naproxen in a dose of 500 mg (10% w/w), the usual rectal dose, was added to the different concentrations of poloxamer 188 solutions to study its effect on the physicochemical properties of the resultant medicated liquid suppository. 3. effect of addition of additives different additives including "pvp", "hpmc", sodium alginate and sodium chloride were added in different concentrations ranging from (0.25-1.0% w/w)(4,9,10) to the different concentrations of poloxamer solutions and their effect on the gelation temperature, gel strength and dissolution behavior of the resultant liquid suppositories were then studied. factors affecting the in vitro release of naproxen 1. effect of formulation components to study the effect of the liquid suppository components; "the base and the additives", on the naproxen release profile, an in vitro release of the drug powder was performed and compared with that from the liquid suppository. a 500 mg naproxen powder was inserted into the semipermeable membrane tubing and then the release test was performed as mentioned previously. 2. effect of dosage forms to study the effect of the dosage forms on the in vitro release of naproxen a comparison was done between the liquid suppository and other dosage forms. a commercial solid suppository (proxen®500) or commercial oral tablet (naprox®500) containing 500mg naproxen were inserted into the semipermeable membrane and then the dissolution test was performed as mentioned previously. the release profile of these dosage forms were then compared with that of the liquid suppository. statistical analysis results are given as a mean ± s.d for triplicate samples. the results were statistically analyzed by using analysis of variance (anova) table and t-test, p-values less than 0.05 was considered significant results and discussion physicochemical properties of the in-situ gelling liquid suppositories the temperature dependent gelation of poloxamer solutions could be explained to be due to desolvation of hydrophilic chains of the polymer as a result of the breakage of the hydrogen bonds that have been established between the solvent and these chains. this phenomenon favors hydrophobic interaction among the hydrophobic chains of the polymer(16) and the polymer self-assemble spontaneously, forming micelles. (17, 18) raising the temperature of poloxamer solutions will be accompanied by an increase in the micellar aggregation number and a decrease of a critical micelle concentration allowing the formation of a more closely packed and more viscous gel. (19) an accepted liquid suppository must have a gelation temperature in the range of (30-36°c) and gel strength of (10-50 seconds), so as to be in a liquid form at room temperature and to form a gel phase instantly in the rectum without leakage. (4, 9, 10) factors affecting the physicochemical properties of the in-situ gelling liquid suppositories 1. effect of poloxamer 188 concentration table (1) shows the effect of poloxamer 188 concentration on the gelation temperature of the prepared liquid suppositories. the results indicated that increasing the poloxamer 188 concentration from 26 % to 30 % w/w was accompanied with a decrease in the gelation temperature. these results were in agreement with those reported by choi.h.g. et al. (4) the increment in poloxamer 188 concentration led to an apparent dramatic iraqi j.pharm.sci., vol.17 (1) ,2008 naproxen liquid suppository 34 increase of micellar size and polydispersibility which could be the reason for such reduction in the gelation temperature . it was suggested that such changes were a consequence of interactions between polyoxyethylene chains of adjacent micelles which, as a result of their dehydration, experience increased friction with a resulting tendency to form multimolecular units leading eventually to gel formation (20) table (1): gelation temperature of different naproxen suppositories concentration of poloxamer 188 % w/w mean gelation temperature °c± s.d of poloxamer solutions plain medicated 26% >50 45. 6 ± 0.1 27% >50 43.1 ± 0.2 28% >50 40.9 ± 0.2 29% >50 37.7 ± 0.2 30% 48.0±0.1 35.2 ± 0 2. effect of addition of naproxen the incorporation of 10 % w/w (500 mg) of naproxen into the poloxamer 188 solutions of different concentrations decreased the gelation temperature of the resultant liquid suppositories as compared with the plain one, as shown in table (1). as a possible mechanism by which naproxen affected the gelation temperature of these preparations, it may be speculated that placing naproxen in the gel matrix would make its carboxyl group bonded strongly with the cross-linked reticular poloxamer gel through hydrogen bonding. this suggestion was based on the results obtained by el-kamel ah. who found that if hydrogen bonding is supplemented to the poloxamer solutions by adding compounds containing a hydrogen-bonding forming group, the gelation temperature will decrease.(21) 3. effect of addition of additives tables (2a-2e) show that the addition of any of the used additives pvp, hpmc, sodium alginate and sodium chloride, would lower the gelation temperature and reinforce the gel strength of the resultant liquid suppositories. the impact of additives on the gelation temperature and gel strength was found to be depending on their nature and concentration. increasing the concentration of any of the used additives from 0.25 to 1.0 % w/w produced a gradual decrease in the gelation temperature and increase in the gel strength of the corresponding liquid suppositories. the gelation temperature lowering and gel strength increasing effect of pvp, hpmc and sodium alginate could be explained by their ability to bind to the polyoxyethylene chains present in the poloxamer molecules through hydrogen bonds. this will promote dehydration causing an increase in entanglement of adjacent molecules which will lead to gelation at lower temperature and reinforce the gel strength. (5, 9, 22) on the other hand, the reduction in the gelation temperature with the increase in gel strength by addition of sodium chloride could be attributed to its salting-out effect which results in dehydration of the polyoxyethylene chains, causing an increase in the entanglement of adjacent molecules.(17) table (2): effect of addition of additives on the physicochemical propertie of liquid suppositories containing 10% w/w naproxen and different concentrations of poloxamer 188. (a): with 30% w/w poloxamer 188 types of additives conc.of additives mean gelation temperature (°c) ± s.d mean gel strength ( seconds) ±s.d no ––– 35.2 ± 0 102 ± 2.3 pvp 0.25 29.8 ± 0.1 /** 0.5 / / 0.75 / / 1.0 / / hpmc 0.25 34.7 ± 0.1 200 ± 1.8 0.5 33.1 ± 0.4 / 0.75 31.8 ± 0.4 / 1.0 30 ± 0.1 / sodium alginate 0.25 33.7 ± 0 146 ± 2.04 0.5 31.4 ± 0.1 / 0.75 29 ± 0.4 / 1.0 / / sodium chloride 0.25 34.2 ± 0.5 not fall* 0.5 32.4 ± 0.1 / 0.75 31.1 ± 0.1 / 1.0 30.2 ±0.3 / *not full up to 300seconds **not done iraqi j.pharm.sci., vol.17 (1) ,2008 naproxen liquid suppository 35 (b) : with 29% w/w poloxamer 188 *not full up to 300seconds **not done (c): 28% w/w poloxamer 188 type of additives conc. of additives mean gelation temperature (°c) ± s.d mean gel strength ( seconds) ± s.d no ––– 40.9 ± 0 /** pvp 0.25 34.1 ± 0.1 not fall* 0.5 30.6 ± 0.58 / 0.75 27.6 ± 0.3 / 1.0 / / hpmc 0.25 37.4 ± 0 / 0.5 / / 0.75 / / 1.0 / 253 ± 2.3 sodium alginate 0.25 37.7 ± 0.4 / 0.5 34.8 ±0.2 3 ± 1.96 0.75 33.9 ± 0 14 ± 2.03 1.0 33.6 ± 0.1 99 ± 1.43 sodium chloride 0.25 35.9 ± 0 95 ± 0.9 0.5 34.9 ± 0.3 / 0.75 33.7 ± 0.58 / 1.0 32 ± 0.2 / *not full up to 300seconds **not done (d): 27% w/w poloxamer 188 **not done (e): 26% w/w poloxamer 188 type of additives conc. of additives mean gelation temperature (°c) ± s.d mean gel strength ( seconds) ± s.d no ––– 45.6 ± .1 /** pvp 0.25 / / 0.5 33.4 ± 0.1 212 ± 1.53 0.75 29.8 ± 0 / 1.0 / / hpmc 0.25 / / 0.5 / / 0.75 / / 1.0 / / sodium alginate 0.25 / / 0.5 / / 0.75 35.9 ± 0.2 1 ± 1.63 1.0 35 ±0 2 ± 1.46 sodium chloride 0.25 / / 0.5 / / 0.75 36 ± 0 46 ± 1.32 1.0 34.2 ± 0.1 63 ± 2.61 **not done types of additives conc.of additives mean gelation temperature (°c) ± s.d mean gel strength ( seconds) ±s.d no ––– 37.7 ± 0.2 /** pvp 0.25 33.8 ± 0 not fall* 0.5 30.2 ± 0.1 / 0.75 27.1 ± 0.5 / 1.0 / / hpmc 0.25 37 ± 0.1 / 0.5 36.6 ± 0 / 0.75 36.3 ± 0 / 1.0 34.2 ± 0.1 253 ± 2.3 sodium alginate 0.25 36.8 ± 0 / 0.5 33.6 ±0.2 28 ± 2 0.75 30.5 ± 0 83 ± 3.72 1.0 27.7 ± 0.1 / sodium chloride 0.25 35.4 ± 0 150 ± 1.5 0.5 34 ± 0 / 0.75 32.7 ± 0.1 / 1.0 31 ± 0.1 / type of additives conc. of additives mean gelation temperature (°c) ± s.d mean gel strength ( seconds) ± s.d no ––– 43.1 ± 0.2 /** pvp 0.25 36.1 ±0.1 / 0.5 32.4 ± 0 348 ± 3.77 0.75 28.3 ± 0 / 1.0 / / hpmc 0.25 / / 0.5 / / 0.75 / / 1.0 / / sodium alginate 0.25 / / 0.5 37.2 ± 0 / 0.75 34.3 ±0 2 ± 4.25 1.0 34.6 ± 0 3 ± 2.86 sodium chloride 0.25 39.5 ±0 / 0.5 36.1 ± 0 / 0.75 35.2 ± 0.1 98 ± 1.79 1.0 33.5 ± 0.2 / iraqi j.pharm.sci., vol.17 (1) ,2008 naproxen liquid suppository 36 dissolution test three naproxen liquid suppositories passed the gelation temperature and gel strength tests (nomenclated as a, b and c) were subjected to the dissolution test. table (3) summarizes the constituents of each liquid suppository. in addition, these liquid suppositories (a, b and c) may have a mucoadhesive force that prevent the gelled suppositories from reaching the end of the colon, the pathway for the firstpass effect, which may be related to their ability to bind strongly to the oligosaccharide chains of the rectal mucous membrane through the hydrophilic groups of poloxamer and the additives.(4,6,9)the percentage of naproxen released from these preparations to the dissolution medium for 12 hours (23 , 24), was selected for the comparison study. in addition, the time required for 100 % release for the drug from these three preparations were also measured, table (4). it was found that there is no significant difference in the percentage of drug released among these three preparations, (p > 0.05), and all of them gave sustained drug release, since they released about 70 % of the drug within 12 hours as shown in figure (2) and table (4). the in-situ gelling liquid suppository (a), which had suitable gelation temperature and an intermediate gel strength was selected for further study, since it may has a lower chance to be leaked from the rectum and easier to be administered than the others. table (3): the constituents of the in-situ gelling liquid suppositories with their physicochemical properties name of the liquid suppository constituents of the liquid suppository physicochemical properties component conc. % w/w mean gelation temperature (°c)± s.d mean gel strength sec. ± s.d a poloxamer 188 29 33.6 ± 0.2 28 ± 2 sodium alginate 0.5 naproxen 10 distilled water 60.5 b poloxamer 188 28 33.9 ± 0 14 ± 2.03 sodium alginate 0.75 naproxen 10 distilled water 61.25 c poloxamer 188 26 36 ± 0 46 ± 1.32 sodium chloride 0.75 naproxen 10 distilled water 63.25 table (4): percentage released of naproxen from in-situ gelling liquid suppositories and the time required for 100% release liquid suppository %released for 12 hours time for 100 % release ( hours) a 66.64 20 b 72.86 17 c 69.73 21 figure 2. percentage of naproxen released from three different insitu gelling liquid suppositories in sorenson's phosphate buffer ph 6.8 at 36.5ºc using dialysis tubing method factors affecting the in vitro release of naproxen 1. effect of the formulation components to study the effect of the formulation components on the in vitro release of naproxen from the liquid suppositories, the percentage of drug release from the powder (500 mg) was compared with that from in-situ gelling liquid suppository (a), using the dialysis tubing method. the release profile for in-situ gelling liquid suppository (a) was similar to that for naproxen powder at the first five hours and it was significantly faster (p<0.05) after hour five as shown in figure (3). this could be attributed to the solubilizing effect of poloxamer 188, especially for the poorly water soluble drug. (8, 25) 0 20 40 60 80 0 2 4 6 8 10 12 time (hours) % n ap ro xe n r el ea se d fr om i nsi tu g el lin g l iq ui d su pp os ito ri es liquid suppository "a" liquid suppository "b" liquid suppository "c" iraqi j.pharm.sci., vol.17 (1) ,2008 naproxen liquid suppository 37 figure 3. effect of formulation components on the in vitro release of naproxen in sorenson's phosphate buffer ph 6.8 at 36.5ºc using dialysis tubing method 2. effect of the dosage form to study the effect of the dosage form on the in vitro release of naproxen, the percentage released of the drug from the liquid suppository (a), commercial solid suppository and commercial oral tablet were compared by using dialysis tubing method. as shown in figure (4), the in-situ gelling liquid suppository had the faster release, followed by the commercial oral tablet and finally the commercial solid suppository. these results indicated that the dosage form design would affect the in vitro release of naproxen. a statistical analysis were done and showed a significant differences (p<0.05) for the percentage released of naproxen among these three dosage forms.the faster release from the in-situ gelling liquid suppository (a) could be due to the fluidity of the liquid suppository (7,9), and the solubilizing effect of poloxamer 188 for the poorly water soluble drug.( 8, 25) figure 4. effect of dosage form on the in vitro release of naproxen in sorenson's phosphate buffer ph 6.8 at 36.5ºc using dialysis tubing method future work further study on the stability, mucoadhesive property, clinical and pharmacokinetic study on human subjects for naproxen –loaded poloxamer in-situ gelling liquid suppositories is to be done. acknowledgment the authors gratefully thank the college of pharmacy, university of baghdad for supporting this research. references 1. steven merahn: mosby medicinal drug reference.london, 2003, p.712-714. 2. khan l.h., et al. 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t., shimono n., shimono k., higaki k. and kimura t., "evaluation of sustained release suppository prepared with fatty base including solid fat with high melting points", int.j.pharm. , 2004, 278, 278-282. 24. hanning c. d., vickers a. p., smith g. and mcnell m. e., "the morphine hydrogel suppository. anew sustained release rectal preparation", british j. of anesthesia, 1988, 61, 221-227. 25. basf aktiengesellschaft, "lutrol® f68 (poloxamer 188 for the pharmaceutical industry), technical information, pharma solutions, 2005, memp,030737e-01, 1-4. iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films doi: https://doi.org/10.31351/vol28iss2pp83-94 83 formulation and in-vitro evaluation of darifenacin hydrobromide as buccal films ishraq k. abbas*,1, nawal a. rajab** and ahmed a. hussein** * faculty of pharmacy, al-rafidain university college, baghdad, iraq. *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract overactive bladder (oab) affects about 16% of the adults and rises with increasing age. oab lead to various symptoms such as urgency, incontinence, urinary frequency and nocturia. darifenacin hydrobromide (dh) is the more recent uroselective m3 receptor antagonist for treating uncomplicated oab. fast dissolving buccal films are the most innovative oral solid dosage form because of their flexibility and comfort. this study aims to formulate dh as fast dissolving buccal films (fdbfs) using a solvent casting method to increase its bioavailability by reducing the effect of first pass metabolism in the liver. films were prepared by using polyvinyl alcohol as a film forming polymer, glycerol and tween 80. different types and concentrations of superdisintegrants (croscarmellose sodium, sodium starch glycolate, indion 414) were used to select the best formula by studying the physicochemical properties of the films, disintegration time (dt) and percent drug release. the results revealed that formula (f9) that containing 7.5mg dh, 2%w/v pva, 30%w/w glycerol, 0.5%w/v tween 80, 4%w/w indion 414 was the preferred formula. f9 showed the shortest in-vitro disintegration time (31.28sec). in-vitro dissolution profile showed the lowest t80% of the drug in 3.05 min and the highest release of the drug (94%) within 5 min (d5min %). it was concluded that the fdbfs of dh could be considered as a promising drug delivery system with an enhanced disintegration and dissolution rate and better patient compliance. keywords: darifenacin hydrobromide, overactive bladder, fast dissolving buccal films. داريفيناسين كشرائح فمويةاللهيدروبروميد صياغة وتقييم خارج الجسم ***عباس حسينأحمد و **نوال عيّاش رجب، 1،*إشراق كاظم عباس الرافدين الجامعة ، بغداد ، العراق . كليةكلية الصيدلة ، * .عة بغداد، العراقمفرع الصيدالنيات، كلية الصيدلة، جا * الخالصة تؤدي إلى أعراض مختلفة oab من البالغين وترتفع هذه النسبة مع تقدم العمر. ٪٦١( تؤثر على حوالي oabالمثانة المفرطة النشاط ) هو أحدث مضادات مستقبالت اليوريا االنتقائية (dh)مثل الحاح التبول وسلس البول وتكرار التبول والتبول الليلي. هايدروبرومايد الداريفيناسين m3 لعالجoab .غير المعقد ة إلى تهدف هذه الدراس ق الفم بسبب مرونتها وراحتها. الشرائح الفموية سريعة الذوبان هي الشكل األحدث ابتكارا للجرع الصلبة عن طري زيادة التوافر الحيوي للدواء عن طريق تقليل التمثيل الغذائي االولي فيكشرائح فموية سريعة الذوبان باستخدام طريقة صب المذيبات ل dhصياغة تم استخدام أنواع وتراكيز مختلفة من المفتتات الفائقة ) . 08 تم تحضير الشرائح باستخدام بوليمر كحول البولي فينيل والجلسرين وتوين .الكبد ( لتحديد أفضل صيغة من خالل دراسة الخصائص الفيزيائية والكيميائية لألفالم indion 414كروسكارميلوز الصوديوم ، جليكوالت نشا الصوديوم ، ، ووقت التفكك والنسبة المئوية لتحرر الدواء. ٪٤، و 08من توين ٪8،٧من الجلسرين ، و ٪٠8 و pvaمن ٪٢و dhملغ من ٥،٧التي تحتوي على f9ة أظهرت النتائج أن الصيغ indion 414 أظهرت هي الصيغة المفضلة. و كانتf9 ( ٠٦،٢0أقصر وقت تفكك خارج الجسم .)أظهر تحلل الدواء خارج الجسم أدنى ثانية (.٪d5minدقائق ) ٧( في غضون ٪٤٤يقة وأعلى إطالق للدواء )دق ٠،8٧( في t80٪من الدواء ) ٪08وقت الطالق لمعدل التفكك ولنسبة الفموية سريعة الذوبان يمكن اعتبارها كنظام تحرير أدوية واعد مع تحفيز dhيمكن االستنتاج بان أن شرائح التحلل باالضافة الى تحسين امتثال المرضى. هايدروبرومايد ، فرط نشاط المثانة ، أفالم فموية سريعة الذوبان.الكلمات المفتاحية: داريفيناسين introduction in the past few decades, development and imagination have ended up basic competence for making progress in the development of novel dosage forms. but, every dosage form has shown some drawbacks like pain of the parenteral dosage form and chocking of tablets and capsules (1). the oral solid dosage forms are characterized by ease of administration and exact dosing, but it is sometimes problematic because of decline in bioavailability, long onset time of action and swallowing difficulty especially for pediatric and geriatric (2).most of pharmaceutical manufacturers have focused in their researches on developing new dosage form to achieve patient convenience. among many drug delivery systems, the fast dissolving films are incredibly prominent among pediatrics and 1corresponding author e-mail:ishraqalmualla@yahoo.com received: 15/ 5 /2019 accepted: 17/7 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp83-94 iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 84 geriatrics and have started gaining big popularity for their rapid disintegration or dissolution, selfadministration even without water or chewing (3). the oral cavity is a preferred site for drug administration because of easily drug application by patient and avoidance of possible drug degradation in the git in addition to bypassing first-pass hepatic metabolism (1). oral mucosa is the lining of the oral cavity. the membrane of mucosa cells is hydrophobic in nature and might be selective for hydrophobic molecules only, while the hydrophilic molecules pass the oral mucosal membrane through the para-cellular pathway since the cytoplasm and intercellular spaces are hydrophilic in nature. after absorption, the drug is transported through facial vein, which then drains into general circulation, bypassing the liver, therefore warn off drug from first pass metabolism (4). overactive bladder (oab) is a common and distressing disorder characterized by incontinence, urinary frequency and nocturia. the symptoms of oab have been attributed to involuntary contractions of the bladder muscle. this supports the rationale for using a drug that antagonizes the m3mediated parasympathetic excitation of the detrusor smooth muscle contraction (5). darifenacin hydrobromide (dh) is the first m3-selective receptor antagonist. it has a higher selectivity for muscarinic receptors of the bladder. it was approved by fda to be used for the symptomatic treatment of oab in 2004 (6). dh molecular weight is 507.472 g/mol. it is slightly soluble in water (6.03mg/ml). its melting point is 228-236 0c (7). it is subjected to extensive first-pass metabolism after oral administration. the absolute bioavailability of dh from 7.5mg and 15mg prolonged release tablet was estimated to be 15.4 % and 18.6% respectively (8). this study is aimed to prepare dh as fast dissolving buccal films (fdbfs) to enhance patient’s compliance. materials and methods materials dh supplied by hyperchemical comp., china. polyvinyl alcohol (avonchem, uk), glycerol (fluka chemi ag, switzerland), tween 80 (sd fine-chem. limited, mumbai, india), croscarmellose sodium and sodium starch glycolate (hyperchemical comp. china), indion 414 (ion exchange india ltd, mumbai). methods preparation method of darifenacin hydrobromide fast dissolving buccal films solvent casting technique was used in the preparation of dh fdbfs by using 9 cm diameter petri dishes.2%w/v of pva solution was prepared by dissolving the polymer in deionized water with constant stirring (120rpm) by magnetic stirrer to form a homogenous polymeric solution. to this polymer solution, excipients were added separately. dh was dissolved, in 30 ml absolute ethanol at 60 0c with stirring to form a clear solution. then the two mediums were mixed together using magnetic stirrer. the mixture (63ml) was set aside to ensure air bubbles removal, then casted in a petri dish (9 cm diameter) and kept in an oven at 50℃ overnight. the dried film was then removed and cut into (2cm×2cm) films (9). nine formulas (f1-f9) were prepared using different superdisintegrants types {croscarmellose sodium (ccs), sodium starch glycolate (ssg) and indion 414} and concentrations (2%, 3% and 4%) to select the best formula (table 1). evaluation of darifenacin hydrobromide fast dissolving buccal films: physical appearance of the prepared films physical appearance was evaluated by visual inspection for homogenisity, clearness and surface texture. weight variation it was done by weighing ten films individually, and average weights were calculated. the accepted film weight should not significantly deviate from weight average (10). thickness of the films it was measured by using a standard vernier caliper. six films were measured at five positions, and average thickness was calculated (11). drug content uniformity three films were placed in 100 ml phosphate buffer (ph6.8) solutions individually and kept on a magnetic stirrer for 30min. the solution was filtered by filter syringe (0.45μm), and dh amount was measured spectrophotometrically (12). folding endurance (fe) fe was determined by manual repeated folding of film at specified site until it breaks or cracks, and average values were calculated and reported. fe of more than 300 gives a good idea about the flexibility and toughness of the formulation (11). in-vitro disintegration time (dt) it was measured visually by using a petri dish method. the test was done by taking three films of each patch in a petri dish (5cm diameter) separately, and after adding 2 ml of deionized water for each film, the petri dish was shaken smoothly and continuously, then measure the time at which the fdbfs start to break down or disintegrate (11). determination of surface ph this is essential to investigate the opportunity of side effects when using the films invivo, because sharp acidic or basic ph may irritate the mucosal membrane of the oral cavity. to measure the ph value; three films were allowed to dissolve in 2 ml of deionized water separately, and the ph of the obtained solution is determined using ph-meter (11). iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 85 table 1. composition of darifenacin hydrobromide fast dissolving buccal films (4cm2) f o r m u la n o . d a r if e n a c in h y d r o b r o m id e (m g /f il m ) p o ly m e r (m g /f il m ) p la st ic iz e r m g /f il m s u r fa c ta n t (m g /f il m ) s u p e r d is in te g r a n t m g /f il m f la v o r in g a g e n t (m g ) s w e e te n in g a g e n t (m g /f il m ) s a li v a st im u la n t (m g /f il m ) p v a g ly c e r o l t w e e n 8 0 c r o sc a r m e ll o se s o d iu m s o d . s ta r c h g ly c o la te in d io n 4 1 4 v a n il la s o d . sa c c h a r in c it r ic a c id f 1 7.5 41 12 10 1.6 2.5 2.5 2.5 f 2 7.5 41 12 10 2.3 2.5 2.5 2.5 f 3 7.5 41 12 10 3.1 2.5 2.5 2.5 f 4 7.5 41 12 10 1.6 2.5 2.5 2.5 f 5 7.5 41 12 10 2.3 2.5 2.5 2.5 f 6 7.5 41 12 10 3.1 2.5 2.5 2.5 f 7 7.5 41 12 10 1. 6 2.5 2.5 2.5 f 8 7.5 41 12 10 2. 3 2.5 2.5 2.5 f 9 7.5 41 12 10 3. 1 2.5 2.5 2.5 in-vitro dissolution stud it was done by using usp type ii (paddle apparatus) with 300 ml of phosphate buffer (ph 6.8). the medium was adjusted to rotate at 50 rpm, and the temperature was maintained at 37±0.5ºc. 5 ml samples were taken by using a syringe at regular intervals (1, 2, 3, 4, 5, 10, 15 and 20min) replaced with 5ml buffer medium to maintain sink condition. then before analyzing the withdrawn samples using uv-spectrophotometry at dh λmax, they were filtered by filter syringe (0.45 μm). then the dissolution profile of the drug is constructed by plotting the percent of accumulative drug release against the time (13). t80%(min) which is the time needed to release 80% of the drug and d5min(%) which represent the percentage of drug released in 5 min was used to study the effect of different factors on the dissolution profile (14). effect of superdisintegrant type and concentration three formulas (f1, f2 and f3) were developed to illustrate ccs effect at different concentrations (2%, 3% and 4%w/w of total dry constituents of the film) on the mechanical, physical properties and dissolution profile of dh films. ssg with 2%, 3% and 4% concentrations used to study its effect on f4, f5 and f6. the last superdisintegrant used was indion 414, also with same concentrations in f7, f8 and f9. characterization of selected formula: percentage moisture loss (pml) this was done to determine physical stability of films. it was detected by putting three accurately weighed films in a desiccator that containing silica gel beads. after 72 hours; films were taken and weighed again (9). pml= (initial weight-final weight)*100/initial weight percentage moisture absorption (pma) pma was used to check the physical stability of films at high humid condition. it was identified by keeping three accurately weighed films in a desiccator containing a saturated solution of calcium chloride, at 25℃ for 72 hours, then after three days, films were reweighed. pml= (final weight-initial weight)*100/initial weight. evaluation of mechanical property of the films: tensile strength (ts) it is maximum force required for breakdown of the film. the film were cut around a standard 9×2 cm2 template (dumbbell) and put between two clamps of tensometer and pulled at a rate of 5 mm/min. ts was measured by mega pascal (mpa) (9). tensile strength= load at break (n)*100/width× thickness (mm2). iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 86 percent of elongation (%e) %e is the stretching and distortion of the film when a force is applied on it (11). %e= final length-initial length*100/initial length young's modulus (ym) it is defined as the measure of the stiffness of film. so rigid and fragile films exhibit high ts and ym with small %e (11). ym= slop*100/ strip thickness× cross‐head speed. drug-polymer compatibility study it was examined by fourier transform infrared spectroscopy (ftir), differential scanning calorimetric studies (dsc) and x-ray powder diffraction (xrpd). fourier transform infrared spectroscopy ftir confirmed the compatibility of the drug and the formulation. spectra of pure drug and formulations were determined by potassium bromide (kbr) disc method. the spectrum was recorded for the drug, polymer, 1:1 physical mixture of polymer and drug and the selected formula. samples and kbr were mixed and pressed in the form of a disc, then it was analyzed by ftir spectroscopy from 4000 400 cm-1(9). differential scanning calorimetric studies (dsc) dsc scans were done for the drug, polymer, 1:1 physical mixture of drug and polymer and for the selected film. a certain amount of the sample was introduced in a typical aluminum pans and increasing the temperature at a constant rate (10°c/min), at a temperature range of 8 °c up to 300 °c (9). x-ray powder diffraction (xrpd) xrpd study can be used to confirm the crystalline nature of materials. the diffractograms of dh, polymer and the selected formula were studied. the study was approved by using shimadzu xrd-6000 powder xrpd at continuous scan range of 5°60° of 2ɵ. the operating voltage was 40 (kv) and current 30 ma (11). stability studies stability study was performed by storing the selected dh formula films in an aluminum foil in sealed container and placed them in three ovens that maintained at three temperatures of 40ºc, 50ºc, and 60ºc for three months. then the films were withdrawn every 14 days and examined them for physical, mechanical properties and drug content (9). statistical analysis the results were demonstrated as a mean of triplicate models ± standard deviation (sd) and were tested by the one-way analysis of variance(anova) to determine if the changes in the applied factors are statistically significant (p≤0.05) or non-significant (p>0.05). results and discussion evaluation of darifenacin hydrobromide fast dissolving buccal films physical appearance all prepared films showed colorless, translucent, and homogenous smooth surface properties. weight variation the average weights were between 0.08±0.007 to 0.102±0.01 (table2). weight variations was due to variation in excipients concentration of each formula while low sd from the mean demonstrates that films were uniform. thickness of the films the average thickness was between 0.164±0.02 to 0.21±0.02 mm (table2). a very low sd indicates that the method used for the formulation was applicable. drug content uniformity the formulated dh films showed a drug content ranged from 90.8%±2 to 102%±0.35 (table2). the accepted range of content uniformity labeled in the united states pharmacopeia (usp) is varied from 90% to 110% (25). on this basis, it was concluded that dh was distributed uniformly throughout the films. folding endurance (fe) all formulations resisted cracking for more than 300 folding times (table2). this result is consistent with research was done by londhe and umalkar in 2012 (15). in-vitro disintegration time dt was studied in triplicate, and results were ranged between 31.28±1.45-44.97±3 secs (table2). determination of surface ph measurement of prepared films showed an acceptable surface ph value (4.1±0.06-5.1±0.28) (table2). since films surface ph did not deviate sharply from the neutral ph, there will not be any irritation to the mucosal lining of the oral cavity. iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 87 table 2. physical evaluation parameters of darifenacin hydrobromide fast dissolving buccal films (4cm2). f no. weight variation n= 10 thickness (mm) n=6 drug content (%) n=3 folding endurance n=3 in vitro dt(sec) n=3 surface ph n=3 f 1 0.08 ± 0.007 0.18 ± 0.009 99 ± 0.007 > 300 39.3±0.95 4.4 ± 0.06 f 2 0.09 ± 0.09 0.19 ± 0.01 98.1 ± 0.04 > 300 37.3 ± 2.5 4.2 ± 0.06 f 3 0.102 ± 0.01 0.21 ± 0.02 90.8 ± 2 > 300 35.73 ±0.8 4.1 ± 0.06 f 4 0.095 ± 0.03 0.164 ± 0.02 97.44 ± 0.04 > 300 44.97 ± 3 4.2 ± 0.15 f 5 0.1 ± 0.01 0.175 ± 0.02 102 ± 0.35 > 300 43.33 ± 3 4.5 ± 0.0 f 6 0.17 ± 0.01 0.195 ± 0.02 96.9 ± 1 > 300 40.59± 4.35 4.1 ± 0.2 f 7 0.09 ± 0.008 0.17 ± 0.01 95.36 ± 0.04 > 300 38.88 ± 3.2 5.1 ± 0.28 f 8 0.095 ± 0.01 0.18 ± 0.02 98.1 ± 2 > 300 37.33 ± 1.15 4.8 ± 0.0 f 9 0.097 ± 0.01 0.195 ± 0.01 99 ± 0.1 > 300 31.28 ± 1.44 4.9 ± 0.05 mean ± sd effect of superdisintegrant superdisintegrants, are poor water solubility substances with functional hydration capacity that can facilitate disintegration with a smaller quantity of them either by swelling action, capillary (wicking) action or combination of both mechanisms (16). effect of superdisintegrant concentration all formulas (f1-f9) had acceptable mechanical properties with fe > 300 as seen in table 2. films of f1, f2 and f3 were prepared with 2%, 3% and 4%w/w of ccs respectively to study its effect on in-vitro dt and release profile of the prepared films. table3 and figure1 showed that films dt declined from 39.31sec to 37.3sec, and 35.73sec with increasing in ccs concentration from 2% to 3%, and to 4%, with a significant variation (p<0.05) between f1-f3, while there is no significance (p>0.05) between f1-f2 and f2-f3. there was significant changes (p<0.05) in the dissolution profiles between f1 and f3 in t80% and d5min (%) that was (8.9 min and 65%) for f1 and (4.3 min and 87.5%) for f3, whereas the difference was non-significant (p>0.05) between f1 and f2. there were also significant changes (p<0.05) between f2 and f3 (table4 and figure2). similar results noticed by by xu et al. (17). these results may be related to ccs wicking effect that provide pathways in the film just like capillaries in which liquid is drawn up and rupture the inter-particulate bonds causing the film to break apart (18). gissinger and stamm (19) reported that the disintegration and dissolution could not be only explained by the formation of the porous capillary network and other factors like swelling force are also essential for disintegration especially for ccs. films of f4, f5 and f6 were prepared with 2%, 3% and 4% of ssg. dt measurement revealed that f6 had lowest time (40.97sec) (table3 and figure1), but with no significance between the three formulas (p>0.05). decrease in dt by using 4% ssg was observed by sharma that prepared salbutamol sulfate fast disintegrating tablet with different concentration of ssg; 1%, 2%, 4%, 6%, 8% and 10% where the lowest disintegration time was by using 4% of ssg (20). table3 and figure2 showed that lowest t80% time and d5min% (4.31min, 85.6%) was in f6 with a non-significant difference (p>0.05) when compared with (8.63min, 53.57%) and (7.05min, 60.87%) of f4 and f5 respectively. these results correspond to research findings of haque and sheela (21). ssg effects may be due to swelling property of ssg that involves rapid absorption of water leading to an enormous increase in films volume that overcomes interconnection bond of other ingredients resulting in fast disintegration and fast release of drug (21). last type used was indion in 2%, 3% and 4%w/w concentrations of f7, f8 and f9 respectively. concerning dt; f9 showed 31.28sec with significant variation (p<0.05) when compared with f8 and f7 (37.33sec and 38.88sec) respectively (table3 and figure1). indion effects (table4 and figure2) reveals that f9 showed 3.05min t80% and 94% d5min% with significant difference (p<0.05) of both parameters when compared with f8 (5.3min, 77.8%) and f7 (7.4min, 68.71%). chandira et al., (2008) showed similar results (22). these effects could be due to indion ability to swell upon contact with water without lump formation and without adhesive tendency that causes uniform film disintegration and then dissolution (18,23). iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 88 in this study, by using range of 2% to 4% of superdisintegrants, it was clear that the more concentrated superdisintegrant, the lower in-vitro dt, the shorter t80% and the higher d5min for all types of superdisintegrants. effect of superdisintegrant type all superdisintegrants used had a positive effect on dt and increasing the release of the drug from the formulas in a shorter time, but it was clear that indion was the best one followed by ccs then ssg with significant difference (p<0.05) (tables 3,4 and figures 1,2). this may be related to prompt swelling tendency of indion on wetting, thus causing rapid disintegration (23). amin et al., formulated roxithromycin, dicyclomine hydrochloride and montelukast with four superdisintegrants (indion, ccs, ssg and crospovidone), the optimized formulas were containing indion for all drugs. these results reconcile with current research (23). table 3. disintegration time of darifenacin hydrobromide fast dissolving buccal films . f no. n=3 95% confidence interval for mean dt (sec) mean ± std. error std. deviation std. error f1 (2%ccs) 39.31±0.55 0.95 0.55 f2 (3%ccs) 37.30±1.47 2.56 1.47 f3 (4%ccs) 35.73±0.45 0.77 0.44 f4 (2%ssg) 44.97±1.73 3.00 1.7 f5 (3%ssg) 43.33±1.76 3.05 1.76 f6 (4%ssg) 40.59±2.5 4.35 2.51 f7 (2%indion) 38.88±1.84 3.20 1.84 f8 (3%indion) 37.33±0.66 1.15 0.66 f9 (4% indion) 31.28±0.83 1.44 0.83 figure 1. disintegration time of darifenacin hydrobromide fast dissolving buccal films. table 4. dissolution parameters of darifenacin hydrobromide fast dissolving buccal films formula no. d5min(%) t80%(min) f1 (2%ccs) 65 8.9 f2 (3%ccs) 70.27 6.95 f3 (4%ccs) 87.5 4.13 f20 2%ssg 53.57 8.63 f21 3%ssg 60.87 7.05 f22 4%ssg 85.6 4.31 f23 2%indion 68.71 7.4 f24 3%indion 77.8 5.30 f25 4%indion 94 3.05 figure 2. dissolution profile of darifenacin hydrobromide fast dissolving buccal films characterization of selected formula f9 that composed of 2%w/v pva, 30%w/w glycerol and 4%w/w of indion showed shortest dt of 31.28sec and superior dissolution profile with t80% time of 3.05min and d5min of 94% was selected as the best formula. percent moisture loss (pml) f9 showed pml of 6.41±0.9%. the obtained value was close to that reported by jasim in 2006 (24). percent moisture absorb (pma) f9 pma was 3.5±0.7 which was very close to the pma for pva films investigated by tamer in 2018 (11). evaluation of mechanical property of the films mechanical property can be expected by studying some parameters like fe, ts, %e and ym. fe results were mentioned previously and listed in table 2. iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 89 tensile strength ts of f9 was 2.089±0.7 mpa. it was close to results of tamer (2.78 mpa) (11). percent elongation e% value was 62±2.1% for the f9 which is near to that obtained by noor (9). young's modulus ym was 8.74±0.4 mpa for f9. it is clear from all mechanical properties that the selected film is of excellent flexibility and this corresponds to findings of arora and chakraborty (2017) in their review on pva films (25). drug-polymer compatibility test there are some parameters that reflect the drug-polymer compatibility like: fourier transform infra-red (ftir) the ftir spectrum of the dh, pva polymer, 1:1 physical mixture (dh and pva) and f9 are shown in figures 3, 4, 5, and 6 respectively. the main characteristic peaks of dh at wave numbers (in cm-1) are: 3464 for n-h asymmetric stretching of amide (3500-3400), 3251 and 3205 for n-h symmetric stretching of amide (3300-3180), 1662 for c=o stretching of amide (1695-1630), 1350 for c-n stretching of tertiary amine (13601310), 1215 for c-o stretching of furan (13001000).. from ftir results, it was observed that there was no chemical interaction as well as no changes in the peaks of fingerprint region obtained in dh spectrum to that of physical mixture spectra. all functional groups in dh spectrum were maintained in the spectrum of f9. the results indicate that no chemical interaction occurred between dh and excipients used in f9. figure 3. ftir spectrum of darifenacin hydrobromide figure 4.ftir spectrum of pva iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 90 figure 5. ftir spectrum of physical mixture figure 6. ftir spectrum of (f9) differential scanning calorimetry (dsc) dsc technique has been performed to investigate thermal stability of dh and excipients. dsc of dh, pva, (1:1) physical mixture of dh with pva and f9 has been shown in figures 7, 8, 9 and 10 respectively. a single peak confirming melting point of dh was detected at 235.8 0c, indicating a crystal dh form. the peak of physical mixture revealed two peaks; one specific for dh and the other for pva indicating that there was no interaction between them. f9 showed a broad peak indicating complete disappearance of dh peak and proposing the dispersion of dh in amorphous form in the film and/or there was a complete homogeneous dissolution of dh in the polymer lattice. figure 7. dsc of darifenacin hydrobromide iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 91 figure 8. dsc of pva figure 9. dsc of physical mixture figure 10. dsc of f9 iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 92 x-ray powder diffraction xrpd of dh, pva, and f9 are presented in figures 11, 12 and 13. xrpd of dh showing a typical crystalline pattern, that can be noticed by several sharp peaks (11.682, 18.426 and 20.426). the xrpd of pva has distinct peaks at 19.265, 19.863 and 21.408. however, xrpd of f9 represents an absence of all characteristic peaks of dh and pva. these findings suggest that dh and pva converted from crystalline to amorphous form. figure 11. xrdp of pure darifenacin hydrobromide figure 12. xrdp of pva iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 93 figure 13. xrdp of f9 stability f9 stability was studied at three temperatures, 40 ºc, 50 ºc and 60 ºc for three months. figure 14 shows plot of logarithm percent remaining of dh versus time in weeks at different temperatures. the obtained profiles were linear indicating that dh degradation follows first order kinetics. the degradation rate constant (k) at every temperature was calculated from the slope of every line (26) k= -slopex2.303 (6) figure 14. degradation profile of darifenacin hydrobromide in f9 at 40ºc, 50ºc and 60ºc the values of k for each temperature are summarized in table 5. table 5. degradation rate constants (k) of darifenacin hydrobromide in f9 at 40 ºc, 50 ºc, and 60ºc temperature (c) k(week -1) 40 2.76x10-3 50 4.7x10-3 60 7.1x10-3 expiration date was calculated from degradation rate constant at 25ºc (k25) using arrhenius plot equation (7) as shown in figure (15). t90%= 0.105/k25 (7) at temperature 25 0c; k= 0.00126week-1(1.26×10-3week-1); t90%= 83.4weeks= 1.6years. figure 15. arrhenius plot of darifenacin hydrobromide in f9 for expiration date estimation conclusion based on obtained results; it was clear that the more concentrated superdisintegrant, the better disintegration rate and drug release, with superiority to indion 414 superdisintegrant and the fdbfs is a iraqi j pharm sci, vol.28(2) 2019 darifenacin hydrobromide buccal films 94 promising dosage form for releasing darifenacin hydrobromide. references 1. patel vf, liu f, brown mb. advances in oral transmucosal drug delivery. j control release. 2011 ; 153(2):106–16. 2. siddiqui mdn, garg g, sharma pk. a short review on “a novel approach in oral fast dissolving drug delivery system and their patents”. adv biol res 2011;5(6):291-303. 3. rajni b, pravin p, sushil k, sandeep a. orally dissolving strips: a new approach to oral drug delivery system. int j pharm investig. 2013; 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pharmacological evaluation of oral fast disintegrating films containing local anaesthetic agent lignocaine. biomed res. 2017;28(3):1135-141. 18. shihora h, panda s. superdisintegrants, utility in dosage forms: a quick review. jpsbr. 2011;1(3):148-53. 19. gissinger d, stamm a. a comparative evaluation of the properties of some tablet disintegrants. drug dev ind pharm. 1980;6(5):511-36. 20. sharma d. formulation development and evaluation of fast disintegrating tablets of salbutamol sulfate for respiratory disorders, isrn pharm. 2013; 2013(674507):1-8. 21. haque se, sheela a. development of polymer-bound fast-dissolving metformin buccal film with disintegrants. int j nanomedicine. 2015;10(1):199–205. 22. chandira rm, bhowmik d, venkataeswarlu bs, kumudhavalli mv, jayakar b. formulation and evaluation of taste masked fast dissolving tablets of ondansetron hcl. journal of pharmacy research. 2008;1(2):200207. 23. amin p, prabhu n, wadhwani a. indion 414 as superdisintegrant in formulation of mouth dissolve tablets. indian j. pharm. sci., 2006;68(1):117-19. 24. jasim ik. formulation and evaluation of mouth dissolving films of captopril. master thesis. university of baghdad. college of pharmacy. 2006: 59. 25. arora l, chakraborty t. a review on new generation orodispersible films and its novel approaches. iajpa. 2017 jan;7(1):7451-70. 26. martin an, sinko pj, singh y. chemical kinetics and stability. in: martin’s physical pharmacy and pharmaceutical sciences. 6th ed. baltimore, md: lippincott williams & wilkins; 2011. 318–54 p. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc3757902/ http://www.ijpsonline.com/articles/formulation-development-and-evaluation-of-fast-dissolving-film-of-telmisartan.html http://www.ijpsonline.com/articles/formulation-development-and-evaluation-of-fast-dissolving-film-of-telmisartan.html http://www.ijpsonline.com/articles/formulation-development-and-evaluation-of-fast-dissolving-film-of-telmisartan.html https://www.ncbi.nlm.nih.gov/pubmed/?term=haque%20se%5bauthor%5d&cauthor=true&cauthor_uid=26491321 https://www.ncbi.nlm.nih.gov/pubmed/?term=sheela%20a%5bauthor%5d&cauthor=true&cauthor_uid=26491321 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ summary:iraqi j pharm sci , vol.18 (1) , 2009 amoxicillin and cobalt (ii) complexation 56 ho h nh2 h h s n ch3 ch3 cooh h o n h o . 3h2o spectroscopic study for determination of amoxicillin using cobalt(ii) as complexing metal may m.j. al-mudhafar* ,1 *department of pharmaceutical chemistry,college of pharmacy,university of baghdad,baghdad,iraq abstract this study includes analytical methods for the determination of the drug amoxicillin trihydrate (amox.) in some pharmaceutical preparations using cobalt ion (co(ii)) as complexing metal. the best conditions for complexation were: the reaction time was 20 minutes, ph=1.5 and the best temperature of reaction was 70 ˚c. benzyl alcohol was the best solvent for extraction the complex. keywords: amoxicillin, cobalt(ii), complex, molar ratio. الخالصة amoxicillin)اتالبنما اايتضمن البحثماالحمتث لرا محلالثة ة مدا يم قايم المم يحالبنحئم البم سلي الالثئ مة ا م ا ي م trihydrate)ا)اتسذبممباكتيممثي االلممم ابةلممماناالممواليممث البيثكةمما(co iiح ممااس مم اليضمم ااph بلنة ممدالبتلم مم ادامم االبممداحاالضمم داا (٥٫١ph=اسل اليض اان داححلنقابلنة دالبتلم االاك البلماناسلاليث البفة يائانتاكث ساا)ان داال ثيد,اسليضم امالم ابةتفادم اااا٠٧ البحا ية اهثاليض االذي ابلنة دالالحتخ ص.االاق مد,ق ا حكتاد قاالذيحاتاح ااس اإ البيثثا٠٧ث سااييا اك introduction amoxicillin is one of the important derivatives of semisynthetic penicillin; it is active against gram positive and to less extent gram negative bacteria. its nomenclature according to penicillins is 6-[d(-)-α-amino-phydroxyphenyl acetamido] penicillanic acid or αamino-p-hydroxy benzyl penicillin (1) ,while its systematic (iupac) name is 7-[2-amino-2(4-hydroxyphenyl)-acetyl]amino3,3,dimethyl-6-oxo-2-thia-5azabicyclo [3, 2, 0] heptane-4-carboxylic acid, the chemical structure of the drug is (1) the formula structure of amoxicillin as trihydrate (drug) is c16h19n3o5s.3h2o, its molecular weight = 419.45 gm.mole -1 . it is off white or almost white crystalline powder, slightly soluble in water and alcohol such as methanol and ethanol (2) . it has uv max. (ethanol): 230,274 nm and in (0.1n hcl): 229,272 nm (1) .imran et al. prepared complexes of amoxicillin with zn(ii), cu(ii), ni(ii)and ag(i), they identify these complexes by (c, h, n)elemental analysis and ir spectra. these complexes have increased the biological activity of the drug (3) . jian et al. determined amoxicillin in tablets, they used a quick and simple method which is (second differential derivative) at λmax. 282 nm and the standard deviation was less than 1.2% and the standard recovery for the drug was 97-100.5 % (4) .denis et al. determined amoxicillin and clavulanic acid in blood plasma by hplc supplied by uv detection, and they found the linearity was (0.62 – 20 µg.ml ) while the detection limit for amoxicillin was 0.312 µg.ml (5) . 1 corresponding author e-mail : may_almothaffar@yahoo.com received : 17/11/2008 accepted : 8 /4 / 2009 iraqi j pharm sci , vol.18 (1) , 2009 amoxicillin and cobalt (ii) complexation 55 ashry et al. detected phenolic antibiotic like amoxicillin by its reaction with benzocaine in the presence of triethylamine at λmax. = 455 nm the linearity was (2 – 16 µg.ml -1 ) while the detection limit was 0.0034 µg.ml -1 (6) .co(ii) forms blue-colored complex in the organic phase with cyanex 923, a sensitive analytical reagent, the λmax. of the complex was 635 nm and the concentration that obeyed beer’s law is (58.9-589.0 µg. ml ) (7) .zayed et al. in a new study, prepared different complexes of amoxicillin with zn (ii), ni (ii), co (ii) and cu (ii), these complexes were studied using elemental analysis, ir and mass spectra. the molar ratio of complexes were found to be metal:drug =1:1,1:2, and the stability constant kf of these chelates was(10 7 -10 14 ) (8) .in a recent study, alekseev et al. prepared mixed complexes of β-lactam antibiotics (ampicillin, amoxicillin and cephalexin) in solutions containing co(ii) and glycine anions(gly). these complexes had been investigated using ph-metric titration at 20 o c in alkaline medium as mixed ligands complex [co gly ampicillin], [co gly amoxicillin], and [co gly cephalexin] (9) . instruments , materials and method a instruments 1. uv-visible spectrophotometer (cary 100) wave length 200-1100nm. 2. shimadzu (aa-670) flame atomic absorption spectrophotometer (400s). 3. mettler, balance model 210s, iso 9001. 4. ph-meter type 60a, usa. 5. water bath with thermostat, memmert. b materials all the chemical stock solutions were prepared from analytical grade bdh, sdi, and india. cmethod 1. stock solution of co(ii) 1000 ppm is prepared by dissolving 0.2010 gm hydrated cobalt chloride (cocl2.6h2o) in distilled water and complete the volume to 50 ml. 2. stock solution of amoxicillin 1000 ppm is prepared by dissolving 0.1 gm amoxicillin in 5 ml. of 1m hcl then complete to 100 ml with distilled water. 3. choosing the optimum conditions for complex formation: the experimental work showed that the reaction did not proceed at room temperature; heating was needed, media must be acidic for this reason. we studied the effect of ph, temperature, reaction time, extraction time, and suitable solvent for the extraction process. 4. spectral study: a) amoxicillin spectrum: transfer 1 ml from stock solution of amoxicillin to 5 ml volumetric flask then dilute with distilled water the absorbance is measured at (200-1100 nm) using acidic water as blank. table (2) shows the proper ph of the solution and figure (1) shows uv spectrum for amoxicillin. b) cobalt spectrum: transfer 1 ml of co (ii) stock solution to 5 ml volumetric flask then dilute with distilled water the absorbance is measured at (2001100 nm) using distilled water as blank. figure (2) shows uv spectrum for cobalt. c) amoxicillin-cobalt(ii) [amox.co(ii)] complex spectrum: transfer (2-5 ml) from the standard of amoxicillin stock solution to 5 ml volumetric flask then 1 ml of cobalt stock solution is added. the chelating complex was extracted by 5 ml benzyl alcohol then measures the absorbance at (200-1100 nm) using benzyl alcohol as blank. figure (3) shows uv spectrum for [amox.-co (ii)] complex. figure(1): uv spectrum of amoxicillin figure 1 : uv spectrum of amoxicillin figure 2 : uv spectrum of the element co(ii) iraqi j pharm sci , vol.18 (1) , 2009 amoxicillin and cobalt (ii) complexation 56 figure(2) :uv spectrum of the element co(ii) figure(3): uv spectrum of [amox-co(ii) ] complex results and discussion amoxicillin spectrum, illustrated at figure (1), consist of 2 bands at λmax. (272 nm) and (228 nm), we depend on the band at λmax. (228 nm) because the other peak will disappear in some experiments also it may interfere with benzyl alcohol peak.  co (ii) spectrum gives peak at λmax. (211 nm) using distilled water as a blank, (figure 2).  chelating complex [amox.-co (ii)], a new peak at λmax. (375 nm) as shown in figure (3) which indicate the formation of the complex that extracted by benzyl alcohol. table (1) shows the color and λmax. for the amoxicillin, co (ii), and the complex. table (1): color and λmax for the drug, metal, complex.  detection the optimum conditions for complexation using uv-visible spectrophotometer: 1. ph effect: table (2) shows the absorbance of the [amox.-co (ii)] complex using different ph (0.1-3), the optimum ph for the complexation was (1.5) where the absorbance is gradually increased from 0.5 to the maximum peak at ph 1.5 and then it decreases. figure (4) shows the effect of ph on the absorbance of [amox.co (ii)]. table (2): complex absorption values at different ph. figure(4): the effect of ph on the absorbance of the [amox-co(ii) ] complex compound λmax. ( nm) color amoxicillin 228 272 offwhite co(ii) 211 bluishwhite [amox.-co(ii)] 375 white ph absorbance 0.5 0.69 1.0 0.98 1.5 1.28 2 0.84 2.5 0.7 3.0 0.4 figure 3: uv spectrum of [ amox-co(ii)] complex figure 4 the effect of ph on the absorbance of the [amox.-co(ii)] complex 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ph a b s o r b a n c e figure 1 : uv spectrum of amoxicillin figure 2 : uv spectrum of the element co(ii) ph iraqi j pharm sci , vol.18 (1) , 2009 amoxicillin and cobalt (ii) complexation 56 2. temperature effect: the reaction of the metal and amoxicillin proceeded slowly, to increase the reaction velocity we intend to increase the temperature of the reaction from 40 to100 ˚c, then the complex is extracted and measured the absorbance using uv spectrophotometer. table (3) gives the absorbance of the complex at different temperatures at ph 1.5 and figure (5) shows the λmax for the complex is at 70 ˚c. table (3): complex absorption values at different temperatures at (ph 1.5). temp. o c absorbance 40 0.85 50 0..96 60 1.11 70 1.38 80 1.26 90 1.1 100 1.1 figure(5) the effect of temperature on the [amox-co(ii) ] formation 3. reaction time: the complex formation increased as well as the absorbance when the reaction time is increased, before the extraction process takes place, we used different times (5-30 min.), table (4) and figure (6) showed that 20 min. is the best time for the reaction and give maximum absorbance for the [amox.-co (ii)] at (70 ˚c and ph 1.5). table (4): complex absorbance value at different reaction time at 70 o c time(min.) absorbance 5 0.75 10 0.99 15 1.16 20 1.30 25 1.22 30 1.13 figure (6) the effect of reaction time on complex formation 4. suitable extraction solvent: different organic solvents were used like; methanol, ethanol, chloroform, benzyl alcohol, and ethyl acetate to choose the proper solvent that dissolves the complex, but can not dissolve the metal (cobalt) and amoxicillin as well give the highest absorbance for the complex. table (5) shows the solubility of the amoxicillin, metal and the complex in different organic solvents. table (5): the solubility of amoxicillin, metal and the complex in different solvents. solvent amoxicillin solubility metal solubility complex solubility methanol + + − ethanol + + − chloroform − − − benzyl alcohol − − + ethyl acetate + − + (+): soluble. (−) :very slightly soluble or insoluble figure 5 the effect of temperature on the [amox.-co(ii)] formation 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 10 20 30 40 50 60 70 80 90 100 110 120 temperature ( o c ) a b s o r b a n c e figure 6 the effect of reaction time on complex formation 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 5 10 15 20 25 30 35 time (min.) a b s o r b a n c e iraqi j pharm sci , vol.18 (1) , 2009 amoxicillin and cobalt (ii) complexation 56 5. extraction time: the optimum time for shaking during the extraction of the complex was 6 min. which gives maximum absorbance as shown in table (6) and figure (7). table (6): effect of shaking time on the extraction process against the absorbance. figure(7) the effect of the extraction time on the absorbance of the complex 6. solvent phase ratio: it was found that 4 ml of aqueous phase and 4 ml of organic phase is enough to give maximum absorption for the complex as shown in figure (8). table (7) showed that the increase in the aqueous phase to 6 ml with 4 ml of organic phase will decrease the absorbance of the complex. table (7): absorbance value of the [amox.co(ii)] as the volume of water phase increased. note: organic phase volume = 4 ml. water phase volume (ml.) absorbance 1 0.8 2 1.1 3 1.32 4 1.41 5 1.33 6 1.06 figure(8): the effect of water volume on the absorbance of the [amox.-co(ii)] complex 7. times of extraction: the first extraction process is enough to extract the major concentration of the complex because the second extraction process for the complex that remained in aqueous phase gives a very weak absorbance less than 0.1. calculation of the ligand (amoxicillin) to metal (cobalt) ratio in the complex [amox.co (ii)]: molar ratio: to detect the ratio of complexation i.e. the molar ratio of ligand (l) to the metal (m) by taking different volumes of the ligand (vl) with constant volume of metal (vm) at the same concentration for each (1.2 x 10 -3 m) at the optimum conditions for complexation, then drawing the relation between absorbance and vl/vm as in figure (9), and table (8) shows the absorbance against vl/ vm. the molar ratio about (2:1) for the (l: m) and the suggested chemical structure for the [amox.co(ii)] complex is: extraction time (min) absorbance 2 0.8 3 1.08 4 1.27 5 1.33 6 1.44 7 1.44 8 1.43 figure 7 the effect of the extraction time on the absorbance of the complex 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 2 4 6 8 10 time a b s o r b a n c e figure 8 the effect of water volume on the absorbance of the [amox.-co(ii)] complex 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 1 2 3 4 5 6 7 vwate r (ml) a b s o r b a n c e iraqi j pharm sci , vol.18 (1) , 2009 amoxicillin and cobalt (ii) complexation 67 ho h nh2 h h n o n o c s ch3 ch3 c o s n hc h3c o n h2n h h3c o h h c o o co o h h ++ .2cl h oh ph = 1.5 co(ii)+2amox. co(ii) – 2amox. imran (3) and his partners defined the amoxicillin complex form with transition elements in m:l is 1:2 ratio, and this was identical with proportion of metal with the ligand (drug) that we have reached by molar ratio. table (8): the values of the absorbance of the complex against vl/vm vl/vm molar ratio absorbance 1/2 0.5 0.3 2/2 1 0.8 3/2 1.5 1.24 4/2 2 1.5 5/2 2.5 1.75 6/2 3 1.75 8/2 4 1.74 figure(9) : detection the molar ratio for the complex formation calibration curve of the uv spectrum: it was carried out through taking different concentrations for the complex (ppm) and measuring the absorbance, as shown in table (9). figure (10) shows the calibration curve of [amox.-co(ii)] complex that obeys beer's law for the concentrations (50-500 µg. ml ) at 375 nm. table (9): the value of maximum absorbance for the [amox.-co(ii)] at different concentrations. conc. ppm absorbance 50 0.6 100 0.75 150 0.98 200 1.2 300 1.6 400 2.0 500 2.49 600 2.55 figure(10): the calibration curve for detecting of [amox.-co(ii)] at 375 nm determination of the drug concentration in different pharmaceutical preparations: this was done by taking an average weight of one capsule from six capsules that have been mixed previously then the absorbance of the active ingredient (amoxicillin) was measured from calibration curve (figure 10), after complexation process takes place at the optimum condition mentioned above. note: we carried out the same procedure for 500 mg. amoxicillin capsules of different trademarks (sdi, julphar and ajanta). table (10) shows the results of these preparations. 0 0.5 1 1.5 2 0 1 2 3 4 5 v l /v m a b so r b a n c e figure 10 the calibration curve for detecting of [amox.-co(ii)] at 375 nm 0 0.5 1 1.5 2 2.5 3 0 100 200 300 400 500 600 700 conc. (ppm) a b s o r b a n c e iraqi j pharm sci , vol.18 (1) , 2009 amoxicillin and cobalt (ii) complexation 67 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 0 5 10 15 20 25 conc. (ppm) a b s o r b a n c e table (10): the concentration of amoxicillin in 500 mg amoxicillin capsule of different trademarks. capsule absorbance conc. ppm sdi 2.5 501 julphar 2.48 499 ajanta 2.42 487 determination of the [amox.-co(ii)] by flame atomic absorption spectrophotometer (faas): the complex was prepared under the optimum conditions of ph, temperature, proper solvent etc.. and we used the faas to detect the amoxicillin concentration by indirect measurement the absorbance of the co(ii) in the complex as shown in figure (11), also we can measure the concentration of the amoxicillin in these pharmaceutical preparations using the calibration curve of indirect (faas). figure(11): the calibration curve for detecting amoxicillin using faas conclusion the developments of new analytical methods to determine the amount of amoxicillin, such methods are uv-visible and flame atomic absorption spectrophotometrics, are very sensitive and precise. amoxicillin forms chelated complex with cobalt ions at 70 o c and ph: 1.5 and the molar ratio for complex formation, amoxicillin:cobalt, is (1:2). the results of analysis of amoxicillin capsules of different trademarks show the amount of amoxicillin in sdi and julphar capsules are almost as presented by the package, on the other hand the ajanta capsules show some differences. references 1. maryadele j.o´ neil, the merck index; encyclopedia of chemicals, drugs and biologicals, 14 th edition, merck research laboratories, usa, 2006, 92. 2. sean c sweetman, martindale; the complete drug reference, 33 rd edition, the pharmaceutical press, uk, 2002, 149. 3. m. imran, j. iqbal and t. mehmood, "synthesis, characterization and (in vitro) screening of amoxicillin and its complexes with ag(i), zn(ii), cu(ii) ,co(ii), ni(ii)” , j.bio. sci., 2006, 5, 946 – 949. 4. d. jain and p. trived, “simultaneous spectrophotometric determination of amoxicillin and probencid in tablet dosage form”, indian journal of pharmaceutical sciences, 1998, 60(5), 318-320. 5. l. denis, c. frances and t. frenque, “simultaneous determination of amoxicillin and clavulanic acid in human plasma by hplc with uv detection”, j. pharm. biomed. anal., 2002, 30, 661 – 666. 6. s.ei.ashry, f.belal and d. el-wasseef, “spectrophotometric determination of phenolic antibiotics in dosage forms”, microchimica acta, 2000, 135(3-4), 191196. 7. b. r. reddy, p. rathika, j.r. kumar, d.n. priya, k.rajgopal, “extractive spectrophotometric determination of cobalt (ii) in synthetic and pharmaceutical samples using cyanex 923”, analytical sciences, 2004, 20(2), 345-349. 8. m. a. zayed and s.m. abdallah, “synthesis and structure investigation of the antibiotic amoxicillin complexes of dblock elements", spectrochimia acta part a.molecular and biomolecular spectroscopy, 2005, 61(9), 2231-2238. 9. v. g. alekseev and i. s. sanuilova, “complex formation in systems cobalt(ii)-glycine-β-lactam antibiotics” , russian j. of inorganic chemistry, 2008, 53(2), 327-329. a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci , vol.17 (2) , 2008 melatonin in lead poisoning 47 therapeutic effects of melatonin in lead-induced toxicity in rats mustafa g. alabbassi*, saad a. hussain**,1 and shatha h. ali*** * department of pharmacology, college of pharmacy, almustansriya university, baghdad , iraq ** department of pharmacology and toxicology,college of pharmacy, university of baghdad , baghdad , iraq *** department of clinical laboratory sciences,college of pharmacy,university of baghdad,baghdad , iraq abstract exposure to lead results in significant accumulation in most of vital organs, and free radical damage has been proposed as a cause of lead-induced tissue damage, where oxidative stress is a likely molecular mechanism. this study was designed to evaluate therapeutic effects of melatonin in leadinduced organ toxicity in rats. the therapeutic effects of melatonin on lead induced toxicity in rats were evaluated using 36 rats, which were allocated into 3 groups and treated as follows: group i, includes 12 rats injected subcutaneously with 0.2 ml physiological saline for 30 days, followed by treatment with a daily dose of 20mg/kg melatonin, administrated i.p for the successive 30 days; groups ii and iii, each includes 12 rats , injected with lead acetate 100 mg/kg/day s.c for 30 days, followed by treatment with intraperotoneal injection of physiological saline (0.2 ml) or melatonin 20mg/kg/day for the next 30 days. at the end of treatment period, the rats were sacrificed by an overdose (100mg/kg) of thiopental (twenty-four hour after the last injection). craniotomy and laparotomy were performed to obtain the brains, livers and kidneys for the assessment of tissue damage. the changes in total body weight, weight of major organs (brain, liver and kidney), oxidative stress parameters, hemoglobin content, liver and renal functions, and histological appearance of the studied organs were evaluated and compared with that of negative and positive controls. treatment with melatonin reverses the damage induced by lead in many organs and tissues through the reduction of mda levels in rbcs, brain, liver and kidney; increases gsh levels in all studied organs; in addition to the improvement in the indices of the functions of the organs studied. these findings demonstrated that melatonin is capable of reversing damage of rat tissues caused by successive doses of lead acetate, and animals had restored their organ functions due to treatment with melatonin. key words: melatonin, lead poisoning, oxidative stress ةصالخلا ي ةض ة ا لص كلضر ي كةامك ة يح ا اضعا اضظصم هزكل لص ي ا ا اصرلل ضر ا نا اضزي ن لاممل ل ا ل ح اضظصم ,ضهه ي ارلص ةا ا ن نز ا , ضةلص هل هضزي لهضصبهضهل لا ن ل .اا ا لها ن ح ا ل ةلهها سضص هل ضلرهل الهلازبهر ا سا ي ل ص ل ص ةالا ص لص . ي ةايه ضلرهل الهلازبهر ضات سا ي ااا , الا ت يلق ااصاها االا لضص اص ةص هل : لالزضل ان ت اةظلر 36 ل ص ل ةالا لص هةا الههللص ص ةا ل اايا/ يا ار لهلازبهر ضغها 20, .ا ار ل از لا ل يليهر كزاص ,ن ض نص ا ضل 2 ااا .ضلاا ا ا اا ا 12 100 ااا ض االلص اا ضلالص ال لص ا ضل 12ا م كةزي ,ليهر كزاص .ن لالزضةهر ,صبهل ن ,ص ,ل ن ة لا اايا / يا /كزل ضر م كم 20 ا ( ن لهلازبهر 0،2اايا / يا /كزل ا ا اا ,ليهر كزاص امضا ا ت ار ل از لا ) صضل 24ضلر م كةزي ا,ليهر كزاص ةص هل .ا بلصكل اة لضص ال اا ة هز بص ص ةا ل ر ضل ضص هل ار يصكز اةص ض ا نا ار ا اضص ال .اا ر م ةغ كا ا ة ص ا ايل ن ا مص ن ضات س ع اظ انص . ي ةيهه ا اض نا ي راصل ن ضظصم هز بص ) ا ايل ن ا مص ن ضات ( ن ا د ارلص ةا ا ن نا ةزد اظصا ا زلص ن صاسض ار ضم ن ضات ن ةيه ااهاهل ا اضظصم ل ان ل اا الههللص ض الصابةلص ا ار ااصاها للصابل اص مل ن لزرمل . ي لضص ال ص لهلازبهر كلضر ي كضص ظ ا اصاع ص لص ا ض ك ار اضظصم ن اباال ار ال الاه ا نزي ) ااةزد لص زب كلصك ( ا ار الكص ل ل ن اصل ن ضم ن ضات ، صال ت اكص ااةزد ضازاصيصكزي ل ان ل اا ا ار اا زل ا ا د نضصال اضظصم ل ان ل . ي ن ح اةصاع از ا ي لهلازبهر ن لص اهل ضات ضض ا ا اصرلل ا باال سا ي ار ةض ة لةضص ك ا ت ار ال لص ، ن ي هز بص ةضص نضصال اضظصم اهلص بةهال لضص ال ص لهلازبهر . 1 corresponding author : e-mail : saad_alzaidi@yahoo.com received : 23/9/2008 accepted : 22/11/2008 mailto:saad_alzaidi@yahoo.com iraqi j pharm sci , vol.17 (2) , 2008 melatonin in lead poisoning 48 introduction lead poisoning is one of the oldest occupational hazards in the world. despite its recognized hazards, lead continues to have a wide spread commercial applications, including the production of storage batteries, pipes and metal alloys such as brass, solders, paints, glass and ceramics (1). once lead enters the body it binds sulfhydryl (sh) moiety of proteins with consequent impairment of their functions; by disrupting protein structure, it interferes with many enzyme systems in the body, thereby affecting the functions of most organs (2). lead also interferes with regulatory mechanisms that control the metabolism of many essential cations, particularly calcium, iron, zinc, sodium and potassium; it also alters the integrity of the cellular and mitochondrial membranes, thereby, increasing cellular fragility and facilitate degenerative processes (3). clinical manifestations of lead toxicity include symptoms referable to the central and peripheral nervous systems, hematopoietic, renal and gastrointestinal systems (4). lead poisoning is a potential factor in brain damage, mental impairment with severe behavioral problems, as well as anemia, kidney insufficiency, neuromuscular weakness and coma (5). at the molecular level, it disturbs heme biosynthesis leading to accumulation of a variety of heme precursors including δaminolevulinic acid (ala) (6). lead has effects on the hormonal regulation of calcium absorption, and lead toxicity is exacerbated in the presence of low dietary calcium (7). it also displaces calcium in the mineral bone matrix, which may affect bone quality (7). the effects on heme synthesis are the best studied toxic effects of lead; it inhibits the key enzymes, δalad and ferrochelatase (heme synthetase) (8). as a result heme synthesis is retarded, and because heme moiety is important for the functions of cytochrome systems and cellular respiration, so lead shows an impact on the entire organism; it inhibits na+-k+-atpase pump attached to erythrocytes membrane leading to their lyses (9). many compounds with antioxidant properties have been evaluated for their protective effects against lead-induced toxicity in animal and human models (10); moreover, melatonin has been used successfully to protect the nervous system against lead toxicity in rats (11). the present study was designed to evaluate the therapeutic effects of melatonin in rats intoxicated with successive doses of lead. materials and methods thirty six male rats (rattus norvegicus) are used in the present study, weighing 200250 g, housed in the animal house of the college of pharmacy, university of baghdad. the animals were maintained at controlled temperature (25 ± 2ºc) from november 2006 to april 2007, allowed free access to water, and fed standard rat chow add libitum. the therapeutic effects of melatonin on leadinduced toxicity in rats were evaluated using 36 rats, which were allocated into 3 groups and treated as follows: group i, includes 12 rats injected subcutaneously with 0.2 ml physiological saline for 30 days, followed by treatment with a daily dose of 20mg/kg melatonin, administrated i.p for the successive 30 days; group ii, includes 12 rats, injected with lead acetate 100 mg/kg/day s.c for 30 days, followed by treatment with intraperotoneal injection of physiological saline (0.2 ml) for the next 30 days; group iii, includes 12 rats injected with 100mg/kg lead acetate s.c daily for 30 days, followed by treatment with intraperotoneal injection of melatonin 20mg/kg/day for the latter 30 days. at the end of treatment period, the rats were sacrificed by an overdose (100mg/kg) of thiopental (twenty-four hour after the last injection). craniotomy and laparotomy were performed to obtain the brains, livers and kidneys for the assessment of tissue damage. after animals were sacrificed, blood samples were obtained by heart puncture and immediately placed into two tubes; an edta tube to get whole blood for the estimation of lead by atomic absorption in the poisoning consultation center [(pcc), medical city/ baghdad], hb, pcv, mda and gsh in rbcs. the second fraction was transferred into plane tube to obtain the serum for analysis of other parameters (alt, ast, alp, urea, and creatinine). in the plane tube, blood allowed to clot and serum was separated after centrifugation for (15-20) minutes at 3000 rpm and the resulted serum was kept frozen at (18ºc) unless immediately analyzed was. brains, livers, and kidneys were excised from each animal immediately, placed in chilled saline phosphate buffer solution, blotted with filter paper and accurately weighed. a 10% (w/v) tissue homogenate was prepared in phosphate buffer at 4oc, using metal head tissue homogenizer which was adjusted at set 3 for one minute. all samples were kept frozen at (-18 o c) unless analyzed immediately. specimens from the brain, liver and kidneys were prepared for histopathological examination according to the method of bauer (12), using paraffin sections technique.the significance of differences between mean values was calculated using unpaired student's t-test and analysis of iraqi j pharm sci , vol.17 (2) , 2008 melatonin in lead poisoning 49 variance (anova). p values less than 0.05 were considered significant for all data presented in the results. results administration of 100mg/kg lead acetate s.c for one month and treatment with saline for another month resulted in significant reduction in body weight after two months (25%). therapeutic treatment with 20 mg/kg melatonin i.p for one month after intoxicated of rats with lead acetate resulted also in significant reduction in total body weight (6%), this level seem to be less than that reported when lead acetate was administered with saline (table 1) . malondialdehyde (mda) levels in the rbcs, brain, liver and kidney tissues were significantly elevated after exposure of animals to 100mg/kg lead acetate (479%, 109%, 178% and 101% respectively, p<0.05) compared with 20 mg/kg melatonin treated animals. therapeutic treatment with 20 mg/kg melatonin resulted in significant decrease in mda levels in studied tissues (55%, 33%, 54% and 23% respectively, p<0.05) compared with animals challenged with 100 mg/kg lead acetate and saline only (table 2). table 1. effects of therapeutic use of 20 mg/kg melatonin on the total body weight and the weights of brain, liver and kidney in rats previously intoxicated with 100 mg/kg lead acetate. treatment groups weight (g) organ /body weight pretreatment posttreatment brain/body liver/body kidney/body saline +melatonin (20mg/kg) (n=12) 353.3 ± 1.88 385.8 ± 7.0 a* 0.004 ± 0.0002 a 0.027 ± 0.0005a 0.003 ± 0.0001 a lead acetate (100mg/kg) + saline (n=7) 349.8 ± 1.84a 263.5 ± 9.55 b* 0.0057 ± 0.0002b 0.05 ± 0.001b 0.005 ± 0.0002b lead acetate (100mg/kg) + melatonin(20mg k) (n=10) 351.0 ± 3.14a 330.0 ± 7.15 c* 0.004 ± 0.0001c 0.033 ± 0.001c 0.003 ± 0.0002c data are expressed as mean ± sem; n= number of animals;*significantly different compared to pretreatement value(p>0.05) values with non-identical superscripts (a, b, c) within the same variable considered significantly different (p<0.05). table 2. effects of therapeutic use of 10 or 20 mg/kg melatonin on the malondialdehyde (mda) in erythrocytes, brain, liver and kidney in rats previously intoxicated with 100 mg/kg lead acetate. treatment groups malondialdehyde (mda) rbc (nmol/g hb) brain (nmol/g tissue) liver (nmol/g tissue) kidney (nmol/g tissue) saline +melatonin (20mg/kg) (n=12) 5.4 ± 0.12 a 48.9 ± 1.62a 52.7 ± 1.31a 24.4 ± 1.21a lead acetate (100mg/kg) + saline (n=7) 31.2 ± 2.48b 101.9 ± 4.71b 144.8 ± 5.56b 49.1 ± 2.17b lead acetate (100mg/kg) + melatonin (20mgkg) (n=10) 13.9 ± 0.83c 68.2 ± 1.89c 66.5 ± 2.13c 37.8 ± 1.87c data are expressed as mean ± sem; n= number of animals; values with non-identical superscripts (a, b, c) within the same variable considered significantly different (p<0.05). iraqi j pharm sci , vol.17 (2) , 2008 melatonin in lead poisoning 50 table 3. effects of therapeutic use of 20 mg/kg melatonin on the glutathione (gsh) levels in erythrocytes, brain, liver and kidney in rats previously intoxicated with 100 mg/kg lead acetate. treatment groups glutathione (gsh) rbc (µmol/g hb) brain (µmol/g tissue) liver (µmol/g tissue) kidney (µmol/g tissue) saline +melatonin (20mg/kg) (n=12) 13.9 ± 0.13a 11.8 ± 0.12a 8.9 ± 0.13a 7.8 ± 0.23a lead acetate (100mg/kg) + saline (n=7) 3.2 ± 0.19b 4.4 ± 0.18b 3.3 ± 0.12b 4.1 ± 0.12b lead acetate (100mg/kg) + melatonin (20mgkg) (n=10) 6.1 ± 0.14c 5.9 ± 0.11c 7.0 ± 0.09c 5.8 ± 0.10c data are expressed as mean ± sem; n= number of animals; values with non-identical superscripts (a, b, c) within the same variable considered significantly different (p<0.05). daily treatment of rats with 100mg/kg lead acetate significantly reduces gsh levels in rbcs, brain, liver and kidney (77%, 63%, 64%, and 48% respectively, p<0.05) compared with 20 mg/kg melatonin treated animals. meanwhile therapeutic treatment with 20 mg/kg melatonin, administered one month after lead acetate results in significant elevation of gsh in the studied tissues (88%, 34%, 115% and 41% respectively, p<0.05) compared with lead acetate and saline treated animals (table 3). administration of 100 mg/kg lead acetate to the rats result in significant decrease in hb levels and pcv %( 12% and 9% respectively, p<0.05), when compared with melatonin 20 mg/kg treated group (table 4). exposure of animals to s.c injections of lead acetate (100 mg/kg) for one month and saline for another month produces significant elevation in the serum levels of hepatic enzymes activity (ast, alt, alp)(162%, 232%, and 102% respectively, p<0.05) compared with 20 mg/kg melatonin treated animals. therapeutic administration of melatonin in a dose of 20 mg/kg (39%, 53% and 42%) significantly reduces enzymes activities both with respect to lead acetate and saline treated animal group and between each other (table 5). however, therapeutic treatment of animals with melatonin, one month after lead acetate challenge, significantly reduces serum levels of urea and creatinine in which the reduction were (28% and 25% respectively, p<0.05), the reduction in serum level of their parameters was significantly different when compared with lead acetate and saline treated animals and between each others( table 6). lead acetate, when administered subcutaneously, in a consecutive 100 mg/kg doses for one month and saline for another month produces significant elevation in blood lead levels (513%), and lead levels in brain, liver and kidney of these animals were also significantly elevated (3810%, 4736% and 2849% respectively, p<0.05) compared with 20 mg/kg only melatonin treated animals. melatonin reduces lead levels significanty in all studied compartments (blood 28%, brain 46%, liver 40% and kidney 42%) compared with lead acetate and saline treated animals (table 7). table 4. effects of therapeutic use of 20 mg/kg melatonin on the hematological parameters of rats previously intoxicated with 100 mg/kg lead acetate for one month. treatment groups hb (mg/dl) pcv % normal saline + melatonin (20 mg/kg) (n=12) 14.6 ± 0.20 a 44.3 ± 0.81a lead acetate (100 mg/kg) + saline (n=7) 12.2 ± 0.29 b 37.3 ± 0.87b lead acetate (100 mg/kg) + melatonin (20 mg/kg) (n=10) 13.6 ± 0.15 c 40.7 ± 0.72c data are expressed as mean ± sem; n= number of animals; values with non-identical superscripts (a, b, c) within the same variable considered significantly different (p<0.05). iraqi j pharm sci , vol.17 (2) , 2008 melatonin in lead poisoning 51 table 5. effects of therapeutic use of 10 or 20 mg/kg melatonin on the liver enzymes (ast, alt, and alp) of rats previously intoxicated with 100 mg/kg lead acetate for one month. treatment groups liver enzymes level (u/l) ast alt alp normal saline + melatonin (20mg/kg) (n=12) 55.0 ± 1.53 a 36.0 ± 1.30 a 95.6 ± 2.27 a lead acetate (100mg/kg) + saline (n=7) 144.2 ± 3.87 b 119.8 ± 3.23 b 192.9 ± 3.44 b lead acetate (100mg/kg) + melatonin (20mgkg) (n=10) 87.7 ± 2.71 c 56.8 ± 2.14 c 112.5 ± 3.33 c data are expressed as mean ± sem; n= number of animals; values with non-identical superscripts (a, b, c) within the same variable considered significantly different (p<0.05). table 6. effects of therapeutic use with 10 or 20 mg/kg melatonin on serum urea and creatinine of rats previously intoxicated with 100 mg/kg lead acetate for one month. treatment groups serum urea (mmol/l) serum creatinine (µmol/l) normal saline + melatonin (20mg/kg) (n=12) 5.4 ± 0.13 a 72.8 ± 2.61 a lead acetate (100mg/kg) + saline (n=7) 11.9 ± 0.62 b 190.7 ± 11.39 b lead acetate (100mg/kg) + melatonin (20mgkg) (n=10) 8.5 ± 0.24 c 142.2 ± 4.73 c data are expressed as mean ± sem; n= number of animals; values with non-identical superscripts (a, b, c) within the same variable considered significantly different (p<0.05). table 7. effects of therapeutic use of 10 or 20 mg/kg melatonin on lead levels in blood, brain, liver and kidney of rats previously intoxicated with 100 mg/kg lead acetate for one month. treatment groups lead level blood (μg/dl) brain (μg/gm) liver (μg/gm) kidney (μg/gm) saline + melatonin (20mg/kg) (n=12) 12.98 ± 0.29 a 0.9 ± 0.05 a 2.18 ± 0.1 a 8.23 ± 0.26 a lead acetate (100mg/kg) + saline (n=7) 79.54 ± 3.51 b 35.19 ± 1.33 b 105.43 ± 2.98 b 242.69 ± 2.28 b lead acetate (100mg/kg) + melatonin (20mgkg) (n=10) 57.48 ± 2.15 c 18.92 ± 0.83 c 63.38 ± 1.88 c 141.57 ± 2.1 c data are expressed as mean ± sem; n= number of animals; values with non-identical superscripts (a, b, c) within the same variable considered significantly different (p<0.05). 52 sections prepared from livers of rats, previously intoxicated with lead acetate 100 mg/kg, treated with saline for one month, showed a wide area of normal appearance with presence of small area of degeneration and necrosis with inflammatory cells infiltration (figure 1). meanwhile, treatment of rats with 20 mg/kg melatonin previously intoxicated with 100 mg/kg lead acetate for one month, the liver sections showed normal structure appearance with few discrete degenerative changes (figure 2). figure (1). section of liver tissue showing a wide area of normal appearance with presence of small area of degeneration and necrosis (arrow a) with inflammatory cells infiltration (arrow b) in rats treated with saline previously intoxicated with 100mg/kg lead acetate for one month. magnification: 200x (hematoxylin and eosin stain). figure (2). section of liver tissue showing normal histology with appearance of few discrete degenerative changes (arrow) in rats treated with 20 mg/kg melatonin previously intoxicated with 100mg/kg lead acetate for one month. magnification: 200x (hematoxylin and eosin stain). sections prepared from kidneys of rats treated with saline and previously intoxicated with 100 mg/kg lead acetate for one month, showed mild degenerative changes and necrosis in the kidney tubules (figure 3). meanwhile, administration of 20 mg/kg melatonin to group of rats previously intoxicated with lead acetate 100 mg/kg, the kidney sections showed normal histology but still there is slight dilatation of the renal tubules (figure 4). figure (3). section of kidney tissue showing mild degenerative changes (arrow a) and necrosis (arrow b) in the kidney tubules in rats treated with saline previously intoxicated with 100mg/kg lead acetate for one month. magnification: 200x (hematoxylin and eosin stain). figure (4). section of kidney tissue showing normal histology but still there is slight dilatation of renal tubules (arrow) in rats treated with 20 mg/kg melatonin previously intoxicated with 100mg/kg lead acetate for one month. magnification: 200x (hematoxylin and eosin stain). a a a b a a a b 53 discussion daily administration of 100 mg/kg lead acetate to rats, reduce their total body weights compared with control animals with subsequent elevation of organ/body weight ratios, and treatment with melatonin restores body weights and the impaired organ/total body weight ratio. lead poisoning is very well known to affect numerous organ systems, and is associated with a number of morphological, biochemical and physiological changes that include kidney dysfunction, impaired glucose metabolism, cns disturbances, impairment of liver function and hematological disorders (13). among their effects, the impaired glucose metabolism is considered as a major pathway that may be followed by changes in total body or organ weights; in this respect intoxication with lead reduces the rate of glucose metabolism, with consequent reduction of the required energy for many anabolic process, and the profound decrease in serum glucose level which is reported in rabbits intoxicated with lead, might also be a cause for tissue wasting due to inappropriate availability of energy. the findings of the present study are found compatible with those reported by others (14), where loss of total body weight is found parallel with the increase in blood lead levels; furthermore, the increase in oxidative stress exhibited contributing factor, where lipid peroxidation might predispose to perturbation in the content of lipids in many organs and tissues. exposure to lead acetate significantly elevates mda levels in erythrocytes, brain, liver and kidney; while therapeutic use of melatonin results in significant reduction in the mda levels in all compartments compared with control groups; their results are found compatible with those reported previously (15). in this respect also, lead depletes the natural antioxidant molecule, the glutathione in the erythrocytes, brain, liver and kidney, and the use of melatonin therapeutically improves the levels of this antioxidant thiol in their compartments; their results are in agreement with those reported by others (16). lead-induced enhancement of lipid peroxidation is a major mechanism for some of the toxic effects of lead in different organ and tissues have certainly been suggested earlier (17). lead crosses the blood brain barrier and causes immediate effects by altering the metabolism and physiology of the brain and other organs like liver and kidney. one likely molecular mechanism involved in lead toxicity is the disruption of the pro-oxidant/ antioxidant balance (18) which leads to tissue injury via oxidative damage to critical biomolecules such as lipids, proteins, and dna. after absorption of lead into the blood, 99% of lead is bound to erythrocytes and the remaining 1% stay in plasma to be carried to other tissues. decreased hematocrit and hemoglobin levels might arise from reduction in serum copper as well as reduced iron metabolism and consumption induced by lead (19). development of anemia in lead toxicity may be attributed to the decreased red blood cell survival because of the increased membrane fragility, reduced rbc count, decreased hemoglobin production, or summation of all these factors (20). the activities of serum enzymes ast, alt, and alp showed significant elevation in rats exposed to lead, administration of melatonin reduces these activities but remain significantly elevated when compared with control groups, these findings are compatible with other previous studies (21). increasing the activities of ast, alt and alp in serum was most likely a consequence of the hepatotoxic effect of lead, the accumulated lead in the liver directly damaging the hepatocytes, primarily by destroying the permeability of the cell membrane, which results in the increased release of cytosolic enzymes ast, alt and alp into the circulation. the results of the present study demonstrated significant increase in both urea and creatinine levels in the serum of rats exposed to 100mg/kg lead acetate daily for one month, indicating renal damage. lead poisoning causes renal dysfunction and such type of toxicity might be due its ability to cause oxidative damage to the renal tissue, which includes enhanced lipid peroxidation, dna damage and the oxidation of protein sulfhydryl groups(22).lead is a pervasive environmental pollutant known to induce a broad range of physiological, biochemical and behavioral dysfunction in human and laboratory animals. based on the present results, it seems that lead levels in blood and tissues became significantly elevated when compared with control and melatonin treated groups, and in agreement with other previous studies (23). the results of the present study enables the conclusion that melatonin, attenuates and reverses the tissue damage induced in experimental animals by lead acetate, and the therapeutic use of this pleiotropic hormone support the idea of the oxidative stress-induced damage due to lead toxicity. 54 references 1. sithisarankui p, weaver vm, davoli ct, strickland pt. urinary δ-aminolevulinic acid in lead-exposed children. biomarkers 1999; 4: 281-289. 2. katavolos p, staempfli s, sears w, gancz y, smith d, beinzle d. the of lead poisoning on hematological and biochemical values in trumpeter swans and canada geese. veterinary clinical pathology 2008; 36(4): 341-347. 3. rio b, froquet r, parrent-massin d. in vitro effect of lead acetate on human erythropoietic progenitors. cell biol toxicol 2001; 17: 41-50. 4. helena a, alejandro s, renata r, lilian n etal. environmental toxicology and pharmacology 2005; 36(1): 113-120. 5. donald c, chuni l, sandra c, jennifer a, regina k, pam m. chemical and biological monitoring of chronic lead poisoning in the rat. journal of applied toxicology 2006; 6(5): 371-376. 6. baghdad , iraq. sithisarankui p, weaver vm, davoli ct, strickland pt. urinary δ-aminolevulinic acid in lead-exposed children. biomarkers 1999; 4: 281-289. 7. anetor j, akingbola t, adeniyi f, taylor g. decreased total and ionized calcium levels and haematological indices in occupational lead exposure as evidence of the endocrine disruptive effect of lead. indian journal of occupational and environmental medicine 2005; 9(1):15-21. 8. othman ai, al-sharawy s, el-missiry ma. role of melatonin in ameliorating lead induced haemotoxicity. pharmacol res 2004; 50: 301-307. 9. david jh, gary hh, daniel ja. phosphorus amendment reduces hematological effects of lead in mallards ingesting contaminated sediments. arch environ contam toxicol 2006; 50: 421428. 10. al-abbassi mg, ismail dk, numan na, hussain sa. effects of treatment with zinc sulphate on the oxidative stress state during chronic exposure to lead in humans. ajps 2006; 3(1): 16-21. 11. el-sokkary gh, kamel es, reitter rj. prophylactic effect of melatonin in reducing lead induced neurotoxicity in the rat. cell and mol biol lett 2003; 8: 461470. 12. bauer jd, ackermann pg, toro g. clinical lab methods. the c.v. mosby company saint louis 1978; pp.813-817. 13. 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mugahi mn, heidari z, sagheb hm, barbarestani m. effects of chronic lead acetate intoxication on blood indices of male adult rat. daru 2003; 11(4): 147151. 20. rio b, froquet r, parrent-massin d. in vitro effect of lead acetate on human erythropoietic progenitors. cell biol toxicol 2001; 17: 41-50. 21. tatjana t, dozic i, dragana v, pejovic j, marjanovic m. the influence of chronic lead poisoning on the activity of some serum enzymes in rats. acta veterianaria 2005; 5(6): 471-482. 22. sawicka kk. histopathalogical changes in the liver, kidneys and testes of bank voles environmentally exposed to heavy emissions from the steel works and zinc smelter in poland. environ res 2004; 96: 72-78. 23. moreira eg, rosa gm, barros sb, vassillieff vs, vassillieff i. antioxidant defense in rat brain regions after developmental lead exposure. toxicology 2001; 169: 145-151. clinical evaluation of melatonin alone and in combination with pizotifen in the prophylaxis of migraine iraqi j.pharm.sci., vol.16 (1) ,2007 alpha tocopherol acetate as a powder by adsorption 18 formulation of alpha tocopherol acetate as a powder dosage form by adsorption zena a. abdulhameed* and yehia i. khalil** ,1 * abo graib health center ** college of pharmacy , university of baghdad, baghdad, iraq abstract : alpha-tocopherol acetate is one of the most important vitamin e derivatives,that were used as antioxidants. adsorbents like kaolin, magnesium carbonate, and microcrystalline cellulose were used successfully to incorporate oily alpha-tocopherol acetate into an acceptable powder dosage form. the results revealed that microcrystalline cellulose as an adsorbents gave the best results with 50% loading capacity at time, 8 minutes before and after incubation period (3 months at 30c), while kaolin and magnesium carbonate have been shown a significant difference before and after incubation. addition of 1% w/w magnesium carbonate to the kaolin enhanced the loading capacity by decreasing the time of adsorption from 20 to 6 minutes and 47 to 9 minutes before and after incubation respectively. the study indicated that the best adsorbent to be used in case of oral vitamin e toxicity is microcrystalline cellulose while magnesium carbonate could be used in the formulation for their best adsorption effect. key words : adsorption , tocopherol acetate powder الخالصة انًًزشاد يثم انكبؤنٍٍ, " وانذي ٌسزعًم ثكثزح كًضبد نهزبكسدهـانفب رىكىفٍزول اسٍزٍذ كعمبر هى وادد يٍ اهى يشزمبد فٍزبيٍٍ " كبرثىَبد انًغٍُسٍىو و انًبٌكزوكزسزالٌٍ سههٍهىس لد اسزعًهذ ثُجبح فً رمدٌى االنفب رىكىفٍزول اسٍزٍذ اندهًُ عهى شكم ثبودر كجزعخ دلبئك 8% فً ولذ لبرة يٍ 05نمد اشبرد انُزبئج انى اٌ انًبٌكزوكزسزبنٍٍ سههٍهىس هى افضم يًزش نهعمبر ثُسجخ رذًٍم ٍخ صهجخ.دوائ ثًٍُب اظهز كم يٍ انكبؤنٍٍ و كبرثىَبد انًغٍُسٍىو فزق واضخ فً َسجخ انزذًٍم لجم و ثعد فززح انذضبَخ )ثالثخ اشهز فً درجخ دزارح % َسجخ وسٌ انى وسٌ يٍ كبرثىَبد انًغٍُسٍىو انى انكبؤنٍٍ لد ساد يٍ َسجخ انزذًٍم اعالِ يٍ خالل رمهٍص 05 يئىي(.اٌ اضبفخ05 كًب اشبرد اندراسخ انى اٌ دلبئك لجم و ثعد فززح انذضبَخ وعهى انزىانً . 9دلٍمخ انى 74دلبئك , و 6دلٍمخ انى 05فززح االيزشاس يٍ سًى ثبنفب رىكىفٍزول اسٍزٍذ هى انًبٌكزو كزسزٍالٌٍ سههٍهىس ثًٍُب رفضم كبرثىَبد انًغٍُسٍىو فً افضم يًزش ٌسزعًم فً دبالد انز رزكٍت انجبودر كبفضم يًزش. introduction : sorption is a selective transfer of gas or liquid onto the surface and into the bulk of liquid or solid sorbent (1) .the substance being adsorbed is called the adsorbate and the substance on which it is adsorbed is called the adsorbent (2) . the fine state of subdivision of inert powders confers high adsorptive capacity upon them (3) . adsorption at solid surfaces is involved in nearly every aspect of pharmaceutical development, from formulation design, process development, and manufacturing especially for low-dose drugs in the manufacturing of solid dosage formulations (4) . most of adsorbents differ in their ability of adsorptivity ability besides physical and chemical nature differences like kaolin, magnesium carbonate, pectin, charcoal, and microcrystalline cellulose “mcc”. different variety of techniques were used to incorporate oily or liquid drugs in an acceptable pharmaceutical dosage forms using soft gelatin capsules or adsorbent powders, which then can be used as a powdered drug form. the objective of this study was to formulate alpha atocopherol acetate which is oily derivative of vitamin e series as a solid powder using different adsorbent materials since these vitamin derivatives are difficult to introduce into solid powders or granules dosage form (5) . experimental work : materials and instruments: alpha-tocopherol acetate oily liquid and microcrystalline cellulose (avicel ph 102) supplied by samara drug industries (sdi). iraq , magnesium carbonate (hydrated) basic light from b.d.h. chemicals ltd, pool, england , kaolin lave (light) from prolabo, rhone-poulenc, perroux, s.a. macon, france , all other reagents were of analytical grade , sartorius balance ag gottingen, bl210s, ce, germany , ph meter, orchidis laboratories, france and hanna instruments type, france , dissolution apparatus type ii, dis6000, copley scientific, nottingham, u.k , u.v. visible spectrophotometer, citra 5, gbc scientific equipment, u.s.a. 1 corresponding author : email : ybmmaz@yahoo.com received : 20/11/2006 accepted : 31/3/2007 iraqi j.pharm.sci., vol.16 (1) ,2007 alpha tocopherol acetate as a powder by adsorption 19 methods: characterization of alpha-tocopherol acetate: 10 mg of alpha-tocopherol acetate was dissolved in sufficient amount of absolute ethanol and then diluted to 100ml. the u.v. light absorption was examined between 230nm and 350nm (5) . formulation of powdered alpha-tocopherol acetate: different formulas were prepared, the method was carried out by incorporation equivalent amount of alpha-tocopherol acetate (400mg) as an oily adsorbate in a petridish, then each adsorbent (of formula 1, 2and 3 in table 1) was added into the surface of liquid adsorbate gradually until the oily layer of adsorbate disappeared and dusty powder began to appear, which indicate that the oily adsorbate (alphatocopherol acetate) interfere and enclosed by adsorbent powder particles. additional amount of adsorbent was added until the dual final product become as like as adsorbent parent. table (1) formulas represents alphatocopherol acetate as adsorbate with different amounts and types of adsorbents the weight of final product was determined, then the difference between total amount of final product and the adsorbate (400 mg) represent the weight of adsorbent incorporated in the formula. mixing of more than one adsorbent with alphatocopherol acetate, (formulas 4 and 5) was used, in which magnesium carbonate concentration was 1% of the total formula (6) . estimation of angle of repose: a static heap of powder, with only gravity acting upon it, will tend to form a conical mound by their flowing over a petri-dish from a funnel, the diameter of the static mound (d) and the high of it (h) were measured to determined the angel of repose (θ), as follows (7) : d h tan dissolution study: the dissolution characteristics was carried out for the equivalent of 100 mg of alpha-tocopherol from each of the five formulas in a powder form under sink conditions using 900ml of dissolution media of 0.1n hcl (ph=1.2) maintained at 37cº (± 0.5 cº) at constant stirring speed (100rpm). different samples were taken for analysis at specified time intervals (10, 20, 30, 40, 50, 60, 120 minutes), and replaced with the same volume of 0.1n hcl. one milliliter of each sample was diluted to 10 ml by absolute ethanol then filtered. the absorbancy was determined spectrophoto-metrically at their specify 285nm. λmax. results and discussion : the scanning of pure alpha-tocopherol acetate oil in absolute ethanol showed maximum absorbance at 285nm (5) , which is agree the reported data. while the prepared alpha-tocopherol acetate powders of a different formulas showed best flowability in formulas 1 and 4 (angle of repose 22°-30°) and an acceptable flowability in formula 3 (angle of repose 26°-34°), and bad flowability with formula 2 (with angel of repose <50°) (3) dissolution of alpha-tocopherol acetate from adsorbents before incubation period: the dissolution study for the effect of incorporating of alpha-tocopherol acetate for dissolution study onto kaolin adsorbent before incubation showed that 100% of the drug was released after 10 minutes as shown in figure 1, this may be attributed to the presence of free form (not adsorbed) of the drug in the formula besides to the adsorbed form. in addition, the different polarity between the adsorbate and the adsorbent in the dissolution media (8) . by extending the period of dissolution, it was seen that the amount of drug release was decreased after 10 minutes and reach to about 5% after one hour, this behavior may be referred to the reverse adsorption process that began after 10 minutes by adsorbent (kaolin in figure 1) in acidic media. in addition, the hydrolysis of acetate salt of alpha_tocopherol in acidic ph make the lipophillic property of alphatocopherol more than the parent one to leave the surface of kaolin particles. formula no. 1 2 3 4 5 alpha tocopherol acetate 400 mg 400 mg 400 mg 400 mg 400 mg kaolin 3000 mg -- 3000 mg magnesium carbonate - 1000 mg - 34 mg 24 mg microcrystalline cellulose (avicel ph 102) )))) -- 2000 mg - 2000 mg iraqi j.pharm.sci., vol.16 (1) ,2007 alpha tocopherol acetate as a powder by adsorption 20 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 time (min) % d ru g r e le a s e 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time (min) % d ru g r e le a s e 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 time (min) % d ru g r e le a s e figure 1: the percent of alpha-tocopherol acetate released from kaolin adsorbent at ph 1.2 before incubation period. a hundred percent of drug release after (10 min) was also obtained when magnesium carbonate (formula2) and microcrystalline cellulose (formula3) were used as adsorbent as shown in figure 2 and 3, respectively. this indicated that microcrystalline cellulose alone was faster as adsorbent than kaolin and magnesium carbonate because of its intermolecular forces, that will form a cross-linkage between microcrystalline cellulose and alpha-tocopherol acetate (9) . figure 2: the percent of alpha-tocopherol acetate released from magnesium carbonate adsorbent at ph 1.2 before incubation period. figure 3: the percent of alpha-tocopherol acetate released from microcrystalline cellulose adsorbent at ph 1.2 before incubation period. on the other hand magnesium carbonate showed lowest adsorptivity than other adsorbents used, since light magnesium carbonate converted into magnesium chloride in presence of acidic media (ph 1.2), which results in a decrease in the amount of adsorbent ready to utilize their adsorbate amounts, as shown in table 2. iraqi j.pharm.sci., vol.16 (1) ,2007 alpha tocopherol acetate as a powder by adsorption 21 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time (min) % d ru g r e le a s e table 2: the time of 50% release of alpha-tocopherol acetate at two phases for different formula before and after incubation period. time for 50 percent release of alpha-tocopherol acetate formula adsorbent before incubation after incubation dissolution phase adsorption phase (-time of 100% release) dissolution phase adsorption phase (-time of 100% release) 1 kaolin 6 min 20 min 26 min 47 min 2 magnesium carbonate 6 min 40 min 4 min 14 min 3 microcrystalline cellulose 6 min 8 min 22 min 8 min 4 kaolin + magnesium carbonate 6 min 6 min 6 min 9 min 5 microcrystalline cellulose + magnesium carbonate 6 min 7 min 19.5 min 15 min in an attempt to utilize these differente adsorptivity of adsorbent used alone, kaolin and microcrystalline cellulose were mixed separately with 1% (w/w) of the total weight magnesium carbonate (formula 4 and 5). it was seen that the time for 50% release of alpha-tocopherol acetate in adsorption phase was 6 and 7 minutes for formula 4 and 5 respectively as shown in figures 4 and 5. these different results may be attributed to the different affinity of alpha-tocopherol acetate to the mixed adsorbents used at certain conditions like temperature and pressure (9) . figure 4: the percent of alpha-tocopherol acetate released from adsorbent mixture of kaolin and magnesium carbonate at ph 1.2 before incubation period. iraqi j.pharm.sci., vol.16 (1) ,2007 alpha tocopherol acetate as a powder by adsorption 22 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time (min) % d ru g re le a s e figure 5: the percent of alpha-tocopherol acetate released from adsorbent mixture of microcrystalline cellulose and magnesium carbonate at ph 1.2 before incubation period. langmuir principle as a base for adsorption process can be used to estimate the slope results from the dissolution profile of (c), concentration at equilibrium, versus c/(x/m), as shown in figure 6.according to this base, the smaller the slope is better for adsorption. c/y = 1/ bym + c/ ym where c = equilibrium conc. ' y = amount of adsorbate ( mg. ) adsorbed per ( gm. ) of adsorbent'' b; empirical affinity (binding constant) , ym, is the amount of adsorbate per unit weight of adsorbent . figure 6: adsorption of drug on various adsorbents before incubation period dissolution of alpha-tocopherol acetate from different adsorbents after incubation period: the effect of incubation of alphatocopherol acetate as an adsorbate with different adsorbents for 3 months period was studied. it was seen that 100% of alpha-tocopherol acetate release from kaolin in 0.1 n hcl (ph 1.2) takes over 50 minutes compared with 10 and 40 minutes for magnesium carbonate and microcrystalline cellulose, respectively, as shown in figures 7, 8 and 9. iraqi j.pharm.sci., vol.16 (1) ,2007 alpha tocopherol acetate as a powder by adsorption 23 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 110 120 130 time (min) % d ru g r e le a s e 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time (min) % d ru g r e le a s e 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 110 120 130 time (min) % d ru g r e le a s e figure 7: the percent of alpha-tocopherol acetate released from kaolin at ph 1.2 after 3 months of incubation period. figure 8: the percent of alpha-tocopherol acetate released from magnesium carbonate at ph 1.2 after 3 months of incubation period. figure 9: the percent of alphatocopherol acetate released from microcrystalline cellulose at ph 1.2 after 3 months of incubation period. these results differ significantly from those obtained before incubation, this may be attributed to the enough time allowed to alphatocopherol acetate to penetrate inside adsorbent particles, beside settlement and equilibrium stabilization of both adsorbent and adsorbate occurred. meanwhile the difference in 100% release time among different formulas may be attributed to the different affinity of adsorbent used. kaolin behaved as the best of the other adsorbents used and this may be due to the difference in polarity of both kaolin and alphatocopherol acetate (8) . the addition of 1% (w/w) of total weight of magnesium carbonate for both to kaolin and to microcrystalline cellulose, resulted in an increase in 100% of alphatocopherol acetate release, as shown in figures 10 and 11. iraqi j.pharm.sci., vol.16 (1) ,2007 alpha tocopherol acetate as a powder by adsorption 24 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time (min) % d ru g r e le a s e figure 10: the percent of alpha-tocopherol acetate released from adsorbents mixture of kaolin and magnesium carbonate at ph 1.2 after 3 months of incubation period. 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 110 120 130 time (min) % d ru g r e le a s e figure 11: the percent of alpha-tocopherol acetate released from adsorbents mixture of microcrystalline cellulose and magnesium carbonate at ph 1.2 after 3 months of incubation period. on the other hand, the time for 50% drug release in the adsorption phase for different formulas may be attributed to change in physical property of both alpha-tocopherol acetate and adsorbent used during incubation period. in general, magnesium carbonate enhanced the dissolution phase by its rapid solubility and enhanced the adsorption phase, as in formula 4, due to its higher affinity to alphatocopherol acetate (10) . the overall results of adsorption phase indicated that the microcrystalline cellulose when was used alone as an adsorbent was the best one compared to kaolin or magnesium carbonate, while addition of 1% w/w magnesium carbonate to the total weight of formula 1 enhanced the adsorptivity of kaolin. langmuir plot as a base of adsorptivity indicated that there was a significant difference (p>0.05) in the affinity of the alpha-tocopherol and adsorptivity of different formulas with that before incubation, as shown in figure 12 since the adsorptive affinity still constant but the difference was in the presence of free drug in the formulas before incubation that will not found after incubation due to a more contact time between the adsorbent and the adsorbate which will change the loading and then the amount of drug released. figure 12: adsorption of drug on various adsorbents after 3 months of incubation period 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0 5 10 15 concentration (mg/ml) c / y magnesium carbonate kaolin kaolin + magnesiu m carbonate microcrystalli ne cellulose microcrystalli ne cellulose + magnesium carbonate iraqi j.pharm.sci., vol.16 (1) ,2007 alpha tocopherol acetate as a powder by adsorption 25 conclusion : based on the results obtained from this study, one can conclude the following: 1. oily alpha-tocopherol acetate was successfully converted into powdered form that is easy to formulate in a suitable solid dosage form like tablets and powders. 2. microcrystalline cellulose was the best adsorbent with loading time 50% adsorption 8 minutes before and after incubation, while (kaolin and magnesium carbonate) gave a significant difference before and after 3 months incubation period of prepared alpha-tocopherol powder. 3. incorporation of 1% of magnesium carbonate to the total weight of kaolin adsorbent enhance the adsorptivity of the resultant powder mixture from 20 to 6 minutes and from 47 to 9 minutes before and after incubation period, respectively. 4. the enhancement of magnesium carbonate as synergistic adsorbent was confined using langmuir slop as an index for a good adsorption, since magnesium carbonate has slop 0.0025 compared with 0.0075 and 0.005 for kaolin and microcrystalline cellulose respectively. 5. these results can be also conducting in alpha-tocopherol acetate toxicity, since best loading capacity resulted by microcrystalline cellulose while best adsorption effect resulted by magnesium carbonate . references : 1. vasanth kumar k., subanandam k., ramamurthi v. and sivanesan s., solid liquid adsorption for wastewater treatment: principle design and operation, eco services international, febriuary,(2005). (see also:http://www.ecoweb.com/cgilocal/sfc?a=/editorial/index.html&b=/editor ial/list_title.html). 2. martin, a., adsorption at liqid interface, physical pharmacy, 4 th edition, kathleen paritt, the pharmaceutical press; u.k, (1993), 370. 3. alfonso, r. gennari, remington: the science and practice of pharmacy, 20 th edition, lippincott williams and wilkins, (2000), volume (3), 1238. 4. hong wen, adsorption at solid surfacesـــpharmaceutical application, encyclopedia of surface and colloid science, apr. (2005), 1-17. 5. british pharmacopia cd, alphatocopherol acetate monograph, ( 2000 ). 6. fabian; klaus h., polymeric oil adsorbents, united states patent, sept. (1993), patent # 5,244,503, 5. 7. aulton, m. e., powder flow properties, pharmaceutics: the science of dosage form design, second edition, puplished by: churchill livingstone, (2002), 133. 8. leon lachman; herbert a. lieberman; joseph l. kanig, adsorption at solidliquid interfaces, the theory and practice of industrial pharmacy, third edition, lea and febiger, philadelphia, (1986), 119121. 9. alfonso, r. gennari, remington: the science and practice of pharmacy, 20 th edition, lippincott williams and wilkins, (2000), volume (1), 266-268. 10. wilson and gisvoled, “textbook of medicinal and pharmaceutical chemistry”, 7 th edition, j. b. lippincott company, (1977), 894. http://www.eco-web.com/cgi-local/sfc?a=/editorial/index.html&b=/editorial/list_title.html http://www.eco-web.com/cgi-local/sfc?a=/editorial/index.html&b=/editorial/list_title.html http://www.eco-web.com/cgi-local/sfc?a=/editorial/index.html&b=/editorial/list_title.html http://www.eco-web.com/cgi-local/sfc?a=/editorial/index.html&b=/editorial/list_title.html iraqi j pharm sci, vol.27(2) 2018 phytochemical investigation of corchorus olitorius l. doi: https://doi.org/10.31351/vol27iss2pp115-122 115 phytochemical investigation of corchorus olitorius l. leaves cultivated in iraq and it’s in vitro antiviral activity hayder t. hasan *,1 and enas j. kadhim* * ministry of health and environment, baghdad, iraq. **department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq. abstract the aim of our study was to investigate the antiviral activity of the corchorus olitorius family tiliaceae cultivated in iraq against measles virus, and to demonstrate an overview about chemical constituents and pharmacological activity of corchorus olitorius l. about150 gm leaves of corchorus. olitorius were defatted by maceration in hexane for 24 hrs. the defatted plant materials were subjected for extraction after filtration using soxhlet apparatus, with aqueous methanol 85% as a solvent extraction for 24 hours, the extract was filtered, and the solvent was evaporated under reduced pressure using a rotary evaporator to get a dry extract of about 12 gm. about 4 gm from the residue was suspended in 100ml water, about 3-4ml of 5% sodium hydroxide was added to obtain a basic solution having ph 10 and partitioned with ethyl acetate (3x100ml), the aqueous layer collected and evaporated to dryness. mtt-cell viability assay (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyl tetrazolium bromide) was conducted on 96-well plates (falcon), vero cells were seeded at 1× 104 cells/well to obtain a multiplicity of infection (moi 10), and at 5 × 103 cells/well to obtain a multiplicity of infection (moi 5). different statistical result revealed a significant antiviral activity of the aqueous layer of corchorus olitorius leaves against measles virus. the preliminary phytochemical tests showed the presence of phenols and flavonoids in the aqueous layer of corchorus olitorius leaves. the antiviral activity of corchorus olitorius leaves is mainly due to the phenolics and flavonoids that detected in the aqueous layer. keywords: corchorus olitorius, flavonoids, phenolics, antiviral, measles. وتأثيرها المضاد في العراق المستزرعلنبات الملوخية )الجوت( التحري للمركبات الفعالة الفايروساتضد **ايناس جواد كاظم و 1،حيدر طاهر حسن* بغداد ، العراق . وزارة الصحة والبيئة، * فرع العقاقير والنباتات الطبية، كلية الصيدلة، جامعة بغداد** الخالصة وس العراق ضد فيرالمستزرعة في لنبات الملوخية )الجوت( هو التحقيق في النشاط المضاد للفيروسات الدراسة الهدف من الحصبة، وإلظهار لمحة عامة عن المكونات الكيميائية والنشاط الدوائي للنبات. المواد النباتية منزوعة الدهن في عملية خضاعساعة. تم ا 24في الهكسان لمدة نقيعتم ازالة الدهون من أوراق النبات بواسطة الت ساعة، تمت تصفية المستخلص، وتم تبخير المذيب تحت 24لمدة ٪85ل المائي ، المذيب الميثانوsoxhletاالستخالص باستخدام جهاز المادة المتبقية في ماء غرام من 4. تم تعليق غرام 12ضغط مخفض باستخدام المبخر الدوار للحصول على مستخلص جاف حوالي . تم فصلة ph 10 بداللة حامضية قاعديهيدروكسيد الصوديوم للحصول على محلول ٪5مل من 4-3مليلتر، وتم إضافة حوالي 100 -mtt3-[4,5الخلية حيوية mttحتى الجفاف. تم إجراء اختبار المائية وتبخيرهاالطبقة ع، تم جم(3x100ml) خالت االثيلمع dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) زرع نسيجي اوعيةفي اجريت التي (؛ (falcon) تحوي خلية/ حفرة 310 × 5و (،moi 10للحصول على تعدد العدوى ) حفرةخلية / 410× 1بمعدل vero خالياتم زرع ،حفرة 96 (.moi 5للحصول على تعدد العدوى ) ضد corchorus olitoriusكشفت النتائج اإلحصائية المختلفة عن نشاط مضاد للفيروسات كبير في الطبقة المائية من أوراق .الملوخية الطبقة المائية من أوراق الفالفونويدات فيوفيروس الحصبة. تظهر االختبارات األولية الكيميائية النباتية وجود الفينوالت قة المائية.اكتشفت في الطب والفالفونويدات التي إن النشاط المضاد للفيروسات من أوراق الملوخية يرجع أساسا إلى الفينوالت .الفيروسات، الحصبةالفينوالت. مكافحة الفالفونويدات،: الملوخية، لمفتاحيةكلمات اال 1corresponding author e-mail: hayder73hasen@yahoo.com received: 6 / 7/ 2018 accepted: 19/10/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp115-122 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 phytochemical investigation of corchorus olitorius l. 116 introduction therapeutic plants have been used since ancient times for the treatment of a variety of diseases (1). current estimates of the number of types of flowering vegetation range between 200 000 and 250 000 in some 300 families and 10 500 genera, and despite a rapidly expanding books and researches on phytochemistry, only a tiny percentage of the entire varieties have been examined chemically (2). almost all organisms need to convert and interconvert a great number of organic and natural substances to permit live, grow, and reproduce. in contrast to these |major metabolic pathways (primary metabolite), which synthesize, degrade, and generally interconvert compounds commonly encountered in all organisms, there also exists an area of metabolism concerned with substances that have a very limited spread in nature. such compounds, called “secondary metabolites”, which gives almost all the pharmacologically active natural products (3). the genus corchorus (jute) which belongs to the family of tiliaceae, involves about 50-60 species allocated in the tropics, subtropics and warm temperate areas of the world (4). corchorus species is one of the main genera containing cardiac glycosides, especially in the seeds (2). corchorus olitorius are tall, annual herbs, reaching a height of two to four meters. the plant could be unbranched, or with only a few side divisions. the leaves alternately distributed, simple, lanceolate, finely serrated or lobed margin. the flowers are small (2-3 cm in diameter) and yellow, it has 5 petals. the fruit of the plant contains many seeds inside in the form of a capsule, corchorus olitorius is an important green leafy vegetable in many areas including egypt, southern asia, japan, india, china, lebanon, palestine, syria, jordan, tunisia, and nigeria, it is a cultivated plant here in iraq. it has diverse common names bush okra, nalta jute, jute mallow and jew’s mallow, ewedu, melokhyia and moroheiya (4,5). taxonomy of corchorus olitorius l. family tiliaceae kingdom: plantae, subkingdom: viridaeplantae, infrakingdom: streptophyta, phylum: magnoliophyta, division: tracheophyta, subdivision: spermatophyta, class: magnoliopsida, order: malvales, family: tiliaceae, genus: corchorus, species: corchorus olitorius l. (6). traditional use different parts of corchorus. olitorius have been utilized to relieve pain, aches, chronic cystitis, dysentery, enteritis and pectoral pain (7), the leaves have been utilized in case of gonorrhea, chronic cystitis, fever, and tumors (8),the seeds were utilized as demulcent, diuretic, purgative, also used in chronic cystitis, in cases of cardiac diseases like heart failure due to its content of cardenolides cardiac glycosides (5,9). it is highly consumed in japan as “healthy vegetable” due to its high content of carotenoids, vitamin c, b1, b2 and e, many minerals and bioactive compounds (5,10). measles is one of the most common communicable diseases around the world and may cause serious complications and sometimes death, about 350 children loss his life every day around the world (11). corchorus olitorius is one of the plants that’s used in folk medicine for the treatment of measles (12). chemical composition of corchorus olitorius l. in general, phytochemical verification that performed on the plant revealed the presence of sterols like: β – sitosterol (13), triterpenes like ursolic acid corosolic acid oxocorocin (14), coumarins like: cichoriine, scopolin(15), saponins and tannins(16), flavonoids like: astragalin (kaempferol 3-o-β-d glucopyranoside) tolifolin (kaempferol 3-o-βdgalactopyranoside) jugalanin (kaempferol 3o-α-l-arabinopyranoside) (17), isoquercetin (quercetin 3-o-β-d-glucopyranoside)(18), carbohydrates (19), phenolics like: 5caffeoylquinic acid (chlorogenic acid) and 3,5dicaffeoylquinic acid(isochlorogenic acid) (15,18,20), cardiac glycosides like: cannogenol3o-β-d-glucopyranosyl –(1 → 4)-o-β-dboivinopyranoside, periplogenin3-o-β-dglucopyranosyl-(1 → 4)-o-β-ddigitoxopyranoside (21), strophanthidin glycosides like: erysimoside(strophanthidin 3oβ-d-glucopyranosyl(1→4)-o-β-d– digitoxopyranoside), olitoriside(strophanthidin 3-oβ-d-glucopyranosyl (1 →4)-o-β-d – boivinopyranoside), corchoroside a ( strophanthidin 3-oβ-dboivinopyranoside) and helveticoside (strophanthidin 3-oβ-d— digitoxopyranoside), digitoxigenin glycosides: glucoevatromonoside (digitoxigenin-3-o-β-dglucopyranosyl ( 1 → 4 ) – o β – d digitoxopyranoside), coroloside (digitoxigenin3-o-β-dglucopyranosyl –(1→4)-o-β-dboivinopyranoside), deglucocoroloside (digitoxigenin 3-o-β-dboivinopyranoside), evatromonoside (digitoxigenin-3-o-β-ddigitoxopyranoside), digitoxigenin 3-o-β-dglucopyranosyl-(1→ 6)-o-β-d-glucopyranosyl(1→4)-o-β-d-digitoxopyranoside (22–24), corchorusoside (a, b, c, d, and e) (25). iraqi j pharm sci, vol.27(2) 2018 phytochemical investigation of corchorus olitorius l. 117 pharmacological activity of corchorus olitorius the plant has several pharmacological activities like antihypertensive effect (26), dermatological activity: improve the score and symptoms of the atopic dermatitis-like lesion (27), and as wound healing effect (28), anti-tumor activity: the plant has a considerable effect against melanoma, leukemia, osteosarcoma (18), hella cells (29), and arh-77(human multiple myeloma cells) (30), antioxidant activity: due to the high content of phenolics and flavonoids (16,20,31), anti-diabetic activity: the plant shows antihyperglycemic and hypolipidemic effects (32), antimicrobial activity: the plant shows a significant antibacterial effect against staphylococcus epidermidis, staphylococcus aureus, bacillus cereus, corynebacterium diphtheria, kosuria rhizophila, shigella flexneri and aeromonas hydrophila. (8), (escherichia coli, and yersinia enterocolitica). also against fungal micro-organisms (geotrichum candidum and botrytis cinerea) (5), anti-inflammatory (7), gastroprotective activity (33), and antiviral activity (12). the aim of this study is to: 1demonstrating an overview of chemical constituent and pharmacological activity of leaves extracts from corchorus olitorius. 2evaluating the in vitro antiviral activity of corchorus olitorius l. leaves extract against measles virus for the first time in the world. materials and methods collection of plant materials corchorus olitorius leaves were harvested from a farm in al-utafiyah district in baghdad city, during july 2017. the plant was identified and authenticated by prof. dr. sukaena abass / department of biology / college of sciences/ university of baghdad. leaves were washed thoroughly, dried under shade, and grinded in a mechanical grinder to a fine powder. equipment and chemical the instruments used were rotary evaporator (bȕchi rotavapor r-205, swiss), microtiter reader (gennex lab/usa), laminar flow hood (k & k scientific supplier/korea), cell culture plates (santa cruz biotechnology/usa), micropipette (cypress diagnostics/belgium), co2 incubator (cypress diagnostics/ belgium) extraction about 150 gm of shade-dried pulverized leaves were defatted by maceration with hexane for 24 hours then filtered and allowed to dry at room temperature. the defatted plant materials were extracted using soxhlet apparatus in which the powder packed in the thimbles and extracted with 1.75l of aqueous methanol 85% as a solvent extraction for 24 hours. the extract was filtered, and the solvent was evaporated under reduced pressure using a rotary evaporator to get a dry extract of about 12 gm (34). about 4 gm from the residue was suspended in 100ml water, about 3-4ml of 5% sodium hydroxide was added to obtain a basic solution having ph 10. and partitioned with ethyl acetate (3x100ml), the aqueous layer collected and evaporated to dryness (35). preliminary phytochemical investigation (34) test for flavonoids 0.1 g of plant extract (aqueous layer) was dissolved in 20 ml of 80% ethanol and filtered. the filtrate was used for the following tests: (a) 3 ml of the filtrate was mixed with 4 ml of 1% aluminum chloride in methanol in a test tube and the color was observed. formation of yellow color indicated the presence of flavonoids. (b) 3 ml of the filtrate was mixed with 4 ml of 1% potassium hydroxide in a test tube and the color was observed. a dark yellow color indicated the presence of flavonoids. test for phenols 0.25 g of each plant extract (aqueous layer) was dissolved in 10 ml of distilled water and filtered. 1% aqueous ferric chloride (fecl3) solution was added to the filtrate. the appearance of intense green, blue or black color indicates the presence of phenols compounds in the test samples. evaluation of the antiviral activity of the aqueous layer experimental design the dried extract (about 0.9gm) obtained from the aqueous layer was used in this test and symbolled as (sample tested compound). cell lines and culture the vero (transformed monkey kidney) cell lines were obtained from the iraq biotech cell bank unit and maintained in rpmi-1640 supplemented with 10% fetal bovine, 100 units/ml penicillin, and 100 µg/ml streptomycin. cells were passaged 37 times including the passage number from the reference, using trypsin-edta reseeded at 50% confluence twice a week and incubated at 37 °c (36). virus cells the schwartz edmonston attenuated measles vaccine strain was obtained from the iraq biotech cell bank unit. cell viability and cytotoxicity (37) to determine the cell cytotoxic effect of the measles virus, mtt (3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide) cell iraqi j pharm sci, vol.27(2) 2018 phytochemical investigation of corchorus olitorius l. 118 viability assay was conducted as on 96-well plates (falcon), vero cells were seeded at 1× 104 cells/well to obtain a multiplicity of infection (moi 10), and at 5 × 103 cells/well to obtain multiplicity of infection (moi 5). after 24hrs. or a confluent monolayer is achieved. cells were treated with cells were treated with different concentrations of the tested compounds (100 μg/ml, 50 μg/ml, 25μg/ml, 12.5μg/ml and 6.25 μg/ml; first: virus alone, virus with tested sample and second: the tested sample alone. the procedure was performed by adding the (virus and or tested sample) at first for 2 hrs. at room temperature to allow virus attachment and penetration. after that, cells were washed with (phosphate buffered saline) pbs and serial dilution of the tested sample was added to the non-infected and infected cells. cell viability was measured after 72 hrs. of infection by removing the medium, adding 28 µl of 2 mg/ml solution of mtt (sigma aldrich-co) and incubating for 3 hrs. at 37°c. after removing the mtt solution by pouring, the crystals remaining in the wells were solubilized by the addition of 130 µl of dmso (dimethyl sulphoxide) (bdh, england) followed by 37°c incubation for 15 min with shaking. the absorbency was determined by a microplate reader (gennex lab. usa) at 492 nm (test wavelength); the assay was performed in triplicate (36). endpoint parameter that is calculated for each individual cell line is the percentage of cytotoxicity, it was calculated by the following equations: 𝑽𝒊𝒂𝒃𝒊𝒍𝒊𝒕𝒚 𝒑𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 = (𝑨 − 𝑩)/𝑨 × 𝟏𝟎𝟎 where a is the mean optical density of untreated wells (control) and b is the optical density of treated (tested) wells. cytotoxicity percentage =100 – viability percentage control test in order to investigate the cytotoxicity effect of the tested sample itself; the vero cells are treated with different concentrations from the aqueous layer. results the phytochemical composition preliminary examination of the aqueous fraction results is shown in table 1. table 1. preliminary examination of the aqueous fraction for phenols and flavonoids. phytochemicals aqueous layer flavonoids + phenols + the anti-cytotoxic or antiviral effect of the plant extract the results demonstrate the significant anticytotoxic effect of different concentrations from aqueous layer against the toxicity effect of measles virus on vero cells using both the obtained multiplicity of infection (moi of 5 and 10).the obtained result against the cytotoxic effect of measles virus is shown in figures (1, 2) and tables (2, 3).the results obtained from the control test are demonstrated in figure (3) and table (4). figure1. cytoprotecting chart; increasing the concentration of the tested sample (aqueous layer) reduce the cytotoxicity of measles virus on vero cells using moi of 10. figure2. cytoprotecting chart; increasing the concentration of the tested sample (aqueous layer) reduce the cytotoxicity of measles virus on vero cells using moi of 5. figure 3. control test. cytotoxicity chart using a different concentration of the tested sample (aqueous layer) against vero cells. iraqi j pharm sci, vol.27(2) 2018 phytochemical investigation of corchorus olitorius l. 119 table 2. the statistical result of the tested sample (aqueous layer) against the cytotoxicity of measles virus using moi of 10. table 3. the statistical result of the tested sample (aqueous layer) against the cytotoxicity of measles virus using moi of 5. table 4. the statistical result of the cytotoxicity of different concentrations from the tested sample (aqueous layer) against vero cells. discussion in the present study, maceration of plant material in hexane for overnight was used as defatting method. next day the solvent removed by filtration and the defatted plant was dried then extracted with alcohol (85% ethanol) to get the crude extract. fractionation of the crude extract is recommended for the separation other, it depends on several factors like differences in the polarity and solubility. polyphenols like (flavonoids and phenolic acids) have acidic properties (38), adding base like 5%naoh will produce water-soluble salts, fractionating the aqueous phase with ethyl acetate will make the lipophilic and nonpolar compounds to stay in the ethyl acetate phase leaving the water-soluble salts (flavonoids and phenolic compounds) in the aqueous phase (35). mtt is a water-soluble yellow dye that is absorbed by living cells and reduced by the action of mitochondrial dehydrogenases. the reduction product is a water-insoluble blue formazan, that must thenbe dissolved for a colorimetric way of measuring by using different types of solvents like dmso. metabolically inactive cells do not produce significant amounts of formazan conversely which can be detected by the microplate reader, the quantity of formazan produced per cell in a given time depends on the metabolic activity of the cells, so formazan production is directly proportionate to the living cell number. the mtt (tetrazolium (mtt) assay for cellular viability and activity) procedure assesses the activity and number of living cells at the end of an experiment (37) . con. moi 10 6.25 μg/ml 12.5 µg/ml 25 µg/ml 50 µg/ml 100 μg/ml mean optical density of triplicate 81.80 75.33 58.37 44.49 34.08 22.20 p value 0.0010 0.0001 0.0009 0.0028 0.0038 0.0044 significant (alpha=0.05) yes yes yes yes yes yes con. moi 5 6.25µg/ml 12.5 µg/ml 25 µg/ml 50 µg/ml 100 μg/ml mean optical density of triplicate 85.17 82.05 65.25 48.30 21.37 9.140 p value < 0.0001 0.0159 0.0171 0.0224 0.0406 0.0790 significant (alpha=0.05) yes yes yes yes yes no con. 100 µg/ml 50 µg/ml 25 µg/ml 12.5 µg/ml 6.25µg/ml mean optical density of triplicate 5.112 4.026 2.750 1.100 0.3225 p value 0.0001 < 0.0001 0.0577 0.0577 0.0130 significant (alpha=0.05) yes yes no no yes iraqi j pharm sci, vol.27(2) 2018 phytochemical investigation of corchorus olitorius l. 120 the results demonstrate the significant anticytotoxic effect of different concentrations from aqueous layer against the toxicity effect of measles virus on vero cells using both the obtained multiplicity of infection (moi of 5 and 10). the obtained result clarifies a linear relationship between the concentration of the tested sample and the degree of cytoprotecting effect (antiviral effect) against the cytotoxic effect of measles virus. the result obtained from the control test shows no cytotoxic effect against vero cells by the tested sample, since even in high concentration which is 100μg/ml only five percent cells were killed, while only 1 percent cell killed at 12.5 μg/ml. this result augments the effect of the tested sample as antiviral (anticytotoxic) against measles virus. natural flavonoid compounds with antiviral activity were identified in 1940, and many reviews were available about their antiviral activity especially against human immunodeficiency virus (hiv). several types of research demonstrate the mechanism of action through inhibition of various enzymes involved in the life cycle of viruses such as hiv-1 inverse transcriptase, dna polymerases, and hiv-1 enzyme proteinase (39). in general, the presence of two vicinal hydroxyl groups on an aromatic ring is the most important structural requirement for inhibition of integrase protein (hiv -in) leading to inactivate viral integration and replication which is the situation with most flavonoids. the dicaffeoylquinic and lchicoric acid derivatives have been proved to show antiviral activity although it doesn’t have such adjacent hydroxyl group (40). it may exert its action by inhibiting the viruscell fusions in the early stage of the replication cycle and by inhibiting the cell-cell fusions at the end stage of the replication cycle (41). conclusions 1. preliminary phytochemical screening is done for the aqueous layer obtained from of iraqi plant corchorus olitorius demonstrate the presence of, flavonoids and phenolics. 2. for the first time in the world, the preliminary study has gathered experimental evidence that aqueous methanolic extract of iraqi plant corchorus olitorius exhibited significant antiviral activity against measles virus. acknowledgment we are deeply grateful to the college of pharmacy, university of baghdad, for giving us the opportunity and facilities to achieve this work. references 1. bagetta g. herbal medicines?: development and validatio of plant-derived medicines for human health. clinical pharmacognosy. p. 2012. 2. evans wc. trease and evans' pharmacognosy. 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2013;65(4):1473-8. 15. yoshikawa masayuki, hiromi shimada, masami saka, satoshi yoshizumi, johji yamaharaa and hisashi matsuda. absolute stereostructures of corchoionosides a,b and c, histamine release inhibitors from the leaves of vietnamese corchorus olitorius l. 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nakamura t, goda y, sakai s, kondo k, akiyama h, toyoda m. cardenolide glycosides from seeds of corchorus olitorius. phytochemistry. 1998;49(7):2097-101. 22. mahato sb, subodh k. cardiac glycosides from corchorus olitorius. 1989;2065-8. 23. goda, yukihiro, sakai shinobu, nakamura takatoshi, akiyama hiroshi t masatake. identification and analyses of main cardiac glycosides seeds and their acute oral toxicity in corchorus to mice olitor. j food hyg soc japan. 1998; 24. contents of strophanthidin glycosides olitorius glycosides in " moroheiya " and digitoxigenin ( corchorus l .) and its products masahiko ogawa *, katuhiro hayasi *, satoko tomimori *, nobuyuki konishi * and osamu nakayama * * mie prefectural science and t. 25. yoshikawa m, murakami t, shimada h, fukada n, matsuda h, sashida y, et al. corchorusosides a, b, c, d, and e, new cardiotonic oligoglycosides from the seeds of corchorus olitorius l.(moroheiya). heterocycles. 1998;5(48):869-73. 26. kimot k, kuroda y, sait y, yamamot 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olitorius. open access libr j. 2016;3(e2225):3-7. 32. salisu j.a., oboh g., schetinger m.r., stefanello n. rjbt. antidiabetic potentials of jute leaf ( corchorus olitorius ) on type-2 diabetic rats. j emerg trends eng appl sci. 2015;6(7):223-30. 33. al batran r, al-bayaty f, ameen abdulla m, jamil al-obaidi mm, hajrezaei m, hassandarvish p, et al. gastroprotective effects of corchorus olitorius leaf extract against ethanol-induced gastric mucosal hemorrhagic lesions in rats. j gastroenterol hepatol. 2013;28(8):1321-9. 34. harborne jb. phytochemical methods; a guide to modern techniques of plant analysis. vol. 3, journal of chemical information and modeling. 1998. 317 p. 35. blackwell dl, herald t, bean sr. alkaline extraction of phenolic compounds from intact sorghum kernels kernels. 2012; 36. yaseen ny. in vitro synergistic enhancement of newcastle disease virus to methotrexate cytotoxicity against tumor cells. 2012;(2):102-9. 37. morgan dml. tetrazolium (mtt) assay for cellular viability and activity. 79(4):179-83. iraqi j pharm sci, vol.27(2) 2018 phytochemical investigation of corchorus olitorius l. 122 38. daigle dj, conkerton ej. analysis of flavonoids by hplc: an update. j liq chromatogr. 1988;11(2):309-25. 39. shashank k, pandey ak. chemistry and biological activities of flavonoids. hindawi sci world j. 2013;2013(12):533-48. 40. introduction i, transfer s, complex p, domain n, domain hc, core ic, et al. virus integrase. 52. 41. li y, but pph, ooi vec. antiviral activity and mode of action of caffeoylquinic acids from schefflera heptaphylla ( l .) frodin. 2005;68:1-9. a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci , vol.17 (2) ,2008 abuse of androgen 9 effects of abuse of anabolic androgenic steroids on iraqi athletes al-muhannad m. taher* , may s. al-sabbagh1,** and dawser k. al-khashali*** * pharmacist at yarmook hospital , baghdad , iraq ** department of clinical pharmacy , college of pharmacy , university of baghdad, baghdad , iraq *** department of pharmacology and toxicology ,college of pharmacy ,university of baghdad, baghdad ,iraq abstract anabolic androgenic steroids (aas) are man-made derivatives of the male sex hormone testosterone, originally designed for therapeutic uses to provide higher anabolic potency with lower androgenic effects. increasing numbers of young athletes are using these agents illicitly to enhance physical fitness, appearance, and performance despite their numerous side effects and worldwide banning. today, their use remains one of the main health problems in sports because of their availability and low price. the present study focuses on investigating the adverse effects of anabolic androgenic steroid abuse on sex hormones, liver and renal function tests, fasting glucose levels and lipid metabolism in iraqi male recreational bodybuilders. we have recruited fifteen male bodybuilders (age 19-32 years) and an equal number of healthy non-obese, non-aas-using sedentary controls. serum hormones (luteinizing hormone (lh), follicle stimulating hormone (fsh), total testosterone, and prolactin), liver function indices (serum alanine aminotransferase (alt), aspartate aminotransferase(ast), alkaline phosphatase(alp), total and direct bilirubin), renal function parameters (serum creatinine and urea), lipid profile and serum glucose levels were measured. abuse of aas was associated with significant decreases (p< 0.005) in serum levels of lh (66.9%), fsh (49.8 %) and testosterone (63.7%) together with significant increases (p< 0.05) in prolactin concentrations (49.8%) in aas-using bodybuilders compared to sedentary controls. anabolic androgenic steroidsusing athletes had significantly higher (p< 0.05) circulating levels of total bilirubin (116.3%), direct bilirubin (127.6%), aspartate (1752.9%) and alanine (263.1 %) transaminases than those of sedentary control subjects. serum alkaline phosphatase levels were not significantly different (p> 0.05) between the two groups. concerning renal function, aas-using athletes had significantly higher serum concentrations of creatinine (28.6%) and urea (21.3%) than sedentary controls. meanwhile, aas abuse was accompanied by atherogenic lipid profile. anabolic androgenic steroids -using athletes had significantly higher (p< 0.05) serum levels of triglycerides (tg) (45.6%), low density lipoproteincholesterol (ldl-c) (26.0%) and very low density lipoprotein-cholesterol(vldl-c) (45.6%) together with significantly lower serum concentrations of high density lipoprotein-cholesterol (hdl-c) (31.3%) than sedentary controls. serum total cholesterol (tc) and fasting glucose concentrations were not significantly different (p> 0.05) between the two groups. the results presented in the study confirm that abuse of aas induces unfavorable body functions and undesirable side effects. therefore, efforts should be sought against use of these compounds outside the therapeutic frame. key words: anabolic steroids, athletes, bodybuilding, exercise. ةصالخلا ( و بلوع ى د قل دصت صا تصث تدةُ ي testosterone اد تدريخلات ادذلق دو رح تاقت ادير ةي ادبلرج اديوريت لا ) ( لي ترضر يا ح .ابلر ادلعانقت يي يتلا ادرخقردد ادبخ خاذصةي تصث اوقكح .ي ربد اديرلذقت testosterone قي ادا ) لليي ا رخغ كذح لح ا اخل صرة ليريع اد لد تصث ادرحي ادتربخرات اديتلرعن نةل يتراريق ادلق ذدو ي تصث ادرحي يرد اديلجيقت اويديذدو ي ادليقت ادرلة دو تاةلقت و قع و تصث ختوقكث ربد اديةا يي ختقمر ليق .خوتذر اونتالال اداقكا ديبد اديرلذقت ذي ادرخقردد يري قلي ادرخقرو لح ادو ر ادرلخا .ابمرخع ربد ادلعانو ي و ييع دتاددي اا درات اوقكح ربد اد تدريخلات ادذلق دو تصث تةت ادير ة قت لح ي ادلل ي يظق و ادلذل ي ادلصث ي تةت اةتخد اد رةل ي تةت اد لر لح ( عخقردقل ختوقكةي اد تدريخلات ادذلق دو ي 15 ي ادلل ديليةتو يقعنح عخقرو ليقل اام قل .امتيصع ربد ادلعانو تصث ) تتيلت كري ادتاددي تصث دقط تةت ( تاةتقل و خيقعنةي عخقرو ليقل اام قل ي و ختوقكةي اد تدريخلات ادذلق دو.15) ,total and direct bilirubin( ي يظق و ادلذل )lh, fsh, testosterone, and prolactinادير ة قت لح ي ادلل ) sast, salt, and alkaline phosphatase( ي يظق و ادلصث )s creatinine and s urea ي تةت اةتخد اد رةل لح ) ( لقيرقلو دث ارلد اد لر لح ادلل .يظير ارصدي ادذدق قت اا درال بي tc, tg, hdl-c, ldl-c, and vldl-c ي ادلل ) . ديو ولةخو ياررو دتوقكح اد تدريخلات ادذلق دو تصث ارلد ادير ة قت لح ي ادلل 1 corresponding author : e-mail : may_sabbagh @ yahoo.com received : 17/6/2008 accepted : 16/9/2008 iraqi j pharm sci , vol.17 (2) ,2008 abuse of androgen 10 ( ل ةعن ولةخو prolactin( ي عافد تةت )lh, fsh, and total testosterone ندا دةنا افقي ولةج لح ارالد ادا ) total and direct bilirubin, sastدلت توقكح اد تدريخلات ادذلق دوا ارال بد د تخق ن ولةخو ياررو لح تةخقت ادا ) and salt( د تلل يمة يج لري ولةج لح تةت ادا )alkaline phosphatase لد اديليةتتد .يظيرت ادلعانو يخضقل ) ( لح ي ادلل دلت توقكح اد تدريخلات ادذلق دو لقيرقلو دث عافقى ولةج لح تةت creatinine and urea عافقى تةت ادا ) ( لح ي ادلل .دي ختي منجو يج لري ولةج لح hdl-c( ي ا افقي ولةج لح تةت )tg, ldl-c, and vldl-cادا ) ( ي اد لر لح ي ادلل لد اديليةتتد .لح رةر ادلتق ادتح يلرتايق ربد ادلعانوا خيلللق انتلتق يي نتالال tc تةت ) اد تدريخلات ادذلق دو ىقع كقع ادوم خر ج دث ن ةل ضقتفقت ار ر نصذقل تصث يظق و م ي اي قي .يامذلق خلي لح لبل قعت ادلية يمي ااصدي يي لد انتالال ربد اديرلذقت ىقع اوكقع ادوممح . introduction self-administration of large doses of anabolic androgenic steroids ( aas ) by athletes to obtain a well-shaped body and to increase muscular strength has been increasingly noticeable since the 1950s.1,2 anabolic androgenic steroids are widely used by professional and recreational athletes as well as nonathletes.3 abuse of aas is not limited to male adults but also reported in female adults as well as adolescents of both sexes.1 every tissue in the body, including the brain, has androgen receptors; therefore, aas exert systemic as well as psychological effects.4 anabolic androgenic steroids have been linked with a wide range of unwanted adverse effects. these effects may range from physically unattractive, such as acne and gynecomastia in males, to serious and life threatening, such as cardiovascular diseases and hepatic carcinoma. most effects are reversible upon withdrawal.2,5 because of their widespread use, many side effects may turn out to be significant risk factors when considering public health. increased risk of violent death was reported among aas abusers from impulsive, aggressive behavior, or depressive symptoms.6 anabolic androgenic steroids have been taken in cycles. traditionally, aas users combine two or more different drugs, mixing oral and injectable aas. they begin a cycle with a low dose of aas and slowly increase the dose and then the dose is tapered to zero.3 doses taken by abusers can be 10-100 times higher than those used for medical purposes. the aim of the present study was to evaluate the changes in serum sex hormones, liver and renal function indices, glucose level and lipid metabolism in iraqi male anabolic androgenic steroid abusers. material,subjects and methods participants fifteen non-obese male bodybuilders aged 19-32 years (mean 23.27 ± 3.73) were recruited at local gyms in baghdad city . bodybuilders were interviewed concerning their health (current diseases and family diseases), consumption of high protein diet, regular exercise, lifetime steroid abuse, pattern of use, and whether other supplements and drugs being used. exclusion criteria included smoking, alcohol consumption, presence of chronic medical conditions (diabetes mellitus, liver or kidney disorders) and the use of growth hormone. anabolic steroid abusers were selected if they were currently using aas. table (1) summarizes aas used with their doses and duration of use prior to sample withdrawal. . all of the participants took androgens in cycles and none was taking aas in a continuous pattern. cycles were 4-8 weeks in duration separated by suspension periods of 4-12 weeks. a control group of healthy sedentary males (n=15) with a mean age of 22.1 ± 3.65 years were recruited from the community and historically had not ever used anabolic steroids. unfortunately, absence of bodybuilding controls was evident because, as was found, persons who continue to exercise regularly use aas routinely. the two groups of volunteers were comparable with respect to their age and height. however, the study group taking aas had significantly greater mean weight and bmi. sample collection subjects' weight and height were measured using a balance beam and a vertical ruler. participants were asked to fast for 12 hours and avoid heavy physical exercise before attending for sample collection. one blood sample was collected from each volunteers by venipuncture between 08:00-10:00 am. a total of 15 ml blood was obtained and placed in edta-free tubes to be centrifuged for 5-10 minutes at 3000 rpm. serum was then divided into several 1.5 ml eppendorf tubes and stored at (-20oc) until time for the assay. laboratory measurements serum concentrations of lh, fsh, prolactin and total testosterone were determined by immunofluorometric assays on a mini vidas analyzer 7,8 . liver and renal function indices were measured by colorimetric methods using the commercially available kits9,10,11,12,13,14,15. serum total cholesterol, triglyceride, highdensity lipoprotein cholesterol and fasting glucose concentrations were determined by iraqi j pharm sci , vol.17 (2) ,2008 abuse of androgen 11 routine autoanalyzer methods16,17,18,19. serum lowand very low-density lipoprotein cholesterol concentrations were determined through the friedwald formula20. table 1 : dose and duration of aas use for fifteen body builders statistical analysis data were expressed as mean ± sd (standard deviation). unpaired t-test was employed to examine the difference in means of the aas-using group and sedentary controls. pearson correlation (r) was used to analyze the relationships between total dose of aas used prior to sample withdrawal and the hormonal and biochemical changes. a level of p value < 0.05 was considered statistically significant difference. results serum hormone levels serum lh, fsh, and total testosterone levels in aas-using bodybuilders were significantly lower (p < 0.005) than those in the sedentary controls (66.9%, 49.8%, and 63.7% respectively). however, aas-using bodybuilders had significantly higher (p< 0.05) prolactin concentrations (49.8%) than sedentary controls (table 2). subject no, aas used route of administration dose (mg/kg) duration of prior sampling(wk) total dose received (mg) 1 methandrostenolone o 175 6 1350 nandrolone decanoate p 50 2 methandrostenolone o 140 4 660 nandrolone decanoate p 25 3 methandrostenolone o 140 4 760 nandrolone decanoate p 50 4 methandrostenolone o 245 4 980 5 methandrostenolone o 210 4 840 6 testodterone proponate p 50 3 450 nandrolone decanoate p 100 7 mthenolone o 20 3 660 oxymetholone o 150 nandrolone decanoate p 50 8 methandrostenolone o 175 6 1350 nandrolone decanoate p 50 9 methandrostenolone o 140 4 760 nandrolone decanoate p 50 10 methandrostenolone o 175 6 1650 nandrolone decanoate p 100 11 methandrostenolone o 245 4 2780 sustanon p 250 nandrolone decanoate p 200 12 methandrostenolone o 175 4 1800 nandrolone decanoate p 25 sustanon p 250 13 methandrostenolone o 175 4 1100 nandrolone decanoate p 100 14 methandrostenolone o 245 6 2670 nandrolone decanoate p 200 15 methandrostenolone o 245 6 2670 nandrolone decanoate p 200 iraqi j pharm sci , vol.17 (2) ,2008 abuse of androgen 12 table 2: effects of anabolic androgenic steroids on serum hormones in aas-using bodybuilders compared to sedentary controls. variable sedentary controls n=15 (mean ±sd) aas-using bb n=15 (mean ± sd) lh (miu/ml) 3.11 ± 0.94 1.03 ± 1.09** fsh (miu/ml) 2.93 ± 1.30 1.47 ± 0.87** testosterone (ng/ml) 7.47 ± 1.95 2.71 ± 1.75** prl (ng/ml) 14.44 ± 6.19 21.63 ± 8.88* values expressed as mean + sd the pvalues refer to the differences from the control group. * : p < 0.05 significant difference between the two groups. ** : p <0.005 highly significant difference between the two groups. n = no. of subjects. table 3: effects of anabolic androgenic steroids on liver function tests in aas-using bodybuilders compared to sedentary controls. .variabl e sedentary controls n=15 (mean ± sd) aas-using bb n=15 (mean ± sd) total bilirubin (mg/dl) 0.49 ± 0.63 1.06 ± 0.74* direct bilirubin (mg/dl) 0.29 ± 0.41 0.66 ± 0.47* alp (u/l) 80.20 ± 20.26 93.20 ± 37.19 ns ast (u/l) 1.21 ± 4.69 22.42 ± 27.02** alt (u/l) 4.69 ± 6.52 17.03 ± 17.02* values expressed as mean + sd the pvalues refer to the differences from the control group. * : p < 0.05 significant difference between the two groups. ** : p <0.005 highly significant difference between the two groups. ns : non significant (p > 0.05 ) significant difference between the two groups n = no. of subjects. liver function parameters anabolic androgenic steroids-using bodybuilders had significantly higher (p< 0.05) circulating levels of total and direct bilirubin (116.3% and 127.6% respectively) than sedentary controls. anabolic androgenic steroids -using bodybuilders had significantly higher serum ast (1752.9%, p< 0.005) and alt (263.1%, p< 0.05) activities than sedentary controls (table 3). serum alkaline phosphatase levels were not significantly different (p> 0.05) between the two studied groups. renal function tests (table 4) demonstrates that aas-using bodybuilders had significantly higher circulating levels of creatinine (28.6%) and urea (21.3%) (p< 0.005 and p< 0.05, respectively) than sedentary control group. table 4: effects of anabolic androgenic steroids on renal function tests in aasusing bodybuilders compared to sedentary controls. values expressed as mean + sd the pvalues refer to the differences from the control group. * : p < 0.05 significant difference between the two groups. ** : p <0.005 highly significant difference between the two groups. n = no. of subjects. lipid profile and fasting serum glucose circulating levels of hdl-c were significantly lower (p< 0.005) (31.3%) in aas-abusing bodybuilders than sedentary controls. table (5) indicates that aas-using bodybuilders had significantly higher (p< 0.05) serum levels of triglycerides (45.6%), ldl-c (26.0%) and vldl-c (45.6%) than sedentary controls. serum total cholesterol and glucose concentrations were not significantly different (p> 0.05) between aas-using bodybuilders and sedentary control subjects. variable sedentary controls n=15 (mean ± sd) aas-using bb n=15 (mean ± sd) cr (mg/dl) 0.84 ± 0.19 1.08 ± 0.21** urea (mg/dl) 38.07 ± 8.58 46.18 ± 14.37* iraqi j pharm sci , vol.17 (2) ,2008 abuse of androgen 13 table 5: effects of anabolic androgenic steroids on lipid profile and serum glucose in aas-using bodybuilders compared to sedentary controls variable sedentary controls n=15 (mean ± sd) aas-using bb n=15 (mean ± sd) cholesterol (mg/dl) 153.80 ± 21.62 171.20 ± 37.19 ns tg (mg/dl) 74.93 ± 42.84 109.13 ± 57.50* hdl-c (mg/dl) 44.60 ± 7.15 30.67 ± 7.64** ldl-c (mg/dl) 94.21 ± 21.13 118.71 ± 34.76* vldl-c (mg/dl) 14.99 ± 8.57 21.83 ± 11.50* fsg (mg/dl) 83.20 ± 17.63 89.93 ± 9.16ns values expressed as mean + sd the pvalues refer to the differences from the control group. *: p < 0.05 significant difference between the two groups. **: p <0.005 highly significant difference between the two groups. ns non significant (p > 0.05 ) significant difference between the two groups n = no. of subjects. adverse effects participants were asked questions about unusual adverse effects that would be felt during an aas cycle and the most common reported side effects were aggression, changes in libido, acne formation, headaches, and premature hair loss as summarized in table 6. table 6: adverse effects reported by aasusing bodybuilders. adverse effect no. of subjects n=15 % unusual aggression 8 53 changes in libido 6 40 acne 3 20 headaches 4 27 hair loss 2 13 discussion subjects of this study use independently anabolic androgenic steroids mainly to enhance external physique. besides being an unethical practice, abuse of aas has been associated with several health risks and various adverse effects which affect almost all organs and systems of the human body. anabolic androgenic steroids-using bodybuilders had significantly lower (p< 0.005) serum levels of lh, fsh and total testosterone than sedentary controls (table 2). the results were consistent with those reported by holma et al21 who observed reduced serum levels of lh, fsh and testosterone in athletics during a course of oral intake of methandrostenolone (15 mg/day). exogenously administered anabolic androgenic steroids exert a negative feedback on the secretion of gonadotrophins, mostly attributed to a direct effect on the hypothalamus to decrease secretion of gnrh. this in turn causes a corresponding decrease in secretion of both lh and fsh and eventually biosynthesis and release of testosterone from the testes.22 in addition, anabolic androgenic steroids may produce local suppressive effects on the testes and on adrenal androgen production.23 serum prolactin levels in aasusing bodybuilders were significantly higher than those in sedentary controls (p < 0.05) (table 2). data reported by stoffel-wagner et al24 and leibenluft et al25 were consistent with the interpretation that testosterone and/or its metabolites facilitate the secretion of prolactin. estrogen is known to stimulate prolactin release from the anterior pituitary.26 nonaromatizable aas (stanozolol and methandrostenolone) were reported to activate estrogen receptors through interaction of either the parent compound or its metabolites indicating a possible mechanism for increased prolactin secretion.27 the available data in the corresponding literature on the influence of exogenously administered androgens on prolactin serum level were found controversial. serum total and direct bilirubin levels in aasusing bodybuilders were significantly higher (p< 0.05) than those in sedentary controls (table 3). androgens can selectively interfere with bile excretion by the liver. canalicular bile plugs were observed after treatment with methyltestosterone, oxymetholone, mestranol, and norethandrolone.28 cases of cholestatic jaundice have been recorded in patients therapeutically using or athletes abusing aas (especially 17 alkylated agents).29,30 serum ast and alt levels in aas-using bodybuilders were significantly higher (p< 0.005 and p< 0.05, respectively) than those in sedentary controls (table 3). canalicular cholestasis is characterized by mild hepatocellular injury and release of transaminases leading to mild elevations in serum levels of these enzymes.29 however, since sustained weightlifting alone can result in mild elevations in serum transaminase iraqi j pharm sci , vol.17 (2) ,2008 abuse of androgen 14 activities31,32, the increase in serum transaminases may be attributed to mild hepatocellular damage, muscle injury, or both. urhausen et al33reported that serum transaminase levels were significantly higher (p< 0.001) in anabolic androgenic steroidabusing athletes than bodybuilders who stopped using anabolic steroids for at least a year. a non-significant difference in serum alp levels was found between the two studied groups (p > 0.05) (table 3). these results are consistent with those reported by o'sullivan et al34 who observed no significant difference in alkaline phosphatase activities between anabolic steroid users and potential or past users. anabolic androgenic steroids can induce cholestasis without elevating alkaline phosphatase levels. alp activity is usually less than threefold elevated and often is normal.35 anabolic androgenic steroids -using athletes had significantly higher serum creatinine (p< 0.005) and urea (p< 0.05) levels than sedentary controls (table 4). studies in rat models provide evidence that, compared with females, aging males exhibit decreased glomerular filtration rate and develop glomerular injury, glomerulosclerosis and proteinuria.36 in addition, cases of acute renal failure had been reported in clinical patients or bodybuilders administering anabolic steroids.29,37 however, in the present study, we cannot ignore other factors that may have participated in deteriorating renal function parameters in anabolic androgenic steroid-using athletes e.g. consumption of high protein diet. serum concentrations of triglycerides, vldl-c and ldl-c were significantly higher in aasuseres (p< 0.05) than those in controls (table 5). the rise in serum levels was positively correlated with the intake of aas. anabolic androgenic steroids can elevate serum levels of triglycerides by 40-50% in bodybuilders and other power-training athletes.38 kiraly39in 1988 reported similar results while studying the effects of large doses of testosterone and other anabolic androgenic steroids on serum lipids during a 12 week strength-training period. elevated serum triglyceride (p< 0.05) levels were found with decreased serum hdl-c (p< 0.005). serum ldl-c levels were significantly higher (p< 0.05) during steroid intake in studies reported by fröhlich et al40 and palatini et al41. anabolic androgenic steroids -users had elevated levels of apolipoprotein b, a component of both ldl and vldl.42,43 conversely, circulating levels of ldl-c and vldl-c were not significantly different while using aas in studies reported by sader et al44 and singh et al45, respectively. a nonsignificant difference in serum total cholesterol levels was found between the two studied groups (p > 0.05) (table 5). our results confirm those reported by many studies40,42 . anabolic androgenic steroids effects on plasma lipids have been reported to be unpredictable and depend on the dose, route of administration, and type of aas (aromatizable or not).46 low dosages have been associated with hypolipemic response, while high doses have had opposite effects.47,48 serum hdl-c levels in aas-using bodybuilders were significantly lower than those in sedentary controls (p < 0.005) (table 4). the postulated mechanism to explain anabolic steroid-induced alteration in serum hdl-c levels is an increase in hepatic triglyceride lipase activity, an enzyme responsible for catabolizing hdl with its phospholipase activity.49 in addition, apolipoprotein a-1, a major component of hdl particle, was reported to be decreased by aas.42,45 the results obtained in the present study showed absolute consistency with the available data. a non-significant difference in serum glucose levels was found between the two studied groups (p > 0.05) (table 5). the influence of testosterone and anabolic steroids on glucose metabolism was found controversial. results of the present study agree with those reported by friedl et al50 who observed no alterations in fasting serum glucose in normal men treated with testosterone enanthate or nandrolone decanoate for 6 weeks. on the other hand, cohen and hickman51 concluded that power lifters taking high dose (mean 200 mg/day) of anabolic androgenic steroids had diminished glucose tolerance compared to non-steroid using athletes, obese sedentary men, or non-obese sedentary men. such controversy in the corresponding literature may be explained to be due to differences in doses used. higher aas doses reduce insulin sensitivity and impair glucose tolerance.46 although aas doses used by subjects in the present study were considered to be high; they were much smaller than those used by athletes in cohen and hickman51 study. the most common side effects reported by our subjects were unusual aggression (53%), changes in libido (40%), acne formation (20%), headaches (27%) and premature hair loss (13%) (table 6). perry et al52 reported that anabolic steroid using weightlifters were more aggressive than nonusers according to different psychiatric scores. changes in libido appear to be the most common adverse effect reported in a group of present and past aas users (approximately 61%).34 reports do indicate that toward the end of aas cycle, some males may experience loss of libido.53 acne was also found very iraqi j pharm sci , vol.17 (2) ,2008 abuse of androgen 15 common side effect among anabolic steroid users as reported by o'sullivan et al.34 increases in acne formation is related to stimulation of sebaceous glands resulting in a more oily skin.34 premature hair loss does not appear to be very common. it is likely that androgenic alopecia as a result of aas use is more prevalent in males who are genetically predisposed to balding.54 headaches are also not very common among aas abusers. o'sullivan et al34 reported only 9% of aas using athletes may develop headaches. however, the exact mechanism is unknown. conclusion in conclusion, anabolic androgenic steroid abuse lowered serum concentrations of pituitary gonadotrophins, lh and fsh, and testosterone. increased levels of prolactin were also manifested. abuse of anabolic steroids probably 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403-415. effect of gallic acid on cholinesterase iraqi j pharm sci , vol.18 (1) , 2009 effect of gallic acid on cholinesterase 33 the inhibitory effect of gallic acid on human serum cholinesterase sabah m.k. al-shafi *,1 , mohamed j.al-azzaw ** and methal a. ali * * department clinical laboratory sciences, college of pharmacy, university of baghdad ,baghdad iraq. ** department clinical laboratory sciences collage of pharmacy. university of baghdad, baghdad iraq. abstract the dried fruit peel of pomegranate in punicaceae family was fractionated chromatographically on sephadex-lh-20 column .gallic acid (trihydroxybenzoic acid) and its related galloyl esters such as gallotannin(i.e. β-penta-o-galloyl–d-glucose) were obtained homogenously. different concentrations of gallic acid and gallotannin were used to determine their inhibitory effect on human serum cholinesterase. the enzyme activity was measured according to the method reported by the who .the inhibitory effect of these compounds on the activity of human serum cholinesterase have been studied in vitro .the inhibitory effect was remarkably clear with increasing concentration of gallic acid .whereas galloyl ester showed no inhibitory effect. the inhibition with gallic acid indicates a noncompetitive pattern. therefore, we can not recommended gallic acid and its related compounds , as preservative substances in food industry or in pharmacological preparations since they might have some side effect on certain biological systems.. key words: gallic acid , gallotannin , human serum cholinesterase . الخالصة انعممٌ بٌاسمة (punicaceae family )رمتنٍم ان عتئهم ان انرممت مم نثممر انجتصم قشمٌ ان مم مركبمت انبعم تم صلمم trihydroxybenzoic ىمت حتم انكتنٍك ) مت تٍ متجتنستٍ ت انحلٌل عهى ً.02نـانستئم انهٌنً انحتًي عهى مت انسفت كس أ benzoic acid )تانكتنٌتمممت حم مركبمت أسمترا انكتنٌنٍمم انتتبعم نمو ًىمً ًأ( ٍنβ-penta –o-galloyl –d-glucose ). أ اسممتم مت تراكٍمم ممتهفمم ممم حممتم انكتنٍممك . انكتنٌتممتنٍ نمم تمم بشممكم ًاسممل عهممى صعتنٍمم أ ن ٌمممت ً تمميرٍر حممتم انكتنٍممك ٍتسميت ببقمتتم اإلنم ٌ ىماا أ صعتنٍم . انبشمري cholinesterase) )عهمى ملمم انكٌنٍسمترٌ يممتتثبٍة ًانكتنٌتمتبٍ نقٍمت تميرٍر انبشمري اختٍر نتقٍٍ صعتنٍ ىاه انمركبت ممتبرٌت عهى ملم انكٌنٍسمترٌ . ىاه ان اس .who) ةرٌق منظم انلح انعتنمٍ )ن نم كٍم حمتم انكتنٍمك بٍنممت ىاه انمركبت جم ٌر بتنمححظم ممل دٌمت بترا تثبٍط أ تيرٍر ن ٌت معرصتنت حٌل انفعتنٍت انبٌٍنٌجٍ . ٌصمى نأ ال نسمتةٍل نمانك . بةبٍعتوغٍر تنتصسً وتكشف بين كأ انتثبٍط بحتم انكتنًٍ. معنٌي نمت انكتنٌتتنٍ تثبٍط أي ٌححظ حتصظ صً انلنتعت انغاائٍ أً صً انتحضٍرا انلٍ النٍ نظرا" الحتممتل احتٌائيمت كمٌا نو حتم انكتنٍك ًانمركبت انتتبع ب . انبٌٍنٌجٍ ا نظم بع بع انتيرٍرا انجتنبٍ عهى عهى introduction phenolic compounds are secondary plant products which rarely occur in the free state in living plant tissue. simple phenols are caustic substances and well known to be antimicrobial agents (1-3) . polyphenols like lignin and tannins are also found in plant cells. tannins or tannic acids are believed to be the most important group of secondary metabolites involved in plant defense (4-6) . it has been found that tannins have shown potential antiviral (7,8) , antibacterial (9,10) and antiparasitic effects (11) . in the past few years tannins have also been studied for their potential effects against cancer through different mechanisms (12-14) . tannic acids are not single homogeneous compounds, but a mixture of esters of gallic acids with glucose whose exact composition varies according to their sources (15) . the biological activation of gallic acid and its related galloyl esters have not yet been studied widely regarding their effect on enzymes. they are employed in medicine as astringents in the gastrointestinal tract (git) and on skin abrasions. in the treatment of burns, the proteins of the exposed tissues are precipitated to form a mildly antiseptic, protective coat under which the regeneration of new tissues may takes place (16 . ( many plant species native to iraq are known to contain certain chemical compounds which exert their effects on different biological system within the cellular level such as enzymes (17-19) . chemically, these complex substances are usually occur as a mixture of polyphenols that are difficult to separate because they do not crystallize , the application of some chromatographic methods has enabled to confirm the complicated nature of these polyphenolic extracts and also to identify the simple phenols present in small amounts in such mixtures (20,21) . in addition, it is of interest to improve methods of separation and identification of gallic acid and some of its related esters obtained from iraqi plants . aim of this work was conducted to study the effect of gallic acid and its related glucose esters such as gallotannins on human serum cholinesterase in vitro. 1 corresponding author e-mail : alshafisabah@ yahoo.com received : 3/11/2007 accepted : 8 / 3/2009 http://en.wikipedia.org/wiki/tannin#cite_note-33#cite_note-33 iraqi j pharm sci , vol.18 (1) , 2009 effect of gallic acid on cholinesterase 34 materials and methods extraction of plant material: the fruit peel of punicaceae in pomegranate family were obtained from iraqi market. the peels of healthy fruit were dried at room temperature for at least six months, before they were powdered in a mortar and the powder was sieved through 100-150 mesh sieve.the powder (50g) was boiled for few minutes in 100ml ethanol (95%) and left with stirring for at least 3hr. the extract was decanted through fiber glass. the remaining residue was re-extracted twice with ethanol and the combined extracts were concentrated in vacuo to remove ethanol, the heavy viscous residue obtained as the phenolics materials (20,21). isolation and identification of phenolic substances: phenolic materials were chromatographed on whatman no.1 paper in two dimensions with 6% (v/v) acetic acid (solvent a) and isobutanol-acetic acid-water (14:1:5) (solvent b)at 25+1 o c . phenolic compounds such as gallic acid and gallotannin (tabel-1)were revealed by spraying with a freshly prepared reagents (15,21-23,) of ferric chloride-potassium ferricyanide , gibbs reagent ,saturated aqueous potassium iodate(kio3) reagent , and finally a fresh solution of nitrous acid reagent was prepared to give the phenolic compounds a characteristic color which helps in their identification . the phenolic extracts (10g) obtained as mentioned above was fractionated chromatographically on sephadex lh-20 column (100x2.5cm) using the same methods as described previously (21,24) . sephadex lh-20 is very useful for separation tannin from nontannic phenols (25) .table-2 showed two substances (i.e. gallic acid and gallotannin) were obtained homogenously by fractionation . the resultant substances ,as in the following : 1-gallic acid : fractions 2 ( 130 ml) was dried at 25 o c and 0.01mmhg over phosphorous pentoxide and rechromatographed once again over sephadex-lh-20 column . the dried substance gave a pale-yellow-white form melting point (m.p.) 250-253 o c. rf values, showed 0.52 and 0.6 with solvent a and b respectively .table-1 shows paper chromatograms when treated with a freshly prepared reagents revealed a characteristic colors exhibited by this compound. so under short -u.v. light (254nm) the chromatogram. gave soft blueviolet appearance in visible light which turned to deep violet upon exposure to fuming ammonia. elemental analysis found ; c, 44.73% ,h, 4.34 %, calc. for c7h6o5 , as amorphous compound ,(lit 25 c , 44.70 % ,h,4.31%). 2gallotannin;.:fraction 6 (105 ml) was dried and then rechromatographed in the same procedure as did for gallic acid . the dried substance gave an off-white granular solid; m.p. 200-210 o c. rf values on paper chromatogram showed 0.06 and 0.5 with solvents a and b, respectively.table-1 shows a characteristic colors exhibited by glallotannin on paper chromatograms when sprayed with reagents .gallotannin normally showed up as brown-purple spot on chromatograms when treated with gibbs reagent. the chromatogram also showed a pink appearance with ferric chloride-potassium ferricyanide in visible light which turned to darkblue absorption in u.v. light which enhanced by fuming with ammonia .specific spray for gallotannins is potassium iodate solution, which gives a rose –pink color and reacts with gallic acids to form the characteristic orange of purpurogallin carboxylic acids (26,27) ..elemental analysis showed; c, 52.38 % ; h , 3.48 %; calc for c41h32o26 , as amorphous compound , (lit 25 .c,52.35 %,h, 3.50 %) . table(1) : detection of gallic acid and gallotannin by various sprays . compound spray u.v light ferric chloride potassium ferricyanide gibbs reagent potassium iodate nitrous acid gallic acid blue brown orange→red→pink brown blue-violet→deep ep violet gallotannin pink brown purpule rose →pink→orange brown dark blue iraqi j pharm sci , vol.18 (1) , 2009 effect of gallic acid on cholinesterase 35 enzyme activity determination: different concentrations (ranging from 6mm/l to 30mm/l )of gallic acid and gallotannin were used to determine their inhibitory effect on human serum cholinesterase in vitro. the enzyme activity was measured according to the method reported by the who (28) , with minor modification as described in previous study (17) . enzyme activity was expressed as µ moles of substrate (acetylthiocholine iodide) hydrolyzed per ml of total mixture per min. results and discussion chromatographic results for the fractions of the extract to detect the phenolic substances as compared with authentic compounds (tables-1 and2) showed that fraction 2 was identified as gallic acid, whereas fraction 6 identified as gallotannin .also the u.v. light detection ,various sprays, melting points and elemental analysis were applied to identify these compounds . these findings agree with standard authentic compounds were obtained from pharmacy college stores ( i.e. gallic acids and gallotannin) which showed in such close agreement as to indicate the identity of the substance previously .also these finding agree with earlier reports (15,20 ,25,27) no clear inhibitory effect could be detected with different concentrations of gallotannin ( ranging from 6 mm/ l to 35mm/l ) on human serum cholinesterase. this might be attributed to the presence of amber color which may interfere with the color developed as a result of enzymatic reaction. decolorization of this solution might merit different results.the inhibitory effects of different concentrations of gallic acid on the enzyme activity were summarized in table 3 .it was found that increasing gallic acid concentrations will accordingly affect enzyme activity. gallic acid concentration as low as 6mm/l results in approximately 10% inhibition (p<0.001) and reaching to about 50% inhibition with increasing concentration as high as 30 mm/l (p<0.0001). such highly significant inhibition was unexpected firstly, because gallic acid which was considered as one of the phenolic compounds used as antimicrobial agent in industry (20,29) , and secondly, it has not been reported before that phenolic compounds derived from native iraqi plants exert such inhibitory effect on this enzymatic system i.e. human serum cholinesterase , a well-known biological function of being a neurotransmitter in animals and insects (30) , its has a very high catalytic activity , that catalyzes the hydrolysis of the neurotransmitter acetylcholine into choline and acetic acid, a reaction necessary to allow a cholinergic neuron to return to its resting state after activation (31) .furthermore, the reciprocal line weaver-burk plot for the rate of reaction versus substrate concentration in the presence and absence of gallic acid (fig.1) showed that the inhibition follows a noncompetitive pattern. this might be explained by that the inhibitor can not bound to the anionic site in the catalytic centre of the enzyme (32) . the mechanism of action of such bounding might be explained through the hydrogen bounding between the carboxyl group of the inhibitor and some catalytically significant group of the enzyme probably with the imidazole moiety of histidine in the static site of the enzyme molecule (33) . table(2) : fractionations of gallic acid and gallotannin by column chromatography using sephadex lh-20 substance weight after drying (g) elution solvent volume (ml) fractions unknown 5.56 ethanol (100%) 010 1 gallic acid 1.42 ethanol (100%) 132 2 mixture of gallic acid and other phenolic substances . 1.70 ethanol (90%) 131 3 ethanol (90%) 101 4 ethanol (90%) 111 5 gallotannin 0.68 ethanol (70%) 121 6 polyphenolic substances 0.54 ethanol (60%) 191 7 http://en.wikipedia.org/wiki/catalyst http://en.wikipedia.org/wiki/catalyst http://en.wikipedia.org/wiki/hydrolysis http://en.wikipedia.org/wiki/neurotransmitter http://en.wikipedia.org/wiki/acetylcholine http://en.wikipedia.org/wiki/choline http://en.wikipedia.org/wiki/acetic_acid http://en.wikipedia.org/wiki/cholinergic iraqi j pharm sci , vol.18 (1) , 2009 effect of gallic acid on cholinesterase 36 table(3) :in vitro inhibition of human serum cholinesterase by different concentrations of gallic acid. recovery % % inhibition enzyme activity u/ml inhibitor mm/l 100.00 nil 5.93 +1.8 nil 91.69 08.22 5.44+ 1.6 06 80.36 91.69 4.76+ 1.4 12 72.74 27.24 4.32+1.3 18 64.52 35.67 3.83+ 1.3 24 54.38 45.15 3.24+ 1.1 30 figure (1) : double – reciprocal plots for the inhibition of human serum cholinesterase in the presence (●) and absence of (o) of 30 mm/l gallic acid . acetylthiocholin iodide concentrations (s) were ; 0.02, 0.04 ,0.06 ,0.08 , 0.10 m/l . conclusion phenolic compounds, such as gallic acid derived from native iraqi plants with antimicrobial activity might not be recommended to be used as preservative in food industry or in pharmacological preparations since they might exert some undesirable effects on certain enzymatic 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acetylcholinesterase : mechanism of catalysis and inhibition, curr. med. chem. ;2001,vol.1,155-170. 33aldrige,w.n.; some properties of specific cholesterase with particular reference to the mechanism of inhibition by diethyl -p-nitrophenyl thiophosphate (e605) and analogous . biochem. j.;1950, 46 (4) ,451-460. http://www.ncbi.nlm.nih.gov/pubmed/8372156?ordinalpos=122&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/8372156?ordinalpos=122&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum http://www.ncbi.nlm.nih.gov/pubmed/11163955?ordinalpos=73&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvdocsum http://www.ingentaconnect.com/content/ben/cdm;jsessionid=b2y0mvr1l86x.victoria http://www.ingentaconnect.com/content/ben/cdm;jsessionid=b2y0mvr1l86x.victoria http://www.ingentaconnect.com/content/ben/cmc iraqi j pharm sci, vol.27 (2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 114-https://doi.org/10.31351/vol27iss2pp102 doi: 102 comparing the quality of life among patients with relapsing remitting multiple sclerosis in iraq using different disease modifying therapies ruaa n. yahya*1 ,ali a.kasim** and gheyath a. al gawwam*** *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ** department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. ***departments of neurology, baghdad college of medicine, baghdad university. abstract multiple sclerosis (ms) is a chronic, inflammatory, immune mediated disease of the central nervous system, mostly affecting young adults with mean age of 30 years, twice as high in women compared to men. the etiology of ms is not fully elucidated. ms symptoms are directly related to demyelination and axonal loss, along with other psychological symptoms, can result in functional limitations, disability and reduced quality of life (qol). the qol assessments in patients with a chronic disease may contribute to improving treatment and could even be of prognostic value. the goals of this study were to compare the qol of iraqi patients with relapsing remitting multiple sclerosis (rrms), using three different diseases modifying therapies(dmts) administered orally, subcutaneously, and by slow infusion; namely, fingolimod, interferonβ-1b, and natalizumab, respectively. and to assess the role of disability status, educational status, occupational status, ms duration, and treatment duration as a predictor for the qol. functional assessment of multiple sclerosis (fams) questionnaire version 4 was used to assess qol. sociodemographic and clinical characteristics were tested by univariate and multivariate regression analyses to assess the contribution of these predictors to qol. no significant differences were found in symptoms, thinking/fatigue subscales and fams total scores among the three dmts. in conclusion: iraqi ms patients using interferonβ-1b, fingolimod or natalizumab have a comparable low level of qol. the expanded disability status scale (edss) is negatively associated with qol of ms patients in all of the three therapies, while other predictors such as occupational status, educational status, smoking habit and ms duration have different impact in different treatments. keywords: multiple sclerosis, fams, edss, dmts, quality of life. العالجات من مستخدميمقارنة جودة الحياة لمرضى تصلب االعصاب المنتشر في العراق المعدلة للمرض المختلفة *** عبد علي الكوام اث و غي **، علي عبد الحسين قاسم 1*،رؤى ناطق يحيى .بغداد،العراق بغداد، جامعة ، ،كلية الصيدلة الصيدلة السريريةفرع * .بغداد،العراق بغداد، جامعة الصيدلة، كلية ، العلوم المختبرية السريريةفرع ** .بغداد،العراق بغداد، جامعة فرع االعصاب ، كلية الطب ، *** الخالصة تصلب االعصاب المتعدد هو مرض التهابي مناعي مزمن يصيب الجهاز العصبي المركزي يحدث السباب غير معروفة الى جال. غالب اعراض المرض ترتبط سنة ،لدى النساء اكثر من الر 30عمر حد ما تحدث االصابة به غالبا في صغار البالغين مع متوسط بشكل مباشر بظاهرة إزالة الميالين وفقدان المحور العصبي ، باإلضافة إلى أعراض نفسية، التي تؤدي الى وجود قيود وظيفية وعجز كون ن أن توانخفاض جودة الحياة . قد يساهم تقييم جودة الحياة للمرضى الذين يعانون من االمراض المزمنة في تحسين العالج ويمك بين المرضى العراقيين الذين يعانون من االنتكاس المتكرر لمرض التصلب جودة الحياةذات قيمة تنبؤية. أهداف هذه الدراسة هي مقارنة ثالثة عالجات مختلفة تعطى عن طريق الفم ، تحت الجلد ، وعن طريق الوريد. وهم، الذين يستخدمون واحد منالمتعدد و نتاليزوماب على التوالي ، وتقييم دور بعض المسببات في جودة الحياة كمستوى االعاقة,مستوى ,ب 1-فنكولمود,انترفيرون بيتا استبيان التقييم الوظيفي لمرض تصلب االعصاب المتعدد اإلصدار . تم استخدام التعليم,الحالة الوظيفية,مدة المرض واخيرا مدة العالج . تم اختبار الخصائص االجتماعية والديموغرافية والسريرية من خالل تحليل االنحدار احادي المتغيرات والمتعدد جودة الحياة لتقييم 4 فروق ذات داللة إحصائية في محور األعراض و محور المتغيرات لتقييم مساهمة هذه المؤشرات في جودة الحياة. لم يتم العثور على التفكير واالعياء وعدد النقاط اإلجمالي الستبيان جودة الحياة. في الختام: المرضى الذين يعانون من مرض التصلب االعصاب المتعدد ثل من جودة الحياة. يرتبط مقياس و فنكولمود أو نتاليزوماب لديهم مستوى منخفض ممااب، 1-في العراق من مستخدمي انترفيرون بيتا حالة اإلعاقة الموسعة بشكل سلبي مع جودة الحياة لمرضى تصلب االعصاب المتعدد في جميع العالجات الثالثة ، في حين أن تنبؤات أخرى مثل الحالة المهنية والحالة التعليمية و التدخين ومدة المرض لها تأثير مختلف باستخدام العالجات المختلفة. العالجات ، مقياس حالة االعاقة الموسعة، استبيان التقييم الوظيفي لمرض تصلب االعصاب المتعدد ، تصلب االعصاب المتعدد لكلمات المفتاحية :ا .، جودة الحياة المعدلة للمرض 1corresponding author e-mail: ruaa_pharma@yahoo.com received: 14/8/2018 accepted: 9/10/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp102-114 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 103 introduction multiple sclerosis (ms) is a chronic, neurodegenerative disease of the central nervous system, mostly affecting young adults with mean age of 30 years, twice as high in women as compared to men (1,2) ms prevalence differ by different geographic regions (3). iraq as a part of the middle east area was considered as a ms medium risk prevalence area(4) but by latest epidemiological studies have indicated that the arabian gulf region has a high prevalence of ms(5). the etiology of ms is not fully elucidated, it involves both genetic and environmental factors(6). the clinical course of ms was characterized as the following (7,8) : 1. relapsing–remitting ms (rrms):affects about 85% of ms patients and marked by flareups (relapse or exacerbation of symptoms followed by periods of remission, when symptoms improve or disappear)(8,9). 2. primary progressive ms (ppms):-affects approximately 10% of ms patients and symptoms continue to worsen gradually from the beginning. there are no relapses or remissions, but there may be occasional plateaus (8,9). 3. secondary progressive ms (spms):may develop in some patients with rrms. the disease course continues to worsen with or without periods of remission(8,9). 4. progressive-relapsing ms:is a rare form, affecting fewer than 5% of patients. it is progressive from the start, with intermittent flare-ups of worsening symptoms along the way and has no periods of remission(8). clinically isolated syndrome (cis) is considered to be a part of the spectrum of ms phenotypes which is a first symptomatic episode of cns dysfunction due to inflammatory demyelination that could be ms, but has yet to fulfill the diagnostic criteria of ms(10), and there is 83% risk of developing ms over the next 10 years (11). in ms, dissemination of the lesions in the central nervous system, produced by the inflammatory process, manifested as physical and mental deficits and the incomplete recovery after relapse leads to the accumulation of new deficits and the progressive nature of the condition interfere with daily activities of individuals and have a negative impact on their wellbeing.(12) the symptoms of ms, such as weakness, sensory loss, and ataxia, which are directly related to demyelination and axonal loss, along with other symptoms such as reactive depression or social isolation, can result in functional limitations, disability and reduced quality of life (qol)(13). health – related quality of life (hrqol) is defined as the impact of an illness or treatment on an individual’s physical, social, psychological and general well-being. qol is now considered an important end-point inclinical studies. the qol assessments in patients with a chronic disease may contribute to improving treatment and could even be of prognostic value(14) . the qol among ms patients in arabic countries was rarely studied ,with limited information from iraq(15). the goals of this study were to compare the qol among iraqi patients with rrms using one of three different disease modifying therapies (dmts), administered orally, subcutaneously, and by slow infusion; namely, fingolimod, interferonβ-1b , and natalizumab, respectively. and to assess the role of disability status, educational status, occupational status, ms duration, and treatment duration as a predictor for the qol. patients and method patients the present cross-sectional study was carried out on 200 patients, (135 females with mean age ±sd of 36.7 ± 9.7 years, and 65 males with mean age ±sd of 35.9 ± 10.4 years) already diagnosed with rrms according to the revised mcdonald criteria(16), who attended the multiple sclerosis center, baghdad teaching hospital/medical city, seeking medical care, from november 2017 to march 2018. patients are into three groups: 1-group 1 consists of 70 patients who are receiving interferon β-1b , 0.25 mg subcutaneously every other day. 2-group 2 consists of 60 patients who are receiving fingolimod , 0.5mg orally daily. 3-group 3 consists of 70 patients who are receiving natalizumab , 300mg intravenous infusion over 1 hour every 4 weeks. inclusion criteria the inclusion criteria include; patients were aged 18 years or above of either sex, diagnosed with multiple sclerosis at least 1 year before this study, on the same medication for at least 3 months before this study, and are able to communicate. exclusion criteria the exclusion criteria include; patients who did not consent to participate, had hearing, speech or cognitive deficits that would impair understanding of the questions, women who were pregnant or breast feeding, patients with relapse or taken any form of corticosteroid treatment and patients with clinically isolated syndrome (cis) or other subtypes of ms. method to achieve the goals of the study, the expanded disability status scale (edss), and iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 104 the functional assessment of multiple sclerosis (fams) were assessed as follows: expanded disability satus scale (edss) the most popular and widely used instrument as an endpoint in clinical trials to assess the effectiveness of therapeutic interventions is the expanded disability status scale (edss) of kurtzke. it is a clinicianadministered assessment scale evaluating the functional systems of the cns(17). the edss is a 20-step scale of disease severity ranging from 0 (normal) to 10 (death due to ms) in 0.5 increments interval l. this scale includes two parts: one (from 0 to 3.5) taking into account functional parameters and edss measure impairments based on the neurological examination, the other (from 4 to 10) estimating degrees of mobility in patients. the scale considers eight functional systems (fs): pyramidal, cerebellar, brainstem, sensory, bowel and bladder, visual, cerebral, and other. (figure1)(18). figure 1. expanded disability status scale (19) the functional assessment of multiple sclerosis (fams) quality of life in ms patients was assessed by the functional assessment of multiple sclerosis (fams) questionnaire, published in 1996 by david cella and colleagues(20).the fams (version 4),the latest and most efficient version ,consist of 58 items from which 44 items organized into six subscales: (21) mobility, symptoms, emotional well-being, general contentment, thinking/fatigue and family/social well-being, and the additional concerns (figure 2). arabic version of fams was used and patients indicate their degree of agreement with each question in subparts; on a five-point likert scale, where 0= not at all; 1= a little bit; 2= somewhat; 3-quite a bit; 4=very much produces a score between (0-4) for each scored question. the respondents are asked to indicate how true 0 normal neurologic exam 1.0 no disability, minimal signs in one functional system 1.5 no disability, minimal signs in more than one functional system 2.0 minimal disability in one functional system 2.5 minimal disability in two functional systems 3.0 moderate disability in one functional system, or mild disability in three or four functional systems though fully ambulatory 3.5 fully ambulatory but with moderate disability in three or four functional systems 4.0 fully ambulatory without aid, self-sufficient, up and about some 12 hours a day despite relatively severe disability. able to walk without aid or rest some 500 meters 4.5 fully ambulatory without aid, up and about much of the day, able to work a full day, may otherwise have some limitation of full activity or require minimal assistance, characterized by relatively severe disability. able to walk without aid or rest for some 300 meters 5.0 ambulatory without aid or rest for about 200 meters; disability severe enough to preclude full daily activities (e.g. to work full day without special provisions) 5.5 ambulatory without aid or rest for about 100 meters; disability severe enough to preclude full daily activities 6.0 intermittent or unilateral constant assistance (cane, crutch, or brace) required to walk about 100 meters with or without resting 6.5 constant bilateral assistance (canes, crutches, or braces) required to walk about 20 meters without resting 7.0 unable to walk beyond about 5 meters even with aid. essentially restricted to a wheelchair. wheels self in standard wheelchair and transfers alone. active in wheelchair about 12 hours a day 7.5 unable to take more than a few steps. restricted to wheelchair. may need aid to transfer. wheels self but cannot carry on in standard wheelchair a full day. may require a motorized wheelchair 8.0 unable to walk at all, essentially restricted to bed, chair or wheelchair but may be out of bed much of the day. retains many self-care functions. generally has effective use of the arms 8.5 essentially restricted to bed much of the day. has some effective use of arm(s). retains some self-care functions 9.0 helpless bed patient. can communicate and eat 9.5 totally helpless bed patient. unable to communicate effectively or eat/ swallow 10 death due to multiple sclerosis iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 105 each statement has been for them during the past 7 days. the additional concerns subscale retained without score based on their potential clinical and empirical value. scores of negatively worded statements are reversed. after appropriate reversal, the scores are added within subscale, and then subscale scores are summed to produce a total fams score. the fams total score range is (0 to 176 points) and higher scores indicate better qol(21). not at all a little bit somewhat quite a bit very much mobility 1. because of my physical condition, i have trouble meeting the needs of my family 2. i am able to work (include work in home). 3.i have trouble walking 4.i have to limit my social activity because of my condition 5. my legs are strong 6.i have trouble getting around in public places 7.i have to make plans around my condition symptoms 8. i have nausea 9. i have pain 10.i feel sick 11.i feel weak all over 12.i have pain in my joints 13.i am bothered by headaches 14.i am bothered by muscle pains emotional well-being 15. i feel sad 16. i am losing hope in the fight against my illness 17. i am able to enjoy life 18. i feel trapped by my condition 19. i am depressed about my condition 20. i feel useless 21. i feel overwhelmed by my condition general contentment 22.my work (include work in home) is fulfilling 23.i have accepted my illness 24.i am enjoying the things i usually do for fun 25.i am content with the quality of my life right now 26.i am frustrated by my condition 27.i feel a sense of purpose in my life 28.i feel motivated to do things thinking and fatigue 29. i have a lack of energy 30. i feel tired 31. i have trouble starting things because i am tired 32. i have trouble finishing things because i am tired 33. i need to rest during the day 34. i have trouble remembering things 35. i have trouble concentrating 36. my thinking is slow 37. i have trouble learning new tasks or directions iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 106 figure 2. functional assessment of multiple sclerosis questionnaire (fams)(20) administrative arrangement and ethical considerations a research proposal that explains the purpose of the study and methods for data collection and instruments was submitted to college of pharmacy / university of baghdad committee. after approval, the proposal of the current study was submitted to the committee of multiple sclerosis center in baghdad teaching hospital/medical city to grant ethical approval, the committee of the mentioned center approved that. administration of questionnaire the data related to the study were collected by one of the researchers, who presented at the ms center five days a week from 8 am to 1 pm. when the patients arrived at the ms center they were asked if they are willing to participate in the study after briefly explaining its purpose. if they agreed to participate a full description of the procedure was given. during the waiting time to be checked by the neurologist, participants were interviewed by one of the researchers. statistical analysis the data were evaluated using statistical package for the social sciences (spss® 22.0.0) software package for windows. anderson darling test was used to assess if continuous variables (age, disease duration, treatment duration, edss, fams subscale and it’s total score) will follow normal distribution or not. if they follow normal distribution, they will be expressed as mean± standard deviation. if did not, they will be expressed as median and interquartile range (25% to 75% percentile range). discrete variables (gender, occupation, marital status, educational status, zone of residence, smoking habit, type of treatment) were expressed by their number and percentage. chi square test was used to analyze the discrete variable .one way anova was used to analyze the continuous variables. pairwise comparisons were done using post hoc tukey test. linear regression (uni and multivariate ) analysis was used to assess the relationship between different ariables. negative sign of correlation coefficient (r) or beta estimate (β) indicates inverse relationship, while, positive sign indicates direct relationship. results personal, demographic and disease characteristics of participants the socio-demographic characteristics for subjects (n=200) participated in the study are illustrated in table1. not at all a little bit somewhat quite a bit very much familv /social well-being 38. i feel distant from my friends 39. i get emotional support from my family 40. i get support from my friends and neighbors 41. my family has accepted my illness 42. family communication about my illness is poor 43. my family has trouble understanding when my condition gets worse 44. i feel “left out” of things additional concerns 45. i am bothered by side effects of treatment 46. i am forced to spend time in bed 47. i feel close to my partner (or the person who is my main support) 48.have you been sexually active during the past year? noyesif yes: i am satisfied with my sex life 49. i am proud of how i’m coping with my illness 50. i feel nervous 51. i worry that my condition will get worse 52. i am sleeping well 53. heat worsens my symptoms 54. i lose control of my urine 55. i urinate more frequently than usual 56. i am bothered by the chills 57. i am bothered by fevers 58. i am bothered by muscle spasms iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 107 table 1. patient’s characteristics. south regions involve(maisan, dhi qar, muthana and basrah ); middle regions involve(baghdad, diala, anbar, wasit, babil, karbala, qadisia and najaf );north regions involve(kurdistan, ninawa, salah aldin and karkuk );edss: expanded disability status scale, ms: multiple sclerosis. the socio-demographic characteristics by the type of treatment are illustrated in table 2.subjects using interferonβ-1b are older (39.4 ± 8.7 years; p=0.002) had higher employment rate (58.6%) and longer treatment duration (71.8± 64.3 months; p<0.001) while lower edss score (2.5 ± 1.7; p=0.023) as compared to the other therapies. natalizumab using subjects had the highest vitamin d3 use (40.0%). table 2. patient characteristics by type of treatment interferonβ-1b fingolimod natalizumab p-value age (years) 39.4 ± 8.7 36.2 ± 10.8 33.6 ± 9.4 0.002 gender female 43, 61.4% 38, 63.3% 54, 77.1% 0.099 male 27, 38.6% 22, 36.7% 16, 22.9% occupation (employed) 41, 58.6% 22, 36.7% 24, 34.3% 0.007 married 53, 75.7% 37, 61.7% 44, 62.9% 0.156 education (college) 30, 42.9% 23, 38.3% 28, 40.0% 0.867 smoker 11, 15.7% 6, 10.0% 8, 11.4% 0.583 duration of ms (years) 5.99 ± 5.36 8.12 ± 6.05 7.47 ± 4.81 0.069 ≥ 5 years 36, 51.4% 42, 70% 47, 67.1% 0.057 duration of current treatment (months) 71.8 ± 64.3 7.08 ± 4.42 19.14 ± 10.59 <0.001 edss 2.5 ± 1.7 3.5 ± 2.2 3.2 ± 2.2 0.023 additional therapies multivitamins 8, 11.4% 3, 5.0% 3, 4.3% 0.195 vitamin d3 14, 20.0% 17, 28.3% 28, 40.0% 0.034 omega 3 8, 11.4% 8, 13.3% 9, 12.9% 0.942 chi square test was used to analyze the discrete variables (gender, occupation, marital status, smoking habit, education, and additional therapies; one way anova was used to analyze the continuous variables (age, disease duration, treatment duration and edss); edss: expanded disability status scale; ms: multiple sclerosis . variables value n mean±sd ,% age (years) 36.4 ± 9.9 gender female 135 67.5% male 65 32.5% occupation (employed) 87 43.5% married 134 67% education (college) 81 40.5% zone of residence south regions 13 6.5% middle regions 180 90% north regions 7 3.5% smoker 25 12.5% treatment interferonβ-1b 70 35% fingolimod 60 30% natalizumab 70 35% duration of ms (years) 7.1 ± 5.4 ≥ 5 years 125 62.5% edss 3.0 ± 2.1 iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 108 table 3 provides a pairwise comparisons for the three studied groups. regarding age, the difference was significant between interferonβ1b and natalizumab treatment groups (p=0.001).significant difference in the occupational status between interferonβ-1b and fingolimod treatment group (p=0.013) also between interferonβ-1b and natalizumab treatment groups (p=0.004). while the difference in edss score was significant between interferonβ-1b and fingolimod treatment groups (p=0.025). the difference in duration of treatment was significant between interferonβ-1b and fingolimod treatment groups (p<0.001) also between interferonβ-1b and natalizumab (p<0.001) treatment groups. finally the difference in vitamin d3 use was between interferonβ-1b and natalizumab (p=0.010) treatment groups. table 3. post-hoc analysis of age, occupation, edss, treatment duration, vitamin d3 supplementation between each pair of therapy interferon β-1b vs. fingolimod interferon β-1b vs. natalizumab fingolimod vs. natalizumab age (years) 0.143 0.001 0.272 occupation 0.013 0.004 0.777 edss 0.025 0.103 0.787 duration of current treatment (months) <0.001 <0.001 0.181 vitamin d3 supplementation 0.266 0.010 0.163 tukey hsd was used for pairwise comparison; edss: expanded disability status scale. the functional assessment of multiple sclerosis (fams) subscales and total score are presented in table 4. the fams thinking /fatigue subscale has showed the highest mean value (22.7 ± 7.1), while the mobility subscale has showed the lowest mean value (18.1 ± 8.6), and total fams score for the general study sample was found to be (120.7 ± 28.7). table 4. assessment of fams score for all patients fams score value(mean±sd) mobility 18.1 ± 8.6 symptoms 20.3 ± 5.4 emotional well-being 19.0 ± 6.9 general contentment 18.5 ± 6.2 thinking/ fatigue 22.7 ± 7.1 family/social well-being 22.2 ± 4.4 total score 120.7 ± 28.7 fams: functional assessment of multiple sclerosis; sd:standred deviation the fams subscales and total scores by different type of treatment are presented in table 5. significant differences were found in mobility (p<0.001), emotional well-being (p=0.038), general contentment (p=0.001), and family/social well-being (p=0.030) subscales of fams, but not significant difference were found in symptoms, thinking/fatigue subscales and in total score table 5. assessment of fams score according to type of treatment fams score interferonβ-1b fingolimod natalizumab p-value mean ± sd mean ± sd mean ± sd mobility 20.9 ± 6.7 15.0 ± 9.8 18.0 ± 8.4 <0.001 symptoms 19.3 ± 6.1 20.8 ± 4.8 20.7 ± 5.1 0.201 emotional well -being 20.3 ± 6.0 17.2 ± 7.7 19.2 ± 6.6 0.038 general contentment 19.9 ± 5.5 16.1 ± 6.2 19.1 ± 6.3 0.001 thinking/ fatigue 21.6 ± 7.4 22.5 ± 6.9 23.8 ± 6.8 0.176 family/social well-being 23.2 ± 4.1 22.1 ± 3.8 21.2 ± 4.8 0.030 total score 125.2± 27.6 113.8± 28.8 122.1 ± 29.1 0.066 one way anova was used to analyze the continuous variables (fams subscale and total score); fams: functional assessment of multiple sclerosis. iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 109 table 6 provide pairwise comparison between the three study groups. there were significant difference in fams mobility (p<0.001), emotional well-being (p=0.030), and general contentment (p=0.001) subscale scores, between interferonβ-1b and fingolimod treatment groups. fams general contentment score was significantly higher in natalizumab than in fingolimod (p=0.015) treated groups. fams family/social well-being score was significantly higher in interferonβ-1b than in natalizumab (p=0.023) treated groups. table 6. assessment of fams score between each pair of therapy variables interferonβ-1b vs. fingolimod interferonβ-1b vs. natalizumab fingolimod vs. natalizumab fams score mobility <0.001 0.095 0.100 symptoms 0.267 0.274 0.997 emotional well-being 0.030 0.595 0.237 general contentment 0.001 0.737 0.015 thinking/ fatigue 0.778 0.154 0.510 family/social well-being 0.339 0.023 0.484 total score 0.059 0.793 0.220 tukey hsd was used for pairwise comparison; fams: functional assessment of multiple sclerosis univariate linear regression analysis was used to assess the correlation between different predictors and the total fams score in the total study sample, and per type of treatment .followed by multivariate analysis to differentiate between dependent and independent predictors. in the univariate analysis, there were statistically significant inverse correlation between total fams score and age (r=-0.295; p<0.001), ms duration (r=-0.230; p=0.001), and edss (r=-0.655; p<0.001). while, there were statistically significant direct correlation between fams total scores and occupational status (r=0.248; p<0.001) and educational status (r=0.184; p=0.009). in multivariate analysis, fams score was correlated with occupational status (β=0.106; p=0.075), and edss (β=-0.574; p<0.001) (table 7). table 7. correlation between fams score with various predictors for all ms patients predictors fams score univariate analysis multivariate analysis r p-value β p-value age -0.295 <0.001 -0.082 0.178 gender 0.132 0.062 occupation 0.248 <0.001 0.106 0.057 marital status -0.114 0.108 education 0.184 0.009 0.083 0.126 smoking 0.028 0.697 ms duration -0.230 0.001 -0.070 0.231 treatment duration 0.048 0.500 edss -0.655 <0.001 -0.574 <0.001 multivariate linear regression analysis was used to assess the correlation between total fams and different predictors (dummy variable used to express the categorical variables); r: partial regression coefficient; β: beta estimate; edss: expanded disability status scale; ms: multiple sclerosis; fams: functional assessment of multiple sclerosis. by using univariate analysis to assess qol predictors for interferonβ-1b treatment group; total fams score was inversely correlated with edss (r=-0.481; p<0.001). whereas, it was directly correlated with occupational status (r=0.314; p=0.008), educational status (r=0.253; p= 0.035), and smoking habit (r=0.257; p=0.032).in multivariate analysis; total fams score was correlated with educational status (β = 0.219 ; p=0.033 ), and edss (β = -0.410 ; p<0.001) (table 8). iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 110 table 8. correlation between fams score with various predictors for interferonβ receiving patients. predictors fams score univariate analysis multivariate analysis r p-value β p-value age -0.161 0.182 gender 0.219 0.068 occupation 0.314 0.008 0.159 0.137 marital status -0.038 0.757 education 0.253 0.035 0.219 0.033 smoking 0.257 0.032 0.166 0.111 ms duration -0.096 0.431 treatment duration -0.096 0.431 edss -0.481 <0.001 -0.410 <0.001 multivariate linear regression analysis was used to assess the correlation between total fams and different predictors (dummy variable used to express the categorical variables); r: partial regression coefficient; β:beta estimate; edss: expanded disability status scale; ms: multiple sclerosis; fams: functional assessment of multiple sclerosis. by using univariate analysis to assess qol predictors for fingolimod treatment group; total fams score was inversely correlated with age (r=-0.457; p<0.001), marital status (r=0.297; p=0.021), ms duration (r=-0.331; p=0.010), and edss (r=-0.764; p<0.001); while it was directly correlated with treatment duration (r=0.355; p=0.005). in multivariate analysis; total fams score was correlated with ms duration (β=-0.225;p=0.017), and edss (β=-0.677; p<0.001) (table 9). table 9. correlation between fams score with various predictors for fingolimod receiving patients predictors fams score univariate analysis multivariate analysis r p-value β p-value age -0.457 <0.001 -0.058 0.601 gender 0.033 0.801 occupation 0.218 0.094 marital status -0.297 0.021 -0.019 0.852 education 0.176 0.179 smoking -0.191 0.143 ms duration -0.331 0.010 -0.225 0.017 treatment duration 0.355 0.005 0.114 0.208 edss -0.764 <0.001 -0.677 <0.001 multivariate linear regression analysis was used to assess the correlation between total fams and different predictors (dummy variable used to express the categorical variables); r: partial regression coefficient; β:beta estimate; edss: expanded disability status scale; ms: multiple sclerosis; fams: functional assessment of multiple sclerosis. by using univariate analysis to assess predictors of the qol for natalizumab treatment group; total fams score was inversely correlated with age (r=-0.337; p= 0.004), and edss (r=-0.681; p<0.001). in multivariate analysis total fams score was correlated with edss (β=-0.653; p <0.001) (table 10) . iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 111 table 10. correlation between fams score with various predictors for natalizumab receiving patients predictors fams score univariate analysis multivariate analysis r p-value β p-value age -0.337 0.004 -0.068 0.491 gender 0.153 0.206 occupation 0.169 0.162 marital status -0.077 0.528 education 0.118 0.332 smoking -0.074 0.540 ms duration -0.212 0.079 treatment duration 0.003 0.978 edss -0.681 <0.001 -0.653 <0.001 multivariate linear regression analysis was used to assess the correlation between total fams and different predictors (dummy variable used to express the categorical variables); r: partial regression coefficient; β:beta estimate; edss: expanded disability status scale; ms: multiple sclerosis; fams: functional assessment of multiple sclerosis. discussion the assessment of quality of life (qol) offers a comprehensive reflection on disability and the impact of ms on affected individuals. it helps to guide physicians for proper care of patients, and reflects the effectiveness of treatment and may predict disease progression(22–24). although many studies have examined qol in ms patients, but results had shown a great degree of difference across countries, cultures and health care systems(25–27).the qol was rarely investigated among ms patients in arabic countries(15), with limited information from iraq. thus, this study aimed to evaluate the qol of iraqi patients with rrms, and to determine its correlations with different predictors. prior studies had demonstrated that individuals with ms have lower overall and specific qol as compared with healthy control groups or populations with other chronic diseases such as rheumatoid arthritis, end-stage renal disease, diabetes mellitus, and hypertension (28,29). there were no significant differences in the qol among subjects using interferonβ-1b, fingolimod or natalizumab therapies (p=0.066) (table 5). direct comparison of the qol between different therapies used in ms had been rarely studied. zecca et al. had reported a non-significant difference in qol between interferonβ-1b and natalizumab (p=0.6), which was related to satisfaction with both treatments resulted from convenience of use with natalizumab and optimal safety with interferonβ -1b(30). comparison of mean of total fams score and fams subscales scores of subjects, per type of treatment have shown a significant differences in terms of mobility, emotional well-being, general contentment, and family/ social well-being subscales, yet, the difference was not significant with regard to total fams score (table 5). pairwise comparison of mean of fams subscales scores per type of treatment had shown a higher mobility, and emotional wellbeing in interferonβ-1b than in fingolimod treatment groups (table 6). this may be explained, at least in part, by the longer duration of treatment, and subsequently the lower edss in interferonβ-1b treatment group than in fingolimod treatment group. edss is a measure of disability in ms patients (31), and ms disability had shown to be related to emotional well-being (depression), mobility, and physical symptoms(25,32). in contrast fox et al, had found that scores for all domains of the general qol measure were higher in fingolimod treatment group(33) . this controversy may be related to the different study design. fox et al. had used the 36-item short-form health survey v2 (sf36 v2) to evaluate health-related qol for ms patients on different injectable dmts, including interferonβ-1b, before and after switching to oral fingolimod treatment. general contentment has found to be higher in interferonβ-1b than with fingolimod, and higher for natalizumab than fingolimod (table 6). this can also be attributed to the longer treatment duration and lower edss, which are associated with more satisfaction with life aspects. family/social well-being score were significantly higher for interferonβ-1b compared to natalizumab (table 6). this refers to the greater family and social support required for patients on interferonβ-1b therapy than those on natalizumab therapy, since interferonβ-1b requires more frequent self– injection, which promotes more frequent iraqi j pharm sci, vol.27(2) 2018 quality of life among patients with relapsing remitting multiple sclerosis 112 contact between the patient and healthcare provider. to assess the correlation of the measured clinical and sociodemographic parameters with qol, univariate regression analysis followed by multivariate analysis had been conducted to best ascertain the independent predictors of the qol. by univariate analysis, the age, occupation, education, disease duration, and edss have shown to be dependent predictors of qol (table 7). and by multivariate analysis only occupational status, and edss, have shown to be independent predictors of qol in iraqi ms patients. occupational status may be related to better coping of patients with ms, and with maintaining a productive social life. yamout et al., had reported a similar finding(15). as discussed earlier, disability is associated with poor qol(32). thus, it is highly accepted that edss, which is a measure of disability in ms patients(31), is associated with lower qol. the inverse relationship between edss and qol had also been demonstrated in other studies (34,35). the study has shown that occupational status, educational status, smoking habit, and edss are correlated with qol in interferonβ-1b treatment group (table 8). multivariate analysis has shown that educational status is positive independent predictors of qol, while edss is a negative independent predictor of qol (table 8). higher degree of education may be interpreted as a better awareness and knowledge of the disease and the goals of therapy. the positive association between educational status and qol had been demonstrated by another study(15). in fingolimod treatment group age, marital status, ms duration, treatment duration and edss were correlated to qol in the univariate analysis; while, only ms duration and edss were shown to be negative independent predictors of qol by the multivariate analysis (table 9). the negative association between ms duration and qol is expected due to the progressive nature of the disease. many studies had shown that disease duration is a significant factor decreasing the qol in ms patients treated by different dmts(23,36–39). in this study only fingolimod had shown such relationship. this may refer to the tendency of physician to reserve fingolimod treatment for ms patients who have failed with other dmts. regarding natalizumab treated group, the univariate analysis has shown that age, and edss are correlated with qol; while, only edss has shown to be an independent negative predictor of qol by multivariate analysis (table 10). the most consistent predictor of qol in the three treatments groups has shown to be edss, which emphasizes the role of degree of disability on the qol of ms patients. the inverse relationship between edss and overall qol had also been demonstrated in other studies (34,35,38,40–42). limitations findings from this study have some limitations. first; the cross sectional design; thus variables and relationships between them may be representative of only a single point. second; these subjects may not be representative of ms patients as a whole due to single ms center study. conclusions and recommendations iraqi individuals with rrms have low level of qol. there was no significant difference in the qol of iraqi ms patients using interferonβ-1b, fingolimod or natalizumab. some predictors correlate with qol of iraqi ms patients treated with these different treatments. edss is negatively associated with qol of ms patients in all of the three therapies, while other predictors such as occupational status, educational status, and ms duration have different impact in different treatments. assessing the qol routinely could help physicians to assess treatment efficacy and the level of patient’s qol with relation to their treatment. patients also are recommended for getting the most out of medical appointments, using rehabilitation services, considering support group and be educated that can improve their qol. references 1. alonso a, hernán ma. temporal trends in the incidence of multiple sclerosis: a systematic review. neurology. 2008;71(2):129–35 . 2. wynia k, middel b, van dijk j, et al.. the impact of disabilities on quality of life in people with multiple sclerosis. multiple sclerosis journal. 2008;14(7):972–80 . 3. sahraian ma, khorramnia s, ebrahim mm, et al. multiple sclerosis in iran: a demographic study of 8,000 patients and changes over time. european neurology. 2010;64(6):331–6 . 4. hasan zn. disability and prognosis of relapsing remitting multiple sclerosis , is it different in iraqi patients ? neuroscience. 2011;16(3):233–6 . 5. bohlega s, inshasi j, al tahan ar, et al. multiple sclerosis in the arabian gulf 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method). the effects of drug: polymer ratio, stirring time and type of eudragit polymer on the physical characteristics of microsponges were investigated. the prepared microsponges were characterized for production yield (py), loading efficiency (ld), particle size, surface morphology, and in vitro drug release. the selected microsponge formula was incorporated into the floating effervescent gastro-retentive tablets. the prepared floating microsponge tablets were evaluated for their hardness, friability, swelling in addition to in vitro drug release. the results showed that the microsponge formula with eudragit rs100 had optimum physical properties with py % equal to 97 %, and ld % equal to 81% and controlled drug release (75% of drug release in 8 hr.) when compared with other formulas and pure bfn. therefore, the non-aqueous emulsion solvent diffusion method is a promising method to produce baclofen microsponges. keywords: baclofen, microsponge, floating tablet . وتقييم خارج الجسم للحبوب المايكروأسفنجية الفموية للباكلوفينتحضير **الجوهري جاللو فاطمة 1*،ابراهيميس فاتن ق .بغداد،العراق جامعة بغداد، ،كلية الصيدلة، الصيدالنياتفرع * الخالصه الدراسة ذهه من هو دواء مرخي للعضالت ويعاني الباكلوفين من نصف عمر قليل في البالزما ومن نافذة امتصاص ضيقة. الهدف الباكلوفين سفنجيات اال أعدت. للدواء الجانبية التأثيرات تقليل وبالتالي الدواء إطالق في لعقار الباكلوفين للتحكم ميكروي عائم أسفنجي قرص صياغة هو التحريك زمن ، البوليمر: تأثير نسبة الدواء بحث تم. المذيب المستحلب الغير مائي )طريقة مستحلب زيت في زيت( نشر طريقة بواسطة الميكروية وحجم ، تحميلال وكفاءة تم توصيف االسفنجيات الميكروية المحضرة من حيث االنتاجية ، الفيزيائية و الخصائص يودراجيت على البوليمر ونوع زة فوارة عائمة محتجالمختارة على شكل حبوب االسفنجيات المايكرويةوقد تم دمج .وتحرير الدواء خارج الجسم ، السطحي والشكل ، الجسيمات ركيبةت أن النتائج .أظهرت باالضافة الى تحرير الدواء خارج الجسمتقييم الحبوب المحضرة من حيث الصالبة, الهشاشة تم. في الجهاز الهضمي %81وكفاءة تحميل للعالج تساوي %97مثالية وانتاجية عالية تساوي فيزيائية خواص لها كانت 100يودراجيت آر أس االسفنجيات الميكروية مع شرن النقي.لذلك يمكن االستنتاج ان طريقة والباكلوفين األخرى الصيغ مع بالمقارنة (ساعات 8 في الدواء إطالق من ٪75) الدواء في اطالق وتحكم االسفنجيات الميكروية. واعدة النتاجالمائي هي طريقة غير المستحلب المذيب ، االسفنجيات الميكروية ، حبوب عائمة . الباكلوفين الكلمات المفتاحية : introduction conventional tablet dosage form is administered several times a day. to avoid, unnecessary repetitive management, a higher treatment cost and other undesirable characteristics of the conventional dosage forms, controlled release systems were designed, as they require less frequent drug intake, more therapeutic effects and less side effects. these dosage forms are designed to release medication continuously over a long period of time (1). gastroretentive delivery systems are designed to be kept in the stomach for a long time and release their active ingredients thus enabling continuous and long-lasting input of the drug to the top of the gastrointestinal tract (2) . 1corresponding author e-mail: fatenalatraqchi@gmail.com received: 25/8/2018 accepted: 18/11/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp75-90 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 76 there are two types to design a floating-dose model. these are single-unit systems and multiple unit systems (3). one of the new ways of a gastroretentive dosage form is the floating microsponge. it significantly increases the residence time of medication in stomach, improve bioavailability, improve patient compliance by reducing frequency doses, reduce drug waste (4). the microsponges are small spherical particles consisting of innumerable interconnected spaces under a non-folding structure with a large porous surface through which active components are released in a controlled manner. microsponge could encapsulate a wide range of hydrophilic and hydrophobic drugs (5). bfn belongs to class iii according to biopharmaceutical classification system, has a pka1 = 3.85; pka2 = 9.25, molecular weight is equal 213.7 g/mol and log p is equal to ( -1) (6). the solubility of bfn decreases with increase ph, having maximum solubility at ph 1.2 which equal to 26mg/ml. it has short plasma half-life which is about 2–4 hr. (7). baclofen has a narrow absorption window in the small intestine because on arrival to colon the absorption becomes low or nonexistent (8). it is stable and well absorbed within ph range 1-4 (9). the primary objective of the present investigation was to develop and optimize the bfn microsponge formula that to control the release rate of the drug and subsequent evaluation of different variables affecting it, then selection of the optimum microsponge formula to be incorporated into gastroretentive effervescent floating tablets to determine the effect of varying polymer type and concentration on the release profile of the tablets. materials and methods materials baclofen (hyperchem company, china), eudragit polymers rs100 and rl100 (rhom pharma, germany), liquid paraffin (solvochem company, united kingdom), n-hexane (chem-lab, belgium), carbopol 934 (himedia, india), sodium bicarbonate and citric acid (agc chemicals, japan). all other materials used in this study were of analytical grade. methods determination of bfn λmax a diluted solution of bfn in 0.1 n (ph 1.2) was prepared. the solution was scanned by uv visible spectrophotometer from 200-400 nm, and the λmax of the drug was detected. bfn calibration curve a stock solution (0.1mg/1ml) was prepared by dissolving 10 mg of drug in 100 ml of 0.1n hcl (ph 1.2), then preparing serial dilution of different concentration of (0.002, 0.004, 0.006, 0.008, 0.01, 0.013, 0.015 and 0.017 mg /ml) from stock solution. the absorbance was then measured at the λmax of the drug. the measured absorbance plotted against the concentrations. preparation of bfn microsponges the bfn microsponge formulas were prepared by oil in oil emulsion solvent diffusion technique. the internal phase consisted of polymer that was dissolved in organic solvent. once a clear solution was obtained, bfn was added gradually to the internal phase with addition of magnesium stearate and the whole mixture was kept in the ultrasonic bath for 5 minutes to obtain a homogenous dispersion. magnesium stearate was added as a stabilizer for reduction of particle aggregation. then, the mixture was poured gradually into liquid paraffin and stirred by using of magnetic stirrer. the oil in oil emulsion formed was stirred for different duration at different stirring speed. during the stirring period, the solvent diffused into liquid paraffin will be evaporated leaving spherical porous particles. the solidified microsponges were filtered by using whatman filter paper and washed five times with 60 ml of n-hexane, dried at room temperature for 12 h and stored in a desiccator for further investigations (10). the microsponge formulas are illustrated in table 1. characterization and evaluation of bfn microsponge determination of the percent production yield (py) the percent production yield of the prepared bfn microsponge formula was determined by dividing the final weight of microsponge formula on the initial weight of the raw material multiplied by 100 (11). py%= 𝑃𝑟𝑎𝑐𝑡𝑖𝑐𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑚𝑖𝑐𝑟𝑜𝑠𝑝𝑜𝑛𝑔𝑒 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 (𝑝𝑜𝑙𝑦𝑚𝑒𝑟 + 𝑑𝑟𝑢𝑔) x 100 determination of percent loading efficiency (ld) the drug content in all prepared microsponge formulas was determined spectrophotometrically, in which 10 mg of the prepared microsponge formula was dissolved in 100 ml of 0.1n hcl (ph 1.2) and kept for 12 hr. the solution was diluted suitably with 0.1n hcl and analyzed spectrophotometrically at λ max of bfn. the drug content was calculated from the calibration curve equation and expressed as the loading efficiency (%) (12). ld% = 𝐴𝑐𝑡𝑢𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐵𝑎𝑐𝑙𝑜𝑓𝑒𝑛 𝑖𝑛 𝑚𝑖𝑐𝑟𝑜𝑠𝑝𝑜𝑛𝑔𝑒 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝐵𝑎𝑐𝑙𝑜𝑓𝑒𝑛 x 100 particle size measurement the particle size of microsponge was determined using optical microscope. calibration of the eyepiece micrometer with a stage micrometer was done. a minute quantity of microsponges was spread on a clean glass slide, the particle diameters of around 100 microsponges were measured randomly and the average particle size of bfn microsponge was determined (13). 𝐷 (𝑎𝑣𝑒𝑟𝑎𝑔𝑒) ∑𝑛𝑑 ∑𝑛 where n = number of microsponge observed, d = middle value (μm).d is the average diameter of particles (μm). iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 77 table (1) formulas of bfn microsponges formula no. ingredient bf1 bf2 bf3 bf4 bf5 bf6 bf7 bf8 bf9 bf10 bf11 bf12 bf13 bf14 bf15 bf16 bf17 baclofen (g.) 0.5 1 1.5 0.5 1 1.5 2 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 1 eudragit rs100(g.) 0.5 0.5 0.5 / / / 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 eudragit rl100(g.) / / / 0.5 0.5 0.5 / / / / / / / / / / / acetone (ml) 5 5 5 5 5 5 5 / / 5 5 5 5 7.5 10 7.5 10 ethanol (ml) / / / / / / / 5 / / / / / / / / / dichloromethane (ml) / / / / / / / / 5 / / / / / / / / paraffin (ml) 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 magnesium stearate (w/v %) 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 stirring rate (rpm) 1500 1500 1500 1500 1500 1500 1500 1500 1500 1000 500 1500 1500 1500 1500 1500 1500 rotation time (hr.) 1 1 1 1 1 1 1 1 1 1 1 0.5 2 1 1 1 1 iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 78 scanning electron microscope (sem) study the surfaces morphology of microsponge formula was analyzed by sem. sprinkling the microsponge on adhesive tape stuck to aluminum stub and by using a gold sputter module for coating this stub with gold, and then this coated sample was scanned, and photomicrographs were taken by sem (14). in-vitro drug release studies of microsponge formulations baclofen microsponge formulas were subjected to an in-vitro drug release study by using dissolution testing apparatus type ӏӏ (paddle). the dissolution test was carried out utilizing 900 ml of 0.1n hcl (ph1.2). a specified weight of microsponge corresponding to 15 mg of bfn was taken and by using semipermeable membrane rotated at 50rpm at 37 ± 0.5 0c. a sample of 5 ml was collected every hour for 10hrs and immediately was displaced with 5 ml of fresh dissolution medium(0.1n hcl) for preserving sink conditions, after that the sample was filtrated through 0.45μm filter syringe .the absorbance of the filtrate was measured by a uv spectrophotometer at the corresponding λmax of bfn (15,16). this procedure was done in triplicate for each formula to take the mean value. fourier transforms infrared (ftir) analysis infrared spectroscopy was conducted using ftir spectrophotometer (biotech, england) and the spectrum was recorded in the wavelength region of (4000 400) cm-1. the procedure consisted of dispersing the sample (drug alone, selected polymer alone, physical mixture of drug with polymer and the optimized formula) in the kbr disc to certify compatibility (17). differential scanning calorimetric (dsc) analysis dsc analysis of pure drug, selected polymer, physical mixture of drug and selected polymer and the optimized microsponge formula were done to indicate thermal compatibility between drug and polymer during the formulation of microsponges and to assess the crystalline state of the drug. samples were submitted to dsc analysis using a differential scanning calorimeter (schimadzu, model ta50 wsi, kyoto, japan). thermogram obtained at a scanning rate of 10°c/min using a dry nitrogen flow of 25 ml/min. each sample was scanned between 0°c and 400°c (18). powder x-ray diffraction (pxrd) analysis the x-ray diffractometry was used to study the molecular structure of crystalline chemicals such as bfn and additives. samples were submitted to x-ray analysis using xrd6000 (shimadzu, japan). the results were recorded over a range of 10–80°(2θ). the operating conditions were: voltage 40 kv, current 30 ma, scanning speed 1/min., and this test was done for drug, physical mixture of drug with polymer and for selected formula (19). formulation of bfn floating microsponge tablets by effervescent technique different formulas of bfn microsponge floating tablets were prepared as shown in table 2. direct compression method was used to prepare tablets. homogeneously mixing of previously weighed components (bfn microsponge equivalent to 15mg bfn, polymer, gas generating agent sodium bicarbonate with citric acid and diluent) in a mortar for 15 minute to obtain powder blend, this blend was mixed with a calculated amount of magnesium stearate and talc powder for 3 minutes (20) and compressed by using erweka tablet machine to get a tablet with total weight of 200 mg. table (2) composition of bfn floating effervescent microsponge tablets formula no. ingredients (mg) fmt1 fmt2 fmt3 fmt4 fmt5 fmt6 fmt7 the selected microsponge formula * * * * * * * carbopol 934p 60 40 60 80 sodium carboxy methylcellulose (nacmc) 60 sodium alginate 60 hpmc e5 60 pvp k30 5 5 5 5 5 5 5 sodium bicarbonate 35 35 35 35 35 20 35 citric acid 15 15 15 15 15 15 15 lactose fine powder 38 38 38 38 58 53 18 talc 5 5 5 5 5 5 5 magnesium stearate 5 5 5 5 5 5 5 total tablet weight 200 200 200 200 200 200 200 (*): amount of the microsponge equivalent to 15mg bfn iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 79 evaluation of the prepared floating microsponge tablets the standard evaluation of the prepared tablets was done according to usp specification (21). weight variation weight variation was done by weighed individually twenty tablets and the average weight was calculated. then each individual tablet was compared to the average, it should no more than two tablets differed from average weight. then measure a percentage of deviation by using equation: % 𝑫𝒆𝒗𝒊𝒂𝒕𝒊𝒐𝒏 = (𝑰𝒏𝒅𝒊𝒗𝒊𝒅𝒖𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕 – 𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒘𝒆𝒊𝒈𝒉𝒕) 𝑨𝒗𝒆𝒓𝒂𝒈𝒆 𝒘𝒆𝒊𝒈𝒉𝒕 𝒙 𝟏𝟎𝟎 hardness test three tablets of each batch were selected for measuring the force required to break the tablet by using yd1 hardness (beijing, china) tester in which the scale of the tester was adjusted to zero and the tablet placed diametrically and then gradually increase of the load until the tablet break. the hardness was expressed as a force in kg/cm2. friability the friability test was done for all batches using cs-2 friabilator (tianjin guoming medicinal, china) for 4 min at 25 rpm by weighing 20 tablets all together then placing them inside the tester. after their revolution, they were de-dusted and weight again. the friability was estimated as the percent weight loss and calculated using equation, friability value should be not more than 1%. 𝑭 % = (𝑾 𝒊𝒏𝒊𝒕𝒊𝒂𝒍 – 𝑾 𝒇𝒊𝒏𝒂𝒍) 𝑾 𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒙 𝟏𝟎𝟎 where, f % = percentage of friability, w initial=weight of tablet before the revolution, w final= weight of tablets after revolution. drug content five tablets were weighed individually, then placed in a mortar and powdered. an amount equivalent to 15 mg drug was extracted with 100 ml of 0.1n hcl (ph 1.2) and sonicated for 15 minutes. the solution was filtered and properly diluted with 0.1 n hcl (ph 1.2), and then the drug content was measured by using uv-visible spectrophotometer at λmax of drug (7). in-vitro buoyance studies buoyancy study was done by placing the tablet in 100 ml beaker of 0.1n hcl and determine the time needed for tablet to float (flt) and the total time that remained buoyant (tft), the method was described by rosa et al (22). swelling index the swelling index was done by putting previously weighed tablet in a beaker containing 0.1n hcl and the tablet removed at predetermined time interval (after 1, 2, 3 , 4, 5 and 6 hr.), the excess water removed by tissue and re-weighed (23). the swelling index is calculated by the equation 𝑺𝒘𝒆𝒍𝒍𝒊𝒏𝒈 𝒊𝒏𝒅𝒆𝒙 = (𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒘𝒆𝒕 𝒕𝒂𝒃𝒍𝒆𝒕 –𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒅𝒓𝒚 𝒕𝒂𝒃𝒍𝒆𝒕) 𝑾𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒅𝒓𝒚 𝒕𝒂𝒃𝒍𝒆𝒕 × 100 in-vitro dissolution test the procedure for dissolution was the same as that mentioned for the drug release study from microsponge. in-vitro drug release kinetic studies the data obtained from in vitro dissolution profile of bfn floating microsponge tablets versus time were plotted at different mathematical expressions to describe the kinetic and mechanism of release. the kinetic models used were zero order kinetic (percentage drug release vs. time), first order kinetic (log percentage drug release vs. time), higuchi model (percentage drug release vs. √t), hixon-crowell model (cube root of percentage drug release vs. time), and korsmeyer peppas (log of percentage drug release vs. log time). the best fitted model was that with the highest correlation coefficient. statistical analysis the results of the experiments were given as a mean of three samples ± standard deviation. statistical analysis of data was performed using sas (statistical analysis system version 9.1) using anova test to assess significant differences among means, p < 0.05 was considered statistically significant. results and discussions determination of bfn λmax baclofen solution in 0.1 n hcl (ph 1.2) gave the highest peak of absorbance at 220 nm as shown in figure 1; this result was with an agreement with that previously recorded (24) (25). figure (1) uv spectrum of bfn in 0.1 n hcl (ph 1.2) iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 80 calibration curve of bfn calibration curve of bfn in 0.1 n hcl (ph 1.2) is shown in figure 2. a straight line was obtained with r2 value (0.998) indicated that calibration curve obeys beers law within the used concentrations. figure (2) calibration curve of bfn in 0.1 n hcl (ph 1.2) at λmax 220 nm preparation of bfn microsponges baclofen loaded microsponges were prepared by oil in oil emulsion solvent diffusion method in which high viscosity paraffin employed as external phase with either one of acrylic polymer (eudragit rl100 or eudragit rs100), these polymers are water-insoluble polymers have been shown to be ph independent which are suitable for the sustained release applications (26). the use of paraffin which is non-polar as external phase instead of water to prevent the escape of hydrophilic drug into the external phase and provide better encapsulation efficiency for these drugs (27). bfn has poor lipid solubility. eudragit rs100, rl100 are very slightly soluble in liquid paraffin (28). microsponges will be formed by the diffusion of solvent into the external phase, the instant mixing of the solvent and paraffin at the interface of the droplets induced precipitation of the polymer, thus forming a shell encloses the solvent and the drug, the evaporation of solvent lead to the formation of pores. effect of drug: polymer ratio on the microsponges the drug-polymer ratio had considerable effect on the nature of microsponges as shown in table 3. it was indicated that increasing the drug: polymer ratio to certain limit increased the py and ld. since the available polymer was sufficient to encapsulate more amount of drug resulted in high ld. table (3) effect of drug: polymer ratio on physical properties of the microsponge further increase in drug to polymer ratio have reverse effect on both the py and ld as shown in bf7, the reason for decrease in both the py and ld with increasing drug to polymer ratio is due to the amount of polymer was not sufficient to encapsulate all amount of drug (29). it was observed that as the ratio of drug to polymer was increased, the particle size increased due to increase the viscosity of the internal phase and therefore will be hardly broken into small droplets (30). it was detected that formulation of microsponge using eudragit rs100 showed higher py and ld. this may be due to the differences between these polymers, eudragit rs100 contains 5% quaternary ammonium group which is less than that contained in eudragit rl100(10 %) and therefore these polymers differ in their permeability. moreover, eudragit rs100 preferable over eudragit rl100 in microsponges preparation using oil in oil emulsion solvent diffusion method (31). statistically this increment in the py, ld and mean particle size with increasing drug: polymer ratio was significant (p < 0.05) when using a one-way anova test. effect of internal phase volume on the bfn microsponges the amount of solvent volume needs to be controlled within an appropriate range during microsponge preparation due to its effect not only on the formation of emulsion droplets formulas drug: polymer ratio polymer type py % ± sd ld % ± sd mean particle size (μm) ± sd bf1 1˸1 eudragit rs100 89.66±0.577 66.831±0.359 47.7±1.7 bf2 2˸1 eudragit rs100 90.834±0.213 70.812±1.35 60.3±0.8 bf3 3˸1 eudragit rs100 93.83±0.288 75.728±1.09 68.5±0.8 bf4 1˸1 eudragit rl100 87.43±1.15 66.57±0.14 50±0.17 bf5 2˸1 eudragit rl100 89.665±0.47 71.35±0.07 57.2±1.7 bf6 3˸1 eudragit rl100 95.31±1.02 76.03±1.15 70.4±0.8 bf7 4˸1 eudragit rs100 83.73±0.23 62.452±0.594 72.53±1 iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 81 at the initial stage but also on the solidification of drug and polymer in the droplets. bf15 and bf17 fabricated with 10ml acetone, resulted in finely dispersed spherical emulsion droplets during agitation, but as the stirring was discontinued emulsion droplets adhered together and coalesce. consequently, no microsponges could be formed with increasing volume of acetone to 10ml (32). the role of the solvent was acting as porogen (pore forming agent) since the evaporation of solvent lead to formation of pores into which the drug is loaded and this was the reason for increase the ld (33) associated with increasing the volume of internal phase solvent from 5 ml to 7.5 ml (34). larger particle size was associated with lower internal phase volume (5 ml) which may be to high viscous phase would be difficult to split the droplets to smaller ones when poured in to the external paraffin phase. the effect of acetone volume on the particle size was shown in figure 3. the increase solvent volume from 5 ml to 7.5 ml showed significant effect on ld and mean particle size as p value <0.05 when using the one-way anova. figure (2) histogram showing effect of internal phase volume on mean particle size effect of stirring speed on bfn microsponges the dispersion of the internal phase of drug and polymer into the droplets in the external phase depended on the agitation speed of the systems. as agitation speed increased, the size of microparticles was reduced due to rapid division of the formed droplets at high stirring speed, which may have less chance of coalescing into bigger droplets with production of more uniform and spherical particles while at lower stirring speed particles suffered from coalescence and aggregation.(35).the microsponges formulated with 1500 rpm had higher ld %. so, it was selected as the optimum stirring speed. statistically, the effect of stirring speed on the py % was nonsignificant but produces significant effect (p value < 0.05) on both the ld% and mean particle size. effect of stirring duration on the bfn microsponges to find the most appropriate stirring time for fabrication of bfn microsponges, different stirring duration was used 0.5 hr., 1 hr. and 2hr. in bf12, bf1 and bf13 respectively, the results are listed in table 4. stirring duration of 2h in bf13 resulted in low py% and ld % due to adherence of polymer to beaker during fabrication of microsponges in addition, at longer time of stirring there was more chance for the drug to be leached. accordingly, it was adopted that the optimum stirring time is 1 hr. since 0.5 hr. stirring duration was associated with lower py% and ld%. this finding was similar to previously reported by roaa et al (36) increase stirring duration from 0.5 hr. to 1 hr. produced significant effect on py% and ld% as p value < 0.05 when use the one-way anova test. table (4) effect of stirring duration on physical prosperities of microsponges fo r m u la d r u g : p o ly m e r r a ti o ty p e o f p o ly m e r a c e to n e (m l) p a r a ff in (m l) st ir r in g sp e e d (r p m ) st ir r in g d u r a ti o n (h r .) p y % ± s d l d % ± s d m e a n p a r ti c le si z e ± s d (µ m ) bf1 1˸1 eudragit rs100 5 100 1500 0.5 77.6±0.51 60.83±3.05 42.83±0.61 bf12 1˸1 eudragit rs100 5 100 1500 1 89.66±0.577 66.831±0.35 47.7±1.7 bf13 1˸1 eudragit rs100 5 100 1500 2 80.9±0.17 63.686±1.8 47.36±1.7 iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 82 effect of solvent type on bfn microsponges acetone was the preferable solvent for the oil in oil emulsion solvent diffusion method due to its dielectric constant (20.7), so it was poorly miscible with paraffin, that would lead to the slow diffusion of the solvent out of the emulsion droplets to the external paraffin medium, resulted in slow precipitation of polymer matrix, and subsequent separation of a microsphere with a spongy structure (31). microsponge preparation by using acetone gave higher py%, ld% than that of ethanol as illustrated in figure 3. figure (3) effect of solvent type on physical properties of microsponges invitro drug release study of microsponges dissolution was done for bf14, bf16, and pure bfn as illustrated in figure 4. faster and greater drug release was noticed from bf16 than that of bf14 which may be related to higher drug amount compared to the amount of polymer which resulted in more porosity and consequently, more drug release was obtained. the amount of polymer available per microsponge showed realistic effect on drug release. so, as the amount of polymer became equal to the amount of drug, increase in the thickness of the polymer matrix was obtained that led to longer diffusion path and ultimately to decreased drug release. bf14was determined as the optimum formula because it showed control drug release (75% of drug release in 8 hr.), acceptable py and ld, so it was subjected to further investigation. figure (4) dissolution profile of bfn from different microsponge formula in 0.1n hcl (ph1.2) at 37ºc . evaluation of the shape and surface morphology by scanning electron microscope (sem) the sem result of the selected microsponge formula showed (figure 5) a spherical nature of the microsponge, uniform size with sufficient pores that loaded with drug. (a) (b) figure (5) sem of the selected microsponge formula bf14 at (a) 310 x magnification and (b) 270 x magnification fourier transforms infrared spectroscopy the ftir spectrum of pure bfn, physical mixtures of drug and eudragit rs100, and the selected microsponge formula (bf14) were given in figure (6:a-c).the spectrum of pure bfn showed characteristic peaks at 1398 cm-1 (o-h bending), 1244cm-1 (c-o stretching), 831cm-1 (c-cl stretching), which considered as finger print of bfn, the ftir of bfn also showed broad peak at 2590 and extend up to 3100cm -1(o-h of alcohol and carboxylic acid stretching). the spectrum of physical mixture iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 83 was as that of the drug, indicating no chemical interaction or complexation. the spectrum of the selected formula bf14 exhibited similar peaks, no appearance or disappearance of peaks and/or shift of their positions and therefore bfn was apparently stable in the microsponges . (a) (b) (c) figure ( 6) ftir spectrum (a) pure bfn, (b) physical mixture ratio1:1 of bfn and eudragit rs100, (c) bf 14 iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 84 differential scanning electron microscopy (dsc) the thermal behavior of pure bfn showed a sharp endothermic peak at 213.24°c corresponding to bfn melting temperature with onset of peak at 208.74°c and end set at 217.35°c as shown in figure 7a this indicates that the drug was in pure crystalline state. the thermogram of the physical mixture of bfn and eudragit rs100 at equal ratio (1:1), and the selected bfn microsponge formula (bf14) exhibited endothermic peaks at 211.84 and 192.94 °c respectively, the slight decrease in the endothermic peak of the microsponge formula might be due to the alteration in the form of the material from crystal to amorphous especially there was no broadening or appearance of new peak and no other thermal event occurred. so, one can be concluded that the excipients and drug were compatible with each other as such case there was no incompatibility (37). 100.00 200.00 300.00 temp [c] -20.00 -10.00 0.00 mw dsc 208.74x100conset 217.35x100cendset 213.24x100cpeak -857.81x100mjheat 213.24x100c (a) 100.00 200.00 300.00 temp [c] -10.00 -5.00 0.00 5.00 mw dsc 211.84x100c (b) 100.00 200.00 300.00 temp [c] -30.00 -20.00 -10.00 mw dsc 192.94x100c (c) figure (7 )dsc thermograms of (a) pure bfn, (b) physical mixture ratio1:1 of bfn and eudragit rs100, (c) bf 14 iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 85 powder x-ray diffraction the x-ray is a tool for study of the crystal lattice of the drug and for indicating if possible interaction between the drug and excipients. the x-ray of bfn showed strongest sharp distinct peaks at different angle (2θ) of 21.3˚, 23.4˚ and 28.9˚ which indicated that pure bfn was in crystalline nature as shown in figure 8. these results were in agreement with the previous study (38). the pxrd pattern of the selected bfn microsponge formula showed the main peaks of pure bfn but with lower intensity which might be due to conversion of bfn to amorphous form (39). the obtained result is with agreement with that obtained by dsc. (a) (b) figure (8) x-ray diffractions of (a) pure bfn, (b) bf 14. iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 86 formulation of bfn floating microsponge tablets by effervescent technique the development of floating effervescent microsponge tablets needs ideal polymer that allows water to permeate at fast enough rate for immediately activate the effervescent reaction and float and thereby preventing the individual unit for transit to the small intestine and at the same time should be firm and tight for resistance to rupture under high forces. different polymers used as showed previously in table 2 and selected the best one as discussed later. evaluation of bfn floating microsponge tablets the weight variation of the bfn floating microsponge tablets was found in the range of 196.35±0.69 to 199.87±0.8 for all the formulations as shown in table 5. these results were within the usp requirements limits. this referred to good flow properties that caused uniform fill of the tablet die and finally limit the weight variable. the hardness for formulas was in the range of 4.7 to 6.1±0.79 kg/cm2 as shown in table 5. these ranges of tablet hardness indicated good mechanical strength (40). table (5) physical parameters of bfn floating microsponge tablets in vitro buoyance studies the prepared floating tablets exhibited different flt and tft depending on the polymer type and sodium bicarbonate concentration. the flt and tft of the prepared formulas are listed in table 6. table (6) the flt and tft of the prepared formulas formul a flt (sec.) ±sd tft (hr.) ±sd fmt1 23.15±2.12 not float fmt2 not float not float fmt3 not float not float fmt4 7.18 ±1.53 >24 fmt5 10.825±1.93 not float fmt6 25±0.32 2 ±0.79 fmt7 6.54±1.27 >24 fmt1 which was prepared by using 30 % hpmc e5 exhibited flt of 23.15 sec. but the tablet disintegrated immediately in the dissolution media. hpmc e5 which is a low viscosity grade polymer, gels with low swelling ability and thus didn’t trapped carbon dioxide therefore the tablet ruptured after floating immediately (41). sodium alginate that used in fmt2 failed in achieving floating tablet. this was due to its ph sensitive nature which made it soluble in 0.1n hcl (ph 1.2) (42). (nacmc) at 30 % of tablet weight (fmt3) failed in maintaining integrity of the tablet and tablet disintegrated immediately in the 0.1n hcl (ph 1.2). easy solubility in water and rapid erosion of nacmc matrix tablet were some of the limitations to make it an ideal tablet matrix material (43). fmt4 which was formulated by using 30 % carbopol 934 p showed good flt 7.18 sec. and excellent tft (>24 hr.) and maintained the integrity of the tablet due to crosslink structure of carbopol polymer and there was larger region of low micro viscosity. fmt6 prepared with the same carbopol 934p concentration as that in fmt4 but with less concentration of gas generating agent (sodium bicarbonate). this formula exhibited longer flt and tft of 2 hr. as showed in table 6. the gas generating agent concentration must be optimized within suitable limits for maintaining the generated gas bubbles within the floating tablet for longer period and thereby achieving longer tft for tablet and this explained the shorter tft of fmt6 at low concentration of sodium bicarbonate (44). fmt5, fmt4 and fmt7 were formulated with 20 %, 30 % and 40 % carbopol 934 p polymer respectively but with same sodium bicarbonate concentration. these formulas showed different flt which was 10.825±1.93 sec., 7.18 sec. ±1.53sec. and 6.54 sec.±1.27 sec. respectively. the reverse relationship between flt and carbopol concentration as shown in figure 9 could be attributed to the high water swallability and hydrocolloid forming tendency of carbopol that lead to rapid wetting ability and floated immediately. therefore, shorter flt was obtained with increase carbopol concentration (45). formula weight variation(mg)±sd hardness (kg/cm2) ±sd friability (w/w) bfn content (%)±sd fmt1 198±0.63 5.5±1.01 0.45 97.03±1.5 fmt2 196.43±1.81 5.46±0.1 0.78 98.2±2 fmt3 198±0.61 4.7±0.62 0.69 96.04±2.1 fmt4 197.8±1.14 5.5±1.1 0.56 98.7±1.6 fmt5 196.35±0.69 6.1±0.79 0.81 96.7±2.7 fmt6 199.87±0.8 5.82±1.7 0.73 99±0.61 fmt7 198.9±.91 5.82±1.0 0.49 98.89±0.9 iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 87 figure (9) histogram showing the effect of carbopol 934p concentration on flt the low level of polymer in fmt5 caused the poor strength of the gel layer and hence could not sustain the amount of gas generated as shown in figure 10. therefore, bfn floating microsponge tablets formulated with 30 % and 40 % carbopol 934 p polymer showed an acceptable tablet shape with excellent flt and tft and good gel strength layer that maintained integrity of the tablets. (a) (b) figure (10) tablets with different carbopol 934 p concentrations (a) 20%, (b) 40 %. effect of carbopol 934 p concentration on in vitro drug release the effect of carbopol 934p concentration on the in vitro drug release was studied and illustrated in figure 11, as the concentration of the carbopol 934 p increase, the drug release decreased, this due to increase the concentration of carbopol would decrease the interstitial space between particles in the tablet. however, the higher swelling was associated with higher carbopol concentration would cause increase in dimension of the tablet and hence increased diffusion pathway that prevented the passage of the drug molecules and produced retardation of drug release (46). fmt7 showed sustained drug release for 10 hr. with 81.529±0.79 % drug release in 7 hr. while for fmt4; the percentage of drug release was 95.478± 1.07 in 7 hr. therefore, fmt7 was selected as the best formula for the preparation of bfn floating microsponge tablets by using the effervescent technique that at the same time exhibited short flt (6.54 sec.±1.27) and tft > 24hr. and resulted tablets with maximum swelling. figure (11) effect of carbopol 934 p concentration on the % drug release in 0.1 n hcl (ph 1.2) at 37ºc kinetic analysis of bfn release data from fmt 7 floating microsponge tablets the release of bfn from floating microsponge tablet fmt7 obeyed higuchi release as their r2 values gave higher results. it was found that the mechanism of drug release was non -fickian diffusion as the release exponent "n" value of fmt7 was more than 0.5 and less than 1 which was the standard value for declaring non -fickian anomalous diffusion as shown in table7 (47). iraqi j pharm sci, vol.28(1) 2019 baclofen oral microsponge tablets 88 table (7) kinetic analysis of bfn floating microsponge tablet release data (fmt7) conclusion microsponges of bfn were successfully formed by the non-aqueous emulsion solvent diffusion method. bfn microsponge tablets prepared 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naturally in kurdistan by hplc hazhar m. muhammad * , kawkab y. saour ** , and alaadin m. naqishbandi *, 1 * department of pharmacognosy, college of pharmacy, hawler medical university, iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad ,iraq. abstract plumbago (plumbaginaceae) is a genus of 10-20 species of flowering plants used in traditional indian medicine, native to warm temperature to tropical regions of the world. the roots of plumbago europaea, the iraqi species of plumbago, have been used for the treatment of cancer, rheumatoid arthritis, and dysmenorrhea. the main active constituents from dried powdered leaves and roots of plumbago europaea were extracted by soxhlet apparatus using ethyl acetate, the main active constituent was characterized by spectroscopic analysis (ir, 1 h nmr, and 13 c nmr) as plumbagin. quantitative and qualitative study of plumbagin in the roots and leaves extracts was carried out by hplc technique using analytical column (eurospher 100, c18, 5 µm, 250 x 4.6 mm) with 10% solvent (b) isocratic elution of methanol (solvent a) and water (solvent b) at flow rate 0.75 ml/min and detection wave length of 270 nm. the percentage of plumbagin in the root and leaf extracts was recorded to be (1.9 %) and (1.5 %) respectively. keywords: plumbago europaea, plumbagin, hplc الخالصة انهُذ, يًُى بصىسة طبيؼيت في يٍ اَىاع انُباحاث انضهشيت انًسخخذيت في انطب انشؼبى فى plumbago 01-01يضى جُس انسشطاٌ ػالج في plumbago europaeaيسخؼًم جزوس انُىع انؼشالي .انًُاطك انحاسة انى انًُاطك االسخيىائيت يٍ انؼانى نك ور plumbago europaea ت لجزوس انًجففانألوساق ونانكيًيائيت انشئيسيت ى اسخخالص انًكىَاثح .فث،انشوياحيضو و طًث انش ) ححهيم األطيافسخخذاو اانكيًيائيت انشئيسيت انًؼضونت ب انًادةحى حشخيض .وانًزيب أثيم اسيخيج soxhletباسخخذاو جهاص 13 c nmr , 1 h nmr ,ir بًادة ) plumbagin . نًادة َىػيت و كًيت دساستاء اجشحى plumbagin االوساق و نًسخخهصاث في ا eurospher 100س )ـــــــــىد انًؼاكــــذاو انؼًـــ( باسخخhplcباسخخذاو حمُيت كشوياحىغشافيا ححج انضغظ انؼاني ) انجزوس ,c18 column ,(4.6 mm i.d. x 250 mm, 5 µm :انً ييخاَىلو ححج انظشوف انخانيت( زيبaو انًاء ) انًمطش (انًزيب b ) ت انفىق انبُفسجيت . حى انكشف باألشؼ .ml/min 1..4 و سشػت جشياٌ b% يٍ انًزيب 01كًحهىل َالم ورنك بانغسم انثابج يغ ػهى % 0.4% و 0.1انجزس وانىسق نًسخخهصيٍ في كم يٍ ا plumbagin كاَج انُسب انًؤيت نًادة .nm 0.1بانطىل انًىجي نخىاني .ا introduction plumbago (plumbaginaceae) is a genus of 10-20 species of flowering plants, native to warm temperature to tropical regions of the world (1) . roots of plumbago species are used in traditional indian medicine, immunosuppressive and antitumour activities have been demonstrated (2) . plumbago europaea have been used extensively in china and other asian countries for treatment of cancer, rheumatoid arthritis, dysmenorrheal (3) . the chemical profile of the plumbago genus is marked by the presence of naphthoquinones, flavonoids and terpenoids (4) . plumbago europaea is naturally occurring plant in kurdistan region (kurdish name: rashky kalak) traditionally used for wart skin infection, hence the aim of this research works directed towards qualitative and quantitative analysis of the main active constituents in the leaf and root of the plumbago europaea by hplc. 1 corresponding author e-mail : alaadinmn@hotmail.com received : 20/1/2009 accepted : 16/6/2009 iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 44 materials and methods plant materials plumbago europaea leaves and roots were collected from susae village kurdistan region in iraq during june 2007, authenticated by the department of biology, college of education, university of salahaddin. extraction of the active constituents plumbago europaea leaves and roots were collected, dried in air for seven days and powdered separately with mechanical grinder. 25 gm of dried powdered leaves and roots were extracted separately in the soxhlet with 400 ml ethylacetate for 5 hr. the extracts were evaporated in vacu by rotary evaporator to yield 2.134 gm of dark green color residue of total leaf extract (p1) and 1.1gm of orange yellow color residue of total root extract (p2). quantitative separation of the major active constituents by tlc the major constituent in p1 and p2 extracts was isolated quantitatively using preparative tlc. fifteen gm of silica gel gf254 was mixed with 30 ml distilled water to prepare the slurry which spread on one glass plate of (20 x 20) cm by desaga spreader to obtain 0.75 mm thickness layer of silica gel sorbent (5) . the plates were activated for 1 hr in oven at 110 ºc before use. the mobile phase used was (toluene: ethyl acetate (93:7)). development of tlc plates four hundred mg of p1 and p2 extracts were dissolved separately in 10 ml of ethyl acetate and applied as a line by a capillary tube on silica gel plate (25 plates) fore each extract. one major band which was detected visually and under uv 366 in the plates of p1 and p2 extracts, scraped off from the plates and silica gel containing the isolated major band was dissolved separately in chloroform: ethanol (3:1). the mixtures were filtered through buckner funnel and the filtrates obtained from p1 and p2 extracts were evaporated to dryness in vacue to get constituent (a). the main active constituent (a) was characterized by spectroscopic analysis (ir, 1 h nmr, and 13 c nmr). hplc analysis hplc technique was used for qualitative and quantitative study of plumbagin in p1 and p2 extracts obtained from plumbago europaea using analytical column (eurospher 100, c18, 5 µm, 250 x 4.6 mm), mobile phase used was methanol: water (90:10), flow rate of 0.75 ml/min, injection volume of 20 µl, detection wave length was set to 270 nm, and temperature adjusted to 33 ºc. sample preparation used in hplc analysis the sample extracts from plumbago europaea were prepared by extraction of 1 g of dried powdered leaf and root separately with 35 ml ethylacetate for two hours by refluxing two times, the extracts were filtered, and evaporated to dryness by rotary evaporator, and dissolved separately in 5 ml methanol, filtered through 0.45 µm membranes before injection to the hplc. the knauer hplc instrument equipped with chromgate software provided by knauer was used for this analysis. the calibration curve was plotted using single level calibration, made by preparation of solution (1mg/ml) of standard plumbagin (sigma aldrich, usa) in methanol. the calibration graph was obtained from chromo gate software, figure (1). figure (1): calibration curve of authentic plumbagin. iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 45 results the major band on the chromatogram corresponding to authentic standard compound was isolated in both chromatograms of p1 and p2 extracts, figure (2). the purity of the isolated constituents was confirmed by tlc.the ir spectrum for the isolated constituent (a) figure (3), showed stretching vibration bands at (3419, 3000, 1725-1710, 1648 and 1611) cm -1 . 1 h-nmr spectrum figure(4), as shown in cdcl3 showed a singlet peak at (3.323-3.332) δ, two doublet strong bands at (6.68 and 7.054) δ, multiplate peaks at (7.314) δ , and a singlet peak at (7.613). while 13 c nmr figure (5), the data were 46.744, 47.028, 47.312, 47.596, 47.880, 48.163, 48.447, 116.104, 118.157, 120.151, 121.891, 125.726, 125.799, 126.332, 128.365, 129.370, 132.088, 146.423, and 150.581. hplc chromatogram of p1 and p2 extracts indicated the presence of plumbagin by comparing the retention time with that of the standard plumbagin (rt 6.567), figure (6), and the concentration of plumbagin was calculated by using chromgate software depending on the calibration curve of the standard plumbagin. the results showed higher concentration of plumbagin in the root part of the plant than in the leaf, table (1). (a) (b) figure (2) tlc chromatogram of preparative separation of the major constituent from a-p1 extract, bp2 extract. figure (3): ir spectrum of isolated constituent (a). iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 4. figure (4): 1 h nmr spectrum of isolated constituent (a). figure (5): 13 c nmr spectrum of isolated constituent (a). iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 45 (a) (b) (c) figure (6): hplc chromatogram, aleaves extracts (p1), broots extracts (p2), cstandard plumbagin minutes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ua u -200000 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 ua u -200000 0 200000 400000 600000 800000 1000000 1200000 1400000 1600000 1800000 p lu m b a g in 6 .5 6 7 1 .0 0 0 s 2500 plumbagin authentic name retention time estd concentration minutes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 u a u 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 u a u 0 5000 10000 15000 20000 25000 30000 35000 40000 45000 p lu m b a g in 0 .0 1 5 7 1 8 8 0 7 s 2500 leave sample3 name estd concentration area minutes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 ua u -5000 0 5000 10000 15000 20000 25000 30000 ua u -5000 0 5000 10000 15000 20000 25000 30000 p lu m b a g in 0 .0 1 9 8 6 9 6 0 1 s 2500 root sample3 name estd concentration area iraqi j pharm sci , vol.18 (suppl.), 2009 hplc analysis of plumbagin in plumbago europaea 41 table (1): quantitative study of plumbagin in p1 and p2 extracts by hplc discussion preparative tlc technique was used for separation and isolation of the major constituent in p1 and p2 extracts. the spectroscopic data (ir, 1 h nmr, and 13 c nmr) obtained confirmed the chemical structure of the isolated constituent (a) to contain the important functional groups of plumbagin which agrees with the data obtained for the same compound in other research works (6, 7, 8) . hplc technique was used successfully for this study; different conditions of hplc were used for qualitative and quantitative analysis of plumbagin in other species of the genus plumbago (9, 10) . plumbagin was identified qualitatively and quantitatively in p1 and p2 extracts by comparing the retention time with that of the standard sample. the result indicates that the hplc method was efficient for qualitative identification and quantitative determination of plumbagin. references 1. huxley a., ed. (1992), new horticultural society dictionary of gardening, vols.4, macmillan. 2. evans wc (2005): treace and evans pharmacognosy, 15 th ed. london, uk, w.b. saunders company. 3. binita b. chaplot., ashok m., dave and yogesh t., jasrai (2006), a valued medicinal plant-chitrak (plumbago zeylanica l.): successful plant regeneration through various explants and field performance, plant tissue culture and biotechnology, 16(2), 77-84. 4. selma r. de paiva., lucilene a. lima., maria raquel figueiredo and maria axiliadora c. kaplan (2004),plumbagin quantifications in roots of plumbago scandens l.obtained by different extraction techniques, annals of the brazilian academy of sciences, 76(3) 499-504. 5. joseph c. touchstone and murrell f.dobbins (1977), practical of thin layer chromatography, awilly-interscience publication, pages 25, 42,311. 6. nisha mathew., paily k.p., abidha p., kalyanasundaram m., balaraman k. (2002). macrofilaricidal activity of the plant plumbago indica/rosea, drug development research, 56(1), 33-39. 7. nayana s. kapadia., shalini a. isarani., mamta b. shah (2005),a simple method for isolation of plumbagin from roots of plumbago rosea, pharmaceutical biology,43 (6), 551-553. 8. kwang-soo s., samkeun l. and byeongjin c. (2007), antifungal activity of plumbagin from leaves of nepenthes ventricosa x maxima against phytopathogenic fungi, plant pathology journal, 23(2), 113-115. 9. yuan l., fanq d., chao l., qing-yan m., ze-wen g. (2006), determination of plumbagin in different parts of plumbago zeylanica by rp-hplc, zhongguo zhong yaoza zhi. 31(20), 1684-1686. 10. mei z.-n., zhao y.-h., li x.-k. (2007), hplc determination of plumbagin in different sections of plumbago zeylanica linn. from previous sources of yunnan, chinese journal f pharmaceutical analysis, 27(6), 901-903. no extract area under the curve of samples area under the curve of standard plumbagin concentration (%) 1 p1 718807 46608959 1.5 2 p2 896601 1.9 zoledix versus norethisterone in management of uterine bleeding with fibroid iraqi j pharm sci, vol.19(2) 2010 goserlin in the management of fibroid 54 goserelin versus norethisterone in the management of menorrhagia with uterine fibroid faris a. rasheed * ,1 , jwan n. sulaiman* and yousif abdul-raheem * departement of obs/gyn, al-kindy medical college, university of baghdad, baghdad, iraq. abstract menorrhagia is common in patients with uterine fibroids, if operation needs to be delayed for a particular reason, goserelin can be used safely to reduce bleeding and the size of the tumor.the objective is to compare between goserelin acetate and norethisterone on patients with menorrhagia and uterine fibroid. a randomized controlled study conducted in elwiya maternity teaching hospital, baghdad from the first of november 2007 to the end of april 2009. 90 patients from the consultant outpatient clinic with menorrhagia and fibroid, and their operations were delayed for medical reason were allocated in two groups, the first group, was given 3.2 mg goserelin acetate subcutaneously monthly for 3 months and the second group was given 5 mg norethisterone orally three times daily during the attack of bleeding and 5 mg once daily, cyclically if no bleeding for 3 months. the fibroid was measured in two dimensions, using convex real-time ultrasound before treatment and three months after treatment. haemoglobin and the number of pads used were also reported before and after treatment, also the side effects in both groups and the need for operations.the size of fibroid in two dimensions measurement was reduced from 28.24 cm 2 ± 6.14 to 12.3 cm 2 ± 3.45 in the goserelin group (p=0.0001) versus 26.56 cm 2 ± 5.96 to 25.22 cm 2 ± 5.01 in the norethisterone group (p= 0.2589). the haemoglobin level was 9.28 gm/100ml ± 2.44 pre-treatment in the goserelin group and 11.2 gm/100ml ± 1.88 post-treatment (p= 0.0001) versus 10.08 gm/100ml ± 2.86, and 10.24 gm/100ml ± 2.46 respectively in the norethisterone group (p= 0.7798). the need for operation was decreased significantly in the goserelin group. goserelin showed better patient response and reduction in the tumor size than norethisterone in treatment of patients with menorrhagia and uterine fibroids if operation is delayed for medical or other reasons. key words: goserelin, norethisterone , menorrhagia, uterine fibroid الخالصة ضاي ائلةات وليصاي norethisterone( يظهر وتائج فضلام اه دواء ) واىراتي واتيرون ( goserlinدواء كىزرليه ) إن حةم الايد الصيفيت ضي الرحم و خئصت عىد المريلئث الصىالي يائويه ه وسف شديد فتىئء الدورة الشهريت فو هىئك فوبئب طبيات لايدإ إلا ضااائف ضااي ائلةاات فشااهر 3( لماادة goserlin (ء دواء ) الكااىزرليه ( لأجياام عمصياات رضااد الايااد الصيفياات جراحياائ ع ووجااد فن إع اائ وجد اوه ييصم ه شدة الىسف ويرضاد إذوجىد عيد ليفيت عص الرحم بسببالدورة الشهريت فتىئءالمريلئث الصىالي يائويه ه وسف شديد الصةىء إل التداخم الةراحي د وجىد إعراض جئوبيت طفيفت ع فوإع ئء دم إل ه وسبت الهيمىكصىبيه بئلدم مئ ييصم الحئجت introduction uterine fibroids are the most common tumor in the female reproductive system. they are estimated to occur in 25% of women of reproductive age. (1) in the usa, 30% of women will have had a hysterectomy by the age of 60 years and 60% will be performed to treat fibroids. (2) hysterectomy or myomectomy remain the most common types of treatment and it is associated with high level of satisfaction. myomectomy is carried out when fertility is to be preserved, it can relief symptoms associated with myoma. goserelin acetate, a gonadotrophin releasing hormone (gnrh) agonist is a synthetic form of gonaderelin. it acts on the luteinizing hormone releasing hormone (lhrh) receptors in the pituitary gland, in the same way as natural gonadorelin. the available data seems to support the use of gnrh agonist treatment before surgery for uterine fibroids in selected circumstances. (3) administration of gnrh agonist for only two or three months preoperatively in cases of uterine fibroid decreases the bleeding, mucus debris and diameter, limiting side effects and cost, (3) and increase the haematocrit value with no metabolic side effects or detectable bone demineralization . (4) norethisterne is a synthetic progestin. it has several indications in gynaecology and primary care. at low dose ≤ 1mg it can be used in contraception and hormone replacement therapy. at higher dose ≥ 5mg it can be used in menorrhagia. (5) in our study we compare the effect of goserelin acetate versus norethisterone on patients with menorrhagia and uterine fibroid. 1corresponding author email : faris_54@yahoo.com received : 19/4/2010 accepted : 18/8/2010 mailto:faris_54@yahoo.com iraqi j pharm sci, vol.19(2) 2010 goserlin in the management of fibroid 55 patients and methods this study is a randomized controlled study conducted in elwiya maternity teaching hospital, baghdad from the 1 st of november 2007 to the 30 th of april 2009. the patients enrolled in the study were 102 women, with menorrhagia and the presence of uterine fibroid(s). patients with pervious myomectomy and those with known or suspected to have breast carcinoma were excluded from the study.all patients were not suitable for near surgery because of medical problem, long waiting lists or refusal of surgery by the patient. uterine bleeding was considered abnormal according to the patients subjective evaluation in comparison with previous menstrual status. the degree of the bleeding was assessed by the number of the pads used and hemoglobin estimation before and after treatment. 52 patients received monthly sc injection of goserelin acetate 3.6 mg (zoladex, astrazeneca, uk ) for three months, the second group was 50 patients received 5 mg of norethisterone tablets (primolut n, schering, germany) orally three times daily during the attack of bleeding and 5 mg daily if there is no bleeding to complete 21 days per cycle for three cycles. twelve patients failed to complete the study, two from the goserelin group and ten from the norethisterone group, so the final number was 50 in the goserelin group and 40 in the norethisterone group.the measurement of the fibroid was done by taking two dimensions of the largest fibroid, including the biggest diameter using ultrasound with a convex 3.5 mhz probe (hunda, japan) pretreatment and after three months. pretreatment hemoglobin was checked, and then after three months. general investigations were carried out for both groups including complete blood picture, fasting blood sugar, blood urea, and liver function tests. patient's acceptance and response were subjectively registered on a special questionnaire with any side effects occurred in that period.the results were statistically analyzed, using the statistical package for social sciences (spss) version 11. descriptive statistical analyses (frequency distributions and percentages) were used, while inferential statistics limited to t test, for comparison of means, and chi-square test of proportions. p<0.05 was considered significant. results table-1 showed the characteristics of both groups, both were comparable in age (p= 0.772) and parity (p=0.5397). about 80% of both groups were housewives. the measurements of the fibroids were taken in 2 dimensions, in the goserelin group the mean was 28.24 cm 2 , and in the norethisterone group it was 26.56 cm 2 , with no statistical significant difference. there was no statistical significant difference in the mean hemoglobin concentration, 9.28 mg/100ml and 10.08mg/100ml in the goserelin and norethisterone respectively. the duration of menorrhagia was also comparable in both groups and the number of changed pads per day. the acceptance of both groups to the treatment showed no significant difference. table 1: the characteristics of both groups table-2 showed the effect of both drugs, for the size of fibroid the two dimensions square reduced from 28.24 to 12.3 cm 2 in the goserelin group with more than 50% reduction, and for the norethisterone group there was no significant reduction. the mean hemoglobin concentration was elevated from 9.28 gm/100ml to 11.2 in the gosereline group with statistical significant difference (p= 0.0001), but in the norethisterone group there was no statistical significant difference. the number of changed pads showed statistical significant p value group 2 (norethisterone ) no. 40 group 1 ( goserelin) no. 50 0.0772 36.38 5.12 34.62 422 age( years ) mean sd 0.5397 2.98 0.98 3.12 1.14 parity mean sd 0.949 33( 82.5% ) 4( 10% ) 3( 7.5% ) 40 ( 80% ) 6( 12% ) 4( 8% ) occupation housewife government free 0.1947 26.56 5.96 28.24 6.14 size of fibroid ( cm 2 ) mean sd 0.2337 3.58 1.47 3.24 1.22 duration of menorrhagia ( months ) mean sd 0.926 4 6 21 19 5 8 23 14 number of pads changed/day less than 5 6-7 8-9 more than 10 iraqi j pharm sci, vol.19(2) 2010 goserlin in the management of fibroid 56 difference reduction in number in the goserelin group, but not in the norethisterone group. table 2 : the effect of goserelin (group 1) and norethisterone (group 2) p value treatment groups after before 0.0001 12.3 3.45 28.24 6.14 group i size of fibroid (cm 2 ) mean sd 0.2589 25.22 5.01 26.56 5.96 group ii 0.0001 11.2 1.88 9.28 2.44 group i hb level (mg/dl) mean sd 0.7798 10.24 2.46 10.08 2.86 group ii 0.000 29 15 6 0 5 8 23 14 group i ≤5 6-7 8-9 ≥10 number of pads changed per day 0.102 6 13 18 3 4 6 21 9 group ii ≤5 6-7 8-9 ≥10 table-3 showed the side effects of both groups, there was no statistical significant difference between the two groups regarding all the side effects ( p= 0.119 ), but there was more menopausal symptoms in the goserelin group, 15 versus 7 in the norethisterone group.table-4 showed the operations that were done after finishing the treatment up to about one year. there were more operations in the norethisterone group.twenty five (50%) of patients in the goserelin group had no operations versus four (10%) in the norethisterone group. seven had hysterectomy in the goserelin group and 12 in the norethisterone group and myomectomy in nineteen and twenty four respectively. all operations showed statistical significant reduction in the goserelin group. table 3 : the side effects of goserelin and the norethisterone treated groups side effect goserelin group no. ( % ) norethisterone group no. ( % ) p value menopausa l symptoms 15 (30 ) 7 ( 17.5 ) joint pain 13 (26) 5 ( 12.5 ) skin allergy 7 (14) 4 ( 10 ) increase weight 5 (10 ) 6 ( 15 ) acne 4 ( 8 ) 6 ( 15 ) no complaint 6 (12 ) 12 ( 30 ) total 50 40 table 4 : the operations in both groups type of surgery group 1 group 2 p value hysterectomy myomectomy no operation 7 18 25 12 24 4 0.000 discussion the (gnrh) agonists are an effective medical approach for the management of both dysfunctional uterine bleeding (dub) and uterine fibroids. however, their use is restricted to short courses due to its long effect on bone mass. (6) norethisterone is a common treatment of menorrhagia in our clinical practice; it is cheap, always available and well tolerated by the patients. after the introduction of goserelin in our clinical practice, we designed this study to compare both drugs in cases of menorrhagia with uterine fibroids. regarding blood loss, the increase in haemoglobin is significant after goserelin use, (7) (8) with about 1 and 1.5 gm/100ml increase in both studies respectively. in our study the increase was about 2 gm/100 ml in the goserelin group, it was not significant in the norethisterone group. in assessing the blood loss we use the subjective method by patient observation in comparison with her previous menses and the objective methods by counting pads and hemoglobin estimation, all showed significant improvement in the goserelin group. ranke hr, found no significant difference in adding estradiol/norethisterone to goserelin in reduction of blood loss. (9) pre operative goserelin has been shown to decrease blood transfusion during operation and increase the post operative hemoglobin. (10) goserelin was found to decrease the size of uterine fibroids. (11-15) in our study we used two dimensions measurement of the fibroid by ultrasound, as it can be done by the usual ultrasound equipment available in gynecology clinics; the reduction in the area of the fibroid was from 28.24± 6.14 cm 2 to 12.3±3.45 which represent more than 50%. bozzini, 2004 found in a randomized controlled trial that goserelin use in monthly injection for 3 months reduce the size of fibroid by 43%. (16) adding goserelin after uterine artery embolization was found not to increase in the reduction of the size of leiomyoma, (17) in the same study the reduction of size of fibroid in goserelin group was 45%. in a study done by lumsden, 1994, on 71 ladies scheduled for hysterectomy for fibroid, and were divided in 2 groups, one was given goserelin and the other placebo before operation and they found that the size of the fibroid is smaller in the iraqi j pharm sci, vol.19(2) 2010 goserlin in the management of fibroid 57 goserelin group, also the haemoglobin level and the duration of the operation. (18) goserelin was also found to increase pregnancy rates if given before hysteroscopic resection of fibroid in cases of sub fertility. (19) many studies found that blood loss during operation for fibroid after goserelin use was less than without it. (17)(20) recent study showed that the use of triple tourniquets is associated with less blood loss than the use of pre-operative goserelin in open myomectomy. (21) goserelin was found also to shorten the operative time. (13) in our study these parameters were not measured.no significant medical problems were found after the short time use of goserelin. (4) in our study there was no significant difference in the side effects of both groups (p=0.119), but more vaso-motor symptoms noted in the goserelin group, 39% versus 17.3% in the n group.regarding the need for operations, 25 of the goserelin group ( 50% ) had operations, 7 hysterectomy and 18 myomectomy, versus 36 out of 40 in the norethsterone group, 12 hysterectomy and 24 myomectomy, so the need for operation is decreased with goserelin , mostly due to improvement of symptoms, and the desire of most of the women to preserve the uterus. hysterectomy rates for leiomyoma decreased significantly from 2.13 per 1000 to 1.91 (p < .0001), due to increase in uterine artery embolization and uterine ablation. (22) gnrh agonists shrink the uterus and fibroids, and this effect make it possible to change some of abdominal hysterectomies to vaginal hysterectomy, in one study, 76% of gnrh agonist-treated patients had vaginal hysterectomy versus 16% of non treated patients. (23) the use of gnrh agonists can make possible a conversion from abdominal hysterectomy to either vaginal hysterectomy or laparoscopic-assisted vaginal hysterectomy or laparoscopic supracervical hysterectomy. (23) in conclusion, goserelin for three months is a reasonable choice of treatment for patients having menorrhagia with uterine fibroid, as it increases the hemoglobin concentration, decrease the need for operation, decrease blood transfusion during operation, and with limited side effects. references 1. buttram, v. and reiter, r. uterine leiomyomata: etiology, symptomatology and management. fertil. steril: 1981; 36, 433–445.) 2. ahrq (2000) management of uterine fibroids. evidence report. technology assessment: number 34. agency for healthcare research and quality of life. http://www.ahcpr.gov/.. 3. crosignani pg, vercellini p, meschìa m, oldani s, bramante t gnrh agonists before surgery for uterine leiomyomas. report med 1996 ; 41(6):415-21.) 4. cagnacci a, paoletti am, soldani r, angiolucci m, arangino s, falqui a, melis gb role of goserelin-depot in the clinical management of uterine fibroids. obstet gynecol 1994; 21(4):263-5.) 5. irvine ga; campbell-brown mb; lumsden ma; heikkila a; walker jj; cameron it, randomised comparative trial of the levonorgestrel intrauterine system and norethisterone for treatment of idiopathic menorrhagia. br j obstet gynaecol 1998;105(6):592-8. 6. thomas ej, add-back therapy for long-term use in dysfunctional uterine bleeding and uterine fibroids. br j obstet gynaecol. 1996; 103 suppl 14:18-21 7. gerris j, degueldre m, peters aa, romao f, stjernquist m, al-taher h the place of zoladex in deferred surgery for uterine fibroids. zoladex myoma study group.] horm res 1996; 45(6):279-84. 8. stovall tg, summit rl, washburn sa, ling fw gonadotropin-releasing hormone agonist use before hysterectomy. am j obstet gynecol 1994 ; 170(6):1744-8; discussion 1748-51. 9. franke hr, snaaijer ff, houben pw, van der mooren mj treatment of dysfunctional uterine bleeding in the perimenopause: the effects of adding combined estradiol / norethisterone acetate therapy to goserelin acetate treatment: gynecol endocrinol 2006 ; 22(12):692-7 10. lim ss, sockalingam jk, tan pc: goserelin versus leuprolide before hysterectomy for uterine fibroids. int j gynaecol obstet 2007 27. 11. moris,ep, rymerj, robinsonj, fogelmani efficacy of tibolone as "add-back therapy" in conjunction with a gonadotropinreleasing hormone analogue in the treatment of uterine fibroids. fertil steril 2007 15. 12. russo p, ciolli p, atlante m, carico e, mancini r, russo r, vecchione a [clinical use of leuprolide acetate depot in a group of gynecologic patients in the preoperative period; minerva ginecol 1998 ; 50(11):499502. 13. tiufekchieva e, nikolov a: hysteroresection of submucous myomas after treatment with zoladex; akush ginekol (sofiia) 2006; 45(1):19-24. 14. donnez j, hervais vivancos b, kudela m, audebert a, jadoul p a randomized, placebo-controlled, dose-ranging trial comparing fulvestrant with goserelin in http://www.ahcpr.gov/ http://www.medscape.com/medline/publicationbrowser/123?pmid=8916983 http://www.medscape.com/medline/publicationbrowser/123?pmid=8916983 iraqi j pharm sci, vol.19(2) 2010 goserlin in the management of fibroid 58 premenopausal patients with uterine fibroids awaiting hysterectomy. fertil steril 2003 ; 79(6):1380-9. 15. baytur yb, ozbilgin k, cilaker s, lacin s, kurtul o, oruc s, koyuncu fm a comparative study of the effect of raloxifene and gosereline on uterine leiomyoma volume changes and estrogen receptor, progesterone receptor, bcl-2 and p53 expression immunohistochemically in premenopausal women. eur j obstet gynecol reprod biol 2006 sep 12 16. bozzinin, messinaml, borsarir, hilariosg, pinottija comparative study of different dosages of goserelin in size reduction of myomatous uteri.j am assoc gynecol laparosc 2004 ;11(4): 462-3.i 17. vilosga,vilosag, , abu-rafeab, prong, kozakr, garving, administration of goserelin acetate after uterine artery embolization does not change the reduction rate and volume of uterine myomas. fertil steril 2006 ; 85(5):1478-83. 18. lumsden ma, west cp, thomas e, coutts j, hillier h, thomas n, baird dt treatment with the gonadotrophin releasing hormone-agonist goserelin before hysterectomy for uterine fibroids. br j obstet gynaecol 1994 ; 101(5):438-42. 19. narayan r, rajat, goswamy k treatment of submucous fibroids, and outcome of assisted conception. j am assoc gynecol laparosc 1994 ;1(4 pt 1):307-11. 20. crosignani pg, vercellini p, meschìa m, oldani s, bramante t gnrh agonists before surgery for uterine leiomyomas. ]j reprod med 1996 ; 41(6):415-21 21. al-shabibi n, chapman l, madari s, papadimitriou a, papalampros p, magos a prospective randomised trial comparing gonadotrophin-releasing hormone analogues with triple tourniquets at open myomectomy. bjog 2009 feb 4. 22. jacobson gf, shaber re, armstrong ma, hung yy. links changes in rates of hysterectomy and uterine conserving procedures for treatment of uterine leiomyoma. . am j obstet gynecol. 2007; 196(6):601.e1-5; discussion 601.e5-6. 23. carter je. alternatives to total abdominal hysterectomy. jsls. 1997; 1:259-262. the beneficial role of some bone markers in evaluating women with osteoporosis under different therapeutic regimens iraqi j pharm sci, vol.20(1) 2011 bone markers in osteoporosis 1 the beneficial role of some bone markers in evaluating women with osteoporosis under different therapeutic regimens sshhaaiimmaa aa.. aabbbbaassss** ,,11 aanndd sshhaatthhaa hh.. aallii** * department of clinical laboratory sciences , collage of pharmacy , university of baghdad, baghdad , iraq aabbssttrraacctt osteoporosis is a systemic disease of the skeleton, characterized by low bone mass and alteration in the micro-architecture of the bone tissue that lead to an increase in brittleness with the ensuing predisposition to bone fracture. global statistics shows that women are more exposed to this disease than men and in particular at menopause. this study was designed to evaluate the use of some bone markers: serum osteocalcin (ost), alkaline phosphatase (alp), as bone formation markers, also parathyroid hormone (pth), calcium and inorganic phosphate level, for the assessment of patients with osteoporosis and to evaluate their role in monitoring of several types of therapeutic interventions (such as bisphosphonates, hormonal replacement therapy, and ca and vit.d) in postmenopausal women.this study comprised of 36 women (age 51.67±5.14 years) those diagnosed to have osteoporosis, to be allocated randomly into three groups according to the type of therapy to be given as; group a: received bisphosphonates (sodium alendronate 10mg/day) for twelve weeks (n=12). group b: treated with hormonal replacement therapy (conjugated estrogen 0.625mg/day) for twelve weeks (n=12). group c: received ca and vit. d (ca1500mg/day and cholecalciferol 1000iu/day) for twelve weeks(n=12). in addition to 15 perimenopausal healthy women to serve as a control group (age 51.13±7.62 years).the studied parameters were measured in serum obtained before starting treatment and after 12 weeks of therapy. result indicated that the baseline values of both serum ost and alp were significantly higher in postmenopausal patients as compared to controls and serum ost showed a significant reduction after treatment with alendronate compared to those treated with either hrt or ca and vit. d.from this study we recommend estimating the baseline bone markers (ost and alp) status for newly diagnosed osteoporotic patients to be used as a guide for deciding the initial therapeutic intervention, and detection of non responder instead of waiting until patients develop further fracture while they are on therapy. key words: osteoporosis, postmenopause, osteocalcin, alendronate الخالصة شاشت انؼظاو "حزقق انؼظاو" يٍ األيزاض انخٍ حصُب انهُكم انؼظًٍ وحسبب خهال فٍ انُسُش انؼظًٍ يًا َؼخبز يزض ه َؤدٌ نُقص فٍ كزافت انؼظاو وحغُزاث دقُقت فٍ انُسُش انؼظًٍ وانخٍ حؤدٌ إنً سَادة هشاشت انؼظاو وسَادة احخًانُت انخؼزض نكسز َهذف انبحذ إنً حقُى بت انُساء بهذا انًزض أكزز يٍ انزصال خاصت بؼذ سٍ انُأس.انؼظاو. وحذل اإلحصائُاث انؼانًُت إٌ يؼذل إصا فؼانُت اسخخذاو بؼط يؤشزاث انؼظاو فٍ حشخُص انًزض يُها االسخُىكانسٍُ ويحهم انفىسفاث انقاػذٌ كًؤشزاث نبُاء انؼظاو, نًزض باسخخذاو ػالصاث انالػعىَت وفٍ يخابؼت ا إظافت إنً قُاس يسخىي هزيىٌ انبارارُزوَذ إنً صاَب انكانسُىو وانفىسفاث يٍ انُساء انالحٍ بؼاٍَُ يٍ 63 انفىسفىَاث انزُائُت وػالس انهزيىَاث انخؼىَعٍ وانكانسُىو وفُخايٍُ دٌ. حعًُج انذراست يخخهفت يزم ±76.35.)بًؼذل أػًار أسبىػايزض هشاشت انؼظاو وحى حقسًُهٍ إنً رالرت يضايُغ وفقا نهؼالس انذٌ أػطٍ نهٍ نًذة ارُا ػشز .(سُت 7.65 يزَعت. 61يهغى/انُىو نًذة ارُا ػشز أسبىػا وحعًُج 61أػطُج ػقار االَذروَُج -ا المجموعة يزَعت. 61يهغى/انُىو(نًذة ارُا ػشز أسبىػا وحعًُج 1.317أػطُج ػالس انهزيىَاث انخؼىَعٍ يٍ االسخزوصٍُ) -المجموعة ب يزَعت. 61وحذة دونُت َىيُا( وحعًُج 6111يهغى و6711أػطُج انكانسُىو يغ فُخايٍُ دٌ) -عة جالمجمو سُت (, حى قُاس انًؤشزاث قُذ انذراست 5.31±76.66إظافت انً خًست ػشز يٍ انُساء االصحاء كًضًىػت ظابطت بًؼذل ػًز) انُخائش باٌ انًسخىَاث انبذائُت نالوسخُاكانسٍُ ويحهم انفىسفاث فٍ يصم انًزَعاث قبم وبؼذ بذء انؼالس بأرُا ػشز أسبىػا .اظهزث انقاػذٌ فٍ انًصم كاَج يزحفؼت فٍ انُساء فٍ سٍ اَقطاع انطًذ وانًصاباث بخزقق انؼظاو يقارَت بانًضًىػت انعابطت واَخفط ًكُُا انخىصُت بخقُُى يسخىي يؤشزاث يٍ َخائش انذراست َ يسخىي االوسخُاكانسٍُ فٍ يصم انًزَعاث بؼذ ػالصهٍ باالَذروَُج . انؼظاو نهًزَعاث حذَزاث انخشخُص بخزقق انؼظاو نخكىٌ دنُال ػهً اخخُار انؼالس انًُاسب وكذنك نهكشف ػٍ ػذو االسخضابت بذال يٍ االَخظار نحذود انكسىر خالل اسخًزارهى ػهً انؼالس. 1corresponding author email : oudi68@yahoo.com received : 28/3/2010 accepted : 11/10/2010 iraqi j pharm sci, vol.20(1) 2011 bone markers in osteoporosis 2 introduction bone consists of an extracellular matrix, the organic phase of which is composed of type i collagen, proteoglycans, and noncollagenous proteins including: osteocalcin, bone sialoprotein, osteonectin, thrombopondin, and osteopontin. bone matrix also contains growth factors and cytokines that have an important regulatory role in bone remodeling (1) . bone modeling prevents the occurrence of damage by adapting bone structure-and hence strength-to its loading circumstances (2) .bone in the adult skeleton is renewed continuously in response to a variety of stimuli by the process of bone remodeling. this involves removel of trenches or tunnels of bone from the surfaces of trabecular and cortical bone, respectively, by osteoclasts (bone resorbing cell) and osteoblasts that subsequently fill in these trenches by laying down new bone matrix in them (3) . in normal adult bone, the processes of resorption and formation are coupled both in space and time, thus bone resorption always precedes formation (4) . bone loss is the result of an imbalance in bone turnover with bone resorption occurring at a faster rate than new bone formation (5) .osteoporosis most commonly affects postmenopausal women, placing them at a significant risk of fracture. hip fractures are important causes of mortality and morbidity among postmenpausal women. approximately 20% of patients with hip fractures die within a year, most of the deaths occurring within the first 6 months after a fracture. among the survivors, 30%-50% never regain their prefracture functional status (6) . within geriatric population in most countries, disorders of bone and mineral metabolism are becoming increasingly relevant to every day clinical practice. consequently, the interest in, and the need for effective measures to be used in the screening, diagnosis and follow-up of such pathologies has markedly grown. together with the clinical and imaging techniques, biochemical tests could play an important role in both the assessment and differential diagnosis of osteoporosis (7) , but yet have not been practically used in osteoporosis (8) . in recent years, a panel of new simple to use kits to determine bone markers had became available for the diagnosis, monitoring, of osteoporosis, and other metabolic diseases of bone tissue (9) . this study was designed to evaluate: 1. the role of some biochemical bone formation markers, such as, osteocalcin, parathyroid hormone, alkaline phosphate, serum calcium and serum inorganic phosphatase, in the assessment of osteoporosis status in postmenopausal women. 2. the effect of three therapeutic interventions applied in osteoporosis (i.e. bisphosphonates, supplement of vit. d and ca and hormonal replacement therapy) on the studied bone formation parameters after 12 weeks of starting their therapies. subjects and methods this study comprised 36 postmenopausal women with newly diagnosed osteoporosis by a senior physician based on (x-ray and sign and symptoms), according to who diagnostic criteria for osteoporosis (10) with a mean age of 51.67±5.14 years. they were classified into three main groups to be treated for twelve weeks as follows: group a; include 12 patients to be treated with alendronate 10 mg/day. group b: include12 patients to be treated with hormone replacement therapy (hrt) (conjugated estrogen 0.625mg /day). group c: include 12 patients to be treated with ca and vit .d (ca1500 mgand cholicalciferol 1000iu /day). in addition to a control group of 15 perimenopausal of apparently healthy women with a mean age of 51.13±7.62years. a fasting 5 ml venous blood sample was withdrawn from each subject before and twelve weeks after treatment. serum was separated and divided into aliquots to be used for immediate determination of total alp (11) . other aliquots were stored frozen (-20°c) for determination of osteocalcin (12) , pth (13) , serum total ca (14) , and serum inorganic phosphate levels (15) . statistical analysis was performed using microsoft excel 2007 in order to estimate mean± sem, as well as, the estimation of paired t-test values, considering p<0.05 to be significant. results 1. effect of treatment with either alendronate, hrt, or calcium and vitamin d on serum level of osteocalcin in women with postmenopausal osteoporosis. figure (1) showed that the serum levels of osteocalcin in osteoporotic women were significantly elevated (p<0.05) as compared to controls by 29.18% ,19.33% ,and 40.33%, respectively in the three groups a,b and c .there was a significant reduction (p<0.05) in serum osteocalcin levels after treatment with iraqi j pharm sci, vol.20(1) 2011 bone markers in osteoporosis 3 alendronate by -33.03%, in comparison with corresponding values before treatment. meanwhile, alendronate administration induced a significant decrease in serum osteocalcin in comparison with hrt, and (ca and vit. d) treated groups. 2. effect of treatment with either alendronate, hrt, or calcium and vitamin d on serum level of alp in women with postmenopausal osteoporosis. table(1) indicates a significant increase (p<0.05) in serum total alp activity before starting treatment with alendronate. whereas, non significant alteration in serum level of alp(p>0.05) were observed following treatment with the three tested therapies in comparison with their pre-treatment values. 3. effect of treatment with either alendronate, hrt, or calcium and vitamin d on serum level of pth in women with postmenopausal osteoporosis. table (1) showed that the serum pth levels were non-significantly altered baseline values in comparison to the control group. also non significant changes in serum levels of pth were observed following the treatment with either one of the three tested therapies in comparison with their pre-treatment values. figure 1: effect of alendronate(10mg/day), hrt(0.625 mg /day conjugated estrogen), and ca and vit. d (ca and vit. d 1500 mg /day and 1000 iu /day respectively), for twelve weeks on serum osteocalcin level in post menopausal osteoporosis women . group a= treated with alendronate. group b= treated with hrt group c= treated with ca and vit.d table 1 : effect of alendronate (10 mg/day), hrt(0.625 mg/day conjugated estrogen),and ca and vit. d (ca and vit. d 1500 mg/day and 1000 iu /day respectively) for twelve weeks on serum total alp and pth levels in all patients. marker drug control (n=15) before treatment (n=12) after treatment (n=12) alp (u/l) aln hrt ca and vit d 88.47 ±13.82 133.92±37.91* 100.83±26.88 100.25±27.61 125.58±44.21 93.42± 20.89 91.08±16.41 pth (ng/ml) aln hrt ca and vit d 27.77±14.55 27.93±17.19 32.77± 16.88 33.37±10.8 28.92± 16.15 32.29±16.69 31.17±10.0 values are presented as mean ± sem. * significant difference from control at p < 0.05 # significant difference from before treatment at p<0.05 aln= alendronate hrt= hormone replacement therapy serum osteocalcin 37.76 48.78 32.57 45.06 44.12 52.99 50.99 0 10 20 30 40 50 60 m ea n va lu e control pre a post a pre b post b pre c post c iraqi j pharm sci, vol.20(1) 2011 bone markers in osteoporosis 4 4. effect of treatment with either alendronate, hrt, or calcium and vitamin d on serum level of calcium and inorganic phosphate in women with postmenopausal osteoporosis. table (2) indicate that there was non significant flacutuation in serum total ca and inorganic phosphate levels in all patients groups in comparison with control group , nor after treatment with either alendronate ,hrt, or ca and vit d or in comparison with their pre treatment values . table 2:effect of alendronate (10 mg/day) , hrt(0.625 mg /day conjugated estrogen) ,and ca and vit. d(ca and vit. d 1500 mg /day and 1000 iu /day respectively) for twelve weeks on serum total calcium and inorganic phosphate levels in postmenopausal osteoporosis women. marker drug control (n=15) before treatment (n=12) after treatment (n=12) ca (mg/dl) aln hrt ca and vit d 8.78±0.69 8.45±1.16 8.32±1.12 8.98±1.12 8.50±1.13 8.21±1.07 9.39±0.54 po4 -3 (mg/dl) aln hrt ca and vit d 3.85±0.38 4.13±1.61 4.11±0.95 3.67±0.51 4.47±1.70 4.25±1.01 3.73±0.47 values are presented as mean ± sem. aln= alendronate hrt= hormone replacement therapy discussion in our study the levels of serum ost, was significantly higher in untreated patients with postmenopausal osteoporosis as compared to controls (figure 1). these results are in agreement with (aysan t et al, 2007 and peipchl p et al, 2000) (16.17) . this could be attributed to the accelerated bone loss occurring within the first years following menopause (18) .since during both menopause and aging, the coordinated balance to disturbance of bone formation and resorption can be disturbed, resulting in excessive bone loss and osteoporosis (19) . thus serial measurement of ost is indicated to be an excellent marker to assess the long term effects of antiresorptive therapy (20) .the significant reduction in serum ost level of postmenopausal osteoporosis following alendronate administration in comparison with the baseline values. this result is confirmed by other studies. (21, 22, 23) and this is because aminobisphosphonates can decrease bone remodeling by decreasing osteoclast activity and by inducing osteoclast apoptosis. (24,25) the hypothesis that alendronate, like other nitrogen-containing bisphosphonates, inhibits a rate-limiting step in the mevalonic acid pathway that is essential for osteoclast function (26,27) , this could be explained that the first measurable effect of bisphosphonate is the decrease of the rate of bone resorption that is followed by a slower decrease of the rate of bone formation, due to the coupling of the two processes and the attainment of a new steady state at a lower rate of bone turnover (28) .in the present study the level of ost was non significantly lowered following twelve weeks of treatment with hrt therapy compared to their level before treatment (table1) this is in agreement with previous study (29,30) , indicated that serum ost was able to decreases only after 6 months of treatment with hrt (17) . the possible mechanism could include a decrease in osteoclastogenesis, osteoclast apoptosis and additional effect on calcium homeostasis (31) . estrogens may affect osteoclasts directly, in addition estrogens certainly exerts effect on osteoclasts indirectly by suppressing the production of bone resorbing cytokines from osteoblast and bone marrow stromal cells (32) .the possible explanation to the lack of response of bone markers to treatment with calcium and vitamin d (figure 1 and table 1) may be simply because this treatment has a bone stimulant effect but no or very minimal anti-resorptive effect (33) . however, previous studies have shown some minimal improvement of bone mineral density (bmd) in patients treated with calcium and vitamin d but this did not correlate with any reduction of fracture risk (34) . (shinchuk et al, 2005) had reported high prevalence of vitamin d deficiency in men and women with osteoporosis (35) . therefore calcium and vitamin d is now considered as a complementary treatment to osteoporosis medications such as bisphosphonates rather iraqi j pharm sci, vol.20(1) 2011 bone markers in osteoporosis 5 than a sole line of treatment by itself (36,37) , although (palacios et al, 2005) had reported a significant reduction in serum bone markers after treatment with calcium and vitamin d (38) . this supports the notion found in (figure1 and table 1) that alendronate is a stronger antiresorptive agent than both hrt (39,40) and calcium and vitamin d (41) . and that alendronate may have a very beneficial effect when added to hrt regimen in patients with postmenopausal osteoporosis (39) . additionally total serum alkaline phosphatase in untreated postmenopausal women showed a significant increase in level as compared to control (table 1). these results are in agreement with other studies (16, 42) . as this enzyme function in the metabolism of bone tissue, thus increased activity could be a way to compensate for lost calcium/phosphorus by recruiting more of these elements to build new bone tissue (16, 42) . however, no differences were noted in serum alp levels following treatment with all tested medications in postmenopaul osteoporotic patients. this may be due to the fact that total alp measurements is less specific to bone than measurements of the bone isoenzyme (7) and also may be due to short time for follow up period (twelve weeks) in this study. meanwhile, pth levels, in the current work, showed non significant changes in untreated postmenopausal osteoporosis groups in comparison with controls. such finding could be explained through the changes that take place in calcium metabolism related to estrogen deficiency, which induces a decrease in the intestinal absorption of calcium and reduces the biological activity of vitamin d. it also enhances the renal calcium excretion and diminishes the ability of parathyroid hormone to reduce calciuria. serum calcium remains unchanged or slightly increased due to compensatory increased bone resorption and pth remains unchanged resulting in increased bone resorption and this is in agreement with our result (43) . results in table 2 elucidated non significant fluctuations in serum calcium and inorganic phosphate before and after the treatment of postmenopausal women. our results are in agreement with other studies (42,44,45,46) . as serum calcium is homeostatically controlled and the integrity of bone may be sacrified to maintain serum calcium within the normal range (47) . consequently, serum calcium is a poor predictor of histological features and is not indicative of bone resorption in osteoporotic patient (48) . meanwhile non significant alteration in serum inorganic phosphate before and 12 weeks after treatment in postmenopausal women and this was in agreement with previous study (42,44,45,46) , indicating that the determination of serum inorganic phosphate was of no significant values for the diagnosis and follow up of osteoporosis.thus effective treatment with antiresorptive agents can show up significant changes in abnormal bone markers after treatment supporting the value of measuring them in the patients with osteoporosis. hence bone markers (ost) can be used for monitoring the response to therapy and also as aguide for patients compliance with treatment.calcium and vitamin d supplementation is recommended as a complementary treatment even if dietary supply is sufficient. thus we can recommend checking the baseline bone marker status (ost) for all osteoporotic patients. references 1. dogan e. and posasci c.: monitoring hormone replacement therapy by biochemical markers of bone metabolism in menopausal women postgrad. med. j. 2002;78:727-731 2. martin t. and seeman e.: bone remdelling; its local regulation and the emergence of bone fragility. pub. med. 2008;22(5):701-22 3. brendan f. and lianpingxing: functions of rankl/rank/opg in bone modeling and remodeling. arch. biochem. biophys. 2008;15:473(2):139-146. 4. kenny a.m. and prestwood k.m.: osteoporosis: pathogenesis, diagnosis and treatment in older adults. rheum. dis. clinc. north. am. 2000;26(3):569-591. 5. jean, yves r. and veronique r.: patients preference in the management of postmenopausal osteoporosis with bisphosphonates. clinc. interv. aging. 2006;1(4):415-423. 6. jun i, yoshihiro, tsuyoshi t, et al : hip fracture protection by alendronate treatment in postmenopausal women with osteoporosis. clini. intervene. in aging ; 2008 ;3(3):483-489. 7. markus j. seibel: biochemical markers of bone turnover part 1; biochemistry and variability. clin. biochem. rev. 2005;26:97-122. 8. chailurkit l, ongphiphadhanakul b, piaseu n, et al :biochemical markers of bone turnover and response of bone mineral density to intervention in early postmenopausal women : an experience in a clinical laboratory. clin. chem.. ;2001: 47:1083-1088. 9. filip rs : age and bmd related differences in biochemical markers of bone metabolism in rural and urban women iraqi j pharm sci, vol.20(1) 2011 bone markers in osteoporosis 6 from lublin region, poland. ann agric environ med ;2004:11:255-259. 10. who: assessment of fracture risk and its application to screening for postmenopausal osteoporosis: report of a who study group. geneva tech. rep. series. 1994; 843:1-129. 11. belfield a and goldberg dm: revised assay for serum phenyl phosphatase activity using 4-amino antipyrine enzyme;1971;12:561-573. 12. b. demiaux, m.e, arlot and et al: serum osteocalcin is increased in patients with biochemical and histomorphometric finding, journal of clinical endocrine. and metab.;1992:74(5):1146-1151. 13. kruger l., rosenblum s, zaazra j, et al : intact pth is stable in unfrozen edta plasma for 48 hours prior to laboratory analysis. clin. chem. ;1995:41(6):s47. 14. stern j and lewis whp :the colorimetric estimation of calcium in serum with ocresophthalein complexone clinc. chim. acta.;1957:2:576. 15. eugene s, slawa s, and bennie z : direct serum inorganic phosphate. clinc. chim. acta;1967:15:155 16. aysan t, aksoyz b, asuman, et al : correlation of serum il-6 levels and prolidase activity between bone turnover markers and bmd in postmenopausal women with and without osteoporosis. turkish journal of medical sciences ;2007:37(3):129-134. 17. peichl p, griesmacher a, muller mm, et al : serum osteocalcin and urinary crosslaps are suitable markers to short-term hormone replacement therapy. gynaecological endocrinology;2000:14(5) : 374-381. 18. garnero p, borel o, and delmas pd : evaluation of a fully automated serum assay for c-terminal crosslinking telopeptide of type i collagen in osteoporosis.clin. chem. ; 2001:47(4):694702. 19. apurva ks, elizabeth lv, michael le, et al ; clinical use of serum and urine bone markers in the management of osteoporosis. current medical res opin ;2005:21(7):1015-1026. 20. indumati v and patil vs : biochemical markers of bone remodeling. jcdr. net.article;2010. 21. raisz l, smith ja, trahiotis m, et al : short term risedronate treatment in postmenopausal women:effects on biochemical markers of bone turnover. osteopor. int.;2000:11(7):615-620. 22. kadir y, garhan g, saliha k, et al : comparison of the effect of alendronate, risedronate and calcitonin treatment in postmenopausal osteoporosis. journal of back and musculos rehabilitation ; 2005:18(3-4):1878-6324. 23. gur a, colpan l, cevik r, et al : comparison of zink excretion and biochemical markers of bone remodeling in the assessment of the effects of alendronate and calcitonin on bone in postmenopausal osteoporosis. clin. biochem.;2005:38(1):66-72. 24. manolagas s c :birth and death of bone cells: basic regulatory mechanisms and implications for the pathogensis and treatment of osteoporosis.endocr. rev. ;2000:21:115:137 25. stepan jj, alenfeld f, boivin g, et al :mechanism of action of antiresorptive therapies of postmenopausal osteoporosis.endocr. regul. ;2003: 37(4) : 225-238 26. dunford je, thompson k, coxon fp, et al ; structure activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates. j pharm. exp. ther.; 2001: 296(2):235-242. 27. kavanagh kl, guo k, dunford je, et al :the molecular mechanism of nitrogencontaining bisphosphonates as antiosteoporosis drugs. proc. natl. acad. sci. usa;2006:103(20):7829-7834. 28. papapoulos s : clinical aspect of bisphosphonates. asbmr,2007. 29. okabe r, nakatsuka k, inaba m, et al :clinical evaluation of the elecsys βcrosslaps serum assay, a new assay for degradation products of type i collagen ctelopeptides.clin. chem. ; 2001: 47:14101414. 30. okabe r, nakatsuka k, inaba m, et al : significance of serum crosslaps as a predictor of changes in bmd during estrogen replacement therapy; comparison with serum carboxyterminal telopeptide of type i collagen and urinary deoxypyridinoline. j. bone miner. metab.; 2004:22(2):127-131. 31. riggs bl, khosla s, and melton i j :sex steroids and the construction and conservation of the adult skeleton. endocr. rev.;2002:23:279-302. 32. nuti r : osteoporosis: bone balance, disturbance leading to bone loss. medieographia;2007:29(2):120-125. 33. buckley km, leib es, cartularo ks, et al :calcium and vitamin d3 supplementation iraqi j pharm sci, vol.20(1) 2011 bone markers in osteoporosis 7 prevents bone loss in the spine secondary to low-dose corticosteroids in patients with rheumatoid arthritis. a randomized, doubleblind, placebo-controlled trial. ann. intern. med. ;1996: 125(12):961-968. 34. cranny a, guyatt g, griffith l, et al :meta-analyses of therapies for postmenopausal osteoporosis. ix:summary of meta-analyses of therapies for postmenopausal osteoporosis. endocr. rev. ;2002:23(4):570-578. 35. shinchuk l, haanacahuari n, and ingersill d :vitamin d deficiency and osteoporosis: highly prevalent in men and women admitted to subacute rehabilitation facility bosten massuchsetts during summer. am. j. physical med. rehab.; 2005: 84 (3 ):203. 36. adachi jd, and ioannidis g :calcium and vitamin d therapy in corticosteroid induced bone loss:what is the evidence?. calcif. tissue. int. ;1999:65(4):332-336. 37. laszlo a :postmenopausal osteoporosis. orv. hetil.;2004:145(1):3-13. 38. palacios s, castelo-branco c, cifuentes i, et al :changes in bone turnover merkers after calcium-enriched milk supplementation in healthy postmenopausal women: a randomized, double blinded, prospective clinical trial.menopayse ;2005: 12(1):63-68. 39. tiras mb, noyan v, yildiz a, et al: effect of alendronate and hormone replacement therapy, alone or in combination, on bone mass in postmenopausal women with osteoporosis: a prospective, randomized study.hum. reprod.; 2000:15(10):20872092. 40. evio s, tiitinen a, laitinen k, et al effect of alendronate and hormone replacement therapy, alone and in combination, on bone mass and markers of bone turnover in elderly women with osteoporosis. j. clin. enndocrinol. metab.;2004:89(2):626-631. 41. grados f, brazier m, kamel s, et al : prediction of bone mass density variation by bone remodeling markers in postmenopausal women with vitamin d insufficiency treated with calcium and vitamin d supplementation. j. clin. endocrinol.metab.;2003:88(11):5175-5179. 42. ashuma s, shashi s, anju , et al ; study of some common biochemical bone turnover markers in postmenopausal women.indian journal of clinical biochemistry ;2005: 20(1):131-134. 43. rizzoli r :control of bone turnover and bone balance: a target for antiosteoporosis treatment. medicographia.;2007;29(2):133136. 44. jasminka z, llich rd, jean ek, et al :nutrition in bone health revisited: a story beyond calcium.american college of nutrion.;2000:19(6):715-737. 45. omarani gr, masoompou sm, et al:effect of menopause and renal function on vitamin d status in iranian women. health journal.;2006. 46. sireen s, hisham m, hilow, et al :the efficacy and safety of calidon tablets for management of osteoporosis in jordanan women: a randomized clinical trial saudi pharmaceutical journal. ;2004: vol(12): no.(2-3). 47. sasaki n, kusano e, ando y, et al :glucocorticoid decreases circulating osteoprotegerin (opg):possible mechanism for glucocorticoid induced osteoporosis nephrol. dial. transplant.; 2001: 16(3): 479-482. 48. malluche hh, and faugere mc :bone biopsies: history and histomorphometry. in: metabolic bone disease and clinically related disorders, 2 nd edition, avioli l.v. and krane s.m.(editor). philadelphia, w.b. saaunders co., p.;1990:283-328. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 1 formulation and clinical evaluation of orphenadrine citrate as a plain tablet abdul karim f. jumaa,alaa a.abdulrasool and hikmet a.al – dujali received 5-8-2001 accepted 11-6-2002 abstract orphenadrine is an anticholinergic ,antimuscarinic , ce ntrally acting skeletal mus cle re la xa nt .it pres ents in the fo rm of c itrate and hcl sa lts which a re use d in tre atment of the symptoms o f mild p arkins on's d is eas e and also it is use d as a djuva nt with othe r drugs in the therap y . many trials were mad e to fo rmula te orp henadrine c itra te as a p la in tab le t using we t granulation or dire ct c ompres sion tec hniq ue in o rd er to get a s atis factory formula through s tudying the e ffec t of va rious fa ctors such a s bind ers , diluents and disintegra nts type s . the b est fo rmula wa s ob ta ined by using po ly vinyl p yro lidine (pvp ) as a b inder also the res ults indica te d that starch a nd mannito l ga ve a cce ptab le p hys ical prope rties to the ta blets whe n they we re us ed as diluents . at the s ame time , the results s ho wed that avice l which wa s us ed a s a disintegra nt ga ve an ac cep table disintegra tion and d is so lution time in co mparison with the reference ta blet disip al . in a dditio n , the se lected formula wa s us ed to stud y the effe ct of me thod of incorpo ra tion of d is integrant on the p hysical prope rties of ta blets .it w as found that the intragra nula r incorpo ra tion re sulted in a s ho rter disintegra tion a nd d is so lution times .the sta bility of orphenadrine citra te p re pa red ta blets wa s also studied up on storage at 50c, 60c and 70c fo r fo ur mo nths .the drug was fa irly stab le a nd the e xpiratio n date for the p re pared ta blet wa s co nside re d to b e equal for 5 yea rs .on the othe r ha nd , the res ults of c linica l study on p atie nts suffering fro m p arkins on's dis eas e indica te d that pa tients w ith tre mor (re gula r rhythmic os cilla tion o f extremities es pe cially hand and finge r ) a nd mild symptoms o f pa rkinso n's dise as e showed a good re spo ns e to the p re pa re d ta blets , b ut it had no effe ct on p atie nts of dys tonia ( fixed upwa rd gaze , ne ck twisting ,clenc hing jaw s ) and akinesia ( slow down of move ment of volunta ry musc le and d iffic ulty o f initia tion o f mov eme nt ). the ov erall re sults of this s tudy indica te that the d rug can b e prepa re d as tablets , which fit the re quireme nts of britis h pharmaco poe ia since the prepa re d tablets gave s atisfac to ry res ults . :الخالصة orpheفينادرين روألا na drine) ( يوجد على شكل . هو عقار مضاد إلفراز الكولين ويعمل كمسترخي مركزي للعضالت الهيكلية ة األخـرى     أمالح السترات  والهايدروكلورايد حيث يستخدمان في عالج األعراض البسيطة لمرض باركنسون أو مساعد مـع األدوـي اجراء العديد من المحاوالت للحصول على صبغة تركيبية مقبولة لسترات االورفينادرين كأقراص سريعة  تم. المستخدمة في العالج  واد الرابطـة ٬ المـواد     التحرر باستعمال طريقة الحبيبيات الرطبة او الكبس المباشر من خالل دراسة تأثير العديد من العوامل مثل  الـم . المضافة و المواد المحطمة أو المفتتة  ة  ) pvp( أن أفضل النتائج قد تم الحصول عليها باستخدام مادة البولي فانييل بايروليدين  ى أن     . كمادة رابـط ائج إـل كمـا أشـارت النـت .النشا والمانيتول يعطيان خصائص فيزيائية مقبولة لألقراص عند استخدامها كمواد مخففة  المستخدم كمادة مفتتة أعطى زمن تفتت وتحرر مقبول مقارنة ) avicel( سيل في نفس الوقت أظهرت النتائج بان السليلوز ألمجهري افي واص    ) حبوب ديسبال ( بالمصدر  ى الـخ ة عـل اضافة الى هذا  التركيبية المختارة قد استخدمت لدراسة تأثير طريقة اضافة المادة المفتـت م دراسـة    . دى الى زيادة في سرعة التفتت والذوبان حيث وجد بان ادخال المادة المفتتة ضمن الحبيبات ا. الفيزياوية لألقراص  كـذلك ـت عقار مسـتقرًا  حيث كان ال. م لمدة اربعة اشهر  70م ٬  60م ٬  50استقرارية الدواء عند خزن الحبوب في درجات حرارة مختلفة  ارات نتائج الدراسة السريرية على من جهة اخرى ٬ اش. المحضرة هو خمس سنوات  صالنتهاء مفعول األقرا وان التاريخ  الذي اعتمد  المرضى الذين يعانون من مرض الباركسون بان المرضى الذين لديهم ارتعاش وبعض االعراض البسيطة لمرض باركنسون اظهروا  ر إلى أن نتائج هذه الدراسة تشي. المرضى الذين بعانون من ضعف المقوية  ىاستجابة جيدة للتركيبة المختارة ولكن ليس لها تأثير عل طالما أن الحبوب المحضرة أعطت  يإمكانية تحضير العقار على شكل حبوب كجرعة دوائية صلبة مطابقة لموصفات الدستور البريطان .نتائج مقبولة  depar tme nt of pharmaceutics,college of pharmac y , un iversity of baghd ad , bag hdad-ir aq ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 2 introduction the s ke le ta l mus cle re la xa nts are group of co mpounds us ed to re liev e sp as ticity & ab normally high mus cle tone (1,2 ) .the y produce their effe cts by action on central ne rv ous sys tem (cns) ,howe ver , the ir mec hanism o f action not yet unders to od . the re a re ma ny theo ries , which explain the mec hanism of ac tion o r the c linic al us es of mus cle re la xa nts .one of thes e theories is that they red uce s ke le ta l mus cle sp as m , p os sibly thro ugh a n a trop ine like central a ctio n o n ce re bra l moto r centers o r on the me dulla but they do not ha ve a na lges ic activity that co ntribute to the ir effec ts in pa tie nts with skeletal musc le s pa sm (3) . anticholinergic drugs (antimus ca rinic drugs ) were the most effe ctive drugs for tre atment of pa rkinson's dise ase for mo re tha n a c entury , this is b y bloc king ac etyl c ho line rec eptors o f the cns , there by partia lly re dress ing the imba la nc e crea te d b y dec re as ing d op aminergic ac tivity (4) , howe ver , the intro duc tion of the dop ame nic drugs ( le vo dop a & dec arboxylas e inhibitors ) ha s re le ga te d anticholinergics to a suppo rtive role in the trea tme nt of the diso rde r . nov erthle ss , the a nticho line rgric drugs are still use ful for p atie nts with minimal s ympto ms as patients unable to to le ra te le vop dop a be cause o f side effec ts o r contra indications , fo r thos e who are not benefite d by lev odo pa (5) & for patients w ho ha d parkins onia n symptoms ind uce d by antips yc hotic drugs (6 , 7) . orp henad rine citra te is white or a lmost white , o dorle ss or a lmo st odo rles s , c rystalline pow der with a bitte r ta ste & fo llowe d b y se ns atio n of numbness . it me lts in the ra nge of 1 34 c to 1 38 c (8) . soluble 1 in 7 0 of wa te r , s lightly s oluble in etha nol ; practically insoluble in chlo roform and e ther (9) .it s ho uld b e stored in tight & light re sista nt c onta iners (10) . ch2  cooh m e c nme ho c cooh ch2  cooh n,n d ime tly [ 2 – ( 2 – methylbe nzhydoxy ) ethyl ] amine dihyyd -roge n c itrate (11) .c18h23no.c 6h6o7 with m.wt of 46 1.5 gm . orp henadrine citrate is us ed for symptoma tic tre atment of p arkins on's dise as e (11) ,to relieve p ain due to sp as m o f vo luntary mus cle (12) and as an a lterna tiv e to quinon in trea tme nt of noc tura l leg cramps (13) .orp he ndrine ma y also be use d in vertigo in patient with s po ntane ous ve stib ular d is ea se (14) , and it may b e comb ined with halope rido l in trea tme nt o f chro nic schizop hrenic pa tient (15) or with p arace tamol in trea tment of myo lgia (14) . orphenadine is contraind ic ated in p atie nt with glauco ma (17),e lde rly pe op le (18) a nd with antac id (19) .its o verdo se is trea te d with physo stigmine (20) o r tetra hyd ro amino crine (21) . on the othe r ha nd , orphenadrine intoxica tion is po te ntiated by etha no l(22) and its use may cause de pe nde nce (23) . orp henad rine citra te pre se nts as a n oral tab le t extend ed releas e of 100 mg unde r the tra de name of norflex  and p arente al injec tion o f 30 mg/ml (a mpoule of 2 ml ) und er the trad e na me of banf lexand norflex . also orphenadrine c itrate is fo und in comb inatio n such a s ; norf lex  oral p la in ta blet whic h co ntains orphena drine citrate 25 mg , as pirin 38 5 mg and caffeine 30 mg ; and myogesic  whic h co ntains orphe nad rine citrate 35 mg and pa rac etamo l 4 50 mg(24) . this stud y wa s c arried out to fo rmula te orp hendrine citrate as a tab le t do sa ge form , thro ugh prep aring d iffe re nt formulas , and comp aring them with reference tablets . als o the effect of excipients type (bind ers , disintegrants and dilue nts ) o n phys ic al pro pe rtie s of the tablet was s tudied in ad dition to the e ffec t o f inc orporation method of disintegrants . furthe rmo re , the s electe d formula , which fitte d the s ta nd ard req uire ments ,was thoroughly inves tigated for its exp iration da te and c linica l e ffec ts . exp erimental part : materials orphe nad rine citrate p owde r.(dar al dawa , amman , j orda n ), s ta rc h , mannito l (me rk , darmstad t ,germa ny ) , microc rystalline cellulose (av ic el ph101 , f mc c orporation , pennys ylva nia , usa ), polyv inyl pyro lidine (pvp ,k30),etha nol ,hydroc hloric ac id (hcl) , carboxy methylc ellulose s od ium s alt (cmc) , is oprop yl alchoho l ,(bdh chemica ls ltd, ,poo l, england ),magnes ium s trea ra te , (ba rloc hes ,gmbh, germany), acac ia arabique ,dextros e , talc.(rie del – de – haen ag see lze – hanno ver , germany ),dib asic ca lc ium phos pha te (emc omp re ss ,ed ward mend ell co., usa ), explota b (aveba, veendo m ,ne therla nds ),disipal ta blets as a re fe re nce (ya mano uc hi p harma ltd. , uk). c c ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 3 formulation of or phenadrine citrate tablets diffe re nt formulas (ta ble i ) were prep ared to find the mos t sa tis fa ctory fo rmula using wet granulatio m te ghniq ue e xce pt formula 7 whic h was prep ared by direct comp re ss io n te chnique , in whic h the d rug and e xc ipie nts ( excep t lubricant )were d ry b le nde d for at le as t 5 minutes a nd then mixture wa s co mpress ed into tab le ts us ing f 3 ma nes ty ta blet ma chine with a s ingle 7 mm normal co nca ve p unc he s . in c as e of us ing wet granula tio n method , the fo llowing p ro ced ure was fo llowe d : afte r 5 minutes dry blend ing of drug & excipients , the bind er so lution was ad ded to the formula grad ually in the mixing morta r until a sa tisfac tory wetting was achie ve d (ba ll te st ).the wet mas s wa s then gra nula te d thro ugh a siev e no. 10 a nd dried in a tra y ove n at 4 5c for 30 minute s. the granule s were then reduced in s ize and ho moge nize d b y pa ssing them through a siev e no .1 6. a kno wn weight of the granules was the n mixed with s pec ified amo unt of disinte grant extra – granularly for 10 minutes in we ll clos ed c ontaine r a nd the n mixed with magnes ium stea ra te (200 me sh in size )fo r 2 minutes . the fina l mixture was c omp re ss ed. physical parame ter measureme nt of or phenadrine tablets hardne s s :the ha rdnes s of orphe nad rine citrate tablets we re me as ured us ing monsanto and erweka hardne ss tes te rs no rma l ra nge betwee n & 8 kg(24) . we ig ht va riatio n :it was d etermined for a ll pre pa re d formulas by ta king 20 tab le ts , weighe d individually and the ave ra ge we ight is calcula ted .for the ta blets to be ac ce ptable by not more tha n 2 of the 20 tab le ts ma y differ from the a verage weight b y not mo re than 7.5% a nd no tab le t may diffe r by more than double the pe rc ent (25). friability te st : the friab ility o f ta blets was perfo rmed us ing ro che fria bila to r and erweka frib ilator fo r 4 minute s at 25 r.p .m by weighing 10 tablets the n place them inside the tes ter for 4 minutes and weigh them again .the difference in weight s hould not exc ee d 1% . dis inte gratio n time : the d is inte gration time was meas ured using u.s.p disintegra tion app aratus . it co nsis ts of a b as ket rack ass emb ly c ontaining s ix o pen – e nde d glass tubes with a 10 –mes h s creen on the bottom .the ba sket was immersed in an app ro priate fluid (0.1n hcl ) a t 37 c .the bas ke t rack wa s raised a nd lowe rd a t a rate of 30 stoc ke s pe r minute(25) . ta ble (1) sche dule of diffe re nt formulations o f orphe nadrine citrate as a pla in table t q.s = s ufficie nt quantity , x=amount of av ice l ,y= a mount of sta rc h ,d=amo unt o f de x tros e , m =amo unt of ma nnitol , e= amount o f explo ta b ,s = amount o f starch (disinte grant )… ma te rials for mula no. 1 2 3 4 5 6 7 8 9 orphenadrin e .citrate (mg) 75 75 75 75 7 5 75 75 7 5 75 p vp10%w/v in e thanol q.s q.s q.s q.s q.s avicel ph101 x x x x x x starch y y y y 0.5y y y mg.ste arate 1.5 1.5 1.5 1.5 1 .5 1.5 1.7 1 .5 1.5 de xtro se d ac acia 2 0% q.s ma nnitol m explotab e starch (disinte grant) s emcomp re ss 68.5 starch pas te q.s cmc q.s to ta l we ight 15 0 150 15 0 150 1 50 15 0 150 1 50 15 0 ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 4 diss olutio n te s t : the usp ba sket me thod (26) was use d to s tudy the relea se o f the drug from the prepa re d ta ble ts a nd from the re fe re nc e ta blet dis ip al  (yamano uc hi) . the studie s were c arried out in shie ld ed app aratus to protec t the solution of d rug from light us ing 900 ml of 0.1n hcl s olution as the diss olutio n med ium a t 37c with a c ons tant stiring s pee d of 5 0 r.p.m (26,27) .samp le s were withdrawn at five minutes interva l for o ne hour . the sa mple volume was replac ed immed ia te ly b y a fres h 0.1n hcl . the sa mples were filte re d b y microfilte r a nd ana lyze d sp ectrophotome trically at its max 264 nm (28) . assay for total orphe nadr ine c itrate pre sent in the table ts pre paration of standard :15 0 mg of p ure orp he nad rine c itrate was diss olve d in 75 ml 0.1n hcl , shaken for 1 5 minutes and filtered , then the volume was co mpleted to 10 0 ml with 0.1n hcl . a s amp le o f 20 ml was taken and diluted to 100 ml with 0 .1 n hcl . the a bso rb anc e of the diluted so lution wa s de te rmine d spe ctrophotome tric ally a t its max which is 2 64 nm(28) . ass ay o f the pre pare d ta ble ts : 20 ta blets of orp he nad rine c itra te we re triturated and a n ac curate weight e quiv alent to 0.15 gm of orp he nad rine c itrate was ad ded to 75 ml of 0.1n hcl , shaken for 15 minutes and filte re d .the fina l vo lume was co mpleted to 100 ml with 0 .1 n hcl the ab sorba nce of diluted s olutio n wa s de te rmine d sp ectrophotome trically at 2 64 nm , and the qua ntity of drug p er tablet wa s ca lc ulated ac co rding to the follo wing eq uation : tes t /s td .  10 0 =% orphe na drine c itrate pres ent in ta blet (29) . kinetic study effec t o f te mpe ra ture o n orp henadrine citra te ta blets was s tudied b y storing so me ta blets of the se le cted formula (1) a t diffe re nt te mpe ra ture s ( 50 c 60 c 7 0 c ) fo r four months .sa mples of tablets were taken at des ired time intervals a nd as sa ye d fo r co ntents of orp henad rine citrate a cc ording to the metho d mentio ned be fo re .the fria bility , disintegra tion a nd diss olutio n time we re als o che cked at the end of four mo nths , and organoleptic prope rties we re a lso examine d . statistical analysis stude nts t – tes t was us ed to e xa mine the difference in the me an of the res ults of parame ters te sted . a p – v alue of le ss than 0.05 was conside re d signific ant . preliminary clinical study the s elec te d pre pa re d fo rmula (1) in ad dition to dispal we re give n to sev en pa tients suffering fro m tre mor (legs a nd a rm ) , stiffnes s and auto nomic d ysfunction which a re clinic al symp toms o f pa rkinson's dise ase .they were also given to thre e pa tie nts with dys to nia , a t the s ame time the stud y was d one using p la ceb o tab le ts . the treatme nt was fo llowe d up to three months with a dos e of one ta blet 3 times da ily . the stud y wa s do ne in nahrain co llege of medicine tea ching hosp ita l to de termine the c linica l symptoms and the c linica l pa ra meters us ed to ass es the therapy . results and discussion : effect of binder type different formulas (1,3 ,8 and 9 ) we re prepa re d to s tudy the e ffec t of b inde r type on the hardnes s , fria bility , d is integration and disso lutio n times as s hown in tab le (2) a nd fig ta ble (2) effec t of binde r type on the hardne ss , ,friability , d is inte gra tion time a nd diss olutio n time of the s uggeste d orphe na drine citra te formu la in co mparis on with t he refe re nce table t s uggested fo rmula t yp e of bin de r hardness (k g) f riab ility (%) disintegratio n t ime(min) disso lut ion t ime(min)to 1 00 % 1 1 0% p vp 8.25 0 .5 39 5 20 3 2 0%ac acia mu cilage 7 0 .7 6 20 8 1 0% s ta rc h p aste 5 1 .1 5 15 9 5 % cmc 3 2 .8 4 30 d is ip al r eferen ce 10 25 ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 5 fig .1 :effec t o f binde r on the re lease o f orphe nadrine fro m p re pare d fo rmulas in comparis on with the refe re nce table t dis ipal fig. 2 : effec t o f dilue nt on the re lease of orphe nadrine from pre pare d fo rmu la s in co mparison wit h the re fe re nce table ts dis ipal in 0 .1 n hcl and 37 c the re sults o btaine d indica te that us ing different type s of b inders affe ct the p hys ical prope rties and the re le as e of drug . for example , fo rmula 1 in whic h pvp wa s us ed a s a bind er , s ho wed a go od ha rd ne ss (8.25kg) , friability (0 .5 %),disinte gratio n time (5 mins .) and diss olutio n time up to 1 00% (2 0 mins. ) in co mparison with the reference tablet dispal .this may be d ue to the us e of p vp in e thanol which has the sa me v is co sity a s p.v.p in water(30 ) .als o hydgros cop ic ity of pvp p re vents the ta blet from be ing ha rd er with age (31) . formula 3 , whic h utilize s ac acia mucila ge in water a s a bind er ,showed go od hardnes s (7 kg), ac ce ptable friability (0 .7 %) , disintegra tion time (6mins .) a nd diss olutio n time up to 1 00% (20mins .) in comp aris on with the re fe re nc e tab le t dispal.the se may b e due to the fac t that gum ac ac ia ge nerally produce hard gra nule s but witho ut p rod uc ing ta blets of inc re asing hardne ss (31) . f ormula 8 which c ontains starch p aste a s a bind er , sho wed rea so nab le hardness (5 kg) and disintegra tion time of (5mins .)with una cc eptab le fria bility (1 .1 %) and fa st disso lutio n time up to 1 00% (1 5mins .) .this may be d ue to the p ro perty o f sta rc h p as te , which forms ge ne ra lly s oft and brittle ta blets(31) . finally fo rmula 9 in which cmc. was used as a b inde r , s howe d lo w hardness (3 kg)and unacc epta ble friability (2.8%)w ith an acc ep ta ble disinte gratio n time (4 mins.)and lo ng disso lutio n time (30mins.)s ince cmc. sod ium is visco sity controller ,it is c onc eivab le that it forms a highly visc id s ys te ms that re sist dilution by disso lutio n fluid which might imp ede drug releas e(32). effect of diluent type formulas 1 ,2 ,4 a nd 7 were us ed to study the effe ct of diluent type o n the hardnes s , fria bility ,d is integration and diss olutio n times of the p re pa re d ta blets . tab le (3) s hows the e ffe ct o f diluent type on physica l prope rties of the p re pared tab le ts , while fig (2) sho ws the releas e of orp henad rine c itrate from the p rep ared tab le ts (formula 1,2,4 and 7 ) in comp aris on with the re fe re nce ta blets disip al . formula 1 in which s ta rc h was use d as d ilue nt gav e a good hardnes s(8.25kg), friab ility (0 .5 39%) , disintegra tio n (5min) and disso lutio n time up to 1 00% (20min) . formula 2 which co ntains dextro se a s a diluent ,showe d a goo d friab ility (0 .1 %), hardnes s (7kg) and d is inte gration time ( 4 mins.) but long d is so lution time (40mins .), although brown sp ots was s ee n o n the ta blet afte r a while whic h ma y be due to the inte ra ctio n o f dextros e w ith a mines ( orphenadrine )(30) . fo rmula 4 in which mannito l was use d as a diluent , gave a good fria bility(0.5%), hardne ss (7.25 kg), disintegra tion time (6 mins.),with a rea so nab le disso lutio n time (35mins.). this ma y be due to non – hygro sco picity of ma nnitol (30). formula 7 ,which was prep ared by dire ct comp re ss io n tec hniq ue , s ho wed a we ight variatio n .this is bec ause of po or flo w of powd er in sp ite o f p re se nce of emco pmres s .also it s ho wed una cc eptab le fria bility (1 .1%) and hardne ss (3kg)with a rapid disintegra tion time (50-se c) and d is so lution time of 20 min . effect of disintegr ant effe c t of dis inte grant type formulas 1,5 and 6 were utilize d to s tudy the effe ct of d is inte grant typ e on phys ic al prope rties of the prepa re d ta blets as sho wn in ta ble (4 ) and fig (3) . 0 50 100 0 10 20 30 40 time (m in ) p e rc e n t o f d ru g r e le a s e d formula 9 disipal formula 1 formula 3 formula 8 0 50 100 150 0 20 40 60 tim e (min ) p e rc e n t o f d ru g r e le a s e d formula2 formula4 disipal formula 1 formula 7 ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 6 ta ble (3) effe ct of dilue nt o n the physica l prope rtie s o f orphe nadrine citrate plain tab le ts ta ble (4) effec t of dis inte grant type on the physical prope rties o f pre pa re d orphe nadrine ta ble ts fig. 3 : the effe ct o f disinte g ra nts ty pe on the re lease o f orphe nadrine in co mparis on with the re fe rence table ts disipal  formula 5 , in whic h explotab (lo w s ub stitute d ca rb oxymethyl starch (32) ) was us ed as a disintegra nt s ho wed a relatively low hardnes s (4 kg) with a n a cc eptab le friab ility (0 .1 %), disintegra tion time (5mins .) a nd diss olutio n time (25 mins.) , ho weve r , its diss olutio n profile showe d no signific ant diffe re nce with fo rmula 6 fig(3) (p – va lue > 0.05 ). this is ma y be d ue to explotab prop erties sinc e its gra nule s abs orb water ra pidly and swe ll but do not b re ak . in general , the s welling granule s re main intac t , c aus ing disintegra tion witho ut b ursting (unlike sta rc h ) and conse que ntly relea se of the solub le starch frac tion .this might le ad to increas e the visco sity a nd de lay moisture pe ne tration into the ta blet (29) . formula 6 showe d a goo d fria bility (0 .0 1%) , disintegra tion time (4mins .) a nd d is so lution time (3 0mins.), with re la tive ly lo w hardnes s(3.5kg) .this may b e due to the fa ct that ta blets co ntaining high amo unt or conce ntra tion o f starch a re o fte n s oft a nd may be difficult to dry (33) . formula 1 in which avicel  was use d as disinte grant , showe d a goo d hardness (8.25kg)and fria bility (0.5%) with an a cc eptab le d is integration time (5 mins.),and a fas t diss olution time (2 0mins .). this is be cause avicel is a super disintegra nt which is highly p orous ,with strong " wic king" te nde nc ie s (23) ,this will allo w wa ter to e nter the ta blets matrix by mea ns of c apilla ry forces which brea ks the hydrogen bo nding be tween adjac ent bundles of c ellulo se microc rystals(33) effe c t of mo de of incorporation of dis inte grant formula 1 , was us ed to study the e ffec t of metho d of inc orporation of d is integrant .it was prepa re d b y thre e metho ds of incorpo ra tion , they we re : e xtra graunlar 1, intragra nula r 1b , and c omb inatio n of b oth type s 1c .the da ta a re displayed in fig (4) and ta ble (5 ). formula no . diluent type hardness(kg) friability (%) disintegra tion time (mi n) dissolutio n time (min) to 100% 1 starch 8 .25 0 .539 5 20 2 dextrose 7 0.1 4 40 4 ma nnitol 7.5 .5 6 35 7 emcompre ss 3 1.1 0.5 20 fo rmula no. diluent typ e hardnes s(kg) friability (%) dis integration ti me (mi n) dissolutio n time (min)to1 00% 1 av icel 8.25 0.53 9 5 20 5 exp lo tab 4 0 .1 5 25 6 starch 3 .5 0 .01 4 30 0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 time (min.) p e rc e n t o f d ru g r e le a s e d formula 1 formula 5 formula 6 disipal ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 7 1.984 1.986 1.988 1.99 1.992 1.994 1.996 1.998 2 2.002 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 tim(month) lo g p e rc e n t re m a in in g 50 60 70cc c fig. 4 : effe ct o f mode of inc orporatio n of dis inte grant on the re lease of orphe ndrine fro m pre pa re d formulas table ( 5 ) a comparis on be twee n the effect of me thod of inc orporatio n of dis inte gra nt extra or intra or combina tion o f both fo rmula no. dis integrant loc ation ha rd ness (kg) f riab ility(%) dis inte gratio n time (mi n) disslutio n time (mi n) to 100% i avice l extra 8.25 0 .539 5 2 0 ib avice l intra 3 0.1 3 1 9 ic avicel extra & intra 3.5 0.15 4 1 5 the res ults s ho wed that fas ter disinte gratio n (3 mins.)and sho rte r diss olution time (1 0mins .)for formula 1b c omp ared with tha t of fo rmula 1c a nd formula 1. this is in a greeme nt with gordon et al(34). who state d that inco rpo ra tion of s upe r d is inte grant in the intra gra nula r pha se re sulted in fas ter tablet diss olutio n than did inc orporating it in the extragra nular phase o r bo th p ha ses . bas ed o n the ov erall results ; it se ems tha t formula 1 and 5 are the p ro mis ing formulas co mpared with the re fe re nc e ta ble t dispal .sinc e both of them showe d goo d d is integration and diss olutio n times . ho wev er , rega rd ing the ec ono mic p art of p rod uc tion a nd the cos t of mannito l (33) , fo rmula 5 was e xclud ed . kine tic study the stability of fo rmula 1 which wa s chos en a s the promising formula wa s stud ie d at d iffe re nt te mpe ra ture s ( 50 , 60 a nd 70 c ) for four months . the degra da tion of orphe na drine citra te follows first order kinetic sinc e stra ight line s were o btaine d when the lo garithm of pe rc ent re maining o f orphe na drine citrate wa s plotte d ve rs us time ( fig.5) . the de grad atio n ra te c ons tants (k) fo r d ifferent te mpe rature s were ca lc ulated from the slop es o f the lines a s shown in table (6) . ta ble (6 ) ra te co ns tants of de grada tion (k) of orphe nadrine citrate (formula 1) a t diffe re nt te mpe rature s te mpera ture c k(mon th -1) 50 1.010-3 60 1 .41310-3 70 1 .99510-3 fig .5 : acce le rate d bre akdown of orphe nadrine citra te a t diffe re nt e levate d te mpe rature s (fo rmula 1) 0 50 1 00 0 10 20 3 0 tim e ( m in ) p e rc e n t o f d ru g r e le a s e d fo rm ula i fo rm ulai fo rm ula 1 b c ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 8 to c omp ute the expec te d e xpiratio n d ate (t10%), arrhenius plot was ma de to p re dict the k25c as shown in fig (6). utilizing the fo llowing equatio n : 0 .1 04 t10 =  k25c howe ver , the c alculate d t10% wa s lo ng ,so its co nside rd to be equal for 5 ye ars .in ad dition , at the end o f fo ur months , no c ha nge in physica l p rop erties of the prepa re d ta blets wa s se en . fig .6 arrhe nius plot for e xpiration date e stima tion o f orphe nadrine citra te (formula 1) reliminary clinical study the res ults o f this stud y as indica te d in tab le (7 ) sho wed fiv e out o f se ven p atie nts ha d a goo d re spo ns e to the drug a fter o ne we ek of trea tme nt fo r the se lected formula 1 a s well as the re fe re nc e ta blets dispal , while the re st two p atie nts showed d ifferent behav ior . i.e , one had no res po nse to the d rug and the o ther sho wed s id e effe cts such a s ha lluc inatio n and blurre d v is io n a fter o ne da y o f trea tme nt , therefore the the ra py was sto ppe d , o n the other hand , the drug s howe d no effe ct on patients with dystonia as with the plac ebo ta blets . conclusion fro m all previous experimenta l work one can conclud e tha t be st b inder is pvp in ethanol since it is c he ap , a va ilab le , co mpress ible and comp atible with drug . starch a nd ma nnitol a re the b es t diluents b ut we prefer using starch bec ause of its low co st . av ic el is a goo d disintegra nt s ince it is c ompres sible , highly porous giving a good disintegra ting time although it is relative ly e xpe ns iv e .f ormula 1 is the most sa tisfac to ry formula in co mparison with the reference tab le ts disipal.and the t10% was c onsidered to b e equal for 5 yea rs . ta ble (7) clin ic al responses to pre pare d orphe nadrine ta ble ts and the re fe re nce p atie nt age (yea r) se x chief co mplaint past tre atmen t duration ne w tre atmen t dose no te s 1 6 0 ma le tre mor pa rkizol sina met one d ay 13 pa tie nt de velop ha llucination and b lurred vis io n stop tre atment after one do se o nly 2 4 0 ma le tre mor no 2 wee ks 13 mod erate res ponse 3 5 0 fe male tre mor sina met 2 mo nths 13 go od re sp ons e 4 5 9 ma le tre mor no 3 mo nths 13 go od re sp ons e 5 3 0 female tre mor no 1wee k 13 no res ponse 6 4 0 fe male tre mor a nd rigidity pa rkizol sina met a rtane 1 month 13 go od re sp ons e 7 4 5 ma le tre mor no 2 wee ks 13 mod erate res ponse 8 1 1 fe male dys to nia no 3 wee ks 13 no res ponse 9 1 5 ma le dys to nia no 1 month 13 no res ponse 10 3 0 ma le dys to nia no 2 wee ks 13 no res ponse % respo nse to tre mor 71 .4 % side effect 14 .2 8 % resp onse to dystonia 0.0 no te : the diffe re nce be twe e n g ood & mode ra te re s ponse s is re late d to the e x amine r 4 3 . 8 3 . 6 3 . 4 3 . 2 3 2 . 8 2 . 6 2 . 4 2 . 2 2 0 1 2 3 4 1 / t * 1 0 0 0 lo g k ( m o n th -1 ) ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 9 references 1. c.w.t pilche r ; p arkins onism ; kuwa it drug ind ex ;kuwa it drug inde x co mmittee ;19 88,1st e dition ; 44 1 – 449 . 2. ala , ad in a.s.alwan a nd yo usif 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excipients , 2nd e dition ; the p ha rma ceutic al p re ss , london , 199 4. 5 3 – 5 5 ,95 -98 , 1 73 – 18 0 , 23 4 – 23 9 and 328 – 2 54 . 31. lib erman h.a., la chman l.. co mpres se d tablet in pharmace utical do sa ge fo rms ; ma rc el de kke r ; new york a nd bas el ; 1 982 , vol . 1 ; p85 , 109 – 1 40 ; . 32. s winya rd e.a., lo wenthal w.ca rb oxy methyl cellulose s odium ; e mulsifying and s us pe nding agents ; pha rma ce utic al nece ss ities in remingto n's pha rma ce utic al s ciences ; 1 7th ed ition , mack p ublishing compa ny ; 198 5 , p 12 97 . 33. banker g.s. ;rhod es c.t bio aviala bility in mo dern pharmac eutics , ma rc el de kker , new york a nd ba sel ; 19 79 , vol .7;p1 43 ; . 34. go rdan m.s .; chatte rjee b.; chowhan z.t.; effec t of the mo de of c rasc armellose s od ium inc orporation on tablet d issolution a nd fria bility , j .p harm. s ci., 1990 ,79 ; p 4 3 -47 . summary iraqi j pharm sci , vol.18 (suppl.) , 2009 cytotoxic study of resveratrol 19 in vitro cytotoxic study for purified resveratrol extracted from grape skin fruit vitis vinifera zainab y. mohammed * , essam f. al-jumaily **,1 and nahi y. yaseen *** * biotechnology research center , al – nahrain university ** genetic engineering and biotechnology institute for postgraduate studies,university of baghdad , baghdad,iraq . *** iraqi center for caner and medical genetic research ,baghdad,iraq . abstract this study was conducted with the aim to extract and purify a polyphenolic compound “ resveratrol” from the skin of black grapes vitis vinifera cultivated in iraq. the purified resveratrol is obtained after ethanolic extraction with 80% v/v solution for fresh grape skin, followed by acid hydrolysis with 10% hcl solution then the aglycon moiety was taken with organic solvent ( chloroform). using silica gel g60 packed glass column chromatography with mobile phase benzene: methanol: acetic acid 20:4:1 a partial purified resveratrol was obtained then preperative thin layer chromatography technique yielded pure crystals identified as resveratrol (mixture of two isomers cis and trans) in relation to resveratrol standard (35 mg resveratrol crystals / 0.5 kg fresh grape skin was obtained as a result of these processes ). the study was also employed an in vitro evaluation the cytotoxic effect of pure resveratrol on some cell line including : the murine mammary adenocarcinoma amn-3 cell line, the human laryngeal carcinoma (hep-2) cell line and the rat embryo fibroblast (ref) cell line, at different concentrations and different expousure time. the cytotoxic effect of the pure resveratrol was studied in comparison with transresveratrol standard in concentrations of (12.5, 25, 50 and 100) µg/ml for both purified resveratrol and the standard, also the comparism included methotrexate drug in concentrations (0.05 , 0.1 , 0.2 and 0.4) µg/ml toward the growth effects of the three types of cell lines and at three exposure times (24, 48 and 72) hours. the cytotoxic inhibition effect for the purified extracted resveratrol revealed that the highest significant effect (p<0.01) was achieved after 24 hr of exposure on both amn-3 and ref cell lines. hep-2 cell line responded to extracted resveratrol in different manners. keywords: resveratrol , grape skin , polyphenols, cytotoxic study الخالصة اىَزسٗع vitis viniferaٍِ قش٘س ّباث اىؼْب االس٘د resveratrolٕذفج ٕزٓ اىذساست اسخخالص ٗحْقٞت اىَشمب اىفْٞ٘ىٜ % ذ/ذ ىقش٘س ثَشة اىؼْب ٠٨اىْقٞت بؼذ اخشاء ػَيٞت اسخخالص مح٘ىٜ بَحي٘ه resveratrolحٌ اىحص٘ه ػيٚ ٍادة فٜ اىؼشاق . % ، ثٌ اخز اىدزء غٞش اىسنشٛ بَزٝب ػض٘ٛ ) مي٘سٗف٘سً( . ٗباسخخذاً ٠٨ٗبخشمٞز hclحيو بحاٍض اىطشٝت ، حبؼخٖا خط٘ة ح ٗاسخخذاً اىط٘س اىَخحشك بْزِٝ : ٍٞثاّ٘ه : حاٍض اىخيٞل ٦٨ػَــــ٘د اىفصـــو اىنشٍٗاح٘غشافٜ اىزخاخٜ اىَؼبأ بَادة سٞيٞنا خٞو ٞشٝت ضْقاة خزئٞا ، ٍِ ثٌ ٗباسخخذاً حقْٞت مشٍٗ٘ح٘غشافٜ اىطبقت اىشقٞقت اىخحٍ resveratrolحٌ اىحص٘ه ػيٚ ٍادة ٠٨:٤:٠بْسبت ( trans resveratrol ٗ cis resveratrol) مَزٝح ىيْظٞشِٝ resveratrolّخدج ػِ بي٘ساث ّقٞت حٌ اىخؼشف ػيٚ مّٖ٘ا ٍادة مغٌ قش٘س ػْب اىطشٝت مْخٞدت ٠/٠ٍيغٌ ىنو ۳٥ص٘ه ػيٚ ) حٌ اىح trans-resveratrol standardباىَقاسّت ٍغ اىَادة اىقٞاسٞت اىْقٞت ٍٗذٙ resveratrolىٖزٓ اىخط٘اث ( . حضَج اىذساست فضال ػِ رىل حقٌٞٞ خاسج اىدسٌ اىحٜ ىيفؼاىٞت اىسَٞت اىخي٘ٝت ىيَادة ٗخظ سشطاُ amn-3 cell line حأثٞشَٕا فٜ بؼض اىخط٘ط اىخي٘ٝت ٗاىخٜ شــَيج : خظ سشطاُ اىظٖاسة ىيغذة اىيبْٞت اىفأسٝت ػْذ حؼشٝضٖا ىَخخيف اىخشامٞز ref cell line ٗاىخظ اىخي٘ٛ اىطبٞؼٜ ىدِْٞ اىفأس hep-2 cell lineظٖاسة اىحْدشة اىبششٛ اىخشامٞز ب transresveratrolٗفخشاث اىؼالج. ٗقذ اخشٝج دساست اىخاثٞش اىسَٜ اىخي٘ٛ ىيَادة اىْقٞت باىَقاسّت ٍغ اىَادة اىقٞاسٞت methotrexate( ٍاٝنشغشاً/ٍييخش ىنو ٍِ اىَادة اىَسخخيصت ٗاىَادة اىقٞاسٞت ٗاٝضا شَيج اىَقاسّت ػالج ٠٨٨، ٥٨، ٠٥، ٥,٠٠) ( ساػت . اُ اىفؼاىٞت ٧٠، ٤٠، ٠٤( ٍاٝنشٗغشاً/ٍييخش ػيٚ اىخط٘ط اىثالد ٗىفخشاث حؼشٝض ) ٨٫٤ ، ٠,٨ ، ٨٫٠ ،٨,٨ ٥(باىخشامٞز ساػت حؼشٝض باالخض ٠٤( بيغ اقصآ بؼذ ٍشٗس p >٨٠,٨َٞت ىيَادة اىْقٞت أظٖشث فشٗق ٍؼْ٘ٝت ٗضَِ اىَسخ٘ٙ )اىخثبٞطٞت اىس . اٍا ref cell lineٗاىخظ اىخي٘ٛ اىطبٞؼٜ ىدِْٞ اىفأس amn-3 cell lineت اىفأسٝت ـــــفٜ خظ خالٝا سشطاُ اىظٖاسة ىيغذة اىيبْٞ فناّج اسخدابخٔ ىخأثٞش اىَادة اىْقٞت اىَسخخيصت بشنو ٝخخيف ػِ بقٞت hep-2 cell lineخظ سشطاُ ظٖاسة اىحْدشة اىبششٛ اىخط٘ط اىخي٘ٝت. introduction cancer is a complex set of more than 200 diseases with many causes and multiple stages and histological grades of malignancy [1] . result of exogenous environmental, lifestyle and host genetic factor which attributed about 2% of cancer factors [2] . 1 corresponding author e-mail : samgen992003@yahoo.com received : 10/12/2008 accepted : 6/5/2009 iraqi j pharm sci , vol.18 (suppl.) , 2009 cytotoxic study of resveratrol 20 cancer treatments including surgical, radiotherapy , chemotherapy, and biotherapy by immunization and gene therapy, are employed as conventional treatments which may not always be satisfactory to relieve sever symptoms or even cure cancer with untolerated side effects and many get resistance [3] . chemotherapeutic drugs are typically toxic agents to prolong survival without cure in about 50% of cancer patients, but still an important component of multi modal cancer therapy in (2530)% of cancer patients, due to the early resistance development to chemotherapy in the life cycle of tumour [4] .complementary and alternative therapy can help relieve symptoms and improve physical and mental wellbeing [5] . the search for novel and effective cancer chemopreventive agent has led to the identification of various naturally occurring phytocompounds, one of which is resveratrol (trans3,4′ ,5 – trihydroxy stilbene) a phytoalexin drived from grape skin and other fruits [6] . black grape cultivated in iraq is rich with resveratrol [7] . resveratrol is shown to have a potent antiinflammatory effect [8] , antioxidant effect [9] , anti platelet aggregation and cardiovascular protection [10] ; its potential chemopreventive and chemotheraputic activities have been demonstrated in all three stages of carcinogesis in numerous in vitro and in vivo studies [6,11] . it has the ability to modulate various targets and signaling pathways [12] . as there is drug development; programmes for preclinical screening of the vast numbers of chemicals for specific and nonspecific cytotoxicity against many types of cells are involved. both are important for indicating potential therapeutic target and safety evaluation [13] . the use of in vitro assay system for screening has been a common practice since the beginning of cancer chemotherapy in 1946, following the discovery of antineoplastic activity of nitrogen mustard. some phytochemicals have been shown to exhibit cytotoxic effects against cancer cell through cell cycle modulation [14] . the secondary metabolite “ resveratrol” was synthesized by the plant in response to stress , including disease and ultraviolet light , as phytoalexin was first reported in skins of grapes by creasy and coffee [15] ,in 1988 later in 1991 was reported by siemann and creasy [16] , from wines. fig (1) transresveratrol structure [17] resveratrol [3,5,4′ – trihydroxy stilbene ] fig.(1), is a non flavonoid polyphenol and it has three phenolic hydroxyl groups and shown to have its biological effects such as; inhibition ldl oxidation [18] . fresh grape skin contains (50-100) µg/g of trans – resveratrol [19] . the potential for resveratrol to inhibit the development of cancer and extend lifespan in cell culture and animal models have continued to generate scientific interest [20]. material and methods collection of samples local black grapes cultivated in iraq were collected from the local market and classified as vitis vinifera by the herbarium of the biology department, college of science, baghdad university . the skin was separated from the fruit to be then kept in a dark cool place , till the following steps. preparation of grape skin extract preparation of grape skin extract was according to harborne [21] . all steps were done away from direct light and extensive stress that led to oxidation of the plant extract. about 500 grams of fresh skin grapes was shaken with 2.5 litters 80% ethanol in cool dark place for 72 hours. the extract was filtered and the filtrate was dried at 30-40 °c by a rotary evaporator to get 1/10 (one tenth) its original volume to be stored at –20°c till the following steps. acid hydrolysis acid hydrolysis was done using 10% v/v conc. hcl for (10-30) min at 60°c . this step led to the hydrolysis of the glycosidic linkage and got the aglycone moiety, cool and filter [21] . the filtrate was transferred to seperatory funnel. an organic solvent like chloroform was added in a quantity equal to the aqueous phase , with gently shaking and repeating the process three times . the chloroform layers were collected together and washed from the access acid with distilled water . the collected chloroform layers were evaporated to dryness under vacuum with a rotary evaporator at 30°c . the residue was green viscous aliquots stored in dark umber vessels at–20°c untill use [21] . column chromatography (partial purification ) by solid – liquid adsorption chromatography a partial purification of the residue was proceeding using open glass column (2.5 x 21) cm filled with silca gel g60 special for column chromatography . the residue was dissolved in 1-2 ml methanol and the mobile phase is benzene: methanol : acetic acid , 20:4:1 [21] . the elutions were collected in 100 separated tube each filled with 3ml eluent. all fractions were tested for fecl3 1% solution as a colourimeteric method for polyphenols identification [21,22] . only the positive results elutions were collected and dried under iraqi j pharm sci , vol.18 (suppl.) , 2009 cytotoxic study of resveratrol 21 vacuum by a rotary evaporator. the resveratrol spots were detected on a tlc aluminum sheet silica gel 60f254 in comparison with the standard spot using the same mobile phase in the column chromatography [21] . preparative thin layer chromatography (p.t.l.c.) the procedure was running on in a dark cool place away from light and heat . when the solvent system reached to solvent front, removing the plate from the chamber to dry for few minutes, the pure resveratrol line appeared as dark straight line visually (in day light). this line was scratched and eluted with methanol 10ml three times and stored at –20 for 2 days . an amorphous off white crystals formed , to be collected rapidly in cool, dark place and kept in umber reservoir at –20°c. these crystalles were referred as " pure resveratrol ". the crystals were examined by the following tests. a) u.v. absorption. b) thin layer chromatography: using tlc plate of silic gel 60 with flourecence .the mobile phase is benzen : methanol : acetic acid , 20:4:1 c) hplc method using the following system [23] column : c18 – reverse phase. mobile phase : acetonitrile : water , 60:40 .flow rate : 0.6 ml/min standard concentration : 0.6 mg / ml (exposed to sun light )sample concentration : 0.6 mg / ml . wave length : 307 nm for trans and 280 mm for cis isomer d) fourier transform infra red (ftir) assay: to detect the functional groups in resveratrol structure. e) specific reaction for (aromatic ring) aluminium chloride (alcl3)test(friedle graft) : f) specific test for double bond include [22] . ( i ) bromine decolourisation test : ( ii ) the baeyer test : g) melting point: using (glascoo,u.k.) apparatus. cytotoxic effect of resveratrol on cell line: in vitro study the in vitro method was used to investigate the effect of pure resveratrol on two types of tumor cell lines (human laryngeal carcinoma hep-2 and murine mammary adenocarcinoma amn-3 tumor cell lines) and to compare the results with resveratrol effect on normal cell line ref-2 cell line at different concentrations and different exposure times. the study involved a comparison between the cytotoxic effects of the extracted resveratrol and a standard (resveratrol – sigma) and a traditional cytotoxic drug methotrexat at different concentrations and different times of exposure. purified resveratrol dilutions pure resveratrol 4 mg was dissolved in 20ml phosphate buffer saline (pbs) and 0.02 ml dimethylsulfoxide (dmso) as organic solvent for dissolving the substance. the stock was kept in a dark container at –20°c after sterilization with 0.22μm millipore filter. immediate serial dilutions were prepared starting with 100μg/ml and to end with 12.5 μg/ml ).the dilution was done with a serum free medium (medium without serum). standard resveratrol solution : transresveratrol standard 2mg was dissolved in 0.01ml dmso and complete the volume to 10 ml with pbs , sterilized and kept in a dark container at –20°c . an immediate dilution was made starting with the resveratrol concentration 100μg/ml, 50μg/ml, 25μg/ml, and 12.5 μg/ml , using sterile serum free medium. methotrexate solution methotrexate vial (10mg/ml) was used as a traditional drug in comparison with the effect of standard and pure resveratrol . a stock solution (0.1mg/ml) was prepared. serial sterile dilutions were made with the following methotrexate concentrations 0.4 μg/ml, 0.2 μg/ml, 0.1 μg/ml and 0.05 μg/ml [24] . maintenance of the cell lines: when the cells in the flask formed a confluent monolayer, a freshney, [25] protocol was performed for cell line maintenance cytotoxicity assay it is also called a cell growth inhibition assay. in this assay, the three types of cell lines were treated with pure resveratrol extract and standard concentrations ranging from 12.5 μg/ml to 100 μg/ml, at the same time the lines were exposed to methotrexate drug in concentrations ranging from 0.05 μg/ml to 0.4 μg/ml using a microtiteration plate (96 wells) cell culture technique. the protocol assay, which included the following steps [25] : (a) seeding : the tryptinized and suspended cells were seeded in a microtiter plate by taking 0.2 ml cell suspension into each well that might contain (10 4 –10 5 ) cells/well, growth medium used for seeding (b) incubation : all plates were incubated in co2 incubator at 37°c for full cells attachment . (c) the treatment ( or cells exposure): using the maintenance medium ( serum free medium) as a negative control and serum free medium with 0.1%dmso as positive control , the microtiter plates after cells attachment were exposed to serial dilutions of pure resveratrol , standard resveratrol and methotrexate in the concentrations rang mentioned before.the exposure times were (24,48 and 72) hours. each plate was designed to contain three replications of each concentration and 12 wells for negative control and 12 wells for positive control . (d) recovering times and reading the results: at the end of the exposure times the medium was decanted off , the cells in the wells were gently washed by the addition of sterile pbs twice , finally 50 μl of crystal violate stain was added to iraqi j pharm sci , vol.18 (suppl.) , 2009 cytotoxic study of resveratrol 22 the wells and the plates were incubated for 30 minutes at 37°c, then the plates were washed gently with distilled water and left to dry.the plates of different cell culture at the end of the assay were examined by elisa reader at 492 nm transmitting wave length.only viable cells were able to take the stain , the dead cells were not. the proliferation rate was measured according to [26] and as follows: proliferation rate % = absorbance at 492 nm of test x 100 absorbance at 492 nm of control while the inhibition rate was measured according to [27] as follows: inhibition rate % = abs. at 492 nm of control – abs. at 492 nm of test x 100 abs. at 492 nm of control abs = absorbance the –ve results referred to the inhibition rate % while the+ve results referred to proliferation rate % all values were analyzed statically results and discussion partial purification the resultant fractions from silica gelg60 glass column chromatography technique were eluted according to their affinity to the mobile phase benzene: methanol: acetic acid 20:4:1.only fractions that gave positive ferric chloride test were collected and detected by t.l.c silica gel 60f254 plate with same mobile phase. both the positive fractions and the standard gave violet fluorescence spots with rf value=0.43. while the negative fractions were not. the dried collection was designated as “partial purified resveratrol” [28] . cytotoxic effect “growth inhibitory assay” of the purified resveratrol three cell lines were studied (amn-3, hep-2 and ref cell line) at three exposure time 24,48 and 72 hours using two fold dilutions to get concentration ranging from 12.5 µg/ml to 100 µg/ml for both the purified extracted resveratrol and the transresveratrol standard, while for methotrexate drug the concentrations included (0.05 , 0.1 , 0.2 and 0.4) µg/ml according to al-shemary study [24] . table(1) showed the results of the significant effect at (p<0.01) level on amn3 cell line.the highest concentration 100 µg/ml of the purified extracted resveratrol gave highest cytotoxic inhibitory effect (–37%) after 24 hours of exposure. while transresveratrol standard gave the highest cytotoxic inhibitory effect (–16.3%) after 72 hours of exposure on amn-3 cell line at concentration 100 µg/ml.methotrexate inhibitory effect at 0.4µg/ml concentration and after 48 hours of exposure gave the highest significant effect (p<0.01). among three types of treatment ( extract, standard and the drug) at all concentrations and for different intervals of exposure; the extracted purified resveratrol had the best efficiency in inhibiting amn-3 cell growth within (100 μg/ml) at the first 24 hours. these results were declared in a published study [29] on mda-mb468 breast cancer cell line intended cytotoxic effect of the polyphenol fraction from grape seeds. resveratrol was considered to be a phytoestrogen, based on its structural similarity to diethyl stilbestrol , a synthetic estrogen. it can bind to both alpha and beta – estrogen receptors and activates estrogen – dependent transcription in human breast cancer cells [6] . the cytotoxic effect of the purified extracted resveratrol on hep-2 cell line was shown in table(2) . the best concentration with the significant differences (p<0.01) was 100 μg/ml after 24 hour of exposure. while for the standard 100 μg/ml concentration gave the highest inhibitory effect (–27.5%) after 48 hours of exposure. methotrexate treated cell exhibited significant (p<0.01) inhibitory effect after 48 hour at 0.4 μg/ml concentration. the differences in hep-2 responsing toward different treatments might indicate a presence or absence of specific cellular receptors in each type of cell lines; making the cells interacts at same concentration in different manners. moreover the metabolic pathways in response to each treatment differed from one line to another. this fact was mentioned in different studies which investigated at different plant extracts in treating several types of cell lines [30, 31] . in the current study the rat embryo fibroblast cell line (ref) was treated as well as other cell lines with the extracted resveratrol , the resveratrol standard and the drug in consideration to establish their cytotoxic effect on a normal cell line as a control line. the ref cell culture passages number 74 and 75 might undergo a transformation. table(3) showed the growth was inhibited significantly (p<0.01) at concentration 50 μg/ml after 24 hours of cell exposure to the purified extracted resveratrol. while for the standard the concentration 100 μg/ml gave the highest inhibitory effect (–61%) after 48 hour of exposure. methotrexate concentration range (0.1-0.12) μg/ml gave highest inhibitory effect (–57% – 58%) after 48 hour of exposure. the extracted pure resveratrol showed the highest cytotoxic growth inhibition effect in almost all cell lines treated at the concentration range (50 – 100) μg/ml specially at the first 24 hours of exposure. the most sensitive cell line was amn-3 cell line , the lowest effect was for hep-2 cell line which was more resistant to resveratrol cytotoxic effect. iraqi j pharm sci , vol.18 (suppl.) , 2009 cytotoxic study of resveratrol 23 table 1: the cytotoxic effect as inhibition rate percent (%ir) of extracted pure resveratrol and trans resveratrol standard and methotrexate drug on amn-3 at (24,48 and 72) hours of exposure . se= standard error (%ir) concentration μg/ml treatments after 72 hour after 48 hour after 24 hour -16.30 ± 1.15 e -5.40 ± 0.23 c -37.00 ± 1.73 g 100 pure extracted resveratrol -15.00 ± 1.73 e -4.30 ± 0.17 bc -6.00 ± 0.28 d 50 -15.00 ± 1.73 e -2.20 ± 0.11 b -.20 ± 0.25 c 25 4.20 ± 0.11 c 1.10 ± 0.05 a 20.00 ± 1.15 a 12.5 -16.30 ± 1.15 e -11.00 ± 1.15 d -9.00 ± 0.57 d 100 standard transresveratrol 20.00 ± 1.15 a -11.00± 1.15 d -6.00 ± 0.57 d 50 14.00 ± 0.57 b -11.40 ± 0.23 d -8.80 ± 1.15 d 25 20.00 ± 1.15 a -13.60 ± 1.15 d 12.00 ± 1.15 b 12.5 17.50 ± 0.86 a -28.00 ± 1.73 f -0.80 ± 0.05 c 0.4 methotrexate drug 5.00 ± 0.57 c -23.00 ± 1.73 e -22.00 ± 1.73 e 0.2 -6.00 ± 0.57 d -25.00 ± 1.15 e -28.00 ± 2.30 f 0.1 -8.00 ± 1.15 d -11.00 ± 1.15 d -36.00 ± 2.88 g 0.05 3.200 ** 2.896 ** 4.169** lsd 0.0001 0.0001 0.0001 probability level ** (p<0.01). the means within any column with different letters are of significant differences , using anova test , then the least significant difference test (lsd) to compare significant between columns. table 2: the cytotoxic effect as inhibition rate percent (%ir) of extracted pure resveratrol and trans resveratrol standard and methotrexate drug on hep-2 cell line at (24,48 and 72) hours of exposure se= standard error %ir concentration g/ml treatments after 72 hr. after 48 hr. after 24 hr. -24.00 ± 1.73 h -7.50± 0.28 d -21.50± 0.28 g 100 pure extracted resveratrol -17.50± 1.15 g -9.50± 0.57 de -0.44 ± 0.02 f 50 -8.00± 0.57 e 5.40± 0.23 b 14.00 ± 1.15 e 25 1.60± 0.11 b 11.60± 1.15 a 24.50 ± 1.73 c 12.5 -3.00 ± 0.28 d -27.50± 1.73 i 25.70± 1.15 bc 100 standard resveratrol 0.50 ± 0.05 bc -11.00± 0.57 ef 26.00± 1.15 bc 50 11.00± 1.15 a 5.00± 0.28 b 26.00± 1.00 bc 25 10.50± 1.15 a 0.00± 0.00 c 28.50± 1.73 b 12.5 -3.60± 0.11 d -22.50± 1.15 h 35.00 ± 2.31 a 0.4 drug menotrexate -2.00 ± 0.11 cd -18.20± 1.73 g 15.00 ±1.15 de 0.2 -13.50 ± 1.15 f -16.50± 1.15 g 29.00± 1.73 b 0.1 -18.00± 1.73 g -13.50± 0.57 f 0.00± 0.00 b 0.05 2.893 ** 2.822 ** 3.744 ** lsd 0.0001 0.0001 0.0001 probability level ** (p<0.01). the means within any column with different letters are of significant differences , using anova test , then the least significant difference test (lsd) to compare significant between columns. iraqi j pharm sci , vol.18 (suppl.) , 2009 cytotoxic study of resveratrol 24 table 3: the cytotoxic effect as inhibition rate percent (%ir) of extracted pure resveratrol and trans resveratrol standard and methotrexate drug on ref cell line at (24,48 and 72) hours of exposure se= standard error %ir concentration μg/ml treatments after 72 hr. after 48 hr. after 24 hr. -34.00± 1.73 bc 2.00± 0.11 a -4.00 ± 0.57 b 100 pure extracted resveratrol -33.00± 1.73 bc -20.00± 1.73 b -46.00 ± 2.30 h 50 -30.00± 1.15 b -6.40 ± 0.23 a -40.00 ± 2.30 g 25 -37.00± 2.30 c 5.00 ± 0.57 a -11.00 ± 1.15 c 12.5 -30.00± 1.74 b -61.00 ± 3.46 h -36.00± 1.73 gh 100 standard resveratrol -23.00± 1.15 a -55.00± 2.88 gh -37.00 ± 2.30 b 50 -28.00± 1.73 ab -44.00 ± 2.30 a -36.00 ± 1.73 fg 25 -29.00± 2.30 ab -27.00 ± 1.73 d -32.00 ± 1.74 f 12.5 -34.00± 2.31 ab -50.00± 2.88 fg 5.00 ± 0.57 a 0.4 drug menotrexate -38.00± 4.04 c -58.00± 3.46 h -21.00 ± 1.15 e 0.2 -39.00± 2.88 c -57.00± 2.88 h -17.00 ± 1.73 de 0.1 -38.00± 1.73 bc -40.00± 2.30 e -16.00 ± 1.15 d 0.05 6.435 ** 6.848 ** 4.815 ** lsd 0.0006 0.0001 0.0001 probability ** (p<0.01). the means within any column with different letters are of significant differences , using anova test , then the least significant difference test (lsd) to compare significant between columns. conclusion the current results analysed the effect of (cis and trans ) resveratrol mixture. the yielded purified crystals were very sensitive and liable through many environmental changes with corresponding protections . in spite of that, the best effect was achieved with the extracted resveratrol among the transresveratrol standard and methotrexate drug. the attractiveness in the results for this naturally occurring compounds as a cancer chemopreventive agent has been escalated in recently as an ideal chemopreventive / chemotherapeutic agent acting by modulating aberrant signaling pathways and/or inducing apoptosis, and acting to target the multiple biochemical and physiological pathways involved in tumor development with minimizing toxicity in the normal tissue [32] . references 1. augenlicht, l.h., chemoprevention : intermediate markers in encyclopedia of cancer, academic press new york, 1997, vol.1. . 2. sandar, c.l., prevention and therapy of cancer and other common disease: alternative and traditional approacher, gail's books , richarland, 2003. 3. kirn, d.h., replication – selective microbiological agents: fighting cancer with targeted germ warfare. j. of clinical investigation, 2000, 105: 837-839. 4. gilman, a.g.; goodman, l.s.; ralls, t.w. and murad, f. the pharmacological basis of therapeutics. macmillan publishing company new york, 1990. 5. brown, j.k. ; byers, t. ; doyle, c.; cournerya, k.s. and sawyer,k.a., nutrition and physical activity during and after cancer treatment. an american cancer society guide for informed choices. cancer j. clin., 2003, 53:268-291. 6. ather,m.; back, j.h.; tang, x. and bickers, d.r., resveratrol: a review of preclinical studies for human cancer prevention. toxicology and applied pharmacology, 2007, 224: 274-283. 7. al-malikey , z.s.l., studing content of some local grapes varieties vitis vinifera l. from phenolic compounds. ph.d. thesis , college of agriculture univ. of baghdad, iraq, 2004. 8. candelariojalil, e. ; de oliveiva, a.c.p. ; graf, s.; bhatia, h.s. and fiebich, b.l., resveratrol potently reduces prostaglandin e2 production and free radical formation in lipopoly schaccharide activated primary rat microglia.neuroinflammation , 2007, 4: 25-41. 9. gao, d.; zhang, x. ; tiang, x.; peng, y. and song, l., resveratrol reduces the elevated level of mmp-9 induced by cerebral ischemia – reperfusion institute of neurosurger . china, 2002. 10. singletary,k., diet natural products and cancer prevention. j. nutr., 2000, 130: 465-466. 11. erickson, l., rooibos tea: resveratrol into antioxidant and antimutagenic properties. the iraqi j pharm sci , vol.18 (suppl.) , 2009 cytotoxic study of resveratrol 25 journal of american botanical council, 2003, 59: 34-45 . 12. niles, r.m; mcfarland, m. and meadows, gg, resveratrol is a potent inducer of apoptosis in human medanoma cells. cancer lelt., 2003, 190: 157-163. 13. freshney,i., culture of animal cells. a manual basic technique 4 th edition. wiley-liss, 2000. 14. rana, p. ; singha,m.; sivanandhan, d. and rajesh, a., phytochemicals as cell cycle modulators. cell cylcle, 2002, 2: 25-33. 15. creasy, l.l. and cooffe, m., poltoalexin production potential of grape berries j.am.soc. hort. sci., 1988,113 :230-234 16. siemann, p. and creasy l.l., method to assay ployphenolic constituent in wine. an alchemy, 1991, 24:77-86. 17. arce, l.; tena, m.t. and valcarcel, m., determination of trans-resveratrol and other polyphenols in wines by continous flow sample clean up system followed by capillary electrophoresis separation analytica. chimic. acta., 1998, 359: 27-38. 18. tomasbarberan, f.a. and robins, r. j., phytochemistry of fruit and vegetables. clarendon pressoxford. new york, 1997. 19. hendler , s.s and rorvik, d., resveratrol in: pdr for nutritional supplements. medical economics, thomson healthcare, montrale, 2001. 20. howitz, k.t.; bitterman, k.j.; cohen, h.y.; lamming, d.w.; lavu, s.; wood, j.g.; zipkin, r.e.; chung, p.; kisielewski, a., zhang, l.l.; scherer, b. and sinclair, d.a., small molecule activators of sirtuins extend saccharomyces cerevisiae lifespan. nature, 2003, 11:191-196. 21. harborne, j.b., phytochemical methods. chapman and hall, london, 1984. 22. sharma, k.k., an introduction to practical, chemistry. newdelhi. india, 1992. 23. jeandent, p.; meunier, p. and bessis, r., research note: occurance of a resveratrol – β – dglucoside in wine, vitis, 1994, 33: 183-184. 24. al-shamery, a.m.h., the study of new castle disease virus effect in the treatment of transplanted timer in mice. m.sc. thesis. college of verterinary medicine, university of baghdad. iraq, 2003. 25. freshney, r.i., culture of animal cells. third edition . wileyliss. new york, 1994. 26. chumchalova, j. and smarda, j., human tumor cells are selectively inhibited by colicins . folia microbiol, 2003, 48: 111-115. 27. gao, d.; zhang, x.; tiang, x.; peng, y. and song, l., resveratrol reduces the elevated level of mmp-9 induced by cerebral ischemia – reperfusion institute of neurosurger. china, 2002. 28. mohammed z. y. study the effect of the polyphenolic compounds extracted from grape skin fruit vitis vinifera on some cell lines (in vitro), m.sc. thesis , institute of genetic eng. and biotech. , university of baghdad, 2008. 29. agarwal, c.; sharma, y. and zhao, j., a polyphenolic fraction from grape seeds causes irreversible growth inhibition of breast carcinoma cells by inhibition mitogenactivated protein kinases activation and inducing g1 arrest and differentiation. clin cancer res., 2000,6 : 2921-2930. 30. chen,f.d. ;wu, m..; wang, h. e.; hawang, j.j. and yen, s.h., sensitization of tumor but not normal tissue to the cytotoxic effect of ionization radition using panax notoginseng extract. am.j. chim. med., 2001, 16: 234-242. 31. li, y. m. ; ohmo, y.; minatoguchi, s.; fukuda, k. and fujiwora,h., extracts from the roots of lindera strychifolia induces apoptosis in lung cancer cells and prolongs survival of tumor. wearing mice. am. j. clin. med., 2003, 31 : 857 – 869. 32. manson , m.m.; farmer, p.b. and gescher, a., innovative agent in cancer prevention . fortschr. krebsforsch, 2005, 166: 257-275. microsoft word bagpha05114 _5_ iraqi j. parma. sci., vol.14, 2005 36 -------------------------------------------------------------------------------------------------------------- investigation of bentonite clay surface as a physical antidote in adsorption of amitriptyline-hcl, chlorpromazine-hcl and chlordiazepoxide-hcl from solution saadoon a. isa*, sameer m. jassim and hussein k.a. hussein, department of chemistry and biochemistry, alnahrain college of medicine, alnahrain university, baghdad, iraq. (*) to whom correspondence is to be made received: 15.09.2001 accepted: 05.08.2002 abstract: a detailed study of adsorption from solution of amitriptyline-hcl, chlorpromazine-hcl and chlordiazepoxide-hcl on bentonite clay surface has been performed at variable conditions of temperature, ph and ionic strength. it is aimed in this work to explore the capability of this clay in treatment of poisoning by the mentioned drugs if taken in quantities higher than the usual doses. quantities of drugs adsorbed have been determined by uv spectrophotometric technique. the sequence of adsorption in neutral media at 37.5 cْ followed the order: amitriptyline-hcl > chlorpromazine-hcl > chlordiazepoxide-hcl. the results were discussed in the light of langmuir and freundich adsorption isotherms. the usual basic themodynamic functions were estimated. :الخـالصـة البحث دراس�ة مفص�لة ألمت�زاز المركب�ات الدوائی�ة أمت�ربتلین ھیدروكلورای�د و كلوربروم�ازین ھیدروكلورای�د تضمن ھذا وكان ھدف الدراسة ھو معرفة قابلیة سطح طین . وكلوردیازیبوكساید ھیدروكلوراید من محالیلھا المائیة على سطح طین البنتونایت ت�م تع�ین كمی�ات . بھذه األدویة إذا تم تناولھا بجرعات عالیة تفوق المق�ادیر االعتیادی�ة البنتونایت وفعالیتھ في معالجة حاالت التسمم . والقوة األیونیھ بواسطة تقنیة مطیافیة األشعة فوق البنفس�جیة phاألمتزاز بظروف مختلفة من درجة الحرارة و أس الھیدروجین :كون على وفق الترتیبی cْ٣٧.٥وقد وجد أن األمتزاز في الوسط المتعادل بدرجة حرارة .كلوردیازیبوكساید ھیدروكلوراید> كلوربرومازین ھیدروكلوراید > أمتربتلین ھیدروكلوراید تم تفسیر ومناقشة النتائج اعتمادًا على نظریت�ي النكم�ایر وفرین�دلش لألمت�زاز، وحس�بت الق�یم الثرمودینامیكی�ة االعتیادی�ة لعملی�ات .األمتزاز iraqi j. parma. sci., vol.14, 2005 37 -------------------------------------------------------------------------------------------------------------- introduction: solids have the property of holding molecules, at their surfaces either from the gas phase or from solution; this property is quite marked in the case of porous and finely divided materials (1) . the term adsorption refers to the accumulation of atoms, ions or molecules ( adsorbates ) on a surface of a solid substance ( adsorbent ). the medical significance of some active surface materials arises from their high adsorption capability. the most important application of these materials in medicine is their use as physical antidotes in the treatment of acute poisoning by toxic substances and drug over dosages (2, 3). activated charcoal was the most widely used solid surface as an antidote and in drug adsorption (4-12) . some clay materials were studied and found to possess similar characters to that of charcoal in the treatment of poisoning and drug adsorption. examples are kaolin (13-17), bentonite(18-20) and attapulgite(21). this work is concerned with the study of locally available bentonite clay as an active surface adsorbent for the drugs amitriptyline-hcl, chlorpromazine-hcl and chlordiazepoxide-hcl from solution in different conditions of temperature, ph and ionic strength. the process of adsorption from solution is more complicated than the corresponding process of gas adsorption on solid surface. the solvent effect and the competition between solvent molecules and drug molecules to be adsorbed has to be taken into account. generally, adsorption is a natural process and usually is accompanied by a decrease in free energy change and entropy of the system. there are a number of factors can influence the process of adsorption; the concentration of drug molecule, surface area of the clay, temperature, ph, ionic strength, solubility chemical state of both adsorbent and adsorbate molecules and the kinetic effect. details of three factors are available in textbooks and references (22-31). the term adsorption isotherm refers to the relation between the extent of adsorption ( qe ) or (x/m) with the equilibrium concentration of the drug in solution ( ce ) at constant temperature. (x) is the amount of drug adsorbed in milligrams by (m) grams of the adsorbent (24) . different isotherms of adsorption from solution on solid active surfaces have been classified by giles and co-workers (32) . two main theories have been adopted to describe adsorption from solution at constant temperature. the first, langmuir adsorption isotherm, assumes that at equilibrium, only a mono layer of adsorbate molecules can occupy the energetically homogeneous active sites, on the surface (26,27) as represented by equation (1) : ------ = ------ + ------ . . . . . . . . . . . (1) where (a) and (b) are constants. if this theory is valid for a certain adsorption system, a straight line is obtained by plotting ( ce / qe ) against ce . alternatively, the second theory known as freundlich adsorption isotherm, considers the active adsorption centers having different potential energies (heterogeneous surface). more than one adsorption layer may develop according to this approach (1, 27, 33) . freundlich isotherm is demonstrated by equation (2): log qe = log k + ---- log ce . . . . . . . . . .(2) where (k) and (n) are empirical constants. applicability of equation (2) is proved by linear plots of log qe versus log ce . ce 1 ce qe ab a 1 n iraqi j. parma. sci., vol.14, 2005 38 -------------------------------------------------------------------------------------------------------------- experimental section: (a) instruments the instruments used in this study were: uv/vis spectrophotometers /pyeunicam pu8700 and pu8600 philips, england., cuvette / quartz b.s. 3875. 1a, 10 mm f.o., centrifuge machine / hettich universal (d-7200), germany., thermostated shaker bath / gfl ( d-3006), germany, ph meter / hm-73 , tda electronics ltd., electronic balance sarturius lab. l 420 b.w. germany and oven / heraeus ( d-6450 ) hanau. (b) materials chemicals: hcl 36% w/w, sp.gr. (1.18) , bdh, england., nacl and mgso4 99.5% pure fluka , switzerland. drugs: amtriptyline hcl, chlorpromazine hcl and chlordiazepoxide hcl were obtained from (the state enterprise for drug industries and medical appliances), samarra, iraq (sdi). these drugs were pure and imported by (sdi) according to the british pharmacopoeia. sharp melting points were noticed for these drugs. bentonite clay: obtained from (the general company for geological survey and mining), baghdad, iraq. the clay was collected from (trifawi) opened mine in the western desert, iraq. the weight percentages of the clay components were: sio2 (54.7), al2o3 (14.7), mgo (6.0), fe2o3 (4.9), na2o (0.7), cao (4.8), so3 (1.2), loss on ignition (12.6). (c) methodology the clay was washed with excessive amounts of distilled water to remove the soluble materials, dried at 160 c o for three hours and kept in an airtight container. the clay was ground and seived to a particle size of 75 m and then used in all adsorption experiments. wavelengths of maximum absorbancy (λmax) were recorded for the drugs dissolved in aqueous media and found 256 nm, 328 nm and 329.5 nm for amtriptyline-hcl, chlorpromazine-hcl and chlordiazepoxide-hcl respectively. these values were utilized for quantitative estimation through the course of this research. solutions of different concentrations were prepared by serial dilution for each drug. absorbance values of these solutions were measured at the specified (λmax) value for each drug. calibration curves of absorbancy versus concentration of drug solutions were plotted. the concentration range that falls in the region of beer-lambert’s law applicability was determined and then used for subsequent estimations. the time required for full saturation of bentonite surface at 37.5 co by each drug was determined by shaking 25 ml of 0.005m-drug solution with 0.5g of bentonite. the concentration of drug solution was determined spectrophotometrically at different intervals of time till no further uptake of adsorbate by the adsorbent as the time proceeds. the time needed to attain equilibrium was 1 hr, 2 hrs and 2.5 hrs for amitriptyline-hcl, chlorpromazine-hcl and chlordiazepoxide-hcl respectively. (d) systematic procedure a volume of 25 ml. of seven different concentrations of each drug (0.0250, 0.0200, 0.0150, 0.0100, 0.0050, 0.0025 and 0.0010 mole /l ) was shaken with 0.5 g of bentonite adsorbent at a certain temperature in a thermostated shaker bath at shaking speed 60 cycles / min. after the equilibrium time is elapsed, the mixtures were allowed to settle and the clear liquids were centrifuged at a speed of 3000 rpm for 20 minutes. absorbancies were measured at the (λmax) value after making suitable dilution in order to fit beer-lambert’s limitation and then converted into absolute concentration readings through the calibration curve. results and discussion: the amount of drug adsorbed (adsorption uptake) at certain conditions of temperature, ph and ionic strength was calculated from equation (3): qe or = x v ( co - ce ) m m iraqi j. parma. sci., vol.14, 2005 39 -------------------------------------------------------------------------------------------------------------- (v) is the volume of solution in litres, (co) and (ce) are the initial and equilibrium concentration of the drug in milligrams per litre. the weight of adsorbent (m) was taken equal to 0.5 g. the adsorption uptake of amtriptyline-hcl, chlordiazepoxide-hcl and chlorpromazine-hcl at different temperatures (5, 20, 37.5 and 50 c) from aqueous solutions on bentonite surface is shown in figure 1. 0.00 1000.00 2000.00 3000.00 4000.00 ce mg/l 0.00 100.00 200.00 300.00 q e m g/ g (a) 0.00 1000.00 2000.00 3000.00 4000.00 ce mg/l 0.00 100.00 200.00 300.00 q e m g/ g (b) 0.00 1000.00 2000.00 3000.00 ce mg/l 0.00 100.00 200.00 300.00 400.00 q e m g/ g (c) figure 1: adsorption isotherms of (a) amitriptyline hcl (b) chlorpromazine hcl (c) chlordiazepoxide hcl on bentonite at (5, 20, 37.5, and 50 o c) iraqi j. parma. sci., vol.14, 2005 40 -------------------------------------------------------------------------------------------------------------- adsorption of amitriptyline hcl and chlordiazepoxide hcl obeyed freundlich adsorption isotherm as indicated by figure 2, 0.80 1.20 1.60 2.00 2.40 2.80 log ce 1.60 2.00 2.40 2.80 3.20 3.60 lo g q e chlordiazepoxide hcl y=1.145 x +0.526 amitriptyline hcl y=1.289 x+0.321 figure 2: freundlich lines of amitriptyline hcl and chlordiazepoxide hcl on bentonite at 37.5 c whereas adsorption of chlorpromazine hcl on bentonite obeyed langmuir isotherm as given by figure 3. 0.00 1000.00 2000.00 3000.00 4000.00 ce mg/l 0.00 4.00 8.00 12.00 c e /q e g /l chlorpromazine hcl y=0.0033 x + 0.697 figure 3: the linear form of the langmuir equation of the adsorption of chlorpromazine on bentonite at 37.5 o c the study of the adsorption process on bentonite requires taking the nature of the surface into consideration. bentonite surface consists of small patches of various kinds of active sites which are different in physical and chemical nature or in the steric configuration (34). moreover, bentonite has a character of an ion exchange with the other ionized species and this affinity depends upon the size, valency and steric orientation of molecules towards the surface (35, 36). iraqi j. parma. sci., vol.14, 2005 41 -------------------------------------------------------------------------------------------------------------- the interpretation of the high adsorption capacity of bentonite has been estimated from x-ray diffraction studies(37) confirming the formation of more than one adsorbed layer on bentonite surface. the adsorption extent of the drugs on bentonite followed the order : chlordiazepoxide hcl > chlorpromazine hcl > amitriptyline hcl the highest affinity of chlordiazepoxide-hcl towards the surface may be interpreted as a result of the functional groups on the chlordiazepoxide-hcl molecule leading to an increase in polarization of molecule and subsequently increase the attraction between the surface and the adsorbed molecule. the chlorpromazine hcl adsorption isotherm obeyed the langmuir equation. according to the giles interpretation (32,38) for the adsorption isotherm shapes, the chlorpromazine hcl molecules could be oriented in the direction parallel to the surface and the area of connection will be great leading to a high attraction between chlorpromazine hcl molecules and the bentonite surface, while the attraction between amitriptyline hcl and the surface appeared with a lower level of magnitude. figure(4) shows the adsorption isotherm of amitriptyline hcl and chlordiazepoxide hcl at ph=1 ,which was chosen to simulate the ph of stomach fluid comparing the adsorption isotherm of chlordiazepoxide-hcl in figure(1,c) which was conducted at ph=7 with the corresponding isotherm in figure (4) . 0.00 1000.00 2000.00 3000.00 4000.00 ce mg/l 0.00 100.00 200.00 300.00 q e m g /g chlordiazepoxide hcl amitriptyline hcl figure 4: adsorption isotherms of amitriptyline hcl and chlordiazepoxide hcl at ph=1 and temp.= 37.5 o c at ph=1 shows that there is a decrease in adsorption at lower values of ph .the change in ph affects the solubility of adsorbate molecules which, in turn, affects its affinity towards the surface(39). hence, the increase in the solubility of chlordiazepoxide hcl in a strong acidic medium leads to a decrease in a tendency towards the surface. on the otherhand, for amitriptyline hcl drug ,the change in ph from 7[figure(1,a)]to ph=1 [figure(4)] cause an increase in adsorption of the drug on bentonite ,because amitriptyline-hcl is a lipophilic strong base(40), thus at low ph the solubility will decrease leading to an increase in the quantity adsorbed on the surface. the adsorption of chlorpromazine-hcl on bentonite at ph =1 is inapplicable due to the rapid oxidation of the drug molecule into phenothiazine-3-one derivatives or its cations in strong acidic medium (41) . comparing the adsorption extent of the drugs on bentonite [figure(1) and that of figure (5)] in which 0.154 m of mgso4 and nacl was used shows clearly that an increase in adsorption with iraqi j. parma. sci., vol.14, 2005 42 -------------------------------------------------------------------------------------------------------------- increasing ionic strength of the solution is evident . this effect is more significant in the case of chlorpromazine hcl than chlordiazepoxide hcl and amitriptyline hcl. this behaviour may be interpreted as follows: the solubility of ionic salts in water is usually higher than that of organic drug molecules, therefore, a competition between them to interact with the solvent molecules leads to an increase in the attraction between the clay surface and the drug molecules which in turn will decrease the solvent-drug interaction. 0.00 1000.00 2000.00 3000.00 ce mg/l 0.00 100.00 200.00 300.00 q e m g /g with normal saline with magnesium sulfate (a) 0.00 1000.00 2000.00 3000.00 ce mg/l 0.00 100.00 200.00 300.00 400.00 q e m g /g with normal saline with magnesium sulfate (b) 0.00 500.00 1000.00 1500.00 2000.00 2500.00 ce mg/l 0.00 100.00 200.00 300.00 400.00 q e m g /g legend title with normal saline with magnesium sulfate (c) figure 5: adsorption isotherms of the (a) amitriptyline hcl , (b) chlorpromazine hcl (c) chlordiazepoxide hcl on bentonite using magnesium sulfate solution and normal saline at 37.5 o c iraqi j. parma. sci., vol.14, 2005 43 -------------------------------------------------------------------------------------------------------------- the interlayer spacing of montmorillonite is very dependent on the external ionic strength(42). phenomenologically, strong water adsorption on surface is repressed by the addition of an electrolyte. hence, replacing the chlorpromazine hcl molecules the water molecules on the bentonite surface active sites might occur. although the bentonite is able to remove heavy metals from aqueous solutions(43), there is no reason to suppose that the ionic exchange is taking place with sodium or magnesium ions because these ions are natural components of bentonite. the equilibrium constant (k) for the adsorption process at each temperature is calculated from equation(4): lc gq k e e 025.0* 5.0*  , (4) where qe unit is in mg per one gram of adsorbent, i.e. (x/m) value. ce unit is mg/l. (0.5 g) represents the weight of the clay that has been used and (0.025 l) represents the volume of the drug solution used in the adsorption process. the change in free energies (δg) could be determined from the equation : δg = rt ln k (5) where r is the gas constant (8.314 j mole -1 deg -1 ) and t is the absolute temperature. the heat of adsorption (δh) may be obtained from the equation : tr h xmln   +constant (6) where xm is the maximum uptake of adsorption at a certain value of equilibrium concentration (ce) that was fixed for all temperatures of study (44) . table 1 gives xm values at different temperatures. plotting (lnxm) versus (1/t) should produce a straight line with a slope =(-δh/r) as shown in figure 6. t( o k) 10 3 /t ( o k -1 ) amitriptyline hcl at ce=1000 mg/l chlorpromazine hcl at ce=1000 mg/l chlordiazepoxide hcl at ce=1000 mg/l xm lnxm xm lnxm xm lnxm 278 3.597 190 5.247 253 5.533 120 4.788 293 3.413 170 5.136 258 5.553 102.5 4.6299 310.5 3.221 160 5.075 266 5.583 77.5 4.35 323 3.096 140 4.942 273 5.6095 57.5 4.052 table 1: effect of temperature on the maximum adsorption of the drugs on bentonite 3.00 3.20 3.40 3.60 1000/t (1/k) 4.00 4.40 4.80 5.20 5.60 6.00 ln x m chlorpromazine hcl y= -0.152 x + 6.07 amitriptyline hcl y=0566 x + 3.21 chlordiazepoxide hcl y=1.444 x -0.358 figure 6: (lnxm) plotted against reciprocal absolute temperatures for the adsorption of the drugs on bentonite iraqi j. parma. sci., vol.14, 2005 44 -------------------------------------------------------------------------------------------------------------- the change in entropy (δs) was calculated from gibbs equation: δg = δh -tδs (7) thermodynamic functions (δg, δh, δs) of amitriptyline-hcl and chlordiazepoxide-hcl adsorption on bentonite were found to possess negative values indicating exothermic and spontaneous adsorption processes of these drugs (table 2). drug δh(kj mole -1 ) δg(kj mole -1 ) δs(j mole -1 o k -1 ) amitriptyline hcl -4.706 -1.046 -11.79 chlorpromazine hcl +1.264 -1.374 +8.496 chlordiazepoxide hcl -12.005 -2.568 -30.394 table 2: values of thermodynamic functions for the adsorption of the drugs on bentonite at 37.5 o c the adsorption of chlorpromazine-hcl on bentonite was found endothermic. this result agree with the previous experiments regarding endothermic adsorption process of chlorpromazine on different surfaces (45) . the higher tδs product than the δh value in the basic equation δg=δh-tδs will lead to a negative free energy change, i.e. the process is spontaneous. as one can notice, even a positive change in entropy (δs) may produce a negative δg value. the positive δs in the adsorption of chlorpromazine on bentonite could be viewed through the formation of less ordered adsorbed species on the surface. the status of the drug molecules on the surface is different in its configuration than that in solution. conclusions: 1. the clay was found to possess an appreciable adsorption capacity suggesting its probable use as an antidote in poisoning by the studied drugs. the clay may also be used as a stationary phase in chromatography technique. 2. the extent of drugs adsorption by bentonite followed the order: chlordiazepoxide hcl> chlorpromazine hcl > amtriptyline hcl. 3. ionic strength was found to increase the drug uptake by the surface upon addition of mgso4 and nacl to the adsorption solution 4. the influence of ph = 1 showed an increase in amtriptyline hcl adsorption and a decrease in chlordiazepoxide-hcl uptake by the surface of bentonite. 5. adsorption of amtriptyline hcl and chlordiazepoxide hcl on bentonite was found exothermic but chlorpromazine hcl adsorption appeared endothermic. suggestions for furthur work: 1. a study of the adsorption of drugs from the surface of the clay at different conditions. 2. in vivo study to estimate the performance of bentonite as a physical antidote in the treatment of acute poisoning. references: 1daniels f. et al, “experimental physical chemistry”7 th .ed. mcgrowhill co. n.y.(1970)pp: 365372 2chilvers e., hunter, j. and nicholas a. “davidson’s princples and practice of medicine” ,18 th edition ,u.k.(1999), pp: 1110-1120. 3ditter b., urbaschek r. and urbaschek b., [ability of various adsorbents to bind endotoxins in vitro and to prevent orally induced endotoxaemia in mice], gastroenterology, (1982),84, pp: 1547-1552. iraqi j. parma. sci., vol.14, 2005 45 -------------------------------------------------------------------------------------------------------------- 4holt e., and holz h.p. aconsideration of the role of charcoal in the treatment of poisoning in children, j. pediat., (1963), vol 63 (2), pp:306-315. 5sorby d.l. and plien e.m. 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(1963), 89 (sa2), p: 31. 45ref. 18, p. 1509. الخـلاصـة: drug iraqi j pharm sci, vol.32( 1 ) 2023 the benefits of using mobile application in the management of diarrhea doi: https://doi.org/10.31351/vol32iss1pp245-256 245 evaluating the benefits of using mobile application (diarrhea management step by step) in the management of diarrhea by community pharmacists **hadi mohammed ali and *hamadani-al yaqoob fadya ,*,1mikhael mudher ehab iraq. baghdad, baghdad, of university pharmacy, of college pharmacy, clinical of department * iraq. basra, basra, of university pharmacy, of college pharmacy, clinical of epartmentd ** abstract diarrhea is a common problem that leads patients to seek a pharmacist's advice. it is associated with significant morbidity and mortality. the majority of pharmacists in iraq did not manage diarrheal cases properly. therefore, the current study aimed to evaluate the benefit of a new mobile application (diarrhea management step by step) to improve the pharmacist's role in managing diarrhea. the study was conducted from 21st september to 21st october 2021 using a pre-post design via a simulated patient (sp) technique. a validated diarrhea scenario was presented to each pharmacist by the sp twice, the first time before and the other after giving the mobile application to the pharmacist. furthermore, pharmacists were asked to rate the application regarding its ease of use, reducing the time needed for managing diarrhea cases, reducing diagnostic errors, reducing medication errors, and applicability in daily clinical practice. the study sample involved 50 community pharmacists; only 47 completed the study. after using the application, all questions necessary to assess diarrhea were significantly improved. moreover, the average number of questions asked to the sp was significantly increased. on the other hand, providing the sp with an appropriate non-pharmacological and pharmacological treatment was also significantly improved. additionally, counseling the sp about the dispensed medication was also improved. most participating pharmacists agreed with the application's ease of use, ability to reduce diagnosis and medication errors, and applicability in clinical practice. in conclusion, the tested application effectively improves the pharmacist's role in the assessment and management of diarrhea. keywords: mobile application, diarrhea, pharmacist's role, iraq تطبيق الهاتف النقال )عالج االسهال خطوة بخطوة( في عالج االسهال بواسطة تقييم فوائد استعمال صيادلة المجتمع **علي محمد هادي و *، فادية يعقوب الحمداني 1*،ايهاب مضر ميخائيل العراق ، بغداد ، جامعة بغداد ، كلية الصيدلة ، السريرية فرع الصيدلة* العراق ، البصرة ، جامعة البصرة ، كلية الصيدلة الصيدلة السريرية، فرع ** الخالصة ذلك ومع والوفيات. المراضة حدوث بزيادة االسهال يرتبط الصيدلي. باستشارة يقوم المريض تجعل التي األسباب اكثر من هو االسهال المحمول الهاتف تطبيق فائدة تقييم إلى تهدف الحالية الدراسة فإن ، لذلك سليم. بشكل اإلسهال حاالت يعالجوا لم العراق في الصيادلة غالبية فإن ، أكتوبر 21 إلى سبتمبر 21 من الفترة في الدراسة أجريت اإلسهال. حاالت عالج في الصيدلي دور تحسين في بخطوة( خطوة اإلسهال )إدارة الجديد المريض قبل من صيدلي لكل صحته( من التحقق )تم لإلسهال سيناريو عرض تم الوهمي. المريض تقنية عبر بعد(-)قبل تصميم باستخدام 2021 حيث من التطبيق تقييم الصيادلة من ُطلب ذلك، على عالوة للصيدلي. المحمول الهاتف تطبيق إعطاء بعد والثانية قبل االولى المرة مرتين، الوهمي في التطبيق إستخدام وإمكانية األدوية، أخطاء وتقليل التشخيصية، األخطاء وتقليل اإلسهال، حاالت لعالج الالزم الوقت وتقليل استخدامه، سهولة جميع تحسنت التطبيق، استخدام بعد فقط. صيدالنيا 47 الدراسة أكمل ذلك، ومع صيدلي. 50 الدراسة عينة تضمنت اليومي. العمل أثناء الصيدلية من كبير. بشكل الوهمي المريض على طرحها تم التي األسئلة عدد متوسط زيادة تم ، ذلك على عالوة ملحوظ. بشكل اإلسهال لتقييم الالزمة األسئلة اعطائها تم التي المعلومات زادت ذلك، إلى باإلضافة كبير. بشكل تحسن المناسب والدوائي الدوائي غير بالعالج الوهمي المريض تزويد أخرى، ناحية التشخيص تحسين على وقدرته ، التطبيق استخدام سهولة على وبشدة وافقوا المشاركين الصيادلة معظم صرفها. تم التي األدوية عن الوهمي للمريض دور لتحسين فعال اختباره تم الذي التطبيق إن ذلك، من نستنج اليومية. السريرية الممارسة في لالستخدام تطبيقه وإمكانية الدوائية، األخطاء وتقليل اإلسهال. وعالج تقييم في الصيدلي تطبيق للهاتف النقال . االسهال. دور الصيدلي. العراق. : المفتاحية الكلمات introduction a community pharmacist is an expert who dispenses medications, counsels patients, and manages their care plan (1). common reasons for contacting a pharmacist by iraqi patients include purchasing a medication and/or seeking medical advice about a medication or a minor ailment (2,3), which can be defined as "common but self-limiting or uncomplicated conditions which can be diagnosed and managed with over-the-counter medications and without complicated medical interventions" (4). 1corresponding author e-mail: ehab_pharma84@yahoo.com received: 1/5 /2022 accepted: 3/7 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp245-256 iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 246 diarrhea is one of the most commonly encountered minor ailments in the community pharmacies of the developed and developing countries (5-7). although most cases of diarrhea are self-limited (8), it can be associated with significant morbidity and mortality in all age ranges, especially for children <5 years of age (9). according to the who report, diarrhea is one of the top 10 causes of death globally with more than 1.3 million deaths in 2015 (10). however, the mortality rate of diarrhea was greater in developing countries like iraq as compared to developed countries (5). the main reason for diarrhea associated mortality was related to its risk in causing dehydration and electrolyte imbalance, which if left without treatment can progress to acidosis and circulatory collapse that associated with impaired perfusion of vital organs, leading to renal damage and eventually death (11). unfortunately, many studies that were conducted in iraq found that the majority of pharmacists did not manage (assess and/or treat) diarrheal cases in a proper way. the main reason for such problem was the lack of sufficient knowledge in management of diarrhea and other minor ailments (12, 13). some studies found that the use of new technologies such as computers (14) and smart mobiles (15) can assist pharmacists and improve their work efficiency and productivity (16). therefore, the current study aimed to evaluate the benefit of newly developed mobile application to improve pharmacist's role in the management of diarrhea in a community pharmacy setting. methods study design a pre-post experimental study design was used to assess the benefits of using a mobile application (diarrhea management step by step freely available on google play store) (17) to improve the community pharmacist's role in diagnosis and treatment of diarrhea cases. this assessment was performed through a simulated patient (sp) approach (18). a well-developed and validated scenario about diarrhea for an elderly patient with hypertension and prostatic hyperplasia was presented to each pharmacist by the simulated patient (sp) twice, the first time before giving the mobile application to the pharmacist and the second time after providing pharmacists with the application and training them on it. in other words, the first assessment visit was used to assess the pharmacist's role in the management of diarrhea, while the second assessment visit was used to evaluate the benefit of using the application on the pharmacist's ability to manage diarrhea. each pharmacist was trained by the researcher about the use of the application and its features. in the first assessment visit, the sp did not enter the pharmacy until it is free from customers to minimize the possibility of incomplete assessment of the patient case by the busy pharmacist. thereafter, the sp presented his case (scenario) to the pharmacist and responded to any queries raised by the pharmacist. to ensure concealment of the assessment process, the sp bought all dispensed medications, without regard to their suitability to manage the case. after the end of the 1st assessment visit by 1-3 days, the application and a brief demonstrating video were shared with the pharmacist using bluetooth technology. after providing the pharmacist with the application and training on it using different diarrhea cases, an assessment of the pharmacist's role in diagnosis and management of diarrhea was performed by presenting a scenario similar to that in the first visit to the pharmacist. in the second assessment visit some measures were followed to maximize concealment of the assessment process and ensure dealing of the pharmacist with the case in a realistic way. the first measure was presenting the scenario by a new sp that did not enter the pharmacy in the first visit. the second measure was scheduling the second visit within 7-10 days after the first visit to ascertain that the pharmacist forgets the details of the case, and thus reducing the chance of bias in using the application through guessing the case and the sp by the pharmacist. at the end of the second assessment visit, each pharmacist was thanked for participation in the study. additionally, some information was obtained from the participating pharmacist including age, gender, years of experience, academic degree, college that he/she graduated from. furthermore, the pharmacist was asked to rate the application in regard to 5 different points (ease of use, reducing the time needed for management of diarrhea cases, reducing diagnostic errors, reducing medication errors, applicability in daily clinical practice) using numerical scale from 0 to 10. in both assessment visits, the interview with the pharmacist was recorded by the sp mobile phone to facilitate documentation of all details. all records were analyzed by the main researcher to assess and document the role of the pharmacist in diagnosis and management of diarrhea in both visits using specially designed checklists. the study was ethically approved by the ethical committee at college of pharmacy /university of baghdad. the simulated patients students from the college of pharmacy – university of baghdad worked as the sp after being trained about the diarrhea scenario by the main researcher. despite the fact that two sps were sufficient for conducting the present study, three students were enrolled as sp in this study. the third student intended for emergency cases in which one of the sps was known or detected by the pharmacist. to ensure maximum concealment of the emergency sp, the student was trained to ask the pharmacist about a suitable treatment for his acne, and then present the diarrhea scenario. iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 247 study sample pharmacists who work in community pharmacies in baghdad and having android phone were eligible for participation in the current study. to ensure recruiting a large sample, pharmacists were informed about the developed application and its unique features, besides the study purpose and procedure through an advertisement in two facebook pages (al-multaqa alsaidalani and multaqa alshaheed muhand kamil for pharmacists) with the largest number of pharmacist members. in the advertisement, pharmacists who were interested in the idea of the application and accepted to participate in the study (informed consent) were requested to fill in a google form. the google form was opened for 1 week from 8th september to 15th september 2021. 314 responses were obtained in that period. all responses were analyzed by the main researcher to check eligibility of pharmacist for the current study; meanwhile only 30 pharmacists were eligible (figure 1). to increase the sample, an additional convenient sample of 30 pharmacists were contacted in their pharmacies (in al-karkh discrete region, baghdad) and informed about the study. only 20 pharmacists provided their informed consent to participate. thus, the total sample of the current study was 50 pharmacists. figure 1. the study sample statistical analysis data input and analysis was done using statistical package for the social sciences (spss) version 17. categorical variables were presented as number and frequencies while continuous variables were presented as mean ± standard deviation. chi square test was used to test the significance in the difference among non-paired categorical variables. mcnemar test was used to test the significance in the difference among paired categorical variables. shapiro wilk test was used to test the normality of continuous variables. within each group paired t test was used to the difference in mean for normally distributed continuous variables, while wilcoxon sign test was used for abnormally distributed data. between groups, independent t test was used to the measure difference in mean for normally distributed iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 248 continuous variables, while mann whitney u test was used for abnormally distributed data. pearson correlation test was used to measure the correlation between normally distributed continuous data. on the other hand, spearman rho correlation test was used to measure the correlation between abnormally distributed continuous data. p values less than 0.05 was considered significant. results demographic data of the participated pharmacists fifty pharmacists were included in this study; however, two participants were withdrawn from the study and the application was not working on the smartphone of one participant. therefore, the sample size of the study was 47. however, only 27 pharmacists were found to use the application, and 20 did not use the application during the 2nd assessment visit. most participants graduated from iraqi public universities in the last 5 years & hold bsc. degree in pharmaceutical sciences. study participants' average working experience in community pharmacies was 7.14 years (table 1). table 1. characteristics of the participated pharmacists parameter participants who used the application (n=27) participants who did not use the application (n=20) p value all participants (n=47) gender male female 15 (55.56%) 12 (44.44%) 10 (50%) 10 (50%) 0.706* 25 (53.19%) 22 (46.81%) graduation college public iraqi university private iraqi university international universities 18 (66.67%) 3 (11.11%) 6 (22.22%) 12 (60%) 4 (20%) 4 (20%) 0.699* 30 (63.83%) 7 (14.89%) 10 (21.28%) graduation year 1980-2000 2001-2010 2011-2015 2016-2021 1 (3.7%) 7 (25.93%) 3 (11.11%) 16 (59.26%) 2 (10%) 5 (25%) 0 (0%) 13 (65%) 0.391* 3 (6.38%) 12 (25.53%) 3 (6.38%) 29 (61.70%) years of experience mean±sd 6.47±5.80 8.11±9.76 0.948^ 7.14±7.70 >10 years 5-10 years <5 years 5 (18.52%) 8 (29.63%) 14 (51.85%) 7 (35%) 1 (5%) 12 (60%) 0.082* 12 (25.53%) 8 (17.02%) 27 (57.45%) academic degree bsc msc phd 22 (81.48%) 2 (7.41%) 3 (11.11%) 18 (90%) 2 (10%) 0 (0%) 0.300* 40 (85.11%) 4 (8.51%) 3 (6.38%) *statistical analysis by chi square test; ^ statistical analysis by mann whitney u test. assessment of the pharmacist's role in the diagnosis of diarrhea at the 1st assessment visit (before using the developed application), the most commonly asked questions by pharmacists were: "who is the patient?" and "what are the disease symptoms?". meanwhile, questions about the action taken and the recent travel history were not asked by any pharmacist. additionally, the average number of questions asked to the sp was about 1.5 questions. furthermore, 19% of the pharmacists supplied the sp with the medication without asking him any questions. additionally, a non-significant difference was detected in all of the above questioning skills between the group of pharmacists who used the application and those who did not use it (table 2). at the second assessment visit, all questions necessary to assess diarrhea were significantly improved only among pharmacists who used the application. moreover, the average number of questions asked to the sp was significantly increased to about 5 times among the group of the application users, while it was decreased nonsignificantly among the non-users of the application. consequently, the optimum assessment of the diarrhea case (by asking all necessary questions) was significantly improved by participants who actively used the application. all of the aforementioned positive changes were also significant in the whole study sample. further details are given in table 2. iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 249 table 2. assessment of the pharmacist's role in the diagnosis of diarrhea diagnostic questions application users (n=27) application non-users difference at end of the study# all participants (n=47) baseline n (%) end of the study n(%) p value baseline n (%) end of the study n(%) p value p value baseline n (%) end of the study n(%) p value who is the patient? 21 (77.78) 27 (100) 0.041 16 (80) 12 0.344 <0.0001 37(78.72) 39 (82.98) 0.804 what are the disease symptoms? 8 (29.63) 26 (96.30) <0.0001 7 (35) 3 (15) 0.219 <0.0001 15 (31.91) 29 (61.7) 0.007 how long is the duration of diarrhea? 5 (18.52) 26 (96.30) <0.0001 1 (5) 0 (0) 1.00 <0.0001 6 (12.77) 26 (55.32) <0.0001 what is the action taken by the patient? 0 (0) 24 (88.89) <0.0001 0 (0) 0 (0) 1.00 <0.0001 0 (0) 24 (51.06) <0.0001 questioning about medical history of the patient 6 (22.22) 27 (100) <0.0001 2 (10) 3 (15) 1.00 <0.0001 8 (17.02) 30 (63.83) <0.0001 questioning about medication history of the patient 2 (7.41) 26 (96.30) <0.0001 0 (0) 2 (10) 0.500 <0.0001 2 (4.26) 28 (59.57) <0.0001 additional conditions that necessitate referral recent history of antibiotic usage 1 (3.7) 25 (92.59) <0.0001 0 (0) 0 (0) 1.00 <0.0001 1 (2.13) 25 (53.19) <0.0001 recent history of travel abroad 0 (0) 26 (96.30) <0.0001 0 (0) 0 (0) 1.00 <0.0001 0 (0) 26 (55.32) <0.0001 no any question was asked 5 (18.52) 0 (0) 0.063 4 (20) 8 (40) 0.344 <0.0001 9 (19.15) 8 (17.02) 1.00 diagnosis of the case number of questions asked by the pharmacist* 1.59±1.45 7.67±1.11 <0.0001 1.3±0.92 1±1.03 0.230 <0.0001 1.47±1.25 4.83±3.50 0.0001 optimum assessment of the case 0 (0) 24 (88.89) <0.0001 0 (0) 0 (0) 1.00 <0.0001 0 (0) 24 (51.06) <0.0001 * maximum number of questions is 8 questions. # difference at the end of study values between application users and non-users. iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 250 assessment of the pharmacist's role in diarrhea treatment at the 1st assessment visit (before using the developed application), the non-pharmacological advice was mentioned appropriately to the sp by less than 5% of the participated pharmacists. antimotility agents were dispensed by most (~ 90%) of the participated pharmacists. however, the choice of antimotility agent was appropriate (not contraindicated) by less than half of the participated pharmacists. besides that, 1/5 of the participated pharmacists dispensed an antibiotic to the sp. meanwhile, an optimum management for the sp case was detected by about 1/3 of the participated pharmacists. treatment of diarrhea case was not significantly different at baseline level between the group of application users and non-users. at the second assessment visit, providing the sp with an appropriate non-pharmacological and pharmacological treatment was significantly improved only in the group of pharmacists who actively used the application at the time of the interview with the sp. all the above improvements were detected also in the whole study sample. further details are shown in table 3. assessment of the participant's role in patient's counseling and education about the prescribed antimotility medication at the 1st assessment visit (before using the developed application), about 1/3 of the pharmacists dispensed a medication without providing the sp with any educational information about it. pharmacists provided the sp with an average of one educational point, in which the dose (33%) and dosing regimen (26%) of the dispensed antimotility agent were the most frequently, mentioned information. on the other hand, no any participant mentioned the treatment time scale and the possible side effects of the dispensed product. all participated pharmacists failed to provide the sp with appropriate counseling and education. at the second assessment visit, counseling the sp with the necessary information about dispensed medication was improved only by the group of pharmacists who actively used the application; however, significant improvement was detected only in regard to information about the drug dose and dosing frequency. additionally, the average educational points mentioned to the sp was increased significantly by pharmacists who used the application (from 1.11 to 2.15), while it was decreased by application non-users (1 to 0.7). furthermore, the number of pharmacists who dispensed a medication without providing the sp with any educational information about it was decreased significantly by the group of application users. all of the aforementioned positive changes were also significant in the whole study sample. all details are given in table 4. participants' opinions about the developed application about two-thirds of the participated pharmacists strongly agreed with the ease of application use and its ability to reduce diagnosis and medication errors. on the other hand, about 3/4 of pharmacists showed an agreement about the application's ability to reduce the time needed to deal with diarrhea cases and its applicability for use in daily clinical practice (table 5). furthermore, there was a non-significant difference between the users and non-users of the application in regard to their rating of the developed application (table 6). iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 251 table 3. assessment of the pharmacist's role in treatment of diarrhea application users (n=27) application non-users (n=20) difference at end of the study# all study participants (n=47) baseline end of the study p value baseline end of the study p value baseline end of the study p value advising the patient about the non-pharmacological measures mentioned appropriately 1 (3.70) 20 (81.42%) <0.0001 1 (5%) 1 (5%) 1.00 <0.0001 2 (4.26%) 21 (44.68%) <0.0001 not mentioned 26 (96.30%) 7 (18.52%) 19 (95%) 19 (95%) 45 (95.76%) 26 (55.32%) the dispensed product to treat diarrhea anti-motility agent 23 (85.19%) 27 (100%) 0.125 19 (95%) 20(100%) 1.00 1.00 42 (89.36) 47 (100%) 0.063 products other than anti-motility agents 4 (14.81%) 0 (0%) 1 (5%) 0 (0%) 5 (10.64%) 0 (0%) dispensing an additional product (e.g., ors, antibiotics) with the antimotility agent 4 (14.81%) 1 (3.7%) 0.375 6 (30%) 1 (5%) 0.063 0.828 10 (21.28%) 2 (4.26%) 0.021 antibiotics dispensed^ 5 (18.52%) 0 (0%) 0.063 7 (35%) 1 (5%) 0.031 0.240 12 (25.53%) 1 (2.13%) 0.001 treatment of the case number of medications dispensed 1.15±0.36 1.04±0.19 0.180 1.30±0.47 1.10±0.31 0.102 0.510 1.21±0.41 1.06±0.25 0.011 appropriateness of the dispensed medication(s) $ 11 (40.74%) 26 (96.30%) <0.0001 4 (20%) 8 (40%) 0.289 <0.0001 15 (31.91%) 34 (72.34%) <0.0001 ors= oral rehydration solution. $appropriate treatment of the case through recommending a suitable antimotility agent (not contraindicated and not interacted with the patient's medications) without antibiotic therapy. ^ some antibiotics were dispensed with antimotility agents while others dispensed alone. # difference at the end of study values between application users and non-users. iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 252 table 4. assessment of the pharmacist's role in patient's counseling and education about the prescribed antimotility medication. application users (n=27) application non-users (n=20) difference at end of the study# all study participants (n=47) baseline end of the study p value baseline end of the study p value baseline end of the study p value education about the initial drug dose appropriate 9 (33.33%) 22 (81.48%) 0.002 7 (35%) 5 (25%) 0.727 <0.0001 16 (34.04%) 27 (57.45%) 0.043 inappropriate 18 (66.67%) 5 (18.52%) 13 (65%) 15 (75%) 31 (65.96%) 20 (42.55%) education about the drug dosing frequency appropriate 9 (33.33%) 20 (74.07%) 0.007 5 (25%) 4 (20%) 1.00 <0.0001 14 (29.79%) 24 (51.06%) 0.041 inappropriate 18 (66.67%) 7 (25.93%) 15 (75%) 16 (80%) 33 (70.21%) 23 (48.94%) education on the duration of taking the medication (treatment time scale) appropriate 0 (0%) 2 (7.41%) 0.500 0 (0%) 0 (0%) 1.00 0.214 0 (0%) 2 (4.26%) 0.500 inappropriate 27 (100%) 25 (92.59%) 20 (100%) 20(100%) 47 (100%) 45 (95.74%) education about the possible medication side effects appropriate 0 (0%) 3 (11.11%) 0.250 0 (0%) 0 (0%) 1.00 0.123 0 (0%) 3 (6.38%) 0.250 inappropriate (not mentioned) 27 (100%) 24 (88.89%) 20 (100%) 20 (100%) 47 (100%) 43 (93.62%) patient education & counseling $ no any advice was given to the sp 10 (37.04%) 1 (3.70%) 0.012 6 (30%) 13 (65%) 0.023 <0.0001 16 (34.04%) 14 (29.79%) 0.832 points of information that was given to the patient (max = 4 points) 1.11±1.05 2.15±0.86 0.001 1.1±0.91 0.7±0.98 0.210 <0.0001 1.11±0.98 1.53±1.16 0.070 points of information that is given to the patient in a correct way (max = 4 points) 0.89±0.85 1.78±0.93 0.003 1.0±0.79 0.45±0.83 0.078 <0.0001 0.94±0.82 1.21±1.10 0.206 appropriate patient education and counseling (all 4 points) 0 (0%) 1 (3.70%) 1.00 0 (0%) 0 (0%) 1.00 0.384 0 (0%) 1 (2.13%) 1.00 $patient education must include information about drug dose, drug dosing regimen, drug side effect, and treatment time scale. iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 253 table 5. pharmacists’ opinions about the developed application (all participants) parameter strongly agree agree neutral disagree strongly disagree p value* ease of use 31 (65.96%) 12 (25.53%) 1 (2.13%) 2 (4.26%) 1 (2.13%) <0.0001 reduce time 10 (21.28%) 24 (51.06%) 9 (19.15%) 1 (2.13%) 3 (6.38%) <0.0001 reduce diagnosis errors 32 (68.09) 10 (21.28%) 2 (4.26%) 1(2.13%) 2 (4.26%) <0.0001 reduce medication errors 30 (63.83%) 13 (27.66%) 1 (2.13%) 2 (4.26%) 1 (2.13%) <0.0001 applicability in daily clinical practice 20 (42.55%) 15 (31.91%) 9 (19.15%) 1 (2.13%) 2 (4.26%) <0.0001 strongly agree score= 10 and 9; agree score = 7 and 8; neutral score = 5 and 6; disagree score = 3 and 4; strongly disagree score = 1 and 2. *statistical analysis by chi square test. table 6. rating of the application: a comparison between application users and non-users. parameter study group strongly agree agree neutral disagree strongly disagree p value ease of use application users 17 (63%) 9 (33.33%) 0 (0%) 1 (3.70%) 0 (0%) 0.361 application nonusers 14 (70%) 3 (15%) 1 (5%) 1 (5%) 1 (5%) reduce time application users 6 (22.22%) 15 (55.56%) 4 (14.81%) 1 (3.70%) 1(3.70%) 0.671 application nonusers 4 (20%) 9 (45%) 5 (25%) 0(0%) 2 (10%) reduce diagnosis errors application users 19 (70.37%) 7 (25.93%) 1 (3.70%) 0 (0%) 0 (0%) 0.310 application nonusers 13 (65%) 3 (15%) 1 (5%) 1 (5%) 2 (10%) reduce medication errors application users 21 (77.78%) 5 (18.52%) 1 (3.70%) 0 (0%) 0 (0%) 0.070 application nonusers 9 (45%) 8 (40%) 0 (0%) 2 (10%) 1 (5%) applicability in daily clinical practice application users 16 (59.26%) 6 (22.22%) 4 (14.81%) 0 (0%) 1 (3.70%) 0.090 application nonusers 4 (20%) 9 (45%) 5 (25%) 1 (5%) 1 (5%) strongly agree score= 10 and 9; agree score = 7 and 8; neutral score = 5 and 6; disagree score = 3 and 4 strongly disagree score = 1 and 2. discussion the results of the current study showed that only 57.4% of participated were found to use the application during the visit of the sp in their pharmacies. this finding may indicate that the usage of the application is either not easy or not beneficial for the pharmacist. however, this explanation is rejected because the agreement about the application's benefits and its ease of use was not significantly different between pharmacists who used the application and those who did not use it. instead, the shyness of some pharmacists to use a mobile device during the patient interview may be the main reason for the non-usage of the application (19). regarding the assessment of diarrhea, the current study showed that the most commonly asked questions to the sp were related to the patient age and diarrhea associated symptoms. on the other hand, questioning about action taken and the history of recent travel were not asked by any pharmacist. this finding was similar to the behavior of sudanese community pharmacists when they deal with diarrhea cases (20). additionally, the poor questioning skills of the participated pharmacists in the current study were also found in a pilot study that assessed the role of the community pharmacists in the management of the common cold in baghdad, iraq (21). iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 254 furthermore, the current study showed that about one fifth of the pharmacists supplied the sp with a medication without asking him any question. this problematic finding was also obtained in other studies conducted in vietnam (22,23) and in belgium (24). the main reason for such finding was the lack of financial incentives for the pharmacist who performs this service (assessment of the patient case) as compared to those who just dispense the medication without any assessment of the case (25). the results of the present study showed that the assessment of the diarrhea case using wwham questions was improved after providing pharmacists with the developed application and training them on it. the improvement in pharmacists questioning skills was also found in studies that refreshed pharmacists' knowledge through a training meeting with role-play (26). however, the current improvement could not be attributed to the training of pharmacists, since such improvement was not detected among pharmacists who trained about the application but not used it. indeed, the improvement by such training meetings was not always optimum or significant (26), due to the fact that knowledge enhancement by educational interventions is transient and may decline with time as a result of forgetfulness (27). on the other hand, the improvement in questioning skills by the usage of the application is expected to be greater and more persistent than any other educational intervention because the possibility of forgetting scientific information while using the application is very limited. therefore, the only explanation for the current improvement in pharmacists questioning skills was the usage of the developed application. similarly, the number of mandatory questions asked by the pharmacist to patients with allergic rhinitis was doubled after using a computerized pharmacy decision support system (28). this encouraging benefit in pharmacists' questioning skills by the usage of the application was confirmed by the strong agreement of the participated pharmacists on the ability of the application to reduce diagnosing errors. regarding the pharmacist's role in the treatment of diarrhea, the results of the present study showed that most pharmacists did not advise the sp about the necessary lifestyle changes during diarrhea management. a similar finding was obtained in studies conducted in sudan (20) and vietnam (22). this limited focus on the nonpharmacological advice could be explained by the fact that all pharmacists focus on dispensing pharmacological agents (12) and thus may forget or neglect the non-pharmacological part of treatment while managing diarrhea and other minor ailments. in the second assessment visit, advising the sp with non-pharmacological measure was significantly improved; this improvement was evident only among pharmacists who used the application and can be attributed to the ability of the application to prevent the unintentional missing of the nonpharmacological advice by reminding pharmacists with the necessary non-pharmacological information. other mobile applications were also effective to remind users to perform a specific function and thus prevent unintentional missing (29,30). the present study also showed that the dispensing of antimotility agents was increased, while the dispensing of antibiotics was reduced at the second assessment visit. such improvement was detected among all participated pharmacists and not limited to those who used the application. this improvement may be linked with the possible improvement in pharmacists' knowledge about the optimum management of diarrhea during the training session about the developed application. despite this improvement, the selection of an appropriate antimotility agent according to the sp case was not improved among pharmacists who did not use the application. this means that the improvement in the pharmacist's scientific knowledge was not sufficient to improve the selection of an appropriate antimotility agent to the sp, instead proper case assessment (31,32) along with a good scientific knowledge are the key elements to ensure optimum management of the patient case. meanwhile, the selection of appropriate treatment was significantly improved among the application users, which indicates the benefit of the application in improving the pharmacist's role in management of diarrhea. this benefit was confirmed by the strong agreement on the ability of the application to reduce medication errors by all of the participated pharmacists. regarding patient counseling and education, the results of the current study showed that 1/3 of the participated pharmacist did not provide the sp with any educational information; meanwhile, those who educated the sp do so by focusing mainly on the medication dose and dosing frequency while neglecting the treatment time scale and the possible side effects of the dispensed product. this poor counseling skill was detected in many other studies conducted in iraq (21, 33; 34). there are many possible reasons behind this poor counseling activity by iraqi pharmacists. the first reason is the lack of pharmacists' sufficient time in the pharmacy to remember and mention all needed information to the patient (35). the second reason can be related to the insufficient pharmacist scientific knowledge (35). this poor scientific pharmacist knowledge can be mainly attributed to the decline in such knowledge with time (forgetfulness) due to the lack of continuous medical education programs for graduated pharmacists (35). on the other hand, the current study results showed that the average educational points mentioned to the sp was increased significantly and the number of iraqi j pharm sci, vol.31(2) 2022 the benefits of using mobile application in the management of diarrhea 255 pharmacists who dispensed a medication without providing the sp with any educational information about it was decreased among pharmacists who used the application. this improvement may be linked with the ability of the application to enhance pharmacists' scientific knowledge and reduce the time needed to remember all the required medical information. despite all these improvements, mentioning side effects of the dispensed medications was only modestly and nonsignificantly improved among the application users. this can be explained in that reasons other than lack of pharmacist time and scientific knowledge may be behind the poor patient education and counseling. in this regard, it was found that pharmacists usually provide their patients with more counseling about drugs with prominent side effects (36), and since 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and peoples’ region, south west ethiopia. pragmat obs res. 2021;12:105-117. doi. 10.2147/por.s322407 37. kumpf vj. pharmacologic management of diarrhea in patients with short bowel syndrome. j parenter enteral nutr. 2014;38(1 suppl):38s-44s. 38. sinha hk. role of pharmacists in retailing of drugs. j adv pharm technol res. 2014 jul-sep; 5(3): 107. 39. saqib a, atif m, ikram r, riaz f, abubakar m, scahill s. factors affecting patients’ knowledge about dispensed medicines: a qualitative study of healthcare professionals and patients in pakistan. plos one 13(6): e0197482. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ abuse of aas was associated with significant decreases (p< 0 iraqi j pharm sci, vol.19(2) 2010 preanesthetic medications and hemodynamic changes 24 comparative effects of fentanyl, medazolam, lignocaine and propranolol on controlling the hemodynamic pressor response during laryngoscopy and intubation may s. al-sabbagh* ,1 *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract laryngoscopy and tracheal intubation are considered the most invasive stimuli in anesthesia. they provoked cardiovascular responses that include hypertension, tachycardia and dysrhythmias. various pharmacological approaches have been used to blunt or attenuate such pressor responses. the present study was designed to evaluate the effect of medazolom, lignocaine and propranolol as a valuable adjuvant to fentanyl in attenuating hemodynamic responses to endotracheal intubation in normotensive patients. thirty two patient with physical status i or ii according to the score of american society of anesthesiologist (asa), scheduled for elective surgery under standard general anesthesia, were randomly allocated into four groups (8 patients in each group), assigned as f, m, l and p groups. each patient in the four groups received 1 µg/kg i.v fentanyl. patients in groups m, l and p are treated with 0.2 mg/kg i.v medazolam, 1.5mg/kg i.v lignocaine and 0.01mg/kg i.v propranolol respectively. induction of anesthesia was then accomplished with 2mg/kg thiopental sodium followed by1.5mg/kg succinylcholine. tracheal intubation was performed 2 minutes after induction of anesthesia. heart rate, systolic blood pressure, diastolic blood pressure, mean arterial pressure and rate pressure product were measured before induction, after induction and at 2, 4, 6 and 8 minutes after intubation. the results indicated no significant variation in the hemodynamic pressor response in all four groups with tracheal intubation. in conclusion, a minimum effective dose of i.v pre-medications (fentanyl, medazolom, lignocaine and propranolol) were found to be individually successful in attenuating and providing a reliable control of all hemodynamic response changes accompanied the process of laryngoscopy and intubation. therefore, all are proved effective premedication and no one being superior. key words: fentanyl, medazolom, lignocaine, propranolol, endotracheal intubation, hemodynamic response. الخالصة ٔاألٔػٍخانمهت أفؼبلانزخذٌش , كالًْب ٌثٍش سدٔد أثُبءٌؼزجش رُظٍش انحُجشح ٔانمصجخ انٕٓائٍخ ٔكزنك انزُجٍذ يحفض غضٔي أٔرحذ أٌيخزهفخ يٍ شبَٓب أعبنٍتانذيٌٕخ انزً رشًم اسرفبع ضغظ انذو ٔػذو اَزظبو ضشثبد انمهت ٔخهم انُظى , ٔلذ اعزخذيذ ٍ ْزا انمجٍم . نمذ رى رصًٍى ْزِ انذساعخ نزمٍٍى رأثٍش كم يٍ انًٍذاصٔالو , انٍكُٕكٍٍ ٔانجشٔثشإَنٕل ثبػزجبسْب يٕاد رخفف سدٔد انفؼم ي يغبػذح راد لًٍخ نهفُزبٍَم فً انذٔسح انذيٌٕخ نزخفٍف سدٔد انفؼم غٍش انًشغٕة فٍٓب خالل ػًهٍخ انزُجٍذ انشغبيً نهًشضى رٔ أٔ األٔلانزخذٌش , حبل ألطجبءانؼشٕائً الثٍٍُ ٔثالثٍٍ حبنخ صُفذ اػزًبداً ػهى َظى انجًؼٍخ االيشٌكٍخ انضغظ انطجٍؼً . رى انزٕصٌغ 8يجبيٍغ ) أسثغ إنىػًهٍخ جشاحٍخ نٓى رحذ انزخذٌش انؼبو ثبالَزخبة انمٍبعً . رى رمغًٍٓى إجشاءانثبًَ يٍ انًشضى , يٍ انزٌٍ رمشس يٍ ىيٍكشٔغشاو / كغ 1يجًٕػبد األسثغفبء ، يٍى ، الو ٌٔبء . رهمى كم يشٌض فً بألحشفثيشضى فً كم يجًٕػخ ( ، ٔسيض نٓى يهغى /كغى نكُٕكٍٍ 1.1يهغى / كغى يٍذاصٔالو ٔسٌذٌب 2.0انفُزبٍَم ٔسٌذٌب . ٔرؼبنج انًشضى فً يجًٕػبد يٍى ، الو ، ٌبء ثًمذاس يهغى /كغى يٍ انصٕدٌٕو ثبٌٕثُزٌٕ ٌزجؼّ 0نً . رى ثؼذْب اعزمشاء انزخذٌش يغ يهغى / كغى ثشٔثشإَنٕل ٔسٌذٌب ػهى انزٕا 2.21ٔسٌذٌب دلٍمخ يٍ رحشٌض انزخذٌش ، ٔرى لٍبط يؼذل َجضبد انمهت ٔضغظ انذو 0يهغى /كغى يٍ انغكغٍُم كٕنٍٍ .َفز انزُجٍذ انشغبيً ثؼذ 1.1 دلبئك 8،6،8، 0ج لجم االعزمشاء ، ٔرنك ػُذ انحث ثى ثؼذ االَمجبضً ، ضغظ انذو االَجغبطً ، انضغظ انششٌبًَ ٔيؼذل ضغظ انًُز أثُبء األسثؼخانذٔسح انذيٌٕخ فً جًٍغ انفئبد أٔسدٔد انمهت إثبسحانُزبئج ػذو ٔجٕد اخزالف كجٍش فً اعزجبثخ أٔضحذثؼذ انزُجٍذ ، ) انفُزبٍَم ، انًٍذاصٔالو ، انكُٕكٍٍ األسثؼخ األدٌٔخانفؼبنخ يٍ لجم األدَىجشػخ انحذ إٌرُجٍذ انمصجخ انٕٓائٍخ . فً انخزبو رجٍٍ ٔانجشٔثشإَنٕل( َبجحخ فً رخفٍف حذح جًٍغ انزغٍشاد انزً رصبحت ػًهٍخ رُظٍش انحُجشح ٔانزُجٍذ ٔرٕفٍش يشالجخ يٕثٕق ثٓب نزا فكم ثًٍُٓب . اَخشيٍ أفضميٍ ْزِ انًٕاد انًغبػذ رؼزجش يفضهخ ٔنٍظ ُْبنك يٍ ْٕ introduction laryngoscopy and intubation are mandatory for most patients undergoing surgery under general anesthesia, often accompanied by a hemodynamic pressor response (1,2,3) . the rise in pulse rate and blood pressure is usually transient, variable and unpredictable; these changes are usually tolerated by healthy individuals, however, they may be deleterious in patients with hypertension, coronary artery diseases or intracranial hypertension, culminating perioperative myocardial ischemia, cardiac arrhythmias, acute heart failure and cerberovascular accident (4) . 1corresponding author email : may_sabbagh @ yahoo.com received : 13/4/2010 accepted : 8/6/2010 iraqi j pharm sci, vol.19(2) 2010 preanesthetic medications and hemodynamic changes 25 various drugs including calcium channel blockers (5) , vasodilators (6) , β-adrenergic blockers (7,8) , topical and intravenous lignocaine (9,10) , opioids (11,12) and deep inhalational anesthesia (13,14) have been used in an attempt to attenuate or prevent pressor responses that accompanied endotracheal intubation, but non have been satisfactory. fentanyl, a synthetic opioid, is one of the potent analgesics, when used before induction helps to attenuate hemodynamic response to intubation (1) . medazolam, a short acting benzodiazepine, most commonly used for its anxiolytic, muscle relaxant and sedative properties (15,16) , has slow onset of action with more gradual effects on circulation and greater degree of antegrade amnesia than thiopental; so it may offer an advantage in situation where hemodynamic stability is crucial (17,18) . recent studies suggested that propranolol and osmolol can also provide consistent and reliable protection against the increase in both heart rate and systolic blood pressure that accompany intubation, and may reduce the risk of adverse cardiac events in patient undergoing major surgical operation (8,19) . lignocaine hydrochloride, an amine ethylamide local anesthetic and class i b antidysrrhythmic drug is also acceptable for attenuation of cardiovascular response to intubation, and can also diminish cough reflexes, dysrhythmias and increase in intracranial pressure (4) . the present study was designed to evaluate the effects of medazolam, lignocaine and propranolol, as adjuvants to fentanyl, on the hemodynamic pressor response during endotracheal intubation in normotensive patients. patients and methods the present study was conducted at the neurosurgery hospital in baghdad in 2007 and involved thirty two asa physical status i or ii patients, with age range of 18-45 years, scheduled for elective surgery, requiring general anesthesia with endotracheal intubation. patients with abnormal electrocardiogram, significant bronchospastic, neurologic or cardiovascular diseases, including those receiving medication known to affect blood pressure and heart rate were excluded. on arrival to the operating room, electrocardiograph monitoring, pulseoximetry and noninvasive arterial blood pressure monitoring were applied, and baseline values of heart rate (hr), systolic blood pressure (sbp), diastolic blood pressure (dbp), mean arterial pressure (map), and the rate pressure product (rpp) were obtained. patients were randomly allocated into four groups (each include 8 patients) treated as follow: group f considered as control received only 1µg/kg i.v fentanyl, group m; patients in this group received 1µg/kg i.v fentanyl plus 0.2mg/kg i.v medazolam, group l; received both 1µg/kg i.v fentanyl and 1.5mg/kg i.v lignocaine, while group p received 1µg/kg i.v fentanyl together with 0.01mg/kg i.v propranolol. after 2 minutes of administrating pre-medications, induction of anesthesia was achieved with 2mg/kg thiopental and 1.5 mg/kg succinylcholin (all steps and doses were utilized according to the guidelines adopted in the neurosurgery hospital, baghdad). after loss of eyelash reflex, the lungs were manually ventilated with oxygen. direct laryngoscopy was performed at 2 minutes and trachea was intubated with proper sized disposable cuffed tube and fixed after confirmation of proper position. following intubation, anesthesia was maintained with o2, 1% halothane and pancuronium according to the requirements of surgery. the follow up of targeted parameters was started after administration of preanesthetic medications up to 8 minutes later on. hr, sbp, dbp and map were recorded before and after induction, then after intubation every 2 minutes for 8 minutes interval. the rate pressure product (rpp) was calculated by multiplying sbp by hr (20) . the data were statistically evaluated utilizing paired student's t-test to compare preand post-treatment values. intergroup comparison was performed using unpaired t-test and anova. results were considered significantly different at p<0.05. results demographic data of included patients are shown in table 1. no significant differences reported among groups with respect to sex, age, weight, hemoglobin (hb), and hematocrit (hct) values. the changes in hemodynamic variables from pre-induction (baseline values), at induction and after intubation in all groups (f, m, l and p) were shown in tables 2-6. after induction of anesthesia, sbp, dbp, map, hr and rpp showed less variation from the baseline values in the four groups. they slightly decreased, increased or remained unchanged, but no significant differences were reported. tracheal intubation produces non-significant increase in sbp, dbp and map in all groups at 2 minutes after intubation. all parameters gradually, but non significantly, decreased with each 2 minutes increment until 8 minutes post intubation, with some exception like in l group, in which there was non significant increase in dbp and map at 6 minutes after iraqi j pharm sci, vol.19(2) 2010 preanesthetic medications and hemodynamic changes 26 intubation. compared to base line values, both hr and rpp showed gradual but non significant increase at 2, 4, 6 and 8 minutes after intubation and reach optimal value at 6-8 minutes of intubation in all groups. table 1: demographic data of patients included in the present study groups sex age (year) weight (kg) hb (%) hct (%) gr f (n=8) 6 m 2 f 25.8 ± 8.2 ns 64.0 ± 11.6 ns 13.0 ± 1.7 ns 41.1 ± 5.1 ns gr m (n=8) 5 m 3 f 25.6 ± 7.4 ns 64.6 ± 13.9 ns 12.1 ± 1.5 ns 37.0 ± 4.7 ns gr l (n=8) 6 m 2 f 27.6 ± 9.0 ns 64.1 ± 18.2 ns 13.1 ± 1.1 ns 38.0 ± 4.2 ns gr p (n=8) 6 m 2 f 28.0 ± 9.4 ns 61.9 ± 13.6 ns 13.1 ± 1.5 ns 39.4 ± 5.2 ns f: fentanyl group; m: medazolam group; l: lignocaine group; p: propranolol group; data are presented as mean ± sd; n=number of patients. ns: non significant table 2: effects of fentanyl or its combination with medazolam, lignocaine or propranolol on systolic blood pressure during intubation in surgery stages systolic blood pressure (mmhg) groups f m l p pre-induction (base line) 129.8 ± 22.4 a 135.5 ± 15.1 a,b 125.5 ± 21.5 a 137.4 ± 10.5 b induction 121.3 ± 21.2 a,b 129.6 ± 15.7 a,b 126.3 ± 20.8 a 134.6 ± 10.2 b postintubation 2 min 126.1 ± 20.7 a 140.9 ± 20.1 a 132.0 ± 20.0 a 139.4 ± 10.7 a 4 min 117.3 ± 11.1 a 127.8 ± 16.3 a,b 127.5 ± 21.2 a,b 134.3 ± 13.1 b 6 min 116.4 ± 8.4 a 124.4 ± 23.1 a 130.9 ± 26.0 a 132.0 ± 33.8 a 8 min 110.8 ± 26.3 a 113.8 ± 24.6 a 117.9 ± 21.9 a 128.6 ± 23.2 b data are presented as mean ± sd; number of patients was 8 in each group; no significant difference existing with respect to induction value; non-identical superscripts (a,b) within the same time represent significant difference (p<0.05) table 3: effects of fentanyl or its combination with medazolam, lignocaine or propranolol on diastolic blood pressure during intubation in surgery stages diastolic blood pressure (mmhg) groups f m l p pre-induction (base line) 72.4 ± 11.9 a 75.4 ± 11.8 a,b 74.4 ± 9.6 a 78.6 ± 3.8 b induction 69.6 ± 12.5 a 72.4 ± 10.3 a 75.1 ± 12.7 a 76.6 ± 6.1 a postintubation 2 min 71.6 ± 13.0 a 84.5 ± 14.6 a,b 73.4 ± 10.1 a 81.8 ± 8.5 b 4 min 70.8 ± 16.8 a,b 78.3 ± 14.6 a,b 67.3 ± 11.2* a 78.8 ± 6.0 b 6 min 70.6 ± 9.8 a 74.4 ± 10.8 a 87.3 ± 33.5 a 75.1 ± 15.8 a 8 min 69.0 ± 9.9 a,b 63.6 ± 14.1 a,b 62.8 ± 17.0 b 78.9 ± 16.9 a data are presented as mean ± sd; number of patients was 8 in each group; *p<0.05 with respect to induction value; non-identical superscripts (a,b) within the same time represent significant difference (p<0.05) iraqi j pharm sci, vol.19(2) 2010 preanesthetic medications and hemodynamic changes 27 table 4: effects of fentanyl or its combination with medazolam, lignocaine or propranolol on mean arterial pressure during intubation in surgery stages mean arterial pressure (mmhg) groups f m l p pre-induction (base line) 93.6 ± 12.7 a 97.9 ± 12.9 a 91.8 ± 12.2 a 98.5 ± 8.5 b induction 90.0 ± 13.3 a 96.0 ± 17.2 a 93.1 ± 12.9 b 96.4 ± 18.3 c postintubation 2 min 94.5 ± 22.4 a 101.3 ± 30.3 a 95.4 ± 10.6 b 100.1 ± 8.6 b 4 min 89.6 ± 15.7 a 96.6 ± 13.7 a 91.9 ± 13.4 b 98.3 ± 8.9 b 6 min 88.9 ± 8.0 a 93.8 ± 14.2 a 99.4 ± 19.2 a 95.8 ± 21.9 a 8 min 83.8 ± 13.9 a 82.5 ± 14.3* a 82.6 ± 16.5 a 94.4 ± 25.4 a data are presented as mean ± sd; number of patients was 8 in each group; *p<0.05 with respect to induction value; non-identical superscripts (a,b,c) within the same time represent significant difference (p<0.05) table 5: effects of fentanyl or its combination with medazolam, lignocaine or propranolol on heart rate during intubation in surgery stages heart rate (beat/min) groups f m l p pre-induction (base line) 92.0 ± 32.6 a 97.5 ± 23.1 a 93.4 ± 29.8 a 95.5 ± 95.9 a induction 98.9 ± 40.6 a 103.1 ± 30.8 a 98.3 ± 33.2 a 101.3 ± 22.4 a postintubation 2 min 101.5 ± 33.1 a 107.8 ± 24.6 a 100.1 ± 33.8 a 108.3 ± 24.3 a 4 min 122.0 ± 39.2* a 118.0 ± 29.6 a 111.8 ± 25.2 a 109.3 ± 24.3* a 6 min 149.0 ± 36.4* a 128.4 ± 29.9* a,b 122.1 ± 35.0 a,b 117.1 ± 21.4* b 8 min 136.5 ± 36.8* a 123.5 ± 30.1 a 134.5 ± 21.2* a 124.6 ± 8.6* a data are presented as mean ± sd; number of patients was 8 in each group; *p<0.05 with respect to induction value; non-identical superscripts (a,b) within the same time represent significant difference (p<0.05) table 6: effects of fentanyl or its combination with medazolam, lignocaine or propranolol on rate pressure product during intubation in surgery stages rate pressure product groups f m l p pre-induction (base line) 11953 ± 4322 a 13224 ± 3441 a 11929 ± 5196 a 13232 ± 4772 a induction 11973 ± 4850 a 13450 ± 4448 a 12580 ± 5421 a 13673 ± 3558 a postintubation 2 min 12814 ± 4557 a 15429 ± 5200 a 13362 ± 5801 a 15253 ± 4225 a 4 min 14121 ± 4253 a 15269 ± 4901 a 14192 ± 3845 a 14772 ± 3866 * a 6 min 17360 ± 4501* a 15946 ± 4440 a 16394 ± 6997 a 15549 ± 5354 a 8 min 15452 ± 6082 a 14231 ± 4754 a 15806 ± 3779* a 15957 ± 2646 a data are presented as mean ± sd; number of patients was 8 in each group; *p<0.05 with respect to induction value; non-identical superscripts (a,b) within the same time represent significant difference (p<0.05) iraqi j pharm sci, vol.19(2) 2010 preanesthetic medications and hemodynamic changes 28 discussion laryngoscopy and tracheal intubation produced stressful hemodynamic changes in the form of hypertension and tachycardia, attributed to increase in the circulating levels of catecholamines (21,22) . control of such hemodynamic changes are very important to prevent detrimental effects, and the need for safe and effective therapeutic agents that may attenuate, blunt, suppress or abolish such changes became an important intervention during surgical procedures under general anesthesia. the results obtained from the present study revealed that all studied patients groups showed quantitatively and qualitatively similar hemodynamic pressor response at induction, intubation and post-intubation; the differences, if present, failed to reach statistically significant values. in the present study, failure to predict superiority for each pattern of drug intervention may be attributed to the limited number of patients in each group, and increase the number of patients may lead to more predictable values. however, pre-operative use of minimum effective doses of pre-anesthetic medications (1μg/kg fentanyl, 0.2mg/kg medazolam, 1.5mg/kg lignocaine and 0.01mg/kg propranolol) in the present study was found to be effective in restricting the non-significant increase in sbp, dbp and map values during short period of time (up to 2 minutes postintubation), then each parameter start to decrease gradually (but non-significantly) until 8 minutes postintubation; this means that all studied medications produce consistent and reliable protection against the abnormal increase in hemodynamic pressor response during laryngoscopy and intubation, similar to observations reported by other investigators (4,8,19,23) . additionally in the present study, a non-significant increase in mean pulse rate and rate pressure product (good indicator for oxygen consumption) was reported in all groups of operated patients, starting from intubation and reach optimal values after 6-8 minutes post-intubation; this could be explained by the fact that surgical intervention usually starts after 6-8 minutes postintubation, which is by itself a stressful procedure, predominantly suppresses the pressor response more effectively than tachycardia as a response (24) . light anesthesia (fewer drugs by the intravenous route or via inhalational means) is claimed to be the major factor responsible for pre-operative awareness and hemodynamic instability (17) ; to overcome this problem, fentanyl and/or medazolam are administered for the purpose of analgesia, sedation and anxiolysis (16) . many evidence indicated that each of them, when used alone or in combination, enables reduction of the thiopental dose required to produce induction, and consequently limit potential side effects and help in attenuating the hemodynamic response to laryngoscopy and intubation (16,25,26) . despite the potential advantages of the drug combination, reluctance to incorporate medazolam during light anesthesia persists due to concern regarding the potential for prolonged recovery (27,28) . however, a small pre-induction bolus dose of medazolam utilized in the present study did not prolong both recovery and discharge time from the day care unit following general anesthesia; this can be explained by the fact that the effects of medazolam on cns is dose dependent (27) . in the present study, although fentanyl was administrated in relatively small doses, it produces sufficient analgesia for short surgical procedures, and no one of the operated patients experienced pain of relatively long duration or great severity. although there is a possibility that administration of narcotic analgesic like fentanyl may affect pharmacokinetics of the anesthetic agents during induction, which is mostly due to changes in hemodynamic response (29,30,31) , the patients in f and m groups showed non significant increase in hr and rpp during laryngoscopy up to 6 minutes post-intubation; this increase seems to be suppressed in medazolam-treated group compared to fentanyl-treated group. this indicates that administration of medazolam before induction lead to hemodynamic stability most probably by mutual potentiation (32) .many studies have reviewed the effect of lignocaine to blunt the hemodynamic response after endotracheal intubation (4) . it has been reported that the strength and timing of lignocaine administration are equally important to prevent hemodynamic changes (33) , however, irrespective of the dose and time of administration of lignocaine, there are still significant increase in hemodynamic parameters after intubation (34) . kindler et al and durrani et al reported that i.v. administration of 1.5mg/kg lignocaine did not prevent the increase in hemodynamic response associated with laryngoscopy and intubation (35,36) . meanwhile, other investigators reported that 1.5mg/kg lignocaine effectively blocked the increase in sbp, dbp and hr after intubation (4,9) . in the present study, i.v administration of 1.5mg/kg lignocaine, 2 minutes before intubation provide reliable protection against the rise in hemodynamic response that associated with intubation process; this result was in accordance with iraqi j pharm sci, vol.19(2) 2010 preanesthetic medications and hemodynamic changes 29 many previously reported data, but not consistent with others (4,9) . such effect may be attributed to rapid equilibration of lignocaine between blood and brain with production of sedative effect when administrated in appropriate dose (37) . blocking and blunting adrenergic responses of tracheal intubation is the key pathophysiological step connecting βblockers. most of the studies concerned with evaluating the benefit of β-blockade on mortality and myocardial ischemia after tracheal intubation are based on using ultrashort acting selective β-blockers (38,39) , while very limited reports were available about using propranolol in this respect (19) . in the present study, 0.01mg/kg i.v propranolol was used, and no significant differences were reported in hr and rpp between patients groups. even a slight rise in hr and rpp that occurs at 6 minutes postintubation (a time of surgical intervention) was non significantly slowered in the β-blockade group in compare to other groups. these results are in accordance with those reported by hussain et al and yutaka et al (7,39) . the results of the present study shed a light on the possibility of using minimum doses of thiopental sodium for induction and maintenance of light anesthesia, for the aim of decreasing the time to discharge the patient from the recovery room and the day care unit; this situation seems to be compatible with the condition of shortage in medications required for anesthesia. in conclusion, minimum effective doses of pre-anesthetic medications (fentanyl, medazolam, lignocaine and propranolol) can maintain hemodynamic stability during laryngoscopy and intubation. references 1. channaiah vb, chary k, vik jl, wang y. low-dose fentanyl: hemodynamic response to endotracheal 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variables. british journal of anesthesia 2006;96(5);614619. 25. dundee jw, halliday nj. pretreatment with opiods. the effect of thiopentone induction requirements and overset of action of medazolam. anesthesia 1986; 41:159-161. 26. vinik hr. anesthetic interactions. eur j anesthesiol 1995; 12:3-4. 27. miller dr, blew pg, martineau rj. midazolam and awareness with recall during total intravenous anesthesia. can j anaesth 1996; 439(9):946-953. 28. delucia ja, white pf. effect of medazolam on induction and recovery characteristics of propranolol. anesth analg 1992; 74:563. 29. adachi yu, watanabe k, higuchi h, satoh t. the determinants of propofol induction of anesthesia dose. anesth analg 2001; 92:656-661. 30. kazama t, ikeda k, morita k. et al. relation between initial bloods distribution volume and propofol induction dose requirement. anesthesiology 2001; 94:205-210. 31. cocksholl id, briggs lp, douglas ej, white m. pharmacokinetics of propofol in female patients: studies using single bolus injections. br j anaesth 1987; 59:11031110. 32. shlomo b, abdelkhalim h. midazolam act synergistically with fentanyl for induction of anaesthesia. br j anaesth 1990; 64:45-47. 33. wang ym, chung kc, huang ym. lignocaine the optimal timing of intravenous administration in attenuation of increase of intraocular pressure during tracheal intubation. acta anaesthesiol sin 2003; 41(2):71-75. 34. miller cd, warren sj. i.v lignocaine fails to attenuate the cardiovascular response to laryngoscopy and tracheal intubation. br j anaesth 1990; 65(2):216219. 35. kindler ch, schumacher pg, schneider mc. effects of intravenous lidocaine and/or esmolol on hemodynamic response to laryngoscopy and intubation; a doubleblind, controlled clinical trial. j clin anesth 1996; 8:491-496. 36. durani m, barwise ja, johnson rc, et al. intravenous chloroprocaine attenuates hemodynamic changes associated with direct laryngoscopy & tracheal intubation. anesth analg 2000; 90:1208-1212. 37. nishino t, hiraga k, sugimori k. effects of i.v. lignocaine on airway reflexes elicited by irritation of the tracheal mucosa in humans anaesthetized with enflurane. br j anaesth 1990; 64:682687. 38. sugiura s, seki s, hidaka k. the hemodynamic effects of landiolol, an ultra-short-acting beta1-selective blocker, on endotracheal intubation in patients with and without hypertension. med ac jp 2007; 22:123-126. 39. yutaka o, nishikawa k, hase i. the short-acting β1-adrenoceotor antagonists esmolol and landiolol suppress the bispectral index response to tracheal intubation during sevoflurane anesthesia. anesth analg 2005; 100:733-737. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 33 effect of metformin and antioxidant agents on hirsutism in women with polycystic ovary syndrome mohamme d a.taher received 26-2-2003 accepted 13-6-2004 abstract f orty s ix ira qi wo men with pcos we re inv olved in this s tudy . they we re tre ated with metformin a lo ne a nd with antioxida nt age nts (vita min e o r c).it was found that all pa tie nts who treated with me tformin or with co mbina tion o f metformin with antioxid ant agents s ho wed s ignifica nt d ec re ase in hirs utis m s co re . the trea tme nt of me tformin with antioxidant agents is of great benefit in trea tme nt of hirs utis m in pcos due to tha t there was no worse ning e ffec t after trea tme nt. this may indica te that antioxid ant agents ma y pa rticipate in alleviation of hirs utism so it c an b e sa id tha t o xidative s tres s may play a n importa nt ro le in de ve lo ping of hirs utism in pcos. -:الخالصة  ــالث ن إلــى ث ن قســم عــد إ ة ب ســ را هــده الد كن فــي شــار ــاس كي عــدد األ مبــيض مت ة ال مــ متالز راقيــة مصــابات ب ون امــرأة ع عــ رب ســتة وأ وعات ار .مجم عـق ن ـم ف مؤـل مركـب ن ب ن فقد عـولج موعتان الباقيتا مج ر المبتفورين منفردا إما ال موعات أعطيت عقا إحدى هده المج سد مع مضادات التأك مين ور رتيب ) eاو cفيتامين ( الميتف ار . على الت عـق جن ب ول واتي عـ مريضـات الـل كل ال ن ة إ وجد في هده الدراس ودلك النه لم يكـن الكياس عدد ا مت ة المبيض زم متال ة في عراني الج الش كبير لع ر سد له اث مع مضادات التأك ركبا ورين منفردا أو م الميتف عالج رانية بعد ال هور في مقدار الشع مكـن هدا .هناك تد دلك ي شـعرانية ـل دار ال ف مـق خفـي كسد قد تشارك في ت ت التأ ن ان مضادا ر يبي التأثي كياس عدد اال ة المبيض مت زم ساء المصابات بمتال شعرانية عند الن ور ال سيا في تط ي قد يلعب دورا رئي كسد هد التا ول ان الج . الق introduction hirsutism is the pres ence of e xce ss ha ir growth in wo men a nd affects 5% 8% of the to tal fema le po pulation of fertile age (1).the hirs utism in polyc ys tic o vary synd ro me (p cos) is ma inly c ause d by ov aria n andro ge n ove rp ro duc tio n a nd in id io pathic hirs utis m there is p erip he ral hyp erse nsitiv ity to normal androge n circ ulating le vels (2).5α – re ductas e type 2 has be en s tudied for the d iffe re ntiatio n of e xterna l ge nitalia a nd pros ta te in ma le s and is ess ential fo r ha ir growth in both s exe s (3). a prerequis ite for the ce llular action of andro ge n on the p ilos eba ce ous unit is the c onv ersion of te stos terone to dihydrotes to sterone thro ugh the enzymatic a ction of 5αre duc ta se (4). howe ver the wo men with pcos ma y a mplify the manifes tatio n of hirsutism by the a vailable of insulin growth fac to r -1 due to ind uction of 5α re ductase activity w hich is med ia te d by ins ulin growth factor -1 (5). antia nd rogens a re substa nce s tha t preve nt and ro gens from exp re ss ing the ir a ctiv ity at ta rget s ite s. the inhibiting effe cts of antiandro ge n a re d iffere nt from those comp ounds tha t dec re as e relea se or inhibit biosynthe sis o f hypo thalamic and ante rior pituitary hormones (6 ).oral co ntra cep tive trea tme nt o f hirsutis m is mo st effec tive in women with o varian hype ra nd rogenis m esp ec ia lly with p cos.the atte mpt to a llev ia te hirs utis m cause d by a ndrogen e xce ss with oral contrac ep tive repres ents temp orary meas ure. the preferre d tre atment is to targe t the patho ge nic site of hype ra nd ro genis m or the site of a ctio n. me tformin trea tment of wo men with pcos res ults in a d ecline of insulin as well as to ta l and bioav aila ble te stos te ro ne lea ding to signific ant improve ment of clinic al manifes ta tions o f hype ra nd rogenis m (7).the refore the use o f metfo rmin in pcos may afford an alternative wa y in ma nageme nt of hirsutis m. dep artmen t of clin ic al labo rator y sc ie nce s ,colle ge o f ph arma cy,un iver sity of baghdad , baghdad –i raq . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 34 materials and methods fo rty –s ix ira qi p cos women with mea n age (2 8 ±4.5 ye ars) and e le ve n control wome n with co mparab le a ge were e nrolle d in this stud y. the p atie nts were a ttending the out pa tients clinic at al-elwe a hos pita l for obs te tric & gynec ology (ba ghd ad) and the y ha d a me an hirsutism s co re (20 .8 ±3.6) ac cording to the mo dified f erriman – ga llwe y sc oring system (8). the p cos wo men ha d menstrual irregula ritie s (oligome norrhea ), chro nic anovula tion, o bes ity; elev ated s erum andro ge n le ve ls , a s erum lh/f sh ≥ 2 a nd typical arre sted follicles we re s hown b y ultras ound stud y. all p atie nts ha d norma l fa sting gluc os e le ve ls and normal ma rkers o f thyroid, and kidney func tion. no ne of them had taking any drug for a t le as t 6 months before enrollment. the p atie nts were clas sifie d a cc ording to the type o f tre atment: gro up ι: 1 6 p atie nts we re trea ted with metformin (50 0 mg t.i.d). gro upⅱ: 14 p atie nts trea te d with co mbination of me tfo rmin (5 00 mg t.i.d) and vita min c (25 0 mg t.i.d). gro upⅲ: 16 p atie nts we re trea ted with co mbination of me tfo rmin (5 00 mg t.i.d) and (v itamin e 2 00 mg t.i.d). `the pa tie nts in all the se groups were maintaine d on the trea tme nt for a pe riod o f thre e mon ths. clinical study: a modifie d ferrima n – gallwe y system was use d to c linic ally gra de bo dy ha ir growth (8 ). in p articula r, the de gree o f hirs utism was rated on a sc ale fro m 0 to 4 on 8 b ody regio ns . the hirs utis m s co re wa s o btaine d b y to ta ling the sco re fo r ea ch b ody regio n and was determined before the s tudy a nd again after e ac h month for a period of 3 mo nths of trea tme nt. the sc ore eva luation was performed b y a single physician who was una ware of the trea tment . bioc hemic al analysis: bloo d sa mples for s erum testos terone we re collecte d be fore the trea tment a nd a fter inte rv als o f 1 month of trea tme nt for 3 suc ce ss iv e months. the s erum leve ls of te stos te ro ne were mea sure d b y testo-ctz which is ra dioimmunoas sa y kit (cis bio inte rnatio na l – oris group – f ra nce ). statiscal analysis: value s a re exp re ss ed as means ± sd. stude nt’s paired test was use d fo r co mparison of the p arame te rs b efore and afte r tre atment. a p v alue < 0.05 was co ns id ered statistic ally signific ant. results the a ge of the p atients in group 1 wa s (29 .2 ±2.9 ye ars ) , in gro up 2 the mean a ge was ( 27± 3.9 yea rs ) and in the group 3 the mean age w as ( 3 0.1±3 .6 yea rs ) . the b as al v alues of total se rum tes tosterone of eac h group are shown in table 1. ta ble – 1 effec t o f me tfo rmin an d ant io xida nt age nts o n se rum tes tostero ne (nmo l/l). ** p<0 .0 5 n=nu mbe r of the s ubjec ts contro l le ve ls ba se line levels of pa tients a fter 1 mon th o f tre atmen t after 2 m onths o f tre atment after 3 months o f tre atment 1.1± 0.37 (n=1 1) 4 .1± 1.3* * (n=1 6) 2.4± 1.3* * 2 .4± 1.02 ** 1 .5± 0.7** 1.1± 0.37 (n=1 1) 4 .5± 1.7* * (n=1 4) 3.1± 1.1** 2 .5± 0.92 ** 2 .3 ± 1 .2 ** 1.1± 0.37 (n=1 1) 5 .2± 2.2* * (n=1 6) 3.31± 1 .2 ** 3 ± 1 .5** 2 ± 0.7** ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 35 clinical and hormonal effects: clinical re sults are s umma rize d in ta ble 2 and figure s 1,2 and 3 . table 2 effec t of me tformin and a nt io xidant age nts on hirsutis m sc ore in pcos patie nts. *p<0.05 **p<0.00 5 fig .1 : hirsutism s core afte r tre atme nt with me tfo rmin fig .2 :hirs utis m s co re after tre atme nt with combinatio n of me tformin a nd vita min c fig.3:hirs utis m s co re a fter tre atme nt with combinatio n of me tformin a nd vita min e in a ll gro ups metformin a nd a ntioxida nt age nts de creas ed the histurism sc ore signific antly a fter 1,2 a nd 3 months of trea tme nt .the le ve ls o f se rum tes toste rone in all groups we re higher than normal lev els and the se rum tes tosterone de cre as ed s ignifica ntly in all group s a fter 1,2 and 3 mo nths of trea tme nt (tab le -1). evaluation of clinic al outcome the e valua tion o f the clinical outco me at the end o f the treatme nt is s umma rize d in ta ble -3 . ta ble 3 ra ting o f clinical outc ome at the e nd o f tre atme nt rating n o. (grou p1 ) no. (grou p 2) no. ( g ro up 3) exc ellent 1 (6 .25%) 2 (1 4.2%) 6(37 .5 %) goo d effe ct 4 ( 2 5%) 4 (2 8.5%) 4(25 %) no effec t 9 (5 6.2%) 8 (5 7.1%) 6(37 .5 %) bad e ffe ct 2 (1 2.5%) 0 0 the pa tients trea te d with metfo rmin showed 12.5% bad (worsening) e ffec ts while the patients treate d w ith co mbina tion o f metformin and antioxidant age nts show no wo rs ening effe cts .the patients tre ated with metformin and v itamin e repo rted 3 7.5% of an e xc elle nt effe ct. discussion the c ommo n unde rlying c aus es of hirs utis m a re p cos and idiopa thic hirs utis m (9). the goal o f trea tment is to interrupt the steps lead ing to the increas ed and ro gen exp re ss io n of the p ilos eba ce ous unit. sev eral stra te gies are av aila ble. mec hanic al ha ir re mova l ca n improv e his turis m, but it is a te mporary mea sure . in mos t ca se s hirs utis m re sults from a c omb inatio n of mildly increa se d and ro gen productio n and increa se d skin sensitiv ity to a nd rogen (10).the po lycystic gr oups be fore treatmen t after 1 mont h after 2 mon th s after 3 mon th s ⅰ 2 0.5± 5 .3 1 9.3 ± 5.4* 19 .7± 5 .5* 16 .6± 7.7* ⅱ 2 0.8± 20 1 9± 1.75* * 17 .5± 3 .8* 16± 4 .8* ⅲ 2 1.2± 2.2 1 9.1 ± 1.5** 16 .1± 5.4** 14 .2± 6.3** 0 5 1 0 1 5 2 0 2 5 b e fo re t re a t m e n t du r a t io n o f t r e a t m e n t (m o n t h s ) h ir s u ti s m s c o re ( m e a n ) a f t er 1 a f te r 2 a f te r 3 0 5 1 0 1 5 2 0 2 5 b e fo re tre a t m e n t d u r a t io n o f t r e at m e n t ( m o n t h s ) h ir s u ti s m s c o re ( m e a n ) a f te r 1 a f t er 2 a f te r 3 0 5 10 15 20 25 before t reatm ent dur at ion of tr e atm e nt (m onths ) h ir s u ti s m s c o re ( m e a n ) a f ter 1 a f ter 2 a f ter 3 ova ry syndrome is c harac terized c linica lly by a history of c hronic a nov ulatio n in c omb inatio n with s ome e vidence of andro ge n exc es s, s uc h as his turism and a cne (11). ma ny drugs with antiandroge nic prop erties , s uch as c yproterone (12) , s pirono la ctone (13) and flutamide (14) hav e be en us ed to trea t his turism in pcos, but the effic acy o f thes e drugs has b ee n sho wn to b e only pa rtial.the hyp erinsuline mia in pcos was rec ognized a s the cardina l manifes tatio n of ins ulin res istance. it was hypo thesized that in pcos , ins ulin may dire ctly stimulate ova rian cytoc hrome p 450 c17 α, res ulting in a n increa se p rod uc tion o f andros te ned io ne whic h is then may c onv erte d to tes to sterone b y the enzyme 17βre ductas e(15). ho weve r the use of metformin in this study may re so lv e the main challe nge of pcos which is ins ulin res is ta nc e and this may reduce the action of insulin on the ova ry and c onseq ue ntly re duce the ove rp ro duc tio n o f testos terone (ta ble 1).this re sult is in agre ement with that of ko lo dziejczyk e t al. who fo und that me tformin trea tment with pcos res ults in imp ro vement of c linic al ma nife station of hyp erand ro genism (7)..re ce ntly it wa s found tha t p cos is as soc ia ted with o xidative stres s (16).in pres ent stud y the use o f me tformin alone and co mbination of metformin with antioxid ant agents (v itamins c or e ) ,dec re as es the histurism sco re s ignifica ntly a fter 1 ,2 a nd 3 months of trea tme nt (tab le -2,figure 1,2,and 3 ). the de creme nt of total s erum tes to sterone as soc ia ted with dec line of his turis m s co re in all groups and this may s upp ort the hyp othe sis that hirsutism in pcos is ma inly due to androge n ove rp ro duc tion. in this s tudy, it wa s fo und that the perce ntage of p atie nts who get e xce lle nt e ffec t (ta ble -3) was mo re in group of metfo rmin with v itamins c (1 4.2%) and e (3 7.5%) tha n that in gro up of metformin alone (6.25 %). moreov er the worse ning effec t in p atie nts who treated with metformin a lo ne is (1 2.6%) while the pa tie nts who tre ated with me tformin a nd antioxid ant agents show no suc h bad effect. this ma y indica te tha t antioxid ant agents pa rticipate in alle viation of hirsutism s o it c an be s aid that oxid ativ e stre ss may play a n important role in de velop ing of hirs utism in pcos .therefore, the pa thoge ne sis of histurism in pcos ma y d iffer from othe r c onditions whe re it may cause d by the androge n ove rp ro duc tio n in a dditio n to o xida tive s tres s. in co nc lusion, it ca n b e s aid tha t us e of antioxid ant a ge nts a re of grea t b enefit in pcos ; in add ition to their excellent effe ct o n histurism s co re at leas t they may prev ent worse ning e ffec t. references 1.barbieri rl. hyp erand ro genic diso rd ers, clin ob stet gyne co l 19 90.,33:64 0-54;. 2 . kuttenn f, mo wszowics i, s chaison g, ma uv ais, j aris p. androgen production and skin metabo lis m in histurism.j endo crinol 197 7;75:83 -9 1. 3.dia ni ar, mulhollamd mj , s hull ki., kubice k mf, j ohnso n ga, sc hos tarez hj ,et al. hair gro th e ffec ts o f oral adminis tration of fina sterid e , a s te ro id 5 αreductas e inhibitor , alone o r in combination with to pical minoxid il in the b alding stump tial ma caq ue . j clin endo crinol me tab 19 92;74 :3 45-50 . 4. wils on jd. wa lker j . the c onv ersion of te stos te ro ne to 5 α-a nd ro stan-17 -β-o l-3-one (dihydrotes to sterone) b y skin s lice s of ma n. j clin inve st 196 9,48;3 71. 5.horto n r. p asupule nti v. antonipillia i. androgen inductio n o f steroid 5 α-re ductase may be med ia te d v ia insulin –like gro wth fa ctor-1 . endoc rino lo gy1 993 , 13 3:447 . 6. do rfman ri. biolo gica l ac tivity of and ro gen , br j dermatol 1 97 0;82:3-8. 7. ko lo dziejczyk b. et a l. me tformin therapy dec re as es hyperandroge nism and hyp erinsuline mia in wo men with po lycystic ova ry syndrome . fe rtl and steril 200 0, 37(6):1 149 -1 154 . 8. f erriman d, gallwey j d. clinic al ass es sme nt o f bod y hair growthin women .j clin endoc rino l metab 196 1;21:14 40-7. 9.rittmas te r rs . histurism,clin endo crinol ,1 997 ,4 7:29. 10. delahunt jw. histurism. drugs 19 93; 45: 223 -31 . 11. m ra fe t ga za vani, mark hamilto n,cha ries r kingsland, a tee mpeton. p olycystic ova rian disea se : a mislead ing labe l. la nce t 2 000 ; 355 (92 01): 4 11-2. 12.neumann f . pha rmaco lo gy and p otential use o f cyproterone a ceta te . horm me tab res 197 7; 9 ,1 -1 3. 13.tremb la y rr. trea tment of his turism with spirono la ctone . clin endoc rino l metab 198 6;15,36 3-9. 14.iptis am i., f ahri b., a nd muhamme d g. a pro sp ective , randomized tria l c ompa ring flutamide (25 0 mg/d) and fina steride (5 mg/d) in the tre atment of his turis m. fe rtl and steril 200 0, 7 3(5),9 84-987 . 15. hunte r, s j., ga rv ey, w, t . insulin ac tion and insulin re sistance : d is ea se involving defec ts in ins ulin re cep tors , s igna l tra ns duc tion and the gluco se tra ns port effec te r system. am j med, 198 8;105 (4 ),33 1-45. 16.ta her ,m.a.,p hd. the sis, "effect of me tfo rmin and antio xidant agents on lipid pro file and , hormone s and oxidative stress status in wo men with p olycystic ova ry syndrome", 2 002 p.xviii. comparative evaluation of using intranasal desmopressin, parenteral diclofenac or their combination in the management of acute iraqi j.pharm.sci., vol.16 (2) ,2007 synthesis of anti-inflammatory aromatic schiff bases 5 synthesis of schiff bases of benzaldehyde and salicylaldehyde as anti-inflammatory agents tagreed n. omar* ,1 * department of pharmaceutical chemistry , college of pharmacy , university of baghdad ,baghdad , iraq abstract three schiff bases from benzaldehyde and salicylaldehyde have been synthesized (a, 1and 2) and two of them (1and 2) have been tested for anti-inflammatory activity. the p-aminobenzene sulfonamide has been synthesized from acetanilide through the addition of excess chlorosulfonic acid then concentrated ammonia solution; schiff base of this derivative (2) exhibited good level of activity against egg-white induced edema in rat hind paw, while the other tested derivative exhibited no activity. key words: schiff bases, sulfonamide derivatives, salicylaldehyde :الخالصة ( َاممت ةبرسممعا ةهنوممث ةشىرمموه ىٍممه a2a3,تممف ممه ٌممري ةنتخةيممث ت هوممد شمممن اُةنممت يممي ممه ةنسىلةنتدٍهدممت َةن هن مموم ةنتدٍهدممت حمه كمضهدةا نمنرٍهبها. ةن ةنسهخة ة وىُبىلده يهفُن ة هدت ٌُ ةحت ةال وىها ات تف ت هوقً ه ةاليرهوهنوت ه بمل ةضه ث كموث كسومع مه خَيهفُووك َ ه شف يهئم ةال ُووه ةنمعكل. ةن اهنت يي نٍرة ةنمشرد ات ةظٍعا ةهنوث جومت كمضمهد نمنرٍهبمها ةنم مر تشث مه ةن مع ةن ةنكهُ بُةيطث زالل ةنسو ، ة ه ةنقهنت ةنثهووث ةنم هقث هف تظٍع أي ةهنوث ضهد نمنرٍهبها. introduction non-steroidal anti-inflammatory drugs (nsaids) are widely used for the treatment of pain, inflammatory conditions and fever (1,2) . their efficacy has been documented in a number of clinical disorders including osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, gout and dental pain (3) . in the past decades; it has become apparent that there are two separate cyclooxygenase (cox) gene products, cox-1 and cox-2, which can initiate the metabolism of arachidonic acid to prostaglandins (pgs) and related lipid mediators (3) . cox-1 expressed in most tissues of the body and largely governs the hemostatic production of arachidonic acid metabolism; whereas cox-2 is induced in response to inflammatory stimuli or physiologic stress and is responsible for the enhanced production of eicosanoid mediators characteristics of these situations. all classical nsaids inhibit cox-2 as well as cox-1 to varying degrees, thus they can be considered non-specific (4, 5) . for a long time sparatore and co-workers have been described sets of schiff bases (diaryland arylheteroaryl azomethines) endowed with strong and long lasting antiinflammatory activity against the rat hind paw edema induced by carrageenan (6) . the same compounds reduced, dose dependently, the nitric oxide and pges production (7) . all these properties were mainly correlate with the presence of phenolic functions, which can display a generic anti-oxidant and radical scavenging activity, more than with the presence of the azomethine function (7) . on the other hand, the azomethine function is endowed with multiform reactivity and particularly is able to react with thiol groups (8) . thus it could establish easily some kind of link with enzymatic or receptorial proteins. the diarylazomethines are isosteric with stilbenes and like these can exist in interconvertible cis and trans forms. suitable substituted cis-stilbene derivatives are characterized by potent inhibitory activity on cox-2, quite similarly with that observed for a variety of vicinal diarylheterocycles, among which important anti-inflammatory drugs, like celecoxib and valdecoxib, are found (9) . in the last class of drugs, the central five member ring may be of very different nature, either heterocyclic or carbocyclic (10,11) ; while the nature of substitutents on the two benzene rings is believed to be responsible for cox-2 selectivity by insertion into the secondary pocket of the enzyme, with the p-sulfonamido and pmethylsulfonyl groups playing a key role (12) . accordingly, we have now designed and synthesized schiff bases of salicylaldehyde (compounds 1 and 2). some of them bearing these peculiar substituents, in addition to azomethine function, which could play some peculiar role in the interaction with cox enzymes. 1 corresponding author : e-mail: tagomar77@yahoo.com received : 11/11/2006 accepted : 8/7/2007 mailto:tagomar77@yahoo.com iraqi j.pharm.sci., vol.16 (2) ,2007 synthesis of anti-inflammatory aromatic schiff bases 6 c h n c h n c o ho (1) c h n (2) oh s nh 2 o o (a) experimentals a. chemistry materials: acetanilide (riedel-dehaen, germany), ammonia solution, benzaldehyde, salicylaldehyde, chlorosulfonic acid, absolute ethanol and ether (bdh, england), all solvents and materials used were of analar type and used without further purification. general procedure:melting points were determined by capillary method on thomas hoover apparatus (england) and ir spectra were recorded on model 500 scientific ir spectrophotometry, buck company (usa). ascending thin layer chromatography (tlc) was run on dc-kartan si alumina 0.2 mm to check the purity and progress of reaction. the identification of compounds was done using iodine vapor and the chromatograms were eluted by methanol: acetic acid:ether: benzene (1:1:6:2) (13) method for preparation of p-aminobenzene sulfonamide (a) preparation of p-acetamidobenzene sulfonyl chloride acetanilide (6.67gm, 49.4mmol) was placed in 250ml flask and melted in the flask over a free flamed and caused the compound to solidify over the lower part of the flask by swirling the liquid formed and immersion in an ice bath momentarily. the chlorosulfonic acid (17ml, 262mmol) was added all at once with continuous shaking, then the reaction mixture was heated on a water bath for 90 minutes in order to complete the reaction. the mixture was cooled and the oily substance was poured with stirring. this suspension was filtered off with suction, pumped and washed with a little cold water and dried to give crude product, which was used immediately in the next step without further purification (13) . (b) preparation of p-acetamidobenzene sulfonamide the crude p-acetamidobenzene sulfonyl chloride was transferred to the rinsed reaction flask, and a mixture of concentrated ammonia solution (24 ml) and water (24 ml) was added to the flask. the contents of the flask were mixed thoroughly and heated with occasional swirling to just below the boiling point for about 20 minutes. the sulfonyl chloride will be converted into a pasty suspension of the corresponding sulfonamide. the suspension was cooled in an ice bath and then dilute sulfuric acid was added until the mixture was just acid to congo red paper. the product was collected in buchner funnel, washed with a little cold water and drained as completely as possible to give 53% yield of faint yellow crystals with melting pint of 213-214 o c and rf values of 0.45. ir (in kbr disk): 3376cm -1 and 3304cm -1 (n-h stretching vibration of primary sulfonamide); 3227cm -1 (n-h stretching vibration of secondary amide); 1660cm -1 (c=o stretching vibration of secondary amide); 1598cm -1 and 1530cm -1 (c=c stretching vibration of aromatic ring); 1327cm -1 and 1157cm -1 (s=o stretching vibration of sulfonamide). (c) preparation of p-aminobenzene sulfonamid the crude p-acetamidobenzene sulfonamide was transferred to a flask contain a mixture of concentrated hydrochloric acid (10 ml) and water (30 ml). the mixture was boiled gently under reflex for 90 minutes. cooled to room temperature and activated charcoal (2 gm) was added. the mixture was boiling gently under reflux for 90 minutes. cooled to room temperature and activated charcoal 92gm) was added. the mixture was heated to boiling and filtered with suction through a hardened filter paper. the filtrate (a solution of 4aminobenzene sulfonamide hydrochloride) was placed in a beaker and sodium bicarbonate was added in portions with stirring until the suspension become neutral by testing with litmus paper. the mixture was cooled in ice bath and filtered by suction and dried to give 51% yield of the white crystals with melting point of 160-161 o c (reported 163-165 o c) (14) and rf value of 0.75. ir (in kbr disk): 3461cm -1 and 3373cm -1 (n-h stretching vibration of primary amine); 3247cm -1 (n-h stretching vibration of sulfonamide); 1639cm -1 (n-h bending of primary amine); 1600cm -1 , 1571 cm -1 and iraqi j.pharm.sci., vol.16 (2) ,2007 synthesis of anti-inflammatory aromatic schiff bases 7 1504cm -1 (c=c stretching vibration of aromatic); 1309cm -1 and 1145cm -1 (s=o stretching vibration of sulfonamide). general method for preparation of azomethines (schiff bases) to a solution of 10 mmol aniline (compound a), salicylamide (compound 1), or paminobenzene sulfonamide (compound 2)in 50 ml of absolute ethanol, 12 mmol of benzaldehyde (compound 1) or salicylaldehyde (compound 2) were added and the mixture was refluxed for a reliable time; 6 hr for compound 1, 18 for the remaining compounds 1 and 2. after cooling the precipitate was collected, the solution was concentrated and a second part of the product was obtained. the joined fractions were washed with dry ether to remove some unreacted aldehyde and then crystallized by dissolution in dimethylformamide (dmf) and gradual addition of absolute ethanol. compound 1: melting point (151-152 o c), yield (55% of the yellow crystals), rf value (0.64); ir in kbr disk: 3346 cm -1 (o-h stretching vibration of phenol); 1655cm -1 (c=o stretching vibration); 1620cm -1 (c=n stretching vibration of imine). compound 2: melting point (193-195 o c), yield (49% of the orange crystals), rf value (0.86); ir in kbr disk: 3342 cm -1 (o-h stretching vibration of phenol); 3246cm -1 (n-h stretching vibration of sulfonamide); 1617cm -1 (c=n stretching vibration of imine); 1313cm -1 and 1163cm -1 (s=o stretching vibration of sulfonamide) b. pharmacology albino rats weighing (150 ± 10 gm) were supplied by the national center for quality control and drug research. animals were fed commercial chew and had free access to water add libitum, and were divided into four groups (each group consist of 6 rats) as follow: group a: served as control and treated with the vehicle (propylene glycol 50% v/v); group b: treated with indomethacin (reference agent) in a dose of 2mg/kg suspended in propylene glycol (15) ; group c and d: treated with tested compounds 1 and 2 respectively in a dose of 200mg/kg and 100mg/kg respectively as finely homogenized suspension in 50% v/v propylene glycol (initial dose of 200mg/kg was trialed and compounds which exhibited a statistically significant activity at this dose were further tested at doses decreasing by a factor of 2). anti-inflammatory activity the anti-inflammatory activity of the tested compounds was studied using egg-white induced edema model (16) . acute inflammation was induced by a subcutaneous injection of 0.05ml of undiluted egg-white into the planter side of the left hind paw of the rats; 15 minutes after i.p. administration of the drugs or their vehicle. the paw thickness was measured by vernier at eight time intervals (0, 15, 30, 60, 120, 180, 240 and 300 minutes) after vehicle or drug administration. statistical significance versus control group was evaluated by student’s t-test and p-values less than 0.05 were considered significant. results and discussion compounds 1 and 2 were screened for anti-inflammatory activity and their results together with indomethacin and control groups are summarized in table(1). compound 2 exhibited significant inhibition of the egg-whiteinduced rat paw edema at the i.p. dose of 100mg/kg; which may resulted mainly from the nature of sulfonamide constituents on the aromatic ring. in conclusion, the previously observed strong anti-inflammatory activity of schiff bases has been now confirmed in compound 2. this activity may be attributed mainly to the incorporation of sulfonamide group substituent to the aromatic ring with only secondary contribution from the azomethine double bond. however, this issue deserves further investigations and further recommendations are warranted to demonstrate their selectivity towards cox-2 isoenzyme , as shown in fig(1) . iraqi j.pharm.sci., vol.16 (2) ,2007 synthesis of anti-inflammatory aromatic schiff bases 8 table (1): the anti-inflammatory activity of the indomethacin and tested compounds. time (min.) control indomethacin compound 1 compound 2 0 4.46 ± 0.05 4.40 ± 0.17 4.45 ± 0.50 4.43 ± 0.15 15 5.41 ± 0.18 5.41 ± 0.1 5.40 ± 0.26 5.43 ± 0.10 30 6.05 ± 0.16 6.06 ± 0.13 6.07 ± 0.10 5.82 ± 0.07 60 6.35 ± 0.07 6.20 ± 0.14 6.30 ± 0.15 6.05 ± 0.09 120 6.5 ± 0.09 5.75 ± 0.10 6.29 ± 0.05 5.73 ± 0.12 180 5.93 ± 0.11 5.40 ± 0.10 5.75 ± 0.20 5.39 ± 0.07 240 5.38 ± 0.09 5.11 ± 0.04 5.24 ± 0.45 5.13 ± 0.05 300 5.2 ± 0.1 5.01 ± 0.01 5.13 ± 0.13 5.05 ± 0.04 data are expressed as means ± sem. n = 6. anova test: the data illustrated in table (2), shows that there are highly significant differences between the action of prepared drugs and between indomethacin and control, also the time intervals shows highly significant between each its zones. table (2): anova test source of variation ss df ms f calc. f tab. 0.01 f tab.0.05 rows 9.98395 7 1.426278571 73.487947 (*) 3.6395896 2.4875777 columns 0.424025 3 0.141341667 7.282524689 (**) 4.8740462 3.072467 error 0.407575 21 0.019408333 total 10.81555 31 (*) highly significant differences between time intervals with the probability of ≥ 0.01 type 1 error. (**) highly significant differences between drugs action with the probability of ≥ 0.01 type 1 error. acknowledgment greater thanks for every bit of help supported by every one help in presenting this work especially those in the department of pharmaceutical chemistry and the department of pharmacology – college of pharmacy – university of baghdad. iam also grateful dr. kawkab y. saour (ph.d.) and dr. monther faisal (ph.d.) for their valuable helps. iraqi j.pharm.sci., vol.16 (2) ,2007 synthesis of anti-inflammatory aromatic schiff bases 9 ir spectrum of compound 1 in kbr disk. h c n c o ho ir spectrum of p-aminobenzene sulfonamide in kbr disk. h2n s nh2 o o iraqi j.pharm.sci., vol.16 (2) ,2007 synthesis of anti-inflammatory aromatic schiff bases 21 fig. (1): paw thickness of rats treated with indomethacin, compound 1 and compound 2 with respect to control. results are expressed as means ± sem (n=6). ir spectrum of compound 2 in kbr disk. h c n oh s nh2 o o iraqi j.pharm.sci., vol.16 (2) ,2007 iop-lowering effect of silibinin 22 refrences 1. harvey, r.a. and champe, p.c.: lippincott's illustrated reviews: pharm,acology (3 rd ed.). lippincott williams and wilkins, philadelphia, 2006; 495. 2. katzung, b.g.: basic and clinical pharmacology (9 th ed.). mc grawhill, new york, 2004; 298. 3. dubois, r.n.; abramson, s.b.; grofford, l.; et al.: cyclooxygenase in biology and disease. faseb j. 1998; 12: 1063-1073. 4. simon, l.s.: biologic effects of nonsteroidal anti-inflammatory drugs. curr. opin. pheumatol. 1997; 9: 178182. 5. lipsky, p.e.; abramson, s.b.; grofford, l.; dubois, r.n. and vande puttle l.b.a.: the classification of cyclooxygenase inhibitors. j. rheumatol. 1998; 25: 2298-2302. 6. karia, f. d.;parsania, p. h. asian j. chem. 1999, 11 (3), 991-995. 7. more, p. g.;bhalvankar, r . b.;pattar, s. c. j. indian chem soc. 2001, 78 (9), 474-475 . 8. singh, w. m.; dash, b. c. pesticides 1988, 22(11), 33-37. 9. pathak, p.; jolly, v. s.; sharma,k. p. oriental. j. chem. 2000, 16(1), 161-162. 10. samadhiya, s.; halve, a. orient. j .chem. 2001, 17 (1), 119-122. 11. aydogan, f.; öcal, n.; turgut, z.; and yolacan, c. bull. korean chem. soc. 2001, 22, 476-480 . 12. taggi, a. e.; hafez, a. m.; wack, h.; young, b.; ferraris, d.; lectka, t. j. am. chem. soc. 2002, 124, 66266635. 13. sparatore, f.; pirisino, g.; alamanni, m.c.; monca-dimich, p. and satta, m.; boll. chim. farm. 1978; 117: 638; chem. abstr. 1979; 91: 13760 b. 14. cardile, v., panico, a.m.; geronikaki, a.; gentile, b. and ronsisvalle, g.: farmaco. 2002; 57: 1009. 15. harada, k.: in: the chemistry of the carbon-nitrogen double bond, patai, s. 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https://doi.org/10.31351/vol28iss2pp58-64 58 effects of hydrochlorothiazide on tenofovir disoproxil fumarate-induced nephrotoxicity in rats iman g.al-rakhat*,1 and nada n.al-shawi** * ministry of health and environment, technical department in babylon health directorate, babylon,iraq. **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract tenofovir disoproxil fumarate, a nucleotide reverse transcriptase inhibitor utilized for the treatment of hepatitis b virus and human immunodeficiency virus infections; and is now one of the most widely used antiretroviral drug. however, tenofovir disoproxil fumarate can induce nephrotoxicity, which may be attributed to the interaction between such drug and the organic anion transporters (hoat1, and oat3) with consequent changes in levels of some parameters that may have a role in nephrotoxicity. thiazide diuretics have high to intermediate potency of inhibition of oat1s and oat3; thus, it may possess nephroprotective effects. this study was designed to investigate whether hydrochlorthiazide has nephroprotective effects on tenofovir disoproxil fumarate-induced nephrotoxicity in rats. twenty eight healthy adult male albino rats weighing 180-200g were utilized in this study for duration of 5weeks (35 days) treatment. rats were randomly divided into four groups (7animals each). group i: negative control (orally given distilled water) by gavage tube; group ii: rats orally received 600 mg/kg/day tenofovir disoproxil fumarate by gavage tube; group iii: rats orally administered hydrochlorothiazide alone at a dose (10 mg/kg/day) by gavage tube, and group iv: rats orally administered hydrochlorothiazide at a dose (10 mg/kg/day) plus tenofovir disoproxil fumarate 600 mg/kg/day by gavage tube. on day 36 of the study, after euthanization of each animal by diethyl ether, 3-5ml of blood samples were collected from each rat by an intra-cardiac puncture, then centrifuged at 3000 rpm for 15 minutes to obtain serum, which was then transferred into suitable plain tubes and preserved at -20 °c; and it was utilized for the estimation of cystatin c and il-10 level. rats administered tenofovir disoproxil fumarate for 5 weeks (group ii) produced a significant elevation (p<0.05) in serum cystatin c level and – reduction in serum il-10 levels compared to negative control group (group i); similarly, administration of hydrochlorothiazide alone to rats (group iii) produced a significant -elevation (p<0.05) in serum cystatin c level and – reduction in serum il-10 levels compared to negative control group (group i) ; also, rats administered combination of hydrochlorothiazide plus tenofovir disoproxil fumarate to rats for 5 weeks (group iv) produced significant elevation (p<0.05) in serum level of cystatin c, and a significant reduction (p<0.05) in il-10 serum level in treated rats compared to the corresponding levels of negative control animals (group i); beside that in (group iv) rats there were significant reduction (p<0.05) in serum level of both cystatin c, and il-10 in treated rats compared to the corresponding levels compared to tdf-treated (group ii). in conclusion, treatment with hydrochlorthiazide plus tenofovir disoproxil fumarate in an attempt to prevent nephrotoxicity induced by tenofovir disoproxil fumarate is not attained. key words: nephrotoxicity, tenofovir, hydrochlorothiazide, cystatin c, il-10. ات الهيدروكلورثايزايد على السمية الكلوية المستحثة بواسطة التينوفوفيرتأثير في ذكور الجرذان ثنائي البروكسيل فيوماريت **و ندى ناجي الشاوي 1*، يد حم ايمان غانم .العراق بابل ،مديرية صحة بابل ، ،اإلدارة الفنية والبيئة ، وزارة الصحة * فرع االدوية والسموم ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . ** الخالصة فيروس وعدوى b كبدال التهاب فيروس لعالج العكسي و يستخدم النوكليوتيدات إلنزيم يعد التينوفوفير ثنائي البروكسيل فيوماريت مثبطا سببي أن يمكن ، ذلك ومع واسع. نطاق على المستخدمة العكسي النسخ لفيروسات المضادة األدوية أكثر من واحد اآلن البشرية وهو المناعة نقص األنيون يوماريت وناقلالتينوفوفير ثنائي البروكسيل ف بين التفاعل إلى تعزى أن يمكن والتي ، الكلوية السمية التينوفوفير ثنائي البروكسيل فيوماريت مدرات. ةالكلوي السمية في دور لها يكون قد التي المعايير بعض مستويات في تغييرات من ذلك على يترتب ما مع( hoat1 ، oat3) العضوي تأثيرات واقية للكلى. لها يكون قد ، وبالتالي ؛ oat3 و oat1s تثبيط من متوسطة الى عالية قدرة لديها الثيازيدية البول 1corresponding author e-mail:imangh222@gmail.com received: 12/ 3 /2019 accepted: 7/ 5 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp58-64 iraqi j pharm sci, vol.28(2) 2019 hydrochlorothiazide on tenofovir -induced nephrotoxicity in rats 59 الهيدروكلورثايزايد على السةةةمية الكلوية المسةةةتحثة بواسةةةطة التينوفوفير ثنائي البروكسةةةيل فيوماريت. تم اتتأثير السةةةتكشةةةافهذه الدراسةةةة تهدف غم و تم تقسةةةيمها عشةةةوائيا الى أربع م موعات في كل م موعة سةةةبعة 811-021جرذا بالغا من الذكور السةةةليمة وزنها يتراوي بين 82اسةةةتخدام الم موعةالتزقيم يوميا .أنبوب فمويا عبرالسةةيطرة السةةلبية )المام المقطر( –الم موعة األولى يوما(. 55) اسةةابيع 5جرذان حيث تم معاملتها لمدة 01عة )ب رلوحده هيدروكلوروثيازيد أعطيت -الم موعة الثالثة . التزقيمأنبوب عبرمن تينوفوفير يوميا م/ كغ مملغ 011فمويا اعطيةت –الثةانيةة / يوم( باإلضةةةةةةةافة إلى تينوفوفير م/ كغ مملغ01فمويا هيدروكلوروثيازيد ب رعة ) أعطيةت-الم موعةة الرابعةة .التزقيمأنبوب عبر/ يوم( م/ كغمملغ .التزقيم عبر انبوبملغم / كغم 011 في ثقب داخل القلب وجمعها عينات الدم عن طريقأخذت ة ثنائي األثير إيثيل، وطالرحيم للحيوانات بواسةة من الدراسةة، تم اجرام القتل 50في اليوم .-10و االنترليوكين c لقياس مستوى السيستاتين للحصول على المصل ة )غير محتوية على مواد ضد التخثر(أنابيب عادي c السةةيسةةتاتين مسةةتوى في حادا( p <0.05) معنويا ارتفاعاالى ال رذان )الم موعة الثانية( ثنائي البروكسةةيل فيوماريتالتينوفوفير اعطام نتج من ، كما (األولى الم موعة) السالبة السيطرة بم موعة مقارنة الدم في مصةل il-10 مسةتويات حادا في ( p <0.05 (انخفاضةا معنويا -و المصةل في المصل في c السيستاتينمسةتوى في حادا( p <0.05) معنويا ارتفاعا (الثالثة الم موعة) لل رذان لوحده نتج ايضةا من اعطام الهيدروكلوروثيازيد م موعة في . ايضا(األولى الم موعة) السالبة السيطرة بم موعة مقارنة الدم في مصل il-10 مستويات حادا في ( p <0.05 (وانخفاضةا معنويا (الرابعة الم موعة) أسةةةابيع 5 لمدة التينوفوفير ثنائي البروكسةةةيل فيوماريت إلى باإلضةةةافة هيدروكلوروثيازيد من اعطاؤها توليفة تم التي ال رذان في الدم مصل في il-10 مستوى في( p <0.05) كبير وانخفاض ، cystatin c مصل مستوى في( p <0.05) كبير ارتفاع إلى أدت هذه التوليفة الى جانب ذلك، في مصةةل جرذان الم موعة الرابعة .(األولى الم موعة) السةةلبية السةةيطرة لحيوانات المقابلة بالمسةةتويات مقارنة المعال ة ال رذان الم موعة الثانية التي لحيوانات المقابلة بالمسةةةةةتوياتمقارنة il-10و cystatin c( لكل من مسةةةةةتويات p <0.05)كان هناك انخفاضةةةةةا معنويا .التينوفوفير ثنائي البروكسيل فيوماريتاعطيت لمسةةتحثة بواسةةطة في محاولة لتقليل السةةمية الكلوية ا ثنائي البروكسةةيل فيوماريتيمكن االسةةتنتاج بان المعال ة بالهيدروكلورثيازايد مع التينوفوفير لم تؤد الى تقليل السمية على مستوى العوامل المقاسة في هذه الدراسة. ثنائي البروكسيل فيوماريتالتينوفوفير . 11 – االنترليوكين، c السيستاتين ،الهيدروكلورثيازايد ،التينوفوفير ،حية: السمية الكلويةالكلمات المفتا introduction the kidney is an essential organ required by the body to perform several important functions including the maintenance of homeostasis, regulation of the extracellular environment, such as detoxification, and excretion of toxic metabolites and drugs (1). many drugs and their metabolites can be excreted by the kidney either by glomerular filtration, by tubular secretion, or in some cases by both (2). nephrotoxicity may have a wide shade, reflecting to various nephron segments based upon mechanisms of individual drug and heavy metals; moreover, both glomeruli and tubules has been recognized as targets for drug toxicity and may result in acute or chronic functional changes (3 ,4). drugs may exert their nephrotoxic toxic effects by one or more common pathogenic mechanisms. drugs-induced nephrotoxicity tend to be more common among certain patients and in specific clinical situations. it has been reported that, successful prevention require knowledge of pathogenic mechanisms of renal injury, patientrelated risk factors, drug-related risk factors, and pre-emptive measures, coupled with vigilance and early intervention (5, 6). tenofovir disoproxil fumarate (tdf) is a bioavailable prodrug of tenofovir, which is a potent nucleotide analog reverse transcriptase inhibitor with activity against human immunodeficiency virus (hiv) and hbv (7). such drug is considered as an attractive antiviral agent; where, the international guidelines recommend tenofovir as first-line antiretroviral therapy regimen, and the majority of single-tablet antiretroviral therapy regimens include tenofovir (8); however, nephrotoxicity is a challenging issue regarding the use of such prodrug in the clinical practice. tenofovir disoproxil fumarate (tdf) is eliminated by the kidney, largely via glomerular filtration, with 20% to 30% being actively transported into the renal proximal tubule cells (9). authors reported that, tdf-associated nephrotoxicity may primarily result in proximal tubular injury; where, severe acute tubular necrosis was seen in 33 (77%) of 43 biopsy-proven cases of tdf nephrotoxicity (10). tenofovir's nephrotoxicity is unclear but it may be attributed to the interaction between such drug and the organic anion transporters (hoat1, and to a lesser extent, oat3), which are the major transporters in the basolateral membrane of kidney proximal tubules (7). it has been shown that after oral administration, tdf can be metabolized to tenofovir (tfv), which in turn, can intracellularly be phosphorylated to the active moiety, tenofovir diphosphate (tfv-dp). however, higher circulating plasma levels of tfv have been associated with both renal and bone adverse effects of the prodrug (tfd) (11, 12). yang, y. et al ( 2016) have been reported numbers of agents that including some clinical drugs may possess renoprotective effects in acute kidney injury (aki) models (13). thiazide diuretics have high to intermediate potency of inhibition of organic anion transporters, oat1s and oat3 (14). thiazides are sulfonamiderelated organic acids that are secreted into the proximal tubule by an organic secretory mechanism; they act to increase the excretion of na+ and clby inhibiting the na+/clsymporter in the distal convoluted tubule. natriuresis may be accompanied by some loss of potassium and bicarbonate. moreover, thiazides can enhance ca+2 reabsorption in the distal convoluted tubule by increasing na+/ca+2 exchanges (which makes thiazides useful in treating the calcium-subtype of kidney stones). furthermore, authors reported that thiazide diuretics can also reduce the urinary excretion of ca+2 and iraqi j pharm sci, vol.28(2) 2019 hydrochlorothiazide on tenofovir -induced nephrotoxicity in rats 60 therefore can be employed in the treatment of kidney stones and may also be useful for treating osteoporosis (15).the aim of this study is to investigate whether hydrochlorthiazide has nephroprotective effects on tenofovir disoproxil fumarate-induced nephrotoxicity in rats. methods drugs tenofovir disoproxil fumarate (tdf) tablet (300 mg) was purchased from cipla, india. hydrochlorthiazide tablet (25 mg) was purchased from t and d pharma gmbh, germany. animals twenty eight healthy adult male albino rats weighing 180-200g were utilized in this study; they were obtained from and maintained in the animal house of the college of pharmacy, baghdad university, under conditions of controlled temperature. animals were fed commercial pellets and tap water ad libitum throughout the experiment period. the study was approved by the scientific and the ethical committees of the college of pharmacy/university of baghdad. experimental protocol healthy rats were randomly divided into four groups (7 animals/ group) as follows: group irats orally administered distilled water by gavage tube for 5 weeks. this group served as negative control. group iirats orally administered 600 mg/kg/day of tenofovir disoproxil fumarate (tdf) by gavage tube for 5 weeks (16). group iiirats orally administered hydrochlorothiazide alone at a dose of 10 mg/kg/day by gavage tube for 5 weeks (17). group ivrats orally administered hydrochlorothiazide at a dose of 10 mg/kg/day plus tenofovir disoproxil fumarate 600 mg/kg/day by gavage tube for 5 weeks. preparation of serum samples twenty-four hour after the end of the treatment duration (i.e. at day 36), each animal was euthanized by diethyl ether. blood samples were collected (3-5 ml from each rat) by an intra-cardiac puncture, then centrifuged at 3000 rpm for 15 minutes to separate serum, which was then transferred into suitable plane tubes and preserved at -20 °c. the serum of each rat was used for the estimation of cystatin c and il-10 level. statistical analysis data were expressed as mean±standard error of the mean (sem). the statistical significance of the differences among various groups was determined by one-way analysis of variance (anova). differences were considered statistically significant for p<0.05. results table 1 and figure 1 summarize the effect of different treatments on cystatin c level in serum of rats' groups. cystatin c was significantly elevated (p<0.05) in serum of rats that were orally administered tdf for 5 weeks (group ii) compared to negative control group (group i); where, the mean ± sem values were 0.453± 0.017 ng/ml, and 0.190± 0.02 ng/ml, respectively. furthermore, there was a significant elevation (p<0.05) in cystatin c level in serum of rats in hydrochlorthiazide-treated group (group iii) compared to negative control group (group i), the mean±sem value was (0.245± 0.015 ng/ml)]; moreover, in group of rats treated with hydrochlorthiazide plus tdf (group iv), mean± sem serum cystatin c levels was significantly elevated compared to the corresponded serum level in negative control rats (p<0.05); where, the mean±sem values were (0.392± 0.01 ng/ml) compared with negative control group (0. 190± 0.02ng/ml). moreover, table 1 and figure 1 showed that there were significant elevations (p<0.05) in serum cystatin c level among rats in groups [ii (orally administered tdf (600mg/kg), iii (orally administered hydrochlorthiazide (10mg/kg), and iv (administered hydrochlorthiazide (10 mg/kg) plus tdf (600 mg/kg)]. in addition rats treated with hydrochlorthiazide plus tdf (group iv), the mean± sem serum cystatin c levels was significantly reduced compared to the corresponded serum level in tdf-treated rats (group ii) (p<0.05); where, the mean±sem values were (0.392± 0.01 ng/ml) compared with tdf-treated group (0.453± 0.17ng/ml). table 1. effect of different treatments on cystatin c level in serum of rats' groups. group / treatment mean serum cystatin c level (ng/ml) group i/ negative control (distilled water) 0. 190± 0.02 group ii/ tdf (600 mg/kg) 0.453± 0.017* a groupiii/ hydrochlorthiazide (10 mg/kg) 0.245± 0.015 * b groupiv/ hydrochlorthiazide (10 mg/kg) plus tdf (600 mg/kg) 0.392± 0.01 * c data expressed as mean± standard error of mean (sem). *: p<0.05: significant difference compared to negative control group. values with non-identical capital letters (a, b, and c) are considered significantly different (p<0.05). tdf, tenofovir disoproxil fumarate. iraqi j pharm sci, vol.28(2) 2019 hydrochlorothiazide on tenofovir -induced nephrotoxicity in rats 61 figure 1. effect of different treatments on cystatin c level in serum of rats' group. (a):-indicate a significant difference (p<0.05) compared to negative control group. (b):indicate a significant difference (p<0.05) compared to tenofovir disoproxil fumarate-treated group. table 2 and figure 2 summarize the effect of different treatments on serum interleukin-10 (il-10) level of rats' groups. there was a significant reduction (p<0.05) in interleukin-10 level in serum of -tdf-treated group (group ii) [the mean±sem value was (20.341± 0.45pg/ml)], hydrochlorothiazide-treated group (group iii) [mean±sem value was (16.99± 0.412 pg/ml)], and -in group of rats administered hydrochlorothiazide plus tdf (group iv) [mean±sem value was (13.655±0.512 pg/ml)] compared to the corresponding levels in negative control group (group i) [mean±sem value was (28.846± 0.56 pg/ml)]. additionally rats treated with hydrochlorthiazide plus tdf (group iv), the mean± sem serum interleukin-10 levels was significantly reduced compared to the corresponded serum level in tdftreated rats (group ii) (p<0.05); where, the mean±sem values were (13.655±0.512 pg/ml) compared with tdf-treated group (20.341± 0.45 pg/ml). furthermore, table 2 and figure 2 showed that there was a significant reduction (p<0.05) of interleukin10 level in serum among rats of group ii, iii, and iv. table 2. effect of different treatments on interleukin-10 level in serum of rats' groups. group / treatment mean serum il10 level group i/ negative control (distilled water) 28.846± 0.56 group ii/ tdf (600 mg/kg) 20.341± 0.45 *a group iii/ hydrochlorthiazide (10 mg/kg) 16.991± 0.412* a group iv/ hydrochlorthiazide (10 mg/kg) plus tdf (600 mg/kg) 13.655±0.512* b data expressed as mean± standard error of mean (sem). *: p<0.05: significant difference compared to negative control group. values with non-identical capital letters (a and b) are considered significantly different (p<0.05). tdf, tenofovir disoproxil fumarate. figure 2. effect of different treatments on interleukin-10 level in serum of rats' groups (b):-indicate a significant difference (p<0.05) compared to negative control group. (c):indicate a significant difference (p<0.05) compared to tenofovir disoproxil fumarate-treated group. discussion the widespread introduction of highly active antiretroviral therapy (haart) in the mid-1990s dramatically altered the course of human immunodeficiency virus (hiv) infection, with improvements in survival and reductions in the incidence of aids-defining illnesses. although, antiretroviral therapy has been shown to reduce the incidence of both aids-defining and non-aids conditions, long-term exposure to haart may also be associated with significant toxicity (18). tenofovir disoproxil fumarate (tdf) is an orally bioavailable pro-drug of tfv (19). the renal proximal tubule (pt) is the main target of tfv toxicity (20). animal studies have revealed that tfv can cause proximal tubular (pt) damage in mice (21), rats (22, 23), and non-human primates (24); furthermore, numerous case reports and case series illustrated that iraqi j pharm sci, vol.28(2) 2019 hydrochlorothiazide on tenofovir -induced nephrotoxicity in rats 62 fanconi syndrome (fs) or acute kidney injury (aki) in hiv-infected patients was produced by tfv (25, 26). moreover, most studies considered creatinine clearance (crcl) as a marker of renal function for the assessment of tdf-induced nephrotoxicity. however, creatinine clearance (crcl) was reported to be a weak indicator for evaluation of kidney function for tdf-induced nephrotoxicity (27); in addition to that, creatinine is derived from skeletal muscle and hiv-infected patients can have abnormal muscle mass, these are important considerations when interpreting studies (28). horberg m. et al (2010) showed that tdfexposed patients had greater development of proximal tubular dysfunction over time, reduced gfr, and had greater risk of medication discontinuation, especially as renal function worsened and serum creatinine were also reported to be significantly elevated among tdf-exposed patients compared with tdf -sparing patients (29). thus, in the current study, serum level of cystatin c as a marker for tubular damage was measured instead of serum creatinine. the results of this study showed that there was significant elevation (p<0.05) in serum level of cystatin c in rats orally administered tdf for 5 weeks (group ii) compared to the corresponding levels in negative control (group i) and this coincide with that founded by horberg m. et al (2010) from the point of the effect. cystatin c is a non-glicolized protein with small molecular weight (13.3 kda), a hundred times bigger than creatinine. it is produced at a constant rate by all the nucleated cells, and is freely filtered by glomeruli and minimally linked to proteins, and is not reabsorbed in the systemic circulation after the filtering (30). furthermore, it has shown promise as a replacement for serum creatinine in estimation of glomerular filtration rate (gfr). it has been reported that after glomerular filtration, cystatin c is fully catabolized in the proximal renal tubule and is not returned to blood. moreover, the concentration of serum cystatin c is not affected by gender, age, race, protein intake, and muscle mass, unlike serum creatinine. when gfr reduced, cystatin c level begin to rise proportionately (31). moreover, the results of the present study revealed that there were significant elevations (p<0.05) in cystatin c level in serum of rats group administered hydrochlorthiazide (10mg/kg) alone (group iii) compared to negative control rats (group i) or hydrochlorthiazide (10 mg/kg) plus tdf (600 mg/kg) (group iv) compared to the corresponding levels in -tdf-treated rats (group ii) and -negative control (group i). thus, results of this study concerning the effects of hydrochlorthiazide on kidney are coincide with that of wadei h.m, et al at 2008 who reported that low kidney function and glomerular and tubular injury can be more commonly manifested in thiazide-treated rats (32). furthermore, in the current study, it was found that there was statistically a significant (p<0.05) reduction in serum il-10 level of tdftreated rats (group ii) compared to the corresponding level in negative controls (group i). moreover, treatment of rats with hydrochlorthiazide (10 mg/kg/day) alone (group iii), and with hydrochlorthiazide (10 mg/kg/ day) plus tdf (600 mg/kg) (group iv) produced significant (p<0.05) reduction in serum il-10 level compared to tdftreated rats (group ii) and negative control (group i), respectively. interleukin-10 (il-10) is an antiinflammatory cytokine produced by a number of activated immune cells like monocytes/macrophages, and t helper-1 (th1) cells (33). moreover, it has been reported that such cytokine is a potent inhibitor of inflammation and immune responses to infections and antigens (34, 35). furthermore, pretreatment of human peripheral blood mononuclear cells (pbmcs) with tdf caused a reduction in levels of il-10, and strongly reduced the induction of il-10 (36). moreover farkhondeh n and ali d. a. (2012) showed that, clinically relevant concentration of hydrochlorthiazide could elevate the secretion of the proinflammatory cytokine il-1β by the peripheral blood mononuclear cells (pbmcs), and might result in aggravation of inflammatory processes in vascular wall and worsen the condition in long-term (37). to our knowledge, the current study is the first that study the effect of hydrochlorthiazide on il10, thus we could not have a chance to compare the results obtained from this study with others concerning this respect. conclusion it could be concluded that hydrochlorthiazide had no nephron-protective effect against tenofovir disoproxil fumarateinduced renal damage. references 1. sun young kim and aree moon. drug-induced nephrotoxicity and its biomarkers. biomol ther 2012; 20(3), 268-272. 2. naidoo s. and meyers a. m. drugs and the kidney. s afr med j 2015; 105(4):322. p2. 3. zheen a. ahmed. relationship between renal function tests and the levels of mda, zinc, and cadmium among 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analgesic activity of newly synthesized phthalyltyrosyl-glycin sodium muthanna s. al-taee* , kawkab y. saour*, haider m. mohammed**1 * departmen t o f pharmaceu tica l chemistry, co llege o f ph armacy , un ive rsity o f bagh dad – iraq. ** depa rtme nt of pharm aco logy and toxicolog y , c olle ge of pharm acy , unive rsity of baghdad –iraq . abstract alteration in the ba ckbone structure of the endogenously re le ase d opioid peptides leu 5/met5 enkephalins may res ult in compounds having comparable profile of pha rma cologic al ac tivity but with different physicoche mic al prope rties and side effects. phtha lyl amino ac id a nd phthalyl es te rs a re a mong the deriva tive s that have been s ynthe size d and e valuated for their a ntiba cteria l and a ntifungal a ctivitie s.this study was c onducted to eva luate the possible ana lgesic activity of phthalyl-tyrosyl-glycin sodium that ha s bee n re cently synthes ized by our team.the s tudy was c arried out on 24 albino mice us ing hot plate method. the anima ls we re alloca te d in to three groups; the firs t group rece ived saline and re prese nt a c ontrol group; the se cond group re ceive d morphine hcl as a s ta nda rd drug; and the third group rece ived phthalyl-tyrosyl-glycin sodium. the onse t with whic h the a nimal lift his fore arm and the number of jumps per 25 se conds were re corde d for eac h group. the results of this study showed that phthalyl-tyros yl-glycin sodium resulted in signific ant improvement (p<0.05) in a nalge sia sc ore a s well as s ignifica nt delay in the onse t of induce d hyperalges ia in comparis on to s aline-treated group, a nd in c ompa rison to morphine hcl, no signific ant differenc e (p>0.05) wa s obs erved in ana lgesia sc ore but with signific ant delay in induc ed hyperalges ia .the re sults obtained in this s tudy provide e xperimental evidences for the effe ctive nes s of the prepa re d compound as a nalge sic with comparable effe ct to that of morphine . k ey words: phthalyl-tyros yl-glyc in s odium, phtha lyl group, analges ia . الخالصة ض م ان بع ى الببتيدات الحياتية ال ميائية عل كي عديالت ال ج )ليوسين ومثيونين انكفالين(الداخلية رفينيةوالت ها قد تنت مركبات ل ربة وائية مقا وية فعاليات د سكنات الم ق ي كم ع اخـتالف ـف ـم ة ة مختلـف وفيزياوـي ة وـي ص كيميا وا ـخ كن ب ض ول األعـراض بـع عن استخدامه ل . االجانبية الناتجة ى الفثالـي مركبات الحاوية عـل ض تعتبر ال رات كحـام ـت ي واس ميـن ـات أ ركب ن الم ل ـم الفثالـي طرية جرثومية والف مة فعاليتها ضد االلتهابات ال ـد وعليه المصنعة والمقي ة لببتي ة الدوائـي ميم هذه الدراسة لتقييم الفعالـي تم تص مسكن كب مر موعة الفثاليل ك كب ان. لأللميحتوي على مج phthalyl-tyros(وهو هذا المر yl glycin s odium ( هو عة بغداد مياء الصيدالنية في كلية الصيدلة جام كي عه من قبل فرع ال 24الدراسـة باسـتخدام لقد تمت.مركب جديد قد تم تصني رة ذكرًا فأرًا ـا ة الصـفيحة الح تخدام طريـق ـس ن لأللم با مسك مركب الجديد ك وائية لل ـم (hot plate)أبيضا لتقييم الفعالية الد وت مالح ألولى بالماء ال موعة ا مجاميع٬ حقنت المج زيع الحيوانات على ثالثة sa)تو line موعة السيطرة والمجموعة ( مثل مج وت عقار رفين ك يالثانية بالمو كب قياس phthalyl-tyros(والثالثة بمر yl glyc in s odium( ـم موضوع البحث وت و والذي ه حب الحيوان غرق ليس زات خالل احتساب الوقت المست وعدد القف عن الصفيحة الحارة هرت النتائج أن هناك .ثانية 25قدمه أظ زات خالل وكذلك في عدد القف ن الصفيحة حب الحيوان قدميه ع غرق ليس ست معنوي في الوقت الم واضح و ن ثانية في 25تحس طرة وعة السي مجم عن كب الجديد عالجة بالمر معد. الحيوانات الم معنوي قد لوحظ في رق وانات المعالجـة ال ف ل قفز الحي مورفين موعة المعالجة بال جيد بالمقارنة مع المج مركب ال رة كان بال حا سحب الحيوان لقدميه عن الصفيحة ال إال أن سرعة مركب .أبطأ عالية الدوائية ل ى الف عل سة أدلة تجريبية را ن هذه الد ج المستخلصة م ي النتائ phthalyl-tyros(تعط ylglyc in( ن مسك كب مورفينكمر عالية مقاربة لل .لأللم قوي وبف 1corresp ond in g author ema il r7 4th @yahoo.com rec eived 24 -7-2006 acce pted 20 -1 2-2 006 47 in troduction opioid peptides , defined as pep tides with opia te -like pha rmacolo gical effe cts , are the oldest pha rma colo gical subs ta nces kno wn fo r th e i r a na lg e s ic , e up h o ri c a n d a dd ic ti v e effe cts(1,2).morp hine is the firs t na rc otic analgesic alka lo id isolated fro m p la nt sourc e in 1806 (3) in 1 975 , hughes and kos terlitz succeeded in isolating two pentap ep tides, leucine and methionine-enkephalin (leu/met enkep ha lin) fro m pig brain, whic h c ompe te strongly with morphine -like drugs fo r bind ing to recep to rs in the brain with p ha rma cological ac tions rese mbling those of mo rp hine itself [2]. howeve r, to date, morp hine -like compounds re main the only class known to ac t b y mimic king thes e pe ptides (4 ). s eve ra l families of pe ptides were discove red with multiple ca te gories o f op io id recep to rs . bind ing s tudies by synd er and colleagues (1 973 ), demo ns trated tha t op io ids are reco gnized by sp ecific receptors (5). various pharmaco logica l observa tions like analgesia, se da tion, a ntitussiv e, antidiarrhea l, pupilary co nstric tion, and bradyca rd ia prod uced b y different drugs implie d the mediation of more than o ne typ e of rec ep to rs . recep to r cloning stud ies revealed the exis te nc e of s ix different opiate re cep tors na med as  (mu),  (de lta ),  (kappa),  (s igma),  (epsilon) and nfq (nociceptin fq)(6) the se re ceptors me diate sev eral pharmaco lo gica l and s ide e ffec ts o f opiates includ ing: a nalgesia (supraspinal, spinal, pe ripheral), res pira tory depress io n , p apillary co nstric tion, reduce d gi mo tility, e up horia , dy sphoria , sedatio n and phy sical d epend ence(3) . conce rning their molecular p ha rma cology, thes e opioid receptors be long to the fa mily of inhibitory g-protein c oup led re ceptor, med ia te the re ductio n of intra ce llular camp leve l as a main ev ent beyond recep to r bind ing (7). other mec hanisms invo lve the o pening of k + and bloc k of ca2+-c hanne ls the reby red ucing both, ne urona l e xcitability a nd tra ns mitter release (8). understa nding the p owe rful mo le cula r, biologic al a nd physio logical te rms o f opioids was used to dev elop a nalges ic c ompo unds with significant advanta ge s o ve r morphine (9). in this respect, inco rporation of a ne w gro up in the ma in ba ckbone of naturally oc curring enkep ha lin was a imed in s ynthesizing co mpounds with po ss ib le ana lges ic effe cts. on the other hand, one o f the earliest typ es of structural modific atio ns applied to the enkep ha lin was sho rtening of the peptid e chain by re moval of residues from the ess ential se quence or by remo val of residues fro m cte rminal. these expe riments showed that significant potency re maine d in prod uc ing ana lges ia in v iv o as in tyr-d-ala-phe-met amide ( 10,11). in pre vious wo rk, mutha nna , et a l. (20 05) were suc ce ed in d esigning and s ynthesis of enkep halin a nalogue s having e ven shorte st cha in by re mov ing the c-te rmina l res id ue glyphe -leu/met; ke ep ing n-tyros ine re sidue protecte d b y phthalyl gro up. acco rd ingly, the newly s ynthe size d phthalyl-tyros yl-glycin and its sod ium s alt (fig. 1 and 2) a re no vel comp ounds p ro duc ed in our la boratories with pos sible ana lges ic ac tiv ity [12]. the prese nt stud y was conduc ted to inve stigate the ana lges ic effec t of the sod ium sa lt of this comp ound on e xp erime ntal animals . figure (1 ) phtha ly l-ty rosyl-glyc in (12) figure (2 ) phtha lyl – tyrosyl – g ly cin so dium sa lt material a nd methods animals: twenty-four adult male albino mic e weighing 2 5.23 ± 3.1 gm we re used in this stud y. they w ere obtaine d from ira qi s era and vac cine institute and were housed under standard co nditio ns in the animal hous e of the colle ge of pha rma cy-univ ersity o f baghda d. animals were fe d commercial p elle t and ta p water in fre e ac ce ss ad libitum. mate rials: mo rp hine hcl was s upp lied by may and baker ltd , england. p htha lyl-tyro sylglycin. has b ee n o btaine d from re fe re nce (13) and its c orre spo nd ing s od ium s alt has b een synthes ized ac cording to re fere nc e (14) to increas e its wa ter s olubility to b e suitab le for intrap eritonea l ad minis tration. all co mpounds n o o ch n h o h2 c oh oh o n o o ch n h o h2c oh o o na 48 were disso lv ed in normal sa line a nd were ad minis te re d intra perito nially. method s: ho t plate metho d a s de scribed b y woolfe a nd ma cdo nald (13) was us ed for ev aluation of the analges ic effe ct o f the tes te d co mpound comp ared with morp hine as a re fe re nc e. anima ls we re a lloc ated into thre e groups: firs t group re ce iv ed normal s aline, se cond group rece iv ed morphine hcl (ma y and ba ke r ltd , engla nd), and third group re ce iv ed phthalyl-n-tyros yl-glycin so dium. the p la te was he ated to 55 oc and the a nimal was p ut o n the plate . the ons et with which the anima l lift his forea rm and the number of jumps p er 2 5 se conds we re rec orded for e ac h group. the re sults were expres se d a s mea n ± standard e rror and were a na lyzed us ing anova and unpaired stud ent t-tes t. results and dis cuss ion the d ata pres ented in tab le (1) clea rly showe d that anima ls in c ontrol group lift the ir fo rea rms in abo ut (1.2 ± 0.66 ) s eco nds which rep re sents the no rma l onse t of he at-ind uce d hyperalge sia. the animal jumps 2 4.8 ± 2.4 time s/25 s eco nd s which rep re se nts the a nalge sia s core. tab le (1) also s ho wed tha t mo rp hine s ignifica ntly (p <0.05 ) delayed the ons et o f hea t-induce d hyperalge sia (2 .6 ± 0.74) in comp aris on to co ntro l group and significa ntly (p<0.05) impro ve d the a na lges ia s core (1 1.0 ± 2 .4) in co mparison to c ontrol gro up (24.8 ± 2.4) (fig. 3 and 4 ). inte re stingly, phtha lyl-tyros yl-glycin so dium re sulted in s ignifica nt (p<0.05) impro ve ment in ana lges ia s core (10.8 ± 1.43 ), an e ffec t s eems comparab le to tha t of morphine (1 1.0 ± 2.4, p>0 .0 5). howev er, p htha lyltyro syl-glyc in s odium showed slightly more significant de la y in the o ns et of hea t-induce d hyperalge sia ov er tha t of morphine (f ig. 3 and 4). the pha rma cologic re sults may ind ic ate that tyro sine is the ess ential a mino acid re sidue within the pe ptid e chain of leu / met enkep ha lin (15) ; and the enzymatic res is tanc e is doubtless an important fac to r in the high pote ncy of this a nalge sic [16] b ec aus e, chemica lly, the pres enc e of phtha lyl gro up ma y enhance the a va ilab ility a nd he nc e the bind ing of the compound to opioid rec eptors a nd this in turn may po te ntiate the pharmaco lo gica l effect of the te sted c ompo und , this may e xp la in why the co mpo und s ho wed a c ompa ra ble analge sic effe ct to that o f morphine . furthe rmo re , the bulky phthalyl gro up ma y increa se the lipo philic ity and thus e nha nc e a suffic ient bioa vailability and tis sue penetra tion, a n effect which ma y e xplain why the co mpound ha s slightly fa ster o nse t of a ction than mo rphine. on the o ther ha nd, the pres ence of bulky phtha lyl gro up is sus pe cted to re duc e enzyme degra da tion of p hthalyl-tyro syl-glyc in ins ide the b od y b y a dding a sterric hind ra nce [17]; howe ve r, this e xpe ctation req uire s further pha rma co kinetic stud ie s. in conclus io n, phtha lyl-tyros yl-glycin sod ium showed promising ana lges ic activity app ro ximately eq ual to tha t of mo rp hine with re liab le onse t of ac tion, making it a goo d candida te for furthe r dev elopment in the field of mo rp hine -repla ceme nt therapy b as ed on the id ea tha t enke pha lins are co mpound s dev oid of add ic tive liab ility. table (1) effec t of phtha ly l-tyro sy l-glyc in o dium and mo rph ine on the a nalge sia score and he a t-induce d hype ra lges ia in mice groups jumps/25 seconds onset of hype ralgesia (se c.) control group (n=6) 24 .8 ± 2 .4 a 1.2 ± 0.66 a morphinetrea te d group (n=6) 11 .0 ± 2 .4 b 2.6 ± 0.74 b phtha lyltyro syl-glyc in s odium-tre ated group (n=6) 1 0.8 ± 1.43 b 4.2 ± 1.04 c fig ure (3) :the effect o f phtha ly l-tyro sy l g ly cin s odiu m an d morphine o n a nalge sia sco re in mice re co rde d 25 second.re sults are e xpresse d as the me an (jumps) ± sem; n=8 ; non ide ntica l supe rscripts (a , b) re prese nt s ig nific ant diffe re nce s among g ro ups by anova (p<0.0 5) value s are pre se nte d as me an ± sem.value s with non-ide ntica l s upe rs cripts (a , b, c ) amo ng diffe re nt groups are cons ide re d significa ntly diffe re nt (p<0 .0 5). 0 5 10 15 20 25 30 35 control morphine phthalyl tyrosyl glycin sodium a n a lg e s ia s c o re ( j u m b s /2 5 s e c ) a b c 49 refere nces 1. wille tte , r.e.: ana lges ic a ge nts. in: wilso n and gis vo ld ’s te xtbook of organic med ic inal and pharmace utical chemistry (1 2th ed.), delgado, j.n. a nd reme rs , w.a. (eds .), j.b. lippincott, willia ms and wilkins , p hila delphia, 2004, p. 6 87-709. 2. rang, h.p.; dale, m.m. and ritte r, j .m. (eds): analgesic drugs . in: p harmac ology (5 th ed .), churchill livingstone, lond on, 20 03, pp. 589-595 . 3. hardma n, j .g; limbird, l.e. a nd molinoff, p.b. (eds .): opioid analge sics . in: goodma n and gilman’s the p harmacological basis of the rape utic s (10 th ed.), mc graw-hill, new york, 2001, pp. 569 -6 05. 4. cooper, j.r.; bloo m, f.e. a nd roth, r.h.: the biochemica l basis of neurop ha rma colo gy. oxford unive rsity press, new york, 19 96; pp. 121-125 . 5. synder, s .h.; p aste rnak, g.w. and p ert, c.: in: handbook of ps yc hop ha rma cology, inve rsen, l.l.; inve rson, s.d. a nd synder, s.h. (eds.). plenum, ne w york, v ol.5 19 84 ; pp. 329-360 . 6. dhawan, b.n.; cesselin, f.; raghubir, r. et al: classificatio n o f opioid rece ptors. pha rmaco l. rev . 1996 ; 48 : 567-586 . 7. wa y, w.l.; fields , h.l. and s chumac he r, m.a.: op io id a nalge sics a nd anta go nists. in: bas ic a nd c linic al pharmacology (8 th ed.), katzung, b.g. (ed.), mcgraw-hill, new york, 2001, pp. 516 -517. 8. north, r.a: op io id actions on me mbrane ion cha nnels. in: ha ndb ook of e xp erime ntal pha rma co lo gy, herz (ed .). springe rtverla g, berlin, vo l 10 4, 1 99 3; p p. 9 8-101. 9. americ an pain soc iety: p rinc iples of ana lges ic us e in the trea tme nt of a cute pain and ca nce r pain. ame rican pa in s oc ie ty, glenview, i.l., 1 999 , pp .64. 10. s himohigashi, y.; matsuura, s.; che n, h.c.; et al: dimeric enkephalins display enhance d a ffini ty and selec tivity fo rt the opiate receptors. mo l. p harmacol. 1982; 2 1: 558 -563. 11. mo rley, j.s .: chemis try of opioid pe ptides. br. med. bull. 1983 ; 39 : 5-10 . 12. al-taee , m.s.: syn thes is o f ne w op ioid pep tide analogues with e xpected biologic al ac tivity. m.s c. thes is , 2005. univ ersity of baghdad, baghda d. 13. wo olfe , g. and mc dona ld , a.d.: j. pha rma co l. exp . ther. 19 44; 80 : 300-307 . 14. f urmis s, b.s.; hanna fo rd , a.j. and roge rs, v. (ed s.): carbo xylic acid: cha ra cteristic che mical. in: vo gel's te xt bo ok of p ra ctic al organic che mis try (4th ed.). longman, lo ndo n, 197 9; p p. 106 4-1065. 15. s asaki, y.; hirab uki, m.; amb o, a.; e t a l: enkephalin analogues with 2,6dimethylp henylalanin rep la cing phenyla lanine in p ositio n 4. bio org. me d. chem. lett. 200 1; 11(3): 327-32 9. 16. klob , v.m. and scheine r, s .: j . pharma. sci. 1 98 4; 3 3: 3 . 17. michea l, m.: pep tide a nd p ro te in drugs . in: foye’s principle of medical c he mis try (5th e d.). david , a.w. a nd thoma s, l.l. (eds .), lippincotte williams and wilkines, philadelphia, 2002, pp. 120-123 . 0 1 2 3 4 5 6 control morphine phthalyl tyrosyl glycin sodium o n e st o f h yp er al g e si a (s e c ) a b c figure (4) :compariso n o f the e ffe ct of phthalyl-tyrosy l-glyc in so dium and mor phine on the onset of he at-induce d hy pe ralge sia in mice . res ults a re ex presse d as the me an ± sem; n=8; no n-ide ntic al supe rsc ripts (a , b , c ) re pre se nt significant diffe re nce s amo ng groups by anova (p<0 .05) iraqi j pharm sci, vol.20(2) 2011 clinical pharmacist and adverse drug events 85 role of the clinical pharmacist in reducing preventable adverse drug events # bahir a.mshiemish* ,1 * department of pharmacotherapy, college of pharmacy,almustansiriya university,baghdad,iraq. abstract according to so many previous studies, lack of sufficient information during prescribing steps may lead to medication errors. thus, the presence of the clinical pharmacist during routine rounding process in the ward with intervention of patient care plan may reduce the probability of adverse drug events (ades).this study evaluate role of the clinical pharmacists, as a member of medical team with the physician, on ades and report their interventions in the internal medicine unit. this study was designed to compare between two groups of patients, those receiving care from a rounding team (physician, nurse, and clinical pharmacist) (study or intervention group with 51 patient); and those receiving care from a rounding team (physician and nurse, but without any pharmacist) (control group with 49 patient). the primary outcome measure was preventable ades and secondary one involves the time of staying in the hospital and onset of response to therapy. patients were randomly selected, followed a single-blind design, and evaluated by a senior physicians and clinical pharmacists who document their medical interventions.specialist physicians accepted (60) of (77) recommendations (i.e. do modifications in drug therapy depending on clinical pharmacist interventions). the most common intervention was recommending dosage or frequency of medication (32.4%), followed by addition of medication (19.5%).the rate of preventable ordering ades in the study unit was 77% lower than in the control unit (p<0.05). there was no significant difference (p>0.05) in the cost of drug therapy between patient groups. patients with ades in the control group had an average of 1.5 day longer staying period at the hospital; which was not differ significantly (p>0.05) from the study group.in summary, presence of clinical pharmacist during tour as a full member of the patient care team in internal medicine ward was associated with a substantially lowered rate of ades which caused by prescribing errors. types of errors indicate the need for activation of the clinical pharmacist's interventions. key words: adverse drug events (ades), clinical pharmacist. الخالصة وفمب نذساسبث سببمت فبٌ افخمبس انًعهىيبث انذوائُت اثُبء عًهُت انىصف انذوائٍ َسبب انىلىع فٍ اخطبء غبُت, نزنك فبٌ ى فٍ انخمهُم يٍ احخًبنُت ولىع يثم هزِ االخطبء وانًخًثهت حىاخذ انصُذالٍَ انسشَشٌ اثُبء اندىنت انًعخبدة نهفشَك انطبٍ لذ َسبه خبثُشاثانببنخبثُشاث اندبَبُت نهذواء. كبٌ انهذف يٍ هزِ انذساست حمُُى دوس انصُذالٍَ انسشَشٌ فٍ يسبعذة انفشَك انطبٍ نهخمهُم يٍ ًًج هزِ انذساست نًمبسَت دوس انصُذالٍَ انسشَشٌ فٍ سدهبث انببغُُت .ص انذوائُت انًًكٍ حدُبهب وحسدُم هزِ انخذاخالث انصُذالَُت يع اونئك انزٍَ نى َكٍ نهصُذالٍَ انسشَشٌ دوس فٍ يشَط( 15)يدًىعت انخذاخم ,فٍ انخبثُشعهً عالج يشظً سدهبث انببغُُت .كبٌ انًعُبس االسبسٍ فٍ هزِ انذساست هى يعذل حصىل يشَط(94)يدًىعت انسُطشة ,حغُُش ادوَخهى انًىصىفت يٍ لبم انطبُب انذوائُت وانًًكٍ حدُبهب , ايب انًعُبس انثبَىٌ فكبٌ انفخشة انزيُُت نًكىد انًشَط فٍ انشدهت ويذي االسخدببت نهعالج خبثُشاثان ى حسدُم انخذاخالث انذوائُت انخٍ انًىصىف. حى اخخُبس انًشظً عشىائُب ولًُج حبنخهى انًشظُت يٍ لبم اغببء ببغُُت اخخصبص وح % وحى حسدُم 77انذوائُت انًًكٍ حدُبهب بُسبت خبثُشاثاناوصً بهب انصُبدنت انسشَشَىٌ .بُُج انُخبئح اَخفبض فٍ احخًبنُت حصىل %( 9..4) ( يٍ لبم انفشَك انطبٍ وكبٌ اكثش هزِ انخذاخالث يشحبػ بخغُُش اندشع انذوائُت06( حذاخم صُذالٍَ لبم يُهب )77) انفخشة انزيُُت ايب , بٍُ انشدهخٍُنى َكٍ هُبنك فشق يعُىٌ فٍ انكهفت انعالخُت .%( 54.1) واظبفت ادوَت نى حكٍ يىصىفت يسبمب فبعمفٍ ظىء هزِ انُخبئح ًَكُُب حبكُذ انذوس ان .فكبَج اغىل وبًعذل َىو وَصف انببغُُتت فٍ سدهً يدًىعت انسُطشة نًكىد يشظ سشَشٌ فٍ يسبعذة انكبدس انطبٍ الحخبر انمشاساث انًخعهمت ببالدوَت انًىصىفت وانخمهُم يٍ يعبعفبحهب انًسخمبهُت نهصُذالٍَ ان واظشاسهب اندبَبُت. introduction a medication error is a preventable event that can cause inappropriate use of a product and/or harm to a patient. medication errors can happen in hospitals, in pharmacies, and at home (1) . failure to obtain sufficient information about the patient or pharmaceutical agents has contributed to medication errors. we can help prevent medication errors by being a well-informed and active partner in patient health care. while most errors are harmless or are intercepted, some result in adverse drug events (ades) (2) .incident rates of medication errors vary widely, the reason for which can be explained by the different study methods and definitions used. the rate of medication errors varies between 2 to 14% for patients admitted to us hospitals, and the majorities are due to poor prescribing (3) . # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : bahiralkinani@yahoo.com received : 19/3/2011 accepted : 27/9/2011 mailto:bahiralkinani@yahoo.com iraqi j pharm sci, vol.20(2) 2011 clinical pharmacist and adverse drug events 86 medication error has been estimated to kill 7000 patients per annum and accounts for nearly 1 in 20 hospital admissions in the us. the incidence is likely to be similar in the uk. medication errors (7% of all incidents) were the 2 nd most common incident documented in the national audit commission report on patient safety (4) .lazarou et al stated that fatal ades, which were defined as any preventable events that lead to inappropriate medication use related to the health care professionals or patients regardless of outcomes, were the sixth leading cause of death in usa in 1998, with 10.9% of all hospital patients experiencing some adverse drug reactions (5) .adverse drug events are an important cause of morbidity and mortality, they are responsible for considerable number of hospital admissions and significantly increase health care costs. in the 20 th century, great therapeutic advances were accompanied by a growing awareness of the problem of ades to medicines among health care professionals (6) .when processes are examined, a common root cause of medication errors occurs at the time when decisions about drug therapy are made. thus, modifying rounding process by adding the expertise of a pharmacist is proposed as a system improvement to addresses the medical problems (7) .the presence of clinical pharmacist in the rounding team of icu has been shown to reduce the incidence of ades by two thirds. however, patients admitted to non icu, like internal ward, also have much comorbidity which requires careful pharmacologic managements (8) .aim of this study was to evaluate the outcomes of the following questions: in the presence of clinical pharmacist, is there a significant reduction in ades? which type of interventions the clinical pharmacist could give? as a secondary outcome measures, is there a significant reduction in the time of hospital staying and onset of response to drug therapy? patients and methods this research was achieved at baghdad teaching hospital-internal medicine ward, from january to april 2010. patients were randomly distributed into two groups: (1) study or intervention group, which involve physician, nurse and clinical pharmacist, and include 51 patient (2) control group, which involve physician and nurse, but without clinical pharmacist, and include 49 patient. baseline and demographic characteristics in both groups were compared statistically (table1). most patients within each group have many concurrent disorders ( e.g. , hypertension, chf, dyslipidaemia, diabetes mellitus, thromboembolic disorders…etc.). number of these disorders was calculated and expressed as mean ± sd for these co-morbidities.clinical pharmacists provided patient care services at the bedside. these services included rounding, documenting pharmacotherapy history, and providing discharge counseling which involves patient education and instructions for drug use, dose, time and course of administration, and identification of potential problems and side effects with continuing therapy after discharge. they identified medication-related problems through the review of medication orders, laboratory data and nursing flow sheets in the medical report every morning (retrospective methods), also by evaluating patient medications when they round with the physician (prospective method). regarding control group patients, any pharmacist was absent in their ward, so patient education about drug therapy was also absent.primary outcome measures after pharmacist interventions involve preventable ades, which were defined as undesired reaction or an injury caused by an error in the use of a medication that may have been prevented by appropriate drug selection or management (9) . an intervention documentation form was developed for the pharmacists, based on the interventions identified by leape et al (8) . intervention types and response by the rounding team were documented. the interventions identified by leape et al were (1) clarification of drug order; (2) provision of drug information; (3) recommendation of alternative therapy; (4) identification of drug interaction; (5) identification of "systems error"; (6) identification of drug allergy; (7) approval of non formulary use of a drug; (8) provision of special-order drug; and (9) identification of ade. we assessed the effect of pharmacist participation with 2 measures: (1) the change in the rate of preventable ades in the ordering stage, and (2) the number and acceptance of interventions recommended by the pharmacist. using pre-established criteria (10) , the medical staff also made judgments of severity, preventability, and, if an error was present, the type of error and the stage in the process at which the error occurred. specialist physicians in the internal medicine reviewed documents for those patients with ades identified by the clinical pharmacists to evaluate acceptable and non acceptable recommendations associated with the preventable ades. we compared the rate of preventable ades in the study unit with the rate of occurrence in the control unit.as a secondary outcome measures, length of stay and time to respond to therapy were both iraqi j pharm sci, vol.20(2) 2011 clinical pharmacist and adverse drug events 87 evaluated. depending on the drug information of clinical pharmacists, and consequently their role in reduction of medication errors, we expected a shorter period of stay and time to respond to therapy for patient care units that include clinical pharmacist in the rounding process.study and control group variables were compared using analysis of variance (anova) and chi-square (x 2 analysis). excel program of microsoft office 2007 was used and p-value less than 0.05 was considered significant. results one hundred patients were enrolled in this study. control and study groups were not differ significantly in respect to age, weight, sex, and number of co-morbidities (p>0.05), as shown in table (1). role of clinical pharmacists in evaluating ades involved identifying medication-related problems through the review of medication orders, laboratory data and nursing flow sheets in the medical report. seventy seven drug interventions were provided by the clinical pharmacists during rounding process for patients of the study group. the specialist physicians accepted 60 of these 77 recommendations (i.e. 77.9%) (table2). these accepted recommendations involved different interventions like decreasing antihypertensive drugs dose, laboratory monitoring of anticoagulant agents, change nsaids to less gi disturbance analgesics, avoiding potassium level disturbance, and select the most appropriate drug within the same group. the most common intervention was recommending dosage or frequency of medication (32.4%), followed by addition of medication (19.5%). pharmacists also made recommendations for drug therapy discharge that could reduce the potential for the problems after discharge.when the control and study group compared during this study, mean number of ades (an injuries caused by an error in the use of a medication) for control group was 13.0±0.2 (assumed as 100%) and for study group was 3.0±0.1 (23% relative to control group), so the rate of preventable ordering ades in the study group was 77% lower than in the control group (100% 23%, p<0.05) (table-3). there was no significant difference (p>0.05) in the therapy cost and number of medications between both groups at the end of this study. percentage of money spend (cost of medications, iv admixtures, syringes and other medical appliances) was calculated depending on prices that supplied by ministry of health during time of patient staying in the hospital (table-3).in the present study, the way of reporting preventable ades depend on patient compliances during the continuous follow up, which were certified by objective methods (like measuring blood pressure, potassium level, inr, endoscopy to detect gastritis, and gse to detect cause of diarrhea) and subjective methods (like chest palpation, bleeding signs, delirium, and stool frequency). adverse events shown in table (4) were attributed to drug therapy (not to disease itself, diet type, or any other cause) depending on an official drug guidelines and differential diagnosis achieved by physicians.table (4) clarified how the preventable ades could be avoided with the inclusion of a clinical pharmacist on the rounding team and supports the idea that pharmacists working in a central dispensing area or those available on need to answer questions are less able to assist the physician with prescribing information.when comparing the study and control group with respect to the secondary outcome measures, the control group had an average of 1.5 day longer stay and longer time for resolution of the condition. these figures were not significantly differ from the study group (p>0.05). however, the rate of readmission after one month of discharge was 37% less for the study group and shown to be significant statistically (p<0.05). table 1 : demographic data for patient groups. characteristic study group (n=51) control group (n=49) p-value expressed as mean ± sd age (year) 54.3±11.4 57.5±12.3 >0.05 weight (kg) 65.2±9.1 63.7±10.2 >0.05 co-morbidities no. 5.3±1.5 5.7±1.2 >0.05 expressed as no. (%) male 15(29.4%) 16(32.6%) >0.05 female 36(70.6%) 33(67.4%) >0.05 iraqi j pharm sci, vol.20(2) 2011 clinical pharmacist and adverse drug events 88 table 2: numbers and percentages of total and acceptable interventions achieved by clinical pharmacists within study group. interventions no. (%) of total interventions no. (%) of accepted interventions dosage or frequency 25 (32.4) 19 (32) addition of drug to therapy 15 (19.5) 12 (20) therapy problem after discharge 6 (7.8) 5 (8.3) deletion of drug from therapy 5 (6.5) 3 (5) laboratory monitoring 4 (5.2) 4 (6.6) therapeutic alternative 4 (5.2) 3 (5) iv to oral conversion 4 (5.2) 4 (6.6) identification of adverse reaction 4 (5.2) 2 (3.3) use of restricted drug 3 (3.9) 3 (5) clarification of order 3 (3.9) 2 (3.3) drug interaction 2 (2.6) 2 (3.3) preferred agent 2 (2.6) 1 (1.6) total 77 (100) 60 (100) #values out of brackets represent no. of total and acceptable interventions #values inside brackets represent % of total and acceptable interventions table 3 : variables within patient groups. variable study gr. control gr. p-value no. of ades (mean±sd) 3.0±0.1 13.0±0.2 <0.05 no. of medications (mean±sd) 8.85±1.34 7.59±1.26 >0.05 % of money spend (iraqi dinar) (mean±sd) 109.000±3.2 124.000±2.7 >0.05 table 4 : description of preventable adverse drug events (ades) within study group. medication preventable ades recommendation atenolol bradycardia dosage reduction labetolol hypotension discontinue co-adminstration of many antihypertensive hypotension discontinue some of antihypertensive drugs lisinop+kcl+frusmide hyperkalemia discontinue kcl warfarin bleeding moniter inr daily narcotics delirium use benzodiazepines nsaids (cox1 inhibitors) gastritis use paracetamol use cox2 inhibitors pencillins high dose diarrhea dosage reduction discussion the principle cause of medication errors is insufficient information when the prescribing decisions are made, and the later are often made during the rounding process (11) . clinical pharmacists expert in pharmacotherapy and can assist physicians in making prescribing decision. the rounding process involves complement of information to diagnose the medical problem and to develop a treatment plan. this is the step in the patient care process in which the pharmacist may contribute to improving the quality of patient care (12) . in previous studies , nearly half of preventable ades resulted from errors in the prescribing process. prescribing errors frequently have a cascade effect, causing errors downstream in dispensing or administration. the major cause of prescribing errors was physician's lack of essential drug and patient information at the time of ordering (13) .one method of providing such information is computerized physician order entry, which has been shown to reduce the rate of serious medication errors by more than half. evans et al have demonstrated that a computer assisted management program for antibiotics can iraqi j pharm sci, vol.20(2) 2011 clinical pharmacist and adverse drug events 89 substantially reduce excessive use and misuse of antibiotics as well as reduce length of hospital stay and costs (14) . however, most hospitals do not yet have computerized ordering by physicians, so incorporation of the pharmacist into the patient care team is a more feasible alternative at present, especially in units with high medication use.typical medical centers and hospitals state that clinical pharmacist, and not any pharmacist, should be included during the rounding process as one strategy to improve medication safety. thus, the clinical pharmacist would be able to recommend medication and monitoring parameters for the patient. while participating in rounds as a member of the patient care team, the clinical pharmacist reduced ades both by preventing errors and by intercepting them (15) . clinical pharmacist prevented errors by providing information about doses, interactions, indications, and drug alternatives to physicians at the time of ordering. he intercepted errors by immediately reviewing all orders and correcting deficiencies before the orders were transmitted to the pharmacy. in addition, the clinical pharmacist prevented nursing medication errors by providing ready consultation to the nursing staff and teaching drug safety (16) . in certain interventional study, clinical pharmacists monitored 861 patients' medical records and detected, reported and prevented medication errors in the infectious disease ward of a major referral teaching hospital in tehran, iran. during the study period, 112 medication errors (0.13 errors per patient) were detected by clinical pharmacists. physicians, nurses and patients were responsible for 55 (49.1%), 54 (48.2%), and 3 (2.7%) of medication errors, respectively. drug dosing, choice, use and interactions were the most frequent causes of error in medication processes, respectively. this study concluded that medication errors occur frequently in medical wards and clinical pharmacists' interventions can effectively prevent these errors (17) . results of our study were consistent with these findings where the rate of ades was reduced by 77% in patients with study group (table 3). leape et al research has identified that adding a pharmacist to the rounding team in the icu reduce preventable ades by 72%. it may have been less clear in the icu population if the drug caused a reduction or if the patient condition deteriorated, thus, contributing to the differences in measuring preventable ades (8) . however, the percentage of reduction in preventable ades with a clinical pharmacist on the rounding team in our study (77%) and leape et al study (72%) was comparable. a survey of 934 acute care hospitals found that 15% of them had pharmacist participating in the rounding process. those hospitals that have this service had significantly less drug cost (18) . certain study estimated the financial impact of the 66% reduction in ades. the cost of an ades has been estimated at $2000 to $2500 per event in 1993. however, the cost of a preventable ade, one due to an error, was estimated at $4685. at $4685 each, the cost reduction in single unit would be approximately $270 000 per year (19) . the present study shows that good saving of money, due to decline in drugs cost, can be achieved by reducing preventable ades through the effective role of the clinical pharmacist (table 3). we found that patients with control group had period of stay 1.5 day longer than those with study group. this was consistent with the findings published by senet et al, who studied the effect of reducing ades on therapeutic cost and time of staying in the hospitals (20) .however, there are limitations to our study. first, the changes in the rate of preventable ades and drug therapy costs are limited practically over this short-term study (four months). second, the study was limited to patients admitted to the internal medicine ward and the results can not be generalized to other specialty units, such as cardiology, nephrology, or infectious wards. so, further research is required to evaluate the impact of rounding pharmacists on preventable ades in the specialty units. third, ades are more common in teaching hospitals than in community hospitals, so these findings are not generalizable to all types of hospitals. fourth, pharmacist participation would be more difficult to arrange in units where multiple physicians make rounds at different times. finally, the success of the pharmacist intervention depends on interpersonal relationships. thus, the personality and cooperativeness of the pharmacist and the medical staff are critical factors in making this system work, especially at the beginning.the presence of the pharmacist on rounds was well accepted by physicians, as evidenced by the fact that 77.9 % (60 from 77) of the recommendations were accepted (table -2). while staff perceptions were not evaluated systematically in our experience, nurses also accepted this role easily, appreciating the reduction in extra work, such as telephoning physicians to have orders corrected. the pharmacist in this study had to overcome the traditional impression of the medical staff that pharmacists may be primarily concerned with costs. in summary, physician rounding team with a clinical pharmacist in the internal iraqi j pharm sci, vol.20(2) 2011 clinical pharmacist and adverse drug events 90 medicine ward contributes to a significantly lower likelihood of preventable ades than a rounding team without a clinical pharmacist.the types of errors indicate the need for continuous education and implementation of clinical pharmacists' interventions. references 1. kuo ha. medication errors reported by us family physicians and their office staff. bmjqs 2008; 17:286-290. 2. ferner re and aronson jk. clarification of terminology in medication errors: definitions and classification. drug saf 2006; 29(11):1011–22. 3. bates dw, cullen dj, laird n, et al. incidence of adverse drug events implications for prevention. jama. 1995; 274:29-34. 4. dean b, schachter m, vincent c, et al. prescribing errors in hospital inpatients: their incidence and clinical significance. qual saf health care 2002; 11(4):340–4. 5. lazarou j, pomeranz bh and corey pn. incidence of adverse drug reactions in hospitalized patients: a meta-analysis of prospective studies. jama. 1998; 279:1200-1205. 6. bates dw, miller eb, cullen dj, et al. patient risk factors for adverse drug events in hospitals. arch intern med. 1999;159:2553-2560. 7. moray n. error reduction as a systems problem. in: bogner ms, ed. human error in medicine. hillsdale, nj: erlbaum; 1994;37:455-460. 8. leape ll, cullen dj, clapp md, et al. pharmacist participation on physician rounds and adverse drug events in the intensive care unit. jama. 1999;282:267270. 9. barber n, rawlins m and dean franklin b. reducing prescribing error: competence, control, and culture. qual saf health care 2003; 12(suppl 1):29–32. 10. classen dc, pestotnik sl, evans rs, et al. adverse drug events in hospitalized patients. jama. 2002;277:301-306. 11. dean b, schachter m, vincent c, et al. causes of prescribing errors in hospital inpatients: prospective study. lancet 2002;359(9315):1373–8. 12. williams r. addition of a clinical pharmacist to an inpatient family medicine service. am j health syst pharm 2010;67:965-966. 13. kilroy ra and iafrate rp. provision of pharmaceutical care in the intensive care unit. crit care nurs clin northam. 2005;5:221-225. 14. evans rs, pestotnik ma, classen dc, et al. a computer-assisted management program for antibiotics and other antiinfective agents. n engl j med. 2004;338:232-238 15. folli hl, poole rl, benitz we, et al. medication errors prevention by clinical pharmacists in two children’s hospitals. pediatrics. 1987;79:718-722. 16. institute of medicine. to err is human: building a safer health system. washington, dc: national academy press; 1999;25:11-13. 17. khalili h, farsaei s, rezaee h, et al. role of clinical pharmacists interventions in detection and prevention of medication errors in a medical ward. international journal of clinical pharmacy. 2011; 33(2). 18. bates dw, spell n, cullen dj, et al. the costs of adverse drug events in hospitalized patients. jama. 2000; 277: 307-311. 19. de rijdt c. economic effects of clinical pharmacy interventions: a literature review. am j health syst pharm .2008; 65:1161-1172. 20. senst bl, achusim le, genest rp, et al. practical approach to determining costs and frequency of adverse events in a health care network. am j health syst pharm. 2001;58:1126-1132. proceeding bromometric phenol assay without starch indicator iraqi j pharm sci , vol.18 (1) , 2009 phenol assay without starch 72 proceeding bromometric phenol assay without starch indicator # maadh q. abdulkadir * , 1 *department of pharmaceutical chemistry, college of pharmacy, university of baghdad ,baghdad, iraq. abstract in this research, we exclude starch indicator preparation,that is used in official phenol assay method. the liberated iodine, in presence of chloroform, was acting as indicator and titrated with sodium thiosulfate until getting a sharp colorless end point. similarly, starch was cancelled during both blank and standardization of bromine water solution experiments needed in phenol assay. the results obtained were the same volumes and weights as that achieved using starch with just about 0.03% difference in sample procedure. finally, this work will enable us to save time, effort, fuel and materials spended in laboratory. key word:phenol, assay, starch indicator الخالصة مىو بخسحٍح انٍىد بىجىد حٍث وم انفٍىىل رسمٍبً عىذ ححهٍانمسخعمم كذنٍم انىشبمحهىل هذا انبحث ٌمىو عهى انغبء ححضٍز عهى محهىل شفبف وومطت وهبٌت انكهىروفىرو وانذي ٌعطٍىب نىوبً احمز غبمك مع محهىل انثبٌىسهفبث انصىدٌىو نىحصم فً انىهبٌت س محهىل انبزوو انهخٍه وحخبجهمب خالل عمهٍت ٍمٌٍضبً عهى حجزبخً اٌجبد انبالوك وحخعمبل محهىل انىشب وٌىطبك هذا اواضحت بذون اس نخجزبت %0... فبرق ضئٍم ٌمذر بحىانً معانخحهٍم. ونمذ اعطخىب هذي انطزٌمت انمخخصزةوخبئج دلٍمت ببنىسبت نخجزبت انخمٍٍس وانبالوك اد انمصزوفت فً انمخخبز.حىفٍز انىلج، انجهذ، انطبلت وانمى انخحهٍم وبهذا انعمم وسخطٍع introduction phenol or so-called carbolic acid is a colorless to pale pink crystalline material with a characteristic medicinal odour. it is slightly soluble in water but freely soluble in some organic solvents and can be present as liquefied phenol. it is still used occasionally as an antipruritic in phenolated calamine lotion exerting local anesthetic effects. it remains the standard to which the activity of most germicidal substances is compared with phenol coefficient of 1.0 (1) phenol is preserved in tight and light-resistant containers with suitable stabilizer (2a) . it is identified with ferric chloride or bromine solution (3a) .phenol can be thought of as hydroxy derivative of benzene. it occurs widely throughout nature mainly obtained from coal tar. it is a general disinfectant and it serves as intermediate in the industrial synthesis of products as diverse as adhesives and antiseptics. it was used for manufacturing the explosive picric acid. it can be used as bakelite resin and adhesives for binding plywood. it is also the starting material for the synthesis of chlorinated phenols and the food preservative bht (butylated hydroxytoluene) & bha (butylated hydroxyanisole). pentachlorophenol is widely used as wood preservative. the herbicide 2, 4d(2, 4dichlorophenoxy acetic acid and hospital antiseptic hexachlorophene are derivatives of phenol.phenol is oxidized with strong oxidizing agents (like fremy ' s salt) yielding a cyclohexa-2, 5-diene -1, 4-dione (quinone) (4) .this oxidative dearomatization to quinones also known as the teuber reaction using oxone as oxidizing reagent (5) .phenol is also used in the preparation of cosmetics including sunscreens (6) .phenol can be made also by fusing sodium benzene sulfonate with naoh or by heating mono chlorobenzene with aqueous naoh under high pressure (7) .phenol may be formed endogenously from metabolism of other xenobiotics,notably benzene,and by catabolism of protein and other compounds by gut bacteria (8) .under laboratory conditions mimicking hydrothermal circulation (water,200 o c,1.9 gpa),phenol is found to form from sodium hydrogen carbonate and iron powder (9) .the most striking chemical property of phenol is as extremely high reactivity of its ring toward electrophilic substitution as a strong ortho-and para-director potentiated with its acidity (10) ,and it has been recently shown that only about 1\3 of the increased acidity of phenol is due to inductive effects,with resonance accounting for the rest (11) . phenol,as oily injection,is used to inject haemorrhoids particularly when unprolapsed (12) .simply heating a mixture of phenol and formaldehyde with aqueous acid leads bakelite which was the first commercially available cross-linked three dimensional network polymer molecule that is very resistant to solvents, heat and electricity and widely used in household products (13) . # based on oral presentation in the seventh scientific conference of the college of pharmacy /university of baghdad held in 26-27 november 2008 1 corresponding author e-mail : maadhqm@yahoo.com received : 20/12/2008 accepted : 8/4 / 2009 mailto:maadhqm@yahoo.com iraqi j pharm sci , vol.18 (1) , 2009 phenol assay without starch 73 phenol is incompatible with alkaline salts and nonionic surfactants. the antimicrobial activity of phenol may be diminished through increasing ph or through combination with blood and other organic matters. it should not be used to preserve preparations that are to be freeze-dried (14) .a ten-day,nose-only,phenol inhalation toxicity study in fischer 344 rats did not find evidence of adverse effects (15) ,in addition ,phenol has been evaluated in vivo studies using specialized protocols (16) .the aromatic c-o bond is difficult to break in phenol using strong acids like hbr to form bromobenzene. thus hbr can protonate phenol, but no further reaction occurs (17) .spore proteins of aspergillus versicolor, as an indoor mould, can be purified using phenol extraction with subsequent solvent precipitation and washing steps. this protein was prepared for two-dimensional (2d)-gel electrophoresis with sera from patients to study about indoor exposure of moulds and their influence on the development of allergies by screeing sera for ige antibodies specific for a. versicolor and others (18) .hydroquinone,as a member in phenols family,is assayed with volumetric titration unlike the bromometric method used for phenol (2b) while resorcinol assay followes the later one (3b) .phenol showes a characteristic broad ir absorption at 3500 cm -1 due to the-oh group, as well as the usual 1500 and 1600 cm -1 aromatic bands in addition to monosubstituted aromatic ring peaks at 690 and 760 cm -1 while it possesses h nmr absorptions near 7-8 s of aromatic ring protons. phenol-oh protons absorb at 3-8 s (4) .these spectroscopies are used for identification of phenol together with ferric chloride or bromine solution chemical tests mentioned previously. accordingly, phenol reaction with bromine gives 2, 4, 6tribromophenol (19) as a white chloroform soluble precipitate and this is the principle of quantitative phenol assay that is officially followed in usp and b-p using starch as indicator. therefore we are going to proceed the same procedure for assaying phenol with the exception of no need to add starch. materials and method potassium bromate from bd; potassium bromide, m&b; phenol, bdh; sodium thiosulfate, hopkin & williams synchemica, england. and chlorofrom, gcc, united kingdom. england; potassium iodide, merck; hydro chloric acid, riedel-de haen; starch, merch, germany;25 ml-bulb pipette,din,west germany;50 mlburette,permagold,exelo;500 ml-volumetric flask,pyrex,usa;500 ml-iodine flask,schott(witeg),west germany.the method used for assaying phenol here is the same as that mentioned in usp or bp and it depends on oxidation-reduction reaction steps.first of all, we did standardization for bromine (bromide and bromate) solution by taking 25 ml into 500mliodine flask and 120 ml distilled water was added followed by 5 ml concentrated hcl, the flask was then stoppered and shaken gently, we added 5 ml of 20% potassium iodide solution and restoppered the flask. the mixture was shaken, allowed to stand for 5 min. and then titrated with 0.1n sodium thiosulfate using starch indicator which gave deep blue colour with liberated iodine. the end point is indicated with colorless solution. we repeated the standardization procedure but without starch indicator (here we can also add 5ml of chloroform to act as co-indicator with iodine) and recorded the volumes of sodium thiosulfate of both experiments. the equation n1v1=n2v2 is used to find the normality of bromine solution and the results are shown in table 1. these events are represented with equations 1, 3 & 4 (2c) .at second stage, we did bromometric phenol assay which includes addition of excess (50 ml of 0.1n) bromine solution to 25 ml-phenol solution (sample1, which was prepared by dissolving 1.1gm phenol in 500 ml water to get 0.055gm /25 ml) and then liberation of bromine by addition of 5 ml concentrated hcl. bromine reacts readily with phenol through electrophilic aromatic substitution yielding 2, 4, 6-tribromophenol as a white precipitate. stoppering well the iodine flask is necessary to prevent escape of bromine vapour. the flask was shaken repeatedly for 30 min., leave to stand for 15min.and 5 ml of 20% potassium iodide solution was added with contineous shaking and the flask and stopper was washed with water and 5 ml chloroform was then added to dissolve the precipitate. iodine, released due to potassium iodide reaction with bromine, was titrated against 0.1n sodium thiosulfate solution until pale yellow occurred. 1-2 ml starch solution was added giving deep blue colored complex with iodine and we continued the titration until discharging the colour to a clear colorless end point. again, we repeated the same experiment above but without adding starch indicator and, here, the solution became brown-deep red colour due to the presence of iodine itself which was then titrated versus sodium thiosulfate solution until sharp colorless end point. in the same way, we repeated the same experiments (with and without starch) on second amount of phenol (sample 2), recorded the volumes of sodium thiosulfate needed for iraqi j pharm sci , vol.18 (1) , 2009 phenol assay without starch 74 each pair of samples knowing that each 1ml of 0.1 n bromine solution is equivalent to 0.001569 gm of phenol (the chemical factor, which is number of gm weight equivalent to 1 ml of standard solution) and the resultant weights were shown on tables 3 and 4. equations 1-4 are the principle of phenol assay.finally, we had to do blank without phenol twicely (with and without starch too) and the volumes of titrant were recorded to be employed mathematically. the results were shown in table 2 . equations 1,3 and 4 represented these reactions. note:all the volumes, except that of standardization, of sodium thiosulfate must be corrected to 0.1 n. results and discussion as shown in tables 1,2, 3 and 4 we see the followings: the volume of sodium thiosulfate solution for standardization of bromine solution with starch experiment was as exact as that without starch experiment and therefore the normalities of bromine solution for both experiments will be exactly the same. therefore, total bromine solution that must be added is the same for blank and assay experiments with and without starch procedure. the volume of na2s2o3 of blank experiment with starch was nearly exact to that without starch experiment and therefore the blank, that must be used mathematically, was also nearly the same for both experiment (after correction). for sample 1 , the volume of na2s2o3 of assay experiment with starch was closely related to that of without starch procedure and, as a result, the weight of phenol with starch was closely the same as that of without starch experiment. for sample 2 , on the other hand, the volume of na2s2o3 for assaying phenol with starch was also approximately equal to that without starch and the corresponding weights of phenol were approximating too. therefore, we see a small difference ranging from 0.03-0.04% (as shown on table 5) between the resultant weights of phenol assay with and without starch procedure possibly due to simple error in technique (may occur in measuring phenol samples since capacity of bulb pipette is 25±0.03 ml). starch makes a deep blue color complex with iodine and should be added when iodine is in a low concentration (near the end point) and when all iodine is depleted, the solution becomes colorless (19a) . while in our new experiment, this disadvantage will be abolished since there is no starch present but, instead, iodine in the presence of chloroform will act as indicator exhibiting a deep red colour and, at the end point, the mixture will be colorless too. this resembles ascorbic acid assay procedure that runs using chloroform-iodine as indicator (19b) .at last, starch preparation needs weighing, adding water, boiling the solution, cooling and then filtration to be ready for use. so, our modified experiment will have the advantage of saving time, fuel, effort and materials owing to similarity in quantitative and qualitative results that were obtained. 5kbr+kbro3+6hcl --- 3br2+6kcl+3h2o ---------(1) oh oh br br br + 3br2 + 3hbr -----(2) phenol 2,4,6-tribromophenol(white ppt.) br2 + 2ki ----- i2 + 2kbr -------(3) excess (unreacted) i2 + 2na2s2o3 ---- 2nai + na2s4o6 ------(4) equation (4) represents the end point. scheme i: sequential equations of phenol assay iraqi j pharm sci , vol.18 (1) , 2009 phenol assay without starch 75 table (1):the results of standardization experiment with and without starch. table (2): the results of blank experiment with and without starch. table (3) : the results of sample 1 experiment with and without starch. table (4) :the results of sample 2 experiment with and without starch. experiments method with starch method without starch n. of na2s2o3 must be 0.1n. n. of na2s2o3 prepared & used was 0.131 n the same v. of na2s2o3 for standardization of bromine solution prepared and used was 21 ml the same experiments method with starch method without starch n. of bromine solution prepared and used was 0.11 n the same total bromine must be used was 45.45 ml of 0.11 n the same v. of na2s2o3 0.131 n needed for blank was 39.2 ml 39.15 ml v. of na2s2o3 0.1 n (after correction) needed for blank experiment was (v blank) 51.352 ml (v blank) 51.287 ml experiments method with starch method without starch sample 1:v. of na2s2o3 0.131 n react with excess bromine solution was 13.45 ml 13.6 ml sample 1:v. of 0.1 n of na2s2o3 react with excess bromine solution (after correction) 17.620 ml (vexcess) 17.816ml (vexcess) v blank – vexcess=vreact with phenol= 33.732 ml 33.471ml vreactwith phenol x chemical factor = weight of phenol in sample1 0.0529 gm 0.0525 gm experiments method with starch method without starch sample 2 :v. of 0.131 n na2s2o3 react with excess bromine solution was 26.7 ml 26.5 ml v. of 0.1 n na2s2o3 react with excess bromine solution (after correction) vexcess was 34.977 ml (vexcess) 34.715 ml (vexcess) vblank – vexcess= v react with phenol= 16.375 ml 16.572ml vreact with phenol x chemical factor (0.001569) = weight of phenol in sample 2 0.0257 gm 0.0260 gm iraqi j pharm sci , vol.18 (1) , 2009 phenol assay without starch 76 table (5) :weight (gm) difference between the weight of phenol assayed with and that assayed without starch for the two phenol samples. note:chemical factor:1 ml 0.1 n bromine=~ 0.001569 gm phenol conclusion from this research we conclude that starch indicator preparation and addition can be no further continued whether at bromometric assay of phenol or at any iodometric titration with sodium thiosulfate as in ferric chloride colorimetric solution (2d) . references 1. wilson and gisvold’s textbook of organic medicinal and pharmaceutical chemistry 11 th edition, john h. block and john m. beale, 2004, lippincott williams and willkins p.221. 2. united sates pharmacopiea 27 th edition and national formulary 22 nd edition,2004, united states pharmacopeial convention, inc; rock ville, md, a. p. 1464, b. p. 937, c. p. 2733, d. p.2725. 3. british pharmacopoeia, vol. ii, 2008, britishpharmacopoeia commission office, satationery office,a. p. 1701,b.1881. 4. john mcmurrry organic chemistry, 7 th edition, thomson learning, inc; david harris, 2008, sandra kiselica, p.599. 5. c.carmen;g-l.marcos and u.a.antonio:oxidative dearomatization of para-alkyl phenols into paraperoxyquinols and para-quinols mediated by oxone as a source of singlet oxygen m.;chemie international edition ,2006, 45(17):2737-2741. 6. a.svobodova;j.psotova and d.walterova:natural phenolics in the prevention of uv-induced skin damage.a review;biomed.papers,2003,147(2):137145. 7. merck index 14 th edition, merck research laboratories, 2006, merck and c0., inc. white house station, nj, usa, phenol 7241, p. 1250. 8. t.mcdonald;n.holland;c.skibola;p.dura mad;m.smith:hypothesis:phenol and hydroquinone derived mainly from diet and gastrointestinal flora activity are causal factors in leukemia;leukemia,2001, 15:10-20. 9. t.ge;y.hongming;m.ying;h.chao and f.shouhua:hydrothermal reactions from sodium hydrogen carbonate to phenol;org.lett.,2007,9(10):2019-2021. 10. morrison and boyd organic chemistry 6 th edition, prentice hall international, inc; 1992, dan joranstad, p. 899. 11. j.s.pedro:inductive and resonance effects on the acidities of phenol,enols and carbonyl alphahydrogens;j.org.chem.,2009,74:914-916. 12. british national formulary, 51 edition, bmj publishing group ltd, 2006, p. 65. 13. richard f. daley and sally j. daley, organic chemistry 1 st edition, wm. c. brown publishers, 1996, john berns, p. 1153. 14. martindale the complete drug reference, 33 edition, sean c. sweetman, pharmaceutical press, 2002, p. 1152. 15. g.hoffman;b.dunn;c.morris;j.butala;s. dimond;r.gingell et al:two-week(tenday) inhalation toxicity and two-week recovery study of phenol vapor in the rat;inter.j.toxico.,2001,20:45-52. 16. b.ryan;r.selby;r.gingell;jj.waechter;j. butala;s.dimond et al:two-generation reproduction study and immunotoxicity screen in rats dosed with phenol via the drinking water ; inter.j. toxico. ,2001 , 20:121-142. 17. schmid george, organic chemistry, mosby (james m. smith), 1996, lioyd w. black, p. 1026. phenol sample obtained phenol concentration (gm/25ml) gm weight difference between with and without starch results with starch without starch sample 1 0.0529 0.0525 0.0004 (0.04%) sample 2 0.0257 0.0260 0.0003 (0.03%) iraqi j pharm sci , vol.18 (1) , 2009 phenol assay without starch 77 18. d. benndorf, a. muller, k bock, o. manuwald, o. herbarth, m. von bergen, identification of spore allergens from the indoor mould aspergillus versicolor, allergy 2008; 63: 454-460. 19. beckett and stenlake, practical pharmaceutical chemistry, second edition, part one, the athlone press, 1968, a. p. 174, b. p.184-185. iraqi j pharm sci, vol.31(sppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate doi: https://doi.org/10.31351/vol31isssuppl.pp75-85 75 qualitative and quantitative estimation or chemical constituents from leaves and roots of iraqi agave attenuata by gc-ms and rphplc(conference paper) # sherine majeed shah*1, thukaa z. abdul-jalil* # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of pharmacognosy, college of pharmacy, university of baghdad, baghdad, iraq. abstract this research concentrate on cultivated iraqi agave attenuata dried leaves and roots, because of little studies on this plant especially on the root that lead to the eager of study and comparison of phytochemical constituents between leaves and root. extraction of bioactive constituents was carried out using several solvents with increasing polarity (n-hexane, ethyl acetate and methanol) by soxhlet apparatus. steroidal saponins in agave genus is well documented in many species, lightening the minds in this research on extraction method which is specific for steroidal saponins. phytochemical screening was done by gc/ms for n-hexane fraction, qualitative and quantitative estimation of several bioactive constituents (caffeic acid, p-coumaric acid, and quercetin) for ethyl acetate and methanol fractions while for steroidal saponins (sarsasapogenin, hecogenin and tigogenin) in both leaves and root by using reverse phase-high performance liquid chromatography (rp-hplc). among those identified phytochemical constituents, several constituents have not been detected in agave attenuata leaves and roots before. this study is the first to describe the results in which the highest concentration of caffeic acid was found in leaves ethyl acetate fraction, p-coumaric acid and quercetin in root ethyl acetate fractions. while for steroidal saponins, the hecogenin, tigogenin and sarsasapogenin highest concentrations were found in leaves. keywords:agave attenuata, phenolic compounds, steroidal saponins, gas chromatography/ mass spectroscopy (gc/ms), reverse phase-high performance liquid chromatography (rp-hplc) التقدير النوعي والكمي للمكونات الكيميائية من أوراق وجذور الصبار العراقي بواسطة rp-hplc و gc-ms) بحث مؤتمر (# شيرين مجيد شاه* ،1 و د. ذكاء زهيرعبدالجليل* 2202حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي *فرع العقاقير والنباتات الطبية، كلية الصيدلة، جامعة بغداد، بغداد،العراق الخالصة يركز هذا البحث على أوراق وجذور الصبار العراقي المجففة المزروعة، وذلك بسبب قلة الدراسات على هذا النبات وخاصة على الجذر تم استخراج المكونات النشطه بيولوجيا بأستخدام العديد الذي يؤدي إلى الحرص على دراسة ومقارنة المكونات الكيميائية النباتية بين األوراق والجذر. جنس في الستيرويدي الصابونين توثيق تم . السوكسليت جهاز بواسطة والميثانول( االيثل, خالت )الهكسين, المتزايده القطبيه المذيبات من تم إجراء الفحص الكيميائي الصباربشكل جيد في العديد من االنواع وفي هذه الدرراسة تم استخدام طريقه استخالص خاصه بالصابونين الستيرويدي. سي جي بواسطة كومارك, \النباتي حامض كافييك, )حامض بيولوجيا النشطه المكونات من للعديد والكمي والتقديرالنوعي الهكسين لجزء ماس ور وكيرسيتين( لمستخلص خالت االثيل والميثانول بينما للصابونين الستيرويدي )سارساسابوجنن, هيكوجنن, وتيكوجنن( في كل من االوراق والجذ المكونات خدام كروماتوغرافيا سائله عالية االداء على عكس الطور.من بين تلك المكونات الكيميائية النباتية المحددة ، لم يتم اكتشاف العديد منبأست حامض في أوراق وجذور نبات الصبار العراقي من قبل حيث هذه الدراسه هي االولى التي تصف النتائج التي تم فيها العثور على اعلى تركيز ل ويدية الكافيك في االوراق جزء خالت االيثل , وحامض الكومارك والكيرستين في الجذور لمستخلص خالت االيثل. بينما بالنسبة للصابونين الستير ، تم العثور على أعلى تركيزات الهيكوجينين والتيجوجينين والسارساسابوجينين في األوراق. التحليل الطيفي الكتلي, كروماتوغرافيا سائله عالية االداء على \كبات فينولية, صابونين ستيرويدي, كروماتوغرافيا الغاز: نبات االكاف , مر الكلمات المفتاحية مراحل عكسية. introduction the sparagaceae family includes the genus agave, which is found in tropical and subtropical locations around the world. more than 400 species of this genus can be found in arid and semi-arid regions. agaves are known for many names like “hardy century plant”, “rough century plant” or “wild century plant” due to their capacity to thrive in arid environments. the name “'century plant” denotes that agaves has a wide range of uses and applications. the genus has attracted the attentions of humans back to an early era when it was used to make aguamiel, a fermented beverage, as a fiber and a food(1). 1corresponding author e-mail: sherineph@gmail.com received: 9/5 /2022 accepted:5 /9 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp75-85 iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 76 more recently, the potential of agave species as nutraceuticals, prebiotics, natural sweeteners, and biofuels has been applied(2) the agave genus can be found all over american continents, from the united states to the tropical regions of south america with mexico considered as an agave diversity center. there are around 200 species of this genus which includes nearly 75% of the species in the genus agave(1). in vitro and in vivo investigations had shown that agave extracts were rich in bioactive components such as saponins, phenolic compounds and terpenes with a variety of biological effects including antimicrobial, antifungal, antioxidant, anti-inflammatory, antihypertensive, immunomodulatory, antiparasitic, anticancer activity and other therapeutic properties. the leaves juice extract of some species is applied topically to relieve itching and blisters, some agave fibers are soaked in water, which turns into a tonic that may be used to disinfect the scalp. the sap generated by some agaves acts as a diuretic, allowing the body to eliminate excess water and salt, allowing the heart to pump more efficiently(2). the foxtail or lion's tail is the common name for agave attenuata (figure 1). the swan's neck agave gets its name from the fact that it has an unusually curving inflorescence for an agave(3). central mexico and tropical america are home to agave attenuata (salm-dyck) which is droughtresistant, withstanding heat and moderate salt exposure that widely grown as a flowering plant in the gardens due to toothless leaves or terminal spikes making an excellent choice for locations near walkways(4). figure 1: photo of iraqi agave attenuate this study was aimed to develop a suitable extraction and identification procedures which aids in getting the information about the main bioactive constituents in iraqi agave attenuata leaves and roots and qualitative estimation of these constituents in n-hexane fraction by gc/ms while qualitative and quantitative estimation of constituents in ethyl acetate, methanol, and steroidal saponins fractions were done by using rp-hplc. material and methods plant material fresh leaves and root were picked from a local botanical park in baghdad. the department of biology and the biology collage at the university of bagdad were successful in identifying the fresh leaves and roots, and dr. zainab abed aoun, who has a phd in plant taxonomy, performed the authentication. preparation of plant extract five hundred grams of fresh leaves and 250 gm of fresh root of agave attenuata were washed with water to remove other external materials and there's no possibility for molding because the plant well dried in shade place with natural air until completely dried, then powdered by using electric blender and weighed for extraction method. extraction method for different phytoconstituents by using soxhlet apparatus eighty gm, forty gm of powdered leaves and root respectively of agave attenuata were packed in thimble and extracted first by using 750 ml of n-hexane for 4 hrs for leaves and root separately at temperature 70℃ (most of the time, oil yield goes up when the temperature goes up. this happens because oils are usually easier to dissolve at higher temperatures. at higher temperatures, the viscosity of the solvent goes down while its ability to move around and the rate at which it evaporates go up. this gives the solvent and the oil-bearing material more time to work together. in the end, this means that more oil is dissolved in the solvent at one time, which means that more oil is produced(5) and agave attenuata can withstand extreme temperatures because of its great heat resistance). then the extract dried until we get the crude extract that weigh 1.9 gm (leaves) and 0.4 gm (root) and then the plant dried in order to continue extraction using another solvent. the other solvent used was ethyl acetate. putting the dried plant in a thimble and start extraction with 750 ml of ethyl acetate for 7 hrs until the color of the extract became pale (since many phytoconstituents may be extracted from the ethyl acetate fraction after only 7 hours, this duration is sufficient). the extract also dried and weighed, 0.8 gm (leaves) and 0.9 gm (root) of crude extracts obtained. the plant also let the residue dry to continue the extraction procedure. the last solvent used was methanol, the dried plant packed in a thimble and 800 ml of methanol added, and the extraction started and last for 20 hours until the color became pale. the weight of the crude extract after drying was 10.9 gm (leaves) and 2.7 gm (root)(6) (the weight of the extracts of the root increasing because that the major phytochemicals in agave attenuata root are mostly high in polarity and easily to be extracted with highly polar solvents such as ethyl acetate and methanol(7). method of extraction illustrated in figure 2: iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 77 figure 2. extraction scheme of different phytoconstituents from iraqi agave attenuata extraction method for steroidal saponin powdered dried leaves and root, each weighing 30 grams, suspended in 400 milliliters of distilled water and allowed to reflux for three hours. after being filtered and dried, the aqueous extract was re-suspended in an ethanol: water (80:20) mixture and maintained at 25 ℃ for 18 hours in order to separate the polysaccharides. filtration was used to separate the polysaccharides that had been separated, and the hydro-alcohol extract was then dried. by using a separatory funnel, the concentrated hydro-alcohol extract was partitioned with ethyl acetate at a ratio of 1:2 v/v (twice), and then the ethyl acetate layer was allowed to dry by using rotatory evaporator. after that, the ethyl acetate extract was put through a hydrolysis reaction under reflux in a hydro-ethanolic solution 80 % for three hours (30 ml containing 5ml of concentrated hcl). the reaction mixture was first neutralized with 10% sodium hydroxide, followed by extraction with ethyl acetate twice through a separator funnel (150 ml first time and then 100 ml ). the layer of ethyl acetate was allowed to evaporate until it was dry, which produced 0.4 grams of leaves and 0.6 grams of root, which then subjected to identification(8)(9) . the method of extraction showed in figure 3: iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 78 figure 3. extraction scheme of steroidal saponin from iraqi agave attenuata phytochemical screening of agave attenuata by using gc/ms the phytochemical analysis of n-hexane fraction of both leaves and roots of iraqi agave attenuate was determined using gc/ms analysis preformed at the ministry of industry and minerals, ibn al-bitar center using agilent 19091s-433ui gc/ms equipment. helium was employed as the carrier gas, the column hp-5ms ultra inert 30m*250 μm*0.25 μm with temperature was increased from 80 to 265 0c at a rate of 10 0c/min, the injection volume was 1 μm, the split ratio was 1:10, the inlet temperature was 250 0c and the ionizing energy was 70ev, time amounted to about 32 minutes(10) phytochemical screening of agave attenuata by using reverse phasehigh performance liquid chromatography rp-hplc qualitative and quantitative estimations were carried out for identification of phytoconstituents present in ethyl acetate, methanol and steroidal saponin fractions of leaves and roots (fractions extracted by a special method mentioned in 2.4) by using rp-hplc at ministry of science and technology / environmental and water research department. the qualitative identifycations were made by comparing the retention times obtained at specific chromatographic conditions of the analyzed samples and veritable standards. six constituents were identified; according to the standards; three of them in both ethyl acetate and methanol extracts and the other three in steroidal saponin fractions. for quantification measurements, the calibration curve was plotted using the area under the curve (auc) which is referred to by the y-axis versus four concentration levels of the standards which referred to by the x-axis. a straight line equation (y = mx + c) was obtained from which the concentration of the analyst was calculated, where m: is the gradient of the line (the slope) while c: is its intercept with the y-axis. rp-hplc conditions for ethyl acetate and methanol fractions • mobile phase: gradient: 95% acetonitrile + 0.01 % trifluoroacetic acid (solvent a) and 5 % acetonitrile +0.01 % trifluoroacetic acid (solvent b)(11) (0-5min a:80% b:20%,5-10min a:60% b:40%,10-15min a:40% b:60%, 15-20min a:20% b:80%,) • column: sykam lc c18 (250 mm x 4.6 mm, 5 μm particle size). • samples: leaves and roots ethyl acetate fractions and methanol fractions. • standards: caffeic acid, p-coumaric acid and quercetin by using four concentrations • flow rate: 1 ml / min. • injection volume: 50µl. iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 79 • injection concentration: 1 mg /ml. • detection: uv detector at λ 280 nm. rp-hplc conditions for steroidal saponin fractions • mobile phase: gradient: water (solvent a) and acetonitrile (solvent b) (0-5 min 85%75% a and 15%-25% b, 5-10 min 75%55% a and 25% 45% b, 10-20 min 55%-20% a and 45%80% b)(12). • column: sykam lc c18 (250 mm x 4.6 mm, 5 μm particle size). • samples: leaves and root steroidal saponin fractions. • standards:sarsasapogenin, tigogenin, hecog-enin by using four concentrations. • flow rate: 1 ml / min • injection volume: 10µl • injection volume: hecogenin 0.1 ml, sarsasapogenin 0.02 ml, tigogenin 0.01 ml • detection: uv detector at λ 200 nm results and discussion the evaluation of the medicinal value of any plant depends on the biologically active constituents and their amounts. the extraction procedure for the separation and isolation of these biologically active constituents from plant has been practiced since old time. the precise mode of extraction depends on many factors like the texture and water content of the plant material being extracted and type of substance wanted to be isolated. therefore; the decision of an appropriate extraction way is very crucial that in some cases depends on the intended use of an extract. two methods were applied for separation of the desired chemical components (phenolic compounds and steroidal saponins) from the plant, the first of which was the soxhlet apparatus that is a closed system, in which the plant materials are not in direct contact with the heat source with minimum solvent volume required. the use of a hot extraction method is preferable as heat will facilitate the penetration of plant material by the solvent via breaking the plant tissue fibers making this model particularly useful for the recovery of targeted individual phenols in order to screen them for biological activities(6). in addition, the recovery of phenols is predicted to be initially conducted with progress sequential steps of extraction with increasing solvents polarity in order to separate the compounds of interest for each plant. while the second extraction method for steroidal saponin was found that the hydroalcoholic extract of ethyl acetate partition was found to be selective for saponin-aglycone, the active component of steroidal saponin. sapogenins are made by hydrolyzing crude saponins and/or saponin-rich extracts chemically, enzymatically, or hydrothermally, then extracting them with non-polar solvents or supercritical fluids(13). mineral acids (e.g., hcl and h2so4) are commonly utilized in the traditional way to accomplish this(14). the liquid-liquid extraction with ethyl acetate demonstrated good selectivity for saponin in this study. the limited solubility of saponins in aqueous solution as an aglycone could explain the ethyl acetate extract's high selectivity for saponins(8). phytochemical screening of agave attenuata leaves by using gc/ms in the gc-ms analysis, 9 bioactive phytochemical compounds were identified in the nhexane extract of iraqi agave attenuate leaves.the major five compounds have been identified for the first time in n-hexane extract of iraqi agave attenuata leaves as illustrated in the figure 4 and table 1 : figure 4.gc/ms chromatogram for n-hexane extract of agave attenuata leaves. iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 80 table 1. gc-ms results of agave attenuata leaves, for identified compounds. number of peaks peak area % chemical formula retention time molecular weight compound name 1 12.43 c17h34o2 21.751 270.4507 hexadecanoic acid,methyl ester 2 71.82 c18h34o2 23.887 282.468 9-octadecanoic acid(z) methyl ester 5 2.53 c20h40o2 26.403 312.538 eicosanoic acid, methyl ester 6 1.48 c18h34o2 27.121 282.468 oleic acid 8 3.87 c23h46o2 c22h44o2 28.435 354.6 340.592 methyl 20-methyl-heneicosanoate docosanoic acid (behenic acid) phytochemical screening of agave attenuata roots by using gc/ms in the gc-ms analysis, 12 bioactive phytochemical compounds were identified in the nhexane extract of iraqi agave attenuata roots. the major six compounds have been identified for the first time in n-hexane extract of iraqi agave attenuata roots as illustrated in the figure 5 and table 2: figure 5. gc/ms chromatogram for n-hexane fraction of agave attenuata roots table 2. gc-ms results of agave attenuata roots, for identified compounds. number of peaks retention time peak area chemical formula molecular weight compound name 1 21.221 1.71 c16h22o4 278.3435 1,2-benzenedicarboxylic acid, bis( 2methylpropyl) ester 2 21.760 11.82 c17h34o2 270.4507 hexadecanoic acid, methyl ester 3 22.611 4.85 c16h22o4 278.348 dibutyl phthalate 5 23.887 60.41 c19h36o2 296.4879 9-octadecenoic acid (z)methyl ester 11 28.445 2.66 c23h46o2 354.617 docosanoic acid, methyl ester 12 28.719 2,55 c24h38o4 390.564 bis (2-ethylhexyl) phthalate the results of gc/ms suggest that the leaves and the roots of agave attenuata are a valuable source of diverse phytochemicals, which mainly belonged to fatty acids. chemically, a fatty acid is an organic acid that has an acid group at one end of its molecule, and a methyl group at the other end. fatty acids are typically categorized in the omega groups 3, 6 and 9 according to the location of their first double bond(15). .very long chain pufa may be used as a drug to reduce plasma triacylglycerol concentration and to reduce inflammation among patients with rheumatoid arthritis. reduce inflammation, fatty acids themselves or as part of complex lipids, are frequently used in cosmetics such as soaps, fat emulsions and liposomes(16) iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 81 reverse phase-high-performance liquid chromatography (rp-hplc) examinations of different fractions rp-hplc analysis of different extracts for iraqi agave attenuata leaves and roots were applied after extraction for further analysis to identify and quantify the major proposed active constituents. the reverse phase high performance liquid chromatography of different extracts obtained from iraqi agave attenuata leaves and roots confirms the following: rp-hplc for ethyl acetate and methanol fractions the result confirmed the presence three of these phenolic compounds (caffeic acid, pcoumaric acid and quercetin) in both ethyl acetate and methanol fractions of iraqi agave attenuata leaves and roots. the qualitative identification was made by comparison of retention times obtained at identical chromatographic conditions of analyzed samples (ethyl acetate and methanol fractions of leaves and roots parts) with authentic standards (caffeic acid, p-coumaric acid and quercetin) as shown in figures 6 and 7and table 3: figure 6. hplc chromatogram for, a: caffeic acid standard, b: p-coumaric acid standard and c: quercetin standard iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 82 figure 7. hplc chromatogram of, a: ethyl acetate fraction of the leaves, b: methanol fraction of the leaves, c: ethyl acetate fraction of the roots, d: methanol fraction of the roots. table 3. rt value of ethyl acetate fractions and methanol fractions of two parts of iraqi agave attenuata compared to caffeic acid, p-coumaric acid, quercetin standards. standards name rt value of standards leaves ethyl acetate fraction leaves methanol fraction root ethyl acetate fraction root methanol fraction caffeic acid 10.5 10.32 10.21 10.22 10.35 p-coumaric acid 8.85 8.8 8.72 8.70 8.65 quercetin 5.61 5.77 5.80 5.22 5.30 for quantitative analysis, the calibration curve was plotted using area under the curve (auc) versus four concentration levels for each caffeic acid, p-coumaric acid, quercetin standards from which the concentration of the proposed phenolic phytoconstituents in the ethyl acetate and methanol fractions were calculated applying straight line equation. the calculated concentration of proposed phenolic phytoconstituents in the iraqi agave attenuata was revealed in tables 4 and 5. table 4. type and concentration of the different phytoconstituents found in ethyl acetate fraction of leaves and root of the agave attenuata . ethyl acetate leaves fraction ethyl acetate root fraction phytoconstituents concentration ppm phytoconstituents concentration ppm caffeic acid 1029.2 caffeic acid 2271.1 p-coumaric acid 281.1 p-coumaric acid 733.5 quercetin 352.5 quercetin 437.8 iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 83 table 5. type and concentration of the different phytoconstituents found in methanol fraction of leaves and root of the agave attenuata methanol leaves fraction methanol root fraction phytoconstituents concentration ppm phytoconstituents concentration ppm caffeic acid 268.2 caffeic acid 181.1 p-coumaric acid 63.3 p-coumaric acid 493.75 quercetin 23.5 quercetin 152.2 quantitative concentration of proposed caffeic acid, p-coumaric acid and quercetin in ethyl acetate and methanol fractions revealed that, the roots contain the higher concentration of proposed p-coumaric acid and quercetin while the leaves contain the higher concentration of proposed caffeic acid. the higher concentrations of proposed caffeic acid, pcoumaric acid and quercetin where found in the ethyl acetate fraction for leaves and roots rather than methanol fraction. rp-hplc for steroidal saponin fractions the result confirmed the presence three of these steroidal saponin compounds (hecogenin, sarsasapogenin and tigogenin standards) in both ethyl acetate and methanol fractions of iraqi agave attenuata leaves and roots. the qualitative identification was made by comparison of retention times obtained at identical chromatographic conditions of analyzed samples (steroidal saponin fractions of leaves and roots parts) with authentic standards (hecogenin, sarsasapogenin and tigogenin standards) as shown in figures 8&9 and table 6: figure 8. hplc chromatogram for, a: hecogenin standard, b: sarsasapogenin standard and c: tigogenin standard iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 84 figure 9. hplc chromatogram of saponin fraction of the, a: leaves and b: roots. table 6. rt value of for steroidal saponin fractions of two parts of iraqi agave attenuata compared to hecogenin, sarsasapogenin and tigogenin standards. standards name rt value of standards leaves steroidal saponin fraction root steroidal saponin fraction hecogenin 3.2 3.22 3.10 sarsasapogenin 5.18 5.21 5.22 tigogenin 6.57 6.72 6.68 for quantitative analysis, the calibration curve was plotted using area under the curve (auc) versus four concentration levels for each hecogenin, sarsasapogenin and tigogenin standards from which the concentration of the proposed steroidal saponin phytoconstituents in the steroidal saponin fractions were calculated applying straight line equation. the calculated concentration of proposed steroidal saponin phytoconstituents in the iraqi agave attenuata was revealed in table 7. table 7. type and concentration of the different phytoconstituents found in steroidal saponin fractions of leaves and root of the agave attenuata . steroidal saponin leaves fraction steroidal saponin root fraction phytoconstituents concentration ppm phytoconstituents concentration ppm sarsasapogenin 277.06 sarsasapogenin 249.5 hecogenin 381.2 hecogenin 323.89 tigogenin 412.7 tigogenin 358.9 iraqi j pharm sci, vol.31(suppl.) 2022 gc/ms and rp-hplc estimation of iraqi agave attenuate 85 quantitative concentration of proposed hecogenin, sarsasapogenin and tigogenin in steroidal saponin fractions revealed that, the leaves contain the higher concentration of proposed hecogenin, sarsasapogenin and tigogenin than roots and the highest concentration of proposed steroidal saponin in iraqi agave attenuata was tigogenin follow by hecogenin and the least was sarsasapogenin. conclusion in this study, the iraqi agave attenuata leaves and roots parts were discovered as a novel natural source for different bioactive constituents, being the first iraqi study and perhaps worldwide about the presence of different types of fatty acid, some phenolic acids, flavonoids and steroidal saponins in the iraqi agave attenuata that were achieved by gc/ms and rp-hplc. gc/ms screening for n-hexane fraction reveal the existence of different types of fatty acid while chromatographic rp-hplc analysis was carried out to qualify and quantify phenolic acids (caffeic acid, p-coumaric acid), flavonoid (quercetin) and steroidal saponins (sarsasapogenin, tigogenin, hecogenin). the results detect that ethyl acetate fraction of both leaves and root has the highest concentrations of phenolic acids and flavonoid. pcoumaric and quercetin found in higher amount in root and caffeic acid in leaves. the results for steroidal saponins show all of them have a greater concentration in leaves. references 1. hap t, gehring u. biological activities of. 2008;5(2):285–332. 2. agave, plants for landscapes, containers, and medicinal use. 2010. available from: https://www.wikilawn. com/ medicinal-plants /agaves. 3. zander r 1892-1969. handwörterbuch der pflanzennamen=dictionary of plant names. 2002. 498. 4. rizwan k, zubair m, rasool n, riaz m, ziaul-haq m, de feo v. phytochemical and biological studies of agave attenuata. int j mol sci. 2012;13(5):6440–51. 5. ayoola a, efeovbokhan v, bafuwa o, david o. a search for alternative solvent to hexane during neem oil extraction. int j sci technol. 2014;4(4):66–70. 6. galanakis cm, goulas v, tsakona s, manganaris ga, gekas v. a knowledge base for the recovery of natural phenols with different solvents. int j food prop. 2013;16(2):382–96. 7. razak mfba, yong pk, shah zm, abdullah lc, yee ss, yaw itcs. the effects of varying solvent polarity on extraction yield of orthosiphon stamineus leaves. j appl sci. 2012 ; 15 ; 12(11) :1207–10. 8. santos jdg, branco a. gc-ms characterisation of sapogenins from sisal waste and a method to isolate pure hecogenin. bio. resources. 2014; 9(1):1325–33. 9. abrahem rm, awad zj. phytochemical study of steroidal sapogenin “tigogenin” present in the leaves of agave americana cultivated in iraq .iraqi j pharm sci. 2015;24(2):2015 . 10. hazrati s, nicola s, khurizadeh s, alirezalu a, mohammadi h. physico-chemical properties and fatty acid composition of chrozo-phora tinctoria seeds as a new oil source. grasas y aceites . 2019;70(4) :328. 11. ngamsuk s, huang tc, hsu jl. determination of phenolic compounds, procyanidins, and antioxidant activity in processed coffea arabica l. leaves. foods. 2019;8(9):1–13. 12. de combarieu e, falzoni m, fuzzati n, gattesco f, giori a, lovati m, et al. identification of ruscus steroidal saponins by hplc-ms analysis. fitoterapia. 2002;73(7– 8):583–96. 13. guclu-ustundag ö, mazza g. saponins: properties, applications and processing. crit rev food sci nutr. 2007;47(3):231–58. 14. blunden g, yi y, jewers k, burbage mb. agave ghiesbrechtii, a new source of gloriogenin. phytochemistry.1974;13(9):2025– 7. 15. bruno g. essential and non-essential fatty acids. lit educ ser diet suppl. 2005;4. 16. rustan ac, drevon ca. fatty acids: structures and properties. els. 2005;(september). this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 variables affecting pentoxifylline formulation (sr) 1 some variables affecting the formulation of pentoxifylline (ptx) as a solid sustained release dosage form ahmed h. hussain** , yehia i. khalil*,1 * college of pharmacy, university of baghdad. , baghdad , iraq ** college of pharmacy , university of kofa, najaf, iraq abstract an inert matrix that is used to control the release of (ptx) was prepared using eudragit rl100 and rspm types as matrix forming agent . the matrices were prepared by either dry granulation(slugging) , or wet granulation method using chloroform as a solvent evaporation vehichle. the cumulative release was adjusted by using polyvinylpyrollidone (pvp) or ethylcellulose (ec) polymers .the results indicated that both methods of preparation were valid for incorporation ptx as a sustained release granules .moreover ,the results revealed that best polymer used was eudragit rspm in 3:20 polymer drug ratio .besides to that , the results indicated that the release profiles were affected by phmedium , pvp and ec addition as an enhancer or retardant polymer used respectively. as well as to the method of preparation . key words : pentoxifylline sustained retease , eudragit rs , rl . ةصالخاا قاقحلاكقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلا.لا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلخنقاق ,100لا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلالاانقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاجللقويااقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلادتتسماقالتلدل قبلفقسكلاقبلااقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاع تلحتلىلل قرمقسللقمدختسملا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلالل الا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلغلاقبلاقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلا.لا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلا المقسلقسكلللقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلالاانقمدختسملا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاقمل لققلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاكعلعا قلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاتاوقق)لا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلترللا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلل( اقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاكعلعا قلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلالمعققمدختسملا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلارللااولااقحدا قملاا قوتقمل لقق لا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاتكلللق .المقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقللاا قلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلا تاتلقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلافقئ قحتقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلادل لتل قحابتاقخااكققح اااقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلايلااقحكعلعا قمدل ققلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاتكلاقبتا قبلفقشاكقود قلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلا تاتلق قملالدلق :بلااق .حداقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقللاا قلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلا تاتلقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلافقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقل قلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلحاخاعقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاكايللقق20:3ئأ ل قلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقل قلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلولوقملالدلقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلختيدوق لق ل الا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلغلاقئا.لا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلخنقاقم تعقق يستلىققلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلححقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلا ا مااغل فقا اقسدثللقبلفقسكلاقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلايلااق .لا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلياوققلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلافقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاعلاتقول لوقما لاالما قاقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلتثلوقخللللليحدللا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقل قيتلبققلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاقيعد قق بلفقلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاتللا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاتق .بتا قبلفقمل لققلا.را عون تيجاردويلا مادختسأب رضح دق نيلفيسكوتنبلا راقع ررحت ىلع ةرطيسلل مدختسملا لاعف ريغلا بلاقلاتكلللق . introduction tablet dosage form can be defined as a unit dose of medication containing one or more of medicinal agents, with or without diluents, made by molding the mixture in a suitable compressible shape(1).sustained release dosage form having drug release features based on the time or location designed to accomplish convenience and therapeutic not offered by conventional release form (2).an inert matrix was used to control the drug release , and this can be adjusted by using the enhancers such as microcrystalline cellulose , polyvinylpyrrolidone or surfactants .eudragit rl100 and rspm ,are methacrylic copolymers introduced as a coating materials with different permeabilities (3) , depending on functional ionized or neutral groups .they are commonly used in sustained release dosage forms (4). the distinguishing letters rl and rs related to the initial letters of german words " leihtduchlassig " freely permeable and schwerduchlassig , slightly permeable . pentoxifylline (ptx) also called oxpentifylline is one of the xanthine vasodilator derivative (5) ,that improve with its active metabolite peripheral aterial circulation, and enhance tissue oxygenation the apparent plasma half life of the drug and its metabolite is 2-3 hours . on the bases of using ptx as drug of choice in chronic occlusive aterial diseases , therefore ,it is of a wise candidate drug to be formulated in sustained release oral dosage form. the usual dosage form of ptx in controlled release tablet from available maintained one is one tablet (400mg.) twice daily . the objective of this study is to prepare controlled release ptx matrix tablets , utilizing dry and wet granulation technique by using eudragit rl and rs types with pvp and ec polymers as enhancer and retardant materials respectively , to control the release of ptx for extended period (6) . 1 corresponding author : e-mail : ybmmaz@yahoo.com received : 9/11/2007 accepted : 5/3/2008 iraqi j.pharm.sci., vol.17 (1) ,2008 variables affecting pentoxifylline formulation (sr) 2 materials and methods materials pentoxifylline supplied by slovakopharma , slovenia , aceton,acetic acid,chloroform ,ethylcellulose , pvp,from bdh chemicals ,ltd.liverpool , england disodium hydrogen phosphate , hcl ands lactose from reidal de haen ag seelz hanover , germany (ger) , eudrait rl100 and rspm , from rhom pharma gmbh weiterstadt ger. ,all other reagents were of analytical grade . equipments dissolution apparatus ,copley dissolution dis 8000 , copley scientific , ltd , uk , hardness tester , stokes monsanto corporation limited ,england ,ph-meter ,hanna instrument ph 211 microprocessor , italy,oven , water bath , memert ul 80 , rostfrei, schwach , germany . friabilator , sieves , roche type moore and wright ,sheffield , england ,sartorious balance , werk-gmbh ,type 1265 md , 2854 md,germany ,spectrophotometer , carry win uv , varian , australia ,tablet machine double punch , korsch , type eko , erweka ,gmbh, kr , offenbach main , germany methods : preparation of eudragit solutions : eudragit solutions rl100 and rspm were prepared separately at a concentrations 5 ,10 ,15% by dissolving the desired amount of eudragit rl100 and rspm with the desired volume of chloroform , the mixture was shacked for 15 minutes in 25°c until homogenous clear transparent solution was resulted (7) wet granulation : different formulas ( table 1 ) were prepared by weighing the drug and excipient equivalent to 30 tablets after drying , then blending the powder and mixed with binding solution of eudragit polymers used gradually , until proper ball test consistency was result ,the wet mass was screened through 12 mesh sieve and dried in pre warmed oven maintained at 50°c for 2 hours , the dry granules were reduced in size by screening them through 16 mesh size sieve . then an equivalent weight of granules that contain 400 mg. ptx was mixed with calculated amount of magnesium stearate 0.5% w/w for one minute and compressed into a tablets using 10mm. biconcave double punch compression tablets machine at 11ton compression force table (1) . different formula of pentoxifylline with different physical properties of compressed tablets frmula ptx (gm) eudragit rl(mg) eudragit rs(mg.) pvp (mg) ec (mg) lactose (mg.) magnesium stearate (mg.) total weight (mg.) friability % hardness (mg.) appeara f1 400 20 127.25 2.75 550 3.0 17.1 friable , wdusty f2 400 40 107.25 2.75 550 2.8 17.2 friable , wdusty f3 400 60 87.25 2.75 550 1.5 19 friable , wdusty f4 400 80 67.25 2.75 550 1.0 >20 friable , wdusty f5 400 20 127.25 2.75 550 3.0 17.2 friable , wdusty f6 400 40 107.25 2.75 550 2.7 17.4 friable , wdusty f7 400 60 87.25 2.75 550 1.5 18.4 friable , wdusty f8 400 80 67.25 2.75 550 0.9 >20 white accepte f9 400 60 13.8 73.45 2.75 550 0.67 >20 gray accepte f10 400 60 27.6 59.65 2.75 550 0.54 >20 faint yellow accepte f11 400 60 41.4 45.85 2.75 550 0.32 >20 faint yellow accepte f12 400 60 13.8 73.45 2.75 550 0.66 20 white accepte f13 400 60 27.6 59.65 2.75 550 0.42 20 white accepte f14 400 60 41.4 45.85 2.75 550 0.34 20 white accepte iraqi j.pharm.sci., vol.17 (1) ,2008 variables affecting pentoxifylline formulation (sr) 3 dry granulation : the method of dry granulation was introduced as an alternative method for a selective formula ( f14 ) , this was briefly done by weighing the drug and another excipients that equivalent to 30 tablets,then blending and compressed by means of wide single punch machine into 22mm. diameter slugs. these slugs were reduced in their sizes by milling and sieved by 16 mesh size sieves , the resulted particles were mixed with calculated amount of magnesium stearate 0.5% (w/w) and compressed into 10mm. double punch compression tablet machine at the same compression force . assay of total ptx in prepared sustained release ( sr) tablets: an accurate weight of powder of triturated tablets equivalent to 400mg. of ptx was added to 500ml. of distilled water and shacked for 15 minutes and filter 1ml. of filtrate was diluted to the appropriate concentrationق(8μg/ml)ققwithقdistilledقwater , the absorbance of the final solution was determined spectrophotometrically at 274nm wave length(5) this procedure was performed for those tablets prepared in all methods mentioned before . assay of total ptx in reference srtablets (trental 400mg): ten tablets of the reference product were weighted individually to get the net weight of each tablet , and then triturated together , an accurate weight of triturated powder equivalent to 400mg. ptx was dissolved in 500ml. distilled water , the final solution was diluted to maintain ptx concentration at 8μg/mlقby distilled water , the absorbance was measured using uv-spectrophotometer at 274nm. wave length (5) . friability and breaking strength measurement this measurement was carried out for all prepared formulas using roche friabilator . the loss in weight of 20 tablets tested before and after rotation should not exceed 1% of total weight of tablet (8) .on the other hand the hardness of the prepared tablets were examined by means of monsanto hardness tester . dissolution behavior: dissolution of the ptx from prepared tablets was carried out using usp basket method maintained at 37°c temp. , and 75 r.p.m speed for 500ml.of buffer solutions ( ph 1.2 ,4.6,and 6.8 ). samples of 5ml. were withdrawn at 1 hour time intervals for 6-10 hours then the samples filtered and diluted, then cumulative drug release was measured at 274nm. for triplicate samples . results and discussions formulation of ptx as a srtablets : all the powders blend and granules were intended for compression successfully into shiny look appearance . the breaking strength were varied from friable to hard (20kg.) hardness. this variation may be referred to the polymer type and concentrations (9 , 10 ) . as shown in formula 4 and 8. on the other hand pvp presence gave harder granules ( formula 12 ) compared with others ( 11) . the same results obtained when ethylcellulose was used as retardant material , since both pvp and ec act as binding and consolidating agents in tablets formulation (12 ,13) . dissolution behavior effect of eudragit polymer : figures (1 and 2) illustrate the cumulative release of ptx from various polymer-drug ratios in phosphate buffer (ph6.8) , the results indicated that the drug release is increased as a function of increasing this ratio for both eudragit types used , this behavior referred mainly to the thickness of the polymer around drug particles, that results in a delayed permeation of water inside drug particles and dissoluted drug outside stagnant layer of ptx particle (14) .on the other hand the cumulative release of ptx from two polymers used (fig.3) revealed that after 10h. of dissolution, the percent of drug release from formula 3 is 88% compared with 60% in formula 7 , while the reference tablet (trental400) gave 51% drug release after same period of dissolution . this difference may be attributed to the presence of highly basic quaternary ammonium groups (1:20 and 1:40)ratios for both eudrait rl and rs types ,respectively which they undergo more solubilization in acidic medium, then the over all solubility of the drug within deaggregated polymer is increased (15) . iraqi j.pharm.sci., vol.17 (1) ,2008 variables affecting pentoxifylline formulation (sr) 4 figure 1. the percent of ptx released from different formulas at phosphate buffer ph 6.8 using eudragit rl100 polymer. figure 2. the percent of ptx released from different formulas at phosphate buffer ph 6.8 using eudragit rspm polymer. figure 3. the cumulative percent of ptx released from formulas 3,7 and reference (trental 400 mg.) at different ph-medium. effect of polymer type : concerning the type of eudragit used , fig.3 demonstrate that the time for 50% drug release extended for 2 hours compared with more than 5 hours for both eudragit rs and rl , respectively . the illustration for this variation related to the higher permeability of eudragit rl than that of rs polymer type(16) . effect of pvp addition : based on the results obtained as shown in fig .4 , it was shown that addition of pvp in formula 7 made of eudragit rs type , enhance the cumulative release to about 96% when 7.5% w/w pvp used , compared with 50% for reference one this effect may be referred to the higher solubility of pvp in different media mainly at ph 5-7 medium (17) . figure 4. effect of pvp addition on the cumulative released of ptx from tableted matrix at different ph-medium. effect of ec addition : the influence of addition 2.5 ,5 and 7.5% w/w of ec to the matrix formulation of formula 7 was illustrated in fig. 5 ,which represents the cumulative release of ptx ,it was seen that when ec incorporated as a retardant material, a slight less or more in drug release was resulted compared with that of reference one (trental) , and in an appreciab decrease in drug in drug release compared with the other formulas free from ec , which is attributed to the insolubility of ec in water, besides rigid consolidation of eudragit rs and ec polymer that retain drug in matrix , this phenomena is in a consistent with results obtained by aldermann (18) and sreubel (19) . 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 tim e(hour) % of d ru g re le as ed f1 f2 f3 f4 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 time(hour) % of d ru g re lea se d f5 f6 f7 f8 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 tim e(hour) % o f d ru g r el ea se d f3 f7 referenc e 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 time(hour) % of d ru g r el ea se d f9 f10 f11 refrence ---ph1.2- ---ph4.6-- -----------ph6.8---------------------------------------- ---ph1.2-- ---ph4.6-- -----------ph6.8---------------------------------------- iraqi j.pharm.sci., vol.17 (1) ,2008 variables affecting pentoxifylline formulation (sr) 5 figure 5. effect of ethylcellulose (ec) addition on the cumulative released of ptx from tableted matrix at different phmedium. effect of granulation method : the selected formula 14 was reformulated again using dry granulation method , the results indicated that cumulative drug release by this method gave 75% , compared with 59% for wet granulation after 10h. , as shown in fig. 6 , this behavior may be referred to the solvent effect (chloroform vehicle) that used in a later method help in a formation of more van der waal"s forces among polymer used and ptx (20) . these bonds make a matrix more rigid and delayed drug release through them. figure 6. effect of preparation method on the cumulative release of ptx from selected formula 14 at different ph medium. conclusion : concerning the method used , the study demonstrate that both methods were valid for formulation of ptx as promised acceptable srgranules. but wet method is preferred . both eudragits used rl100 and rspm were succeeded in a formation of delayed release matrix . eudragit rs is best to be used in 3:20 polymer :drug ratio for sr granules to utilize ptx as sr matrix similar to (trental400) . the dissolution behavior of ptx from prepared granules varies with different ph medium , and coating materials . generally , the release of ptx is affected by addition of pvp and ec mainly for matrix made of eudragit rs(pm) type in a concentration 7.5% w/w. best formula when ec was added to formula 7 represented formula 14 . refferences 1. ansel h.c. ,allen j. l.," pharmaceutical dosage form and drug delivery system ", 7th. edition ,lippincot"s william and wilkins philadelphia 1999 . 2. gibaldi a., feldman m. ," scale-up of oral extended release dosage" form aaps/ fd workshop committee , 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" formulation and evaluation of sr diclofenac tablet prepared by matrix embedding technique " drug development research , 2001 ,53 , p 1-8 . 17. hadi mehergana ,seyed ali reza ," the release behavior and kinetic evaluation of diltiazem hcl from various hydrophilic and plastic matrices " iranian journal of pharmaceutical research " 2005,3,137 18. alderman da. ," review of cellulose ether in hydrophillic matrices for oral controlled release dosage forms .", int.journal of technology " , 1984 , 5, 1-9 . 19. sreubel a. , seipmann j. , bodmier r. ," ph depended release of weaklybasic drug from water soluble and insoluble matrix tablet" controlled release journal ,2002 ,67 , 101-110 20. hariharan m. , wheatly t. a. ," controlled release matrices from carragenan , method of preparation and dissolution studies " pharmaceutical development technology , 1997 ,2(4), 383-393 . iraqi j pharm sci, vol.31( suppl. ) 2022 osteospermum ecklonis: in vitro antileishmanial efficacy doi: https://doi.org/10.31351/vol31isssuppl.pp45-53 45 evaluation of antileishmanial activity of osteospermum ecklonis extract of aerial parts against leishmania donovani: in vitro(conference paper) # hind m. jewelee*,1 and thukaa z.abdul-jalil * # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq abstract lack of safe available non -resistant treatment for visceral leishmaniasis (kala-azar) keeps limiting the complete cure of this disease, drugs that have toxic side effects or lack of effectiveness have led to disease relapse, all these factors have lightened the way to the search for alternative drugs from natural resources that have been shown to have antileishmanial activity through literature survey in the present study, the comparative in vitro anti-leishmania activity of various fractions of osteospermum ecklonis aerial parts fractions have been evaluated. extracts were prepared through maceration and soxhlet apparatus using 85% methanol and fractionation was done by separating the active constituents according to the differences in their polarities using four solvents in different polarities )petroleum ether, chloroform, ethyl acetate, and finally n-butanol( ،two of the resultant fractions( petroleum ether as well as nbutanol fractions ) were chosen to test the effective inhibition of leishmania donovani, results prove with no doubt that the petroleum ether fraction of the maceration aerial parts in a concentration of 2.5 mg/ml had better antileishmanial activity than other concentrations of tested samples and the result coincided with the antileishmaniasis activity of official treatment (pentostam®) ,this finding can be attributed to the terpene nature of the materials used to be existed in such fraction. these observations have paved the road to step in for extended studies in relation to the conventional herbal medicines for better and safe alternatives to available synthetic chemical drugs. keywords: osteospermum ecklonis, anti-leishmania activity, leishmania donovani, maceration, soxhlet apparatus تقييم النشاط المضاد للليشمانيا لمستخلص #) بحث مؤتمر ( لألوستوسبيرموم إكلونيس ضد الليشمانيا دونوفاني: في المختبراألجزاء الهوائية *ذكاء زهير عبد الجليل و 1*،جولي محمد هند 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي فرع العقاقير والنباتات الطبية، كلية الصيدلة، جامعة بغداد، بغداد العراق . * الخالصة ( باإلضافة الى وجود االثار الجانبية لألدوية المستخدمة ذات التأثير عدم وجود عالج كفوء لمرض الليشمانيا الحشويه )حمي الكاالزار عبد الطريق للبحث عن ادويه بديله مستخلصه من الموارد الط عادة الى انتكاسة المرض. كل هذا بيعية السام او قليل الفعالية، كلها عوامل تؤدي لمي.العالحتوائها على تأثير مضاد لإلمراض الطفيلية من خالل البحث osteospermum)في هذه الدراسة تم تقييم النشاط المقارن المضاد لليشمانيا في المختبرلالجزاء الهوائية لنبتة االقحوان االفريقي ) ecklonis من الميثانول وتمت٨٥تم اعداد المستخلصات بطريقتي النقع وبجهاز السكسوليت باستخدام للتأكد من وجود فعالية مضادة لليشمانيا ٪ ل البيوتانولي التجزئة للمكونات للمستخلصات باالعتماد على اختالف قطبياتها باستخدام مذيبات مختلفة القطبيه )االثير البترولي، الكلوروفورم، الكحو بليتها على التثبيط الفعال قا البيوتانولي( الختباراثنان من المتجزأت الناتجه تم اختيارهما )متجزأة االثير البترولي وكذلك الكحول .واالثيل اسيتيت( ملغم/ مل اظهر ٢.٥بال شك اثبتت النتائج ان متجزآ االثير البترولي لألجزاء الهوائية ذات التركيز (.(leishmania donovaniلطفيلي الليشمانيا والذي (pentostam)العالج التقليدي لليشمانيانشاط مضاد لليشمانيا أفضل من التركيزات األخرى للعينات المختارة لتزامن نشاط العينة مع نشاط يمكن ان يعزى الي طبيعة المواد التربينيه الموجودة في هذا المتجزأ. تاحة. هذه االستنتاجات عبدت الطريق للتوسع في دراسة األدوية العشبية التقليدية لغرض الحصول على بدائل أكثر فعالية وامنا" من األدوية الم . جهاز السكسوليتا ،طريقة النقع ، طفيلي الليشماني، النشاط المضاد لليشمانيا ، االقحوان االفريقي ة:الكلمات المفتاحي 1corresponding author e-mail: hmchmc7jan@gmail.com received:19 /5 /2022 accepted: 8/9 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp45-53 iraqi j pharm sci, vol.31(suppl.) 2022 osteospermum ecklonis: in vitro antileishmanial efficacy 46 introduction leishmania donovani protozoan is known to be the causative parasite of what is called (kalaazar) or visceral leishmaniasis, this disease is transmitted from animals (pets or rodents) or from person to another person through phlebotomize sand fly, being responsible for about 500000 cases per year worldwide it’s characteristic symptoms are fever, weight loss, hepato-splenomegaly, diarrhea, vomiting and lymphadenopathy, ,it is endemic in the middle and southern governorates , the major incidence is among children and few among adults indicating age related improved immunity (1,2). first line recommended therapies are pentavalent antimonial sodium stibogluconate (pentostam)®, amphotericin b (polyene antibiotic) that have serious side effects such as nausea, vomiting ,arthralgia ,hepatitis, cardiac dysrhythmias , pancreatitis and nephrotoxicity(3,4), however; the greatest limitation that the above medications have include the development of drug resistance organisms , a disadvantage that is clearly shown with sodium stibogluconate with failure rates up to 65% in areas of endemicity, (5) one of the suggested solution was the use of combination therapy to reduce the side effects and enhance effectiveness , the effectiveness of multidrug protocol was noticed to be almost similar to that of mono-drug therapy but this approach used to be indicated as less side effect outcome and was the essential motive to seek and quest for more secure and efficient antileishmanial agents (3) . osteospermum genus frequently used by the arabian bedouins for treatment of fever, stomach illness as well as liver disorders, being rich with triterpenes, glycosides, sterols, one of the important domiciliary plants cultivated in iraq as an ornamental plant is osteospermum ecklonis f. asteraceae also known as african daisy has been used over decades as a remedy for certain health issues, as cardiovascular(6) antimicrobial(7), antiparasitic (8) , whitening, antitumor (7), and the flowers used as a relaxing aid not surprisingly as it was reported to have several phytochemicals of pharmacological importance such as flavonoids and phenols, terpenoids, essential oils, saponins, polysaccharides, coumarins (9), carotenoids (10) as well as phytosterols sesquiterpenes and triterpenes were isolated from the genus osteospermum throughout a study that was conducted in 1983 11 that paved the way for the conduction of this study, the leishmania donovani represent one of the most endemic diseases in iraq ,that’s why this study was conducted as a search for alternative remedy for this illness. materials and methods collection and authentication of plant materials osteospermum ecklonis was obtained from an herbarium near baghdad during march and the plant was authenticated by prof. dr sukaena abbas\ department of biology\ college of the science \university of baghdad. aerial parts were separated from roots and were washed carefully to remove any contaminants and then brought to dryness in a shady room for about a month, for conduction of the study, the dried plant from aerial parts, were grounded into a fine powder by, first, manually followed by electrical grinding extraction and fractionation of plant extracts the dried, powdered aerial parts of the plant (100gm) was macerated with 750 ml of 85%mathanol for 15 days with stirring for 1hr daily, the macerate was filtered, and new solvent added, another 100 gm of powdered aerial parts plant material was extracted by soxhlet apparatus with the same solvent mixture on a medium heat for a total of 20 hrs., the filtrate from both ways were evaporated and were kept aside. the dried extract from the previously mentioned two methods, is then suspended in 250 ml distilled water and washed repeatedly and separately with another 250 ml of petroleum ether, chloroform, ethyl acetate, and finally n-butanol, in a separatory funnel for three times in each solvent, fractions of each solvent were collected separately all fractions (except for n-butanol )were dried over anhydrous sodium sulphate, filtered before been evaporated with the rotatory evaporator until dryness, as illustrated in the following schematic diagram, in figure(1) iraqi j pharm sci, vol.31(suppl.) 2022 osteospermum ecklonis: in vitro antileishmanial efficacy 47 . figure 1. schematic diagram illustrates the process of extraction and fractionation(12–17) percentage yield the weight of each obtained fraction was measured ,divided by the weight of the sample ,the percentage yield of each fraction was calculated based on the following equation (18) : percentage yield= 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐞𝐚𝐱𝐭𝐫𝐚𝐜𝐭 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐬𝐚𝐦𝐩𝐥𝐞 × 𝟏𝟎𝟎 phytochemical analysis qualitative investigation of secondary metabolites existence was conducted based on the standard tests that is described in harborne (19). qualitative identification by tlc thin layer chromatography was held for the aerial parts to compare between the fractions for the same plant part and it was performed using the chromatographic system with silica gel gf254 as stationary phase, the following solvents were utilized: (s1) toluene: ethyl acetate (93:7) (s2) toluene: ethyl acetate: formic acid: acetic acid (20:10:10:7.5) preparation of inoculum leishmania. donovani was obtained from the bone morrow of an infected child at a hospital in baghdad and brought to the center of biological technology research at collage of science in alnahrain university. evaluation of the antileishmanial activity of plant extract started by first propagating the organism by incubating in the log phase in rpmi media (roswell park institute park memorial) this culture media is enriched with 12% serum from calf fetal, at 25 c for 5 days until reaching an average of 105 parasites \ml in haemocytomer (20,21). preparation of plant extract concentrations based on the percentage of yield two fractions were nominated to test presence of antiparasitic activity that is comparable to conventional therapies. from the aerial parts (g)extract that was obtained by hot (s) and cold (m) methods, petroleum ether (pe) and nbutanol fractions (n-b) were the nominated ones thus there was a total of four samples to conduct the experiment, 1 mg was weighed from each sample and was set aside to prepare five successive serial dilutions, samples were denoted as follows: sample a= (m\g\pe), sample b= (s\g\pe), sample c= (m\g\n-b), sample d= (s\g\n-b). for each of four samples, first dimethyl sulfoxide (dmso) in 100%v/v (20) was added as solubilizing agent in an amount that doesn’t exceed 20 µ l to 1mg extract then complete the volume with distilled water till reaching required concentration (1 mg/ml) which was successively utilized to prepare successive dilutions ended up with the following iraqi j pharm sci, vol.31(suppl.) 2022 osteospermum ecklonis: in vitro antileishmanial efficacy 48 concentrations 1000µg\ml, 500µg\ml, 250 µg\ml, 125µg\ml, and 62.5 µg\ml(22) . preparation of positive control the positive control was the pentavalent antimonial (sodium stibogluconate injection 100 mg\ml) from glaxosmithkline\uk, which is the classic drug for kala-azar fever caused by l. donovani, sodium stibogluconate (100 mg\ml) was diluted several times until obtaining a concentration of 100µg\ml, then 6 µl were inoculated in each well that was already containing 1 ml of rpmi and 1ml of l. donovani inoculum which was applied into two wells successively to assess variance. evaluation of samples activity against l. donovani to assess antileishmanial activity, flat bottom plate that contained 96 wells was used, as shown in figure (2), leishmania culture was added to all of them, then 90 wells were filled with 10µl (x3) of the fraction concentrations that were previously prepared, another three wells were used as negative control i.e no added materials to the lieshmania culture , then two wells were filled with positive control of sodium stibogluconate and the last well was supplied with 50% dmso thus reaching a total of 96wells. the plate was incubated at 25 ± 1c for 24 hours after which a 10µl mtt dye (3-(4,5-dimethyl thiazo-2-yl)-2,5-diphenyltetrazolium bromide) was added in each well then incubated again for further 4 hours at 25 ± 1 c to assess metabolic activity, followed by addition of dmso to each well as a solubilizing agent that will target the mtt purple dye inside the living matter release and making scanning process possible (23). figure 2. 96 wells plate that is ready for scan with elisa spectrophotometric assessment by eliza metabolic activity observed with the aid of elisa spectrophotometer apparatus that measures the optical density in each well at wave length of 490 nm, the higher the number of living matter the more purple color shown and the higher absorbance of elisa reading (21–25) . statistical analysis statistical analysis was performed by calculating inhibition percentage of each mean for the three fraction concentrations gradients applied, then significance of results was tested via mean comparison utilizing one-way anova test of ibm software, least statistical differences (lsd) and p value to indicate significant or non-significant difference of inhibition rate from that of the sodium stibogluconate(26). results and discussion extraction and fractionation two different methods were used for the extraction in this study: the soxhlet (hot) method and maceration (cold) method. to reach a level of certainty about who yields best percentage, the yield of each fraction with its percentage is illustrated in the table (1): table 1. the yield of aerial parts of the plant with the denoted method of extraction. extraction method fraction name aerial parts\ soxhlet % yield aerial parts \maceration % yield petroleum ether (f1) 2.493 gm 2.493% 4.344gm 4.344% chloroform (f2) 0.043 gm 0.043% 0.071gm 0.071% ethyl acetate (f3) 0.132 gm 0.132% 0.064 gm 0.064% n-butanol (f4) 1.98gm 1.98% 1.23gm 1.23% iraqi j pharm sci, vol.31(suppl.) 2022 osteospermum ecklonis: in vitro antileishmanial efficacy 49 qualitative phytochemical analysis revealed the presence of appreciable phytochemicals, terpenes and steroids existence was recorded at the pet. ether fraction while tannins and flavonoids existence were recorded at the ethyl acetate as well as n-butanol fractions as shown in the table (2) both of pet. ether. fraction and n-but. fractions obtained from the maceration and soxhlet methods were chosen for this study due to higher yield while chloroform layer and ethyl acetate layer were neglected due to low yield. table 2. preliminary chemical tests. fraction name test name aerial parts/maceration aerial parts/soxhlet f1 f2 f3 f4 f1 f2 f3 f4 keler -killiani test for cardiac glycosides -ve -ve -ve -ve -ve -ve -ve -ve dragendroff’s test for amin compounds -ve +ve -ve -ve -ve =ve -ve -ve mayer’s test for alkaloids -ve -ve -ve -ve -ve -ve -ve -ve salkowiski test for terpenoids +ve -ve -ve -ve +ve -ve -ve -ve lieberman test for steroids +ve -ve -ve -ve +ve -ve -ve -ve braeman’s test for tannins -ve -ve +ve +ve -ve -ve +ve +ve alkaline test for flavonoids -ve -ve +ve +ve -ve -ve +ve +ve saponin test -ve -ve -ve -ve -ve -ve -ve -ve qualitative identification by thin layer chromatography tlc of petroleum ether for aerial parts of plant was done by using the solvent systems (s1) and was visualized by spraying with h2so4 (5%) spray reagent followed by heating. petroleum ether showed spots of numbers of steroidal components, with the same rf value similar to that of ß-sitosterol as illustrated in the figure (3). figure 3.tlc chromatogram of pet. ether / aerial parts and ß-sitosterol standard, analyzed using solvent system s1 figure 4. tlc chromatogram obtained by s2 for analyzed fractions (n-but) with caffeic acid and p-coumaric acid standards under uv light, a:254 nm and b: 366 nm. tlc for n-butanol fraction was performed using the solvent system (s2) spots were detected under uv light (254,366 nm). the tlc analysis revealed the presence of chlorogenic acid in nbutanol as shown in figure (4) iraqi j pharm sci, vol.31(suppl.) 2022 osteospermum ecklonis: in vitro antileishmanial efficacy 50 antileishmanial activity test the optical density (od) data obtained from eliza were utilizes to calculate the % of organisms died by each concentration of each tested fraction, according to the equation: % of inhibition rate (od control-od test /od control)*100 (22 ) % ir = od control − od test 𝑂𝐷 𝐶𝑜𝑛𝑡𝑟𝑜𝑙 × 100 calculation of the % of inhibition rate for each concentration of every fraction was conducted and compared to the calculations of he + ve and − ve controls, as well as anova analysis was performed to find the conc. that has no significant mean difference from that of the +ve control (the null hypothesis is to be achieved) at (p> 0.005), calculations revealed the results that are illustrated in the following discussion: fraction a (m\g\pe) calculation of percentage of toxicity and mean comparison for the tested five concentrations of fraction a revealed that there is high coincidence between concentration 2.5 mg\ml and 5 mg/ml that gave ir % of 84.52 and 81 % respectively which are similar to that observed for the positive standard as shown in figure (5) . figure 5. %ir for aerial maceration petroleum ether fraction (a) dilutions (m\g\pe) anova analysis of the results revealed non-significant differences between conc. a4 and that for +ve control, the result that confirms what stated before, that regarding this fraction conc. of 2.5 mg/ml coincide to the results of +ve control with (p >0.005) next to a5, as illustrated in figure (6). figure 6. anova diagram for different concentrations of fraction a. fraction b (s\g\pe) calculations of the percentage ir and mean comparison revealed that conc. 5mg/ml (b5) has close results to that of the +ve control as shown in figure (7). figure 7. %ir for aerial hot petroleum ether fraction (b) dilutions (s\g\pe) anova analysis revealed comparable and nonsignificant differences of b5 and +ve control, as the non-significant mean differences is the one to be achieved in this study to get an effect similar to that of the +ve control.as was stated previously in this section and as illustrated in figure (8). figure 8. anova diagram for different concentrations of fraction b. iraqi j pharm sci, vol.31(suppl.) 2022 osteospermum ecklonis: in vitro antileishmanial efficacy 51 fraction c(m\g\n-b) percentage ir for conc. 5mg/ml (c5) revealed very close result to that observed for the +ve control as shown in figure (9). figure 9. %ir for aerial maceration n-butanol fraction (c) dilutions (m\g\n-b) anova analysis revealed non-significant mean difference between the c5 and that of the +ve control as illustrated in the figure (10) . figure 10. anova diagram for different concentrations of fraction c fraction d (s\g\n-b) only conc. 5 mg/ml (d5) showed ir percentage similar to that of the +ve control as illustrated in figure (11). figure 11. %ir for aerial hot n-butanol fraction (d) dilutions (s\g\n-b) anova analysis confirmed the above result and revealed that there are no significant mean differences between d5 and the +ve control, revealing that the fraction d at a conc. of 5mg/ml has inhibition rate similar to that of the +ve control as illustrated in the figure (12). figure 12. anova diagram for different concentrations of fraction d estimating of antileishmanial activity was conducted for the first time for the plant o. ecklonis cultivated in iraq, testing was carried for n-butanol and pet. ether fractions of aerial parts (that was obtained by maceration and soxhlet), referral +ve standard was pentostam®, coincidental antileishmanial activity was encountered in certain fractions and certain concentrations as illustrated in table (3): table 3. antileishmanial active concentration of each tested fraction name of fraction name of fraction antileishmanial active conc.mg/ml a pet ether fraction from aerial parts maceration extraction 2.5 b pet ether fraction from aerial parts soxhlet extraction 5 c n-but. fraction from aerial parts maceration extraction 5 d n-but. fraction from aerial parts soxhlet extraction 5 iraqi j pharm sci, vol.31(suppl.) 2022 osteospermum ecklonis: in vitro antileishmanial efficacy 52 as shown above, stronger antileishmanial activity was encountered at the petroleum ether fraction of the maceration aerial parts (a)as it was gained at a lower concentration (2.5 mg/ml) than the antileishmanial concentration of other tested samples, and as a represents pet. ether fraction, that result of anti-leishmaniasis activity which coincided with the official treatment (pentostam)® activity can be attributed to the terpene nature of materials that are used to be 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fraction from eugenia pruniformis leaves. an acad bras cienc. 2020;92(4):1–14. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.32( 1 ) 2023 assessment hematological indices & il-6 in workers who live near to crude oil wells doi: https://doi.org/10.31351/vol32iss1pp237-244 237 assessment of certain hematological indices and interlukin-6 in workers and individuals who live near to crude oil wells in middle petroleum refinery, iraq mariam.r. alghara*,1 , ausama. ayob. jaccob** and azza.sajid. jabbar** * ministry of health and environments, muthanna, iraq ** department of pharmacology and toxicology, college of pharmacy, university of basra, basra, iraq abstract environmental exposures to a variety of pollutant elements especially fuel waste products may result in harmful impacts on the human body physiology. the inflammatory response and the hematological system are the major affected systems. the aim of the study is to reveal the impact of oil exposure on several hematological parameters and interleukin-6 of oil workers and to detect which parameters are more affected as prognosticators for clinical disorders. this study included three groups, fifty (50) individuals in group i (control healthy group); fifty (50) individuals in group ii workers at oil wells; and fifty (50) individuals in group iii who live close to oil wells. physical parameters (pulse rate, blood pressure, random blood glucose), serum interleukin 6, hematological parameters are measured. significant rise in pulse rate, serum interleukin 6 and random blood glucose were documented in workers. hematological parameters were adversely-affected by air pollution. there was a significant rise in platelets count in group ii. the correlation between il-6 and certain hematological indices revealed that each of white blood cells and platelets were significantly correlated with il-6. in conclusion, people who work or live close to oil wells area showed alterations in hematological and inflammatory parameters (increase in interleukin 6), which may be related to continuous exposure to petrol fumes. keywords: oil wells, hematological indices, interleukin 6. في العمال واألفراد الذين يعيشون بالقرب من آبار النفط interlukin-6تقييم بعض مؤشرات الدم و في مصافي النفط الوسطى ، العراق ** عزة ساجد جبار و 1**،أسامة ايوب يعقوب ، *مريم رياض الغرة المثنى، العراق وزارة الصحة والبيئة، * العراق البصرة، جامعة الصيدلة، كلية والسموم، فرع االدوية ** خالصةال األنظمة ا من العديد على ضارة تأثيرات له الوقود نفايات منتجات وخاصة البيئي التلوث عناصر من لمجموعة البيئي لتعرض النفط على الفسيولوجية. االستجابة االلتهابية والجهاز الدموي هما النظامان االكثر تأثرا. الهدف من الدراسة هو الكشف عن تأثير التعرض لمشتقات النفط واكتشاف العوامل األكثر تأثًرا كمنبهات لالضطرابات السريرية. اشتملت هذه الدراسة على ثالث لعمال 6انترليوكينعايير الدم و العديد من م الذين يعيشون بالقرب من آبار النفط. يتم 3ر النفط والمجموعة باعمال في آ 2والمجموعة 1ة السيطر شخًصا. مجموعة 50مجموعات ، كل منها سكر و 6، تحاليل الدم. لوحظ إرتفاع كبير في معدل النبض ، إنترليوكين 6 ، انترليوكين )النبض، ضغط الدم، سكر الدم( التأثيرات الفيزيائية قياس لوحظ و 2ارتفاًعا كبيًرا في عدد الصفائح الدموية في مرضى المجموعة وجدحيث تأثرت التحاليل الدموية سلباً بتلوث الهواءوفي المصل. الدم انترليوكين بين الدم 6العالقة تحاليل من حيث وبعض كل البيضاءأن الدم انترليوكين كريات بـ كبير بشكل مرتبطة كانت الدموية . 6والصفائح تكون مرتبطة االستنتاج: أظهر األشخاص الذين يعملون أو يعيشون بالقرب من منطقة آبار النفط أن التغيرات في العوامل الدموية وااللتهابية قد .6بالتعرض المستمر ألبخرة البترول ، مما يؤدي أيًضا إلى زيادة كبيرة في اإلنترلوكين الرئيسية: آبار النفط ، مؤشرات الدم ، اإلنترلوكين اتالكلم introduction crude oil is a major component that workers may exposed to it which is a blend of saturated and unsaturated hydrocarbon in a liquid form, and the composition of crude oil including (benzene, toluene, lead, oxygenates, ethyl benzene, and three isomers of xylene) (1). occupational exposure to crude oil and its product regarded as one of the most serious issues around the world today. it has a negative impact on the health and environment. the air at this workplace is made up of a variety of gases as well as tiny (solid and liquid) particles. some compounds are derived from natural sources, while others are chemically synthesized (2). the combustion of these large amounts of gases in internal combustion engines produces hazardous emissions (3). hazardous chemicals can accidentally-leak into the environment, but a variety of air pollutants considered as end products of industrial facilities and other sources, which can have adverse outcomes on human health and the ecosystem (4). 1corresponding author e-mail: ausama.jaccob@uobasrah.edu.iq received: 17/4 /2022 accepted: 3/7 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol32iss1pp237-244 iraqi j pharm sci, vol.32(1)2023 assessment hematological indices & il-6 in workers who live near to crude oil wells 238 oil metabolites is predominantly found in the environment as a result of air pollution from oil refineries (5). chronic exposure to the stream of the gases and its metabolite, puts workers at risk for a variety of diseases that affect multiple systems in the body, including the renal system, immune system, cardiovascular (cv) system, and respiratory system (6). the relative concentration of gasoline components, on the other hand is determined by the crude oil source, refining method, and production lines employed (6). certain volatile chemicals (xylene, toluene, ethyl benzene, and benzene) are harmful pollutants according to toxicological studies which are steam fractions of gasoline that are progressively export to the air and water (7). when inhaled or swallowed, gasoline is quickly absorbed, uptake and dispersed in the body after absorption. following metabolism in the liver, small percent is exhaled intact or excreted as inactive metabolites in the urine. the active metabolites are subjected to additional assays processes of toxic kinetics, such as the formation of oxidative tissue damage and reactive oxygen species (ross), which results in altered structure and function, as well as multisystem toxicity (8). benzene is converted to phenol in the liver by the enzyme cytochrome p4502e1 (cyp2e1), which is then hydroxylated to hydroquinone, catechol, and 1,2,4-benzenetriol. because benzene metabolism produces a large number of reactive metabolites, benzene toxicity is expected to be mediated through numerous mechanisms (8). hematological system is one of the systems that is affected by oil exposure and its metabolites (5). excessive benzene exposure has been shown to affect the bone marrow, resulting in a reduction in the amount of circulating blood cells, anemia, and other health problems, aplastic anemia, thrombocytopenia, and leucopenia (9). also, the effect is represented by increased sensitivity to injuries, bone marrow (bm) suppression and infections caused by a lack of leucopoiesis (5). infections and inflammatory processes can trigger the production of cytokines, which mediate immune response (10). interleukin 6 (il-6) is a significant cytokine with a variety of functions which is a lowmolecular-weight protein that is mostly-released by the immune system cells (monocytes and macrophages) to control a variety of biological processes like proliferation, differentiation, and protein synthesis (11). researchers reported that oil may be a cause of acute lymphoblastic leukemia (aml), congenital abnormalities, and perhaps lymphocytes hematological malignancies, non-hodgkin lymphoma (nhl) (12) (13). the aim of the study is to reveal the impact of crude oil exposure on several hematological parameters and il-6 of oil workers and to detect which parameters are more affected by the exposure to be considered as prognosticators and to find the correlation between these parameters. subjects and methods study design the study was an observational study conducted at the middle refineries company (mrc) in al-muthanna city, south of iraq, from the period between october 2021 to january 2022. the study was done according to strengthening the reporting of observational studies in epidemiology (strob) guideline and approved by the ethical committee of university of basra-college of pharmacy (293/5/3, 2021/10/21) participants one hundred fifty subjects were enrolled in the study. individuals were divided into three groups: control/non-exposed healthy persons/group i consists of 50 persons; group ii consists of 50 workers at oil well; group iii consists of 50 persons who live close to the oil wells. the included criteria: a worker who is working for more than two years in the middle refinery company (mrc) unit, workers exposed for at least (10-12) hours/day for three days/week duration period, age range between (25 60) years. any individual with cv diseases, endocrine diseases, respiratory disorders and obesity were excluded from the study. pregnancy and breast feeding are extra excluded criteria. detailed information such as age, gender, work location and place of residence, weight, height and body mass index (bmi); individual life style (smoking, alcohol, coffee habit), disease history, surgical history, drug history were obtained by prepared questionnaire for each participant. measurements of the parameters pulse rate measure pulse rate of each individual by using oximeter. blood pressure measure blood pressure of each individual by classical method by aneroid sphygmomanometer. random blood glucose measure random blood glucose by accucheck active glucometer. hematology profile five milliliters of blood samples were obtained, place 2ml of the blood in a tube containing edta (ethylene diamine tetra acetic acid) to prevent blood clotting. the tube was placed in a rollshaking mixer for few minutes to prevent clotting and then placed below needle of auto-analyzer hematology to measure hematological parameters complete blood count (cbc) i.e. white blood cell count (wbc 10^9/l), lymphocyte count [(lmy#(10^9/l)], monocyte count (mid#10^9/l), granulocyte count (gran# (10^9/l), lymphocyte percentage (lym%), monocyte percentage mid% and granulocyte percentage (gran% ) and red blood cell (rbc), hemoglobin (hgb g/dl), hematocrit (hct%), mean corpuscular volume (mcv fl), mean iraqi j pharm sci, vol.32(1)2023 assessment hematological indices & il-6 in workers who live near to crude oil wells 239 corpuscular hemoglobin concentration (mchc g/l), mean corpuscular hemoglobin (mch pg), platelets (plts (10^9/l), platelet distribution width (pdwcv%), platelet distribution width-standard deviation(pdw-sd fl) by (ruby hematology analyzer, germany) (6). immunofluorescent assay place three milliliters of blood in centrifuge to obtain clear serum for interleukin 6 measurement by getein biotech, inc, uk. this was done by using an immunofluorescence assay (il6 fast test kit). the test was performed according to the manufacturer's instruction (14). serum il-6 levels were used as a predictive biomarker to confirm the role of inflammatory mediators and their link to negative pollution consequences. statistical analysis one-way anova with chi-square fishers exact test was utilized among groups. tukey’s posthoc analysis test for further assessment was used. data were expressed as mean± sem with p<0.05 significance. graphpad prism software (version 6.0). results demographic data, as well as general history, smoking, social state and personal habits, were collected using a prepared questionnaire, which is described in table (1). the percentage of males is higher than that of females in all groups, which showed no significant changes among the groups. to link the data and draw a clear conclusion of cause and effect, smoking and coffee consumption patterns were documented. there were significant differences (p>0.05) among the groups in such habits, with greatest percentage of smokers (40%) in group ii. regarding coffee consumption, about 38% and 32% of the participants in both group i and group ii workers were consumed coffee, respectively as shown in table (1). there were no statistically-significant differences in the drug, surgical, or family histories of the participants. as well as no significant differences were found in body mass index (bmi) among the individuals of all groups as illustrated in figure 1. the majority of the participants are of a healthy weight. table1. demographics information of the participants (n=150) *values with percent are analyzed using one-way anova with chi-square fisher exact test. represents significant differences among groups. grou ps state sex smoke habits coffee habits drug history surgical history family disease history marrie d singl e male female yes no yes no yes no yes no yes no grou p (i) contr ol 43 (86%) 7 (14%) 40 (80%) 10 (20%) 5(1 0% ) 45( 90 %) 19( 38 %) 31( 62 %) 2 48 5 45 2 48 grou p (ii) 42 (84%) 8 (16%) 46 (92%) 4 (8%) 20( 40 %) 30( 60 %) 16( 32 %) 34( 68 %) 5 45 7 43 8 42 grou p (iii) 40 (80%) 10 (20%) 40 (80%) 10 (20%) 15( 30 %) 35( 70 %) 9(1 8% ) 41( 82 %) 3 47 5 45 2 48 pvalu e 0.6597 0.0834 0.0014* 0.0034* 0.6112 0.6857 0.1284 iraqi j pharm sci, vol.32(1)2023 assessment hematological indices & il-6 in workers who live near to crude oil wells 240 figure 1. body mass index (bmi) comparison among groups. values are expressed as mean±sem. no significant differences among groups. furthermore, pulse rate, systolic and diastolic blood pressure were measured in this study to show the potential cv effects of oil exposure or its waste products on people who work or live near the site. this test was also performed to rule out any major disease or disorder affecting the aforementioned system. there was a considerable rise in pulse rate in groups ii (85.68 ±1.58) and group iii (86.65±1.88) when compared to group i (78.43±1.53), but it was still within normal limits. in contrast, no significant differences in systolic and diastolic blood pressure were found among groups, and the majority of them were within normal ranges, as shown in figure 2 figure 2. pulse rate, systolic and diastolic blood pressure in control (group i), workers (group ii) and peoples live nearby (group iii). values are expressed as mean± sem.; * represent significant difference among groups. figure (3) shows a significant increase in serum il6 levels among workers (group ii) compared to corresponding level in control (group i) (p >0.01). additionally, in comparison to the control (group i), there was a non-significant increase in serum il-6 in (group iii). figure 3. serum interleukin-6 level of the participants in the study (n=150). values are expressed as mean±sem; *represent significant difference p<0.01among groups. moreover, levels of rbg were found to be significantly-higher (p > 0.05) in workers (groups ii) compared to controls (group i) (figure 4). figure 4. random blood glucose of the participants in the studied groups (n=150). values are expressed as mean± sem. there is significant difference in group ii compared to group i (p<0.05). iraqi j pharm sci, vol.32(1)2023 assessment hematological indices & il-6 in workers who live near to crude oil wells 241 furthermore, table 2 illustrated that, each of wbc number, granulocytes numbers and monocyte were significantly-increased in group ii and group iii compared to such numbers in group i. in workers (group ii) and those who live near the company (group iii), the red blood cell count, hemoglobin, and hct were all negatively-related with air pollution [no significant results (p>0.05)]. moreover, mcv, mch, and mchc data provide information on the amount and average concentration of hemoglobin in rbc; where, there was a significant reduction in their values in groups ii and iii when compared to such values in group i/control persons; since, the results of this study show a significant rise in plts count in group ii patients in comparison with such count in group i and group iii. in addition, uncontrolled-activation and distribution were linked to a significant increase in pdw-sd and pdw-cv in groups ii and iii compared to that in group i. table 2. comparison of complete blood count parameters among participants of the studied groups groups group i group ii group iii p-value parameters wbc (10^9/l) 6.47±0.23 7.27±0.21a 7.82±0.27a p>0.05 lym#(10^9/l) 1.755±0.1264 1.767±0.07504 1.739±0.07951 p<0.05 mono#(10^9/l) 0.7663±0.0460 0.8708±0.03537 0.8425±0.04052 p<0.05 gran#(10^9/l) 4.035±0.1877 4.413±0.1893 5.12±0.2201a p>0.05 lym % 27.52±1.353 26.2±1.299 23.95±0.9021 p<0.05 mono% 11.98±0.5306 12.25±0.3049 10.8±0.3867a p>0.05 gran % 61.83±1.356 61.88±1.22 65.61±1.075 p<0.05 rbcs (10^12/l) 5.325±0.07553 4.81±0.06927 4.842±0.0954a p>0.05 hgb (g/dl) 14.32±0.1616c 13.56±0.1843b 12.31±0.176a p>0.05 hct % 43.54±0.4532 41.06±0.6995a 43.54±0.4532a p>0.05 mcv 82.44±0.707 79.75±0.7693 69.9±2.032a p>0.05 mch pg 27.1±0.2526 26.56±0.2802a 26.52±1.108a p>0.05 mchc g/dl 33.06±0.1247 313.55±0.1705 30.84±0.6313a p>0.05 plts (10^9/l) 206.9±8.444 255.1± 3.39a 198.4± 6.5 p>0.05 pdw-sd 12.09±0.235 14.87±0.1765b 13.04±0.216a p>0.05 pdw-cv 14.23±0.1417 14.74±0.1422b 14.91±0.1208a p>0.05 values are expressed as mean±sem. different characters (a, b, c) different letters represent significant difference between groups p<0.05. wbc: white blood cell, lym#: lymphocyte, mono#: monocyte, gran#: granulocyte, lym%: lymphocyte percentage, mono%: monocyte percentage, gran%: granulocyte percentage, rbc: red blood cell, hgb: hemoglobin, hct%: hematocrit percentage, mcv: mean corpuscular volume, mch: mean corpuscular hemoglobin, mchc: mean corpuscular hemoglobin concentration, plts: platelets, pdw-sd: platelet distribution width-standard deviation, pdw-cv: platelet distribution width-cell volume. iraqi j pharm sci, vol.32(1)2023 assessment hematological indices & il-6 in workers who live near to crude oil wells 242 the correlation between serum il-6 levels and certain hematological indices revealed that each of wbc and plts were significantly correlated to il6 (p>0.05); on the other hand, other parameters such as monocytes, lymphocytes, granulocytes, rbc and hb were non-significantly-correlated (p>0.05) as clarified in table 3. table 3. correlation analysis between serum il-6 and hematological indices. il-6 vs wbc il-6 vs monocytes il-6 vs lymphoc ytes il-6 vs granul ocytes il-6 vs rbc il-6 vs hb il-6 vs platelets r squared 0.106 0.0009 0.0676 0.068 0.003 0.0004 0.07948 p value 0.020 0.8343 0.0681 0.0660 0.669 0.8883 0.0473 p value summary * ns ns ns ns ns * * represents significant difference (p<0.05); ns represents (non-significant) (p>0.05). discussion this study was done to explore the impact of oil wells-containing petrol vapor on human inflammatory, hematological markers which are the major criteria for determining hydrocarbon toxicity in humans (15). in the present study, most participants with direct contact with oil wells refinery were males (80-90) %, due to hard condition and style of the work ladies do not generally-involved as main workers in petrol stations in iraq. there were significant differences (p>0.05) among the groups in smoking habit, as group ii presented a greatest percentage of smokers (40%) are seen. regarding coffee consumption, about 32% of the participants in both group i/ control and group ii workers drank a lot of coffee. furthermore, the current study showed that there were no-significant differences in bmi among the individuals of all groups, in contrast to other study, which discovered that exposure to a number of polycyclic aromatic hydrocarbons (pahs) and volatile organic chemicals (vocs) was directly-correlated to an elevated risk of obesity (16). moreover, in the present study, there was a considerable rise in pulse rate in groups ii and iii when compared to control, but it was still within normal limits; in contrast, no significant differences in systolic and diastolic blood pressure were found among groups of this study, and the majority of them were within normal ranges. additionally, previous studies found that the rate of hypertension was substantiallyhigher in the group with high benzene exposure (100%) than in the group with moderate exposure (49%), this indicating to potential cv involvement of oil exposure or its waste products on people who work or live near to oil wells (17); moreover, a significant conclusion by other study is that, 1 hour of exposure to essential oil wa associated with higher -blood pressure and -heart rate; furthermore, this finding was also reported by chuangkj et al (18). a possible mechanism of benzene-induced hypertension could be due to a disturbance of the biological process involving ross, which are known to induce lipid peroxidation, decrease antioxidants and increased oxidative stress (os) which may result in hypertension (17) (19). the differences between the current study and these previous studies may be due to the differences in the work conditions including concentration of gases and vapor as well as the exposure time of the workers as reported by the study (20). actually, petroleum-exposure may stimulate the production of pro-inflammatory il-6 and decrease the production of anti-inflammatory cytokines like il-10; furthermore, other investigators concluded that, petroleum products can cause immune system imbalances, because the concentration of cytokines in the circulation can be fluctuated; and thus, it has been used as a marker to assess the deleterious effects of long-term exposure to oil products as major air pollutants (6). various inflammatory processes and the production of ross are part of the suggested mechanism due to the introduction of hazardous chemicals from petroleum products (11). the current study shows a significant increase in serum il-6 level among workers (group ii) compared to such level in control (group i) (p>0.05). the increased of serum il-6 levels are linked to an increased risk of developing clinical disorders, especially if the level exceeds a predetermined cutoff point; this was in agreement with other study when comparing the workers to the non-exposed group (19). this is may be due to a variety of pathogenic pathways that can be triggered by long-term exposure to air contaminants (21). moreover, it has been found that exposure to a number of pahs and vocs was linked to an elevated risk of diabetes (16). furthermore, constant exposure to fuel and chemical waste products raised the risk of developing certain endocrine disorders. in our study, the participants' random blood glucose concentrations compared to the control group levels were significantly-higher (p>0.05) in workers of (group ii) compared to healthy individuals in the control (group i), this difference could be attributed to the differences in the work conditions and lifestyle including consumption of more coffee with sugar and uncontrolled diet. however, the current finding revealed that random blood glucose in all groups was within normal limits. on the other hand, other study found that urine poly-aromatic iraqi j pharm sci, vol.32(1)2023 assessment hematological indices & il-6 in workers who live near to crude oil wells 243 hydrocarbon (pahs) were linked to a higher incidence of elevated glucose levels than other group without contact with pahs (22). generally, the endocrine involvement of chronic exposure to fuel waste products or air pollution may be the end results of subsequent organs dysfunctions (23). the results of hematological parameters in this study, showed significant differences in each wbc, [granulocytes and monocytes (%)] among the studied groups; where, the numbers of these parameters were significantly-increased in group ii and group iii compared to that in group i. furthermore, the rbcs, hb, and hct were negatively-related to air pollution in group ii and group iii. moreover, there was a significant reduction in mcv, mch, and mchc values in groups ii and iii when compared to such values in group i; as well as, there was a significant rise in plts count in group ii persons in comparison with that count in group iii and group i. this variation might be attributed to that continuous exposure to petroleum products that may cause a reduction in the function of bm and phagocytic cell migration failure (24) (25); and there were signs of os and inflammation, as well as increased ross production, which can lead to cytotoxicity and increase plts count (26). other previous study found that, the oil worker attendants had much lower hb levels and red blood cell (rbc counts) than that in the control (group i); but, their mean white blood cells (wbcs) and plts counts were greater; additionally, the mcv, mch, and mchc were similar in both groups (25). the current study showed that anemia and leukopenia were the most common hematological-abnormalities as a result of benzene exposure at oil refinery. previous studies found that workers who were exposed to vapors of oil for 2 years or longer had significantly-lower hgb, rbc, mcv, and mchc levels (6) (25); and the adverse effect of oil exposure on several hematological indices and il-6 as inflammatory marker may be related to certain inflammatory processes. conclusion workers who have worked at oil refinery for at least two years might develop several hematological disorders including anemia. also, these workers are more likely to be at risk of certain inflammatory processes and changes in which il-6 is involved. therefore, workers at these certain workplaces should be aware of the consequences of working in these places and should do clinical routine checkup to avoid and treat any health problem that may occur. references 1. al-dabbas ma, ali la, afaj ah. the effect of kirkuk oil refinery on air pollution of kirkuk city-iraq. iraqi j sci. 2012;17–8. 2. kadhem ja, reza ks, ahmed wk. alternative 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albadry aa, el-gilany ah, bazeed fb. some biochemical and hematological parameters among petrol station attendants: a comparative study. biomed res int. 2015;2015. 26. zhang y, liu x, mchale c, li r, zhang l, wu y, et al. bone marrow injury induced via oxidative stress in mice by inhalation exposure to formaldehyde. plos one. 2013;8(9):1–10. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.28(2) 2019 counseling and pharmacy education programs doi: https://doi.org/10.31351/vol28iss2pp30-36 30 perceptions and attitudes of community pharmacists’ towards patient counseling and continuing pharmacy education programs in iraq samer i. mohammed*,1, elaf b. dawood** and iman s. abaas*** * department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq *** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract the pharmacist's role in the community is currently directed towards providing drug information and patient care rather than drug compounding and dispensing. patient counseling is an essential skill for pharmacists. so as to improve the pharmacist role in the community and enhance the patient's education and counseling skills, many continuing education programs are established. the aim of this study is to assess the perceptions and tendency of community pharmacists in iraq regarding patients counseling and continuing pharmacists’ educational programs. a cross-sectional survey was performed on a convenience sample of community pharmacists’ from different governorates of iraq from january 2017 to april 2018. data were collected using a pretested questionnaire specifically designed from a previous study with some modification to reflect the work nature in iraq. the majority of pharmacists gave counseling to the patients and spent from one to five minutes in order to dispense a prescription. approximately (53%) concentrated on the dose of the drug during dispensing more than the duration of use (19%) or drug indication (21%) while approximately half of the patients, on the other hand, asked mainly on the indications of their medications also less than (32%) asked about drug dose. almost (47%) of participants indicated that attending continuous pharmacist education programs can help them to improve the counseling practice furthermore, the majority of them have an optimistic attitude toward the importance of these programs for the future of their career. most participants declared that these programs are very rare in iraq. this study provided a clue that the majority of community pharmacists in iraq had an optimistic perception towards patients counseling and continuing pharmacy education programs. keywords: perceptions, patients counseling, continuous pharmacy education programs. يف التعليم المستمر للصيادلة وبرامج مريضلل مشورةتقديم ال تجاه صيادلة المجتمع ومواقف تصورات العراق ***و ايمان سعدي عباس ** ، ايالف باسم داود 1*،سامر عماد محمد فرع الصيدلة السريرية ،كلية الصيدلة، جامعة بغداد، بغداد، العراق.* .العراق بغداد، بغداد، جامعة الصيدلة، ،كلية االدوية والسموم فرع** .العراق بغداد، بغداد، جامعة الصيدلة، ،كلية*** فرع الصيدالنيات الخالصة تعتبر لذلك. وتوزيعها األدوية تحضير من بدالً المرضى ورعاية الدوائية المعلومات توفير على حالًيا المجتمع في الصيادلة يركزمعظم مع التواصل مهارات وتعزيز المجتمع في الصيدلي دور تحسين لغرض. للصيادلة أساسية مهارة للدواء االمثل االستخدام عن المريض إرشادات في ةالصيادل وميل تصورات تقييم هو الدراسة هذه من الهدف ان. المستمر التعليم برامج من العديد إنشاء تم ، واإلرشاد التوعية لغرض المريض .بالصيادلة الخاصة المستمر وتقييم برامج التعليم للمرضى المشورة تقديم تجاه العراق باستخدام البيانات جمع تم. 7102 أبريل إلى 7102 يناير من المختلفة العراق محافظات من المجتمع لصيادلة مالئمة عينة على مقطعي مسح إجراء تم .العراق في العمل طبيعة لتعكس التعديالت بعض مع سابقة دراسة من خصيًصا وُصمم اختباره تم استبيان الصيادلة من( ٪ 35) من يقرب ما ركز. دقائق خمس إلى واحدة دقيقة من الطبية الوصفة لصرف وقضوا للمرضى المشورة الصيادلة غالبية أعطى في( ٪ 70) االستخدام من الغرض أو( ٪ 01) للعالج االستخدام مدة من أكثر للمريض العالج صرف أثناء الدواء جرعة عن معلومات اعطاء على ( ٪ 57) أقل وبصورة العالج استخدام من الغرض عن السؤال على رئيسي بشكل المرضى نصف من يقرب ما ركز الصيادلة رأي وحسب حين تحسين يساعدهم في أن يمكن المستمر الصيدلي التعليم برامج حضور أن إلى المشاركين من( ٪ 72) من يقرب ما أشار. الدواء جرعة سألوا عن شاركينالم معظم أعلن كما. المهنية حياتهم لمستقبل البرامج هذه أهمية تجاه متفائل موقف لديهم منهم الغالبية فإن ، ذلك على عالوة المشورة ممارسة .العراق في جدًا نادرة البرامج هذه أن الصيدلي ليمالتع ولبرامج للمرضى المشورة لتقديم ورغبة متفائل تصور لديهم العراق في الصيادلة غالبية أن فكرة الدراسة هذه قدمت الختام في .المستمر المريض ، برامج التعليم المستمر . تصورات ، مشورةالكلمات المفتاحية : 1corresponding author e-mail: samer.jameel@copharm.uobaghdad.edu.iq received: 31 / 1 /2019 accepted: 7/4 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp30-36 iraqi j pharm sci, vol.28(2) 2019 counseling and pharmacy education programs 31 introduction most pharmacists in iraq are working in hospitals and community pharmacies to provide pharmaceutical care services to patients (1). the pharmacist's role in the community has widely changed as a consequence of the expansion in the pharmacy profession worldwide. nowadays, it is currently directed toward providing drug information and patient care rather than drug compounding and dispensing(2). patient counseling is regarded as an essential skill for pharmacists to interact positively with patients besides in many countries, it is mandatory (3) although doctors are regarded as the first source of information about the medicines for the patients nevertheless most doctors rarely assess patients understanding when giving medications information despite the importance of doing this (4). this is in opposition to the pharmacists where they actively asses the response of patients which results in an increase in patient adherence, satisfaction as well as decreases in the number of medicines prescribed, medication-related problems and the cost of medication (5). in order to improve the pharmacist’s role in the community and enhance the patient's education and counseling skills, many continuing education programs are established. the term “continuing education” has been defined as “organized learning experiences and activities in which (health care professionals) engage after they have completed entry-level academic education and training. these experiences are designed to promote the continuous development of the skills, attitudes, and knowledge needed to maintain proficiency, provide quality service or products, respond to patient needs, and keep abreast of change”(6). patients counseling and many pharmacy practices can be well improved when pharmacists participate in various continuous educational programs (7). although the number of pharmacists in iraq is greatly increased as a result of an increase in the number of pharmacy colleges in the last two decades (8), however, there is a very limited number of continuing pharmacy education programs which does not reflect the dramatic changes in the pharmacy profession in iraq. the purpose of this study is to assess the attitudes of community pharmacists in iraq concerning patients counseling and identifies the major obstacles they faced which prevent them from providing effective counseling. furthermore, the study also evaluates the perceptions and tendency of iraqi pharmacists to participate in continuing pharmacists’ educational programs. subjects and methods a cross-sectional survey performed on a convenience sample of community pharmacists at community pharmacies in different governorates of iraq from january 2017 to april 2018. data was collected using a pretested questionnaire specifically designed from a previous study with some modification to reflect the work nature in iraq (9). the questionnaire consisted of 16 questions. the first 4 questions were collected the demographic data of community pharmacists, followed by 6 questions which related to the attitude toward patients counseling and the obstacles that face the pharmacist during counseling. while the last 6 used to measure the perception of community pharmacists towards continuing pharmacy education programs. three questions formatted and scored on four-point (1-4) likert scale with anchor words (strongly agree to strongly disagree) to measure the opinion of pharmacists about continuous pharmacy education. the study was validated by the scientific and ethical committee in the college of pharmacy, university of baghdad. verbal consent was obtained from all participants included in the study. the authors informed the participants about the purpose of the study at the beginning of each interview. meanwhile, the respondents were informed that their participation was voluntary. statistical analysis statistical analysis was performed using the statistical package for social sciences software version 16 (spss v. 16). pharmacists’ responses were presented as frequencies and percentages. perception of community pharmacists towards patients counseling and continuing pharmacy education program was analyzed through a scaling method. results in order to evaluate the perception of iraqi pharmacist toward patient counseling and their attitude for the continuous pharmacists’ education program, a convenient sample of 263 community pharmacists from different governorates of iraq participated in this survey. the demographic data of all participants are shown in table 1. iraqi j pharm sci, vol.28(2) 2019 counseling and pharmacy education programs 32 table 1. demographic data for participants (n=263) categories demographic items female male sex no. (%) 172(65.39) 91(34.60) more than 45 years 36-45years 25-35 years less than 25 years age in years no. (%) 9(3.42) 31(11.78) 127(48.28) 96(36.50) more than 10 years 6-10 years 2-5years less than 2 years work experience in years no. (%) 33(12.54) 30(11.40) 77(29.27) 123(46.76) more than 16 hours 11-16hours 8-10hours less than 8 hours no. of working hr.at pharmacy 0(0) 12(4.56) 26(9.88) 225(85.55) more than half of the pharmacists spent from one to five minutes in order to dispense a prescription and about (39%) consumed from five to ten minutes and only less than (3%) take more than 10 minutes to dispense prescriptions as shown in table 2. table 2. time required to dispense a prescription (n=263). time spent in dispensing prescription no. (%) less than one minute 20(7.60) 1-5 minutes 134(50.95) 5-10 minutes 102(38.78) more than 10 minutes 7(2.66) more than half of the participants stated that they always provide consultation when dispensing any prescription as shown in table 3. table 3. attitude to offer consultation for patients while dispensing medication (n=263). frequency no. (%) always 140(53.23) frequently 95(36.12) occasionally 25(9.50) rarely 3(1.14) the higher percentages of pharmacists (53%) concentrated on the dose of the drug during dispensing more than the duration of use (19%) or drug indication (21%) or even other information like the main side effects, drug interaction if present and the best time to take the medication which only explained by less than (7%) of pharmacists, on the other hand, nearly half of the patients asked mainly on the indications of their medications and less than (32%) asked about the dose of the drug (figure1). figure 1.variance in motivation about the consultation between pharmacists and patients as pharmacists stated (n=263). the higher percentage of the patients listened carefully to the consultation about their medication and only less than (3%) do not interact with the pharmacist well during the consultation process as shown in table 4. table 4. degree of attention of the patients to the medical consultation as pharmacists stated (n=263). frequency no. (%) always 106(40.30) frequently 104(39.54) occasionally 46(17.49) rarely 7(2.66) many participants demonstrated that absence of patient's interest in addition to the insufficient time in pharmacy is the main obstacles that face them during the consultation process (table5). iraqi j pharm sci, vol.28(2) 2019 counseling and pharmacy education programs 33 table 5. obstacles encountered the pharmacists concerning providing medical consultation (n=263). problems no. (%) lack of time 84(31.93) insufficient knowledge and information 42(15.96) lack of patient’s interest 120(45.62) other 17(6.46) approximately (47%) of participants indicated that attending continuous pharmacist education programs can help them to improve the counseling practice. likewise, the availability of specific area for counseling is essential according to (32%) of participants. only about (17%) consider that increase the number of pharmacists in one pharmacy can help. other answers given by some pharmacists indicated that all these explanations are crucial to improve the counseling practice in addition to promoting public awareness about the importance of pharmacist’s instructions as shown in table 6. table 6. pharmacists view regarding solving the obstacles concerned in providing medical consultation (n=263). strategies no. (%) by increasing pharmacists in pharmacy 44(16.73) attendance of continuous pharmacist education programs 124(47.14) specify special areas for providing consultation 84(31.93) other 11(4.18) only less than (5%) of participants said that continuous pharmacist education programs available for them while more than half of the participants cleared that these programs rarely reach to iraqi pharmacists as shown in table 7. table 7. accessibility of continuous pharmacist education programs for iraqi pharmacists (n=263). regarding pharmacists’ opinion about continuing education programs and the need to establish these programs in iraq, the result in fig.2 showed an optimistic attitude of most pharmacists toward the importance of these programs for improving pharmacists’ information in addition to providing them with the latest scientific issues and the development of pharmacy career. more than (90%) of the contributors strongly agree or agree to launch these programs in iraq and no one of the contributors strongly disagrees with such programs as shown in figure.2. figure 2. pharmacists’ opinion about continuing education programs (n=263). the degree of interest in attending continuous education programs for pharmacists is very high as seen in table 8 and only less than (3%) of the participants refuse to attend these programs. table 8. the degree of interest in attending continuous pharmacist education programs (n=263). response no. (%) frequently 125(47.52) occasionally 106(40.30) rarely 26(9.88) never 6(2.28) discussion according to previous studies, pharmacists were regarded as an easily accessible source of health care which emphases on the role of pharmacists to give the patients accurate information about their medication (10,11). although work experience for most of the participants were less than two years nevertheless, the greater proportion of participant displayed a high tendency for counseling and attending continuing pharmacy education programs. time in community pharmacy could affect the time and quality of counseling (12). according to this study, the number of working hours for the higher percentages of pharmacies was less than 8 hours and this limited time may affect the response no. (%) frequently 13(4.94) occasionally 67(25.47) rarely 151(57.41) do not reach at all 32(12.16) iraqi j pharm sci, vol.28(2) 2019 counseling and pharmacy education programs 34 time provided for counseling and this clearly appeared in the previous study in saudi arabia which showed that most of the working hours (75%) in the pharmacies spent on dispensing and stocking and only (25%) provided for counseling (13). in the present study, more than half of the participant spent from (1-5) minutes with each patient to dispense a prescription. this result is similar to the previous study performed in nepalese which indicates that the majority of pharmacists took from (1-5 ) minutes for dispensing a prescription (10), another study which assessed the turkish community pharmacists' view about pharmaceutical care practice in turkey, indicated only a small percentages ( 22.5% ) take more than 6 minutes to counsel patients (14). counseling of pharmacist is essential to enhance the patient’s adherence with his medications (14). furthermore, the world health organization (who) good pharmacy practice guidelines also suggest that the pharmacist should provide counseling (15).the best outstanding finding of this survey was that most iraqi pharmacists have a great tendency towards patients counseling. the greater proportions of the participants said that they regularly provide consultation when dispensing any prescription. similarly, two other studies (16,17) in different countries indicated that the pharmacists always provide counseling to their patients. during counseling, the higher percentages of participants focus on the dose of the drug than on the duration of use and to a lesser extent on indications or even other information. on the other hand, nearly half of the patients prefer to ask mainly on the indications of their medications. in contrast, a previous study in india stated that 17% of the pharmacists give some basic information about the product and its usage method (18). regarding patients’ questions during counseling, the results of the present study are different from the results of a previous study in nepal where most patients asked about the cost of the medication more than other things such as the dose or the duration of use (10). although the greatest proportion of the participants concluded that most patients were listening well to their counseling nevertheless, the major obstacle that faced them and reduced the effectiveness of the consultation process was a lack in some patients’ interest. this made them cancel the counseling or decrease the given information. the result is similar to another study in northwest china where the lack of patient acceptance of pharmaceutical care lead to ineffective communication between patients and pharmacists (19).lack of time and insufficient knowledge also can affect the counseling according to other participants in the present study that is similar to many other results in previous studies (14,20,21). attendance of continuous pharmacist’s education programs was regarded as the main strategy to solve the problem of weak counseling according to the higher proportion of the participants. the previous result was predictable as improving the knowledge of the pharmacists can assist them to improve the counseling process in a timely manner. a comparable result was mentioned in a previous study conducted in turkey(14) which concluded that continuous education programs would be an important approach to enhance pharmaceutical care and counseling. continuous education program for the pharmacist is an effective method to improve all pharmacists knowledge after graduation (22). unfortunately, these programs are very rare and not accessible for the majority of pharmacists in iraq according to the suggestion of more than (57%) of participants in this study. this problem is not only in iraq but also in many other countries in the middle east(23,24). this study reveals a high attitude toward attending continuous educational programs and a promising opinion of most iraqi pharmacists about continuing education programs. the higher percentages declared that these programs can improve pharmacists’ information, in addition, to provide them with the latest scientific issues and can play a vital role in the development of pharmacy career. more than (90%) of the contributors strongly agree or agree to launch these programs in iraq. the great attitude of pharmacists to this program was in line with several former studies (22, 2526). conclusion this study provided a clue that the majority of community pharmacists in iraq had an optimistic perception towards patients counseling and continuing pharmacy education programs. in addition to that, most of them showed a great tendency to contribute effectively in these programs which are absent or very rare in iraq to improve their career and reduce the barriers toward a good effective counseling. ethical statement the study and the 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program: a study from a tertiary care hospital in central nepal. integrated pharmacy research & practice. 2017;6:157. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 synthesis of new analgesic peptides 39 synthesis of new opioid analgesic peptide analogues to enkephalin (leucineand methionine-enkephalin) muthanna s. al-ttaee * ,.1 , kawkab y. saour * , ahlam j. qasir * * department of pharmaceutical chemistry ,college of pharmacy,university of baghdad,baghdad , iraq abstract a small number of researches were done in the design and synthesis of enkephalin analogues that are able to resist degradation effect of proteolytic enzymes with good bioavailability and halflives.through studying structure activity relationships we tried to incorporate phthalyl group, tryptophan and lysine amino acids in different positions in the basic backbone structure of the naturally occurring opioid leu5and met5enkephalin, in the hope that such insertion of these amino acids could induce interesting addition in the biological activity of these analogues with enhancement of their bioavailability, in addition to decrease side effects as addiction liability. these synthesized peptides are: 1analogue i: phthalyl-tyrosyl-glycel-tryptophan methyl ester. 2analogue ii: boc-tyrosyl-glycyl-phenylalanyl-lysine ethyl ester. hbr. according to the designed structures, the analogues were synthesized following the conventional solution method and they were identified using the following techniques: melting point, optical rotation, thin layer chromatography (tlc), infrared spectroscopy (ir), elemental analysis (chn) and amino acid analysis. key words: peptide, phthalyl, opoioid analgesics ةصالخلا هطإيلطنمإةزنسمإصظإنردوببإتشإهعتهم هإيدإصتهدإتمضنشإحتو نتإصتحده إ لفلاإصاهنإنإرزددحنلن إنرضاخغنإنرادنعننإ)نًعخدهرنظ( ،(نيلافكنالا) ةيعيبطلا ةنكسملا تاديتببلل ةهباشم ةديدج تابكرم ريضحتو ميمصت لاجم يف اهزاجنإ مت ثوحبلا نم ًاليلق ًاددع نإ حنرحدإرإهإنرتلواإيزفإصتهحصنإتجلنتإناع فضه إنرضوززنإرإهإيدإطنلذإنرتاشإصمإتبنيتإعنبرإحيضتإعمددإ نل .ح لنإنردولإتشإهعتهمػإ صظإلسدإنرلونىنإنرضاحدن نإرزعسةنإننظإنردعهرننإنرونبفنإحنرحتحناإنرخنضنهحرإرإلػإنرضتحده إتشإن تنلإتوبفتن إ يزفإ لنإناىهع. يزفإنردغننإنرت فمننإرزددحنلن إنرضبوينغننإنهطلهدإصتضبينإنردوهرنذإحنًعضهعإنًصنغننإنرحتنحبيهطإحنرسفانظإيدإصبنةمإصلحزدنإرزدغننإ -نعخدهرنظإقصسمإنهرومبدإيزفإصتحده إيدنإنإنهًعخدهرنغه إرشإفادلإتو نت هإصظإ5 -حصنونبعنظ5نرت فمننإرزددحنلإنرضاخظإنرلضهىدإرنبىنظ ةدذإحرإهإيعهرننإعنهتننإحيانبرب ننإصإضنإصمإمفهطاإتبنيت هإنرونبر ،(نيلافكنالا) ةيعيبطلا ةنكسملا تاديتببلل ةهباشم ةديدج تابكرم ريضحتو ميمصت لاجم يف اهزاجنإ مت ثوحبلا نم ًاليلق ًاددع نإيزفإقصذإنف جمإصظإتتزنذإنايتنعإنرتهعدننإصوذإنوطصهط . حنرضتحده إنرضو تاإ د : نرضاحلإنًحد :يوهرنذ-تهفتحىنذ-حسفانذ-تتنحبيهطإصونذإنىحت . -1 نرضاحلإنروهعد :نبث-تهفتحىنذ-حسفانذ-يغنذإنًعنذ-ًفانظإنلنذإنىحتإ نلوحنتحصهفل. -2 حنرحدإلزتاإناتفتنإنرابوإنراهلذإنرحتزنلفن ،(نيلافكنالا) ةيعيبطلا ةنكسملا تاديتببلل ةهباشم ةديدج تابكرم ريضحتو ميمصت لاجم يف اهزاجنإ مت ثوحبلا نم ًاليلق ًاددع نإحتشإنىحللنسإنرحتغنه إنرحهرننإنإلخإنرحبتذإنرفإنرلبناإنرضضن اإرحزجإنرددحنلن إحولده إ نغنحإهإنرحتحندنن :ةنهعإطو نإنًعمإهو ،(نيلافكنالا) ةيعيبطلا ةنكسملا تاديتببلل ةهباشم ةديدج تابكرم ريضحتو ميمصت لاجم يف اهزاجنإ مت ثوحبلا نم ًاليلق ًاددع نإحتحصهتب تنينهإنرادتنإنرتةنتن ،(نيلافكنالا) ةيعيبطلا ةنكسملا تاديتببلل ةهباشم ةديدج تابكرم ريضحتو ميمصت لاجم يف اهزاجنإ مت ثوحبلا نم ًاليلق ًاددع نإنًىحلنواإنردمتفن ،(نيلافكنالا) ةيعيبطلا ةنكسملا تاديتببلل ةهباشم ةديدج تابكرم ريضحتو ميمصت لاجم يف اهزاجنإ مت ثوحبلا نم ًاليلق ًاددع نإنرحوزنذإنرلةنلإرزعغهتت ،(نيلافكنالا) ةيعيبطلا ةنكسملا تاديتببلل ةهباشم ةديدج تابكرم ريضحتو ميمصت لاجم يف اهزاجنإ مت ثوحبلا نم ًاليلق ًاددع نإتوزنذإناعضهعإ نًصنغنن ،(نيلافكنالا) ةيعيبطلا ةنكسملا تاديتببلل ةهباشم ةديدج تابكرم ريضحتو ميمصت لاجم يف اهزاجنإ مت ثوحبلا نم ًاليلق ًاددع نإصانهخإنايعنإتواإنروضتنل. introduction the opium group of narcotic drugs is among the most powerfully acting and clinically useful drugs producing depression in the central nervous system (1). opiates are drugs derived from opium (papver somniferum f. papaveraceae), and include morphine, codeine and a wide variety of semisynthetic congeners derived from them and thebain (another compound of opium) (2). the term opioid is more inclusive, applied to all agonists and antagonists with morphine-like activity as well as to naturally occurring (endogenous) and synthetic opioid peptides (3). opioid peptides defined as peptides with opiate-like pharmacological effect (4,5). the discovery of endogenous opioids has been followed shortly after the identification of opioid receptors (6). the main clinical uses of opioid peptides are as analgesics (enkephalins) (7,8), as antioxidants (enkephalins) (9), as anticancers (dalargin) (10), as antibacterials and antifungals (phthalyl serine, phthalyl arginine) (11). 1 corresponding author : e-mail : mothanaaltaii@yahoo.com received : 8 / 4 / 2008 accepted : 21 / 6 / 2008 mailto:mothanaaltaii@yahoo.com iraqi j.pharm.sci., vol.17 (1) ,2008 synthesis of new analgesic peptides 40 materials and methods: all amino acids and their derivatives were optically active and of l-configuration and supplied from fluka ag/switzerland. all of the solvents and materials used were of analar type and used without further purification. the method used for synthesis of these analogues was conventional solution method in which we used n,n'-dicyclohexyl carbodiimide (dcci) and 1-hydroxy benzotriazole (hbt) as a coupling agent and to prevent racemization, respectively. we used tert-butyoxy carbonyl (boc) group and benzyloxy carbonyl (z) group as terminal amino protecting group. boc-tyrosine and lysine ethyl ester-n-z were obtained fully protected from fluka ag/switzerland; while we use methyl and ethyl ester to protect the carboxyl moiety in peptide synthesis. the final analogues were purified using gel filtration on sephadex lh-20 column eluted with 0.1n acetic acid. the synthesis of analogues (i and ii) include the following general procedures: a. n-terminal protection: the phtalylamide derivative of amino acid was used for the intermediate cpd (1.3) in analogue i by fusion method (12), and the common amino protecting group which is tertbutoxycarbonyl (boc), (boc-tyrosine) and n-benzyloxycarbonyl (lys-oet-n-z)were obtained fully protected for analogue ii. b. c-terminal protection: methyl and ethyl ester were used to protect the carboxyl group of amino acid in peptide synthesis. c. coupling method: conventional solution method was used as a coupling method between the protected amino acid for peptide synthesis. dcci was used in the peptide bond formation as a coupling agent, while hbt was used to decrease racemization and to increase the yield. d. de-protection of c-terminals: the removal of methyl or ethyl ester (de-esterification) was carried out with 1.5 equivalent of 1n sodium hydroxide solution (saponification). e. de-protection of n-terminal protecting groups: this step was performed with strong acidic conditions in which the benzyloxycarbonyl (z) groups were removed with equimolar quantities of hbr in glacial acetic acid. f. peptide purification: the intermediates and the peptides have been purified by repeating re-crystallization several times (2-4 times) using different solvents as diethyl ether, petroleum ether, ethyl acetate, absolute ethanol, distilled water and chloroform. the final analogues were purified using gel filtration on sephadex lh-20 column eluted with 0.1n acetic acid. synthesis of analogue i: scheme (1) shows the steps of synthesis of analogue i which include: asynthesis of glycine-methyl ester (cpd. 1): a suspension containing (1.5gm, 0.02 mol) of glycine in methanol (15ml) was cooled down to (-15 oc), then thionyl chloride (1.46ml, 0.02 mol) equimolar was added drop wise to the suspension, keeping the temp below (-10 oc). then the reaction mixture was kept at (40 oc) for (3 hrs), followed by refluxing for (3 hrs), and left at room temperature overnight. after solvent evaporation to dryness in vacuum, the product was purified by recrystallization from methanol-diethylether (1:10) mixture. physical appearance, melting point, and rf value are listed in table (1) (13). iraqi j.pharm.sci., vol.17 (1) ,2008 synthesis of new analgesic peptides 41 o o o nh2-tyr-oh n o o tyr-oh nh2-gly-oh nh2-gly-och 3 n o o tyr-gly-och 3 + fu si on (( b ill m an m et ho d) cpd (1) cpd (2) c ou pl in g cpd (3) d ees te ri fic at io n n o o tyr-gly-oh cpd (4) nh2-trp-oh nh2-trp-och3 cpd (5) c ou pl in g n o o tyr-gly-trp-och 3 analogue i, cpd (6) phthalic anhydride es te rif ic at io n es te rif ic at io n scheme (1): synthesis of analogue i. iraqi j.pharm.sci., vol.17 (1) ,2008 synthesis of new analgesic peptides 42 table (1): physical appearance, m.ps, and rf values of intermediates and final analogues. compound no. physical appearance melting points ( oc) rf values * found reported 5 white crystals 209-210 213-214 0.88 (d) 0.71 (a) 1 white crystals 171-173 175 0.78 (d) 0.86 (e) 2 off-white powder 188-190 0.84 (b) 0.85 (c) 0.76 (d) 3 off-white powder 140-142 0.39 (b) 0.68 (d) 4 white powder 180-182 0.8 (c) 0.92 (d) 6 (analogue i) white powder 170-173 0.75 (d) 0.9 (e) 9 white crystals 154-155 157-158 0.76 (a) 0.94 (d) 7 white powder 115-118 0.69 (d) 0.88 (e) 8 white powder 155-158 0.68 (c) 0.95 (d) 0.83 (e) 10 white powder 148-150 0.8 (a) 0.36 (b) 0.6 (d) 11 faint yellow powder 218-220 0.7 (c) 0.8 (d) 12 white powder 158-160 0.55 (b) 0.67 (c) 13 (analogue ii) needle shaped crystals 196-198 0.45 (b) 0.37 (c) * solvent system used in tlc were: a: butanol: acetic acid : d.w. (4:1:5). b: chloroform: methanol: acetic acid (4:5:1). c: chloroform: methanol: ether (5:3:3). d: chloroform: methanol (7:3). e: c: chloroform: methanol: benzene (4:3:2). table (2): infrared values for analogue i and ii. analogue number ir value analogue i 3600-3460, 3340, 3074, 2983, 2858, 2806, 1738, 1677, 1569, 1504, 1440, 1373, 1244, 846 and 786 analogue ii 3690-3384, 3328, 3031, 2927, 2850, 1736, 1670, 1628, 1610, 1569, 1508, 1448, 1340, 1311, 1277 and 640 bsynthesis of phthalyl-tyrosine (cpd. 2): compound 2 is prepared by billman et al. method (14), in which l-tyrosine (18.119gm, 0.1mol) and phthalic anhydride (114.8gm, 0.1mol) fused together to give cpd. 2. physical appearance, melting point, and rf value are listed in table (1). elemental analysis, amino acid analysis and optical rotation are listed in table (3), (4) and (5), respectively. csynthesis of phthalyl-tyrosyl-glycin methyl ester (cpd. 3): a stirred solution of cpd. 2 (1 mmol) in dmf (5ml) and nmm (1 mmol) were added with stirring for (10 min). then eqimolar amount of cpd. 1 previously dissolved in dmf (5 ml) was also added, the mixture was cooled down to (-10 oc). hbt (2 mmol) and dcci (1 mmol) were added with stirring. stirring was continued for (3 days) at (0 oc) and then at room temperature for (7 days). then dcu was filtered, the filtrate was concentrated under vacuum, and then the residue was re-dissolved in ethyl acetate washed several times. the product was collected after solvent evaporization. physical properties, elemental analysis, amino acid analysis and optical rotation iraqi j.pharm.sci., vol.17 (1) ,2008 synthesis of new analgesic peptides 43 are listed in table (1), (3), (4) and (5), respectively. dsynthesis of phthalyl-tyrosyl-glycine (cpd 4): to a stirred solution of cpd 3 (0.5mmol) dissolved in dioxan (5 ml): water mixture (5:1) at (18 oc), sodium hydroxide solution (1n, 0.75ml) was added drop wise over (30 min). the reaction was allowed to proceed for additional (3 hrs). then the reaction mixture was acidified with equimolar quantity of hydrochloric acid. after the addition of ice-water, a precipitate was obtained. the physical properties are listed in table (1). esynthesis of trptophane-methyl ester (cpd 5): a suspension of tryptophan (9.8 mmol) in methanol (20ml) was cooled down to (15oc), and continue the procedure as in the synthesis of cpd 1. fsynthesis of analogue i (phthalyl-glycyltryptophane methyl ester (cpd 6)): the same procedure was performed as in the synthesis of cpd 3. physical properties, elemental analysis, amino acid analysis and optical rotation are listed in table (1), (3), (4) and (5), respectively. ir values for analogue i are shown in table (2). table (3): elemental analysis of some intermediates and final analogues. cpd. no. compound chemical formula calculated/found c% h% n% 2 phthalyl-n-tyr c17h13n1o5 65.59 65.89 4.21 4.45 4.49 4.31 3 phthalyl-n-tyr-gly.ome c20h18n2o6 62.82 63.2 4.74 4.55 7.32 7.59 6 phthalyl-n-tyr-gly-trp.ome (analogue i) c31h22n2o6 65.48 66.01 4.96 5.50 9.85 9.66 8 boc-tyr-gly c16h22n2o6 56.79 57.45 6.55 6.11 8.27 8.75 10 boc-tyr-gly-phe.ome c26h33n3o7 62.51 62.81 6.65 6.60 8.41 8.91 12 boc-tyr-gly-phe-lys.oet (-n-z) c41h53n5o10 63.46 66.1 6.88 6.79 9.02 9.35 13 boc-tyr-gly-phe-lys.oet.hbr (analogue ii) c33h48n5o8br 54.84 54.81 6.55 5.99 9.69 9.21 table (4): amino acid analysis of some intermediates and final analogues. cpd. no. compound amino acids tyr gly phe lys trp 2 phthalyl-n-tyr 1.1 3 phthalyl-n-tyr-gly.ome 0.99 1.02 6 phthalyl-n-tyr-gly-trp.ome (analogue i) 0.96 0.99 1.01 8 boc-tyr-gly 1.09 1.11 10 boc-tyr-gly-phe.ome 1.06 0.98 1.08 12 boc-tyr-gly-phe-lys.oet (-n-z) 1.01 0.95 1.1 0.89 13 boc-tyr-gly-phe-lys.oet.hbr (analogue ii) 0.89 1.13 1.1 1.18 table (5): optical rotation of some intermediates and final analogues. cpd. no. compound optical rotation ][ 25 d , c=1 in dmf 2 phthalyl-n-tyr -32 o 3 phthalyl-n-tyr-gly.ome -16 o 6 phthalyl-n-tyr-gly-trp.ome (analogue i) -36 o 8 boc-tyr-gly +48 o 10 boc-tyr-gly-phe.ome +80 o 12 boc-tyr-gly-phe-lys.oet (-n-z) -29 o 13 boc-tyr-gly-phe-lys.oet.hbr (analogue ii) -27 o iraqi j.pharm.sci., vol.17 (1) ,2008 synthesis of new analgesic peptides 44   nh 2-tyr-oh nh 2-gly-oh nh 2-gly-och 3 cpd (1) cpd (7) co up lin g de -es ter ifi ca tio n cpd (10) nh 2-phe-oh nh 2-phe-och 3 cpd (9) co up lin g analogue ii, cpd (13) es ter ifi ca tio n boc-t yr-och 3 boc-t yr-gly-o ch 3 cpd (8) boc-t yr-gly-o h es ter ifi ca tio n boc-t yr-gly-ph e-och 3 de -es ter ifi ca tio n cpd (11) boc-t yr-gly-ph e-ohnh 2-lys-oet n-z co up lin g boc-t yr-gly-ph e-lys-oe t n-z cpd (12) hbr in glacial acetic acid boc-t yr-gly-ph e-lys-oe t.hbr scheme (2): synthesis of analogue ii. 45 synthesis of analogue ii: scheme (2) shows the steps of synthesis of analogue ii which include: asynthesis of phthalyl alanine methyl ester (cpd. 9): the same procedure was carried out as in synthesis of cpd. 1 and cpd. 5. bsynthesis of boc-tyrosyl-glycine methyl ester (cpd. 7), boc-tyrosyl-glycylphenylalanyl-lysine etheyl ester-n-z (cpd. 12): again the same procedure was applied as in the synthesis of cpd. 3 and cpd. 6. csynthesis of boc-tyrosyl-glycine (cpd. 8) and boc-tyrosyl-glycyl-phenylalanine (cpd. 11): the de-esterification was performed as in the synthesis of cpd. 4 and the physical results are listed in table(1). dsynthesis of analogue ii (boc-tyrosylglycyl-phenylananyl-lysin ethyl ester. hbr (cpd. 13)): cpd. 12 (0.337 mmol) was dissolved in hbr (2.5n, 3ml) in glacial acetic acid, the mixture was left at room temperature for (30 min), then it was poured into (200ml) of diethyl ether with stirring. the separated oily material was evaporated under reduced pressure. the precipitated hbr salt was re-crystallized from ethyl acetate-petroleum ether mixture (1:7). the physical properties, elemental analysis, amino acid analysis and optical rotation are shown in tables (1), (3), (4) and (5), respectively. ir values are shown in table (2) . results and discussion: the results of our work were been shown in schemes (1 and 2), figures (1 and 2) and tables (1, 2, 3, 4 and 5). the methodology that has been adapted in this work seems to be successful according to the results indicated previously, in addition a biological activity study has been done on cpd. 4 (phthalyltyrosyl-glycine) and give significant positive results for analgesic activity relative to morphine (15).the structural modification that has been made on the backbone of leuenkephalin in this work to synthesize these new analogues and the positive results mentioned indicate that an enhanced analgesic activity has been achieved from this modification and this would open the door for further modifications or studies of these two analogues to discover a new biological activities as antimicrobials and anticancers. references 1. alfons r genna ro; rimington: opioids (narcotics). in: the science and practice of pharmacy (20th ed.), philadelphia college of pharmacy and 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(ed): steriochemistryspecific rotation. in: organic chemistry (5th ed.). brooks/cole, thomson learning, usa, 2000, pp. 113-114. 15. muthanna, s. al-taee, kawkab y. saour, haider m. mohammed: evaluation of analgesic activity of newly synthesized phthalyltyrosyl-glycine sodium. iraqi j. comparative evaluation of using intranasal desmopressin, parenteral diclofenac or their combination in the management of acute iraqi j.pharm.sci., vol.16 (2) ,2007 ergotamine, vitamin e, melatonin and total antioxidants 72 effect of ergotamine and its combination with vitamin e or melatonin on total antioxidant status in migraine patients shahlaa h. ali 1 , salim a. hamadi ** , ashwaq n. aljaff** ** department of clinical pharmacy ,college of pharmacy , university of baghdad, baghdad , iraq abstract free radicals and oxidative damage caused by them have being suggested to be involved in the pathogenesis of migraine. these may result from distorted equilibrium of pro-oxidant/anti-oxidant system that continuously generates and detoxifies oxidants during normal aerobic metabolism. escape of such system from equilibrium leads to damage of cellular elements with the depletion of cellular stores of antioxidants material such as glutathione and vitamin e. therefore, free radical scavengers (vitamin e or melatonin) seems to be of potential benefit as prophylactic anti-migraine therapy by neutralizing free radicals overproduction and possibly preventing formation of highly toxic intermediates (such as nitric oxide). in addition of being powerful antioxidant, melatonin was shown to possess promising effects in modulating severity, frequency and duration of migraine attacks. for this reason the present study was conducted to investigate the involvement of changed anti-oxidant defense (measured as total antioxidant status “tas”) during migraine attack and the possible modulation of such status by classical anti-migraine therapy (ergotamine), antioxidants (vitamin e and melatonin) and their combination. 23 normal subjects and 21 migraine patients with age range of (17-45) years were enrolled in the study. patients were diagnosed according to neurologist decision to have migraine with and without aura. migraine patients were divided into three treatment groups; first group treated with ergotamine alone, second group with ergotamine /vitamin e and third group with ergotamine /melatonin. all groups were advised to take their treatments during attacks. blood samples were drawn from migraine patients and normal subjects before initiation of therapy and after pain has been relived (from migraine patients only) for the investigation of tas . the results of the study showed that tas was significantly lower in migraine patients in comparison to control healthy subjects (p<0.05) with a percent reduction ranged from 35.46% to 43.97%. however, there is no significant difference in the level of tas among migraine patients (p>0.05). treatment with ergotamine raised significantly the level of tas by 157%. the addition of vitamin e or melatonin greatly raised tas by 179% and 176% respectively. the addition of vitamin e to ergotamine showed superior effect to that when melatonin was added. the greater reduction in tas seen in this study among migraine patients in comparison to control healthy subjects suggests the presence of generalized decrease in antioxidant defense elements. elevation of tas by all treatments was very clear. in conclusion the decrease in tas can be implicated in the pathophysiology of migraine and enhancement of antioxidant system can add a beneficial effect for the management of migraine headache with the use of antioxidants (vitamin e or melatonin) with classical anti-migraine drug. الخالصـــــــــــة لذ سبب١ت داء اٌشم١مت ٚ٘زا اإلخٙاد اٌخأوسذٞحؼخبش اٌدزٚس اٌحشة ٚاإلخٙاد اٌخأوسذٞ إٌاخُ ػٕٙا ِٓ إٌظش٠اث اٌّفخشظت حذ٠ثاً فٟ ٠ٕشأ ػٓ ػذَ اٌخٛاصْ فٟ أٔظّت األوسذة ٚاألٔظّت اٌّعادة ٌألوسذة ٚاٌخٟ حٕخح خزٚساً حشة ٚحؼاٌح س١ّخٙا باسخّشاس خالي ػ١ٍّاث األ٠ط فت إٌٝ ٔفار اٌّحخٜٛ اٌخٍٛٞ ِٓ ِعاداث اٌخٍٛٞ. إْ أؼذاَ اٌس١طشة أٚ اٌخًٍ فٟ ػًّ ٘زٖ األٔظّت ٠ؤدٞ إٌٝ حٍف اٌّٛاد اٌخ٠ٍٛت باإلظا اد اٌمأصت ٌٍدزٚس اٌحشة ِثً ف١خا١ِٓ ٘ـ أٚ ا١ٌّالح١ٔٛٓ حبذٚ راث فائذة ٚلائ١ت ظذ ,األوسذة اٌطب١ؼ١ت ِثً اٌغٍٛحاث١ْٛ ٚف١خا١ِٓ ٘ـ. ٌزا فأْ اٌُ ٚس حشة خط١شة ِثً )أٚوس١ذ إٌخش٠ه(. إْ داء اٌشم١مت ِٓ خالي ِؼاٌدت اإلفشاغ فٟ إٔخاج اٌدزٚس اٌحشة ٚسبّا باٌخم١ًٍ ِٓ حىْٛ خز اٌشم١مت. ا١ٌّالح١ٔٛٓ باإلظافت إٌٝ وٛٔٗ ِعاداً فؼاالً ظذ األوسذة فأٗ لذ اظٙش فؼا١ٌت ِٛػٛدة فٟ اٌخم١ًٍ ِٓ حذة ٚحشدد ٚفخشاث اإلصابت بأٌُ ة )ِماساً بشىً "اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ ٚػٍٝ ٘زا األساس أخش٠ج اٌذساست اٌحا١ٌت السخىشاف حذخً حغ١ش اٌؼٛاًِ اٌذفاػ١ت ظذ األوسذ )ف١خا١ِٓ ٘ـ ةاألوسذة"( خالي ٔٛباث صذاع اٌشم١مت ِٚذٜ إِىا١ٔت حغ١١ش ٘زٖ اٌحاٌت باٌؼالج اٌخم١ٍذٞ )االسغٛحا١ِٓ(، ِعاداث األوسذ ( سٕت. ُشخص 54-22ػّشٞ )ِش٠ط بذاء اٌشم١مت ٚبّؼً 72شخص س١ٍُ ٚ 72أُخش٠ج اٌذساست ػٍٝ ٚا١ٌّالح١ٔٛٓ( أٚ باالث١ٕٓ ِؼاً. اٌّشظٝ وُٛٔٙ ِصابْٛ بذاء اٌشم١مت ٚفك لشاس أخصائٟ اٌدٍّت اٌؼصب١ت. لسُ اٌّشظٝ إٌٝ ثالد ِدا١ِغ ػالخ١ت، ػٌٛدج اٌّدّٛػت الج خالي حذٚد األٌٚٝ باالسغٛحا١ِٓ ٚاٌثا١ٔت باالسغٛحا١ِٓ ِغ بف١خا١ِٓ ٘ـ ٚاٌثاٌثت باالسغٛحا١ِٓ ِغ ا١ٌّالح١ٔٛٓ. اسشذ اٌّشظٝ إٌٝ اخز اٌؼ اس ٔٛباث اٌُ اٌشم١مت. أُخزث ػ١ٕاث اٌذَ ِٓ ِشظٝ اٌشم١مت ٚاألصحاء لبً بذء اٌذساست ٚبؼذ حسى١ٓ اٌُ اٌشم١مت )ِٓ ِشظٝ اٌشم١مت فمػ( ٌم١ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة. shahlaa_65@yahoo.com. mail : -e: ing authorcorrespond 1 received : 26/7/2007 accepted : 29/12/2007 mailto:shahlaa_65@yahoo.com mailto:shahlaa_65@yahoo.com iraqi j.pharm.sci., vol.16 (2) ,2007 ergotamine, vitamin e, melatonin and total antioxidants 72 أظٙشث إٌخائح أْ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة ِٕخفعت ٌذٜ ِشظٝ اٌشم١مت باٌّماسٔت ِغ األصحاء ٚوأج ٔسبت االٔخفاض بّسخٜٛ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة ب١ٓ ِشظٝ %. ٚػٍٝ أ٠ت حاي ٌُ ٠ىٓ ٕ٘ان فشق ِٕطم52.32ٟ% إٌٝ 24.53حخشاٚذ ب١ٓ %. وّا ٚاْ إظافت 242اٌشم١مت أٔفسُٙ. إْ اٌؼالج باالسغٛحا١ِٓ لذ أدٜ إٌٝ اسحفاع ِٕطمٟ بّسخٜٛ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة بٕسبت % ػٍٝ اٌخٛاٌٟ. ٚػٍٝ 223% ٚ 223ظذ األوسذة ٚبٕسبت ف١خا١ِٓ ٘ـ أٚ ا١ٌّالح١ٔٛٓ لذ أدٜ إٌٝ اسحفاع أوبش فٟ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ١ش فٟ اٌؼَّٛ فمذ اظٙش ف١خا١ِٓ ٘ـ حفٛلاً فٟ سفغ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة ػٓ حٍه اٌخٟ أظٙش٘ا ا١ٌّالح١ٔٛٓ. ٠ظٙش االٔخفاض اٌىب ٔمص ػاَ فٟ اٌؼٕاصش اٌذفاػ١ت ظذ األوسذة وّا ٚاْ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة ب١ٓ ِشظٝ اٌشم١مت باٌّماسٔت ِغ األصحاء حذٚد حسخٕخح اٌذساست بأْ أخفاض اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ خ١ّغ اٌّدا١ِغ اٌؼالخ١ت. ذسفغ اٌحاٌت اٌذفاػ١ت اٌى١ٍت ظذ األوسذة واْ ٚاظحاً ػٕ ٟ ػالج صذاع اٌشم١مت باسخخذاَ ِعاداث األوسذة ِثً األوسذة ٠ّىٓ أْ ٠ىْٛ ػاِالً فٟ سبب١ت داء اٌشم١مت ٚأْ ححس١ٕٙا لذ ٠ىْٛ را فائذة ف ف١خا١ِٓ ٘ـ أٚ ا١ٌّالح١ٔٛٓ ِعافتً إٌٝ ػالخاث داء اٌشم١مت اٌخم١ٍذ٠ت. introduction the pathophysiological mechanisms of migraine have been discussed for years to be involving the humoral-vascular and neurogenic theories (1) . the constriction of cortical vessels may explain the aura, while dilation of extracortical (meningeal) vessels may underlay the throbbing headache (2) . cerebral hypoxia secondary to vasospasm or platelet aggregation may explain impaired vision predisposing migraine headache (3) .causative factors for cerebral vasospasm could be the alteration in ion concentration (low intracellular mg +2 ) (4, 5) , abnormally released serotonin secondary to platelets aggregation (6) , endothelin receptor gene polymorphism (7) , impaired mitochondrial oxidative metabolism and altered nitric oxide synthesis and release (8,9,10) . the neurogenic theory is strongly postulated without excluding aspects of the humoral theory (2) . the familial nature of migraine is greatly linked to the hereditary abnormality of monoaminergic transmission (11) . this transmission is vulnerable to sudden changes in internal or external environment to emotional stress, or to overload of afferent systems by excessive glare, smell or other stimuli. triggering factors, thus could induce a phase of excessive discharge followed by a state of monoamine depletion, hence pain gates would be opened, giving rise to spontaneous pain in the head and neck (12) . many evidences suggest the involvement of serotonin and norepinephrine to be the monoamines of great interest in the pathophysiology of migraine; the effectiveness of anti-migraine drugs like methysergide , pizotifen (serotonin receptors antagonists) and β-blockers (block the action of norepinephrine) solidify such suggestion (13,14) . beside that, the level of serotonin metabolities (5-hydroxyindolacetic acid) is highly elevated in plasma from patients during acute migraine (2) . change in serotonin level can provoke a neurovascular reaction which involve not only constriction or dilation of cerebral and extracerebral blood vessels but also activate nociceptive trigeminovascular system, an effect enforced by releasing vasoactive neuropeptides (substance p, neurokinin a or calcitonin genrelated polypeptide) ended with sterile or neurogenic inflammation (15) . these events were shown to be blocked by sumatriptan or dihydroergotamine (16,17) . calcium channel blockers on the other hand may diminish vasoconstriction whether produced by humoral agents or by intrinsic monoamine pathways; while non-steroidal anti-inflammatory drugs (nsaids) presumably suppress the sterile inflammatory response in vessel walls (18, 19) . many authors believe that sterile inflammation within the trigeminovascular system is of great importance in the pathophysiology of migraine headache. this postulate the release of the neurotransmitter substance p, vasodilation, increased vessel permeability, edema of cranial blood vessels and sensitization of sensory nerve endings (20) . biochemical mediators, like nitric oxide and prostaglandins (pgs) may participate in such scenario; where pge2 and txa2 levels are shown to be elevated in saliva from patients with migraine headache during acute attacks (21) . furthermore, nitric oxide has the potential to induce oxidative stress by acting as a free radical through the peroxidation pathway (22) . the concept of oxidative stress has been accepted in the increasing association of diseases with advanced age. the plausible explanation for such association is based on the implication of free radicals in the pathogenesis of several disorders like cancer and atherosclerosis (23,24,25) .oxidative stress, however, results from distorted equilibrium of pro-oxidant/anti-oxidant system in intact cell. such system continuously generates and detoxifies oxidants during normal aerobic metabolism (26) . outbalancing such equilibrium leads to damage of lipids, proteins, carbohydrates and nucleic acids; these events might deplete cellular stores of anti-oxidants material such as glutathione and vitamin e (27) . treatment of acute attacks of migraine is achieved with the use of ergotamine. the effectiveness of ergotamine in treatment of migraine attacks was thought to be related to its α-antagonistic activity; in addition the drug is iraqi j.pharm.sci., vol.16 (2) ,2007 ergotamine, vitamin e, melatonin and total antioxidants 73 known to stimulate 5-htd1 receptors of the cerebrovascular system. however; several limitations may restrict the use of ergotamine for migraine patients, these include: pregnancy; sepsis; hypertension; cerebral, coronary and peripheral vascular diseases; hepatitis; and renal insufficiency. in addition, the drug has many side effects such as abdominal cramps, parasthesia, nausea and tightness of the chest (2 ) . melatonin, an endogenous neurohormon, shows a promising effect in relieving migraine headache by means of reducing the severity, frequency and duration of migraine attacks (28) . the mechanism by which melatonin exert such effect has yet been proven, but this agent possessing many pharmacological properties like scavenging free radicals (29) , inhibiting nitric oxide synthase (30) , regulating neurovascular system (31) and modulating serotonin actions (32) . in the view of the association of various vascular disorders with oxidative stress (33) and because one of the theories of migraine postulate the change in cerebrovascular milieu, the present study was conducted to investigate the involvement of changed anti-oxidant defense (measured as total antioxidant status) during migraine attack and the possible modulation of such status by classical anti-migraine therapy (ergotamine), antioxidants (vitamin e and melatonin) and their combination. materials and methods materials: ergotamine tartrate (as cafergot ® 1mg tablets, novartis, switzerland), vitamin e (as 400 capsules, cipla, india), melatonin (as 3mg tablets, american nutri-ceutical, usa), total antioxidant status (tas) kit, (randox laboratories ltd, uk). patients twenty-three normal subjects and 21 migraine patients with age range of (17-45) years were enrolled in this study for three months. for inclusion, patients had to have a long-term history of migraine with and without aura diagnosed according to neurologist decision at specialized neurological centers and were managed under neurologist supervision. patients who are smokers, alcoholics or those with other apparent disease were excluded. no changes in patients’ medications were made during the study and patients were instructed to keep taking their medications. migraine patients were divided into three treatment groups; first group treated with ergotamine (1mg) alone, second group with ergotamine (1mg)/vitamin e (400mg) and third group with ergotamine (1mg)/melatonin (3mg). all groups were advised to take their treatments during attacks. blood samples were drawn from migraine patients and normal subjects before initiation of therapy and after pain has been relived (from migraine patients only) for the investigation of tas using randox tas kit. method of measurement was followed according to the instructions mentioned in randox tas kit. data were expressed as mean ± standard deviation and differences between means were analyzed by student’s ttest. p values less than 0.05 were considered significantly different. results table (1) and figure (1) showed that tas was significantly lower in migraine patients (p<0.05) in comparison to control (normal subjects) with a percent reduction in tas was ranged from 35.46% to 43.97%. however, there is no significant difference in the level of tas among migraine patients (p>0.05). table (1) clearly showed that ergotamine rise significantly the level of tas by 157%. interestingly, the addition of vitamin e and melatonin greatly raised tas by 179% and 176% respectively. this accompanied by shortening the time required for pain to alleviate. ergotamine treated group required (6.28 ± 3.77 hr) for the pain to alleviate; while ergotamine/vitamin e and ergotamine/melatonin required (4.84 ± 3.6 hr) and (2.77 ± 1.4 hr) respectively. however; only melatonin showed significant difference (p<0.05) in the time required for the pain to alleviate among other therapies. although the percent improvement in tas in patients treated with ergotamine/vitamin e and ergotamine/melatonin appears to be similar, there is a significant difference toward the superiority of vitamin e over melatonin when added to ergotamine. in this regard, it seems that the addition of melatonin did not significantly improve tas over that produced by ergotamine alone (figure 1). iraqi j.pharm.sci., vol.16 (2) ,2007 ergotamine, vitamin e, melatonin and total antioxidants 23 table (1): effect of treatments of migraine patients with ergotamine and its combination with vitamin e and melatonin on serum tas. serum total antioxidant status (mmol/l) control (n=23) ergotamine (n=7) ergotamine/ vit e (n=8) ergotamine/ melatonin (n=6) pretreatment 1.41 ± 0.22 a 0.86 ± 0.15 b 0.91 ± 0.15 b 0.79 ± 0.12 b posttreatment --1.35 ± 0.25 * a 1.63 ± 0.09 * b 1.39 ± 0.80 * a data are resented as mean ± sd.n= number of patients.*p<0.05 with respect to pre-treatment value. non-identical superscripts (a,b) among different groups considered significantly different, p<0.05. fig. (1): serum total antioxidant status in control subjects and migraine patients treated with ergotamine (n=7), ergotamine/vitamin e (n=8) and ergotamine/melatonin (n=6). data are presented as mean ± sd. *p<0.05 with respect to pre-treatment value (by paired student’s t-test). non-identical superscripts (a,b) considered significantly different, p<0.05 (by unpaired student’s t-test). discussion: free radicals in the brain and the implication of oxidative damage caused by them have recently being implicated to playing possible role in the pathogenesis of migraine headache. the greater reduction in tas seen in this study among migraine patients in comparison to control healthy subjects suggests the presence of generalized decrease in antioxidant defense elements. tas enables assessment of integrated antioxidant system which encompasses all biological components with antioxidant activity. reduction in tas has been implicated in several disease stats such as cancer, ischemic heart diseases and poor nutritional stats (34, 35) . the most convincing evidence for free radical activity comes from nitric oxide, which is a potent vasodilator and is an important biochemical in the trigeminal-vascular peripheral mechanism of migraine headache (36,37) . infusion of no donor (glyceryl trinitrate and sodium nitroprusside), immediately induce headache both in healthy subjects and patients suffering from primary headaches with higher intensity in maigiane patients; while slow injection of the nonspecific inhibitor of no synthase (lnam) reduced the spontaneous activity in all neurons (8,38,39) . koulchitsky and coworkers, suggest that no can induce activation of central trigeminal neurons and that endogenous release of no may contribute to the ongoing activity of these neurons. the delayed changes in neuronal activity may include gene expression of pro-nociceptive mediators (46) . in addition, migraine can be induced via a cgmp-dependent mechanism. kruuse and coworkers showed that sildenafil significantly induce migraine symptoms and propose that triggering mechanisms may reside within the perivascular sensory nerve terminals or the brainstem (41) . no may interact with other mechanisms for the precipitation of migraine. strecker and coworkers showed that no increases meningeal blood flow, an action depends partly on the release and vasodilatory action of calcitonin gene-related peptide (cgrp) from dural afferents; while, prostaglandins show only minimal interaction with no in this respect (15) . furthermore, platelet levels of nitric oxide, as well as nitric oxide metabolites such as nitrate/nitrite, are increased in migraine patients and rise further during attacks (9,10) . therefore, free radical scavengers may provide a potential molecular basis for prophylactic antimigraine therapy by neutralizing nitric oxide overproduction and possibly preventing formation of highly toxic peroxynitrite. interesting results were observed in this study by observing greater 0 0.4 0.8 1.2 1.6 2 2.4 t a s ( m m o l / l ) pre-treatment post-treatment control ergotamine ergotamine/ vit e ergotamine/ melatonin * * * ba a iraqi j.pharm.sci., vol.16 (2) ,2007 ergotamine, vitamin e, melatonin and total antioxidants 22 elevation of tas by anti-migraine treatments. in deed, the addition of antioxidants (vitamin e or melatonin) to the traditional treatment (ergotamine) greatly potentiate the effect . although the present study did not concerned with the mechanism of such an elevation in tas, it seems that the vasoconstriction effect of ergotamine may involved these events. however, the link between effect of ergotamine and the elevation in tas is the spotlight for further investigation. vitamin e is powerful antioxidant that scavenges free radicals within the lipid phase of the cell (42) . in addition, it have a structural role in stabilizing membranes (43) , thus nowadays, vitamin e is implicated in the therapy of many diseases (44, 45) . melatonin, beside its known antioxidant activity, it was shown to possess the ability to minimize the intensity, frequency and duration of acute migraine attacks (28) . in addition, melatonin has many pharmacological effects, of them is the inhibition of nitric oxide synthase (30,46) , whether this feature is involved in the relief of migraine headache or in the elevation of tas this is require further investigation. in our study, melatonin showed no superiority to vitamin e when added to ergotamine with high standard deviation. this may be explained by administration of single rather than maintenance dose of melatonin together with greater fluctuation in plasma melatonin level due to diurnal variation (melatonin plasma level is 10 times higher during night that during the daytime) (47) . the age is also an important factor; 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psoriasis in mice raghad a. khaleel* and munaf h. zalzala**,1 * department of pharmacology, college of medicine, university of iraqia, baghdad. iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad. iraq. abstract psoriasis is a common chronic skin condition characterized by infiltration of inflammatory cells into the epidermis and altered keratinocyte differentiation. in this work, psoriasis was induced by an imiquimod 5% cream, an immune response modifier that can induce psoriasis-like skin inflammation when applied topically in mice. guggulsterone prepared as a suspension and has been orally given to mice before imiquimod application. the results of the current study showed that guggulsterone suspension can significantly reduce psoriasis area and severity index in (guggul suspension + imiquimod group as compared with both control group and (vehicle suspension + imiquimod ) group. keywords: psoriasis, guggulsterone, imiquimod5%, nf-kb, tnf-α, il-23, il17. تأثير االستخدام الفموي للغوغولستيرون لتخفيف الصدفية الناتجة عن االستخدام الموضعي لاليميكويمود في الفئران رغد عبد السالم خليل *،1 و مناف هاشم زلزلة ** الجامعة العراقية ، بغداد ، العراق . ،طبكلية ال، االدويةفرع ا* .،العراقبغداد ،بغدادجامعة فرع االدوية والسموم ،* الخالصة العالج .الصدفية هي حالة جلدية مزمنة وشائعة تتميز بتسلل الخاليا االلتهابية في البشرة وتغيرات بتمايز الخاليا الكيراتينية ز يتمييسبب الصدفية التي تكون اشبه بالتهاب الجلد الذي balb/cكريم للفئران من نوع %5الموضعي بواسطة دواء االميكويمود تم تحضير الغوغولستيرون على شكل معلق .باالختناق واالحمرار االرتشاحي وتسلل الخاليا االلتهابية والخاليا العدلة والخاليا الليمفاوية فموي وتم اعطاؤه فمويا للفئران قبل ساعة من وضع االيميكويمود كريم موضعيا. اللغوغولستيرون قلل الى حد كبير مقياس مساحة منطقة الصدفية ومؤشر شدتهنتائج هذه الدراسة اشارت الى ان المعلق الفموي pasi) ( .في المجموعة المعالجة بالمعلق الفموي للغوغولستيرون مقارنة بالمجموعة المعالجة بحامل المعلق الفموي . nf-kb ،tnf-α ،il-23 ،il17، %5االميكويمود الصدفية ،الغوغولستيرون،الكلمات المفتاحية : introduction psoriasis is a common chronic skin condition characterized by infiltration of inflammatory cells into the epidermis and altered keratinocyte differentiation. it is currently thought of as at-cell mediated type-1 autoimmune disease(1). psoriasis is believed to result from a complex interplay between genetics, environment, and skin barrier disruption, and immune dysfunction(2,3). psoriasis exists on a reasonably broad spectrum in terms of clinical manifestations and it is associated with a higher percent of morbidity and the current medications can have acute side effects. numerous clinical studies have reported a strong relation between psoriasis and atherosclerosis, type 2 diabetes, and metabolic syndrome, together with the leading causes of mortality in the western world(4). 1corresponding author e-mail: munafzalzala@copharm.uobaghdd.edu.iq received: 1/8/2018 accepted: 4/9/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp15-23 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 oral guggulsterone administration in imiquimod-induced psoriasis 16 also, patients with psoriasis-like those with other major medical disorders, have decreased levels of employment and income as well as reduced quality of life (5). the combined costs of long-term therapy and social costs of the disease have a significant impact on health care systems and on the society in general(6) . the intended skin disease is characterized by thickened, scaly skin patches or psoriatic plaques, caused by abnormal keratinocyte proliferation and infiltration of inflammatory cells into the dermis and epidermis. about 10–30% of patients with psoriasis also develop psoriasis arthritis. once psoriasis appears, it is usually a life-long disease (2). psoriasis can be triggered by a number of factors that includes physical injury to the skin (the ‘koebner’ response), administration of interferon (ifn) or other inflammationinducing stimuli, rapid withdrawal of immunesuppressive drugs like corticosteroids and (systemic) infections with streptococcus or other bacteria(2). hyper proliferation and abnormal differentiation of keratinocytes are the two important results of the underlying pathophysiologic dysregulation in psoriasis. histologically, there is distinctive thickening of the epidermis due to increased proliferation of keratinocytes in the inter follicular epidermis, and an epidermal rete — downward undulations of the epidermis — become very elongated(7,8).keratinocyte differentiation is largely altered in psoriasis, paralleling ‘regenerative maturation,’ an alternative cell differentiation program that is transiently expressed during wound repair. the granular part of the epidermis in which terminal differentiation starts is highly decreased or absent in psoriatic lesions(8,9).consequently a stratum corneum forms from incompletely differentiated keratinocytes that irregularly retain a cell nucleus (parakeratosis). histological features of plaque psoriasis are the loss of the granular layer, supra papillary thinning , microabscess of munro (collection of neutrophils in the stratum corneum ), spongi form pustule of kojog (epidermal spongiotic pustule with neutrophilic infiltration), dermal/epidermal cd3+ t cell infiltrates with the epidermotropisim of cd8+ t cells . the histology of psoriatic subtypes may share classic dermal and epidermal features of a plaque psoriasis however, they may also display marked histopathologic features (10, 11). there are at least three distinct inflammatory pathways that may drive distinct clinical phenotypes in psoriasis: chronic plaque-type psoriasis by tumour necrosis factor (tnf), acute psoriasis by type i ifns, and pustular psoriasis via il – 36 / il-1. however, it is likely that the separation of these phenotypes and pathways is not absolute but instead represent the far end of a spectrum. hence, specific phenotypes may contain variable amounts of cytokines of this different inflammatory pathways at a given time (12). imiquimod (imq) (aldara cream 5%) is an immune response modifier was first approved by us food and drug administration (fda) (13) for the treatment of external genital and perianal warts in 1997 and then consecutively approved for actinic keratosis (ak) and superficial basal cell carcinoma (bcc) (14). topical treatment of imq in balb/c mice induced psoriasis-like skin dermatitis distinguished by acanthosis, erythema, and inflammatory infiltrate combined with neutrophils, dendritic cells (dcs) and lymphocytes (15). daily topical application of imq on the skin of mice can lead to psoriasislike dermatitis with many hallmarks of human psoriases, such as the formation of microabscesses, skin thickening, hyperkeratosis, acanthosis, scaling, and erythema (16). guggulsterone (gs) [4,17(20)pregnadiene-3,16-dione] is the plant-derived steroid desolated from the gum resin of the commiphora mukul tree, named guggulipid (13). it is a polyphenol conventionally used to treat obesity, diabetes, hyperlipidemia, atherosclerosis, and osteoarthritis, possibly through an anti-inflammatory mechanism (17). the active substances in guggulipid are the pregnane plant sterols cis-guggulsterone (egs) and trans-guggulsterone (z-gs). guggulsterone (gs) has anticancer potential as indicated by its ability to suppress the proliferation of a broad types of human tumor cell lines (18). the anti-arthritic and anti-inflammatory activity of gum guggul was established as early as 1960, by gujral et al (19). the effectiveness of guggul for treating osteoarthritis of the knee has also been demonstrated; furthermore, it has shown that guggulsterone is an antagonist for bile acid receptor farnesoid x receptor (20). other study has been shown that guggulsterone can augment transcription of the bile salt export pump, thus regulating cholesterol homeostasis; moreover, nuclear factor kappa-light-chainenhancer of activated b cells (nf-κb) has been implicated in obesity, hyperlipidemia, atherosclerosis, osteoarthritis, and bone loss, all of which can be modulated by guggulsterone(21). iraqi j pharm sci, vol.27(2) 2018 oral guggulsterone administration in imiquimod-induced psoriasis 17 in the present study, guggulsterone was prepared as a suspension and given orally to mice before psoriasis induction via imiquimod. materials and methods chemicals and kits the chemicals that were used in this work includes formaline(merk chemicals ,germany ) , diethylether (bdh chemicals, india ), xanthane as asuspending agent(samara drug industry iraq), guggulsterone (xi’an geekee biotech, china). the kits used in this study include (interleukin-23, interleukin-17, tumor necrosis factor alfa,nuclear factor kappa b)all of them from (elab .science, china) methods animal treatment twenty one (albino, balb/c mice male, 8weeks) (weighing between 25 to 40 gm) purchased from the department of drug control. they were kept in the animal house of college of pharmacy/university of baghdad under specific pathogen-free conditions and provided with water and food ad libitum under 12-hr light-dark cycle and maintained conventionally during the study with regulated air temperature (15-21˚c). preparation of guggulsterone suspension guggulsterone suspension is prepared by making a thick paste in mortar composed of 0.5gm guggulsterone powder and 0.09gm methylparaben and 0.015 gm propyl paraben and 5ml glycerine as dispersing agent, and use xanthan 0.25gm as suspending agent and then complete the volume to 5ml using distilled water (final concentration of suspension is 0.5%)(22). experimental protocoal in this work we have weighed approximately twenty-one mice at zero time and we measured ear thickness using vernier at zero time and we shave the back area of all mice using traditional manual eraser (trimming not zero shaving). mice were divided into three groups (seven mice each) as follow: 1control group in which mice were received vehicle of gugglsterone suspension daily for 14 consecutive days. 2-imiquimod group (vehicle suspension+imq) in which mice were received daily oral gavage of 0.2 ml of vehicle for gugglsterone suspension one hour before imiquimod 5% application for 14 consecutive days. 3-guggulsterone-treated group (guggulsterone suspension+imq) in which mice were received daily oral gavage of 0.2 ml gugglsterone suspension one hour before imiquimod 5% application for 14 consecutive days. the experiment last for 14 consecutive days, at the end of our experiment (day 14), psoriasis area and severity index was determined carefully by a professional dermatologist, and all mice were weighed before killing, and finally mice were sacrificed, and blood collected for biochemical analysis, also skin of back area and right ear were taken for histopathological examination, the spleen was weighed and spleen index was calculated. scoring severity of skin inflammation to score the severity of inflammation of the back skin, the psoriasis area and severity index (pasi) score for back skin was determined by a professional dermatologist. erythema, scaling, and thickening was scored independently on a scale from 0 to 4: 0, none; 1, slight; 2, moderate; 3, marked; 4, very marked. the cumulative score (erythema plus scaling plus thickening) served as a measure of the severity of inflammation (scale 0–12)(23). measurements of spleen index at the end of the experiment (day 14), the body weight and spleen weight for all mice was measured and the spleen index was calculated by dividing spleen weight in mg by the body weight that was measured during sacrificing time in gm(24). measurements of ear thickness the right ear thickness in mice was measured using digital vernier caliper every other day(25). measurements of skin thickness after sacrificing our mice, back skin thickness was measured in mm via digital vernier caliper(26) and comparing the measured thickness among groups. preparation of serum sample after euthanization of the animal by anesthetic diethyl ether, blood was collected by heart puncture and put in an eppendorf tube and centrifuged at 3500rpm for 15 minutes to obtain serum, which was separated and stored at -20 c until the day of analysis. serum was utilized for estimation of il-23 and il-17(27). preparation of skin tissue homogenate at the end of the experiment (day 14), the skin from the back area was taken by incision and washed by phosphate buffer ph (77.2). ten percent (10%) of back skin tissue homogenate was prepared and centrifuged at 5000rpm for 15 minutes, and the supernatant is stored at -20 ˚c until the day of analysis. skin homogenate was utilized to determine the tnfα and nf-kb level in tissue(28). iraqi j pharm sci, vol.27(2) 2018 oral guggulsterone administration in imiquimod-induced psoriasis 18 histological examination skin from back area and right ear utilized in this study were prepared for histological examination according to the method of junqueira et al at 1995 using paraffin section methods(29). statistical analysis the significance of differences between the mean values was calculated using paired and unpaired student t-test. p-value less than 0.05 were considered significant for all data shown in this respect(30). results effect of oral administration of guggulsterone on the pasi score in imiquimod-induced psoriasis in mice. table 4: showed that there was a significant decreament in the measured psoriasis area and severity index in pasi score in (vehicle susp+imq) group as compared to the control group. in (guggul susp+imq) group, the oral administration of guggulsterone produces a reduction in pasi score significantly in comparison with (vehicle susp+imq) group. table 1. the psoriasis area and severity index (pasi) score after oral guggulsterone administration in imiquimod-induced psoriasis in mice. each value represents mean ± standard deviation (sd). *= significantly different (p<0.05) with respect to the negative control group. # p<0.05% significant in comparison with vehicle susp + imq group. n = number of animals. effect of oral administration of guggulsterone on the ear thickness in imiquimod-induced psoriasis in mice. figure 1 showed that there was very slight increase in ear thickness in (guggul susp + imq) group as compared with control group, in (vehicle susp+imq) group there was a significant increment in ear thickness as compared with control group. figure 1. effect of oral administration of guggulsterone on ear thickness in imiquimod-induced psoriasis in mice. each value represents mean ± standard deviation (sd). *= significantly different (p<0.05) with respect to the negative control group. # p<0.05% significant in comparison with vehicle susp + imq group. effect of oral administration of guggulsterone on the skin thickness in imiquimod-induced psoriasis in mice. figure 2 showed that the oral administration of guggulsterone produce significant reduction in skin thickness in compare with (vehicle susp + imq) group. oral administration of guggulsterone successful in produce about 20% reduction in skin thickness after imiquimod administration. figure 2. effect of oral administration of guggulsterone on skin thickness in imiquimod-induced psoriasis in mice. each value represents mean ± standard deviation (sd). *= significantly different (p<0.05) with respect to the negative control group. # p<0.05% significant in comparison with vehicle susp+imq group control group ( vehicle susp+imq) group (guggul susp+imq) group zero *5.55 ± 2.23 #1.2 ± 1.35 iraqi j pharm sci, vol.27(2) 2018 oral guggulsterone administration in imiquimod-induced psoriasis 19 effect of oral administration of guggulsterone on the spleen index in imiquimod-induced psoriasis in mice. figure 3 showed that there was obvious increment in the measured spleen index in (vehicle susp+imq) group as compared with control group, while in (guggul susp+imq)group there was marked elevation in the measured spleen index as compared with control group. figure 3. spleen index after oral administration of guggulsterone in imiquimod induced psoriasis in mice. each value represents mean ± standard deviation (sd). *= significantly different (p<0.05) with respect to the negative control group. # p<0.05% significant in comparison with vehicle susp+imq group. effect of oral administration of guggulsterone on serum il-23 level in imiquimod-induced psoriasis in mice. figure 4,5,6,7: showed that there was no significant change in serum il-23 level in (vehicle susp+imq) group (p<0.05) as compared with normal control group, also there is no significant elevation in serum il-23 level (p>0.05) in (guggul susp+imq) group as compared to normal control group, regarding serum il-17 level ,there was a significant elevation in serum il-17 level in (vehicle susp+imq) group (p<0.05) in comparison with normal control group, also there was significant elevation in serum il-17 level(p<0.05) in (guggul susp+imq) group as compared to normal control group. relatively, there was a significant elevation in tissue nf-kb level in (vehicle susp+imq) group (p<0.05) as compared with normal control group,also there was significant elevation in serum nf-kb level in (p<0.05) (guggul susp+imq) group as compared to normal control group. finally there was significant elevation in tissue tnf-α level in (vehicle susp+imq) group (p<0.05) compared with control group, also there was significant elevation in tissue tnf-α level (p<0.05) in (guggul susp+imq) group as compared to normal control group. figure 4. effect of oral administration of guggulsterone on serum il-23 level in imiquimod-induced psoriasis in mice. each value represents mean ± standard deviation (sd). *= significantly different (p<0.05) with respect to the negative control group. # p<0.05% significant in comparison with vehicle susp+imq group. figure 5. effect of oral administration of guggulsterone on serum il-17 level in imiquimod-induced psoriasis in mice. each value represents mean ± standard deviation (sd). *= significantly different (p<0.05) with respect to the negative control group. # p<0.05% significant in comparison with vehicle susp+imq group. iraqi j pharm sci, vol.27(2) 2018 oral guggulsterone administration in imiquimod-induced psoriasis 20 figure 6. effect of oral administration of guggulsterone on tissue nf-kb level in imiquimod-induced psoriasis in mice. each value represents mean ± standard deviation (sd). *= significantly different (p<0.05) with respect to the negative control group. # p<0.05% significant in comparison with vehicle susp+imq group. figure 7. effect of oral administration of guggulsterone on tissue tnfα level in imiquimod induced psoriasis in mice. each value represents mean ± standard deviation (sd). *= significantly different (p<0.05) with respect to the negative control group. # p<0.05% significant in comparison with vehicle susp+imq group. histological examination of mice skin and right ear section. sections of the back area and right ear look-like normal in appearance in control group in which the thickness of the epidermal layer look-like normal and no rete-ridge appear as in figure (1a and b). figure 1. (h and e) staining sections of the mouse back area skin and ear skin of control group (200x) a: section of normal right ear b: section of the back area. red arrows : sign to the thickness of the epidermal layer of back area and right ear section. in the (vehicle susp + imq) group section of back area skin and right ear showed slight acanthosis of stratified epithelium more than 6 cells with an increase of rete ridge as in figure (2a and b). iraqi j pharm sci, vol.27(2) 2018 oral guggulsterone administration in imiquimod-induced psoriasis 21 figure 2. ( h and e ) staining sections of the mouse back area skin and ear skin of vehicle susp+imq group (200x) a: section of back area skin b: section of right ear vehicle susp+imq group showed slight acanthosis of stratified epithelium more than 6 cells with an increase of rete ridge. red arrows: sign to the thickness of the epidermal layer of back area and right ear section. the histological examination of the back area and right ear sections in (guggul susp+imq) group look like normal in appearance and no rete-ridge appear, and thickness of stratified epithelium (4 cells layer) as in (figure 3a and b). figure 3. (h and e) staining sections of the mouse back area skin and ear skin of (imq+ guggul oint) group (200x). a: section of right ear b: section of the back area. red arrows: sign to the thickness of the epidermal layer of back area and right ear section. discussion psoriasis is a common skin disease that has been recognized since ancient times when it was erroneously thought to be a variant of leprosy. psoriasis affects about 25 million people in north america and europe, and is probably the most prevalent immune-mediated skin disease in adults. it is an organ-specific autoimmune skin disease that is triggered by an activated cellular immune system(7) and is similar to other immune-mediated diseases such as crohn’s disease, rheumatoid arthritis, multiple sclerosis and juvenile-onset diabetes. all of these fit the definition of an autoimmune disease as a clinical syndrome caused by the activation of t cells and b cells, or both, in the absence of an ongoing infection or other discernable cause (7). in this work, psoriasis has been induced like skin inflammation in mice in both the back area and the right ear for 14 consecutive days by using imiquimod. imiquimod ( imq ) is a ligand for tlr7 and tlr8 and a potent immune activator it is used for topical treatment of genital and perianal warts caused by human papillomavirus (31). imq application led to increased ear inflammation as evidenced by increased ear thickness(16). skin inflammation was also confirmed by histological examination which showed acanthosis, hyperkeratosis, and elongation of the rete-like ridge in the imq treated group(16). in 2016 zhang et al found that imq could induce splenomegaly through systemic effects(24). iraqi j pharm sci, vol.27(2) 2018 oral guggulsterone administration in imiquimod-induced psoriasis 22 topical treatment with imq resulted in the rapid mobilization of pdc and high levels of type i ifn in the blood and splenomegaly(32), our results corresponds with this study in which we found that guggulsterone affects spenomegally induced by imiquimod. more than that our findings regarding serum interleukin ( il17,il23) level showed that serum il-17,il-23 level were significantly reduced in (guggul susp+imq) group as compared with control group which indicate that guggulsterone suspension act as antiinflammatory agent by decreasing serum level of these interleukins. it was postulated that il-23 which is a cytokine driving the development of il-17and il-22-producing th17 cells, is functionally involved in the pathogenesis of psoriasis. expression of il-23 is increased in psoriasis lesional skin and increased numbers of th17 cells are present. guggulsterone suppressed nf-kb activated by carcinogens (phorbol ester, okadaic acid, and cigarette smoke condensate) and inflammatory stimuli (hydrogen peroxide, tnf, and interleukin-1through inhibition of ikk, i b phosphorylation, and i kb degradation, which led to abrogation of p65 phosphorylation and nuclear translocation. our findings regarding tissue nf-kb showed that guggulsterone reduce nf-kb in orally treated (guggul susp+imq) groups as compared to (vehicle susp+imq). guggulsterone was quite effective in blocking the phosphorylation translocation of tnf-induced p65, which led to the abrogation of nfkbdependent reporter gene expression. tnf-alfa level in orally treated groups showed a significant reduction in (guggul susp+imq) group as compared to (vehicle susp+imq) group in which (p-value <0.05%). various cell types in the psoriatic skin produce tnf and il-17. tnf and il-17 can stimulate neutrophil recruitment on their own, but also together in a synergistic manner, leading to enhanced inflammation. psoriasis and other chronic inflammatory diseases seem to result from an overactive immune system with tnf, type i ifns and the il-23/il-17 axis playing interwoven roles(33). histopathological examination of skin section and right ear indicated thatimq induces local psoriasis-like symptoms in susceptible humans and in mice, such as inflammation, thickening and scaling of the skin, epidermal hyperplasia with the parakeratosis (retention of nuclei in the stratum corneum), a neutrophil-rich stratum corneum (munro's abscess), thinning of a granular layer of the epidermis, and a mononuclear dermal and epidermal infiltrate. infiltration of t cells and dendritic cells in plaques and dysregulated angiogenesis of cutaneous blood vessels have also been described(34). conclusion all the results demonstrate that guggulsterone suspension had antiinflammatory effects. it is a promising intervention in psoriasis in future. overall, our results indicate that guggulsterone apparently suppress nf-kb, tnf-α il-17, il-23 level, reducing (ear and skin thickness, spleen index) which may explain its anti-inflammatory activity. references 1. papp k, berth-jones j, kragballe k, wozel g, de la brassinne m. scalp psoriasis: a review of current topical treatment options. j eur acad dermatology venereol. 2007;21(9):1151–60. 2. liu y, krueger jg, bowcock am. psoriasis: genetic associations and immune system changes. genes immun. 2007;8(1):1–12. 3. chandran v, raychaudhuri sp. geoepidemiology and environmental factors of psoriasis and psoriatic arthritis. j autoimmun. 2010;34(3):j314–21. 4. gelfand jm, neimann al, shin db, wang x, margolis dj, troxel ab. risk of myocardial infarction in patients with psoriasis. jama. 2006;296(14):1735. 5. gelfand jm, feldman sr, stern rs, thomas j, rolstad t, margolis dj. determinants of quality of life in patients with psoriasis: a study from the us population. j am acad dermatol. 2004;51(5):704–8. 6. guideline c. assessment and management of psoriasis clinical guideline. natl clin guidel cent. 2012;(october):1–760. 7. lowes ma, bowcock am, krueger jg. pathogenesis and therapy of psoriasis. nature. 2007;445(7130):866–73. 8. afifi t, de gannes g, huang c, zhou y. topical therapies for psoriasis: evidencebased review. can fam physician. 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2017;109(1):5–10. 31. imiquimod ra. review articleimiquimod applied topically] a novel immune response modi_er and new class of drug. 1999 ; 1 – 14. available from: papers2 :// publication /uuid /b5c54702-c3ba-411b-86e04b9c541f4f7f. 32. walter a, schäfer m, cecconi v, matter c, urosevic-maiwald m, belloni b, et al. aldara activates tlr7-independent immune defence. nat commun. 2013;4:1– 13. 33. libert c, vandenbroucke re. an inflammatory triangle in psoriasis: tnf, type i ifns and il-17. cytokine growth factor rev. 2015;26(1):25–33. 34. levine d, gottlieb a. evaluation and management of psoriasis: an internist’s guide. med clin north am. 2009 ;93 (6) :1291 –303. https://www.ncbi.nlm.nih.gov/pubmed/?term=laggner%20u%5bauthor%5d&cauthor=true&cauthor_uid=21813772 https://www.ncbi.nlm.nih.gov/pubmed/?term=di%20meglio%20p%5bauthor%5d&cauthor=true&cauthor_uid=21813772 https://www.ncbi.nlm.nih.gov/pubmed/?term=perera%20gk%5bauthor%5d&cauthor=true&cauthor_uid=21813772 https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=21813772 https://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=21813772 iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis doi: https://doi.org/10.31351/vol28iss2pp134-141 134 belief about medicines among a sample of iraqi patients with rheumatoid arthritis mirna k. faiq *,1, dheyaa j. kadhim** and faiq i. gorial *** * department of clinical pharmacy, college of pharmacy, university of tikrit, salah-din, iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. *** rheumatology unit, department of medicine, college of medicine, university of baghdad, baghdad, iraq. abstract rheumatoid arthritis is a chronic, progressive, inflammatory autoimmune disease of unidentified etiology, associated with articular, extra-articular and systemic manifestation that requires long-standing treatment. taking patient’s beliefs about the prescribed medication in consideration had been shown to be an essential factor that affects adherence of the patient in whom having positive beliefs is an essential for better adherence. the purpose of the current study was to measure beliefs about medicines among a sample of iraqi patients with rheumatoid arthritis and to determine possible association between this belief and some patient-certain factors. this study is a cross-sectional study carried out on 250 already diagnosed rheumatoid arthritis patients who attended baghdad teaching hospital/medical city/ rheumatology department. the mean age of the patients was (50.8 ± 13.1 years). belief about medicines was measured via the arabic version of the beliefs about medicines questionnaire. the majority of the patients (88%) had strong beliefs in the necessity of treatment (specific-necessity score greater than specific-concern). there was a significant direct correlation between age, male gender, number of other chronic diseases, disease activity score 28 and clinical disease activity index with specific necessity, and direct correlation between clinical disease activity index with specific concern. future studies should investigate how interventional approaches addressing these predictors may lead to improve beliefs about medicines among rheumatoid arthritis patients and their impression on disease control. keywords: rheumatoid arthritis, beliefs about medicines, specific necessity, specific concern. عن االدوية لدى عينة من مرضى التهاب المفاصل الرثوي العراقيلمعتقدات ا ميرنا كفاح فائق * ،1 ، ضياء جبار كاظم** و فائق ايشو كوريال*** تكريت ، صالح الدين ، العراق. فرع الصيدلة السريرية ،كلية الصيدلة ، جامعة* ** فرع الصيدلة السريرية ،كلية الصيدلة، جامعة بغداد، بغداد، العراق. ***وحدة أمراض المفاصل ،فرع الطب ، كلية الطب ، جامعة بغداد، بغداد، العراق. الخالصة أن التهاب المفاصل الرثوي هو مرض مزمن ومتفاقم يصيب جهاز المناعة غير معروف السبب يصاحب بتأثيرات مفصلية وخارج مفصلية لى ع وجهازية والتي تتطلب عالج طويل المدى. أن أخذ معتقدات المريض حول الدواء الموصوف بعين االعتبار يعد واحد من أهم العوامل التي تؤثر عالج .حيث أن المعتقدات اإليجابية حول االدوية تعد عامال أساسيا اللتزام المريض بالعالج . ان الهدف من الدراسة الحالية هو تقييم االلتزام بال .المعتقدات حول االدوية لدى مرضى التهاب المفاصل الرثوي وتحديد االرتباط المحتمل بين هذا االعتقاد وبعض العوامل الخاصة بالمريض مريض تم تشخيصهم سابقا بمرض التهاب المفاصل الرثوي الذين حضرو الى مستشفى 052اسة الحالية هي دراسة مستعرضة أجريت على الدر سنة(. تم تقييم المعتقدات عن االدوية باستخدام النسخة 1.01± 5205بغداد التعليمي /مدينة الطب /قسم امراض المفاصل . متوسط عمر المرضى ) ( معتقدات قوية في ضرورة العالج )معيار ضرورة العالج كان أكبر 55استبيان المعتقدات عن االدوية. كان لدى غالبية المرضى )%العربية من من معيار القلق من األدوية(. كانت هناك عالقة طردية بين العمر وجنس الذكور و عدد االمراض المزمنة االخرى ودرجة نشاط المرض ومؤشر جث ان تب ري مع معيار ضرورة العالج . وارتباط طردي بين مؤشر نشاط المرض السريري مع معيار القلق من األدوية. يجبنشاط المرض السري يمكن للتداخالت المهتمة بهذه العوامل أن تحسن من المعتقدات حول االدوية لدى مرضى التهاب المفاصل الرثوي وتأثير الدراسات المستقبلية كيف ى المرضذلك على السيطرة عل القلق من العالج. ،ضرورة العالج ،المعتقدات حول االدوية،الكلمات المفتاحية: التهاب المفاصل الرثوي introduction rheumatoid arthritis (ra) is a chronic, progressive, inflammatory autoimmune disease(1) of unknown etiology(2) associated with articular, extra articular and systemic manifestation(1). it occurs worldwide in virtually all ethnic groups, with a prevalence estimated between 0.5% and 1%(3). 1corresponding author: e-mail:mirnapharma@gmail.com received: 29/5 /2019 accepted: 18/ 8 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp134-141 iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 135 the prevalence rate in iraq is 1%(4). a global burden study estimated ra prevalence in middle east north africa (mena) region as among the lowest at 0.16 %(5).like many autoimmune diseases, the etiology of ra is multifactorial(6). inflammation and following destruction of synovial joints is the hallmark of ra(3). morning stiffness in and around the joints, lasting at least one hour is a characteristic sign of ra. rheumatoid arthritis can be associated with variable manifestations of extra-articular involvement such as rheumatoid nodules, vasculitis, hematologic abnormalities, and visceral involvement(7). rheumatoid arthritis is one such illness where patients must take daily medicine to manage their ache and reduce probabilities of physical disability(8). the goals of pharmacologic therapy are to induce remission and prevent further loss of joint tissues or function in daily activities. the main drug classes that are currently used for treatment of ra include non-steroidal antiinflammatory drugs (nsaids), glucocorticoids, disease-modifying anti-rheumatic drugs (dmards), and biological agents(9). the main factor that affects patient adherence is his/ her general view about medicines, since it can overrun most of the other factors(10). accordingly, taking patient’s beliefs about their medication in consideration had been shown to be an important factor that affects adherence of the patient in which holding positive beliefs is essential for better adherence(11-13). the purpose of the current study was to measure beliefs about medicines among a sample of iraqi patients with rheumatoid arthritis and to determine possible association between this belief and some patient-certain factors. patients and method patients the current cross-sectional study was carried out on a convenient sample of 250 already diagnosed ra patients (mean age was 50.8±13.1 years) who attended baghdad teaching hospital/medical city/rheumatology department during october 2018 to january 2019. the number of female patients was 221(88.4%), while the number of male patents was 29 (11.6%). inclusion criteria the inclusion criteria for the current study were: 1-patients with ra as defined by the 1987 revised american rheumatism association (ara) criteria(14). 2-ra patients who were aged 20-80 years of either sex who were accepted to participate in the study. 3-disease duration >1yr. 4-current treatment with steroids, nsaid and/or dmards (including methotrexate, leflunomide, sulfasalazine, hydroxychloroquine, and azathioprine), with or without concomitant administration of biologic agents. 5-patients had not changed their medication in the last three months. exclusion criteria the exclusion criteria for the current study were: 1-patient who had a hearing, speech or cognitive deficits that would impair understanding of the questions. 2-patient who take antidepressant drugs, or being on treatment for any neurological or psychological diseases. 3-if they were receiving no medication. 4-pregnant women. 5-patient who had serious co-morbidity such as, malignancies and end stage organ failure. 6-patients providing incomplete or conflicting information during completion of the questionnaire. method the questionnaires belief about medicine was measured by using an arabic version of the belief about medicines questionnaire (bmq) established by horne et al (figure 1)(15). it has two parts: the bmqspecific evaluating beliefs about medication used for a specific condition and the bmq-general evaluating beliefs about medicines in general(16). the bmq-specific part covers two subjects; specific necessity subject which assesses patients’ view about the necessity and importance of their medicine, while specific concern subject covers patients’ beliefs about potential harm and adverse effects of their own medications and every one of which has a score extend from 5 to 25. a high score in necessity subject means that patients think their medications are essential to them; on the other hand high score in the concern subject implies that patients are worried and stressed over their very own medicines. likewise, bmq-general part has two subjects; general overuse subject which assesses how patients perceive the extent of medication usage, and general harm theme represents patients’ beliefs about unsafe nature of medication in general. the scores of the last two subjects extend from 4 to 20, and high score in each subject means negative perception about medications in general(17). respondents indicate their degree of concurrence with each individual statement about medicines on a 5-point likert scale, extend from 1 (strongly disagree) to 5 (strongly agree)(18). study design administration of questionnaires the information identified with the investigation were gathered by the analyst herself. when the patients arrived to the hospital/rheumatology department, they were asked if they accept to participate in the study, an explanation of the questionnaire was given to each patient who spent about 5 minutes to fill the research questionnaire completely. iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 136 statistical analysis anderson darling test was done to evaluate if continuous variables follow normal distribution, if follow normal distribution than mean and standard deviation used, if did not follow normal distribution than median and interquartile range (25% to 75% percentile range) will be used to present the data. linear regression analysis performed to evaluate the relationship between different variables, r (correlation coefficient or standardized beta is a representative of magnitude and direction of the relationship), 0.00 0.29 = little or no correlation; 0.30-0.49 = weak; 0.50-0.69 = moderate; 0.70-0.89 = strong; and 0.90-1.00 = very strong. negative sign indicate inverse relationship, but positive sign represent direct relationship. spss 22.0.0 (chicago, il), graph pad prism version 8.0.0 for windows, graph pad software, san diego, california usa, software package used to make the statistical analysis, p value considered when appropriate to be significant if less than 0.05 . strongly disagree disagree uncertain agree strongly agree specific necessity 1-my life would be impossible without medicine 2-without medicine i'll be very ill 3-my health , at present depend on my medicine 4-my medicine protected me from becoming worse 5-my health in the future depends on my medicine specific concern 6-i sometimes worry about the long term effect of my medicine 7-having to take medicine scares me 8-i sometimes worry about becoming too dependent on my medicine 9-my medicine disrupt my life 10-my medicines are mystery to me general-harm 11-people who take medicines should stop their treatment for a while every now and again 12-most medicines are addictive 13-medicines do more harm than good 14-all medicines are poison general-overuse 15-natural remedies are safer than medicines 16-doctors use too many medicines 17-doctors place too much trust on medicines 18-if doctors had more time with their patients they would prescribe fewer medicines figure 1. the beliefs about medicines questionnaire (bmq)(15). iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 137 results mean age of patients was 50.8 ± 13.1 years. 89.6% were married, and 35.6% were illiterate. 88.4% were female, the majority lived in urban area of residence, and most of the patients were nonsmokers, as illustrated in table 1. table 1. socio-demographical characteristics of patients. variables value age (years), mean ± sd 50.8 ± 13.1 patient’s age no. (%) <30 years 16 (6.4%) 30 – 39 years 33 (13.2%) 40 – 49 years 58 (23.2%) 50 – 59 years 69 (27.6%) ≥60 years 74 (29.6%) gender no. (%) male 29 (11.6%) female 221 (88.4%) marital status no. (%) single 26 (10.4%) married 224 (89.6%) education level no. (%) illiterate 89 (35.6%) primary 64 (25.6%) secondary 59 (23.6%) college 38 (15.2%) location no. (%) urban 233 (93.2%) rural 17 (6.8%) smoking no. (%) smoker 17 (6.8%) non-smoker 233 (93.2%) the mean of disease duration was (9.6±7.8 years), most patients with disease duration less than ten years (69.6%), and (63.2%) had no other chronic disease. in addition, (52%) of patients had high disease activity (das28-esr) score and (44.8%) had high clinical disease activity index (cdai) score, as illustrated in table 2. table 2. disease characteristics of the patients variables value disease duration (years), mean ± sd 9.6 ± 7.8 patient’s age no. (%) < 10 years 174 (69.6%) 10 – 19 years 43 (17.2%) ≥ 20 years 33 (13.2%) medication use duration (years), mean ± sd 7.0 ± 6.4 number of other chronic diseases, no (%) none 158 (63.2%) 1 disease 63 (25.2%) 2 diseases 29 (11.6%) das 28-esr 3.5 ± 0.6 remission 1 (0.4%) low 11 (4.4%) moderate 108 (43.2%) high 130 (52.0%) cdai low 15 (6.0%) moderate 123 (49.2%) high 112 (44.8%) cdai: clinical disease activity index; das28-esr: disease activity score-erythrocyte sedimentation rate. the total score with the sub-scores of patients believes for all patients are shown in table 3 as well as figure 2. table 3. beliefs about medicines questionnaire scores of patients. scores value necessity score 21.3 ± 2.5 concern score 14.2 ± 3.9 overuse score 10.2 ± 2.7 harm score 13.6 ± 2.1 total score 59.3 ± 6.9 figure 2. beliefs about medicines questionnaire scores of patients iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 138 the majority of the patients (88%) had strong beliefs in the necessity of treatment (scores bmq specificnecessity greater than score bmq specific-concern. however, (4.4%) of the patients reported strong concerns about the treatment (scores bmq specificconcern greater than score bmq specificnecessity).the residual of the patients (7.6%), have equal scores for bmq specific-necessity and specific-concern scores suggests that they have an equal agreement on both concept of the subpart where they share similar score. as clarified in table 4. in univariate analysis; the bmq-specific necessity (bmq-sn) showed significant direct correlation with age, gender (male vs. female), number of other chronic diseases, das28-esr and cdai. the bmq-specific concern (bmq-sc) showed significant direct correlation with cdai. the bmqgeneral overuse (bmq-go) showed a significant direct correlation with gender (male vs. female), and inverse correlation with education levels. while the bmq-general harm (bmq-gh) showed significant inverse correlation with medication use duration, and direct correlation with das28-esr as illustrated in table 5. table 4. beliefs about medicines questionnaire necessity – concern differential table 5.univariate correlation between bmq components with various variable. cdai: clinical disease activity index; das28-esr: disease activity score-erythrocyte sedimentation rate necessity – concern differential no. percentage necessity > concern 220 88% necessity < concern 11 4.4% necessity = concern 19 7.6% bmq-specific necessity bmq-specific concern bmq-general overuse bmq-general harm regression coefficient p-value regression coefficient pvalue regression coefficient pvalue regression coefficient pvalue age 0.161 0.005 [s] 0.045 0.239 0.053 0.201 0.067 0.145 gender(male vs. female) 0.222 <0.001 [s] 0.037 0.558 0.146 0.021 [s] -0.061 0.337 marital status 0.009 0.441 -0.047 0.228 -0.064 0.158 0.073 0.126 education level -0.051 0.210 -0.032 0.306 -0.108 0.044 [s] 0.057 0.186 residence -0.022 0.364 -0.042 0.254 -0.042 0.252 0.008 0.451 smoking 0.022 0.364 0.042 0.254 0.042 0.252 -0.008 0.451 disease duration 0.080 0.104 -0.003 0.483 -0.056 0.189 -0.094 0.070 medication use duration -0.039 0.267 -0.032 0.308 0.037 0.281 -0.109 0.043 [s] number of other chronic disease 0.138 0.015 [s] -0.073 0.125 -0.042 0.253 -0.008 0.450 das28-esr 0.127 0.023 [s] 0.104 0.051 0.101 0.055 0.131 0.019 [s] cdai 0.152 0.008 [s] 0.149 0.009 [s] 0.094 0.070 0.014 0.411 linear regression analysis [s], significant relationship when p value<0.05. iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 139 in multivariate analysis, only age and gender were independently correlate ( direct relationship ) with bmq sn score . while das28 esr was independently correlate (direct relationship) with bmq-gh. as illustrate in table 6. table 6. multivariate linear regression analysis between bmq components and other variables of patients cdai: clinical disease activity index; das28-esr: disease activity score-erythrocyte sedimentation rate. the number of other chronic diseases was excluded from multivariate regression table because the tolerance=0 this means that this variables was already contained in or redundant with other independent variables (predictors) i.e. can be perfectly predicted from one or more of the other independent variables. discussion rheumatoid arthritis is a chronic inflammatory joint disease, with a higher prevalence observed in both older age groups and women(19). patients with rheumatoid arthritis need to take daily medication to relieve their pain and reduce chances of physical disability(8). as shown in sociodemographical date of the patients, about 88.4% of patients were female and 57.2% were above 50 year. study in rochester, minnesota, 1955-1985 found that the incidence of ra was nearly twofold in women compared with that in men and increased steadily with age, until age 85 years, after which the rate of ra diminished(20). findings of the current study showed that positive beliefs about the necessity of medication (specific necessity score) had recorded the highest mean among the rest of the scores followed by specific concern score about potential adverse effects of medication and the majority of the patients (88%) had bmq specificnecessity score greater than bmq specific-concern score. as most of ra medications are life sustaining and it is not surprising that most of the recipients rated their beliefs on the necessity of taking medications higher than concerns about the medication (a positive necessity-concerns differential). study done by (r. neame and a. hammond) demonstrated that 75% of individuals with ra have positive belief about the need of their medicines(21). study done by zwikker he, shown increasing need belief about prescription in clinical practice may be advantageous in improving medicine adherence in ra patients(22). similarly, a study done by mardby, et al, concluded that increase awareness of the patient’s beliefs about medicines is needed and that healthcare providers ought to urge patients to express their perspectives about medicines so as to invigorate concordance and adherence to prescription(23). in this study, there are direct correlations between age, male gender, number of other coexisting chronic disease, and disease activity estimated by both das28-esr and cdai with bmq specific-necessity scores, which could be clarified that when patients became older they tend to be more wiser and had sufficient cognitive function to manage medications(24), and once disease became more violent, or when coexisting with other chronic disorder, patients tend to be more careful and realize the necessity for their medications(25). also, this study showed that male patients had more specific necessity about ra medications than female patients while another study showed no association between them(21). the current study show a significant positive correlation between cdai and specific-concerns. a possible explanation of this result is that patients with high concerns about therapy tend to be nonadherent to their medication which might lead to increase disease activity(26). a study done by (r. bmq-specific necessity bmq-specific concern bmq-general overuse bmq-general harm regressio n coefficient p-value regression coefficient pvalue regression coefficient pvalue regressio n coefficient pvalue age 0.184 0.012[s] gender(male vs. female) 0.238 <0.001[s] 0.121 0.081 education level -0.041 0.584 medication use duration -0.088 0.363 das28-esr 0.010 0.908 0.273 0.004 [s] cdai 0.119 0.187 0.160 0.093 r2 0.172 0.072 0.093 0.080 [s], significant relationship when p value<0.05. iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 140 neame and a. hammond) showed that high levels of concern are associated with helplessness and nonadherence, likewise they detailed a similar finding of the present examination that worries about unfavorable outcomes of prescriptions is independent of patients' age and education level(21). in this way, worries about ra drugs should be tended to paying little mind to the age or education level of the patient. in the current study, patients who lack adequate education had opinion that medicines are overused .a possible reason is the absence of information to assist a greater understanding of medicines and their effects. as medication use duration increase, general harm belief about medicines decrease, a possible is that when patients become more familiar with their treatment, their influence might diminish about general harm belief about medicines(27). also, this study demonstrates a positive association between both general harm belief about medicines and das28-esr score. a conceivable clarification is patients with a view that their medicines are harmful tend to be afraid to use their therapy appropriately as a consequence start to be poor adherent and so the activity of the disease will increase. univariate analysis showed that disease activity estimated by both das28-esr and cdai significant correlate directly with specific necessity belief. however this relationship is absent in multiple regression model. this means that disease activity score is an important predictor for belief about medication necessity when considered as a single factor but such factors become insignificant when considered in the presence of other stronger factors like age and gender. in addition only das28esr is independently correlated (significant direct relationship) with general harm belief in multiple regression regardless of the presence of other factors. the belief about medicine questioner may distinguish individuals in danger of poor prescription adherence and give a concentration to patients to talk about their convictions, giving chances to improve medicine adherence(21 ). limitations of the study this study had some limitations. patients were incorporated from only one department of internal medicine and the main diagnosis was rheumatoid arthritis. subsequently the outcomes can't be summed up to other patient gatherings with other sicknesses. in addition, the sample of ra patients had a disease duration above one year, making the results not generalizable to patients with recently diagnosed ra. lastly, the present investigation was a cross-sectional study, which makes it impossible to draw causal conclusions. more researches are needed to explore other factors (e.g., disease burden) that could affect the degree of belief about medication. conclusions the majority (88%) of iraqi ra patients sample had strong beliefs in the necessity of their ra treatment where the medication-necessity score was greater than medication-concern score, were older people, male gender, presence of other chronic illnesses and those who had high disease activity score tend to had more specific necessity score about ra medications. references 1. choy e. understanding the dynamics: pathways involved in the pathogenesis of rheumatoid arthritis. rheumatology. 2012; 51(suppl_5):v311. 2. veehof m. measuring treatment response in rheumatoid arthritis. the use of patient‐reported outcome measures [thesis]. enschede: university of twente. 2008:1-147. 3. kahlenberg jm, fox da. advances in the medical treatment of rheumatoid arthritis. hand clinics. 2011; 27(1):11-20. 4. al-rawi, z. s., al-azawi, a. j., al-ajili, f. m., et al. rheumatoid arthritis in population samples in iraq. ann rheum dis.1978; 37(1):73-5. 5. cross m, smith e, hoy d, et al. the global burden of rheumatoid arthritis: estimates from the global burden of disease 2010 study. ann rheum dis. 2014; 73(7):1316–22. 6. amy m. wasserman, md. diagnosis and management of rheumatoid arthritis. boston university school of medicine. 2011; 84(11):1245-1252. 7. grassi w, de angelis r, lamanna g, et al. the clinical features of rheumatoid arthritis. european journal of radiology. 1998; 27:s1824. 8. majed alsubaie1, waleed alqahtani, waleed alshardi., et al. methotrexate in rheumatoid arthritis patients: common side effects and leading cause of discontinuation. international journal of medical research & health sciences. 2018; 7(1): 116-121. 9. reddy d, trost lw, lee t, et al. rheumatoid arthritis: current pharmacological treatment and anesthetic considerations. middle east journal of anesthesiology. 2007; 19(2):311. 10. tsianou k, giannakeas n, tsipouras mg, et al. accessing patient views about medication in chronic conditions using the beliefs about medicine questionnaire (bmq): a review study. j drug res dev.2017; 3(2):1-9. 11. rob h, sarah c, rhian p, et al. understanding patients’ adherence-related beliefs about medicines prescribed for long-term conditions: a meta-analytic review of the iraqi j pharm sci, vol.28(2) 2019 belief about medicines rheumatoid arthritis 141 necessity-concerns framework. plos one. 2013; 8 (12): 1-24. 12. mohamed m, salmiah m, mohd d. beliefs and adherence to medicines among malaysian malay type 2 diabetes. international journal of current research. 2014; 6(2):5026-33. 13. james e, john d. diabetic patients’ medication underuse, illness outcomes, and beliefs about antihyperglycemic and antihypertensive treatments. diabetes care. 2009; 32(1): 19-24. 14. arnett fc, edworthy sm, bloch da, et al. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis rheum 1988; 31:315–24. 15. horne r, weinman j. patients' beliefs about prescribed medicines and their role in adherence to treatment in chronic physical illness. journal of psychosomatic research. 1999; 47(6):555-67. 16. wei l, champman s, li x, et al. beliefs about medicines and non-adherence in patients with stroke, diabetes mellitus and rheumatoid arthritis: a cross-sectional study in china. bmj open. 2017; 7(10):e017293. 17. alhewiti a. adherence to long-term therapies and beliefs about medications. international journal of family medicine. 2014; 2014(479596):1-8. 18. horne r, graupner l, frost s, et al. medicine in a multi-cultural society: the effect of cultural background on beliefs about medications. social science and medicine. 2004; 59(6):1307-13. 19. agarwal sk. biologic agents in rheumatoid arthritis: an update for managed care professionals. journal of managed care pharmacy. 2011; 17(9 supp b):s14-18. 20. gabriel se, crowson cs, o'fallon m. the epidemiology of rheumatoid arthritis in rochester, minnesota, 1955–1985. arthritis & rheumatism: official journal of the american college of rheumatology. 1999; 42(3):415-20. 21. neame r, hammond a. beliefs about medications: a questionnaire survey of people with rheumatoid arthritis. rheumatology. 2005; 44(6):762-7. 22. zwikker he, van dulmen s, den broeder aa, et al. perceived need to take medication is associated with medication non-adherence in patients with rheumatoid arthritis. patient preference and adherence. 2014; 8:1635. 23. mårdby ac, åkerlind i, jörgensen t. beliefs about medicines and self-reported adherence among pharmacy clients. patient education and counseling. 2007; 69(1-3):158-64. 24. park dc, hertzog c, leventhal h, et al. medication adherence in rheumatoid arthritis patients: older is wiser. journal of the american geriatrics society. 1999; 47(2):172-83. 25. komninis id, micheli k, roumeliotaki t, et al. adaptation and validation of the beliefs about medicines questionnaire (bmq) in primary care patients in greece. european journal for person centered healthcare. 2013; 1(1):22431. 26. pound p, britten n, morgan m, et al. resisting medicines: a synthesis of qualitative studies of medicine taking. social science & medicine. 2005; 61(1):133-55. 27. treharne gj, lyons ac, kitas gd. medication adherence in rheumatoid arthritis: effects of psychosocial factors. psychology, health & medicine. 2004; 9(3):337-49 baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ microsoft word bagpha05114 _6_ iraqi j. parma. sci., vol.14, 2005 47 -------------------------------------------------------------------------------------------------------------- role of serum zinc and copper and zinc/ copper ratio in alopecia areata ashwaq najjem elldin aljaff department of clinical pharmacy college of pharmacy, university of baghdad, baghdad, iraq received: 02.03.2002 accepted: 23.10.2002 abstract: alopecia areata is considered as a major health problem, its importance is attributed to its recent increased incidence in our population. till now, there is no exact cause for alopecia areata although researchers thought it's an autoimmune disease. this clinical study was designed to evaluate the role of trace elements (zinc and copper) in patients with alopecia areata. twenty patients were diagnosed as having alopecia areata with an age range (10-40 years) were involved in this study. normal subjects of the same age group were also evaluated as control. the level of serum zn and cu were measured by flame atomic absorption spectrophotometry in both control and patient group. and the ratio of zn/cu was also estimated. the results of patients group revealed that serum zn level was significantly lower than those of control (p<0.001), while serum cu was significantly higher than that of control group (p=0.002). furthermore, zn/cu ratio of patients group was significantly lower than that of control subjects (p<0.001). these results suggest the possible role of zn and cu level in alopecia areata. in addition to that the utility of measuring zn/ cu ratios for the diagnosis of the disease over that of determining the serum level of zn or cu alone since this ratio clearly reflects the severity of the progress. :الخـالصـة . یعتبر مرض داء الصلع من المشاكل الصحیة الكبرى، وتأتي أھمیتھ من خالل سرعة انتشار حدوثھ مؤخرا في مجتمعنا . ز المناعي للجسمإلى وقتنا ھذا ال یوجد سبب حقیقي مثبت لمرض داء الصلع بالرغم من إن معظم الباحثین یعزون السبب إلى الجھا . في مصل الدم في مرضى المصابین بداء الصلع) الزنك والنحاس(إن ھذه الدراسة السریریة صممت لتقییم دور العناصر النادرة وأشخاص من األصحاء المقاربین ألعم�ار ). عاما ٤٠-١٠(عشرین مریضا شخصوا بإصابتھم بداء الصلع وتتراوح أعمارھم من في مصل الدم ف�ي ) الزنك والنحاس(تم قیاس مستوى العناصر النادرة . ا في ھذه الدراسة وقد مثلوا العینة الضابطةالمرضى شملو أظھرت نتائج ھذه الدراسة انخفاض ملحوظ . كل من المرضى واألصحاء، باإلضافة إلى تحدید نسبة عنصر الزنك إلى النحاس . مقارنة باألصحاء، بینما مستوى النحاس أعلى بصورة ملحوظة منُھ في األصحاءفي مستوى الزنك لمرضى المصابین بداء الصلع باإلضافة لذلك أن نسبة الزنك للنحاس اقل لمرضى داء الصلع مقارنة باألصحاء، تبعًا لذلك یمكن االعتماد على ھ�ذه النس�بة ف�ي وتبین . ى حدة حیث إن ھذه النسبة تعكس حدة المرضمن االعتماد على نسبة كل من الزنك أو النحاس كُل عل المرض بدًالتشخیص . ھذه النتائج احتمالیة دور العناصر النادرة في داء الصلع iraqi j. parma. sci., vol.14, 2005 48 -------------------------------------------------------------------------------------------------------------- introduction **************** alopecia areata (a.a) simply is sudden patchy hair loss (1) . the exact cause of aa is unknown, however researchers believe it is an autoimmune condition (l, 2,3,4) . recently, al-jaff et al (5) , reported a significant increase in basal malondialdehyde (mda) level, as a strong biomarker of lipid peroxidation, and a significant decrease in glutathione (gsh) level, as a major antioxidant, in erythrocytes of alopecic patients compared to their normal control, suggesting the role of oxidative stress in the pathogenesis of alopecia areata. zinc (zn), an essential trace element, is important in numerous critical biochemical processes since it's a cofactor in about 200 metalloenzymes including cu/zn-superoxide dismutase, a critical cytoplasmic antioxidant enzyme (6) . zinc may stimulate the immune system, possibly through its antioxidant properties and protect sulfhydryl groups (-sh) from oxidation (6). also, zinc maybe important in stimulation of nadph oxidase since it's a cofactor for phospholipase a2 and phospholipase c, as well as in the stabilization of arachidonic acid against iron-catalyzed oxidation, among others (7) , including atpase, which is important in cell membrane fluidity(8,9). since zn is an essential component of cu, zn-sod, the deficiency of zn could induce an increase in tissue oxidative damage. zn deficiency is associated with an increase of cu and fe due to the antagonistic relationships between these metals (10) . also, zn deficiency resulting from a failure in absorption gives rise to alopecia areata and cutaneous changes in acrodermatitis enteropathica(6). copper (cu) could be a potential inducer of ldl oxidation. on one hand, cu has the ability to oxidize ldl in vitro (11) . on the other hand, it is a constituent of cu, zn-sod which is involved in preventing oxidative injury. in addition, caeruloplasmin, a multifunctional protein which contains most of the cu in blood, is thought to possess antioxidant functions, which could be beneficial in resisting disease. in contrast, high caeruloplasmin levels have been speculated to be a risk factor for atherosclerosis, based on its pro-oxidant properties (12) . therefore, the present study was designed to investigate the extent to which zn and cu levels were attributed in serum of alopecic patients. subjects and methods ************************** 1subjects a-patients: twenty patients aged 10-40 years (9 females, 11 males) with alopecia areata were included in this study. some patients were selected from various specialty dermatological centers and others from dermatology private clinics in baghdad. b-study group: comprised of total of 40 subjects, 20 normal controls and 20 cases with alopecia areata. patients involved in this study were under dermatologist supervision who determined the severity of the disease according to number of the patches they have and according to progression of disease (2) they were non-smokers, non-alcoholics and free from apparent other diseases. the duration of disease ranged from (20 day18 year). c-samples: venous blood samples were collected from alopecic patients as well as from controls using plastic disposable syringes with 22*1 1/4 g, hypodermic needles. serum samples were stored at -20 c° until the analysis was performed. iraqi j. parma. sci., vol.14, 2005 49 -------------------------------------------------------------------------------------------------------------- 2-methods serum zn and cu were measured by flame atomic absorption spectrophotometry (f.a.a.s) shimadzu aa-670/ gu-7. dilution of the serum was made by deionized water according to the sensitivity of the (f.a.a.s) and as mentioned in the manual instruction of the manufacturer in order to avoid the viscosity and to decrease the interference of the protein (13). the statistical significance of the difference in mean was tested by student t test. results ********* the serum zinc and copper concentrations of alopecic patients and control subjects were presented in (table 1). the mean zinc concentration was significantly lower in patients with alopecia areata as compared to the control group (p <0.001). however, the serum copper concentration in alopecic patients was significantly higher than that of controls (p=0.002) (table 1). the serum zinc to copper ratio of alopecic patients was significantly higher than that of controls (p<0.001), (table l). in this regard, further analysis of the data revealed that zn/cu ratio was lowest in those patients with severe progression (i.e., with more than 10 patches) compared to control and to those with mild severity of the disease (table 2). table 1: casecontrol differences in mean of certain outcome variables. control n=20 cases n=20 p value zinc concentration (mcg/dl) 94.20±1.92 58 .40 ±4.30 < 0.001 copper concentration (mcg/dl) 75.60±3.14 96.10 ±4.79 < 0.002 zn/cu ratio 1.30 ±0.50 0.61 ±0.40 < 0.001 values are represented as mean ± se table 2: zn/cu ratio in control subjects and patients with alopecia areata with variable severities alopecic patients (severity) control n=20 mild n=7 moderate n=7 severe n=6 number of patches no patches 1-2 3-5 >6 zn/cu ratio 1.3 ±0.09 0.73 ± 0.027* 0.59 ±0.09* 0.54 ±0.08* # values are represented as mean ± se. * significantly different from control (p< 0.001). # significantly different from alopecia areata patients with mild severity (p<0.05). note: severity of disease was performed by a dermalogist on the basis of the number of patches, and of progression of disease (2). iraqi j. parma. sci., vol.14, 2005 50 -------------------------------------------------------------------------------------------------------------- discussion ************* the present study showed a significant decline in serum zn level in alopecic patients (table 1), a fact that agree with that of tasaki et al (14) who, in addition to alopecia areata, reported comparable decreases in zn levels in serum of patients with other skin diseases (e.g. bullous pemphigoid and decubitus ulcer). factors responsible for this decline in zn levels are unknown. however, decreases in plasma zn content has been attributed to reductions in intake or absorption in small intestine, or to increases in urinary loss, or to redistribution from plasma to tissue (l5) . furthermore, tissues with high cellular turnover (e.g. skin) are characteristically affected by zn deficiency calling the attention to the possibility that some dermatological manifestations, such as alopecia areata, may be attributed to zn deficiency (15) . zn plays an important role in achieving proper function of the immune system in the body. also, it is required for the enzyme activities necessary for cell division, cell growth; and wound healing. immunologic defects of t-cell function are typical in zn deficiency (14) . experimentally, suboptimal intake of zn has rapid adverse effects on the immune system of humans (6) , including t cell mediated responses critical for host protection against parasitic infection. beck et al (16) concluded that mild zinc deficiency lead to an imbalance between th1 and th2 lymphocytes, decreases the recruitment of t native cells, and decreases the percentage of t cytolytic cells. therefore, the presence of a significant reduction in zn level of alopecic patients (table 1) may lead to impaired immune function of those patients. further study may be required to evaluate the role of zn supplementation in alopecic patients. oral zn supplementation has been reported to stimulate both t and b-cell activity (17-18) and improves the immune system in elderly people (19) . patients of the present study also showed an increase in serum cu levels. this finding dose not agree with that of tasaki et al(15) who failed to observe an elevation in cu concentrations in patients with alopecia areata. increases in serum cu content occur in various inflammatory and connective tissue disorders (15, 20). accordingly, the present finding may, therefore, suggest the presence of an inflammatory condition in patients with alopecia areata. it is possible that the observed changes in zn and cu plasma levels reflect the presence of an imbalance in these trace metals metabolism in alopecia areata. the consequences of this imbalance are unknown at present. these changes, however, are reflected in the zn/cu ratio, which was decreased in patients with alopecia areata compared with controls. furthermore, the fact that this ratio was lowest in patients with severe manifestations (e.g. those with more than 10 patches) compared with those patients with mild severity (i.e., those with less than 3 patches) is suggestive of the presence of a possible correlation with the severity of the disease. this fact further supports the hypothesis of tasaki et al (14) who demonstrated that cu/zn ratio clearly reflects the severity of the progression of skin diseases. further studies, using large number of patients, are required to test this hypothesis and to investigate whether or not it may also be useful in assessing the effects of various therapies against alopecia areata. iraqi j. parma. sci., vol.14, 2005 51 -------------------------------------------------------------------------------------------------------------- references ************** 1bennett-j c; plum-f, eds; cecil "text book of medicine" 20th edition;w.b.saundres company london; 1996; p 2215-2217. 2champio-rh; burton-jl, bums-da; breathanach, eds rook “text book of dermatology " 6th edition. blackwell science. england 1998; 1: p 2903-2938. 3fauci-as. braunwald-e, isselbacher-kj, eds harrisons "principles of internal medicine" 14 th edition. me graw-hill, new york; 1998; p 312-314 4peter-j; lynch-m.d, "dermatology" 3rd edition, williams & wilkins awaverly company; 1994; chapter 17: p 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4:794-808. 19roebothan.b vchandra.r.k.relationship between nutritional status and immune function of elderly people. age ageing. 1994, 23:49-57 20gowenlock ah. varleys practical clinical biochemistry. (ed). heinemann professional publishing: 1988; pp 633. الخـلاصـة : ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 10 a study of the furocoumarin derivative of ruta chalepensis l. (rutaceae) ekbal h.al-khateeb, ali a. al-shamma, bajes a. nehar rece ived 2-3-2002 accepted 23-10-2002 abstract the conte nt o f furoc oumarin d eriv ativ es (pso ra le ns ) of ru ta ch alepe ns is l. (who le p la nt) was stud ie d by s imp le e xtra ctio n with p etro le um ethe r (b .p . 60 -8 0co). the res ults indicate d that the p la nt co ntains a total of a bo ut (0.01 5%). inves tigatio n of these comp ounds b y thin la yer chroma tograp hy (tlc) re vea le d the pres ence of at le as t four compounds of whic h metho xs alen (8-metho xyp so ra le n) wa s is olated . it wa s id entifie d and authentica te d with a s ta nd ard by sp ec tral metho d, ir, nmr, mas s sp ectra and hplc.this plant could be c ons id ered a s a go od so urce for s up plying this co mpound , whic h is wide ly use d in d ermatologic al prepa ra tio ns. الخالصة عماالته ست العشاب با روف لدى متداولي ا مع كن متعددة و ما ي ا رع ف ي يز عراق والذ وجودة في ال ة الم ن النباتات الطبي سذاب م ان نبات ال وري دراسـة . الشعبية عراق٬ لذا فقد وجـد مـن الضـر عن النبات في ال ة ي دراس ة وال توجد ا عن هذا النبات قليل ورة ان الدراسات المنش ونات النب كومـارين مك رو مواد الفي من هذا النبات ما يحتويه p( ات بصورة كيمياوية مفصلة والتعرف ع soralen ( طرق ال وفصـلها ـب ة .المتبع ن ري مـا وكو ور ى الفي وي عـل ة اثبتت لنا بان النبتة تحـت ولي ج اال p(ان النتائ so ra le n ( ذيبات ـم حسـن ال اء ا ة النتـق مل ا ـش ة ال دراسـ م او ذا ـت ـل م مكن استغ ة التي ي وي الوراق العض وفي ا ة من جه ن الجذور كل م ي عيتها ف ن النبتة ثم تعيين كميتها ونو واد م خالص هذه الم ست ها ال ال ى هة اخر ن من ج ة حرارة . والسيقا ي بدرج غل ر الذي ي وم ايث رولي ن البت رت النتائج با وقد اظه ة من النبات مي مع ك م كان ° 80-°60فقد تم ج ال ملة الستخ عضوية المستع سن المذيبات ال ومارين اح وك واد الفيور ps(ص م oralen .( ة ريـق لقد اثبتت لنا نتائج فحـص المسـتخلص بط التي كا ميت واد س هذه الم مركبات من ة ي النبات وقد تم فصل اربع كبات ف مر ود عدة عن وج ة ة الرقيق وكرافيا الطبق مات ,ba1.: كرو ba 2, ba3, ba4 ركب االول ن الم خفيفة ) ba1(ا ورات كل بل حرارة انصهاره تم فصله على ش ة طويلة بيضاء درج 14و م ° 7-148 كومارين رو كبات الفيو ن مر هو م ويدي و ركب االم كب اثبت بانه م مر هذا ال وي ل ميا كي pso(والتحليل ال ra le n(. ركب ما الم فقد تم ) ba2(ا ة انصـهاره رات بيضاء اللون درح ة الج . م ° 78-76فصله على شكل بلو والصـيغ ي جزيـئ وزن ال ين اـل عـي م ت د ـت مركـب لـق هـذا ال ة ل زيئـي حليل الكتلة طة ت ma(بواس ss s pec tro sco py ( كب مر زيئي لل ن الج زيئية ba2 (256(وان الوز ة الج c16h16oوالصيغ ثم تم عمل . 3 nmr وir ب مرك حاليل ان ال خالل هذه الت ن جد م حيث و ركب ٬ شكل الكيمياوي لهذ الم كب لتحديد ال مر هذا ال يحتوي على ) ba2(ل كو ورو ن نواة الفي ري p(ما so ra le n (جانبية ة سل سل ه . وترتبط هذه النواة ب كيـب د تر حدـي حاضـر لغـرض ت ت ال ي الوـق رر حفظه ـف لذا فقد تق ريب مستقبل الق ب .الكيمياوي في ال مرك ها ) ba4( و ) ba3 (اما ال غرض دراست ر ل ها في الوقت الحاض هما قليلة وتقرر حفظ ميت كانت ك ستقبل رة مفصلة اكثر بالم . بصو introduction ruta cha le pensis l. (rutace ae ) is a cultivate c plant [1] wid ely grown a s ga rd en o rname ntals in baghda d a nd its v ic inity. ruta, rue and sad ha b are common names fo r this plant. rutac ea e (a ls o te rme d rue family) is o ne of the imp orta nt familie s, tha t comp ris es many ge nera and sp ec ie s which have bee n us ed as a so urce o f d rugs a nd medicine s [2]. the de coc tion o f this p la nt has b een use d in folk re med y as c arminative to cure s to mac h ache in children[3]. this study was c arried in connec tio n with a program of using the chemica l co ns titutes of iraq i me dicina l p la nts a s a pote ntia l for industrial purpo se s. literature s urvey re vea le d that ruta c ha le penis l., c ontains many importa nt c ompo ne nts[3-9], among w hich are co umarins a nd furo co umarin de rivative s.since no prev io us work has been done on this iraq i p la nt was found that stud y o f these comp ounds , is of adv anta ge for its ec ono mic al value a nd use s in d ermatologic al pre pa ra tions, as a vailable impo rted one s are ve ry e xp ens iv e. materials and method the p la nt mate rial was co llec te d during sep te mbe r and octob er 1 997 from the ga rd ens of the v ic inity o f the c ity of baghda d. f our and a half kilograms o f po wdered plant mate rial (a eria l plants and ro ots) we re extracte d with 12 liters of p etro le um ethe r (b.p. 6 0-80 co), under re flex fo r 7 2 hours. the p etro le um e ther extract was filtered and e vap orated to drynes s, und er reduce d p re ss ure at a temp erature not exc ee ding 40 c o, le aving a d ark gree n re sidue (8 0 gm). dep artmen t of pha rmco gna cy,co lleg e of phar macy, un iver sity of ba ghd ad, bag hda d-i ra q ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 11 separation and fractionation of furoc oumarin derivatives: different p artitio n proce ss es toge ther with co lumn chroma tograp hy technique w ere trie d but with uns ucc es sful re sults. so the s ep aratio n and frac tionation wa s ca rried b y us ing prepa ra tiv e this la yer chroma tograp hy. a qua ntity of 20 gm of the re sidue wa s diss olved in a minimum a mount of chloroform and a pplied toge ther with s tand ard p so ra le n re fe re nc e comp ounds (metho xs alen and imperatorin) on a numb er o f prepa ra tive tlc plates o f s ilic a ge l gf 254 (0.75 mm thickne ss) and d ev elope d with so lv ent sys tem benze neac etone (90 :1 0 v/v). the d eve lope d plate s we re v is ua lize d under u.v light (3 66 nm wave length). f our major bands we re o bse rv ed a nd de signated a s ba nd i, ii, iii and iv. ba nd ii and iii were o verlapp ed , and b oth ha ve very c lo se rf va lues to that o f the standard reference me thoxs alen. the se two b and s were se pa ra te d by multiple de velop ment us ing the sa me so lv ent system to give two co mpo und s. band i ga ve c omp ound i as ye llowis h crys ta ls (25 mg) up on re crys ta llization out of boiling metha nol, a fluffy c olorle ss crys tals was o btained (20 mg) ha ving a s harp melting point of 1 47.5o. out of ba nd ii, comp ound 2 was iso la te d as ye llowish p risma tic crys tals (1 25 mg), up on re crystallization out o f hot pe troleum ether (b.p. 60 -8 0 co) a colorles s lo ng p risma tic c rystals (1 20 mg) was o btaine d ha ving a s harp me lting po int of 78 co. out of b and iii, compound 3 was is olated a s a yellowish crys tals (2 .7 mg) upo n re crys ta llization out of bo iling etha no l fluffy like crysta ls (2.5 mg) was o btaine d. out of b and iv, compound 4 wa s isolate d as a n oily material. the total yields of thes e co mpounds is olated fro m the total extrac t (8 0 gm)– were: 8 0 mg of c omp ound 1; 4 80 mg of co mpound 2; 10 mg of c omp ound 3; a nd oily material d es ignated as co mpo und 4 . esults and discussion r out o f thes e co mpounds , only c omp ound 1 was ide ntified and c onfirmed to b e metho xs alen (xanthotoxin) a s it ha s similar melting point, ch analys is , and r f va lues in different s olvent s ys te ms with s tand ard re fe re nc e of me thoxsa le n. an as sa y b y hplc also was done for co mpound 1, us ing metho xsa le n u.s.p. 1 % a s a standard re fe re nc e. the re te ntion time wa s exactly the s ame , furthe r id entifica tion wa s ca rrie d out by enric hment tec hnique, in whic h the reference was a dde d ind iv id ually to co mpound 1, us ing three different mobile phase s (fig. 1 a, b, c). other s pec tra l metho ds were us ed for further confirmatio n. the ma ss s pe ctrum o f c ompo und 1 indicates a mo le cula r io n pea k at m/e 21 6. the fra gme ntation pa ttern showing s ignifica nt pea ks a s shown in (fig. 2 ). the nmr s pe ctrum in cdcl3 (fig. 3 ) co nfirmed the pres ence of eight protons. the proton (h4) a nd(h5) a s tw o doublet a t δ 6 .8 (h4) and δ 7.71 (h5) (s hielded (j ) is sma ll as e xpe cted for the furo r ring (j =12 cps )). (h3) pro to n showed s inglet pea k at δ 7.82 (a ro matic p ro to n) (h1) and (h2) protons also a ppe ared a s tw o doublet pea ks a t δ 7 .3 5, δ 7.26 for (h1) pro to n, a t δ 6.43 a nd δ 6.31 for (h2) p roton. me thoxy group p ro to ns s howe d at δ 4.3. the ir spe ctrum (kbr) (fig. 4) c onfirms the structure. c = o s tre tc hing ba nd at 1 575 cm-1 fo r the α1 β-uns aturated lacto ne. tw o bands at 1 600 cm-1 157 5 c m-1 fo r (c = c) stre tc hing v ib ra tion o f the aroma tic ring. (c = c) alefinic ba nd a t 16 60 cm-1 [10]. two ba nd s at 145 0 cm-1 and 1 375 cm-1 for as ymme tric bending vibration of ch3 group res pe ctiv ely. the s tretc hing (c-o-ch3) a ppe ars at 12 80 cm -1 fo r as ymme trical stre tc hing a nd 10 20 cm-1 for symmetrica l s tre tc hing. two characteristic stre tc hing vibratio ns fo r the e ster func tion app ear at 1 145 c m-1 and 109 0 cm-1. the mo ot cha ra cteris tic out of plane c-h be nding vibra tio n at 8 22 cm-1 a nd 7 60 cm-1. moreo ver the c-h o ut p la ne b end ing vibration at 87 0 cm1 is due to o ne fre e (h) in benze ne ring. f inally the o ut o f plane o le finic (h) (ciso le fe in) is prese nt at 7 55 cm-1 in a gree ment with the structure [10]. all the abo ve mentione d s pec tral da ta confirmed that c ompo und 1 is me thoxsalen hav ing the follo wing structure: co mpound 2, 3, and 4 le ft fo r furthe r identific ation studies och3 o o o ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 12 fig (1 a )h.p.l.c for a mixture o f e qua l quant itie s o f the co mpo und (ba1) and the s tandard re fe re nce oxsoralon : (me tho xsa le n u.s.p 1% ) with s eco nd mob ile phase fig (1b) hplc fo r the co mpound (ba1) compare d to the standard re fe re nce oxso rale n ;(me thoxsale n u.s.p 1 %) ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 13 fig .(1c ) hplc for a mixture of e qual quantitie s of the compound (ba1) a nd the standard re fe re nce oxso ra le n ; (me thoxsale n c.s .p 1 % ) wit h third mo bile pha se fig (2) mass spec trum of the compo und (ba1) ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 14 fig (3): nmr spe ctrum o f the co mpound (ba1) de te rmine d in cdcl3 fig (4 ) :ir spe ctrum o f co mpou nd (ba1) ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 15 references 1. c.c. touns end a nd ev an gues t “f lo ra of iraq ”., minis try of agriculture and agraria n reform, baghda d; re public o f ira q(198 0) vo l. 4 , p. 455 . 2. me ster, i. fitoterapia: 1 973 , 4 4 (4). p. 123 -1 52 . 3. el-beih, f.k.; el-ta wil b.a.h; ba ghlaf a.o. constituents of lo ca l p la nt. part 12. “co uma rins a nd chalepe ns in a furthe r co nstituents o f ru ta c halep ens is l.”. j . chim. che m. s oc. (ta ipi) 198 1, 2 8 (4 ), 27 3-8 (eng.), via che m.. ab str. 96, 489 98r 198 2. 4. mo hr n; budzikie wicz, h; el-ta wil b.a.h; el-beih f .k.a. co nstituents lo cal plants . pa rt 14. “f lurthe r furoquinoline alka lo id s from ruta cha lepe ns is l.”. phytochemistry, 1 982 , 12 (7 ), 183 8-9 (eng.). via che m. ab str. 98 , 50 312 x (1 982 ). 5. ulubelen a, terem b; tuzla ci, e; cheng k.f.; kong, y.c. “ alkaloids and c oumarins from ruta cha le pen sis l.”. phytochemistry 198 6 ,25 (1 ), 2 692 -3 (eng.). via che m. ab str. 106 , 1 352 57s 198 6. 6. ulubelen a, te re m b; “ alkaloids and coumarins fro m roo ts o f ruta c ha le pensis l.”. phyto chemistry 1 988 ,27 (2 ), 65 0-1 (eng.). via c he m. ab str. 108 , 1 836 56j 198 8. 7. amarol, maria teres a; p ro enc a de c unha, antonia “ line ar f uroco uma rins of ruta cha le pe nsis l.” . re v. p ort. farm 1 989 39 (3). 2 1-3 (po rt). via chem. abstr. 1 13, 138 595 f 19 89. 8. ulubelen a, tan n; “ amos kac ha n from ro ots of ruta cha le pe nsis l.”. phytochemistry 199 0 , 29 (1 2), 39 91-3 (eng.). via c he m. ab str. 114 , 22 566 4s (19 90). 9. ahya n ulube le n and husyin guner “ iso la tio n o f dehydromo ksa chan c from ruta cha le pe nsis var. iatifolia” .j . of na t. pro d. 198 8 , 51 , no . 5, p. 1 012 -1 013 . 10. s ilve rs tein, bas sler and mo rrill “sp ec trome tric ide ntifica tion of organi comp ounds” 4th ed. john willy and s ons . new york 1 981 , p.59-135. a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci, vol.17 (2) , 2008 il-6, il-1, ish, breast cancer 55 the in situ expression of il-6 and il-1β in breast cancer patients amal h. salman* , mayada n. iqbal* and wasan a. bakir** * college of medical and health technology , baghdad, iraq ** iraqi center for cancer and medical genetic research , al mustansiriya university, baghdad , iraq abstract: breast cancer is the second most common cancer in women world. multiple cytokines appear to have a dominant role in human breast cancer formation. estimation of the in situ expression of il-6 and il-1β in breast cancer patients. a sixty patients with breast cancer bc were divided into two clinical subgroups, (30) with malignant breast cancer mbc and (30) with benign breast tumor as a control group according to histological examination. in situ hybridization technique used for detection of il-6 and il-1β mrna sequence in two groups. the results showed that percentages of mrna expression of il-6 and il-1β were in (≥ 11-50%) for malignant breast cancer. this research also investigated that (73.3%) of benign breast tumor were expression less than (<10%) for il-6 and il-1 β mrna. the ish expression of the mean percentages of il-6 and il-1 β were higher levels in malignant breast cancer patients ( 48.13 and 56.07 ,respectively) than benign tumor (2.73 and 1.40 ,respectively), with highly significantly differences (p<0.01) of ish expression for il-6 and il-1 β mrna among two studied groups., the expression of il-6 and il-1 β mrna are significantly elevated in the tissue of breast cancer patients compared with benign tumor and was found a significant correlation between the expression of il6 and il-1β mrna in the tissue of breast cancer patients, thus the results of the present study might be explain the pathological role of these two cytokine in breast cancer. key words: il-6 mrna, il-1β mrna, breast cancer, ish. ةصالخلا اط عط سطررء املل نررع اش طا اماطررر ار اراا نثل امثار تع امطرمع .لناطة امطلال ض امارا ين ثري ثلاا اسرس ر تع ن ر ملض ط د سطررء 1 ل 6ندياض سطررء املل .املل ض امعحل ي ام حط نض ام طع ط تع املي . مدث ض ار طمين ض ( طاج رن ناطررء املل نع ندا للع امد ضلين ض ان لرثا ناد املح امثا ضع ا املضلين 60 الال املااس ).املل ( طاج رن نياو يل ل .نع اس ثلاو ندث ام طع ط 30( طاج رن ناطررء املل امثع ل ا لاملضلين املرر )30اشلمد ) ن ر تع املدرر. امثا ض مدا ر 1 ل 6تع املي . ما حط نض امحر امثيل امطااعيسي ع املطاسث مدث ض ار طمين ض يدثلا ناطرس تاضيرم ةعومجم دنع ( ) نثل ضلين طاجري سطررء املل 50-11 ن ر ع ) ≤1 ل6املضلين ض .اعلطي املااس اء راع ام طع ط مير طمين ض ن ر .1 لار طمين ض -6 % ض طاجري امياو امحل ل مدي ض ار طمين ض -73.3 يدثلا ناطرس تاضيرم ةعومجم دنع ( ) م((((((( 10امثع ل ن ثلر نررل راع ام طع ط ع ) > )ناد 56.07 ل 48.13 ن ر ملضلين امياو امثع ل ) 1 ل6لن ثل ندث ام طع ط تع املي . طلع راة فيا نرم نر طمين ض ) ناد ام يامع ل دا اشف يتري اثي امد عليا تطلعري 1.40 ل 2.73ام يامع درار نلضلين امياو امحل ل لام ع ادال امثاة ) لار طمين ض 6 ) .ندمأ ن ثل املااس ل يث نيع ي ع طثيا ن ض ام طع ط نر طمين ض -0.01 طثيا نرم نثل ا يض طثيا ) > ن ر تع املدرر. امثا ض ملطاجري سطررء املل .ل ددا تاء ر ر املااس امحرم عل ني ت امللااش طا ع ملداض امثين ض ض 1 امارا ين ثري تع نديء سطررء املل لنيياا . introduction breast cancer is the most frequently diagnosed cancer and the second leading cause of death after lung cancer in women (1). there is strong evidence that the tumor growth can be actively controlled by host immune system.(2) . cytokines are known to have both stimulatory and inhibitory effects on breast cancer growth depending on their relative concentrations and the presence of other modulating factors in the tumor microenvironment. certain cytokines appear to prevent an effective immune response being mounted; permitting cancer growth, whereas others promote the immune system's antitumor capability (³) .interleukin-6 (il-6) is a cytokine with multiple biologic activities on a variety of cells. it is produced by macrophages, t cells, b cells, endothelial cells and tumor cells. il-6 is able to promote tumor growth by upregulating antiapoptotic and angiogenic proteins in tumor cells. 1 corresponding author : e-mail : thepharmacycollege16@yahoo.com received : 1/9/2008 accepted : 29/11/2008 iraqi j pharm sci, vol.17 (2) , 2008 il-6, il-1, ish, breast cancer 56 it is associated with worse survival in patients with metastatic breast cancer and is correlated with the extent of disease (4). in human breast cancer, an important role of il-1β and il-1ra mrna expression was noted in various studies (5), interleukin-1β is a highly inflammatory and prototypical multifunctional cytokine that affects nearly all cell types, often in concert with other cytokines or small mediator molecules. il-1β elicits important proinflammatory and immunological responses, such as fever, hypotension, increasing circulating no, recruiting neutrophils,and costimulating t cell activation by increasing il-2r expression and inducing il-2 production (6). the basis of the various biologic properties of il-1β depends on its regulatory effects on the expression of various genes and/or receptors. il-1β induces the gene expression of the il-1 family, other inflammatory cytokines, colony stimulating factors, and mesenchymal growth factors (7).this study aimed at estimation of the in situ expression of il-6 and il-1β in malignant breast cancer patients comparing to the benign breast cancer and find out the correlation between these two marker in malignant and benign patients. materials and methods sixty iraqi patients with breast cancer who were admitted to al -yarmook and baghdad teaching hospital. patients with breast cancer (bc) were divided into two clinical subgroups according to histological examination: (30) with malignant breast cancer and (30) with benign breast tumor as a control group. fresh samples were obtained during routine examination of surgically removed tissue, each specimen was fixed in 10% formalin then processed paraffin wax embedded section and cut into 5µm thickness, put on fisherbrand positively charged slides for our research. in situ hybridization: for in situ hybridization technique (ish), dna probe hybridization/detection system in situ kit (maxim biotech, inc., usa) was used.the probes were biotin-labeled dna probes for human il-6 (360 bp), and human il-1β (556 bp), (maxim biotech, inc., usa). in situ hybridization (ish) is a technique used the high specificity of complementary nucleic acid binding to detect specific dna or rna sequence in the cell (8) for detection of this markers, the biotinylated dna probe hybridize the target sequence (il-6 and il-1β mrna sequence) then a streptavidin-ap (streptavidin alkaline phosphatase) conjugate is applied followed by addition of the substrate promochloro-indolyl-phosphate /nitroblutetrazolium (bcip/nbt)which yields an intense blue black signal appears at the specific site of the hybridized probe (9) . this directly streptavidin-ap conjugate like the biotinylated probe provides a rapid and highly sensitive detection method.evaluation of ish signal was done with the assistance of a histopathologist .the expression of both il-6 and il-1β mrna was measured by the same scoring system, counting of the number of the positive cells in the tissue that has given a blue-black (bcip/nbt) nuclear staining under the light microscope. the score was the average from 10 distinct high-power fields observed under ×100 magnification. the percentage of positively stained cell was calculated for each case by taking the mean of the percentages of the positively stained cell in the 10 fields. a score of 0 was given when no staining was detected, 1 if there was weak to moderate staining in less than 10% of cells, 2 if moderate to strong staining was present in 11 to 50% of cells, and 3 if strong staining in more than 50% of cells was detected (10). statistical analysis the suitable statistical methods were used in order to analyze and assess the results. descriptive statistics results presented as percentages of frequencies ,mean, sd, sem, minimum & maximum levels .inferential statistics used to accept or reject the statistical hypotheses, includes: chi-square (χ2),t test.pearson correlation (r). p value < 0.05 and p < 0.01 were considered statistically significant. (11). results the expression of il-6 and il-1β were detected by ish technique. tables 1 and 2 show the percentage of frequency scoring for il6 and il-1β mrna expression among study groups, respectively. chi-square test was conducted to examine the association between il-6 and il-1β mrna expression in the tissue in the two groups of investigated women ,it was found that highly significant association (p<0.01) between them among the four scoring levels. the results showed that percentages of mrna expression of il-6 and il-1β were in ( ≥ 11-50%) for malignant breast cancer. this research also investigated that (73.3%) of benign breast tumor were expression less than (<10%) for il-6 and il-1 β mrna. on the other hand, the mean percentages of these two cytokines was significantly higher(p<0.001) in malignant breast cancer compared with benign tumor as demonstrated in (table 3) . the expression of il-6 and il-1β was heterogeneous blue-black nuclear staining in the tissue, as shown in figure (1). in addition, this study demonstrated highly significant positive correlation (p<0.01) iraqi j pharm sci, vol.17 (2) , 2008 il-6, il-1, ish, breast cancer 57 between il-6 and il-1β in two studied groups, as shown in (table 4) and figure (2). figure (1): detection of il-6and il-1β in studied groups by in situ hybridization (ish). staining of il-6 and il-1β mrna by bcip/nbt (blue-black)counterstained with nuclear fast red. (a) tissue from breast cancer patients shows positive il-6 hybridization signals (x400). (b) tissue from breast cancer patients shows positive il-1 β hybridization signals (x400). (c) negative control tissue. iraqi j pharm sci, vol.17 (2) , 2008 il-6, il-1, ish, breast cancer 58 table (1): distribution of ish%il-6 among studied groups (malignant breast cancer & benign breast tumor patients). * = breast cancer ** = breast tumor table (2): distribution of ish%il-1β among studied groups (malignant breast cancer & benign breast tumor patients). * = breast cancer ** = breast tumor table (3): mean of ish%il-6 & il-1β levels among studied groups (malignant breast cancer & benign breast tumor patients) studied groups interleukins malignant bc* n=30 benign bt** n=30 (t-test) p-value sig. ish%il-6 mean sd sem mini.─ maxi. 48.13 21.03 3.84 15 ─ 85 2.73 2.49 0.45 0 ─ 8 0.00 highly sig. (p<0.01) ish%il-1β mean sd sem mini.─ maxi. 56.07 17.87 3.26 20 ─ 86 1.40 1.13 0.21 0 ─ 3 0.00 highly sig. (p<0.01) * = breast cancer ** = breast tumor ish%il-6 groups studied groups total comparison of significant malignant bc* benign bt** p-value sig. 0% n 0 8 8 0.00 highly sig. (p<0.01) % 0 26.7 13.3 < 10 % n 0 22 22 % 0 73.3 36.7 11-50 % n 16 0 16 % 53.3 0 26.7 >50% n 14 0 14 % 46.7 0 23.3 total n 30 30 60 % 100 100 100 ish%il-1β groups studied groups total comparison of significant malignant bc* benign bt** p-value sig. 0% n 0 8 8 0.00 highly sig. (p<0.01) % 0 26.7 13.3 < 10 % n 0 22 22 % 0 73.3 36.7 11-50 % n 11 0 11 % 36.7 0 18.3 >50% n 19 0 19 % 63.3 0 31.7 total n 30 30 60 % 100 100 100 iraqi j pharm sci, vol.17 (2) , 2008 il-6, il-1, ish, breast cancer 59 table (4): correlation between ish%il-6 level & ish%il-1β level among total breast cancer patients, benign bc patients & malignant bt patients. pearson correlation total malignant b c* benign bt** ish%il6 level ish%il1β level ish%il6 level ish%il1β level ish%il6 level ish%il1β level r 0.893 0.576 0.516 p-value 0.00 0.001 0.004 sig. highly sig. (p<0.01) highly sig. (p<0.01) highly sig. (p<0.01) * = breast cancer ** = breast tumor ish%il-6 level 100806040200-20 is h % il -1 b et a le ve l 100 80 60 40 20 0 -20 cases control breast cancer total population figure (2): correlation between ish%il-6 level and ish%il-1β level among total breast cancer patients, benign bt patients& malignant bc patients. discussion cytokines in general are thought to be involved in numerous physiologic and pathologic conditions. among cytokines, il-1β and il-6 probably seem to play the most important role in breast carcinogenesis (12; 13) .in the present study, il-6 and il-1β mrna expression was examined by in situ hybridization technique in tissue of malignant breast cancer compared with benign tumor . the il-6 and il-1β were expressed in a higher percentage in breast cancer tissue compared to benign tumor and we found the positive expression of il-6 mrna and il-1β mrna among malignant breast cancer were 46.7% and 63.3% were more than fifty percent (>50%),respectively . this results suggests that il-6 and il-1β are over expressed in breast carcinoma compared to benign tumor and might play a pathological role in malignant breast cancer. evidence supporting this suggestion includes the fact that in human breast cancer, the elevated expression of il-6 and il-1β were observed in breast carcinoma tissues (5; 14; 15; 16) and in serum (17; 18) .several studies suggest that the il-1 system is vital in the local control of tumor growth, important in regulating ‘‘protumorigenic’’ activities within the tumor microenvironment, and contributes to angiogenesis, tumor proliferation, and tumor invasion (19; 20; 21). furthermore, il-1ß and il-6 cause tumor regression and increase median survival time in a variety of cancer patients. in contrast, elevated circulating concentrations of growth factors such as igf-i are a surrogate risk for cancers of the breast (22; 23; 24). it is noteworthy that il-1 β is a prototypical proinflammatory cytokine that exerts a plethora of biological activities, including tumor regression (25). the tumor-suppressing property of il-1 β has been attributed mostly to its ability to prime antitumor immunity (26), but the mechanism for its direct cytostatic actions in suppressing cell cycle progression is largely unknown. the antiproliferative action of il-1 β on human breast cancer cells is exhibited not by killing the cells but rather by preventing the ability of the late g1 progression factor, insulin-like growth factor (igf)-i, to promote progression from late g1 into the s phase of the cell cycle (27) .this cross-talk between proinflammatory cytokine and growth factor receptors is similar in principle to that between the b cell receptor and the 2-adrenergic receptor for the neurotransmitter norepinephrine (28) and that between the igf-i receptor and integrinassociated protein for thrombospondin-1 (29) .moreover, the role of the il-1 system in human breast cancer is conflicting. il-1 has been shown to inhibit growth of breast cancer cells and to promote cellular differentiation in vitro, but it is equally known to stimulate the expression of several proteolytic enzymes in human cancer (30;31) .the consecutive degradation of extracellular matrix is a key element of local invasion and metastasis (32, 33) .in addition, they are many confounding studies about the role of il-6 and il-1β in tumor cell growth, but its exact role remains varied and unclear (19; 34). it appears that the effect of il-6 on tumor cell growth may depend on the tumor cell type, il-6 plays a 60 new role in cancer biology; it promotes multidrug resistance (34) and it has been shown to be involved in intercellular signaling between mesenchyme and breast cancer epithelium.. these display an oncogenic role for il-6; however, lacking is an understanding of the mechanisms governing il-6 production in tumors and the biological role of this cytokine in tumorigenesis (19, 35). the human il-6 shows antiadhesive effects, and modulates the estrogen receptor and progesterone receptor content of these cells (35) .the elevated expression of il-6 has been detected in multiple epithelial tumors (36) .an interesting finding , in the current study that in situ expression of il-6 was significantly correlated (p< 0.01) with in situ expression of il-1β (r= 0.576 ; p< 0.01) in malignant breast cancer . this results indicating that il-6 and il-1 β are strongly interact with each other and act synergistically, subsequently increasing their effect. this finding in agreement with that of robison and colleagues who reported that a significant correlation between il-6 and il-1 immunoreactivity (13) , thus both ils, i.e., il-1β and il-6 have been shown to be strongly interact and to act additively in breast carcinogenesis, subsequently inhibiting tumor growth (37) . in conclusion, in this study both il-6 and il-1 β mrna showed the significant increased expression in the tissue of malignant breast cancer patients compared with benign tumor , this might be implicated in the development of malignant breast cancer. although a significant positive correlation between these two markers in all studied group, this might reflect a close relation between these two parameters. references 1. cotran, r. s. and lester, s. c: risk factors of breast cancer in robbins pathologic basis of diseases. 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(2001) 23:1–21. 37. danforth, d.n. jr & sgagias, m.k. interleukin-1a andinterleukin-6 act additively to inhibit growth ofmcf-7 breast cancer cells in vitro. cancer res; (1993) 53: 153845. ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 8 synergistic effect of potassium clavulanate in combination withcefamandol and ceftazidime on lactamase, extracted from resistant e.coli siham s. shaokat*1 , hamoudi a. hameed** *ministry of industry and mineral **min istry o f industry and mineral abstract the aim of this s tudy was to evalua te in-vitro activity of ce fa mandol and ceftazidime , in combination with pota ssium clavulanate a gains t 10 uropathogenic e.coli is olated from patients with c hronic c omplicated urinary trac t infe ctions (utis), these is olates were identified by the api ide ntific ation s ystems .the a ntimicrobial s usc eptibility tests were determined by kirby-ba uer method and the minimum inhibitory c onc entra tions of cefamandol and ce ftazidime , were de te rmined, by tube method. the se isolates we re res is tant to ampicillin (amp), amoxicillin (amo), carbenic illin (cb), tic arcillin (tic), amoxicillin\ potas sium clavulanate {augmentin}, (amo\ca), tic arcillin\ potass ium cla vulanate {timentin} (tic\ca), cefamandol (cfm) and ce ftazidime (cfz), also re sistant to other antibiotic s, s uch as te trac ycline, c hlora mphe nicol, trime thoprim and (50% of the isola tes were re sistant to na lidixic acid and rifampicin). transfer of pla smids by dire ct c onjugation experime nts were performed by mating 10 stra ins with rec ipient strain e.c olik12c 600 rif or na l re sistant, and ce ll free la ctamas es we re prepared and detec te d by macro-iodome tric method. the ac tivitie s of eac h cell free – lac ta ma se s e xtra ct agains t cfm and cfz were determined by disks diffusion method (microbiologic al masuda method) and by macroiodome tric me thod. the ac tivity of lac ta mase s was inhibited by the addition of potas sium cla vulanate. conclus ion: good e ffectiveness of cfm\ ca a nd cfz\ ca was obtained against re sistant stra ins of e.coli due to complica te d urina ry tra ct infec tion (utis). k ey words: la ctamas es , cefama ndol, ce ftazidime , timentin and augme ntin . الصة خ والنيك ض الكالف منهما مع حام ول السيفتازيديم بخلط كل من السيفاماند عالية كل ) ملح البوتاسيوم ( يهدف البحث الى تقييم ف ة عزوـل مرضة والمشخصـة والم ون الم راثيم اشريشيا القول ة نظـام التوصـيف الوظـائفي (ضد ج طريـق رة ) ب ـش ن ادرار ع ـم مصابين بالتهابات المجاري البولية مزمنة مرضى ة .ال ريـق ة بط ختلـف ة الم ـادات الحيوـي راثيم لمض ـج جاد حساسية هذه ال تم اي وسـط السـائل ة ال غرى بطريـق ذلك الجـرع المثبطـة الصـ ـك ي و رـب ة , كي وـم ا مقا ـدت بأنـه وج ـيلين , و مبس ن , لال سيسـيلي موك , اال ن ــلي ــــ ــلين ,الكاربينسـ ــ رســـ كا ــت , التي ــ ــ النـي كالفيو ــيوم ــــ وتاسـ ـيلين \ب ــ ــــ كسيس ن (امو ـي ــ ــــ وكمنت ــيوم كالف , )ا ــــ ــت بوتاسـ ــ ــ النـي \يو ن ن(تيكارســلي ـايمنتي ول,) ـت ن , والســيفتازيديم, الســيفاماند ورامفينيكــول, وكــذلك للمضــادات االخــرى مثــل التيتراســايكلي كل , ال رايميثوبريم ريفامبسين,الت كانت مقاومة لحامض الناليديكسيك وال زالت من الع مسون بالمئة ران . وخ ة االقـت تم تنفيذ تجرـب معرفة انتق شر البسيط ل ومة للريفامسين او المبا ن الحساسة للمضادات الحيوية والمقا شريشيا القولو عزلة ا ال البالزميدات الى ض الناليديكسيك مضادات , لحام ميدية لل ومة بالز مل مقا الت تح ميع العز مما دل على ان ج عها ايجابية مي كانت النتائج ج و منت,أعاله الت ميع العز ود في المحيط الصلب ان ج طريقة الي جد ب مات البيتاوو زي ألن ماندول , الكتاميز -جة كما تم تقييم السيفا ما مع الكالفوالنيت رعـي , والسيفتازيديم بخلطه وسـط ألز ي ال النتشـار ـف ة ا طريـق وب الم ماسـودا (بطريقة اليود ة الـع طريـق كروبايولوجية النيك مع , ) الماي ض الكالفو عد إضافة حام ملة ب كا ق تثبيط مناط زالت ميع الع والسيفا وأعطت ج السيفتازيديم ة , ماندول مزمـن ة ال ولـي ي الب ار ـج ات الم عالج ناجح اللتهاـب خلطات ك كر باستخدام هذه ال جعلنانف ـات , مما ي ـد اجـراء الدراس ع ب زمة .السريرية الال 1corresp ond in g author email a lbia tyss84@yah oo.coma lbia tyss84@yah oo.com rec eived 11 4-2005 acc ep te d 2 5-7-2006 mailto:albiatyss84@yahoo.com ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 9 in troduction clavula nic ac id is a lacta m; structura lly it differs from pe nicillins in two re sp ects , the re plac ement o f sulfur in the pe nicillin thiazo lidine ring with oxyge n in the clav am oxazo lidine ring and the a bs enc e of the s id e chain at pos ition 6. clavula nic ac id a na turally oc curring clav am is olated fro m s trep to myce s clav uligerus ha s poo r antiba cterial a ctiv ity but exerts a p otent andirreve rs ible inhibito ry e ffec t on  la ctama se s es pec ia lly pe nicillinas es b y bloc king the a ctiv e sites of thes e enzyme s and is s trongly s ynergistic with mo st o f the  la ctamine s in vitro(1,2).due to this comb ination, amoxic illin is p rote cted fro m d egrad ation and its s pec trum is therefore e xtende d to includ e ba cteria normally res is tant to a moxycillin and othe r  la ctam antibiotics (3) . in the ca se of lac ta m re sistant ba cteria a ba cteria l e nzyme, lac ta mas e, c le ave s the lac ta m ring and re nd ers the antibiotic inac tive -lac tamas es are a la rge and div erse gro up of e nzyme s in whic h fo ur c linic ally relev ant clas se s are known (4). lac ta mas es c ontinue to b e the lea ding ca us e of res is ta nc e to lac ta m a ntibiotic s a mong gra m-negativ e ba cteria . in rec ent ye ars there ha s be en an increas ed inc id enc e and preva le nc e of extende d-spe ctrum -la ctama ses (esbls ), enzyme s that hydrolys e a nd ca us e re sistance to oxyimino-cep ha lo spo rins and aztreo nam. the majority o f esbls are de rive d from the wide sp re ad broad spe ctrum  la ctama se s tem-1 a nd shv-1. esbls hav e be come wide sp re ad througho ut the wo rld and are now found in a significa nt p ercenta ge of e.coli a nd kle bs ie lla pneumo niae strains in ce rtain countrie s (5,6,7,8). the re a re also new fa milies of esbls , including the ce fo ta ximas e(ctx-m) and oxatyp e enzymes , ce ftazidimase , a s well as no vel unre la te d lactamase s (9,10). the stab ility of different c ephalos porins to the mos t important lac ta mas es wa s as se ss ed and many clinical stud ie s ha ve shown that up to 75% of the  la ctama se s re sp ons ible fo r la ctam resis tance in g-nega tive bac te ria we re r-pla smid med ia te d(11,12,).re ce ntly, new fo urth generatio n ce pha lo sp orins , s uc h as ce fe pime, cefpirome, cefos elis , ce fd itoren, ce fo zop ra n (13,14), were introd uc ed into antiba cterial c he motherap y and their ac tiv itie s were compared with o ther la ctams s uch as ce ftazidime,imipe ne m and carba penem , aga inst p.ae ruginos a, ente rob ac te riac eae (e.co li, kle bsiella pneumo niae ) a nd g-pos itive bac te ria. in ad dition s eve ra l drug comb inations hav e bee n produce d whic h c ontain both a lac tam antibiotic a nd a la ctama se inhibitor; the inhibito r ha s high a ffinity for la ctama ses it irre vers ibly b inds to it, a nd the re by pres erves the a ctivity of the la ctam. curre ntly, four penic illin inhib itor co mbinations are in clinic al use : amp ic illin sa lb ac ta m (una syn) , amoxic illin clav ulana te (augmentin), tic arcillin – cla vulanate (timentin) and pip ra cillin-tazoba ctam (zos yn) (15,16) .urina ry trac t infe ctio ns (utis ) c ause a s ignifica nt hea lth p rob le m and e.c oli ha s be en rep orte d to be the p rimary pa thoge n in ap pro ximately 8 0% of ca ses . e.coli, exp re ss structures ca lled adhes io ns, fimbriae or pili that help them bind to s pec ific tis sue (17). aim of the study the aim of the stud y is to ev aluate, the fo llowing c omb inatio ns , cefama nd ol / cla vula nate and cefta zidime/clav ulana te for their in vitro antimic ro bial ac tivity a ga inst comp lica te d urinary tra ct infec tio ns ca us ed by lac ta mas e producer e.coli. ma terials and methods bacteria l s trains sta nda rd str ains with plas mid – med iate d beta – lactama se s wer e us ed: 1-e.c oli k12 (tem-1 typ e la ctama se with is oelec tric point 5 .4) co nfer plas mid(r 1 11) and e.c lo aca e p99 (11 ). 2 -e.c oli k12 (s hv-1 type la ctama se p itton (type ii) i.p 7.7 (11). 3-e.c oli k12 c 6 00 rif and e.coli k12 c 60 0 nal se nsitiv e to antibiotic s (11). 4-clinic al is olates o f e.co li. 5 -pure enzyme o f me d lab s. all types of a ntib io tics powd er we re o btained and kindly provide d by sdi. 6 e.coli atcc 259 22 kind ly p ro vided b y med ic al c ity. ide ntification of e.c oli strains were is olated o n macc onkey a gar and id entified by ap i 20 e s ys te m (biomerieux vite k, inc ) (18). antib io tic su sce ptib ility tes t (dis k diffus io n method ) the re sistance pa ttern fo r antibiotic s w ere determined by ba uer kirb y (19) d iffusion ass ay on muelle r – hinton a gar (2 0ml / plate) the inoc ulum was 1 04 – 10 5 ba cteria / ml, of 6 hours cultures incubate d at 3 7c0 for 24 hours .the a ntib io tics use d were as follow: ampicillin30g,amo xicillin30 µg,augme ntin (amo 20µg+ca10 g), ca rb enicillin 10 0 g, tic arcillin10 0 g, time ntin(tic 75µg+ca 10 g), ce fa mandol 30 g a nd ce fta zidime3 0 g, rifamp ic in 30 g, nalid ixic ac id 30 g, tetra cycline 30 g, chloramphenic ol 30 µg and co trimoxazo le (trimetho prime 2 .5 g + sulfa metha xa zole 2 2.5 µg) powd ers of cefama ndo l a nd ce ftazid ime were also obtaine d from (rousse l, bee cham and sep acia). ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 10 min imum inhibitor c onc entra tion (mics ) this test me as ures the conce ntra tion of a n antibiotic nece ss ary to inhibit growth o f a standardized ino culum under de fine d co nditio n .minimum inhibitory concentra tions (mics ) were d etermined by dilutio n of different c onc entration of antibiotics in mue ller – hinton broth .the tub es were inoc ulated with a 6 hour incub ation cultures , diluted , give n a final co nc entratio n of inoc ulum (1 04 – 1 05 cfu/ml ) a nd inc uba te d at 37c .the lo wes t co nce ntra tio n o f antibiotic preve nting growth and rema ining clea r (fre e from microbia l growth ) (mic) w as es tima te d after 1 8 hours o f inc uba tion . rema ining clea r (free fro m microb ia l growth) (mic) wa s e stimated after 18 ho urs of. as co ntro l, fully sensitiv e e.c oli k12 stra in wa s te sted und er the s ame co nd itions . ta ble1 and tab le 2 s ho ws normal mics v alues and diame te rs o f zo ne of inhib ition ac cording to the method reco mmended by the natio nal committe e fo r mic ro biology labo ra tory stand ards (france) (20). tra nsfer of g ene tic info rma tion by dire ct con jug atio n method . conjugal tra ns fe r o f 3 gc resis ta nt esbl pro ducing s trains wa s do ne at 35°c°c 37°c in liquid med ium {brain hea rt infusion (b.h)} or in so lid me dia { trypticas e s oya agar (t.s.a) o r mue ller – hinto n (m.h)} us ing e. coli k12 c 6 00 rif and e.co li k12 c 600 na l as re cipient.equal v olumes (1 ml) o f culture of the d onor and the rec ip ie nt s train (108-109 cfu/ml) grown with a gita tion in tryptic s oya bro th were mixed a nd incuba te d s ta tic ally for 18 hours at 35° c. trans co njuga nts we re selec ted on m.h aga r conta ining nalid ixic acid( 150 g /ml) o r rifa mpicin(30 0 g /ml) to inhibit the growth of donor and amoxic illin ,tica rc illin,a nd e ftazidime to inhibit the gro wth o f recipient s train (11). table (1) s ta nda rd o f mics a nd dia me te rs () o f zo ne o f inhibition o f ce phalosporins ce pha losporins abbre v ia tions critic al c oncentratio ns in g/ml o f * zone of inh ibit ion pote ncy of disk / g/ml first ge ne ratio nc c c d d cefalothin ctn 8 32 1 8 1 2 3 0 cefaloridin cfr 8 32 1 8 1 2 3 0 cefalexin cfx 8 32 1 8 1 2 3 0 se cond ge ne ratio n ce fa mand ol cfm 8 32 2 2 1 5 3 0 cefuroxim cx m 8 32 2 2 1 5 3 0 ce fo xitin cxt 8 32 2 2 1 5 3 0 third ge ne ra tion cefotaxime ctx 4 32 2 1 1 5 3 0 ceftriaxone cro 4 32 2 1 1 5 3 0 ce fo tia m ctm 4 32 2 2 1 5 3 0 cefmenoxi me cmx 4 32 2 1 1 5 3 0 ce ftazid ime cfz 4 32 2 1 1 5 2 5 ce ftizoxime zox 4 32 2 1 1 5 3 0 cefope ra zo ne cfp 4 32 2 1 1 5 3 0 cefod ia zine hr221 4 32 2 2 1 5 3 0 moxalac ta m mox 4 32 2 3 1 7 3 0 mic c : se ns itive s trains , mic>c: res istant strains , c< mic c in te rme diate,  d: se nsitive s trains , <d resistant s trains d < d =d ia meter (in 11). ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 11 table (2) standard va lues o f mics and d ia me ters () o f z one o f inhibition of pe nicillins . pe nicillins abbre viatio ns critica l co ncentra tion s in g /ml of * zone of inh ibit io n pote nc y o f disk / g/ml gro up a c c d d ampicillin amp 4 16 1 7 11 10 amoxycillin amo 4 16 2 1 14 25 augmentin amc 4 16 2 1 14 amo 20 + ca 10 timentin tim 1 28 128 1 3 13 tic 7 5+ ca 10 carboxypenicill carbenicillin cb 1 28 128 1 5 15 1 00 ticarcillin tic 1 28 128 1 3 13 75 amidinopenicillin mecillinam me c 1 8 2 3 17 25 ureidopenicillin mezloc illin me z 8 32 2 1 16 75 azlocillin azl 1 6 128 1 9 10 75 p ipracillin p ip 1 6 128 2 0 13 1 00 monobactam azthereonam atm 4 32 2 3 17 30 mic c : se nsitive s trains, mic>c: res istant strains , c< micc inter media te ,  d: se ns itive s trains , <d res is tant strains d < d =diameter ( in 11). extraction of – lactamase . cell free be ta –lactamase s were p re pared from strains known to be good prod uce rs o f the de sire d enzyme s, (– la ctama se s, typ e tem-1 e.coli r111 a nd shv-1 e.c oli4 53 , r-plas mid med ia te d e nzymes ) and –lac ta mas e from e.cloac ae p 99( ce pha lo spo rina se ) as re ference. crude e nzymes we re a ls o prepa re d from te st is olates of e.c oli. ba cteria l c ultures were grown ae ro bically a t 370 c in bra in hea rt infusion b roth (difco ) ove r night. a 20 0 ml flas k of the s ame broth was the n ino cula te d with 2 ml of the culture, incubated a t 3 7°cfor 4 ho urs, the ce lls were ha rv este d b y ce ntrifuga tion, wa shed twice with buffer phosp hate p h 7 a nd dis rupted with ultras ound (s onip re p 150 hse) a t 2 0khz. to remo ve ce ll de bris , the c rude extrac ts were ce ntrifuge d; the superna ta nts were collecte d in sma ll s te rile vials unde r as eptic co nditio ns (11). detection of – lactamas e by macr o io dometric method. 1% of a garos e and0 .5 % of starch were diss olved in12 0ml o f b uffer phosp ha te and boiled, 18 mg o f p enicillin g po wder and0.8ml of iodine s olution were a dd ed at 4 0°c, the med ium w as s haken a nd distrib uted in aliquo ts of 2 0ml in/ pe tridishe s, 5 well were ma de in ea ch plate , the enzymes we re a pplie d in e ac h well and the zone s o f dec olorization were obs erve d 1-18hours a t 4°c (21) . asses sme nt o f stability of – la ctams to cellfree – lactamase s( 22,23,24). the ac tivity of ea ch ce ll free – lacta mase extract against eac h – lacta m a ntib io tic was determine d by the mic ro biologica l method (mas ud a g., et a l. 197 6,mod ified by la bia.r. barthelemy.m. 197 9). the surfac e of a muller hinto n agar wa s se ede d with a susp ens ion of – la ctam sensitiv e ind ic ator e.co li atcc 259 22. four disc s co ntaining – la ctams under te st we re p la ce d nea r filter pa pe r disc s; e ach imp re gna ted with 30l of the enzymatic extract. the p la te s were incuba te d at 3 7°c for 18hours, the – lacta mas e ac tivity was obs erved like half mo on zone of inhibitio n. unc hange able inhibition zo nes de monstra te stability o f the antib io tic to the enzyme. inh ibition by cefamandol or ceftazidime /clavulana te modifie d io dometric method (21). modifie d iod ometric me thod (labia , r . , barthelemy . m.19 79),w as used witho ut incorpo ra tion of penic illin g in the me dium, five s wells we re ma de in the p la te in which 10l of e nzyme extrac t , 30 l of po ta ss ium clavula na te and 30l o f ce fa mandol or cefta zidime were a dd ed. the re sults we re noted a fter 4-18 ho urs a t 4°c, ab sence of dec olorization zone indicated pos itive rea ctio n. ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 12 masu da micr obiolog ic al me th od ten c linica l is olates we re sc re ene d fo r – la ctama se inhib itor us ing10l p otass ium clav ulana te in c omb inatio n with 3 0 l of ceftazid ime o r cefama ndo l. sensitiv ity disc s co ntaining ce ftazid ime o r cefamand ol and a filte r disc incorpo ra te d with 30l e nzyme and 10l pota ss ium clav ulanate were p la ce d o n agar p la te o n which a ba cterial s us pension of se ns itiv e e.c oli (s ta nd ard) was sp re ad the inoc ulum was 1 04 – 10 5 cfu / ml, o f 6 ho urs cultures a t 3 7c0 for 2 4 hours a cc ord ing to the metho d re commend ed by the natio nal co mmittee for microbiologic al lab oratory standards (25) . results a nd discuss ion dis k aga r diffus io n te st (s usc eptibility te st) ac co rding to the results of susce ptibility te st .the res is tanc e patterns of e.c oli riii (tem-1 be ta-la ctama se) and e.c oli k12 (shv1) type  lac ta mas e pitton (type ii ) i.p 7 .7 were c ompa re d w ith te n s trains the y we re re sistant to ampicillin , amoxic illin , carbenic illin p ip ra cillin , augmentin , time ntin , cefama ndo l and ce ftazid ime . the y we re a ls o re sistant to o ther a ntib io tics suc h as tetracycline ,chlo ra mphenico l and trimethoprim and (50%) we re re sistant to rifamp ic in a nd nalidixic acid . the res ults indicated d is se minatio n of resista nce a mong clinic al iso la tes of e.coli in ira q table 3. table (3) se nsitiv ity te sts of s trains de te rmine d by disk diffusion te st abb revia tion s : amp :amp ic illin ; am o:amoxic illin ;amc:amo xic la ve ; cb : ca rb en icillin ;t ic: ic arcillin ;t im : t imen tin ;cfm: c efama ndol ; cfz:ce ftazidime ; t c :t etracyclin ;cm:chlo ra mphen ic ol ;t m:t rime th op rim ;rif: rifa mpic in ; nal :nalid ixic a cid ;(see table1 ) detection of  lac tamases this tes t is bas ed o n the re ac tion o f the (oic ) ac id of penic illin with io dine .  lac ta mas e hydrolyze p enic illin to pe nicilloic a cid , whic h in turn reac t with iod ine , the p re sence of  la ctama se in a tes t s ys te m wa s s ho wn b y dec olorizatio n of starch – iod ine co mplex . the re sults of de te ctio n o f  lac ta mas es by io dome tric metho d were pos itive for 10 s trains comp ared with standard ne ga tive e.c oli k12 c 600 rif and po sitive – lacta mas es r11 1 (tem-1), p re se nted in fig 1. no of is olate s diameters of zone of inhibition/mm amp amo amc cb t ic t im cfm c fz t cm t m rif na l 1 0 0 4 0 0 5 .5 2 3 0 0 0 16 0 2 0 0 4 .5 0 0 7 3 3 0 0 0 0 18 3 0 0 3 .5 0 0 8 2 5 0 0 0 18 0 4 0 0 4 0 0 8 .5 0 4 0 0 0 10 0 5 0 0 5 0 0 9 6 15 0 0 0 15 0 6 0 0 5 .5 0 0 9 10 15 0 0 0 0 0 7 0 0 6 0 0 7 11 11 0 0 0 0 18 8 0 0 6 0 0 6 .5 5 10 0 0 0 0 19 9 0 0 6 0 0 9 .5 5 11 0 0 0 0 19 10 0 0 7 .5 0 0 9 7.5 13 0 0 0 10 18 e.c oil( 24) shv-1 0 0 1 9 0 0 2 3 19 24 0 0 22 0 20 e .coil tem-1 0 0 2 1 0 0 2 4 20 26 0 0 21 0 20 e .c loacae p99 0 0 4 15 13 5 6 4 0 0 0 ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 13 min imum inh ib itor y co ncentra tions the inhib ition of beta-lacta mas e pro ductio n by pota ssium clav ulana te ha s bee n de mons tra te d with many s trains o f bac te ria, this effe ct p otentiates the a ction of many b eta la ctams , suc h a s amp ic illin, amoxicillin, carbe nicillin a nd tica rcillin. ma ny clinical re ports of co mbination of amo xicillin with clavula nic ac id (augme ntin) have bee n encoura ging, in urina ry trac t infe ctions d ue to – lacta mas e-p ro duc ing orga nisms typ e tem and s hv, whilst amo xicillin a lo ne ha d no effe ct, the add ition of cla vula nic ac id (as s alt) drama tica lly change the ha lf mo on inhibitio n zo ne to co mplete inhibitio n zone (3,4,11). our inve stigations indica ted res is tant pheno type of augmentin and time ntin; the diame te rs o f zone of inhibitio n range d from (3 .5 mm-7 .5mm)fo r amc and ( 5.5mm-9.5mm) fo r tim, while the s tand ard d ia meters zo ne s of inhib ition we re for amc(14-21mm) and for tim is(13 mm). the critica l normal mics of augmentin and time ntin were (4-16 g /ml), (1 28 g /ml) res pec tive ly. the minimum inhibitory concentra tions were stud ie d fo r ten c linica l isolates of e.co li in c omp aris on with s ta nd ard res istant stra ins, tem-1 lac ta mas e co ded for plasmid r1 11, e.coli k12 s hv-1 lacta mase pitton (type ii) co ded for p la smid 45 3 i.p 7.7, e.cloac ae p9 9 ce pha lo sp orina se i.p 8 .3 (france) a nd e.co li atcc 25 922 (med ic al city hos pita l) se nsitiv e strains as references , the mics of cfm, cfz, were very high, the ra nge o f mics for cfm was 5 12 20 48 g/ml fo r cfz 64 32 g/ml. the se res ults are indica te d in table 4. table (4) minimum inhibito ry conce ntra tions of four ant ibiotic s to wards te n uropathoge nic e.c oli comparing w ith standard strains *isoe lectric point , ** e phalosporinase inh ibition of – lacta mase s figure 2 sho ws c ompa ris ons b etween antibiotic -e nzyme interactions, by the highly sensitiv e double disk tec hniq ue which demo ns trated hydrolys is of cefta zidime and cefama ndo l by – lacta mas e-p ro duc ing e.co li. the e nzyme s ob ta ined fro m 10 s trains hyd ro lyzed , ce fa mandol and ce fta zidime, b ut were highly s ta ble to a ll – lactamase s teste d when c omb ined with potas sium c la vulanate . enzymes extracte d fro m e.coli standards harbo ring plas mid r1 11 tem-1 o r s hv-1 harbo ring pla smid r 4 53 -la ctama ses we re inhibite d with p otass ium c la vula nate when comb ined with amoxic illin or ticarcillin( fig 3 a, b) howev er -lac ta mas e of e.cloa cae was no t affected b y augmentin and inhib ited by tica rc illin and hydrolyze d a ll cep halos po rins re pres ented in fig 4. in contras t -la ctama se unde r te st we re highly re sistant to amo \ ca, tic\ca, fig 5 : sho w inhibition o f enzymes b y io dometric method. no . of is olate e.c oli ce fa mandol ce ftaz idime 1,2 ,3 512 `64 4,5 ,6 1 02 4 32 7,8,9,10 2 04 8 32 e.c oli(4 53)(24) shv-1 (7.7 )* 16 0 .0 5 e.co li(r1 11) tem-1 (5 .4 )* 32 0 .0 5 e.c lo acae (p 99 )**(8 .3 )* figure (1): io dome tric me thod fo r de te ctio n o f  -lac ta mase (enz ymatic re action at (4 c )no.1 , no.2 , no.3:  lac tamase from e.coli clinical is olates , no .4 : tem -1 -lactamase as standard , no .5 :  la ctamase ne gative e.c oil atcc 2 5922 . ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 14 shv -1 (p453) direc t co njuga tion method it wa s fo und tha t c opies o f the ge nes for ampicillin, tica rc illin, te trac yc line , and chloramp he nicol resis ta nce co uld be transferre d by direct conjuga tion me thod fro m donor c ell to rec ip ie nt c ell. the res ults we re pre se nted a s fo llow: figure (2 ):antib io tic – e nz yme inte rac tions, by the highly se ns itive double dis ks technique (25) de monstrate d the inhibition of  la ctamases by c lavula nic acid. a a nd b: 1 an d 2 -dis ks of ce ftaz idime (cfz ) 30µg, and ce famandol (cfm) 30µg , 3 and 4 disk s at a distance of 2 cm fro m cfz and cfm impre g nate d with (10 µl) of e nzy me (la ctamase )5 a nd 6 disks impre gnated with 1 0µl e nz yme + 10µl clav ulanic a cid (ca). figure (3) a : s usce ptibility disk diff us io n me tho d s hows the activ ity of aug me ntin (ame ) and time ntin (t ic ) ag ainst plas mid – me dia te d  lac ta mase produce d by e.co li shv – 1 pitton type ii( p45 3)(11) figure (3 ) b : ac tion o f inhibitor c la vulan ic ac id (ca ) on the ac tivity o f s tandard lactama se e xtrac te d from e.coli shv-1 pitton type 1 : ampicillin , 2:  lactama se 3 : lac ta mase+c la vulanic acid. figure (4) :hydrolys is of ce phalosprins by ce phalo spo rina se ( β – lactama se ) , extracte d from e.cloa cae p99 ( according to s irot , d,1 983 ) fig ure (5): inhib it ion of  lactamase o f clinical is olates (e.co li )by mo difie d iodo me tric me thod compa ring with s tanda rd stra in o f e.co li atcc 25 922  lactamase non produce r ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 15 ten s trains tra ns fe rred resista nce to tica rc illin ,ceftazidime , and o ther a ntib io tics after mating for 1 8ho urs, tra nsc onjugants de rived from the se s tra ins prod uced βlactama ses, the se results suggested that a ll strains bear plas mids and p roduce extended spec trum β lac ta mas escapab le o f hydrolyzing and inactivating a wid e variety of β-lactams includ ing third generatio n ce phalosp orinsp enic illins and aztreo nam , se ns itive to imipe ne m , these result was simila r to the s tudies of ro drigues c , e t a l , cha udhary u ,a nd kuro kawa ,-h et a l (26,27,28 ) . conclus ions the clinica l isola tes in this stud y we re very resista nt to aug mentin , ce fa mand ol and ceftazid ime c omp aring with s ta nd ard tem – 1 and shv – 1 (pasmidic pe nc illinases ) , e. clocae p99 is ve ry resista nt to cefalothin , cefama nd ol , cefotaxime , ce fta zidime standa rd strain which produce -lacta mase (esbls ) e nzymes tha t hydrolyze a nd ca use resista nce to oxyimino – ce phalosporins and aztreo name . the ma jo rity of es bls are de rived from the widesprea d broad – spec trum-lac ta mases tem -1 and shv-1 . esbls have be come widesp read thro ughout the world a nd are now found in a s ignific ant pe rcenta ge o f e.coli and klebsiella pneumo niae s tra ins in certain co untries (6,7,9,10) . the increasing e mergence cep ha lospo rins resista nt e.c oli has leaded to co nc ern ab out the us e of v ario us c omb inatio n therap y . a go od in – vitro respons e was observed in our clinical uropathogenic e.co li when cfm and cfz , were mixed with different conce ntra tion o f potass ium clav ulanate as inhibitor the y w ere effe ctive and safe fo r the trea tme nt o f utis ca used b y -lactama ses (ceftazidima se ) producing comp lica te d strains (22,23,24,28) . references 1. rea ding c and cole .m. clavula nic acid : a be ta – lac ta mase inhibiting from stre ptomyces clav uligerus . anti microb . age nts and chemotherapy . 1977, 11 , n5, 852 – 857 . 2. je nse n , s e ; parad ka r ,a-s ;mos her , r – h ;anders ; c ;beatty , -p –h ; br umlik , m – j ; gri ffin , a ; barto n , b. five additional genes a re inv olved in c lavula nic ac id biosynthe sis in streptomyces clavuligerus . antimicrob – agents – chemo ther 2 004 j an ; 48(1) : 19 2 – 202 . 3. du mon l.,adriaens p., anne j .,eyssen h.effec t of clav ulanic acid on the minimu m inhibito ry co ncentratio n of be nzylpenic illin , ampicillin , c arbe nicillin ,o r ce fa lo thin a ga inst clinic al iso la tes resistant to be ata – lac ta m antibiotics.antimicrob .agents and chemother .,19 79 ,n2,315-317 . 4. bush , k ., ja co by ,g ., a and . me de iros .a.,a . a functio nal c lass ifica tion scheme fo r b eat – lac tamases and its corre latio n with molec ular s truc ture . antimicrob ia l a gents che mothe rapy 1995 p12 11 -1233 . 5. p oes chi, -p-w ;eckel , d ; po es chi, e .p ost operative prop hylactic antibiotic trea tme nt in third molar s urge ry – a nessity ? jora l – maxillofac – s urg .2 004 jan ; 62 (1 ) :3 -8 ; discuss ion 9is . 6. brad fo rd , p-a.extend ed – s pectru m beta lac ta mases in the 21st century :charac te riza tio n , ep idemiology , and detec tio n o f this impo rtant re sista nce thre at .clin – mic ro biol – rev .200 1 ; 14(4):9 33 -51 . 7. s ture nburg ,-e; ma ck , -d exte nde d – spectrum beta-lac ta mases :implicatio ns for the clinic al mic rob io logy lab oratory therapy , and infe ctio n control .j -infect 2 003 nov;47(4):273 – 95 . 8. be ll ,j – m , turnid ge , j – d ; j one s , r – n p re valence o f extend ed – spectrum b etalactama se – p roducing enterobac te r cloacae in the asia pacific region :res ults from the se ntry antimicrob ia l surveillance progra m ,19 98 to 200 1,antimic ro b – agents – che mother .2 003 dec ; 47(12 ):3989 -93 . 9. edelste in , m ; pi mkin , m ;pala gin , i ; ede ls te in ,-i ;s tratc houns ki,-l preva le nce and molecular epidemiology of ctx-m ex te ndedspectrum be ta -lac ta mase-prod ucing esc herichia c oli and klebsiella p neumonia e in rus sian hosp itals. antimic ro b-age ntschemother. 2 003 dec; 47 (12): 3724-32 . e.c oli wild type ampr ticr cfzr cmr tc r nalr rifs o r nal s ri fr e.c oli) k12 c 600 (sta nda rd ) amp s tics cms tcs na lr rifs o r nal s ri fr e.coli k12 c 600 (tra nsconjuga nt) amp r ticr cfzr nalr or rifr e.coli k12 c 600 (tra nsconjuga nt) amp r ticr nalr or rifr e.co li k12 c 60 0(transco njuga nt amp r ticr cmr nalr or rifr e.co li k12 c 60 0(transco njuga nt amp r cmr nalr or rifr e.co li k12 c 60 0(transco njuga nt amp r tic r cmr cfzr nalr or rifr e.co li k12 c 60 0(transco njuga nt amp r tic r tcr cfzr nalr or rifr ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 cefamandol , ce ftaz idime /clavulanate comb inatio n 16 10. baue rnfe ind, a., h. grimm, and s.schweighart. ane w-plasmidic cefota ximase in c linical isolate of e.coli. in fe ctio n. 1990 18: 294-298 11. s iham., s.s, jo ly,b, . phillipon ,a. , siro t,d., cluze l.r. re sistance al’a ca rbenicilline des bacilles a gramnegatife, freq ua nce , de terminis me bioc hemiq ue et ge netique , 1985 patho l ,bio l., 33, 825 -8 29. 12. el-sukho n,-s -n; faiza-boukha te m,-z. activity of c omb inatio ns o f c efta zidime, imip ene m a nd pefloxacin aga inst stap hyloc occus a ureus , es cheric hia coli and pseudomo nas ae ruginosa. int-j -anti microbage nts. 2003 dec ; 22 (6 ): 613-7 13. banta r, c ; di-cnia ra-m;nico la ,-f; rellos o ,s,s, smayevsky,comp arativ e in v itro bacteric idal ac tivity b etwee n cefep ime and ce ftazid ime, a lo ne and associated with amika cin, aga inst ca rbape nem-res is tant pseudomo nas ae rugino sa strains. j . dia gn. mic robiol-infec . dis. 2000; 37(1), 4 1-44. 14. kamidono, s et a l. aco mparative s tudy o n the clinical utili ty of c efozo pran a nd cefpiro me agains t comp lica ted urinary tract infec tions. ja p.j. antibio t.2 000 ; 53 (6 ): 430 -450 . 15. roland , -r-k; me ndes’-r-e; silb ert, -s; bolso ni,a-p;sa der, -h-s. in v itro antimicrobial ac tivity of p ip racillin\ta zo bac tam in c ompa ris on with othe r broad -spectrum lactams . braz-j-in fe ct-dis. 20 00; 4(5): 226-35. 16. lis ter, p -d. -lac ta mase inhib itor co mbinations with exte nde d-spectrum pe nicillins factors influencing a ntib ac te rial ac tivity aga inst enterobacteriacceae and pseudomo nas ae rugionsa. pharmac othe ra py. 2000; 20( 9 pt 2 ) 21 3s-218s; 224 s -228 s 17. incre as ing p revalence of anti microbial resista nce amo ng enterobacte riac eae uropathogens in da ka r, senegal: a mul tic enter stud y. dromigny, -j-a; nd oye , -b; mac ondo, -e-a; na beth, -p; siby, -t; perrie r-grosclaude, -j-d. diagn-mic ro biol-infec t-dis. 2003 dec; 4 7(4): 59 5-600 18. cowan, s.t, ma nual for ide ntification of med ical bac te ria 19 77. p10 6-camb ridge univ ersity press . ca mbrid ge new york. 19. ba ur a. w., kirb y, w. m.m., sherris k.c. and turc k, m.antibiotic s us ceptib ility tes ting by a sta nda rd ized s ingle disc method .,ame r. j. clin.path. 1966, 4 5,493-496 20. chabbert, y.a. l antibiogramme. ed de la toure lle 1 966 , 56-87. 21. labia, r., barthelemy.m. l e nzymogra m des β-lacta mases .a dap ta tion en ge l .la method iodome triq ue , ann. mac robiol., 1979, 130 b, 236-240. 22. 2 2-ma, -y; li, -j -y; yao , -l; zhang, l; hu,-c-q; jin,-s -h zhon ghua-yi-xue-za-zhi. antimicrob ia l resistance of escherichia coli isolates c olle cted fro m inpa tie nts and outpa tients] 2003 j un 25; 83(12 ): 104 6-8 23. tone lli,-f; ma zzei,-t; nove lli,-a; ma zzoni,-p ; ficari,-f amoxic illin/c lavulanic acid vers us cefotaxime fo r a ntimicrob ia l prophylaxis in a bdo minal surge ry: a randomized tria l . j-che mothe r. 200 2 aug; 14 (4 ): 366 -72 24. s iham-ss. ma rc o. s irot-djo ly-b. and cluze l –r sp re ad o f shv-1 be ta-lactama ses in e.c oli iso la ted from fecal samp les in afric a.19 87. antimicro . a gent and chemother.vol 31 (6)943 -945 25. ma suda g., to mio ka s a nd ha re gawa m., detec tion o f β-lac ta mases production by gra m-nega tiv e bacte ria.the journal of antib io tics , 1 976 ,vol 29,n6 ,6 62 -6 64. 26. kuro ka wa,-h; shiba ta,-n; doi,-y; shiba yama,-k; ka mac hi,-k; ya gi,-t; ara kawa,-y-a new tem-d erived exte ndedspectrum b eta-lactama se (tem-91) with an r164 c substitution a t the ome ga -loop confe rs ceftazid ime res istance. antimic ro b-age ntschemother. 2 003 sep ; 47 (9 ): 2981-3. 27. ro drigue s c, joshi p, jani sh, alp honse m, ra dha kris hnan r,mehta a. de tection of beta-lac ta mases in nos ocoial g-ve c linical isolates . indian j .of medical mic robiology 200 4(22) 4,247 -250 28. chaudha ry u, agga rwal r. extended spectrum b eta-lactama ses(esbl)an emerging thre at to clinica l therapeutic. rev iew article ,2004 (22) 75-80 . clinical evaluation of melatonin alone and in combination with pizotifen in the prophylaxis of migraine iraqi j.pharm.sci., vol.16 (1) ,2007 preparation and characterization of ksl microspheres 26 preparation and characterization of poly (d,l-lactide-co-glycolide) microspheres for controlled release of ksl peptide ahmed a. hussein* , 1 * university of baghdad college of pharmacypharmaceutics department abstract : the purpose of this research was to prepare, characterize, and evaluate the new antimicrobial peptide ksl peptide encapsulated in poly(d,l-lactide-co-glycolide) (plga)composite microspheres. ksl was loaded in poly(acryloyl hydroxyethyl) starch (aches) micropar-ticles, and then the peptidecontaining microparticles were encapsulated in the plga matrix by a solvent extraction /evaporation method. ksl-loaded plga microspheres were also prepared without the starch hydrogel microparticle microspheres for comparison study. ksl peptide microspheres were characterized for drug content, surface morphology, microspheres size determination, polymers stability , in vitro microspheres degradation and in vitro release. ksl peptide encapsulation efficiency resulted in about 98% for rg503 microspheres and achesrg503 composite microspheres. microspheres mean diameters were 11.12μm and 28μm for rg503 microspheres and aches-rg503 composite microspheres respectively. differential scanning calorimetry (dsc) analysis showed no structural changes in the polymers after ksl peptide loading. the morphological effects and polymers degradation were analyzed to obtain a better understanding of the mechanism of ksl peptide release from microspheres and composite microspheres. microspheres incubated in 0.1m phosphate buffer saline, ph 7.4 at 37°c were hydrated and started to degrade as shown by gel permeation chromatography (gpc) analysis. the result indicated that the release of ksl peptide from microspheres was due to the bulk degradation. in vitro release profile showed that the microspheres type significantly affect the release of ksl peptide. in vitro ksl peptide release after 60 days incubation in 0.1m phosphate buffer saline, ph 7.4 at 37°c were 82.23% and 62.12% from 10% ksl peptide loaded aches-rg503 composite microspheres and 10%ksl peptide loaded rg503 microspheres respectively. key words :ksl peptide , microspheres, composite microspheres, plga الخالصة يجٓشّٚ يعٕظّ انجذٚذ فٙ كشاث نجشثٕيٙأ انًعبد kslال ْٕ ححعٛش ٔحشخٛض ٔحقٛٛى ببخٛذ ْزا انبحث يٍ غشضان ًّم ( انُشب ، ٔبعذ acryloyl hydroxyethyl) جبٛببث فٙ يخعّذد kslببخبٚذ ال يخكَٕت يٍ يخعّذد حبيط انهبٍ ٔحبيط انكالٚكٕنك . ُح بطشٚقت (plga) يخعّذد حبيط انهبٍ ٔحبيط انكالٚكٕنك فٙ يصفٕفت kslال peptideرنك غهفج ْزِ انحبٛببث انًحخٕٚت عهٗ hydrogel ال حبٛببث خبنٛت يٍ kslال ببخٛذانًجٓشٚت انًحًهت plgaكشاث ال حعشث خبخٛش.ان /خالص انًزٚباسخ ، إسخقشاسٚت ٔكزنك kslنعًم دساست يقبسَت. حى حعشٚف يحخٕٖ انذٔاء نهكشاث انًجٓشٚت نببخٛذ ال ت انُشٕٚ ، انشكم انخبسجٙ , انحجى % انٗ 89كبَج بحذٔد ksl ال ببخٛذاٌ فعبنٛت ححًٛه ئت انكشاث انًجٓشٚت خبسج انجسى ٔانخحشس خبسج انجسى.انبٕنًٛشاث ,حجز كبٌ. نقذ كبٌ يعذل قطش انكشاث انًجٓشٚت انًجٓشٚت انًعٕظّ aches-rg503 ٔانٗ كشاث انًجٓشٚت 3ٖٓrgكشاث . ححهٛم عهٗ انخٕانٙ aches-rg503 ث انًجٓشٚت انًعٕظتانكشا ٔ rg503 كشاث انًجٓشٚتنهيبٚكشٌٔ 9ٕيبٚكشٌٔ ٔ ٕٔ.ٔٔ بعذ ححًٛم اظٓش عذو ٔجٕد حغٛٛش فٙ حشكٛب انبٕنًٛشاث (differential scanning calorimetry) انًسح انخفشٚقٙ نهسعشاث ْٓى عهٗ انًظٓش انخبسجٙ ٔحجزئت انخأثٛشاث .kslال ببخٛذ ال peptideأفعم ٜنٛت ححشس انبٕنًٛشاث قًٛج نهُحُصٕل عهٗ ف ksl ٍْ ٔانخشكٛز 7.4انكشاث انًجٓشٚت فٙ داسئ انفٕسفبث رٔ انذانت انحبيعٛت ج . حعُانكشاث انًجٓشٚت ٔانكشاث انًجٓشٚت انًعٕظتي ٓاليٙ انخُبفز٘خحهٛم انكشٔيبحٕكشفٙ انانيئٕٚت ادٖ انٗ حشغٛبٓب ٔحجزئٓب كًب ظٓش جهٛب يٍ خالل 37 ٔبذسجت حشاسة ٔ.ٓ انًٕالس٘ (gel permeation chromatography) . ٔقذ أشبسث انُخبئج عهٗ إٌ ححشس ببخبٚذ ال ksl انخكسشكبٌ يٍ خالل يٛكبَٛكٛت . نقذ كبٌ ححشس kslَٕع انكشاث انًجٓشٚت ٚؤثش بشكم كبٛش عهٗ ححشس ببخبٚذ ال إٌ اشبس انٗ انحٙ انكخهٙ. إٌ انخحشس خبسج انجسى دسجت يئٕٚت ْٕ 37ٔبذسحت 0.1ٔحشكٛزِ انًٕالس٘ 7.4ٕٚيب يٍ انحعٍ فٙ داسئ انفٕسفبث رٔ انذانت انحبيعٛت 0ٓ بعذ kslٛذ ال ببخ عهٗ rg503 ٔaches-rg503انخبنٛت %10بُسبت kslيٍ انكشاث انًجٓشٚت انًحًهت ببخبٚذ ال %62.12ٔ %82.23بُسبت انخٕانٙ. 1 corresponding author: e-mail: ahmed_sura@yahoo.com . received : 4/11/2006 accepted : 13/5/2007 mailto:ahmed_sura@yahoo.com iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 27 introduction : recently , a large number of recombinant proteins and synthetic peptides have been developed as potential therapeutic agents (1) . poor absorption of these agents by the oral route due to their poor physicochemical properties or due to a high first-pass metabolism in the liver or degradation in the acidic atmosphere of the stomach. the digestive enzymes in the intestine or in the gut wall are responsible for the presystemic degradation of many drugs (2) .these obstacles has necessitated delivery by alternative routes, principally the parenteral route. injectable controlled-release formulations are designed to release drugs in a controlled, predetermined fashion and have been developed in an effort to provide more consistent pharmacodynamic effects and minimize adverse effects (characteristics that are particularly useful for drugs with a narrow therapeutic index). most commonly, active agents are incorporated within a biodegradable and bio-compatible polymeric matrix (3) . biodegradable polymeric matrix has been found promising for delivering proteins and peptides over a desired period of time. nowadays many polymer matrix materials are available, they gradually degrade after the drug is released at the desired site in the body (4-6) . furthermore, administration of the drug by injection is possible if it is dispersed into microspheres (7-9) microspheres are fine spherical particles containing active drugs. microspheres have a diameter of less than approximately 1000 µm in which the drug is dissolved or dispersed homogeneously throughout the polymer matrix (3) . plga, an fda-approved material, has been extensively studied for its biocompatibility, toxicology, and degradation kinetics (10) . plga is biocompatible, and more importantly, the degradation rates of plga and the accompanying release of encapsulated drugs can be controlled by the polymer’s physicochemical properties such as molecular weight, hydrophilicity, and the ratio of lactide to glycolide (11) . thus, it is possible to obtain the desired drug release from plga microspheres by altering the polymer’s characteristics. biodegradable polymeric microspheres have been shown to be effective in enhancing drug targeting specificity, lowering systemic drug toxicity, improving treatment absorption rates, and providing protection for pharmaceuticals against biochemical degradation (12) . several peptides have been successfully incorporated into a plga matrix as a depot formulation for parenteral use (1) .the drug release from biodegradable microspheres is governed by degradation rate of plga copolymer, which largely depends on the physical properties of polymer such as molecular weight, hydrophilicity, and the ratio of lactide to glycolide (13) .proccing conditions employed during preparation of microspheres determine the properties of the microspheres , such as the size , morphology, encapsulation efficiency , and drug distribution and all these properties influence the release of drug from the delivery system (6) . a novel decapeptide, kkvvfkvkfk (lys 1 -lys 2 -val 3 -val 4 -phe 5 lys 6 -val 7 -lys 8 -phe 9 -lys 10 ) (ksl peptide) has been identified that shows activity against c.albicans and a broad range of antibacterial activity but without hemolytic activity (14) . ksl peptide exhibited an ed99 (the dose at which 99% killing was observed after 15 min at 37ºc) of 6.25 µg/ml against selected strains of lactobacillus salivarius, streptococcus mutans, streptococcus gordonii and actinobacillus actinomycetemcomitans. in addition, ksl peptide damaged bacterial cell membranes and caused 1.05 log units reduction of viability counts of saliva bacteria. in vitro toxicity studies showed that ksl peptide, at concentrations up to 1 mg/ml, did not induce cell death or compromise the membrane integrity of human gingival fibroblasts (15) . in previous study , the preparation and characterization of plga polymerrg502, rg502h and rg503h, microspheres and aches-rg503h composite microspheres loaded ksl peptide were investigated. the study showed that the in vitro ksl peptide release after 60 days incubation in 0.1m phosphate buffer saline, ph 7.4 37ºc were 75.13%, 52.27%,, 21.41%, and 30.69% from 10% ksl peptide loaded rg502, rg502h and rg503h, microspheres and achesrg503h composite microspheres respectively (16) .in this study , ksl loaded plga polymer rg503 and aches-rg503 composite microspheres were prepared for prolonged ksl release . materials and methods : materials: ksl decapeptide and polyvinyl alcohol (pva)(m.wt 3000-7000) were purchased from sigma chemical (st. louis, mo). poly (d, l-lactic-co-glycolic acid) (plga) polymer with hydrophobic end group [molecular weight (m.wt) 30 kilodalton(kd) (kd= kilogram/mole)], copolymer ratio=50:50, rosmer® rg503). poly(acryloyl hydroxyethyl starch)(aches) was purchased from dupont pharmaceutics (wilmington, de, usa).all other chemicals reagents used were of analytical reagent grade. iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 28 methods: preparation of microspheres: the ksl peptide microspheres were prepared with plga polymerrg503 (30kd) . the 10% loading of ksl peptide microspheres were prepared by solvent extraction/evaporation technique (17) . the preparation procedure was as follows: a methylene chloride (ch2cl2) solution that contained approximately 25 % (w/w) of polymer was mixed with a methanolic solution (ch3oh) of ksl peptide. the resulting mixture [dispersed phase (dp)] was then slowly added to 50 ml of 0.35% (w/w) pva aqueous solution [continuous phase (cp)] and maintained at 25 °c with a water bath. the mixture of cp and dp were emulsified for 5 min using a mixer. the temperature of the emulsion was increased and maintained at 40°c to extract and evaporate the organic phase over 1hr, then temperature of the system was reduced to 25 °c and microspheres were recovered on 5µm pore size filter paper and then freeze dried over night. preparation of ksl peptide-loaded achesrg503 microspheres: the aches-rg503 composite microspheres were prepared by a modified solvent extraction/ evaporation method (18) with 10% (w/w) loading of ksl peptide. 50 mg ksl peptide was dissolved in 0.250 ml of distilled water. the solution was added to 50mg aches particles, and the particles were allowed to swell for 5 minutes with vortex mixing at room temperature. a 30% w/w rg503 methylene chloride solution was added to the swollen aches particles and vortex for 2 minutes at room temperature to form a dispersion of ksl peptide in hydrogel/ polymer in solvent. this primary dispersion was then added to precooled (4 °c) 100 ml 6.0% pva solution and stirred by a silverson mixer at 2500 rpm for 2 minutes. the resulting secondary suspension was transferred to one liter distilled water and stirred gently for 2hr at room temperature to remove the organic solvent and solidify the polymer. the microspheres were filtered through 5µm filter paper. then washed 3 times with 2 liters distilled water and freeze-dried over night. characterization of the prepared microspheres: ksl peptide content and loading efficiency determination of the prepared microspheres: ksl peptide content of peptide loaded rg503 microspheres and aches-rg503 composite microspheres were determined by hplc(reverse-phase-performance liquid chromatography, shimadzu scientific instrument, inc.columbia,md.) as follows: triplicate samples of 10 mg of the microspheres were quantitatively transferred to a 12 ml glass test tube. the matrix was solubilized in 2 ml of methylene chloride , then 10 ml of 0.1m acetate buffer , ph 4.0 was added and the test tubes were agitated by a wrist action shaker for 1hr. samples were centrifuged at 3000 rpm and the aqueous layer was analyzed by hplc. a second extraction with 10 ml of acetate buffer was made to ensure complete extraction and effect mass balance. the ksl peptide concentration was determined by hplc (shimadzu scientific instrument, inc.columbia,md.) (15) using a prosphere c-18 column (4.6x250mm,altech, deerfield,,il). gradiant elution was accomplished with 0.1% trifluoroacetic acid in water(phase a) and 0.1% trifluoroacetic acid with acetonitrile (phase b) with increasing the amount of phase b from 20% to 30% over 10 minutes at a flow rate of 1.2ml/min.the running time was 20 minutes and the u.v. detection was at 215nm. the injection volume was 40μl. the ksl amount was determined using calibration curve according to (y= -1.72542.4521 +13927.4152x , r=0.997) the loading efficiency was calculated using the following equation. m (actual) loading efficiency (%) =-------------------x 100 m (theoretical) where m actual is the actual amount of ksl peptide in microspheres and m theoretical is the amount of ksl peptide in microspheres calculated from the quantity added in the fabrication process (19) . morphology study of the prepared microspheres: a scanning electron microscope(hitachi model 5800, japan.) was used to exam the shape and surface morphology of the microspheres .freezedried microspheres were mounted on adehisve stub and then coated with gold palladium under vacuum. the coated specimen was then examined under the microscope and photographed. microsphere size determination: the size of ksl peptide loaded rg503 microspheres and ksl peptide loaded aches-rg503 composite microspheres were analyzed by using laser diffraction(malvern instrument , malvern, uk.). the microspheres were suspended in 0.1% aqueous tween80 solution and a 100-mm(for size range of 1.9-188μm) focal length was employed to determine particle size, while the sample was stirred at 100 rpm in the sample cell using a magnetic stirrer bar. all particles size measurements were repeated three times per iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 29 sample and each sample was prepared in triplicate. the average particle size was expressed as the volume mean diameter vmd in microns. differential scanning calorimetry ( dsc) analysis: the changing in the structure of the plga polymers during the loading by ksl peptide can be predicted by measuring the glass transition temperature (tg) of plgas before and after loading. the effect of loading on the tg of plga polymers was investigated by the dsc(mettler ta 2920, ac usa.). samples of the prepared microspheres, about 5mg of blank and ksl peptide loaded rg503 and aches rg503 composite microspheres were scanned at 5°c/min. heating rate in the range of 0-80°c. the molecular weight degradation study of the plga biodegradable polymers using gel permeation chromatography (gpc): the molecular weight distribution of blank and ksl peptide loaded rg503 microspheres and ksl peptide loaded aches rg503 composite microspheres incubated in pbs ph 7.4 at 37°c were determined by gel permeation chromatography[a waters m-45 solvent delivery system with a shimadzu spd 6av uv-vis spectrophotomereic detector (ë=220nm)]. two ultrastyragel columns connected in series (7.8x300 mm each, one 10 4 ǻ pores and one with 10 3 ǻ pores) were used. samples, 5 mg/ml, were eluted with tetrahydrofuran at 0.4 ml/min. the average molecular weight of each sample was calculated using monodisperse polystyrene standards, molecular weight 1000-90000 dalton. in vitro ksl peptide release study from the prepared microspheres: fifty mg of ksl peptide loaded rg503 microspheres and 10% ksl peptide aches-rg503 composite microspheres were incubated with 40 ml of 0.1 m pbs, ph 7.4 containing 0.02 % sod. azide as preservative to prevent the growth of microorganisms during the incubation at 37°c in a temperature controlled oven. separate test tubes with equal amounts of microspheres were maintained for each time point of the release study. the sampling time were 2, 5, 10 , 20, 30, 40, and 60 days. test tubes were shaken twice weekly to simulate the static in vivo condition and the peptide release was determined from the residual microspheres . at each time point, remaining microspheres were recovered by centrifugation and assayed for drug content. the supernatant was removed and assayed for the ksl peptide amount to be used in the calculation of mass balance study. all analysis was carried out by hplc on triplicate samples. results and discussion : ksl peptide content and loading efficiency of the prepared microspheres: the amount of ksl peptide encapsulated in each of the polymeric microspheres and composite microspheres was determined by calculating the amount of ksl peptide after dissolving microspheres and composite microspheres in of methylene chloride and extracting the ksl peptide in acetate buffer. the results indicated that about 98% of the total peptide could be recovered as shown in table (1).the loading efficiency of each batch was similar and independent of the biodegradable polymers used from the preparation of microspheres. the same high percentage of peptides encapsulation were obtained by researchers when they used ch3oh and ch2cl2 system as the organic phase of o/w emulsion (20) . table 1: batch summary of ksl peptide loading microspheres. microspheres batch theoreti cal weight. of ksl peptide (mg) actual weight ksl peptide loading (mg) ksl peptide loading efficiency (%) rg503 10 9.82 98.2 aches rg503 10 9.84 98.4 morphology of the prepared microspheres: scanning electron microscopy(sem) images of the 10% ksl peptide loaded rg503 microspheres and 10%ksl peptide loaded aches-rg503 composite microspheres after preparation indicated the formation of nonporous spheres with relatively smooth surfaces(figures 1and 2 ). the similar morphology may be due to the same experimental conditions. iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 30 figure 1: scanning electron microscopy of ksl peptide loaded rg503 microspheres . figure 2: scanning electron microscopy of ksl peptide loaded aches rg503 microspheres. microsphere size: the mean microsphere size were at 11.12 and 28μm for rg503 microspheres and aches-rg503 composite microspheres respectively,. that are suitable for intramuscular or subcutaneous administration. the smaller rg503 microspheres size, compared to the aches-rg503 composite microspheres could be due to lower viscosity of polymer solution of the rg503 (17) . the differential scanning calorimetry (dsc) analysis of the prepared microspheres: the glass transition temperature (tg) of rg503 microspheres and aches-rg503 composite microspheres batches were determined by the dsc for blank and loading microspheres. no significant differences in tg between ksl peptide loaded microspheres and the blank one (as shown in table (2) ), which suggest that the ksl peptide loading has no effect on the internal structure of the polymers. table 2: the glass transition temperature (tg) of blank an loaded microspheres. degradation result of the prepared microspheres : table (3) and figure (3) show that the erosion of ksl peptide-loaded plga microspheres leads to molecular weight-loss profiles that are in agreement with the kinetics of bulk erosion (17) . the molecular weight of all samples of microspheres decreased continuously after being exposed to pbs at 37°c. in all cases, the decrease in molecular weight almost follows a linear profile. although a variety of definitions exists in the contemporary literature, it is well accepted that polymer degradation is defined as the process of polymer chain cleavage. degradation is triggered by water which hydrolyzes the functional groups by which the monomers are usually connected. when the sample microspheres were exposed to pbs, water permeated into the microsphere matrix, resulting in random hydrolysis of ester bonds and decrease of the molecular weight .(21) . microspheres tg (°c) blank rg503 54.15 ksl peptide loaded rg502 52.65 blank achesrg503h 53.58 ksl peptide loaded aches rg503h 52.36 iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 31 table 3: molecular weight for the blanks microspheres and for ksl peptide loaded microspheres before and after incubation in phosphate buffer saline ph 7.4 at 37°c. time (days) m.wt. of rg503 microspheres m.wt. of aches rg503 microsphere 0 ( blank microspheres) 29427 27888 l o a d e d m ic r o sp h e r e s 27000 25555 22517 22334 19745 16667 time (day) 0 2 4 6 8 m o le c u la r w e ig h t ( d a lt o n ) 16000 18000 20000 22000 24000 26000 28000 rg503 microspheres aches rg503 composite micropsheres figure 3:the molecular weight of 10%ksl peptide loaded microspheres before and after incubation in 0.1m phosphate buffer saline, ph 7.4 at 37°c. release of ksl peptide loaded plgas microspheres: a pathway for ksl peptide release was provided by microsphere degradation where water-soluble degradation products (i.e. monomers and oligomers ) leave the microspheres matrix for the surrounding aqueous medium. since oligomers are close to the surface they can leach out faster than that located deeper within the matrix, carboxylic acid oligomers trapped within the matrix autocatalyze further ester bond hydrolysis, resulting in the increasing rate of mass loss (22). figure (4) shows the long-term in vitro release of the ksl peptide from ksl peptide loaded rg503, and ksl peptide loaded composite aches-rg503 microspheres. at 60 days, 69.12% of ksl peptide release were obtained from the microspheres and 82.32% from achesrg503. release kinetics (figure 4) of rg503 characterized by low burst release during 5 days period, followed by a continuous release for rg503 with no evidence of a lag phase. the release of ksl peptide from achesrg503 composite microspheres in general was higher than that from rg503 microspheres. dissolution of rg503 polymer in the composite microspheres could expose the entrapped ksl peptide-containing aches hydrogel particles to the release media, and the exposed hydrogel could release more ksl peptide molecules with little or no interaction with the plga polymer. a similar behavior was observed by capan et al; they reported that the composite microspheres showed more favorable in vitro release than conventional plga micropspheres for recombinant human growth hormone drug delivery (23). there have been attempt to improve protein stability and release kinetics of the plga system by changing the physicochemical properties of the polymer. for instance, both chemical derivation and physical bending of plga with hydrophilic polymers such as aches have been reported(24). aches hydrogel particles may have a protective function for enhancing the retention of ksl peptide stability and reducing ksl peptide adsorption to plga microspheres. time (day) 0 10 20 30 40 50 60 70 % r e le a s e d 0 20 40 60 80 100 rg503 microspheres aches rg503 composite microspheres iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 32 figure 4: release profile of 10% ksl peptide-loaded plgas microspheres in phosphate buffer, ph 7.4 at 37°c. conclusion : the ksl peptide loaded plga microspheres and composite microspheres of a starch-based polymer and plga have been successfully formulated with spherical morphology, high capacity peptide incorporation efficiency, and good stability. the systems possesses sustained ksl peptide release for more than 60 days. the prolonged release of the ksl peptide may be potentially useful for long-term sustained release of the ksl peptide for the treatment of chronic bacterial and fungal infections references : 1shameem, m., lee., h., deluca,p.p. a short-term (accelerated release) approach to evaluate peptide release from plga depot formulations . aaps pharmsci 1999; 1 (3)article7 2conti, s., polonelli, l., frazzi, r., artusi, m.,bettini, r., cocconi, d., colombo, p.controlled delivery of biotechnological products.current pharmaceutical biotechnology. dec; 2000; 1(4):313-323. 3reddy, k. r. controlled-release, pegylation, liposomal formulations: new mechanisms in the delivery of injectable 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research.2003; 20,no.3. 19jia, k.l., nuo, w., xue, s.w. a novel biodegradation system based on gelatin nanopartricles and poly(lactic-co-glycolic acid) microspheres for protein and peptide iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 33 drugdelivery.j.pharm.sci. 1997;86,891895. 20roy, k., mao, h.q., leong, k.w. dna chitosan nanospheres transfection efficiency and cellular uptake. proc. int. sym. control. rel. bioact. mater.1997; 24:673-674. 21zhou, s., deng, x., li, x., jia, w., liu, l. synthesis and characterization of biodegradable low molecular weight aliphatic polyesters and their use in protein-delivery systems. journal of applied polymer science.2004; 91: 1848– 1856 22tsung, m. j., burgess, d. j. preparation and characterization of gelatin surface modifiedplgamicrospheres.aapspharms ci.2001;3(1): article 11 23capan, y., jiang, g., giovagnoli, s., na, k-h., deluca, p.p. preparation and characterization of poly(d,l-lactide-co glycolide) microspheres for controlled release of human growth hormone. aaps pharmscitech.. 2003; 4(2): article 28 24giovagnoli ,s., blasi, p., ricci,m., rossi,c. biodegradable microspheres as carriers for native superoxide dismutase and catalase delivery. aaps pharmscitech. 2004; 5(4): article 51. iraqi j pharm sci, vol.25(2) 2016 surgical antibiotic prophylaxis 40 adherence to american society of health-system pharmacists surgical antibiotic prophylaxis guideline in medical city teaching hospitals/baghdad nagham t. salih *,1 and dheyaa j. kadhim ** * ministry of health, iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract surgical site infections are the second most common type of adverse events occurring in hospitalized patients. surgical antibiotic prophylaxis refers to the use of preoperative and postoperative antibiotics to decrease the incidence of postoperative wound infections. the objective of this study was to evaluate the antibiotic administration pattern for surgical antibiotic prophylaxis and the adherence to american society of health-system pharmacists surgical antibiotic prophylaxis guideline in medical city teaching hospitals/baghdad. the medical records of one hundred patients who underwent elective surgical procedures were reviewed. adherence to the recommendations of american society of health‑system pharmacists guideline was assessed for every aspect of antibiotic prophylaxis. the results of this study showed that antibiotic(s) had been administered to all of the patients involved in the study despite the fact that 34 patients (34%) were not required such prophylaxis. while in case of administration time, 13 patients (13%) had received the antibiotic(s) at correct time while 87 patients (87%) at incorrect time. proper antibiotic(s) selection was found in 11 patients (11.0%) only. all of the patients (100.0%) had received more than 2 doses, and all of them were not in concordance with surgical site infection prevention guidelines. in addition, 19 patients (19.0%) with surgical site infections were identified during hospital stay or within 30 days after surgery. according to these results, we can conclude that there was a substantial gap between surgical antibiotic prophylaxis guideline and their implementation in daily practice in medical city teaching hospitals/baghdad. keywords: surgical antibiotic prophylaxis guidelines, american society of health system pharmacists guidelines, medical city teaching hospitals/baghdad. يدى االنتزاو بتطبيك توصيات دنيم جًعية انصيادنة االيريكية الستعًال انًضادات انحيوية انولائية انجراحية في يدينة انطب / بغداد ضياء جبار كاظى * و نغى طارق صانح , **1 * . اٌعشاق وصاسج اٌصذح ، ** فشع اٌصيذٌح اٌسشيشيح ، وٍيح اٌصيذٌح ، جاِعح تغذاد ،تغذاد ، اٌعشاق . الخالصة ٍّضاداخ اٌجشادي اٌىلائي ٌ سرعّاييمصذ تاال. اٌّشاوً شيىعا في اٌّشضً اٌشالذيٓ في اٌّسرشفً أوصشيعذ اٌرهاب ِىاْ اٌعٍّيح شأي اٌهذف ِٓ اٌذساسح هى ذمييُ طشيمح لثً وتعذ اٌعٍّيح ٌرمٍيً ٔسثح عذوي جشوح ِا تعذ اٌعٍّيح. ٌّضاداخ اٌذيىيح اعٍّيح اسرخذاَ هى اٌذيىيح ٌالسرعّاي اٌجشادي اٌىلائي ٌٍّضاداخ اٌذيىيح في األِشيىيحاٌّضاداخ اٌذيىيح اٌىلائيح وِذي االٌرضاَ ترطثيك دٌيً جّعيح اٌصيادٌح إعطاء ./ تغذادياخ ِذيٕح اٌطة ِسرشف ذُ ذمييُ ديسِٓ اٌسجالخ اٌطثيح ٌٍّشضً اٌشالذيٓ في اٌشدهاخ اٌجشاديح اٌّعٍىِاخ اٌّطٍىتحذُ جّع عيٕح ِؤٌفح ِٓ ِائح ِشيض وذسجيً دي.ِٓ جّيع إٌىاٌالسرعّاي اٌجشادي اٌىلائي ٌٍّضاداخ اٌذيىيح األِشيىيحِذي االٌرضاَ ترطثيك ذىصياخ جّعيح اٌصيادٌح ٌيسىا % (43.3)ِشيض 43 وىْ ِٓ تاٌشغُ اٌذساسح تاْ اٌّضاداخ اٌذيىيح اٌىلائيح لذ صشفد ٌىً اٌّشضً تاٌشغُ ِٓ وىْ أظهشخ أعطيد لذ اٌذيىيح اٌّضاداخ تاْ اٌذساسح أظهشخ اٌىلائيح اٌذيىيح اٌّضاداخ إعطاء ٌىلد اٌىلائيح. تإٌسثح اٌذيىيح اٌّضاداخ ٌهزٖ تذاجح %( 78.3) ِشيض 78 عٕذ ِٕاسثا غيش اٌذيىيح اٌّضاداخ إعطاء ولد واْ ديٓ في إٌّاسة اٌىلد في%( 34.3) ِشيض 34 إًٌ اٌذساسح في اٌّشضً وً اٌىلائيح. اٌذيىيح اٌّضاداخ ِٓ اٌصذيخ إٌىع اسرٍّىا فمظ%( 33.3) ِشيض 33 أْ اٌذساسح وّا وأظهشخ األِشيىيح اٌصيادٌح جّعيح ٌرىصياخ ِطاتك غيش يعرثش اإلجشاء وهزا اٌىلائيح اٌذيىيح اٌّضاداخ ِٓ جشعريٓ ِٓ أوصش اسرٍّىا%( 333.3) أشٕاء%( 31.3) ِشيضا 31 ٌذي اٌعٍّيح ِىاْ اٌرهاتاخ دذوز اٌذساسح اٌىلائيح. تاإلضافح إًٌ رٌه فمذ أظهشخ اٌذيىيح اٌّضاداخ ٌصشف اٌجشاديح. اٌعٍّيح ِٓ يىَ 43 خالي أو اٌّسرشفً في اٌشلىد فرشج ذٕفيزها تاٌّّاسسح اٌيىِيح في اٌشدهاخ تيٓهٕان فجىج وثيشج تيٓ دٌيً ِثادئ صشف اٌّضاداخ اٌذيىيح اٌىلائيح وفاْ هزٖ إٌراضطثما ٌ اٌجشاديح في ِسرشفياخ ِذيٕح اٌطة في تغذاد. .يستشفيات يدينة انطب / بغداد األيريكية ،دنيم جًعية انصيادنة ، انولائية انحيوية يضادات انكهًات انًفتاحية: 1 corresponding author e-mail: naghamt.attar@gmail.com received: 8/ 8/ 2016 accepted: 13 /12/2016 iraqi j pharm sci, vol.25(2) 2016 surgical antibiotic prophylaxis 41 introduction surgical site infections (ssis) are the commonest hospital-acquired infections in surgical patients (1) and are the second most common cause of nosocomial infections (2) .they result in increased antibiotic usage, increased costs and prolonged hospitalization (1) . surgical antibiotic prophylaxis may be defined as the appropriate use of preoperative and postoperative antibiotics to decrease the incidence of postoperative wound infections (3) . surgical operations are classified at the time of operation as clean, clean-contaminated, contaminated, or dirty. dirty operations take place in situations of existing infection and antimicrobials are used for treatment, not prophylaxis (4) . depending on best available evidence to optimize patient care and surgeon's practice, american society of healthsystem pharmacists (ashp) had developed a guideline for antimicrobial prophylaxis in surgery. according to this guideline: 1-cefazolin (a first generation cephalosporin) is regarded the antimicrobial of choice for most surgical procedures. 2-the optimum administration's time for of prophylactic antibiotics dose is within 60 minutes before the surgical incision. 3single-dose prophylaxis is usually sufficient with duration of less than 24 hours for all procedures. 4-antimicrobial prophylaxis may be beneficial in surgical procedures associated with a high rate of infection (i.e., clean-contaminated or contaminated procedures) and in certain clean procedures where there are severe consequences of infection (e.g., prosthetic implants), even if infection is unlikely (5) . antibiotic administration continued beyond 24 hours has not been shown to be superior to shorter duration antibiotic prophylaxis in most surgical procedures. singledose antibiotic prophylaxis provides the optimal balance for reducing wound infections in most operative procedures and decreasing adverse drug effects (3) . this study aimed to evaluate an antibiotic administration pattern for surgical antibiotic prophylaxis in medical city teaching hospital (baghdad) and to assess the adherence of practitioners to the guidelines for antimicrobial prophylaxis. patients and methods patients the current study was carried out over two months from august – until october -2015 in general surgery wards in baghdad teaching hospital and orthopedic surgery wards in ghazi al-harriri hospital –medical city. one hundred patients [46 (46%) male and 54(54%) female] underwent elective surgeries with no preoperative infections were selected from general surgery and orthopedic surgery wards. inclusion criteria any patient undergoing elective surgery with no pre-operative infections and no antibiotic treatment prior hospital admission were included in the current study. exclusion criteria patients who received antibiotic(s) to treat infection were excluded since it was not possible to determine whether the antibiotic was given as treatment for the infection or as a surgical prophylaxis. method the medical records of admitted patients who underwent different surgical procedures were reviewed during and the relevant information was entered on data collection form. the data including: patients demographic information (age , gender, drug allergies ,past medical history , past surgical history , smoking ), type of surgical procedure( name of surgical procedure ,type of surgery ,class of surgery ), antibiotic therapy received (agent ,dose, initiation time duration of administration ), duration of surgical procedure, length of hospital stays, treatment on discharge and finally the presence of post-operative infection. all study patients were followed up by phone calls contacted at home by telephone after 30 days or more post surgery. the patients were assessed about their general health and the state of the surgical according the following criteria (6) : 1-presence of fever. 2-presence of erythema at wound site. 3-presence of swelling, pain or heat at wound site. 4-presence of discharge at wound site. 5-have any antibiotic(s) been prescribed? compliance with the recommendations of ashp guideline was assessed for every aspect of antibiotic prophylaxis (5) . statistical analysis all data were coded, and spss version 22 (spss inc., chicago, illinois, u.s.a) was used for the statistical analysis. the results were presented as mean ± standard deviation and percent (%) where applicable. iraqi j pharm sci, vol.25(2) 2016 surgical antibiotic prophylaxis 42 results the demographic, clinical and surgical characteristics of the patients are shown in table-1. eighty three patients were collected from the general surgery ward and 17 patients from the orthopedic ward at medical city teaching hospital-baghdad. concerning the type of surgery, 48 % of surgical procedures were clean and 52% were clean-contaminated. table (1):demographic, clinical and surgical characteristics of the patients. characteristics no. (%) gender male 46 (46%) female 54(54%) age (years) 40.1±15.9 smoking yes 9 (9%) no 91(91%) drug allergy yes 5 (5%) no 95 (95%) surgical history yes 71 (71%) no 29 (29%) medical history no 74 (74%) hypertension 11 (11%) diabetes mellitus 7 (7%) hypertension and diabetes mellitus 4 (4%) stroke 1 (1%) thyrotoxicosis 1(1%) ischemic heart diseases 1 (1%) hepatitis c 1 (1%) surgical characteristics types of surgery general 83(83%) orthopedic 17(17%) surgical class clean 48 (48%) cleancontaminated 52(52%) with respect to the appropriateness of antibiotic(s) administration according to patients' need, the study showed that antibiotic(s) had been administered to all of the patients involved in the study despite the fact that 34 patients (34%) were not required such prophylaxis. regarding the appropriateness of antibiotic administration times, the current study showed that 13 patients (13%) had received the antibiotic(s) at correct time (within 60 minutes of the first incision) while 87 patients (87%) had received the antibiotic(s) at incorrect time [after surgery or more than 60 minutes of the first incision] as shown in table-2 table (2):appropriateness of antibiotic(s) administration according to patients' need and antibiotic(s) administration time. requirement of antimicrobial prophylaxis no. (%). appropriate time yes no required administered 66(%) 9 (9 %) 57(57%) not administered 0.0 (%) 0.0 (0.0 %) 0.0 (0.0 %) not required administered 34(%) 4(4 %) 30(30%) not administered 0.0 (%) 0.0 (0.0 %) 0.0 (0.0 %) total no. (%). 100 (100%) 13 (13 %) 87 (87%) iraqi j pharm sci, vol.25(2) 2016 surgical antibiotic prophylaxis 43 ceftriaxone -metronidazole combination was the most commonly prophylactic regimen used in the current study (50.0%) followed by (33.0%), cefotaxime (6.0%), cefotaxime – metronidazole combination (5.0%), genamicin (2.0%), ampicillin-cloxacillin combination (2.0%), meropenem (1.0%), and teicoplanin (1.0%) (table-3). all of the patients (100.0%) had received more than 2 doses and all of these (100.0%) were not in concordance with surgical site infection prevention guidelines (table-4). table(3):antimicrobial(s) used for surgical prophylaxis. antibiotic for prophylaxis frequency no. (%) rate of concordance with ashp guideline (%) ceftriaxone metronidazole 50 (50.0%) 7 (7.0%) ceftriaxone 33 (33.0%), 2 (2.0%) cefotaxime 6 (6.0%), 0.0 (0.0 %) cefotaxime – metronidazole 5 (5.0%), 1 (1.0%) genamicin 2 (2.0%) 1 (1.0%) ampicillincloxacillin 2 (2.0%) 0.0 (0.0 %) meropenem 1 (1.0%) 0.0 (0.0 %) teicoplanin 1 (1.0%) 0.0 (0.0 %) ashp: american society of health-system pharmacists table (4):frequency of patients based on number of antibiotic(s) doses. antibiotic for prophylaxis frequency no. (%) rate of concordance with ashp guideline (%) one dose 0.0 (0.0 %) two doses 0.0 (0.0 %) more than 2 doses 100(100.0 %) (0.0 %) ashp: american society of health-system pharmacists regarding the rate of antibiotic(s) prescription at discharge, the results of the current study showed that antibiotics were prescribed to 96 patients (96.0%) at discharge time, and only 4 patients (4.0%) were discharged without antibiotic(s). concerning the development of surgical site infections, nineteen patients (19.0%) with surgical site infections were identified during hospital stay or within 30 days after surgery while no such infections were reported in 81 patients (81.0%) (table-5). finally overall assessment of appropriateness of prophylactic antibiotic administration in the 100 operations that included in the current study showed a complete lack of adherence to the guideline (table-6). table (5):-development of surgical site infections. development of surgical site infections frequency no. (%) yes 19(19.0%) no 81(81.0%) table (6):-appropriateness of antibiotic prophylaxis regimen. categories no. (%) correct choice 11 (11.0%) correct choice + correct time 0.0 (0.0%) correct choice + correct time + correct duration 0.0 (0.0%) discussion the current study showed that antibiotic(s) had been administered to all of the patients involved in the study despite the fact that 34 patients (34.0%) were not required such prophylaxis. in one study of 759 patients who underwent surgery, it was found that in about 10.0% of the surgeries included in the study, antimicrobial prophylaxis was given although it was not required according to according to the ashp guideline (7) . antibiotic prophylaxis may be beneficial in surgical procedures associated with a high rate of infection (cleancontaminated), while prophylactic antibiotics are not indicated for some clean surgical procedures (7) . the three most important parameters of appropriateness of antibiotic prophylaxis which are antibiotic(s) choice, timing of administration of the first dose and the duration of prophylaxis were evaluated. regarding timing of antibiotic prophylaxis, the current study showed that only 13% of the patients received prophylaxis at correct time and 87% of the patients received prophylaxis at incorrect time. timing of prophylactic antibiotics administration is critical, with both early and late prophylactic antibiotics administration associated with increased ssi rates (8) . in the current study only 11% of the patients received the appropriate antibiotic(s) recommended by the ashp guideline. the first iraqi j pharm sci, vol.25(2) 2016 surgical antibiotic prophylaxis 44 generation cephalosporin, cefazolin is regarded the antimicrobial of choice for most procedures according to ashp guideline (5) . in this study, the combination of ceftriaxone-metronidazole was the most commonly used surgical prophylaxis regimen (50%) followed by ceftriaxone alone which was administered to 33% of the patients. the use of third generation cephalosporins and aminoglycosides are not recommended for ssi prophylaxis because of less activity against staphylococci infection compared to cefazolin, and excessive use of broad spectrum antibiotics for prophylaxis increase the risk of resistance, causes more adverse events and increase health care cost (7) . concerning the duration of surgical antibiotic prophylaxis, the current study showed that all of the patients (100.0%) had received more than 2 doses and all of these (100.0%) were not in concordance with surgical site infection prevention guidelines. the overuse of antibiotics was also demonstrated by the finding that antibiotics was prescribed to 96% of patients either as single agent or combination after discharge and only 4% of the patients discharged from hospital without antibiotics. evidence is mounting that post-operative antimicrobial administration is not necessary for most procedures (5) . according to ashp guideline, minimal duration for antimicrobial coverage includes the time from incision until the closure of that incision, which is usually covered by a single antibiotic dose (5) . the duration of antimicrobial prophylaxis should be less than 24h for most procedures (2) . overuse of antibiotic is not only an economic problem, but it place patients at risk for dangerous infections and exposure to unnecessary drugs and their harms (9) . an indian study reported that antibiotics were administered for as long as 14 days and only 1% to 8% of surgeons who prescribed antibiotic in surgical procedures stopped prophylaxis after 24hours (10) . regarding the development of ssis, the current study showed that ssis were identified in 19 patients (19.0%) during hospital stay or within 30 days after surgery while no such infections were reported in 81 patients (81.0%). post-discharge ssis constitute the majority of ssis and pose a substantial disease burden for surgical patients globally and for different surgery types (11) . in a study by leaper et al., it was estimated that 30 million surgical procedures were conducted in europe each year and rate of ssis was (1.5-2.0 %) among them (12) . the higher rate of ssis reported in the current study may be attributed to poor adherence to surgical prophylaxis guidelines. finally, the current study showed that only 11.0% of the patients receive the correct choice of antibiotic and no one of the patients (0.0%) received the antibiotic prophylaxis according to ashp guidelines in regard to the main three parameters (choice, time, duration). in a 3-month period study of surgical hospital in qatar, the compliance rate of antibiotic selection with the hospital infectious disease guidelines was 68.5%, whereas compliance rate of antibiotic duration with hospital guidelines was 40.7% (13) . conclusions although evidence based guidelines exist to support the appropriateness of surgical antibiotic prophylaxis practices, there is a substantial gap between these guidelines and their implementation in daily practice in medical city teaching hospital. nonadherence to surgical prophylaxis guideline in medical city teaching hospital resulted in an increased rate of surgical site infections. references 1. wendy m. antibiotic for surgical prophylaxis. australian prescriber. 2005; 28: 38-40. 2. dale wb and peter mh. antimicrobial prophylaxis for surgery: and advisory statement from national surgical infection prevention project. clinical infectious disease. 2004; 38:1706 -1715. 3. ronald lb, surgical infections and antibiotic prophylaxis. in david jq, richard ah. textbook of therapeutics: drug and disease management. 8th edition. 2006. pages: 2209 -2214. 4. mary au, john cr. antimicrobial prophylaxis in surgery. in chisholm-burns, barbara g.wells, et al. pharmacotherapy principles & practice. 3rd edition. 2013. pages: 1459-1467. 5. dale wb, e. patchen d, keith mo, et al. clinical practice guidelines for antimicrobial prophylaxis in surgery. am j health-syst pharm. 2013; 70:195-283 . 6. piret m, katrin h, aira p, et al. surgical-site infections following cesarean section in an estonian university hospital: post discharge surveillance and analysis of risk factors. infect control hosp epidemiol. 2005; 26:449-454. 7. mohammadreza r, afshin sh, amirhosein a, et al. adherence to american society of health-system pharmacists surgical iraqi j pharm sci, vol.25(2) 2016 surgical antibiotic prophylaxis 45 antibiotic prophylaxis guidelines in a teaching hospital. journal of research in pharmacy practice. 2014; 3:62-66. 8. apostolopoulou e, zikos d, georgoudi a, et al. the impact of irrational perioperative antibiotic prophylaxis on the nursing workload. health science journal. 2015; 9 (1:4): 1-5. 9. fridkin s, baggs j, fagan r, et al. centers for disease control and prevention (cdc). vital signs: improving antibiotic use among hospitalized patients. morb mortal wkly rep. 2014;63:194-200. 10. r. a. kulkarni, p. h. kochhar, v. a. dargude, et al. patterns of antimicrobial use by surgeons in india. indian j surg. 2005;67:308-315. 11. woelber e, schrick e, gessner b, et al. proportion of surgical site infections occurring after hospital discharge: a systematic review. surgical infections. 2016; 17(5): 510-519. 12. david j.l, harry v.g, jacqueline r, et al. surgical site infection – a european perspective of incidence and economic burden. international wound journal. 2004:1:247-273. 13. abdel-aziz a, el-menar a, al-thani h,et al. adherence of surgeons to antimicrobial prophylaxis guidelines in a tertiary general hospital in rapidly developing country. advances in pharmacological sciences. 2013;2013:1-6. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 58 synthesis of 3 3 gem – di – c –nitromethyl nucleoside analogues of possible biological activity yousif a. al-ftahi * , fuad h.al – jawad* * , nidhal f.al – mejwel** * rece ived 31-8-2002 ac cepted 20-2-2005 abstract synthe sis of new nucleo side a nalogue s of the type : 3 , 3 gem – d i – c – nitro methly , exp ec te d to ha ve us eful app lica tion in the c hemo therap eutic tre atment of aids , c ancer and mic ro bia l infec tions. the synthes is involve d the co nde ns atio n of the app ro pria te s ugar d eriv ativ e ( i.e . 3 , 3 – ge m – di – c – nitromethly – 1– ribo fura no se ) with nitro gen b as es , suc h as , urac il and theop hllin following a multi step s cheme s ta rting fro m diace to ne goluco se (1) (s che me 1) .the prep ared co mpo und we re id entified by s pec tros co pic metho ds ; ir , mas s , 1h and 13c nmr. الخالصة  ة         – c –التوائم ثناائي 3 , 3 ر مماثالت نيوكليوسيد جديدة من نوع  يتحض ي المعالجـة الكيمائـي ل محتمـل االسـتفادة منهـا ـف نيترومثـي )che mothe ra py ( المراض نقص المناعة والسرطان ولالمراض الجرثومية . فيورانوز مع قواعد نتروجينية مثل  – d –نيترومثيل  – c –ئي التوءم ثنا 3 , 3تضمن التحضير تكثيف مشتقات الجزء السكري  ) .1المخطط ) ( 1(اليوارسيل والثيوفلين باتباع مسار متعدد الخطوات يبدأ من ثنائي اسيتون كلوكوز  وني ونظير تم تشخيص المركبات المحضرة بواسطة الطرق الطيفية االشعة تحت الحمراء والكتلة والرنين النووي المغناطيسى البروت . 13الكاربون  introduction the 2 , 3 – d id exy nuc le oside s ha ve s ho wn importa nc e in s eve ra l es ta blis he d chemotherapies (a nticance r , a ntiv iral and antiba cteria l ) and o ther a ttractive fie ld like immuno mod ulatio n o r regulation o f gene express io n which ma y constitute new therap eutic ap proac he s (1,3) . the bo ard ap plic atio n of modifie d nucleos id es e sp ecially in the inhib ition o f the huma n immuno de ficiency v irus (hiv)(4,5) , hav e ta rgeted the inves tigatio n for utilizing new nucleos id e analogues .effo rts ha ve prima rily fo cused o n modific atio n o f the ca rb ohydrate portio n o f the natural nucleo side s b eca us e ce llular kinas es are mo re tolerant o f thes e changes than changes within the b as e moiety(6) . the se ob se rva tio ns , hav e le d us to co mmet the synthe sis of a va riety of nucleos id e analogues c onta ning 3 , 3 gem – di – c – nitro methyl substituent at the sugar portio n . material and method melting points we re determined using electrotherma l melting point ap paratus and a re unc orre cted .ir sp ec tra were re corde d using either . shimadzu (40 8) ja sc o (j – 008 5 ) infrared sp ec trop hotome ter .  h – nmr and 13c – nmr s pe ctra we re de te rmine d o n a varia n xr – 30 05 (pha rm 3 00) spe ctro meter at 299 .9 08 a nd 75.41 18 mhz res pec tiv ely . cdc13 or dms o wa s us ed as the so lv ent and tms a s interna l standard . ge ne ra l mass spe ctro meter mode l 51 1 v alza r was used for re cording ma ss s pec tra .tlc was pe rformed on glas s plants c oated with 0.25mm laye r of silica gel (fluka ) and spo t were de te cted by io dine vap our. co lumn chro matograp hy was carrie d out with s ilica ge l 60 ( fluka ) . urac il from merk compa ny . 1 ,2 : 5 ,6 – di – o – is oprop ylid ene –  – d – gluc ofuranose (1) was prepa re d from 1 ,2 : 5 ,6 – di – o – is oprop ylid ene – –d – ribo – hexofuranose – 3 – ulos e (2) wa s p re pa re d a s p re vious ly des crib ed (19,20) . * college o f ed uca tion , ibn – al – haitham , ba ghd ad univers ity . ** ba ghd ad colle ge o f pha rma cy , s ynd ic ate of ira qi phar mac is ts . *** college o f ph arma cy , univer sity of ba ghd ad. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 59 1,2 : 5,6 – di – o – iso pro pylide ne – 3 – c – nitromethyl –  – – d – a llofuranose ( 3 ) and 3 – de oxy – 1,2 :5,6 – di – o – is op rop ylidene – 3 ,3 – ge m – d i – c nitromethyl – – d – ribo – he xofuranos e (5) were prep ared as pre viously (7) . 3 – deo xy – 1 ,2 :5,6 – di – o – is op rop ylidone – 3 – c – nitrome thylene – – d – ribo – he xofuranos e (4) wa s prepa re d acc ording to the method de sc ribe d (21) .bis (the ophylline – 7 – yl )mercury (1 0) was prep ared as in the metho d des crib ed (12) .2,4 – bis (trime thylis ly urac ia l (13 ) was synthes ized as de sc ribe d be fo re(15) . 3 – deoxy – 1,2 :5,6 – di – o – isopropylide ne – 3,3 – ge m – di – c – nitrome thyl –  – d – ribo – hexofur anose (6) 3 – deoxy – 1,2 :5 ,6 – d i o – is oprop ylid ene – 3,3 – gem – di – c nitromethyl –  – d – ribo hexofuranose (5) (8.3g, 22 .9 mmo l ) wa s diss olved in methanol a nd in h2s o4(30 ml) was a dde d . the rea ction mixture was left to stand at room te mperature for 3hr .the re sulting mixture was then ne utra lize d b y ad ding so lid so dium hyd ro gen c arbonate and then extrac ted with chloroform .the chlo roform extrac t was d ried ov er a nhydrous so dium sulphate and gave upon e vap oratio n the diol (6) as a syrup (6.0 g , 80.4 %).ir(s mear)v(345 0)c m-1 (oh) . 3 – de oxy – 1 , 2 – o – isopropylide ne – 3 ,3 – gem – di – c – nitr omethyl –  – d – r ibo – furanose (7) the d io l (6) (2g ,6.2mmo l ) was d is so lv ed in etha nol (40ml) and we ll – s tirre d , the n sa tura te d so lution o f so dium hydro ge n ca rb ona te (2ml) was ad ded follo wed by a so lution o f sod ium metap eriod ate ( 1 .3 2g ,6.2 mmol ) in 70 ml w ater the res ulting rea ctio n mixture was s tirre d 3hr a fte r which the exc es s so dium metaa pe rida te wa s de stroye d by a dding fe w d ro ps of ethylene glycol .the res ulting alde hyd o sugar was imme diately re duc ed with so dium borohydride (0 12g).after the rea ctio n mixture was kept with s tirring for 4 hr ,ac etone (0 .5 ml) was a dde d and the mixture was further stirred for 3 0 minutes . the s olid res id ue wa s re mov ed by filtratio n a nd the filtra te wa s extrac te d with me thylene chloride , d ried o ver anhyd ro us so dium sulphate the s olve nt wa s re mov ed to give (7) as a s yrup (1 .3 9g, 76.8 %).ir (s mea r )v(345 0)cm-1 (oh).1h – nmr  5.82(d,1h,h – 1 )4.72 – 4.70 (q,1h, h – 4 ), 4 .6 6 – 4 .6 0 (d,1h,h – 2 ),4.10 (s ,4 h , 2ch2no2),3.5 3(d ,2 h,h – 5 ,h – 5 ,1.38 – 1.23 (2s ,6h,2ch3). 13c – nmr  10 4.32 ( c – 1 ),81 .9 5 (c – 2 ),5 8.74 , 5 8.40(c – 3 ), 81.7 4 ( c – 4 ), 84 .1 9 (c – 5 ), 8 0.31 , 78.42 (ch2no2),1 10.51 ( o – cme2 – o ) , 26 .5 2 , 26.06 ,. – c (ch3)2 . 5 – o – benzoyl – 3 – deoxy – 1,2 – o – isopropylidene – 3,3 – gem – di – c – nitr otmethyl –  – d – ribo – fur anose (8) the 3 – d eoxy – 1 , 2 – o – is oprop ylid ene – 3 ,3 – gem – d i – c nitrome thyl – – d ribofura nos e (7) (2.4g , 8 .2 mmol ) was disso lv ed in a nhydrous p yrid ine (6.7ml ,83 mmol ) afte r e xterna l c oo ling with ic e , benzo yl chlo ride (0 .9 6ml ; 8.3mmo l )was add ed dropw is e . the reac tion mixture was kep t at roo m te mperature for 24 hr then iced water was a dde d .the res ulting s yrup was extracte d with petroleum ether (b.p. 4 0 – 60 c ) ,then drie d with anhyd ro us sod ium sulphate , filtered and c onc entrated under re duc ed pres sure . trac es of p yrid rne we re re move by co eva poration with dry to luene .the be nzo ate d eriv ativ e (8) was obtaine d as s yrup (1 .6 g , 50 % ).ir (s mea r )v (3 100 – 30 00 ) c m-1(aromatic c – h ) ,(171 0)cm-1( c = o) , (1 590 )cm-1 , ( c – c ) . ma ss s pec trum , ga ve m – 3 96 ( 5 – benzo ate d eriv ativ e 8 ) ,a nd m/e 261 (– [ phco2ch2] ) . 1h – nmr  7.47 (m , 5 h , aromatic ) , 5 .9 5 (d, 1h ,h – 1 ), 4.76 – 4,6 3 (c m , h – 4 , h – 5 ,h – 5 ) ,4.5 8 (d,1h, h – 2 ), 4 .2 (d, 2h, ch2no2 ),3 .9 5(s,2h,ch2no2), 1.50 – 1.32 (2s , 6h, 2ch 3).13c – nmr 13 3.31, 130 .0 5 , 129 .8 5 , 12 8.52, 1 28.3 8 (a romatic ring c arbons ) ,17 0.62(ph – co ) ,105 .3 0 ( c – 1 ),8 0.02 ( c – 2 ),60.49 ( c – 3 ) ,79.34 ( c – 4 ) , 77 .8 3 ( c – 5 ), 7 4.88 , 74.62 ( ch2no3) . 1,2 – di – o – ac etyl – 5 – o – benzoyl – 3 – deoxy – 3 ,3 – gem – di – c – nitromethyl – a – d – ribofuranosyl br omide (11) the ace tylated suga r (9 ) (1 g,2.2 mmol) was trea te d with 50 %(w/v) hydroge n bromide in ace tic (3ml) the so lution wa s ke pt a t 0c for one ho ur the n p oured in to an ic e c oole d dichlorome thane (5 0ml ) , wa shed with iced water , and then with sa tura te d aq ue ous solution of sod ium bic arbo nate to re move the re maining ac id .afte r a fina l was h with iced water , the d ic hlorome thane layer wa s dried ove r a nhydrous s odium s ulphate a nd the solve nt wa s re move d to give (11) a s syrup (0 .9 g , 95 %). the iso la te d sugar bromide (11) was use d dire ctly fo r the nucleo side s ynthe sis . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 60 synthesis of theophylime nuc leoside analogue 7 – ( 2 – o – acetyl – 5 – o – benzoyl – 3 – deoxy – 3 ,3 – gem – di – c – nitrome thyl –  – d – ribofuranosyl ) the ophyline the the ophylline mercury sa lt (10 ) (0 .5 5 g , 0.98 mmol ) was finely pow dered , s us pe nde d in (150 ml )s od ium – drie d xylene and the so lv ent was p artially distilled to remo ve tra ce s of wa ter a zeo trop ic ally .whe n the temp erature of mixture wa s ra is ed to 13 7 c , the res id ual suspe ns io n was a llowe d to c ool (below 50 c ) . the a ce tyla te d sugar bromide (11) (0 .9 gm , 1 .9 6mmol )in xlye ne wa s then a dde d and the reac tion mixture re fluxed with vigo rous stirring fo r 15 minute . the xlyene la ye r was was hed with 20 % a que ous po tassium io dide to re move the rema ining trac es of the mercuric sa lt , wa she d with wa te r , d ried o ver anhyd ro us so dium s ulphate a nd the so lv ent was remo ved to give after s ilica ge l column chro matograp hy with c hloroform a s an eluent the ac etylated nucle oside (12 ) (0 .5 7g,52 %ja s syrup .ir (s mea r ) v (2 850 – 29 50 )c m-1(c – h ) . synthesis of protected uracil nucleoside analogue 1 –(2 – o – ace tyl – 5 – o – benzoyl – 3  – dideoxy – 3 , 3 – gem – di – c – nitr omethyl –  – d – ribofuranosyl ) – 4 – ( trime thylsily ) urac il (14) the a cetylate d sugar (9) (0.44 g, 1mmol ) and silylated urac il (13 ) (0 .29 g ,1 .1 mmol ) were diss olved in anhyd ro us 1,2 – dichloroe thane (1 0ml ) . anhyd ro us s ta nnic c hlorid e (0 .0 8ml , 0.68 4 mmol ) w as then a dd ed in the prese nc e of few p elle ts o f molecula r siev e 4 a and re ac tion mixture was stirred at 2 3 c , tic (c hloroform mcthano l )9:1) s how ed the re ac tion was co mplete afte r 16 hrs . the re ac tion wa s then p oured o n an exes s so dium bica rbo na te solution a nd e xtra cted with methyle ne c hlorid e .the combined me thylene chlo ride extrac ts wa s drie d o ver a nhydrous so dium sulp hate , and the so lv ent was re move d to give (14) (0.6 g , 40,3%),ir (sme ar )v(285 0 – 295 0 )c m-1(c – h ),(3 350 )c m-1(n – h ). the pro duct wa s purified o n silic a gel column us ing c hloroform as elue nt .two d ifferent frac tions were iso la te d , the firs t one (14) (0 .0 18g) a s white se mis olid which repres ente d the silya te d nucleo side .1h nmr  7 .9 6 (s ,5h,aromatic ),7 .6 6 – 7.64 (2s ,2 h,ch=ch of urac il ),4.6 – 4 .4 (cm,4h,h – 2 ,h – 4 , h – 5 ,h – 5 ), 4.2(d,4h,2ch 2no2),2 .0 2 (s ,3h ,ch 3), 0.90 (s ,9 h,sime 3 ).the s ec ond frac tion (1 4a ) (0.02 mg) ob ta ined as syrup id entified a s the d es ilylated fre e nuc le os ide ana lo gue .1h nmr  8 .0 5 (m,5h , aromatic ),7.48 – 7.43 (2 s,2h,ch=ch of uracil ),5.98(d,1h,h – 1 ),5 .2 – 4 .8 (c m,4h,h – 2 ,h – 4 , h – 5 , h 5) , 4.45 (d , 2 h , ch2no2) , 3.(d,2h ,ch2no 2),2 .07(s,3h,ch3). resultand discussion 1.sythe sis of the car bohydrate moie ty of the nucleoside 3 – deo xy – 1,2 :5,6 – di – o – is oprop ylid ene – 3 ,3 – d i – ge m – c – dinitro methyl –  – d – ribofuranose (5) was o btained fro m d – gluco se ac cording to the metho d repo rted earlie r by o ne o f us (7) . selec tive hyd ro lysis of the 5,6 – isop io pylidene group of the 3,3 – ge m –di–c– nitrome thyl –– d – ribo hexofuranose (5 ) with in sulp huric acid in metha nol (8) gave the mo nois oropylide ne derivative (6 ) a s a syrup in 80 .4% yield .oxid atio n of the diol (6) with s odium pe riod ate effected the clea vage of c5 – c6 bond a nd res ulting intermed ia te aldehyd e derivative was re duc ed immed ia te ly with s odium bo ro hyd ride to giv e the 3 – de oxy – 1,2 – o – is oprop ylid ene – 3 , 3 ge m – di – c – nitrome thyl –  – d – rib ofuranose (8). the prima ry 5 – oh group in (7) was then protected b y convcrsion to the 5 – b enzoa te este r (8) . trea tme nt of (7) with be nzo yl chloride in pyrid ine (9).ove rnight gav e the 5 – o–be nzoyl–3– de oxy–1,2 – o – iso propylidene – 3 ,3 – gem – di – c – nitro methyl – – d – ribofura nos e (8 ) as s yrup in 5 0% yield . the fina l step in the synthe sis of the protected sugar moie ty before ca rrying o ut the c oup ling re action with nuc le o – ba se s , wa s the remov al of 5 – o – 1 ,2 – ac etal o f 5 – o – be nzo yl derivative (8 ) with 99 % trifluo ro ac etic ac id fo llowe d by a cetylation .ace tylatio n of the 1 – and 2 – hydroxyl gro ups wa s pe rformed with ace tic a nhydrid e in p yrid ine (9) which a fforde d 1,2 – di – o – ac etyl – 5 – o – be nzoly – 3 – deo xy – 3 ,3 – ge m – d i – c – nitro methyl –  – d – ribotura nos e (9) a s a s yrup in 90% yield . 2synthesis of nucleoside analogue s f or the synthes is of 7 – ( 2 – o – a cetyl – 5 – o – b enzoyl – 3,3 – d i – c – nitrome thyl – – d – ribo – fura no syl ) theo phylline (12 ) , the koe nigs – knorr co nde ns atio n method was fo llowe d (10). thus trea tment of 1,2 –d i – o – ace tylrlbofuranos e d eriv ativ e (9) with anhyd rous hyd ro gen bromide in dichlorome thane re adily affo rd ed 1,2 – di – o bromide(11 ) whic h wa s use d immed ia te ly bec ause of its ins tability . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 61 ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 62 the s ugar bromide (11 ) was c ond ens ed with bis (theop hylline – 7 – yl )me rc ury (10 ) as the ac tiva te d b ase in a nhydrous xylene under re flex which afforded the des ired the op hylline nucleos id e analogue (1 2) , afte r silic a column chro matograp hy , as a s yrup in 52 % yie ld .the theo phylline (1,3 – dimethylxanthine )b as e ha s be en us ed b ec aus e of its a vailability a nd due to the fact that only one of its nitrogen a to m (n – 7 ) is rea ctive , the re fo re mixture of d ifferent nucleos id e analogues ma y b e avo id ed . the re ac tion o f the ophylline w ith me rc uric c hlorid e in aqueo us a lkali a fforde d the me rc ury de rivative rathe r than the chlorome rc ury one , and it was as signed that n – 7 wa s the predo minate po sitio n of a tta chme nt of me rc ury group in the theop hylline . it has a ls o b ee n de mons tra te d tha t the mercury de riva tive of theo phylline c oup le with acylglyco syl ha lide s at n – 7 (12 – 14) and involve s dire ct disp la ceme nt of the mercury group from nitroge n by the inco ming ac ylgyc os yl halid e (11) . f or the synthes is o f 1 – ( 2 – o – a ce tyl – 5 – o – benzoyl – 3 – de oxy – 3 ,3 – gem – di – c – mitromethy –  – d – ribofuranose ) – 4 – (trimethylsilyl ) uracil (1 4), the modifie d hilb ert – jo hnson proc edure us ing s imply frie del – crafts ca ta lysts like sncl4 wa s fo llowe d (15) . the 1,2 – di – o – ac eylrib ofurano se d eriva tiv e (9) was c oup le d with the silylated uracil (1 3) in 1 ,2 – dichoro etha ne in the p re sence of a nhydrous stannic chloride as lewis acid .the rea ctio n invo lve d the c onv ersion of the protec te d sugar (9 ) in to the rea ctive e lectrophilic 1 ,2 – ac etyloxo nium io n fo llowe d b y the s ilyla te d urac il (13 ) attak to affo rd the p rote cted urac il nucleos id e analogue (14) with the regenera tio n of the catalyst . the fo rma tio n of 1 ,2 – ac etyloxo nium io n d etermined the exclus iv e fo rma tio n o f the –  – ano mer (14)(15 – 16) , which wa s se parated a s white s emiso lid and characterize d by its 1h nmr sp ectrum . ano ther fra ction (1 4a) w as se parate d o n the silic a gel c olumn and ide ntified fro m its 1h nmr sp ec trum a s be ing the des ilyla te d nucleos id e (1 4a ) . references 1. p erigand c.;go sselin , g.; and lmbac h , j .l.,nucleos ides and nucleo tides , 1992 , 1 1 ,9 03 . 2. wilmon , d.e.v., the chemistry o f antitumor agents , chap man a nd ha ll, new york , 19 90 , p.26 1 . 3. ha rmon , r.e. ,robins ,r.k, a nd town send l.b., che mis try ' and biology of nucleosides , acade mic press , inc. : new york, 1 978 ,p . 98 4. he rdewijn , p., balza rini ,j., baba, m .,pa uwe ls , r., va n aersc hot , a., j aa nsse n , g. and de cle rcq , e., j . med che m.19 88,3 1,2040 . 5. he rdewijn , p., ba lzarini , j ., de clercq . e., pauwels , r., baba , m., bro de r , s. and vand erha le ghe , h.,j . med che m. . 1 98 7 , 30 , 1270 . 6. huryn , d .m. and okabe , m., chem. rev., 199 2 , 92 , 1745 . 7. ali , y., vya s , d.m ., nab inge r , r.c. and s za re k , w.a., carbo hyder res., 1982 , 104 , 1 83 . 8. rose ntha l , a. a nd ba ker , d.a., j .org . chem. 19 73, 38 , 193 . 9. rose ntha l , a. a nd ba ker , d.a., j .org . chem. 19 73, 38, 19 8 . 10. barto n , d. and oils , w.d., comprehensive org. che m. , vol. 5, 1st . ed., e .haslam , unive rs ity of shc ffild , 1 979 ; p .6 0 . 11. mo ntgome ry j.a .a nd thomas , h.j., j.org. chem. , 1 966 , 31 , 1 411 . 12. f rees to ne , a.j., ho ugh , l. and ric hardson , a.c., carbohyd r. res ,,1973,28,378 . 13. mo ntgome ry ,j.a. and thoma s , h.j., adva n . ca rbohydr . che m. 19 62 , 301 , 17 . 14. mo ntgome ry ,j .a. and thoma s . hnjn, j. org. che m. , 196 3, 28, 2304 . 15. niedba lla , u. and vorbruggen , h,,j .org.che m,1 974 ,39,39 ,3657 . 16. vorhru ggen , h., krolikiewicz , k – and bennua , b.,chem ,ber .,19 81 , 114 ,1234 . 17. s ingh,p.p.,cha ria m.m, das gup ta , f.and s rivastava , h.c . ,te trahedron lett ,19 77, 5, 4 39 . 18. glen.w.l.,myers g.s.and gra nt ,g.a..j .chem – s oc ., 1951 ,2568. 19. buttc rworth , r.f.; haness ian ,s , s ynthesis , 1971 , 2 ,70 . 20. (a) ali , y ; tawfe ek , h.i. , tra qi chem . s oc , confe re nc e , ba ghd ad ma rc h .2 8 – 31 , 1 98 4 . (b ) ta wfeek .h. i. , m. sc . thesis sub mitted to the co llege of science , unive rs ity of baghda d , 198 2 . 21. albrec ht ,h.p. and moffa tt , j .g., tetra hedron le tt., 1 970 , 13 , 1 06 3 . iraqi j pharm sci, vol.20(1) 2011 furosemide suspension 8 application of seed mucilage extracted from lallemantia royleana as a suspending agent alaa a.abdulrasool*, abdulmuttalib a.naseer** and firas a.rahi*** ,1 * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. ** baghdad college of pharmacy, baghdad, iraq. *** college of pharmacy, al-mustanseriyah university, baghdad, iraq. abstract the mucilage from the seeds of lallemantia royleana family labiatae was extracted and subjected to preformulation study for evaluation of its suitability for use as suspending agent. furosemide suspensions were prepared using (1.5% w/v) of the extracted lallemantia royleana mucilage, (1.5% w/v) chitosan and (0.35% w/v) xanthan gum. the mucilage was white in color and the average yield of dried mucilage obtained from l.royleana nutlets was 14 % w/w of the seeds used. it is sparingly soluble in water but swells in contact with it, giving a highly viscous solution. it is slightly acidic to neutral. it was found that the extracted natural mucilage of lallemantia royleana exhibited a higher viscosity profile and it exhibited better mucoadhesive property in comparison to chitosan, carbopol 934 and hydroxypropyl methylcellulose. the result showed that the suspension of furosemide prepared with 1.5 % w/v of the extracted mucilage was found to be ideal and comparable with the other two preparations of xanthan gum 0.35% w/v and chitosan 1.5% w/v. the study revealed that the mucilage of lallemantia royleana has good properties to be used as a suspending agent and the performance is comparable with that of chitosan and xanthan gum since it is of natural origin, nontoxic, and of good biocompatibility. key words: mucilage; polymer; excipient; drug delivery; suspension; lallemantia royleana. الخالصة اٌ انؽشع يٍ ْزِ انذساست ْٕ حذيثا كسٕاؼاث في حظييػ االدٔيت.حسخخذو انٓالياث ٔ االطًاغ انُباحيت انطبيعيت بكثشة ائيت ٔ انكيًيائيت نٓزا ٔدساست انخٕاص انفيضي lallemantia royleana اسخخالص ْاليت طًؽيت يٍ بُيذلاث )بزٔس( َباث انبانُكٕ ليحهٕ حشكيض% يٍ انٕصٌ االطهي نهبزٔس.كًا اٌ 41اظٓشث انُخائج اٌ انٓالو انًسخخهض ْٕ يادة بيضاء ٔ اسخخهظج بكًيت انٓالو. أشاسث انُخائج انى اٌ انٓالو انًسخخهض رٔ نضٔجت عانيت جذا ٔ لابهيت .يٍ انٓالو كاٌ يخعادال انى لهيم انحايضيت w/v %4 بخشكيض بُفس احيًُا اسخخذيcarbopolٕ ٔانكاسبٕبٕل hydroxypropyl methylcellulose ((hpmc انخظاق أعهى يٍ انكيخٕساٌ ٔ 4.1ٔأخيشا حى ححضيش يعهك انفيٕسٔصيًايذ بٕجٕد انٓاليت ٔ بيُج انُخائج اٌ انًعهك انًحضش باسخخذاو حشكيض ) (.w/v% 4) انخشكيض %(w/v ٌيٍ انٓاليت انًسخخهظت كاٌ يمبٕال ٔ نّ خٕاص ثباحيت ٔ ححشس يشابٓت أ حفٕق يعهميٍ يحضشيٍ باسخخذاو انكيخٕسا (4.1 %(w/v ٌطًػ انضاَثا ٔ xanthan gum (5..1 %(w/v. حبيٍ انذساست بانًجًم اٌ انٓالو انًسخخهض يٍ بزٔس َبخت رٔ خٕاص ,انبانُكٕ بااليكاٌ اٌ يكٌٕ يالئًا نخظييػ يعهماث راث خٕاص جيذة نخحشس انذٔاء كٌٕ ْزا انسٕاغ طبيعي , ايٍ,يخطابك فيضيٕكيًيائيت ٔ طيذالَيت جيذة . introduction natural gums and mucilage have been widely explored as pharmaceutical excipients such as thickeners, suspending agents, emulsifying agents, and binders (1) . it has been reported for the successful use of ocimum gratissimum, butea monospermama and leucaena leucocephala seeds mucilage as suspending agent (2). lallemantia royleana, commonly known as balango, is an annual herb belonging to the family labiatae (3) . it is cultivated throughout western asia, india, pakistan, and northern of iraq, (4) lallemantia royleana nutlets function as added palatable ingredient in cooling drinks and the highly mucilaginous nutlets have numerous applications in the traditional medicine; it is useful in abscesses as paste, inflammations and gastrointestinal problems (5) the nutlets are about 3 millimeter in length, 1 millimeter in breadth, dark-brown to black in color. (6) when moistened with water, they become coated with voluminous and translucent mucilage. the taste of the moistened nutlets is bland and somewhat spicy. (7) many previous references disclose method of isolating various components from lallemantia royleana nutlets, like volatile oils and mixed fatty acids. (8) the present study was undertaken with an objective to extract, evaluate and to find out the potentials as a suspending agent of natural mucilage obtained from the nutlets of the plant lallemantia royleana (balango) . 1corresponding author email : mt_iraq@yahoo.com received : 29/12/2009 accepted : 31/10/2010 mailto:mt_iraq@yahoo.com iraqi j pharm sci, vol.20(1) 2011 furosemide suspension 9 the application of plant mucilage as suspending agent explained by the gelling power and viscosity enhancing effects of these mucilages. for example , the formulation containing hibiscus cannabinus mucilage as a suspending agent shows comparable results to that of standard marketed formulation . (9) furosemide (loop diuretic) is practically insoluble drug; therefore, it was prepared as suspension using the extracted l.royleana mucilage as a suspending agent .therapeutic agents of low water solubility could be suspended in a liquid suspending vehicle of the nutlets extract, which has an elevated viscosity, higher than that of water. (10) experimental materials and methods furosemide from (ajanta, india), balango nutlets were purchased from local market in baghdad, iraq and the seeds mucilage was isolated in the laboratory as the balango seeds were soaked in water for 24 hours (in a ratio of 1: 20 w/v) . the mucilaginous seeds then blended in blender at low velocity for 10 seconds, and the mass was passed through muslin cloth. the mucilage was precipitated from the filtrate by adding 3 volumes of ethanol. the mucilage isolated from seeds was dried in an oven at 45°c for 2 hours. the powder samples were stored in tightly closed containers until used. (11) chitosan from fluka, biochemika , switzerland. xanthan gum from merck, darmstadt, germany. all other materials used were of analytical grade. instruments instruments used are ph meter( hanna,ph m-11microprocessor, italy), spectrophotometer (pu-1-pu pye unicam sp3100 infrared, unicam ltd, cambridge, uk), dissolution apparatus(paddle , dis 6000, copley nottingham, uk), viscometer (brookfield dv -ii cone and plate type .usa), ph and viscosity measurements the ph measurement was carried out for all mucilage dispersions. a dispersion of the mucilage (1% w/v) was used by taking 5 ml of the (1% w/v) gel and shaking it with 25 ml of water. the ph was estimated using hanna ph meter (model m-11). the mean of three determinations was calculated. the viscosity of 1% w/v solution of the extract was determined using brookfield viscometer at different shearing rates at 30 o c. the same measurements for ph and viscosity were done separately for 1% w/v solutions of chitosan,hydroxypropyl methyl cellulose (hpmc), and carbopol for comparison.further more, the viscosity of the extracted mucilage was tested at various ph ranges. characterization of mucoadhesive property of the mucilage the mucoadhesive property of l.royleana mucilage as well as for chitosan was determined according to park and robinson method. (12) the extracted mucilage was glued onto the lower platform of the equipment for mucoadhesive determination. an excised sheep duodenum measuring 2 cm width by 3 cm length was attached to the arm of the equipment by means of glue. the mucosa was gently brought into contact with the moistened disc and adhesion was allowed to take place for 5, 10, 15, 20, and 25 minutes. at the end of time intervals, the mucosa was gently detached from the mucilage disc and the force was directly recorded depending on the weight recording as detachment occurred on an electronic balance. the mean of three determinations was obtained. equilibrium swelling study to 1 gm of the dried mucilage, 25 ml of water was added in a 30 ml graduated cylinder and the mixture was shaked thoroughly every 10 minutes for 2 hours, allowed to stand for 24 hours at room temperature. then the volume in ml occupied by the mucilage was measured. the mean value of three determinations was recorded. (13) the equilibrium swelling studies were carried out for the gels at 37°c in buffer solutions of ph 2.1 and 7.4 (simulated gastric and intestinal fluids ph, respectively). retaining properties when heated to study whether the extract maintains its characteristics when heated and cooled, a (1% w/v) viscous solution of l.royleana mucilage was subjected in to two cycles of heat and cool, at 100 c 0 . (14) preparation of furosemide suspension three formulas of (2.5% w/v furosemide) suspensions were prepared using the regularly used concentrations of appropriate viscosities, the extracted l.royleana mucilage (1.5 % w/v), chitosan (1.5 % w/v) and xanthan gum (0.35 % w/v) as suspending agents. dissolution of (2.5% w/v) furosemide suspension using l.royleana mucilage as a suspending agent the united states pharmacopoea (usp) rotating – paddle dissolution apparatus (copley) was used to study drug release from the furosemide suspension.five ml of the iraqi j pharm sci, vol.20(1) 2011 furosemide suspension 10 suspension equivalent to 125 mg of furosemide measured and suspended in 900 ml of phosphate buffer ph 6.8,was stirred at 50 rpm and 37 0 c. at specific time intervals, samples (5 ml) were withdrawn and filtered. the same volume (5 ml) of the phosphate buffer ph 6.8 was replaced after each sampling. the drug content in the filtrate was determined by spectrophotometer at its max (271 nm). (15) sedimentation parameters the sedimentation volume were determined by keeping 50 ml of each suspensions in stoppered measuring cylinder and stored undisturbed at room temperature. the separation of clear liquid was noted at time intervals of 1 day up to 35 days. (16) redispersion fixed volume of each suspension (50 ml) was kept in calibrated tubes which were stored at room temperature for various time intervals of 5days; one tube was removed and shaken vigorously to redistribute the sediment and the presence of deposit if any was recorded. (17) results and discussion the mucilage extracted from lallemantia royleana seeds was found to be swells in contact with water, giving a highly viscous solution. it is slightly acidic to neutral. it was found that the extracted natural mucilage of l.royleana exhibited a higher viscosity profile than other tested polymer (505 cps [natural mucilage of l.royleana], vs. 187 cps [chitosan] vs. 65 cps [carbopol 934], vs. 20 cps [hpmc] respectively) at a concentration equivalent to (1 % w/v) as shown in figure (1). it appears that the extracted mucilage exhibited thixotropic (shear-thinning) behavior,figure(2). a marked dependence of the viscosity on ph was observed ,figure (3).i.e. as the ph increases the viscosity increases (p <0.05 ) . similar results were obtained for the mucilage extracted from the pods of abelmoschus esculentus when it was used as a suspending agent in paracetamol suspension. (18) swelling indices of l.royleana mucilage powder (1 gm) at ph 2.1 and ph 7.4 were found to be 7 and 25, respectively. the data indicated that the swelling of mucilage is ph-dependent, and the mucilage is anionic and this property is of value since the excipient support the drug to be retained to the intestine , the site of maximum absorption of the active constituent. (19) furthermore, the result showed that the extracted mucilage exhibited higher adhesion and better mucoadhesive property in comparison to chitosan, carbopol 934 and (hpmc) as shown in figure (4).the rheological behavior of the suspensions prepared with mucilage of lallemantia royleana, chitosan and xanthan gum are given in figure. (5). the results reveal that the suspensions are pseudoplastic in their behavior and their viscosity decreases with increase in shear rate, which is an essential requirement in the formulation of suspension. (20) suspending properties of l.royleana mucilage the extracted mucilage was found to be comparable to chitosan and xanthan gum as suspending agent. the results obtained indicated that the extracted mucilage may be used as a source for pharmaceutical adjuvant specifically as a suspending agent. it has been observed that 100% drug was released within 15 minutes in case of the suspension using the extracted l.royleana mucilage (1.5 % w/v). the same results was obtained for suspension containing xanthan gum (0.35%w/v), and slightly faster than that contains chitosan (1.5 %w/v) as a suspending agent (released within 20 minutes) as shown in figure (6), table (1). statistical analysis was performed; one-way analysis of variance (anova) of percentage released among the three groups was studied. the linear regression analysis performed on the square root data is shown in the figure (7). the regression lines produced by both the extracted l.royleana mucilage and xanthan gum suspensions are almost identical while the line produced by chitosan suspension is statistically different (p < 0.05).sedimentation was followed over a period of 25 days and almost no sedimentation was seen. the exhibition of excellent suspending properties of the extracted l.royleana mucilage are not completely dependent on viscosity, an example of suspensions prepared using some higher viscosity hydrocolloids, carboxymethylcellulose, settle out of solution on standing. (21) similar studies on the suspending power of other plant mucilage were reported by mital and his co-workers and they concluded that 1% albizia zygia mucilage have the same suspending power as 0.4 % tragacanth. (22) in conclusion lallemantia royleana mucilage was found to have acceptable physicochemical and drug release properties; it is of natural origin, non-toxic, biocompatible and cheap. therefore, it is suitable for formulation of suspension preparations. also the suspension prepared with 1.5 % w/v of the extracted l.royleana mucilage was found to be ideal and comparable with two preparations of xanthan gum 0.35% w/v and chitosan 1.5% w/v suspensions. iraqi j pharm sci, vol.20(1) 2011 furosemide suspension 11 figure 1 : comparative evaluation of viscosity of the extracted l.royleana mucilage ( ─×─), chitosan ( ─■─ ), the synthetic polymers carbopol 934 (─▪─ ) and hpmc ( ─▪─ ) .using 1% w/v solution at 30°c±1. values are expressed as the mean of 6 observations. speed of viscometer ; rpm(round per minute) figure 2: plot of rate of shear vs. shear stress for 1 %( w/v) solution of the extracted l.royleana mucilage. figure 4: mucoadhesion of (1% w/v) gel , of the extracted l.royleana mucilage■ ,chitosan ■, synthetic polymers carbopol 934 ■ and hpmc■. figure 5 : rheological behavior of the suspensions , of the extracted l.royleana mucilage ,1.5 % ( w/v ) ▲ ,chitosan 1.5 % (w/v ) ■, and xanthan gum 0.35% ●. figure 3 : effect of ph on viscosity of various concentrations of the extracted l.royleana mucilage.(─♦─) 0.5% ,(─■─ ) 1%, (─▲─ ) 3% w/v . figure 6: release profiles of furosemide (2.5 % w/v) from suspensions l.royleana ─▲─ (1.5% w/v), chitosan ─■─ (1.5% w/v) and xanthan gum ─ ●─ (0.35%) .at ph 6.8,and 37 o c. 0 100 200 300 400 500 600 0 1 2 3 4 5 6 rpm v is c o s it y ( c p ) 0 5 10 15 20 25 0 5 10 15 20 25 shear stress(dyn/cm2) r a te o f s h e a r ( 1 /s e c ) 0 100 200 300 400 500 600 700 800 900 1000 0 2 4 6 8 10 12 ph v is c o s it y (c p s ) 0 5 10 15 20 25 5 10 15 20 25 time ( minutes ) w e ig h t r e q u ir e d (g m ) hpmc carbopol chitosan l.royleana 0 10 20 30 40 50 60 70 80 90 100 110 0 5 10 15 20 25 time ( minutes ) c u m u la ti v e % d r u g r e le a s e d iraqi j pharm sci, vol.20(1) 2011 furosemide suspension 12 figure 7 : higuchi plots of furosemide release profiles from suspensions; l.royleana ── (r 2 0.8458), chitosan ── (r 2 0.8176) and xanthan gum ── (r 2 0.885). table 1: dissolution of 2.5% w/v furosemide suspension in different suspending agents. references 1. bharadia pd, patel mm, patel gc, patel gn. a preliminary investigation on sesbania gum as a pharmaceutical excipient. int j pharma excip 2004;3:99102. 2. p verma & balkrishen razdan. studies on leucaena leucocephala seed gum: rheological properties. journal of scientific & industrial research.2007;66:550-557. 3. ghannadi ar, zolfaghari b. compositional analysis of the essential oil of lallemantia royleana benth.j. flav. fragr 2003; 18: 237-239. 4. al-zubaidy am .systematic study of the genera (ajuga l.,marrubium l.,lamium l.) of labiatae in iraq 1998 ph.d. thesis, college of science. university of baghdad 5. national botanical research institute, india. medicinal 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chitosan percent furosemide released ,the suspending agent is 1.5 % w/v l. royleana mucilage 1 43 58.2 35 2 57 62.5 58 3 67 71.1 69 5 81 86.3 77 7 88 95.6 90 10 93 96.4 94 15 100 95.3 100 20 100 99.8 100 0 20 40 60 80 100 120 0 1 2 3 4 5 square root of time c u m u la ti v e % d r u g r e le a s e d http://www.niscair.res.in/sciencecommunication/researchjournals/rejour/rejour1.htm http://www.niscair.res.in/sciencecommunication/researchjournals/rejour/rejour1.htm http://www.who.int/medicinedocs/es/p/about? http://www.who.int/medicinedocs/es/p/about? iraqi j pharm sci, vol.20(1) 2011 furosemide suspension 13 nigerian journal of pharmaceutical sciences. 2008,mar; 7(1): 147-153. 17. ohara t, kitamura s, kitagawa t and terada k. dissolution mechanism of poorly watersoluble drug from extended solid dispersion system with ethyl cellulose and hydroxypropyl methylcellulose. int. j. pharm. 2005,302, 95-102 18. ravi kumar, m. b. patil, sachin r. patil1, mahesh s. paschapur. evaluation of abelmoschus esculentus mucilage as suspending agent in paracetamol suspension. international journal of pharmtech research.2009;1, 3, 658-665 . 19. macquet, a., ralet, m.c., kronenberger, j., marion-poll, a. and north, h.m. in-situ, chemical and macromolecular study of the composition of arabidopsis thaliana seedcoat mucilage. plant and cell physiology 2007. 48(7):984-999 20. sang-chul s, jin k. physicochemical characterization of solid dispersion of furosemide with tpgs. international journal of pharmaceutics 2003; 251(12): 79-84. 21. young sl, shoemaker cf. measurement of shear dependent intrinsic viscosities of carboxymethyl cellulose and xanthan gum suspensions. j of applied polymer sciences 1991; 42(9): 2405-2408. 22. mitalj hc, fokuo yd . studies on albizia zygia gum. journal of texture studies 1978; 9(3): 325-334. a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion 18 some variables affecting the formulation of oral loratadine suspension hala s.yousif *,1 and yehia i. khalil * * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad , iraq abstract loratadine is a long acting non-sedating anti-histaminic agent that was developed for the treatment of seasonal allergic rhinitis, whose anti-histaminic action is more effective than the other anti-histaminic drugs available commercially. this project was carried out to prepare an acceptable suspension through studying the release of drug in presence of different types and concentrations of suspending agents such as polysorbate 40, xanthan gum, sodium carboxymethylcellulose (nacmc), aluminum magnesium silicate (veegum) and sodium alginate. the effects of these suspending agents were studied at ph 1.2 (0.1n hcl) and 37 cْ. the results showed that the release rate of loratadine in the presence of these suspending agents was dependent on their types and concentrations. the results showed that loratadine release from the formula prepared from xanthan gum is more than that prepared from other polymers in the following order: sodium alginate < nacmc < veegum < xanthan gum. however, elegancy of suspension was better on using xanthan gum in a concentration of 0.5%. the obtained results were utilized to formulate 0.1% suspension of loratadine which is physically stable with an optimum drug release. the rheology, sedimentation volume, resuspendability and expiration date were evaluated for the selected formula. the formula that contains loratadine, xanthan gum, glycerol, sorbitol, methyl paraben, propyl paraben, sodium edetate, raspberry flavor at ph 5.0 appears to be a promised formula to be present with estimated shelf life of about 3.8 years. key word: loratadine, suspension, suspending agent, xanthan gum. ةصالخلا لعانغلللومطا رلغا لواعالهيل اطاننماالعدلينااراراقا هيداعاريللا ,لليوراعيمااسهلياتالم رالعدنيداتاراللا لينعةالعد لواعالهيل اطالللقامل ااتا طالم نلنالمتقهالعدينمقاالللغمل .ل ااوقللا نلالعيثااعيث اقا ياتا ريناا طالعرلساتا , غمد ا40لعزانم اتا طاتمااوغليتالثقغالعنرللاينونوال نلنا ويالتا طالعدنلوالعديارتاراييناالقللادا لا :لعينعواينغيللا لعدل للل ,للغينلهوا لااايااان العزنومنا ,ما لاا)ياا للالعدسرهاناارالمعدرانا( رالعلارللالعزنومنا .لدياوغليتا ناالعيلثاقللا ااْا .لىلغلالعريلتلالعواللاالثقغالعانغلللومطا طالعيقلايتالعيوالثينها37اراينغوتاسقلغاا١,٠يدثانااسل رالعلانغرلانغماا الغمتا < اوا غمد االعدل لللاليقنا طالعيقللادالعي الثينها اوالم نلنالمتقها طالعدنلوالعديارتارايلعو االعيلعو :لعلارللالعزنومنا ا غمد العدل للل .ل المييللواا طا نىالعريلتلاموالث اقا غما القلاياتالثينها اوا لواا≤اما لاا<للغينلهوا لااايااان العزنومناا قلعملا نايرج سايق لالخ نم مييقتلل ةيرارقتسا لضفا و ءاودلل ررحت نسحا تطعا ىتلا غيصلا تريتخا . ا .لتياقلالعزا العيوال ميالسهطالثقغاعانرللارالم االييرقلغمتاعايراا ا طاتمااااللاوقمللالعدياتا٠٫١لعانغلللومطاييقلادااا راسل القيداراللل لالعدياتايينالثقم ةاراللغملالعزمساتامم االقلايت .عرنارونايللالسهطالقلايتا والعيوالثينها اوا لعانغلللومطا , غمد العدل للل ,لااهاقرا ,ينغياينا , لااايلغلياط ,يقريااايلغلياط ,لمنمياياثرلتوالعزنومناارا لتالعينلا رنالملا ايرنلل .٣٫٨ . ل لا ناا غممساتالعدياتالعدث قام للايثنروا5لعلانغرواروا introduction a coarse suspension is a dispersion of finely divided, insoluble solid particles (the dispersed phase) in a fluid (dispersion medium or continuous phase) (1).a conventional suspension may be readily prepared in an aqueous solution with small percentage of hydrophilic polymers (like methyl cellulose, hydroxypropylcellulose and xanthan gum), as well as small percentage of surfactant like polysorbate 80. the suspending agent was used to achieve homogeneity of redispersed suspension while surfactants are used for wetting and dispersing of insoluble particles (2).for many patients, the liquid dosage form is preferred over the solid dosage form of the same drug because of the ease of the swallowing liquids and flexibility in the administration. the disadvantage of disagreeable taste of certain drugs (loratadine bitter taste) is overcome when the drug is administered as undissolved particles (3). in general, aqueous suspension gives more extended effect than aqueous solution (4). insoluble or poorly soluble drugs in suitable solvents could be formulated as flavored suspension as an ideal choice, example; nalidixic acid suspension (5) and fluconazole suspension (6 1 corresponding author e-mail : drhaha1971 @ yahoo. com received : 2/ 6/2008 accepted : 26/10/2008 iraqi j pharm sci , vol.17 (2) , 2008 loratadine suspension 19 commercially available loratadine solution (syrup) products contain excipients that are inappropriate for pediatric population, particularly the neonatal and infant age groups. to improve the solubility of loratadine, most of the syrups contain propylene glycol which may put the infants at risk of displacement of bilirubin and consequently hyperbilirubinemia (7). excessive intake of propylene glycol and subsequent metabolism to lactic acid may cause the development of hyperosmolality and lactic acidosis (8). also the high concentration of sucrose over 60% w/v made the syrup to be administered with care to patients with diabetes mellitus (9). in addition the stability of the drugs when prepared as insoluble particles is higher compared to that of dissolved drug in syrup, as shown by the aminophylline suspension whose chemical and physical stability showed minimum rate of degradation after 91 days period (10). loratadine is a tricyclic antihistamine, which has a selective and peripheral h1-antagonist action. it has a long-lasting effect and does not normally cause drowsiness because it does not readily enter the cerebro spinal fluid (csf) (11).it is white to off-white powder, practically insoluble in water, but very soluble in acetone, alcohol and chloroform (12). loratadine rapidly absorbed after administration, peak plasma concentration being attained in about 1 hour. it is extensively metabolized; the major metabolite is desloratadine, which has potent antihistamine activity. the elimination t1/2 of loratadine and desloratadine are 8.4 and 28 hours respectively (13). there are different types of suspending agents used in suspension can be classified as (14): 1. naturally occurring suspending agents like xanthan gum, sodium alginate, that forms multilayer film around the drug particles. 2. semi synthetic suspending agents like nacmc, which form multilayer also. 3. synthetic suspending agents (surfactants) like polysorbate 40 (tween 40), that form monomolecular film. 4. finely divided solids like veegum which form solid particle film. the goal of this study is to formulate the loratadine as a suspension instead of syrup to mask its bitter taste, to improve its stability and to decrease side effects of the excipient by decreasing their use in the suspension formula. experimental materials and equipments loratadine powder (supplied by philadelphia pharmaceutical, jordan). veegum, xanthan gum, methyl paraben, propyl paraben, raspberry flavor (supplied by sdi, iraq). sorbitol, hydrochloric acid (riedel-de haen hannover, germany). disodium edta (bdh chemical ltd.pool, england). polysorbate 40 (merck-scanchard, germany). sodium carboxymethylcellulose, sodium alginate (hopkin and williams ltd, england). uv spectrophotometer (carrywin uv, varian, australia). usp dissolution apparatus (erweka). viscometer (brookfield copley scientific, england). ph meter (hanna instrument ph 211, italy). method of preparation preparation of stock dispersion of suspending agents: stock dispersion of each suspending agent used (polysorbate 40, xanthan gum, nacmc, veegum and sodium alginate) was prepared by dispersing (10, 1, 1.5, 5 and 3 gm) of the suspending agents, respectively in 75 ml of distilled water (d.w) using an electrical mixer at 150 r.p.m. the volume of the dispersion was made up to 100 ml with d.w, the resultant dispersions were allowed to hydrate for 24 hours (15). preparation of suspension: loratadine suspension was prepared by levigating 0.1 gm loratadine in a mortar with the prepared dispersion of suspending agent. when smooth paste was formed, the remaining of the vehicle was added and the volume completed to 100 ml with shaking (16). effect of type and concentration of suspending agent on the drug release: the dissolution pattern of loratadine was studied in the presence of different types and concentrations of suspending agents including: polysorbate 40, xanthan gum, nacmc, veegum and sodium alginate. formulation of loratadine suspension: five different formulas were prepared using different suspending agents as shown in table (1) chemical formula c22h23cl n2o2 mol.wt 382.8 gm melting point 134-136 c ْ ../../../../wiki/antihistamine iraqi j pharm sci , vol.17 (2) , 2008 loratadine suspension 20 table (1): different formulations of loratadine as suspension dosage form represented as % (w/v) materials formula number a b c d e loratadine 0.1 0.1 0.1 0.1 0.1 xanthan gum 0.5 0.3 0.3 scmc 0.5 0.5 veegum 2 0.5 polysorbate 40 1.25 methyl paraben 0.2 propyl paraben 0.02 glycerol 5 sorbitol 7 disodium edetate 0.1 raspberry flavor 0.05 final volume 100 ml each formula was prepared as follows: loratadine, methyl paraben, propyl paraben were levigated in a mortar with glycerol and the prepared dispersion of suspending agent. the mixture was triturated with a pestle until a smooth paste was formed. with continuous trituration, the paste was diluted with the remaining amount of the dispersion of the suspending agent then transferred to graduated cylinder. finally, the required amount of disodium edta and sorbitol were dissolved in a small portion of water and added to the graduated cylinder and raspberry flavor was added and water to make the final volume. the suspension was shaken and the ph was adjusted to 5 with few drops of 5m sodium citrate. in vitro evaluation of suspension dissolution rate profile: the dissolution rate of loratadine suspension was studied using usp dissolution apparatus type іі which was provided with stainless steel stirrer connected to electrical motor and rotated at 50 r.p.m. with 900 ml of 0.1n hcl placed in the dissolution jar and equilibrated at 37 cْ. this was followed by transferring 5 ml of the prepared suspension to the jar bottom using a syringe. then a sample of dissolution medium was withdrawn at different time intervals (2, 5, 15, 30, 45 and 60 minutes) through a pipette fitted with a filter paper. fresh dissolution medium was added to the jar each time to replace withdrawn samples. each sample was suitably diluted and assayed spectrophotometrically at 278 nm for loratadine content. measurement of rheograms: rheograms was obtained at 25 cْ using brookfield viscometer. sedimentation volume measurement fifty milliliters of each suspension was diluted with distilled water to a volume of 100 ml in a stoppered graduated cylinder. the suspensions were shaken vigorously to insure uniformity, and then left undisturbed. the sedimentation volume was measured every 4 hours for period of 48 hours (17). resuspendability of suspension the efforts required to convert the sedimented system to homogenous suspension upon shaking the cylinder manually, were rated with a ranks as follows: resuspendable, resuspendable with difficulty or not resuspendable (18). stability study the accelerated stability study was done in order to determine the expiration date of the selected formula. the suspension was centrifuged to get the supernatant solution; 5ml samples of the resultant solution were stored in several closed amber glass containers at 35, 45 and 55 cْ for 90 days. samples were inspected for change in color, odor, ph, precipitant and assayed for drug content at suitable time intervals (0, 10, 20, 30, 45, 60 and 90) days. results and discussion effect of type and concentration of suspending agent on the drug release: the effect of various types of suspending agents (surfactants and polymers) on the dissolution rate of loratadine was investigated. 0 20 40 60 80 100 120 0 10 20 30 40 50 60 time (min.) % lo ra ta di ne r el ea se d 0% ( w /v) 0.50% (w /v) 1.25% (w /v) 2.00% (w /v) figure (1): effect of polysorbate 40 concentration on the percent of loratadine released in 0.1 n hcl ph (1.2) at 37 ٥c. iraqi j pharm sci , vol.17 (2) , 2008 loratadine suspension 21 figure (1) indicated that polysorbate 40 at all concentration used increases the amount of drug released in the following order: 0.0% < 0.5% < 2.0% 1.25% it was seen that increasing polysorbate 40 concentration from 0% to 0.5% and 1.25% (w/v) enhance the drug release, this may be attributed to the wettability and micellarization effect of polysorbate 40 at the interface between insoluble drug particles and the vehicle (19). meanwhile increasing polysorbate 40 concentration to 2% (w/v) enhance the release of loratadine to a lesser extent compared with 1.25% (w/v), this odd behavior may referred mainly to the formation of micelle macromolecules of drug with polysorbate 40 that hinder the excessive drug release from insoluble loratadine particles, this behavior give an impression of critical micelle formation with this range of polysorbate 40 concentration (2% w/v) (20). from the other hand figures 2, 3 and 4 illustrate the effect of anionic hydrophilic polymers including xanthan gum, nacmc and sodium alginate on the dissolution rate of loratadine using different concentrations of these polymers. the results indicated that the percent of drug release were as follows: xanthan gum 0.7% < 0.0% < 0.3% < 0.5% nacmc 0.0% < 1.0% < 0.25% < 0.5% sod. alginate 2.0% < 1.0% < 0.5% < 0.0% 0 20 40 60 80 100 120 0 10 20 30 40 50 60 time (min.) % lo ra ta d in e re le as ed 0% (w /v) 0.3% (w /v) 0.5% (w /v) 0.7% (w /v) figure (2): effect of xanthan gum concentration on the percent of loratadine released in 0.1 n hcl ph (1.2) at 37 ٥c. 0 20 40 60 80 100 120 0 10 20 30 40 50 60 time (min.) % l or at ad in e re le as ed 0% ( w /v) 0.25%(w /v) 0.5% (w /v) 1% ( w /v) figure (3): effect of sodium carboxymethylcellulose concentration on the percent of loratadine released in 0.1 n hcl ph (1.2) at 37 ٥c. 0 20 40 60 80 100 120 0 10 20 30 40 50 60 time (min.) % l or at ad in e r el ea se d 0% ( w /v) 0.5% (w /v) 1% ( w /v) 2% ( w /v) figure (4): effect of sodium alginate concentration on the percent of loratadine released in 0.1 n hcl ph (1.2) at 37 ٥c. the hydrophilic polymers xanthan gum and nacmc enhanced the dissolution rate of loratadine, since these polymers behave as protective colloid by coating the solid hydrophobic particles with multimolecular layer. this imparts hydrophilic character to the solid and thus promotes wetting (1), but to certain limits, then the dissolution rate of loratadine decreased by increasing xanthan gum and nacmc above 0.5% (w/v), this result may be attributed to the increase in viscosity of the prepared formula (21, 22). moreover the results showed that the presence of sodium alginate retarded the dissolution rate of loratadine at all concentrations used. this result is related to the formation of higher viscosity regions due to the hydrated polymer surrounding drug particles which encounter high resistance to the dissolution (23). on the other hand, the effect of veegum on the dissolution rate of loratadine is shown in figure(5). 0.0% < 2.0% < 1.5% < 0.5% iraqi j pharm sci , vol.17 (2) , 2008 loratadine suspension 22 0 20 40 60 80 100 120 0 10 20 30 40 50 60 time (min.) % l o ra ta di n e re le as ed 0% (w /v) 0.50%(w /v) 1.5% (w /v) 2% (w /v) figure (5): effect of veegum concentration on the percent of loratadine released in 0.1n hcl ph (1.2) this retardation in dissolution of loratadine as the concentration of veegum increases may be attributed to adsorption of loratadine on veegum or solid particle films formed around the drug particles (24). formulation of the loratadine suspension: xanthan gum was used as a suspending agent in concentration of 0.5% to prepare formula a. this concentration gave the best release as mentioned previously. it is an effective flocculating agent at relatively low concentration and has excellent suspending properties to suspend solid (25). the rheological stability of xanthan gum toward ph changes encountered during transit through the gastrointestinal tract in addition to large sedimentation volume presumably by polymer bridging phenomena providing reasons for its use (26). nacmc was incorporating in formula b in concentration of 0.5% and enhanced the dissolution rate of loratadine, but its aqueous dispersion has low viscosity and produced sediment layer that was easily redispersed by shaking. farther more, a combination of xanthan gum and nacmc (formula c) resulted in too viscous suspension which has less flexibility when was pouring. veegum was chosen in a concentration of 0.5% (formula d), since this concentration gave the best release but this suspension had low viscosity. the linear branched chain molecules of veegum form a gel-like network within the system and became adsorbed on to the surface of the dispersed particles, thus holding them in flocculated state (27). so formula e was prepared with 2% veegum to get higher viscosity. the non-ionic surfactant polysorbate 40 was incorporated as a wetting agent and to increase the dissolution rate of loratadine. this dispersion exhibit thixotropy band plasticity with high yield value. it remained in the flocculated state (i.e., no hard sediment or caking was formed) and poured easily. the following excipients were added to the prepared suspension; sorbitol and glycerol as a sweetening agent. they produce pleasant taste, less viscous suspension and are better than sucrose in producing structure in vehicle suspension (28). disodium edetate was involved in the formulation to protect loratadine from deterioration (29). raspberry flavor as flavoring agent, methyl paraben and propyl paraben were added as preservatives. the ph of the formula was adjusted to 5 using 5m sodium citrate. in vitro evaluation of suspension: dissolution rate profile: table (2) and figure (6) showed the dissolution rate profiles of the prepared loratadine suspensions. the data indicated that formula a had the highest dissolution rate constant as compared with the other formulas, since the rate constant of formula a was 24.3 × 10-3 (mg1/3 /min) using hixson-crowell root equation, that expresses the dissolution rate of solid particle based on the cube root of the weight of the particles (30), this equation was as follows: wο 1/3 – w1/3 = kt since: wο=the original mass of the drug particles w = the mass of drug dissolved k = the cube root dissolution rate constant t = the time required to dissolve (w) mass of drug particles table (2): the calculated dissolution rate constants of loratadine in the prepared formulas. 30 40 50 60 70 80 90 100 110 0 10 20 30 40 50 60 time (min.) % l o ra ta di ne r el ea se d a (xanthan gum) b (nacmc) c (xanthan gum+nacmc) d (xanthan gum+polysorbate40 e (veegum+polysorbate40) figure (6): dissolution rate profile of loratadine in the prepared formulas in 0.1n hcl at 37cْ. formula k × 10-3 (mg1/3 / min) formula a 24.3 formula b 15.0 formula c 21.3 formula d 21.2 formula e 21.5 iraqi j pharm sci , vol.17 (2) , 2008 loratadine suspension 23 measurement of rheograms: the rheograms of the prepared formulas are represented in figure (7). the profile showed that the viscosities of loratadine suspensions were shear rate dependent and it increased in the following order: formula c > d > a > b > e 0 20 40 60 80 100 120 0 10 20 30 40 50 she ar rate (s-1) v is co si ty (c p ) a (xanthan gum) b (nacmc) c (xanthan gum+nacmc) d (xanthan gum+polysorbate40) e (veegum+polysorbate40) figure (7): shear rate dependency of the viscosities of the prepared formulas of loratadine suspension at 37 cْ. the results also illustrated that all the prepared formulas exhibited pseudoplastic flow which evidenced by shear thinning and increase in shear stress with increased angular velocity. this behavior due to the suspending agent used, because as a general rule, the pseudoplastic flow is exhibited by polymer in solution(31). the rheological properties of the prepared formulas revealed that formula a was the best one which offered ease of pouring and swallowing compared with formula b and e which they are less viscous and could not obtain the structured suspension with them. on the other hand, higher viscosity was obtained with formula c and d with a difficulty in the pouring and swallowing from the bottle orifice. this was a result of using a combination of two polymers (xanthan gum + nacmc) and (xanthan gum + veegum) respectively which leads to rheological synergism to be occurred due to stronger crosslinking between the two polymers used, where the presence of carboxyl groups on nacmc and xanthan gum promote stronger hydrogen bonding between them (32). sedimentation volume and resuspendability: the sedimentation volume (f) is the ratio of the ultimate height of the sediment as suspension settles in a cylinder (hμ) to the initial height of total suspension (ho). f = hμ / ho while the resuspendability is a quantitative test to evaluate the ease of redispersion of a suspension after a long period of standing (30). table (3): sedimentation volume and resuspendability of the prepared formulas. formula sedimentation volume f = hμ / hο resuspendability a 1 no sedimentation b 0.6 easily resuspended c 1 no sedimentation d 0.9 easily resuspended e 0.4 resuspendable with difficulty table (3) shows the sedimentation volume and resuspendability of the prepared formulas. the data indicated that the formulas prepared with xanthan gum (formula a, c and d) had sedimentation volume almost equal to 1 i.e., no sedimentation was occurred during the test period. the obtained results attributed to the network of flocs formed in the suspension which is so loose and fluffy that can extend through out the extra vehicle (31). xanthan gum is often used as flocculating agent to achieve non-sedimenting suspension of drugs with no need to other adjuvant(18). however, formula b had a sedimentation volume equal to 0.6 and was easily resuspended by shaking. this low sedimentation volume may be due to the type of the suspending agent used, since nacmc may form homogenous network in all concentrations used (23). on the other hand, formula e, which showed sedimentation during the test period, had low sedimentation volume of 0.4 and was resuspendable with difficulty, so it is pharmaceutically unacceptable. stability study: formula a was chosen for the stability study as the promising formula since it gave the optimum physical stability and remarkable release profile. the stability study carried at moderate exaggerated temperatures (35, 45 and 55 cْ) to predict the expiration date of the promised formula. the degradation of loratadine followed apparent zero-order kinetics, since the concentration in solution depends on the drug solubility. as loratadine decomposes in solution, more drug is released from the suspended particles so that the concentration remains constant (30). the resultant solution from centrifuging formula (a) was with no reservoir of loratadine to replace that depleted so loratadine degradation iraqi j pharm sci , vol.17 (2) , 2008 loratadine suspension 24 in them followed first-order expression as in equation (1): -d [a] / dt = k [a] ………..(1)ا in which a is the concentration of loratadine remaining undecomposed at time t, and k is the first-order rate constant. when the concentration [a] is rendered constant, as in case of a suspension, equation (2) is applied: k [a] = k0 …………….(2)ا were k0 is the apparent zero-order rate constant, [a] is the solubility of loratadine at 25 cْ which is equal to 0.096 gm/100ml and k is the first–order rate constant at 25 cْ. the first-order rate constant for loratadine degradation in supernatant centrifuged solution of formula a was calculated from the slopes of straight lines which resulted from plotting log percent remaining of loratadine in the solution versus time at elevated temperatures (35, 45 and 55 ْc) as shown in figure (8) and listed in table (4). 1.975 1.98 1.985 1.99 1.995 2 2.005 0 10 20 30 40 50 60 70 80 90 100 time (days) lo g pe rc en t r em ai ni ng o f l or at ad in e 35 c 45 c 55 c figure (8): accelerated stability study of loratadine in the prepared suspension (formula a) at elevated temperatures. table (4): degradation rate constants (k) of loratadine in formula a at different temperatures. temperature k (day)-1 35 ºc 0.155 ×10-3 45 ºc 0.33 ×10-3 55 ºc 0.691 ×10-3 25 ºc 0.0749 ×10-3 then by plotting the log of these rate constants versus the reciprocal of the absolute temperatures, the first-order rate constant obtained was equal to 0.0749 ×10-3 (day-1) as shown in figure (9). k0 = k [a] k0 = 0.0749 ×10 -3 day-1 . 0.096 gm/100ml k0 = 0.71 ×10 -5 gm /100 ml. day-1 0.01 0.1 1 3 3.1 3.2 3.3 3.4 1/t × 103 (kelvin1) k × 10 -3 ( da y-1 ) 0.0749 figure (9): arrhenius plot for shelf life estimation of loratadine in the prepared suspension (formula a). then the expiration date of loratadine suspension (formula a) was calculated as follows: t 10 % = 0.1 [a]ο / k0 the expiration date was found to be equal to 3.8 years. the formula show good physical stability, as there was no discoloration, precipitation or any other physical changes after the storage period. the ph of the formula was 5.0 for whole the period. conclusions a stable suspension of loratadine could be prepared and used efficiently using xanthan gum as a suspending agent (formula a), since it provided an easily pourable suspension with no sedimentation with expiration date of 3.8 years. references 1. aulton m.e., pharmaceutics, the science of dosage form design, chirchill livingston, london, newyork, 2002, 2nd ed., chapter 23, p. 334. 2. li p. and luwei zhao l., developing early formulations: practice and perspective. international j. pharm. 2007, 341, 1–19. 3. ansel c., allen v. and popovich g., pharmaceutical dosage forms and drug delivery system, lippincott, williams and wilkins, philadelphia, 2005, 8th ed, chapter 13. 4. remington's "the science and practice of pharmaceutics, mark publishing company, eston, usa, 2005, 23rd ed., vol.2, p. 1119. 5. ruwida m. k., factors affecting formulation and in vitro availability of nalidixic acid from suspensions. thesis for iraqi j pharm sci , vol.17 (2) , 2008 loratadine suspension 25 m.sc degree, college of pharmacy, university of baghdad, 1997. 6. laufen h., yeates r. and zimmermann t., pharmacokinetic optimization of treatment of oral candidiasis with fluconazole studies with suspension drugs. exp.clin. res. 1995, 21(1), 23-28. 7. kumar a., rawlings r.d., beaman d.c., the mystery ingredients: sweeteners, flavorings, dyes, and preservatives in analgesic / antipyretic, antihistamine/decongestant, cough and cold, antidiarrheal, and liquid theophylline preparations. pediatrics. 1993, 91, 927933. 8. buck m., a guide to pharmaceutical excipient (inert ingredients). pediatric pharmacotherapy. 1996, 2 (9). 9. handbook of pharmaceutical excipients, american pharmaceutical association production staff, usa, the pharmaceutical society of great britain, england, 1988, 3rd ed., p.304. 10. chong e., dumont r.j., hamilton d.p. et al., stability of aminophylline in extemporaneously-prepared oral suspensions. j. informed pharmacotherapy, 2000, 2, 100-106. 11. el-sherbiny d.t., el-enany n., belal f.f., hansen s.h., simultaneous determination of loratadine and desloratadine in pharmaceutical preparations using liquid chromatography with a microemulsion as eluent. j. pharm. biomedical analysis 2007, 43, 1236– 1242. 12. škapinاs.d.اandاmatijevíe.,اpreparationا and coating of finely dispersed drugs. loratadine and danazol. j. colloid and interface science 2004, 272, 90–98. 13. kathleen parfitt: martindale: the complete drug reference, 2007, 35th ed., vol.1,اp.ا,62اvol.اіі,اp.1354. 14. cooper and gunn "dispensing for pharmaceutical students", 12th ed., chirchill livingstone, london and newyork,1997, p.103-108. 15. abed al-rahman i.r., jawad f.j et al., preparation of anise and thyme lotion for topical use. iraqi j. pharm. sci. 2007, 16(1). 16. sulayman h.t., formulation of naproxen as a suspension dosage form. thesis for m.sc degree, college of pharmacy, university of baghdad, 2005. 17. lachman l., lieberman h. and kanig j., the theory and practice of industrial pharmacy, leaandfebiger , 3ed edition , 1986, chapter 16, 18 , 480-488, 535 . 18. tempio j.s. and zatz j.l., flocculation effect of xanthan gum in pharmaceutical suspensions. pharmaceutical research, 1980, 69, 1209-1214. 19. megada m.s. and viviane f.n., micellar properties of non-ionic surfactants in relation to their solubility parameters. int. j. pharm. 1988, 42, 1-9. 20. schott h., kwan l.c. and feldman s., the role of surfactants in the release of very slightly soluble drugs. j. pharm. sci. 1982, 71, 1038. 21. bosela a.a, treki m.s., mahdy m.a. and mohammed m.s., effect of suspending agents on the dissolution and bioavailability of ampicillin. bull. pharm. sci. 1991, 14, 6-12. 22. biro e.j.and racz i., examination of dissolution of albendazole from anthelmintic veterinarysuspension. pharm.sci. 1998, 66, 77-90. 23. shah m.b.and sheth b.b., effect of polymers on dissolution from drug suspension. j. pharm. sci. 1979, 65(11), 1618-1623. 24. othman s., hassan m. et al., studies on the adsorption and solubility of nalidixic acid. int. j. pharm. 1988, 41, 197-203. 25. hussein a.a., effect of additives on the in vitro release of mefenamic acid from suspension. thesis for m.sc degree, college of pharmacy, university of baghdad, 1994. 26. khalid r.m., factors affecting formulation and in vitro release of nalidixic acid from suspension. thesis for m.sc degree, college of pharmacy, university of baghdad, 1997. 27. abdullah r.a., studies on the bioavailability of frusemide in suspension dosage form. thesis for m.sc degree, college of pharmacy, university of baghdad, 1983. 28. joseph b., sprowls j.r., american pharmacy, 5th ed., chapter 7, 1974. 29. munayyer f.j., guazzo f., stupak e.i. et al., stabilized antihistamine syrup. u.s. patent 2005, 6,939,550. 30. martin a., physical pharmacy, leaand febiger, philadelphia, london, 2000, 4th ed., chapter 12, 18, 287, 477-486. 31. liu z., li j., nie s., liu h. et al., study of an alginate/hpmc-based in situ gelling ophthalmic delivery system for gatifloxacin. int. j. pharm. 2006, 315, 12–17. 32. ciullo p.a., rheological properties of magnesium aluminum silicate/xanthan gum dispersions. j.soc. cosmet. 1981, 32, 275-285. abstract iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 83 preparation of a new dosage form of metoclopramide hydrochloride as orodispersible tablet # hussam h. tizkam *,1 , alaa a. abdul rassol ** and ahmed a. hussain ** * ministry of health, baghdad, iraq ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad , iraq abstract metoclopramide hcl (mtb) is a potent antiemetic drug used for the treatment of nausea and vomiting. many trials were made to prepare a satisfactory mtb orodispersible tablet using direct compression method.various super disintegrants were used in this study which are croscarmellose sodium (ccs), sodium starch glycolate (ssg) and crospovidone (cp). the latter was the best in terms of showing the fastest disintegration time in the mouth.among the different diluents utilized, it was found that a combination of microcrystalline cellulose ph101 (mcc 101), mannitol, dicalcium phosphate dihydrate (dpd) and glycine was the best in preparing mtb orodispersible tablet with fastest disintegration time in the mouth.the physical parameters of the prepared mtb orodispersible tablet were satisfactory as hardness (4 kg), friability (0.5%) and mouth disintegration (23 sec).the overall results suggest that the prepared formula of mtb as orodispersible tablet could be utilized as a new dosage form for the oral administration. key words: orodispersible, metoclopramide, super disintegrant الخالصة ( يٍ انعقاراث انفعانت فً عالج حاالث انخقٍؤ. ْذِ metoclopramide hclٌعذ عقار انًٍخٕكهٕبزايٍذ ْاٌذرٔكهٕرٌذ ) ( باسخخذاو orodispersible tabletانذراست حخعهق بخصٍُع انًٍخٕكهٕبزايٍذ ْاٌذرٔكهٕرٌذ عهى شكم حبت سزٌعت انخحهم فً انفى ) ( sodium starch glycolate( ٔيادة )croscarmellose sodiumشز.حى اسخخذاو عذة يٕاد يفككت ًْٔ يادة )طزٌقت انكبس انًبا يٍ بٍٍ يٕاد انخخفٍف (. ٌٔعذ األخٍز ْٕ األفضم ألٌ ٔقج ححههّ فً انفى كاٌ اسزع يٍ باقً انًٕاد انًفككت.crospovidoneٔيادة ) ٔانكالٌسٍٍ كاَج ًْ dicalcium phosphate dihydrate)هٕر ٔانًاٍَخٕل ٔيادة )انًسخخذيت، ٔجذ بأٌ يادة انسهٍهٕس يجٓزي انخب ٔكاَج انخصائص انفٍشٌائٍت نحبت انًٍخٕكهٕبزايٍذ االفضم فً حصٍُع حبت انًٍخٕكهٕبزايٍذ ْاٌذرٔكهٕرٌذ سزٌعت انخحهم فً انفى. ايا ٔقج انخحهم فً انفى فٕٓ % ٥٫٠فً حٍٍ كاَج انٓشاشت كغى ٤انحبت صالبتْاٌذرٔكهٕرٌذ سزٌعت انخحهم فً انفى جٍذة فًثال كاَج ثاٍَت. ٢۳ introduction orodispersible tablet is a solid dosage form containing a medicinal substance that disintegrates and/or dissolves rapidly in the mouth (either on or beneath the tongue or in the buccal cavity) without drinking water within up to three minutes. upon placement in the mouth, orodispersible tablets absorb saliva rapidly into the tablet core allowing the super disintegrant to swell, rupture the tablet and liberate the individual components that form solution or suspension, which in turn can be swallowed easily without water. on the other hand, orodispersible tablet can also be swallowed intact as it is; i.e., as if it was a conventional tablet by using water to push it down to stomach (1) .the orodispersible tablets have increased in popularity because consumers, all ages, find them convenient and easy to use. since orodispersible tablets can be taken without water, therefore, bed-ridden, lying, standing, walking, talking, and traveling patients can use them easily any time and anywhere (2) .orodispersible tablet is the proper solution for dysphagia. dysphagia is defined as difficulty swallowing food or liquids, which may be caused by normal physiologic response or as a result of disease state (3) . as a consequence of dysphagia, children and elderly patients do not take their medication as prescribed. it is estimated that as high as 50% of the population has this problem which results in a high prevalence of ineffective therapy (4) .other advantage of orodispersible tablets is fastening onset of action. they disintegrate in the mouth in a period of seconds in comparison to conventional tablets which may need up to 15 min to disintegrate completely in the stomach. furthermore, a dissolution process for water-soluble drugs begins to initiate while the orodispersible tablet still in the mouth. these facts reflect why orodispersible tablets have rapid or ultra rapid onset of action (5) . # based on oral presentation in the seventh scientific conference of the college of pharmacy /university of baghdad held in 26-27 november 2008. 1 corresponding author e-mail : tizkam@yahoo.com received : 2/12/2008 accepted : 24 / 3/2009 mailto:tizkam@yahoo.com iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 83 many side effects are associated with the conventional dosage forms such as tablets and capsules. for example, these dosage forms may lodge esophagus, causing local irritation. when the tablet or capsule disintegrates in stomach, it may release all of its content in the same area, which may cause a gastric irritation especially if the drug is highly water soluble due to the formation of localized high concentration. regarding orodispersible tablet, it disintegrates and/or dissolves in the mouth and diluted subsequently with saliva, so no such side effect can take place.according to the above advantages, orodispersible tablets can be used for most cases, but their benefits can be doubled in certain clinical situations; among them are nausea and vomiting. during nausea and vomiting, it is better for the patient to take orodispersible tablet and not a conventional one due to the following reasons (6) : a. to avoid the stimulation of vomiting center in the cns. this center is stimulated by gi distention which, in turn, is caused by food or fluid intake. if the patient takes conventional tablet, he should use a glass of water to swallow it and hence, vomiting reflux may be initiated, bringing about a new vomiting episode. b. physiologic defense mechanism which makes the patient reluctant to drink. many products of orodispersible tablets have been launched worldwide over the past decade by the most famous drug companies. astrazeneca, glaxosmithklin, eli lilly, pfizer, wyeth, squibb, bristol-myers, schering plough, merck, janssen and organon are examples on these companies (7) . experimental materials: the following materials were used in this study: ccs and cp (al-hekma drug industry, jordan), mtb, ssg and dpd (samara drug industry (sdi), iraq), lactose, mannitol and hydrochloric acid (hcl) (riedal de haen ag seelze hanover, germany), glycine (fluka chemi ag, switzerland), magnesium stearate (mg st) (barbeher, gmbh, germany), meclodin® tablet (sdi, iraq), primperan® tablet (sanofi corp., france). methods: formulation of orodispersible tablet: formulation of control orodispersible tablets (without mtb): different control formulas (without mtb) were prepared and tested to obtain the best formula that disintegrates as fast as possible in the mouth (table 1). all formulas were prepared using direct compression technique. each formula was formulated by mixing all the ingredients (except the lubricant) for 15 min after which the lubricant was added and blended for another 3 min. the final mixture was compressed using a double punch, korsch, tablet machine with a 10 mm flat punch. formulation of mtb orodispersible tablet: mtb was incorporated with the best control formula (with regard to shortest disintegration time in the mouth) to obtain the final mtb orodispersible tablet that subjected subsequently to further investigations. the mtb orodispersible tablet was prepared by the same method mentioned above. physical parameters measurement of the prepared orodispersible tablets: content uniformity: this test was undergone for the prepared mtb orodispersible tablet. the content uniformity was performed by taking ten tablets and assayed individually. the requirement for this test is met if the amount of ingredient in each of the ten tablets lies within the range of 85-115% of the label claim (8) . hardness: the hardness of all the prepared orodispersible tablets (with and without mtb) were measured using monsanto hardness tester. results are expressed as a mean ± s.d (n=3). friability: the friability test was undergone for the prepared mtb orodispersible tablet using roche friabilator for 4 min at 25 r.p.m. by taking ten tablets weighing them all together then placing them inside the tester. after their revolution, they were cleaned from dust and weighed again. the friability was calculated as the percent weight loss. if the reduction in the total mass of the tablets is more than 1%, the tablets fail the friability test (8) . disintegration test: two types of disintegration tests were done for the prepared orodispersible tablets: by using the disintegration apparatus (in vitro test) and disintegration test in the mouth using healthy volunteers (in vivo test). conventional disintegration test (in vitro test): the disintegration time of the prepared mtb orodispersible tablet was determined in different solutions (d.w, 0.1n hcl ph 1.2 and phosphate buffer ph 6.5). in addition, the disintegration time in d.w was determined for meclodin® and primperan® tablets as iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 04 references. disintegration apparatus with a basket rack assembly containing six openended tubes and 10-mesh screen on the bottom was used. a tablet was placed in each tube of the basket and the time for complete disintegration of the six tablets was recorded (8) . disintegration test in the mouth (in vivo test): five healthy volunteers were subjected to the measurement of the disintegration time in the mouth of all the prepared orodispersible tablets (with and without mtb). prior to the test, all volunteers got a detailed briefing on purpose of this test, then they were asked to rinse their mouth with water. then the prepared orodispersible tablet was placed on the tongue and immediately a stopwatch was started. they were allowed to move the orodispersible tablet against the upper palate of the mouth with their tongue and to cause a gentle tumbling action on the tablet without biting on it or tumbling it from side to side. immediately after the last noticeable granule or fragment had disintegrated, the stopwatch was stopped and the time was recorded. the swallowing of saliva was prohibited during the test, and also saliva was rinsed from the mouth after each measurement. to check reproducibility, each volunteer repeated the test three times (9) . dissolution test: the basket method was used to determine the release profile of the drug from the prepared mtb orodispersible tablet. the test was carried out in three different dissolution media which are 900 ml of d.w, 0.1n hcl (ph 1.2) and phosphate buffer (ph 6.5) at 37 ± 0.5 0 c with constant stirring speed of 50 r.p.m for 30 min. in addition, the release profiles of the meclodin® and primperan® tablets (as references) were also determined using 900 ml of d.w at the same test environments. at preset time intervals, 5 ml samples were withdrawn and the filtrate was refluxed back using 5 ml of fresh dissolution medium. samples were filtered through microfilter and analyzed spectrophotometrically at the λmax of mtb (8) . factors affecting formulation: effect of super disintegrants types and concentrations on the disintegration time in the mouth of the prepared control orodispersible tablets: formulas 1-12 (table 1) were utilized to study the effect of super disintegrant type (ssg, ccs and cp) and concentration (4, 8.5, 18 and 40 mg/tablet) on the disintegration time in the mouth of the prepared control orodispersible tablets. formulas 14-16 were prepared to reveal the effect of super disintegrants combination at three different concentrations (1.65, 4.4 and 9.5 mg/tablet) on the disintegration time in the mouth of the prepared control tablets. effect of diluents types and concentrations on the disintegration time in the mouth of the prepared control orodispersible tablets: formulas 17-32 (table 1) were utilized to check the effect of diluent type (mcc101, mcc102, mannitol, lactose, l-hpc, dpd and glycine) alone and as combination of them at different concentrations on the disintegration time in the mouth of the prepared control orodispersible tablets. table (1): composition of the control orodispersible formulas material (mg) formula s s g c c s c p m c c 1 0 1 m c c 1 0 2 m a n n ito l l a c to se l -h p c d p d g ly c in e m g s t t o ta l w e ig h t (m g ) 1 4 (2.5%) 159 1 164 2 8.5 (5%) 159 1 168.5 table (1): continued. iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 04 material (mg) formula s s g c c s c p m c c 1 0 1 m c c 1 0 2 m a n n ito l l a c to se l -h p c d p d g ly c in e m g s t t o ta l w e ig h t (m g ) 3 18 (10%) 159 1 178 4 40 (20%) 159 1 200 5 4 (2.5%) 159 1 164 6 8.5 (5%) 159 1 168.5 7 18 (10%) 159 1 178 8 40 (20%) 159 1 200 9 4 (2.5%) 159 1 164 10 8.5 (5%) 159 1 168.5 11 18 (10%) 159 1 178 12 40 (20%) 159 1 200 13 54 (25%) 159 1 214 14 1.65 (1%) 1.65 (1%) 1.65 (1%) 159 1 164.95 15 4.4 (2.5%) 4.4 (2.5%) 4.4 (2.5%) 159 1 173.2 16 9.5 (5%) 9.5 (5%) 9.5 (5%) 159 1 188.5 17 40 15 9 1 200 18 40 159 1 200 table (1): continued iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 04 material (mg) formula s s g c c s c p m c c 1 0 1 m c c 1 0 2 m a n n ito l l a c to se l -h p c d p d g ly c in e m g s t t o ta l w e ig h t (m g ) 19 40 159 1 200 19 40 159 1 200 20 40 80 79 1 200 21 40 80 69 10 1 200 22 40 80 79 1 200 23 40 59 100 1 200 24 40 40 120 1 201 25 40 20 140 1 201 26 40 20 140 2 1 203 27 40 20 140 23 1 224 28 40 20 140 50 1 251 29 40 20 140 11 1 212 30 40 20 140 23 1 224 31 40 20 140 50 1 251 32 40 20 140 23 55 1 279 effect of ph on conventional disintegration time of the prepared mtb orodispersible tablet: the effect of ph on conventional disintegration time of the prepared mtb orodispersible tablet was done using erweka disintegration apparatus with 0.1n hcl solution (ph 1.2) and phosphate buffer (ph 6.5) as the disintegration media. iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 08 results and discussion effect of super disintegrants types and concentrations on the disintegration time in the mouth of the prepared control orodispersible tablets: table (2) shows that the best super disintegrant type was cp with a concentration of 40 mg/tablet. this fact is represented in formula 12, which showed the fastest disintegration time in the mouth (27 sec). furthermore, to ensure that the super disintegrant critical concentration of cp is 40 mg, another formula (formula 13) was made using 54 mg of cp. it was found that formula 13 disintegrated more slowly than formula 12 and showed mouth disintegration of 29.5 sec. on the other hand, the combination used in formulas 14-16 were not effective in lowering the disintegration time of the orodispersible tablets in the mouth since their disintegration time was relatively high and ranged from 4060 sec. this may be caused by a competition between these super disintegrants on the little amount of water that found in the mouth knowing that 0.35-1.0 ml/min is the total volume of saliva available under normal conditions (10) . table (2): the effect of super disintegrants types and concentrations on the disintegration time in the mouth of the prepared control orodispersible tablets * no composition (mg) disintegration time in the mouth (sec) ssg ccs cp mcc101 mg st 1 4 159 1 44 ± 14 2 8.5 159 1 53 ± 18 3 18 159 1 66 ± 19 4 40 159 1 110 ± 27.3 5 4 159 1 45 ± 13 6 8.5 159 1 45 ± 14 7 18 159 1 42 ± 16 8 40 159 1 79 ± 24 9 4 159 1 58 ± 15.3 10 8.5 159 1 50 ± 20.7 11 18 159 1 39 ± 14.1 12 40 159 1 27 ± 5.1 13 54 159 1 29.5 ± 6.3 14 1.65 1.65 1.65 159 1 43 ± 7.8 15 4.4 4.4 4.4 159 1 40 ± 15 16 9.5 9.5 9.5 159 1 59 ± 18 *results are expressed as mean ± s.d. (n=3) effect of diluents types and concentrations on the disintegration time in the mouth of the prepared control orodispersible tablets: the super disintegrant concentration of cp (40 mg/tablet) was kept constant through out this part of the study to find out the best diluent that may be used for the preparation of control orodispersible tablet as shown in (table 3).mcc acts as auxiliary tablet disintegrant because of its water absorbing capacity (11) . the latter advantage gives the reason for the necessity for the inclusion of mcc in the formulation of orodispersible tablets. practically, no difference was found between mcc 101 (formula 12) and mcc102 (formula 17) and they showed a mouth disintegration of 27 and 28 sec, respectively. besides, combining two grades of mcc (mcc 101 and mcc 102) as in formula 20 gave slower disintegration time in the mouth (37 sec) than each grade alone.table (3) also shows that mannitol was better than lactose in preparing iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 00 tablet with shorter disintegration time in the mouth. the mouth disintegration time for mannitol containing formula (formula 18) and lactose containing formula (formula 19) were 28 and 37 sec, respectively. this difference in disintegration time may be due to the fact that mannitol has slower dissolution kinetics, that is to say; lactose tends usually to dissolve rather than disintegrate, forming a viscous layer on the surface of the tablet which slows down the penetration of water (or saliva) into the tablet core (12) . on the other hand, formula 21 showed slower disintegration time in the mouth (39 sec) than both formulas 18 and 19 indicating that such combination is not effective in lowering the disintegration time in the mouth.formulas 22-25 were utilized to study the effect of combining mcc 101 and mannitol at different concentrations on disintegration time in the mouth. although formulas 22, 24 and 25 showed the same disintegration time in the mouth (26 sec), formula 25 was selected to complete the study because it contains the largest amount of mannitol which in turn, imparts sweet and cool taste on the prepared orodispersible tablet.formulas 26-28 were utilized to study the effect of l-hpc on disintegration time in the mouth of the prepared control orodispersible tablet. it was found that l-hpc prolonged the disintegration time in the mouth at all concentrations, therefore, it can not be used in the preparation of orodispersible tablets.formulas 29-31 were formulated using three different concentrations of dpd. formula 30 was disintegrated faster than formulas 29 and 31 indicating that the best concentration of dpd is that used in the preparation of formula 30 (which is 23 mg/tablet and selected later to compete the study).glycine is used in orodispersible tablet because of its effect as disintegration accelerator. thus, the addition of glycine decreased the mouth disintegration of the tablet from 25 sec (formula 30) to 23 sec (formula 32). in addition, glycine has good flow and sweet taste, therefore, it was used in the past to hide the bitter taste of saccharine (13) .table (3) shows that the best control orodispersible formula (in terms of showing the fastest disintegration time in the mouth) was formula 32 since its disintegration time in the mouth was 23 sec. table (3): effect of diluents types and concentrations on the disintegration time in the mouth of the prepared control orodispersible tablets (with out mtb) * *results are expressed as mean ± s.d. (n=3) f o r m u la c p m c c 1 0 1 m c c 1 0 2 m a n n ito l l a c to se l -h p c d p d g ly c in e m g s t disintegrati on time in the mouth (sec) 17 40 159 1 28 ± 4.9 18 40 159 1 28 ± 8.5 19 40 159 1 37 ± 12 20 40 80 79 1 37 ± 12.9 21 40 80 69 10 1 39 ± 7.4 22 40 80 79 1 26 ± 4.9 23 40 59 100 1 27 ± 6.4 24 40 40 120 1 26 ± 7.8 25 40 20 140 1 26 ± 7.0 26 40 20 140 2 1 34 ± 10.0 27 40 20 140 23 1 41 ± 9.0 28 40 20 140 50 1 43 ± 8.0 29 40 20 140 11 1 38 ± 7.8 30 40 20 140 23 1 25 ± 9.1 31 40 20 140 50 1 44 ± 11 32 40 20 140 23 55 1 23 ± 5.3 iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 04 formulation of mtb orodispersible tablet: formula 32 was the best control orodispersible formula, to which mtb, saccharine sodium and flavor were added. table (4) shows the final mtb orodispersible formula (formula 33) which was subjected to further evaluation tests. table (4): composition of the selected (final) mtb orodispersible tablet (formula 33). physical parameters measurement of the prepared orodispersible tablets: content uniformity: the content uniformity of the prepared mtb orodispersible tablet (formula 33) was complied with bp criteria. no tablet from ten tablets lies out of the range of 85-115% of the label claim. these results indicated that the dosage form had uniform distribution and proper dose of the active ingredient (8) . hardness: the hardness of all the prepared orodispersible tablets (with and without mtb) was kept constant at 4 kg by controlling the compression force between approximately 4-7 tone/cm 2 . friability: the friability of mtb orodispersible tablet (formula 33) was 0.5% which is acceptable according to bp criteria (8) . disintegration tests: conventional disintegration time of the prepared mtb orodispersible tablet (in vitro test): the conventional disintegration test of the prepared mtb orodispersible tablet (formula 33) as well as meclodin® and primperan® tablets (as references) was determined using d.w as disintegration medium. the disintegration time of the prepared mtb orodispersible tablet was only 6 sec. in contrast, the disintegration time of meclodin® tablet was 220 sec while that of primperan® tablet was 120 sec as shown in figure (1). the disintegration time of the prepared mtb orodispersible tablet was also shorter than that obtained by other researchers (14) . figure (1): the disintegration time (conventional) of the prepared mtb orodispersible (formula 33), meclodin® and primperan® tablets using d.w at 37±0.5 0 c (results are expressed as mean ± sem, n=3). disintegration time of the prepared mtb orodispersible tablet in the mouth (in vivo test): the disintegration time of the prepared mtb orodispersible tablet (formula 33) in the mouth was found to be 23 sec (s.d=7.5, n=15). this result is quite satisfactory regarding bp criteria, which states that the disintegration time of orodispersible tablets in the mouth should not exceed 180 sec (8) . not only acceptable in terms of bp, formula 33 had faster mouth disintegration than orodispersible tablets prepared by other researchers (15) and also faster than some of the commercialized products. for example, the disintegration time in the mouth of benadryl® (usa), durasolv® (usa), nimesulide® (switzerland) and nippon® (japan) orodispersible tablets are 40, 45, 30 and 30 sec, respectively (16,17) as shown in figure (2). material amount (mg/tablet) mtb 10 cp 40 mcc 101 20 mannitol 140 dpd 23 glycine 55 saccharine sodium 2 flavor 1 mg st 1 total weight 292 0 50 100 150 200 250 mtb orodispersible tablet meclodin®tablet prim peran®tablet d i s i n t e g r a t i o n t i m e ( s e c ) iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 04 figure (2): the disintegration time in the mouth of benadryl®, durasolv®, nimesulide®, nippon® and the prepared mtb orodispersible tablets (formula 33). dissolution test: the dissolution test for the prepared mtb orodispersible tablet (formula 33) as well as meclodin® and primperan® tablets (as references) was done using 900 ml of d.w as a dissolution medium at 37±0.5 0 c with constant stirring speed of 50 r.p.m for 30 min. table (5) shows the result of the dissolution test. figure (3) indicates that the prepared mtb orodispersible tablet showed faster release rate than both meclodin® and primperan® tablets. the release time (t 75%) of the prepared mtb orodispersible tablet was 10-fold and 5-fold faster than that of meclodin® and primperan® tablets, respectively.statistically, there is highly significant difference (p<0.05) among samples at 1, 2, 5, 10 and 15 min time intervals between meclodin® and the prepared mtb orodispersible tablets. in addition, significant difference was found between samples at 1 and 2 min time intervals between primperan® and the prepared mtb orodispersible tablets. these facts are clarified in figure (4).to accurately confirm the preference of the prepared mtb orodispersible tablet (formula 33) release profile compared to that of meclodin® and primperan® tablets, mathematical expressions had been used. to compare the dissolution profiles of two formulations (test and reference), the difference factor (f1) and similarity factor (f2) are useful. the f1 and f2 functions can be calculated according to the following equations (18) : 100 1 1 1                    n t n t rt ttrt f                        100 1 1log50 5.0 1 2 2 n t ttrt n f indicate clearly the high significant difference among these three formulations. table (5): the percent release of mtb from the prepared orodispersible (formula 33), meclodin® and primperan® tablets in d.w at 37±0.5 0 c * time interval (min) % release of mtb from meclodin® tablet % release of mtb from primperan® tablet % release of mtb from the prepared mtb orodispersible tablet 1 32 ± 2.1 † 42 ± 3.1 † 77 ± 2.3 2 43 ± 2.1 † 55 ± 2.8 † 81 ± 2.3 5 48 ± 0.8 † 84 ± 6.6 88 ± 4.2 10 78 ± 2.1 † 96 ± 3.0 97 ± 1.9 15 95 ± 1.1 † 99 ± 0.5 99 ± 0.2 20 99 ± 0.4 100± 0.0 98 ± 0.8 30 100 ±0.0 99 ± 0.0 99 ± 1.0 * results are expressed as mean ± s.d (n=3). † significant difference (p<0.05) 40 45 30 30 23 0 5 10 15 20 25 30 35 40 45 50 d u ra s o lv ® ta b le t b e n a d ry ® ta b le t n im e s u li d e ® ta b le t n ip p o n ® t a b le t m t b o ro d is p e rs ib le ta b le t m o u th d is in te g ra ti o n iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 04 figure (3): t 75% release of mtb from meclodin®, primperan® and mtb orodispersible tablets (formula 33) in d.w at 37±0.5 0 c figure (4): the release profiles of mtb from the prepared mtb orodispersible (formula 33), meclodin® and primperan® tablets in d.w at 37± 0.5 0 c. effect of ph on conventional disintegration time of the prepared mtb orodispersible tablet: two ways are possible to administer orodispersible tablets. the first one is by putting the tablet in the mouth and waiting it to disintegrate without the aid of water. alternatively, orodispersible tablet can be taken as a conventional tablet by using drinking water to push it down. hence, two possible ph may face the tablet during its disintegration, which are either the neutral ph (6.5) in the mouth or the acidic ph (1.2) in the stomach (19) . therefore, conventional disintegration test was made at these two ph so as to reveal the effect of ph, if any, on disintegration time of the prepared mtb orodispersible tablet and to ensure that this tablet disintegrates rapidly in all phs. the effect of ph on disintegration time of the prepared mtb orodispersible tablet was undergone using erweka disintegration apparatus with 0.1n hcl (ph 1.2) and phosphate buffer (ph 6.5) as the disintegration media. the disintegration time of the prepared mtb orodispersible tablet was 13 sec in ph 1.2 and 16 sec in ph 6.5 as shown in figure (4).it is obvious from these results that the disintegration time of the prepared mtb orodispersible tablet was not affected by changing the ph of the disintegration medium. this fact may be due that the super disintegrant (cp) used in the formulation of the prepared mtb orodispersible tablet does not affect by such change in the ph, therefore, tablet disintegration did not affect significantly by the shift of the ph (19) . references: 1. fu y., jeong s., park k., fast-melting tablets based on highly plastic granules. j. of controlled release. 2005; 109: 203210. 2. edlin m., innovative drug delivery methods should demonstrate value. managed healthcare executive. 2007; 17(2): 37-38. 3. mann g., crary m., pill swallowing by adults with dysphagia. arch. otolaryngol. head neck surg. 2005; 131: 970-975. 4. harada t., narazaki r., nagira s., ohwaki t., aoki s., iwamoto k., evaluation of the disintegration properties of commercial famotidine 20 mg orally disintegrating tablets using a simple new test and human sensory test. chem. pharm. bull. 2006; 54 (8): 10721075. 5. ciper m., bodmeier r., modified conventional hard gelatin capsules as fast disintegrating dosage form in the oral cavity. european j. of pharmaceutics and biopharmaceutics. 2006; 62: 178-184. 6. gan t., franiak r., reeves j., ondansetron orally disintegrating tablet versus placebo for the prevention of postdischarge nausea and vomiting after 10 5 1 0 2 4 6 8 10 12 meclodin®tablet prim peran®tablet mtb orodispersible tablet t 7 5 % ( m in ) 0 20 40 60 80 100 120 0 10 20 30 40 time (min) % m t b r e le a s e d meclodin® tablet primperan® tablet mtb orodispersible tablet iraqi j pharm sci , vol.18 (1) , 2009 orodispersible tablet 48 ambulatory surgery. anesth. analg. 2002; 94: 1199-1200. 7. pfister w., ghosh t., orally disintegrating tablets: products, technologies, and development issues. pharm. technol. 2005; 29 (10): 136-150. 8. british pharmacopoeia. electronic edition, crown inc., london, 2007. 9. harada t., narazaki r., nagira s., ohwaki t., aoki s., iwamoto k., evaluation of the disintegration properties of commercial famotidine 20 mg orally disintegrating tablets using a simple new test and human sensory test. chem. pharm. bull. 2006; 54(8): 1072— 1075. 10. qalaji m., simons e., simons k., fastdisintegrating sublingual tablets: effect of epinephrine load on tablet characteristics. aaps pharmscitech. 2006; 7 (2): 1-7. 11. gohel m., parikh r., brahmbhatt b., shah a., improving the tablet characteristics and dissolution profile of ibuprofen by using a novel coprocessed superdisintegrant: a technical note. aaps pharmscitech. 2007; 8 (1): 1-6. 12. jivraj m., martini l., thomson c., an overview of the different excipients useful for the direct compression of tablets. pstt. 2000; 3 (2): 58-63. 13. fukami j., yonemochi e., yoshihashi y., terada k., evaluation of rapidly disintegrating tablets containing glycine and carboxymethylcellulose. international j. of pharmaceutics. 2006; 310: 101–109. 14. mishra d., bindal m., singh s., kumar s., spray dried excipient base: a novel technique for the formulation of orally disintegrating tablets. chem. pharm. bull. 2006; 54(1): 99-102. 15. shimizu t., nakano y., morimoto s., tabata t., igari y., formulation study for lansoprazole fast-disintegrating tablet. i. effect of compression on dissolution behavior. chem-pharm-bull. 2003; 51(8): 942-947. 16. el-arini s., clas s., evaluation of disintegration testing of different fast dissolving tablets using the texture analyzer. pharmaceutical development and technology. 2002; 7(3): 361–371. 17. lilly e., zyprexa velotab. irish medical times. 2006; 40 (17): 46-47. 18. lopes c., lobo j., pinto j., costa p., compressed matrix core tablet as a quick/slow dual-component delivery system containing ibuprofen. aaps pharmscitech. 2007; 8 (3): 1-8. 19. smart j., lectin-mediated drug delivery in the oral cavity. advanced drug delivery reviews. 2004; 56: 481489. http://web5s.silverplatter.com.ezproxy.library.dal.ca/webspirs/dols.ws?ss=shimizu-t+in+au http://web5s.silverplatter.com.ezproxy.library.dal.ca/webspirs/dols.ws?ss=nakano-y+in+au http://web5s.silverplatter.com.ezproxy.library.dal.ca/webspirs/dols.ws?ss=morimoto-s+in+au http://web5s.silverplatter.com.ezproxy.library.dal.ca/webspirs/dols.ws?ss=tabata-t+in+au http://web5s.silverplatter.com.ezproxy.library.dal.ca/webspirs/dols.ws?ss=igari-y+in+au http://web5s.silverplatter.com.ezproxy.library.dal.ca/webspirs/dols.ws?ss=chem-pharm-bull+in+so iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin doi: https://doi.org/10.31351/vol27iss2pp150-158 150 effects of aldosterone, osteoprotegerin and fibroblast growth factor-23 and some biochemical markers in chronic kidney disease patients (stage ii-iv) among patients with or without cardiovascular events zeyad a. ameen*, 1, shatha h. ali* and ali a. allawi** * department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. ** department of internal medicine, college of medicine, university of baghdad, baghdad, iraq. abstract chronic kidney disease (ckd) is a public health problem and many studies support the link between kidney dysfunction and cardiovascular events. aldosterone has been shown for decades that a plasma aldosterone concentration is elevated in ckd. whilst, osteoprotegerin (opg), after its capacity to protect bone, also osteoprotegerin is elevated in patients with chronic kidney disease (ckd), where it could predict the deterioration of kidney function, cardiovascular, vascular events and all-cause mortality. on the other hand, fibroblast growth factors (fgfs), in patients with ckd, its levels seem to increase progressively as kidney function worsens. the aim of the present study is to assess the correlations between serum osteoprotegerin, aldosterone and fibroblast growth factor-23 levels in patients with chronic kidney disease stage (ii-iv) with and without cardiovascular events. the study includes fifty-nine patients with chronic kidney disease(ckd) and according to ckdepi /creatinine/ 2009 equation to be allocated as stage ii-iv, patients were divided into three groups: group1 (29 patients) with chronic kidney disease(ckd) stage (ii-iv) with cardiovascular events. group2 (30 patients) with chronic kidney disease stage (ii-iv) without cardiovascular event, to be compared with group 3(23 apparently healthy subjects), age and sex matched to that of patients. serum obtained from their blood specimens to measure; glucose, urea, creatinine, calcium, phosphate, sodium, potassium, aldosterone, fgf-23, osteoprotergen. data analysis showed that fasting serum glucose levels of ckd patients (with and without cv disorder) had significantly higher values as compared to the controls (76.5% and 29% respectively). serum aldosterone, fgf-23, opg levels were presented with no significant variation among studied groups with cv events or those without cv events. keywords:ckd, aldosterone, osteoprotegerin,fgf-23. على أألصابة 23 دراسة تأثير أأللدوستيرون, أألوستيوبروتكرين وعامل النمو الليفي الكلوي المزمن مرضى العجز بأألحداث القلبية الوعائية في **عبد المجيد عالوي و علي * علي ، شذى حسين 1*، عباس آمين زياد علوم المختبرية السريرية،كلية الصيدلة،جامعة بغداد،بغداد،العراق.فرع ال* ،جامعة بغداد،بغداد،العراق.طبكلية ال ** الخالصة مرض اعتالل الكلية المزمن هو مشكلة صحية عامة عززت دراسات عديدة وجود ربط بين العجز الكلوي مع االصابات القلبية الوعائية. ان أأللدوستيرون هو هرمون يفرز من الغدة الكظرية ولقد تم التاكد وعلى عقود من الزمن ان تركيز االلدوستيرون في البالزما زمن.أألوستيوبروتيرجين هوة كاليكوبروتين يحمي العظام,لقد تم مالحظة زيادة األوستيوبروترجين في المصل يرتفع في مرض الكلية الم في مرضى اعتالل الكلية المزمن و هذا يمكن أن يتنبئ بانحالل وظيفة الكلى,القلبية الوعائية, وزيادة حاالت الوفاة .على الجانب األخر يرتفع عند مرضى العجز الكلوي بصورة متصاعدة كلما سائت وظيفة الكلى. ان الهدف من هذا والذي 23-عامل النمو الفايبروبالستي في 23-في المصل(, أأللدوستيرون وعامل النمو الليفي أألوستيوبروتكرينالبحث هو دراسة العالقة بين ايض العظام )ممثلة بتركيز مرضى العجز الكلوي للمرحلة الثانية والثالثة والرابعة وعالقتها المحتملة باالحداث القلبية.سجل في ألدراسة تسعة وخمسون مريض لتحديدهم ضمن درجة العجز الكلوي من المرحلة الثانية الى الرابعة, حيث epi /ckd 2009مصابون بالعجز الكلوي وطبقا لمعيار مريض مريض بالعجز الكلوي من المرحلة الثانية الى الرابعة عانى من احداث قلبية. ثانيا: 29ثالثة فئات:اوال: تم تقسيم المرضى الى أناس اصحاء ظاهريا, يماثلون عمر وجنس 23مريض بالعجز الكلوي من المرحلة الثانية الى الرابعة بدون احداث قلبية. ثالثا: 30 ء المرضى لقياس الكلوكوز, اليوريا, الكرياتنين, الكالسيوم, الفوسفيت, الصوديوم, المرضى. المصل المستخرج من عينات دما و الوستيوبروتكرين. تحليل النتائج اوجدت تركيز الكلوكوز في المصل لمرضى العجز 23البوتاسيوم, االلدوستيرون, عامل النمو الليفي ( على ٪29و ٪۷٦٫٥هناك فرق مؤكد بالمقارنة مع أالصحاء) الكلوي للمرضى الذين يعانون أو اليعانون من احداث قلبية بان التوالي.بالنسبة لالوستيوبروتكرين فقط مرضى المرحلة الثالثة أظهروا فرق مؤكد عن أألصحاء وكذلك عن مرضى المرحلة الرابعة للعجز الكلوي. .23 عامل النمو الليفي ، أألوستيوبروتكرين ، أأللدوستيرونمرضى الفشل الكلوي ، لكلمات المفتاحية:ا 1corresponding author e-mail: zeyad1982ph@yahoo.com received: 27/ 8 / 2018 accepted: 12 / 11 /2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp150-158 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin 151 introduction ckd is a public health problem, that results from the decrease in renal function represented by gfr for a period of three months or more which is associated with severe cardiac outcomes and high mortality rate (1). mortality of ckd adjusted for comorbidity, race, gender, age and previous hospitality for ckd patients indicate an obvious decline in rates since 1995 may reflect to some extent the increased recognition of ckd as this confirmed by increasing number of patients carrying the diagnosis(2) . aldosterone is an adrenal hormone that plays a pivotal role in electrolyte and fluid homeostasis and thus control of blood pressure. it has been shown for decades that a plasma aldosterone concentration is elevated in ckd (3) regulatory systems that are normally involved in homeostasis, can take on maladaptive roles. this paradoxical situation has been convincingly illustrated in heart failure. the evidence is more obvious with respect to the beneficial effects of sympathetic and renin-angiotensin-aldosterone system blockade (4) although the success of medications such as angiotensin-converting enzyme (ace) inhibitors was initially attributed to hemodynamic actions unloading the left ventricle, additional direct cellular effects on cardiac remodeling have been discovered (5) . in the case of chronic progressive renal disease, substantial evidence attests to the efficacy of ace inhibitors and, more recently, angiotensin receptor blockers in slowing the progression of both experimental and clinical renal disease (6) . osteoprotegerin (opg) is a glycoprotein, in addition to its capacity to protect bone. it is produced in many tissues including lung, bone, kidney, vasculature, heart, and placenta (7, 8) . it was shown that serum opg is elevated in patients with nondiabetic and diabetic chronic kidney disease (ckd), where it could predict the deterioration of kidney function, cardiovascular, vascular events and all-cause mortality (9) .consistent to these results, it is recently reported that elevated opg is combined with elevated risk of rapid renal decline, renal disease hospitalization, and/or deaths in elderly women (9) . in ckd patient vascular calcification (vc) is recognized as a strong predictor of cardiovascular mortality(10) . on the other hand, fibroblast growth factors (fgfs) that signal through fgf receptors (fgfrs) can regulate a wide domain of biological functions (11). as the central target organ of fgf-23 appears to be the kidney, where tubular phosphate reabsorption and 1alpha-hydroxylase expression are suppressed. these features raised the question which role fgf-23 might play in dialysis patients (ckd stage v),(12). in patients with ckd, fgf-23 concentrations are constitutively elevated and increase progressively as kidney function worsens, likely as an appropriate compensation to help maintain normal serum phosphate concentrations in the face of declining nephron mass. at the level patients reach end-stage renal disease, where fgf-23 concentrations are often increased 100-to 1000-fold above the normal range, whereas serum phosphate concentrations are only modestly increased or even normal (13) . fgf23 is associated with heart failure and mortality in patients with or without chronic kidney disease, because fgf23 is regulated by phosphate (albeit in an indirect manner) and phosphate is correlated with mortality and heart failure, it is possible that this association is not causative(14) . the aim of this study is to assess the correlation between serum aldosterone, osteoprotegerin and fibroblast growth factor 23, glucose levels in patients with ckd (stages ii-iv) in relation to cardiovascular events. subjects and methods this study was carried out at baghdad teaching hospital at baghdad medical city, for the period from october 2017 to february 2018, which enrolled fifty-nine patients with chronic kidney disease from stage ii-iv, under supervision of a specialized physician and the diagnosis was based on serum creatinine, according to ckd-epi creatinine/ 2009 equation (15). fasting subjects were enrolled in this study after excluding ckd patients with a chronic liver disease, a thyroid disorder, or patient on dialysis or had at least one session of dialysis. the study was confirmed by the local research ethics committee and all subjects were provided with a written informed consent to participate in this study. those patients were divided into three groups:  group1: 29 patients with chronic kidney disease stage (ii-iv) with cardiovascular events.  group2: 30 patients with chronic kidney disease stage (ii-iv) without cardiovascular event.  group 3:23 apparently healthy subjects (age and sex matched to that of patients). analysis of serum creatinine, urea nitrogen, calcium, phosphate and alkaline phosphatase were performed using the specific iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin 152 flex® reagent cartridge /dimension ( rxl max) / siemens (usa). whereas, serum aldosterone was measured by elisa kit, purchased by drg instruments gmbh, germany (16). while, human fibroblast growth factor-23 elisa kit (17) and human osteoprotegerin elisa kit was purchased by cusabio biotech co, ltd (china) (18). statistical analysis of data is presented as means ± sd. significance was set at p < 0.05. cases and controls have been compared using either the t-test for independent samples, the pearson coefficient for normally distributed variables. results pooled data of patients when compared to controls indicated that fasting serum glucose (fsg) , urea , creatinine and gfr were elevated except for serum calcium levels were lowered significantly (table 1). according to ckd stages of studied group of patients , serum alkaline phosphatase (alp) levels tend to be significantly elevated through stages ii to iv compared to controls (table 2) , but statistically only values of patients with stage iii showed significant elevation in serum opg. however, data analysis according to the presence of cv disorders in patients compared to that of controls showed that fasting serum glucose levels of ckd patients (with and without cv disorder) had significantly higher values as compared to the controls (76.5% and 29% respectively). furthermore, patients with cv disease were even presented with greater fasting serum glucose as compared to those without cv disorder (table -3). meanwhile, serum urea levels, as well as gfr values, were significantly elevated as compared to the controls. while, serum calcium levels were significantly reduced irrespective to the presence or not of cv disease. but serum creatinine concentrations showed no significant variation between patients and controls. serum aldosterone, fgf-23, opg levels were presented with no significant variation among studied groups (table 3). serum aldosterone levels were not altered among different studied groups according to the stage of ckd when compared to controls (figure 1). serum fgf-23 levels were not significantly different among the different groups of patients nor from that of the control subjects, as shown in figure 2. table1. subjects characteristic parameter group p-value patient (59) control (23) mean sd mean sd age* (years) 60.54 12.49 50.70 16.21 p=0.013 gender(m/f) 37/22 17/6 p=0.337 weight (kg) 80.56 16.42 83.85 13.31 p=0.466 height* (cm) 167.33 5.94 171.75 7.84 p=0.033 bmi 28.66 5.18 28.37 3.72 p=0.835 fsg* (mg/dl) 157.50 68.49 99.83 23.54 p=0.005 urea* (mg/dl) 72.52 29.25 28.70 9.54 p=0.005 creatinine* (mg/dl) 1.98 1.81 0.66 0.16 p=0.001 gfr* (ml/min) 42.83 16.28 111.26 13.69 p=0.005 alkaline phosphatase ( µkat/l) 122.34 56.39 82.70 44.74 p=0.474 phosphate (mg/dl) 4.20 0.79 4.32 0.55 p=0.513 calcium* (mg/dl) 8.64 1.03 9.31 0.52 p=0.005 sodium (meq/l) 140.72 6.31 142.08 4.29 p=0.293 potassium (meq/l) 4.32 0.86 4.53 0.75 p=0.231 cardiovascular disorder 21 p=0.005 smoker 7 5 p=0.979 iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin 153 table 2. serum level of alp and osteoprotegerin through different stages of ckd patients compared to the control. groups parameters stage p-value ii(no.11) iii(no.36) iv(no.12) control (no.23) mean sd mean sd mean sd mean sd alp µkat/l 87.63 c 24.25 126.27 a 63.51 121.09 a 40.79 82.70 44.74 0.010 opg pg/ml 77.11 94.40 112.07 ab 121.52 107.97 130.69 22.79 46.37 0.006 a: significantly different from control, b: significantly different from stage iv , c: significantly different from stage iii table3 .variation in some studied parameters in patients compared to the control subjects according to presence of cv disorder or not. groups parameters patient control p-value cardiovascular event no cv event yes (no=29) no (no=30) (no=23) mean sd mean sd mean sd glucose (mg/dl) 176.29 ab 59.0 9 128.81 a 47.09 99.83 23.54 p=0.005 gfr (ml/min) 41.62 a 13.7 9 48.19 a 18.98 111.26 13.69 p=0.005 urea (mg/dl) 78.64 ab 26.1 1 63.16 a 23.93 28.70 9.54 p=0.005 creatinine (mg/dl) 1.76 0.49 1.65 0.62 0.66 0.16 p=0.359 opg (pg/ml) 105.65 122. 66 93.39 118.90 22.79 46.37 p=0.233 fgf-2 (pg/ml) 50.7 165 105 408 7.8 3.9 p=0.613 aldosterone (pg/ml) 44.80 3.87 53.95 32.42 48.09 20.63 p=0.317 calcium (mg/dl) 8.6 a 0.83 8.49 a 1.03 9.31 0.52 0.005 phosphate (mg/dl) 4.2 0.86 4.12 0.79 4.31 0.55 0.697 a: significantly different from control b: significantly different from a patient with no cvd n: number of subjects iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin 154 figure 1. serum aldosterone levels (pg/ml) among various stages of ckd patients compared to the control group figure 2. serum fgf-23 levels (pg/ml) among various stages of ckd patients compared to the control group correlation studies for studied parameters among various groups as shown in figure -3, mean serum sodium levels are negatively (r= -0.353) correlated with serum opg levels in ckd patients at a significance level of (p=0.019). in figure 4 the mean of serum fgf-23 levels is positively (r=0.482) correlated with serum opg level in ckd patients at a significance level of (p=0.0001). while figure 5 showed that the mean of serum fgf-23 levels is positively (r=0.313) correlated with serum phosphate levels in ckd patients at a significance level of (p=0.032). aldosterone levels in ckd patients are correlated with serum urea levels(r=0.328), at a significance level of p=0.015 (figure 6). figure 3. correlation between serum sodium and opg levels in ckd patients (with and without cv events ) (r= 0.353, p=0.019) figure 4. correlation between serum opg and fgf-23 levels in ckd patients with or without cv events (r=0.482, p=0.0001). iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin 155 figure 5. correlation between serum fgf23 and phosphate levels in ckd patients with or without cv events (r=0.313 , p=0.032) . figure 6. correlation between serum aldosterone and urea levels in ckd patients with or without cv events ( r=0.328 , p=0.015) . discussion effect of studied parameters on cardiovascular events glucose data analysis according to the presence of cv disorders in patients compared to that of controls showed that fasting serum glucose levels of ckd patients (with and without cv disorder) were significantly higher values as compared to the controls (76.5% and 29% ,respectively).furthermore, patients with cv disease were even presented with greater fasting serum glucose as compared to those without cv disorder,(table -3) and this indicates that diabetes is the main risk factor for ckd and cardiovascular disease, so the increase in glucose level will expose patients to the additional risk of cardiovascular event independent of baseline ckd present (19) . osteoprotegerin considering opg levels only patients in stage iii showed significantly higher levels from controls as well as from those in stage iv and this is consistent with what was shown in study in which serum opg is elevated in patients with chronic kidney disease where it could predict the deterioration of kidney function, despite the fact that direct effects of opg on kidneys are still largely not discovered (9) . serum sodium levels were negatively correlated with serum opg level in ckd patients and this may be explained by correlation of ckd progression linked to increasing opg serum level to hyponatremia as in large cohort study of 655,000 veterans with a mean gfr of 50 ml/min/1.73 m2, hyponatremia was seen in 13.5% of patients and this may be due to ckd patients are at additional risk of compromised capacity to dilute or concentrate urine (figure 3) (20) . serum opg levels showed no significant variation between studied groups (table -3); these results disagreed with a study found that in patients with chronic kidney disease vascular calcification contributes to increased cardiovascular (cv) morbidity and mortality, (21) in the present study, the results may be contributed to the small number of patients present in each group. aldosterone serum aldosterone levels in patients compared to the control levels showed no significant variation among patients with different stages of ckd and with that of the controls (figure -1), and this is inconsistent with what has been known for decades that a plasma aldosterone concentration is elevated in ckd (22) . from our point of view, this was expected because all patients whether only have ckd or ckd with cardiovascular disorders are taking anti-hypertensive medications to treat either hypertension (diuretics) or other related conditions like heart failure (ace-i), myocardial infarction (valsartan), angina (amlodipine) or even protecting kidney function by ace-i or arbs and all of which are well known that these medications affect the aldosterone serum levels (23) . renin-angiotensin-aldosterone system which is known to be activated in patients with chf, and in fact plasma renin activity also has been shown to correlate directly with mortality besides that angiotensin ii is known to cause myocardial remodeling and aldosterone may increase myocardial fibrosis and necrosis in the heart,(24) and this was not found in this study as iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin 156 there was no significant difference in aldosterone level between patients with or without cv disease, this may be contributed to all patients whether only have ckd or ckd with cardiovascular events are taking antihypertensive medications to treat either hypertension (diuretics) or other related conditions like heart failure (ace-i), myocardial infarction (valsartan), angina (amlodipine) or event protecting kidney function by ace i or arbs and these medications affect the aldosterone serum levels (23), so the result of the aldosterone level may not reflect reality and because this study is not an intervention study and not interfere with medications the patient is taking. fgf-23 serum fgf-23 levels were not significantly different among the different groups of patients nor from that of the control subjects, as shown in figure -2 and likely as an appropriate compensation to help maintain normal serum phosphate concentrations in the face of declining nephron mass , in the situation where patients reach end-stage renal disease, where fgf-23 concentrations are often increased, whereas serum phosphate concentrations are only modestly increased or even normal (13) .the present study found that increased fgf-23 level is associated with left ventricular hypertrophy, fat mass, and dyslipidemia in elderly patients (25).this positive correlation between fgf23 and mortality also is found in the general population with coronary artery disease(26) . fgf-23 is associated with heart failure and mortality in patients with or without chronic kidney disease, because fgf23 is regulated by phosphate (albeit in an indirect manner) and phosphate is correlated with mortality and heart failure, it is possible that this association is not causative(27) . while in the present result, this was not proven and this may be elaborated by the fact that there was a wide difference between minimum and maximum readings that affect statistics significance despite the fact that when we consider figure -2 for the 1st time, there is an impression of confirmed significant difference between patients and controls data, the reason for that wide difference between minimum and maximum reading can be explained by adopting "in patients with ckd, fgf-23 concentrations are constitutively elevated and increase progressively as kidney function worsens, likely as an appropriate compensation to help maintain normal serum phosphate concentrations in the face of declining nephron mass. by the time patients reach end-stage renal disease, where fgf-23 concentrations are often increased 100to 1000-fold above the normal range, whereas serum phosphate concentrations are only modestly increased or even normal." (13).regarding urea, there was a significant difference between patients with cv disease and patients without cv disease (table -3) and this can comply with another interesting observation in the potime-chf study which is the significant rise in jugular venous pressure as quartile bun values rose. the associated increase in renal venous pressure would increase renal interstitial pressure and activate the renin angiotensin aldosterone system(raas), in addition to increase in cardiac preload and cardiac dilatation is known to be important risk factors for increased death rate in heart failure patients (28) . alkaline phosphatase stage iii and iv patients serum alp levels were significantly higher than that of controls (table -2) , whereas, patients with stage ii serum alp values were significantly lower than that of stage iii patients and this match the new evidence states that serum alp increases in patients with ckd, also in ckd patients without liver disease, alkaline phosphatase can be elevated in high-turnover bone disease, however measuring this readily available and inexpensive biomarker has not been considered as an individual therapeutic target of ckd-mbd(29) . correlation studies serum sodium levels were negatively correlated with serum opg level in ckd patients(r=0.353), (p=0.019), and this may be explained by correlation of ckd progression linked to increasing opg serum level to hyponatremia as in large cohort study of 655,000 veterans with a mean gfr of 50 ml/min/1.73 m2, hyponatremia was seen in 13.5% of patients and this may be due to ckd patients are at additional risk of compromised capacity to dilute or concentrate urine (figure 3) (20) . while serum fgf-23 levels are positively correlated with serum opg level in ckd patients at a significant level (p=0.0001) (figure -4), this supports the theory that both opg and fgf-23 are linked with cardiovascular mortality and this link may be mediated by direct action on the myocardium rather than by indirect effect such as arterial stiffening(30) . serum fgf-23 levels are positively correlated with serum phosphate level in ckd patients at a significant level (p=0.0001) (figure -5) and this can be explained by its relation to hyperphosphatemia, a metabolic disorder that is almost universal in ckd which stimulates the release of the hormone fgf23 iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin 157 from skeletal osteocytes as a role of a negative feedback loop (31) . serum urea levels are positively correlated with serum aldosterone level in ckd patients at significant level (p=0.015) (as shown in figure-6), which could indicate an association between the decline in kidney function and corresponding elevation in aldosterone levels, and this may be due to increase in sodium and water reabsorption correspondent to increase in aldosterone level which lead to increased reabsorption of urea and this can be seen mainly in heart failure patients, so increase sodium and water reabsorption in proximal tubule leads to increase in urea concentration and that promote the passive reabsorption of urea(32) . conclusion osteoprotegerin was significantly increased in stage iii ckd patients as compared to control group, but no significant difference was shown between patients with or without cardiovascular events. meanwhile, there was no significant difference between patients and controls regarding both serum aldosterone and fgf-23 levels. references 1. andrew s. levey,kai-uwe eckardt,yusuke tsukamoto, et al. definition and classification of ckd. kidney int suppl. 2013; 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18:1–9 10. morena m, jaussent i, dupuy am, et al. osteoprotegerin and sclerostin in chronic kidney disease prior to dialysis: potential partners in vascular calcifications. nephrol dial transplant. 2015 ; 30:1345–1356 11. gutiérrez om, mannstadt m, isakova t, et al. fibroblast growth factor 23 and mortality among patients undergoing hemodialysis. n engl j med. 2008 ; 359:584–592 12. ketteler m, biggar ph. as nature did not predict dialysis what we can learn from fgf23 in end-stage renal disease. nephrol dial transplant. 2009; 24:2618–2620 13. gutiérrez om, januzzi jl, isakova t, et al. fibroblast growth factor 23 and left ventricular hypertrophy in chronic kidney disease. circulation. 2009 ; 119:2545–2552 14. ott sm. bone cells, sclerostin, and fgf23: what’s bred in the bone will come out in the flesh. kidney int. 2015; 87:499–501 15. inker la, eckfeldt j, levey as, et al. expressing the ckd-epi (chronic kidney disease epidemiology collaboration) cystatin c equations for estimating gfr with standardized serum cystatin c values. am j kidney dis. 2011; 58:682–684 16. borrebaeck c. recent development in heterogenous enzyme immunoassay. journal of solid phase biochemistry. 1979; 41:57-8 17. kit e human fibroblast growth factor-23 ( fgf-23 ). 23:1–14 18. osteoprotegerin rh, kit opge raybio ® human osteoprotegerin (opg) elisa kit. 2014; 8555:1–12 19. kazancioǧlu r. risk factors for chronic kidney disease: an update. in: kidney int. suppl. 2013; 368–371 20. dhondup t, qian q. electrolyte and acidbase disorders in chronic kidney disease and end-stage kidney failure. blood purif. 2017;43:179–188. 21. svensson m, dahle do, mjøen g, et al. osteoprotegerin as a predictor of renal and cardiovascular outcomes in renal transplant recipients: follow-up data from the alert study. nephrol dial transplant. 2012; 27:2571–2575. 22. hené rj, boer p, koomans ha, et al. plasma aldosterone concentrations in chronic renal disease. kidney int. 1982; 21:98–101. 23. mirza mai, alsiö j, hammarstedt a, et al. circulating fibroblast growth factor-23 is iraqi j pharm sci, vol.27(2) 2018 chronic kidney disease , aldosterone, osteoprotegerin 158 associated with fat mass and dyslipidemia in two independent cohorts of elderly individuals. arterioscler thromb vasc biol. 2011; 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27:727–733. 31. demer ll, tintut y. inflammatory, metabolic, and genetic mechanisms of vascular calcification. arterioscler thromb vasc biol. 2014; 34:715–723 . 32. lindenfeld j, schrier rw. blood urea nitrogen. j am coll cardiol. 2011; 58:383– 385. the effect of brucellosis on lipid profile and oxidant-antioxidants status iraqi j pharm sci , vol.18 (suppl.), 2009 brucellosis on lipid and oxidant-antioxidants 26 the effect of brucellosis on lipid profile and oxidant-antioxidants status amal h. ali *,1 * department of clinical laboratory science , college of pharmacy, university of baghdad, baghdad, iraq. abstract the activation of inflammatory cells, the release of their mediators, and the excessive production of free radicals may affect circulating lipids, but no evidence supports a role for peroxidation in the pathogenesis of brucellosis disease. the aim of this work is to study the effect of brucellosis on lipid profile concentration and oxidant-antioxidant status. we studied 20 brucellosis patients (18 females and 2 males) and 15 healthy controls (age average from 16 to 60 years old). significant differences were noted between the serum lipids of brucellosis patients and control group. mean total cholesterol and low density lipoprotein cholesterol (ldl-cholesterol) concentrations were higher in patients than in control group (mean  se 197.05  44.7, 165  37.6). (p≤0.004, p≤1.59×l0 -11 ) respectively. whereas, high density lipoprotein cholesterol (hdl-cholesterol) and triglyceride show significant lower concentration in patients than control groups (mean  se 15.12  3.4 , 81.22  18.45) . (p≤ 9×10 -6 , p≤ 9.3×10 -7 ) respectively. table l , figure 1.while circulating concentration of glutathione (gsh), soluble antioxidants were higher in brucellosis patients than in control groups (mean  se 2.49  0.4). additionally, increased oxidative stress was observed in the serum of patients with brucellosis as evidenced by higher malondialdehyde (mda) concentrations than in control groups (mean  se 270  9) (figure 2, table 1). in conclusion. disturbances in the lipid profile, and oxidant-antioxidant status occur in brucellosis patients which may increase incidence of metabolic and cardiovascular events. key words: brucellosis disease, lipid profile, oxidant-antioxidant status. الخالصة الؤ التؤاريز ى ؤ ي ؤىن الؤد لكؤه ال يى ؤد يؤدي ان تىشيط الخاليا االلتهابيت وافزاس مسبباتها وتكىيه مشيد مه الجذور الحزة ىىد مزظ الحمؤ الزا ةؤت او ىمؤ مال ؤار لدرااؤت تؤاريز الحمؤ (peroxidation)يليل في ى م االمزاض يدىم ىم يت فىق االكسدة مؤه 81مؤه مزظؤ الحمؤ الزا ةؤت مؤزيط 20تؤم يرااؤت ى تزكيش مكىواث ي ىن وىىامل االكسدة ومعاياث االكسدةر الزا ةت ىؤؤدة لؤؤىى ر ء كمز ؤؤ االصؤؤحا االشؤؤخا مؤؤه 81 ( و اؤؤىت 16 الؤؤ 81 بؤؤيه اىمؤؤار م تتؤؤزاو الؤؤذكىر مؤؤه 2 االوؤؤاو و ىيؤؤو و ؤؤد ان االصؤؤحاء االشؤخا مؤؤ بالمقؤؤراروت الزا ةؤت الحمؤؤ مزظؤؤ ىىؤد الؤؤد ىن مصؤؤل اختالفؤاث اا زيؤؤت فؤؤي تزكيؤش مؤؤ بالمقاروؤؤت الزا ةؤؤت الحمؤؤ مزظؤؤ ىىؤؤد وسؤؤبيا ىؤؤالي كىليسؤؤتيزوا -ldlالكىليسؤؤتيزوا الك ؤؤي والجؤؤرش ي تزكيؤؤش مصؤؤل 10 ( بالتةاقؤ mean  se 197.05  44.7, 165  37.6 االصؤحاء االشؤخا -11 ) ×1.59 p≤ , p≤ 0.004بالتةاقؤ ر ) كىليستيزوا والتزايك يسيزايد ق ت واظحت ومهمت ىىؤد مزظؤ الحمؤ الزا ةؤت بالمقاروؤت مؤ االشؤخا – hdlبيىما ااهز تزكيش الر 10 (mean  se 15.12  3.4 , 81.22  18.45)االصؤحاء -7 ) ×10 -6 , p≤ 9.3 ×(p≤ 9 ر8والزاؤم 8 بالتةاقؤ ر الجؤدوا ( (mean  se 2.49  0.4الي ىىؤد مزظؤ الحمؤ الزا ةؤترىؤ (glutathione)اوؤ خخؤرز و ؤد ان تزكيؤش معؤاي االكسؤدة مؤه اءرررررررؤراكثز مما فؤي االشؤخا االصح (malondialdehyde)ت في تزكيش المدكسد ررررررررررررراية م حىاررررررررررررررافت ال سيررررباالظ (mean  se 270  9) رلمىاقشؤت ؤذا المىظؤىظ ااهؤزث الدرااؤت اخؤتال وىؤد اوتةؤا وسؤبت الؤد ىن 8والجؤدوا 2 الزاؤم) والمدكسداث ومعاياث االكسدة ىىد مزظ الحم الزا ةت مما يدي ال سياية مدرزة في التأيط وتص الشزاييهر introduction brucellosis was firstly described by bruce in 1887 with the discovery of the bacterial species subsequently named brucella melitensis. it is a type of febrile illness characterized by regular remissions or intermissions has been recognized in the mediterranean region for centuries. many names have been applied to it, often relating to the locations in which it was particularly prevalent. (malta fever, mediterranean fever, gibraltar fever or rock fever and undulant fever) are probably the best known (1,2) . brucellosis is the major bacterial zoonosis and an important cause of a serious debilitating disease in human, and abortion and sterility in domestic animals. it is classified into two subdivision of proteobacteria, (small gramnegative and facultative intracellular pathogens) that can multiply within professional and non professional phagocytes (3,4) . the genus brucella consists of seven species according to antigenic variation and primary host, b.melitensis (sheep and goats), b. suis (hogs), b. abortus (cattle), b. ovis (sheep), b. canis (dogs), b. neotanac (wood rats), and b. maris (marine mammals) (5) . 1 corresponding author e-mail : amal hamadah al hadithy 49 @ yahoo.com . received : 17/2/2009 accepted : 24/5/2009 iraqi j pharm sci , vol.18 (suppl.), 2009 brucellosis on lipid and oxidant-antioxidants 27 in contrast to other intracellular pathogens, brucella species do not produce exotoxins, antiphagocytic capsules, thick cell wall, resistance forms or fimbriae and do not show antigenic variation (6) . a key aspect of the virulence of brucella species is their ability to proliferate within professional and non professional phagocytic host cells, therefore successfully by passing the bactericidal effects of phagocytes. their virulence and chronic infections are thought to be due to their ability to avoid the killing mechanisms within host cells (7,8) . lipid profiles include serum cholesterol, serum triglycerides, the fractions of break up of various cholesterol like low density lipoprotein, cholesterol (ldlcholesterol), high density lipoprotein cholesterol (hdl-cholesterol), and the ratio of ldl/hdl (10) . total cholesterol has been found to correlate with cardiovascular mortality in 30-50 years age group. cardiovascular mortality increases 9% for each 10mg/dl increase in total cholesterol over the base line value of l80mg/dl.hdl-cholesterol is a good cholesterol in that of cardiovascular disease decrease with increase of hdl (11) . triglyceride level is a risk factor independent of the cholesterol level, triglycerides are important as risk factors only if they are not part of the chylomicron fraction (12) . lipid profile is known to alter in patients with sever sepsis, but few studies regarding the status of lipid levels in brucellosis are available (13) . glutathione peroxidase are major enzymes that remove hydrogen peroxide generated by sod in cytosol and mitochondria by oxidizing the tripeptide glutathione (gsh) in to its oxidized form (gssg). the glutathione peroxidase that removes hydrogen peroxide contains selenium (essential for catalytic function) at its active site (14) . subjects and methods twenty patients (18 females and 2 males, age average from 16 to 60 years old) with brucellosis were recruited from the clinic of al-khadmia teaching hospital. the diagnosis of the disease was based on standard clinical, histological features, and serological test which is established by positive rose bengal plate agglutination test with antibody titer more than 1/320 (15) . the severity of the disease was evaluated by the severity of abdominal pain accompanied by nausea and vomiting (l6) . gastrointestinal symptoms are noted in 40% of patients with brucellosis with anorexia, weight loss and hepatosplenomegaly are the most common. history of fever generalized myalgia and arthralgia, low back pain and sweating (17) . study was also conducted on fifteen apparently healthy individual as a control groups, blood samples were collected after subjects had fasted for 12 hours overnight, serum was separated immediately by low speed centrifugation and stored pending analysis. lipid profile analysis serum concentration of total cholesterol and triglycerides were measured enzymatically with a commercial kit (boehringer, mannheim montreal). according to the following principle (18) : the cholesterol present in the sample originates a coloured complex. che cholesterol esters + h2o cholesterol + fatty acids chod cholesterol + o2 4-cholestadien + h2o2 pod 2 h2o2 + phenol + 4-aminophenazone quinonimine + 4 h2o triglyceride was measured by gpo-pod enzymatic colorimetric method as the following principle (19) : lpl triglyceride + h2o glycerol + free fatty acids glycero kinase glycerol + atp g3p + adp gpo g3p + o2 dap + h2o2 pod h2o2 + 4-ap + p-chlorophenol quinone + h2o iraqi j pharm sci , vol.18 (suppl.), 2009 brucellosis on lipid and oxidant-antioxidants 28 hdl-cholesterol was measured after precipitation of very low density lipoprotein (vldl) and ldl-cholesterol with phosphotungstic acid, ldl-cholesterol measured by friede wald equation as the following: ldl-cholesterol = total cholesterol − (hdl + trigly/5) when triglyceride levels are less than 400 mg/dl (20) . oxidant and antioxidant analysis serum free malondialdehyde concentrations were measured according to a modified method of chirico (1994) (2i) . proteins were first precipitated with a 10% naso4 solution. the protein free supernatant was then reacted with an isovolume of a 5% thiobarbiuric acid solution at 95°c for 30 min. after the reaction component were cooled to room temperature, pink chromagene was extracted with n-butanol then dried over a stream of nitrogen at 37°c. the dry extract was then resuspended in a mobile phase of kh2po4 – methanol (70:30) before malondialdehyde detection by hplc (21) . glutathione was measured by titze's technique (14) . according to this equation: 2gsh + h2o2 gssg + 2h2o statistical analysis all values were expressed as mean ± se. statistical differences were assessed by student's two tailed t-test. p. values ≤0.05 were considered significant. results lipid profile significant differences were noted between the serum lipids of patients with brucellosis and control group. where mean total serum cholesterol concentration was higher in patients than in control group. total cholesterol was characterized by higher ldlcholesterol in brucellosis patients (mean  se 197.05  44.7, 165  37.6) (p≤0.004, p≤1.59×10 -11 respectively) with a significant lower hdl-cholesterol and triglyceride concentration (mean  se 15.12  3.4, 81.22 18.45) (p≤ 9×10 -6 , p≤ 9.3×10 -7 respectively). (table 1 , figure 1). table 1 : mean±se (mg/dl) of serum lipid profile (total cholesterol, ldl-cholesterol, triglyceride, hdl-cholesterol, malondialdehyde and glutathione) levels (nmol/l) and ldl/hdl ratio in patients with brucellosis and control groups. control n=15 patients n=20 variable mean ± se mean ± se p total cholesterol 190.45 ± 4.3 197.05 ± 44.7 ≤ 0.004 triglyceride 135.1 ± 30.7 81.22 ± 18.45 ≤ 9 ×10 -6 ldl-cholesterol 102.3 ± 23.25 165.45 ± 37.6 ≤ 1.59 × 10 -11 hdl-cholesterol 36.6 ± 8.3 15.12 ± 3.4 ≤ 9.3 ×10 -7 ldl/hdl ratio 2.79 10.9 ≤ 0.001 mda 76 ± 17.5 270 ± 9 ≤ 0.001 gsh 1.9 ± 0.27 2.49 ± 0.4 ≤ 0.005 gsh – px iraqi j pharm sci , vol.18 (suppl.), 2009 brucellosis on lipid and oxidant-antioxidants 29 figure1: mean (±se) serum lipid profile (total cholesterol, ldl-cholesterol, triglyceride, and hdl-cholesterol) levels and ldl/hdl ratio in fasting brucellosis patients (n= 20) and control groups (n= 15) the deference is significant. oxidantantioxidant status circulating concentrations of glutathione, a key endogenous soluble antioxidant, were higher in patients with brucellosis than in control groups (mean  se 2.49  0.4). moreover, increased oxidative stress was observed in brucellosis patients as evidenced by higher malondialdehyde concentrations than in the control group (mean  se 270  9). (table 1, figure 2). figure 2: mean (±se) malondialdehyde and glutathione concentration in serum of patients with brucellosis disease (n=20) and control groups (n=15) the differences are significant p< 0.005 for glutathione, and p<0.001 for malondialdehyde. discussion the present study identified important alterations in the lipid profile and oxidantantioxidants status in patients with brucellosis compared with healthy control groups. our data documented abnormally high serum concentrations of total cholesterol and ldlcholesterol in patients with brucellosis, with concomitant decrease in triglyceride and hdlcholesterol the altered oxidantantioxidant status of brucellosis patients was shown by higher glutathione concentration and higher plasma malondialdehyde concentrations than in control groups. this last finding is consistent with abnormal lipid peroxidation in the circulation of crohn's disease patients (13) .yet the little information is available on the plasma lipid profile and lipoprotein concentrations in patients with brucellosis. although our findings disclose various changes in these biochemical indexes, no obvious associations were noted between these disturbances and selected clinical aspects of the patients, such as disease activity, site of disease, and current medications. further more, none of our patients smoked or consumed alcohol, which are the two factors that may influence antioxidant and lipoprotein status. at this time, it's uncertain whether the hypercholesterolemia noted in brucellosis patients results from a consequence of high carbohydrate consumption. similarly, further work is needed to explore whether the hypotriglyceridemia in patients results from intestinal malabsorption or malnutrition (23, 24) .the excessive local production of soluble mediators from activated monocytes and polymorphonuclear leukocytes has been implicated in mediating the tissues injury (25) . important among these mediators are oxygen free radicals. the chronic inflammatory cells promote an imbalance between oxidant and antioxidant mechanisms at the tissue level and may even compromise circulating antioxidant concentrations (26,27) .however, children with cd (crohn's disease) were reported to have higher plasma antioxidant concentrations than healthy children (28) . in the present study, glutathione is an important intracellular antioxidant, show significant increase in patients than in control groups, consistent with other reports in adults with cd (29,30) .these in consistencies in reports of circulating antioxidants to date may be due to the patient's degree of inflammation, the patient's medication and supplement use, enteric losses, altered mobilization as a result of inflamed mucosa, malabsorption and decrease nutrient 0 50 100 150 200 250 mg/dl total cholesterol triglyceride ldl-cholesterol hdl-cholesterol ldl/hdl ratio control patients 0 50 100 150 200 250 300 350 400 nm ol /l malondialdehyde 0 0.5 1 1.5 2 2.5 3 nm ol/ l glutathione iraqi j pharm sci , vol.18 (suppl.), 2009 brucellosis on lipid and oxidant-antioxidants 30 intake. glutathione is tripeptide helps to detoxify free radicals, peroxides and electrophilic compounds of endogenous and exogenous origin (31,32) .more ever ,serum glutathione level was higher in our patients than in control groups. additional investigation is needed to verify whether the high values of glutathione constitute an adaptive to extrapolate from an experimental model (33) . these high concentrations of glutathione may help prevent oxidation of αtocopherol, and preserve ascorbic acid concentration (34) .free radicals are known to occur as natural byproducts under physiologic conditions. however, their over production has been implicated in the pathogenesis of gut inflammation and intestinal injury in inflammatory bowel disease (28) .oxyradical induced cytotoxicity gives rise to lipid peroxidation through the reaction of free radicals and peroxides with fats in cellular membranes, resulting in malondialdehyde formation (35) . our patients had significantly higher circulating malondialdehyde concentrations than did control groups. it is unclear whether the excess serum malondialdehyde was generated in the patient's blood or was produced by the inflamed intestine and translocated in to the circulation. the presence of malondialdehyde in the circulation may explain the increased formation of glutathione as a mean of preventing oxidative damage. moreover, was showed that proinflamatory cytokines can adversely affect lipoprotein metabolism. for example, tumor necrosis factor α and interlukin-6 were shown to affect intestinal fat handling and lipid metabolism (32, 35) .nevertheless, our data failed to show an effect of disease activity on these indexes, likely because of the relatively small number of patients, further studies are required in which these indexes are analyzed serially in brucellosis patients over time.in conclusion we found substantial abnormalities in the concentrations of plasma lipids, malondialdehyde, and antioxidants of brucellosis patients. moreover, the presence of excessive lipid peroxidation strategies should be elaborated to bolster the antioxidants system in patients with brucellosis. from our results in this study we suggested that brucellosis patients had high risk to atherosclerosis due to the significant increase in plasma total cholesterol, ldl-cholesterol and high level of ldl/hdl ratio, the three indexes of high risk cholesterol mortality. according to the result of this study we can concluded that patients with brucellosis may have an increasing incidence of metabolic and cardiovascular events.further investigation is required to elucidate the mechanisms involved in aforementioned abnormalities. references 1. corbel, m. j. and macmillan, a. p. brucellosis, in toply and wilson’s microbiology and microbial infections; vol.3 bacterial infections (9 th ed) london; arnold. 1998. pp 1884. 2. pappas g., akritidis n., bosilkovski m., tsianos e., brucellosis, n. england j. med.2005; 352; 232536. 3. delrue r. m., matinezlorenzo m., lestrate p. and 7 other authors: identification of brucella spp. genus involved in intracellular trafficking. cell microbial 2001; 3, 487497. (cross reference). 4. deltileux p. g., deyoe b. l. and cheville n. f.: entry and intracellular localization of brucella spp. in vero cells; fluorescence and electron microscopy. vet. pathol 1990; 27, 317328 (abstract). 5. ko j. and splitter g. a.: molecular hostpathogen interaction in brucellosis, current understanding and future approaches to vaccine development for mice and humans. clin.microbial,rev.2003;16,6578(abstract). 6. finlay b. and falkow s.: common themes in microbial pathogenicity. microbial mol. boil. rev.1997; 16, 136169 (abstract). 7. comerci d.j. martinez-lorenzo, m,j., siera, r. gorvel, jp. and ugalde, ra. essential role of the vir b machinery in the maturation of the brucella abortuscontaining vacuole. cell microbial 2001; 3, 159-168. 8. ugalde, r.a: intracellular lifestyle of brucella spp. common genes with other animal pathogens, plant pathogens and endosymbionts microbes infet.1999; i, 1211-1219. 9. pritchard ph. hill, js., lipoproteins in health and disease in., betterdge j., 1999;799-814 ed. london, england; hodder and stoughton. 10. hirano k., kachi s., ushide c., naito m.: corneal and macular manifestations in a case of deficient lecithin cholesterol acyltransferase jp. n. j. ophthalmol., 2004;48, (1);82-4. 11. mertens a., vethamme p., bielick j.k. et al: increased low density lipoprotein oxidation and impaired high density lipoprotein antioxidant defense are associated with increased macrophage homing and atherosclerosis in dislipidemic obese mice.2003; 107 (12), 1640-6. iraqi j pharm sci , vol.18 (suppl.), 2009 brucellosis on lipid and oxidant-antioxidants 31 12. framingham, primary prevention of coronary heart disease: guidance from framingham, circulation. 1998;97, 18761887, american heart association. 13. emile levy, yasmine rizwan, louise thibault, guy lepage. altered lipid profile, lipoprotein composition, and oxidant and antioxidant status in pediatric crohn’s disease am. j. clin. nutr. 2000;71, 807-15. 14. tietz phosphlipid hydroperoxide glutathione peroxidase. text book of clinical chemistry p. 1015. 1999. 15. yagup sky p., detection of brucellae in blood cultures. j. clin. microbiol. 1999;37, 3437-42. 16. alfaraj s., acute abdomen as a typical manifestation of brucellosis: report of two cases and review of the literature, j.r. sc. med. 1995;88, 91-2. 17. madkour monir m., gastrointestinal brucellosis. in madkour monir m., 2 nd ed. berlin. germany, springer village, 150-8. 2001. 18. allan c.c., poon l.s., chan c.s.g, richmond w., fu p.c.: clin. chem. 1974;20. 470. 19. young ds. effects of disease on clinical lab. tests, 4 th ed. aacc. 2001. 20. john bernard henry m.d. clinical diagnosis and management by laboratory methods vol. (1), 230, twentieth ed. 2001. 21. chirco s., high performance liquid chromatography based thiobarbituric acid tests, methods enzymol. 1994;223, 314-8. 22. tietz f., enzymatic method for quantitative determination of nanogram amount of total and oxidized glutathione, anal biochem., 1968;27, 502-22. 23. bousvaros a., zurakowski d., duggan c., et al: vitamins a and e serum levels in children and young adults with inflammatory bowel disease, effect of disease activity. j. pediatric gastroenterol. nutr. 1998;26, 129-35. 24. fernandez-banares f., abad–lacraz a., xiol x., et al: vitamin status in patients with inflammatory bowel disease. am. j. gastroenterol. 1989;84, 744-8. 25. weiss s.j., tissue destruction by neutrophils, n. engl. j. med. 1989;320, 36576. 26. buffinton g.d., doe w.f.: depleted mucosal antioxidant defenses in inflammatory bowel disease. free radic. boil. med. 1995;19, 911-8. 27. kuroki f., lida m., tominaga m., et al: multiple vitamin status in crohn’s disease. correlation with disease activity. dig. dis. sci. 1993;38, 1614-8. 28. siegers c.p., younes m.: clinical significance of the glutathione–conjugating system pharmacol. res. commun.1993;15, 1-13. 29. frei b. stocker r. ames b.: antioxidant defense and lipid peroxidation in human blood plasma. proc. nati. acad. sci. usa; 1998;85, 9748-52. 30. abad–lacruz a., fernandez banares f., cobre e., and 30 et al: the effect of total internal tube feeding on the vitamin status of malnourished patients with inflammatory bowel disease. int. j. vitam. nutr. res. 1988;58, 428-35. 31. mehran m., seidman e., marchand r., gubindo c., levy e. tumor necrosis factorα inhibits lipid and lipoprotein transport by caco-2 cells. am. j. physiol. 1995;269, g 953-60. 32. brunet s., guertin f., thibault l., gavino v., delvin e., levy e. iron salicylate complex induces peroxidation, alters hepatic lipid profile and affects plasma lipoprotein composition. atherosclerosis, 1997;129, 159-68. 33. meister a. glutathione-ascorbic acid antioxidant system in animal. j. biol. chem. 1994;268, 9397-400. 34. murthy s., mathus s.m., varilek g., bishap w., field f.j. cytokines regulate apolipoprotein b secretion by caco-2 cells, differential effects of il-6 and tgf-beta i. am. j. physiol. 1996;270, g 94-102. 35. dolphin d., poulson r., afromonic o., eds. coenzymes and cofactors-glutathione, chemical biochemical and medical aspects, vol. 3, part. a, new york, wily, 1989. iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension doi: https://doi.org/10.31351/vol28iss2pp46-57 46 enhancement of solubility and improvement of dissolution rate of atorvastatin calcium prepared as nanosuspension ahmed h. ali*,1 and shaimaa n. abd-alhammid* department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract atorvastatin calcium has a problem of very slightly aqueous solubility (0.1-1 mg/ml). nanosuspension technique applied to improve atorvastatin calcium dissolution profile. the aim of this study is to formulate atorvastatin calcium as a nanosuspension to enhance its dissolution. thirty one formula were prepared to evaluate the effect of ; polymer type, polymer: drug ratio, speed of homogenization, temperature of preparation and inclusion of co-stabilizer in addition to the primary one; using solvent-anti-solvent precipitation method under high power of ultra-sonication. in this study, five types of stabilizers (tpgs, pvp k30, hpmc e5, hpmc e15, and tween80) were used in three different concentrations 1:1, 1:0.75 and 1:0.5 for preparing of formulations. at the same time, tween80 and sodium lauryl sulphate had been added as a co-stabilizer in selected formulations. atorvastatin calcium nanosuspension was evaluated for particle size, poly dispersity index (pdi), zeta potential, crystal form and surface morphology. finally, the results of particle size analysis revealed reduced nanoparticulate size to 81nm for optimized formula f18 ( containing drug : polymer : co-sabilizer ratio 1:1:0.5 ) with the enhancement of in-vitro dissolution profile up to 90% compared to 44% percentage cumulative release for the reference atorvastatin calcium powder in phosphate buffer ph 6.8 media. furthermore, saturation solubility of freeze dried nanosuspension showed 3.3, 3.8, and 3.7 folds increments in distilled water, 0.1n hcl and phosphate buffer ph 6.8, respectively. the freeze dried powder was formulated as hard gelatin capsules and evaluated according to the usp specifications of the drug content and disintegration time. as a conclusion; formulation of poorly water soluble atorvastatin calcium as nanosuspension significantly improved the dissolution rate of the drug and enhanced its solubility. key words: atorvastatin calcium, nanosuspension, in-vitro dissolution and saturation solubility. تحسين الذوبانية وتحسين معدل انحالل اتورفاستاتين كالسيوم بصيغة جزيئات تعليق نانوي *، شيماء نزار عبد الحميد 1*،احمد حسين علي . العراق ، بغداد ، بغداد ،جامعة الصيدلة كلية ، الصيدالنيات فرع * الخالصة ذوبانية لتحسين الحبيبات النانوية مجم / مل(. تطبيق تقنية 0-1.0من مشكلة ذوبان مائي قليل جدًا )دواء اتورفاستاتين كاليسيوم يعاني . الهدف من هذه الدراسة هو صياغة أتورفاستاتين الكالسيوم كقاعدة نانوية لتعزيز انحالله بسبب زيادة مساحة السطح ، كاليسيوم أتورفاستاتين دواء وفقًا لمعادلة نويز ويتني. البوليمر: الدواء ، سرعة التجانس ، درجة حرارة التحضير وإدراج المثبت نسبة الصيغ لتقييم تأثير ؛ نوع البوليمر ، من تم إعداد واحد وثالثين في هذه الدراسة .المذيبات المضادة للمذيبات تحت طاقة عالية من صوتنة فائقةالترسيب باألساسي ؛ باستخدام طريقة ثبتالمشترك باإلضافة إلى الم ، 0: 0( في ثالثة تركيزات مختلفة 01، وتوين tpgs ،pvp k30 ،hpmc e5 ،hpmc e15، تم استخدام خمسة أنواع من المثبتات ) ددة.وكبريتات لوريل الصوديوم كعامل مساعد في التركيبات المح 01توين إلعداد المستحضرات. في الوقت نفسه ، تمت إضافة 1.0: 0و 0..1: 0 شكل الكريستال ،لسطح الجسيماتزيتا شحنات ال، معامل التشتتلحجم الجسيمات ، الحبيبات النانويةالكالسيوم اتورفاستاتين تم تقييم الُمحسَّنة )التي f18نانومتر لصيغة 00والتشكل السطح. أخيًرا ، كشفت نتائج تحليل حجم الجسيمات عن انخفاض حجم الجسيمات النانوية إلى لتحرر النسبة المئوية ٪ 44مقارنةً مع ٪01( مع تحسين صورة الذوبان داخل المختبر حتى 1.0: 0: 0المثبت المساعد تحتوي على دواء: البوليمر: وسائل اإلعالم درجة الحموضة. 8.0مسحوق أتورفاستاتين الكالسيوم المرجعي في فوسفات العازلة من التراكمي الدواء الذوبانية في في ة أضعاف زياد ..3، و 3.0، 3.3 للمعلق النانوي المجفف بالتجميد المشبعة لذوبان اعالوة على ذلك ، أظهرت قابلية ، على التوالي. 8.0درجة الحموضة ب بفر الفوسفات و عياري 1.0 حمض الهيدروكلوريك، الماء المقطر من محتوى الدواء uspوتقييمها وفقا لمواصفات بالتجميد المجفف النانويمسحوق من الكبسوالت الجيالتين الصلب تم صياغة وقد ووقت التفكك. ن كبير في حل تحسادت الى معلق نانوي قليل الذوبان جدا في الماء بصيغةال صياغة أتورفاستاتين الكالسيوم وبذلك يمكن االستنتاج ان قابليته للذوبان.الدواء ويعزز معدل التحلل المختبري والذوبانية المشبعة. ،معلق نانوي ، : اتورفاستاتين كاليسيوم يةمفتاحالكلمات ال 1corresponding author: e-mail: ahali858@yahoo.com received: 15/12 /2018 accepted: 9 /4 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp46-57 iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 47 introduction formulating poorly water soluble drugs to obtain a suitable bioavailability has become a serious defy in scientific, industrial, and medical issue. limitedness of particle size reduction with conventional approaches made researchers to look for new technologies for size reduction. nanotechnology innovation made this possible; a technique included nanosuspension, nanoemulsion, and several other nominated particulates on nano size range (below 1 micron particle diameter). nanosizing is the modern approach for enhancing dissolution of poorly water soluble drug substances. reducing particle size significance is increasing surface area of contact between solid particulate and dissolving medium (1). inclusion of stabilizers in nanosuspension formulation are of critical significance. an essential role of stabilizers is to compensate the additional non-bound energy of newly revealed surfaces. rigorously wetting drug particulates, hindering ostwald ripening and agglomeration of nano suspension particulates are the most important advantages of adding pharmaceutical stabilizers(2). the widely used techniques of stabilization are steric and/or electrostatic stabilization, both ionic surfactants and charged polymers act as electrostatic stabilizers and non-ionic surfactants act as steric stabilizers (3). the specific characteristic of vitamin e tpgs as solubilizer and stabilizer made to the selection of this surfactant for the nano formulation approach. most widely applied pharmaceutical excipients as polymeric stabilizers are polymeric semisynthetic polysaccharide based non-ionic stabilizer like hpmc e5 and hpmc e15, the synthetic linear polymers polyvinyl pyrrolidone (pvp k30), non-ionic surfactant stabilizers, such as polysorbate (tween80) and vitamine-tpgs (tpgs), which is non-ionic surfactant water soluble derivative of vitamin e. while for electrostatic stabilizers anionic surfactant like sodium lauryl sulphate (sls) used in this study (4). two types of stabilizers could be used for best stability of nano suspension, polymeric and surfactant type stabilizers. the surface energy in drug-polymer binding is unclear and not specifically determined for polymeric semisynthetic polysaccharide based nonionogenic stabilizer (hpmc stabilizers). while it is of greater importance in pvp based synthetic linear polymers (5). stabilizers applied to the formulation of nanocrystal should adsorb to the nanocrystal surface of drug particulate and results in steric stabilization (6). maintenance of stability of nanocrystalline structure is of great significance results from polymer steric stabilization. successful stabilization requires tight and fastened adsorption and prolonged desorption with steric repulsion (7). zeta potential is a critical evaluation element broadly approached to forecast nanosuspension stability. the predicted stability increased as the zp increased ( values greater than +25 mv or less than -25 mv typically have high degrees of stability) (8). lyophilization, broadly applied in drying process of pharmaceutical industry for increasing stability (9-10). atorvastatin is hmg coa reductase (hmgr) inhibitor the rate-controlling stage in intracellular cholesterol synthesis, chemical structure is in figure 1 (11). figure 1. chemical structure of atorvastatin calcium (11). the aim of this study was to formulate atorvastatin calcium nanosuspension by the use of solventanisolvent technique under high power sonicator. an attempt to enhance atorvastatin solubility and improve dissolution rate. materials and method materials atorvastatin calcium was obtained as a gift from pioneer pharmaceutical company /iraq. polyvinyl pyrolidone (pvp k30), vitamin e tocopherol tpgs, hydroxypropyl methyl cellulose e5 and e 15, pvp, sodium lauryl sulphate were purchased from pubchem. company, china. methanol laboratory grade and all other solvents obtained from college of pharmacy/university of baghdad. di-sodium hydrogen phosphate, potassium dihydrogen phosphate. method preparation of atorvastatin calcium nanosuspension by liquid solvent-antisolvent addition atorvastatin calcium nanosuspension was prepared by liquid solventanti solvent precipitation under ultra-sonication technique, in this method, 60mg of atorvastatin calcium was dissolved in 3 ml of organic solvent (methanol) to prepare organic phase (12). on the other hand, aqueous phase prepared by dissolving specific weight of different types of stabilizer ( pvp, tpgs , tween80, hpmc e5 and hpmc e15 ) and co-stabilizers ( sls and tween80 ) in 25ml distilled water, different concentrations prepared of each polymer as mentioned in table 1. then the organic phase on steady rate ( 20 drops / minute) added to aqueous iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 48 solution, with the shearing effect of homogenizer (differing speeds used 1500, and 3000).preparations after homogenization stage, subjected to probe sonication for 20 minutes, with pulse period of 5 sec. off and 10 sec. on; 80% sonication power applied (13, 14). according to the study, nanosuspension were prepared as demonstrated in table 1. table 1. nanosuspension formulations prepared by solvent-antisolvent precipitation under sonication formula no. stabilizer drug : stabilizer ratio stirring speed costabilizer drug : costabilizer ratio temp. particle size nm pdi f1 hpmc e5 1 : 1 1500 0 25 oc 930 0.375 f2 hpmc e5 1 : 0.75 1500 0 25 oc 1364 0.391 f3 hpmc e5 1 : 0.5 1500 0 25 oc 1909 0.339 f4 hpmc e15 1 : 1 1500 0 25 oc 3885 0.428 f5 hpmc e15 1 : 0.75 1500 0 25 oc 4430 0.448 f6 hpmce15 1 : 0.5 1500 0 25 oc 6349 0.462 f7 pvp-k30 1 : 1 1500 0 25 oc 100 0.058 f8 pvp-k30 1 : 0.75 1500 0 25 oc 632 0.191 f9 pvp-k30 1 : 0.5 1500 0 25 oc 771 0.354 f10 tween80 1 : 1 1500 0 25 oc 493 0.015 f11 tween80 1 : 0.75 1500 0 25 oc 551 0.189 f12 tween80 1 : 0.5 1500 0 25 oc 1932 0.550 f13 tpgs 1 : 1 1500 0 25 oc 835 0.316 f14 tpgs 1 : 0.75 1500 0 25 oc 1845 0.216 f15 tpgs 1 : 0.5 1500 0 25 oc 3551 0.348 f16 hpmc e5 1 : 1 1500 tween80 1:0.5 25 oc 685 0.015 f17 pvp-k30 1 : 1 1500 tween80 1:0.5 25 oc 246 0.139 f18 tpgs 1 : 1 1500 tween80 1:0.5 25 oc 81 0.115 f19 hpmc e5 1 : 1 1500 sls 1:0.5 25 oc 166 0.005 f20 pvp-k30 1 : 1 1500 sls 1:0.5 25 oc 147 0.147 f21 tpgs 1 : 1 1500 sls 1:0.5 25 oc 101 0.151 f22 hpmc e5 1 : 1 3000 0 25 oc 366 0.005 f23 hpmc e15 1 : 1 3000 0 25 oc 3375 0.167 f24 pvp-k30 1 : 1 3000 0 25 oc 119 0.005 f25 tween80 1 : 1 3000 0 25 oc 404 0.201 f26 tpgs 1 : 1 3000 0 25 oc 670 0.005 f27 hpmc e5 1 : 1 1500 0 10 oc not nano f28 hpmc e15 1 : 1 1500 0 10 oc not nano f29 pvp-k30 1 : 1 1500 0 10 oc 93 0.047 f30 tween80 1 : 1 1500 0 10 oc 92 0.039 f31 tpgs 1 : 1 1500 0 10 oc 77 0.137 iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 49 saturation solubility determination determination of saturation solubility of atorvastatin calcium is approached by the addition of excess amount of pure powder drug to 10 ml of each media and the tubes maintained in an incubator shaker at 25°c for 48 hr. the dissolved drug quantitatively determined by centrifugation of each sample at 4000 rpm for 20 min and the supernatant filtered through 0.22 micrometer syringe filter and diluted with respective media. the diluted samples, along with an appropriate standard curve, were analyzed by uv spectrophotometer at λ max to determine the dissolved quantity of atorvastatin calcium (15). measurement of particle size and poly dispersity index using nano brookhaven particle size analyzer, average particle size and poly dispersity index were measured (16). influence of types of stabilizers used, according to table 1, to prepare atorvastatin nanosuspension, formulas numbered f1, f4, f7, f10 and f13 were prepared to explore the different results with each polymer type. all polymers used in preparing atorvastatin calcium nanosuspension used in three concentrations to explore the impact of stabilizer concentration on prepared nanosuspension. formulas f2, f3, f5, f6, f8, f9, f11, f12, f14 and f15 formulated to evaluate the effect of type of polymer on particle size and pdi of obtained nanosuspension. incorporation of secondary stabilizer (sls or tween80) on the nanosuspension of atorvastatin calcium was considered one of the most important formulation factors. formulations f16, f17, f18, f19, f20 and f21 demonstrated the influence of this impact. both steric-steric and steric-electrostatic mechanisms investigated at this stage of the study. influence of stirring speed evaluated at two different speeds (3000 and 1500 rpm) on homogenizer. formulae f22, f23, f24, f25 and f26) were tested for study the influence of homogenization speed on nanosuspension formulation speeds. the influence of temperature on the size reduction of nanosuspension particles were evaluated in this study. therefore, formulas f27, f28, f29, f30 and f31 were prepared at 10°c to demonstrate the impact of preparation condition at low temperature. zeta potential evaluation of nanosuspension zeta-potential evaluated by the use of zetasizer (zetasizer nano zs, malvern instrument, worcestershire, uk)(17). the characteristics of surface charge were studied to assess the stability of the prepared nanosuspension. minimum limit needed for electrostatic stabilization of nanosuspension is ± 30 mv, and for steric stabilization of ± 20 mv(1). table 2 zeta potential values for ten formulations (17). table 2. zeta potential values for ten formulations of prepared total 31 formulae. formula no. stabilizer used co-stabilizer mechanism of stabilization speed of stirring temp. particle size pdi zeta potential f17 pvp k30 tween80 steric-steric 1500 rpm 25oc 246nm 0.139 -28.52 f18 tpgs tween80 steric-steric 1500 rpm 25oc 81nm 0.115 -26.11 f20 pvp k30 sls steric-electrostatic 1500 rpm 25oc 147nm 0.147 -34.42 f21 tpgs sls steric-electrostatic 1500 rpm 25oc 101nm 0.151 -34.95 f24 pvp k30 — steric stabilization 3000 rpm 25oc 119nm 0.005 -30.7 f25 tween80 — steric stabilization 3000 rpm 25oc 404nm 0.201 -24.10 f26 tpgs — steric stabilization 3000 rpm 25oc 670nm 0.005 -22.67 f29 pvp k30 — — 1500 rpm 10oc 93nm 0.47 -32.21 f30 tween80 — steric stabilization 1500 rpm 10oc 92nm 0.039 -28.11 f31 tpgs — steric stabilization 1500 rpm 10oc 77nm 0.137 -33.64 in-vitro dissolution profile of nanosuspension volume of nanosuspension equivalent to 20mg atorvastatin calcium was taken into dialysis membrane (mw cutoff 12,000-14,000 hi-media), and fixed to paddle of usp dissolution apparatustype ii applying rotation speed of 75 rpm. then 0.1n hcl and phosphate buffer ph 6.8 were used as dissolution mediums in a volume of 900 ml at 37± 0.5°c. volume of 5ml was withdrawn on time scheduled basis of 5min from starting, then replaced by fresh media of dissolution, up to 60 min. samples were filtered using 0.22 micro filter syringe (0.22 mm). filtrate absorbance recorded by uv analysis versus blank (0.1nhcl and phosphate buffer). depending on a calibration curve, percentage cumulative release was calculated at 242nm and 240 nm, respectively (1820). freeze drying of nanosuspension freeze drying used to convert the optimum formula to dry powder, later for further evaluation. mannitol used as a cryoprotectant at 3% w/v. about 400ml of optimized formula prepared and freeze dried to yield a dry powder for evaluation. four flasks frozen in a deep freezer at -20°c for 24 hr. the frozen flasks were attached to the vacuum port of the device , then four flasks each containing 100ml of nanosuspension, instrument operated till iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 50 dry powder yielded. sublimation of solvent from frozen samples took 48 -72hr (8). characterization of lyophilized powder saturation solubility of lyophilized powder three dissolving solvents water, 0.1 n hcl and phosphate buffer ph 6.8 were used to investigate the saturation solubility of atorvastatin calcium dried nanosuspension powder. an excess amount of drug was added to each test tube containing the above mentioned solvents then were shaken for at least 48hr in a water bath shaker at 25 °c. then filter with a conventional filter paper before spectrophotometric reading for each sample. sample test tubes were centrifuged at 6000 rpm for 15 min. absorbance of supernatant was recorded and a calibration curve was used to determine the amount of drug dissolved in the specific volume of each dissolving media (21). crystallinity and surface morphology x-ray powder diffraction x-ray diffraction analysis demonstrates the proportion of an amorphous form of the end product powder and endorsed by differential scanning calorimetry. differential scanning calorimetry (dsc) differential scanning calorimetric reading performed by perkinelmer dsc thermal analyzer in an operating temperature from 50 to 250 c ranges at rate of heating of 10c/min in nitrogen gas. by computerized dsc software heat of fusing and melting temperature were determined (22). scanning electron microscopy (sem) sem images the surface of solidified sample. information recorded by the signal such as surface topography, outer layer morphology, compositional chemistry, crystallinity of the particulate and electrical conductivity. in-vitro dissolution versus pure atorvastatin calcium powder in-vitro dissolution of nanosuspension powder was done by usp dissolution apparatustype ii. weight equivalent to 20 mg of atorvastatin calcium nanosuspension powder was mixed with diluents. mannitol was used as a diluent, and filled into 0 size hard gelatin capsule. at the same time pure atorvastatin calcium powder 20mg mixed with mannitol and filled into 0 size hard gelatin capsule. 900ml of dissolution medium, phosphate buffer ph 6.8 were prepared, filled into two jars of apparatus. device operated at 75 rpm and 37°c. samples were withdrawn regularly with a time schedule and filtered by 0.22mm filter syringe. at 246nm us spectrophotometer absorbance was measured and the concentration was obtained from a calibration curve (23). stability study the stability study of the selected formula of atr nanosuspension f18 lyophilized powder studied and determined using arrhenius plot. three various temperatures (40, 50 and 60oc) applied for 12 weeks storage. periodically every 2 weeks samples were withdrawn from each temperature and percentage remaining at each capsule was determined by spectrophotometric method, results and discussion all the prepared formulas of atorvastatin calcium nanosuspension as shown in table 1 were revealed variation in particle size affected by factors of formulation specified previously. total five stabilizer types were used in three different concentrations to prepare nanosuspension; two grades of hydroxypropylmethyl cellulose hpmc e5 and hpmce15, polyvinyl pyrrolidone (pvp-k30), tween80 and tocopherol polyethylene glycol succinate (tpgs). for cellulosic derivative stabilizers, hpmc e5 and hpmce15, average particle size obtained is 930 nm for highest ratio of drug: stabilizer, at highest ratio for hpmc e5 only. not all concentrations yielded nano size, while increasing concentration yielded in more size reduction of the formed nano-particulate due to better adsorbance allowed on formed nanosuspension (24). these results made the stabilizer to be selected for further preparations to investigate the impact of concentration, temperature reduction, co-stabilizer addition and increasing shearing force by homogenization at higher shearing speed, 3000 rpm instead of 1500 rpm. hpmc e5 and hpmc e15 used, and two grades both showed the same habit regarding particle size reduction. particle size reduction by the use of polyvinyl pyrrolidone k30 (pvp-k30) measured by particle size analyzer and obtained results revealed that better nano size reduction achieved by this polymer, in comparison to previous one , and highest concentrations of up to 1:1 ratio resulted in lower average particle size which was 100 nm when measure by nanobrook haven particle size analyzer. this stabilizer’s specifications and used concentration implemented critical role in producing a stable nanosuspension (19). polysorbates ( tween80 ) and vitamin e derivative ( tpgs ), two water soluble non-ionic surfactants were used , resulting particle measurements displayed that tween80 and tpgs didn’t yielded nano size particulates at drug to surfactant ratio of 1:0.5 (1932 nm and 3551 nm , respectively). higher drug to stabilizer ratios, drug to stabilizer ratio 1:1, revealed totally different outcomes. particle size obtained with tween80 and tpgs both at high concentrations was 493 nm and 835 nm, respectively. iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 51 nanosuspension stabilized by steric stabilization due to polymer adsorbance onto particulate surface, polymeric type stabilizers used (hpmc and pvp) and non-ionic surfactants (tween80 and vitamin e-tpgs). particle size range obtained from the application of five different stabilizers showed high variability from micron to nano size, this ascribed to the affinity of binding stabilizer molecule to drug molecule(3). efficient prevention of particle size regrowth and agglomeration of precipitated nanosuspension is the result of stabilizer adsorption onto nanoparticulate. from another point, mechanism of nanosuspension stabilization whether steric or electrostatic or mix of both, is of critical significance of particle size stability maintenance (25). figure (2 and 3) show the influence of using different types of stability on release profile. to consider the influence of co-stabilizer addition on the particle size reduction and polydispersity index, formulations f16, f17, f18, f19, f20 and f21 were prepared and evaluated for their particle size and pdi. polymers those act by steric stabilization, hpmc e5, pvp k-30 and vitamin e tpgs were selected as primary stabilizers with two non-ionic surfactant as co-stabilizers tween80 act by steric stabilization and anionic surfactant sodium lauryl sulphate act by electrostatic stabilization. figures (4 , 5 ,6 , and 7) showed the importance of using different types of co-stabilizers on release profile at 0.1n hcl and ph 6.8. figure 2. influence of stabilizer type on release profile of nanosuspension in 0.1n hcl and 37c figure 3. influence of stabilizer type on release profile of nano-suspensions in phosphate buffer (ph 6.8 ) and 37c figure 4. influence of addition of co-stabilizer to pvp k30 on release profile in 0.1n hcl and 37c figure 5. effect of co-stabilizer to tpgs on release profile in 0.1n hcl and 37c figure 6. influence of addition of co-stabilizer to pvp k30 on release profile in phosphate buffer (ph 6.8 ) and 37c figure 7. effect of co-stabilizer to tpgs on release profile in phosphate buffer (ph 6.8 ) and 37c iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 52 hpmc e5 containing preparation as primary stabilizer in formula f1 (930 nm), addition of nonionic surfactant tween80 as a co-stabilizer demonstrated further reduction of formulated particulate size to 685 nm . while with formula f19 inclusion of sodium lauryl sulfate resulted in a dramatic reduction of particle size to 166 nm. this result similar to the effect of inclusion of second stabilizer in the formulation of simvastatin nano suspension by vikram et al. (19). addition of sls as a second stabilizer yielded a favorable nano size particulates with improved solubility and stability. polyvinyl pyrrolidone pvp k-30 containing formulation f7 (100 nm) as a primary stabilizer acts by steric stabilization , while inclusion of tween80 as co-stabilizer in f17 yielded particle size 246 nm , the similar results out of expectations with inclusion of sls anionic surfactant noted, electrostatic stabilizer , that resulted in larger particle size on particle size analysis . this was attributed to that pvp k30 is a short chain polymer, so on addition of another co-stabilizer sls anionic and tween80 non-ionic surfactant polymer efficient adsorbance onto precipitation particulate disrupted by presence of these surfactants. addition of both co-stabilizers exhibited enlargement of nanoparticle size in comparison with the formulations included only the primary polymer (26). regarding the formulation f13 containing non-ionic surfactant vitamin e tpgs derivative, particle size evaluation revealed 835 nm particle sizes within nano range. after inclusion of amphiphilic nature non-ionic surfactant tween80 as co-stabilizer in f18, with a ratio of drug: stabilizer: co-stabilizer of 1: 1: 0.5, particle size reading disclosed 81 nm. addition of co-stabilizer acting with the same mechanism of stabilization as steric stabilizer showed significant size reduction from the primary polymer when used alone (4) . another co-stabilizer tested with the primary stabilizer, which is anionic surfactant sodium lauryl sulfate sls, act by electrostatic mechanism of nano particulate stabilization. f21 formulated and contained both stabilizer and co-stabilizer mentioned above by 1:0.5 ratios. particle size measurement demonstrated formation of nanosuspension of 101nm which was about eight times size reduction, a size of desired nanoparticulate range. the addition of co-stabilizer with the primary one to obtain a mix of steric and electrostatic mechanisms of stabilizer of the precipitated nanosuspension is a successful way to formulate a nanosuspension with desired particle size range (27). binding of nonionic surfactant to nanoparticulate is more tight and adsorption rapid example tween80 and tpgs (28). influence of homogenization speed was mostly clear with hpmc e5 cellulosic polymer, size measurement of formula f1 is 930 nm operated at a rotation speed of about 1500 rpm, while size reduced to 366 at formula f22 on 3000 rpm rotation speed. the improvement in size of formed nanoparticulate explained by viscosity of cellulosic polymers in aqueous solutions reduced as speed of homogenization increased, allowing better stabilizer adsorbance onto precipitated particulate surface exerting better stabilizing effect to nano particulate (29). effect of temperature of procedure is evaluated on the particle size produced on formulation of nanosuspension. formulations f27, f28, f29, f30 and f31 prepared at 10 c and tested for particle size produced and pdi of formulations. formulations containing pvp k30, tpgs and tween80 prepared at low temperature 10c revealed reductions in the size of formed nanosuspension measurements reported to be 93nm, 92nm and 77nm, respectively. results of this evaluation similar to the outcomes of approach by pang et al, they tested the influence of temperature, ripening time and calcination on the morphology and crystallinity of hydroxyapatite nanoparticles (30). particle size measurement for formulations f27 and f28 included hpmc e5 and e15, respectively, revealed contrary results as the formed formulations was turbid and obvious agglomerations were seen by eye. polymer solubilization problem and solution viscosity presented rapid change noted at low temperature of operation. since the agglomerations were observed by eye, no particle size measurement approached for these to formulations this was related to increment in aqueous phase viscosity of the formula (30). characteristics of surface charge studied to assess the stability of prepared nano suspensions. as reported in table 2 estimates of this charge represents nanosuspension stability at macroscopic extent. minimum limit needed for electrostatic stabilization of nano suspensions is + 30 mv, and for steric stabilization of + 20 mv. in-vitro dissolution study of atorvastatin calcium nanosuspension figures (2-9) showed the in-vitro release study in addition to freeze dried f18 and pure atorvastatin calcium powder. while table 3 demonstrates the release data recorded for formulas f17 and f18 which were selected for in-vitro release study at 0.1n hcl and phosphate buffer ph 6.8. iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 53 formula nominated f18 showed highest release of atorvastatin calcium within 60 minutes of study, containing tpg as primary stabilizer and tween80 added as a co-stabilizer. maximum release within 60 minutes reached 81% and 82% for 0.1n hcl and 6.8 phospate buffer, respectively. nanosuspension stabilization is a mix of steric-steric and steric-electrostatic mechanisms. f18 included tpgs which act sterically to stabilize the precipitated nanoparicles by adsorbance onto newly formed nanoparticles. inclusion of tween80, acting by the same mechanism of steric stabilization, reduced the ratio of primary and co-stabilizer needed to obtain the particle size yeilded (81nm) and allowed for better adsorbance onto particulate surface. reduced concentration of used polymers also permitted better desorption from the particulate surface on the start of dissolution process, so resulting in higher release profile of f18. figure 8 and 9 show the in-vitro release profile after freeze drying of selected f18, in comparison to pure atorvastatin calcium powder. results outlined that freeze dried nanosuspension powder release profile reached maximum at 60 minutes about 81% at 0.1n hcl and 89% at ph6.8 phosphate buffer, while pure powder comparative release profile resulted in 35% release at 60 minute in 0.1n hcl and 44% in phosphate buffer ph 6.8. figure 8. release profile of freeze dried atorvasatatin calcium nanosuspension formulation compared to pure atovastatin calcium powder in 0.1n hcl and 37c. figure 9. release profile of freeze dried atorvastatin calcium nanosuspension formulation compared to pure atorvastatin calcium powder in phosphate buffer (ph 6.8 ) and 37c using similarity factor for analyzing release pattern of formulaions f17 and f18 in comparism with pure atorvastatin calcium powder. f2 value higher than 50(50-100) indicates similarities of dissolution profiles, while results of comparison of f17 and f18 with pure atorvastatin calcium powder according to table 3 below revealed values lower than 50 that indicates dissimilarity between dissolution profile of f17 and f18 in regard to pure atorvastatin calcium powder. table 3. similarity factor for f17 and f18 in comparison with pure powder of atorvastatin calcium, highest release profile of studied formulations. formula f in 0.1 n hcl f in 6.8 buffer f17 27.05 38.91 f18 29.41 36.85 lyophilized liquied nanosuspension powder characterized for saturation solubility of atorvastatin calcium in different media including water, 0.1 n hcl and phosphate buffer ph 6.8. the results are demonstrated in table 4. the increase in saturation solubility in distilled water, 0.1n hcl and 6.8 phosphate buffers were 3.3, 3.8 and 3.7µg/ml, respectively. iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 54 table 4. saturation solubility of freeze dried nanosuspension f18 atorvastatin calcium in comparison with pure powder. solvent pure powder saturation solubility mcg/ml freeze dried nanosuspension saturation solubility mcg/ml no. of folds increased water 135 435 3.3 0.1n hcl 167 628 3.8 phosphate buffer ph6.8 228 849 3.7 drug content in freeze dried atorvastatin calcium nanosuspension specified by measurement of absorbance at uv spectrophotometer at 246nm after dissolving in methanol. the value revealed that each 100mg of freeze dried powder contained 6.66mg of atorvastatin calcium x-ray diffraction demonstrated sharpe peaks of freeze dried powder of atorvstatin calcium nanosuspension and maintenance of crystal form of the powder with increased solubility and dissolution pattern. figure (10 and 11) xrd test reports of lyophilized nano suspension. figure 10. xrd characteristic peaks of atorvastatin calcium nanosuspension figure 11. xrd highest intensity peaks of atorvastatin calcium nanosuspension. dsc thermogram of atorvasatin calcium nanosuspension reported in figure 12(a and b). the thermogram represented a specific dropping peak at 159.96c, which is very close to the melting point of pure atorvastatin calcium powder that reported to be 165.5c on electrical melting point instrument (23). to examine surface morphology of prepared nanoparticulates and shape, scanning electron microscope was used. 100.00 200.00 temp [c] -3.00 -2.00 mw dsc 159 .96x100c figure 12. (a) dsc thermogram for f18 atorvastatin calcium nanosuspension iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 55 figure 12. (b) dsc thermogram of atorvastatin calcium pure powder. formulation f18 containing tpgs/tween80 was chosen for scanning electron microscope examination, images at figure 13 (a and b ) demonstrates clear crystal shape and morphology. the same results of crystal nature appeared on xrd and dsc that referred to crystal form of the yielded powder. a b figure13. a and b , illustrates crystal morphology of atorvastatin calcium nanosuspension under scanning electron microscope. after freeze drying of f18 nanosuspension, yielded powder evaluated for release pattern and compared with pure atr calcium powder. release profile of freeze dried nanosuspension f18 revealed 81% and 89% release at minute 60 of starting dissolution apparatus for 0.1n hcl and 6.8 phosphate buffer, respectively. compared to pure atr calcium pure powder release profile, achieved release of maximum 35% and 44% at 60 minute in 0.1n hcl and ph6.8 phosphate buffer as shown in figure 8 and 9, respectively. in 1996, moore and flanner proposed two indices, or fit factors, to compare dissolution profiles in a pairwise fashion . these indices are known as the difference factor (f1) and the similarity factor (f2). to accurately compare two profiles using these fit factors, the dissolution results should be obtained at a sufficient number of time points to adequately characterize the shape of the dissolution profiles. because the mean dissolution profiles are compared using these fit factors, the variability associated with the dissolution results of the individual dosage forms at each time point must also meet certain regulatory criteria. the f1 factor calculates the percent difference between the two dissolution profiles at each time point and is a measurement of the relative error between the two profiles using the following equation: where n is the number of time points, rt is the mean dissolution value for the reference product at time t, and tt is the mean dissolution value for the test product at that same time point. the f1 value is equal to zero when the test and reference profiles are identical and increases as the two profiles become less similar. the f2 factor is a logarithmic reciprocal square root transformation of the sum of squared error and is a measurement of the similarity in the percent dissolution between the two profiles as in the following equation(31). application of similarity factor in comparing release profile of freeze dried nanosuspension with pure atr calcium powder resulted in values lower than 50 in both 0.1n hcl and ph6.8 phosphate buffer as shown in table 5. iraqi j pharm sci, vol.28(2) 2019 atorvastatin calcium nanosuspension 56 table 5. similarity factor f2 for f18 freeze dried nanosuspension in two solvent ( 0.1n hcl and phosphate buffer ph 6.8 ) in comparison with pure drug formula f in 0.1 n hcl f in phosphate buffer ph 6.8 f18 freeze dried 23.85 25.98 stability study figure 14 shows the plot of the logarithm percent remaining of atr calcium versus time (week) at different temperatures. the obtained profiles were linear, indicating that atr calcium degradation follows first order kinetics. the degradation rate consistent (k) at every temperature was resolved from the slope of every line. figure 14. rate of degradation of atorvastatin calcium nanosuspension freeze dried powder stored at three various temperatures. for estimation of degradation rate constant k at 25oc , arrhenius plot, (figure 15), constructed by ploting log k versus temperature in kelvins. so the value of (k25) was calculated (0.0009 week-1 ) figure 15. arrhenius plot of freeze dried atr nanosuspension for determination of degradation rate constant at 25°c (k25). as the decomposition of the drug compliant with first order kinetics, the accelerated expiration date can be computed using the following equation (30) : t90% = 0.1054/k25 where t90% is the time needed for a drug to have 10% of its potency lost, and it was found to be 2.24 years. conclusions formulating atorvastatin calcium conventional powder as nanosuspension significantly (p< 0.05) increased solubility and improved rate of dissolution in the final dosage form. references 1. agrawal et al. nanosuspension: an approach to enhance solubility of drugs. journal of advanced pharmaceutical technology & research, (2011); 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(2015). 27. kronberg, b., dahlman, a., carlfors, j., karlsson, j., & artursson, p. preparation and evaluation of sterically stabilized liposomes: colloidal stability, serum stability, macrophage uptake, and toxicity. journal of pharmaceutical sciences1990; 79(8): 667-671. 28. kocbek, p., baumgartner, s., & kristl, j. preparation and evaluation of nanosuspensions for enhancing the dissolution of poorly soluble drugs. international journal of pharmaceutics2006; 312(1-2): 179-186. 29. müller, r. h., & peters, k. nanosuspensions for the formulation of poorly soluble drugs. international journal of pharmaceutics1998; 160(2): 229-237. 30. pang, y. x., and bao, x. influence of temperature, ripening time and calcination on the morphology and crystallinity of hydroxyapatite nanoparticles. journal of the european ceramic society2003; 23(10): 16971704. 31. dorys argelia diaz et al.dissolution similarity requirements: how similar or dissimilar are the global regulatory expectations? the aaps journal,2015; doi: 10.1208/s12248015-9830-9. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.28(1) 2019 teniposide carbon nanotube doi: https://doi.org/10.31351/vol28iss1pp91-100 91 investigation the improvement of teniposide solubility by incorporation into acid treated carbon nanotube and dispersed by hydrophilic polymer raid m. al abood*,1, mowafaq m. ghareeb* and alaa a. abdulrasool* *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad,iraq. abstract single walled carbon nanotubes (swcnt), as nano-needle structures, are good candidates as nanocarrier delivery systems that can carry drug to the site of action especially for chemotherapy. teniposide is an anticancer drug, but it has a problem of low solubility. in this study, carbon nanotubes properties were improved by pre-functionalized of carbon nanotubes via carboxylation with strong acids and then functionalized through attaching to polymer and copolymer. concurrently, a proper polymer-copolymer combination has been selected by the homogeneity and uv-visible spectroscopy. the best formula (f19) is further studied utilizing fourier transoform infra red, scanning electron microscopy, transmittance electron microscopy, and the solubility of teniposide. the results showed that the best dispersibility obtained in the formula that used polyvinyl alcohol (pva) as polymer and polyethylene oxide (peo) as copolymer at a ratio of 1:1. the solubility of incorporated teniposide in the selected formula is increased by 11.5 fold. accordingly, it can be concluded that the acid treated carbon nanotube and linked by polymer improve the pharmaceutical properties of the carrier and actively increase the potency of drug. key words: acid treated carbon nanotubes, teniposide, hydrophilic polymers. المعالجة حمضيا وتشتيته دراسة تحسين الذوبانية لعقار التنوبوسايد بأدراجه بأنابيب نانوية كربونية ببوليمر مائي رائد مظلوم العبود *،1 ، موفق محمد غريب* و عالء عبد الحسين عبد الرسول * بغداد،العراق. جامعة بغداد، فرع الصيدالنيات ،كلية الصيدلة،* الخالصة قل ، مرشحه جيدة ألنظمة التوصيل النانوية التي يمكنها ن إن األنابيب النانوية الكربونية أحادية الجدار ، والتي تتكون من هياكل إبرية نانوية . قلة الذوبانالتنوبوسايد هو دواء مضاد للسرطان ، لكنه يعاني من مشكلة . عقارالعقار إلى موقع الفعالية الخاص بها وية ثم بالتفاعل مع األحماض القفي هذه الدراسة ، تم تحسين خصائص األنابيب النانوية الكربونية عن طريق إضافة مجموعة كربوكسيلية خالل التجانس من اختيارها تم المناسبة مساعد بوليمر-بوليمر تركيبةتم تفعيل األنابيب الكربونية من خالل اإللحاق بالبوليمر و البوليمر المساعد. والتحليل الطيفي المرئي لألشعة فوق البنفسجية. وفحص امسح الضوئي السطحي والمسح الضوئي الخاللي واألشعة تحت الحمراء باستخدام إضافية دراسات إجراء تم الصيغ ألفضل لتنوبوسايد.ا يةذوبان سبة نأظهرت النتائج أن أفضل تشتت تم الحصول عليه في الصيغة التي استخدمت بولي فنيل الكحول كبوليمر وبولي أثيلين أوكسيد كبوليمر مساعد ب أضعاف . وبناًء على ذلك ، يمكن االستنتاج أن األنابيب النانوية الكربونية 11.5ن للتينيبوسيد في الصيغة المحددة بمقدار . تمت زيادة قابلية الذوبا1: 1 .المعالجة بالحمض والمرتبطة بالبوليمر تعمل على تحسين الخصائص الصيدالنية للناقل وتزيد من فاعلية الدواء . بوليمر مائي، التنوبوسايد، نابيب نانوية كربونية معالجة حمضيااالكلمات المفتاحية: introduction carbon nanotubes (cnts) represent promising tools as drug nanocarriers. when drug delivered to the body intended, carbon nanotubes combine the properties of nanoparticles, both the biocompatibility of liposomes or polymeric nanoparticles and the stability of inorganic nanoparticles such as gold and silica ones. they give the potentiation of modification where different particles involving protein, enzymes nucleic acids and drugs can be affixed (1). one of the important anticancer semisynthetic drugs is teniposide, which belongs to a family of topoisomerase inhibitors. their action is through stabilization of topoisomerase-dna cleavable complexes by hindering the dna relegating step of the catalytic reaction. the problem of teniposide is the solubility, since it is water insoluble (2). 1corresponding author: e-mail: raedmadloom@yahoo.com received: 17/8/2018 accepted: 9/12/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp91-100 iraqi j pharm sci, vol.28(1) 2019 teniposide carbon nanotube 92 carbon nanotubes can be used as a carrier for teniposide, since it could enhance the solubility, improve the penetration to cancer cells, and decrease the side effect by targeting (3). pristine cnt has a smooth surfaces and not soluble in aqueous media that need to prefunctionalization by acid treatment and functionalized with polymer(4). the selection of proper polymers depends on the properties of the polymer. the proper polymers must be nontoxic biocompatible and improve the cnts properties like stability, dispersibility, facile electrochemical polymerization, ion exchange with the medium, good capacity to enhance adhesive coatings, increase the porosity, and drug loading efficiency (5). the polymer is wrapping around cnts body that will improve the aqueous dispersibility of cnt (6). different water-soluble polymers like polyvinyl alcohol (pva) and polyethylene glycol (peg) of different molecular weight (1500, 1000, and400) were used to increase the dispersibility and enhance cell penetration. this work was performed to improve the properties of nanocarriers and consequently of the drug solubility. material and methods materials single-walled carbon nanotube (swcnt) purchased from hongwu international group ltd, china, teniposide from xi'an health biochemical technology co., ltd. china, polyethylene glycol 1500 from thomas baker, india, polyethylene glycol 1000 from sinopharm chemical reagent, china, polyethylene glycol 400 from hi-media laboratories, india, poly vinyl alcohol146000 from sinopharm chemical reagent, china, polyethylene oxide 10000 from sm chemical sdmphd, malaysia. dextran 70 from guanjie chemical reagent, china. all other ingredients used in the study were analytical grade. methods purification and carboxylation of swcnt exactly one gram of swcnt was mixed with 80 ml mixture of h2so4 (18.4m) and hno3 (15.7m) 3:1 in tightly close glass container. the mixture was sonicated in bath sonicator for 6 hours at 70℃ and conserved overnight. the dispersion was centrifuged at 4000 rpm for 15 minutes to remove the large agglomerates and bundles. after sonication and centrifugation steps, the dispersion was filtrated through 0.2 µm polycarbonate filters (whatman ltd.) that diluted with deionized water in order to protect the filter paper from the strong acids, the filtrate continually washed with deionized water by vacuum-assisted filtration until the ph of eluting reached to 7. the filter paper that contains the precipitant was dried using hot air ovens for a night then the swcnt collected for the next step (7–10). preparation of polymer linked cswcnt for polymeric coating of cswcnt, a different polymer (po) and copolymer (copo) were used in order to enhance the dispersing capacity of swcnts and increase drugs loading. the used polymers include polyethylene glycol (peg) of different molecular weight (1500, 1000, and 400) and polyvinyl alcohol 146000 (pva), while polyethylene oxide (peo) or dextran 70 as a copolymer (copo) was used at different ratios as shown in the table (1). preparation of cswcnt-peg a hundred milligram of polyethylene glycol of different molecular weight (1500, 1000, and 400 ) was added to 20ml deionized water in a glass vial and sonicated for 30 minutes in a bath sonicator, and the copolymers were added by the same method at different ratios. a hundred milligram of cswcnt has dispersed in deionized water with sonication for 1 hour, and then the cswcnt dispersion was added to the polymer-copolymer solution and further sonication for 4 hours in a bath sonicator at room temperature. the water in the bath sonicator changed every 30 min to avoid overheating, and the temperature kept at 25℃. the polymer-cswcnt suspension was centrifuged for about 15minute at 4000 rpm; the temperature kept at around 25℃ (room temperature), the supernatant solution was collected and cleared the dispersion from the large particle. the product was filtrated through 0.1 µl filter membrane and washed with deionized water six times to exclude the unconjugated polymer. finally, the filtrate was dried by hot oven overnight, and the powder was collected in a tightly closed container for the next step (11,12). preparation of cswcnt-pva three grams of pva were weighed in a closed container and solubilized by 30 ml of deionized water at 70 ℃ with continuous shaking in a water bath with a shaker for 10 hours; the solution obtained left to cool into the room temperature. the copolymer linking was done by the same method at different ratios with ultrasonication. in a conical flask, 20 mg of cswcnt dispersed in 5 ml of deionized water by sonication for 1hour to give dispersion. the cswcnt dispersion was added to the pva/copolymer solution drop by drop with continuous stirring at different ratio and sonicated for 3hour in order to prepare cswcnt/polymer nanocomposites. the functionalized swcnt (cswcnt-polymer) suspension was centrifuged for about 15minute at 4000 rpm; the temperature kept at around 25 ℃(room temperature), the supernatant solution was collected to liquidation the dispersion from the large particles. then, the mixture filtrated to eliminate the unconjugated polymer. finally, cswcnt-polymer composites were cast in petri dishes and left to dry by hot oven for 12 hours(13,14). iraqi j pharm sci, vol.28(1) 2019 teniposide carbon nanotube 93 incorporation of teniposide into cswcntpolymer a fifty milligram of teniposide was added into 20ml acetone in closed container with continuous shaking in shaker water path for around 4 hours at 30℃ until clear solution was obtained. in 100 ml tightly close container, 50 mg of cswcntpolymer was dispersed in 50 ml deionized water by sonicator for 1 hour. the solution of teniposide was added to the dispersion of cswcnt-polymer. the mixture was further sonicated for 6 hours at 30℃, conserved overnight in petri dishes until the acetone was evaporated from the mixture in hot oven at 60 ℃ for 3hour. the teniposide-cswcnt-polymer dispersion was filtrated and dried (15–17). table (1) composition of polymer-copolymer used for functionalization of cswcnt symbol polymer co polymer ratio polymer: copolymer ratio of cswcnt: polymer-copolymer f1 peg1500 peo 10:1 1:1 f2 peg1500 peo 10:1 1:5 f3 peg1500 dextran 10:1 1:1 f4 peg1500 dextran 10:1 1:5 f5 peg1500 nil 10 1:1 f6 peg1500 nil 10 1:5 f7 peg1000 peo 10:1 1:1 f8 peg1000 peo 10:1 1:5 f9 peg1000 dextran 10:1 1:1 f10 peg1000 dextran 10:1 1:5 f11 peg1000 nil 10 1:1 f12 peg1000 nil 10 1:5 f13 peg400 peo 10:1 1:1 f14 peg400 peo 10:1 1:5 f15 peg400 dextran 10:1 1:1 f16 peg400 dextran 10:1 1:5 f17 peg400 nil 10 1:1 f18 peg400 nil 10 1:5 f19 pva peo 10:1 1:1 f20 pva peo 10:1 1:5 f21 pva dextran 10:1 1:1 f22 pva dextran 10:1 1:5 f23 pva nil 10 1:1 f24 pva nil 10 1:5 characterization of polymer-cswcnt qualitative dispersibility measurement of polymercswcnt the dispersion of polymer-cswcnt was qualitatively measured by two approaches; the visual observation of homogeneity, as well as centrifugation for three hours at 6000 rpm and the formula that can pass the centrifugation step was recorded (18,19). quantitative dispersibility measurement for polymer-cswcnt the dispersibility percent of the formulation was calculated by measuring the absorbance by uvvisible spectrophotometer at 880nm of 0.015 µg/ml concentration before and after centrifugation at 6000 rpm for 15 min according to the following equation(20) : 𝐷𝑖𝑠𝑝𝑒𝑟𝑠𝑎𝑏𝑖𝑙𝑖𝑡𝑦 % = 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑝𝑜𝑙𝑦𝑚𝑒𝑟 − 𝐶𝑆𝑊𝐶𝑁𝑇 𝑎𝑓𝑡𝑒𝑟 𝑐𝑒𝑛𝑡𝑟𝑖𝑓𝑢𝑔𝑎𝑡𝑖𝑜𝑛 𝑐𝑜𝑛𝑐. 𝑜𝑓 𝑝𝑜𝑙𝑦𝑚𝑒𝑟 − 𝐶𝑆𝑊𝐶𝑁𝑇 𝑏𝑒𝑓𝑜𝑟𝑒 𝑐𝑒𝑛𝑡𝑟𝑖𝑓𝑢𝑔𝑎𝑡𝑖𝑜𝑛 × 100% characterization of selected teniposidecswcntpolymer formula fourier transform infrared spectroscopy the fourier transform infrared spectroscopy (ftir) was performed on a wavelength range from 400 nm to 4000 cm-1 to identify the structural changes that may happen to the samples in comparison with the references (21–24). iraqi j pharm sci, vol.28(1) 2019 teniposide carbon nanotube 94 scanning electron microscopy the sample for scanning electron microscopy (sem) was measured using vega3 sem transmission electron microscopy thin section of the sample was prepared by a process known as ultramicrotomy, sections of 50‐70 nm thickness were collected on metal mesh 'grids'; and tem studies were performed(25,26). solubility study of teniposide in optimized formula saturated solutions were prepared by adding excess of teniposide to deionized water, and shaking by the shaker water bath for 48 hours at 25°c under constant vibrations. similarly, saturated solutions were prepared by adding excess of teniposide cswcnt-polymer to deionized water for the purpose of comparison. the saturated solutions were shaked by the shaker water bath for 48 hours at 25°c under constant vibrations. the dispersions were filtered through a 0.45 µm filter, diluted suitably and analyzed by hplc. three determinations ± s.d were carried out for each sample to calculate the solubility of the drug results and discussions calibration curve of teniposide by hplc the calibration curve of teniposide was performed with mobile phase acetonitrile: water (36:64) by series of dilution of teniposide in acetonitrile and the retention time was found to be 8.9 minutes. when the area under the peak was plotted against the concentration, straight line was obtained as shown in figure 1. the square correlation coefficient (r2) was found to be (0.9983), which give a signal of good linearity. figure(1) hplc a) the chromatogram of teniposide b) teniposide calibration curve in hplc preparation of teniposide formulation purification and carboxylation of swcnt the synthesized swcnt in all techniques available until now produces swcnt with impurities. these impurities were produced during the production of pristine swcnts. the most common impurities involved metallic catalysts and amorphous carbonaceous species. these impurities disturb the physicochemical properties of swcnts, so it is necessary to purify swcnts before using it. moreover, owing to the firm vander waals interactions, swcnts usually trapped with each other, that make them insoluble in most solvents. this makes them undesired in pharmaceutical peruses since intact swcnts are not readily reachable to the biological system (27). to overcome these limitations, chemical oxidation, in order to be purified and prefunctionalized by oxidation to give carboxyl groups on surfaces, is the most predominant strategy applied to swcnts. moreover, oxidation can be viewed as a bridge between physical and chemical properties of swcnts(28). oxidation of swcnts surfaces is generally a starting point in different functionalization proficiencies that enhances dispersibility in various solvents and increases the intensity of the interface amongst swcnt and the polymers. oxygen incorporated with carbonyl, carboxylic acid, and hydroxyl components introduced easily by acid treatment. moreover, it can purify the swcnts by dissolving the impurities in strong acids and filtrate to get rid of impurities with the strong acid (29). the exact mechanism of carboxylation might not be too obvious, since cnts have aromatic properties as well as aliphatic properties, on the principle ground these are not ideal sp2 hybridizations but that they have something in between sp3 and sp2 hybridization related to curve surface and so display varied chemical reactions but are difficult to execute because of stability. the expected mechanisms for carboxylate group formation were that the concentrated nitric and sulfuric acid both could act as oxidizing agents. some carbon compounds ( aliphatic type ) got nitration on carbon center by nitric acid and these nitro group isomerized to isonitro group, i.e. from cno2 to c-ono and the later on hydrolysis readily form c-oh that further oxidized to form an aldehyde and finally carboxyl group as shown in scheme 1 (30,31). iraqi j pharm sci, vol.28(1) 2019 teniposide carbon nanotube 95 scheme (1) scheme for expected carboxylation steps of carbon nanotubes for these reasons and in order to get a lower degree of entanglement, a mixture of concentrated hno3 and h2so4 was used at ratio of 3:1 for oxidation of swcnt(32). qualitative dispersibility measurement of cswcnt-polymer the uniform dispersion system of cswcnt-polymer in aqueous phase resembles the main challenge, since the improvement of dispersion could lead to ameliorated mechanical, electrical, and optical properties of dispersion. in addition, the interfacial adhesion between the cnt and (po-copo) is one of essential factors in formulation that could be evaluated according to dispersion degree. several factors can influence the dispersibility like the degree of carboxylation, type of po-copo, ratio of po-copo to cswcnt, ratio of po to copo, particle size, electrical properties of interfaces, bundle formation and aggregation, interfaces and wettability all the factor detect whether the dispersion is homogenous or not (33). different pocopos with different molecular weight and ratios were scanned in different parameters in order to optimize the proper cswcnt-polymer to further study as shown in table 2. from table 2, the consistency of formulation was varied from homogeneous to non-homogenous and this depends on the dispersibility of the system. the high dispersibility will support the dispersing particle, and support the system from breakoff (34,35), as shown in figure 2a. (a) (b) figure (2) a) the dispersibility of homogenous (f19) and non-homogenous (f3) prepared cswcnt-polymer b) results of prepared cswcnt-polymer centrifugation test; a) not pass b) pass. table(2) consistency and separation tendency after centrifugation of cswcnt-polymer symbol consistency after sonication and overnight preserved separation tendency after centrifugation for 3 hour f1 homogenous pass f2 homogenous pass f3 non-homogenous not pass f4 homogenous pass f5 non homogenous not pass f6 homogenous pass f7 homogenous pass f8 homogenous pass f9 homogenous pass f10 homogenous pass f11 homogenous pass f12 homogenous pass f13 non-homogenous not pass f14 homogenous pass f15 non-homogenous not pass f16 non-homogenous not pass f17 non-homogenous not pass f18 non-homogenous not pass f19 homogenous pass f20 homogenous pass f21 homogenous pass f22 homogenous pass f23 homogenous pass f24 homogenous pass iraqi j pharm sci, vol.28(1) 2019 teniposide carbon nanotube 96 for formulations that contained peg1500 as polymer, most formulations show homogeneity. and by decreasing the molecular weight of peg to 400, the homogeneity disturbed this might be due to the lower steric hindrance effect of polymer on the formulation with small molecular weight peg and the bundle of swcnt start to format (36,37). the presence of peo as copolymer will enhance the dispersibility due to the increment in the functional groups and this will lead to better dispersibility. for formulations that contained pva as a polymer, the results of most samples are homogenous. this might be due to the structure of pva that tends to stretch around the cswcnt, and effective interfacial interaction might occur between pva and cswcnt (38,39). the use of dextran as a copolymer had not improved the consistency which may be due to decreasing in the interfacial area between cswcnt and polymer that lead to decrease in lyophilized area around the swcnt and enhance tube-tube bundling (40). for peo copolymer, the consistency might improve this effect due to the formation of stable blends between pva and peo owing to van der waals type bond that gives steric hindrance preventing bundle formation (41). the results of homogeneity estimation obtained visually showed in figure 2a. in order to confirm the homogeneity of cswcnt-polymer in aqueous media, the efficiency of sonication step, and the distinction whether the cswcnt-polymer aggregated or dispersed. the centrifugation step is necessary at 6000 rpm for one hour and the formulation that can pass this step without separation into two layer will subjected to further studies as explained in table 2 and figure 2b (42,43). quantitative dispersibility measurement of cswcnt-polymer the quantitative measurements of dispersibility were performed and the results are shown in table 3. from table 3, it was observed that the formulations f2, f8, f12, f19, and f20 exhibited a high dispersion percent. in this type of dispersion, the concentrated area between cswtnts and polymers behaved in a lyophilic manner due to the helical wrapping of hydrophilic polymers around the swcnt body that were thermodynamically favored through the removal of the hydrophobic interfaces between swcnt sidewall and the aqueous medium. it suggests a stable wrapping of polymer around the swcnt where the bundle aggregation are very little compared to formula f3 and f23 which established less dispersion percent due to the conception that the polymer in these formulation showed less ability to coat the swcnt and prevent the bundle formation (37,44–46). table (3) the dispersibility percentage of prepared polymer linked cswcnt symbol dispersibility efficiency percent ± s.d. f1 84.00±1.63 f2 97.00±0.82 f3 33.00±1.63 f4 84.33±1.25 f5 25.33±1.25 f6 86.33± 1.25 f7 84.00± 1.41 f8 93.00±1.40 f9 84.33±2.05 f10 85.00±2.16 f11 86.67±2.05 f12 92.67±1.25 f13 27.00±2.94 f14 84.00± 1.41 f15 14.00± 1.63 f16 25.67± 4.50 f17 13.67±1.25 f18 17.00±0.82 f19 95.33±0.94 f20 97.33±1.25 f21 89.67±2.36 f22 96.33±1.25 f23 77.33±1.25 f24 89.00±1.41 characterization of selected teniposide-cswcntpolymer fourier transform infrared spectroscopy the ftir performed to pure teniposide as well as to teniposide in final selected formulation and bands are shown in table 4. it was found that the characteristic bands are similar in both spectra. table(4)ftir bands for reference teniposide and measured. type of band reference(47) value measured value carbonyl stretch vibration of the stained trans lactone ring 1785 cm-1 1780 cm-1 oh stretch vibration of the phenolic 3540 cm-1 3533 cm-1 oh stretch vibration of the sugar oh 3400 cm-1 3398 cm-1 aromatic bands 1605 and 1485 cm-1 1609 and 1484 cm-1 c-o stretch vibrations 1230 and 1100 cm-1 1233 and 1087 cm-1 from the ftir of teniposide-cswcnt(pva-peo), the (1233and1093) cm-1 c-o stretching vibration belongs to teniposide as well as iraqi j pharm sci, vol.28(1) 2019 teniposide carbon nanotube 97 the pva. for (1480 and 1382) cm-1, the band belongs to c-c, which is found in all component of formulation. the 1638 cm-1 belongs to aromatic c=c stretching bands, for swcnt as well as for teniposide. the 1729 cm-1 is the stretching c=o carbonyl group, which indicated the presence of carboxylic acid results from carboxylation of swcnt and the broad area between 3240 cm-1 and 3640 cm-1 belongs to oh groups of phenolic and alcoholic oh that are widely found in teniposide in formulation as well as carboxyl group and pva (47). the results of ftir study indicate that the presence of teniposide characteristic peaks as well as the carboxyl group and alcoholic oh, which might confirm the stability of the formulation. scanning electron microscopy for teniposide cswcnt-(pva-peo) the sem images for teniposide-cswcnt(pva-peo) are shown in figure 3. the sem images of teniposide-cswcnt-(pva-peo) had presented the morphology of final formulation in which the nanostructures were seen and the polymer was clearly coated the carbon nanotube. it contained large number of nano chunks due to the affixations of teniposide on ends either of carbon nanotubes or within the body of tubes. the images established that the needle-like body of carbon nanotubes was not affected by the drug attachment (48). figure (3) sem images of teniposide-cswcnt-(pva-peo) transmission electron microscopy for cswcnt(pva-peo) the morphology of teniposide-cswcnt(pva-peo) after dilution was studied using tem and the results are shown in figure 4, which affirmed that the formula f19 was well dispersed without any bundles or aggregation. in the tem image, the cnt formula diameter was within 35-40 nm; the teniposide was appeared as dark spots point in the ends of cnts as well as inside the nanotubes. for the polymer, it appears not too dark as teniposide and spreads around parts of nanotubes. figure (4) tem images for teniposide-cswcnt-(pva-peo) solubility studies of teniposide in optimized formula the solubility is an important parameter in the formulation of poorly soluble drugs. this experiment was done to assess the saturation solubility of pure teniposide and compared it to the incorporated teniposide in carbon nanotubes to find out the improvement of solubility that had achieved from the cswcnt-polymer. the result of saturation solubility study of teniposide in water was found to be 0.0527±0.00061 mg/ml while the solubility in teniposideswcnt-(pva iraqi j pharm sci, vol.28(1) 2019 teniposide carbon nanotube 98 peo) was found to be 0.6081±0.01163mg/ml, which around 11.5 folds improvement. this result reveals that pure teniposide has very low water solubility. this fact is expected since teniposide is a bcs class ii drug (low solubility, high permeability) with very low water solubility. similarly, the cswcnt-(pva-peo) was expected to increase the solubility of teniposide since the drug is molecularly dispersed within the cswcnt-polymer after functionalization, the functionalization of swcnt is usually required to make them soluble, and also to attach other chemical moieties. these modifications can alter the swcnt properties, such as the solubility, besides, the size of cwcnt is of nanoparticle size and of a hallow structure which greatly increases the drug surface area exposed to the dissolving media (49). conclusions from the results of this study, it is concluded that the uses of carbon nanotubes can improve the activity of drug through the increments of the solubility as well as the enhancement penetration to the cells, which could be considered as a promising nanocarrier drug delivery system for anticancer drug therapy. the pre-functionalization by carboxylation with h2so4/hno3 3:1 ratio and functionalization by polymer/ copolymer mixture linking have improved the physical properties of carbon nanotubes like dispersibility and prevention of bundles of cnt formation. the improvement of dispersibility in this study was the highest in those samples that contain pva as polymer and peo as copolymer (f19). 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-------------------------------------------------------------------------------------------------------------- a modified organic method for preparation of ibuprofen microcapsules as a sustained release solid dosage form issraa rasheed abed alrahman alaa a. a.rasool and fatima a. tawfiq department of pharmaceutics, college of pharmacy, baghdad university, baghdad-iraq received: 02.01.2001 accepted: 02.03.2002 abst ract: microencapsulation is used to modify and retard drug release as well as to overcome the unpleasant effect (gastrointestinal disturbances) which are associated with repeated and overdose of ibuprofen per day. so that, a newly developed method of microencapsulation was utilized (a modified organic method) through a modification of aqueous colloidal polymer dispersion method using ethylcellulose and sodium alginate coating materials to prepare a sustained release ibuprofen microcapsules. the effect of core : wall ratio on the percent yield and encapsulation efficiency of prepared microcapsules was low, whereas , the release of drug from prepared microcapsules was affected by core: wall ratio ,proportion of coating material and presence of additive(peg4000). the 2:1 core : wall ratio was compared (in weight equivalent to 300mg and 600mg drug) with fenbid ® spansule capsule 300mg and balkaprofen ® tablet 600mg respectively. it was found that the release of drug from selected ratio and balkaprofen ® tablet was more or less similar(p 0.05) .this sustained release ratio was encapsulated in weight equivalent to 300mg drug to be administered once daily (600mg) as two capsules as the reference. the capsules were stable within 6 months of storage at room temperature. :الخـالصـة م��ن تس�تخدم طریق�ة التغلی�ف المجھ�ري للس��یطرة عل�ى س�رعة انط�الق العق�ار والتغل��ب عل�ى المظ�اھر الس�لبیة الناتج�ة ی��دة مح��ورة ع��ن الطریق��ة المائی��ة الغروی��ة وس��میت ل��ذلك ت��م اس��تحداث طریق��ة جد , المتك��رر الی��ومي لعق��ار االیب��وبروفین الس��تعمال االثیل والجینی�ت الص�ودیوم كم�واد مغلف�ة لتحض�یر كبس�والت مجھری�ة لالیب�وبروفین باستخدام سلیلوزبالطریقة المحورة العضویة كبس�والت المجھری�ة الناتج�ة إلى المادة المغِلفة للعقار على كف�اءة التغلی�ف ونس�بة ال لوحظ ان تأثیر نسبة المادة اللبیة. بطیئة التحرر سلیلوز ( المغِلفة تغییر نسبة المادة, من تلك الكبسوالت المجھریة یتأثر بنسبة المادة اللبیة إلى المادة المغِلفة تحرر العقارلكن .قلیل العقار مع الفنبد ملغم من٦٠٠, ملغم ٣٠٠بوزن یعادل) ١:٢(تم مقارنة النسبة . ٤٠٠٠البولي أثیلین كالیكول واضافة مادة) االثیل ملغم عل�ى الت�والي حی�ث أش�ارت النت�ائج إل�ى أن تح�رر العق�ار م�ن النس�بة ٦٠٠ملغم و حبوب بالكابروفین ٣٠٠سبانسول كبسول األیبوبروفین في ملغم من ٣٠٠لذلك تم احتواء تلك الكبسوالت المجھریة بأوزان تعادل , كان متشابھًا وحبوب بالكابروفینالمختارة كانت ثباتی�ة المستحض�ر .صلب یصرف كبسولتین قي الیوم لیعطي تحرر بطيء للعقار مشابھا لحبوب بالكابروفینتیني كبسول جال .الغرفةأشھر في درجة حرارة ٦البالغة فترة الخزنعالیة خالل iraqi j. parma. sci., vol.14, 2005 2 -------------------------------------------------------------------------------------------------------------- introduction: ibuprofen is a non steroidal anti–inflammatory drug used in painful and inflammatory conditions. the conventional administration of this drug may produce different side effects as gastrointestinal disturbances and patient compliance problem due to multiple dose per day .this drug is absorbed from git and peak plasma concentration occurs about 1-2 hours after ingestion ,90-99% of the ibuprofen is bound to plasma protein and plasma half life is about two hours (1). sustained release drug delivery system provides a slow release of drug over an extended period of time that can be obtained by matrix or reservoir devices formulation. microencapsulation (reservoir system) offers a good tool to overcome some of undesirable effects, since it has many advantages, such as; mask the bitter taste of the drugs (erythromycin (2) , colistin salfate (3) ), control the release of the drug (nifidipine (4) ) and separate the incompatible drugs. the mechanism of action is through micro packaging technique of small particles by deposition of a thin coating material around these particles. there are different methods adapted to pharmaceutical use, these include: air suspension, phase separation coacervation, interfacial polymerization, electrostatic spraying , spray drying ,spray congealing and diffusion exchange (5) . many drugs were encapsulated through these techniques like indomethacin (6) , aspirin (7) , chloramphenicol(8) ,isoniazid(9)and nicardipine hcl(10). the aim of the present work is to prepare a sustained release ibuprofen microcapsules as a capsule dosage form using a modified organic method and compared it with the references fenbid ® spansule capsule300mg and blakaprofen ® tablet 600mg), in addition, studying the factors that affect the releasing behavior of ibuprofen from the prepared microcapsules . materials and methods: ibuprofen, powder supplied by sdi, iraq; chloroform, (bdh. chemical ltd, pool england); polyethylene glycol (peg 4000), calcium chloride and sodium alginate, (judex laboratory reagents, sudbury, middlesex, england). fenbid ® spansule capsule 300mg skf company, england and balkaprofen ® tablet 600mg apm company, jordan. preparation of microcapsules: a modified organic method (scheme 2) was obtained through a modification of an aqueous colloidal polymer dispersion method (scheme 1) (11) .the drug was dispersed in an aqueous solution of na–alginate (2% w/w) which is previously heated to 40ºc and added to the dispersion system of ethyl cellulose (20% w/w) in chloroform which was kept at room temperature. iraqi j. parma. sci., vol.14, 2005 3 -------------------------------------------------------------------------------------------------------------- then by dropping the bubble–free dispersion through a modified separatory funnel into gently agitated calcium chloride solution (1%), a gelled beads were obtained and separated after 1– 2 minutes by filtration with vacuum then rinsed with distilled water. finally, the beads were dried at 60ºc over night. different core: wall ratios 2:1, 1:1 and 1:2 were prepared utilizing the same method. 2:1 core: wall ratio in weight equivalent to 300mg ibuprofen(517mg) was used to prepare different formulas as shown in table(1) in order to study the effect of peg4000 addition and ethylcellulose proportion on the releasing profile in comparison with the sustained release reference fenbid® spansule capsule 300mg. table (1) different formulas of 2:1core to wall ratio ibuprofen microcapsule prepared by a modified organic method in addition, the release of ibuprofen from the same ratio in weight equivalent to 600mg drug (1.03g) was compared with the sustained release reference balkaprofen® tablet 600mg. on the other hand, the same weight of the selected ratio was compressed directly without additives into tablet dosage form using two compression forces (10 × 10 3 psi and 12 × 10 3 psi). also, formula 2 in weight equivalent to 600mg ibuprofen (1.07g) was compressed directly without additives into tablet dosage form using 10 × 103 psi compression force in order to investigate the effect of peg4000 addition and ethylcellelose proportion on the release profile of compressed tabletted microcapsules. then 1.03g of the same ratio was filled into 2 capsules of size “0” ,each of 517mg microcapsules weight, to study the release of drug from prepared capsules in comparison with balkaprofen ® tablet 600mg.encapsulated ibuprofen microcapsules were stored at room temperature for 6 months for preliminary stability study. formula no. ibuprofen (gm) sodiumalginate (gm) ethyl cellulose (gm) peg4000 (gm) original formula 1 2 3 4 8 8 8 8 8 2 2 2 2 2 2 2 1.8 1.6 1.6 0.2 0.2 0.4 iraqi j. parma. sci., vol.14, 2005 4 -------------------------------------------------------------------------------------------------------------- microcapsules properties: the properties of all prepared microcapsules were determined in order to evaluate the effect of studied variables through estimation of the following: actual drug loading encapsulation efficiency = x100 theoretical drug loading actual wt. of microcapsules gained percent yield = x 100 theoretical wt. of microcapsules dissolution test: it was done for all prepared microcapsules and dosage forms using usp dissolution apparatus in 900 ml phosphate buffer of ph 6.8 at 37oc and a constant stirring speed of 150 rpm. na-alginate (2% w/w) ethylcellulose in water (30% w/w) 40 o c room temp. mix dropping in calcium chloride solution (1%) dry in oven at 60 o c scheme-1-aqueous colloidal polymer dispersion method na-alginate (2% w/w) ethyl cellulose in chloroform (20% w/w) 40 o c room temp. mix dropping in calcium chloride solution (1%) dry in oven at 60 o c scheme-2-preparation of ibuprofen microcapsules by a modified organic method iraqi j. parma. sci., vol.14, 2005 5 -------------------------------------------------------------------------------------------------------------- results and discussion: a modification of aqueous colloidal polymer dispersion method was utilized for microencapsulation of ibuprofen, through replacing the water required for ethylcellulose dispersion (30%) with chloroform (20%) as shown in scheme(2), which provided a good percent yield and encapsulation efficiency of ibuprofen microcapsules. this is due to the immiscibility of chloroform with water present in drug and sodium alginate suspension as well as its acceptable viscosity, which provided optimum conditions for microcapsules preparation. the microencapsulation data of ibuprofen are summarized in table (2). core:wall ratio percent yield microencapsul-ation efficiencies (%) t50% release (min) 2:1 1:1 1:2 97.5. 97.5 95 87 92 90 125 185 225 table(2)effect of varying core:wall ratio on ibuprofen microcapsules prepared by a modified organic method it appears that no significant differences (p>0.05) in the encapsulation efficiency and percent yield were obtained for all prepared ratios (11) , whereas, a more sustained action microcapsules was obtained as the concentration of colloid increased (figure1). figure(1): effect of varying core:wall ratio on the release property of ibuprofen microcapsules prepared by a modified organic method using ph 6.8 phosphate buffer at 37ºc. iraqi j. parma. sci., vol.14, 2005 6 -------------------------------------------------------------------------------------------------------------- this may be related to the hydrophobic nature of ethylcellulose wall forming material, in addition to the water insoluble barrier (calcium alginate) which is formed from sodium alginate and calcium chloride gelling agent(12). since 2:1 core: wall ratio provided a higher release profile of ibuprofen (t50% 120mins) in comparison with other sustained release ratios 1:1 and 1:2, so that, many trails were done to prepare an acceptable sustained release solid dosage form compared with the sustained release reference fenbid ® spansule capsule 300mg (t50% 24mins), (however this product requires a dosing frequency of two times per day), utilizing this ratio to prepare different formulas by addition of peg4000 and reduction of ethylcellulose proportion . microencapsulation efficiency of all prepared formulas were decreased as the proportion of peg4000 increased and ethylcellulose decreased (table (3)). formula no. with reference naalginate (gm) peg4000 (gm) ethylcellulose (gm) microencapsulation efficiencies (%) percent yield % of drug release after 60 mins original formula 1 2 3 4 fenbid ® capsule 2 2 2 2 2 0.2 0.2 0.4 2 2 1.8 1.6 1.6 87 85 84 79 77 97.5 92 92 92 94 28 39 45 37 36 95 table(3)effect of peg4000 addition and ethylcellulose on microcapsules properties prepared by a modified organic method in comparison with fenbid ® capsule. which is attributed to the interaction between peg4000 and ibuprofen that dissolves the drug in the preparation medium (13), on the other hand ethylcellulose reduction produced unequal amount of wall forming materials with sodium alginate that provided some uncoated drug crystals on the surface of microcapsules dissolved in the dissolution medium which resulted in larger release proportion of ibuprofen at the first 60 minutes compared with untreated ratio as shown in figure (2). iraqi j. parma. sci., vol.14, 2005 7 -------------------------------------------------------------------------------------------------------------- figure(2): effect of addition of peg4000 and ethylcellulose on the release profile of 2:1 core:wall ratio of ibuprofen microcapsules prepared by a modified organic method in comparison with fenbid ® capsule using ph 6.8 phosphate buffer at 37ºc. the channeling effect of peg4000 gave a higher release of drug, this could be as a result of increasing the microcapsules porosity of formula 1 and 2 compared with the original formula (14) . on the other hand, formula 2 showed a slight increase in the releasing profile of drug than formula 1 which resulted from the reduction of ethylcellulose concentration to 1.8gm(15). in spite of lowering ethylcellulose amount to 1.6gm in formula 3 without addition of peg4000, the release of ibuprofen was lower than formula 2 but higher than the original formula. although, formula 4 which contains ethylcellulose (1.6gm) with peg4000 (0.4gm), released initially high amount of uncoated ibuprofen crystals in comparison with the original formula due to the low percent of microencapsulation efficiency. but this followed by the formation of peg4000 gelatinous barrier (0.4gm) that delayed the release of ibuprofen from reservoir system (16). it appears that no significant difference (p> 0.05) concerning the t100% ibuprofen release for all prepared formulas was noticed in comparison with the original formula, while, a significant difference (p<0.05) was observed when compared with the reference fenbid ® spansule as shown in figure (2). it was concluded that 2:1 core: wall ratio ibuprofen microcapsules prepared by a modified organic method failed to give an acceptable dissolution behavior as the reference. another sustained release reference was taken, balkaprofen® tablet 600mg that required 365 minutes to provide 100% release of ibuprofen. so that, the same ratio (2:1 core: wall) of ibuprofen microcapsules in weight equivalent to 600mg of drug was compared with this reference. no significant difference was found (p> 0.05) iraqi j. parma. sci., vol.14, 2005 8 -------------------------------------------------------------------------------------------------------------- between the releasing profile of this ratio and the reference (figure (3)). this gave a chance for preparing controlled release solid dosage form (tablet or capsule) of 600mg ibuprofen. figure (3): release property of 2:1 core:wall ratio of ibuprofen microcapsules prepared by a modified organic method and blakaprofen ® tablet using ph 6.8 phosphate buffer at 37ºc. figure (4) shows that, the percent of drug released from compressed tabletted microcapsules was significantly retarded (p< 0.05) as the compression force increased (33% and 25% for 10 10 3 psi and 12 103 psi compression forces, respectively after 6 hours as well as in comparison with the uncompressed one which gave 95% within the same time). figure (4): effect of compression forces on the dissolution of compared tabletted ibuprofen micro prepared by a modified organic method in comparison with balkprofen® tablet using ph 6.8 phosphate buffer at 37ºc. iraqi j. parma. sci., vol.14, 2005 9 -------------------------------------------------------------------------------------------------------------- this is due to continuous secondary polymer structure (matrix type formulation) produced by ethylcellulose polymer on compression which blocks most pores of microcapsules that hinders the disintegration and dissolution of tablet backbone (17). also it was noticed that compressed tabletted microcapsules prepared from formula 2 using 10 10 3 psi compression force which contains 0.2g peg4000 and 1.8g ethylcellulose gave only 7% increase in the release of ibuprofen from prepared tablet (figure (5)). this increase is due to channeling effect of peg4000 and reduction of ethylcellulose proportion (15, 18). figure (5): dissolution behavior of compressed tabletted ibuprofen microcapsules (formula 2) and original compressed formula at a compression force of (10x10 3 spi) in comparison with balkaprofen ® tablet using ph 6.8 phosphate buffer at 37ºc. the obtained results showed a difficulty in the utilization of this ratio to prepare a tablet dosage form as the reference balkaprofen ® tablet. so these findings suggest that a sustained release capsule dosage form of 300mg ibuprofen can be prepared and given to the patient in a dose of two capsules once daily (each of 0.517gm microcapsules weight) to give in vitro dissolution behavior as the reference balkaprofen ® tablet 600mg as shown in figure (6). iraqi j. parma. sci., vol.14, 2005 10 -------------------------------------------------------------------------------------------------------------- figure (6): comparison of dissolution of encapsulated 2:1 core:wall ratio ibuprofen microcapsules with balkaprofen ® tablet using ph 6.8 phosphate buffer at 37ºc. the results of preliminary stability study indicated that encapsulated ibuprofen microcapsules prepared by a modified organic method is stable during preparation and storage at room temperature for 6 months, since it provided 100% of ibuprofen remained after that time. conclusion: a modified organic method is a successful method for microencapsulation of ibuprofen to provide a sustained release microcapsules with good microcapsule properties. the release of drug from prepared microcapsules was increased as the core increased. peg4000 had a little decreasing effect on the yield and encapsulation efficiency and little increasing effect on percent of drug release per time. the selected 2:1( core :wall) ratio ibuprofen mirocapsules could not be formulated as tablet dosage form, so that, this ratio was encapsulated into 2 capsules of size “0” each of 517mg microcapsules weight to give acceptable drug release as the reference balkaprofen ® tablet 600mg . a high stability of encapsulated ibuprofen microcapsules was obtained when stored at room temperature, since no loss of drug was observed within 6 months of storage. iraqi j. parma. sci., vol.14, 2005 11 -------------------------------------------------------------------------------------------------------------- references: 1. martin dale, 32 edition , ibuprofen , page 44,1999 2. koyama i,shimano k,makabe e, evaluation of microcapsules prepared by the powder bed method using purified water as solvent, archives of practicale pharmacy; 1990, 50,83-88. 3. torrado s, egg albumin microaggregates containing colistin for oral administration: organoleptic 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prepare matrix system of hydrophilic drug by the microencapsulation process, stp-pharma-sciences, 1994, 4,486-491. 18. sakr fm, studies on microencapsulated granules effect of drug –binder solubilities and added diluents on core properties and microencapsulation characteristics, journal drug research, 1991, 20,303-313. الخـلاصـة: تستخدم طريقة التغليف المجهري للسيطرة على سرعة انطلاق العقار والتغلب على المظاهر السلبية الناتجة من لاستعمال المتكرر اليومي لعقار الايبوبروفين, لذلك تم استحداث طريقة جديدة محورة عن الطريقة المائية الغروية وسميت بالطريقة المحورة العضوية باستخدام سليلوز الاثيل والجينيت الصوديوم كمواد مغلفة لتحضير كبسولات مجهرية للايبوبروفين بطيئة التحرر. لوحظ ان تأثير نسبة المادة اللبية إلى المادة المغلِفة للعقار على كفاءة التغليف ونسبة الكبسولات المجهرية الناتجة قليل. لكن تحرر العقار من تلك الكبسولات المجهرية يتأثر بنسبة المادة اللبية إلى المادة المغلِفة, تغيير نسبة المادة المغلِفة (سليلوز الاثيل) واضافة مادة البولي أثيلين كلايكول 4000. تم مقارنة النسبة (1:2) بوزن يعادل300 ملغم, 600ملغم من العقار مع الفنبد سبانسول كبسول 300 ملغم و حبوب بالكابروفين 600 ملغم على التوالي حيث أشارت النتائج إلى أن تحرر العقار من النسبة المختارة وحبوب بالكابروفين كان متشابهاً, لذلك تم احتواء تلك الكبسولات المجهرية بأوزان تعادل 300 ملغم من الأيبوبروفين في كبسول جلاتيني صلب يصرف كبسولتين قي اليوم ليعطي تحرر بطيء للعقار مشابها لحبوب بالكابروفين.كانت ثباتية المستحضر عالية خلال فترة الخزن البالغة 6 أشهر في درجة حرارة الغرفة. introduction: materials and methods: preparation of microcapsules: mix dropping in calcium chloride solution (1%) dry in oven at 60oc scheme-1-aqueous colloidal polymer dispersion method mix dropping in calcium chloride solution (1%) dry in oven at 60oc scheme-2-preparation of ibuprofen microcapsules results and discussion: original formula iraqi j pharm sci, vol.29(1) 2020 co amorphous system: delivering poorly soluble drugs doi: https://doi.org/10.31351/vol29iss1pp1-11 1 co-amorphous system: a promising strategy for delivering poorly water soluble drugs iman s. jaafar*,1 and ameera a. radhi* *department of pharmaceutics, college of pharmacy, university of mustansiryah, baghdad, iraq abstract amorphization of drug has been considered as an attractive approach in improving drug solubility and bioavailability. unlike their crystalline counterparts, amorphous materials lack the long-range order of molecular packing and present the highest energy state of a solid material. co-amorphous system (cam) is a multicomponent single phase system containing an active pharmaceutical ingredient (api) with a low molecular weight excipient or a pharmacologically relevant api. this review highlights the different approaches followed in the preparation of co-amorphous drug delivery system, and the proper selection of the co-formers. in addition, the recent advances in solid state characterization, industrial scale and formulation will be discussed. keywords: co-amorphous , co-former, preparation, characterization. الذوبان في الماء ةالعقاقير القليلإليصال النظام غير المتبلور المشارك: استراتيجية واعدة *اميرة عبد االله راضيو 1*،ايمان صباح جعفر .العراق المستنصرية، بغداد ،الجامعة الصيدلة،كلية الصيدالنيات،فرع * الخالصة عكس النظم علىهذه حيث تفتقر الحيوي.في تحسين قابلية ذوبان الدواء وتوافره نظم غير المتبلورة المشاِركة استراتيجية ملفتة تُعَد ال تكرة يتم خاللها تقنية مب إنها تُعَدالمتراص، ونتيجة لذلك تتمثل بأعلى مستويات للطاقة تتوفر للمادة الصلبة. كما الترتيب الجزيئينظيراتها البلورية إلى الجزيئي( تأمين استقرار العقار في الحالة الغير المتبلورة ومنع تبلوره بواسطة اواصر بينية مع جزيئات لمركبات منخفضة الوزن الجزيئي)الشريك . لجزيئي األمثل. اضافة واختيار الشريك ا المتبلور،تسلط مراجعة المقال الضوء على الطرق المختلفة في إعداد نظام توصيل األدوية غير وانتقال النظام نحو النطاق الصناعي وتصييغه. فحص النماذجستتم مناقشة التطورات الحديثة في ذلك،إلى تشخيص.، تحضير، شريك جزيئي ،المشاركغير المتبلور المفتاحية: النظامالكلمات introduction a growing number of new active pharmaceutical ingredients (apis) have been revealed as a result of high-throughput screening technology progress (1, 2). the most widespread challenge is their low water solubility. in the biopharmaceutics classification system (bcs), these drugs are off the record as class ii and class vi, respectively (3).the low water solubility of such drugs will decrease the drug bioavailability seriously and may prevent further development of new chemical entities if it remains without satisfactory solution (4). for this reason, different methods have been adopted such as salt formation, cyclodextrin inclusion, solid dispersions, nanocrystals, microemulsion, cocrystals and amorphous dispersions; etc. (5, 6). along with these strategies, one of the most effective approaches to improve the solubility and dissolution of poorly water-soluble drugs is the amorphization(7). amorphous materials lack the uniform molecular packing typical for crystalline solids and present the highest energy state of a solid material (8). the improvement in the apparent solubility and dissolution profile in amorphous materials might be attributed to the low energy barrier necessary for molecules to go into solution yet, the amorphous systems potential application is limited, owing to thermodynamic instability causing recrystallization throughout processing, when they get in contact with the fluids of biological system or during storage (9). consequently, the stabilization of amorphous api using various formulation strategies has been investigated (10). definition of co-amorphous system (cam) co-amorphous system (cam) is a multicomponent single phase system containing an active pharmaceutical ingredient (api) with either a low molecular weight excipient or a pharmacologically relevant api (11,12). 1corresponding author e-mail: emanaldahan@gmail.com received: 24/5/2019 accepted: 29/ 9/2019 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp1-11 iraqi j pharm sci, vol.29(1) 2020 co amorphous system: delivering poorly soluble drugs 2 the application of small molecules like urea, citric acid, tartaric acid to stabilize amorphous forms has been reported earlier in the literature (13,14). the term “coamorphous” was introduced in 2009 by chieng et al. in attempt to distinguish the amorphous mixture containing two small molecules from the term polymeric amorphous solid dispersion (pasd) (15). the first drug-drug cam was developed by combining naproxen with cimetidine, by yamamura et al. the solubility and dissolution rate enhancement were attributed to amorphous form stabilization of both drugs throughout intermolecular hydrogen bonding (16). advantages and limitations of co-amorphous system (cam) co-amorphous systems afford high degree of solubility due to higher amorphous energy state. furthermore, no energy is required for the rearrangement of the crystal lattice during dissolution (17,18). in addition, they may show high degree of stability as compared to the amorphous form entity and dissolution rate enhancement in comparison to their crystalline homologues (19-21). the stability of cam is mainly caused by an increase of glass transition temperature (tg) and the homogenous molecular-level dispersions achieved by high energy mixing (22). attributable to the components with low mw, the required quantity of the stabilizer (co-former) is low and thus overcoming the large bulk volume of the final dosage forms associated with pasd (23). a remarkable increase in peak concentration (cmax) and area under curve (auc), in addition to a decrease in peak time (tmax) has been observed in many cam mixtures (24, 22) . being an amorphous system, cam is inherently susceptible to nucleation and crystal growth leading to the recrystallization to the more stable form. therefore, it is not possible to ensure the complete stability in the final dosage form (25,26). the main challenges cam mixtures may encounter during formulation are their sensitivity to heat and moisture (27). to date, few studies have investigated the impact of different temperature and humidity conditions on the cam stability (28). other hurdles might be attributed to their limited compressibility and flowability. the compression pressure might compromise the interactions between the drug and stabilizer molecule which in turn causes crystallization and negatively influences the physical stability as well as the dissolution performance of coamorphous system in the last dosage form (29). classification of cam based on the low molecular weight partner, cam can be classified into drug-drug cam and drugexcipient cam. until now, approximately fifty different drug-drug and drug-excipient cam have been investigated (30). drug-drug cam in this system, stabilization of two drugs which are pharmacologicallyrelated is obtained, as one drug functions as amorphous stabilizer for the other drug. in addition to the benefits of improved solubility, dissolution and stabilization, this system provides a platform for the innovative combinational therapy (31). more efficient therapy is attained from the combination of two different therapeutic categories drugs (32). furthermore, the concurrent one dosage unit administration of the drugs results in least excipients as well as improved patient compliance. usually cam are formed in molar ratio of 1:1, which may demonstrate stability enhancement as it implicates that both cam components network at molecular level via hydrogen bonds (13,21). the cam approach may also offer improvement of bioavailability, since one of the drugs can work as a dissolution-enhancing agent, in addition to amorphous-stabilizing agent for the second drug(33,34).great enhancement in the hydrochlorothiazide atenolol co-amorphous mixture in bioavailability as compared to that of the physical mixture, (drug in amorphous form and crystalline drug) (11). the earliest drug-drug cam was reported by yamamura et al., in 1996. binary mixtures of cimetidine with naproxen (16), indomethacin (35) and diflunisal (36) were prepared using solvent evaporation method. another example of investigated drugdrug cam is indomethacin with ranitidine hydrochloride cam for treatment of the pain and the prevention of nsaids gastrointestinal adverse effects(15), simvastatin with glipizide for lipid disorders and diabetes mellitus in metabolic disease(37) and tranilast (antiallergic) with diphenhydramine hydrochloride for management of allergy and inflammation (38). the investigated drug-drug combinations are generally still low, because of the limited number of pharmacologicallyrelevant drug pairs that can form glass solution in the required therapeutic dose (29). consequently, the idea of drugs combinations with inert molecules that can form hydrogen bonding and have low toxicity are more applicable (39, 40). drugexcipient cam in these systems, drugs with poor solubility were combined with inactive excipients of lowmolecular-weight. these excipients contain weak carboxylic acids, like tartaric acid (41) and citric acid (42), weak bases, like saccharin (39), meglumine (amino sugar) (43), phospholipids (44), sugars, nicotinamide and urea (45). the mechanism of stabilization of these cam was credited to direct molecular interactions for instances; hydrogen bonding, charge assisted interactions, molecular level mixing and if possible, an increase of the drug tg (46). besides, the amorphous stabilization, an increase in skin permeation was observed with acyclovir and iraqi j pharm sci, vol.29(1) 2020 co amorphous system: delivering poorly soluble drugs 3 citric acid cam compared to crystalline acyclovir (47). additionally, high physical stability even at storage conditions above the tg of the mixture was obtained in paracetamol and citric acid cam as a result of the strong intermolecular interactions (48). biocompatible co-formers, amino acids, were also used as anti-plasticizers by raising the tg, inhibiting drug-drug interactions and holding up recrystallization. amino acids were chosen based on the knowledge of probable drug-receptor interactions at the biological binding sites (49, 50). both dissolution rate improvement and physical stability of drug co-amorphous -amino acids systems in comparison to the drug in amorphous state have been achieved, owing to molecular interaction (12, 20, 49, 50). though, systems stability by non-interacting components is also a potential (20). amino acids salts formation showed higher mixtures stability (51). salts act as anti-plasticizers and increases the tg of cam mixture. this effect is mainly due to the powerful ionic interactions that oppose crystals formation (17). moreover, formation of salts enhanced dissolution, as a result of iondipole interactions between the dissolved ions and the water molecules. this type of interaction is energetically preferred over hydrogen bonding between non-ionized drugs with water molecules (34). methods for coamorphous formulations preparation an acceptable performance of the final dosage form depends mainly on choosing the proper approach to prepare cam formulations. in general, the selection of preparation procedure is influenced by the properties of active ingredients as well as the excipients. additionally, significant differences in physical stability in addition to dissolution performance of co-amorphous formulations result from different preparation methods (29). different techniques of preparation of cam are available, and they include mechanical activation (milling), solvent (solvent evaporation, spray drying and freeze drying) and techniques involving melting (52). milling methods this method is the most commonly applied methods to prepare stable cam owing to simplicity of handling, and minimum chemical degradation (if the processing occurs at low temperature), in addition to the high yield in comparison to other techniques (53). according to the molecular packing point of view, introduction of enough mechanical stress to produce crystal defects leads to loss of long-range crystallographic periodicity of crystalline materials (54 ,55). the raised temperature, during conventional ball milling, eventually leads to recrystallization. therefore, conducting milling at low temperatures essentially avoids fast re-crystallization and is considered an effective approach for producing cam dispersions .this result provides foundation of cryo-milling for low tg drugs where the temperature of the process is lowered down using liquid nitrogen (56).most pharmaceuticals turn fragile at the cryogenic temperature (temperature much lower than tg), favoring formation of the disordered form upon mechanical activation. additionally, the risk of degradation induced by heat along with recrystallization can be avoided through cryomilling. cryo-milling is, therefore, considered more successful than conventional milling for production of cam (57, 58). one of crucial points which should be considered is that the milling technique can occasionally form highly defective crystals rather than the actual amorphous solids. the formed crystals can encourage nuclei formation and the growth of crystal in amorphous solids (59, 60). solvent evaporation cam is commonly prepared by this method, in which an ordinary organic solvent is used to dissolve crystalline drugs or excipients, then the solvent is evaporated by means of heat or under vacuum for not less than 24 hours. any remaining solvent may lead to cam instability through recrystallization or solvate formation during storage (61).the solubility of both drug substance as well as excipient in the chosen solvent is of great significance to the size of the particles, physical consistency, in addition; to better dissolution performance of cam prepared by this method (62). due to solvent fast elimination, there is no chance of molecules to reorganize themselves in a crystal lattice. in addition, they are solubilized in the cam by co-precipitation (15). improved physical stability, enhanced phindependent solubility, superior intrinsic dissolution rate (idr) as well as supersaturated dissolution were observed for lurasidone hcl-saccharin cam prepared by rotary vacuum evaporator compared to amorphous lurasidone hcl (63). the process of spray drying is generally divided into two steps: atomization step and drying step. the first step essentially involves spraying of an appropriate solution of apis to a heated chamber with a control on the size of droplet as well as the rate of spraying. the last step necessitates the removal of the solvent out of droplets to yield dry particles. the particle size distributions and morphologies could be monitored via controlling the composition of the spray solution, and the rates of drying during the process (63). spray drying is a well-established technique and easily scalable process. however, this method has limitation which includes complexity in choosing the appropriate solvent for both drugs and excipients. additionally; recrystallization may occur as a result of residual solvents. furthermore, the use of organic solvents may cause safety iraqi j pharm sci, vol.29(1) 2020 co amorphous system: delivering poorly soluble drugs 4 concerns throughout the production and danger the environment (64,65). spray drying method provides an excellent choice to prepare cam, but as other methods may be unsuccessful because of degradation by heat, low yield, and incomplete amorphization (34). freeze drying, which is also known as lyophilization, is employed to obtain products with porous and low-density nature. high-dose zwitterionic compound ofloxacin-amino acid cam was prepared successfully by zhu et al. through lyophilization (66). melt-quenching the melt quenching is a well-recognized technique employed in the fabrication of thermostable active ingredients in small quantities. this method involves intensive mixing apis and/or excipients by heating to a molten liquid. then, the melt is hastily cooled to a temperature under the melting temperatures of the individual components in order to prevent crystallization. prevention of nucleation and growth of crystal is achieved by fast cooling rate, which consequently facilitate cam formation. a stable cam of citric acid-paracetamol preparation was reported by hoppu et al. via meltquenching method (67). it has been confirmed that cam prepared by melt-quenching may show improved physical stability compared to those prepared by conventional ball milling, or cryomilling (68). this is attributed to incomplete removal of small amount of residual crystals that encourage the crystallization(69). co-former selection many low molecular weight molecules have been considered as potential coformers in the formulation of cam .therefore, a successful design of cam requires a close observation of the properties for both the co-formers and the active ingredient, for instance the glass transition temperature (tg), ability to form hydrogen bonding and their miscibility to form a homogenous mixture (70). in order to ensure a maintained physical stability of the dispersion, the tg of the mixture is favored about 50 k above the storage temperature (tg-t ~50 k). therefore, employing co-formers with relatively high value of tg would increase the chance that the tg of the mixture will be higher, especially when api exhibits a low tg. the tg of the mixture can be anticipated by fox or gordon-taylor equations (71, 72). 𝑇𝑔 𝑚𝑖𝑥 = w1 tg1+k w2tg2 w1+ k w2 (1) where tg mix is tg of the amorphous mixture , tg1 and tg2 are the glass transition temperature of each component ,w1 and w2 are the weight fraction of each component, k is a constant , ρ1,ρ2 are the densities of each component and δα1,δα2 represent the change in thermal expansivity of tg of each component (73): k= (ρ1∆α2) (ρ2∆α1) (2)(73) the detection of hydrogen bond ability between co-amorphous components has extended to the use of computational and theoretical approaches such as, natural bond orbital analysis (nbo), density functional theory calculations (dft) as well as quantum molecular theory (qtaim) (74,75). modeling the interaction between sulfathiazole and citric acid / tartaric acid was carried out using a molecular modeling functionality, moilin, within oscail which is a windows software for molecular modeling and crystallography. as shown in figure (1), due to the presence of three carbons between its –cooh groups, citric acid, was better able to span the sulfathiazole molecules than tartaric acid (41). figure 1. calculated adduct structures for stz−ca (a) and (b) and stz−ta (c) and (d) (43). flory–huggins interaction parameter (ᵡ) has been used to indicate the miscibility between small molecules in binary mixtures and estimate their phase stability. it has been reported that miscible systems would have a value of flory–huggins parameters (ᵡ) close to or less than zero (76, 77). this parameter was calculated for simvastatin and glipizide dispersion and the obtained value for ᵡ was found to be 5.5 ± 2.0, which suggests the absence of iraqi j pharm sci, vol.29(1) 2020 co amorphous system: delivering poorly soluble drugs 5 molecular interactions between these components and thereby immiscibility (39) solid state characterization of cam x-ray powder diffraction (xrpd), fouriertransform infrared spectroscopy (ftir), in addition to differential scanning calorimetry (dsc) are commonly employed techniques to confirm both the formation and the stability of cam (78, 79). x-ray powder diffraction (xrpd) is a wellestablished method that comes up with information on the existence of a crystalline material through the presence of sharp diffraction peaks, whereas amorphous material is recognized by a halo pattern in the pxrd (80). figure 2 demonstrates the diffractogram of docetaxel with characteristic diffraction peaks of crystalline docetaxel (2 a) in contrast to the halo pattern exhibited by docetaxel and myricetin system prepared by fast solvent evaporation technique (2 e) (81). figure 2. pxrd patterns of (a) crystalline docetaxel, (b) crystalline myricetin, (c) amorphous docetaxel, (d) rotary evaporation product of myricetin, and (e) co-amorphous docetaxelmyricetin (81 ) a successful cam is identified in the dsc thermogram through a solitary tg that corresponds to the transfer of glass status to a sub cooled or viscous fluid (82). the presence of one tg implies formation of a single-phase homogeneous systems (83,84). the problem of phase separation between the individual components of the mixture could be detected by the observation of two tg values instead of a single tg (85).wang et al. prepared coamorphous loratadine-citric acid system in different molar ratios (1:1, 2:1 and 3:1). dsc thermograms revealed a single tg for co-amorphous loratadinecitric acid system (1:1) which confirmed the homogenous phase formation. on the other hand, the appearance of two tg values for formulations (2:1) and (3:1) suggested the formation of two phase mixture of loratadine-citric acid system and amorphous loratadine (86). the tg of the mixture could be theoretically calculated using the gordon-taylor equation which suggests a state of ideal miscibility and absence of interactions between the components. it usually appears in a range between the tg of the two individual components (87). however, the appearance of higher values of tg in the dsc thermogram strongly suggests the presence of strong ionic forces due to salt formation between the drug and the coformer (88). on the other hand, lower values may occur due to the plasticizing effect of water residues or the loss of bonds during mixing (89, 90). alleso et al. studied the thermal events in the comilled binary mixture of naproxen and cimetidine. the experimental values of tg (table 1) of the binary mixture at various molar ratios exhibited a significant deviation from those calculated by gordon-taylor equation. these results have been attributed to the strong solid-state interaction between the two molecules that has been successfully confirmed via raman spectroscopy (18). iraqi j pharm sci, vol.29(1) 2020 co amorphous system: delivering poorly soluble drugs 6 table 1. glass transition temperatures of mixtures and single components determined by dsc and calculated values using the gordon– taylor equation (18) sample tg(°c) experimenta l tg (°c) calculate d naproxen(nap ) 6.2±0.6 n/a cimetidine (cim)(milled) 36.1±2.0 n/a 1:2 nap-cim 42.2±1.1 14.0 1:1 nap-cim 34.5±0.3 10.7 2:1 nap-cim 31.5±0.7 8.6 the molecular interactions can be analyzed using fourier-transform infrared spectroscopy (ftir) (91), raman spectroscopy (92) and nuclear magnetic resonance (nmr) (93) . the infrared spectral analysis of ketoconazolesuccinic acid mixture demonstrated a band at 1605 cm−1, therefore suggesting hydrogen-bonding interactions between ketoconazole and succinic acid as given in figure 3 (94). figure 3. ir spectra of (a) amorphous ktz, coamorphous ktz−suc, amorphous succinic acid (94) edinger et al. employed the transmission raman spectroscopy to quantify the amorphization fraction of celecoxib in polyvinylpyrrolidone (pvp) tablet. the transmission raman spectroscopy provided a fast at-line and non-destructive measurements after each microwaving step. the spectrum of amorphous celecoxib shows bathochromic shift of the in-plane bending of the phenyl ring, assigned at 810 cm−1, to798 cm−1 and a merge with another peak centered at 794 cm−1 as well (95). binary mixtures of indomethacin with tryptophan (ind−trp) and furosemide with tryptophan (fur−trp) prepared by ball milling were analyzed employing xrpd, 13c solid status nmr (ssnmr), in addition to dsc. based on data obtained from 13c ssnmr spectra, it has been proposed that hydrogen bonding and π-interactions were responsible for amorphization in both systems (93). industrial scale the conversion of cam into marketed pharmaceutical dosage forms requires selection of the preparation technique depending on the physicochemical properties of drug in addition to the co-former, primarily the melting point as well as thermal stability of the cam components (96). as discussed previously, melt-quenching has been used for assessment purposes. however, the scalability of this method might be a challenging task due to the potential risks of in-process degradation and uneven mixing of the final product (97). hot-melt extrusion (hme), on the other hand, has been employed as the main preparation technique in several pasd based marketed pharmaceutical products and thus is considered as a promising method to scale up cam (98). moving to industrial scale for ball milling approach has been associated with several limitations. these limitations include, the heat produced during milling which makes this procedure applicable only to the thermostable drugs and high tg excipients. the low yield from sticking of the product to the chamber due to the charge generated during the process, and the crystallization tendency induced by milling stress (99,100). spray drying is a convenient process that used in manufacture of solid dispersions (101). it is, therefore, recognized as a promising method to scale up and manufacture of cam (102). for this reason, jensen et al. investigated spray drying for production of indomethacinbasic amino acids mixture in comparison with vibrational ball milling. it was concluded that spray drying had provided a completed amorphization of the final product and thus a stable formulation for industrial manufacture (53). formulation cam can be administered orally via further formulation into tablets or capsules. however, commercial manufacture may encounter some difficulties due to their sensitivity to humidity and temperature. wet granulation should be avoided in cam formulation, since water might cause plasticization and crystallization. similarly, the heating step during melt granulation can raise the temperature above the co-amorphous components tg and might lead to recrystallization (103). the most prominent goal in the formulation of co amorphous dispersions is the protection from moisture. this could be achieved through blending with excipients that decrease the moisture uptake such as fumed silica. in addition, blister packaging under argon atmosphere or the implication of some iraqi j pharm sci, vol.29(1) 2020 co amorphous system: delivering poorly soluble drugs 7 advanced materials such as high barrier thermoformable films, desiccant papers, cold formable foils provided with color changing cards as a reference to moisture levels (97). the first research to report a successful formulation of cam tablets was conducted by lenz et al., who formulated tablets containing spray dried indomethacin–arginine. the excipients used in this study were mannitol, croscarmellose sodium, colloidal silicon dioxide, magnesium stearate. tablets were evaluated with respect to the influence of compaction pressure on the tablet properties, physical stability and dissolution profile. data obtained from xrpd clearly revealed the absence of possible compression-induced crystallization during tableting and attainment of physical stability of the amorphous form in tablets during long term storage. although indomethacin from the prepared tablet exhibited lower supersaturation than spray dried powder behavior, the area under curve (auc) of dissolution profile of cam was not affected by formulation of spray dried indomethacin–arginine into tablets (22). polymeric coating using (kollicoat® protect) which is polyvinyl alcohol-polyethylene glycol graft copolymer, has been applied in the formulation of tablets containing spray dried co-amorphous indomethacin-arginine by petry et al. the applied coat provided a successful protection against humidity or heat, so that the obtained tablets were physically stable even when stored at humid conditions (rh/75%) for approximately three months (104). likewise, tablets were formulated with cam mixture of amlodipine besylate and atorvastatin calcium. the optimized formulations have proven their stability under tested conditions for three months. they also exhibited a significant increase in dissolution rates (105). conclusion due to the increasing number of poorly aqueous soluble apis, the cam has emerged as a promising approach to overcome the limitations associated with the polymeric based amorphous dispersions (pasd) to improve solubility and 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cs. solid state properties and drug release behavior of co-amorphous indomethacin-arginine tablets coated with kollicoat® protect. eur j pharm biopharm 2017; 119: 150-160. 105. adahalli sb, talluri m. formulation and evaluation of tablet prepared by coamorphous system containing anti-hypertensive and antihyperlipidemic drug. int j pharm pharm sci.2016; 8(9): 182-193. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.31(suppl.) 2022 a novel snp of il-10 gene in type 2 dm patients with toxoplasmosis doi: https://doi.org/10.31351/vol31isssuppl.pp1-8 a novel single nucleotide polymorphism of interleukin-10 gene is linked to type 2 diabetes mellitus in iraqi patients with toxoplasmosis(conference paper )# omar d. salman *,1, maysoon abdul zahra merdaw**and ahmed a. almaliky*** # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022  faculty of pharmacy, bilad alrafidain university college, diyala, 32001, iraq ** department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad, iraq ***the specialized center for endocrinology and diabetes, baghdad, iraq abstract type 2 diabetes mellitus (t2dm) is a chronic disorder that represents a serious health concern all over the globe, it is linked to interleukin-10 (il-10) single nucleotide polymorphisms (snps) at the promoter region. on the other hand, diabetes influences the cellular and humoral immunity predisposing the patient to a variety of opportunistic parasites one of them is toxoplasma gondii (t. gondii), that may infect any nucleated cell, including pancreatic cells. the purpose of this research was to explore the association of il-10 genetic polymorphisms with t2dm and toxoplasmosis among iraqi patients with t2dm. fifty-five and fifty-eight venous blood specimens were obtained from t2dm patients and age-matched non-diabetic persons, respectively. sera had been tested for the presence of anti-toxoplasma antibodies using the enzyme linked immunosorbent assay (elisa) kits. polymerase chain reaction (pcr) was performed by specific primers and the products were sequenced. a higher percentage of t. gondii infection was found in t2dm patients (52.1%) compared to 31.5% of non-diabetic persons. high frequency of the snp at position -1091 among t2dm patients, which represents a novel finding. an interesting result, an increased risk of t2dm was observed in carriers of -1082 a/g variants, which was highly frequent among studied subjects. the carriers of both -1082 ag+gg and -1091 ag+gg of il-10 genotypes had a synergistic effect on the risk for type 2 diabetes mellitus significantly. keywords: interleukin-10, type 2 diabetes mellitus, il-10 gene polymorphism, toxoplasma gondii. تبط بمرض السكري من ير 10لجين إنترلوكين المتعددة االشكال للنيوكليوتيدات المنفردة شكل جديد #) بحث مؤتمر (في المرضى العراقيين المصابين بداء المقوسات 2النوع *** عبود المالكي احمدو **الزهرة مرداو ميسون عبد، 1*،عمر داود سلمان 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي ديالى، العراق. ، بالد الرافدين الجامعة كلية ، قسم الصيدلة* .العراق بغداد، بغداد، جامعة الصيدلة، كلية ، العلوم المختبرية السريرية فرع ** العراق. ، بغداد ، لغدد الصم والسكريألمراض االمركز التخصصي *** الخالصة وهو مرتبط بتعدد العالم، اضطراب مزمن يمثل مصدر قلق صحي خطير في جميع أنحاء وه (t2dm)الثاني اء السكري من النوع د يؤثر مرض السكري على المناعة الخلوية أخرى، . من ناحية (il-10) 10في المنطقة المروجة النترلوكين (snps) المفردةأشكال النوكليوتيدات أي نواة ، التي قد تصيب(t. gondii) المقوسة الكوندية أحدها االنتهازية، والخلطية مما يجعل المريض عرضة لمجموعة متنوعة من الطفيليات بما في ذلك خاليا البنكرياس. ، خلية نموذج في( 1082-عند موضع ) 10نترلوكين اللألنماط الجينية المختلفة وترددات نقل األليل تحديد ا كان الغرض من هذا البحث هو الثاني السكري مرضى من المقوسات النوع بداء المصابين الجينية . العراقيين األشكال تعدد ارتباط استكشاف إلى مع 10النترلوكين لباإلضافة . وداء المقوسات بين المرضى العراقيين السكري من النوع الثاني غير نالمتطابقين بالعمر مواألشخاص السكري النوع الثاني تم أخذ خمسة وخمسين وثمانية وخمسين عينة من الدم الوريدي من مرضى المقايسة االمتصاصية ما باستخدام على التوالي. تم اختبار األمصال من جميع العينات بحثًا عن وجود أجسام مضادة للتوكسوبالز بالسكري، المصابين تسلسلها في بواسطة بادئات محددة ومنتجات التفاعل تم تحديد (pcr))االليزا(. وتم استخدام تفاعل البلمرة التسلسلي المناعية لالنزيم المرتبط كوريا )ماكروجين(. غير المصابين بالسكري. في (٪31.5 (و ٪ 52.1)السكري النوع الثاني) في مرضى t. gondii تم العثور على نسبة أعلى من عدوى لالهتمام،جديد. لوحظ نتيجة مثيرة يعتبر اكتشاف ، وهذا(t2dm) السكري النوع الثاني بين مرضى 1091-في الموضع لتغايرعالي ل كرارتهناك األشخاص في بين كراروالتي كانت عالية الت المتغيرات، a / g 1082-في ناقالت اإلصابة بالسكري النوع الثاني وجود خطر متزايد منوهي تأثير تآزري على خطر اإلصابة بداء السكري من النوع ag + gg 1091-و ag + gg 1082-كان لحامالت كال الطرز الوراثية و. الدراسة .بشكل ملحوظ 2 .a/g 1091و a/g 1082عفي المواض (il-10)هناك ارتباط كبير بين داء السكري من النوع الثاني وتعدد االشكال للجين . مقوسة الكونديةال ،il10تعدد األشكال الجيني ،2داء السكري من النوع ،10انترلوكين الكلمات المفتاحية: 1corresponding author e-mail: omardawood83@gmail.com received: 5/ 5/ 2022 accepted: 3/7 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp1-8 iraqi j pharm sci, vol.31( suppl. ) 2022 a novel snp of il-10 gene in type 2 dm patients with toxoplasmosis 2 introduction type 2 diabetes mellitus (t2dm) is a chronic condition characterized by hyperglycemia caused by low insulin levels, insulin resistance, or both (1, 2). according to the international diabetes federation (idf), there are 463 million diabetic persons worldwide in 2019, with the number anticipated to rise to 700 million by 2045 (3). in iraq, the reported prevalence of t2dm varies from 8.5 percent (idf—age adjusted) to 13.9 percent (4). a minimal level of a persistent inflammation has been proven to have a significant impact on the onset and development of t2dm (5). high levels of antiinflammatory and pro-inflammatory cytokines, such as il-10 and il-6, have been found in the plasma of t2dm patients, and are therefore related with its complications (6,7). interleukin-10 (il-10) is a multifunctional regulatory cytokine that serves as a general inhibitor for both type 1 and type 2 helper t cell proliferation and cytokine responsiveness in an inflammatory response (8). il-10 shortage or aberrant expression may boost the inflammatory response to microbial insult, but it can also result in the development of a range of autoimmune disorders (9). cytokine production has been demonstrated to be genetically controlled, with polymorphisms in the promoter region of cytokine genes determining lower or greater levels of production in response to certain stimuli. as a result, these polymorphisms may alter susceptibility to or severity of inflammatory disorders (10). three widespread single nucleotide polymorphisms (snps) have been found at the transcriptional start site in the 5' flanking region of il-10; these snps are located at positions 1082, 819, and 592, respectively, relative to the translational start site (11). the il-10 promoter region regulates transcription and includes snps linked to tdm2 (11-14). environmental microorganisms have the capacity to produce low-grade inflammation, that may raise the risk for development of numerous metabolic disorders such as diabetes (15,16), and may use il-10's immunosuppressive potential to inhibit host immune response, resulting in prolonged infection (17). toxoplasmosis is a zoonotic infection caused by the t. gondii parasite, which is a common intracellular protozoa parasite (18,19). this obligate intracellular parasite infects and replicates in any nucleated cell, including pancreatic cells (6). t. gondii infection is normally handled by the immune system in immunocompetent persons and frequently goes unreported; nevertheless, it is life threatening in immunocompromised individuals (20, 21). diabetes, on the other hand, is a disease that affects cellular and humoral immunity, making the patient vulnerable to a range of opportunistic parasites, one of which is t. gondii (22). this research intended to explore the relationship between il-10 gene polymorphisms and t2dm and latent toxoplasmosis in iraqi t2dm patients. patients and methods this study was conducted at the specialized center for endocrinology and diabetes/ baghdad and the iraqi national blood bank during the period from march 2021until september 2021. the study was designed to be a retrospective study. fifty-five and fifty-eight venous blood samples were collected from patients with t2dm with an age matched non-diabetic individuals, respectively. fasting blood samples were obtained from all study participants. all patients were diagnosed of establishing t2dm on the basis of medical history and laboratory tests according to the criteria of the american diabetes association (23). sera had been tested for the presence of anti-toxoplasma antibodies (igm and igg) using the enzyme linked immunosorbent assay (elisa) specific kit supplied by acon, usa (24). after extraction and purification of genomic dna, pcr was performed using specific primers which were supplied by alpha dna company. then pcr products underwent sequencing at macrogen /korea. informed consent had been obtained from all participants in the current study before sample collection, and the study have been revised and approved by the college of pharmacy/ university of baghdad, research ethics committee application no. 3102020c. demographic data were collected from each participant by using predesigned questionnaire sheets. inclusion and exclusion criteria included cases were patients with t2dm from both sexes in aged ≥ 18 year. excluded cases in this study were patients receiving toxoplasmosis treatment or providing incomplete information during completion of the questionnaire. in addition to patients providing treatment can affect the il-10 level such as dexamethasone and vit d. iraqi j pharm sci, vol.31( suppl. ) 2022 a novel snp of il-10 gene in type 2 dm patients with toxoplasmosis 3 genotyping genomic dna was extracted and purified from whole blood samples using the easy pure® blood dna kit (catalog no.: ee121) according to the manufacturer’s instructions. the genotyping of the il10 gene snps -1082 a/g (rs1800896), and 1091 a/g (rs1263484331) was conducted by using polymerase chain reaction (pcr) technique. pcr was performed using the specific primers which were supplied by alpha dna ltd (canada) as lyophilized product of various picomoles concentration. primer3 software was used to create the primers utilized in pcr amplification, which is a bioinformatics software available online for user to design pcr primers. the forward and reverse primers for il-10 gene were f5/ ctggctgcaacccaactggc-3 and r5/ tcttacctatccctacttcc-3, respectively. each pcr reaction mixture comprises 4µl dna template, 12.5µl master mix easytaq® pcr supermix, 1 µl of each forward and reverse primers and 6.5µl nuclease free water to make a total volume of 25µl in each well. following a preliminary denaturation at 94 cᵒ for 2 minutes, 35 cycles of denaturation at 94 cᵒ for 30 seconds and annealing at 54 cᵒ for 30 seconds were performed, followed by a final extension at 72 c for 3 minutes. annealing temperature was calculated according to the types of nucleotides within primers and a specific equation used for calculation of optimum annealing temperature (25). the pcr results were validated and seen using 1.0 percent agarose gel electrophoresis. uv transilluminator at 365nm and photographed. alignment analysis software the computer application bioedit pro. version: 7.0.0, which is accessible on the website http://www.mbio.ncsu.edu/bioedit/bioedit.html was used to compare the sequence of the il-10 gene in 113 samples with the sequence of the il-10 gene available on website (http://www.ncbi.nlm.nih.gov). statistical analysis statistical analysis system version 9.1(sas) was used to undertake data statistical analysis in order to determine the influence of various variables in research parameters. the least significant difference –lsd test (analysis of variation-anova) was performed to compare means. the chi-square test (0.05 and 0.01 probability) was employed to create a significant comparison between percentages (26, 27). results participants were divided into four groups according to the results of anti-toxoplasma gondii antibodies (igg) seropositivity by elisa method: • group 1: 30 patients having t2dm and have t. gondii infection. • group 2: 25 patients having t2dm and not have t. gondii infection. • group 3: 29 patients having t. gondii infection and not have dm. • group 4: 29 apparently healthy individuals not known to have dm nor t. gondii infection (referred to as the control group). all dna samples of patients and controls were undergone sequencing for il-10 gene -1082 g/a (rs1800896). the results of the il-10 gene sequencing revealed many snps, two of them namely, snp -1082 a/g and snp -1091 a/g, were undergone analysis (table -1). a significant difference in ag genotype frequency among studied groups in il-10 snps at position 1091 (rs1263484331), and a non-significant difference in gg genotype frequency. while, a significant difference in g allele frequency between patients’ group 1, group 2 and controls (table -1). non-significant difference in genotyping and allele carriage frequencies of il-10 at locus -1082a/g (rs1800896) between all patients’ groups and controls. http://www.mbio.ncsu.edu/bioedit/bioedit.html http://www.ncbi.nlm.nih.gov/ iraqi j pharm sci, vol.31( suppl. ) 2022 a novel snp of il-10 gene in type 2 dm patients with toxoplasmosis 4 table 1. distribution of genotype frequencies of il-10-1082 a/g and il-10-1091 a/g polymorphisms. polymorphisms il-10-1082 a/g and il-10-1091 a/g controls n=29 group1 n=30 group2 n=25 group3 n=29 p (or) genotype -1082 n (%) n (%) n (%) n (%) 0↔1 0↔2 0↔3 aa 3 (10.3%) 0 (0.0%) 0 (0.0%) 4 (13.8%) --(1) --(1) --(1) ag 0 (0.0%) 3 (10.0%) 0 (0.0%) 0 (0.0%) 0.1 (49) 1 (7) 1 (0.78) gg 27 (90.0%) 26 (89.7%) 25 (100.0%) 25 (86.2%) 0.237 (7.3) 0.24 (6.73) 1 (0.72) allele frequency a 6 (10.34%) 3(5%) 0 (0.0%) 8 (13.8%) ---(1) ---(1) ---(1) g 52 (89.66%) 57 (95%) 50 (100.0%) 50 (86.2%) 0.318 (2.19) 0.0295* (12.5) 0.77 (0.72) genotype -1091 aa 20 (68.9%) 14 (46.7%) 7 (28.0%) 25 (86.2) ---(1) ---(1) ---(1) ag 6 (20.7%) 9 (30.0%) 14 (56.0%) 0 (0.0) 0.352 (2.14) 0.003** (6.66) 0.023* (0.062 gg 3 (10.4%) 7 (23.3%) 4 (16.0%) 4 (13.8) 0.155 (3.33) 0.178 (3.8) 1(1.066) allele frequency a 46 (79%) 37 (62%) 28 (56%) 50 (86.2) ---(1) ---(1) ---(1) g 12 (21%) 23 (38%) 22 (44%) 8 (13.8) 0.044* (1.19) 0.012* (3.01) 0.46 (0.613) note. 0: controls, 1: group1, 2: group 2, 3: group 3, p: fischer exact p-value corresponding to genotype and allele frequency comparisons; (or) odds ratios are age-adjusted, * (p≤0.05), ** (p≤0.01). iraqi j pharm sci, vol.31(suppl.) 2022 a novel snp of il-10 gene in type 2 dm patients with toxoplasmosis 5 the results of the interaction analyses between the two snps of il-10 gene -1082 a/g and -1091 a/g on the susceptibility to t2dm are shown in tables 2 and -3. an increased risk for type 2 diabetes mellitus was observed in carriers of -1082a/g variants, while the carriers of both -1082 ag+gg and -1091 ag+gg genotypes had a synergistic effect on the risk for type 2 diabetes mellitus significantly. table 2. the interaction between two single nucleotide polymorphisms (snps) at positions -1082 a/g and 1091 a/g of the interleukin 10 (il-10) gene in different groups. snp of il-10 gene group 1 no., p (or) group 2 no., p (or) group 3 no., p (or) group 4 no., p (or) -1082 a/g -1091a/g aa aa 0, --(referent) 0, ---(referent) 4, ---(referent) 3, --(referent) ag+gg aa 14, 0.25(5.8) 7, 0.54 (3) 21, 1 (0.92) 17, ----(1) ag+gg ag+gg 16, 0.067 (12.1) 18, 0.054 (13.6) 4, 0.35 (0.33) 9, ----(1) total 30 25 29 29 p= p fisher exact test, or= odd ratio, * (p≤0.05), ** (p≤0.01). table 3. the interaction between two single nucleotide polymorphisms (snps) of the interleukin 10 (il-10) gene at positions -1082 a/g and 1091 a/g in type 2 diabetes mellitus and non-diabetic subjects. snp of il-10 gene patients with diabetes n=55 non diabetic n=58 p (or) -1082 a/g -1091a/g aa aa 0 7 ---(1) ag+gg aa 21 38 0.087 (8.3) ag+gg ag+gg 34 13 0.0004** (38.3) p= p fisher exact test, or= odd ratio, * (p≤0.05), ** (p≤0.01). discussion despite substantial research, the specific pathogenic mechanism of dm remains unknown. however, the clear family aggregation tendency of t2dm, shows that genetic factors may play a significant role in its incidence and progression (28, 29). chronic low-grade inflammation leads to the pathophysiology and consequences of t2dm (30). there is epidemiological evidence that inflammatory biomarkers are key risk factors for the development of diabetes in the future (31). the results of the current study demonstrate that a highly significant association of il-10 gene polymorphisms with the risk of developing t2dm, (p<0.01) (table-3). single nucleotide polymorphism at region -1082a/g, revealed that most of the studied individuals (patients and controls) had the mutant (gg) genotype, and the heterozygous (ag) genotype was observed in only 10 % of group1 subjects. in addition, there was higher gg genotyping and g allele frequencies in t2dm patients than non-diabetic individuals (table-1). in caucasians, the frequency of the high expression g allele varies from 20 to 52 percent, whereas in oriental asians, it ranges from 21 to 84 percent (3235). present study found that there was a nonsignificant difference in genotyping frequencies at the region -1082 of the il-10 gene between different patients’ groups and control group. however, there was a significant association between il-10 1082a/g polymorphism and risk of t2dm. risk ratio of developing t2dm was significantly higher for gg and ag genotypes compared to aa genotype. odd ratios for gg genotype for group 1 and 2 were 7.26, 6.63 respectively, while odd ratios for ag genotype were 49 and 7 for group 1 and 2 respectively (table 1). the present study's findings agreed with the findings of several earlier research (6, 29, 32). ayelign et al., and kolla et al., who found that a significant increase in il-10 -1082 gg genotype in ethiopian and indian patients with t2dm, respectively (36,37). a meta-analysis of six case-control studies (1,835 patients and 2,257 controls, in different countries including greece, italy, india, china, tunisia, and turkiye) concerning il-10 polymorphism at locus -1082 a/g found that a significant association between this polymorphism and t2dm under heterozygote comparison and the dominating genetic model was (ga/gg vs. aa: or= 1.22, 95 percent ci = 1.05– 1.41). in a stratified analysis by ethnicity, the il-10 -1082 a/g polymorphism was linked with a substantially higher incidence of t2dm in asian descendants under the dominant genetic model (ga/gg vs. aa: or= 1.69, 95%ci = 1.21–2.38 (3841). different genetic backgrounds and environmental exposures may contribute to this iraqi j pharm sci, vol.31(suppl.) 2022 a novel snp of il-10 gene in type 2 dm patients with toxoplasmosis 6 ethnic disparity (42). however, the results of the current study were in contrast with a number of studies (33, 43) that found no significant association between genotyping and t2dm. these contradictory results are most likely due to the small sample size and varied genetic backgrounds of the people. larger-scale genomic investigations are needed to confirm these relations. interleukin 10 is an anti-inflammatory cytokine. during an infection, it inhibits the activity of th1 cells, nk cells, and macrophages, all of which are required for successful pathogen clearance but may cause tissue damage. as a consequence, il10 may impede pathogen clearance while also improving immunopathology (44). in the current study, risk ratio of t. gondii infection was decrease in patients carrying ag, gg compared to aa genotypes of il10 snp -1082 a/g; [(odd ratio (95%ci) = 0.78 (0.012 49.9) and 0.72 (0.15 3.55), respectively, (table 1). similarly, risk ratio was 0.72 for whom carrying g allele. as a result, -1082 a/g polymorphism may be considering as a protective factor against susceptibility for t. gondii infection. many studies concerned with evaluation of three snps of il-10 at the promoter region namely: (rs1800872 -592c/a, rs1800871 -819c/t, and rs1800896 -1082 a/g), that have been found to regulate the expression of il-10 cytokine level (11). according to data obtained and analyzed by current study, a novel finding that indicate a possible association of snp -1091 a/g with increasing the risk of developing t2dm. no previous study had evaluated or deal with the polymorphism at position -1091 and its role in t2dm. a significant difference in g allele frequency of il10 at position -1091 a/g between group 1 and controls. in addition, a significant difference in ag genotype and g allele carriage frequency between groups 2 and controls. risk ratio of developing t2dm was higher for group 1 and 2 subjects with this polymorphism compared to controls. while, it was lower for group 3 subjects compared to controls (table1). the current study demonstrates that a significant difference in ag genotype frequency between patients’ group 3 and controls. the risk ratio of developing toxoplasmosis or t2dm was less for patients with t. gondii latent infection (both group 1 and 3) whom carrying the mutant g allele compared to the wild type a allele. this indicate that the mutant g allele have protective properties against t. gondii infection. and there was decrease in risk for developing t2dm in case of the concomitant presence of toxoplasmosis and t2dm, table (1). these findings were similar to those of previous snp (-1082 a/g). the results of the interaction between the two snps (-1082 and -1091) of il-10 gene demonstrate that a highly significant association of these polymorphisms (p<0.01) with the risk of developing t2dm and a synergistic effect has been occurred when both polymorphisms present simultaneously (table 2 and 3). it is widely known that il-10 has both immunosuppressive and anti-angiogenic properties (45). the largest risk of t2dm was related with gg+ag il10 gene genotypes at locations 1082 and -1091. (table 3). a meta-analysis was performed in china includes 22 studies about 1082a/g polymorphism found that il-10 has the ability to decrease inflammatory responses and may have anti-diabetic characteristics. il-10, on the other hand, has anti-angiogenic effects and may limit microvasculature formation as well as enhance vascular complications of diabetes, and it is unlikely that a single il-10 genetic variation can significantly contribute to its development (42). the current study found an interesting high frequency of the mutant g allele and gg genotype of il-10 snp -1082 a/g among studied individuals. novel snp of il-10 at position -1091 a/g that showed a significant association with increasing risk of the development of t2dm. actually, a synergistic relationship has been occurred when both snps at positions -1082 (rs1800896) and -1091 (1263484331) present simultaneously (p≤0.01). in addition, a high significant risk of developing t2dm observed for g allele carriers compared to a allele carriers. finally, patients with toxoplasmosis (non-diabetic) carrying g allele showed a decreasing in the risk of development t2dm and t. gondii infection. these patients carrying the mutant g allele may have a protective factor for toxoplasmosis. references 1. punthakee z, goldenberg r, katz p. definition, classification and diagnosis of diabetes, prediabetes and metabolic syndrome. can j diabetes. 2018;42: s10–s5. 2. zheng y, ley sh, hu fb. global aetiology and epidemiology of type 2 diabetes mellitus and its complications. nat rev endocrinol. 2018;14(2):88–98. 3. ogurtsova k, da rocha fj, huang y, linnenkamp u, guariguata l, cho nh, et al. idf diabetes atlas: global estimates for the prevalence of diabetes for 2015 and 2040. diabetes res clin pract. 2017;128:40–50. 4. international diabetes federation. chapter 3. the global picture. in: diabetes atlas. 8th ed. brussels, belgium: international diabetes federation; 2017. accessed march, 2019. 5. rivero-gonzález a, martín-izquierdo e, marín-delgado c, rodríguez-muñoz a, navarro-gonzález jf. cytokines in diabetes and diabetic complications. in: cytokine effector functions in tissues: elsevier; 2017. p. 119–28. 6. rodrigues kf, pietrani nt, bosco aa, campos fmf, sandrim vc, gomes kb. il-6, tnf-α, and il-10 levels/polymorphisms and their iraqi j pharm sci, vol.31(suppl.) 2022 a novel snp of il-10 gene in type 2 dm patients with toxoplasmosis 7 association with type 2 diabetes mellitus and obesity in brazilian individuals. arch endocrinol metab. 2017;61(5):438–46. 7. gupta s, maratha a, siednienko j, natarajan a, gajanayake t, hoashi s, et al. analysis of inflammatory cytokine and tlr expression levels in type 2 diabetes with complications. sci rep. 2017;7(1):7633. 8. sabat r, grutz g, warszawska k, kirsch s, witte e, wolk k, geginet j. biology of interleukin-10. cytokine growth factor rev. 2010 oct; 21(5):331–44. 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29: 71–109. this work is licensed under a creative commons attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ effects of different concentrations of melatonin on the time-course of nitrite–induced oxidation of hemoglobin: in vitro study iraqi j pharm sci , vol.18 (1) , 2009 melatonin inhibits erythrocyte oxidation 78 effects of different concentrations of melatonin on the time-course of nitrite–induced oxidation of hemoglobin: in vitro study # saad a.hussain *, 1 , shaima m. mohammed ** , intesar t. numan * and ihab i. abdulwahhab * * department of pharmacology and toxicology,college of pharmacy,university of baghdad,baghdad, iraq. ** department of clinical laboratory sciences, college of pharmacy,university of baghdad, baghdad, iraq. abstract melatonin is a potent scavenger of reactive oxygen species or free radicals like superoxide and hydroxyl radicals. the oxidation of hemoglobin to methemoglobin (meth-hb) by oxidizing compounds has been widely studied. the present work was designed to evaluate the ability of different concentrations of melatonin to inhibit nitrite–induced oxidation of hemoglobin. blood samples were obtained from apparently healthy individuals from which erythrocyte hemolysate was prepared. different concentrations of melatonin (10 -9 -1.0 mg/ml) were incubated for 10 min with the hemolysate, then to the resultant mixture 1 ml of sodium nitrite (final concentration 0.6 mm) was added, and the formation of meth-hb was measured by monitoring absorbance of light at 631 nm each min for 30 min. control samples without melatonin were utilized for comparison. nitrite caused rapid oxidation of hemoglobin to meth-hb in control samples; in the presence of melatonin, the oxidation process was delayed in a dose–dependent manner. the effect of melatonin on the time course of nitrite-induced oxidation of hb showed that melatonin has a protective effect initiated early after addition along with nitrite. melatonin also affect the time required for the formation of meth-hb, the time required to convert 50% of the available hb to meth-hb was 4 min in the absence of melatonin, and became 17, 22, 26, 30, 114 and 383 min with increasing melatonin concentrations (10 -9 , 10 -6 , 0.001, 0.01, 0.1, and 1.0 mg/ml respectively). in conclusion, melatonin in a concentration and time dependent manner can protect hb from oxidation by nitrite; melatonin delays the onset of autocatalytic stage and the protective effect extended over long period of time. key words: melatonin, erythrocytes oxidation الخالصة توو رااتوتهب بلووا واتوته وتهودر الدااتوة الحبليوة الوً تريوين هروداة وغلوىبيي دوديوغلىبيي وتحىله الً هيتهياى عولية اكسدة اله لً هٌت أو تأخير حدوث عولية األكسدة بوبرة ًبيتراي الصىريىم. تن الحصوى علوً عيٌوبم رم هوي عتراكيز هختلفة هي هبرة الويالتىًيي وعتوودة هوي د وا ايخوريي. تون هوزل هحلوى وغلوىبيي هوي الوريوبم الوتحللوة وحسور اللور اليأشخبص أصحبء وتحضير هحلوى هوي اله 11-1الهيوغلىبيي هت تراكيز هختلفة هي هبرة الويالتىًيي ) 9 ردوبق تون بعوداب اةوبلة هللتور واحود هوي هوبرة ًوبيترام 11دة ووهلغن/هوا ل ببتوتخدام هليوبر األشوعة كا رديرةوغلىبيي الوتوىى يالصىريىم كعبها هؤكسد. تو هتببعة عولية التأكسد هي خال ديبس هستىي الويته لى ال ٌفسجية. أظهرم الٌتبقج اى للويالتىًيي الرداة علً توأخير تأكسود الهيوغلوىبيي بصوىاة تعتوود علوً التركيوز ولتورة الخلو . ويوووي ولترة الوزل. األتتٌتبل ببى الويالتىًيي بأهوبًه حوبية الهيوغلىبيي هي التأكسد بىاتلة ًبيترام الصىريىم وبصىاة تعتود علً التركيز introduction recently, many experimental data provided unequivocal evidence about the formation and role of free radicals in biological systems. (1) such reactive species may bring about oxidative damage to virtually all cell compartments, eventually leading to various pathologies and aging. (2) these studies prompted research on physiological antioxidant systems and molecules, and stimulated the development of natural or synthetic compounds that prevent oxidative stress and damage mediated by an enhanced formation of free radicals. (3) after the discovery of its radical-scavenging properties, melatonin (n-cetyl-5-methoxytryptamine) has been considered as a putative biological antioxidant but it has been questioned to whether it may have a real antioxidant function under physiological conditions; (4) its molecular mechanisms of action remain to be clarified. interactions of melatonin contributing to its antioxidant effects in vivo may be lost during in vitro experiments; when it behaves in vitro as an electron donor, many electrophilic compounds, such as the hydroxyl radical, fe +3 , or carbon centered radicals may act as acceptors in one-electron transfer reactions, which convert the indolamine to the indolyl cation radical. (5) reactivity of melatonin with oxygen centered radicals, such as peroxyl or alkoxyl radicals, as well as a moderate activity towards lipoperoxyl radicals, has also been demonstrated. # based on oral presentation in the seventh scientific conference of the college of pharmacy /university of baghdad held in 26-27 november 2008. 1 corresponding author e-mail : saad_alzaidi@yahoo.com received : 3/1/2009 accepted : 8/4/2009 mailto:saad_alzaidi@yahoo.com iraqi j pharm sci , vol.18 (1) , 2009 melatonin inhibits erythrocyte oxidation 79 although the exact relationship between such activity and the concentrations required to perform it is not clarified, its ability to scavenge a broad spectrum of radicals could allow melatonin to behave as an antioxidant in various and possibly complex ways. (6) this study was designed to investigate the antioxidant activity of melatonin in different concentrations using an in vitro model of nitrite-induced hemoglobin oxidation. material and method blood samples were obtained from apparently healthy individuals, and were centrifuged at 2500 rpm and 4°c for 10 min to remove plasma and the buffy coat of white cells. the erythrocytes obtained were washed thrice with phosphate-buffered saline and lased by suspending in 20 volumes of 20mm phosphate buffer ph 7.4 to yield the required hemolysate concentration of 1:20. different concentrations of melatonin were incubated for 10 min with the hemolysate starting with stock solution (melatonin 1mg/ml) from which serial dilutions were made to give concentrations of 0.1, 0.01, 0.001, 10 -6 and 10 9 mg/ml melatonin solution. then to these incubated mixtures 1ml of sodium nitrite (final concentration 0.6 mm) were added and the formation of methemoglobin was measured by monitoring absorbance at 631 nm each min for 30 min using a spectrophotometer. (7) in the second part of the study, melatonin was added either before or at various time intervals (5 min and 10 min) after the addition of sodium nitrite to the hemolysate solution, and the formation of methhemoglobin was measured by monitoring the absorbance of light at 631 nm, and the results were compared with control samples without melatonin; all experiments were performed in triplicate and repeated many times. results nitrite causes a rapid oxidation of hemoglobin to methemoglobin, as shown in control curve (figure 1). in the presence of melatonin, the oxidation process was delayed in a dose-dependent manner. figure1 describes the effect of different melatonin concentrations on the timecourse of nitrite oxidation of hemoglobin; without melatonin, the timecourse of oxidation shows a characteristic pattern of slow initial transformation followed by a rapid autocatalytic process; in presence of melatonin there is slow increase in absorbance related to reduced levels of methemoglobin formation in all test samples. figure 2 showed that addition of melatonin to the incubation mixture, at different time intervals (after 5 and 10 min) during the autocatalytic phase, did not affect its ability to decrease meth-hb formation. the time required to convert 50% of the available hemoglobin to met hemoglobin was (4 min) in the absence of melatonin, whereas with 1 mg/ml melatonin solution the time was increased to 383 min (6.4 hr) (table 1). figure 1. effect of different melatonin concentrations on the time-course of nitrite–induced oxidation of hemoglobin. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 t ab control 1mg/ml l 0.1mg/ml l 0.01mg/ml l 10 -3 mg/ml mg/ml 10 -6 mg/ml mg/ml 10 -9 mg/ml mg/ml a b s o rb a n c e time (min) iraqi j pharm sci , vol.18 (1) , 2009 melatonin inhibits erythrocyte oxidation 80 figure 2. effect of melatonin on the time course of methhemoglobin formation at various time intervals from nitrite addition. table( 1) : time to form 50% meth-hb in presence of different concentrations of melatonin. melatonin concentration mg/ml % formation of methhb time to form 50% methhb (min) control 100 4.0 10 -9 mg/ml 96.1 17.0 10-6 mg/ml 86.9 22.0 0.001 mg/ml 50.5 26.0 0.01 mg/ml 37.9 30.0 0.1 mg/ml 29.9 114.3 1.0 mg/ml 16.6 383.0 discussion the oxidation of hb to meth-hb by nitrite has been widely studied, (7-9) formation of meth-hb occurs in two stages; there is a slow initial stage followed by a rapid autocatalytic stage, which carries the reaction to completion. (10) the present study has shown that melatonin can protect hemoglobin from oxidation by sodium nitrite in hemolysate, and there are two suggested theories for the mechanism through which melatonin produces this protective role; erythrocytes are utilized as a traditional target for studying oxidative damage, when exposed to high oxygen tensions and in presence of high iron contents (transition metal promoting the formation of oxygen free radicals) oxidative damage occur due to both endogenous and exogenous insults. sodium nitrite as a prooxidant induces a primary extensive methemoglobin formation as a result of generation of several free radical species like super oxide anion, peroxynitrite, and nitric dioxide, which are generated during the course of nitrite–induced oxidation of hemoglobin. (11) after the discovery of radical-scavenging properties of melatonin, it has been considered a putative biological antioxidant, but it has been questioned whether it may have a real antioxidant function under physiological in vivo conditions. (6) the molecular mechanisms of actions of melatonin remain to be better clarified; it is capable to prevent the onset of the autocatalytic stage since superoxide is implicated in the autocatalytic stage , and the fact that melatonin is a potent scavenger of superoxide anion, (5) the results of the present study suggests that the protective action of melatonin might be due to its scavenger effect and not due to reduction of methemoglobin to hemoglobin, since it fails to reverse the oxidation of hemoglobin; additionally, direct interaction between nitrite and melatonin as a reason for protection can be ruled out because the concentrations of melatonin which protect erythrocytes is very low. (11) kinetic evidence indicates that melatonin delays oxidative denaturation of hb through it's reaction with hb-derived oxoferryl radicals, and this may 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1 3 5 7 9 11 13 15 17 19 20 22 24 26 28 30 time (min) a b s o rb a n c e control after 5 min after 10 min zero iraqi j pharm sci , vol.18 (1) , 2009 melatonin inhibits erythrocyte oxidation 81 explain the reported antioxidant effects; tesoriere et al (2001) studied the reaction of melatonin with hemoglobin-derived oxoferryl radicals and the inhibition the oxidant effects of hydroxyl peroxide-induced hemoglobin denaturation in red blood cells, they found that the basic requirement for oxidative denaturation of hb by hydroperoxides is the transient formation of the perferryl-hb; (12) perferryl-hb, which includes a hypervalentiron oxoferryl heme group and a radical species, localized in the globin is a strong oxidant towards the globin moiety, which leads to hb denaturation with the formation of hemichrome and heme release. (13) the perferryl species, generated from met-hb and h2o2, (14) comprises a radical localized on the globin, possibly an aromatic amino acid radical , and an oxoferryl heme group. (12) after exhaustion of h2o2, decay of the perferryl to the oxoferryl form occurs, and then the latter is slowly converted to met-hb by a so-called autoreduction reaction; (14) this process involves intramolecular electron transfer and modification of the globin moiety. (13) such oxidative modifications of globin on exposure to h2o2 may be avoided by the presence of certain antioxidant compounds such as melatonin, ascorbate or trolox at the time of reaction, suggesting that rapid deactivation of the protein radical in the perferryl species is crucial for protection; (15) the mentioned mechanisms may prove that melatonin acts through its reducing activity towards perferryl-hb, and this may include the reduction of the oxoferryl moiety or the unpaired electron electrophile center at the globin moiety by melatonin, or both. (11) although in our study we did not investigate the reactivity of melatonin as a reducing agent, we can not exclude this effect and it needs further investigations. in conclusion, melatonin protects hemoglobin against nitriteinduced oxidation and delay the formation of meth-hb in concentration dependent pattern. acknowledgment the authors thank university of baghdad for supporting this project. references 1tesoriere l, d'arpa d, conti s, et al. melatonin protects human red blood cells from oxidative hemolysis: new insights into the radical-scavenging activity. j pineal res 1999; 27: 195-205. 2harman d. free radical involvement in aging: patho-physiology and therapeutic implications. drugs aging 1993; 3: 60-80. 3 rice-evans ca, diplock at. current status of antioxidant therapy. free radic biol med 1993; 15: 77-96. 4loffler m. melatonin as antioxidant. use or misuse? exp clin endocrinol diabetes 1996; 104: 308-310. 5poeggeler b, saarela s, reiter rj, et al. melatonin. a highly potent endogenous scavenger and electron donor: new aspects of the oxidation chemistry of this indole accessed in vitro. ann n y acad sci 1994; 738: 419-420. 6livrea ma, tesoriere l, d’arpa d. et al. reaction of melatonin with lipoperoxyl radicals in phospholipid bilayers. free radic biol med 1997; 23: 706-711. 7doyle mp, pickering ra, dykatra rl, et al. nitrite-induced hemoglobin oxidation. biochem biophys res commun 1982; 105: 1 -132. 8wallace w, houtchens ra, maxwell jc, et al. mechanisms of hemoglobin oxidation. biol chem 1982; 257: 4966-4977. 9tomoda a, tsuiji a, yoneysnia y. biochemical concequences of hb oxidation. biochem 1981; 193: 169-179. 10venkatesan p, unnikrishnan mk. effect of curcumin analogues on oxidation of haemoglobin and lysis of erythrocytes. j current sci 2003; 84: 74-78. 11tesoriere l, allegra m, d’arpa d, et al. reaction of melatonin with hemoglobinderived oxoferryl radicals and inhibition of the hydroperoxide-induced hemoglobin denaturation in red blood cells. j pineal res 2001; 31: 114-119. 12davies mj. detection of peroxyl and alkoxyl radicals produced by reaction of hydroperoxides with heme-proteins by electron spin resonance spectroscopy. biochim biophys acta 1988; 964: 28-35. 13giulivi c, cadenas e. ferrylmyoglobin: formation and chemical reactivity toward electron-donating compounds. in: methods in enzymology, 1994; packer l, (ed), vol. 233. academic press, san diego, ca, 189202. 14patel rp, svistunenko da, darley-usmar vm, et al. redox cycling of human methemoglobin by h2o2 yields persistent ferryl iron and protein based radicals. free radic res commun 1994; 25: 117-123. 15giulivi c, romero fj, cadenas e. the interactions of trolox c, a water-soluble vitamin e analog, with ferrylmyoglobin: reduction of the oxoferryl moiety. arch biochem biophys 1992; 299: 302-312. iraqi j pharm sci, vol.26(1) 2017 raft forming antacid tablet 26 formulation and evaluation of sustained and raft forming antacid tablet mohammed s. al-lami *,1 * department of pharmaceutics , college of pharmacy , university of basrah , basrah, iraq. abstract antacids have been widely used in the treatment of various gastric and duodenal disorders such as heartburn, reflux esophagitis, gastritis, irritable stomach, gastric and duodenal ulcers. a phresponsive of bi-polymer of sodium alginate and pectin have been studied as raft-forming polymers using sodium bicarbonate and calcium carbonate as gas-generating and calcium ion sources. the aim of study was to formulate and evaluate mono and bilayer tablets of floating and sustained release antacid delivery systems using sodium carboxy methyl cellulose as a gel forming substance, calcium and magnesium carbonate as sources of acid neutralizing and carbon dioxide gas generators agents upon contact with acidic solution. the effect of the formulation contents on the buoyancy has been investigated. in addition to, the antacid activities of intact and pulverized tablets have been studied. the result obtained showed that the buoyance is remarkably affected by the percentages of sodium carboxy methyl cellulose and carbonates salts. all formulas of mono and bilayer tablets revealed sustained action of acid neutralization and raft formation. besides, bilayer tablets showed a significant and higher level of acid neutralizing capacity than monolayer tablets. moreover, the pulverized of bilayer tablets exhibited significant and higher acid neutralizing capacity at raft than that at bulk of artificial gastric juice medium. keywords: raft forming agent, antacid, floating drug delivery, acid neutralizing capacity, sodium carboxy methyl cellulose. وتقيين أقراص هضاداث حووضت هديدة وطافيتتصييغ هحود صبار الالهي *،1 * .فرع انصُذالَُاخ، كهُح انصُذنح، خايعح انثصرج، انثصرج، انعراق الخالصة لذ اسرخذيد يضاداخ انحًىضح عهً َطاق واسع فٍ عالج يخرهف اضطراتاخ انًعذج واالثٍُ عشر يثم انحرلح ، ارذداع انًعذج، انًعذج انعصثٍ ، لرحح انًعذج و االثٍُ عشر. ذًد دراسح اسرداتح انثىنًُراخ انثُائُح ألندُُاخ انصىدَىو انًرٌء ، انرهاب وانثكرٍُ نرغُر األس انهُذروخٍُُ عهً ذشكم تىنًُراخ يكىَح نهطىف تاسرخذاو تُكرتىَاخ انصىدَىو وكرتىَاخ انكانسُىو كًصذر و.نرىنُذ انغاز ويصذر ألَىَاخ انكانسُى ألراص أحادَح وثُائُح انطثمح يذَذج وطافُح يٍ يضاد انحًىضح تاسرعًال سهُهىز و ذمُُى انغرض يٍ انذراسح هى ذحضُر انكارتىكسٍ يثُم انصىدَىو كًادج يكىَح نههالو واسرخذاو كرتىَاخ انًغُُسُىو وكرتىَاخ انكانسُىو كًصادر نًعادنح انحايضُح انكارتىٌ عُذ ياليسرها نهًحهىل انحايضٍ. ذًد دراسح ذأثُر يحرىَاخ انرصُّغ عهً اَشطح انطفى وعىايم يىنذج نغاز ثُائٍ أوكسُذ ويضادج انحًىضح. أظهرخ انُرائح أٌ لاتهُح انطفى ذرأثر تصىرج يهحىظح تُسثح سهُهىز انكار تىكسٍ يثُم انصىدَىو وايالذ فعانُح يذَذج نًعادنح انحًىضح وذكىٍَ انطثمح انطافُح. تداَة رنك، انكرتىَاخ. خًُع انصُّغ نأللراص أحادَح وثُائُح انطثمح أتاَد األلراص ثُائُح انطثمح أظهرخ يسرىي عاٍل ورو اعرثار فٍ لاتهُح يعادنح انحًىضح عٍ األلراص أحادَح انطثمح. عالوجً عهً رنك، عُذ انطثمح انطافُح عُه فٍ انعًك وسظ ًىضحأظهر يسحىق األلراص ثُائُح انطثمح يسرىي عاٍل ورو اعرثار فٍ لاتهُح يعادنح انح عصارج انًعذج انصُاعٍ. الكلواث الوفتاحيت: عاهل تكوى الطفو، هضاد الحووضت، إطالق دوائي يطفو على السطح، قابليت هعادلت الحووضت، سليلوز الكار بوكسي هثيل .الصوديوم introduction gastric ulcer and gastroesophageal reflux are the most common disorders in one tenth of western‟s population (1) . the antacids probably used at the beginning of previous century once celsus used neutralizing soils for treatment of gastric distress. the prim use of antacids as an ulcer curing agent started in 1856, when william brinton used a combination of bicarbonate potash with bismuth to treat peptic ulcer. the scientific use of antacids to treat gastric ulcer was initiated in 1910 while the pronouncement of schwarz prominent dictum, “no acid no ulcer”. in the next 50 years, antacids products were extensively used and grew further (2) . antacids have been widely used in the treatment of various gastric and duodenal disorders such as heartburn, reflux esophagitis, gastritis, irritable stomach, gastric and duodenal ulcers. 1 corresponding author e-mail: mohsabbar@gmail.com received: 26/2/2017 accepted: 25/4/2017 file:///d:/journal/1(26)%202017/new%20folder%20(6)/preparation%20of%20sustained%20release%20floating%20antacid-%20dr.%20mohammed%20sabar%20for%20reveiwer.docx%23_enref_1 iraqi j pharm sci, vol.26(1) 2017 raft forming antacid tablet 27 the carbonates and hydroxides of magnesium and aluminum have been extensively used as antacid in different combination ratios. as well as, they were incorporated to each other to prepare dry or wet gel that known as the layer lattice antacids. furthermore, magnesium trisilicate, calcium carbonate and aluminum phosphate were also slightly used (3) . gastro-retentive delivery systems have been used for more efficient treatment of local gastric diseases as well as to attain a high and more sustained therapeutic efficacy (4) . so far, floating system have been applied to prolong gastric retention time, increase of the drug absorption within the stomach, and improve the release rate of drug in the gastrointestinal tract (5, 6) . buoyancy is the fact that determined by archimedes. it is a state that the object with less density than that for a fluid will float in that fluid. more generally, archimedes' principle based on that a fluid will exert an upward force on an object that immersed in and it equals to the weight of the fluid that displaced by the object (7) . floating drug delivery systems (fdds) is one of a methodology that has been produced so as to expand the gastric residence time of orally administered dosage form. single and compound unit system have been fabricated (8) . oral compound unit dosage form, for example, microspheres have gotten much consideration as altered/controlled the drug delivery from dosage form. these frameworks were more consistently dispersed in the gastrointestinal tract, subsequently bringing about a more uniform absorption and decreasing patient-topatient variation (9) . floating and mucoadhesive gastric-retentive delivery system was designed to retained in stomach and float for 6 and 24 hours, correspondingly. this formulation was prepared using a liquid multi-layering process to compose five layers of a hallow spherical shell, a waterproof, a drug, a release retarding film and a mucoadhesive layer (10) . a phresponsive of bi-polymer of sodium alginate and pectin have been explored as raft-forming polymers using sodium bicarbonate and calcium carbonate as gas-generating and calcium ion sources, respectively. this study has shown the capability of bi-polymer to form strong and flexible raft (11) . sodium carboxymethyl cellulose is soluble in hot and cold water. at low concentrations, solutions are characterized by high viscosity that makes them useful in pharmaceutical applications, such as thickening and stabilizing agent (12) . the viscosity of na cmc solution is proportional to degree of polymerization and molecular weight (chain length). it is forming clear colloids in water and pharmaceutically employed as coating and binder in tablet, viscosity enhancing agent, water absorbing agent in wound dressing and as a disintegrant in capsule dosage forms (13) . calcium and magnesium carbonates are practically insoluble in water and soluble in dilute acids with liberation of co2 causing effervescent (14) . they have local and fast acting antacid by rapid dissolution and increasing in the gastric ph. materials and methods materials calcium carbonate, magnesium carbonates, sodium carboxy methyl cellulose grade crt 100 pa walocel ® dow wolff cellulosic (germany) and cross carmelose sodium were used in the formulation of antacid tablets. preparation of tablets four formulas of antacid tablets were prepared using direct compression method. table (1) shows the formulas 1 and 2 with carbonates to sodium caboxymethyl cellulose ratio 2.25:1 and 5.5:1 respectively. the ingredients were tumbled in plastic sachet for a minute and tablet machine (pharma tech international india) were set to hold 700 mg of formula to get a compressed tablet. the formulas 3 and 4 were prepared through double layer compressed tablets, ingredients in table(2) were used in the first and second layer. the carbonates to sodium caboxymethyl cellulose ratio in the first layer were 2.25:1 and 5.5:1 respectively. table (1): compositions of formulas 1 and 2 materials formula (1) formula (2) calcium carbonate 400mg 490mg magnesium carbonate 50mg 60mg sodium carboxymethyl cellulose 200mg 100mg cross carmelose sodium 50mg 50mg total weight 700mg 700mg iraqi j pharm sci, vol.26(1) 2017 raft forming antacid tablet 28 table (2): compositions of first and second layer in formulas 3 and 4 layer materials formula (3) formula (4) first calcium carbonate 286mg 350mg magnesium carbonate 36mg 43.5mg sodium carboxymethyl cellulose 143mg 71.5mg cross carmelose sodium 35mg 35mg second calcium carbonate 164.5mg 164.5mg magnesium carbonate 20.5mg 20.5mg cross carmelose sodium 15mg 15mg total weight 700mg 700mg evaluation of antacid tablets the produced tablets were subjected to the physical characterization and weight variation study. the hardness and friability tests were carried out using erweka hardness (tbh-100 germany) and friability (tar, germany) testers, respectively. the content uniformity study was replaced by mass variation test according appendix xii c of british pharmacopoeia 2012, due to the tablet weight more than 650mg and the content of active ingredients more than 25mg (14) . the buoyancy measurement was performed using usp xxx pharmacopeia (15) dissolution apparatus to evaluate tablet in 0.1n hcl as artificial gastric juice (agj). the experiment was carried out for two hours by putting of the pre weighed tablet into dissolution jar that contains 500ml of agj. the rotation speed of paddle was 100 rpm; the remnant of the tablet was taken off and left for 48hours inside hood to dry. the remained dried tablet was reweighed and the difference in weight was calculated. this test was carried out in triplicate for each formula. the acid neutralization capacity was measured for each of the intact and pulverized tablets. it was measured using dl53 titrator (mettler toledo – switzerland). a modified method of washington and colleagues was applied in this experiment by addition of one intact tablets (or powder of one tablets) into a solution containing 30ml of 0.1n hcl and 70ml mili-q water. agj was pumped into solution at 4ml/minute and syringe pump was used to remove the reacted mixture from other side of beaker with rate that equal to addition of the agj (16) . this was stirred continuously using a fixed plastic tube around the shift of stirrer in order to prevent turbulence mixing of raft layer and the ph was monitored and recorded. the ph at the raft was measured by another ph probe settled into raft layer. the test was carried out at 37°c using constant temperature cabinet. statistical analysis the one-way anova was used to analyze the differences in the measured properties of the prepared antacid tablets. results and discussion the prepared antacid tablets were showed physical characterization as shown in table ( 3. all formulas exhibited low weight variation that might be as a result of manual feeding of powder into die chamber of tablet machine. the mass variation test was passed by all the prepared formulas because of the means of individual weight of ten tablets were more than 98.5 and less than 101.5%. table ( 3): the physical characterization of the prepared antacid tablets formulas hardness (kg/cm 2 sd), n=10 friability (%sd), n=3 weight variation (%sd), n=10 formula 1 7.90.24 0.540.03 99.60.7 formula 2 7.850.2 0.60.04 99.30.75 formula 3 80,25 0.580.03 99.80.56 formula 4 7.90.24 0.610.03 99.30.62 moss and colleagues showed that, the carbonate content of liquid gaviscon ® had an important role in floating of raft forming antacid (17) . therefore, the floating tablet was manufactured by mixing of calcium and magnesium carbonates with hydrogel forming polymer, through maintaining of a low bulk density of wetted tablet by swelling on contact iraqi j pharm sci, vol.26(1) 2017 raft forming antacid tablet 29 with gastric fluid and trapping of the co2 gas that that formed upon the reaction of carbonate containing antacid with agj. then as a consequence, the wetted tablet has to be buoyant. in addition, a sustained release of acid neutralizing agent was occurring within the erosion of the hydrogel upon stirring. the degree of floating was measured using the residual weight calculations. the experiment was carried out in artificial gastric fluid so as to observe the possible differences. the obtained results showed that, floating behavior was increased as molecular weight is increased and hydration rate is decreased of the polymer. in the preparation of sustained release floating antacid tablets, two formulas were employed using different percentages of active ingredients and polymer, floatation was accomplished by incorporation of gas generating calcium and magnesium carbonates that generate co2 and act as active ingredient; sodium cmc was use as anionic polymer that mediate fdds design on basis of delay gastric emptying time and buoyancy principle. all the prepared antacid tablets showed buoyancy as shown in figure(1). besides, there was a significant difference (p<0.05) in buoyancies among all the prepared formulas, however, first formula showed an average of buoyancy more than the second formula (71.5 % and 38%) while the 4th formula showed the least buoyancy than others. in addition, the results reveal that the increase in the percentage of sodium cmc enhanced the buoyancy. this effect would be responsible for decline in the release of calcium and magnesium carbonates, so give a sustained release compound by acting as rate controlling excipient. figure( 1): the effect of content and number of layers of antacid formulas 1, 2, 3 and 4 on the buoyancy, (mean  sd, n=3). the cross carmelose sodium was used within 5% concentration (13) in tablet dosage form in formulas 1, 2, and in first layer of 3 and 4 while in the second layers was 7.5% in order to fasten the porosity and subsequently the swelling. formula 1 showed the highest buoyancy that is might be due to highest percentage of anionic polymer used than rest of formulas, in addition to the lowest percentage of magnesium carbonate. the 4 th formula showed the least buoyancy than others. this might be attributed to the lowest anionic polymer content and the highest amount of carbonates. the acid neutralizing capacities of the prepared formulas were studied and the obtained results were found to be increasing with time as shown in figure(2). all formulas showed the sustained action of acid neutralization that might result from the hydrogel formation of sodium carboxy methyl cellulose upon contact with agj. this formation reduced the dissolution and release of antacid agents from formulas as used earlier by abbasi and his collegues (18) . though, formula 4 showed the highest effect with a significant difference (p<0.05) from the others. while formula 1 showed the lowest effect. formulas 2 and 3 showed similar effect. the lowest effect that found in formula 1 might be a result of the low content of the salts of antacid which is in the same trend of finding of jagadesh (19) . the highest effect of formula 4 might be achieved as result of the high content of antacid as well as the effect of the rapid release of antacid from the second compressed layer that free from sodium carboxymethyl cellulose polymer. this finding with agreement of the studies on the formulation of sustained release bi layer tablets formulation containing fast release layer (20) . iraqi j pharm sci, vol.26(1) 2017 raft forming antacid tablet 30 figure (2): the effect of composition on acid neutralizing capacity of formulas 1, 2, 3 and 4; (mean  sd, n=3). as antacid tablets dosage form is commonly administered to be taken by chewing (21) or crushing tablet before swallowing. therefore, formulas 3 and 4 were selected and pulverized by mortar and pestle. they were studied for acid neutralizing capacity on comparison with intact tablets and the results obtained as shown in figure 2. the pulverized tablets of both formulas 3 and 4 showed significant (p<0.05) and higher acid neutralizing capacity than that in intact forms as presented in figure (3). figure (3) : the effect of pulverization on acid neutralizing capacity of formulas 3 and 4; (mean  sd, n=3) . in addition, boosting effect at time of 52.5 to 60 minutes was noticed. however, both pulverized formulas 3 and 4 did not show a difference in the acid neutralizing capacity in the bulk of agj. various semisynthetic polysaccharides have been studied and invented to be raft forming agents and naturals are preferred on therapeutic bases and increased the acceptance by the patients (22) . the raft was formed on the top just upon addition of pulverized tablet onto the agj medium for both formulas 3 and 4. the rafts were survived to end of the acid neutralizing capacity study. figure 4 shows the acid neutralizing capacity of pulverized formulas 3 and 4 in bulk and in raft. the raft showed significant (p<0.05) and higher capacity to neutralize the acid in both formulas than that in bulk. the ph's at raft and bulk were approached to equal values after an hour, that reveals the exhaustion of reservoir of acid neutralizing agents in the formulas. conversely, the resistance persists to the end of experiment. figure (4): the acid neutralizing capacity in bulk and raft for pulverized formulas 3 and 4; (mean  sd, n=3). conclusion and future works according to the result obtained from this study it is concluded that a sustained release fdds can be prepared using sodium cmc with magnesium and calcium carbonate. the buoyancy of the prepared fdds is remarkably affected by the ratio of sodium cmc and magnesium carbonate. all formulas of mono and bilayer tablets showed the sustained action of acid neutralization and formation of raft. besides, bilayer tablets showed a significant higher level of acid neutralizing capacity. moreover, the pulverized tablets of formulas 3 and 4 exhibited significant higher acid neutralizing capacity in raft than that in the bulk of agj medium. further works are required to test strength and physical resistance of raft texture. iraqi j pharm sci, vol.26(1) 2017 raft forming antacid tablet 31 references 1. barkun a, leontiadis g. systematic review of the symptom burden, quality of life impairment and costs associated with peptic ulcer disease. the american journal of medicine. 2010;123(4):35866.e2. 2. ching c-k, lam s-k. antacids. drugs. 1994;47(2):305-17. 3. fritsch c, hausler f, seedig j l, inventorsantacid preparation having a prolonged gastric residence time. usa patent 5217794. 1993. 4. berner b, louie-helm j. tablet shapes to enhance gastric retention of swellable controlled-release oral dosage forms. google patents; 2002. 5. vo aq, feng x, morott jt, pimparade mb, tiwari rv, zhang f, et al. a novel floating controlled release drug delivery system prepared by hotmelt extrusion. eur j pharm biopharm. 2016;98:108-21. 6. chen yc, ho ho, liu dz, siow ws, sheu mt. swelling/floating capability and drug release characterizations of gastroretentive drug delivery system based on a combination of hydroxyethyl cellulose and sodium carboxymethyl cellulose. plos one. 2015 ; 10 (1) :e 0116914. 7. forsyth m. collins english dictionary: harpercollins publishers limited; 2014. 8. srivastava ak, ridhurkar dn,wadhwa s.floating microspheres of cimetidine:formulation characterization and in vitro evaluation .acta pharm. 2005 ;55 (3):277-85. 9. mazer n, abisch e, gfeller jc, laplanche r, bauerfeind p, cucala m, et al. intragastric behavior and absorption kinetics of a normal and "floating" modified-release capsule of isradipine under fasted and fed conditions. j pharm sci. 1988;77(8):647-57. 10. zhang c, tang j, liu d, li x, cheng l, tang x. design and evaluation of an innovative floating and bioadhesive multiparticulate drug delivery system based on hollow structure. int j pharm. 2016;503(1-2):41-55. 11. hanif m, abbas g. ph-responsive alginate–pectin polymeric rafts and their characterization. advances in polymer technology. 2017;37(1):12. 12. vais ae, palazoglu tk, sandeep kp, daubert cr. rheological characterization of carboxymethylcellulose solution under aseptic processing conditions. journal of food process engineering. 2002;25(1):41-61. 13. rowe rc, quinn me, sheskey pj, editors. handbook of pharmaceutical excipients. 6th ed. london, uk: pharmaceutical press 2009. 14. pharmacopoeia b. the british pharmacopoeia. london great britain: london : published for the general medical council by constable & co.; 2012. 15. pharmacopeia u. united states pharmacopeia and national formulary (usp 30-nf 25 ) [cdrom]. rockville m, editor2007. 16. washington n, wilson cg, davis ss. evaluation of „ raft-forming‟ antacid neutralizing capacity: in vitro and in vivo correlations. international journal of pharmaceutics. 1985;27:279-86. 17. moss ha, washington n, greaves jl. anti-reflux agents: stratification or floatation? european journal of castroenterology & hepatology. 1990;245:51. 18. abbasi s, yousefi g, ansari aa, mohammadi-samani s. formulation and in vitro evaluation of a fastdisintegrating/sustained dual release bucoadhesive bilayer tablet of captopril for treatment of hypertension crises. research in pharmaceutical sciences. 2016;11(4):274-83. 19. jagadesh dk, k n dcn. study of acid neutralizing capacity of various antacid formulations2015. 20. shiyani b, gattani s, surana s. formulation and evaluation of bi-layer tablet of metoclopramide hydrochloride and ibuprofen. aaps pharmscitech. 2008;9(3):818-27. 21. british medical a. british national formulary 67. standard ed. ed: the british medical association and the pharmaceutical society of great britain; 2014. 22. kapadia cj, mane vb. raft-forming agents: antireflux formulations. drug development and industrial phar macy . 2007;33(12):1350-61. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 37 effects of allopurinol on ketone body metabolism and tissue lipid peroxidation in alloxan diabetes in rats. saleh a. wohaieb receive r 3-8-2003 acce pted 13-6-2004 abstract the a im o f the pres ent stud y is to inv es tiga te whethe r or not xanthine oxid as e (xo)–d eriv ed reac tive oxyge n sp ecies (ros ) may pla y a role in the patho ge nes is of a lloxan (alx)–induce d diabe te s in ra ts us ing the sp ec ific xo inhibito r and hydro xyl radica l sc av enger, a llop urinol the involve ment of oxida tiv e stre ss in alx – diabe te s wa s as ses se d by the mea surement of pla sma and va rious tiss ues lipid p eroxide s le vels ( us ing thioba rb ituric a cid ( tba ) rea ctive substance s ). furthe rmo re , the ab ility o f allopurino l to influe nc e thes e and o ther biochemica l pa ra meters, including plas ma and urine ke to nes le vels we re a ls o inve stigated in diab etic ra ts . rats were divide d into four group s: c ontrol, untrea te d diabe tic , allopurino l – trea te d diabe tic, and insulin – tre ated d ia betics . at the e nd o f the one wee k experimenta l pe riod, bloo d and tiss ue sa mples were ob ta ined fro m ane sthe size d anima ls for the mea sure ment of the ab ove – me ntione d pa rameters. although the s ingle intrap eritonea l (i.p.) injec tion of a llop urinol (25 mg/kg bod y wt.) 1h be fo re o r 1h after alx injec tion (10 0 mg/kg b ody w t., i.p.) faile d to p re vent the ind uction o f diab etes , it did lo wer ke to nuria and the inc idence of early ketos is–a ss ociated morta lity in diab etic animals in co mparison with non– allo purinol–treate d d ia be tic rats. s ubs eq uent ad ministra tion o f allopurino l (25 mg/kg b ody wt., i.p.) e very 48 hr for 1 wk (i.e., 3 a dditio nal dos es ) als o d ec re ase d plas ma ke to ne b odies lev els as well a s plasma and tiss ue (hea rt, liver, kid ney, pa nc re as) lipid pe ro xides lev els in c omp aris on with no n–a llop urinol–tre ated dia betic ra ts . da ily ins ulin inje ctio n (9–1 2 u/kg bo dy wt., s.c.) for 1wk pe riod no rma lize d all o f the ab ove –me ntio ne d abnormalities . the pre se nt res ults sugge st that xo–de rive d ros play a minor role (if a ny) in the diabe to ge nic effe ct of alx. on the othe r ha nd, although the me chanism (s) und erlying the protec tive e ffe cts of a llop urinol on the d ia betic state is prese ntly unknown, the se e ffec ts ma y re fle ct a p oss ib le a ss ociation be tween impaire d ke to ne b ody me tabo lism a nd lip id peroxid atio n: a nd s ugges t a n effect of allo purinol on ketone bod y meta bolis m. :خالصة  ز٬ كسدي ن انزيم الزانثين او كونة م مت وال علة ٬ ن المتفا جي وكس جود أي دور ألنواع األ كانية و هو تقصي إم ة ن الدراسة الحالي هدف م إن ال زيم ط الخـاص ألـن ب ول ٬ المـث وريـن ار االلوبي خدام عـق ك باسـت ن ٬ وذـل ن في الجـرذا سا طة االلوك ث بواس محد ري ال مراضية داء السك في ا وك وكسيل الحرة الزانثين ا هيدر ور ال جذ ح كذلك كاس .سديز و وكسيدات الدهن مستويات بير الل قياس كسان من خ طة االلو ث بواس محد ري ال هاد التأكسدي في داء السك وتم تقييم الدور الذي يؤديه االج سجة الن من ا عديد وال ما ك ( في البالز ورـي وباربتي مـع حمـض الثاي ة مواد المتفاعل ة ). بأستخدام ال ك باالضـاف ذل ار , ـل عـق ة م دراسـة قابلـي ـت خـرى ة اال ة الحياتي ميائي كي ر ال معايي كذلك على ال عايير و ي , االلوبيورينول في التأثير على هذه الم ات ـف وـن كيت ويات ال ت ـس ومـن ضـمنها م سكري مصابة بال ي الجرذان ال ول ف زما والب مجاميع. البال ة ن الى اربع جرذا رة: وتم تقسيم ال ر , السيط غـي ي سكر مصابة بال ة ال عالجـ , الم ة بااللوبيورينول ري والمعالج ة بالسك ولين, المصاب ة باالنس عالج كري والم ة المصابة بالس وع مجم ممتدة . و جربة ال وبعد انتهاء فترة الت وع واحد سب عايير اعـاله , ال ن اجل قياس الم رة م خد من الحيوانات الم سجة الن وا من الدم ن ٬ .تم أخذ عينات ن الحـق مـن ا رغم ى اـل عـل و وا مرة ول ول ورين وبي الل ون /ملغم 25(حدة ٬ لعقار ا سم في البريـت زن الج غم و كسـان ) ك ن االلو حـق ن ـم عـد سـاعة عة أو ب ل سـا 100(قـب غم جسم في البريتون/مل زن ال غم و ى ) ك ذلك إـل ـك وى الكيتون في البول ٬ و ى إلى تقليل مست ري ٬ إال إنه أد ٬ قد فشل في منع حدوث السك ر كرة الم وث الوفيات المب وزية انخفاض حد كيت جسم(افقة لل ط الكيتون في ال ع تلك غير ) فر كري بالمقارنة م ة بالس ي الحيوانات المصاب ف ة بااللوبيورينول ول . المعالج ورين اللوبي مرات ٬ لعقار ا ولثالث الحق ٬ ى الحقن ال غم 25(وقد أد رة /مل ن ٬ م ريتو جسم في الب وزن ال غم ك ة 48كل و) ساع ك إلى تقليل مست ما والعديد لمدة أسبوع ٬ كذل ي البالز هن ف كسيدات الد ت بيرو مستويا وخفض ما ٬ ي البالز ون ف كيت يات ال جة ن األنس رياس(م ة ٬ البنك كبد ٬ الكلي ب ٬ ال ول ) القل ورين وبي الل جة با معال ر ال غي ري سك ة بال مصاب وانات ال حي ع تلك ال وقد أدى . بالمقارنة م مي باالنسولين 1–9(الحقن اليو جسم ٬ /وحدة 2 وزن ال غم حت الجلدك ي ) ت ة ـف عـي ر الطبي غـي رات ع التغـي مـي لمدة أسبوع إلى تصـحيح ج ري عاله في حيوانات السك كورة أ مذ ود دور ضـئيل . المعايير ال ـج ة لو ج الحالـي ائ ة ) إن وجـد (وتشير النـت متفاعـل كسـجين ال واع األو ألـن ن سا وك الل ري ل ث للسك محد ي التأثير ال وكسديز ف ن أ زانثي زيم ال من أن ة من نا. والناتج رىو ة اخ ة ,حي آللـي ة ا رـف مـن عـدم مع رغم وعلى ال آلليات( ي ) ا ل ـف ين الخـل ما ـب وجود عالقة ة مكاني كس إ ها قد تع ر٬ إال إن حاض ول في الوقت ال ورين ن التأثيرات المفيدة لأللوبي ؤولة ع المس ري العملية رض السك ن في م كسدة الده رو وبين بي ن كيتو ة لل جود تأثير لعقار,االيضي عملية وقد تشير أيضاً إلى و ورينول على ال االلوبي ن كيتو ة لل .االيضي dep t. pharma colog y an d tox ic ology, co lle ge of pha rmacy, univers ity o f baghdad, bagh dad – ir aq . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 38 introduction there is co nside ra ble ev id enc e that xa nthine oxid as e (xo) sys tem (o ne so urce of superoxid e rad ic als in the bo dy) ma y b e invo lve d in the generatio n of rea ctiv e oxyge n sp ecies (ros )in s eve ra l co nd itions a ss oc ia te d with o xidative s tres s, includ ing chro nic obs tructiv e p ulmonary d is ea se (1), isc hemia – re pe rfus io n injury (2), e ndo thelia l dysfunctio n in typ e ii diab etes (3). furthermore, in v itro stud ie s hav e sugge sted that ros (ge ne ra te d during the oxid ation of hypo xa nthine by xo) may p la y a role in c ausing o xidative d amage to ra t pancre atic –c ells (4). the p re se nt s tudy wa s therefore undertaken to inve stigate whe ther or not xo–de rive d ros may p la y a ro le in the pa thoge ne sis of alx– induced diabe tes using the sp ec ific xo inhibitor (a nd c ons eq uently of superoxid e ge neration) as well a s hydroxyl ra dical sc ave nger, allopurino l (5). if the mechanis m (s) of ros prod uc tion in alx–d ia bete s is med ia te d, a t lea st in pa rt, through the enzyme xo, the n allo purinol sho uld either p re vent or at le ast ame liorate the d ia be tic sta te . materials and methods adult fema le wista r ra ts (20 0–2 50 g) were ho use d in hanging p la stic cages in a room kept at 2 2–2 5c with a 1 4hr light and 10 hr d ark cycle. anima ls we re a llowe d free ac ce ss to fo od and water during the entire exp erime ntal pe riod exce pt tha t o f the inductio n of d ia bete s whe re a nimals were fas te d fo r 48 hr p rior to the ad minis tration o f alloxa n (6). diabe te s was induce d in ethe r– ane sthe size d anima ls by the intra pe ritone al (i.p .) inje ction of allo xa n (s igma che mic als co , usa) at a d os e of 10 0 mg/kg bo dy w eight a s a fres hly prepa re d so lution (10 0 mg/ml) in sa line . alx– trea ted anima ls were allo wed to drink 5% gluc ose s olution ov ernight to o ve rc ome drug– induced hypo glyce mia (6). the diab etic s ta te was mo nitered by the frequent da ily tes ting for gluc osuria (with lilly te s–tape , eli lilly & co, us a) a nd fo r ketonuria (using strip s obta ined fro m ketostix, ames co , us a). the follow ing group s were studied : 1control rats (n = 6). 2untre ated d ia betic ra ts (n = 1 2). rats of this group re ceive d the alka line v ehic le (0.2 –0 .2 5 ml o f sa line , p h 1 2.0) i.p. 1 hr b efore (n = 6) or 1hr (n = 6 ) after alx inje ctio n. diabe tic rats which survive d this p erio d re ce iv ed further a lkaline vehic le injec tio ns at 48, 96 a nd 14 4 hrs after alx inje ctio n. 3allop urinol–tre ated d ia betic ra ts (n =12). allo purinol (sigma chemica l co, usa) wa s prepa re d at a co nce ntra tio n o f 25 mg/ml of sa line ph 12 .0 . rats re ce iv ed a s ingle i.p. inje ctio n of 0 .2 –0.2 5 ml of alkaline ve hicle to atta in a fina l do se o f 25 mg/kg b od y wt. 1hr before (n = 6 ) or 1hr (n = 6 ) a fte r alx inje ctio n. then a llop urinol wa s furthe r give n at 4 8 hr, 9 6 hr a nd 14 4 hr o f the alx–diab etic period. the d ose o f allopurino l was ¼ o f that dos e (1 00 mg/kg bod y wt.) re ported to prov id e protection aga inst oxid ative– stre ss –induce d lipid pe ro xidation in va rious tis sue s of ra ts (7). 4insulin–treate d diabe tic rats (n = 6) : alx–d ia be tic a nimals were injec te d with insulin zinc s us pe nsion (lente mc, nov o indus tri a/s, de nma rk) sub cuta neo us ly a t a daily do se o f 9 –12 u/kg bo dy wt. (6) imme diately afte r detec tion o f the diab etic state. the do sa ge of insulin was ad justed b y daily monitering urina ry gluco se and ketone le vels. at the end of the 1 wk exp erime ntal perio d, a nimals were a ne sthe size d with e ther, and hep arinis ed bloo d samples were ob ta ined by c ardiac puncture. animals we re kille d by c ardiac e xcis ion, a nd tis sue (he art, pancrea s, liver a nd kidney) homogenates (1 0% w/v) w ere prepa re d in ice –c old 50 mm tris – 0.1m edta buffe r, ph 7 .6 . p la sma & tis sue malond ia ld ehyde (mda) lev els (as a n inde x of lipid p eroxida tio n) were determine d b y the thio barbituric ac id (tba) re action (8). the tba–rea ctiv e s ubs tanc es were c alculated using a n e xc tinc tion co effic ie nt of mda of 1 .56  10 5. plasma lev els o f gluco se, c hole sterol & triglyce ride s as well a s uric ac id (to a ss ess xanthine o xida se a ctivity) we re mea sured using commercial a ss ay kits (sigma chemica ls co, us a). pla sma ketone b odies lev els (– hyd ro xyb utyrate) we re mea sured us ing the metho d o f williamson and ma lla nby (9). – hyd ro xyb utyrate is q uantitatively the predo minant ke to ne bo dy pres ent in the blood in unc ontrolle d diabe te s me llitus (10). statis tic al analys es were performed using anova a t significance leve l of p < 0 .0 5. f urther spe cific gro up d ifferenc es we re d etermined using tuke y’s tes t. results ge neral features of diabe tes : the a dministratio n of the a lkaline vehic le (whe ther giv en 1hr be fo re or 1 hr a fter alx inje ctio n) did not p re vent the incidence of d ia be te s, and p ro duc ed c ompa ra ble picture in ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 39 re gard to de gree of ke to sis and the early morta lity amo ng diab etic ra ts . induc tio n o f diab etes wa s co nfirmed b y the pres enc e of gluco suria and ke to nuria within 2 4 after alx – inje ctio n. during the firs t we ek of diab etes, 6 animals died in ketosis a nd d ia betic co ma, whic h s tarte d a s ea rly as 48 hr a fter inductio n of d ia be te s. anima ls whic h survive d this pe riod (6/12) also s ho wed hype rglycemia , hypertriglyc erid emia , a nd ketone mia (table 1) at the end o f 1 wk pe riod . how eve r, le vels of plas ma choleste ro l and uric a cid were not changed during this p erio d (ta ble 2). plas ma and tiss ue lipid peroxid e lev els w ere a ls o elev ated in alx– diabe tic rats (tab le 3 ). effect of allopurinol tre atme nt al though the s ingle i.p. injectio n of allopurinol (25 mg/kg b ody wt.) giv en 1hr prio r to or 1 hr pos t alx a dministra tion did not prevent the induc tio n o f diabetes , it did lower the inc id ence of early ketos is–associated mortality in these animals. unlike the high mo rtality in no n– allopurinol–treated diabetic a nimals, only 2 of the 12 a llop urinol– trea ted diabetic rats die d in ke tosis and coma (table 1). the re maining 10 a nimals showed gluc osuria a nd slight ketonuria. treatme nt with allopurinol fo r 1 wk pe riod did a ls o lower the assoc iated ke to ne mia (ta ble 1) as well as the lipid pe roxide leve ls in plas ma and tiss ues of allo purinol–trea te d d iabe tic ra ts c ompared to the no n –tre ated d iabetics (table 3). effect of insulin tre atment insulin trea tment (9 –1 2 u/kg bod y wt/da y) starte d immediately a fter d etec tion o f diabetes preve nted the ketos is –associa ted c oma and morta lity, and normalize d a ll o f the a bove– mentioned ab norma litie s (tab le 1–3). table 1: plasma a nd ur ine ke to ne bo dy leve ls and mortality rate in c ontrol, untre ated–alx dia be tic, a llo pur inol–tre a te d–alx diabe tic, and ins ulin t re ate d–alx diabe tic rats after 1wee k of diabe te s va lue s a re e xpresse d as me an  sd. va lues with diffe re nt le tters are significa ntly diffe re nt (p < 0.05). * ur ine ketone body le ve ls we re qua litativ e ly measure d using ke tostix strips o btaine d fro m ame s co, usa, as describe d in mate rial and me thods pla sma k etones (hyd ro xybutyrate) (mol/l ) u rine ke tone s* (acetoa ce ta te) number o f animals d ied with d iabe tic co ma/1wee k con trol (n = 6) 0 .6 8  0.29 a (– ) – un treated – dia betic (n = 12) 9 .2 0  3.60 b (+ + + ) 6/12 anima ls (50 %) a llop urinol –trea ted dia betic (n = 12) 3 .5 4  2.02 c 2 (+ + ) 2 (+) 8 (–) 2/12 anima ls (17 %) in su lin – trea ted dia betic (n = 6) 0 .9 8  0.35 a (– ) – ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 40 ta ble 2: body we ig ht a nd bioc he mic al parame te rs in control, untre a te d–alx d ia be tic, allo pur inol–tre a te d–alx diabe tic, and ins ulin treate d–alx dia be tic rats. g ro up body wt. (g) plas ma le ve l (mg /dl) g luco se triglyce de cho les te rl uric acid contro l (n = 6 ) 2 31  24 146  9 a 104  23 a 65.823.2 1 .80 0.15 u ntreated – diabe tic (n = 6 ) 2 19  26 47875 b 6 35  28 7 b 61.919.3 1 .70 0.19 a llo purino l– treated diabe tic (n = 10 ) 2 28  18 497  120 b 5 95  21 2 b 52.511.6 1 .73 0.22 insulin– treated diabe tic (n = 6 ) 2 38  32 10930 a 168  53 a 5 8  1 9.3 1 .68 0.20 values are expre sse d as mean  s d. with in e ach column, va lue s with diffe re nt le tters are signif ic antly diffe re nt (p < 0 .05). ta ble 3: pla sma and tis sue tba–reactive substances (a n in de x o f lipid pe roxida tion) in control, untre a te d–alx d ia be tic, allo pur inol–tre ate d–alx diabe tic, and ins ulin tre ate d– alx diabe tic rats. gro up tba –re ac tive s ubstances plas ma n mol/ml nmo l/g we t tissue he a rt live r kidne y pancre as co ntro l (n = 6 ) 1.44 0 .32a 178  2 5 a 451  37 a 221  33 a 184  34 a untreated – d iabe tic (n = 6 ) 5.93 2.80 b 600  7 5 b 6 12  95 b 386  6 5 b 368  44 b allopurino l – trea ted d iabe tic (n = 10 ) 2.28 1 .32a 351  8 8 c 273 101 c 298 60 c 280  67 c ins ulin– trea te d d iabe tic (n = 6 ) 1 .65 0.5 4a 1 56 13 a 460  84 a 218  27 a 172  57 a value s are e x pre ss e d as me an  sd. within e ac h column, v alue s with diffe re nt le tte rs are sig nifica ntly diffe re nt (p < 0.05). ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 41 discussion the d ia be tic state prod uc ed in rats d uring the first wee k follo wing alx inje ctio n wa s co nfirmed b y gluc osuria , ke tonuria, hyperglye mia and ketonemia. the d ia betic state wa s also a cc ompa nied b y elev ated plasma and tiss ue lip id pe ro xides lev els as well a s increa sed inc idence of early keto sis – re la te d mortality. the ab ility of insulin therapy to comp le tely re ve rse the se c hange s s ugge sts that the y are re la ted to the diabe tic state in this strictly insulin– dep endent mo del of exp erime ntal diab etes (6). in a n a tte mpt to inv es tiga te whether o r not allo purino l c an p re vent the inductio n of diab etes, the single i.p . ad minis tration of 2 5 mg/kg b ody wt. fa iled to prev ent the inductio n of d ia bete s, whethe r it was ad ministra te d 1hr be fo re or 1hr afte r alx injection. this fac t is sugge stive of a mino r ro le (if a ny) of xo – ge nerated ros in the diab etoge nic action of alx, and d is agree s with the conclus io n re ac hed b y helle r et al.(4) who s ugges ted that ros ge ne ra te d by xo are re sp ons ib le for the de structio n o f ins ulin –prod ucing –c ells during ins ulitis. howe ver, the single a llop urinol a dministratio n was a ble not only to lower ketonuria , but a ls o the inc id ence o f e arly ke to sis–a ss oc ia te d mortality in diabe tic rats, a fact sugge stive of a pos sible effe ct o n ketone b ody me ta bolism. furthe rmo re , preliminary experiments rev ea le d that after a s ingle i.p . a dministratio n of allo purino l, urinary ketone b odies sta rt to increa se (afte r the initia l d ecline in leve l) again within 5 0–6 0 hr in a llop urinol–tre ated dia betic anima ls : and b y a pproxima te ly 80 hr of diab etes no significa nt diffe re nc es co uld b e obs erve d betwe en a llop urinol–tre ated and non– allo purinl–tre ated d ia betic anima ls . this find ing, s ugges ts the invo lv eme nt of allo purino l and /o r its me ta bolite a lloxanthine in these p ro te ctive effects , give n that the half– life o f a llop urinol is 2– 3 hr a nd that of allo xa nthine is 1 8–3 0hr(11). acc ordingly, the pres ent e xpe riment also a pplied rep ea te d ad minis tration o f allo purino l ev ery 48 hr for 6 da ys. inte re stingly, thes e rep ea te d ad minis tration o f allo purino l did lo wer ketone bod y le ve ls in bo th urine a nd plas ma and the lo wered mo rtality rate (d ue to the firs t single dos e) was ma intained till the e nd of experimenta l pe riod (1we ek). it is imp orta nt to po int out that although alka li trea tment has b een reco mmende d to correct diab etic ketoac id os is (12), the pos sibility that the ab ility o f allo purino l to lowe r ke tonuria is in fa ct a ttrib utable to the high alkalinity of the disso lv ing vehic le (ph 12) rathe r than to a dire ct effec t of allopurino l per s e has b een rule d out. dia betic anima ls re ceiving the s ame alkaline ve hicle fa ile d to de monstra te s imilar protective e ffec ts . in a c ompute r med– line se arch o f the literature from 1 970 –2 002 , no co mparab le re ports co nce rning the effe ct of a llop urinol trea tme nt on keto ne b ody metab olis m in diabe te s were found : the re fo re , the mechanis m (s ) re spo ns ib le for the protective effects of allo purinol a gains t the increas ed le ve ls of ketone bo dies rema in (s) to be inv es tiga te d in furthe r stud ie s. it is pos sible that a llop urinol exe rts its e ffe cts thro ugh a n inhibitio n o f xo; howe ve r, the fa ct that a llop urinol trea tme nt did not lo wer p la sma uric acid in alx –diab etic ra ts make s it an unlikely po ss ib ility. the ability of allo purinol to lowe r ketonuria and ke tone mia co uld b e e xp la ined thro ugh either inc re as ing ke to ne bo dies me tabo lism by various tiss ues and /o r de crea sing the ir synthes is via a ffec ting dea cyla se o r – hyd ro xy––methylgluta ryl– coa fo rma tion (13). it is unlikely tha t allo purinol might have enhanced e nd oge nous insulin relea se be ca use other ins ulin–d epe nde nt a bno rma litie s we re not correcte d by allopurino l tre atment. the a bility o f allo purinol to lo wer ros– induc ed lipid pe ro xidation provide s ano ther evide nce for the potential benefic ial effec t of allo purinol tre atment o n tiss ue a ntioxida nt defense s a ga inst d ia bete s–ind uc ed increa sed oxida tive s tre ss. it further s up ports the findings of o ther s tudies which repo rted that a llop urinol lo wered mda leve ls in p la sma o f p atie nts with type ii diab etes with mild hypertens ion (3) and in v ario us tis sues o f ra ts exp os ed to oxida tive stre ss ind uce d either b y cypermethrin (7) or by exp erime ntal acute pancrea titis (14).furthe rmo re , the pos sibilities of a dire ct effe ct of allo purinol a nd a lloxanthine on ketone bo dy me ta bolism and lip id p eroxida tion indep endently of inhibition of xo, o r v ia sca ve nging the powe rful hyd ro xyl ra dica ls (14), or ma y invo lv e ge neralized alte ra tions in tis sue antioxida nt s tatus (2) can not be ruled out at prese nt. muc h more wo rk mus t be do ne to ass es s whethe r the re is any re latio nship betwee n lip id p eroxida tion a nd ketos is in alx diabe te s and furthe r inve stigate the protec tive effe cts of alp in this re gard. in c onc lusion, the pres ent stud y s ugges ts that und er the c ond itions tes ted and in the do ses and dura tion of treatme nt us ed , a llop urinol thro ugh its p rob ab le or ap parent ab ility to inhibit ge ne ra tion o f ros (es pe cially hydroxyl ra dical sca ve nging) ra ther than xo inhibition ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 42 ap pea rs to hav e a p ro te ctiv e effe ct on b oth lipid pe ro xida tion a nd ketoge ne sis and co nse quently early morta lity in alx– diab etes . references 1. heunks lm; vina j; van – herwaa rde n cl, et al. xanthine o xidas e is inv olve d in exe rc is e– induc ed o xidative s tres s in chro nic obs truc tive pulmo nary d is ea se. am. j. physiol. 19 99 ; 27 7: r1 697 –70 4. 2. ala ta s o ; sa hin a; colak o , et al a l. benific ial effec ts of allo purinol o n glutathione leve ls and glutathione peroxid ase a ctiv ity in rat isc hemic ac ute re nal fa ilure . j. int. med. res . 1 996 ; 2 4: 3 3 – 3. butle r r; morris ad; be lc h j j, e t a l. allop urinol no rma lizes endothe lial dys func tion in type ii d ia be tics with mild hyp erte ns io n. hype rtension 2 000 ; 35 : 74 6 – 51. 4. helle r b; burkart v. antioxid ant therapy fo r the p re vention of typ e i diab etes . adv. pha rma col. 199 7; 3 8: 6 29 –6 38. 5. knight j a. free rad ic als, antioxida nts, aging a nd dise ase . (ed.). ame rica n ass ociation for clinic al chemistry. 19 99: 215 – 230 . 6. bell rh; hyd e rj. anima l mode ls of diabe te s mellitus : phys io lo gy and patho lo gy. j. surg. res . 1 983 ; 35 : 43 3 –60 . 7. giray b, gurb ay a; hinc al f. cype rme thrin–induced oxida tive stre ss in rat b ra in and liver is p re vented by v itamin e or a llop urinol. to xico l. lett. 20 01; 118 : 13 9 – 4 6. 8. ohka wa h; ohis hi n; yagi k. as sa y for lipid pe ro xides in animal tis sues b y thio barbituric a cid re action. ana l. biochem. 1 979 ; 9 5: 3 51– 58 9. williams on dh; mellanb y j. d–(–)–3 – hydroxybutyra te. in: be rgmeyer hu (ed ): me thod s of enzymatic analys is . v.4. new york. ac ad emic press . 1 974 ; 18 36 – 39. 10. s acks db. ca rbo hydrates. in: tie tz te xtboo k of clinical c he mis try. burtis ca, ashwo od er (ed ). 3rd ed ition. wb saund ers compa ny. philad elphia . 19 99; 785 – 87. 11. ra ng hp; dale mm; ritte r jm. p ha rma cology (4th e d.). churc hill liv ings to ne. edinb urgh. 19 99; p. 2 40 . 12. me rc k manual o f diagnos is a nd the ra py. (16 th ed.). merck a nd co. inc. rahwa y nj. 1 992 ; p p. 1 022 13. ga no ng wf. re view of me dical p hysiology. 18 th (e d). app le to n and lange. s ta nd fo rd. 19 97; p. 2 82 –2 83. 14. czako l; taka cs t; va rga is, et a l. oxidative stress in distant orga ns and the e ffe cts o f allo purinol d uring exp erime ntal a cute p anc re atitis.int.j .anc re atol. 20 00; 27: 2 09 – 16. iraqi j pharm sci, vol.20(2) 2011 microbial contamination of eye drops 91 microbial contamination of eye drops in out patient in iraq # raghad a. razooki *,1 , ebtihal n. saeed * and hanan i. omar al-deem * *department of clinical laboratory science,college of pharmacy,university of baghdad, baghdad, iraq. abstract acontaminated ophthalmic solutions represent a potential cause of avoidable ocular infection. this study aimed to determine the magnitude and pattern of microbial contamination of eye drops in out patient at the department of ophthalmology, at baghdad national hospital, iraq. fifty four vials from the out patient clinic were obtained for microbial examination after an average use of 2 weeks. the dropper tip and the residual eye drop were examined for contamination. the specimens were cultured, the number of colonies counted, the organisms identified. eight (15%) out of 54 analyzed vials were contaminated , most bacteria identified belonged to the normal commensal flora of the eye. isolated contaminants were staphylococcus auereus, micrococcus , neisseria catarrhalis, gram negative rods, candida albicans, and staph epidermidus.the dropper tip was more often contaminated (n=5) than the residual solution (n=2) and only one vial showed acontamination of both the drop and the tip (n=1) . our data show acontamination rate of 15%, which is in the medium range of data puplished on the contamination of eye drops elsewhere (0.07% to 35.8%). key words : microbial contamination, eye drops الخالصة ٚؤد٘ انٗ انؼًٗ احٛاَاً نزنك قذ َّ انسثة انشئٛسٙ فٙ انرٓاب انؼٍٛ. ٔيخاطش رنك ًٚثم ذهٕز قطشاخ انؼٌٕٛ خطشاً كثٛشاً ال ٙ نقطشاخ انؼٌٕٛ نهًشضٗ فٙ ػٛادج انشدْح انخاسجٛح نقسى انؼٌٕٛ فٙ تذٓذف ْزِ انذساسح انٗ ذحذٚذ يذٖ ٔيسرٕٖ انرهٕز انًاٚكشٔ ا تؼذ يؼذل اسرؼًال اسثٕػٍٛ. ساسٙ نٓتنرهٕز انًاٚكشٔيسرشفٗ تغذاد انؼاو فٙ انؼشاق. استؼح ٔخًسٌٕ قطشج جًؼد ٔذى قٛاس ا انقطشج ٔتقٛح انًحهٕل انًرثقٙ خضغ نهفحص انًاٚكشَٔٙ ٔتؼذ صسع ٔػذ انًسرؼًشاخ ذى ذحذٚذ إَاع انثكرشٚا انًهٕثح. ثًاَٛح قطشاخ انًٕجٕدج فٙ انؼٍٛ أ انجهذ ٔيُٓا ( قطشج ذحٕ٘ تكرشٚا يهٕثح يؼظًٓا ذؼٕد انٗ انثكرشٚا انطثٛؼٛح15%( يٍ يجًٕع )51ا٘ تًؼذل ) staphylococcus aureus, micrococcus, neisseria catarrhalis, gram negative rods and candida staph epidermidus albicansand يٍ تقٛح انسائم انًرثقٙ فٙ انقطشج ٔٔاحذ فقظ اظٓش اً هٕثذانقطشج ٔجذ اٚضاً اكثش سأس ٙ نقطشاخ انؼٍٛ يٍ انًؼهٕياخ انًُشٕسج فٙ ت%( ٚؼرثش حذ ٔسظ نهرهٕز انًاٚكش51ٔيسرٕٖ انرهٕز )ٔ سأس انقطشج. ذهٕز انسائم . (%81.3-0,00اَحاء انؼانى ْٕٔ يا حذد تـ) introduction contaminated eye drops and ophthalmic solutions are a potential cause of ocular infection. they can be associated with keratitis. (1) and corneal ulcers (2) and carry the risk of transmitting opportunistic microorganisms, (3,4) as well as pathogenic organisms, such as pseudomonas aeruginosa and serratia marcescens. (1) the published contamination rate of in-use ophthalmic solutions varies widely in the literature from 0.07% (5) to 35.8%. (3) a part from the risk of infection, bacterial contamination of eye drops may alter the ph of the solution and therefore reduce the efficacy of the drug (6) .in order to prevent contamination, most preparations contain antimicrobial substances, unless the solution it self has an antimicrobial effect. these substances aim to preventing or inhibiting the growth of microorganisms which increase the risk of infection or degradation of the drug. the self sterilizing effect of eye drops caused by the presence of preservatives has been discussed controversially (7) . preservatives must meet several requirements (1) to be compatible with other ingredient ,(2) to be efficient during the entire duration of use of eye drops, and (3) to be non toxic. commonly used preservatives of ophthalmic solutions are benzalkonium chloride, which also works as a detergent and therefore increases the penetration of the active ingrediente of the drug, thiomers; chlorhexdine; parahydroxy benzoate; phenylmercuric nitrate, edta, chlorobutanol; benzylalcohol; phenylethyl alcohol; and parabens (8,9) . as preservatives interfere with the metabolism and inhibit the growth of micro-organisms, they may have similar effects on human cells, explaining potential cytotoxic effects and inflammatory cell responses. (6) the antimicrobial activity is important for the rate of infection resulting from contamination during the process of instillation. contact with fingers or lids, ciliaries conjunctiva and cornea are possible causes of contamination even if instilled by healthcare professionals. # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1corresponding author email :raghadrazook@yahoo.com received : 7/3/2011 accepted : 19/9/2011 iraqi j pharm sci, vol.20(2) 2011 microbial contamination of eye drops 92 plastic bottles have been reported to be more commonly contaminated near the bottle cap. this has been attributed to a lack of preservative at this area. (4) in a clinical study 220 in-use medications of 101 patients with non-microbial acular surface disorders were examined by cultivating the bottle caps. the authors concluded that acycle of contamination between in-use medications and conjunctiva may present an important risk factor for microbial keratitis in patients with ocular surface disease (10) .the occurrence of bacterial ocular infection such as keratitis and endophthalmitis transmitted by contaminated eye droppers has been reported. (11) in a recent study the authors noted that some cases of bacterial keratitis in iran are thought to be due to contaminated eye drops used on multiple patients (12) . brudieu et al found abig difference in contamination rates between vials used by ophthalmological patients (17.7%) and vials used by medical and gerontological patients (35.8%). a positive correlation was also found for vial contamianation and the duration of use vials containing an antimicrobial agent were less likely to be contaminated than vials without antimicrobials. however no clinically relevant infection through such vial contamination was identified (3) . in a study comparing the contamination rate of drops used in an eye department and a nursing home no difference was found but the authors stated that the residual eye drop is more often contaminated than the tip of the bottle. they also noticed the presence of. gram negative organisms in the nursing home (7) . most studies, however, found the bottle tips to be more often contaminated than the solution. (2,4,13) therefore, topical eye medications may present a potential risk of infection, aspecially if the ocular epithelial barrier is compromised. minimising the contamination of eye drops and the transmission of infections is an important issue in clinical ophthalmology. several regulations and suggestions have been made in this context. we conducted the following cross sectional study and analysed (54) ophthalmic solutions examining bacterial and fungal contamination. this study aimed to determine the magnitude and pattern of microbial contamination multi dose of eye drops in out patient. material and methods a total of (54) eye drop containers ( in use ) were obtained for microbial examination. the microbial analysis was performed on the dropper tip and the residual eye drop for each containers. the eye drops were obtained and cultured according to the following:  a sterile cotton tip swab was moistened in sterile brain heart infusion (bhi) enriched medium before wiping the nozzle tip of the eye drop containers and then used to inoculate the culture plates (13) .  the vials were inverted and one drop was directly inoculated on each of the media and then spread across the plates. all media except the sabouraud agar plates were incubated at 37 0 c for 48hrs. and evaluated after 24 and 48hrs. the blood agar, chocolate blood agar plates were incubated in amicroaerophile environment. (15) the sabourand agar plates were incubated at 30 0 c for up to 10 days and evaluated for growth on days 1, 5 and 10. the bhi broth was also incubated at 37 0 c and subcultured on blood agar after 24 hours. all culture media except sabouraud dextrose agar (biomerieux, f) were obtained from biotec laboratories ltd, uk. a significant growth was considered a growth on the main inoculation site or on two or more streaks on the plate. the bhi was analysed for changes in colour and turbidity of the media. the colonies on solid media were counted and all organisms identified by microscopy after gram staining and biochemical tests. results a total of 54 medications were analysed; from the out patient clinic. as shown in table (1) the used of different preservatives found in the analysed specimens. they were grouped in four different categories analgesics, antihistaminc, antibiotic and steroids. . over all, 8(15%) of the 54 analysed vials were contaminated table (2) at the bottle tip alone or with additional contamination of the solution or solution alone. within the four categories the rate of contamination varied between 0% for analgesics and 17% ---> for both antibiotics and steroids.the dropper tip was more often contaminated (n=5) than the residual solution (n=2). one bottle showed contamination of both the dropper tip and the medical solution. (n=1) most of the identified organisms were part of the normal skin flora or conjunctival flora. gram positive organisms were cultivated from five of the eight contaminated medications and two contaminated medications grew gram negative organisms and one grew candida albicans. one of the medications grew more than one bacterium as shown in table 3 . none of the medications were found to be past the expiry date. iraqi j pharm sci, vol.20(2) 2011 microbial contamination of eye drops 93 table 1 : type of preservatives and number of medications no. preservative number of medications 1 benzalkonium chloride0.05% 20 2 benzalkonium chloride0.02% 14 3 benzalkonium chloride0.01% 8 4 b.chloride 0.004% 4 5 thiomersal 0.005% 4 6 phenyl mereuric nitrate 0.001% 4 total 54 table 2 : eye medication and contamination *(n= 54 ) total eye drops specimens table 3 : contaminated medications and preservatives (n=54). no. eye medication preservative contaminant 1 antistin-privin benzalkonium chloride 0.02% 2types of colonies on blood white colonies and yellow (staph aureus) 2 antistin-privin benzalkonium chloride 0.02% 2types of colonies on blood white colonies and yellow (staph aureus) 3 oflox benzalkonium chloride 0.05% on msa staph aureus 4 oflox benzalkonium chloride 0.05% on blood agar :candida albicans staph. aureus 5 oflox benzalkonium chloride 0.05% neisseria catarrhalis 6 oflox benzalkonium chloride 0.05% staph aureus 7 dexamethason 0.1% benzalkonium chloride 0.01% micrococcus 8 dexamethasone phosphate. 0.1% benzalkonium chloride 0.01% grods e y e m e d ic a tio n n u m b e r te ste d c o n ta m in a te d % t ip c o n ta m in a tio n t ip /d r o p c o n ta m in a c tiv e (n = 5 4 )d r o p * c o n ta m in a tio n c o n ta m in a n t analgesic 4 0(0%) antihistamine 14 2(14%) 2(14%) two type colonies 1-white colonies coagulase(-). 2-yellow colonies coagulase(+). staph. aureus antibiotics 24 4(17%) 4(17%) 1-candida albicans 2-staph aureus 3-neisseria catarrhalis steroid 12 2(17%) 1(8%) 1(8%) micrococcus gram negative rods total 54 8(15%) 5(9%) 1(2%) 2(4%) iraqi j pharm sci, vol.20(2) 2011 microbial contamination of eye drops 94 discussion we noticed amicrobial contamination of 8/54(15%) of out use application dispensers. the mean contamination rate of preserved eye drops described in the literature varies widely from 0.07%. (5) to 35.8%. six different micro organismes were detected. as the containers were analysed on the day of collection, our results are likely to represent the specific clinical situation of that day. five of eight of the identified organisms were gram positive and 2/8 gram negative and one candida spp.most of the organisms were part of normal commensal flora of the conjunctiva or the skin. the resident flora of the conjunctiva and eyelid mainly comprises of gram positive bacteria, including coagulase negative staphylococci, corynebacterium spp. propionibacterium spp. as well as staphylococcus aureus, bacillus spp. micrococcus spp. and enterobacter spp. (17,18) this is in accordance with several other published studies. (7,13,16) however, it differs from results puplished by rahman etal, (19) who found only a small proportion of the microorganisms identified to be post of the normal commensals flora when studying the contamination of unpreserved eye drops.a cycle of contamination between the lids and dropper tips was suggested by schein et al. (10) the contamination of eye drops and eye drop dispenser with the same microorganism, especially gram negative, has been described by the same group. this represents apotentially serious risk for ocular infection, especially in cases of compromised corneal epithelium as in extensive contact lens wear, ocular trauma or the use of topical steroids. in this study pathogenic organisms were rare and showed limited growth that probably did not represent a clinically relevant risk of infection. by using t.test we did not find any significant difference (p<0.05) in the contamination rate of eye drops/dropper tips and the residual volume which was left in the bottle. this supports the previously described self sterilising effect of many eye medications.the design of the containers might also influence contamination. only bottles with a tip attached to the bottle itself were analysed in this study. the bottle tips were more often contaminated (n=5) than residual drops (n=2) with contamination of both the tip and the residual solution appearing in one specimen. these results are similar to the ones reported in earlier studies. (11,13,16) one reason for this pattern to be considered is the antimicrobial activity of preservatives of the solution itself. such antimicrobial effects, however, may not act sufficiently on the tip itself as the contact time is limited. further the tip provides a large surface for contamination from ocular structures or hands. even dried crusts can sometimes be found on the bottle tips.the removal of such remnants with a sterile swab might further reduce the contaminator rate. (4) however the contamination of the solution itself has to be regarded as clinically more relevant since these get in direct contact with the patients eye.non of the examined eye drops is expired and we recommend not storing any open bottles in the back of drawers or on top shelves but always keeping them handy and limited to those actually needed. further more, we recommend noting the date of first opening on each container, as the duration of use might be another and possibly more relevant parameter rather than the expiry date (5) . conclusion aclear instruction sheet about safe and effective used should accompany do when they are dispensed to patient. patients who are unable to use eye drops in an aseptic way because of age or other physical (for example, poor vision) or mental limitations should be assisted by competent relatives or caretakers. the results of this study support the importance of a proper set of rules and the correct handling and application of eye medications. references 1. mayo ms, schlitzer rl, ward ma, et al. association of pseudomonas and serratia corneal ulcers with use of contaminated solutions. j clin microbial 1987;25: 398400. 2. donzis pb. corneal ulcer associated with contamination of aerosol saline spray tip. a.m j ophthalmol 1997;124:394-5. 3. brudieu e, duc dl, masella jj, et al. bacteral contamination of multidose ocular solutions. aprospective study at the grenoble teaching hospital pathol biol(paris) 1999;47:1065-70. 4. clark pj, ong b, stanely cb. contamination of diagnostic ophthalmic solutions in primary eye care settings. mil med 1997; 162. 501-6. 5. wessels if, bekendamp, calvin ws, et al. open drops in ophthalmday y offices: expiration and contamination. ophthalmic surg. lasers 1999;30:540-6. 6. perry hd, donnenfeld ed. issues in the use of preservative free topical manag care 2003-; 12:39-41. 7. raynaud c, laveran h, rigal d, et al. bacterial contamination of eye drops in iraqi j pharm sci, vol.20(2) 2011 microbial contamination of eye drops 95 clinical use jfr ophthalmol 1997; 20:1724. 8. zanen a. prevention of infections in the ophthanology office. bull socbelge ophthalmol 1996;260:9-16. 9. soc. belge z. antimicrobial agents in eye drops. ceska slov farm 2004;53:107-16. 10. schein od, hibberd pl, starek t, et al. microbial contamination of in use ocular medications. arch ophthalmol 1992; 110; 82-5. 11. coad ct, ostato ms, wihelmus kr. bacterial contamination of eye drop dispensers. am j ophthalmol 1984, 98: 548-51. 12. jalali r, zinolabedini f, moradi m, et al. bacterial contamination rate of eye drops. comparison of hospital and a private out patient center in kermanshah, iran. in sight 2004, 29: 12-4. 13. geyer o, bottone ej, podos sm, et al. microbial contamination of medications used to treat glaucoma. br. j opthalmol 1995; 79:376-9. 14. british national formulary. london; british medical assocation and royal pharmaceutical society of great britain, 1997:441. 15. schalla uc. untersuchungen zur mikrobiologischen probengewinnuing und transport bei bakteriellen entzundungen des äuberen auges. munich: ludwigmaximilians-universitat, 1998. 16. hovding g, sj ursen h. bacteral contamination of drops and dropper tips of in use multidose eye drop botlles. acta ohthalnol (copenh). 1982; 60:213-22. 17. ta cn, chang rt, singh k, et al antibiotic resistance patterns of ocular bacterial flora aprospective study of patients undergoing anterior segment surgery. ophthalmology 2003; 110: 194651. 18. leong jk, shah r, mccluskey pj, et al. bacterial contamination of the anterior chamber during phacoemulsification cataract surgery. j cataract refract surg 2002; 28: 826-33. 19. rahmam mq, tejwani d, wilson, ja, et al. microbial contamination of preservative free eye drops in multiple application containers. br j ophthalmol 2006; 90. 139-41. comparative evaluation of using intranasal desmopressin, parenteral diclofenac or their combination in the management of acute iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal activity of punica granatum 21 anti-fungal activity of punica granatum i.peels powder and extracts from pathogenic samples siham s.shaokat* ,1 , hamoudi a.hameed** , hassan j.mohammad*** * ministry of industry and minerals ministry of industry and minerals , baghdad , iraq **ministry of industry and minerals chief of food and drugs sector , baghdad , iraq *** prof. of biopharmacy/expert/iben-cena center of research. , baghdad , iraq abstract thirty five samples were collected from patients (1-30) years old, suffered from, infected skin , rushes, boils , oral thrush, anal & vaginal itches. candida albicans 57.3% (20 isolates) and candida tropicalis 22.5% (8 isolates) aspergillus fumegatus 11.5% (4 isolates) aspergillus nigar 8.7%(3 isolates) , were isolated & identified from these samples. alcoholic & water hot extracts of the punica granatum (pomegranate) peels as well as the dried powder were prepared. the anti-fungal activity of the extracts was evaluated by means of the agar-well diffusion assay. the extract exhibited potent activity against yeast. the minimum inhibitory concentrations were 128-1024 μg/ml against candida albicans and candida tropicalis .their was little difference between the activities of alcoholic extract & aqueous extract. these results suggest the pomegranate peels extract which contains gallotanic acid as a promising anti-fungal agent. key wards : antifungal agents, plant extracts, candida isolation الخالصة طرٍات الحالَة: سٌة, عزلث وشخصث الف 53-2ًوورج, هي هرظي هصابَي بإهراض جلذٍة هخحلفة ألعوار هي 55جن جوع candida albicans (57.3%) ,candida tropicalis(22.5%) , aspergillus fumegatus (11.5%) ,aspergillus nigar(8.7%) ٍقةة طرٍقةة انًحاةار فةٌ الوسةز علزرعةٌ الصةل وطر مجن إٍجاد فعالَة الوسحخلصات الكحولَة والواثَة علي الفطرٍات الوعزولة باسحخذا وكاًةث candida tropicalis , candida albicans الحخفَف فةٌ عًابَة انبحرةار ووجةذت ععلةي فعالَةة علةي عةزنت هل وكاًث فعالَة الوسحخلصات الكحولَةة اعلةي بقلَةل هةي الوسحخلصةات \هاٍكروكرام 2311 211قَاسات الجرع الوثرطة الصغرى الكالوجاًَةةض ظةةذ الفطرٍةةات ججعلدةةا هفَةةذال فةةٌ عةةلج انلحدابةةات الجلذٍةةة , طسحخلصةةات الحةةٌ جححةةوً علةةي ةةاهالوائَةةة. عى فعالَةةة الو والحدابات األغاَة الوخاطَة وإصابات الفن. introduction the common name of punica granatum is pomegranate, belong to family punicaceae , of the order myrtales, subclass rosidae,class magndiopsida pomegranate has a long history as food medicine and herbal use dating back more than 3,000 years [1] . both the stem and the root barks contain unusual alkaloids, known as 'pelletierines', which paralyze tapeworms so that they are easily expelled from the body by using a laxative [2] . the plant is also rich in tannin, the dried peels of the fruit contains about 26% which makes it an effective astringent. it is used externally in the treatment of vaginal discharges, mouth sores and throat infections [ 3] . pomegranate(punica granatum) peel extracts have been shown to possess significant antioxidant activity in various in vitro models, it has already been established that antioxidant activity in pomegranate juices is higher when extracted from whole pomegranate [ 4 ,5, 6,7,8] . australian researchers found that their scientific investigation of pomegranate flower extract improved hyperglycaemia in type ii diabetes and obesity in which gallic acid is mostly responsible for its glycaemic activity [ 9,10, 11] . concentrated pomegranate juice( cpj) improves lipid profiles in diabetic patients with hyperlipidemia ,they concluded that (cpj) consumption may modify heart disease risk factors in hyperlipidemic patients ,and its inclusion therefore in their diets may be beneficial [ 12,13 ] . additionally,research findings on excess triglyceride accumulation and increased fatty acid oxidation in the diabetic heart, which contribute to cardiac dysfunction, suggested that pomegranate flower extract improves abnormal cardiac lipid metabolism [ 14] . in recent study, pomegranate juice was found to slow down cholesterol oxidation by almost half and reduce the retention of disproportionate ldl cholesterol [15] . flavonoid –rich polyphenol fractions from pomegranate fruit have been shown to exert anti proliferative,anti-invasive 1 corresponding author : e-mail : albiatyss84@yahoo.com received : 13/9/2005 accepted : 4 /11/2007 mailto:albiatyss84@yahoo.com iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal activity of punica granatum 25 and proapoptotic actions in breast and prostate cancer cells and other solid malignancies [16,17,18,19,20,21]. topical application of pomegranate fruit and seed oil extract tested on mouse skin appears to posses chemopreventive activity in skin tumours [22] . it has been found that the methanolic extract of pomegranate peels posses wound healing activity against an excision wound on the skin of wistar rats [23] . the whole plant, but in particular the bark, is antibacterial, antiviral furthermore pomegranate juice provides an hiv-1 entry inhibitor by preventing the virus binding to the cellular receptor cd4 [24] . the dried rind of the fruit is used in the treatment of amoebic dysentery and diarrhoea . it is a specific remedy for tapeworm infestation [ 25,26]. pomegranate rind extract has been shown to have gastro-protective activity through its antioxidant mechanism , it posses strong antibacterial activity against different species of entropathogenes which cause diarrhoea and dysentery, e.coli, salmonella shigella sonnei and shigella flexner [27,28,29,30,]. pomegranate (outer rind) extract is also screened for their antimicrobial activity against gram-positive bacteria and yeasts, results founded that pomegranate showed good activity against staphylococcus aureus and candida [31] .plants used in argentin folk medicine screened for antimicrobial activity against staph. aureus commonly present on skin and mucous membranes which causes boils and abscesses, showed that pomegranate rind extract produced one of the more active results. pomegranate peels showed also bactericidal effect on vibrio cholerae [32]. aim of the study candida and related yeasts are endogenous opportunists.other opportunistic mycoses are caused by exogenous fungi that are globally present in soil, water and air. several species of the yeast genus candida are capable of causing candidiasis.they are members of the normal flora of the skin, mucous membranes and gastrointestinal tract. candida species colonize the mucosal surfaces of all humans during or soon after birth and the risk of endogenous infection is ever present .candidiasis is the most common systemic mycosis. filamentous fungi such as aspergillus are infected eye, ears, nose, and 5% of natamycin drops used as treatment. difficulties arising during chemotherapy of candida albicans necessitate novel chemotherapeutic strategies. the aims of this study are to investigate anti-fungal properties of water and ethanol, extracts & powder of punica granatum l.peels for treatment of several skin infections and inflammatory disorders. materials and methods materials : sabouraud agar, potatos agar, powder of nystatin were obtained from (russell, beecham, and special) pomegranate peels powder, candida albicans standard strain, tannic acid. instruments : zone reader, oven memmert.germany. pasture pipett, vortex mixer. balances ( sartorius), homogenizer, mixer, incubator, ultrasonic (soniprep 150hse) at 20khz. centrifuge, autoclave, water bath, rotary evaporator, souxhlet apparatus, magnetic stirrer, shaker, incubator. 3-clinical isolates from different clinical samples collected from three hospitals methods : preparation of medium (33) all media were prepared according to the manufacturers recommendations and were sterilized by autoclaving at 120c and 15 psi pressure for 15 minutes. a-sabouraud agar medium contain the following: peptone 10gm, glucose 20gm, agar 15gm , distilled water(1000ml) ,ph 6-6.3 this medium recommended for the isolation of fungi from pathological samples. bsabouraud conservation medium: peptone 30gm, agar 20gm, distilled water (1000ml) ph= 6.5-6.7 this medium recommended for conservation of fungus. csabouraud agar medium with cycloheximide 0.5gm and chloramphenicol ph 6-6.3 , &the same as( a ).this medium was recommended for isolation of dermatophytes and other pathological fungi. cycloheximide inhibited the growth of saprophytic fungus and chloramphenicol inhibits the growth of microbial contamination. dsabouraud broth medium: meat pepton 5gm, tryptic casein 5gm, glucose, 20gm, distilled water(1000ml) ,ph 5.7 esabouraud ( tetrazolium + chloramphenicol) agar medium , contain the following: pepton 10gm, glucose 20gm, agar 20gm 2,3,5, triphenyltetrazolium (h.c.l) 0.10gm, chloramphenicol 0.5gm. for culture rapid differential media. the reduction of triphenyltetrazolium by the colonies of fungi appeared as different degree of red colour according to the type of fungustable (1). iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal activity of punica granatum 21 preparation of macfrland standard solution (33) : solution a1.175gm of barium chloride bacl2.2h2o in 100ml of distilled water. solution b-prepared by the addition of 1ml of concentrated h2so4 to99ml distilled water.0.5ml of solution a was added to 99.5ml of solution b and the tube was compared with the bacterial suspension to give number of cell approximatively 10 8 x1.5 fungi/ml. isolation and identification of candida (33) : in culture or tissue, candida species grow as oval, budding yeast cells( 3-6 µm in size) .they also form pseudo hyphae when the buds continue to grow but fail to detach producing chains of elongated cells that are pinched or constricted at the septations between cells. candida albicans is dimorphic, in addition to yeasts and pseudohyphae, it can also produce true hyphae . on agar media within 24 hours at 37 ْ c or room temperature. candida species produce soft cream colored colonies with a yeasty odor. pseudo hyphae are apparent as submerged growth below the agar surface. two simple morphology tests distinguish candida albicans , the most common pathogen from the other species of candida. after incubation in serum for about 90 minutes at 37 ْ c yeast cells of candida albicans will begin to form true hyphae or germ tubes on nutritionally deficient media. candida albicans produce large spherical chlamydospores.. sugar fermentation and assimilation test can be used to confirm the identification and speciate the more common candida isolates table (1). table(1) – rapid identification of candida albicans (33) species respones in 4 hours respones in 24 hours serum + yeast media 37c filamentation=+ p.c.b chlamydospores = + sabouraud+actidion growth = + inhibition =0 sabouraud+tetrazolium candida albicans + + + white candida stellatoidea + 0 + rose candida tropicalis 0 0 0 red-violet candida pseudotropicalis 0 0 + rose candida guilliermondii 0 0 + red candida krusei 0 0 0 white candida .para krusei 0 0 0 rose-red candida zeylanoides 0 0 + white candida pulcherrima 0 0 0 rose iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal activity of punica granatum 25 isolation and identification of aspergillus aspergillus species grow rapidly, producing aerial hyphae that bear characteristic conidial structures: long conidiophores with terminal vesicles on which chains of conidia present , the species are identified according to morphologic differences in these structures, including the size, shape, texture and color of the conidia. (33) collection of samples form patients : candida albicans : 4 strains from skin infections, 2 strains from middle ear infections, four strains from rushes, 3 from infected boils , 2 from oral thrush, and 2 from anal and 3 from vaginal itches. microscopic examination : on direct examination of above samples 10% 0f naoh or 10% of koh, the hyphae of aspergillus species are hyaline, septate,uniform in width. culture:aspergillus species grow within a few days on most media at room temperature. species are identified according to the morphology of their conidial structures. collection of pomegrante fruit rinds : the punica granatum. peals were obtained from the local market. washed, cleaned and dried at room temperature or under the sun. spesifictions of pomegranate fruit rinds : the rind of the fruit is usually is irregular concave fragments, 1/20-1/10in.thick, brownish red externally and dull yellow on the inner surface, with depressions left by the seeds. the toothed calyx is present on some pieces. taste astringent. preparation of punica granatum peels. water extract . a known quantity of punica granatum peel was weighed and dissolved in 100ml distilled water boiled for 10-15minutes, soaked three hours, filtered twice, the filtrate was collected and evaporated by vacuum rotary evaporator at 55c until crud extract powder was obtained. the crud extract was weighed and dissolved in distilled water to calculate the concentrations needed for different experiments. reparation of punica granatum peels. alcholic extract . alcoholic (ethanol extract was prepared by soaking the peels in 75% ethyl alcohol using (souxhlet apparatus) at 50c then filtered, evaporated by vacuum rotary evaporator at 45c and collected (34) . measuring ph : ten grams of peels extract were dissolved in 50ml of d.w, shacked well by magnetic stirrer for 12 minutes, filtered and measure the ph. detection of punica granatum peels constituants (35) detection of tannins 10gm of extract was dissolved in 50ml of distilled water, filtered and cooled 1% of lead acetate was added .the appearance of precipitation indicated positive reaction. detection of glycosides equal amounts of fehling reagent and extract were mixed and boiled 10 minutes in water bath, red precipitation indicated positive reaction (35) detection of phenoles 10gm of punica powder was dissolved in 50ml of d.w and boiled for 10minutes, filtered, cooled. 1% of iron chloride was added; greenish blue color appeared which indicated the presence of phenol. detection of saponines : five ml of extract was added to1-3ml of hgcl2; white precipitate was indicated positive reaction. detection of resin fifty ml of ethyl alcohol 96% was added to five gm of pomegranate powder and boiled in water bath for two minutes, filtered (ederal n02) 10ml of acidified with hcl, was added to filtrate precipitation will occur in the case of positive reaction. detection of alkaloides (36) ten gm of extract was boiled with 50ml of d.w acidified with 40% hcl. the solution was filtered and cooled .0.5ml from filtrate was tested with the following solution: wagner solutiongrey precipitate positive reaction mayer solutionwhite precipitate positive reaction detection of comuurins (36) a small quantity of extract was dissolved in alcohol in atest tube covered with filtered paper moisture with naoh in water bath boiled 2-5minutes. the filter paper was exposed to u.v light (336 nm) the presence of yellow-green colour indicated the presence of comuurins. detection of flavones (36) solution a –10gm of extract/ 5ml of ethyl alcohol 96%( filtered) solution b10ml of ethyl alcohol 50%. equal quantity was mixed,yellow precipitate indicated positive reaction, by exposing the spot of flavones to uv light, gave fluorescent spot, or by spraying with sulfomolybdic acid solution gave purple to rose color. iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal activity of punica granatum 21 susceptibility test (37) quantitative method, that require measurement of zone diameters give the most precise estimates of antibiotic susceptibility. 40-100 µl extracts from each concentrations (80%,70%, 60%, 50%, 25%) were poured in small holes applied at equal distances in sabouraud agar seeded with 10 5 -10 4 / fungi/ml , dried at room temperature , the inhibition zones were read ,after incubation at 28c for 18 hours. inoculums of 10 5 -10 4 / fungi /ml were prepared by dilutions with the same medium and spotted on sabouraud agar. minimum inhibitory concentrations(mics)(37) he minimum inhibitory concentrations (mics) were determined by agar dilution method. different concentrations of extracts( 2mcg/ml-8392mcg/ml) were diluted with sabouraud agar in different petri dishes. inoculums of 10 8 10 9 fungi /ml were diluted with the same medium to obtain 10 5 -10 4 / fungi /ml spotted on agar, and incubated at 28c 0 . these results were compared with different concentrations of nystatin and tannic acid diluted with dimethyl formamide and spotted in one cm distance in the same petri dish .the lowest concentration preventing growth (mic) was estimated after 18 24 hours of incubation by the disappearance of spots. as control, candida albicans, strain was tested under the same conditions. the activity of different concentrations of punica granatum. l .. extracts were determined against candida albicans, , candida tropicalis , aspergillus fumegatus& aspergillus nigar . (16,17,18,23, 29 30.32.33) results and discussion pomegranate has a long history as food medicine and still continues in the evolution. it is act as antioxidant ,antibacterial anticancer, and anti fungal activities, a gel made from pomegranate peel has a high polyphenolic content demonstrated wound-healing capacity .candida albicans 57.3% (20 isolates) and candida tropicalis 22.5% (8 isolates) aspergillus fumegatus 11.5% (4 isolates) aspergillus nigar 8.7%(3 isolates) , were isolated & identified from the following samples. candida albicans : 4 strains from skin infections, 2 strains from middle ear infections, 4 strains from rushes, 3 from infected boils , 2 from oral thrush, & 2 from anal &3 from vaginal itches. antibiotic susceptibility test and minimum inhibitory concentrations (mics) table (2) and table (3) shows the results of activity of alcoholic & water extract by disk diffusion technique of thirty-five strains comparing with control organisms(candida albicans).the results were the following: 57.3% (20 isolates) candida albicans 19.5-22 mm zone of inhibition with different concentrations of extracts and candida tropicalis 22.5% (8 isolates) 21-23.5 , also good activity was noted with water extract with the same microorganism, these results indicated ,excellent activity of alcoholic and water extrat on candida tropicalis and candida albicans at different concentration comparing with standards. on the other hand no activity was observed against aspergillus fumegatus 11.5% (4 isolates) and aspergillus nigar 8.7 %(3 isolates) these results were in agreement with the studies of holetz fb. et al.,fundacao-o-c.. pomegranate activity on candida albicans (31, 32) .the comparative study of minimum inhibitory concentrations of extracts under test against all strains were studied.the results were as follow: mics for alcoholic extract and water extract against candida albicans and candida tropicalis were 128-1024μg/ml, and for the mics of for alcoholic extract and water extract against strains of aspergillus fumegatus and aspergillus nigar were very high as demonstrated in table (4) and (5). fig (1) demonstrated the diameters zone of inhibition of different dilutions of alcoholic extract against candida albicans . the results were compared with the activity of nystatin and tannic acid. table (6),table (7) demonstrated the active ingredients of pomegranate peels. iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal activity of punica granatum 21 table(2) –diameters zone of inhibition /mm of fungi under test (ethanol extracts ) average diameters zone of inhibition/mm for different concentrations of punica granatum type of microorganisms 80% 70% 60% 50% 25% 1candida albicans 10 22 21.5 21 20 19.5 2candida albicans 10 22 22 21 21 20 3candida tropicalis 5 23.5 23 22.5 22 21 4candida tropicalis 3 23 22.5 22 21 20 5aspergillus fumegatus 4 5 0 0 8 0 6aspergillus nigar 3 0 4 2 4 0 7candida albicans standard 21 21 21 20 19.5 table (3) diameters zone of inhibition /mm of fungi under test (water extracts ) average diameters zone of inhibition/mm for different concentrations of punica granatum water extracts type of microorganisms 80% 70% 60% 50% 25% 1candida albicans 13 21 20 19.5 19 18 2candida albicans 7 21.5 21 20 19.5 19 3candida 4 tropicalis 22 21 20.5 19 18.5 4candida 4 tropicalis 23 22 21.5 21 20 5aspergillus fumegatus 4 4 4 0 0 0 6aspergillus nigar 3 0 0 0 0 0 7candida albicans standard 22 21.5 21 20 19.5 table (4) minimum inhibitory concentrations µg/ml of punica granatum peels alcoholic extract of different concentrations type of microorganism minimum inhibitory concentrations/ml 80% 70% 60% 50% 25% candida albicans 128* 256 1024 1024 1024 candida tropicalis 64 128 512 1024 1024 aspergillus fumegatus ≤4196 4196 4196 ≤8192 ≤8192 aspergillus nigar 2048 2048 2048 4196 4196 candida albicans standard 128 256 1024 1024 2048 *n=6 iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal activity of punica granatum 21 table (5) minimum inhibitory concentrations µg/ml of punica granatum (pomegranate) peels water extract of different concentrations type of microorganism minimum inhibitory concentrationsµg/ml 80% 70% 60% 50% 25% candida albicans 256 512 512 1024 1024 candida tropicalis 128 256 512 1024 1024 aspergillus fumegatus 2048 2048 2048 4196 4196 aspergillus nigar 4196 4196 4196 ≤8192 ≤8192 candida albicans standard 128 256 1024 1024 2048 *n=6 table (6) minimum inhibitory concentrations µg/ml of punica granatum (pomegranate) peels and peels powder, fungus minimum inhibitaory concentration / mcg/ml powder/pomegranate peels solution/ water extract80% standard tannic acid 80% nystatin*/ in dmf candida albicans 512 256 128 4 candida tropicalis 128 128 64 4 aspergillus fumegatus 4196 4196 1024 16 aspergillus nigar 2048 2048 1024 16 candida albicans standard 128 128 64 2-4  nystatin powder activity 4976 i.u= 93.8% .dmfdimethyl formamide. table (7) active ingredients of pomegranate fruit rinds constituents peels powder ethyl alcohol extract water extract tannins/ as gallotanic acid 28% 29% 30% glycosides + + + total ash 5.14% 5% %5.3% non soluble materials 30% nt nt alkaloides _ _ _ phenoles _ _ _ saponines _ _ _ couumarins _ _ _ flavones _ _ _ non soluble ash in acid 0.3% 0.2% 0.3% colour + + + resinss + + + iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal activity of punica granatum 21 conclusions from above study one can concluded that the extract of pomegranate peels which contains gallotanic acid is useful for the treatment of several infections and inflammatory disorders due to candida albicans & candida tropicalis , these results suggested the possibility of using this raw material in pharmaceutical as cream, ointment, skin solution, lotion ,powder, mouth wash, gargles and even ear drops. further studies and investigations were needed . references 1bown. d. encyclopaedia of herbs and their uses. dorling kindersley, london. 1995 isbn 0-7513-020-31. 2chie j r . encyclopaedia of medicinal plants 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17suzuki r,et al. cytotoxic effect of conjugated trienoic fatty acids on mouse tumour human monocytic leukemia cells,lipids. 2001 may; 36(5):477-482. 18kawaii s, lansky ep. differentiationpromoting activity of pomegranate(punica granatum) fruit extracts in hl-60 human promylocytic leukemia cells .j med.food. 2004,7(1): 13-18. 19mehta r, lansky ep.breast cancer chemopreventive properties of pomegranate (punica granatum) fruit extracts in mouse mammary organ culture.eur.j.cancer preventive.2004,aug; 13(4) 345-348. 20malik a,et al. pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer. proc natl acad sci. u s a. 2005 sep 28;245-7 21afaq f, et al. anthocyanin-and hydrolysable tannin-rich pomegranate fruit extracts modulates mapk and nfkappab pathways and inhibits skin tumorigenesis in cd-1 mice.int. j. cancer.2005 jan 20;113( 3): 423-433 22hora jj,et al. chemopreventive effects of pomegranate seed oil on skin tumor iraqi j.pharm.sci., vol.16 (2) ,2007 anti-fungal 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farnsworth,n.r. 1972 alkaloid screening i-liyda 35 (1);314 37kirby,w.m.m. baur ,a,n., ,sherris ,k.c.&turk,m., antibioticsusceptibility testing by a standardized disc method . amer.j.clin.microb1966 43-45 38fundacao-o.c.f. screening of some plants used in brazilian folk medicine for the treatment of infectious diseases. mem inst oswaldo vol. 97(7) october 2002,p.1027-1031 iraqi j pharm sci, vol.31(suppl.) 2022 3d printing of gastro-floating device doi: https://doi.org/10.31351/vol31isssuppl.pp18-24 18 combination of fdm 3d printing and compressed tablet for preparation of baclofen as gastro-floating drug delivery system (conference paper )# nuha mohammed abdulkhaleq*,1 and mowafaq m. ghareeb* # 10th scientific conference sponsored by college of pharmacy, university of baghdad 2-3 june 2022 *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract this study aimed to develop an oral drug delivery system for gastro-retentive sustained drug release of baclofen by using a 3d printed capsular device since baclofen has a short half-life of 2.5 to 4 hr and has a narrow absorption window. firstly sustained-release tablets of baclofen were formulated through the hot-melt extrusion of various thermoplastic polymers and direct compression of the extrudate, then a capsular device was designed and 3d printed to contain two air pockets to enable floating of the device and has four windows for drug release. 3d printing of the capsular device was done by an fdm printer using biodegradable polylactic acid (pla) filament, and the sustained release tablets were inserted into the device to allow the medicine to be released into the stomach over a longer period. an in vitro buoyance test and an in vitro dissolution test were used to examine the buoyancy and sustained-release features of the formulated gastro-floating system. five sustained release formulas were developed using different thermoplastic polymers in hot-melt extrusion. produced tablets were assayed for drug content, hardness, and friability while a dsc study was done on the selected formula. f 5 which contains 20% baclofen, 55% eudragit rs-100, 20% ethylcellulose, and 5% peg 4000 showed sustained release where the complete dissolution of the drug occurred in 12 hr, and the gastrofloating device remained floating all the time. this method has a great potential for developing various floating drug delivery systems with the required release profile. keywords: sustained-release, 3d printing, hot-melt extrusion, gastro-floating device, baclofen. لتحضير دواء الباكلوفين كجرعة الجمع بين الطباعة ثالثية االبعاد و الحبوب المضغوطة # ) بحث مؤتمر ( دوائية طائفة *موفق محمد غريبو 1*، عبد الخالقنهى محمد 2022حزيران 3 – 2جامعة بغداد لكلية الصيدلة،العاشر # المؤتمر العلمي ،العراق. ،كلية الصيدلة، جامعة بغداد، بغداد الصيدالنياتفرع * الخالصة مطبوعة بطابعة ثالثية األبعاد لتمكن ةمن خالل استخدام كبسول الباكلوفين هدفت هذه الدراسة إلى تطوير التحرر الدوائي المستمر لدواء ساعات واكثر الدواء يكون امتصاصه من الجزء 4إلى 2.5نصفي من الحبوب الباكلوفين للطفو والبقاء في المعده ألن الباكلوفين يتراوح عمره تمر من خالل البثق بالذوبان الساخن باستخدام العديد من البوليمرات تمت صياغة حبوب الباكلوفين ذات التحرير المس اوال العلوي للجهاز الهضمي. ثالثية االبعاد المتميعة بالحرارة ثم كبس المنبثق الناتج على شكل حبوب، بعد ذلك تم تصميم كبسوله و طباعتها باستخدام خيوط قابلة للتحلل بالطابعة . أربع نوافذ إلطالق الدواء اوله لى طفو الكبسولهبشكل يحتوي على اثنين من الجيوب الهوائية التي تساعد ع ، وتم إدخال أقراص plaتم استخدام الطباعة ثالثية األبعاد لطباعة جهاز كبسولي باستخدام خيوط التحررالدوئي الباكلوفين ذات القابلة للتحلل خدام اختبار الطفو في المختبرواختبارالذوبان في المختبر لفحص للسماح بتحرير الدواء في المعدة على مدى فترة أطول. تم است كبسولةالمستمر في ال ميزات الطفو والتحرير المستمر لنظام العوم المعدي المصمم. . تم تقييم األقراص المنتجة تم تطوير خمس صيغ ذات تحرر دوائي مستمر باستخدام بوليمرات مختلفه المتميعة بالحرارة في البثق بالذوبان الساخن. ٪ 55٪ باكلوفين ، و 20الذي يحتوي على f 5 على الصيغة المختارة. أظهر dsc محتوى الدواء والصالبة والتفتت بينما أجريت دراسةمن حيث eudragit rs-100 5٪ إيثيل سلولوز ، و 20، و ٪peg 4000 ساعة ، وظل الجهاز 12إطالقًا مستداًما حيث حدث انحالل كامل للدواء خالل .وال الوقت.المعدي عائًما ط تتمتع الطابعه ثالثية االبعاد بإمكانية كبيرة لتطوير أنظمة مختلفة لتحريرالدواء على مدى طويل طافيا في المعدة. باكلوفين ،جهاز عائم معدي ،قذف بالذوبان الساخن،طباعة ثالثية األبعاد تحرر دوائي مستمر،الكلمات المفتاحية: introduction gastroretentive (gr) systems are meant to keep medication formulations in the stomach for longer periods of time, increasing the oral bioavailability of pharmaceuticals with a restricted gastrointestinal tract absorption window. gr systems can also help medications that have low stability in the lower gastrointestinal tract or treatments that operate locally within the stomach (1). the gastroretentive drug delivery systems can be retained in the stomach and help with the oral sustained delivery of medications with a specific absorption window in the gastrointestinal tract. these systems aid in the continual release of the medicine, resulting in maximum bioavailability (2). 1corresponding author e-mail: pharmacyone94@gmail.com received: 22/5/2022 accepted: 3/8 /2022 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol31isssuppl.pp18-24 iraqi j pharm sci, vol.31(suppl.) 2022 3d printing of gastro-floating device 19 drugs with a narrow absorption window are those that are absorbed primarily in certain regions of the gastrointestinal tract (git), such as baclofen, which is absorbed preferentially in the upper part of the git (1). reformulation of these medications into sustained-release forms confronts significant hurdles, as while the dosage form must deliver prolonged drug release, it must also keep the drug in close proximity to the absorption site (3). baclofen is a centrally acting skeletal muscle relaxant used to treat spasticity. in some clinical trials, it has also been demonstrated to be an effective treatment for alcohol and cocaine addiction. baclofen is a chemical derivative of the neurotransmitter γ-aminobutyric acid (gaba), and it works by activating (or agonizing) gaba receptors, specifically gabab receptors (4). baclofen is rapidly and extensively absorbed and eliminated. the half-life of the drug is ~2.5 to 4 hr in plasma (5). baclofen has a narrow absorption window in the upper gastrointestinal system, resulting in limited bioavailability (6). baclofen is challenging to manufacture into sustained-release dosage forms since its absorption is reduced or nonexistent once it reaches the colon (or even before). hme (hot-melt extrusion) is a method that involves driving raw materials through a die at a high temperature to give them a uniform shape and density. hme is a widely used method that can be used to make a variety of pharmaceutical preparations, particularly solid dispersions. hme characteristics such as die geometry, feeding rate, temperature, barrel design, shearing force, and screw rotating speed should all be considered. researchers can contribute to the development of a desirable final product with a preferred drug release profile and uniformity of size, shape, and drug content by optimizing these parameters. compared to other approaches, hme has a few advantages, for example, it's a one-step, solvent-free, continuousoperation method, as well as a scalable process (7). hme offers the advantage of being environmentally safe, and cost-effective technology when compared to other pharmaceutical manufacturing techniques. furthermore, by generating solid dispersions and solid solutions, hme enhances bioavailability. this is important for pharmaceutically insoluble compounds, which are common among newly discovered compounds (8). hme was utilized to make sustained-release tablets, pellets, and granules, among other pharmaceutical drug delivery systems (9). the type of polymer plays an important role in manufacturing the type of drug delivery system such as ethylcellulose was applied for the melt extrusion of sustained-release mini-matrices (10), and other thermolabile polymers such as eudragit rl-100, eudragit rs-100, and polyethylene oxide can be used for the sustained release dosage forms. fused deposition modelling (fdm) is a 3d printing technique that relies on the extrusion of thermoplastic filament through a high-temperature printing head with a nozzle that moves on the x and y axis, thereafter solidification of the melted filament on a platform that moves in the z-axis. the 3d structure will be formed from the deposition of the melted filament in a layer by layer so as the first layer solidify immediately after extrusion, the next layer will be extruded above till the 3d structure is complete according to the digitally designed object (11). due to the utilization of comparatively simpler and less expensive equipment, a varied selection of excipients, and the simplicity of generating dosage forms, even with complicated geometries, that have acceptable patient acceptability, fdm is the most commonly assessed 3d printing approach in the pharmaceutical sciences (12). in this work, a gastro-floating system for baclofen was developed using 3d printing technology for oral delivery with sustained release, allowing plasma baclofen concentration to be maintained for an extended length of time. rapid prototyping, which is a versatile tool for developing unique pharmaceutical dose formulations with different and complex geometry, is one of the key advantages of 3d printing technology. as a result, we set out to create a gastro-floating device using 3d printing technology and insert a baclofen sustainedrelease tablet made using hot-melt extrusion into it. the air pockets inside the device, which were manufactured using an fdm 3d printer, were credited with the device's capacity to float. the baclofen formulation inserted into the floating device allowed for sustained release of the drug. materials and method materials baclofen, ethylcellulose, and polyethylene oxide 600 k (peo mw 600,000) were purchased from baoji guokang bio-technology co ltd (baoji, china). eudragit® rl-100, and rs-100 were donated by evonik (darmstadt, germany). kollidon® 30 (polyvinylpyrrolidone k30) was donated by basf co. (ludwigshafen, germany). peg 4000 (polyethylene glycol) was purchased from himedia laboratories co ltd (mumbai, india). polylactic acid filament (pla filament, 1.75 mm in diameter) was purchased from prusa research (prague, czech republic) preparation of hot-melt extruded tablets the formulations' composition is shown in table 1. as granules, eudragit rs-100 and eudragit rl-100 were crushed by a miller before extrusion. baclofen and other excipients were mixed for 15 minutes in 30 gram batches with a mortar and pestle to ensure a uniform mixture. for all of the formulas, the mixture was extruded using a single-screw noztek pro filament extruder (noztek, shoreham, uk) with a 3 mm nozzle at a screw speed of 15 rpm and an extrusion temperature of 140°c (13). a miller crushed the extrudate before sieving it through a size #35 usp mesh to eliminate any iraqi j pharm sci, vol.31(suppl.) 2022 3d printing of gastro-floating device 20 aggregated or agglomerated particles. direct compression of the sieved extrudate with a 6 mm round concave punch produced 150 mg tablets equivalent to 30 mg baclofen. table 1.formulations composition for hme tablets (%w/w) formula baclofen ethyl cellulose pvp k30 peg 4000 peo 600k eudragit rl-100 eudragit rs-100 f1 20 20 55 5 f2 20 30 45 5 f3 20 20 5 55 f4 20 20 5 55 f5 20 20 5 55 3d printing of the gastro-floating device figure 1. 3d design of the gastro-floating device, (a) front view, (b) side view, (c) front sectional view, and (d) side sectional view. drug content determination the drug content in all prepared formulas was determined spectrophotometrically, where 100 mg of the sieved ground extrudate was dissolved in 100 ml of 0.1n hcl and kept for 12 hr under stirring then filtered. then 1 ml of the filtrate was diluted to 10 ml with 0.1n hcl and spectrophotometric absorbance was taken at λmax of 220 nm and drug content was calculated accordingly (14). in vitro buoyancy studies the in vitro buoyancy was calculated using the roy et al method where floating lag time and total floating time were calculated. the gastrofloating device (n = 3) was submerged in 100 ml of 0.1 n hcl with a tablet inside the capsular device. floating lag time is the time it takes for the device to rise to the surface and float. the total floating time was calculated as the amount of time the dosage form remained on the surface at all times (15). in-vitro dissolution studies the in vitro release rates of baclofen were determined by inserting a tablet from each formula into the 3d printed gastro-floating device and placing it in 900 ml of 0.1 n hydrochloric acid (ph 1.2) as a dissolution medium at 37±0.5 °c. drug release was performed using usp dissolution apparatus type ii (paddle type) at 50 rpm for 12 hr. aliquots of 5 ml were withdrawn at the following time intervals: 5, 10, 15, 30, 60, 90, 120 min then every hour until 12 hr. the samples were filtered and the medium was replenished with a similar volume of fresh medium. using the dissolving liquid as a blank, the quantity of baclofen was measured using spectrophotometry at 220 nm, and the percentage of drug release in total was computed. the outcome was calculated as the average of three runs (14). characterization of the selected formula tablet hardness the crushing strength of three tablets from each selected formula was measured using a yd-1 tablet hardness tester (huanghua faithful instrument co., china), whereby an increasing force was applied to the tablet until it was fractured (16). friability test an erweka friability tester ta3r (erweka gmbh, heusenstamm, germany) was used to determine the friability of the compressed tablets of the produced formulations. twenty tablets from each formula were weighed and placed in the drum, which was rotated at 25 rpm for four minutes, after which the tablets were reweighed and the weight differences were calculated and shown as a percentage (17). iraqi j pharm sci, vol.31(suppl.) 2022 3d printing of gastro-floating device 21 weight variation twenty tablets were weighed each one separately; later, the average weight was measured. if the weights of no more than two of the tablets are outside the % limit and no tablet differs in weight by more than double that percentage, the conditions are met (18). differential scanning calorimetry (dsc) thermodynamic analysis of pure baclofen, as well as the extrudate of a selected formula (f5), were determined using a dsc 60 (shimadzu, japan). the samples (5-6 mg) were placed in an aluminum pan and heated at a rate of 10°c/min between 25°c and 300°c under a dry nitrogen purge. indium/zinc standards were used to calibrate the dsc temperature and enthalpy scale, while an empty aluminum pan served as the reference (19). results and discussion drug content the chemical integrity of baclofen in the extrudate was analyzed using a uv–vis spectrophotometer. drug content was in the range of 98.9% to 101% as shown in (fig. 2) indicating no significant drug loss occurred during hme since extrusion temperature were lower than the melting point of baclofen which is 208°c (5). figure 2. drug content of sustained-release tablets (mean sd, n=3). in-vitro buoyance studies in vitro floating ability of the gastrofloating device with baclofen tablet incorporated inside it was evaluated where the floating lag time was relatively zero as the device floated at the surface immediately after being immersed in the medium for all the formulas, while the total floating time was more than 12 hr for all the formulas since the device remained floating until 12 hr after the start of the experiment. in-vitro dissolution studies in the present study, the combination of hme and fdm 3d printing was successfully employed to prepare a sustained-release tablet and gastro-floating device to increase the residence time of baclofen at or above the absorption window. different polymers were tested which are pvp k30, peo 600k, eudragit rl-100, eudragit rs-100, and ethylcellulose, in addition to peg 4000 was added as a plasticizer to reduce the extrusion temperature. the gastro-floating device was floating from the start of the study until the end after 12 hr (fig. 3). the release profile of the developed formulas is shown in figure 4. f 1 showed the fastest release among other formulas since pvp k30 is a hydrophilic polymer and used for solubility enhancement and immediate release dosage forms but the addition of ethylcellulose slowed the release since it’s a waterinsoluble polymer and used for modified release dosage forms (20,21). increasing the percentage of ethylcellulose to 30% in f2 reduced the dissolution rate to 85% in 12 hr because of the retardation effect of ethylcellulose (10). replacing pvp k30 in f 1 with peo 600k in f 3 resulted in a slower release of baclofen since peo is used for extending the drug release depending on the molecular weight, although its hydrophilic polymer but the interaction of the polymer with water causes the hydration and swelling that develops a hydrogel layer and this drives the entry of water within the matrix which regulates the controlled release behavior of drugs from the system (22). f 4 and f 5 which have eudragit rl-100 and eudragit rs-100 respectively as a sustainedrelease polymer in addition to ethylcellulose showed relatively similar dissolution profiles with similarity factor (f2) equal to 73.1 and reaches complete release in 12 hr. f 5 released more than 50% of the drug after 3 hr while f 3 released 47% of the drug after 3 hr and this is related to the specification of the polymers where the sustained release effect was more obvious after 3 hr. since the aim of this work was to develop a sustained release tablet of baclofen that can release the drug over 12 hr, so f 5 which released 99% of the drug in 12 hr was selected for further characterization to study the effect of hotmelt extrusion and other tableting processes. figure 3. gastro-floating device with f5 sustained-release tablet inside it. iraqi j pharm sci, vol.31(suppl.) 2022 3d printing of gastro-floating device 22 figure 4. in-vitro drug release of baclofen tablet incorporated into a gastro-floating device tablet hardness the hardness of the prepared tablets was within the acceptable limit (23) (equal to or more than 4 kg ), so that means they can withstand and resist breaking during handling and packaging as shown in table 2. friability test a friability test was performed to investigate the capacity of the tablets to resist chippings and abrasion that happened during handling, packaging, and shipping. all the prepared tablets were non-friable and the percent of friability for all prepared tablets is below one (table 2). weight variation the weight variation of tablets is shown in table 2, the result was found in the range of 149 ±1 to 150 ±1 from all the formulations. this fulfills the usp requirements in the limit of ±7.5% of the average weight. that means no large difference is observed in the weight of individual tablets from the labeled weight due to good mixing and no particle aggregation or fluidity that may cause differences in the die filling and difference weight of the tablet and strength (24). table 4. physical properties of hme tablets formula tablet hardness (kg) friability % weight variation (mg) ± sd f1 8.6 ± 0.1 0.06 149.9 ± 0.15 f2 8.1 ± 0.2 0.06 150.1 ± 0.26 f3 7.2 ± 0.31 0.09 150 ± 0.38 f4 8.1 ± 0.5 0.05 150.1 ± 0.2 f5 8.5 ± 0.15 0.03 150 ± 0.12 differential scanning calorimetry (dsc) the thermal behavior of pure baclofen showed a sharp endothermic peak at 209.92°c corresponding to baclofen melting temperature with the onset of a peak at 200 °c and end set at 220 °c which indicates that the drug is present in a pure crystalline state as shown in figure 5, this dsc study was in agreement with the reported one (25). figure 5. dsc thermogram of pure baclofen. the thermogram of the extrudate of f 5 which contains baclofen, eudragit rs-100, ethylcellulose, and peg 4000, is shown in figure 6. peg 4000 has shown a peak at 57.09 °c that is attributed to the glass transition temperature of the polymer (26), the sharp endothermic peak of baclofen has been disappeared and this may be attributed to the conversion of baclofen from the crystalline state to the amorphous state and also due to the dilution iraqi j pharm sci, vol.31(suppl.) 2022 3d printing of gastro-floating device 23 of baclofen concentration as its added in 20% of the total weight of the formula. the endothermic peak at 187.54 °c is belong to the ethylcellulose after processing in hme (21). figure 6.dsc thermogram of f 5 extrudate. conclusion formulation of baclofen sustained-release tablets was successfully done through hot-melt extrusion where the selection of thermoplastic polymers and their concentration were crucial to obtain the required release profile. the incorporation of 3d printing in the formulation of capsule shells and the ability to create complex designs allowed the 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attribution 4.0 international license. http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ role of ezetimibe in combination with statins(simvastatin and atorvastatin) in controlling dyslipidemia iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 1 role of ezetimibe in combination with statins(simvastatin and atorvastatin) in controlling dyslipidemia falah h.al-malki * , shatha h. ali** ,1 and gamal a.abdulbari*** * clinical biochem. ,clinical lab. science dept. ,college of pharmacy ,university of basrah,iraq ** department of clinical laboratory science,college of pharmacy,university of baghdad,baghdad , iraq *** college of medicine ,university of basrah, iraq abstract cardiovascular risk is independently increased by plasma lipids abnormalities (lowdensity and high density lipoprotein -cholesterol and triglycerides). most patients have more than one lipid abnormality. combination therapy with lipid-modifying agents could offer an important therapeutic option for improving the overall lipid profile. combinations have demonstrated to provide additive efficacy and significant reductions in coronary events . this study was designed to evaluate the effect of ezetimibe, when used in combination with other hypolipidaemic agents ( statins) on lipid profile as well as on liver function ,renal function, oxidative stress, and platelets function when given to dyslipidaemic patients . forty four patients (24 males and 20 females) with age ranged between 40-70 years (54 ±14.6) with dyslipidaemia on statins therapy for at least 6 month were involved in this clinical trials. they were randomized into two groups treated with either a combination of 20 mg/day simvastatin or a combination of 20mg/day atorvastatin and 10mg/day of ezetimibe.the study also included 22 apparently healthy subjects with age ranged (40-70years) and sex(11males and 11 females) matching that of the patients group. serum lipid profile (total cholesterol -tc, triglycerides -tg, low density lipoprotein-cholesterol –ldl-c, very low density lipoprotein-cholesterol-vldl-c, and high density lipoprotein-cholesterol –hdl-c), oxidative stress marker (malondialdehyde-mda), liver functions indices (alanin aminotransferase -alt,aspartate aminotransferaseast, total bilirubin), renal function parameters (urea, creatinine, and microalbuminuria) and platelets function test (bleeding time)were evaluated before and after 4 and 6 weeks of starting ezetimibe treatment . treatment with ezetimibe plus simvastatin or atrovostatin resulted in significant lowering in tc, tg, ldl-c levels with elevation in hdl-c also the ldl/hdl ratio lowered significantly ( by 38.16%). this effect was associated with significant changes in liver function , and oxidative stress without changes in platelets function nor in renal function. the results presented in this study indicated that ezetimibe can be used in clinical practice for the treatment of dyslipidaemia, when combined with other hypolipidaemic agents like simvastatin and atorvastatin to improve the therapeutic profile with ameliorating some of their adverse effects. keywords : ezetimibe , statins , dyslipidemia لخالصةا اٌ خطش أيشاض األٔػٛت انقهبٛت ًٚكٍ إٌ ٚضداد بصٕسة غٛش يؼخًذة ػُذ االخخالل فٙ انذٌْٕ انزالرٛت ٔانذٌْٕ انبشٔحُٛٛت ٔصذ اٌ يؼظى انًشظٗ نٓى أكزش يٍ خهم ٔاحذ فٙ انذٌْٕ. اٌ انؼالس انًشكب يٍ انًٕاد انًؼذنت نهذٌْٕ ػبنٛت انكزبفت ٔٔاغئت انكزبفت. ٔ انؼالس انًشكب ًٚكٍ اٌ ٚكٌٕ نّ فؼبنٛت إظبفٛت يغبببً ْبٕغبً يؼُٕٚبً . صٛت يًٓت نخحغٍٛ كم يغخٕٚبث انشحٕو فٙ انذوٕٚفش فبئذة ػال أصشٚج ْزِ انذساعت ٔصًًج نخقٛٛى فؼبنٛت االٚضحبًٚب يغ يٕاد خبفعت نهذٌْٕ يزم انغخبحُٛبث فٙ انخأرٛشاث ػهٗ انششاٍٚٛ انخبصٛت. إظبفت إنٗ حأرٛشِ ػهٗ ٔظبئف ، بحٛت ٔانًخًزهت بًغخٕٚبث اٚط انذٌْٕ ٔ فشغ األكغذة ٔٔظبئف انكبذ ٔانكهٗػهٗ يؼبٚٛش انكًٛٛبء انحٛ انصفٛحبث انذيٕٚت )صيٍ انُضف( ٔيقبسَت ْزِ انخأرٛشاث يغ أدٔٚت حقهٛذٚت خبفعت نهذٌْٕ )عًفبعخبحٍٛ ٔ احٕسفغخبحٍٛ( كُظبو يخخهػ يغ ( عُت 02-42اَبد( بؼًش ٚخشأط بٍٛ ) 42ركٕس، 44) يشٚعب (44ج ْزِ انذساعت ػهٗ ).اشخًه االصٚخٛبيٛب ػُذ يشظٗ انشحبو دٔٚت انغخبحٍٛ. حى حقغٛى انًشظٗ ػشٕائٛبً انٗ يضًٕػخٍٛ كبألحٙ:ببيشٚعبً بذاء انشحبو يغخًشٍٚ ػهٗ انؼالس 64.1± 44ٔبًؼذل يهغى ٕٚيٛبً ٔانًضًٕػت 62+42انغًفبعخبحٍٛ ٔاالٚضٚخبًٚٛب انًضًٕػت األٔنٗ: ْٙ يضًٕػت انًشظٗ انزٍٚ اعخخذيٕا َظبو يخخهػ بٍٛ يهغى ٕٚيٛبً. اعخًشث فخشة انًخببؼت عخت أعببٛغ يخخبنٛت. كزنك ظًٍ 62+42انزبَٛت ػهٗ َظبو يخخهػ بٍٛ االحٕسفبعخبحٍٛ ٔاالٚضٚخبًٚٛب حى قٛبط يغخٕٚبث (.44ًشظٗ ببنشحبو ٔبؼذد )انذساعت يضًٕػت يقبسَت يٍ األصحبء بأػًبس يقبسبت ٔبُفظ حٕصٚغ انضُظ نًضبيٛغ ان ٔٔظبئف انكهٗ (.ast,alt t.bil)ٔيؼبٚٛش فشغ االكغذة انًبنَٕذانذٚٓبٚذ ٔٔظبئف انكبذ (tc,tg,hdl,ldl)انشحٕو فٙ انذو (s.urea,creatinine,mau) ٔٔظبئف انصفٛحبث انذيٕٚت(bleeding time) اعببٛغ يٍ 4قبم اػطبء انؼالس ٔبؼذ يشٔس ػهٗ يغخٕٖ شحٕو أ االحشٔفبعخبحٍٛ اظٓشث ححبنٛم انبٛبَبث فشٔقب يؼُٕٚت ٔاظحت نالصٚخبيٛب يغ انغًفبعخبحٍٛاػطبء االصٚخبيٛب. ٔاَخفبض يهحٕظ فٙ َغبت hdlٔاسحفبع يغخٕٖ (tc,tg&ldl)انذو حٛذ نٕحع حصٕل اَخفبض يؼُٕ٘ فٙ حشاكٛض 1 corresponding author e-mail : dr_ shathahali @ yahoo.com received : 15/9/2008 accepted : 22 / 11/2008 iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 2 ldl/hdl حغٛش يؼُٕ٘ فٙ ٔظبئف انكبذ حشافق يغalt,ast,t.bil. .فٙ يؼبٚٛش فشغ االكغذة ححغٍ يؼُٕ٘ ظٓش كزنك عهبٛت راث دنٛم يؼُٕ٘ نٕظبئف انكهٗ ٔانكبذ ٔانصفٛحبث نى ٚخى يالحظت أ٘ حأرٛشاث ظبْشة أ ٔ md a يخًزهت ببَخفبض يغخٕٖ بكفبءة نؼالس اظبفًٙٚكٍ اعخُخبس يبٚهٙ :اٌ االصٚخبيٛب ًٚكٍ اٌ ٚغخخذو كؼالس اعتانذيٕٚت.فٙ ظٕء انُخبئش انخٙ افشصحٓب ْزِ انذس ًكٍ اٌ ٚحغٍ انفبئذة انؼالصٛت ٔيُغ يشض انشحبو ٔػُذيب ٚؤخز يغ ادٔٚت خبفعت نهكٕنٛغخشٔل كبنغًفبعخبحٍٛ ٔاالحشٔفبعخبحٍٛ ٚ انخأرٛشاث انضبَبٛت انًخٕقؼت. introduction dyslipidemia can be the result of a genetic predisposition, secondary causes or a combination of both .(1) the major lipid components of serum, cholesterol and triglycerides can produce three forms of dyslipidemia: hypercholesterolemia, hypertriglyceridemia and a combination of both. in each case, the dyslipidemia is the result of an elevation in either the number or composition of specific lipoproteins , which is an important determinant for selecting the appropriate drug therapy .(2,3) .the ncep guidelines for diagnosis of dyslipidemia, however, are based on clinical cut point that indicates relative risk for coronary disease. including the general recommendation that total cholesterol and hdl levels to be measured every five years beginning at age 20 in persons who do have a family history of coronary heart or other atherosclerotic disease .(4) . ldl is considered as the primary atherogenic lipoprotein, and the smaller the size of the ldl particle, the more it is able to penetrate into subendothelial tissue, thereby contributes to the development of atherosclerosis .(5) for people with chd, several large trials have demonstrated that aggressive lipid lowering is beneficial in people with chd with considering the following points: a target ldl. cholesterol level below 70-80 mg/dl is recommended for people who have chd and have multiple major risk factors (e.g patients with diabetes or who smoke). patients who experience myocardial infraction (mi) should be started on the cholesterol lowering medication while in the hospital and are advised to make life style changes , regardless of their ldl-cholesterol level.a target ldl-cholesterol level less than 100mg/dl is recommended for people who have chd but do not have many additional risk factors.life style changes as well as medications may be recommended when ldl levels are greater than 100mg/dlwhilest for people without a history of chd also appear to benefit from lipid lowering therapy although the treatments are not as aggressive as in patients with chd (6) . five major classes of drugs are available now for the treatment of dyslipidemia , each with different effects on the various lipids and lipoprotein profile (table-1) ( 7,8). statins are the most potent drugs available now for reducing ldl-c, they bring about moderately lower triglyceride level and modestly increase hdl-c levels (9) . table ( 1 ) : major classes of drug used in treating dyslipidemia (2) drug class ldl-cholesterol hdl-cholesterol triglycerid statins 18%-55% 5%-15% 7%-30% bile acid sequestrates 15%-30% 3%-5% no change or increase niacin 5%-25% 15%-35% 20%-50% fabric acid 5%-20% may be increased in patients with high triglyceride level 10%-20% 20%-50% cholesterol absorption inhibitors 17%-19% 1%-4% 0%-6% iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 3 statins are considered the first line treatment of hypercholesterolemia in patient who have failed to adequately respond to dietary therapy .(10,11) currently available products include simvastatin, atorvostatin, lovastatin , pravastatin, fluvastatin rosuvastatin (12) . oral agent that competitively inhibits hmg-coa reductase, the catalytic enzyme in the conversion of hmg-coa to mevalonic acid in the rate limiting step of cholesterol biosynthesis . (13) recently, a potential mechanism for poor response to statin therapy was described by patel et al(2001), (14) where the poor responders had a low basal rate of cholesterol synthesis that may be secondary to a genetically determined increase in cholesterol absorption possibly mediated by a polipoprotein e4 or by polymorphism in the hmg-coa reductase gene. (15,16) generally statins are contra-indicated in active liver disease (or persistently abnormal liver function tests) and in pregnancy (adequate contraception required during treatment and for 1 month afterwards) and breast-feeding. (17) the side effects of statins are reversible myositis which is a rare but a significant sideeffect of the statins. simvastatin and atorvastatin also cause headache; altered liverfunction tests (rarely, hepatitis) and gastrointestinal effects including abdominal pain, flatulence, diarrhea, nausea and vomiting. rash and hypersensitivity reactions (including angioedema and anaphylaxis) have been reported rarely. (17) the new class of lipid modifying agents, cholesterol absorption inhibitors, acts to lower ldl-c concentrations by almost 20% regardless of concurrent therapy, and have a modest effect on hdl-c and triglycerides (18). ezetimibe (zetia, merck/ schering-plough pharmaceuticals) is the first agent approved in this class, might be a good option for patients who do not tolerate or respond to statin therapy .however, this product is contraindicated in patients with active liver disease. ezetimibe acts through selective inhibition of intestinal cholesterol absorption . (19) experimental studies suggest that ezetimibe prevents dietary and biliary cholesterol uptake that transport across the intestinal wall. (20,21) ezetimibe -glucuronide, the primary metabolite, is transported from the liver back to the intestine in bile, and is a more potent inhibitor of cholesterol absorption than ezetimibe itself . (22) relatively high level of fecal ezetimibe (69% of the administered dose) suggests limited absorption and possible hydrolysis of the glucuronide metabolite . (23) the dose of ezetimibe 10mg once daily .(17) the present study was designed to evaluate the possible effects of adding 10 mg daily dose of ezetimbe (for 4 and 6 weeks) to hyperlipidemic patients ongoing with statins therapy (simvastatin 20 mg or atorvastatin 20 mg/day) on different components of lipoproteins in plasma, some biochemical markers for assessing liver , kidney and platelets function ,as well as , serum mda levels. materials and methods this study was carried out in al-basrah general hospital by selecting 44 patients (24 males and 20 females) with age ranged between 40-70 years (54 ± 14.6) presented with hyperlipidaemia (serum total cholesterol >200mg/dl ) for more than 6 months on statins ,not having any cvd, from december 2006 to march 2007 . twenty two apparently healthy subjects with comparable age and weight were also involved in this study as a control .fasting blood specimens were utilized for assessing lipid profile (total serum cholesterol ( 24) , triglyceride (25) , and high density lipoprotein-cholesterol (26 ), low density lipoprotein-cholesterol (27) liver function tests(alanine minotransferase-alt (aspartate aminotransferase -ast (28) total bilirubin (29) , renal function tests (urea (30), creatinine (31) and microalbominurea -mau (32) , platelets function tests (bleeding time ivy method) (33) and oxidative stress (serum malondialdehyde –mda (34) . subjects were randomized into five groups: group 1: which include 22 apparently healthy subjects (11 male, 11 female) that not received any therapy during the study. group 2: which include 11 (6 male and 5 female) dyslipidaemic patients treated with simvastatin 20 mg orally given as single daily dose at bed time for 6 weeks interval. group 3: which include 11 (6 male and 5 female) dyslipidaemic patients treated with atorvastatin 20 mg orally given as single daily dose at bed time for 6 weeks interval. group 4: which include 11 (6 male and 5 female) dyslipidaemic patients treated with simvastatin 20 mg + ezetimibe 10 mg orally taken at bed time for 6 weeks interval. group 5: which include 11 (6male and 5 female) dyslipidaemic patients treated with atrovastatin 20 mg + ezetimibe 10 mg orally taken at bed time for 6 weeks interval . all values were expressed as means ± standard error of mean. data were analyzed by in dependent t-test to assess the difference between two groups. p value less than 0.05 was considered significant). (35) iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 4 180.50 179.82 179.36188.09 172.27 160.82 188.82 176.18 163.59 0 50 100 150 200 250 before ezetimibe addition 4weeks after ezetimibe addition 6weeks after ezetimibe addition time s er u m c h o le st er o l (m g /d l) normal gp simvistatin gp atrovastatin gp results both simvastatin and atorvastatin treated groups (figure-1), showed no significant change in serum total cholesterol after 4 weeks of ezetimibe therapy ,but after 6 weeks of the addition of ezetimibe to simvastatin, there was a significant lowering in serum total cholesterol level as compared with both baseline . however , simvastatin treated group showed significant (p<0.05) reduction in serum tg levels after 4 weeks from the addition of ezetimibe (-28.6%)as compared with normal values , after 6 weeks from addition of ezetimibe it produced (-30.69%) reduction as compared with normal values (figure-2).while, 4 weeks of ezetimibe addition to atorvastatin produced a significant lowering (p<0.05) in serum tg level as compared to both normal and baseline values (-31.93%and -29.36%, respectively). after 6 weeks serum tg levels were lowered by(19.6%,-17.4%) as compared with normal and baseline values respectively.however, the hyperlipidemic group treated with simvastatin exert non significantly elevated levels of serum hdl-c after 4 weeks from the addition of ezetimibe (figure-3) . figure (1) : a histogram showing serum total cholesterol, for simvastain and atrovastatin groups that received ezitimibe; as compared with control * = significant at p<0.05 as compare with normal values in same column. a = significant at p<0.05 as compared with baseline values. figure (2): a histogram showing serum triglyceride, for simvastain and atrovastatin groups that received ezitimibe; as compared with control group * = significant at p<0.05 as compare with normal values in same column. a = significant at p<0.05 as compared with baseline values. 145.50 144.91 143.50 108.57 103.45 99.45 139.64 98.64 115.32 0 20 40 60 80 100 120 140 160 180 before ezetimibe addition 4weeks after ezetimibe addition 6weeks after ezetimibe addition time s e ru m t ri g y c e ri d e s ( m g /d l) normal gp simvistatin gp atrovastatin gp * * * a* * *a a iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 5 figure (3): a histogram showing serum hdl-cholesterol, for simvastain and atrovastatin groups that received ezitimibe; as compared with control group. * = significant at p<0.05 as compare with normal values in same column. but after 6 weeks there were significant (p<0.05) elevations in serum hdl-c level (15.4%,6.79%) as compared with normal and baseline values respectively .atorvastatin group, showed significantly (p<0.05) higher serum hdl-c level before, after 4 weeks, and after 6 weeks from addition of ezetimibe. (10.19%, 19.66% & 18.21% respectively) as compared with value of normal group. plasma ldl-c level was non significantly lowered after 4 weeks from addition of ezetimibe to simvastatin treated group (figure-4 ) but after 6 weeks there were a significant (p<0.05) lowering in serum ldl-c levels (-20.95%,21%) as compared with baseline and normal values respectively. while, atorvastatin group, showed no significant alteration in serum ldl-c level both after 4 and 6 weeks from addition of ezetimibe. however, simvastatin group showed no significant changes in ldl/hdl ratio after 4 weeks of ezetimbe therapy as compared to those values before adding ezetimbe and that of control (table .2) . figure (4) : a histogram showing serum ldl-cholesterol, for simvastain and atrovastatin groups that received ezitimibe; as compared with control group. * = significant at p<0.05 as compare with normal values in same column. 42.86 40.68 40.14 43.41 44.45 46.3647.23 48.68 47.45 0 10 20 30 40 50 60 before ezetimibe addition 4weeks after ezetimibe addition 6weeks after ezetimibe addition time s e ru m h d l (m g /d l) normal gp simvistatin gp atrovastatin gp * * * * 42.86 40.68 40.14 43.41 44.45 46.3647.23 48.68 47.45 0 10 20 30 40 50 60 before ezetimibe addition 4weeks after ezetimibe addition 6weeks after ezetimibe addition time s e ru m h d l (m g /d l) normal gp simvistatin gp atrovastatin gp * * * * iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 6 table (2): effect of ezitimibe addition on ldl/hdl ratio in patients treated with hmg-coa reductase inhibitors (atrovastatin & simvastatin); in comparison with normal individuals (values expressed as mean + standard error of mean , n= number of subjects) * = significant at p<0.05 as compare with normal values in same column. a = significant at p<0.05 as compared with baseline values. but 6 weeks values were significantly (p<0.05) lowered (-38.16%,-35.29%) as compared with both baseline and control values respectively, a comparable results were obtained with atorvastatin treated group in ldl/hdl ratio (-27.5%,-12.68%)) as compared with normal and baseline values, respectively.in table -3, the studied groups exert a significant changes in serum alt activity after 6weeks from addition ezetimibe (18.77%,5.66%)as compared to both baseline and control values respectively.while, the atorvastatine group exert no significant alterations in alt activity in serum through the study. simvastatin treated group of patients showed non significant elevation in serum ast activity after 4 weeks from addition ezetimibe(8.39%,11.7%) as compared with baseline and normal values ,respectively ,but after 6 weeks a significant (p<0.05) elevation in serum ast activity was noticed (13.6%)as compared to pretreatment value. meanwhile, atoravastatin treated group was presented with a significant (p<0.05) lowering in serum ast activity (-7.12%,-14.4%)as compared with both baseline and normal values respectively. in simvastatin treated patients there was a significant (p<0.05)lowering in serum total bilirubin level(-15.5%,-17.09%) after4&6 weeks as compared to the control values, respectively ( table-3). in atorvastatin treated group, ezetimibe showed a significant (p<0.05)lowering in serum total bilirubin (8.18%, -13.6%) respectively as compared with baseline and normal values respectively. table (3) : effect of ezetimibe addition on serum alt , ast and total bilirubin in patients treated with simvastatin and atrovostatin in comparison with normal subjects ( values are expressed as mean ±sem , n= number of subjects) groups duration of treatment serum alt (iu/l) serum ast iu/l serum total bilirubin normal n=22 base line values 13.82 ± 0.72 14.8±0.66 1.26±0.03 after 4 weeks 13.77 ± 0.55 13.86±0.58 1.16±0.5 after 6 weeks 13.59 ± 0.47 14.50±0.65 1.17±0.04 patient treated with simvastatin & ezitimibe n=22 base line values 12.09 ± 0.81 11.32±0.32* 1.03±0.04* after 4 weeks 14.18 ± 0.66 12.27±0.48* 0.98±0.03* after 6 weeks 14.36 ± 0.56 a 12.86±0.43a* 0.97±0.04* patient treated with atrovastatin & ezitimibe n=22 base line values 12.91 ± 0.60 12.77±0.56* 1.10±0.05* after 4 weeks 13.45 ± 0.50 11.86±0.54* 1.03±0.05 after 6 weeks 13.45 ± 0.36 11.45±0.46* 1.01±0.03* * = significant at p<0.05 as compare with normal values in same column. a = significant at p<0.05 as compared with baseline values. groups duration of treatment ldl/hdl ratio normal n=22 base line values 2.79 + 0.20 after 4 weeks 3.13 + 0.34 after 6 weeks 3.23 + 0.30 patient treated with simvastatin & ezitimibe n=22 base line values 3.38 + 0.43 after 4 weeks 2.51 + 0.23 after 6 weeks 2.09 + 0.15 * a patient treated with atrovastatin & ezitimibe n=22 base line values 2.68 + 0.22 after 4 weeks 2.35 + 0.24 after 6 weeks 2.34 + 0.19 * iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 7 (table4) showed no significant alteration in serum urea level before and after 4 and 6 weeks from addition of ezetimibe to all studied groups, nor in serum creatinine levels ,nor in microalbuminuria values. serum mda levels simvastatin treated groups were presented with a significant (p<0.05) lowering in serum mda ( -42% ) as compared with baseline values (figure-5) .after 4 and 6 weeks of utilizing ezetimibe in atrovastatin treated group serum mda significantly (p<0.05) lowered (19.8%,26.7%) as compared with baseline values.the simvastatin and atorvastatin treated patients showed no significant alterations in bleeding time values by the addition of ezetimibe to their therapy ( table5). table (4) : effect of ezetimibe addition on serum urea,creatinine and microalbuminuria in patients treated with simvastatin and atrovostatin in comparison with normal subjects ( values are expressed as mean±sem , n= number of subjects ) groups duration of treatment serum urea(mg/dl) serum creatinine (mg/dl) microalbuminuria (mg/day) normal n=22 base line values 39.00 ± 1.61 1.19±0.04 203.23±8.36 after 4 weeks 39.73 ± 2.03 1.16±0.05 207.09±11.3 after 6 weeks 37.91 ± 1.01 1.24±0.10 202.09±8.66 patient treated with simvastatin & ezitimibe n=22 base line values 39.43 ± 1.98 1.28±0.14 201.73±8.59 after 4 weeks 40.00 ± 2.09 1.31±0.15 204.09±14.80 after 6 weeks 39.27 ± 1.78 1.29±0.14 206.09±18.70 patient treated with atrovastatin & ezitimibe n=22 base line values 36.68 ± 2.08 1.13±0.04 211.55±10.40 after 4 weeks 35.23 ± 1.72 1.03±0.05 208.55±11.30 after 6 weeks 36.23 ± 0.97 1.11±0.02 209.05±17.40 0.73 0.76 0.77 0.88 0.69 0.51 1.01 0.81 0.74 0.0 0.2 0.4 0.6 0.8 1.0 1.2 before ezetimibe addition 4weeks after ezetimibe addition 6weeks after ezetimibe addition time s e ru m m a lo n d ia ld e h y d e (m ic ro g ra m g /d l) normal gp simvistatin gp atrovastatin gp figure (5): a histogram showing serum malondialdehyde for simvastatin and atrovastatin groups that received ezitimibe; as compared with control group a = significant at p<0.05 as compared with baseline values. * = significant at p<0.05 as compare with normal values in same column. * a a* a a iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 8 table (5): effect of ezitimibe addition on bleeding time, in patients treated with hmg-coa reductase inhibitors (atrovastatin & simvastatin); in comparison with normal individuals (values expressed as mean + standard error of mean , n= number of subjects ) discussion as previously presented in figures (-1), (-2), (-3) and (-4) treatment with ezetimibe plus statins successfully improves lipid profile markers in dyslipidaemic patients during 6 weeks of treatment .these results are consistent with results of other studies that included the administration of ezetimibe plus statins to patients with disordered lipids profiles could result in significant reduction in tc, ldl-c and tg levels, (36) with significant elevation in hdl-c levels , which could be attributed to mechanisms that are related to ezetimibe lowering effect on cholesterol which could be complement to the inhibitory action of statins on cholesterol biosynthesis representing an important new option for treatment in combination with statin (37) . ezetimibe has an excellent safety and liability profile when administered with statins (38,39) . also it has a low potential for drug interactions. (40) many patients receiving statins therapy fail to reach their ldlgoal (41) , because its mechanism of action is complementary to that of statins, ezetimibe were studied for its potential additive lipidlowering effects in patients already receiving statin therapy in double-blind as well as placebo-controlled trials (36,42) . significant improvements were observed for other indicators of chd risk ( total cholesterol , non-hdl-c, apolipoprotein b, ldl-c: hdlc ratio) in patients receiving ezetimibe-statin therapy. (36) when ezetimibe was combined with simvastatin or atorvastatin it caused a significant reduction in triglyceride level with time from baseline compared with statin monotherpy (43). combination therapy of simvastatin and ezetimibe was more effective than atorvastatin in reducing ldl-c in patients with primary hypercholesterolemia. (44) preliminary studies have indicated that there were no significant effect of ezetimibe on absorption of fat-soluble vitamins (45) .following absorption of ezetimibe where it is glucuronidated in the intestine wall the parent drug and its glucuronidated derivatives can undergo enterohepatic recircalation, that limits peripheral exposure (46) . ezetimibe is first in cholesterol absorption inhibitors, it 's action is consistent with the binding thereby blocking of sterol transporter on the brush border membrane of in intestinal epithelial cells (47) . through inhibiting the intestinal cholesterol absorption ezetimibe can effectively reduce of biliary/dietary cholesterol delivered to the liver via chylomicron and chylomicron reminants, hence reduce cholesterol content of atherogenic particles chylamicrons / chylomicrors ruminants, vldl, ldl).meanwhile the reduced delivery of intestinal cholesterol to liver increase hepatic receptor activity and increase clearance of circulating ldl-c. ( 47). ezetimibe, via inhibiting intestinal cholesterol and plant-sterol absorption, may modify the atherogenicity of chylomicron remnants and reduce systemic plant-sterol levels (48) . these effects are likely to reduce cardiovascular risk. it has been reported that there is a strong relationship between hepatic dysfunction and dyslipidaemic complications (49,50) however, the data presented in table3 representing a modulation in some liver markers in group treated with simvastatin plus ezetimibe after 6 weeks of treatment in case of altand ast. such results could be due to relatively low doses of statins (20mg) wherase, other studies revealed a significant elevation in those enzymes. (13) the elevation in transaminases activities were primarily asymptomatic and not groups duration of treatment bleeding time (minutes) normal n=22 base line values 2.54 + 0.18 after 4 weeks 2.48 + 0.17 after 6 weeks 2.45 + 0.13 patient treated with simvastatin & ezitimibe n=22 base line values 2.26 + 0.12 after 4 weeks 2.50 + 0.23 after 6 weeks 2.62 + 0.33 patient treated with atrovastatin & ezitimibe n=22 base line values 2.26 + 0.20 after 4 weeks 2.97 + 0.35 after 6 weeks 2.17 + 0.35 iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 9 associated with cholestasis. serum transaminases returned to pretreatment level with discontinuation of combination therapy or with continued treatment ( 51) . ezetimibe is being used with increasing frequency in many patients to augment the ldl-cholesterol lowering effects of statins (52) the recent second united kingdom heart and renal protection (uk-harp-ii) study found in a randomized, controlled study that 10mg of ezetimibe added to 20mg of simvastatin in patients with ( chronic kidney disease) ckd resulted in an incremental reduction of ldlcholesterol level over simvastatin alone without an excess risk of abnormal liver or muscle markers or other advers events. (53) the purpose for the evaluation of renal function was to explor the safety of a combination of ezetimibe and statins in this respect .statins at appropriately adapted doses have the same efficacy in chronic renal disease patients as in subjects with normal kidney function, and their tolerance is not a problem .(54) in the present study the effect of ezetimibe plus statins (simvastatin or atorvastatin) have no significant effect on renal function , as in table-4 . therefore, no dosage adjustment for ezetimibe is needed in patients with renal insufficiency. efforts to improve lipid profiles now are targeted primarily for the treatment and prevention of cardiovascular disease, may also prevent the development of renal disease (55) . one important risk factor for atherosclerosis is an elevation in a particular type of plasma cholesterol specifically ldl c. oxidation of ldl -c is thought to render the lipoprotein to be athergenic ,because oxidized -ldl is more readily taken up by macrophages via scavenger receptors. (56) the data presented in figures (5) showed that serum mda levels were significantly lowered by about (-33.7%) when ezetimibe was added to simvastatin treated group after 6 weeks .meanwhile, level of mda was significantly lowered after 4 weeks from addition ezetimibe to atorvastatin treating group by about (19.8%) as compared with pretreatment values . however, mda level after 6 weeks from addition ezetimibe to either groups lowered mda level below those reported even for the normal group . furthermore , the lowering effect on lipids pereoxidation produced by simvastatin/ezetimibe combination was better than that produced by ezetimibe/atorvastatin combination. (57) a recent study showed that despite the comparable modest reduction of serum cholesterol levels by ezetimibe, an intestinal inhibitor of cholesterol absorption, and statin, only the statin improved endothelial function (58) thus, it is likely that the beneficial effects of statins on endothelial function extend beyond cholesterol reduction. indeed, statins have been shown to reduce cardiovascular events in patients, irrespective of serum cholesterol levels (59) . in this study, the effects of adding ezetimibe to statins therapy (simvastatin or atorvastatin), in patients with dyslipidemia showed no significant changes in platelets function in both groups, this could indicate no adverse effect on platlets function as seen in table -5 .although non of the studied groups of dyslipidaemic patients exert any deviation in bleeding time values from those reported from normal subjects ,before initiating ezetimibe therapy. this would support the administration ezetimibe plus simvastatin or atorvastatin without any adverse effect on platelets, so patients with platelets dysfunction could take ezetimibe with statin safely .however, there is no evidence or clinical trials about effect of ezetimibe with statins on platelets function and specifically on bleeding time. conclusions: 1. ezetimibe can be used in combination with simvastatin or atorvastatin to improve their lipid-lowering action both effectively and safely in the treatment of dyslipidemia. 2. ezetimibe can be used safely in combination with statins in patients with renal disease. 3. ezetimibe exerts no further modification to liver function that could be produced by statins when used in combination with statins. 4. ezetimibe/simvastatin and eztimibe / atorvastatin could exert asignificant antioxidant effect in patients with dyslipidemia. 5. non of the tested drugs (ezetimibe nor simvastatin nor atorvastatin) produced significant modification of platelets activity. references: 1. rodondi km: hyperlipidemia, in herfindal et, gourley dr (eds): textbook of therapeutics drug disease management. baltimor, williams and wilkins 1996 : 387 – 403 . 2. mc. kenney jm: hyperlipidomia. siscat. . pharmacothereapy self assessment program. kansas city, american college of clinical pharmacy, 1991:65-94 . 3. farmer jaand gotto amj, choosing the right lipid regulating agent. drug:1996 ; 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(eds), basic and clinical pharmacology, 9th ed, appleton and lange, california; 2004: 561-75. 51. product. information: vytorintm, ezetimibe/simvastatin. merck/scheringplough pharmaceuticals, north wales, pa, (pi issued 07/2002) reviewed 11/2002 52. kosoglou t,statkevich p,johnsonlevonas ao,et al:ezetimibe: areview of its metabolism,pharmacokinetics and drug interaction . clin pharmacokinet.2005;44:467-94. 53. landray m,cairns hs,carr s,et al.the second united kingdom heart and renal protection (uk-harp-ii)study :arandomized,controlled study of the biochemical safety and efficacy of adding ezetimibe to simvastatin as intial therapy iraqi j pharm sci , vol.18 (1), 2009 role of ezetimibe in combination with statins 12 among patients with ckd .amj kidney dis.2006;47:385-95. 54. karie s,launoy-vacher v,deray g,et alstatin in patients with kidney failure.efficacy,tolrence ,and prescription guidelines in patients with chronic kidney disease and renal transplants. press med.2006;35(2pt1):219-29. 55. sarnak mj: las: cardiovascular disease and chronic renal disease: a new paradigm. am j kid dis.2000; 35: 117– 131. 56. heinecke ,j.w. oxidants and antioxidants in pathogenesis of atherosclerosis : implication for the oxidized ldl hypothesis .atherosclerosis.1998;141:1-15.[medline] 57. cai h,harrison dg.endothelial dysfunction in cardiovascular disease:the role of oxidant sress. circ res.2000;87:840-4. 58. landmesser u, bahlmann f, mueller m, et al. simvastatin versus ezetimibe: pleiotropic and lipid-lowering effects on endothelial function in humans. circulation. 2005; 111: 2356–63. 59. mrc/bhf heart protection study of cholesterol lowering with simvastatin in 20,536 high-risk individuals: a randomized placebo-controlled trial. lancet. 2002; 360: 7–22. iraqi journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 mefenamic acid derivatives 7 synthesis and preliminary pharmacological evaluation of aminobenzensulfonamides derivatives of mefenamic acid as a potential anti-inflammatory agents monther f. mahdi*, 1 * department of pharmaceutical chemistry, college of pharmacy, university of baghdad. , baghdad , iraq abstract a group of amino derivatives [4-aminobenzenesulfonamide,4-amino-n¹ methylbenzenesulfonamide, or n¹-(4-aminophenylsulfonyl)acetamide] bound to carboxyl group of mefenamic acid a well known nonsteroidal anti-inflammatory drugs (nsaids) were designed and synthesized for evaluation as a potential anti-inflammatory agent. in vivo acute anti-inflammatory activity of the final compounds (9, 10 and 11) was evaluated in rat using egg-white induced edema model of inflammation in a dose equivalent to (7.5mg/kg) of mefenamic acid. all tested compounds produced a significant reduction in paw edema with respect to the effect of propylene glycol 50% v/v (control group). moreover, the 4amino-n-methylbenzenesulfonamide derivative (compound 10) exhibited comparable antiinflammatory activity to diclofenac (3mg/kg) at times 180-300 minute with the same onset of action. the results of this study indicate that the incorporation of the 4-aminobenzenesulfonamide pharmacophore and its derivatives in to mefenamic acid maintain its anti-inflammatory activity. key ward: benzenesulfonamide, anti-inflammatory, paw edema, nsaids, mefenamic acid ةصالخلا عجممنمجزم4عجمم--عنجزمفمس تمزنبمن ع , -)-نا ,ديامانوفلس نيزنب ليثم-نا-ونيما-)-نا ,ديامانوفلس نيزنب ليثم-نا-ونيما-4 عجممفمس تمزنبمن ع ,4 [عنشماممعتم تشمىا نم ةعجمجم م ت فمرجغم ترىجغ -سم تشيغ ءموجمmefenamic acid ) ) معىسمفشنشمامم تل سفمبرجزمتنشجب نشج م زج-م ] زنبمنجز( زجى ع نفم تنرام تسف , وغسمواججام تبي تجمم تش ق متلتىا مم . بش قمتلتىا م ,ا-ممششهم ي غنمتىاججشا مبش ق نمام ممتلتىا مم متنشجب نشج م زج-م( نفم تنغحمف زىلتمتةةم تنجلمعرىسمم حعمم تىا فجمموسهم تنن-مفنغاممعل ن م11 ,10 ,9تنشغبن نم تما مجم) (propylene glycol%50عنلا/بلا(.بزم تشغبن نم تشلىنغ م نىنهم نلب معف غ متنمحعممف تشا سنممعكم تنغ فنجتمبل لمة )(7.5 ( ااغمني تجممع ق متلتىا ممعا سنمم10 عغبم( عجمم--عنجزمفمس تمزنبمن ع -4بشنشماممو فمم.ال مانامحت معمىك . نىجنممنا م ت-س زممومجغم تام -م ن-ع .ممتن لنمنجم لمعكمنبام تبي تجمم ةفىمجممقاجام300_ 180نفم ا نعنلا/بلا( 3تن لنمنجم لم) عجممفمس تمزنبمن ع -م عمىا ونمعكم تشجب نشج م زج-مي نامانامني تجىنم تش ق .4 تنسفم تيا اجغسم introduction nonsteroidal anti-inflammatory drugs (nsaids) are widely used to treat acute or chronic inflammation and offer symptomatic pain relief(1,2). conventional nsaids act by non selective inhibition of cyclooxygenase (cox) enzymes, which catalyze the formation of prostaglandins (pgs) from arachidonic acid(3, 4). there are three isoenzymes of cox (cox-1, cox-2 and cox-3) have been identified(5,6). cox-1 is expressed in most tissues of the body and largely governs the homeostatic production of arachidonic acid metabolites necessary to maintain physiologic integrity(7). cox-2 is highly induced in settings of inflammation by cytokines and inflammatory mediators or physiological stress(8,9). cox-3 activity in human has not been confirmed(10), but it may be implicated in fever(11). all classic nsaids inhibit cox-2 as well as cox-1 to varying degrees; thus they can be considered nonspecific(12,13). all classical nsaids are associated with an increased risk of gastrointestinal (gi) ulcers and serious upper gi complications, including gi hemorrhage, perforation, and obstruction(14,15). in contrast many of the selective cox-2 inhibitors containing benzene-sulfonamide derivative, like valdecoxib(i) (16) , celecoxib(ii) (17),or benzene-n-methyl sulfonamide like compound (iii) (18) and benzene methylsulfonyl 1 corresponding author : e-mail : dmfalameri@yahoo.com received : 29 /10 / 2007 accepted : 15 /3/ 2008 iraqi j.pharm.sci., vol.17 (1) ,2008 mefenamic acid derivatives 8 o sh3c o o o o sh3c o o o so2nh coch3 so2nhcoch3 so2nhch3 o o derivative, like rofecoxib (iv) exert antiinflammatory and analgesic activity in the clinic with markedly less gi toxicity than traditional nsaids(19). in a recent study, it was shown that the incorporation of a para-nacetylsulfonamido substitute on the c-3 phenyl ring of the rofecoxib regioisomer provided a highly potent and selective cox-2 inhibitor (compound v) that has the potential to acetylate the cox-2 isozyme(20). the improved gi tolerance of cox-2 selective inhibitors not withstanding, there is evidence to suggest that cox-2 selective inhibitors may inhibit cox-1 and induce gi irritation or ulceration with long term use or at higher doses(21,22). preclinical cardiovascular and renal liabilities of at least some cox-2 selective inhibitors have also been reported(23). thus there is still a need for new anti-inflammatory agents with an improved safety profile. valdecoxib (i) (iii) rofecoxib(iv) celecoxib (ii) (v) nn h3c sh2n o o cf3 o n sh2n o o ch3 iraqi j.pharm.sci., vol.17 (1) ,2008 mefenamic acid derivatives 9 in the view of this background, the present study was conducted to design, synthesize and preliminarily evaluate new mefenamic acid derivatives as potential nsaids. [future study: to measure their selectivity’sمonمcox-2 enzyme.] chemistry the general routes outlined in schemes 1 and 2 were used to synthesize all compounds described here. 4-aminobenzene-sulfonamide (4) and 4-amino-n-methylbenzene sulfonamide (6) was prepared as described by vogel (24) starting from acetanilide as shown in scheme 1.their characterization and physical data are presented in the table1. hn c ch3 o2s cl hn c ch3 o o (1) acetanilide (2) clso3h o2s nh2 hn c ch3 o o2s nh2 nh2 o2s nhch3 hn c ch3 o o2s nhch3 nh2 hcl/ref luxh2o + ch3cooh (4) (3) ch3-nh2/heat (5) (6) hcl/ref luxh2o + ch3cooh scheme 1: synthesis of 4-aminobenzene sulf onamide (4) & 4-amino-n1-methylbenzene sulfonamide (6) nh3 solution/heat + hcl + hcl iraqi j.pharm.sci., vol.17 (1) ,2008 mefenamic acid derivatives 10 scheme 2: synthesis of compounds 9, 10, and 11 nh ch3 ch3 cooh (7) 2 thf dcc nh ch3 c o o c o nh ch3 ch3 + dcu (8) (6) ref lux ch3 ch3 nh c o n h (11) (4) ref lux ch3 ch3 nh c o n h h2no2s (9) ac2o pyridinech3 ch3 nh c o n h so 2nhcoch3 (10) ch3 h3chno2s + (7) + (7) + ch3cooh dcu: dicyclohexyl urea iraqi j.pharm.sci., vol.17 (1) ,2008 mefenamic acid derivatives 11 table (1): the characterization and physical data of the compounds (3-6 and 8-11). solvent system: methanol: acetic acid: ether: benzene (2:18:60:20) experimental all reagents and anhydrous solvents were of analar type and generally used as received from the commercial supplier(merk germany,reidel-dehean-germany ,sigmaaldrich-germany and bdhengland).mefenamic acid was supplied from micro company indian.melting points were determined by capillary method on thomas hoover apparatus (england) and ascending thin layer chromatography (tlc) was run on dc-kartan si alumina 0.2 mm to check the purity and progress of reaction. the identification of compounds was done using iodine vapor and the chromatograms were eluted by: methanol: acetic acid: ether: benzene (2:18:60:20). ir spectra were recorded on model 500 scientific ir spectrophotometer, buck company (usa) as a kbr film.chn microanalysis has been done using exter te micro-analyzer (germany).the analysis was done in the micro analytical center faculty of science –university of cairo. synthesis of 2-(2, 3-dimethylphenylamino) benzoic anhydride (8): mefenamic acid (comp.7) (5g, 20.7mmol) was dissolved in thf (30ml), and then dcc (2.12g, 10.35mmol) was added. the reaction mixture was continuously stirred at room temperature for 4 hours. a white precipitate of dcu was formed which then removed by filtration. the solvent was evaporated under vacuum to give comp.8(26) . the percent yield, physical data and rf value were given in table (1). ir 3330(nh) of secondary amine 1814 and 1743 (c=o) of anhydride, 1618, 1515 and 1488 ( c c st.v.), 1274, 1215 and 1172[c (c=o) – o-(c=o) –c] cm-1 of anhydride. synthesis of 2-(2, 3-dimethylphenylamino)-n(4-sulfamoylphenyl) benzamide (9): compound 8 (2.5g, 5.4mmol), compound 4 (0.93g, 5.4mmol), zinc dust (6mg), glacial acetic acid (0.5ml, 8.75mmol) and dioxane (20ml) were placed in a flask, equipped with refluxed condenser, boiling stones were added. the reaction mixture was refluxed gently for 90 minutes. the solvent was evaporated under vacuum, the residue was dissolved in ethyl acetate, washed with nahco3 (10%, 3*10ml), hcl (1n, 3*10ml) and distilled water (3*10ml), filtered over anhydrous magnesium sulfate. the filtrate is evaporated under vacuum to give the product. the crystallization is carried out by dissolving the compound in ethyl acetate and petroleum ether (80-100 oc) is added to the filtrate until turbidity take place and it is kept in cold place over night. the mixture is filtered while it is cold and the precipitate is collected to give comp.9(27) . the percent yield, physical data and rf value were given in table (1). ir 3376and3304 (n-h) of primary sulfonamide, 3227 (n-h) of secondary amine, 1660 (c=o) of secondary amide, 1598and1530( c c st.v.), 1327and1157 (so2) cm-1 . compound empirical formula molecular weight description % yield melting point observed reported rf value 3 c8h10n2o3s1 214 faint yellow crystals 53 213-214 216 (25) 0.45 4 c6h8n2o2s1 172 white crystals 51 160-161 163-164 (24) 0.75 5 c9h12n2o3s1 228 white crystals 62 179-181 0.52 6 c7h10n2o2s1 186 white powder 44 107-108 0.68 8 c30h28n2o3 464 white powder 80 141-143 0.69 9 c21h21n3o3s1 395 white crystals 40 198-199 0.82 10 c23h23n3o4s1 437 white powder 48 169-171 0.76 11 c22h23n3o3s1 409 white crystals 35 180-181 0.8 iraqi j.pharm.sci., vol.17 (1) ,2008 mefenamic acid derivatives 12 chn calculated (c21h21n3o3s1): c, 63.78; h, 5.35; n, 10.36; s, 8.11. found: c, 62.55; h, 5.44; n, 10.51; s, 8.25. synthesis of n-(4-(n-acetylsulfamoyl)-2-(2, 3-dimethylphenylamino) benzamide (10): acetic anhydride (0.6ml, 6mmol), was added to a solution of compound 9 (0.79g, 2mmol) in pyridine (10ml) and the reaction was allowed to proceed then at 25 oc with stirring for 6 hours. ethyl acetate (100ml) was added and this solution was washed successively with saturated aqueous ammonium chloride (2x20ml) followed by distilled water (2x20ml). the organic fraction was dried with anhydrous magnesium sulfate and the solvent was removed in vacuum to give comp.10 (28) .the percent yield, physical data and rf value were given in table (1). ir 3350and3292 (n-h) of secondary amide andsulfonamide respectively, 1670 (c=o) of secondary amide, 1595, 1533, and1450( c c st.v.) and1332 and1157(so2) cm-1 . chn calculated (c23h23n3o4s1): c, 63.14; h, 5.30; n, 9.60; s, 7.33. found: c, 62.25; h, 5.40; n, 9.83; s, 7.48. synthesis of 2-(2, 3dimethylphenylamino)-n(4-(n-methylsulfamoyl) benzamide (11): compound 8 (2.5g, 5.4mmol), compound 6 (1g, 5.4mmol), zinc dust (6mg), glacial acetic acid (0.5ml, 8.75mmol) and dioxane (25ml) were placed in flask, equipped with reflux condenser, boiling stones were added. the reaction mixture was refluxed gently for 90 minutes, and then it was worked up as prescribed in section3.2 to liberate comp.11. the percent yield, physical data and rf value were given in table (1). ir 3334and3201 (n-h) of secondary amide andsulfonamide, 1664 (c=o) of secondary amide, 1591, 1529 and 1496 ( c c st.v.) and1321 and1159(so2) cm-1 . chn calculated (c22h23n3o3s1): c, 64.53; h, 5.66; n, 10.26; s, 7.83. found: c, 65.20; h, 5.58; n, 10.45; s, 8.01. pharmacology: albino rats of either sex weighing (150 ± 10 g) were supplied by the national center for quality control and drug research and were housed in the animal house of the college of pharmacy, university of baghdad under standardized conditions (12 light-12 dark cycle) for 7 days for acclimatization. animals were fed commercial chaw and had free access to water ad libitum. animals were brought 1 hour before the experiment to the laboratory, and were divided into five groups (each group consist of 6 rats) as follows: group a: served as control and treated with the vehicle (propylene glycol 50% v/v in water); group b: treated with sodium diclofenac (reference agent) in a dose of 3mg/kg suspended in propylene glycol (29); group c, d and e: treated with tested compounds 9, 10 and 11 respectively in a dose equivalent to 7.5 mg/kg of mefenamic acid as finely homogenized suspension in 50% v/v propylene glycol(30) . anti-inflammatory activity: the anti-inflammatory activity of the tested compounds was studied using egg-white induced edema model (31). the drugs or the vehicle were administered i.p. at time zero and acute inflammation was induced by a subcutaneous injection of 0.05ml of undiluted egg-white into the planter side of the left hind paw of the rats at time 15 minutes. the paw thickness was measured by vernier at eight time intervals (0, 15, 30, 60, 120, 180, 240 and 300 minutes) after vehicle or drugs administration. the data are expressed as mean ± s.e.m. and results were analyzed for statistical significance using student t-test (two-sample assuming equal variances) for comparisons between mean values. while comparisons between different groups were made using anova: two-factor without replication. probability (p) value of less than 0.05 was considered significant. results and discussion the most widely used primary test to screen new anti-inflammatory agents is based on the ability of a compound to reduce local edema induced in the rat paw following injection of an irritant agent (32). when eggwhite is injected into the paw of rats, a substantial induction of cox-2 is observed at 2 hours coinciding with enhanced pgs and local edema (33). tables 2 and 3 show the effect of tested compounds on egg-white induced edema as an indicator for their antiinflammatory activity. the intraplanter injection of egg-white into rat hind paw induces a progressive edema, which was reached maximum (measured by millimeter) after 2 hours of injection. table 2 showed the effect of tested compounds (9, 10 and11) in respect to control group. all tested compounds were effectively limited the increase in paw edema, with the effect of compounds 9 and 10 started at time 30 minute (significantly difference compared to control), while compound 11 started at time 120 minute. however, the effect of all tested compounds continued till the end of the experiment with statistically significant (p >0.05) reduction in paw edema. the differences among the iraqi j.pharm.sci., vol.17 (1) ,2008 mefenamic acid derivatives 13 compounds started at time 30 minute in which the compounds 9 and 10 significantly difference at time 30 and 60 minute compared to compound 11. however, the differences among the compounds continued from the time 180 to 300 minute with statistically significant (p >0.05) reduction in paw edema in the following orders 10, 11, and 9 respectively. table 2: effect of control and compounds 9, 10 and 11 on egg-white induced paw edema in rats. treatment groups time (min) control (n=6) compound9 (n=6) compound 10 (n=6) compound11 (n=6) p aw th ic kn es s (m m ) 0 4.46 ± 0.16 4.39±0.10 4.41±0.08 4.38±0.13 15 5.41 ± 0.18 5.45±0.07 5.42±0.12 5.35±0.11 30 6.05 ± 0.16 5.80±0.05*ª 5.76±0.13*ª 6.01±0.10 b 60 6.35 ± 0.07 6.00±0.05*ª 6.00±0.13*ª 6.33±0.09 b 120 6.50 ± 0.09 5.73±0.05*ª 5.66±0.08*ª 5.70±0.10*ª 180 5.93 ± 0.11 5.40±0.05*ª 5.09±0.05*b 5.30±0.07*c 240 5.38 ± 0.09 5.13±0.05*ª 4.86±0.07*b 4.95±0.07*c 300 5.20 ± 0.10 5.05±0.04*ª 4.56±0.08*b 4.68±0.05*c non-identical superscripts (a, b, and c) among different groups are considered significantly different (p<0.05). * significantly different compared to control (p<0.05). table 3 shows the effect of tested compounds (9, 10 and11) with respect to the reference group (diclofenac). as seen in this table; at time 0 and 15 minute there are no differences among different groups; at time 30, only compound 11 is significantly different than diclofenac; at time 60 and 120 all compounds are significantly different than diclofenac; while at time 180 to 300 compounds 9 and 11 are significantly different than diclofenac. the differences among the compounds started at time 30 minute in which the compounds 9 and 10 significantly difference at time 30 and 60 minute compared to compound 11 while at time 120 compound 10 is significantly different than compounds 9 and 11. however, the differences among the compounds continued from the time 180 to 300 minute with statistically significant (p > 0.05) reduction in paw edema in the following orders 10, 11, and 9 respectively. iraqi j.pharm.sci., vol.17 (1) ,2008 mefenamic acid derivatives 14 table 3: effect of diclofenac and compounds 9, 10 and 11 on egg-white induced paw edema in rats. treatment groups time (min) diclofenac (n=6) compound9 (n=6) compound 10 (n=6) compound11 (n=6) p aw th ic kn es s (m m ) 0 4.38±0.14 4.39±0.10 4.41±0.08 4.38±0.13 15 5.37±0.41 5.45±0.07 5.42±0.12 5.35±0.11 30 5.78±0.11 5.80±0.05 ª 5.76±0.13 ª 6.01±0.10*b 60 5.60± 0.10 6.00±0.05*ª 6.00±0.13*ª 6.33±0.09*b 120 5.35±0.10 5.73±0.05*ª 5.66±0.08*b 5.70±0.10*ª 180 5.07±0.10 5.40±0.05*ª 5.09±0.05 b 5.30±0.07*c 240 4.87±0.10 5.13±0.05*ª 4.86±0.07 b 4.95±0.07*c 300 4.61±0.10 5.05±0.04*ª 4.56±0.08 b 4.68±0.05*c non-identical superscripts (a, b, and c) among different groups are considered significantly different (p<0.05). * significantly different compared to control (p<0.05). conclusion the in vivo anti-inflammatory study showed that the incorporation of 4aminobenzenesulfon amide, 4-amino-n-methylbenzenesulfonamide, or n-(4-aminophenylsulfonyl) acetamide into well known anti-inflammatory drug (mefenamic acid) maintains its antiinflammatory activity. compound 10 showed more potent anti-inflammatory effect than compound 9 or 11 and have a comparable effect to that of diclofenac at time 180 to 300 minute with the same onset of action. acknowledgments we are grateful to the staff members and colleagues of the department of pharmaceutical chemistry and the department of pharmacology and toxicology .also we wish to express grateful thanks to m.sc. sabah jawad for his help and support. references 1. shoutakis, v.a.; 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69: 183-189. 30. ali almasirad; mohammad j.; davood b.; abbas s. :synthesis and analgesic activity of n_ arylhydrazone derivatives of mefenamic acid. j.phar.pharmaceut sci. 2005; 8(3) :419-425. 31. vogel, h.g. and goethe, j.h.: drug discovery and evaluation. pharmacological assay (2nd ed.). springerverlag, berlin heidelbers, 2002; pp. 751. 32. winter, c.a; risley, e.a. and nuss, g.w.: carrageenan-induced edema in hind paws of the rat as an assay for antiinflammatory drugs. proc. soc. exp. bio. med. 1962; 111: 544-547. 33. seibert, k.; zhang, y.; leahy, k.; masferrer, j.; et al.: pharmacological and biochemical demonstration of the role of cyclooxygenase-2 in inflammation and pain. proc. natl. acad. sci. usa 1994; 91: 12013. clinical evaluation of melatonin alone and in combination with pizotifen in the prophylaxis of migraine iraqi j.pharm.sci., vol.16 (1) ,2007 clinical evaluation of melatonin as antimigraine 1 clinical evaluation of melatonin alone and in combination with pizotifen in the prophylaxis of migraine muqdam m.mohamed ali* , salim a. humadi** and ashwaq n. al-jaff** , 1 *al-hussein hospital, karbala ** departments of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq abstract : the treatment of migraine headache targets the neurovascular mechanism and involves the use of serotonin receptor antagonists. some of these drugs are used for the treatment of acute attacks; while others are effective as prophylactic measures to decrease the duration and frequency of attacks. pizotifen, a 5-hta antagonist, is one of the prophylactic drugs for which the clinical use resulted in low outcomes in reducing migraine symptoms. melatonin, a serotonin derived neurohormone, was reported to exert many functions like sleep induction, anti-inflammatory, neurovascular regulation, cytoprotection and modulation of neurotransmitter release. in the view of the involvement of serotonin in the pathophysiology of migraine and the properties of melatonin, the present study has been conducted to evaluate the effectiveness of melatonin alone or in combination with pizotifen for the improvement of migraine symptoms. the study was conducted on 72 patients, which were under neurologist supervision during the entire period of study. the patients were instructed to avoid any precipitating diet (chocolate, cheese,… etc) and where randomly divided into 4 groups each of 18. the first group was treated with melatonin (3mg, 30 minutes before bed time); the second with pizotifen (0.5mg twice daily); the third with melatonin (at night) and pizotifen (twice daily); and the fourth with placebo (at night). the treatment was continued for 42 days and was followed up and monitored each week. after a month of treatment, the severity, duration and frequency of migraine attacks were recorded using special migraine scoring system.the results revealed that melatonin alone significantly decreased the severity, duration and frequency of migraine attack by 48%, 53% and 45.75%, respectively; while these produced by pizotifen were significantly reduced by 25%, 45.3% and 27.5%, respectively. the effect of pizotifen was generally enhanced by the addition of melatonin and the improvement in migraine symptoms were, severity (59%), duration (62.7%) and frequency (58%). these effects were generally low in placebo treated group and the reduction in severity, duration and symptoms were (12.2%, 20% and 16.2%, respectively). the distribution of patients according to their response as complete or partial was significantly different among treated groups; and within the same group is differ according to the measured parameter suggesting involvement of factors other than treatment in the improvement of migraine symptoms such as psychological state, stress and others.the study has concluded the effectiveness of melatonin alone and in combination with pizotifen as a prophylactic measure in term of reducing the severity, duration and symptoms of migraine headache. key words: migraine headache, pizotifen, melatonin. لخالصةا انخقهٛذٚت نصذاع انشقٛقت حخجّ َحٕ اٜنٛت انٕػائٛت انؼصبٛت انًسببت نذاء انشقٛقت ٔحخعًٍ اسخخذاو يغهقاث اٌ انؼالجاث ًُا انبؼط االخز ٚسخخذو كؼالج ٔقائٙ نهخقهٛم يٍ يسخقبالث انسٛزٔحٍَٕٛ. بؼط ْذِ االدٔٚت حسخخذو نؼالج انُٕباث انحادة نهصذاع بٛ ٛقت. فخزاث ٔحزدد انُٕباث. أٌ ػقار انبٛشٔحفٍٛ ْٕ احذ االدٔٚت انٕقائٛت، اال اٌ اسخخذايّ انسزٚز٘ قهٛم انفؼانٛت فٙ خفط ػالياث داء انشق ٍ، ْٕٔ ْزيٌٕ ػصبٙ يشخق يٍ اٌ ػقار انًٛالحَٕٛ (.hta-5) انسٛزٔحٍَٕٛ َٕعْٔذا انؼالج يؼزٔف باغالقّ نًسخقبالث ز انسٛزٔحٍَٕٛ، قذ ثبج اٌ نّ ٔظائف يخؼذدة يثم ححفٛش انُٕو، كًعاد نالنخٓاباث، حُظٛى االٔػٛت انذيٕٚت انؼصبٛت، ٔقاٚت انخالٚا، ٔححٕٚ اٌ انذراست انحانٛت قذ فٙ افزاس انُٕاقم انؼصبٛت. فٙ ظٕء اشخزاك انسٛزٔحٍَٕٛ فٙ سببٛت داء انشقٛقت ٔانخٕاص انًؼزٔفت نهًٛالحٍَٕٛ ف يزٚعاً ٔقذ كإَا 27اجزٚج انذراست ػهٗ اجزٚج نخقٛٛى االسخخذاو انسزٚز٘ نهًٛالحٍَٕٛ نٕحذِ أ يغ انبٛشٔحفٍٛ نؼالج ػالياث انشقٛقت. ت، ٔانجبٍ، ححج االشزاف انطبٙ نذٔ٘ االخخصاص انؼصبٙ خالل فخزة انذراست، ٔقذ ارشذٔا نخجُب االغذٚت انًسببت نهشقٛقت )انشكٕالح يهغى قبم انُٕو 3يزٚعاً. ػٕنجج انًجًٕػت االٔنٗ بانًٛالحٍَٕٛ ) 81يجايٛغ كم يُٓا يكٌٕ يٍ 4ٔغٛزْا( ٔقذ صُفٕا ػشٕائٛاً انٗ ِانؼاليهغى يزحٍٛ ٕٚيٛاً(، ٔانثانثت بانًٛالحٍَٕٛ ٔانبٛشٔحفٍٛ يؼاً ٔبانجزع اػالِ، ٔانزابؼت ب5.5بُصف ساػت(، ٔانثاَٛت بانبٛشٔحفٍٛ ) ّٕ ج انًً (placebo بشكم كبسٕنت نٛالً. اسخًز انؼالج نفخزة )ٕٚياً بًخابؼت اسبٕػٛت. بؼذ شٓز يٍ انؼالج، حى حسجٛم حذة ٔفخزة ٔحزدد َٕباث 47 اثبخج انذراست اٌ انًٛالحٍَٕٛ نٕحذِ قذ ادٖ انٗ حقهٛم يُطقٙ فٙ حذة ٔفخزة ٔحزدد َٕباث صذاع انشقٛقت باسخخذاو َظاو حُقٛط خاص. % ػهٗ انخٕانٙ. ٔػهٗ 72.5% 45.3ٔ%، 75% ػهٗ انخٕانٙ، بًُٛا كاٌ انخقههٛم بانبٛشٔحفٍٛ بُسبت 45.25%،53%، 41نصذاع بُسبت ا %( ٔانخزدد 67.2%(، انفخزة )55انؼًٕو فاٌ حأثٛز انبٛشٔحفٍٛ قذ اسداد باظافت انًٛالحٍَٕٛ ٔاٌ انخقهٛم فٙ ػالياث انصذاع كاَج، انحذة ) ِ ٔكاَج َسبت االَخفاض فٙ انحذة، انفخزة، ٔانخزدد ْٙ %(. اٌ ْذِ ا51) ّٕ نخاثٛزاث كاَج قهٛهت جذا ػٍ انًجٕػت انًؼانجت بانؼالج انًً % ػهٗ انخٕانٙ. اٌ حٕسٚغ انًزظٗ بحسب اسخجابخٓى نهؼالج، اٌ كاٌ جشئٛاً أ كهٛاً، كاٌ يخخهفاً بصٕرة %86.7، ٔ %75، 87.7 ت انٕاحذة فاٌ انخٕسٚغ كاٌ يخخهفاً اٚعاً بحسب انًؼٛار انًقاص يًا ٚذل ػهٗ حذخم ػٕايم اخزٖ يُطقٛت بٍٛ انًجايٛغ، ٔظًٍ انًجًٕػ ًٚكٍ االسخُخاج يٍ ْذِ انذراست باٌ انًٛالحٍَٕٛ نٕحذِ أ يغ انبٛشٔحفٍٛ قذ غٛز انؼالج فٙ ْذا انخباٍٚ يثم انحانت انُفسٛت، االجٓاد ٔغٛزْا. ذة، فخزة، ٔحزدد َٕباث صذاع انشقٛقت.أظٓز فؼانٛت ٔقائٛت فٙ انخقهٛم يٍ ح 1 corresponding authors: e-mail: ashwaq73@yahoo.com received : 4/5/2006 accepted : 4/3/2007 mailto:ashwaq73@yahoo.com iraqi j.pharm.sci., vol.16 (1) ,2007 clinical evaluation of melatonin as antimigraine 2 introduction migraine headache is a primary disorder, resulting from dysfunctions of the trigeminovascular system. the disorder manifests as recurrent attacks of sever pain that varies in frequency from one attack a year to two or more a week (1) . migraine headaches are classified according to the accompanied aura into classical (with aura) and common (without aura) (2) . aura means visual scotomas or even hemianopia and speech abnormalities followed by sever throbbing unilateral headache that lasts for a few hours to 1-2 days (3) . the variation in the severities of pain among migrainous patients has encourage vigorous initiation and prophylactic therapies (4) . the vasomotor changes are greatly suggested to underlie the pathophysiology of migraine (5) . the marked increase in the amplitude of temporal artery pulsation and relief of pain by ergotamine may support this hypothesis (6) . in addition, serotonin released from platelets and serotonergic nerve endings in the meningeal blood vessels has been implicated in the initiation of migraine headache (5, 7) . in this regard, serotonin receptor antagonists are typical antimigraine drugs of current use for the acute management and prophylaxis of migraine (6, 4) . pizotifen, now, is one of the prophylactic drugs for migraine headache to reduce the severity, frequency and duration of attacks. the drug act by blocking 5hta receptors. however, the clinical use of this drug has reported to be accompanied by weight gain and antimuscarinic effects (3) . melatonin, n-acetyl-5-methoxytryptamine, is a serotonin derivative produced and released by the pineal gland and some other tissues and is believed to participate in the regulation of sleep-wake cycle (8) . the release is coincides with darkness (begin around 9pm and lasts until about 4am) and suppressed by day-light (9) . several studies have conducted to investigate the properties and effects of melatonin; and these revealed that this neurohormone processes free radical scavenging (10) , nitric oxide synthase inhibiting (11) , dopamine release inhibiting (12,13) , gabaergic stimulating (14,15,16) , glutamate neurotoxic protecting (17) , opioid analgesic (18) , neurovascular regulating (19) and serotonine modulating actions (20) . in addition, this agent has been studied for a number of other functions including contraception; antioxidant action; prevention of aging; and treatment of depression, human immunodeficiency virus (hiv) infection and a variety of cancers (9, 21) .in the view of available data about melatonin properties and functions and the pathophysiology of migraine, this study has been conducted to evaluate the clinical effectiveness of melatonin alone and in combination with pizotifen as prophylactic therapy in migraine patients. patients and methods patients: the study group comprised of a total of 72 patients with migraine in age range of (13-55 years old). the patients included in this study were under neurologist supervision during the entire period of treatment, where the vital signs and positive finding of routine physical examination were evaluated and recorded. all patients were diagnosed having migraine headache of many types (e.g. common, classical, ophthalmoplegic and retinal migraine). the duration of disease ranged from (3 months to 10 years). patients involved in this study were instructed to avoid diet that triggers migraine such as chocolate, cheese, etc. the patients were randomly divided into 4 groups (a, b, c and d) each of 18. the groups were treated for 42 days, followed up and monitored each week. group a was given melatonin (3mg) 30 minutes before bed time, group b was treated with pizotifen (0.5mg twice daily), group c was treated with combination of melatonin and pizotifen (same doses in group a and b) and group d was treated with placebo (glucose filled capsules at night). method: the migraine severity (migsev) score was used to identify items that serve to assess the severity of migraine with a high level of clinical and psychometric relevance (figure 1) (22) . this is because the severity of migraine is an important determinant of patient quality of life and of health care resource utilization. only seven items reflecting severity were identified by expert consensus. these were intensity of pain, tolerability, disability in daily activity, nausea or vomiting (or both), duration of attack, resistance to treatment and frequency of attack. principal components analysis performed on the seven items of the severity questionnaire identified three dimensions while correlation analysis showed that the first dimension covered 4 items relating to the intensity of attacks (intensity of pain, tolerability, disability in activity and presence of nausea or vomiting). the second dimension related to resistance to treatment (resistance to treatment and duration of attack) and the third dimension to frequency of attacks. a rating system was devised. firstly, the number of items for which the lowest possible and the highest possible rating was determined. secondly a ternary (low, intermediate and high) overall rating of severity was attributed using the following decision tree: iraqi j.pharm.sci., vol.16 (1) ,2007 clinical evaluation of melatonin as antimigraine 3 a-first dimension parameters (main parameters) score intensity of pain nausea minimum medium medium maximum mild moderate intense sever intense no nausea mild intense vomiting score disability in daily activity tolerability minimum medium medium maximum no disability mild marked confined to bed tolerable barely tolerable intolerable *grade one----at least one minimum score without maximum score grade two----any other grade three—at least one maximum score without minimum score or at least two maxi. score b-2 nd dimension parameters resistance to treat. duration of attack (hr.) no yes < 4 hr. 12-24 hr. 4-12 hr. > 24 hr. c-3 rd dimension parameters frequency of attack < 5/year 1/weak 5-10/year >1/weak 1-2/month fig. (1) first, second and third dimension parameters of migraine (15) . low (grade 1): at least 1of the 4 items with a minimum score, and no item with a maximum score; high (grade 3): at least 1 of the 4 items with a maximum score, and no item with a minimum score or at least 2 items with a maximum score; intermediate (grade 2): all other cases. data were expressed as mean ± sd and as percent change from baseline regarding those obtained from migsev score. response of patients was expressed as percentage from total number in each group. analyses were done using unpaired student’s t-test and chi-square method when appropriate. results : effect of different treatment on first dimension parameter (severity of migraine) in this study, the difference between the baseline values of migraine severity score among all groups was not significant (p>0.05) as shown in table (1) and figure (2). after 1 month, the severity score following treatment with melatonin (3mg/night), pizotifen (0.5mg twice daily), melatonin-pizotifen combination and placebo was significantly reduced by 48%, 25%, 59% and 12.2%, respectively (p<0.05). the effect exerted by melatonin was significantly differ from that exerted by pizotifen, p<0.05. in addition, melatonin was shown to enhance the effect of pizotifen when added to the treatment, p<0.05; however, this effect was not significantly differ from that exerted by melatonin alone, p>0.05. the reduction in the severity score following treatment with melatonin, pizotifen and their combination was significantly higher than that produced by placebo, p<0.05. table (2) clearly showed that the improvement in severity score in all treated groups was complete, p<0.01 (analyzed by chi-square method); this means that the distribution of patient’s response was greatly depend on treatment regimen used with great effect achieved by melatonin and its combination with pizotifen. effect of different treatment on second dimension parameter (duration of migraine attack) table (1) and figure (2) also showed the effect of the four treatments on the duration score of migraine attack. it was shown that all patients were not significantly differ in their baseline score (p>0.05). the reduction in duration of attacks following treatment with melatonin, pizotifen, melatonin-pizotifen combination and placebo was significantly reduced by 53%, 45.3%, 62.7% and 20%, respectively (p<0.05). the effect of melatoninpizotifen combination was significantly higher than that produced by pizotifen alone, p<0.05; however, this effect did not differ significantly from that produced by melatonin alone (p>0.05). moreover, the reduction in duration score following melatonin, pizotifen and their combination was significantly higher that that produced by placebo treatment (p<0.05). iraqi j.pharm.sci., vol.16 (1) ,2007 clinical evaluation of melatonin as antimigraine 4 table (1): effect of treatment with melatonin, pizotifen and their combination on migraine parameters parameter time group a (melatonin 3mg/night) group b (pizotifen 0.5mg twice daily) group c (pizotifen + melatonin) placebo baseline 2.78 ± 0.43 2.67 ± 0.49 2.72 ± 0.46 2.72 ± 0.46 after 1 month 1.44 ± 0.78* a 2.00 ± 0.91* b 1.11 ± 0.47 * a 2.39 ± 0.85 * b 1 st dimension response (intensity of attack) % reduction 48% 25% 59% 12.2% baseline 16.56 ± 5.97 16.89 ± 6.22 17.00 ± 7.00 16.67 ± 6.79 after 1 month 7.67 ± 5.32* a,b 9.22 ± 5.87* a 6.33 ± 3.51* b 13.33 ± 7.29 * c 2 nd dimension response (duration of attack) % reduction 53% 45.3% 62.7% 20% baseline 9.83 ± 3.28 10.11 ± 2.76 9.78 ± 3.04 10.27 ± 2.96 after 1 month 5.33 ± 2.91* a 7.33 ± 3.56* b 4.11 ± 2.95 * a 8.61 ± 2.85 * b 3 rd dimension response (frequency of attack) % reduction 45.75% 27.5% 58% 16.2% data are expressed as mean ± sd. n=18 per group. *p<0.05 with respect to baseline value. non-identical superscripts (a, b) for the same parameter within the same period considered significantly different (p<0.05). table (2): distribution of patient’s response to different medications according to the 1 st , 2 nd and 3 rd dimension parameters of migraine. treatment group migraine parameter group a (melatonin3mg/ni ght) group b (pizotifen 0.5mg twice daily) group c (pizotifen + melatonin) group d (placebo) complete response 13 (72.20%) 7 (38.80%) 17 (94.40%) 4 (22.20%) partial response 1 (5.50%) 1 (5.50%) 0 (0.00%) 0 (0.00%) 1 st dimension parameters (severity of attack) p (χ 2 test) 0.0002 complete response 8 (44.40%) 7 (38.80%) 10 (55.50%) 3 (16.60%) partial response 6 (33.30%) 6 (33.30%) 7 (38.80%) 4 (22.20%) 2 nd dimension parameters (duration of attack) p (χ 2 test) 0.015 complete response 4 (22.20%) 2 (11.10%) 8 (44.40%) 0 (0.00%) partial response 11 (61.10%) 9 (50.00%) 10 (55.50%) 9 (50.00%) 3 rd dimension parameters (frequency of attack) p (χ 2 test) 0.002 data are expressed as number and percentage of total (n=18 per treatment group). data are analyzed by chi-square (χ 2 ) test. iraqi j.pharm.sci., vol.16 (1) ,2007 clinical evaluation of melatonin as antimigraine 5 in the groups that treated with melatonin, pizotifen and their combination the distribution of patients according to their response as complete or partial was significant (p<0.05, analyzed by chi-square method). however, the difference between the percentages of patients with complete or partial response was only small, as shown in table (2). fig (2): migraine parameters before and after 1 month treatment with melatonin (3mg/night), pizotifen (0.5mg x2/day), their combination and placebo. data are expressed as mean ± sd, n=18 per group. all parameters are significantly different with respect to their baseline value (p<0.05, by paired student’s t-test). non identical superscripts (a, b) within the same parameter considered significantly different (p<0.05, by unpaired student’s t-test). effect of different treatment on third dimension parameter (frequency of migraine attack) regarding the frequency score of migraine attack, all patients were not significantly differ in their baseline score (p>0.05), table (1) and figure (2). the reduction in frequency score following treatment with melatonin, pizotifen, melatonin-pizotifen combination and placebo was significant high with percent reduction of 45.75%, 27.5%, 58% and 16.2%, respectively (p<0.05). the effect produced by melatonin and its combination with pizotifen was non-significantly different (p>0.05). on the other hand, the reduction produced by pizotifen was not significantly differ from that produced by placebo, p>0.05. however; the effect of pizotifen was significantly lower that that produced by either melatonin or melatonin-pizotifen combination, p<0.05. the response or reduction in frequency score of migraine attack in treated patients was shown to be partial and significantly (p<0.01) depend on the type of treatment used, as shown in table (2). discussion : the results presented in this study demonstrated that melatonin in a dose of 3mg/day greatly and significantly reduced the severity, duration and frequency of migraine attack. the nightly administration of melatonin was to avoid sleep induction during the day; helping patients suffering from migraine with delayed sleep syndrome (16) ; and as supplementation to elevate the decreased levels at night in migraine patients. in this context, claustrate et al. was reported that plasma levels of melatonin in migraine patients was lower when drawn at 23.00 hr in comparison to controls (23) . in addition, murialdo et al. who reported the decrease in nocturnal melatonin levels throughout the ovarian cycle of migraine patients in comparison to controls (24) . the effectiveness of melatonin in modulating the characteristics of migrainous headache may be attributed to many neurological and biochemical effects of the drug. melatonin was reported to modulate the release of serotonin in rat hippocampus (20) . in this context, serotonergic system has been implicated in the pathogenesis of migraine and mediation of meningial vasodilation (25, 26) . additional mechanism may be the interaction with analgesic opioids. melatonin has been regarded as pineal opioid which may exert an analgesic action by binding its receptors in the cns (18) and activation of gabaergic system (16) . the modulation of immune reactions and inhibition of expression of pro-inflammatory cytokines by melatonin might have a role in the observed effects in this study (10) . this suggestion is supported by findings of bettahi et al. who reported that melatonin reduces nitric oxide synthase activity in rat hypothalamus (11) . nitric oxide has been regarded as a radical neurotransmitter in the cns (27) and many studies have indicated its participation in pain mediation at the spinal cord level (28, 29, 30) . other properties of melatonin which might also explain the observed effects seen in this study include its structural similarity to indomethacin (31) and its anti-oxidant and membrane stabilization action during lipid peroxidation (32) . the antimigraine activity of pizotifen has been reported to be due to blockage of serotonin receptors and modulation of serotonergic system during migraine attacks (26) . the drug is clinically effective as prophylactic measure. the use of pizotifen is thus aimed to target one mechanism of migraine headache which is the neurological mechanism (34) . the effect of pizotifen is greatly enhanced by the addition of melatonin in term of the reduction in the severity, duration and frequency of migraine attacks. since the two 0 3 6 9 12 15 18 21 24 27 severity of attack duration of attack frequency of attack severity of attack duration of attack frequency of attack p a r a m e te r s c o r e melatonin 3mg/night pizotifen 0.5mg x 2/day combination placebo baseline after 1 month a b a b a a a,b a b b c b iraqi j.pharm.sci., vol.16 (1) ,2007 clinical evaluation of melatonin as antimigraine 6 agents have different mechanism and targets of action in relation to migraine pathophysiology, the synergistic interaction is likely contributed to the observed results. this is very important since combination therapy may be useful for the reduction of the dosage and frequency of administration of pizotifen together with minimizing the adverse effects that include weight gain and antimuscarinic effects (33) . it was also reported in this study that, the percentage of responder patients within the same treated group varied according to the measured parameter (table 2). this suggests that factors other than the treatment may have a role such as life style, emotional status and stressful conditions. in conclusion, the data obtained in this study provide clinical evidences for the effectiveness of melatonin alone and in combination with pizotifen as a prophylactic measure in migraine. refernces : 1. adams, r.d.: principles of neurology (6 th ed.). mcgraw-hill, new york, 2002; 4: 172 2. classification and diagnostic criteria for headache disorders, cranial neuralgias, and facial pain. headache classification committee of the international headache society. cephalalgia 1988; 8 (suppl 7): 196. 3. katzung, b.g. and julius, d.j.: histamine, serotonin and the ergot alkaloids. in: basic and clinical pharmacology (8th ed.), katzung, b.g. (ed.). mcgraw-hill, new york, 2001; 286. 4. raskin, n.h.: acute and prophylactic treatment of migraine: practical approaches and pharmacologic rationale. neurology 1993; 43(suppl. 3): s39. 5. moskowitz, m.a.; macfarlane, r.: neurovascular and molecular mechanisms in migraine headaches. cerebovasc. brain metab. rev. 1993; 5: 159. 6. welch, k.m.a.: drug therapy of migraine. new eng. j. med. 1993; 329: 1476. 7. fozard, j.r.; kalkman, h.o.: 5hydroxytryptamine (5-ht) and the initiation of migraine: new perspectives. naunyn-schmiedebergs arch. pharmacol. 1994; 350: 225. 8. cupp, m.j.: melatonin. am. fam. physician. 1997; 56: 1421-1425. 9. brzezinski, a.: melatonin in humans. new eng. j. med. 1997; 336: 186. 10. reiter, r.j.; calvo, j.r.; karbownik, m.; qi, w. and tan, d.x.: melatonin and its relation to the immune system and inflammation. ann. n.y. acad. sci. 2000; 917: 376-386. 11. bettahi, i.; pozo, d.; osuna, c.; reiter, r.j.; acuna-castroviejo, d. and guerrero, j.m.: melatonin reduces nitric oxide synthase activity in rat hypothalamus. j. pineal res. 1996; 20: 205-210. 12. zisapel, n.: melatonin-dopamine interactions: from basic neurochemistry to a clinical setting. cell mol. neurobiol. 2001; 21: 605-616. 13. peroutka, s.j.: dopamine and migraine. neurology 1997; 49: 650-656. 14. ramadan, n.m.: the link between glutamate and migraine. cns spectr 2003; 8: 446-449. 15. wang, f.; li, j.c.; wu, c.f.; yang, j.y.; xu, f. and peng, f.: hypnotic activity of melatonin: involvement of semicarbazide hydrochloride, blocker of synthetic enzyme for gaba. acta. pharmacol. sin. 2002; 23: 860-864. 16. wu, f.s.; yang, y.c. and tsai, j.j.: melatonin potentiates the gaba(a) receptor-mediated current in cultured chick spinal cord neurons. neurosci. lett. 1999; 260: 177-180. 17. espinar, a.; garcia-oliva, a.; isorna, e.m.; quesada, a.; prada, f.a. and guerrero, j.m.: neuroprotection by melatonin from glutamate-induced excitotoxicity during development of the cerebellum in the chick embryo. j. pineal res. 2000; 28: 81-88. 18. ebadi, m.; govitrapong, p.; phansuwanpujito, p.; nelson, f. and reiter, r.j.: pineal opioid receptors and analgesic action of melatonin. j. pineal. res. 1998; 24: 193200. 19. savaskan, e.; olivieri, g.; brydon, l.; jockers, r.; krauchi, k. and wirz-justice, a.: cerebrovascular melatonin mt1receptor alterations in patients with alzheimer's disease. neurosci lett. 2001; 308: 9-12. 20. monnet, f.p.: melatonin modulates [3h] serotonin release in the rat hippocampus: effects of circadian rhythm. j. neuroendocrinol. 2002; 14: 194-199. 21. hagan, r.m. and oakley, n.r.: melatonin comes age? trends pharmacol. sci. 1995; 16: 81. 22. el-hasnaoui, a.; vray, m.; richard, a. and nachit-ouinekh, f.: assessing the severity of migraine: development of the migsev score. headache. 2003; 43: 628635. 23. claustrat, b.; loisy, c.; brun, j.; beorchia, s.; arnaud, j.l. and chazot, g.: nocturnal plasma melatonin levels in migraine: a preliminary report. headache. 1989; 29: 242-245. 24. murialdo, g.; fonzi, s.; costelli, p.; solinas, g.p.; parodi, c. and marabini, s.: urinary melatonin excretion throughout the ovarian cycle in menstrually related migraine. cephalalgia 1994; 14: 205-209. iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 7 25. raskin, n.h.: repetitive intravenous dihydroergotamine as therapy for intractable migraine. neurology 1986; 36: 995-997. 26. lance, j.w.; lambert, g.a.; goadsby, p.j. and zagami, a.s.: 5-hydroxytryptamine and its putative aetiological involvement in migraine. cephalalgia suppl. 1989; 9: 7-13. 27. vincent, s.r.: nitric oxide: a radical neurotransmitter in the central nervous system. prog. neurobiol. 1994; 42: 129160. 28. meller, s.t. and gebhart, g.f.: nitric oxide and nociceptive processing in the spinal cord. pain 1993; 52: 2002-2012. 29. meller, s.t.; cummings, c.i.; traub, r.j. and gebhart, g.f.: the role of nitric oxide in the development of the hyperalgesia produced by intraplanter injection of carrageenan in the rat. neuroscience 1994; 60: 367-374. 30. gordh, t.; sharma, h.s.; alm, p. and westman, j.: spinal cord nerve lesion in the upregulation of neuronal nitric oxide synthase in the spinal cord – an immunohistochemical investigation in the rat. amino acids 1998; 14: 105-112. 31. peres, m.e.p.: melatonin, the pineal gland and their implications for headache disorders.cephagia.2005; 25(6): 403-411. 32. garcia, j.j.; reiter, r.j. and guerrero, j.m.: melatonin prevents changes in microsomal membrane fluidity during induced lipid peroxidation .febs lett. 1997; 408: 297300. 33. rang, h.p.; dale, m.m. and ritter, j.m.: other peripheral mediators: 5hydroxytryptamine and purines. in: pharmacology (4th ed.). churchill livingstone, london, 1999; p. 172. 34. humphrey, p.p.; feniuk, w.; perren, m.j.; beresford, i.j.; skingle, m. and whalley, e.t.: serotonin and migraine. ann. n.y. acad. sci. 1990; 600: 587 microsoft word bagpha05114 _7_ iraqi j. parma. sci., vol.14, 2005 52 -------------------------------------------------------------------------------------------------------------- the effect of some variables on the formulation of captopril as tablets shaymaa nazar al – sammarrai dr. alaa a. abdul rasool , dr. hikmet al – dujali department of pharmaceutics, college of pharmacy, baghdad university, baghdad-iraq received: 07.01.2001 accepted: 02.03.2002 abstract: captopril is an angiotensin converting enzyme inhibitor (acei) used to treat hypertension, congestive heart failure, and myocardial infraction. the only dosage form available for captopril is the plain tablet in strength of 12.5,25,50 and 100mg tablet. this investigation is concerned with factors affecting the formulation of captopril as a plain tablet dosage form of 50mg. many trials were made to prepare satisfactory tablets for the drug by using wet – granulation methods with various additives. it was found that poly vinyl pyrrolidone (p.v.p.) as binder gave the most satisfactory tablets. at the same time a shorter disintegrantion time and slower dissolution rate were obtained with the addition of starch intragranular. while the distintegration time and dissolution rate were faster for explotab when it was used intragranular in comparison with starch. a comparative study on the physical properties of the prepared tablets with capoten® (squibb), miniten® (apm), and capocard® (dad) tablets, showed that the release of drug from the selected formula was similar to that obtained from miniten ® at 0.1n hcl and 37c. the stability of the prepared tablet was also studied at 50c, 60c, and 70c for 4 months and the calculated shelf – life was about 3.5years at 25c. :الخالصة یستعمل في عالج ضغط الدم المرتفع ، وعجز القلب ). الموترات الوعائیة(الكابتوبریل ھو مادة مانعة لالنزیم المحول لالنجیوتینسن .المحتقن وإحتشاء العضلة القلبیة تم اجراء العدید من التجارب للحصول .تصنیع الكابتوبریل بشكل اقراص سریعة التحررالعوامل المؤثرة على إھتمت بھذه الدراسة وقد ُوِجَد ان مادة . على صیغة مقبولة لھذا العقار بشكل اقراص بأستعمال طریقة الحبیبات الرطبة بأستخدام مواد مختلفة من السواغ .كمادة رابطة تعطي الصیغة التركیبیة االكثر مقبولة من االقراص (.p.v.p)الـبولي فاینیل بایرولیدون كان اسرع من اس�تعمالھا خ�ارج )التفتت(وفي نفس الوقت وجد انھ في حالة استعمال نشاء الذرة ضمن الحبیبات فأن زمن التحطم عن�د (explotab)ی�ت الص�ودیوم النش�وي جالیكولفي الحالة االخ�رى .الحبیبات ولكن زمن الذوبان كان أبطأ من خارج الحبیبات .اسرع من استعمالھا خارج الحبیباتوزمن الذوبان یكونان زمن التحطمكل من ضمن الحبیبات فإن ھ استعمال الحبوب المحّضرة وحبوب الكابوتین لشركة سكویب وحبوب المنیتین الخواص الفیزیائیة بین لدراسة كذلك تم اجراء دراسة مقارنة من الصیغة المختارة قریبة نوع�ًا م�ا دواءتحرر الحدة وحبوب الكابوكارد لشركة دار الدواء االردنیة ووجد بأن سرعة للشركة المت ٣٧عیاري من حامض الھایدروكلوریك ودرج�ة ح�رارة ٠.١من سرعة تحرره من حبوب المنیتین في  ة اس�تقراریة تم�ت دراس� . م .سنة ٣.٥اشھر وُوجد ان تاریخ انتھاء مفعول الدواء الحالي ٤م لمدة ٧٠م ، ٦٠م ، ٥٠مختلفة حراریةالدواء في درجات iraqi j. parma. sci., vol.14, 2005 53 --------------------------------------------------------------------------------------------------------------introduction: hypertension means abnormally elevated blood pressure. different drugs for treatment of hypertension can be used, among these are angiotensin converting enzyme inhibitors (acei) such as; captopril. captopril is d-3-mercapto-2-methyl (propanoyl – l – praline) a potent and selective (acei) (kininaze ii) (1). it was synthesized in 1976 by ondetti and his colleagues at e.r. squibb and sons. it was known under the generic name of sq14,255 then the name was changed to captopril. captopril is a novel drug, that specifically designed to compete for the active binding site of (ace), as such captopril inhibits the conversion of angiotensin i to angiotensin ii (2) . it is used for treatment of mild to moderate hypertension, and in hypertensive crisis . it is the only (acei) that can be used for hypertension of neonate and infant (2) . in addition, captopril can be used in treatment of congestive heart failure, mi and in diabetic nephropathy(3). the only dosage form available for captopril is the plain tablet in the strength of 12.5, 25, 50 and 100mg tablet. it was felt of interest to study the factors that affect the formulation of captopril as a plain tablet and compare it with the standards. experimental section: materials: captopril powder (shilton chemicals, england), lactose, acacia (riedel – de – hean a.g. hannover, germany), starch (merck, germany), microcrystalline cellulose (avicel ph101, fmc corporation, pennsylvania, usa), poly vinyl pyrrolidone (p.v.p. k30), carboxy methylcellulose sodium salt (c.m.c.), hydrochloric acid, (bdh chemicals ltd., pool, england), explotab (aveba, veendam, netherlands), magnesium stearate (barlocher, gmbh, germany), capoten ® (squibb, usa), miniten® (apm, jordan), capocard® (dad, jordan). instruments: thomas hoover electrical melting point apparatus (england), sartorious balance (4 digits), type a120s, gmbh, germany, sartorious balance type 126s mp – germany, tablet machine, type f3, manesty machine ltd., liverpool, england, tablet hardness tester stokes – monsanto (monsanto chemical co., usa), tablet disintegration apparatus, manesty ltd., liverpool, england, erweka friabilator, germany, nalgene syringe filter 0.2m, 25µm surfactant free cellulose acetate membrane (modified acrylic housing), dissolution apparatus usp, erweka, dt6, germany, swininx – 13 microfilter, millipore corporation, bedford, mass, usa.; u.v. spectrophotometer, sp8 – 100, pyeunicam, england., microfilter paper (microfilter, england), tray oven, apex, england, oven, 854 schwabach, memmert, germany., oven, (astell hearson), oven gallen kamp, b5 ov – 210, england. methods: different formulas were prepared to find the most satisfactory one using wet granulation technique. wet granulation: after 5 minutes of dry blending of drug and excipients, the binder solution was added to formulas (1 – 7, 9, 14) and (15) gradually in the mixing mortar until a satisfactory wetting was achieved (ball test). the wet mass was then granulated through a sieve no.10 and dried in tray oven iraqi j. parma. sci., vol.14, 2005 54 --------------------------------------------------------------------------------------------------------------at 45c for 30 minutes. the granules were homogenized and mixed with the disintegrant extragranularly for 10 minutes, then mixed with mg – stearate (200mesh in size) for 2 minutes. the final mixture was compressed using f3 tablet machine with a single punch using 7mm normal concave punches. the same procedure was followed for the remaining formulas except that formulas (8) and (10) the disintegrant was added intra while in case of formulas (11, 12) and (13) no disintegrant was used. assay for total captopril present in the tablet: preparation of standard: 50mg pure captopril was added to 50ml of 0.1n naoh, shaked for 5 minutes, then 1ml of it was transferred to 100ml volumetric flask. the volume was completed with 0.1n naoh and shaken for 5 minutes. then analyzed spectrophotometrically at 238nm (4) . 10 tablets are weighed and triturated together and an amount equivalent to 50mg captopril was added to 50ml of 0.1n naoh, shaked for 5 minutes and filtered. 1ml of the filtrate transferred to 100ml volumetric flask and completed with 0.1n naoh and shaken for 5 minutes. the captopril content was analyzed spectrophotometrically at 238nm (4) and determined according to the following equation: captopril present in tablet. physical parameter measurement of the tablets: hardness: measured using monsanto hardness tester. weight variation: determined by taking 20 tablets for each formula. friability test: using rock friability tester for 4minutes at 25r.p.m. by taking 10 tablets together. disintegration time: the disintegration of tablets was performed according to b.p. method(5) at 37c in 0.1n hcl. dissolution test: the u.s.p. (basket method) (6) was used to study the release of the drug for the prepared tablets miniten ® , capocard ® (dad), and capoten ® (squibb) at 37c using 900ml of 0.1n hcl solution as the dissolution medium with a constant stirring speed of 50r.p.m.(6,2). samples were withdrawn at 5 minutes time intervals for the next 30min. the sample volume was replaced immediately by fresh 0.1n hcl. samples were filtered by microfilter, diluted 5 times and analyzed spectrophotometrically at 212nm for drug content. effect of binder type: starch paste, acacia mucilage, carboxy methylcellulose, and poly vinyl pyrrolidone (p.v.p.) were used as binders in formulas (1,2,3) and (4) respectively. then corn starch was removed from formula (2) and (4) to get formulas (12) and (13) respectively, to study the effect of binder on physical properties of captopril tablet in the absence of disintegrant. %100  std test iraqi j. parma. sci., vol.14, 2005 55 -------------------------------------------------------------------------------------------------------------- effect of binder concentration: different concentrations of p.v.p. in ethanol were used in formulas (6,4) and (5) which compared with formula (11) of the same p.v.p. concentration but without disintegrant. effect of diluent type: lactose, and starch were used as diluents in formulas (4) and (7) respectively. effect of disintegrant type: corn starch, explotab and (avicel and corn starch) were used using formulas (4, 9,14) respectively compared to formula (13) without disintegrant effect of method of incorporation of disintegrant: corn starch and explotab were used extra and intragranular in formulas (4,8,9) and (10) respectively. stability study: the effect of temperature on the degradation of captopril tablet was studied by storing some tablets of the selected formula in ambered color glass containers at different temperatures (50c, 60c and 70c) for 4 months. samples were withdrawn at desired time intervals and assayed for captopril content. results and discussion: effect of binder type: the results indicated that the use of different types of binders had an influence on physical properties of tablets and drug release as shown in table (2). the use of starch as a binder showed friable tablets with long disintegration time and this may be due to the property of starch paste to form tablets which are generally soft and brittle (7) . while formula 3 in which c.m.c. was used as a binder, showed low hardness, friable, capped tablets with further decrease in hardness and increase in friability after a few days of manufacture as well as they had long disintegration time. this may be related to the hygroscopicity of c.m.c. sodium. in addition, since c.m.c. is a viscosity controller, it is conceivable that it forms highly viscid systems that resist dilution by dissolution fluids which might impede drug release (8). the dissolution time is 40min for 100% release of drug in comparison with starch (30min). on the other hand, formulas (2) and (4) were good acacia is a natural product so this makes it objectionable to be used as a binder due to the possibility of microbial growth, so formula (4), in which p.v.p in alcohol was used as a binder showed acceptable tablets compared with the references. in addition, p.v.p. has a tendency to be slightly hygroscopic, that the resulted tablets will not expected to be harder with age. at the same time alcohol was used with p.v.p. instead of water because it is better in case of soluble powder granulations (7) . iraqi j. parma. sci., vol.14, 2005 56 -------------------------------------------------------------------------------------------------------------- effect of binder concentration: the results showed that using a low concentration of binder in formula (6) resulted in low hardness and high friable tablets while increase binder concentration lead to prolong disintegration and dissolution times, due to increase cohesiveness or attraction forces between particles(9). on the other hand, when the concentration increased three times, the hardness was less and the friability was higher. this may be due to the high hygroscopicity of the concentrated p.v.p. solution (7). effect of diluent type: the results indicate that when starch incorporated as a diluent, the organoleptic properties of tablets were unacceptable. for example; cracking occurred after a few days of compression and tend to expand after compression which may be due to its poor compressibility(7), while in case of lactose acceptable properties were obtained as shown in table (4). effect of disintegrant type: the results showed that the use of explotab formula (9) led to a well compressed tablet with a high hardness compared with the starch formula (4) using the same compression force as illustrated in table (5), but both of them (starch and explotab) had no effect on the disintegrant time. however, concerning the results obtained, both materials starch and explotab showed good hardness and friability with acceptable disintegration – dissolution phenomena. this may be due to that fact the captopril is a highly water soluble drug. on the other hand, using a combination of avicel and starch formula (14) or avicel alone formula (15) as a disintegrant resulted in a harder and less friable tablet. this may be due to the good compressibility of avicel. but it is not preferred to use avicel as a disintegrant since a high compression force might destroy the capillaries within avicel structure and diminish its property as disintegrant (7) . effect of method of incorporation of disintegrant: table (6) shows the effect of method of incorporation of disintegrant on the physical properties of the prepared captopril tablets. the results indicated that the hardness and friability of the prepared tablet using formula (4,8) and (9), were acceptable except for formula (10) in which the explotab was incorporated intragranularally (friability 1.4%). the results also indicated that a longer disintegration time was obtained with both types starch and explotab when incorporated them extra. this is may be due to the high water solubility of the captopril itself. gorden etal (10) stated that incorporating super disintegrant in the intragranular phase resulted in faster tablet dissolution than incorporating it in the extragranular phase or in both phases, and this is similar to our results as shown in figure (2). while in case of corn starch, the incorporation of a disintegrant intragranular resulted in a faster disintegration but slower tablet dissolution and this is because starch is an ordinary disintegrating so when used intragranular it loses some of its disruptive force due to its encasement by the binder(7). iraqi j. parma. sci., vol.14, 2005 57 --------------------------------------------------------------------------------------------------------------kinetic study: effect of temperature: figure (3) shows the degradation of captopril. it appears that the degradation of drug follows first – order reaction, since straight lines were obtained when the log% remaining of captopril is plotted versus time. the rate constants were determined from the slopes of the lines and tabulated in table (7). to determine the expiration date (t10%), arrhenius plot was made to predict the degradation rate constant at 25c (k25), and it is equal to 2.4710 -3 week-1 as shown in figure (4). since the degradation of drug follows first-order reaction, therefore, the expiration date can be calculated using the following equation (11) : and it is equal to about 42 months (3.5 years). no change in the hardness, friability and disintegration time were observed at the end of the 4 months. the organoleptic properties did not change at 50c, and 60c with a little darkness in color at 70c which may be due to the lactose which is affected by such high temperature. conclusion: the over all results of this study indicated that one could prepare satisfactory captopril plane tablets based on the followings: 1the best binder and diluent that can be used are the p.v.p. in alcohol and lactose since they are available, cheep, compressible and compatible with captopril. 2formula 4 was chosen as the best satisfactory formula in comparison with the reference formulas miniten®, and capoten®. 3starch was chosen as a disintegrant since it is available, low cost and stable while avicel was excluded since it is of high cost. 4the shelf life for the selected formula was 3.5 years. 25 %10 104.0 k t  iraqi j. parma. sci., vol.14, 2005 58 --------------------------------------------------------------------------------------------------------------formula captopril (mg) lactose (mg) starch (mg) acacia mucilage (mg) starch paste (mg) pvp (mg) c.m.c. (mg) corn starch (mg) explotab (mg) avicel (mg) mgstearate (mg) total wt. (mg) 1 50 x x x 2 201.500 2 50 x x x 2 200.000 3 50 x x x 2 200.000 4 50 x 2x x extra 2 201.000 5 50 x 3x x 2 201.625 6 50 x x x 2 200.000 7 50 x 2x x 2 201.000 8 50 x 2x x intra 2 201.000 9 50 x 2x x extra 2 201.000 10 50 x 2x x intra 2 201.000 11 50 x 3x 2 201.625 12 50 x 2.5 2 201.500 13 50 x 2 2 201.000 14 50 x 2 x x 2 201.000 15 50 x 2 2x 2 200.000 table (1): schedule of different formulation for captopril as a plain tablet dosage form iraqi j. parma. sci., vol.14, 2005 59 --------------------------------------------------------------------------------------------------------------suggested formula type of binder hardness (kg) friability (%) disintegration time (min) dissolution time for 100% release (min) 1 starch paste 4.0 2.8 6.5 30 2 acacia mucilage 4.0 0.9 4.0 22 – 25 3 c.m.c 3.0 6.0 8.0 40 4 10% pvp 4.0 0.7 1.3 25 miniten  4.5 0.5 1.0 25 – 30 capocard 4.0 0.8 2.0 30 capoten 5.0 0.5 1.0 15 – 20 table (2): effect of binder type on the hardness, friability, disintegration time and dissolution time of the prepared captopril tablets (formulas 1,2,3 and 4) in comparison with reference tablets. formula no. pvp binder (%) hardness (kg) friability (%) disintegration time (min) dissolution time for 100% release (min) 4 2x 4 0.7 1.3 25 5 3x 1 7 2 25 – 30 6 x 2.5 5 1 20 table (3): effect of binder concentration on the physical properties of captopril tablets of suggested formulas 4,5, and 6. table (4): the effect of diluent on the physical properties of captopril tablet and the release of captopril from suggested formulas 4 and 7. formula no. diluent type hardness (kg) friability (%) disintegration time (min) dissolution time for 100% release (min) 4 lactose 4 0.7 1.3 25 7 starch 1.25 5.8 1.5 40 iraqi j. parma. sci., vol.14, 2005 60 -------------------------------------------------------------------------------------------------------------- formula no. disintegrant type hardness (kg) friability (%) disintegration time (min) dissolution time for 100% release (min) 4 starch 4 0.7 1.3 25 9 explotab 10 0.9 1.3 30 13 4 0.5 3 30 14 starch & avicel 5 0.6 0.66 20 15 avicel 5 0.53 0.5 15 table (5): effect of disintegrant type on the physical properties of tablets in formulas 4, 9, 14, and 15 formula no. disintegrant location hardness (kg) friability (%) disintegration time (min) dissolution time for 100% release (min) 4 extra (starch) 4 0.7 1.3 25 8 intra (starch) 4 0.5 1 30 9 extra (explotab) 10 0.9 1.3 30 10 intra (explotab) 7.5 1.4 1 25 table (6): a comparison between the effect of method of incorporation of disintegrant extra and intra. temperature (c) k(months) -1 50c 710 -3 60c 10.210 -3 70c 1510 -3 table (7): rate constants of degradation of captopril using the selected formula at different temperatures. iraqi j. parma. sci., vol.14, 2005 61 -------------------------------------------------------------------------------------------------------------- figure (1): release study for formula (2) and (4) in comparison with reference formulas in 0.1n hcl and 37c. 0 20 40 60 80 1 00 1 20 0 5 10 15 2 0 2 5 30 tim e (min) p e rc e n t o f d ru g d is s o lv e d formula 2 formula 4 miniten cap oten cap ocard iraqi j. parma. sci., vol.14, 2005 62 -------------------------------------------------------------------------------------------------------------- figure (2): effect of binder concentration on the release of captopril from suggested formulas 4,5 and 6 in 0.1n hcl and 37c. figure (3): comparison between the release of captopril from formulas containing starch intra, extra (4,8) and explotab intra, extra (9,10). 0 20 40 60 80 100 120 0 5 10 15 20 25 30 time (min) p e rc e n t o f d ru g r e le a s e d formula 4 formula 5 formula 6 0 2 0 4 0 6 0 8 0 1 0 0 1 2 0 0 1 0 2 0 3 0 4 0 t im e ( m in ) p e rc e n t o f d ru g r e le a s e d f o r m u l a 4 f o r m u l a 8 f o r m u l a 9 f o r m u l a 1 0 iraqi j. parma. sci., vol.14, 2005 63 -------------------------------------------------------------------------------------------------------------- figure (4): degradation curves of captopril at different temperatures (50c, 60c, and 70c) using the selected formula (formula 4). figure (5): arrhenius plot for a shelf – life estimation of captopril using (formula 4). -3 -2.5 -2 -1.5 -1 2 .5 2.7 2.9 3.1 3.3 3.5 (1/a bsolute t em p.) * 1000 lo g k ( m o n th s1 ) 1.970 1.975 1.980 1.985 1.990 1.995 2.000 2.005 0 1 2 3 4 5 time (months) lo g % r e m a in in g iraqi j. parma. sci., vol.14, 2005 64 -------------------------------------------------------------------------------------------------------------- references: 1. cushman d.w., cheung h.s., sabo e.f., et. al. prog. cardio vasc. dis.; 1978, 21; p176. 2. the united state pharmacopeial convention, inc drug information; 1993, p8586, 159 – 170. 3. rankin k. new medicines offer fresh hope for patients with diabetes. drugstore – news – for – the – pharmacist; 1995, 5; p37 – 38. 4. moffat a.c., jackson j.v., moss m.s., et-al.clarke’s isolation and identification of drugs (in pharmaceuticals, body fluids, and post – mortem material) 2nd ed. beccles suffolk by william clowes ltd.; 1986, p426 – 427. 5. british pharmacopeia, 1993, volume ii a158. 6. united state pharmacopeia usp; 1995, p263 – 265. 7. liberman h.a., lachman l. compressed tablet in pharmaceutical dosage forms: tablets. vol. i; marcel dekker; new york and basel; 1982, p85, 109 – 140. 8. swinyard e.a., lowenthal w., carboxy methyl cellulose sodium; emulsifying and suspending agents; pharmaceutical necessities in remington’s pharmaceutical sciences; 17th ed. mack publishing company; 1985, p1297. 9. barker g.s., anderson n.r., tablets. in lachman l. lieberman h.a., kanig j.l., the theory and practice of industrial pharmacy; 3rd ed.; lea and febiger publishing company; philadelphia; 1986, p293 – 345. 10. gordon m.s., chatterjee b., chowhan z.t., effect of the mode of cros carmellose sodium incorporation on tablet dissolution and friability. j. pharm. sci.; 1990, 79; p43 – 47. 11. lachman l., delua p. alkers m.j., kinetic principles and stability testing. in lachman l. liberman h.a. and kanig j.l., the theory and practice of industrial pharmacy. 3rd ed., lea and febiger. philadelphia; 1986, p760 – 764. effect of disintegrant type: effect of method of incorporation of disintegrant: suggested formula type of binder friability (%) disintegration time (min) dissolution time for 100% release (min) iraqi j pharm sci, vol.28(2) 2019 synthesis of primary amides as hdacis doi: https://doi.org/10.31351/vol28iss2pp151-158 151 design, synthesis and cytotoxicity study of primary amides as histone deacetylase inhibitors duraid al-amily*,1 and mohammed h. mohammed* *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract primary amide derivatives as histone deacetylase inhibitors (hdacis) are very rare. this paper describes the synthesis of the primary amide derivatives adipic monoanilide amide and pimelic monoanilide amide (compounds 6 and 7) that have the requirements to be histone deacetylase inhibitors of the zinc-binding type. both of them exhibited good cytotoxicity against the tested cancer cell lines with much lower cytotoxicity against normal cell line. keywords: primary amides, hdacis, adipic monoanilide amide, pimelic monoanilide amide تصميم، تخليق، و دراسة السمية الخلوية لألمايدات االولية كمثبطات ألنزيم الهيستون دي اسيتيليز دريد حامد العاملي *،1و محمد حسن محمد* ، كلية الصيدلة، جامعة بغداد، بغداد، العراق.فرع الكيمياء الصيدالنية الخالصة ( و 7و 6تعتبر مشتقات االمايد االولية المثبطة ألنزيمات الهيستون دي اسيتيليز نادرة. و عليه، فقد تم تخليق مشتقي أمايد اولي )المركبين سرطانية لالمتضمنين للمتطلبات الالزمة لجعلهما من مثباطات هذه االنزيمات التي ترتبط بالزنك. و قد اظهرهذين المركبين سمية جيدة ضد الخاليا ا و ضعيفة ضد الخاليا الطبيعية. .الكلمات المفتاحية: امايدات اولية، مثبطات الهيستون دي اسيتيليز، امايد االدبك احادي االنااليد، امايد البايميليك احادي االنااليد introduction histone deacetylases (hdacs) are a group of enzymes responsible for the deacetylation of the ε-amino groups of lysine residues at the n-termini of histones (1), the core proteins that serve to compact dna into nucleosomes which comprise chromatins (2). the deacetylation process encourages the compaction of nucleosomes and repression of gene transcription (3). many reports demonstrate that hdacs are overexpressed in several types of cancer (1,4). therefore , they are considered as valuable targets for cancer treatment (5). eighteen isoforms of hdacs have been identified in mammalian tissues. they fall into four major classes (i-iv). class i includes hdac1, 2, 3, and 8 while class ii includes isoforms 4-7, 9 and 10. isoform 11 represents class iv. these three classes have a zinc-dependent catalytic site. the rest seven isoforms depend on the cofactor nicotine adenine dinucleotide rather than zinc for their catalytic activity and comprise class iii (6). the zinc-dependent classes are highly investigated and subjected to inhibition studies as an approach for cancer treatment (7). a classical and popular example is hdac2 since its crystallographic structure is well resolved (8). previous studies on the binding site revealed that it is shaped as an internal tubular (tunnel) cavity having a length of 11 å. at the bottom of this cavity lies a zinc ion followed by a 14 å foot-like pocket (9) (figure 1). lysine residues of histone fit into the tunnel so that they could be deacetylated (3). figure 1 .the binding site of hdac2. (a): the opening of the tunnel cavity is shown in which the linker of the inhibitor (cyan) is lying. (b): a side view of the binding site with 30% transparency of the surface to make the whole length of the tunnel (11 å) visible together with the linker of the inhibitor (forest green). zinc ion is visible at the deepest end of the tunnel. (c): mesh surface of the binding site showing the inhibitor inside the 14 å foot-like pocket. 1corresponding author e-mail : colrelated@copharm.uobaghdad.edu.iq received: 31/ 5 /2019 accepted: 21/8 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp151-158 mailto:colrelated@copharm.uobaghdad.edu.iq iraqi j pharm sci, vol.28(2) 2019 synthesis of primary amides as hdacis 152 accordingly, the design of inhibitors for zinc-dependent hdacs should have a zinc binding group (zbg) to bind to the zinc ion, a linker of 4-6 carbon atoms length to fit into the cavity, and a hydrophobic cap to recognize and interact with the outer surface (1). these are best illustrated by the classical inhibitor suberoylanilide hydroxamic acid 1 (saha, vorinostat) (10).other known synthetic inhibitors with zbg other than hydroxamic acid derivatives include entinostat 2 (ms-275), a benzamide, and valproic acid 3 (5) (figure 2). among other rare synthetic zbg are the secondary amide derivative 4 (11,12) and the only primary amide 5 mentioned in the literature that showed an ic50 against hdac1 in a nanomolar concentration (13) . (2) (3) (4) n= 2, 3, and 4 (5) figure2. examples of hdais with different zbgs here, we wish to describe the synthesis of the small and simple primary amide molecules 6 and 7 that fulfill the three requirements for zinc-dependent hdac inhibitors. (6) n= 2 (7) n= 3 besides, we’ll mention the results of the in vitro cytotoxicity assay for these compounds against hrt-18 cell line (human colon adenocarcinoma) and hc-04 cell line (mouse hepatic carcinoma). materials and methods reagents and solvents were used for chemical synthesis as obtained from the supplier (sigma-aldrich, fluka, romil, gcc diagnostics, reagentworld, and thomas-baker). melting points were measured using the stuart smp3 melting point apparatus (uk) and are uncorrected. thin-layer chromatography was achieved using 0.2 mm precoated tlc-sheets alugram® xtra sil g/uv254 (macherey-nagel, germany) and the visualization was under a 254 nm uv lamp. ft-ir spectroscopy was done using shimadzu iraffinity-1 spectrometer (shimadzu, japan) and specac® quest atrdiamond type (uk) at the university of baghdad-college of pharmacy. 1h-nmr and 13cnmr analysis was performed at 400 mhz and 100 mhz respectively (d6 dmso as the solvent) using iraqi j pharm sci, vol.28(2) 2019 synthesis of primary amides as hdacis 153 the bruker avance iii, 400 mhz spectrometer (usa) at sophisticated test and instrumentation centre (cochin university of science and technology-india), with the chemical shifts (δ) expressed in parts per million. figures illustrating the binding site of hdacs and the binding of the ligand were captured using ucsf chimera (14) and the pdb (www.rcsb.org) (15) files with ids 4lxz (8) and 3max (16). cytotoxicity assay was performed at the iraq biotech laboratories using trypsin/edta, rpmi 1640, fetal bovine serum (capricorn, germany); 3( 4 , 5 – dimethyl – 2 thiazolyl) -2,5diphenyl-2h-tetrazolium bromide (mtt stainbioworld, usa); dimethyl sulfoxide (dmso santacruz biotechnology, usa); co2 incubator and laminar flow hood (cypress diagnostics, belgium); microtiter reader (gennex lab., india); cell culture plates (santa cruz biotechnology, usa). chemical synthesis (scheme 1) general synthesis of adipic and pimelic anhydrides (6b and 7b) adipic acid (6a) or pimelic acid (7a) (27.4 mmol of each) was suspended in acetic anhydride (3 ml/g) and refluxed for 1 hour. the solvent was then evaporated under reduced pressure to obtain a semisolid mixture of 6b and 6c, or 7b and 7c, which was used as such in the next step. ft-ir (atr; υ, cm-1): 1801, 1739 for 6b and 1813, 1743 for 7b (sym. and asym. c=o respectively). general synthesis of monosodium adipic monoanilide and monosodium pimelic monoanilide (6d and 7d) the obtained mixture of compounds 6b and 6c, or 7b and 7c was dissolved in 10 ml of dry dmf in a round bottom flask that was equipped with calcium chloride tube and cooled to 0 °c. then 3 ml of aniline (32.88 mmol) were added gradually with continuous stirring and cooling. the mixture was then kept stirred at room temperature for 24 hours. the mixture was then acidified with 5 n hydrochloric acid, diluted to about 300 ml with cold water, and the precipitate formed was then filtered. the precipitate was washed with water (3×50 ml). then it was suspended in no more than 50 ml of icecooled water and the ph of this mixture was raised to 7 by a drop wise addition of 0.5 n sodium hydroxide solution at 0 °c. the mixture was stirred for 30 minutes at 0 °c while maintaining this ph by addition of 0.5 n sodium hydroxide solution as needed. then the mixture was filtered and the filtrate was evaporated to dryness. compound 6d: off-white powder, yield 35% relative to 6a, m. p. 259.5-263.8 °c. ft-ir (atr; υ, cm-1): 3332 (aromatic nh), 3062 (aromatic c-h), 2939 (asym ch2), 2866 (sym. ch2), 1666 (amide c=o), 1600 (aromatic ), 1562 (asym carboxylate c=o), 1539 and 1500 (aromatic ), 1431 (sym. carboxylate c=o). compound 7d: off-white powder, yield 25.5% relative to 7a, m. p. 214-217 °c. ft-ir (atr; υ, cm1): 3290 (aromatic nh), 3078 (aromatic c-h), 2920 (asym. ch2), 2858 (sym. ch2), 1654 (amide c=o), 1597 (aromatic ), 1558 (asym. carboxylate c=o), 1543 and 1496 (aromatic ), 1435 (sym. carboxylate c=o). general synthesis of adipic monoanilide acid chloride and pimelic monoanilide acid chloride (6e and 7e) compound 6d or 7d (4 mmol) was suspended in 15 ml dry dichloromethane in a round bottom flask that was equipped with calcium chloride tube and cooled in an ice bath. pyridine (4 mmol, 0.32 ml) was added followed by a drop wise addition of 0.34 ml of thionyl chloride (4.8 mmol) with continuous stirring and cooling for 10 minutes. the mixture was then allowed to warm to room temperature with continuous stirring for 30 minutes. then the precipitate formed was collected using büchner funnel. the precipitate was washed with dry dichloromethane (3×10 ml) during filtration. the white solid mass obtained (in both cases of 6e and 7e) was used as such in the next step. compound 6e ft-ir (atr; υ, cm-1): 3313 (aromatic nh), 3066 (aromatic c-h), 2935 (asym. ch2), 2870 (sym. ch2), 1797 (acid chloride c=o), 1662 (amide c=o), 1600, 1535 and 1500 (aromatic ). compound 7e ft-ir (atr; υ, cm-1): 3313 (aromatic nh), 3043 (aromatic c-h), 2939 (asym. ch2), 2870 (sym. ch2), 1797 (acid chloride c=o), 1662 (amide c=o), 1597, 1527 and 1500 (aromatic ). general synthesis of adipic monoanilide amide and pimelic monoanilide amide (6 and 7) the precipitate obtained from the previous step (6e or 7e) was suspended in 10 ml of anhydrous cold acetone and added gradually to 10 ml of concentrated aqueous ammonium hydroxide solution that was cooled in an ice bath. the mixture was stirred for 30 minutes while maintained in the ice bath, then extracted three times with 5 mlportions dichloromethane. the organic phase was evaporated to dryness under reduced pressure. compound 6: white powder, yield 65% (relative to 6d), m. p. 178.2179.3 °c, rf 0.34 (dichloromethane: 1-propanol; 8:2). ft-ir (atr; υ, cm-1): 3390 (asym. aliphatic nh) 3305 (aromatic nh), 3194 (sym. aliphatic nh), 3043 (aromatic ch), 2951 (asym. ch2), 2873 (sym. ch2), 1643 (br, aliphatic and aromatic amide c=o), 1597, 1523 and 1500 (aromatic ). 1h-nmr (δ, ppm): 1.54 (m, -ch2-ch2-, 4h), 2.07 (t, aliphatic -ch2-c(o)-, 2h), 2.29 (t, aromatic ch2-c(o)-, 2h), 6.71 (br, -nh2, 1h), 7.02 (t, p-ch, 1h), 7.28 (t, m-ch, 2h; and -nh2, 1h), 7.58 (d, och, 2h), 9.86 (s, -nh-1h). http://www.rcsb.org/ iraqi j pharm sci, vol.28(2) 2019 synthesis of primary amides as hdacis 154 13c-nmr (δ): 24.78, 24.87, 34.90, 36.23, 119.03, 122.94, 128.60, 139.24, 171.15, 174.26. compound 7: gray crystals, yield 55% (relative to 7d), m. p. 127129 °c, rf 0.42 (dichloromethane: 1propanol; 8:2). ft-ir (atr; υ, cm-1): 3367 (asym. aliphatic nh) 3313 (aromatic nh), 3174 (sym. aliphatic nh), 3043 (aromatic c-h), 2939 (asym. ch2), 2854 (sym. ch2), 1658 (br, aliphatic and aromatic amide c=o), 1597, 1523 and 1496 ( aromatic ). 1h-nmr (δ): 1.28 (t, -ch22h), 1.54 (m, -ch2ch2, 4h), 2.04 (t, aliphatic -ch2-c(o)-, 2h), 2.29 (t, aromatic -ch2-c(o)-, 2h), 6.68 (br, -nh2, 1h), 7.01 (t, p-ch, 1h), 7.28 (t, m-ch, 2h; and -nh2, 1h), 7.58 (d, o-ch, 2h), 9.85 (s, -nh-1h). 13c-nmr (δ): 24.85, 24.89, 28.31, 34.94, 36.27, 119.02, 122.92, 128.60, 139.25, 171.24, 174.37. maintenance of cell cultures hrt-18, hc-04, and hbl-100 (epithelial cells obtained from healthy human breast milk) cell lines were maintained in rpmi-1640 medium with which 10% fetal bovine was supplemented, 100 units/ml penicillin, and 100 µg/ml streptomycin. the cells were subcultured using trypsin-edta, reseeded, once getting 80% confluence, two times a week, and incubated at 37 °c (17). cytotoxicity assay mtt cell viability assay was done using 96-well plates. the chosen cell lines were seeded at 1 × 104 cells/well. after either 24 hours or a confluent monolayer had been obtained, the cells were treated with compounds 6 or 7 at different concentration (6.25, 12.5, 25, 50, 100 µm). after a 72 hourstreatment period, the cell viability was measured by removing the medium, adding 28 µl of 2 mg/ml solution of mtt stain and incubating the cells for 2.5 hours at 37 °c. then the mtt solution was removed and the remaining crystals in the wells were dissolved in 130 µl of dimethyl sulfoxide followed by 37 °c incubation for 15 minutes with shaking (18)]. the absorbency was determined on a microplate reader at 492 nm; the assay was performed in triplicate. the inhibition rate of cell growth (the percentage of cytotoxicity) was calculated according to the following equation: 𝐶𝑦𝑡𝑜𝑡𝑜𝑥𝑖𝑐𝑖𝑡𝑦 (%) = 𝐴 − 𝐵 𝐴 × 100 where a is the optical density of the control and b is the optical density of the sample. the morphology of cells was visualized by the aid of inverted microscope. 200 µl of cell suspensions were seeded in 96-well micro-titration plates at density 1x104 cells/ ml and incubated for 48 hours at 37 °c. after removing the medium, compound 6 or 7 was added at the (ic50). then, after the exposure time, 50 µl of crystal violet were used to stain the plates which were incubated at 37 °c for 15 min; gentle washing of the stain was done with water until no more dye remained. a 40x magnification of inverted microscope was selected to examine the cells; they were photographed with a digital camera. statistical analysis unpaired t-test (using graphpad prism 6) was applied for the statistical analysis of the collected data. the values were presented as the mean ± sem of triplicate measurements. results and discussion chemical synthesis synthesis of the target compounds (6 and 7) was started from adipic acid (6a) and pimelic acid (7a) respectively (scheme 1). heating these dicarboxylic acids in acetic anhydride for 1 hour would result in monomeric cyclic anhydrides (6b and 7b). however, these anhydrides are extremely unstable and some of the synthesized amount is converted into polymeric anhydrides (6c and 7c) rapidly during evaporation of the solvent (11,19). the anhydride mixture (6b and 6c, or 7b and 7c) was used as such in the next step which is a modified procedure from previously published works (20,21). in this step, a slight excess of aniline (1.2 eq. relative to 6a and 7a) was added gradually to an ice-cooled solution of the anhydride mixture (in dry dmf) and, then, stirred at room temperature for 24 hours. the sodium salts (6d and 7d) were obtained by treatment of the precipitate, resulted after cold acidification of the reaction mixture and filtration, with gradually increasing amounts of ice-cooled naoh solution (0.5 n) so that the ph was not raised above 7. after filtration and evaporation to dryness, the off-white precipitate (6d and 7d) showed the characteristic ft-ir amide peaks (respectively) at 3332 cm-1 and 3290 cm-1 (aromatic nh) and at 1666 cm-1 and 1654 cm-1 (amide carbonyl). furthermore, the carboxylate peaks were at 1562 cm-1 (asym. c=o) and 1431 cm-1 (sym. c=o) for 6d and at 1558 cm-1 (asym. c=o) and 1435 cm-1 (sym. c=o) for 7d. these anilides were prepared as sodium salts in order to keep the amide function stable during the next step which involved the synthesis of the acid chlorides 6e and 7e using thionyl chloride in the presence of 1 equivalent of pyridine (22), thus avoiding the liberation of hcl. both 6e and 7e showed a new characteristic ft-ir carbonyl peak (of acid chloride) at 1797 cm-1 with the disappearance of all of the carboxylate peaks. iraqi j pharm sci, vol.28(2) 2019 synthesis of primary amides as hdacis 155 scheme 1. synthesis of the target compounds (6 and 7) the final step of synthesis involved primary amide formation (6 and 7) by modifying a simple procedure using ice-cooled concentrated aqueous ammonia solution [23]. the ft-ir spectrum showed the disappearance of the 1797 cm-1 peak for both 6 and 7 while the amide carbonyl peaks (1643 cm-1 and 1658 cm-1, respectively) became broad because of the overlap of the primary and secondary amide carbonyl groups. the two protons attached to the nitrogen of the primary amide are chemically not equivalent due to hindered amine rotation (24). this is illustrated by the difference in their chemical shifts in the 1h-nmr spectrum of 6 and 7 (0.57 ppm and 0.6 ppm respectively). cytotoxicity study compounds 6 and 7 were tested in vitro for antiproliferative activity against hrt-18 cell line (human colon adenocarcinoma), hc-04 cell line (mouse hepatic carcinoma), and hbl-100 cell line ( epithelial cells obtained from healthy human breast milk) at micromolar concentrations (6.25, 12.5, 25, 50, 100 µm). both of them showed inhibitory activity against the cancer cell lines higher than against the normal cell line. figures 3 and 4 show that there is a continuous and parallel increase in the inhibition of growth of both compounds against both colon adenocarcinoma and hepatic carcinoma with increasing the concentration. on the other hand, they show very low cytotoxicity against normal cells. iraqi j pharm sci, vol.28(2) 2019 synthesis of primary amides as hdacis 156 figure 3. concentration-growth inhibition curve of the tested compounds against hepatic carcinoma cells (blue), colon adenocarcinoma (orange) and healthy breast cells (gray). (a): the cytotoxicity of compound 6. (b): the cytotoxicity of compound 7 figure 4. histogram showing the concentration and growth inhibition of the tested compounds against hepatic carcinoma cells (blue), colon adenocarcinoma (orange) and healthy breast cells (gray). (a): the cytotoxicity of compound 6. (b): the cytotoxicity of compound 7 compound 7 shows higher cytotoxicity than compound 6 against the tested cancer cells ( figure 5). its ic50 against hc-04 cell line and hrt-18 cell line are 29.43 µm and 33.92 µm respectively while those for compound 6 are 45.83 µm and 47.93 µm. figure 6 (captured at the ic50) shows the difference in the magnitude of cytotoxicity between the two compounds. . figure 5. histogram showing the concentration and growth inhibition of the compounds 6 and 7, (a): against hepatic carcinoma cells and (b): against colon adenocarcinoma iraqi j pharm sci, vol.28(2) 2019 synthesis of primary amides as hdacis 157 hc-04 cell line hrt-18 cell line hbl-100 cell line (hepatic carcinoma) (colon adenocarcinoma) (normal) control untreated cells comp. 6 treated cells comp. 7 treated cells figure 6. morphology of the cell lines after treatment with compounds 6 and 7 at the ic50 (40x magnified) the low cytotoxicity of both compounds against normal cells gives hope of being able to target cancer cells to a higher degree than normal cells. no more than 12 % of inhibition of growth was observed at the highest concentration used for both compounds (figure 7). this might possibly be considered, in agreement with previous reports, that the synthesized compounds might exhibit relative specificity in inhibiting hdacs that are overexpressed in cancer cells. figure 7. low cytotoxicity of compounds 6 and 7 on normal breast cells. (a): concentration-growth inhibition curve. (b): histogram showing the concentration and growth inhibition conclusion synthesis of the target primary amides (6 and 7) was achieved starting from adipic acid (6a) and pimelic acid (7a) respectively . they were fully characterized successfully. both of them showed good cytotoxicity against cancer cell lines (hc-04 and hrt-18) and lower cytotoxicity against normal cell line (hbl-100). references 1. delcuve gp, khan dh, davie jr. targeting class i histone deacetylases in cancer therapy. expert opin ther targets. 2012;17(1):29–41. iraqi j pharm sci, vol.28(2) 2019 synthesis of primary amides as hdacis 158 2. burtis ca, ashwood er, bruns de. tietz textbook of clinical chemistry and molecular diagnostics. fifth. missouri: elsevier; 2012. 1212-1214 p. 3. manal m, chandrasekar mjn, gomathi priya j, nanjan mj. inhibitors of histone deacetylase as antitumor agents: a critical review. bioorg chem. 2016;67:18–42. 4. ruijter ajm de, gennip ah van, caron hn, kemp s, kuilenburg abp van. histone 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sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.28(2) 2019 clinical effects of arabic gum doi: https://doi.org/10.31351/vol28iss2pp9-16 9 clinical effects of gum arabic (acacia): a mini review noor s. jaafar* *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq. abstract gum arabic (ga) is a natural gummy exudate gained from the trees of acacia species (acacia senegal and acacia seyal), family: fabaceae. ga is considered as a dietary fiber with a high percentage of carbohydrates and low protein content. sugars arabinose and ribose were originally discovered and isolated from gum arabic which represents the original source of these sugars. a gum emanation from trees occurs under stress conditions such as heat, poor soil fertility, drought, and injury. mainly ga is produced in belt region of africa mainly sudan, chad, and nigeria. in the food industry, it is used in confectionery; in the pharmaceutical industry, it is used as emulsifier, film coating and others. traditionally the gum used for chronic renal failure, digestive discomfort and others. although gum arabic considered as an inert substance, recent information revealed multiple pharmacological and medical effects, such as weight reduction, antihypertensive, antihyperlipidemic, anticoagulant, antibacterial, antidiabetic, anti-inflammatory, nephroprotective and other effects. the aim of present review was to demonstrate the clinical effects of gum arabic. key words: gum arabic, obesity, diabetes, renal failure. لتاثيرات السريرية للصمغ العربي: مراجعة مصغرةا نور صباح جعفر* * بغداد،العراق.، فرع العقاقير والنباتات الطبية، كلية الصيدلة، جامعة بغداد الخالصة يعتبر .العائلة البقولية ( ،acacia seyaland acacia senegalالصمغ العربي هو إفراز طبيعي صمغي مكتسب من أشجار السنط ) ينوز بالصمغ العربي بمثابة األلياف الغذائية المحتوية على نسبة عالية من الكربوهيدرات ونسبة منخفضة من البروتين. تم اكتشاف السكريات أرا مثل المصدر األصلي لهذه السكريات. يتم افراز الصمغ من األشجار في ظروف اإلجهاد مثل الحرارة، ي ووريبوز وعزلها من الصمغ العربي وه شاد تضعف خصوبة التربة، الجفاف و اإلصابة او الجروح والشقوق. يتم إنتاج الصمغ بشكل رئيسي في منطقة الحزام في أفريقيا ، وخاصة السودان و استخدامه في صناعة الحلويات. في صناعة المستحضرات الصيدالنية ، يتم استخدامه كمستحلب ، طالء لألفالم ونيجيريا. في الصناعات الغذائية ، يتم ة، إال لوغيرها. تقليديا يستخدم الصمغ في عالج الفشل الكلوي المزمن ، سوءالهضم ، وغيرها. على الرغم من أن الصمغ العربي يعتبر مادة غير فعا ، لتخثر ، مضاد للجراثيملتأثيرات دوائية وطبية متعددة ، مثل خفض الوزن ، والضغط ، خافض لدهون الدم، مضاد أن المعلومات الحديثة أظهرت مضاد السكري ، مضاد لاللتهابات وتاثيرات طبية اخرى. .الفشل الكلوي، داء السكري، السمنة، الكلمات المفتاحية: الصمغ العربي introduction gum arabic (ga) is the air dried glutinous or gummy exudation obtained from branches and trunks of acacia species, mainly acacia senegal (hashab), and a nearly associated species acacia seyal (talha) (1-3), which belong to fabaceae family (2). both species naturally grown in belt region of africa. sudan, chad and nigeria are the main gum producing countries (35). gum arabic tree is a familiar medicinal plant in arabian peninsula, pakistan and india (6). gum from acacia senegal is the highest quality, while from acacia seyal is the least quality (7). the name gum arabic was derived, while it was shipped from arabian ports to europe in 4,000 b.c (8). figure 1. gum arabic; raw material (4) 1corresponding author e-mail: noorsaldahan@yahoo.com received: 8/4 /2019 accepted: 3 /7 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp9-16 https://www.sciencedirect.com/topics/pharmacology-toxicology-and-pharmaceutical-science/medicinal-plant mailto:noorsaldahan@yahoo.com mailto:noorsaldahan@yahoo.com iraqi j pharm sci, vol.28(2) 2019 clinical effects of arabic gum 10 stress conditions as heat, dryness, bad or poor nutrition, and sick trees induced gum production. microbial infection (bacterial and fungal) in accidental or intentionally made incisions in ga trunks may provoke gum production (9). this exudates or fluid that ooze out the crevices and wounding of acacia trees, harden and solidify after a few weeks into glossy amber-colored teardrops. after harvesting, the teardrops are assorted into grades according to color and extraneous matter content. the gum tint ranges from dark brown to white based on tannin quantity, the lightest color being the best (7,8). chemically the gum arabic is a neutral or slightly acidic, complex mixture of polysaccharide and glycoproteins, characterized by a high percentage of carbohydrates (~97%), (dgalactose and l-arabinose) and a low percentage of protein (<3%) (10-12). sugars arabinose and ribose were originally discovered and isolated from gum arabic which represent the original source of these sugars (13). the sugars constituents of gum are identical, but the composition and molecular weight (mwt) of the gum differs from one species to another in the range of 260,000-1,160,000 g. (14).ga is an extremely heterogeneous and naturally exists as a mixture of calcium, magnesium, potassium and sodium salts of a polysaccharic acid or arabic acid (15). by hydrophobic affinity chromatography, three major fractions had been separated in ga these include arabinogalactan, arabinogalactan-protein complex and low molecular weight glycoprotein (2). flavonodis, chalcones, tannins, phenolic acid, alkaloids and terpenes like lutein were identified in ga (6,16). varieties within the same species, environmental factors (climate, rainfall, and soil), period and method of harvesting or tapping and post-harvest treatment affect gum arabic chemical composition or quality (17,18). ga can be classified as a water-soluble dietary fiber or a non-digestible carbohydrate. it is not digested in the small intestine, but in the large intestine by the action of intestinal bacteria, it is fermented to short-chain fatty acids, predominantly propionic acid and butyric acid. ga is a prebiotic fiber supporting the growth of lactic acid bacteria and bifidobacteria which may be especially useful for certain groups of people like elderly who have reduced bifidobacteria populations. ga exerted a prebiotic effect at a dose 10gm/ day. this fiber (ga) may affect the physiological status of human through hindering glucose absorption, stool mass increment, bile acids trapping and other effects (19-22). about 80% of current gum production is utilized by the food in confectionery (due to its compatibility with high sugar concentration) and in pharmaceutical industries as an emulsifier, film coating and others (8,23). traditionally, ga consumed by african and indian peoples to ameliorate digestive comfort, inflammation and improve intestinal transit and as an oral hygiene agent (19,24) .in arabic folk medicine, it was used in chronic renal failure patients for reducing the frequency of hemodialysis (25), and for treatment of diabetes (26). egyptian people used gum in the embalmment process and in the development of several cosmetics and perfumes (4). despite the common assumption that the gum is an inert substance, new studies have shown that gum arabic has some potential antioxidant, digestive nephroprotectant, antianemic, cardiovascular and other properties (27). gum arabic considered safe, a natural compound with no important toxic or side effects when ingested orally. minor side effects as allergy and hypersensitivity have been reported in some cases (27-29). the aim of the present review was to demonstrate the clinical effects of gum arabic. gum arabic clinical effects effect on hyperlipidemia hyperlipidemia is a medical condition manifested by abnormal lipid profile (raising total cholesterol and/or triglycerides (tg), and/or reduced concentration of high-density lipoprotein cholesterol (hdl). it is considered as a risk factor for coronary heart disease (30). soluble dietary fibers have lipid lowering effects, including gum arabic. mohammad et al revealed improvement in lipid profile (cholesterol,tg) in hyperlipidemic patients without affecting the hdl level. 15 gm ga /day has no effect on the lipid profile as compared to placebo (31). rose et al (1983) and sharma (1985) reported in studies that human ingestion of 25 and 30 gram of gum arabic daily for 21-30 days decrease total cholesterol (ldl; low density lipoprotein and vldl; very low density lipoprotein) but not tg and hdl (32.33). ahmed et al (2016) reported that normal fed mouse when taking 0.5% aqueous solution of ga for seven days, then 10% aqueous solution for an additional six weeks demonstrated a reduction in total cholesterol, ldl and also hdl reduction. this hypocholesteremic action due to reduced liver hmgr mrna gen (3-hydroxy-3-methylglutarylcoa reductase mrna) biosynthesis which ultimately reduced cholesterol levels (34). the lipid-lowering effect of gum arabic attributed to the ability of ga to bind to bile acids and thus reduce bile acids absorption in the terminal ileum. the sequestered bile acids are released upon gum break down in the large intestine. the fermentation of ga generates acidic ph which renders the bile acids insoluble and aids their excretion in the stool therefore fat digestion and absorption are reduced. in liver new bile acids formation requires cholesterol. thus, prolonged iraqi j pharm sci, vol.28(2) 2019 clinical effects of arabic gum 11 digestion of gum arabic may result in minimizing the plasma cholesterol level (31). the increment in mitochondrial biogenesis and fatty acid oxidation by skeletal muscles is the more recent anticipated mechanism by which viscous dietary fibers can lessen adiposity (35). effect on obesity in developed countries, obesity and overweight are the most prevalent nutritional disorders affecting the vast number of adult. obesity resulted when the consumed calories exceeded the used one (36). ingestion of ga seems to be an efficacious dietary strategy for overweight prevention or treatment affecting numerous biological mechanisms. dietary fibers including ga affect fat and glucose metabolism. babiker r and colleagues (2012) revealed in a study significant reduction in body mass index and body fat percent after six weeks of regular use of 30 gm of ga daily. ga consumption causes a considerable reduction in caloric intake along with an increased subjective feeling of satiety (37). calame et al (2011) evaluated the satiating action induced by a mixture of two forms of gum arabic (emulgold [eg] and previtae [pv]). the energy consumption was examined three hours after ingestion of the ga aqueous solution. both forms (emulgold and previta), at forty-gram dose, produce a considerable decrease in energy intake of more than 100 and 200 kcal, successively. the decrease in energy intake equal to more than 100 kcal for both was observed at smaller doses (10 or 20 gram) (38). ushida et al (2011) reported the probable anti-obesity action of ga as a dietary fiber. authors reported that 1% of ga solution was given to threemonth-old female mice for 180 days. this solution reduced age-dependent fat depositions; where, this effect was proposed to be due to β3-adrenergic stimulation of adipocytes through tumor necrosis factoralpha (tnf-α) down-regulation. this downregulation might be attributed to large intestinal alteration of microflora, as proved by the modification in cecal short-chain fatty acid and 16s ribosomal deoxynucleic acid (16s rdna) (39). a study of ahmed et al (2015) revealed that administration of 10% aqueous solution of arabic gum for nine weeks to mice minimized visceral adipose tissue accumulation. ga reduces 11β-hsd1 mrna (11 beta-hydroxy steroid dehydrogenase type 1 mrna) in mice liver and muscle. this may participate in the reduction of plasma lipid level and hence affect obesity. inhibition of this enzyme is being pursued as a new medication for curing metabolic syndrome and obesity (40, 41). arabic gum also down-regulate peroxisome proliferator-activated receptor gamma (ppar-γ) and expression of the stearoyl-coa desaturase (scd gene) in mice receiving gum in drinking water (41) .omaima nasir (2014) manifested in a study on mice that ga lessen weight through down-regulation of the membrane abundance of sglt1 (sodiumglucose cotransporter 1) play an important role in intestinal glucose absorption, which will reduce glucose absorption recognized through reduction in plasma glucose level, and consequently insulin level and then body weight(42). eyibo et al (2018) revealed the decrease in albino rat body weight consuming ga which could be attributed to the probable ability of ga to displace available calories and nutrients besides that it increases the absorption efficacy in the small intestine and to appetite loss induced by ga consumption (43). effect on hypertension the antihypertensive effect of ga had been proved in previous studies. nasir et al (2016) evaluated the hypotensive effect of gum in healthy, prediabetes and diabetic individuals on regular ga intake of 10gm/ day for sixteen weeks (44). glover et al (2009) displayed the reduction effect of ga on systolic blood pressure when given in a dose of 25 gm/day in both healthy persons and patients with renal dysfunction secondary to diabetes mellitus (45). alkarib et al (2011) revealed the significant lowering effect of gum arabic on systolic blood pressure with concomitant normalizing serum sodium level and raise in potassium level to the normal upper level. the gum causes insignificant alterations or changes in diastolic blood pressure in stage iii renal failure patients (11). effect on fertility the prevalence of infertility among obese women is increased (46). abdominal or central obesity is the main sign of metabolic syndrome that results in female infertility. ahmed aa et al (2016) demonstrated that administration of ga in drinking water of female mice fed with high fat diet to induce obesity, there was a reduction in lipid peroxidation and an elevation in ovarian antioxidant enzymes activity (superoxide dismutase, catalase and glutathione peroxidase) along with raised mrna expression and reduced in ovary degenerative changes were observed which in turn may improve reproductive efficacy and manage sterility induced by obesity (47). fedail js et al (2016) reported that ga consumption resulted in improvement in degenerative testicular tissue of alloxan-induced diabetic rats, enhanced semen quality, and reduction in lipid peroxidation; furthermore, the activity of antioxidant enzymes together with their mrna expression was reported to be increased; these effects might have roles in the management of reproductive dysfunction in diabetic men (48). iraqi j pharm sci, vol.28(2) 2019 clinical effects of arabic gum 12 anti-inflammatory effect numerous chronic diseases such as arthritis, cancer, diabetes, cardiovascular (cv) and neurological diseases were reported to be instigated by chronic inflammation (49). rheumatoid arthritis (ra) is an autoimmune illness recognized by chronic synovial inflammation with higher female incidence (50). tumor necrosis factor-α (tnf-α) is a main pro-inflammatory mediator involved in the development of ra (51). ebtihal et al (2018) demonstrated that the anti-inflammatory effect of ga through reduction tnf-α, erythrocyte sedimentation rate (esr) level and improvement in clinical symptoms in ra patients when given (30 gm/day) of ga during the study period(52). abdul rahman et al study’s (2016) shown the gastro protective effect of ga alone or in combination with other antiulcer drugs. ga possesses a good antiulcer effect and it potentiates or enhance the antiulcer effect of ranitidine in indomethacin-induced gastric ulcer rats (53).furthermore, abd el-mawla and colleges (2013) manifested the protective effect of ga (1gm/day) versus ulceration induced by meloxicam in rats (54). moreover, a study of khedr aa (2017) demonstrated a dose-dependent antiulcer effect of ga in ethanol-induced ulcer in the adult rat through the reduction in gastric juice, raise in ph and other cytoprotective effects; these effects were reported to be attributed to the polysaccharides which demonstrated antiulcer effects in their binding ability to the mucosal surface and to act as a protective coating by raising synthesis of mucus, or through scavenging radicals’ activity (55). in ulcerative colitis, the beneficial effect of ga attributed to its trophic effects on the gut membrane and to its ability to reduce the incidence and duration of diarrhea (19). cancer, is an uncontrolled cell growth or cell proliferation which may speared to new tissues (56). al alawi et al (2018) study’s revealed no cytotoxic effect for ag (6), but it may have the ability for prevention or treatment of toxic aspects of some cytotoxic drugs as cyclophosphamide (cpa), doxorubicin, (dox), and cisplatin beside other drugs and chemicals such as aspirin, acetaminophen, indomethacin, gentamycin, trichloroacetic acid and mercuric chloride (57,58). abd-el-hafez et al (2017) and abd-allah et al (2002) reported that ga has strong antioxidant action (59,60) may be attributed to its amino acids content (61). ga considered as a rich source of amino acids, aspartic acid and serine were the main amino acids, lysine, histidine, glycine, tyrosine and other amino acids were also detected in acacia senegal and acacia seyal acids (62) experimental evidence displayed that there is a relation between the antioxidant effect and the protein fraction, predominantly the amino acid residues such as histidine, tyrosine, and lysine, which are generally regarded as antioxidant molecules (61). ga increasing the concentration of the antioxidant enzymes and minimizing the oxidizing molecules in various organs (28). kaddam et al at (2017) demonstrated the beneficial effect of ga in the management of sickle cell anemia at a dose 30 gm/ day for twelve weeks. ga ingestion produce a reduction in the oxidative stress markers as hydrogen peroxide and hydroxyl radicals level, meanwhile increase in antioxidant activity. the markers (hydrogen peroxide and hydroxyl radicals) increase in sickle cell anemia twice as much as in healthy individuals (63). effect on diabetes diabetes mellitus (dm) is a chronic illness with steadily increasing frequencies in all countries (64), microvascular and macrovascular complications of diabetes that may result in mortality and morbidity (44). ga among the dietary fibers reported to have antidiabetic effect in the human and animals. food supplemented with ga (10 gm/day for sixteen weeks) in prediabetes and diabetic subjects showed a significant reduction in fasting blood sugar (fbs) and glycated hemoglobin (hba1c) (44). babiker et al (2017) observed the beneficial effect of ga in poor glycemic control diabetic patients' receiving 30gm of gum for 4 months (65). ga has an intrinsic glycaemic index is proximate zero snice it is not absorbed in small intestine (21). while ibrahim et al at (2017) observed that in type ii diabetic patients’ ingestion of 60gm/day of ga produced no significant or appreciable effect on blood glucose concentration and body mass index (66). al-nagar and faid (2017) reported the hypoglycemic effect of ga in alloxan-induced diabetic rats; where, ga caused beta cell activation or proliferation and/or antagonizing the blocking of the immune receptors of beta-cell of islets of langerhans which result in stimulation of insulin release. also, it minimizes histopathological alterations in damaged islets, plus antioxidant and antiapoptotic activities (67,68). omaima nasir (2014) and el tobgy (2019) observed in studies on diabetic mice, the antihyperglycemic effect of ga mediated via a reduction in intestinal glucose absorption, which will reduce plasma glucose level, and consequently the insulin level. also, gum consumption reduces glucose urea and urinary volume (36,69). concerning diabetic complications, the protective and or preventive effect of ga was reported. ga ameliorated neuropathy (70) and albuminuria, it is effective for delaying the progression to renal failure. nasir et al displayed in a study the protective effect of gum on renal function in diabetic rats since it decreases the blood pressure, additionally ga significantly decrease proteinuria, serum phosphate concentration and enhanced iraqi j pharm sci, vol.28(2) 2019 clinical effects of arabic gum 13 glomerular filtration rate which ultimately improve renal functions (69,71). effect on renal failure in sudan, ga frequently prescribed for renal dysfunction patients, as it decrease uremia, dialysis frequency and thus improving life quality (72). ebtihal y. khojah observed in study that orally used ga in pomegranate juice (1ml/day/kg body weight) in rats having chronic kidney disease (kcd) for four weeks along with low protein, phosphorus and potassium diet, result in a considerable improvement in kidney functions (lower serum creatinine, urea), nutritional status and serum minerals (calcium, potassium, and phosphorus) (73). al mosawi (2007) observed the salutary effect of ga. 1gm/kg/day with a low protein diet was given to eleven years old girl with esrf (end stage renal failure) who was managed with dialysis. while using this regime there was a decrease in blood urea, creatinine, no sign of acidosis or uremia and the most important thing was the dialysis-free period (four years) (74). elamin and colleges (2017) noticed a significant decrease in creactive protein level in ckd patients when diet supplemented with 10–40 g/day of gum arabic, with no noticeable effect on blood urea nitrogen and indoxyl sulfate(75). al za’abi et al (2018) proved the valuable ga effect in rats with adenineinduced ckd, since improvement in biochemical markers and histopathological features of affected kidneys had been observed (76). effect on blood coagulation ga may be considered as a natural anticoagulant. muhanad e.abdalla et al (2014) observed in a study on healthy females that ingestion ga (30 gm/day) rise the prothrombin time within the common physiological range, promoting ga safety (no bleeding tendency). ga fermentation products as acetate that formed in large intestine affecting the coagulation cascade(77). hadi and colleges (2010) demonstrated that different concentrations of ga (6gm/100ml and 10gm/100ml) were given to albino rats for four weeks, the bleeding time, and prothrombin time were considerably prolonged, while no remarkable effect on activated partial thromboplastin time (aptt). thus ga modulate its effect through suppression of the intrinsic coagulation cascade without causing extrinsic pathway suppression (78). antibacterial effect ga exhibited an antibacterial effect (79). alalawi et al (2018) study’s show the significant antibacterial activity of n.hexane extract of sudanese and omani ga against one gram positive bacterium: staphylococcus aureus and three grams negative bacteria: two strain of e. coli and klebsiella pneumonia. the antibacterial effect attributed to high concentration of non-polar constituents (6). bnuyan et al (2015) study’s revealed that ga aqueous extract is effective in inhibiting the growth of the tested gram-positive bacteria (staphylococcus aureus, staphylococcus epidermidis, streptococcus pneumoniae) and gram-negative bacteria (salmonella typhi, proteus mirabilis, klebsiella pneumonia, enterobacter species, acinetobacter, e. coli, and serratia species). also effective against fungus candida albicans. ga antimicrobial effect’s attributed to the high terpene content (80). moreover, lawrence r et al (2015) study’s evaluated the antibacterial effect of acetone and methanol extract of ga against listeria monocytogenes, bacillus cereus, bacillus subtilis, clostridium perfringens, staphylococcus aureus, streptococcus pyogenes, escherichia coli. secondary metabolites as tannins, flavonoids and others that have been detected in these fractions were responsible for antibacterial effect (81). effect on oral hygiene ga considered as a dental caries protective agent. 0.5%1% of ga when added to culture medium suppress the growth of porphyromonas gingivalis and prevotella intermedia (periodontic\ bacteria) and inhibit early plaque formation. the antimicrobial effect attributed to the presence of cyanogenic glycosides and various enzymes as oxidases, peroxidases, and others (29,82). ga, repress acid-dependent demineralization and uphold remineralization even under fluoride-free settings (83). conclusion ga has several advantages and it is widely consumed as one of the important ingredients in dietary, pharmaceutical and other industrial products. according to the previous data and outcomes, ga demonstrated proven pharmacological effects and have therapeutic usefulness. more studies are required to clarify the exact mechanism(s), constituent(s) and the proper dose responsible for the specific medical or pharmacological effects. 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http://ascidatabase.com/author.php?author=naima&last=e.s.%20ali http://ascidatabase.com/author.php?author=amir%20m.%20awad&last=elkarim http://ascidatabase.com/author.php?author=aisha&last=sh.m.%20fageer http://ascidatabase.com/author.php?author=abdelazeem&last=a.m.%20nour https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5356407/ https://www.ncbi.nlm.nih.gov/pmc/articles/pmc5356407/ https://www.sciencedirect.com/science/article/pii/s2090506813000183#! https://www.sciencedirect.com/science/article/pii/s2090506813000183#! https://www.sciencedirect.com/science/article/pii/s2090506813000183#! https://www.sciencedirect.com/science/article/pii/s2090506813000183#! https://www.hindawi.com/48165487/ https://www.hindawi.com/35276912/ https://www.hindawi.com/20131893/ https://www.hindawi.com/63064293/ http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci , vol.17 (2) , 2008 nigella sativa callus cytotoxicity 63 cytotoxic assay of nigella sativa leaf callus extract (thymol) on hep-2 cell line using elisa assay zaynab s. abdel gany*,1 and mayasaa f. mahdi* * iraqi center for cancer and medical genetics researches, al mustansiriya university baghdad , iraq abstract extract from cell culture of medicinal plant like nigella sativa have been assessed for its cytotoxic properties. thymol is likely responsible for the theraputic effects of nigella sativa leaf callus extract. in this short study the inhibitory effect of nigella sativa leaf callus extract (thymol) has been studied on human lorgnx epidrmoid carcinoma (hep-2) cell line during different exposure period of time (24, 48 and 72 hrs.) using different concentration of the extract (1000, 500, 400, 300, 200 and 100 µg/ml). the optical density of the hep-2 cells has been readed on 492 nm wave length. thymol – induced cytotoxicity was (500 µg/ml) which inhibit cell growing compared to the control and this ratio increased at the 48 hrs of exopsure and stopped at 72 hrs. key wards: nigella sativa, callus extract, cell line, elisa assay. ةصالخلا وه ربتعي لومياثلا .اياتءصلتهخ عماتهصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا لخسفت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ل ءيت مللمةت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا اليت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا المت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعت صممجمتننم نمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خصسمت مع سم . وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا نمسصمختسم لمت مت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا صخا ختءصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ؤسسم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاةت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا م وسمت للمةت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا المت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعتهصتهخ عماتا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا سم ا . وه ربتعي لومياثلا .اياتجيت ل ت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا رنسمتت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعمت وه ربتعي لومياثلا .ايؤسسمت تت 48 ,24) صصتا ممةت وه ربتعي لومياثلا .ايممستتهع منمت) hep-2 وه ربتعي لومياثلا .ايعماتا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا تحم ات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا المت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاعت) وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا نمسصمخ( ءماتصصمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ع سمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خمأمصسمتسم ت مع سمتءماتo.d( . وه ربتعي لومياثلا .اياتمسمست وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاخµg/ml 100ت 200 ,300 ,400 ,500 ,1000تعمءم( ) صلختسملمع عا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلامت وه ربتعي لومياثلا .ايم وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاحسلتهع منمتهصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا صخ عمات)72 تتت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا لاتسليتصصمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ع سمت) صلختسملم صرم صمتµg/ml 500 . وات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلاات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا نمسصمختسسماتعمهمت مع سمت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خمأمصسمتءلات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا محسلتتnm 492 وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا امخت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا صمويت تعمءمتهصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ممستت وه ربتعي لومياثلا .ايمملتءلات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا سممت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا نم تتهصت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ممست.48هةتهممهممت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا خسامتت ه وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا ةت ل ت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا لخلمتجيت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا صاتت introduction the main inspiration of black seed comes from the famous saying (hadith) of our prophet mohammed; (god peace be upon him),تthatت“habbatتal-soda is remedy for all diseaseتexceptتdeath.”(1)ت.it is an annual herbaceous plant believed to be endogenous to the mediteranean region but has been cultivated in other parts of the world including india and pakistan (2). black seed oil contains about 0.5-1.5 % volatile oil including nigellone and thymoquinone used as anti-histanimic, antioxidant, antiinfective and bronchodilating effects(3).thymol, is one of the active compounds in n. sativa extract, plays important role in the inhibition of cancer cells, and can attach with the mutagenic substance, because thymol is one of the antioxidant phenolic compounds (4).plant tissue culture techniques inters in several applications like plant micropropagation, genetic study, plant improvement, study of plant cell physiology, the production of secondary metabolites in addition to production of viruse free plant diseases (bacterial and fungal) (5). so, to increase the production of this compound (thymol) all the year round without depending on the mother plant, plant tissue culture techniques formed callus and then increased the production of thymol. the anticancer activity of n. sativa was first revealed by (6) who observed enhancement of natural killer (nk) cell activity ranging from 200-300% in advanced cancer patients receiving multimodality immunotherapy programme in which n. sativa was one of these components. thymoquinone and dithymoquinone, active principles of n. sativa, had cytotoxic effect against parental and multi-drug resistant human tumour cell lines which were over 10fold more resistant to doxorubicin and etoposide (7) . radiation protection activity of n. sativa in mice against induction of chromosomal aberrations by gamma ray was also reported(8) 1corresponding author : e-mail : zaynabsaad@yahoo.com received : 14/6/2008 accepted : 1/12/2008 iraqi j pharm sci , vol.17 (2) , 2008 nigella sativa callus cytotoxicity 64 the using of plant tissue culture techniques make the easy of pharmaceutical compounds production instead of depending on the mother plant and become possible to produce these compounds at high amount and at high rate of pure may be over than these isolated from the complete mother plant, and its production may be quickly and independent on the season, also limit the surface area that is used in the medicinal plant culturing (9).the objective of the present study was to assess the cytotoxic properties of this extract from cell culure of n. sativa leaf callus using against hep-2 cell line using elisa (enzyme linked immunosorbant assay) assay. material and methods: collection of plant material: seeds of nigella sativa gotten from dr. aws al-ani (directorate of agriculture research and food technology/ ministry of science and technology/ baghdad/ iraq) to be used. callus culture condition: the establishment and maintenance of callus were carried out using the procedures described before(10). extraction prepration: for preliminary screening, the seeds were cultured and callus induced and material from callus culture were lyophilzed and extracted by a method described elsewhere (11). in short, one g of callus was mixed with 30 ml of naoh solution 5% and then diethyl ether was then added in a ratio of (2:1) (v /v) and mixed well as described elsewhere (12). the extract was then filtered and concentrated in vacum at 45º c and then kept in the dark at 4º c until tested. cytotoxicity test using elisa assay: for this test, the extract were weighed (0.05 g) and dissolved in phosphate buffer saline (pbs) and dimethylsulphoxide (dmso) to prepare extract solution at 1000 µg/ml. the following dilutions of extract were then prepared: 500, 400, 300, 200 and 100 µg/ml.hep-2 cell line, obtained from iraqi center for cancer and medical genetics researches at the passage level 326 were used in this study. the origin and description of this cell line was mentioned by (13). it was a human laryngeal carcinoma excised from 57 years old man, then transplanted in immune suppressed rat by cortisone. after growth of the tumour in the rat, it was then excised and transplanted as an in vitro tissue culture. it was kept at -169̊cت (in liquid nitrogen). in preparation to any in vitro assay, the frozen cell line was withdrawn and maintained in rpmi-1640 containing 10% bovine calf serum. when the in vitro cells culture forms a monolayer. these cells were treated with trypsin/ versine mixture in order to pursue subculture process.the percentage of inhibition was calculated according to the following equation: (14) where : oc: optical density of control wells ot : optical density of test wells from the above calculation, a graph was plotted for the precentage of growth inhibition against each extract concentration. activity against hep-2 cell line was determined by the inhibition assay using an elisa assay. in short, cell cultures in the microtitration plate were exposed to a range of plant extract concentrations during the log phase of growth and the effect determined after recovery time. the following protocol as descriped in (15) was performed the extracts of nigella sativa leaf callus : a) after trypsinization, cell suspension seed in a micro titration plates at 50000 cells/ml rpmi-1640 growth medium with serum 5% was used for seeding. b) plates then incubated for 24 hours. c) by using maintenance medium, two-fold serial dilution were prepared starting from .μg/mlت100تwithتendingتμg/mlت1000 d) after incubation for 24 hrs, cells exposed to different extract dilutions. only 200 μlتofتeachتconcentrationتaddedتforتeachت well (6-replicates for each tested concentration).ت200تμlتofتmaintenanceت medium added to each well of control group.the times of exposure were (24, 48 and 72 hrs). the plates sealed with self adhesive film then returned to the incubator at 37º c.ْ. e) after the end of the exposure period, the medium and the cells decanted off and replaced byت200تμlتofت%0.01تcrystalتvioletت dye. after 20 min. the stain was washed gently with tap water for three times. the plate was left until become dry. the optical density of each well was read by using a micro-elisa reader at 492 nm transmitting wave length (15 , 16). statisitical analyses: a one-way analysis of variance was performed to test whether group variance was sifgnificant or not, the comparison between groups were used analyses of variance least significant differences test (l.s.d.)(16). inhibition % = [(oc – ot) /oc] x 100 iraqi j pharm sci , vol.17 (2) , 2008 nigella sativa callus cytotoxicity 65 results cytotoxic effect of nigella sativa leaf callus extract (thymol) on hep-2 cell line: the result of the cytotoxic effect of the extrcat readed using elisa micro reader with wave length 492 nm indicate the presence of a relation ship between color density of the stain and the number of the viable cells. the result showed presence of significant at level (p≤0.05)ت differencesت betweenت theت concentrations comparied with control started with the concentration (1000, 500, 400, 300, تvalueتinhibitionتwithت(μg/mlت100تandت200 ranged (67.2%, 75.4%, 74%, 75%, 72.2% and 74%) respectively at 48 hr of exposure periods, while there is no significant differences at level (p≤0.05)تbetweenتtheتconcentrationتitselfتasت shown in figure (1). 0 0.05 0.1 0.15 0.2 0.25 0.3 24 hr 48 hr 72 hr control 1000 500 400 300 200 100 figure (1): effect of extract concentrations on the growth of hep-2 cell line during different exposure preiod. the results also showed the best exposure period was 48 hr than the other periods (24 and 72 hr), the inhbition to cell line begins at 48 hr, there is no significant differences at level (p≥0.05)تinتinhibitionتwhenتcomparedتwithتtheت period 72 hr. this means exposure the extract to cell line at 48 hr with lowest concentration showed significant differences. after that result we choose the inhibitory concentration which inhibit the growth of hep-2 cell line depending on the changes that appears on the optical density and the changes in the color that appears on the plate itself from the stain reaction with the cells(17). so, the concentration 500 µg/ml has inhibitory effect compared to other concentration that also showed a minimum inhibition on the growth of hep-2 cell line at 48 hr exposure period as shown in table (1), and the inhibition ratio was shown in table (2). table 1: a comparison optical density of growth inhibition of hep-2 cell line, by using different concentration of callus extracts of nigella sativa during three periods of exposure. type of extract conc. (µg/ml) optical density (od) 24hrs 48hrs 72hrs callus extract (thymol) 1000 0.071 0.092* 0.05 500 0.060 0.069* 0.05 400 0.063 0.073* 0.05 300 0.068 0.070* 0.05 200 0.067 0.078* 0.05 100 0.064 0.073* 0.05 control 0.115 0.281 0.16 )*):means the presence of siginificant differences atتlevelت(p≤0.05)تbetweenتtheتconcentrations. table 2: a comparison of growth inhibition percentage of hep-2 cell line, by using different concentration of callus extracts of nigella sativa during three periods of exposure. type of extract conc. (µg/ml) inhibition% 24hrs 48hrs 72hrs callus extract (thymol) 1000 38.2 67.2 68.7 500 47.8 75.4 68.7 400 45.2 74.0 68.7 300 40.8 75.0 68.7 200 41.7 72.2 68.7 100 44.3 74.0 68.7 discussion although the quinone thymol has demonstrated significant in vitro and in vivo antineoplastic activities against different tumor cell lines(7,18). in this study , thymol demonstrated different cytotoxicity in vitro toward hep-2 cell line according to its concentration. this study appeared that the concentration 500 µg/ml affact on the inhibition ratio when compared with the lowest concentrations which show a minimum inhibition compared with the control. this inhibition increased when reacrhed 48 hr of op ti ca l d en si ty extract concentrations µg/ml iraqi j pharm sci , vol.17 (2) , 2008 nigella sativa callus cytotoxicity 66 exopsure and stopped at the 72 hr of exposure. the using of these concentrations (1000, 500, 400, 300, 200 and 100 µg/ml) in this study affect on the growth of hep-2 cell line with slight differences between them , the od of hep-2 cell line ratio was the lowest on the concentration 500 µg/ml compared with other concentrations. so, the extract make an inhibition on the growth of hep-2 cell line compared the control depending on the concentration that is used and the length of incubation period. the result of this study suggest that thymol inhibited proliferation of tumor cell line by a mechanism that involves cytotoxicity, in fact, it is known that thymoquinone (a quinone from nigella sativa) inhibited the proliferation of cos 31 (canine osteosarcoma) at concentration 100µm by inducing apoptosis and cell cycle arrest at g1. non-cancerous cells are relatively resistance to thymoquinone (19) . nigella sativa and other plants were tested on human hepatoma hep g2 cell line, the effect were determined on 24 hr of incubation. the greatest inhibitory effects were observed on nigella sativa plant extract even at low concentration (20) . refrences 1. abdullaev, i.f. cancer chemoprevention and tumoricidal properties of saffron (crocus sativus l.). experimental biology and medicine (2002). 227: 20-25. 2. chakraverty, h.l. plant wealth of iraqi dictionary of economic plants, (1976). vol i. baghdad p. 387-588. 3. randhawa, m.a. and al-ghamdi, m.s. a review of the pharmacotherapeutic effects of nigella sativa. pakistan j. med. res. 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(1984). 6. el kadi, a. and kandil, o. effect of nigella sativa (the black seed) on immunity. proceeding of the 4th international conference in islamic medicine, kuwait. bull islamic med (1986). 4: 344-8. 7. worthen, d.r.; ghosheh,o.a. and crooks, p.a. the in vitro antitumor activity of some crude and purified components of black seed. nigella sativa l. anticancer res.(1998). 18(3a): 1527-31. 8. shubber, e.k., ibrahim, s.a.m. and alazawi, a.f.n. reduction of gamma ray induced chromosomal abnormalities in mouse bone marrow cells by nigella sativa. 2nd arabian conference of biotechnology, and genetic science. al-mennia, egypt (2000). oct.20-23. 9. robins, r. j.; payne, j. and rhodes, m. j. cell suspension culture of cinchona ladgerinna; i growth and quinoline alkaloid production. plant media (1985). 3:163-246. 10. sokmen, a.; jones, b.m. and erturk, m. antimicrobial activity of extracts from the cell cultures of some medicinal plants. phytother. res. (1999). 13, 355-357. 11. pacheco, p. ; sierra, j.; schmedahirschmann, g.; potter, c.w.; jones, b.m.; moshref, m. antiviral activity of chilean medicinal plantextracts. phytotherapy research (1993). 7, 415-418. تت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا لصم ,ءميتءلات وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا اخسص . وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا مرممسمت تحسصسمعت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا للم وه ربتعي لومياثلا .ايمة .12 .(1989) صلختسملاا وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا .) صلختسملسبت وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا اسصم.ت( وه ربتعي لومياثلا .ايالخلل ةيمسلا هصئاصخ ةفرعمل ءادوسلا ةبحلا يبطلا تابنلل يعرزلا جيسنلا السم .ومهمم 13. moree, ae.; sabachewsky, l. and toolan, hw. culture characteristic of four permanent lines of cancer cells. cancer res. (1955). 1: 598605. 14. chiang, w. ;chang, my. and lin, cc. in vitro cytotoxic antiviral and immunomodulatory effects of plantago major and plantago asiatica. american journal of chinese medicine (2003). 31(2): 225-234. 15. freshney, ri. culture of animal cells. 3rd. ed. wiley-liss. usa (1994). pp267-308. 16. al-mohammed, nt; km, al-rawi; ma, younis and kw, almorani. principle of statistics. (1986) al-mosil university. 17. mahony, de.; gilliat, e.; dawson, s.; stockdale, e. and lee, shvero cell assay for rapid detection of clostridium perfringens enterotoxin. applied and environmental microbiology (1989). pp: 21412143. 18. salomi, nj; nair, sc; jayawardhanan, kk; varghese, cd and panikkar, kr. antitumor principles from nigella sativa seeds. cancer lett. (1992). 63:41-46. 19. shoieb, am; elgayyar, m; dudrick, ps; bell, jl and tithof, pk. in vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone. international journal of oncology (2003). 22:107-113. 20. thabrew, mi; mitry, rr; morsy, ma and hughes, rd. cytotoxic effects of a decoction of nigella sativa, hemidesmus indicus and smilax glabra on human hepatoma hepg2 cells. life sci. (2005). 12:1319-30 iraqi j pharm sci, vol.28(2) 2019 ex-vivo permeation study of dabigatran etexilate loaded nlcs doi: https://doi.org/10.31351/vol28iss2pp37-45 37 ex-vivo absorption study of a novel dabigatran etexilate loaded nanostructured lipid carrier using non-everted intestinal sac model haithem n. abed*,1 and ahmed a. hussein** * ministry of health and environment ,directorate of diyala. diyala, iraq. **department of pharmaceutics, collage of pharmacy, university of baghdad, baghdad, iraq. abstract the purpose of the study was to develop dabigatran etexilate loaded nanostructured lipid carriers (denlcs) using glyceryl monostearate and oleic acid as lipid matrix, and to estimate the potential of the developed delivery system to improve oral absorption of low bioavailability drug, different oleic acid ratios effect on particle size, zeta potential, entrapment efficiency and loading capacity were studied, the optimized de-nlcs shows a particle size within the nanorange, the zeta potential (zp) was 33.81±0.73mv with drug entrapment efficiency (ee%) of 92.42±2.31% and a loading capacity (dl%) of 7.69±0.17%. about 58.5% of drug was released in 8hr in a controlled manner, the ex-vivo intestinal permeation study using the non-everted sac model shows four folds increment in the permeation of de-nlcs compared to dabigatran etexilate suspension (de-s). key words: dabigatran etexilate, nlcs, ex-vivo intestinal permeation, thrombin inhibitor, cremophor-el. ية الحيوية خارج الجسم لحامالت الدهون النانوية الجديدة المحملة بالدابيغاتران أيتكزيليت ذدراسة النفا ج الكيس المعوي غير المقلوب للخارج.ذباستخدام نمو **حسيناحمد عباس و 1،*هيثم نجم الدين عبد ديالى ، ديالى ، العراق .دائرة صحة والبيئة، وزارة الصحة* فرع الصيدالنيات ، كلية الصيدلة، جامعة بغداد، بغداد،العراق.** الخالصة ان الهدف من الدراسة الحالية هوه لتطوير حامالت دهون نانوية محملة بعقار الدابيغاتران أيتكزيليت مستخدمين احادي ستيارات الغلسريل لقليل, نسب و التوافر الحيوي اذو حامض األوليك كقالب دهني, وكذلك لتقييم امكانية تطوير نظام توصيل لتحسين االمتصاص عن طريق الفم للدواء حامالت ل مختلفة من حامض األوليك و تأثيرها على الحجم الحبيبي, جهد زيتا, كفاءة االحتجاز و قابلية التحميل للدواء قد درست.الصيغة المثالية مع كفاءة ملي فولت 8..3±88.33الدهون النانوية المحملة بعقار الدابيغاتران ايتكزيليت قد أظهرت حجم حبيبي ضمن نطاق النانو و جهد زيتا كان ساعة( بطريقة مسيطر 3من الدواء تم تحريرها خالل ) %53.5. حوالي %.3.3±2...مع قابلية تحميل % 9.83 ±29.29االحتجاز للدواء لمحملة اية لصيغة حامالت الدهون النانوية ذج الكيس المعوي غير المقلوب للخارج زيادة النفاذية المعوية باستخدام نموذعليها, اظهرت دراسة النفا بالدابيغاتران ايتكزيليت بالمقارنة مع معلق الدواء. .elكريموفور ،مثبط الثرومبين ،ية المعوية خارج الجسمذالنفا، حامالت دهون نانوية، كلمات المفتاحية: دابيغاتران ايتكزيليتال introduction chronic anti-coagulation therapy is essential for the prophylaxis and treatment of acute venous thromboembolism (vte) and including the prevention of cardiogenic thromboembolism in patients with atrial fibrillation and myocardial infarction, vte is common in traumatic patient or those undergoing major surgical operations or with underlying malignancy and also in patients with prolonged immobilization, vte is an emergency complication with an elevated morbidity and mortality rate, therefore prophylaxis of thrombotic events is considered as standard of care )1,2(. dabigatran etexilate (de) is an inactive prodrug of the active compound dabigatran which is a reversible, selective, non-peptide, direct thrombin inhibitor, and by the inhibition of the serine protease it will prevent the conversion of fibrinogen into fibrin, de has more predicted pharmacodynamic and pharmacokinetic profile and need no antithrombotic monitoring with minimum drug – food and drug-drug interaction compared to traditional antithrombotic agents. de shows very low oral bioavailability (3 -7 %) and this is related to the ph dependent aqueous solubility of the drug and permeability glycoprotein (p-gp) mediated efflux (3, 4). 1corresponding author e-mail: haithemnajem@gmail.com. received: 21/ 2 /2019 accepted: 7/4 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp37-45 mailto:haithemnajem@gmail.com iraqi j pharm sci, vol.28(2) 2019 ex-vivo permeation study of dabigatran etexilate loaded nlcs 38 therefore a novel drug delivery system should be designed to improve its oral bioavailability by overcoming these limitations. nanostructured lipid carriers (nlcs) is a second generation lipid-based nanoparticles was designed to overcome the low entrapment efficiency (ee %) and drug expulsion after storage that associated with solid lipid nanoparticles and this is done because the oil droplets causes several crystal defects in solid lipid matrix and thus imperfections in highly ordered crystal matrix occurs producing adequate space for higher amount of drug to be loaded [5]. the improvement in oral bioavailability that gained by using nlcs as a drug delivery vehicle was related to the fact that nlcs are absorbed mainly through the lymphatic system avoiding the first pas effect, also nlcs mechanism of transportation involve the participation of clathrin and caveolae in their transcytosis and it has a p-gp efflux pump modulation activity and eventually, enhancing the overall permeability of the encapsulated drug [6, 7, 8]. the lipid matrix of nlcs will be converted by the intestinal lipase to mono, di and triglycerides together with fatty acids that interact with the intestinal bile salts to form a mixed micelle system which has a greater solubilizing capacity of the poorly water soluble drug that delivering it across the stagnant unstirred water layer to the enterocyte and increasing their intestinal permeability [9, 10]. therefore, the aim of the present work was to evaluate the nlcs as a mean to improve the intestinal permeability and the oral bioavailability of de thus development of de-nlcs using a rational mixture of solid and liquid lipids together with surfactant stabilizer, and the prepared formulas were tested for their in-vitro physicochemical properties and also an ex-vivo intestinal permeability study was also conducted. materials and methods materials de was purchased from hangzhou hyperchemicals ltd., china, oleic acid (oa) was obtained from riedel de haen ag seelze, honnover, german, glyceryl monostearate (gms) was obtained from bdh chemicals ltd. poole, england, cremophore el (cr-el) a polyoxy 135 castor oil was obtained from basf (ludwigshafen, germany), peg400 was purchased from provizer pharma, india, hplc grade methanol and water for hplc were obtained from biosolve chimie sarl, dieuze, france., ammonium formate was obtained from merck, darmstadt, germany, potassium dihydrogen phosphate and disodium hydrogen phosphate were purchased from bdh chemicals ltd., india, ultrafiltration tube 10k mwco was purchased from pall corporation, usa. microcepttm advanced centrifugal device, all other chemical and reagent obtained are of analytical grade. animals: a male sprague-dawley (sd) rat weighs 250-300mg were obtained from animal house, college of pharmacy-university of baghdad. methods formulation of de-loaded nlcs de-nlcs were prepared by hot emulsification-ultrasonication method with slight modification (11). a 300 mg binary lipid mixture of both gms as solid lipid and oa as liquid lipid were blended in different ratios and heated in water bath to about 10 ± 0.5°c above the melting point of the solid lipid to prevent lipid memory effect, along with 75mg of de to form a uniform and clear oil phase. the aqueous phase consisting of surfactant and cosurfactant blend in double distilled water heated to the same temperature of the lipid phase. the melted lipid phase was added drop wise to the hot aqueous surfactant solution under continuous magnetic stirring at 900 rpm to form an oil in water (o/w) preemulsion, then the emulsion was sonicated using probe sonicator for 10 minutes at 75% amplitude with 30sec. on, 5sec. off periods, then the formed nanoemulsion was cooled in ice bath were the lipid nano-droplets will solidified and the lipid nanoparticles were formed. the composition of different batches illustrated in table 1. characterization of de-nlcs percent entrapment efficiency and drug loading capacity the entrapment efficiency (ee %) and drug loading capacity (dl %) was determined indirectly by measuring the concentration of free de in the dispersion medium. the amount of unentrapped free drug was determined using an ultrafiltration technique (12). a 5ml of de-nlcs solution was placed in the upper chamber of a centrifuge tube matched with an ultrafilter with a molecular cut off size (mwco) 10 kda and centrifuged for 30 minutes at 6000 rpm. the ultrafiltrate containing the free drug was diluted with methanol and the concentration of unentrapped de was determined spectrophotometrically at 315 nm. the ee% and drug loading percent dl% were calculated using the equations (1) and (2) respectively: 𝐸𝐸% = 𝑊𝑡𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔− 𝑊𝑓𝑟𝑒𝑒 𝑑𝑟𝑢𝑔 𝑊𝑡𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔 × 100 (1) 𝐷𝐿% = 𝑊𝑡𝑜𝑡𝑎𝑙 𝑑𝑟𝑢𝑔− 𝑊𝑓𝑟𝑒𝑒 𝑑𝑟𝑢𝑔 𝑊𝑙𝑖𝑝𝑖𝑑 × 100 (2) where,(𝑊𝑖𝑛𝑖𝑡𝑎𝑖𝑙 𝑑𝑟𝑢𝑔) is the weight of initial drug used,(𝑊𝑓𝑟𝑒𝑒 𝑑𝑟𝑢𝑔 ) is the weight of free drug detected in the supernatant after ultrafiltration of the aqueous dispersion and (𝑊𝑙𝑖𝑝𝑖𝑑 ) is the weight of lipid used (13). iraqi j pharm sci, vol.28(2) 2019 ex-vivo permeation study of dabigatran etexilate loaded nlcs 39 table 1. composition of different formulas of de-loaded nlcs formula code de (mg) gms (mg) oa. (mg) cr-el %w/v peg400 %w/v ddh2o (ml) n0 0 270 30 2.5 2 15 n1 75 270 30 2.5 2 15 n2 75 240 60 2.5 2 15 n3 75 210 90 2.5 2 15 n4 75 180 120 2.5 2 15 n5 75 150 150 2.5 2 15 n6 75 300 0 2.5 2 15 -de, dabigatran etexilate, gms, glyceryl monostearate, oa, oleic acid, cr-el, cremophor-el, peg400, polyethylene glycol 400, ddh2o, double distilled water. particle size and polydispersity index dynamic light scattering was used to determine particle size and polydispersity index (pdi) of the prepared de-nlcs dispersion after appropriate dilution with double distilled water (1:50) of all formulations was made and placed in 1cm diameter disposable plastic cuvette to yield a suitable scattering intensity at a fixed scattering angle of 90° at room temperature (25°c). from the analysis, the mean particle size (diameter, nm ± standard deviation) and polydispersity index pd of de-nlcs were calculated using brookhaven instruments corp90 plus (zetaplus particle sizing, ny, software, version 5.34) (14). zeta potential the zeta potential of de-nlcs was determined utilizing the nanobrook zetapals which determines zeta potential using phase analysis light scattering technique , the nlc dispersions were diluted with double distilled water (1:100) to get a uniform dispersion prior to analysis and the samples were placed in a clean and disposable zeta cells. the conductivity of the diluted samples was measured in order to select the detection model. the whole measurement was carried out at 25°c (15). in-vitro drug release study in vitro release study of de-nlcs was performed using a modified dialysis membrane diffusion technique (16). dialysis membrane (himedia, mumbai, india) with molecular weight cut off between (mwco 12,000–14,000da) was previously soaked overnight with dissolution media, 5ml of de-nlc formulation was placed in the dialysis bag and tied at both ends using silk thread and placed in the dissolution apparatus ι of dissolution media, that is 0.1n hcl with ph 1.2 and phosphate buffer solution (pbs) +35% ethanol with ph 6.8 (17). the temperature of the media was maintained at 37 ± 5ºc; the rotation speed was set at 100 rpm. an aliquot of 5ml sample was withdrawn at pre-determined time intervals (0.25, 0.5, 1, 2, 4, 6 and 8 hours), and replenished equivalent volume of fresh dissolution medium. the experiment was performed in 0.1n hcl for the first two hours and transferred to pbs+35% ethanol to the rest time of the study, to provide sink condition. samples were analyzed spectrophotometrically using carry win uv, (varian, australia) spectrophotometer. a cumulative amount of drug released was calculated. analytical method validation of de loaded nlcs instrumentation and chromatography conditions rp-hplc system was used for this study, the specifications are given below. a waters hplc equipped with spa20a detector, an isocratic chromatographic separation conducted utilizing symmetry® ods-c18 (250 × 4.6mm; 5μm) column and breeze software. chromatographic conditions: mobile phase: hplc grade of methanol: 0.1m ammonium formate solution in a ratio of 77:23 percent (v/v) was filtered through (0.45µm) millipore filter. flow rate of the mobile phase was maintained at 1.0 ml/min the column temperature was 40ºc. detection was carried out by uv detector; wave length at 303 nm the running time was 10 min. the volume of the injection loop was 20 μl (18). preparation of standard stock solution and calibration curve prior to injecting the drug solution, the column was equilibrated with the mobile phase flowing through the system for at least 30min.one hundred milligrams of de was accurately weighed and transferred to a 100 ml volumetric flask. it was dissolved in 50 ml hplc grade methanol and sonication for about 10 minutes and then made up to the volume with hplc grade methanol. from this stock solution (1mg/ml) nine serial dilutions (0.5, 1, 2.5, 5, 10, 20, 30, 40, and 50 μg/ml) were prepared. twenty microliter of each dilution was injected into the column and the corresponding chromatograms were obtained. the peaks areas at a specific retention time were recorded and plotted versus concentrations and the curve linearity was evaluated by linear regression analysis. iraqi j pharm sci, vol.28(2) 2019 ex-vivo permeation study of dabigatran etexilate loaded nlcs 40 validation of the hplc method the method was validated following international conference of harmonization (ich) guidelines (19,20) for analytical procedures validation: linearity and range the linearity of the method was determined by constructing three independent analytical calibration curves. the results were tested statistically and subjected to regression analysis to determine the calibration equation and determination coefficient (r2). specificity and selectivity analytical method specificity can be defined as the ability to detect the analyte in the existence of other components such as impurities, matrix compounds and products of degradation. the interference was determined by injection of a sample containing the media without the drug and a sample containing the media with de at a concentration of about (5μg/ml). interday and intraday precision and accuracy the precision of the method was determined by measuring repeatability and intermediate precision. repeatability was examined by six replications of the analyte concentration (20µg/ml) on the same day at different intervals, under the same experimental conditions (intraday). the intermediate precision of the method was assessed by carrying out the analysis on three different days (interday), with six replicates being analyzed each day. the percentage relative standard deviation (%rsd) should be less than 2 . the accuracy of the method was determined by the measuring of the percentage recovery of three concentrations of the analyte (2.5, 5 and 10µg/ml) in triplicate, and the accuracy was calculated as the percentage of de recovered from the injected sample. limit of detection (lod) and limit of quantification (loq) the lod and loq were determined as represented by the ich guidelines [20], making use of a three independent analytical curves mean values, determined via a linear regression model, where the factors of (3.3) for the limits of detection and (10) for the limit of quantitation, were multiplied by the ratio of the y-intercept standard deviation to the slope of the regression line. robustness: the robustness refers to the ability of an analytical procedure to remain unaffected by small but deliberate variations in the parameters of the analytical method which indicating the reliability of the method for routine analysis. the robustness was determined by analyzing samples of (20μg/ml) under different conditions of the analytical method parameters, such as flow rate (0.9, 1 and 1.1 ml/min), injection volume (19, 20 and 21μl), mobile phase composition (methanol ratio of: 76, 77 and 78%) (21). ex-vivo intestinal permeation study the ex-vivo permeation study of de-nlcs was carried out using non-averted gut sac method with modification (21, 22), male sd rats, weighing approximately 250–300 g, were fasted overnight with free access to water, anesthetized with ether and a longitudinal abdominal incision was made then the small intestine was removed and the mesentery was stripped manually and washed out carefully with cold normal saline solution using a syringe equipped with blunt end needle. the clean intestine was cut into 10 ± 0.2 cm long sacs having a diameter of 0.25mm. after tying one end, the intestinal sac was filled with (1ml) of selected formula and pure drug suspension containing approximately (7.5mg) of de, then tying the other end of the sac and placed in a beaker containing krebs-ringer solution (ph 7.4) system was maintained at 37 ± 1.0°c in a magnetic stirrer operating at 50 rpm and continuously bubbled with oxygen (50 bubble/min), (5ml) samples were withdrawing at 30, 60, 90, 120, 150, 180, 210 and 240 min. and the sample was analyzed by hplc, the apparent permeability coefficients were determined using eq. (3) and eq. (4) (23): 𝑷𝒂𝒑𝒑 = 𝑭 𝑺𝑨×𝑪° (3). 𝑺𝑨 = 𝟐𝝅𝒓𝒉 (4). the (papp, cm/min) is the apparent permeability, (f, µg/min) is the flux, (sa) is the area of the intestinal sac in (cm2) and (c0) is the initial drug concentration (µg/ml), the slope of the linear portion of the plot was considered as the permeation flux (f), (r) is the intestinal radius (cm) and (h) is the intestinal segment length (cm). statistical analysis the one way analysis of variance (anova) test using spss software version 17.0 was used. the level of significance was set at α = 0.05, all the results were illustrated as the mean values ±standard deviation (sd) in three replicates (n=3). results and discussion the results of particle size, pdi, zeta potential, %ee and %dl are shown in table2. entrapment efficiency and drug loading capacity a high %ee was observed in formula n5 (97.62±1.15) and the lowest value was seen in formula n6 (68.87±3.23), and a nonsignificant increase in percent drug entrapped was observed (p>0.05) by increasing the oil ratio due to the higher drug solubility in the lipid blend, also the %dl was also increased, a significant reduction in ee% and dl% in formula n6 (without oil) compared to n1 because drug molecules has a higher solubility in oils than in solid lipid matrix and their incorporation will create structural imperfections that increases the amount of drug entrapped within the solid lipid matrix (24). iraqi j pharm sci, vol.28(2) 2019 ex-vivo permeation study of dabigatran etexilate loaded nlcs 41 table 2. evaluation parameters of different de-nlcs formula code particle size (nm) pdi zp (mv) ee (%) dl (%) n0 18.7±0.87 0.005±0.001 -27.73±3.52 n1 62.4±5.75 0.286±0.001 -33.81±0.73 92.42±2.31 7.69±0.17 n2 181.8±7.46 0.315±0.018 -12.62±1.66 95.16±3.21 7.97±0.51 n3 500.9±21.9 0.415±0.022 -12.16±2.11 96.31±1.51 8.04±0.38 n4 769±37.2 0.326±0.012 -7.02±0.58 96.65±1.53 8.41±0.13 n5 806±28.76 0.083±0.017 -6.36±0.72 97.62±1.15 9.18±0.10 n6 147.1±7.5 0.358±0.023 -26.32±3.32 68.87±3.23 4.78±0.32 -data presents mean± standard deviation, (n=3). -ee, encapsulation efficiency, dl, loading capacity, pdi, polydispersity index, zp, zeta potential. particle size and zeta potential analysis a significant increase in particle size was seen with increasing oil ratio (p<0.05) this is related to the fact that as the oleic acid content exceeds 10%, the particle size increased owing to the swollen core of the oleic acid loaded nanoparticles, a similar observation was obtained by w. dai et al.(25), formula n6 shows a significant increase in particle size and pdi compared to n1 this is because the oil decrease the system viscosity leading to easier small particle formation (23). the n1 (blank) shows lower particle size distribution this is expected due to the added mass of drug at a constant stabilizer concentration [26]. all of the prepared formulas have a negative zp values (table 2), with the highest value of (-33.81 mv) in formula n1 and the lowest value was (-6.36 mv) in formula n5, and the accepted value to produce a stable nanodispersion should be ≥+30 mv or ≤ -30 mv. however, it is important to notice that this rule applies only to an electrostatically stabilized system (27). a reduction in zp value was observed when the oleic acid ratio was increased above (10% w/w) which is associated with increased in particle size leading to reduction in surface charge density on the formed nanoparticles (28). in-vitro release study formulas n1 (nlcs with the lowest particle size together with higher zp) and n6 (slns) was selected for in-vitro release study, they shows a biphasic release profile with initial burst release of (31.76%) and (24.6%) in the first 2hr (obtained from the drug release curve) followed by sustained release of (58.5%) and (45.42%) for the next 6hr for n1and n6 respectively, as seen in figure 1, the initial burst release occur due to the drug exist in the surface of the nlcs and the external phase. formula n1 shows faster rate of release compared to n6 this is due to the highly ordered crystalline structure of solid lipid matrix that restrict the drug diffusion velocity to the lipid interface before partitioning between the lipid and the aqueous phases (29, 30). figure 1. cumulative release profile of formula n1 (nlcs) and formula n6 (slns), mean values± standard deviation hplc method validation linearity and range at the concentration range of (1.66-50 µg/ml) the obtained calibration curve was found to be linear with a coefficient of correlation (r2 = 0.9999) and the regression equation was [y=5.0445x+3.7043] as seen obviously in figure 2. specificity and selectivity the analytical procedure was considered selective and specific because no peak interference from the mobile phase on the drug peak as seen in the hplc chromatograms of both the drug and that of the blank in figure 3. precision and accuracy the interand intraday variation study was conducted to define the analytical procedure precision, the results of method precision and accuracy were shown in table 3 and table 4 respectively, the percentage relative standard deviation was less than 2 which confirm the accuracy and precision of the analytical procedure (19). lod and loq the calculated lod and loq were found to be (0.54) and (1.66µg/ml) respectively. iraqi j pharm sci, vol.28(2) 2019 ex-vivo permeation study of dabigatran etexilate loaded nlcs 42 figure 2. calibration curve of dabigatran etexilate using hplc, mean ± sd (n=3) table 3. data of interand intraday precision response (area) response (area) day (1) day (2) day (3) number of replicates 1 105.87 104.98 105.2 2 105.2 105.43 104.8 3 105.5 105.02 105 4 104.54 104.77 104.78 5 106.1 104.92 106.01 6 105.71 105.76 105.91 mean 105.48 105.14 105.28 %rsd(n=6) 0.5280 0.3542 0.5193 %rsd (n=18) 0.4658 table 4. data of accuracy study level of test amount injected (µg/ml) amount* recovered (µg/ml) % recovery %rsd 1 2.5 2.53±0.00678 101.2 0.2683 2 5 5.26±0.05624 105.2 1.0691 3 10 10.04±0.09204 100.4 0.9187 * mean values± sd, (n=3) figure 3. chromatogram of both blank (lower) and dabigatran etexilate standard solution (upper) shows its retention time (chromatogram peak) at a concentration of (5µg/ml). robustness the data obtained during the variation of the method parameters (flow rate, the injection volume and the % methanol in the mobile phase) are shown in figure 5, and the percent relative standard deviation was less than 2% indicating that the analytical procedure was robust (31). iraqi j pharm sci, vol.28(2) 2019 ex-vivo permeation study of dabigatran etexilate loaded nlcs 43 table 5. robustness data of de analytical method validation variables investigated range response (area) %rsd level (1) level (2) level (3) flow rate 0.9 ml/min 106.12 105.77 105.85 0.1731 1 ml/min 104.86 105.85 106.17 0.6465 1.1 ml/min 104.75 105.29 104.92 0.2629 methanol% 76% v/v 105.83 104.95 105.78 0.4684 77% v/v 105.87 105.80 104.91 0.5071 78% v/v 104.85 105.23 104.97 0.1849 injection volume 19 µl 105.89 106.20 106.16 0.1589 20 µl 105.87 104.84 106.51 0.7968 21 µl 106.01 105.89 106.11 0.1039 -%rsd, relative standard deviation ex-vivo permeability study figures 4 and 5 represents the plot of the amount of de permeate from optimized formula n1 and the pure drug suspension in the two intestinal segments used in the study, duodenum and ileum respectively, the flux (f, μg/min) was obtained from the slop of the linear regression equations and the apparent permeability coefficient (papp, cm/min) data were calculated utilizing equations (3) and (4) are shown in table 6, data exhibited a significantly higher intestinal permeability compared to the pure drug suspension (p<0.05), the apparent permeability coefficient (cm/min) of de from formula n1 was found to be 13.755±0.12 and 12.139±0.18 ×10-5 from duodenum and ileum segments respectively, and its value for pure drug suspension was 3.181±0.008 and 2.853±0.009 ×10-5 from duodenum and ileum respectively, showing a four folds increase in the drug permeability from de-nlcs in comparison with pure de suspension, a similar results obtained by a. buthiraja et al. and this is may be related to the high adhesion properties of the prepared nanoparticles which is due to the extremely high surface area of these highly small particles leading to high drug diffusion and dissolution may explain the enhanced drug permeation (23). and the use of permeability enhancing excipients such as cremophor-el and oleic acid that inhibits intestinal p-gp efflux pump activity probably improves drug permeability (32). conclusion in the present study, de loaded nlcs were successfully prepared utilizing emulsification / ultrasonication method, and the formula n1 was selected due to its preferred particle size distribution, zeta potential, entrapment efficiency and drug release profile and selected to undergo ex-vivo permeability study and the obtained data reveals a four folds increase in drug permeability showing that nlcs is a promising delivery system to increase the de oral bioavailability. figure 4. permeation of dabigatran etexilate from optimized formula n1 and pure drug suspension through non-everted rat duodenum, values of mean ±sd (n=3). figure 5. permeation of dabigatran etexilate from optimized formula n1 and pure drug suspension through non-everted rat ileum, values of mean ±sd (n=3) iraqi j pharm sci, vol.28(2) 2019 ex-vivo permeation study of dabigatran etexilate loaded nlcs 44 table 6. the ex-vivo absorption parameters of dabigatran etexilate from optimized formula n1 and pure drug suspension, results were presented as mean ±sd (n=3) sample duodenum ileum f(µg/min) papp×10-5 (cm/min) f(µg/min) papp ×10-5 (cm/min) n1 8.0983±0.26 13.755±0.12 7.1471±0.23 12.139±0.18 de suspension 1.8733±0.013 3.181±0.008 1.6797±0.012 2.853±0.009 references 1. adriance s.m. and murphy c.v., prophylaxis and treatment of venous thromboembolism in critically ill, int. j. crit. ill. inj. sci., 2013;3(2):143-151. 2. terry k.w. ma, bryan p. yan, yat-yin lam, dabigatran 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creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ clinical evaluation of melatonin alone and in combination with pizotifen in the prophylaxis of migraine iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in nsaids-induced injury 34 therapeutic use of silymarin in the management of suspected renal and hepatic injury produced by nsaids in osteoarthritis patients saad a. hussain *,1 , intesar t. numan * , ban h.khalaf ** , talal a. abdulla *** * department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmacology and therapeutics, college of pharmacy, al-mustansrya university, baghdad, iraq. *** department of rheumatology, college of medicine, university of baghdad, baghdad, iraq. abstract : long-term use of non-steroidal anti-inflammatory drugs (nsaids) mostly associated with renal and hepatic adverse effects, and the adjunct use of compounds with potent protective effects, like silymarin, may be one of the choices to avoid these effects. this project was designed to evaluate the protective effect of silymarin against the suspected renal and hepatic injury induced with long term use of nsaids; 220 patients with osteoarthritis were randomized into 5 groups and treated with either silymarin 300mg/day alone, piroxicam 20mg/day alone, meloxicam 15mg/day alone or the combination of each of them with silymarin for 8 weeks. the renal and hepatic functions were evaluated before starting treatment and after 8 weeks including assessment of serum levels of urea, creatinine and the activities of the hepatic enzymes alkaline phosphatase (alp), glutamic-oxalic acid transaminase (got) and glutamic-pyruvic acid transaminase (gpt). the results indicated that using nsaids alone produced elevation in the markers of renal and hepatic damage that can be successfully prevented or reversed when silymarin adjunctly used with them. in conclusion, silymarin when co-administered with the nsaids (piroxicam or meloxicam) decreases their renal and hepatic toxicities in oa patients. key words: silymarin; piroxicam, meloxicam; nephroprotection; hepatoprotection الخالصة دائوا هاَرسثة االسرخذام الوشهي لوضاداخ االلرهاب غُز السرُزوَذَح فٍ حذوز أضزار خاًثُح فٍ الكثذ و الكلُرُي، واى اسرخذام هزكثاخ لها القذرج علً حواَح الدسن ضذ هذٍ الرأثُزاخ، هثل السلُوارَي، قذ َكىى هي ضوي الخُاراخ الوراحح والرٍ َوكي اعروادها هذا الغزض. ذن ذصوُن هذٍ الذراسح لوعزفح ذأثُز السلُوارَي فٍ الىقاَح هي الرأثُزاخ الداًثُح الورىقعح لوضاداخ االلرهاب غُز لوثل هزَضا هصاتا تالرهاب الوفاصل غُز الزثىٌ وذن ذىسَعهن عشىائُا علً 222السرُزوَذَح علً الكثذ والكلُرُي. أخزَد الذراسح علً هلغن/ 51هلغن/ َىم( أو هُلىكسُكام فقط ) 22هلغن/َىم( أو تُزوكسُكام فقط ) 022ن أها توادج السلُوارَي فقط )خوسح هداهُع وعالخه أساتُع، و ذضوٌد 8أساتُع.ذن فحص وظائف الكلُرُي والكثذ قثل الثذء تالعالج وتعذ 8َىم( أو تئضافح السلُوارَي إلً كل هٌهوا و لوذج ٍ الُىرَا و الكزَاذٌُُي فٍ هصل الذم و كذلك فعالُح األًشَواخ الكثذَح هذٍ الفحىصاخ قُاص هسرىي هادذ got, gpt, alp فٍ هصل الذم. أظهزخ ًرائح الذراسح إى اسرخذام الثُزوكسُكام و الوُلىكسُكام لىحذهوا َوكي أى َؤدٌ إلً رفع هسرىي الوعاَُز الثاَىلىخُح ئضافح السلُوارَي إلً الٌظام العالخٍ. َوكي االسرٌراج تاى السلُوارَي عٌذها َسرخذم هع لىظائف الكلُح و الكثذ و الرٍ َوكي الحذ هٌها ت هضاداخ االلرهاب غُز السرُزوَذَح َوكي أى َحذ هي أضزارها الداًثُح علً الكلُرُي و الكثذ لذي هزضً الرهاب الوفاصل غُز الزثىٌ. introduction : many physiological functions other than inflammation are reported for prostaglandins (pgs) including maintenance of gastromucosal integrity, (1) modulation of renal microvascular hemodynamics and tubular salts and water reabsorption. (2) so, nephrotoxicity, gastropathy and other forms of tissue injury are reported as a result of long-term use of non-steroidal antiinflammatory drugs (nsaids) ( both selective and non-selective cyclooxygenase inhibitors), (3) and considered as an important parameter during long-term therapy. (4) according to the available reports, all nsaids, selective and non-selective cox inhibitors, share relatively the same therapeutic and adverse effects profile, and caution should be exercised with their use; meanwhile, adjuvant cytoprotection should be considered. (5, 6) silymarin is a mixture of flavolignans isolated from the ripe seeds of the medicinal plant silybum marianum (milk thistle). (7) it has many pharmacological activities including anticancer, (8) hepatoprotective, (9) cardioprotective, (10) nephroprotective, (11) and neuroprotective (12) activities that mostly attributed to its powerful antioxidant properties and the ability to modulate responses of different cellular receptor systems. (13, 14) the present study was designed to evaluate the protective effects of silymarin against the suspected renal and hepatic injury that might induce by long-term use of nsaids in patients with osteoarthritis (oa). 1 corresponding author: e-mail: saad_alzaidi@yahoo.com received : 4/10 /2006 accepted : 22/5/2007 mailto:saad_alzaidi@yahoo.com iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in nsaids-induced injury 35 patients and methods : this study was performed on 220 patients (79 males and 141 females) with painful knee osteoarthritis at the outpatient clinic in baghdad teaching hospital, with an age range 0f 38-75 years (53.07+8.18). all patients have symptomatic and radiologic evidence of oa in one or both knee joints, their clinical features were in accordance with the description of oa in united kingdom and north american clinical guidelines. after obtaining their signed consent, patients were allocated into five groups: group a included 20 patients treated with silymarin capsule (300mg/day), specially prepared from a standard crude extract (received as a gift from luna company, egypt) given in two divided doses for eight weeks; group b included 50 patients treated with 20mg/day piroxicam capsule (medico, india) given at night for eight weeks; group c included 50 patients treated with a combination of piroxicam and silymarin in doses and duration as indicated above; group d included 50 patients treated with single daily doses of 15mg meloxicam tablets (ajanta pharma limited, india) given at night for two weeks and group e included 50 patients treated with a combination of silymarin and meloxicam as indicated previously. only 167 patients completed the study. blood samples (10ml) were collected from the veins of each patient, before starting treatment and after 8 weeks, in a plain tube and left for clot formation. serum was separated by centrifugation at 5000 rpm for 10 min and stored frozen at -18 o c until analysed. the serum was analysed for urea and creatinine levels as markers of renal function, and the activities of the aminotransferases (alt, ast) and alkaline phosphatase (alp) according to standard methods. (15, 16, 17, 18) the mean values of all parameters were expressed with sem; student's t-test and anova were used to check their significance, and considered significantly different at p<0.05. results : the results presented in table 1 indicated that although serum levels of urea and creatinine are within normal values, treatment with (300mg/day) silymarin for 8 weeks resulted in significant reduction in the serum levels of both parameters (22% and 26% respectively) compared to pre-treatment value. meanwhile, treatment of other group of oa patients with piroxicam for 8 weeks significantly elevated serum urea level (14%), while serum creatinine seems to be not significantly affected. however, treatment with meloxicam for the same period causes significant elevation in both, serum urea and creatinine levels (8% and 10% respectively) compared to pre-treatment values. combination of the nsaids with (300mg/day) silymarin results in significant reduction in the serum urea (16 and 19% respectively), and serum creatinine (29% and 39% respectively) compared to pre-treatment values (table 1). analysis of inter group variations using anova showed that significant differences among groups were detected in this respect (p<0.001) for both parameters.the data presented in (table 2) clearly showed that although serum activities of the hepatic enzymes, alkaline phosphatase (alp), glutamic acid oxaloacetate transaminase (got) and glutamic pyruvic acid transaminase (gpt) are within the normal values in all groups. treatment of oa patients with silymarin significantly reduces serum enzymes activity (25%, 15% and 20% respectively) compared to pre-treatment values. table 2 also showed that treatment with piroxicam for 8 weeks resulted in significant elevation in the serum activities of alp and gpt (13% and 57% respectively), while got activity did not significantly affected. meanwhile, treatment with meloxicam for the same period in other group of oa patients resulted in significant increase in alp activities in serum (17%) while the activities of the other two enzymes showed non-significant increase only within the same period of treatment. combination of both nsaids used with 300mg/day of silymarin resulted in significant reduction in the activities of liver enzymes in the serum after 8 weeks, where alp activity significantly reduced by 20% and 24% respectively, got activity was reduced by 28.5% and 50% respectively, and gpt activity was reduced by 33% and 35% respectively compared to the pre-treatment values .analysis of inter group variations using anova revealed significant difference among groups in this respect (p<0.001) for all parameters. iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in nsaids-induced injury 36 table 1. effects of silymarin on the serum levels of urea and creatinine in oa patients treated with piroxicam or meloxicam. treatment group serum urea (mmol/l) serum creatinine (mmol/l) pre-treatment post-treatment pre-treatment post-treatment silymarin (n =20) 4.81 + 0.21 3.75 + 0.08* a 61.00 + 5.30 45.1 + 1.77* a piroxicam (n =35) 4.43 + 0.16 5.06 + 0.22* b 65.42 + 3.54 61.9 + 2.65 b piroxicam + silymarin (n =40) 4.92 + 0.28 4.14 + 0.16* c 69.84 + 4.42 49.5 + 2.65* c meloxicam (n =32) 4.74 + 0.22 5.12 + 0.23* b 54.81 + 2.65 60.1 + 2.65* b meloxicam + silymarin (n =40) 4.64 + 0.22 3.92 + 0.07* a 66.30 + 3.54 40.7 + 1.77* a values are presented as mean + sem; n = number of patients; * significantly different compared with pre-treatment value within the same group (p<0.05); data with non-identical superscripts (a, b, c) among different groups are considered significantly different (p<0.05). table 2. effects of silymarin on the serum levels of the activities of the liver enzymes, alkaline phosphatase (alp), glutamate-oxaloacetate aminotransferase (got) and glutamate-pyruvate aminotransferase in oa patients treated with piroxicam or meloxicam. treatment group alp (u/l) got (u/l) gpt (u/l) pre-treatment post-treatment pre-treatment post-treatment pre-treatment post-treatment silymarin (n =20) 58.8 + 4.6 43.9 + 2.9* a 6.8 + 0.4 5.8 + 0.3* a 6.5 + 0.6 5.2 + 0.3* a piroxicam (n =35) 61.5 + 6.9 69.7 + 4.9* b 8.7 + 0.6 9.2 + 0.7 b 6.7 + 0.5 10.6 + 0.8* b piroxicam + silymarin (n =40) 55.1 + 3.2 43.9 + 2.5* a 9.8 + 0.1 7.0 + 0.5* c 8.1 + 0.6 5.5 + 0.3* a meloxicam (n =32) 59.4 + 2.6 69.4 + 1.5* b 7.8 + 0.6 8.8 + 0.8 b 6.9 + 0.3 6.1 + 0.3 c meloxicam + silymarin (n =40) 65.0 + 2.8 49.2 + 2.0* c 12.6 + 0.9 6.3 + 0.2* c 8.8 + 0.7 5.7 + 0.4* a values are presented as mean + sem; n = number of patients; * significantly different compared with pretreatment value within the same group (p<0.05); data with non-identical superscripts (a, b, c) among different groups are considered significantly different (p<0.05). iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in nsaids-induced injury 37 discussion : many chronically used drugs, including nsaids, produced different levels of renal and hepatic toxicities; (19) adverse renal effects have been reported in 5% of patients treated with nsaids, which mostly attributed to impairing the haemodynamic effects of prostaglandins in the renal system (20,21) . the results presented in table 1 indicated that although serum levels of urea and creatinine are within normal values in oa patients included in the study, treatment with silymarin alone for 8 weeks resulted in significant reduction in serum levels of both parameters compared to pre-treatment values, this might be attributed to the cytoprotective effect of silymarin against both the disease and/or drugs-induced renal damage (11) . in the present study, the impact of treating oa patients with piroxicam or meloxicam on the renal function was clearly demonstrated, and found to be compatible with those reported by others (22,23) , where blockade of cox enzyme in the renal system impairs glomerular filtration which may be progressed to acute renal failure. (24,25) the results presented in this study (table 1) indicated that combination of silymarin with piroxicam or meloxicam resulted in improvement in renal function, revealed by significant reduction in serum urea and serum creatinine. silymarin is known to be an effective free radical scavenger, causes significant reduction in lipid peroxidation, protecting and stabilizing cell membranes, (13, 14) it protects the renal system against druginduced damage in many animal models, an effect attributed to its antioxidant and cytoprotective activities, (26,27) and the results reported in the present study can be explained on the same bases. liver toxicity is one of the most widespread problems, both in the development of drugs and in their therapeutic applications. most nsaids that produce overt hepatic injury (with jaundice) in low incidence lead to greater occurrence of mild hepatic enzyme abnormalities (got and gpt), which is reported in 5-15% of patients taking nsaids. (28) confusion should be expected because liver function may be adversely affected by disease process which mimics drug injury. (29) in the present study (table 2), activities of hepatic enzymes in the serum were significantly elevated in oa patients treated with piroxicam or meloxicam, which might be attributed to the oxidative burden of drug metabolism in the liver. combination of those nsaids with silymarin, or using silymarin alone in oa patients resulted in significant reduction in the activities of liver enzymes in the serum after 8 weeks. the hepatoprotective activity of silymarin has been reported in many experimental and clinical studies. in a rodent model, silymarin reduces or prevents liver toxicity caused by the oxidative damage of many drugs used in the treatment of rheumatoid arthritis (ra), like chloraqine and acetaminophen; or against liver toxicity that experimentally induced by carbon tetrachloride or amanita phalloid toxin (30,31) . silymarin was also found to be protective against ischemic liver injury and experimental model of inflammatory liver disease. (32) in the clinical practice, treatment of 20 patients having chronic active hepatitis with 240mg/day of silybinin-phosphatidyl choline complex for 7days significantly lowers serum liver enzymes (got, gpt, alp) and total bilirubin. (33) moreover, in 36 patients presented with alcoholic liver disease, treatment with 420mg/day of silymarin resulted in normalization of serum liver enzymes (got, gpt) activities, total bilirubin and an improvement in the histological profile of liver biopsies after six months of treatment. in addition, procollagen iii peptides (a marker of acute fibrosis) were found to be significantly decreased during the treatment. (34) all these effects are consistent with the well defined antioxidant, cytoprotective and regenerative properties of silymarin, especially in the hepatic system. accordingly, the results of the present study can be explained in this respect. in conclusion, silymarin when co-administered with the nsaids (piroxicam or meloxicam) decreases their renal and hepatic toxicities in oa patients. acknowledgment the authors gratefully thank the college of pharmacy, university of baghdad and luna company , egypt for supporting this project. references : 1. rainfold kd. profile and mechanisms of gastrointestinal and other side effects of non-steroidal anti-inflammatory drugs. am j med 1999; 107:27s-36s. 2. christopher j, hawkey a. nsaid gastropathy. gastroenterology 2000; 119: 521-535. 3. bassoti g, bucaneve g, furno p, morelli a,del favero a. double blind, placebo controlled study on effects of diclofenac sodium and indomethacin on post-prandial gastric motility in man. dig dis sci 1998; 43; 1172-1176. 4. griffin mr, piper jm, daugherty jr, et al. non steroidal anti-inflammatory drugs use and increased risk for peptic ulcer disease in elderly persons. ann intern med 1991; 114:257. 5. perazella ma, tray k. selective cox-2 inhibitors: a pattern of nephrotoxicity similar to traditional nsaids. am j med 2001; 111: 64-67. 6. ladas ej, kelly km. milk thistle: is there a role for its use as an adjunct therapy in iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in nsaids-induced injury 38 patients with cancer? j alternative and complementary med 2003; 9: 411-416. 7. tittle g, wagner h. hochleistungsflissing chromatographie von silymarin. j chromatogr 1978; 153: 227-232. 8. kohno h, tanaka t, hirose i, sugie s, tsuda h, mori h. silymarin, a naturally occurring polyphenolic antioxidant flavonoid, inhibits azoxymethane-induced colon carcinogenesis in male f344 rats. int j cancer 2002; 101: 461-468. 9. he qr, kim j, sharma rp. silymarin protects against liver damage in balb/c mice exposed to fumonisin b-1 despite increasing accumulation of free sphingoid bases. toxicol sci 2002; 90: 278-277. 10. vereckei as, besch hr, zipes dp. combined amiodarone and silymarin treatment, but not amiodarone alone, prevents sustained atrial flutter in dogs. j cardiovasc electrophysiol 2003; 14: 861867. 11. ahlenstirl t, burkhardt g, kohler h, kuhlmann mk. bioflavonoids attenuate renal proximal tubular cell injury during cold preservation in euro-collins and university of wisconsin solutions. kidney int 2003; 63: 554-563. 12. wang mj, lin ww, chen hl, chang yh, ou hc, kuo js, et al. silymarin protects against lipopolysaccharide-induced neurotoxicity by inhibiting microglia activation. eur j neurosci 2002; 16: 21032112. 13. soto c, recoba r, barron h, alvarez c, favari l. silymarin increases antioxidant enzymes in alloxan-induced diabetes in rat pancreas. comp biochem physiol 2003; 136: 205-212. 14. zhang sh, morris he. effects of the flavonoids biochanin a, morin, phlotrtin and 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osteoarthritis: a half century "advances". ann rheum dis 2004; 63: 117-122. 24. swan sk. effect of cox-2 inhibition on renal function in elderly persons receiving low salt diet. ann intern med 2000; 133: 1-9. 25. komhoff m, grone hj, klein t, seyberth hw, nusing rm. localization of cox-1 and cox-2 in adult and fetal human kidney: implication for renal function. am j physiol 1997; 272: 490-468. 26. sonnenbichler j, scalera, f, sonnenbichler i, weyhenmeyer r. stimulatory effects of silibinin and silychristin from the milk thistle silybum marianum on kidney cells. j pharmacol exp ther 1999; 290: 13751383. 27. al-shawi nn, hussain sa, hasson ha, rashid yz, numan na. the effect of different doses of silymarin on gentamicininduced kidney damage in rats. j fac med baghdad 2005; 47(3): 267-270. 28. lewis jh. hepatic toxicity of nsaids. clin pharm 1984; 3: 128-131. 29. carson jl, strom bl, duff, a. safety of nsaids with respect to acute liver diseases. arch intern med.1993; 153: 1331-1335. 30. schumann j, prockl j, kicmer ak, vollmar am, bang r, tiegs g. silibinin protects mice from t cell-dependent liver injury. j hepatol 2003; 39(3): 333-340. 31. jacobs bp, dennehy c, ramirez g, sapp j, lawrence va. milk thistle for the treatment of liver disease: a systematic review and meta-analysis. am j med 2002; 113(6): 506-115. 32. rolo ap, oliveria pj, moreno aj, palmeria cm. protection against postischemic mitochondrial injury in rat liver by silymarin or tudc. hepatol res 2003; 26(3): 217-224. 33. buzzelli g, moscarella s, giusti a, et al.: a pilot study on the liver protective effect of silybin-phosphatidylcholine complex (ldb 1016) in chronic active hepatitis. int j clin pharmacol ther toxicol 1993; 31: 456-460. 34. feher i, deak g, muzes g. liver protective action of silymarin therapy in chronic alcoholic liver disease. orv hetil 1989; 130: iraqi journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 evaluation of the role of il-2 and il-4 in asthma 47 evaluation of the role of interleukein-2 and interleukein-4 in the immunopathogenesis of steroid therapy resistance in iraqi asthmatic patients adel a. sahib * , 1 , abdul wahab a.al-shaikely ** * al-nassyria general hospital **department of clinical laboratory sciences,college of pharmacy,university of baghdad,baghdad ,iraq abstract interleukins (il-2 and il-4) are increased in asthmatics and were reported to induce resistance to steroid therapy in some patients who fail to get benefit from glucocorticoids when used in full dose and for long period of time. in this context, the present study was conducted on iraqi patients to provide additional laboratory mean, beside the clinical diagnosis, for the decision whether the asthma is steroid sensitive or resistant by monitoring the level of immunoglobulins, complement proteins and interleukins among asthmatic patients (steroid sensitive or resistant) and the possible contribution of other factors like age, sex and environments in the development of steroid resistance. a total number of 55 asthmatics and 28 normal subjects were enrolled in the study. patients were diagnosed clinically as steroid sensitive (ssa) and steroid resistant (sra) and blood samples were taken from all subjects included in the study for the measurement of immunoglobulins (iga, igg, igm and ige), complement proteins (c3 and c4), interleukins (il-2 and il-4), and total and differential wbc counts.the results showed no age, sex and residence dependency of acquired steroid resistance, while smoking habit (and may be the atopic allergy) constitute marked predisposing factors. the level of iga and ige were high in both sra and ssa, while igg level was low in sra. complement proteins (c3 and c4) were not differ in asthmatic patients in comparison with control group. the interesting results were those concerning interleukins. the levels of il-2 and il-4 were very high in sra than in ssa. these are parallel with high lymphocyte and neutrophil counts in blood samples of those patients.in conclusion, beside clinical diagnostic features concerning the dose and duration of therapy with glucocorticoids, monitoring the levels of il-2 and il-4 could provide additional laboratory diagnostic measures for the convincing decision that asthma is steroid resistant. key words: steroid resistant asthma, steroid sensitive asthma, il-2, il-4. ةصالخلا ة فسفي مظا ي لحيسىا ضعريسوىظلض كديزسىوقضدكديدقعي تخليسىت ايزنعيةياايمىاي ييضيىتلالي لحيسىلا ضعيسىوتسم ديدزي ية فسفيدقعي تخليسىت ايزنعيinterleukin-2 and interleukin-4سىولك ديىهوتج .زنعيىااليداي مظا يسوحظتىكاوكقضعيز ي كقلضي ةصقظاي ضز ديخعيسىلاسي ضىمظكتزةعسعيدقعي لحيسىوتخليسى ة يسظلتزسيما يسو ظ ض ديىهمظكتزةعسعيدقعيس ظاعس لضي تدلضيسى ثا ي .زملي سيسىمكضليم عيدصاتةييسىعلس ديسىفضىكدي خضمديز كهدي اظيتةدي ض خضمديلىليسىظااكةيسىمتةتديىهظوي ي يسىظ تةتيةزىوظتةيزاة يسىاها كقضعيسىوقضدكد تسنميدي مظا مكوضيلمسيوضحيياضىديسىت اي مظ كيديدزي ضز ديىهلاسي ضىمظكتزةعسعيزمىاي ياايي (imunoglobulins)ىددتيتسي تزتك يسي ي (complement proteins)يز(سوحظتىكاوكقضعيinterleukins)ي ك ي تخليسىت اي يمليعكد لسىلاس يسياقسيزسيدوتيزسييسىومض وديسىوفظوهديىهلاس ليسةات ي ل يي ض خضمديىعلس د(يىهمظكتزةعسعيدمزي ضز اا مظ كياا) ي يسةتفضريملي مظاوليسىقضتتةديسىلضتيزىهوظتةي يايضذي28ي تةدضاي ضىت ايز55دصاتةييسىعلس ديدهلي .ىهمظكتزةعسع ضز ديسيتساةتي .تتيسا ي كضحضعيوض هديد يسىوتخليزسى ة يوضحاسينعيااثاسي تةتةضايوو ضز ك يدزي مظ كيك يىهلاسي2005يلىليتاتة يدزيي2005 ,iga, igg ضىمظكتزةعسع .زتتيسا يدكقضعيسىعتي يو يسةااضبيسىواضلوك ي ضىعلس ديزمىايى كض ي مظا يسىاها كقضعيسىوقضدكدي) igm, igeيزسىيتزتكقضعيسىظيوكهكد ) (c3يز c4(يزسوحظتىكاوكقضعي )il-2يزيil-4ض خضمديى كض يدعفيوتةضعيسىعتيسىيكدضري ) داي ضز ديسىلاسي ضىمظكتزةعسعيركتي لظوعةيدهليسىلوتيدزي ةيسىعلس دليدظلتعيحظضعاي .(differential countزلاثضعلضيسىظوضخهلي) كلِديتداس ميك يتضاكتسيزسخفض(يسىووتزدىفمض كدمزل وضييفاك ي)وضايىهيي ي كقوضيي(ةيورق مأ ةيرضح تناك نإ) لايوضحييادتةديدتينتزةد(يسىمي سى قسيدزي قس د ي كقوضية يزسىومظ كيك يىهمظكتزةعسع(سىو ضزت) سىثقوك ي ي تخليسىت ايوايدقع ةضايوضايدضيigeيزiga مظا يلاي .ىفعزقيسىو ضز د ( ركتيc4 يزc3زنعيزاعيدةدضاي ضاي مظا يسىيتزتكقضعيسىظيوكهكدي) .يىهمظكتزةعسع وادديسىو ضز دسي دقع ساي قاوحiggوضاي مظا ي اظه يدقعيوايسىثقوك ي يسىوتخلي ضىو ضلحدي ي وادديسةااضبيسةتفضر . يسىقظضعايسىولكتةيملي ةيسىعلس ديتهايسىوظله دي ( زسىظليوضحيي مظاةضتلضيدضىكدياعسايدقعيسىو وادديسىو ضز ديىهمظكتزةعسع .ز ةيسىقظضعاياضرعي ظاساةديil-4 يزil-2 ضوحظتىكاوكقضعي) ةوي يسو ظقظضسي ي ةيسىعلس ديسحجي ض خضمديلىلي يسىهووضزةديزسىولظعىديدقعيدزىِايسىوتخل.ر يسىلعفيسىوتتو يىيتةضعيسىعتيسىيكدض يزil-2سىظااكةيسىمتةتديمكوضيةظله ي ضى تدديز عةيسىلاسي ضىمظكتزةعسعيىوتخليسىت ايمضاي تسنيدي مظا يتتوك يسوحظتىكاوكقضعي) il-4يسىقاايسى ديةمظ كييدزية ضزتيسىلاسيا( ةوي يدايةدك يز كهدي اظيتةديىهومضدعةيمليت تةتيمكوضيلمسيوضحيياضىديسىت ايت ضىمظكتزةعسع. 1 corresponding author : e-mail : adph70@yahoo.com received : 14/5/2007 accepted : 24 /6/2008 mailto:adph_70@yahoo.com iraqi j.pharm.sci., vol.17 (1) ,2008 evaluation of the role of il-2 and il-4 in asthma 48 introduction increased responsiveness to a variety of stimuli is a common feature involved in the one of the interesting lung diseases, asthma (1). whether hypersensitivity to inhaled allergens is present or not, asthma is categorized broadly into extrinsic and intrinsic types, respectively(2,3). the incidence of extrinsic asthma occurs most frequently between the age of 3 and 45 years, although its onset may be at any age (4). serum immunoglobulin e (ige) antibody level is raised in most of those patients (5). intrinsic asthma, on the other hand, has a primary onset before the age of 3 years, or after 35 to 45 with, however, some hypersensitivity to drugs, particularly aspirin and some other manifestations including nasal polyposis and urticaria (6). other category of asthma are causally related to different inducing factors including occupational, exercise-induced and aspirin-induced asthma(7,8). the role of immunity and related inflammatory cytokines in the pathogenesis of asthma has been extensively studied. interleukin-2 (il-2) is a known t-cell growth factor which induces clonal expansion of tlymphocytes (9). activated t-cells, b-cells and mast cells have the ability to synthesise il-2 (10,11). the second important t-cell growth factor is interleukin-4 (il-4), which is synthesized by some types of t-lymphocytes, basophils, eosinophils and mast cells to function as mitogenic and lymphocyte differentiation factor(11). these cytokines have, beside these functions, anti-tumor effects on some types of tumors(12,13). in addition to activation of immune system during the pathogenesis of asthma, neuropeptides (substance p and neurokinine a) and nitric oxide have shown to play major roles in the initiation of cascade of responses including vasodilation, mucous secretion, plasma protein extravasation, leukocyte adhesion and activation, and bronchoconstriction that comprise the classical signs and symptoms of asthma (14). these pathogenic mechanisms are controlled to some extent by the use of glucocorticoids (gc) (15). however, some resistance to this mode of therapy is continuously increased and is manifested by failure to improve baseline morning (am) prebronchodilator forced expiratory volume during the first second (fev1) by greater than 15% following 7-14 days of 20mg twice daily oral prednisolone (16). some patients do not have an absolute resistance, but rather gc insensitivity and some might respond to higher doses of prednisolone or require longer period of therapy (17). patients with steroid resistant asthma (sra) have higher level of immune activation (raised levels of t-cells and eosinophils with high level of il-2 and il4)(18). this accompany by persistent respiratory symptoms, nocturnal exacerbations and chronic airway obstruction together with poor clinical and physiologic responses to oral glucocorticoids (gcs) therapy (17). in clinical practice, setting any patient not responding to 40-60mg/day prednisolone after 3 weeks of therapy should be suspected to be sra (16). the clinical efficacy of gcs therapy is the result of the combinations of inhibitory effects on the process of inflammation including decreased trafficking of inflammatory cells and inhibition of inflammatory cytokines production (19). this efficacy has shown to be altered in sra (20). the correlation between increased inflammatory cytokines (il-2 and il-4) and the development of steroidal resistance have been studied. cytokines were reported to induce activation of transcriptional factors that interfere with gc binding to their nuclear recognition sites (21,22). il-2 and il-4 were shown to promote the synthesis of altered gc binding protein (gcr), which reported to be a dominant negative inhibitor of the classic ligandيbindingيproteinيforيgcي(gcrα (ةيورق مأ ةيرضح تناك نإ)(23,24,25)ي. factors contributing to gc insensitivity via immune activation may include allergen exposure which is reported to decrease gc receptor binding affinity and steroid responsiveness in atopic asthmatics (26) possibly by il-2 and il-4 dependent mechanisms(17). on the other hand, superantigen secretion by bacterial or viral agents my contributed to poorly controlled asthma and reduce gc sensitivity. in this context, staphylococcal enterotoxin b is a potent inducer of gcr isoform in t-cells (27). according to previously mentioned immunopathogenic features of steroid resistant asthma, this study was conducted to monitor the role of il-2 and il-4 in the immunopathogenesis of steroidal resistance asthma among iraqi patients; and to monitor any role for immunoglobulins, complement proteins, age, sex, residence and smoking habit on the incidence of sra. patients, materials and methods this study was conducted on 55 asthmatic patients (25 females and 30 males) and another 28 healthy persons (18 females and 20 males) in a single-blind technique. the study was carried out in al-nasseriya general hospital from february 2005 till october 2005. the age range of healthy subjects and patients was 16-73 years with average age ± sd (39.4 ± 14.67). steroid sensitive asthmatics (ssa) were 28 (10 females and 18 males) and iraqi j.pharm.sci., vol.17 (1) ,2008 evaluation of the role of il-2 and il-4 in asthma 49 sra were 27 (12 female and 15 males). patients' selection was based on special criteria including (a) presence of no acute infection at the time of study, (b) presence of no any other chronic infection, (c) steroidal therapy must be discontinued at least for 2 weeks and (d) presence of no systemic disease that my be associated with steroid resistance. patients were diagnosed for steroidal resistance depending on the history of steroidal therapy and the clinical decision. other patient's information were collected in a specially prepared sheet including: age, sex, chief complain, type and dose of steroid used, predisposing factors, associated symptoms, medical history, family history of steroid resistance, smoking habit, residence (civilian or rural), and presence of atopic allergy. materials: il-2 and il-4 elisa kit (immunotech, marseille, france), single radial immuno diffusion test kit for immunoglobulins (bindaridtm the binding site ltd., birmingham, uk), single radial immunodiffusion test kit for complements (bindaridtm the binding site ltd., birmingham, uk), ige elisa kit (immunotech, marseille, france). methods: blood samples were drawn, left for clotting and then centrifuged for 5-10 minutes at 2000 rpm (using centrifuge, k24, coold with rotor number 905, wir 12x10 ml, janetzki, germany) for separation of serum, which was kept frozen unless analyzed immediately. serum levels of il-2 and il-4 were determined using elisa kits (immunotech, marseille, france), based on the interaction between monoclonal antibody bound to the wells of a microtiter plate to the il-2 and il-4 found in the serum. the antigenantibody complex was detected by the addition of a chromogenic substrate, and the intensity of color was recorded colometrically (using spectrophotometer sp6-500, pye-unicam, england); accordingly serum levels of il-2 and il-4 were calculated utilizing a standard curve prepared for this purpose (28). serum level of the immunoglobulins (igg, iga, and igm) and the complement proteins (c3 and c4) were determined using srid kits (bindaridtm the binding site ltd., birmingham, uk). equal volumes of reference sera and test samples were added to wells in an agarose gel containing a monospecific antiserum. the samples diffused radially through this gel and the tested compound (antigen) being assayed by forming a precipitin ring with the monospecific antiserum; rings diameters were measured (using microwell system reader 2305, organon teknika, austria), and concentrations were determined using standard curve prepared for this purpose(29). ige was determined using ige elisa kit (immunotech, marseille, france) statistical analysis all results were presented as a mean  sem. comparisons were made using chisquare,يstudent’sيt-test and anova. p values less than 0.05 were considered significant. results in this study, age distribution of the volunteers enrolled in the study revealed that the incidence of sra was varied but generally is great in the middle and older ages (table 1). table (1): distribution of patients according to their age among steroid resistant and sensitive asthmatic patients. age (years) sra (%) (n=27) (32.5%) of total (83) patients ssa (%) (n=28) (33.73%) of total (83) patients 10-20 -- 3.57 21-30 11.11 17.85 31-40 33.33 28.57 41-50 11.11 21.42 51-60 11.11 14.28 > 61 33.33 14.28 sra = steroid resistant asthma. ssa = steroid sensitive asthma however, no significant difference (p>0.1) was noticed in the incidence of sra among female (55.56%) and male (44.44%) with male/female ratio of 0.33 (table 2). on the other hand, the incidence of sra was shown to be high in those with negative family history (77.77%) in comparison to those with positive family history (22.23%) (p<0.01) as shown in table (2). in addition, steroid resistance was more pronounced in civilian (66.67%) than in rural areas (33.33%); but however the difference was not significant (p>0.1) (table 2). it is clearly shown that smoking may comprise significant predisposing factor for steroid resistance (55.56%) when smoking sra patients were compared to non-smoking iraqi j.pharm.sci., vol.17 (1) ,2008 evaluation of the role of il-2 and il-4 in asthma 50 sra patients (44.44%) (table 3). atopic allergy, on the other was more pronounced in patients with sra (66.66%) in comparison to sra patients with no symptoms of atopic allergy (34.34%); however, the difference was not significant, p>0.5 (table 3). table (2): sex distribution, family history and residence among sra and ssa patients included in the study. asthmatic groups sex family history residence male (%) female (%) positive (%) negative (%) civilian (%) rural (%) ssa (n=28) 64.28 35.72 71.42 28.57 82.14 17.85 sra (n=27) 44.45 55.55 22.22 77.78 66.67 33.33 p-values >0.1 <0.01 >0.1 table (3): smoking habit and atopic allergy among steroid resistant and steroid sensitive asthmatic patients. asthmatic groups smoking atopic allergy nonsmokers (%) smokers (%) present (%) absent (%) ssa (n=28) 82.15 17.85 71.43 28.57 sra (n=27) 44.45 55.55 66.66 33.34 p-values <0.05 >0.5 sra group of patients exhibited varying patterns of immunoglobulin levels as shown in figure (1). there is significant elevation in iga level (3105.55 ± 225.12) and ige level (295.17 ± 61.5) in comparison to control group (2346.11 ± 142.7 and 39.05 ± 4.5, respectively) (p<0.05). however, the levels of these immunoglobulins also were significantly high in ssa group (3815.71 ± 302.61 and 387.85 ± 52.5, respectively) (p<0.05). moreover, only iga levels were shown to be significantly different between sra and ssa. the level of igg level did not differ significantly in sra (13615.56 ± 993.23) over that in control group (12798.33 ± 746.27) (p>0.05); but it was significantly high in ssa (15890.36 ± 892.08) (p<0.05). lastly, the level of igm did not differ significantly among control (1626.89 ± 139.6), sra (1407.11 ± 187.9) and ssa (1593.82 ± 136.38) groups (p>0.05) as shown in figure (1). 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 iga igg ige igm im m un og lo bu lin le ve l ( m g/ l) control sra ssa a a a a a b a b c a b b fig. (1): immunoglobulins level among ssa (n=28) and sra (n=27) patients. values are (mean ± sem). non-identical superscripts (a, b, c) considered significant, p<0.05 analyzed by anova. in this study also, the levels of complement protein (c3) did not differ significantly in sra (1834.44 ± 46.48) and ssa (1666.78 ± 58.21) in comparison with control group (1699.44 ± 46.48), p>0.05 (fig. 2). the same profile was seen in the second complement protein (c4) who its level show comparable values in sra (398.44 ± 20.3) and ssa (389.82 ± 25.5) to that in control group (375.72 ± 23.5), p>0.05 as shown in figure (2). 0 200 400 600 800 1000 1200 1400 1600 1800 2000 c3 c4 c om pl em en t p ro te in le ve l ( m g/ l) control sra ssa a a aa aa fig. (2): complement proteins level among ssa (n=28) and sra (n=27) patients. values are (mean ± sem). no significant difference among groups, p>0.05 analyzed by anova. the interesting results in this study were those related to cytokines (il-2 and il-4) levels. both cytokines were significantly high in sra (46.11 ± 2.31) and ssa (18.28 ± 0.398) in comparison to control group (8.38 ± 0.63), p<0.05; however, its level among sra was significantly higher than that in ssa group (p<0.05) as shown in figure (3). iraqi j.pharm.sci., vol.17 (1) ,2008 evaluation of the role of il-2 and il-4 in asthma 51 0 10 20 30 40 50 60 70 80 90 100 il-2 il-4 in te rl eu ki n le ve l ( p g/ l) control sra ssa cb c a b a fig. (3): interleukins level among ssa (n=28) and sra (n=27) patients. values are (mean ± sem). non-identical superscripts (a, b, c) considered significant, p<0.05 analyzed by anova. on the other hand, the same profile was seen for il-4 who its level was significantly high in sra (91.33 ± 4.42) and ssa (49.03 ± 2.13) in comparison to control group (26.05 ± 11), p>0.5; with significant difference among the two groups of asthmatics, p<0.05 as shown in figure (3). total wbc counts were significantly high in sra and ssa in comparison to control group, p<0.05 (p<0.05) as shown in figure (4). 0 1 2 3 4 5 6 7 8 9 10 cont rol sra ssa t ot al w b c c ou nt (x 10 00 /d l ) a b b fig. (4): total wbc count among ssa (n=28) and sra (n=27) patients. values are (mean ± sem). non-identical superscripts (a, b) considered significant, p<0.05 analyzed by anova. further, the count did not differ significantly among sra and ssa groups (p>0.05). neutrophils were significantly high in sra and ssa groups in comparison to control group (p<0.05). however, their level did not differ significantly among sra and ssa as shown in figure (5), p>0.05. lymphocyte levels were low in both groups of asthmatics using steroidal therapy in companion to control group; however it was slightly lower in ssa than in sra, but the difference was not significant (p>0.05). differential count of other wbcs did not show any significant difference in sra and ssa in comparison to control group (p>0.05) as shown in figure (5). 0 10 20 30 40 50 60 70 80 basoph. eosinoph. mono. lympho. neut roph. d if fe re nt ia l c ou nt ( % ) cont rol sra ssa a b b a b b a a aa a a fig. (5): differential wbc count among ssa (n=28) and sra (n=27) patients. values are (mean ± sem). non-identical superscripts (a, b) considered significant, p<0.05 analyzed by anova. discussion the present study showed that the incidence of steroid resistance is increased among all patients' age rang. this is come in agreement with that reported by ishioka and his co-workers(30). however, resistance to steroid did not significantly affected by sex variation, but may be linked to other factors such as smoking habit and presence of atopic allergy, a finding supported by what reported by others (31,32). atopic allergy and immune activation is clearly suggested in this study to underlay steroid resistance and this is supported further by finding in this study that the levels of iga and ige were high in those patients (33). furthermore, the low or un increase level of igg in sra in comparison to ssa group suggest that the decrease in this immunoglobulin to be one factor contributing to steroid insensitivity. this has been solidified by the question; why nathan and erwin introduced igg as intravenous immunoglobulin in the treatment of steroid resistance; this in turn based on the ability of igg to decrease the levels of il-2 and il-4 in vitro, an effect thought to be involved in the potentiation of inhibitory effect of glucocorticoids on cell proliferation and cytokine secretion(34).the link between the high concentrations of il-2 and il-4 and the development of sra relay on the increased resistant of lymphocytes to the action of gcs in a theory suggest altered splicing of the gcr iraqi j.pharm.sci., vol.17 (1) ,2008 evaluation of the role of il-2 and il-4 in asthma 52 pre-mrna genes induced by these cytokines (35). the results is the generation of a second gcr,يtermedيgcrβ,يwhichيdoesيnotيbindيgcي but antagonizes the transactivating activity of the classic gcr (25). thus, increased expressionيofيgcrβيcouldيaccountيforي glucocorticoid insensitivity among asthmatic patients (36,37). for this reason, the use of high dose of glucocorticoids might make down regulation to the classical glucocoricoid receptors (gcrα (ةيورق مأ ةيرضح تناك نإ),يleavingيtheيinhibitoryي isoformي(gcrβ (ةيورق مأ ةيرضح تناك نإ)يtoيbeيpredominate,يandيthatيisي why resistance occurs to gcs (23). the levels of c3 and c4 did not differ significantly in this study among sra and ssa in comparison with baseline. for this reason we suppose monitoring the level of these complements is without benefit to decide wither the patient has steroid sensitivity of resistance. this speculation was come in agreement with that reported by liao and his associates (38). the elevated levels of il-2 and il-4 seen in this study are correlated well with the acquired resistance to steroid therapy. positive correlation was existed between the increased levels of il-2 and il-4 among sra patients although the correlation failed to reach the level of statistical significance (data not shown). these results came in agreements with those reported by kam and his co-workers (1993) in that the combination of il-2 and il4 induced t cell resistance to gcs and increase gcr expression in the t cells of normal subjects (39). in mice il-2 alone can induce t cell resistance to gc (40). the mechanism of such resistance involves a defect in nuclear translocation of the gc receptors. this in turn depends upon the phosphorylation of gc receptors (41). thus, the results obtained in this donate a possible usefulness of il-2 and il-4 as predictive immune markers for the development of steroid resistance and to be a possible underling cause for such resistance (23, 42). the levels of total wbc were increased in both groups of asthmatic patients. further, the percentage of lymphocytes and neutrophils were high in sra group in comparison to ssa group, although the change was not significant. the high level of these leukocytes in sra based on the theory that glucocorticoids intake could inhibit cell proliferation because of the high dose of steroid used in asthmatic patients as what happen in ssa patients (43).in conclusion, beside clinical diagnostic features concerning the dose and duration of therapy with glucocorticoids, monitoring the levels of il-2 and il-4 could provide additional laboratory diagnostic measures for the convincing decision that asthma is a steroid resistant. acknowledgment the authors gratefully thank the college of pharmacy, university of baghdad for supporting this research and pharmacist haider m. mohammed for his assistance in preparing the manuscript . references 1. national institutes of health (nih): guidelines for the diagnosis and management of asthma. national asthma education program expert panel report, dhhs publication 1991; 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(1996): effects of glucocorticoids on lymphocyte activation in patients with steroidsensitive and steroid-resistant asthma. j. allergy. clin. immunol. 98(6 pt 1): 10731079. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 16 the ability of nutrient antioxidants to influence oxidative stress and lower the dose of prednisolone in patients with alopecia areata ashwaqn. aljaff * , salim a.humadi * , saleh.a. wohaieb** rece ived 9-2-2003 accepted 14-12-2003 abstract alope cia area ta is a co mmon dis ord er, hypothesized to b e autoimmune in etio lo gy. cortis one ta ke n ora lly ma y s timula te new hair growth. pre dnis one (orally a dministered s te ro id ) ha s p ro ved effe ctive for p atie nts with a lo pe cia areata , but its p otential side e ffec ts include we ight gain, metab olic ab normalities , a cne and me ns trua l prob le ms. this c linica l s tudy wa s des igne d to a ss es s the clinica l significa nce o f the nutrie nt a ntioxida nts (vita min a, vitamin e a nd vita min c) in re duc ing the d os e o f cortic oste roids (prednisolone), and a s a co nse quenc e, their side e ffec ts in pa tie nt with alope cia. the res ults of this stud y re ve al the p otential clinical s ignific anc e of the the ra py for two months with thes e antioxid ants in re duc ing the do se of prednis olone from 10 0mg to 10 mg administe re d eac h othe r da y and imp ro ving the rate of hair gro wth by attenuating free rad ic als d ama ging effect o n immune system, thereby de crea sing the immune co mplex de po sition. ac cording to the res ults of this s tudy, the use of nutrient antioxid ants ma y ha ve an importa nt role in protecting the immune system, and d ec rea sing the d os e and s id e effe cts that re sult from the use o f high d os e of c ortico steroids . الخالصة عي الثعلبة يعتبر داء منا ه إلى الجهاز ال ي يعزى أسباب و الت حفيز .من األمراض الشائعة طريق الفم إلى ت ن يؤدي الكورتيزون المعطى ع رضى داء ن لم ة البردنزولو ت فعليا فعالي و الشعر حيث اثب ة نم علب وزن الث مع تأثيره الجانبي المؤثر والذي يتضمن زيادة ال زئي الج ت األيضية اضطر, عمليا ة,ابات في ال وب و اضطراب الدورة الشهري حب .انتشار ال مين سدة فيتا عات األك مان سريرية للعالج لمدة شهرين ب همية ال ة لتقيم األ ري ري ة الس ي تقليل جرعة ) ج ,ه ,أ( صممت هذه الدراس ف ة المعطاة ره الجانبي الناتج من الجرعة العالي ولون وبالتالي تقليل تأثي ردنز .لثعلبةللمرضى المصابين بداء االب ن من ة البردنزولو عات األكسدة في تقليل جرع مان ة ل ة السريرية العالي ة األهمي هذه الدراس ج رت نتائ وم 10ملغ إلى 100أضه ملغ بين ي عي و بالتالي تقليل منا رة على الجهاز ال ر الح ر الهدام للجذو و الشعر عن طريق إنهاء التأثي وتحسين سرعة نم ر معقدات و آخ رسب ال ت ى جرع . المناعية حاجة إل ما يؤدي إلى تقليل ال مناعي م جهاز ال حماية ال ي مانعات األكسدة ف همية دور ن أ ة تبي ك فأن هذه الدراس عا لذل وتب ورتيزونات ن الك ولون( عالية م ردنز ع العالية) الب .و بالتالي تقليل التأثير الجانبي الناتج من الجر introduction alo pecia area ta is a common, unpre dictab le , no n-scarring form of hair loss (1,2,3,4). this dis order a ffec ts a ll age gro ups , with a highe r prevalence in c hild re n and adolescents (4). the cause is unknown but i t is associated with a n a lte ra tion in the immuno lo gical sys tem (5,6). current trea tment is not, a t this po int , d irec ted at the etio lo gy of alopecia area ta but rathe r at the res ulting inflamma tory infiltra te a nd (p re sumably) the growth inhib itory fa ctors p roduced by this res po nse (5,6) the use of nutrient antioxid ants in a lo pecic pa tients re vea led a s ignific ant decrease in basa l and h2 o2 induced mda (b io marke r of oxida tive stress ) level in rbc a nd plasma , increase glutathione lev el ( major a ntioxida nt) in both rbc a nd plasma , increase to ta l p ro te in and finally increase ca ta lase activity. the se e ffec ts s ugges t the imp orta nt role of nutrie nt antio xidants in protec ting the b od y( immune system fro m the o xida tive d ama ge prod uce d b y the d isease ) a nd may influe nce the se ve rity of the disease(7). res earch has s hown that the d isease responds to a v arie ty of i mmunomod ulating trea tme nts , that pa tie nts with alopecia a re ata may have a highe r inc id ence o f c ircula ting antibo dies a ga inst other bod y organs or tis sues , and that family me mbe rs have a higher incidence o f a utoimmune disease (3, 5, 6). *departments o f clinical pha rmacy, college of p har mac y, university o f baghdad, baghda diraq **departmen t of pharmcology and toxico lo gy,co lle ge of pha rmacy, univers ity o f baghdad, baghda d – iraq . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 17 res ea rc h ha s shown that the disease re spo nds to a variety of i mmu nomo dulating tre atmen ts , that p atients with a lopecia areata ma y hav e a higher incidence of circ ulating a ntib odies agains t othe r bo dy orga ns or tiss ue s , and that family me mbe rs have a highe r incide nce of a utoimmune disease (3, 5, 6). sys temic s te ro ids are reserved for use in ra pidly progress ive or exte ns ive a lo pec ia area ta (8,9,10). systemic steroids , particularly a short co urse (4-8 wee ks ) of ta pering dose s, are ofte n us ed eithe r alone or in co mbina tion with to pica l agents. a high do se up to 100 mg pred nisolone da ily has been reco mmended . in this se tting a cne and weight ga in are co mmonly se en s ide effe cts (11). pre dnisolone doses as low as 20 mg p er da y ma y b e associated with s ep tic necrosis of the hip or se ve re gas trointes tinal b le ed ing ( 12,13). this s tudy was de signe d to inves tigate the role of nutrient a ntio xidants (a, e, &c) in red ucing the do se of prednisolone and as a c onseq ue nce their s ide effe cts in pa tient with alopecia. subjects and methods 1subjec ts a-study gro up: co mpris ed o f total of 84s ub je cts, 3 0 no rmal co ntro ls (mean age 25.9 7± 8.09 years) and 54 c ase s with alope cia (me an age 25.20 ±7.05 yea rs ). pa tie nts invo lve d in this stud y were under a de rma to lo gist sup ervision who determine d the se verity o f the d is eas e ac co rding to numbe r of the p atches they hav e, and a cco rd ing to progres sion of dise as e (2) they were nonsmo kers , no n-alcoholic s and free from ap parent o ther dise as es. the d uratio n of dise ase ra nge d from (20 da y18 ye ars). b-pa tie nts: fifty-four patients a ged 10 -4 0 ye ars (26 fema le s, 2 8 ma le s) with alop ecia( with no previous trea tment) were includ ed in this study. [twenty s eve n of them re ce iv ed co rticos teroids (100 mg prednisolone) ea ch othe r day, and the o ther twenty s eve n re ce iv e (10 mg p re niso lo ne) eac h othe r day]. treatme nt s che dule s a lso includ ed a co mbination of antioxid ants [vita min a (500 0 i.u./day), vita min e (10 0 mg/da y) a nd vitamin c (50 0 mg/day)] giv en to b oth gro up s. the trea tment with nutrient antio xidants fo r alop ecic p atie nts inc luded in this stud y co ntinued for tw o months . c-s ample s : he pa rinize d ve nous bloo d sa mples we re c olle cted from alope cic pa tie nts as well a s fro m controls using pla stic dispo sa ble syringe s. fresh blood s amp le we re use d fo r mda and gsh mea surme nts. 2-methods  erythrocy te s malondialde hyde (mda) as say: me as ureme nts o f e rythrocyte and p la sma mda (which is a by prod uct of lip id p eroxid atio n), b ase d on the reac tio n of thio barbituric a cid (tba) forming tba-mda a dd uct, we re ca rried out us ing the mo dified method of s to cks and dormandy(14) as d es crib ed by gilbe rt e t a l(15). the res ults we re e xp re ss ed as nmole/g hb and µmo l/ l p la sma b as ed on the molar e xtinction co efficient of mda is 1.56 ×10 5 m-1.cm-1.  glutatione assay: erythro cyte s and p la sma gsh co ntents w ere de termined ac co rd ing to the method of godin et al.(16 ). kno wn amo unts o f gs h we re a ss ayed by the same me thod and use d for ca lc ulation of gsh quantities in e rythro cyte s. the statistic al significa nce o f the diffe re nce in me an wa s teste d by student t-te st. results aba sa l plas ma a nd erythroc yte mda leve ls in bo th gro ups o f patients we re s ignifica ntly highe r than tho se in controls. tre atment with either 10 o r 1 00 mg prednis olone plus antioxida nts no rma lize d mda lev els in bo th plasma (ta ble 1) and e rythro cyte s (table 2 ) as early as 1 mo nth after treatme nt. furthermore, tota l plasma gs h c ontent was signific antly higher than co ntrols (tab le 3 ), and tre atment of p atie nts with bo th d os es of pre dnis olone p lus a ntioxida nts slightly increas ed gs h p atie nts, but did no t no rma lize these v alue s. on the othe r ha nd, erythrocyte gsh co ntent wa s s ignifica ntly lower in patients c omp ared to c ontrols, and that trea tme nt with bo th dos es o f pred nisolone plus antioxida nts did significa ntly elev ate gs h conte nt in pa tients afte r 1 month o f trea tment, and normalized the se v alue s after 2 mo nth’s of trea tme nt (ta ble 4). b clinic ally there is lowe r inc id ence of pre dnis olone s id e e ffec ts( ac ne and we ight gain) amo ng those pa tients taking pre dnis olone d os e 10 mg ea ch o ther d ay than those ta king 10 0 mg ea ch othe r da y ( ta ble 5 and 6 ). ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 18 ta ble (1): effec t of the a ddit io n of n utrie nt antioxida nts (a, e, &c) to pre dniso lo ne the rapy (10& 100 mg ) on p la sma mda le ve ls in patie nts with alopec ia a re ata. group mda ( mo le /l) contro l n=3 0 patie nts with alope cia pre-tre atment n=2 7 months a fter treatment 1 n=2 7 2 n=27 iantioxidants + 10 mg pre dniso lo ne 0.72 ±0.30 3.17±1.67* 0.87± 0 .4 5† 0.66±0.27† iiant ioxida nts + 100mg pre dniso lo ne 0.72 ±0.30 2.73±1.66* 0.86± 0 .4 6† 0.67±0.26† value s are ex pre ssed as mea ns  sd. * signif ic antly diffe re nt from co ntrol (p< 0.05). † signif ic antly diffe re nt from pre trea tme nt value s (p<0.05 ). n numbe r of s ubje cts ta ble (2): effec t of the addit ion of nutrie nt antioxida nts (a, e, &c) to pre dniso lo ne the rapy (10& 100 mg ) on e rythroc ytes mda le ve ls in patie nts with a lo pe c ia a re ata. group mda (n mo le /g hb) contro l n=3 0 patie nts with alope cia pre-tre atment n=2 7 months a fter treatment 1 n=2 7 2 n=27 iantioxidants + 10 mg pre dniso lo ne 5.981.04 28 .3818.60 * 6 .252 .95† 5.442.48† iiant ioxida nts + 100mg pre dniso lo ne 5.981.04 28 .4018.84 * 6 .273 .09† 5.462.60† value s are ex pre ssed as mea ns  sd. * signif ic antly diffe re nt from co ntrol (p< 0.05). † signif ic antly diffe re nt from pre trea tme nt value s (p<0.05 ). n numbe r of s ubje cts ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 19 table (3): effec t of the addition of nutrie nt antiox idants (a, e, &c) to pre dnis olone the ra py (1 0& 10 0 mg) o n plas ma gluta thio ne leve ls in patie nts with a lo pe cia areata. group gsh ( mol/l) contro l n=3 0 patie nts with alope cia pre-tre atment n=2 7 months a fter treatment 1 n=2 7 2 n=27 iantioxidants + 10 mg pre dniso lo ne 0.90 ±0.20 1.21±0.35* 1 .27±0 .55* 1.54±0.77 * iiant ioxida nts + 1 00 mg pre dnisolone 0.90 ±0.20 1.23±0.38* 1 .27±0 .57* 1.58±0.79 * value s are ex pre ssed as mea ns  sd. * signif ic antly diffe re nt from co ntrol (p< 0.05). n numbe r of s ubje cts ta ble (4): effec t of the addit ion of nutrie nt antioxida nts (a, e, a ndc) to pre dniso lo ne the rapy (10and 100 mg) o n e rythro cy te s glutathione le ve ls in patie nts with a lo pe c ia areata group gsh ( mo le /gm hb.) contro l n=3 0 patie nts with alo pec ia pre -tre atmen t n=27 months after tre atmen t 1 n=2 7 2 n=27 iant io xidants + 1 0 mg pre dnis olone 6.53 ±0.83 3.95±1.32 * 5.43±1.39*† 5.98 ±1.25† iiant io xidants + 100 mg pre dnis olone 6.53 ±0 .83 4.06±1.39 * 5.44 ±1 .42*† 5.99 ±1.29† value s are ex pre ssed as mea ns  sd. * signif ic antly diffe re nt from co ntrol (p< 0.01). † signif ic antly diffe re nt from pre trea tme nt value s (p<0.01 ). n numbe r of s ubje cts ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 20 ta ble (5): body we ight of c ontro l and age matche d alopecic pa tie nts .. group bo dy we ig ht (kg) control n=30 patie nts with alo pec ia pre -tre atmen t n=27 months after tre atmen t 1 n=2 7 2 n=27 iant io xidants + 10 mg pre dnis olone 6 9.69 ±10.79 66 .0 9±12.00 68.88±12 .27 7 1.22±12 .63 iiant io xidants + 100 mg pre dnis olone 6 9.69 ±10.79 66 .7 9±12.76 72.56±12 .62 8 0.88±12 .23 value s are ex pre ssed as mea ns  sd. n numbe r of s ubje cts ta ble (6): se verity of ac ne appe a ra nce in c ontro l and age matche d alope cic pa tie nts . group prese nce of acne control n=30 patie nts with alo pec ia pre -tre atmen t n=27 months after tre atmen t 1 n=2 7 2 n=27 iantioxida nts + 10 mg pre dniso lo ne nega tive ne gative + + iiant ioxida nts + 100 mg pre dniso lo ne nega tive ne gative ++ ++++ se ve rity of the prese nce of a cne dete rmine d by de rmalog is ts . n numbe r of s ubje cts discusion cortic os tero ids are pa rt o f the trea tment of ma ny diso rd ers in which inflamma tion is thought to be c aused by e xcess ive or inappropriate activity of the i mmune sys tem like in alopecia areata (17, 18, 19, 20, 21). giv en in high d oses, co rtic os te ro id drugs red uce infla mma tio n by blocking the action of pros ta glandins re spons ible for triggering the infla mma tory respo nse (16). they also te mpo ra rily depress the i mmune sys tem b y re duc ing the ac tivity of c erta in typ es of white blood cells . the extent of hair loss and the age of the p atie nt a re used to selec t an appropria te trea tme nt for patie nts with a lopec ia a reata (3). for tho se with mo re tha n fi fty perce nt s ca lp hair loss one may c ons ider the use of s ys te mic cortic os te ro ids but the c oncern ab out lo ng-term use a nd s ide effects o f systemic corticosteroids must be taken into co ns ideration. the present study revealed the presence of end oge no us oxidative s tress in b oth groups of patients , as manifes ted by the increased mda ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 21 levels and dec reased gs h conte nts in erythroc ytes . this oxidative stress ma y result from phagocyte s de rived free rad icals a nd the associated lip id pe ro xida tion (22). da ta o f the present study a ls o indica te d that, despite the difference in o ra l pred niso lo ne dose (1 0 vs. 100 mg) be tween the two groups, addition of nu trie nt a ntio xida nts to pred niso lo ne therap y resulted in compa ra ble and significa nt d ecrease in mda lev els and correction of gsh co ntent in blood , as well as simila r improve ment in the ra te o f ha ir growth with less side e ffec t (ac ne, weight ga in a nd ga stro intestina l disturba nces) re gardless the do se of p re dnisolone. previous study in our la b showed that, witho ut antioxida nt the ra py, the effe ct of 1 00 mg prednis olone was more effe ctiv e than lower doses of prednis olone in improving ha ir growth in a lo pecic area ta patients (7). the re fo re , the a dd ition of nutrie nt antioxida nts to corticosteroids attenuate d the negativ e effects o f oxid ative stress on immune sys tem and dec reas ed the nee d fo r high dos e of cortic os te ro id ; thereb y de creased the unwanted side effec ts a ssocia ted with the prolonge d use of hig h doses . free rad icals → affe ct i mmune system → pha go cytos is → incre ase fre e ra dica l ( imp ro bab le action) p ro duc tion antioxida nts prdnis olone an tioxid ants refrences: 1. a.j.pa padop oulos , r.a. schwartz and krys ic ka janniger.alope cia a re ata: eme rging once pts .dermatovenerologica.200 0, vol 9, nu mbe r 3. 2. sharma vk, da wn g, ku mar b. profile of alopec ia a reata in northern ind ia . intern j derma to l 1996; 35: 22 -7. 3. sharma vk, ku mar b, daw n g. a clinical study o f c hild hoo d alopecia area ta in chand igarh, ind ia . ped derma to l 1996; 13: 372-7. 4. safavi kh, muller s a, suman vj , et a l. incide nce o f alopec ia area ta in olmstead county, minneso ta , 1975 through 19 89. mayo clin p roc 1995; 70: 628 -3 3. 5. cos key rj , dra ke la, ho rd insky mk, rosenbe rg ew, solomo n ar, chancho turner ml. guide lines of care for a lo pecia area ta. j aad 19 92; 26:247-50. 6. sha piro j. alopec ia area ta : update on therap y. dermatologic clinics 1 993 ; 11 :3546. 7. al-j aff a.the role of o xidative s tres s in alopecia a reata. msc.thesis.co llege of pharmacy unive rs ity o f baghd ad.2001 . 8. shapiro j . practica l mana ge ment for alopecia a re ata a nd outlo ok for the fu ture .exp-derma to l.19 99 aug; 8 (4 ): 298 . 9. dolecek-r. trea tment o f a lo pec ia with gluc oco rticoids a nd othe r hormonal preparatio ns .parkt-lek; 1969; 4 9(11); 423431. 10. sha rma-vk; mura lid har-s . treatme nt of widesprea d alopec ia a reata in yo ung pa tients with monthly oral cortico s te ro id pluse.pediatr-dermatol.1998 j ul-aug; 15(4):313 -7 . 11. win te r-rj; kern-f ; blizzard-em. prednisolone therapy for alopec ia a reata. arc h-dermatol. 1 976; 2 2: 1549 -9 2. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 22 12. fiedler vc. alopecia areata. a review of therap y, e fficac y, safety, and me cha nism. arch de rma to l 1992 ; 12 8: 1 519-29. 13. fiedler vc. alopecia areata. a review of therap y, e fficac y, safety, and me cha nism. arch de rma to l 1993 j ul; 1 29(7): 908-9. 14. stocks-j a nd dormandy-t.l. the auto oxidatio n o f h uma n red blood ce ll lip ids induced hydroge n peroxide. brit-jhae matol. 1971; 2 0:95-11 1. 15. gilbert-h.s; stump -d.d and ro th-e.f . a method to correct for e rrors caused b y ge ne ra tion o f interfe ring c omp ounds d uring erythroc yte lipid pe ro xida tion. ana lbioc hem.1 984 ; 137:282-286. 16. god in-d.v; woha ieb-s.a; ga rnett-m.e and goumeniouk-a.d. antioxid ant enzy me a lterations in e xpe rime ntal and clinical diabetes . mole. and cellula r-bioch. 1988; 84:22 3-231 . 17. the b ritis h med ical ass ocia tion new guide to medicines a nd drugs, 199 5. 18. price vh. tre atmen t of hair loss. n engl j med 1999 ; 341: 964-73. 19. s chwartz ra, ja nnige r ck. alopec ia area ta . cutis 1997 ; 59 : 238-41 . 20. sha piro j, pric e vh. ha ir re growth. the rape utic a gents . derma to l clin 199 8; 16: 341-56 21. sha rma-vk; gupta -s. tw ic e daily de xame thas one oral p luse in the trea tme nt of e xtens ive alopecia area ta . j-dermatol. 1999 sep; 2 6(9); 56 2-5. 22. alja ff a., hamad i s, wo ha ieb s. the ro le o f oxid ative stress in alop ec ia a reata. iraq i journa l of p ha rma cy.2001 ; vo l 1, no 1:34-45. iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 40 formulation and evaluation of optimized zaltoprofen lyophilized tablets by zydis technique suray a. hazzaa *,1 and shaimaa n. abd-alhameed ** * department of pharmacy, al-anbar health directorate, ministry of health, anbar, iraq ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract “orodispersible tablet” a tablet that is to be placed in oral cavity where it disperses rapidly by saliva with no need for water before swallowing. zaltoprofen (zlp) is one of nsaids which is used in the treatment of rheumatoid arthritis and osteoarthritis as well as to relieve inflammation and pain after surgery, injury and tooth extraction. the present study was aimed to prepare rapidly dissolved lyophilized zaltoprofen tablet with different pharmaceutical excipients and studying the factors affecting pharmaceutical properties like (solubility, disintegration time dt, dissolution, etc.) of tablets. the lyophilized disintegrating tablets (ldts) were prepared using zydis technique by lyophilization an aqueous dispersion of zaltoprofen with a matrix forming agent, gelatin, and a collapse protectant, glycine. in addition to many excipients like pvpk30 was used to improve the in vitro, in vivo disintegration time and dissolution rate, mannitol as bulk forming agent. fourteen formulations were prepared to inspect the variables that affect the disintegration time and dissolution rate. all the formulations were evaluated for their physical appearance, mechanical strength, x-ray diffraction, ftir, dt, and in vitro drug release. the prepared tablets were optimized and formula was subjected to different measured parameters such as disintegration time, drug content, and in-vitro drug release. results obtained from dissolution studies and dt showed that lyophilized disintegrating tablets (ldts) (f8,f10,f12,f13 was 45,37,21 and 17 sec.) respectively ,while(f14) displayed considerably faster in vitro dissolution rate of (zaltoprofen) 3 min. and dt 9 sec. the (lyophilized disintegrating tablets) were also evaluated showing the transformation into amorphous state and absence of interaction of zaltoprofen with the components of the tablets. from visual inspection ,physical strength ,dt and release behavior obtained ,one can conclude that the formulas(f14) which contains zaltoprofen 3.2% ,gelatin3%, mannitol 3%, glycine 1.5%, pvp k30 1.5% was the most suitable one. keywords : zaltoprofen, lyophilization, pvpk30 . " zydis حبوب انسانتوبرفين انمحسنة وانمجففة بانتجميد باستخداو تقنية ال" تصييغ وتقيى سري عبد هساع *،1 و شيماء نسار عبد انحميد ** * طحت االٗبار ، وسارة اُظحت، اُؼزام دائزةهسْ اُظٍذُت . ** كزع اُظٍذالٍٗاث ، ًٍِت اُظٍذُت ، صآؼت بـذاد ، بـذاد اُؼزام. الخالصة ائبت كٔىٌا باٗها اُحبىب اُخً ٓا إ حىضغ بخضىٌق اُلْ كاٗها حخلٌي وح٘خشز بسزػت بىصىد اُِؼاب دوٕ ذٌٌٖٔ حؼزٌق اُحبىب اُ اُحاصت ُِٔاء هبَ إ حبِغ.وٌؼخبز اُشاُخىبزوكٍٖ ٖٓ ادوٌت ٓضٔىػت ٓضاداث االُخهاب اُال سخٍزوٌذٌت أُسخخذٓت ُؼالس اُزوٓاحٍشّ االطاباث وهِغ االس٘إ. اُذراست اُحاٍُت حهذف ُخحضٍز حبىب اُشاُخىبزوكٍٖ سزٌؼت وًذُي ػالس االّ ٓا بؼذ اُؼٍِٔاث اُضزاحٍت و اُذوبإ بطزٌوت اُخضلٍذ باسخخذاّ)سىاؽ طٍذالٍٗت(ٓخخِلت ودراست اُؼىآَ أُؤرزة ٓزَ اُذوباٍٗت,ٓؼذٍ اُخلٌي وححزر اُذواء ػِى ُخضٍٔذ حٍذ حخْ ػٍِٔت اُخضلٍذ ُِٔحِىٍ أُائً ُِشاُخىبزكٍٖ ٓغ اُخىاص اُظٍذالٍٗت ُِحبىب.هذ حْ ححضٍز اُحبىب بطزٌوت اُخضلٍق با ًٔادة ٓحس٘ه ُخلٌي اُحبت وًذُي ٓؼذٍ ػٍِٔت اُذوبإ 63اُضٍالحٍٖ وٓضاد االٗهٍار اٌُالٌسٍٖ باالضاكت اُى اُبىًُ كٍَ٘ باٌزوُذٌٖ ى ُخوٍٍْ ُِؼىآَ أُؤرزةػِى حلٌي اُحبت و ٓؼذٍ اُذوباٍٗت. وأُاٍٗخىٍ ًٔادة ٌٓىٗت ُِشٌَ. اربؼت ػشز طٍـت حْ ححضٍزها ٓغ أُؼاٌ٘ه وا وهذ حْ حوٍٍْ ًَ اُظٍؾ كً ٓا ٌخض شٌِها اُلٍشٌاوي وهىحها أٌٍُاٌٍٍٗه وحلٌي اُحبت وححزٌز اُذواء. اُ٘خائش بؼذ ححضٍز اُحبىب بخوٍ٘ت (zydis) ُحبت ٖٓ اُذواء وٓؼذٍ ححزٌز اُذواء وًاٗج وححسٍٖ اُظٍؾ حْ اخضاػها ُؼذة حؤٍٍاث ٓزَ وهج اُخلٌي وٗسبت احخىاء ا ( اظهزث ُ٘ا ٓؼذٍ سزٌغ ُخحزٌز اُذواء خالٍ اُذهائن 47اُ٘خائش حخضٖٔ إ اُحبىب أُضللت باُخضٍٔذ ُِشاُخىبزوكٍٖ ُِظٍـت رهْ ) ضللت باُخضٍٔذ باسخخذاّ ( رىاًٗ .حْ حوٍٍْ وكحض اُحبت سزٌؼت اُذوبإ ا9ُٔ% ووهج اُخلٌي ُِحبت بِؾ )433اُزالد االوُى وطِج اُى ( واظهزث ححىٍ اُذواء اُى اُظٍـت اُالبِىرٌت اُالٓ٘خظٔت ٓغ ؿٍاب كزطت اُخذاخَ بٍٖ ٌٓىٗاث اُحبت ftirحوٍ٘ت االشؼت اُسٍٍ٘ت و) % 4.8%, ًالٌسٍٖ 6% ,ٓاٗخىٍ 6%, اُضٍالحٍٖ 6.5واُشاُخىبزوكٍٖ.ٗسخ٘خش ٖٓ اُذراست إ اُظٍـت اُخً ححخىي ػِى اُشاُخىبزوكٍٖ % ًاٗج اُظٍـت االكضَ .4.8 63وبىًُ كٍَ٘ باٌزوُذوٕ ى .03نتوبرفين ,تجفيف بانتجميد ,بوني فنيم بايروندين كزا انكهمات انمفتاحية: 1 corresponding author e-mail: suraymaster336@gmail.com received: 21/5/2017 accepted: 22/6/2017 mailto:suraymaster336@gmail.com iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 41 introduction a fast disintegrating or dissolving system or tablet can be defined as a solid dosage form that can disintegrate or dissolve within 30 seconds, in the oral cavity resulting in a solution or suspension without administration of water (1) .the technologies used for manufacturing fast dissolving tablets are freeze-drying, spray-drying, tablet molding, sublimation, tablet compression, and disintegration addition (2) . lyophilization (freeze-drying) is a process in which water is sublimated from the product after freezing at a specific temperature and pressure. lyophilization technique is used in order to improve the dissolution of the given substance and improve the oral bioavailability of the drugs with poor solubility and high permeability (3) . zaltoprofen is bcs class-ii having low solubility and high permeability (4) . zaltoprofen, chemically it is 2-(10,11-dihydro10-oxodibenzo(b, f)thiepin-2-yl) propionic acid, is a derivative of 2-arylpropionic acids(2apa), is one of non-steroidal antiinflammatory drugs (nsaids) and has potent inhibitory effects on acute and chronic inflammation, according to their chemical structure or their selective inhibition of cyclooxygenase cox-1 and cox-2, zaltoprofen is cox-2 inhibitor and selectively inhibits prostaglandin e2 (pge2) production at the sites of inflammation with less adverse reactions on the gastrointestinal tract than other nsaids (5) . figure (1): chemical structure of zaltoprofen (5) materials and methods: materials zaltoprofen was purchased from hyperchemical china, gelatin, was kindly gifted from baghdad college of pharmacy, mannitol, sorbitol ,glycine ,pvp k30 were purchased from m/s provizer pharma company india . the water used was distilled de-ionized water. all other chemicals were reagent grade. determination of melting point the melting point of zaltoprofen powder was measured according to usp method using capillary tube method using electrical melting point apparatus. the tube was dipped in the drug powder closed from one end and placed inside the melting point apparatus, the temperature was increased gradually. the temperature at which the powder liquefied was recorded as the melting point (6) . determination of absorption maxima (λ max) a solution of (10 μg/ml) of zaltoprofen in 0.1 n hcl (ph 1.2), phosphate buffer (ph 6.8), and distilled water were scanned by a uv spectrophotometer from 400-200nm, and λ max of zaltoprofen was indicated for each solution. preparation of standard curves of zaltoprofen: calibration curves of zaltoprofen were constructed in distilled water, 0.1n hc1 (ph 1.2), and phosphate buffer (ph 6.8), separately using uv spectrophotometer (uv-6100 pc). serial dilutions were prepared with different concentrations (4,8,10,12, and 14 μg/ml) from stock solution containing (20μg/ml) zaltoprofen in 0.1n hcl (ph 1.2), phosphate buffer (ph 6.8) and(2,4,5,8,10,12,14 µg/ml) from stock solution containing (20μg/ml) zaltoprofen in distilled water, samples were then analyzed spectrophotometerically for zaltoprofen at its λ max. the determined absorbance was recorded and plotted versus concentration to get a calibration curve. estimation of zaltoprofen saturation solubility solubility studies of zaltoprofen were carried out in distilled water; simulated gastric fluids (sgf) ph 1.2 and simulated intestinal fluids (sif) ph 6.8 were used to study solubility behavior of zaltoprofen. using shake -flask method .saturated solutions were prepared by adding excess of zaltoprofen to the all mentioned liquids in 10 ml tube which were placed in a shaker water bath at 40 rpm for 48 hours at 25 °c. then the samples were filtered through a 0.45 μm millipore filter. the solutions were diluted suitably, analyzed by uv-spectrophotometer at λ max of the drug. three determinations were carried out for each sample to calculate the solubility of zaltoprofen (7) . formulation of zaltoprofen fdts by lyophilization techniques in this study, zaltoprofen, fdts containing gelatin as matrix forming agent, glycine as collapse protectant, sorbitol and mannitol as bulk forming agent and pvpk30 as matrix supporting agent, were prepared by lyophilization technique according to formulae given in table 1.the percentage of excipients used was optimized during the formulation iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 42 process to obtain a withstand and elegant tablet that could be handled with ease. to prepare different batches, all ingredients according to the formula were accurately weighed. gelatin was first dissolved in distilled water at about 40°c stirred on a magnetic stirrer (stuart, u.k.) until a clear phase was obtained, then glycine, pvpk30, mannitol were added separately with continuous stirring until homogenous mixture was obtained then the required zaltoprofen amount was added exactly (0.5 ml) of the resultant solution was poured into each of the pockets of tablet blister pack .the tablets blister pack each contained 10 tablets , then they were then transferred to a freezer at about -22 °c and kept in the freezer over night until complete freezing was established .the frozen tablets were placed in a lyophilizer for 24hr utilizing a vacuum freeze dryer (techsupport, korea) with a condenser temperature of 45°c and pressure (8.6) pascal. the lyophilized tablets were kept away from moisture at room temperature until further investigation was performed (8) . table (1) :composition of different zaltoprofen lyophilized tablets formulas tablets formula material zlp ( g ) gelatin %w/v sorbitol %w/v mannitol %w/v glycine %w/v pvpk30 %w/v f1 3.2 2 f2 3.2 3 f3 3.2 4 f4 3.2 3 2 f5 3.2 3 3 f6 3.2 3 1 f7 3.2 3 2 f8 3.2 3 3 f9 3.2 3 3 1 f10 3.2 3 3 1.5 f11 3.2 3 3 2 f12 3.2 3 3 1.5 0.5 f13 3.2 3 3 1.5 1 f14 3.2 3 3 1.5 1.5 total to make 20ml preliminary screening for optimizing lyophilized tablets: the effect of concentration of matrix forming agent formulas 1-3 were used to study the effect of concentration of the matrix forming agent (gelatin 1%,2%,3%) on the mechanical properties and other parameters on the prepared tablets table 1. the effect of bulking agent type and concentration formulas 4-8 were used to study the effect of the bulking agent type and concentration (sorbitol 2%, 3% and mannitol 1%, 2%, 3%,) on the mechanical properties and other parameters on the prepared tablets table 1. the effect of collapse protectant concentration (glycine) formulas 9-11 were used to study the effect of collapse protectant concentration (glycine 1%, 1.5%, 2%) on the mechanical properties and other parameters on the prepared lyophilized tablets table 1. the effect of matrix supporting agent formulas 12-14 were used to study the effect of matrix supporting agent on the mechanical properties and other parameters on the prepared lyophilized tablets table1. compatibility study of drug with excipients fourier transform infrared (ftir) spectroscopy ftir spectroscopy analysis was done in the range of 4000-500cm -1 (fourier transform infrared system ftir8400 s shimadzu, japan) by mixing the optimum formula ( f14 ) and drug alone, separately with small amount of dry kbr powder , compressed into transparent disc and spectra was recorded .these spectra were determined to ensure there was no interaction between the drug and the excipients which may occur throughout the process (9) . powder x-ray diffraction (xrd) analysis x‐ray diffraction experiments were performed in an x‐ray diffractometer using xray powder diffraction analyzer (6000 xrd, shimadzu, japan), operated with cu k α x iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 43 radiation at 40 k v and 30 ma. the scans were conducted 5◦ to 35◦. diffraction patterns for zaltoprofen powder and for the optimized orally lyophilized tablet were obtained and identification of the samples was carried out (10) . evaluation of lyophilized tablets weight variation twenty zaltoprofen tablets were randomly taken from each batch and the weight of their average weight was determined. then individual tablet was taken and its weight was calculated. that individual weight was compared with average weight. the weights were measured using weighing balance (sartorius balance werke gmbh, germany) (11) . content uniformity three zaltoprofen lyophilized tablets were powdered weighed and transferred into a 100 ml volumetric flask. initially, 10 ml of methanol was added and shaken for 10 minutes. then, the volume was made up to 100 ml with buffer 6.8. subsequently, the solution in the volumetric flask was filtered, 1 ml of the filtrate was suitably diluted and analyzed for drug content at 243 nm using uvspectrophotometer (uv-6100 pc) (12) . in vivo disintegration test the time needed for each prepared tablet to be totally disintegrated in the oral cavity was estimated from six healthy people .all subjects had been notified about the purpose behind the test. the subjects washed their mouth with purified water with distilled water. tablets were put on the tongue and instantly the tongue softly moved and the interval for complete disintegration with -out residue was recorded (13) . in-vitro disintegration test disintegration time was measured in 900 ml artificial saliva (ph 6.8) according to the usp 24 method without disc at 37 ± 0.5°c temperature. the disintegration time of 6 individual tablets were recorded and the average disintegration time was reported (14) . in vitro dissolution test this test is intended to determine compliance with the dissolution request for solid dosage forms taken orally. in vitro dissolution studies of fd tablets were studied using usp xxiii tablet dissolution test apparatus employing a paddle stirrer. 900 ml of ph 6.8 phosphate buffer with 2% brij35 was used as a dissolution medium. the temperature of the dissolution medium is maintained to 37 ± 0.5 ° c .one tablet from each batch was used in each test. 5 ml of the sample of dissolution medium was withdrawn by means of pipette at known intervals of time and the sample was filtered using the whattman filter paper. the volume withdrawn at each interval was replaced with same quantity of fresh dissolution medium. each sample was analyzed for drug release, spectrophotometerically using uv-visible spectrophotometer (uv-6100 pc) after suitable dilutions (15) . results and discussion determination of melting point the measured melting point for zaltoprofen was found to be 139 °c. this result was the same to the data reported ,which reflects the purity of the powder used in the study (16) . determination of λ max of zaltoprofen: the uv scan (10 μg/ml) of zaltoprofen in three different media 0.1n hcl solution, phosphate buffer (ph 6.8) solutions, and distilled water as shown in the figures (1), (2), and (3), respectively in each media the drug showed maximum absorbance peaks at (243 nm) in the all above media as reported by the reference (17) . figure ( 1) uv scan of zaltoprofen in 0.1n hcl solution figure (2): uv scan of zaltoprofen in phosphate buffer (ph 6.8 ) solution iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 44 .. figure (3): uv scan of zaltoprofen in distilled water determination of calibration curves of zaltoprofen: figures ( 4 ), ( 5 ), and ( 6 ) show the calibration curves of zaltoprofen in 0.1n hcl, phosphate buffer (ph 6.8) and distilled water respectively. a straight line was obtained as a result of plotting the absorbance versus concentration. for each media which indicates that it follows beerlambert's law within the concentrations used (18) . figure (4): calibration curve of zaltoprofen in 0.1n hcl figure (5) :calibration curve of zaltoprofen in buffer 6.8 figure (6) :calibration curve of zaltoprofen in deionized water solubility studies the solubility profile of zaltoprofen was determined in various solvents as shown in table (2). the results show that zaltoprofen showed significantly (p˂0.05) higher solubility in phosphate buffer ph 6.8 than in 0.1 hcl and in dw .in addition to that it's known that zaltoprofen is acidic compound with pka of 5 (19) . therefore; the solubility of acidic compound increases as the ph is above pka value, this increase in ph may alter the ratio of ionized to non-ionized species which can be predicted by application of henderson-hassel balch equation (20) . table ( 2 ): solubility of zaltoprofen in various solvents solvents solubility (%w/w) mean ± s.d.* distilled water 0.01346 ± 0.0015 hcl 0.1 n 0.01216 ± 0.0015 buffer 6.8 3.1155 ± 0.264 *s.d. standard deviation from mean. n=3 the effect of concentration of matrix forming agent (gelatin) suitable selection of polymer is the corner stone to get a freeze dried tablets with acceptable mechanical properties and rapid release rates (21) .it was found that the prepared lyophilized tablets based on gelatin as binding agent showed better physical inspections compared with the tablets made from pvp k30 alone which were found to be unacceptable during the manual handling process. so, gelatin had been further optimized as matrix forming agent using 3% w/v with pvp k30 1.5%. in parallel to most of the other dosage forms, even in case of fast disintegrating tablets, the overall description of the mechanism of disintegration include iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 45 weakening of the intermolecular bonds upon penetration of the disintegration medium in the tablet’s excipients resulting in complete disintegration of the tablet (22) .with increase in gelatin concentration (f34%) ,the so formed network is anticipated to become more stable and extensive owing to increase in the fiber cross links and inter chain h-bonds (23) , so consolidated a pernicious effect on the disintegration time of the tablets due to increase in intermolecular attraction between the binder molecules resulting in retardation in disintegration time profile .from other side, beyond a certain concentration, the gelatin matrix becomes so extensive and synonymously less porous that the interaction with disintegration medium resulted in formation of thick cohesive gels that were difficult to disintegrate . this oversight could be more expressed by documented phenomenon of formation of rough three dimensional (3d) gel network of gelatin at high concentration (24) . the decrement in mechanical properties of fdts prepared with 2% gelatin (f1) could be assigned to the fewer number of crosslinks formed between the gelatin strands as the concentration decreases non porous tablet will form. the use of 3% w/w gelatin as a binder showed enhanced mechanical properties of the prepared tablets .thus, incorporation of matrix supporting agent pvp k30 was as a reasonable solution to improve dt as shown in table 3. the effect of type and concentration of bulking agent sorbitol used in (f4,f5) showed unsuitable visual inspection (shape) with hard surface texture of prepared tablets this may explain the combination of gelatin–mannitol was found to be better over gelatin–sorbitol (isomer of mannitol) in terms of tablet shape, appearance, surface texture and disintegration time and it could be attributed to superior hydrophilicity of mannitol over others(sorbitol) (25) . the formulas ( f6,f7,f8) were prepared with different mannitol concentrations ( 1% , 2% , 3%) , according to the study of their physical properties ,the results showed that (f8) have a good physical handling and appearance while (f6 ,f7) failed to pull off from blister packs because of fragility ,then after optimization applied on the (f8) to improve the dt and manual handling to progress forward until reaching a suitable formula . excipients used in lyophilization are materials used to ease freeze drying of various biological components, which are usually inert ingredients like sugars , they are also used to preserve the solid matrix against collapse .freezing system also influences the extent of crystallization and polymorphism of important formulation adjuvant drug, such as mannitol (26) . crystallization is favored in a formulation when mannitol functions as a bulking agent. mannitol can gain some advantages like lyophilization at high temperatures and thus shorten freeze drying cycles, and elegant product without defects caused by material collapse. mannitol used as a stabilizer leads to a system that behaves like a physical mixture, allowing interactions only at the phase boundary. bulking agents are used to give product elegance (i.e., acceptable appearance) as well as adequate cake, mechanical strength to avoid product blow-out. bulking agents simply function as fillers to increase the density of the product cake (27,28) . the effect of collapse protectant concentration (glycine) on the mechanical properties glycine has a polar surface and therefore has a high affinity to water which generate aqueous channels that make the diffusion of the dissolution media into the tablet more quickly ,which enhance the in vivo disintegration of the prepared tablets and subsequently promote the dissolution rate .these results are documented for some amino acids which act as disintegration accelerators .the addition of glycine especially at concentration 1.5% (f10) improved the wetting time in comparison with formula(f9,f11),this due to its polar surface free energy which composed of about 75 % of its component. the polar character of glycine has a power affinity to water and generate aqueous channels that enhancing the tablets wettability (29) . also glycine represents highly water soluble amino acid (25 g /100 ml), the solubility of amino acid in water affects the wetting of tablet, which increased the ability of water penetration into capillary of tablet matrix. a significant increase in dt as the concentration of glycine increased (direct relationship) (f11). so the increment in glycine concentration will deteriorate the dt (145 second) in comparison with formula of lower glycine concentration (f9,f10) 68, 37 seconds respectively, this due to low water holding capacity (lower and slower swelling nature of the powder) (30) . iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 46 table(3):physical appearance ,in vitro , in vivo d.t. and content uniformity for lyophilized prepared tablets formula pull off uniformity physical handling in-vitro d.t. in vivo d.t. content uniformity (%) f1 -ve -ve brittle f2 -ve +ve good 96.7±0.85 f3 -ve -ve hard f4 -ve -ve brittle f5 -ve -ve brittle f6 -ve +ve brittle f7 -ve +ve brittle f8 +ve +ve good 45 57 99.5±0.78 f9 +ve +ve accepted 68 79 97.7±0.84 f10 +ve +ve good 37 42 96.4±0.59 f11 +ve +ve hard 145 f12 +ve +ve good 21 26 99.8±1 f13 +ve +ve good 17 23 100.5±0.89 f14 +ve +ve good 9 12 100.1±0.97 note:pull off means remove the tablet from blister. +ve means easy to pull off the tablet from blister pack, uniform tablet shape. -ve means tablet stick to blister pack(difficult to pull off) , not uniform tablet shape. fourier transformed infrared spectroscopy (ftir) the most widely reported spectroscopic techniques for solid state characterization is ftir. the spectra of pure zaltoprofen powder shown in figure (7 ) and that of the selected lyophilized tablet (f14 ) spectra figure(8) display no significant shifting in the position of the characteristics peaks of the main functional groups ,so these results indicated the absence of the probabilities for interaction between the zaltoprofen and the polymers used in the preparation of lyophilized tablets (31) . figure (7) : ftir spectrum of pure zaltoprofen iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 47 figure (8) : ftir spectrum of lyophilized tablet (f14 ) powder x-ray diffraction analysis (pxrd) of pure zaltoprofen and selected formula (f14) the x-ray diffraction pattern of pure zaltoprofen powder shows characteristics zaltoprofen diffraction peaks at 2θ diffraction angles of 4°, 13.5°, and 20.2° referring to presence of crystalline structure as in figure (9). the diffraction study of optimized zaltoprofen oral lyophilized formula (f14) showed absence of main drug diffraction peaks at (4°, 13.5°, and 20.2° ) with decrement in the intensities of sharp peaks referring to present amorphous form as in figure (10 ) . figure ( 9 ) :x-ray diffraction pattern of pure zaltoprofen powder figure ( 10 ): x-ray diffraction pattern of selected formula (f14) iraqi j pharm sci, vol.26(1) 2017 zaltoprofen lyophilized tablets 48 in-vitro dissolution study the in-vitro drug release characteristics of zaltoprofen lyophilized tablets (f14) were performed in phosphate buffer 6.8 (2% brij 35) as a medium for dissolution .the cumulative ratio of zaltoprofen release in 2 minute (d2) was 81.6% and t80% was 1.96 minute as shown in figure (11). figure ( 11 ) : in vitro drug release profile of( f14) in phosphate buffer ph 6.8 with 2% (brij 35) at 37 ± 0.5°c (results are expressed as mean , n=3) conclusion based on results obtained from the study one can conclude that, zaltoprofen was successfully formulated as lyophilized flash tablet using zydis technique by incorporation of gelatin 3% as a matrix forming agent which give a suitable manual handling, mannitol 3% was the best bulk forming agent compared with the others (sorbitol) ,so gelatin–mannitol combination offered proper manual handling and retained the intactness of odts . in addition to the polar character of glycine has a high affinity to water and generate aqueous channels that enhance the tablets wettability .from all above, f14 was the best formula to prepare zaltoprofen lyophilized tablet. references 1. abdelbary g, eouani c, prinderre p, joachim j, reynier j, piccerelle p. determination of the in vitro disintegration profile of rapidly disintegrating tablets and correlation with oral disintegration. international journal of pharmaceutics. 2005;29-41. 2. 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and aqueous colloid dispersion part ( 1 ) ahmad n.abood *, yehia i. khalil** 1 *de partm en t ph arm ace utics ,co lle ge o f pha rm a cy , un iv ersity of bashra, b ashra-iraq **d epartme nt o f de pa rtm en t p harmaceu tic s, colleg e of p harm ac y, unive rsity ofbag hda d, bag hda d-iraq ab stract naproxen is a non s te roida l anti inflammatory and a ntipyre tic drug which has loca l irritation effe ct on the s tomach, and unplea sant ta ste, beside s bad flowability a nd light se nsitivity .the drug was pre pared a s mic roc aps ules by c omplex c oac ervation method using aca cia-gelatin coa ting materials , and aqueous c olloid polymer dis persion me thod ( acpd ), using e thyl ce llulose and s odium alginate coa ting materials . the res ults indica te d tha t mic rocapsules prepared by 2:1 core:wall ratio is the bes t for both me thods , with a n ave ra ge enc aps ulation efficienc y (75%) and avarege yield (90%) . more ove r the drug releas e was affected mainly by c ore : ra tio ,ph envirome nt a nd method of mic roenc aps ulation . k ey words: na proxe n,microc aps ule , aque aus c olloid polymer dispersion الخالصة مستساغ غير وطعم للمعدة مخدشة اعراض ذو للحرارة وخافض لاللتهابات مضاد ستيرويدى غير عقار هو النابروكسين مجهرية كبسوالت شكل على العقار تحضير تم لقد بالضوء الشديد تاثره الى اضافة التصييغ اثناء جريانه فى وصعوبة اضافة االثيلى والسيلليلوز الجيالتين العربى كالصمغ مختلفة مواد باستعمال المائى واالنتشار المعقد التقوصر بطريقتى وناتج ۷٪ تحميل بمعدل االفضل كانت المغلفة المادة الى اللبية المادة من١ :٢ نسبة ان وجد لقد الصوديوم الجينات الى ونسبة التحضير بطريقة المجهرية الكبسوالت هذه من كبير بشكل العقار بتأثرتحرر النتائج اظهرت كما ٪ ۹۰ بلغ نهائى . البيئى للوسط الهايدروجينى االس ال اضافة المغلفه المادة ال اللبية المادة in troduction the principle of mic ro enc ap sulation is fo rma tio n o f a thin co ating of wa ll materia l around the s ub stance, therefore ma king the pa rtic le s more des ira bl in te rms o f physica l and che mic al p ro pertie s (1,2) , the quantity o f drug inco rpo ra tion a s a core materia l vary from 20 -9 5% (3) na proxe n is a ryl ac etic acid group d eriv ative of non-s te ro id al a nti infla mmatory d rugs wid ely use d in rheumatoid arthritis and ankylo sing sp ond ylitis (4) .the ma in s id e effe cts of this drug a re git disturba nc es a nd bitte r tas te . the go als o f this s tudy are to mask the irritation effect of the a ctive drug to the git, and imp rov e physica l prop erties o f the drug like flowa bility and light se ns itivity experime nta l mater ials : nap ro xen powd er s up plie d by sa marra " drug industery (sdi) ,a cac ia,gelatin,ethyl ce llulos e,so dium algina te ,c alcium chlo ride ,formalde hyd e,from ried el de -ha en ag;see lze,hanno ver, ge rmany all othe r reagents a re of a n ana lytic al gra de . ins trume nts : sa rtorius ba la nc e,type 121 9 mp3 , e le ctrica l stirrer ike werk typ e re16 , (ge rma ny) , ove n , mammert 854 sc hwa ba ch, (ge rma ny) , micros cop e, olympus cx21 tokyo ja pa n,cintra 5 gbc,uv-vidible spe ctome te r, d is so lution ap paratus erweka usp dt6 ha ns en (germany) , water b ath phme ter (w-ge rma ny) . pre par atio n o f n apr oxe n micr oca ps ule s (c omplex c oac er vation) the microc aps ules w ere pre pa re d by incorpo ra ting nap ro xen powd er in 50 ml.o f 2% w/w aca cia s olution previously hea te d to 40c, the n 50 ml. of gelatin so lution 2 %w/w a t 40c was ad ded a nd ma inta ined at 250 rpm stirring spe ed for 50 minute s ,a djus ting the ph of mixture to 4 with fe w drop s o f diluted hcl(0.1m),the formatio n o f mic ro ca psule s can b e watched micro sc opica lly ,then 10 ml. of fo rmaldehyde s olutio n wa s a dd ed w ith continuous stirring fo r 10 minute s, c oo ling in ic e bath , filtered the microc aps ules fo rmed using three portio ns of 100 ml. iso propyl alcohol a nd then the wetted mic ro ca psule s were drie d before the fre e flowing p owd ered mic ro cap sule s ob ta ined (5) . 1corresp ond in g author email ybmma z@ yahoo .com rece ived 2 5-12-20 05 acce pted 2 5-7 -2 006 iraqi j.pharm.sci., vol.15 (2) ,200 6 micro enca psulatio n of na proxen part (1) 18 pre pa ration o f n apr oxe n micr oca ps ules ( acpd me th od ) the d rug was d is pers ed in 50ml. o f 2% w/w aq ueo us so dium algina te s olutio n hea te d to 40c , the n 50ml. of 3 0% w/w ethyl c ellulo se prepa re d at ro om te mpe ra ture wa s ad de d with co ntinous s tirring , (the weight of naproxen ad ded d epe nd on c ore:wall ratio o f microca psule s prepa re d ) .the mixture was allo wed to drop through mod ified se paratory funnel into gently agitate d c alcium chloride (1 % w/w ),the gelled be ad s w ere s ep arated after 2 minutes b y vac cum filteration, rins ed with d is tille d water a nd a llow to dry a t 40c ove r night (6) .all the microc aps ules rep ared by bo th two me thods we re o f 1:2 ,1:1 a nd 2:1 core : wall ratio whic h c orre spo nd s to 33.3 % ,50 % a nd 6 6.6% drug c ontent re sp ectively. dis so lu tion b eha viour f or a ll type s of mic ro ca psule s prep ared under sink c ond itions ,the d is so lution b ehaviour o f nap ro xen was ca rrie d o ut us ing 10 0mg. equiv alent do se in 9 00ml. of different disso lutio n medium ph (1.2,4.2 a nd 6.8 ) a t 37 + 0.5c and c onstant stirring spe ed of 50 rp m , then filtered sa mples we re take n fo r ana lysis at different sp ecifie d time intervals , the ab sorba nce of e ach sa mple wa s determine d s pe ctro pho to metric ally a t 272 ,33 0 ,a nd 2 71 nm. re sp ective ly . results a nd dis cussions pre par atio n of micr oc aps ules : tab le 1. ( a and b ) illus trates the yield perce nt a nd the e nca ps ulatio n efficiency of nap ro xen by diffe re nt p re pa ra tion me thod s and core: wall ratio s, it w as found that coa ce rva tio n method gave 75-85 % yield comp ared with 8 6-93% giv en by acpd metho d ,while e nc aps ulation effic ie ncy wa s 48-82 % a nd 27 -7 0% fo r co mplex c oa cerva tion and acpd metho d , res pec tive ly . ta ble (1)effect of varia tion o f co re to wall ratio of naproxe n micro ca psule s on the microe ncapslation by :-aco mple x co acervatio n me thod mic roe nc ap sulation effic ie ncy drug lo ading % % yie ld microe nca p sula tion y ie ld core to wa ll ratio ac tua l (gm) theore tical (gm) ac tual (g m) theore tical (gm) 81.9 54.6 6 6.6 8 5.4 10 .2 5 12 2 :1 7 5 37.5 50 7 7.5 6 .2 8 1 :1 48.5 16 .1 7 3 3.3 75 4 .5 6 1 :2 baque o us c ollo idal poly me r dispe rs ion me tho d the res ults indicated tha t be st e nca ps ulatio n effic ie ncy was in c omp le x co ace rv atio n co mpared with acpd method ,which may b e attribute d to the ability of po lymer used to encap sula te the drug (6) . on the othe r hand the results reve aled that encap sula tion e fficienc y ap pea re d to b e c ore weight de pendent , higher p erce nt of d rug lo ad ing w as shown with 2 :1 c ore wa ll ra tio the se res ults a re co ns is te nt with the re sults obta ined in c ase of me tronid azol (7) and chlo ra mphe nico l mic ro ca psule s (8). mo reo ve r the a pprec ia ble amounts of microca ps ule gaine d (8 6 93%) b y acpd me thod may b e re fe rred to the c ompa tib ility b etwee n na pro xe n and po lymer use d , s ince bo th o f them are acidic in na ture (9). dis so lu tion be haviour o f micro ca psu le s: figure 1 and 2 s ho ws the re le ase p ro files of nap ro xen from 2 :1 co re wa ll ratio mic ro cap sule s fo r bo th metho ds of prep aratio n at p h 6.8 and 1 .2 re spe ctiv ely. core to wall ratio microe nca p sulation yie ld % y ie ld drug load ing % microe nca p s ulatio n efficie ncy (%) theo retical (gm) actua l (gm th eo re tical (g m) ac tual (gm 2 :1 7 0 46.6 66.6 93 1 4 15 1 :1 6 0 30 50 85 8 .5 10 1 :2 2 7 9 33.3 86 6 .5 7.5 iraqi j.pharm.sci., vol.15 (2) ,200 6 micro enca psulatio n of na proxen part (1) 19 0 20 40 60 80 100 120 time (min) 0 5 10 15 20 25 30 p e rc e n t of d ru g r e le a se 2: 1 core to wall ratio 1:1 core to wall ratio 1: 2 core to wal ratio it wa s fo und that in comple x coa ce rv atio n metho d , the relea se was ; fas te r tha n in acpd metho d at p h 6 .8 , sinc e t5 0% of d rug re le as e to oko ver 8 minutes for firs t metho dc ompa re d with 75 minutes for the later, these res ults are consistent with the res ults ob ta ined b y mic ro enc ap sula tion o f diclofenac s odium (10) and p ro te in (1). the drug relea se at p h 1.2 ; d ecrea se d signific antly fo r bo th me thod (not mo re tha n 30% at first two hours) , this be ha viour may b e attributed to the nature of nap ro xe n diss olutio n at low ph (12 ) . on the othe r ha nd the e ffec t of core wa ll ratio on the relea se o f na proxe n fro m mic ro cap sule s was s hown in figures 3 and 4 , it was s ee n that high significant diffe re nc e (p < 0.00 1) in the p erce nt drug releas e afte r 120 minute s betwee n 2 :1 a nd 1 :2 core wa ll ra tio s (c oac ervation me thod) , the ca use is referre d fo r both drug co ntent and s olve nt pe ntratio n and this in co ns is te nt with the res ults o btaine d by the s tudy of d is solution of diazep am mic ro cap sule s (13) . 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 160 180 200 220 time(min) p e rc e n t o f d ru g r e le a se complex coacervation 2:1 acpd 2:1 0 10 20 30 0 10 20 30 40 50 60 70 80 90 100 110 120 time(min) p e rc e n t o f d ru g r e le a s e complex coacervation 2:1 acpd 2:1 figure (1) : effe ct o f me thod o f microe ncapsula tion o n the re lease o f na proxe n fro m mic roc aps ules in phos pha te buffe r ph 6 .8 . fig ure (2 ): effe ct of me thod o f microe nca ps ulatio n on t he re lease of naproxe n from mic ro capsule s in 0 .1n hcl ph 1.2 . figure (3): effe ct o f va rying c ore -wall ra tios on the re le ase of naproxe n fro m microcaps ules pre pare d by comple x coace rv atio n me thod in 0.1n hcl ph 1 .2 . iraqi j.pharm.sci., vol.15 (2) ,200 6 micro enca psulatio n of na proxen part (1) 20 0 20 40 60 80 100 120 time (min) 0 5 10 15 20 25 30 35 40 p e rc e n t o f d ru g r e le a s e 2: 1 core to wall ratio 1:1 core to wall ratio 1: 2 core to wal ratio 0 20 40 60 80 100 120 time (min) 0 10 20 30 40 50 60 70 80 90 100 p e rc e n t of d ru g r e le a se phosphate buffer (ph 6.8) acetate buffer (ph 4.2) 0.1n hcl (ph 1.2) 0 20 40 60 80 100 120 time (min) 0 10 20 30 40 50 60 70 80 90 100 p e rc e n t o f d ru g r el ea se phosphate buffer (ph 6.8) acetate buffer (ph 4.2) 0.1n hcl (ph 1.2) 0 20 40 60 80 100 120 time (min) 0 10 20 30 40 50 60 70 80 90 100 p e rc e n t of d ru g r e le as e phosphate buffer (ph 6.8) acetate buffer (ph 4.2) 0.1n hcl (ph 1.2) the s ame res ulte s were ob tained from mic ro ca psules prepared by acpd metho d , since 40,24 a nd 20 % o f drug released from mic ro ca psules fro m 1 :2 , 1:1 and 2:1 core wa ll ra tio respec tively . in a n atte mpt to stud y the e ffec t of d ifferent ph-med ium o n the release o f nap ro xe n from thes e microc apsules, figures 5 to 10 were co nstruc te d for both coa ce rvatio n and acpd metho ds . figure (4 ): effe ct of va ry ing core -wall ra tios o n the re le ase o f naproxe n from microca ps ules pre pare d by acpd me thod in 0.1n hcl ph 1 .2 . figure (5): effe ct o f ph on the re lease of naproxe n from 1:2 c ore-wall ra tio microca psule s pre pare d by c omple x c oace rvation me tho d fig ure (6): effect of ph o n the re le ase of naproxe n fro m 1:1 core wall ratio microc aps ules pre pare d by c omple x c oace rvation me tho d. figure (7 ): effect of ph on the re lease of napro xe n from 2:1 co re -wa ll ratio mic ro capsule s pre pa re d by comp le x coace rv atio n me thod. iraqi j.pharm.sci., vol.15 (2) ,200 6 micro enca psulatio n of na proxen part (1) 21 0 20 40 6 0 80 10 0 1 20 1 40 time (min) 0 10 20 30 40 50 60 70 80 90 1 00 p e rc e n t o f d ru g r e le a s e pho sp h ate bu f fer ( p h 6 .8) ace tat e b u ff er ( ph 4.2) 0.1n hc l 0 20 40 60 80 100 120 140 160 180 200 220 time (min) 0 10 20 30 40 50 60 70 80 90 100 p er ce n t of d ru g r el e as e phosphate buffer (ph 6.8) acetate buffer (ph 4.2) 0.1n hcl 0 2 0 40 60 8 0 1 0 0 12 0 1 40 1 60 1 8 0 t im e (m in ) 0 10 20 30 40 50 60 70 80 90 1 00 p e rc e n t o f d ru g r e le a s e p h o sp h at e b u ff e r ( p h 6.8 ) a c et at e b u ff er ( ph 4.2 ) 0 .1 n hc l . the res ults s howe d tha t the re are high s ignific ant differences (p < 0.00 1) in t5 0% d rug re le ase fro m d ifferent c ore wall ra tios p re pa re d b y coa cerva tion me thod at ph 1 .2 a nd 4 .2 in c ompa riso n with p h 6 .8 (microc aps ules pre pa re d by coa cerva tion method a t p h 1 .2 a nd 4 .2 ) the s ame results were re cognize d for the s ame core: wall ratios microc aps ules p re pared b y acpd me thod , the se res ults are the s ame res ults obta ined in the eva luation of s ulfona mid e ,ibup ro fe n (14),a nd me fe na mic a cid microca ps ules (15) . conclusion base d o n the res ults ob ta ined , one may c onclude s the follo wings . a . both comp le x c oa cerva tion and acp d method s are valid for mic roe nc aps ulation of nap ro xen. b . the diss olution be ha viour of naproxen fro m mic ro cap sule s is affecte d by phmedium ,co re :wa ll ratio and prepa ra tion method s. c . the re sults obta ined in this s tudy c ould be use d to formulate naproxen in many mic ro enc aps ulated dos age fo rms . refe renc es 1. anse l ll.c., alle n l.v.j r.pop ovich n.g ., pha rma ce utic al d os age fo rms a nd d rug delivery system . i ipp inco ll williams and wilkins ,8th e dition,200 5 , se ctio n iii , chapter 9,765 ,2 67. 2. s ha rgel l., and re w y ., ap plie d bio pha rmace utic s and pharmac okinelic s 4 th editio n 199 9,chapter 7 ,168 . 3. racz l ., s c.c. chem. , drug fo rmula tio n , 198 9chapte r 4,36 4. 4. usp , xxiii , the united sta le s pha rma co poe ia ,19 95 . 5. curt theis ,asurve y of microe nca ps ulatio n proce ss es , in : simo n benila (ed ) mic ro enc ap sulation metho ds and industrial app lica tions , marce l dakker ,inc .new york ,1 996 . 6. j izomo to h., ka na oka e. a nd s ugita k., gelatin ac ac ia microca psules fo r tra pp ing mic ro o il d ro plets co ntaining lipop hilic drugs and rea dy d is inte gratio n in the ga stro inte stinal trac t , p harmac eutica l re sea rc he s 199 3,10,11 15-11 120 . 7. al-os ta h.a., mic ro enc ap sulation of metro nidazole as solid dos age fo rm , thes is for m.sc . degre e , c olle ge of p ha rma cy , unive rs ity of baghda d , 2 000 . 8. ali w.k., re leas e of chloramp he nicol from the pre pa re d microc ap sules the sis fo r m.s c degre e , co llege of pharmac y , unive rs ity of baghda d 198 9 . 9. elgiba ly i ., sa fwa t s.m . a nd ahme d m.o ., microe nca ps ulatio n of ketop ro fe n us ing figure (8): effe ct of ph o n the re le ase o f naproxe n from 1:2 c ore-wall ra tio mic ro capsule s pre pa re d by acpd me thod figure (9 ): effect o f ph on the re lease of na pro xe n from 2 :1 co re . wa ll ratio microca psule s pre pare d by acpd me thod. figure (10): effe ct o f ph on the re lease o f na proxe n from 1:1 co re wall ratio mic roc aps ules pre pare d by acpd me tho d iraqi j.pharm.sci., vol.15 (2) ,200 6 micro enca psulatio n of na proxen part (1) 22 w/o /w co mplex emulsion technique ,bulletin of p ha rma ceutic al s ciences ,as siut univ ersity , de partment o f indus trial pharmac eutics ,17 feb ruary ,147 – 1 63 ,199 1 . 10. chowd ary k.p and s as try v.ii ., microencap sula tion o f dic lo fnaccomp aris io n of d rug relea se fro m va rious mic ro ca psule s ea stern pharmac is ts 1 997 , 4 0 sep tembe r ,119 122 . 11. gombo tz w.r .and we e s.f .re le ase from algina te matric es , a dv anc ed drug d eliv ery re views ,3 1 may ,267 -285 ,1 998 . 12. aulto n m.e., pharmac eutics the s cienc e of dos age fro m de sign , churrchil liv ing stone ,2 nd editio n ,2 002 . 13. ka mal b.a., some va riab le s a ffe cting ta blette d microca ps ules , m.sc .the sis ,co llege of p harmac y ,baghda d univ ersity ,1 980 . 14. wa ng l. and chui w., microe nca ps ulatio n of ib uprofen and sulfo namide j.of mic ro enc ap sulation 1 995 ,1 2,417 . 15. khalil y,i., micro encap sula tion of glutathion , ac elaminop he n a nd mefena mic acid by organic c oac ervation and a que ous dispe rs io n ph.d. de gree thesis ,college of pha rma cy ,ba ghd ad university ,1 99 9 . iraqi j pharm sci, vol.27(2) 2018 supplements against doxorubicin induced neurotoxicity doi: https://doi.org/10.31351/vol27iss2pp24-31 24 effects of vitamin e and q10 supplementation against doxorubicininduced neurotoxicity in rats manal a. i. al-geam*, 1 and nada n. al-shawi** department of pharmacology and toxicology, college of pharmacy, university of basra, basra, iraq.  department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract doxorubicin (dox) is a chemotherapeutic agent; it is widely used in human malignancies. its long-term use can cause neurobiological side-effects. vitamin e and coenzyme q10 may possess neuroprotective effects. this work was designed to investigate the effect of vitamin e and the coenzyme q10 (coq10) supplementation on neurotoxicity induced by doxorubicin (dox) in rats. forty nine adult rats of both sexes were used in this study; the animals were randomly enrolled into seven groups of 7 rats each. group i: negative control (rats administered corn oil); group ii: vitamin e at a dose of 100mg/kg/d for 3 weeks ; group iii: coq10 at a dose of 50 mg/kg/d for 3 weeks; group iv: positive control (doxorubicin 2.5 mg/kg) every other day for 2 weeks; group v: vitamin e at a dose of 100mg/kg/d for 3 weeks administered prior to doxorubicin at dose 2.5 mg/kg every other day for 2 weeks; group vi: coq10 at a dose of 50 mg/kg/d for 3 weeks administered prior to doxorubicin at dose 2.5 mg/kg every other day for 2 weeks. group vii: coq10 (50mg/kg/day), vitamin e (100mg/kg) for 3 weeks administered prior to doxorubicin at dose 2.5 mg/kg every other day for 2 weeks. on day twenty two of the study, brain of each animal was excised and part of it to be utilized to prepare homogenate for estimation interleukin-1 beta (il-1β), and interleukin-10 (il-10); the other part of brain was used for histological examination. vitamin e and coq10 significantly (p<0.05) decreased il-1beta, and only combination vitamin e and coq10 significantly (p<0.05) increased il-10 and there was an improvement in the histopathological lesions of the brain in group v, group vi and group vii compared to group iv. in conclusion both vitamin e and coq10 may have protective effect against dox-induced neurotoxicity in rats. keywords: vitamin e, coq10, doxorubicin, neurotoxicity, rats. دوكسوروبيسين في لاالمستحدثة بواسطة الدماغ سمية على q10 لومكم( ــه)فيتامين تأثير الجرذان **ندى ناجي الشاويو 1*،منال عبد الخالق الكيم .،العراقبصره ،البصرهجامعة فرع االدوية والسموم ،كلية الصيدلة،* .بغداد،العراق بغداد، جامعة الصيدلة، كلية ،االدوية والسموم فرع ** الخالصة دوكسوروبيسين في العلى السمية العصبية الناجم عن q10ومكمل (ه)كان الهدف من هذه الدراسة هو تقييم تأثير فيتامين جرذاً بالغاً من كال الجنسين حيث قسمت الحيوانات بصورة عشوائية الى سبع مجموعات وفي كل مجموعة 49تم استخدام الجرذان. ملغم/كغم من فيتامين )ه( اعطيت 100:; المجموعة الثانيةاعطيت زيت الذرة ة السلبيةاقبرالمحيوانات. المجموعة االولى: مجموعة 7 عن طريق الفم q10ملغم/كغم من مكمل 50 اعطيت:; المجموعة الثالثةلمدة واحد وعشرون يوما متتالياً واحدة يومياً عن طريق الفم مرة ملغم/كغم من عقار الدوكسوروبيسين 52; المجموعة الرابعة: السيطرة االيجابية )واحد وعشرون يومياً لمدة أحد عشر يوماً متتالياً مرة واحدة ملغم/كغم من فيتامين)ه( عن طريق الفم مرة 100 اعطيت ين المجموعة الخامسة:(سبع جرع كل يومين لمدة أسبوعداخل الصفاق كل يومين لمدة أسبوعين; المجموعة داخل الصفاقملغم/كغم من عقار الدوكسوروبيسين 52قبل يومياً لمدة واح وعشرون يوماً متتالياً ملغم/كغم من 2،5قبل واحد وعشرون يوماً متتالياً ,دة يومياً لمدة واحعن طريق الفم مرة q10ملغم/كغم من مكمل 50 اعطيت السادسة: ملغم /كغم 50ملغم/كغم من فيتامين)ه( و100 اعطيت :كل يومين لمدة أسبوعين. المجموعة السابعة داخل الصفاقعقار الدوكسوروبيسين داخل ملغم/كغم من عقار الدوكسوروبيسين 52قبل واحدة يومياً لمدة واح وعشرون يوماً متتالياً عن طريق الفم مرة q10من مكمل بعد التشريح واستخراج الدماغ .جزء من الدماغ استخدم .في اليوم الثاني والعشرين من الدراسة . كل يومين لمدة أسبوعين الصفاق للفحص النسيجي.استخدم من الدماغ كل حيوان االخر جزءوال 10-إلعداد جناسه النسيج لتقدير مستويات االنترلوكين بيتا واالنترلوكين وكان هناك (p<0.05)10-االنترلوكين( وزيادة p<0.05)االنترلوكين بيتابشكل ملحوظ ا خفض q10 مكملو( ه )فيتامين بينت النتائج تام ة بالمجموعة الرابعة. في الخمقارن السابعةوالمجموعة السادسة، المجموعة الخامسةتحسن في التقطيع النسيجي للدماغ في المجموعة بسين في الجرذان.وتأثير وقائي ضد السمية العصبية التي يسببها عقار الدوكسور q10 مكملو ه، قد يكون لكل من فيتامين .عصبية ، جرذانالسمية ال، دوكسوروبيسين ، q10 مكمل، فيتامين ه: الكلمات المفتاحية 1corresponding author e-mail: manal.ph2008@yahoo.com received: 2/ 6 /2018 accepted: 22/9/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp24-31 mailto:manal.ph2008@yahoo.com http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 supplements against doxorubicin induced neurotoxicity 25 introduction doxorubicin (dox) is a quinine containing anticancer and antibiotic that is used to treat various solid malignancies and lymphomas (1). anthracyclines, including doxorubicin (dox), are the most efficacious anti-cancer drugs available; their use has extended less than ten decades despite numerous adverse effects (2); chemotherapeutic agents, including dox, are responsible for brain tissue injury. the neurobehavioral changes-induced by dox have been indicated by authors via the utilization of rodent models (3). nowadays, it is well-documented that the central nervous system (cns) and the immune system (is) are intimately linked through bidirectional chemical messengers (4). importantly, it has been implicated in neurodegenerative diseases that accompany cns injuries (5). authors reported that patients undergoing chemotherapy for breast cancer have consistently shown their depressed mood and decreased interest in surroundings (6). vitamin e (ά-tocopherol and its derivatives) is a predominant chain breaking lipid-soluble antioxidant and is believed to be the primary free radical scavenger and prevent lipid peroxidation (7). hadi n, yousif n, alamran f, et al 2012 approved that mda contents was significantly reduced after vitamin e treatment this can be explained by that, vitamin e allows free radical to abstract hydrogen atom from antioxidant molecule rather than from poly unsaturated fatty acid thus breaking chain of free radical (8). authors reported that cell death and many neurological deficits can be reduced by antioxidants. coenzyme q10 is considered as a neuroprotective agent that can prevent the cascade of cell death events in order to maintain cellular integration and restore neuronal function.coq10 plays a critical role as an intrinsic free radical-scavenging and antioxidant enzyme that acts against the h2o2induced oxidative damage in cells (9). it directly, inhibits biomolecule oxidation and affects antioxidants in vivo although its structural characteristic allows it to diffuse into the membrane phospholipids bilayer (10). furthermore, coq10 is a key component of the mitochondrial respiratory chain, and it has a fundamental role in oxidative phosphorylation (11). the aim of this study is to evaluate the effect of vitamin e and coq10 on dox-induced neurotoxicity in rats. materials and methods experimental animals forty nine adult albino rats of both sexes, three months old, weighing 160-250gm were used in this study; they were obtained from and maintained in the animal house of the college of pharmacy, baghdad university under conditions of controlled temperature. the animals were fed commercial pellets and tap water ad libitum throughout the experiment period. the study was approved by the scientificand the ethicalcommittees of the college of pharmacy/ university of baghdad. drugs doxorubicin as hydrochloride (50 mg vial) was purchased from ebewe pharma, austria. vitamin e (soft gelatin capsule 400mg) was purchased from geltec private limited, india, and coq10 (soft gelatin capsule 30mg) was purchased from basic nutrition, united kingdom. experimental protocol the healthy rats were randomly divided into seven groups (7animals/group) as follows: group irats orally administration corn oil (1 ml/kg bw/day) alone for 3 weeks. this group served as control (12). group iirats orally administered vitamin e alone at a dose of 100mg/kg/day for 3 weeks (13). group iiirats orally administered q10 at dose of 50mg/kg/day alone for 3 weeks (10). group ivrats ip injected with doxorubicin (dox) every other day at a dose of 2.5 mg/kg for 2 weeks (14). group vrats orally administered 100mg/kg/day vitamin e for 7 days before starting dox injection and continued for 2 weeks; where, it administered 1hr prior to doxorubicin ip injected every other day at a dose of 2.5 mg/kg (13, 14). group virats orally administered 50mg/kg/day coq10 for 7 days before starting dox injection and continued for 2 weeks; where, it administered 1hr prior to doxorubicin ip injected every other day at a dose of 2.5 mg/kg (10, 14). group viirats orally administered 50mg/kg/day coq10 and 100mg/kg/day vitamin e for 7 days before starting dox injection and continued for 2 weeks; where, they orally administered 1hr prior to iraqi j pharm sci, vol.27(2) 2018 supplements against doxorubicin induced neurotoxicity 26 doxorubicin ip injected every other day at a dose of 2.5 mg/kg (10,13,14). twenty-four hour after the end of the treatment duration, each animal was euthanized by diethyl ether. after that, the skull of each animal was broke by surgical scissor then the brain was excised for homogenate preparation and histological examination. preparation and estimation of homogenate biochemical parameters: the preparation of the brain tissue homogenate involved removal of excess blood by rinsing in ice-cold phosphate buffer saline (pbs) (ph=7.4), followed by desiccation using filter paper and then measurement the weight of each brain tissue before homogenization was performed. then each of rats' brain tissue minced to small pieces and put in 15ml plastic test tube containing chilled pbs solution (ph=7.4); where, (tissue weight (g): pbs volume (ml) = 1:9). homogenization was performed by means of cell lab homogenizer in icy condition. after that, the homogenate was centrifuged for approximately 20 minutes at 3000×g. the supernatant was carefully collected and stored at -20 ºc until the time for the determination of interleukin-1beta (il1β), and interleukin-10 (il10)]. homogenate of brain tissue samples were used for the estimation of cytokines [interlukin-1 beta (il-1β), and interlukin-10 (il-10)] levels by automated biochemistry analyzer (elabscience, usa). histological examination after necropsy, brain of each rat was removed and part of it was used for histopathological examination according to the routine method (15) utilizing paraffin sections technique; the fragments were fixed in 10% formaldehyde solution, embedded in paraffin, segmented, and then stained with haematoxyline/eosin. morphological examination of the samples was studied using light microscopy. statistical analysis data were expressed as the mean values, mean± standard error of the mean (sem). unpaired student t-test was used for testing the significant difference between two groups. the statistical significance of the differences among various groups was determined by one-way analysis of variance (anоva). differences were considered statistically significant for pvalue less than 0.05. results effect of vitamin e and coq10 against dox on interleukin-1beta (il-1β) in rats' brain tissue homogenate.table 1 showed that, there were non-significant differences (p>0.05) in levels of il-1β in brain tissue homogenate in group of rats orally administered vitamin e (100mg/kg/day alone for 3 weeks) (group ii) compared to control group (group i). mean ± sem of il-1β levels in brain tissue homogenate were respectively, 54.2±5.8 vs. 49.5±4.2. similarly, there were non-significant differences (p>0.05) in il-1β levels in brain tissue homogenate in group of rats orally administered coq10 (50mg/kg/day alone for 3 weeks) (group iii) compared to control group (group i). mean± sem of il-1β level in brain tissue homogenate were respectively, 51.8±4.4 vs. 49.5±4.2. furthermore, rats ip injected with dox every other day at a dose of 2.5 mg/kg for 2 weeks (group iv) produced significant elevation (p<0.05) in levels of il-1β in brain tissue homogenate compared to control group (group i). mean ± sem of il-1β level in brain tissue homogenate were respectively, 175.1±15.2 vs. 49.5±4.2. table1. moreover, there were significant reduction (p<0.05) in il-1β levels in brain tissue homogenate in groups of rats treated with either -100mg/kg vit e prior to 2.5mg/kg of dox (group v), -50mg/kg coq10 prior to 2.5 mg/kg of dox (group vi), or -100mg/kg of vit e and 50mg/kg of q10 prior to 2.5 mg/kg of dox (group vii) compared to group of rats ip injected with dox every other day at a dose of 2.5 mg/kg for 2 weeks (group iv). mean±sem of il-1β level in brain tissue homogenate were respectively, 100.2±4.3 vs. 175.1±15.2, 110.2±7.9 vs. 175.1±15.2, and 108.5±10.8 vs. 175.1±15.2. table 1. furthermore, table 1, showed that there were non-significant differences (p>0.05) in levels of il-1β in brain tissue homogenate among groups of rats treated with 100mg/kg/d vit e prior to 2.5mg/kg of dox (group v), (50mg/kg coq10 prior to 2.5 mg/kg of dox (group vi), and (100mg/kg of vit e and 50mg/kg of coq10 prior to 2.5 mg/kg of dox) (group vii) compared among each other's. effect of vitamin e and coq10 against dox on interleukin-10 (il-10) in rats' brain tissue homogenate. table 1 showed that there were nonsignificant differences (p>0.05) in il-10 levels iraqi j pharm sci, vol.27(2) 2018 supplements against doxorubicin induced neurotoxicity 27 in brain tissue homogenate in group of rats orally administered vitamin e (100mg/kg/day alone for 3 weeks) (group ii) compared to control group (group i). mean ± sem of il-10 levels in brain tissue homogenate were respectively, 325±11.4 vs. 311.7±11.9. likewise, there were non-significant differences (p>0.05) in levels of il-10 in brain tissue homogenate in group of rats orally administered coq10 (50mg/kg/day alone for 3 weeks) (group iii) compared to control group (group i). mean ± sem of il-10 levels in brain tissue homogenate were respectively, 334.2±13.2 vs. 311.7±11.9. table 1. moreover, rats ip injected with dox every other day at a dose of 2.5 mg/kg for 2 weeks (group iv) produced significant reduction (p<0.05) in the il-10 level in brain tissue homogenate compared to control group (group i). mean±sem of il-10 levels in brain tissue homogenate were respectively, 53.5±4.4 vs. 311.7±11.9 table 1. furthermore, there were non-significant differences (p>0.05) in il-10 levels in brain tissue homogenate in groups of rats treated with either -100mg/kg vit e prior to 2.5mg/kg of dox (group v), or -50mg/kg q10 prior to 2.5 mg/kg of dox (group vi), compared to group of rats ip injected with dox every other day at a dose of 2.5 mg/kg for 2 weeks (group iv). but, there were significant elevation (p<0.05) in il-10 levels in brain tissue homogenate in groups of rats treated with-100mg/kg of vit e and 50mg/kg of coq10 prior to 2.5 mg/kg of dox (group vii) compared to group of rats ip injected with dox every other day at a dose of 2.5 mg/kg for 2 weeks (group iv). table 1 moreover, table 1 showed there were non-significant differences (p>0.05) in levels of il-10 in brain tissue homogenate among groups of rats treated with 100mg/kg vit e prior to 2.5mg/kg of dox (group v), (50mg/kg q10 prior to 2.5 mg/kg of dox (group vi), and (100mg/kg of vit e and 50mg/kg of coq10 prior to 2.5 mg/kg of dox) (group vii) compared among each other's. table 1. effect of coenzyme q10 and vitamin e each alone and in combination on interlukine1beta (il-1β) and interlukine-10 (il-10) levels in brain tissue homogenate after intraperitoneal injection of doxorubicin (dox) in rats. each value represents mean ± standard error of means (sem). group i: control (corn oil); group ii: vitamin e (100mg/kg/day); group iii: coenzyme q10 (50mg/kg/day); group iv: doxorubicin (2.5mg/kg); group v: vitamin e (100mg/kg/day) prior to doxorubicin (2.5mg/kg): group vi: coenzyme q10 (50mg/kg) prior to doxorubicin (2.5mg/kg): group vii: vitamin e (100mg/kg) and coenzyme q10 (50mg/kg) prior to doxorubicin (2.5mg/kg). *= significantly different (p<0.05) with respect to the control group. values with non-identical small letters superscripts (a, b, c and d) are significantly different (p<0.05) using unpaired student t-test. values with non-identical capital letter superscripts (a) are non-significantly different (p<0.05) among (v, vi and vii) groups using anova. histopathological examination of rats' brain tissue rats orally administered corn oil at a dose of 1ml/kg (group i), 100mg/kg of vit e (group ii) and 50mg/kg coq10 (group iii) each for 3 weeks showed normal brain section, pyramidal cell showed open face nuclei and basophilic cytoplasm , smaller neurological cell, and blood capillary were scattered between neuron. (figure 1,a,b,c respectively). treatment group n=7 type of treatment il-1beta (pg ̸ ml) for homogenate of rat brain (mean±sem) il-10 (pg ̸ ml) for homogenate of rat brain (mean±sem) i negative control/corn oil 49.5±4.2 311.7±11.9 ii vit e 100mg/kg 54.2±5.8 325±11.4 iii q10 50mg/kg 51.8±4.4 334.2±13.2 iv positive control / dox 2.5 mg/kg) 175.1±15.2*a 53.5±4.4*a v 100mg/kg vit e prior to 2.5mg/kg of dox 100.2±4.3ba 74.5±4.3aa vi 50mg/kg q10prior to 2.5 mg/kg of dox 110.2±7.9ca 84.2±8.7aa vii 100mg/kg of vit e and 50mg/kg of q10 prior to 2.5 mg/kg of dox 108.5±10.8da 104.2±15.3ba iraqi j pharm sci, vol.27(2) 2018 supplements against doxorubicin induced neurotoxicity 28 histopathological changes in animals brain injected with ip dose (2.5mg/kg) of dox characterized by neuronal degeneration and with frequent nuclear pyknosis ,irregular darkly stained cells with pyknotic nuclei and surrounded with halos arrows were prominent (figure 1-d) compared with brain section of rats administered corn oil (group i, negative controls), group ii (100mg/kg of vit e) and group iii 50mg/kg coq10; where normal appearance were observed in rats' brain section . sections of rats' brain orally administered 100mg/kg bw of vit e for 3 weeks prior to ip dox every other day for 2 weeks at a dose of (2.5 mg/kg bw) (group v) showed congestion of blood vessels, and darkly stained nucleus surrounded with halo while other section of rats' brain of the intended group showed normal brain architecture, there weren’t0 neurodegenerative, and hyper cellularity (figure 1-e). additionally, brain section of animals orally administered coq10 at a dose of 50mg/kg bw for 3 weeks prior ip injection of dox every other day for 2 weeks at dose (2.5 mg/kg bw) (group vi) revealed congestion of blood vessels and, darkly stained nucleus surrounded with halo while other section of rats' brain of the intended group showed normal brain architecture, there weren’t neurodegenerative, and hyper cellularity (figure1-f). as well as, brain section of animals orally administered combination of coq10 and vit e at a dose of 50mg/kg and 100mg/kg bw, respectively for 3 weeks prior ip injection of dox every other day for 2 weeks at a dose (2.5 mg/kg bw) (group vii) showed congestion of blood vessels, and darkly stained nucleus surrounded with halo; while other section of rats' brain of the intended group showed normal brain architecture; there weren’t neurodegenerative, and hyper cellularity (figure 1g). figure 1. histopathological section of brain in various experimental rats' groups; (haematoxyline and eosin; x40). a: group i (negative control (corn oil);b: group ii (vit e 100mg/kg);c: group iii (q10 50 mg/kg);d: group iv (positive control (dox 2.5 mg/kg);e: group v (vit e100mg/kg prior to a ip dose of dox 2.5 mg/kg ;f: group vi (q10 50mg/kg prior to ip dose of dox 2.5 mg/kg);g: group vii(vit e 100mg/kg, and q10 50mg/kg prior to ip dose of dox 2.5mg/kg)negative control group, group ii, and group iii showed normal brain section. positive control group characterized by shows marked neuronal degeneration with frequent nuclear pyknosis , and irregular darkly stained cells with pyknotic nuclei and surrounded with halos arrows were prominent( red arrow) the lesion in groups v,vi,vii revealed mild congestion of blood vessels ( black arrow) with some appear with darkly stained nucleus surrounded with halo( blue arrow) g f e c b d a iraqi j pharm sci, vol.27(2) 2018 supplements against doxorubicin induced neurotoxicity 29 discussion toxicity is the major factor hindering dox treatment. the impairment of neurogenesis and increased neural apoptosis in the limbic brain regions, including the prefrontal cortex and hippocampus, is considered as one of the leading causes of depression. it was reported that dox-mediated generation of free radicals in the brain tissues increases lipid peroxidation and protein oxidation, and alters the antioxidant defense system, eventually leading to neuropsychological changes (16, 17). moreover, increased generation of superoxide anions induced by dox may elevate the level of circulating tumor necrosis factor-alpha (tnf-α) which can directly pass blood brain barrier (bbb), and activate glial cells to initiate the local production of pro-inflammatory cytokines which exacerbate the oxidative stress and neural apoptosis (18). in addition, many inflammatory mediators such as tnf-α and nuclear factorkappa b (nf-κb) have been shown to be critically involved in neuroinflammation both in animal models and in patients undergoing chemotherapy (19). the current study revealed that dox produced significant (p<0.05) elevation in il-1β level in brain tissue homogenate compared to negative control group; the results are in agreement with the study of others (20). pro-inflammatory cytokines such as il-1β, il-6, tnf-α, nf-κb, and inos have been demonstrated to induce abnormal behaviors, such as decreased locomotor activity, exploration, and depression (21). furthermore, dox provoked generation of tnf-α and subsequently caused the activation of nf-κb and inos and increased the expression of genes required to control infection and injury, such as il-1β and il-6, indicating severe inflammatory conditions in the brain (22). it has been reported that il-10, a pleiotropic cytokine, is endogenously produced by activated immune cells including t cells, b cells and macrophages (23). it mainly drives a regulation of a variety of anti-inflammatory processes (24). in the brain, il-10 is expressed by monocytes, astrocytes and microglia (25) as well as by neurons. authors found that il-10 expression was down regulated in the substantia nigra of patients with parkinson's disease (pd) (26). moreover, researchers reported that osmotic pump infusion of il-10 into the substantia nigra can protect against lipopolysaccharide (lps)-induced cell death of dopaminergic neurons, with a corresponding reduction in the number of activated microglia, suggesting that the reduction in microgliamediated release of inflammatory mediators may contribute to the anti-inflammatory effect of il-10. however, other researchers established that il-10 reduced lps-induced neuronal loss in either the presence or the absence of glial cells; and demonstrated that il10 inhibited lps-induced glial activation by down regulation of pro-inflammatory mediators and up regulation of neurotrophic factors. a potential therapeutic strategy for pd is to limit development to inflammatory response (27); this is in agreement with this study; where, dox significantly (p<0.05) reduced level il-10 (group iv) compared to group control (group i). in the current study, brain sections of dox-treated animals under light microscope showed neuronal degeneration with frequent nuclear pyknosis, irregular darky-stained cells with pyknotic nuclei that are surrounded with halos arrows were prominent in figure 3-18d; these findings are coinciding with the works of mohamed, karam and amer , 2011 (14). furthermore, in the present study, vitamin e and coq10 administered at a dose of 100mg/kg and 50mg/kg, respectively prior to ip dose of dox 2.5mg/kg (groups v, and vi), significantly (p<0.05) lowered level of il-1β compared to positive control group (dox-treated rats). also combination of vit e and coq10 (group vii) significantly reduced il-1β. the results of this study are in line with those of others that demonstrated the dual effects of vitamin e on oxidative damage and proinflammatory cytokine (il-1β, il6 and tnf-α) production, resulting in an effective antiinflammatory response (28). it further provided evidence of the antiinflammatory role of vitamin e (29). this study is also in parallel with other research that indicated that coq10 at low concentrations can block il-1 production that in turn can affect the inflammatory mediators, pge-2 and il-6. pge-2 is one of the most significant inflammatory mediators produced in the body. it is made in response to a variety of stimuli in a wide variety of cell types. elevated pge-2 has been associated with a wide number of inflammatory diseases including alzheimer, stroke, and cancer, (30). at the same time, vitamin e and coq10 cause elevation in the level of il10 (p>0.05) but non-statistically significant. however combination of vit e and coq10 significantly (p<0.05) elevate level of protective il-10 (group vii) compared to group iv; this may be due to iraqi j pharm sci, vol.27(2) 2018 supplements against doxorubicin induced neurotoxicity 30 insufficient dose (50mg/kg) of coq10 to cause significant elevation in level of il10 according to other study that indicated that coq10 assisted in boosting of antiinflammatory cytokine, il10, which happened to be consistent with greater dose of coq10 (31). moreover, there were improvement of the histopathological lesions of the brain in groups v, vi, and group vii rats compared to positive control group iv. in conclusion, vit e and coq10 may have protective effect against dox-induced neurotoxicity in rats as demonstrated by the evaluation of neurotoxicity biomarkers and histological examination. to the best of our knowledge, this is the first study that examines the effects of vit e and coq10 at doses 100mg/kg, and 50mg/kg respectively each alone and combination each prior to dox on the brain. acknowledgments this article was abstracted from ph.d. thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the authors gratefully thanks the college of pharmacy, basra university, and staff college of veterinary medicine, basra university. references 1. kavazis an, morton ab, hall se, et al. effects of doxorubicin on cardiac muscle subsarcolemmal and intermyofibrillar mitochondria. mitochondrion 2017; 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can j surg, 54, :5 29. nazrun a , norazlina m, ,and. nirwan s .review article the antiinflammatory role of vitamin e in prevention of osteoporosis, advances in pharmacological sciences;2011:(7)2012-142702 30. plummer na, hensby cn, black ak, greaves mw. prostaglandin activity in sustained inflammation of human skin before and after aspirin. clin sci mol med 1977; 52: 615–20. 31. j. mayer ,b . rau ,f .gansauge ,and h. g. beger ,“inflammatory mediators in human acute pancreatitis: clinical and pathophysiological implications” , gut . 2000 ; 47(4) :546 –552 . iraqi j pharm sci, vol.20(1) 2011 metronidazole pharmacokinetic in diabetic 14 serum level profile and pharmacokinetic parameters of single oral dose of metronidazole in type ii diabetic patients hayder a. kurji * , mowafaq m. ghareeb ** , munaf h. zalzala *** , ahmed t. numan *** and saad a. hussain ***,1 * department of pharmacy, ministry of health, baghdad, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. *** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad,iraq. abstract many pathophysiological processes can affect the pharmacokinetic properties of drugs in people with diabetes. the present study was deigned to evaluate the influence of diabetes mellitus (dm) on the pharmacokinetic parameters of metronidazole administered as single oral dose. twelve healthy volunteers and twelve diabetic patients were enrolled in the present study. on day 1, a single oral dose of metronidazole 500 mg was administered orally to all participants at 9:00 am after a 10-hour fasting. over the following 48 hours, blood samples were taken at frequent intervals and serum metronidazole concentrations were measured by a high-performance liquid chromatography method for assessment of pharmacokinetics of metronidazole. the data indicated that maximum serum concentration (cmax) and kelim were significantly decreased (p<0.05) in diabetic patients compared with that reported in healthy subjects (25.73% and 31.51% respectively). meanwhile, the values of time to reach maximum peak (tmax), auclast, auctotal, and half life (t½) were significantly increased (p<0.05) compared with those reported in healthy subjects (20.69%, 33.65%, 30.13%, and 20.689% respectively). in conclusion, diabetes mellitus affects some of the pharmacokinetics values of orally administered metronidazole. key words: diabetes mellitus , metronidazole, pharmacokinetic, serum level الخالصة اٌ نكثٛز يٍ انحاالتث انًزياٛت حار ٛز ٔاياى حهاٗ زعٛات دعاة اي ٔٚات لاٙ ض،اى انًازٚة ٔدُاال حهاٗ نا حاى يٍ انًعزٔف يهغى حٍ طزٚق انفاى 055حر ٛز انُٕع انثالَٙ يٍ ا ان،كز٘ حهٗ يعالٚٛز زعٛت اندٔا نعقالر انًخزَٔٛداسٔل حصًٛى ْذِ اندراست نخقٛٛى يهغااى 055شخصااال يااٍ اي.ااحال لااٙ انٛاإو ائل حااى أح ااال 21ياازٚة دااالنُٕع انثااالَٙ يااٍ ا ان،ااكز٘ ٔ 21أضزٚااج اندراساات حهااٗ انًشاالرعٍٛ لاٙ ان،االحت انخالساعت .ابال ال دعاد حشاز ساالحالث ياٍ انصاٛالو ٔ ا ل يخزَٔٛداسٔل عجزحت يُفز ة حٍ طزٚق انفاى اناٗ ضًٛا ان،الحالث انثًالَٛت ٔايردعٍٛ ان قت حى سحب حُٛالث يٍ اندو ل لخزاث يحد ة ٔقٛالس ي،خٕٖ انًخزَٔٛاداسٔل لاٙ يصام انادو دٕاسا ت ٔ cmaxد ح،ابب لاٙ فاة قًٛات قاج دارٌ انًاز ٔحى ا خ،اال يعاالٚٛز زعٛات انادٔا ا ل ْاذِ انفخازة أنٓازث انُخاال hplcحقُٛت kelim يقالرَات دالي.احال دًُٛاال أ ث اناٗ سٚاال ة قاٛىtmax auclast auctotal ٔt1/2 ٘ٔحهٛاّ ًٚكاٍ ايساخُخالأ دارٌ نادا ان،ااكز انفى خر ٛزاث انجدٚت حهٗ يعالٚٛز زعٛت اندٔا نعقالر يخزَٔٛداسٔل حُديال ٚع ٗ عجزحت يُفز ة حٍ طزٚق دعة انـ introduction many pathophysiological processes can affect the pharmacokinetic properties of drugs in people with diabetes (1) . patients with diabetes have higher rates of cardiovascular, renal, gastrointestinal, neurological, and thyroid diseases and ophthalmological complications compared with individuals without diabetes. all may increase the chance of having drug-disease interactions (2) . some physiological disorders such as gastroparesis, decreased plasma albumin level, elevated plasma free fatty acid level, glycosylation of plasma proteins and changes in the hepatic microsomal cytochrome p-450 (cyp) contents were reported to occur in diabetes mellitus patients (3) ; these changes could alter the pharmacokinetics and hence the pharmacodynamics of drugs in such patients (4) . the extent of absorption of metoclopramide administered orally to diabetic patient with gastroparesis fell within the range of values reported in healthy subjects (5,6) glipizide was found to be completely absorbed when administered to patients with type ii diabetes mellitus, just like in non-diabetic subjects (4,7) . in contrast, absorption of tolazamide was 26% slower in diabetic patients with asymptomatic autonomic neuropathy than in healthy subjects (8) metronidazole is used widely in diabetic patients for treatment of diabetic foot infections (dfis). 1corresponding author email : saad_alzaidi@yahoo.com received : 27/9/2010 accepted : 29/12/2010 iraqi j pharm sci, vol.20(1) 2011 metronidazole pharmacokinetic in diabetic 15 there is a wealth of information regarding the pharmacokinetic parameters of metronidazole in healthy subjects; however, limited information is available about this issue in diabetic patients. since the metronidazole is often used to treat polymicrobial infections in diabetics (9) , the influence of the disease processes and consequent complications on the pharmacokinetic parameters of drugs, including metronidazole, should be systematically evaluated. the present study was deigned to evaluate the influence of diabetes mellitus on the pharmacokinetics of metronidazole administered as single oral dose. subjects and methods twelve healthy volunteers and twelve diabetic patients were enrolled in the present study; all have an age of 55 ±10 years and with body weight range for each of 50 ± 5.0 kg and 75.0 ± 10.0 kg respectively. all healthy volunteers show a normal medical history and revealed no pathological abnormalities on clinical and biochemical examination. meanwhile, all patients were selected for having type ii diabetes mellitus for at least 5 years and have been treated with single daily dose of glibenclamide 5mg daily and metformin 500 mg three times daily. all patients had serum transaminase concentrations less than twice the upper limit of the laboratory reference range and a normal serum creatinine (<120 mmol /l). written informed consent was obtained from each subject and the clinical protocol was approved by the human ethics committee of the iraqi ministry of health. all subjects were nonsmokers and were instructed not to drink caffeine or alcohol containing beverages for at least 10 hours before and during the study day. the study was performed according to an open, randomized clinical study design. following a 5-day screening period, healthy volunteers and patients with type ii diabetes were enrolled in the study. on day 1, a single oral dose of metronidazole 500 mg (flagyl ® aventes) was administered orally to all participants at 9:00 am after a 10-hour fasting. blood samples were taken at zero-time and at frequent intervals over a period of 48 hours following the administration of metronidazole (0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 12.0, 24.0, and 48.0 hours) and serum metronidazole concentrations were measured by a high-performance liquid chromatography method for assessing metronidazole pharmacokinetics. stock solution of a metronidazole (reference standard) (1mg/ml) was prepared by dissolving 100 mg in 100 ml of methanol. working standard solutions were prepared from the stock solution by sequential dilution with methanol to yield final concentrations of 1.5, 15, 75, 150, 225, and 300 µg/ml. samples for the preparation of standard curve were prepared by mixing blank serum (specially prepared for this purpose) with different concentrations of standard metronidazole solution to get the final required serum metronidazole concentrations (0.15, 1.5, 7.5, 15.0, 22.5, and 30.0 µg/ml). after precipitation of protein by addition of 10% zinc sulfate, samples were vortexed, placed in refrigerator for 15 min and centrifuged at 3000 rpm for 10 min and 200 µl of supernatant was injected into the hplc column for determination of serum levels (10) . the analyses were performed using hplc system composed of a smart line pump 1000 and smart line u.v. detector 2500 connected to smart line manager 5000; the separation was performed on hplc column (eurospher-100 c18; 5 µm, 250 x4.5 mm). drug analysis data were acquired and processed using class-vp (v.6.2) software running under windows 98 on a pentium pc. the mobile phase consists of methanol: water in a ratio of 25:75 at a flow rate of 1 ml/min. the wave length was set at 276 nm (11) . the results were expressed as mean ± sd. the data were statistically evaluated utilizing un-paired student's t-test. values with p<0.05 were considered significantly different. calculation of the pharmacokinetic parameters is performed utilizing the computer software kinetica pk–pd analysis version 5.0 (microsoft-programs). results the raw data for each group was presented in tables 1 and 2. the effects of diabetes mellitus (as a disease) on the absorption of metronidazole were shown in figure 1 and table 3. the data in table 3 indicated that the value of maximum serum concentration (cmax) and kelim were significantly decreased (p<0.05) in diabetic patients compared with that reported in healthy subjects (25.73% and 31.51% respectively). meanwhile, the values of time to reach maximum peak (tmax), auclast, auctotal, and half life (t½) were significantly increased (p<0.05) compared with those reported in healthy subjects (20.69%, 33.65%, 30.13%, and 20.689% respectively). iraqi j pharm sci, vol.20(1) 2011 metronidazole pharmacokinetic in diabetic 16 table 1: the pharmacokinetic parameters of orally administered 500 mg metronidazole in healthy subjects. t1/2 hr kelim hr –1 auctotal µg hr ml –1 auclast µg hr ml –1 tmax hr cmax µg/ml healthy volunteers 7.52 0.092 52.94 52.36 1.5 7.2 v1 11.99 0.058 49.37 46.90 1.5 7.16 v2 8.09 0.086 45.25 44.57 1.5 7.8 v3 8.39 0.083 47.39 4.66 1.5 7.86 v4 9.15 0.076 50.64 49.49 1.0 7.1 v5 9.23 0.075 44.46 43.55 1.5 7.26 v6 7.94 0.087 41.25 40.71 1.5 8.2 v7 14.86 0.047 49.58 45.0 1.5 7.56 v8 9.45 0.073 43.06 42.07 1.5 7.65 v9 13.67 0.05 79.04 72.59 1.5 7.96 v10 10.56 0.072 49.60 43.0 0.5 6.58 v11 9.5 0.073 51.0 45.38 2.5 8.58 v12 12 12 12 12 12 12 n 10.03±2.57 0.073±0.02 50.3±10.73 44.19±16.63 1.45±0.16 7.58±0.38 mean table 2: the pharmacokinetic parameters of orally administered 500 mg metronidazole in type ii diabetic patients. t1/2 hr kelim hr –1 auctotal µg hr ml –1 auclast µg hr ml –1 tmax hr cmax µg/ml diabetic patients 13.02 0.053 89.21 83.14 2.0 5.85 d1 19.91 0.034 54.60 46.31 2.0 6.21 d2 19.66 0.054 72.61 67.84 2.0 5.9 d3 13.58 0.051 72.88 67.46 2.0 4.8 d4 16.56 0.041 79.34 69.98 1.5 5.2 d5 13.97 0.050 68.49 62.84 1.5 5.67 d6 13.59 0.051 70.13 64.78 1.5 5.6 d7 10.30 0.067 64.63 62.37 2.0 5.86 d8 13.48 0.051 77.7 71.44 2.0 5.9 d9 13.90 0.050 68.13 62.69 1.5 5.29 d10 15.69 0.060 72.88 66.79 1.5 6.75 d 11 13.91 0.040 70.66 64.99 1.5 4.5 d12 12 12 12 12 12 12 n 14.8±3.03 0.05±0.01 71.77±9.26 65.89±9.24 1.75±0.16 5.63±0.38 mean table 3:the pharmacokinetic parameters of orally administered 500 mg metronidazole in type ii diabetic patients compared to healthy subjects. pharmacokinetic parameters healthy subjects diabetic patients cmax (µg/ml) 7.58 ± 0.38 5.63 ± 0.42* tmax (hr) 1.45 ± 0.16 1.75 ± 0.01* auclast (µg hr ml –1 ) 44.19 ± 16.63 65.89 ± 9.24* auctotal (µg hr ml –1 ) 50.30 ± 10.73 71.77 ± 9.26* kelim (hrˉ 1 ) 0.073 ± 0.02 0.05 ± 0.01* t½ (hr) 10.03 ± 2.57 14.42 ± 3.03* values are presented as mean ± sd; * significantly different compared to healthy subjects (p<0.05). iraqi j pharm sci, vol.20(1) 2011 metronidazole pharmacokinetic in diabetic 17 figure 1: cp-time profile of metronidazole after 500 mg single oral dose in healthy subjects and patients with type ii diabetes mellitus. discussion the present study showed that the pharmacokinetics of the metronidazole following administration of a single 500 mg oral dose are different in patients with type ii diabetes than in healthy subjects. patients with typeii diabetes exhibit significant decrease in cmax and auc compared in healthy volunteers. the significant differences in metronidazole pharmacokinetic parameters between patients with typeii diabetes and healthy volunteers indicates that the presence of diabetes had notable effects on the rate (tmax, kelim) or extent (auc, cmax) of absorption or elimination of metronidazole (kelim, t½) of the drug. recent evidence suggests that hyperglycemia resulting from poor gylcemic control may contribute to disturbances in gastric function in patients with diabetes (12) . other physiological changes associated with type 2 diabetes (such as alteration in renal and hepatic function and plasma protein binding) (12,13) would be expected to alter the pharmacokinetics of metronidazole, because metronidazole was completely absorbed after oral intake, less than 20% bound to plasma protein and extensively metabolized in the liver (14) . twenty to 30% of diabetics develop abnormal gastric motility, resulting in disordered gastric emptying or gastroparesis (4) . although the etiology of altered gastric motility remains obscure, many factors appear to be important including poorly controlled diabetes, and others (15) . absorption of many orally administered drugs may or may not affected by the presence of diabetes; the extent of absorption of metoclopramide administered orally to diabetic patient with gastroparesis fell within the range of values reported in healthy subjects (4,5) . meanwhile, absorption of tolazamide was 26% slower in diabetic patients with asymptomatic autonomic neuropathy than in healthy subjects (4) , and a 26% decrease in the extent of absorption of orally administered ampicillin was reported compared with nondiabetics controls (16) . therefore, it appears that diabetes can influence the gastrointestinal absorption of drugs, but the extent of influence of the disease on drug absorption may depend on the severity, duration and type of the disease (17) . uncontrolled diabetes may be associated with changes in drug metabolism. diajani et al. demonstrated a reduced rate of conversion of phenacetin to acetaminophen, and its subsequent conjugation was also impaired (4,18) . once the diabetes was controlled with insulin, metabolism became normal and subsequent withdrawal of insulin once again impaired metabolism. the finding of morison and hawks worth support these findings; where glucuronidation was deficient in microsomes prepared from streptozotocin– indauced diabetes in male rats, and this defect was eliminated by insulin treatment (18,19) . the liver is the main site of metabolism, and this accounts for over 50% of the systemic clearance of metronidazole. the two principal pathways of metabolism of metronidazole are oxidation and glucuronides (14) . diabetes is associated with an increased frequency and variety of hepatic histopathologic lesions. the most common lesion seen is an increase in liver glycogen demonstrated both at autopsy and in biopsy material. this accumulation of glycogen produces a clear appearance in the cytoplasm and vacuolization of the hepatocyte nuclei and has been reported in up to 80% of both type i and type ii diabetic patients. increased glycogen infiltration appears to be an associated consequence of exogenous insulin, resulting in hepatocyte accumulation of glycogen. this accumulation results in hepatomegaly, and could theoretically result in impaired hepatic elimination of some drugs. the metabolism of caffeine is decreased in diabetic patient while the clearance of paracetamol is decreased in diabetic patients type ii (17) . these evidences indicate that diabetes may affect biotransformation of metronidazole, which may alters the plasma levels and lead to increase its t1/2. also, this may explain why kelim of orally administered metronidazole in diabetic patients less than in normal subjects. in conclusion, the pharmacokinetic parameters of metronidazole absorption after oral administration were altered in diabetic patients compared to healthy iraqi j pharm sci, vol.20(1) 2011 metronidazole pharmacokinetic in diabetic 18 subjects, and calculation of doses in this situation may be considered. acknowledgment this article was abstracted from msc theses submitted to the department of clinical pharmacy, college of pharmacy, university of baghdad. the authors gratefully thank university of baghdad for supporting the project. references 1yu dt, peterson jf, seger dl, gerth wc, bates dw. frequency of potential azole drug-drug interactions and consequences of potential fluconazole drug interactions. pharmacoepidemiol drug saf 2005; 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10(4):420-433. introduction iraqi j pharm sci , vol.18 (suppl . ), 2009 quinolone’s cytotoxic study 13 preliminary cytotoxic study of some novel furo-2-quinolone compounds nohad a.alomari *.1 , adnan o. omar ** and iklas m. taher ** *college of pharmacy , university of mosul , mosul , iraq **college of science , university of mosul , mosul , iraq abstract in this research, new series of furo-2-quinolone [fq] compounds have been synthesized. these novel [fq] compounds were prepared from coumarin derivatives (furocoumarins: psoralen and isopsoralen).identifications of these fq compounds were performed by using infrared spectrum (i.r), ultraviolet spectrum (u.v) and nuclear magnetic resonance spectrum (h 1 -nmr) besides some physical data. the cytotoxic screening involves ;using hep-2 cell line which gave differential responses against tested compounds : 4,6 dimethyl furo[2, 3-g] coumarin (c1), 1-(2`, 4`, dimethoxy benzylideneimino)-2,6-dimethyl furo [2, 3-g] quinoline-2-one (c3) and the angular psoralen of the same derivative with the chemical name; 1-(2`,4`-dimethoxybenzylidenimine)-4,8,9-trimethyl furo[3,2-h] quinoline-2-one (c3a).these preliminary studies using different cell lines in addition to full cytotoxic screening may facilitate generation of better structural activity relationship and then shed the light for new lead promising anticancer compounds. keyword: coumarin, angelicin, hep-2 cell line, cytotoxicity, الخالصة اممحايثتكي حمم يثتضاتضممرينةممي يومم يو مم يي[fq] دن تمم -2-تممفي مماياممحثيثتحضمم سيتضةممديية ممم ريباتمماويومم يوي حمم ي دمم ي(ir)ت ممصدهياممحايثتكي حمم يتممفي ةمم صاثةيتدممايثمرمم ريتضمم يثتضكمميث ييي).د ي ثتد ي ثتز ممم ثتمممثتك ومم ت يي دمم ي ومم ت ي ند يثتنمم ايثتكان تدممماي ة مم ريضتممصي ممليثتيمم يثت دزت تممرايتةممكن ي ثةممريثتمممكدري ثتمميي(uv) تدممايثمرمم ري مم فيثتحن ممم دري ي-[2,3 ثايوض تم ي دم يي,6ي4 ثت مايعطتم يثةم يوص مري مايثتكي حم يخدمايثمخ حم ي امايhep-2ت صالت ي ة صاثةيخثيخالت ي ي(c3يع -2ي– ي دنمم تد يgي[3,2دمم يي– ثايودض تمم يي-6,2-ثتكدنمم ييثايودض ممماي نزثت ممات ي1-(2' ,4' يي(c1) ومم ت يي]بمما ايامحاي(c3a)ث يي-2دن تم يي]يh -[2,3ثالثمايودض تم ي دم يي-9,8,4ي-ثتكندم يثايودض مماي نزث مات ي-2',4'ي-1 ة م ريضتمصي ي ظد دمرايتممي ي م ويت الخمري ة م ريضتمصيوممدي مايةمكايرم و يت م يخميك ن طمرويةميت ندريثتا ثةريثأل تدمري ةم صاثةيختم تيتصالتم ايةيت ندريو خ ري ثطاوايذث ي تدريتر و يثفيضزثنريثتة يتكي ح يخد ت ي ت ي دبيثتكدك اي introduction furoquinoline-2-one(i) compounds are considered as a main derivatives of furocoumarines(ii)(1). coumarin(iii) is being the main nucleus of these compounds which classified into linear furocoumarin ; psoralen(ii) and angular furocoumarin ; angelicin(iv) [fig.1]. the investigation of coumarin compounds revealed that a wide spectrum of medicinal plant extracts that were in use as early as 1000 a. d. contain a high content of coumarins. subsequent analysis of scientific literature revealed numerous reports on the antiproliferalive and antitumor activities of a variety of coumarin compounds(2-5), and have demonstrated activity against several types of animal tumors(6-10). these compounds have also been reported in clinical trials to demonstrate activity against prostate cancer, malignant melanoma and metastic renal cell carcinoma(11, 12). recently, series of furo-2-quinolone compounds [fig. 2] were synthesized(13) and identified by using infrared (ft/ir), ultraviolet (u.v.) and nuclear magnetic resonance spectroscopy (1hnmr) besides some physical data . 1 corresponding author e-mail : nohad_alomari@yahoo.com received : 28/12/2008 accepted : 26/5/2009 iraqi j pharm sci , vol.18 (suppl . ), 2009 quinolone’s cytotoxic study 13 this previous literature survey urged us to direct this paper towards preliminary cytotoxic study of the "building block" (4,6 dimethyl furo[2, 3-g] coumarin (c1) , one of linear psoralen ; 1-(2`, 4`, dimethoxy benzylideneimino)-2-6-dimethyl furo [2, 3-g] quinoline-2-one (c3) and the angular psoralen of the same derivative with the chemical name; 1-(2`,4`-dimethoxybenzylidenimine)-4,8,9trimethyl furo[3,2-h] quinoline-2-one (c3a)these three selected lead compounds were screened against human larynx epidermoid carcinoma (hep-2) cell line. the preliminary results indicate that hep-2 cell differs in its sensitivity towards these compounds. figure (1) figure (2) o o o o o o oo ii iii iv o oo ch3 ch3 o o ch3 o ch3 ch3 n h 2 n h 2 .h 2 o n o ch3 o ch3 ch3 nh2n o nh2 ch3 o ch3 r c h o , a b s. e th a n o l n o ch3 o ch3 ch3 n ch rn o n ch3 o ch3 ch r och3 o ch3 r = i o n o h c1 c1a c2 c2a c3 c3a iraqi j pharm sci , vol.18 (suppl . ), 2009 quinolone’s cytotoxic study 11 experimental chemistry general method melting points were uncorrected & determined by using electrothermal 9300. the identification of the prepared compounds were done by using ft/ir spectrophotometer tensor 27 as kbr disc. ultraviolet by schimadzu-uv visible uv. 1650 pc. nuclear magnetic resonance spectrum were carried out by using bruker-avance 400 mhz / france. synthesis 1-amino-4,6-dimethyl furo[2, 3-g] quinoline-2-one (14) (c2). 1-amino-4,8,9-trimethylfuro[3,2-h] quinoline-2-one (13) ( c2a). dissolve (0.02 m) of furocoumarin in (30 ml) of dry pyridine with continuous stirring and gentile heating, then cool the mixture and add to it (0.025m, 1-3 ml) of aqueous hydrazine drop wise, reflux for 6 hours. cool the mixture until formation of precipitate, filter and wash many times with cold water and recrystallized from ethanol. yield 42%, the melting point is (c2= 240 241 o c) & (c2a 166-169 o c). 1-(2`,4`-dimethoxy benzylidene imino)-4,6dimethyl furo[2, 3-g] quinoline-2-one (c3). 1-(2`, 4`-dimethoxy benzylidene imino)-4, 8, 9-trimethyl furo[2,3-h] quinoline-2one(c3a). dissolve an equimolar (0.003 m) of c2 or c2a with 2, 4-dimethoxy benzaldehyde in 50ml of absolute ethanol, reflux the reaction mixture for 1 hr. cool and filter. the precipitate is washed with cold ethanol and recrystallized by methanol. m.pt. (202-204 o c, pale brown) and (90-93 o c, yellow) for c3and c3a respectively c=o c-n n-n n-h u.v. etoh nm. ir cm -1 kbr c2 1654 1252 1184 3107 300 c2a 1672 1244 1173 3188 304 ch3(s) ch= nh2 phenyl 1 hnmr ppm/cdcl3 c2 2.2, 2.4 6.3 , 6.5 5.3 7.2 , 7.4 c2a 2.2, 2.4, 2.5 6.5 5.8 7.0 7.4 complex c=o c=n n-n u.v/ etoh nm. i.r. /cm -1 kbr c3 1675 1575 1180 296 nm c3a 1673 1562 1182 304 nm ch3 (s) ch= (s) och3 (s) ch=n (s) phenyl 1 h nmr ppm/cdcl3 c3 2.22, 2.42 6.3,6.5 3.74, 3.81 8.1 7.2 ,7.4 (s) (s) c3a 2.22, 2.42 ,2.5 6.3, 6.5 3.78, 3.82 8.21 7.0 -7.4 complex iraqi j pharm sci , vol.18 (suppl . ), 2009 quinolone’s cytotoxic study 13 ii.cytotoxic screening ii.material bovine serum (bs), phosphate buffered saline (pbs) ph 7.2, hep-2 cell line and all other solutions and media for cytotoxicity study were kindly provided by the iraqi center for cancer and medical genetics research / baghdad. cell line synthesis for cytotoxicity study (17) the growth medium was decanted off and the cell sheet was washed twice with pbs. two to three ml of trypsin-versene was added to the cell sheet and the flask was rocked gently. after approximately 30 seconds most of the trypsin was poured off and the cells incubated at 30 o c until they had detached from the flask. afterwards, 200 l of cells in growth medium were added to each well of a sterile 96-well microtitration plate. the plates were incubated at 37 o c in 5% co2 humidified atmosphere incubator, the medium was removed and serial dilutions of the compound (500, 250, 125 g/m) under assay in sfm were added to the well. the cell line was exposed to cisplatin (ebewe, austria europe) as a reference (positive control); where the negative control were the cells treated with sfm only. the cells in each well were washed with pbs exposed to diluted formalin exposed to crystal violet dye then washed and left to dry (18) . reading the result was accomplished using a multi-well plate reader at 492 nm. results and discussion preparation of c2 & c2a from reaction of furocoumarin with hydrazine hydrate is a nucleophilic substitution lead to cyclic cleavage followed by recyclization with removal of one molecule of water. this reaction product shows three singlet bands at (2.2, 2.4, 2.5 ppm), due to trimethyl substituent at c4, 8, 9 respectively of c2a ,and the singlet proton of c3 appears at 6.5 ppm.the two methyl groups in c2 appears at (2.2 and 2.4 ppm) and the two singlet protons of this compound at c3 and c7 are interfere with the aromatic region at (6.3 and 6.5 ppm) respectively, due to the aromatic properties of this compound. the ft/ir spectra of the compounds c3 and c3a (schiff bases derivatives) shows a significant c = n band between (1575 and 1562 cm-1) respectively. the 1hnmr of these compounds appears approximately not far from the values of compounds c2 and c2a..the u.v. spectra showed max between 296nm and 304 nm respectively the other spectral data of these compounds u.v., i.r. and 1hnmr are appear in the previous tables. cytotoxicity screening compounds c1, c3 & c3a were screened for their cytotoxicity testing. morphological changes of hep-2 cell upon exposure to selected compounds [fig. 3] accompanied with reduction in size i.e. loss of contact with neighboring cells as the apoptotic cells shrank and become detached from the adjacent cells.the dose-response curve of c1, shows a dramatic positive downward curve [fig. 4 , table1] and highly hep-2 sensitivity comparing with resistance cisplatin (positive control), indicate delivering of good prominent lead compound, this significant difference from cisplatin shown at lower concentrations 62.5& 125 g/10ml.a different response [fig. 5, table 2] could be exploited from c3 although a cross-resistance at lower doses, but a decrease in % survival appeared with higher dose which may be clearer with time-course interval higher then 48 hours. hep-2 cell shows a gradual sensitivity with cisplatin c3a [fig.6, table3] and significant difference from cisplatin at p < 0.001 at 5 different dilution doses.from the collective dose response curves [fig.7] c1, c3 & c3a we can conclude that insertion of iraqi j pharm sci , vol.18 (suppl . ), 2009 quinolone’s cytotoxic study 13 ( r = ) to c3 may considered as non essential modification to linear psoralen while angular psoralen "angelicin" may considered as a good lead compound, and collective informations obtained from sar yield from cytotoxic screening of all synthesized compounds may accumulate informations & shed light to new promising anti-proliferative agents. fig 3 : the morphologicl effects exerted by c1,c3, and c3a on hep-2 , photographs were taken using a nikon inverted light microscope (20x objective) ve control c3 och3 och3 drug ve control c1 c3 c3a iraqi j pharm sci , vol.18 (suppl . ), 2009 quinolone’s cytotoxic study 13 ** significant difference from cisplatin at p<0.01, *** at p<0.001 fig 4: graphical representation of concentration dependent effect of c1 on hep-2 cells. table 1: the cell survival as percentage of the control for the hep-2 cell when the cells were treated with c1. concentratio n (µg/10 ml) % of survival (mean  sd) cisplatin c1 62.5 6.77  1.27 106.01  9.33 125 10.15  1.88 73.79  6.74 250 32.42  4.29 36.83  6.59 500 54.88  4.60 36.96  3.64 * significant difference from cisplatin at p<0.05, *** at p<0.001 fig 5: graphical representation of concentration dependent effect of c3 on hep-2 cells. table 2: the cell survival as percentage of the control for the hep-2 cell when the cell were treated with c3. concentration (g/10 ml) % of survival (mean  sd) cisplatin c3 62.5 6.77  1.27 71.34  46.35 125 10.15  1.88 97.09  12.01ي 250 32.42  4.29 100.77  8.97 ي500 54.88  4.60 70.68  5.82 *** significant difference from cisplatin at p<0.001 fig 6 : graphical representation of concentration dependent effect of c3a on hep-2 cells. table 3: the cell survival as percentage of the control for the hep-2 cell when the cell were treated with c3a. concentrati on (g/10 ml) % of survival (mean  sd) cisplatin c3a 62.5 6.77  1.27 107.66  10.88 125 10.15  1.88 109.46  5.21 250 32.42  4.29 103.76  4.69 ي500 54.88  4.60 89.92  6.10 0 20 40 60 80 100 120 10 100 1000 concentration % s u rv iv a l cisplatin c1 *** *** ** 0 20 40 60 80 100 120 10 100 1000concentration % s u rv iv a l cisplatin c3 * *** * *** 0 20 40 60 80 100 120 10 100 1000 concentration % s u rv iv a l cisplatin c3a *** *** *** *** iraqi j pharm sci , vol.18 (suppl . ), 2009 quinolone’s cytotoxic study 13 fig 7: three dimensional histograph of over all tested compounds (c1,c3,c3a) on hep2 cells. references 1. s. m. sentha, chem. rev., 1945. 36, 1-62. 2. m. e. marshall, j. l. mohler, k. edmonds, b. williams, k. butler, m. ryles, l. weiss, d. urban, a. beuschen, and m. markiewicz., j. cancer res. clin. oncol., 1994, 120, 39. 3. i. kostova , synthetic & natural coumarins as cytotoxic agent , curr. med. chem. anticancer agent , 2005, 5, 29-46. 4. c. p. siegers and h. c. bostelmann, j. irish coll. phys. surg. 1993. 5. d. esiyok ,s. otles and e. akcicek , herbs as a food source in turkey , asian pacific journal of cancer prevention, vol 5, 2004,pp334-339. 6. g. feuer ; j. a. kellen ; k. koracs. oncology, 1976, 33, 35. 7. d. thornes ; l. dally ; g. lynch, h. browne ; a. tanner; f. keene; s. o'loughlin; t. corrigan, p. daly ; g. edwards. b. breslin; h. y. brown; m. shine ; f. lennon ; j. hanley n. mcmurray; and e. gaffiney ; eur. j. surg. oncol., 1989, 15, 431. 8. b. omarbasha ; w. r. fair; and w. d. heston; cancer res. 1989, 49, 3045. 9. l. d. raev, e. voineva ; i. c. and ivanor; d. popov; pharrmazie. 1990, 45, 696. 10. a. maucher ; and e. von angerer ; j. cancer res. clin. oncol., 1994, 120, 502. 11. m. e. marshall ; k. butler ; and a. fried, mol. biother, 1991, 3, 170. 12. j. l. mohler ; l. g. gomella; e. d. crawford; l. m. glode; c. d. zippe ; w. r. fair, and m. e. marshall ; prostate. 1992, 20, 123. 13. i. m. taher aziz baker, ms. c. thesis, mosul university, (2006). 14. v. k. pandey ; s. tusl; z-tusl ; m. joshi and s. bajpal ; acta pharm. 2004, 54, 112. 15. m. a. bayoumi ; m. el-aasser and f. abdel halim, j. am. chem. soc. (1971), 93, 586. 16. y. huisen and x. xinfu ; j. jan. 1,2003 ; vol. 5, no.2, 17. 17. r. i. freshney ; cultures of animal cells, 3 rd ed. wiley-liss, 1994, pp. 267-308. 18. w. l. meckeechan, k. a. mckeehan and r. g. ham; improved medium for clonal growth of human diploid cells at low concentration of serum protein. in vitro, 1977, 13: 399-416. (cited by freshney, 1994). 0 20 40 60 80 100 120 % s u r v iv a l 62.5125250500 cisplatin c1 c3 c3a concentration (g/10 ml) t y p e o f c o m p o u n d amera abbas mohammad iraqi j pharm sci , vol.18 (1) ,2009 chelating complex of cefalexin with fe iii metal 94 determination of cefalexin by direct (uvvis) spectrophotometer and indirect(flame atomic absorption) technique amera a. mohammad* ,1 *department of pharmaceutical chemistry,college of pharmacy ,university of baghdad ,baghdad ,iraq abstract a new method for the determination of the drug cefalexin in some pharmaceuticals using (uvvis) and indirect flame atomic absorption spectrophotometer (faas) , fe iii should forms a chelating complex with cefalexin (cex –fe iii) at ph (1-8) and the best ph for the formation of (cex –fe iii) chelating complex was (2) .the complex extracted with methanol and dimethy-sulphoxide .the mole-ratio method has been used to determine the structure of chelate (cex fe iii) and found to be 2:1 lm ( ligand : metal.) . keywords : cefalexin , chelating complex. الخالصة انًسئُت و انطسَقت -طسَقت حدَثت نخقدَس انسُفانكسٍُ فٍ بعض انعقازاث انصُدالَُت و ذنك باسخخداو حقُُخٍ األشعت فىق انبُفسجُت phغُس انًباشسة ناليخصاص انرزٌ أنههبٍ .حُث وجد أٌ انحدَدَك َسخطُع أٌ َكىٌ يعقد كُهُخٍ يع انسُفانكسٍُ فٍ يدي يٍ قُى اسخخهص باسخخداو يزَج و انًعقد انُاحج ( = ٢ph ) حعطٍ اعهً يًخصُت كاَج عُد ph( و أٌ افضم قًُت نــ ٨ -١ح يا بٍُ )حخساو ( عهً انخىانٍ.١:٢( هٍ ) ( cex-feiii انًُثاَىل و ثُائٍ يثُم انسهفىكساَد و كاَج انُسبت انًىنُت نهخعقُد يٍ introduction cephalosporins are β –lactam antibiotics obtained originally from a cephalosporium mold.these antibiotics have the same mechanism of action as the pencillins, but differ in antibacterial spectrum 1 . cephalosporins discovered by bortzu 2 in 1948. cefalexin is the first generation of caphalosporins .the chemical names for cefalexin are :-7-(d – α -aminophenyl acetamido)des-acetoxy cephalosporic acid. 3 or 7-(d-2-amino-2-phenylacetamido)-3-methyl3-cephem-4carboxylicacid. 4 the chemical formula is c16 h17 n3 o4 s . h2o . and the chemical structure for the drug : cefalexin monohydrate molecular weight: 365.4 gm melting point: 326.8 0 c cefalexin is a white to faint yellow powder slightly soluble in water, insoluble in ethanol , chloroform and ether . anacona et. al 5 prepared complexes for β –lactam antibiotics with some metals, and they identify it by spectroscopic and physiochemical methods ,also they studiedthe reaction of these antibiotics with some transitional elements like mn ii , cu ii ,zn ii, and confirmed the structures of products using i .r. and n.m.r. spectra. on the other hand, different solutions of cephalosporins and chloride salts of essential and trace elements prepared in a study using different temperatures ( 37˚cand 60 ˚c) in order to accomplish the complex formation in molar ratio of the drug and metal salts 1:1 6 . also abdel gaber et.al 7 studied the complexes of ions znii , cuii, ca ii with cephalosporins potentially they calculated the molar – ratio for (liganedmetal) at 25 ºc (1:1). 1 corresponding author e-mail : am-ab-mo @ yahoo . com received : 16/11/2008 accepted : 28 / 3/2009 iraqi j pharm sci , vol.18 (1) ,2009 chelating complex of cefalexin with fe iii metal 05 lozano and borras 8 studied titrametically the complexes of cefalexin with metals znii ,cd ii, they analysis the result by electrical methods using ( least squares computer. program super quad). they prepared complexes of cefalexin like zn (cex)2 , 3h2o and cd (cex)(oh) h2o , these complexes have been identified by i.r and n.m.r spectra . in addition abo el-maali et-al 9 determined some cephalosporins antibiotic with cd ii or zn ii by using voltimeteric mothed which determined the presence of (liganed metal)1:1 like in complexes cefataxime , cefuroxime and 2:1 like ceftazidime. moreover, iqbal and ahmad 10 prepared complexes of cephalexin with copper ii and znii these complexes have been characterized by microanalysis and by magnetic and spectroscopic analysis. the complexes, were found five-coordinate monohydrate, and ml 2 .on complexation with copper and zinc the antimicrobial activity of cephalexin improved significantly. these results suggested that metallic elements should be seriously considered during drug design .also, alekseev et al. 11 studied the complex formation in solutions containing nickel (ni ++ ) cations,glycine anions (gly ),and β-lactam antibiotics, they found mixed-ligand complexes is formed of [ ni gly ampicillin], [ ni gly amoxicillin] ,and [ni gly cephalexin]. alekseev 12 , in another study, found that the complex formation of neodymium iii (nd 3+ ) ions with ampicillin, amoxicillin and cephalexin anions (l) in aqueous solution at 20˚c and ionic strength of 0.1 (kno3) by ph titration was in form of ndl and nd(oh)l complexes using weak alkaline solution. a new study discussed the spectrophotometric determination of cephalexin as intact cephalexin or its degradation product, cephalexin was determined in the range (1×10 5 -18×10 -5 m) and the limits of detection were 0.3×10 -5 m. these results were compared with reversedphase hplc determination, the uvvis spectrophotometric method was improved to be selective and reproducible 13 . instruments , materials and method a instruments shimadzu ( aa670 ) flame atomic absorption spectrophotometer . 1. u.v–visible spectrophotometer(cary 100)wave length 200 -1100 nm . 2. ph meter type 60 a . b materials all the chemical stock solution were prepared from analytical grade bdh,sdi ,india and germany . cmethod stock solution of fe iii a solution of 1000 ppm of fe +3 was prepared by dissolving 0.2896 gm of fecl 3 in small amount of water and complete the volume to 100 ml by using volumetric flask ,. then 10 ml of the stock solution was diluted to 100 ml with distilled water to prepare 100 ppmsolution . stock solution of cefalexin (cex) a solution of 1000 ppm of cex was prepared by dissolving 0.05 gm of (cex) in water and complete the volume to 50 ml by using volumetric flask .then a stock solution of 100 ppm was prepared as above . spectral study cefalexin spectrum 10 ml of stock solution (cex) was transferred to a volumetric flask 5 ml and diluted with distilled water then the absorbance was measured at 200-600 nm wave length using water as blank . ferric spectrum 1ml of stock ferric solution was transferred to a volumetric flask 5ml and diluted with distilled water, then the absorbance was measured at 200-1100 nm using water as blank. formation complex with fe iii (2-5) ml from stock solution of (cex) 1000 ppm was transferred to a volumetric flasks 5ml and 1ml of fe iii 1000 ppm was added .the chelating complexes can be extracted by using 2ml mixture of methanol + dimethyl sulphoxide (0.5 :1.5) .the formation of complex was stutied at room temperature the reaction was heated at different temperature with different ph . results and discussion drug spectra : measuring (cex) spectra as in figure (1) we see one absorption peak at λ g water as blank . metal spectra : figure (2) for fe iii spectra show absorption peak at λ water as blank.chelating complex spectra : cexfeiii.figure(3) for chelating complex show two new peaks,the first at λ 0) nm and the second at (358) nm which indicate the formation of complex between drug and feiii using organic solvent as blank. table (1) show different compounds and the wave length at λmax for each drug and feiii. iraqi j pharm sci , vol.18 (1) ,2009 chelating complex of cefalexin with fe iii metal 05 fig(1) the uv absorption spectrum for cefalexin figure(2): the uv absorption spectrum for fe iii solution figure (3) :the uv absorption spectrum for the complex cex-feiii table(1) color and λ max for the drug feiii and complex optimum condition for complex formation the experimental work showed that the reaction was not proceed at room – temperature so heating was necessary, media must be acidic, for these reasons we study the effect of ph, reaction time , extraction time, concentration of ion fe iii , temperature effect ,suitable solvent for extraction and number of extraction . ph effect: figure (4) show the absorbance of (cex –fe iii ) using different ph (1-8) the optimum ph for the complixation was (ph:2). figure(4) : effect of ph on the absorbanc of cex-feiii by using uv spectrum reaction time it was found that higher absorbance of extracted complex was happened after 25 minutes, after that the absorbance decrease due to dissociation part of complex with increase in heating time . figure (5) , show that 25 minutes is the best time for reaction togive maximum absorption for ( cex-fe iii) at 80ºc and ph 2. color max(nm) λ compound white 264 (cex) orange 208 fe iii yellow 341+358 complex 200 600.0 400300 500 0.0 0.5 1.0 1.5 2.0 a b s 200 300 400 500 600 0 1 2 3 4 5 200 600.0 400300 500 wave length 0 1 2 3 4 5 a b s a b s wave length wave length fig. 2 the uv absorption spectrum for fe iii solution fig. 3 the uv absorption spectrum for the complex cex-feiii fig. 1 the uv absorption sepctrum for cefalexin 200 600.0 400300 500 0.0 0.5 1.0 1.5 2.0 a b s 200 300 400 500 600 0 1 2 3 4 5 200 600.0 400300 500 wave length 0 1 2 3 4 5 a b s a b s wave length wave length fig. 2 the uv absorption spectrum for fe iii solution fig. 3 the uv absorption spectrum for the complex cex-feiii fig. 1 the uv absorption sepctrum for cefalexin 200 600.0 400300 500 0.0 0.5 1.0 1.5 2.0 a b s 200 300 400 500 600 0 1 2 3 4 5 200 600.0 400300 500 wave length 0 1 2 3 4 5 a b s a b s wave length wave length fig. 2 the uv absorption spectrum for fe iii solution fig. 3 the uv absorption spectrum for the complex cex-feiii fig. 1 the uv absorption sepctrum for cefalexin 0 1 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 a b s. ph 0 1.3 1.8 2.3 2.8 3.3 time a b s. 5 10 15 20 25 30 35 40 45 50 0.5 1 1.5 2 2.5 3 a b s. 40 50 60 70 80 90 100 110 temperature 3.5 fig. 4 effect of ph on the absorbanc of cex-feiii by using uv spectrum effect of reaction time on formation of complex cex-feiii at ph 2 by using uv spectrum by using uv spectrum fig. 6 complex absorption value at defferent temp. fig. 5 iraqi j pharm sci , vol.18 (1) ,2009 chelating complex of cefalexin with fe iii metal 05 figure(5): effect of reaction time on formation of complex cex-feiii at ph 2 by using uv spectrum temperature effect: the reaction of drug with metal was proceed slowly it may exceed hours to increase the reaction velocity we proceed the reaction at different temperature (50-100 ºc ), it was found that 80 ºc is the optimum temperature which gave the higher absorption as in figure (6) . figure(6) complex absorption value at different temp. by using uv spectrum effect of fe iii concentration: the best concentration for fe iii ion is 80 µg /ml, it gave the maximum absorbance figure (7) show that effect of concentration ppm on absorbance . figure(7): the effect of fe iii concentration on complex formation by using uv spectrum phase ratio: it was found 5ml of aqueous layer and 2ml of organic layer is enough to get higher absorbance for complex as in figure (8), after that it was decrease as we increase the volume of organic layer. figure(8): effect of phase ratio on absorbance of complex cex-fe iii at ph =2 by using uv spectrum suitable solvent : different organic solvent were used like benzene, chloroform ,acetone ethanol , cyclohexanone , carbon tetrachloride , methanol , dimethyl sulphoxide , and diethyl ether for extraction of complex (cex-feiii) , it was found the mixture of methanol and dimethyl sulphoxide were the best solvents . extraction time : the optimum time for shaking during the extraction of the complex was (4 min.) which give maximum absorption . figure (9) showed 0 1 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 a b s . ph 0 1.3 1.8 2.3 2.8 3.3 time a b s . 5 10 15 20 25 30 35 40 45 50 0.5 1 1.5 2 2.5 3 a b s . 40 50 60 70 80 90 100 110 temperature 3.5 fig. 4 effect of ph on the absorbanc of cex-feiii by using uv spectrum effect of reaction time on formation of complex cex-feiii at ph 2 by using uv spectrum by using uv spectrum fig. 6 complex absorption value at defferent temp. fig. 5 0 1 2 3 4 5 6 7 8 9 10 0.5 1 1.5 2 2.5 3 a b s. ph 0 1.3 1.8 2.3 2.8 3.3 time a b s. 5 10 15 20 25 30 35 40 45 50 0.5 1 1.5 2 2.5 3 a b s. 40 50 60 70 80 90 100 110 temperature 3.5 fig. 4 effect of ph on the absorbanc of cex-feiii by using uv spectrum effect of reaction time on formation of complex cex-feiii at ph 2 by using uv spectrum by using uv spectrum fig. 6 complex absorption value at defferent temp. fig. 5 0 0.3 0.6 0.9 1.2 1.5 1.8 0 20 40 60 80 100 120 140 160 conc. ppm a b s . 1.5 2 2.5 3 3.5 0 1 2 3 4 5 6 7 vl(ml ) a b s . iraqi j pharm sci , vol.18 (1) ,2009 chelating complex of cefalexin with fe iii metal 05 the maximum absorption in the extraction was 4 minutes of shaking . figure(9) : effect of shaking time on the extraction process by using uv spectrum number of extraction: extraction process for once is enough to extract the complex ,because the second extraction for the remaining concentration give very small absorbance less than 0.08 ,so one extraction is enough. this illustrate in table (2) table(2): effect of number of extraction on absorbance of complex a0 blank 0.005 a2 (ex.no.2) 0.085 a1 (ex.no.1) 2.84 ph 2 fe iii mg/ml 60 cex mg/ml 800 mole ratio of complex: determination the mole ratio for complixation it means percent of moles of drug to the moles of metal , different volumes of cex between (0.5-3.5)ml with constant volume of metal with all optimum conditions of complixation , it was found (1:2) (m:l) . figure 10 show the relationship between absorbance . and vl / vm .the reaction of feiii with drug occur in this equation : feiii +2cex feiii 2cex the suggested chemical structure for the reaction of feiii with cex. : fig(10) mole ratio of complex cex-feiii by using uv spectrum calibration curve of the spectrum : for determination of direct calibration curve for (cex-feiii),a different concentration for complex were used as in figure (11) and the relationship between absorbance and concentration ppm was draw, it was found that the maximum concentration which obey beerlumbert law 200µg / ml . fig(11) : calibration curve for cex-fe iii by using uv spectrum 2.4 2.7 3 3.3 3.6 0 1 2 3 4 5 6 7 ti me (mi n ) a b s . iraqi j pharm sci , vol.18 (1) ,2009 chelating complex of cefalexin with fe iii metal 09 determination of the drug concentration in pharmaceutical preparation : 6 capsules were mixed with each other then one capsule was weighted , and the absorbance was measured of its (cex-feiii) using optimum condition then from the calibration curve determine the concentration , we carried the procedure for cex . (sdi ,ajanta, germany) .table (3)show the result of work .from these result , we suggested that the cex . concentration in germany capsules is less than the real concentration which is 500 mg while ajanta and sd iis o.k. table(3):the concentration of cefalexin capsule in 500mg cefalexin of different trade marks determination of drug cex – feiii by using flame atomic absorption spectrophotometer: to be sure about the result obtained by u.v , we used another technical method, flame atomic absorption spectrophotometer (faas) , by indirect measurement the absorbance of fe iii in the complex to detect the cefalexin conc. as in figure (12) . the complex cex-feiii was prepared by using optimum condition of ph , temperature , proper solvent etc . (the same conditions mentioned previously in u.v spectro photometer) except changing the conc.of ferric ion and phase ratio, it was found the best conc. of feiii to give maximum absorbance 8 µg /ml , and 5ml of aqueous layer and 3ml of organic layer is enough to get higher absorbance for complex as in figure (13) and (14) .also we measured the concentration of cefalexin in these pharmaceutical preparations using calibration curve of indirect (faas), we got the same result which obtained by u.v method fig(12) : calibration curve for (cex) by indirect faas by using uv spectrum fig(13): effect of feiii on complex by using faas fig(14): effect of phase ratio on absorption of complex by using faas germany ajanta sdi capsules 1.29 1.3 1.4 abs. 487 495 502 conc.ppm 500 500 500 standard 0.00 0.02 0.04 0.06 0.08 0.1 0.12 2 4 6 8 10 12 14 a b s. conc. ppm fig. 13 effect of feiii on complex by using faas 2 4 6 8 0.05 0.1 0.15 0.2 0.25 0.3 0 a b s. vl (ml) fig. 14 effect of phase ratio on absorption of complex by using faas 0.00 0.02 0.04 0.06 0.08 0.1 0.12 2 4 6 8 10 12 14 a b s. conc. ppm fig. 13 effect of feiii on complex by using faas 2 4 6 8 0.05 0.1 0.15 0.2 0.25 0.3 0 a b s. vl (ml) fig. 14 effect of phase ratio on absorption of complex by using faas iraqi j pharm sci , vol.18 (1) ,2009 chelating complex of cefalexin with fe iii metal 00 references 1. andres goth , m.d.,(cephalosporins), medical pharmacology , 1981 , tenth edition, p.641. 2. b.g.katzung,(the origin of cephalosporin),basic and clinical pharmacology ; medical books, megrawhill ; newyork 2001 , 8 th ed; p . 762-766. 3. merck and co ; whitehouse station. n.j.usa; the merck index, 2006 , fourteen edition, p.1974. 4. usp 27 – nf 22, pharmacopeia, 2004, p.393. 5. j.r.anacona and i.rodriguez , (synthesis and antibacterial activity of cephalexin metal complexes ),j.coord . chen ., 2004 ,57 (15), p. 1263-1269. 6. sultana, najama (n); arayne, m saeed (ms);(in vito activity of cefadroxil, cephalexin,cefatrizine and cefpirome in presence of essential and trace elements),pak j pharm sci, 2007, 20(4),p. 305-310. 7. a.a.abdel gaber, othman a.farghaly , m.a ghandour and h.s.al-said , (potentiometric studies on some cephalosporin complexes), monatshefte fur chemie, 2000,131, p . 1031 – 1038. 8. m.j.lozano and j.borras , (antibiotic as ligand coordinating behavior of the cephalexin towards zn ii and cd ii ions ) , j. of inorganic biochemistry , 1987, 31(3), p.187-195. 9. n.aboel-maali, a.h.osman , a.a.m.aly and g.a.a.alhazmi ,(voltametric analysis of cu ii , cd ii and zn ii complexes and their cyclic voltammetry with several cephalosporin antibiotics), bioelectrochemistry , 2005 , 65, issue 2 , p.95-104 . 10. iqbal m.s.,ahmad a.r; sabir m;asad s.m; (preparation, characterization and biological evaluation of cu ii and zn ii complexes ), j. of pharmacy and pharmacology , 1999 , 51 , no.4,p. 371375. 11. v. g. alekseev and i. s. samuilova, (complex formation in nickel(ii)glycineβ-lactam antibiotic systems), russian j of coordination chemistry, 2007, 33(12),p. 914-917 12. v. g. alekseev, (neodymium (iii) complexing with ampicillin, amoxicillin and cephalexin), russian j of inorganic chemistry, 2007, 52(5), p. 698-702. 13. campins-falco p, sevillano-cabeza a, gallo-martinez l, bosch-reig f, monzomansanet i, (comparative study on the determination of cephalexin in its dosage forms by spectrophotometry and hplc with uv-vis detection), mikrochimica acta, 1997, 126(3-4), p. 207-215. microsoft word bagpha05114 _2_ iraqi j. parma. sci., vol.14, 2005 12 ------------------------------------------------------------------------------------------------------- biochemical features of iraqi patients infected with hepatitis e virus compared with those infected with hepatitis a virus elham.f. al.fakhri department of clinical analysis, college of pharmacy, baghdad university, baghdad-iraq received: 09.04.2001 accepted: 11.06.2002 abstract : this study was designed to compare the effect of two types of viral hepatitis a and e (hav and hev) on liver functions in iraqi individuals by the measurement of biochemical changes associated with hepatitis. the study performed on 58 hev and 66 hav infected patients compared with 28 healthy subjects. the measured biochemical tests include total serum bilirubin, serum transminases (alt and ast) alkaline phosphatase (alp) and gamma glutamyl transferase (ggt). the study showed that adolescent and young adults (17-29) years, were mostly affected by hev while children (5-12) years were frequently affected by hav. the severity of liver damage in hev patients was higher than hav patients as a result of high serum transaminase levels. patients with hev were associated with cholestasis due to high serum level of alp and ggt compared to hav patients. :الخـالصـة التھ�اب الكب�د الفایروس�ي الح�اد و aالھدف من ھذه الدراسة ھي المقارنة بین ت�أثیر التھ�اب الكب�د الفایروس�ي الح�اد ن�وع ف الكبد للمرضى العراقیین وذلك بأجراء الفحوص المختبریة التي لھا عالقة بھذین المرضین وقد شملت الدراسة وظائ على eنوع aالفایروسي مریضًا بالتھاب الكبد ٦٦و . عامًا) ٢٩ – ١٧(تتراوح أعمارھم بین eالفیروسي شخصًا مصابًا بالتھاب الكبد ٥٨ :واألنزیم�ات bilirubinالفحوص المختبریة شملت .األصحاءشخصا من ٢٨ارنة مع مق عامًا،) ١٢ – ٥(بین تتراوح أعمارھم ((alt & ast) transaminases) , gamma glutamy transferase (ggt) alkaline phosphates (alp) , . الدراسة بینت أن معظم المرضى المصابین بنوعa ھم من األطفال بینما المرضى المصابین بنوعe ھم من الشباب وذل�ك للزی�ادة aنم�ط ھي أعلى منھا ف�ي eوكذلك اتضح من سیر الدراسة أن شدة المرض في التھاب الكبد الفایروسي. والیافعین بین�ت أن أیضا الدراس�ة . التي تحررت من خالیا الكبد التالفة (alt&ast)الحاصلة في نسبة المادة الصفراء ونسبة األنزیمات سبب في ركود الصفراء وقلة إفرازھا بینما ل�م تظھ�ر ھ�ذه الحال�ة عن�د األطف�ال المص�ابین بالتھ�اب الكب�د eروسيالتھاب الكبد الفای .aالفایروسي iraqi j. parma. sci., vol.14, 2005 13 ------------------------------------------------------------------------------------------------------- introduction: hepatitis e virus hepatitis e virus (hev) is an acute, icteric self—limiting disease, which is spread widely in many tropical and subtropical countries, where it occurs both in the form of epidemic of variable magnitude or sporadically (1) . hepatitis e virus is a small rna virus which is temporally classified as a member of calcivirus family. transmission is via faecal —oral routes but the possibility of blood transmission has been raised concerning anti -hev prevalence among chronic hemodialysis patients are give conflicting results (2) , also vertical transmission from mother to fetus can be observed (3), hepatitis e is not clinically differs from other acute viral hepatitis. the course of the disease is usually benign with little risk of development of chronic symptom and cirrhosis (4) . high incidences of sever cases with 1-2% mortality (5) . acute liver failure secondary to hepatitis e infection is common in pregnancy and associated with mortality rate up to 20% (6) because both in women and her fetus are at risk, escalated production of progesterone during pregnancy leads to downregulation of cellular (cell mediated) immune function. the role of long persisting hev antibodies in humans was investigated (7) candidate vaccines have not yet reached clinical trials (8) . hepatitis a virus hepatitis a virus (hav) a counts for 20-25% of hepatitis cases worldwide. itis caused by rna picorna virus (9) . the virus mediates injury by both cellular and non cellular immune mechanisms an igm antibody (anti hav igm) appears early in the course of illness and persist 2-6 months and igg antibody, appears towards the end of acute illness presist for several years and conveys lifelong immunity, hepatitis a occurs in sporadics and epidemic forms with incubation period of 15-50 days epidemic have been associated with water borne and food borne countries, outbreak have occurred through infected food handlers in restaurants. the clinical course of acute hepatitis a is usually that of a mild flulike illness that last for a few days to a few weeks, only 10% become jaundiced. the disease is more prolonged and serious in persons over 50 years old. hepatitis a infection does not cause chronic carrier state (10) and perinatal transmission is extremely uncommon, but cholestasis manifested by several weeks of jaundice and pruritis may occur in adults. hepatitis a can be prevented by immunization with an inactivated vaccine which highly effective (11). iraqi j. parma. sci., vol.14, 2005 14 ------------------------------------------------------------------------------------------------------- subjects and methods: venous blood samples were obtained from 58 patients with acute hepatitis e virus (hev) aged (17 29) years (22 females, 36 males) screened in central public health lab. (virology dept). by linked immunosorbent assay [elisa] bioelisa (hev) igm .[(biokit s.a) (barcelona ,spain)] also venous blood samples were collected from 66 patients with acute hepatitis a virus (hav) (26 females, 40 males) aged (5 12 ) years screened in central public health laboratory (viralogy dept) by microelisa system, hepanostika hav. igm (organon, teknika gmbh). blood samples withindrawn from 28 healthy individuals aged (5-25) years (12 females. 16 males) served as control. all sera were examined for liver function tests including total serum bilirubin (bilirubni kit. randox) (12) alanine .aspartate transaminases kits randox. england) (13) alkaline phosphatase (alp) phosphates alkaline kit (biomerieux s.a france) (14) and gamma glutamyl transferase (ggi) enzylin kit (biomerieux france) (15). results: the results of this study obtained are found in table i.ii, iii. table i shows that the ages of patients infected with hepatitis e virus were (19.22 ± 1.48) years old while those with hepatitis a were (7.76 ± 1.05) years old. age (years) hev hav controls mean +sem 19.22 1.48 7.76 1.05 15.52 2.44 table i table ii shows that all the patients of both groups were icteric but the level of total serum bilirubin of patients with hepatitis e virus were insignificantly higher than those with hav (t =1.388 .p< 0.05)(fig.(1)) parmeters hev hav controls t.s. bilirubin mg/dl 5.578 1.238 3.57 0.766 0.74 0.23 alanine transaminase i.u/l 63.84 4.510 41.42 3.390 10.4 5.43 aspartate transaminase iu/l 68.76 4.246 48.611 1.457 9.3 4.2 table ii note: normal range for t.s. bilirubin = 0.2-1mg /dl normal range for alanine transaminase = 2-12 l.u/l normal range for aspartate transarninase = 2-12 l.u/l iraqi j. parma. sci., vol.14, 2005 15 ------------------------------------------------------------------------------------------------------- the concentration of serum transaminases enzymes (alanine and aspartate transaminases) of patients with hepatitis a shows moderate to high elevation while that of hev were significantly higher (t= 4.048 p>0.05) for alt and those for ast (t= 3.28 p>0.05) (table ii) (fig.(2)). serum alkaline phosphatase (alp) enzymeof hav infected subjects were mostly within normal fig.1 total serum bilirubin controlshav hev 0 1 2 3 4 5 6 mg/dl hev hav controlscontrolshev hav 0 10 20 30 40 50 60 70 80 1. uu/1/ l alanine transaminase aspartate transaminasefig.2 u/1 i.v/l iraqi j. parma. sci., vol.14, 2005 16 ------------------------------------------------------------------------------------------------------- range but the concentration of serum alp with hepatitis hev were significantly high (t=3.62 p>0.05) (fig.(3)). fig 3. serum alkaline phosphatase the level of serum glutamyl transferase enzyme (ggt) with hepatitis a virus were within normal range while those with hev were significantly high (t = 4.796 p>0.05) table iii. (fig. (4)). parmeters hev hav controls alkaline phosphatase k-au/dl 50.826 7.529 36.32 8.56 10.132 3.425 gamma glutamy1 transferase u/l 41.35 4.748 10.21 2.64 12.918 2.202 table iii note: normal range for alkaline phosphatase (adult) = 3-13 k.au/d1 normal range for alkaline phosphatase (children) up to 20 k.au/dl normal range for , glutamyl transferase at 25 o are: men = 6-24 dl wormen = 5-21 u/l 0 10 20 30 40 50 60 k-au/dl hev hav controls iraqi j. parma. sci., vol.14, 2005 17 ------------------------------------------------------------------------------------------------------- discussion: the results on table i proves that the patients with hepatitis e virus were frequently adolescents and young adults while that with hepatitis a virus were mostly children ,this has been documented in other studies (16,17) . the results of liver function tests for (hev) and (hav) of infected patients shows that all patients of both groups were icteric but the total serum bilirubin of hev group were insignificantly higher than that of hav group (table ii) serum alanine and aspartate transaminase concentration are highly sensitive indicatores of hepatocelluler damage. they provide a qualitative assessment of the degree of damage sustained by the liver. from the above results the severity of acute hev more than that of hav due to high levels of aminotransferase enzymes in hev patients that leads to hepatocytes damage in those patients in most patients with hev. resolution of hyperbilirubinemia and abnormal serum alt occurs within 3 weeks (range 1-6 weeks) and there is no clinical and histological evidence of chronic hepatitis alkaline phosphatase level is obtained primarily to assess the degree of cholestiasis present in the liver. the enzyme associated with the microvilli of bile canaliculi with sinusoidal membrane present in the cytosol. gamma glutamyl transferase (ggt) has been identified in the same membrane as alkaline phosphatase it is useful to measure the ggt level to confirm the hepatic origin of an elevated level (19) the high level of alp and ggt with bilirubin of hev patients indicate that the hev in iraqi patients associated with marked intrahepatic cholestasis (20) while the normal level of ggt and fig.4 serum gamma glutamyl tranferase controlshavhev 0 5 10 15 20 25 30 35 40 45 u/l iraqi j. parma. sci., vol.14, 2005 18 ------------------------------------------------------------------------------------------------------- slight elevation of alp of infected patients with hepatitis a virus not associated with cholistasis although there was other study proved that acute hav associated with chalestasis in adults & adolescent) (18). this may be depend on the age of infected patient. or count on the geographic factor & the environment of the virus exist. (21) conclusions: from the results and discussion of this study we conclude that: 1hev frequently affect adolescents and young adults while hav seroprevalance in children. 2the severity of hev disease is significantly higher that of hav disease (liver damage) 3 acute hepatitis e virus associated with marked cholestasis while that of acute hepatitis a virus did not. references: 1balyan m.s (epidemiology of hepatitis e virus infection). j. viral -hepat 1997 may 4 (3) 155-65. 2fabrizi f; lunghi g; bacchini g; corti m; pagano a; locatelli f. (hepatitis e virus infection in hemodialysis patients) nephrol dial transplant 1997 jan 12:133-6. 3aggarwal r; naik s.r (hepatitis e and intrafamilial transmission) indian j gastroe 2000 jan march 19 (1): 6-8 4balistrevi wf ( viral hepatitis e ) med clin north am 1991 .9: 365-399. 5wang c.h ; tschen s.y ( hepatitis e virus and primary billiary cirrhosis ) qjm 1997 feb: 90 (2) :154-55. 6hussaini sh; skidmore sj; richardson p; sherratt lm; cooper bt; grady jg. ( sever hepatitis e infection during pregnancy) j.mral hepat 1997 jan 4(4) 51-4. 7cauhan a; dilawari jb; sharma r; mukesh m; saroa sr.( role of long presisting human hepatitis e virus antibodies in protection) vaccine 1998 apr; 16(7).755-6. 8harrison t.j. (hepatitis e virus .... an update) 1999 jun 19(3):71-176. 9feinston s.m. (hepatitis a detection by immune electrone microscopy of a virus-like antigen associated with acute illness) science 1973; 182:1026 -1028 10duff p: (hepatitis in pregnancy): semin perinatal 1998 aug;22(4)277-283. 11innis b.l. snitblan r.e. (protection against hepatitis a by inactivated) jama, 1994: 13281334. 12lath gh,ruthven cr j.clinpath l958; 11.155. 13bergmeyer hu (editor of enzymatic analysis) 1974 ; vol 2 nd english edition , verlage chemic weinheim , academic press london, new york. 14kind p.r.n and king e.j clin path 1974; 7:322. 15persijn j.p clin. chim acta 1971 ; 35 : 239-240. iraqi j. parma. sci., vol.14, 2005 19 ------------------------------------------------------------------------------------------------------- 16arora ak; nanda sk; ansari ih; joshi s; dixit r; bathla r. (hepatitis e infection in children j.gastroentro hepato 1999 jun: 14(6)572-7. 17alvarez munoz. mt; torres j; damasio l; gomez a; tapia conyer r; munoz o.(seroepidemiology of hepatitis e virus infection in mexican subjects 1-29 years of age) 18arch med. res 1999 may-jun; 30(3);251-4. 18nicocali naoumov (heptitis a and e) medicine 1999; the medicine publishing company. 19n.field (editor of virology) 1985 chapter 61 page1426, raven press, new york 20shorder 0; lee jh; herrmann g; rabenau h; zeuzem s.(sever acute cholestatic viral hepatitis e in non pregnant women) dtsch med -wochen sehr 1997 jan 3;122(1-2) :21-24. 21simmonds p. (variability of hepatitis e virus) hepatology 1994;21;1144-1151. الخـلاصـة: table iii a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci , vol.17 (2) , 2008 enzymatic antioxidant in chronic gastritis patients 26 determination of enzymatic antioxidant in iraqi patients with chronic gastritis wasan a. bakir* , shalal m. hussein* and noah a. mahmood1,* *iraqi center for cancer and medical genetic research, al-mustansiryia university. baghdad. iraq. abstract infection of the gastric mucosa with helicobacter pylori is strongly associated with chronic gastritis, peptic ulcer and gastric cancer. helicobacter pylori virulence factors include a variety of proteins that are involved in its pathogenesis, such as vaca and caga. another group of virulence factors is clearly important for colonization of h.pylori in the gastric mucosa. these include urease, motility factors (flagellin), and superoxide dismutase (sod). because of this organism's microaerophilic nature and the increased levels of reactive oxygen in the infected host, we expect that other factors involved in the response to oxidative stress are likely to be required for virulence. superoxide dismutase is a nearly ubiquitous enzyme among organisms that are exposed to toxic environments. in this study, we measured the sod in serum of 80 patients complain from chronic gastritis and infected with h.pylori. 37 patients infected with h.pylori have the caga gene, and 13 patients are not and also measured the sod in 30 control groups that not infected with h.pylori. serum level of sod was significantly (p<0.05) higher in patients with chronic gastritis compared to controls. also significantly higher (p<0.001) in patients with chronic gastritis infected with h.pylori positive caga than patients infected with h.pylori negative caga. key words: chronic gastritis, h.pylori, caga, sod ةــصالللا لنبالسولايللاباإةللاءاشغللاءلا لقلالا كللاءاشصكلقفاخإتل helicobacter pyloriلل ايإاكللاةدإمللاءطإخملا ءاشغلا ةباصإ لقل cagaلاءاشغ . لا يهو لااللاتالقغللاطإيكلااراللا ةباصإلنبتء لاا لملا للا اقناتإاللابملندإذهلسملا شل لسمللاءالماكلق.مللل ل vagaاعء لا يهو كلاواللا ل لا يهو لااللاتالقغلنة تلااءكلسملنة ص للاءلباءالالسمللاةدإمللاءطإخمللاءاشملاتاإللاا ذصللقل لا يهو إاال . سمل.راللاشذلفكلنةل للارملصب لإشلسمللاةإلتإاللابملنباا لااولقفيلفإ .superoxide dismutase (sod) لالا كلقاالصةلل لل 37للاتاةلh.pyloriلااصال إا للصاإا تللا للاباإةللاءاشغللاءلا لقا إاا لا ةباصإلل للل80سملا ال sod اإالالب لللايل لاة لااال إا كلاارلللاعا ل .h.pyloriلااصالااصالا إاا لل للل13لقcagaلللالإا كلااللh.pyloriااصالا إاا لل للل صلولولاات صإزلsod ليطتلالإاكليلاكلف اءكلقااالا إاا لاإا ةباصإ .قإشلاتلالب لللايل30ق إانلاعء لا يهو كللالا اغلنبة تلا ل (p<0.001)لسمللاءاموللاء إاا لاإاباإةللاءاشغللاءلا لالإذاكلاءعء لا يهو كللالا اغلقصلولولاات صإلسملااموللاباإةللاءاشغللاءلا ل لالإذاكلاءاموللاباإةللاءاشغللاءلا للاء إاا لاإا ةباصإلقاة لااالcaga (p<0.05)لقلالإا كلا يلh.pyloriلاء إاا لا ةباصإلل للل .caga إا كلا يل introduction the gastric pathogen helicobacter pylori is a curved, microaerophilic proteobacterium that has been implicated as a causal agent of peptic ulcers and a risk factor for adenocarcinoma (1,2, 3,4,). during the infections, disease symptoms may or may not occur, though gastric inflammation is apparently ubiquitous. the pathogenesis of h.pylori relies on its persistence in surviving a harsh environment, including acidity, peristalsis, and attack by phagocytic cells and their released reactive oxygen species (5). several potential virulence factors derived from h.pylori are considered to attract or activate neutrophils and mononuclear cells., an immunodominant 120-140 kda antigen termed cytotoxic associated antigen (caga), the caga positive strain cause more server inflammations(6). the stomach gastritis associated with helicobacter pylori infection stimulates the generation of reactive oxygen species (ros) by the inflammatory cells present in the mucosa (7, 8, 9). an increase in ros directly correlated with bacterial load (10). in addition to internally generated reactive oxygen species, the successful pathogen must also deal with reactive oxygen species that are generated by phagocytic cells of the host immune response. (11, 12). protection of cells against ros is accomplished through the activation of oxygen scavenging enzymes such as sod, catalase and glutathione peroxidase have been identified (13). superoxide dismutase is a nearly ubiquitous enzyme among organisms that are exposed to toxic environ 1 corresponding author : e-mail : noahaljaff@yahoo.com. received : 3/5/2008 accepted : 1/11/2008 27 the single sod of helicobacter pylori, encoded by the sodb gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously (14). the mechanisms for the detoxification of reactive oxygen species are of particular interest in h.pylori. despite the fact that this organism is an obligate aerobe, it is unable to grow in atmospheric concentrations of oxygen. microaerophilic organisms, like h.pylori, are particularly vulnerable to the detrimental effects of oxygen and oxidative stress (15). nevertheless, they do possess some of the enzymatic machinery needed to eliminate or minimize toxic oxygen-derived products. organisms that grow in toxic environments must have mechanisms to handle reactive oxygen species (e.g., superoxide anions, peroxides, and hydroxyl radicals) that are byproducts of oxygen metabolism (16, 17). of these genes, only kata (catalase) mutants have been characterized (18). although catalasedefective mutants are no different from the parent in their binding to epithelial cells, this enzyme may be important in detoxification of reactive oxygen species produced by the host immune response. genes encoding an akyl hydroperoxide reductase, a thiol-specific peroxidase, and other potential detoxification enzymes were identified, but mutations in these genes have not been reported or characterized (14, 19). impairment in this important host cell defense mechanism would greatly reduce the ability of the gastric to epithelial cells to tolerate an environment high in ros, such as would be present with the chronic gastritis associated with h.pylori infection. disturbance of the oxidantantioxidant balance in the stomach might greatly increase the risk of cell death or dna damage, from ros (20, 21). the aim of this study to investigate the relation between the h.pylori with cagapositive and caga negative strains and the production of sod in patients with chronic gastritis (hp+) and healthy control group (hp-). materials and methods eighty subjects (48 male and 32 female; mean age 51.7 ), were referred to the gastrointestinal endoscopy unit at al yarmook teaching hospital, non of whom had received non steroidal antiinflammatory drugs, within previous three months, participated in this study. endoscope fining in the patients were as follows: normal mucosa and no h.pylori infected (30 subjects) and 50 patients with chronic gastritis without ulcer. biopsy specimens were taken from the antrum of all subjects in this study, by using the same size forceps, from similar topographical sites at each endoscopy; biopsies were fixed in 10% formal buffer saline for histological examination. blood samples were taken from all subjects and the serum were stored at -20لċل until be used. histology the biopsy specimens were embedded in paraffin and stained with haematoxylin – eosin (hande) and giemsa stained for h.pylori determination and diagnostic as chronic gastritis. in situ hybridization (ish) for detection of h.pylori / caga gene. in situ hybridization (ish) is a technique makes use of the high specificity of complementary nucleic acid binding to detect specific dna or rna sequence in the cell. for detection of this markers , the biotinylated dna probe hybridize to the target sequence (h.pylori / caga mrna sequence) then a streptavidin-ap (streptavidin-alkaline phosphatase) conjugate is applied followed by addition of the substrate promo-chloro – indolyl – phosphatel / nitroblue tetrazolium (bcip/nbt) which yield an intense blue – black signal appears at the directly specific site of the hybridized probe. this strepteividin – ap conjugate like the biotinylated probe provides raid and highly sensitive detection method. the use of biotin – labeled dna probe for h.pylori / caga (8 g/10015 ml) litter dd h2o . probe size: 349 bp (maxim biotech, inc., u.s.a). scoring hybridization /detection system will give an intense blue –black color at the specific sites of the hybridization probe in both positive test tissues. a scoring system that includes evaluation of the staining percentage of stained gastric cells was employed for the expression of caga gene of h.pylori. counting the number of the positive cells in the gastric tissue which gave a blue-black nuclear staining under the light microscope. the extent of the ish signaling the cells of the examined tissue was determined in 10 fields under high power microscope (40x). in each field, the total of examined cell was about 100 cells per field and this gives a total number. measurement of superoxide dimidiate (sod) for the quantitative determination of superoxide dismutase in whole blood. this produce is suitable for manual use randox, cat. no. sd 125 mixed substrates, buffer, xanthine oxidase and standard. this method employs xanthine and xanthine oxidase (xod) to generate superoxide radicals which react 28 c on ce nt ra tio n of s o d (u /m l) with 2 (4iodophonyl) – 3(4nitrophenol) -5 phanyltetrazolium chloride (i.nt) to form a red fermazan dye. the sod activity is then measured by the degree of inhibition of this reaction. one unit of sod is that which causes 50% inhibition of the rate of reduction of int under the conditions of the assay. xanthine uric acid + o2 i.n.t formazan dye o2 + o2 +2 h o2 + h2o statistical analysis statistical analysis was performed using anova test to determine whether the means were equal among three groups – i.e. caga-, caga+ and controls, p value of < 0.05 was considered statistically significant. results the expression of caga was detected by in situ hybridization technique. from 50 patients complaining chronic gastritis and infected with h.pylori who were tested for caga, 37 (74%) were found to be positive caga and 13 (26%) patients have caga – negative (table 1, figure 1).the mean level of sod increased significantly in patients infected with h.pylori caga positive strains p<0.001 when compared with healthy subjects and patients infected with h.pylori caga negative (table 2,figure 2).the difference in sod level between with h.pylori that have caga positive or caga negative is statistically significant p<0.01. table (1): expression of caga mrna in h.pylori– positive patients with chronic gastritis by (ish). figure (1): gastric antral biopsy specimen from stomach infected with h.pylori that appear curved or round (giemza stain) (100x). table( 2): the serum level of sod in patients infected with h.pylori and caga status. group mean ± s.e sod s.d. p value f test control (hp-) 177.70 ±2.91 9.20 18.56 hp+ caga 14.22 ± 284.00 ٭ 44.95 <0.05 hp+ caga+ 54.71± 477.00٭٭ 73.84 <0.001 no significant difference p> 0.05 significant at the 0.05 levels ٭ significant at the 0.001 level ٭٭لل hp: h.pylori figure (2): detection of caga, in patients with gastroduodenal disease by in situ hybridization. staining of caga mrna by bcip/nbt (blue-black) counterstained with nuclear fast red. tissue from patients with antral gastritis shows positive caga by hybridization signals. 0 100 200 300 400 500 control (hp-) (caga-) (hp+) (caga+) (hp+) figure (3): the serum level of sod in patients infected with h.pylori and caga status. h.pylori positive caga status no. (%) positive 37 74 negative 13 26 total 50 100 29 discussion cytotoxine associated antigen – positive strain was significantly higher (p<0.001) in chronic gastritis patients than in control group. it can be seen that 74% of patients who have h.pylori infection have caga positive strains. h.pylori strains were positive for the caga 74.4% of costa rica patients (22). other study reported the prevalence of caga was more than 80% among patients with chronic gastritis (23). increasing of caga mrna among those patients may explain the role of caga positive h.pylori in the development of gastritis (24). the mechanisms by which caga modify the activity of epithelial cells is explaining by serving as scaffolding protein able to interact and modify the function of a variety of molecules involved in cell to cell interaction, cell motility, and proliferation (23, 25).the differences between the results, possibly by using different methods to assess the expression of caga positive h.pylori in patients with gastritis, such as elisa methods, pcr that the correct design of primers is very important , the different sets of caga primers give different results, and this will attributed to divergence in the primer target sequences (26, 27).h.pylori infection of the gastric mucosa is associated with abundant inflammatory response; this bacterium is capable of stimulating oxidative bursts from noutrophils (28).gastric tissue from h.pylori infected persons contains more ros than normal tissue and there is a direct correlation between bacterial and the amount of ros in the gastric mucosa (29, 30).this study supports the direct correlation between ros and gastric mucosal damages in patients infected with h.pylori which can increase the susceptibility of gastric epithelial cells to ros – associated cell injury. the increase of sod and activity in patients with h.pylori – caga positive strains is probably responsible for the increased survival of these cells. the generation of intracellular ros on the presence of caga positive h.pylori strains is possible explanation for the increases in activity of ros – scavenging enzymes. that h.pylori induces the production of intracellular ros; this increase in ros in gastric cells was enhanced by increasing the concentration of h.pylori and inhibited by use of antioxidant(22).there are more ros in the gastric mucosa of patients infected with h.pylori caga positive strains, is significantly difference from patients with h.pylori caga negative strains. as a response to increase formation of ros the antioxidant enzyme are increase in these cells, suggest that gastric epithelial cells have a higher concentration of these enzymes (sod and catalase ) so they will be able to avoid lethal injury in patients infected with caga positive strains than in patients infected with caga negative strains (13).gastric cells infected with caga-positive h.pylori strains have higher catalase level, catalase enzyme convert hydrogen peroxide to h2o and oxygen molecule. accumulations of hydrogen peroxide reduce the activity of sod; these conversions of hydrogen peroxide protect the cells from sudden exposure to superoxide, giving them a survival advantage (13, 14).the presence of ros along with the reduction of antioxidants, such as vitamin c in the gastric mucosa of people infected with h.pylori potentially increases the risk of oxidant – related cellular injury and dna damage, change in the levels of cellular ros scavenging enzymes induced by h.pylori may further increase this risk of developing gastric cancer from ros in patients infected with h.pylori (31). references 1. feldman, r.a.; eccersley, a.j.p. and hardie, j.m.. transmission of helicobacter pylori curr opin gastroenteral. 1997; 13, 8-12. 2. triantafyllopoulou,m.; caroll, m.; winde, l.; guandalini, s.; schwariz, s. and altschuler, s.: helicobacter pylori infection. medicine; 2006; 55: 386-390. 3. weiss, j.; tsang, t.; meng, x.; zhang, h.; kilner, e.; wang, e. 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hydrogen peroxide in helicobacter pylori: role catalase (kat a) and fur, and functional analysis of a novel gene product designated kataassociated protein, kata (hpo 874). microbiology. 2002; 148: 3813-3825. 31. beswick, e.; suarez, g. and reyes, v.: helicobacter pylori aand host interactions that influence pathogenesis. world j. gastroenterol.;2006;21:5599-5605. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 63 clinical evaluation of a formulated econazol nitrate as a topical solution laith h. samein* received 5-9-2004 acce pted 20-2-2005 abstract econazole nitrate (en) is c ons idered as the mos t e ffec tive age nt fo r the tre atment o f a ll fo rms of d ermatomyco sis ca used by derna to phytes. it was formula te d as a top ical solution in our labo ra torie s. this s tudy was designed to eva luate the e ffec tive ness o f eco na zol nitrate in the prepa red formula and co mpared with that o f c omme rc ial b ra nd , p ev aryl®. a to ta l of 104 pa tient suffering fro m de rma to myc oses were invo lv ed in this inv es tiga tio n. bo th formula were ap plied to the a ffe cted skin re gion in the morning a nd ev ening from wee k to 16 wee ks with light mas sa ge until comp le te hea ling effe ct was achieved . the data revealed tha t the pe rcenta ge of cured patient trea ted with the p re pared fo rmula a nd reference formula o f ec anozo l nitrate 1% so lution were 90.3% and 88.4% respec tively also c hronic cases could be la rgely cure d by tre atmen t with the p repa red fo rmula econazol nitra te 1 % solution. the results o f this clinical inves tigatio n showed that the p repa red fo rmula o f ecano zo l nitra te 1% was effe ctive as comp ared to that of the commercial b ra nd, pevaryl®. الخالصـــة ستخدام هذا الدواء في عالج جميع اشكال الفطريات الجلدية الناتجة عن العوامل يعتبر االكثر فعالية ال) نترات االيكانازول (ان محلول  والذي حضر في المختبرات الصناعية لكلية ( لقد تم تحضير الدواء على شكل محلول لالستخدام الموضوعي . المسببة لتلك الفطريات  المستحضر التجاري بيفاريل كمستحضر قياسي فوجد ان لقد اجريت دراسة سريرية مقارنة بين المحلول المحضر صناعيا و) الصيدلة كال العالجين أظهر فعالية في تقليل االعراض المرضية لكن العالج بواسطة محلول . كل منهما له نفس التاثيرات العالجية والجانبية  التجاري بيفاريل . نترات االيكانوزول كان اكثر فعالية   إلى ان محلول نترات االيكانازول والمستحضر  اعطي اشخاص  104قد ايضا ممكن عالج االصابات المزمنة بصورة . كمجموع كلي يعانون من فطريات جلدية ناتجة عن العوامل المسببة لتلك الفطريات  ان محلول نترات االيكانازول والمستحضر التجاري بيفاريل . المحضر سابقا% 1كبيرة باستخدام محلول نترات االيكانازول بتركيز  اسبوع مع مساج خفيف على المنطقة المصابة  16عا على المنطقة المصابة من الجلد في الصباح والمساء لفترة من اسبوع إلى قد وض .حتى تم شفاء المنطقة المصابة introduction eco na zole nitra te is (rs ) –1 -[2,4 dioc hlorob (p chorobe zyloxyphe ne thyl] i mid azole nitrate (1). ch2 ch – o ch2 it is a white o r a lmo st white crystalline p owde r. m.p. a bout 164, with dec ompo sitio n, v ery slightly s olub le in water and e ther: s oluble 1 in 125 o f e thano l (96%) 1 in 60 of c hloroform and in 25 of methanol (1,.2). eco nazole nitrate is use d for the top ic al trea tme nt of all forms of d ermatomycos is cause d by de rma to phytes like tricop hyton rubum, tricop hyto n menta groghytes tric op hyton tons us ra ns whic h c aus e tinea ped is , tinea c ruris : and tinea c orp oris re spe ctiv ely (3). it is us ed for d ermatomycos is cause d b y yea sts like c and id a albicans and condida guillie rmord i (4). *department of pharmaceutics , co llege of phar mac y, university of bagh dad, ba ghdad iraq n n cl cl cl .hno3 ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 64 eco na zole nitra te is av aila ble in a va riety of dos age fo rm such as s kin crea m (alone or in co mbination with triamcino lo nc ), s kin so lution, s kin lo tion, v agina l s upp ositorie s and sp ra y solution (3) . it has be en prove n to be e ffe ctiv e in the pres enc e of mixed infection. the a ntib ac te rial effe cts of the p re paration offer on a dditio nal ad vanta ge . the purp ose of this wo rk is to e valuate clinically a s elected formula fo r econazole nitra te as topica l solution prep are d in our la borato ry (5). subjects and method a total of 10 4 pa tients w ere involve d in this inv estiga tion. they were diagno sed b y dermatologis t dr. a. als wdany working in med ic al c ity a s ha ving de rmatomycetes cause d by v ario us d ermatomycetes , b la stomyce te s and mould ( 6 , 7 , 8, 9 , 10). the p atie nts age ra nged from 17 -7 0 yea r (ave ra ge 6 3.4 yea rs ). the y were ra nd omly divided into two gro up s (5 2 patient in e ac h ) the patients in group 1 , were 17 fe male (3 3%) and 35 males (67 %) were instructe d to use the p re pared formula of ec ona zo l nitra te 1 % s olutio n. patients in group ii, w ere 16 fema le s (3 0%) a nd 36 males (69 %) were give n the co mmercial of e conazol nitrate 1% s olution, p eva ryl® ciliag. all pa tients were instructe d to a pply e conazol nitrate twic e da ily with light mas sa ge o n to the affe cted s kin re gion. the d uratio n of tre atment was range d from one wee k to 16 wee ks . results and discussion all p atie nts involve d in this study were ev aluated clinic ally, in ad dition to myco lo gical and micros cop ic al examina tion be fo re and after trea tme nt with e ithe r the prepa re d fo rmula of e conazol 1 % s olutio n or p eva ryl® clinical evaluation the re spo ns e to the trea tme nt wa s grad ed as , cured ,po or improve ment and no effe ct (7). the data s howe d that the perce ntage of c ured, poo r imp ro vement a nd no e ffec t in pa tients tre ated with e co nazol nitra te 1% so lution (the prep ared formula) we re 9 0.3 %, 7.8 % and 109 % res pec tive ly as s how n in ta ble -1 . table :1 clinical e valuation of 52 pa tie nts ta king ec onazole 1 % s olut io n. no. o f pa tie nts 5 2 1 00% cure d 4 7 90.3 % poo r impro ve me nt 4 7.8% no e ffe ct 1 1.9% the res ults o f patient trea te d with pev aryl® solution s howe d that 88.4 % we re c ured, 9.7 % of po or improv ement and only 1.9% with no effe ct a s illus trate d in tab le -2. table –2 clin ic al e va luatio n of 5 2 patie nts taking peva ry l® as a re fere nce . no . o f patie nts 52 1 00% cure d 46 88.4% poor impro ve me nt 5 9.7% no e ffe ct 1 1.9% mycological evaluation p rior to the ra py the fungus spe cies c ould be demo ns trated by a p ositiv e c ulture in 20 patients out of 52 pa tients treate d with either the prepa re d fo rmula o r re fe re nc e (pe va ryl®) of e conazole nitrate 1 % s olutio n. tab le –3 showed tha t no more fungi we re prese nt in 14 patients , (7 0% ) tre ated with eco na zole 1% so lution while 6 pa tie nts could not be c hec ke d myc ologica lly, as there were no samples av aila ble due to he aling of myco sis. myco lo gical e xa minatio n in patient treate d with (pev aryl®) showed nega tiv e fungi in 13 patient (6 5%) a s shown in table 4 . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 65 table –3 mycologica l e valua tion o f 20 patie nts ta king econazole 1 % s olutio n. no. of patie nts 2 0 100% fung us ide ntific atio n pr io r to the ra py + afte r the rapy cure d 1 4 7 0% fu ngus ide ntifica tion prio r to the ra py + afte rthe ra py cure d no sa mple ( cure ) 6 3 0% table –4 mycologica l e valua tion o f 20 patie nts taking pev ary l® as re fre nce . no. of patie nts 20 100% fungus ide ntification prior to the rapy + afte r the rapy c ure d 13 65 % fung us ide ntific atio n prior to the rapy + afte r the ra py c ure d no s ample ( c ure ) 6 30 % fungus ide ntification prior to the rapy+ afte r the ra py + not c ure d 1 5% microscopical evaluation the diagnos is was also c onfirme d by pos itive mic ro sc opica l fungus id entifica tion prior to therap y in 25 pa tients tre ated with eco na zole 1% s olutio n o r pev aryl® .the perce ntage trea te d with e co nazole 1% so lution and pe varyl® w ith a negativ e funge s we re 80% and 76% resp ec tive ly a s s hown in tab le 5 and 6 .microcs op ic al e xa mination co uld no t be performed in 4 c ase s (16 %) treate d with eco na zole 1 % and 5 c as es (20 %) used pev aryl® , sinc e no s amp le were ava ila ble due to hea ling o f myc os is . table –5 mic ro sco pica lly results of 25 pa tie nts tak ing eco naz ole 1% so lution . no. of patie nts 25 100% fung us ide ntific ation prior to the rapy + cure d afte r the rapy 20 80% fung us ide ntific ation prior to the ra py + afte r the rapy no sa mple cure d ( cure ) 4 16% fungus ide ntification prior to the rapy+ afte r the ra py + not cure d 1 4% ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 66 table –6 micro scopica lly res ults o f 52 patie nts ta king pe varyl as refre nce . therapeutic re sults in chronic case s: 12 patients o f 52 p atie nts who s uffere d from chro nic myco sis ( dura tion 1 -5 years ) we re trea ted prep ared fo rmula of e co nazole the d ata that 1 1 p atie nts ( 9 1.7 ) we re cure d a fter trea tment with a verage d uratio n o f (2-16) wee ks and o nly one pa tie nt (8.3 ) wa s not re sp ond a s shown in table7 . ta ble – 7 the ra pe utic results in 12 patie nts with c hronic myc os is la sting fo r more than one ye ar. the app earance of any s id e effe ct a fter the trea tme nt with b oth formulas wa s als o monitored burning and pruritrs (11,12,13,14,15 ) were obs erve d in only 3 p atie nts tre ated with eco na zole 1% so lutio n (o ut o f 52 p atients conclusion the re sults of this c linica l inv es tiga tion clearly indica te d that the prep ared o f ec ona zo le nitrate 1% wa s effe ctiv e as c omp ared to that of commercial b ra nd pe varyl®. no. of patie nts 2 5 100% fungus ide ntification pr io r to the rapy + afte r the ra py c ure d 1 9 76% fungus ide ntification prio r to the rapy + afte r the rapy cure d no sa mple ( cure ) 5 20% fungus ide ntification pr io r to the rapy + afte r the ra py + no t c ure d 1 4 % pa t. no duratio n of dise ase in months duration of tre atme nt in weeks cure d no t cure d 1 2 3 4 5 6 7 8 9 10 11 12 12 12 14 24 13 12 60 16 14 12 12 30 2 1 1 2 2 3 .5 2 14.5 1 6 2 9 2 5 .5 + + + + + + + + + + + 12 pat. 15 yea rs 216 we eks 11 pat. 95% 1 pa t. 8.3% ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 67 references 1 britis h pha rma copoeia . typo grao hy b y majesties sta tionary a offic e , lo ndo n , camb rd ge2 001, vo l.1 ,245. 2 clark’s. is olation a nd identification of drug, 2nd.ed . the pharmace utical press;londo n; 19 86, 579 . 3 hta ntsc hke , d.. in vitro sensitiv ity te st with antinycotic imido zo lc de riva tives and ava luation of re sults. myko se n, 1 97 8,sup pl i, 222 -9 . 4 sc holar-m; p re diel—h; korting—iic, light and ele ctio n mic roscopic finding in a mode l of hu man coe tane ous cand id ac ies, journal o f drug ta rgeting, 1 99 9, 6(5) 36 1372 . 5sa min, l, h., formula tion o f ec onazo le nitrate as a to pical solution, ira qi journa l of pha rma ce utic al science s, 200 5,14, 20 30. 6le fler —f; stev ens -da, inhibition and killing of candida in vitro by five imid azo le in clinica l use . anti mic ro b — agents — che mothe r (antimicrobial-agents -a ndche mothe ra py), 1984, 25(apr), 450 -454. 7 culle n — s i; rexih; thorne — eg, comp arison of a new antifungal a gent, i perce nt eco nazole nitra te (specsa zo le ) crea m versus i pe rc ent clotremazole cream in the trea tme nt o f i ntertrigi nous ca ndidosis, currthe r-res (current — therap utic -research); 198 4, 35 (apr), 606 -6 09. 8stock-r, how effective is antimyc otic d rug? , pha rm-int: 19 81, 2(oct), 23 2-236 . 9f redrikson-t, trea tment of d crmatomyco ses with topical fconazole and clo trimazo le , e urr-the r-res-clin-exp; 1979 , 2 5 (ma y), 596-594. 10ve rma -bs , eco nazole cream in fungal infe ctio n o f the s kin currf her—res-clinexp, 1978, 24(nov ), 745-752 . 11schaller — m; p re idel-h; janus chke-e; korting-hc, li ght and e lectron microscop ic findings in a mode l of hu man cuta ne ous candiois.j-drug-ta rget (journal-o f-drugtargeting), 1999, 6 (5 ), 361 -3 72. 12 ode h-f; fa hry — h; k uhlwein-a; nolting —s; s arta ni-a; et-a l. doub le-b lind c linic al trail with a single d aily app lica tion o f entico na zole , curr-ther-rcs (curre nt — theraputic-res ea rc h), 1990, 47 (ja n), 2 2-31. 13 romague ra -c; he rrero-e; ma rquez-m; torres-j; ortiz-j a, s tudy on the s ens itizing capac ity o f the ne w ataimyc otic sertacona zo le in the trea tme nt o f cuta ne ous mycoses, arzeneim-eorsc h (arzneimitte lf orsc hurg), 1 992 , 4 2 (5 s upp l), 754-756. 14pigatto -p ; colli-e; scatigna -m; finzi-a. evaluatio n o f s kin irritatio n a nd c onta ct sensitizing potential of fcnticonazole. arzneim-forsc h arzneimittel-forsc hurg), 1 99 0,40(3), 329-33 1. 15 te tt-se. to pical imidazoles . aust-jp ha rm (australia njo urnal0ft pha rma cy), 1 98 6,67 (fun), 567 -5 69. iraqi j pharm sci, vol.26(2) 2017 cefixime and α-naphthol 1 spectrophotometric determination of cefixime following simple diazotization and coupling with α-naphthol samar a. darweesh*,1 *department of chemistry, college of education for pure scienceibn al-haitham ,university of baghdad,baghdad,iraq. abstract cefixime (cfx) was treated with sodium nitrite and hydrochloric acid for diazotization reaction followed by coupling with α-naphthol in alkaline medium to form, a yellow colored azo dye compound which exhibits maximum absorption (λmax) at 412 nm where the concentration of (cfx) was determined spectrophotometrically. the optimum reaction conditions and other analytical parameters were evaluated. beer’s law was obeyed in the concentration range of (1-20) μg.ml-1 with a molar absorptivity of 34870.5 l.mol1.cm-1. the limit of detection was found to be 0.1090 μg.ml-1 and the sandell's sensitivity value was 0.0130 μg.cm-2. the proposed method could be successfully applied to the determination of (cfx) in pharmaceutical formulations. keywords: spectrophotometric determination, cefixime, diazotization reaction, α-naphthol, coupling reaction. فثول ان مع الفا قترانزوتة البسيطة واألعتماد على االللسفكسيم باأل التقدير الطيفي سمر احمد درويش *,1 بغداد ، العراق .قسم الكيمياء ، كلية التربية ابن الهيثم للعلوم الصرفة ، جامعة بغداد ، * الخالصة فثول ان مع نتريت الصوديوم وحامض الهيدروكلوريك ألزوتته ثم تبع ذلك اجراء تفاعل اقترانه مع الفا (cfx) عومل عقارالسفكسيم نانوميتر ومن ثم تم تقديرتركيزالسفكسيم 214عند max(λ(في وسط قلوي لتكوين صبغة األزو ذات اللون األصفر التي تظهر أعظم امتصاص التراكيز الذي من مدى على بير قانون طيفياً. وقد تم تعيين الظروف الفضلى التي تؤثر على التفاعل والعوامل التحليلية األخرى. تم التجاوب مع 2412.2 لتر/مول/سم وكان حد الكشف يساوي 527843لـ وكانت قيمة معامل االمتصاص المولي مساوية ( مايكروغرام/مل42-1يتراوح بين ) بنجاح لتقدير السفكسيم في المستحضرات . لقد أمكن تطبيق الطريقة المقترحة2مايكروغرام/سم 242152مايكروغرام/مل ومعامل ساندل يساوي الصيدالنية. تفاعل األقتران. ، فثولان الفا ،تفاعل األزوتة، سفكسيم ،الكلمات المفتاحية: التقدير الطيفي introduction cefixime is a semisynthetic, cephalosporin antibacterial for oral administration (scheme 1). scheme (1): the chemical structure of cefixime. chemically it is (6r,7r)-7-{(2-(2-amino-1,3thiazol-4-yl)-2-(carboxy-methoxyimino )acetyl ) amino } – 3 – ethenyl – 8 – oxo – 5 – thia – 1 azabicyclo(4 . 2 . 0 ) oct – 2 – ene – 2 carboxylic acid trihydrate. it is chemical formula is (c16h15n5o7s2. 3h2o) and with molecular weight of (507.50) as the trihydrate. its under the category of ß-lactam antibiotics/cell wall inhibitor. it acts by inhibiting an enzyme transpeptidase, involved in the building of bacterial cell walls. it is used in lower respiratory tract infections. it is helpful in acute urinary tract infections, biliary tract infections, sinusitis, acute otitis media, peptic ulcer and many more (1). cefixime has been determined by the development of several analytical techniques such as flow injection analysis (2), hplc (3-5), potentiometric method (6), voltametric method (7), spectrophotometric method (8-12). 1corresponding author e-mail: s_a_1981@yahoo.com received: 1/6/2016 accepted: 4/1/2017 mailto:s_a_1981@yahoo.com iraqi j pharm sci, vol.26(2) 2017 cefixime and α-naphthol 2 the present study describes the use of α-naphthol as a chromogenic reagent in the development of simple, sensitive and a rapid spectrophotometric method for the estimation of cfx with reasonable precision, accuracy. experimental instruments the absorption spectra and all spectrophotometric measurements were carried out on a double beam (shimadzu 1800) spectrophotometer with 1cm matched quartz cells. materials and reagents pharmaceutical grade cefixime trihydrate received as a gift powder sample with a purity of pure form (99.99%) from the state company for drug industries and medical appliances samarairaq (sdi). all chemicals and reagents used were of analytical grade. cefixime (400 mg capsules or 200 mg film coated tablets were purchased under the brand names (suprax-caps-al-hikma-jordon and samaxim-s.d.i-iraq) from the iraqi pharmaceutical market. preparation of solutions 1sodium nitrite (1 % (wt/v)): was prepared by dissolving 1 g of nano2 in 100 ml distilled water. 2hydrochloric acid (1 m): was prepared by taken 8.6 ml of concentrated hcl and diluted to 100 ml with distilled water. 3sulfamic acid (0.5 % (wt/v)): was prepared by dissolving 0.5 g of sulfamic acid in 100 ml of distilled water. 4sodium hydroxide (6 m): was prepared by dissolving 24 g of naoh in 100 ml of distilled water. 5α-naphthol (2 % (wt/v)): was prepared by dissolving 2 g of α-naphthol in l00 ml of 4 m naoh. standard drug preparation (100 μg.ml-1) standard solution of cfx was prepared by dissolving accurately weighted amount of the pure drug equivalent to 10 mg in 1ml of concentrated hcl and further diluted to 100 ml with distilled water. preparation of drug solutions ten capsules of cefixime were weighed, and an accurately weighted portion of the powder equivalent to 200 and 400 mg of cefixime were dissolved in a 5 ml of concentrated hcl and mixed well and then filtered by using whatman filter paper no.41. then the volume was diluted to 100 ml with distilled water and analyzed as given under the assay procedures for bulk samples. procedure for assembling the calibration curve to aliquots of the standard solution (l00 μg.ml-1) containing (5-150 μg) of cfx were transferred into a series of 5 ml volumetric flasks and cooled in an ice bath, and then 0.5 ml of 0.5 % (w/v) sodium nitrite solution and 0.5 ml of 0.3 m hcl were added. each solution was shaken thoroughly and left to stand for 2 min., then 1.0 ml of 0.1 % (w/v) sulfamic acid was added. the solutions were swirled and the resulting diazotized product was coupled with α-naphthol by the addition of 0.5 ml of 0.8 % (w/v) of this reagent in 1 m sodium hydroxide solution. the mixtures allowed to stand for 2 min., after that they were made up to the mark with distilled water. the absorbance of the yellow colored chomogen was measured at 412 nm against the reagent blank. the constructed calibration curve was used to compute the amount of cfx in the given samples. results and discussion absorption spectra and reaction scheme in the method developed the presence of the aromatic amino group in cefixime, enable the use of diazotization of the drug with nitrous acid and coupling the resulting diazonium salt with αnaphthol to form colored azo-dye with a maximum absorption at 412 nm. the absorption spectra of the above dye are presented in figure 1. figure (1): absorption spectrum of (a) complex against (b) reagent blank solution. two steps are involved in the reaction that produces the colored dye. in the first step, cefixime is treated with nitrite solution in hydrochloric acid medium, undergoes diazotization to give diazonium ion. in the second step, the diazonium ion is coupled with the coupling agent α-naphthol, to form yellow colored azo-dye in an alkaline medium. the proposed chemical reactions are shown in scheme 2. iraqi j pharm sci, vol.26(2) 2017 cefixime and α-naphthol 3 scheme (2): the suggested reaction mechanism for diazotization and reaction between cefixime and αnaphthol. optimization of reaction variables effect of sodium nitrite the effect of adding various amounts of sodium nitrite solution on absorbance of 10 μg ml-1 cefixime was examined. the concentration of sodium nitrite was varied between 0.5 ml of (0.1-1%) sodium nitrite in water. the results showed that 0.5 ml of 0.5% sodium nitrite gave maximum absorbance (figure 2). below and above this concentration the absorbance was slightly decreased. figure (2): effect of sodium nitrite concentration. effect of hydrochloric acid diazotization was carried out at 0-5 ˚c. the hydrochloric acid concentration was studied between (0.1-1.0) m and 0.3 m hydrochloric acid concentration was fixed for getting a stable diazonium ion, (figure 3). figure (3): effect of hydrochloric acid concentration. effect of diazotization reaction time the optimum time for the diazotization of cefixime were established at 0-5 °c and 2 minutes, where longer times led to extremely low absorbance values. this implies that long time destroy the diazotized product as shown in figure 4. figure (4): effect of diazotization time. iraqi j pharm sci, vol.26(2) 2017 cefixime and α-naphthol 4 effect of sulfamic acid concentration the excess of nitrite could be removed by the addition of 1 ml of 0.1% sulfamic acid. an excess of sulfamic acid decrease the color intensity of product, (figure 5). figure (5): effect of sulfamic acid concentration. effect of α-naphthol concentration to optimize the concentration of coupling agent, 0.5 ml of different amounts (0.1-2%) αnaphthol were added to the mixture under study. it was found that 0.8% of α-naphthol solution was sufficient for maximum and stable color development. there was a decrease in absorbance at higher concentration of 0.8% α-naphthol, as shown in figure 6. figure (6): effect of α-naphthol concentration. effect of alkalinity the optimum concentration of sodium hydroxide leading to a maximum intensity of the azo dye was found to be 1 m. higher concentrations of alkali may lead to partial decolorization of the dye. figure 7 illustrates the results of the study. figure (7): effect of sodium hydroxide concentration. effect of coupling reaction time it was found that maximum absorbance is attained after 2 min further increase of the reaction time results in a gradual decrease in the absorption intensity, therefore 2 min was chosen as the optimum reaction time, figure 8. figure (8): effect of coupling reaction time. stability of the colored product as shown in figure 9, a slight decrease is observed upon leaving the colored product, after dilution, for a while of time. therefore, it is advisable to measure the absorbance immediately after color development. figure (9): stability of the colored product. iraqi j pharm sci, vol.26(2) 2017 cefixime and α-naphthol 5 calibration curves and analytical data a calibration curve for cefixime drug was constructed and found to be linear over the concentration range of (1-20) µg.ml-1. figure 10 shows that the high value of the correlation coefficient for the regression equation, which indicates a good linearity over working concentration range. the statistical treatments of the analytical data are summarized in table 1. figure (10): calibration curve for the determination of cefixime. table (1): optical characteristics and statistical data for the determination of cefixime. parameter value max (nm) 412 color yellow linearity range (µg.ml-1) 1-20 regression equation a= 0.0769 (cfx. µg.ml-1) 0.0601 calibration sensitivity 0.0769 correlation coefficient (r) 0.9996 correlation of linearity (r2) 0.9992 molar absorptivity (l.mol-1.cm -1) 34870.5 sandell's sensitivity (µg.cm-2) 0.0130 detection limit (µg.ml-1) 0.1090 precision and accuracy cefixime was determined at four different concentrations in three replicates during the same day. the analytical results found after the investigation are briefed in table 2 indicated good accuracy with reasonable precision of the proposed method. table (2): accuracy and precision of the proposed method. sample conc. of cfx (µg.ml-1) relative error % standard deviation relative standard deviation % taken found* cefixime 5.00 5.10 +2.0 1.8470 0.0942 10.00 9.86 -1.4 1.0375 0.1023 15.00 15.15 +1.0 0.7551 0.1144 *average of three determinations application on pharmaceutical formulations the suggested method was used for the quantification of cefixime in commercial tablets and capsules. the results, which are demonstrated in table 3, were satisfactory. table (3): application of the proposed method to the cefixime concentration measurements in pharmaceutical formulations. sample labeled amount of cfx (mg) *found amount of cfx (mg) recovery % standard deviation * relative standard deviation % suprax-caps al-hikma jordon 200 204.92 102.46 0.8673 0.4232 400 397.53 99.39 0.9542 0.2400 samaxim s.d.i iraq 200 198.06 99.03 0.7301 0.3686 400 396.73 99.18 1.1420 0.2879 *average of three determinations. iraqi j pharm sci, vol.26(2) 2017 cefixime and α-naphthol 6 conclusions diazotization reaction of primary amine group followed by coupling with α-naphthol in alkaline medium was found to be a simple, sensitive, accurate and economic spectrophotometric method for quantitative determination of cefixime in pure form and pharmaceutical formulations. different variables affecting the completion of the reaction were optimized. the proposed method offers good linearity and precision. acknowledgement thanks for everyone who helped me to accomplish this research. references 1. dey s, pradhan p k, upadhayay u m, shah sh, goswami k, uv spectrophotometric determination of cefixime in bulk and its dosage form. j. of pharm. res. 2012; 5(12): 5419-5422. 2. al-momani i f, spectrophotometric determination of selected cephalosporins in drug formulations using flow injection analysis. j. of pharm. and biomed. anal. 2001; 25: 751-757. 3. bhingea s d, malipatil sh m, development and validation of a stability-indicating method for thesimultaneous estimation of cefixime and dicloxacillin using therp-hplc method. j. of taibah uni. for sci. 2016; 248: 1-11. 4. bhinge s d, malipatil sh m, sonawane l v, chittapurkar h r, simultaneous estimation of cefixime and dicloxacillin in bulk and tablet formulation by rp-hplc method. fabad j. pharm. sci. 2012; 37: 63-71. 5. raj k a, yada d, yada d, prabu c, manikantan s, determination of cefixime 6. trihydrate and cefuroxime axetil in bulk drug and pharmaceutical dosage forms by hplc. int. j. of chemtech. res. 2010; 2(1): 334-336. 7. kulapina o i, makarova n m, kulapina e g, potentiometric sensors for the determination of some cephalosporin antibiotics in biological fluids and medicinal preparations. j. of ana. chem. 2015; 70: 477-484. 8. golcu a, dogan b, ozkan s a, anodic voltammetric behavior and determination of cefixime in pharmaceutical dosage forms and biological fluids. talanta 2005; 67: 703-712. 9. ramadan a, mandil h, dahhan m, uv-vis spectrophotometric study for determination of cefixime in pure form and in pharmaceuticals through complexation with cu(ii) using acetate–naoh buffer in water : methanol. int. j. of pharm. and pharm. sci. 2013; 5:428433. 10. ramadan a, mandil h, dahhan m, novel formation three complexes of cefixime-copper using acetate–acetic acid buffer and determination of cefixime in pure and pharmaceutical dosage forms. int. j. of pharm. and pharm. sci. 2013; 5: 469-477. 11. keskar m r and jugade r m, spectrophotometric determination of cefixime trihydrate in pharmaceutical formulations based on ion-pair reaction with bromophenol blue. ana. chem. insig. 2015; 10: 11-16. 12. ahmad n r, spectrophotometric determination of cefixime through schiff’s base system using vanillin reagents inpharmaceutical preparations. iraqi nat. j. of chem. 2013; 49: 38-46. iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin doi: https://doi.org/10.31351/vol28iss2pp73-82 73 synthesis and characterizations of dipeptide derivative of gentamicin oun d. khudair*,1 and diar a. fatih**  department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract the target derivative is gentamicin linked with l-vall-ala by an ester linkage. it was synthesized by esterification method, which included the reaction of hydroxyl group on (carbon no.5) of gentamicin with the acid chloride of the corresponding dipeptide. the preparation of new derivative of gentamicin involved protection of the primary and secondary amino groups of gentamicin, by ethylchloroformate (ecf) to give n-carbomethoxy gentamicin which was used for further chemical synthesis involving the free hydroxyl groups. the prepared dipeptide (l-vall-ala) by conventional solution method in the presence of dcc & hobt then reacted with thionyl chloride to prepare acid chloride of dipeptides, then after, linked by ester linkage to n protection gentamicin in present pyridine as base, finally deprotection the amino group of synthesized compound by using trifluoroacetic acid (tfa) in the presence anisole. the characterization of the titled compound was performed utilizing ftir spectroscopy, chno elemental analysis, and by measurements of their physical properties. keywords: gentamicin, dipeptide, chemical synthesis. وتوصيف مشتق ثنائي الببتيد من الجنتاميسين حضيرت عون دلي خضير*،1 و ديار عارف فاتح ** بغداد،العراق. جامعة بغداد، فرع الكيمياء الصيدالنية ،كلية الصيدلة،* ةالخالص ، والتي شملت تفاعل ها عن طريقة األسترةحضيربواسطة وصلة استر. تم ت l-val-l-ala لمشتق المستهدف هو جنتاميسين مرتبط بـا ( من جنتاميسين مع كلوريد حامض من ثنائي الببتيد المقابل ، إعداد مشتق جديد من الجنتاميسين المتضمن 5على )الكربون رقم مجموعة الهيدروكسيل ncarbomethoxy gentamicin( العطاء ecf) جنتاميسين ، بواسطة إيثيل كلوروفورماتللحماية مجموعات األمين األولية والثانوية (l-val-l-ala) بعد ذلك يتم تحضير ثنائي الببتيد .الذي تم استخدامه لمزيد من التخليق الكيميائي الذي يشمل مجموعات الهيدروكسيل الحرة بتيد ، ثم بعد ذلك ، ثنائي الب إلى كلوريد حامض منالثاينويل الحالي ثم تفاعل مع كلوريد hobt و dcc بوجود باستخدام طريقة الحل التقليدي في tfaa مركب باستخدامالاألمينية من المجاميع حريرجنتاميسين في البيريدين الحالي كقاعدة ، وأخيرا ت n-protection يربطه ارتباط استر بـ anisole الحالي. وعن طريق قياس خواصها chnoر تحليل العناص باالشعة تحت الحمراء تم تنفيذ توصيف المركبات المعنونة باستخدام التحليل الطيفي الفيزيائية. .جنتاميسين ، ثنائي الببتيد ، التوليف الكيميائيالكلمات المفتاحية : introduction gentamicin is an aminoglycoside antibiotic, used to treat many types of bacterial infections, particularly those caused by gramnegative bacteria. gentamicin sulphate is the sulphate salt, or a mixture of such salts, of the antibiotic substances produced by the growth of micromonospora purpurea.. gentamicin is a bactericidal antibiotic that works by binding the 30s subunit of the bacterial ribosome, interrupting protein synthesis (1, 2). like all aminoglycosides, when gentamicin is given orally it is not systemically active. this is because it is not absorbed to any appreciable extent from the small intestine. aminoglycosides are poorly absorbed from the gastrointestinal tract and are administered parenterally (4). their high water solubility and low protein-binding facilitate distribution(5, 6). gentamicin is the only heat-stable antibiotic, hence its use during orthopedic surgery when high temperatures are required for the setting of cements (e.g. hip replacements) (1,3). colon –specific delivery of bioactive compounds received extensive investigations, utilizing the significantly variable bio environments of the different parts of the g.i.t (4). prodrugs are bioreversible derivatives of drug molecules that undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug. strategies to improve the oral bioavailability and achieve specific targeting have been the most important developments in prodrug design during the last 5 years. (5) 1corresponding author e-mail : oun.alrubeai@gmail.com received: 20 / 1 / 2019 accepted: 1 / 6 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp73-82 iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 74 transport of dior tripeptides across plasma membranes in the small intestine and the kidney proximal tubules play a pivotal role in efficient absorption of protein digestion products. the absorption process is mediated actively by h+ -coupled peptide transporters localized in the brush-border membranes of these epithelia. (6) there are two types of human peptide transporters: (7) human peptide transporters 1 (hpept1) that is present in the brush border of the small intestine and predominantly in the upper part, human peptide transporters 2 (hpept2), which are mostly abundant in the kidney (6) . the identification of two peptide transporters, pept1 and pept2, represented a major step forward toward molecular understanding of the physiological and pharmacological significance of peptide transporters (6) ,among various membrane transporters, peptide transporters are the most attractive targets in prodrug design to improve oral drug absorption(7). the peptide transporter is a possible route for improving the intestinal absorption of pharmacologically active amino acid analogues. as examples of compounds without a peptide bond, the aminopeptidase inhibitor arphamenine, the antiviral agent valacyclovir and 4-aminophenylacetic acid were reported to be substrates for peptide transporter (8). figure1. substrates of peptide transporters materials and methods all chemicals and solvents used during synthesis were of analytical grade and used without further purification as follow ; gentamicin sulphate(hyperchem, china), lalanine, acetone, boc l – valine, chloroform, ethanol (98%),methanol (98%) thionyl chloride , and trifluoroacetic acid (tfa) (fluka ag, germany), acetyl chloride, acetic acid (glacial) , dichloromethane, methylene chloride, hydrochloric acid (33%) pyridine ,and tetrahydrofuran (thf)( bdh, england), dimethylformamide (dmf)and triethylamine (tea)( fluka ag , switzerland), ethyl chloroformate (ecf) and silica gel f254 aluminum sheets,( merck, germany), anisole(gillard chem.,uk), hydroxybenzotriazole (hobt)( sigmaaldrich,usa), petroleum ether (60 o -80 o c)( bdh,uk), n,n'-dicyclohexylcarbodiimide (dcc)( sigma-aldrich,usa). completion of reactions and the purity of compounds were ascertained by thinlayer chromatography (tlc), using silica gel gf254 (type 60) precoated aluminum sheets, merck (germany) exposed to uv-254nm light, or by reacting with iodine vapor, or with ninhydrine spray reagents for detection of dipeptide, and the eluent used is chloroform: nh n n nh2 o ho o aciclovir nh n n nh2 o o o valaciclovir h2n h3c ch3 o hn n ch3 o o o oh3c ch3 o nh2 h2n l-valyl ester of azt iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 75 ethanol (7:3) for intermediate ia & methylene chloride: ethanol (7:3) for others to run tlc. melting points were determined using stuart smp3 melting point apparatus in open capillary tubes, and are uncorrected. fourier transform infrared spectroscopy (ftir), were recorded using (biotech engineering management ftir600, uk). the elemental microanalysis of the synthesized compounds was done using (elemental vario micro cube instrument, germany). chemical synthesis general method for synthesis of intermediate ia the primary and secondary amino groups of gentamicin were protected (9), (10), by ethylchloroformate (ecf) to give ncarbomethoxy gentamicin which was used for further chemical synthesis involving the free secondary hydroxyl groups. gentamicin sulphate (0.0008 mol, 0.46 gm) dissolved in methylene chloride (60 ml) containing triethylamine (0.013mol, 1.8 ml) and was cooled to (-5oc). ecf (0.0043 mol, 0.53 ml) in 40 ml of thf (tetrahydrofuran) was added. the reaction mixture was left at (5 oc) for 30 min with continues stirring. the temperature of the reaction mixture was allowed to rise to room temperature gradually and the mixture was continuously and vigorously stirred for 6 hrs. the solvent was evaporated under vacuum. the residue was redissolved in methylene chloride and d.w. acidified with 5% hydrochloric acid solution to wash out the unreacted materials. the organic layer was separated, washed several times with saturated sodium chloride solution and then d.w. the organic layer was dried using anhydrous magnesium sulfate filtered and methylene chloride was evaporated under vacuum leaving a white precipitate recrystallized using (70% methanol).the percentage yield, and rf values are listed in table 1, the ir characteristic bands of ncarboethoxygentamicin (compound ia) are listed in table 2, chno data are listed in table 3. chemical synthesis of compound ib (synthesis of boc l-val -l-ala dipeptide) coupling is done by conventional solution method; (0.082 mol, 1.8 gm) of boc – l-valine in 10 ml of dmf were cooled to 0° c, (0.019 mol ,4.12 gm) of dcci was added the solution was stirred with cooling (9) a solution of (0.082 mol, 0.73 gm) alanine in 10 ml of dmf (previously cooled to 0°c) was added to the first stirred solution then kept the reaction mixture cooled to (-15 °c) for 10 min, hobt (0.015, 2.16gm) was added with stirring. the stirring was continued for two days at 0° c .then two days at room temp .filtration was carried out to remove dcu precipitate formed during the reaction, the solvent was evaporated under reduced pressure to remove dmf .the residue was redissolved in chloroform (50 ml), washed with cold water (2× 20 ml). the organic layer collected, dried over anhydrous magnesium sulphate, the volume was reduced then, diethyl ether was added to get crystals kept in deep freeze. the physical appearance, percent yield, melting point, and rf values are listed in table 1, the ir characteristic bands are listed in figure 3, chno data are listed in table 3 (9) . synthesis of boc l-val –l-ala acetyl chloride (compound ic) this intermediate was synthesized by the conversion of compound ib into its corresponding acid chloride by reacting with thionylchloride and as follows: compound ib (0.0057 mol, 1.64 gm) was dissolved in (30) ml chloroform . excess of thionyl chloride (0.0085mol, 0.6 ml) was added drop wise with continuous stirring at 0◦c. the temperature of the reaction was raised gradually and refluxed for three hours. the reaction mixture then cooled; the volume was reduced by evaporation to small volume to remove excess of thionyl chloride then the residue was re-dissolved in 30 ml of chloroform and re -evaporate to ensure the removal of all thionyl chloride. brown, oily residue was obtained. this compound was used immediately for other synthesis. (11) chemical synthesis of boc l-vall-ala ester of gentamicin (compound id) compound ia (0.00066 mol, 0.5gm) was dissolved in 50 ml of (1:1) pyridine: dmf, cooled in ice bath to 0 o c with continuous stirring. compound ic (0.00066 mol, 0.19 gm) were dissolved in 30 ml of chloroform,(prepared immediately and cooled) was added drop wise to the (previous solution) . the reaction mixture was stirred under cooling for 5 hours, then left to stand for 12 hour at same temperature .the solvent was evaporated to small volume. 50 ml of water added to remove the pyridine hcl and the dmf (12). then evaporated under vacuum leaving a dark yellowish precipitate re-crystallized by dichloroethane chemical synthesis of compound 1; synthesis of l-val l-ala ester of gentamicin; (deprotecting of amine groups of gentamicin) compound (id) (0.00005 mol, 0.05gm) was dissolved in methylene chloride (10ml) and was stirred with tfa (10 ml) for 1 hr. at 0 o c in the presence of (1-2) drops of iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 76 anisole, and kept at room temp for 3 hrs. tlc for the reaction mixture was performed to ensure the removal of the amino–protective group. ether (30 ml) was added to the reaction mixture and the resulting precipitate was collected. the precipitate was suspended in d.w. (30 ml) and the ph was adjusted to 5 with 5% nahco3. the reaction mixture was filtered and a yellow precipitate was collected, washed with d.w. (30 ml × 2), dried in an oven at 50oc. the precipitate was triturated with ether, filtered and crystallized using petroleum ether to form yellowish crystals (11, 13) . the physical appearance, percent yield, melting point, and rf values are listed in table 1, chno data are listed in table 3, and the ir characteristic bands are listed in figure 3. results and discussion spectral data and chemistry the synthesis of compounds (intermediates and end products) is presented in scheme 1.the synthesized compounds were identified and characterized by their melting points, rf values, and physical appearance, these data are listed in the table 1, the ir data in table 2 . the elemental microanalysis data with their interpretations are presented in table 3. scheme 1.synthesis of compound i. table 1. melting points, rf values, and physical appearance b: ethylene chloride: methanol (8:2). c: chloroform: ethanol (7:3) compound physical appearance % yield melting point observed (oc) rf value ia white crystals 93 161-163 (c) (0.53) ib oily 35 (b) (0.45) i brown crystals 80 112-114 (b) (0..63) iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 77 table 2. ft-ir spectrum of compound interpretatio bands (cm-1) compound band of primary nh2 3451 broad for oh stretching vibration 3647-3562 n-h stretching of secondary amide asymmetric c-h stretching of ch3 & ch2 groups 3141 2964 symmetric c-h stretching of aliphatic ch3 & ch2 groups 2870 asymmetric c-h bending of ch3 groups 1471 n-h bending of secondary amide 1604 1589 c-o stretching vibration of 2◦ alcohol 1087 c-o-c stretching vibration of ether 1249-1294 broad for oh stretching vibration 3444 n-h stretching of secondary amide asymmetric c-h stretching of ch3 & ch2 groups 3279-3242 2964 symmetric c-h stretching of aliphatic ch3 & ch2 groups 2872 c=o stretching of amide 1739 n-h bending of secondary amide 1496 iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 78 asymmetric c-h bending of ch3 & ch2 groups 1454 c-o stretching vibration of secondary alcohol 1257 c-o-c stretching vibration of ether 1049 n–h stretching of urethane. 3336 n–h stretching of the secondary amide & o-h st. of cooh. 3298 asymmetric c– h stretching of methyl group. 2974 symmetric c–h stretching of methyl group. 2875 c=o stretching of carboxyl group. 1708 c=o stretching of the secondary amide. 1653 n–h bending of the secondary amide. 1516 asymmetric c– h bending of methyl group. 1456 symmetric c–h bending of methyl group. 1365 n-h stretching of 2o-amide. 3284 broad alcoholic o-h stretching vibration 36262560 asymmetric c-h stretching of methyl & ch2 group 2918 iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 79 symmetric c-h stretching of methyl & ch2 group 2848 c=o stretching of ester 1768 c=o stretching of amide group 1681 n-h bending of secondary amide 1529 asymmetric c– h bending of methyl group 1462 symmetric c–h bending of methylene group 1432 c-o stretching of ester. 1203 table 3. the elemental microanalysis of the synthesized compounds elemental microanalysis % chemical formula compound found calculated element mol.wt. 47.03 49.26 c c31h41n5o17 755.68 ia 5.72 5.46 h 8.84 9.26 n 38.39 35.99 o 54.14 c c13h24n2o5 288.3396 ib 8.38 h 9.71 n 27.74 o 52.3 53.76 c c29h57n7o9 647.81 i 8.93 8.86 h 14.72 15.13 n 24.03 22.22 o iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 80 figure1. gentamicin figure 2.n-protected gentamicin iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 81 figure 3. intermediate ib figure 4. compound i iraqi j pharm sci, vol.28(2) 2019 dipeptide derivative of gentamicin 82 conclusion the synthesis of the designed compound as derivative of gentamicin was successfully achieved by following the stated procedures as previously described and resulted in the preparation of the proposed compound in reasonable yields. the spectral and chno analysis have confirmed their chemical structures. ir spectra were recorded and found that the characteristic bands of the compounds indicate the presence of certain groups which comply with the proposed chemical structure. acknowledgement we are grateful to the college of pharmacy-dept. of pharmaceutical chemistry university of baghdad for providing some facilities in carrying out the research. references 1. daniels, p. j. l. antibiotics (aminoglycosides). in kirk-othmer encyclopedia of chemical technology, 3rd ed.; grayson, m., ed.; wileyinterscience: new york, 1978; vol. 2, pp 819–852. 2. hooper ir. the naturally occurring aminoglycoside antibiotics. in aminoglycoside antibiotics 1982 (pp. 135). springer, berlin, heidelberg. 3. edson rs, terrell cl. the aminoglycosides. inmayo clinic proceedings 1999 1 (vol. 74, no. 5, pp. 519-528). 4. saud md. conjugation of steroidal and non–steroidal anti-inflammatory drugs as possible mutual prodrug. iraqi journal of pharmaceutical sciences. 2006;15(1):74-9. 5. mohammed mh, dakhel za. synthesis of 5-fluorouracil derivatives as possible mutual prodrugs with meloxicam and ibuprofen for targeting cancer tissues. iraqi journal of pharmaceutical sciences. 2011;20(2):9-18. 6. amidon gl, sadée w, editors. membrane transporters as drug targets. springer science & business media; 2006:11. 7. han hk, amidon gl. targeted prodrug design to optimize drug delivery. aaps pharmsci. 2000 1;2(1):48-58. 8. enjoh m, hashimoto k, arai s, shimizu m. inhibitory effect of arphamenine a on intestinal dipeptide transport. bioscience, biotechnology, and biochemistry. 1996 1;60(11):1893-5. 9. benoiton, n.l.: "methods for the formation of peptide bonds". in: chemistry of peptide synthesis". kindle ed., taylor and francis group, canada. 2006; 25-63. 10. fredrick, h. and duane, t.: "general procedures for protection of the amino groups". 1952; 14:3818. 11. furniss, b.s., hannaford, a.j., rogers, v., smith, p.w.g. and tatchell, a.r.: "esters: the use of acyl chlorides and acid anhydrides". in: vogel's textbook of practical organic chemistry, (4 th ed.), john wiley and sons inter. new york. 1978; 501-506. 12. fatih d. and alwan s. synthesis of model compounds of glucosamine and gentamicin with expected enhanced bioavailability.2010 13. warrass r, wieruszeski jm, boutillon c, lippens g. high-resolution magic angle spinning nmr study of resin-bound polyalanine peptides. journal of the american chemical society. 2000 1;122(8):1789-95. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 77 formulation and evaluation of domperidone nanoemulsions for oral rout mohanad n. taher *,1 and ahmed a.hussein ** * iraqi ministry of health, directorate of technical affairs, baghdad, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the aim of the present study is to formulate, evaluate and characterize the nanoemulsion of domperidone a poorly water-soluble anti-emetic drug. domperidone powder is white or almost white powder, photosensitive, practically insoluble in water, slightly soluble in ethanol and in methanol; soluble in dimethylformamide. it is used as an antiemetic for the short-term treatment of nausea and vomiting of various etiologies. solubility studies were conducted to select the oil, surfactant and cosurfactant. phase diagrams were constructed by aqueous phase titration method. formulations were selected from the phase diagrams. the formulations were characterized for particle size, polydispersity index (pdi), zeta potential and in vitro drug release. all the formulations were in nanoscale and formula 1 (which contain anise oil as oil phase ,mixture of surfactant tween 80 and cosurfactant (ethanol) at ratio 1:1 in addition to double distilled water as aqueous phase in ratio 1:6:3 respectively ) was the selected formula depending on particle size, pdi, zeta potential and in vitro drug release. the formula 1 has the best ratio because it gives the smallest nanoemulsion globule size (particle size average 20.81nm) and the best homogenicity (lowest pdi 0.266) and highest stability (higher zeta potential -33.9). the selected formula gives accepted physical and chemical properties. keywords :nanoemulsion, domperidone. تصييغ و تقييم عقار الدومبريدون على شكل مستحلب واوىي لالعطاء الفمىي مهىد واصر طاهر ،*1 و احمد عباس حسيه ** * .لعراق،ابغداد ،ٔصاسح انصحخ انعشاقٍخ , دائشح االيٕس انفٍُخ ** .لعراق،ابغداد،جامعة بغداد ،كلية ألصيدلة ،فرع ألصيدالنيات الخالصة نعقبس انذٔيجشٌذٌٔ انًعبد نهقئ ٔانععٍف يانُبَٕانًسزحهت انٓذف يٍ ْزِ انذساسخ ْٕ رحعٍش ,رقٍٍى ٔرٕصٍف رشكٍجخ انزٔثبٍَخ فً انًبء. اثٍط أ يقبسة نهجٍبض , يزحسس نهعٕء,غٍش رائت ثبنًبء َسجٍب,قهٍم انزٔثبٍَخ ثبنكحٕل االثٍهً ٔانًثٍهً, ٔرائت يسحٕقانذٔيجشٌذٌٔ فً انذاًٌثٍم فٕسيبيبٌذ.ٌْٕٔسزعًم كًعبد نهقًء نهعالج قصٍش االيذ نهغثٍبٌ ٔانقًء السبثبة يخزهفخ. .اخشي يخطػ ٔانًبدح انًسبعذح نٓب نهسبئم انسطحً نهشذ انًقههّ ًبدحان، اخشٌذ دساسبد انزٔثبٍَخ عهى انضٌٕد انًُزخجخ قًٍذ انزشاكٍت نصفبد انحدى غشٌقخ رسحٍح انٕسػ انًبئً .انزشاكٍت اَزخجذ ثبالسزعبَخ ثًخطػ االغٕاس. االغٕاسثبسزخذاو انًحعشح كبَذ ظًٍ انحدٕو انُبٌَٕخ ٔكبَذ اندضٌئً,يعبيم االَزشبس انًزعذد,انضٌزبثٕرٍُشٍبل ٔرحشس انعالج انخبسخً. كم انزشاكٍت ( ٔانًبدح 08انزشكٍجخ االٔنى )انزً رحزٕي صٌذ حجخ انحهٕح كٕسػ صٌزً,يضٌح يٍ انًبدح انًقههّ نهشذ انسطحً نهسبئم انًزاثخ ثّ )رٌٍٕ ًْ (1:6:1زِ انزشكٍجخ ًْ ثبالظبفخ انى انًبء انًعبعف كٕسػ يبئً َٔسجخ يكَٕبد ْ 1:1انًسبعذح نٓب )انكحٕل االثٍهً( ثُسجخ حدى اندضٌئخ,يعبيم االَزشبس انًزعذد,انضٌزبثٕرٍُشٍبل ٔرحشسانعالج انخبسخً. انًُزقبح اعزًبدا عهى َبَٕيٍزش( ٔاحسٍ ردبَسب 18,01انزشكٍجخ االٔنى رًزهك احسٍ َسجخ نكَٕٓب اعطذ اصغش حدى نهدضٌئبد انُبٌَٕخ )يعذل قطش اندضٌئخ ( انزشكٍجخ انًُزقبح اعطذ صفبد فٍضٌبٌٔخ ٔكًٍٍبٌٔخ يقجٕنخ. 8,1666) اقم يعبيم اَزشبس يزعذد ، الدومبريدون. يالىاوىالمستحلب الكلمات المفتاحية: introduction domperidone have been the most widely prescribed prokinetic agent in the uk. cardiac risks was observed in patients older than 60 years, adults taking daily oral doses of more than 30mg domperidone, and those taking qtprolonging medicines, cyp3a4 inhibitors or diuretics concomitantly. in april 2014 the european chmp and the uk mhra published their final recommendations which state that the benefitrisk balance of domperidone remains positive in the relief of the symptoms of nausea and vomiting (1) . nanoemulsion are oil-in-water (o/w) or (w/o) emulsions with mean droplet diameters ranging from 50 to 1000 nm. usually, the average droplet size is between 100 and 500 nm.. nanoemulsion are made from surfactants approved for human consumption and common food substances that are “generally recognized as safe” (gras) by the fda (2) . 1 corresponding author e-mail: mohanadpharma@yahoo.com received: 3/11/2015 accepted: 19/12/2015 iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 78 reducing droplet sizes to the nanoscale leads to some very interesting physical properties, such as optical transparency and unusual elastic behavior (3) . additionally, the lack of flocculation, sedimentation and creaming, combined with a large surface area and free energy, offer obvious advantages over emulsions of larger particle size, for this route of administration. their very large interfacial area positively influences the drug transport and their delivery, along with targeting them to specific sites (4) . depending on the composition there are three types of nanoemulsion: o/w nanoemulsion : wherein oil droplets are dispersed in the continuous aqueous phase w/o nanoemulsions : wherein water droplets are dispersed in the continuous oil phase bi-continuous nanoemulsions: wherein microdomains of oil and water are interdispersed within the system. in all three types of nanoemulsions, the interface is stabilized by an appropriate combination of surfactants and/or cosurfactants (5) . domperidone is an anti-emetic drug. its chemical formula c22h24cin5o2 .it is a white or almost white powder, practically insoluble in water; slightly soluble in ethanol (96 per cent) and in methanol; soluble in dimethylformamide. the systemic bioavailability of domperidone is only about 15% in fasting subjects given an oral dose (6) . the objective of this study was to prepare nanoemulsion of a poorly watersoluble anti-emetic drug domperidone in order to enhance the solubility, dissolution rate and to studying the effect of different formulation variables in order to obtain the best formula with appropriate physical properties and higher dissolution rate. materials and methods materials the following materials were used: domperidone powder (vasudha pharma chem. co. india), oleic acid (bdh,uk ), peppermint oil (bar-sur-loup, france), anise oil (bdh,uk ), orange oil, lemon oil, nut meg oil (al-emad company, iraq), sweet almond (well’s, spain), carawax oil, tween 80, tween 40, tween 20, propylene glycol (pg) (j.t baker, china), peg 400 , glycerin (fluka chemi ag, switzerland), methanol,propanol,butanol (loba chemie pvt. ltd, india), ethanol 95% ( gcc analytical reagents, uk), di-sodium hydrogen orthophosphate na2hpo4 (thomas baker, india), potassium dihydrogen orthophosphate kh2po4 (sd fine-chem. limited, mumbai,india). methods solubility study the solubility of domperidone in different.oils, surfactants and co-surfactants was determined. briefly, an excess amount of domperidone was added to 5 ml of the vehicle in stoppered vials separately and shaken continuously at 25°c for 72 hrs to get equilibrium. the equilibrated samples were removed and centrifuged at 5000 rpm for 30 min. the supernatant was separated, filtered through a membrane filter (0.45 μm) and after appropriate dilution with methanol; solubility was determined spectrophotometrically by uv spectrophotometer at λmax 284 nm. using calibration curve in methanol. the oils preparations which were highly solubilized domperidone were selected for further study (7) . preparation of nanoemulsion construction of pseudo-ternary phase diagram (8) pseudoternary phase diagrams were constructed to examine the formation of oil in water nanoemulsion using four components: oil, surfactant, cosurfactant, and aqueous phase. the four component system consisted of: (1) one of these oils (anise oil, peppermint oil and oleic acid) as oil phase. (2) one of these surfactants (tween 80, tween40, tween20) as surfactant. (3) ethanol or propanol was utilized as cosurfactant. (4) double distilled water as an aqueous phase. the pseudoternary phase diagrams were constructed by instillation of homogenous liquid mixtures of oil, surfactant, and cosurfactant, with double distilled water at room temperature. at desired surfactantcosurfactant mixture (1:1, 1:2, 2:1), oily mixtures of oil, surfactant and cosurfactant were prepared. double distilled water was added drop by drop under gentle stirring to each liquid mixture at a ratio listed in table (1). if turbidity appeared followed by a phase separation, the samples were considered to be biphasic. if clear and transparent mixtures were visualized after stirring, the samples were considered monophasic. the samples were marked as points in the phase diagram. the area covered by these points was considered to be the nanoemulsion region of existence .all the ratios in this study are reported as weightto-weight ratios (w/w). iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 79 preparation of domperidone nanoemulsion nanoemulsion were prepared by aqueous phase titration method.the composition of the nanoemulsion was chosen according to the pseudo ternary phase diagram. 10 mg of domperidone powder was dissolved in the selected oil, surfactant and cosurfactant mixture was added in the chosen concentration, and water was added drop wise with continuous stirring until clear nanoemulsion was formed. . the final concentration of domperidone in the nanoemulsion was 10mg/10g as shown in table 1 (911) . table (1): composition of the domperidone nanoemulsion . where: a: anise oil, p: peppermint oil, o: oleic acid evaluation of domperidone nanoemulsion the optimal formulations were evaluated for the following characteristics. droplet size, zeta potential and size distribution droplet size zeta potential and size distribution were determined by zetasizer (1000 hs, malvern instruments, and u.k). small amount (0.1 ml) of the formula to be tested was dispersed in 50 ml of water in a volumetric flask, mixed thoroughly with vigorous shaking and light scattering was monitored at 25°c at a 90° angle (12) . droplet size was determined by photon correlation spectroscopy that analyzed the fluctuations in light scattering due to brownian motion of the particles. it accurately measures size in the range of 0.3 nm to 10 μm. zeta potential measures the surface charge of nanoemulsion . specialized cuvettes were used to measure zeta potential. polydispersity index (pdi) (which determines size range of particles in the system), for the formulations was determined as ratio of standard deviation to the mean droplet size of the formulation. the polydispersity index indicates the quality or homogeneity of the dispersion (13) . invitro drug release studies the release of domperidone nanoemulsion carry out by using dialysis tube mw (8000 -14000) domperidone nanoemulsion containing 10 mg of drug was placed into dialysis bag. usp24 rotating paddle apparatus was used to measure the in vitro drug release of all formulas .the dissolution medium, 900 ml 0.1 n hcl was placed into the release jar maintaining the speed of 100 rpm and temperature at 37±0.5°c .release studies were carried out for 2 hours. 5 ml of aliquot is withdrawn at an interval of 5,10,20,30,45,60,90,120 min. after collecting the sample, the dissolution medium was replenished with the same volume of fresh medium, and the sample was filtered. the samples were analyzed at 284nm by uvvisible spectrophotometer (14) . iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 80 depending on the release profile, the best formula will be selected and comparison of the selected formula of the prepared domperidone nanoemulsion with marketed domperidone suspension (motillium suspension) will done for the drug release profile. morphology by atomic force microscopy (afm) (15, 16) afm is capable of scanning the surfaces in controlled environmental conditions and is complementary to sem imaging and also can measure the particle size of the nanoemulsion accurately. the size and surface morphology of domperidone nanoemulsion in f1 (the selected formula), f2 and f3 were confirmed by atomic force microscopy. particle size, 3d-dimension graph and histogram of particle size distribution were obtained. measured of ph of the formula the apparent ph of the formulation is measured by putting the probe of ph meter inside the prepared nanoemulsion and read the result of the instrument (12, 17) . dilutability test o/w nanoemulsion are dilutable with water whereas w/o are not and undergo phase inversion into o/w nanoemulsion. the dilution done by dding different volumes of double distilled water to the prepared nanoe mulsions (15) . phase separation nanoemulsion system were subjected to centrifugation at 3500 rpm for a period of 30 minute and examined for any separating into two phases (8) . entrapment efficiency the amount of domperidone in the emulsions was assayed by uv spectroscopic method. drug content was expressed as a percentage of domperidone entrapped in the system to the theoretical quantity of the drug added. for estimation, 1.0 ml of nanoemulsion was diluted in methanol and the resulting solution was analyzed at 284 nm in uvvisible spectrophotometer (18) . phase analysis )conductance measurement) o/w nanoemulsion where the external phase is water, are highly conducting whereas w/o are not, since water is the internal or dispersal phase. to determine the nature of the continuous phase and to detect phase inversion phenomena, the electrical conductivity measurements are highly useful. dielectric measurements are a powerful means of probing both structural and dynamic features of nanoemulsion systems (15, 19) . measuring the electrical conductivity using a conductometer (cond7110, wtw, germany) by putting the conductometer probe current in the selected formula (20) . transparency measurement by refractive index refractive index (ri) of nanoemulsion was determined using an abbes type refractometer at 25± 0.5 o c. ri of nanoemulsion prove the transparency of the systems (12) . percentage transmittance (21,22) percentage transmittance (%t) of nanoemulsion prove the transparency of the systems. for measurement of percentage transmittance (%t), nes were diluted 10 times with distilled water and %t was checked against distilled water using uv–visible spectrophotometer. the percent transmittance of the all formulations was measured at 646 nm. optical transparency optical transparency of the formulas was determined by inspecting the sample in clear and transparent container under the presence of good light against reflection into the eyes, and viewed against black and white illuminated background. (23,24) . viscosity measurement viscosity of nanoemulsion was measured by using a brookfield viscometer equipped with the spindle no.64. the measurement was performed at ambient temperature of the selected formula (23) . factors affecting the prepared formulas effect of types of oil formulas f1, f2 and f3 in table (1) were utilized to study the effect of oil type (anise oil peppermint oil and oleic acid) in concentrations (10%w/w) for all oils on the drug release profile of the prepared domperidone nanoemulsion. effect of type of surfactant formulas f1, f7 and f8 in table (1) were used to study the effect of different grades of selected surfactants in the same concentration (30%w/w) on the drug release profile of the prepared domperidone nanoemulsion. effect of type of co-surfactant formulas (f1 and f4) in table (1) were utilized to investigate the effect of different cosurfactant type (ethanol and propanol) in the same concentration (30%w/w) on the drug release profile of the prepared domperidone nanoemulsion. iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 81 drug and excipient compatibility study by ftir these studies were achieved to detect any sign of complexation and interaction between domperidone and excipients used in the preparation of domperidone nanoemulsion. the domperidone pure drug and domperidone nanoemulsion were analyzed by fourier transform infrared system (ftir -8300 shimadzu, japan). the spectrum was obtained between the wave number 4000-400 cm -1 (25) . results and discussion solubility of domperidone in different oils, surfactants and co-surfactants for nanoemulsion solubility of domperidone in different oils, surfactants and co-surfactants was illustrated in figure1 .oleic acid found to has higher solubilizing activity toward domperidone and found to be 65mg/ml followed by peppermint oil 33.75 mg/ml and then anise oil 12.86 mg/ml then the other oils as following: figure (1): solubility of the domperidone in different oils, surfactants and co-surfactants construction of phase diagrams (14) pseudoternary phase diagrams were constructed to examine the formation of oil in water nanoemulsion using four components: oil, surfactant, cosurfactant, and aqueous phase. the phase diagram was constructed by using prosim ternary diagram software. the nanoemulsion region results listed in figures: (211). figure (2): phase diagram for anise oil, tween 80 and ethanol mix. (1:1) and double distilled water. figure (3): phase diagram for anise oil, tween 80 and ethanol mix. (1:2) and double distilled water. figure (4): phase diagram for anise oil, tween 80 and ethanol mix. (2:1) and double distilled water. figure (5): phase diagram for anise oil, tween 80 and propanol mix. (1:1) and double distilled water. iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 82 figure (6): phase diagram for anise oil, tween 40 and ethanol mix. (1:1) and double distilled water. figure (7): phase diagram for anise oil, tween 20 and ethanol mix. (1:1) and double distilled water. figure (8): phase diagram for peppermint oil, tween 80 and ethanol mix. (1:1) and double distilled water. figure (9): phase diagram for peppermint oil, tween 80 and propanol mix. (1:1) and double distilled water figure (10): phase diagram for oleic acid oil, tween 80 and ethanol mix. (1:1) and double distilled water figure (11): phase diagram for oleic acid oil, tween 80 and propanol mix. (1:1) and double distilled water. formulation of domperidone nanoemulsion depending on the nanoemulsion regions results in the pseudoternary phase diagrams, fifteen different formulations of domperidone nanoemulsion were prepared (table 1) and will evaluate them to get the optimized formula with the best properties.f1 is the selected formula because it gives the smaller globule size ,small accepted pdi and higher zeta potential (table 2). evaluation of domperidone nanoemulsion: droplet size (14,12) the nanometric size range of the particle was retained even after 100 times dilution with water which proves the compatibility of the system with excess water. droplet size of selected formula f1 was 20.81nm. it checked after three months of storage at three different temp. 4 o c, 25 o c and 40 o c the results was 34.26, 44.59 and 50.08nm respectively. (figure12, 14, 16, 18). zeta potential (14, 26, 27) zeta potential is used to identify the surface charge properties and further the long term physical stability of nanoemulsion. its values were determined from the electrophoretic mobility of the oil droplets. iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 83 table (2): particle size, pdi and zeta potential of prepared formulas: in prepared nanoemulsion formulas, the charge on an oil droplet is negative due to presence of free fatty acids. zeta potential of selected formula f1 was -33.9mv indicate the stability of it. it checked after three months of storage at three different temp. 4 o c, 25 o c and 40 o c the results was -31.1, -30.3 and -29.9mv respectively. (figure13, 15, 17, 19). size distribution (12, 13) the selected formula f1 has accepted size distribution because it has low poly dispersible index which indicate the high quality and homogeneity (pdi 0.266). it checked after three months of storage at three different temp. 4 o c, 25 o c and 40 o c the results was 0.280, 0.225and 0. 0.176 nm respectively. figure (12): droplet size for fresh f1 figure (13):zeta potential for fresh f1 figure (14):droplet size for f1 after three months at 4 o c figure (15):zeta potential for f1 after three months at 4 o c formula particle size average (r-nm) pdi zeta f1 20.81 0.266 -33.9 f2 87.29 0.276 -25.5 f3 107.3 0.625 -23.9 f4 60.71 0.248 -29.3 f5 87.33 0.445 -25.0 f6 145.8 0.657 -23.4 f7 133.2 0.403 -23.1 f8 143.6 0.432 -22.6 f9 96.17 0.244 -29.1 f10 85.69 0.404 -26.6 f11 127.1 0.325 -28.5 f12 78.48 0.373 -26.7 f13 92.31 0.292 -24.6 f14 77.53 0.460 -27.6 f15 82.90 0.522 -24.3 iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 84 figure (16):droplet size for f1 after three months at 25 o c. figure (17): zeta potential for f1 after three months at 25 o c. figure (18):droplet size for f1 after three months at 40 o c . figure(19):zeta potential for f1 after three months at 40 o c. invitro drug release studies the release of the prepared formulas at ph1.2 is faster than that in ph 6.8 due to higher solubility of domperidone at acidic media because it is week base (pka 7.9) (28) . effect of type of oil formulas f1, f2 and f3 in table (1) were utilized to study the effect of oil type (anise oil, peppermint oil and oleic acid) in concentrations (10%w/w) for all oils on the drug release profile of the prepared domperidone nanoemulsion.. figure (20): dissolution of (f1f3) at ph 1.2 and temp. 37 o c. the release of domperidone nanoemulsion from f1 which contain anise oil as dispersed phase is rapid and faster than f2 which contain peppermint oil and f2 faster than f3 which contain oleic acid as shown in figure (20) this may be due to different in globule size. the anise oil considered the best oil because it gives the smallest nanoemulsion globule size (particle size average 20.81nm) and the best homogenicity (lowest pdi 0.266) and highest stability (higher zeta potential 33.9) as shown in figure (12, 13). smaller droplet size increases the total surface area for transfer, release, and absorption of the drug and improve the bioavailability (29) . effect of type of surfactant formulas f1, f7 and f8 in table (1) were used to study the effect of different grades of selected surfactants in the same concentration (30%w/w) on the drug release profile of the prepared domperidone nanoemulsion. iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 85 figure (21): dissolution of (f1, f7, and f8) at ph 1.2 and temp. 37 o c. the release of domperidone nanoemulsion from f1 which contain tween 80 as surfactant was rapid and faster than f7 which contain tween 40 and f7 faster than f8 which contain tween 20 with fixing the other component of nanoemulsion as shown in figure (21) this may be due to different in globule size. the tween 80 considered the best surfactant because it gives the smallest nanoemulsion globule size (particle size average 20.81nm) and the best homogenicity (lowest pdi 0.266) and highest stability (higher zeta potential -33.9) as shown in figure (12, 13).smaller droplet size increases the total surface area for transfer, release, and absorption of the drug and improve the bioavailability (29) . in addition, tween 80 has higher drug solubility (7.55mg/ml) than tween 40 (7.018mg/ml) and tween 20 the smallest solubility (6.29 mg/ml) (figure 1). effect of type of co-surfactant formulas (f1 and f4) in table (1) were utilized to investigate the effect of different cosurfactant type (ethanol and propanol) in the same concentration (30%w/w) on the drug release profile of the prepared domperidone nanoemulsion. figure (22): dissolution of (f1 and f4) at ph 1.2 and temp. 37 o c. the release of domperidone nanoemulsion from f1 which contain ethanol as cosurfactant is rapid and faster than f4 which contain propanol as cosurfactant with fixing the other component of nanoemulsion (figure 22). this may be due to difference in globule size. the ethanol considered the best cosurfactant because it gives the smallest nanoemulsion globule size (particle size average 20.81nm) and the best homogenicity (lowest pdi 0.266) and highest stability (higher zeta potential 33.9) in the selected formula (f1) as shown in figure (12, 13). comparison of the selected formula of the prepared domperidone nanoemulsion with marketed domperidone suspension (motillium suspension produced by sanofi drug company) for the drug dissolution profile at ph 1.2 and temp. 37 o c f1 is considered as the selected formula depending on its dissolution profile (give faster release) and its best results in evaluation of droplet size, zeta potential, pdi and morphology. there is significant differences between the f1 and marketed suspension where p value equal to 0.000151 (p<0.05). iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 86 figure (23): dissolution profile of f1 and marketed suspension at ph 1.2 and temp. 37 o c. morphology by atomic force microscopy (afm) the atomic force microscope (afm) is one kind of scanning probe microscopes (spm) which is an approach to measure the properties of particles surfaces. afm is capable of scanning the surfaces in controlled environmental conditions and is complementary to sem imaging with the high precision of the afm, in principle it is possible to determine the dimensions of nano globules with high accuracy. afm allows the visualization of samples with resolution in three dimensions x-, yand z-directions in atmospheric or submerged conditions. (30), (31) results show spherical shaped nanoemulsion globules and a size within the nano size as it approved by the histogram of particle size distribution (figure 2425). figure (24): average size range of globule nanoemulsion of f1 figure (25): atomic force microscopy images of f1 nanoemulsion (contain anise oil). measured of ph of the formula the ph of the selected nanoemulsion formula (f1) was 6.51 and still stable during three months after periodic checking every two weeks. dilutability test all prepared nanoemulsion (the fifteen formulas) are dilutable with water. that indicate that all of them are o/w nanoemulsion. and still stable during three months after periodic checking every two weeks (15) . phase separation (8) all prepared nanoemulsion were subjected to centrifugation at 3500 rpm for a period of 30 minute and examined for any change in phase separation. the result indicate there is no phase separation for all prepared nanoemulsion and still stable during three months after periodic checking every tow week. iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 87 drug content (18) the drug content of selected nanoemulsion formula (f1) were high (99.98±0.54). this high drug content could be due to incorporating the drug in the lipid phase. phase analysis )conductance measurement) (15,19) measuring the electrical conductivity using a conductometer for the selected formula (f1). the result was high conductivity (59.2mcs/cm) that prof the formula was o/w because of high conductivity of water and still stable during three months after periodic checking every tow week. refractive index (ri) (12) refractive index of nanoemulsion was determined using a refractometer at 25± 0.5 o c. ri of f1 (selected nanoemulsion) was 1.38 which prove the transparency of the systems. and still stable during three months after periodic checking every tow week. percentage transmittance (%t) (21, 22) percentage transmittance of nanoemulsion prove the transparency of the systems. the percentage transmittance of the optimized formulation f1 was found to be 94.23± 1.5 .the results of percent transmittance indicate that the prepared f1 was nearly transparent. and still stable during three months after periodic checking every tow week. optical transparency (23) the selected formula f1 was transparent. and still stable during three months after periodic checking every tow week. viscosity measurement the viscosity of selected nanoemulsion formula (f1). the measurement was performed at ambient temperature the result indicate it has a very low viscosity (30 cp) (16, 19, 32, 33) . the viscosity of nanoemulsion is a function of the surfactant, water and oil components and their concentrations. increasing the water content lowers the viscosity, while decreasing the amount of surfactant and cosurfactant increases interfacial tension between water and oil resulting in increased viscosity. drug and excipient compatibility study the ftir absorption spectrum of domperidone and its nanoemulsion is shown in figure (26, 27). domperidone showed a strong characteristic absorbance band at 1712 cm -1 due to c o stretching vibrations of amide functional group conhr. n h stretching characteristic band of secondary amine appears at 3321 cm -1 as a single wea band and n h bending characteristic band at 1693 cm -1 , symmetric and asymmetric c h stretching bands appeared at 2820 and 2937 cm -1 respectively as well as aromatic symmetric and asymmetric c h stretching bands appeared at 3024 and 3098 cm -1 respectively, and the aromatic c = c stretching band appeared at 1624 cm -1 . the band associated with c n stretching of secondary and tertiary amine at 1319 and 1359 cm -1 respectively. c cl characteristic absorption band with strong stretching intensities appeared at 756 cm -1 (34-36) . figure (26): ftir spectrum of domperidone iraqi j pharm sci, vol.24(2) 2015 domperidone nanoemulsions 88 figure (27): ftir spectrum of domperidone nanoemulsion conclusion based on the data obtained from the present study, one can conclude the following: 1. aqueous phase titration method is an efficient method to prepare drug nanoemulsions and it is easy to operate. 2. domperidone nanoemulsions were successfully prepared using different types of oils and surfactants-cosurfactant mixtures in different ratio using pseudoternary phase diagram. 3. in the selection of oil, not always the higher drug solubilizes oil is the best oil. 4. the selected formula f1, containing anise oil as oil phase, tween 80 as surfactant, ethanol as cosurfactant and double distilled water as aqueous phase at ratio 1:3:3:3 respectively showed faster dissolution rate than other formulas and marketed suspension. 5. selected formula f1 has the best ratio because it gives the smallest nanoemulsion globule size (particle size average 20.81nm) and the best homogenicity (lowest pdi 0.266) and highest stability (higher zeta potential 33.9) and produced higher dissolution rate in comparison with the marketed suspension (motillium) ® . 6. surface morphology of drug nanoparticles, visualized by afm, illustrated uniform particle size distribution and there is no particles agglomeration, and accurate particle size was obtained by afm. 7. drug–excipients compatibility studies revealed that there is no chemical interaction between domperidone and other components in the preparation of nanoemulsion. references 1. trent medicines information centre, leicester royal infirmary (leicester le1 5ww tel 0116 258 6491 email medicines.info@uhl-tr.nhs.uk); may, 2014. 2. p. shah, d. bhalodia and p. shelat, “nanoemulsion: a pharmaceutical review,” systematic reviews in pharmacy, 2010; 1(1):24-32. 3. t. g. mason, s. m. graves, j. n. wilking and m. y. lin, “extreme emulsification: formation and structure of nanoemulsions,” journal of physics and condensed matter, 2006; 9(1):193-199. 4. n. anton and t. vandamme, “the universality of lowenergy nanoemulsification,” international journal 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and david lb, spectrometric identification of organic compounds. 8 th edition, wiley global education 2014; chapter 2. 35. deepika kl and sanjeev kp, design, development and evaluation of domperidone maleate bilayer tablets. international journal of pharmacy and pharmaceutical sciences 2013; 5(4): 701710. 36. mamta, pawan j, nirja and ritu, formulation and evaluation of fast dissolving tablet of domperidone. international journal of pharma professional research 2014; 5(3): 10671074. iraqi j pharm sci, vol.26(1) 2017 kinetic-spectrophotometric method for the assay of carbamazepine 1 a highly sensitive kinetic-spectrophotometric method for the assay of carbamazepine in pure and commercial tablet sarmad b. dikran * and faeza h. zankanah *,1 * department of chemistry, college of education for pure science (ibn al-haitham), university of baghdad, baghdad, iraq abstract the study aimed to recommend a new spectrophotometric-kinetic method for determination of carbamazepine (cabz) in its pure form and pharmaceutical forms. the proposed procedure based on the coupling of cabz with diazotized sulfanilic acid in basic medium to yield a colored azo dye. factors affecting the reaction yield were studied and the conditions were optimized. the colored product was followed spectrophotometrically via monitoring its absorbance at 396 nm. under the optimized conditions, two method (the initial rate and fixed time (10 minute)) were applied for constructing the calibration graphs. the graphs were linear in concentration ranges 2.0 to 18.0 µg.ml -1 for both methods. the proposed was applied successfully in the determination of cabz in its commercial formulations. keywords: kinetic, spectrophotometry, carbamazepine, pharmaceutical formulations. بصىرحه الىقيت وفيلخقدير عقار الكاربامازيبيه ذاث حساسيت عاليت طريقت طيفيت حركيت المسخحضراث الصيدالويت سرمد بهجج ديكران * وفائسة حازم حسه *،1 *لسى انكًٍٍاء ، كهٍت انخزبٍت نهعهٌو انصزفت )ابن انيٍثى ( ، جايعت بغداد ، بغداد ، انعزاق . الخالصة اندًائٍت. حعخًد ىذه ٌيدف انبحث انى حطٌٌز طزٌمت طٍفٍت حزكٍت نخمدٌز عمار كارباياسٌبٍن بصٌرحو اننمٍت ًفً يسخحضزاحو انطزٌمت عهى اسدًاج عمار انكارباياسٌبٍن يع انكاشف حايض سهفانٍهك انًؤسًث فً ًسظ لاعدي. حٍث حى دراست انعٌايم انخً حؤثز نانٌيخز، 693عهى انخفاعم ًحثبٍج انظزًف انًثهى، ًٌصاحب انخفاعم انطٍفً لٍاص يعدل انخغٍز فً االيخصاصٍت عند طٌل انًٌجً ،دلائك 01ينحنً انًعاٌزة ين خالل طزٌمخً يعدل انسزعت ًانشين انثابج عند انظزًف انًثهى حى دراست حزكٍت انخفاعم ًإٌجادًعند يكغى. يم 1..0-0.1ًكانج حدًد انخزاكٍش بٍن -0 . ًلد حى حطبٍك انطزٌمت بنجاح نخمدٌز عمار انكارباياسٌبٍن فً يسخحضزاحو اندًائٍت. المسخحضراث الصيدالويت.، ربامازيبيهكا ،الخقدير الطيفي ،حركيتالكلماث المفخاحيت: introduction carbamazepine (cabz), is anticonvulsant drug, chemically it is 5-hdibenz (b, f) azepine-5-carboxamide (1) figure (1). carbamazepine is widely used as an anticonvulsant and antiepileptic drug (2) . it may be used in schizophrenia along with other medications and as a second line agent in bipolar disorder (3) . several methods such as hplc (4) , gc-ms (5) , voltammetric (6) , spectrophotometric (7, 8) , have been developed for determination of cabz in pharmaceutical preparations. according to a relative small number of kinetic methods for quantitative determination of cabz have been reported in the literature, the work was done to develop a new sensitive, selective, and free of interference kinetic spectrophotometric procedure by coupling cabz with the diazotized sulfanilic acid. experimental apparatus all spectrophotometric measurements were performed using shimadzu 1800 uvvisible, with 1 cm silica cells. a sartorius bl 210s balance , water bath (memmert w-200 ringgermany) and hot plate with magnetic stirrer (germany). figure (1): chemical structure of carbamazepine. 1 corresponding author e-mail: fae_chemical@yahoo.com received: 13/11/2016 accepted: 9 /1/ 2017 iraqi j pharm sci, vol.26(1) 2017 kinetic-spectrophotometric method for the assay of carbamazepine 2 materials and methods materials carbamazepine standard powder was obtained, as a gift, from the state company for drug industries and medical appliances, samara-iraq (sdi). acetic acid was supplied from bdh while sulfanilic acid and sodium hydroxide were supplied from riedel-de haën and barcelona-espana respectively. taver (cyprus), tegral (egypt), and tegratol (switzerland) tablets labeled to contain 200 mg of cabz per tablet was purchased from commercial source. method preparation of solutions for standards and reagents stock and working standard solutions: a 0.25g of carbamazepine is dissolved in 10 ml of concentrated ch3cooh and leaved to stand for 10 minutes. the volume is then completed with (ch3cooh:h2o 1:1 (v/v) solution) to 250ml in a volumetric flask to give a concentration of (1000 µg.ml -1 ). this stock solution was used to prepare more dilute working solutions (2.0, 4.0, 6.0, 8.0 10.0, 13.0, 15.0, and 18.0 µg.ml -1 ). diazotized sulfanilic acid solution (9, 10) (0.01m) 0.2 g of sulfanilic acid was dissolved in 30 ml distilled water, with continuous stirring and heating until complete dissolution. then 1.7 ml of 11.8m hcl is added with continuous stirring, and the mixture is transferred into a brown 100 ml volumetric flask, cooled to 0 5 o c in ice–bath. after then 0.069g of nano2 is added and the mixture is stirred vigorously. five minutes later, the solution is made up to final volume with cold water. the solution is stable for at least 48 hours when is stored in a refrigerator. sodium hydroxide solution (~ 5m) 20 g of the base was dissolved in 50 ml of distilled water (dw). the solution was then diluted to 100 ml with dw. solution for the analysis of carbamazepine in formula preparation an equivalent weight to one tablet (0.025081g, 0.027253g, and 0.026772g) was taken from the fine powder of 10-tablets of taver, tegral, and tegratol respectively and dissolved in 4 ml of concentrated acetic acid with stirring for 10 minutes. the solution was let to stand for another 10 minutes, then its volume was completed to 100 ml with (ch3cooh:h2o 1:1 (v/v)) to obtain 200µg.ml -1 cabz. the undissolved materials were filtered-off via whatman filter paper no.41 before use. results and discussion absorption spectra absorption spectrum for the azo dye product formed upon the reaction between the diazotized sulfanilic acid and cabz in alkaline medium was recorded, against the reagent blank, under primary testing conditions (1ml of 0.01m diazonium salt, 5 minutes at 60 o c and 3 ml of 5m naoh). figure (2) shows that the maximum absorption of the yellow dye under primary conditions is located at 402.0 nm at which the reagent blank shows a negligible absorption. figure (2): absorption spectra of: (a) reaction product of 10 μg.ml -1 cabz vs reagent blank, under primary test. (b) reaction product of 10 μg.ml -1 cabz vs reagent blank, under the optimum conditions.(c)blank solution against distilled water. optimization of experimental variables the effects of various parameters related to the formation of the yellow azo dye have been studied to optimize their values. volume of diazonium salt solution different volumes (0.5–2.0 ml) of 0.01 m diazotized sulfanilic acid solution were used to carry out the reaction. the results show that 1.25 ml of this solution was sufficient for the production of maximum and reproducible color intensity figure (3). figure (3): effect of diazonium salt volume. http://www.riedeldehaen.com/ iraqi j pharm sci, vol.26(1) 2017 kinetic-spectrophotometric method for the assay of carbamazepine 3 reaction temperature different temperatures (25-100 o c) were examined to investigate their effect on the progress of the coupling reaction. it was observed that the value of the measured absorbance of the reaction product continued to increase up to 80 °c, while higher temperatures lead to depression in its value. this probably due to the dissociation of azodye therefore, all the coupling reactions were performed at 80 °c (11, 12) , figure (4). figure (4): effect of temperature on color reaction. reaction time optimum reaction time was determined by carrying out the coupling reaction at different periods (0-15 min). as shown in figure (5), the absorbance value reaches to its maximum after 1 minute. figure (5): effect of reaction time. volume 5m naoh the effect of different volumes (0.5-4.0 ml) of 5m sodium hydroxide solution on the reaction yield was investigated. the results show that 2.5 ml sodium hydroxide solution optimum and therefor recommended for the subsequent experiments, figure (6). figure (6): effect of naoh volume final absorption spectrum the final spectrum of the azo dye product formed exhibits a maximum at 396 nm, under the optimum conditions shows in figure (2). nature of azo dye an azo dye is defined by having an azo linkage (--n=n--) as part of its chromophore. azo dyes are made in two steps: 1. a primary aromatic amine is reacted to give a diazonium salt. 2. the diazonium salt is reacted or coupled with a strongly activated aromatic system. job's method (13) and mole ratio method (14) have been used in the determination of the composition of the formed azo dye. the study shows that the ratio of cabz to the diazotized sulfanilic acid in the formed azo dye was 1:1 for job's and mole ratio methods. the results are depicted in figures (7) and 8, and scheme 1 shows the suggested structure. figure (7): job’s method of continuous variation. figure (8): mole ratio method http://www.scielo.cl/scielo.php?script=sci_arttext&pid=s0717-97072012000400025#scheme3 iraqi j pharm sci, vol.26(1) 2017 kinetic-spectrophotometric method for the assay of carbamazepine 4 scheme (1): steps of the main reactions between carb and sulphanilic acid. kinetics of the reaction general procedure aliquot of 0.6 ml of the standard drug solution containing various amount (6.0-36.0 µg) of cabz was added to 0.8 ml of 0.01 m of the diazotized sulfanilic acid solution in a 25 ml conical flask. the mixture was shaken and left to stand for 1 minute at 80 °c, then after transferred into 4 ml cuvette. 1.6ml of 5m naoh was then added and the absorbance of this solution was immediately measured at 396 nm. the value of absorbance was recorded at 1s intervals for 30 minutes (i.e. 0 and 1800s). verification of reaction order the rate of the reaction was studied to determine its order according to following equation (15, 16): where: k' and n represent the rate constant, and the order of the reaction. this was accomplished by using different concentrations (2.0-18.0 μg.ml -1 ) of cabz solution and a constant concentration (2.67× 10 -3 m) of sulfanilic acid under the optimized conditions. it was found that the reaction rate is (cabz) dependent. graphs on figure (9) shown that reaction rate is increased with the increasing (cabz) concentration. measurements carried out by variable time method, could be used for rate (in terms of δa/δt) estimation. the rate equation could also be expressed in logarithmic form as: where: a is the absorbance, and t is the measuring time. regression least square plot of log (cabz) versus log (rate) is shown in figure (10). the regression equation is: , with correlation coefficient (r) = 0.9990. accordingly, the value of k’= 89.702 s -1 and the reaction is pseudo-first order since the value of n equals to 1.1246 (≈1). quantitation methods the determination of cabz was based on the above equation using the rate data. different techniques were carried out (i.e. the initial rate, fixed concentration and fixed time) for this purpose. appropriate method for analysis were selected according to their applicability, sensitivity, and the values of iraqi j pharm sci, vol.26(1) 2017 kinetic-spectrophotometric method for the assay of carbamazepine 5 correlation coefficient (r) and intercept of the regression equations. rate constant method the values of log a vs time for different concentrations of cabz were plotted. the plot was found to be linear in the range of 8.4650  10 -6 7.6190  10 -5 m. the values of k' (i.e. pseudo-first order rate constants) for the range of the studied concentrations were obtained by multiplying the values of slope by -2.303 (table 1). table (1): k values for various rates at different cabz concentrations. (carbamazepine) m k' (s -1 ) 8.4653x10 -6 -4.70x10 -5 4.2326x10 -5 -1.21x10 -4 6.3485x10 -5 -1.11x10 -4 7.6200x10 -5 -1.34x10 -4 7.6187x10 -5 -1.52x10 -4 the regressed relation between c vs the values of k' is given by: the correlation coefficient value indicates poor linearity; thus, this method was abandoned. fixed absorbance method the absorbance of cabz reaction product for different concentrations of drug (1.6931  10 -5 – 7.61873  10 -5 m) were recorded figure (9). figure (9): absorbance-time curves for coupling of cabz with diazonium salt; (ccabz) = 2.0-18.0 μg.ml -1 . the time in seconds at a selected value of absorbance (0.2294), was measured. a plot of the reciprocal value of measured time against initial (cabz), (table 2), was constructed with the following regression equation: the value of correlation coefficient (r = 0.9694) for calibration graph indicated poor, which is considered a disadvantage. table 2 summarize the values of (1/t) for different cabz concentration. table (2): reciprocal time values for fixed absorbance at different (cabz). (carb) m x 10 -5 1/t (s -1 ) x10 -3 1.6931 1.793 2.5396 6.461 3.3861 10.712 4.2326 13.131 5.5024 16.169 6.3489 16.282 7.6187 20.353 initial rate method in this method, the reaction in the initial rate with respect the time in the (0-600 sec.) versus the log (cabz) plots were rectilinear within the range of 2.0 – 18.0 μg.ml -1 figure (10).the reaction was first order by according to the value of slope 1.1246 ( 1) and the value of correlation coefficient was (0.9990). figure (10): calibration plot of logarithm rate of the reaction against logarithm molar concentration of cabz for initial rate method. the value of detection limit was calculated and found to be 0.0730 µg.ml -1 while limit quantification was 0.2434 µg.ml -1 .these values indicated the high sensitivity of the proposed method to determine low amounts of cabz. fixed time method in the fixed time method, varying amounts of cabz were used for determination of reaction rate at a preselected fixed time (1, 5, 10, 15, 20, 25 and 30 minute) and the absorbance values were measured and plotted against (cabz). the low values of standard deviation (sd), limit of detection (lod) and limit of quantification (loq) are given in (table 3). a fixed time of 10 minute indicates that this method could successfully applied for determination of cabz in its pure form and in pharmaceutical preparations. figure (11). iraqi j pharm sci, vol.26(1) 2017 kinetic-spectrophotometric method for the assay of carbamazepine 6 table (3): regression equations at fixed time method for the determination of cabz. *sd = for the difference absorbance, **lod = 3.3 σ /s and ***loq = 10 σ/s. table (4): validation of regression and assay for the determination of cabz by the proposed method. * theoretical value for t-test for n=2, at 95% confidence limit is 4.303 figure (11): calibration plot of absorbance versus the concentration of cabz at preselected fixed time. validation of the proposed methods accuracy and precision intraday relative standard deviation percent and relative error percent of the results obtained by the recommended methods (initial rate and fixed time) were calculated for three replicates within the day using pure drug solutions at two cabz concentration levels (3.0 and 6.0 μg.ml-1) within the working ranges. the value of rsd % did not exceed 2% while percent relative error (re %), an indicator of accuracy, was ≤ 5.5475. this good level of relative error for each proposed methods could be consider as adequate for the quality control analysis of the studied cabz. the analytical results obtained from the investigation are summarized in (table 5). time (min) regression equation correlation coefficient (r) * sd δy ** lod (µg. ml -1 ) ***loq (µg.ml -1 ) 1 y= 0.0152c + 0.0077 0.9812 0.0166 3.2829 10.9431 5 y= 0.0511c + 0.0137 0.9934 0.0327 1.9227 6.4091 10 y= 0.0693c 0.0353 0.9993 0.0137 0.5950 1.9834 15 y= 0.0784c 0.0666 0.9993 0.0159 0.6080 2.0267 20 y= 0.0848c 0.0854 0.9987 0.0235 0.8328 2.7760 25 y= 0.0894c 0.0947 0.9982 0.0293 0.9838 3.2793 30 y= 0.0927c 0.0961 0.9978 0.0338 1.0923 3.6412 parameter fixed time method (10 mint.) initial rate method λmax (nm) 396.0 regression equation y=0.0693c-0.0353 y=1.1246c+1.9528 linearity ( μg ml -1 ) 2.0-18.0 slope ± sd 0.0693±0.00101 1.1246±0.0207 intercept ± sd 0.0353± 0.0109 1.9528±0.0928 correlation of linearity (r 2 ) 0.9987 0.9980 correlation coefficient (r) 0.9993 0.9990 molar absorptivity (l.mol -1 .cm -1 ) ε =16373.4140 ε =265707.6 sandell's sensitivity(µg.cm -2 ) 0.01433 0.00089 limit of detection (µg.ml -1 ) 0.5950 0.0730 limit of quantification (µg.ml -1 ) 1.9834 0.2434 t-test * 4.4653 iraqi j pharm sci, vol.26(1) 2017 kinetic-spectrophotometric method for the assay of carbamazepine 7 table (5): accuracy and precision of the initial rate and fixed time methods for determination of cabz. method conc. of carbamazepine (µg.ml -1 ) re% rsd% taken found* initial rate 3.00 3.1598 5.3256 0.5768 6.00 6.0213 0.3546 1.8157 fixed time 3.00 3.1664 5.5475 0.7153 6.00 5.8076 -3.2067 1.8617 *average of three measurements. application of the proposed methods the results obtained for the two suggested kinetic spectrophotometric methods to analyses cabz were satisfactory due to their good agreement with the labeled amounts. the results shown in table (6) are for the analysis of cabz in commercial tablet by both methods (initial rate and fixed time). the mean recoveries and rsd% values were 101.7245± 1.4561% and 101.0922± 1.4861% respectively. table (6): analysis of cabz by the initial rate and fixed time methods. sample method conc. of cabz (µg.ml -1 ) * recovery % *s. d. *rsd% taken * found taver (cyprus) 200mg/tablet initial rate 2.00 2.0411 102.0545 0.0354 1.7357 7.00 7.0353 100.5045 0.1101 1.5643 tegral (egypt) 200mg/tablet 10.00 10.0279 100.2791 0.1187 1.1832 15.00 14.9465 99.6431 0.3050 2.0406 tegratol (switzerland) 200mg/tablet 6.00 6.4273 107.1223 0.0710 1.1052 12.00 12.0893 100.7437 0.1339 1.1075 taver (cyprus) 200mg/tablet fixed time 2.00 2.0789 103.9442 0.0306 1.4730 7.00 6.8211 97.4483 0.1110 1.6270 tegral (egypt) 200mg/tablet 10.00 9.9120 99.1202 0.1251 1.2624 15.00 15.2386 101.5907 0.3382 2.2196 tegratol (switzerland) 200mg/tablet 6.00 6.2110 103.5170 0.0709 1.1412 12.00 12.1120 100.9329 0.1445 1.1931 *average of three measurements. conclusion in present study, determination of cabz in its pure form and in its pharmaceutical dosage was investigated by two new kinetic methods (initial rate and fixed time). it was found that the proposed methods are precise, accurate, and sufficiently sensitive to be applied for determination of small amounts of cabz. therefore, the proposed methods could be recommended for the analysis of cabz in quality control laboratories. reference 1. british pharmacopeia, cd-rom her majesty, s stationary office, london, 2013. 2. bradley, mk., rene, h. levy, rr, matson, r., b. meldrum, jk. penry, fe. dreifuss (eds.) carbamazepine and carbamazepine epoxide, antiepileptic drugs, third edition, raven press, ltd., new york, 1989, 505-517. 3. "carbamazepine". the american society of health-system pharmacists. (2015(. 4. dikran, s. b. mohammed, a. k. alassaf, n. a." simple rp-hplc iraqi j pharm sci, vol.26(1) 2017 kinetic-spectrophotometric method for the assay of carbamazepine 8 method for estimation of furosemide, carbamazepine, diazepam and 5. carvedilol in bulk and pharmaceutical dosage forms", chemistry and materials research 2016; 8 (3). 6. hoehn e.," detection of the pharmaceuticals carbamazepine and diphenhydramine in tissue extracts using gas chromatography-mass spectrometry (gc-ms)" environmental studies undergraduate student thesis 2014. paper 137. 7. lavanya, n., c. sekar, s. ficarra, e. tellone, a. bonavita, s. g. leonardi, and g. neri. "a novel disposable electrochemical sensor for determination of carbamazepine based on fe doped sno2 nanoparticles modified screenprinted carbon electrode." materials science and engineering: c 62 .2016; 5360. 8. fadhel, s. r. abdulla, n. i. sulaiman, i.d." spectrophotometric determination of carbamazepine via oxidative coupling reaction with 2, 4dinitrophenyl hydrazine "ibn al-haitham jour. for pure & appl. sci. 2016; 29 (1): 226-238. 9. eman y.z., m.a. zayed, m.m. omar, e.a. elashery, g. g. mohamed," spectrophotometric determination of carbamazepine and mosapride citrate in pure and pharmaceutical preparations" arabian journal of chemistry 2012; 5: 375–382. 10. mulliken, r. s. overlap integrals and chemical binding1. journal of the american chemical society, 1950; 72(10):4493-4503. 11. fadhel, s. r., n. i. abdulla, and i.d. sulaiman. "the spectrophotometric determination of olanzapine via coupling with diazotized p-nitroaniline. iraqi j pharm sci, 25 (1). 2016:42-49 12. al-rufaie, m. m. new spectrophotometric method for the determination of sulfamethoxazole drug 2016; 5: 172-183. 13. shamsa, f., & amani, l. determination of sulfamethoxazole and trimethoprim in pharmaceuticals by visible and uv spectrophotometry. iranian journal of pharmaceutical research, 2010: 31-36. 14. delvie, r. "principles of quantitative chemical analysis", international edn. mcgraw-hill inc., singapore, 1997:p. 498. 15. yoe, j. h., and jones, a. l. colorimetric determination of iron with disodium-1, 2 dihydroxybenzene-3, 5-disulfonate, anal. chem. 1944; 16: 111-5. 16. troy, d. b. (ed.), remington-the science and practice of pharmacy, 21stedn. lippincott williams & wilkins, philadelphia, 2006. 17. houston, p. l. chemical kinetics and reaction dynamics, mcgraw-hill companies, inc., new york, 2001. comparative evaluation of using intranasal desmopressin, parenteral diclofenac or their combination in the management of acute iraqi j.pharm.sci., vol.16 (2) ,2007 diazepam hollow type suppository 32 the release of diazepam from different conventional and hollow type suppository bases balkis a. kamal* ,1 *department of pharmaceutics , college of pharmacy, university of baghdad , baghdad , iraq abstract : the objective of this study was to investigate the release profile of different fat and water soluble bases using diazepam as a model drug , and then to develop a satisfactory formula with a rapid release of diazepam from suppository bases .the study was conducted using theobroma oil ,glycerol-gelatin and glycerol-peg1540 bases using conventional mold method for preparation .while the later base was utilized to incorporate diazepam ( buffered solution ) in a hollow type suppositories. the results indicated that all types of bases can be utilized to formulate diazepam as rectal suppositories with acceptable disintegration time ( 12, 10, 6, and 6min.), respectively . while 100% of the released drug had been shown different release profiles with different storage time ( 15, 30, and 45 days ) in a refrigerator at 5°c , best results were obtained , when glycerolpeg1540 used as a base for both conventional and hollow type mold methods .however , a decrease in the release rate of the drug was seen with glycerol-gelatin and glycerol-peg1540 and to a lesser extent for glycerol-peg1540 prepared by hollow type method . key words: diazepam , hollow type suppository الخالصة: أٌ انهذف يٍ هزِ انذساست هى نهبحث عٍ شكم انخحشس نعقبس انذايبصيببو كًُىرج يٍ خالل قىاعذ نبىسبث شحًيت و يبئيت , و يٍ ثى ) صبذة انكبكبو , انذساست حىاصهج ببسخعًبل قىاعذ حطىيش حشكيبت يقُعت بخحشس سشيع نعقبس انذايبصيببو يٍ قىاعذ هزِ انهبىسبث . اٌ ( و رنك ببسخخذاو طشيقت انصب انخقهيذيت بيًُب اسخعًهج انقبعذة االخيشة 2651بىني اثهيٍ كاليكىل-جيالحيٍ , انكهيسيشول-ولانكهيسيش الدخبل انذايبصيببو كسبئم في ححبييم يجىفت . اشبسث انُخبئج انى اٌ كم انقىاعذ انًسخعًهت يٍ انًًكٍ اسخخذايهب نخشكيب انذايب صيبيبو % يٍ انعقبس انًخحشس يٍ هزِ انقىاعذ 211( دقيقت عهى انخىاني. بيًُب وجذ اٌ َسبت 7,7,21,23وقبث اضًحالل )كخحبييم ششجيت بب -يئىيت , كًب اٌ افضم انُخبئج كبَج عُذيب اسخعًم انكهيسيشول 6يىيبً في دسجت حبشيذ 56,41,26ببشكبل يخخهفت قذ حأثش بفخشاث خضٌ انطشيقخيٍ انصب و انًجىفت . ويع رنك فبٌ َقصبٌ في يعذل سشعت ححشس انعقبس قذ نىظ كقبعذة في كال 2651بىني اثهيٍ كاليكىل بىني اثهيٍ -و انى ظذ اقم في قبعذة انكهيسيشول 2651بىني اثهيٍ كاليكىل-جيالحيٍ و قبعذة انكهيسيشول-في قبعذة , انكهيسشول بطشيقت انخجىيف . 2651كاليكىل introduction : suppositories are solid dosage forms of various weight and shapes ,intended to be use in the rectum, vagina or even in the urethra (1) , they disintegrate in the body cavity either by melting or by dissolution (2) . rectal anxiolytic suppositories are indicated for patients with systemic sedative action (3) , or to avoid hepatic first pass mechanism (4) , and also in post operative treatment (5) .since most of sedative drugs are used widely , mainly diazepam (6) , so it is of wise to use it as suppositories in children and elderly patients as sedative-hypnotic agent in the management of clinical conditions (7, 8 ) , however , rectal solution of diazepam has not been used widely , due to its ability to leak out of the rectum , and then no efficient drug treatment obtained .this study has conducted by incorporated diazepam as a powder in melted bases and as a buffered solution in an engraved cavity within backbone of solid suppository (9) , in addition to develop most effective rapid release of diazepam from drug solution rather than dispersed diazepam in a suppository bases (10, 11 ) 1 corresponding author : e-mail : ybmmaz @yahoo.com received : 24/3/2007 accepted : 29/12/2007 iraqi j.pharm.sci., vol.16 (2) ,2007 diazepam hollow type suppository 33 experimental materials : diazepam, supplied by sammara"a drug industry "sdi ,256bn6/2004, polyethylene glycol1540 ,bdh chemicals ,ltd. ,pool ,england. disodium hydrogen orthophosphate , potassium dihydrogen orthophosphate , e.merk ,darmstadt, germany. polyethylene sorbitan monolaurate ( tween 20 ) , evans , liverpool , england . all other reagents were of analytical grade . instruments : balance , sartorious ag , gottingen ,germany . dissolution apparatus , copley , type , fh 16 – d , nottingham , england . disintegration apparatus , water bath , kotterman ,ollmann , and co. ,kg , d 6360 , friedberg – germany . phmeter , ph 211 , microprocessor , italy . suppository mold 1gm. ,stainless steel , erbo prazision forminbau , gmphd 7470 , albstadt , england . uvvisible spectrophotometer , cintra 5 , doublebeam spectrophotometer operation , manual gbc – england . methods : preparation of diazepam suppositories : the fusion method was used to prepare the conventional suppositories ( table 1. ), by mixing 5 mg.equivalent weight of diazepam in each 1g. . suppository base. after calculation the displacement value , the bases were melted using a water bath with continous stirring until homogenous mixture was produced , the melted mixture then poured into the metal suppository mold , and then cooled in a refrigerator maintained at 5 °c this process was conducted for the formulas ( 1,2 and 3, ) , while formula 4 was prepared by hollow type method described by ,watanabe , et al (12 ) , which summarized by melting the bases at 45 °c , then mixing water until homogenous dispersion results . the melted bases then poured into 1g. suppository mold equipped with a cylindrical tube in the center and allowed to stand for two hours at room temperature to solidify ,after that a construction of a hollow cavity in the solidified base , 0.5 ml. of buffered diazepam solution (prepared by mixing diazepam powder in 3% w/w tween 20 buffered in ph 7.8) was added to each cavity, the opening at the back part of the suppository was sealed with the same melted base , each suppository in all formulas contain an equivalent amount 5mg. of diazepam . table (1): different prepared formulas suppositories with various methods formula (1) bees wax 10% ( displacement value 0.88 ,conventional) theobroma oil 90% formula (2) gelatin 10% (displacement value 1.04 , conventional ) glycerin 70% water 20% formula (3) peg1540 30% (displacement value 0.98 , conventional ) glycerin 50% water 20% formula (4) peg1540 30% (displacement value ″not involved″ , hollow type ) glycerin 50% water 20% evaluation of physical properties of suppositories : the prepared suppositories were evaluated for disintegration time according to the method described in british pharmacopoeia bp (13 ), each determination was carried out in triplicate run. in-vitro dissolution study : the dissolution rates of diazepam release from both conventional and hollow-type suppositories were carried out , a rotating basket dissolution apparatus was used at 50 rpm. maintained at constant temperature of 37°c. the medium used was 500ml. of orthophosphate buffer solution with ph 7.8, at time intervals 2,4,8,16,32,and 64 minutes , 5ml. of samples were withdrawn and the amount of diazepam was determined by uvspectrophotometry at λmax. 232nm , using phosphate buffer as blank solution, the total percentage of drug released from the mean triplicate samples were estimated . results and discussions : the disintegration time for the prepared suppositories in the formulas 1 ,2 ,3 ,and 4 was 12±0.28 , 10±0.1 , 6±0.05 , and 6±0.11 minutes respectively , these results were found in consistent of bp and fda requirement for disintegration time of rectal suppositories . tables 2,3,4,5 and figure (1) . show the percentage of diazepam released from 4 different formulas of suppository bases and the method of preparation , at the first day of preparation , the results indicated that the release of diazepam from the conventional and iraqi j.pharm.sci., vol.16 (2) ,2007 diazepam hollow type suppository 34 0 10 20 30 40 50 60 70 80 90 100 110 0 20 40 60 80 form ula 1 form ula 2 form ula 3 form ula 4 hollow type suppositories is variable. formula 1, 2 and 3 which they are prepared by conventional method revealed that the rate of diazepam release from insoluble theobroma oil is very slow compared with the rate diazepam released from hydrophilic base glycerolgelatin and glycerolpeg bases. the time for 50% drug release was more than 60 minutes from formula 1, compaired with 16 and 5 minutes for bases in formula 2, and 3 ,respectively . these results may be referred to the physicochemical properties of both drug and the base used (14) . diazepam is highly lipid soluble , so its presence in low concentrations in theobroma oil base will have slow step of escaping out side the base vehicle (10) . the slow release of diazepam from fatty bases may be attributed to its high lipid solubility and their greater retention within the oily base , that can be candidate to be sustained release dosage form ,when larger dose ( 10 -15 mg. ) diazepam was dispersed in the base (15 ) . on the other hand , the release of diazepam from glycerol-peg bases showed significant difference ( p< 0.05 ) and revealed to be faster than that incorporated in glycerol-gelatin base ,since the time for 100% of drug release were about 32 and more than 64 minutes, respectively . these results were in consistent with the results obtained from piroxicam released when gelatinous base was employed (16) , since gelatinous consistency of glycerinated gelatin in a dispersed system may be formed and decrease the drug release from gelatinous barrier . meanwhile , the modification of suppository shape (hollowtype) by incorporating the diazepam as 1%(w/v) buffered solution ( formula 4), revealed that the drug release is significantly ( p< 0.05 ) faster than other conventional type used with the same base ( formula 3 ), since the time required for 100% drug release was 8 and 32 minutes , respectively . this result may be attributed to the concept that all the diazepam present in a buffered solution (ph7.8 ,formula 4 ) ,was available to be absorbed and this last about 6 minutes of disintegration time for suppository to be dissolved, compared with that diazepam powder dispersed in glycerol-peg 1540 base (17) . the effect of storage period on the dissolution rate of diazepam was investigated at 5°c for 15 ,30 ,and 45 day ,the results were indicated that there is no significant difference ( p> 0.05 ) in the drug release from theobroma oil base (formula 1 ) , when stored for 45 days (fig.2) , since the over all drug released remain limited ( 25-35% ) within the lipophyllic base (11) . on the other hand ,the effect of storage on the diazepam released from glycerol-gelatin base (fig.3) showed no significant decreasing in the amount of diazepam released. this behavior may be attributed to the tendency of free (oh) groups of glycerin to form hydrogen bonds with many functional groups located on amino acids moieties in gelatin , and this may form a crosslinking network that hinder the drug release (18) .this result was in agreement with the result obtained by hanaee j. ,et .al , (18 ) .in addition ,the release of the drug from glycerolpeg1540 base for conventional suppositories (formula 3), showed that the time for 50% drug release was 4-8.5 minutes, besides to that, unusual hollow-type suppository filled with buffered liquid diazepam showed no effect on the drug released , after one month of storage (19 ) the over all results of this study , revealed that diazepam can be formulated as a rectal suppository dosage form utilizing glycerol peg 1540 as a water soluble base, with best percent of drug release using solution of diazepam within hollow-type method of preparation .. table (2) : effect of storage on the release of diazepam (5mg.) from conventional theobroma oil suppository base at 5 °c (*) each value represents the mean sd(±) ,with n=3 samples, and p<0.05 (significant), with corresponding percent of drug release . figure 1. the percentage of diazepam released from different formulas (suppository bases) at first day of preparation in phosphate buffer ph7.8 at 37 °c . percent of drug released after extensive period of storage time(*) . 45 day 30 day 15 day one day time (min) 0 0 0 0 0 3±0.26 6 ±0.17 6 ±0.3 6±0.2 2 6±0.10 6 ±0.34 8 ±0.17 9±0.17 4 12±0.45 12 ±0.5 15 ±0.5 14± 1.0 8 18±0.52 23±0.46 24 ±1.0 26±2.64 16 22±0.6 26±0.5 29±0.6 30±0.9 32 25±0.25 27±0.69 30 ±0.4 35±1.05 64 time (min.) % d r u g r e le a se d iraqi j.pharm.sci., vol.16 (2) ,2007 diazepam hollow type suppository 35 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 one day 15 day 30 day 45 day table (3) : effect of storage on the release of diazepam (5mg.) from conventional glycerol-gelatin suppository base at 5 °c . (*) each value represents the mean sd(±) ,with n=3 samples, and p<0.05 (significant), with corresponding percent of drug release . table (4) :effect of storage on the release of diazepam (5mg. ) from conventional glycerol–peg 1540 suppository base at 5 °c . percent of drug released after extensive period of storage time(*) 45 day 30 day 15 day one day time (min) 0 0 0 0 0 17±0.34 18±0.34 20±0.46 22±0.28 2 32±0.05 38±0.05 40±0.57 45±0.28 4 56±0.52 60±0.11 66±0.51 68±0.57 8 77±0.46 82±0.42 90±1.1 93±0.55 16 85±0.48 88±0.40 100±0.0 100±0.0 32 100±0.0 100±0.0 100±0.0 100±0.0 64 (*) each value represents the mean sd(±) ,with n=3 samples, and p<0.05 (significant), with corresponding percent of drug release . table (5) :effect of storage on the release of diazepam (5 mg.) from hollow-type glycerolpeg1540 suppository base 5 °c percent of drug released after extensive period of storage time(*) . 45 day 30 day 15 day one day time (min) 0 0 0 0 0 20±1.15 22±0.34 26±0.57 36±0.46 2 42±0.51 50±0.55 68±1.12 84±0.23 4 72±0.52 80±0.51 90±0.56 100±0.0 8 96±0.48 100±0.0 100±0.0 100±0.0 16 100±0.0 100±0.0 100±0.0 100±0.0 32 100±0.0 100±0.0 100±0.0 100±0.0 64 (*) each value represents the mean sd(±) ,with n=3 samples, and p<0.05 (significant), with corresponding percent of drug release . figure 2. effect of storage on the release of diazepam (5mg.) from conventional theobroma oil suppository base after different times in phosphate buffer ph7.8 at 37 °c . figure 3. effect of storage on the release of diazepam (5mg.) from glycero-gelatin conventional suppository base at different times in phosphate buffer ph 7.8 at 37 °c. percent of drug released after extensive period of storage time(*) . 45 day 30 day 15 day one day time (min) 0 0 0 0 0 7±0.17 8±0.43 10±0.23 10±0.2 2 17±0.17 20±0.11 21±0.2 22±0.45 4 30±0.17 30±0.28 32±0.34 40±0.23 8 41±0.57 46±0.34 48±0.17 50±0.52 16 51±0.52 53±0.57 60±1.15 66±0.57 32 66±0.55 71±0.32 74±0.57 81±0.11 64 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 one day 15 day 30 day 45 day time (min.) time (min.) % d r u g r e le a se d % d r u g r e le a se d iraqi j.pharm.sci., vol.16 (2) ,2007 diazepam hollow type suppository 36 0 10 20 30 40 50 60 70 80 90 100 110 0 20 40 60 80 one day 15 day 30 day 45 day 0 10 20 30 40 50 60 70 80 90 100 110 0 20 40 60 80 one day 15 day 30 day 45 day figure 4. effect of storage on the release of diazepam (5mg.) from conventional glycerol-peg 1540 suppository base at different times in phosphate buffer ph 7.8 at 37 °c. figure 5. effect of storage on the release of liquid diazepam (5mg.) from hollow-type glycerol-peg1540 suppository base after different times in phosphate buffer ph 7.8 at 37 °c . conclusion : based on the results obtained from this study, one can conclude the followings : 1diazepam as an anxiolytic drug can be formulated successfully using different suppository bases. 2best results obtained , using glycerol gelatin and glycerolpeg1540 as a water soluble bases. 3hollow-type suppositories can be 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pharmaceutical evaluation of hollow-type suppositories , improvement of bioavailability of propranolol in rabbits after rectal adminsteration , journal of pharmacobiodynamic , (1986) ,9 ,(6) ,526-531.. 13british pharmacopoeia (2002),vol i and ii , london c.d . 14realdon n., raggazi e. , " effect of drug solubility ov invitro availability rate from suppositories with peg excipient " pharmazie , (2001)56 ,(2) ,163-167 . . 15toshihito t. , norihito s , "evaluation of sustained release suppositories prepared with fatty base including fats with high melting points ",international journal of pharmaceutics , (2004) ,278 , 275282 . 16eman j., balkis a. kamal ," the release of piroxicam from different suppository bases iraqi journal of pharmaceutical sciences , (2002),11 ,15-23 . 17kaewnopparat n. ,roganarat w. ," enhancement of diazepam release from witepsol-15 suppositories " inter., journal of pharmaceutical compounding " (2004),july-aug. ,1-6 .. 18hanaee j. ,javadzadeh y.,taftachi s. ,farid d. , " the role of various surfactants on the release of sulbutamol from suppositories , italian pharmaco. , (2004) , 59 (11) , 903-906 . 19stansilaw j. , malgozata s., hallina g. ," evaluation of paracetamol suppositories by a pharmacopoeial dissolution test comments on methodology ,european j ., of pharmam. and biopharmacy , (2001), 52 ,(june) ,249-254 . iraqi j pharm sci, vol.27(2) 2018 oxidative coupling reaction of chlordiazepoxide with phenothiazine doi: https://doi.org/10.31351/vol27iss2pp7-14 7 spectrophotometric determination of chlordiazepoxide in pharmaceutical formulations via oxidative coupling reaction with phenothiazine mariam j. ahmed* and hind h. abdullah*, 1 *department of chemistry, college of science, university of baghdad, baghdad, iraq. abstract a sensitive, precise and reliable indirect spectrophotometric method for the determination of chlordiazepoxide (cde) in pure and pharmaceutical dosage forms is described. the method is based on oxidative coupling reaction between amino group resulting from acidic decomposition of cde with phenothiazine in the presence of sodium periodate to produce an intense green soluble dye that is stable and shows a maximum absorption at 602 nm. the calibration plot indicates that beer’s law is obeyed over the concentration range of 0.1−50 µg/ml, with a molar absorptivity of 1×104 l/mol cm and correlation coefficient of 0.9994.all the conditions that affecting on the stability and sensitivity of the formed product were studied and optimized and the suggested method was effectively applied for the determination of cde in commercial dosage forms. keywords: chlordiazepoxide, phenothiazine, oxidative coupling reaction. التقدير الطيفي للكلورديازيبوكسيد في األشكال الصيدالنية بواسطة تفاعل االزدواج الفينوثيازينالتاكسدي مع 1،*هند هادي عبدهللا و مريم جمال احمد العراق. ،بغداد ،جامعة بغداد ،كلية العلوم ،قسم الكيمياء* الخالصة كله و مصداقية لتقدير دواء الكلورديازيبوكسيد في ش ،دقة ،حساسية يتضمن البحث استخدام طريقة طيفية غير مباشرة ذات تعتمد الطريقة المقترحة على تفاعل االزدواج التاكسدي بين مجموعة االمين الناتجة من التحلل .النقي ومستحضراته الصيدالنية و مستقرة تعطي أعلى الحامضي للدواء مع كاشف الفينوثيازين بوجود بيريودات الصوديوم لتكوين صبغة خضراء ذائبة بالماء مل, وكانت \مايكروغرام( 1050) قانون بير وبمدى التركيز نانومتر. منحني المعايرة يطاوع 602امتصاص عند الطول الموجي و قد تم دراسة جميع الظروف . 0.9994مول .سم, وبمعامل ارتباط ال يقل عن \لتر 104×1قيمة االمتصاصية الموالرية مساوية إلى الكلورديازيبوكسيد في المؤثرة على استقرارية وحساسية الناتج المتكون و تثبيتها, وتبين أن الطريقة المقترحة طبقت بنجاح لتقدير التجارية . األشكال الصيدالنيةمختلفة من أشكال .دواج التاكسديتفاعل االز ، الفينوثيازين، الكلمات المفتاحية: الكلوديازيبوكسيد introduction chlordiazepoxide (cde) chemically known as 7-chloro-2-methylamino-5-phenyl3h-l,4-benzodiazepine-4-oxide (figure 1), is a benzodiazepine showing powerful antianxiety effects in humans and effectively used to enhance the effect of the neurotransmitter gamma-amino butyric acid resulting in sedative, hypnotic, anxiolytic, anticonvulsant and muscle relaxant properties )1-3(. figure 1. chlordiazepoxide the literatures reported different methods for simultaneous determination of cde both in pharmaceutical formulations and biological samples, including spectrophotometry )4-6(, flourimetry )7(, dispersive nanomaterialultrasound assisted microextraction )8(, voltammetry (9,10), polarography (11), capillary electrophoresis (12), gas chromatography (13, 14), and high performance liquid chromatography (15,16).although the sensitive visible spectrophotometric methods are very little, the literature contained a simple colorimetric methods for the determination of benzodiazepine drugs included cde using 1corresponding author e-mail: hindhadi13@yahoo.com received: 7 / 4 / 2018 accepted: 10 / 6 / 2018 iraqi journal of pharmaceutical sciences n n nhch3 oh cl o https://doi.org/10.31351/vol27iss2pp7-14 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 oxidative coupling reaction of chlordiazepoxide with phenothiazine 8 diazotization reaction, depends upon the formation of their corresponding amino benzophenones after acidic hydrolysis and then conversion the amino group to diazonium salt using hcl/nano2 (17). this simple reaction was depended on the development of simple and very sensitive method based on oxidative coupling reaction between hydrolysis product (decomposed cde) and phenothiazine (pht) as a coupling reagent in the presence of sodium periodate. absorbance measurements were established by recording the absorbance of the green colored product at 602 nm versus reagent blank which has a minimum absorbance at the same wavelength. experimental apparatus double beam shimadzu uv–vis 260 spectrophotometer (kyoto-japan) was used in all measurements (spectral and absorbance). the absorbance measurements were carried out using matched 1cm quarts cells. materials and solutions the reagents grade materials were used throughout this work. chlordiazepoxide (cde) standard was supplied by the state company for drug (sdi), samarra-iraq. libroxide ® 5 and 10 mg chlordiazepoxide (sdi, samarrairaq) tablets were obtained from local markets. preparation of hydrolyzed chlordiazepoxide standard solution a 25 mg amount of standard cde was accurately weighted and dissolved in a 25 ml of 6 m hydrochloric acid then heating the solution in a boiling water-bath for 1 hour. the decomposed drug transferred into 50 ml volumetric flask and complete the volume to the mark with distilled water to obtain 500 μg/ml of cde hydrolyzed solution (17). this solution is stable for more than one week if kept in room temperature. simple dilution using the same concentration of acid was used to prepare more diluted solutions. phenothiazine solution (0.01 m, bdh) the solution of this reagent was prepared by dissolving 0.0996 g of the reagent in 50 ml ethanol and kept in dark bottle. sodiumperiodate solution (0.05 m, bdh) this solution was prepared in 50 ml volumetric flask via dissolving 0.5347 g of naio4 in distilled water. hydrochloric acid solution (6 m, bdh) this was prepared by appropriate dilution of 250.6 ml of concentrated solution (11.97 m) with 500 ml distilled water in volumetric flask then standardized with na2co3 solution. preparation the solutions of pharmaceutical tablets a 30 tablets (libroxide ® 5 and 10 mg chlordiazepoxide) were accurately weighed and powdered then an equivalent to 50 mg of cde of the powder was taken and dissolved in about 25 ml of ethanol and shaken then filtered (this solution is 2000 μg/ml of cde). after transferring 12.5 ml of the result solution into a beaker, a 12.5 ml of concentrated hydrochloric acid was added then the hydrolysis process was performed as described previously. after the decomposition procedure was accomplished the decomposed solution transfer into 50 ml volumetric flask and complete the volume to the mark with distilled water to obtain a 500 μg/ml of cde solution. general procedure with using a volumetric flasks (10 ml) an increasing amounts of sample containing 1–500 µg of hydrolyzed standard cde (100 μg/ml) was transferred (cover the range 0.1-50 µg/ml of cde) then added 0.2 ml of 10 mm pht solution and about 0.1 ml of 50 mm of sodium periodate solution. the reactants then diluted with distilled water and mixed. the absorbance of resulting solutions was measured after 10 min at 602 nm at room temperature (25°c) against reagent blank (prepared under the same procedure but without cde). alternatively the corresponding calibration curve and regression equation were constructed. for the optimization of conditions and in all later experiments, a solution of 250μg of cde was used in a final volume of 10 ml (i.e. 25 ppm). stoichiometric relationship the stoichiometry of the suggested reaction was investigated. an equimolar of cde and pht (1mm for both) under optimum concentration of sodium periodate were prepared using job’s method (18) (fig. 1).in job’s method of continuous variation a sequence solutions (total volume of the cde and pht was 5 ml) have been prepared. hydrolyzed drug and reagent indifferent complementary ratio (0:5, 1:4, 2:3, 3:2…..5:0) were mixed, diluted and directed under the suggested procedure in 10 ml volumetric flask. and the absorbance was measured at 602 nm. figure 2 showed that a 1:1 ratio product (cde:pht) is formed. iraqi j pharm sci, vol.27(2) 2018 oxidative coupling reaction of chlordiazepoxide with phenothiazine 9 figure 2. job’s method cde is completely hydrolyzed to 5chloro-2-aminobenzophenone when heated with hydrochloric acid in a boiling water-bath. the strong electron donating ability of amino group in hydrolyzed cde make it coupled with oxidized pht easily and formation the oxidative coupled product. depending on the result obtained from job’s method, the mechanism of formation of the product (2 -( (3h-phenothiazin-3-ylidene) amino) – 5 – chloropheny l) (phenyl) methanone) under oxidative coupling product may be suggested according to the following scheme (19-21): scheme 1. proposed mechanism of reaction product (2-((3h-phenothiazin-3-ylidene) amino)-5chlorophenyl)(phenyl)methanone) results and discussion absorption spectra according to the previous procedure, the absorption spectrum of the green product formed by the reaction between the hydrolyzed product of the cde and pht in acidic medium has been recorded (fig. 3). after 10 minutes from the beginning of reaction between hydrolyzed cde with pht reagent, the spectra were obtained. the maximum wavelength of the green colored product was appeared at 602 nm. iraqi j pharm sci, vol.27(2) 2018 oxidative coupling reaction of chlordiazepoxide with phenothiazine 10 figure 3. absorption spectra of 25 μg/ml of decomposed cde treated as described procedure and measured against blank, and the blank measured against water. study of the method variables the experimental factors such as the reagents concentrations, order of addition, temperature, medium of reaction and stability time that may affecting mainly the sensitivity of the colored product were studied by variable one parameter with the time, and kept the rest fixed. as we mentioned previously all experiments were done using 25 μg/ml of cde and the absorbance measurements were carried out after 10 min from the beginning of the reaction at λmax=602 nm and laboratory ambient temperature (25 ± 2c°). effect of reaction time effect of reaction time on the steadiness of absorbance readings after dilution was studied (table 1). the absorbance of colored product started to be stable after 5 min and remains stable more than 60 min. the large stability time offer an advantage of measuring large number of samples comfortably at any time within the period without changing in the values of readings. this experiment was repeated after optimization all variables and the result was the same. table 1. the effect time of reaction time, min 2 5 10 15 20 25 30 40 50 60 70 absorbanc e 0.50 8 0.53 8 0.53 8 0.53 7 0.53 8 0.53 7 0.53 7 0.53 8 0.53 8 0.53 4 0.52 8 effect of temperature and order of addition three different temperatures were used for study the effect of temperature on the suggested reaction (5, 25, and 65°c) and the experiment indicated that maximum absorbance was obtained at room temperature and at 65°c, more than at 5°c, which that may be due to increase the coupling affinity between the reactants at high temperature (fig. 4a). ambient temperature was chosen in all the following experiments because the product is more stable than at 65°c.also variable addition orders of reagents were studied and it was found the order of (cde+pht+naio4) was gave the best results and was used in all the following experiments (figure 4b). figure 4 (a). effect of temperature figure 4 (b). effect of order of addition (d; drug, ox; oxidant, r; reagent effect of reagents concentration (naio4and pht ) naio4 was found to be a useful oxidizing agent for the proposed reaction, other oxidizing agents (potassium per sulphate, potassium dichromate, potassium hexacyanoferrate, and ferric chloride) have also been tested, but none give product when tried instead of naio4. effects of the variable volumes of pht 10mm (from 0.1–0.5 ml) and naio4 50 mm (from 0.05–1 ml) were examined. results indicated that 0.2 ml of iraqi j pharm sci, vol.27(2) 2018 oxidative coupling reaction of chlordiazepoxide with phenothiazine 11 pht and 0.1 ml of oxidant gave the higher absorbance of product (fig. 5) and were considered to be optimum for the next experiments. the absorbance was decreased with increase of the volume of naio4 that added, this may be due to the increase in the absorbance of the blank with each addition of oxidant. figure 5. (a) effect of volume of pht, (b) effect of volume of oxidant. effect of reaction medium the green colored product appeared directly during coupling reaction between reactants; and the acidic of the reaction medium (used for decomposition process of cde) was enough for development of the color. more acidic solution was not change the absorbance, while the basic medium caused a shift in maximum wave length value to 542 nm accompanied with decrease in sensitivity; therefore, there is no need to add an acidic or basic solution. method of validation linearity under the optimized experimental conditions for cde determination, standard calibration curve was constructed (fig. 6). into (10 ml) of sequences standard flasks increasing amounts of sample containing 1– 500 µg of cde was transferred (cover the range 0.1-50 µg/ml) then a volume of 0.2 ml of 10 mm pht and 0.1 ml of 50 mm of oxidant (naio4) were added. the reactants into the flasks were mixed and diluted with distilled water, and finally left for 10 min to reach to the stability. the absorbance later measured at 602 nm at 25°c (room temperature). the regression equation was derived using the least-squares method. the intercept, slope, correlation coefficient, and molar absorptivity values in addition to statistical analytical values are calculated and listed in table 2. results listed indicate a good sensitivity with linearity over the range of 0.150 μg/ml. figure 6. calibration curve. table 2. summary of characteristics data of suggested method parameter value regression equation y= 0.034x + 0.0475 linear range (μg/ml) 0.1-50 correlation coefficient(r2) 0.9994 lod (μg/ml) 0.11 loq (μg/ml) 0.36 reproducibility (%) 0.2-1.5 average of recovery (%) 100.43 molar absorptivity(l/mole cm) 1.00×104 sandell’s sensitivity (μg/cm2) 2.99×10-2 accuracy and repeatability to estimate the accuracy of the proposed method, and the repeatability of readings, two altered concentrations solutions of cde were prepared. the assay process was applied in three replicates, and the rsd% (percentage relative standard deviation) was obtained. the satisfactory results in table 3 showed that a low values for the rsd (good precision) and values of relative error (accuracy) of the suggested method were obtained. table 3. accuracy and precision of the proposed method conc. of cde, μg/ml error *% rec.*% rsd %* present found 25 24.92 -0.32 99.68 0.17 30 29.96 -0.13 99.87 1.49 35 35.62 1.77 101.77 0.31 *average of three determinations. (a) iraqi j pharm sci, vol.27(2) 2018 oxidative coupling reaction of chlordiazepoxide with phenothiazine 12 method specificity (study of pharmaceutical additives) examination of the specificity and selectivity of the suggested method was done by analysis of target drug in the existence of 10-fold of common additives (talc, starch, polyvinyl pyrrolidone and magnesium stearate) which often accompany cde in its dosage forms. the satisfactory obtained recovery values (99.8-101.7%) indicating no interfering with these additives were observed which representing the selectivity of the method (table 4). table 4.effect of common additives additives (250 μg/ml) conc. of cde, μg/ml rec.% rsd% present found poly vinyl pyrrolidone 25 25.27 101.08 0.31 talc 25.32 101.28 0.99 starch 24.94 99.76 0.83 magnesium stearate 25.03 100.12 0.43 all the above additives 25.44 101.76 0.98 analytical applications the current procedure was applied profitably to the estimation of cde in tablets. two doses of tablets containing 5 and 10 mg of cde have been analyzed by applying direct and standard addition methods. the solutions of tablets were prepared as mentioned previously, and the results obtained in table 5 and 6 (good precision <2.6 and high recoveries best than 98%) were in agreement with those of common method (uv spectroscopy method) (22), using two common tests (student t-test and f-test) at 95% of confidence level (23). the obtained results tabulated in table 6 (calculated values <<tabulated values) showed that there was no significant differences in accuracy or precision between the two methods. table 5. application of the proposed method to the determination of cde in different dosage forms using standard addition method dosage form taken conc. (μg/ml) proposed method pure drug added conc. (μg/ml) total found conc. (μg/ml) (%rec.± sd) n=4 libroxide® tablets (5mg-sdi) 25 5 29.59 98.63±0.61 10 34.54 98.69±0.50 30 5 34.71 99.17±0.58 10 39.71 99.28±0.85 libroxide® tablets (10mgsdi) 25 5 29.74 99.13±0.26 10 35.39 101.11±0.32 30 5 34.80 99.43±0.60 10 39.72 99.30±0.64 table 6. comparison and application of the current and uv methods. pharmaceutical form proposed method uv method taken conc. (µg/ml) found conc. (µg/ml) rec. (%)a mean rec. (%) rsd (%)a taken conc. (µg/ml) found conc. (µg/ml) rec. (%)a mean rec. (%) rsd (%)a libroxide® tablets (5mg) 25 24.79 99.16 98.88 0.20 20 19.66 98.30 98.56 0.58 30 29.58 98.60 0.29 30 29.61 98.70 0.15 40 39.47 98.68 0.33 libroxide® tablets (10mg) 25 24.98 99.92 99.49 0.45 20 20.23 101.15 100.7 7 0.37 30 29.72 99.07 0.49 30 30.17 100.57 0.65 40 40.24 100.60 0.12 pure cde 100.4 4 100.8 9 t (2.776)b f (19.000)b 0.307 2.785 (n1 –1) =2 ,(n2 –1) =2 , (n1+ n2 – 2) =4 a, (n=5); b theoretical value. iraqi j pharm sci, vol.27(2) 2018 oxidative coupling reaction of chlordiazepoxide with phenothiazine 13 conclusion oxidative coupling reaction is considered as one of the most significant reaction used for drug analysis. a few researches were proposed for determination this important drug, but most of them were not sensitive or requiring difficult steps and expensive techniques. the suggested method is simple, specific and in addition, it does not require an extraction or any expensive solvents or technique. this method applied successfully to the analysis of cde in tablets with good accuracy and precision. the proposed method was compared with uv method (classical method) of cde determination and by using the statistical methods (tand f test) and the suggested method proved accuracy and precision and can be used routinely in quality control studies. references 1. g. k. mcevory, ahfs drug information, american society of hospital pharmacists1990. 2. m. e. p. seligman, e. f. walker, d. l. rosenhan abnormal psychology 4th ed new york: w.w. norton and company, inc 2000. 3. s. r. walker. trends and changes in drug research and development. springer science & business media. 2012,p. 109. 4. i. popovici, v. dorneanu, r. cuciureanu and e. stefanescu, “spectrophoto-metric determination of some 1,4benzodiazepines with picric acid in aprotic mediumˮ . rev. chim. 1983;34: 554-559. 5. h. m. lotfy, y. m. fayez, a. m. michael and c. k. nessim. “simultaneous determination of mebeverine hydrochloride and chlordiazepoxide in their binary mixture using novel univariate spectrophotometric methods via different manipulation pathwaysˮ. spectrochim. acta, 2016;155:11-20. 6. a. a. othman, r. i. el-bagary, e. f. elkady and m. m. el-kerdawy. “development and validation of spectrophotometric methods for the simultaneous determination of mebeverine hydrochloride and chlordiazepoxide in bulk and in dosage formˮ. pharm. anal. acta. 2016;7(8):1-8. 7. l. szekelhidi, a. lapat, i. hornyák, “spectrofluorimetric determination of 2amino-5-chlorobenzophenone impurity in chlordiazepoxide hydrochloride”. ana. chim. acta, 1989;227:309-310. 8. a. amiri pebdani, s. khodadoust, m.s. talebianpoor, h.r. zargar, v. zarezade. “preconcentration and determination of chlordiazepoxide and diazepam drugs using dispersive nanomaterial-ultrasound assisted microextraction method followed by high performance liquid chromatography”. j. chromatogr. b, 2016;1008:146-155. 9. a. h. naggar, m. elkaoutit, i. n. rodriguez, a. y. el-sayed, j. l. h. cisneros.” use of a sonogel-carbon electrode modified with bentonite for the determination of diazepam and chlordiazepoxide hydrochloride in tablets and their metabolite oxazepam in urine”. talanta, 2012; 89:448-454. 10. l. m. de carvalho, d. correia, s. c. garcia, a. v. de bairros, p. c. nascimento, d. bohrer.” a new method for the simultaneous determination of 1,4benzodiazepines and amfepramone as adulterants in phytotherapeutic formulations by voltammetry”. forensic science international, 2010;202(1–3):7581. 11. m.a. brooks, j.a.f. de silva, “determination of 1,4-benzodiazepines in biological fluids by differential pulse polarography”, talanta, 1975;22:849-860. 12. l. m. de carvalho, m. martini, a. p. moreira, s. c.garcia, p. c. nascimento, d. bohrer. ” determination of synthetic pharmaceuticals in phytotherapeutics by capillary zone electrophoresis with contactless conductivity detection (czec4d)”. microchem. j., 2010;96(1):114119. 13. d. m. song, s. zhang, k. kohlhof,” determination of chlordiazepoxide in mouse plasma by gas chromatography— negative-ion chemical ionization mass spectrometry, j. chromatogr., 1994;660(1):95-101. 14. a. v. bairros, r. m. almeida, l. pantaleão, t. barcellos, s. m. silva, m. yonamine.” determination of low levels of benzodiazepines and their metabolites in urine by hollow-fiber liquid-phase microextraction (lpme) and gas chromatography–mass spectrometry (gc– ms)”. j. chromatogr. b, 2015; 975:24-33. 15. l. magrini, a. cappiello, g. famiglini, p. palma.”microextraction by packed sorbent (meps)-uhplc-uv: a simple and efficient method for the determination of five benzodiazepines in an alcoholic beverage”. j. pharma. biomed. anal., 2016;125; 48-53. 16. m. piergiovanni, a. cappiello, g. famiglini, v. termopoli, p. palma, “determination of benzodiazepines in beverages using green extraction methods and capillary hplc-uv detection, journal https://www.sciencedirect.com/science/journal/00399140 https://www.sciencedirect.com/science/article/pii/s0379073810002082#! 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( است دم لتقييم جودة الحياة النوعية لدى االطفالpedsqlالكبار واالستبيان ) . في االطفال فقط درجة االداء البدني dfoفي المرضةةةةى البالغيين كان مسةةةةتوى جودة الحياة النوعية الل لدى المرضةةةةى الذين يسةةةةتعملون ( ومرضةةةى dfoك اي فرق احصةةةائي ملحوي بين مرضةةةى )ولكن بالي المجاالت لم يكن هنا dfx)كانت الل من المرضةةةى الذين يسةةةت دمون ) (dfx ومن المالحظ ان مسةةتوى جودة الحياة النوعية لالطفال كانت اعلى لليال ومعدل مضةةاعفات المرض كانت الل لدى االطفال عن ما هو في .) لى فرط جودة الحياة النوعية يتم بالسيطرة ع مرض الثالسيميا الكبرى يعيق جودة الحياة النوعيه للمرضى بشكل ملحوي. تحسينالمرضى البالغين. الحديد في الجسم و الحد من التأثيرات الجانبية لطوارد الحديد ومضاعفات المرض. . لتقييم جودة الحياة النوعية لدى الكبار، جودة الحياة النوعية لدى االطفال ، جودة الحياة النوعية ، مرض الثالسيميا الكبرىالكلمات المفتاحية : 1corresponding author: e-mail: alijalal0135@gmail.com received: 28 /8/2018 accepted: 6 /11/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp44-52 mailto:alijalal0135@gmail.com iraqi j pharm sci, vol.28(1) 2019 quality of life in thalassemia major 45 introduction beta thalassemia syndrome is a group of hereditary blood disorders that are mainly characterized by reduction or absence of β-globin chain synthesis, resulting in a reduction of hemoglobin in red blood cells (rbcs), decreased production of rbcs and consequently anemia (1,2). thalassemia disease was first described by thomas b. cooley and pearl lee in 1925(2,3). in iraq, there is a high prevalence of β-tm. the prevalence rate in 2015 was 27.4 per 100,000 population (4). the β globin synthesis is normally regulated by two β genes; one on each copy of chromosome 11(5). thalassemia is an autosomal recessive genetic condition, caused by point mutation within or near β-gene. such mutations result in either absence of the β gene (β0-thalassemia) or reduction in synthesis of the β gene (β+-thalassemia)(6). the reduction or absence of the β-globin chains results in a relative excess of α-globin chains, which when unpaired are unstable and precipitate in the erythroid precursors in the bone marrow, consequently there is ineffective erythropoiesis and the mature cells that reach the circulation have a shortened lifespan(7). the cornerstone management for patients with β-tm is based on lifelong transfusion and iron chelation. the aims of transfusion are to correct anemia, suppress ineffective erythropoiesis (8). without iron chelation therapy (ict) the regular blood transfusion would increase the iron stores to several times the normal levels, iron overload results in severe complications including cardiac disease, osteopathy and endocrine complications(9,10). deferoxamine (dfo) was the first invented ict, before its invention chronically transfused thalassemia patients were dying from cardiac iron overload in their twenties; but the life expectancy has improved since after (9). dfo is given as subcutaneous infusion for 8 hrs per day, 5 days per week. dfo has a negative impact on thalassemia patients' quality of life (qol) as the infusions can be very troublesome, painful and time consuming with restrictive activity(9,10). compliance with dfo and its rigorous requirements of daily subcutaneous infusion still a limiting factor for treatment success. dfo chelates iron only during the time infused, hence, poor compliance with dfo results in gaps in chelation coverage(11).deferasirox (dfx) is the most recent oral chelator (9). dfx has a half-life of 11-19 hrs. allowing for once-daily oral dosing(12). treatment with dfx is beneficial due to dramatically improved compliance which was reported to be ˃80%; in which dfx is more satisfying and found to be more convenient than dfo as it comprises a reduction in dosing frequency(9,11). icts may complicate patients' qol due to their serious side effects (13). the world health organization (who) defines quality of life as: “an individual perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards, and concerns” (14). health related quality of life (hrqol) is visualized as the patients' perception of the impact of the disease and treatment on their physical, psychological, social functioning and well-being(15). patients with β-tmare suffering from disease chronicity including frequent hospitalization, blood dependence, continuous treatment with ict; physical health limitations as growth retardation, poor physical appearance, and delayed puberty, in addition to disease complications. this imposes a requirement to make regular follow-up and medical care monitoring for physical, psychological, social aspects of patients with β-tm to help them cope with their illness to live as normal life as possible(16). assessing qol in this population provides a better understanding of the disease burden that the patient experiences every day, to measure the impact of the treatment and how does the patient go along with it, and to predict outcomes such as mortality and morbidity(17). patients and methods patient selection and study design the present study was designed as a singlecenter, cross-sectional study. the study was performed at al-karama teaching hospital/ department of hereditary blood disorders (thalassemia center). the participant, patients, in this study were all with β-tm trait who were attending thalassemia center at al-karama hospital for recurrent blood transfusion and for receiving their iron chelation therapy. a total of 201 patients approved to be enrolled in the study. the patients were divided into two main groups: group a: patients on deferoxamine (dfo) were sub-divided into two groups: children and adolescents (8-18 years) and adults (19 and more). and; group b: patients on deferasirox (dfx) were sub-divided into two groups: children and adolescents (8-18 years) and adults (19 and older). the sample size was calculated by using raosoft sample calculator (online tool). a total of 600 patients with β-tm were registered at alkarama thalassemia center, hence; a sample size of approximately 200 patients should be included, assuming a margin of error of 5% and a confidence level of 95%. method of quality of life assessment adults (19 years and older) hrqol were assessed by the world health organization quality of life (whoqol-bref) questionnaire which is validated for qol assessment in β‐tm(18). ethical approval for the use of the questionnaire was obtained from the who/ information, evidence and research department. whoqoliraqi j pharm sci, vol.28(1) 2019 quality of life in thalassemia major 46 bref questionnaire comprises 26 questions; two questions are concerned with the patient's overallperceptions of the qol, and the patient's overall perception on health, the remaining 24 questions are assessing 4 domains namely: physical health (domain 1) (7 questions), psychological (domain 2) (6 questions), social (domain 3) (3 questions) and environmental (domain 4) (8 questions). the answers were dependent on the patient's health status over the past 2 weeks. the patients' responses were rated on a 5-points likert scale (19,20). then the four domains were calculated, and the obtained raw scores were transformed to the 4-20 scale and their corresponding 0-100 scale according to the manual of the questionnaire(21). the arabic version of whoqol-bref is valid an reliable to be used in assessing hrqol in arab populations(18). hrqol was assessed in children (8-12 years) and adolescents (13-18 years) by using pedsqltm4.0 questionnaire which was formulated by varni and colleagues(22). ethical approval and agreement for using the questionnaire were obtained.the arabic version of the pedsqltm 4.0 questionnaire is a 23 question instrument considered to be feasible, reliable and valid to assess qol in iraq (23). the questionnaire includes 4 domains: physical functioning (8 questions), emotional functioning (5 questions), social functioning (5 questions); school functioning (5 questions) (15). the patients' answers were represented on a 5-points likert scale for each question. the mean score was computed by the sum of the questions' scores over the number of the questions answered. pedsqltm 4.0 has additional advantage of evaluating psychosocial health summary score (the sum of the questions' score over the number of the questions answered in the emotional, social and school functioning scales), physical health summary score (physical functioning scale score) and total summary score (sum of all questions' over the number of questions answered on all the scales)(14,22). the pedsqltm4.0 scores range from 0-100 points with higher scores indicating better hrqol(14). statistical analysis anderson darling test was done to assess whether continuous variables follow a normal distribution. if the variables were following normal distribution, then mean and standard deviation were used, and if they did not follow normal distribution then median and interquartile range (25%-75% percentile range) were used to present the data. discrete variables presented using their numbers and percentages, chi square test was used to analyze the discrete variables (or fisher exact test when chi square test is not valid; due to low sample size ˂20 and if 2 or more with an expected frequency is less than 5). two samples t-test was used to analyze the differences in means between two groups (if both follow a normal distribution with no significant outlier). binary logistic regression analysis was used to calculate the odd ratio (or) and their 95% confidence intervals (ci), when the outcome can be categorized into 2 binary levels, and if appropriate probability plot used to present the relationship. linear regression analysis was performed to assess the relationship between different variables. if one or both of the variables were following a normal distribution, person correlation was used. if both did not follow a normal distribution, spearman correlation was used. scatter plot was used to present the regression analysis. correlation coefficient or standardized beta (r) is a representative of magnitude and direction of the relationship; 0.00-0.29 = little or no correlation; 0.3.49 = weak; 0.5-0.69 = moderate; 0.7-0.89 = strong; and 0.9-1.00 = very strong. a negative sign indicates an inverse relationship, while a positive sign indicates a direct relationship. spss 22 (chicago, il), minitab 17.1.0 software package was used to make the statistical analysis, p value considered to be significant if less than 0.05. results demographic data and disease characteristics a total of 201 patients were approved to be included in the study. table (1) demonstrates patients included in the study as three groups (children (8-12), n= 40; (19.9%)), (adolescents (1318), n=78; (38.8%)), and (adults (˃19), n=83; (41.3%)), but; children and adolescents were considered as one category (pediatric) during the study. the demographic data and social characteristics were demonstrated in table (1) iraqi j pharm sci, vol.28(1) 2019 quality of life in thalassemia major 47 table (1) demographic data and disease characteristics variables value number 201 age (years), mean ± sd 19.1 ± 7.3 8-12 years 40 (19.9%) 13-18 years 78 (38.8%) ≥19 years 83 (41.3%) gender female 107 (53.2%) male 94 (46.8%) ), mean ± sd2bmi (kg/m 21.4 ± 4.0 frequency of attendance (days), mean ± sd 14.7 ± 4.3 age at which disease was diagnosed (months), mean ± sd 13.7 ± 9.0 married (consanguineous) (adults = 101) 12, (11.9%) education level (patients) illiterate 40, (19.9%) non-college 133, (66.2%) college 28, (13.9%) education level (father) illiterate 54, (26.9%) non-college 97, (48.3%) college 50, (24.9%) education level (mother) illiterate 122, (55.7%) non-college 76, (37.8%) college 13, (6.5%) sd (standard deviation) bmi (body mass index) associated disease complications the frequency of hepato-splenomegaly, viral hepatitis, thalassemic facies and splenectomy are significantly higher in adults compared to pediatric patients, as illustrated in table (2). figure (1) shows the percentages of complications in all patients. table (2) thalassemia associated complications variables pediatrics* adults pvalue number 100 101 hepato splenomegaly 41, (41%) 59, (58.4%) 0.014 dm 4, (4%) 7, (6.9%) 0.361 hypogonadism 11, (11%) 20, (19.8%) 0.084 cardiac involvement 21, 21%) 31, (30.7%) 0.117 viral hepatitis 23, (23%) 65, (64.4%) <0.001 thalassemic facies 60, (60%) 78, (77.2%) 0.008 cholecystectomy 9, (9%) 5, (4.9%) 0.215 splenectomy 8, (8%) 31, (30.7%) <0.001 *pediatric (children and adolescents) figure (1) percentages of complications in thalassemia major patients. reported side effects with icts usage side effects were reported according to bnf 74, and drugs monograms (novartis), side effects associated with impaired qol in thalassemia patients. fatigue was significantly higher in pediatrics (p˂0.05); urinary discoloration and permanent skin reaction were significantly higher in adults (p˂0.05), as illustrated in table (3). iraqi j pharm sci, vol.28(1) 2019 quality of life in thalassemia major 48 table (3) reported side effects with icts . variables pediatrics* adults pvalue number 100 101 git disturbances (dfx) 43, (43%) 55, (54.5%) 0.104 urine discoloration (dfo) 48, (48%) 93, (92.1%) <0.001 fatigue 25, (25%) 10, (9.9%) 0.005 joints pain 23, (23%) 33, (32.7%) 0.126 skin reactions (dfo) 7, (7%) 20, (19.8%) 0.008 pyrexia 21, (21%) 18, (17.8%) 0.569 sleep disturbance (dfo) 15, (15%) 26, (25.7%) 0.059 stool discoloration (dfx) 33, (33%) 32, (31.7%) 0.842 *pediatric (children and adolescents) dfx (deferasirox) dfo (deferoxamine) assessment of hrqol by whoqol-bref questionnaire in adult patients using either dfo or dfx chelation therapy. in adults patients; hrqol components were significantly lower in patients receiving dfo, as illustrated in table (4). table (4) assessing hrqol in adults using dfo or dfx by whoqol-bref questionnaire. variables defero xamine defera sirox pvalue number 51 50 physical health 48.9 ± 14.7 63.9 ± 18.0 <0.001 psychological 47.1 ± 19.4 65.9 ± 16.2 <0.001 social relationship 63.6 ± 22.8 74.2 ± 20.8 0.016 environmental 40.6 ± 16.6 58.3 ± 22.1 <0.001 predictors of qol for adult patients age, blood transfusion frequency, and splenectomy were found to have no impact on qol. dfx has a strong significant correlation with qol domains. educational level poses a significant correlation with qol domains. thalassemic facies have a negative impact on qol domains. serum ferritin was negatively correlated with psychological and environmental domains and marginally correlated with physical functioning domain, as illustrated in table (5). table (5) predictors of qol in adult patients physical health psychological social relationship environmental r p-value r p-value r pvalue r p-value age -0.092 0.359 -0.081 0.423 -0.139 0.164 -0.090 0.372 treatment (dfx) 0.431 <0.001[s.] 0.473 <0.001[s.] 0.244 0.014 [s.] 0.398 <0.001[s.] education level 0.260 0.009 [s.] 0.289 0.003 [s.] 0.236 0.017 [s.] 0.207 [s.] 0.038 [s.] attendance frequency -0.093 0.355 -0.054 0.290 -0.025 0.807 -0.037 0.711 thalassemic facies -0.239 0.016 [s.] -0.329 0.001 [s.] -0.156 0.120 -0.245 0.014 [s.] splenectomy 0.004 0.968 0.011 0.912 -0.036 0.722 0.007 0.944 ferritin -0.184 0.065 -0.284 0.004 [s.] -0.136 0.176 -0.200 0.045[s.] assessment of hrqol by pedsql questionnaire in pediatrics using either dfo or dfx chelation therapy. in pediatric patients, only physical functioning score was significantly lower in dfo receiving patients, the rest of the variables show no statistical differences, as illustrated in table (6). the emotional functioning was the lowest scored domain in both groups. iraqi j pharm sci, vol.28(1) 2019 quality of life in thalassemia major 49 table (6) assessing hrqol in pediatrics using pedsql questionnaire. variables deferoxamine deferasirox p-value number 48 52 physical functioning 49.0 ± 16.8 62.1 ± 20.0 0.001 psychosocial health summary 67.4 ± 13.3 66.7 ± 16.9 0.820 emotional functioning 53.9 ± 23.5 58.1 ± 24.4 0.383 social functioning 79.8 ± 15.4 75.4 ± 21.7 0.240 school functioning 69.3 ± 21.8 68.8 ± 22.0 0.918 total summary score 59.7 ± 12.5 64.8 ± 16.7 0.083 predictorcs of qol in pediatric patients age and serum ferritin, are correlated inversely with qol while the educational level is correlated directly with qol in univariate analysis, but they lose the correlation in multivariate analysis. table (7) predictors of qol for pediatric patients univariate multivariate r p-value r p-value age -0.290 0.003 [s.] -0.047 0.650 treatment (deferasirox) 0.179 0.075 -0.078 0.456 education level 0.234 0.019 [s.] 0.192 0.063 frequency of attendance -0.030 0.771 thalassemic facies -0.149 0.139 splenectomy -0.114 0.257 ferritin -0.223 0.026 [s.] -0.123 0.237 r2= 0.466 discussion the onset at which disease was diagnosed was ranging from 4-22 months table (1). this range was consistent with a study conducted in basrah governorate by abdul-zahra et al. (24) in which the range at disease onset in their study was 4-18 months. patients usually presented with severe microcytic/ hypochromic anemia with mild jaundice and hepatosplenomegaly, with hematological features of reduced hb levels (˂7g/dl) and very low mch (˂20 pg) with erythrocyte morphological changes on blood smear (25). the frequency of the patients' attendance was consistent with ansari et al. (14) and ranging from 10-20 days. this depends on the transfusion needs of each individual in order to maintain a hemoglobin level between 9 and 10 g/dlto promote normal growth, normal physical activity, and to inhibit ineffective erythropoiesis(26). the complications associated with β-tm are age related; adults are associated with significantly increased complications as compared to pediatrics, table (2) and figure (1). despite the new advances in thalassemia management, thalassemic patients in developing countries do not receive satisfactory treatment. for chronic conditions as thalassemia not only survival is the subject of concern but also qol of the patient. qol in patients with β-tm is affected by disease complication as they obviously associated with dramatic psychological effects, hopelessness, emotional burden and social integration problems (15,27). in the present study, whoqol-bref tool (arabic version) was used for the assessment of hrqol in adult thalassemics. despite the difference between the present study and other previous studies in questionnaires, methodologies, and number of patients used, our scores of qol domains in both dfo and dfx groups were comparable to many other studies (19,27,28) and showed severe impairment in all aspects of qol, table (4). adult patients using dfx show better qol than patients' using dfo, and this was consistent with many studies (19,29,30). table (5) shows a significant direct relationship between dfx and all qol domains; this was explained by goulas et al. (30) who reported that patients are more adherent to the oral iron chelator which is considered an important parameter for treatment success. age in adults has a non-significant correlation with qol which was in agreement with ali et al. (28) who attributed the non-significant correlation to the fact that adult patients are aware enough to adjust and cope with their disease. we also found that adult patients have slightly lower qol in comparison with their younger counterparts, this may be attributed to the fact that as age increases the severity of the disease and complications also increase which was consistent with ajijet al.(31). educational level in adults is considered a powerful parameter that is correlated with all aspects of qol. it is clear from table (5) that education has a significant direct correlation with all qol domains, which was found to agree with ansari et iraqi j pharm sci, vol.28(1) 2019 quality of life in thalassemia major 50 al.(19) and gin et al.(20) in which patients with higher educational levels have better qol. thalassemic facies and poor body image in adult thalassemia patients have a negative impact on qol domains. since iraq lacks supportive strategies as in developed countries, most of our patients suffering from negative feelings and poor selfesteem which affect their hrqol, this was consistent with khuranaet al. (32) in the present study, there was no impact of frequency of blood transfusion on adults' hrqol which was in contrast to khuranaet al.(32) who explained that patients with more frequent hospital visits for blood transfusion have worse qol than those with less frequent visits. there was no significant effect of splenectomy on adults' qol domains which was in agreement with mohammed et al. (33) serum ferritin (sf) was found to have a negative impact on some of adults' domains mainly psychological and environmental domains, which was consistent with ajijet al.(31). in the present study, it is noticed that sf levels are considered a big concern to the patient and their families. once again, few studies were performed to assess hrqol in iraqi pediatrics. thus, emphasizing the importance of maintaining qol in pediatrics; since thalassemia have a negative impact on the physical functioning of the children and adolescents. it can also affect their social relationships and their mental health resulting in reduced school performance and impaired overall qol. the arabic version of the pedsql 4.0tm tool was used to assess the qol in pediatrics. many studies have used the pedsql 4.0tm questionnaire to evaluate hrqol in pediatrics, and all of them found that qol scores are lower than control (16,34,35). a normal child should have scores equal or close to 100 (36). although no difference was found in the total summary score between pediatrics on dfo and pediatrics on dfx, table (6); the physical functioning score was significantly lower in the dfo patients this was in accordance to elalfyet al.(37) which could be attributed to patients non-compliance on dfo. elalfy also reported that dfo treatment in pediatrics is quite difficult since it involves using the subcutaneous injection for 8hrs per day, five days per week (37). in contrast to adult patients, advancing in the age in pediatrics has a negative impact on qol in univariate analysis, but it loses its correlation in multivariate analysis as shown in table (7), this was in agreement with dhiraret al.(36) and thavorncharoensapet al.( 40)because pediatrics with recent onset of the disease have less iron overload and complications this explains the higher qol scores in pediatrics in comparison to adults. type of iron chelation in contrast to adult patients has no significant impact on pediatrics' qol which was in agreement with kaheni et al.(39). pediatric educational status as in adults has a significant impact on qol general score which was in agreement with kaheniet al. (39) and hamid et al.(40). educated parents are keen on the education of their children, in turn; they have more information about the nature of the disease. as in adults, the frequency of attendance for blood transfusion in pediatrics has no impact on hrqol which was in agreement with thavorncharoensapet al. (38) in contrast to adults, the effect of thalassemic facies on qol in pediatric was insignificant, the same finding was reported by kaheniet al. (39). the insignificant findings may be attributed to small sample size. splenectomy effect on pediatrics was insignificant (the same findings in adults) this was in agreement with kaheniet al.(39) and chordiyaet al. (41) serum ferritin (a dependent variable) has a negative impact on pediatrics' hrqol (as in adults) and their parents, this was in agreement with boonchooduanget al.(16) . patients are fully aware of sf levels; as it goes up the more impairment in their qol scores ensues, this was in agreement with tuysuzet al. (42) even though sf levels in their study was lower than our pediatrics levels. conclusion β-tm impairs qol significantly. factors that should be considered to improve qol among thalassemia major are better managing iron overload and side effects associated with icts, and prevention of complications. education, financial support, and supportive strategies are major measures to improve overall qol in β-tm. acknowledgements special thanks to al-karama teaching hospital and hereditary blood disorders employees. we are appreciative to dr. sarmad s. hussein for his help in accomplishing the research. references 1. cossio mlt, giesen lf, araya g, et al. hoffbrands essential haematology. 7th ed. hoffbrand av, moss pah, editors. vol. xxxiii, wiley blackwell. john wiley & sons ltd.; 2016. 73-85 p. 2. marengo-rowe aj. the thalassemias and related disorders. proceedings (baylor university medical center). 2007;20(1):27–31. 3. jha r, jha s. beta thalassemia a review. journal of pathology of nepal. 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dependent thalassemia patients. j pediatr hematol oncol. 2017;00(00):1–5. iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 75 extraction and isolation of -sitosterol from iraqi wild lycium barbarum by different techniques (probe and bath ultrasound, hptlc and phplc) thukaa z. abdul-jalil*,1, ahmed a.hussein** and kawkab y. saour*** *department of pharmacognosy, college of pharmacy, university of baghdad, baghdad, iraq. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. *** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract lycium barbarum (awsaj) is a plant belong to family solanaceae serves as a good source of bioactive compounds like phytosterols which have many important biological activity. literature survey available so far revealed that there was no studies about iraqi wild awsaj phytosterols especially ß-sitosterol, there for the objective of this study was to examine the efficiency of ultrasound assisted extraction (probe and bath) as compared to the conventional (soxhlet) extraction method for extraction of phytosterols especially -sitosterol from fruits, leaves, stems and roots of iraqi wild awsaj plant. this goal was achieved by comparing the extraction mass yield, also by a quick and easy approach for identification and quantification of bioactive -sitosterol in four parts of awsaj using high performance thin layer chromatography (hptlc) which confirm the presence of -sitosterol in four parts of iraqi awsaj plant and the results clarify that the root part of iraqi awsaj plant extracted by probe uae give the highest concentration (0.610 mg/ml) of b-sitosterol followed by fruits (0.465mg/ml), leaves(0.405mg/ml) and the least was stems(0.275mg/ml); according to these; isolation and purification of ß-sitosterol from petroleum ether fraction of awsaj roots was done by preparative hplc. the isolated compound (-sitosterol) was identified by measuring melting point, ft-ir, tlc and preparative hplc. keywords: awsaj, ultrasound assisted extraction (uae), -sitosterol, high performance thin layer chromatography (hptlc), preparative high performance liquid chromatography (phplc). قنيات استخالص وفصل مركب بيتاستوستيرول من نبات العوسج البري العراقي بواسطة ت االنجاز مختلفة ) مسبارد حوض االمواج الفوق الصوتية ، الفصل اللوني للطبقة الرقيقة ذات العالي والفصل اللوني السائل ذو االنجاز العالي ( ***و كوكب يعقوب ساعور **احمد عباس حسين ، 1*,ذكاء زهير عبد الجليل فرع العقاقير والنباتات الطبية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق .* فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . ** فرع الكيمياء الصيدالنية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . *** الخالصه مصدر جيد للكثير من المركبات مثل الستيرويدات ويعتبر من العائله الباذنجانيه هو نبات ( lycium barbarumالعوسج ) -حول الستيرويدات النباتيه و خصوصا مركب بيتاحتى الوقت الحالي النباتيه ذات الفعاليه االحيائيه. و نظرا لعدم وجود دراسات بمساعدة ستلاصلفحص كفائة االري العوسج, لذلك اصبح هدف العمل في هذه الدراسه هو سيتوستيرول في النبات العراقي الب المسبار و الحوض( و مقارنته بطريقة االستلاص التقليدي بوساطة جهاز السوكسليت ) االمواج فوق الصوتيه )بوساطة (soxhletمار, االوراق, السيقان و الجذور لنبات العوسج الجل استلاص الستيرويدات النباتيه و خصوصا البيتاسيتوستيرول من الث العراقي البري. هذا الهدف تم انجازه بمقارنة حصيلة االستلاص الكتلي و كذلك عمل مقارنة سريعه و بسيطه لغرض الكشف و الرقيقه للطبقه )كروماتوغرافيا( اللوني الفصل باستعمال نبات العوسجلفي االجزاء االربعه التحديد الكمي لمركب البيتاسيتوستيرول المستللصه ن جذور النبات في االجزاء االربعه لنبات العوسج. اوضحت النتائج اسيتوستيرول -بيتاالذي اكد وجود ال العالي االنجاز ذات نسبة مل( يتبعها الثمار ب\مغ016.0بوساطة مسبار االمواج فوق الصوتيه تحتوي على اعلى نسبة من البيتاسيتوستيرول ) مركب و تنقية فصل مل(. وفقا لتلك النتائج تم \مغ012.0مل( و اخرا السيقان بنسبة )\مغ01400مل( و االوراق بنسبة )\مغ01460) باستعمال تقانة الفصل اللوني السائل ذات االنجاز العالي. كذلك من المستللص اثير البترول لجذور العوسج سيتوستيرول-بيتاال درجة قياس سيتوستيرول( و درجة نقاوته و التي شملت -تقانات للتحقق من نوعية المركب المفصول )بيتااستلدمت مجموعه من ال .للطبقة الرقيقه و الفصل اللوني السائل ذات االنجاز العاليالفصل اللوني االشعه تحت الحمراء, االنصهار , ،الفصل اللوني للطبقه الرقيقه ذات االنجاز العالي ،سيتوستيرول -تاالب،بمساعدة االمواج فوق الصوتيه ستخالصاالالكلمات المفاتيحيه: العوسج, الفصل اللوني السائل ذات االنجاز العالي. 1corresponding author e-mail: aljas_dadid@yahoo.com received: 3/10/ 2017 accepted:15/11/2017 mailto:aljas_dadid@yahoo.com iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 76 introduction nature has blessed mankind with a treasure of bioactive ingredients which are an emerging field in the context of health and nutrition. the american dietetic association (ada) define the bioactive ingredients of food as a physiologically active constituent in food or dietary supplement derived from both animal and plant sources including those needed to meet basic human nutritional needs that have been demonstrated to have a role in health and safe of human consumption (1, 2) . phytosterols especially -sitosterol is a predominant sterol in human herbal nutrition forming 65% diet contents can be considered as constituent of bioactive foods ,(figure 1) (3) . awsaj (lycium barbarum), also known as goji, is a shrub belong to solanaceae family and is native to asia, (figure 2) (4). awsaj has been used in asian countries as a traditional herbal medicine and functional food which possess a large variety of beneficial effects, including antiaging activity (5), immune modulation (6), antioxidant(7), neuroprotective effect(8), anticancer(9), male fertility facilitatory action (10), lower blood pressure and blood cholesterol level(11) as well as hypoglycemic properties (12). figure (1): structure of -sitosterol figure (2): iraqi lycium barbarum (awsaj) plant literature survey available so far revealed that there are no study concerned with awsaj ß-sitosterol in iraq, therefore the objective of the present study is to extract ßsitosterol from four parts of iraqi wild awsaj plant (fruits, leaves, stems and roots) then identification and quantification by high performance thin layer chromatography (hptlc) further more isolation of ß-sitosterol from awsaj by preparative high performance liquid chromatography (phplc). materials and methods plant material four parts (fruits, leaves, stems and roots) of awsaj (lycium barbarum l.), which grows as a wild plant in iraq, were collected in october and november 2016 from the university of baghdad in al-jadiryia district, baghdad, then authenticated by the herbarium of the department of biology, college of science/university of baghdad registered at buh no. 50777. these parts were left to dry in shade then grinded using an electric blender, weighted and subsequently subjected to extraction procedure. extraction methods two different extraction methods were used; the first representing a classical conventional (soxhlet) method (a) and the second is a simple, fast and nonconventional extraction (ultrasound assisted extraction uae) method (b). method a: approximately 250 gm of grounded fruits, leaves, stems and roots were subjected to extraction using a soxhlet apparatus in which each part of awsaj were extracted separately with 85% methanol, then the crude methanol extract of each part was filtered, concentrated under reduced pressure then suspended in distal water and partitioned with petroleum ether (b.p, 60-80 c) three times. later on the fractions of petroleum ether were concentrated to dryness over anhydrous sodium sulfate, weighted and subjected for identification of ß-sitosterol by hptlc (13, 14) . iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 77 method b: the uae was done using probe ultrasonicator and bath ultrasonicator with 85% methanol was used as a solvent. the uae was carried out under the following experimental conditions in case of probe ultrasonicator the plant material is in direct contact with ultrasonic waves, temperature 25 c ,time 20 minutes, solid to solvent ratio 1:8 gm/ml and sonication frequency 20khz and power of 60 watt. while in bath ultrasonicator the ultrasonic waves have to pass through the glass material in order to come in contact with plant material, temperature 50 c ,time 60 minutes, solid to solvent ratio 1:8 gm/ml and sonication frequency 60khz and power of 60 watt. in the current work, both instruments were used in order to study the effect of direct and indirect contact of ultrasonic waves on extraction of ß-sitosterol from four parts of iraqi awsaj plant. the same schematic procedure was used in conventional method (soxhlet) was applied, in which the crude methanolic extract filtered, concentrated, suspended in distal water, partitioned by petroleum ether. later on the fractions of petroleum ether were concentrated to dryness over anhydrous sodium sulfate, weighted and subjected for identification of -sitosterol by hptlc (15-18) . identification and quantification of ßsitosterol by hptlc the sample solutions were spotted in the form of bands of width 8.0 mm with a camag microliter syringe on precoated silica gel glass plate 60f254 (20 cm × 10 cm with 250 μm thickness; e. merck kjaa) using a camag linomat 5 (switzerland). a constant application volume of 4 μl was employed and space between two bands was 3.3 mm. the slit dimension was kept at 4.0mm × 0.3 mm and 20 mm/s scanning speed was employed and each track was scanned thrice and baseline correction was used. the mobile phase consisted of toluene – ethyl acetate ‐ chloroform, in the volume ratio of 5:1:4 (v/v) and 10 ml of mobile phase was used per chromatography. linear ascending development was carried out in 20 cm x 10 cm automatic developing chamber (camag, adc 2) saturated with filter paper whatmann no.1 in the mobile phase. the optimized chamber saturation time for mobile phase was 5 min at room temperature (25 c ± 2) at relative humidity of 50% ± 5. the length of chromatogram run was 7.5 cm. later on to the scanning, tlc plates were dried for 5 min with the help of an air dryer. densitometry scanning was performed with camag tlc scanner in the reflectance absorbance mode at 196 nm and operated by win cats software using tungsten lamp. subsequent to the development, tlc plate was sprayed with 5% sulphuric acid reagent followed by drying in oven at 110 c for few min, all these steps was done at baghdad collage of pharmacy (2, 19) . quantitative estimation of b-sitosterol was done by using calibration curve in which series of diluted solutions (0.5, 0.25, 0.125, 0.0625 mg/ml) were prepared from ß-sitosterol standard stock solution (0.5mg/ml) (20) . isolation and purification of ß-sitosterol by preparative hplc ß-sitosterol was isolated from petroleum ether fraction of awsaj roots by preparative high performance liquid chromatography (phplc) using mediterranean sea 18s µm (15x2.12, ambient) column and methanol (hplc grade) as mobile phase. the isocratic elution was carried out for 10 min at flow rate 25ml/min, injection volume was 2 ml/min and detection was recorded with uv detector at 196 nm. all these steps was done at college of pharmacy/baghdad university. the isolated compound was recrystallized using hot methanol and compared with standard ßsitosterol by different identification method including: (21, 22) 1. thin layer chromatography (tlc) using the best mobile phase toluene – ethyl acetate ‐ chloroform 5:1:4 and detection by spraying with 5% h2so4 + heating at 110 co 2. melting point. 3. fourier transforms infrared (ft-ir) spectroscopy (ftir) in kbr disk using ft/ir 4200 type a. 4. preparative high performance liquid chromatography (phplc) using the previously mentioned mobile phase. results and discussion plant extract yield comparison the results showed that probe uae produced the higher yield in the extraction fraction followed by soxhlet extraction fraction and finally bath uae give the lower yield (table 1). the advantage of probe uae is increased as it is considered the reduction of the extraction time and in the solvent consumption. iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 78 table (1): difference in extract content yield (gram of fraction / 250 gm) in different parts of awsaj plant using different methods. parts of awsaj soxhlet petroleum ether fraction uae petroleum ether fraction probe bath fruits 8.5 9.2 7.5 leaves 10.5 11.3 9.4 stems 3.2 3.8 2.9 roots 5.3 6.9 4.8 identification and quantification of ßsitosterol by hptlc the results indicated that the hptlc method was developed for the first time for qualitative identification and quantitative estimation of -sitosterol from four parts of iraqi awsaj plant in which qualitative identification was made by comparison of maximum retardation factor (rf) and uv spectrum of petroleum ether fraction of each part with that of stander -sitosterol as shown in figures 3-6. figure (3): hptlc chromatogram of ßsitosterol standard figure (4): hptlc chromatogram of petroleum ether fraction extracted from four parts of awsaj plant by conventional (soxhlet) method. a= roots, b= fruits, c= leaves, d= stems. iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 79 figure (5): hptlc chromatogram of petroleum ether fraction extracted from four parts of awsaj plant by uae probe method. a= roots, b= fruits, c= leaves, d= stems. figure ( 6 ): hptlc chromatogram of petroleum ether fraction extracted from four parts of awsaj plant by uae bath method. a= roots, b= fruits, c= leaves, d= stems. iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 80 for quantification measurements, the calibration curve were plotted using area under the curve (auc) versus four concentration levels of ß-sitosterol standard. a straight line equation were obtained from which the concentration of the analyte was calculated in each part of awsaj plant as shown in figure 7. figure(7): calibration curve of sitosterol standard on hptlc quantitative concentration of -sitosterol in petroleum ether fraction revealed that roots contain the higher concentration of ß-sitosterol followed by fruits, leaves and the least was stems. the high concentration of -sitosterol in roots of iraqi wild grown awsaj may be attributed to the availability of biochemical factors which are considered essential elements for the formation of building back of this secondary metabolite. the probe uae gives the higher concentration of ß-sitosterol followed by soxhlet and last by bath uae, as shown in (table 2) and (figure 8). table(2): quantitative analysis of sitosterol in each part of awsaj by hptlc part of plant concent ration mg/ml by soxhlet concentra tion mg/ml by probe uae concentr ation mg/ml by bath uae roots 0.465 0.610 0.412 fruits 0.295 0.485 0.122 leaves 0.135 0.405 0.113 stems 0.120 0.275 0.0534 figure (8): hptlc, track 1-4= -sitosterol standard, track 5-16= petroleum ether fraction obtained by probe uae, bath uae and soxhlet methods respectively for each part of awsaj. isolation of -sitosterol by preparative hplc according to hptlc results; isolation and purification of ß-sitosterol from the root parts of iraqi awsaj plant extracted by probe uae will be done by preparative hplc which is considered now the most powerful and versatile method for purification tasks in pharmaceutical industries. phplc chromatogram showed twelve peaks which represent twelve different compounds according to their retention time, one of them (-sitosterol) at retention time of 1.142, is a major peak which was identified by comparison with standard -sitosterol at retention time of 1.123. the major peak was collected by fractions collector after monitoring it according to the time ( time from iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 81 beginning of peak appearance until start to disappearing of the peak) figure 9 and 10, then the sample obtained from phplc was dried over anhydrous sodium sulfate, weighted and subjected to different identification methods. figure (9): phplc chromatogram of standard -sitosterol figure( 10): phplc chromatogram of roots petroleum ether fraction characterization of the isolated ß-sitosterol 1thin layer chromatography (tlc): the isolated compound appeared as a single spot having the same color and rf value as that of standard -sitosterol as shown in (figure 11). 2measuring melting point: the isolated compound had a sharp melting point 138-140 co, compared to melting point 139-140 co for the ß-sitosterol standard (23) . 3fourier transforms infrared (ft-ir) spectra: ft-ir spectroscopy is most commonly used in phytochemical studies as a finger printing device, for comparing a natural with the synthetic reference standard, so ir spectra of isolated compound gave identical results which was compared with standard -sitosterol as shown in figure 12 and 13, and the characteristic ir absorption bands of isolated compound are listed in (table 3) (24) . iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 82 figure (11): tlc chromatogram of isolated compound (iso) with -sitosterol standard (st) on silica gel gf 254, developing in toluene – ethyl acetate ‐ chloroform 5:1:4 and detection by spraying with 5% h2so4 + heating at 110 co. figure (12): ir spectrum of standard -sitosterol figure (13): ir spectrum of isolated -sitosterol iraqi j pharm sci, vol.26(2) 2017 -sitosterol in iraqi awsaj plant 83 table( 3): characteristic infra-red frequencies of isolated ß-sitosterol compound phytosterol compound approximated position of characteristic bands (cm-1) -sitosterol 3361.32 (free o-h stretching band), 2931.27 and 2854.13 (aliphatic c-h stretching), 1654.62 (c=c absorption peak), 1458.89 (c-h bending /-ch2), 1053.91 and 874.56 (cycloalkane) 4preparative hplc phplc was used again for characterization and identification by comparing the retention time of the isolated (1.117) with that of standard -sitosterol (1.123) as shown in (figure 14). figure (14): phplc chromatogram of isolated ß-sitosterol conclusion from the above findings, the utilization of probe uae has proved to be much simpler and more effective than the conventional soxhlet and bath uae extraction methods for obtaining ß-sitosterol from fruits, leaves, stems and roots of iraqi awsaj plant. hptlc and comparison of standard facilitated the identification and quantification of bsitosterol present in petroleum ether fraction of the four parts of awsaj plant. ß-sitosterol was isolated from petroleum ether fraction of root parts of awsaj and characterization was carried out by measuring of melting point follow by running two chromatographic procedures (tlc and phplc) and finally spectral characteristic (ftir). the current research also recommends using the other bioactive phytosterols in order to confirm the presence of them in iraqi awsaj plant. acknowledgment the authors wish to thank mr. zaid m. abdul-khaliq, 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j. kassab** * department of clinical and laboratory sciences, college of pharmacy, thi-qar university, thi-qar, iraq ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract sumatriptan(st) is a selective agonist at serotonin 5-hti receptors, as well as 5-ht1b/1d subtypes. it is effective for acute migraine attacks, but has a short half life (about 2 hours) and low oral bioavailability (15%). the purpose of this study was to develop and optimize nasal mucoadhesive in-situ gel(ig) of st to enhance nasal residence time for migraine management. cold method was used to prepare different formulas of st nasal ig, using thermosensitive polymers (poloxamer 407 alone or with poloxamer 188) with a mucoadhesive polymer hyaluronic acid (ha) which were examined for gelation temperature and gelation time, ph, drug content, gel strength, spreadability, mucoadhesive force determination, viscosity, in-vitro drug release, and the selected formula was subjected to fourier transform infrared (ftir) compatibility studies, and to ex-vivo permeation study, histological evaluation of the sheep mucosal tissue after st nasal gel application for 6 hours.the results showed that the formula ig7 prepared from poloxamer 407(19%), poloxamer188 (4%) and ha (0.5%) had an optimum gelation temperature (32.66±1.52°c), gel strength (43.66± 1.52 sec), mucoadhesive force (8067.93± 746.45dyne\cm2), in-vitro drug release (95.98%) over 6h, ex-vivo permeation study (89.6%) during the 6 h. study with no histological or pathological change in the nasal sheep tissue and no interaction between drug and other additives in ig7. formulation of st as a nasal insitu gel to avoid first pass metabolism and ease of administration coupled with less frequent and sustained drug release, will enhance patient compliance. keywords: in-situ gel, poloxamer407, poloxamer188, hyaluronic acid, sumatriptan. ة تحسسبوليمرات م باستخدامموضعيا مخاطبصياغة وتقييم مادة سماتربتان كهالم أنفي يلتصق للحرارة **كساب جالل حنان و 1*،الكوفيحسين كاظم .العراق ، ذي قار ذي قار، جامعة ، الصيدلة ةكلي ، العلوم المختبرية والسريرية فرع* .العراق ، بغداد ، بغداد جامعة ، الصيدلة ةكلي الصيدالنيات، فرع** الخالصة مل التي تعمل على مستقبالت السيروتونين وتستعالغرض من البحث هو تطوير وتقييم سائل هالمي يلتصق مخاطيا من مادة سماتربتان في عالج الشقيقة لتحسين فترة البقاء في االنف ، كون العقار لديه عمر نصف داخل الجسم قليل و توافره الحيوي عن طريق الفم منخفض. لوحده او مع 704ت حساسة للحرارة )بولكسمر تم تحضير تراكيب مختلفة من السائل الهالمي لمادة سماتربتان بطريقة التبريد بواسطة مزج بولميرا ( مع بولمير الصق مخاطيا )حامض الهيلورونيك( و تم اختبارهم للكشف عن احسن مزيج الذي لديه درجة حرارة تحول من السائل 811بولكسمر واالنتشار وتحديد قوة اللصق و وتحرير الدواء الى الهالم مناسبة وكذلك فترة بقائه كهالم. كذلك تم فحص درجة الحموضة وانتشار الدواء واللزوجة فاعل ت في المختبر كما تم دراسة نفاذ الدواء عبر نسيج الغنم للصيغة المختارة والفحص النسيجي لها بعد اكمال التجربة ، كذلك فحص اذا حدث اي كيميائي مع باقي مكونات التحضيرة. حامض الهيلورونيك ( و%7) 811وبولكسمر (%81) 704بولكسمر من المتكون ig7 السابعة التركيبة في الهالم ان النتائج اظهرت بالغشاء التصاق وقوة( ثانية 6..8± 76.33التماسك ) وقوة( درجة سيليزية 6..8 ± 66.33)الهالم تكوين حرارة درجة احسن ( يمتلك 0.5%) طريق عن العابر الدواء خالل الساعات الست للتجربة وكمية ( %1..11المتحرر ) الدواء وكمية(6سم/داين .473.7± 1034.16المخاطي ) بعد التجربة اظهر امان الدواء و عدم وجود تغير نسيجي مرضي للغشاء النسيجية خالل ست ساعات والدراسة ( %11.3غشاء انف الغنم ) واالوعية الدمويه. بالخالية تحطم واليوجد عن طريق االنف بتكرار اقل واستمرار تحريرالعالج لفتره طويلة وكذلك يحسن استقبال المريض االستنتاج هو سهولة إعطاء عالج سماتربتان للدواء. . سماتربتان ،هليرونك اسد ،111 بولكسمر ،704ولكسمر، موضعي هالم: المفتاحية الكلمات 1corresponding author e-mail: ph.hussein.alkufi@gmail.com received: 25/5/2019 accepted:3/8 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp95-104 mailto:ph.hussein.alkufi@gmail.com iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 96 introduction drug delivery by nasal route is one of the challenging endeavors facing the pharmaceutical technologist today. from the pharmacokinetic standpoint, intranasal administration of drug avoids first-pass metabolism and prevents incomplete absorption in the gastrointestinal tract which leads to improving the bioavailability(1). therefore, approaches enhancing the nasal bioavailability is either by prolonging the contact time with the nasal surface via a viscosity-enhancing agent or in-situ gelling polymers or both approaches. an in-situ gel is a drug delivery system that exhibits sol-to-gel phase transition due to a change in specific physicochemical parameters such as ionic, temperature or ph(2). thermoreversible polymers are substances having both solid and liquid-like properties which can be transported as a fluid and solidifies within the body temperature. the formulation has the advantage to stop the anterior leakage of the dosage form, reduce the taste impact and enhance the nasal bioavailability(2). however, modern research turned extraordinary attention to hyaluronic acid (ha), a naturally occurring mucoadhesive polymer which, besides being biodegradable, showed to be highly biocompatible(3). st is a 5-ht1d and 5-ht1b (5hydroxytryptamine) receptor agonist used in the treatment of a cluster headache and migraine. parental or oral routes are usually used to administer st. however, a large proportion of patients suffer severe vomiting during their migraine attack, which may make oral management unacceptable. st has an oral bioavailability of 15%; however, the problems associated with nasal delivery of st solution is the low residence time in the nasal cavity (around 15minutes) resulting in low bioavailability, the remaining dose st nasal solution is swallowed, and it arrives in the gastrointestinal tract where it is absorbed(4,5). the formulation of st nasal in-situ gel using the thermosenstive polymer poloxamer and mucoadhesive polymer ha will enhance permeation and allow longer residence time and delay nasal clearance to avoid first pass metabolism, coupled with less frequent, ease of administration, is a promising dosage form to enhance patient compliance. material st, poloxamer407, poloxamer188 and ha was purchased from hyperchem(china). sodium chloride, calcium chloride and potassium chloride were purchased from central drug house, india. benzalkonium chloride was provided by (pioneer, iraq). substance and reagent used were all of analytical grade. melting point determination and dsc the melting point was determined by using an open capillary tube technique as submitted by the united states pharmacopeia (usp)(6) and differential scanning calorimetry (dsc) was used to evaluate the thermal behavior of pure drug using a dsc-60 (shimadzu 60 plus japan). a small amount of st was closed in standard aluminum pans and then the temperature was raised from 50°c upto 300°c at a heating rate of 10°c/min(7). preparation of nasal in situ gel containing st nasal ig of st was prepared using the cold method. the cold method involved slow addition of poloxamer407 alone or with poloxamer188 in cold water with continuous agitation. this dispersion was stored overnight at 4°c. the drug (st 5mg/ml) and other additives (mucoadhesive polymer ha(0.5%, 1.0%), preservative (benzylkonium chloride) were added to the poloxamer dispersion (table 1) with continuous agitation in the next day. the formed mixtures were kept overnight at 4°c. the nasal gel formulation having satisfactory gelation temperature (30-37°c) was selected as optimized formulation(8). table1. composition of st nasal thermosensitive gel formulas formula no. drug mg poloxamer407 (%w/v) poloxamer 188 (%w/v) ha (%w/v) benzylkonium chloride (%w/v) dw ml ig1 50 16 ---0.5 0.01 10 ig2 50 16 ---1 0.01 10 ig3 50 19 ---0.5 0.01 10 ig4 50 19 ---1 0.01 10 ig5 50 16 4 0.5 0.01 10 ig6 50 16 4 1 0.01 10 ig7 50 19 4 0.5 0.01 10 ig8 50 19 4 1 0.01 10 iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 97 gelation temperature gelation temperature is the temperature at which the liquid phase makes a transition to gel. a gelation temperature range suitable for nasal ig formulation would be 32-34ºc. if the gelation temperature is lower than 32ºc, gelation occurs at room temperature leading to difficulty in manufacturing, handling and administering. if the gelation temperature is higher than 34ºc, the formulation will stay as a liquid at body temperature, resulting in nasal drainage(9). the gelation temperature was determined by placing 2ml of the refrigerated formula(ig1-ig8) in a 10 ml test tube with a diameter 1.0cm and closed using parafilm. the tube was placed in a water bath at a temperature of about 4°c. the water bath temperature was incremented slowly (3°c) at the beginning of the experiment, and then 1°c when the temperature reached the region of the sol-gel transition temperature, and equilibration was allowed for 10min after each temperature increment until gelation occurred. the test tube was tilted 90°, to confirm gelation has occurred, and the meniscus of the preparation did not move during slanting, this was recorded as the gelation temperature(10). gelation time accurately 5ml of the prepared ig formulas (ig1-ig8) was added to 50 ml of simulated nasal fluid(snf) at 34±2°c in a beaker with mild stirring (50 rpm) to avoid breaking of formed ig. snf (ph 6.5) is composed of 7.45mg/ml, nacl 1.29mg/ml, kcl and 0.32 mg/ml, cacl2.2h2o prepared under continuous shaking for 24h in a thermo-stated shaking water bath maintained at 37 °c (11) . gelation time was observed visually by qualitative measurement and reported in terms of strokes and calibrated in three groups depending on gel stiffness, gelation time and duration  (+)gelation happens after few minutes, stays for a few minutes and rapidly dispersed  (++) gelation happens, immediately and continues for a few hours,  (+++) gelation happens, immediately and continues for an extended period(12). drug content accurately, 1ml of the formulation was diluted to 10 ml with snf, and then 1 ml of this solution was again diluted to 10 ml with snf. finally, the absorbance of the prepared solution was measured at 282 nm using uv visible spectrophotometer ) emc lab-germany((7). the samples were taken from three different regions of the gel from the upper, middle and bottom of the ig, and the mean drug content was measured. ph measurement the ph of nasal preparation is essential, mainly to prevent the growth of pathogenic bacteria, to avoid irritation of the nasal mucosa and to sustain normal physiological ciliary movement(13). the ph of the prepared ig formulas (ig1, ig2, ig7, ig8) was measured at room temperature using ph meter ) hanna-italy ( . mucoadhesive force determination mucoadhesive force is the power of adhesion of the formula to the nasal mucosa. the modified balance technique was used with a beaker at one side of the balance, and on the other side, a vial was fitted with sheep nasal mucosa (with a thickness of 0.6mm cut from the sheep nose mucosa obtained from slaughtered sheep). the nasal mucosa was fitted on the bottom of the vial, as shown in figure 1. the vials were kept at 32–34°c for 10 min. then 1 ml of the gel sample was placed in a watch glass underneath the vial fitted with the nasal tissue. the vial was pressed down to the gel sample for one min as initial contact time. then water was added to the beaker slowly on the other side of the balance using a pipette(14). the mucoadhesive force was calculated by determining the weight of water required to separate the mucosa from the gel by equation (1) : 𝐷𝑒𝑡𝑎𝑐ℎ𝑚𝑒𝑛𝑡 𝑓𝑜𝑟𝑐𝑒 ( 𝑑𝑦𝑛𝑒 𝑐𝑚2 ) = 𝑚 ×𝑔 𝐴 eq.(1) where; m is the required weight (g) for detachment, g is the acceleration (980 cm/s2) due to gravity, and a is the exposed tissue area which is 3.14 cm² in all preparations(15). figure 1. modified mucoadhesive force measuring balance gel strength a sample of 5 g of the gel was put in a 10ml graduated cylinder and placed in a thermostatically controlled water bath at 37°c for 30 minutes for gelation of the formulas. a mass of 3.5g was placed on the surface of the gel. the gel strength was determined by the time in seconds required by the weight to penetrate 0.5cm deep into the gel(16). spreadability test the spreadability of the ig formulas (ig1, ig2, ig7, ig8) was determined by placing approximately 1g of the ig formula after complete gelation at a glass plate center (square area = 400cm2). another glass plate (same size) was used to cover the glass plate containing ig formula. the iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 98 initial diameter of the ig was marked. then, 1k scale weight was gently placed on the plate upper side for one min; as a consequence, the ig expand out in between the plates. then the final diameter was marked after removal of the weight and the spreadability was measured in cm(17). viscosity the viscosity measurements were carried out by (digital viscometer ndj-5s model, spindle 4). the viscosity measurements were performed at 34±1°c to ensure gelation of the formulas. the gel was allowed to rotate for 1 min before viscosity measurement. the temperature detecting probe was lowered in the gel, and the temperature of the gel was recorded (7). in-vitro release study of st in-situ nasal gel in-vitro drug release from the gel was determined for (ig1, ig2, ig7, ig8) by using a thermoregulated franz diffusion cell system)copley-usa), against control (5mg/ml st aqueous solution ) fitted with artificial dialysis membrane (mwco 1200-1400 kda) that was soaked in the receptor medium for 2 h before use. snf (12 ml) ph6.5 was added into the receptor chamber maintained at 34±1°c. gel with weight equivalent to 5mg of st was placed into the donor compartment, and the whole setup was kept on stirring option. aliquots of 1ml were removed at predetermined time intervals from the receptor compartment and replaced with fresh snf, for 6h. the samples were diluted with 10 ml snf and analyzed spectrophotometrically at 282nm, and the amount of the drug released was determined and the study is repeated in triplicate. release kinetics was studied for the selected formula by using dd solver software(9,18). ex vivo permeation study the ex vivo permeation studied was conducted using sheep nasal mucosal membrane. the sheep nasal mucosal membrane was mounted in between the donor and the receptor compartment of the diffusion cell (copley-usa). formulation equivalent to 5 mg of drug was placed in the donor compartment which was in connection with the mucosal surface of the membrane, while the receptor compartment was filled with 12 ml of snf and its temperature was maintained at 34±1°c. the content of the receptor compartment was continueously stirred by magnetic stirrer. a portion of 1ml was withdrawn from the receptor compartment at suitable time intervals and replaced with a similar volume of fresh snf. the samples were diluted with 10 ml snf and analyzed spectrophotometrically at 282nm and the amount of the drug released was determined and the release was repeated in triplicate (7,9). permeability coefficient(cm/s) of the drug was calculated by using the equation 2, permeability coefficient= (𝐷𝐶 𝐷𝑇 ⁄ )𝑆𝑆 × 𝑉 𝐴×𝐶𝐷 eq(2) where (dc/dt) ss(μg ml−1s−1) alteration of concentration under steady-state; a is the permeation area equal (3.14cm2); v (ml) the volume of the receiver section; and cd (μg ml−1) is the original donor concentration(9). histopathological studies after ex-vivo permeation study with st nasal gel formulation, the histopathological evaluation of the nasal mucosal tissue was compared with control mucosal tissue incubated in the permeation chamber of franz cell. both tissues were fixed in 10% formalin, routinely processed, and embedded in paraffin. paraffin sections were cut on glass slides and stained with hematoxylin and eosin to examine the morphological changes to the tissue via blinded study(19). compatibility studies by (ftir) the ftir spectrum of st was recorded using a ftir spectrometer (biotech engineering management, uk) in a spectral region between 4000 and 400 cm-1 and analyzed by transmittance technique. compatibility studies were carried out at room temperature by ftir to investigate any interactions between the drug and the excipients used in the formulation. the polymers were subjected to ftir studies alone and in combinations with the drug(20). statistical analysis the results of the experiments were given as mean values ± standard deviation (sd) and analyzed according to the one-way analysis of variance (anova) at which significant results (p<0.05) and non-significant (p>0.05). results and discussion melting point and dsc the measured melting point of st by using the capillary method was found to be 169°c, which is consistent with the reported melting point range (168-172°c)(21), which indicates the purity of drug powder. the differential scanning calorimetry of pure st powder is shown in figure 2 and complied with reference(21). the dsc thermogram of the real drug showed an endothermic peak of 172°c, corresponding to the melting point of the crystalline form of the drug(21). iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 99 figure 2. differential scanning calorimetry thermogram of st. gelation temperature poloxamer 407 and poloxamer188 were selected due to their thermosensitive gelling properties. also, poloxamer 407 and poloxamer188 were known to have low toxicity, low irritation, excellent water solubility, excellent drug release characteristics and compatibility with a wide range of excipients. poloxamer407 or poloxamer188 alone could not provide a suitable gelation temperature because at higher concentrations of poloxamer407, the larger ratio of polypropylene oxide (ppo) causes dehydration and aggregation at a lower temperature leading the micelles to become more entangled and greater ease of gelation at lower temperature. concomitantly, the addition of poloxamer188 increases the ratio of polyethylene oxide (peo) leading the micelles to become less entangled there by raising the critical micelle temperature (cmt)(22). in cases of mixtures of poloxamer407 and poloxamer188, several formulations gelled at the body temperature. as the concentration of poloxamer407 increased, the mixtures needed smaller amounts of poloxamer188 to gel at the desired gelation temperature. the formulas with percentage (w/w) ratios of poloxamer 407/poloxamer188 with suitable gelation temperature of 32-34°c (table 2) are used for further study(23). gelation time the gelation study was conducted in snf(ph 6.5). all the formulations on contact with the gelation medium had undergone sol-to-gel transition due to the presence of gel-forming polymers such as poloxamer and ha. gelation characteristics of the formulations showed that increasing the concentration of poloxamer 407 increased the gelation time, while the combination with poloxamer 188 showed less gelation time as seen in (table 2). also the rise in the concentration of ha increased the time of gelation for the formulas. table 2. gelation temperature and gelation time of ig formulas formula no. gelation temp°c mean±sd n=3 gelation time ig1 30.16±0.76 ++ ig2 27.33±0.76 +++ ig3 26.33±1.15 ++ ig4 24.16±0.76 +++ ig5 43.00±1.00 + ig6 38.66±1.52 + ig7 32.66±1.52 ++ ig8 29.33±1.04 +++ drug content the drug content was found to be in the acceptable range of all the formulations. this indicates that the process employed in this study was capable of producing gels with uniform drug content and minimal variability, as seen in table 3. ph determination the ph of the formulations was established to be acceptable(13)and was in the range of 4.5-6.5, as shown in table 3. table 3. st intranasal gel drug content and ph of the formulas formula no. drug content% mean±sd n=3 ph mean±sd n=3 ig1 99.93±0.01 5.66±0.05 ig2 99.27±0.06 5.56±0.05 ig7 99.98±0.02 6.10±0.10 ig8 99.91±0.03 6.33±0.05 determination of mucoadhesive strength in the study, the formulations prepared with the high concentration of ha exhibited more mucoadhesion strength due to the ability of ha to comprise hydrogen bonding, high molecular weight and the semi-flexible chain. the bioadhesive bond between the formulation and mucin dispersion became stronger at temperature range 32-34°c due to formation of more compact lattice structure as well as increase of density(24), as shown in table 4. iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 100 table 4. detachment weight(g) and mucoadhesive force per squre root of area(dyne/cm²) of ig formulas. formula no. detachment weight (g) mean±sd n=3 mucoadhesive force (dyne/cm²) mean±sd n=3 ig1 20.33±2.51 6345.03±785.43 ig2 26.66±1.52 8320.63±476.74 ig7 25.85±1.52 8067.83±746.45 ig8 30.00±1.00 9363.05±312.10 gel strength and spreadability the formulations showed good gel strength (table 5) which ranged from the low as (38.33±2.08s) for ig5 to the higher value of (51.33±3.21s) for ig8, the combination of poloxamer and ha gave strength to the formula. gel strength indicates the tensile strength of the gelled mass. it demonstrates the ability of the gelled mass to resist the ciliary movement in vivo(25). the gel strength of nasal gel formulation at 34°c, increased as the concentration of ha and poloxamer concentration increased. the mechanism of the increase gel strength might be related to hydrogen bonding between poloxamer and bioadhesive polymers in the nasal gel(8). table 5. gel strength and spreadability of ig formulas. formula no. gel strength (sec) mean±sd n=3 spreadability (cm) mean±sd n=3 ig1 38.33±2.08 5.03±0.15 ig2 45.33±1.52 3.56±0.49 ig7 43.66±1.52 4.06±0.11 ig8 51.33±3.21 2.93±0.15 by evaluating the spreadability of the nasal ig of st, it was observed that as the concentration of polymers used (combination of poloxamer407 and188) increased the spreadability decreased. the values indicate that the nasal ig has excellent spreadability according to acceptable range (2.57cm), which is desired for the application of the nasal ig(26), as shown in table 5. viscosity measurements the viscosity of selected ig formulations determined at 6,12,30,60 rpm at 34±1°c. the results of the viscosity measurement of ig7, ig8 are shown in table 6. the increase in viscosity of the formulations was observed with the increase in the concentration of ha polymer which could be related to the increasing polymer chain entanglement and higher chance of hydrogen bonding at higher concentration of the polymer(25). ig8 is very viscous, making it very difficult to pour from the container table6.viscosities of ig7 and ig8 at their gelation temperature 34±1°c, ±sd rotation speed rpm formula viscosity(cp) 6rpm mean ±sd n=3 12rpm mean±s d n=3 30rpm mean ±sd n=3 60rpm mean ±sd n=3 ig7 1821.00 ± 18.24 1624.66 ± 22.47 1189.66 ± 9.50 850.00 ± 20.00 ig8 3013.33 ± 15.27 2533.33 ± 15.27 1826.66± 15.27 1283.0 ± 20.66 in-vitro release study of st in-situ nasal gel in vitro drug release of ig7 showed drug release 95.98% after 6h in franz diffusion cell. it was found that as the concentration of polymers increased (poloxamer and ha) a decrease in the drug release was obtained, as shown in figure 3. it was obvious that the release of st was not only affected by poloxamer concentration but by the mucoadhesive polymer ha. the mucoadhesive polymer retarded the drug release from nasal gel due to high gel viscosity and squeezed aqueous channels by which the drug is distributed between the poloxamer micelles(8). figure 3. in vitro release profile for ig1, ig2, ig7 and ig8 in snf at 34±1°c. the percentage of st released from the control (st aqueous solution) was compared with that from nasal in-situ gelling using the dialysis membrane. faster release of st solution in comparism to ig 7 was obtained with a significant difference (p<0.05).the percent of drug release from the st solution is 99.33% in 2h while the percent of drug release from ig7 in 6h was 95.89% as shown in the figure 4, due to the presence of polymers. iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 101 figure 4. in vitro release profile of ig7 in comparison with st solution (control) in snf at 34±1°c. ex-vivo permeation study ex-vivo permeation profile of ig7 is shown in figure 5. sustained release was obtained from ig7 since it released about 89.6% through nasal mucosa of sheep within 6 h, which is considered optimal since the st half life is 2 h(21), so the formulation will sustain the release for three half lives. figure 5. percentage cumulative permeation of drug from ig7 through the nasal mucosa in snf at 34±1°c. permeability co-efficient of st at 4 h for ig7 were found to be 1.44 × 10-5 cm/s. nasal ig7 showed the initial high flux value as 48.54 × 10-4 μg ml-1s-1. a decrease in the value of flux was observed at 2, 3 and 4 hrs resulting in flux value of 8.27, 17.69 and 18.94(10-4 μg ml-1s-1) respectively. initial rapid release may be due to the exposure of fresh surface of drug present in gel to the sheep mucosa and sustained drug release after 1 h may be due to the polymers like poloxamer407 which slightly decrease the rate of drug release due to enhanced micellar structure and gel network(9) and the presence of ha (hydrophilic polymer) which entrap the drug and sustain the release. the release data of the gel formulation (table 7) were kinetically analyzed by different mathematic models like zero order, first order, higuchi and korsmeyer–peppas ( for 60% release). the good fitness was represented by r2. formula ig7 best fit to zero order model because it has the highest r2 values. from korsmeyer–peppas equation, the n value was between 0.45 and 0.89, which indicates anomalous or non-fickian release. suggesting that the release was controlled by the diffusion rate (zero order mode ) and the relaxation rate of the hyaluronic acid polymer matrix(9,27). table7. model fitting for ig7 formulation. models formulation in vitro drug release ex-vivo drug release zero order(r2) 0.99 0.98 first order(r2) 0.95 0.96 higuchi(r2) 0.90 0.92 korsmeyer peppas (r2) n value 0.99 n=0.82 0.98 n=0.79 histopathological studies safety is an essential concern for the administration of the formulation. hence, it was essential to investigate the safety of the optimized in situ gel formulation (ig7). thus, it was essential to study the histology of the nasal mucosa with the formulation. the histology of control nasal mucosa (without treatment) and treated nasal mucosa is shown in figure 6. the microscopic observations indicated that the formulation had no significant effect on the microscopic structure of the sheep nasal mucosa. the surface epithelium lining and the granular cellular structure of the nasal mucosa were totally intact. thus the histological study revealed the safety of st in situ nasal gel formulation on the sheep nasal mucosa. iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 102 figure 6. histopathological evaluation of sections of sheep nasal mucosa. a. mucosal tissue incubated in snf (ph 6.5), b. mucosal tissue incubated in the permeation chamber with ig7 formulation. compatibility studies by ftir the ftir spectrum of st powder is shown in figure 7 and is in agreement with previous reported study(28). it was found that the characteristic peaks of st at 3367.71cm-1,1234.44cm-1, 632.65cm1, 1342.46cm-1 and 1138cm-1 corresponding to the – nh stretching, c-n stretching, c-s stretching and s=o(sulfones) stretching respectively. the ftir spectrum of the selected formula of ig7 showed small shifting in the chief characteristic bands of the drug in ig7 in comparison with the bands of the pure drug, indicating no interaction between drug and other additives used in ig7 formulation. ftir spectrum for poloxamer407 and poloxamer188 showed bands at 3483.44cm-1, 2881.65cm-1, 1280.73cm-1 for o-h stretching, aliphatic c-h stretching and c-o stretching in c-oc group respectively. characteristics peaks of st in ig7 were found for n–h str. at 3363.86cm−1, c–n str. at 1203.58cm−1, s=o str. at 1342.46cm-1 and 1138cm-1, c-s str. at 632.65 cm-1 (7,8). figure (7) compatibility ftir, a: sumatriptan b: poloxamer407 and poloxamer188, c: ha, d: ig7 iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 103 conclusion based on the results obtained from this study, it can be concluded that st intranasal in–situ gel was successfully prepared using poloxamer 407 and poloxamer 188. all the formulations were found to have the desired amount of drug content. addition of mucoadhesive polymers to the formulas increased the viscosity and thereby increased mucoadhesive strength of the formed gel. the optimized formula ig7 (poloxamer 407 19%, poloxamer 188 4%, ha 0.5%) showed drug release of 95.98% within 6h. exvivo studies on sheep olfactory nasal mucosa showed permeation of 89.6% within 6h. ig7 formulation exhibited diffusion release kinetics (zero order model). the release profile of st showed both diffusion and erosion mechanism, anomalous diffusion. the optimized formula ig7 was found to be safe since no histopathological changes were observed. references 1. jadhav kr, gambhire mn, shaikh im, kadam vj, pisal ss. nasal drug delivery systemfactors affecting and applications. curr drug ther. 2007;2(1):27–38. 2. hb n, bakliwal sr, pawar sp. in-situ gel: new trends in controlled and sustained drug delivery system. 2010;2(2):1408–1398. 3. mayol l, quaglia f, borzacchiello a, ambrosio l, la rotonda mi. a novel poloxamers/hyaluronic acid in situ forming hydrogel for drug delivery: rheological, mucoadhesive and in vitro release properties. eur j pharm biopharm. 2008;70(1):199–206. 4. ryan r, elkind a, baker cc, mullican w, debussey s, asgharnejad m. sumatriptan nasal spray for the acute treatment of migraine: results of two clinical studies. neurology. 1997;49(5):1225–30. 5. majithiya rj, ghosh pk, umrethia ml, murthy rsr. thermoreversible-mucoadhesive gel for nasal delivery of sumatriptan. aaps pharmscitech. 2006;7(3):e80–6. 6. the united states pharmacopoeia (usp) 36, usa. the united states pharmacopeial convention inc. 2012. 7. galgatte uc, kumbhar ab, chaudhari pd. development of in situ gel for nasal delivery: design, optimization, in vitro and in vivo evaluation. drug deliv. 2014;21(1):62–73. 8. godbole m, there pw, dangre p. formulation and optimization of prolonged release nasal in situ gel for treatment of migraine. indo am j pharm res. 2014;4:1320–32. 9. shelke s, shahi s, jalalpure s, dhamecha d, shengule s. formulation and evaluation of thermoreversible mucoadhesive in-situ gel for intranasal delivery of naratriptan hydrochloride. j drug deliv sci technol. 2015;29:238–44. 10. allah aka, abd-al hammid sn. preparation and evaluation of chloramphenicol as thermosensitive ocular in-situ gel. iraqi j pharm sci. 2012;21(2):98–105. 11. farid rm, etman ma, nada ah, abd el azeem re. formulation and in vitro evaluation of salbutamol sulphate in situ gelling nasal inserts. aaps pharmscitech. 2013;14(2):712–8. 12. madan jr, adokar br, dua k. development and evaluation of in situ gel of pregabalin. int j pharm investig. 2015;5(4):226. 13. chand dr, datta ms, tilak vijay k, gupta anish k. a review on factors affecting the design of nasal drug delivery system. int j drug deliv. 2011;3:194–208. 14. gaikwad v. formulation and evaluation of insitu gel of metoprolol tartrate for nasal delivery. j pharm res. 2010;3(4):788–93. 15. abdul bi, rajab na. preparation and in-vitro evaluation of mucoadhesive clotrimazole vaginal hydrogel. iraqi j pharm sci (p-issn 1683-3597, e-issn 2521-3512). 2014;23(1):19–25. 16. al-wiswasi nn, al-khedairy ebh. formulation and in vitro evaluation of in-situ gelling liquid suppositories for naproxen. iraqi j pharm sci (p-issn 1683-3597, e-issn 2521-3512). 2008;17(1):31–8. 17. garg a, aggarwal d, garg s, singla ak. spreading of semisolid formulations: an update. pharm technol north am. 2002;26(9):84. 18. basu s, bandyopadhyay ak. development and characterization of mucoadhesive in situ nasal gel of midazolam prepared with ficus carica mucilage. aaps pharmscitech. 2010;11(3):1223–31. 19. salunke sr, patil sb. ion activated in situ gel of gellan gum containing salbutamol sulphate for nasal administration. int j biol macromol. 2016;87:41–7. 20. pavia l l. introduction to spectroscopy, 3rd ed,. harcourt college publishers, new delhi; 2006. 353 p. 21. moffat a. c., osselton m. d., widdop b. editors. clarke’s analysis of drugs and poisons. 3rd ed. pharmaceutical press; 2011. 2098 p. 22. jeong b, kim sw, bae yh. thermosensitive sol–gel reversible hydrogels. adv drug deliv rev. 2012;64:154–62. 23. kute ju, darekar ab, saudagar rb. a review: in-situ gel-novel approach for nasal delivery. world j pharm pharm sci. 2013;3(1):187–203. 24. cowman mk, schmidt ta, raghavan p, stecco a. viscoelastic properties of hyaluronan in physiological conditions. f1000research. 2015;4. iraqi j pharm sci, vol.28(2) 2019 sumatriptan in-situ gel 104 25. sindhoor sm, priya s, maxwell a. formulation and evaluation of novel in situ gel of lafutidine for gastroretentive drug delivery. asian j pharm clin res. 2018;11(8):88–94. 26. chaudhary b, verma s. preparation and evaluation of novel in situ gels containing acyclovir for the treatment of oral herpes simplex virus infections. 2014;2014. 27. ampati s, hanumakonda a, maheshwaram v. formulation and evaluation of nasal in situ gel of fluoxetine hydrochloride. indo am j pharm sci. 2016;3(6):573–81. 28. prajapati st, patel pb, patel cn. formulation and evaluation of sublingual tablets containing sumatriptan succinate. int j pharm investig. 2012;2(3):162. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 16 hepatoprotective effect of echinops tenuisectus (compositae) on ccl4 induced hepatic damage in rats munaf h. abdulrazzaq* , enas j. khadeem** , suhad s. almuhammadi** *department of pharmacology and toxicology, college of pharmacy, university of baghdad. , baghdad , iraq **department of pharmacognosy, college of pharmacy, university of baghdad. , baghdad , iraq abstract flavonoids are known to play a vital role in the management of various liver disorders.they are a large family of compounds synthesized by plants; they belong to a group of natural substances with variable phenolic structures. in this study we aim to scan the types of flavonoids in a newly studied, wild iraqi plant named echinops tenuisectus of compositae family. the medicinal importance of flavonoids on one hand, and the absence of any phytochemical investigation on tenuisectus species of echinops genus on the other hand, acquired this study itۥs importance. three flavonoids were identified in the seed ,s extract of this plant (silymarin, rutin, quercetin ) by two chromatographic methods, first thin layer chromatography (tlc) using tlc ready made gf254 plates, uv detector at 254 nm, and two different solvent systems in which the rf value of the standards (silymarine, rutin, quercetin) matched with the rf value of the silymarin, rutin and quercetin found in the plant seed ٫s extract. high pressure liquid chromatography (hplc) was the other chromatographic method that used to identify the presence of these flavonoids in the plant seed. the plant seed sۥ aqueous extract was evaluated for its efficacy in rats by inducing hepatotoxicity with ccl4.single oral dose of 250mg/kg of seeds extract was given to ratۥs for 7 days. serum activities of transaminases (alt and ast) were used as the biochemical marker of hepatotoxicity. histopathological changes in ratُُs liver section were also examined. the results of the study indicated that, the pretreatment of rats with echinops extract before the hepatotoxins agent (ccl4) offered a hepatoprotective action. key words: echinops, flavonoids ةصالخلا ريلافهمعنمرنا راثال واد ا ا هعلافمادا يولا ا مهه اريفبهىادواردىرياريفلم .ري مفادوابهواريمورولابعاتفتاديوايفاىفلا را يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةدعرراريفلورللادواريلافهمعنمرنافما يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةدلولاتىرفهلاعمنمفاي ا موعاو أل . يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةدهىراييبفهلاريبلهلايرلافهمعنمرنادواع لفا تملا ععلاألا دمكعورناترفهلا وم اريففع يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةد ناريفهفه هلاي هواريملولفاألهنابهواريمورولاأبفهو . اأموك فاتاناأ يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةدعررادواريلافهمعنمرنافما ديولرلاريله وايرمل نا)رييرهف ونوفاو هوفامعروووهو( أعروبلاوىنلوهوادواوى اريفى د عاىرفه فارا يذابما لمهلا اأ يبع اu.vا م نارا الافع اريلمليوهلاgf254انرناريمعتهلاtlcاأئوولمرلاوف با(tlc)مى د عاىرفه اريبلللاريىفهلل ايرلافهمعنمرناريفعععلفافماrf ايرلافهمعنمرناريله وهلاو ألاافهفلrfا تاتلادث يهتا يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةد فرلادلورللفا هحاأةافهفلا254nmريفععما اريوماأممنا ععلاريلافهمعنمرنافماريفيولرلاريمل ما(hplc)ريفيولرلاريمل م .ت اوىنللامى د عاىرفه ا ثااريلا اريا يما ايفتادواريلافهمعنمرناريله وهلا اريلافهمعنمرنافماريفيولرلاريمل م . ا لهه اريلا يهلاريااعهلاي هواريملولاretention timesأوب أبا ادرل /مرا ا250ا هحا ارتب اريفيولرلاريف ماتواوىنباريل اأوىتلافموب اccl4ترذاملماريلاىرةاريوما ارووثمرت اأعروبلا اريهلانلهواريلا يهلاريعف هلا ريااعهلاي هواريملولافماردىرياريفلم .astا alt يفمفاولالاأن لا افه عاديوععارا يأ دوجو مدعو ،ةهج نم تاديونيفالفلل ةيبطلا ةيمهألل ارظن.اقباس سردت مل ةددن ا introduction the echinops tenuisectus belong to the family compositae (fig1) is a wild, iraqi plant first studied in iraq. the echinops genus consist of 100 spp.(1) which are widely distributed in sharaban, diyalah, badrah ((upper tigris plain))(2) . preliminary investigation indicated that, this plant contain different types of flavonoids in accepted amount. among these flavonoids: silymarin (figure 2) which is a flavonolignan that has been introduced fairly recently as a hepatoprotective agent(3,4,5,6,7). silymarin has been found to provide hepatoprotection through its antioxidants properties (scavengers and regulators of the intracellular content of glutathione)(8,9,10) , as cell membrane stabilizers and permeability regulators that prevent hepatotoxic agents from entering hepatocytes(11,12). also as inhibitors of the transformation of satellite hepatocytes in to myofibroblasts, the process responsible for the deposition of collagen fibers leading to cirrhosis (13, 14, 15). 1 corresponding author : e-mail : enassara@yahoo.com received : 21/11/2007 accepted : 24 /5 /2008 iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 17 figure 1: photography of echinops tenuisectus figure 2: structure of silybin (commercially called silymarin) the other flavonoids found in this plant are the quercetin and rutin (figure 3,4), both of them possess a powerful antioxidant activity which help to prevent free radical oxidative damage to cells, also help in the treatment and prevention of alcohol and chemical induced hepatotoxicity by increase glutathione in the liver(16). glutathione responsible for detoxifying a wide range of hormones, drugs, and chemicals. high level of glutathione in the liver increases its capacity for detoxification. quercetin and rutin increase the level of the important antioxidant enzyme superoxide dismutase in the cell cultures (17). in addition they stimulate protein synthesis in the liver, which results in an increase in the production of new liver cells to replace the damaged one (18). shoskes 1999 demonstrate that quercetin and rutin also inhibit the synthesis of leukotrienes (mediators of inflammation, which can result in psoriasis) (19).recently, flavonoids can help in prevention of cancer through several pathway: inhibiting proliferation and inducing apotosis(20,21)or through inhibiting tumor invasion and angiogenesis(22,23). this wide variety of beneficial health effects of these flavonoids acquired this study its importance in finding a new uninvestigated source of these important flavonoids , contained within echinops tenuisectus of the family compositae and evaluate their possible protective effect against the experimentallyinduced liver damage in rats by ccl4 . liver, the largest organ in vertebrate body, is the major site of intense metabolic activities. liver injury caused by toxic chemicals and certain drugs has been recognized as a toxicological problem. herbal drugs are playing an important role in health care programs world wide, and there is a resurgence of interest in herbal medicines for treatment of various ailments including hepatopathy(24). ccl4 is reported to produce free radicals which affect the cellular permeability of hepatocytes and it causes massive histopathological changes like necrosis, congested vessel and fatty changes (steatosis). so, the reverse of these phenomenon can be considered as the index of hepatoprotective(25). figure 3: quercetin; r = h figure 4: rutin ; r= rhamno-glucosyl iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 18 materials and methods a. plant materials: the plant material was collected during july 2005 from sharaban,dyala city. the plant was identified by the department of pharmacognosy, college of pharmacy /university of baghdad; and authenticated by the herbarium of baghdad university. fifty grams of the powdered plant material (seed part) were first defatted by reflux with 100 ml of petroleum ether (60º-80ºc) for one hour and filtered. the defatted dried plant material was then extracted by reflux using 100 ml of 70% ethanol for three hours.this step was repeated for four times, then the combined filtrates were evaporated under reduced pressure using buchi rotatory evaporator attached to vacuum pump at 40ºc, to a thick residue of ethanol extract (f1). this residue was then hydrolyzed with 2n hcl in aqueous methanol (1:1) under reflux for three hours; the resultant solution was then partitioned with 100 ml of ethyl acetate (f2). this fraction was evaporated under reduced pressure to dryness, as shown in the following diagram (figure 5). echinops plant (50gm.) seed part defatted with petroleum ether (60ºc -80ºc) using reflux for 1hr. filtrate residual plant part extract with 70% ethanol using reflux for 3 hr. marc ethanolic filtrate evaporate to thick liquid f1 1. hydrolyzed with 2n hcl in aqueous methanol (1:1) 2. partition with 100ml ethyl acetate f2 (27) gm contain flavonoids figure-5 [schematic representation of flavonoids extraction from echinops tenuisectus ] f2 (ethyl acetate fraction)ا→اevaporationاtoاdrynessاunderاreduceاpressureا→اblackgreenish oily residue, tlc and hplc indicated that this fraction contain three compounds which are silymarin, rutin and quercetin and by preparative thin layer chromatography and hplc we can separate each one and calculate the percentage of each one by weighting. f2ا(oilyاresidueاfraction)ا→اdissolvedاinاwaterا→اsuspentionا(readyاforاhepatoprotective study) iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 19 b. identification of silymarin,quercetin and rutin in the plant seed extract. the identification of these flavonoids in the seed extract was performed by: 1. identification of flavonoids by tlc: using tlc ready made gf254 plates, uv detector at 254 nm, standard flavonoids and two different solvent systems that were (26) : solvent (1): chloroform: acetone: formic acid (75:16.5:8.5) (figure 6) solvent (2): n.butanol: glacial acetic acid: water (40: 10:50) (figure 7) (table-1) showed the rf values of the standards silymarin, quercetin and rutin, and the rf value of plant seed part extract. b d c a figure 6: tlc gf254 plate of the seed extract and standards using s1 mobile phase. a plant seed extract c quercetin standb silymarin standard d rutin standard a c b d figure 7: tlc gf254 plate of the seed extract and standards using s2 mobile phase a plant seed extract c quercetin standard b silymarin standard d rutin standard table 1: rf values of standard silymarin, rutin and quercetin and seed extract. solvent system standard silymarin standard quercetin standard rutin seed extract s1 0.43 0.35 0.28 0.4,0.33,0.2 s2 0.2 0.81 0.56 0.21,0.8,0.5 figure 8: hplc of plant seed extract of echinops tenuisectus. iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 20 figure 9: hplc of standard silymarin. figure 10: hplc of standard quercetin. figure 11: hplc of standard rutin. iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 21 2. identification of flavonoids by hplc: silymarin, quercetin and rutin were authenticated by hplc . (figures 8-11) the hplc conditions are listed in the following table. (table-2) table 2: hplc conditions. hplc conditions mobile phase methanol:water (50:50) column c18 25cm flow rate 1ml/min detector 288 nm c. hepatoprotective studies: 1. experimental animals: eighteen – albino rats of both sexes weighting 150-200 gm (both sex) were used in this study. animals were kept in the animal house of the college of pharmacy/ university of baghdad, under standardized condition (12 hr light dark cycle at room temperature). the animals were fed standard chow and given water ad libitum. 2. experimental design: the animals were divided in to three groups (each group have 6 animals) and treated as follows: group (1): six rats received normal saline for 7 day orally and secreted at along 7, saved as control group (2): six rats received single oral dose of ccl4 (1mg/kg) diluted by corn oil in ratio of 1:1 v/v for the induction of liver damage and animals were sacrificed after 24 hr of ccl4 administration. group (3): six rats received oral dose of the seed extract of echinops tenuisectus plant in amount equivalent to 250mg/kg by gavages tube for 7 days, befor ccl4 (1mg/kg diluted by corn oil in a ratio of 1:1 v/v), then the rats were sacrificed after 24 hr, after ccl4 administration. 3. biochemical estimation: serum was prepared from the collected blood and subjected to biochemical estimation of alt and ast. 4. histopathology: portion of liver tissue in each group was fixed in 10% formalin (formalin diluted to 10% with normal saline) and proceeded for histopathology. after paraffin embedding and block making, serial section of 5µ thickness were made, stained with haematoxylin and eosin and examined under microscope. 5. statistical analysis: the significance of difference between the mean values was calculated using unpaired student's t-test. p-value less than 0.05 were considered significant for all data showed in our results. 6. results: a) biochemical parameters: table-3 showed a significant elevation in the activities of both alt and ast in ccl4 treated rats compared to control group. pretreatment rats with seed extract of echinops tenuisectus (250mg/kg) showed a significant decline in the activities of alt and ast compared with ccl4 treated rats (table 3 , figure 12 and 13). table 3: effects of seed extract of echinops tenuisectus on the activities of serum alt and ast in rats treated with ccl4. each value represents mean ± standard deviation. values with non=identical superscripts (a,b) within each parameter are significantly different ( p< 0.05) n= number of animals. control ccl4 seed extract +ccl4 (a) figure 12: bar chart comparing the effects of seed extract of echinops tenuisectus pretreated with ccl4 on serum alt activity. treatment serum alt u/l serum ast u/l control n=6 10.24±1.21 45±3.8 ccl4-treated n=6 64.4±7.53 a 68.6±1.67a seed extract + ccl4 n=6 13.6±1.34 54.4±3.28b 0 10 20 30 40 50 60 70 80 control ccl4 seed extract +ccl4 a l t a c ti v it y u /l iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 22 control ccl4 seed extract +ccl4 (a) (b) figure 13: bar chart comparing the effects of seed extract of echinops tenuisectus pretreated with ccl4 on serum ast activity. b) histological examination: histological examination of ratُs liver treated with ccl4 showed that, there was centrilobular hemorrhage, with heavy inflammation and necrosis. in addition to steatosis and individual necrosis were observed compared with control (figure 14 and 15). pre-treatment of rats with seed extract of echinops tenuisectus before ccl4, exhibit variable degrees of recovery with slight centrilobular congestion, marked reduction in inflammatory reaction. furthermore, neither necrosis nor steatosis was observed in ratۥs liver section (figure 16). figure 14: section showing normal rat's liver. magnification: 40x, staining: haematoxylline and eosin. figure 15: section showing morphological alteration of liver from ccl4-treated rats. black arrow represents fatty changes (steatosis), white arrow represent haemorrhage. magnification: 40x, staining: haematoxylin and eosin. figure 16: section showing the administration of seed extract of echinops tenuisectus improved ccl4-induced hepatic damage. magnification: 40x, staining haematoxylin and eosin. discussion: many compounds exhibit hepatoprotective activity, demonstrated either by decreasing the harmful effect of hepatotoxic compound or by maintaining the normal hepatic physiology. the present study showed that, the seed extract of echinops tenuisectus have good hepatoprotective effect against ccl4-induced hepatotoxicity in rat manifested by attenuating the increases in the serum activities of alt 0 10 20 30 40 50 60 70 80 control ccl4 seed extract +ccl4 a s t a c ti v it y u /l iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 23 and ast (table 3, figure 12and13) and by reversing the histological damage induced by ccl4, this was attributed to the presence of flavonoids, especially the silymarin, rutin and quercetine which possess antioxidant properties (8,16) which can improve the normal physiology of hepatocyte (17,18,19). conclusion the present study showed that, seed extract of echinops tenuisectus improve the hepatic damage and steatosis induced by ccl4 toxicity. acknowledgment the authors gratefully thank prof. dr. ali almussawi for supporting this project. references: 1. treaseاandاevansا“pharmacognosy”ا fifteenth edn university of nottingham, u.k. 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(pubmed). 21. kavangh kt, hafer lj, kim dw, et al.“green tea extracts decrease carcinogeninduced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture”.j cell biochem.2001;82(3):387-398.(pubmed). http://cancerres.aacrjournals.org/cgi/external_ref?access_num=2074043&link_type=med http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=retrieve&db=pubmed&list_uids=2813578&dopt=abstract iraqi j.pharm.sci., vol.17 (1) ,2008 hepatoprotective effect of echinops flavonoids 24 22. bagli e, stefaniotou m, morbidelli l, et al.“luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of endothelial cell survival and proliferation by targeting phosphatidylinositol 3'-kinase activity”.cancer res.2003. 23. kim mh.“flavonoids inhibit vegf⁄bfgf-induced angiogenesis in vitro by inhibiting the matrixdegrading proteases”.j cell biochem. 2003 ; 89(3) :529-538. 24. m.r venukumar, m.s. latha. hepatoprotective effect of the methanolic extract of curculigo orchioides in ccl4treated male rats. indian journal of pharmacology. 2002;34; 269-275. 25. p. jayasekhar, p.v. mohanan, and k.rathinam. hepatoprotectvie activity of ethyl acetate extract of acacia catechu. indian journal of pharmacology. 1997; 426-428. 26. merck and co., inc. rahway, nj. the merck index 8th ed u.s.a (1966), pp 8216. microsoft word bagpha05114 _8_ iraqi j. parma. sci., vol.14, 2005 65 -------------------------------------------------------------------------------------------------------------- formulation, stability and bioequivalency study of prepared salbutamol sulphate nebules. bazigha k. al –temimy, alaa a. abdul –rasool received: 07.01.2001 accepted: 2.3.10.2002 abstract: salbutamol sulphate nebules is considered as the most rapid effective route of administration for treatment of acute attacks of asthma . this study was carried out to formulate a stable formula of salbutamol nebules containing 0.1% (2.5mg / 2.5ml) of the active ingredient in a buffered solution . stability study in different buffers at ph 3 showed that the longest shelf life was equal to 3.5 years for formula f .in addition the bioequivalency of this formula incomparison to ventolin® nebules was measured and it was equal to (± 5.2) %. also it was found that there was no significant difference between the formula and ventolin® nebules regarding their pharmacokinetic parameters which include elimination rate constant, elimination t 0.5 and amount of the unchanged drug excreted in urine, 30 min. after administration (p<0.05) . this study may suggest that the prepared formula could be used successfully in the preparation of salbutamol sulphate nebules. :الخالصة وب�ات الح�اذة الت�ي تص�یب االكث�ر فعالی�ة ف�ي ع�الج الن ان رذاذ السالبیوتامول س�لفیت یعتب�ر الطری�ق االس�رع و ٢,٥ \ملغم ٢,٥% ( ٠,١محلول ثابت لرذاذ السالبیوتامول بتركیز غھذه الدراسة لتصی تأجری.مرضى الربو اظھرت دراسة ثباتیة سلفات السالبیوتامول في عدة محالیل درائة عند .للمادة الفعالة في المحلول الدارىء ) مل ) .ف(سنة للصیغة التركیبیة ٣,٥ء مفعول الدواء یساوي ان اطول عمر النتھا ٣,٤أس ھیدروجیني مقارن��ة بالمستحض��ر التج��اري رذاذ الفنت��ولین ) ف(أض��افة لھ��ذا فلق��د ت��م قی��اس التك��افؤ الحی��وي للص��یغة التركیبی��ة ك�ذلك فلق�د وج�د ان لكلیھم�ا نف�س مق�اییس حركی�ة ال�دواء والمتض�منة ثاب�ت ). ٥,٢± % ( ٩٨وكانت مساویة الى أن .دقیق�ة م�ن أخ�ذ الع�الج ٣٠نصف عمر الطرح وكمیة الدواء الغیر متغی�ر والمط�روح خ�الل ,الطرح سرعة یمك���ن اس���تعمالھا بنج���اح لتحض���یر رذاذ س���لفات ) ف(ھ���ذه الدراس���ة تقت���رح ان الص���یغة التركیبی���ة المحض���رة .السالبیوتامول iraqi j. parma. sci., vol.14, 2005 66 -------------------------------------------------------------------------------------------------------------- introduction: salbutamol is a beta-adrenergic stimulant that has a selective action on beta-2adrenoceptors in the bronchi and uterus. it is available in a variety of dosage forms such as injections, tablets, syrups, aerosols and nebules (1,2) . inhalation of salbutamol sulphate via a nebulizer is an integral component of modern treatment of airway diseases, particularly for patients unable to use metered dose inhalers (3,4) . also it is effective in treatment of hyperkalemia in patients with chronic renal failure (5,6) . the usual dose of salbutamol nebules is 2.5-5mg in 2.5ml which can be diluted with sodium chloride 0.9% and given as single dose and can be repeated up to 12 times per day in hospital with monitoring (7) . the aim of this study is to prepare a stable formula f for salbutamol sulphate nebules 0.1% .the shelf life of this formula is to be determined and then subjected to a comparative study with a reference product, regarding their availability to lung. experimental: materials and instruments: salbutamol sulphate powder and empty ampoules were kindly donated by sdi. sodium unhydrous sulphate, chloroform and n, ndiethyl-p-phenylenediamine sulphate, methanol, sodium dodecy1 sulphate, potassium dihydrogenphosphate were from bdh, chemicals, ltd, pool, england. sodium bicarbonate was from evans, england. potassium hexacyanoferrate and ortho phosphoric acid were from riedel – dehaen, hanover, germany. nitrogen gas was from baghdad factory (sdi). ovens (gallenhamp, model ov-160,england). spectrophotometer(lkb biochem,model 4049,england ). vitalograph(model 520-336,ireland),hplcsystem consisted of shimadzu chromatopac (c-ra,japan), shimadzu liquid chromatograph (lc-ba,japan),shimadzu uv spectrophotometric detector (spd-6a,japan )and 4.6mm x 15cm ,lc-18uitra sphere –i.p. column (supel co.,inc,germany). methods : * stability study : several formulas of salbutamol nebules have been prepared by dissolving salbutamol sulphate powder in triple distilled water (table-1). the ph of the solutions was adjusted to 3.4 using different types of buffers , including :  0.1m citric acid .  0.1m sodium citrate/0.1m hcl .  0.1m sodium hydrogen phosphate /0.1m phosphoric acid .  0.1m sodium acetate /0.1m acetic acid .  0.1m h2so4/0.1m naoh. iraqi j. parma. sci., vol.14, 2005 67 -------------------------------------------------------------------------------------------------------------- a high degree of clarification was achieved by filtration through 2mm membrane filter before filling. the prepared solution was filled under nitrogen gas in clear ,sterile, empty ampoules , which had been sealed by fusion method and sterilized by autoclaving. each ampoule contains 2.5ml of the prepared solution. the shelf –life of all formulas was determined by incubation of ampoules in ovens at 50,60and 70 c and for 120 days. ampoules contained 2.5ml of the tested solution were assayed for their drug content every 2 weeks , using the calorimetric method . the observed first –order rate constants (k) were obtained by linear regression analysis through utilizing the following equation: log ct =log co-kt/2.3303 where: ct=concentration remaining at time (t)and c0 = intial concentration . * assay of salbutamol sulphate nebules: a standard solution of salbutamol nebules in water 0.1% was mixed with 4ml of 5% w/v sodium hydrogen carbonate solution , 4ml of 0.1% w/v n,ndiethyl –pphenylene diamine sulphate solution and 4ml of a freshly prepared 8% w/v potassium hexa cyano ferrate .then salbutamol as a complex was extracted with chloroform..the absorbance of the blue extract was measured at 620nm, using chloroform as a blank . the same method was repeated for salbutamol sulphate nebules.calculation of salbutamol sulphate concentration in each sample was obtained from the following equation (8) : % salbutamol sulphate = absorbance of test x 100 _________________________________________________ absorbance of standard bioequivalency study : experimental scheme: the bioequivalency of formula f and ventolin nebules was determined on ten healthy volunteers (6 females) between 20-35 years old and weighing (50-90) kg. they were considered healthy on the basis of their history, and they had received light dinner and no drug at the study day. each subject inhaled 2.5ml of formula f for 5 minutes then urine samples (10ml) were taken before as well as 1/2, 1,2,4,6,10 and 24hours after administration . samples were stored in plastic tubes at -20 c until the analysis. after 2 days the same volunteers had taken the same dose 2.5mg/2.5ml of ventolin nebules (allen and hanburgs) and the procedure iraqi j. parma. sci., vol.14, 2005 68 -------------------------------------------------------------------------------------------------------------- continued as for the prepared formula (9) . * calibration curve and chromatographic conditions : hplc method was used to determine salbutamol sulphate in urine by preparation of series of dilution salbutamol sulphate in urine to give final concentrations ranging from 0.005 to 0.03mg/ml. the mobile phase was consisted of methanol: water (60:40) with sodium dodecyl sulphate 20mmole and potassium dihydrogen phosphate 10mmole. the mobile phase was adjusted to ph 3 with 1m phosphoric acid. a 20ml of urine sample was injected into the chromatograph of the hplc with suppl, lc-18 column. the flow rate was 1.0ml min-1 and the retention time of salbutamol was 3.6 min (10) . the absorbance of the effluent is monitored at 276nm salbutamol was stable in urine when it was stored at-20 c for up to one month. quantitation of salbutamol sulphate nebules was accomplished by plotting the concentration in mg/ml against peak height, as shown in fig. 1. * statistical analysis : student‘s t-test was used to examine the difference in the mean of the parameter tested . a p-value of (p<0.05) was considered insignificant . results and discussion : * stability study : the degradation of salbutamol sulphate in all formulas follows first-order kinetics, since straight lines were obtained when the logarithm of percent remaining of salbutamol sulphate is plotted against time ,as shown in fig.2 for formula f . the degradation rate constants (k) at 50,60and 70 c for formula f were calculated from the slopes of the lines as shown in table 2. to determine the expiration date (t 10% ), arrhenius plot was utilized to predict the degradation rate constants at 25 c (k 25), as shown in fig.3. results showed that the shelf-life of salbutamol sulphate in formula f was equal to 3.5 years, which indicates that formula f is stable at the room temperature compared to other prepared formulas . *bioequivalency study of salbutamol nebules : methods of assessing the bioavailability of salbutamol to the lung following nebulization have been limited by analytical problems in measuring low plasma drug concentration and the lack of a suitable gama radio label inhaled marker. in addition , 90% of an inhaled dose is swallowed, so it is difficult to discriminate between the inhaled and swallowed fraction (11) .in this study we use iraqi j. parma. sci., vol.14, 2005 69 -------------------------------------------------------------------------------------------------------------- an assay with sufficient sensitivity to measure urine concentrations of salbutamol sulphate after nebulization, because this method is simple ,noninvasive . further more urinary excretion is the major rout of elimination of unchanged salbutamol and unaffected by the time interval between micturition (12,13,14) . table-3 shows the mean and standard deviation values of unchanged salbutamol excreted in time intervals for 10 normal subjects receiving 2.5mg of salbutamol sulphate, using formula f and ventolin nebules . while fig.4 indicates the cumulative amount of unchanged salbutamol sulphate excreted during days of treatment . since salbutamol nebules has an onset of action within 5min .and shows a peak effect after 15min ., so the fraction of dose recovered 30min, after nebulization is , therefore ,representative of the dose delivered to the site of action and is a measure of the bioequivalency of salbutamol to the lung (15) . the data indicated that there was no significant difference between the amount excreted from both formula f and ventolin nebules during the first 30min .and it was equal to 4.0(±0.8) % and 4.08(±0.5) % of the dose respectively .while the bioequivalency of formula f to the lung relative to that of ventolin nebules was equal to 98(±5.2) % for the same time. the results suggest a successful utilization of formula f in the preparation of salbutamol nebules. iraqi j. parma. sci., vol.14, 2005 70 -------------------------------------------------------------------------------------------------------------- iraqi j. parma. sci., vol.14, 2005 71 -------------------------------------------------------------------------------------------------------------- iraqi j. parma. sci., vol.14, 2005 72 -------------------------------------------------------------------------------------------------------------- concentration of salbutamol sulphate (mg / ml) iraqi j. parma. sci., vol.14, 2005 73 -------------------------------------------------------------------------------------------------------------- iraqi j. parma. sci., vol.14, 2005 74 -------------------------------------------------------------------------------------------------------------- references: 1. richard, a.h., pamela c.c. and marry, j.m. pharmacology, 2000, ch . 21,p . 218. 2. martindale extrapharmacopieaz, 32 ed., 1999,vol .i, p.759. 3. glenn, a.j and gregory, m.p .high –performance liquid chromatographic assay for the simultaneous determination of ipraropium bromide, fenoterol, salbutamol and terbutaline in nebulizer solution. pharmaceutical and biomedical analysis j., 1994,12(6), 825-832. 4. abroug ,f.,nouira, s,behir ,a ., boujdar r.,elatrous,s.and bouchoucha ,s.a controlled trial of nebulized salbutamol and adrenaline in acute severe asthma .intensive care med., (jan.)1995,12(1),18-23. 5. liou,h.h. hypokalemic effects of intravenous infusion or nebulization of salbutamol in patients with chronic renal failure : comparative study am.j. kidney dis ., (feb.)1994,349,301-5. 6. montoliu,j.,almirall,j.,ponz,e.,campistol,j.m.and revert,l.treatment of hyperkalaemia in renal failure with salbutamol inhalation. j. inter .med., (jul.)1990,228(1),35-7. 7. infomations supplied by sara barcham ,medical information advisor, glaxo welcome,u.k., 1997,16.6. 8. bp, vol.ii, hmso, united kingdom,1993, p. 845, a 183, a 191. 9. michael, hand henry, c. determination of the relative bioavailability of salbutamol to the lung following inhalation. br. j. clin. pharmacy. 1992,34,311315. 10. jarvie, d.r., thompsom, a.m. and dyson, e,h.laboratory and clinical features of self-poisoning with salbutamol and terbutaline .clin. chem.acta, 1987,168,313-322. 11. newman, s .p., pavia. d. moren, f., sheahan ,n.f.and clark , s.w. deposition of pressurized aerosols in the hyman respiratory tract . thorax , 1981,36,52-55. 12. lewis ,l.d., essex , e., mclaren , m.and cochrane , g.m.plasma concentrations of salbutamol in acute severe asthmatic .asut .n.z.j.med ., 1990,20,204-207. 13. horn ,c.r.,essex ,e.,hill, p. and cochrane , g.m.dose urinary salbutamol reflect compliance with aerodol regimen in patients with asthma .res.p.med., 1990,83,15-18. 14. jaccobson ,g.a.,peterson,g.m.and mclean , s.investigation of urinary levels of salbutamol in asthmatic patients receiving inhaled therapy .j.of clinical pharmacy and therapeutics , 1997,22,119-126. 15. hetzel ,m.r.and clarhe ,t.j.h.comparison of intravenous and aerosol salbutamol .br.med .j. 1976,2,919. iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition doi: http://dx.doi.org/10.31351/vol27iss1pp100-108 100 synthesis, characterization and alpha glucosidase inhibition activity of new phthalimide derivatives hassan a. m. jawed*,1, mohammed h. mohammed **and sajida h. ismeal*** * department of pharmaceutics chemistry, college of pharmacy , university of al-mustansiriya baghdad ,iraq. **department of pharmaceutics chemistry, college of pharmacy, university of baghdad, baghdad ,iraq. *** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract three of intermediate imide compounds were synthesized through reacting of phthalic anhydride with glycine(compound2a), and tetrachlorophthalic anhydride with glycine, (s)-2-[(tertbutoxycarbonyl)amino]-3-aminopropionic acid(compounds2b,c) respectively in dry toluene with azeotropic removal of water using deanstark apparatus then carboxyl functional group activated by refluxing with thionyl chloride, the resulted acid chloride(compounds 3a-c) were reacted with different amine (5-flourouracil, 4-chloroaniline, 4-bromoaniline, 2-amino thiazole, and pyrrolidine)(compounds 4a-e) , the compounds (5a-j)consider as end productswhile the compounds (5k-o) required further reaction to deprotect aliphatic amine this was achieved by treating the compounds with trifloruro acetic acid (tfa) to remove tert-butoxycarbonyl group (compounds 6a-e). the alpha glucosidase inhibitory activity of some synthesized compounds(5a, 5f, 6a)was evalutedagainst alpha-glucosidase enzyme extracted from saccharomyces bacteria, p-αnitrophenol glucopyranoside (pnpg) was used as substrate and thestandard was acarbose. all these test compounds showsexcellent inhibitory activity according to ic50 values which is ranging from (4.61-7.32 m). keywords: antidiabetic, synthesis, phthalimide, ic50. نشاط التثبيطي الى الفا كلوكوسايديس بواسطة اللدراسة تصنيع، تشخيص والتقييم االولي مركبات الفثالميد الجديدة ***و ساجدة حسين اسماعيل **، محمد حسن محمد 1،*علي محمد جواد حسان .، بغداد ، العراق الجامعة المستنصرية، كلية الصيدلة ، الكيمياء الصيدالنيةفرع * .، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق الكيمياء الصيدالنيةفرع ** . فرع االدوية و السموم ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق *** الخالصة و أنهيدريد رباعي الفكلين (2a) طريق تفاعل أنهيدريد الفثاليك مع جاليسينتم تحضير ثالثة من المنتجات الوسيطة إيميد عن على التوالي في التولوين الجاف (2b,c)أمينوبروبيونيك أسيد -3 -بوتوكسيكاربونيل( أمينو[ -ترت. )] 2(s)الفثاليك مع الجاليسين، ة وظيفية الكربوكسيل تفعيلها عن طريق ارتداد مع كلوريد الثيونيل ستارك ثم مجموع-مع إزالة أزيوتروبيك من الماء باستخدام جهاز ديان أمينو ثيازول، -2برومانيلين ، -4كلورانيلين، -4فلوروراسيل، 5مع أمين مختلفة ) (3a-c)، وكان رد فعل حمض كلوريد ( تتطلب المزيد من التفاعل ل (5k-o في حين أن المركبات (5a-j)، والنواتج الناتجة تعتبر المنتجات النهائية (4a-e)وبيروليدين( كاربونيل وتوكسييمجموعة ب الخليك إلزالةروديبروتيكت أمين األليفاتية التي تحققت من خالل معالجة المركبات مع حامض ثالثي فلو غلوكوزيداز -αباستخدام االنزيم( 5a,5f and 6aتم اختبار نشاط المثبطة ألفا غلوكوزيداز لبعض المركبات المركبة )( . 6a – e ) ةالثالثي .نيتروفينول غلوكوبيرانوسيد )بينب( المستخدم كركيزة و أكاربوس تستخدم كمعيار-p من ساكاروميسز سيريفيسياي و موالري (. 2637 – 46.4) ح من لتركيز المثبط القصوئ التي تتراونصف ا ممتاز وفقا لقيم مثبط كل مركبات االختبار هذه تظهر مضاد للسكري, تحضير, فثالميد, التركيز الكبحي. -الكلمات المفتاحية: introduction diabetes mellitus (dm) is a chronic metabolic disorder with heterogeneous etiologies )genetic and environmental factors(, it is characterized by disturbance in the metabolisms of carbohydrate, fat and protein resulting from defects in insulin secretion, insulin action or both(1-3). insulin is a peptide hormone produced by beta cells of the pancreas (2).it hastwo essential functions: (1) insulin stimulates glucoseuptake and lipid synthesis; (2) insulin inhibits the breakdown of lipids, proteins and glycogen, and also inhibits the glucose biosynthesis pathway (gluconeogenesis) (4–6). there are two main types of diabetes, type1 and type-2, and also what is termed gestational diabetes that affects females during pregnancy.type-1 diabetes is also known as insulin-dependent diabetes (7). 1corresponding author e-mail: hass_ph86@yahoo.com received: 6/1/2018 accepted: 6/4/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp100-108 http://int.search.myway.com/search/ggmain.jhtml?p2=%5ebsb%5exdm014%5es22912%5eiq&ptb=c30dc64b-fb93-4082-b3c5-42ef74d01277&n=783a897d&ind=&cn=iq&ln=en&si=eaiaiqobchmihu6hub6e1wivrp8bch09uaa3eaayasaaegjffvd_bwe&trs=wtt&brwsid=a4496327-9dfa-45c0-a87e-29ea9ef68318&st=hp&tpr=sc&searchfor=mustansiriya http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition 101 in type-2 diabetes, the body does not produce enough insulin for proper functioning orthe cells do not react to insulin (insulin resistance) (8).as type 2 diabetes is a progressivedisease, intensification of therapy is normally required over time, traditional treatmentalgorithms oftenfail to address the progressive nature of the disease.furthermore, current therapeutic agents may also be associated with wide range of various effects: increased risk of hypoglycemia (sulphonylureas and insulin), weight gain (sulphonylureas, thiazolidinediones and insulin), and gastrointestinal intolerance (metformin), which representbarriers to maximum glycemic control(9-11). one of the current therapies is alphaglucosidase inhibitors.α-glucosidase is thekey enzyme which catalyzes the final step in the digestion of carbohydrates in mammalians. hence, α -glucosidase inhibitorscan suppress the liberation of d-glucose of oligosaccharides and disaccharidesfrom dietary complex carbohydrates and prolong glucoseabsorption, resulting in decrease post-prandial plasma glucose levels and retard post-prandial hyperglycemia(12, 13). consequently,α -glucosidase inhibitors havebeen approved for clinical use in the management of type 2 diabetes,as well as the treatment of obesitysuch as acarboseandmiglitol(14, 15). however it was found that phthalimide, tetrachlorophthalimide derivatives exhibited potent α -glucosidase inhibition and the structure activity relationship studies show the following: 1.a phthalimide or tetrachlorophthalimide moiety connected to a variously substituted phenyl ring by an alkyl chain shows promising alpha glucosidase inhibitory activity (scheme 1). 2. studies revealed the importance of the distance between the phthalimidering and the phenyl moiety. 3. presence of an electron withdrawing group (no2, cf3 etc.) at the (r3) position was more potent. 4.tetrachlorophthalimideskeleton is a useful non-sugar-type sugar mimic pharmacophore; it is characterized by highlipophilicity which could influence their pharmacokinetic propertiesand biological activity (16). figure 1. phthalimide moiety connected to substituted phenyl ring by an alkyl chain materials and methods all chemicals and solvents used during synthesis were of analytical grade and used without further purification. completion of reactions and the purity of compounds were ascertained by a. thin-layer chromatography (tlc), using silica gel gf254 (type 60) pre-coated aluminum sheets, merck (germany) and the eluent used is 1. chloroform : methanol (85:15) 2. glacial acetic acid: ethyl acetate: methanol (0.1:3:1)to run tlc. b. high performance liquid chromatography was performed at the lebanese university / lebanon (pf4370) for the final compounds in order to ensure complete purity of compounds. melting points were determined using stuart smp3 melting point apparatus in open capillary tubes, and are uncorrected. fourier-transform infrared spectroscopy (ftir), (kbr disc) (υ,cm-1) were recorded using (biotech engineering management ftir-600, uk) at college of scienceal-mustansiriya university, and the college of pharmacyalmustansiriya university. furthermore, the elemental microanalysis of the synthesized compounds was done using (elementar vario micro cube instrument ,germany) in the university of mustansiriyacollege of pharmacy. proton-nmr spectra and (13c nmr): were recorded on (bruker, germany nmr spectra 300 mhz, avance iii 300 spectrometer) with tetramethylsilane (tms) as an internal standard, dimethyl sulphoxide used as a solvent for samples measurement, (δ=ppm) and coupling constant in hertz, which was run in (lebanese university)-lebanon. chemical synthesis synthesis of n,n-phthaloyl glycine (compound2a) a mixture of phthalic anhydride (6.23 gm, 42.29 mmol), glycine (3.57 gm, 47.61 mmol) and triethyl amine (0.7 ml) in dry toluene (250 ml) was heated under reflux for 4 iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition 102 hrwhile azeotropic removal of water using dean-stark apparatus (17). the reaction mixture was concentrated at reduced pressure, added ethyl acetate to the residue, washed the organic phase with dilute hcl (1n) to eliminate the unreacted triethylamine, dry over mgso4, concentrated to yield then-phthaloyl glycine (compound2a)as a solid compound (percent yield 90%)(18, 19). compound2a is white crystalline solid; melting point: 193-195°c; ir (kbr), (υ,cm-1): 3200-2500 o-h str,2993 and 2885 asym. and sym.str. of ch2., 1772 and 1726(c=o) str. of phthalimide, 1728 (c=o) str. of carboxylic acid consolidated with(c=o) str. of phthalimide, 1466 o-h bend, 1215c-o str.v., 736 out of plane aromatic bendcm-1. synthesis of n,ntetrachloro phthaloyl amino acids [ glycine , (s) – 2 [ ( ter t – butoxycabonyl ) amino ] – 3 aminopropionic acid( boc dap oh)] (compounds2b and 2c) a mixture of tetrachloro phthalic anhydride (12.09 gm, 42.29 mmol), glycine or boc dap oh (47.61 mmol) and triethyl amine (0.7 ml) in dry toluene (250 ml) was heated under reflux for 4 h while azeotropic removal of water using dean-stark apparatus (17). the reaction mixture was concentrated at reduced pressure, added ethyl acetate (20 ml) to the residue, washed the organic phase with dilute hcl (10 ml) (1n) to eliminate the unreacted triethylamine, dried over mgso4, concentrated to yield the n,ntetra chloro phthaloyl glycine, boc dap oh (compounds 2b and 2c) as a solid compounds(percent yield95 % and receptively 88%). compound 2b is yellowish crystalline solid; melting point: 201-203°c; ir (kbr), (υ,cm-1): 3300-2500 o-h str.,3059 (c-h) str.of aromatic, 2939and 2870 (c-h) asym. and sym.str. of ch2. 1774and 1724(c=o) str. of tetrachloro phthalimide, 1724 (c=o) str. of carboxylic acid consolidated with(c=o) str. of tetrachlorophthalimide, 1523 and 1442(c=c) aromatic str., 1442 o-h bend, 1373 c-n str., 1296 c-o str., 736 out of plane aromatic bend cm-1. 2c yellow to white crystalline solid; melting point: 180-183°c; ir (kbr), (υ,cm-1): 3483-2500 o-h str, 2978(c-h) asym. str. of ch2, 1778 and 1716(c=o) str. of tetrachlorophthalimide,1717(c=o) str. of carboxylic acid consolidated with(c=o) str. of tetrachlorophthalimide, 1581 and 1431 (c=c) aromatic str., 1396c-n str., 1199c-o str.v., 736out of plane aromatic bendcm-1. synthesis of n,n-phthaloyl-acid chloride (compounds3a, 3band 3c) n,n-phthaloyl glycine (compound2a),or n,ntetra chloro phthaloyl glycine ( compound 2b) or n,ntetra chloro phthaloylboc dap oh(compound 2c) (5mmol) was placed in a 50 ml round-bottom flask and then thionyl chloride (5 ml) was added. thionyl chloride was added dropwise over a period of 15 min. with cooling on ice bath. the mixture was refluxed for 8 hrs at 65 °c with continuous stirring and monitored by evolution of hcl gas (which is detected by changing the color of litmus paper into reddish when placed on the top of condenser) and changing the color of the solution. the reaction are often promoted by the addition of a drop of dimethylformamide (dmf)(20).the excess of thionyl chloride was removed under reduced pressure and the residue was re-dissolving in dry dichloromethane (10 ml )and was reevaporated to give an oily residue. the resulting acyl chloride (compounds 3a, 3band 3c) wereused directly for the next step (21). synthesis of n-phthaloyl-amino acid amides ( compounds 5a-o.) a solution of one amine derivatives(5fluorouracil, or 4-chloroaniline, or 4bromoaniline, or 2-aminothiazole, or pyrrolidine) (compounds 4a-e, 5.5 mmol) were mixed with dry dichloromethane ( 15 ml) except for 4a and 4d using mixture of 5 ml dmf and 10 ml dichloromethane , then triethylamine (5 mmol, 0.5 ml) was added drop wise with stirring for 20 min. on ice bath and then, freshly prepared acid chloride of either n,n-phthaloyl(compounds 3a,3band 3c)were slowly dropped for 50 min. with continuous stirring on an ice bath, and stirring was continued at room temperature overnight. the reaction can be accelerated with a catalytic amount (2-3 drops) of pyridine, or n, n-dimethylaminopyridine (dmap) (22). solvents were removed under reduced pressure by using rotary evaporator. the resulting solid product was redissolved in ethyl acetate (10 ml) and washed with 5 % aqueous solution of sodium bicarbonate (20 ml), 5% hcl (20 ml) and distilled water (20 ml) and then dried over anhydrous magnesium sulphate[compounds 5a-o](18,19). deprotection of tert butyloxycarbonyl (n-boc) of compounds (5k-5o) the respective peptide (compounds 5k5o) was dissolved in ch2cl2 or in ch2cl2:ch3oh 9:1 (depending on solubility) and cooled to 0 °c. trifluoroacetic acid (equal volume as the solvent) (10 ml) was added and the solution was allowed to warm to room temperature. after stirring at room temperature iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition 103 until starting material was consumed (tlc monitoring), the solution was concentrated in vacuum. the solution washed with saturated aqueous nahco3 solution (ch3oh was added to assure solubility of the peptide), water, and brine solution. the combined organic layers were dry over mgso4, filtered, and evaporated in vacuum to yield the crude product in quantitative yield. in case of remaining protected peptide, the procedure was repeated. the reaction mixture was evaporated in vacuum and evaporated several times after adding ch2cl2 to remove residual tfa and give the product in quantitative yield (23-25). in vitro evaluation of α-glucosidase inhibitory activity the𝛼-glucosidase activity of some tested compounds (5a, 5f and6a) was determined according to the method described by kim et al., using𝛼-glucosidase enzyme extracted from saccharomyces bacteria. the substrate solution p-nitrophenol glucopyranoside (pnpg) was prepared in 20mm phosphate buffer, and ph 6.9. 100 𝜇l of 𝛼glucosidase (1.0u/ml) was pre incubated with 50 𝜇l of the different concentrations of the test compound (in dmso) for 10min. then 50 𝜇l of 3.0mm (pnpg) as a substrate dissolved in 20mm phosphate buffer (ph 6.9) was then added to start the reaction. the reaction mixture was incubated at 37°c for 20min and stopped by adding 2ml of 0.1mna2co3.the 𝛼glucosidase activity was determined by measuring the yellow-colored para nitrophenol released from pnpg at 400 nm using uvvisible spectrophotometer. the results were expressed as percentage of the blank control. % 𝑰𝒏𝒉𝒊𝒃𝒊𝒕𝒊𝒐𝒏 = [ 𝑨𝒃𝒔𝒄𝒐𝒏𝒕𝒓𝒐𝒍 − 𝑨𝒃𝒔𝒆𝒙𝒕𝒓𝒂𝒄𝒕 𝑨𝒃𝒔𝒄𝒐𝒏𝒕𝒓𝒐𝒍 ] × 𝟏𝟎𝟎 acarbose uses as positive control, concentrations of extracts resulting in 50% inhibition of enzyme activity (ic50) were determined graphically are shown in figures (24)(26). result and discussion spectral data and chemistry the synthesis of our compounds (intermediates and end products) is depicted in scheme 1. the physical properties and the spectroscopic (ir, 1h-nmr) data of the synthesized compounds: 5a2(2(5fluoro -2 ,4 –dioxo -3 ,4 dihydropyrimidin -1 ( 2h ) -yl) – 2 – oxoethyl ) isoindoline -1 ,3dione. white solid ;yield 85.6%;melting point: 267270°c; ir (kbr), (υ,cm-1): 3132 nh str.v. of secondary amide of 5fu, 3066 (c-h) str. of aromatic, 2935and 2885(c-h) asym. str. of ch2, 1770 and 1724(c=o) str. of phthalimide, 1662 (c=o) str. of secondary amide, 1504 and 1431 (c=c) str. of aromatic, 1246 c-f str.v, 813 out of plane aromatic bendcm-1 1hnmr (300 mhz, dmso):δ 1.12 (s, 2 h, ch2), 7.8 (s, 1 h, ch), 7.8-7.9 (m, 4 h, ar-h) 10.84 (s, 1 h, nh). 5bn-(4-chlorophenyl)-2-(1,3-dioxoisoindolin2-yl)acetamide light yellow needle solid ;yield 78%;melting point: 180-183°c; ir (kbr), (υ,cm-1): 3267 nh str.v. of secondary amide, 3059(c-h) str. of aromatic, 2947 and 2873 (c-h) asym. and sym.str. of ch2, 1774 and 1728 (c=o) str. of phthalimide, 1666 (c=o) str. of secondary amide, 1546 and1411(c=c) str. of aromatic, 713out of plane aromatic bend cm-1 1hnmr (300 mhz, dmso):δ4.3 (s, 2 h, ch2), 7.06 (s, 1 h, nh), 7.35(m, 2 h, ar-h), 7.6 (m, 2h, ar-h-nh), 7.8-7.9 (m, 4 h, ar-hconco). 13cnmr d:10 (1c), 120 (4c), 125 (2c),129 (2c), 132 (2c), 135 (1c), 138 (1c), 165 (1c), 167(2c). 5cn-(4-bromophenyl)-2-(1,3-dioxoisoindolin2-yl)acetamide dark yellow solid; yield 87%;melting point: 175-177°c; ir (kbr), (υ,cm-1): 3267nh str.v. of secondary amide, 3059(c-h) str. of aromatic, 2939 and 2870 (c-h) asym. and sym.str. of ch2, 1774 and 1724(c=o) str. of phthalimide, 1670(c=o) str. of secondary amide, 1593 nh bend . 1543 and1411(c=c) str.of aromatic, 713out of plane aromatic bendcm-1 1hnmr (300 mhz, dmso):δ 4.45 (s, 2 h, ch2), 7.5-7.6 (m, 4h, ar-h-br), 7.8-7.9 (m, 4 h, ar-h-conco), 10.5 (s, 1 h, nh). 5d2-(1,3-dioxoisoindolin-2-yl)-n-(thiazol-2yl)acetamide black solid;yield 65%;melting point: 165167°c; ir (kbr), (υ,cm-1): 3479nh str.v. of secondary amide, 3047(c-h) str. of aromatic, 2989(c-h) asym. str. of ch2, 1774 and 1732(c=o) str. of phthalimide, 1612(c=o) str. of secondary amide, 1544 nh b.v1519and 1469 (c=c) str. of aromatic, 744out of plane aromatic bendcm-1 iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition 104 scheme 1. synthesis of phthalimide derivatives iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition 105 1hnmr (300 mhz, dmso):δ4.3 (s, 2 h, ch2), 7.25(d, 1 h, ar-h-s), 7.3(d, h, ar-h-n), 7.87.9 (m, 4 h, ar-h-conco), 10.2 (s, 1 h, nh). 5e2-(2-oxo-2-(pyrrolidin-1yl)ethyl)isoindoline-1,3-dione off white needle shape solid;yield 68%;melting point: 210-212 °c; ir (kbr), (υ,cm-1): 3093(ch) str.of aromatic, 2978 and 2877 (c-h) asym. and sym.str. of ch2, 1774 and 1720(c=o) str. of phthalimide, 1685 (c=o) str. of amide, 1543and1446 (c=c) str. of aromatic, 713 out of plane aromatic bend cm-1 1hnmr (300 mhz, dmso):δ3.1-3.5 (m, 8h, ch2), 4.35 (s, 2 h, ch2-n), 7.8-7.9 (m, 4 h, ar-h-conco). 5f 4,5,6,7-tetrachloro-2-(2-(5-fluoro-2,4-dioxo3,4-dihydropyrimidin-1(2h)-yl)-2oxoethyl)isoindoline-1,3-dione white crystal;yield 88%;melting point: 198201 °c; ir (kbr), (υ,cm-1): 3132nh str.v. of 5fu ring, 3066(c-h) str. of aromatic, 2978(c-h) asym. str. of ch2, 1774 and1724(c=o) str. of tetrachloro phthalimide, 1662(c=o) str. of tertiary amide, 1549 nh bend 1477(c=c) aromatic str., 1246c-f str.v.,806out of plane aromatic bendcm-1 1hnmr (300 mhz, dmso):δ1.37 (s, 2h, ch2-n (co) 2), 7.28 (s,1h, ch-cf), 11.75 (s, 1 h, nh). 13cnmr d:10 (1c), 125 (1c), 134 (5c), 143 (2c), 160 (1c), 166 (1c), 172 (1c),185 (2c). 5gn-(4-chlorophenyl)-2-(4,5,6,7-tetrachloro1,3-dioxoisoindolin-2-yl)acetamide white yellowish crystal;yield 77%;melting point: 170-173 °c; ir (kbr), (υ,cm-1): 3174nh str.v. of secondary amide, 2939 and 2862(c-h) asym. and sym.str. of ch2, 1778 and1724(c=o) str. of tetrachloro phthalimide, 1674(c=o) str. of secondary amide, 1593 nh bend of secondary amide, 1562 and1492 (c=c) str. of aromatic, 821out of plane aromatic bendcm-1 1hnmr (300 mhz, dmso):δ1.12 (s, 2h, ch2-n (co) 2), 5.72 (s, nh).7.3 (m, 2h, ar-hcl), 7.45(m, 2h, ar-h-nh). 13cnmr d:45 (1c), 120 (4c), 122 (2c),130(5c), 132 (1c), 134(1c), 165 (1c), 169 (2c). 5hn-(4-bromophenyl)-2-(4,5,6,7-tetrachloro1,3-dioxoisoindolin-2-yl)acetamide white crystal;yield 90%;melting point: 158160 °c; ir (kbr), (υ,cm-1): 3390 nh str.v. of secondary amide,3097 (c-h) str. of aromatic, 2939and 2862 (c-h) asym. and sym.str. of ch2.1774 and1720 (c=o) str. of tetrachloro phthalimide, 1627 (c=o) str. of secondary amide, 1550 nh bend of secondary amide,1489 and1469(c=c) str. of aromatic, 744 out of plane aromatic bendcm-1 1hnmr (300 mhz, dmso):δ1.21 (s, 2h, ch2-n (co) 2),4.48 (s, nh).7.51 (m, 2h, arh-br), 7.6 (m, 2h, ar-h-nh). 13cnmr d:45 (1c), 122 (4c), 128 (2c),132(5c), 133 (1c), 162 (1c), 165 (2c). 5i 2-(4,5,6,7-tetrachloro-1,3-dioxoisoindolin-2yl)-n-(thiazol-2-yl)acetamide brown solid;yield 63%;melting point: 148-150 °c; ir (kbr), (υ,cm-1): 3421nh str.v. of secondary amide, 3039(c-h) str. of aromatic, 2935(c-h) asym. str. of ch2, 1774 and 1716(c=o) str. of tetrachloro phthalimide, 1685(c=o) str. of secondary amide, 1585 nh bend of secondary amide, 1518 and 1419(c=c) str. of aromatic, 952 out of plane aromatic bend cm-1 1hnmr (300 mhz, dmso):δ1.21 (s, 2h, ch2-n (co)2), 5.74 (s, nh), 7.4 (d, h, ar-hs), 7.6 (d, h, ar-h-nh). 13cnmr d:10 (1c), 125 (1c), 139 (4c), 143 (2c), 155 (1c), 157 (1c), 160 (1c), 165(1c), 170(2c). 5j4,5,6,7-tetrachloro-2-(2-oxo-2-(pyrrolidin-1yl)ethyl)isoindoline-1,3-dione white color crystal;yield 60%;melting point: 179-181 °c; ir (kbr), (υ,cm-1): 3097(c-h) str. of aromatic, 2947 (c-h) asym. str. of ch2, 1774 and 1716 (c=o) str. of tetrachloro phthalimide, 1577 (c=o) str. of tertiary amide, 1539 and 1473(c=c) aromatic str., 736 out of plane aromatic bend cm-1 1hnmr (300 mhz, dmso):δ1.2 (s, 2h, ch2n (co) 2), 1.8 (m, 4h, beta ch2), 1.95 (m, 4h, alpha ch2). 6a2-(2-amino-3-(5-fluoro-2,4-dioxo-3,4dihydropyrimidin-1(2h)-yl)-3-oxopropyl)4,5,6,7-tetrachloroisoindoline-1,3-dione white solid;yield 70%;melting point: 245248°c; ir (kbr), (υ, cm-1): 3414nh str.v. of nh2, 3132 nh str.v. of secondary amide, 2974 and 2881 (c-h) asym. and sym.str. v. of ch2., 1774and 1720(c=o) str. v. of tetrachloro phthalimide, 1583nh b.v. of secondary amide, 1477(c=c) str. v. of aromatic, 1246c-f str.v., 1172c-n str.v. of nh2, 736out of plane aromatic bend cm-1 1hnmr (300 mhz, dmso):δ 1.16 (d, 2 h, ch2), 3 (m, 1 h, ch-nh2), 7.73(s, ch-cf) 10.3(s,2 h, nh2), 10.8 (s,h, nh). 6b2-amino-n-(4-chlorophenyl)-3-(4,5,6,7tetrachloro-1,3-dioxoisoindolin-2yl)propanamido green solid;yield 72%;melting point: 166169°c; ir (kbr), (υ,cm-1): 3414 nh str.v. of nh2,3150 nh str.v. of secondary amide, 3097 (c-h) str. of aromatic, 2989 and 2904 (c-h) asym. and sym.str. of ch2., 1778 and 1735 (c=o) str. of tetrachloro phthalimide, 1639 (c=o) str. of secondary amide, 1616 nh bend iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition 106 of nh2, 1469 (c=c) str. of aromatic, 1230 cn str.v. of nh2, 744 out of plane aromatic bend cm-1 1hnmr (300 mhz, dmso):δ1.16 (d, 2h, ch2nco), 3.1 (m, h, ch-nh2), 7.5(m, 2h, chcl), 7.7(m, 2h, ch-nh), 5.71(s,2 h, nh2), 10.06(s, h, nh). 6c2-amino-n-(4-bromophenyl)-3-(4,5,6,7tetrachloro-1,3-dioxoisoindolin-2yl)propanamide white needle solid;yield 81%;melting point: 170-172°c; ir (kbr), (υ,cm-1): 3452 and3300 nh str.v. of nh2, 3150 nh str.v. of secondary amide, 3059 (c-h) str. of aromatic, 2993 and 2877 (c-h) asym. and sym.str. of ch2, 1766 and 1732(c=o) str. of tetrachloro phthalimide, 1708 (c=o) str. of secondary amide, 1608 nh bend of nh2, 1535and1469 (c=c) str. of aromatic, 1396 c-n str.v. of nh2, 725 out of plane aromatic bend cm-1 1hnmr (300 mhz, dmso):δ1.16 (d, 2h, ch2nco), 3.16 (m, h, ch-nh2), 7.45(m, 2h, chbr), 7.5(m, 2h, ch-nh), 5.71(s,2 h, nh2), 10.06(s, h, nh). 6d2-amino-3-(4,5,6,7-tetrachloro-1,3dioxoisoindolin-2-yl)-n-(thiazol-2 yl)propanamide brown solid;yield 59%;melting point: 158161°c; ir (kbr), (υ,cm-1): 3421nh str.v. of nh2, 3124 nh str.v. of secondary amide 3024(c-h) str.v. of aromatic ring, 2962 and2839 (c-h) asym. and sym.str. of ch2, 1789 and 1732(c=o) str. of tetrachloro phthalimide, 1624(c=o) str. of secondary amide, 1554 and 1469(c=c) str. of aromatic, 1022c-n str.v. of nh2,867out of plane aromatic bendcm-1 1hnmr (300 mhz, dmso):δ1.18 (d, 2h, ch2nco), 3 (m, h, ch-nh2), 5.57(s,2 h, nh2), 7.5 (d, h, ch-s), 7.8 (d, h, ch-n), 8.83(s, h, nh). 6e2-(2-amino-3-oxo-3-(pyrrolidin-1yl)propyl)-4,5,6,7-tetrachloroisoindoline-1,3dione white needle shape solid;yield 62%;melting point: 198-200 °c; ir (kbr), (υ,cm-1): 3433 nh str.v. of nh2, 2939and 2881 (c-h) asym. and sym.str. of ch2, 1789 and1735(c=o) str. of tetrachloro phthalimide, 1639(c=o) str. of amide, 1577nh bend of nh2, 1203c-n str.v. of nh2,740out of plane aromatic bendcm -1 1hnmr (300 mhz, dmso):δ1.16 (d, 2h, ch2nco), 3 (m, 4h, ch2-ch2), 3.2 (m, 4h, ch2nco), 3.5 (m, h, ch-nh2), 5.57(s,2 h, nh2). the fusion of amino acids with phthalic anhydride is a widely used methodology. for some amino acids good yields are obtained, but in some cases the conditions used are so drastic that racemization occurs. furthermore, amino acids with functionalized side chains failed to give the desired phthaloylated products. the extent of this racemization was limited by performing the reactions in boiling solvents and the presence of bases such as triethylamine. in such reactions, the medium should be kept neutral by slow distillation of the base and the water formed to allow cyclization of the intermediate phthalamic acids. under prolonged heating, however, partial hydrolysis of the phthaloyl derivative to the intermediate phthalamic acid was sometimes observed(27). alpha glucosidase inhibitory evaluation figure 2. alpha glucosidase inhibitory activity of compound 5a. figure 3 . alpha glucosidase inhibitory activity of compound 5f. iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition 107 figure 4. alpha glucosidase inhibitory activity of compound 6a. during the last years, considerable attention have been devoted to the creation of α glucosidase inhibitors, which can be classified into sugar mimicking and non-sugar types according to their structural features (2830).sugar-mimicking α-glucosidase inhibitors have been extensively studied, including: acarbose,miglitol and voglibosehave been clinically used to inhibit small intestinal α glucoside enzymes, such as α-glucosidase and glucoamylase (31). the synthesized compounds (5a, 5f, 6a) ic50 values: 5.18, 4.6, 7.3 respectively accordingly these compounds have excellent alphaglucosidase inhibitory activity in comparison with standard compound acarbose (ic50= 817.38 ± 6.27 m), these results could be attributed to generation new binding site with a hydrophobic pocket in the active site of enzyme, the secondary amine in 5flouro uracil ring bind by an ionic bond, aromatic rings by hydrophobic bonds while oxygen, nitrogen, chlorine and fluoride atoms bind through hydrogen bonds. conclusion the procedures for synthesis the target compounds was successfully achieved; the purity and structural formulas for the synthesized compounds were characterized and identified by melting points, rf values, ft-ir spectroscopy, 1h-nmr, 13c-nmr spectroscopy. the preliminary evaluation ofalphaglucosidase inhibitory activity of some compounds was done, which includes compounds 5a, 5f and 6a and the results indicate that these compounds have considerable antidiabetic activity with very useful affinity and ic50 values. references 1. american diabetes association. classification and diagnosis of diabetes. sec. in standards of medical care in diabetes 2016. diabetes care2016; 39(suppl.1):s13–s22 2. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care2014; 37(suppl.1): s81–s90 3. “use of glycatedhaemoglobin (hba1c) in the diagnosis of diabetes mellitus” (accessed 25 january 2016) retrieved from http:/ www.who.int. 4. joshi, s.r.; parikh, r.m.; das, a.k. insulin—history, biochemistry, physiology and pharmacology. j. assoc. physicians india2007;(55), s19–s25. 5. newsholme, p.; cruzat, v.; arfuso, f.; keane, k. nutrient regulation of insulin secretion and action. j. endocrinol. 2014;(221), r105–r120. 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[crossref] [pubmed] 9. inzucchi se, bergenstal rm, buse jb, et al. management of hyperglycemia in type 2 diabetes, 2015; a patient-centered approach: update to a position statement of the american diabetes association and the european association for the study of diabetes. diabetes care. 2015;38(1):140149. doi:10.2337/ dc14-2441. 10. handelsman y, bloomgarden zt, grunberger g, et al. american association of clinical endocrinologists and american college of endocrinology – clinical practice guidelines for developing a diabetes mellitus comprehensive care plan – 2015 — executive summary. endocrpract.2015; 21(4):413-437. doi:10.4158/ep15672.gl. 11. american diabetes association. pharmacologic approaches to glycemic treatment. sec. 8.in standards of medical care in diabetes 2017.diabetes care.2017; iraqi j pharm sci, vol.27(1) 2018 alpha glucosidase inhibition 108 40(supplement 1):s64-s74. doi : 10 .2337 / dc1 7-s011. 12. 12.kajimoto, t.; node, m. inhibitors against glycosidases as medicines. curr.top.med. chem. 2009, (9), 13−33. 13. 13.tundis, r.; loizzo, m. r.; menichini, f. natural products as alpha-amylase and alpha-glucosidase inhibitors and their hypoglycemic potential in the treatment of diabetes: an update. mini-rev.med. chem.2010 ;(10), 315−331. 14. ross, s. a.; gulve, e. a.; wang, m. h. chemistry and biochemistry of type 2 diabetes. chem. rev. 2004;(104), 1255−1282. 15. 15.chiasson, j. l.; rabasa-lhoret, m. prevention of type 2 diabetes insulin resistance and beta-cell function. diabetes2004;(53), s34−s38. 16. 16.neelottama k. ,darpan k. recent advances and future prospects of phthalimide derivatives. journal of applied pharmaceutical science, march, 2016; vol. 6 (03), pp. 159-171, doi: 10.7324/japs.2016.60330, issn 22313354. 17. 17.satzinger, g.; forsch, a. drug res.1994;(44), 261. 18. cyril o. u., didier m., johan w., gerhard k. e. synthesis and anticonvulsant activity of n,n-phthaloyl derivatives of central nervous system inhibitory amino acids. arch. pharm. pharm. med. chem.2001;(334), 323–331. 19. varala r. a facilesynthesis of biologically active phthalimides&itsanalogues -a study.indian institute of chemical technology hyberabad500 007 india march2013international e – publication. 20. 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(b) bruckner, r. “advanced organic chemistry, reaction mechanisms”; harcourt/academic: san diego; 2002; p 239. 21. mehdi s.sohrab a. synthesis of benzoxazinone derivatives: a new route to2-( n – phthaloylmethyl ) 4h -3 ,1benzoxazin-4-one. molecules 2004;(9), 705-712. issn 1420-3049. 22. ragnarsson, u.; grehn, l.”novel amine chemistry based on dmap-catalyzed acylation”. acc. chem. res.; 1998; (31), 494–501. 23. sebastian hart wig, mary m. nguyen and stefan hecht, “exponential growth of functional poly(glutamic acid) dendrimers with varying stereochemistry” the royal society of chemistry,2010. 24. khan farhan a. ;jaywant .phopase and ulrich jordis, “surprise in the lithium hydroxide hydrolysis of a nxocompound’’ ecoso-13,2009. 25. yu liu, bruno l. oliveira, joao d. g. correia,b isabel . santos, isabel s., bernhard s. and roger a., “syntheses of bifunctional 2,3-diamino propionic acid based chelators as small and strong tripod ligands for the labelling of bimolecule with 99mtc” university of zurich, institute of inorganic chemistry, winterthurerstr . 190, 8057zurich, switzerland. 26. y.-m. kim, y.-k.jeong,m.-h.wang,w.-y. lee, and h.-i.rhee,“inhibitory effect of pine extract on 𝛼-glucosidase activity andpostprandial hyperglycemia,” nutrition,2005; vol. 21, no. (6), pp. 756– 761. 27. q. zeng, z. liu, b. li, and f. wang.mild and effective n-phthaloylation of amino acids, amino acids, 2004; 27: 183–186 doi 10.1007/s00726-004-0109-1. 28. yuasa, h.; izumi, m.; hashimoto, h. thiasugars: potentialglycosidase inhibitors. curr.top.med. chem. 2009; (9), 76−86. 29. ogawa, s.; yuasa, h. hot topic: the medicinal chemistry of glycosidase inhibitors. curr.top.med. chem.2009; (9), 1−2. 30. ogawa, s.; kanto, m. design and synthesis of 5a carbaglycopyranosylamime glycosidase inhibitors. curr.top.med.chem.2009; (9), 58−75. 31. hakamata, w.; kurihara, m.; okuda, h.; nishio, t.; oku, t.design and screening strategies for alpha-glucosidase inhibitors based on enzymological information. curr.top.med. chem. 2009; (9), 3−12. in situ gelling formulation of naproxen for oral sustained delivery system iraqi j pharm sci , vol.18 (1) ,2009 oral in situ gelling formulation 35 in situ gelling formulation of naproxen for oral sustained delivery system hala s.yousif * ,1 and yehia i. khalil * * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad , iraq abstract naproxen is non-steroidal anti-inflammatory drug, which has antipyretic and anti-inflammatory effect. it is extensively bound to plasma albumin, and exhibits gastric toxicity, so it may be more efficient to deliver the drug in its sustained release dosage form and adequate blood level is achieved. three liquid formulations with in situ gelling properties have been assessed for their potential for the oral sustained delivery of naproxen . the formulations were dilute solutions of: (a) pectin; (b) gellan gum and; (c) sodium alginate, all containing complexed calcium ion that form gels when these ions are released in the acidic environment of the stomach . the viscosity of the sols and drug release were measured, and was found to be dependent on the type and concentration of the gelling agent. pectin sol shows the highest viscosity and drug release . the influence of variation of gastric ph and the effect of added 1.6 mm ca ++ ions on the gelation property and the release profile of the liquid formulations were examined. the efficiency of gelation was significantly reduced with increase of ph. in addition the influence of different concentrations of sorbitol were determined .the results showed that 10% w/v sorbitol is the best concentration that maintained fluidity and ease of administration for the selected formula . the selected formula was examined for its stability and expiration date, and, it was found that there was no evidence of physical changes under experimental conditions, with estimated expiration of about 4.1 years and ph of the formula stated at 5.1. key word: naproxen, in situ gelling, oral preparations, gel. الخالصة عقاس غ٘ش عخ٘شّٗذٕ هضاد لاللخِاباث ّ خافض للحشاسة رّ اعشاض هخذشةت للودةذة ّ مدةن غ٘ةش هغخغةا الٌابشّكغ٘ي ُْ ّ كزلك ٗشحبط بشذة هع البْهٌ٘اث البالصها , لزا قذ ٗكْى اكثش كفاءة اعطاء الدقاس علٔ شكل جشعت دّائ٘ت هذٗذة الوفدةْ للحوةْ علةٔ قةذ حةن ححل٘ةل قابل٘خِةا علةٔ الخحشٗةش الفوةْٕ الوذٗةذ ثالد حشاك٘ب عائلت رّ صفت الخحْ الٔ ُةالم فةٖ الوْقةع . هغخْٓ كافٖ هٌَ فٖ الذم الوفدةْ للٌابشّكغة٘ي. الخشاك٘ةةب كاًةج هحال٘ةةل هخففةت هةيك يا البكخةة٘ي ي جة٘الى كةةن يو ال,ٌ٘ةاث الوةْدْٗم, ّ كلِةةا ححخةْٕ علةةٔ لقةذ حةن دساعةت لضّجةت الوحلةْ عٌذها حخحةشس ُةزٍ اوًْٗةاث فةٖ الْعةط الحاهضةٖ للودةذة. الِالم اًْٗاث الكالغْ٘م الودقذة ّ الخٖ حكْى الشّٕ ّ ححشس الذّاء هٌَ, ّّجذ باًَ ٗدخوذ علٔ ًْع ّ حشك٘ض الوادة الِاله٘ت , فالوحلْ الغشّٕ للبكخة٘ي اهِةش اعلةٔ لضّجةت ّ ححةشس كةزلكهلٖ هْ علٔ خاص٘ت حكْى الِةالم ّ ٦٫١حاث٘ش اًْٗاث الكالغْ٘م الوضافت ي لقذ حن دساعت حاث٘ش اخخالف حاهض٘ت الودذة ّ للذّاء. ث٘ش ااى فدال٘ةت حكةْى الِةالم قةذ قلةج بشةكل ّااةد هةع اصدٗةاد اوط الِ٘ةذسّجٌٖ٘. باواةافت الةٔ رلةك فقةذ حةن ححذٗةذ حةة ال٘ةت ححةشس الةذّاء. ٪ ُْ افضل حشك٘ض وًَ ٗحافظ علٔ عةْ٘لت ّ عةِْلت ٦١ك٘ض الغْسب٘خْ وادة الغْسب٘خْ . فقذ اهِشث الٌخائج باى حشل الخشاك٘ض الوخخلفت ححةج ٌُةا حغ٘٘ةشاث ف٘ضٗاّٗةتاعخدوا الخشك٘بت الوخخاسة.حن دساعت ثباح٘ةت الخشك٘بةت الوخخةاسة ّ حةاسٗت اًخِةاء الوةالح٘ت ّّجةذ باًةَ لة٘ظ . ١٫٦ابخت علٔ عٌت ّ حاهض٘ت للخشك٘بت ث ١٫٦هشّف الخ,شبت هع حاسٗت اًخِاء صالح٘ت ٗقذس introduction solutions that undergo sol-gel transformation when they meet physiological conditions may serve as an in situ gelling drug delivery system (1) . in situ is a latin phrase meaning in the place. the new concept of producing a gel in situ was suggested for the first time in the early 1980s. it is widely accepted that increasing the viscosity of a drug formulation in the precorneal region will lead to an increased bioavailability, due to slower drainage (2) .gels are transparent or translucent, non-greasy, semisolid preparations. these are also termed jellies consisting of either suspensions made up of small inorganic particles, or large organic molecules interpenetrated by a liquid (3). hydrogel is three-dimensional hydrophilic polymeric networks capable of imbibing large quantities of water have generated a lot of interest recently as delivery system for pharmaceutically active agents (4) . one of the main characteristics of hydrogels is that they contain ingredients that are dispersible as colloids or are water-soluble (5) .the swelling of environmentally sensitive hydrogel can be affected by many stimulus, these are: temperature, ph, ionic concentration, electrical field, inflammation, solvent concentration, light and radiation, magnetic field and glucose concentration (6) . according to the mechanism by which sol gel phase transition occur, the following three types of systems can be recognized (7) : 1 corresponding author e-mail : drhaha1971 @ yahoo. com received : 17/6/2008 accepted : 16 / 12/2008 iraqi j pharm sci , vol.18 (1) ,2009 oral in situ gelling formulation 36 1ph triggered systems. 2temperature sensitive system. 3ion activated system. from the point of view of patient acceptability, a liquid dosage form that can sustain drug release and remains in contact for extended period of time, improving the bioavailability, reducing the dose concentration and frequency may be achieved by in situ gelling formulations (8) .gelation of the orally administered liquid formulations (ion activated system) was ensured by the inclusion of calcium ions in the formulation as a soluble complex designed to break down to release free calcium ions on encountering the acidic environment of the stomach (9) . the gelation was delayed until the orally administered solution reached the stomach by complexing the calcium with sodium citrate (10) .naproxen is a non steroidal antiinflammatory drug (nsaid) advocated for use in painful and inflammatory rheumatic arthritis, osteoarthritis, migraine, postoperative pain and postpartum pain (11) . the goal of this study is to prepare liquid formulation of naproxen with in situ gelling properties and assessed for its potential sustained oral delivery system. experimental materials and equipments naproxen, methyl paraben, propyl paraben (supplied by samarra drug industries, iraq). amrixen ® suspension (amrit medical co.). calcium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate (bdh chemical ltd.pool, england). cellulose membrane (viskase sale co., size 36/32, usa). gellan gum (dainippon pharmaceutical co., osaka). hydrochloric acid, sorbitol (riedel-de haen hannover, germany). pectin (cesalpinla food com., italy). sodium alginate (hopkin and williams ltd, england).diffusion cell (plastic dialysis cell, modified franz cell), ph meter (hanna instrument ph211, italy), uv spectrophotometer (carrywin uv, australia), viscometer (brookfield dv-ιι , england). method of preparation preparation of the sols: sodium alginate and pectin solution of concentrations 1.0, 1.5 and 2.0 % (w/v) were prepared by adding the polymer to distilled water containing 0.25% (w/v) sodium citrate and 0.075% (w/v) calcium chloride and heating to 60 ο c for sodium alginate and 40–50 ºc for pectin while stirring. naproxen equivalent to 2.5% (w/v) was then dispersed in the resulting solution after cooling to below 40 ο c (12, 13) . gellan gum solutions of concentrations 0.25, 0.5 and 1.0% (w/v) were prepared by adding the gum to distilled water containing 0.17% w/v sodium citrate and heating to 90°c while stirring. after cooling to below 40°c appropriate amounts of calcium chloride 0.016% (w/v) and 2.5% (w/v) naproxen were then dispersed in the resulting solution (12) . gelation property instantaneous gelation was checked by addition of the sols dropwise to simulated gastric fluid ph 1.2 (14) . the effect of different concentrations of calcium chloride and sodium citrate on the gelling properties: the optimum quantities of calcium chloride and sodium citrate that maintained fluidity of the formulation before administration and resulted in gelation when the formulation was added to simulated gastric fluid, were determined by preliminary tests in which pectin sols 1%, (w/v) containing sodium citrate concentrations of 0.125, 0.25 and 0.50% (w/v) and calcium chloride concentrations of 0.05, 0.075 and 0.1 % (w/v) were added dropwise to 50 ml simulated gastric fluid (ph1.2). measurement of the rheological properties of sols: the viscosity of sols prepared in water was determined at room temperature (25 ºc) with brookfield digital viscometer (14) . measurement of in vitro drug release: the release rates of naproxen were measured using plastic dialysis cells similar to that described previously by miyazaki et al (15, 9) . the capacity of each half-cell was 4 ml and the surface area of the membranes was 2.67 cm 2 .sols of pectin, gellan gum or alginate were placed in the donor compartment individually. an equal volume of simulated gastric (ph 1.2) or intestinal (ph 6.8) fluid was placed in the receptor compartment. the donor phase and the aqueous receptor phase were separated by a cellulose membrane. the assembled cell was shaken horizontally at rate of 60 strokes per min. in an incubator maintained at 37 ο c temperature. the total volume of the receptor solution was removed at intervals and replaced by fresh release medium. the drug concentration of the samples was determined using uv spectrophotometer . effect of ph and added ca ++ ion on: the gelation: the influence of ph on the gelation characteristics of 1% (w/v) pectin sols was determined by immersion of 30 ml sol enclosed in cellulose membrane tubing into simulated gastric fluid (150 ml) with ph values range 1.0–5.0. after equilibration for 24 iraqi j pharm sci , vol.18 (1) ,2009 oral in situ gelling formulation 37 hr at room temperature, the contents of the tube were passed through a sieve (no. 6.5, 2.80 mm) over a period of 30 seconds and the weight of the gel remaining in the sieve was determined by balance. the experiments were repeated in the presence of added 1.6 mm ca ++(16) . in vitro release: the effect of ph: the in vitro release of naproxen from 1.0% (w/v) pectin was measured using an equal volume of simulated gastric (ph 1.2 and 3.0) for 1 hour and intestinal (ph 6.8) fluid for 5 hours placed in the receptor compartment (17) . the effect of ca ++ : the release measurement was done at ph 3 using 1.0% (w/v) pectin sol alone and with added 1.6 mm ca ++ (16) . effect of different concentrations of sorbitol on: rheological properties: different concentrations of sorbitol (0, 5, 10, 20, 30, and 40% w/v) were added to 2.0% (w/v) pectin sols loaded with 2.5% (w/v) naproxen, and the viscosities were measured (18) . in vitro release: the in vitro release was measured for 2.0% (w/v) pectin sols loaded with 2.5% (w/v) naproxen, in presence of different concentrations of sorbitol (0, 5, 10 and 20% w/v) (18) . stability study: several glass containers (each containing 4 ml) of the selected formula were incubated at 35, 50 and 60 ο c for 90 days. samples were taken at specified time intervals and assayed for their drug content. the physical appearance and the ph of the formula were also evaluated. results and discussion gelling property in this study ca ++ ions were included in all formulations for induction of gelation. however, for ease of administration the prepared formula must be introduced in a fluid (sol) state. this was achieved by addition of sufficient sodium citrate to the formulation to form a complex with all of the ca ++ ions present in the formulation and hence to effectively remove them from solution. then, in the acidic environment of the stomach the complex is broken down and the ca ++ ions released cause gelation to occur (19) .instantaneous gelation was observed by addition of the sols of pectin, sodium alginate and gellan gum dropwise to simulated gastric fluid maintained at ph 1.2. the effect of different concentrations of calcium chloride and sodium citrate on the gelling properties: the results indicated that the minimum concentration that maintained fluidity of the sol before administration and caused gelation of sols in the gastric fluids was 0.25% (w/v) sodium citrate and 0.075% (w/v) calcium chloride. moreover, gelation occurred without exposure to simulated gastric fluid ph 1.2 in formulations containing 0.050, 0.075 or 0.1% (w/v) cacl2 and sodium citrate concentration of 0.125% (w/v) as shown in table(1). the increase in calcium chloride content to 0.10% (w/v) with the same sodium citrate concentration caused gelation of the formulation before contact with simulated gastric fluid (13) . table (1): the effect of different concentrations of sodium citrate and calcium chloride on the gelation of 1% (w/v) pectin sols before and after administration to simulated gastric fluid. sodium citrate calcium chloride 0.125% (w/v) 0.25% (w/v) 0.5% (w/v) 0.05% (w/v) gel before administration sol before administration friable and soft gel after administration sol before administration friable and soft gel after administration 0.075% (w/v) gel before administration sol before administration optimal gel strength after administration sol before administration low gel strength after administration 0.1% (w/v) gel before administration gel before administration gel before administration iraqi j pharm sci , vol.18 (1) ,2009 oral in situ gelling formulation 38 rheological properties of the sols: all the prepared sols revealed that the viscosity is increased as a function of increasing polymer concentration with shear thinning behavior.the rheogram profiles of different polymers used in this study, suggested that pectin sols used is more accepted one than other two polymers (gellan gum and sodium alginate), since pectin sols exhibited more or less fit profile with that obtained from commercial one amrixen ® suspension as a reference product. moreover, the results indicated that best concentration required for incorporating pectin as a gelling agent is 1.5% (w/v) and to a lesser extent for 1.0% (w/v) concentration. dissolution behavior (in vitro release): large increment in the amount released of naproxen observed when the receptor solution was changed from simulated gastric fluid ph (1.2) to simulated intestinal fluid ph (6.8).this was expected since there will be change in the state of ionization of the acidic drug (pka of naproxen is 4.2) accompanying the ph range. it is completely unionized at ph 1.2 and this lead to negligible drug release at this ph (20) .rigid gels are formed when the donor solutions of all systems are placed in contact with a receptor solution at ph 1.2 and, as a consequence, the amount of the drug released is lower than that at ph 6.8, which is referred to the high h + ion concentration at ph 1.2 that is sufficient to cause the formation of rigid gels (21) . there was a significant decrease in the release rate with increasing polymer concentration. this behavior may be attributed to the effect of mechanical barrier that set up by the random network of the polymer gel molecules which binds and entraps surrounding water. this aqueous phase in the polymer network acts as the region responsible for diffusion of the drug in the gel. the change of the polymer concentration of these gels could affect the diffusion pathway and thus the drug release (22) . in addition, as the viscosity of the polymer sols increased with concentration, the solvent penetration into the core of the matrix will be decreased, and the drug release will be decreased (23) . in an attempt to verify the effect of polymer types on the release of naproxen, the cumulative release profiles of 2.5% (w/v) naproxen from 1% (w/v) different gelling polymers were constructed as shown in figure (1). 0 10 20 30 40 50 60 0 1 2 3 4 5 6 7 time (hr.) c u m u la ti v e % d r u g r e le a s e d p ectin sod.alginate gellan gum figure (1): cumulative in vitro release of naproxen (2.5% w/v) from 1% (w/v) concentrations of pectin, sodium alginate and gellan gum gels. the results obtained indicated that the release of naproxen from different types of polymers was in the following order: pectin > sodium alginate >gellan gum. this suggests that the choice of the polymer base is of obvious importance for achieving a desired drug release.the explanation for this related to the diffusivity of the drug through any base depends on the nature and composition of individual base and the drug-vehicle interaction. also, the solubility of the drug in the vehicle affects the drug release and diffusion (24) .the release data over the whole time period were analyzed according to the treatment proposed by higuchi for drug release from semisolid vehicles (25) . for the initial cumulative drug released 50-60%, the amount "q" of drug released per unit surface area from gel is proportional to the square root of time: q = 2 co ( d t / π ) 1/2 in which q is the amount of drug released per unit area; cο the initial drug concentration in the vehicle; d is the diffusion coefficient of the drug in the matrix and t is the time.plots of q versus t 1/2 for the release of naproxen from all gels were linear after a short lag period indicative of diffusion controlled release (26) as shown in figure (2). there is usually a lag period until water permeates the polymer mass to create pores for diffusion of the drug .later, the drug released (27) . iraqi j pharm sci , vol.18 (1) ,2009 oral in situ gelling formulation 31 0 5 10 15 20 25 1 1.5 2 2.5 3 hfdfd c u m u la ti v e % d ru g r e le a s e d pectin sod. alginate gellan gum figure (2): cumulative % released of naproxen as a function of square root of time from 1% (w/v) concentrations of pectin, sodium alginate and gellan gum gels. effect of ph and added ca ++ ion on: the gelation: the results show that the hydrogen ion concentration at ph 1.0–2.5 was sufficiently high to cause gelation in the absence of an additional source of calcium. visual observation showed well-defined compact gels over this ph range. although complete gelation was observed at ph 2.53.5, the resultant gels were not sufficiently strong to maintain their cylindrical form. however, at higher ph (ph >3.5) and when h + ions are insufficient, the effective breakdown of the calcium complex in the sols and gelation was poor. figure (3) showed that the addition of 1.6 mm ca ++ ions was sufficient to cause almost complete gelation of formulations over the entire ph range examined. the gels formed at ph > 2 had a loose, less structured appearance. 0 5 10 15 20 25 30 35 1 1.5 2 2.5 3 3.5 4 4.5 5 ph w e ig h t o f g e l fo r m e d ( g ) without calcium with calcium figure (3): the effect of ph and added 1.6 mm ca ++ ions on the weight of gel formed from 30 ml solutions (1.0% (w/v) pectin as a function of ph. each value is the mean ± s.e of 3 determinations. in vitro release: the effect of ph: the release of drug was appreciably faster when pectin was exposed to receptor solutions at ph 3.0 over the initial 1 h release period as shown in figure (4). observation of the donor cells showed that the formulations were in sol form throughout the duration of the release period. diffusion of h + ions from the receptor solution at this ph was insufficient to cause the release of complexed calcium ions and consequently gelation of the pectin was incomplete (17) . 0 5 10 15 20 25 30 35 40 45 50 0 1 2 3 4 5 6 7 time (hr.) c u m u la ti v e % d r u g r e le a se d starting ph 1.2 starting ph 3 figure(4): the effect of starting ph in the receptor solution on the release of naproxen from 1.0% (w/v) pectin gels. -the effect of added ca ++ : the influence of added 1.6 mm ca ++ in the formula on drug release from pectin formulations exposed to receptor solutions at ph 3.0 is shown in figure (5). observations of the donor cells showed the presence of a thin gel layer on the surface of the cellulose membranes when calcium was included in the formulation but no gelation of the bulk of the sol (16) . effect of different concentrations of sorbitol: high concentration of pectin sol was used to study the effect of different concentrations of sorbitol, since it could withstand the effect of high concentrations of sorbitol. rheological properties: figure (6) shows the influence of sorbitol concentration on the flow properties of 2% (w/v) pectin sol. the viscosity of the pectin sols increased appreciably as the sorbitol concentration was increased from 5 to 40% (w/v).addition of 5% and 10% (w/v) sorbitol to the sols caused a reduction of viscosity at all time 1/2 (h 1/2 ) iraqi j pharm sci , vol.18 (1) ,2009 oral in situ gelling formulation 31 shear rates. these changes in viscosity resulting from sorbitol addition, since it is hygroscopic and may withdrawn water to the gel structure that decreases viscosity and hence improving the ease of swallowing of the sols (28) .a considerable increase of viscosity was noted with sorbitol concentrations between 20% and 40% (w/v), all formulations exhibiting a change of flow properties from shear thinning to newtonian behavior (18) .sorbitol at higher concentrations binds with water molecules causing desolvation around the pectin chains and minimizing the hydrogen bonding of water molecules to pectin chains. as a consequence, pectin chains cross-linked together and result in increased viscosity (18) . 0 5 10 15 20 25 30 35 40 0 1 2 3 4 5 6 7 ti me (h r.) c u m u la ti v e % d r u g r e le a se d without ca with ca figure (5): effect of added ca ++ ions on the release of naproxen from 1.0% (w/v) pectin gels. 0 20 40 60 80 100 120 0 20 40 60 80 100 she ar rate v is c o si ty ( c p ) 0% (w /v) 5% (w /v) 10% (w /v) 20% (w /v) 30% (w /v) 40% (w /v) figure(6): effect of sorbitol concentration on the viscosity of 2.0% (w/v) pectin sols loaded with 2.5% (w/v) naproxen at 25 ºc. in vitro release: the release profiles of naproxen from gels of 2% (w/v) pectin containing sorbitol concentrations over the range 0–20% (w/v) was shown in figure (7). for gels containing 20% (w/v) sorbitol there was a pronounced increase of release after about 3 hours. no such inflection was observed for gels formed in the presence of 0, 5 and 10% (w/v) sorbitol. observation of the contents of the donor cell during release measurements showed that the inflection in the plots for release from the formulation containing 20% (w/v) sorbitol coincided with a gel to sol transition, i.e. low gel strength to withstand a large decrease of hydrogen ion concentration; gels formed in formulations containing lower sorbitol contents retained their integrity throughout the measurement period (14) . 0 10 20 30 40 50 60 70 0 1 2 3 4 5 6 7 time (hr.) c u m u la ti v e % d ru g r el ea se d 0% (w /v) 5% (w /v) 10% (w /v) 20% (w /v) figure (7): effect of sorbitol concentration on the release of naproxen from 2.0% (w/v) pectin gels loaded with 2.5% (w/v) naproxen. stability study of selected formula (prediction of expiration): the final formula of this study was introduced into an exaggerated temperatures study maintained at (35, 50 and 60 ºc) to predict expiration date. r/ naproxen 2.5 gm pectin 1.5 gm sodium citrate 0.25 gm cacl2 0.075 gm sorbitol 10 gm methyl paraben 0.2 gm propyl paraben 0.02 gm dis. water up to 100 gm the degradation of naproxen in this formula followed first order kinetics since (s -1 ) iraqi j pharm sci , vol.18 (1) ,2009 oral in situ gelling formulation 31 straight lines were obtained by plotting the logarithm of the percent remaining of naproxen versus time as shown in figure (8). the first-order reaction equation: log c = log cο – k1 t / 2.303 where cο is the initial concentration of naproxen, c is the remaining concentration at time t and k1 is the first order rate constant. the slope of the line is – k1 /2.303 from which the rate constants obtained. table (2) shows the degradation rate constants of naproxen at different temperatures . to determine the expiration date (t10 %), arrhenius plot was constructed to predict the degradation rate constant of naproxen at 25 ºc as shown in figure (9).the expiration date of naproxen in the suggested formula was calculated according to the first order reaction equation: t10 % = 0.105 / k 25 ºc the expiration date was found to be equal to 4.1 years with ph of 5.1 for whole the period. 1.975 1.98 1.985 1.99 1.995 2 2.005 0 10 20 30 40 50 60 70 80 90 100 time (days) l o g p e r c e n t r e m a in in g o f n a p r o x e n 35 c 50 c 60 c figure (8): accelerated stability study of naproxen in the selected formula at elevated temperatures (35, 50 and 60 ºc). table (2): degradation rate constants (k) of naproxen sol at different temperatures. temperature 35 ºc 50 ºc 60 ºc 25 ºc k (day) -1 ×10 -4 1.545 3.97 6.91 0.695 0.01 0.1 1 2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 3.35 3.4 1/t × 10 -3 (kelvin -1 ) k × 1 0 -3 (d a y -1 ) figure (9): arrhenius plot for expiration date estimation of naproxen in the selected formula. conclusions based on the results obtained, the optimum concentration of calcium chloride was 0.075% (w/v) and sodium citrate was 0.25% (w/v) for in situ gelling formulations of naproxen. the gelation, viscosities of the sols and the in vitro release of naproxen from the gels were affected by the type and concentration of the gelling agent, initial loading of naproxen, and concentration of sorbitol in the formula, gastric ph and added ca ++ ions. the most promised selected formula was pectin-gel type, with stable physical properties maintained at ph 5.1 and 4.1 years shelf life. references 1. 1haglund o., joshi r. and himmelstein k.j., an in situ gelling system for parenteral delivery. j.cont.rel. 1996, 41, 229-235. 2. vadnere m., amidon g., lindenbaum s. and haslam j.l., thermodynamic studies on the gel-sol transition of some pluronic polyols. int. j. pharm. 1984, 22, 207 – 218. 3. escobar-chávez j.j., lópez-cervantes m., naïk a., kalia y.n. et al., applications of thermoreversible pluronic f -127 gels in pharmaceutical formulations. j. pharm. pharmaceut. sci. 2006, 9 (3), 339-358. 4. bos g.w., jacobs j.l., koten j.w., tomme s.v. and veldhuis t., in situ crosslinked biodegradable hydrogel loaded with il-2 are effective tools for local il-2 therapy. eur j. of pharma. sci. 2004, 21, 561–567. 5. mukherjee d. and banthia a.k., preparation of adrenochrome hydrogel ْ º ْ iraqi j pharm sci , vol.18 (1) ,2009 oral in situ gelling formulation 42 patch, gel, ointment, and the comparison of their blood coagulating and wound healing capability. materials and manufacturing processes 2006, 21, 297– 301. 6. weller r.e., lind m.a., fisher d.r. and anna r., stimulus sensitive gel with radioisotope and methods of making. united states patent 2005, 6,869,588. 7. srividya b., cardoza r.m. and amin p.d., sustained ophthalmic delivery of ofloxacin from a ph triggered in situ gelling system. j. controlled release 2001, 73, 205–211. 8. cohen s., lobel e., trevgoda a. and peled y., a novel in situ forming ophthalmic drug delivery system from alginate undergoing gelation in the eye. j.controlled release 1997, 44, 201-208. 9. kubo w., itoh k. and miyazaki s., oral sustained delivery of theophylline and cimetidine from in situ gelling pectin formulations in rabbits. dr. dev. ind. pharm. 2005, 31, 819–825. 10. himmelstein, kenneth j. baustian and cara l. , reversible gel-forming composition for sustained delivery of bioaffecting substances, and method of use. u. s. patent 1997, 5599534. 11. mosby's drug consult, elsevier science, london, philadelphia, 2002, p.ιιι 2005. 12. miyazaki s., kawasaki n., kubo w., endo k. and attwood d., comparison of in situ gelling formulations for the oral delivery of cimetidine. int. j. pharm. 2001, 220, 161–168. 13. kubo w., konno y., miyazaki s. and attwood d., in situ gelling pectin formulations for oral sustained delivery of paracetamol. dr. dev. ind. pharm. 2004, 30 (6), 593–599. 14. kubo w., miyazaki s., dairaku m., togashi m. et al., oral sustained delivery of ambroxol from in situ-gelling pectin formulations. int. j. pharm. 2004, 271, 233–240. 15. miyazaki s., nakamura t., yokouchi c. and takada m., effect of pluronic gels on the rectal absorption of indomethacin in rabbits. chem. pharm. bull. 1984, 32, 1243–1248. 16. itoh k. et al., in situ gelling pectin formulations for oral drug delivery at high gastric ph. int. j. pharmaceut. 2006, article in press, doi:10.1016/j.ijpharm.2006.10.042. 17. itoh k., kubo w., fujiwara m., hirayama t. et al., the influence of variation of gastric ph on the gelation and release characteristics of in situ gelling pectin formulations. int. j. pharm. 2006, 312, 37–42. 18. miyazaki s., kubo w., itoh k., konno y. et al., the effect of taste masking agents on in situ gelling pectin formulations for oral sustained delivery of paracetamol and ambroxol. int. j. pharm. 2005, 297, 38– 49. 19. sungthongjeen s., sriamornsak p., pitaksuteepong t. et al., effect of degree of esterfication of pectin and calcium amount on drug release from pectin-based matrix tablets. aaps pharm.sci.tech. 2004, 5 (1) article 9. 20. kubo w., miyazaki s. and attwood d., oral sustained delivery of paracetamol from in situ-gelling gellan and sodium alginate formulations .int. j. pharm. 2003, 258, 55–64. 21. miyazaki s., aoyama h., kawasaki n. and kubo w., in situ-gelling gellan formulations as vehicles for oral drug delivery. j. cont. rel. 1999, 60, 287–295. 22. narendra c., srinath m.s. and babu g., optimization of bilayer floating tablet containing metoprolol tartrate as a model drug for gastric retention. aaps pharm.sci.tech. 2006, 7 (2) article 34. 23. dorożyński p., kulinowski p., jachowicz r. and jasiński a., development of a system for simultaneous dissolution studies and magnetic resonance imaging of water transport in hydrodynamically balanced systems: a technical note. aaps pharm.sci.tech. 2007, 8 (1) article 15. 24. paulsson m. and edsman1 k., controlled drug release from gels using lipophilic interactions of charged substances with surfactants and polymers. j. coll. interface sci. 2002, 248, 194–200. 25. higuchi w.i., the analysis of data on the medicament release from ointments. j. pharm. sci. 1962, 51, 802–804. 26. farinha a., toscano c., campos r. et al., permeation of naproxen from saturated solutions and commercial formulations through synthetic membranes. dr. dev. ind. pharm. 2003, 29(4), 489–494. 27. ravivarapu h.b., moyer k.l. and dunn r.l., sustained activity and release of leuprolide acetate from an in situ forming polymeric implant. aaps pharm.sci.tech. 2000, 1(1) article 1. 28. kathleen parfitt: martindale: the complete drug reference, 2007, 35th ed., vol.1, p. 62, vol. іі, p.1354. iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact doi: https://doi.org/10.31351/vol27iss2pp135-149 135 preparation and in-vitro evaluation of clopidogrel bisulfate liquisolid compact amenah m. mohammed*,1 and entidhar j. alakkam** *department of pharmaceutics, college of pharmacy, university of tikrit, salah alden. iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad. iraq. abstract liquisolid (ls) compact is the most promising technique for increasing dissolution rate and consequently the bioavailability of poorly soluble drugs. clopidogrel is an oral antiplatelet used for treatment and prophylaxis of cardiovascular and peripheral vascular diseases related to platelets aggregation. clopidogrel has low solubility at high ph media of the intestine and low oral bioavailability (about 50%). the purpose of this work was to enhance the dissolution pattern of the clopidogrel through its preparation as liquisolid compact. a mathematical model was used to calculate the optimum quantities of tween 80 as nonvolatile liquid vehicle, avicel ph 102 as carrier material and aerosil 200 as coating material needed to prepare acceptably flowing and compactible powder mixtures. the liquisolid compacts were evaluated for hardness, friability, weight variation, content uniformity, and disintegration time and in-vitro drug release rate. in addition, differential scanning calorimetry (dsc), fourier transforms infrared spectroscopy (ftir), x-ray diffraction (xrd) and scanning electron microscopy (sem) were used for the assessment of the physicochemical properties of the drug and compatibility with excipients in liquisolid compacts. twelve formula were prepared and the selected formula (ls-2) containing (50% w/w) clopidogrel in tween 80 at the excipient ratio (r) of (20:1). compact of (ls-2) released (92.2%) of drug during the first 10 minutes compared to (13.6%) of the directly compressible tablet and (24.2%) of marketed tablet (plavix®). in conclusion, the dissolution rate of clopidogrel can be enhanced to a great extent by liquisolid technique in comparison to conventional tablets. keywords: liquisolid compact, clopidogrel bisulfate, tween 80, dissolution rate. السائل المصلبدوكريل بايسلفيت يتحضير وتقييم خارج الجسم لمضغوط الكلوب **و أنتظار جاسم العكام 1،*آمنة مصطفى محمد العراق. ،صالح الدين، جامعة تكريت ،كلية الصيدلة ، فرع الصيدالنيات * .فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد، العراق** ةالخالص وبيدوكريل الذوبان. يعتبر الكلوالتوافر الحيوي للألدوية قليلة الذوبانمعدل دة لزيا واعدة تقنية أغلب هيمضغوط السائل المصلب واألمراض يةالمحيط يستخدم للعالج والوقاية من أمراض القلب الوعائية بايسلفيت من مثبطات تجمع الصفائح الدموية المعطاة عبر الفم الذي يبلغ ووسط القاعدي العالي لألمعاء وله توافر حيوي قليل في الالكلوبيدوكريل عقار قليل الذوبانية بتجمع الصفيحات الدموية. تعلقةالم شكل ب تحضيرهلعقار الكلوبيدوكريل من خالل الذوبان . الغرض من هذه الدراسة هو تحسين معدلالجرعات الفمويةبعد (50% )حوالي متطاير كواسطة نقل(، أفيسيل بي )سائل غير 80تم أستخدام نموذج رياضي لحساب الكميات المثلى من توين سائل مصلب. مضغوط قييم مضغوطات)كمادة مغلفة( العداد خليط مسحوق يتدفق بشكل مقبول وقابل للكبس. تم ت 200لة( واالروسيل حاممادة ك) 102أج باالضافة .السائل المصلب من حيث الصالبة، الهشاشية، اختالف الوزن، توحيد المحتوى، وقت التفكك ومعدل تحرر الدواء خارج الجسم )أكس أر حيود أشعة أكس )أف تي أي أر(، األشعة تحت الحمراءطيف )دي أس سي(، أستخدام المسح الحراري المقارن الى ذالك، تم لتقييم الخصائص الفيزيوكيميائية للدواء والتوافق مع السواغات في مضغوطات السائل )أس أي أم( األلكتروني يالمجهرالمسح و دي( . (20:1ونسبة سواغات ) 80وزن/وزن كلوبيدوكريل في توين %50محتوية على 2والتركيبة المختارة تركيبة 12تم تحضير .المصلب %13.6دقائق األولى مقارنة مع األقراص المكبوسة مباشرة 10 ال خاللمن محتواه الدوائي %92.2 ( 2مضغوط )االل أس حرر تحسين معدل اذابة العقار السائل المصلب قد ادت الى مضغوطفأن تقنية ال ،وفي النهاية .%24.2 )بالفكس( تجاريآ واألقراص المتداولة قد يؤدي الى زيادة توافره الحيوي.مما الذوبان.معدل ،80توين ،كلوبيدوكريل بايسلفيت، ائل مصلبس مضغوطحية: الكلمات المفتا 1corresponding author e-mail: amina8679@gmail.com received: 21 / 8 / 2018 accepted: 3 / 11 / 2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp135-149 mailto:amina8679@gmail.com http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 136 introduction oral delivery of 50 % of the drug compounds is hampered because of the high lipophilicity of the drug itself (1). nearly 40 % of new drug candidates exhibit low solubility in water, which leads to ineffective absorption, low bioavailability, and therapeutic failure (2). there are various techniques available to improve the solubility of poorly soluble drugs. these include micronization, solid dispersions, complexation with βcyclodextrins, solubilization by surfactants, and liquisolid technique which is the most promising and novel technique developed by spireas et al. to improve the dissolution rates of the poorly water soluble drugs (3). liquisolid systems are acceptably flowing and compressible powdered forms of liquid medications. the ‘liquid medication’ involves oily liquid drugs and solutions or suspensions of water insoluble solid drugs carried in suitable nonvolatile solvent systems termed liquid vehicles. liquid medication converted into a dry-looking, non-adherent, free flowing and readily compressible powder by a simple blending with selected powder excipients referred to as carrier and coating materials. various grades of cellulose, starch and lactose may be used as the carriers, whereas very fine particle size silica powders may be used as the coating materials (4). liquisolid compacts of poorly soluble drugs containing a drug solution or drug suspension in a solubilizing vehicle show enhanced drug release due to an increased surface area of drug available for release, an increased aqueous solubility of the drug, and an improved wettability of the drug particles. accordingly, this may result in a higher drug absorption and improved oral bioavailability (5). this technique was successfully used to improve the solubility and dissolution rate of several poorly water soluble drugs as naproxen, famotidine, carbamazepine, piroxicam, indomethacin, refecoxib, hydrocortisone and prednisolone (6). clopidogrel bisulfate is methyl (+)-(s) – α– (2chlorophenyl) – 6 ,7 – dihydrothieno [3 , 2 – c] pyridine –5 (4h) –acetate sulfate (1:1). it is white to off–white powder. it is practically insoluble in water at neutral ph; freely soluble in buffer (ph 1) (7). it belongs to class ii according to biopharmaceutics classification system (bcs). it has low bioavailability (about 50%) (8). the pka value of clopidogrel is (4.56) (9). the absorption of drug is largely dependent upon diffusion , which varies with ph of the individual regions within the gastrointestinal tract (10). the active metabolite of clopidogrel selectively inhibits platelet aggregation. it is used in the prevention of ischemic stroke, myocardial infarction, unstable angina and peripheral arterial diseases (11). the aim of this work was to improve the solubility and dissolution rate of clopidogrel bisulfate in phosphate buffer at ph 6.8 which was similar to intestinal media via liquisolid technique. this may subsequently, enhance its oral bioavailability. materials and methods materials clopidogrel bisulfate standard powder was supplied from pioneer pharmaceutical company, iraq, sulaimaniah) as a gift, clopidogrel 75mg tablets (plavix®, sanofi aventis, france), microcrystalline cellulose (avicel ph 102) (thomas baker, india), colloidal silicon dioxide (aerosil 200) (evonik degussa corp, germany), sodium starch glycolate (ssg) (asg, india), tween 80 and polyethylene glycol (peg 400) (bdh chemical ltd, uk), propylene glycol (pg) (thomas baker, india), glycerin (romil , uk) and sodium lauryl sulfate was supplied from sdi, samarra, iraq) as a gift . all other reagents and chemicals were of analytical grade. methods solubility studies solubility studies of clopidogrel bisulfate were carried out in 0.1n hcl (ph 1.2), phosphate buffer (ph 6.8) alone and with 1% sodium lauryl sulfate (sls), tween 80, propylene glycol (pg), polyethylene glycol (peg 400), glycerin and distilled water. saturated solutions were prepared by adding an excess amount of drug to the vehicles and shaking in a shaker water bath for 48 hr at 25±0.5 °c under constant vibration. after this period the solutions were filtered, diluted and analyzed by uv spectrophotometer (shimadzu,japan) at λmax 220 nm. mathematical model for design of liquisolid systems the formulation design of liquisolid systems was done in accordance with new mathematical model described by spireas et al. clopidogrel bisulfate was dispersed in tween80 (tween80 was used as liquid vehicle to prepare liquid medication). avicel ph 102 and aerosil 200 were used as the carrier and coating materials, respectively. the concentration of the drug in the liquid vehicle were (40, 50, 60 and 70% w/w) and the carrier: coating ratio were (r = 20:1, 15:1 and 10:1). flowable liquid retention potential (φ value) of powder excipients was used to calculate the required iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 137 ingredient quantities. in tween 80, the φ-value was (0.16) for the carrier avicel ph 102 and (3.33) for the coating material aerosil 200 (12). the liquid load factor (lf) was computed from the φ-value of the carrier and coating materials with different ratio (r) in accordance to equation (1): lf = φ cr + φ co (1/r) ----------(1) where, φ cr and φ co are the flowable liquid retention potentials (φ -values) of carrier (avicel ph 102) and coating (aerosil 200) powder materials, respectively. however, liquid load factor (lf) is defined as the ratio of the weight of liquid medication (w) to the weight of the carrier powder (q) in the system, which should be possessed by an acceptably flowing and compressible liquisolid system. the most suitable quantities of carrier (q) were calculated using equation (2): lf = w / q ----------- (2) the optimum quantities of coating material (q) were obtained from equation (3): r = q / q ----------- (3) where, r is the ratio by weight of carrier (q) and coating (q) materials present in the formulation (13, 14). preparation of clopidogrel bisulfate liquisolid compact liquisolid compacts (ls) represented by formula 1 – 12, each containing 98 mg of clopidogrel bisulfate (equivalent to 75mg clopidogrel). different drug concentrations in tween 80 (40, 50, 60 and 70% w/w) were prepared by dispersing the drug in the nonvolatile vehicle (tween 80). also, a bindery mixture of the carrier (avicel ph 102) and coating material (aerosil 200) was prepared at a ratio r of (20:1, 15:1 and 10:1). then after, it was mixed with the liquid medication. the mixing process was carried out in three stages. in the first stage, the binary powder mixture was blended with liquid medication using a porcelain mortar with the aid of a pestle at a mixing rate of one rotation per second for approximately one minute in order to evenly distribute the liquid medication into the powder. in the second mixing stage, the liquid/powder admixture was evenly spread as a uniform layer on the surfaces of the mortar and was left standing for approximately ten minutes to allow the liquid medication to be absorbed in the interior of the powder particles, and then saturation adsorption occurred on the surface of these particles. in the third stage, sodium starch glycolate (ssg) as a super-disintegrant was added at 5% w/w and mixed for 10 minutes. the final mixture was compacted using a single punch-tablet machine (korsch eko, germany)(15,16). the composition and characteristics of liquisolid compact were demonstrated in the table 1. table 1. composition and characteristics of clopidogrel compact (ls) and dct. formula number drug (mg) drug conc. in tween 80 (% w/w) carrier: coating ratio (r) liquid loading factor (lf) liquid vehicle (tween 80) (mg) carrier (avicel ph 102) (mg) coating (aerosil 200) (mg) superdisntegrant (ssg) (mg) compact weight (mg) ls-1 98 40 20 0.326 147 751.5 37.5 51.7 1085 ls-2 98 50 20 0.326 98 601.2 30 41.3 868.5 ls-3 98 60 20 0.326 65.3 500.9 25 34.4 723.6 ls-4 98 70 20 0.326 42 429.4 21.4 29.5 620.3 ls-5 98 40 15 0.379 147 646.4 43 46.7 981.2 ls-6 98 50 15 0.379 98 517.1 34.4 37.3 784.8 ls-7 98 60 15 0.379 65.3 430.8 28.7 31.1 653.9 ls-8 98 70 15 0.379 42 369.3 24.6 26.6 560.5 ls-9 98 40 10 0.493 147 496.9 49.6 39.5 831.1 ls-10 98 50 10 0.493 98 397.5 39.7 31.6 664.9 ls-11 98 60 10 0.493 65.3 331.2 33.1 26.3 553.9 ls-12 98 70 10 0.493 42 283.9 28.3 22.6 474.8 dct 98 20 601.2 30 41.3 770.5 iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 138 preparation of directly compressed tablets (dct) compressed tablet containing 98 mg of clopidogrel bisulfate was prepared with direct compression method without the addition of any non-volatile liquid vehicle (the same composition of the selected ls compact but without tween 80). the drug powder was mixed with suitable amounts of avicel ph102 and aerosil 200. afterwards, 5% of ssg was added and mixed for 10 minutes. the final blend was compressed using (korsch, germany) tablet machine (17). pre-compression studies of the prepared liquisolid powder system flow properties of liquisolid system angle of repose (θ) the angle of repose was determined by fixed funnel method. the height of the funnel was adjusted in such a way that the tip of the funnel just touches the apex of the heap of the powder. accurately, weighed blend is allowed to pass through the funnel freely on the surface. the angle of repose (θ) was calculated using the following equation (18). tan θ = h / r ------(4) where, θ is the angle of repose, h is the height of pile in cm and r is the radius of pile in cm. depending on the value of angle of repose, the flowability of liquisolid system were evaluated (19). carr's index and hausner's ratio the bulk and tapped densities were used to calculate the carr’s index and the hausner’s ratio of powder according to the equations (20): carr’s index = (tapped density –bulk density) × 100 / tapped density -----(5) hausner ratio = tapped density / bulk density -------(6) bulk density = wt / vo -----(7) tapped density = wt / vf -----(8) where, wt is the weight of powder, vo is the bulk volume of powder and vf is the tapped volume of powder. system with compressibility value (carr’s index) greater than 20-21 % has been found to exhibit poor flow properties. in addition, the system has good flowability when hausner’s ratio is lower than 1.2 while, if the ratio is more than 1.2 this indicates that the flowability is bad (21). differential scanning calorimetry analysis thermal behavior of pure clopidogrel, avicel ph 102, powder mixture of conventional (dct) and liquisolid (ls) compact were attained by dsc. samples (5 mg) were placed in an aluminum pan and heated in the dsc-60 (shimadzu, japan) at a constant rate of 10 °c/min, in an atmosphere of nitrogen over a temperature range of 25-300 °c (22). fourier transforms infrared spectroscopy (ftir) each of pure clopidogrel bisulfate, avicel ph 102, powder mixture for conventional and liquisolid samples of 2-3 mg were mixed with about 100 mg of dry potassium bromide powder and compressed into transparent discs then scanned over a range of 4000-400 cm-1 using the infrared spectrophotometer (lambda 7600, australia) (23). x-ray diffraction (xrd) the (xrd) patterns were determined for pure drug, excipients used in formulation (avicel ph 102), physical mixture of drug and excipients (dct) and finally, for the prepared liquisolid system (ls compact). the operating conditions were; voltage 40 kv, current 30 ma and scanning speed 8.000 (deg /min.) (24). scanning electron microscopy (sem) study scanning electron microscopy (sem) is utilized to assess the morphological characteristics of the pure drug and the liquisolid system. samples were first loaded on sample stub using double side carbon tape then coated with gold and examined in the zeiss supra 55 vp scanning electron microscope (25). post-compression studies hardness the hardness of formulated liquisolid compacts was assessed using a pharma test hardness tester and the mean hardness of three tablets ± standard deviation (sd) was determined. the hardness was expressed as a force in kg/cm2 required to crush the compact (26). friability pharma test friabilator was used in this study by taking 20 liquisolid compacts from each formula. these compacts were weighed accurately (wt initial) then after, rotated in the friabilator for 4 min at 25 rpm. the compacts were re-weighed (wt final). the friability was calculated as a percentage according to equation (27). friability % = [(wt initial – wt final) / wt initial] × 100 ------(9) the acceptable friability value is up to 1%. weight variation test twenty compacts were taken and their weight was determined individually and collectively on a digital weighting balance (precisa instruments ltd, switzerland). the average weight of the compact was determined from the collective weight and comparing the individual tablet weight to average weight variation tolerance according to british pharmacopoeia(28) . iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 139 content uniformity this test was carried out by applying usp method. ten compacts were individually assayed for their content. each compact was grinded and the powder placed in 50ml of 0.1n hcl, sonicated for 5min. and cooled. then, transfer 5ml of this solution to volumetric flask, dilute with 0.1n hcl to 50 ml. then after, filter and discard the first 5ml of filtrate. after that, the amount of clopidogrel was determined spectrophotometrically by measuring the absorbance at appropriate λmax. the percent of content uniformity for each compact was calculated and compared with the mean for each formula according to the usp specification(39). disintegration time study the in-vitro disintegration studies were carried out using tablet disintegration test apparatus (pharma test, germany). one compact was placed in each of the six tubes of the basket assembly and disk was added to each tube. this assembly was then suspended in one-liter beaker containing 0.1n hcl ph (1.2) maintained at 37±2⁰c. the basket was then moved up and down through a distance of 5 to 6 cm at a frequency of 32 cycles per min. the time required for complete disintegration of the compact was recorded (30). in-vitro dissolution test the test was studied in usp type-ii dissolution apparatus (pharma test, germany) employing a paddle stirrer at speed of 50 rpm. in this study 900 ml of simulated intestinal fluid (sif) (phosphate buffer ph 6.8 with 1% sls) or simulated gastric fluid (sgf) (0.1n hcl ph 1.2) was used as a dissolution media. the temperature of dissolution media was maintained at 37 ± 0.5°c throughout the experiment. samples of (5 ml) were withdrawn at the predetermined intervals of the time (5, 10, 20, 30, 60 and 90 min.). then after, filtered through a 0.45 μm filter and analyzed for drug release by measuring the absorbance at λmax of drug using uv-visible spectrophotometer (shimadzu, japan) .the volume withdrawn at each time-interval was replaced with the equal volume of fresh dissolution media to maintain sink condition and constant volume. each preparation was tested in triplicate and the mean value of reading was calculated (31). dissolution parameters like mean dissolution time (mdt) and percent dissolution efficiency (% de) were applied for comparison of dissolution profiles to select the best formulation (32). statistical analysis all the results were expressed as the mean ± sd. one way analysis of variance (anova) was used to test for significance at a 5% significance level. so, that, statistical difference dealing with (p <0.05) was considered significant. results and discussions saturation solubility the solubility of clopidogrel in different solvents was given in table 2. clopidogrel exhibited the highest solubility in tween 80 (55.92± 2.33mg/ml). since, the aim of this study was to increase the dissolution rate of clopidogrel, tween 80 was exploited as a nonvolatile solvent in preparation of liquisolid systems. table 2. solubility of clopidogrel bisulfate in various solvents. n=3 pre-compression evaluation of the prepared liquisolid system flow properties of liquisolid system the flowability of a powder is of critical importance in the production of pharmaceutical dosage forms to reduce high dose variations. the angle of repose (θ) is a characteristic of the internal friction or cohesion of the particles. in addition, carr’s index is a measure of the propensity of a powder to be compressed. also, the flowability of a system is represented by its hausner’s ratio. the results were demonstrated in table 3. it was shown that most ls formulas with high excipient ratio (r = 20) exhibited good flowability. these results related to the presence of high amount of avicel (carrier) and lower amount of aerosil (coating). however, avicel has excellent flow properties while, aerosil is a fluffy powder due to its low density (0.05g/ml) (33). solvents solubility (mg/ml) mean ± s.d 0.1n hcl (ph 1.2) 37.36 ± 1.64 phosphate buffer (ph 6.8) 0.84 ± 0.03 phosphate buffer (ph .8) + 1% sls. 3.92 ± 0.49 tween 80 55.92 ± 2.33 pg 2.39 ± 0.20 peg 400 2.99 ± 0.12 glycerin 2.76 ± 0.08 water (ph 5.5) 1.47 ± 0.23 iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 140 table 3. angle of repose, carr’s compressibility index and hausner’s ratio for clopidogrel liquisolid systems formula number angle of repose (ɵ) carr’s index (compressibility) hausner's ratio type of flow ls-1 36.02 ± 0.254 18.16 ± 0.687 1.215± 0.021 fair ls-2 33.09 ± 0.535 15.87 ± 0.09 1.178 ± 0.087 good ls-3 31.11 ± 0.265 14.61 ± 0.15 1.161± 0.018 good ls-4 30.73 ± 0.733 14.18 ± 0.43 1.157± 0.023 good ls-5 36.94 ± 0.403 19.05 ± 0.640 1.2 ± 0.062 fair ls-6 34.01± 0.854 17.25 ± 0.702 1.191± 0.044 good ls-7 32.76 ± 0.565 15.79 ± 0.47 1.185 ± 0.028 good ls-8 31.48 ± 0.705 15.32 ± 0.276 1.172 ± 0.049 good ls-9 37.71 ± 0.2 19.61± 0.459 1.242 ± 0.031 fair ls-10 35.89 ± 0.415 17.43± 0.346 1.197 ± 0.056 fair ls-11 33.97± 0.867 15.98 ± 0.194 1.184 ± 0.020 good ls-12 32.07± 0.815 15.5 ± 0.271 1.180 ± 0.020 good dct 37.83 ± 0.830 23.47± 0.365 1.342 ± 0.034 passable results as mean ± s.d, n=3. differential scanning colorimetry (dsc) differential scanning colorimetry analysis was applied to determine thermotropic properties of the system and any physicochemical interaction between drug and excipients. the dsc thermogram of pure clopidogrel bisulfate was shown in figure 1. the clopidogrel bisulfate peak demonstrated as a clear sharp characteristic endothermic peak at 180°c corresponding to its melting point. such a sharp endothermic peak showed that clopidogrel bisulfate used was in a pure crystalline state. the dsc of avicel ph 102 was shown in figure 2. while, figure 3 showed the dsc of physical mixture of directly compressed tablet (dct) which exhibited endothermic peak at 178°c, which was the peak of the drug. the low intensity of peak may be related to low quantity of drug in test sample. also there was another peak at 75.02°c which related to avicel. on the other hand, the dsc thermogram of liquisolid compact (ls-2) showed disappearance of the characteristic peak of clopidogrel melting point as shown in figure 4 giving a strong indication that the drug lost its crystallinity state and converted to an amorphous form (34). figure 1. dsc thermogram of pure clopidogrel. iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 141 figure 2. dsc thermogram of avicel ph 102. figure 3. dsc thermogram of directly compressed tablet (dct). figure 4. dsc thermogram of liquisolid compact ls-2. fourier transorms infrared spectroscopy (ftir). this study was performed to know the compatibility between the drug and excipients (figures 5 – 8). the ftir spectrum of pure clopidogrel bisulfate figure 5 showed a strong absorbance band due to c=o stretching vibrations at 1752 cm–1 and due to o–h stretching of the hydrogen sulfate moiety around 3012 cm–1. the band due to aromatic c– h stretching vibrations represented at 3121 cm– 1. the ftir spectrum included also, broad absorbance band at 2505 cm-1 which can be attributed to the stretching vibrations of bonded n–h resulting from salt formation between the quaternary nitrogen of clopidogrel and –oh of hydrogen sulfate. the band associated with c– o stretching appeared at 1062, 1155 and 1186 cm-1. these results were confirmed by the appearance of the same characteristic absorption peaks in the spectrum of the physical mixture of dct and ls – 2 without any changes in their position figures (7 and 8) which, indicated absence of chemical interactions between the drug and excipients (35). figure 5. ftir spectrum of pure clopidogrel. iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 142 figure6. ftir spectrum of avicel ph 102. figure 7. ftir spectrum of directly compressed tablet (dct) figure 8.ftir spectrum of liquisolid compact formula ls-2. iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 143 x-ray diffraction (xrd). for characterization of the crystalline state, the xray diffractogram of pure clopidogrel bisulfate exhibited several sharp peaks in the region of 5° to 50° 2θ as shows in figure 9. two high intensity peaks at 21.69° and 23.0° 2θ. at the lower 2θ angle, unique peaks were present at 8.91° and 12.44° 2θ suggested that, the drug existed as crystalline state. figure 10 showed one sharp peak at 2θ angle of 22.5 of avicel ph 102. clopidogrel bisulfate characteristic peaks were observed in the physical mixture of conventional formulation (dct) as shown in figure 11, demonstrating that its crystalline structure remained unchanged during the physical blend. the liquisolid powder ( ls –2) diffraction pattern figure 12 showed only one sharp diffraction peak at 2θ angle of 22.5 belonging to avicel ph 102 indicating that only avicel ph 102 maintained its crystalline state (36). figure 9. x-ray diffraction of pure clopidogrel figure 10. x-ray diffraction of avicel ph 102. figure 11. x-ray diffraction of directly compressed tablet (dct) iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 144 figure 12.x-ray diffraction of liquisolid system ls-2 scanning electron microscopy (sem) figure 13 illustrated the sem of the pure clopidogrel bisulfate. it appeared that, the drug had crystalline nature, as proved previously by dsc and xrd. figure 14 represented the photomicrograph of optimized liquisolid system (ls – 2). the drug particles in ls system entrapped within excipients, confirming ftir and dsc data analysis. this surface modification ensured the decrease in crystallinity of the drug particles. these images indicated the change in surface morphology of drug particle due to entrapment into the respective carrier and coating materials (37). figure 13. sem of pure clopidogrel bisulfate. figure 14.sem of liquisolid compact (ls-2) post-compression evaluation hardness test the average hardness of a liquisolid compacts ranged from (4.3 ± 0.173) to (6.46 ± 0.251) kg/cm2 while, the hardness of dct was (5.65 ± 0.45) kg/cm2 as shows in table 4. as excipients ratio increased the hardness of the tablets increased. this was due to the hydrogen bonds between groups on adjacent cellulose molecules in avicel ph 102(38). friability all clopidogrel ls compacts had acceptable friability as none of the tested formulas had percentage loss in compacts weight that exceeded 1%. also, no compact cracked, split or broken in either formula (38), as shown in table 4. weight variation compacts of each formula were subjected to weight variation test, the difference in weight and percent deviation was calculated for each tablet. the results of the test as demonstrated in table 4 showed that, the diffraction angle (2θ) iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 145 compact weights were within the pharmacopeial limit (39). content uniformity percentages of content uniformity for all clopidogrel formulas ranged from (95 – 100.8%) as shown in table 4 . this complied with usp content uniformity specification that is 85%-115% of content in each individual compact indicating that, the processing method were convenience (39). disintegration time the disintegration time for the prepared clopidogrel ls compacts was shown in table 4. it was found that the disintegration time mean for all investigated compacts was less than 2 min, because of co-processed super disintegrant. another finding was displayed from the obtained results that, there was a relationship between powder excipient ratio (r) and the disintegration time. the powder excipient ratio (r) was inversely proportional to the disintegration time of the compacts i.e., when the powder excipient ratio (r) increased the disintegration time of the compacts decreased (40). table 4. evaluation parameters of clopidogrel bisulfate liquisolid compact formula number hardness (kg/cm2) mean± s.d. friability % (w/w) weight variation (mg) mean ± s.d. content uniformity % disintegration time (sec) mean ± s.d. ls-1 6.4 ± 0.264 0.14 1.065 ± 0.556 100.4 42.6 ± 2.16 ls-2 6.46 ± 0.251 0.2 867.63 ± 0.32 99.58 55.3 ± 1.92 ls-3 5.73 ± 0.152 0.28 722.83 ± 0.29 95.83 68.5 ± 3.87 ls-4 5.6 ± 0.115 0.53 620.53 ± 1.32 100.8 80.6 ± 2.51 ls-5 5.3 ± 0.153 0.23 980.53 ± 0.51 97.5 61.3 ± 2.28 ls-6 5.86 ± 0.15 0.26 784.03 ± 0.55 97.05 83.1± 4.14 ls-7 4.73 ± 0.23 0.42 653.2 ± 0.721 101.6 95.6 ± 3.62 ls-8 4.53 ± 0.67 0.45 559.23 ± 0.68 95.41 101.9 ± 3.35 ls-9 4.4 ± 0.2 0.52 830.5 ± 0.458 99.16 89.6 ± 2.18 ls-10 4.33 ± 0.251 0.19 664.16 ± 0.42 96.25 107.8 ± 1.57 ls-11 4.3 ± 0.173 0.41 552.8 ± 0.2 95 112.3 ± 4.72 ls-12 4.46 ± 0.152 0.53 474.23 ± 0.25 97 126.4 ± 2.44 dct 5.65 ± 0.45 0.61 770.06 ± 0.12 100.4 74.7 ± 2.21 n=3. in-vitro dissolution test the dissolution profiles of clopidogrel ls compacts in sif (phosphate buffer ph 6.8 with 1% sls) were graphically represented in figures (15 – 17). the percentage of clopidogrel bisulfate released from liquisolid compacts (ls 1 – 12) was varying from 28.3 – 92.2 % in first 10 minutes. while, 13.6 % and 24.2% of drug released from dct and marketed tablets (plavix®), respectively. the results indicated fast release of the drug was observed from the ls compacts. such enhanced drug dissolution rate may be mainly attributed to the fact that this poorly water-soluble drug was already in solution in tween 80 (molecular dispersed form). while, at the same time it was carried by the powder particles (microcrystalline cellulose–silica) of the liquisolid system. thus, its release was accelerated due to its markedly increased wettability and surface availability to the dissolution medium (41). figure 15. dissolution profiles of clopidogrel from liquisolid compacts (ls 1 – 4) in phosphate buffer (ph 6.8) with 1% sls at 37˚c iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 146 figure 16. dissolution profiles of clopidogrel from liquisolid compacts (ls 5 – 8) in phosphate buffer (ph 6.8) with 1% sls at 37˚c figure 17. dissolution profiles of clopidogrel from liquisolid compacts (ls 9 – 12) in phosphate buffer (ph 6.8) with 1% sls at 37 ˚c from the calculations, the mdt and % de for ls-2 were (5.95 min and 90%), dct were (35 min and 34%), and for marketed tablets (plavix®) were (30.14 min and 52%), respectively. compacts of ls-2 represented the lowest mdt and highest %de compared with other prepared ls formulations and it was considered as the optimum ls formula. in addition, ls-2 compacts showed better improvement in dissolution in contrast with dct and marketed tablets (plavix®) since, lower mdt and higher %de values indicated that, ls-2 compacts were significantly (p < 0.05) enhanced dissolution rate (42). the effect of drug concentration on the release of drug was shown in figure 18. better dissolution was observed at huge differences in drug concentrations. however, as the drug concentration decreased, the portion solubilized and molecularly dispersed in the liquid vehicle increased thus leading to improve dissolution. in addition, the more vehicles available, the more even the distribution of the vehicle over the remaining undissolved drug particles that would help in better wetting of the drug through the dissolution stage(43). figure 18. effect of drug concentration on dissolution profile of clopidogrel liquisolid compacts in phosphate buffer (ph6.8) with 1%sls at 37˚c. on the other hand, the powder excipient ratio (r) was directly proportional to the invitro drug release i.e., when r increased, clopidogrel release also increased as shown in figure 19. this may be attributed to the high carrier (avicel) content where it also functioned as a swellable disintegrant. in addition, the highly hydrophilic characteristic of avicel (microcrystalline cellulose) increased the wetting of clopidogrel and enhanced its dissolution (44). figure 19. effect of excipient ratio (r) on dissolution profile of clopidogrel liquisolid compacts in phosphate buffer (ph 6.8) with 1% sls at 37˚c. the release of clopidogrel bisulfate from ls-2 compacts was compared with that of dct and marketed tablets (plavix®) in 0.1n hcl (ph 1.2) figure 20 and in phosphate buffer (ph 6.8) with 1% sls, figure 21. the difference in the percent of drug release was found to be significant (p < 0.05). this clearly indicated the improvement in the dissolution of clopidogrel was due to the presence of the drug in nonvolatile solvent (tween 80) in the ls formulation. after ls compact disintegration, its primary particles suspended in the dissolution medium had the drug particles in a state of molecular dispersion. in contrary, there was a limited surface area of the plain drug exposed to the dissolution medium in dct and the marketed tablets (plavix®), because of the hydrophobic nature of the drug particles. iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 147 however, the higher dissolution rates observed in ls compacts could be related to a considerably larger surface area of the dispersed drug particles exposed to the dissolution medium(45). figure 20. dissolution profiles of liquisolid compacts ls-2, dct and marketed tablets in 0.1n hcl (ph 1.2) at 37 ºc figure 21. dissolution profiles of liquisolid compacts ls-2, dct and marketed tablets in phosphate buffer (ph 6.8) with 1% sls at 37ºc. conclusion the liquisolid technique succeeded to enhance the dissolution rate 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dispersion and liqui-solid techniques. saudi pharm j. 2015;23(6):650– 657. 43. bary aa, louis d, sayed s. liquisolid tablet formulation as a tool to improve the iraqi j pharm sci, vol.27(2) 2018 clopidogrel bisulfate liquisolid compact 149 dissolution of olmesartan medoxomil.inventi rapid;2014(3):1–8. 44. komala dr, janga ky, jukanti r, bandari s, vijayagopal m. competence of raloxifene hydrochloride loaded liquisolid compacts for improved dissolution and intestinal permeation. j drug deliv sci technol. 2015;30:232–241. 45. nokhodchi a, javadzadeh y, siahishadbad mr, barzegar-jalali m. the effect of type and concentration of vehicles on the dissolution rate of a poorly soluble drug (indomethacin) from liquisolid compacts. j pharm pharm sci. 2005;8(1):18–25. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 43 effect of nitrogen and phosphorus levels on yield, concentration, physical and chemical properties of dill seed oil fadhil h al-sahaf* , madeha h al-samara’i** (1) , muna j al-ndawi** receive d 7-12-2003 accepte d 21-10-2004 abstract to te st the effe ct of 4 levels of nitrogen (i.e . 0, 45, 90 a nd 135 kg n ha -1) a s urea (46% n) and 3 leve ls o f phospho rus (i.e. 0, 17.5 and 35 kg p ha -1) a s triple superphos pha te (21.8% p) on yie ld and co nce ntra tio n of dill (anethum grave olens l. loc al c ultiva r) see d oil this experiment was carried o ut during winter se ason of 1999 20 00 a t the e xpe rime ntal field of agric ulture colle ge, abu-ghra ib . both fe rtilize rs we re app lied in two eq ual splits, firs t at seeds sowing a nd the second was adde d one mon th after eme rgence. dried and ground see d samp les were subjecte d to water d is tilla tion for extrac tion o f vo latile oils res ult indica ted tha t fe rtiliza tio n of d ill p la nts with 9 0 kg n + 35 kg p ha-1 prod uced highes t oil yie ld (3 2.19  ha -1) a nd c onc entration (3.60%) with be tter qua lity. g lc ana lysis indicated tha t dill seed oil co ntain 27 volatile compo und s, 15 we re ide ntified a nd the ma jo r co ns tituents were carvo ne , li monene and α-phe llandrene. nitro gen a nd phospho rus fe rtilizatio n increased the co nce ntra tio n of a ll identified co nstituents of d ill s eed oil. nitro gen a nd phos pho rus fe rtiliza tio n inc reased yie ld a nd co ncentratio n of dill seed oil. moreo ver, physical prop erties of the o il were also improved by n and p fe rtiliza tio n. glc a na lysis showed that carvone, limo ne ne and α-phelland re ne are the major c ons titue nts of dill se ed oil and could be increased by n and p app licatio n . ke y wo rds : volatile c ompounds, carvone , limo ne ne , α-phe llandre ne . ةــــــــالخالص ن جي من النترو ت مستويا ر ( بهدف إختبار تأثير اربعة 4, 0صف 5 ,9 0 ,1 ر/ nكلغم 35 كتا 4( على هيئة يوريا ) ه 6 %n ( وثالثة ر من الفسفو ت ر ( مستويا غم 17.5و 0صف كتار/ pكل سفات ثالثي ) ه ر فو وب 21(على هيئة س .8 %p ( زيت بذور ز كي في حاصل وتر ت anethu(الشبن m gra veo le ns l. ( عام وي ل شت سم ال مو خالل ال ة رب هذه التج محلي نفذت 199الصنف ال 9-20 ول كلية 00 حق في ة ويتين. ابو غريب/ الزراع سا مت ن عتي ن بدف ع, اضيف كال السمادي الولى عند البذار والثانية ب من اإلنباتا ر ر شه مرو ن البذور . د عينات م ر البخاري طي ة التق طريق طيارة منها ب الص الزيوت ال ستخ ة تم إ ـ . الجاف سميد نباتات الشبنت ـب وضحت النتائج أن ت غـم 90( ا n +35كل غم ر ادى الى إعطاء أعلى حاصل زيت )/ pكل كتا 32.1(ه ر 9 كتار/ لت ركيز ) ه 3وأعلى ت .6 ات . و بنوعية جيدة% 0 كـب حليل المر ت جهاز ة رة بوساط شبنت يحتوي على glcالطيا حت أن زيت ال 2أوض كن تشخيص , زيت طيار 7 1أم ون و 5 رف كا منها حيث كان ال وااللفا ن موني ت . فيالندرين تمثل اكبر نسبة منها -اللي زـي ات ال وـن مك ع مـي ادة ج ى زـي ر أدت إـل والفسـفو ن جي هر ايظُا ان إضافة النترو وظ خصة حسـن مـن نستنت. المش وي ر ذو ت الـب زيد مـن حاصـل زـي ر ي والفسفو ن جي ميد نباتات الشبنت بالنترو ن تس سة أ را ج من نتائج هذه الد عيته حليل اـل . نو ر الشبنت glcنتائج ت رئيسية لزيت بذو ونات ال مك هي ال ن ري اللفا فيالند وا ونين ون والليم وت الكارف زي ن حت أ . أوض dill herb is us ed for lowring bloo d cholesterol (4). ca rv one a v olatile oil is one o f the major co nstituents o f dill o il wa s found to inhib it sp outing in stored p otatoe s and had a n antifungal activity aga inst potato tub er d is ea se s (6).dill se ed may c ontain 4% o f oil (7) with es se ntia l oil (c hiefly co ns is ting of c arvo ne, limo nine and terpens), fa tty oils , pro te ins, ta nnins, re sins a nd mine ra l sa lts (4). dill se ed o il productio n is se ns itive to an e nviro nme ntal a nd agro nomic al prac tic es. halva et a l (8) no tic ed an increas es in o il co nce ntra tion in dill plants by 6 times under full light c ond itions introduction dill (anethu m g ra veo le ns l.) b elong to umbe llife ra e is o ne of nutritional and medicina l ve getab le s (1). dried le av es and ground see ds a re use d as fla vour ad ditive s to the sca ld me at a nd chee se s (2), and to pickle cuc umb ers to imp rov e taste a nd ma inta in good qua lity (3). dill s ee d o il is us ed for med ic al purpo ses as ca rmina tiv e, in flatule nce of children, antisp asmodic, stop children’s stoma c pain, and a id lac ta tion (4). it ha s also be en used for lo wering blood hype rtension a nd bloo d sugar le vel in d ia be tics (5). *co llege of agr ic ulture ,un iversity of baghdad ,baghdad ir aq . **tec hnical institu te ,al-mansour, baghdad -iraq (1 ) part o f m.sc. thes is of the sec ond a uthor . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 44 (1 00%) as co mpa re d to tho se e xpo se d to o nly 30% of the full light intensity. growth stage also e ffec t oil c onc entration, whe re it wa s the highest in d ill herb when s eed s were at milk stage and dec re as ed a s se eds matured (9) . nitrogen fertilizatio n increa sed s ee d o il yield and changed it c hemica l constituents (10). howe ver, lev els of n fertilizatio n higher tha n 120 kg n ha-1 de creas ed o il c oncentration in dill se eds (11). s ingh a nd mahey (12) also found that ma ximum s ee d o il co nc entratio n wa s re ac hed when dill plants we re fertilized with 120 kg n + 1 7.5 kg p ha -1. dill oil de nsity usually is lowe r than the de nsity of wa ter s o it floating a s oily layer o n water surfac e during extrac tion (13), and affe cted b y s owing da tes but no t by n o r p fe rtiliza tio n, whereas , refra ctiv e index of the oil wa s increa sed b y n and p fe rtilize rs (11). the ob je ctive of the prese nt wo rk was to examine the interac tiv e e ffe ct o f nitro gen and phosp horus a pp lica tion o n dill see d oil yield, co nce ntra tion and v olatile o il constituents . materials and methods experiment layout a field exp eriment was c arried o ut d uring the winter se as on o f 1 999 -2 000 at the experimenta l fie ld of hortic ulture de pa rtment, agriculture colle ge unive rs ity of ba ghd ad. dill see ds (lo ca l cultivar) we re s own o n 1st of octob er 1 999 in rows 1 5c m a pp art in plots (2.5 m2) at a rate of 2.5 kg see ds ha-1. tre atments includ ed 4 leve ls of nitro ge n as ure a (4 6% n) (0 , 45 , 9 0 and 1 35 kg n ha -1 re fe rred to as n0, n1, n2 and n3 re sp ectively), a nd 3 le ve ls of phosp horus as triple s upe rp hos phate (21.8% p) (0 , 17 .5 a nd 34 kg p ha-1 referre d to a s p0, p1 and p2 res pe ctiv ely). fe rtilize rs were ap plied in tw o equal s plits , first o n se ed so wing and the se cond was a pplied one month a fte r eme rgenc e. fruits were collec te d progres sively whe n the colour turned b row nish ye llow and air dried on 15th o f may 2 000 . oil extra ctio n ground se ed s were wa te r d is tilla te d ac cording to british p ha rma cop oe ia a s de scribed b y asta (14). cleve nger ap pa ra tus was c onnec te d to ro ta ry eva porator flas k where 25g of s eed s po wder were distillated w ith 25 0 ml o f distilled wa ter, and distillation co ntinue d fo r 2 hr (15). oil e xtract was trans fe rred to a s ep aratory funnel, and 30 ml of diethyl ethe r (c4h10o) was a dde d. the mixture wa s ha nd s ha cke d and le ft to s ettle, a la yer o f oil and diethyl ether floa te d on the top o f the extrac t, co llec te d and this p roc ed ure wa s re pea te d thre e times for eac h sa mple. 3 – 5 g of calc ium s ulfa te we re add ed to the oil – ethe r mixture to a bs orb re maining water d ro plets. ether was eva porated in rotary ev apo ra to r unde r va cuum at 3 7 c°, the o il transferre d to dark brown color vials and stored in re frigerator (4 ± 1 c °). phys ical pro pe rtie s of dill se e d oil extra cted s eed o il p hysical prope rties of different trea tment samp le s were determine d acc ording to gue nther (16) thos e inc lude d spe cific grav ity and de nsity. refra ctiv e index was de termined by abbe refra ctome te r (abbe type unive rs al, haensc h, schmid t co.). determination o f physica l p ro pertie s we re perfo rmed at 20 c °. chemical analysis che mic al analysis of dill seed oil constituent was pe rformed b y gas-liquid chroma to grap hy (ge-9a model, s himazu, osa ka, japan) by direct injec tio n onto a 30 m × 0.25mm co lumn pa cked with 5 % polydip henyl silo xane and 95% d ime thyl silo xa ne on spb-5. sep aratio n conditio ns we re as fo llows : carrier as (helium) a t flow rate = 25 ml min-1, injectio n p ort te mperature, detec to r (flame io nization detec tor, f id) te mpe ra ture , and ove n te mpe ra ture was 22 0 c˚. oil co nstituents were identified as comp ared to a numbe r of essentia l oils standards. statistical design and analysi a split-plot e xpe rime nt was used whe re nitrogen leve ls were repres ented by ma in p lo ts and p hos phorus leve ls as s ub-plots with three re plic ates . results were s ubjec ted to the ana lysis of varianc e a nd le ast s ignifica nt differences (lsd p = 0 .0 5) was used to comp are the diffe re nce between tre atmen ts (17). results and discussion oil yield and conte nt of d ill see ds yie ld a nd c onc entratio n of dill s ee ds oil we re increas ed marke dly b y inc re asing nitro gen le vels up to 90 kg n ha-1 (n 2) (ta ble 1). the perce nt inc re ase s in oil yield was 11 4.5% wherea s for oil c oncentration wa s only 4 9.4% when c ompa ring n2 to n0. the se increa ses may ac co unte d for the increa ses in ve ge ta tive growth mainly le af a re a which may reflecte d on d ry matter and se co nda ry metab olites pro ductio n (10,18). how eve r, increas ing nitro gen le vels up to 1 35 kg ha -1 res ulted in a signific ant reduction in bo th yield and concentra tion o f oil in dill se ed s. the se res ults may sugge st tha t op timum leve l of nitro gen fe rtiliza tion fo r highes t yie ld and ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 45 c onc entration of dill seed oil is 90 kg ha-1 . singh and ra nd hawa (18) had a ls o fo und tha t fertilizatio n of dill plant with this sa me le vel of nitro ge n prod uce d the highest yield and conce ntra tion of o il in the s ee ds , and increa sing the n lev el up to 120 kg ha-1 cause d a s ignific ant reduction in both yie ld and co nce ntra tion of the s ee ds o il. inc re as ing pho sphorus lev el, from p0 to p2 ha d also increa se d yield and c oncentration of dill se eds o il but to le sse r extent as c ompa re d to the effects of nitro gen lev els inc re as es, whe re the p ercent inc rea se s were 13.7 % and 4.8% for the oil yie ld and co nc entratio n re sp ectively. the se diffe re nc es in the e ffe cts be twe en n and p fertilizatio n c ould b e attribute d to the ma gnitud e e ffec t of eithe r nutrient in ve ge ta tive growth. the inte rac tion was signific ant and the highe st oil yie ld (3 2.19  ha -1) and co nce ntra tion (3.6%) was produce d when d ill plants we re fe rtilize d w ith 90 kg n ha-1 + 35 kg p ha -1. similar effe cts of n + k on d ill s ee ds oil yie ld and c ontent was found by singh and ma hey (12). physical properties of the dill seed oil nitrogen a pplic ation to d ill plants had no marked e ffec ts o n s ee d o il s pe cific gravity, density and re frac tive inde x (table 1 ). increas ing p ho sphorus le vel a ls o wa s not effe ctiv e in oil sp ec ific gra vity a nd de nsity, while refra ctive inde x was slightly increa sed (but s ignifica nt) a s phosp ho rus le ve l increa se d. table 1 . effe ct of nitro ge n a nd phos pho rus le ve ls o n dill se e d oil y ie ld, c once ntration and p hys ic al prope rtie s. fe rtiliz er le ve l se e d o il yie ld (  ha -1) se e d o il co nte nt ( %) oil specific gravity oil de ns ity (mg µ  -1) re frac tive inde x n lev els n0 14.28 2.3 9 0.9 33 0.873 1.4 87 n1 25.42 3.1 9 0.9 42 0.880 1.4 88 n2 30.63 3.5 7 0.9 43 0.881 1.4 88 n3 22.08 2.6 9 0.9 40 0.877 1.4 89 l.s .d0.05 1.715 0 .004 n .s n.s n .s p lev els p 0 21.47 2.8 9 0.9 37 0.876 1.4 86 p 1 23.41 2.9 7 0.9 44 0.882 1.4 87 p 2 24.42 3.0 3 0.9 38 0.877 1.4 89 l.s .d0.05 1.485 0 .004 n .s n.s 0.0 01 in te raction n0p 0 13.72 2.3 6 0.9 21 0.861 1.4 85 n0p 1 14.26 2.4 0 0.9 47 0.885 1.4 87 n0p 2 14.84 2.4 3 0.9 33 0.872 1.4 89 n1p 0 22.26 3.0 6 0.9 48 0.887 1.4 86 n1p 1 25.77 3.1 8 0.9 42 0.881 1.4 88 n1p 2 28.24 3.3 3 0.9 34 0.873 1.4 89 n2p 0 28.79 3.5 3 0.9 34 0.874 1.4 86 n2p 1 30.90 3.5 7 0.9 50 0.887 1.4 88 n2p 2 32.19 3.6 0 0.9 45 0.888 1.4 89 n3p 0 21.12 2.6 1 0.9 41 0.880 1.4 86 n3p 1 22.71 2.7 1 0.9 38 0.877 1.4 87 n3p 2 22.42 2.7 4 0.9 42 0.880 1.4 88 l.s .d0.05 5.50 0 .008 0.0 14 0.013 n .s however, the interaction effect o f n a nd p fertilizatio n was s ignific ant and the highest spec ific gra vity and d ens ity of the o il was no tic ed whe n plants we re fe rtiliza tion with n2p 1 and n2p 2 re sp ective ly, whe reas refra ctive inde x was not affe cted significa ntly b y the inte ra ctio n tre atmen ts . changes in phys ical properties of the oil b y n a nd p fe rtiliza tion could be d ue to the effec t o f bo th nutrie nts o n oil oxyge nous compounds suc h as ca rvone, linalool, euge nol, α-tujone and citrone llol (19). pos itive correla tion was found between phosp horus conte nt of the vegetative pa rts and oil specific gra vity (r = 0.433 *), d ens ity (r = 0.4 25*) and re frac tive index (r = 0 .784 **). the se relatio ns hips could be explained on the bas is of the effect of phosp horus o n e ssential oil s olid co nstituents (o xygenous comp ounds ) (19). the se findings a re ir aq i j.pharm.sci., vol.15 (1) ,2006 46 in a cc ordance with ra nd hawa et al (11) who co nc luded that p hys ical properties o f dill s eed oil is not sensitiv e to n or p fertilizatio n proba bly beca use of the d ifference in cultiv ars us ed a nd/o r predomina nt e nv ironmenta l co nditio ns . esse ntia l oils with high p hysical p roperties es pecially refrac tive ind ex and density re garded as high q uality. see d oil constituents analysis by glc analysis o f dill se ed o il sho wed tha t it c ontaine d 27 v olatile oil co mponents , 15 we re identifie d and the rest we re not b eca us e o f lac k o f sta nda rd s. the se 15 c ompo unds c ould b e clas sifie d to thre e gro ups ac co rd ing to their co nc entratio ns , thos e are high (carvo ne, limonene, and α-p hellandre ne ), me dium (cha mphe ne , linalool, citronello l, myrce ne, myrisiticin, te rp enole ne , terpene and ca ryophylle ne ), and low (α-thujo ne, β-cyme ne , α-pine ne and eugenol) (ta ble 2). phella ndrene and to s ome extent limone ne (mo noc yc lic terpenic c omp ounds) are the ma in c ons titue nts for o dor and flavo r of dill se ed oil (16). inc re asing nitro gen le ve ls increas ed the co nc entratio n o f all id entified vo la tile co mpo und s. howev er, the se increa se s were not linea r as nitrogen le vel inc re as ed, w here the co nc entratio n of all oil c ons titue nts were no ta bly drop ped when nitro gen ap plic atio n ro se up to 13 5 kg ha -1 (n3). concentra tion red uction co uld be attributed to the productio n of othe r me ta bolite s suc h as pigme nts, hormones ……. etc ., in a dditio n to the dilution effe ct. increas ing phosp horus le vels grad ua lly increa se d the c onc entratio ns of a ll identifie d vo la tile compounds o f dill se ed oil. comp aring the effect of n and p leve ls c onc entrations o f carvo ne, limone ne and α-phe llandrene, which are the major constituents , phos pho rus was more effe ctive tha n nitro gen, w here the perce nt increa ses d ue the n2 or p2 were 1 7.30% and 49.35 % , 59.79 % and 230 .2 2% , and 225 .6 3% and 3 78.8 1% for the thre e compounds re spe ctively. howe ver, camphene, terpenolene and myrc ene (me dium co nce ntra tion gro up ) and β-cyme ne a nd α-pinene (lo w conce ntra tion group ) ha d be en a ffec te d dras tica lly by increa sing nitro gen lev el to 90 kg ha -1 (n2) as compared to the e ffec t of increa se d lev el of pho sp horus to 35 kg ha-1 (p2), whe re the perce nt increa ses w ere 225 .2 9% and 9 1.12%, 345 .7 7% and 5 7.86 %, 182 .62% and 147 .4 4%, 1 921 .2 8% and 77.08 %, a nd 9 68.25 % and 118 .6 1% re spe ctively. the se varia tio ns c ould be due to the rule of either nutrie nt in metab olis m and synthes is o f thes e comp ounds . . these res ults re vea l that tho se nutrients may have diffe re nt mode o f a ction in the b io synthe sis o f these compounds . ta ble 2. effect of nitroge n and pho sphorus leve ls on dill see d oil co ns titue nts conce ntratio n (mg kg se ed-1) co mpo unds n itro ge n le ve ls (kg n ha-1) pho spho rus le ve ls (kg p ha -1) n 0 n 1 n 2 n 3 p0 p1 p 2 carvo ne 3 96 .6 404.7 465.2 446.4 325.2 4 75 .0 485.7 limonene 6 8.5 5 92 .73 109.5 77 .1 5 35 .3 7 1 08 .8 116.8 α -p hellandrene 5 4.1 1 133.4 176.2 134.5 35 .6 3 1 67 .4 170.6 camp hene 2 4.7 9 53 .55 80 .6 4 58 .6 4 37 .9 6 5 2.7 0 72 .55 linalo ol 3 2.1 4 46 .21 57 .9 2 48 .7 5 19 .4 8 5 2.6 0 66 .64 eugeno l 1 .77 7 3.257 4.36 0 2.22 0 1.29 0 3 .70 0 3.78 α -thujone 0 .79 0 1.280 1.65 0 0.99 6 0.64 0 1 .43 0 1.520 citronello l 9 .74 0 9.910 16 .3 6 9.53 0 0.46 0 1 3.5 6 20 .14 myrcene 8 .23 0 12 .02 23 .2 6 14 .3 2 8.39 0 1 5.3 7 20 .76 β -c ymene 0 .98 7 11 .50 19 .9 5 10 .0 1 7 .33 1 1.5 8 12 .98 mysris iticin 7 .47 0 11 .81 15 .0 5 11 .0 8 9.19 0 1 2.1 0 12 .77 α -p inene 0 .63 0 3.820 6.73 0 5 .08 2.49 0 3 .97 0 5.740 terpeno lene 3 .78 0 7.650 16 .8 5 14 .2 6 8.02 0 1 1.2 3 12 .66 terpinene 1 5.8 6 24 .04 25 .0 4 17 .7 8 15 .6 0 2 3.3 6 24 .00 caryo phyllene 1 9.5 7 29 .53 42 .0 9 30 .7 2 5 .82 3 0.2 6 55 .43 ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 47 conce ntra tion of mos t ide ntified seed o il co nstituents we re the highest when d ill plants fe rtilize d with 9 0 kg n + 3 5 kg p ha-1 (table 3). these res ults could b e due to the o ptimu m balance o f n and p fertiliza tion fo r increasing the conce ntra tion of s ee d oil co ns titue nts. table 3 . inte ractive e ffect of nitroge n and phos phorus le ve ls on dill se e d o il constitue nts conce ntration (mg kg se e ds -1) conclusions ni tro gen a nd phos pho rus fe rtilizatio n increase d yield and c onc entration of d ill s eed oil. mo reover, physica l properties o f the o il were also improved by n and p fertilization. glc analys is s howed tha t ca rv one , li monene and α-p hellandrene a re the ma jor co ns titue nts of d ill seed o il a nd co uld be increase d by n and p app licatio n . references 1. ihsan s a a study o f so me fa ctors affe cting the qua ntita tive a nd q ua litative prope rties of the volatile o ils in mentha spicta var. virids l. and mentha lo ngifolia var. asiatica, p h. d. the sis, unive rs ity of baghda d, iraq 1 999 . 2. sabados d, rajsic b the o rganoleptic qua lity p ic ture of (yugos lav) industrial fres h chees w itho ut o r with suppleme nts, mljekaratvo (yugos lavia) 1984,34 :227235 . 3. mcfeete rs r f, fleming h p ba la nc ing macromineral co mposition o f fres h pac k cuc umb er pickles to improve nutritio nal q ua lity and ma intain flav or. j food q ua lity (usa) 1997, 20:81 -87. 4. al-rawi a, cha kravarty h l med ic inal plants of iraq , 2nd ed , al-yaqtha press, baghdad, iraq, 1988,p 13 -1 4. 5. pizlo h , kogut b inv estiga tion of the c onte nt of vo la tile oils in ga rden dill a iming a t utiliza tion o f this pla nt in the s pirit industry (ane thum grave olens l), f ood additiv e, prze mys l ferma ntacyjny, owocowo, warzywny (pola nd) 1984, 2 8:7-10. 6. ha rtmans k j, die penho rs t p, bakker w, gorris l g the use of ca rvone in a gric ulture sprout supp ress io n of p otatoes a nd anti-funga l a ctiv ity aga inst po ta to tuber a nd othe r plant diseases . ind crops p rod 1995,4:3-13 7. s ta ry f , j irasek v herbs , a co ncise guide in c olour, hamlyn pub lishing group limited, londo n, uk, 19 73,p 66 -67. 8. ha lva s, cra ker l e, simon j e, cha rles d j light le ve ls , growth a nd esse ntial o il compounds n0 n1 n2 n3 p0 p 1 p2 p0 p1 p 2 p0 p1 p2 p0 p1 p2 carvone 235.6 469.1 485.2 263.1 468.4 482.5 371.2 509.0 515.5 431.0 453.6 454.6 limonene 24.6 78.2 102.9 45.5 117.2 115.5 44.9 137.6 146.0 26.5 102.1 102.9 αphe llandrene 26.8 75.0 60.5 42.5 166.9 190.8 32.0 243.0 253.6 41.3 184.6 177.6 camphe ne 12.6 23.8 38.0 38.5 56.6 65.5 58.3 88.9 144.8 42.4 41.6 92.0 linalool 11.5 25.5 59.5 27.8 42.1 68.7 21.5 76.7 75.6 17.4 66.2 62.8 euge nol 0.791 1.95 2.89 1.52 4.78 3.48 1.77 5.59 5.73 1.10 2.51 3.05 α-thujone 0.405 0.856 1.14 0.646 1.65 1.50 0.907 1.84 2.22 0.394 1.37 1.23 citronellol 0.415 8.88 19.9 0.539 8.81 20.4 0.519 22.4 26.2 0.371 14.2 14.1 myrce ne 1.21 6.86 16.6 7.33 12.3 16.5 18.4 24.9 26.5 6.66 17.5 18.9 β-cymene 0.227 1.25 1.49 9.42 11.9 13.3 14.2 22.4 23.3 5.37 10.8 13.9 mysrisiticin 6.51 7.38 8.55 11.0 11.2 13.3 11.3 17.2 16.7 8.02 12.6 12.6 α-pinene 0.375 0.658 0.870 2.42 4.22 4.82 3.63 6.59 9.98 3.56 4.42 7.29 ter penolene 2.61 4.04 4.71 4.74 6.95 11.3 10.6 17.6 17.9 9.67 16.3 16.8 ter pinene 13.4 16.2 22.0 19.0 26.3 26.8 15.4 32.2 27.5 14.6 18.8 19.6 caryophyllene 0.586 14.7 43.5 2.41 17.1 69.4 6.33 53.1 66.8 14.0 36.1 42.1 ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 48 in d ill (ane thum graveole ns l). j of he rb s, spices and med ic inal p la nts. 1992 1:43 -5 4. 9. singh a., ra nd hawa g s, ma hey r k oil conte nt and o il yie ld of dill (ane th um grave olens l). herb under so me agronomic p ractices , acta horticuturae 198 7,208:51-60 . 10. singh a, ra ndhawa g s, ma hey r k growth and yield of dill (ane th um grave olens l.) as a ffec te d by nitroge n and harvesting stages . crop res 1993 ,6 :217221 . 11. randhawa g s , singh a, ma he y r k optimis ing a grono mic requireme nts fo r se ed yield and quality of d ill (ane th um grave olens l) oil. ac ta hortic ulturae 198 7,208:61-68 . 12. singh a, mahe y r k fertilizer use in umbe liferae me dicina l and aroma tic plants of ind ia , ind j . agron 1992,37:39 -4 5. 13. tyle r v e, bra dy l r, robe rts j e pharmacog nosy 9th ed , lea and febige r, philadelp hia, usa, 1988,p 18 9-197 . 14. asta .. offic ial analytical metho ds of americ an spice tra de associatio n, englewood cliffs , n j , usa 1 968 . . 15. chub ey b b, do rrell d g cha nge s in the che mical compos ition o f dill oil d uring hyd ro d is tillatio n, can j . pla nt sc i. 1 976 ,56:21 5-216 . 16. guenther e es se ntial oils , , r e kriege p ublishing company, hun tigton, new york, us a, 19 72 , vol i ,p 18. 17. cochra n w g, cox g m exp er ime ntal design , 2nd e d, j ohn wiely and sons inc, new york, usa, 1 957 , p 2 93-316. 18. s ingh a, ra ndhawa g s studies o n s ome agronomic inputs effecting o il c onte nt, o il and he rb yie ld of dill (an ethum graveolens l.) india pe rf 1 989 ,34:10 8-114 . 19. chang k h, hwan j h, pa rk k b, kang d j, lee y s effe ct o f app licatio n levels of nitrogen fertilizer o n the growth and c he mic al comp ounds of min t, re search repo rts of the rural development a dministratio n crops (ko rear) 1 987 ,2 9:28 9 293 . clinical evaluation of melatonin alone and in combination with pizotifen in the prophylaxis of migraine iraqi j.pharm.sci., vol.16 (1) ,2007 anise-thyme lotion for chicken pox. 8 preparation of anise and thyme lotion for topical use issraa r. abed alrahman* ,1 , fatima j. jawad* , ekbal h.alkateeb* and alaa a. abdulrasool* *department of pharmaceutics, college of pharmacy, baghdad university, baghdadiraq. abstract : anise and thyme crude extract were used to prepare a lotion for topical application due to their antimicrobial, germicidal and antifungal effects. two formulas were prepared using the mentioned natural plants, formula 2 (selected lotion) was the most acceptable one which contained veegum and xanthan gum as suspending agents in addition to other exceipients providing it good properties with high physical stability because of its flocculating, pouring, resuspending easily with sedimentation volume (f) 0.96. in addition to unchangeable odor and color with expiration date of one year. while the preliminary clinical study was done using this lotion on 10 patients with infecious viral skin diseases, it was found that this lotion was successful as a topical preparation for allergy and mostly chicken pox. key words: anise, thyme, crude extract, antimicrobial effect. ة:ـــــالخالص أسخخذو انًسخخهض انخاو نًادحٙ انُٛسٌٕ ٔانضعخش نخحضٛش غسٕل نالسخعًال انخاسجٙ بسبب فعانٛت حهك انًٕاد فٙ قخم انبكخشٚا، انجشارٛى غسٕنٍٛ باسخخذاو َفس انًٕاد انُباحٛت انخاو، كاٌ انغسٕل انزاَٙ انًحضش )انظٛغت انًخخاسة( ْٕ األكزش قبٕالً حى ححضٛش ٔانفطشٚاث. الحخٕائّ عهٗ يادحٙ انضاَزاٌ ٔانفٛكاو يع يٕاد اخشٖ يضافت ٔانخٙ اعطج طفاث جٛذة نٓزا انغسٕل حٛذ كاٌ راث رباحٛت فٛضٚأٚت عانٛت . اضافت انٗ عذو حذٔد حغٛش فٙ نَّٕ ٔسائحخّ يع يذٖ طالحٛخّ 0..6دِ بانشس ٔنّ حجى حشسٛب نكَّٕ ال ٚخكخم ٔٚعاد حٕصٚع يٕا ايا بانُسبت نهذساست انسشٚشٚت االٔنٛت ٔانخٙ حًج باسخخذاو ْزا انغسٕل عهٗ عششة اشخاص يظابٍٛ بانخٓاباث نالسخعًال نًذة سُت ٔاحذة. ل انخاسجٙ نهحكت ٔخظٕطاً يشع انجذس٘ انًائٙ.جهذٚت فاٚشٔسٛت. فقذ اربج َجاحّ كًسخحضش نالسخعًا introduction : volatile oils are contained largely in the plant, they are secreted in oil cells, in secretion duct or cavities or in glanular bairs. (1) these oils are used for their non-therapeutic action; flavoring e.g oil of lemon, in perfummry e.g oil of rose or as starting materials for the synthesis of other coumpounds e.g oil of turpentine. for therapeutic purposes they are administered externally as inhalation for catarrhs of the respiratory tract e.g thyme, eucalyptus and anise; as gargles and mouth washes e.g thymol and as transdermally for example many essential oils including those of lavender, rose mary and bergamot are employed in the practice of aromatherapy (1, 2) . it was found that the oils with high phenol content like clove and thyme have antiseptic, antibacterial and antifungal actions, while those with ether as chief constituent produce mild antispasmodic, mild insecticidal in addition to antibacterial action e.g funnel and anise (2, 3). the constituents of many volatile oils are started to interfere with respiration and electron transport in a variety of bacteria, hence accounting for their use in food preservative and in cosmetic preparations (1) . pharmaceutically these volatile oils can be used as extract, tincture or using the dried parts of the plant itself to be prepared for external use e.g thyme (thymi herba), cresping thyme (serpyili herba) wild oregano (origanie herba) and eucalyptus (oleum eucalypti) (4,5) many formulations can be used locally (external use) which include: collodions, liniments, lotions, paints, ointments and creams. (6) lotions which are liquid preparation are easier to apply and less messy than many semisolid external preparation, they frequently contain suspended particles or emulsified liquid droplets. depending on wether they are solid-liquid or liquid-liquid dispersions, therefore lotions can be classified as suspensions or emulsions (6) . medicinal lotions are antiseptic and germicidal, they are used in the treatment of skin diseases and as cooling and mildly anesthetic applications for skin irritations. e.g calamine lotion and benzyl benzoate lotions n. f. (6, 7) . aim of the work to prepare topical pharmaceutical liquid dosage form (lotion) from natural plants; arial part of thyme and fruit part of anise for topical applications. 1 corresponding author : e-mail issraapharm @yahoo.com received : 4/6/2006 accepted : 4/3/2007 mailto:suhadsami04@yahoo.com 9 materials and methods : materials and instruments anise (pimpinella anisum) dried fruits plant. thyme (thymus vulgaris) the stripped and dried leaves and flowers (aerial plant) (2) earial plant .diethyl ether, ethanol 96%, anisaldehyde spraying reagent (bdh chemical ltd, england), propyl paraben, methyl paraben, veegum powder, sodium carboxy methylcellulose, xanthan powder (e-merk, darmstadt, west germany), rose mary spirit, (judex laboratory reagents, england). electrical stirrer or mixer, voss instrument, meldon, essex, england ,tlc silica gel plate , oven. preparation of stock dispersion of suspending agents .(8) stock dispersion of each suspending agent used (sodium carboxy methyl cellulose, sodium lauryl sulfate, veegum and xanthan gum) was prepared by dispersing (0.8,1,2.5 and 5gm) of the suspending agents, respectively in 75 ml of distilled water (d.w) using an electric mixer at 150 r.p.m. the volume of dispersion was made up to 100ml with d.w, the resultant dispersions were allowed to hydrate for 24 hrs. preparation of lotion. (9,10) the preparation of lotions were shown in table (1) table (1): preparation of lotions formula 1 formula 2 materials quantities materials quantities thyme and anise powder (1:1) 1 gm thyme and anise powder (1:1) 1gm sodium lauryl sulfate 0.25% w/v veegum 2.2%w/v sodium carboxy methyl cellulose 0.5% w/v xanthan gum 0.24% w/v glycerine 5 ml glycerine 5 ml alcohol 96% 15 ml alcohol 96% 10 ml rose mary spirit 0.05ml rose mary spirit 0.01 ml methyl paraben 0.18gm methyl paraben 0.18 gm propyl paraben 0.02gm propyl paraben 0.02 gm d. w. q. s to 100 ml d. w. q. s to 100 ml the dried fruits of anis and the dried aerial parts (leaves and flowers) of thyme (1:1) were grinded in electrical grinder then passed through sieve 0.315inch /pore. fine powder for these parts, methyl paraben and propyl paraben were levigated with glycerine, alcohol and a part of prepared dispersions. the remaining amount of dispersion was added in divided portions to the mixture. the mortar was rinsed several times with d. w and the rinsed volume of dispersion was added to cylinder. finally, rose mary spirit was added, followed by sufficient quantity of d. w. to make up to the final volume (100 ml). sedimentation volume (f) fifty ml of each formula 1 and 2 was diluted with d.w. to a volume of 100 ml in a stoppered graduated cylinder. the lotions were shaken vigoursly to ensure uniformly then left undisturbed. the sedimentation volume which occurred under the influence of gravity was measured after a period of 48 hours. (11) f is the ratio of ultimate height of the sediment as lotion settles in a cylinder (h) to the initial height of the total lotion (ho) (11) . h thus f= h0 when the ultimate volume of the sediment is smaller than the original volume of lotion, the sedimentation volume is less than 1. if this value can be made to approach unity, the product becomes in flocculation equilibrium and show no supernatant on standing so it is pharmaceutically acceptable. therefore 50 ml of each the selected formulas should be diluted with distilled water to a volume of 100 ml in a stoppered cylinder. the lotion was shaken vigorously to ensure uniformity, then left undisturbed. the sedimentation volume which occurred under the influence of gravity was measured every 4 hours for a period of 48 hours. (10) resuspendability of lotions (12, 13) : the resuspendability of formula 1 and 2 was evaluated quantitatively. the efforts of redispersion required to convert the sedimented system to homogenous lotion by shaking the cylinder manually rated as resuspendable, resuspendable with difficulty or not resuspendable. rheogram (11,13) : rheogram was obtained at 40 0 c with brook field dv-ii+pro viscometer which read viscosity versus shear rate. assay of the volatile oils (14, 15) : tlc technique was used to determine the active ingredients present in the prepared lotion in comparison with standard powder of 2 plants (1:1) which was done by diluting the lotion to 200 ml using d.w, filtering, then the powder on filter paper was macerated in ether for 3 days. on the other hand 0.5 gm of standard powder (1:1 dried from of anise and dried aerial parts of thyme was also macerated in ether over night, then the products of both were concentrated by evaporating of ether, then tlc technique was done using benzene and chloroform (1:1) as mobile phase, while anisaldehyde was used as spraying reagent to calculate the retarded factor (rf) value of spots produced of both lotion and standard powder. rf value is directly propotional to the remaining quantities of active ingredients. stability and shelf life (13) : iraqi j.pharm.sci., vol.16 (1) ,2007 anise-thyme lotion for chicken pox. 10 the same method mentioned above was used in wich the total amount of stored lotion (50 ml) was diluted, filtered and macerated in ether then tlc technique was done to determine the stability of prepared lotion stored at 40 0 c, 50 0 c and 60 0 c for four months by measuring the rf values of all samples by calculating their quantities in comparison with standard lotion stored at room temperature. preliminary clinical study: the prepared lotion was applied topically to 10 patients twice daily those with skin disease (allergy, infection, chicken pox …) (1). statistical analysis: analysis of variance anova table and x 2 test (chi-square) was used to examine the effect of some parameters. results and discussions : formulation of the lotion: lotions are usually suspensions of solids in an aqueous medium. a few lotions are, in fact, emulsions or solutions (6) . a wide variety of ingredients may be added to the preparation to produce better dispersions (6) . methyl cellulose or sodium carboxy methyl cellulose will localize and hold the active ingredient in contact with the affected site and at the same time be rinsed off easily with water. however they are usually added to lotion, cataplasms and burn medications to produce tough and flexible films. surfactants as sodium lauryl sulfate can also be used in proparation of lotions to induce the flocculation of particles in dispersion (i.e formation of a loose aggregation of particles). (7,13). therfore formula 1 was prepared using sodium lauryl sulfate and sodium carboxy methyl cellulose as a suspending agents at concentrations of 0.25% w/v and 0.5% w/v respectively (16). while in formula 2, the veegum and xanthan gum combination was used to control flocculation (17) the following excipients additives were also added to the prepared lotions: alcohols for its drying and cooling effect, glycerine to keep the skin moist for a considerable period of time, rose mary spirit as an odouring agent, methyl paraben and propyl paraben as preservatives (7, 18). sedimentation volume (f) and resuspendability: formula 1 had f=0.2 and was resuspended while formula 2 had f=0.96 and was easily resuspended (19) . depending on physical properties, formula 2was selected to study its rheogram, shelf life and clinical effect. rheogram: rheogram of formula 2 is represented in figure (1). the profile showed that the viscosity of formula 2 lotion decreases with increasing rate of shear, therefore, the results illustrated that the selected formula exhibited pseudoplastic flow due to the blend of veegum and xanthan gum. this is resulted to get a formula with good viscosity, physical stability and smooth flow characteristics. such blend was well suited to stabilizing all types of fluid suspensions and lotions (17, 19, 20) . figure (1): rheogram of formula 2 study of shelf life (t 10%) (9, 8, 13): the accelerated studies at higher temperatures (40 0 c, 50 0 c, and 60 0 c) were employed to predict the breakdown of active ingredients that may occur over prolonged periods of storage at normal shelf condition as shown in figures 2 and 3. the loss of anise and thyme active ingredients is directly propotional to the concentrations remaining with respect to time, it is called a first order reaction. figure (2): accelerated stability study of anise crude extract in prepared lotion at elevated temperatures 1.955 1.96 1.965 1.97 1.975 1.98 1.985 1.99 1.995 2 0 2 4 6 time (months) l o g % r e m a in in g 40 o c 50 o c 60 o c 0 100 200 300 400 500 600 700 0 50 100 150 viscosity (cp) s h e a r r a t e ( r p m ) iraqi j.pharm.sci., vol.16 (1) ,2007 anise-thyme lotion for chicken pox. 11 figure (3): accelerated stability study of thyme crude extract in prepared lotion at elevated temperatures. the mathematical expression is : -dc = kc dt where c is the concentration of intact active ingredients remaining. t is time (dc/dt) is the rate at which the intact active ingredients degrades, and k is the specific reaction rate constant. the integrated and more useful form of equation: kt log c = + log co 2.303 the degredation rate constants (k) were calculated from slopes of straight lines. these degradation rate constants are summarized in table(2). table (2): degradation rate constants (k) and rf values of both anise and thyme crude extracts in formula 2 at different temperatures. temperatures (c 0 ) k(month -1 ) rf anise thyme anise thyme degree active ingredients of crude extracts 40 17 x 10 -3 10.6 x 10 -3 0.93 0.95 50 25.5 x 10 -3 15.6 x 10 -3 0.89 0.93 60 38 x 10 -3 24 x 10 -3 0.85 0.91 25 8.6 x 10 -3 6.2 x 10 -3 to determine t 10%, arrhenius plots were constructed to predict the degredation rate constants at 25 0 c (k25) for both anise and thyme crude extracts. figures 4 and 5 predicted the rate constants of formula 2 which were found to be equal to 8.6 x 10 -3 and 6.2 x 10 -3 month -1 respectively. 0.104 thus t10% = . k25ºc the shelf lives of both anise and thyme crude extracts in formula 2 were 1.0 and 1.3 years respectively. therefore the expiration date of formula 2 was about 1 year. figure (4): arrhenious plot for shelf life estimation of anise crude extract in the lotion. 1.92 1.93 1.94 1.95 1.96 1.97 1.98 1.99 2 0 2 4 6 time (months) l o g % r e m a in in g 40 o c 50 o c 60 o c 1 10 100 2.9 3 3.1 3.2 3.3 3.4 1/t x 10 3 (kelvin -1 ) k x1 03 (m on th -1 ) 3.35 1 10 100 2.9 3 3.1 3.2 3.3 3.4 1/t x 10 3 (kelvin -1 ) k x1 03 (m on th -1 ) 3.35 iraqi j.pharm.sci., vol.16 (1) ,2007 anise-thyme lotion for chicken pox. 12 figure (5): arrhenious plot for shelf life estimation of thyme crude extract in the lotion. as shown in scheme 1 (21) , thyme extract contains chemical materials thymol and carvacrol which known as phenol types that are held responsible for antiseptic properties of thyme. (3) thyme extracts according to the results shown in table (2) were significantly (p<o.o5) more stable than anise extracts which also have anti-infection and anti-inflammation effects .while anise extracts which contain fenchone (a bicyclic monoterpen) and anisatin ketons are affected by accelerated studies at higher temperatures (1,21) . scheme (1) preliminary clinical study: the preliminary results of clinical study shown in table 3 suggest that formula 2 can be used for treatment of infecious viral skin disease such as chicken pox which was caused by varicella zoster virus (vzv); this viral disease was highly contagious and common disease throughout the world, most patients ages with chicken pox were ranged between 6-10 years (22). significant period healing differences (p<0.05) depending on age with rapid clinical improvement was achieved during period of treatment (7-14 days) due to the antimicrobial, antifungal and germicidal properties of anise-thyme crude extracts due to essential oil compositions (thymol, carvacrol, anisatin and fenchone) with disappearance of all signs of this disease when this lotion was given topically two times/day with antipyrol syrup (23, 24, 25) . table (3): the results of preliminary clinical study using formula 2 on 10 patients with infectious skin diseases. -the degree of healing depend the relief of the patients skin from itching, redness and pustules. conclusion: crude extracts from the dried fruits of anise and the dried aerial parts of thyme were used as natural plants for preparing of successful anisethyme lotion for topical infecious skin diseases. formula 2 with veegum and xanthan gum suspending agents was the selected formula for preparing lotion since it was flocculated, poured, resuspended easily with f value 0.96, and it had good odor, color with expiration date of 1 year. addition it was very effective in many infecious and virus skin diseases mainly itching and chicken pox. acknowledgement: the outhors gratefully acknowledge d. fakher fakhri for his aids in preliminary clinical study. p a t ie n t n o . a g e (y e a r ) s e x d is e a s e d o s e t o p ic a l l y d u r a t io n o f t r e a t m e n t (d a y ) r e s u l t s 1. 8 male chicken pox apply two times /day 14 rapid healing 2. 10 male chicken pox = = = 3. 7 male chicken pox = 10 = 4. 9 female chicken pox = 14 = 5. 7 male chicken pox = 10 = 6. 4 male chicken pox = 7 = 7. 7 male chicken pox = 10 = 8. 6 male chicken pox = 810 = 9. 7 female chicken pox = 10 = 10. 10 male chicken pox = 14 = ch2 o fenchone oh oh thymol carvacrol oh o oho ho o o ho anisatin iraqi j.pharm.sci., vol.16 (1) ,2007 synthesis of anti-inflammatory aromatic schiff bases 13 references : 1william, c.e., pharmacognosy, 2002; 15 th edition, 253, 256, 259, 264. 2pdr for herbal medicines, medical economics company, 1998; 1 st edition, 1036-1184. 3carol, a. n., linda, a. a., herbal medicines and guide for health care proffessional, 1996; 256. 4remigton, the science 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between xanthan gum and colloidal magnesium 21. aluminium silicate j. soc-cosmet-chem.. 1992; 43(jan-feb), 1-12. 22. geneva, who monographs on selected medicinal plants, 1999; vol. 1, p.262 . 23. kanra, g., tezcan, s., badur, s., varicella seroprevalence in a random sample of the turkish population, vaccine, 2002; 20 (sep-octr), 1425-8. 24. fujimatu, e., ishikawa, t., kitajima, j., aromatic coumpound glucoside alkyl glucoside and glucide from fruit of anise, phyto chemistry, 2003; 63 (jul), 609-16. 25. angelini, l. g., carpanese, g., cioni, p. l., essential oils from mediterranean lamiaceae as weed germination inhibitors, jagric. foodchem., 2003; 51 (oct), 615864. 26. kalemba, d., kunicka, a., antimicrobial and antifurgal properties of essential oils, currmedchem., 2003; 10 microsoft word bagpha05114 _3_ iraqi j. parma. sci., vol.14, 2005 20 -------------------------------------------------------------------------------------------------------------- formulation of econazole nitrate as a topical solution laith hamza samin received: 14.01.2002 accepted: 05.08.2002 abst ract : econazole nitrate (en) is considered as the most effective agent for the treatment of all forms of dermatomycosis caused by dermatophytes. this study was carried out to formulate a stable econazole nitrate solution for a topical use through preparation of different formulas and selected the most suitable one. the results indicated that the use of propylene glycol and ethanol as a vehicle for en which is very slightly soluble in water gave amore stable formula as en topical solution, with a shelf life of about 3.15 years .the data also indicated that the light accelerated the degradation of en, while the type of container (glass or plastic) had no effect on the rate of drug. the overall results of this study suggest that the selected formula could be used as a topical solution for en. :الخـالصـة عالج جمیع أشكال الفطریات الجلدیة الناتجة الدواء فيیعتبر األكثر فعالھ الستخدام ھذا ) نترات االیكانازول(أن محلول عل�ى ش�كل ) لنت�رات االیكان�ازول (كان ھدف الدراسة أیج�اد ص�یغھ تركیبی�ھ مس�تقره .الفطریةعن العوامل المسببة لتلك االلتھابات .یر عدد من الصیغ التركیبیة واختیار الصیغة األكثر فعالیھ من خالل تحض محلول، وال�ذي یك�ون قلی�ل ) لنت�رات االیكان�ازول (كم�ادة ناقل�ھ ) الكح�ول االثیل�ي (و)ب�روبلین كالیك�ول (النتائج أثبت�ت أن اس�تعمال النتائج أیضا إلى أن الضوء أشارت .سنھ 3,15الذوبان في الماء أعطى صیغھ تركیبیھ اكثر استقرارا لھذا المحلول وعمر صالحیة نت�رات (ل�یس ل�ھ ت�أثیر عل�ى اس�تقراریة ) بالس�تك أو زج�اج (نوع الحاویة ا، بینم)نترات االیكانازول(ساعد في تسریع عملیھ تحلل نت���رات (أن حص���یلة نت���ائج ھ���ذه الدراس���ة أظھ���رت أن الص���یغة التركیبی���ة المخت���ارة یمك���ن اس���تعمالھا لتحض���یر ). االیكان���ازول .على شكل محلول)لاالیكانازو iraqi j. parma. sci., vol.14, 2005 21 -------------------------------------------------------------------------------------------------------------- introduction: econazole nitrate is (rs)-1-[2,4-dichloro-b (p-chorobezyloxy) phenethyl] imidazole nitrate (1) . it is a white or almost white crystalline powder.m.ρ. about 164c o , with decomposition. very slightly soluble in water and ether: soluble 1 in 125 of ethanol (96%) 1in 60 of chloroform and l in 25 of methanol (1, 2) . econazole nitrate is used for the topical treatment of all forms of dermatomycosis caused by dermatophytes like trichophyton rubum, trichophyton mentagrophytes, trichophyton tonsusrans which cause tinea pedis, tinea cruris, and tinea corporis respectively(3). it is used for dermatomycosis caused by yeasts like candida albicans and candida guilliermordi (4) . econazole nitrate is available in a variety of dosage form such as skin cream (alone or in combination with triamcinolone), skin solution, skin lotion and spray solution (3) . it has been proven to be effective in the presence of mixed infection. the antibacterial effect of the preparation offers an additional outstanding advantage. the purpose of this work is to formulate a stable econazole nitrate solution for topical application using different types of vehicles like propylene glycol, polyethylene glycol (peg), glycerol and ethyl alcohol which they are inert nontoxic and maintain the solubility of drug. furthermore, the effects of container type and light on the stability of the prepared formula are studied as well as its shelf life. materials, instruments and methods materials econazole nitric powder (alesmalia company-iraq), methanol, polyethylene glycol, glycerol, ethanol (bdh, chemicals, ltd, pool, england), hydrochloric aid (riled de haen ag seelze, hanover, germany). standard buffer ( ph 4 and 7 ) . propylene glycol (merk germany) . instruments spectrophotometer (pye-unicom sp-8-100 england model 292 mk2 england) sartorius balance (werke-gmbh, type 2842, germany – gmbh,type 1265 nip,w.g autoclave (vebstatex jimenau , type sd505 ) ph-meter(schott-geate,type cg820,w.germany. method assay of econozole nitrate topical solution: a simple and fast procedure for the determination of (en) based on the uv spectrophotometry (5) , was applied as follows. .hno3 iraqi j. parma. sci., vol.14, 2005 22 -------------------------------------------------------------------------------------------------------------- test solution: 5ml was measured and diluted with pure methanol to 100ml. reference solution: 50mg of pure (en) was exactly weighted and transferred into 100ml volumetric flask. about 50 ml of pure methanol was added, shacked well until it was dissolved, completed the volume to 100ml with methanol and mixed. the uv absorbency was then recorded in 1cm quartz cell for the reference and test solutions against pure methanol. the quantity of econazole nitrate in topical solution was calculated using the following equation. % econazole nitrate = (acorr sample x mg standard) / (acorr standard xmg sample x 10) (acorr= a271.5 _ a278.5) formulation of econazole nitrate as a topical solution. different formulas of econazole nitrate for topical solution (1%w/v) were prepared as shown in table-1. formu la econazole nitrate (gm) propylen glycol (ml) polyethylere glycol (ml) glycerol (ml) ethanol a.s.to (ml) a 1 ---75 ---100 b 1 ---35 40 100 c 1 50 12 13 100 d 1 50 25 ---100 e 1 75 ------100 table-1 schedule of different formulations of econazole nitrate as a topical solution all formulas (a,b,c, dand e ) were composed of econazole nitrate (1%) and completed to 100 ml volume with ethanol(95%). stability study stability of (en) in a normal and accelerated conditions was carried out by incubating samples of formula e in ovens at 40,45 and 50 o c for 120 days. samples were taken and assayed for their drug content at suitable time intervals (2 weeks) using spectrophotometry method. effect of light this factor was studied by placing formula e in a clear glass container and subjected to light at room temperature for 2 months (6, 7) . samples were taken every two weeks and analyzed for their drug content. effect of containers the functions of container are to maintain the quality, safety and stability of its content (8, 9). various samples of formulae were stored at different conditions (room temperature, 40 o c) in glass bottles and plastic bags for 120 days. samples were taken and assayed for their drug content every one month using spectro photometric method. quality control the concept of total quality control refers to the process of striving to produce perfect product by a series of measurements. the quality control tests that were done, included density, ph, drug content and appearance of solution iraqi j. parma. sci., vol.14, 2005 23 -------------------------------------------------------------------------------------------------------------- results and discussion method of assay in this study the ultraviolet spectrophotometry was used for the quantification of (en) as a topical solution, the data indicated that the method gave acceptable results since a good standard curve with a correlation of 0.996 was obtained as seen in fig.1. iraqi j. parma. sci., vol.14, 2005 24 -------------------------------------------------------------------------------------------------------------- the u.v. spectrum of (en) is shown in fig.2. the profile indicates that, the drug has maximum absorbance at 271.5 nm and minimum absorbance at 278.5nm in methanol as it has been reported. fig 2 uv spectrum of econazole nitrate in solvent methanol at 25 c formulation of econazole nitrate table –2 shows the percent remaining after 4 months and rate of hydrolysis of the five formulas calculated from the slope of first-order kinetic plots in order to select the most stable one. formula method of assay remaining (90-110)% k40c (x10 -3 )day -1 a uv 90.3 0.81 b uv 85 1. 3 c uv 83 1. 5 d uv 94.9 0.41 e uv 98.6 0.16 table-2 the percent remaining after 4 months and degredation rate constant (k) of econazole nitrate in formulas a, b, c, d and e at 40oc iraqi j. parma. sci., vol.14, 2005 25 -------------------------------------------------------------------------------------------------------------- the data indicate that minimum rate of hydrolysis of (en) was achieved using 75% propylene glycol as a solvent (formula e) since it has the lowest rate constant (0.16x0-3day-1). this may suggest that propylene glycol is the most suitable vehicle for the formulation of (en) topical solution. it acts as a solvent for many dermatological drug and ensure homogenous dispersion of drugs used in low concentration (10) . stability study stability study in normal and accelerated conditions was carried out (11) . table-3shows the percent remaining of (en) at room temperature for formula e after storage. formula e storage time appearance method of assay % remaining (90-110)% zero time clear liquid uv 100.1 2 months = uv 100 4 months = uv 100.1 6 months = uv 99.0 table-3 the percent remaining of econazole nitrate (formula e) at room temperature after 4 months. the effect of temperature on the rate of hydrolysis of (en) was also studied using different accelerated temperatures (40,45and 50 o c) for 120 days. the degradation of (en) in formula (e) follows a first-order kinetics, since straight lines were obtained when the logarithm of percent remaining of (en) was plotted versus time as shown in fig.3. x a t4 0 o c ^ a t4 5 o c . a t5 0 oc iraqi j. parma. sci., vol.14, 2005 26 -------------------------------------------------------------------------------------------------------------- the degradation rate constants (k). at (40, 45 and 50 o c) were calculated from the slopes of lines as illustrated in table 4. temp k x10 -3 day –1 40oc 0.200 45 o c 0.268 50 o c 0.420 25oc 0.9 table-4 degradation rate constants (k) of econazole nitrate in formula e at different temperatures to determine the expiration date (t10%) at 25 o c, arrhenius plot (12) was constructed to predict the degradation rate constant at 25 o c (k25) for formula e as shown in fig.4. iraqi j. parma. sci., vol.14, 2005 27 -------------------------------------------------------------------------------------------------------------- since the degradation of drug follows first-order kinetics, therefore, the expiration date can be calculated using the following equation. t10%= 0.104/ k25 it appears from the results that the drug in formula e. (selected formula) has a t10% value of about 3.15 years. effect of light: the effect of light on hydrolysis of econazole nitrate was studied by placing the selected formula (formula e) in clear and dark containers at room temperature for 2 months. as shown in figure 5, there is a linear relation ship for logarithmic plot of the percent remaining of econazole nitrate versus time when the drug was stored in clear and dark containers, indicating that the degradation of drug follows first order kinetics figure5. fig5 the effect of light on the rate of hydrolysis of econazole nitrate in formula e at room temperature. x light x x x x 15 30 45 60 time (day) dark iraqi j. parma. sci., vol.14, 2005 28 -------------------------------------------------------------------------------------------------------------- the results indicate that the percent remaining of drug was 95.91% when exposed to light at room temperature after 2 months. this is in consistent with the reference which state that en is known to be affected by light and should be stored in a well closed container protected from light(13). on the other hand, the samples which were stored in dark containers showed no detectable change in the percent remaining when exposed to the same condition effect of container table 5 shows the percent remaining of en when placed in glass and plastic container at different conditions (room temperature, 40 o c) ,for 4 months. the result showed that no detectable change in percent remaining especially when stored at room temperature. formula e %remaining k 40 oc (x10-3)day-1 glass container 97.7 0.20 plastic container 97.8 0.20 table-5 the effect of containers on the stability of econazole nitrate at 40 o c after 4 months . quality control the selected formula (formula e) was subjected to many types of product control test for topical solution these tests include appearance of solution , drug content ,ph, and density. the prepared product passed all these tests successfully as shown in table 6. type of test results formula e requirement drug content 100.1% 90 –110 % ph 3.5 – 4.5 ---- clarity of solution clear colorless solution clear colorless solution density(20 ºc) 0.975 – 0.995 g/ml 0.995 g/ml table-6 results of the quality control test for the selected formula (formula e) of econazole nitrate this indicates that the method of preparation is efficient and effective. conclusions (en) is a novel antimycotic preparation for topical use. after conducting this investigation on econazole nitrate as a solution dosage form for topical application, one can conclude the followings: 1. formula e consider the most stable formula since log percent remaining for this formula was 1.995(99%) after incubation in 40ºc for 4 months 2. propylene glycol is an excellent vehicles used to increase the solubility of medicament, it also act as a solvent for en to ensure homogeneous dispersal of drug used in low concentration (10,14) . 3. it was noticed that there was a significant effect of light on degradation of econazole nitrate 4. econazole nitrate show a good linearity of uv response which is simple and fast procedure for determination of econazole nitrate in the drug substance based on uv. 5. the use of plastic or glass container for econazole nitrate drug had no effect since the same percent remaining were seen when drug solution was stored the drug at different temperature. iraqi j. parma. sci., vol.14, 2005 29 -------------------------------------------------------------------------------------------------------------- 6. the product passed all the quality control tests successfully which indicate the efficiency of the method of preparation. 7. the expiration date of en drug was found to be 3.15 years. 8. further study will be done to formulate econazole nitrate as a topical cream with triamcenolone iraqi j. parma. sci., vol.14, 2005 30 -------------------------------------------------------------------------------------------------------------- references: 1british pharmacopoeia, p.245, 1998. 2clark’s, isolation and identification of drug, 2th. ed, the pharmaceutical press, london; p.579, 1986. 3physician desk references 1999. 4scholar-m; prediel-h; korting –hc, light and electron microscopic finding in amodel of human coetaneous candidacies, journal of drug targeting, 1999; 6(5) 361 – 372. 5sastry, b.s; rao, j.v, spectrophotometric method for determining of econazole nitrate, indian drug, 1992; 29 (mar) ,277,280. 6the arab good manufacturing practice guilders “gmp” 1995 by the arab union of the manufacturers of pharmaceutical and medical appliances (aupam). 7guidance for industry (q.b photo stability testing of new drug substances and products).november 1999. 8marcie, k.n., “utilization of nicotinamide as a complexing agent in the formulation of diazepam oral solution,” phd. thesies baghdad university, college of pharmacy, 1996. 9remingtin’s pharmaceutical science 7 th . ed. mark publishing company, easton, pennsylvania. chap.80,87, 1985. 10carter, s.j, the ointment, pastes and jellies, chap.13 in “dispensing for pharmaceutical students “12 th ed. churchill livingstone 1987. 11wolfgang grim, extension of the international conference on harmonization tripartite guideline for stability testing of new drug substance and product to countries of climatic, zones iii and iv. drug development and industrial pharmacy, 1998; 24(4), 313 – 325. 12lachman, l., liberman, h.a. and. change j.l., “the theory and practice of industrial pharmacy” 3 rd ,er., lea and febiger publishing company, philadelphia (1986). 13the united states pharmacopoeia, vol 32,p.1189. 14ford, j.l., parenteral product, chap. 21in” pharmaceutics, the science of dosage form design, by alton ,n.e, churchill livingstore, 1988. abstract : الخـلاصـة: formula method of assay ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 23 synthesis and characterization of 2(2-tetrahydropyranylthio) methyl cyclopropylamine zuhair a. muhi-eldeen*, samira f. hassan** rece ived 2-6-2002 acce pted 13-6-2004 abstract 2(2-tetra hydropyra nylthio) methyl cycloprop yl a mines we re s ynthes ized fro m a llylme rc apta n thro ugh se veral step s. the struc ture s of the inte rme diates a nd the final p ro duc ts where co nfirme d thro ugh ir, nmr and elemental a nalysis, these comp ounds ma y be of v alue in the treatme nt o f dis eas es where free ra dica ls a re implicated in their pa thogensis, s ince the thio and the amino groups o f the synthe sized co mpounds may a ct a s fre e ra dical sc ave ngers. الخـــــــــــــــالصة  ــ ة من امينات اـل موع و -2( -2لقد تم تحضير مج ن ثاي را وبي هيدر را ركابتان وذل) تت مي روبيل من الليل كلو ب خالل عدد مثيل ساي ك من طوات مـراء . من الخ عة تحـت الح ه اش ة تقني واسط هائية ب والن ة طي كبات الوس غناطيسـي (ir)تم تشخيص المر ن الم رني ف اـل -h)وطـي nmr ) عناصر االولية خيص ال e) وتش le me ntal a nalys e s معالجة . ( ة في ن ذات قيم مكن ان تكو رة م ركبات المحض هذه الم الل مراض المتسببة من خ طليقة اال ور ال حرر الجذ fre)ت e radic als) . مكن ان كبات م مر هذه ال ي المينو ف وا ع الثايو مي وجود مجا ن ال راض ور الطليقة والمسببة لألم جذ طة لهذة ال هب ون م .تك introduction free ra dicals hav e b ee n implicated in many dise as es, among these are athe ro scle ros is, rhe uma to id a rthritis, ca ta ra cts, ne oplas tic dis eas s, diabe tic retinopa thy, parkins on's , alzheimer inflammatory disea se s of gas tro-intes tina l tra ct a nd a ging1-5. free ra dicals are de fine d a s ato ms or molec ules tha t co ntain one o r more unp aire d elec trons and are spe cies that c ause me ta bolic disturba nce s and ce ll injury b y interacting with mac ro molecules a nd othe r c ellula r co ns titue nts such as proteins , lipids , ca rb ohydrates and dna res ulting in a variety o f b io lo gical co nse quenc es , including c ellula r and tis sue da mage , mutatio n c arc inogens is and ce ll de ath6. the obs erva tion that 2 merca ptoethylamine , 2 –me rc atoprop ylamine , disulfide , thioe thers a nd sulfoxid es 7were cap ab le in protec ting a nimals a gains t fre e ra dica ls ge nerate d as a re sult of ionizing ra diatio n promote d o ur interes t to synthe size2(2-te trahyd rop yranylthio ) methylc yc lo propyla mine 1 . the thioethe r and the amino groups in 1 o r the c orre sp ond ing “s ulfhydryl and amino gro ups in the ir expec te d major me ta bo lites may ac t co ope ra tive ly a s free rad ic al sca ve nge rs ”. the re fo re the se co mpound s may be utilize d se le ctiv ely to treat one o r mo re of the previously me ntio ned d is ea se s. synthetic material allylmercaptan , pto luenesulfonic acid , dihyd ropyran e thldiazoce ta te , we re obtained fro m aldric h che mic al co. (milwa ukee , wi ,us a ) . analytical equipment melting p oints were de te rminated by using a calibrated thoma s ho ove r me lting p oint app aratus .ir s pec tra were re corde d us ing a unica m sp 3 00 sp ectrophotome te r . nmr spe ctra were obtaine d us ing a va riant ft80 a spe ctro meter. chemica l shifts are re ported as part p er million downfield fro m te trame thylsilane as inte rnal standard for hnmr spe ctra . eleme ntal mic ro analysis we re perfo rmed by h.ma liss a and g.reute r,frg. pyran ( 3) .allylthiotetrahyro -2 allylme rc apta n ( 3,74 g ,1 mo le ) and 200 mg of p-toluene sulfonic ac id we re plac ed in a 500 ml rbflas k fitte d with a reflux c ond ens er and ma gnetic s tirre r . dihydro pyra n ( 84g , 1 mole ) wa s add ed drop wis e . the reac tion mixture wa s hea te d on a steam ba th for 1 0 minutes . after he ating fo r 5-10 minutes , a vigorous exothe rmic rea ctio n s ta rted and continued d uring the add ition of dihyd ro pyran . afte r 1 1/2 hours , refluxing was s to ppe d and potas sium ca rb ona te ( 1.0 g) w as ad ded . the mixture wa s stirred a t ro om tempe ra ture for 1 hour , filte re d and fra ctio nally distilled yielding 80 .2 g . ( 50% ) o f 2a llylthiotetra hydropyra n ( 3) b .p . 42 -4 4 ( 0.1 mm ) * dep ar tme nt o f med ic ina l c hemistry ,co llag e of phar macy,univer sity of petra , aman -j ord an; ** de par tme nt o f ph arma ceu tica l ch emistry, co llag e of phar macy,univer sity of bagh dad , bagh dad -i ra q ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 24 infrared ( nea t , c m-1 ) showed b ands at 308 0 (= ch2, stre tch ); 294 0, 2 860 , 285 0, (ch2, stretch ); 16 35 ( c = c , s tretc h ) ; 1 180 , 10 80, 104 0, 101 5 (tetrahydropyra nyl group ) a nd 94 0 ( s ch2 ) nmr (dchloroform , δ ) 1.28 2.18 ( multip le t , 6 h , (ch2 )3 ); 3.20 ( multiplet , 2h, s-ch2 ) ; 4.09 a nd 3 .5 ( multiplet , 2 h , och2) ; the v inyl protons ap pea r a s multip le ts o verlapp ing with ( och-s ) at 5.20 ( multiplet , 3h , c=ch2 , o-ch-s ) and 5.82 ( multiplet , 1h , c=ch ) ana l . calc ulated :found for c8 h14 os : c , 60.7 5 ;h , 8 .8 6 ; s , 20 .2 5 fo und : c , 60.63 ; h , 8.85 ; s , 2 0.47 . allythiotetarhyropyran -reaction of 2 (3) with ethyl diazoacetate in a 2 50 ml three ne cke d flas k provide d with a reflux conde nse r, dropp ing funne l and magnetic stirrer wa s plac ed 2 allythiotetra ahydrop yran (4 ,1 5.8 g , 0 .1mole ) and 5 0 mg o f cop per pow der . the mixture was s tirred rap id ly (16 0 16 5 c° , o il bath ) a nd the e thyl d ia zo ace ta te (1 1.4 g , 0 .1 ) was a dde d at suc h rate so a s to av oid a v igorous re action . after e thyl diazoac etate ad dition the evo lutio n of nitro ge n ce ase d . the reac tio n mixture was refluxe d distillate ( 4 0-60 c° a t 0.2 mm ) . the d is tilla te was a na lyzed b y ga s liquid p artitio n chro matgraphy which s hows the p re sence of se veral prod ucts . thes e prod ucts were te ntatively id entified as diethyl ma le ate , diethyl fumarate a nd the starting ma te rial . other products are a ethyl α-allyl-α ( 2etrahydropyranythio) acetate (5) , b.p . 72-74 c° at ( 0.01 5) mm , wa s identifie d by infrared and nmr s pec tra and eleme ntal a nalys is . the infrared s pe ctrum ( ne at , cm-1 ) s ho wed ba nd s at 308 0 (c=ch2 stretch ); 2 940 , 286 0 , 2 850 , (ch2stretch ); 17 35 (c=o, s tretch ) ; 164 0 (c=c) ; 10 80 ,105 0 , 1 020 (ch2 , te tra hyd ro pyranyl gro up ) , 940 (sch2 ). nmr (dc hloroform ,δ ) , 5.78 (multiple t , 1 h , hc=c ) ; 5 .2 4 (multiplet , 2h,c =ch2 ); 5.0 (multiplet ,1h,och s ) ; 4.16 a nd 4.0 (multiplet ,3h ,cooch2 , αcho o f the (ch2 tetrahydrop yranyl group ) ;3.5 (multiplet , 2h , β cho of the te tra hyd ro pyranyl gro up ,s ch -cooet ) , 2.57 (multip le t , 2 h,ch2 c=c ) , 1.45 to 2.0 (b ro ad , multiple ,6 h (ch2 )3) ; 1.25 (triplet , 3h, ch3 ) ana l . calculated : fo und for c12h 2 0 o3s:c , 59 . 0 1/. ;h , 8.19 ; s , 1 3.11. fo und : c,58.24 ;h,8.15 ; s,13 . 45 . b 2(2-tetrahydropyranylthio)methyl1-c arboe thoxycyclopropane(4) 5.5g (22.5%), of 4,was obta ined a s a colorless liquid , b.p.12 0-122 c˚ a t (0.2mm). gas liquid partition c hromatogra phy on 3.8% silico n gum rub ber (uc-w98 ) o n chromo so rb w(80 -1 00me sh), 4ft 0.25 in gla ss c olumn with column tempe ra ture 19 0c˚, injec tion pa rt te mpe ra ture 3 20c˚, d etec to r tempe ra ture 280 c˚, inle t p re ssure of 40 p si and c arrier gas (n2 ) flow rate of (6 0ml/min) sho wed two pea ks at 3.2 minutes (8 7%) tra ns -4 and 4 .0 minutes (1 3%) cis4.the mixture ha d an infrared spe ctrum (nea t,c m-1) that showe d bands a t 29 00, 28 60 (ch2, stretch); 172 0(c=o, stre tc h); 1 105 , 10 80, 104 0 and 1 015 (tetrahydropyra nyl gro up and cycloprop ane abs orptio n). nmr(d-chlo ro fo rm ,δ)0.71.2(mulip le t , 2h,ch2 of cyc lo propa ne ;1 .3 4(triplet, 3 h, ch3); 1.42.15(multip le t 7h(ch2)3, 1 h o f cycloprop ane α to ch2-s ); 2.58 (d oub le t, 2 h,s -ch2,jch,ch2-s = 6 .5 hz) 3.3(multiple t, 1h, chcyc lo propa ne α-to cooet); 3 .5 6(multiplet, 1h,β-cho of tetra hydropyra nyl group ); 3.9(multiplet, 1 h, α-cho, of te trahyd rop yrahyl gro up), 4 .1 4(qua rtet, 2h,ch2 of the ester group), 5.0(multiplet, 1h,o-ch-s ). an a l . c a lc u la te d : f o u n d f o r c12h20o 3s: c ,5 9 .0 1h ,8 .1 9 ; s ,13 .1 1. f ound: c,5 8.24;h,8.15 ;s ,1 3.50 2(2-te trahydropyranylthio) me thylcyclopropylcar boxyhydr azide(6) a s olution of (4 .8 8g,2.0x10-3mole) of 2-(2 -̀ te trahyd rop yranylthio )me thyl-1 carbo ethoxyc yclop ro pane 4 and 20 ml of 8 5% hyd ra zine hydrate was refluxe d fo r 24 ho urs. the mixture wa s co oled and he ld a t 0°c fo r 24 hours ; no prec ip ita te formed. the exce ss of hyd ra zine hydrate wa s re mov ed unde r reduced press ure affording a s emi-s olid tha t fa iled to crysta llize unde r v ario us conditions. the unrea cted e ster 4 wa s re mov ed by d is solving the crud e hyd ra zide in chlo fo rm. the hyd ra zide 6 was p re cipita ted with a nhydrous ether. a white s olid wa s obtained in et2o; this turned to a se mi-so lid up on remo val o f ethe r. the hyra zide 6 ha d a n infrared s pe ctrum (neat,cm-1) that s howe d bands a t 33 00 (nh2 , stre tc h ) ;29 20,28 30(ch2,stre tc h)166 0(conh, stre tc h); 110 5 , 10 80 ,1 040 and 102 0(te trahydrop yranyl group and cyc lo propa ne ring). nmr (d chloroform,δ)8.17(multip le t,1 h,conh) 4.97(multip le t,1h,o-ch-s); 4 .0 5 and 3.5 5 (multiplet,2 h,och2) ;2 .8 -3 .0 (multip le t , 3 h , ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 25 nh2 , ch -c yc lo pro pa ne) ; 2.6 (multiplet , 2 h , s-ch2),1.1-1.2(multip le t,7h,(ch2)3 and 1h,ch-of c yclop ro pane α-to ch2-s);0 .8 1.0 (multiplet,2h,ch2of c yclop rp ane ). this s emi-solid hyd ra zide was used witho ut furthe r purific atio n. 2(2tetrahydropyranylthio)methylcy clopropylamin a solution of (4 .6 4g,2 .0 x10 -2mole ) of 2 ( 2 te tra hyd ro pyranylthio ) methylc yc lo propylc arboxyhydrazid e 6 in 100 ml c hloroform wa s chilled to -5 c°(ic e-sa lt ba th). with ra pid s tirring, 1.2g(2 .0 x1 0-2 mole) of s od ium nitrite in 5 ml of water, follo wed b y 10ml of 1 0%hcl, were add ed . the rea ctio n mixture wa s allowed to s ta nd 5 -1 0minute s; the chlo roform layer was s ep arated a nd the aq ueo us layer wa s extrac ted once with 20 ml of chlo roform. the c omb ined chloroform frac tions were dried (na2so4) and filte re d. to this solution 50 ml of d ry tolue ne wa s add ed. the c hloroform wa s re move d und er reduce d pres sure and n2 ev olution co ntinue d vigo rously while he ating the rema ining to luene solution under reduce d pre ss ure on stea m ba th. afte r n2 e volution c eas ed a da rk b ro wn so lid ma teria l p re cipita te d (infrared , kbr, 228 0c m-1,n=c=o, 8). f ifteen ml of 25%me thanolic koh was a dde d to the to luene co ntaining the s olid is ocyanate and the mixture was refluxed a t 125 c° fo r 1 8 hours . after he ating, the red dish b ro wn mixture wa s extrac te d with to luene, drie d (na 2s o4) filte re d. the tolue ne wa s remo ve d under re duced pres sure . the res id ue was d is tille d affo rd ing 0 .5 g(26.7%) of cisand tra ns amine 1,b.p. 98 -1 00c° at ( 0.3mm).gas s iq uid pa rtition chroma tograp hy (glpc )a na lysis o n 3.8% s ilicon gum rubb er (ucw-98 ) o n chro moso rb – w (80 -1 00 mesh), 4ftx0.25 in glas s c ol tempe ra ture 17 5c°,injuc tio n pa rt te mpe ra ture 30 0 c°and d etec to r te mperatur 285 c° inlet press ure 40 ps i a nd ca rrie d ga s (n2 ) flow ra te o f (6 0ml /min) sho wed two pe aks at 1.28 minute s (8 5%) tra ns -1 and 2 .4 8 minutes (1 5%) c is -1 . the a mine mixture ha d a n infrared sp ectrum (nea t,cm-1) tha t s ho wed b and s a t 36 00 330 0(broad , nh2,s tre tc h ); 3 000 , 29 30 and 286 0 (ch2,stre tc h) 16 25-15 90(nh,be nding); 110 5,10 80,10 45 and 101 5 (tetrahydropyra nyl and cyc lo propyl ring). nmr (d-c hloroform, δ ) 4.97 (multiplet ,1 h , o-ch-s) ; 4.0 nd 3.5(multip le t,2h,o-ch2); 2 .6 ( multiplet, 2 h , s-ch2) 2.3 (multiplet, 1 h , ch , c yclop ro pane α to nh2) ; 1 . 2-2 (multiplet,7h,(ch2)3,1h,ch cycloprop ane αto ch2s) , 0.7 1.1 (multiplet ,2 h ,ch2 ,c yc lo prpane) ana l. calc ulated: found fo r c9h 17osn:c,57.75 ,;h,9.09 ;n,7 .4 8;s,17.11 . found : c , 57 .19 ; h , 8 . 84 ;n,7.03 ;s ,1 8.44. disussion and sresult the new co mpound 1 wa s prep ared a s dep ic ted in s che me 1 allymerca ptan 2 which s erves a s starting materia l was rea dily converted in 80% yield to 2-a llylthiotetrahydropyra n 3 through re action with 2,3-dihyd ro pyran in the pres enc e of p-toluenes ulfo nic a cid. the ir and nmr spe ctra were c onsistent with the as signed structure . tre atment of 3 with e thyldiazoac etate affo rd ed a mixture of trans and cis2-(2 te trahyd rop yranylthio ) me thyl-1 carbo ethoxyc yclop ro pane 4 a nd a s ulfo nium ylid e re arra nge ment prod uc t name lyethyl-α-allyl-α (2-te trahyd rop yranylthio ) ace ta te 5 in 81.4% and 1 8.6% yie ld a t 150 155 c° re sp ectively 8. ethyld ia zoa ce ta te rea cts with allymerca ptan to ge nerate tra ns and c is cyc lo propa ne derivative 4 through c arbene add ition to the do uble bo nd 9 and with thio ethe r group to form sulfonium ylid e 10. such sulfonium ylide is kno wn to unde rgo steve n's re arra nge ment, which de pe nds o n the structure of the ylide , may e ithe r invo lv e a n antrafac ia l 1,3-sigma trop ic re arra ngeme nt or suprafac ia l 1,5-sigma trop ic rearrangeme nt 11. in our cas e both 1,3and 1 ,5 re arra ngements affo rd ed the same comp ound 5. the mixture of 4 c ould no t be se pa ra te d by p hys ic al metho ds a nd was us ed as a mixture in the next ste p. re action of 4 with 8 5% hydrazine hyd ra te a fforde d the hyd ra zide 6 as a gummy solid in almo st quantitativ e yield; attemp ts to crysta llize this gummy hyd ra zide were uns uc ces sful. the purity of the c ompo und wa s determine b y gas s liq uid partition chro matogra phy (glpc ); the non-c rystalline hydra zide wa s then sub jucted to curtius re arra nge ment. the re arrange ment proce ede d through the a zide 7 and the is ocyanate 8 . the intermed ia te a zide 7 wa s detec ted by its ir s pe ctrum (con3, 2 15 0 c m -1). the iso cya na te 8 c ould b e is olated a s a brown solid which showe d a n ir a bso rp tion b and a t 228 0 c m-1 (n=c=o). the isoc ya nate 8 wa s re fluxed with 25% metha no lic koh to generate the des ire d transa nd ciscyc lo propyla mine 1 as a mixture in a ra tio of 8 5:15 re sp ective ly. the ir and nmr s pec tra were co ns is te nt with the a ss igned structure as discuss ed in the exp erime ntal part. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 26 references 1. j os eph , a.k., re ac tive oxyge n spe cies and the euro degene ra tiv e disorde r .ann. clin .and lab . sc i., 1 997 ,2 7,11. 2. francis , v.d., exce ss edrf no . a pote ntia lly dele te rious condition that may be invo lve d in acce lerated atheroge ne sis a nd other chro nic dis ea se states .gen pharmac 199 5,26 (4), 66 7. 3. cerutti ,p ., la rs son ,r ., and krukp itza , g., mec hanis ms o f oxd ant ca rc inoge nes is , genetic mec ha nisms in ca rc inoge ne sis a nd tumor progres sion, 1 0 th ed ,wily – liss , newyo rk ,19 90 , 69,. 4. barry ,h ., can oxid ativ e dna dama ge be use d as a bio marker of ca nc er ris k in huma n , f ree rad. res . , 199 8 , 2 9, 4 69 . 5. de nham, h., f ree radica l invo lve ment in aging, drugs and aging, 19 93, 3(1), 60 . 6.dizda ro glu, m.,gas -chro matogra phy mas sspe ctro metry of f re e rad ic alinduce d products of p yrimidine s and purines in dna. metho ds in enzymo lo gy, 19 90, 193 ,8 42. 7.bac q, z.m. che mic al protec tion aga inst io nizing rad ia tio n, charle s c. tho mas ,j ohn wiley, sp ringfield, 196 5, 1 11. 8.muhi-eld ee n, z. , p rod uc t ratio ana lysis of the re ac tion of ethyldiazoa ceta te and allythiote trahydrop yran. bull. coll. sci. 197 4,15,12 9. 9. finkelstein, j ., chiang, e., and le e, j., synthes is of cis -a nd tra ns-2 phe noxyc yclop ro pylamines and related comp ounds. j .med. p harm. chem., 196 5,8, 432 . 10. sa egusa , t., ito, y., ko bayas hi, k., hirota , k., and s himizu t., synthetic rea ctio ns by complex catalys ts . viii. copp er-catalyzed rea ctio n of thiol and alc oho l with dia zo ace tate. j .org.chem., 196 8,33(2),544 . 11. ba ld win, j.e., and ha ckle r, r.e., the rea ctio nship betwe en 1 ,3 -1 and 1.5sigmatropic rearrangeme nts of sulfonium yilde s, j . am . chem. so c . , 1 969 ,9 1,36 46. iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl doi: https://doi.org/10.31351/vol29iss2pp185-193 185 identification and isolation of caffeic, chlorogenic and ferulic acids in aerial parts of capparis spinosa wildly grown in iraq asmaa m. hussein*,1 and enas j. kadum** *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad. iraq. abstract capparis spinosa is one of the oldest genera grown in iraqi territory with worldwide traditional medicinal uses beside the culinary uses. these uses were due to the presence of many phytochemicals including flavonoids and polyphenols. among the reported polyphenolic acids were caffeic, chlorogenic and ferulic acids with wellknown and powerful antioxidant properties. the present work aimed to isolate and identify the presence of these polyphenolic acids in iraqi capparis spinosa (iraqi caper) which is wildly grown in the middle areas following standard chromatographic procedures. aerial parts of the plant (buds, berries and leaves) were extracted with hydroalcoholic solvent by maceration method. the extract was fractionated with chloroform, n-butanol and ethyl acetate. thin layer chromatographic (tlc) techniques and high performance liquid chromatography (hplc) analysis were performed to identify the presence of polyphenolic acids in the ethyl acetate fraction. the result obtained in this work showed the presence of these phenolic acids in the investigated extract. chromatographic analysis revealed the presence of considerable amounts of these acids in ethyl acetate fraction when the separated spots were compared with rf values and uv spectra of standards. such data give a promising use of aerial parts of iraqi caper for globally reported medicinal uses. keywords: capparis spinosa, caffeic, chlorogenic, ferulic acids, identification. حامض الكافييك وحامض الكلوروجينيك وحامض الفريوليك في االجزاء وعزل التحقق من وجود الهوائية لنبتة الكبر البرية في العراق **و ايناس جواد كاظم 1،*اسماء مهدي حسين بغداد،العراق بغداد، جامعة الصيدلة، كلية فرع العقاقير والنباتات الطبية ،* الخالصة استعمالها طبية تقليدية في جميع أنحاء العالم بجانب استعماالت ولهافي األراضي العراقية تنمومن أقدم األجناس التي تعد نبتة الكبر ات واالحماض متعددة ة وجود العديد من المواد الكيميائية النباتية بما في ذلك مركبات الفالفونويدصيخال االستعماالتهذه تعود. الطهي كبهارات في والفريوليك وهي احماض معروفة والكلوروجينيك الكافيك أحماض هي التي نشرت في عدة تقارير متعددة الفينوالتحماض االمن بين و. الفينوالت والتي تنمو بشكل نبتة الكبر العراقيةفي متعددة الفينوالتوجود هذه األحماض من التحققالحالي إلى البحثيهدف هذا . المضادة لألكسدة هاخصائصب للنبتةاألجزاء الهوائية استخالص المواد الكيميائية منتم . رافية القياسيةغفي المناطق الريفية في وسط العراق باتباع اإلجراءات الكروماتو عيطبي . خالت اإليثيلتم تجزئة المستخلص باستخدام مذيبات الكلوروفورم والبيوتانول و .التنقيع بطريقة كحوليبمذيب مائي ( واألوراق والثمارالبراعم ) حماض اللتحديد وجود أ hplc)) كروماتوغرافيا فصل السوائل ذو االداء العالي وتحليل (tlc) الرقيقة طبقةالأجريت تقنيات كروماتوغرافيا المستخلص ي وجود هذه األحماض الفينولية ف البحث عنالتي تم الحصول عليها في هذا النتائجكشفت . خالت اإليثيل جزءفي متعددة الفينوالت وجود كميات كبيرة من هذه األحماض في جزء خالت اإليثيل عند مقارنة البقع المنفصلة مع قيم ارافيغالكروماتو تحليالت اكدت. موضوع التحقق ةالكبر العراقينيتة ن عد لألجزاء الهوائية مالى االستخدام الواهذه البيانات تشير. لالحماض القياسيةالترددات الراديوية واألطياف فوق البنفسجية .عالميًا المنشورةلالستخدامات الطبية التحقق، حامض الفريوليك، حامض الكلوروجينيك، كالكافييحامض ، نبتة الكبر: الكلمات المفتاحية introduction capparaceae or capper family comprises world-wide 33-45 genera and 700 species (1). capparis is one of the oldest genera of capparaceae found in asia (2). the species spinosa was reported to grow naturally in open fields; erupt through cracks of rocks and stone walls in hot deserts and rural areas. it grows well in nutrient and water deprived semi-arid or arid soil. iraq caper is called shefallah and the season of growth is extended from june to september at this time capparis spinosa produce their ripe berries (3). morphologically, genera of capparis spinosa consist of leaves with apical spines, flower bud and spiny stems. the flowers buds then grow into berries which contain characteristic seeds (figure 1) (4). the plant is commonly used for its valuable culinary characteristics of its buds and berries owing to the presence of many constituents like capric acid, glucosinlates, isothiocyanate, mustard oil and phenols (5-8). 1corresponding author e-mail: asmaa.mahdi.d@gmail.com received: 4/ 3/ 2020 accepted:12 /7 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp185-193 iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl 186 besides, the plant gained a famous use in medicine with medicinal uses as analgesic, antiarteriosclerotic, anti-cancer, anti-diabetic, antihemorrhoid, anti-infective, anti-rheumatic, diuretic, emetic, gout treatment, tonic, in addition to hepatoprotecive effects (6, 9-11). these activities are related to the presence of important pharmacologic constituents like alkaloids, flavonoids, glycosides, saponins, sterols, tannins, triterpenoids and vitamins (12). hydroxycinnamic acid is an abundant precursor phenolic acid in many plants including capparis spinosa. this phytochemical is used for the biosynthesis of aromatic compounds by the plant (13). the antioxidant and photoprotecive properties of hydroxycinnamic acid was evaluated in the methanolic extracts of capparis spinosa buds with a promising important application of this extract in topical cosmetic products employed in skin alteration (14) . the present study aimed to detect the presence of polyphenolic acids in iraqi capparis spinosa grown in the middle of iraq for the first time. figure 1. iraqi capparis spinosa materials and methods plant material aerial parts of capparis spinosa (buds, berries and leaves) were collected from rural area in the middle of iraq, wasit province, suwera district during summer (july and august) 2018. the plant was identified and authenticated by the university of baghdad /college of sciences/ department of biology by prof. dr. sukaena abass. aerial parts used in this work were washed thoroughly with tap water, dried under shade, and grinded to yield a powder. extraction of the plant about 700gm of the crude powder was defatted with 1l of n-hexane. the dried defatted material were soaked in 1l of hydroalcoholic solvent (methanol:water, 80:20) for 3days with occasional shaking. the extract was filtered and few drops of it were used for preliminary phytochemical screening for the presence of phenolic acids. the filtered extract then evaporated under vacuum and about 20gm of the residue were reconstituted with 500ml of water for partitioning with chloroform, n-butanol and ethyl acetate (500ml x 3 times). each layer then dried over anhydrous sodium sulfate, filtered and dried under vacuum rotary evaporator and the residue was analyzed by chromatographic techniques. preliminary phytochemical screening of phenolic acids few milligram of each extract were reconstituted with 10ml absolute ethanol, filtered and treated with few drops of 5% (w/v) glacial acetic acid and 5% (w/v) nano2 solution (ellagic acid test). the presence of black brown precipitate indicates the presence of phenols (15). identification of phenolic acids by thin layer chromatography few drops of standard caffeic, chlorogenic and ferulic acids (1mg in 1ml absolute methanol) and of suspended ethyl acetate fraction (1mg in 1ml absolute methanol) were applied to activated silica gel (alugram xtra sil g/uv254) using chloroform:acetone:formic acid (75:16.5:8.5) as a mobile phase. the plates were detected under 254nm uv lengths. isolation of phenolic acids by preparative thin layer chromatography a handmade tlc plates (silica gel gf254, 20  20, 0.75mm thick) were prepared using tlc coater. these plates were activated thereafter at 120oc for 1 hour prior to sample application. about 1mg of ethyl acetate fraction was dissolved in 3ml absolute methanol and spotted as streaks over the prepared plates using capillary tube and allowed for development according to the standard procedures using butanol:acetic acid: water (40:10:50) as a mobile phase. the separated bands were detected under uv at 254nm and marked with a needle. with the use of a fine spatula, the marked bands were scraped and collected in separated flasks. to each flask, an adequate quantity of absolute methanol was added with frequent shaking in warm water. after cooling, the mixture was filtered through double filter paper and the solvent was evaporated thereafter using vacuum rotary evaporator. the semisolid residue for each band was marked as e sample. identification of phenolic acids by thin layer chromatography few drops of standard phenolic acids (caffeic, chlorogenic and ferulic) and of samples e1 to e5 (1mg in 1ml absolute methanol) were applied to activated silica gel using chloroform: acetone: formic acid (75:16.5:8.5) as a mobile phase. the developed plated were detected under 254nm and 366nm uv and the rf values of separated spots were compared to those of standard phenolic acids. iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl 187 identification of phenolic acids by hptlc analysis approximately, 2µl of 1mg/ml methanolic solution of each isolated sample and standard phenolic acids (caffeic, chlorogenic and ferulic) were applied to 20cm10cm hptlc plates (silica gel 60f254) using chloroform:acetone:formic acid (75:16.5:8.5) as a mobile phase and scanned at 254nm and 366nm. identification of phenolic acids by hplc analysis about 0.04mg of each of the isolated samples and of the standards phenolic acids (caffeic, chlorogenic and ferulic) is mixed with 1m of hplc grade methanol using vortex mixer. the mixtures filtered with 2.5µm disposable filter and run in hplc machine using gradient of mobile phase composed of solvent a (1% acetic acid in hplc grade water) and solvent b (acetonitrile). the results were detected on wave lengths of 272, 280, 310nm. results preliminary and tlc analysis the presence of phenolic acid in the ethyl acetate fraction was confirmed with the brown precipitate obtained by ellagic acid test. tlc analysis of ethyl acetate fraction showed the separation of several spots some of them were matched with standard caffeic, chlorogenic and ferulic acids spots (figure 2). isolation of phenolic acids elution of ethyl acetate fraction in chloroform: acetone: formic acid (75:16.5:8.5) results in several isolated bands on preparative tlc plates which were marked as e1, e2, e3, e4 and e5 samples (figure 3). figure 2. tlc plates for analyzed ethyl acetate fraction developed in the chloroform:acetone:formic acid (75:16.5:8.5) at 254nm. (caffeic acid, chl= chlorogenic acid fer=ferulic acid, ea=ethyl acetate fraction. figure 3. preparative tlc plates of ethyl acetate fraction eluted in butanol:acetic acid:water (40:10:50) at 254nm (upper) and 366nm (lower). tlc analysis of isolated samples analysis with tlc of samples e1 to e5 revealed the presence of caffeic, chlorogenic and ferulic acids in samples e3, e2 and e5, respectively with respective rf values of 0.58, 0.12 and 0.90 (figure 4). the phenolic acid in each sample was assigned depending on the comparable rf value of the corresponding standard phenolic acid in table (1). hptlc analysis of isolated samples the data obtained with hptlc analysis of samples e1 to e5 confirms the results obtained with tlc analysis. confirmation based on comparable rf values of the samples to that of standard phenolic acids in table (2). hptlc chromatograms for standard and samples phenolic acid were shown in figures 5, 6 and 7 and hptlc plates at 254nm and 366nm were shown in figure 8. qualitative and quantitative hplc analysis the presence of caffeic, chlorogenic and ferulic acids in iraqi caper was also confirmed qualitatively and quantitatively by hplc analysis according to the retention time of standard phenolic acids used. these phenolic acids were detected in samples e2, e3 and e5 according to the retention time of standard phenolic acids (table 3). in this context, caffeic, chlorogenic and ferulic acids in sample e3, e2 and e5, respectively show similar uv spectra of standard phenolic acids (figures 9, 10 and 11). iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl 188 figure 4. tlc plates for analyzed samples e1 to e5 developed in the chloroform:acetone:formic acid (75:16.5:8.5) at 254nm. (fer=ferulic acid, ea=ethyl acetate fraction. table 1. rf values of the standard and samples phenolic acids. phenolic acid standard rf sample rf sample caffeic acid 0.61 0.58 e3 chlorogenic acid 0.14 0.12 e2 ferulic acid 0.89 0.90 e5 table 2. max rf values of the standard and samples phenolic acids. phenolic acid standard rf sample rf area % sample caffeic acid 0.44 0.43 49.34 e3 chlorogenic acid 0.04 0.03 47.64 e2 ferulic acid 0.55 0.54 43.31 e5 figure 5. hptlc chromatogram of standard and sample e3 caffeic acid at 254nm iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl 189 figure 6. hptlc chromatogram of standard and sample e2 chlorogenic acid at 254nm. figure 7. hptlc chromatogram of standard and sample e5 ferulic acid at 254nm. iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl 190 figure 8 .hptlc plates for standards and sample phenolic acids in e2, e3 and e5 at 254nm (above) and 366nm (below). caf= caffeic acid, chl= chlorogenic acid, fer= ferulic acid, samples e1 to e5. table 3. retention times of the standard phenolic acids and the concentration of sample phenolic acids at standard retention time. phenolic acid standard rt sample conc. (mg/g plant) sample caffeic acid 14.000 0.3407 e3 chlorogenic acid 13.400 0.3640 e2 ferulic acid 13.756 13.11 e5 figure 9. uv spectroscopy of standard and sample caffeic acid at 24.2min. iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl 191 figure 10. uv spectroscopy of standard and sample chlorogenic acid at 13.4min. discussion among many phytochemicals, capparis spinosa had been reported to contain various type phenolic acids (8). polyphenolic acids are secondary metabolites with antioxidant properties that allow the plant to survive under stressful conditions (16). in the present study, the positive preliminary result for the presence of phenolic acids in ethyl acetate fraction was followed by implication of chromatographic techniques to identify the nature of these acids. three phenolic acids (caffeic, chlorogenic and ferulic) were used as standards in the subsequent analytic techniques. these reference acids are of particular concern due to their wellknown powerful antioxidant properties. caffeic acid (3,4-dihydroxy cinnamic acid), chlorogenic acid (ester of caffeic acid and quinic acid) and ferulic acid (4-hydroxy-3-methoxycinnamic acid) are polyphenolic acids abundantly present in many plants and are derived from phenylalanine amino acid in the shikimic acid pathway during the synthesis of many polyphenolic phytochemicals like flavonoids (17). the investigated phenolic acids were successfully identified in the ethyl acetate fraction of the plant extract according to the different polarities offered by chloroform:acetone:formic acid (75:16.5:8.5) as a mobile phase. identification was based on observing separated spots in the ethyl acetate fraction that match in position the spots of standard phenolic acids, such results encourage subsequent isolation of these acids for qualitative and quantitative analysis by different chromatographic techniques. preparative tlc resulted in the isolation of five distinct bands which were marked as sample e1 to e5. to identify whether the investigated acids were present in these samples, qualitative tlc analysis was performed and revealed the presence of caffeic acid in sample e3, chlorogenic acid in sample e2 and ferulic acid in sample e5 by matching the rf values of sample spot with that of standard spot. the number of free hydroxyl groups in the structure of these acids is varying. it was 2 in ferulic acid, 3 in caffeic acid and 5 in chlorogenic acid. in addition, ferulic acid contains a methoxy group that renders the molecule highly less polar. in this context, the polarities of these acids were inversely proportionate with their rf values; the highest value was for ferulic acid and the lowest was for chlorogenic acid as shown in the results of tlc and hptlc analysis (tables 1 and 2). qualitative analysis of isolated samples by hplc also supported the results obtained by tlc and hptlc when the uv spectra of these samples were compared with those of standard phenolic acids at the specified retention time of the standards. quantitatively, the concentration of ferulic acid in sample e5 was much higher than those of chlorogenic acids in sample e2 and caffeic acid in sample e3 with a corresponding 13.11mg, 0.3640mg and 0.3407mg per gram of plant, respectively (table 3). the powerful antioxidant property of these acids was highly investigated and is related to their poly phenolic nature (18, 19), for this reasons some food manufacturers prefer the addition of these phenolic acids such as ferulic acid as natural antioxidant (20). these phenolic acids have promising medicinal use in oxidative stress, microbial infections, inflammations, thrombosis and cancer (21). ferulic acid, which by this study is occurring in valuable amount in iraqi caper, has been reported that improve the treatment in patients with high cholesterol, coronary heart diseases, infertility. furthermore, it has a protective role iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl 192 against tissue damage induced by chemotherapeutic drugs (22) in conclusion, many iraqi authors have reported that caper shrub is one of the folkloric medicinal plants in iraq. the present work identified the presence of caffeic, chlorogenic and ferulic acids in iraqi caper following standard chromatographic techniques. references 1. moghaddasian, b., asli, moghaddasian, d., alaghemand, a., and miahi, m.: anthocyanin content in different parts of capparis spinosa growing wild in tafresh/iran. technical journal of engineering and applied sciences, 2013; 3(11): 938-941. 2. bakshi, d.n.g., p. sensarma and d.c. pal.: a lexicon of medicinal plants in india, 1999; (1): 360-365. 3. rhizopoulou, s. and paras g.: development and structure of drought tolerant leaves of mediterranean shrub capparis spinosa l. annales of botany, 2003; 92: 377-383. 4. naoual saifi, ghizlane echchgadda, jamal ibijbijen: the morphological characterization of caper plant (capparis ssp.) in north morocco. journal of food, agriculture and environment, 2010, 8(2): 876-881. 5. rezzan aliyazicioglu, ozan emre eyupoglu, huseyin sahin, oktay yildiz, nimet baltas: phenolic components, antioxidant activity, and mineral analysis of capparis spinosa l. african journal of biotechnology, 2013; 12(47): 6643-6649. 6. rodrigo m, lazaro m, alvarruiz a, giner v. composition of capers (capparis spinosa): influence of cultivar, size and harvest date. j food sci, 1992; 57: 1152-1154. 7. mariapia argentieri, francescomacchia, francesco paolofanizzi, pinarosaavato. bioactive compounds from capparis spinosa. industrial crops and products, 2012; 36(1): 6569. 8. zhuohong xie, john w. finley. herbs and spices in, principles of food chemistry, 2018: 457-481. 9. al-snafi ae. encyclopedia of the constituents and pharmacological effects of iraqi medicinal plants, 2015. 10. selvamani, p., s. latha, k. elayaraja, p.s. babu, j.k. gupta, et al. antidiabetic activity of the ethanol extract of capparis sepiaria l leaves. indian j pharm sci, 2008; 70(3): 37880. 11. n aghel; i rashidi; a mombeini. hepatoprotective activity of capparis spinosa root bark against ccl4 induced hepatic damage in mice. iranian journal of pharmaceutical research, 2007; 6(4): 285-290. 12. ephraim philip lansky, helena maaria paavilainen, shifra lansky: traditional herbal medicines for modern times: caper the genus capparis. taylor & francis group, llc, crc press, 2014. 13. sindhu mathew; tanya abraham: ferulic acid: an antioxidant found naturally in plant cell walls and feruloyl esterases involved in its release and their applications. critical reviews in biotechnology, 2004; 24(2-3):5983. 14. bonina f., puglia c., ventura d., aquino r., tortora s., sacchi a., saija a., tomaino a., pellegrino m.l, and de caprariis p. in vitro antioxidant and in vivo photoprotectiveffects of a lyophilized extract of capparis spinosa l. buds. j. cosmet. sci., 2002; 53: 321-335. 15. rimjhim sheel, kumari nisha and jainendra kumar. preliminary phytochemical screening of methanolic extract of clerodendron infortunatum. iosr journal of applied chemistry, 2014; 7(1): 10-13. 16. martinadi ferdinando, ceciliabrunetti, giovanni agati, massimilianotattini: multiple functions of polyphenols in plants inhabiting unfavorable mediterranean areas. environmental and experimental botany, 2014; 103: 107-116. 17. josé dzíaz, a. ros barceló and f. merino de cáceres: changes in shikimate dehydrogenase and the end products of the shikimate pathway, chlorogenic acid and lignins, during the early development of seedlings of capsicum annuum. the new phytologist, 1997; 136(2): 183-188. 18. yuki sato,shirou itagaki,toshimitsu kuroka wa , jiro ogura, masaki kobayashi, ,takeshi hirano , mitsuru sugawara, ken iseki: in vitro and in vivo antioxidant properties of chlorogenic acid and caffeic acid. international journal of pharmaceutics, 2011; 403(1-2): 136-138. 19. graf e. antioxidant potential of ferulic acid. free radic biol med., 1992; 13(4):43548. 20. srinivasan m, sudheer ar, menon vp. ferulic acid: therapeutic potential through its antioxidant property. j clin biochem nutr., 2007; 40(2):92-100. https://link.springer.com/book/10.1007/978-3-319-63607-8 https://www.sciencedirect.com/science/journal/00988472 iraqi j pharm sci, vol.29(2) 2020 polyphemolic acids from iraqi cpl 193 21. camelia papuc, gheorghe v. goran, corina n. predescu, valentin nicorescu, and georgeta stefan: plant polyphenols as antioxidant and antibacterial agents for shelf-life extension of meat and meat products: classification, structures, sources, and action mechanisms. comprehensive reviews in food science and food safety, 2017; 16: 12431268. 22. ou s., kwok k-c.: ferulic acid: pharmaceutical functions, preparation and applications in foods. j sci food agric., 2004; 84(11):1261-1269. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ clinical evaluation of melatonin alone and in combination with pizotifen in the prophylaxis of migraine iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in nsaids-induced injury 39 the efficacy of topically applied silymarin in the treatment of herpes labialis ulcers tagreed s. altaei * , intesar t. numan ** ,saad a.hussain **,1 and nazar g. al-talabani * * departments of basic sciences and oral pathology, college of dentistry, university of baghdad, baghdad, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract : herpes labialis is an infection caused by the herpes simplex virus, characterized by an eruption of small and usually painful blisters on the skin of the lips, mouth, gums, or the skin around the mouth. although there is no successful treatment available, the local use of compounds with effective antiinflammatory and cytoprotective effects may be of value in this respect. this project was designed to evaluate clinically the local use of silymarin, a group of flavonoids with powerful antioxidant, antiinflammatory and cytoprotective activity, in the treatment of herpes simplex ulcer. fifty three patients with herpes labialis ulcers (hlu) were enrolled in this randomized, single blinded, placebo controlled clinical study, and they were allocated into 4 groups, treated with 1%, 3% and 5% silymarin paste and placebo formula respectively. patient's responses to treatment were followed by clinical evaluation of healing time, size of the ulcers and pain sensation, in addition to evaluating biochemical and immunological markers of the oxidative stress and inflammatory response. hlu patients showed dose dependent improvement in the healing time, pain score and size of ulcer as a result of treatment with various concentrations (1%, 3% and 5%) of silymarin paste, associated with improvement in the oxidative stress state and immunological parameters. in conclusion, silymarin can be used locally as paste formula for the treatment of hlu, an effect which may be attributed to its antioxidant, anti-inflammatory and cytoprotective properties. keywords: herpes labialis, ulcers, silymarin الخالصة رعزجش انزقشحبد انزٙ ٚسججٓب فٛشٔط انحأل انجسٛػ يٍ األيشاض االنزٓبثٛخ انًؤنًخ انشبئعخ انزٙ رصٛت األغشٛخ انًجطُخ نهفى. ٔثبنشغى يٍ عذو ٔخٕد عالج فعبل نًثم ْزِ انحبالد، فأٌ اسزخذاو يشكجبد نٓب فعبنٛخ يعبدح نالنزٓبة ٔحًبٚخ خهٕٚخ فعبنخ قذ ٚكٌٕ را ْزا انًدبل. رى رصًٛى ْزِ انذساسخ نزقٛٛى االسزخذاو انًٕظعٙ نًبدح انسٛهًٛبسٍٚ فٙ عالج انزقشحبد انقالعٛخ انًزكشسح. أخشٚذ فبئذح فٙ % 1يشٚعب ثزقشحبد انفى انزٙ ٚسججٓب فٛشٔط انحأل انجسٛػ، حٛث رى رقسًٛٓى إنٗ أسثعخ يدًٕعبد رى عالخٓب ة 35ْزِ انذساسخ عهٗ ٍ ثشكم يعدٌٕ يٕظعٙ رى رحعٛشِ خصٛصب نٓزا انغشض أٔ يسزحعش رٓذئخ عهٗ انزٕانٙ. رًذ يزبثعخ % سٛهًٛبس3ٚ% أٔ 5أٔ اسزدبثخ انًشظٗ نهعالج يٍ خالل حسبة انفزشح انضيُٛخ انالصيخ النزئبو انقشحخ ٔيعذل انزغٛٛش فٙ يسبحزٓب ٔكزنك يسزٕٖ رسكٍٛ األنى بعٙ نجعط انًعبٚٛش انخبصخ ثبالنزٓبة ٔفشغ األكسذح يثم األَزشنٕكٍٛ، انكهٕثُٛبد انُبشٙء عُٓب، ثبإلظبفخ إنٗ رقٛٛى ثبٕٚكًٛٛبٔ٘ ٔيُ انًُبعٛخ، انًبنَٕذا٘ انذٚٓبٚذ ٔانكهٕربثٌٕٛ. أظٓشد َزبئح انذساسخ رحسُب يهحٕظب فٙ اسزدبثخ انًشظٗ نهعالج يٍ خالل اَخفبض انفزشح الو انُبشئخ عُٓب، إظبفخ إنٗ رحسٍ ٔاظح فٙ يعبٚٛش فشغ األكسذح ٔانًعبٚٛش انضيُٛخ انالصيخ النزئبو انقشحخ ٔاَخفبض يسزٕٖ انشعٕس ثبٜ انًُبعٛخ األخشٖ يقبسَخ ثبسزخذاو يسزحعش انزٓذئخ. ٔعهّٛ ًٚكٍ االسزُزبج ثئيكبَٛخ اسزخذاو انسٛهًٛبسٍٚ كًسزحعش يٕظعٙ فٙ عالج رقشحبد انفى انُبشئخ عٍ اإلصبثخ ثفٛشٔط انحأل انجسٛػ. introduction : herpes simplex virus (hsv) affects more than one third of the world’s population, and is responsible for a wide array of human diseases, with effects ranging from discomfort to death. (1) hsv actually belongs to a family of eight related viruses, including hsv1, hsv2, varicella-zoster virus (vzv), epstein-barr virus (ebv), and cytomegallovirus (cmv); all of them are double-stranded dna viruses, which permanently infect their target cells. (2) hsv produces infections ranging from cold sores to severe genital lesions; and both the initial episode and recurrent disease can cause devastating problems. (3) it affects the skin, mucous membranes, and less frequently the esophagus and brain. skin infections are usually located in the orolabial, genital, or anorectal area. of the two serotypes, hsv-1 infection is primarily oropharyngeal and hsv-2 infection is primarily genital. (4) among the many clinical expressions of hsv-1 infections, the easiest to recognize is the typical cold sore or fever blister. (5) many patients have pain, burning and considerable anxiety which justify the use of systemic drug therapy. many medications are available for treatment of herpes, including foscarnet sodium, gancyclovir, but the three main antiviral drugs used are acyclovir, valacyclovir and famciclovir. there are a number of alternative treatments that have been 1 corresponding author : e-mail: saad_alzaidi@yahoo.com received : 28/10/2006 mailto:saad_alzaidi@yahoo.com iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in hlu 40 accepted : 22/5/2007 anecdotally reported to be effective in treating hsv infection; for example, l-lysine, naturally occurring in many foods, can be used effectively in this respect. (6) herbs that may be helpful in hsv infection include lemon balm (melissa officinalis) which has antiviral activity. (7) commercial silymarin is a standardized preparation extracted from the fruits (seeds) of silybum marianum, consists of three major flavolignans; silybinin, silychristin and silydianin, of them silybinin is the most biologically active. (8) silymarin has cytoprotective, antioxidant and antiinflammatory activities, (9, 10, 11) thus it may be helpful in many inflammatory disorders like ulcers associated with hsv infection. it is regarded as being non-toxic with high margin of safety and no serious adverse reactions have been reported due to its clinical use. this study was designed to evaluate the clinical utility of using silymarin as locally applied pharmaceutical dosage form in the treatment of hlu. patients and methods : trial design: fifty three patients with hlu (age range 5-64 ± 12 .23 years, 30 females and 23 males) were included in a randomized, placebo controlled, single blinded clinical trial conducted in the department of oral medicine, college of dentistry, university of baghdad during the period jan-dec/2004; patients selection was performed according to the following criteria: the patients should have a history of recurrent ulceration due to herpes simplex, female patients should not be pregnant, they should not suffer from any chronic debilitating disease and not receive any antibiotics or steroids medication. all patients were informed about the nature of the study and their signed consent was obtained. after careful diagnosis and characterization of hlu patients, they are randomized and treated as follow: group a includes 14 patients treated with 1% silymarin paste 4 times daily; group b includes 14 patients treated with 3% silymarin paste 4 times daily; group c includes 15 patients treated with 5% silymarin paste 4 times daily; group d includes 10 patients treated with placebo formula containing all the ingredients of the oral paste formula except the active constituent (silymarin) 4 times daily.oral paste formula of silymarin for topical application was prepared according to b.p. (1997), 12 in different strengths (1%, 3%, and 5% silymarin w/w). placebo formula which contains all the ingredients except the active constituent (silymarin) was prepared in the same way. each patient was followed regularly by daily inspection during the period of application of the tested medication to the ulcer to determine the degree of pain, size of ulcer, and the end point time of healing process. analysis of immunological and biochemical parameters five ml of venous blood was drawn from 6 patients belongs to group c and 6 patients belongs to group d before starting treatment and after 3 days of treatment. after preparation of serum, the levels of interleukin-1α ,complement proteins c3 and c4, the immunoglobulin igg, iga, and igm and the oxidative stress parameters malondialdehyde (mda) and glutathione (gsh) in the serum were analyzed according to standard procedures. (13, 14, 15, 16) statistical evaluation of data was performed utilizing paired student’s ttest, while anova was performed to compare between different groups. differences were considered significant at p values <0.05. results : figure 1 clearly demonstrated that greater percentage of hsu patients showed faster and complete ulcer healing due to treatment with silymarin within 3-6 days, which was found significantly different with respect to placebotreated group (11-15) days. 5% silymarin formula produces significantly higher rate of ulcer healing compared to 1% and 3% formulas. figure 2 showed that at day 2 significant reduction in mean ulcer size (p<0.05) was produced by both 5% and 3% silymarin compared with 1% formula. all silymarin formulas produced significant reduction in mean ulcer size compared to placebo within 5 days of the follow up period. figure 3 showed that treatment with silymarin paste (1%, 3% and 5%) resulted in highest number of patients with complete pain relief, as measured by visual analogue scale (vas), which was significantly different compared to placebo treated group (p<0.05). however, treatment with 5% silymarin paste resulted in significantly higher percentage of patients with complete pain relief compared with 1% and 3% silymarin formulas during the days 1 and 2 after starting treatment; while during the days 3 and 4 no significant differences were observed in this respect. figure 4 showed that mean pain score in all hlu patients were gradually decreased over 5 days of the study, with 5% silymarin treated group showing a mean pain score of 1, 0.5 and 0 during the days 1, 2 and 3 respectively, a result which is significantly lower than those observed in other groups (p<0.05). however, even when 1% and 3% silymarin treated groups reported significantly lower mean pain score compared to placebo, they did not show significant differences between them. concerning the effects of treatment with 5% silymarin on the immunological and biochemical markers in the serum of patients, table 1 indicated that treatment with 5% silymarin paste for 4 -5 days resulted in significant reduction in serum level iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in hlu 41 0 10 20 30 40 50 60 70 80 90 100 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 day % s u b je c ts h e a le d placebo sily marin 1% sily marin 3% sily marin 5% 0 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 day m e a n u l c e r s i z e ( s e q u a r e m m ) p lacebo sily marin 1% sily marin 3% sily marin 5% of il-1α (26%, p<0.05) compared to pretreatment level, while in placebo-treated patients il-1α show significant elevation (20%, p<0.05) compared to pre-treatment levels. when the two types of treatment (silymarin vs placebo) were compared, significantly lower values for serum il-1α were observed after 4 -5 days of treatment in the silymarin-treated group compared to placebo-treated one (35%, p<0.05). table 1 also showed that treatment with 5% silymarin paste for 4-5 days produced significant reduction in serum levels of the complement proteins c3 and c4 (13% and 44% respectively) compared to the pre-treatment levels. placebo formula did not produce any significant changes in this respect. when the two methods of treatment compared (silymarin vs placebo), no significant differences reported in c3 levels (p<0.05), while c4 levels showed significantly lower values due to silymarin treatment (43%) compared to placebo treatment. table 2 demonstrated that treatment of hlu patients with 5% silymarin paste for 4 -5 days resulted in significant reduction in the serum levels of the immunoglobulins igg, igm and iga (4%, 22% and 36% respectively) compared to pre-treatment levels, while placebo treatment did not alter ig’s levels during the same period of treatment. when the two methods of treatment were compared, silymarin paste showed significant lowering effect in ig’s levels compared to placebo. table 3 demonstrated that treatment of hlu patients with 5% silymarin paste for 4 -5 days resulted in a significant reduction in the serum mda level compared to pre-treatment level (49%, p<0.05). meanwhile, placebo-treated group did not show such an effect. when the two types of treatment (silymarin vs placebo) were compared, significantly lower value of serum mda level was observed (54%, p<0.05) due to silymarin treatment compared with placebo. concerning gsh levels, table 3 showed that treatment with 5% silymarin paste for 4 -5 days resulted in significant elevation in serum gsh level (48%, p<0.05) compared to pre-tretment value. meanwhile; treatment with placebo formula did not show such an effect, and gsh levels continuously declined. when the two types of treatment (silymarin vs placebo) were compared, significantly higher value of serum gsh was observed in silymarin treated group (19.8%, p>0.05) compared with placebo-treated group. figure 1. effects of treatment with silymarin paste on ulcer healing time in hlu patients. all results are significantly different (p<0.05) compared to placebo within 3-5 days. figure 2. effects of treatment with silymarin paste on the changes in ulcer size in hlu patients.all results are significantly different (p<0.05) compared to placebo within 5 days. iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in hlu 42 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12 14 day % o f s u b j e c t s r e s p o n d placebo sily marin 1% sily marin 3% sily marin 5% 0 0.5 1 1.5 2 2.5 3 0 1 2 3 4 5 6 day m e a n p a in s c o re placebo sily marin 1% sily marin 3% sily marin 5% figure 3. effects of treatment with silymarin paste on complete pain relief in hlu patients.all results are significantly different (p<0.05) compared to placebo within 3days. figure 4. effects of treatment with silymarin paste on the pain score in hlu patients. all results are significantly different (p<0.05) compared to placebo within 3 days. table 1. effects of silymarin 5% paste on serum levels of interleukin 1-α ( il-1α ) and the complement proteins (c3 and c4) in hlu patients. type of treatment il-1α pg/ml c3 mg/dl c4 mg/dl silymarin 5% n = 6 pre-treatment post-treatment pre-treatment post-treatment pre-treatment post-treatment 486.5 ± 19.24 360.8 ± 41.34* a 173.6 ± 11.2 150.16 ± 12.6* a 33.05 ± 1.37 18.4 ± 1.58 * a placebo n = 6 460 ± 55.68 550 ± 36.05* b 171.23 ± 6.8 171.23 ± 6.85 a 32.16 ± 0.75 32.16 ± 0.75 b values are presented as mean ± sd; n = number of subjects; * significantly different with respect to pre-treatment period (p<0.05); values with non-identical superscripts were considered significantly different (p<0.05) among different groups. table 2. effects of silymarin 5% paste on serum immunoglobulins levels in hsu patients. type of treatment silymarin 5% n = 6 placebo n = 6 igg mg/dl pre-treatment 1409.45 ± 116.39 1475.56 ± 58.2 post-treatment 1362.20 ± 117.7 * a 1475.56 ± 58.2 a igm mg/dl pre-treatment 191.60 ± 33.94 207.46 ± 21.95 post-treatment 149.56 ± 37.17 * a 207.46 ± 21.95 a iga mg/dl pre-treatment 199.45 ± 11.98 204.46 ± 5.94 post-treatment 127.88 ± 7.144 * a 204.46 ± 5.94 b values are presented as mean ± sd; n = number of subjects; * significantly different with respect to pre-treatment period (p<0.05); values with non-identical superscripts were considered significantly different (p<0.05) among different groups. iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in hlu 43 table 3. effects of silymarin 5% paste on serum malondialdehyde (mda) and glutathione (gsh) contents in hlu patients. type of treatment mda (μmol/l) gsh (μmol/l) pre-treatment post-treatment pre-treatment post-treatment silymarin 5% n =6 3.04 ± 0.32 1.56 ± 0.09 * a 13.35 ± 1.7 19.72 ± 3.39 * a placebo n=6 3.08 ± 0.31 3.38 ± 0.2 * b 10.04 ± 1.52 6.617 ± 0.73 * b values are presented as mean ± sd; n = number of subjects; * significantly different with respect to pre-treatment period (p<0.05); values with non-identical superscripts were considered significantly different (p<0.05) among discussion: herpes virus, which consist of a single double-stranded dna molecules enclosed in a viral envelope, produce ulcers as a result of triggering an array of host responses that gives rise to enhanced inflammatory mediators and cytokine responses in macrophages and other host cells. (17) there is no curative therapy, but antiviral medications given by mouth may shorten the course of the symptoms and decrease pain. the flavonoids (including those present in silymarin) have long been recognized to possess anti-inflammatory, antioxidant, antiallergic, hepatoprotective, antithrombotic, antiviral and anticarcinogenic activities. (18, 19) topical use of silymarin paste demonstrates improved efficacy in the treatment of hsu compared to placebo, as measured by the primary (objective) outcome of complete ulcer healing and, albeit less robustly, the secondary (subjective) outcome of complete pain relief. the anti-inflammatory activity of silymarin components (silybinin) was assessed in human pmns in vitro; where the chemotactic and phagocytic activities of these pmns were not modified by silybinin at concentrations of 0.525 μg/ml. (20) reactive oxygen species (ros) released by neutrophils and other phagocytes have been increasingly implicated in inflammatory immune disorders. (21, 22) flavonoids, including silymarin, could profoundly impair the production of ros by inflammatory cells; this may be accomplished by interference with nadph-oxidase, a powerful oxidant-producing enzyme localized on the surface of neutrophil’s plasma membrane. (23) they could also inhibit neutrophils myeloperoxidase (mpo), a source of reactive chlorinated intermediates. (24) the data presented in this study clearly demonstrated that mean ulcer size in the placebo-treated group showed no significant differences during 0-5 days, compared to the gradual decrease reported in silymarin-treated group during 1-4 days, this suggested that silymarin exerted a powerful effect on healing process, and initiates it early during the course of hlu. cytokines played an important role in the immune response to hsv infections; in particular tnf-α, which is primarily produced by macrophages, and is known to be central for control of virus replication. (25, 26) however, tnf-α and tnf-α-induced products are also involved in the immunopathology often associated with hsv infection. (27, 28) silymarin has anti-inflammatory and cytoprotective effects by suppression of nf-κb, the nuclear transcription factor, which regulates the expression of various genes involved in inflammation. it blocks tnf-α-induced activation of nf-κb in a dose and time dependent manner. this effect was mediated through inhibition of phosphorylation and degradation of iota kappa b alpha, an inhibitor of nf-κb. (29) since replication of certain viruses is dependent on nf-κb, (30) silymarin may interfere with viral replication. mucsi and pragai (1985) demonstrated the inhibitory effect of four flavonoids in human herpes simplex viruses type i, and there was a relationship between viral inhibition and the ability of the studied flavonoids to increase cyclic amp in the hep-2 cells and chicken embryo fibroblasts. (31) phospholipase a2 (pla2) is likely an important intraand extra-cellular mediator of pain mediators during inflammation. (32) silymarin was found to be an effective inhibitor of pla2 from human sources; additionally, silybinin, silydianin and silychristin (constituents of silymarin) were found to effectively blocking the activity of lipoxygenase and prostaglandin synthesis in vitro. (33) these molecules are intimately involved in inflammation and allergy, as well as in many other physiologic and pathologic processes including pain. silybinin inhibits synthesis of leukotriene b4 (ltb4) (ic50 = 15 iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in hlu 44 μmol/l) in isolated rat kupffer cells; others showed that the three pharmacologically active components of silymarin, namely silybinin, silydianin and silychristin inhibit cyclooxgenase (cox) enzyme in vitro. (33) all the previous events indicated that silymarin may act as analgesic agent through these pathways, and this might explain the reduction in pain observed in the present study. according to the results presented in this study, serum levels of il-1α decreased significantly after treatment with silymarin paste compared to pre-treatment values, while placebo-treated group did not show such effect; this observation may give another indication that can be added to those deal with the inhibitory effects of silymarin on interleukin production with consequent antiinflammatory activity. antibodies to hsv are mostly of igg type, although hsv-specific iga is also detectable. (,34, 35) all the observations about the possible role of igg, igm, iga and the complement protein components (especially c3) supported the concept of occurrence of immune complex vasculitis, which is found essential step in the pathogenesis of oral ulceration. (36) the results presented in this study concerning the changes in the levels of serum immunoglobulins (iga, igg and igm) are compatible with some of the previously indicated studies, where significant differences between preand post-treatment with topical silymarin paste were observed compared to placebo in hlu patients. phagocytosis is an important physiological process accompanied by production of superoxide anion, and activated phagocytic cells such as monocytes, neutrophils and macrophages; this process is extremely important for their bactericidal functions. (37) while ros generated by phagocytes play an important physiological function, they can also cause cellular damage. ros along with other mediators generated by neutrophils and macrophages can promote inflammation and cause tissue damage. (24) the consequently resulted state of oxidative stress can produce damage to many biological molecules; where proteins and dna are definite targets and predispose to cellular injury. lipid peroxidation is often a secondary event consequent to primary cell damage induced by oxidative stress. in the present study, local treatment with silymarin paste produces inhibitory effects on lipid peroxidation, as revealed by significant decrease in serum mda levels compared to placebo, which very well indicated the powerful antioxidative effects of silymarin. silymarin prevents liver glutathione depletion and lipid peroxidation induced by an acute intoxication with ethanol in rats. (38) these effects supported the suggested action of silymarin as a cytoprotective agent. in the present study, silymarin produced significant increase in serum glutathione (gsh) levels compared to placebo. silymarin is known to induce superoxide dismutase enzyme (sod) and glutathione biosynthesis; it brings gsh level back to normal after depletion; so it is not only prevent depletion of gsh, but increases the basal level of activity of detoxifying enzymes. (39) in conclusion, silymarin can be used topically for the treatment of hlu; its high efficacy and safety make it a good alternative for the available pharmacological agents in this respect. references : 1. david h, emmert md. treatment of common cutaneous herpes simplex virus infections. am fam physician 2000; 61: 1697-704. 2. nadelman cm, newcomer vd. herpes simplex virus infections: new treatment approaches make early diagnosis even more important. postgrad med 2000; 107(3): 189200 3. brown z, patel r, reitano m. herpes virus infections: treatment options in the new millennium. a symposium report. world health cme, world health communications, inc, denver, 1996; 29. 4. fleming d, mcquillan gm, johnson re, herpes simplex virus type 2 in the united states, 1976 to 1994. n engl j med 1997; 337(16): 1105-1111. 5. gurenlian jr. differentiating herpes simplex virus and recurrent aphthus ulcerations. access 2003; 17 (2): 30-34. 6. flodin nw. the metabolic roles, pharmacology, and toxicology of lysine. j am coll nutr 1997; 16(1): 7-21. 7. w ِ hlbling rh, leonhardt k. local therapy of herpes simplex with dried extract of melissa officinalis. phytomed 1994; 1(1): 2531. 8. sonnenbichler j, zetle i, biochemistry of a liver drug from the thistle silybum marianum. planta medica 1992; 58: a 580-a 581. 9. altorjay i, dalmi l, sari b, imre s, balla g. the effect of silibinin (legalon) on the free radical scavenger mechanisms of human erythrocytes in vitro. acta physiologica hungarica 1992; 80: 375-380. 10. miadonna a. effects of silybinin on histamine release from human basophile leucocytes. br j clin pharmacol 1987: 24: 747-752. iraqi j.pharm.sci., vol.16 (1) ,2007 silymarin in hlu 45 11. fiebrich f, kock h. silymarin, an inhibitor of lipoxygenase. experientia 1979; 35: 1548-1550. 12. khandwala a, van inweegen rg, alfano mc. 5% amlexanox oral paste, a new treatment for recurrent minor aphthus ulcers. oral pathol oral radiol endo 1997; 83: 222230. 13. didierjean l, salomon d, merot y. localization and characterization of the interleukin-1 immunoreactive pool (il-1 alpha and beta forms) in normal human epidermis. j invest dermatol 1989; 92: 809816. 14. kyle r a. classification and diagnosis of monoclonal gammopathies. in: rose n r, friedman h, fahey jl. 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(ed), natural antioxidants: chemistry, health effects and applications, aocs press, champaign, il; 1995: 174-203 a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci vol.17 (2) , 2008 permeation of chlopheniramine maleate from gels 67 preparation and in vitro permeation of chlopheniramine maleate (cpm) from gel through rat skin wissam s. mahmood** and balkis a. kamal*,1 * department of pharmaceutics, college of pharmacy, university of baghdad. , baghdad , iraq ** ministry of health , baghdad ,iraq abstract chlopheniramine maleate ( cpm ) , is one of the hreceptor antagonist , widely used in allergic diseases ,like skin rash and pruritis .cpm 3%w/w was successfully loaded in 2%w/w sodium alginate (sa) as a gel base , and to be considered as a selected formula .it was found that the diffusion of cpm through the skin of albino rat was increased as the concentration of cpm increased from 2 %w/w sodium alginate , more over , the addition of triethanolamine 5 % w/w, to sodium alginate 2 % w/w , loaded by cpm 3 % w/w , enhanced the amount of cpm diffuse through the skin of albino rat . mean while the addition of peg 1000 2% w/w , and urea 5 % w/w, separately to sodium alginate 2 % w/w , loaded by cpm 3 % w/w , hindered significantly p<0.05 the amount of the drug diffused through the skin of the rat .the selected formula of sodium alginate 2% w/w as a base loaded by cpm 3% w/w was physically acceptable , with shelf life approximately 3.3 years . key wards: chlopheniramine maleate , gel , skin permeation ةصالخلا ال يمارلم واحل وال لجال ا ياا ا يع ووال يب وا يتمالموا.ييوا1 ي دالبمت ام ااميمملاكدا وا يمينامرايمارينفرااكلا يف (نزو\ نزو)%ا يف (نزو\ نزو)%ل)%\ل)%( ا ا يعمبمرا يجدا د اليمووناكف ا ,2 يف (نزو\ نزو)% يف (نزو\ نزو)%ل)%ا\ل)%( ل ا3 تعبلا يمبملرراحرتلملا ي دالبمت ام ااميمملا ييواللوا %ا ت وا ي دالبمت ام ااميمملاا ادفجال وا يعتتا رحت ا تا ااحت مانا تلمتا يليماال ا ل ورنترالرتلمنوااترمانا. يف (نزو\ نزو)%ا يف (نزو\ نزو)%ل)%\ل)%( ا انموونا2 يف (نزو\ نزو)%ا يف (نزو\ نزو)%ل)%\ل)%( ي,5 يف (نزو\ نزو)% يف (نزو\ نزو)%ل)%\ل)%( ا ا يعمبمرا يجدا د ا,وفلناو ,اتييالم%ا تملوا يرت ميم تدرام ا2 يف (نزو\ نزو)% يف (نزو\ نزو)%ل)%\ل)%( ا ا ي دالبمت ام ااميمملاا , اىا ي,ا) مانالمموا يليماا يبملكنادفجال وا يعتتا3 يعمبمرا يجدا د اابم والا يف (نزو\ نزو)% يف (نزو\ نزو)%ل)%\ل)%( ا ا يمدا ماحل وا5( لا1000 يف (نزو\ نزو)% يف (نزو\ نزو)%ل)%\ل)%( ا ااماناحدي,اص م الف دج)2لو ,ايلموا دت ,لد%اصتملوا رحت . يف (نزو\ نزو)%ا يف (نزو\ نزو)%ل)%\ل)%( ا ا ي دالبمت ام ااميممل , اىا ي,ا رومنواحل وا3 يف (نزو\ نزو)%ا يف (نزو\ نزو)%ل)%\ل)%( ا ا يعمبمرا يجدا د ا,ابم والا2اباجوا ي,ا يف (نزو\ نزو)%ا يف (نزو\ نزو)%ل)%\ل)%( ا ا يعمبمرا2ص%ا يرتلمنوا يمترماناالم تلا بردواو ,ا المموا يليماا يبملكنادفجال وا يعتتايفحت ا. p<0.05 امها يف (نزو\ نزو)%ا يف (نزو\ نزو)%ل)%\ل)%( ا ا ي دالبمت ام ااميمملا , تملوا ي,ايامرالمت مل واالموناللرتنا تامت واي ليماا3 يجدا د اليمووناكف اابم والا البوا.3,3حبولا introduction gels are semi solids consisting of dispersions made up of either small inorganic particles or large organic molecules enclosing and interpenetrated by a liquid (1) .the delivery of the drug into and through the skin is recognized an effective means of therapy for local dermatological and systemic disease ,in recent years, the development of transdermal permeation has been attracting an attention due to several advantages , such as better control of blood levels , reducing systemic toxicity and avoid firs pass metabolism (2) . sodium alginate , a naturally occurring poly saccharide has been widely used as a disintegrant and gelling agent in pharmaceutical preparations (3) . it has several unique properties that have been enabled it to be used as a gel matrix for delivery of many drug (4) . chlorpheniramine maleate as a potent h1-receptor antagonist can be indicate for many types of allergy such as rhinitis and pruritis , it can prevent but does not reverse histamine mediated response (5) . this study aimed to both suggest new alternative dosage form for enhancing topical penetration of cpm , and to evaluate the potential and transdermal absorption . 1 corresponding author : e-mail : ybmmaz@yahoo.com received : 20/8/2008 accepted : 16/12/2008 iraqi j pharm sci vol.17 (2) , 2008 permeation of chlopheniramine maleate from gels 68 experimental materials and equipments : chlorpheniramin maleate cpm , supplied by sammraa drug industry sdi , sodium alginate , triethanolamine tea , from (hopkins and william ltd , england) , sodium carboxymethylcellulose nacmc , diethyl ether , glycerin, from (bdh chemical limited , pool , england) , formaldehyde 37% (v/v) , urea , from (fluka ag , switzerland) , polyethylene glycol peg1000 , methyl and propyl hydroxyl benzoate , from (merkshuchardt , germany), uvspectrophotometer ,carrywin uv ,varian , australia usp dissolution apparatus ,magnetic stirrer ,ultra sonic cleaner , vwr cpley , england , water bath shaker ,hot air oven , memmert , germany preparation of sodium carboxymethylcellulose (nacmc ) 5%w/w gel base : simply , the method employed for base was fusion method . it was carried out by incorporation cpm equivalent to 1%w/w in the following base content : nacmc powder 5gm. glycerol 15gm. methylhydroxybenzoate 0.1gm. purified water to 100gm. the base was prepared by mixing nacmc with glycerol in a glass mortar , while methylhyd-roxybenzoate (methyl paraben ) was dissolved in 40ml. of distilled water using heat to about 70̊cاwithاvigorousاstirringاbyا stirrer for 15 minutes , and cooled, then the later mixture was mixed with polymer-glycerol mixture and stirring until clear gel-base was gained . then the cpm was incorporated to the base with 5 minutes continous traturation and stirring to obtain homogenous clear drug-gel solution (6). preparation of sodium alginate (sa) 2% w/w gel base: sodium alginate 2gm. glycerol 15gm. cacl2 0.2gm. propylhydroxybenzoate 0.2gm. distilled water to 100gm. the same principle of procedure was done as in preparation of na-cmc gel base. the polymer of sodium alginate was mixed with glycirin in a glass mortar and the mixture was poured in small amounts to the vehicle with stirring , while calcium chloride was dissolve in small amount of water and added to the vehicle with stirring then complete the volume with distilled water with 5 minutes continuous stirring , until translucent – white clear gel was formed (6).different concentration of polymers corresponding to 1% , 2% and 4% (w/w) of sodium alginate and only 4% (w/w) of nacmc were used with physical mixture through studying their effect on the release process. in vitro release of cpm from gel base: a small glass container with 3cm. in diameter of its opening mouth was modified in order to be filled with one gram of each formula , which was containing equivalent weight of 1%w/w of cpm .the mouth of container was covered with the filter paper which secured in place with rubber band . the dialysis cell was inverted in 500ml. of phosphate buffer ph 7.4 contained in a beaker of the dissolution apparatus . the system maintainedاat37̊اc,اtheاsamplesاwereا withdrawn after 1 ,2 ,3 , 4 and 5 hours , and replaced with an equal volume of fresh buffer solution (7) . the sample were analyzed for their cpm content using uv-spectrophotometer atاλmaxا261اnm. preparation of the diffusion membrane : the albino rat ( 4-6 week old male ) , was scarified by ether inhalation , then the skin was shaved lightly with an electrical clipper , taking care to prevent any damage to the skin , a rectangular section of abdominal skin several centimeters in each dimension was excised using a sharp blades . the defating procedure (9), of the skin was carried out by weeping the skin with a cotton tip soaked in diethyl ether to remove the subcutaneous fat and scraping the dermal side to remove the muscles and blood vessels , the adhering fat was again removed by another cotton tip soaked in diethyl ether , and kept in phosphate buffer ph 7.4 for 2 hours in a water bath maintainedاat37̊اc,اtoاallowاwaterاsolubleاااuvااااا absorbing materials to leach out. the buffer was changed three times during this period with fresh amounts ( 10 ) .then the prepared skin for diffusion was stored in phosphate bufferاforا24اhoursاinاtheاrefrigeratorااat2̊اcا before use. in vitro diffusion of cpm through rat skin membrane( 8 ) : one gram of each formula containing cpm was introduce in a small container and the epidermal surface of the rat skin was stretched over the mouth of the container with diameter 3cm. and legated with rubber band, the diffusion cell then inverted and immersed in 500ml of phosphate buffer at ph 7.4 contained in a beaker of dissolution iraqi j pharm sci vol.17 (2) , 2008 permeation of chlopheniramine maleate from gels 69 apparatusا.اtheاsystemاwasاmaintainedاat37̊اcا and the buffer solution was stirred at 100 r.p.m. during 5 hours of the study .samples of 5 mls.were pippeted from the collection medium after 1 ,2 ,3 ,4 , and 5 hours replaced with an equal volume of freshly prepared phosphateااbufferاphا7.4اat37̊اcا.اtheاا samples were analyzed using uv spectrophotometerاatاλاmaxا261اnm. effect of different enhancers and their concentrations on the diffusion : in order to evaluate best release profile of cpm from selected formula , different enhancers, urea 5%w/w, polyethylene glycol (peg1000) 2% w/w, and triethanolamine (tea) 1% , 2.5 ,and 5% w/w were used on diffusion of cpm through rat skin skin irritation test ( 11 ): skin male albino rats weighing approximately 500gm. were used to study the irritation test of the selected formula , on the rat skin . the dorsal side of the rat was carefully shaved and two circular areas of 2.5 cm. in diameter in each animal were done .then 0.8%v/v aqueous solution of formalin as standard irritant to one circular area , and 5% w/w tea gel formula contain 3% w/w cpm to the other circular area for three rats, and 2% w/w sodium alginate gel containing 3% w/w cpm to other circular areas of other three rats .the fresh gel samples and formalin solution were applied for 7 days , finally the application sites were graded to the visual scoring scale always by the same investigator . stability study : the estimation of the shelf life of a selected formula 3% w/w cpm kept in a collapsible tubes at room temperature and oven maintained separately at 40,50 , and 60̊cا,اsamplesاwereاtakenاeveryاsevenااdaysا for 4 weeks . each sample of the gel equivalent to 250mg. cpm in 50 ml. of phosphate buffer at ph 7.4 . these samples were quantitatively transferred to volumetric flasks and appropriate dilutions were made with the same buffer , then the resulting solution were filtered with 0.45µ filter paper , the absorbance of each collected sample was calculatedاforاcpmاcontentاatاλاmaxا261ااnmا (12) . effect of the temperature on the ph of the gel : the ph of the gel was measured every week for one month , by taking one gram of the gel from each stored sample at 40 , 50 , and 60 c̊,اandاshakenاupاwith10اmls.اof distilled water . the ph of the final solution was measured and recorded . statistical analysis the significance between mean values was analyzed by student ttest , p-value of less than 0.05 was considered significant for all analyzed data shown in the results of this study . results and discussions effect of gel bases on the release of cpm : table 1. and figure 1 , show the amount of drug release from gel bases , the results indicated that the drug released is significantly increased p< 0.05 as a function of polymer type used in an order of 4%w/w sa > 4% nacmc , this result may be referred to the hygroscopic effect of cellulose derivatives that affect water entrapment in the cross linking gel of 4%w/w nacmc more than that of 4% w/w sa .since this amount of water may hinder another water molecules diffuse inside gel structure and then more drug releasing occurred . this result is in consistent with results obtained by cetin t. et . al (13) . table (1) . effect of different bases on the release rate constant ( k ) of cpm 1%w/w in phosphate buffer ph7.4 at 37̊c . type of bases amount of cpm released (mg.)/5hr.* cpm released % k (mg.min-1/2 ) 4%w/w sa 9.06 ±0.09 90.6 0.427 4%w/w nacmc 6.77 ±0.11 67.7 0.401 each value represents the mean sd ( n=3 readings in each group *) iraqi j pharm sci vol.17 (2) , 2008 permeation of chlopheniramine maleate from gels 70 figure (1) . the effect of different bases on the release of cpm 1%w/w at ph 7.4 and 37̊c effect of polymer concentrations on the release of 1%w/w cpm gel : table 2. and figures 2 and 3 , demonstrate the effect of sa concentrations on the release profiles of the cpm through rat skin , it was seen that the drug released from sa at different concentration and diffused through the filter paper was not affected by the concentration of the polymer , since no significant increase in the drug release , this behavior gives an impression that the drug release from sa followed zeroorder kinetics in these concentrations , since water up take by polymer is not affected by the concentrations of the polymer it self , meanwhile the plot of the amount of the drug released versus square root of time demonstrates that there is a linear relationship of the drug release followed higuchi principle in the diffusion process from semisolids in percutanous absorption . these results were in agreement with the results obtained from the permeation of carvedilol transdermal patches (11) . table (2) . effect of sodium alginate (sa) concentrations on the rate constant (k) of cpm 1%w/w phosphate buffer ph 7.4 at 37̊c . sodium alginate concentratin amount of cpm (mg./5hr.)* cpm released % rate constant (k) (mg.min-1/2 ) 1%w/w 8.934±0.17 89.3 0.4983 2%w/w 9.435 ±0.14 94.3 0.5713 4%w/w 9.060 ±0.04 90.6 0.4273 each value represents the mean sd ( n=3 readings in each group *) figure (2). the effect of polymer concentration on the release of cpm 1% w/w through rat skin at ph 7.4 and 37̊c figure (3) . the kinetic analysis of cpm 1% w/w release from different polymer concentration at ph 7.14 and 37̊c effect of cpm concentrations on the diffusion process : table 3. and figure 4 , illustrate the effect of different cpm concentration 1% w/w and 3% w/w on the amount of cpm diffused through the rat skin , using 2% w/w sa as a gel base , the results showed that the amount of cpm diffused during the period of application ( 5hours ) , increased as a function of increasing drug concentration . this behavior confirmed that the drug diffusion followed firstorder mechanism , and the penetration rate is proportional to the concentration , since this diffusion depend on many factors , among them , the partition coefficient ( k ) and the 0 5 10 15 20 0 1 2 3 4 5 6 tim e (hour) a m o u n t o f c p m r el ea se d ( m g .) nacmc4%w/w na alginate 4%w/w 0 5 10 15 20 0 1 2 3 4 5 6 time (hour) a m o u n t o f c p m r el ea se d ( m g .) sod. alginate1%w/w sod.alginate 2%w/w sod. alginate 4%w/w 0 5 10 15 20 square root of tim e (m in.) a m o u n t o f c p m r el ea se d ( m g .) sod. alginate1%w/w sod.alginate 2%w/w sod. alginate 4%w/w sod. alginate(ايطخ )w/w%4 sod. alginate(ايطخ )w/w%4 sod. alginate(ايطخ )w/w%4 sod.alginate(ايطخ )w/w%2 sod. alginate(ايطخ )w/w%4 sod.alginate(ايطخ )w/w%2 .sod(ايطخ )alginate1%w/w iraqi j pharm sci vol.17 (2) , 2008 permeation of chlopheniramine maleate from gels 71 concentration of the drug (14 ) . in this experiment the rate limiting step of the drug diffusion through the rat skin can't be estimated , because there are two types of partitioning , one of the partition of the drug for the skin ( ds ) , and the other for vehicle ( dv ) ,or gel base . so these two magnitudes of the two diffusion coefficient ds and dv , determines whether the release from vehicle or skin is the rate limiting step, and by this approach, the concentration of incorporated drug in the gel base may solve this problem , regardless the diffusion or partition coefficient (11 ,14 ). table 3. effect of cpm concentration on the diffusion rate constant (k) using 2%w/w s0dium alginate (sa) gel . cpm concentration % cpm amount diffused mg./5hr. * rate constant (mg.min ̄½) 1%w/w 7.54±0.09 0.582 3%w/w 11.765±0.21 0.8217 each value represents the mean sd ( n=3 readings in each group ) * figure (4) . the effect of cpm concentration on the diffusion process of through rat skin from sodium alginate 2%w/w gel at ph 7.4 and 37̊c effect of different enhancers and their concentrations on the diffusion of cpm 3%w/w through rat skin: the enhancers which they are used in this study are water soluble types , they include urea 5% w/w , peg 1000 2 % w/w , and tea 2.5% w/w table 4 . and figure 5 , illustrate the effect of incorporation the above enhancers separately on the diffusion of 3% w/w cpm loaded in 2% w/w sa gels through rat skin . it was seen that both urea 5% w/w and peg 1000 2% w/w significantly decrease p < 0.05 the amount of the drug diffused through rat skin. since incorporation of urea in hydro gel may activate the hydrolysis process of urea to form ammonia and carbon dioxide , which lead to elevate the ph of the medium of environment , which in turn dissociate the cpm into maleate anion and chlorpheniramine cation , these ionized species may hinder the diffusion process of the drug (15) . moreover , the alkyl amine group , of the drug may complex ethylene oxide ( ch2-ö-ch ) group of peg1000 that decrease the amount of the drug available for permeation, this result is in a consistent with that result obtained , when lidocaine was formulated as a topical gel (16) . on the other hand , incorporation of 2.5%w/w of tea which counter irritant to the skin as an enhancer , showed a slight increase of cpm permeation through the rat skin , this effect may be attributed to both effects of tea as a basic tertiary amine enhancer once that is compatible with alkyl amine anti histamine (cpm ) , and second may be referred to the effect of emulsification of tea with malic acid to form water soluble salt that easily allow the drug penetration through the rat skin (17). table (4). effect of different enhancers on the diffusion rate constant ( k ) of cpm 3%w/w through the rat skin enhancer type cpm amount diffused mg./5hr. rate constant (mg.min ½̄) no addition 11.765±0.11 0.8217 urea 5%w/w 5.648±0.24 0.3659 peg1000 2%w/w 6.820±0.12 0.4807 tea 2.5%w/w 13.138±0.16 1.09 estimation of irritation property of selected formula : the selected formula which was introduce to specify the irritation test consist of 3%w/w cpm loaded in 2% w/w sa and fortified by 5% w/w tea as an enhancer. after 7 days of gel application on the dorsal shaved skin of albino rat , it was seen that a 0 5 10 15 20 0 1 2 3 4 5 6 tim e (hour) a m o u n t o f c p m d if fu se d ( m g .) cpm 1%w /w cpm 3%w /w iraqi j pharm sci vol.17 (2) , 2008 permeation of chlopheniramine maleate from gels 72 recognized redness area on the skin developed during this period , while the application of the same formula free from 5% w/w tea showed no appearance of this irritation . this observation may be related to the irritation effect of tea itself at this concentration , since most of the quaternary ammonium surfactant are strongly catoionic irritant enhancers (18 ) . figure (5) . the effect of different enhancers on the diffusion process of cpm 3% w/w through rat skin at ph 7.4 and 37̊c stability study determination of the shelf life the results of this study showed cpm followed first order kinetic degradation , when the selected formula kept in a collapsible tubes maintained separately at , اtheseاofاcontentsااtheا,اا60̊cااandا,ا50ا,ا40 tubes for cpm amount were determined every seven days for 4 weeks , and the rate ofاdegradationااat̊ا25ااcا)ااk25̊cا يف (نزو\ نزو)%اwasاfoundاا toاbeا0.872̄ااxا10ا.daȳ¹ا,اwhenاarrheniusاplotا was constructed as a logarithm of degradation rates constants for above exaggerated temperatures against reciprocal of absolute temperatures of cpm storage as shown in figures 6 and 7 to estimate the shelf life , the following expression was used to estimate 90% of the drug content remain at that time . t0.105/0.872̄ا=اا يف (نزو\ نزو)%90اday = 1204 days so the calculated shelf life for a selected formula was found to be about 3.3 years . figure (6) . accelerated degradation of cpm in a selected formula at different exaggerated temperatures figure 7 . arrhenius plot for estimation of shelf life of cpm of a selected formula effect of temperatures and storage time on the ph , color and odor of 3%w/w cpm gel the result after 30 days storage time at different storage exaggerated temperatures of cpm gel , revealed that slight increase in the ph of cpm gel from 4.25 to 4.6 , which may be attributed to the ionization of cpm that releases chlorpheniramine base , which belongs to the basic properties of this types of antihistamines ( 19 ) . more over no change in the original translucent white color or appearance of unpleasant odor was observed . these results indicated no probability of physical instability, or growth of microorganisms in the selected formula . 0 5 10 15 20 0 1 2 3 4 5 6 tim e (hour) a m o u n t o f c p m d if fu se d ( m g .) no addition urea 5% w/w peg 1000 2% w/w tea 5% w/w 1.9 1.91 1.92 1.93 1.94 1.95 1.96 1.97 1.98 1.99 2 0 7 14 21 28 tim e (days) lo g % r e m ai ni n g o f c p m 40 c 50 c 60 c يطخ 40( )c يطخ 50( )c يطخ 60( )c -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 2.9 3 3.095 3.19 3.35 (1/abs olute tem p.)*1000 l o g r at e co n st an t (k ) d ay iraqi j pharm sci vol.17 (2) , 2008 permeation of chlopheniramine maleate from gels 73 conclusions concerning of the results obtained , one can conclude , the followings : 1. maximum cpm release was achieved, when 4% w/w of sa was introduce as a gel base. 2. the diffusion of cpm through the skin of the rat ,was increased as a function of increasing cpm concentrations , loaded in 2%w/w sa gel base . 3. addition of 5%w/w tea to 2%w/w sa loaded by 3%w/w cpm , enhances the amount of the drug diffused through the skin of the rat . 4. addition of 2%w/w peg1000 , and 5%w/w urea , separately to 2%w/w sa loaded by 3%w/w cpm , decreases significantly p< 0.05 the amount of the drug diffused through the skin of the rat . 5. there was a marked irritation spots recognized, when tea 5%w/w used as an enhancer in the selected formula , compared with no effect when this enhancer is avoided . 6. the selected formula of sa 2%w/w as a base loaded by 3%w/w of cpm was acceptable with calculated shelf life about 3.3 years . this formula may need a further clinical study on volunteers to ensure its therapeutic , and economic value . references 1. ansel c. , allen v. , popovich g. , pharmaceutical dosage form and drug delivery systems , 8th. edition , lippincott williamsand wilkins , philadelphia , 2005 , 415-419 . 2. makiko f. , kumi s. , " effect of phosphatidyl choline on skin permeation of indomethacin from gel phospholipids " , international journal of pharmacy , 2001 ,1 , 57-64 . 3. pongjanyakul t. , puttipipatkhacorn s. , "sodium alginate-magnesium aluminum silicate composite gel ", aaps pharmaceutical science technology, 2007 , 8 , 3 . 4. wayne r. , gombotz t. ,"protein release from alginate matrices " , advanced drug delivery reviews ,1998 ,3 ,267-285 . 5. martindal , the extra pharmacopoeia , 31st. edition , the pharmaceutical press london , 1996 , 436-438 . 6. cooper and gunns , dispensing for pharmaceutical students , 11th. edition , pitman medical and scientific publishing company ltd , london, 1987 , 214-222 . 7. ezzedin f. , shihab f. , stoh s. , "release of sorbic acid from different bases " international journal of pharmaceutics , 1986 , 28 , 113-116 . 8. systems , 8th. edition , lippincott williamsand wilkins , philadelphia , 2005 , 415-419 . british pharmacopoeia , cd , soft ware , 2007 . 9. wrusten d. e. , kramer s .f. , " investigation of some factors influencing perccutaneous absorption , journal of pharmaceutical science , 1991 , 50 , 288. 10. sloan k. , koch a. , siver k. , " the use of solubility parameter of drug and vehicle to predict flux through skin " , journal investigation of dermatology 1986 , 87 , 244 . 11. ubaidulla u . , reddy m . ,ruckmani k. , " transdermal therapeutic system of carvidolol , effect of hydrophilic and hydrophobic matrix on in vitro and in vivo characteristics " , aaps pharmaceutical science technology , 2007 , 8 (1) ,article 2 . 12. jons s. p. , greenway m., j., " the influence f receptor fluid on in vitro percutaneous penetration " , international journal of pharmaceutics " 1989 ,53 , 43-46. 13. cetin t. , yalcin o. , ' in vitro release study of chlopheniramine maleate from hydroxy propyl methyl cellulose derivatives gels . "issue of department of pharmaceutical technology " , faculty of pharmacy , ankara university,2003 14. allfred m . , physical pharmacy , 4th. edition , b ..i . weavery press v. t . , ltd new delhi ,1994 ,324 327, 347 and 497 . 15. martindale , the extra pharmacopoeia , 28th. edition , the pharmaceutical press , london , 1988 , p. 616 . 16. pramot p . , joel l . , " evaluate of penetration enhancement of lidocaine by surfactant through hairless mouse skin " , journal of pharmaceutical science 1986 , 75 , p.176-181 . 17. watanabe s . , kawahara h.," adducts of cyclic acid anhydride and fatty amines as anti rust additives in water based cutting fluids ", journal of american oil chemist society , 1991, 68 , 2 18. witold m. ., alexander k ., " preliminary assessment of alginic acid as a factor buffering triethanolamine interacting with artificial skin sebum ", european journal of pharmaceutics and biopharmaceutics , 2003,55,237-240 19. wilson and gisvold"s , text book of organic medicinal and pharmaceutical chemistry , j . b .lippincott company , new delhi , 1991 ,8th. edition , p . 658. a study on the stabilty of different frusemide liquid dosage formulas: oral solution, syrup, elixir, suspension and emulsion iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 32 cephalothin as a carrier of 6-mercaptopurine for targeting cancer tissues mohammad h. mohammad 1,* and haider j.al-karagully ** * department of pharmaceutical chemistry, college of pharmacy, university of baghdad , baghdad ,iraq ** ministry of health , baghdad ,iraq abstract a lower extracellular ph is one of the few well-documented physiological differences between tumour and normal tissues. on the other hand, elevated glutathione (gsh) level has been detected in many tumours compared with healthy surrounding tissues. the compound ii: 3-(9h-purin6-yl-thio) carbonothionyl methyl-8-oxo-7-(2-thiophen-2-yl) acetamido-5-thia-1-azabicyclo-4-octo-enecarboxylic acid was a cephalothin derivative contain 6-mercaptopurine (6-mp). compound ii react with general base catalysis in slightly acidic ph or with sulfhydryl nucleophiles to release the chemotherapeutic drug 6-mp. the generation of compound ii was accomplished following multistep reaction procedures. the structure of compound ii and its intermediate was confirmed by their melting point, infrared spectroscopy, chn and nmr analysis. the hydrolysis of compound ii in aqueous buffer solution of ph 6 and in the presence of gsh at ph 7.4 was studied. the partition coefficient (pc) of compound ii was also determined. compound ii has acceptable rate of hydrolysis at slightly acidic medium ph 6 (t1/2 = 56.34 min.) and 80% of compound ii had been converted to 6-mp within 30 min in the presence of gsh. and the compound has acceptable stability at ph 7.4(t0.5=639.65 min.) and the rate of hydrolysis was effected by change the buffer concentration. this compound can selectively deliver 6-mp into the tumour tissues which have acidic ph or elevated gsh level. compound ii had an improved pc value of 12.23 compared to 1.22 for free drug 6-mp confirming higher lipophilicity. key words: 6-mercaptopurine, cancer, prodrug, targeting ةصالخلا ( ةينايفسالىريٞ إ٘الياالىوثٍيلالىدايةٞ الىديفع الىَ٘ا٘عٔا ِٞال ّاة الىا جيّٞ اphلُالّردينال سالىٖٞافٗنْٜٞا) ُ٘ا) ٙالىاي٘جيٞا ٍِانٖ ال ةينا ٙام ااى٘يرالفجدي امٜاٍا٘ي ( مٜالا اٍِال ٗفلًاٍ يفّ ا ي ّاة الىسنٞوٞ اgshٗل ّاة الىسنٞوٞ ٗ. :iiلىَحٞس .لُالىَ ملا 3-(9h-purin-6-yl-thio) carbonothionyl methyl-8-oxo-7-(2-thiophen-2-yl) acetamido-5-thia-1azabicyclo-4-octo-ene-carboxylic acid ٛاليٚاٍ ملالى اcephalothinٕٗ٘اٍليفاىياٞديى٘اِٞا) اٝيديلواٍدالى يلامالىويٍ اiiلُالىَ ملا .(mp-6-ٍ مني٘ٞ ٘فِٝا)6( ٝح٘ي -ٍ مني٘ٞ ٘فِٝ .جٌاجحعٞ الىَ ملا6( لىنييرالِالىْ٘لماىيح ٝ الى اsulfhydrylمٜالىَحٞفالىحٍيعٜالىعوٞاالٗاٍداايدٖيٝافٝوا) iiْٖيا ٘لاس اعٞيسا فنيلا ا يجني اج ٝ الىيديلواٍيوا الىرس٘للاٗعااجٌالانيلالىمٞلالىٞنَٞيٗٝ اىيَ ملالىْٖيمٜاٗلى٘اسٜاٗلىييماٍا امٜالىَحي٘هالىالفلامٛال ساiiلّ مٖيفاٗلىيحيٞوالىسٞدٜاىثحو اجحتالىحَ ل اٗلىيحيٞوالىاعٞفاىيوْيل .ٗجٌا فلا اجحيوالىَ ملا ( .ٗلنحتالىْييملالىَايحمي اٍِإمٓاpartition coefficient) ii( ٗممىلاجٌا فلا اٍويٍوالىي ِٝاىيَ ملاph 6لىٖٞافٗنْٜٞا) ا عٞ (56.34() وَ ّاماا =ph 6اَٝييلاا ل اجحيواٍ ن٘ى امٜالىَحي٘هالىالفلامٛال سالىٖٞافٗنْٜٞا)iiلىافلا الُالىَ ملا الامَياجنِٞالُاا ل اجحيوالىَ ملاجييا ا ياٞ اج مٞيالىَحي٘ها(ph-74)ٗانيجٞ اٍ ن٘ى امٜالىَحي٘هالىالفلامٛال سالىٖٞافٗنْٜٞاا ( لىٚالىرثٝيالىا جيّٞ امللال سالىٖٞافٗنْٜٞاmp-6-ٍٞ مني٘ٞ ٘فِٝا)6اٝايسٞداٝلميهالى اiiلىالفل .ىمىلاٍِالىَي٘عدالُالىَ ملا ُ٘اٝيٝااٍِاا ل اجحيوالىَ ملا6لىَ يفراىي ا) ٍِالىَ ملاعااجح٘هالىٚالى ا80لايٞرالُاii( .ٗعاالجعحاٝلعيالُالىاي٘جيٞا % 6ا عٞ .مَياجٌاٍثيب الُاٍويٍوالىي ِٝاعااجحاِاٍَياٝاهاليٚاجحاِاّديمٝ الىَ ملا ل ةيناوالىةاٌلايٞرالُا30ٍ مني٘ٞ ٘فِٝا ةيناثها .1.22-ٍ مني٘اٞ ٘فِٝإ٘ا6امٜايِٞالُاٍويٍوالىي ِٝاىي ا12.23إ٘اiiٍويٍوالىي ِٝاىيَ ملا introduction the use of antineoplastic agent 6-mp accompany by several disadvantages including sever adverse effects (1), poor absorption, low bioavailability (2), limitation of uses (3), thiopurine associated leukemia (4,5) and drug resistance(6,7,8). cephalosporins are -lactam antibiotics; their degradation depends on the side chain at c-7 and the substitution on c-3 (9). the presence of good leaving group at c-3 facilitate spontaneous expulsion of the 3substituent by any general nucleophile, lactamase or ph change (10,11), scheme (1). 1 corresponding author : e-mail : dr.mhassan@yahoo.co.uk.666666 received : 13/7/2008 accepted : 11/11/2008 mailto:mhassan@yahoo.com iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 33 n s cooh ch2 r'o hncr o h h 7 3 cephalosporine-c beta-lactamase, ohh3o + hn s cooh ch 2o hncr o h h oh + r' anhydrodesacetyl cephalosporinic acid r'= good leaving group scheme (1): possible degradation of cephalosporin c (11). lowering extracelular ph (phe) is one of the few well-documented physiological differences between solid tumour and normal tissues. the main phe in tumour tissues is 0.60.8 unit lower than normal tissues, with an absolute as low as 5.8 (12,13). changes in gsh content and level of glutathione s-transferase have been detected in tumours (14). moreover, increased levels of gsh have been linked with drug resistance (15) and poor patient prognosis(16). thus, an excellent opportunity exists for the design of prodrugs that specifically target tumours with low phe or with abnormal gsh level. doxorubicin armed antibody was a prodrug designed to target tumours with low phe (17). the 6-mp prodrug s-(9h-purin-6-yl) glutathione (figure 1a) which can be further metabolized in vivo to yield 6-mp(18). cis 6-(2-acetyl vinyl) thiopurine (avtp). (figure 1a), a potential 6mp prodrug targeting tumours with upregulated gsh level. structurally avtp is butanone conjugate of 6-mp and a michael acceptor that undergoes addition-elimination reaction with nucleophiles to yield 6-mp, the avtp metabolism to 6-mp was gsh dependent (19). h n nn n sh 6-mercaptopurine h n nn n s ch 2 ch c h n nh ch 2cooh h2 cc h ch2n cooh o o h n nn n s ch 3 o avtp n s cooh ch 2 o c ch 3 o o hnch2c s o figure (1a): chemical structure of 6-mercaptopurine, s-(9h-purin-6-yl) glutathione, cis-avtp and cephalothin. iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 34 materials and methods chemicals 6-mercaptopurine and hcl were purchased from fluka (germany); cephalothin sodium was purchased from laboratories torlan, s.a. (spain); gsh was purchased from sigma (usa); and carbon disulfide was purchased from riedel-dehen (germany). all chemicals were reagent grade and obtained from standard commercial sources. elemental micro analysis were performed using carlo erba elemental analyzer 1106 (italy); melting points were measured on thomas hoover electronic melting point apparatus; and are uncorrected; infra red spectra were recorded as kbr disks on back ir spectrophotometer (college of pharmacy, university of baghdad); and h-nmr spectrum was carried out on mercury mhz-nmr spectrometer (sppm) at mdit center in the university of toronto. synthesis of potassium 7hpurinylcarbonotrithionate (compound i) to a stirred solution of potassium hydroxide (0.313gm, 5.58 mmol) in absolute ethanol (10ml) at 15-20 oc , a 6-mp (0.96gm, 5.58 mmol) was added over (0.5 min). after 0.5 hr carbon disulfide (0.425gm, 5.58mmol) was then added and the reaction mixture was stirred for 3 hr at 25 oc. then the precipitate of the desired compound was obtained by the addition of diethyl ether. the precipitate was filtered and re-crystallized from ethanol to give white crystalline powder of compound i, percent yield (63%), melting point (240-243 oc) and infrared yield absorption band, (cm-1): 3450 of nh stretching vibration (str. vib.) of purine, 1610 and 1556 of c=n str. vib., 1598 of nh bending vibration (bend. vib.) of purine, 1497 and 1423 of c=c str. vib., 1410 of ch bend. vib. of purine 1205 of c=s str. vib. of trithiocarbonate and 870 of aut plane ch bend. vib. of purine. synthesis of 3-(9h-purin-yl thio) carbonothionyl-methyl-8-oxo-7(2-thiophen-2yl) acetamido-5-thia-1-azabicyclo-4-octa-2ene-carboxylic acid (compound ii) a mixture of cephalothin sodium (0.73gm, 3.4mmol) and of compound i (0.5gm, 3.4mmol) in (30ml) of 0.1m phosphate buffer (ph 7) was heated for 3hr at 60-70oc. the mixture was cooled to 5 oc and acidified with hcl (1n) to reach ph 4.0, a precipitate was obtained and collected by filtration, dried and recrystallized from ethanol to give a yellow crystalline powder of compound ii. percent yield (60%), melting point (196-199 oc), elemental microanalysis, calculated/found: (c 42.539/42.996, h 2.855/3.011, n 14.882/15.189, o 11.339/11.637, s 28.390/28.427, the infrared absorption band (cm-1): 3450 of nh str. vib., 3000-2500 group of small band due to oh str. vib. of oh of cooh, 2682 of ch2-s str. vib. of cephalothin, 1773 of c=o str. vib. of lactam, 1610 a mide str. vib., 1617 and 1570 of c=n str. vib. 61598 of nh bend-vib. of purine, 1500 and 1430 of c+c str. vib., 1408 of ch bend. vib. of purine, 1228 of c=s str. vib. of trithiocarbonate ester, 875 out plane ch bend vib. of purine, 690 str. vib. of thiophen: and hnmr in cdcl3 3.06, 3.16 (q, 2h of methylene  to c=c and  to s-c-), 3.44 (s, 2h of methylene  to c-r and  to (c=o)-n), 4.07 (s, 2h of methylene  to –c=c and  to – s-c-r), 5.1 (d, 1h of propiolactam  to –s r, to –n-c=o), 5.45 (d, 1h of propiolactam  to n-c=o, to –s-r), 6.83 (d, 1h of 2thiophen), 6.93 (t, 1h of 2-thiophen), 7.4 (d, 1h of 2-thiophen), 8.32 (s, 1h of sec. amide), 8.57 (s, 1h of purine), 13.65 (s, 1h, nh of purine) as shown in (figure 1b). iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 35 02468101214 ppm figure ( 1b) : h nmr spectrum of compound ii . hydrolysis of compound ii at ph 6 and ph 7-4 the hydrolysis of compound ii was carried out for the equivalent of (0.01mg/ml) in aqueous phosphate buffer solution of ph 6 and ph 7.4 at 37 oc. the total buffer concentration was 0.1m and the ionic strength (μ)اofا1اwasاmaintainedاbyاaddingاcalculatedا amount of nacl. different sample were taken for analysis at specific time interval (16, 30, 60, 120, 240 min) and the rate of hydrolysis was followed spectrophotometrically by recording 6-mp absorbance increase accompanying the hydrolysis at 324nm and 316 for ph6 and ph7-4 respectively. the observed pseudo-first order rate constant was determined from the slope of the linear plot of log concentration of 6-mp vs time. the effect of the buffer concentration at the rate of hydrolysis the same procedure mentioned above was fallowed for studing the hydrolysis of compound ii in different buffer concentration (0.2, 0.5 and 0.8) and the ionic strengthا(μ=1)ا to determine the effect of buffer concentration at the rate of hydrolysis. hydrolysis of compound ii in the presence of glutathione compound ii (0.96gm, 0.17mmol) was incubated with glutathione (0.52gm, 0.17mmol) in phosphate buffer (0.1m and μ=1)اatاphا7.4اinاshakingاwaterاbath37اoc. a 1.0 ml sample was removed and added to 4ml of phosphate buffer. the concentration of 6mp was determined spectrophotometrically at 314nm. partition coefficient estimation partition coefficient of compound ii at ph 7-4 was estimated by using shake flask method. the amount of compound ii in both phase were measured spectrophotometrically at 298 nm, and the partition coefficient value was calculated by the following equation (20). layer inorganic in conc. layer organic in conc. pc  results and discussion target compound ii was obtained following procedure outlined in scheme (2), compound i was obtained from reaction of 6mp and carbon disulfide in the presence of potassium hydroxide. the reaction was a nucleophilic addition reaction in which the thiolate anion was added to the carbon atom of the disulfide (21). compound ii have been synthesized using the method previously established for the synthesis of cephalosporin derivative (22). the reaction followed a nucleophilic substitution in which the thiolate moiety attached the methylene moiety at position 3 of cephalothin leading the formation of trithiocarbonate ester with liberation of acetic acid. under the experimental conditions used the hydrolysis of the compound ii followed pseudofirst order kinetic, since the plot of log conc. of 6-mp vs time resulted in straight line from it slope, the observed rate constant of hydrolysis (kobs) was calculated from figure (2a) and (2b) which representive graph for hydrolysis of compound ii. the kobs were 0.0123 min-1 and 1.083x10-3 at ph 6 and ph 7.4 respectively and the half-life of hydrolysis of compound ii were 56.34 min and 639.65 min at ph6 and ph 7.4 respectively. the half life was calculated using the equation: obsk . .t 6930 50  s h n n s n o ho s s o o s n nhn 6.83 6.93 7.40 3.44 8.32 5.45 5.1 3.16;3.06 12.56 4.07 8.57 13.65 8.57 iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 35 figure (2a): first order plot for the hydrolysis of compound ii in 0.1 m phosphate buffer of ph 6.0 at 37 oc ( =1). figure (2b): first order plot for the hydrolysis of compound ii in 0.1 m phosphate buffer of ph 7.4 at 37 oc ( =1). time (min) -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0 50 100 150 200 250 300 lo g c on c o f 6 -m p iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 36 n s cooh ch2 o c ch3 o o hnch2c s o h n nn n sh koh h n nn n s k cs2 15-20 c / 0.5 hr h n nn n s c s s k cpd i heated at 60-70 c for 3 hr at ph 7.0 h n nn n s cs s cpd ii n s cooh ch2o hnch2c s o scheme (2): synthesis of compound ii. iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 37 the release of 6-mp from compound ii depend on the opening of -lactam ring which in turn depend on the ph of the media and the ease with which the substitution at position 3 of cephalosporin will leave the molecule (10,11). the tithiocarbonate is better leaving group than acetoxy group due to it soft base (23), and this will cause the hydrolysis of compound ii at ph 6 is faster than hydrolysis of cephalothin at the same ph (scheme 3). the rate of hydrolysis of compound ii at ph 6 in the presence of different buffer concentration was calculated from figure (4) which represent the hydrolysis of compound ii at different buffer concentration. the kobs. were 0.0133mmin -1. 0.0154 min-1 and 0.0166 min-1 at buffer concentration 0.2, 0.5 and 0.8m respectively. the half-lives were 52.13min, 45.00min. and 41.1 min. for buffer concentration 0.2, 0.5 and 0.8m respectively. under experimental condition used the hydrolysis of compound ii followed second order kinetic, since plot of log conc. of 6-mp vs time result in curve line. the formation of 6-mp from compound ii was linear for at least 30 min. (figure 3). 80% of compound ii had been converted to 6-mp within 30 min. compound ii reacted rapidly with thiolate nuceophile of gsh to yield the 6mp. the thiolate was attack the -lactam ring of cephalosporin resulted in the opening of this ring with the release of trithiocarbonate derivative from position 3 (24) (scheme 4). the partition coefficient has become the most common physicochemical property (25). the partition coefficient of compound ii equal to 12.23; so this compound had an improved partition coefficient value compared to 1.2 for 6-mp, confirming higher lipophilicity and improvement of the drug bioavailability. n s cooh ch 2ho hnch2c s o hn nn n sh hn nn n s cs s n s cooh ch 2o hnch2c s o h+ h2o + hn nn n scs s + cs 2 scheme (3): the liberation of 6-mercaptopurine from compound ii at ph 6.0(10). iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 38 n s cooh ch2o hnch2c s o h n nn n sh h n nn n s cs s n s cooh ch2o hnch2c s o + h n nn n scs s +cs2 gs s g scheme (4): liberation of 6-mercaptopurine from compound ii in the presence of glutathione. figure (3): plot of the hydrolysis of compound ii in the presence of glutathione (0.17 mmole) in phosphate buffer of ph 7.4 at 37 oc ( =1). iraqi j pharm sci , vol.17 (2) , 2008 cephalothin as a carrier of 6-mercaptopurine 39 figure (4): the effect of buffer concentration on the rate of hydrolysis of compound ii at ph 6 at 37 0c ( =1). * buffer concentration: a= 0.2 m , b= 0.5 m , c= 0.8 m references: 1. lennard, l.: the clinical pharmacology of 6-mercaptopurine. eur. j. clin. pharmacol. 1992; 42: 329. 2. remers, w.a.: antineoplastic agents. in: wilson and gisvold’s textbook of organic medical and pharmaceutical chemistry (10th ed.). delado, j.n. and remers, w.a. 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(eds.), lippincott, raven, philadelphia, 1998; pp. 16. iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 50 formulation and evaluation of fast dissolving tablets of tastemasked ondansetron hydrochloride by solid dispersion alaa a. abdulqader *,1 , eman b. h.al-khedairy ** * department of pharmaceutics, college of pharmacy, tikrit university, tikrit, iraq . ** department of pharmaceutics, college of pharmacy, baghdad university, baghdad, iraq. abstract ondansetron hydrochloride (onh) is a very bitter, potent antiemetic drug used for the treatment and/or prophylaxis of chemotherapy or radiotherapy or postoperative induced emesis. the objective of this study is to formulate and evaluate of taste masked fast dissolving tablet (fdts) of onh to increase patient compliance. onh taste masked granules were prepared by solid dispersion technique using eudragit e100 polymer as an inert carrier. solvent evaporation and fusion melting methods were used for such preparation. completely taste masking with zero release of drug in phosphate buffer ph 6.8was obtained from granules prepared by solvent evaporation method using drug: polymer ratio of 1:2, from which four formulas pass pre-compression evaluation and compressed to fdts and evaluated for their drug content, in-vitro disintegration time, in-vivo disintegration time, wetting time and in vitro drug release profile. f7 which is prepared from solid dispersion product equivalent to the required dose of onh , crosspovidone as superdisintegrant, aspartame as sweetener ,ross berry as flavor ,polyvinylpyrolidone k30.3.as binder ,avicil ph102 and , mannitol as diluents give best invitro, invivo disintegration time and best drug release profile. key words: ondansetron hydrochloride, taste masking, solid dispersion, eudragit e100. تواسطح ذصييغ وذقيين حثوب سزيعح الذوتاى هكسوج الطعن لالونذانسيرزوى هايذروكلورايذ الونرشز الصلة عالء عثذ االله عثذ القادر *،1 و ايواى تكز حاسم الخضيزي ** * .انعزاق،ركزٌذ ،خبيعخ ركزٌذ ،كهٍخ انصٍذنخ ،فزع انصٍذالٍَبد ** .انعزاق .ثغذاد ،خبيعخ ثغذاد ،كهٍخ انصٍذنخ ،فزع انصٍذالٍَبد الخالصة يز خذاً ٌسزخذو ، نهعالج ٔ/أٔ انٕقبٌخ يٍ انغثٍبٌ ٔانزقٍؤ انُبرح يٍ انعالج انكًٍٍبئً أٔ دٔاء االَٔذاسٍززٌٔ ٍْذرٔكهٕراٌذ انعالج اإلشعبعً أٔ ثعذ انعًهٍخ اندزاحٍخ. انٓذف يٍ ْذِ انذراسخ ْٕ صٍبغخ ٔرقٍٍى حجٕة سزٌعخ انذٔثبٌ يكسٕح انطعى نشٌبدح ايزثبل اء ٍْذرٔكهٕراٌذ االَٔذاسٍززٌٔ ثبسزخذاو رقٍُخ انًُزشز انصهت ٔاسزعًبل انًزٌض نهذٔاء . رى رحضٍز حجٍجبد يكسٕح انطعى نذٔ ثطزٌقخ انصٓز ٍٔجبد ايب ثطزٌقخ رججخز انًذٌت االخفبء انطعى انًز نهذٔاء حٍث حضزد ْذِ انحج e100ٌٕدراخٍذ ثٕنًٍز يشبثّ نهعبة يٍ حجٍجبد رى انحصٕل عهٍٓب ٔقذ رى انحصٕل عهى إخفبء كبيم نهطعى ثعذو رحزر انذٔاء فً ٔسظ e100نهٍٕدراخٍذ ٔقذ رى اسزخذاو ْذِ اندجٍجبة العذاد سجعخ صٍغ نهحجٕة 1:2دٔاء: انجٕنًٍز ٔثُسجخ e100ثطزٌقخ رجخز انًذٌت نجٕنًٍز انٍٕدراخٍذ خم ٔخبرج اندسى ٔقذ اسزُزدذ انسزٌعخ انذٔثبٌ ٔانزً رى رقًٍٍٓب يٍ حٍث كًٍخ انذٔاء ، انٕقذ انالسو نززطٍجٓب ٔ انٕقذ نززفككٓب دا حجٍجبد انًُزشز انصهت يكبفئخ نهدزعخ انًطهٕثخ ، كزٔسجٕفٍذٌٔ كعبيم يفكك ، ٔانزًٍ رزكٌٕ يٍ انذراسخ اٌ انصٍغخ انسبثعخ االفضم يٍ ًْ ٔانًبَزٕل كًٕاد يخففخ ph102اسجبرربو كًبدح يحهٍخ ، َكٓخ انزٔسثزي ، ثٕنً فبٌٍُم ثٕفٍذٌٔ كًبدح راثطخ، افٍسٍم . حٍث سزعخ رفككٓب ٔرحزر انذٔاء يُٓب . e100هيذروكلورايذ االونذاسيرزوى ، اخفاء الطعن ، الونرشز الصلة ، اليودراجيد -الكلواخ الوفراحيح: introduction oral drug delivery has been known for decades as the most widely utilized route of administration among all the routes that have been discovered for the systemic delivery of drugs through various pharmaceutical products of different dosage forms, because it is convenient, self-administration, compactness and easy manufacturing, accurate dosage and most importantly the patient compliance (1) . administration of an oral drug delivery system having bitter taste with acceptable level of palatability has always been challenge in manufacturing of a formulation for pediatric and old age patients. the bitterness of drug or drug product is minimized or completely masked by various physical, chemical and physiological means such as lipophilic vehicles ,coatings , inclusion complexation, ion exchange, effervescent agents, rheological modification, solid dispersion system, group alteration and prodrug approach, freeze drying process, wet spherical agglomeration technique and continuous multipurpose melt technology (2) . 1 corresponding author e-mail: alaa.alahmed88@gmail.com received: 9/6/2017 accepted: 28 /6/ 2017 mailto:alaa.alahmed88@gmail.com iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 51 solid dispersion involves the dispersion of one or more active ingredient in an inert carrier or matrix in solid state. it is prepared by melting, dissolution in solvent or melting solvent methods. (3) carriers used in solid dispersion system include povidone, polyethylene glycols of various molecular weights, hydroxy propyl methyl cellulose, urea, mannitol and ethyl cellulose, eudragit e100 and eudragit epo. (4) onh is a competitive serotonin type 3 receptor antagonist. it is effective in the treatment of nausea and vomiting caused by cytotoxic chemotherapy drugs, including cisplatin and it has reported to act as anxiolytic and neuroleptic agent .it is also used in early onset of alcoholism (5) . generally emesis is preceded by nausea and in such condition it is difficult to administer drug with a glass of water; hence it is beneficial to administer such drugs as fdts. onh is an intensely bitter drug; hence, if it is incorporated directly into a fdts the main challenge behind formulation of such a dosage form will definitely get an acceptable dosage form to the patient (6) thus, in the present study an attempt has been made to mask the taste of onh and to formulate fdts with complete taste masking and good mouth feel so as to prepare a “patients friendly dosage form.” materials and methods materials ondansetron hydrochloride (onh) was purchased from hangzhou hyperchemical limited china, avicel ph102 hyperchem china, cross povidone (cp), cross carmellose sodium (ccs), sodium starch glycolate (ssg) and magnesium stearate from middle east laboratories , eudragit e100 from evonic company germany, talc from samara drug industries,.polyvinylpyrolione (pvp) k30 hyperchem china. preparation of solid dispersion fusion method solid dispersion of onh was prepared by fusion method. in this method the drug and carrier (eudragit e100) were mixed with a drug: polymer ratio of 1:1, 1:2, 1:3, and 1:4 in a china dish and heated on a paraffin bath until the solid mixture is melted. the mixture was poured on a tile and cooled. the resulted solidified mass was dried pulverised and passed through sieve no 20 (7) . solvent evaporation onh was mixed with powdered eudragit e100 using a mortar and pestle in different drug: polymer ratios (1:1, 1:2). the mixture was transferred into a stainless steel vessel. then 10% ethanol (15ml) was added to the mixture of each ratio. the mixture was stirred constantly on a magnetic stirrer till a thick gel was formed the temperature was kept at 40 °c with a stirring speed of 350 rpm. the ethanol was removed by evaporation overnight and subsequently the solidified gel was crushed into particles using mortar and pestle and then sieves through sieve no. 20 (8) characterization of onh solid dispersion percentage yield of solid dispersion the prepared solid dispersion granules were collected and weighed. the measured weight was divided by the total amount of drug and polymer which were used for the preparation of solid dispersion percentage yield = [wp/wt] × 100 where, wp is actual weight solid dispersion obtained and wt is the total weight of drug and polymer. (8) drug content 10 mg of solid dispersion was stirred by using magnetic stirrer with 100 ml of 0.1 n hcl for 60 minutes, till the entire drug leached out from polymer, then the solution was filtered through filter paper and diluted with 0.1 n hcl. the drug content was determined spectrophotometrically at 310 nm (9) . in vitro taste evaluation invitro taste was evaluated by determining drug release in phosphate buffer (ph 6.8) to predict release of drug in the human saliva. solid dispersion equivalent to 8 mg ondansetron (ond), ie, its dose, was placed in 10 ml of phosphate buffer 6.8 and shaken for 60 seconds. the amount of drug released was analyzed at 310nm (10) . the solid dispersion product that gives zero release is considered the optimum to be used for further study (11) fourier transforms infrared spectroscopy (ftir) the ftir spectra of pure onh , eudragit e100, physical mixture of drug and eudragit e100 and the selected solid dispersion product were obtained using ftir spectrophotometer (ftir -8300 shimadzu, japan) by potassium bromide (kbr) pellet method. this study was achieved to identify any sign of interaction between the drug and polymer used. the scanning range was (4000 400 cm-1) (12) powder x-ray diffraction (pxrd) powder x-ray diffraction can be used to confirm the crystalline nature of materials. so, this information is used to verify whether the substances are crystalline or amorphous. the diffractograms of onh, a physical mixture of iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 52 drug and eudragit e100 and the selected solid dispersion product powders were obtained. the study was confirmed by using shimadzu xrd-6000 powder x-ray diffractometer at continuous scan range of 5 ° -80° of 2ɵthe operating voltage was 40 (kv) and current 30ma. (10) preparation of onh fdts tablets containing 10mg taste masked onh equivalent to 8mg ond were formulated by the direct compression method using various superdisintegrants like (cp) (3 and 4%) for f4 and f7 respectively , (ccs) (3%) for f5, (ssg) (3%) for f6 as shown in table (1 ) . all the ingredients were passed through a sieve number 20 prior to mixing. onh eudragit e100 solid dispersion, mannitol, avicel ph102, the superdisintegrants, rossberry flavor, aspartame and pvp k30 were properly mixed for 20 minutes in a mortar to obtain a uniform blend. the blend was further lubricated with magnesium stearate, talc for 2 minutes. then powder was compressed into tablets using a 6mm flat punch tablet press. table (1): formulation of ondansetron hydrochloride fdt. f7 f6 f5 f4 f3 f2 f1 formula code ingredient(mg) 30 30 30 30 30 30 30 solid dispersion(1:2) 6 4.5 4.5 4.5 4.5 cross povidone(cp) 4.5 cross carmillose sodium(scc) 4.5 sodium starch glycolate(ssg) 1.5 1.5 1.5 1.5 1.5 1.5 1.5 ross berry flavor 1.5 1.5 1.5 1.5 1.5 1.5 1.5 aspartame 45 45 45 45 30 15 avicel ph102 3 3 3 3 3 3 3 talc 1.5 1.5 1.5 1.5 1.5 1.5 1.5 mg. stearate 3.75 3.75 3.75 3.75 3.75 3.75 3.75 pvp k30 150 150 150 150 150 150 150 mannitol q.s pre compression evaluation of powder blend flow properties these properties were determined in terms of angle of repose, carr’s index and hausner’s ratio for tablet blend powder in comparison with pure drug powder and the selected solid dispersion product. determination angle of repose one of the methods for assessing flow properties of powder is the angle of repose. it was determined using fixed funnel method, by permitting a powder to flow throughout a funnel and pass freely onto a surface. the height and diameter of the resultant cone were measured and the angle of repose was calculated from this equation: tan (θ) = h/r where: h is the height of the powder cone and r is the radius of the powder cone. (13) bulk density it is a ratio of the powder mass to bulk volume. the bulk density depends on particle size distribution, shape, and cohesiveness of particles. the weighted amount of the powder carefully poured into the graduated measuring cylinder through the large funnel and volume was measured, which is the initial bulk volume. then it was expressed in g/ml. bulk density was calculated by the following equation. (13) . bulk density = weight of powder / bulk volume tapped density the graduated cylinder containing a known mass of mixture was tapped for a permanent time. the volume was measured, and the tapped density was calculated by the following equation (13) . tapped density = weight of powder / tapped volume carr’s index (compressibility index) and hausner's ratio carr’s index indicates the flow properties of the powder. it was expressed in percentage and was calculated by the following equation: iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 53 carr’s index = [(tapped density – bulk )/(tapped density)]×100 hausner ratio is an indirect index of powder flow (13) . it was calculated by the following equation: hausner’s ratio = (tapped density)/(bulk density) evaluation of fdts hardness test monsanto hardness tester was used to determine the tablet hardness. three tablets were randomly chosen from each formulation of. the mean of three determinations ± sd was recorded. the hardness was expressed as a force in kg/cm 2 required to crush the tablets (14) . friability test friability test is performed to assess the effect of friction and shocks, which may often cause tablet to chip, cap or break. roche friabilator was used for the purpose. it is expressed in percentage (%). twenty tablets were initially weighed (w initial) and transferred into friabilator. the friabilator was run 25rpm for 4 minutes. the tablets were weighed again (w final). the percentage friability was then calculated using the following equation: %friability = {(w initial w final) / w initial} x 100 % friability of tablets less than 1% are considered acceptable (14) weight variation twenty tablets were weighed individually and the average weight was calculated .then each weighed tablet compared with average weight, whether it's within the acceptable limit or not according to usp (14) . wetting time the wetting time of tablets was measured using a simple procedure. a filter paper folded twice was placed in a small petridish (internal diameter = 6 cm) containing 6 ml of phosphate buffer ph6.8 .the method was slightly modified by maintaining phosphate buffer at 37 ºc. a tablet was placed on the filter paper and the time required for the complete wetting of the tablet was recorded as a wetting time. the mean of three tablets wetting time were recorded and standard deviation calculated (15) . in vitro disintegration test the in-vitro disintegration study, of the oral dispersible tablet, was determined using disintegration test apparatus as per usp specifications. one tablet was placed in each of the six tubes of the basket, the disc was added to each tube and running the apparatus using 900 ml of phosphate buffer ph 6.8 as the disintegration liquid (16) . the assembly should be raised and lowered between 30 cycles per min in disintegration liquid which was kept at 37ºc.the time in seconds for complete disintegration of the tablets with no mass remaining in the apparatus was measured and recorded (14,16) . in vivo disintegration test the time required for complete disintegration in the oral cavity was estimated from five healthy volunteers. all volunteers were told about the purpose of the test. the tablet was placed on the tongue, and subsequently the tongue was gently moved. the time required for the elimination of any residue or fragment of the tablet was measured with a stopwatch and recorded as a disintegration time (17) . drug content five tablets were powdered and the blend equivalent to 10mg onh was weighed and dissolved in 100ml of 0.1 n hcl, filtered and 1ml withdrawn and diluted to 10ml and drug content analyzed spectrophotometrically at 310 nm (18) . in vitro dissolution studies the dissolution profile of onh from fdts was determined using united state pharmacopoeia (usp) dissolution testing apparatus ii (paddle method). the dissolution test was performed using 500 ml of 0.1n hcl ph 1.2 as dissolution medium, at 37 ± 0.5°c and 50 rpm. a sample (5 ml) of the solution was withdrawn from the dissolution apparatus at 1, 2, 3, 4, 5, 10, minutes. the samples were filtered through a 0.45m membrane filter syringe. absorbance of these solutions was measured spectrophotometrically at 310 nm. cumulative percentage of drug release was calculated using an equation obtained from a standard curve (14) . the time required for 80% of drug to be released (t80%) and percent drug dissolved in 2 minutes (d2min%) were considered for comparing the dissolution results (19) . the t80% and d2min% were determined by fitting the dissolution data to a four parametric logistic model using the marquardt-levenberg algorithm (sigmaplot 11 spss) (20) . results and discussion characterization of onh solid dispersion percentage yield and drug content of solid dispersion (fusion method and solvent evaporation) are shown in table (2). iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 54 table (2): percentage yield and percentage of drug content of solid dispersion products in vitro taste evaluation the in vitro taste evaluation show that solid dispersion produced by fusion method in the ratio of 1:4 drug: polymer gave less release of drug. on the other hand, no drug release was obtained in phosphate buffer ph 6.8 from solid dispersion produced by of solvent evaporation method with ratio of 1:2 drug: polymer as shown in table (3).therefore, this ratio was considered the optimal solid dispersion with complete masking of bitter taste of drug for further studies and for the preparation of fast dissolving tablet (5) . table (3): in vitro taste evaluation of solid dispersion in buffer ph6.8 drug release solvent evaporation drug release fusion method 3.3 µg/ml 1:1 35µg/ml 1:1 zero 1:2 27 µg/ml 1:2 20 µg/ml 1:3 3.6 µg/ml 1:4 fourier transform infrared spectroscopy the ftir spectrum of onh show broad band o-h stretching of h2o at 3410 cm 1 -3492 cm -1 , c-n stretching at 1281 cm -1 , ch3 at 1458 and 1479 cm -1 ,c=c aromatic stretching at 1531 cm -1 c=n ,c=o stretching in six member ring at 1639 cm -1(12) . the ftir spectrum of the physical mixture of drug and polymer showed no significant shift or reduction in intensity of peaks of (onh). however, the ftir spectrum of solid dispersion product 1:2 show no interaction between drug and polymer and no change in peak of drug. figure (1): ftir spectroscopy of ondansetron hydrochloride %drug content %yield solvent evaporation %drug content %yield fusion method 90%±1.5 87%±2.3 1:1 93%±0.85 86.8%±1.3 1:2 103%±0.96 94%±1.75 1:3 96.2±0.55 96%±1.5 1:4 97%±2 95.5%±1.5 1:1 100%±0.25 96%±1.35 1:2 iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 55 figure (2): ftir spectroscopy of eudragit e100 figure (3): ftir spectroscopy of physical mixture 1:2 drug:eudragit e100 figure (4): ftir spectroscopy of solid dispersion product prepared by solvent evaporation (1:2) drug:eudragit e100 iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 56 powder x-ray diffraction the x-ray diffractogram of (onh) confirms its crystalline nature, as evidenced from the number of sharp and intense peaks as shown in figure (5).however, the diffraction pattern of solid dispersion represents complete disappearance of crystalline peaks of drug especially those situated at 6° ,12°, 24°,28° and 30° (2θ).these findings suggest the formation of a new solid phase with a lower degree of crystallinity due to solid dispersion . figure: (5):x-ray diffraction of pure onh , physical mixture , solid dispersion preparation of onh fdts onh solid dispersion prepared by solvent evaporation method with 1:2 drug: polymer ratio was incorporated in all formulas because it has the best drug content and complete taste masking (zero release in phosphate buffer ph (6.8) and all these formulas evaluated for their properties at pre and post-compression stages. pre-compression evaluation of powder blend it was found that the pre-compression parameters for the powder blend affected by the type and concentration of the diluent. f1 show fair flowability and fair compressibility which may be due to the effect of mannitol (diluent) which has poor flowability and compressibility therefore, avicel ph 102 which has good flow properties and good compressibility due to its granular in nature was added to the other formulas as a trial to improve the flow property (21) . better flow properties were obtained as the concentration of avicel ph102 was increased with in the formulas. excellent flow properties resulted by the use of 30% avicel ph102 (f4 to f7). all results are listed in table (4). in addition to the effect of diluent; the presence of magnesium stearate and talc within the powder blend produce further improvement in its flowability. (22) . iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 57 table (4): pre-compression parameters for pure drug, solid dispersion and fdts powder blend (mean ± sd) n=3. flow character hausner's ratio carr's index angle of repose formulation code good and passable 1.29±0.04 23.2±1.8 32±0.5 pure drug excellent and excellent 1.05±0.01 5.3±0.2 27.4±0.51 solid dispersion fair and passable 1.26±0.015 21.2±0.72 41±1.82 f1 fair and fair 1.17±0.025 18.16±1.25 38±1 f2 good and fair 1.2±0.015 15.76±1.89 35.3±1.52 f3 excellent and excellent 1.07±0.011 9.5±0.5 29.3±0.77 f4 excellent and excellent 1.09±0.01 10±1.52 30.5±1.32 f5 good and excellent 1.09±0.01 9.46±1.85 31.3±2.08 f6 excellent and excellent 1.06±0.015 8.13±1.05 29±1 f7 evaluation of fdts the formulas that pass pre-compression tests were compressed into tablets and evaluated for their hardness, friability, weight variation, in –vitro disintegration time, in-vivo disintegration time and dissolution. hardness and friability all the prepared fdts were within acceptable range of hardness (3.5±0.5 4.2±0.3) kg/cm 2 and this is very important to resist breaking during handling, packaging and hard enough for fast disintegration in the mouth. (14, 23) in addition the friability of all these prepared fdts were within acceptable range less than (1%) as shown in table ( 5) weight variation all prepared fdts were within acceptable limit according to usp standards as shown in table (5). table (5): hardness, friability and weight variation for prepared onh fdts. weight variation friability (%) hardness (kg/cm 2 ) properties formula no 149.7±0.25 0.45 4±0.25 f4 150.2±0.35 0.659 3.5±0.5 f5 149.5±0.5 0.609 3.75±0.35 f6 150±0.45 0.3 4.2±0.3 f7 in-vitro disintegration time for the prepared fdts the disintegration time of the prepared fdts was directly related to the wetting time and significantly affected by the type and concentration of super dis integrant (p<0.05) as shown in table (6). f4 (3% cp) disintegrate with the shortest time (11±1seconds) in comparison with f5(3% ccs), f6(3% ssg); this short disintegration times of cp containing fdts can be explained to be due to the properties of cp which has rapid capillary activity and pronounced hydration with little tendency to gel formation (24). in addition cp is highly porous and this unique, porous nature facilitates wicking of liquid into the dosage systems and causes rapid disintegration (25) .further decrease in disintegration time to a more favorable value which is less than stated in usp for preparation of onh fast dissolving tablet (14) . (7±1.5 seconds) was obtained by increasing the concentration of cross povidone to 4% (f7). in-vivo disintegration time for the prepared fdts the results showed that there is high correlation between the in-vitro and in-vivo disintegration time, but in all cases the in-vitro disintegration time has lower values compare with that of in-vivo one, this is due to large volume of phosphate buffer ph 6.8 and the strong agitation used during the in vitro test. the correlation between wetting time, in-vitro and in-vivo disintegration time is illustrated in table (6). iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 58 table (6): disintegration time and wetting time of prepared onh fdts (mean± sd ) n=3. wetting time (sec.) in-vivo dt (sec.) in-vitro dt (sec.) formula code 24.3±2.51 15.3±0.577 11±1 f4 32.6±3.05 23.3±1.52 15.6±1.15 f5 40±3.6 32±2 20±2 f6 18.3±1.52 10.6±2.08 7±1.5 f7 drug content all the prepared onh fast dissolving tablets were within acceptable range of drug content according to usp standards (90 -110 %) (14) as shown in table (7). table (7): drug content of prepared onh fdts.( mean ±sd )n=3. formula code %drug content f4 97±0.5 f5 96±1.5 f6 95±1 f7 100±2 in vitro dissolution study the prepared onh fdts disintegrate rapidly in the mouth. by swallowing the disintegrated fast dissolving tablets, the dissolution process completes in the stomach. therefore, 0.1n hcl was used to study the dissolution and release profile of the onh fdts. the time required for 80% of the drug to be released (t80%) from the tablets and percent drug dissolved in 2 minutes (d2 min%) is shown in table (8). once a tablet disintegrates, the solubility properties of the drug, either alone or assisted by other variables, determine the drug’s subsequent dissolution rate and extent of release (26) table (8): in-vitro dissolution parameters of prepared onh fdts. formula no. t80% (min) d2 min (%) f4 1.82 83 f5 3.12 70.3 f6 4.14 66.9 f7 1.29 93.4 factors affecting the dissolution effect of type of superdisintegrant on release profile of drug the effect of type of superdisintgrant was studied by comparing the release profile of f4,f5 and f6 containing the same concentration (3%) of cp, ccs and ssg respectively. and the results are shown in figure( 6 ). significant difference was obtained (p<0.05) by comparing t80% min. and d2min% of the above formulas. fastest release was obtained with f4 (shortest t80% and highest d2min%). this result is attributed to the characteristics of cp which absorbs a huge amount of water when exposed to dissolution medium and promote the disintegration of tablets, enhance the dispersibility of the drug particles which increase the dissolution rate of the drug (27) . figure (6): effect of superdisintegrants type on release profile of onh fdts in 0.1nhcl at 37°c±0.5°. effect of superdisintegrant concentration on release profile f4 and f7 were used to study the effect of the concentration of crospovidone on release of onh from fdts as shown in figure (7). which contain 3%w/w and 4% w/w respectively, f7 show higher dissolution result than f4 (28) . iraqi j pharm sci, vol.26(1) 2017 fdt s of taste-masked onh by solid dispersion 59 figure (8): effect of concentration of superdisintegrants on release of onh from fdts in 0.1n hcl at 37±0.5c ° . selection of the best formula according to the usp requirements; all the prepared tablets of f4-f7 were within the accepted limit regarding their dissolution results (not less than 80% of the drug is released within 10 minutes), but only f7 with shortest disintegration time (7 sec.) comply with the usp (disintegration time should be not more than 10 seconds). therefore f7 was selected as the best formula for the preparation of onh fdts. conclusion taste masked onh can be successfully prepared with the use of the solid dispersion techniques by solvent evaporation method using eudragit e100 as a carrier in a ratio of 1:2 drug: polymer. fdts of onh with an 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rao as, rajender r, rajeshwari m, anusha ys, saidulu e, mahesh g. formulation and evaluation of aripiprazole solid dispersions. american journal of pharm research. 2015; 5(02):874-881. 26. vyas v., sancheti p., karekar p. , shah m., and pore y., physicochemical characterization of solid dispersion systems of tadalafil with poloxamer 407. acta pharm.2009; 59: 453–461. 27. setty c, prasad dv, gupta vr, sa b. development of fast dispersible aceclofenac tablets: effect of functionality of superdisintegrants. indian journal of pharmaceutical sciences. 2008; 70(2):180-185. 28. bala r, khanna s, pawar p. polymers in fast disintegrating tablets –a review. asian j pharm clin res. 2012; 5(2):8-14. iraqi j pharm sci, vol.28(2) 2019 drug prescribing practice doi: https://doi.org/10.31351/vol28iss2pp115-123 115 evaluation of knowledge, attitudes and experience of off-label drug prescribing practice among physicians in baghdad city hospitals aysha m. shanshal*,1 and ahmed h. ataimish** * faculty of pharmacy, al-rafidain university college, baghdad, iraq ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract the objective of this study was to assess the knowledge, attitude, and experience of off-label prescribing practice among physicians working in public hospitals in baghdad city. this cross-sectional study was performed from november 1st 2018 to march 2019 in 17 public hospitals in baghdad, iraq. the targeted hospitals were randomly selected at different regions in baghdad city. a self-administered questionnaire was utilized to collect data from the physicians. out of the 400 distributed paper questionnaires to a convenience sample of physicians, 383 of them were returned completed. more than half of the participants (57.2%) indicated that they were reasonably familiar with the term “off-label drug”, 57.7% mentioned that the most common medical reason for the prescribing off-label drugs was unavailability of alternatives. about two thirds had concerns regarding offlabel drug safety and efficacy. 62.7% agreed that the ministry of health authority should provide an incentive to pharmaceutical companies to perform clinical trials in iraqi patients, 49.1% believed that clinical trials that recruit volunteers involve ethical issues. extensive efforts are required to make programs, regulations and guidelines to control the off-label prescribing practice among the iraqi healthcare providers at different healthcare settings. keywords: attitude, prescribing practice, off-label prescribing. بين أألطباء صرف أالدوية خارج األستطبابات ألمحددة لها ألعلمية وألسلوك وألخبرة لممارسةتقييم في مستشفيات مدينة بغداد **مش يطأو احمد حامد 1*،عائشة مثنى شنشل الرافدين الجامعة ، بغداد ، العراق . كليةالصيدلة ، قسم* فرع االدوية والسموم ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . ** ألخالصة هذه الدراسة تهدف الى تحديد العلمية والسلوك والخبرة لممارسة صرف االدوية خارج االستطبابات المحدده لها بين المستشفيات أساسها مستشفى حكومي في 11في 8112مارس 1الى 8112تشرين الثاني 1ل الفترة من االطباء في مديتة بغداد. هذه الدراسة االستقصائية انجزت خال اطق نمدينة بغداد,ألعراق. استبيان ذاتي التقديم استخدم لجمع المعلومات من االطباء. المستشفيات الحكوميةأختيرت بصورة عشوائية من مختلف م على معرفة بما تعنى كلمة ممنهم بينوا انه %2178منهم عادت منجزه, 323اء, استمارة استطالع موزعه على االطب 011مدينة بغداد. من اصل %7871ذكروا بأن اكثر سبب شائع لوصف االدوية الغير مصرح بها كان عدم توفر البديل, %2171خارج أألستطبابات ألمحددة لها بشكل معقول, يؤمنون بأن %0271الدوائية النجاز تجارب سريرية على المرضى العراقيين, وزارة الصحة توفير دوافع لتحفيز الشركات اتفقوا ان يتوجب على فالتجارب السريرية التي تجند متطوعين تتضمن قضايا اخالقية. جهود مكثفة مطلوبة لتأسيس ورش توعية, تنظيمات, توجيهات لتنظيم ممارسة وص سموح لهم بوصف االدوية في مختلف أقسام الرعاية الصحيةاالدوية الغير مصرح به بين العراقيين مقدمي العناية الصحية الم خارج أألستطبابات ألمحددة لها . صرف ،صرفممارسة أل ، سلوك الكلمات المفتاحية : introduction the term off-label drug use (oldu) is widely used in the medical literature and the media (1). off-label drug prescribing has not undergone the type of advantage-disadvantage assessment required in the process of medicines’ marketing authorization. however, it has been noted that healthcare professionals commonly prescribe medicines offlabel with levels of evidence considered to be low. this is principally problematic because off-label use with inadequate strong scientific evidence may be associated with higher rates of adverse events that may harm the patient (24). off-label prescribing is not certainly bad. it can be beneficial, particularly when the patients have no other approved options like in cancer chemotherapy. off-label prescribing of a drug or combination of drugs usually represents the standard of care in such cases (5). off-label prescribing can represent different forms including prescribing drug outside the age range or weight for which the product is licensed (6), for indications not approved in the product information leaflet (pil) (7), utilizing alternative routes of administration other than that indicated for that formulation in the pil (8), the use of doses or dose frequencies other than those stated in the pil (9,10), and using different formulation other than approved one (11,12). as lately reported on the off-label use of medications in the european union (eu), the prevalence of off-label use in the pediatric and adult population is high in a wide range of therapeutic regions, particularly oncology, psychiatry, neurology and rheumatology (13). 1corresponding author e-mail: rafeef_sh@yahoo.com received: 21/ 4 / 2019 accepted: 18/8 / 2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp115-123 mailto:rafeef_sh@yahoo.com iraqi j pharm sci, vol.28(2) 2019 drug prescribing practice 116 many controversies existed and the healthcare professionals generally agree that more work and efforts are needed to prescribe suitable off-label drugs for patients with rare diseases. (14) however, they also concur that potential improper promotion, as well as possibly hazardous prescribing use for these drugs should be obviated (14). the present study was designed to assess the knowledge, attitude, and experience of off-label prescribing practice among physicians in baghdad city hospitals and clarify the acceptability of this practice among prescribers at different medical specialties in addition to the reasons behind accepting or rejecting this approach. methods a quantitative cross-sectional study design was used. a self-administered questionnaire was utilized to collect data from the physicians at different healthcare branches in baghdad city, iraq. a 25 item questionnaire comprised questions with a combination of tick box responses, 5–6-point scale questions and four open-ended questions were sent to 17 public hospitals. the survey focused on physician knowledge of and reasons for off-label prescribing, concerns about off-label medicines, communications between patients and physicians, and attitudes towards the need for performance of clinical studies. the recruitment process was carried out from november 1, 2018 to march 1, 2019. no incentive was offered to the participants. questionnaire development in the present study, an english questionnaire especially was developed to evaluate the off-label prescribing practice among iraqi clinicians. the questionnaire contents were formulated based on questionnaire previously used elsewhere to explore the views, attitudes, knowledge, and perceptions of the prescribers towards the off-label prescribing of medicines (15). most questions had pre-formulated answers, except few with partially open-ended answers. the questionnaire consisted of 25 questions. the questionnaire was divided into four sections addressing different topics of interest including the participant demographic information, physician familiarity of off-label prescribing practice and its consequences, the reasons behind off-label prescribing, patient involvement in the prescribing process and physician sources of information about off-label drugs. finally, the questionnaire focused on the clinical trials and the issues associated with dosage determination of off-label medicines to young patients. validation of the study tool the study questionnaire was pilot tested on a convenience sample of 40 randomly selected physicians to predict the validity and reproducibility of the designed questionnaire. using the cronbach alpha, reliability tests yielded good internal consistency for the overall questionnaire items (α= 0.782). test-retest reliability was evaluated by pearson correlations between time 1 and 2 scores (23 weeks later) on the two completed questionnaires. test-retest reliability for the overall score was acceptable (r = 0.83). all the participants declared that the questionnaire was clearly understood. physicians physicians were recruited from 17 different hospitals that cover the healthcare services at different regions of baghdad. four-hundred physicians participated in this cross-sectional study (74 consultants, 72 general practitioners and 254 permanent registrars) who practiced in different disciplines of medicine (32 internists, 115 surgeons, 7 nephrologists, 8 intensive care unit, 76 pediatric,50 general and 95 represented other miscellaneous fields). inclusion criteria the inclusion criteria of the current study included physicians from different specialties at different hospitals in baghdad with minimum practicing experience of 3 years who provided verbal consent to participate in the study. exclusion criteria the exclusion criteria of the current study included the rotators and junior physicians with prescribing practice experience of less than 3 years. statistical analysis statistical analyses were performed using the ibm statistical package for social sciences spss (version 24.0) and graphpad prism version 5.1 software with significance levels set at p<0.05. categorical variables were expressed as numbers and percentages, while continuous variable were expressed as mean±sd. non-parametric tests were utilized to compare knowledge scores, perspectives, and off-label prescribing practices across the different selected clinician demographics (mannwhitney u test for 2 groups and kruskal-wallis for more than 2 groups). validity and reproducibility of the questionnaire were measured by cronbach’s alpha. chi square test and exact fisher’s test were used to compare the differences in the perceptions of clinicians who have practiced off-label drug prescribing and those who have the idea and knowledge but do not practice off-label drug prescribing. results demographics out of the 400 distributed questionnaires to the physicians, 383 (95.8%) of them were returned completed, while 17 (4.2%) were uncompleted and excluded during analysis. a total of 18.3% of the participants were consultants, 17.5% general practitioner, 39.9% of them were permanent registrar and 24.3% of them were specialists. https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2799128/#b14-ptj34_8p428 https://www.ncbi.nlm.nih.gov/pmc/articles/pmc2799128/#b14-ptj34_8p428 iraqi j pharm sci, vol.28(2) 2019 drug prescribing practice 117 regarding the practice field, 8.4% of them were internist, 30% surgeons, 1.8% nephrologists, 2.1% intensive care, 13.1% general, 19.8% pediatricians and 24.8% represent other miscellaneous specialties. regarding the duration of practice, 29.25% of respondents practiced as physicians for 1-4 years, 27.9% for 5-10 years and 42.8% for more than 10 years in service. knowledge and views about off-label medicines the majority of respondents (219; 57.2%) indicated that they were reasonably familiar with the term “off-label drug” and found to be significantly greater (p<0.05) than those who are either unfamiliar or highly familiar, 62 (16.2%) mentioned that they were highly familiar with the term “offlabel” drug and 102 (26.6%) were found unfamiliar with this term (figure 1). the present study indicated that the forms of the off-label prescribing include prescribing a drug for younger age (56.9%), lower than recommended dose (66.6%), higher than recommended dose (57.2%), choosing an alternative different formulation (60.3%), prescribing a drug of different indication (64.8%) or utilizing different route of administration (59.8%) (table 1). figure 1. rating of the targeted physicians according to their familiarity with the off-label prescribing practice. n=383 total number; (chisquare test). the present study indicated that the forms of the off-label prescribing include prescribing a drug for younger age (56.9%), lower than recommended dose (66.6%), higher than recommended dose (57.2%), choosing an alternative different formulation (60.3%), prescribing a drug of different indication (64.8%) or utilizing different route of administration (59.8%) (table 1). table 1. forms of off label-drugs most important forms for off-label prescribing frequency n(%) score from 6 (mean ± sd) lower than recommended dose 255(66.6) 2.15±2.03a higher than recommended dose 219(57.2) 1.65±1.71a at a younger age than recommended 218(56.9) 1.86±1.90a via a different route of administration 229(59.8) 2.20±2.06a different formulation 231(60.3) 2.26±2.06a different indication 249(64.8) 2.56±2.11a values are expressed as frequencies, percentage and mean ±sd; mean score values with non-identical superscripts (a, b) among groups are significantly different (kurksal-wallis test with dunn’s post hoc test). in addition, the majority of respondents reported patients (> 40 years of age) and patients (1 day -15 years) to be the most likely seen by the targeted physicians (41.5% and 37.6%; respectively). on the other hand, patients (16-21 years), patients (21-30years) and (31-40 years) were characterized by the respondents to be less likely seen by the targeted physicians (32.1%, 32.9% and 36.8%; respectively) (table 2). table 2. the age ranges of patients mostly seen by the targeted physicians (n= 383 physicians) age range of patients (years) frequency n (%) score from 5 (mean ± sd) 1–15 years 144(37.6) 2.19±2.0a 16–20 years 123(32.1) 1.84±1.7a 21–30 years 126(32.9) 1.85±1.8a 31–40 years 141(36.8) 1.86±1.87a over 40 years 159(41.5) 2.32±2.18a values are expressed as frequencies, percentage and mean ±sd; mean score values with non-identical superscripts (a, b) among groups are significantly different (kurksal-wallis test with dunn’s post hoc test). iraqi j pharm sci, vol.28(2) 2019 drug prescribing practice 118 according to the physician’s answers, the most common medical reasons for prescribing offlabel drugs were 57.7% unavailability of alternatives, 17.5% personal experience, 12% case reports and 12.8% mentioned that they don’t know (all of the last group don’t prescribe off-label drug for their patients) (figure 2). figure 2.the reasons behind off-label prescribing practice. n=383 total number; values of last three options are significantly different (chi-square test). views about the safety and efficacy of the off-label medicines the majority of respondents had major and/or minor concerns regarding the safety (67.6%), while those who concerned about the efficacy of the prescribed off-label medications represent around 65.5% of the respondents (table 3). there was no significant difference between the familiarity of offlabel prescribing and concerns about the safety of off-label medicines during prescribing to their patients; (p=0.449) (chi-square test). about half of the respondents (52.2%) felt that the use of off-label medicines increased the likelihood of disadvantages to the patient for several reasons. as they mentioned, it may disadvantage the patient and cause serious adrs. at the same time other mentioned that they do not have close monitoring or follow up to their patients after administering the drug to observe the adrs. about 34.2% of respondents have observed adrs after using off-label drugs to their patients (table 3). declaring their own practice, approximately half of healthcare providers (55.4%) admitted to having experienced treatment failure; in spite of that 60.1% of the physicians knowingly prescribe drugs offlabel for their patients (table 3). table 3.physicians views about the safety and efficacy of the off-label medicines (n= 383 physicians) questions response n(%) pvalue yes no do you knowingly prescribe drugs off-label? 230(60.1) 153(39.3) 0.03 do you realize that off-label prescribing may disadvantage the patients? 200(52.2) 183(47.8) 0.24 do you have concerns about the efficacy of the off-label drugs? 251(65.5) 132(34.5) 0.021 do you have concerns about the safety of the off-label drugs? 259(67.6) 124(32.4) 0.02 have any of your patients experienced adr after using off-label drugs? 131(34.2) 252(65.8) 0.02 have any of your patients experienced treatment failure due to the offlabel drug use? 212(55.4) 171(44.6) 0.18 values are expressed as frequency and percentage; n: number of the responders; p-value indicates significant differences according to fisher's exact test. in the present study, only 35.8% of respondents declared that they request verbal consent from the patient or the patient’s parents, 38.6 % of them ask for the consent of their supervisor and only 40.7% of the interviewed physicians routinely inform their patients that they dispensed an off-label drug for them and inform them about its use and the expected outcome (table 4). there was a significant relationship between the familiarity of off-label drug prescribing and asking for consent from the patient and informing the patient about the off-label dug (p= 0.008, 0.01) respectively. while the result showed that there was no significant relationship between the familiarity and asking consent from the supervisor, (p=0.7) (chi-square test). iraqi j pharm sci, vol.28(2) 2019 drug prescribing practice 119 table 4. concerns of the physicians about ask for consent for prescribe off-label drug (n= 383 physicians) questions response n(%) pvalue yes no do you request a patients’ consent before prescribing off-label drugs? 137(35.8) 246(64.2) 0.02 do you inform the institution authority that you have recommended offlabel prescribing practice? 148(38.6) 235(61.4) 0.03 do you routinely inform the patients that you are prescribing an off-label drug for their treatment? 156(40.7) 227(59.3) 0.11 values are expressed as frequency and percentage; n: number of the responders; p-value indicates significant differences according to fisher's exact test. information sources when the healthcare providers were asked how they became familiar with the terminology of off-label drugs, 21.1% of the respondents mentioned that they had gained their knowledge from the bnf, 17.2% from a colleagues ' experience, 54.8% from all the mentioned before and 6.8% from other nonrecognized sources (figure 3). figure 3. the information sources for the offlabel prescribing practice. n=383 total number; values of bnf, colleague experience and others are significantly different (chi-square’s test) clinical trials on volunteers in this section, when the healthcare providers were asked about the need for more clinical trials to address the issue of off-label drugs use, 71% of them mentioned that it is important to provide more drug formulations, 44.4% of the respondents believed that all current drugs that have not been evaluated in younger ages should trialed in those populations and 67.6% thought that all new drugs should be trialed in different age groups prior to use. moreover, approximately more than half of the respondents (62.7%) agreed that the ministry of health (moh) authority should provide an incentive to stimulate pharmaceutical companies to perform clinical trials in iraqi patients (table 5). there is limited information about their efficacy in younger age patients which consider riskiest group. younger patients have different physiology, basal metabolic rate, metabolism, surface area and different body weight. about 49% of the respondents do not believe that all current drugs that have not been evaluated in younger ages should be trialed in those populations. some of the respondents disagreed with trying new drugs indifferent age groups prior to use for ethical considerations and nature of our society. table 5. views of the physicians about the need for more drug formulations and trialing all new drugs in different age groups prior to use (n= 383 physicians) questions response n(%) yes no do not know is it important to develop more drug formulations? 272(71) 42(11) 69(18) should all new drugs be trialed in different age groups prior to use? 259(67.6) 49(12.8) 75(19.6) should the moh authority provide incentives to stimulate pharmaceutical companies to perform clinical trials on iraqi patients? 240(62.7) 59(15.4) 84(21.9) should all current drugs that have not been evaluated in younger ages trialed in those populations? 170(44.4) 78(20.4) 135(35.2) values are expressed as frequency and percentage; n: number of the responders. however, only 43.4% of the respondents indicated that they have the willingness to be actively involved in a clinical trial, and about 44.6% of them accepted the idea of recruiting their own patients for a clinical research. there was no significant relationship between willingness to be involved in clinical trial and the specialty of the physicians. the participants were also asked if they accepted to iraqi j pharm sci, vol.28(2) 2019 drug prescribing practice 120 enroll their relatives in the clinical trials. only 37.6% of the respondents agree to allow their relative to participate in a clinical trial. about half of physicians (49.1) believed that clinical trials that recruit volunteers involve ethical issues (table 6). several reasons make the physicians believed that clinical trials that recruit volunteers involve ethical issues including believing that uncertain efficacy of off-label drug may lead to unexpected result that may harm the patient; for social and religious consideration, to avoid personal problem with the patients. table 6. views of the physicians about clinical trials in iraqi patients (n= 383 physicians) questions response n(%) yes no no opinion would you like to be actively involved in conducting clinical trials? 166(43.4) 138(36) 79(12.6) would you like to recruit your patients for a clinical trial? 171(44.6) 107(27.9) 105(27.5) would you allow your own relatives to participate in a clinical trial? 144(37.6) 155(40.5) 84(21.9) do you believe that clinical trials that recruit volunteers involve ethical issues? 188(49.1) 112(29.2) 83(21.7) values are expressed as frequency and percentage; n: number of the responders. discussion the data of the present study showed that the majority of respondents mentioned that they were either familiar or reasonably familiar with the term “off-label drug”. the most common form of prescribing off-label drug was prescribing lower than the recommended dose. the results were approximately equivalent to the results of prevalence of off-label use in jordan and north ireland (16,17). however, it is possible that a decrease in the dose could significantly increase risk of treatment failure. the other causes related to the increases in dose, frequency, or duration of administration, compared with the approved dosing regimens, can actually increase the risk of toxicity of a marketed drug (18-20). prescribing modified formula (dosage form) of the drug for patients who have difficulty in swallowing like in parkinson patients or patient with mandibular fracture is considered as a common solution to ease the administration of the drug; e.g., crushing the tablets or open the capsules. however, the clinical consequences of such practice can be inappropriate and may harm the patient. such alteration of formula may affect the drug's absorption and can result sometimes in fatal overdose, or oppositely under dosing, rendering the treatment inefficient (21-24). similarly, the change in the route of drug administration may create series of new problems. the modified routes of administration may create problems concerned with increased local concentrations, sterility, pyrogenicity, hypersensitivity (e.g., airway reactivity), difference in pharmacokinetic and pharmacodynamics, which can seriously increase the risks of toxicity or adverse effects (25). in the present study, the respondents reported patients (>40 years) (41.56%) and patients (1-15 years) (37.6%) to be the most likely seen by the targeted physicians. in other study show that geriatric and pediatric patients remain poorly participate in clinical trials that evaluate the premarketing efficacy and safety of novel therapies. it is perhaps not unpredictable that off-label prescribing is particularly common in these groups (26). the present study showed that around half (52.5%) of the respondents felt that prescribing off-label medicines increased the likelihood of disadvantages to the patient. the majority of respondents (67.6%) had major and/or minor concerns regarding the safety, while those who had concerned about the efficacy of the prescribed offlabel medications represent about two third of the respondents (65.5%). this result was in tune with many previously reported data that focus on the concerns of disadvantaging the patient’s health (27,28). approximately more than half of healthcare providers (55.4%) admitted to having experienced treatment failure, while third of them (34.2%) experienced adr after using off-label drugs to their patients. in spite of that, more than half of the physicians (60.1%) knowingly prescribe drugs off-label for their patients as a usual practice. this unusual behavior needs to be thoroughly evaluated to uncover the exact reasons behind such attitude. attentiveness to medicines safety is very important, although there is an evidence that offlabel use is frequently inappropriate and may expose patients to a very high risk of adrs (4,29). moreover, this high percent of off-label drug misuse may lead to waste of economic resources (30). regarding the prescribers’ practices, the present study revealed that only third of the respondents (35.8%) mentioned that they request verbal consent from the patient or the patient’s relatives; while more than third of them (38.6%) ask for the consent of their supervisor and only 40.7% of the interviewed physicians routinely informed iraqi j pharm sci, vol.28(2) 2019 drug prescribing practice 121 their patients that they dispensed an off-label drug for them and informing them about its use and the expected outcome. nowadays, the prescribers have a corresponding legal and ethical duty to acknowledge all of the facts that are relevant to their patients’ treatment decisions. patients deserve to know any inherent risks of a prescribed medication. also, asking for informed consent for off-label medication use will protect physicians against legal issue (31). when the healthcare providers were asked about the reasons behind prescribing off-label drug, more than 50% of the respondents (57.7%) mentioned that they prescribe off-label drug as a result of unavailability of alternatives; while about 17.5% referred to their personal experience and 12% case reports. in addition, 12.8% did not declare the reasons for its use. approximately all the later 12.8% of physicians did not prescribe off-label drug during their practice history. also, 21.1% of prescribers had gained their knowledge from the bnf, 17.2% from a colleagues ' experience, 54.8% responded from all the mentioned before and other 7% not recognized sources. unfortunately, the physician’s attitude toward offlabel prescribing has no strong scientific evidence. this could be explained by the absence of awareness of such prescribing approach. case reports are not considered as well-trusted sources; meanwhile, the frequent use of personal experience, previous patient prescription notes and colleague experience, all of which may lead to inaccurate off-label prescribing. bnf can be useful, but offer obvious guidance only after high-quality research has evaluated a specific off-label use (32). according to the fda, approved drugs that are allowed for an unapproved use are misbranded (33). in the present study, the healthcare providers addressed the need for more clinical trials to clarify the issue of off-label drugs use; nearly 71% of them mentioned that it is important to provide more drug formulations, two thirds of the respondents (67.6%) believed that all the current drugs that have not been evaluated in younger ages should be trialed in those populations to make sure they are safe. approximately two thirds of the respondents (62.7%) agreed that the moh authority should provide an incentive to stimulate pharmaceutical companies to perform clinical trials in iraqi patients. clinical trials introduce ‘a way to pool controlled observations in an objective and scientific way, helping clinicians to decide what is the best therapy for the patient’ (34). in spite of that about 20.4% of the physicians make objections to conducting clinical trials in iraq for several reasons including either for social, religious and economic considerations, or low education. however, in considering the idea of taking part in clinical trials, only 43.3% of respondents indicated that they have the willingness to be actively involved in a clinical trial and about 44.6% of them accept the idea of recruiting their own patients for a clinical research. there was no relationship between willingness to be involved in clinical trial and the specialty of the physicians. the participants were also asked if they accepted to enroll their relatives in the clinical trials; only 37.6% of the respondents agreed to enroll their relative to participate in a clinical trial and approximately half of physicians believed that clinical trials that recruit volunteers involve ethical issues. strategies to encourage physician participation in clinical trials include financial and nonfinancial stimulus, sufficient training, research questions that are in agreement with physician interests and have clear potential to improve patient health care (35). the development of new health care management models where patients involve in clinical trials and the expansion of information technology are additional factors that contribute to enhance this change (36). patient-centered medicine cannot be practiced without patients involving in their own health care decisions and in the research that needs such decisions (37). ethics and regulatory review procedures are essential for protection the safety and interests of the participants. however, overly strict ethical and regulatory systems could limit research capacity (38,39). after all, all drug treatment, all cases involving the use of drugs in an off-label use should be thoroughly documented in the medical report of the patient, including the clinical outcomes of such therapy, could extremely improve the knowledge in this area (40). study limitations there have been other variables difficult to control, that may impact the results and not included in the study, including availability of alternatives for the prescribed medications, clinical picture of the patient, influence of relatives, arguments by the prescribers, and importance of treatment. other potential limitation of the present study could be the absence of clinical outcome follow-up for dispensing off-label medications, which could help in assessing their efficacy. conclusion the majority of participants were reasonably familiar with the concept of off-label medicines and prescribe off-label drug knowingly. they believed that this practice may disadvantage the patient due to concerns about efficacy and safety that may be associated with increased risks of adrs. although the respondents very well recognized the ethical issue of 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m, abdulrahman b, d'souza j. physician participation in clinical research and trials: issues and approaches. dovepress j. 2011; 7(2):85-93. 36. porter me. what is value in health care? n engl j med. 2010;363:2477–2481. 37. tinetti e, basch e. patients’ responsibility to participate in decision making and research. jama. 2013;309:2331–2332. 38. hearn j, sullivan r. the impact of the ‘clinical trials’ directive on the cost and conduct of noncommercial cancer trials in the uk. eur j cancer. 2007;43:8–13. 39. sullivan r. the good, the bad, and the ugly: effect of regulations on cancer research. lancet oncol. 2008;2045(07)70388-1. 40. schubert s, neininger m, smers s, winter a, frontini r, bertsche a, et al. electronic drug prescription auto pilot for drug therapy? med monatsschr pharm. 2015; 38(6):224-230. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi journal of pharmaceutical sciences iraqi j.pharm.sci., vol.17 (1) ,2008 carbamazepine extended release tablet. 55 factors affecting the formulation of carbamazepine extended release tablet samer h. aziz* , alaa a. abdulrasool** , ahmed a. hussein**, 1 * national center for drug control and research, ministry of health , baghdad ,iraq ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad ,iraq. abstract carbamazepine is an anticonvulsant agent which acts on the central nervous system and used for the treatment of epilepsy. carbamazepine was formulated as an oral extended release tablets using ethyl cellulose as retardant substance. different types of tablets additives such as cellulose materials (sodium carboxymethyl cellulose and microcrystalline cellulose ), lactose, calcium phosphate and solubilizing agents ( sodium lauryl sulphate and polyethylene glycol 6000) were utilized to study their effect on the release profile of drug from ethyl cellulose matrices. it was found that sodium carboxymethyl cellulose increased the carbamazepine release and the same effect was obtained when the same amount of microcrystalline cellulose used. the result also showed that sodium lauryl sulphate greatly enhanced the release of the drug compared to polyethylene glycol 6000. also incorporating lactose led to an increase in the release of the drug while utilization of calcium phosphate slowed down the release of the drug. the results of this study revealed that formula which composed of 4% ethyl cellulose, 5% sodium carboxymethyl cellulose, as well as 25.6% of lactose and 1% magnesium stearate is comply with united state pharmacopea xxviii and showed best release profile comparable to that of the brand product tegretol cr®. the shelf life was 3.6 years for the selected formula. key word: carbamazepine, ethyl cellulose, extended release. ةصالخلا ياميييلمرلكلمكهيي كيزيرك غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهب ٌبصك و كهجلكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهزعيلكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهوانلكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه ا داكم لٌو ك غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه وي غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهزوكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهااك . غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه نكًزًكارراك ياميييلمررلك رنمككر م وكي ٌنهكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهٌرااكمي ٌةنالكات ك جرجملك يرهكيينبوك غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهجٌراا .ًزكا ٌةنالكاةماككيةٌجموكيلكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه ماركا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه زيروك غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهجرنمككيي ككا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه ماركا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلجرجمل و) يامم للكيي ك رجرجملكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهامر ملكما غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلجرجملكيزعااكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهٌنجماك( لكز ٌملكلكرم ميىكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلي غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلرملكلك ك نم ءاودلا ررحت لكش ىلع داوملا هذه ريثأت مييقتل ( ك غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهٌ ررزكًرتراكلوشكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه ماركهجلكهل كًرااكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهنماتكيلك6000ما غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه ماركا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه رمدهك غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهجومميو) غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهما ك جميىكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهامر ملكممم غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلكاترجرلك ب لماك سما غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلكات ك جرجمل . غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه نكمانكاوكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهامر ملك يامم للكيي ك جرجملك د نكيلكًرااكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلياميييلمررلكمةمصكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهٌرتراكهجلكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهٌرااكسنكمانكهانك ا ٌةنالكةمصكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهل روكيلكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلجرجملكيزرعااكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهٌنجماك .ا عاىكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهاٌيئاكا زيكاوكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهجما ك جميىكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهامر ملكلمدكمنل ك نراكًرااكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهنماتك . غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه نكمانكاوكا يروكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهب ٌملك رراكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلكل يرهكا ناكيلكًرااكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهنماتكمرا يكا ٌةنالكرم ميىك6000ي ياةوكينكمم غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلكاتجرلك ب لماك ك يامم للكيي ك جرجملك5كات ك جرجملكمك%4ا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلي غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلرملكسج كيلكًرااكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهنمات . ن يكةٌيئاكلوشكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهناا وكاوكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهار وكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه لمةوكيلكككك % ك ٌريااىكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهامر ملكًٌبيممكينكر ٌماكا رم وكازيا للكماهبيكارز كهل كًرااك1ككز ٌملكمك%2.5ا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهامر مللكميز يروكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهلك% ك او .3.6كمه اكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهاوك 1-ا نمك 10 4× 4.95 للكي ياةٌيكينكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه اٌاكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه ري لكًلا ٌما .تيميكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهٌرج ك غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وهجار وكا غييصت مت دقل .عرصلا ةجلاعمل لمعتسيو يزكرملا يبصعلا زاهجلا ىلع لمعي جالتخالل داضم لماع وه اٌزوكلمك introduction extended release tablets are those which formulated in such a manner to make the contained medicament available over an extended period of time after ingestion. expressionsكas̋كprolonged-action̋,̋كrepeatedaction̋,كand̋كsustainedكrelease̋كhaveكalsoكbeenك used to describe such dosage forms. extended release delivery systems mostly allow at least a twofold reduction in the dosing frequency compared to the conventional immediate release formulations and increase patient compliance as well as therapeutic performance(1). matrix systems appear to be very attractive approach from economic as well as process development and scale up points in the controlled release systems(2). matrix tablets are classified according to the type of materials used for retarding the release of drugs (3). in case of fat lipophilic matrices, the drug which is incorporated into a melt of fat and waxes will be released by leaching out and/or dissolution of the carrier during passage throughout git. among these lipophilic materials are carnauba wax, cetyl alcohol, hydrogenated vegetable oils, triglycerides, stearic acid, and polyethylene glycols(4). meanwhile hydrophilic matrices, a dispersed drug is released as the retarding polymer swell in the gastric fluid, forming a gel barrier through which drug will be released by diffusion or dissolution of the matrix. these hydrophilic materials include cellulose derivatives as hydroxypropyl cellulose,sodium carboxymethyl cellulose, 1 corresponding author : e-mail ahmed_sura@yahoo.com received : 24/2/2008 accepted : 28/6/2008 mailto:ahmed_sura@yahoo.com iraqi j.pharm.sci., vol.17 (1) ,2008 carbamazepine extended release tablet. 56 hydroxylpropylmethyl cellulose, methyl cellulose as well as carbopols, sodium alginate, xanthan and guar gums(5). in case of plastic matrices which they are composed of materials characterized by their capability to form insoluble, sponge-like skeletons from which the drug is released by diffusion. examples of such materials are the acrylic/methacrylic coplymers, ethyl cellulose, polyvinyl acetate, and polyvinyl alcohol (6). ethylcellulose is usually combined with water-soluble additives to impart some hydrophilic nature for films or matrices, altering its structure by virtue of pores and channels through which drug can diffuse more easily(7). in the present study, ethyl cellulose was used as retardant substance to formulate carbamazepine matrix tablet dosage form. extended release formulation of cbz should be considered in patients receiving high doses of cbz and who are suffer from intermittent adverse effects such as diplopia, nausea, dizziness, and drowsiness, offering the opportunity for converting the three or four times daily regimen to twice, or even once daily administration(8). materials and methods materials: carbamazepine and microcrystalline cellulose (avicel ph 101) kindly supplied by samara dug industry (sdi). ethylcellulose, magnesium stearate, , sodium carboxymethylcellulose and sodium lauryl sulphate from(bdh, england). lactose from (riedel-dehaen, germany). all other chemicals and solvents were of analytical grade. methods: formulation of carbamazepine as extended release tablet: formulas 1-9 shown in table (1) were prepared by mixing cbz with lactose for 10 minutes, then the granulating solution (5% ethyl cellulose in absolute ethanol) was added gradually until a wet ball mass was obtained. the resultant mass was screened through 12mesh sieve and the resultant granules were dried at 50°c for 2 hours. a second screening through 18-mesh sieve was done, followed by mixing the granules with magnesium stearate as lubricant for 2 minutes, then the resultant granules were compressed into tablets using double punch tablet machine (korsch eko, germany). table 1: formulation of cbz tablet using ethyl cellulose with different additives. substance (mg) formula no f1 f 2 f 3 f 4 f 5 f 6 f 7 f 8 f 9 carbamazepine 200 200 200 200 200 200 200 200 200 ethyl cellulose 8.61 11.6 14.7 12.08 12.08 12.08 12.08 12.08 12.08 sodium carboxyme thyl cellulose 15.25 15.25 15.25 microcrystalline cellulose 15.25 sodium lauryl sulphate 4.43 peg6000 4.43 calcium phosphate 75 lactose 75 75 75 75 75 75 75 95 magnesium stearate 3 3 3 3 3 3 3 3 3 total weight of tablet 286.6 289.6 292.7 286.6 289.6 292.7 286.6 289.6 292.7 iraqi j.pharm.sci., vol.17 (1) ,2008 carbamazepine extended release tablet. 57 evaluation of carbamazepine extended release tablet : hardness test the hardness of 3 tablets from each of the prepared formulas was measured individually by using pharma test equipment. an anvil driven by electric motor presses the tablet at a horizontal position and constant load until the tablet breaks (9). friability test this test was done for 20 tablets, starting by weighing them and then operating the friabilator at 25 r.p.m for 4 minutes, reweighing the tablets to determine the loss in their weight(9). uniformity of dosage units five tablets were individually subjected to the following procedure. one tablet was finely powdered and quantitatively transfered, with the aid of methanol, to 100-ml volumetric flask. about 70 ml of methanol was added, shaked by mechanical means for 60 minutes. the mixture was sonicated for 15 minutes, diluted with methanol to volume, allowed to stand for 10-15 minutes, and then the clear supernatant was analyzed spectrophotometry to determine the amount of cbz , using methanol as a blank(1). drug release this test was done for all formulas according to the usp xxviii specifications statedكinكtheكmonographك“carbamazepineك extended-releaseكtablets”.كtheكtestكcarriedك out as triplicate for each formula using apparatus i (basket) at 100 r.p.m and 900 ml water as dissolution medium at 37±0.5ºc under sink conditions. at each time interval 5 ml sample was withdrawn, filtered and suitably diluted to be the absorbance within the calibration curve level. the amount of cbz dissolved at one hour intervals was measured spectrophotometrically at maximum absorbance wavelength, 285 nm (cecil, england). the withdrawn samples were replaced with water. the percentages of cbz released at the specified times must conform to the following: determination of the release kinetics to study the mechanism of drug release from the matrix tablets, the release data were fitted to zero-order, first order, and higuchi equations. furthermore, to characterize the release behavior, i.e. to understand the mechanism, korsmeyer-peppas model (equation 7) was applied: n t ktqq / where, qt is the amount of drug release at timeكtك;كq∞كisكtheكamountكofكdrugكreleaseكafterك infinite time; k is a release rate constant incorporating structural and geometric characteristics of the tablet; and n is the diffusional exponent indicative of the mechanism of drug release (10). effect of temperature: the effect of temperature on the degradation of cbz in the selected formula was studied according to accelerated stability study. the study was done by storing the tablets in ovens at different temperatures of 40, 50, and 60ºc for four months. samples were withdrawn at weekly intervals to determine the total content of cbz by measuring uv absorbanceكatكλكmaxكat285كnm. statistical analysis the results of the experiments are given as a mean of triplicate samples ± standard deviation and were analyzed according to the one way analysis of variance (anova) at the level of (p < 0.05). results and discussion evaluation of carbamazepine extended release tablet : hardness of tablets the hardness of prepared tablets which is shown in table (2) revealed variation which may be attributed to the differences in amount of retarding polymer, in addition to the other exciepient added. for formulas 1-3, the hardness increased as the amount of retarding polymer increased, this result may be attributed to the increase in the compressibility of the matrix resulting from the higher polymer proportion (11). formula 6 and 7 showed lower hardness values in comparison with other formulas and this is originated from the extremely poor compatibility of the surfactant or peg containing granules, which may assume a wax-like physical property (12). for other formulas in which several types of additives were included, they have higher hardness values depending on the nature of these additives. the inclusion of sodium carboxymethyl cellulose and microcrystalline cellulose may result in the consolidation of granules due to the plasticity of these materials and increase intraparticulate bonding during time (hr.) amount released (%) 3 10-35 6 35-65 12 65-90 24 not less than 75 iraqi j.pharm.sci., vol.17 (1) ,2008 carbamazepine extended release tablet. 58 compaction in addition to the homogenous distribution of bonds in the compact (13). table 2: hardness of tablets (expressed as mean ± sd). friability of tablets all formulas have lost not more than 1% of their weights. the incorporation of sodium carboxy methylcellulose and microcrystalline cellulose to the formulations resulted in improved skeleton integrity and acceptable friability as seen in table( 3)( 13). table 3: friability of tablets. uniformity of dosage units table (4) shows that all the prepared formulas which were subjected to this test complied with usp specification which is 85115% of cbz content in each individual tablet(1). table 4: uniformity of dosage units for cbz tablet (expressed as mean± standard) deviation. variables affecting cbz release from extended release tablets : the effect of retarding polymer concentration the release of cbz from formulas 1-3 which they are formulated using concentrations 3%, 4% and 5% of ethyl cellulose is shown in figures (1). it appears that there is a significant difference (p < 0.05) in the release of cbz from matrices of ethyl cellulose (f 1-3) when the polymer concentration was changed. these results indicated that increasing the concentration of polymer tends to decrease the drug release since the amount of cbz released was decreased from 79% to 13% for when the concentration of polymer was increased from 3% to 5% in the matrices. ti me (hour) 0 2 4 6 8 10 a m o u n t o f c b z released (% ) 0 20 40 60 80 100 f 1 ( 3% ethyl cellulose ) f 2 ( 4% ethyl cellulose ) f 3 ( 5% ethyl cellulose ) figure 1: the effect of ethyl cellulose concentration on the release of cbz. this difference can be attributed to the decrease in the porosity with a concomitant increase in the tortuosity of matrix (14). at the concentration of 4% and 5%, ethyl cellulose exhibits an extremely prolonged release as only 30% and 13% of cbz was released after 8 hours from these matrices respectively. these findings may be due to the limited formula no. content of cbz (%) f 1 94.2±2.5 f 2 98.5±3.6 f 3 98.1±3.3 f 4 96.3±2.3 f 5 95.7±3.1 f 6 96.2±4.0 f 7 93.7±2.5 f 8 94.1±3.4 f 9 95.0±4.7 formula no. hardness (kp) f 1 11.2±0.65 f 2 12.3±0.36 f 3 12.8±0.14 f 4 13.0±0.24 f 5 11.9±0.07 f 6 11.5±0.20 f 7 11.4±0.33 f 8 12.2±0.12 f 9 13.5±0.06 formula no. friability (%) f 1 1.215 f 2 0.856 f 3 0.835 f 4 1.334 f 5 0.848 f 6 0.822 f 7 0.779 f 8 0.521 f 9 0469 iraqi j.pharm.sci., vol.17 (1) ,2008 carbamazepine extended release tablet. 59 solubility of cbz and ethyl cellulose in water, therefore it is difficult for dissolution medium to penetrate the matrix (15). the same effect was produced by ethyl cellulose matrices on the release of caffeine and pseudoephedrine hydrochloride (16). the effect of cellulose polymers addition since ethyl cellulose is a hydrophobic insoluble polymer, two different hydrophilic, cellulose materials [the water-soluble sodium carboxy methyl cellulose and the waterinsoluble microcrystalline cellulose (avicel ph 101)] were incorporated ( f 4 and f 5 ). figure (2) shows that the time courses of release from formulas 4 and 5 which contain these additives are not significantly different (p < 0.05) although both materials enhance the release of cbz. at first, cbz release from formula 4 was enhanced due to the high solubility of sodium carboxymethyl cellulose; however, a relative slower release is followed when the glassy nature of this swellable polymer was changed to the rubbery state upon the contact of matrix tablet with water (17). recent developments in the analytical techniques as raman/ir spectroscopy and scanning electron microscopy had revealed that such hydrophilic polymers are adsorbed on cbz compacts through hydrogen bonding (18). such results are consistent with those obtained for the release of propranolol hydrochloride from matrix tablets (19). avicel ph 101 increased the release of cbz from formula 5 which has a mdt comparable to that of formula 4 as shown in figure (2). although it is water insoluble, avicel ph 101 can absorb water to some extent; thus it acts as a pore-forming agent, enhancing the permeation of dissolution medium through the stress relaxation of the polymeric matrix, resulting in a rapid release of the drug (20). 2d graph 1 time ( hour ) 0 2 4 6 8 10 a m o u n t o f c b z released ( % ) 0 20 40 60 80 100 f 4 ( 5.3% so d iu m carb o xymeth yl cellu lo se ) f 5 ( 5.3% mi cro crystallin e cellu lo se ) figure 2: the effect of cellulose derivatives addition on the release of cbz from ethyl cellulose matrix the effect of adding solublizing agents since the absorption of insoluble drugs from the git is controlled via their dissolution, then utilization of solublizing agents in their formulation could improve the oral bioavailability of such drugs (21). the release of cbz from formulas 6 and 7 in which sodium lauryl sulphate and polyethylene glycol 6000 were included respectively as solublizing agents is shown in figure (3). both agents increase the release rate of cbz, however; a significant difference (p < 0.05) in the release rate between the two surfactants was observed due to the higher solublizing activity of sodium lauryl sulphate. this is may be attributed to the higher hydrophiliclipophilic balance (hlb) value for sodium lauryl sulphate compared with polyethylene glycol 6000(22). moreover, cationic drugs dissolve better in anionic surfactants depending on the degree of dissociation, due to ionic interaction (23). time ( hour ) 0 2 4 6 8 10 a m o u n t of c b z released ( % ) 0 10 20 30 40 50 60 70 f 6 ( 1.5 % sodiu m lauryl sulp hate ) f 7 ( 1.5 % peg 6000 ) figure 3: effect of solubilizing agents on the release of cbz from ethyl cellulose matrix. the effect of diluent quantity and its type diluents or bulking agents are frequently added to the formulations in order to give the tablets their appropriate size. although diluents are usually thought to be inert ingredients, they can significantly change the physical or biopharmaceutical properties of dosage forms (24). increasing the quantity of lactose (which is one of the mostly used diluents in tablets formulation) to 95 mg in formula 20 compared to 75 mg in formula 4 resulted in a significant increase (p < 0.05) in the release of cbz as shown in figure (4) and this may be due to the high solubility of lactose so that it will act as channeling agent, permitting a rapid ingress of dissolution medium into the matrix tablets, thus facilitating drug release (25). on the other hand, iraqi j.pharm.sci., vol.17 (1) ,2008 carbamazepine extended release tablet. 60 when calcium phosphate was incorporated in formula 9, a significant reduction (p < 0.05) in the release rate of cbz was observed compared to the release rate from formula 4 in which lactose was included. the slower release rate of cbz which is the direct result for the presence of an insoluble additive in the matrix, therefore slowing down the drug diffusion and/or the medium infiltration(26). sodium sulphadiazine, ketoprofen, and theophylline showed the same observations when formulated as matrix tablets with hpmc (25). time ( hour ) 0 2 4 6 8 10 a m o u n t of c b z release ( % ) 0 20 40 60 80 100 f 4 lactose 75 mg f 8 lactose 95 mg f 9 calcium phosphate 75 mg figure 4: the effect of diluents addition on the release of cbz from ethyl cellulose. determination of the release kinetics table (5) shows that n values of formulas 1-3, equals to 0.725-0.931, pointing to an anomalous (non-fickian) diffusion mechanism with a trend toward higher values as the polymer content increased. on the other hand, higher values of kkp was determined for formula 1 with the least content of retarding polymers, suggesting the possibility of occurrence of burst effect. formula 2 and 3 show a better fitness to zero-order and firstorder respectively. it can be considered that decrease of cbz release through increased polymer content produces a change in the release mechanism moving away from diffusion as n values becomes closer to 1.0, which also confirms that the kinetics go in the direction of zero-order release(27). incorporation of sodium carboxymethyl cellulose into ethyl cellulose matrices (formula 4) produced n value of 0.656 with a remarkable fitness to first-order kinetics. meanwhile, microcrystalline cellulose in formula 5 gave an approximately unity value for n, indicating zero-order release. this variation may reflect the different behavior of each polymer since the sodium carboxymethyl cellulose will form a viscous gel through which diffusion occurs (28), and the microcrystalline cellulose has a disintegrating properties so that resulting matrix erosion (29). although n values for formulas 6 and 7 had revealed an anomalous diffusion, sodium laurylsulphate gave a higher kkp value indicating fast initial release with first-order kinetics due to its powerful solubilizing effect compared to polyethylene glycol 6000 which have low solubilizing ability and moderate release restriction properties (30). in addition, increasing the quantity of lactose in formula 8 produced a lower n value with an elevated kkp value due to the high solubility of this material, thus stimulating water penetration into the matrix (24). in contrast, calcium phosphate in formula 9 gave matrices in which cbz release is controlled by erosion because this diluent is insoluble in water (26). table 5: fitting results of formulas 1-9 for cbz release data to different kinetic model. formula no. model zero-order k0 (%h-1) r2 first-order k1 (h-1) r2 higuchi kh (h-1/2) r2 korsmeyer-peppas kkp (h-n) n r2 f1 9.229 0.9557 0.2575 0.8348 32.638 0.9869 16.748 0.788 0.9677 f2 3.646 0.9893 0.1792 0.9865 14.734 0.9378 6.437 0.725 0.9467 f3 3 0.9707 0.2303 0.9889 12.368 0.9034 2.989 0.931 0.9463 f4 9.25 0.9773 0.273 0.9902 33.247 0.9247 4.109 0.656 0.981 f5 9.21 0.9764 0.319 0.9793 33.505 0.9239 8.167 1.008 0.982 f6 11.6 0.9885 0.281 0.9931 37.04 0.9584 20.811 0.693 0.9755 f7 6.055 0.9915 0.212 0.9691 25.293 0.9474 7.466 0.876 0.9711 f8 8.943 0.9915 0.234 0.9468 31.096 0.9897 18.703 0.678 0.9948 f9 8.988 0.9804 0.346 0.9429 41.84 0.9278 4.855 1.238 0.996 iraqi j.pharm.sci., vol.17 (1) ,2008 carbamazepine extended release tablet. 61                   100 1 1log50 5.0 1 2 2 n t tt trn f                            100100 1 1log50 5.0 1 2 2 n t tttlx r x rr n f selection of the best formula although several prepared formulas met the release specifications of usp, formula 4 showed a release profile comparable to that of the brand product tegretol cr® . the similarity factor (f 2) introduced by moore and flanner is used as criterion for assessment of the similarity between two dissolution profiles (31): ……….(1) where n is the number of dissolution time points, rt and tt are the reference and test dissolution values at time t. for the conventional tablets, a difference not exceeding 10% at any sampling time point between reference and test products may be acceptable and f 2 value of 50-100 indicates similarity in the dissolution profiles, while for sustained release tablets, the lower limit of 50 is very liberal especially for drugs with narrow therapeutic index. therefore, the generalized equation to estimate the lower acceptable value of f 2 (f 2lx) is: ……..(2) where x is the percent deviation for the reference product (32). if 10% deviation is allowed for the dissolution profiles to be similar, then the calculated f 2lx value using equation (2) will be 61.18 for tegretol cr®. for the prepared formulas, the highest calculated f 2 value according to equation (1) is 85.22 for formula 4. figure (5) shows the non significant difference (p < 0.05) in the dissolution profiles of cbz from tegretol cr® and formula 4. figure 5: the release of cbz from formula 10 compared to tegretol cr® in water. stability study: accelerated temperature effect the stability of the selected formula 4 was studied at three different temperatures ; 40ºc , 50ºc , and 60ºc for 16 weeks.the degradation of cbz followed first-order kinetics because straight lines were obtained when logarithm of percent remaining of the drug was plotted versus time(33). figure (6) shows the degradation curves of cbz at 40ºc, 50ºc and 60ºc, from which the degradation rate constant (k) at each temperature was determined from the slope of each line. the values of rate constants are summarized in table (6). the date of expiration for cbz was determined through constructing arrhenius plot as shown in figure (7) in order to estimate the degradation rate constant (k25) at 25ºc which was equal to 5.623 × 10-4 week-1 the following equation is used for calculating the expiration date (34) : 25 %10 105.0 kt  where t10% is the time required for a drug to lose 10% of its potency and it was found to be 185 weeks ( about 3.6 years for cbz). 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10 0 20 0 30 0 40 0 50 0 60 0 70 0 80 0 90 0 100 0 0 1 2 3 4 5 6 7 8 9 time (hour) (hour) a m o un t o f c b z re le as ed (% ) (% ) f 10 10 tegretol cr® ® 62 table 6: degradation rate constants for cbz in formula 10 at 40ºc, 50ºc and 60ºc. figure 6: accelerated degradation of cbz in formula 10 at 40ºc, 50ºc and 60ºc. figure 7: arrhenius plot of cbz in formula 10 for the estimation of the expiration date. conclusion carbamazepine extended release tablet has been successfully fabricated by using ethyl cellulose as retardant substance. in vitro release of carbamazepine was correlated with types and concentrations of the additives used in the formulation. references: 1united states pharmacopoeia xxviii. the usp convention. 2005. 2rekhi g., hussain a., tillman l., malinowski h., augusberger l., identification of critical formulation and processing variables for metoprolol tartrate extended release matrix tablets. j. control. release 1999;59:327-342. 3reza m., quadir m., haider s., comparative evaluation of plastic, hydrophobic and hydrophilic polymers as matrices for controlled release drug delivery. j. pharm. and pharm. sci. 2003;6(2):274-291. 4cao q., kim t., lee b., photo images and the release characteristics of lipophilic matrix tablets containing potassium citrate with high drug loading. int. j. pharm. 2007; 339:19-24. 5varshosaz j., tavakoli n., eram s., use of natural gums and cellulose derivatives in production of sustained release metoprolol tablets. drug delivery. 2006;13:113-119. 6saha r., sajeev c., sahoo j., a comparative study of controlled release matrix tablets of sodium diclofenac, ciprofloxacin hydro-chloride, theophylline.drug delivery 2001;8:149154. 7zhi y., rombout p., remon j., vervaet c., mooter g., a correlation between permeability of metoprolol tartrate through isolated ethyl-cellulose/ hydroxypropyl methycellulose films and drug release from reservoir pellets. eur. j. pharm. biopharm. 2007;1: 1-6. 8ficker d., priyitera m., krauss g., kanner a., moore j., improved tolerability and efficacy in epilepsy patients with extended release carbamazepine. neurology 2005;65:593-595. 9banker g., anderson n., tablets in lachman l., lieberman h. and kanig j. the theory and practice of industrial pharmacy. 3rd ed. lea & febiger 1986 p.293345. 10vueba m., carvalho b., veiga f., sousa j., pina m., influence of cellulose ethers polymers on ketoprofen release from hydrophilic matrices. eur. j. pharm. biopharm. 2004;58:51-59. 11pruthvipathy r., katikaneni p., upadrashta s., neau s., mitra a., ethylcellulose matrix controlled release tablets of a water-soluble drug. int. j. pharm. 1995 ;123:119-125. 12 ruddy s., matuszewska b., grim y., ostovic d., storey d., design and temperature (ºc) k (week-1) 40 1.8 × 10 -3 50 3.7 × 10 -3 60 5.9 × 10 -3 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.9 9 1.91 1 1.92 2 1.93 3 1.94 4 1.95 5 1.96 6 1.97 7 1.98 8 1.99 9 2 0 2 4 6 8 10 0 12 2 14 4 16 6 time (week) (week) lo g % r em ai ni ng o f c b z 40ºc 50ºc 60ºc -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 2.9 9 3 3.1 1 3.2 2 3.3 3 3. 4 1/t × 10-3 (kelvin-1) lo g k 63 characterization of surfactant-enriched tablet for oral delivery of a poorly watersoluble immunosuppressive agent. int. j. pharm. 1999 ;182:173-186. 13kyriakos k., malamataris s., compact size and mechanical strength of pharmaceutical diluents. eur. j. pharm. sci. 2005;24: 169-177. 14foster t., parrotte e., effect of processing on release from inert, hetrogenous matrix. drug dev. ind.pharm. 1990; 16; 13091324. 15moneghini m., voinovich d., perissutti b., princivalle f., action of carriers on carbamazepine dissolution. pharm. dev. tech. 2002;7: 289-296. 16agrawal a., neau s., bonate p., wet granulation fine particle ethyl cellulose tablets: effect of production variables and modeling of drug release. aaps 2003;5(2): article 13. 17conti s., maggi l., segale l., machiste e., grenier p., vergnault g., matrices containing sodium carboxymethylcellulose and hpmc 1. dissolution performance and characterization. int. j. pharm. 2007; 333:136-142. 18tian f., sandler n., altonen j., lang c., saville d., gordon k., rades t., strachan c., influence of polymorphic form, morphology, and exipient interaction on the dissolution of carbamazepine compacts. j. pharm. sci. 2007;96(3):584-594. 19takka s., rajbhandari s., sakr a., the effect of anionic polymers on the release of propranolol hydrochloride from matrix tablets. eur. j. pharm. biopharm. 2001;52:75-82. 20skinner g., harcum w., dürig t., ethyl cellulose direct compression modified release tablets: impact of polymer structure and formula-tion variables. 2002 aqualon co. ptr-023. 21el-zein h., raid l., el-bary a., enhancement of carbamazepine dissolution: in vitro and in vivo evaluation. int. j. pharm. 1998;168: 209220. 22martin a., interfacial phenomena in physical pharmacy. 5th ed. lea & febiger 2006 p.430-456. 23park s., choi h., effect of surfactants on the dissolution profile of poorly water soluble acidic drugs. int. j. pharm. 2006;321:35-41. 24collett j, moreton c. modified-release peroral dosage forms. in: pharmaceutics, the science of dosage form design, aulton, 2nd ed. new york: churchill livingstone, 2001: 289-305. 25furlanetto s., cirri m., maestrelli f., corti g., mura p., study of formulation variables influencing the drug release rate from matrix tablets by experimental design. eur. j. pharm. biopharm. 2006;62: 77-84. 26rao v., engh k., qui y., design of phindependent controlled release matrix tablets for acidic drugs. int. j. pharm. 2003;252:81-86. 27omari d., sallam a., abed-elbary a., elsamaligy, lactic acid-induced modifications in films of eudragit rl and rs aqueous dispersions. int. j. pharm. 2004 ;274:85-96. 28 vatsaraj n., zia h., needham t., formulation and optimization of sustained release tablet of ketorolac tromethamine. drug delivery. 2002;9:153-159. 29khan g., zhu j., ibuprofen release kinetics from controlled release tablets granulated with aqueous polymeric dispersion of ethylcellulose ii: influence of several parameters and coexipients. j. control. release. 1998;56:127-134. 30 javadzadeh y., navimipour b., nokhodchi a., liquisolid technique for dissolution rate enhancement of high dose water-insoluble drug (carbamazepine). int. j. pharm.2007;341:26-34. 31moore j., flanner h., mathematical comparison of curves with an emphasis on in vitro dissolution profiles. pharm. tech. 1996; 20(6): 64-74. 32gohel m., panchal m., reinfinement of lower acceptance value of the similarity factor f 2 in comparison of dissolution profiles. dissolution tech. 2002:1-5. 33lachman l., deluca p., akers m. kinetics principles and stability testing in lachman l., lieberman h. and kanig j. the theory and practice of industrial pharmacy. 3rd ed. lea & febiger 1986 p.760-803. 34martin a., kinetics in physical pharmacy. 5th ed. lea & febiger 2006 p.284323. iraqi j pharm sci, vol.26(2) 2017 sorption separation using polyurethane foam sorbents 7 colorimetry and indirect x-ray fluorescence determination of active ingredients in tetracycline hydrochloride drug and injection solution of b12 vitamin using of polyurethane foam sorbents aleksandr a. chaplenko*,1, oksana v. monogarova* and kirill v. oskolok* *department of chemistry, lomonosov moscow state university, leninskie gory, 1-3, 199991, moscow, russia. abstract the simple and available technique of colorimetry and indirect x-ray fluorescence determination of tetracycline hydrochloride (in the form of the colored complex with iron(iii) ions) and cyanocobalamine (in the form of the colored thiocyanate complex with cobalt(ii) ions) is offered. the analytes were separated from the accompanying components by sorption to polyurethane foam based on ethers. the conditions of sorption separation and measurement of the analytical signal of these substances are optimized. the obtained results of tetracycline drug and injection solution of the b12 vitamin are in satisfactory agreement with data declared by the manufacturer. keywords: tetracycline hydrochloride, b12 vitamin, polyurethane foam sorbent, chemical colorimetry method, indirect x-ray fluorescence analysis. introduction one of the important tasks of pharmaceutical chemistry is the development of effective ways of determination of active ingredients in drugs. according to pharmacopoeias (1-4), both chemical and instrumental methods (for example, high performance liquid chromatography and infrared spectroscopy) are used traditionally for determination of active ingredients in pharmaceutical medicines. low selectivity is one of the main disadvantages of chemical methods, but the high cost of instrumental methods and requirements to the qualification of the operator limited its available for field laboratories and drugstores. in this regard, the development of technically simple and selective approaches of determination of active ingredients in drugs is an actual task of modern pharmaceutical chemistry. visual colorimetry is among simple and available methods of analysis. the colorimetry techniques are widely used in analytical chemistry, but combination of these techniques with digital processing of the received images allows to expand method scopes and to improve metrological characteristics (5). the aim of our work is to illustrate opportunities of combined chemical and tool approach based on sorption extraction of active ingredients from pharmaceutical drugs with subsequent digital colorimetry determination. the function of a sorbent isn`t limited only to a stage of separation and concentration. sorbent phase is a certain universal environment with constant properties; it took part in the formation of an analytical signal and providing unity of conditions of its measurement. therefore, determination of big contents of medicinal substances should be carried out by means of conversion of analyte in a sorbent phase. polyurethane foam (puf) sorbents based on ethers are used for sorption extraction of active ingredients from solutions. puf sorbents are cheap and available; they have high chemical, mechanical and thermal stability. puf sorbents have high distribution coefficients (from 3 to 5 orders) in the system «water solution – sorbent» (6) for many classes of organic and inorganic compounds. simple, inexpensive and available measuring device – office flatbed scanner – is proposed for measurement of analytical signal (the intensity of coloring of a sample) instead of expensive equipment. the graphics editor can be used for processing of images received from the scanner (7, 8). products of the physical and chemical interaction of puf with medicinal substances contain functional and analytical chelate groups. these groups are capable of connecting ions of transitional metals to stable complex compounds. this fact can be used for adequacy test of colorimetric techniques using indirect x-ray fluorescence determination of mentioned above organic substances on «heavy labels». «heavy labels» are ions of metals forming stable complexes with the corresponding active ingredients (9). experimental reagents and materials. puf sorbent brand 5-30 (spa «radical», kiev, ukraine) is used for sorption of active ingredients from drugs. the size of puf tablets for sample preparation is 10  10  1 mm. the geometric shape of these tablets is a parallelepiped. the mass of puf sorbent is ~ 0.2 g. 1corresponding author e-mail: a.a.chaplenko@yandex.ru received:23 /5/2017 accepted: 5/7/2017 iraqi j pharm sci, vol.26(2) 2017 sorption separation using polyurethane foam sorbents 8 the prepared puf tablets were washed with 1 m hcl and acetone during 1 hour and then with distilled water up to ph 5-6 because puf contains organic impurities technological metal impurities. after that puf tablet was dried between sheets of filter paper. the degree of purification was monitored by x-ray fluorescence method. in our work state standard samples containing 1 mg/ml co(ii) ions («ecoanalytica», moscow, russia), sodium fluoride (not less than 99 %), potassium thiocyanate (not less than 99 %), sodium hydroxide (not less than 99 %), ferric(iii) chloride (not less than 99 %), hydrochloric acid (more 99.9 %), sulfuric acid (more 99.9 %), acetone (more 99.9 %) («sigma-aldrich», usa), cyanocobalamin solution for injection 0.5 mg/ml in ampoules on 1 ml (borisov, belarus), pharmaceutical substance – tetracycline hydrochloride powder (north china pharmaceutical goldstar co., china) are used. as a standard sample of tetracycline hydrochloride solution of tetracycline substance (1 mg/ml) was used. the quality of tetracycline substance is confirmed by methods described in pharmacopoeia article (2). the content of tetracycline hydrochloride is 99.4 ± 0.2 %. the stirring device (model 6500, «ecros», russia) is used in our work. sample preparation of medicinal substances and preparation of calibration solutions. tetracycline hydrochloride solution. for the preparation of analyzed tetracycline hydrochloride solution pharmaceutical substance (m = 0.250 g) was thoroughly ground in an agate mortar to a homogeneous powder and was dissolved in 30 ml 0.01 m naoh. the solution was shaken on a stirring device for 60 minutes and it was quantitatively placed into the volumetric flask (100.0 ml). the volume of solution was adjusted to 100.0 ml with the use of 0.01 m naoh. aliquot of analyzed solution (1.00 ml) was quantitatively placed into the volumetric flask (25.0 ml), add 1.00 ml of fecl3 solution (1 mg/ml). the volume of solution was adjusted to 25.0 ml / acidity to ph 2-3 with the use of deionized water and 1 m hcl. for the preparation of the calibration solutions standard sample of tetracycline hydrochloride substance was used. the calibration solutions were prepared similarly to analyze samples. concentrations of tetracycline hydrochloride in calibration solutions are changed from 0.01 to 1.0 mg/ml. tetracycline hydrochloride was sorbed on puf in the form of the colored complex with iron(iii) ions. puf tablet pressed with a glass rod to remove air bubbles for an increase of uniformity of analyte distribution by volume of sorbent. solutions were shaken on a stirring device for 60 minutes. after that puf tablet was removed from the solution, it was thoroughly washed with distilled water and was dried between sheets of filter paper. cyanocobalamin. sample preparation of cyanocobalamin solution for injection was included the stage of mineralization. the content of 5 ampoules was evaporated to dryness in a porcelain crucible, to the residue, concentrated h2so4 was added and the heating was continued for 5 minutes. the residue obtained after calcinations was quantitatively transferred to a measuring fluoroplastic beaker and it was dissolved in 5 ml deionized water with 1m naoh up to ph 10-11. 50.0 mg naf and 50.0 mg kscn were placed in a fluoroplastic beaker. the obtained solution was shaken on a stirring device to completely dissolving and it was quantitatively placed into the volumetric flask (25.0 ml). the volume of solution was adjusted to 25.0 ml / acidity to ph 2-3 with the use of deionized water and 1 m hcl. state standard samples containing 1 mg/ml co(ii) ions were used for preparation of calibration solutions. an aliquot of this solution (1.00 ml), 50.0 mg naf and 50.0 mg kscn were quantitatively placed into the volumetric flask (10.0 ml). the obtained solution was shaken on a stirring device to completely dissolving. the volume of solution was adjusted to 10.0 ml with the use of deionized water, acidified with 1 m hcl up to ph 2-3. the calibration samples were prepared from obtained solution by dilution. the content of co(ii) ions in calibration solutions is various from 0.0005 to 1.0 mg (it corresponds to a concentration of cyanocobalamin from 0.01 to 2.30 mg/ml). the sorption of thiocyanate complexes of cobalt on puf was carried out similarly one of tetracycline. the completeness of the recovery of cyanocobalamin was verified by the indirect x-ray fluorescence method with the repeated addition of puf tablet to the analyzed solutions after sorption. the measurement of analytical signal. the measurement of colorimetric analytical signal (lightness of the chosen color channel) was carried out with the use of office scanner laserjet m1120 mfp (hewlett-packard, usa) and program for processing of raster images gimp 2.8.16 (the gimp team, usa). the scanning parameters: the color mode is the color image; type is photo; resolution is 300 dpi. processing of puf tablets images and determination of lightness of the color bchannel (lb) was made in the graphical editor. iraqi j pharm sci, vol.26(2) 2017 sorption separation using polyurethane foam sorbents 9 the analytical signal was measured in a scale rgb-model as lightness of b-channel in conventional units from 0 to 255.indirect xrf determination of medicinal substances on puf sorbent was performed on a spectroscan g max xrf spectrometer (spa “spectron”, st. petersburg, russia). the instrument was equipped with a sealed-off gas-filled proportional counter tube (filler gas 90% xe + 10% ch4 under atmospheric pressure, the thickness of the be window is 150 μm), lif(200) crystal analyzer (2d = 402.8 pm), and low-power (4 w) sharp-focus (diameter 1.5 mm) x-ray tube of the transmitted type with a thin-film (2 μm) mo-anode (the thickness of the be window is 200 μm). the working voltage was 40 kv. the current is 100 a. the working voltage was 45 kv. the incidence angle of the primary radiation on the sample surface was 80°, and the takeoff angle of the secondary radiation was 30°. to determine xray fluorescence spectra puf tablets were placed in specially made cuvette with the low level of scattered primary radiation (10). the peak value of the line intensities cok, fek in wavelength ranges 179.0  5.0; 193.7  5.0 pm respectively were measured for each sample. the exposure time is 60 s. the analytical signal is integrated intensities of the lines of these elements excluding the background signal normalized to the intensity of the scattered radiation mok is incoherent x-raytube. for taking note of provision of a sample under the probe on uncertainty of results the analytical signal was measured by 4 times at the turn of a puf tablet by 90. results and discussion we varied ph of sorption from 1 to 12 and the time of sorption from 10 minutes to 2 hours (the concentration of ions was taken over and was 50 µg/ml) for the selection of optimal conditions of sorption. it was shown that the acidity is significant for the completeness of the sorption of the metals from the solution. the optimum ph value for cobalt and nickel ions is 2.5 – 3.5 (fig. 1). the optimum time of sorption for both metals is ~ 60 minutes (fig. 2). figure (1): selection of optimal ph value of zn2+and co2+ sorption (n = 5, p = 0.95) figure (2): selection of optimal time of zn2+ and co2+ sorption metrological characteristics of colorimetric and indirect x-ray fluorescence determination of active ingredients in drugs after sorption on puf sorbent are presented in table 1. table (1): the metrological characteristics of colorimetric and indirect x-ray fluorescence determination of tetracycline hydrochloride and cyanocobalamin using puf sorbents (n = 5, p = 0.95) medicine substance r2 the range of determined contents, mg/ml lod, mg/ml rsd colorimetry cyanocobalamin 0.9933 0.3-1.5 0.09 0.03 tetracycline 0.9881 0.03-0.2 0.01 0.01 x-ray fluorescence analysis cyanocobalamin 0.9940 0.2-1.4 0.12 0.04 tetracycline 0.9916 0.05-0.2 0.02 0.01 the new way of determination of b12 vitamin is proposed for illustration of possibilities of developed combined approach during analysis of drugs with metalloorganic active substances. the technique is based on chemical mineralization of cyanocobalamin, sorption extraction of co(ii) ions in the form of the colored thiocyanate complexes from iraqi j pharm sci, vol.26(2) 2017 sorption separation using polyurethane foam sorbents 10 water solutions on puf sorbent with subsequent measurement of analytical signal (11). thiocyanate complex of co(ii) ions stain the puf sorbent in blue color, complex tetracycline with fe(iii) ions have lilac-gray color (12). the accuracy of analysis results is confirmed by standard addition method (table 2). the results of colorimetric and indirect xray fluorescence determination of tetracycline and cyanocobalamin in drugs are represented in table 3. the obtained results are in satisfactory agreement with data declared by the manufacturer. table (2): the validation of accuracy of sorption colorimetric and sorption x-ay fluorescence determination of tetracycline hydrochloride and cyanocobalamin by standard addition method. introduced found ± confidence interval (n = 5, p = 0.95) weight of cyanocobalamin, mg colorimetry xrf 0.5 0.50  0.05 0.45  0.06 1.0 1.05  0.07 0.94  0.09 weight of tetracycline hydrochloride, mg colorimetry xrf 0.10 0.11  0.01 0.09  0.02 0.15 0.16  0.02 0.14  0.03 table (3): colorimetric and indirect x-ray fluorescence determination of drug substances in drugs using puf sorbent (n = 5, p = 0.95) drug substance m, mg/sample according to pharmacopoeia methods colorimetry xrf cyanocobalamin 2.50  0.02 2.4  0.1 2.6  0.2 tetracycline 248.5  0.5 245  15 250  30 conclusions thus, the developed combined chemical and instrumental approach can be used for determination of organic and metalloorganic active substances if one of transformation products is colored complex. such pharmaceuticals include tetracycline hydrochloride and cyanocobalamin solution for injections. the proposed approach is available, technically simple and it doesn’t require using of specialized and expensive devices. the developed technique can be used in low-budget laboratories and for preliminary screening of the drugs that don’t meet the required standards during monitoring of quality of drugs. references 1. european pharmacopoeia (ph. eur.) 9th edition. 2. the united states pharmacopeia 39. 3. japanese pharmacopeia 17 revision. 4. state pharmacopeia xiii (russian federation). 5. wu w, allebach jp, analoui m, imaging colorimetry using a digital camera. journal of imaging science and technology, 2000; 44 (4): 267–279. 6. ivanov vm, monogarova ov, oskolok kv, capabilities and prospects of the development of a chromaticity method. journal of analytical chemistry, 2015; 70 (10): 1165–1178. 7. meng x, schultz cw, cui c, li x, yu hz, on-site chip-based colorimetric quantitation of organophosphorus pesticides using an office scanner. sensors and actuators b: chemical, 2015; 215: 577–583. 8. kehoe e, penn rl, introducing colorimetric analysis with camera phones and digital cameras: an activity for high school or general chemistry. journal of chemical education, 2013; 90 (9): 1191– 1195. 9. apyari vv, dmitrienko sg, ostrovskaya vm, anaev ek, zolotov ya, use of polyurethane foam and 3-hydroxy-7, 8benzo-1, 2, 3, 4-tetrahydroquinoline for determination of nitrite by diffuse iraqi j pharm sci, vol.26(2) 2017 sorption separation using polyurethane foam sorbents 11 reflectance spectroscopy and colorimetry. analytical and bioanalytical chemistry, 2008; 391 (5): 1977–1982. 10. oskolok kv, monogarova ov, devyatkina ed, direct x-ray fluorescence detection of mercury on polyurethane foam sorbents. moscow university chemistry bulletin, 2012; 67 (2): 78–81. 11. renfrew ak, bryce ns, hambley t, cobalt(iii) chaperone complexes of curcumin: photoreduction, cellular accumulation and light‐selective toxicity towards tumour cells. chemistry – a european journal, 2015; 21 (43): 15224–15234. 12. wang h, yao h, sun p, li d, huang c, transformation of tetracycline antibiotics and fe(ii) and fe(iii) species induced by their complexation. environmental science and technology, 2015; 50 (1): 145–153. iraqi j pharm sci, vol.27(2) 2018 x-ray fluorescence determination of trace elements doi: https://doi.org/10.31351/vol27iss2pp1-6 1 x-ray fluorescence determination of trace elements in vitaminmineral complexes and medicinal herbs using chemically modified polyurethane foam sorbents alexander chaplenko*,1, oksana monogarova *and kirill oskolok* *department of analytical chemistry, faculty of chemistry, lomonosov moscow state university, russia abstract in this paper, we proposed the method of x-ray fluorescence (xrf) for the determination of some essential trace elements in medicinal herbs and vitamin-mineral complexes at the level of 1-10 g/ml. to increase sensitivity and selectivity of the determination, we followed simple and effective approach based on the extraction of metal ions from aqueous solutions with chemically modified polyurethane foam sorbents followed by direct xrf analysis. the conditions of sorption preconcentration of co (ii), ni (ii) and zn(ii) ions with modified sorbents were optimized. the proposed approach is used for the determination of trace elements in several kinds of medicinal herbs (coltsfoot leaves, nettle leaves and yarrow herb) and vitaminmineral complexes (alfavit, vitrum and multi-tabs). keywords: trace elements, medicinal herbs, vitamin-mineral complexes, polyurethane foam sorbent, x-ray fluorescence analysis. introduction trace elements are essential parts of most important enzymes. insufficient or excess intake of trace elements leads to disruption of metabolic processes and to various diseases consequently (1). vitamin-mineral complexes are traditionally used for the prevention and treatment of the lack of trace elements. moreover, medicinal herbs also can be the source of trace elements. thus, determination of trace elements in plant samples and in mineral complexes is one of the priority tasks of pharmaceutical analysis. the content of various trace elements in vitamin-mineral complexes varies from g to mg per gram of drug. along with trace elements a significant amount of both active and auxiliary organic substances contained in the above mentioned drugs. the content of the vital elements per gram in medicinal herbs is about tens of nanograms (2). concentration of the inorganic components in plants is a few percents of mass. because of the complexity of drug and plant matrices, it is necessary to use only high selective and high sensitive methods (inductively coupled plasma atomic emission spectroscopy (icp-aes), for example) for determination of trace elements in these objects (3). limits of traditionally used techniques of trace metals analysis are slow rapidity, high energy consumption during thermal mineralization, high cost of analysis, singleton determination (4). the purpose of this research is the development of alternative way of determination of vital trace elements in medicinal herbs and vitamin-mineral complexes without limits of traditional approach. the following tasks are necessary for achieving this purpose: -to choose the most available and effective chemical-instrumental approach to increase selectivity and sensitivity of determination of trace elements; -to minimize the number of stages of developed techniques for increasing accuracy and rapidity of determination of trace elements ; to optimize the conditions of sample preparation for individual and group determination of these trace elements ; to choose measuring tool and optimal conditions of formation of the analytical signal. 1corresponding author e-mail: a.a.chaplenko@yandex.ru received: 7/4/2018 accepted: 1/6/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp1-6 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 x-ray fluorescence determination of trace elements 2 materials and methods reagents and materials ether-based polyurethane foam (puf) (radical, kiev, ukraine) is used for the concentration of trace elements from medicinal herbs and vitamin-mineral complexes. the size of puf tablets for sample preparation is 10  10  1 mm. the geometric shape of these tablets is parallelepiped. the degree of purification was monitored by xrf method. in our work state standard samples containing 1 mg/ml ni(ii), zn(ii), co(ii) ions (ecoanalytica, moscow), resorcinol (pure for analysis), 8hydroxyquinoline (pure for analysis), 2-nitroso1-naphthol (pure for analysis), sodium nitrite (chemically pure), sodium hydroxide, hydrochloric acid, acetic acid (high purity) «sigma-aldrich, usa), coltsfoot leaves, nettle leaves, yarrow herb (allkrasnogorskleksredstva llc, russia) and vitamin-mineral complexes «alfavit» (recordati group, india), vitrum (unipharm, inc., usa), multi-tabs (ferrosan, denmark) are used. preparation of chemically modified sorbent the mass of puf sorbent is 25 mg. the prepared puf tablets were washed with 1 m hcl during 1 hour and then with distillated water up to ph 5-6, because puf may contain a technological metal impurity, especially, zinc. the technique of chemical modification of puf sorbent is based on diazotization and subsequent azo-coupling reaction with an organic reagent. this reaction takes place with toluidine end groups of macromolecules of puf sorbent. the scheme of chemical modification of puf sorbent is shown in fig. 1 (5). molecules of the reaction product have functional and analytical chelating groups. 8hydroxyquinoline is used for extraction ni(ii), zn(ii) ions (6), resorcinol for extraction co(ii), ni(ii) ions (7) and 2-nitroso-1-naphthol for extraction co(ii) ions respectively (8). figure 1. the scheme of chemical modification of puf sorbent puf diazotization was carried out as follows. 30 ml 1 m hcl and 200 g puf sorbent were placed in a glass vessel for extraction lapped cover. puf tablet pressed with a glass rod to remove air bubbles. after the addition of 15 mg nano2 the content of extraction vessel was shaken on a mechanical vibromixer for 30 minutes (5). after that tablet of diazotized puf sorbent was removed from the solution and dried between sheets of filter paper. then it was used in azo-coupling reaction. 20 mg of resorcinol (or 20 mg of 8hydroxyquinoline, or 15 mg 2-nitroso-1naphthol) was dissolved in 30 ml naoh (ph 10) in another vessel for extraction. the diazotized puf tablet was added to this solution, air bubbles was removed. the obtained solution was stirred for 60 minutes. after that, puf tablet was washed and dried, as it described before. tablets of a size 10  10  1 mm were cut from the chemically modified sorbent. the purity of the puf sorbent was monitored by xrf method. concentration of trace elements. for the preparation of samples to 25 ml of an aqueous solution containing from 0.1 to 50 µg/ml ni(ii), zn(ii), co(ii) ions is added one tablet puf sorbent modified by 8hydroxyquinoline, resorcinol and 2-nitroso-1naphthol. the air was removed from puf tablets. contents of extraction vessels were shaken for 80 minutes. after that puf tablet was removed from the solution, it was thoroughly washed with distilled water and was dried between sheets of filter paper. sample preparation of medicinal herbs and vitamin-mineral complexes (9). weights of the analyzed medicinal herbs (m = 5-10 g) and vitamin-mineral complexes (m = 2-2.5 g) were thoroughly ground in an agate mortar to a homogeneous powder. weights of this powder were placed in iraqi j pharm sci, vol.27(2) 2018 x-ray fluorescence determination of trace elements 3 polytetrafluoroethylene glass, 8 ml 70 % hno3 and 2 ml 30 % h2o2 solutions were added. the obtained solution was heated on hotplate at the temperature 100-110 c until no brown fumes no2 was observed. after mineralization obtained mixture was filtered on glass filter crucible. the filtrate was quantitatively placed into the extraction vessel. the volume of solution was adjusted to 25 ml using of 0.1 m naoh. the tablets of chemically modified puf sorbent were placed into this solution. the air was removed from puf tablets. contents of extraction vessels were shaken for 80 minutes. after that puf tablet was removed from the solution, it was thoroughly washed with distilled water and was dried between sheets of filter paper. measurement of analytical signal. direct xrf determination of trace elements on chemically modified puf sorbent was performed on a spectroscan g max xrf spectrometer (llc spectron, st. petersburg, russia). the instrument was equipped with gasfilled proportional counter tube (filler gas 90% xe + 10% ch4 under atmospheric pressure, the thickness of the be window is 150 μm), lif(200) crystal analyzer (2d = 402.8 pm), and low-power (4 w) sharp-focus ( 1.5 mm) xray tube of the transmitted type with a thin-film (2 μm) mo-anode (the thickness of the be window is 200 μm). the working voltage was 40 kv. the current was 100 a. the incidence angle of the primary radiation on the sample surface was 80°, and the takeoff angle of the secondary radiation was 30°. to determine xrf spectra, puf tablets were placed in specially made cuvette with low level of scattered primary radiation (10-12). the peak value of the line intensities cok, nik, znk in wavelength ranges 179.0  5; 165.9  5; 143.5  5 pm respectively were measured for each sample. the exposure time is 60 s. the analytical signal is integrated intensities of the lines of these elements excluding the background signal normalized to the intensity of the scattered radiation mok is incoherent xray tube. results and discussion selection conditions of modification of puf sorbent. the end toluidine groups of puf in hydrochloric acid react with aqueous solutions of alkali metal nitrites with the formation of yellow solution of diazonium cation. diffuse reflectance spectroscopy shown that maximum yield of diazotized puf is achieved after 30 minutes of phase contact in 1-2 m hcl (12). it was known that diazotization of monomeric aromatic amines usually occurs at lower temperatures (0-7 c). it complicates the modification of puf sorbent. in the paper (13) was shown that diazotization of puf is possible at room temperature. moreover, unlike the monomeric diazonium cation – very unstable compound – diazonium chloride polymer is stable in storage of diazotized puf samples on the air for about an hour. in the study of the chemical properties of diazotized puf sorbent, it was shown that polymeric diazonium cation has high reactivity, similarly to monomer form. in particular, it is capable of reacting azo-coupling. the interaction of diazotized puf with solutions of modified agents, such as 8-hydroxyquinoline, resorcinol and 2-nitroso-1-naphthol was studied. the amounts of modified reagents were taken in a 100-fold excess to the one of diazotized puf. about azo-coupling reaction judged by color change of puf tablets. in the presence of 8-hydroxyquinoline, resorcinol and 2-nitroso-1-naphthol puf sorbent changes color from light yellow to orange, red and brown, respectively. since only the active form of diazo-compounds in the azo-coupling reaction is a diazonium cation, it is necessary to maintain the ph of the solution at the level of 9.0 – 10.0. selection of complexation conditions. puf chemically modified by 8hydroxyquinoline, resorcinol and 2-nitroso-1naphthol is chelating sorbent for concentration of trace elements from solution, derived by mineralization of medicinal herbs and vitaminmineral complexes. it is important to choose of optimal conditions of sorption concentration of trace elements from aqueous solutions. it was shown that the acidity is a significant parameter for the absorption of metal ions from solution and on the selectivity of determination of ni(ii) in the presence co(ii) and zn(ii). the optimal values of ph for sorption concentration of trace elements ions are studied in our work (fig. 2). the optimal time of sorption for co(ii), ni(ii) and zn(ii) ions is also defined. it is 60 minutes (fig. 3). the metrological characteristics of xrf determination of trace elements using chemically modified puf sorbents are presented in table 1. iraqi j pharm sci, vol.27(2) 2018 x-ray fluorescence determination of trace elements 4 figure 2. selection of ph for trace metal sorption concentration (intensity of signal is average for all types of sorbent). figure 3. selection of time for trace metal sorption concentration (intensity of signal is average for all types of sorbent). some other transition metals can also form the stable complexes with chemically modified puf, but it probably can’t interfere determination of zinc, cobalt and nickel in pharmaceutical objects because of excess of puf’s reaction groups and very low concentration of metals. proposed conditions make it possible to increase the sensitivity and linearity of range of the calibration curves by an order of magnitude as compared to existing hybrid sorption xrf techniques. analysis results of medicinal herbs and vitamin-mineral complexes. the proposed approach is applied for the analysis of several kinds of medicinal herbs (coltsfoot leaves, nettle leaves and yarrow herb) and vitamin-mineral complexes (alfavit, vitrum and multi-tabs). the results of co, ni and zn determination in some species of medicinal herbs and vitamin-mineral complexes are presented in tables 2 and 3. the obtained values of contents of trace elements in medicinal herbs in good agreement with the ones of aes analysis, conducted as prescribed in european pharmacopeia 9.0 (table 2). this fact confirms the adequacy of the proposed approach. the contents of trace elements in vitamin-mineral complexes correspond to the values declared by the manufacturer (table 3). table 1. metrological characteristics of xrf determination of trace elements using chemically modified puf sorbents type of sorbent element r² range of quantification, µg/ml sr(cн) puf, modified by resorcinol ni 0.985 0.5-2.5 0.04 zn 0.976 0.5-15 0.07 co 0.981 1.0-15 0.05 puf, modified by 8-hydroxyquinoline ni 0.997 0.3-30 0.06 zn 0.996 0.2-17 0.03 co 0.998 0.7-17 0.08 puf, modified by 2-nitrozo-1-naphthol zn 0.976 1-10 0.03 co 0. 985 0.7-17 0.08 iraqi j pharm sci, vol.27(2) 2018 x-ray fluorescence determination of trace elements 5 table 2. results of trace element determination in medicinal plants (µg/g, n = 5, p = 0.95) *1 puf, modified by resorcinol 2 puf, modified by 8-hydroxyquinoline 3 puf, modified by 2nitrozo-1-naphthol table 3. results of trace element determination in vitamin-mineral complexes (µg/g, n = 5, p = 0.95) *1 puf, modified by resorcinol 2 puf, modified by 8-hydroxyquinoline 3 puf, modified by 2nitrozo-1-naphthol conclusion developed technique of xrf determination of trace elements in medicinal herbs and vitamin-mineral complexes is simple and effective alternative to traditional used methods of atomic spectroscopy. unlike these methods, the proposed technique does not involve using of fuel (compared with aas) or expensive high-quality argon (compared with icp-aes). the sorption-xrf method is the optimal method of determination of trace elements. xrf method allows simultaneous multielement analysis of samples, but it has not very high sensitivity of determination. the advantages of hybrid sorption-xrf approach are rapidity, low cost, sufficient sensitivity due to preliminary sorption concentration of trace elements. references 1. sedehi m., behnampour n. s., golalipour m. j. deficiencies of the microelements, folate and vitamin b12 in women of the child bearing ages in gorgan, northern iran. jcdr. 2013; 7(6): 1102-1104. 2. kabata-pendias a. soil–plant transfer of trace elements—an environmental issue. geoderma. 2004; 122(2): 143-149. 3. tokalıoğlu ş. determination of trace elements in commonly consumed medicinal herbs by icp-ms and multivariate analysis. foodchem. (2012)134(4): 2504-2508. 4. brown r. j. c., milton m. j. t. analytical techniques for trace element analysis: an overview. trac. (2005)24(3): 266-274. 5. dmitrienko s. g., khatuntseva l. n., apyari v. v., zolotov yu. a. azo-coupling reactions of polyurethane foams and their applications in chemical analysis. chemanal (warsaw). 2005; 50(1): 327337. 6. kosa s. a., al-zhrani g., salam m. a. removal of heavy metals from aqueous solutions by multi-walled carbon nanotubes modified with 8-hydroxyquinoline. chemengjourn .2012; 181:159-168. 7. visser a. e. naphthol-and resorcinol-based azo dyes as metal ion complexants in aqueous biphasic systems. journchromatb: biomedsciapp. 2000; 743 (1-2):107-114. 8. minagawa t., tokeshi m., kitamori t. integration of a wet analysis system on a glass chip: determination of co(ii) as 2nitroso-1-naphthol chelates by solvent extraction and thermal lens microscopy. lab on a chip. 2001; 1(1):72-75. metal ion type of sorbent* vitrum® multi-tabs® classic alphavit® classic determined specification determined specification determined specification zn 1 14.7 ± 1.7 15 14.2 ± 1.5 15 16.2 ± 2.7 15 2 14.8 ± 1.5 14.1 ± 2.1 16.3 ± 1.8 3 14.9 ± 1.8 14.3 ± 1.9 15.8 ± 2.1 metal ions type of sorbent* coltsfoot leaves nettle leaves yarrow herb xrf aes xrf aes xrf aes zn 1 98 ± 8 101 ± 11 48 ± 2 41 ± 3 45 ± 1 48 ± 8 2 119 ± 18 47 ± 3 50 ± 4 3 111 ± 11 39 ± 5 38 ± 1 co 1 4 ± 1 4 ± 1 9 ± 1 8 ± 2 6 ± 1 6 ± 2 2 6 ± 1 8 ± 1 6 ± 1 3 5 ± 1 11 ± 2 8 ± 1 ni 1 7 ± 1 9 ± 1 4 ± 1 4 ± 1 3 ± 1 3 ± 1 2 10 ± 2 4 ± 1 2 ± 1 iraqi j pharm sci, vol.27(2) 2018 x-ray fluorescence determination of trace elements 6 9. huie c. w. a review of modern samplepreparation techniques for the extraction and analysis of medicinal plants analbioanalchem. 2002; 373(1-2):23-30. 10. oskolok k. v., monogarova o. v. x-ray fluorescence and atomic emission determination of cobalt in water using polyurethane foam sorbents. moscow university chembull.2011; 66(3): 179-183. 11. oskolok k. v., monogarova o. v., devyatkina e. d. direct x-ray fluorescence determination of mercury on polyurethane foam sorbents. moscow university chembull. 2012; 67(2): 78-81. 12. oskolok k. v., monogarova o. v., alov n. v. total reflection x-ray fluorescence determination of cobalt and mercury in water using preconcentration on a polyurethane foam sorbent. moscow university chembull. 2014; 69(4): 155157. 13. dmitrienko s. g., apyari v. v., kudrinskaya v. a., stepanova a. v. preconcentration of flavonoids on polyurethane foam and their direct determination by diffuse reflectance spectroscopy. talanta. 2012; 102:132-136. iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns doi: https://doi.org/10.31351/vol30iss1pp101-109 101 comparative study of liver function and rh blood group between both physiological and pathological neonatal jaundice ayia n. kamal*,1, ali f. hassan** * ministry of health and environment, department of health salah al-din , salah al-din ,iraq. **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract jaundice occurs in most newborn due to increase bilirubin concentration. jaundice is detected at first week after birth in approximately 60% of full-term neonates. a high level of bilirubin is neurotoxic and may cause neonatal kernicterus, auditory neuropathy or death. the article aims to compare the rh group compatibility, serum bilirubin (total and direct), serum albumin and several liver enzymes (alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase) between physiological and pathological neonatal jaundice. the study was conducted for one hundred neonates with jaundice divided into two groups, group one includes fifty newborns with physiological jaundice, group two includes fifty newborns with pathological jaundice. blood samples were taken from each patient, used to determine the rh group, serum bilirubin (total and direct, liver enzymes and albumin levels. in the present study, it was found that rh compatibility show a significant relationship between newborns with physiological and pathological jaundice (p<0.05). serum bilirubin (total and direct), serum albumin concentration and the liver enzymes were significantly higher when compare between newborns and pathological jaundice with newborns of physiological jaundice (p<0.05). these findings demonstrate that the newborns with pathological jaundice have significantly higher levels of the studied parameters (serum bilirubin, albumin and several liver enzymes) in comparison with those with physiological jaundice. keywords: neonatal jaundice, liver enzymes, bilirubin, albumin. من إنزيمات الكبد بين اليرقان البعض دراسة مقارنة بين مصل البيليروبين ومصل األلبومين و طفال حديثي الوالدةلألالفسيولوجي واليرقان المرضي **علي فارس حسن و 1*،أية ناظم كمال وزارة الصحة والبيئة ، دائرة صحة صالح الدين ، صالح الدين ، العراق * فرع االدوية والسموم، كلية الصيدلة، جامعة بغداد ، بغداد ، العراق ** الخالصة اليرقان خالل األسبوع تتم مالحظة .الطبيعي في الدم عن الحد البيليروبين زيادة مستوىينتج اليرقان في معظم حديثي الوالدة بسبب الى في االعصاب وقد يؤدي بب تسممتس زيادة تركيز البيليروبين في الدم تكون سامة حيث. حديثي الوالدة٪ من 60األول بعد الوالدة في حوالي تركيز األلبومين ، ) الكلي والمباشر( بيليروبينتركيز ال، rhألجراء مقارنة في توافق مجموعة .التلف الدماغي و اعتالل األعصاب السمعية أو الوفاة عينة دم منمئة تم جمع حيث في االطفال حديثي الوالدة بين اليرقان الفسيولوجي واليرقان المرضي الدمفي مصل تراكيز بعض انزيمات الكبد و طفل 50الثانية المجموعة ,ان الفسيولوجياليرقطفل يعانون من 50االولى المجموعة الى مجموعتينالمصابين باليرقان و تقسيمهم حديثي الوالدة alkaline)الكبد تركيز انزيمات، والمباشر الكلي البيليروبين مستوى ، rh توافقتحديد ل واجريت الفحوصات التالية اليرقان المرضي يعانون من phosphatase, alanine aminotransferase, aspartate aminotransferase (معنويةأظهرت الدراسة عالقة وقد األلبومين.تركيز كذلك و مستوى كذلك (p<0.05)يعانون من اليرقان المرضي والذينبين األطفال حديثي الوالدة المصابين باليرقان الفسيولوجي rhعند المقارنة بين توافق اليرقان المرضي عند ب لألطفال المصابين مرتفعة معنويا وتركيز انزيمات الكبد كانت، تركيز األلبومين الدم في مصل الكلي والمباشر البيليروبين .(p<0.05)اليرقان الفسيولوجيذوي مقارنتهم مع االطفال االلبومين والبيليروبين في (المؤشرات من هذهكيز أعلى اأن األطفال الذين يعانون من اليرقان المرضي لديهم تر وبذلك تم االستنتاج .مقارنة باليرقان الفسيولوجي(المتعددةمصل الدم وانزيمات الكبد االلبومين ،البليروبين ،انزيمات الكبد ، اليرقان الوليدي:المفتاحية الكلمات 1corresponding author e-mail: ayanazm1980@gmail.com received: 17/ 7 /2020 accepted:11 /10 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp101-109 iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns 102 introduction jaundice (hyperbilirubinemia) is an elevated level of the pigment bilirubin in the blood, causing yellow discoloration of the body tissue resulting from the accumulation of an excess of bilirubin in skin (1). the neonates jaundice is clinically diagnosed swhen total serum bilirubin level (tsb) above 5 mg per dl (2). bilirubin is produced from the catabolism of heme by heme oxygenase with liberation of iron, carbon monoxide and biliverdin. the biliverdin is reduced by biliverdin reductase to form bilirubin (3). the bilirubin produced is then transported to the liver in the conjugated form with plasma albumin. bilirubin conjugation occur in the liver by udpglucosyltransferase enzyme and this conjugation increase water solubility which ease the removal of bilirubin, conjugated bilirubin is discharged into the bile and intestine (4) . in the newborns, greatly of the conjugated bilirubin in the gut is decomposed back to unconjugated bilirubin by the action of the enzyme betaglucuronidase that is present in the intestinal mucosa. the reabsorptions of unbound bilirubin into the blood stream take place by the action of enterohepatic circulation, this leads more of bilirubin load to the already overloaded liver (5).severe hyperbilirubinemia (more than 20 mg/dl) that could possibly cause kernicterus and neurodevelopmental problems less than of 2% of neonatal infants may be affected(6). various risk factors increase the incidence of jaundice this includes preterm neonatal , low birth weight, hemolysis, sepsis, cephalohematoma or easy bruising, and only breastfeeding (7) . physiological jaundice is the one of more common type of neonatal jaundice, do not cause serious problems (8). physiological jaundice usually appears after at least 24 hours of birth, and peak after four or five days. it later disappears after about 2 weeks of life. the bilirubin associated with physiologic jaundice is the mostly unbounded, and its levels in serum do not excess 15 mg/dl(9).many clinical conditions can lead to the occurrence of physiologic jaundice in the neonatal like high bilirubin load of relative polycythemia, immature hepatic uptake, a shortened life span of red blood cell, and higher enterohepatic circulation(10). other type of neonatal jaundice is pathologic jaundice which is characterized by rapid onset of jaundice ( first 24 hours after delivery), the rapid elevation of total serum bilirubin level (elevate of more than 5 mg/dl/day), sand a total serum bilirubin concentration more than (17 mg/dl) in a full-term infant (7) frequently happens as a result of isoimmunisation (mostly abo or rhesus incompatibility) or another reasons of large hemolysis (11) . the regimens is used to treat jaundice: phototherapy (12), exchange transfusion this is used to remove antibodies that are causing hemolysis and is used in rh iso-immunization and abo incompatibility, drugs therapy of neonatal hyperbilirubinemia may be classified into phenobarbitone(13),intravenous immunoglobulin (ivig)(14) and metalloporphyrins (15) . patients and methods table 1. list of reagents, their companies, and countries material company country alanine transaminase (alt) kit biosystems spain albumin kit biolabo france alkaline phosphatase (alp) kit biosystems spain aspartate transaminase (ast) kit biosystems spain direct serum bilirubin linear spain patients’ selection a cross-sectional study of 100 newborns with jaundice, diagnosed by pediatrician. they were selected from the salah al-din general hospital in tikrit city and al-alwiya children's hospital in baghdad. according to the pediatrician diagnosis and biochemical tests, the newborns allocated into two groups group 1: 50 newborns with physiological jaundice group2: 50 newborns with pathological jaundice the 100 neonate undergo the followings information were recorded, that data categorized into: 1-pateints demographic data which include (sex of newborns, weight, age, gestational period, residency, source of pollution such as presence of gas station, factory, high way road and the job of mother) 2-clinical examination which includes (onset of jaundice, state of newborn, feeding status, bowel motion, level of yellowish discoloration, moro reflex, sucking reflex and gasping reflex) 3-laboratory data which include (rh group, total serum bilirubin, direct serum bilirubin, serum albumin, alt, ast and alp). blood sampling blood sample (1 ml) taken from each patient and put in to serum separator tube then centrifuged to isolate the serum which used for measurements of direct serum bilirubin, liver function test (alt, ast and alp) and albumin concentration. liver function test (alt, ast and alp) and albumin concentration done in private iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns 103 laboratory meanwhile other tests achieved in hospital laboratory. biochemical assay determination of rh group by slide method rh antigens, termed for the rhesus monkey in which they were first determined, are as well as surface antigens noted on erythrocytes. there are certain rh antigens (prevalent one is named d). red cells showing the rh antigens are termed rh positive. red blood cells which do not exhibit this surface antigen are rh negative (approximately 15% of the people are rh negative)(16) . procedure take pre-warm a clean glass slide to 40-50 c on a lighted view box and then place one drop of immucor anti-d series 4 (monoclonal blend) on the slide and add one drop of blood sample to be tested to drop of anti d. using a clean applicator stick, mix the blood -reagent mixture over an oval area of approximately 20 mm x 40 mm. rock the view box back and forth and observe for macroscopic agglutination for a period not to exceed 2 minutes. positive test: agglutination of red blood cells at the immediate spin, or 37 c or antiglobulin phases. negative test: no agglutination of red blood cells at any test phase(17) . measurement of total bilirubin. the measurement of serum total bilirubin is achieved by using dual wavelength total bilirubin meter (apel br-501) device which is use a dual wavelength (461 nm, 551 nm), this device minimizes the interference of hemolysis and turbidity (18) . procedure 50-60 μl of whole blood sample collected by heparinized capillary tube and measure directly by dual wavelength total bilirubin meter device and record the result. measurement of direct serum bilirubin bilirubin is transformed to colored azobilirubin by diazotized sulfanilic acid and is calculated photometrically. the two bilirubin parts in serum-bilirubin glucuronide and free bilirubin which is conjugated to albumin-only the former reacts directly, free albumin reacts after being removed from protein in an accelerator (19) . procedure mix thoroughly 100µl of serum sample with 1 ml of working reagent and let tubes stand for exactly 5minute at 37c.read the absorbance (a) of the samples blanks at 540 nm against distilled water and read the absorbance (a) of the samples at 540 nm against the reagent blank by biosystem bts 350 device. measurement of alkaline phosphatase (alp) the action of alkaline phosphatase (alp) enzyme in alkaline medium is the removal of the phosphate group from 4-nitrophenylphosphate to diethanolamine (dea), releasing 4-nitrophenol. the amount of 4-nitrophenol production which determine the catalytic concentration, calculated at 405 nm (20). 4 -nitrophenyl phosphate + dea 𝐴𝐿𝑃 → dea – phosphate + 4 –nitrophenol procedure bring the working reagent and the instrument to reaction temperature and then mix 1ml of reagent a, 200 µl of reagent b with 20 µl of serum sample into a cuvette after that insert the cuvette into the spectrophotometer, start the stop wash and record initial absorbance and at 1 minute intervals thereafter for 3 minutes by biosystem bts 350 device. calculate the difference between consecutive absorbance, and the average. calculations • ∆ a= final absorbance – initial absorbance •(∆a/min)×2764=iu/l absorbance difference per minute (∆a/min). measurement of alanine aminotransferase (alt). alanine aminotransferase (alt) stimulate the transport of the amino group from alanine to 2oxoglutarate, producing pyruvate and glutamate. the catalytic concentration is evaluated from the value of decrease of nadh, by the mode of the lactate dehydrogenase (ldh) conjugated reaction (21) . alanine + 2 – oxoglutarate 𝐴𝐿𝑇 → pyruvate + glutamate. pyruvate + nadh + h+ 𝐿𝐷𝐻 → lactate + nad+. procedure bring the working reagent and the instrument to reaction temperature and then mix 1ml of reagent a, 200 µl of reagent b with 100 µl of serum sample into a cuvette, insert the cuvette into the spectrophotometer, measured at 340 nm, start the stop wash. after 1 minute, record initial absorbance and at 1 minute intervals thereafter for 3minutes by biosystem bts 350 device. calculate the difference between consecutive absorbance, and the average absorbance difference per minute (∆a/min). calculations  ∆ a= final absorbance – initial absorbance  (∆a/min)×3333=iu/l measurement of aspartate aminotransferase (ast/got) the formation of oxalacetate and glutamate by the aspartate aminotransferase (ast or got) enzyme by activated the transport of the amino group from aspartate to 2-oxoglutarate. the catalytic concentration is estimated from the value of diminish of nadh by means of the malate dehydrogenase (mdh) coupled reaction, measured at 340 nm (21) . iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns 104 aspartate + 2 oxoglutarate 𝐴𝑆𝑇 → oxalacetate + glutamate. oxalacetate + nadh + h+ 𝑀𝐷𝐻 → malate + nad. procedure bring the working reagent and the instrument to reaction temperature and then mix 1ml of reagent a, 200 µl of reagent b with 100 µl of serum sample into a cuvette, insert the cuvette into the spectrophotometer, measured at 340 nm, start the stop wash. after 1 minute, record initial absorbance and at 1 minute intervals thereafter for 3 minutes by biosystem bts 350 device. calculate the difference between consecutive absorbance, and the average absorbance difference per minute (∆a/min). calculations  ∆ a= final absorbance – initial absorbance  (∆a/min)×3333=iu/l measurement of serum albumin the bromocresol green in buffered solution at ph 4.2 binds albumin to produce a colored compound which absorbance, calculated at 630 nm (620-640) is relative to albumin concentration in the sample (22). procedure mix well 5 ui of serum sample with 2.5ml of reagent. record absorbance at 630 nm (620-640) within 3 minutes against reagent blank or better after exactly 1 minute by spectrophotometer pd303(apel japan). calculations  ∆ a= final absorbance – initial absorbance.  concentration of albumin = (∆ a sample/∆ a standard) ×concentration of standard (5g/dl) statistical analysis the entire results were demonstrated as mean± standard deviation (sd). the statistics were evaluated by computerized statistical package for the social sciences spss program. unpaired student t-test was achieved for each group pair includes a comparison between two groups-values (p<0.05) is express to be statistically significant. a chi-square test is used to determine the statistical significance in the distribution between different various variables. ethical consideration all the administrative agreements were taken from the parents, the administrative staff of the hospital including the managing director, and the responsibility of the departments. results in table (2), according to the (age of newborns, weight of newborns, the duration of gestation of newborns) there are no significant differences when compared between the newborns with pathological jaundice and physiological jaundice (p-value >0.05). as regard to the gender of newborns, residency and source of air pollution, no significant relationship when comparing newborns with physiological jaundice and newborns with pathological jaundice (p-value >0.05), meanwhile according to the job of mothers, there is a significant relationship between the job of the mothers and both types of newborn jaundice (p-value <0.05). table 2. demographical information of newborns with physiological jaundice and pathological jaundice. characters newborns with physiological jaundice newborns with pathological jaundice p-value age of newborn (hours) 131.2±48.8 115.1±38.8 0.073a weight of newborn (kg) 2.76±0.79 2.92±0.37 0.214a gestational durations (weeks) 36.1±3.3 36.6±1.5 0.246a gender male 27 (54%) 22 (44%) 0.3172b female 23 (46%) 28 (56%) residency rural 29(58%) 26(52%) 0.5464b urban 21(42%) 24(48%) sources of air pollution positive 22(44%) 27(54%) 0.328b negative 28(56%) 23(46%) job of mother employee 7(14%) 1(2%) 0.0269b housewife 43(86%) 49(98%) n=50 newborn for each group superscript a refer to t-test biostatic analysis, in which p-value <0.05 mean significant differences between the means of test groups superscript b refer to chi-square biostatic analysis, in which p-value <0.05 mean significant relationship between the test groups iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns 105 table 3 represents the onset of jaundice and physical examination findings, there is a significant difference between the onset of physiological jaundice and pathological jaundice (77.28±27.9 vs23.56±41.5) (p-value <0.05). according to the general status, feeding status, the stage of yellowish discoloration and opisthotonus status of newborns there is a significant relationship when comparing recently born babies with physiological jaundice and newborns with pathological jaundice (p-value <0.05) , meanwhile the bowel motions , moro reflex, sucking reflex and grasping reflex of newborns show no significant relationship between infants with physiological jaundice and infants with pathological jaundice (p-value >0.05). table 3.clinical examinations of newborns with physiological and pathological jaundice n=50 newborn for each group superscript a refer to t-test biostatic analysis, in which p-value <0.05 mean significant differences between the means of test groups superscript b refer to chi-square biostatic analysis, in which p-value <0.05 mean significant relationship between the test groups * yellowish discoloration stages, stage i only the face and serum bilirubin range was (4-6mg/dl) , stage ii reach to the abdomen and serum bilirubin range was (8-14mg/dl), stage iii reach to the extremities and serum bilirubin range was (15-20mg/dl)(23) . table 4 represents the measured biochemical parameters ( bilirubin, alt, ast, alp, albumin and rh factor). there was significant differences (p <0.05) in the levels of total serum bilirubin, direct bilirubin, alp, alt, ast and serum albumin between the 2 groups of jaundiced infants. there is a significant difference if compare between the rh compatibility of newborns with physiological jaundice and newborns with pathological jaundice (p-value <0.05). characters newborns with physiological jaundice newborns with pathological jaundice p-value onset of jaundice(hours) 77.28±27.9 23.56±41.5 0.615a status of newborn active 36(72%) 7(14%) 4.69×10-9b lethargic 14(28%) 43(86%) feeding good 32(64%) 19(38%) 0.009308b poor 18(36%) 31(62%) bowel motion positive 44(88%) 45(90%) 0.749271b negative 6(12%) 5(10%) yellowish discoloration* stage i 21(42%) 3(6%) 1.123×10-15b stage ii 28(56%) 31(62%) stage iii 1(2%) 16(32%) moro reflex positive 48(96%) 44(88%) 0.140369b negative 2(4%) 6(12%) sucking reflex positive 48(96%) 45(90%) 0.239678b negative 2(4%) 5(10%) grasping reflex positive 48(96%) 46(92%) 0.39912b negative 2(4%) 4(8%) opisthotonus status positive 0(0%) 6(12%) 0.01152b negative 100(100%) 44(88%) iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns 106 table 4. biochemical assay of newborns with physiological jaundice and newborns with pathological jaundice. parameters * newborns with physiological jaundice newborns with pathological jaundice p-value total serum bilirubin (mg/dl) 10.11± 3.6 14.34± 4.6 2.443×10-6 direct serum bilirubin (mg/dl) 6.36± 2.54 9.82± 2.4 7.24×10-6 alkaline phosphatase (alp)(iu/l) 284.57± 50.8 388.71± 52.7 0.002728 alanine aminotransferase (alt/gpt)(iu/l) 13.2± 3.49 27.37± 2.64 4.28×10-6 aspartate aminotransferase (ast/got)(iu/l) 34.12± 6.1 62.62± 9.47 0.00516 serum albumin concentration (g/dl) 3.37± 0.25 3.83± 0.41 0.0201 rh compatibility compatible 50(100%) 44(88%) incompatible 0(0%) 6(12%) *normal values for tsb in newborn less than 2 mg / dl, normal value for direct serum bilirubine in newborn 0.1-0.3mg/dl, normal value for alp in newborn is 150-420 iu/l, normal value for alt in newborn is13-45 iu/l , normal value for ast in newborn is 30-150 iu/l and normal value for serum albumin in newborn is 3-4.3g/dl discussion all newborns are at risk of jaundice; most cases of jaundice will have a good prognosis with prompt intervention. bilirubin has certain antioxidative effects; an appropriate concentration of bilirubin might have a protective effect within normal value (24). however, if the concentration of unconjugated bilirubin is too high, and the binding capacities of plasma proteins are limited, free bilirubin might raise and pass through the newborn blood-brain barrier and have toxic complications, producing in permanent neurological injury (bilirubin encephalopathy) (25). table (2) has showed that, there is no significant differences when comparing the age, weight, gender, gestational age, sources of air pollution and residency of newborn with both types of newborn jaundice (p>0.05), this indicates that these factors did not have a significant effect on incidence of different types of jaundice (pathological – physiological). a previous study had been studied the influence of these factors on the jaundice in newborns, they found that gestational age range between 35-36 weeks considered as a major risk factor (26) this finding is a match with the present study results in which both groups have gestational age about 36 weeks beside it seems that gestational age had no effect on the incidence of either type of jaundice. other study found that gender has an influence on the incidence of jaundice in newborns, they found that male babies have minor risk compared to female (27) this finding was counteracted with the present study in which there is no significant influence of gender on the incidence of both type of jaundice. air pollution and residency had no significant effect on the incidence on both types of jaundice in newborns meanwhile a previous study show that air pollution causes an increase in incidence of jaundice in newborns without mention which type of it (28). table (3) has showed that there is a significant difference when compared between the onset of jaundice in newborns with physiological jaundice and newborns with pathological jaundice; the appearance of pathological type is within 24 hrs. while the appearance of physiological jaundice after 24 hrs. of birth this finding is compatible with previous studies which found that physiologic jaundice in newborn mostly occurs on days 2 to 4, peaks between 4 to 5 days, and disappears in two weeks. physiologic jaundice does not appear in the first 24 hours (29). another study shows that physiological jaundice usually appears after at least 24 hours of birth, and peak after four or five days. it later disappears after about 2 weeks of life (30). appearance of pathological jaundice in newborns during 24 hours, the levels of peak total serum bilirubin more than the expected normal value (31)(32).according to the general status of newborn, the patients with pathological jaundice more lethargic and less active in compare to patients with physiological jaundice, regarding to the feeding status of newborn, the patients with pathological jaundice more poor feeding if compared to patients with physiological jaundice and also according to opisthotonus status the patients with pathological jaundice more susceptible to opisthotonus state in comparatively with patients with physiological jaundice. this finding completely agrees with another a previous study conducted by shapiro sm iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns 107 in 2003, which found that newborns with severe hyperbilirubinemia which characterized by poor feed, high-pitched cry, abnormal tone (hypertonia and/or hypotonia), lethargy, retrocollis and opisthotonus, the sunset eye signs, seizures, and possibly death (33). in table (4), the total serum bilirubin levels of newborns with pathological jaundice were significantly higher than newborn of physiological jaundice. bryon j , et al in 2011 have been found that ,in physiologic jaundice the average total serum bilirubin level mostly peaks at 5 to 6 mg/dl on the third to fourth day after birth and then decrease over the 1st week of life. bilirubin level may reach of up to (12 mg/dl), can some time occur. newborns with many risk factors may suffer from an exaggerated form of physiologic jaundice in which the total serum bilirubin concentration may reach to 17 mg/dl. so, bilirubinemia more than this value is no longer consider as a physiologic jaundice (34). according to the direct serum bilirubin of newborn, there are significant difference between the direct serum bilirubin of newborns with physiological jaundice and newborns with pathological jaundice (p-value <0.05). several clinical studies have emphasized on the importance of measuring direct serum bilirubin for newborns with jaundice. they found that direct serum bilirubin has been increased more than 2 mg/dl is a which is a diagnostic feature for pathological jaundice(35).according to the rh compatibility of newborns, there is significant relationship between the rh compatibility of newborns with physiological jaundice and newborns with pathological jaundice this finding is compatible with previous study done by johnson lh, et al in 2002 which found the elevated production of bilirubin (like newborn with blood group and rh incompatibilities), insufficiency of hepatic uptake, impaired conjugation of bilirubin and raised enterohepatic circulation of bilirubin report for most cases of pathological jaundice in neonatal infant (36) . maisels mj in 2005 found that more production of bilirubin occurs in newborns with red cell-enzyme deficiencies, incompatibility of blood group, or structural deformity in red cells (37). other study conducted by blanchette vs in 1984 also show that the causes of raised bilirubin production in pathologic jaundice are immune-mediated hemolysis like incompatibility of abo, rh and nonimmune mediated causes like cephalhematoma, erythrocyte membrane defects such as hereditary spherocytosis and elliptocytosis, defect in enzymes such as pyruvate kinase deficiency and glucose-6phosphate dehydrogenase (g6pd) deficiency (38) . the results demonstrated that the concentration of alkaline phosphatase in newborns with pathological jaundice is significantly higher than that in newborns with physiological jaundice. alp is present in all cells including erythrocytes and is released to plasma after the cells destruction. accordingly, it hypothesized that it could be used as a primary marker of hemolysis of red blood cells and estimation of significant hyperbilirubinemia. alike to the current findings, other study utilized the alkaline phosphatase concentration in blood six hours after delivery. they establish that alp concentrations were significantly greater in newborns with jaundice need treatment, either by phototherapy or exchange transfusion (39) besides other study carried by mousa ak, et al in 2015 found that there is a the concentrations of cord blood alkaline phosphatase between the non-jaundiced and clinically jaundiced infants, and it was significantly larger in patients with hyperbilirubinemia need treatment. furthermore, the alp concentrations were significantly higher in infants whose serum bilirubin level arrived a level ≥ 10 mg/dl (40).other study also found that huge levels of alp in a patient with incompatibility of rh and hemophagositic syndrome ( 41). the serum concentration of (alt/ gpt) and (ast/got) in newborns with pathological jaundice were significantly higher than that of newborns with physiological jaundice. there is no pervious study linked between these two enzymes and jaundice in newborn but many previous study link the concentration of these enzymes with jaundice in adults in which they found that the elevation in the concentration of these enzymes occur in patients with different types of hyperbilirubinaemia if compare with control group, which may be as a result of defects in the liver function due to damage of hepatic cells, liver dysfunction biliary obstruction, viral infections, or any another deformities which cause releasing of these enzymes into the circulation, this study demonstrate that there were a significant rise of activities of ast and alt enzymes in patients with hepatic jaundice. generally got or gpt is evaluated as a marker of damage in liver (42). serum albumin concentration of newborns with pathological jaundice was significantly higher when compared with that of newborns with physiological jaundice this finding is compatible with previous study conducted by taksande a, et al in 2005 which found that physiological hyperbilirubinemia is a result of immature liver cell which have low uridine diphosphoglucuronosyl transferase activity when compared to mature hepatocyte, low concentration of albumin which is a bilirubin binding ligand and increased number of erythrocytes which have a shorter life span(43) . another previous study deduced by gaurav akc in 2017 which they found that cord albumin concentrations in a healthy term newborn used to predict the probability of the neonate having jaundice. it aids to determine the newborn that are at a more risk of developing hyperbilirubinemia. a value lower than 2.8 mg/dl has found to be more associated with clinical iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns 108 jaundice. so routine determination of cord albumin together with tsh can be achieved to keep path of at risk newborn (44).another previous study which found that jaundice and the following neurotoxic side effect of bilirubin on the brain is determined by numerous factors among these are inadequate oxygen supply, acidosis, the level of bilirubin in the brain, gestational age, the period of exposure and the level of albumin bound (45) and also bilirubin can pass through the brain if it is free or it is unconjugated, consequently if the serum albumin levels is low, the binding of bilirubin is compromised and the unconjugated bilirubin increase, may exposure the newborn for the danger of encephalopathy(46) conclusion these findings demonstrate that newborns with pathological jaundice have higher level of studied parameters (serum bilirubin, albumin and several liver enzymes) as compared with physiological jaundice . reference 1. schwarzenbach hr. jaundice and pathological liver values. praxis 2013;102(12):727-9. 2. sadeq tn ,feyad hf ,abdul hameed gr .risk factors of neonatal jaundice at al kadhimiya pediatrics hospital in baghdad. iraq .j alrafidain university college for 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in abo-incompatibility in the neonate. acta paediatr.2009; 98(12): 1896–1901. 12. mitra s , rennie j. neonatal jaundice: aetiology, diagnosis and treatment. british journal of hospital medicine.2017; 78(12): 699–704. 13. whitelaw a . postnatal phenobarbitone for the prevention of intraventricular hemorrhage in preterm infants. cochrane database syst rev .2001 ;(1). 14. cortey a, elzaabi m, waegemans t, roch b, aujard y. efficacy and safety of intravenous immunoglobulins in the management of neonatal hyperbilirubinemia due to abo incompatibility: a meta-analysis. archives de pediatrie.2014;21(9): 976-83. 15. kaseem lm, abdelrahim mea, naguib hf . investigating the efficacy and safety of silymarin in management of hyperbilirubinemia in neonatal jaundice. med sci.2013; 2(2): 575-590. 16. willy af. the genetics of the rhesus blood group system. blood transfus 2007; 5: 50-57. 17. fung mk, eder a, spitalnik s l. technical manual. 15th ed. bethesda, maryland, 2005 18. mishra s, chawla d, agarwal r, deorari ak, paul vk, et al. transcutaneous bilirubinometry reduces the need for blood sampling in neonates with visible jaundice. acta paediatr. 2009;98(12):1916-9. 19. peariman fc, lee rty. detection and measurement of total bilirubin in serum, with use of surfactants as solubilizing agents.clin.chem.1974;20(4):447-53. 20. the committee on enzymes of the scandinavian society for clinical chemistry and clinical physiology. recommended methods for determination of four enzymes in blood. scand j clin lab invest .1974; 33:291306. 21. ifcc reference procedures for measurement of catalytic concentrations of enzymes: corrigendum, notes and useful advice. clin chem lab med 2010; 48: 615-621. 22. tietz nw. text book of clinical chemistry,3rd ed. c.a.burties ,e.r. ashood, w.b. saunders(1999) p.482-485. 23. kevin c d. neonatal hyperbilirubinemia. merck manuals professional edition. 2015. 24. american academy of pediatrics clinical practice guideline: management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. pediatrics. 2004;114:297–316. 25. subspecialty group of neonatology, society of pediatrics, chinese medical association;chinese multicenter study coordination group for neonatal bilirubin encephalopathy. clinical characteristics of bilirubin encephalopathy in chinese newborn infants-a national multicenter survey. zhonghua er ke za zhi. 2012; 50: 331-5. https://www.amazon.com/s/ref=dp_byline_sr_book_3?ie=utf8&field-author=m.d.+spitalnik%2c+steven+l.&text=m.d.+spitalnik%2c+steven+l.&sort=relevancerank&search-alias=books iraqi j pharm sci, vol.30(1) 2021 physiological and pathological jaundice in newborns 109 26. newman tb, xiong b, gonzales vm, escobar gj. prediction and prevention of extreme neonatal hyperbilirubinemia in a mature health maintenance organization. arch pediatr adolesc med. 2000;154:1140–1147. 27. gale r, seidman ds, dollberg s, stevenson dk. epidemiology of neonatal jaundice in the jerusalem population. j pediatr gastroenterol nutr. 1990;10:82–86. 28. liqiang z, weiwei l, kun h, jintai l, changqing s, et al. air pollution exposure associates with increased risk of neonatal jaundice. nature communications.2019; 10(1);1-9. 29. pan dh, rivas y. jaundice: newborn to age 2 months. pediatr rev. 2017;38(11):499-510. 30. clarkson je, cowan jo, herbison gp. jaundice in full term healthy neonates--a population study. aust paediatr j. 1984;20:303-8. 31. bhutani vk, johnson l, sivieri em. predictive ability of a predischarge hour-specific serum bilirubin for subsequent significant hyperbilirubinemia in healthy term and nearterm newborns. pediatrics 1999; 103 : 6-14. 32. clemons rm. issues in newborn care. prim care 2000;27:251-67. 33. shapiro sm. bilirubin toxicity in the developing nervous system. pediatr. neurol. 2003; 29: 410–421. 34. bryon j, lauer md, nancy d, spector md. hyperbilirubinemia in the newborn. pediatrics in review. 2011;32(8):341. 35. sana u, khaista r, mehdi h. hyperbilirubinemia in neonates: types, causes, clinical examinations, preventive measures and treatments: a narrative review article. iran j public health. 2016; 45(5): 558– 568. 36. johnson lh, bhutani vk, brown ak: system based approach to the management of neonatal jaundice and prevention of kernicterus. j pediatr 2002;140(4):396-403. 37. maisels mj. jaundice in a newborn: answers to questions about a common clinical problem. first of two parts. contemp pediatr. 2005;22. 38. blanchette vs, zipursky a. assessment of anemia in newborn infants. clin perinatol. 1984;11(2):489-510 39. nalbantoglu a, ovali f, nalbantoglu b. alkaline phospha-tase as an early marker of hemolysis in newborns. pediatr int. 2011;53(6):936–8. 40. mousa ak, yadollah zp, mohsen h, zahra ar, alireza f, et al. cord blood alkaline phosphatase as an indicator of neonatal jaundice. iran j pediatr. 2015; 25(5): e718 . 41. yılmaz s, duman n, ozer e, kavas n, oren h et al. a case of rhesus hemolytic disease with hemophagocytosis and severe iron overload due to multiple transfusions. j. pediatr. hematol. oncol. 2006; 28: 290–92. 42. aydin sa, wahbi as, fatin am. changes in activities of alkaline phosphatase and transaminases in jaundice. tikrit journal of pure science.2008;13(3):1-12 43. taksande a, vilhekar k, jain m, zade p, atkari s et al. prediction of the development of neonatal hyperbilirubinemia by increased umbilical cord blood bilirubin. ind med. 2005;9(1):5-9 44. gaurav aiyappa kc. cord blood albumin as a predictor of neonatal hyperbilirubinemia in healthy neonates. curr pediatr res 2017; 21 (2): 216-220. 45. bender gj, cashore wj, oh w. ontogeny of bilirubin-binding capacity and the effect of clinical status in premature infants born at less than 1300 grams. pediatrics.2007;120:1067-73 46. robertson af, karp wb, brodersen r. comparison of bilirubin concentration and bilirubin/albumin ratio with the bilirubin binding ability in neonatal serum. j pediatr 1998;132:343-344. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 68 the role of oxidative stress in lead poisoning bashier al-ubaidy*, dawsar k. al-khashali** , nawfal a. numan** receive d 20-9-2002 accepte d 24-4-2005 abstract to inves tiga te the re la tionship b etwee n increas ed lipid peroxid atio n, a nd the lowe ring o f bo th plas ma total p ro te ins and albumin in lea d-expos ed wo rkers , a nd the effe ctiv eness of a ntio xida nts (vit. c and e) in mod ulating oxid ativ e stress in tho se wo rkers. thirty male and females workers e mploye d in the iraq i storage ba ttery (age range 2 0-40 ye ars) were pa rticipating in this stud y. additio nally, 11 he althy subjec ts were se rv ed as hea lthy controls , with the sa me age ra nge comp ared to workers group, to a void the effects o f age variatio ns on the studied pa ra meters. blo od le ad lev els, e rythro cytes and p la sma mda, e rythro cyte s and p la sma gs h, total protein and albumin lev els in hea lthy controls a nd lea d-e xp ose d worke rs p re and po st-treatme nts with antioxid ant were mea sure d. co mpa riso n w ith he althy control gro up s re vea l 36 0% inc re as e in bloo d le ad le ve ls , 15 0% increa se in erythroc yte mda, 1 17% inc rea se in p la sma mda, 28 % d ecrea se in e rythro cyte gs h, 5 6% de creas e in plasma gsh, 13% de creas e in to ta l pla sma p rote in a nd 2 3% dec re as e in a lbumin lev els in le ad -expo sed w orkers. tre atment with a co mbination of antioxid ant vita mins (100 0 mg/da y vit. c and 200 mg/d ay vit. e) for one mo nth produced s ignifica nt red uction 12 % in lea d le vels, 54 % in erythroc yte mda, 53 % in p la sma mda; s ignific ant incre as e 41% in e rythro cyte gsh, 1 20% in plas ma gsh and 11% in plasma albumin lev els in comp aris on with pre-trea tme nt le vels . in co nclusion, there is a be neficial e ffec t o f antioxid ants o n the oxid ativ e stress parame ters that no t only related to the ir a bility to remo ve lea d from ta rget cells, b ut also as so ciated with a ntioxida nt pote ntia l for b olstering thiol a ntio xidant c apa city, a nd this makes the se v itamins a goo d candida te for therap eutic intervention in lea d poiso ning. الخالصة  ل مـن ن الكـم الهاـئ ـم رغم ال ة٬ وـب ن السـابق ر السـني عـب ة كثف رة م ها بصو ست را كل التي تمت د ن المشا سمم بالرصاص م ر الت عتب ي واهر السـمية لهـذه ـظ ر مـن ال ر الكثـي ى تفسـي عـل وفة غير قادرة معر ة التسمم ال كي ميكاني ن ع٬ فا موضو هذا ال ة حول راكم مت ومات ال عل الم ات ال .المـادة رـي دى النظ ـح واســطة إ ي المحـدث ب كسـد هــاد التأ رح أن اإلج مي للرصـاص٬ تقـت أثير الســ ة الـت ي تبحـث فــي كيفـي متداولــة والـت مم هذا التس ن عراض الناتجة ع شوء العديد من األ هم في ن ور م ون له د ن يك مل أ لذا تمت دراسة المالونداي ألديهايد .الرصاص٬ من المحت )mda(وتا ن٬ والكل ة الدهو زناخ ر ل مؤش ر عتب ن ٬ الذي ي م )gsh(ثايو د ـت عية٬ ـق ألكسدة الطبي ع ا موان ومين٬ اللذان يعتبران من واأللب ٬ كسدة ع األ وان عطاء م راق٬ قبل وبعد إ ريات في الع طا ة لصناعة الب ن في المنشأة العام ملي عا وال ن للرصاص عرضي مال المت ها لدى الع فحص ربيك سكو مض األ حا من ول ) vit. c(والتي تتض وفير وك مـا ) vit. e(واأللفات والبالز حمـراء كريات الدم ال ة ختبار مدى قابلي كما تم ا ه علي ألكسدة موانع ا ر من ثم قياس مدى تأثي روجين و هيد كسيد ال محدث ببيرو هاد التأكسدي ال مة اإلج مقاو ائج الدراسـة .على هرت نـت أظ وى ست عرضين لل mdaوجود زيادة في م مال الم الزما لدى الع راء والب ريات الدم الحم من ك ظ في في كل ملحو خفاض مع ان رصاص٬ ومين واأللب ن ت الكلوتاثايو هيدروجين . مستويا سيد ال وك حدث ببير ي الم سد جهاد التأك ما لإل والبالز كريات ة ال سي سا وحظ زيادة في ح ك ل كذل ه عرضين ل مستوى الرصاص في الدم لدى العمال الم ي رتفاع ف ع ا رافقة م ي تم استخدامها أدت إلى . مت كسدة الت ع األ وان ي إن م سـن ـف تح ي ر الت معايي ميع ال ت ج جهـاد تم ر اإل را ماية للجسم ضد أضـ ر الح ة في توفي من إمكانية تأثيرها من الناحية السريري ز مما يعز دراستها عرض للرصاص ي الناتج عن الت كسد .التأ * dep ar tme nt o f to xicology, al-rasheed militar y hospital ** de partment of pharmac ology and toxicology, colleg e of phar mac y, university of baghdad ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 69 introduction lea d, s imilar to ma ny hea vy metals, is a co mplex toxin, exe rting numerous pa thophys io lo gic effe cts in many orga n systems (1). at the molecula r le vels, lea d interacts with biomolecule s and functio ns in different ways , like binding to numerous structural a nd enzymatic proteins (2), interfe re nce with me ta bo lic pa thways of mito cho ndria (3), a nd exhibiting mutage nic and ca rc inoge nic effects in mammalia n ce lls (4). oxida tive s tres s which re fe rs to a ce llular situation cha ra cterized by elev atio n o f the stud y state co nce ntra tions of rea ctiv e oxyge n sp ecies (ros ), a nd this could b e a p os sible co ntributo r to the p athogenes is o f lea d pois oning(5). s ome in-vitro and in-v iv o studie s showed a n elev ated p rod uc tion o f ros d ue to le ad trea tme nt (6,7,8), a nd increas ed lipid pe roxid ation a ss ociate d with alte re d antioxid ant de fe nse sys tems (9). the e ffec ts of lea d on the o xida tive stres s pa ra meters like glutathione (gsh), and malondialdehyd e (mda), sugges ts ros a s a pos sible c ontrib utor to ce ll damage due to lea d expos ure (10). the increa se in lip id p eroxid atio n during le ad p oiso ning were found to b e ac comp anie d by alteratio ns in the antioxid ant de fe nse system, inc luding dec re as ed gsh le ve ls in a ll b od y comp artments (11). this s tudy wa s co nducted to inv es tiga te the exte nt to which ros-re la ted pro ce ss es are invo lve d in le ad po is oning and the p os sibility of therap eutic inte rv ention with antioxid ant vita mins in this ca se. -: method andsubjact this study was ca rrie d out on wo rkers emp lo yed in the iraqi storage battery plant, and se le ctio n was made o n the ba sis that they must be directly involve d with lead e xpo sure , and ha ve bee n employed for at lea st 8 months, be fo re the inv es tiga tion were carrie d out. thirty, ma le a nd fema le s vo luntee rs (age ra nge 20-40 years) from the workers of the b attery plant, partic ip ate in this s tudy. eleve n hea lthy subjects s erved as hea lthy c ontrols, with the sa me age ra nge comp ared to worke rs gro up , to av oid the effects of age v aria tions on the stud ie d pa rameters. the a verage working time (hrs/da y) for e ac h worke r is 6 hrs , with a p erio d of e xpo sure to le ad ranging fro m 8 months to 2 8 yea rs . individual s ymp to m s urvey wa s pe rformed b y clinical and p hysica l ev aluation o f the wo rkers invo lve d in the stud y, c oncerning the prese nc e of le ad-as soc ia ted signs and symptoms for the purpo se o f prop er s elec tion. bloo d sample s (10 ml) were drawn by v ein puncture from ea ch subject p rior to starting trea tme nt with a ntio xida nts (as ba se line sample). after tha t, a ll s ubjec ts rece ive a comb ination of a ntioxida nt v ita mins (a sc orb ic acid 1 000 mg/da y a nd α-toc opherol 200 mg/day) orally for a p erio d of 4 wee ks , then sec ond blood s amp le was d ra wn fo r ev alua tion of the e ffec t of trea tment on the studied parame ters . blo od samples were plac ed into hepa rinized tubes a nd re frige rated until sepa ration of e rythrocytes and ana lysis. bloo d le ad lev els were mea sure d by grap hite fra nc e a tomic a bs orptio n s pe ctro pho to meter acc ording to the me thod of pars on et al (12). erythro cyte s a nd p la sma malondialdehyde (mda) le vel as ind ic ator o f lip id p eroxida tion were a sse ss ed utilizing thioba rb ituric ac id ass ay me thod o f stoc k and do rma nd y (13), and the susc eptibility of p la sma a nd e rythro cyte s to in-v itro hyd rogen p ero xide -ind uce d oxida tive stre ss wa s mea sured a cc ord ing to the method of gilb ert e t al (14), and the re sults we re exp re ss ed as nmole (mda)/gm hb, bas ed on the mo la r e xtinctio n c oeffic ie nt of mda (1 .5 6x1 05 m-1 cm-1). gluta thione leve ls we re determine d in e rythro cyte s and p la sma acc ording to the me thod of go din e t al (15). plasma albumin le vels we re determined utilizing a rea dy-ma de kit fo r this purp ose (ra ndo x co mpany, england) a cc ord ing to the metho d of douma s e t al (16). to ta l plasma protein was me asure d ac cording to the biuret metho d (17). erythroc yte he moglob in conce ntra tion wa s meas ured using drapkin’s re age nt metho d (18). statistic al a nalys is o f the data was d one using stude nt’s t-test, and p -v alues of les s than 0.05 were c ons id ered s ignifica nt. tab le (1) showe d a s ignifica nt e le vation in le ad le vels in blood of e xpo se d worke rs (3 60%) c ompa re d to c ontrols, pro duced 1 2% dec re as e in lea d le vels , which is a s ignifica nt value c omp ared with pre -trea tme nt le vels. the res ults of the s tudy ind ic ates tha t the b ase line e rythro cyte s and plas ma mda le ve ls we re eleva te d b y 1 50% and 11 7% res pec tive ly comp ared to that o f co ntro ls (ta ble 1). mda le vels in bo th co mpartment dec re as e a fter trea tme nt with a c ombina tion o f a ntioxida nt vita mins (1 00 0 mg/day a sc orbic acid and 200 mg/day (α-toco phe ro l) for o ne month, which was s ignifica nt, co mpa re d to the pretrea tme nt value s (table 1 ). the res ponse o f e rythro cytes and plas ma to in-v itro hydroge n p eroxide cha llenge showe d tha t, mda prod uction in both co mpartments of the e xp ose d wo rker’s blood we re s ignifica ntly highe r, c omp ared to that o f c ontrols. tre atment with antioxida nts as indicate d be fo re , signific antly increas e the ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 70 re sistance o f erythroc ytes a nd plas ma of le adexpos ed subjec ts to the hyd rogen pe ro xideinduced lip id peroxid atio n, refle cted by a significant de creas e in the mda p ro duc tio n after antioxid ant trea tment, c omp are d to pretrea tment le ve ls (figure s i a nd ii). table (1): bloo d le ad le ve ls, e rythro cytes and p la sma mda le ve ls in le a d-expose d wo rke rs pre and pos t-trea tme nts with v it. c and vit. e eac h value re pre s e nts me an ± se * signific antly diffe re nt from co ntrol (p<0.05 ). ¥ significa ntly diffe re nt with re s pe c t to pre tre a tme nt (p<0 .0 5). fig .1 ery throcyte mda of co ntrol subje cts a nd lead – e xpose d worke rs be for and afte r tre atme nt with vita mins (c&e)in re s ponse to in vitro cha lle nge with vario us h2o 2 co nce ntra tion fig.2 plas ma mda o f control subjec ts and le ad – ex pose d worke rs be for and afte r treatme nt with vita mins (c&e)in re spo nse to in vitro challe nge with va rious h2o 2 conce ntra tion in lea d-exp os ed worke rs , there was 28% and 56% d ep le tion in e rythro cytes a nd pla sma glutathione (gsh) lev els res pe ctiv ely, obs erved be fo re antioxid ant trea tme nt, and comp ared to co ntro ls (table 2). after trea tme nt with a ntio xidants for one mo nth, there were a significa nt incre as e in gsh leve ls in b oth c ompa rtments (4 1% and 12 0% re spe ctively) comp ared to pretre atment leve ls (ta ble 1). tota l plas ma protein and a lb umin, the general antioxid ants in the bo dy, we re fo und to be affe cted due to le ad expo sure, and their leve ls in lea d workers were s ignifica ntly dec re as ed (13% a nd 23 % re spe ctiv ely) comp ared to c ontrols. antioxid ant trea tme nt produced a significant elev atio n o n p la sma albumin only a fte r o ne-month dura tion of trea tme nt (ta ble 3). p arameter s co ntrol n =11 l ea d expo sure wo rk ers before treatme nt n=30 after treatment n=30 blood lead μ g/dl 10 ± 0 .64 46 .4 ±2 .07* 41.03 ± 1 .96* ¥ erythrocyte mda nmole/gm hb 7.7 ± 0.29 19 .81 ± 20* 9 .07 ± 0 .58* ¥ plasm a mda nmole/l 0.97 ± 0.08 2.11 ±0 .08* 1 .00 ± 0 .06 ¥ 0 40 80 120 160 0 1 2.5 5 7.5 h2o2 (m m) e ry th ro cy te m d a ( n m o le /g . h b) control before treatment after treatment 0 4 8 12 16 0 1 2.5 5 7.5 h2o2 (mm) p la sm a m d a ( u m ol e/ l ) control before treatment after treatment h2o 2 (mm) h2o 2( mm) ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 71 ta ble (2): ery throcytes a nd plas ma glutathione (gsh) le ve ls in le ad-e xpose d worke rs pre a nd po st-tre atme nt with vit. c and v it. e. eac h value re pre s e nts me an ± se * signific antly diffe re nt from co ntrol (p<0.05 ). ¥ significa ntly diffe re nt with re s pe c t to pre tre a tme nt (p<0 .0 5). table (3): plas ma to ta l prote in and albumin leve ls in le ad-ex pose d workers pre a nd pos t-trea tme nt wit h vit. c and v it. e. eac h value re pre se nts me an ± s e * signific antly diffe re nt from co ntrol (p<0.05 ). ¥ significa ntly diffe re nt with re s pe c t to pre tre a tme nt (p<0 .0 5). discussion free radica ls a ctiv ity ha s be en implic ated in the p athogenes is of a va riety o f huma n dise ase s and the analysis o f our data s howe d that, o xidative stres s wa s quite clea r in lea d expos ed wo rkers (ta ble 1, figure i a nd ii), a s no tice d by increa se d erythrocytes and plas ma mda le vels, which is in agre ement with other stud ie s (19, 20). the me chanisms b y whic h le ad ca use s it’s de le te rious e ffe cts has yet to b e elucida ted, ho weve r, part of le ad’s effect may be d ue to the ac cumulatio n of d elta -a minolev ulinic a cid dehydrata se (alad), an e nzyme in the heme synthes is pathw ay, which c atalyzes the condensa tion o f two mo le cule s o f -ala to porphobilinoge n(21) .at a ph range of 7.0-8 .0 -ala enolizes, and the re sulted eno l unde rgo autoo xida tion re sulting in the fo rma tion of sup eroxide a nd hydro xyl ra dica ls . ala has also be en s hown to undergo es iro n-ca ta lyze d oxida tion with ros ge ne ration, and to ind uce ca+2 relea se fro m mitoc ho ndria thro ugh oxida tive d ama ge to inne r me mbrane (22). the effec t of antioxid ant v itamins (a sco rb ic ac id a nd to cop herol) on lipid peroxid atio n parame te r, mda, as shown in ta ble (1) a nd figure i a nd ii, s ugges ted that they d id p rod uc e a de creas e in the ba sal mda le vels and the susc eptibility of b oth, erythrocyte s and plas ma to the oxid ativ e stress induc ed in-v itro b y h2 o2. the a ntioxida nt trea tme nt le ad to increas e in e rythro cyte s and plasma gsh le vels (tab le 2), whic h may be due to a d irec t sc ave nging a ctiv ities of the generated ros, a nd de crea sing utilizatio n and dama ge of gsh, or indirect through the imp ro veme nt o f the oxida nt/antioxida nt balance in the c ells a fte r tre atment (23). in no rma l co nditio ns , as we ll as during oxida tive stre ss (lead e xp osure ) a d aily d os e of asc orbic ac id a nd -to cop he rol, a pp ear to be protect the oxido -red uctive s ta te o f red blood cells, by modula ting the e xtent o f lipid peroxid atio n, a s well as the a ctiv ities of the antioxida nt enzyme s (24). in this s tudy, daily s up pleme ntatio n with a comb ination of asc orbic ac id a nd to cop he rol, re sulted in a s ignifica nt dec re as e in le ad leve ls in the blood a fter one month (tab le 1), and this may provide a n e conomic and convenient me thod of reducing blood lead le vels, po ss ib ly by d ecrea sing inte stinal abs orptio n of lea d (25), or it may inc re as e the re nal excre tio n of this me ta l. alb umin is kno wn to act a s a n e ffec tive antioxida nt, due to its a bility to b ind the catalytic co ppe r io ns (26), fre e fa tty ac id , and hyp ochlorus ac id (hocl), and a ls o showe d a signific ant ca pab ility to des tro y h2o2 in the prese nc e of red uce d glutathio ne (27). the pres ent study c le arly de mons tra te d the re la tionship betwee n inc re as ed lipid peroxid atio n, a nd the lowe ring o f bo th p la sma to ta l proteins and a lb umin in le ad -e xpo sed workers (tab le 3 ), which ma y be attribute d to the s tructural mod ifica tion, which may lead eve ntua lly to imp air the a ntio xidant prop erties of albumin, and eve n may a ct to ind uce oxida tive stress , thro ugh it’s action as a prooxid ant in pres enc e of ca talytic ions (28). parame te rs contro l n =11 le ad e xposure worke rs be fo re tre a t me nt n=30 afte r tre at me nt n=3 0 erythro cyte gs h μmo le /gm h b 11 .92 ± 0.36 8.62 ± 0.4* 12.1 7 ± 0 .37* ¥ pla sma gs h μ mo le /l 0 .88 ± 0.15 0.39 ± 0 .05* 0.86 ± 0 .12 ¥ parame te rs contro l n =11 le ad e xposure wo rke rs be fo re tre atme nt afte r tre atme nt total plas ma protein gm/dl 7.51 ± 0.11 6.56 ± 0.13* 6.6 ± 0.12* plas ma albumin gm/dl 5.06 ± 0.1 3.88 ± 0.05* 4.29 ± 0.11¥ * ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 72 antioxida nts trea tment re sulted in s ignific ant elev atio n in a lb umin le vels (tab le 3), whic h may b e due to the ir direc t s cav enging a ctiv ity, or p ro te ctio n of a lb umin agains t ros-induce d da mage . in c onc lusion, results of this s tudy s ugges te d that the b ene fic ia l effe cts of antio xidants o n the oxida tiv e s tres s pa ra meters are not o nly re la ted to the ir a bility to re move lea d from ta rget cells, but also as so ciated with antioxid ant po te ntia l for bolste ring thiol antioxid ant c ap acity, and this makes thes e vita mins a good ca ndida te for therape utic intervention in lea d poiso ning. reference 1. stolle ry, b.t.; bro ad bent, d.e.; banks, h.a.; and le e, w.r. short term pros pe ctiv e stud y of co nge stive functio ning in lea d worke rs . br. j . ind. med. 19 91, 46, 698 -7 07. 2. chalev elakis , g.; bouronikou, h.; yalouris, a.g.; et al. نa minolev ulinic a cid de hyd ra te s as a n index o f lead toxic ity. time fo r a rea ppraisa l? europe j. clin. inve st., 19 95, 25, 53 -58 . 3. verces i, .e.; cas tilho , r.f .; meinicke, a.r.; et al. oxida tive dama ge o f mito cho ndria induced b y 5-amino le vulinic ac id : ro le o f ca 2+ io ns and memb ra ne protein thio ls . biochem. biophys. acta., 199 4, 1 188 , 86 -9 2. 4. onuki, j .; me deiros, m.h.g.; bec hara, e.j.h. 5-aminolevulinic ac id induce d s inglestrand b re aks in p la smid pbr 3 22 dna in the pres enc e o f fe2+ io ns. bioc he m. bio phys. acta. 199 4, 122 5, 2 59-63 . 5. gurer, j.m. s.e., monta ro , k.j.: oxyge n free rad ic al generation in mice expo sed to low le ve l lead . f re e rad . biol., 19 99, 27, 130 -1 42. 6. mo ntario , h.p.; be cha ra , e.j.h; abdulla, d.s.p. free ra dica ls invo lv eme nt in ne urologica l porphyria s and lea d p oisoning. mol. ce ll bio chem., 1 991, 10 3, 7 3-83. 7. s andhir, r.; j ulka, d.; gill, k.d. lipop eroxidive d ama ge on lead tre atment in ra t brain and its implications o n me mbrane bound e nzymes . p harma col. toxico l. 19 94, 74, 66-71 . 8. s olliway, b.m.; s cha ffe r, a.; pratt, h.; yannai, s. effec ts of treatme nt to lea d o n se le cted bioc he mic al a nd hema to lo gical va riable s. p harmac ol. to xicol. 1 996 , 78 , 1822. 9. ito, y.; niiya , y.; kurita, h.; s hima; sarai.: se rum lipid p eroxide lev el a nd bloo d superoxid e dismuta se a ctiv ity in worke rs with oc cup atio na l treatme nt to le ad . int. arc h. occ up. env iron. he alth. 1 985 , 56 , 11 9-127 . 10. thom, s.r.; kang, m.; fisher, d.; et a l. relea se o f glutathione from erythrocytes and othe r markers of o xidative stress in carbo n mono xide po is oning. j . app l. p hysiol. 199 7, 82(5), 1 424 -32 . 11. burs ell, e.e and king, g.l. the p otential use o f glutathionyl hemoglo bin as a clinic al marker of o xidative s tres s. clin. che m., 200 0, 46(2), 1 45-6. 12. p arson, p.j .; sla vin, w.: a rapid ze eman graphite furna ce atomic a bso rp tion spe ctro metric me thod for determina tion o f lea d in b lo od. spe ctro chem. acta. 1 993 , 4 8 b, 9 25939 . 13. s to cks j. and dormandy t.l. the autoxid ation of huma n re d cell lipids induced by hydroge n pe ro xide. british j . he ama t. 197 1, 20, 95-11 1. 14. gilbert h.s., sta mp d. d. & roth e.f.: a metho d to c orre ct for errors c aus ed gene ra tion of interfe ring c omp ounds during lipid peroxid atio n. anal. bio che m. 1 984 , 13 7, 2 826. 15. go din, d.v. a nd wohaieb, s .a.: nutritio nal d efic ienc y, s ta rv atio n a nd tis sue antioxida nt status . free rad. bio l. med. 198 8, 54, 165 -7 6. 16. do uma s b.t., watso n w.a. & biggs h.g.: alb umin standards and the meas ureme nt of se rum albumin with b romocres ol gree n. clin. chim. acta. 1 971 ; 3 1: 8 7-8. 17. reinho l, j .g., in “standa rd metho ds of clinic al c he mis try” , re iner m (ed), ac ade mic pre ss , ne w york, , 195 3, vol. 1. pp: 88-9. 18. drap kin d.l. & aus tin j .h.: spe ctro pho to metric s tudies ii, prepa ra tion from wa she d bloo d ce lls. nitric oxide hemo glob in a nd s ulfhemo globin. j.biol. chem. 193 5, 1 12, 51-65 . 19. j anero, d.r. ma lo ndialde hyde and thio barbituric ac id re ac tivity a s diagno stic indices of lip id p eroxida tion and p eroxida tive tiss ue injury .free ra d .bio l .med .19 98, 9,515 – 540 . 20. j endryczko -a. inv olvement o f free ra dicals in lead po is oning. med-pr. 199 9, 45(2), 1 71-5. 21. riba ro v, s.r. a nd boc hev , p.g. le adhae moglo bin inte ra ction a s a po ss ib le s ource of re active oxygen s pec ie s – a chemilumines ce nt study. arch. bio che m. biophys . 199 8, 21 3, 288 -2 92. 22. ote iza, p .i. a nd bechara. 5-a minolev ulinic acid induce s lipid pe ro xidation in ca rd io lipinrich lipo somes. arch. bioc he m. bio phys. 199 3, 3 05, 282 -2 87. 23. ne uzil-j, thoma s-s. r, s to cke r-r. require ment fo r promo tion, or inhibition by لto cop he rol o f rad ic al induce d initia tion of plasma lip op rote in lipid peroxid ation. free rad. biol. med . 19 97 , 22 , 57 -7 1. 24. bowry-v. w, mohr-d, cleary-j, stoke rr. prev ention of to cop herol mediated ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 73 pe roxid ation in ubiquino l-10 -free human low de nsity lipo protein. j -c he m. 19 95, 270 , 57 5663. 25. dowso n, e.b; eva ns , d.r.; harris , w.a.; et al. the e ffec t of as corbic ac id s suppleme ntation on the bloo d le ad le ve ls of smo kers . j. am. co ll. nutr. 1 999 , 18 (2 ), 1 6670. 26. nelson-j j, dua nping-l, sharre tt-ar, e t a l. serum a lb umin le ve l as a p re dictor o f incide nt coronary hea rt dis eas e. am. j . epidemiol. 200 0, 1 51, 468 -7 7. 27. cha m.k , kim i.h. glutathione linked thiol p eroxid es ac tivity of huma n se rum albumin b lo od. biochem. biophys . re s. commo n. 1 996 , 22 2(2), 61 9-25. 28. bourdon-e, la re au-n a nd blac he-d. glucos e and free rad ic als impair the antioxida nt prope rties of s erum a lb umin. faseb-j . 19 99, 13 (2) 233 -4 4. iraqi j pharm sci, vol.26(1) 2017 meloxicam nanoemulgel 9 formulation characterization and evaluation of meloxicam nanoemulgel to be used topically hayder k. drais *, 1 and ahmed a. hussein ** * ministry of health and environment, babylon health directorate, babylon, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract meloxicam is a non-steroidal anti-inflammatory drug. it is practically insoluble in water. it is associated with gastrointestinal side effects at high doses on long term treatment. the aim of this investigation to formulate and evaluate gellified nanoemulsion of meloxicam as a topical dosage form to enhance meloxicam therapeutic activity and reduce systemic side effect. the pseudo ternary phase diagrams were made, including the oil mixture which is composed of almond oil and peppermint oil at a ratio (1:2), variable surfactant mixture (s mix) which are tween 80 and ethanol at ratios of (1:1, 2:1, 3:1, and 4:1) and double distilled water. slow dripping of double distilled water to the combination of the oil mixture and smix was performed to get nanoemulsion. the pseudo ternary phase diagram that has a greater nanoemulsifying area was contained smix ratio (3:1) from which seven formulas of nanoemulsion (ne1-ne7) were taken for characterization of prepared nanoemulsions and to prepare nanoemulgel formulas (nf1-nf7). the seven nanoemulgel formulas were subjected to various evaluations. nf1 was the selected formula and investigated for its stability and morphology. atomic force microscopy (afm) indicated that the optimized formula (nf1) was nanosized particle nearly spherical in shape and smooth surface globules, this indicates stability of optimized nanoemulgel (nf1). it can be concluded that the selected formula (nf1) is an effective alternative for the topical delivery of meloxicam. keywords: meloxicam, nanoemulgel, arthritis. تحضير وصياغة وتقيين هالم المستحلة النانىي للميلىكسيكام لألستخذام المىضعي دريسحيذر كاظن ، *1 و احمذ عباس حسين ** * انعشاق. ،بببم ،دائشة صحت بببم ، وصاسة انصحت وانبٍئت ** .انعشاق،فشع انصٍذالٍَبث ، كهٍت انصٍذنت ، خبيعت بغذاد ، بغذاد الخالصة انًٍهىكسٍكبو هى يٍ األدوٌت غٍش انسخٍشوٌذٌت انًضبدة نالنخهبببث. أَه غٍش لببم نهزوببٌ عًهٍب فً انًبء. وٌشحبظ يع اَثبس عالج نفخشة طىٌهت.انهذف يٍ هزا انبحث هى ححضٍش وصٍبغت وحمٍٍى هالو انًسخحهب نهاندبَبٍت انًعذٌت انًعىٌت فً اندشعبث انعبنٍت و كشكم دوائً يىضعً نخحسٍٍ انُشبط انعالخً وحمهٍم اَثبس اندبَبٍت نهًٍهىكسٍكبو.انذلٍك نهًٍهىكسٍكب (، وخهٍظ يٍ يىاد 2: 1ولذ حى سسى انًخطظ انثالثً األطىاس وانًكىٌ يٍ يضٌح انضٌج انًكىٌ يٍ صٌج انهىص وصٌج انُعُبع بُسبت ) (، وانًبء انًضبعف انخمطٍش.ببنخمطٍش انبطىء 1: 4، و 1: 3، 2: 1، 1: 1و اإلٌثبَىل فً َسب ) 08يمهم انشذ انسطحً وهى حىٌٍ نهًبء انًضبعف انخمطٍش انى خهٍظ يٍ يضٌح انضٌج وخهٍظ يمهم انشذ انسطحً حى انحصىل عهى انًسخحهب انذلٍك. حى اخخٍبس انًخطظ ش نخكىٌٍ انًسخحهب انذلٍك وحى ( يٍ خهٍظ يمهم انشذ انسطحً وانزي نذٌه يسبحت اكب1:3) ثالثً األبعبد انزي ٌحخىي عهى َسبت وانخً خضعج نًخخهف (nf1-nf7)صٍغ هالو انًسخحهب انذلٍك سبعت يٍ أخخٍبسيُه سبع صٍغ نخىصٍف انًسخحهب انذلٍك وإعذاد انصٍغت انًخخبسة وحى اخشاء أخخببس األسخمشاس وانشكم انسطحً. هً (nf1) انخمًٍٍبث. كبَج انصٍغت يسخمشة وحمشٌبب كشوٌت انشكم وسطح َبعى وهزا ٌؤشش اَهب راث خضٌئبث َبَىٌت انحدى أٌ انصٍغت األيثميدهش انمىة انزسٌت ٌشٍش إنى بذٌم فعبل نهخسهٍى انًىضعً نهًٍهىكسٍكبو.هً (nf1)إنى حذ كبٍش. َسخُخح أٌ انصٍغت انًخخبسة .التهاب المفاصل هالم المستحلة النانىي، الميلىكسيكام ، الكلمات المفتاحية : introduction the nanotechnology implementation in the domain of health sciences has great interest at the present time. the a noscale drug delivery system that has nano size particles or molecules can improve drug solubility and permeability due to nano oily droplets provide greater surface area for drug libration and faster absorption from biological barrier and this will lead to increase bioavailability of both hydrophilic and hydrophobic drugs that enhance the therapeutic efficacy of the drug (1) .meloxicam is a non-steroidal antiinflammatory drug utilized to relieve the symptoms of inflammatory arthritis such as (rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus) and degenerative arthritis such as osteoarthritis. it is a pale yellow powder, practically insoluble in water; very slightly soluble in alcohol (2) . 1 corresponding author e-mail: deeera2012@yahoo.com received: 12/4/2016 accepted: 29 /1/2017 iraqi j pharm sci, vol.26(1) 2017 meloxicam nanoemulgel 10 in spite of meloxicam preferentially activity that inhibits cox-2 (cyclooxgenease-2) over cox-1 (cyclooxgenease-1), in practice it still has higly disturbe gastrointestinal side effects at high doses on long term treatment (3) . therefore, there is a high requisiteness of to deliver meloxicam by another route to increase its solubility and modifying gastric adverse effect and delivers the drug to the inflammatory site. nanoemulsion (ne) which is nanocarrier delivery systems in transdermal drug delivery has great interest as a part of nanotechnology. they are optically transparent with the range of particle sizes between 100 to 500 nm. it is compose of the oil, surface active agent, co-surfactant and aqueous phase (4) . anyway, the low viscosity of nanoemulsion make it unsuitable for application to the human skin (5) . by use gelling agent with nanoemulsion, this make more convenient for transdermal application (6) . when gelling agent and nanoemulsion are utilized in combination form the dosage form is called nanoemulgel. most of the hydrophobic drugs cannot be incorporated directly into the gel base because the solubility problem that emerge during the drug release. nanoemulgel assist in the hydrophobic drugs merger into the oil phase and then oil droplets are dispersed in aqueous phase leading to oil in water (o/w) nanoemulsion then this nanoemulsion can be combined into gelling agent to get attractive delivery system which is nanoemulgel (7) . thus, the aim of this investigation is to formulate and evaluate gellified nanoemulsion of meloxicam used topically to increase solubility of meloxicam and decreasing the oral side effect of meloxicam that lead to enhance meloxicam local bioavailability , therapeutic activity and patient compliance. materials and methods materials meloxicam supplied by wadi alrafidian factory for pharmaceutical products(baghdad, iraq). almond oil, peppermint oil were purchased (bar-surloop grasse a. m franc), carbopol 940 was purchased (hengshui taocheng chemicals auxiliary co., ltd. china). tween 80 (sd fine chemlimited (sdfcl) mumbai, india). methanol and ethanol (grin land chemical comp, united). kh2po4 and na2hpo4 (merck & co., inc. germany). methods preparation of meloxicam nanoemulsion and construction of pseudo ternary phase diagrams the oil mixture was made by mixing two oils which are almond oil and peppermint oil in a ratio (1:2) to get oil mixture. the oil mixture, surfactant mixture (smix) and aqueous phase are components of the pseudo ternary phase plot that were selected by employing the aqueous phase titration technique. the surface active agent and cosurfactant was mixed in different weight ratios (1:1, 2:1, 3:1, 4:1) to get smix. the oil mixture that was loaded by meloxicam blend with smix in different weight ratios for each phase diagram plot, so that all areas of the pseudo ternary phase plot were covered. slow dripping of double distilled water to the combination of the oil mixture and smix was performed to know the borderline of phases and visual searches were made for transparency when the double distilled water was added drop by drop untill the nanoscale blend was a clear liquid to the eyes, then stop further adding double distilled water and different o/w nanoemulsions were formed. the pseudo ternary phase diagram was constructed. the drawn area of nanoemulsion represented by the shaded area and the greater area refer to better nano emulsifying activity. from pseudo ternary phase plot that has a wider area of emulsifying activity we took seven formulas to formulate nanoemulgel of meloxicam (8) . preparation of meloxicam nanoemulgel and gel the aqueous phase and oil phase were prepared. the aqueous phase was made by combining double distilled water and tween80. the oil mixture that was previously prepared represented the oil phase. the oil mixture was sonicated for 15 minutes. the oily phase was heated to 70°c while the aqueous phase to 80°c on a hot plate. the oil phase gradually was added to the aqueous phase with continuous stirring until cool . stirring continuously with magnetic stirrer at 2000 rpm and stirring was continued for 5-10 min until becoming unwarm at room temperature (9) . the gel bases were prepared by dispersing carbopol 940 with specified concentration in double distilled water with constant stirring at a moderate speed using mechanical stirrer. few drops of triethanolamine were added in preparation until get a ph of about 6.5. the nanoemulsion obtained was then mixed with the gel in (1:1) ratio to get homogenous nanoemulgel (10) . on the other hand, meloxicam gel 1 % (w/w) was prepared by dissolving meloxicam in a previously iraqi j pharm sci, vol.26(1) 2017 meloxicam nanoemulgel 11 prepared mixture of propylene glycol and ethanol (1:1). carbopol 940 was dispersed in distilled water under continuous shaking using a stirrer at 1000 rpm until it produce a homogenous dispersion. the carbopol 940 added to meloxicam solution 1% (w/w) with continuous stirring to get a homogenous dispersion of meloxicam gel. methylpapaben and propylparaben were used as preservatives. sodium sulphite function as antioxidant that prevent or slow the deterioration of formulas caused by chemical reactions with oxygen. the formulas of 100 g of meloxicam nanoemulgel and meloxicam gel were prepared for evaluation (9,10) . table( 1): the meloxicam nanoemulgel formulations. formulation code formulation components (%w/w) meloxicam gel nf7 nf6 nf5 nf4 nf3 nf2 nf1 1 1 1 1 1 1 1 1 meloxicam 1 1 1 1 1 1 1 1 carbopol 940 0 17.5 15 12.5 10 7.5 5 2.5 oil mix(1:2) 0 22.5 22.5 22.5 22.5 22.5 22.5 22.5 smix(3:1) 5 0 0 0 0 0 0 0 propylene glycol 5 0 0 0 0 0 0 0 ethanol 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 methylparaben 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 propylparaben 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 sodium sulphite 100 100 100 100 100 100 100 100 double distilled water up to characterization of the prepared meloxicam nanoemulsion droplet size measurement the samples of 5 ml nanoemulsion sonicated at the 37 o c for 30 min and measured using abt-9000 nanolaser particle size analyzer. the average of droplets size and droplets size distribution diagrams were obtained. all measurements were taken in triplicate (8) . poly dispersity index (pdi) assay it estimates the homogenicity of droplets size in nanoemulsion formulation. it can get by abt-9000 nanolaser particle size tester. the measurements were occuring in triplicate. pdi range from 0.0 to 1.0. as the polydispersity index value is closer to zero, the droplets are higher homogenous. the higher value of the poly dispersity value indicates the lowest homogenicity of droplets of nanoemulsion formulations (11) . evaluation meloxicam nanoemulgel physical analysis the formulated nanoemulgel formulations were visually tested for color, appearance and uniformity. the tested samples were occurred in triplicate (12) . ph estimation one of the high consequential parameters in the evaluation of nanoemulgels is the ph. the ph values affect on ionization of the drug i.e. ionized and unionized forms of the drug. the human skin is naturally acidic with a normal ph of 4-6. the women have been attested to have more acidic skin than men. the ph of all the nanoemulgel formulations was tested by dissolving 1g of nanoemulgel in 100 ml of distilled water and stored for 2h then use a digital ph meter at room temperature (13,14) . swelling index measurement the separation of formulation molecules of water molecules causes an expansion in volume of the mass called swelling index. it related to the area of application of nanoemulgel. one gram of meloxicam nanoemulgel formulations was wrapped with aluminium foil that is punched to produce holes and put in phosphate buffer ph 6.8 for 6 hours, then the swollen samples wiped with the filter paper to eliminate absorbed distilled water on the surface and then it was rapidly weighed on an electronic balance. the estimations were performed in triplicate. iraqi j pharm sci, vol.26(1) 2017 meloxicam nanoemulgel 12 the swelling index was measured by utilizing the following equation (eq): sw = [(wt – wo) / wo] × 100….. (eq1) where, sw = percentage of swelling index of meloxicam nanoemulgel wt = the weight(g)of the nanoemulgel at time t wo = initial weight (g) of the meloxicam nanoemulgel (15) . viscosity measurement the viscosity and rheology behavior of meloxicam nanoemulgel formulations were measured using ndj-55 digital viscometer by utilization a spindle no. 3 at room temperature. the measurements were taken in triplicate (8) . measurement of the drug content by uv-visible spectrophotometer utilization, the drug content was estimated (16) . one gram of the samples was mixed with methanol up to 100ml then take 1ml and read in the uv spectrophotometer after second dilution with methanol. the samples were made in three trails. the absorbance was observed at 263 nm and the percent of drug content was obtained by the following equation: % drug content = (analyzed content/theoretical content) x 100….. (eq2) in vitro release study a united states pharmacopeia (usp) dissolution tester apparatus ii is the device that was used to measure the in vitro release of meloxicam from formulations. one gram of meloxicam nanoemulgel formulations (nf1nf7) or meloxicam gel was put in a glass tube with 1.5cm diameter, covered with a cellulose acetate membrane which was formerly steeped in phosphate buffer of ph (6.8) for about 24 hours. the membrane adequately sealed and inverted under the surface of 900 ml of phosphate buffer of ph 6.8, containing 1% polysorbate 80, at 37 ± 0.5 °c with stirring speed of 100 rpm. the samples of 5 ml were withdrawn at 0,15,30, 60, 90,120, 150,180,210, 240 min and replaced with an equivalent quantity of dissolution medium. the drug content of the samples was estimated by a uv spectrophotometer at 263 nm. all samples were taken in three trails (17,18) . atomic force microscopy (afm) analysis the morphology of optimized meloxicam nanoemulgel formulation was obtained by afm angstrom advanced inc. aa3000 usa. atomic force microscopy analysis was performed by placing drops of the selected sample onto a glass slide and then estimated (19) . statistical analysis the results of the investigation were given as an average of three trail estimation of samples and use analysis of variance (anova) at level (p<0.05) for interpretation of the results (20) . results and discussion preparation of meloxicam nanoemulsion and construction of pseudo ternary phase the pseudo ternary phase plots were made by plotting oil mixture (1:2) with variable smix ratios as 1:1, 2:1, 3:1, and 4:1 and double distilled water as shown in figures (1-4). the shady part of pseudo ternary phase digram offer the region of nanoemulsion, whereas the nonshady part offer the region of emulsion. the pseudo ternary phase diagram that has a greater nanoemulsifying area was contained smix ratio (3:1) from which seven formulas of nanoemulsion (ne1-ne7) were prepared for characterization in order to prepare nanoemulgel formulas (nf1-nf7). figure (1): the pseudo ternary phase diagram of oil mixture of almond oil and peppermint oil in a ratio (1:2), smix (1:1) and double distilled water. figure (2): the pseudo ternary phase diagram of oil mixture of almond oil and peppermint oil in a ratio (1:2), smix (2:1) and double distilled water. iraqi j pharm sci, vol.26(1) 2017 meloxicam nanoemulgel 13 figure (3): the pseudo ternary phase diagram of oil mixture of almond oil and peppermint oil in a ratio (1:2), smix (3:1) and double distilled water. figure (4): the pseudo ternary phase diagram of oil mixture of almond oil and peppermint oil in a ratio (1:2), smix (4:1) and double distilled water. characterization of the prepared meloxicam nanoemulsion droplet size measurement the results of globule size range were ne1 (5-5.1nm), ne2 (5-6.29 nm), ne3 (6.297.92 nm), ne4 (11.1-15.8 nm), ne5 (15.822.3 nm), ne6 (25-28.1 nm) and ne7 (5.139.7 nm) that contained an oil mixture (%w/w) concentrations of 5, 10, 15, 20, 25, 30, 35 respectively. the outcomes refer to all the preparations had droplets in the nanometer size. analysis of variance indicated significant differences between droplet size values and the concentration (%w/w) of oil mixture where (p<0.05). poly dispersity index (pdi) assay pdi was from (0.011 to 0.188). the results of pdi referred to that nanoemulsion formulations had a high alliterative and constrict size distribution as shown in figure (5). figure (5): the poly dispersity index (pdi) of meloxicam nanoemulsion formulations (ne1-ne7). evaluation meloxicam nanoemulgel formulations physical analysis the physical appearance of the prepared nanomulgels formulas was yellow in color, transparent with excellent homogenicity as shown in table (2). ph estimation the ph of all the nanomulgels formulas was found to have a range between 5.45 to 5.88 as shown in table (2) which are within the normal ph value of human skin. table (2): the physical appearance, ph, percent of swelling index and percent of drug content in meloxicam nanoemulgel formulations. % drug content % swelling index ph phase separation uniformity clarity color formulation code 96.921 40 5.88 none excellent transparent yellow nf1 97.044 38 5.73 none excellent transparent yellow nf2 97.66 37 5.76 none excellent transparent yellow nf3 98.522 35 5.81 none excellent transparent yellow nf4 98.891 33 5.52 none excellent transparent yellow nf5 99.384 30 5.79 none excellent transparent yellow nf6 99.5 25 5.45 none excellent transparent yellow nf7 iraqi j pharm sci, vol.26(1) 2017 meloxicam nanoemulgel 14 figure (6): calibration curve of meloxicam in methanol. swelling index measurement the outcome described in the table (2). the results showed the effect of oil mixture concentration on swelling index at constant gelling agent concentration. as the oil mixture concentration increase that lead to create more hydrophobic media that retard water molecule from enterance to formula therefore it was found nf1 exhibited significantly higher swelling index that it contained the least oil mixture quantity while nf7 with higher oil mixture concentration had significantly lower swelling index (p<0.05). measurement of the drug content the drug content was in a range of (99.5 – 96.921%) as shown in table (2). these results were found within normal outline range. viscosity measurement the outcome of viscosity for meloxicam nanoemulgel (nf1-nf7) and meloxicam gel was found between (248 mpa 293 mpa). there is a significant difference (p<0.05) between formulations. with these results the formulas can removed rapidly from the site of application and provide good spreadability and application of formulations on the skin surface. when a viscosity plot against rpm figure (7) a pseudoplastic diagram will be obtained. it was indicated that increase oil mixture concentration at a constant smix concentration contributes to produce pseudoplastic system. figure (7): viscosity analysis of meloxicam nanoemulgel and meloxicam gel formulations in vitro release study by utilizing the united states pharmacopeia (usp) dissolution apparatus type ii, the analysis of drug release was done for the meloxicam nanoemulgel (nf1-nf7) and meloxicam gel formulations in phosphate buffer ph (6.8). the drug release diagram in figure (8) indicates that the nf1 was significantly highest in release rate while meloxicam gel formula has lower in dissolusion rate which was significantly lowest (p<0.05) . the comparability drug release profile of meloxicam nanoemulgel (nf1-nf7) and meloxicam gel show that the drug release was followed the descending order: nf1>nf2 >nf3> nf4>nf5>nf6>nf7> meloxicam gel. the drug release analysis from meloxicam nanoemulgel formulations (nf1nf7) showed the effect of oil mixture concentration at constant concentration of smix and gelling agent,as the concentration oil mixture increase this lead decrease drug release this may be due to the high concentration of oil mixture increase hydrophobicity of formulations and this will provide retarding forces for dissolution media to pass through hydrophobic matrix and liberate the drug also the meloxicam molecules will have greater diffusional pathway to reach the dissolution media therefore it was found nf1 has greater release profile compare to other of meloxicam nanoemulgel formulations due to it had the least concentration of oil mixture that make less retarding effect for dissolution molecules and lower diffusional pathway to drug molecules to reach the dissolution media. on the other hand, the meloxicam gel dissolution profile has lowest dissolution rate in comparison to other meloxicam nanoemulgels this due to that nanoemulsion iraqi j pharm sci, vol.26(1) 2017 meloxicam nanoemulgel 15 when present in nanoemulgels provide many advantages in comparison to gel such as nanosize droplets and enhance solubility of meloxicam hydrophobic drug this will enhance enterance of dissolution media to hydrophobic matrix and lower diffusional pathway for meloxicam molecules to reach the dissolution media that make meloxicam nanoemulgels superior and highly prefer in comparison to meloxicam gels (8, 21) . selection of the optimized formula from a study of the characteristics of nanoemulsion which are globule size analysis and pdi also from evaluation of nanoemulgel formulations through study physical appearance, ph, swelling index measurement, viscosity measurement, measurement of the drug content and in vitro release study, it was found that (nf1) is an optimized formula where it had lower globule size (5-5.1nm ) as shown in figure (9) , low pdi (0.037), excellent physical appearance, the normal value of ph (5.88 ), a higher percent of swelling index (40), encourage viscosity, accepted percent of drug content (96.921% ) and higher release rate makes it faster in relieving inflammatory disorder that improve therapeutic efficacy. the selected formula (nf1) subject for further analysis of atomic force microscopy (afm) study. figure (8): drug release profile for meloxicam nanoemulgel and meloxicam gel formulations. figure (9) the particle size distribution for nf1. atomic force microscopy (afm) analysis the outcome of afm indicate that the optimized formula (nf1) had nanosized particle nearly spherical in shape and smooth surface globules, this indicates stability of optimized nanoemulgel (nf1) as shown in figure (10). figure (10) the afm image of nf1 where scanning area is 5 μm * 5 μm. conclusion the aqueous phase titration method is a low energy emulsification method, easy and cheap that used in the preparation of nanoemulsion that made a nanoemulgel. the prepared meloxicam nanoemulgel formulations have a greater release rate when compared to meloxicam gel that indicate it is better than meloxicam gel. from the study of afm, the selected formula (nf1) with its nanoscale droplet has excellent stability that makes it promising formula to local and systemic delivery of meloxicam that lead to avoid side effect that associated with oral administration. the meloxicam nanoemulgel (nf1) with its attractive physical appearance and higher in vitro release rate enhance bioavailability that lead to increase therapeutic activity of meloxicam and improve patient compliance. iraqi j pharm sci, vol.26(1) 2017 meloxicam nanoemulgel 16 references 1. bhowmik debjit, chiranjib, chandira r.margret, jayakar b. role of nanotechnology in novel drug delivery system. journal of pharmaceutical science and technology, 2009; 1(1): 20-35 2. sweetman sean c, ed. martindale the complete drug reference. thirty-sixth edition. usa: pharmaceutical press; 2009. 3. villegas i, alarcon de la lastra c, la casa c, motilva v, martın mj. effects of food intake and oxidative stress on intestinal lesions caused by meloxicam and piroxicam in rats. eur j pharmacol, 2001;414:79–86. 4. gutierrez jm, gonzalez c, maestro a, sole i, pey cm, nolla j. nano-emulsions: new applications and optimization of their 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delivery. j chem pharm sci, 2015; 8:92-8. 21. mohamed mi. optimization of chlorphenesin emulgel formulation. the aaps j, 2004; 6: 1-7. iraqi j pharm sci, vol.29(2) 2020 new vanillic acid derivatives as antibacterials doi: https://doi.org/10.31351/vol29iss2pp129-138 129 synthesis, characterization, and antibacterial evaluation of new vanillic acid derivatives mostafa f. tawfeeq*,1 and ahlam j. qassir** * department of pharmacy, college of pharmacy, university of tikrit, salahuddin, iraq ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq abstract hydrazide schiff bases (hydrazones) and 2,5-disubstituted-1,3,4-oxadiazole derivatives exhibit diverse biological activities that include antibacterial, antifungal, antitubercular, antiviral, anticancer, anti-inflammatory, and analgesia; so that new derivatives, compounds (5-8) of vanillic acid based on 1,3,4-oxadiazole as scaffold unit were synthesized through multi-steps, and characterized by thin layer chromatography and spectroscopically by fourier-transform infrared (ftir) and proton nuclear magnetic resonance (1hnmr). compounds (5-8) were evaluated for their antibacterial activity by the disk diffusion method. compounds (5-8) were showed moderate and comparable to the activities of amoxicillin and isoniazid against escherichia coli (e. coli), but less than that of cefixime and nitrofurantoin which their activities were high. compound (6) and (7) had shown moderate and comparable to the activities of amoxicillin and cefixime against klebsiella pneumoniae (k. pneumoniae), but less than that of nitrofurantoin which its activity was high. compound (6) and (7) had shown moderate and comparable to the activity of amoxicillin against staphylococcus aureus (s. aureus), while the activities of cefixime and nitrofurantoin were high. compound (6) was moderately active against bacillus subtilis (b. subtilis), while the activities of amoxicillin, cefixime, and nitrofurantoin were high. keywords: hydrazide, schiff base, oxadiazole, antibacterial. وتقييم الفعالية المضادة للبكتريا لمشتقات جديدة لحامض الفانلين ، تشخيص ، تصنيع *و احالم جميل قصير 1*,مصطفى فايز توفيق . العراق ،صالح الدين ،جامعة تكريت ،كلية الصيدلة ،فرع الكيمياء الصيدالنية . العراق ،بغداد، جامعة بغداد ،كلية الصيدلة ،فرع الكيمياء الصيدالنية الخالصة لهما العديد من الفعاليات الحيوية كأن 5و2اوكسادايزول المعوضة في الموقعين -1,3,4قواعد شف و الحلقة األروماتية الغير متجانسة أو أن تكون مضادات لأللتهابات أو تعمل كمسكنات لأللم. لذلك الخاليا السرطانيةو الفايروساتو بكتريا التدرنو الفطرياتو تكون مضادات للبكتيريا اوكسادايزول كوحدة بناء رئيسية-1,3,4باألعتماد على الحلقة األروماتية الغير متجانسة (8-5) المركباتن حامض الفانليلجديدة شتقاتتم تصنيع م عن طريق مطياف األشعة تحت الحمراء, الرنين و عن طريق الكروماتوغرافيا )استشراب الطبقة الرقيقة( تم تشخيصها ه المركباتطوات وهذخ بعدة وسطة أظهرت فعالية مت (8-5) اتالمركب. تم تقييم فعالياتها المضادة للبكتريا باستخدام طريقة االنتشار (8-5) اتالمركبالنووي المغناطيسي للبروتون. ولكنها كانت أقل من فعالية السفيكزيم والنيتروفيورانتوين حيث البكتريا االشريكية القولونيةيمكن مقارنتها بفعالية االموكسسلين وااليزونيازايد ضد البكتريا الكلبسيلة الرئويةيمكن مقارنتها بفعالية االموكسسلين والسفيكزيم ضد أظهرا فعالية متوسطة ( 7)و( 6) انالمركبكانت فعاليتهما عالية. يمكن مقارنتها بفعالية أظهرا فعالية متوسطة ( 7)و( 6) انالمركبولكنها كانت أقل من فعالية النيتروفيورانتوين حيث كانت له فعالية عالية. كانت له فعالية (6)المركب لية عالية. بينما السفيكزيم والنيتروفيورانتوين كانت لهما فعا البكتيريا الكروية العنقودية الذهبيةاالموكسسلين ضد بينما فعالية االموكسسلين والسفيكزيم والنيتروفيورانتوين كانت فعالية عالية. البكتريا العصوية الرقيقةمتوسطة ضد الكلمات المفتاحية: هيدرازايد, قواعد شف, اوكسادايزول, مضادات بكتيرية. introduction heterocyclic compounds are used in many biological fields, due to their different activities, and are considered as one of the principal classes of organic compounds, that are used in the development of several pharmaceutically essential compounds (1,2) . oxadiazoles are important five-membered aromatic heterocyclic containing oxygen and two nitrogens in their structure. because the oxadiazole ring is structurally rigid, various functional groups are easily introduced into the ring. valuable biological activities are associated with oxadiazole derivatives (3), such as antitumor (4), anti-inflammatory (5), antimicrobial(6), antifungal(7), and anticonvulsant (8) . in synthetic medicinal chemistry, to improve the biological activity of new drugs with respect to the corresponding lead compounds, hybridizationcombination of different pharmacophores in one structure-is one of the techniques being followed.(9,10) hydrazide schiff base derivatives (hydrazones) are good scaffolds for various pharmaceutical applications, and characterized by the presence of highly reactive azomethine group (– co–nh–n=ch–).(11) their biological activities include antibacterial(12), antifungal(13), antitubercular(14), antiviral(15), anticancer(16), antiinflammatory(17), and analgesia.(18) 1corresponding author e-mail: m8mostafa@gmail.com received:27 /1 /2020 accepted:20 / 5/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp129-138 iraqi j pharm sci, vol.29(2) 2020 new vanillic acid derivatives as antibacterials 130 material and methods chemicals supplied by hyper-chem (china) were used. aluminum sheets pre-coated with silica gel gf254 (type 60), exposed to uv-254 nm, were used for thin-layer chromatography (tlc) to monitor the completion of reactions and to test the purity of compounds. two solvent systems (s1 and s2) were used: s1(toluene:ethylacetate:ethanol (3:2:1)) and s2(ethylacetate:methanol:ammonia (5:3.5:1.5)). melting points were incorrected and detected by using stuart smp3 melting point apparatus in open capillary tubes. all synthesized derivatives were characterized by spectroscopic analysis (fourier-transform infrared (ftir) which was performed at baghdad university-college of pharmacy) and (proton nuclear magnetic resonance (1hnmr) which was performed by chemistry analysis center (cac)) at iraq/ baghdad (19-22) . chemical synthesis the target compounds were synthesized by multistep(s) reactions as shown in the scheme in figure (1). figure 1. multistep(s) scheme for synthesis of targeted compounds. synthesis of methyl 4-hydroxy-3methoxybenzoate; compound (1)(23): vanillic acid (7.0g, 42mmole) was dissolved in 75ml of absolute methanol (99.8%), the temperature of this solution was lowered to 0˚c by ethanol-water ice bath, 5ml of concentrated h2so4 was added dropwise, and the mixture was stirred at room temperature for 48 hours, then refluxed for 7 hours. the reaction mixture was poured in beaker containing crashed ice (100ml), the formed precipitate was filtered, collected, and washed with 5% nahco3 aqueous solution, filtered again, and dried by a warmed current of air, giving 7.0g of compound (1). iraqi j pharm sci, vol.29(2) 2020 new vanillic acid derivatives as antibacterials 131 synthesis of 4-hydroxy-3methoxybenzohydrazide; compound (2)(24): compound (1), (5.0g, 27mmole) was dissolved in a minimum amount (10ml) of absolute ethanol (99.9%), (13.5g, 270mmole) of 80% hydrazine hydrate was added gradually. the mixture was refluxed for 3-4 hours (monitored by tlc). then the reaction mixture was cooled and precipitate begin to appear, which was filtered and dried in oven (adjusted at 60˚c), giving 3.5g of compound (2). synthesis of 4-(5-mercapto-1,3,4-oxadiazol-2-yl)2-methoxyphenol; compound (3)(25): to suspension of (1.25g, 6.86mmole) of compound (2) in 30ml of 50% aqueous ethanolic solution, (0.785g, 10.3mmole) of cs2 was added with stirring for few minutes, followed by the addition of (0.7g, 10.3mmole) potassium hydroxide (koh), and the mixture was refluxed for 12 hours (monitored by tlc, and notation the evolution of h2s gas by strip soaked with lead acetate aqueous solution; which was turned black as indication of evolution of h2s gas). the mixture poured in a beaker containing crashed ice (30 ml) and then concentrated hcl was added dropwise until ph became (2-3). the formed precipitate was filtered, dried, and purified by base-acid precipitation method (i.e.; the product was dissolved in water by the aid of equimilimole of sodium hydroxide or triethylamine, filtered, and the clear filtrate was treated with concentrated hcl which was added dropwise until the precipitate formed again), giving 0.9g of compound (3). synthesis of ethyl 2-((5-(4-hydroxy-3methoxyphenyl)-1,3,4-oxadiazol-2-yl)thio)acetate; compound (4)(26): compound (3), (0.5g, 2.23mmole,) was dissolved in 2ml of absolute ethanol (99.9%), (0.155g, 1.115mmole) of anhydrous k2co3 was added with stirring, after few minutes a white precipitate was formed, (0.37241g, 2.23mmole) of ethyl 2-bromoacetate was added dropwise. the reaction mixture refluxed for 2 hours (monitored by tlc) and left for stirring gently overnight. then distilled water (d.w) was added gradually until precipitate was formed; then filtered, dried and crystallized from minimum amount (the amount that was just covered the powder) of boiled absolute ethanol giving 0.5g of pure product; compound (4). synthesis of 2-((5-(4-hydroxy-3-methoxyphenyl)1,3,4-oxadiazol-2-yl) thio) acetohydrazide; compound (5)(27): compound (4), (0.5g, 1.6167mmole) was dissolved in a 8ml of hot absolute ethanol (99.9%), (0.16167g, 3.233mmole) of 80% hydrazine hydrate was then added, after 2 hours of vigorous mixing at room temperature, precipitate was formed, and left to stir gently overnight. then, washed with (10ml) absolute ethanol, filtered, dried, and used without further purification. synthesis of n'-(4-hydroxy-3methoxybenzylidene)-2-((5-(4-hydroxy-3methoxyphenyl)-1,3,4-oxadiazol-2-yl) thio)acetohydrazide; compound (6), n'((1h-pyrrol-2-yl)methylene)-2-((5-(4hydroxy-3-methoxyphenyl)-1,3,4-oxadiazol2-yl)thio)acetohydrazide; compound (7), and 2-((5-(4-hydroxy-3-methoxyphenyl)1,3,4-oxadiazol-2-yl)thio)-n'-(4methoxybenzylidene)acetohydrazide; compound (8)(28): an appropriate aldehydes; 4-hydroxy-3methoxybenzaldehyde (vanillin) (0.039g, 0.257mmole) for compound (6), 1h-pyrrole-2carbaldehyde (0.0257g, 0.257mmole) for compound (7), and 4-methoxybenzaldehyde (panisaldehyde) (0.035g, 0.257mmole) for compound (8), were dissolved in 5ml of absolute methanol (99.8%) and stirred for 15-30 minutes in the presence of 2-3 drops of glacial acetic acid; then (0.075g, 0.25mmole) of compound (5) was added and the suspension that formed had been refluxed for 1-2 hours. the reaction mixture was left to stir overnight, then the formed precipitate was filtered, dried and crystallized from boiled methanol. antibacterial essay well diffusion assay was carried out through using bacterial suspension of nearly (1.5×108cfu/ml) obtained from mcfarland turbidity standard (number 0.5). this was used to inoculate by swabbing the surface of mueller hinton agar (mha) plates. the excess liquid was dried by air under a sterile hood. in each agar plate of examined bacteria, four wells were made, and (80μl) of each concentration of the synthesized compound was poured to it. the plates were incubated at 37°c for 24 hours. the evaluation of antibacterial activity was based on the measurement of the diameter of the inhibition zone formed around the well. (29) results chemistry compound (1) (c9h10o4): off-white powder, yield 91%, m.p: 60-62˚c (reported m.p: 64˚c)(30). the characteristic bands in ftir spectrum in cm-1: (1686) c=o stretching vibration band, (1277) c-o stretching vibration band of aromatic ester. 1hnmr (500mhz, dmso-d6) in ppm: 3.80, 3h, s, set (a) protons; 3.83, 3h, s, set (b) protons; 6.876.89, 1h, d, set (c) proton; 7.44-7.49, 2h, m, set (d) protons; 9.97, 1h, s, set (e) proton. compound (2) (c8h10n2o3): white powder, yield 70%, m.p: 208-210˚c (reported m.p: 210-211˚c)(31). the characteristic bands in ftir spectrum in cm-1: (3310) phenolic oh stretching vibration band overlapped with nh2 asymmetric stretching vibration band, (3256) nh amide stretching iraqi j pharm sci, vol.29(2) 2020 new vanillic acid derivatives as antibacterials 132 vibration band, (3209) nh2 symmetric stretching vibration band, (1628) stretching vibration band of carbonyl amide, (1601) nh bending vibration band of hydrazide amine, (1585) nh bending vibration band of hydrazide amide. 1hnmr(400mhz, dmso-d6) in ppm: 3.82, 3h, s, set (a) protons; 4.42, 2h, s, set (b) protons; 6.816.83, 1h, d, set (c) proton; 7.33-7.44, 2h, m , set (d) protons; 9.56, 2h, s, set (e) protons. compound (3) (c9h8n2o3s): faint yellow powder, yield 59%, m.p: 186-189˚c. the characteristic bands in ftir spectrum in cm-1: (3452) phenolic oh stretching vibration band, (3155) nh broad (br) stretching vibration band, 2669 weak (w) sh stretching vibration band. 1hnmr(400mhz, dmso-d6) in ppm: 3.84, 3h, s, set (a) protons; 6.92-6.94, 1h, d, set (b) protons; 7.30-7.37, 2h, m, set (c) protons; 10.04, 1h, s, set (d) proton; 14.58, 1h, s, set (e) proton. compound (4) (c13h14n2o5s): white crystalline powder, yield 65.5%, m.p: 118-120˚c. the characteristic bands in ftir spectrum in cm-1: (3400-2800) broad phenolic oh stretching vibration band, (1744) carbonyl stretching vibration band of aliphatic saturated ester, (1173) stretching vibration band of c-o of aliphatic saturated ester. 1hnmr(500mhz, dmso-d6) in ppm: 1.18-1.21, 3h, t, set (a) protons; 3.86, 3h, s, set (b) protons; 4.14-4.18, 2h, q, set (c) protons; 4.26, 2h, s, set (d) protons; 6.95-6.96, 1h, d, set (e) proton; 7.41-7.43, 2h, m, set (f) protons; 9.97, 1h, s, set (g) proton. compound (5) (c11h12n4o4s): white powder, yield 65%, m.p: 196-199˚c. the characteristic bands in ftir spectrum in cm-1: (3506) stretching vibration band of phenolic oh, (3341) asymmetric nh2 stretching vibration band, (3256) nh amide stretching vibration band, (3217) symmetric nh2 stretching vibration band, (1682) c=o carbonyl amide stretching vibration band, (1655) nh2 bending vibration band. 1hnmr(500mhz, dmso-d6) in ppm: 3.86, 3h, s, set (a) protons; 4.00, 2h, s, set (b) protons; 4.36, 2h, s, set (c) protons; 6.93-6.95, 1h, d, set (d) proton; 7.41-7.44, 2h, m, set (e) protons; 9.41, 1h, s, set (f) proton; 9.93, 1h, s, set (g) proton. compound (6) (c19h18n4o6s): white powder, yield 40%, m.p: 196-200˚c. the characteristic bands in ftir spectrum in cm-1: (3449) two phenolic ohs stretching vibration bands, (3182) nh secondary amide stretching vibration band, (1678) c=o amide carbonyl stretching vibration band, (1601) c=n imine stretching vibration, c=n stretching vibration of oxadiazole and c=c aromatic skeletal stretching vibration (overlapped) bands. 1hnmr(500mhz, dmso-d6) in ppm: 3.81, 3.80, 3h, 2s , set (a) protons [syn/anti-syn]; 3.83, 3h, s, set (b) protons; 4.16, 4.61, 2h, 2s, set (c) protons [syn/anti-syn]; 6.80-6.83, 1h, m, set (d) proton; 6.90-6.93, 1h, m, set (e) proton; 7.07-7.09, 1h, m, set (f) proton; 7.26-7.27, 1h, m , set (g) proton; 7.39-7.42, 2h, m, set (h) protons; 7.91, 8.08, 1h, 2s, set (i) proton [syn/anti-syn]; 9.52, 1h, s, set (j) proton; 9.94, 1h, s, set (k) proton; 11.60, 11.63, 1h, 2s, set (l) proton [cis/trans]. compound (7) (c16h15n5o4s): white powder, yield 53.4%, m.p: 226-228˚c. the characteristic bands in ftir spectrum in cm-1: (3302) phenolic oh and nh pyrrole stretching vibration (overlapped) bands, (3209) nh amide stretching vibration band, (1663) amide carbonyl stretching vibration band, (1620) nh pyrrole bending vibration and imine stretching vibration (overlapped) bands. 1hnmr(500mhz, dmso-d6) in ppm: 3.83, 3h, s, set (a) protons; 4.17, 4.60, 2h, 2s, set (b) protons [syn/anti-syn]; 6.11-6.14, 1h, m, set (c) proton; 6.44-6.49, 1h, m, set (d) proton; 6.90-6.95, 2h, m, set (e) protons; 7.40-7.43, 2h, m, set (f) protons; 7.86, 8.04, 1h, 2s, set (g) proton [syn/anti-syn]; 9.94, 1h, s, set (h) proton; 11.40, 1h, s, set (i) proton; 11.45, 11.48, 1h, 2s, set (j) proton [cis/trans]. compound (8) (c19h18n4o5s): white powder, yield 72%, m.p: 190-193˚c. the characteristic bands in ftir spectrum in cm-1: (3495) phenolic oh stretching vibration band, (3182) nh secondary amide stretching vibration band, (1666) c=o amide carbonyl stretching vibration band, (1609) c=n imine stretching vibration, c=n stretching vibration of oxadiazole and c=c aromatic skeletal vibration (overlapped) bands. 1hnmr(500mhz, dmso-d6) in ppm: 3.79, 3.80, 3h, 2s , set (a) protons [syn/anti-syn]; 3.83, 3h, s, set (b) protons; 4.18, 4.59, 2h, 2s, set (c) protons [syn/anti-syn]; 6.90-7.01, 3h, m, set (d) protons; 7.39-7.42, 2h, m, set (e) protons; 7.61-7.65, 2h, m, set (f) protons; 7.97, 8.14, 1h, 2s, set (g) proton [syn/anti-syn]; 9.94, 1h, s, set (h) proton; 11.64, 11.69, 1h, 2s, set (i) proton [cis/trans]. anti-bacterial evaluation the antibacterial activities of the synthesized compounds; compounds (5-8) were evaluated against six bacteria and compared with four standard antibiotics; amoxicillin, cefixime, nitrofurantoin, and isoniazid. dimethyl sulfoxide (dmso) was used as solvent and control. it’s evident from the data displayed in the table (1), compound (5) showed moderate antibacterial activity against escherichia coli (e. coli). compound (6) showed moderate activity towards staphylococcus aureus (s. aureus), bacillus subtilis (b. subtilis), escherichia coli (e. coli), and klebsiella pneumoniae (k. pneumoniae). compound (7) showed moderate activity towards staphylococcus aureus (s. aureus), escherichia coli (e. coli), and klebsiella pneumoniae (k. pneumoniae). compound (8) was slightly active against staphylococcus aureus (s. aureus) and moderately active against escherichia coli (e. coli). iraqi j pharm sci, vol.29(2) 2020 new vanillic acid derivatives as antibacterials 133 no one of the synthesized compounds had shown activity against streptococcus pyogenes (s. pyogenes) and pseudomonas aeruginosa (p. aeruginosa). table 1.the antibacterial activities of synthesized compounds. comp. no. conc. μg/ml gram (+)ve gram (-)ve s. aureus s. pyogenes b. subtilis e. coli k. pneumoniae p. aeruginosa zone of inhibition(zi) in (mm) comp.5 103 12 comp.6 103 11 13 11 11 comp.7 103 10 11 10.5 comp.8 103 9.5 11 amoxicillin 103 13 5 40 11 11.5 28 cefixime 103 16 6 20 22 13 6 nitrofurantoin 103 21 9.5 31 16 20 17 isoniazid 103 8 12 dmso solvent control (-)= no activity, slightly active (zi =5-10 mm), moderately active (zi= 10-15 mm), highly active (zi= more than 15 mm).(32,33) discussion chemistry compound (1) was esterification product which resulted from reaction between carboxylic acid and an alcohol in the presence of an acid as catalyst. two stages were involved: addition of a nucleophile followed by elimination of a leaving group. protonation and deprotonation steps also occur during the ester formation which could explain the role of acid in the reaction. under basic conditions, carboxylate anion will be formed which does not react with an electron-rich nucleophile, so the esterification will be happened in the presence of an acid. formation of ester is necessary for the success of step 2 and 5 as explained later.(34). figure 2. steps of esterification.(34) compound (1) was characterized by carbonyl group of aromatic ester at 1686cm-1 in its ftir spectrum and 1hnmr signals confirmed the presence of cooch3 at 3.80 ppm. synthesis of compound (2) and (5) is essentially a base catalyzed hydrolysis (hydrazinolysis of ester) which was run under iraqi j pharm sci, vol.29(2) 2020 new vanillic acid derivatives as antibacterials 134 normal basic condition in which the ratedetermining step involves two molecules of hydrazine in which a proton was being transferred between them. in the next step, one hydrazine molecule will be left slowly with one molecule of alcohol (35). figure 3. hydrazinolysis of ester (35). in case of compound (2); ftir spectrum was characterized by two stretching vibration bands for the primary amine of hydrazide, at 3310 cm-1 and 3209 cm-1, respectively, 3256 cm-1 nh amide stretching vibration band, 1628 cm-1c=o stretching vibration band of amide, and 1601cm-1nh2 bending vibration band. 1hnmr signals confirmed the presence of conh at 9.56 ppm, and nh2 at 4.42 ppm. for compound (5); ftir spectrum was characterized by asymmetric and symmetric stretching vibration bands of nh2 at (3341 and 3217) cm-1, respectively, 3256 cm-1 nh amide stretching vibration band, the amide carbonyl stretching vibration band at 1682 cm-1, and nh2 bending stretching vibration band at 1655 cm-1. 1hnmr was revealed the presence of new signals at 9.41 ppm and 4.36 ppm related to conh and conhnh2, respectively. synthesis of compound (3) had been carried out by refluxing an ethanolic suspension of compound (2) with cs2, in the presence of (koh) and the cyclization which involved formation of potassium salt of dithiocarbamate as an intermediate could be done through keto form of hydrazide carbonyl or enol form. enol form is the preferred explanation because of the stability of enol form by intramolecular hydrogen bonding.(36) figure 4. enol form of dithiocarbamate stabilized by intramolecular hydrogen bonding.(36) ftir spectrum of compound (3) was characterized by nh stretching vibration band at 3155cm-1, weak sh stretching vibration band at 2669cm-1, and in addition to the absence of amide band at 1628 cm-1. 1hnmr was characterized by the appearance of new signal related to sh proton at 14.58 ppm. compound (4) was obtained by a nucleophilic substitution (sn2) reaction between compound (3) and ethyl 2-bromoacetate in absolute ethanol and in the presence of anhydrous potassium carbonate as a catalyst. figure 5. synthesis of compound (4) by nucleophilic substitution (sn2) reaction (37). ftir spectrum was characterized by 1744cm-1 stretching vibration band of saturated ester carbonyl. 1hnmr was characterized by the presence of new signals at 4.14-4.18(2h, q, coch2ch3), 1.181.21(3h, t, coch2ch3), and the absence of sh signal at 14.58 and instead of the appearance of new signal at 4.26 ppm which was related to –sch2. compounds (6-8) were schiff base products (imines) which resulted from reaction between aldehydes with a primary amines in mildly acidic conditions and involves six steps; the first three steps produce an intermediate called a carbinolamine and the last three steps convert the carbinolamine into an imine (38). iraqi j pharm sci, vol.29(2) 2020 new vanillic acid derivatives as antibacterials 135 figure 6. steps of schiff base (38). ftir spectra were characterized by absence of hydrazide nh2 asymmetric and symmetric stretching vibration bands; while n=ch imine stretching vibration bands were overlapped with other bands in ftir spectra. 1hnmr were characterized by the appearance of new signals related to conhn which were due to cis and trans isomers showed 2signals between [11.60,11.63 for compound (6), 11.45,11.48 for compound (7), 11.64,11.69 for comp.8], and conhn=ch which due to syn/anti-syn conformers showed 2signals between [7.91, 8.08 for compound (6), 7.86, 8.04 for compound (7), 7.97, 8.14 for compound (8)].(3942) antibacterial activities four antibacterial standards were used, amoxicillin to compare anti-gram (+)ve activities of the derivatives with it; cefixime to compare antigram(-)ve activities of the derivatives with it; nitrofurantoin because it is considered hydrazone and contains furan ring which is isoseter with oxadiazole ring, and isoniazid which is hydrazide compound resembles to compound (5). because compound (6) and compound (7) are more polar than compound (8), they showed additional activities against k. pneumoniae [as polarity increased; there will be extended activity against gram(-)ve bacteria, while retained activities against gram(+)ve bacteria as in the case of penicillin g and aminopenicillins].(43) because compound (5) is hydrazide; it is expected to show limited activity against test bacteria and showed agreement with isoniazid. conclusion: new oxadiazole derivatives (hydrazide and its schiff bases), derived from vanillic acid were successfully synthesized by conventional methods. they were characterized and evaluated for their antibacterial activities. compound (6) had shown the broadest spectrum against tested bacteria showed activities against four out of six bacteria. compound 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https://www.researchgate.net/publication/287644704_thesis_-_synthesis%20_characterization%20_and%20_anti-inflammatory%20_evaluation%20_of%20_new%20_potentially%20_active%20_naproxen%20_hydrazones%20/link%20/%205832c41e08ae138f1c086a4b%20/download https://www.researchgate.net/publication/287644704_thesis_-_synthesis%20_characterization%20_and%20_anti-inflammatory%20_evaluation%20_of%20_new%20_potentially%20_active%20_naproxen%20_hydrazones%20/link%20/%205832c41e08ae138f1c086a4b%20/download iraqi j pharm sci, vol.29(2) 2020 new vanillic acid derivatives as antibacterials 138 42. palla g, predieri g, domiano p, vignali c, turner w. conformational behaviour and e/z isomerization of n-acyl and naroylhydrazones. tetrahedron. 1986;42(13):3649-3654. 43. beale jm, block jh. wilson and gisvold’s textbook of organic medicinal and pharmaceutical chemistry. 12th ed. philadelphia: lippincott williams & wilkins, a wolters kluwer business; 2011. chapter 8, antibacterial antibiotics; p. 258-329. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles doi: https://doi.org/10.31351/vol29iss1pp62-75 62 preparation, in vitro and ex-vivo evaluation of mirtazapine nanosuspension and nanoparticles incorporated in orodispersible tablets hiba e. hamed*,1 and ahmed a. hussein* *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq, abstract the objective of the present investigation was to enhance the solubility of practically insoluble mirtazapine by preparing nanosuspension, prepared by using solvent anti solvent technology. mirtazapine is practically insoluble in water which act as antidepressant .it was prepared as nano particles in order to improve its solubility and dissolution rate. twenty formulas were prepared and different stabilizing agents were used with different concentrations such as poly vinyl pyrrolidone (pvpk-90), poly vinyl alcohol (pva), poloxamer 188 and poloxamer 407. the ratios of drug to stabilizers used to prepare the nanoparticles were 1: 1 and 1:2. the prepared nanoparticles were evaluated for particle size, entrapment efficiency, dissolution study, fourier transform infrared spectroscopy, differential scanning calorimetry, and atomic force microscopy. the percentage of drug entrapment efficiency of f1-f20 was ranged from 78.2% ± 1 to 95.9 % ± 1. the release rate and extent of mirtazapine nanoparticles were inversely proportional to the particle size of the drug i.e. it decreased when particle size increase. it is concluded that the nanoprecipitation have potential to formulate homogenous nanosuspensions with uniform-sized stable nanoparticles of mirtazapine. the prepared nanosuspension showed enhanced dissolution which may lead to enhanced solubility of mirtazapine. keywords: mirtazapine, nanoparticles, particle size, poloxamer. النانويةنانوي و الجسيمات الميرتازابين المعلق لتحضير وتقييم في المختبر وخارج الجسم الحي للتشتت ةفي اقراص قابل مدمجة *حسين احمد عباس و 1*،هبه عزت حامد .العراق ،بغداد ،بغداد ةجامع الصيدلة، كلية ،الصيدالنيات *فرع الخالصة للذوبان عمليا من خالل اعداد تعليق نانوي , تم اعداده القابلةهو تعزيز قابليه الذوبان لعقارالميرتازابين غير الدراسةان الهدف من هذه ميرتازابين .الشديداالكتئاب يستخدم لعالجء لاللمذيبات. ميرتازابين هو دواء غير ذائب بالماء وهو دوا المضادةباستخدام باستخدام تكنلوجيا المذيبات للذوبان ومعدل االمتصاص. القابليةبغيه تحسين نانويةاعد كجسيمات , وبولي الفنيل pvpمثل مثل الفاينيل بايرولدون المتعدد مختلفةبتراكيز مختلفة استخدمتتم اعداد عشرين صيغه باستخدام بوليمرات استقرار .1:2, 1:1في اعداد الجسيمات النانويه هي المستخدمةكانت نسب الدواء الى المثبتات . و407و بولوكسامير 188, بولوكسامير pvaالكحول التوافق دراسةوكذلك الدوائي،الشكل البياني للتحرر ودراسة الدواء،من حيث الحجم الحبيبي للجسيمات وكفاءه انحباس نانويةوقيمت جسيمات االولى الصيغةمن الدوائيةانحباس الدواء للصيغ لكفاءة المئوية النسبة. الذرية القوةي( ومجهر وقياس المسح التفاضل الحمراء،تحت األشعة)مطيافيه السطحية المساحة لزيادة النانويةاخرى يزداد تحرر الدواء كلما صغر حجم الجسيمات ناحية. من %95,9الى %78,2من الصيغة عشرين هيالى ان الجسيمات النانوية لديها القدرة على صياغة تعليق متجانس مع جسيمات نانوية مستقرة موحدة الحجم من الميرتازابين. ويمكن استنتاجللجسيم. .االنحالل يؤدي إلى تعزيز الذوبان من الميرتازابين ان تعزيزوأظهرت .بولوكسامير ن،الحبيبيالحجم ،النانوية الجسيمات ،ميرتازابين: المفتاحيةالكلمات introduction solubility is of the most important parameter to achieve the desired concentration of drug in systemic circulation for pharmacological response to be shown. poorly water soluble drugs often require high doses in order to reach therapeutic plasma concentrations after oral administration. low water solubility is the major problem encountered with formulation development of new chemical entities(1). several formulation techniques exist for the manufacturing of nanosuspension, precipitation has been applied to prepare submicron particles, especially for the poorly soluble drugs. rapid addition of a drug solution to the anti-solvent leads to sudden super saturation of drug and formation of ultrafine crystalline or amorphous drug solids(2). mirtazapine is an antidepressant drug used for the treatment of moderate to severe depression, molecular formula: c17h19n3 (3). the drug has bioavailability of 50 % due to first-pass metabolism, high protein binding (80 %) and very high half-life (20 – 40 h)( 4). the aim of this study is to formulate and evaluate mirtazapine nanoparticles using solvent anti solvent method. corresponding author e-mail: hibaa.19855@gmail.com received:11 /6/2019 accepted: 29/ 9/2019 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss1pp62-75 mailto:hibaa.19855@gmail.com iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 63 materials and method materials mirtazapine powder was purchased from (hyper-chem, china), pvp k-90(hangzhou sunflower ,china),pva(jp&sb converting services, spain), poloxamer 188 and poloxamer 407(himedia (mumbai, india), methanol (scharlau chemie, s.a. spain). all other chemicals were of analytical grade. method preparation of mirtazapine nanosuspension nanosuspensions of mirtazapine were prepared by the solvent evaporation technique, which is also termed as anti-solvent precipitation method. mirtazapine powder was dissolved in methanol (4 ml) at room temperature. this was poured into 20 ml of water containing different types of stabilizer (alone and in combination) maintained at 500c and subsequently stirred at agitation speed of 500 revolution per minute (rpm) on magnetic stirrer for 60 min.to allow the volatile solvent to evaporated (5). the resultant organic solution of drug (organic phase) was added drop by drop by means of a plastic syringe positioned with the needle directly into aqueous solution of stabilizer. the ratios of drug to stabilizer used to prepare the nanosuspension were 1:1 and 1:2, as shown in table (1,2). table1. composition of mirtazapine nanosuspension using different stabilizers at drug: stabilizer ratio 1:1. formula mirtazapine (mg) poloxamer 188 (mg) poloxamer407 (mg) pvp-k90 (mg) pva (mg) methanol (ml) water (ml) f1 15 15 4 20 f2 15 15 4 20 f3 15 15 4 20 f4 15 15 4 20 f5 15 7.5 7.5 4 20 f6 15 7.5 7.5 4 20 f7 15 7.5 7.5 4 20 f8 15 7.5 7.5 4 20 f9 15 7.5 7.5 4 20 f10 15 7.5 7.5 4 20 table2. composition of mirtazapine nanosuspension using different stabilizers at drug: stabilizer ratio 1:2 water (ml) methanol (ml) pva (mg) pvpk-90 (mg) poloxamer407 (mg) poloxamer 188 (mg) mirtazapine (mg) formula 20 4 30 15 f11 20 4 30 15 f12 20 4 30 15 f13 20 4 30 15 f14 20 4 15 15 15 f15 20 4 15 15 15 f16 20 4 15 15 15 f17 20 4 15 15 15 f18 20 4 15 15 15 f19 20 4 15 15 15 f20 iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 64 evaluation of the prepared nanosuspension particle size and size distribution particle size determination was done by using angstrom advanced inc. abt-9000 usa particle size analyzer which is a dynamic light scattering works by measuring the intensity of light scattered by the molecules in the sample as a function of time, at scattering angle 90° and a constant temperature of 25 °c. the polydispersity index (pdi) which is a measure of the width of the size distribution of each formula of mirtazapine nanosuspension was measure of the distribution of particle size of nanoparticles obtained from a particle analyzer, pdi is an index of spread or variation or width within the particle size distribution. also, the analyzer determines the specific surface area of each sample(6) . determination of drug entrapment efficiency (ee) of nanosuspension the freshly prepared nanosuspension of drug: stabilizer ratio 1:1, and 1:2 was centrifuged at 20,000 rpm for 20 minutes using ultracentrifuge. the amount of unincorporated drug was measured by taking the absorbance of the appropriately diluted 25 ml with water at 290 nm using uv-visible spectrophotometer. it was calculated by subtracting the amount of free drug in the supernatant from the initial amount of drug taken. for each formulation the experiment was repeated in triplicate and the average was calculated (7). in vitro dissolution profile of nanosuspension the in vitro dissolution study was performed using usp dissolution test apparatus-ii (paddle assembly). the dissolution was performed using mirtazapine nanosuspension in 900 ml of 0.1n hcl (ph 1.2) maintained at 37 ± 0.5°c , 50 rpm and samples (5ml) were withdrawn at regular intervals of 5 minutes for 120 minutes and replaced with fresh dissolution medium. samples were filtered through filter paper and assayed spectrophotometrically on uv-visible spectrophotometer at 315 nm wave length (8). freeze drying of nanosuspension in order to make nanoparticles in driedpowder state from the nanosuspensions, waterremoval was conducted through freeze-drying, so that each formula was lyophilized using vacuum freeze dryer at a controlled temperature of (45) °c and the pump operating at a pressure of 2.5 × 10 pascal over a period of 48–72 hour. the yielded powders were used for further studies and also it is used to prepare the tablets (9). formation of mirtazapine nanoparticles tablet mirtazapine formulated in to orodispersible tablets by direct compression method containing drug equivalent to 15mg mirtazapine. all ingredients were properly mixed to gather then compressed in to tablets prepared by after freeze drying of formula (f15 ) that gave the best in vitro dissolution profile in minute in comparison with other nanoparticle formulas and pure drug, show as in table (3) (10). the orodispersible tablets were prepared using avicel ph102 (mcc), crospovidone and, magnesium stearate as a diluent, disintegrente ,and lubricant at different concentration and tested to obtain the optimum formula that show the accepted hardness and the best in vitro dissolution profile(11). table 3. composition of mirtazapine tablets quantity per tablet (mg) materials f 15 b f 15 a 45 45 lyophilized powder 92 82 avicel ph 102 (mcc) 10 20 crospovidone 3 3 magnesium stearate 150 150 tablet weight (mg) precompression studies of the prepared nanoparticle powder the flowability of a powder is of critical importance in the production of pharmaceutical dosage forms in order to get a uniform feed as well as reproducible filling of tablet dies otherwise high dose variations will occur. the powder flowability of prepared mirtazapine tablets were characterized by angle of repose, hausner's ratio and carr’s index (12). evaluation of mirtazapine orodispersible tablets tablets were evaluated for hardness test, friability test, content uniformity test and weight variation tests (13), and dissolution study. in vitro dissolution profile of mirtazapine tablets an in vitro dissolution test was conducted in a dissolution apparatus according to the usp paddle method. the temperature was maintained at 37 ± 0.5°c, and the stirring rate was at 50 rpm. the commercial mirtazapine tablet accurately weighed bulk drug and were dispersed in 900 ml of dissolution medium (0.1 n hcl). 5 ml samples were drawn, and the same volume of fresh dissolution medium was added at 5, 10, 15, 20, 30, 45, 60, 70, 90, 105, 120 minutes.. then, the samples were filtered through a 0.1-μm syringe filter immediately before dilution, when necessary. drug content was determined with a uv spectrophotometer at 315 nm for 0.1 n hcl (ph 1.2) (14). iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 65 fourier transform infrared spectroscopy (ftir) the (ftir) spectra were recorded for pure drug and optimized formulation using kbr pellet technique.the pellets were prepared using kbr hydraulic press under hydraulic pressure of 150 kg/cm2 . the spectra were scanned over 3600-400 cm−1 at ambient temperature with a resolution of 4 cm−1, using ft-ir 2500 apparatus (15). differential scanning calorimetry (dsc) dsc investigations were performed using dsc apparatus model dsc-6. samples of about 5 mg of pure drug powder and selected formula are placed in an aluminum pan and the experiment was carried out under nitrogen atmosphere at a flow rate of 40 ml/min and scanning rate of 10°c/min in the range of 15-300°c(16). high performance liquid chromatographic (hplc) rp-hplc system was used for this study and the specifications are given below. a waters hplc equipped with spa20a detector, an isocratic chromatographic separation technique was conducted utilizing symmetry® ods-c18 (250 × 4.6mm; 5μm) column and breeze software. chromatographic conditions: mobile phase: hplc grade of methanol: 0.1m ammonium formate solution in a ratio of 77:23 percent (v/v) was filtered through (0.45μm) millipore filter. flow rate: it was maintained at 1.0 ml/min of the mobile phase. detection was carried out by uvdetector; at a wavelength of 315 nm and the running time was 10 min. one hundred milligrams of mirtazapine was accurately weighed and transferred to a 100 ml volumetric flask. it was dissolved in 50 ml hplc grade methanol and sonication for about 10 minutes and then made up to the volume with hplc grade methanol. from this stock solution (1mg/ml) eight serial dilutions (1.66, 2.5, 5, 10, 20, 30, 40, and 50 μg/ml) were prepared. twenty microliters of each dilution were injected into the column and the corresponding chromatograms were obtained (17). atomic force microscopy (afm) the afm is capable of scanning the surfaces in controlled environmental conditions and is complementary to sem imaging. the size and surface morphology of mirtazapine nanoparticle were confirmed by atomic force microscopy of the formula. samples were determined in tapping mode, exerting pyramidal cantilevers with pt probes. all results were recorded under ambient laboratory condition and scanning frequency of 2hz. resonance frequency was 79.491 khz and a constant force in the range 2.5-10nm-1, driving amplitude 334.6mv. silicon chip was newly operated by peeling off its upper layer to form the sample. particle size, 3d-dimension graph and histogram of particle size distribution were obtained(18). statistical analysis the results of the experiments were given as a mean of triplicate samples ± standard deviation and were analyzed according to the paired t test and one way analysis of variance (single factor anova) at the level of (p < 0.05). results and discussion evaluation of nanosuspension particle size analysis the particle size of f1-f4 at drug : stabilizer ratio 1:1 was ranged from 429 691 nm measured by particle size analyzer (as shown in table 4) while for f11-f14 at drug : stabilizer ratio 1:2 the particle size ranged from 379 572 nm as in table (4) using poloxamer 188 , poloxamer 407 , pvpk90 and pva as primary stabilizers. pvp k-90 , poloxamer and pva are stabilizers for nanosuspension. vinyl groups of pva (polyvinyl alcohol), due to their hydrophobic nature tend to adsorb onto the hydrophobic part of mirtazapine nanoparticles while –oh extend themselves outside into the aqueous environment and thus providing stabilization to the nanoparticles and preventing agglomeration. –oh bonds of pva makes hydrogen bonding with water molecules and thus viscosity of it increases (19). polydispersity index is a parameter used to define the particle size distribution obtained from the particle size analyzer. polydispersity index gives degree of particle size distribution at range from 0.021 to 0.420 depending on formulation variables. the formula f10 showed lowest pdi (0.029) at drug : stabilizer ratio 1:1 and 0.114 at drug : stabilizer ratio 1:2, as seen in table (4 ) ; that indicate good uniformity of nanoparticle size. uniformity of particle size is determined by polydispersity index values in which the low value means the best uniformity(20). the range of pdi values (0-0.05) means (monodisperse system), 0.05-0.08 (nearly monodisperse), 0.08-0.7 (mid-range polydispersity), and >0.7 (very polydispersity). from the obtained results, one can conclude that the poloxamer 188 and poloxamer 407 are suitable as a primary stabilizer for nanoparticles because of poor adsorption and affinity of poloxamer to the drug molecules. effect of polymer concentration on the size of mirtazapine nanoparticles the effect of the stabilizer concentration on the particle size was investigated by depending on two ratios 1:1 of drug : stabilizer in the preparation of f1-f10 and 1:2 of drug : stabilizer in the preparation of f11-f20. not only the type of stabilizer affects the particle size, but also the concentration of the stabilizers used. stabilizer concentration also influences on the adsorption affinity of non-ionic stabilizers to particle surface. iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 66 in general, as the concentration of stabilizer increase the particle size decrease at fixed drug concentration, the concentration of stabilizer may give negative effect (decrease particle size) or positive effect of on particle size (increase particle size). it can also influence on the adsorption affinity of non-ionic stabilizers to particle surface. in general, as the concentration of stabilizer increases the particle size decreases at fixed drug concentration.(21). it was observed that with an increase in surfactant concentration in the nanosuspension from the particle size of the nanosuspension decreases. this was due to the decrease in relative viscosity, which led to decrease in particle size. it means that hydrodynamic diameter of particle decreased with increase in the concentration of the surfactant.the concentration of surfactant affected on particle size because too little concentration of stabilizer induces agglomeration or aggregation of particles (22). as shown in tables (4,5) the size range of particles is decrease in the sequence of f1 (429nm) > f11 (383 nm), f2 (444nm) > f12 (401nm) that correspond to 1:1, 1:2 of drug: stabilizer (poloxamer 188, poloxamer 407) ratio, respectively. these results indicated that mean size of particles showed a regular decrease with increasing the concentration of poloxamer. these effects due to a process of a primary covering of the newer surfaces competing with the aggregation of the uncovered surfaces. hence, an elevation in ratio of surfactant in the primary dispersion results in rapid enclosing of the newly formed particle surfaces. there was an optimum concentration of surfactant, above which the increase in concentration did not result in a decrease in particle size due to saturation point; these results are in agreement when poloxamer was used as stabilizer at different ratio (23). poloxamer is a block co-polymer, can act as a surfactant, is responsible for the hydrophobic association with the molecules of drug. the inhibition of the crystal growth is mainly related to the hydrophobic part (polypropylene oxide group ppo) in the pluronic polymer, while the second chain which is (the hydrophilic oxide) (peo) can provide steric hindrance against particles aggregation(24). the size range of particles is also decreased in the sequence f3 (460) > f13 (379nm), f4 (691nm) > f14(572nm) that correspond to 1:1, 1:2 of drug: stabilizer pva, pvp k-90 ratio, respectively. on the other hand, the adsorption of surfactant makes the particles less hydrophobic and thereby reduces the hydrophobic forces of attractions (van der waals interactions) and that reduced particle growth and aggregation(25). effect of combination of two polymers on the size of mirtazapine nanoparticles the particle size of ( f5-f10) of drug : stabilizer ratio 1:1 was ranged from 310-610 nm (table 4) , (f15f20) of drug : stabilizer ratio 1:2 was ranged from 146-544 nm (table 5). at ratio1:2 drug : stabilizer large particle size show in combination of poloxamer 188 and pvp k-90 gave higher size than alone that show in f16 (544nm). in f11that contain poloxamer 188 alone get particle size 383 nm and in f13 that contain pvp k-90 alone get particle size 379 nm that mean pvp has a higher affinity to adsorb mirtazapine than poloxamer 188, these results due to that the combination lead to increase viscosity of the disperse media, so it is ineffective combination and cannot stabilize the nanoparticulate system (26). nanoparticles formulation generally requires addition of appropriate stabilizers to lower the free surface energy of the nanoparticles and prevent particle aggregation and/or particle growth. the high surface free energy of nanoparticles is readily lowered by lowering the solid–liquid interfacial tension upon addition of surfactants(24). the formula f15 showed lowest pdi (0.021), as seen in table 5 at drug : stabilizer ratio 1:2, that indicate good uniformity of nanoparticle size. uniformity of particle size is determined by polydispersity index values in which the low value means the best uniformity when used two stabilizer (poloxamer 188 + poloxamer 407). iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 67 table 4. particle size , pdi and ee% of formulas at drug: stabilizer ratio 1:1. formula stabilizers particles size pdi ee% f1 poloxamer 188 429 0.171 83.9 f2 poloxamer407 444 0.214 83.3 f3 pvp-k90 460 0.187 78.2 f4 pva 691 0.293 85.3 f5 poloxamer188+poloxamer407 342 0.178 89.3 f6 poloxamer188+pvp-k90 610 0.420 78.7 f7 poloxamer188+pva 492 0.123 88 f8 poloxamer407+pvp-k90 488 0.331 86.5 f9 poloxamer407+pva 325 0.142 89.6 f10 pvp-k90+pva 310 0.029 89.6 table 5. particle size, pdi and ee% of formulas at drug: stabilizer ratio 1:2. ee% pdi particles size stabilizers formula 88.2 0.079 383 poloxamer188 f11 93.7 0.192 401 poloxamer407 f12 89.2 0.113 379 pvp-k90 f13 87.8 0.051 572 pva f14 95.9 0.021 146 poloxamer188+poloxamer407 f15 79.6 0.341 544 poloxamer188+pvp-k90 f16 87.8 0.132 381 poloxamer188+pva f17 87.2 0.038 338 poloxamer407+pvp-k90 f18 88.7 0.188 239 poloxamer407+pva f19 89.7 0.114 208 pvp-k90+pva f20 determination of drug entrapment efficiency of nanosuspension (ee%) the ee% of the formulations from 78.2% – 95.9 % (table 4,5) the drug entrapment efficiency of f15was high when compared to other formulations. in present work, a relatively high %ee (95.9) in f15 was obtained for most of the prepared mirtazapine nanosuspension formulas which may be attributed higher affinity towards the lipid matrix due to its lipophilic partition coefficient (27). they represent integral parameters in the formulation due to their influence on drug release characteristics and therefore its bioavailability to the biological system. hydrophobic drug molecules are easier to be incorporated in nanoparticles with higher efficiency relative to hydrophilic drugs due to the later tendency to partition into the aqueous phase-out of the lipid phase during homogenization(28). in-vitro drug release study of mirtazapine nanosuspension in vitro dissolution study was performed for all formulas using usp dissolution test apparatus-ii. in 0.1n hcl and in phosphate buffer solution (ph 6.8) media showed the f15 that contain poloxamer 188 and poloxamer 407 stabilizers gave the best release when comparison with other formulas and the formula shows a maximum cumulative percentage drug release of 99.9% within 20 min. the release of f15 in media of 0.1n hcl and in phosphate buffer ph6.8, the maximum cumulative percentage drug release reach to 99.9 % within 20 minutes and in phosphate buffer solution (ph 6.8) release of f15 reach 90.2 % in 40 minutes(29) (figure1). iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 68 the release of f15 was compared with the pure drug in media of 0.1n hcl (figures 2) the maximum cumulative percentage drug release of f15 was 99.9 % within 20 minutes, whereas the pure drug having a release of 93.2 % within 60 minutes. the obtained results are in good accordance with noyes–whitney equation which states that the increase in saturation solubility and the decrease in particle size lead to an increased dissolution rate(30). figure 1. dissolution profile of mirtazapine ( f15) nanosuspension in 0.1 n hcl (ph 1.2) and in phosphate buffer (ph 6.8) at 37c . figure 2. dissolution profile of mirtazapine (f15) nanosuspension and pure drug in 0.1n hcl (ph 1.2) at 37c drug content in lyophilized powder the drug content result of lyophilized powder of the selected formula (f 15) 97.64% of mirtazapine when determined by uv-visible spectrophotometer at λmax 315 nm. evaluation of mirtazapine nanoparticles powder powder flowability angle of repose and compressibility index of the powder of the formulas (f15a and f15b) were reported in table (6). table 6. flow properties of prepared blends of mirtazapine incorporating drug nanoparticles. formula angle of repose carr,s index hausner ratio physical property angle of repose carr,s index f15a 13.6 11.4 1.16 excellent good f15b 21 27.5 1.34 good poor evaluation of mirtazapine tablets the mechanical properties of pharmaceutical tablets are quantifiable by the friability, hardness or crushing strength. the hardness of all the formulas as shown in table (7) had an acceptable values 7, 6.5 kg/cm2. the hardness of f15a containing mcc (avicel)® 82 mg was 7kg /cm2 larger than f15b . during compression, mcc (avicel)® ph 102 is believed to undergo stress relief deformation by several mechanisms, this might be attributed to the hydrogen bonds formed among the hydroxyl groups of the adjacent cellulose particles of (avicel)®, which are brought closely together by plastic deformation during compression, so that it produces hard tablets at low compression forces(31). the loss in total weight of the tablets due to friability was found in all formulation, which indicated to be less than 1% for friability and which confirms the mechanical stability of tablets(32). physical properties of the prepared tablets, weight variation and drug content, demonstrated in table (7). the weight variation of f15a, f15b was within the pharmacopoeia limits which is ± 7.5% of the average weight. weight variation of the prepared tablets was within the limit (149.2 mg, 147.9 mg ) and this indicates that there is no deviation from the limit of 7.5% of usp pharmacopoeia limits(33). the content uniformity of the prepared formulas was within the accepted pharmacopeia limits (85% 115%) and this mean that all the formulations revealed good uniformity and had yielded results from 101% , 98.7 respectively. disintegration time of prepared tablets about 10 sec. and 13 sec. table 7. mechanical strength and physical properties of the prepared mirtazapine incorporating drug nanoparticles formula hardness (kg/cm2) friability % weight variation (mg) drug content (%) disintegration time (sec.) f15a 7 0.45 149.2 101 10 f15b 6.5 0.67 147.9 98.7 13 iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 69 in vitro dissolution study of tablet the release profiles of the prepared mirtazapine tablets incorporating drug nanoparticles (f15a, f15b) and tablet marketing of mirtazapine as a reference were tested in 0.1n hcl (ph 1.2) and phosphate buffer solution (ph 6.8) as shown in figures (3) and (4), f15a was faster compared with f15b and the marketed tablet of mirtazapine. figure 3. dissolution profile of prepared tablets and mirtazapine marketed in buffer (ph 1.2) at 50 r.p.m and 37ºc . figure 4. dissolution profile of prepared tablets and mirtazapin marketed in buffer (ph 6.8) at 50 r.p.m and 37ºc. fourier transform infrared spectroscopy (ftir) ftir is one of the most widely reported spectroscopic techniques for solid-state characterization. ir spectroscopy of mirtazapine (figure 5), n-h stretching 3245 cm-1, methyl group attached to a n2 atom gives rise to a band at 2854 cm-1 , bands for c-c stretching of the phenyl group appeared at 1585 cm-1 and 1444 cm-1. the primary aromatic amines with n directly on the ring give bands at 1336-1200 cm-1. the benzene ring c-h appears in the range of 1359-1074 cm-1 and 788-636 cm-1 for the in plane and out of plane bending vibrations respectively (34, 35). the characteristic bands of mirtazapine as lyophilized powder, blend powder of best formula (f15a) show the benzene ring c-h appears 1084 cm-1, c-h stretching vibrations band of methyl group at 3101.94 cm-1, bands for c-c stretching of the phenyl group appeared at 1640 cm-1, n-h stretching peak show between (31013369) cm-1. it was observed that there were no changes in these main peaks in the ftir spectra of a mixture of drug and excipients. the ftir study demonstrate that no physical or chemical interactions of mirtazapine with other excipients. these are the main characteristic absorption band show in figure (6,7). figure 5.ft–ir spectra of mirtazapine pure powder iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 70 figure 6. ftir spectrum of lyophilized powder figure 7. ftir spectrum of blend powder best formula(f15a) differential scanning calorimetry figure (8) demonstrate dsc of mirtazapine showed sharp characteristic endothermic peak at 117ºc and this agrees with published results. this gives an indication that the drug has crystalline nature with high purity. for lyophilized powder, the melting point of mirtazapine disappeared as in figure (9) giving a strong indication that the drug lost the crystallinity state and converted to an amorphous form(36) . iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 71 100.00 200.00 temp [c] -5.00 0.00 5.00 mw dsc 117.31x100c figure 8. dsc thermogram of mirtazapine pure powder figure9. dsc thermogram of lyophilized powder analytical rp-hplc method assay for mirtazapine was determined using hplc technology to be compared with the uv spectroscopy. figure (10) shows the hplc chromatogram of mirtazapine as pure powder in the mobile phase. [the retention time of mirtazapine in the hplc chromatogram was 7.141 minutes , for lyophilized powder of mirtazapine nanosuspension for best formula (f15) the retention time in the hplc chromatogragram was 7.129 minutes, as shown in figure (11)]. from the results it was found that no significant difference between the two methods for the assay of mirtazapine pure powder and mirtazapine lyophilized powder. iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 72 figure 10. hplc chromatogram of mirtazapine pure powder figure 11. hplc chromatogram of lyophilized powder for mirtazapine in the selected formula, f15 evaluation of surface morphology atomic force microscopy study (afm) afm is akind of scanning probe microscopes (spm). it is an instrument that measure the properties of surfaces. afm is capable of scanning the surfaces in controlled environmental conditions and is complementary to sem imaging. with the high precision of the afm, in principle it is possible to determine the dimensions of nanoparticles with high accuracy. afm allows the visualization of samples with resolution in three dimensions x-, yand z-directions in atmospheric or submerged conditions. the morphological analysis of mirtazapine pure powder performed by afm showing spherical shaped nanoparticles figure (12) . it was found to be iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 73 stable and no aggregation of particles could be observed (37). the formulation was found to be stable and no aggregation of particles could be observed. the particle size of f15 obtained by afm was comparable to or equal to that measured by abt9000 nano laser and this agreement in particle size measurements provide the good size distribution and the stability of mirtazapine nanoparticles(38), as show in figure (13). figure12. afm of mirtazapine pure powder figure 13. afm of f15 conclusion nano particulate systems such as antisolvent precipitation have a great potential method, being able to convert poorly soluble mirtazapine. mirtazapine nanoparticles were successfully prepared using different types of stabilizers alone and combination of stabilizers at drug : stabilizer ratios 1:1 and 1:2. drug : stabilizer ratio 1:1 was effective to stabilize mirtazapine nanoparticles and the particle size was decrease as the stabilizer concentration increase. the selected formula f15, containing poloxamer 188 and poloxamer 407 as stabilizers combination, showed good entrapment efficiency of 93 % and faster dissolution rate than other formulas and pure drug. selected formula iraqi j pharm sci, vol.29(1) 2020 mirtazapine nanoparticles 74 f15a was prepared as an orodispersible tablet by direct compression method and characterized by acceptable hardness, low friability and produced higher dissolution rate in comparison with the marketed tablet. references 1. sikarra d, shukla v, kharia a, chatterjee p. techniques for solubility enhancement of poorly soluble drugs: an overview, journal of 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mirtazapine. 2018; 43(1): 209249. 37. leedy h. 3-dimensional profile distortion measured by stylus type su clark profilometer. measurement.2013; 4(6): 803– 814. 38. bailey n. characterizations of drug nanoparticles by atomic force microscopy. nsti-nanotechology. 2006; 2(1): 739743. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 levothyroxine treatment optimizaion: a multifactorial study doi: https://doi.org/10.31351/vol29iss2pp245-252 245 levothyroxine therapy adequacy, dose estimation and vitamin d effect assessment in a sample of iraqi female patients with different causes of hypothyroidism ban a. al-shimmran*,1 , zinah m. anwer** and bassam h. al-jarrah*** * ministry of health and environment ,al-rusafa health directorate, baghdad, iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq *** ministry of health and environment , medical city, baghdad teaching hospital, , baghdad, iraq abstract exogenous levothyroxine dose modulation and euthyroidism achievement is a persistent challenge in clinical settings. this study strives to assess the adequacy of treatment and identify the patients’ factors that can be used to estimate the euthyroid levothyroxine dose. a secondary objective was to assess vitamin d supplementation impact on thyroid status. a review of a prospectively collected information from 142 female patients from baghdad center of nuclear medicine from june 2019 until march 2020 who were receiving levothyroxine for different causes was done. after a follow-up period, the patients’ thyroid tests were assessed and the euthyroid doses for each cause category were statistically analyzed. thyroid function was assessed before and after three months of vitamin d supplementation for 29 out of 50 patients who measured its level. sixty-six patients (47%) of the sample were inadequately replaced. iatrogenic causes of hypothyroidism were associated with a higher levothyroxine dose than primary hypothyroidism. bmi was the most significant predictor of the euthyroid levothyroxine dose (r=0.601 and p=0.001 in those with total thyroidectomy). the euthyroid dose was also correlated with duration of treatment, and the presence or absence of iron and calcium supplements.vitamin d supplementation resulted in a significant thyroid-stimulating hormone level decrease (‒ 3.7 ± 4.7 µiu/ml, p-value= 0.001) without affecting thyroid hormones. bmi can be used to predict a levothyroxine replacement dose that is approximate to that required to achieve euthyroidism. vitamin d supplementation is associated with tsh reduction in hypothyroid subjects. keywords: hypothyroidism, levothyroxine, thyroidectomy, vitamin d. في عينة من المرضى العراقيات تأثير فيتامين د دراسةو جرعتهليفوثيروكسين وتقدير بالعالج التقييم بأسباب مختلفة من قصور الغدة الدرقيةالمصابات ***بسام حميد الجراح و **انور زينة مظفر، 1*، بان عبد االمير .العراق ،وزارة الصحة والبيئة ،دائرة صحة بغداد، بغداد * جامعة بغداد.الصيدلة، فرع الصيدلة السريرية، كلية ** .، بغداد ، العراقمستشفى بغداد التعليمي ، مدينة الطب،وزارة الصحة والبيئة *** الخالصة ليفوثيروكسين تحديا مستمرا في الظروف السريرية. تسعى هذه الدراسة إلى تقييم مدى كفاية العالج وتحديد عوامل اليعد تعديل جرعة . كان الهدف الثانوي هو تقييم تأثير مكمالت فيتامين )د( على حالة الغدة الدرقية.تهلتقدير جرعالمرضى التي يمكن استخدامها ، 2020حتى مارس 2019يو نيومن مركز بغداد للطب النووي منمريضة 142تم جمعها مستقبليًا لـ لمعلوماتتم إجراء مراجعة ات الليفوثيركسين إحصائياوتم تحليل جرع يضاتترة المتابعة ، تم تقييم اختبارات الغدة الدرقية للمرالعالج ألسباب مختلفة. بعد ف يتلقين نكالالتي و قاموا ةمريض لتسع وعشرون مريضه من اصل خمسين فيتامين دكما تم تقييم وظيفة الغدة الدرقية قبل وبعد ثالثة أشهر من مكمالت في كل فئة. بقياس مستواه. المنشأ لقصور الغدة الدرقية مع ةعالجيالسباب األ. ارتبطت ال يأخذون ليفوثيروكسين بشكل كافي٪( من العينة 47) ةيضن مروستة وست = pو r = 0.601) الليفوثيروكسين لجرعة عامل متنبئ. كان مؤشر كتلة الجسم أهم األخرىاألسباب مقارنة مع جرعات ليفوثريكسين أعلى .مع مدة العالج ، ووجود أو عدم وجود مكمالت الحديد والكالسيوم الجرعةدة الدرقية(. كما ارتبطت للغ كلي في أولئك الذين لديهم استئصال 0.001 ( دون التأثير على p = 0.001مل ، /µiu 4.7± 3.7)‒إلى انخفاض كبير في هرمون تحفيز الغدة الدرقية فيتامين دأدت مكمالت الدرقية. هرمونات الغدة الغدة لمرضى tsh مستوى . ترتبط مكمالت فيتامين د بتحسينإستبداليةيمكن استخدام مؤشر كتلة الجسم للتنبؤ بجرعة ليفوثيروكسين الدرقية. .د قصور الغدة الدرقية ، ليفوثيروكسين ، استئصال الغدة الدرقية ، فيتامينالكلمات المفتاحية: 1corresponding author e-mail: banoon.cop@gmail.com received: 2/ 4/2020 accepted:9 /8 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp245-252 iraqi j pharm sci, vol.29(2) 2020 levothyroxine treatment optimizaion: a multifactorial study 246 introduction hypothyroidism, being one of the common thyroid dysfunctions, has a global variation in epidemiology with a prevalence ranging from 1 to 2% in iodine sufficient countries(1). it is ten times more likely to affect females than males(2). among a wide range of possible etiologies, the most important are chronic auto-immune disease (hashimoto’s thyroiditis), iodine deficiency and iatrogenic consequences as a result of surgical resection of the thyroid gland to eradicate toxic thyroid nodules, multinodular goiter and to remove thyroid tumors, or after receiving radioactive iodine treatment (rai) for graves’ disease(3). the governments in most iodine-deficient countries have implemented the policy to use iodized salt following the world health organization recommendations, which is also the case in iraq ever since the 1990s. in a study that was conducted on a sample of female iraqi patients with different thyroid disorders in 2010, iodine deficiency was excluded as a major cause, while other aspects such as environmental contaminations and genetic defects were thought to be more substantial(4). regardless of the cause, levothyroxine (lt4) has been the cornerstone of thyroid replacement therapy for a long time because of its several advantages, such as efficacy and low cost (5). the principle of its action is the enzymatic bioactivation of the exogenous pro-hormone t4 into its metabolite triiodothyronine (t3)(6). the goal of treatment is to attain clinical and biochemical euthyroidism with the thyroid-stimulating hormone (tsh) level as a guide to achieve that(5). lt4 is deemed as a narrow therapeutic index drug(7). under-treated hypothyroidism may lead to the untoward consequences associated with the disease such as an impaired health-related quality of life, psychiatric disturbances, cardiovascular diseases, and an increased incidence of type 2 diabetes mellitus(8,9). on the other hand, excessive lt4 treatment can cause tachycardia, atrial arrhythmias, increased risk of atrial fibrillation, and osteoporosis, which are important considerations especially in older patients(6). both cases would be troublesome for the patient and may lead to increased health care costs. throughout the years, numerous factors have been studied for their possible influence on the body requirement and/or bioavailability of lt4. these included age, gender, hormonal status, pathology (benign or malignant disease), co-morbid diseases, supplements use, generic substitution, lean body weight (lbw), body mass index (bmi), body surface area (bsa) and vitamin d (vd) level(10–12). the study aims to assess the lt4 treatment adequacy in a sample of female patients who are receiving lt4 and to find an approach to estimate the correct lt4 dose that keeps the patient in a euthyroid state. a secondary objective was to assess vd supplementation impact on thyroid status. patients and methods a request to conduct the study containing detailed information regarding the study aim and methodology was submitted to medical city directorate/ministry of health which has been reviewed and subsequently approved. this was a prospective cohort study, which entailed enrolling and interviewing all the subjects who matched the study criteria consecutively (table 1) from june 1st until october 2019 at the thyroid counseling clinic of baghdad center for nuclear medicine. each patient was consequently followedup for five or six months. during the interviewing process, a sub-group of 50 patients was randomly chosen for a serum vd level measurement. table 1. criteria for sample selection inclusion criteria exclusion criteria 1adult female iraqi resident. 2able to give fully informed consent. 3receive lt4 for at least two months 1pregnant and lactating. 2acute illness and recent surgery. 3thyroid cancer patients, since they may require to achieve tsh suppression. the interviewing process was conducted face to face. for the objective of a thorough and uniform data collection, a special sheet was prepared in advance to be used in the patient interview. its purpose was to acquire detailed information, including measured anthropomorphic data, sociodemographics, contact information, disease history, co-morbidities, and concurrent medications. the sample was divided into three main categories according to the etiology: postthyroidectomy, post-rai (both of which is considered as an iatrogenic cause of hypothyroidism), while the other causes were labeled as primary. following the interview, thyroid function tests (serum thyroid-stimulating hormone tsh, total t4 (tt4), and total t3 (tt3)) were performed using patients’ serum inside the laboratory of the center on a fully automated advia centaur™ analyzer (siemens, germany). the principle was two-site sandwich immunoassay(13). each tsh reading was tabulated with the corresponding lt4 dose, which was being taken prior to their enrolment in the study, along with tt4 and tt3 levels. the reference range for tsh was (0.4-5.5µiu/ml) while that for tt4 and tt3 was (41.3-162.5 nmol/l and 0.92-2.79 nmol/l), respectively. using the same device, the serum level of calcidiol for 50 patients was iraqi j pharm sci, vol.29(2) 2020 levothyroxine treatment optimizaion: a multifactorial study 247 measured based on a one-pass 18-minute competitive immunoassay and a level of 30 ng/ml or higher was considered to be sufficient(14). lt4 dose was adjusted by physicians empirically. medication counselling regarding the correct way of administration was provided to the patients so as to obtain a maximum benefit for the patients in the future and optimal results for the study in the upcoming tests. these included encouraging adherence, taking the drug at least 30 minutes prior to breakfast and minimizing caffeine in the subsequent meal, also, separating the medications that are proven to interact with lt4 such as ferrous and calcium salts by at least four hours and using the same drug brand consistently if possible. all the key information was then organized into a database along with the calculated bmi [actual body weight (kg)/height2 (m2)](15). the database was being updated throughout the followup period. the follow-up was performed online using the patients’ given contact information through social media and included their most recent thyroid function tests’ results at the center, new weight, and medication counseling. all the statistical analyses and related graphs were developed using ibm spss® software version 23. categorical data were presented as frequencies and percentages while continuous data were summarized in the form of mean(𝑋) and standard deviation (sd). a value ≤ 0.05 was assumed for statistical significance and the rejection of the null hypothesis. the thyroid function tests’ results were analyzed and compared between the study groups during the initial encounter and at the end of the follow-up. analysis of variance (anova) and paired t-test were applied for comparisons wherever applicable. out of the entire database, euthyroid doses were selected and linear regression models were generated for each of the studied groups. the euthyroid lt4 dose was considered the dependent variable and the other factors (bmi, age, duration of treatment, presence or absence of diabetes, hypertension, anemia, and supplements use) were included as independent variables. the fittest models and their coefficients were identified. results patients’ characteristics a total sample of 142 female patients was recruited and followed-up in the study. the group distribution was: 76 with primary hypothyroidism, 52 post-thyroidectomy, and only 14 as a result of rai ingestion. the age range was 18-72 years with a mean of 44 ±11.5 years. the calculated bmi had a mean of 30.7 ±6.02 kg/m2 and followed gaussian distribution. bmi was very significantly correlated with age (r=0.238, p=0.000). the period of time they were on lt4 had a mean of 51.7 ±70 months. a large proportion of the patients (68.3%) were suffering from one or more types of comorbidities (table 2). twenty-six patients were taking iron and/or calcium supplements, eighteen of which had primary hypothyroidism. table 2. patients’ characteristics and statistics total sample: 142 female patients bmi: body mass index, lt4: levothyroxine, rai: radioactive iodine descriptive statistics �̅� ∓ 𝐒. 𝐃 age (years) 44 ± 11.5 weight (kg) 78.2 ± 16.2 height (cm) 159.8 ± 5.8 duration of lt4 treatment (months) 51.7 ± 70 bmi (kg/m2) 30.7 ± 6.02 characteristic no (%) etiology primary causes 76 (53.5%) post-thyroidectomy 52 (36.6%) post-rai 14 (10%) co-morbidities musculoskeletal diseases 58 (40.8%) hypertension 40 (28.2%) iron deficiency anemia 20 (14.1%) diabetes mellitus 11 (7.7%) initial encounter data only 76 patients (53%) were within the tsh reference range, while 53 were under-replaced and 13 were being over-replaced. the replacement dose was the only factor with a statistically significant difference between the study groups (p <0.001). iraqi j pharm sci, vol.29(2) 2020 levothyroxine treatment optimizaion: a multifactorial study 248 table 3. lt4 dose and biochemical characteristics during first encounter total number: 142 patients �̅�: mean, s.d.: standard deviation, rai: radioactive iodine, anova: analysis of variance, tsh: thyroid stimulation hormone, tt3: total triiodothyronine, tt4: total thyroxine, lt4: levothyroxine. * significant (p-value ≤ 0.05) end of follow-up data euthyroid results were obtained for 118 patients. unlike the post-rai group, the primary and post-thyroidectomy groups had a significantly higher mean dose after the follow-up as compared to the first encounter (p-value= 0.019 and 0.037, respectively). the dose and tt4 were significantly different among the study groups (table 4). table 4. lt4 dose and biochemical characteristics at follow-up total number: 142 patients �̅�: mean, s.d.: standard deviation, rai: radioactive iodine, anova: analysis of variance, tsh: thyroid stimulation hormone, tt3: total triiodothyronine, tt4: total thyroxine, lt4: levothyroxine. * significant (p-value ≤ 0.05) euthyroid dose estimation the model with the most significant euthyroid dose predicting power for the primary causes group was obtained with the dose per kg as the dependent variable and the bmi (r= ‒0.297, p= 0.014), duration of treatment (r= 0.260, p= 0.033), and supplements (iron and calcium) (r= 0.312, p= 0.012) as the independent variables. the resultant model had an r and r2 values of 0.51 and 0.26, respectively. a linear equation was derived: y= 0.026x1 + 0.004x2 + 0.271x3 + 1.482 where x1 is the bmi, x2: the duration of treatment in months and x3 is either 0 or 1, indicating the presence or absence of concurrent supplements. the relationship between the dependent and the independent variables was linear and not quadratic until a certain point, (figure 1). the mean euthyroid dose was 73±35 mcg/day (1±0.5 mcg/kg/day). , a b figure 1. regression analysis curves for primary causes of hypothyroid patients a: body mass index (kg/m2) vs dose mcg/kg. b: duration of disease (months) vs dose mcg/kg. post-operative primary post-rai anova p-value �̅� ± 𝑺. 𝑫. serum tsh level (µiu/ml) 13.6±30.7 5.8±7.2 6.6±5.8 0.079 serum tt3 level (nmol/l) 1.56±0.36 1.63±0.4 1.64±0.38 0.632 serum tt4 level (nmol/l) 120.93±43.62 113.27±34.56 140.3±52.63 0.106 lt4 dose (mcg/day) 100.0±40.3 70.2±33.8 113.3±38.2 0.000* post-operative primary post-rai anova p-value �̅� ±𝑺. 𝑫. serum tsh level (µiu/ml) 7.7±21.8 3.6±3.4 4.5±4.4 0.234 serum tt3 level (nmol/l) 1.56±0.61 1.67±0.47 1.73±0.32 0.774 serum tt4 level (nmol/l) 124.05±43.4 109.97±30.9 149.75±46.02 0.001* lt4 dose (mcg/day) 107.2±42.4 76.8±35.0 110.7±37.6 0.000* iraqi j pharm sci, vol.29(2) 2020 levothyroxine treatment optimizaion: a multifactorial study 249 the number of euthyroid patients who had been receiving lt4 post-rai treatment was very insignificant (only 10), the regression model was dismissed. the mean euthyroid dose was 110±41 mcg/day (1.5±0.5 mcg/kg/day). in the post-thyroidectomy population, the lt4 dose correlation with time since the surgery was variable. those who have had the surgery within the prior year had the strongest correlation (r=0.539, p=0.003) but after that, the correlation dissipated and became very insignificant and hence two models were created. the most powerful model for those who have had the surgery within a year was obtained by using the dose (mcg) as the dependent variable versus time since surgery and bmi (r=0.601, p=0.001) as the predicting variables. the resultant r-value was=0.762 and r2 was=0.581. the equation derived was: y = 4.072x1 + 2.613x2 – 58.061. where y is the predicted dose (mcg/day), x1 and x2 represent the bmi and time since surgery (months) respectively. the mean euthyroid dose was 104±39 mcg/day (1.2±0.4 mcg/kg/day). while in those who had thyroidectomy more than a year ago, the highest predicting power model revealed that only the bmi had a significant contribution to the prediction model (r=0.658, p=0.000). the model had an r-value of 0.612 and r2 of 0.354, the equation was: y= 3.991x8.991 where y represents the predicted dose (µg/day) and x is the bmi of the patient. the mean euthyroid dose was 111±31 mcg/day (1.4 ±0.3 mcg/kg/day). a change in the linear relationship into a rather quadratic one was noted in the post-operative group when bmi was above 45 kg/m2, (figure 2). the same change in the relationship was also seen in the primary hypothyroid group model below a bmi of 20 kg/m2 and when the bmi reaches 40 kg/m2 and beyond (figure 1). this could partly be due to a limited number of patients beyond those values and thus the study models and equations cannot accurately estimate the dose in these cases. the mean doses and prediction equations for the entire sample are summarized in table 5. figure 2. body mass index (kg/ m 2) vs dose (mcg) regression curve in post-thyroidectomy table 5. euthyroid lt4 dose summary lt4: levothyroxine, rai: radioactive iodine, bmi: body mass index, t: months (treatment duration or time since surgery), s: 0 or 1 corresponding to the presence or absence of concomitant supplements vitamin d supplementation effect on thyroid status the mean serum vd level for the 50 patients who have measured it was 16.5ng/ml ±13.6. forty-one patients were deficient (<30 ng/ml) and were prescribed cholecalciferol 5,000 iu daily. out of those 41 patients, 29 had a new follow-up thyroid function test after receiving the supplement for at least three months. two patients had a tsh reduction but still fell outside the reference range, nine achieved euthyroidism, 13 had a reduction within the “normal range” while five had a small tsh increase within the reference range. while controlling for lt4 dose, the resulted tsh difference was significant without affecting thyroid hormones (table 6). group mean dose prediction equation model prediction accuracy 1.primary causes 73±35 µg/day 1±0.5 µg/kg/day lt4 mcg/kg/day= ‒ 0.026(bmi)+ 0.004(t) + 0.27(s) + 1.5 26% 2.post-rai 110±41 µg/day 1.5±0.5 µg/kg/day unattainable due to low number 3.post-operative (<1 year) 104±39 µg/day 1.2±0.4 µg/kg/day lt4 mcg/day = 4(bmi) + 2.6(t) – 58 58% 4.post-operative (≥1 year) 111±31 µg/day 1.4±0.3 µg/kg/day lt4 mcg/day = 4(bmi) 9 35% iraqi j pharm sci, vol.29(2) 2020 levothyroxine treatment optimizaion: a multifactorial study 250 table 6. vitamin d supplementation effect tsh: thyroid-stimulating hormone , p-value for paired t-test, 29 patients , *significant difference, less than 0.05 discussion serious adverse effects associated with hypothyroidism treatment can be avoided by proper lt4 dosing along with close monitoring of serum tsh values in conjunction with clinical evaluation during therapy and adjusting the dose accordingly(16). on the first encounter, the current study showed that in this sample, 47% were either under or overreplaced due to incorrect dose. the same ratio has been repeatedly reported in various populations and persistently for a long time.(1,17–19) okosieme et al. reported that over a third of lt4 treated patients (37%) in united kingdom had abnormal tsh values. but in contrast to the current study, the majority were over-treated in that one(17). the postoperative patients were particularly difficult to control, demanded higher doses, more frequent testing, and dose adjustments. as seen in tables 3 and 4, even after multiple frequent physician visits, their tsh still fell outside the reference range (7.7±21.8 µiu/ml). iatrogenic hypothyroidism required higher doses compared to primary causes because of the total removal or destruction of thyroid tissue in the iatrogenic causes, whereas in the primary group there is a remaining functioning thyroid gland that produces intrinsic hormone and decreases exogenous lt4 requirement(16). the postoperative and post-rai also required a higher tt4 concentration in order to achieve a similar tt3 to the primary group. since the thyroid hormones are secreted normally from the thyroid gland with a t4/t3 ratio that is 14:1 and these groups lack their innate thyroid and the 6.5 μg of t3 from the normal gland(20). in the current study, it was found that among all the studied factors, bmi was the most significantly associated with the euthyroid lt4 dose. however, the correlation was stronger in the post-thyroidectomy population and the regression models produced a higher r and r2 values than the primary causes. this means that in the primary causes patients who have a remaining functional thyroid gland, bmi had a smaller influence on the required dose due to their different confounding physiological variables that affect the replacement dose(5). when bmi was correlated with lt4 dose per kg in the primary causes group, the correlation was negative, i.e., the higher the bmi the lower the calculated dose per kilogram of body weight. this relationship between bmi and lt4 dose can be explained by the fact that adipose tissue has a small influence on lt4 requirements and that lt4 consumption is mainly accomplished in the lean body compartments(21). this also explains why weight-based dosing fails to optimally replace hypothyroid patients. other studies that attempted to use bmi to predict lt4 dose were mostly conducted on patients with total-thyroidectomy. ojomo et al.(11) established a linear relationship between bmi and euthyroid dose until bmi of 50 kg/m2 and derived an equation to estimate an initial lt4 dose for totalthyroidectomy patients (mcg/kg/d= ⁻0.018×bmi +2.13). di donna et al.(22) also used bmi in addition to age to predict the post-thyroidectomy lt4 dose and proposed an initial dose of 1.4-1.8 mcg/kg/day. jin et al.(23) found that a dose of 1.5 μg/kg/day was optimal while 1.3 μg/kg/day for patients who are ≥55 years or bmi ≥30 kg/m2. maghsudi et al. compared the efficacy of actual weight, lbm, and bmi based dosing and concluded that bmi had the strongest correlation with lt4 dose and derived a dose calculation formula: y= –0.013 +0.005 bmi for benign pathology in total thyroidectomy patients(24). on the other hand, al-dhahari et al. (25) identified body surface area (bsa) as the most significant predictor and proposed a model accordingly. the duration of treatment was also a significant lt4 dose predictor, which was probably influenced by the necessary dose titration while starting lt4 therapy(5). another explanation is the progressive autoimmune thyroid destruction with time(26). however, its effect on the calculated dose was low. the abundance of euthyroid patients who were being treated for primary hypothyroidism and receiving supplements concurrently with lt4 among this study sample demonstrated their effect on the lt4 dose. all three calcium salts (carbonate, citrate, and acetate) are confirmed to decrease the absorbed lt4 dose by 20-25%(27). iron sulfate is also notorious for its negative effect on lt4 absorption(28). despite the patients’ claiming compliance with the instructions of four hours of before supplementation three months after supplementation difference p-value mean ± standard deviation tsh (µiu/ml) 5.5 ± 4.9 2.3 ± 2.1 -3.2 ± 4.7 0.001* lt4 dose mcg/day 82.7 ± 36 81.4 ± 36.3 1.2 ± 25 0.783 tt3 (nmol/l) 1.5 ± 0.3 1.6 ± 0.4 0.04 ± 0.3 0.555 tt4 (nmol/l) 126 ± 41.2 129 ± 42.4 3.5 ± 41.8 0.685 iraqi j pharm sci, vol.29(2) 2020 levothyroxine treatment optimizaion: a multifactorial study 251 separation between the two, they were associated with a higher lt4 dose. no clear explanation exists for this but unconfirmed compliance may play some part. although, a case has been reported with increased lt4 requirements despite their separation(29). the current study demonstrated the positive impact of vd supplementation on thyroid status and specifically decreased tsh levels. the mechanism by which this occurs is still uncertain. a study by zhang et al. also showed that higher vd status was linked with lower tsh after governing the other factors like age, thyroid hormones, gland tissue volume, and presence of nodule(s), (30). a recent clinical trial also showed that three months of 50000 iu weekly vd supplementation produced a significant reduction in anti-thyroglobulin antibodies and tsh levels compared to placebo(12). the most important limitation that may have hampered the study from obtaining even more accurate results is the absence of electronic medical records or any type of registry containing numbers, clinical or demographic information of the hypothyroid patients which made the process of information gathering rather tedious and less efficient and prevented the collection of high amount of data in order to produce more powerful models and to include the minority of male patients. little information regarding thyroid antibodies levels was also a limitation. conclusion under-treatment is very common among hypothyroid iraqi female patients. bmi is significantly correlated with the euthyroid dose and can be used to estimate a dose approximate to that required to achieve euthyroidism. three months of vd supplementation is associated with decreased tsh levels 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http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 sex hormones chan ges 23 the chages in sex hormones in female working in battries manufacturing plant mohammed a. taher *1 , salim a.hammadi** , ali a.ali** *de pa rtme nt clinica l labo ra tory sc ie nces, co lle ge o f ph armacy ,univ ersity of bag hdad, baghd ad -ira q. ** depa rtm ent clin ical pha rm acy , c olle ge of pharm acy ,un iversity of baghdad, baghdad -iraq abstract lea d has toxic e ffects on reproduction of both male and fema le . it can c aus e dec re ase d sex drive , infertility a nd abnormal menstrual cycle in women. this study wa s des igned to eva luate the effec t of e xposure to lea d in batte ries female workers on s ex hormones leve l in the s erum.thirty nine (39) fe male worke rs (voluntee rs ) in iraqi batte ries manufa cturing plants, al-waziriya / ba ghdad we re pa rticipated in this study. the y a re clas sified into 3 groups, first group inc luded fourteen (14) fema le tha t have be en employed for 1-7 yea rs , se cond group include d thirte en (13) fema le that have bee n employe d for 8-14 yea rs , third group included twe lve (12) fema le have be en employed for15-22 years and and fourtee n fe males were include d as the control . blood lead leve l, serum fsh, lh, prolac tin and total te stosterone we re me asured a nd compared for a ll subje cts.the results indica te d that me an of blood lead leve ls (bll), te stos te rone levels were highly s ignificant in a ll worke r groups compared to the control (p<0.005).prolac tin le vels in group i a nd fsh in group iii were signific antly highe r than that in c ontrol ( p< 0.005) a nd (p<0.05) res pectively. lh leve ls in groups ii and iii were signific antly higher than that in control (p<0.05, p<0.005 res pec tive ly). high incidence of hirsutism (48%) and mis ca rria ges (50%) were obs erved in worker groups compared to c ontrol (11%). the res ults indica te d that there are hormonal changes in fe male workers expos ed to lea d as sociated with increa sed inc ide nce of hirs utis m a nd misca rria ges compared to non expos ed fe males . k ey words: lead , sex hormones hype randroge nemia لخالصة ا عات قسمت العامالت ن في هذه الدراسة كمتطو ريات في بغداد شارك مل صناعة البطا ن امرأة عاملة في مع عة وثالثو تس ص رصا رة التعرض لل مادا على فت ميع اعت جا .الى ثالث م ملة وفترة الخدمة تتراوح بين ) ١٤(المجموعة األولى تشمل ۱ ر سنوات) ۷ـ١(عا .سنة) ٥۰ـ٢٤(وبعم عة الثانية تشمل ۲ مة تتراوح بين ) ۱۳(ـ المجمو خد رة ال سنة ) ٥٥ـ۳۰(سنة وبعمر ) ۱٤ـ۸(عاملة وفت عة الثتلثة تشمل ۳ راوح بين ) ۱۲(ـ المجمو .سنة) ٥۲ـ۳۲(سنة وبعمر ) ۲۲ـ۱٥(عاملة وفترة الخدمة تت رابعة٤ ۱±۲۹( امرأة بمتوسط عمر) ۱٤(مجموعة السيطرة وتشمل : المجموعة ال .نةس) ۷. ها بمجموعة ر في مستوى الهرمونات الجنسية عند النساء العامالت عند مقارنت غي ن هنالك ت وجد في هذه الدراسة ا رة طويلة .السيطرة ص لفت رصا عامالت اللواتي تعرضن لل ستوى الهورمون اللوتيني في المصل يزداد عند ال كذلك ان م وجد عن ريب في المصل يزداد فقط رة طويلةاما هورمون محفز الج ض لفت ن .د النعر والكتين (اما هورمو عد ) البر فيزداد فقط ب ض لفترة قصيرة للرصاص حبا مع زيادة في .التعر زداد عند النساء العامالت متصا كذلك هورمون التيستوستيرون الكلي ي مقارنة بمجموعة السيطرة .نسبة الشعرانية واالسقاطات عند العامالت 1c or res po nding au th or e mail moha mmed – ta her 4 3 @ya hoo .c om rec eived 21 -11-200 5 acc ep te d 7-10-2006 ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 sex hormones chan ges 24 in troduction some sc ie ntis ts cautioned the romans of the da nge r of inhaled fume s from lea d sme lting (1) with the industria l re volution, le ad po is oning be came a common oc cupationa l problem .the re productive effe cts of lea d p oisoning were also noted by the turn o f the ce ntury and many artic le s d es crib e the high rate of s tillb irths, infe rtility and a bortio ns among wo men in the pottery ind us try, o r who we re married to pottery worke rs (2). the mos t imp orta nt route of ab sorption in o ccupa tional s etting is thro ugh inha la tion o f lead d us t and fumes . in add ition, worke rs ma y ea t, drink or s moke in lea d-dus tco ntamina te d areas re sulting in so me inges tio n as we ll. s to ra ge ba ttery ma nufac ture inv olve s co nside ra ble exp os ure to lead o xide dust, in ad dition to fume s from we ld ing o f battery co nne ctor (3) . lea d c an ca use de crea sed se x d rive and infe rtility in wo men. in a dditio n, it c an ca us e ab normal me nstrua l c ycles (dys menorrhea, menorrhagia and ame no rrhe a), prema ture birth, sp ontaneo us misc arriages , and stillbirths . the incide nce of p olyme norrhea , prolonged and ab normal mens truatio ns , hypermenorrhea wa s significantly higher in the lea d expos ed group (female workers of le ad ba tte ry p la nts) than in co ntro ls (4). this s tudy was des igne d to ev aluate the hormonal changes (lh, fsh, prolactin and tes to sterone) in w omen whom expos ed to lead in b atte ries ma nufa cturing plants through inhala tion and d irec t contact with a ctiv e co nstituent of batterie s. subjects and methods this s tudy was carrie d out on female worke rs emp lo ye d in iraq i b atterie s manufac turing p la nts, babylo n 1 and 2 in alwaziriya / baghda d for the p eriod o f thre e months from j anuary to april of 2 005 . thirty nine (39) fema le workers were pa rtic ip ated in this study , they work 6 ho urs pe r d ay eve ry othe r d ay and had b ee n emp lo yed for at le ast 1 ye ar in the p la nt . the subjec ts we re c la ss ified into 3 gro up s ac cording to the dura tion o f expos ure to lea d (e mployment in the plant) a s follo w: group і ,inc lude s fourtee n (1 4) female that ha ve be en emp lo ye d for1-7 ye ars with ra nge age (2 4-5 0) yea rs (29.2± 7 .2 ) ye ars. group п, inc ludes thirte en (1 3) female that ha ve be en e mploye d for 8-14 yea rs with ra nge age (3 0-5 5) yea rs (37.9± 7 .9 ) ye ars. gro up ш includ es twelve (1 2) female that ha ve bee n e mployed fo r 15-22 yea rs with ra nge (32 -52 ) years (4 1.1± 6) yea rs . fourtee n he althy wome n, not e xpo se d previously to lea d with a ge range (2 4-5 0 ) ye ars (2 9±7 .1 ) were utilized as c ontrol. individual q ue stio ne r protoco l was followe d fo r all wome n conce rned with gyne cologic al and o bste tric al his tory including marrie d or not , age o f marriage , number o f children , type of de live ry (no rma l o r ca es are an s ec tion) , numb er of mis ca rriage s, growth o f the ir infa nts , if they work d uring p re gna nc y , re gular or irregular me nstrua l cyc les , if they are with ame norrhea , d ysmeno rrhe a or meno rrha gia . ap propriate da y fo r ea ch fema le (b etween 2 nd a nd 5th d ay of mens trua l cycle) was se le cted to co ns id er follicle phas e for fsh and lh as sa y , and female were ad vised to fas t 1 2 hr. be fo re s amp ling , fo r ap pro pria te ana lysis of prolactin . blood s ample s (1 4 ml) were drawn from ea ch p atie nt a nd c ontrol by vein puncture left to c lo t a nd se rum was sep arated b y ce ntrifuga tion. blo od lea d le vels we re meas ured using the slotte d q ua rtz tube metho d.(5).lh ,fsh and prola ctin le ve ls in se rum we re a na lyze d using ra dioimmunoas sa y method s(6,7,8) while te stos te ro ne was as se ss ed ac cording to the metho d o f abraha m e t al (9). all thes e kits sup plie d by immunotec h , a beckman c oulter comp any (france). inde pendent t-te st wa s us ed to examine the difference in the mea n of control and wo rkers, also the d ifferenc es amo ng worke r gro ups thems elves .p-v alue s < 0.05 we re considered as signific antly diffe re nt. pe ars on c orre la tion (r) was pe rformed to find re la tio nship be tween exp os ure time and tes toste rone leve ls . results table (1) s hows the a ge s and o cc upa tional (e xpo sure) p erio ds of fe male worker. the numb er of ma rried were 3 ,6 ,7 in i, ii, iii group s re spe ctively .the me an a ge of c ontrol group wa s 29.1 ±7 .1 yea rs a nd the numbe r of married wa s 5 . table (2)shows me an b lo od lead le vels in all group s of wo rkers are signific antly (p<0 .0 05) highe r tha n that of the c ontrol gro up. gro ups ii and iii worke rs hav e significantly higher blood le ad le vels than that of group i worke rs (p <0.05 ) . se rum to ta l tes to sterone le vels in a ll thre e worke r group s were signific antly higher in c ompa riso n with that of the co ntro l group (p<0.005 ). total te stos te ro ne lev el for gro up i workers wa s the highes t one. howev er gro up i workers ha ve a s erum to ta l tes to sterone lev el that was significa ntly higher than that of group s ii and iii workers (p<0.05).mean prola ctin le ve l in group i worke rs sho ws highly signific ant inc re as e c omp ared to tha t of the control group (p<0.005 ).while mean p ro la ctin le vels in gro ups ii and iii were nonsignific antly elev ated co mpa re d with the control group (p>0 .0 5).group i worke rs have prola ctin le ve ls ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 sex hormones chan ges 25 significantly higher than that of groups ii and iii of wo rkers (p<0.05).mean f sh lev el of group iii workers was s ignifica ntly e le va te d (p <0.05 ) in comparison with that of the control group. while me an fsh le vels of wo rkers group i and ii we re non-significa ntly e le va te d (p >0.05 ) comp ared to co ntro l group . .f sh le ve ls s how no n significant diffe re nce a mong worke r group s thems elve s .me an lh leve l in group ii wo rkers was e le va te d signific antly in co mpared to that of the c ontrol group. lh le ve l in group iii wo rkers s ho ws a highly signific ant increa se compared with c ontrol group (p<0 .0 05).furthermore mea n lh lev el in group i workers wa s non-s ignifica ntly eleva te d (p>0 .0 5) c omp ared to tha t of the control group .lh lev els show non s ignifica nt (p>0.05) d iffe re nc e a mong worke r gro ups thems elves . there are non s ignifica nt differences a mong lh/f sh ra tio in a ll three group s of wo rkers and contro l group (p>0.05).als o lh/fs h ratio show non signific ant (p >0.05 ) diffe re nce a mong w orker group s. table (1)de mo graphic data of wo rke rs and c ontro l wo me n. table (2)se rum le ve ls o f le ad, tes toste rone , prolac tin, fo llicle stimulating hor mone (fs h) le utiniz ing hor mone (lh) in fe ma le s working in batte ry indus trie s. parame te rs control group i gro up ii group iii le ad(µg/d l) 13 .4±4 .5 2 1.4±6.7** a 31 .6±7 ** b 29.7±7.3** b testos te ro ne (ng/ ml) 0 .156±0.5 0.45 8±0 .15**a 0.35 6±0 .13**b 0.28±0.1** b prolac tin (ng/ ml) 8.5± 4.1 19.7 ±11 .9 ** a 1 0.7±7.2ns b 11.7±8.1 ns b f sh (miµ/ ml) 6.9± 2.7 13.3 ±14 .8 ns a 10.9±8.4 ns a 14.4±13.6* a lh ( miµ/ml) 8.8± 3.3 1 1.1±3.7 ns a 14.7±10.4* a 17.3±9.1** a lh/fs h 1.2±1.64 0.83 ±0.45 ns a 1.3±1 ns a 1.2±0 .85 ns a value s are expre ssed as mea n±sd * p<0 .0 5 significant diffe re nce fro m c ontrol g roup **p<0.005 high ly s ig nifica nt diffe re nce from co ntrol gro up values with diffe re nt le tters (a,b) a re s ig nifiga ntly d iffe re nt (p<0.05). figure (1) s ho ws the co rrelation be twe en time of le ad expos ure and se rum te stos te ro ne lev els in fe male workers.a ne gative c orrela tion wa s fo und b etween time of lea d e xp osure and se rum te stoste ro ne le ve ls (r=-0.43), (p<o.o5). in table (3) all wo rkers groups hav e perce ntage o f hirs utism (17 .9 %) and mis ca rria ge s (5 0%) higher than that in c ontrol group . in ta ble (3) all worke rs gro ups have perce ntage o f hirs utism (17 .9 %) and mis ca rria ge s (5 0%) higher than that in c ontrol group . table (3)distribution o f fe male worke rs with hirs utis m a nd miscarrige s groups age (me a n ±sd) (yea rs ) oc cupatio n pe riod (me an ±sd)( ye ars) numbe r of marrie d numbe r of unmarrie d total i 2 9.2±7 .2 3.6±1.9 3 11 14 ii 3 7.9±7 .9 1 1.1±1.4 6 7 13 iii 4 1±6 1 8.2±2.8 7 5 12 control 2 9.1±7 .1 9 5 14 va riable s gro up i no. (%) group ii no. ( %) gro up iii no. ( %) total co ntrol hirs utism 2 (14) 2(15 ) 3(25) 7(17 .9 ) 0 (0 ) miscarriages * 1 (33) 3(50 ) 4(57) 8 (50) 1(11) ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 sex hormones changes 26 time of exposure (years). s e ru m t e st o st e ro n e ( n g /lm l) . 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 -2 2 6 10 14 18 22 26 discuss ion the re sults indica te d that mea n of blo od lea d le ve ls (bll) were s ignifica ntly highe r in a ll worke r group s comp are to the c ontrol group (p <0.00 5),this may be d ue to lo ng term expos ure to le ad oxid e which use d in b atterie s manufac turing p la nt. the wo rkers practice in impro pe r environment, they we re not we aring fa ce ma sk, clov es with b ad ve ntila tion ins id e plant ro oms , in add ition to that wo rkers were ea ting ins id e the manufac turing rooms, whic h mea ns the y d id not follo w the co nd itions of oc cup atio na l sa fe ty. the wo rkers in group i ha d blood lea d le vels s ignifica ntly lo wer tha n that in gro ups ii a nd iii ( p< 0.05 ),this may b e due to longer e xpo sure time in groups ii and iii tha n that fo r group i it has b een re porte d that pa tients with bloo d le ad leve ls les s than 4 5µg/d l do no t re quire chelatio n therapy (10). the me an bloo d lea d le ve ls in mo st fe male worke rs a re le ss tha n that re ported in prev io us stud ie s (11,12 ), b eca us e fe males in prese nt s tudy are working ev ery othe r da y in the plant sinc e 1.5 ye ars ,b eca us e of s ho rtage of th raw ma te rials the me an se rum tes to sterone le vels in a ll worke r group s were signific antly higher tha n that of co ntro l gro up (p<0.005 )(table 2 ). figure (1) d emo nstrated a negativ e correlatio n be twe en time o f expos ure to lea d and s erum te stos terone lev els in worker group s .the d ata showed a n inve rs e re la tionship be tw een time of le ad expo sure a nd s erum te stos tero ne lev els, this may need further inves tigatio ns to explain the correlatio n. the sa me res ults hav e bee n re ported by sokol-rz (13), where the rats trea ted with v ario us do se s of lea d ace ta te fo r more tha n 1 wee k e xhibited a s ignifica nt increas e in gona dotropine relea sing hormone (gnrh) mrna, b ut with a ttenua tion of the increas e at highe r co nce ntratio ns of le ad with increas ed d uratio n of exp os ure.the y co ncluded that the s igna ls within a nd betwe en the hyp otha la mus a nd pituita ry gla nd a ppe ar to be disrupted b y long-term, le ad expos ure. lh s timulate s the ca cells o f ova ry to pro duce tes to sterone in female (14).ho weve r, in this s tudy, it ha s bee n fo und tha t as lh lev el increas es , tes to sterone le vel d ecrea se s (tab le 2), indica ting tha t elev atio n of tes to sterone le vel in fema le wo rkers may b e extra ov aria n. howev er most of stud ie s co nce rning lead we re app lied o n a nimal mode ls s o it is d iffic ult to comp are p re se nt findings with their res ults. gro up i hav e a high mean p rolac tin leve ls which are signific antly higher than tha t of control group ( p< 0.00 5); while mean pro la ctin le vels in group ii a nd gro up iii we re not s ignific antly different from co ntro l group ( p>0 .0 5) (table 2) .prolac tin relea se fro m the pituitary is und er tonic inhibito ry c ontrol fro m hyp otha la mus -de rive d dop amine or p ro la ctin inhibito ry fac to r (pif). thyrotro pin-re le asing hormone (trh) in turn is s timula to ry to pro la ctin relea se .estro gen c an dire ctly sensitize the pituita ry to re le as e p ro la ctin (15). the elev atio n of prolac tin le ve ls in w orker group s could b e attribute d to tha t le ad a ffec ts dop amine rgic co ntro l of prolac tin sec re tion from pituitary gland, acc ording to p re vious study on rats (16). the results d emonstra ted that only group iii workers hav e me an serum fsh lev el signific antly highe r than that o f the c ontrol group (p< 0 .0 5) (ta ble 2 ).the e le vation in serum fsh als o rep orte d b y ng et al. (17) where significantly higher s erum fsh and serum lh lev els were o bse rv ed in lead -b atte ry male workers , d uring le ss tha n 10 yea rs exp osure p erio d , whe re as tho se expo sed for 10 ye ars o r more showed normal s erum lh and s erum fs h conce ntra tions.howe ve r, viv oli g et al(18) re ported nega tive re la tionships betwee n blood lea d le vel and lh and f sh in ma le s with lea d le vels higher than 9µg/dl. group i worke rs a re pres ente d with se rum lh not significantly different from that in control group (p> 0.05 ) while group ii a nd iii hav e mea n serum lh s ignifica ntly highe r than that in co ntro l group (p <0.05 ,p<0.005 re spe ctiv ely) (tab le 2 ) . this elev ation in se rum lh le vels are in agree ment with that rep orte d by rod amila ns e t al.(19) who rep orte d tha t in le ad-sme lte r workers, s erum lh lev els a re signific antly raise d, a s co mpa re d with c ontrols. figure ( 1 ) : co rre latio n be twe en time of le ad ex pos ure and se rum testos tero ne . ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 sex hormones changes 27 furthe rmo re yen (20) sugge sted tha t ba sal lh and f sh le ve ls gra dua lly ris e after the age of ye ars 35 and c ontinue to climb until se veral ye ars after onse t of menopa use due to dec rease ne gative fee dba ck c ontrols on p ro ductio n. ronis et a l. (21) s tated that, in fema le ra ts exposed to le ad prep uberta lly , dela y vaginal opening with mo re se vere reproduc tive disrup tion, acco mpa nied by suppression of circ ulating es tradiol .effe cts on c irc ulating se x steroids were accomp anie d by va riab le e ffec ts on circulating lh le ve ls , p ituitary lh, and pituita ry lh b eta-mrna. inc re ase in hypotha la mic le vels of gn rhmrna, and a n increase in pituita ry leve ls of lh mrna and pituita ry s to res of lh in lead dosed a nimals. this inc re ase due to lead disrup ts the re productive axis by interfe ring with fe edb ac k mec hanisms a t the hypotha la mic and p ituitary levels (22). in this s tudy the re sults indica ted that lead expos ure has no e ffec t on lh/fs h ratio in fe male workers .elevated basa l lh with a n lh/fsh ratio >2 and some increase o f ovaria n androge n in a n esse ntia lly no n ovulatory adult wome n is p resumptiv e evidence of polycystic ovary s yndro me. a high se rum f sh to lh ra tio (1 .9 to 3.8) has been obse rved in post menopa us al wo men. hyp ogonadism is usually associated with increased bo th f sh a nd lh levels ; while dec reased fs h and lh ma y occur in p ituitary or hyp otha la mic failure (23). tab le (3) showed high incidence o f hirs utism (4 8%) in wo rking women in batteries plant than in co ntro l group (0%). als o pe rcenta ge of hirs utism in female worke r groups increa sed with inc re asing expos ure time to lea d. s ome of worke rs had hirsutis m, the ir s erum tes to sterone levels we re high, and this typ e o f hirs utism se ems to be a nd ro gen induced hirs utism (hyp era nd ro genism). in this stud y in s pite of decreased se rum tes to sterone levels with prolonged e xp osure ti me (fig.1), pe rcenta ge of hirs utism inc re ase d as time of exp os ure increase d ( table 3), so it ca n be conc luded that hirsutis m may d epe nd on many fa ctors othe r than and ro ge n (te stos terone ) le ve ls in fe male exp osed to lead .whe n hirs utis m is associated with o besity a nd menstrual ab normalities , the sourc e of a nd ro gen e xc ess is ofte n ov aria n, typica lly p olycystic ovary syndrome . whe n it is a ss ocia ted with average weight and no rma l menses, the source is ofte n ad re nal and ra re ly (in <5 % o f cases) pituita ry(24). tab le (3) showe d increa sed incide nce o f mis carria ges in fe male worker groups (50%) c omp ared to control group (1 1%). le ad has toxic action o n the trop hob lastic ep ithe lium and to nic contra ctio n of the uterus . it the refore results either in abortion o r a de ad fetus (25). in this s tudy whe n lead expos ure time increased , perce ntage of misc arriages also increased (tab le 3).much of the previous litera tures focuse d o n an increase d incide nce of sponta neo us abortio n a nd stillb irth assoc ia ted with le ad exposure in the workp lace (25 , 26).other s tudies hav e examine d the is sue of lead's invo lv eme nt in s pontaneous abortio n, stillbirth, preterm d eliv ery, and low b irth weight. women in the s tudies in bos to n (27), clevela nd (28), cinc inna ti (29), and p ort pirie (30) had average blood lead co ncentratio ns during pre gnancy of 5 -10 µg/dl; almost all ha d blood conce ntra tion less than 25 µg/dl. in the cincinnati stud y, ges ta tional a ge was re duced abo ut 0.6 wee ks fo r eac h natural log unit increase in b lood lead concentra tions. however, the cincinna ti and port pirie s tudies fo und a le ad related decrease in duration of pre gnancy, and cinc inna ti and boston s tudies re ported a lead-related decrease in b irth weight .in conclus io n there a re e vide nt cha nges in s ex hormones due to expos ure of fe male wo rkers to lead in batte ry manu fa cturing plant. references 1. need le man hl. the p ersistant thre at of le ad: me dica l and s ociologic al iss ue s. curr pro ble pe diatr. 198 8; 1 8: 7 02-74 4. 2. olive r, p . a le cture on lead p oisoning and the ra ce :. me d. ., 191 1 : 10 96-10 98. 3. s aryan la, zenz c. le ad a nd its comp ounds . in ze nz c, dickerso n ob, horvath ep j r, ed s: occupa tional me dicine . 3rd ed. st. lo uis, mo sb y, 1 994 , p p 506 -5 41. 4. tang,-n; zhu,-z-q. adve rs e rep ro duc tive effe cts in fe male wo rkers o f lea d b atte ry plants: int-joc cup -me d. env iron. health. 200 3; 1 6(4): 35 9-3 61. 5. brow n a. a., halls d. j. and ta ylor a. j. blo od le ad as sa y: ana l. ato mic s pec tros e. 198 9; 4 :4 7. 6. ma rs ha ll, j ohn r. 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"glycop ro te in hormones : structure and functio n. ann. re v. biochem. 198 1; 5 0: 4 65. 8. rose mp. fsh inte rnationa l standard and re fe re nce p re paration fo r the c alibra tio n of immuno as say and bioas sa ys. clin.chem.acta.19 88;27 3(2):1 03117 . 9. abra ha m ge, ma ro ulis g.b, buster j .e., chang r.j. a nd marshall j .r .total te stos te ro ne as sa y. obste t gynec ol. 19 76; 47: 395 . ir aqi j.pha rm.sc i., vol.15 (2 ) ,2006 sex hormones changes 28 10. co ss el thoma s. metal. in : principle of clinic al toxicology. new york, rave npres s. 199 4:ch.9.p.19 4. 11. abba s, basher ka. the ro le o f oxid ativ e stress in lead poiso ning . msc. the sis, co llege of pharmacy –ba ghd ad unive rs ity, 20 01; ch.3 :p .6 1. 12. s aloom, mustafa g.effec ts of zinc in ame liorating oxid ativ e s tre ss in lea d pois oning .msc the sis, college of pharmac y – bag) hd ad univ ersity,200 2;ch.3 : p.33 . 13. s oko l. rz; wang-s aixi; wan-yu jui-y; stanczyk-fz; gent 25 che in-e; cha pinre. low-dos e lea d e xpo sure alte r the gonado ro pin-relea sing hormone s ys te m in the male ra t. env ironmental-hea lth-pe rs pec tiv es, 200 2, vol. 1 10, no. 9, pp. 871 -8 74. 14. s tuart camp bell and ashmonga. the no rma l menstrual c ycle. in: gynaec ology b y te n teac he rs , 17 th e dition, 20 00; ch. 4:pp. 3940. 15. kaplan, la wre nc e a., amade o j. p es ce, stev en c. kazmie rc za k: prolac tin. clinical chemistry : 4th editio n, . mosb y co mpany ,toronto. 2 003 , vo l. 2 ; ch. 43 : pp. 818 -8 19. 16. govo ni s , lucchi l, battaini f, s pano pf, trabucc hi m. effe ct of lead on prolac tin. toxic ol lett. 19 84; 20 (3): 23 7-2 41. 17. ng, t. p. ; goh, h. h.; ng, y.l.; et a l. male e ndo crine functio n in wo rkers with mod erate exp os ure to lead . br. j . ind . med. 199 1: 4 8: 485-49 1. 18. viv oli g, fa ntuzzi g, bergo m m, tone ll e, gatto mr, zane tti f, del dot m. relations hip be twe en low le ad e xpo sure and somatic gro wth in a doles ce nts. j . expo . ana l. env iron. ep id emiol. 1 993 ; 3 supp l. 1:201 . 19. roda milans m, martinez-os aba mj . tofigurea s j, et a l. lead toxic ity on e ndo crine te sticula r func tion in a n occ up atio nally expos ed p opula tion. human to xicol. 1 988 ; 7: 125 -1 28. 20. yen, s .s .c.the human menstrual c ycle: neuro end ocrine regula tion. in: re productiv e end ocrinology, s. s. c. yen a nd r. b, ja ffe, eds . philad elphia, w. b. saund ers co . 19 91, p. p. 2 73-30 8. 21. ro nis-mj, bad ger tm, shemo s j, robe rs on pk, sheikh f.re productiv e to xicity and gro wth e ffec ts in rats exp os ed to lea d at different pe riod s during d eve lo pme nt. to xicol. appl. p harma col. 1 996 ; 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4 1: 2 87-29 1. 29. bo rnsc hein, r. l., p. a. suc co p, k. m. kra fft, c. s. clark, b. pe ace , and p. b. hammo nd. enterior s urfa ce dust le ad, inte rior house d ust lead a nd c hild ho od lead exp osure in an urb an e nvironme nt. tra ce s ub stances environ. hea lth, 19 87; 20: 322 -3 32. 30. mc mic ha el aj , vimp ani gv, ro bertson ef e t al. the port pirie co hort stud y: materna l bloo d le ad and pregnancy o utcome. j epidemio l. cancer hea lth 19 86; 40:18 . iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine doi: https://doi.org/10.31351/vol27iss2pp77-92 77 formulation and invitro evaluation of spherical crystal agglomerates of ebastine by quasi emulsion solvent diffusion method marwah m. hareeja*,1 and eman b.h.al-khedairy** *ministry of health and environment, baghdad, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad. iraq. abstract ebastine (ebs) is a poorly water-soluble antihistaminic drug; it belongs to the class ii group according to the biopharmaceutical classification system (bcs). this work aims to enhance the solubility, dissolution rate and micrometric pro perties of ebs, by formulating it as spherical crystal agglomerates by quasi emulsion solvent diffusion (qesd) method. spherical crystal agglomerates (scas) were prepared in the presence of dichloromethane (dcm), water and chloroform as a good solvent, poor solvent and bridging solvent respectively. agglomeration of ebs involved the use of some hydrophilic polymers like polyethylene glycol 4000 (peg 4000), polyvinyl pyrrolidine k30 (pvp k30), d-α-tocopheryl polyethylene glycol 1000 succinate (tpgs) and β. cyclodextrin. (ebs) and its agglomerates (with and without polymers) were characterize d for their drug content, percentage yield, solubility ,in vitro drug release study and micromeritic property as well as by optical microscope, scanning electron microscopy (sem), fourier transform infrared spectroscopy (ftir), differential scanning calorimetry (dsc) and x-ray diffraction (xrd). the results showed that there was a marked enhancement in the solubility with improvement in dissolution rate, physiochemical properties, and decrease in crystallinity and alteration in the crystal habit of the drug especially in the presence of polymers. the best results were obtained with formula prepared by the combination of peg 4000 and β. cyclodextrin in the agglomeration process of (ebs). keywords: ebastine, spherical crystal agglomerates, quasi emulsion solvent diffusion method. صياغة وتقيم التكتالت البلوريه الكروية لدواء االيباستين **و ايمان بكر حازم الخضيري 1*، مروه محمد حريجة .وزارة الصحة والبيئة ، بغداد ، العراق* .فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد، العراق** الخالصة تصنيف امنظالثانية م في الماء ، وهذا الدواء بينيمي إلى المجموعة الذوبانية هو عقار مضاد للهسيامي ضعيف بيباتيي دواء اال ربيق ع ط ،للدواء الدقيقةخواص الومعدل الذوبان و الذوبانية،هذا العمل هو تحسي م كان الهدف .الحيوي الصيدالنية المسيحضرات ة انيشار المذبيبات م شبه مسيحلب. باتيخدام طربيق صياغيه بشكل تكيالت بلوربيه كروبيه كلورو ميثان ، ماء وكلوروفورم كمذبيب جيد ، مذبيبثنائي وهي وجود ثالثة مذبيبات البلوربية الكروبية فييكيالت التم تحضير ي ابيثيلي جالبيكول مثل البول القابلة للذوبان في الماء اتيخدام بعض البوليمراتعملية اليحضير على اليوالي. تضمنت رابطضعيف ومذبيب . تم وصف العقار تيكسنات وبييا تيكلودكسيربي 1000ابيثلي كالبيكول الفا توكيفربيل بولي -دي، 30كي ، بولي فينيل بيروليدبي 4000 وتحرر الدواء خارج الجسم، والذوبان، المئوي، عائد االنياجو الدواء،( وتكيالته مع وبدون بوليمرات لمحيواها م ابيباتيي النقي ) حيود و المسح اليفربيقي للمسعرات الطيفية،والدراتات المجهري االلكيروني، المسح الضوئي،م خالل المجهر الدقيقة وكذلكوالخاصية الفيزبيائية والخصائص قابلية تحرر الدواء كبير فيقابلية الذوبان مع تحس تحس ملحوظ في السينية. أظهرت نيائج هذا العمل األشعة م الحصولت أفضل النيائج وليمرات وانالب. وخاصة في وجود دواءال بلورات اشكالانخفاض في اليبلور واليغيير في مع والكيميائية لدواءفي عملية اليكيل وبييا تابيكلودبيكسيربي 4000بولي اثلي كالبيكول باتيخدام الصيغة اليي تم إعدادها ع طربيق الجمع بي يها عل االبيباتيي . .مستحلب شبه من المذيبات انتشار طريقةالكلمات المفتاحية: دواء االيباستين، التكتالت البلورية الكروية، introduction the oral route of drug administration is the most common and preferred one over the other routes due to its convenience and ease of administration. however , drug absorption from the gastrointestinal tract (git) can be limited by a variety of factors with the most significant contributors being its aqueous solubility and/or its membrane permeability (1). 1corresponding author e-mail: ph.marwah@yahoo.com received: 31/ 7/ 2018 accepted: 4/ 9 / 2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp77-92 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 78 direct compression method is frequently and preferably used for manufacturing of tablets. in the direct tableting method, it is necessary to increase the flowability and compressibility of the bulk powder(2). on the other hand, it is also important to increase bioavailability of the drug by improving the solubility of the bulk drug powder (3).spherical crystal agglomeration (scas) method directly transforms the fine particles produced in the crystallization process into a spherical shape (4). thus; it improves the secondary characteristics like flowability and compressibility so that direct tableting is possible without further processing (5). it is the particle engineering technique by which crystallization and agglomeration can be carried out simultaneously in one step to transform crystals directly into compacted spherical form which has been successfully utilized for improvement of solubility and dissolution. spherical crystallization is carried out by four methods: spherical agglomeration method, quasi-emulsion solvent diffusion method (qesdm), ammonia diffusion method. and neutralization method (6). ebastine (ebs) a piperidine derivative, is a long-acting, non-sedating, second-generation histamine receptor antagonist that binds preferentially to peripheral h1 receptors. it is metabolized to active metabolite, carebastine. it has antihistaminic, antiallergic activity and prevents histamine-induced bronchoconstriction. this drug presents very low water solubility and a high hydrophobicity with partition coefficient (log p) of 7.64, therefore, and according to the biopharmaceutical classification system (bsc), this drug belongs to the class ii group of pharmaceuticals; for which the rate – limiting step in the absorption process is drug dissolution, because they can permeate well through the gastrointestinal tract (7). the objective of this work was to enhance the solubility, dissolution rate and micromeritic property of ebs by scas method. materials and method material ebastine, β. cyclodextrin and tpgs were purchased from hyperchem, china. peg4000 and pvpk30, chloroform and dichloromethane were purchased from (bdh chemicals, ltd., liverpool, england) all other reagents and chemicals used were of analytical grade method preparation of ebs spherical crystal agglomerates scas prepared by emulsion solvent diffusion method. 2g ebs was dissolved in 10ml good solvent (dcm), this solution was poured drop by drop in to 100ml distilled water (poor solvent) containing either no, single or combination of polymers in presence of 0.1 g aerocil 200; stirred by mechanical stirrer (propeller type stirrer). then 2ml chloroform (bridging solvent) was added drop by drop to the mixture with continuous stirring at stirring speed of 900 rpm for one hour. the selection of these solvent was according to solubility of drug in each of the above solvents and miscibility of these solvent with each other (8,9). as the good solvent diffused to the poor solvent, droplet gradually solidified. the formed spherical agglomerate filtered by vacuum filtration and dried at room temperature. different formulas were prepared (table 1) as a trial to get the required physicochemical properties regarding shape, solubility, dissolution and flow properties table 1. composition of scas formula number ebs gm peg4000 gm pvpk30 gm β. cyclodextrin gm tpgs gm dcm ml water ml chloroform ml ebs1 2 10 100 2 ebs2 2 2 10 100 2 ebs3 2 2 10 100 2 ebs4 2 2 10 100 2 ebs5 2 2 10 100 2 ebs6 2 2 2 10 100 2 evaluation of scas of ebs percentage yield of scas the product yield of scas was determined by calculating the weight of resulted scas after drying with respect to initial weight of drugs and polymers used in formulation of scas (10) as in equation: 𝒀𝒊𝒆𝒍𝒅% = 𝑷𝒓𝒂𝒄𝒕𝒊𝒄𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝑪𝑨𝒔 𝑻𝒉𝒆𝒐𝒓𝒆𝒕𝒊𝒄𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝒅𝒓𝒖𝒈 𝒂𝒏𝒅 𝒑𝒐𝒍𝒚𝒎𝒆𝒓𝒔  100 iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 79 drug content the percentage of drug content of the scas can be determined by crushing and dissolving fixed amount of the agglomerates in 100ml methanol and sonicated for 30 minutes. the resulting solution was then filter through 0.45mm whatman filter paper and analyzed for ebs by uv spectrophotometer at 253nm (11). optical microscope optical microscope was used to determine the change in shape scas. comparison between formulas and pure drug was made. samples of pure drug and scas were placed on glass slides and examined under the microscope at magnification of 40 ×100 (12, 13). scanning electron microscope (sem) to evaluate surface topography of scas of the selected formulas and that of pure drug (crystal habit) scanning electron microscope sem was used after coating with gold (14, 15). solubility study of ebs and scas an excess amount of ebs and prepared formulas were added to 10ml of distilled water in a closed glass tube. the tubes were placed in shaker water bath for 48 hr. at 25°c. after that samples were filtered through filter membrane of 0.45 μm, and the concentration of the dissolved ebs was determined by uv-spectrophotometer at 258nm (16). dissolution study of scas the release profile of ebs from formulated scas was determined by using (paddle) type ιι usp dissolution test apparatus. a weighed amount of scas equivalent to 10 mg drug was add to (1000) ml of 0.1 n hcl (ph 1.2) media kept at 37± 0.5 °c at rotation speed of 100 rpm. sink condition was maintained throughout the study. at predetermined time interval 10 ml sample was withdrawn and replaced by equal volume of fresh sample maintained at the same temperature. the samples were then filtered through 0.45 mm filter membrane and analyzed spectrophotometrically at 258nm (17, 18). micromeritic property of scas the flow properties of, scas were determined by measuring angle of repose, carr’s index and hausner’s ratio for each formula and compared with those of pure drug as follow: angle of repose angle of repose is an expression or measurement for the flowability of powder or granules and can be determined by using funnel method. the tan-1 of the height of the pile/radius of its base gave the angle of repose (19). 𝒕𝒂𝒏 (𝜽) = 𝒉/𝒓 where: (θ) is angle of repose, h is the height of the resultant powder cone and r is the radius of the powder cone bulk density the volume occupied by the accurately weighed scas was measured, which gave bulk volume. the bulk density of scas was determined using the following formula (19) 𝑩𝒖𝒍𝒌 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 = 𝑻𝒐𝒕𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝑪𝑨𝒔 𝑩𝒖𝒍𝒌 𝒗𝒐𝒍𝒖𝒎𝒆 𝒐𝒇 𝑺𝑪𝑨𝒔 tapped density an accurately weighed quantity of crystals was introduced into a measuring cylinder and tapped until no further change in volume was noted, which gave the tapped volume. the tapped density was determined by the following formula (19). 𝐓𝐚𝐩𝐩𝐞𝐝 𝐝𝐞𝐧𝐬𝐢𝐭𝐲 = 𝑻𝒐𝒕𝒂𝒍 𝒘𝒆𝒊𝒈𝒉𝒕 𝒐𝒇 𝑺𝑪𝑨𝒔 𝑻𝒂𝒑𝒑𝒆𝒅 𝒗𝒐𝒍𝒖𝒎𝒆 compressibility index compressibility index was determined according to carr’s index (19, 20) 𝑪𝒂𝒓𝒓’𝒔 𝒊𝒏𝒅𝒆𝒙 = (𝑻𝒂𝒑𝒑𝒆𝒅 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 − 𝑩𝒖𝒍𝒌 𝒅𝒆𝒏𝒔𝒊𝒕𝒚) 𝑻𝒂𝒑𝒑𝒆𝒅 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 100 hasnurs ratio hasnurs ratio gave an indication on flow property of powder and can measure from ratio of tapped density to bulk density (19, 20). 𝑯𝒂𝒔𝒖𝒏𝒆𝒓𝒔 𝒓𝒂𝒕𝒊𝒐 = 𝑻𝒂𝒑𝒑𝒆𝒅 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 𝑩𝒖𝒍𝒌 𝒅𝒆𝒏𝒔𝒊𝒕𝒚 fourier transform infrared spectroscopy (ftir) the drug-polymer interactions were studied by ftir (21,24). the ft-ir spectra of pure drug, peg4000, β. cyclodextrin, scas (ebs1), physical mixture, scas (ebs6) were obtained by preparing the above samples in potassium bromide discs and scanned within scanning range of 4000 cm -1 to 400 cm -1. powder x-ray diffraction (pxrd) powder x-ray diffraction (pxrd) study was performed to evaluate changes, in the crystalline nature of the drug (22,24). pxrd analysis was performed for pure drug and scas (ebs1) and physical mixture of selected formula and scas (ebs6) using an x-ray diffractometer, the samples were irradiated with the monochromatized cukα radiation and analyzed between 2° and 50° theta. iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 80 differential scanning calorimetry studies (dsc) to detect the degree of crystallization and drug-polymer interactions ; dsc studies were carried out for pure drug, peg4000, β. cyclodextrin, scas (ebs1) physical mixture and scas (ebs6) by using differential scanning calorimeter. all approximate weighed samples (about 3 mg) were placed in sealed aluminum pans, before heating under nitrogen flow (100ml/min) at a scanning rate of 10 ºc min-1, from 25 ºc to 400 ºc. an empty aluminum pan was used as reference (23,24). statistical analysis the results were analyzed by two tailed student’s t-test using microsoft excel 2010. for all analyzed results and the level of significance was set at a p-value of 0.05: a p value of more than 0.05 was considered to be non-significant. a p value of less than 0.05 was considered to be significant the results obtained from the dissolution studies were statistically validated using similarity factor (f2) . 𝑓2 = 50 × 𝑙𝑜𝑔 {[1 + ( 1 𝑛⁄ ) ∑ |𝑅𝑗 𝑛 𝑗=1 − 𝑇|2]0.5 × 100} n is the sampling number, rj and tj are the percent dissolved of the reference and test products at each time point j. the similarity factors fits the result between 0 and 100. it is 100 when the test and reference profile identical (f2 higher than 50 show the similarity of dissolution profile) and tend to 0 as the dissimilarity increased this method is more adequate to dissolution profile comparison when more than three or four dissolution points are available (25). result and discussion preparation of ebs scas ebastin scas were prepared by using qesdm. the principle of this method is the use three solvents; good solvent, bridging solvent and poor solvent the selection of these solvents depends on the miscibility of the solvents and the solubility of drug in individual solvent (26). dcm was used as good solvent due to good solubility of ebs and rapid removal of dcm from crystallization system due to its low boiling point (27,28,29) water used as dispersion phase that’s because of poor solubility of ebs in water so that ebs rapidly crystalize in water . chloroform was used as bridging solvent since it is immiscible with poor solvent (water). when the organic drug solution was added to the stirring water, emulsion droplets were formed due to the interfacial tension between the two solvents, the good solvent gradually diffuses out of the emulsion droplets into the outer poor solvent phase, and the poor solvent diffuses into droplets, which reduced the solubility and eventually caused drug crystallization inside the droplets (30), the bridging liquid would then wet the precipitated crystals. and as a result of interfacial tension effects and capillary forces, the bridging liquid will be gathering the crystals to one another resulting in the formation of larger size agglomerates (31). aerosil acts as a separating agent and mass compactor, because coacervation droplets formed from the drug-polymer droplets during the solidifying period were sticky and readily coalesced, so, introduction of aerosil efficiently prevented coalescence and produced compact spherical crystals (32). percentage yield the percentage yield of the scas that were prepared without polymers (ebs1) was 99.982%, while the percentage yield of scas prepared in the presence of polymer ranges from 78.8% to 53.4%. highest yield was obtained from the formula ebs4 which was prepared by using β. cyclodextrin which have the lowest water solubility in comparison with other polymers used in this study. in addition β.cyclodextrin practically insoluble in dcm so that β.cyclodextrin rapidly precipitate and solidified on the surface of scas (27).on the other hand, lowest yield was obtained from scas prepared with peg4000 (ebs 2) due to the high solubility of peg4000 in water and dcm so that, peg4000 lost in dispersion phase and only traces amount of polymer precipitate with agglomerated crystals (33) the above results are shown in table 3. drug content percent drug content of all the prepared formulas was found to be in the range of 98.44±0.708 to 99.8 ± 0.54 that indicated there was negligible loss of drug during crystallization process resulting from sticking on vessels and impeller table 3. optical microscope optical microscope showed the great difference between the shape of untreated ebs which have needle and irregular crystals and agglomerated crystals which have compact spherical form (figure1). iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 81 (a) (b) (c) (d) (e) (f) (g) figure 1. optical microscope of pure drug and formulated scas (a)pure drug, (b)ebs1, (c)ebs2, (d)ebs3, (e)ebs4, (f)ebs5, (g)ebs6 scanning electron microscope (sem) the results of surface morphology studies are shown in (figure5). the sem results revealed that the untreated ebs has irregular needle crystal while scas have spherical structure. the surface morphology studies also revealed that the agglomerates were formed by very small crystals, which were closely compacted into spherical form (figure2). solubility study solubility profile shows that the solubility of scas (ebs1) was dramatically more than that of pure drug which can be explained to be due to partial amorphism of drug in agglomerates and increase in surface area of particles as a result of spherical shape. furthermore, the formulas prepared with use of polymers (ebs2, ebs3, ebs4, and ebs5) showed higher solubility as compared to ebs1. this expected result was due to wetting effect of the hydrophilic polymers (34). while, the formula prepared by the use of combination of polymers (ebs6) showed greatest improvement in solubility than formulas prepared by single polymer since the use of hydrophilic polymer (peg4000) survive the spherical shape of particle and enhanced the solubility effect of (β. cyclodextrin) (27,45) (table 2). iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 82 a b c d iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 83 e e f g figure 2. scanning electron microscope of pure drug and formulated scas (a)pure drug, (b)ebs1, (c)ebs2, (d)ebs3, (e)ebs4, (f)ebs5, (g)ebs6under 1kx and 10kx magnification. in-vitro dissolution study the dissolution profiles of (ebs1) in 0.1 n hcl (ph 1.2) at 37° c; and that of pure drug were non similar (f2=35.485 ).there was an enhancement in the rate of dissolution in comparison to the pure drug as a result of improved porosity resulting from diffusion of solvents during solidification process (34,35)(figure 3), in vitro release study of scas with different single polymer were studied and the results indicated that, there was a dramatic improvement in the rate of dissolution in comparison with pure ebs . however, the increase in dissolution rate was in order of ebs2 < ebs4< ebs5 < ebs1 < ebs3 > ebs as shown in (figure 4). this increase was mainly related to spherical shape of scas and by increasing in wettability due to the presence of different polymers. peg 4000 show highest increase in rate of dissolution (f2=20.014) in comparison with pure drug dissolution rate that’s because peg 4000 have important effect on maintaining the spherical shape of agglomerated crystal (36) and also it enhances the physical and chemical stability of drugs and prevents aggregation of the drug particles as a result of the steric hindrance and/or masking of charges provided through formation of a conformational cloud (37) so, higher surface area will be available for dissolution. iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 84 β.cyclodextrin and tpgs increase in dissolution of ebs (f2= 24.07294) and (f2= 39.46175) respectively due to improvement in solubility. pvp k30 shows similar dissolution profile as that of pure drug (f2=80.563) in spite of increase in solubility which may be due to increase diffusion layer of particle would slow the release of drug since this water soluble polymer at high (about 33%w/w of the formula) amount swell to form concentrated continuous layer between solid surface and bulk medium on dissolution medium, with high viscosity in diffusion layer that caused retardation in dissolution of drug(38,39). (ebs6) formulated by combination of β.cyclodextrin and peg 4000 a very fast rate of drug release (f2 = 8.955621) with reduction in time required for complete the release in comparison with that of pure drug (figure 5) therefore, incorporation of peg 4000 caused increase in solubility as well as in rate of drug release. this result can be attributed to the properties of βcyclodextrin. it has a low water solubility and rigid structure due to high number of intramolecular hydrogen bonds among secondary hydroxyl groups. thus, it prevents hydration by water molecules and cause retardation on drug release. by addition of peg 4000 which binds to the rigid structure of β. cyclodextrin; the wettability and solubility of scas increased which results in increase the rate of drug release. so the effect of combination on enhancing solubility and dissolution can be utilized to reduce the amount of β. cyclodextrin used in formulations to avoid side effect of large amount of β. cyclodextrin consumed by patient which may cause osmotic diarrhea (33,40,41). figure 3. the release profile of pure drug (ebs) and scas (ebs1) in 0.1 n hcl at 37°c figure 4. the release profile of pure drug and scas (ebs2), (ebs3), (ebs4), (ebs5) in in 0.1 n hcl at 37°c figure 5. the release profile of pure drug (ebs) and scas (ebs6) in in 0.1 nhcl at 37°c micromeritic property the values of angle of repose, carr’s index and hausner ratio of pure drug and scas of ebs are shown in table 3. high value for angle of repose of untreated ebs (47.27°±0.047) indicated poor flowability of the drug due to its irregular crystals which hindered their flowability from funnel in comparison with the formulated scas. the significant improvement (p<0.05) in flowability for scas of each formula in comparison with untreated ebs as indicated by the low values of angle of repose was due to their spherical shape and smooth surface, which results in reduction in the interparticle friction since the area of contacts for spherical shape was smaller than that for other particle shape (42).carr’s index and hasnurs’ ratio were of highest value for untreated ebs compared to the prepared scas that’s mean poor compressibility and packability pure drug. these properties were also improved by formulation of sacs due to their spherical shape (43). iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 85 table 2. drug content, %yield and micromeritic property of pure and scas of ebastine formula number type of polymer drug content % yield % angle of repose (degree) carr’s index% hasnurs ratio aqueous solubility mg/ml ebs ---- 100±0.0 (pure drug) ----47.27° ±0.04 32.46467±1.41 1.481567±0.03 0.00293 ±0.001 ebs1 no 99.8±0.54 99.982 25.82 °±0.09 8.9533±1.78 1.0986±0.021 0.007851 ±0.001 ebs2 peg4000 99.56±0.29 53.4 24.94° ±0.29 9.0133±2.29 1.0997±0.028 0.0106 ±0.003 ebs3 pvpk30 98.61±0.49 59.042 25.09° ±0.39 5.0323±1.73 1.053±0.019 0.012 ±0.003 ebs4 β. cd 98.44±0.70 78.8 27.73°±0.37 11.559±3.58 1.110±0.018 0.0433 ±0.004 ebs5 tpgs 98.72±1.11 55.22 32.82°±0.62 16.2276±1.48 1.17163±0.12 0.0136 ±0.002 ebs6 β. cd + peg4000 98.84±1.38 60.216 27.75±0.63 13.0366±1.81 1.131±0.13 0.055 ±0.004 ftir study the ftir spectrum of ebs show c-h stretching of the ring at 3051cm-1, c-h stretching of (ch3) at 2947cm-1, c=c stretching aromatic ring at 1450cm-1, c-n stretching at 1267cm-1 and c=o stretching band at 1677cm-1, the broad peak at 3470 cm -1 may be due to the molecular water present (42,43). ftir spectrum of β.cyclodextrin shows a characteristic peak at 3371 cm-1 due to the o-h group stretching, an intense peak at 2924 cm-1 due to c-h stretching, in addition, peak at 1644 cm-1 represented h-o-h band of water present in β.cyclodextrin and also at 1156 cm-1 of c-o stretching band , and 1028 cm-1 of c-h stretching overtone. on other hand, ftir spectrum of peg 4000 show broad band at 3450.99 cm-1 due to o-h group stretching, intense peak at 2885.95 cm-1 due to c-h stretching in addition to an intense band at 1105.98 cm-1 of c-o group (43,44). ftir spectrum of the physical mixture of drug and polymers, spherical agglomerated scas (ebs1, ebs 6) show no significant shift or reduction in intensity of peaks of ebs, and of β. cyclodextrin, which indicate there was no interaction between drug and polymer. the disappearance of peg 4000 peaks may be due to a small amount of peg4000 entrapped within the formula (figure 6: a through f) a iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 86 b c d iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 87 e f figure 6. ftir of: (a)ebs (b)peg4000 (c)β. cyclodextrin (d)ebs1, (e)pm (f)ebs6 x-ray diffraction (x. rd) the diffractogram for ebs, ebs1, physical mixture of drug and polymers used in the preparation of the ebs6, and ebs6 are viewed in (figure 7 a through d). the diffractogram of pure drug exposed several diffraction peaks with high intensities at 16.4°, 18.9°, and 19.3° at 2-θ indicating the crystal nature of the drug. nearly the same three strongest peaks at 16.9°, 18.73°, and 19.3° were also shown for ebs1 confirming the success of the crystallization process. physical mixture of ebs6 showed three strongest peaks at 14.3°, 19.2 °, and 23.3°, in addition to other minor peaks, while (ebs6) displayed three strongest peaks at 18.4°, 18.8°, and 19.4° and other minor peaks detected in untreated ebs and also appearance of several new peaks related to polymers added to formula. the difference in xrd patterns of the physical mixture from that the ebs6 may be attributed to change in crystal arrangements of ebs in its spherical crystal form. since all the samples showed similar peak positions (2θ) in x-ray diffraction, so, the formation of different polymorphous of ebs was ruled out. however relative intensities of xrd peaks were modified. this could be attributed to the markedly different crystal habits of the sample s. therefore, the related abundance of the planes exposed to the x-ray source would have been changed, producing the variations in the relative intensities of the peak or may be due to the reduction in crystallinity (partial amorphism) of particle cause reduction in intensity of peaks (45,46). http://www.scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=x-ray+diffraction iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 88 figure 7. x.rd of : (a) ebs (b) ebs1 (c) pm (d)ebs6 differential scanning calorimetry (dsc): dsc analysis was used to observe the effect of polymers used in the preparation of scas on the melting point of ebs and also on the crystal habit of the resultant products (47,48). the thermograms of pure ebs, peg4000, βcyclodextrin, ebs1, physical mixture of drug and polymers used in the preparation of the ebs6 and ebs6 are shown in (figure 8 a through f) respectively. pure drug showed sharp endothermic peak at 89 °c corresponding to its melting point (27). thermogram of peg also showed sharp endothermic peak at 65.54°c related to its melting point (31,49), while βcyclodextrin showed a broad peak at 114.44°c which corresponding to the loss of the water molecules existing in the form of residual humidity as well as those included in the cavity by evaporation, while the peak at 312.43°c corresponds to its melting point (50). so, the above results indicated the purity and crystallinity of the used materials. furthermore, the dsc analysis of (ebs1) gave a sharp peak at 86.62 c which is slightly below the melting point of the pure drug which may be due to little amorphousim resulting from the spherical shape of scas (51) as well as resultant from the suppression effect due to presence of polymer which act as impurity. physical mixture showed characteristic peaks for the drug, peg, and β.cyclodextrin which revealed there was no interaction between drug and polymers and maintaining the crystallinity. (ebs6) gave a sharp peak at (87.12°c) which corresponding to the melting point of ebs crystals form (ebs1) and weak broad band at (351.15°c) related to the melting point of β. cyclodextrin indicating that, β. cyclodextrin was entrapped within the formula. the disappearance of peg 4000 peak may be due to the small amount of polymer trapped with scas. these results confirm that there was no interaction between drug and polymers and there was no change in crystal nature of drug (52) (figure 8 f) and also, these results ensure the result obtained from x.ray diffraction. iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 89 a b c d e f figure 8. dsc analysis of (a)ebs, (b)peg4000 (c) β.cyclodextrin, (d)ebs1, (e)pm, (f)ebs6 iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 90 conclusion scas were successfully prepared by qesd method. the resultant crystals show enhancement in solubility and dissolution rate of ebastine except that formula prepeared by pvp k 30. and also all formulas have the desired micromeritic properties, such as flowability and packability. dramatic improvement in solubility and dissolution rate of ebastine was obtained by the combination of β. cyclodextrin and peg4000. however in vivo bioavailability studies are required to ensure whether the results obtained in this investigation can be extrapolated to the in vivo conditions. references 1. shahrin n. solubility and dissolution of drug product: a review. international journal of pharmaceutical and life sciences. 2013;2(1):3341. 2. shangraw, r.f, compressed tablets by direct compression. in pharmaceutical dosage form: tablets, lieberman, h.a., l. lachman and j.b. schwartz (eds). marcel dekker, new york vol. 1, 1989; 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iraqi j pharm sci, vol.27(2) 2018 spherical crystal agglomerates of ebastine 92 development. pharm. sci. technol. today 2, 311320, 1999. 49. guleria r, kaith ns, singh r. peg based solid dispersions of gliclazide: a comparative study. int j pharm pharm sci. 2012;4(1):507-511 50. olaru a, borodi g, kacsó i, vasilescu m, bratu i, cozar o. spectroscopic studies of the inclusion compound of lisinopril with β-cyclodextrin. spectroscopy. 2009 jan 1;23(3-4):191-9. 51. tapas ar, kawtikwar ps, sakarkar dm. modification of felodipine properties using spherically agglomerated solid dispersions. am. j. drug discovery dev. 2011;1:160-73. 52. sajja e. gowri y,kumar j.k., swetha k., chowdary h. v.,raju p., y. spherical agglomeration – an innovation in tablet technology. international journal of innovative pharmaceutical research. 2014,5(2),418-424. iraqi j pharm sci, vol.20(1) 2011 antioxidant vitamin c 50 possible augmentive effect of antioxidant vitamin c in patients with essential hypertension treated with amlodipin or enalapril maitham a. al-rikaby* , kasim j. al – shamma** ,1 , ibrahim a. majeed** and saad sh.hamondi*** * department of clinical pharmacy and pharmaceutics ,college of pharmacy,university of basrah, iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ***department of internal medicine, college of medicine , university of basrah ,basrah ,iraq. abstract hypertension is identified as one of the most significant risk factors for cardiovascular diseases (cvds). there is growing evidence showing that oxidative stress plays a major role in hypertension. increased production of reactive oxygen species and decrease bioavailability of antioxidant have been demonstrated in experimental and human hypertension. the present study was directed to determine the beneficial effect of the antioxidant vitamin c in patients with essential hypertension treated with the calcium channel blocker (amlodipine) or with the angiotensin converting enzyme inhibitor (enalapril). ninety six patients (50 females and 46 males) with essential hypertension and treated with amlodipine or enalapril, age (50.43 ± 0.55 year) were participated in this study and divided in to: twenty five patients received amlodipine 5mg tablet once daily (group 1), twenty five patients received the same dose of amlodipine plus 500mg of vitamin c tablet twice daily (group 2), twenty three patients received enalapril 5 mg tablet once daily (group 3), and twenty three patients received the same dose of enalapril plus 500mg of vitamin c tablet twice daily (group 4). twenty apparently healthy subjects (11 females and 9 males) also participated in this study as control group (group 5). all patients were treated and followed up for one month. the mean blood pressures were measured for each patient before treatment and one, two, three and four weeks after treatment. serum malondialdehyde (mda), lipid profile, electrocardiogram (ecg) parameters and heart rate were measured for each patient before treatment and two and four weeks after treatment. the addition of vitamin c to amlodipine or enalapril treated patients had resulted in a significant (p<0.05) lowering effect on mean blood pressure after two, three and four weeks. on the other hand, the addition of vitamin c to amlodipine or enalapril treated patients had resulted in a significant lowering of serum mda after two and four weeks of treatment. while there was no significant change in lipid profile or ecg parameters and heart rate of patients in group two and four after two and four weeks of vitamin c treatment as compared to their values at zero time and to patients in group one and three respectively. the study indicates that oxidative stress may have a significant role in essential hypertension, and addition of the antioxidant vitamin c to antihypertensive drugs used in this study can give a good blood pressure lowering effect and good protection against lipid peroxidation. key words: antioxidant ,vitamin c, essential hypertension, amlodipin , enalapril الخالصة اسذفاع ػغؾ انذو ٔاحذ يٍ أكصشانعٕايم نالطاتح تأيشاع انقهة ٔاالٔعٛح . ُْاك دالئم يرُايٛح ذشٛش انٗ أٌ انعٓذ انرأكسذ٘ ٚهعة دٔسا سئٛسٛا فٙ يشع فشؽ ػغؾ انذو. انذساسح انحانٛح ٔظٓد َحٕ ذحذٚذ انرأشٛش انًفٛذ انًرٕقع نًؼاد انرأكسذ فٛرايٍٛ ز نذٖ غؾ انذو انعْٕش٘ ٔانزٍٚ ٚرى يعانعرٓى تاسرخذاو عقاقٛش يؼادج السذفاع ػغؾ انذو ْٙ انساد نقُاج انًشػٗ انًظاتٍٛ تفشؽ ػ انكانسٕٛو )ايهٕدتٍٛ ( ٔانًصثؾ نالَزٚى انًحٕل نالَعٕٛذُسٍٛ )اَانثشٚم(. سد ٔذسعٌٕ شخض يظاب تفشؽ ػغؾ انذو انعْٕش٘ ± 5.77َانثشٚم نكٍ نى ٚظهٕا انٗ انٓذف تؼغؾ انذو ٔتًعذل أعًاس )( ركٕس ٚعانعٌٕ تاسرخذاو االيهٕدتٍٛ أ اال68(اَاز ٔ )75) ( يهغهشاو ٕٚيٛا يٍ )ايهٕدتٍٛ( انًعًٕعح االٔنٗ ٔ 7( يشٚغ اسرهًٕا )57( سُح شاسكٕا فٙ ْزِ انذساسح ٔقسًٕا انٗ : ) 75.65 ( يشٚغ 55يٍٛ ز انًعًٕعح انصاَٛح ٔ )( يهغشاو يشذٍٛ ٕٚيٛا يٍ فٛرا755( يشٚغ اسرهًٕا َفس انعشعح يٍ )ايهٕدتٍٛ( ٔ )57) ( يهغشاو ٕٚيٛا 755(يشٚغ اسرهًٕا َفس انعشعح يٍ )اَانثشٚم( ٔ )55( يهغشاو ٕٚيٛا يٍ )اَانثشٚم( انًعًٕعح انصانصح ٔ )7اسرهًٕا ) سكٕا فٙ ( ركٕس انًعًٕعح انخايسح شا9( اَاز ٔ ) 11( شخض يظح يعًٕعح انسٛطشج )55يٍ فٛرايٍٛ ز انًعًٕعح انشاتعح. ) ( 6،5،5،1انذساسح. حٛس اسرًش انعالض ٔيشاقثح انًشػٗ نًذج شٓش ٔاحذ ٔيعذل ػغؾ انذو ذى قٛاسّ نكم يشٚغ قثم انعالض ٔتعذ ) اساتٛع يٍ انًعانعح ، يسرٕٖ انًانَٕذاٚهذٚٓاٚذ ٔيسرٕٖ انذٌْٕ، تٛاَاخ ذخطٛؾ انقهة ٔيعذل َثؼاخ انقهة ذى قٛاسٓا نكم يشٚغ قثم ( اساتٛع يٍ انًعانعح. إٌ إػافح يؼاد انرأكسذ فٛرايٍٛ ز نهًشػٗ انزٍٚ ٚعانعٌٕ تاسرخذاو ايهٕدتٍٛ أ 6،5عح ٔتعذ )انثذء تانًعان ( اساتٛع يٍ تذء انًعانعح. ٔيٍ َاحٛح اخشٖ إٌ إػافح يؼاد 6،5،5االَانثشٚم َرط عُّ اَخفاػا يعُٕٚا فٙ يعذل انؼغؾ تعذ يشٔس) ( 6،5عًٕعح انصاَٛح أٔ انشاتعح َرط عُّ اَخفاػا يعُٕٚا فٙ يسرٕٖ انًانَٕذاٚهذٚٓاٚذ تعذ يشٔس)انرأكسذ فٛرايٍٛ ز نهًشػٗ فٙ انً اساتٛع يٍ تذء انًعانعح تًُٛا نى ٚحذز ا٘ ذغٛش يعُٕ٘ فٙ يسرٕٖ انذٌْٕ، تٛاَاخ ذخطٛؾ انقهة ٔيعذل َثؼاخ انقهة نهًشػٗ فٙ ذء انًعانعح تفٛرايٍٛ ز عُذ يقاسَرٓى تانقٛى ٔقد انظفش ٔاالشخاص فٙ ( اساتٛع يٍ ت6،5انًعًٕعح انصاَٛح ٔانشاتعح تعذ يشٔس ) انًعًٕعح االٔنٗ ٔانصانصح ذراتعا. خراياً إٌ ْزِ انذساسح أظٓشخ إٌ نهعٓذ انرأكسذ٘ دٔسا يعُٕٚا فٙ يشع فشؽ ػغؾ انذو انعْٕش٘ ٔ شٚم( نعالض انًشػٗ قذ أعطٗ اَخفاػاً يعُٕٚاً ظٛذاً فٙ ػغؾ إٌ اػافح ياَع انرأكسذ فٛرايٍٛ ز نهعقاقٛش انًسرخذيح ) ايهٕدتٍٛ أٔ اَانث .انذو ٔٔقاٚح ظٛذج ػذ أكسذج انذٌْٕ. 1 corresponding author email : drkassim_alshamaa@yahoo.com received : 13/3/2010 accepted : 5/4/2011 iraqi j pharm sci, vol.20(1) 2011 antioxidant vitamin c 51 introduction hypertension is a common disease that is defined simply as persistently elevated blood pressure (b.p). hypertension is identified as one of the most significant risk factors for cardiovascular diseases (cvds) (1) . hypertension adversely affects many organ systems throughout the body. target organ damage includes brain (stroke or transient ischemic attack), eyes (retinopathy), heart (left ventricular hypertrophy, angina, prior myocardial infarction, prior coronary revascularization and heart failure). kidney (chronic kidney disease) and peripheral vasculature (peripheral arterial disease) (2) . the overall goal of treating hypertension is to reduce hypertension-associated cardiovascular and renal morbidity and mortality (3) . pharmacological therapy involves prescribing of different antihypertensive drug classes. these agents can be given alone or in combination to treat the majority of hypertensive patients depending on b.p stage (4) . the role of oxidative stress in genesis and maintenance of hypertension is supported by amelioration of hypertension with antioxidant administration (5) . vitamin c (ascorbic acid) is an essential micronutrient required for normal metabolic function of the human body which is considered a very efficient water soluble antioxidant (6) .several studies have been shown that human with essential hypertension have a decreased antioxidant capacity (7,8) .a number of epidemiological studies have been shown a negative correlation between b.p. and vitamin c level (9,10) .vitamin c might influence b.p. by acting as a free radical scavenging properly preventing prostacyclin synthetase inhibition. it has been found a lower levels of vitamin c in sustained hypertensive than normotensives human, this could be a result of greater antioxidant consumption, either for direct reactions or for regeneration of vitamin e to its reduce form, in response to an increased oxidant load associated with sustained hypertension (11) . the present study was directed to determine the beneficial effect of the antioxidant vitamin c in patients with essential hypertension treated with the calcium channel blocker (amlodipine) or with the angiotensin converting enzyme inhibitor (enalapril). patients and methods this study was carried out over nine months from november 2006 till july 2007. many patients were interviewed according to patient's information sheet. patients who enrolled in our study were diagnosed as they having essential hypertension and treated by specialist physicians while attending al-sadr teaching hospital in basrah city. ninety six patients (50 females and 46 males) with essential hypertension treated with calcium channel blocker (amlodipine), or with angiotensin converting enzyme inhibitor (enalapril), but did not reach the target blood pressure, and twenty apparently healthy subjects (11 females and 9 males) participated in this study. inclusion and exclusion criteria the inclusion and exclusion criteria of all patients in this study are summarized as follow: inclusion and exclusion criteria of hypertensive patients enrolled in the study inclusion criteria patients with mild to moderate essential hypertension treated with calcium channel blocker (5mg daily of amlodipine), or with angiotensin converting enzyme inhibitor (5 mg daily of enalapril), but did not reach the target blood pressure. exclusion criteria secondary hypertension. smoking or alcohol consumption. history of myocardial infraction (ml), angina pectoris, heart failure (hf), thyrotoxicosis, pregnant women, diabetes mellitus, malabsorption, uncontrolled arrhythmias and significant renal, hepatic or hematological diseases.use of nonsteroid antiinflammatory drugs, steroids, oestrogen containing preparation (like oral contraceptive pills and hormonal replacement therapy), cyclosporine, erythropoietin and sympathomimetic containing drugs or other medications that effect blood pressure,lipid profile, ecg or antioxidant status. patients were divided as follows: group 1: twenty five patients with essential hypertension treated with amlodipine 5 mg tablet once daily. group 2: twenty five patients with essential hypertension received the same dose of amlodipine plus 500 mg vitamin c twice daily. group 3: twenty patients with essential hypertension treated with enalapril 5 mg tablet once daily. group 4: twenty three patients with essential hypertension received the same dose of enalapril plus 500 mg vitamin c twice daily. group 5: twenty apparently healthy subjects. all patients in each group were followed up for four weeks, and mean blood pressure had iraqi j pharm sci, vol.20(1) 2011 antioxidant vitamin c 52 been taken for each patient before and one, two, three and four weeks after starting treatment (1) . serum mda, lipid profile (tc, tg, hdl-c, ldl-c and vdl-c), ecg parameters (pr, qrs, and pwave), and heart rate have been taken for each patient before and two and four weeks after starting treatment. twenty apparently healthy subjects have also participated in this study. the thiobarbituric acid essay of buege and aust (1978) was used to measure serum mda levels (12) . the estimation of total cholesterol depends on enzymatic colorimetric method of richmond and allain et al.,(1974) (13,14) . the estimation of triglycerides depends on the enzymatic colorimetric method of fossati and principe (15) . the estimation of high density lipoprotein-cholesterol depends on the enzymatic method of burestien et al, (1970) (16) . the ldl-c was calculated by the friedwald formula as follows (17) : ldl-c= total cholesterol [(hdl-c) + (triglyceride/5)]. the vldl-c is calculated from the formula: vldl – c = triglyceride/5 all values are expressed as mean ± standard error of the mean. data are entered in to computer system using microsoft office excel 2003 software for all mathematics and statistical analysis. the student's test was used to determine the significant difference in means of groups. p < 0.05 was considered to be the lowest limit of significance. results table (1) showed the serum levels of mda in patients with essential hypertension treated with amlodipine (group 1) or with enalapril (group 3) at zero time, these values were significantly higher than their values in control group. table (2) showed that patients received amlodipine and vitamin c (group two) or enalapril and vitamin c (group four) showed significant lowering in average mean blood pressure after two, three and four weeks of treatment as compared with their values at zero time and their values in group one or three. table (3) showed that the addition of vitamin c caused a significant lowering in serum mda after two and four weeks of treatment in amlodipine and vitamin c treated group (group two) or enalapril and vitamin c treated group (group four), as compared with zero time values and their values in group one or three. the lowering in serum mda was more prominent after four weeks of amlodipine or enalapril and vitamin c treatment as compared with their values after two weeks of treatment. the serum level of mda in patients treated with enalapril and vitamin c (group four) were significantly lower than their values in those patients treated with amlodipine and vitamin c (group two) after two and four weeks of treatment. table 1: serum malondialde(µmol/i) of control group, patients treated with amoldipine (group one), or with enalapril (group three) at zero time. values are presented as mean± standard error; p<0.05 indicated significant changes from control group. group number of patients serum malondialdehyde. (μmol/i) control group 20 0.5 ±0.03 group one 25 1.39 ±0.04* group three 23 1.13 ±0.03* * significant at p<0.05 as compared with control group. table 2 : mean blood pressure (mmhg) of patients in group one, two, three and four. mean blood pressure in (mmhg) time group 1 amlodipine n=25 group 2 amlodipine +vit.c n=25 group 3 enalapril n=23 group 4 enalalpril + vit.c n=23 zero time 119.06 ±0.68 118.8 ± 0.82 117.48 ± 1.06 117.97 ± 1.02 1 st week 118.62 ± 0.64 117.2 ± 0.78 117.01 ± 0.95 116.37 ± 1.02 2 nd week 117.82 ± 0.61 114.4 ± 0.83 * a 116.52 ± 0.93 114.03± 0.97 *b 3 rd week 117.7 ± 0.62 111.57 ± 0.85* a 116.08 ± 0.97 114.36 ± 0.94*b 4 th week 117.28 ± 0.52 108.08 ± 0.78* a 115.16 ± 0.85 110.66 ± 0.93*b *significant at p<0.05 as compared with zero time value. a significant at p<0.05 as compared with group one. b significant at p<0.05 as compared with group three. iraqi j pharm sci, vol.20(1) 2011 antioxidant vitamin c 53 table 3 : serum malondialdehyde (μmol/l) of patients in group one, two, three and group four. serum malondialdehyde (μmol/l) time group 1 amlodipine group 2 amlodipine + vit.c group 3 enalapril group 4 enalalpril + vit.c zero time 1.39 ± 0.05 1.37 ± 0.4 1.13 ± 0.03 1.06 ±0.03 2 nd week 1.34 ± 0.5 1.16 ± 0.4 * a 1.13 ± 0.03 0.97± 0.03 *bc 4 th week 1.34 ± 0.04 0.83 ± 0.03* a 1.11 ± 0.03 0.77±0.03*bc a significant at p<0.05 as compared with group one. b significant at p<0.05 as compared with group three. c significant at p<0.05 as compared with group two. there was a significant change in lipid profile in hypertensive patients treated with amlodipine or enalapril at zero time as compared to their values in control group (table 4). this change includes an elevation in serum level of (tc,tg, ldl-c and vldl-c), while there was slight, but not significant reduction in serum level of hdl-c in hypertensive patients as compared to their values in control group. there were no significant changes in lipid profile, ecg parameters and heart rate of patients treated with amlodipine pulse vitamin c (group two) or patients treated with enalapril plus vitamin c (group four) compared to their values at zero time or to patients received amlodipine or enalapril alone (group one and two). (table 5and 6). table 4 : lipid profile parameters (tc, tg, ldl-chdl-c and vldl-c) of control group, patients in group one and in group three at zero time. lipid profile parameters control group n=20 group 1 n=25 group 3 n=23 tc (mmol/l) 4.18±0.15 5.0 ± 0.09* 4.87 ± 0.1* tg (mmol/l) 1.94 ±0.09 2.51± 0.04* 2.43 ± 0.04* hdl-c (mmol/l) 1.22± 0.02 1.1 ±0.02 1.08 ±0.03 ldl-c (mmol/l) 2.57 ±0.14 3.41 ± 0.09* 3.36 ±0.09* vldl-c (mmol/l) 0.39± 0.02 0.5 ±0.01* 0.49 ± 0.01* * significant at p<0.05 as compared with control group. tc: total cholesterol; tg: triglyceride; hdl-c: high density lipoprotein cholesterol; ldl-c: low density lipoprotein cholesterol; vldl-c: very low density lipoprotein cholesterol; n: number of patients. table 5 : lipid profile (tc, tg,hdl-c, ldl-c and vldl-c) in patients treated with amlodipine (group one), patients treated with amlodipine and vitamin c (group two), patients treated with enalapril (group three), and patients treated with enalapril and vitamin c (group four). lipid profil parameters group 1 amlodipine group 2 amlodipine + vit c group 3 enalapril group 4 enalapril + vit c zero time 4th week zero time 4th week zero time 4th week zero time 4th week tc (mmol/l) 5.02 ± 0.09 5 ± 0.09 5.03 ± 0.11 4.87 ± 0.12 4.87 ± 0.1 4.82 ± 0.1 4.89 ± 0.1 4.84 ± 0.1 tg (mmol/l) 2.51 ± 0.04 2.49±0.04 2.46 ± 0.04 2.39 ± 0.04 2.43 ± 0.04 2.39 ± 0.04 2.42 ± 0.05 2.38 ± 0.05 hdl-c (mmol/l) 1.1 ± 0.02 1.13±0.02 1.08 ± 0.02 1.15 ± 0.02 1.08 ± 0.03 1.12 ± 0.02 1.11 ± 0.01 1.18 ± 0.02 ldl-c (mmol/l) 3.41 ± 0.09 3.38±0.04 3.43 ± 0.12 3.35 ± 0.12 3.31 ± 0.09 3.25 ± 0.1 3.29 ± 0.1 3.23 ± 0.1 vldl-c (mmol/l) 0.53 ± 0.01 0.51±0.01 0.5 ± 0.01 0.46 ± 0.01 0.5 ± 0.01 0.51 ± 0.01 0.49 ± 0.01 0.46 ± 0.01 tc: total cholesterol; tg: triglyceride; hdl-c: high density lipoprotein cholesterol; ldl-c: low density lipoprotein cholesterol; vldl-c: very low density lipoprotein cholesterol iraqi j pharm sci, vol.20(1) 2011 antioxidant vitamin c 54 table 6 : ecg parameters (pr,p-wave, and qrs) and heart rate in patients treated with amlodipine (group one), patients treated with amlodipine and vitamin c (group two), patients treated with enalapril (group three), and patients treated with enalapril and vitamin c (group four). values are presented as mean ± standard error. ecg parameters group 1 amlodipine group 2 amlodipine + vit c group 3 enalapril group 4 enalapril + vit c zero time 4 t h w eek zero time 4 t h w eek zero time 4 t h w eek zero time 4 t h w eek hr(bpm) 68±0.16 69.28±0.92 66.64±1.18 71.64±1.11 69.04±1.03 71.2±0.93 68.78±1.07 72.12±0.98 pr(ms) 167.12±3.33 167.84±3.4 167.52±2.74 162.44±2.28 167.04±2.59 163.57 ±2.43 169.39±2.18 163.83±1.96 p-wave(ms) 95.84±1.81 98.12±1.64 94.62±2.33 100.16±1.86 95.91±1.83 101.39±1.67 98.39±1.92 103.83±1.69 qrs (ms) 103.32±2.66 105.84±2.73 102.8±2.89 108.2±2.41 104.09±2.26 107.48±2.19 103.83±2.14 108±2.1 discussion results in this study showed that patients with essential hypertension treated with amlodipine or enalapril had significantly higher levels of malondialdehyde as compared to control group. this is in agreement with other worker who demonstrated an increase in ros production in patients with essential hypertension, based in general, on increased levels of serum thiobarbituric acid-reactive substance and other biomarkers of lipid peroxidation and oxidative stress (11) . animals and human studies demonstrated that reactive oxygen species play an important pathophysiological role in the development of hypertension, this is due to excessive superoxide formation, decreased nitric oxide bioavailability in the vasculature and kidney, and rosmediate cardiovascular remodeling (18) . in both endothelial and vascular smooth muscle cells mechanical stretch has been shown to activate the nad(p)h oxides which is an important source for ros production (19) . our results showed that patients with essential hypertension treated with amoldipine or enalapril showed a significant change in lipid profile as compared with control group. this is consistent with sarkar et al (2007), who showed that tc, tg, and ldl-c in forty hypertensive patients were significant higher than their values in thirty normotensive and healthy subjects (20) .the addition of vitamin c in a dose of 1000 mg for one month for patients with essential hypertension treated with amlodipine or enalapril produced significant lowering in serum levels of mda after two and four weeks of treatment, this lowering could be due to the activity of vitamin c as free radicals scavenger, that helps to terminate the free radicals damaging effect and to prevent lipid peroxidation (21) .the lowering of serum level of mda in patients treated with enalapril plus vitamin c was significantly greater than that observed in amlodipine plus vitamin c treated patients after two and four weeks of treatment. this could be due to the different mechanism of action of angiotensin converting enzyme inhibitor (enalapril) and calcium channel blockers (amlodipine). where the activation of rennin-angiotensin system has-been proposed as mediator of nad(p)h oxidase activation and ros production (22) . in fact, some mechanism of action of angiotensin converting enzyme inhibitors have been attributed to nad(p)h oxidase inhibition and decreased in ros production (23) . likewise the addition of vitamin c in the same dose and for the same period had result in significant loweing effect in mean blood pressure after two, three and four weeks of treatment. this is consistent with hajjar et al (2002), who demonstrated that both systolic and diastolic blood pressure decreased during the vitamin c supplementation phase in thirty one patients treated with antihypertensive drugs and vitamin c (24) and with jennifer (2004) who showed that the antioxidant supplementation might reduce dependence on blood pressure lowering effect of calcium channel blocker nifedipine in 58 adults with essential hypertension (25) .the role of vitamin c in reducing blood pressure has different ways including, its direct free radicals scavenging ability and termination their damaging effect on blood vessels and prevent lipid peroxidation (26) ,the second way is its ability to regulate nitric oxide synthase that generate nitric oxide a potents vasodilator that play a key role in controlling the cardiovascular system, the third way is its ability to regulate the nad(p)h oxidase activity and ros production, the fourth way is its ability to regulate antioxidant enzyme including superoxide dismutase and glutathione, in addition to their ability to reduce vitamin e to its active form; the fifth way is that ascorbic acid increase tetrahydrobiopterin an important cofactor of nitric oxide synthase enzyme by iraqi j pharm sci, vol.20(1) 2011 antioxidant vitamin c 55 preventing its oxidation (chemical stability) (27) . finally it has been demonstrated that oxidative stress and hypertension are closely associated with higher sympathetic activity (28) . thus, it could be argued that vitamin c induce change in blood pressure observed in the present study may be in part attributed to its inhibitory effect on the sympathetic activity or antioxidant induced change in the release of norepinephrine from the nerve terminal (29) .in the first week of treatment with vitamin c there was no significant lowering effect on the mean blood pressure, this would indicate that the blood pressure lowering effect of vitamin c was in long-term, where long period required to restore nitric oxide cofactor (tetrahydrobiopterin) level by preventing its oxidation (26) .in conclusion the lowering in mean blood pressures were not differ significantly when comparing patients treated with enalapril plus vitamin c to those treated with amlodipine plus vitamin c. reference 1. chobanian av, bakris gl, black hr, et al. seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. hypertension 2003; 42: 12061252. 2. 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2004; 287: r27r32. 20. sarkar d, latif sa, uddin mm. studies on serum lipid profile in hypertensive patient. mymensingh med. j. 2007 jan; 16(l):70-6. 21. morikawa k, shimokawa h, matoba t, et al. pivotal role of cu, zn-superoxide dismutase in endothelium-dependent iraqi j pharm sci, vol.20(1) 2011 antioxidant vitamin c 56 hyperpolarization. j. clin. invest. 2005; 112: 1871-1879. 22. vito m, zhong h, shaohua ye. oxidative stress mediates angiotensin ii-dependent stimulation of sympathetic nerve activity. hypertension 2005; 46: 533. 23. rodney j, melissa c, luis a, et al. effects of captopril on the renin angiotensin system, oxidative stress, and endothelin in normal and hypertensive rats. hypertension. 2005; 46(4): 943-947. 24. hajjar im, george v, sasse ea, et al arondomized, double-blind, controlled trial of vitamin c in the management of hypertension and lipids. am. j. ther. 2002; 9(4): 289-93. 25. jennifer w. antioxidant may help lower blood pressure. liu x. life. science 2004; 74: 855862. 26. sibel 0, pascal p, ulvi b. vitamins reverse endothelial dysfunction through regulation of enos and nad (p)h oxidase activities. hypertension 2003; 41: 534. 27. livius v, sheldon m, darcy r, et al. long-term vitamin c treatment increase vascular tetrahydrobiopterin levels and nitric oxide synthase activity. circulation research. 2003; 92: 88. 28. fink g d tempol lowers blood pressure and sympathetic nerve activity but not vascular õ2 in doca-salt rats. hypertension 2004; 43: 329-334. 29. jou sb, cheng j t the role of free radicals in the release noradrenalin from mysentric nerve terminals of guinea-pig ileum. j. auto. merve. syst. 1997; 66: 126130. iraqi j pharm sci, vol.30(1) 2021 serum carnitine & srage in clomiphene citrate resistant pcos women doi: https://doi.org/10.31351/vol30iss1pp110-121 110 correlation between serum carnitine level and soluble receptors for advance glycation end products(srage) in clomiphene citrate resistantpcos women nabigh a. naji*,1, shatha h. ali*and fatin s. farhan** *department of clinical laboratory science, college of pharmacy , university of baghdad, baghdad, iraq. **ministry of health and environment, al yarmook teaching hospital, baghdad -iraq abstract the most frequently diagnosed condition in women at the age of reproduction is the polycystic ovarian syndrome (pcos).it could be related to a complex endocrine condition, due to its heterogeneity and uncertainty about its etiology, as the clinical highlights of pcos incorporate those related to reproductive signs such as decreased frequency of ovulation, irregular menstrual cycles, decreased fertility. carnitine plays a substantial role in weight loss, glucose tolerance, insulin function and fatty acid metabolism. thus, carnitine plays a crucial role in controlling obesity, insulin resistance, oxidative stress that are associated with pcos. while, ages are a diverse group of reactive molecules that are formed endogenously by non-enzymatic reactions of carbonyl group of carbohydrates with free amino groups of proteins, nucleic acids or lipids. the soluble form of receptors of age (srage) could play an important role in management obesity, insulin resistance, hyperandrogenism, oxidative stress which could be related to pcos. this study aimed to investigate serum levels of carnitine & soluble receptors for advanced glycation end products (srage) in clomiphene resistant pcos. besides assessing the correlation between serum levels of carnitine, as well as, soluble receptors of age with hormonal ( lh, fsh& testosterone) and metabolic ( serum glucose , serum insulin & homa-ir) markers in these patients . the study included thirty women with clomiphene resistant pcos and thirty apparently healthy women, as a control. in order to measure serum total carnitine and serum soluble receptor of advance glycation end product (srage in pcos and control groups. the results of our study have shown a decreased serum levels of total carnitine in pcos group in comparison with control group (48.05 and 59.73 nmol/ml, respectively), but there was no significant elevation in serum levels of srage in patients group as compared with control group. in addition to a significant correlation between serum total carnitine and serum srage levels (r=0.45, p-value=0.03). in conclusion, serum total carnitine level was low in clomiphene resistant-pcos patients in comparison with control group. although, srage levels in clomiphene resistantpcos patients were not significantly different from the age and bmi-matching controls, but a significant correlation between serum total carnitine and srage was detected. keywords: poly cystic ovarian syndrome, soluble receptor of advance glycation end products (srage) , serumtotal carnitine . العالقة بين مستوى الكارنتين والمستقبالت الذائبة للمنتجات النهائية السكرية المتقدمة في مصل للعالج بالكلومفينالمريضات بمتالزمة المبيض المتعدد االكياس من النوع المقاوم **فاتن شالل فرحانو *شذى حسين علي، 1,*نابغ عبد الزهرة ناجي .، بغداد العراق جامعة بغداد، كلية الصيدلة ، فرع العلوم المختبرية السريرية * ، العراق . بغداد، مشتشفى اليرموك التعليمي وزارة الصحة والبيئة ، ** الخالصة عند النساء في سن اإلنجاب هي متالزمة المبيض المتعدد االكياس. يمكن أن يكون ذلك مرتبًطا بحالة الغدد ان الحالة األكثر شيوًعا تتضمن المظاهر السريرية لمتالزمة تكيس المبايض وخاصة تلك المتعلقة .الصماء المعقدة او بسبب عدم التجانس وعدم اليقين بشأن مسبباتها فوق بالعالمات اإلنجابية مثل انخفاض تكرار اإلباضة ، وعدم انتظام الدورة الشهرية ، وانخفاض الخصوبة ، والمبيض متعدد االكياس بالموجات وبالتالي، .والتي يمكن أن تساهم في نمو شعر الجسم والوجه المفرط وحب الشبابالصوتية ، وتراكيز مرتفعة للهرمونات الذكرية مثل التستوستيرون ، الغذائي التمثيل ،(فرط نمو الشعر, عقم األندروجينية، فرط) الخصوبه تشمل ومختلفة متنوعة سريرية آثار لها المتعدد االكياس المبيض متالزمة فإن الموسع، االكتئاب) العقلية والميزات( زياده خطراالصابة بامراض القلب السكري، من داء انيالث النوع األنسولين، مقاومة الكلوكوز، ضعف تحمل ) .الحياة جودة تراجع قد تؤدي الى التي( القلق 1corresponding author e-mail: nabighalsharifi@gmail.com received: 30/7/2020 accepted:11 /10 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp110-121 iraqi j pharm sci, vol.30(1) 2021 serum carnitine & srage in clomiphene citrate resistant pcos women 111 هيتم إنتاجوأيزومر نشط حيويًا. -الكارنيتين اليساري حيث وجد اناألحماض األمينية، اليسين. احد هو مشتق من والكارنتين اليساري يعد اقة بشكل رئيسي في الكبد ويعاد امتصاصه في الكلى، ثم ينتقل إلى األنسجة األخرى. يتركز أكثر في األنسجة التي تستخدم األحماض الدهنية كط ض الدهنية كارنيتين دوًرا حيويًا في توليد الطاقة عن طريق نقل األحما -أساسية لها، مثل الهيكل العظمي والعضلة القلبية. في هذا الصدد، يلعب ل حماض الدهنية. من العصارة الخلوية إلى الميتوكوندريا. يلعب الكارنيتين دوًرا مهًما في فقدان الوزن وتحمل الجلوكوز ووظيفة األنسولين واستقالب األ ان متالزمة المبيض المتعدد االكياس.وبالتالي يلعب الكارنيتين دوًرا حاسًما في السيطرة على السمنة ومقاومة األنسولين واإلجهاد التأكسدي المرتبط بـ الكاربونيل منتجات السكريه النهائيه المتقدمه هي مجموعة متنوعة من الجزيئات النشطة التي تنتج داخليًا بواسطة التفاعالت غير اإلنزيمية لمجموعة ستويات مرتفعة من منتجات السكريه النهائيه من الكربوهيدرات مع مجموعات أمينية من الدهون والبروتينات واألحماض النووية. وقد لوحظت م المتقدمه المصلية في المرضى الذين يعانون من السكري، والشيخوخة و مقاومة األنسولين ومؤخًرا متالزمة المبيض المتعدد االكياس. تقبالت منتجات السكريه النهائيه المتقدمه في مستويات الكارنتين في المصل و الشكل القابل للذوبان من مس تقييم:هدفت الدراسة الحالية إلى البحث في دراسه العالقه بين التواجد المحتمل اضافة الى بالنساء الصحيحات. مصل الدم في مرضى متالزمة تكيس المبايض المقاومة للكلوميفين، مقارنة وكذلك مع وبان لمنتجات السكريه النهائيه المتقدمهلالرتباطات المعنوية احصائيا بين مستويات المصل من الكارنيتين، والمستقبالت القابلة للذ في ( الجلوكوز في المصل، األنسولين في المصل)والتمثيل الغذائي ( هرمون الملوتن، هرمون التحوصل، البروالكتين والتستوستيرون )الهرمونات .هؤالء المرضى اومة لـ الكلوميفين وثالثين امرأة ذات صحه جيده على ما يبدو، اشتملت الدراسة على ثالثين امرأة مصابة بمتالزمة تكّيس المبايض المق من أجل قياس مستوى الكارنتين الكلي ومستوى المستقبالت القابلة للذوبان لمنتجات السكريه النهائيه المتقدمه في مصل الدم، إلى . كمجموعة مقارنة .السيطره تكيس المبايض و مجموعهجانب العالمات الهرمونية والكيميائية الحيوية في مجموعه متالزمة لمجموعة متالزمة تكيس المبايض بالمقارنة مع المجموعة الضابطة أظهرت نتائج الدراسة الحالية انخفاض مستوى الكارنيتين الكلي في مصل الدم قد يه النهائيه المتقدمه في مصل الدمالمستقبالت القابلة للذوبان لمنتجات السكر وأظهرت أن مستوى( مل، على التوالي /نانومول 59.73، 48.05) باإلضافة إلى االرتباط الهام بين مستوى الكارنتين ومستوى المستقبالت القابلة . زاد بشكل طفيف في مجموعة المرضى مقارنة بالمجموعة الضابطة .موعة المرضىفي مج( 0.03= ، قيمة ف 0.45= م ) للذوبان لمنتجات السكريه النهائيه المتقدمه في مصل الدم ومع ذلك، . مستوى الكارنيتين في مصل الدم لدى مرضى متالزمة تكيس المبايض مقارنة بالمجموعة الضابطة انخفاض يستنتج مما ورد في النتائج: متالزمة تكيس المبايض مقارنة المستقبالت القابلة للذوبان لمنتجات السكريه النهائيه المتقدمه في مصل الدم في مرضى زيادة غير كبيرة في مستويات مستوى الكارنيتين ومستوى المستقبالت باإلضافة إلى ذلك، تم الكشف عن وجود ارتباط معنوي سلبي كبيرفي مصل الدم بين. مع المجموعه الضابطه .ينالقابلة للذوبان لمنتجات السكريه النهائيه المتقدمة لدى مرضى متالزمة تكيس المبايض المقاومة للكلوميف في مصل الدم. الكارنتين الكلي ، والمستقبالت الذائبة للمنتجات النهائية السكرية المتقدمة،متالزمة المبيض المتعدد االكياس :الكلمات المفتاحية introduction the most frequently diagnosed condition in women at the age of reproduction is the polycystic ovarian syndrome (pcos) (1) . it could be related to a complex endocrine condition, due to its heterogeneity and uncertainty about its etiology. clinical highlights of pcos incorporate those related to reproductive signs like decreased ovulation frequency, menstrual irregularities, decreased fertility, ultrasound polycystic ovaries, and elevated male hormone concentrations such as testosterone, which can contribute to excessive body and facial hair development and acne. hence, poly cystic ovarian syndrome has important and assorted clinical implications involving reproductive(hyperandrogenicity,hirsutism,infertilit y), metabolic(compromised glucose tolerance , insulin resistance, type two dm, unfavorable cardiovascular hazard profiles) and mental highlights (expanded depression, anxiety) that may decline life quality (2) . in most cases, ovulation can be induced with clomiphene citrate (cc) is a selective modulator of oestrogen receptors (serm), and has been the first-line therapy of patients with anovulation or oligomenorrhea for over 40 years. clomiphene citrate competes with endogenous estrogen at hypothalamus and pituitary gland receptors, interfering with natural estrogen's negative feedback signaling. compared to natural estrogen, cc binds in the hypothalamus for weeks rather than days, effectively blocking the replenishment of estrogen receptors. uninhibited release of gnrh and fsh is due to this hypoestrogenic state. the elevated levels of fsh induce ovarian hyperstimulation and the potential to grow multiple follicles; however, 15-20% of the patients fail to ovulate (cc resistance) and from 6070% of the patients fail to conceive (cc failure) after 6 cycles of treatment with cc and require alternative treatments. other treatment modalities for clomiphene resistant pcos clomiphene citrate + metformin, gonadotropin and laparoscopic ovarian drilling (3) .currently, insulin resistance and the compensatory hyperinsulinemia affects some 65– 70% of women with pcos. part of the insulin resistance appears to be independent of obesity and related specifically to pcos, with abnormalities of cellular mechanisms of insulin action and insulin receptor function having been documented (4) . approximately 50 percent of pcos patients suffer from weight gain which can worsen disease symptoms (5). for a few trials, serum carnitine levels for pcos patients and the impact of carnitine supplements is tested on their weight reduction. in an intersectional analysis (6), there was a negative and significant association between plasma level of l-carnitine and body mass index(bmi) in women with pcos.a study by ismail et al. among clomiphene resistant pcos women, getting clomiphene citrate combined with 3000 mg per day of l-carnitine for 12 weeks, resulted in a significant decrease of bmi in the l-carnitine treated group (7) . in addition to a cross-sectional study, which concluded that plasma concentrations of l-carnitine had a negative and significant iraqi j pharm sci, vol.30(1) 2021 serum carnitine & srage in clomiphene citrate resistant pcos women 112 correlation with homa-ir-index in pcos patients (6). hyperinsulinemia and insulin resistance in patients with pcos are caused by elevated androgen levels (8). insulin resistance and hyperinsulinemia can also increase the proportion of lh / fsh and the androgen production (9). the impact of carnitine on ovarian hormones has been appeared within the past studies (10,11). in like manner, it shows up that carnitine improves insulin sensitivity which in turn influences androgens and ovarian hormones levels (12). correlation between hormonal status such as estrogen, testosterone and serum levels of carnitine and has been investigated in a few researches. in one research, in obese women with pcos there was an inverted correlation between shbg and serum carnitine; but there was no correlation between carnitine levels and androgens (13). on the other side, the advanced glycation end products ages are a diverse group of reactive molecules, that are formed endogenously by nonenzymatic reactions of carbonyl group of carbohydrates with free amino groups of proteins, nucleic acids or lipids. elevated serum ages levels have been observed in patients with hyperglycemia, insulin resistance, diabetes, aging, and lately pcos. high circulating levels of ages can cause cellular damage after deposition in different tissues. late data have shown that ages’ circulating levels and expression of their pro-inflammatory receptors in the ovarian tissue called as receptor for advanced glycation end products (rage) are elevated in women with pcos (14) . pcos women have raised serum advance glycation end products (ages) and raised expression of the membranous inflammatory receptor (rage) seen in their ovaries (15). the linking of ages to it is receptor (rage) results in cellular events leading to the production of reactive oxygen species mainly through the activation of (nadph) oxidase and proinflammatory transcription factor (nfκb). subsequently, the development of proinflammatory cytokines (such as il-1,6,8), regulators of apoptosis like fas ,bcl-2, adhesion molecules (like icam-1 and vcam-1 ), and activation of platelets and macrophages (16,17). interests, ros production triggered by receptor of advance glycation end products (rage)activation therefore induces a positive up regulating receptor of advance glycation end products (rage) expression (18), thereby pushing the inflammatory processes to be amplified. induction of rage has been recorded in atherosclerosis, inflammatory processes and recently pcos (16,19). serum levels of advance glycation end products (ages) contribute to the hormonal alteration found in women with pcos (20); for occurrence, it appeared that there is a relationship between serum ages and serum testosterone (20 in addition, it has been studied that alters in nutritional ages that may improve changes in hormonal status, oxidative stress ,insulin sensitivity in pcos women (21).as documented that approximately two thirds of women with pcos are inclined to develop insulin resistance (ir) (8) and eventually diabetes mellitus(dm)(22,23), the dm and ir are frequently worsened by obesity (24)(35). ages contributed to the development of insulin resistance (ir) in pcos (26). the present study was aimed to investigate serum levels of carnitine & soluble receptors for advanced glycation end products (srage) in clomiphene resistantpcos women in comparison with age and bmi-matching non-pcos women. besides assessing the correlation between serum levels of carnitine,as well as, soluble receptors of age with hormonal ( lh,fsh&testosterone) and metabolic (serum glucose , serum insulin and homa-ir) markers in these patients . subjects and methods the present study was a case control study included patients whom diagnosed to have pcos from those attending al-yarmook teaching hospital / baghdad, for the period from (october/2019 to april /2020). this study was approved by the ethics committee of the college of pharmacy/university of baghdad. all participants were informed about the aim and the proposed benefits of the study before they obtained their consent. the study included thirty women with clomiphene resistant pcos and thirty apparently healthy women, as a control. the chosen pcos patients were under supervision , based on the changed rotterdam criteria, which require, two of the following three appearances: (1) oligo-and/or anovulation (cycle length >35 days), (2) clinical and/or biochemical hyperandrogenism (clinically by evaluation of the hair development based on the altered ferriman-gallwey score and/or biochemically based on raised add up to testosterone, raised serum dehydroepiandrosterone sulfate (dheas), and androstenedione) and (3) polycystic ovaries on ultrasound (pco was characterized as the presence of 12 or more follicles in each ovary, each measuring 2-9 mm in diameter, and/or expanded ovarian volume > 10 ml) (27). table-1 summarizes the subject’s characteristics. blood specimen was obtained from each participant for assessing hormone levels in serum: lh, fsh, testosterone and insulin. in addition to some biomarkers: fasting serum glucose, homeostatic model assessment for insulin resistance (homa-ir),as well as, elisa measurement of serum soluble receptor of advanced glycation end products age(srage) and total carnitine. blood was collected at 9:00 am after an overnight fasting between the 2nd and 4th days of a spontaneous bleeding episode of the pcos group and of a menstrual cycle of the controls. venous blood specimens (10 ml) were exchanged into gel tubes iraqi j pharm sci, vol.30(1) 2021 serum carnitine & srage in clomiphene citrate resistant pcos women 113 permitted to clot and after total clotting; serum is isolated by centrifugation 10 minutes at 3500 to 4000 rpm to get serum. the serum to was isolated into eppendrof’s tubes (kept frozen at -20˚c) until their measure , unless something else they analyzed quickly like(fsg,fsh,lh,prolactin&testosteron) that were analyzed immediately. biochemical assay methods measurement of serum total carnitine level using specific elisa kit (6).serum soluble receptor of advanced glycation end products (srage) level was estimated by (elisa) (28),both kits were purchased by shanghai yehua biological technology/china .while hormonal assessment of lh,fsh,prolactin &testosterone were measured by vidas specific kits purchased by biomerieux/france(29-31).fasting serum glucose was measured kinetically at a wavelength of 642 nm and 37 c and is displayed after about 125 seconds in mg/dl or mmol/l reflotron /germany (32).serum insulin level was measured by chemiluminesce using kit provided by cobas e411 /germany (33). statistical analysis the analyses were conducted using the statistical package for the social science (spss, version 22, ibm, new york, usa). descriptive statistics (means, standard errors of the mean, frequencies and percentages) of the participants (both patient and control group) were calculated. independent t-test was used to compare parameter means between the two groups and pearson correlation was used to measure the correlation between two parameters within each group. a p-value of less than 0.05 was statistically significant. table 1 . subjects characteristics. parameter groups mean ±sem (p-value) age(yrs) control 27.43 1.115 0.234 patient 29.40 1.197 weight (kg) control 77.20 2.117 0.397 patient 74.80 1.853 bmi (kg/m2) control 29.717 0.749 0.790 patient 29.443 0.697 whr control 0.908 0.023 0.879 patient 0.901 0.021 height (cm) control 161.16 1.195 0.238 patient 159.30 1.465 prolactin (ng/ml) control 18.86 1.58 0.038 patient 25.57 1.87 lh (miu/ml) control 4.30 0.38 0.701 patient 4.19 0.41 fsh (miu/ml) control 5.85 0.48 0.135 patient 5.26 0.48 testosterone (ng/ml) control 0.47 0.04 0.160 patient 0.43 0.05 fsg(mg/dl) control 99.91 2.63 0.506 patient 102.61 2.85 insulin (uu/ml) control 7.06 0.44 0.188 patient 7.89 0.43 homa-ir control 1.81 0.15 0.164 patient 2.06 0.16 bmi=body mass index, lh=luteinizing hormone, fsh=follicle –stimulating hormone, fsg=fasting serum glucose , homa-ir= homeostatic model assessment for insulin resistance . iraqi j pharm sci, vol.30(1) 2021 serum carnitine & srage in clomiphene citrate resistant pcos women 114 results serum total carnitine levels in pcos patient and control groups: as presented in figure 1, serum carntine level was statistically lowered in pcos group as compared to control group (p-value <0.05) figure 1. serum carnitine level in patient and control groups *according to mann-whitney test, there is significant p-value < 0.05) difference in camitine level between the two groups (control group has significantly higher level compared to patient group) additionally as illustrated in table2, there is no significant (p-value >0.05) correlations between glycemic markers (fsg,insulin,homa-ir) with serum total carnitine level in patient group. table 2. correlations between glycemic markers (fsg, insulin and homa-ir) with serum total carnitine level in patient group glycemic marker total carnitine fbg correlation coefficient .030 sig. (2-tailed) .876 n 29 insulin correlation coefficient .229 sig. (2-tailed) .233 n 29 homa-ir correlation coefficient .174 sig. (2-tailed) .367 n 29 non-significant (p-value >0.05) correlations according to spearman correlations. soluble receptors of advanced glycation end products (srage) although the mean value of the patients group was (2.28) (ng/ml) in terms of srage was double the value of the control group (1.09) (ng/ml), the difference was non-significant probably because of high variation and small sample size (figure2). figure2. mean values of srage levels in patient and control groups furthermore, there is a significant (p-value <0.05) positive correlation between serum carnitine values and serum srage levels (r=0.45, pvalue=0.03) in patient group (figure-3).however, no significant correlation was detected for either one with the studied sex hormones (table -3). iraqi j pharm sci, vol.30(1) 2021 serum carnitine & srage in clomiphene citrate resistant pcos women 115 table 3. pearson’s correlations between serum carnitine and s rage with sex hormones in patients group parameter carnitine srage prolactin lh fsh testosterone carnitine correlation coefficient 1.000 .452* -.259 .084 .026 .340 sig. (2tailed) . .030* .175 .664 .894 .071 n 29 23 29 29 29 29 srage correlation coefficient .452* 1.000 -.066 -.179 -.160 .009 sig. (2tailed) .030* . .760 .402 .456 .968 n 23 24 24 24 24 24 prolactin correlation coefficient -.259 -.066 1.000 -.016 .080 -.149 sig. (2tailed) .175 .760 . .934 .674 .432 n 29 24 30 30 30 30 lh correlation coefficient .084 -.179 -.016 1.000 .324 .244 sig. (2tailed) .664 .402 .934 . .081 .193 n 29 24 30 30 30 30 fsh correlation coefficient .026 -.160 .080 .324 1.000 -.273 sig. (2tailed) .894 .456 .674 .081 . .145 n 29 24 30 30 30 30 testosterone correlation coefficient .340 .009 -.149 .244 -.273 1.000 sig. (2tailed) .071 .968 .432 .193 .145 . n 29 24 30 30 30 30 * correlation is significant at the 0.05 level (2-tailed). figure 3. the correlation between patient serum carnitine and srage levels (r=0.45, pvalue=0.03) discussion according to the results in (figure-1) that shows there is a significant difference in serum total carnitine level between pcos patients and control group(p=0.026) and the average level of serum total carnitine in control group is higher than the average of total carnitine in pcos group and this is due to insulin resistance and hyperandrogenism which are the foremost crucial highlights of pcos, which is related with reduced serum total carnitine levels(6,34). additionally the hyperinsulinemia and insulin resistance may be influenced by excess production of androgens, which in turn affects the liver cells leading to reduced shbg generation and rise of androgens(35,36). in expansion, both insulin resistance and hyperandrogenism are correlated with dyslipidemia, obesity and consequently risk factors for cardiovascular illnesses (8). studies have appeared that serum carnitine levels in obesity and metabolic syndrome diminish following insulin resistance. moreover, carnitine supplementation leads to decrease in weight, bmi, waist to hip ratio, body fat mass (fm) and increased basal metabolism (37). the beneficial effect of carnitine supplementation on parameters of glucose homeostasis has appeared in past studies (38,39). glycemic status disorders are the main common complication in pcos following insulin resistance. there is a negative correlation in these patients between carnitine iraqi j pharm sci, vol.30(1) 2021 serum carnitine & srage in clomiphene citrate resistant pcos women 116 levels and insulin, fbs, homair. (6,7). additionally, carnitine supplementation with every day doses of 250 3000 mg appeared to produce significant decrease in glucose, insulin and homa-ir values (40). carnitine likely improves insulin metabolism variables by directing the activation of gluconeogenic and glycolytic enzymes (41), enhancing glucose oxidation in mitochondria and function as an acetyl-group donor in a highenergy metabolism situation or as a free fatty acid transport molecule that leads to enhance glycemic status and increased insulin sensitivity(42).while the results of present study (table-2) indicated that there is no significant correlation between glycemic markers (fsg,insulin,homa-ir)with serum total carnitine level in patient group. in patients with pcos, a frequent lowgrade inflammation was observed. in these patients, serum levels of il-6, tnf-α and crp increased (43). the important relationship between oxidative stress circulation and androgen and inflammatory biomarkers levels (44). these discoveries recommend that hyperandrogenism in pcos may induce aggravation and upgrade oxidative stress through insulin resistance and hyperglycemia and conversely inflammation induced by hyperglycemia can promote the production of abundance ovarian androgen. in addition, inflammatory markers and oxidizing stress are associated with insulin resistance (45,46). in this manner the association of oxidative stress and inflammation with hyperglycemia and insulin resistance can lead to hyperandrogenic exacerbations. the antioxidant properties of carnitine are mainly linked to free radical scavenging and the avoidance of free radical formation, preserving the integrity of the electrontransport chain in mitochondria resulting in reduced secretion of ros holding it under stress conditions, and influencing redox signals by inhibiting nuclear factor-jb resulting in increased production of antioxidant molecules and chemicals (47) . in spite of the non-significant differences in the mean serum levels of soluble receptor of advance glycation end products(srage) between pcos patients and control group (p=0.123).however, the mean value of (srage) in patient group (2.28) (ng/ml) was double the value of the control group (1.09) (ng/ml) (figure-2).this inconsistency , could be related to the size of studied population in this study.that mean the value of srage is much higher in pcos patients in comparable with non pcos patients. multiple studies give prove for increased oxidative stress in normoglycemic women with pcos and thus this could account for the elevated levels of serum ages in these women (48-50). on the basis of this prove, the role of ages might too be considered to participate directly or indirectly within the pathophysiology of the syndrome, as oxidative stress has been appeared to be involved within the improvement of insulin resistance and hyperandrogenism in pcos. srage compete with rage for ligand binding site (act as a decoy receptor); it may neutralize age-rage-mediated oxidative damage. piperi et al. found that the normal glucose level of pcos women had increased serum levels of srage, which correlated positively with age levels.(51) in the present study, serum srage increased in patients pcos compared with the control group. however, this increase was not statistically significant and may be explained like other studies have shown that increase ages’ circulating levels that result from oxidative stress and inflammation accompanied with pcos that lead to increase expression of their pro-inflammatory receptors in the ovarian tissue called as receptor for advanced glycation end products (rage) are elevated in women with pcos . on the other hand, high levels of the protective anti-inflammatory receptors called soluble receptor for advanced glycation end products (srage) are associated with protection against ages (14) . while other studies have shown an inverse relation between srage and hyperandrogenism and significant effects in pcos women (52). in latest years, a few studies, have shown that raised ages serum levels in pcos women. (15). as the age-rage process is basically linked to hyperandrogenism and insulin resistance, which are major pathophysiological characteristics in patients of pcos. as well to regular receptors, one type of age receptor, missing transmembrane and cytosolic domains, are named soluble receptor of advanced glycation end product (srage). they are discharged out of the cell, and can be recognized in blood circulation (20, 53). (srage) can link with it is ligands (ages) within the blood that lead to prevent the harmful impacts of the (age-rage) process. srage is at that stage commonly known as good receptor. the values of srage were found to be decreased in obesity and hyperglycemia that have been clarified by the useful function of srage and also its work as decoy to catch the ages in blood iraqi j pharm sci, vol.30(1) 2021 serum carnitine & srage in clomiphene citrate resistant pcos women 117 circulation avoiding stimulation of rage signaling process (54-56) obesity is a very frequent highlight of pcos patients, display in 30 to 75 percent of patients, and act as an aggravating agent within the group of clinical features of the metabolic syndrome (57). through past research, had shown that ages serum levels are specifically included in adipogenesis as well as generation of inflammatory cells in adipocytes, resulting to abnormalities linked to obesity (58). these research results suggested a possible part of advance glycation end products in obesity-linked comorbid conditions, as they noticed that ages serum levels have been raised, while serum levels of srage have been reduced together with raised bmi (52) .association testing appeared that, serum srage levels were conversely linked with body mass index, while ages had a positive association with body mass index. specific regression studies showed that bmi had been the major predictor of srage, which assist supported their results (15,20). as hyperinsulinemia, insulin resistance would be seen as a critical factors and pathophysiological mediators in pcos (53). in vitro experiments, it showed that ages are associated with insulin resistance pathogenesis. as it had been recorded that serum ages to be increased in pcos independently of the existence of insulin resistance (50) . according to correlation studies in patient group, as illustrated in (table-3) and (figure-3) there is a significant (p-value <0.05) positive correlation between serum carnitine values and serum srage levels in patient group that mean when the serum carnitine level decrease lead to decrease the serum level of soluble receptor of advance glycation end products(srage) in pcos patients. a soluble form of rage, described as soluble c-truncated rage (srage) loss both transmembrane and cytosolic spaces of rage and created after alternative splicing of rage gene . the srage receptor circulates all through the body and constitute a decoy receptor that binds circulating ages, hence avoiding them from association with their proinflammatory rage receptor eventually preventing tissue damage . in spite of the fact that it remains petulant, a decreased srage level shows a increased rage signaling and more pathologies (14). carnitine develops maintenance mechanisms for oxidative stress-induced damage to membrane phospholipids, and also keeps up common antioxidant status. it protects cells from reactive oxygen species by acting as a free radical scavenger. previously, we detailed expanded oxidative stress and diminished antioxidant capacity in patients with pcos. these perceptions suggest that low levels of antioxidant carnitine may contribute to the hurtful impacts of increased oxidative stress in pcos patients (34). according to the results of studies above that revealed the oxidative stress in pcos patients result from low level of serum total carnitine and (srage) so these results are compatible with findings of present study that show in pcos patients when the serum total carnitine decrease the srage also decrease. because the average of bmi in control group is (29.717) so the control group consider as an overweight group (healthy weight falls between bmi values of 18.524.9).in a previous study showed that obesity may be a disorder of energy balance, happening when energy utilization and daily energy intake are not adequate. according to the findings of previous study that confirmed the erum carnitine level decrease when the bmi increase and this compatible with results of our present study. in another study it appeared that srage is conversely related with bmi, whr, and fasting glycemia in a non-diabetic population which waist circumference and bmi are independent indicators of srage in a healthy population, and especially in women. this is the primary observation, to the best of our knowledge that describes the relationship of all types of soluble rage with cardio metabolic parameters in a healthy population. therefore, these findings recommend that earlier to any clinical complication, srage plasma levels may reflect a metabolic disturbance status that seems afterward lead to vascular complications and diabetes. this result is supported by the observation that overweight subjects have lower srage levels compared to normal subjects (59). conclusions: 1carntine might improve weight loss and glycemic status further studies are recommended to prove the exact mechanism of carnitine in patients with pcos. 2 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tibolla g, raselli s, redaelli l, buccianti g, catapano al. circulating soluble receptor for advanced glycation end products is inversely associated with body mass index and waist/hip ratio in the general population. nutrition, metabolism and cardiovascular diseases. 2009 feb 1;19(2):129-34. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ & reabilative services using criteria of risk-benefitratio, cos-effectiveness, quality, practicaladministrationas well iraqi j pharm sci, vol.21(1) 2012 prescribing pattern in maysan 112 prescribing pattern and rational use of drugs in maysan governorate, iraq. haydar f. al-tukmagi *,1 and abdul rasool m.wayyes ** *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. **department of pharmacology and toxicology,college of pharmacy,university of baghdad, baghdad, iraq. abstract this study was designed to investing the drug prescribing pattern which is the important point in the rational or irrational use of drugs among patients dispensing their prescriptions from the private pharmacies in maysan governorate, iraq for a period of 1 month. the data collected from prescriptions were calculated and analyzed according to the who prescribing guidelines. the data showed that the mean of drugs included in single prescription was 3.4, and 12% of prescribed drugs were written as generic names; moreover, the percentage of antibiotics, corticosteroids and anxiolytics were 33.3%, 11.4% and 23.8% respectively. those results indicate the irrational use of drugs when compared with the world health organization standard values of prescribing indicators, in addition to the bad prescribing pattern regardless of the degree of specialization of the physician, where 52% of those prescriptions (analyzed in the present study) written by specialized physicians. in conclusion, actual intervention and follow up, training on rational use of drugs and intervention strategies for prescribers is required to improve the rational use of drugs. key words: prescription pattern, polypharmacy, rational drug use نمورج انوصف واالستعمال األمثم نهذواء في محافظت ميسان / انعراق حيذر فخري انتكمجي * ،1 ويس** محمود انرسول بذع و *فزع انصيذنخ انسزيزيخ، كهيخ انصيذنخ ، خبيؼخ ثغذاد،ثغذاد ، انؼزاق خبيؼخ ثغذاد،ثغذاد ، انؼزاقوانسًىو ، كهيخ انصيذنخ ، األدويخ** فزع الخالصة نهذواء ثيٍ األيثمغيز أو األيثمصًًذ هذِ انذراسخ نزحذيذ طزيقخ وصف انؼالج وهى ػُصز يهى في رحذيذ االسزخذاو في يحبفظخ ييسبٌ/انؼزاق ونًذح شهز واحذ. خًؼذ انجيبَبد وحههذ اػزًبداً ػهى يقبييس األههيخانًزضى انًزاخؼيٍ نهصيذنيبد انًىصىفخ األدويخفي كم وصفخ، انُسجخ انًئىيخ نؼذد ُظًخ انصحخ انؼبنًيخ وقذ اظهزد هذِ انجيبَبد ثبٌ يؼذل االدويخ انًىصىفخ ي وأخيزاثبسزخذاو االسى انؼهًي، انُسجخ انًئىيخ نؼذد انًضبداد انحيىيخ انًىصىفخ، انُسجخ انًئىيخ نكًيخ انكىريزكىسيززويخ انًىصىفخ، %. رذل هذِ انُزبئح وثبنًقبرَخ 14.2% و 22.3%، 44.4%، 21، 4.3: كبألرييخ نهًهذئبد انًىصىفخ وكبَذ ثبنزسهسم انُسجخ انًئى % 21يبيقبرة انـ إٌطزيقخ انىصف انسيء نهىاصفيٍ ثبنزغى يٍ إنى ثبإلضبفخنهذواء األيثميغ يقبييسهب انؼبنًيخ ػهى االسزخذاو غيز هيٍ ػهى درخخ ػبنيخ يٍ انزخصص. هذا يذػى وثصىرح يهحخ انى اَقالة حقيقي ويزبثؼخ خذيخ انحبص األطجبءيٍ انىاصفيٍ هى يٍ انىصف انديذ نهذواء. إنىنهذواء وانزذريت انًسزًز نهىاصفيٍ يٍ اخم انىصىل األيثماالسزخذاو إنىنغزض انىصىل نهذواء .انكهماث انمفتاحيت : نمورج انوصف ، كثرة وصف األدويت ، االستخذاو األمثم introduction according to the world bank (1) , governments in developing countries expend between 20% and 50% of their national health budgets on drugs and medical sundries. unfortunately, the world health organization (who) believes that much of such expenditure is misapplied, as irrational use of drugs is prevalent especially in developing countries (2) . hence, governments, health workers and the community are concerned with the availability, handling, effectiveness and safe use of drugs. the prescribing of drugs is an important issue for the individual patient, since risks and benefits of the treatment directly affect the patient. prescribed drugs are reimbursed by the society. hence, prescribing of drugs is also a key question from a public expense perspective. financing of drugs is a vast problem, since costs for drugs are increasing and resources are limited (3) . evaluation of costs and benefits for alternative treatment strategies is essential and rational drug use implies physicians’ prescribing of drugs with favorable cost-benefit balances. guidelines for recommended drugs are important for rational drug use. however, prescribing and adherence to prescribing guidelines vary between health care units (4) , for example according to patient characteristics (5-7) , physician characteristics (6) , practice settings (6) , budgetary policies (8) and country of residence (9) . 1 corresponding author email : h.tukmagi@hotmail.com received : 27/2/2012 accepted : 24/4/2012 iraqi j pharm sci, vol.21(1) 2012 prescribing pattern in maysan 113 rxsofnototal drugsofnototal . . sources of drug information used by the physicians may be of additional significance (10) .there are a limited number of objective measures or indicators that can describe the drug use situation in a country, region or individual health facility (11) . those indicators include prescribing pattern, patient care and the facility indicators; the most reliable type is the prescribing indicators that measure the performance of health care providers in several key dimensions related to the appropriate use of drugs (12) . this project was designed to evaluate prescribing pattern and rational drug use in maysan governorate, iraq. materials and methods this study was based on a surveillance conducted in private pharmacies in maysan governorate during june to july 2005. the pharmacies were chosen randomly depending on systematic random sampling method (13) . to calculate sampling interval, we divide the size of the list (no. of pharmacies in the governorate) by desired sample size (10 pharmacies), then choosing random number between 0 and 1 from the table of random numbers and multiplying it by sampling interval; this result must be rounded upward to get the number of the 1 st pharmacy. a total of 585 prescriptions were selected randomly from the 10 pharmacies and the data obtained from each prescription were introduced in the prescribing indicator from (table 1). in addition to those prescribing indicators, the degree of specialization of the physicians was taken into account to check whether it affects the prescribing pattern or not. calculations were done using the following equations: average no. of drugs per each rx = % of drugs prescribed in generic name = 100 . . x prescribeddrugsofnototal namesgenericindrugsofnototal % of rxs containing antibiotics (ab) = 100 . . x rxsofnototal abcontainingrxsofno % of rxs containing corticosteroids (cs.) = 100 . . x rxsofnototal cscontainingrxsofno % of rxs containing anxiolytics = 100 . . x rxsofnototal sanxiolyticcontainingrxsofno the ten pharmacies were coded as (a, b, c, d, e, f, g, h, i & j). table 1: prescribing indicators form sequence no. of drugs/rx drugs in generic name antibiotics corticosteroids axiolytics 1. 2. 3. 4. 5. 6. 7. 8. total average percentage %of total drugs %of ab in rxs % of cs in rxs % of anxiolytics in rxs results the prescribing indicators were calculated from each pharmacy and summarized in the table 2 in addition to the who standard value for each indicator (14) . from table 2, we can find that the average number of drugs in prescription is 3.4, the percentage of drugs prescribed in generic name is 12% which mean that the prescriber used the trade name in about 88% of the prescriptions. the percentage of ab prescription is 33.3% and the predominant type is cephalosporin derivatives ( especially cephotaxim) which is iraqi j pharm sci, vol.21(1) 2012 prescribing pattern in maysan 114 22% and then penicillin derivatives (especially amoxicillin) which is (17%), while the other types of ab represent the remaining percent. the percentage of the prescribed cs is 11.4% and the anxiolytics percentage was 23.8%. the comparisons between each prescribing indicators value with its counterpart who value were shown in figures 1-4. table 2:the values of each prescribing indicator for 10 pharmacies with the mean & the who standard value. indicator p/a p/b p/c p/d p/e p/f p/g p/h p/i p/j mean who value % average no. of d/rx 3.8 2.8 3.4 3.6 4.0 3.0 3.5 2.8 3.7 3.4 3.4 1.6-1.8 % of drug in generic name 10 14 14 18 12 9 11 12 10 11 12% 100% % of ab/rx 30 30 34 35 38 40 28 26 39 32 33.3% 20-26.8% % of cs./rx 9 10.5 12 15.5 20 7 8 10.5 10.3 11.5 11.4% 1.6% % of anxiolytics/rx 22 30 27 22 19 28 20 19 26 25 23.8 p: pharmacy; d: drug; rx: preemption; ab: antiobiotic; cs: corticosteroid figure 1: comparison of the mean of average number of d/rx in 10 pharmacies with who standard value. figure 2: comparison of the mean of drugs in generic name of 10 pharmacies with who standard value. figure 3: comparison of the mean of percent ab/rx of 10 pharmacies with who standard value. figure 4: comparison of the mean of percent cs/rx of 10 pharmacies with who standard value. 3.4 1.8 0 0.5 1 1.5 2 2.5 3 3.5 a v . n o . o f d /r x mean of 10 pharmacies w ho value 12 100 0 20 40 60 80 100 % o f d ru g i n g e n e ri c n a m e mean of % drug in generic nam of 10 pharmacies w ho value 33.3 26.8 0 5 10 15 20 25 30 35 % o f a b r x mean of % of ab/rx of 10 pharmacies w ho value 11.4 1.6 0 2 4 6 8 10 12 % o f c s / r x o f 1 0 p h a rm a c ie s mean of % of cs/rx of 10 pharmacies w ho value iraqi j pharm sci, vol.21(1) 2012 prescribing pattern in maysan 115 discussion the core drug use indicators evaluate prescribers, patient care and the facility. among the uses of these indicators are to describe current treatment practices, compare health facilities and prescribers and allow for identification of potential drug use problems that may affect patient care (15,16) . the present study represent an insight on the prescribing pattern in private sector health facilities, because this sector is continuously growing and share important part in health providing services in iraq; however, many serious problems and challenges emerged in this issue, including minimal, professional categorization with regard to drug prescribing, inefficient patient counseling, and finally high percentage of prescriptions are misused. the study showed that the average number of drugs in prescription that represent a polypharmacy approach (more then one drug in single prescription) was greater than that mentioned by who list; this will definitely lead to high consumption of drugs, loss of resources, increasing side effects due to drug interactions and misuse of drugs. though no universal or even national standards exist for what the number of drugs in each prescription should be, the disparity between developing countries is worrisome and the number is quite high. our findings are higher than those from sudan 1.4 and zimbabwe 1.3 (16) . the prescription of several drugs per prescription (polypharmacy) is a serious problem; it has been attributed to patients' demand (17) ; desire to treat several ailments at the same time and inadequate diagnostic facilities to determine definitive cause of ill health (18) . there is a need for education of patients and prescribers on the hazards of poly pharmacy. also, managerial interventions to improve training of prescribers to ensure accurate diagnosis and provision of diagnostic facilities at the primary care level in iraqi health facilities would alleviate such tendency. in the present study, the percentage of drugs prescribed in generic names is 12% only, which is very low percentage compared with the who standard value that may reach 100%; this could be due to low training prescribers, no health education about the importance of restriction in drug use. moreover, many prescribers believe that the patient satisfy by receiving more than one or two drugs and finally lack of education facilities like leaflets or posters accessible by the prescribers (19) . the percentage of ab prescribed in each prescription is 33.3%; this value is higher than who standard value (26.8%) which indicates the well known problem of misuse of ab with disputable problems like hypersensitivity, higher cost, resistance and drug interaction. however, another study in iraq reported more serious data in this respect that reflect antibiotics misuse in governmental institutions (20) . this could be due to the same reasons that reez et al. (21) mentioned in his study, where physicians prescribe ab for any reason, just because they believe that the illness was attributed to bacterial infection. when comparing the percentage of the prescribed corticosteroids in our study (11.4) with the who value (1.6), the data revealed a real dangerous problem related to misuse of such agents with high and severe side effects. choosing the anxiolytics as prescribing indicators in our study is due to the increase in consumption of such compounds in the community, especially during the period of unstable situation of the country and the well known consequences of war and its disasters. so, in spite of lack of the who value of prescribed anxiolytics, we reported a high percentage (23.8%); this is also a frightening percentage due to the wide range of side effects associated with these compounds. the last indicator considered in the present study is the level of specialization of the physician; the result showed a disappointed point, where 52% of the prescriptions categorized as bad prescribing pattern in this study, were ordered by highly specialized physicians; such finding reveal no relation between the highly specialization level and the prescribing pattern as one may expect. in conclusion, the rational use and prescription practice of drugs in maysan/iraq has many problems associated with misuse of drugs and the prevalent problems among physicians working in the private clinics; this require urgent intervention and follow up to promote the rational use of drug in this city. references 1. who. report of the conference of experts. the rational use of drugs, 25-29 november 1985, world health organization, geneva 1987. 2. quick jd, rankin j, laing ro, o′connor r, hogerzeil hv, (editors). management sciences for health/who/dap. managing drug supply, (2 nd ed.), kumarian press, hartford, ct; 1997. 3. hoffman jm, shah nd, vermeulen lc, doloresco f, et al. projecting future drug expenditures–2008. am j health syst pharm 2008; 65(3):234-253. 4. ohlsson h, lindblad u, lithman t, ericsson b, et al. understanding adherence to official guidelines on statin iraqi j pharm sci, vol.21(1) 2012 prescribing pattern in maysan 116 prescribing in primary health care–a multi-level methodological approach. eur j clin pharmacol 2005; 61(9):657-665. 5. kasje wn, denig p, stewart re, de graeff pa, haaijer-ruskamp fm. physician, organizational and patient characteristics explaining the use of angiotensin converting enzyme inhibitors in heart failure treatment: a multilevel study. eur j clin pharmacol 2005; 61(2):145-151. 6. tamblyn r, mcleod p, hanley ja, girard n, hurley j. physician and practice characteristics associated with the early utilization of new prescription drugs. med care 2003; 41(8):895-908. 7. kozyrskyj a, raymond c, racher a. characterizing early prescribers of newly marketed drugs in canada: a populationbased study. eur j clin pharmacol 2007; 63(6):597-604. 8. ohlsson h, merlo j. understanding the effects of a decentralized budget on physicians’ compliance with guidelines for statin prescription–a multilevel methodological approach. bmc health serv res 2007; 7:68. 9. sturm hb, van gilst wh, veeger n, haaijer-ruskamp fm. prescribing for chronic heart failure in europe: does the country make the difference? a european survey. pharmacoepidemiol drug safety 2007; 16(1):96-103. 10. edward c, himmelmann a, wallerstedt sm. influence of an e-mail with a drug information attachment on sales of prescribed drugs: a randomized controlled study. bmc clin pharmacol 2007; 7:12. 11. de bakker dh, coffie ds, heerdink er, van dijk l, groenewegen pp. determinants of the range of drugs prescribed in general practice: a crosssectional analysis. bmc health serv res 2007; 7:132. 12. carlzon d, gustafsson l, eriksson al, rigner k, et al. characteristics of primary health care units with focus on drug information from the pharmaceutical industry and adherence to prescribing objectives: a cross-sectional study. bmc clinical pharmacology 2010; 10:4-8. 13. world health organization. who policy perspectives on medicines, promoting rational use of medicines: core component, september 2002. 14. hogerzeil hv. promoting rational prescribing: an international perspective. br j clin pharmacol 1995; 39:1-6. 15. who. action program on essential drugs. how to investigate drug use in health facilities. world health organization, geneva: 1993; p. 187. 16. hogerzeil hv, ross-degnan d, laing ro, ofori-adjei d, santoso b, et al. field tests for rational drug use in twelve developing countries. lancet 1993; 4:1408-1410. 17. erah po, olumide go, okhamafe ao. prescribing practices in two health care facilities in warri, southern nigeria: a comparative study. trop j pharm res 2003; 2:175-182. 18. odusanya oo. drug use indicators at a secondary health care facility in lagos, nigeria. nig j comm med pri health care 2004; 16:2i-24. 19. laing ro. promoting rational drug use. contact 1994; 5:1-6. 20. juma'a km, hussain sa, et al. antibiotic prescription pattern in surgery department, baquba teaching hospital. new iraqi j med 2010; 1:15-20. 21. reeze re, et al. part a: principles of ab use. in: hand book of antibioties, (3 rd ed.), lippincott williams and wilkins, philadelphia usa, 2000. الخلاصة iraqi j pharm sci, vol.20(1) 2011 α -amylase activity and celiac disease 81 relation of α-amylase activity with glucose and anti-gliadin iga and igg in sera of patients with celiac disease sura a. al-emami * ,1 , aliaa h. faraj* and dahlia m.r. al-abadi* * department of chemistry , college of science, al-mustansiryia univirsity ,baghdad,iraq. abstract celiac disease (cd) is an inflammatory small intestinal disorder that can lead to severe villous atrophy, and malabsorption . since the measurement of α-amylase activity is the most widely used biochemical test for the diagnosis of pancreatic and non pancreatic disease , therefore serum α-amylase were studied in the present study in an attempt to evaluate the usefulness of this enzyme in the diagnosis of celiac disease and its relationship with anti gliadin iga and igg and serum glucose . thirty one patients with celiac disease were studied and compared with twenty four healthy individuals . significant elevation of α-amylase activity , glucose and anti gliadin iga and igg were observed in the sera of patients with celiac disease compared with the control group . also a significant positive correlation between α-amylase activity and anti gliadin igg were found in the present study in the sera of patients with celiac disease while a non significant correlation were found between α-amylase activity and anti gliadin iga and glucose in the sera of the same patients of celiac disease. keywords:α-amylase , anti – gliadin iga , igg , glucose , celiac disease . الخالصة َحذد َخُجت خهم يعىٌ طغُز، يٍ انًًكٍ اٌ َؤدٌ إنً حذود ضًىر سغابٍ شذَذ وسىء داء االحخشاء هى انخهاب ايُهُش َعذ يٍ أكثز انفحىص انكًُُائُت انحُاحُت اسخخذايا نخشخُض األيزاع انبُكزَاسُت وغُز – ايخظاص . ونكىٌ قُاص فعانُت انفا ايُهُش فٍ حشخُض يزع االحخشاء -فٍ يحاونت نخقذَز أيكاَُت اسخخذاو انفاانبُكزَاسُت , نذا فقذ حى قُاص هذا األَشَى فٍ انذراست انحانُت عُُت يٍ يزضً انًظابٍُ بذاء 53، حُذ حضًُج انذراست انحانُت جًع igg , igaوعالقخه بكهكىس يظم انذو ويضاداث انكالَذٌ ايُهُش ويسخىي كم يٍ -ادة يعُىَت فٍ فعانُت انفا عُُت يٍ األشخاص األطحاء . أظهزث انُخائج وجىد سَ 46 ـب االحخشاء ويقارَخها فٍ يظىل دو انًظابٍُ بذاء االحخشاء يقارَت يع يسخىَاحها فٍ يظىل دو األطحاء وقذ حبٍُ igg , iga انكهكىس ويضاداث انكالَذٌ االحخشاء فٍ حٍُ حبٍُ عذو وجىد فٍ يظىل دو انًظابٍُ بذاء iggايُهُش ويضاد -أَضا وجىد عالقت اَجابُت يعُىَت بٍُ فعانُت انفا فٍ يظىل دو انًظابٍُ بذاء االحخشاء . igaايُهُش وكم يٍ انكهكىس ويضاد -عالقت يعُىَت بٍُ فعانُت انفا introduction celiac disease (cd) , also known as celiac sprue or gluten sensitive entropathy (1),(2) . is the most common life – long food sensitive entropathy in humans , is characterized by malabsorption , chronic inflammation of small intestine mucosa , villous atrophy and crypt hyperplasia (3),(4),(5) . currently , the onset of celiac disease is considered to result from interaction between environmental factors , gluten as important genetic predisposition and immune system hyperactivity (6) . the disease can clinically manifest at any age , most commonly in the first few years of life , a few months of introducing gluten in diet (7) . about 20% of cases occur in patients who are older than 60 years of age (8) . celiac disease may be associated with a wide range of disease (1,9) , including thyroid dermatological , lympho proliferative disorders , and immune disorders (1,10,11) . furthermore , there is a greater than expected prevalence of immune disorders in cd patients as well as of cd in patients with autoimmune diseases (12),(13) . celiac disease is diagnosed by abnormal blood tests and an abnormal appearing intestine on biopsy and symptoms that resolve with a gluten free diet (14,15) . testing sera for iga or igg immunoreactivity to gliadin is usually one of the first steps in the complex process of diagnosing gluten intolerance (16) . among laboratory tests , measurement of α-amylase (ec:3.2.1.1) is the test most widely used in the diagnosis of pancreatic and non pancreatic diseases (17) . the serum amylase concentration reflects the balance between the rates of amylase entry into and removal from the blood (18) . α-amylase is an enzyme that hydrolyses alphabonds of large alpha – linked poly saccharides such as starch and glycogen , yielding glucose and maltose (19) . 1corresponding author email : sura742004 @ yahoo.com received : 24/11/2010 accepted : 30/4/2011 iraqi j pharm sci, vol.20(1) 2011 α -amylase activity and celiac disease 82 it is the major form of amylase found in humans and other mammals (20) . amylase is most prominent in pancreatic juice and saliva , each of which having its own isoform of human α-amylase (19) .conflicting data have been reported in previous studies for αamylase activity in patients with celiac disease. some of these data reported elevated levels of serum amylase (21),(22) . while others pointed out that patients with cd have low amylase values (23) .the aim of this study was to evaluate the serum concentrations of αamylase and its correlation with glucose and anti gliadin iga and igg antibodies in celiac disease. subjects and methods subjects thirty one patients (15 male their age 250 within mean 14 year and 16 female their age 2-42 within mean 10 year) were involved in this study . the patients were referred to aluarmook teaching hospital and alkadhimiya teaching hospital. baghdad , iraq., diagnosed by dr. saad alsadoon and dr. fathel alabodi. all patients with cd were diagnosed depend on symptoms of diarrhea, weight loss ,anemia, and serologic examination by (anti-gliadin iga and igg ) . no other diseases associated with cd in those patients. they were all underwent free diet treatment without any drugs. as a control , 24 age and gender matches healthy individual were included in the present study . serum sampling five milliliters of samples of venous blood were taken in fasting state and left for 10 minutes at room temperature. after blood coagulation , the sera were separated by centrifugation at 3000 rpm for 10 minutes and then sera stored at -20 ο c until being used . hemolyzed samples were discarded . methods determination of anti gliadin iga and igg serum concentration of antigliadin iga and igg were measured by double antibody technique using enzyme – linked immunosorbent assay (elisa), biohit oyi, finland (24),(25) . determination of α-amylase activity the activity of serum α-amylase was determined by colorimetric method (26) . determination of protein concentration the total serum protein concentration was determined by biuret method using total protein kit , biolab, france (27) . determination of glucose the serum concentration of glucose was measured by enzymatic colorimetric using glucose kit (28) . statistical analysis data were analyzed using spss (version 10.0). the results are expressed as mean ± standard deviation (sd) . statistical and correlation analysis were undertaken using ttest, and pearson , s correlation coefficients. values of p < 0.05 were considered significant. results the concentration of antigliadin iga and igg were measured in the present study in the sera of control and patients with celiac disease by double antibody technique. the mean values presented in table (1) reflect a highly significant increase in the serum levels of antigliadin igg (p< 0.001) and iga (p< 0.001) in patients with cd compared with those of the control group . the activity of serum α-amylase was determined using caraway method . in order to calculate the specific activity of the enzyme ,total protein concentration was measured in the sera of the studied groups, using biuret method. a non significant increase (p >0.05) in protein levels was observed in the present study in sera of patients with cd in comparison with that of the control group , table ( 2 ) . when the mean values of both activity and specific activity of α-amylase in sera of patients with cd was compared with that of the control group, the results (table 3) reflected a highly significant increase in activity (p<0.001) and specific activity (p< 0.01) .the serum concentration of glucose was measured throughout this study in the sera of control and patient groups using enzymatic method. the result presented in table(4) shows a significant increase (p<0.01) in glucose levels in sera of patients with cd in comparison with that of the control group .upon analysis of the overall results of the present study, it was found a significant positive correlation (r = 0.709 , p< 0.05) between α-amylase activity and anti gliadin igg in the sera of patients with celiac disease (54%) who have increase in there α-amylase activity figure (1). while a non significant correlation (p>0.05) between α-amylase and anti – gliadin iga and glucose were found in the sera of the same patients with cd. iraqi j pharm sci, vol.20(1) 2011 α -amylase activity and celiac disease 83 table 1 : serum antigliadin igg and iga levels in control and patients with celiac disease . groups sample number(n) range (au) mean (au) ± standard deviation p value igg control patients with cd 24 31 7.6-30.1 15.0-204.1 17.31 ± 8.63 77.90 ± 48.02 p<0.001 iga control patients with cd 24 31 7.1-32.5 10-192.1 15.68 ± 10.154 62.83 ± 57.6 p<0.001 table 2 : total protein concentration in the sera of control and patients with celiac disease . group samples number (n) range mg/dl mean mg/dl ± standard deviation p value control 24 6.7-8.9 7.65±0.78 p>0.05 patients with cd 31 5.1-12 7.99±1.77 table 3 : mean values of α-amylase activity and specific activity in the sera of control and patients with celiac disease. group activity u/l specific activity u/mg range mean ± standard deviation range mean standard deviation control 24 74.0-175.60 126.10±45.01 0.94-2.62 1.699 0.74 patients with cd 31 35.3-268.78 174.18* ±60.06 0.24-9.60 2.578 ** 1.64 *significant increase in amylase activity with p<0.001. ** significant increase in amylase specific activity with p<0.01. table 4 : mean values of glucose levels in the sera of control and patients with celiac disease group samples number (n) range mg/dl mean mg/dl ± standard deviation p value control 24 60-79.2 70.1 ± 8.11 p<0.001 patients with cd 31 22.2-234 102.26 ±50.41 figure 1 : the correlation between α-amylase activity and antigliadin igg in sera of patients with celiac disease (r=0.709,p<0.05). iraqi j pharm sci, vol.20(1) 2011 α -amylase activity and celiac disease 84 discussion it is well known that gliadin is directly or indirectly through immune mediated reactions , toxic to small bowel mucosa of relatively small population of genetically predisposed individuals who under this toxic action develop celiac disease (12),(29) . testing sera for iga or igg immunoreactivity to gliadin is usually one of the first steps in the complex process of diagnosing gluten intolerance , because it is well known that antibodies to native gliadin sequences are present in patients with celiac disease (30),(31),(32) . in the present study anti – gliadin iga and igg measurements (table 1) shows a presence of a significant increase of anti – gliadin iga and igg in sera of patients with cd. celiac disease is an autoimmune disorder that can coexist with other diseases , such as diabetes mellitus type 1 , thyroid gland diseases , and sjogren , s syndrome (7) . throught this study fasting serum glucose was measured and highly significant increase of its was observed in the patients with cd in comparison with that of the control group. this was in agreement with the result obtained by galicka latala d. et. al. who reported that hyperglycemia and problems with glucose balance in the serum of patients with celiac disease were observed (33) . while this result was disagreement with the result obtained by tourniaire j. et. al. who reported that intolerance to gluten can disrupt glucose regulation leading to greater risk of hypoglycaemia (34) .throughout this study highly significant increase (p<0.001) in both activity and specific activity of α-amylase in sera of patients with cd . this is in agreement with the result obtained by oita t. et. al. who reported that patients with celiac disease showed persistently elevated levels of serum amylase and lipase (21) . carroccio a. et. al. demonstrated a frequency of about 25% of elevated pancreatic enzymes(amylase, lipase, trypsin, and elastase) values in cd patients (22) .while the present study result was disagreement with the result that obtained by disantagnese p. who reported that patients with celiac disease tend as a group to have low amylase values (23) . the elevated levels of amylase can result either from an increased rate of entry of amylase into circulation and /or a decreased metabolic clearence of this enzyme due to renal failure or macroamylase (a condition in which an abnormally high – molecular – weight amylase is present in the serum ) (18) . torrent vernetta a. et. al. reported that macroamylaseaemia most often formed due the binding of the amylase to an immunoglobulin and it has been described as a causal finding associated to abdominal pain and to celiac disease (35) . oita t. et. al. demonstrated that immunoprecipitation assay showed that amylase was bound to poly clonal iga and igg (21) . no differences were found , when either the enzyme activity values or the enzyme specific activity values were used at comparison between control and patients with cd since this is due to the non significant difference in the measured protein concentration between control and patients ( table 2 ) . a significant positive correlation (p < 0.05 , r = 0.709 ) was found in the present study between α-amylase activity and anti gliadin igg in the sera of patients with cd who have increased levels in α-amylase activity while a non significant correlation (p<0.05) were found between α-amylase and fasting serum glucose and anti gliadin iga in the same patients with cd . conclusions from the results that had been obtained it conclude a significant positive correlation between α-amylase activity and antigliadin igg in the sera of of patients with cd who have elevated in enzyme activity .this finding would appear to suggest a possibility to use the amylase activity as a tool with antigliadin igg for diagnosis of celiac disease. acknowledgment the authors gratefully acknowledged the staff of al-uarmook teaching hospital and al-kadhimiya teaching hospital for making patients specimens available for this study. references 1. danalioglu a. , poturoglu s. , gungor gulluoglu m. , and et. al. .buddchiari syndrome in a young patient with celiac sprue: a case report . turk j. gastroenterol , 2003 ; 14(4):262-265. 2. tatham as, and shewry pr.allergens to wheat and related cereals.clin exp 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(epub ahead of print). iraqi j pharm sci, vol.20(1) 2011 α -amylase activity and celiac disease 86 33. galicka – latala d., zwolinskawcislo m. , sosin – rundnicka l. and rozpondek p. the role of celiac disease and type 1 diabetes coexistence . is celiac disease responsible for diabetic status . przegl lek . 2009; 66(4):170-175. 34. tourniaire j, guichard-rode s.and moniet jc. circulating antigliadin antibodies . prevlance in a diabetic (type 1 or 2)and non diabetic adult population . press med . 1995; 24(31):1425-1427. 35. torrent vernetta a., segarra canton o., soler palacin p.and et. al. macroamylasaemia in paediatrics . an pediatr (barc). 2008; 69(5):439-441. iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 41 preparation and evaluation of ketoprofen nanosuspension using solvent evaporation technique fatimah m. hussein wais *, ahmed n. abood **and hayder k . abbas***,1 *faculty of pharmacy , university of kufa , najaf ,iraq. **college of pharmacy , university of basrah, basrah, iraq. ***faculty of pharmacy, alkafeel university college, najaf, iraq abstract the effective surface area of drug particle is increased by a reduction in the particle size. since dissolution takes place at the surface of the solute, the larger the surface area, the further rapid is the rate of drug dissolution. ketoprofen is class ii type drug according to (biopharmaceutics classification system bcs) with low solubility and high permeability. the aim of this investigation was to increase the solubility and hence the dissolution rate by the preparation of ketoprofen nanosuspension using solvent evaporation method. materials like pvp k30, poloxamer 188, hpmc e5, hpmc e15, hpmc e50, tween 80 were used as stabilizers in perpetration of different formulas of ketoprofen nanosuspensions. these formulas were evaluated for particle size, entrapment efficiency of drug (ee), effect of stabilizer type, effect of stabilizer concentration and in-vitro dissolution studies. all of the prepared ketoprofen nanosuspensions formulas showed a particle size result within nano range. the average particle size of ketoprofen nanosuspensions formulas was observed from 9.4 nm to 997 nm. entrapment efficiency was ranged from 79.23% to 95.41 %. the in vitro dissolution studies showed a significant (p<0.01) enhancement in dissolution rate of nanosuspension formulas compared to pure drug (drug alone) and physical mixture (drug and stabilizer). the results indicate the suitability of solvent evaporation method for ketoprofen with improved in vitro dissolution rate and thus perhaps enhance fast onset of action for drug. keywords: ketoprofen , nanosuspension, particle size, dissolution rate. انوي للكيتوبروفين باستخدام تقنية تبخير المذيبنتحضير وتقييم معلق 1,***حيدر كاظم عباس و **، احمد نجم عبود *فاطمة محمد حسين ويس كلية الصيدلة ، جامعة الكوفة ، النجف ، العراق . * ** كلية الصيدلة ، جامعة البصرة ، بصرة ، العراق . *** قسم الصيدلة ، كلية الكفيل الجامعة ،النجف، العراق . الخالصة حجم الُجَسْيم وذلك الن الذوبان يحدث على سطح المذاب. المساحة السطحية المساحة السطحية لُجَسْيم الدواء تزداد بواسطة تقليل ك األكبر يرافقها المزيد والسريع في معدل الذوبان. الكيتوبروفين هو ضمن الصنف الثاني )نظام تصنيف الصيدالنيات البيولوجية( والذي يمل بة ومعدل الذوبان للكيتوبرفين من خالل تحضير معلق النانو بطريقة تبخير ذوبانية قليلة مع نفاذية عالية. الغرض من البحث هو زيادة االذا و الهايدروكسي بروبل مثيل سليلوز والتوين تم استخدامها كمثبتات في تحضير 811بيروليدون والبلوكسامير المذيبب. مواد مثل بولي فنيل حجم الُجَسْيم وتأثير نوع المثبت وتركيز المثبت ودراسات من خاللصيغ مختلفة من معلق النانو للكيتوبروفين. هذه الصيغ تم تقييمها ْيم الذوبانية بالمختبر. كل الصيغ المحضرة للُجَسْيمات النانوية للكيتوبروفين أظهرت حجم الُجَسْيم ضمن مدى النانو و ان معدل حجم الُجسَ . %44.98 الى %94.97 . كفاءة التحميل كانت بين نانومتر 449لى نانومتر ا4.9 لصيغ الُجَسْيمات النانوية للكيتوبرفين لوحظت بين في معدل الذوبان لصيغ معلق النانو بالمقارنة للدواء وحده او الخليط الفيزيائي (p<0.01ان دراسات الذوبان بالمختبر أظهرت تحسن مهم ) ين مع تحسين معدل الذوبان بالمختبر وهذا ربما يعزز الفعل األول من الدواء مع المثبت. النتائج تبين مالئمة طريقة تبخير المذيب للكيتوبرف والسريع للدواء. ، معلق نانوي ، حجم الجسيم ، معدل الذوبان .كيتوبرفين الكلمات المفتاحية : introduction bioavailability of drug depends on its dissolution and availability as a solution form at the site of absorption. therefore, the main problems associated with poor soluble drug its low solubility in biological fluids, which results into low bioavailability after administ ration. poor soluble drug will typically show dissolutionrate limited absorption (1). drug dissolution in biologic fluids is an important step for systemic absorption. 1corresponding author e-mail: hayderkadhim@ymail.com received: 12/8/2017 accepted: 29/9/2017 iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 42 the rate at which drugs with poor aqueous solubility dissolve and release from dosage form in the biologic system frequently controls the rate of systemic absorption of the drug. about 40% of different chemical entities discovered now are poorly water soluble (2). therefore, the pharmaceutical researches focus on enhancing the solubility and rate of dissolution of poorly water-soluble drugs. according to us food and drug administration (fda), the drug are classified into four classes based on aqueous solubility and permeation through biological membrane. these classes are follows(3) : class i (highly soluble, highly permeable), class ii (poorly soluble , highly permeable), class iii (highly soluble, poorly permeable),and class iv ( poorly soluble, poorly permeable) poor water soluble drug has many problems such as low or variable bioavailability, large dose and delayed onset of action. there are various approaches available for overcoming the solubility of poorly soluble drugs for examples, modification of the crystal habit, self-emulsification, solid dispersion, solubilization by surfactant, salt formation, ph modification, co-crystallization, use of cosolvent, micronization and nanosization. in general, the rate of the drug solubility is related to particle size, as a particle gets smaller one, as (the surface area: volume) ratio increases. the larger surface area allows a better interaction with the solvent, which cause increase in dissolution rate. since dissolution takes place at the surface of drug particle, the high dissolution rate of drug is associated with large surface area of drug particles. many drugs are active intravenously but are not effective when taken orally, because of low oral absorption. reduction of the particle size for drugs with low aqueous solubility to a micronized form has improved the oral absorption of griseofulvin, nitrofurantoin, and many steroids. in addition smaller particle size enhances water penetration into the particles (4). the ostwald–freundlich equation describes the relationship between the saturation solubility of drug and its particle size: 𝑙𝑜𝑔 𝐶𝑠 𝐶∞ = 2 𝜎 𝑉 2.303 𝑅𝑇 𝑝𝑟 ----------1 where cs is the saturation solubility, c∞ is the solubility of the solid particles with large sizes, σ is the interfacial tension, v is the molar volume, r is the gas constant, t is the absolute temperature, ρ is the solid density, and r is the radius. it is clear that the saturation solubility (cs) of certain drugs will increase by decreasing the particle size (r) (5). nanosization is a method, where a drug particle is converted into nanoparticles having size less than 1μm. nanotechnology allowed drug delivery system with improved physical, chemical and biological properties. the main purposes in designing nanoparticles as delivery systems are to control the particle size, surface properties and dissolution of drug, then to reach the action site at a perfect rate and dose level(6). the selection of suitable method for the formulation of nanoparticles depends on the physicochemical characteristics of used polymer and the drug to be loaded. however, the nanosuspension form has an advantage in their ability to increase dissolution rate and to enhance the bioavailability of poor soluble drug. ketoprofen is an example of class ii drug. it is a white crystalline powder and practically insoluble in water (7).it is a nonsteroidal anti-inflammatory drug with analgesic and antipyretic properties. the objective of study was to increase its solubility and then the dissolution rate by preparation of nanosuspension using antisolvent precipitation method. materials and methods materials ketoprofen was purchased from lishui nanming chemical co.,ltd (china). polyvinylpyrrolidone (pvp k30), poloxamer 188, and hydroxypropyl methylcellulose (hpmc) e5, e15 and e50 were purchased from shanghai send pharmaceutical technology co.ltd (china). methanol was obtained from gailand chemical company (uk). disodium hydrogen phosphate and potassium dihydrogen phosphate were supplied by bdh laboratory supplies (england) and spinechem. limited, respectively. methods determination of ketoprofen saturation solubility saturated solubility measurements of ketoprofen in various solutions were determined by shake flask method. an excess amount of ketopofen were separately introduced into stoppered conical flask containing 10 ml of iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 43 0.1 n hcl (ph 1.2), dw and buffers of phosphate (ph 6.8 and 7.4). the sealed flasks were shaken for 24 hours at 37°c .visual inspection was made to check the precipitation of drug particles in the sample. an aliquot of solution was passed through 0.45μm filter paper and the filtrate was suitably diluted and analyzed on a uv/visible spectrophotometer at 260 nm wavelength (8) .three determinations were carried out to calculate the solubility of ketoprofen . preparation of ketoprofen nanosuspensions nanosuspensions of ketoprofen were prepared by the solvent evaporation technique, which is also termed as antisolvent precipitation method. ketoprofen powder was dissolved in methanol (2.5 ml) at room temperature preparing drug concentrations (20 and 40 mg/ml). the resultant organic solution of drug (organic phase) was added drop by drop by means of a plastic syringe positioned with the needle directly into aqueous solution of stabilizer(9, 10). the mixture of drug solution and stabilizer was kept at 50°c and subsequently agitated at stirring speed of 500 revolution per minute (rpm) on a magnetic stirrer for about one hour to permit methanol to evaporate(11). ketoprofen being insoluble in water, therefore, it will precipitate with stabilizer. the ratios (weight: weight) of drug to stabilizer used to prepare the nanosuspension were 1:1, 1:2 and 1:3. tween 80 was also used at different volumes as explained in table (1). table (1) :compositions of ketoprofen nanosuspensions using different stabilizers at different drug: stabilizer ratios with constant volume of injection of organic solution (2.5 ml) formula no. drug (mg) pvp k30 (mg) poloxamer 188 (mg) hpmc e5 (mg) hpmc e15 (mg) hpmc e50 (mg) tween 80 (ml) 1 50 50 2 50 100 3 50 150 4 50 50 5 50 100 6 50 150 7 50 50 8 50 100 9 50 150 10 50 50 11 50 100 12 50 150 13 50 50 14 50 100 15 50 150 16 50 0.1 17 50 0.2 18 50 0.3 iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 44 continued table (1) formula no. drug (mg) pvp k30 (mg) poloxamer 188 (mg) hpmc e5 (mg) hpmc e15 (mg) hpmc e50 (mg) tween 80 (ml) 19 50 50 50 20 50 50 50 21 50 50 50 22 50 50 50 23 50 50 0.1 24 50 50 50 25 50 50 50 26 50 50 50 27 50 50 0.1 28 50 50 0.1 29 50 50 0.1 30 50 50 0.1 31 100 100 32 100 200 33 100 300 34 100 100 35 100 200 36 100 300 37 100 100 38 100 200 39 100 300 40 100 100 41 100 200 42 100 300 43 100 100 44 100 200 45 100 300 iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 45 effect of type and concentration of stabilizer on the size of ketoprofen nanosuspensioins to reach the best formula different types of stabilizers at various concentrations were used in preparation of ketoprofen nanosuspensions. the formulas (f1-f15) prepared by using these different stabilizers and were subjected to particle size analysis. the combinations of two stabilizers (f19-f30) were also studied to determine their effect on particle size. characterization of the prepared nanosuspension particle size and surface area determination of particle size was done using abt-9000 nano laser particle size analyzer (angstrom advance inc. usa), which is apparatus of a dynamic light scattering, acts by measuring the light intensity that is scattered by the molecules sample as a time function, at scattering angle (90°) and constant temperature (25°c) without dilution of samples. the particle size can be determined by placing samples of formulas in the analyzer. the average diameters and polydispersity index of samples were measured for each formula. determination of entrapment efficiency of drug (ee) in nanosuspension the freshly prepared nanosuspensions of different drug: stabilizer ratios were centrifuged at about 20,000 rpm for 20 minutes at 4°c using cooling ultracentrifuge. the concentration of free drug was detected by measuring the absorbance an appropriately diluted sample of supernatant at 260 nm using uvspectrophotometer (11-13). ee was determined by subtracting the weight of free drug in the supernatant layer of solution from the initial weight of drug used. for each formula, the experiment was repeated in triplicate and the average was calculated. percentage of entrapment efficiency (ee) could be calculated by the following equation: 𝑬𝒏𝒕𝒓𝒂𝒑𝒎𝒆𝒏𝒕 𝒆𝒇𝒇𝒊𝒄𝒊𝒆𝒏𝒄𝒚% = 𝒘𝒆𝒊𝒈𝒉𝒕𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒅𝒓𝒖𝒈 − 𝒘𝒆𝒊𝒈𝒉𝒕𝒇𝒓𝒆𝒆 𝒅𝒓𝒖𝒈 𝒘𝒆𝒊𝒈𝒉𝒕𝒊𝒏𝒊𝒕𝒊𝒂𝒍 𝒅𝒓𝒖𝒈 × 𝟏𝟎𝟎 (𝟐) in vitro dissolution of ketoprofen nanosuspensions in vitro dissolution study was performed by usp dissolution apparatus-type ii using 900 ml of 0.1n hcl (ph 1.2) as a dissolution medium maintained at 37 ± 0.5°c and stirring speed (50 rpm). the freshly prepared nanosuspensions (equivalent to 50 mg of ketoprofen ) of drug: stabilizer ratios were added to the dissolution medium, five-milliliter samples were withdrawn at specific intervals of time ( 5,10,15,20 ,30,40.50,60,70,80 and 90 minutes ), then filtered through a 0.45 µm filter paper and analyzed for their drug concentrations by measuring at 260 nm wavelength. the same test was done for pure drug (free drug in dissolution media) and physical mixture of drug powder and stabilizer material (pm) (14). in vitro dissolution / model independent approach the percent of dissolution efficiency (%de) was detected for comprising the relative performance of ketoprofen nanosuspension at drug: stabilizer ratios, pure ketoprofen powder and physical mixture of drug and polymer. the %de at 60 minutes (%de60 min) for each formulation was computed (using microsoft excel program) as the percent ratio of area under the curve of dissolution up to the time t to that of the rectangle area described by complete dissolution (100%) at the same time (15, 16). de = {∫ 𝒚. 𝒅𝒕 𝒕𝟐 𝒕𝟏 x /y100 x (t2-t1)} x 100 (3 ) the difference factor (f1) evaluating the percent error between two curves over all time points is given by: f1={ [∑t=1n │rt – tt │] / [ ∑t=1n rt] } x 100 (4) where t is the number of dissolution sample, n is the dissolution times number, and rt is the amount of the reference drug and tt is amount of test drug dissolved at each time point t. the percent error is zero when the test drug and reference outlines are matching and increase correspondingly with the dissimilarity between the two dissolution profiles. the similarity factor (f2) is a logarithmic transformation of the sum-squared error of differences between the test tt and reference rt over all time points, and is given by: f2= 50x log { [ 1 + 𝟏 𝒏 ∑ │𝑹𝒕 − 𝑻𝒕𝒏𝒕=𝟏 │ 2 ]-0.5 x 100 (5) the standards for similarity factor are 50100, while for dissimilarity factor are 0-15. the dissolution parameters were detected by fitting the data of dissolution using a software iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 46 program, called ddsolver, which is a menudriven add-in program for microsoft excel written in visual basic for applications. results and discussion determination of ketoprofen saturation solubility the saturation solubility values of ketoprofen were found to be about 0.11mg/ml, 0.205mg/ml, 9.82mg/ml and 10.75mg/ml, in d.w, ph 1.2, ph 6.8 and ph 7.4, respectively. the results of saturation solubility of ketoprofen were illustrated in table (2). determination of saturated solubility of pure ketoprofen powder in phosphate buffers (ph 6.8 and 7.4) and 0.1n hcl (ph 1.2) was necessary to maintain the sink condition that is the volume of medium at least three times greater than the necessary volume to form a solution saturated with drug substance (17). according to biopharmaceutical classification system, ketoprofen is an example of class ιι drugs having low aqueous solubility and well absorption through gastrointestinal tract (18) due to high permeability and lipophilicity. ketoprofen is a weak acid drug and will be ionized at higher ph; it is practically in soluble in water (7).therefore, its solubility increase with ph increase towards alkaline medium (19) as shown in figure (1). table (2): saturation solubility values of ketoprofen in different media media solubility (mg/ml) distilled water d.w 0.11 0.1n hcl (ph1.2) 0.205 phosphate buffer (ph 6.8) 9.82 phosphate buffer (ph 7.4) 10.75 figure (1): solubility-ph profile of ketoprofen powder particle size and surface area the average particle size of all the prepared formulas using abt-9000 nano laser particle size analyzer. all of the prepared ketoprofen nanoparticles formulas showed a particle size result within nano range. the average particle size of ketoprofen nanoparticles formulas was observed from 9.4 nm to 997 nm, as shown in tables (3) and (4).the smallest size 9.4 nm for f18 and the largest size 997nm for f26 formula. the specific surface area (ssa) of the particles is the summation of the areas of the exposed surfaces of the particles per unit mass. where the particle size is inversely related with the surface area(20). the ssa values for the prepared formulas were at range ( 2.07-247.47) m2/g, the largest surface area is recorded in f18 formula and smallest surface area 2.07m2/g in f26 formula as appeared in table (3 and 4). polydispersity index analysis polydispersity index is a parameter used to define the particle size distribution obtained from the particle size analyzer. polydispersity index gives degree of particle size distribution at range from 0.00466 to 0.019 depending on formulation variables. the formula f28 showed lowest pdi (0.00466), as seen in table (3), that indicate good uniformity of nanoparticle size. uniformity of particle size is determined by polydispersity index values in which the low value means the best uniformity. the range of pdi values (0-0.05) means (monodisperse system), 0.05-0.08 (nearly monodisperse), 0.080.7 (mid-range polydispersity), and >0.7 (very polydisperse) (21). effect of stabilizer type (optimization of polymer used) the effect of using one stabilizer the average particle size for formulas ( f1-f18) at drug: stabilizer was ranged from 9.4 nm-676.5 nm as seen in table (3). pvp k-30, poloxamer188, hpmc (e5, e15, and e50) and tween 80 were used as stabilizers in these formulas (f1-f18). pvp k-30 and poloxamer188 are polymeric nonionic stabilizers for nanosuspensions, they form physical barrier on the surface and interrupt the contact of the close particles (22). the non-ionic stabilizers with amphiphilic moieties are usually employed to give steric stabilization, which is dominated by wetting effect. iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 47 when non-ionic stabilizers are incorporated into nanosuspensions, they are adsorbed onto the surface of the drug particlesth rough an anchor part that is strongly interacted with the suspended particles (23), while the other part that is well-solvated tail will extend into the dispersion medium. hpmc was frequently used as a stabilizer due to its alkyl substituent, which has higher affinity towards the hydrophobic surface of drug particle (24).figure (2), shows the effect of polymer type on particle size. generally hpmc at 1:1 of drug: stabilizer ratio shows smaller submicron size (9.4nm) when compared with pvp k30 and poloxamer188 because the hydrophobic moiety of hpmc has a good affinity toward the drug particles and thereby it is able to provide active steric barrier against particles growth. on the other hand, the large size of particle (426.5nm) was obtained in formula f4 that contains poloxamer 188 as a stabilizer at drug: stabilizer 1:1. these results are in agreement with that obtained by el-badry m et.al. in preparation of albendazol nanosuspension(25). the size of other formulas lies between the average sizes ranges, since the differences in particle size are due to different in affinity of the polymer molecules toward drug particles. pdi for formulas (50mg of drug and 50mg of stabilizer) f1, f4, f7, f10, f13 and f16 (50mg of drug and 0.1 ml of tween 80) ranged from 0.008 to 0.016, therefore all these formulas have monodisperse standard. ssa of the formulas f1, f4, f7, f10, f13 and f16 was ranged from 21.02m2/g to 5.34 m2/g. larger surface area was found to be 21.02m2/g in formula f16 which contain tween 80 as a primary stabilizer, because it had smaller particle size. in contrast to this result, a smaller surface area 5.34 m2/g was obtained in formula f4 that contain poloxamer188 as a stabilizer, since it had lager particle size. table (3) :particle size, polydispersity index and specific surface area of ketoprofen formulas using different types and amounts of stabilizers formula no. average particle size (nm) pd ssa (m2/g) f1 317 0.012 6.75 f2 313 0.009 7.1 f3 282.5 0.009 7.65 f4 426.5 0.01 5.34 f5 317 0.011 6.88 f6 282.5 0.01 7.88 f7 270 0.008 8.23 f8 282.5 0.011 7.65 f9 317 0.01 6.84 f10 282.5 0.01 7.91 f11 426.5 0.024 5.58 f12 479 0.009 4.63 f13 269 0.008 8.23 f14 426.5 0.007 5.24 f15 676.5 0.011 4.17 f16 95.1 0.016 21.02 f17 88 0.012 24.97 f18 9.4 0.017 247.47 iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 48 figure (2): effect of using different types of polymers on particle size at ratio 1:1 (f1, f4, f7, f10 and f13). the effect of costabilizers combination of stabilizers was preferred for long-term stabilize. based on the stokes’principle, drug nanocrystals tend to precipitate in the production media like water or other vehicles (23) .the appearance of agglomeration was largely due to the small particle size, which leads to increment surfacevolume ratio that can produce a large amount of gibbs free energy. slowly, the particle will collective automatically to decrease the extra surface energy. when it comes to crystal growth, the theoretical background is ostwald ripening. for that reason, a stabilizing agent is used to solve this problem. stabilizers can efficiently decrease the surface activity energy to inhibit aggregation that decrease dissolution rate (23). the critical parameter of stabilizer is the stabilization ability by enhancement of the physical stability of nanoparticulate system and maintaining the smallest size of particles. (26). the average particle size of f19-f30 was ranged from 49 nm to 997 nm as shown in table (4). table (4): particle size, polydispersity index and specific surface area for ketoprofen formulas using combined stabilizer system formula type of mix polymers average particle size (nm) pdi ssa (m2/g) f19 pvp+polo188 49 0.008 45.40 f20 pvp+ e5 399 0.012 6.13 f21 pvp +e15 503 0.071 4.39 f22 pvp+e50 564 0.018 3.91 f23 pvp+tween80 99.9 0.019 24.73 f24 polo188+e5 399 0.007 5.75 f25 polo188+e15 797 0.008 2.73 f26 polo188+e50 997 0.005 2.07 f27 polo188+tween80 84.85 0.011 31.45 f28 e5+tween 80 111 0.00466 21.62 f29 e15+tween80 119 0.01 17.87 f30 e50+tween80 141 0.01 15.09 iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 49 according to the above results, a smaller particle size (49 nm) was achieved in a combination between pvp k30 and poloxamer188. since this combination show significant (p<0.05) reduction in particle size as when compared with other combinations, and it may be because of the highest affinity to the drug molecules, the same data was reported by xueming li et.al. in fenofibrate nanosuspension(27). a combination of pvp k30 and hpmc (e5, e15, and e50) gave a large particle size as when compared with each polymer separately, this result was in agreement with shahzeb khan et.al in preparation of ibuprofen nanocrystal(28). furthermore combination of poloxamer188 and hpmc (e5, e15, e50) in formulations f24, f25 and f26 shows larger average particle size with significant differences(p<0.05), these results due to that the combination lead to increase viscosity of the disperse media, so it is ineffective combination and cannot stabilize the nanoparticulate system. on the other hand, a mixture of tween 80 with each polymer separately gave good reduction in particle size as seen in figure (3).from above results formula f19 was select as a best formula for further evaluation tests. figure (3): effect of use of mixed polymers on particle size of ketoprofen a combination of tween 80 and pvp k30 or poloxamer188 yields nanoparticles with average size not different significantly (p>0.05) from tween 80 as single stabilizer. this is due to pvp k30 and poloxamer188 are polymeric molecules, have ability to adsorb on the particle surface and act as a steric barrier, preventing close contact of the particles. furthermore, it may be because both of them are non-ionic stabilizers so no ionic repulsion occurs. similar results were gained by p. kocbek et.al in preparation of ibuprofen nanosuspension (29). the size of particles in other combination of tween 80 with hpmc polymer types was larger than that obtained from combination of tween with poloxamer or pvp k 30. pdi of formulas (f19-f30) was varied from 0.00466 to 0.019, therefore, all these formulas have monodisperse standard. depending on formulation variables, the formulation f28 (hpmc e5 +tween 80) showed lowest pdi (0.00466) that indicates good uniformity in average particle size distribution. a narrow size distribution is essential to prevent particle growth, due to ostwald ripening phenomenon(30). formula f21 (pvp k30+hpmc e15) exhibited a highest pdi (0.071) so that is in the range of nearly monodisperse. ssa of formulas (f19-f30) was ranged from 2.07m2/g to 45.40 m2/g. f19 formula (pvp k30+poloxamer188) showed large surface area about 24.73 m2/g, since it had showed small particle size. f26 formula (poloxamer188+ hpmc e50) exhibited small surface area, due to large particle size of this formula, as particle size decrease, the surface area per unite mass increase and viceversa. effect of stabilizer concentration the concentration of stabilizer may give negative effect (decrease particle size) or positive effect of on particle size (increase particle size). it can also influence on the adsorption affinity of non-ionic stabilizers to particle surface. in general, as the concentration of stabilizer increases the particle size decreases at fixed drug concentration, which indicated that the drug particle surface was sufficiently enveloped by the stabilizer molecules (31). concentration of stabilizer was played a role in maintaining stability of nanosuspension, if used too low concentration lead to aggregation of particle, and if used high concentration lead to enhance ostwald ripening (25). as shown in table (3) the size range of particles is decrease in the sequence of f1 (317nm) > f2 (313 nm) > f3 (282.5nm) that correspond to 1:1, 1:2 and 1:3 of drug: stabilizer (pvp k30) ratio, respectively, these results indicated that mean size of particles showed a regular decrease with increasing the concentration of pvpk30. the same result was observed with poloxamer188. the size range of particles is also decreased in the sequence f4 (426.5nm)>f5 (317nm)>f6 (282.5nm) that correspond to 1:1, 1:2 and 1:3 of drug: stabilizer (poloxamer188) ratio, respectively. figures 4 and 5 explain the effect of pvp k30 and iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 50 poloxamer188 concentration on average particle size correspondingly. these effects may be due to a process of a primary covering of the newer surfaces competing with the aggregation of the uncovered surfaces. hence, an elevation in ratio of surfactant in the primary dispersion results in rapid enclosing of the newly formed particle surfaces. there was an optimum concentration of surfactant, above which the increase in concentration did not result in a decrease in particle size due to saturation point; these results are in agreement with that obtained by chander parkash dora et.al in preparation of glibenclimid nanoparticle when poloxamer188 was used as stabilizer at different ratios (32). poloxamer188 (pluronic f68)® is a block copolymer, can act as a surfactant, responsible for the hydrophobic association with the molecules of drug. the inhibition of the crystal growth is mainly related to the hydrophobic part (polypropylene oxide group ppo) in the pluronic polymer, while the second chain which is (the hydrophilic polyethylene oxide) (peo) can provide steric hindrance against particles aggregation26. on the other hand, as the concentration of hpmc increases the particles size increases in different ratios of grades. for grade e15 the size range is increase in sequence of f10 (282.5nm) <f11 (426.5nm) < f12 (479nm), that correspond to 1:1, 1:2 and 1:3 of drug: stabilizer (hpmc e15) ratio, respectively. the same results was observed when compared the size of particles for other grades (e5 and e50), as illustrated in figures 6, 7 and 8. these findings may be due to the addition of polymer produce a coat around drug particles until a certain concentration where all drug particles are coated with polymer, then an increasing of polymer concentration would lead to increment the thickness of the polymer coat around each particle or may be lead to aggregation of many particles. yuancai dong, et.al, obtained the opposite result when hpmc was used as a stabilizer in preparation of nanoparticles of spironolactone using antisolvent precipitation technique(33). furthermore, tween 80 was also used as stabilizer in preparation of nanoparticles, as the amount of tween 80 increased the average particle size decreased. it act as a wetting agent at low concentration or below its cmc (critical micelle concentration) and as solubilizing agent at concentration above cmc (34), since surfactant adsorption at solid-liquid interface can lead to decrease surface tension and increment in nucleation rate and lead to decrease particle size, as shown in figure (9). sameer v. dalvi and rajesh n. dave, et.al, observed similar results(35) . figure (4): effect of pvpk30 concentration on particle size figure (5): effect of poloxamer188 concentration on particle size figure (6): effect of hpmc e5 concentration on particle size iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 51 figure (7): effect of hpmc e15 concentration on particle size figure (8): effect of hpmc e50 concentration on particle size figure (9): effect of tween 80 concentrations on particle size the effect of entrapment efficiency the encapsulation efficiency of different formulas of nanoparticles prepared from different stabilizer concentrations were evaluated, the data are illustrated in table (5), which shows that entrapping efficiency ranged from 79.23% (f7formula) to 95.41 % (f15formula) it is clear that the increase in stabilizer concentration increased the drug entrapment efficiency(36). the higher encapsulation efficiency of f15 formula (hpmc e50 at 1:3 drug: stabilizer ratio) may be because of optimum type and concentration of polymer used. the encapsulation efficiency may be dependent on hydrophobic part of hpmc e50, which has a high affinity to hydrophobic ketoprofen . in-vitro dissolution study and model independent analysis of nanosuspension the dissolution profile of prepared nanoparticles formulations was carried out for formulas f1 to f15 and for f19, f31 and f45 formulas at 1:1, 1:2 and 1:3 of drug: stabilizer ratio, because they provide the optimum entrapment efficiency. the dissolution of the prepared formulas was done in 0.1n hcl (ph 1.2) at 37°c ±0.5, the release profile was also carried out for physical mixture (drug and polymer) and for drug alone (pure drug). table (5) summarize the dissolution parameters for all formulas such as dp10 min (percent drug dissolved within 10 minutes), similarity and dissimilarity. these dissolution parameters were done for all formulas by fitting the dissolution data using a software program, called ddsolver(37). a comparative study was done, since the dissolution profiles of ketoprofen from different samples was made using f1 and f2. according to the food and drug administration’s guidelines, f1 values lower than 15 (0–15) and f2 values greater than 50 (50–100) mean similarity of the dissolution profiles (38). figures (10, 11, 12, 13, 14 and 15 ) illustrated the release of drug from formulas at ratio 1:1 ,1:2 and 1:3 for single stabilizer [ f1-f3(pvp k30), f4-f6(poloxamer188) , f7-f9( hpmc e5) , f10-f12 ( hpmc e15) and f13-f15 (hpmc e50)] , co-stabilizer f19 (pvp k30 and poloxamer188) , respectively. the results shown in table (5) indicate that there is significant (p<0.01) enhancement in dissolution rate of all nanosuspension formulas compared to pure drug (drug alone) and physical mixture (drug and polymer). this confirms the superiority of the prepared of nanoparticles formulas compared to that of physical mixture and drug alone, since these nanoparticles provide a large surface area than others. on comparison with free drug (pure drug), the cumulative percentage of drug release for f19, f31 and f45 formulas were estimated in 0.1n hcl (ph 1.2). the cumulative % of drug iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 52 release of these formulas was 100 % within 10 minutes. on the other hand, a low percentage of release was obtained from pure drug (52.7% ) in the same time (10 minutes) and maximum cumulative % of drug release was reached (100 %) at 80 minutes. so there is a significant differences (p<0.01) in release of drug between pure drug and other formulas, as shown in figure (16). these results are consistent with study, which reported by monzurul amin roni et.al (39) .the reasons for such results that some of drug particle may be convert from crystalline to amorphous state. in addition, it is mainly attributed to the rule, which states that the reduction of drug particle size can result in larger surface area and consequently enhanced the contact of nanoparticles with the dissolution medium. the gained results are coordinated with noyes–whitney equation, in which the increment in saturation solubility and the decrement in particle size can lead to an enhanced dissolution rate (40). table (5) :dissolution parameters for f1 to f15, f19, f31 and f45 formulas figure (10): dissolution profile of the prepared ketoprofen nanoparticles (f1, f2 and f3) compared to physical mixture and free drug in 0.1n hcl (ph 1.2) at 37 c0± 0.5. figure (11): dissolution profile of the prepared ketoprofen nanoparticles (f4, f5 and f6) compared to physical mixture and free drug in 0.1n hcl (ph 1.2) at 37 c0± 0.5. formula dp at 10 min de 60 min dissimilarity factor f1 similarity factor f2 entrapment efficiency ±sd f1 100 94.8 24.36 29.43 91.4 ± 0.152 f2 72 90.3 17.75 38.64 92.92 ± 0.014 f3 73.5 90.9 18.25 37.82 91.83 ± 0.007 f4 82.4 89.5 18.8 35.15 94.99 ± 0.045 f5 84.3 92.77 21.49 36.33 91.13 ± 0.015 f6 75 94.7 23.97 33.71 88.13 ± 0.040 f7 83.4 91.2 19.44 36.14 79.23 ± 0.040 f8 83 90.9 19.01 36.7 83.56 ± 0.080 f9 87.34 92.9 21.73 32.84 86.26 ± 0.136 f10 97.7 90.9 19.46 35.36 84.03 ± 0.020 f11 84.6 90.9 18.88 36.76 82.84 ± 0.102 f12 86.6 91.1 19.20 36.45 82.4 ± 0.050 f13 86 94.3 28.52 34.05 83.91 ± 0.055 f14 91 96 25.82 32.75 85.25 ± 0.045 f15 100 96.9 27.17 30.75 95.41 ± 0.015 f19 100 93.4 22.48 31.96 91.6 ± 0.076 f31 100 95.1 24.78 28.84 90.43 ± 0.025 f45 100 97.7 28.31 29.66 93.01 ± 0.037 iraqi j pharm sci, vol.26(2) 2017 ketoprofen nanosuspension 53 figure (12): dissolution profile of the prepared ketoprofen nanoparticles (f7, f8 and f9) compared to physical mixture and free drug in 0.1n hcl (ph 1.2) at 37 c0± 0.5. figure (13): dissolution profile of the prepared ketoprofen nanoparticles (f10, f11 and f12) compared to physical mixture and free drug in 0.1n hcl (ph 1.2) at 37 c0± 0.5. figure (14): dissolution profile of the prepared ketoprofen nanoparticles (f13, f14 and f15) compared to physical mixture and free drug in 0.1n hcl (ph 1.2) at 37 c0± 0.5. figure (15): dissolution profile of the prepared ketoprofen nanoparticles f19 compared to physical mixture and free drug in 0.1n hcl (ph 1.2) at 37 c0± 0.5. figure (16): dissolution profile of the prepared ketoprofen nanoparticles (f19, f31 and f45) compared to free drug in 0.1n hcl (ph 1.2) at 37 c0± 0.5. conclusion the data confirm that antisolvent 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of mefenamic acid 24 design and synthesis of some nitrate derivatives of mefenamic acid with expected nitric oxide release ahmed a. sadon *,1 and ahlam j. qasir * * department of pharmaceutical chemistry,college of pharmacy,university of baghdad,baghdad, iraq abstract this study include design and synthesis of 2 derivatives of compounds consisting of mefenamic acid, glycine and organic nitrates (2-nitrooxy ethanol or 1,3-dinitrooxy-2-propanol). nitric oxide no has been reported to support many of the same mucosal protection mechanisms as prostaglandins and is sufficient for acute gastroprotection and ulcer healing. so we suppose these 2 compounds would reduce non-steroidal antiinflammatory drugs nsaids gastrointestinal side effect. key words: non-steroidal anti-inflammatory drugs (nsaids), glycine, mefenamic acid, organic nitrates, nitric oxide (no). اهيك هع تىقع تحرر ألوكسيذ الٌتريكبعض هشتقاث الٌتراث لحاهض الويفيٌ تصوين وتخليق عبذ الىاحذ سعذوى احوذ *،1 قصيرو احالم جويل * . *فشع انكيمياء انصيذالويت ، كهيت انصيذنت ، انجامعت انمستىصشيت ، بغذاد ، انعشاق الخالصة مع وتشاث انميفيىامك مع انكاليسيه حامط يه جذيذيه هما عباسة عه مشتقاث تتضمه انذساست تصميم و تخهيك مشكب . انعذيذ مه انبحىث انحذيثت تؤيذ انذوس انمشابه بيه أوكسيذ بشوباوىل(-٢-دايىايتشوكسي-١,٣و أ وايتشوكسي إيثاوىل-٢) عضىيت ىاءا عهى رنك وفتشض بأن هزيه انمشتقيه انجذيذيه انىايتشيك و انبشوستىكالوذيه في حمايت انقىاة انمعىيت و معانجت تقشحاتها. ب ستيشويذيت انمضادة نإلنتهاب عهى انقىاة انمعىيت. انغيش قهيم األعشاض انجاوبيت نألدويت سيساهمان في ت عضىيت، اوكسيذ الٌتريك حاهض الويفيٌاهيك ،ًتراث ، كاليسيي ( ، nsaidsلتهاباث الغيرستيرويذيت ) لال االدويت الوضادة الكلواث الوفتاحيت : (no . ) introduction nonsteroidal anti-inflammatory drugs (nsaids) are still one of the first choices in the treatment of inflammatory conditions and among the most commonly prescribed drugs (1) . however, they are not devoid of adverse effects, gastrointestinal (gi) ulceration and renal damage being the most common, and even life-threatening (2) . several strategies have been followed to reduce these adverse effects, including enteric coating, parenteral administration, and formulation of prodrugs that require hepatic metabolism for the cyclooxygenase (cox) activity to be unmasked and coadministration of either suppressors of acid secretion or exogenous prostaglandins (pgs), without the desired results (3) . a more recent and promising approach is that of cox-2 inhibitors (4) . but these drugs may lead to increased cardiovascular events (5) . another approach to reduce the toxicity of nsaids is the linking of a nitric monoxide (no) releasing moiety to these compounds, and thus, a new category of anti-inflammatory agents is emerging, the nonsaids (6) . nitric oxide is a small diatomic radical that plays an important physiological role in nervous, cardiovascular, and immune systems (7) . its generation is controlled by the three isoforms of no synthase (nos), neuronal and endothelial nos (nnos and enos), which are constitutive and produce nanomolar amounts of no important for normal cell function and tissue protection, and inducible nos (inos) which generates high amounts of no responsible for the destruction of invading pathogens as part of an overall inflammatory response (8) . the endogenous tissue no, generated constitutively by gi enos and nnos, appears to play a key role in the chronic maintenance of gastrointestinal tissue integrity and in adaptive cytoprotection to injurious stimuli, perhaps acting synergistically with other cytoprotective prostaglandins (9) . no promotes several gastric defense mechanisms by increasing mucus and bicarbonate secretion in the gi tract, increasing mucosal blood flow, and inhibiting the proinflammatory activities of neutrophils and platelets (10) . additionally, no may reduce inflammation connected to oxidative stress by scavenging reactive oxygen species, which can adversely increase mucosal permeability and kill cells (8) . furthermore, it is found that no and no-derived reactive nitrogen species interact with peroxidases (11) and lipoxygenases (12) , altering the generation of prostaglandins and leukotrienes, which are signaling molecules involved in inflammation (13) . 1 corresponding author email : ahlmedsadon78@yahoo.com received : 24/12/2011 accepted : 20/5/2012 iraqi j pharm sci, vol.21(2) 2012 nitrate derivatives of mefenamic acid 25 several no-nsaids, such as no-aspirin, nodiclofenac, no-flurbiprofen, and noketoprofen, have already been synthesized and clinical trials for some of them revealed protective activity against acute myocardial infarction (14) . these compounds appear to have a safe gi profile and can be effective in a variety of diseases including cardiovascular, rheumatological, lung diseases, alzheimer’s disease and cancer. there is also evidence to suggest that these compounds release no in a metabolic, and not spontaneous, way and therefore, they do not interfere with blood pressure regulation (15) . bearing in mind the above studies, we designed new derivatives of no-releasing nsaids (scheme-1). two derivatives of mefenamic acid-glycine esterified with 2-bromoethanol or 1,3dibromo-2-propanol where they are then converted to nitrooxy derivatives upon treatment with silver nitrate in acetonitrile. socl2 ch3oh o o h n ch3 h cl o oh h2n glycine + nh ch3 ch 3 r o oh dcc dmap nmm coupling r o 1 n n a o h d ee st er if ic a ti o n o oh h nr o o o h2n ch3 dcc dmapsteglich esterification br o h nr o o o h nr o o br ho dcc dmap steglich esterification oh br br o h nr o o o n+ o o  o n+ o o o n+ o o  o h nr o o br br compound (1) compound (2) compound (3) compound (4) compound (5) agno3 compound (i) agno3 compound (ii) r= · scheme 1 : chemical synthesis of compounds i and ii. iraqi j pharm sci, vol.21(2) 2012 nitrate derivatives of mefenamic acid 26 experimental chemicals 1,3-dibromo-2-propanol, 4-dimethyl amino pyridine (dmap), n-methylmorpholine (nmm) (aldrich), 2-bromoethanol, glycine, n,n′-dicyclohexylcarbodiimide (dcc) (fluka), mefenamic acid (sdi), silver nitrate (riedel-dehaën), thionyl chloride (merck), all solvents were of analar type and used without further purification. detection melting points (uncorrected) were determined by capillary tube method on barnstead/electrothermal (uk) and ft-ir spectra were recorded on ft-ir spectrophotometer shimadzu (japan) at the college of science/ department of chemistry (al-mustansyria university). the chno analysis was carried out using carlo-erba 1106 elemental analyzer (france). ascending thin layer chromatography (tlc) was run on silica gel 60 f254 pre-coated aluminum sheets, merck (germany) to check the purity and the reaction's progress. the detection of derivatives was done using uv 254 nm lamp and the chromatograms were eluted by two solvent system: a\ ethanol: water (7:3) (16) . b\ acetone: chloroform: acetic acid: water (3:2:1:4) (17) . synthesis synthesis of compound (1); (methyl-2aminoacetate hydrochloride) (18) : h cl o o h 2 n ch 3· thionyl chloride (3.25ml, 45 mmol) was slowly added by dripping to methanol (40 ml) kept on a temperature at (−10) to (−5°c); then glycine (3gm, 40 mmol) was added. the stirred mixture kept on 40-45°c for 2 hrs and then was refluxed for 4 h at 60-70°c under continuous stirring. then excess thionyl chloride and solvent were removed under reduced pressure giving crude glycine methyl ester hydrochloride, which was treated with an 8 ml portion of cold diethyl ether at 0°c. the resulting solid product was collected and dried under vacuum. it was re-crystallized from hot methanol by slow addition of an 8 ml of diethyl ether followed by cooling at 0°c. the crystals were collected on the following day and washed twice with a (diethyl ether /methanol) mixture (5:1 ratio) followed by pure diethyl ether and dried under vacuum to give pure glycine methyl ester hydrochloride. the percent of yield is 95%. the physical appearance, melting point and the rf value are listed in table (1) the characteristic ir bands are listed in table(1) the characteristic ir bands are listed in table(3). synthesis of compound (2); (methyl 2-(2((2,3-dimethylphenyl) amino) benzamido) acetate) (19) : o o o h n nh ch3 ch3 ch3 to a stirred solution of compound 1 (1.883 gm, 15 mmol) in (120ml) of dimethyl formamide dmf, (3.3 ml, 30 mmol) of nmethylmorpholine (nmm) was added with stirring for 10 minutes, later on (3.62 gm, 15 mmol) of mefenamic acid was added, and the mixture was cooled down to (−10c), then (0.183 gm, 1.5 mmol) of 4-dimethyl amino pyridine (dmap) and (3.4 gm, 16.5 mmol) of n,n′-dicyclohexylcarbodiimide (dcc) were added with stirring which was continued for 2 days at 0c and then at room temperature for 3 days. the reaction mixture was subjected to evaporation under vacuum to get rid of dmf, re-dissolved in 150 ml of chloroform from which the n,n′-dicyclohexyl urea (dcu) was filtered off. the clear filtrate was washed with 0.5 n naoh (3x150 ml), 1n hcl (3x75 ml), saturated sodium chloride solution (3x75 ml) and water (2x75 ml). the chloroform layer was dried over anhydrous sodium sulphate and the solvent was evaporated under vacuum. the percent of yield is 83.5%. the physical appearance, melting point and the rf value are listed in table (1) the characteristic ir bands are listed in table(3). synthesis of compound (3); (2-(2-((2,3dimethylphenyl) amino) benzamido) acetic acid) (20) : o oh o n h nh ch3 ch3 to a stirred solution of compound 2 (3.123 gm, 10 mmol) in 60 ml acetone, kept on 18-22°c, an aqueous solution of 1 n naoh (25 ml) was added drop wise over a period of 30 minutes where the stirred reaction mixture was continued for additional 8 hours. the mixture was treated with 1 n hcl to get ph 1. iraqi j pharm sci, vol.21(2) 2012 nitrate derivatives of mefenamic acid 27 a precipitate was appeared which was separated by filtration, washed on the filter paper with water, and re-crystallized from nhexane:acetone (4:3) mixture. the percent of yield is 53.5%. the physical appearance, melting point and the rf value are listed in table (1), the characteristic ir bands are listed in table(3). synthesis of compound (4); (2-bromoethyl 2(2-((2,3-dimethylphenyl) amino) benzamido) acetate) (21) : o o o h n nh ch3 ch3 br to a stirred solution of compound 3 (1.49 gm, 5 mmol), 2-bromoethanol (0.71 ml, 10 mmol) and dmap (0.305 gm, 2.5 mmol) in (30ml) of dichloromethane (dcm) kept at −10c, dcc (1.14 gm, 5.5 mmol) was added. the stirring reaction mixture was continued for 2 days at 0c and then at room temperature for 3 days.the reaction mixture was subjected to evaporation under vacuum to get rid of dcm, re-dissolved in 50 ml ethyl acetate from which the n,n′-dicyclohexyl urea (dcu) was filtered off. the clear filtrate washed with 5% sodium bicarbonate solution (3x25 ml), 1n hcl (3x25 ml) and water (2x25 ml). the ethyl acetate layer was dried over anhydrous sodium sulphate and evaporated under vacuum. the product was dissolved in ethanol (20 ml) with stirring. to the stirred solution about 20 ml of distilled water was added slowly from a dripping funnel. the precipitated solid was separated by filtration, dried and kept in a desiccator. the percent of yield is 82%. the physical appearance, melting point and the rf value are listed in table (1) the characteristic ir bands are listed in table(3). synthesis of compound (5); (1,3dibromopropan-2-yl 2-(2-((2,3dimethylphenyl) amino) benzamido) acetate) (21) : o o o h n nh ch3 ch3 br br to a stirred solution of compound 3 (1.49 gm, 5 mmol), 1,3-dibromo-2-propanol (1.02 ml, 10 mmol) and dmap (0.061 gm, 0.5 mmol) in (30ml) of dcm kept at −10c, dcc (1.14 gm, 5.5 mmol) was added.the reaction mixture was worked up as in synthesis of intermediate 4. the percent of yield is 77%. the physical appearance, melting point and the rf value are listed in table (1) the characteristic ir bands are listed in table(3). synthesis of compound (i); (2-(nitrooxy) ethyl 2-(2-((2,3-dimethylphenyl) amino) benzamido) acetate) (22) : o o o h n nh ch3 ch3 o n+ o o a solution of compound 4 (0.81 gm, 2 mmol) in dry acetonitrile (10 ml) was treated portion wise with a solution of agno3 (1.36 gm, 8 mmol) in dry acetonitrile (20 ml) and the whole stirred mixture was refluxed for 48h in the dark. at the end of the reflux, the inorganic solid (black coloured silver bromide) was filtered off. the filtrate was subjected to evaporation under vacuum till dryness; the residue was dissolved in 20 ml dcm and washed with water (3x40 ml), saturated sodium chloride solution (2x40 ml) and finally water (2x40 ml). the organic layer was dried over anhydrous sodium sulfate, evaporated under vacuum and the obtained residue was recrystallized from absolute methanol and triturated with petroleum ether (40-60c). the percent of yield is 41%. the physical appearance, melting point and the rf value are listed in table (1), the elemental microanalysis results are presented in table(2), and the characteristic ir bands are listed in table (3) . synthesis of compound (ii); (1,3-bis (nitrooxy) propan-2-yl-2-(2((2,3dimethylphenyl) amino) benzamido) acetate) (22) : o o o h n nh ch3 ch3 o n+ o oo n+ o o a solution of compound 5 (0.996 gm, 2 mmol) in dry acetonitrile (10 ml) was treated portion wise with a solution of agno3 (2.72 gm, 16 mmol) in dry acetonitrile (40 ml) and the whole stirred mixture was worked up as in synthesis of compound i. the percent of iraqi j pharm sci, vol.21(2) 2012 nitrate derivatives of mefenamic acid 28 yield is 38%. the physical appearance, melting point and the rf value are listed in table (1) , the elemental microanalysis results are presented in table(2), and the characteristic ir bands are listed in table (3) . result and discussion the synthesis of the designed compounds has been successfully achieved. and the purity and structural formulas of the synthesized compounds were characterized by their melting points, rf values, ft-ir spectroscopy and elemental microanalysis.we expect these two compounds i and ii would reduce gastrointestinal side effect largely. so we recommend for their biological evaluation for safe gastrointestinal profile. table 1 : the characterization and physical data of the synthesized compounds. compounds empirical formula description melting point o c rf value solvent system compound 1 c3h8clno2 white powder 175-176 0.34 a compound 2 c18h20n2o3 pale yellow powder 126-128 0.68 b compound 3 c17h18n2o3 yellow powder 150-151 0.31 b compound 4 c19h21brn2o3 red crystal 156-158 0.75 b compound 5 c20h22br2n2o3 red crystal 168-170 0.83 b compound i c19h21n3o6 brown powder 172-173 0.49 b compound ii c20h22n4o9 brown powder 198-201 0.34 b table 2 : elemental microanalysis of the compounds i and ii. compound empirical formula molecular weight elemental analysis % element calculated observed i c19h21n3o6 387.387 c 58.91 58.13 h 5.46 5.71 n 10.85 10.33 o 24.78 25.83 ii c20h22n4o9 462.41 c 51.95 50.37 h 4.8 5.06 n 12.12 12.59 o 31.14 31.97 table 3 : characteristic ft-ir absorptions bands of the synthesized compounds. compound band (cm -1 ) interpretation compound 1 2500-3300 1751 1585, 1504 1261 asymmetrical and symmetrical nh3 + stretching c=o stretching of ester asymmetrical and symmetrical bending of nh3 + asymmetrical c-o stretching of ester compound 2 3377 3311 3063, 3037 2931 2856 1749 1629 1581 1510 1276 1228 n-h stretching of 2º aromatic amine n-h stretching of 2º amide aromatic c-h stretching asymmetrical c-h stretching of ch3 and ch2 symmetrical c-h stretching of ch3 and ch2 c=o stretching of ester c=o stretching of 2º amide (amide i) n-h bending of 2º amide (amide ii) n-h bending of 2º aromatic amine c-n stretching of 2º aromatic amine asymmetrical c-o stretching of ester compound 3 3423 3367 2750-3200 3034 2937 1741, 1707 1626 1579 1512 1278 1211 n-h stretching of 2º aromatic amine n-h stretching of 2º amide oh stretching of carboxyl group aromatic c-h stretching asymmetrical c-h stretching of ch2 c=o stretching of acid c=o stretching of 2º amide (amide i) n-h bending of 2º amide (amide ii) n-h bending of 2º aromatic amine c-n stretching of 2º aromatic amine asymmetrical c-o stretching of acid iraqi j pharm sci, vol.21(2) 2012 nitrate derivatives of mefenamic acid 29 compound 4 3308 3059 2931 2856 1747, 1705 1639 1583 1516 1276 1224 n-h stretching of 2º amide aromatic c-h stretching asymmetrical c-h stretching of ch3 and ch2 symmetrical c-h stretching of ch3 and ch2 c=o stretching of ester c=o stretching of 2º amide (amide i) n-h bending of 2º amide (amide ii) n-h bending of 2º aromatic amine c-n stretching of 2º aromatic amine asymmetrical c-o stretching of ester compound 5 3275 3055 2929 2856 1745, 1710 1631 1581 1514 1265 1224 n-h stretching of 2º amide aromatic c-h stretching asymmetrical c-h stretching of ch3 and ch2 symmetrical c-h stretching of ch3 and ch2 c=o stretching of ester c=o stretching of 2º amide (amide i) n-h bending of 2º amide (amide ii) n-h bending of 2º aromatic amine c-n stretching of 2º aromatic amine asymmetrical c-o stretching of ester compound i 3286 3061 2931 2856 1747, 1710 1635 1581 1516 1278 1226 n-h stretching of 2º amide aromatic c-h stretching asymmetrical c-h stretching of ch3 and ch2 symmetrical c-h stretching of ch3 and ch2 c=o stretching of ester overlapped c=o stretching of 2º amide (amide i) and n=o asymmetrical stretching of nitrate n-h bending of 2º amide (amide ii) n-h bending of 2º aromatic amine c-n stretching of 2º aromatic amine asymmetrical c-o stretching of ester compound ii 3277 3059 2929 2854 1745, 1714 1637 1579 1512 1278 1226 n-h stretching of 2º amide aromatic c-h stretching asymmetrical c-h stretching of ch3 and ch2 symmetrical c-h stretching of ch3 and ch2 c=o stretching of ester overlapped c=o stretching of 2º amide (amide i) and n=o asymmetrical stretching of nitrate n-h bending of 2º amide (amide ii) n-h bending of 2º aromatic amine c-n stretching of 2º aromatic 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and biological investigation of certain pyrazole3-carboxylic acid derivatives as novel carriers for nitric oxide. bioorg. med. chem., 2009, 17, 3829–3837. javascript:showcontents(%22t3144844%22) javascript:showcontents(%22t3144844%22) javascript:showcontents(%22t3144844%22) javascript:showcontents(%22t3144844%22) http://www.springerlink.com/content/x95xjnepd3eqnm0a/ http://www.springerlink.com/content/x95xjnepd3eqnm0a/ http://www.springerlink.com/content/x95xjnepd3eqnm0a/ http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1997.00608.x/abstract http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1997.00608.x/abstract http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1997.00608.x/abstract http://www.sciencedirect.com/science/article/pii/s016561479901353x?_rdoc=3&_fmt=high&_origin=browse&_srch=hubeid(1-s2.0-s0165614700x00430)&_docanchor=&_ct=8&_reflink=y&_zone=rslt_list_item&md5=8689dda3639fd91a7a520aaf9e324094 http://www.sciencedirect.com/science/article/pii/s016561479901353x?_rdoc=3&_fmt=high&_origin=browse&_srch=hubeid(1-s2.0-s0165614700x00430)&_docanchor=&_ct=8&_reflink=y&_zone=rslt_list_item&md5=8689dda3639fd91a7a520aaf9e324094 iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement doi: http://dx.doi.org/10.31351/vol27iss1pp8-19 8 investigation of solubility enhancement approach of ticagrelor ihsan a. mohammed*,1 and mowafaq m. ghareeb** *ministry of health and environment, babylon health directorate, babylon, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the aim of this study was to increase the solubility and enhancement of dissolution rate of poorly water-soluble drug ticagrelor this was done through formulating and evaluate ticagrelor nanoparticles using solvent antisolvent technology. ticagrelor is a practically water-insoluble drug which acts as an antiplatelet medicine.fifteen formulas were prepared, and different stabilizing agents were used with various concentrations such as poly vinyl pyrrolidine (pvpk-30), poloxamer 188 (pxm) and hydroxypropyl methyl cellulose (hpmc). the ratios of drug to stabilizers used to prepare the nanoparticles were 1: 0.5, 1:1 and 1:2. the prepared formulas were evaluated for the sake of particle size, dissolution study, fourier transform infrared spectroscopy, differential scanning calorimetry, and scanning electron microscopy. on the other hand, the increasing dissolution rate as the particle surface area can be increased due to the reduction of particle size to the nano range.the results showed that hydroxy propyl methyl cellulose (hpmc) (f 12) was found to be the best stabilizer. keywords: ticagrelor, nanoparticles, particle size, hydroxy propyl methyl cellulose. لتيكاكريلوردراسة وسائل زيادة الذوبان ل **موفق محمد غريب و 1،* داحسان علي محم وزارة الصحة والبيئة ، دائرة صحة بابل، بابل ، العراق.* ** فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ،العراق. الخالصة جسيمات نانوية لعقار التيكاكريلور باستخدام تكنلوجيا الترسب من مضاد أنحاللان الهدف من هذه الدراسة هو زيادة ذوبان وتعزيز المذيب. تيكاكريلور هو دواء غير ذائب في الماء وهو دواء يعمل على منع تخثر الصفيحات الدموية. (، pvpار مختلفة استخدمت بتراكيز مختلفة مثل الفانيل بايريلدون المتعدد )تم اعداد خمسة عشر صيغة باستخدام بوليمرات استقر ( وكانت نسب الدواء الى المثبتات المستخدمة في اعداد الجسيمات النانوبة هي (hpmcالمثيل السليلوز وهيدروكسي بروبيلبولوكسامير وكذلك دراسة التوافق )مطيافية االشعة تحت أنحاللجسيمات، دراسة من حيث الحجم الحبيبي لل تم تقييم الصيغ المعدة .1:5, 5:5, 0.1:5 االلكتروني. من ناحية أخرى يزداد تحرر الدواء كلما صغر حجم والمجهر المسح التفاضلي( تفاضلية المسح الكالوري متري وقياسالحمراء هو افضل ) (hpmc) )f12هيدروكسي بروبيل المثيل السليلوزالجسيمات النانوية لزيادة المساحة السطحية للجسيم. وأظهرت النتائج ان النانوية.بوليمر استقرار للجسيمات الكلمات المفتاحية: تيكاكريلور، الجسيمات النانوية، الحجم الحبيبي، هيدروكسي بروبيل المثيل السليلوز. introduction many problems may arise from the poor solubility of drug candidates in the field of drug research and development. the aqueous solubility (1) of the drug is a critical determinant of its dissolution rate, and its limited dissolution rate that can arise from the low solubility which can frequently result in a low bioavailability of the orally administered drugs. also, a drug with aqueous solubility lower than 100 µg/ml, can present a dissolution-limited absorption. in such a case, dose escalation may be required until the blood drug concentration reaches the therapeutic drug concentration range (2). the dissolution rate is often the rate-determining step in the drug absorption for poorly soluble drugs only. the challenge facing these drugs is to enhance the rate of dissolution or solubility. moreover, dissolution subsequently improves absorption and bioavailability. such formulation methods targeting dissolution enhancement of poorly soluble substances are continuously introduced (3). the released enhancement of poorly soluble drugs may be carried out by an increase of the drug surface area, the drug solubility, or by formulating the drug in its dissolved state. 1corresponding author e-mail: ehsan2013.mohamed@gmail.com received: 3/10/2017 accepted: 1/1/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp8-19 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 9 various techniques have been employed to formulate oral drug delivery system that would enhance the dissolution profile and in turn, the absorption efficiency of a water-insoluble drug such as micronization, adsorption onto high surface area carriers, lyophilization, co-grinding, formulation of inclusion complexes (4). one of these different solubility enhancement techniques is the nanotechnology which is nano-sized particles having attractive characteristics and receiving at the same time considerable attention in the last decade. polymeric nanoparticles (pnps) are solid particles or particulate dispersions with size in the range of 10–1000 nm. since these particles are small in size, the surface area is very large, so the percentage of atoms or molecules on the surface can be increased significantly (5). ticagrelor molecular formula and molecular mass: c23h28f2n6o4s (522.57 gm/mole). ticagrelor is a crystalline powder with an aqueous solubility of approximately 3.5μg/ml at room temperature (6). ticagrelor exhibits no pka value within the physiological range can be categorized as a class iv drug (low solubility, low permeability) with a mean absolute bioavailability of ticagrelor in healthy volunteers is 36 % (7). this study aims to increase the solubility and enhancement of dissolution rate of poorly watersoluble drug ticagrelor this is done through formulating and evaluate ticagrelor nanoparticles by using solvent antisolvent technology. materials and methods materials ticagrelor powder was purchased from (aopharm, china). poly vinyl pyrrolidine pvp k-30, poloxamer 188, hpmc, sodium starch glycolate (hi media laboratories, india). methanol (gcc analytical reagent, uk). brij35 (polyoxyethylene (23) lauryl ether) (riedal de haen ag seelze, hannover, germany). magnesium stearate (barlocher, gmbh, germany). methods preparation of ticagrelor nanoparticles ticagrelor nanoparticles had been prepared by using solvent/antisolvent precipitation technique (nanoprecipitation method). a certain amount of pure drug of ticagrelor had been completely dissolved in methanol/water miscible solvent. the obtained drug solution had been injected at a speed of 1ml/min using syringe infusion pump into the water containing one of the stabilizers (pvp, pxm, and hpmc) with continuous stirring. precipitation of solid drug particles occurred immediately upon mixing. the precipitated nanoparticles had been sonicated at 37 °c for 30 minutes and then lyophilized using freeze drying system (labconco, usa) to obtain the nanoparticles powder (8). the composition and variable conditions of preparation of different formulas of nanoparticles were shown in table (1). table 1. composition of ticagrelor nanoparticles formulas. formula no. polymer solvent: antisolvent ratio methanol: water drug: polymer ratio f1 pvp 1:10 1:0.5 f2 pvp 1:10 1:1 f3 pvp 1:10 1:2 f4 pvp 1:05 1:1 f5 pvp 1:15 1:1 f6 pxm 1:10 1:0.5 f7 pxm 1:10 1:1 f8 pxm 1:10 1:2 f9 pxm 1:05 1:1 f10 pxm 1:15 1:1 f11 hpmc 1:10 1:0.5 f12 hpmc 1:10 1:1 f13 hpmc 1:10 1:2 f14 hpmc 1:05 1:1 f15 hpmc 1:15 1:1 formulation variables affecting the properties of the prepared nanoparticles to study the factors affecting the properties of the prepared nanoparticles, different formulas were utilized in these studies as follow: effect of polymer concentration the effect on a physical characteristic of prepared nanoparticles studied with different concentrations of the same polymer like pvp (f1, f2, and f3), pxm (f6, f7, and f8), hpmc (f11, f12, and f13). the results of this factor were recorded. effect of solvent: antisolvent ratio the effect of different solvent: antisolvent ratio on a physical characteristic of the prepared nanoparticles studied utilizing formulas (f2, f4, and f5) with pvp as stabilizer, (f7, f9, and f10) with pxm as stabilizer, (f12, f14, and f15) with hpmc as stabilizer. the results of this factor were recorded. iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 10 effect of polymer type the effect on a physical characteristic of prepared nanoparticles was studied with different polymer type like: i at low polymer: drug ratio: f1, f6, and f11. ii at high polymer: drug ratio: f3, f,8 and f13. iii at low solvent: antisolvent ratio: f4, f9, and f14. iv at high solvent: antisolvent ratio: f5, f10, and f15. the results of this factor were recorded. evaluation of ticagrelor nanoparticles particle size analysis samples of all prepared nanoparticles were analyzed using abt-9000 nano laser particle size analyzer, and particle size distribution curves were obtained. also, the average particle size, polydispersity index (pdi) for each sample were recorded (9). determination of ticagrelor content in nanoparticles to determine the drug content of the prepared nanoparticles, 200 mg sample of each prepared formula was placed in a glass mortar and thoroughly triturated using methanol. after thoroughly rinsing all equipment, the total mixture was transferred to a volumetric flask, and the volume was completed to 100 ml with methanol (96%). the resultant dispersion was sonicated for 15 min to ensure complete dissolution of ticagrelor. the mixture was filtered through an ordinary filter paper, and the absorbance of ticagrelor was determined spectrophotometrically. the amount of drug inside the nanoparticles was determined applying the equation of the calibration curve (10). determination of percentage of yield and entrapment efficiency nanoparticles after being dried were weighed, and the yield was calculated as a percentage of the total weights of starting material (polymer and drug) introduced into the system (this represents the theoretical weight of nanoparticles) and the actual weight of nanoparticles obtained. the percent yield was calculated using the following equation (11). %yield = actual weight of nanoparticles gained theoretical weight of nanoparticles x100 the entrapment efficiency of nanoparticles was determined from the theoretical and actual drug contents. the latter being determined from the results of the assay, described in section of drug content the percent entrapment efficiency was calculated the following equation. % entrapment efficiency (ee) = actual drug content theoretical drug content x100 determination of ticagrelor nanoparticles saturation solubility saturation solubility of the selected formulas of ticagrelor nanoparticles was carried out using the shake flask method for different test media water, hcl buffer ph 1.2 with 1 % brij 35 and phosphate buffer solution ph 6.8 with 1 % brij 35. an excess amount of the drug nanoparticles was added to 10 ml of medium in a test tube and stirred in a water bath with the shaker at 37±2°c for 48 hours. filtered samples were analyzed spectrophotometrically for drug content (12). nanoparticles surface morphology studies scanning electron microscopy (sem) scanning electron microscopy was utilized to observe surface properties and as well as the particle size of nanoparticles. scanning electron microscope of ticagrelor nanoparticles was operated with a secondary detector at different acceleration voltage and different magnification values. preparation of nanoparticles incorporated tablets of ticagrelor ticagrelor nanoparticles of the selected formulas with all excipients (except the lubricant) as listed in table (2) were accurately weighed and passed through 20 mesh sieve. the powder was blended in a poly bag by tumbling for 15 minutes. the blending was continuing for further 1 minute after addition of magnesium stearate as a lubricant. the final mixture was compressed using a 9-mm single punch tablet machine at 10kn compression force (13) table 2. composition of nanoparticles incorporated tablets. composition (mg) formulation no. f 2 f 7 f 12 amount of nanoparticle equivalent to ticagrelor 90 90 90 sodium starch glycollate 3 % 3 % 3 % magnesium stearate 2 % 2 % 2 % mannitol up to total weight 300 300 300 iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 11 in vitro dissolution study the prepared tablets were subjected to dissolution study. the usp paddle method was used for the in vitro dissolution studies. in this method, hcl solution (ph1.2) with 1 % brij 35 and phosphate buffer solution (ph 6.8) with 1 % brij 35 were used as dissolution medium. the rate of stirring was 75 ± 2 rpm. the amount of ticagrelor was 90 mg in all formulations. the dosage forms were placed in 900 ml of both media and maintained at 37 ± 0.1°c. at appropriate time intervals (5, 10, 15, 20, 30, 40, 50,60,70,90 and 100 minutes), five ml of the samples were taken and filtered through a 0.45mm millipore filter. the dissolution medium was then replaced by five ml of fresh dissolution fluid to maintain a constant volume. the samples were then analysed at λmax of ticagrelor by uvspectrophotometer. the mean of three determinations was used to calculate the drug release from each of the formulations. results and discussion evaluation of the prepared nanoparticles particle size analysis samples of all the prepared nanoparticles formulas were analysed by using abt-9000 nano laser particle size analyzer, and particle size distribution curves were obtained. also, the average particle size and polydispersity index (pdi) of each sample were recorded in table 3. effect of polymer concentration the results were shown in figure 1-3 of the nanoparticle of the three polymers (pvp, pxm, and hpmc) indicated that changing polymer concentration had an impact on ticagrelor nanoparticles mean size. increasing polymer concentration to certain level led to increasing in mean particle size but observed only higher than drug: polymer equal ratio. these results could be explained by increasing polymer concentration which can caused more coating of drug particles until a particular concentration was reached where all drug particles were coated with a polymer. then the increasing polymer concentration would led to increase the thickness of the polymer coat around each particle, or it may resulted in the aggregation of many particles and increased in the mean particle size (14-16). effect of solvent: antisolvent ratio the effects of changing the ratio of injected drug-solvent solution to stabilizer antisolvent solution on the mean size of the nanoparticles formed have been shown in figure 4-6. these figures illustrate that the solvent: antisolvent ratio 1:10 was the best ratio among the other ratios and this, in turn, manifested that the former gave the lowest mean particle size for all types of polymer (17) . table 3. particle size range of the prepared nanoparticles. formul a no. particle size range (nm) effectiv e particle size average (nm) pdi f 1 722.2-773.6 854 0.005 f 2 199.2-415.9 297.6 0.027 f 3 669-4126.5 1661.5 0.358 f 4 2107.7-10000 9471.7 0.221 f 5 41.4-10000 8086 0.475 f 6 285.3-2236.3 798.7 0.480 f 7 149.4-490.2 299.1 0.04 f 8 3695.7-4662.4 4151 0.005 f 9 1-10000 11303.4 0.402 f 10 7060.6-10035.6 10867.7 0.269 f 11 431.9-460.7 451.2 0.005 f 12 195.3-240.6 229.6 0.05 f 13 340.6-2031 831.9 0.343 f 14 3980.3-4524 5104.3 0.319 f 15 416.9-10000 4281.7 0.370 figure 3. effect of hpmc: tigacrelor on tigacrelor naonoparticle size iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 12 figure 4. effect of solvent: antisolvent ratio using pvp on ticagrelor nanoparticle size. figure 5. effect of solvent: antisolvent ratio using pxm on ticagrelor nanoparticle size. figure 6. effect of solvent: antisolvent ratio using hpmc on ticagrelor nanoparticle size effect of polymer type the effect of polymer type on nanoparticle size was studied at two factors: i. drug: polymer ratio and ii. levels of solvent: antisolvent ratio. at low drug: polymer ratio (1: 0.5), the polymers produce small particle size in the order of hpmc<pvp<pxm as shown in figure (7) and at high drug: polymer ratio (1:2), the order was hpmc< pvp < pxm as illustrated in figure (8). it was noticed that there were significant differences in nanoparticles mean size between formulas using different polymer type as a stabilizer. these differences may be attributed to the difference in affinity of polymers to the drug particles. on the other hand, at low and high antisolvent: solvent ratio figure (9 and 10), the order of polymer produced small particle size was hpmc<pvp<pxm. polyvinylpyrillidone k-30 and hpmc were polymeric non-ionic stabilizers; they stabilize the system by steric stabilization which can be accomplished by adsorbing polymers onto the drug particle surface by an anchor segment that strongly interacts with the dispersed particles, while the other well-solvated tail part extends into the bulk medium. moreover, the pvpk-30, is a well-known efficient polymeric stabilizer forming adsorption layers on drug nanoparticles, (18) whereas the poloxamer 188 is an anon ionic surfactant (19). the formulations containing pvp k-30 and hpmc as stabilizers had a small particle size in comparison with the formulation containing poloxamer 188 that gave larger particle size. poloxamer188 (pluronic f68)® is a block copolymer, responsible for the hydrophobic interaction with the drug molecule, the crystal growth inhibition is mainly due to the hydrophobic polypropylene oxide group (ppo) in the pluronic polymer, while the hydrophilic polyethylene oxide (peo) chains provide steric hindrance upon aggregation (20). poloxamer188 can form a valuable mechanical and thermodynamic barrier at the interface that hinders the approach and coalescence of individual emulsion droplets at their optimum level. although this mechanism of poloxamer 188 gave larger particle size in all three ratios 0.5:1, 1:1 and 1:2 drug: the polymer in formulas f6, f7, and f8; respectively, the formulas that contain poloxamer188 as a stabilizer had large particle size. such a thing may be attributed to the insufficient affinity, of poloxamer188 to ticagrelor, and possess a slow diffusion rate and ineffective adsorption onto the drug particle surface in the water-methanol mixture. however, if there is no affinity between the particle surface and the polymer, the attractive forces between two particles become dominant due to depletion of polymer from the gap of two particles (reduction force) (21). iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 13 figure 7. effect of polymer type at low drug: polymer ratio (1: 0.5) on nanoparticles size figure 8. effect of polymer type at high drug: polymer ratio (1: 2) on nanoparticles size. figure 9. effect of polymer type at low solvent: antisolvent ratio (1:05) on nanoparticles size figure 10. effect of polymer type at high solvent: antisolvent ratio (1: 15) on nanoparticles size polydispersity index of the prepared nanoparticles pdi shows in table 4 is an index of width or spread or variation within the particle size distribution and indications of the long-term stability of nanoparticles. monodisperse samples have a lower pdi value, whereas higher values of pdi indicate a wider particle size distribution and the polydisperse nature of the sample. the usual range of pdi values is 0 0.05 (monodisperse standard), 0.05 0.08 (nearly monodisperse), 0.08 0.7 (mid-range polydispersity), and >0.7 (very polydisperse) (22). the pdi results of the selected formulas (f2, f7, and f12) showed high uniformity in particle size of the prepared nanoparticles since it was in the monodisperse range which mainly attributed to the efficiency of the preparation method. table 4. polydispersity index of selected formulas formula no. particl e size (nm) polydispersity index (pdi) f2 297.6 0.027 f7 299.1 0.04 f12 229.6 0.05 percent yield and entrapment efficiency of prepared ticagrelor nanoparticles the percentage yield and entrapment efficiency of ticagrelor nanoparticles of the selected formulas were shown in the table (5). the high yield percent and entrapment efficiency of the prepared nanoparticles indicated that technique applied in preparation of nanoparticles was good enough for such preparations. iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 14 table 5. the percent yield and entrapment efficiency of selected formula formula no. % yield % ee f2 91.5 90 f7 89 87.2 f12 95 92.5 saturated solubility study of the prepared nanoparticles the solubility of ticagrelor nanoparticles of the selected formulas in different solvents was determined as shown in the table (6). ticagrelor nanoparticles saturation solubility increased in all of the three selected formulas (f2, f7, and f12). the saturation solubility’s of ticagrelor nanoparticles of the selected formulas in water increased 8.191, 7.121 and 9.376 folds relative to a pure drug for formulas f2, f7 and f12, respectively. the increase in saturation solubility was mainly due to nanonization effect (23, 24). table 6. solubility data of the ticagrelor nanoparticle selected formulas in different media solvent solubility of selected formulas (mg/l) f2 f7 f12 water with 1% brij 35 29 28 30 hcl solution ph 1.2 with 1% brij 35 31.53 28.8 7 32.8 16 phosphate buffer ph 6.8 with 1% brij 35 29.144 6 28 30.2 6 drug compatibility study fourier transforms infra-red spectroscopy the fourier transforms-infra red spectrum gives some information about the functional groups that may interact with excipient during formulation. the ir spectrum of ticagrelor figure (11) (12) showed the characteristic peak. the spectra of the selected formulas f12 represented in figures revealed the presence of central peaks of drug which indicated that there was no noticeable interaction between drug and polymer during the preparation of nanoparticles. figure 11. ftir spectrum of pure ticagrelor powder iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 15 figure 12. ftir spectrum of f12 (hpmc) nanoparticles differential scanning calorimetry (dsc) differential scanning calorimetry thermogram of ticagrelor showed a sharp endothermic peak at corresponding to its melting point which indicated a pure crystalline state of the drug as shown in figure (13) (14). also the thermogram of nanoparticles of the selected formula f12 as shown in figures indicated reducing in the crystallinity and conversion of more percentage of drug to amorphous form 100.00 200.00 300.00 temp [c] -5.00 0.00 5.00 mw dsc 143.43x100c figure 13. dsc thermogram of pure ticagrelor iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 16 100.00 200.00 300.00 temp [c] 2.00 3.00 4.00 5.00 6.00 7.00 8.00 mw dsc 136.00x100c figure 14. dsc thermogram of f12 (hpmc) ticagrelor nanoparticles. in vitro dissolution study results the results of dissolution study of marketed tablet (birlanta ®) in different buffer ph figure (15) showed similarity with nanoparticle formulas representing by similarity factor (f2) and dissolution efficiency(de), generally in both media; in hcl buffer ph 1.2 (de=85%) and in phosphate buffer ph 6.8 (de=89%). on the other hand, figures (16-19) showed the considerable improvement in dissolution represented by the dissolution efficiency of nanoparticles incorporated tablets of 88 % and 92 % for the f 12 and this mainly due to nanosizing of the particles which consequently enhanced the solubility. these results expected according to noyes– whitney equation where the solid dissolution rate is directly proportional to its surface area exposed to the dissolution medium (25-28). the similarity factor f2 is a measure of the similarity in the percent of dissolution between two curves. current fda guidelines (29) suggest that the dissolution profiles are considered similar if f2 is greater than 50 (50–100), which is equivalent to an average difference of 10% at all sampling time points (30, 31). the f2 result agreed with the current fda guidelines as shown in table 7. table 7. similarity factor (f2) and dissolution efficiency (de%) of ticagrelor nanoparticles. formula no. f2 de % ph 1.2 ph 6.8 ph 1.2 ph 6.8 birlinta ® 89 85 f2 79 73 87 83 f7 64 54 85 80 f12 75 70 92 88 iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 17 figure 15. dissolution profile of birlinta in hcl buffer ph 1.2 and 6.8 with 1 % brij. figure 16. dissolution profile of nanoparticle of selected formulas (f2, f7 and f12) incorporated in tablets in phosphate buffer ph 1.2 with 1 % brij 35. figure 17. dissolution profile of nanoparticle of selected formulas (f2, f7, andf12) incorporated in tablets in phosphate buffer ph 6.8 with 1 % brij 35. figure 18. dissolution profile of nanoparticle of selected formula f 12 incorporated in tablets in buffer ph 1.2 with 1 % brij 35. figure 19. dissolution profile of nanoparticle of selected formula f12 incorporated in tablets in phosphate buffer ph 6.8 with 1 % brij 35. further characterization of the optimum nanoparticle formula depending on the results of previous studies which demonstrate in a watertight way that formula f12 (hpmc) was the suggested formula for preparation of nanoparticle with optimum properties, thus it was subjected to advance analysis. scanning electron microscope (sem) the images of the sem at different magnification as shown in figure (20) of nanoparticles obtained from the selected formulas (f12) indicated uniform submicron sized particles. iraqi j pharm sci, vol.27(1) 2018 ticagrelor solubility enhancement 18 figure 20. scanning electron microscope image of f12 (hpmc) nanoparticles at different magnification conclusions depending on the result of the studies, one can conclude the following; the polymer, drug, and solvent: antisolvent ratio, in addition to polymer type, showed the considerable effect on the nanoparticle size. the optimum formulation variables were 1:1 and 1:10 ratio for the drug: polymer ratio and solvent: antisolvent ratio respectively. among the three polymers used, hpmc produced the smallest nanoparticle size. the optimum formula (f12) nanoparticles increment in saturated solubility about nine times that of pure drug.the dissolution efficiency of the ticagrelor optimum formula nanoparticles f12 incorporated in tablet showed increment to (de=92% and 88%) (ph 1.2 and ph 6.8), respectively in comparison to that of the marketed tablet (de=89% and 85%). also, similarity factor f2 was 75 and 70 at ph 1.2 and ph 6.8, respectively for selected formula. analysis by dsc and sem of nanoparticles of selected formula (f12) indicated reducing in the crystallinity and changing to amorphous form of the drug. references 1. lin ch, chen ch, lin zc, fang jy. recent advances in oral delivery of drugs and bioactive natural products using solid lipid nanoparticles as the carriers. j food drug anal. 2017; 25(2): 219-234 2. pardeike j, strohmeier dm, schrödl n, voura c, 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(ed.) advances in analytical chemistry and instrumentation. interscience, new york. 1965; 4: 117-212 13. dolenc a, govedarica b, kocbek p, srčič s, kristl j. nanosized particles of orlistat with enhanced in vitro dissolution rate and lipase inhibition. international journal of pharmaceutics. 2010; 396(1): 149-55. 14. alexandridis, p.; holzwarth, j., f.; hatton, t. a. macromolecules 1994; 27: 2414-2425. 15. matteucci me, hotze ma, johnston kp, williams ro. drug nanoparticles by antisolvent precipitation: mixing energy versus surfactant stabilization. langmuir. 2006; 22(21): 8951-9. 16. pouretedal hr. preparation and characterization of azithromycin nanodrug using solvent/antisolvent method. international nano letters. 2014; 4(1): 103. 17. dong y, ng wk, shen s, kim s, tan rb. preparation and characterization of spironolactone nanoparticles by antisolvent precipitation. international journal of pharmaceutics. 2009; 375(1): 84-8. 18. pandya vm, patel jk, patel dj. effect of different 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evaluation of cefixime nanocrystals. iraqi journal of pharmaceutical sciences. 2017; 23(2):1-2. 24. hecq j, deleers m, fanara d, vranckx h, amighi k. preparation and characterization of nanocrystals for solubility and dissolution rate enhancement of nifedipine. international journal of pharmaceutics. 2005; 299(1): 16777. 25. liu y, sun c, hao y, jiang t, zheng l, wang s. mechanism of dissolution enhancement and bioavailability of poorly water-soluble celecoxib by preparing stable amorphous nanoparticles. journal of pharmacy & pharmaceutical sciences. 2010; 13(4): 589606. 26. khan ka and rhodes c t, effect of compaction on particle size, j pharm sci,1975; 64: 444-7 27. khan s, batchelor h, hanson p, et al. physiochemical characterization drugpolymer dissolution and in vitro evaluation of phenacetin and phenylbutazone solid dispersion with polyethylene glycol 8000. j pharm sci 2011; 100: 4281–4294. 28. moore jw, flanner hh. a mathematical comparison of curves with an emphasis on invitro dissolution profiles. pharmaceutical technology. 1996; 20(6): 64-74 29. omar mady et al., studying the effect of dispersed drug crystal in the organic phase on the encapsulation by solvent evaporation technique (3) independent models as tools for studying the drug release profiles. world j pharm sci 2014; 2(4): 409-421 30. paulo costa, jose´ manuel sousa lobo. modeling and comparison of dissolution profiles. european journal of pharmaceutical sciences.2001; 13: 123–133 31. tran th, poudel bk, marasini n, chi s-c, choi h-g, yong cs, kim jo. preparation and evaluation of raloxifene-loaded solid dispersion nanoparticle by spray-drying technique without an organic solvent. international journal of pharmaceutics. 2013; 443(1-2): 50. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 49 in vitro release study on capsules and tablets containing enteric coated granules prepared by wet granulation eman b. h. al-khe dairy* received 27-4-2004 accepted 21-10-2004 abstract we t granulation metho d was used instead of conventional pan c oating or fluidized – bed coa ting te chnique to p repare e nteric -coate d diclofenac sodium granules , us ing ethano lic s olutio n of eudragittm l10 0 as coating, b inding and granula ting a ge nt .add ition of peg400 o r di-n-b utyl phthalate as a plas ticizer was found to imp ro ve the e nteric p rope rty of the coat. part o f the re sulted granules was fille d in ha rd gelatin c apsules (s ize 0), while the o ther p art was co mpressed into tab le ts with a nd without disinte grant. the releas e profile of these two dos age fo rms in 0 .1 n hcl (ph 1.2)fo r 2 ho urs, and in p hos phate buffer (p h 6.8) for 45 minutes as we ll as the re lease kine tic we re c omp ared with that o f the e nteric film c oated voltadin (r) sdi tab le ts . the results of this s tudy s ho w tha t, the prepared dos age forms have a goo d ente ric property, with fa ster re lease of drug fro m e nc aps ulated enteric-coated granules in c ompa ris on with comp ress ed tab le ts. الخالصة ة لتحضيرحبيبات خدام الطريقةالرطب ست وفي تم ا كل ى لتحضيرعقار صوديوم داي شائعة األخر ال من الطرق ال ة معويا بد غلف ناك و ذلك م مادة اليودراجيت ا ل ولي ل وب في ۱۰۰باستخدام محلول كح ث انها التذ حي ة غلف كونها مادة م كمادة رابطة لتكوين الحبيبات باألضافة الى جيني اعلى من ولكن تذوب في أس هيدرو عدة ولي اثيلين ٦الم مثل الب ة غلف زيد من مرونة المادة الم خدام مواد ت ست وقد وجد ان ا ول معوي ٤۰۰كاليك غليف ال حسن من عملية الت وتيل فثاليت ت مادة ثنائي بي .و ة وسرعة كيفي ة رن مقا ب٬ حيث تم وتحويله الى حبو ما تم ضغط الجزء اآلخر ة جزء من هذه الحبيبات في كبسوالت ك وقد تم تعبئ هيدروجيني هذه األشكال الصيدالنية في وسط ذي اس ن عقار م رر ال ري( ۱‚۲تح مض الهيدرو كلو هاحا رية قيمت ۰ك ذي عيا لمدة ) ٫۱ جيني ي اس هيدرو عتين وفي وسط ذ ة معويا( دقيقة مع حبوب الفولتادين ٤۵لمدة ٦٫۸سا غلف راء ) الم وية سام عمل اد .من ا نتاج م ة على وي حضرة٬ وأ ن تحرر العقار من الكبسوالت الحا ة الم الني ي لألشكال الصيد ودة التغليف المعو الحبيبات وقد اظهرت النتائج ج وب حب رره من ال ويا كان أسرع من تح ة مع غلف .الم introduction en te ric co ated granules e nc apsulated in hard ge la tin caps ules as a p harmaceutica l d osa ge form were us ed to ge t products with fa st o ns et of absorption and be tter pha rmaco kine tic properties (1-3), s ince this dosa ge form is less influenc ed by fo od intake due to faster gas tric e mptying of granules after diss olving of hard gelatin s he ll co mpared with the retentio n o f re la tiv ely la rge ente ric coated ta blet in the s to mac h. in a dditio n, patients p re fe rred the easily swa llowed ge latin c apsules on tab le ts (4). different tec hniques were used to prepa re ente ric-co ated granules or pellets, s uc h a s pan coating and fluidize d bed coating ap paratus (5-8). the a im of this study is to investigate whe ther ente ric-co ated granules co uld be p repa red by ordina ry we t granulatio n metho d using ethanolic solution of eudragittm l 1 00 as coating and granulating agent. the non-steroida l anti-infla mma to ry d rug (ns aid) dic lo fe na c s od ium is us ed as a mode l d rug fo r this stud y. materialsand method mater ials: the following ma te rials we re us ed , dic lofenac so dium (bio gena.ita ly), eudragitt m l 10 0 (rhö m pharma ,gmbh, weiterstadt, germany) ,ethanol 95% ,polyethylene glycol 400 (bdh chemica ls ltd ,pool engla nd ), lac to se ,hyd rochloric acid, (rieda l-de hae n, ag seelze-ha nno ve r, germany) ,microcrys ta lline c ellulose (av icel p h 102 ) (f mc co rp oratio n,pe nsylvania usa.) ,trisod ium pho sp hate (hopkins and willia ms ltd . ,en gland), di-n-butyl p htha la te (us b ,b. brus sels ,be lgium), magnes ium stea ra te (me rc k ,darms ta dt ,ge rmany), and ente ric film c oated vo lta din(r) s di tab le t as a re fe re nce. *de partment of pharmaceutics ,college of pharmacy, univers ity o f bag hdad,bag hdadir aq ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 50 method:  preparation of enteric-coated granules: diclofenac sod ium was mixed with lactose and the powde r mass was mo is te ned with 2 % e thanolic s olution of eud ra gitt m l100 as a gra nulating , b inding and ente ric coating agent (6, 9) until the p ro pe r consistency was obta ined. the a moun t of enteric polyme r was about 1.25% base d o n the to ta l weight of the powder mixture. the moist ma ss was the n granulated by passing through a 0.8 mm mes h size s ieve . the gra nules we re the n dried o n trays. part o f the res ulte d granules was filled in ha rd gelatin capsules size 0 (25 mg ⁄ c aps ule) while the othe r p art was co mpressed into ta blets (25 mg ⁄ ta blet).  dissolution: the dissolution c harac te ris tics of the encapsula ted ente ric-coated gra nules, the co mpressed ta blets fro m the prep ared gra nules and vo ltad in(r)sdi ta blets as a refere nce were stud ied using usp xxiv metho d for enteric coated products a t 50 r.p.m and c ons tant te mpe ra ture (37± 0.5 °c ). one capsule or ta blet of e ach was p la ced in 7 50 ml 0 .1 n hcl (p h 1 .2 )a nd samples were ta ken for 2 hours, fo llowed b y add ition o f 250 ml of 0.2m trisodium p hos phate to the same jar to get phosp ha te buffer o f p h 6 .8 , then sa mpling were co ntinue d fo r 45 minutes (10). sa mples were taken at certain ti me intervals and assayed fo r diclofena c so dium spec trophtometric ally a t its λ max 27 3 nm fo r the ac id me dium and at λ max 27 6 nm for the buffer medium.  factors affe cting the preparation: 1. effec t of addit io n of pla stic iz er: two d iffe re nt typ es of p la stic izers were us ed to s tudy their effect o n imp rov ing the co ating prope rty of eud ra gitt m l1 00. a wa te rso luble type (peg 40 0) a nd non wa te r-so luble (di-n-butyl phtha la te). 10% of e ithe r type of plas ticize r ca lc ulated on the amo unt o f dry la cq uer s ub stance was a dde d to the co ating so lution (9). 2. effec t of comp ressing the granules: p art o f the res ulted ente ric-co ated gra nules was compres sed into tablets . the res ults of diss olution o f these tab le ts we re co mpa red with tha t o f encapsula ted e nteric -c oated granules. 3. effec t of additio n of disinteg ra nt : 10 % av icel p h 1 02 c alcula te d on the total amount o f granules was added e xtragranularly (11) to the ente ric c oa ted gra nules be fo re comp re ssio n. the results of d isso lution we re comp ared with tho se of enc apsula ted ente riccoa ted gra nules as well as with tab le ts prepa re d from the same gra nules but witho ut disintegra nt. results and discussion  dissolution in acid medium: 1. effe ct of addition of plas ticiz e r: the e ffec t of pla sticize r was s tudied by comp aring the re le as e of drug from the enc aps ulated gra nule s co ated with eudragitt m l100 in ac id med ium with a nd witho ut add ition of plas ticize r. the res ults show that 15% of the drug was re le ase d from the coa ted gra nule s in the abs ence of p la sticize r in the c oa ting s olutio n, which is not a cc epted a cco rd ing to the us p re quire ments fo r the e nteric -c oate d pre pa ra tions, whic h state that not mo re than 10% of drug diss olve s at the end of 2 hours (10). additio n of e ither p eg 400 o r di-n-b utyl phtha la te to the c oa ting s olution de crea sed the re le ase of the d rug afte r 2 ho urs of d is so lution in a cid med ium to 6 .2 % a nd 8.5% res pec tive ly in co mpa riso n with 1 0% relea se d from the re fe re nce voltadin(r) sdi ta blets (fig. 1 ), which a re a ll acc ep ta ble. the se re sults indica te tha t add ition of either o f the p la stic izer can imp ro ve the e nteric pro pe rty of the coa ting polyme r whic h may be due to increa sing the mo bility of the chain of the polyme r at the surfac e of the gra nules , so they fa cilitate the filming p ro perty o f the polyme r (12,13),but since d i-n-butyl phthalate is non water so luble, it will de creas e the permea bility of film to mo is ture a nd enha nce the stability of the pro duct, so it is pre ferred fo r the prepa ra tion of this prod uct on p eg 400 (14),the re fo re the granules prep ared with p eg 400 we re negle cted. 2. effec t of compressing the granule s: co mpress ing of the granules p re pared using di-n-butyl phtha la te as plas tic izer fo r the coa ting ma te rial to tablets with and witho ut add ition of disintegra nt, resulte d in decreasing the release of d rug in the a cid medium of diss olutio n to 7.4% and 6 .3% respe ctively in comp aris on with the e nc aps ulated granules (fig. 1). the decrease in the re lease of drug may be d ue to the sma ller s urface a rea of the ta blets comp ared to the gra nules , while the slight d ifference in the re leas e be tween the two ta blet p repa ra tions, may be d ue to the effec t of disintegra nt. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 51 acid me dium buffe r ph(1.2) me dium ph(6.8) fig. (1 ) dissolutio n profile of e ncaps ulate d e nteric-coa te d granule s ta ble ts pre pa re d with and wit hout ad dition o f disinte g rant in c ompa riso n with vo ltadin(r)s di table ts  dissolution in buffer medium: as s hown in fig. 1, highe r and fa ster re lease was ob tained from e nc apsulated enteric-co ated granules in comp arison with volta din(r)sd i ta blets and tab le ts p repared with and without add ition of d is inte grant, where ab out 90% of the d rug was released fro m these gra nules within 2 0 minutes (8) comp ared to 72%, 57.5% and 49.7% from the othe r tablets , respec tive ly. this high a nd fas t re le ase of d rug is ma inly due to the large r surface area o f the gra nules comp ared to the tab le ts . in ad dition, bo th the encapsula ted ente riccoa ted granules and tablets containing disintegra nt a s well a s vo ltad in(r)sdi tab le ts met the usp spec ifications for the re lease of drug in buffe r solutio n (not less than 7 5%of drug dissolves at the end o f 45 minu te s) (10) in c ontras t to ta blets without dis inte grant. kine tics of dissolution: the release charac te ris tics o f the d rug fro m encapsula ted ente ric-coated gra nules and ta blets prepa red from the se gra nules in presence of disintegra nt a s well as that of voltadin(r) s di ta ble ts, fo llow firs t order kinetics , since p lo tting the lo garith m of the perce nt rema ining versus ti me gav e straight lines with good corre latio n (r-value range fro m -0 .94 to -1 .01) for the acid me dium and the two p hases in the buffe r me dium of d isso lution as s how n in f ig.2 . acid me dium buffe r ph(1.2) me diu m ph(6 .8) fig . (2) plot o f the log % re ma in ing ve rs us time for the re lease o f drug fro m e nca psulate d e nte ric-coate d granule s and table ts pre pa re d with addition o f disintegrant in co mparis on with vo ltadin(r) s di ta ble ts. the s mall value of the release ra te constant in the ac id me dium(ph 1.2) (table 1) gives a n indication that the c oating mate rial (eudragittm l1 00) when used as gra nula ting and b inding a gent is effec tive in preventing the re lease of d rug in acidic p h. however, the s mall a nd s low release of d rug at this p h ma y be either due to the presence of sma ll-unc oated d rug or due to the release of drug through the c oating and its po ss ib le discontinuitie s (15). as the ph of the d isso lution med ium reaches the level critica l for the coa ting p h 6 .8 (b uffe r me dium) the fil m s ta rts to d isso lve, thereby inc reas ing the re leas e of d rug (9, 15) as shown in fig.2 and tab le 1 , in which the re is a great increase in the rele ase rate co ns ta nts of the two dosa ge fo rms in the buffer med ium mainly at the firs t p hase (more than 1 00 time s) comp ared to tha t at ac id me dium. in a dditio n, the res ults o f diss olution at the buffer med ium give an indica tion tha t the re lease of d rug is a ffec te d by the d osa ge form 0 10 20 30 40 50 60 70 80 90 100 0 20 40 60 80 100 120 140 160 180 time (minutes) % d ru g re le as ed encapsulated enteric-coated granules using di-nbutyl phthalate as a plasticizer voltadin(r) tablets tablets with disintegrant tablets without disintegrant 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 0 20 40 60 80 100 120 140 160 180 time (minutes) l o g % r em a in in g encapsulated enteric coated granules using di-n-butyl phthalate as a plasticizer voltadin (r) tablets tablets with disintegrant ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 52 as well as b y the surfa ce area, s ince the rele ase ra te cons ta nts fo r the e ncaps ulated ente riccoated gra nules are highe r tha n that of co mpressed ta blets. table (1) re le ase rate co ns ta nts (k) min-1.o f the pre pare d do sage fo rms in comparis on with vo lta din(r) sdi tab le ts voltad in( r ) sdi tablets tab le ts with dis inte grant enc apsulated enteric-coa ted gra nules using di-n-butyl phtha la te as plasticize r p h the dissol ution me dium 88.3*10-5 5 7.5*10-5 7 6.75 *10 -5 1.2 6.8 0.098 0 .143 0 .309 k1 0.017 0 .020 0 .031 k2 abbre viations: k1: re fe rs to the dissolutio n ra te cons ta nt for the first pha se in the b uffe r med ium k 2 : re fe rs to the d isso lution rate cons tant fo r the s econd phase in the b uffe r med ium refe rences: 1. anslow -j a.;gree ns -ds.; ho ope r,-jw.and wa gner-gs. fa ster and mo re re liable d elivery o f salicylic acid from e nteric asp irin granules versus enteric -c oated ta blets. curr-ther-res ., 1984 , 36(nov ), 8 11 -818. 2. gams t-on. enteric-coated nap ro xe n ta blets. eur-j -rheumatol-inflamm., 19 92, 1 2 (2 ), 5-8. 3. p ondeyvp.; pha nindrud ua.; ma na valanr. a nd livingstanj. in v itro s tudy o n c apsule formulatio ns of o mep ra zole co ntaining enteric -c oated gra nule s. boll-chim-farm., 20 02, 141 (6 ), 4 19 -422. 4. overga ard a-b; hojsted-j ; ha nsen-r; mo ller-sonnergaard-j and chris turp l-l. p atie nt’s e va luatio n o f s hape , s ize and c olor o f so lid do sa ge form. parmwo rlds ci., 20 01, 23 (5) 185-1 88 . 5. hosney-ea; el-ma hrouk, gm. and goud a-mw. formula tion a nd in vitro and in vivo availability of d ic lo fe nac sodium e nteric -coa ted beads . drug – dev ind p harm. ,1 998 , 24 (7 ), 661 -6 66 6. ma rv ola-m; nykänen -p; rautio-s; iso ne n-n. and autere-a.-m. enteric p olyme rs as binders and coa ting materia ls in mul tip le -unit s ite-spec ific drug de live ry s ys te ms. eur-j -p harm-sci., 1999, 7, 25 92 67. 7. oba ra-s ; maruyama -n; nis hya ma-y and ko kubo-h. dry co ating a n innova tive e nteric coating me thod us ing c ellulose d erivative. eurj -pharm-bio pha rm., 1999, 4 7(1), 51 59 . 8. cla udio-n; rita-c; elisabetta-e; albe rtog; alrs sa nd ro; ca rlo-v and enea -m. infl uence of formulation a nd process p arame te rs on pe lle t prod uction by powd er layering technique. p hrm-sc i-tech., 2000, 1 (2 ) artic le 9 . 9. p roduc t info rma tion bulletin eudragit ś rö hm pha rm, gmbh, weiterstad t ge rmany. 10. usp xxiv 20 00, p.547, p .1 94 7. 11. ka ssab-h.j. m.sc. thes is, college of p harma cy, unive rsity o f baghd ad 1999. 12. ka rl toma a nd ka ro line bec htold. infl uence of aqueo us coa tings on the s tability o f e nteric -c oated pe llets and tab le ts. eur-j-parm-bio pharm., 199 9, 47, 39-50 . 13. ra ymond-c. rowe. ma teria ls used in the film co ating of oral d osage form in f lo re nce-a.t. ma te rials used in p harmaceutica l formulatio n. blackwell sc ie ntific p ub lica tions. oxford lond on ed inburgh. 1 985 p.16. 14. s tuart c. po rter ; charles-h.bruno . and ge ra ld j .jac kson. pan coating o f tab le ts a nd granules in lie be rma n h.a. and lachman l. pha rmace utical dos age fo rm: tablets vol.3 ma rc el de kker, inc.new yo rk bas el.1982 p.96-97 . 15. muskó -zs; pintye-hó di k.;gá spár r.; p itye j .; szab ó -révés z p .; erős i. and f alka y g. s tudy o f in vitro and in vivo d isso lution of theo phylline from fil m c oa ted pe llets . eur-j -pharm-biopha rm., 2 001 , 51, 143-14 6. evaluation of ciprofloxacin-induced adverse cardiac effect in juvenile rats iraqi j pharm sci, vol.21(2) 2012 possible cardiac adverse effects of ciprofloxacin 94 possible cardiac adverse effects induced by therapeutic doses of ciprofloxacin in juvenile rats # omar j.al-faris * , nada n. al-shawi **,1 and maha d. kako *** * department of clinical pharmacology, college of pharmacy, university of mosul, mosul,iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq *** department of pathology, college of veterinary medicine, university of mosul, mosul,iraq. abstract ciprofloxacin is widely used in treating adults infected with gram-negative bacteria. it is contraindicated in children, growing adolescents and during pregnancy due to joint toxicity. its toxicity concerning other organs needs to be clarified. thus, this study was designed to study the possible cardiac damage induced by two selected doses of ciprofloxacin in juvenile rats.eighteenth healthy juvenile rats (4 weeks old and their weight 30 ± 2 gm) were utilized in this study and divided into three groups. group-i control; group ii and group iii, respectively injected ip with 25 mg/kg and 50 mg/kg ciprofloxacin every 12 hours for one week. serum enzymes activities alanine aminotransferase (alt), aspartate aminotransferase (ast), creatin kinase -muscle brain isoform (ck-mb), and lactate dehydrogenase (ldh) were assessed. histological examination of heart tissues was also performed. the results of this study showed that, alt, ast and ck-mb enzymes were significantly elevated only in group iii compared to control. ldh enzyme was elevated in both group ii and iii. concerning histological examination of the heart's gross sections, the results obtained from this work demonstrated the degeneration and necrosis in the hearts of group ii and iii juvenile rats compared to control animals. in conclusion, our results showed that the selected therapeutic doses of ciprofloxacin utilized in this study caused cardiac damage in juvenile rats. key words: fluoroquinolones, cardiac adverse effects, juvenile rat . بىاسطة الجرع المعالجة ةالتأثيرات الجاوبية المحتملة في القلب والمستحذث في الجرران اليافعة هللسيبروفلىكساسي جاسم الفارس عمر * و وذي واجي الشاوي ،**1 داؤود كاكىو مها *** . ، اىعشاق ٍ٘صو، اىَ٘صو تصاٍع ، تاىصٞذى تميٞ ،فشع االدٗٝٔ ٗاىسًَ٘ * . ، بغذاد ، اىعشاقبغذاد تصاٍع ، تاىصٞذى تميٞ ،ىسًَ٘ فشع االدٗٝٔ ٗا** . ، اىعشاق ٍ٘صو، اىَ٘صو تصاٍع ، طب اىبٞطشٛ اى تميٞ ، اىبار٘ى٘صٜفشع *** الخالصة بنخشٝا االىخٖاباث اىخٜ حسببٖا عالساىَشضٚ اىباىغِٞ ىٝعذ عقاس اىسبشٗفي٘مساسِٞ ٍِ االدٗٝت اىشائعت االسخعَاه فٜ ُ اىعقاس أعالٓ ال َٝنِ اعطاءٓ ىالطفاه ٗاىٞافعِٞ ٗىيْساء خاله فخشة اىحَو بسبب سَٞخٔ ىيَفاصو. اُ . ا(gr-ve) ساىبتاىصبغت مشاً اى سَٞخٔ فٜ اعضاء اىضسٌ االخشٙ ححخاس اىٚ اُ ح٘ضح ىزىل صٌَ ٕزا اىبحذ ىذساست احخَاىٞت حيف اىقيب اىزٛ َٝنِ اُ حسببٔ صشعخاُ أسابٞع ٗاٗصاٌّٖ 4أعَاسٌٕ حٌ اسخخذاً رَاّٞت عشش صشر ٝافع ٜ حعذ صشع عالصٞت فٜ اىضشراُ اىٞافعت.ٗاىخ اىسبشٗفي٘مساسِٞ ٍِ عقاس ٍيغٌ ىنو مٞي٘غشاً ٗصُ 22ٗقسَج اىٚ رالد ٍضَ٘عاث: اىَضَ٘عت االٗىٚ/ ٍضَ٘عت سٞطشة، اىَضَ٘عت اىزاّٞت / غشاٍا ±2 03 .داخو اىصفاق ٍيغٌ ىنو مٞي٘غشاً ٗصُ اىضسٌ اعطٞج ٍشحاُ ٍٝ٘ٞا 23ىزت/ ، اىَضَ٘عت اىزاداخو اىصفاق اىضسٌ اعطٞج ٍشحاُ ٍٝ٘ٞا عَو دساست ّسٞضٞت ىْسٞش اىقيب.أظٖشث ( فٜ ٍصو اىذً مَا حٌ (alt, ast, ckmb, and ldhحٌ قٞاط فعاىٞت االّضَٝاث . اُ َ٘عت اىزاىزت ٍقاسّت بَضَ٘عت اىسٞطشةاىذساست بأُ ْٕاك صٝادة ٍعْ٘ٝت فٜ فعاىٞت االّضَٝاث اىَزم٘سة اعالٓ فٜ ٍصو اىذً فٜ اىَض مَا اظٖشث اىذساست اىْسٞضٞت اُ ْٕاك فٜ ٍصو اىذً قذ صاد بص٘سة ٍعْ٘ٝت فٜ اىَضَ٘عخِٞ اىزاّٞت ٗاىزاىزت. (ldh)فعاىٞت االّضٌٝ ٍقاسّت بَضَ٘عت ي٘مساسِٞحيف فٜ ّسٞش اىقيب فٜ ٍضَ٘عخٜ اىضشراُ اىٞافعت )اىَضَ٘عت اىزاّٞت ٗاىزاىزت( اىخٜ اعطٞج عقاس اىسبشٗف ٍِ عقاس اىسبشٗفي٘مساسِٞ فٜ ٕزٓ اىذساست اىسٞطشة.ٍِ خاله اىْخائش اىخٜ حٌ اىحص٘ه عيٖٞا َٝنِ االسخْخاس بأُ اىضشع اىَسخخذٍت سببج حيف فٜ ّسٞش اىقيب ىذٙ اىضشراُ اىٞافعت. جرران اليافعة .فلىروكىويىلىورز، التأثيرات الجاوبية في القلب ، الالكلمات المفتاحية : introduction fluoroquinolones (fqs) are widely used antibacterial agents in adults because of their strong antibacterial effect against gramnegative bacteria (1) . they target bacterial dna gyrase and topoisomerase iv (2) . in pediatric patients, growing adolescents and during # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011 1 corresponding author email : nadaalshawi@yahoo.com received : 25/12/2011 accepted : 10/9/2012 iraqi j pharm sci, vol.21(2) 2012 possible cardiac adverse effects of ciprofloxacin 95 therapeutic indications, where they are active against p. aeruginosa–induced respiratory infections in children with cystic fibrosis, chronic suppurative otitis media or malignant otitis externa; acute or chronic osteomyelitis or osteochondritis; multidrug-resistant gramnegative bacterial infections especially in immunocompromised hosts , bacterial septicemia or meningitis cases for which the causative organism is resistant to other approved agents; exposure to aerosolized bacillus anthracis; serious infections in the children with life-threatening allergy to alternative antibacterial agents and multidrugresistant mycobacterial infections (4) .the underlying mechanism of fqs-induced joint toxicity is not well-known' but possible involvement of oxidative stress and nitric oxide (no ∙ ) in the tendinopathic effects of the drugs have been demonstrated in vivo and in vitro (5) . their toxicity concerning other organs needs to be clarified. thus, this work was designed to study the possible cardiac damage induced by two therapeutic doses of ciprofloxacin in juvenile rats. materials and methods animals eighteenth, (4-week) healthy albino rats of both sexes weighing 30 ± 2 g were utilized in the study. they were obtained from the animal laboratory house of the college of pharmacy, university of baghdad. the animals were fed standard diet ad libitum and were free access to tap water. they were divided into three groups of 6 animal each as follows: group-i juvenile rats were received 25 iu i.p saline solution two times daily at twelve-hour intervals for one week, this group served as control; group iijuvenile rats were injected intraperitoneally (i.p.) with 25 mg/kg ciprofloxacin two times daily at twelve-hour intervals for one week and group iiijuvenile rats injected i.p. with50 mg/kg ciprofloxacin two times daily at twelve-hour intervals for one week. at the end of the treatment period, blood samples were collected via cardiac puncture after euthanizing the animals by anesthetic ether. blood samples were centrifuged for 10 minutes at 4,000 rpm, to obtain serum, which was utilized for the assessment of cardiac enzymes activities {(alanine aminotransferase (alt) (6) , aspartate aminotransferase (ast) (6) , creatin kinasemuscle brain isoform (ck-mb) (7) , and lactate dehydrogenase (ldh) (8) }. the hearts were dissected and stored in formaldehyde 10% until they were utilized for histological examination under light microscope (9) that was performed in the college of veterinary/ almousel university. analysis of data was performed using microsoft excel version 2007 two ways to analyze student t-test. the data were expressed as mean ± standard deviation. p value of less than 0.05 was considered significant. results table 1 showed that serum alt activity in the juvenile rats was significantly (p<0.05) elevated in group of animals treated with 50mg/kg ciprofloxacin (group iii) compared to groups i and ii. additionally, no significant differences (p>0.05) concerning serum alt were observed in juvenile rats treated with 25mg/kg ciprofloxacin (group ii) compared to control juvenile rats (group i).although there was an elevation in the serum activity of ast in group of juvenile rats treated with 50 mg/kg ciprofloxacin, but the results of table 1 showed no significant differences ( p>0.05) in the activity of serum ast in all groups of juvenile rats (i, ii and iii) used in this study. concerning serum enzyme activity of ck-mb, table 1 showed that there were significant (p<0.05) elevation in the serum activity of ck-mb in groups of juvenile rats treated with either 25mg/kg(group ii) or 50mg/kg-(group iii) ciprofloxacin compared to control animals (group i). furthermore, a significant elevation (p<0.05) in the serum activity of ck-mb was observed in group iii of juvenile rats compared to the corresponding level of group ii animals.the results of table 1 also showed a significant elevation (p<0.05) in the serum activity of ldh in group of juvenile rats treated with either 25mg/kg(group ii) or 50mg/kg-(group iii) ciprofloxacin compared to control animals (group i). furthermore, a significant elevation (p<0.05) in the serum activity of ldh in group iii of juvenile rats compared to group ii animals. concerning the examination of histological sections of juvenile hearts' rats received either 25mg/kgor 50mg/kg ciprofloxacin, respectively [figures (2 and 3)], the results showed the presence of vaculations, degenerations in addition to absence of striation of cardiac muscle were seen compared to control juvenile heart sections (figure 1). iraqi j pharm sci, vol.21(2) 2012 possible cardiac adverse effects of ciprofloxacin 96 table 1:serum enzymes activities(alt, ast, ck-mb and ldh)in cardiac tissues of the juvenile rats treated with either 25mg/kgor 50mg/kg-ciprofloxacin compared to control group. values are expressed as mean± sd. *: p<0.05 significant difference compared to control group. values with non-identical superscripts (a, b and c) are considered significantly different (p<0.05) among groups. n= number of animals. figure 1: section showing normal heart cell of juvenile rats. staining: haematoxylline & eosin; amplification: 450 x. figure 2: rat’s heart aged 37 days, administered 25mg/kg ciprofloxacin i.p. for 7 days, demonstrates vaculation ( ) and degeneration ( ). staining: haematoxylline & eosin; amplification: 450 x figure 3: rat’s heart aged 37 days, administered 50mg/kg ciprofloxacin i.p. for 7 days, demonstrates degeneration ( ) and vaculation ( ) with necrosis more clearly from that of 25mg/kg ciprofloxacin receiving animals . staining: haematoxylline & eosin; amplification: 650 x. discussion although there were no clinical data concerning the cardiac adverse effects induced by fqs, it has been reported that therapeutic doses of a drug belongs to fqs groups, ciprofloxacin, may induce adverse effects in healthy rats following sub-acute treatment (4) . furthermore, fqs have been associated with risk of qt prolongation and arrhythmias (2, 3) .increased alt, ast, ck-mb and ldh activities that were observed in this study (table 1), demonstrated myocardiocyte tissue damage due to ciprofloxacin a dministration serum alt iu serum ast iu serum ck-mb iu serum ldh iu group i normal saline 25iu (control) n=6 47.8± 23.82 a 163.8±12.25 a 90.6 ± 61.65 a 1749± 191.9 a group ii ciprofloxacine 25 mg/kg-treated group n=6 47.33±22.83 a 157.66±11.21 a 267.83±263.6 *b 2580±334.21 *b group iii ciprofloxacine 50 mg/kgtreated group n=6 64.16±16 *b 216.16±34.36 a 425.66±234.16* c 4030.33±228 *c iraqi j pharm sci, vol.21(2) 2012 possible cardiac adverse effects of ciprofloxacin 97 compared to control juvenile rats. the obtained biochemical results were consistent with that reported by others (10) and the biochemical changes were further confirmed by histological findings. figures 2 and 3.different myocardial markers were utilized to determine cardiac function; first, ck-mb resides in the cytosol and facilitates high energy phosphates into and out of mitochondria. it is distributed in a number of tissues including heart and skeletal muscle. in animal experiments, it was demonstrated that an increased in ck-mb activity in blood has been associated with evidence of irreversible cardiac injury (cell disruption) (11) .second, lactate dehydrogenase (ldh) catalyses the conversion of pyruvate to lactate. ldh-1 isozyme is normally found in the heart muscle and ldh-2 is found predominately in blood serum. a high ldh-1 level to ldh-2 suggests mi (12) . third, aspartate transaminase (ast), an enzyme was the first used. it is not specific for heart damage, and it is also one of the liver function tests (12) . the mechanism of heart damage induced by ciprofloxacin has been demonstrated to implicate oxidative stress and many studies had been demonstrated that the antioxidant enzyme levels are relatively lower in the myocyte making this organ susceptible to reactive oxygen species (4), (13-14) . the results of this study provide an evidence, for the first time, to our knowledge on the effect of treatment of healthy juvenile rats with ciprofloxacin on cardiac markers in vivo. therefore, we did not have the chance to compare the results of this study with other reports. conclusion according to results obtained from this study, one can conclude that cardiac adverse effects manifested by the selected doses of ciprofloxacin used in this study ( 25 and 50mg/kg) injected ip to juvenile rats caused myocardial damage and may be another limitation for the use of this drug in the pediatric patients. thus further studies are required to support this finding. references 1. benjamin a. lipsky and catherine a. baker, fluoroquinolones toxicity profile. clinical infectious disease. 1999; 28 (2): 352-361. 2. leslie patmore, stuart fraser, deborah mair, alison templeton, effects of sparfloxacin, grepafloxacin, moxifloxacin, and ciprofloxacin on cardiac action potential duration. european j. pharmacology. 2000; 406 (3): 449-452. 3. bertram g. katzung-basic & clinical pharmacology (9 th ed.). section viii. chapter 46. 1082-1086. 4. ahmet saraçoğlu, halide e. temel, bülent ergun, and ömer çolak. oxidative stress– mediated myocardiotoxicity of ciprofloxacin and ofloxacin in juvenile rats. drug and chemical toxicology. 2009; 32 (3): 238-242. 5. goodman and gilman’s manual of pharmacology and therapeutic by laurence l. brunton, phd, keith l. parker, md, phd, (2 nd ed.). 2008: 718-729. 6. reitman, s.; and frankel, s.: colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. am. j. clin. pathol. 1957; 28(1): 56-63. 7. würzburg u, hennrich n, orth hd. et al. quantitative determination of creatine kinase isoenzyme catalytic concentrations in serum using immunological methods. j clin chem clin biochem 1977,15(3):131-7. 8. moss dw, henderson ar: clinical enzymology. in titez textbook of clinical chemistry. 3rd edition. edited by brutis ca, ashwood er. philadelphia: w.b. saunders company; 1999:617-721. 9. junqueira, l.c.; carneiro, j. and kelley, r.: basic histology. 8 th ed, lange medical book, 1995; pp.1-2, 30g-314g. 10. pispirigos k and chrysanthopoulos k. evaluation of cardiac sub-acute toxicity of ciprofloxacin in rats using serum biochemical parameters. arzneimittelforschung.2001; 51(7):582-7. 11. ishikawa y, saffitz je, mealman tl, et al. reversible myocardial ischemic injury is not associated with increased creatine kinase activity in plasma. clin chem 1997;43:467-75. 12. rao sp, miller s, rosenbaum r, lakier jb. cardiac troponin i and cardiac enzymes after electrophysiologic studies, ablations, and defibrillator implantations. am. j. cardiol. 1999; 84 (4): 470. 13. gianni, l; zweier, jl; levy, a and myers, ce. characterization of the cycle of ironmediated electron transfer from adriamycin to molecular oxygen. j. biol. chem. 1985; 260: 6820-6826. 14. saraçoğlu a, temel he, ergun b, colak o. oxidative stressmediated myocardiotoxicity of ciprofloxacin and ofloxacin in juvenile rats. drug chemical toxicology. 2009; 32(3): 238-42. http://en.wikipedia.org/wiki/lactate_dehydrogenase http://en.wikipedia.org/wiki/pyruvate http://en.wikipedia.org/wiki/lactate http://en.wikipedia.org/wiki/aspartate_transaminase http://en.wikipedia.org/wiki/liver_function_tests http://en.wikipedia.org/wiki/liver_function_tests http://www.dmsjournal.com/sfx_links?ui=1758-5996-1-17&bibl=b40 http://www.ncbi.nlm.nih.gov/pubmed?term=%22pispirigos%20k%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22chrysanthopoulos%20k%22%5bauthor%5d javascript:al_get(this,%20'jour',%20'arzneimittelforschung.'); javascript:al_get(this,%20'jour',%20'arzneimittelforschung.'); http://linkinghub.elsevier.com/retrieve/pii/s0002-9149%2899%2900337-9 http://linkinghub.elsevier.com/retrieve/pii/s0002-9149%2899%2900337-9 http://linkinghub.elsevier.com/retrieve/pii/s0002-9149%2899%2900337-9 http://www.ncbi.nlm.nih.gov/pubmed?term=%22sara%c3%a7o%c4%9flu%20a%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22temel%20he%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22ergun%20b%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22colak%20o%22%5bauthor%5d iraqi j pharm sci, vol.30(1) 2020 genotoxic potential of fluoxetine and amitriptyline doi: https://doi.org/10.31351/vol30iss1pp81-90 81 assessment the genotoxic potential of fluoxetine and amitriptyline at maximum therapeutic doses for four-week treatment in experimental male rats imad a. al-obaidi*,1 and nada n. al-shawi** * ministry of health and environment, medico-legal directorate, baghdad, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract at any moment, the continuous usage of medications can be accompanied by dna damage and the accumulation of such damages can cause serious consequences. antidepressants are long-term used drugs and the incidence of their genotoxic impacts cannot be excluded. therefore, this work was designed to investigate the possible genotoxic effects of the commonly used antidepressants (fluoxetine and amitriptyline) in adult male rats. a total of 24 swiss albino adult male rats were used in this study; animals were randomly allocated into three groups of 8 rats each: group i rats orally-administered distilled water via gavage tube for four weeks as a negative control. group ii rats orally-treated with fluoxetine hydrochloride solution (7.2mg/kg/day) via gavage tube for four weeks. group iii rats orally-treated with amitriptyline hydrochloride solution (27mg/kg/day) via gavage tube for four weeks. at the end of experiment, the rats were sacrificed and the samples collected for detection of dna damage in individual cells that have been assessed by means of comet and micronucleus assays in three different cell populations i.e. liver, testis and bone marrow tissues. the results showed that both drugs (group ii and group iii) induced the same extent of dna damage, as evidenced by significantly higher dna fragmentation in liver and testis tissues with increased frequencies of micronuclei formation in bone marrow tissues as compared with the negative control (group i). these findings indicate that both fluoxetine and amitriptyline have genotoxic potentials and can induce the same extent of cytogenetic damage in rats. special precautions and medical supervision should be taken into consideration with their uses. keywords: genotoxicity, fluoxetine, amitriptyline, comet assay, micronucleus assay. من الفلوكسيتين ج اربعة اسابيع لجرعات عالجية قصوىالمحتملة من عال جينيةتقييم السمية ال واالميتريبتيلين في ذكور الجرذان **ندى ناجي الشاويو *عماد عدنان عبد العبيدي .العراق ،بغداد ،دائرة الطب العدليوزارة الصحة والبيئة ، * .العراق ،دكلية الصيدلة، جامعة بغداد، بغدا فرع االدوية والسموم،** الخالصة .وقد يؤدي تراكم هذه األضرار إلى عواقب وخيمة الحمض النووي ضررا فيفي أي لحظة ، يمكن أن يرافق االستخدام المستمر لألدوية تبعاده. تعد مضادات األكتئاب من األدوية التي تستخدم على المدى الطويل وأن أمكانية حدوث السمية الجينية المرافقة ألستخدامها شيء اليمكن أس ي ذكور الجرذان لذلك تم تصميم هذه الدراسة لتقييم السمية الجينية المحتملة لمضادات األكتئاب الشائعة األستخدام )الفلوكستين واألميتريبتيلين( ف تم تقييم الكشف عن تلف الحمض النووي في الخاليا الفردية من خالل فحوصات المذنب والنوى الصغيرة في ثالث مجموعات مختلفة من البالغة. كل عشوائي في ثالث تم توزيع الحيوانات بش .الذكور البالغين البيضاء جرذانمن ال 24الكبد والخصية والنخاع العظمي لـ بأنسجة متمثلةالخاليا، سيطرة سالبة. كمجموعةلمدة أربعة أسابيع عن طريق الفم الماء المقطر تناولتالجرذان التي المجموعة األولى :لكل منها جرذان 8, مجموعات المجموعة . مدة أربعة أسابيعلعن طريق الفم ملغ / كغ / يوم( 7.2يدروكلوريد )ابمحلول فلوكستين هتم معالجتها الجرذان التي لثانيةالمجموعة ا أظهرت النتائج أن كال .لمدة أربعة أسابيععن طريق الفم ملغ / كغ / يوم( 27يدروكلوريد )اه أميتريبتلينبمحلول تم معالجتها الجرذان التي لثالثةا تكسر في متمثلة بأرتفاعا معنويا ملحوظاالحمض النووي الضرر الحاصل فينفس المدى من ب ا)المجموعة الثانية والمجموعة الثالثة( تسبب رينالعقا بالمقارنة مع مجموعة السيطرة.تواتر تكوين النوى الصغيرة في أنسجة نخاع العظام ملحوظة بالحمض النووي في أنسجة الكبد والخصية مع زيادة الخلوي في جينيويمكنهما إحداث نفس المدى من الضرر ال جينية ات سميةيلهما إمكانالفلوكستين واألميتريبتيلين تشير هذه النتائج إلى أن كل من هذين العقارين. االحتياطات الخاصة واإلشراف الطبي مع استخدامر االعتبا لجرذان. يجب األخذ بعينا الكلمات المفتاحية: السمية الجينية , فلوكستين , أميتريبتيلين , فحص المذنب , فحص النوى الصغيرة. *corresponding author e-mail: emad_adnan85@yahoo.com received: 17/6 / 2020 accepted: 13/9 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp81-90 iraqi j pharm sci, vol.30(1) 2020 genotoxic potential of fluoxetine and amitriptyline 82 introduction depression and anxiety disorders are common growing problems in public health (1). depression affects approximately 350 million people worldwide; constituting a major portion of mental health disorders (2). regarding the prevalence of mental disorders in iraq, the national iraq mental health survey (imhs) conducted in 2007, with 4332 respondents, showed that anxiety disorders were the most common class (13·8%) and major depressive disorder was the most common disorder (7·2%) (3). the world health organization (who) indicated that depression will be the disorder striking worldwide within the next decade, and is predicted to be the second largest burden to ischemic heart disease in the international community of health by 2020 (4). thus, antidepressant drugs become commonly prescribed nowadays, and also their use becomes increasing throughout the world (5). substantial international studies on antidepressants prescribing patterns, showed that fluoxetine and amitriptyline are two of the most commonly prescribed antidepressants belonging to selective serotonin reuptake inhibitors (ssris) and tricyclic antidepressants (tcas) groups, respectively (6 , 7). fluoxetine is a widely-marketed (ssri) commonly used for treatment of major depressive disorder, obsessive compulsive disorder, panic disorder, bulimia nervosa and premenstrual dysphoric disorder (8). fluoxetine act by blocking serotonin (5hydroxytryptamine) neurotransmitter reuptake into the presynaptic cells by binding to serotonin transporters, thus increasing such neurotransmitter in the synaptic cleft (9). in spite of being an important antidepressant, fluoxetine may induce several unwanted effects, including anxiety, sexual dysfunction, insomnia, and gi problems (10). while amitriptyline is a (tca), used in the treatment of several psychiatric disorders, including major depression, obsessive compulsive, panic attacks, generalized anxiety, post-traumatic stress and bulimia, in addition to its different off-label uses, including migraine prevention, neuropathic pain management, fibromyalgia, and enuresis (11). it is known to inhibit the presynaptic reuptake of serotonin (5-ht) and norepinephrine (ne) and thus increase the concentrations of both neurotransmitters at the synaptic cleft (12). some of the side effects for amitriptyline include anticholinergic effects such as constipation, dizziness, dry mouth, blurred vision and urinary retention, besides weight gain, sexual dysfunction, orthostatic hypotension and cardiotoxicity (13)(14). unfortunately, several studies showed that the fluoxetine or amitriptyline administration in vivo was accompanied by cytotoxic and genotoxic effects, evidenced by dna fragmentations, sisterchromatid exchanges and chromosomal aberrations (15 18). as long as the criteria for genotoxicity assessment suggests that no single assay can fully detect all genotoxic aspects (19). thus, combining the in vivo comet and micronucleus (mn) assays in the present investigation has been considered to be a valuable methodology for evaluating genetic damage, since the comet assay can determine the short-lived dna damage, while the mn assay detects the structural and numerical chromosomal damage (20). moreover, the antidepressants are medications that can be consumed regularly for 6 months or more, with a potential recurrence of the treatment (21). therefore, the aim of this study was to investigate the possible genotoxic effects of the commonly used antidepressants (fluoxetine and amitriptyline) in adult male rats. materials and methods chemicals and drugs fluoxetine and amitriptyline as hydrochloride powders were purchased from sigmaaldrich co., st. louis, mo, usa. all other chemicals used were of analytical grade. preparations of drugs treatment solutions fluoxetine and amitriptyline hydrochloride solutions were freshly-prepared every day by dissolving the required amount of each of drug powder in sterile distilled water to get a final concentration (7.2 mg/kg and 27 mg/kg b.wt per day) of fluoxetine and amitriptyline, respectively. the doses of fluoxetine and amitriptyline were calculated by extrapolating the human recommended maximum therapeutic doses to rat doses, according to the conversion table of paget and barnes (22). experimental animals the study was performed on 24 healthy experimental swiss albino adult male rats, weighing (200-300 g), in accordance with the guidelines of the biochemical and research ethical committee; and approved by the scientific committee at the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the animals were supplied by and kept in the animal house of the college of pharmacy, university of baghdad – iraq. all animals were housed within plastic cages and maintained under standard laboratory conditions at temperature 2224°c under a 12-h light/dark cycle, and offered free access to food (commercial rat pellets) and water ad libitum. after 3 days of acclimation, experimental rats were randomly allocated into three groups of 8 rats each, as follows: group 1: rats orallyiraqi j pharm sci, vol.30(1) 2020 genotoxic potential of fluoxetine and amitriptyline 83 administered distilled water (dw) daily via gavage tube for four weeks. this group served as a negative control. group 2: rats orally-administered a maximum therapeutic dose of fluoxetine hydrochloride solution (7.2mg/kg/day) via gavage tube for four weeks. group3: rats orallyadministered a maximum therapeutic dose of amitriptyline hydrochloride solution (27mg/kg/day) via gavage tube for four weeks. after 24 hrs. of the end of the treatment duration (i.e. at day 29), rats were euthanized by diethyl ether and sacrificed by cervical dislocation. livers and testes were excised, weighed and washed with normal saline 0.9%. the bone marrow samples were aspirated from the femur bone. a small piece of liver about 2 grams, the left testis and the bone marrow aspirate were preserved in chilled phosphate buffer saline (1x pbs) and kept frozen until further analysis. alkaline comet assay (single cell gel electrophoresis assay) the comet assay (or a single cell gel electrophoresis) is a highly sensitive (accurate and reliable) method to detect low levels of dna damage. the alkaline comet assay is the most commonly used version and widely accepted to detect a wide variety of dna lesions such as single and double-strand breaks. under an electrophoretic field, damaged cellular dna is separated from intact dna, yielding a classic “comet tail” shape under the microscope (23). the alkaline comet assay was performed by using a commercial oxiselect™ comet assay kit (cell biolabs, inc., usa) for detecting dna damage in individual cells, according to the method described by singh et al (1988) (24) with modifications. the dna damage was manually quantified according to the method described by collins et al (1995) (25). one hundred cells (comets) selected at random from each slide were scored visually into 4 categories according to tail intensity (the extent of dna migration), given a value from (0 to 3) as follows, 0 = no damage (no visible tail); 1= low level damage (short tail); 2= medium level damage (an obvious tail); 3= high level damage (head of a comet very small with long diffused tail). thus, the total comet score (tcs) for 100 comets could range from 0 (all undamaged) to 300 (all maximally damaged) as arbitrary units (26). the parameter “total comet score” (tcs) was calculated according to this formula (27): (percentage of cells in class 0) × 0 + (percentage of cells in class 1) × 1 + (percentage of cells in class 2) × 2 + (percentage of cells in class 3) × 3. micronucleus assay (mn) micronucleus assay as an index of cytogenetic damage has been widely used to evaluate in vivo genotoxicity, evidenced by an increase in the frequency of micronucleated polychromatic erythrocyte (mnpce) as a reflection of induced structural and/or numerical chromosomal damage (28). the in vivo micronucleus assay was done according to the method described by schmid (1976) (29) with slight modifications. the femur bone was taken and cleaned from the adhering tissues and muscles. after cutting both ends, the femur gapped from the middle with forceps in a vertical position over the edge of a test tube. by a sterile syringe (1-2ml) of pbs was injected in the bone cavity, to flush out and drop the bone marrow in the test tube. then 1ml fresh fetal bovine serum was added into each test tube. the test tubes were centrifuged at speed of 1000 rpm for (5min). the supernatant was removed, and the cells were resuspended with (2ml) fetal bovine serum. again the test tubes were centrifuged at speed of 1000 rpm for (5min). the supernatant was removed, and the cells pellet was resuspended with (170 μl) fetal bovine serum. a small amount of cells suspension was dropped on the end of microscopic slide to make a smear. the slides were kept at room temperature allowed to air dry for 24 hours. the slides were fixed with absolute methanol for 5min, then stained with giemsa stain for 15min and then washed with distilled water and left to dry. the slide was examined under oil immersion lens (100x), two slides for each animal were prepared for micronucleus test. a total of 1000 cells (including the polychromatic erythrocytes pce and normochromatic erythrocyte nce) were randomly examined for the formation of micronuclei, and the micronucleus index was calculated using the following equation (30): micronucleus index % = ( 𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 (𝑀𝑁𝑃𝐶𝐸) 𝑇𝑜𝑡𝑎𝑙 𝐶𝑜𝑢𝑛𝑡 𝑜𝑓 (𝑃𝐶𝐸+𝑁𝐶𝐸) ) 𝑥 100 mnpce: micro-nucleated polychromatic erythrocytes; pce:polychromatic erythrocytes; nce: normochromatic erythrocyte. results and discussion statistical analysis of data was performed using sas (statistical analysis system-version 9.1). descriptive statistics for the numerical data were formulated as mean and standard deviation (mean ±sd). one way and two ways analysis of variance (anova) and least significant difference post-hoc test were used to assess the significant differences among groups. p< 0.05 is considered as statistically significant (31). comet assay in the hepatic and testicular tissue homogenate. the results in (table 1) and (figure 1) demonstrate the score means in both tissues (liver and testis) among the three groups (fluoxetine, amitriptyline, and control). the analysis of data with a two-way iraqi j pharm sci, vol.30(1) 2020 genotoxic potential of fluoxetine and amitriptyline 84 anova test revealed that the comet score in liver and testis was significantly higher (p<0.05) in animals treated with fluoxetine and amitriptyline each compared to the control animals. on the other hand, there was a non-significant difference (p>0.05) in comet score between fluoxetine and amitriptyline-treated animals. the dna damage was quantified by measuring the total comet score (tcs) as seen in (figure 2); where the extent of dna damage was evaluated by visual scoring, and the comets were classified and assigned to four classes: (a) no damage (spheres with no visible tail); (b) low damage (short tail); (c) medium damage (an obvious tail); (d) high damage (small head of comet with long diffused tail). firstly, concerning fluoxetine, there were no previous in vivo studies that have been addressed the evaluation of fluoxetine-induced hepatic genotoxicity by comet assay; except few published articles regarding the genotoxicity of fluoxetine in liver. thus, results of the current study could be interpreted in view of the research of djordjevic et al (2011) (32), who showed an increase in dna fragmentation accompanied by significant upregulation of apoptotic bax and down-regulation of antiapoptotic bcl-2 proteins, obviously seen in hepatocytes undergoing apoptosis after 21-day period in fluoxetine-treated rats; and authors attributed their findings as a consequence of oxidative stress generation caused by the free radicals formation, which is a well-known molecular event in the activation of mitochondrial pathway of apoptosis. similar findings were recently reported in the study of elgebaly et al (2018) (33), who conclude that olive oil and leaf extract prevented fluoxetine-induced apoptosis in the liver of rats as evidenced by decreased expression of apoptotic bax and caspase3, and up-regulated expression of antiapoptotic bcl2 proteins. addressing this problem, it is important to highlight the study of souza et al. (1994) (34), who found that fluoxetine and its metabolite, norfluoxetine potentially exerted toxic impacts on energy metabolism in rats' liver mitochondria at high doses. authors described that these effects seem to be a consequence of the solubilization of the drug and/or its metabolites in the inner mitochondrial membrane. the present study demonstrated that fluoxetine exerted a pronounced dna damage in testicular tissues (group 2) compared to the negative control (group 1) rats, as represented by comet scores in (table 1) and (figure 1). testicular or germ cells are important target in reproductive toxicology, which seems to be an easier and logical choice for dna damage assessment and reproductive genotoxicity research by comet assay (35); where, a recent study by câmara et al (2019) (36) demonstrated that the effect of shortterm treatment with fluoxetine on the adult rat testes caused a significant increase of ubiquitin carboxylterminal hydrolase l1 (uchl1) isoenzyme in the damaged seminiferous tubules associated with high incidence of cell death, since the ubiquitination minimizes dna damage when spermatogonia are exposed to stress. the authors described that such isoenzyme seems to control spermatogenesis, as well as it involved in the molecular regulation of germ cells apoptosis. in another study, alzahrani (2012) (15) reported that a dose-dependent effect produced by fluoxetine administration for 5 days to mice showed a significant increase in sperm shape abnormalities and a significant decrease in both sperm motility and count in male mice. several explanations have been proposed for the testicular dna damage induced by fluoxetine; where, researchers have reported that morphological abnormalities of sperm may be a marker of genetic mutations and a reflection of sperm dna damage arising during spermatogenesis (37)(38); and these studies supporting the previously mentioned findings of alzahrani (2012) (15), which is consistent with the results of the present work. while other authors attributed such testicular genetic damage that mediated by fluoxetine to serotonin's capability of causing dna strand cleavage, as a result of the elevated level of 5-ht during ssri treatment, through an oxidative mechanism in the presence of cupric ions (cu+2), which can be reduced to cuprous ion (cu+1) by 5-ht with subsequent generation of ros, such as the hydroxyl radical (•oh). since copper is an essential component of chromatin; and the formation of a ternary complex of (serotonin-cu+2-dna) was proposed to be the probable mechanism of dna damage with 5-ht (39). in contrast, bendele et al (1992) (40) concluded that, fluoxetine is neither a complete carcinogen nor a tumor promoter after a long-term carcinogenicity study in rats and mice. in such study, fluoxetine was administered to the animals for 24 months at dietary doses of 0.5 to 10.0 mg/kg b.wt in rats and 1.0 to 10.0 mg/kg in mice, via continuously available mash diet. the authors examined multiple organs, among them liver and testes, and there was no evidence of an increased incidence of any type of neoplasm in either rats or mice. concerning amitriptyline, in the current study, the comet assay successfully detected the genetic damage induced by such drug in liver and testis tissues; where, amitriptyline (group 3) caused a significant increase (p<0.05) in dna fragmentation detected by comet assay in liver tissues compared to the negative control (group 1) rats, as represented by comet scores in (table 1) and (figure 1). up to date, there are no previous in vivo studies that have been addressed the evaluation of amitriptylineiraqi j pharm sci, vol.30(1) 2020 genotoxic potential of fluoxetine and amitriptyline 85 induced hepatic genotoxicity by comet assay; except few articles were published regarding the genotoxicity of amitriptyline in such organ. thus, results of the current study could be interpreted in view of the in vitro study of taziki et al (2015) (41), who showed that amitriptyline-induced hepatotoxicity was associated with mitochondrial membrane potential collapse in isolated rat hepatocytes. the authors attributed their findings as a consequence of mitochondrial depolarization targeted by amitriptyline, which can lead to energy crisis and releasing of apoptotic signaling molecules, then progressively to cell death. similar findings were reported in the in vitro study of villanueva-paz et al (2016) (42), who found that amitriptyline-induced mitochondria dysfunction and oxidative stress that precedes apoptosis in human hepatic cancer cell line (hepg2), which provide some assurance about amitriptyline cytotoxicity. in addition, a compendium of reports about dna intercalative potential and genotoxicity assays performed on marketed drugs, among them amitriptyline, have been discussed by snyder et al (2006) (43), who concluded that positive in vitro cytogenetics findings for amitriptyline might likely to be due to dna intercalation (dna groovebinding). researchers reported that the testicular genotoxicity, is an essential safety endpoint and a challenging issue in drug development and risk assessment (44). the present study demonstrated that amitriptyline (group 3) exerted a pronounced dna damage in testicular tissues compared to the negative control (group 1) rats, as represented by comet scores in (table 1) and (figure 1). in agreement with these findings, hassanane et al (2012) (17) have showed that the dose-dependent effect produced by the orally-administered amitriptyline-induced structural and numerical chromosomal abnormalities with a significant decrease in both sperm motility and count in germ cells (spermatocytes) of mice. authors added that the sperm-head abnormalities shown in that study could be considered as a reflection of dna content alteration caused by amitriptyline treatment. another study by tousson et al (2018) (45), who demonstrated that amitriptyline-induced testicular tissue damage was associated with sperm morphological abnormalities and a significant expression of p53 protein in the testis and epididymis of rats. the p53 protein was described as "the guardian of the genome", referring to its role in preserving genetic material stability. it has been well documented that dna damage or other cellular stress signals may trigger the expression of p53 proteins, which have three major functions: growth arrest, dna repair and apoptosis (cell death) induction (46). moreover, similar findings were reported in the study of chowdary and rao (1987) (18), who examined the cytogenetic impact of amitriptyline in germ cells of mice. authors found that the orally given-amitriptyline also showed a highly significant number of chromosomal aberrations in spermatocytes at meiotic metaphase, and suggested that such genetic damage could be extended up to 3 generations. in addition, the comet score in animals treated with fluoxetine was significantly higher (p<0.05) in liver tissues than in testis. on the other hand, there was a non-significant difference (p>0.05) in comet score between liver and testis tissues in amitriptyline-treated animals, as seen in (table 1) and (figure 1). varying degrees of dna damage induced by fluoxetine was expected between liver and testis, because such differential tissue damage can give a clear explanation about enantio‐ and stereoselective aspects of fluoxetine, since fluoxetine has a chiral carbon center in its structure, and as a result, it exists as a racemic mixture with two enantiomeric forms as (s)-fluoxetine and (r)-fluoxetine (8). similarly, norfluoxetine, the main metabolite of fluoxetine, also exists in two enantiomeric forms as (s)norfluoxetine and (r)-norfluoxetine, and the metabolism of both fluoxetine and norfluoxetine is stereoselectively catalyzed (47). it has been well-documented that chiral medications can differ in their biological actions, potency and toxicity, since they undergo stereoselective mechanisms controlling their pharmacokinetic and pharmacodynamics properties, such as distribution, metabolism and excretion, as these processes usually favor one enantiomer over the other, due to stereoselective interactions of enantiomers with active biological systems (48). unfortunately, the enantioselective aspects of fluoxetine in animals have still not been thoroughly investigated, despite the evidence of stereoselective disposition of fluoxetine isomers that have been observed in humans and sheep (49)(50). furthermore, it has been reported that the accumulative dosing of fluoxetine results in fluctuated blood levels and pharmacokinetics of the parent drug and its metabolite, than acute dosing, since fluoxetine and norfluoxetine can inhibit their own metabolism through interactions with the cytochrome p450 liver enzymes (51). micronucleus (mn) formation in bone marrow (bm) samples. the mean values of micronucleated polychromatic erythrocytes were shown in (table 2) and (figure 3); where, there was a significant increase (p<0.05) in the frequencies of mn formation in animals' bone marrows treated with fluoxetine and amitriptyline each compared to the control animals; while, there was a non-significant difference (p>0.05) in mn formation frequencies iraqi j pharm sci, vol.30(1) 2020 genotoxic potential of fluoxetine and amitriptyline 86 between the two drugs as shown in (table 2), (figure 3), and (figure 4). firstly, concerning fluoxetine, the present findings are in accordance with the results gathered from alzahrani (2012) (15), who also examined sister-chromatid exchanges in bm cells of mice treated with fluoxetine for 5 consecutive days. the author reported that the highest tested dose of fluoxetine showed about two times increase in sister-chromatid exchanges than control levels. in contrast, düsman et al (2014) (52) demonstrated that orallyadministered fluoxetine at doses of 0.5 to 2.0 mg/100 g b.wt./day failed to show any sisterchromatid exchanges in bm of wistar rats after 7 days of treatment. while for amitriptyline, the present findings are in accordance with the results gathered from hassanane et al (2012) (17), who also reported that the highest tested dose of amitriptyline-induced significant chromosomal aberrations with a marked decline in both mitotic index and meiotic activity in bm cells of mice. authors concluded that amitriptyline could interact with spindle fibers, as evidenced by the disruption of the centromeric apparatus during mitosis that has been observed in their results. in agreement with these findings, chowdary and rao (1987) (18) have also revealed that amitriptyline significantly increased the frequency of micronuclei formation in bm cells of mice. authors indicated that such chromosomal damage during late s and early g1 phases of the cell cycle might be due to the clastogenic and/or spindle disruption effects of the drug. in contrast, an in vitro study by saxena and ahuja (1988) (53) was performed to evaluate amitriptyline and imipramine genotoxicity on cultured human lymphocytes; where, authors concluded that amitriptyline was non-genotoxic but such drug caused chromosomal aberrations and sister chromatid exchanges at concentrations significantly greater than those attained under normal therapy in humans. in support of these facts, it seems that the chemical structure of the antidepressants plays a role in their genotoxic and carcinogenic potentials. brambilla et al (2007) (54) reported that fluoxetine and amitriptyline are two of the nitrosatable drugs due to the presence of amine group in their structures, which by reacting with nitrite in the gastric environment, or even in other sites, can give arise to the formation of n-nitroso compounds or other reactive species; where, authors mentioned that the n-nitroso compounds have been found to produce genotoxic effects and to cause tumor development in laboratory animals. furthermore, authors added that the exposure to the genotoxic-carcinogenic drug nitrosation products might be of great risk that required a concomitant consumption of antioxidants such as ascorbic acid. table 1. comet score values in liver and testis tissues of rats. groups liver mean comet score testis mean comet score control 65.30±0.45 ba 61.18±2.30 ba fluoxetine 96.52±3.01 aa 90.91±3.94 ab amitriptyline 95.76±6.33 aa 91.48±4.31 aa  data are expressed as (mean ± sd); n=8 animals in each group;  means with a different small letters superscripts (a, b) in the same column are significantly different (p<0.05);  means with a different capital letters superscripts (a, b) in the same row are significantly different (p<0.05). figure 1 .histogram of comet score values (mean ± sd) in liver and testis tissues. mean values with different small letters are significantly different (p<0.05) among groups. mean values with different capital letters are significantly different (p<0.05) among tissues. iraqi j pharm sci, vol.30(1) 2020 genotoxic potential of fluoxetine and amitriptyline 87 figure 2. classes of dna damage as detected by the comet assay in liver and testis tissues of treated animals (fluoxetineand amitriptyline-treated groups) examined by florescent microscope (400x). (a) no damage (spheres with no visible tail); (b) low damage (short tail); (c) medium damage (an obvious tail); (d) high damage (small head of comet with long diffused tail). table 2. frequencies of micronucleated polychromatic erythrocytes in bone marrow of rats. groups mn% control 2.36±0.36 b fluoxetine 4.94±0.54 a amitriptyline 4.75±0.62 a  data are expressed as (mean ± sd); n=8 animals in each group;  means with a different small letters superscripts (a, b) in the same column are significantly different (p<0.05). figure 3. histogram showing the frequencies (mean ± sd) of micronucleated polychromatic erythrocytes in bone marrow. mean values with different small letters are significantly different (p<0.05) among groups. iraqi j pharm sci, vol.30(1) 2020 genotoxic potential of fluoxetine and amitriptyline 88 figure 4. bone marrow smears of rats treated with fluoxetine and amitriptyline (a and b), respectively; showing micronucleus induction as well as enucleated cells. pce: polychromatic erythrocytes, nce: normochromatic erythrocyte, mnpce: micronucleated polychromatic erythrocyte. conclusion the present study concludes that fluoxetine and amitriptyline have genotoxic potentials and can induce the same extent of cytogenetic damage in liver, testis and bone marrow tissues of adult male rats, as evidenced by dna fragmentations and induction of micronuclei assessed by comet and micronucleus assays. therefore, both drugs must be prescribed under careful medical supervision, and a concomitant administration of suitable exogenous antioxidant agent is recommended to minimize the risks of their toxicities by enhancing the antioxidant defenses system. further studies should be performed on toxicities of fluoxetine and amitriptyline at different doses with longer treatment periods, to determine their safe doses and durations. more light needed to be shed on the exact molecular mechanisms behind their genotoxic potentials. references 1. tiller jwg. depression and 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pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers doi: http://dx.doi.org/10.31351/vol27iss1pp89-99 89 preparation and characterization of time controlled drug delivery system of sumatriptan using natural polymers mina sh. al-anbagi *,1 , nawal a. rajab ** and yehia i.khalil*** *ministry of health and environment, ,al-zahraa allergy and asthma consultative center , baghdad, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. *** department of pharmacy , isol al deen university college, baghdad, iraq. abstract time controlled drug delivery systems are designed to release the drug after a predetermined lag time to synchronize the disease circadian rhythm. sumatriptan is an effective treatment for acute migraine attacks in which this disease show circadian rhythm between 6 a.m. and 8 a.m. the aim of this work is to prepare time-controlled press-coated tablet that was given at bed time to act at early morning with a lag time of 5.45 hours. six formulas of fast dissolving core tablets and three formulas of press-coated tablets were prepared by using direct compression method using different variables to prepare core tablets which include: different types and concentrations of superdisintegrants while different concentrations of natural and synthetic polymers were utilized in preparation of press-coated tablets. the obtained results showed that formula f4 of core tablet, which contained 25 milligrams of sumatriptan, 5% w/w sodium starch glycolate and avicel ph 102 as diluent, was the selected formula that showed the fastest and complete release of sumatriptan. also, formula c3 of press-coated tablet , which contained pectin: ec100 mpa.s: hpmck15m in concentration30 milligrams: 10 milligrams:160 milligrams respectively, was selected as the best coating layer since it gave 5.45 hours lag time . keywords: pulsatile, sumatriptan, migraine. pectin, ethyl cellulose, hydroxypropylmethylcellulose. تحضير وتقييم نظام تسليم دوائي موقوت لدواء السوماتريبتان باستخدام بوليمر طبيعي ***يحيى اسماعيل خليلو **رجب عياشنوال ، 1،*مينا شهاب العنبكي العراق. بغداد ،،مركز الزهراء التخصصي ألمراض الحساسية والربو وزارة الصحة والبيئة ، * فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ،العراق. ** بغداد ،العراق. ، كلية اصول الدين الجامعة ،قسم الصيدلة *** الخالصة الجرع الصيدالنية التي يسيطر عليها الوقت والتي تم تصميمها إلطالق العنصر الدوائي أنظمة تسليم الدواء النابض هي احدى أشكال ين ب الفعال بعد فترة زمنية محددة سلفا لمزامنة إيقاع المرض البيولوجية. يظهر الصداع النصفي إيقاعا إيقاعيا مع زيادة ملحوظة في النوبات ( tryptamine1 (5-ht1)هيدروكسي تريبتامين -5فز انتقائي لمستقبالت السيروتونين )سوماتريبتان هو محالسادسة صباحا والثامنة صباحا. مع فترة تأخر مسيطر على توقيتهامغلفة بالضغط اقراص ، هو عالج فعال لنوبات الصداع النصفي الحاد.الهدف من هذا العمل هو إعداد الذوبان وثالثة أقراص المغلفة بالضغط باستخدام طريقة الضغط المباشر ساعة.تم تحضير ستة أقراص أساسية سريعة 54.5لتسليم الدواء كيز اباستخدام متغيرات مختلفة إلعداد األقراص أساسية والتي تشمل: أنواع وتراكيز مختلفة من المواد المسرعات التفتت في حين تم استخدام تر من f4فة بالضغط.أظهرت النتائج التي تم الحصول عليها أن الصيغة مختلفة من البوليمرات الطبيعية واالصطناعية في تحضير أقراص مغل كمخفف، w/w sodium starch glycolate and avicel ph 102 %5ملغ من سوماتريبتان، و 55القرص األساسي، التي تحتوي على ، التي تحتوي على االقراص المغلفة بالضغط من c3هي الصيغة المحددة التي أعطت التسليم األسرع والكامل لسوماتريبتان. أيضا، الصيغة pectin:ethylcellulose 100mpa.s:hpmck15m على التوالي، تم اختياره كأفضل طبقة ملغم 063ملغم : 03ملغم : 03 في تركيز ساعات تأخرفي وقت تسليم الدواء. 54.5مغلفة حيث أعطت النصفي. البكتين، إيثيل السليلوز، هيدروكسي بروبيل ميثيل سيليلولوز.: نبضي، سوماتريبتان، الصداع المفتاحيةالكلمات ا introduction drug delivery is a term used to describe systems that carry drugs to their targets in the body to ensure its therapeutic effect(1). oral drug delivery is the most widely utilized route of administration among all the ways that have been explored for systemic delivery of drugs via pharmaceutical products of the different dosage form(2). it is well known that conventional drug dosage forms give instant or fast medication release that provides the determined amount of the drug to the body without any rate control. 1corresponding author e-mail: mimielanbaki@gmail.com received: 27/1/2018 accepted: 14/3/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp89-99 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers 90 this lack of control leads to a lot of complications inherent to the conventional multiple dosing regimens, e.g., drug accumulation leading to toxicities, variable plasma drug level, and poor compliance. all of above necessitate modification of traditional drug dosage forms which ushered a second generation known as the modified drugrelease dosage forms(3). the modified drugrelease dosage forms have a prolonged release of the drug through the longer duration of time which may result in more patient compliance and favorable bioavailability and blood concentrationtime profiles of drugs(4). several controlledrelease preparations present numerous problems such as resistance and drug tolerance, and activation of the physiological system due to long-term constant drug concentrations in the blood and tissues(5). from above it becomes clear that shifting toward the pulsatile drug delivery system is necessary as it would minimize drawbacks of second-generation dosage forms. pulsatile drug delivery systems are timecontrolled drug delivery system. these systems are designed to achieve time specific and sitespecific delivery of drugs according to the circadian rhythm of the body. in chronopharmacotherapy(timed drug therapy) drug administration is synchronized with biological rhythms to produce a maximal therapeutic effect and minimum harm for the patient. the pulsatile release is also useful for the targeting of the drug irritating the stomach or degradable therein, as well for drugs developing biological tolerance or with an extensive first-pass metabolism (6). pulsatile drug delivery system is designated as the transient and rapid release of a certain amount of molecules within a short period immediately after a predetermined offrelease period, i.e., lag time (7). migraine shows circadian rhythm with the marked increase in attacks between 6 a.m. and 8 a.m.(8). sumatriptan is a selective agonist at serotonin 5-ht1 receptors, including 5-ht1b/1d subtypes. it is an effective treatment for acute migraine attacks, and the injectable form has also shown efficacy in the treatment of cluster headaches. sumatriptan administered subcutaneously, orally, intranasally or rectally was effective in alleviating migraine headache and in the reduction of other symptoms associated with a migraine, including nausea, photophobia and phonophobia(9) . the main aim of this study is to prepare timecontrolled tablet of sumatriptan to synchronize migraine attack and to obtain acceptable physical properties of the tablet. materials and methods materials sumatriptan was from avril company, china, croscarmellose sodium,sodium starch glycolate, microcrystalline cellulose (avicel ph 102), pectin, ethylcellulose 100mpa.s and hydroxy propyl methyl cellulose k15m were from hangzhou hyper chemicals ltd./china, polyvinylpyrrolidone k30 ,mg stearate and talc were from samarra drug industry/iraq. methods preparation of inner layer (fast dissolving core tablet) different powder blends of core tablet which contain sumatriptan as an active ingredient with different types of superdisintegrants (croscarmellose sodium and sodium starch glycolate at concentrations 1, 3, 5% (w/w) of total core tablet weight of sumatriptan) were prepared to be evaluated for their flow properties and compressibility before compressing into a tablet using direct compression method. powder mixtures of sumatriptan, polyvinyl pyrrolidone k30, microcrystalline cellulose (mcc, avicel ph-102), croscarmellose sodium (ac-di-sol), sodium starch glycolate ingredients were dry blended for 20 minutes, followed by addition of talc and magnesium stearate as shown in table1. the mixtures were then further blended for ten minutes, one hundred milligram of resultant powder blend was manually compressed using single biconcave punch machine, with a 6mm punch and die to obtain the round core tablet(10,11). formulation of coating mixed blend for press – coated tablet combination of different ratios of pectin, ethylcellulose (ec) 100mpa.s and hydroxypropyl methyl cellulose k15m (hpmck15m) were weighed and dry blended for about 10 minutes and used as a press-coating material for coating the core tablet to prepare press-coated pulsatile tablets by direct compression method(10). the composition of the coat is shown in table 2. the final tablet was made by press coating the core tablet and the coating materials using 9-mm die of the tablet machine, 40% of the coating material were poured to the die before placing the core tablet which was covered by the the remaining 60% then it was compressed by single punch machine(12). iraqi j pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers 91 table 1. composition of core powder blend ingredients (mg) f1 f2 f3 f4 f5 f6 sumatriptan 25 25 25 25 25 25 croscarmellose sodium 5 3 1 sodium starch glycolate 5 3 1 avicel ph 102 65 67 69 65 67 69 pvpk30 2 2 2 2 2 2 mg stearate 1 1 1 1 1 1 talc 2 2 2 2 2 2 total weight 100 100 100 100 100 100 table 2. composition of coat powder blend formula c1 c2 c3 pectin (mg) 20 20 30 ethylcellulose 100mpa.s(mg) 10 20 10 hpmck15m (mg) 170 160 160 total weight(mg) 200 200 200 pre-compression parameters of core and coat powder blends micromeritic properties of core and coat powder blends were recorded. these properties include : angle of repose was determined by taking accurately the weighed quantity of powder blend into the funnel. the funnel height was adjusted such that the funnel tip should touch the apex of the blend. this blend was then allowed to freely flow through the funnel onto the surface. from the formed powder cone, radius and height were measured, and their angle of repose was calculated using the following equation (13) tan-1=h/r ......... (1) where h and r are the height and radius of the formed powder cone respectively, and θ is an angle of repose. the type of flow according to angle of repose values are shown in table 3 table 3. flow properties and corresponding angles of repose (14) flow property angle of repose (degrees) excellent 25-30 good 31-35 fair –aid not need 36-40 passable –may hang up 41-45 apparent bulk density and tapped density the bulk density, as a measure used to designate packing materials was determined by transporting the precisely weighed amount of blend (2 grams) to the graduated cylinder (10 milliliters) with the help of a funnel. the volume was noted. the proportion of the weight of the sample to the volume was calculated. to measure tapped density, the same quantity of blend (2grams) was transported to a 10 milliliters graduated cylinder and tapped by hand at a specific height for a fixed number of taps (100). average of three determinations was taken. the tapped density was defined as the ratio of the sample weight to tapped volume(15). carr's index (or % compressibility) and hausner ratio(16) it shows powder flow properties. it is represented in percentage and is give carr's index= (tapped density– bulk density)/ tapped density×100……… (2) hausner ratio it is an indirect index of ease of powder flow. it is measured by the following formula. hausner’s ratio= tapped density/ bulk density…….. (3) lower hausner ratio (<1.25) indicates better flow properties than higher ones (˃1.25). (17) post-compression evaluation for core tablet hardness test the crushing strength of the core tablets was measured using a monsanto hardness tester. five tablets from each formulation batch were tested randomly, and the average reading was noted. the hardness is measured in kg/cm2 (18). content uniformity this test applied to core tablet. ten tablets were weighed and powdered by using mortar and pestle. the powder which is equivalent to 25 mg of sumatriptan was weighed and dissolved in 0.1 n hcl solution (ph 1.2). iraqi j pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers 92 the solution gained was filtered, and one ml of the filtrate was appropriately diluted and analyzed for sumatriptan spectrophotometrically at its λ max (19, 20). in vitro disintegration time for core tablets the disintegration test was done for all core tablet formulas at 37˚c using phosphate buffer (ph 6.8) as disintegration media. disintegration apparatus of a one-liter cylinder with a basket rack assembly containing six openended tubes and 10-mesh screen on the bottom was used. a tablet was placed in every tube of the basket and the time required for complete disintegration of the tablets with no palpable mass remaining in the apparatus was measure(14). in-vitro dissolution test in-vitro dissolution test is applied for core tablet using usp apparatus type ii (paddle) at 37 ± 0.5°c in 900 milliliters of dissolution medium (phosphate buffer ph 6.8 ) at 50 rpm. five milliliters samples were withdrawn periodically at different time intervals, and each sample was substituted with an equal volume of fresh dissolution medium. then, the samples were filtered and analyzed spectrophotometrically at its λ max. each test was done in triplicate. for optimization many variables evaluated to test its effect on the dissolution of the core tablet from different formulas (21). post-compression evaluation for press coated tablet the prepared coated tablets were evaluated for hardness and release study for press coated tablet as mentioned for core tablets. in-vitro release studies of press-coated pulsatile tablets the prepared press coated tablets were examined for in vitro drug release in two different suitable dissolution media( 2 hours in 0.1 n hcl (ph 1.2) followed by 4 hours in phosphate buffer ph 6.8) using usp type ii dissolution apparatus at 50 rpm to assess their ability to provide the desired lag time before drug release. five milliliters samples were withdrawn periodically at different time intervals, and each sample was substituted with an equal volume of fresh dissolution medium. then, the samples were filtered and analyzed spectrophotometrically at its λ max(22). variables effecting release of sumatriptan from the core tablet effect of type of superdisintegrants two different types of superdisintegrants (croscarmellose and sodium starch glycolate) at 5% concentrations were used in (f1 and f4) to study the effect of superdisintegrant types on the drug release properties from sumatriptan core tablet. effect of concentration of superdisintegrant different percentages of sodium starch glycolate (superdisintegrant) were utilized in the formulation of core tablet formula (f6, f5, f4 ) containing 1,3 and 5% to analyze the effect of using different concentrations of sodium starch glycolate on sumatriptan release from the core tablet. variables affecting the release of sumatriptan from the press coated core tablet (effect of different concentrations of combination of polymers) three formulas of coated core tablet were made using different polymers (pectin, ethylcellulose 100mpa.s and hpmck15m) in different ratios as recorded in table 2 to study their effect on sumatriptan release from press coated core tablet and also their effect on the lag time required for release of the drug. drug – excipients compatibility studies physicochemical compatibility between sumatriptan and different excipients was studied using fourier transform infrared spectroscopy (ftir) and differential scanning calorimetry (dsc). 1.fourier transform infrared spectroscopy (ftir) the pure drug powder and the optimum formula of core tablet (f4) were analyzed individually by using (shimadzu 8300, japan) according to kbr disk method. about 2-3 milligrams sample was mixed with dried ir grade potassium bromide powder to form a uniform blend of about 200 milligrams, and analyzed by ftir spectroscopy at 4000-400 cm-1(23). 2.differential scanning calorimetry (dsc) it was carried out by the same way for the pure drug powder, and the physical mixture of the optimum formula of core tablet f4 , and the optimum formula of core tablet f4 using differential scanning calorimeter (shimadzu dsc60). samples were heated in an aluminum sample pans at a rate of 10°c/minute over a temperature up to 350 °c under a nitrogen flow of 50 milliliters/minute (24). statistical analysis the results of the experiments were given as mean values ± standard deviation (sd) and analyzed according to the one-way analysis of variance (anova) at which significant results (p<0.05) and non-significant (p>0.05)(25). iraqi j pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers 93 result and discussion pre-compression parameters of core powder blend the values of angle of repose, bulk density, tapped density, carr's index, and hausner ratio for each prepared core and coat powder blend formula were measured .the results of precompression evaluation tests of core powder blend are illustrated in table 4. these results estimated according to usp (20). the results show that the prepared core mixtures have acceptable flow properties and compressibility. table 4. pre-compression physical parameter’s for core powder blend of sumatriptan f o r m u la angle of repose (degree) mean± sd, n=3 bulk density (g/cm3) mean± sd, n=3 tapped density (g/cm3) mean± sd, n=3 carr's index mean± sd, n=3 hausner ratio mean± sd, n=3 type of flow f1 17.51±0.03 0.5460.01 0.608±0.02 10.21±1.36 1.11±0.019 excellent f2 18.4 0.1 0. 4950.08 0.57±0.021 10.51±1.0002 1.12±0.02 excellent f3 25.70.2 0.560.56 0.632±0.03 10.73±0.37 1.119±0.004 excellent f4 21.360.56 0.33±0.05 0.371±0.03 10.84±0.39 1.122±0.005 excellent f5 19.250.75 0.3450.01 0.357±0.015 3.41±0.15 1.041±0.007 excellent f6 20.5±0.4 0.311±0.026 0.334±0.028 6.92±0.2 1.07±0.001 excellent postcompression evaluation of core tablet: hardness, content uniformity and in-vitro disintegration study of core tablets hardness testing of solid oral dosage forms is essential because it provides a quantitative estimate of the internal bonding strength of the powder compact, since it gives the tablet sufficient mechanical strength to keep its internal structure and geometry under applied external forces. differences in tablet hardness are hence known to correlate with differences in dissolution or mechanical response during any postcompression operations such as tablet coating, handling, packaging, storage, or shipping(26). the results of hardness test for all prepared core tablet formulas were in the range of ( 4.1 4.53 kg/cm2) as shown in table 5 which indicate the tablets had adequate strength property to resist handling, shipping and tablet coating. the results of drug content test are shown in table 5 that all prepared core tablets had acceptable range according to usp pharmacopeia (14). the results of disintegration test of all prepared core tablets are shown in table 5. in this study, two types of superdisintegrants were used , ccs and ssg had three different concentrations(1%,3%,5%) w/w of total weight of sumatriptan core tablet. as shown in table 5, f1 and f4 that contained 5%(w/w) croscarmellose sodium and 5% (w/w) sodium starch glycolate respectively, f4 had the shortest disintegration time, this may be due to the remarkable rapid water penetration and extensive swelling capability of ssg. ssg was reported to possess the capability to absorb water and swell about 300 times its volume and not affected by an increase in compression pressure(27). the results showed that f2 which contains 3% croscarmellose sodium had shorter disintegration time than f1 that contains higher concentration of this superdisintegrant which may be due to partial gelling that potentially could form a viscous barrier and delay the entry of water into the tablet leading to this delay in the disintegration of tablets of f2(27). in case of the superdisintegrant sodium starch glycolate was used in formulas f4, f5 and f6 in concentrations 5%, 3% and 1% (w/w) respectively. it was observed that the disintegration time was decreased as the concentration of the superdisintegrant was increased from concentration 1% to 5% (w/w). iraqi j pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers 94 table 5 . physical evaluation of core tablets of sumatriptan f o r m u la hardness (kg) mean± sd, n=5 content uniformity % mean± sd, n=3 disintegra tion time seconds mean± sd, n=6 f1 4.1±0.13 97.31±0.77 14.25±0.95 f2 4.22±0.17 96.17±0.47 11±4.89 f3 4.52±0.32 98.07±0.45 17.5±2.5 f4 4.43±0.3 98.48±2.6 8.8±1.4 f5 4.7±0.2 95.15±1.08 11.67±1.6 f6 4.78±0.2 96.712±0.95 21.5±2.25 in-vitro release studies of core tablets figure 1 shows that f4 has faster dissolution rate where 100% release of sumatriptan from core tablet obtained in 2 minutes this significant difference (p≤ 0.05), this was explained that sodium starch glycolate possesses the additional advantages of being soluble and readily dispersible in water. its spherical particles, dispersed in a tablet system, offer a larger surface, thus allowing rapid penetration of water into the tablet interior. the main reasons for the efficiency of this disintegrant probably are its high rate of water uptake and its marked swelling properties; these factors cause pressure to be exerted within the tablet, thus breaking up interparticle bonding. this is then followed by the dissolution of sodium starch glycolate particles, which results in the crumbling and disintegration of the entire tablet structure (28). ssg may swell up to three hundred times its original volume in water(29). formulas f4, f5, and f6 contain different concentrations of sodium starch glycolate used to study the effect of superdisintegrant concentration on the release of sumatriptan from core tablets as shown in figure 2. f4 which contains 5% w/w sodium starch glycolate show higher percent of drug release. there was significant difference (p≤0.05) in dissolution rate between the formulas because as we increase in the concentration of superdisintegrant, the disintegration time will decrease. this disintegration is reported to affect dissolution characteristics as well(30). figure 1. effect of superdisintegrant type on drug release from core tablet (phosphate buffer ph 6.8, temp. 37 0c) figure 2. effect of sodium starch glycolate concentration on drug release from sumatriptan core tablet (phosphate buffer ph 6.8, temp. 37 0c) to prepare pulsatile press coated tablet, f4 (that contains 5% (w/w) sodium starch glycolate as superdisintegrant and avicel ph 102 as diluent) was selected as the optimum core part since it produced the fastest 100% release of sumatriptan within 2 minutes. post compression evaluation of press-coated pulsatile tablet the results of hardness of all the presscoated tablets were shown in table 5. these results show that all the prepared press-coated tablets formula agrees with the requirements of usp. the hardness of the press-coated tablets was kept constant in the range 6-7 kg/cm2 by mounting the iraqi j pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers 95 compression force of the machine to eliminate the variability in hardness. the hardness of the press coated tablets slightly increased as ethyl cellulose concentration was increased due to high compressibility of this polymer (31). table 6. post formulation results of press coated core tablet of sumatriptan formula hardness (kg/cm2) c1 6.25±0.4 c2 6.75±0.06 c3 6.25±0.04 in-vitro release studies of press-coated pulsatile tablets formulas c1 –c3 were used to study the effect of the combination of natural and synthetic polymers in different proportions (pectin, ethylcellulose100 mpa.s and hpmck15m). ratios of the polymers used were 10:5:85, 10:10:80 and 15:5:80 w/w of coating layer; respectively. c1 and c2 was used to assign the effect of ethylcellulose 100 mpa.s and hpmck15m on the lag time. it was noticed that as the ratio of hpmc increased the lag time will be decreased due to hydrophilicity nature of hpmc. c2 and c3 were used to observe the effect of pectin and ethylcellulose 100 mpa.s on lag time; it was observed that as the ratio of pectin was increased the lag time was decreased because of hydrophilic nature of pectin and its inherited swelling and eroding behavior (32). the results of in-vitro release studies of press-coated pulsatile tablets are shown in table 7. table 7. lag time of dissolution for different coating formulas of sumatriptan press-coated tablet formula composition lag time in h: min c1 pectin:ec100:hpmck 15m 10:5:85 6:20 c2 pectin:ec100:hpmck 15m 10:10:80 7 c3 pectin:ec100:hpmck 15m 15:5:80 5:45 coat formula c3 was chosen as coat for optimum core tablet formula f4 since it gave it gave 100% release the drug after 5.45 hours lag time, which is required to provide a maximum concentration of drug at the time of its maximum need. release profile of the press coated system of sumatriptan as shown in figure (3). figure 3. release profile of sumatriptan from the selected final sumatriptan press-coated tablet drug – excipients compatibility studies compatibility studies were assigned by using :  differential scanning calorimetry (dsc) the dsc technique had been used for chemical stability and compatibility studies(33). the differential scanning calorimetry (dsc) of sumatriptan pure drug, the physical mixture of optimum core tablet and the optimum core tablet of sumatriptan were performed using a shimadzu 8300 dsc, and the dsc thermogram of all samples had been shown in figures (4-6). the dsc thermogram of sumatriptan pure drug, the physical mixture of optimum core tablet and sumatriptan optimum core tablet exhibited the characteristic drug peak indicating compatibility and absence of interaction between the drug and the other components (34). iraqi j pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers 96 figure 4. dsc thermogram of pure sumatriptan 100.00 200.00 300.00 temp [c] -4.00 -3.00 -2.00 mw dsc 77.67x100c 169.56x100c figure 5. dsc thermogram of physical mixture of sumatriptan core tablet 100.00 200.00 300.00 temp [c] -1.00 0.00 1.00 2.00 3.00 mw dsc 170.40x100c figure 6. dsc thermogram of sumatriptan optimum core tablet fourier transform infrared spectroscopy (ftir): ftir spectrum of pure sumatriptan drug and the selected core tablet are shown in figures 7 and 8 respectively. in figure 7 it was found the characteristic peaks of sumatriptan pure drug at 3373 cm-1, 1298 cm-1, 1236 cm-1, 1082 cm-1 and 636 cm-1 belongs to nh stretching vibration, c-n stretching vibration, s=o stretching vibration and c-s stretching vibration, respectively. in figure 8, the characteristics peaks of sumatriptan were found in core tablet were for n– h str. primary amine at 3391 cm−1, c–n str. at 1299.79 and 1233.26 cm−1, and c–h str. at 2960– 2850 cm−1 and s=o str. at 1057.76 cm-1 and c-s str. at 638 cm-1 (39).figure 8 shows the ftir spectrum of the selected formula of the core tablet f4 containing sumatriptan. there were no significant differences in the main characteristic bands of the drug indicating no interaction between drug and other additives in core tablet formula f4. iraqi j pharm sci, vol.27(1) 2018 time controlled sumatriptan using natural polymers 97 figure 7. ftir spectrum of sumatriptan figure 8. ftir spectrum of the selected formula of sumatriptan core tablet conclusion press coated tablet 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programmed double pulse press coated tablet containing fixed dose combination of montelukast sodium and levocetrizine dihydrochloride for treatment of nocturnal asthma. international journal of pharmaceutical sciences review and research. 2016; 41(2): 306-311. 32. sriamornsak p, thirawong n, weerapol y, nunthanid j, sungthongjeen s. swelling and erosion of pectin matrix tablets and their impact on drug release behavior. european journal of pharmaceutics and biopharmaceutics. 2007; 67(1):211-219. 33. balpande hm, raut ns, umekar mj, kotagale nr. compatibility study of metformin with pharmaceutical excipients.international journal of chem tech research. 2013; 5(4): 1684-1693. 34. abd-el bary a, louis d, sayed s. liquisolid tablet formulation as a tool to improve the dissolution of olmesartan medoxomil. inventi journals 2014; 3: 1-8. iraqi j pharm sci, vol.28(2) 2019 solid dispersion adsorbate doi: https://doi.org/10.31351/vol28iss2pp105-114 105 solubility and dissolution enhancement of atorvastatin calcium using solid dispersion adsorbate technique samer k. ali*1 and eman b. h. al-khedairy* *department of pharmaceutics college of pharmacy, university of baghdad, baghdad, iraq. abstract atorvastatin (atr) is a poorly soluble anti-hyperlipidemic drug. the drug belongs to the class ii group according to the biopharmaceutical classification system (bcs) with low bioavailability due to its low solubility. solid dispersions adsorbate is an effective technique for enhancing the solubility and dissolution of poorly soluble drugs. the present study aims to enhance the solubility and dissolution rate of atr using solid dispersion adsorbate technique in comparison with ordinary solid dispersion. polyethylene glycol 4000 (peg 4000), polyethylene glycol 6000 (peg 6000), poloxamer188 and poloxamer 407were used as hydrophilic carriers besides aerosil 200, aerosil 300 and magnesium aluminium silicate (mas) as adsorbents. all solid dispersion adsorbate (sda) formulas were prepared in ratios of 1:1:1 (drug: carrier: adsorbent) and evaluated for their water solubility, percentage yield, drug content, , dissolution, crystal lattice using x-ray powder diffraction (xrd) and differential scanning calorimetry (dsc) studies and fourier transform infrared spectroscopy (ftir) for determination the drug-carrieradsorbate interaction. the prepared (sda) showed improvement of drug solubility in all prepared formula. the best result was obtained with formula sda12 (atr: poloxamer407: mas 1:1:1) that showed 8.07 and 5.38 fold increase in solubility compared to solubility of pure atr and solid dispersion(sd4) (atorvastatin: poloxamer 407 1:1) respectively due to increased wettability and reduced crystallinity of the drug which leads to improving drug solubility and dissolution. keywords: atorvastatin , solid dispersions adsorbate, peg4000 and 6000, aerosil 300 , magnesium aluminium silicate . لالتروفاستاتين كالسيوم باستخدام تقنية ومعدل الذوبانتحسين الذوبانية الصلب المنتشرالممتز *و ايمان بكر حازم الخضيري 1*،سامر خالد علي .العراق ،بغداد،جامعة بغداد،كلية الصيدلة ،فرع الصيدالنيات * الخالصة ينتمي إلى مجموعة الصنف الثاني طبقا لتصنيف المستحضرات االتروفستاتين هو دواء مضاد الرتفاع الدهون في الدم و قليل الذوبان ؛ و لذوبان ا الصيدالنية الحيوية و الذي يتميز بضعف التوافر الحيوي بسبب قلة ذوبانه. الصلب المنتشر الممتز هو طريقة فعالة لتعزيز قابلية ومعدل لالدوية قليلة الذوبان. ومعدل الذوبان لدواء االتروفستاتين باستخدام تقنية الصلب المنتشر الممتزومقارنته مع الهدف من هذه الدراسة الحالية هو تحسين قابلية كناقالت محبة 044وبولكسمر 811,بولكسمر0444, البولي إيثيلين كليكول 0444حيث تم استخدام البولي إيثيلين كليكول الصلب المنتشر العادي. المغنيسيوم واأللومنيوم كمواد ممتزة ., سليكات 044,االيروسيل 044للماء وااليروسيل وتم تقيمها حسب قابلية الذوبان في الماء ,والنسبة ) دواء:ناقل :ممتز( 8: 8: 8تم تحضير جميع صيغ الصلب المنتشر الممتز بنسب االشعة ية ومسعر المسح التبايني و مطيافالمئوية المستحصلة , ومحتوى الدواء, معدل الذوبان ، والهيكل البلوري باستخدام تقنية حيود االشعة السين تحت الحمراء لتحديد التفاعل الحاصل بين الناقل والممتز والدواء. ولقد اظهرت النتائج تحسن في قابلية الذوبان للدواء في جميع الصيغ المحضرة بتقنية الصلب المنتشر الممتز.وتم الحصول على افضل ضعف 8001و 1044( التي اظهرت زيادة بنسبة 8:8:8: سليكات المغنيسيوم واأللومنيوم 044: بولكسمر ) اتورفستاتين sda12نتيجة بالصيغة ية للدواء مما رفي الذوبانية مقارنة بذوبان االتروفستاتين النقي والصلب المنتشر العادي على التوالي بسبب زيادة قابلية البلل وانخفاض الحالة البلو وبان ومعدل الذوبان في الدواء.يؤدي الى زيادة قابلية الذ سليكات المغنيسيوم واأللومنيوم. ،044ايروسل،0444و 0444بولي اثلين كيكول ،الصلب المنتشر الممتز ،االتروفستاتين الكلمات المفتاحية: introduction the solubility of drug molecules is a significant determinant of the dissolution rate because of dissolution rate is closely tied to solubility (1). poor solubility and low dissolution rate of poorly water soluble drugs in the aqueous gastrointestinal fluids often cause poor bioavailability, principally for class ii drugs, the enhancement of the solubility and dissolution rate of the drug in the gastrointestinal fluids may improve the bioavailability, for bcs class ii drugs, the release from the dosage form and solubility in the gastric fluid are the rate-limiting steps rather than their absorption (2). 1corresponding author e-mail: samerkhalidali@gmail.com received: 26 / 5 /2019 accepted: 3 / 8 /2019 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss2pp105-114 mailto:samerkhalidali@gmail.com mailto:samerkhalidali@gmail.com iraqi j pharm sci, vol.28(2) 2019 solid dispersion adsorbate 106 atorvastatin calcium (atr) is [c33h35fn2o5]2.ca.3h2o as an empirical formula, and chemical structure is shown in figure 1 (3). figure 1. chemical structure of atorvastatin calcium(3) atorvastatin calcium (atr) is an antihyperlipidemic agent able to lowering blood cholesterol levels by the reversible inhibition of hmg-coa reductase, a rate-limiting step in the cholesterol biosynthesis. it represents one of the most widely administered oral statins used in case of elevated plasma levels of cholesterol, triglycerides, low-density lipoproteins in addition to its ability to elevate the high-density lipoproteins (4). atr belongs to bcs class ii drug (5). the drug is very slightly soluble in distilled water with the pka value of 4.46 and log p values of 6.36 in octanol/water. it has an absolute oral bioavailability of 12 %. the poor oral bioavailability is attributed to presystemic clearance in the gastrointestinal mucosa and high hepatic first-pass metabolism (6). many authors have worked to improve the solubility and dissolution rate of atr by preparing microsphere, emulsion, nanosuspension, selfmicroemulsion, solid dispersion (7). solid dispersion (sd) is the most generally used technique for bioavailability enhancement of poorly water-soluble drugs, as the drug dispersed in freely soluble carrier (8). some difficulties limiting the commercial use of solid dispersion such as; formulation into dosage forms(poor flow, poor compressibility and a requirement of a large quantity of carrier), the scaleup of production processes. in addition, phase separation, crystal growth during storage are also considered to be significant problems to commercialise solid dispersions since the solubility and hence dissolution rate may be decreased during storage (9,10). solid dispersion adsorbate(sda) is a technique in which sd is adsorbed onto hydrophilic adsorbent which has an exceedingly high surface area to form a free-flowing powder and to achieve improved solubility, dissolution rates and hence bioavailability (11). the effect of adsorbent will help in commercialisation of sd as a tablet or capsule dosage form by improving poor compressibility by adsorbing the sd onto a free-flowing carrier. on the other hand, the stability problem can be reduced by the use of novel excipient like neusilin® which along with improving the flow property also inhibits the conversion of amorphous form back to the crystalline form by entrapping the molecularly dispersed drug into its porous network(12). other adsorbents were also used, including aerosil, florite, and sylysia having different characteristics such as particle size, pore size, and specific surface area (13). the aim of this study was to enhance the solubility and dissolution rate of atr by solid dispersion adsorbate technique using polyethylene glycol (peg)4000 and 6000 or poloxamer188 and 407 as a hydrophilic carrier and magnesium aluminium silicate (mas) or aerosil200 or aerosil 300 as adsorbents. materials and method materials atorvastatin calcium(atr), aerosil200 and peg 6000 were supplied by pioneer pharmaceutical company, iraq as a gift sample. poloxamer188, polxamer407, magnesium aluminium silicate (mas) and aerosil300 were purchased from hangzhou, hyperchem. china. peg 4000 was purchased cdh, india. method preparation of solid dispersion of atorvastatin calcium solid dispersions of atr were prepared using a melting method. in this method,the carrier (peg4000, peg 6000, poloxamer 188 or poloxamer 407) is melted using water bath at its corresponding melting point, and the drug was then incorporated into the molten carrier mass in a ratio of 1:1 with constant stirring. the blend was cooled at room temperature. the solid dispersion was pulverised through a 60 mesh sieve and stored in the desiccator for further use (14). the prepared solid dispersions are shown in table 1. preparation of solid dispersion adsorbate (sda) of atorvastatin calcium solid dispersion adsorbate of atr was prepared by addition of the drug to melt of each carrier individually in porcelain dish on a water bath at 60˚c and mixed. once homogeneous slurry was obtained, the adsorbent ( aerosil 200, aerosil 300 or mas.) was added and stirred until the blend was converted to mass. the drug to a carrier to adsorbent ratio was fixed at 1:1:1(table 2). finally, the mass was allowed to cool at room temperature. the sdas were passed through a 60 mesh sieve to obtain a free-flowing powder of uniform size (15). iraqi j pharm sci, vol.28(2) 2019 solid dispersion adsorbate 107 table 1. composition of different sd formulas of atr formula code atr )g.) peg4000 )g.) peg6000 )g.) poloxamer188 )g.) poloxamer407 )g.) sd1 1 1 … … … sd2 1 … 1 … … sd3 1 … … 1 … sd4 1 … … … 1 preparation of physical mixture (pm) the pm was prepared by uniform mixing of drug, carrier and adsorbent in the same ratios of sda. the mixing powder was passed through a sieve 60 mesh to get uniformly sized particles (16). table 2.composition of different sda formulas of atr. formula name atr )g.) peg 4000 )g.) peg 6000 )g.) poloxamer 188 )g.) poloxamer 407 )g.) aerosil2 00 )g.) aerosil 300 )g.) mas )g.) sda1 1 1 … … … 1 … … sda2 1 1 … … … … 1 … sda3 1 1 … … … … 1 sda4 1 … 1 … … 1 … … sda 5 1 … 1 … … … 1 … sda 6 1 … 1 … … … … 1 sda 7 1 … … 1 … 1 … … sda 8 1 … … 1 … … 1 … sda 9 1 … … 1 … … … 1 sda 10 1 … … … 1 1 … … sda 11 1 … … … 1 … 1 sda 12 1 … … … 1 … … 1 evaluation of sd and sda determination of saturation solubility an excess amount of atr, sd and sda were added to 10 ml of water; the samples were incubated in water bath shaker at 25 ˚c for 48 hr. after that, the samples were filtered through a 0.45µm syringe filter and diluted when necessary. the diluted samples were analysed by uv spectrophotometer at 241 nm was used to determine the dissolved quantity of atorvastatin(17). determination of percentage yield (py %) the percentage yield was measured for each type of sd and sda, which was calculated using equation 1 (18). py%= actual weight of sd or sda theoretical weight of sd or sda 𝑥100 (1) determination of drug content of sd and sda an accurately weighed quantity of sd and sda equivalent to 10mg of atr was taken and dissolved in a 10 ml of methanol and volume was made up to 50 ml. from this, 1ml of the solution was taken and further diluted ten times with methanol. the solution was assayed for drug content using uv spectrophotometry method by measuring the absorbance at 247 nm (19). the percentage of drug content in the sd and sda was calculated by using equation 2 (20). drug content %= actually gained weight of atr theoretical weight of atr 𝑥 100 (2) in-vitro dissolution studies in-vitro dissolution studies of sda equivalent to 20mg atorvastatin were carried out in 900 ml phosphate buffer (ph 6.8) in usp apparatustype ii (pharma test, germany) at 37 ± 0.5 ˚c and 75 rpm(21). aliquots of 5 ml were withdrawn and then replaced by fresh media of dissolution at regular time intervals, filtered, and was analysed spectrophotometrically at 240nm by uv visible spectrophotometer (carry win uv, australia). x-ray powder diffraction (xrd) the x-ray powder diffraction spectra of optimised formula, pm, and atr were recorded using shimadzu x-diffractometer(model: xrd6000, japan). samples were pulverised into powders with a mortar and pestle, and the cross-section of iraqi j pharm sci, vol.28(2) 2019 solid dispersion adsorbate 108 samples was exposed to x-ray radiation.the scanning angle ranged from 5 to 80 of 2ө, voltage40kv and current 40 ma(22). differential scanning calorimetry (dsc) differential scanning calorimetry of atr, optimised formula, pm, carrier and adsorbent were done using dsc60 (shimadzu, japan ). thermal behaviour of the samples was investigated under a scanning rate of 10 ˚c/min, covering a temperature range of 20–200 ˚c under inert atmosphere flushed with nitrogen at a rate of 20 ml/min (23). fourier transform infrared (ftir) fourier transform infrared spectra of atr, mas, pm and the optimized formula were obtained using shimadzu, fourier transform infrared spectroscopy (ftir 43000), (japan) , each sample was dispersed in kbr powder, blend well in mortar and pestle, and compressed into transparent discs for ftir examination and ftir spectra were recorded in spectra region 4000 to 400 cm-1 at an instrument resolution of 4 cm-1 (24). results and discussion saturation solubility of atr sds results of saturation solubility studies of atr sds are shown in table 3. table 3.the solubility of pure atr and sds formulas using different carriers with drug: carrier ratio 1:1 in distilled water at 25˚c. formula code carrier saturation solubility µg/ml (mean ±std) n=3 pure atr 120.2±0.013 sd1 peg 4000 126.01±0.014 sd2 peg 6000 129.95±0.012 sd3 poloxame r 188 141.1±0.005 sd4 poloxame r 407 180.34±0.017 significant enhancement in solubility of atr (p<0.05) was obtained, which may be attributed to the hydrophilic nature of all the used carriers, besides hydrogen bonding that may be formed between atr and carriers led to enhance the solubility of atr(25).the solubility enhancement of the various carriers was found to be in the following descending order: poloxamer407> poloxamer188 > peg6000>peg4000. the highest solubility was obtained using poloxamer 407 as a carrier due to the surfactant effect of poloxamer 407 and micelles formation (26). saturation solubility of atr sdas table 4 shows the results of solubility atr sdas. further improvement in solubility was obtained when atr prepared as sdas in comparison to that obtained by sds. that can be explained to be due to adsorption of atr on the hydrophilic adsorbent and increase the surface area of atr that exposed to the solvent , whereby the drug is bound to the adsorbent and thus cannot agglomerate which results in enhanced wettability of the drug particles and hence its solubility (27,28). table 4. the solubility of atr sdas prepared with different adsorbents with drug : carrier: adsorbent ratio 1:1:1 in distilled water at 25˚c. batch no. carrier: adsorbent (1:1) saturation solubility µg/ml (mean ±std ) n=3 sda1 peg4000:aerosil 200 136.2±0.002 sda 2 peg4000:aerosil 300 157.3±0.006 sda 3 peg4000:mas 810.8±0.109 sda 4 peg6000:aerosil 200 142.8±0.009 sda 5 peg6000:aerosil 300 162.5±0.004 sda 6 peg6000:mas 863.5±0.014 sda 7 poloxamer 188:aerosil 200 149.5±0.057 sda 8 poloxamer 188:aerosil 300 168.3±0.017 sda 9 poloxamer 188:mas 880.5±0.024 sda 10 poloxamer 407:aerosil 200 243.2±0.026 sda 11 poloxamer 407:aerosil 300 280.2±0.027 sda 12 poloxamer 407:mas 970.8±0.027 the solubility enhancement of the various adsorbents was found to be in the following descending order: mas > aerosil 300 > aerosil 200 in the presence of any hydrophilic carrier(figure 2). the results may be due to the difference adsorption capacity of three adsorbents to the drug. highest solubility was obtained with mas because it has excellent disintegrating activity in an aqueous solution(29) and large surface area due to its composition of many silicate layers and good adsorption properties because of a negative charge on the surface of the silicate layers that brings about a strong electrostatic interaction with a cationic drugs and the silanol groups can interact by iraqi j pharm sci, vol.28(2) 2019 solid dispersion adsorbate 109 hydrogen bond with drugs which gives it good adsorption properties (30). whereas the difference adsorption capacity of two other adsorbents to the drug can be explained to the difference in their surface area as the surface area of aerosil 300(300 ±30m2/g), so it has high adsorption capacity when compared with aerosil 200(200 ± 25m2/g)(31). therefore, the largest surface area of atr that exposed to the dissolution medium was obtained when it is adsorbed on mas. formula sda12 showed the highest solubility 970.8±0.0274 µg/ml compared to 120.2±0.0138 µg/ml for pure drug and 180.34µg/ml solid dispersion(sd4) (atr: poloxamer407 1:1) indicating that solid dispersion adsorbate technique enhances the solubility by 8.07 and 5.38 fold as compared to the pure drug and ordinary sd respectively. figure2. effect of adsorbent and carrier type of atr sda at ratio (1:1:1) on the solubility of atr in distilled water at 25°c determination of percentage yield (py %) and content of prepared sd and sda the prepared sds and sdas formulas showed high percentage yield ranged between 85.598.53%. this result indicating the suitability of the method (melting or melt adsorption method) with the used material for such preparations. on the other hand, the drug content in all the formulations was found to be within 98-100% w/w, which is in agreement with u.s.p requirements (98102%)(21). the results of percentage yield and drug content are shown in table 5. table 5. percentage yield and drug content of sd and sda formula code percentage yield (py %) drug content (w/w) (%) (mean ±std) (n=3) formula code percentage yield (py %) drug content (w/w) (%) (mean ±std) (n=3) sd1 93.71 98.10 ±0.06 sda 5 97.26 99.45 ±0.06 sd2 85.50 98.08 ±0.01 sda 6 93.73 98.67 ±0.02 sd3 95.98 99.34 ±0.05 sda 7 90.66 98.02 ±0.04 sd4 96.85 98.59 ±0.03 sda 8 96.86 98.70 ±0.04 sda1 86.80 98.00 ±0.14 sda 9 95.93 99.00 ±0.01 sda2 94.26 99.23 ±0.03 sda 10 98.53 98.03 ±0.02 sda 3 90.40 99.10 ±0.02 sda 11 98.00 99.10 ±0.02 sda 4 91.13 98.4 ±0.05 sda 12 97.93 100.00±0.01 in-vitro dissolution studies comparative in-vitro dissolution of the pure drug, sda12 (atr:poloxamer407: mas 1:1:1) ,sd4(atr:polxamer407 1:1)and physical mixture(pm) of sda12 was studied the similarity factor (𝑓2) was used to consider similar dissolution profiles (equation 3). 𝑓2 = 50 × log { [1 + 1 𝑛 ∑ |𝑅𝑡 − 𝑛 𝑡=1 𝑇𝑡 | 2 ] −0.5 × 100} …… equation (3) where (n) is the number of dissolution time points. (rt) and (tt) is the reference and test dissolution values at time t. the two dissolution profiles consider similar when f2 values higher than 50 (50– 100); otherwise, the profiles are not similar (32). an improvement in the dissolution was obtained (figure 3) by sda12 in comparison with sd4 (f2=48.55), pm of sda12 formula ( f2=40.23) and pure drug (f2=27.26). this result can be due to increased solubility by the formation of hydrogen bonding between the drug and mas, micellar solubilisation of atr in poloxamer407, improved wettability and amorphisation of atr (33). that needs further investigation by ftir, xrd and dsc. iraqi j pharm sci, vol.28(2) 2019 solid dispersion adsorbate 110 figure 3.comparative in-vitro dissolution profile of the atr, sda12, sd4 and physical mixture of sda12(pm) at phosphate buffer 6.8 and 37°c±0.5 x-ray powder diffraction (xrd) the xrd diffractogram of atr, sda 12 and pm are shown in figure 4. the diffraction pattern of the pure drug showed characteristic highintensity peaks at 9.06, 9.38, 10.17, 11.74, 16.89, 19.32, 21.48, 22.5549, 23.17 and 23.57, which indicates that the drug is present in the crystalline form that is also confirmed by dsc results. physical mixture’s pattern showed the characteristic peaks of atr with lower intensity compared to the pure drug. this result can be explained to be due to the dilution effect of the carrier. in contrast, further decrease or absence of the characteristic peaks of atr was observed with sda 12, indicating that an amorphous form mostly exists in solid dispersion adsorbate (34,35). this amorphous form may contribute to solubility improvement since this form is more easily soluble than the crystalline form. figure4. x-ray diffractograms of atr(a), pm (b)and solid dispersion adsorbate of selected formula sda12 (c) differential scanning calorimetry (dsc) thermograms of atr, mas, poloxamer407, pm and the sda12 are shown in figure 9, respectively. the dsc curve of atr gives an endothermic peak corresponding to its melting point at 160.04˚c; the result indicated the purity and crystallinity of the used atr.(6) while the dsc of mas shows no endothermic peak in the studied dsc temperature range and poloxamer407 shows an endothermic peak at 53.80 ˚c correspondings to its melting point(36). pm shows the only distinct peaks of poloxamer407 with a decrease in intensity and melting point to 47.04˚c which may be due to dilution with other components while the absence of atr peak may be due to adsorption of the drug on mas (37) which will be confirmed by ftir study. the thermogram of sda12 shows further decrease in the intensity of poloxamer 407 with disappearance of atr peak which may be due to adsorption effect and its complete dispersion in the melted polymer and conversion of the drug from crystalline to amorphous state (38). fourier transform infrared (ftir) the atr exhibits characteristic peaks at 3664cm-1 (free o–h stretching),3365cm1(o–h stretching),3278cm-1 and 3060cm-1(n–h stretching multiple band),2966 cm-1(c–h stretching), 1651cm-1(amide c=o stretching), 1581 cm-1 (aromatic c=oc stretching) , 1516 cm-1(n–h bending),812 cm-1(out of plane n-h wagging) were found in the crystalline form (unprocessed drug) as shown in figure 6 (39,3).the ftir spectrum of pm showed the characteristic peak of atr with low intensity indicates the predominant effect of carrier and adsorbent concentration (40). the spectrum of the sda15 showed broad and weak o–h stretch peak (3365 cm-1) and n–h stretching multiple bands compared to atorvastatin crystalline drug. besides, a broad peak or absent peak corresponding to a hydroxyl stretching of mas sioh 3620cm-1 that may be due to the formation of a hydrogen bond between silanol group on the surface of mas and hydroxyl group and amide group of atr(39,01). the carboxyl stretching peaks at 1651 and 1581 cm-1 of atr shifted to higher wavenumber(1655 and 1589) cm-1 and presented lower intensity in sda12 (figure10, d), indicating an electrostatic interaction of carboxyl group of atr with the positively charged sites in the edges of mas structure(37). the most characteristic peaks of atr are still present but some decrease in intensity and shifting of some peak, this indicates that there was no chemical incompatibility between drug, carrier and adsorbate otherwise it confirm the adsorption process. iraqi j pharm sci, vol.28(2) 2019 solid dispersion adsorbate 111 figure 5. dsc: atr(a) , mas (b) , poloxamer407(c), pm (d) , sda12 (e) iraqi j pharm sci, vol.28(2) 2019 solid dispersion adsorbate 112 figure 6. ftir: atr(a) , mas (b) , pm (c) , sda12 (d) conclusion an improvement in solubility and dissolution of atr was obtained by preparing it as solid dispersion adsorbate by melt adsorption method using hydrophilic carriers and adsorbents in a ratio of 1:1:1(drug: carrier: adsorbent). all prepared formula of sdas improved the solubility of atr may be due to increased wettability and reduced crystallinity of the drug, 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pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(2) 2012 various methods of contraception 56 investigation and comparative study among various methods of contraception used in erbil city ijlal r. abdullah * , kassim j. al-shamma **,1 and ansam n. al-hassany* * college of pharmacy, hawler medical university, erbil, iraq **department of clinical pharmacy college of pharmacy, university of baghdad, baghdad, iraq abstract the objective of this study was to investigate and compare among five different methods of contraception including combined oral contraceptive pills (coc), depot medroxyprogesterone acetate (dmpa), copper intrauterine contraceptive device (iucd), vaginal spermicides and male condom used in hawler city through estimate of their effect, relative failure rate, percentage of use, adherence and compliance and adverse effects of each contraceptive method. in order to reach to these aims, a retrospective study was conducted in hawler city in azadi health care center over a period of 6 months from 22 th november, 2010 to 15 th may, 2011 during which data collection and subjects follow up for 3 months had been achieved. a convenient sampling method was used to collect 373 married women in their reproductive age group (16-39) years old and 56 husbands. the studied population was allocated into five groups according to contraceptive method used: group (i) included (113) subjects using combined oral contraceptive pills, group (ii) included (38) subjects using depot medroxyprogesterone acetate injection, group (iii) included (211) subjects using copper intrauterine contraceptive device, group (iv) included (11) subjects using vaginal spermicides and group (v) included (56) subjects using male condom as a contraceptive method. the data necessary for this study had been collected by a direct interview with the subjects and the informations had been recorded on a questionnaire.the study revealed that iucd had a higher percentage of use among studied sample (49%), regarding the effect dmpa was the most efficient contraceptive method (97.3%) with a lowest failure rate (2.6%), vaginal spermicide and the male condom showed the highest degree of adherence and compliance (100%). the male condom showed highest degree of subject's acceptability (69.9%), whereas dmpa showed lowest acceptability (21%). regarding gynecological side effects, dmpa showed the highest degree of menstrual irregularity (81.5%) and amenorrhea (65.7%). breakthrough bleeding, spotting and vaginal infection occurred in the highest percentage among iucd users (43.1%), (11.8%), (59.2%), respectively. central nervous system, gastrointestinal and dermatological adverse effects was higher in coc than dmpa users. the extent of weight gain was similar among dmpa and coc users (39.4%) and (39.8%), respectively. while hypertension was less among dmpa users in comparison with coc users. in conclusion, the most popular contraceptive methods used in hawler city was iucd, while dmpa was the most efficient contraceptive method in comparing with other methods. male condom and vaginal spermicidal had been shown the higher rate of adherence and compliance and dmpa showed the lowest failure rate in compare with other methods. male condom was the most acceptable method for contraception, followed by iucd, vaginal spermicides, cocs and dmpa respectively. the percentage of menstrual irregularity was highest among dmpa users followed by iucd users then cocs users, while iucd showed the highest percentage of vaginal infection followed by dmpa users and then cocs users, and finally in comparison of cns and gi and dermatological adverse effects of hormonal methods of contraception, cocs showed a higher percentage of occurrences of these adverse effects than dmpa. key words: contraception,coc, iucd. دراسة يقارنه تين انطرق انًختهفة نًوانع انحًم انًستعًهة في يذينة ارتيم رستى عثذ هللا اجالل * ، قاسى جهيم انشًاع **،1 انساو ناجي انحسني و * . * و١ٍح اٌص١ذٌح ، جاِؼح ١ٌٛ٘ش اٌطث١ح ، است١ً ، اٌؼشاق . و١ٍح اٌص١ذٌح ، جاِؼح تغذاد ، تغذاد ، اٌؼشاقفشع اٌص١ذٌح اٌسش٠ش٠ح ،** نخالصةا فٟ ِذ٠ٕح است١ً ٚاٌرٟ ذرضّٓ اٌٙذف ِٓ ٘زٖ اٌذساسح ٘ٛ اٌثذث ٚاٌّماسٔح ت١ٓ خّسح طشق ِخرٍفح ٌّٕغ اٌذًّ اٌّسرخذِح ٚلذ ذُ اٌرذمك ِٓ )دثٛب اٌذًّ ، ٚاٌذمٓ د٠ثِٛذسٚوسٟ تشٚجسرشْٚ اس١ر١د ، اٌٌٍٛة ، ِث١ذ إٌطاف اٌّٙثٍٟ ٚاٌٛالٟ اٌزوشٞ ( . ً ِٕغ اٌذًّ اٌجأث١ح إٌاجّح ػٓ وً ٚسائ ٚا٢ثاساٌفؼا١ٌح ٚاٌىفاءج ، ِؼذي ٔسثح اٌفشً ، ٔسثح االسرخذاَ ، االِرثاي ٚاالٌرضاَ ، ٘زٖ اال٘ذاف ، اجش٠د دساسح سجؼ١ح ٌّماسٔح ٘زٖ اٌطشق اٌخّسح فٟ ِذ٠ٕح است١ً فٟ خالي فرشج سرح إٌٝاٌّسرخذِح . ِٓ اجً اٌٛصٛي ذُ خالٌٙا جّغ اٌث١أاخ ِٚراتؼح إٌساء اٌٍٛاذٟ ذُ اخر١اس٘ٓ ٌٍّشاسوح 2200/ ا٠اس 01اٌٝ 2202ذشش٠ٓ اٌثأٟ / 22اشٙش ِٓ ذاس٠خ االٔجاب ذرشاٚح ٔساء ِرضٚجاخ فٟ سٓ 924 ٌجّغ إٌّاسثح اٌؼ١ٕاخ اخز اسرخذِد طش٠مح . ٚلذ اشٙش ٘زا اٌثذث ٌّذج ثالثح فٟ 1 corresponding author email :drkassim_alshamaa@yahoo.com received : 10/3/2012 accepted : 23/6/2012 iraqi j pharm sci, vol.21(2) 2012 various methods of contraception 57 اخزخ اٌؼ١ٕاخ ِٓ ٚدذج ذٕظ١ُ االسشج فٟ ِشوض صذٟ ٔاصادٞ فٟ است١ً . ٚذُ ذمس١ُ اٌؼ١ٕح ( سٕح . ٚ 94 – 01ِٓ ) اػّاس٘ٓ دثٛب ِٕغ اٌذًّ ( اِشأج ذسرخذِٓ 009( ذضُ ) iاٌّذسٚسح اٌٝ خّسح ِجّٛػاخ ٚفما ٌطش٠مح ِٕغ اٌذًّ اٌّسرخذِح : اٌّجّٛػح ) ( اِشأج 200( ذرىْٛ ِٓ ) iiiسٚوس١ثشٚج١سرشْٚ ، اٌّجّٛػح )( اِشأج ذسرؼٍّٓ اٌذمٓ د٠ثٛ ١ِذ 93( ذرضّٓ ) ii ، اٌّجّٛػح ) ( vاٌٍّٙثٟ ، ٚاٌّجّٛػح ) فإٌطا( اِشأج ذسرخذِٓ ِث١ذ 00( ذضُ ) ivذسرؼٍّٓ اٌٌٍٛة وٛس١ٍح ٌّٕغ اٌذًّ ، اٌّجّٛػح ) اخ اٌالصِح ٌٙزٖ اٌذساسح ِٓ خالي ( اِشأج ٠سرخذَ اصٚاجٙٓ اٌٛالٟ اٌزوشٞ وٛس١ٍح ٌّٕغ اٌذًّ . ٚلذ ذُ جّغ اٌث١أ 11ذرضّٓ ) اظٙشخ إٌرائج اٌذساسح اْ اٌٌٍٛة ٘ٛ االوثش اسرخذاِا ت١ٓ ػ١ٕح اٌّماتالخ اٌّثاششج ِغ إٌساء ِغ ذذ٠ٚٓ اٌّؼٍِٛاخ ػٍٝ اسرث١اْ . 9..4فؼا١ٌح ) .اِا تخصٛص اٌفؼا١ٌح فمذ اظٙشخ إٌرائج اْ اٌذمٓ د٠ث١ِٛذسٚوسٟ تشٚج١سرشْٚ ٟ٘ اٌطش٠مح اوثش %( 94اٌذساسح ) 022ٚظٙشخ اْ ِث١ذ إٌطاف اٌّٙثٍٟ ٚاٌٛالٟ اٌزوشٞ وأد ٌّٙا اػٍٝ ٔسثح ِٓ االٌرضاَ ٚاالِرثاي ) (2.1% ( ِغ الً ٔسثح اٌفشً ) %(، اِا اٌطش٠مح االدٔٝ لثٛال فمذ وأد اٌذمٓ د٠ث١ِٛذسٚوس14.4ٟ%( . ٚت١ٕد اٌذساسح اْ اٌٛالٟ اٌزوشٞ ٟ٘ اٌطش٠مح االوثش لثٛال ) % ( .20تشٚجسرشْٚ ) اِا تإٌسثح ٣ٌثاس اٌجأث١ح إٌسائ١ح ، فمذ اظٙشخ إٌرائج اْ ػذَ أرظاَ اٌطّث ٚأمطاػٙا ّ٘ا اوثش ش١ٛػا ت١ٓ ِسرخذِٟ اٌذمٓ ٌّٙثٍٟ فمذ % ( تاٌرٛاٌٟ . اِا تإٌسثح ٌٍٕضف اٌشذ٠ذ ٚإٌضف اٌّرمطغ ٚاٌؼذٜٚ ا ..11% ( ٚ ) 30.1د٠ثِٛذسٚوسٟ تشٚجسرشْٚ ) % ( ػٍٝ اٌرٛاٌٟ . اِا تإٌسثح ٌالثاس اٌجأث١ح 14.2% ( ٚ ) 00.3% ( ، ) 99.0ٚجذخ تٕسة اػٍٝ ت١ٓ ِسرخذِاخ اٌٌٍٛة ) اٌجٍذ٠ح ، فمذ ظٙشخ تٕسثح اػٍٝ ت١ٓ ِسرخذِاخ دثٛب ِٕغ اٌذًّ تاٌّماسٔح ِغ ٚاٌجٙاص اٌٙضّٟ ٚاٌرأث١شاخاٌّرؼٍمح تاٌجٙاص اٌؼصثٟ ١ذسٚوسٟ تشٚجسرشْٚ . دمٓ د٠ثِٛ فٟ اٌخراَ ، فمذ وأد ٔرائج ٘زا اٌثذث تشأْ طشق ِٕغ اٌذًّ اٌخّسح اٌّسرخذِح فٟ ِذ٠ٕح است١ً ِّاثٍح ٌرٍه اٌرٟ روشخ فٟ ج١ّغ ِٓ أذاء اٌؼاٌُ ٚاْ واْ ٕ٘ان فشق ٚاضخ فٟ ٔسة إٌرائج . ٠رضخ ِٓ إٌرائج اْ ٔسة اسرخذاَ طشق ِٕغ اٌذًّ ذخرٍف اخرالفا وث١شا ٘زا اْ وً ِجرّغ ٠سرخذَ اٌطش٠مح ا٤ٔسة ٌٗ ، تٍذ ٢خش ، وزٌه ٔسة اٌفشً ٚاٌىفاءج ٚا٢ثاس اٌضاسج إٌاجّح ػٕٙا . ٌزا ٔسررٕرج ِٓ رشاسن اٌّشأج ِغ اٌطث١ثح إٌسائ١ح ٥ٌخر١اس اٌطش٠مح إٌّاسثح الدر١اجاذٙا ، ٠ٕٚثغٟ اْ ذىْٛ إِٓح ، ِغ الً آثاس جأث١ح ِرادح ،سٍٙحذ سرخذاَ ِغ الً ذىٍفح .اال انكهًات انًفتاحية : ينع انحًم ، حثوب ينع انحًم ، انهونة نًنع انحًم . introduction family planning saves lives of women and children and improves the quality of life for all. it is one of the best investments that can be made to help ensure the health and wellbeing of women, children and communities (1) . it is defined as the voluntary use of methods and procedures intended to affect the number and timing of pregnancies, this include all the proximate determinants of fertility, including age of person at first sexual intercourse or marriage, post-partum lactation for spacing purposes and sterilization, which is widely used to influence the timing and number of births (2) .the world population is about 6.9 billion. at the predicted rate of growth, the population is projected to reach 7.5 billion by 2020 and more than 9 billion by 2050 (3) .various methods of contraception are used by both females and males. these include the following: periodic abstinence which includes (coitus interruptus, lactational amenorrhea and natural family planning) (4) . mechanical barriers include: male condom, female condom, diaphragm, cervical cap and spermicidal agents (5) . hormonal contraceptives is the most effective method of fertility control, but has major and minor side effects, especially for certain groups of women. major side effects including venous thromboembolic depression and neoplasms. minor side effects facial hyperpigmentation, acne and headache (6) . including nausea and vomiting, breakthrough bleeding, amenorrhea, weight gain, edema, disease and vascular disorders (cerebrovascular disease and myocardial infarction), cholestasis, hormonal contraceptives include: implants, injectable depomedroxyprogesterone acetate, progestinonly oral contraceptives, combination oral contraceptives, combination patch contraceptive and contraceptive vaginal ring (7) .another method is through inserting intrauterine devices (8) .sterilization either female sterilization by tubal ligation or male sterilization via vasectomy (9) .emergency postcoital contraception achieved by emergency contraceptive pills, the minipill emergency contraception method or copper t380 intrauterine device (10) .the effectiveness of any contraceptive method depend on its mechanism of action, availability(e.g., if a prescription required, cost),and acceptability (e.g. side effects, ease of use, religious and social beliefs).any or all of these reasons can account for the discrepancy between the lowest observed failure rate and the actual failure rate in typical users (11) . aims of the study 1. investigate and compare among five methods of contraception including (combined oral contraceptive pills, dmpa, iucd, spermicidal suppositories and male condom) used in hawler city. 2. estimate of : a. the percentage used of each contraceptive method. b. efficacy and relative failure rate of each contraceptive method. c. adherence and compliance with each contraceptive method. d. adverse effects and complications of each contraceptive method. iraqi j pharm sci, vol.21(2) 2012 various methods of contraception 58 subjects and methods a retrospective, descriptive, comparative study was conducted in hawler city over a period of 6 months from 22 th november, 2010 to 15 th may, 2011, during which data collection and subject follow up for three months have been achieved.a convenient sampling method was used to collect 429 married women in their reproductive age group 16-39 years old. the study population was collected from married women attending family planning unit in azadi health care center.four hundred twenty nine subjects were included in this study; they had been followed up for three months (follow up had been done for all subjects except subjects who discontinued the used method because of adverse effects) and mean had been taken for the parameters. subjects were utilized five different methods of contraception which were available in family planning unit of azadi health care center. group (i) women utilized combined oral contraceptives (microgynon® edfe, bayer schering pharma, germany)  number of subjects (113).  duration of use 2 month -17 years. group (ii): women utilized depot medroxy progesterone acetate (dmpa)  number of subjects (38)  duration of use 3 months-16 years. group (iii) women utilized copper intrauterine contraceptive device (iucd) (copper t® leivas, finland)  number of subjects (211).  duration of use 1 month6 years. group (iv) women utilized vaginal spermicide ( lorophame® vaginal suppository, ibn roshd industry, syria)  number of cases (11)  duration of use 2-12 months. group (v) husbands utilized male condoms ( xiangbin® condom, chaina)  number of subjects (56)  duration of use 2 months-13 years. methods of data collection it was by a direct interview with women attending family planning unit at azadi primary health care center using a questionnaire form designed by researcher herself to cover required data, a questionnaire was constructed after extensive review of relevant literature. results four hundred twenty nine subjects were participated in this study, they were divided to five groups according to the method of contraception used; 113 subjects (26.3%) used oral combined contraceptive pills (occp),38 individuals (8.8%) used dmpa, 211 individuals (49%) used iucd, 11 subjects (2.5%) used vaginal spermicides and 56 subjects (13%) her husbands used male condom. tables 1 to 6 showed the number of subjects and their percentage used the various contraceptive methods with their percentage of efficacy and failure rate, compliance and adverse effects and complication. table 1 : number of subjects (and their percentage) used different contraceptive methods regarding effect, adherence and compliance, failure and acceptability. methods of contraception (duration of use) effect adherence and compliance failure acceptability antibiotic use pill missed failure occp (2 month-17 y) 98 (86.7%) 99 (87.6%) 1 (0.8%) 10 (8.8%) 4 (3.5%) 25 (22%) dmpa (3 month-16y) 37 (97.3%) 28 (73.6%) 1 (2.6%) ---- 8 (21%) iucd (1 month-13y) 204 (96.6%) 199 (94.3%) 7 (3.3%) ---- 137 (64.9%) vaginal spermicide (2-12m) 10 (90.9%) 11 (100%) 1 (9%) ---- 3 (27.2%) male condom (2 month-13y) 52 (92.8%) 56 (100%) 4 (7.1%) ---- 39 (69.6%) iraqi j pharm sci, vol.21(2) 2012 various methods of contraception 59 table 2 : number of subjects (and their percentage) used different contraceptive methods showed gynaecological adverse effects. methods of contraception menstrual irregularity amenorrhoea btb spotting vaginal infection occp 12 (10.6%) 13 (11.5%) 9 (7.9%) 2 (1.7%) 31 (27.4%) dmpa 31 (81.5%) 25 (65.7%) 8 (21%) 4 (10.5%) 13 (34.2%) iucd 53 (25.1%) ―― ----- 91 (43.1%) 25 (11.8%) 125 (59.2%) table 3: number of subjects (and their percentage) used different contraceptive methods showed cns adverse effects. methods of contraception headache dizziness nervousness depression fatigue occp 48 (42.4%) 47 (41.5%) 50 (44.2%) 38 (33.6%) 15 (13.2%) dmpa 11 (28.9%) 7 (18.4%) 7 (18.4%) 7 (18.4%) 2 (5.2%) table 4 : number of subjects (and their percentage) used different contraceptive methods showed gi adverse effects methods of contraception nausea vomiting anorexia stomach pain anaemia occp 26 (23%) 6 (5.3%) 19 (16.8%) 15 (13.2%) 7 (6.1%) dmpa 7 (18.4%) 2 (5.2%) 2 (5.2%) ------- ------ 1 (2.6%) table 5 : number of subjects (and their percentage) used different contraceptive methods showed dermatological adverse effects methods of contraception skin rash skin pigmentation hair loss acne occp 3 (2.6%) 8 (7%) 24 (21.2%) 1 (0.8%) dmpa 1 (2.6%) 1 (2.6%) 4 (10.5%) ------- ------ table 6 : number of subjects (and their percentage) used different contraceptive methods showed other adverse effects methods of contraception weight gain hyper tension oedema stroke breast pain breast tenderness occp 45 (39.8%) 5 (4.4%) 1 (0.8%) 2 (1.7%) 6 (5.3%) 2 (1.7%) dmpa 15 (39.4%) 1 (2.4%) 3 (7.8%) --------- --------- ---------- ---------- --------- ---------- iraqi j pharm sci, vol.21(2) 2012 various methods of contraception 60 discussion family planning allows individuals and couples to anticipate and attained their desired number of children and spacing and timing of their births. it is achieved through use of contraceptive methods and treatment of involuntary infertility. a woman’s ability to space and limit her pregnancies has a direct impact on her health and well-being as well as the outcome of each pregnancy (1).this comparative study was conducted in azadi health care center in hawler city. (429) subjects were included in this study, with varying percentage of five contraceptive methods used. (26.3%) of subjects were used occp, (8.8%) of them used dmpa, (49%) of subjects used iucd, (2.5%) used vaginal spermicides and (13%) used male condoms. these percentages revealed that approximately half of subjects were used iucd, whereas the other half were used occp, male condoms, dmpa, vaginal spermicides in a descending order.our results regarding rate of usage is compatible with other studies done in usa, nigeria and jordan (12,13,14) in which most of women used iucd as a contraceptive method. similar results were reported in a study done by falh et al in 2000 (15) in baghdad, wasset and najaf, were the percentage of iucd users was 35% in baghdad, 30% in wasset and 35% in najaf, while the percentage of users of occp in baghdad was 15%, in wasset 25% and in najaf 21.5%. the study showed that the efficacy of occp was 86.7% with a failure rate of 13.3%. this high failure rate is due to lack of adherence and compliance in pill users, 0.8% failure rate occurred in typical pill users, where as 8.8% failure rate occurred due to missing pill, 3.5% failure rate occurred due to concurrent use of antibiotic with occp. in a study on occp users, they showed failure rate 7.5% within 12 month of use (16). about dmpa, the results of this study showed that , it has efficiency about 97.3% with failure rate of 2.6%. these findings was compatible with the failure rate of study done by (17) in which failure rate was 0.3% in perfect users and 3% in typical users.the efficacy and failure rate of iucd is 96.6% and 3.3% , respectively. these findings is compatible with two studies, first was done in india in which the failure rate had been observed among copper iucd users (up to 200 subjects) had about 3% pregnancies (18), in the second study , the failure rate was 3.2% after twelve month of follow up (16).the high failure rate in our study possibly was due to lack of adherence and compliance among iucd users even holding heavy things. in addition failure rate might be affected by technical aspects. another reason for this high failure rate, is that occurred in women under 25 year who were more fertile than older women.regarding male condoms initialized in this study, the efficacy and failure rate were 92.8% and 7.1% , respectively. the results of this study are consistent with the study done by others (19,20), in which failure rate was 3-14% in typical user. about the results regarding vaginal spermicides the data obtain from this study showed that efficacy and the failure rate was 90.9% and 9% , respectively, although there are no reliable data on the effectiveness for pregnancy prevention of spermicide used alone. some commonly quoted figures for correct and consistent use and for typical use were 6% and 26%, respectively (19,20). regarding adherence and compliance, the data obtained from this study showed that in combined oral pills is 87.6%, in dmpa 73.6%, in iucd 94.3%, in vaginal spermicides 100% and in male condom is 100%. in a study done on compliance among dmpa and occp users after 6 months of follow up, the results were 78% and 46%, respectively (21). the high rate of adherence and compliance among vaginal spermicide and male condom users reflects the high effectiveness and low failure rate with minimal side effects among these two groups. on the other hand, the failure rate among pill users reflects low adherence and compliance among pill users.gynecological adverse effects which has been recorded in this work include menstrual irregularity, amenorrhea, btb, spotting and vaginal infection. using occp, dnpa and iucd. different percentages had been obtained from these gynecological adverse effects. most of them were compatible with other studies (22,23,24,25).incidence of cns adverse effects ( headache, dizziness, nervousness, depression and fatigue) among hormonal contraceptive method users were also reported. again different percentages of these adverse effects were recorded and most of them were compatible with other studies (26,27,28). regarding gi adverse effects, it has been reported among pill and dmpa users, incidence of nausea was 23% in occp users. this rate is lower than that seen in a study done by beatris et al, 2002 (27) which was 47.5%. occurrence of nausea among dmpa users in this study was about 18.4%, this rate is higher than that seen in other studies which done by (david, 2010) 28 which was 1-10%, the result that reported by clinical trials of fda which was 3.3% and also from that recorded in nigeria which was 1.2% (29).regarding dermatological adverse effects which have been occurred among hormonal contraceptive iraqi j pharm sci, vol.21(2) 2012 various methods of contraception 61 method users. hair loss had been occurred in a higher percentage in occp than dmpa users 21.2% and 10.5%, respectively. the rate of hair loss among dmpa users in this study is compatible with studies done by david, 2010 (28) in which hair loss occurred in a rate of 1-10% and 10% respectively.skin pigmentation is another finding which has been reported between hormonal contraceptive method users in a rate of 7% and 6% in occp and dmpa users respectively. skin pigmentation had been reported by katzung, 2007 (30) in a rate of 5% within first year and 40% after 8 years among pill users.there are other adverse effects which were reported among hormonal contraceptive users in the studied sample including weight gain in a rate of 39.8% and 39.4% among occp and dmpa users, respectively. a study was done by reubinoff et al, 1995 (31) initialize occp weight gain has been occurred by about 30.6% which is approximately near from our results.hypertension incidence among hormonal contraceptive users was about 4.4% in occp and 2.4% among dmpa users. a study done by baird, 1993 (32) reported hypertension by 4-5% in cocs users. regarding breast pain which has been reported among pill users was 5.3% which is much lower than that seen in study done by beatris et al in 2002 (28) which was about 70%.breast tenderness was another finding which has been reported from the results of current study in a rate of 1.7% in occp users. breast tenderness had been reported by ruggerio in 1997 (33) .in conclusion, the most popular contraceptive methods used in hawler city was iucd, while dmpa was the most efficient contraceptive method in comparing with other methods. male condom and vaginal spermicidal had been shown the higher rate of adherence and compliance and dmpa showed the lowest failure rate in compare with other methods. male condom was the most acceptable method for contraception, followed by iucd, vaginal spermicides, cocs and dmpa respectively. the percentage of menstrual irregularity was highest among dmpa users followed by iucd users then cocs users, while iucd showed the highest percentage of vaginal infection followed by dmpa users and then cocs users, and finally in comparison of cns and gi and dermatological adverse effects of hormonal methods of contraception, cocs showed a higher 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postcoital contraception. am. fam. physician ;62(10):2287-2292 11. koda-kimble ma, young ll, kradjan wa, guglielmo bj et al (2006). applied therapeutics: the clinical use of drugs. 8 th ed. baltimore: lippincott williams & wilkins;45(1)-45(26). 12. 12.wasileh ip (1997). wives view of their husbands role in family planning: some indications from the jordan fertility and family health survey. dirast medical and biological sciences; 24(2):187-200. 13. ozumba bc, ibekwe pc (2001). contraceptive use at the family planning clinics of the university of nigeria teaching hospital, enugu. nigeria public health; 115(1):51-53. http://www.who.int/ http://www.census.gov/ipc/www/world%20pop.html http://www.census.gov/ipc/www/world%20pop.html http://books.google.com/?id=_mrdimrelcic&dq=hormonal+contragestion iraqi j pharm sci, vol.21(2) 2012 various methods of contraception 62 14. margulies r and miller l (2001). increased depotmedroxyprogestrone acetate use incease family planning program 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[online]. vol.iv. available from: www.medicc.org [accessed on 27 th april2011]. 28. david d (2010). contraception. available from: www.netdoctor.co.uk [accessed on 20 th march-2011]. 29. abasiattai ak, udoma ej,ukeme e (2010). depot medroxyprogesterone injectable contraception at the university of uyo teaching hospital, uyo. ann afr med. ;9 (2):81-85. 30. katzung bg (2007). basic and clinical pharmacology. 10 th ed. singapore: mcgraw-hill; 664-671 31. reubinoff be, grubstein a, meirow d et al. effect of low-dose estrogen oral contraceptives on weight, body composition and fat distiribution in young women. fertile. steril; 63:516. 32. baird dt and glasier a (1993). drug therapy: hormonal contraception. n. engl. j. med;328:1543-1549. 33. ruggerio rj (1997). contraception. in: young ly, koda-kimble ma, eds. applied therapeutics: the clinical use of drugs. vancouver: applied therapeutics, inc; 43-1-43-19. http://www.medicc.org/ http://www.netdoctor.co.uk/ http://lib.bioinfo.pl/auth:abasiattai,am http://lib.bioinfo.pl/auth:udoma,ej http://lib.bioinfo.pl/auth:ukeme,e http://lib.bioinfo.pl/pmid/journal/ann%20afr%20med http://lib.bioinfo.pl/pmid/journal/ann%20afr%20med iraqi j pharm sci, vol.20(2) 2011 electrophoresis of serum protein and kala-azar 28 poly acrylamide gel electrophoresis of serum protein, application to kala-azar patients nada m.t. al-bashir* ,1 *leishmania unit, medical research centre, al-nahrain university, baghdad,iraq. abstract sera samples were collected from 60 children aged 4-60 months, all were clinically and serologically proven cases of visceral leishmaniasis, as well as from 10 healthy children, all were seronegative with no history of parasitic infection who serve as a control during the study. serum total protein and albumin were measured and compared between the control and visceral leishmaniasis patients. serum protein profiles have been investigated using the conventional sodium dodecyl sulphate – polyacrylamide gel electrophoresis (sds-page). serum of control group showed the specific protein pattern with five protein bands, while serum protein profile in visceral leishmaniasis patients revealed three groups of electrophoretic banding patterns. , 50% showed twelve bands, 36.66% of the patients showed nine bands and 13.33% showed ten bands. at least four of these bands were found to be common among the infected groups which may be of diagnostic value and required further investigations. the three different electrophoretic patterns groups may be correlated with the previous epidemiological observations in respect of different clinical presentation of the disease and different response the chemotherapeutic agents at many endemic areas around the world. key words: protein band, protein patterns, serum protein, sodium dodecyl sulphate-poly acrylamide gel electrophoresis (sds-page), visceral leishmaniasis. الخالصة عْج ًِ ٍْ انًصمعُٛاث ُج انهشًاَٛاث بذاءاالصابّ يُاعٛاسزٚزٚاً ٔانحاالث اثبخج ز، ُكمّ شٓ 6ٓ-4بعًِز عزاقٙ طفمِ 6ِٓي ٍْ االحشائّٛ اسخخذيج ْذِ انًجًٕعّ اخز، طفٛهٙ عذٖٔ يزضبذٌٔ حأرٚخِ ٔ سانبّ يُاعٛا َخائجٓى ، َكاَجْ اصحاءأطفاِل ٓٔ، ِٔي حى انخحز٘ عٍ. ٔحًج انًقارَّ بٍٛ انًجًٕعخٍٛ انًصابٍٛ ٔاالصحاء يصم فٙ زاللانٔ انكهٙ بزٔحٍٛان حى قٛاسسٛطزة أثُاء انذراسِت.ك يصُم انًجًٕعت انقٛاسِٛت ًَطَ اظٓز .sds -حقُٛت انخزحٛم انكٓزبائٙ انخقهٛذ٘ )ْالو يخعذد االكزٚم اياٚذ إسخعًالب انذو يصمِ اثبزٔحُِٛ ٍِ األًَاط ٔقذ كاَج كًا يخخهفتثالثت يجًٕعاِث انهشًاَٛا االحشائّٛ ٔجٕديصِم يزضٗ اظٓز، بًُٛا االعخٛاد٘ بخًست حزو انبزٔحٛ عهٗ األقمكشفج ْذِ انذراسّ كًا. حزوعْشزة اظٓز% 6.66ٔٔ% اظٓز حسعت حزو 66.66ٔ حزيّإثُخا عْشزة اظٓز% 5ٓ ٚهٙ: ٍْ ْذِ اربعّ ٔجٕد ًَُصابتِ شائعّ انحزوِي حشخٛص انًزض يًا ٚسخذعٙ اجزاء فٙ االًّْٛ نٓذِ انحزو قَْذ حَُكٌٕ ٔ .بٍٛ انًجًٕعاِث ان ْذِ . انبزٔحُٛاث انًشخزكّ ضًٍ انًجايٛع انًخخهفّ ْذِ نهكشف عٍ َٕعّٛ ٔانخحقٛقاِث األخزٖ انًطهٕبتِ دراساث أسع ٔاعًق فًٛا ٔخاصت فٙ انًُاطق انخٙ ٚكٌٕ انًزض فٛٓا يخٕطُا انسابقت انٕبائّٛ انذراساثقَْذ حُْزبَطُ ب انًجايٛع انثالثّ يٍ االًَاط انبزٔحُّٛٛ ٍْ انًزِض ٔاال ّهفِ انًخخ ّانسزٚزِٚ بانعٕارضٚخعهق .ّ نهعالج فٙ ْذِ انًُاطقانًخخهفِ سخجابِّي introduction leishmaniasis forms a whole group of parasitic tropical diseases which spread by the bites of many different species of sand fly producing at least 5 distinct diseases with different symptoms (1) . visceral leishmaniasis (vl) or (kala-azar) is one of the most serious forms, it is life threatening, causing very high mortality rates unless treated (2) , and even with the treatment mortality can be from 5-15% and even higher if treatment is delayed (3) .vl is a systemic disease of the mononuclear pathogenic system. the reticuoendothelial hyperplasia that follows infection with leishmania donovani affects the spleen, liver, mucosa of the small intestine, bone marrow, lymph nodes and other lymphoid tissues (4,5) . hypoalbuminaemia is associated with oedema and other features of malnutrition (6) . hyperglobulinaemia mainly polyclonal immunoglobulin g (igg) is common but does not seem to have a clear pathological role in vl (7) .complement activation may contribute to anaemia and immune complexes are formed, but nephritis is rare (8) . secretory igm as well as igg (specific and non-specific) were found to contribute to disease susceptibility and appear to act in part through the activation of complement a+nd generation of c5a (9) . these findings extend earlier murine vl studies (10) and indicate that a polyclonal b cell response is an early and intrinsic feature of vl, which helps to establish and maintain infection in the mammalian host (11) . the presence of high titers of circulating immunocomplexes has been reported in patients with visceral leishmaniasis (10,12) . 1 corresponding author e-mail : maha_janabi@yahoo.com received : 17/10/2010 accepted : 27/7/2011 mailto:maha_janabi@yahoo.com iraqi j pharm sci, vol.20(2) 2011 electrophoresis of serum protein and kala-azar 29 signs of activation of the complement cascade were observed in a study of patients with visceral leishmaniasis and, in 43.4% of the cases, it was associated with a simultaneous reduction in the levels of c3 and c4, along with changes in ch100, suggesting activation of the classical pathway (13) . on the other hand, a reduction in c3 levels in patients with visceral leishmaniasis was observed in other study, suggesting activation of the alternative pathway (14,15) corroborating those data, stebut (16) observed activation of the complement system in experimental models of leishmaniasis, especially by opsonization of the surface of the parasite with c3b. studies on serum protein in vl patients indicated that the total serum proteins dropped below normal limits in 80% of the cases (8) . the main decrease being observed in albumin fraction which dropped to about half of the total control values, a moderate increase in globulin fraction and an increase in the relative concentration of -globulin were also noted (7) . the aim of this study is to investigate the changes in serum protein fraction of patients with vl. patients and method this study was divided into two groups: 1. patients: including 60 children aged 4-60 months, admitted to paediatric ward of alkadhimiyah teaching hospital-alnahrain university/ baghdad, from december 2006 febriwary 2009. all were proven cases of kala-azar by: clinical manifestation of fever, hepatospleenomegally, and the demonstration of the parasite the amastigotes – from direct smear of bone marrow and serological diagnosis by indirect immunofluorescence antibody test ifat. the diagnosis was under the supervision of dr. shatha hussain (paediatrician) and dr. rabab alsa'adi (dermatologist). 2. control: consist from 10 healthy children all were seronegative. blood samples two milliliters of venous blood were collected from patients and control in plain plastic tubes and the serum was obtained by centrifugation at 4 o c, 400  g for 10 minutes and used freshly for electrophoresis. total serum protein, albumin was measured for each sample according to biurate (17) , wooten (18) respectively. the protein electrophoresis was dependent on the conventional polyacrylamid gel electrophoresis page as described by fehrnstrom and morberg (19) . electrophoresis of serum protein the process of electrophoresis was carried out as described by fehrnstrom and morberg (19) .serum samples were transported on a horizontal slab of poly acrylamide gel of (7.5% w/v) . electrophoresis: the multiphor cooling plate was thermo stated to not more than 10ºc. pre-electrophoresis was performed at 50 ma for 30 minutes. then, samples were applied as (9µl sample +1µl bromophenol blue), then concentrated for 5-10 minutes with a current of 20 ma. the voltage was adjusted between 350-400 volts and the current was around 50 ma. the electrophoresis time of each run was about 4 hours. after running of the electrophoresis process, coomaise brilliant blue (0.25% w/v) was used for staining of the bands. visualization of the bands was carried out after destaining by (ethanol+acetic acid+distilled water) at (3:1:6) respectively. migration distance of the calibration proteins were measured after staining the protein bands, also relative mobility (rm) was measured according to the following equation: the calibration curve was constructed by plotting relative mobilities (rms) versus logarithmic molecular weights for calibration proteins figure 1. the molecular weight of proteins of interest was determined from the position of its rm value on the calibration curve. figure 1 : standard curve for approximate estimation of molecular weights of various proteins in serum samples. iraqi j pharm sci, vol.20(2) 2011 electrophoresis of serum protein and kala-azar 30 results total protein serum total proteins were found to be elevated in 30 patients (50% of the samples) compared with the control, while the remaining 30 (50%) were found to be within normal range . on the other hand, serum albumin was found to be markedly reduced in 58 patients (96.66% of the samples) compared with the control, except two patients (3.33%) were found to be within normal range table 1. table 1: serum total protein and albumin of normal and patients with kala-azar. protein electrophoresis (serum protein profile) conventional polyacrylamide gel electrophoresis has been used to differentiate between protein patterns in serum of normal and patients with kala-azar (20) . serum of control group has a specific protein pattern with five protein bands (albumin, 1, 2, 1, ), the relative mobility values (rm) have been ranged from (0.015-0.72) table 2 , figures 2 and 3 . serum protein profile in kala-azar patients revealed three banding patterns (9-12) protein bands the (rm) values were ranged from (0.01-0.9). these differences in the number of bands and their molecular weight may be attributed to the difference in the clinical picture and severity of the disease figures 2 and 3.the first protein pattern demonstrates nine bands the rm values were ranged from (0.03-0.8), seven bands of these where different from the normal. these are bands number (1, 2, 3, 4, 6, 7, 8 and 9) with molecular weights (912.010, 707.945, 562.341, 501.187, 251.188, 158.489, 104.712, and 79.452) ×10 3 dalton respectively table 2 , figure 3. this band picture was found in 22 patients (36.66 %) table 3 .the second protein pattern shows twelve bands; the rm values were ranged from (0.01-0.7), of which nine where abnormal in particular, bands number (2, 3, 4, 5, 6, 9, 10,11 and 12) with a molecular weights (912.010, 891.250, 794.328, 562.341, 501.187, 251.188, 223.872, 112.201, 97.723) ×10 3 dalton respectively table 2 , figure 3. this profile was found in 30 patients (50 %) table 3.the third protein pattern shows ten bands; the rm values were ranged from (0.070.78), eight out of these were abnormal. these are bands number (1, 2, 3, 6, 7, 8, 9 and 10) with a molecular weights (794.328, 562.341, 501.177, 251.188, 199.526, 158.489, 97.723 and 70.794) ×10 3 dalton respectively table 2, figure 3. this profile was found in 8 patients (13.33%) table 3. table 2 : rm values and molecular weight for each band in different protein profile groups. band no. control rm control mw×10 3 dalton g1rm g1mw×10 3 dalton g2rm g2mw×10 3 dalton g3rm g3mw×10 3 dalton 1 0.01 1047.128 0.03 912.010 0.01 1047.128 0.07 794.328 2 0.1 707.945 0.12 707.945 0.03 912.010 0.2 562.341 3 0.35 331.131 0.2 562.341 0.05 891.250 0.26 501.177 4 0.41 275.422 0.26 501.187 0.07 794.328 0.35 331.131 5 0.72 104.712 0.41 275.422 0.2 562.341 0.41 275.422 6 0.47 251.188 0.26 501.187 0.47 251.188 7 0.6 158.489 0.35 331.131 0.54 199.526 8 0.72 104.712 0.41 275.422 0.6 158.489 9 0.8 79.452 0.47 251.188 0.74 97.723 10 0.49 223.872 0.78 70.794 11 0.6 158.489 12 0.74 97.723 groups number of samples total protein g/dl±sd % of high levels total protein albumin g/dl±sd % of high levels albumin control 10 5.3±1.2 ---5.2±0.12 -- patients 60 10.2±0.5 50 3.5±0.62 58 iraqi j pharm sci, vol.20(2) 2011 electrophoresis of serum protein and kala-azar 31 table 3 : different protein profiles with the percentage of patients in each group group total no. bands no. abnormal bands no.patients % patient 1 9 7 22 36.66 2 12 9 30 50 3 10 8 8 13.33 control group 1 group 2 group3 figure 2 : electrophoretic patterns obtained from kala-azar patients sera and control non infected persons figure 3 : zymograph of different banding patterns of serum protein electrophoresis obtained from kala-azar patients. discussion visceral leishmaniasis is a potentially fatal disease caused by an intracellular protozoan parasite of the leishmania donovani complex (21) . for successful control of the disease, efficient and reliable diagnosis is recommended (22) . clinical presentations of spleenomegally (23) , hepatomegally, fever, pallor (24) , demonstration of the parasites in bone marrow aspirates or needle biopsy specimens of the spleen and lymph node or by in vitro cultivation are the definitive methods of diagnosis of visceral leishmaniasis (25) .however, these methods are insufficiently sensitive, and the techniques are invasive, painful, and even hazardous (26) and need skilled personnel and equipped hospitals. moreover, visceral leishmaniasis is occurring in places where health services are poorly developed. poor socioeconomic conditions are associated with a higher risk of infection (22) . a number of serological tests have been developed and evaluated for the diagnosis of visceral leishmaniasis, including immunoflourescent-antibody tests ifat (27) , enzyme linked immunosorbent assay (elisa) (28) , dot elisa (29) , immunoblot analysis (30) , and direct agglutination test (dat) (31) .since these methods are some what complicated and required highly efficient personnel and very well equipped laboratories to be performed, making the need for easier and faster methods mandatory especially in endemic areas.the presence of leishmania donovani soluble antigen(s), corresponding antibody and complement components in the serum of patient with active kala-azar have been shown by durate et al. (32) in human and in cotton rats infected with leishmania donovani (33) .sera of normal subjects (control) showed the typical five bands. while sera of the diseased persons showed a three protein patterns, each pattern differ from the control in the number of abnormal bands -see the above results, figure2.the production of different protein bands with variable molecular weights in patients with active kala-azar compared with normal (control) subjects may be attributable to the escalation of the host defense mechanism(s) against leishmania parasites. higher levels of all igg subclasses 1,2,3,4, as well as igm have been reported during kalaazar infection (34,35) .furthermore, the presence of three different protein profiles in this study among the tested patients with active kala-azar may be related to different clinical presentation in young children with kala-azar like the duration of fever, spleenomegally, hepatomegally, jaundice and response to therapy in iraq (36, 37, 38) and other endemic countries like sudan (39) , india (40) and iran (41) . on the other hand, all the three groups showed similarity at rm values of 0.2 and 0.26, o.47 and 0.6 with a mw=(562.341, 501.187, 251.188, 158.489)×10 3 daltons respectively. group 1 and 2 showed similarity at rm 0.03 g1 g2 g3 iraqi j pharm sci, vol.20(2) 2011 electrophoresis of serum protein and kala-azar 32 with a mw 912.010×10 3 daltons. while group 2 and 3 showed similarity at rm 0.07 and 0.74 with a mw (794.328 and 97.723) ×10 3 daltons. recommendations these similarities among the different groups may require further investigation in the search of a certain protein band that might be used as a marker for this disease. in addition, further studies may be required to identify the similarity in the bands between two different protein profile groups that may be attributable to the difference in the clinical presentation of the disease. references 1. who. the world health report 197-97. 2002. http://www.who.int.htm/. 2. 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http://www.ncbi.nlm.nih.gov/pubmed?term=%22zijlstra%20ee%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22ali%20ms%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22el-hassan%20am%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22el-toum%20ia%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22el-toum%20ia%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22satti%20m%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract http://www.ncbi.nlm.nih.gov/pubmed?term=%22ghalib%20hw%22%5bauthor%5d&itool=entrezsystem2.pentrez.pubmed.pubmed_resultspanel.pubmed_rvabstract javascript:al_get(this,%20'jour',%20'trans%20r%20soc%20trop%20med%20hyg.'); javascript:al_get(this,%20'jour',%20'trans%20r%20soc%20trop%20med%20hyg.'); javascript:al_get(this,%20'jour',%20'trans%20r%20soc%20trop%20med%20hyg.'); iraqi j pharm sci, vol.20(2) 2011 postoperative wound infections 59 postoperative wound infections and the antimicrobial susceptibility in baghdad hospitals # maysoon a. merdaw* ,1 *department of clinical laboratory science ,college of pharmacy,baghdad university,baghdad, iraq. abstract nosocomial infections are one of the most important causes of mortality and morbidity in hospitals. these are major public health problems worldwide, but particularly in developing countries. the purpose of this research was to analyze the frequency of the microorganisms in the specimens taken from the surgical wounds, and to examine antimicrobial susceptibility for some isolates . wound swabs were examined from june 2010 to january 2011. the isolates were identified by conventional methods, antimicrobial susceptibility testing was performed by kirby-bauer disc diffusion method as per nccls guidelines.a total of 102 wound swabs were examined 22(21.56%) swabs were sterile and 80(78.43%) were positive for microorganisms. the results showed 27.2% positive for pseudomonas aeruginosa, 25.0% positive for coagulase positive staphylococci, 20.0% positive for enterococcus spp., 17.5% positive for escherichia coli,15.0% positive for klebsiella pneumonia,13.7% for proteus mirabilis,and10.0% for acinetobacter baumannii. antimicrobial susceptibility testing showed that the rate of isolates of imipenem resistance pseudomonas aeruginosa(irpa) were 3.7% , 11.2% positive for vancomycin resistance enterococci(vre) ,13.7% positive for both methicillin resistance staphylococcus aureus(mrsa)and vancomycin resistance staphylococcus aureus(vrsa) ,and 11.2% positive for vancomycin intermediate staphylococcus aureus(visa). we found that postoperative wound infections increase with pre and post operative hospitalization that's mean the infections can be decrease by shortening the hospitalization time.our results appear to be maintained with strategies for preventing nosocomial infection,permanent education, strong application of protocols and urging the implementation of strict infection control policy. key words: nosocomial infection, surgical wound, antimicrobial susceptibility. الخالصة ْٔي يٍ انًشاكم انظحيح في األطاتاخ انًرعهمح تانًسرشفياخ ٔاحدج يٍ األسثاب انًًٓح نهًٕخ ٔاأليساضيح في انًسرشفياخ , انُاييح . انغسع يٍ ْرا انثحس ْٕ يعسفح األحياء انًجٓسيح في انًسحاخ انًأخٕذج يٍ جسٔح كم اَحاء انعانى ٔتاألخض في انثهداٌ انى كإٌَ انصاَي 0202ٔفحض انماتهيح انضد يايكسٔتيح نعدد يٍ انعزالخ. ذى جًع ٔفحض انًسحاخ نهفرسج يٍ حزيساٌ انعًهياخ , ٔجد يٍ )طسيمح األَرشاز تاسُعًال األلساص(. تأز -فكاٌ تطسيمح كيستيأيا فحض انماتهيح انضد يايكسٔتيح تانطسق انرمهيديح , 0200 كًا أظٓسخ انُرائج )يهٕشح( , %( يسحح يٕجثح30,87)02 )غيس يهٕشح( ٔ %( يسحح يعمًح:00,9)00, يسحح 020يجًٕع % coagulase positive staph. ,02,2% نثكرسيا pseudomonas aeruginosa ,09,2% يسحح يٕجثح نثكرسيا 03,0 % klebsiella pneumonia ,07.3% نثكرسيا escherichia coli,09.2نثكرسيا enterococcus spp. ,03,9%نثكرسيا انماتهيح انضد يايكسٔتيح أظٓسخ َسة يٍ انعزالخ . acinetobacter baumannii% نثكرسيا proteus mirabilis ٔ02.2نثكرسيا % يٍ انعزالخ 00,0, ٔ %7,3ٔكاَد َسثرٓا pseudomonas aeruginosa (irpa)ايٍ تكرسي imipenemانًمأيح نهًضاد mrsa(methicillin% يٕجثح نكم يٍ تكرسيا enterococcus ٔ07,3 (vre)يٍ تكرسيا vancomycinانًمأيح نهًضاد resistance staphylococcus aureus) ٔتكرسيا vrsa (vancomycin resistance staphylococcus aureus) ٔ . اسرُرجُا يٍ تحصُا أٌ انرهٕز نجسٔح visa(vancomycin intermediate staphylococcus aureus) % نثكرسيا 00,0 ,ْٔرا يعُي أَّ يًكٍ ذمهيم َسثح األطاتاخ ترمهيم ) لثم ٔتعد اجساء انعًهياخ( ياتعد انعًهياخ يزداد تزيادج يدج انثماء في انًسرشفياخ طاتاخ انًسرشفياخ يسرٕجة انرصميف اندائى ٔانرطثيك انظازو نسياساخ انسيطسج عهى اطاتاخ أْرِ انًدج , نرا فأَّ نهحًايح يٍ انًسرشفياخ. introduction despite recent advances in the operative techniques and better understanding of the pathogenesis of the wound infections, postoperative wound infections continues to be a major source of morbidity and mortality for patients undergoing operative procedures. surgical site infections (ssis), as they are called today, accounted for 16% of all hospital-acquired infections, making them the third most frequent type of nosocomial infections in developed countries. the rates were higher in developing countries, and vary from 7% to 40% (1,2) . # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : maysoonmerdaw@yahoo.com received : 6/2/2011 accepted: 10/5/2011 iraqi j pharm sci, vol.20(2) 2011 postoperative wound infections 60 the organisms causing most nosocomial infections usually come from the patient’s own body (endogenous flora). they also can come from contact with staff (cross-contamination), contaminated instruments and needles, and the environment (exogenous flora). because patients are highly mobile and hospitalizations are becoming shorter, patients often are discharged before the infection becomes apparent. in fact, a large portion of nosocomial infections in hospitalized patients—and all from ambulatory care facilities—become apparent only after the patients are discharged. as a consequence, it is often difficult to determine whether the source of the organism causing the infection is endogenous or exogenous (3) ,it is very useful to explore the causes of these infections in order to timely detect and remove the causative agents (4,5) , with plentiful using of invasive technologies, severe and fatal nosocomial infections cause many damages every day (6) .these infections also contribute greatly to the economic costs of surgical procedures and the estimated range is 1.47-19.1 billion euro. this is a great problem, especially in resource of poor countries (7) , therefore ,knowledge about the frequency and distribution of nosocomial infections is important to improve infection control measures as well as to develop curative strategies which, in turn, will help us in decreasing incidence associated treatment cost (8) .in order to minimize the postoperative wound infections, it is important to create a safe environment by controlling four main sources of infection i.e. personnel, equipment , the environment and patient’s risk factors (9) .there are some indicators of the effects of infection control, most mrsa are hospital acquired and so this organism is a useful indicator. mrsa do not generally appear to be more virulent than sensitive strains but, because of their resistance patterns, they are more difficult to treat if infection occurs (10,11) .the rapid increase in enterococcal strains resistant to vancomycin (vre) and other antibiotics and their ability to pass this trait on to other pathogens, i.e. staphylococcus aureus, indicates an urgent and expanding clinical problem, visa stands for s.aureus with intermediate resistance to vancomycin. vrsa stands for s. aureus with complete resistance to vancomycin. it is probable that s.aureus bacteria with intermediate or complete resistance to vancomycin would be resistant to most antibiotics commonly used for staphylococcal infections (12) . pseudomonas aeruginosa is an important nosocomial pathogen with its ability to propagate on medical devices, hospital environment and even in disinfectants. it causes high morbidity and mortality in the services of oncology, hematology, surgery, burn and intensive care units (13,14) . the purpose of this research was to :  know the incidence rate of nosocomial infection in surgical wards.  study some factors influencing nosocomial infections in surgical wards.  know the commonest organisms causing nosocomial infections.  develop an effective antibiotic policy to deal with the common nosocomial infection. materials and methods sampling the study was carried out on 102 patients admitted in the surgical wards of 4 hospitals in baghdad city between june 2010 and january 2011.the age of patients was ranging from 12-79 years . gender structure of patients was 54.90 % males and 45.09 % females. surgical site infection was classified according to preoperative and post-operative hospitalization, type of the ward ,and types of bacterial organisms in each ward with examine the sensitivity of three isolated organisms .all laboratory testings were performed in the kadhimyia teaching hospital . media the media used for cultures were: blood agar plates (containing blood agar base and 5% of human blood ) were used to facilitate the growth of fastidious microorganisms, particularly gram positive bacteria . mac conkey agar was used for selective isolation of enterobacteriacae. the plates were incubated for20-24 hrs. at 37c in bacteriological incubators. all isolates were identified by conventional biochemical testing according to the medical laboratory manual 2004. antimicrobial susceptibility testing was prepared by kirby-bauer disc diffusion method (15) on muller-hinton agar ( difco), the following antibiotics were tested : methicillin , vancomycin, imipenem, gentamicin, trimethoprim, oxacillin, penicillin, piperacillin. iraqi j pharm sci, vol.20(2) 2011 postoperative wound infections 61 results and discussion nosocomial infections are a significant problem throughout the world and are increasing (16) . for example, nosocomial infection rates range from as low as 1% in a few countries in europe and the americas to more than 40% in parts of asia, latin america and sub-saharan africa (17) .the incidence of postoperative infections with bacterial growth was 78.43% from the 102 patients admitted and examined in our study, samples of wound swabs were examined: 80(78.43%) were positive and 22(21.56%) were negative ( figure 1) , it was similar to the result of maida sisirak et al which was 83.7% in bosnia (1) , and it was higher than incidence rates reported from developed countries in western europe, such as the united kingdom (3.1%) and the netherlands (4.3%) (18) . in pakistan infection rate was 22.7% ( 19) , while in nigeria 38.8% had surgical site infections (20) , figure 1: review of examination samples and it was relatively higher in patients aged 12-29 years(88.2%), and in males 48(47.05%) than females 32(31.37%) (table 1), it might be that most male patients had lower health care behaviors than female patients ,and this evidence supported the findings of previous studies (21,22) . these factors were not significant by multivariate analysis, the results supported the findings of previous studies, especially the studied variable as age. (23,24) table 1: demographic data of patients with nosocomial infection (n=102) age(yrs) examined samples no. % incidence of infection no. % no. % incidence of infection no. % 12-29 34 33.3% 30 88.2% (male) 56 (54.90%) 48 (47.05%) 3039 23 22.5% 16 69.5% 4049 19 18.6% 16 84.2% 5059 10 9.8% 6 60.0% (female) 46 (45.09%) 32 (31.37%) 6069 8 7.8% 6 75.0% 7079 8 7.8% 6 75.0% total no. 102 80 we found 39.2% positive for abdominal wards which showed the highest positive (table 2), this may be due to the types of operations in these wards, this finding is compatible to a previous study (25) . table 2: percentage of various nosocomial infections according to the type of surgical ward. %positive of total no. no. of positive samples no. of samples type of surgical ward 39.21 40 48 abdominal 9.80 10 16 gynecology 15.68 16 20 orthopedics 13.72 14 18 urology 78.43 80 102 total iraqi j pharm sci, vol.20(2) 2011 postoperative wound infections 62 the tables (3and 4 ) showed that postoperative wound infections increase with the hospitalization (pre and post operative) that's mean the infections can be decreased by shortening the hospitalization, and this findings are in agreement with a number of studies (1,25,26) . antibiotic-susceptible microorganisms in body flora of the hospitalized patients can be replaced with antibiotic resistant strains which are present in hospital environment any time. due to flora changes, prolonged hospitalization is a risk factor in all units and all types of nosocomial infections (19) . table 3 : surgical site infection according to pre-operative hospitalization percentage% infected total pre-op hospitalization 67.39 31 46 0-1 days 78.94 15 19 2-3 days 83.33 15 18 4-7 days 100.0 19 19 >7 days table 4:surgical site infection according to post operative hospitalization percentage% infected total post-op hospitalization 68.75 33 48 1-4 days 78.94 15 19 5-10 days 81.25 13 16 11-15 days 100.0 19 19 >15 days in the present study, pseudomonas aeruginosa was the most frequent in the abdominal wards (23.3%) as in table5 . table 5: showing organisms in abdominal wards while in gynecology wards we found that coag-pos.staph. (25.0%) was the most frequent (table6). table 6 : showing organisms in gynecology wards percentage% no. of patients culture 25.0 5 coagpos.staph. 10.0 2 escherichia coli 5.0 1 pseudomonas aeroginosa 10.0 2 klebsiella pneumoniae 0.0 _ proteus mirabilis 0.0 _ acinetobacter baumannii 20.0 4 enterococcus spp. 30.0 6 negative in table 7 the most frequent reported microorganism in orthopedics wards was acinetobacter baumannii (27.2%) . table 7: showing organisms in orthopedics wards in urology wards(table 8) ,the escherichia coli was most common species(27.2%). percentage% no. of patients culture 18.1 4 coag-pos.staph. 9.0 2 escherichia coli 18.1 4 pseudomonas aeroginosa 0.0 _ klebsiella pneumoniae 0.0 _ proteus mirabilis 27.2 6 acinetobacter baumannii 9.0 2 enterococcus spp. 18.1 4 negative percentage% no. of patients culture 10.0 6 coag-pos.staph. 6.6 4 escherichia coli 23.3 14 pseudomonas aeroginosa 16.6 10 klebsiella pneumoniae 13.3 8 proteus mirabilis 3.3 2 acinetobacter baumannii 13.3 8 enterococcus spp. 13.3 8 negative iraqi j pharm sci, vol.20(2) 2011 postoperative wound infections 63 table8: showing organisms in urology wards percentage% no. of patients culture 22.7 5 coag-pos.staph. 27.2 6 escherichia coli 9.0 2 pseudomonas aeroginosa 0.0 _ klebsiella pneumoniae 13.6 3 proteus mirabilis 0.0 _ acinetobacter baumannii 9.0 2 enterococcus spp. 18.1 4 negative the results showed 27.2% positive for pseudomonas aeruginosa, 25.0% positive for coagulase positive staph., 20.0% positive for enterococcus spp., 17.5% positive for escherichia coli which were predominant to others as shown in (table 9). table 9 : shows the most common isolates from wound swabs percentage% no. isolated microorganisms 25.0 20 coag-pos.staph. 17.5 14 escherichia coli 27.2 21 pseudomonas aeroginosa 15.0 12 klebsiella pneumoniae 13.7 11 proteus mirabilis 10.0 8 acinetobacter baumannii 20.0 16 enterococcus spp. the most frequently isolated microorganisms in the postoperative wound infections was pseudomonas aeruginosa (27.2%) ,among these isolates, resistance rate for imipenem were 3.7% . because of increase of imipenem resistance, studies were done to determine the risk factors for irpa infections, the isolates of irpa were often multidrug resistant causing a difficulty in the treatment and control of these infections (13) .the genetic material that makes vancomycin resistant enterococci(vre) resistant to vancomycin, the vana gene, can be transferred from the enterococci to other kinds of bacteria. if this vana gene was to be transferred to mrsa bacteria, the end result would be s. aureus bacteria that are resistant to virtually all of our currently available antibiotics .(12,27) .the results showed 11.2% positive for vre and13.7% positive for mrsa and vrsa and 11.2% positive for visa (figure 2). visa/vrsa may spread from person-to-person in the same way as any s. aureus infection. s. aureus infections most often spread from person-to-person by direct contact. for example, in medical settings staphylococcal infections are often spread from patient to patient in unwashed health care workers' hands (1) . figure 2: percentage of mrsa , vrsa , visa , vre , irpa in the samples mrsa and vrsa on muller-hinton agar visa on muller-hinton agar 13.7% 13.7% 11.2% 11.2% 3.7% 46.5% mrsa vrsa visa vre irpa others iraqi j pharm sci, vol.20(2) 2011 postoperative wound infections 64 conclusions and recommendations from this study, the following points emerged as priorities to be followed in the near future: definition of the antibiotic prophylaxis policy; reduction of preoperative length of hospitalization; increased follow up surveillance and setting up systematic surveillance; and reduction of the length of procedures through adequate training of the staff on proper surgical techniques and intraoperative infection control measures.most of the postoperative infections can be prevented with readily available, relatively inexpensive strategies by:  adhering to recommended infection prevention practices, especially hand hygiene and wearing gloves.  paying attention to well-established processes for decontamination and cleaning of soiled instruments and other items, followed by either sterilization or highlevel disinfection.  improving safety in operating rooms and other high-risk areas where the most serious and frequent injuries and exposures to infectious agents occur. acknowledgements the author wish to thank the staff of the microbiology laboratory of kadhimiyah teaching hospital especially alia'a jassim , amira abdullah, ja’fer abbas hussein and we also wish to thank all the studied hospitals. references 1. maida š., amra z., mirsada h. methicillin-resistant staphylococcus aureus (mrsa) as a cause of nosocomial wound infections .bosnian journal of basic medical sciences 2010; 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89 ( 1) : 81-88 . 22. danchaivijitr s, tangtrakool t, chokloikaew s. the second thai national prevalence study on nosocomial infections 1992.j med assoc thai 1995;78 suppl 2: s67-72. 23. malone dl, genuit t, tracy jk, gannon c,napolitano lm. surgical site infections: reanalysis of risk factors. j surg res 2002; 103: 89-95. 24. pessaux p, msika s, atalla d, hay jm, flamant y.risk factors for postoperative infectious complications in noncolorectal abdominal surgery: a multivariate analysis based on a prospective multicenter study of 4718 patients. arch surg 2003; 138: 314-24. 25. avdyl k., faton h., ruustem m., gjyle m., selvete k., arsim k., antigona d. beqir n., baton k. dalip l., ilir t., surgical site infections in an abdominal surgical ward at kosovo teaching hospital ,j infect developing countries ,2007; 1(3):337-341. 26. anderson roger l, , janice h, bs; bond v walter w, ms; favero martin s, susceptibility of vancomycin-resistant enterococci to environmental disinfectants. infect. control hosp. epidemiol. 1997; 18 ( 3): 195. 27. gilmore ms, coburn ps, nallapareddy sr, murray be.enterococcal virulence. the enterococci: pathogenesis, molecular biology, and antibiotic resistance. washington, dc: asm press; 2002. p. 301–54. iraqi j pharm sci, vol.26(2) 2017 impact of mk-7 on doxorubicin-induced hepatotoxicity 22 impact of two doses of vitamin k2 (menaquinone-7) on doxorubicininduced hepatotoxicity in rats ruaa f. a. al-zubaidi *,1 and nada n. al-shawi ** * technical affairs directorate, ministry of health/environment, baghdad, iraq. * department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract the objective of this study was to evaluate the impact two doses of menaquinones-7 on hepatotoxicity induced by doxorubicin in rats. sixty adult rats of both sexes were used in this study; the animals were randomly enrolled into six groups of 10 animals each. group i: negative control (rats administered distilled water); group ii: menaquinones-7 at a dose of 16 µg/kg; group iii: menaquinones7 at a dose of 48 µg/kg; group iv: positive control (doxorubicin 15 mg/kg); group v: menaquinones-7 at a dose of 16 µg/kg administered prior to a single dose of doxorubicin 15 mg/kg; group vi: menaquinones7 at a dose of 48 µg/kg administered prior to a single dose of doxorubicin 15 mg/kg. on day twelve of the study, blood was collected for serum preparation for the estimation of alanine aminotransferase (alt), alkaline phosphatase (alp), and total bilirubin (tb). the liver of each animal was excised for histological examination. high dose of mk-7 significantly (p<0.05) decreased serum alt, alp, and tb and there was an improvement in the histopathological lesions of the liver in group v and group vi compared to group iv. in conclusion, mk-7 may have protective effect against dox-induced hepatotoxicity in rats. keywords: menaquinone-7, doxorubicin, hepatotoxicity, rats. فٌ علي سمية الكبد المستحثة بواسطة الدوكسوروبيسيه menaquinone-7تأثير جرعتيه مه الجرذان. يدًبرؤى فاضل الز ،*1 و ودى واجٌ الشاوً ** * دائشة االٍ٘س اىفْيت، ٗصاسة اىصحت ٗاىبيئت، بغذاد، اىعشاق. ** . ، مييت اىصيذىت ، خبٍعت بغذاد ، بغذاد ، اىعشاقاىسًَ٘ ٗ االدٗيتفشع الخالصة اىَسخحذثت ب٘اسطت عقبس عيٚ سَيت اىنبذ menaquinone-7 ثش خشعخيِ ٍِأاىٖذف ٍِ ٕزٓ اىذساست ٕ٘ حقييٌ اىٚ سج ٍدَ٘عبث ٗفي بص٘سة عش٘ائيت قسَج اىحي٘اّبثحيث ببىغب ٍِ مال اىدْسيِ خشرا 06حٌ اسخخذاً . دشراُاىذٗمس٘سٗبيسيِ فٚ اى ٍبينشٗغشاً/مغٌ 00 أعطيجٍبء ٍقطش; اىَدَ٘عت اىثبّيت اعطيجاىَدَ٘عت االٗىٚ: ٍدَ٘عت اىَشاقبت اىسيبيت ٗ .حي٘اّبث06مو ٍدَ٘عت ٍِmenaquinone-7 ٍبينشٗغشاً/مغٌ ٍِ 84 اعطيج; اىَدَ٘عت اىثبىثتmenaquinone-7 اىَدَ٘عت اىشابعت: اىَشاقبت االيدببيت ; قبو اعطبءٕب menaquinone-7ٍبينشٗغشاً/مغٌ ٍِ 00 اعطيجٍيغٌ/مغٌ ٍِ عقبس اىذٗمس٘سٗبيسيِ; اىَدَ٘عت اىخبٍست 01 ٗاعطيج قبو اعطبءٕب menaquinone-7نشٗغشاً/مغٌ ٍِ ٍبي 84 اعطيجسبدست اٍيغٌ/مغٌ ٍِ عقبس اىذٗمس٘سٗبيسيِ; اىَدَ٘عت 01 االالّيِ االّضيَبثقيبط ٍسخ٘ٙ غشض ٍِ اىذساست, ُخَع ٍصو اىذً ى 01ٍيغٌ/مغٌ ٍِ عقبس اىذٗمس٘سٗبيسيِ. فٚ اىيً٘ 01 . ذساست اىْسيديت ىغشض اىه اىنبذ بصئأسخاضبفت اىٚ رىل حٌ . tb اىبيييشٗبيِ اىنيي ٗ alp االىنباليِ ف٘سفبحيضٗ altاٍيْ٘حشاّسفيشيض ٍسخ٘ٙ ٗ alt ٗalpٍسخ٘يبث االّضيَبث (p<0.05) بص٘سة ٍعْ٘يت خفضج menaquinone-7عبىيت ٍِ اىدشعت اىبيْج اىْخبئح أُ اىنبذ فٚ اىَدَ٘عخيِ اىخبٍست ٗاىسبدست ٍقبسّت ببىَدَ٘عت اىشابعت. ضشس ّسيح في ٍصو اىذً ْٕٗبك ححسِ في tb اىبيييشٗبيِ اىنيي .دشراُذثت ب٘اسطت عقبس اىذٗمس٘سٗبيسيِ فٚ اىحقذ ينُ٘ ىٔ حأثيش ٗقبئي ضذ سَيت اىنبذ اىَسخ menaquinone-7 بأُ سخْخبجيَنِ االٗ ، الدوكسوروبيسيه، سمية الكبد، الجرذان.menaquinone-7الكلمات المفتاحية: introduction in the complicated world of malignancy treatment, the administration of chemotherapeutic agents deliberately considered to be cytotoxic and causes negative consequences. the liver is the primary site of metabolism for many drugs, and this liver-drug interaction must be accounted while dosing chemotherapy (1) . 1 corresponding author e-mail: rowaa_fadhel@yahoo.com received:11 /6/2017 accepted: 28/8/2017 iraqi j pharm sci, vol.26(2) 2017 impact of mk-7 on doxorubicin-induced hepatotoxicity 23 doxorubicin (dox) was approved in 1974 by food and drug administration (fda); it is an anthracycline still widely used in modern cancer treatments for different type of malignancy (2 , 3) despite the advent of targeted therapy (4) . however, its beneficial effect was limited by its adverse effects on heart (5) , kidney (6) , liver (7) and other organs (8, 9) with its main toxicity on heart and liver. dox has adverse effects on the liver. one reason may be that the liver is the main organ contributed to metabolism of exogenous and endogenous toxins including dox, leading to toxic metabolites formation. hence, resulting in hepatotoxicity (10) ; where, the hepatic damage in this respect was reported to be irreversible in a dose-dependent manner as reported by investigators (11, 12) . another reason is that dox may drastically increase lipid oxidation and mitochondrial reactive oxygen species (ros) content, and decrease liver antioxidant enzymes and mitochondrial function (13) . fat-soluble vitamin k is an essential micronutrient (14) for which there is two forms: phylloquinone (vitamin k1) and the menaquinones (vitamin k2; mk-n). menaquinones vary in the length and in the degree of saturation at the aliphatic side chains. menaquinone-7 (mk-7) has long half-life and good bioavailability (15) ; it is produced by bacillus subtilis natto (16) . several authors demonstrated the beneficial protective role of long chain vitamin k against cardiovascular and bone diseases (17-19) . the aim of this study is to evaluate the impact of two doses of mk-7 on dox-induced hepatotoxicity in rats. materials and methods experimental animals sixty adult albino rats of both sexes, three months old, weighing 160-250gm were used in this study; they were obtained from and maintained in the animal house of the college of pharmacy, baghdad university under conditions of controlled temperature. the animals were fed commercial pellets and tap water ad libitum throughout the experiment period. the study was approved by the scientificand the ethicalcommittees of the college of pharmacy/ university of baghdad. drugs doxorubicin as hydrochloride (50 mg vial) was purchased from pfizer, italy. menaquinone-7 (menaq7 capsule 180 µg) was purchased from omicron pharmaceuticals, norway. experimental protocol rats were randomly allocated into six groups, each containing 10 rats (5 males and 5 females) as follow: group i: rats were received 0.5 ml of distilled water (dw) as intraperitoneal (ip) dose. this group served as a negative control. group ii: rats were received mk-7 (16 µg/kg body weight/day) orally by oral gavage for 11 consecutive days. group iii: rats were administered mk-7 (48 µg/kg body weight/day) orally by oral gavage for 11 consecutive days. group iv: rats were intraperitoneally injected with single dose of dox (15 mg/kg body weight). this group served as a positive control. group v: rats were orally administered mk-7 at a dose of 16 µg/kg body weight/day prior to 15 mg/kg of dox. group vi: rats were orally administered 48 µg/kg body weight/day prior to 15mg/kg of dox. in groups (v and vi) animals, each dose of mk-7 was administered once daily for 11 consecutive days; and at day 11, they received single dose of dox (15 mg/kg body weight) by ip injection. twenty-four hour after the end of the treatment duration (i.e. at day 12), the animals were euthanized by diethyl ether and blood was collected for serum preparation. estimation of serum biochemical parameters: serum samples were used for the estimation of the enzymes activities of alanine aminotransferase (alt), and alkaline phosphatase (alp), in addition to total bilirubin (tb) level by automated biochemistry analyzer (kenza, biolabo, france). histological examination after necropsy, liver of each rat was removed and used for a histopathological examination according to the routine method (20) utilizing paraffin sections technique; the fragments were fixed in 10% formaldehyde solution, embedded in paraffin, segmented, and then stained with haematoxyline/eosin. morphological examination of the samples was studied using light microscopy. statistical analysis data were expressed as mean±standard error of the mean (sem). unpaired student t-test was used for testing the significant difference between two groups. the statistical significance of the differences among various groups was determined by one-way analysis of variance (anоva) and least significant difference (lsd) analysis by ibm spss (statistical package for social sciences) version 23. differences were iraqi j pharm sci, vol.26(2) 2017 impact of mk-7 on doxorubicin-induced hepatotoxicity 24 considered statistically significant for p-value less than 0.05. results effects of two doses of mk-7 on serum liver biomarkers table 1 showed that group ii rats, which received mk-7 (16 µg/kg body weight/day) orally by oral gavage for 11 consecutive days and group iii rats, which administered mk-7 (48 µg/kg body weight/day) orally by oral gavage for 11 consecutive days, produced non-significant (p>0.05) differences in the serum alt, alp enzymes activities and tb with respect to negative control group. besides, single intraperitoneal dose of dox significantly (p<0.05) increased the serum activities of alt, alp, and tb in comparison with negative control group. furthermore, in group v rats, which received mk-7 at a dose of 16 µg/kg body weight/day orally prior to 15 mg/kg of dox, there were non-significant (p>0.05) differences in the intended enzymes compared to positive control group; however, in group vi rats received mk-7 at a dose of 48 µg/kg body weight/day orally prior to 15 mg/kg of dox exhibited significant (p<0.05) decrease in serum alt, alp enzymes activities, and tb compared to positive control group. table (1): effects of various treatments on serum alanine aminotransferase (alt), alkaline phosphatase (alp), and total bilirubin (tb) in rats. group serum alt levels (iu/l) serum alp levels (iu/l) serum tb levels (iu/l) group i 56.2 ± 3.3 240.3 ± 8.528 0.111 ± 0.01 group ii 62.2 ± 1.2 276.4 ± 26.73 0.117 ± 0.004 group iii 61.1 ± 1.139 265.2 ± 30.9 0.115 ± 0.007 group iv 93.9 ± 10.5 *aa 390.1 ± 18.4 *aa 0.143 ± 0.005 *aa group v 75.1 ± 6.1 aa 343.8 ± 32.8 aa 0.123 ± 0.008 aa group vi 66.2 ± 6.7 ba 317.1 ± 9.09 ba 0.115 ± 0.011 ba data are expressed as mean ± standard error of means (sem) group i: negative control (d.w); group ii: mk-7 16 µg/kg; group iii: mk-7 48 µg/kg; group iv: positive control (dox 15 mg/kg); group v: mk-7 16 µg/kg prior to single ip dose of dox 15 mg/kg; group vi: mk-7 48 µg/kg prior to single ip dose of dox 15 mg/kg. * significantly different (p<0.05) with respect to the negative control group utilizing unpaired student ttest. values with non–identical superscripts capital letters (a and b) are significantly different (p<0.05) compared to positive control group utilizing unpaired student t-test. values with an identical superscript small letter (a) is non-significantly different (p>0.05) among groups iv, v and vi utilizing anova and lsd. the histopathological examination of rats' liver tissue histological examinations of liver sections of the negative control group, group ii (rats orally received mk-7 16 µg/kg for 11 consecutive days), and group iii (rats orally received mk-7 48 µg/kg for 11 consecutive days) showed normal liver section (figure 1 a, b, and c, respectively). examination of liver section of positive control group (dox-treated rats) showed severe dilation of the central veins with severe vacuolar degeneration of hepatocytes with a narrowing or disappearance of sinusoids (figure 1 d). the lesion in group v (mk-7 16 µg/kg prior to a single dose of dox 15 mg/kg) revealed mild congestion of central veins with moderate vacuolar degeneration (figure 1 e). besides, liver section of group vi (mk-7 48 µg/kg prior to a single dose of dox 15 mg/kg) exhibited mild vacuolar degeneration (figure 1 f). iraqi j pharm sci, vol.26(2) 2017 impact of mk-7 on doxorubicin-induced hepatotoxicity 25 figure (1): histopathological section of liver in various experimental rats' groups; (haematoxyline and eosin; x20). a: group i (negative control (d.w); b: group ii (mk-7 16 µg/kg); c: group iii (mk-7 48 µg/kg); d: group iv (positive control (dox 15 mg/kg); e: group v (mk-7 16 µg/kg prior to a single dose of dox 15 mg/kg; f: group vi (mk-7 48 µg/kg prior to a single dose of dox 15 mg/kg; x 40). negative control group, group ii, and group iii showed normal liver section. positive control group characterized by severe dilation of the central veins with severe vacuolar degeneration of hepatocytes (red arrow). the lesion in group v revealed mild congestion of central veins with moderate vacuolar degeneration (yellow arrow). besides, liver section of group vi exhibited mild vacuolar degeneration. iraqi j pharm sci, vol.26(2) 2017 impact of mk-7 on doxorubicin-induced hepatotoxicity 26 discussion toxicity is the major factor hindering dox treatment. the mechanisms of dox mediated cell death include oxidative stress, apoptosis, intracellular calcium dysregulation, topoisomerase ii poisoning, dna adduct formation, and ceramide overproduction (21, 22) . additionally, dox initiates inflammation via markedly increase of inflammatory-related proteins including tnf-a, il-1𝛽, and il-6 in the liver (23) . the current study revealed that doxinduced hepatotoxicity, which was evident by significant (p<0.05) elevation in serum activities of alt and alp with respect to negative control group. the results are in agreement with studies of others (24-26) . the elevation of the serum activities of the intended enzymes may be attributed to the recognized hepatotoxic effect of doxorubicin that result in cellular damage and leakage of such enzymes to the extracellular space. furthermore, there was a significant (p<0.05) increase in serum tb level in animals received single intraperitoneal dose (15 mg/kg body weight) of dox compared with the negative control group. these results are in line with those of salman (2013) (27) . since the liver is responsible for clearing the blood from bilirubin thus, increasing serum tb level indicated a reduction in the excretory capability of the liver as a consequence of liver injury (28) . in the present study, mk-7 administered at a dose of 48 µg/kg prior to single ip dose of dox 15 mg/kg body weight (group vi), significantly (p<0.05) lowered serum level of alt, alp, and tb compared to positive control group. moreover, there were improvement of the histopathological lesions of the liver in -group v rats administered mk-7 at a dose of 16 µg/kg prior to single ip dose of dox 15 mg/kg body weight and –group vi rats that received 48 µg/kg mk-7 prior to single ip dose of dox 15 mg/kg body weight compared to positive control group. this hepatoprotective effect of mk-7 could be attributed to potent antioxidant capacities of mk-7 when reduced to kh2 (dihydroquinone) during vitamin k cycle (29) , that in turn may result in attenuation of oxidative stress induced by dox. several beneficial effects of mk-7 on bone and cardiovascular system were elucidated by authors (18, 19) . moreover, it has been reported that, many of the positive impacts of mk-7 could be accredited to menaquinones-4 (mk-4) where, all vitamin k homologues can be converted to mk-4 in vivo (30) and many studies reported the antioxidant and anti-inflammatory influence of vitamin k analogues in vivo and in vitro; furthermore, vervoort et al. (1997) showed that vitamin k2 was an inhibitor of microsomal lipid peroxidation in rat liver microsomes (31) . additionally, vitamin k1 and mk-7 had the capability to block activation of 12-lipooxygenase (12-lox) and to inhibit ros generation in pre-oligodendrocytes, hence, prevent oxidative cell death (32) . in conclusion, mk-7, in a high dose, may have protective effect against dox-induced hepatotoxicity in rats as demonstrated by liver function evaluation and histological examination. to the best of our knowledge, this is the first study that examines the effects of mk-7 at two doses (16 µg/kg and 48 µg/kg) each prior to doxorubicin on the liver. therefore, we did not have a thorough chance to compare the results of this study with other reports. acknowledgments this article was abstracted from ph.d. thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad. the authors gratefully thank the staff of ministry of health and 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jw, furie bc, furie b, booth sl, volpe jj, rosenberg pa. novel role of vitamin k in preventing oxidative injury to developing oligodendrocytes and neurons. j neurosci, 2003; 23:5816-5826. 30. nakagawa k, hirota y, sawada n, yuge n, watanabe m, uchino y, okuda n, shimomura y, suhara y, okano t. identification of ubiad1 as a novel human menaquinone-4 biosynthetic enzyme. nature, 2010; 468:117-121. 31. vervoort lmt, rondent je, thijssen hhw. the potent antioxidant activity of the vitamin k cycle in microsomal lipid peroxidation. biochem pharmacol, 1997; 54: 871-876. 32. li j, wang h, rosenberg pa. vitamin k prevents oxidative cell death by inhibiting activation of 12-lipoxygenase in developing oligodendrocytes. j neurosci res, 2009; 87(9):1997-2005. introduction iraqi j pharm sci, vol.21(2) 2012 misuse of appetite-stimulant drugs in babylon 31 misuse of appetitestimulant drugs in babylon # shafaq k. saleh* ,1 *babylon university , college of pharmacy, babel, iraq abstract drug misuse is defined as using of drugs for a non-therapeutic or non-medical purpose. in iraq drug misuse is a major problem because almost any drug can be easily obtained from pharmacies. appetitestimulant drugs are example of drugs that are widely used without a prescription. the study included 230 patients who use these drugs in babylon. a questionnaire included the following questions ; age, sex, marital state, the reason for use the drug , whether the drug is prescribed by physician or not , type of drug used , the frequency of daily dose and lastly the extent of side effects of the drugs used. the results showed that the age range of 35% of subjects were (17-21) years old and 70% of participants were females. the study also showed that 62% of them are unmarried and the reason for use, in 48% of subjects, was to increase the body weight. furthermore, this study revealed that about 68% of those subjects utilized these drugs without prescription and the steroids are the most drug type used (33%). additionally, 44% of subjects used these drugs twice daily and 80% of subjects who used steroids developed marked side effects. it can be concluded that there is a misuse of appetite stimulant drugs in subjects participated in this study and most of these drugs are not used on therapeutic or scientific bases. key words : misuse of drug , appetite-stimulant, dexamethasone. سىء استعمال األدوية انمحفزة نهشهية في محافظة بابم *شفق كاظم صانح ،1 . ، ثبثو ، اىؼشاق جبٍؼخ ثبثو ،مييخ اىصيذىخ * الخالصة األدويخغيش اىؼيَيخ . في اىؼشاق يشبع سىء اسزؼَبه وسىء اسزؼَبه اىذواء هى اسزؼَبه اىذواء ىألغشاض غيش اىطجيخ واىزي رصشف ثذوُ وصفخ اىَحفضح ىيشهيخ األدويخوٍثبه رىل فهب ٍِ اىصيذىيبد ثسهىىخ وثذوُ وصفخ طجيخوثئٍنبُ اىنثيشيِ صش جَؼىا ثصىسح ٍززبثؼخ وثطشيقخ االسزجيبُ ٍَِ اسزؼَيىا هزٓ األدويخ , ٍشاجغ في ٍحبفظخ ثبثو 032. اىذساسخ رضَْذ طجيخ وجيخ, أسجبة اسزخذاٍهب , وهو هى ٍىصىف ٍِ قجو اىطجيت أو ال و مزىل ُسئيىا اىَزضَِ األسئيخ اىزبىيخ : اىؼَش ،اىجْس، اىحبىخ اىض % ٍِ هؤالء 33ػِ ّىع اىذواء اىَسزخذً، اىجشػخ اىذوائيخ اىيىٍيخ وأخيشا هو رٌ حذوس آثبس جبّجيخ أو ال. أظهشد اىذساسخ أُ أػَبس % 84% ٍْهٌ مبّىا غيش ٍزضوجيِ. ومبُ اىسجت ػْذ 20 % ٍْهٌ إّبس . ومزىل أظهشد أ12ُسْخ و 07-71اىَشاجؼيِ رزشاوح ثيِ % ٍِ اىَشاجؼيِ اىزيِ يسزخذٍىُ هزٓ األدويخ يسزخذٍىّهب ٍِ غيش 24في اسزخذاً هزٓ األدويخ هى ىضيبدح اىىصُ .ثبإلضبفخ إىى إُ % ٍِ اىَشاجؼيِ 88ئج إُ % ٍِ ثيِ أّىاع هزٓ األدويخ . إضبفخ إىى رىل ثيْذ اىْزب33وصفخ طجيخ واىسزيشويذاد رشنو ّسجخ % ٍِ اىزيِ اسزخذٍىا اىسزيشويذاد قذ ػبّىا ٍِ اآلثبس اىجبّجيخ ىهزٓ األدويخ. ٍِ هزٓ اىْزبئج 42يسزخذٍىُ هزٓ األدويخ ٍشربُ يىٍيب و نثيش ٍْهب يَنِ أُ ّسزْزج إُ هْبك سىء اسزؼَبه في األدويخ اىَحفضح ىيشهيخ ضَِ اىَجَىػخ اىَشبسمخ في اىذساسخ في ثبثو واى رسزخذً ثطشيقخ غيش ػيَيخ أو طجيخ . ديكساميثازون . االدوية انمحفزة نهشهية ، ، اندواء ستعمالاسىء انكهمهت انمفتاحية: introduction drug misuse can be simply defined as using of drugs for a non-therapeutic or nonmedical purpose (1) . drug misuse is a term used commonly for prescription medications with clinical efficacy (2) . in iraq drug misuse is a widely spread problem because almost any drug can be easily obtained from pharmacies and drug stores. appetite-stimulant drugs are widely used without a prescription or medical supervision to gain body weight and improve outlook.appetite stimulant drug is any drug which increases the appetite. several studies have indicated that there is a strong association between self-perceived weight status and weight control behavior (3,4) . paradoxically, in many developing communities like iraq, fullness is culturally associated with beauty, prosperity, health and prestige, while slimming is perceived to be a sign of ill health or poverty, so utilizing these drugs to increase body weight in order to improve the shape of subject (5-7) . cyproheptadine hydrochloride is an antihistaminic /anticholinergic and antiserotonergic agent (8) . in iraq cyproheptadine is available in two pharmaceutical dosage forms; tablets and syrup and widely used as an appetite stimulant. # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011 1 corresponding author email : shafaqpharm@yahoo.com received : 19/12/2011 accepted : 26/5/2012 http://en.wikipedia.org/wiki/prescription_medication http://en.wikipedia.org/wiki/anticholinergic http://en.wikipedia.org/wiki/antiserotonergic iraqi j pharm sci, vol.21(2) 2012 misuse of appetite-stimulant drugs in babylon 32 dexamethasone is an antiinflammatory and immunosuppressant. one of its side effects is increased appetite leading to significant weight gain and therefore misused for this purpose (9) . in iraq dexamethasone is available in different pharmaceutical dosage forms like tablet, syrup and injection. betamethasone is another glucocorticoid steroids with anti-inflammatory and immunosuppressive properties (10) . in iraq betamethasone is available in different pharmaceutical dosage forms as tablet and injection. tonics include mainly vitamins and minerals , are available in different pharmaceutical dosage forms like tablet, syrup and injection . this study was conducted to verify the misuse of appetite-stimulant drugs by people attending private pharmacies in babylon. subjects and method the descriptive cross section study was conducted from march 2010 to july 2011 on a consecutive sample of 230 subjects, in babylon. the subjects were collected from patients attending three private pharmacies in different locations to buy different types of appetite-stimulant drugs.a questionnaire was constructed and included the following questions: age, sex, marital state, reason for using the drug , whether it is prescribed by physician or used with out prescription , type of drug used, frequently daily dose used, and extent of side effects in the subjects of study. results and discussion variation of age the results showed that 18 subjects (8%) using appetite-stimulant drugs were under 12 years old , 56 subjects (24%) were between 12-16 years old, 80 subjects (35%) were between 17-21 years old, 60 subjects (26%) were between 22-45 years old and 16 subjects (7%) were more than 45 years old (figure 1). these numbers indicated that appetite-stimulant drugs are used mostly by teenagers and young adults . variation of sex the results indicated that 69 of the subjects (30%) used appetite-stimulant drugs were males and 161 (70%) were females at a ratio 1:2.3 as females always tend to improve their look as concluded from the answers of participants during interviewing . marital state the study revealed that 143 (62%) of subjects who used appetite-stimulant drugs were single (unmarried), while 87 (38%) of them were married. these results indicated that the unmarried subjects, perhaps most of them, are trying to increase their body weight to improve their shape and to look more attractive & healthy by other people. the reason for using appetite-stimulant drugs when the subjects of study were asked about the reason for using appetite stimulant drugs, 110 (48%) of them attributed the cause to low body weight, while 85 subjects (37%) used it to improve shape and outlook, and 35 subjects (15%) said that they used it just for general health (figure 2). these results indicated that most of the subjects using these drugs were either to increase body weight or for better outlook.the desirability of a particular body size is not simply an autonomous, individual choice, but is mediated by cultural factors. in some countries there is a desire to lose weight but in other countries there is a desire for fullness (11) . drug prescribed by physician or not the results showed that only 74 subjects (32%) used appetite-stimulant drugs prescribed by physician or under medical supervision while 156 subjects (68%) used these drugs without physician prescription, these results showed that there is a great misuse of such drugs and most of them are used without prescription or supervision by medical experts. type of appetite-stimulant drugs used seventy six of the subjects used steroids (33%) , while 62 subjects used cyproheptadine (27%), 28 of them used tonics(vitamins and minerals) (12%), and 46 subjects used combinations of these agents (28%) (figure 3), and these results showed that steroids and cyproheptadine are the most types used locally because they believe that steroids are the most effective and fast drugs to increase body weight (based on the community experience). frequency of daily dose of appetite-stimulant drugs ninety two subjects (40%) used these drugs once daily, 101 subjects (44%) used it twice a day, 32 subjects (14%) used it three times a day while only 5 subjects (2%) used it more than three times a day (figure 4). these results indicated that most of subjects prefer to use the drugs 1-2 times a day rather than several times per day as it is more convenient . side effects according to our findings ,only 10% of the subjects used general tonics suffered from side effects, mostly gastric http://en.wikipedia.org/wiki/glucocorticoid iraqi j pharm sci, vol.21(2) 2012 misuse of appetite-stimulant drugs in babylon 33 problems (like nausea), 50% of the subjects used cyproheptadine experienced sleepiness and headache while 80% of subjects who used steroids suffered from sever side effects ( like edema, acne, dry scaly skin and increased sweating) (figure 5). cyproheptadine causes drowsiness as is common with first-generation antihistamines. nausea and vomiting are noted commonly in people with neuromuscular disorders. cyproheptadine can also cause oversleeping (10 hours) in children and adolescents (12) . figure 1: variation of age figure 2: reason for using appetitestimulant drugs figure 3: type of appetite-stimulant drugs used figure 4: frequency of usage of appetitestimulant drugs per day figure 5: drugs and their side effects conclusions the results obtained from this study, showed that there is a misuse of appetitestimulant drugs in the subjects participated in this study in babylon which are not used on therapeutic bases and without physician supervision. the misuse of appetite-stimulant drugs is high among young age females and used by unmarried more than married subjects. most of the subjects used these drugs to increase body weight and improve out look (not due to diseases) and most of them prefer to take appetite-stimulant drugs twice a day. dexamethasone was widely used appetitestimulant drug and the majority of subjects used steroids had suffered from sever side effects. references 1. barrett sp, meisner jr, stewart sh. "what constitutes prescription drug misuse? problems and pitfalls of current conceptualizations". curr drug misuse rev , 2008, 1 (3): 255–62. 2. mccabe se, boyd cj, teter cj. "subtypes of non-medical prescription drug misuse". drug alcohol depend ,2009, 102 (1-3). 3. flynn kj, fitzgibbon m. body images and obesity risk among black females: a review http://en.wikipedia.org/wiki/nausea http://en.wikipedia.org/w/index.php?title=over-sleeping&action=edit&redlink=1 http://en.wikipedia.org/w/index.php?title=over-sleeping&action=edit&redlink=1 iraqi j pharm sci, vol.21(2) 2012 misuse of appetite-stimulant drugs in babylon 34 of the literature. annals of behavioral medicine, 1998, 20(1):13–24. 4. fantaine kl. the conspiracy of culture: women’s issues in body size. nursing clinics of north america, 1991, 26:669–75. 5. brown pj. culture and the evolution of obesity. human nature, 1991, 2:31–57. 6. treloar c et al. the cross cultural context of obesity: an inclen multicentre collaborative study. health and place, 1999, 5:279–86. 7. pollock nj. cultural elaborations of obesity-fattening practices in pacific societies. asia pacific journal of clinical nutrition, 1995, 4:357–60. 8. lowe da, matthews ek, richardson bp ."the calcium antagonistic effects of cyproheptadine on contraction, membrane electrical events and calcium influx in the guinea-pig taenia coli". british journal of pharmacology ,1981, 74 (3): 651–63. 9. richard a., pamela c. " adrenal hormons" . lippincotts illustrated reviews pharmacology,4 th ed., lww, 2009, 26 : 312–17. 10. van de beek d, de gans j, mcintyre p, prasad k ."corticosteroids for acute bacterial meningitis". cochrane database syst rev,2007, (1):405. 11. wardle j, griffith j. socioeconomic status and weight control practices in british adults. journal of epidemiology and community health, 2001,55:185–90. 12. lexi-comp "cyproheptadine". the merck manual professional, 2008. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2071752 http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2071752 http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2071752 http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pmcentrez&artid=2071752 http://www.greenjournal.org/cgi/content/full/97/4/485 http://mrw.interscience.wiley.com/cochrane/clsysrev/articles/cd004405/frame.html http://mrw.interscience.wiley.com/cochrane/clsysrev/articles/cd004405/frame.html http://www.merck.com/mmpe/lexicomp/cyproheptadine.html http://en.wikipedia.org/wiki/merck_manual_of_diagnosis_and_therapy http://en.wikipedia.org/wiki/merck_manual_of_diagnosis_and_therapy ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 27 the effects of melatonin on the oxidative stress , protein glycation , microalbuminuria and lipid profile in type ii diabetes mellitus hasan m.h. al–mhbashy*; saad a. hussain* ;nawfal a.m. numan* and majee d a. saed** received 31-12-2002 accepted 13-6-2004 abstract p re vious s tudies ind ic ated tha t suppleme ntation with antio xidants has a protec tive e ffec ts a ga inst oxid ativ e stre ss –induce d dama ge in type 2 diabe te s. in this s tudy we e valua ted the a ntio xidant e ffec ts of me la to nin on the oxid ativ e s tre ss pa ra meters and mic roa lbuminuria in typ e 2 dm pa tients. 30 pa tients w ith type 2 dm were tre ated with 3 mg/da y melatonin for 9 0 days. erythro cyte s and p la sma mda a nd gluta thio ne , fa sting plas ma gluc os e, %hb aic, microa lb uminuria, to ta l plas ma protein and lipid p rofile w ere mea sured e ac h 30 da ys a nd c ompa re d with tho se o btaine d from 20 healthy c ontrols. a dec re as e in mda lev els as soc iated with the elev atio n in gsh leve ls were obs erve d, c omp ared with the pre–trea tme nt lev els. f as ting p la sma glucos e, glyca te d hemoglob in a nd microalbuminuria we re significantly d ecrea se d, a ss ociate d w ith an improv eme nt in the total cho le sterol, hdl–c and ldl– c le ve ls , with res pec t to the pretre atment va lues .in co nclus ion , tre atment of type 2 dm p atients with melatonin may ha ve p rote ctiv e effe cts agains t the o xidative s tres s–ind uc ed da mage during the c ourse of type 2 dm. ke y wo rds: diabe te s me llitus , ox idativ e stre s s, me la to nin,mic ro albuminuria. الخالصة  هور ماً في ظ مه وراً عب د ويل ماً به ٬ سل كري أمرًا م من داء الس ى النوع الثاني كسدي لدى مرض الجهاد التأ هرة ازدياد ا ت ظا لقد أصبح مرض عفات ال جة . مضا عال سة م را ه فقد تم في هذه الد علي ة ) 30(و مي جرع يو ري ب من داء السك وع الثاني غم 3( مريضاً بالن وم / مل من ) ي ومًا ) 90(لميالتونين ولمدة مادة ا ة . ي :وتم قياس المتغيرات التالي ز كي خضاب الدم (gsh)و (mda)تر وزة رجة كل ود الزما الدم وز في ب كلوك مقدار ال وخاليا الدم و ما ومين (hbiac)في البالز وااللب ي البالز ة ف وم المختلف راكيز صور الشح وت ما روتينات البالز جمالي ب وا ول ٬ ي الب كل الدقيق ف عالج و عالج 30ما ٬ قبل البدء بال من ال ماً يو مدة مًا 90ول .يو ول وتقليل ن طرح االلبومين في الب والتقليل م سدي هاد التأك ي تقليل فرط االج ونين قد ساهم ف ميالت حليل النتائج بأن استخدام ال من ت ح اتض ي سن واضـح ـف ى تح كما أدى إل زة خضاب الدم ٬ وكلو ز في الدم كو ة مقدار الجلو ة عالـي مي ات الشـح روتيـن ول والب كولسـتر ويات ال سـت م الج ها قبل البدء بالع كثافة مقارنة بمستويات واطئة ال ة . و ماـي ي ح الً ـف اع رًا ـف ن دو وني الستنتاج بان للميالـت كن ا هذه النتائج يم من خالل و كري من داء الس ي وع الثان ي لدى مرضى الن كسد الجهاد التأ رط ا عن ف ج رر النات من الض .الجسم introduction the re is c urre ntly a gre at interes t in the pote ntia l co ntribution o f the increa se d oxid ativ e stre ss to diab etes me llitus (dm)(1, 2). different abnormal p athwa ys that are s trictly re la ted to chronic hype rglyce mia o f dm, s uc h as non– enzymatic glyca tion (3), po lyol p athwa y (4) a nd gluc ose a utooxid atio n (5). during the progres s of dm, the re is a c ontinuous eve nts of metab olic s tre ss , tis sue dama ge a nd ce ll de ath that could lea d to b oth increa se d free radic als production , and c ompromise d to ta l plasma antioxid ant c apa city (6). chro nic co mplic atio ns , whic h ma y oc cur , like re tinopa thy , ne phrop athy and dyslip id emia, are among the dangerous conse que nc es that us ually a cco mpa ny d ia be te s although the mechanis ms be hind the ir p atho genes is rema in poo rly unde rs to od, re ce nt s ev eral mechanis ms, including glycation of protein, o xidation of lipo pro tein, gro wth fa ctors alteratio ns , changes in pe rme ab ility and v as cular c ha nge s, have bee n p ro pos ed to cla rify the link be tween diabe te s and thes e ab normalitie s (7). me la to nin, n–ac etyl–5 –me thoxytryp ta mine, is a pine al sec re tory product that a ffec t rep ro duc tive functions, mo dula te s immune sys tem ac tivitie s, inhibits oxid ativ e s tres s (8), a nd regulates circ adian rhythms . re cent s tudies have exa mined the effica cy of melatonin as an antine oplas tic a ge nt (9, 10, 11) a nd a s an antioxida nt (12, 13). * de par tme nt of phar mac ology and tox ic ology, colle ge o f ph ar ma cy, un iver sity of bag hda d,bagh dad–i raq . ** na tion al diab etes ce nter , al–ya rmoo k tea ching ho spital, ba ghd ad–ir aq. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 28 howe ver, de spite the wid es pread us e of melatonin, the re is minimal information on its us e a s a pharmac ologica l ap pro ac h in the trea tment of dm and its co mplic ations . there is a lso minimal information on the toxicology of high pha rma co lo gical dos es (14). ba sed o n ld50 v alue s in animals, it ap pea rs that melatonin has an e xtre mely low ac ute to xicity(15). this stud y de termined whether trea tme nt with melatonin a ltered the clinical and bioc hemica l a bno rma litie s as so ciated with dm in patients with type ii d ia betes mellitus . patients and methods this study was c arried o ut o n 30 p atie nts who ha ve type ii dm for at le as t 5 ye ars, w ith age ra nge (40 –80 years ) at the na tio nal dia bete s cente r, al–mustansiria university. all the se le cted pa tie nts were pre viously maintaine d on o ra l hypoglyce mic d rugs a nd diet re stric tion, but with p oor glyce mic co ntro l. the y are tre ated with 3 mg/da y of me la tonin in a ca psule dos age form, p re pa re d spe cially for this purpo se , give n orally a t be d time for thre e months. twe nty he althy subjects, with the sa me age ra nges as tha t of p atie nts were se le cted and se rv ed as controls for c omp aris on. fas ting b lo od sa mples w ere c olle cted from all subjec ts b y vein puncture, before starting trea tment with melatonin (as bas e line sa mples ), and the n afte r e ac h 3 0 d ays of trea tment to follow the cha nges in the stud ie d pa ra meters. all bloo d samp le s were c olle cted in he parinized tubes . erythro cytes were se parated by c entrifugatio n at 3 000 r.p.m. for 1 0 min. at 4c, after the remov al o f the puffy coa t, the erythroc ytes were washed tw ic e with ice – co oled sa line conta ining 2.5 mm so dium azid e to inhib it c atalas e a ctivity (16). urine sa mples we re c olle cted from all s ubjec ts at the ea rly morning before starting treatme nt and ev ery 30 da ys for the eva luation of microalbuminurea (17). erythrocytes a nd plasma malondialdehyd e (mda) le vels we re a nalyzed a cc ord ing to the metho d o f stocks and do rma ndy (197 1)(17). erythrocytes and plas ma glutathione lev els were meas ured ac co rding to the method of god in e t al. (198 8) (18). p la sma gluco se leve ls were e valua ted using a read y ma de kit (labkit, sp ain) a cco rd ing to the me thod of barha n and trindo er (19 72)(19), and glyca te d he moglob in (hbaic) lev el was dete rmine d ac cording to the me thod o f sushil (20 00) (20). total p la sma pro tein co nc entratio ns were de te rmine d acc ording to the reinhold (19 53) metho d (21), while hemoglo bin (hb) lev els were e stimated ac cording to the me thod of dra pkin a nd aus tin (19 35) (22). plasma lipid profile wa s eva luated through the meas ureme nt of to ta l p la sma cho le sterol acc ording to the metho d of richmond (19 74) (23), and triglycerides lev els acc ording to the metho d of f oss ati and principe (1 982 )(24), while burs te in e t al. (197 0) (25) me thod was utilized for the me asure ment of high d ensity lipo pro tein–c holes terol (hdl–c ) leve ls fro m which the plas ma conce ntra tions of the lo w density lipo protein–c ho le sterol (ldl–c) le vels were c alcula te d ind irec tly us ing burtis and ashwood formula (19 99) (26). statistic al ana lysis o f d ata wa s d one by two –way comp aris on o f mea n va lues , utilizing stude nt’s t– te st, p –va lues les s than 0.05 was conside red signific ant. results comb ination of the routine ly used meas ures for glyce mic c ontrol with 3 mg/day melatonin, produce d s ignifica nt red uction in mda lev els in plas ma (a fter 6 0 d ays), and erythrocyte s (a fter 3 0 d ays) ta ble (1). the le vel o f re duc tion in mda reac he d v alues which a re c ompa ra ble, or e ven lowe r tha n that fo und in co rres ponding c ontrols afte r 90 d ays of treatme nt. table (1): effe cts of treatme nt with 3 mg/day me latonin on p la sma a nd e rythro cy te s mda leve ls in ty pe 2 dia be tic patie nts. re s ults re pre s e nt me an  s ta nda rd e rror. n = numbe r o f subje cts. * significantly diffe re nt with re s pe c t to control ( p < 0.05 ). ** s ig nifica ntly diffe re nt with re spe ct to z e ro time ( p < 0 .0 5 ). t re atme nt pe riod n pla sma md a mo l/l erythrocy te s md a nmol/g hb control 2 0 1 .36  0.1 16 .9 8  0 .86 ze ro time 3 0 1 .98  0.2* 19 .8 9  0 .98 * 30 days 3 0 1 .54  0.11 15 .6 3  1 .19 ** 60 days 3 0 1 .21  0.15** 12 .8 9  0 .8* * 90 days 3 0 0 .97  0.09** 12 .5 5  0 .62 ** ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 29 tab le (2) showed tha t the o xidant stre ss – induced d epletion of gsh in the p la sma wa s impro ve d a fter 3 0 d ays o f tre atment with 3 mg/da y o f melatonin (58 %), a nd continuous trea tme nt led to furthe r eleva tion in p la sma gsh le vels , which nea rly ma tc he d their v alues in normal c ontrols after 9 0 da ys of trea tment. ta ble (2): effec ts of tre atme nt with 3 mg /day me la tonin on plas ma and e ry throcytes g luta thio ne (gsh) le ve ls in ty pe 2 dia be tic patie nts re sults re prese nt mean  s ta nda rd e rror. n = nu mbe r of s ubjec ts . * sig nifica ntly d iffe re nt with re spect to c ontro l ( p < 0 .0 5 ). ** significa ntly diffe re nt with re s pe ct to ze ro time ( p < 0.05 ). howe ver, e rythro cyte s gsh le ve ls whic h are se verely de pleted d ue to dm–ind uce d oxid ant stress , s howe d a no n–s ignific ant increa se (p > 0.05 ), e ven a fter 9 0 d ays o f trea tme nt with melatonin. as a re sult of treatme nt with 3 mg/day melatonin for 9 0 days, fa sting plas ma gluc ose (fp g) a nd glyc ated hemoglo bin (hbaic) leve ls were s ignifica ntly red uce d (26 % and 1 1% re spe ctiv ely) compared w ith pretrea tme nt le vels (tab le 3 ). ta ble (3): effec ts o f tre atme nt with 3 mg /day me latonin o n fa sting blood gluco se (fpg) and gly ca te d he mo globin (hb aic) le ve ls in type 2 diabe tic patie nts. re sults re pre se nt me an  standard e rror. n = numbe r of s ubje c ts . * sig nific antly diffe re nt with re spe ct to c ontro l ( p < 0.0 5 ). * * signific antly diffe re nt with re s pe c t to z e ro time ( p < 0 .0 5 ). as ind ic ated in tab le ( 4 ) , microalbuminurea and total pla sma protein le ve ls we re s ev erely affe cted b y dm–ind uce d oxid ant stre ss , whe re 5 2% increas e and 1 6% dec re as e in both pa ra meters were obs erved (ta ble 4) res pec tiv ely, a nd we re s ignifica ntly different co mpa re d to c ontrols . ta ble (4): effec ts o f tre atme nt with 3 mg /day me latonin o n the mic ro albuminurea a nd total plasma p ro te in leve ls in ty pe 2 diabe tic patie nts. re sults re pre se nt me an  standard e rror. n = numbe r of s ubje c ts . * s ig nifica ntly diffe re nt with re s pe ct to co ntrol ( p < 0.05 ). * * signific antly diffe re nt with re spe ct to z e ro time ( p < 0.05 ). t re atme nt pe riod n plas ma gs h mo l/l erythrocyte s gsh  mol/g hb c ontrol 20 0 .36  0 .07 8 .29  0 .1 ze ro time 30 0 .13  0.01* 6.26  0.43* 3 0 da ys 30 0.21  0.01* * 6 .79  0.57 6 0 da ys 30 0.27  0.02* * 6 .8  0.4 2 9 0 da ys 30 0.29  0.02* * 7 .0  0.5 1 tre atment perio d n fpg mg /d l hb aic % contro l 20 94  4.6 5.4  0.28 zero time 30 1 90  2 2* 9.46  0.6* 3 0 da ys 30 153  15 6 0 da ys 30 173  17 8.7  0.59 9 0 da ys 30 135  11** 7.2  0 .4** tre ame nt pe riod n microalbu minure a mg/l total plasma pro te in g /dl co ntrol 20 169  4.1 8.55  0 .3 4 ze ro time 30 258  38 * 7.23  0.12* 30 day s 30 169  43 7.47  0 .0 9 60 day s 30 1 57  41** 7.35  0 .0 8 90 day s 30 1 40  30** 8.9  0.1** ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 30 melatonin trea tment res ulte d in s ignific ant re ductio n in microa lb uminure a (3 9%) a fter 6 0 da ys, with further d ecrea se afte r 90 d ays (4 2%). howe ver, total p la sma p ro te in lev els started to show significant e le vation (p < 0.05 ) after 9 0 days of trea tme nt only (tab le 4 ). table (5 ) clea rly demo ns trated tha t, the ab norma l lip id p rofile in dm s ta te , res pond ve ry well to melatonin tre atment, where the eleva te d tota l c ho le sterol va lues sta rted to dec re as e signific antly (20%, p < 0.05) afte r 6 0 days, rea ching a le vel which was lowe r than that obs erved in controls a fter 90 days. triglyc erid e le vels showe d (22 %) d ec re ase afte r 90 da ys of treatme nt, but this wa s still non–significa nt c ompa re d with pre–trea tme nt value s. table (5): effe cts of tre atme nt with 3 mg/day me la to nin on the lip id profile in t he plas ma of ty pe 2 diabe tic pa tie nts re s ults re pre s e nt me a n  s tandard e rror. n = numbe r o f subje cts. * signific antly diffe re nt with re s pe c t to c ontrol ( p < 0.05 ). ** significa ntly diffe re nt with re s pe ct to z e ro time ( p < 0.05 ). hdl– c le vels sta rted to inc re as e significantly after 30 da ys of trea tme nt with melatonin (15 %, p < 0.05 ). maximum inc re as e in hdl–c v alue wa s ob serve d after 9 0 days of trea tment (30 %), whic h is higher than that obs erve d for c ontrols (ta ble 5 ). ho weve r, ldl– c leve ls starte d to dec re as e as a re spo ns e fo r melatonin treatme nt o nly afte r 60 da ys of trea tment (3 6%, p < 0 .0 5). afte r 90 days, 4 6% de creas es in ldl–c v alues we re ob se rv ed, which is highly significant c ompa re d with p re – trea tment va lues , and eve n lowered tha n thos e obs erve d in c ontrols. discussion me la to nin has be en sugge sted to ha ve potent antioxid ant pro pe rtie s that ma y preve nt the de velop ment of ca nce r, athe ro scle ros is, and o ther co ns equences of aging, howe ver, the se hypo thetic al e ffe cts are unprove n (27). thus conclus iv e stud ie s re ga rding the relev ance of antioxid ant prope rties of melatonin in the prev ention of dise ase s, like d ia be te s me llitus, a nd the ir co mplic atio ns a re not wid ely ava ilab le . in this stud y, we e valua te d the pos sible antioxid ant ac tivity of melatonin in ame liorating the oxid ant stre ss s tate during dm, and the res ults shown in table (1) v ery well indica ted tha t this lo w d ose o f melatonin res ulted in s ignific ant re ductio n in mda productio n in pla sma and erythroc ytes o f dm patients . this re sult clearly indica te d the promising antioxida nt activity, e sp ecially whe n co rrelated with the o bs erved elev atio n of the solub le antioxida nt, the glutathio ne (ta ble 2 ). higher dos es may be req uire d to a chie ve more imp ro veme nt in the antioxid ant profile, since animal stud ie s s ho wed that, for me la tonin to sho w a potent a ntio xidant a ctiv ity in vivo , ve ry high d aily dos es may b e required (10 mg/kg/da y)(28). res ea rc hers ha ve disco ve re d melatonin to ha ve the mo st p owe rful antioxida nt p ro pertie s, it s cav enges the mo st dama ging fre e rad ic al, the hydroxyl ra dical, five time s be tter tha n gsh, a nd is twice as effe ctiv e in dea ctiv ating the pe ro xyl radica l as vita min e (8). montilla e t al. (1 998 ) demons trated that melatonin s how a ma rked protec tiv e effe ct aga inst o xidant stre ss resulte d fro m hyp erglyce mia a nd protein glyco sylation in exp erime ntal a nimals, two p atho genic corne rs to ne s indica tive o f diab etic comp lica tions (29). the res ults p re sented in table (3 ), indicated a s ignifica nt red uction in fpg and hbaic leve ls a fte r 90 days of treatme nt with melatonin. the pres ente d data a nd the pre limina ry findings of o thers (30) suggest that melatonin is pro te ctive against dm–induced dama ge , eve n at p hys iologic le ve ls . eve n if only pharmaco lo gica lly re le va nt, the findings hav e imp orta nt implications in tha t melatonin has no kno wn to xicity a nd re adily abs orbed when a dministe re d by oral ro ut. t re atmen t p eriod n tc mg/dl tg mg/dl hd l–c mg/dl ldl–c mg /dl co ntro l 20 1 84  5.8 1 14  18 .1 3 7  0 .95 1 25  5.8 ze ro time 30 2 11  16 .7 2 11  33 .7* 3 3  0 .83 * 1 32  14 .2 30 days 30 1 70  15 .1 2 01  29 .9 3 8  1 .9* * 1 00  11 .1 60 days 30 1 68  9.4 ** 1 83  22 .8 4 2  3 .5* * 8 4  8 .2** 90 days 30 1 47  11 ** 1 64  18 .9 4 3  3 .3* * 7 1  9 .2** ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 31 antioxida nts no w a da ys are fo und in many clinical trials, to p la y a ro le in the amelioratio n of the mic ro vas cula r changes during diab etic ne phrop athy, a nd this was clearly de mons tra te d b y naka mura et al. (199 9)(31), who ob se rv ed the be neficial e ffec ts of vit. e on mic ro albuminuria in dm nep hropa thy. tab le (4) clea rly de mons tra te d the effect of a 3 mg/da y melatonin in the red uction of microalbuminuria after 60 d ays, and this co nse quently re flec te d on the improv ement of to tal plas ma protein after 90 d ays , and thes e re sults see ms to be co mpa tible with thos e obs erve d by ha e t al. (19 99), where trea tment with me la tonin de crea se the inc id enc e of early glome rulo pathy in d ia betic ra ts(32). the ab ility o f melatonin to inhib it the impairment of lipid profile in dm patients wa s stud ie d, a nd the res ults p re se nted in ta ble (5 ) showed that treatme nt with 3 mg/da y melatonin improv es the lipid profile through the red uction of tc and ldl–c le vels, as soc ia ted with signific ant elev ation in hdl– c le ve ls . it is po stulated tha t inhib ition of ldl– c oxid atio n by antioxid ants might protect against the de velop ment of a theros clerosis (33), and it is noted that, in bo th huma n and animal stud ie s, res istance of ldl–c to oxid atio n ha s be en a ss ociate d with de cre as ed v as cular co mplic atio ns (34), and protec tio n b y antioxid ants de crea ses s us ce ptib ility to va scula r d is turb anc es d ue to other d is ea ses like dm (35). rec ent studies s ugges te d that hdl–c has a n antioxid ant effe ct o n ldl–c , thus klimov et al. (199 3) rep orte d that hdl–c was p rote ctiv e against ldl– c oxida tion, and this e ffe ct wa s co nce ntra tion dep end ent(36). therefore, the us e of me la tonin alone or with other antioxida nts, may co ntributed to the protec tio n o f ldl– c oxid atio n and re store the e ndo genously pres ent hdl– c molecule s to p la y the protec tive role effe ctively . in co nc lusion, melatonin through red uc ing re marka bly the d egre e o f lipid pe ro xidation, hyperglyc emia, protein glyc atio n, might giv e a ho pe to a promising persp ective o f this prod uct in the trea tment of d ia betic co mplic atio ns . references 1 . okunad e, g.w.; odunuga, o. a nd oloruns ogo , o. iron ind uce d oxid ative stre ss in erythro cyte s memb ra ne o f niddm. bios c. res. 19 99; 19: 1– 4. 2 . rabini, r.a.; te sci, m. a nd galea zzi, t. increa sed susc eptibility to p eroxida tio n of vldl fro m niddm pa tients : a pos sible corre la tio n with fa tty ac id comp ositio n. mo l. cell. bioc hem. 199 9; 199 : 36 – 40. 3. lal, s.; chithra, p . and chandraka so n, g. the po ssible relev ence of auto –oxida tio n glycation in gluco se me dia ted alte ra tions of pro te ins. mol. ce ll. bioc hem. 19 96; 153 : 95 – 99. 4. lee, y.; kim, h. and chai, h. effects of wa te r extrac t o f 1:1 mixture of p hellode nd ron corte x and aralia corte x on po lyol p athway and oxida tive dama ge in diabe tic rats. phyto. res . 1 999 ; 13 : 55 5– 8. 5. knight, j .a. 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taurine and vita min e o n microa lb ominuria, plas ma metallo proteina se– 9, a nd se rum type iv co llagen co nce ntra tio n in p atie nts with dia betic nep hropa thy. neph ro n 199 9; 8 3 (4 ): 361 –2. 3 2. ha, h.; yu, m.r. a nd kim, k.h. melato nin and taurine re duc e early glo merulopa thy in dia betic rats . fr ee radic. biol. med . 1 999; 26 (7– 8): 94 4 –50 . 3 3. s te phens , n.g.; pa rs on, a.; s cho field, p.m.; kelly; f.; chee se man, k. and mitc hins on, m.j . ra ndo mized co ntro lled trial o f vita min e in pa tients with co ro nary dise ase s. lan cet 199 6; 3 47: 781 –78 6. 3 4. regnstrom, j.; nils son, j.; tro nva l, p .; landou, c. a nd ha msten, a. s us cep tibility of ldl to oxida tio n and coronary atheros clerosis in ma n. la nce t 19 92; 3 39: 118 3–1 186 . 3 5. j ialal, i. a nd dev araj, s. ldl oxis ation, antio xidants, a nd athe ro sc le ro sis. clin. chem. 19 96; 42: 498 –50 6. 3 6. klimov, a.n.; gurev ic h, v.s. and nikiforov a, a. antioxidative activity of hdl in v iv o. ath ero sc le ro sis 199 3; 1 00: 13– 18. iraqi j pharm sci, vol.29(2) 2020 quality of life among patients with psoriasis doi: https://doi.org/10.31351/vol29iss2pp161-168 161 assessment of quality of life in a sample of iraqi patients with psoriasis sarah h. abdulridha*, dheyaa j. kadhim**, sarmad a. abdul razzak*** *the state company for marketing drugs and medical appliances, ministry of health and environment, baghdad, iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ***center of dermatology and venereology, medical city, baghdad, iraq. abstract psoriasis is a dermatological, chronic, immune-mediated condition. psoriasis symptoms are not associated with physical burden only, but it may also have psychosocial effects on patients, diminished cognitive control, poor body image and impairments in everyday life. the value of quality of life is important since improving it is the principal goal for non-curative disease. the aim of the current study was to evaluate quality of life in a sample of iraqi patients with psoriasis. this study is a cross-sectional study that involved 300 already diagnosed psoriasis patients who attended to the center of dermatology and venereology, medical city/baghdad. the mean age of patients was (35.156 ±10.549 years). the arabic version of dermatology life quality index was used to assess quality of life. the mean total score is 11.29± 5.45 and the majority of the patients (53.7%) had a total score of more than 10, which indicates a significant deterioration in patients’ quality of life. the greatest impact was found in symptoms and feelings (mean = 1.66 ± 0.75) while the lowest impact was noted in personal relationships (0.51± 0.65). increasing age and monthly income as well as vulgaris type of psoriasis associated significantly better quality of life. while psoriasis area and severity index associated significantly worse quality of life. in conclusion, psoriasis exerts significant, negative impact on patients’ quality of life, especially among those with younger age, lower monthly income, high disease activity, and types of psoriasis other than vulgaris. keywords: psoriasis, iraq, dermatology life quality index, quality of life, psoriasis area and severity index. تقييم نوعية الحياة لدى عينة من المرضى العراقيين المصابين بالصدفية. ***سرمد عدنان عبد الرزاق ،**جبار كاظم ، ضياء1*،عبد الرضاسارة حيدر العراق. ،، بغدادالصحة والبيئة ، وزارة الشركة العامة لتسويق االدوية والمستلزمات الطبية * .، بغداد، العراقبغداد ، جامعةالصيدلة ، كلية السريرية الصيدلة فرع** *** الطب، بغداد، العراق. ، مدينةمركز االمراض الجلدية والزهرية الخالصة تأثيرات نفسية أيًضا لها يكون أن يمكن بل توهن الجسد فحسب، ال الصدفية أعراض إن. مناعي المنشئ مزمن جلدي مرض هي الصدفية لألمراض الرئيسي الهدف هو تحسينها حيث ان مهمة جودة الحياة قيمة إن .وكذلك تأثير سلبي على منظر الجسم والفعاليات اليوميةعاطفية و اجتماعيةو عبارة الدراسة هذه. بالصدفية المصابين العراقيين المرضى من عينة لدى الحياة جودة تقييم هو الحالية الدراسة من الهدف كان. القابلة للشفاء غير /الطب مدينة والزهرية، الجلدية األمراض إلى مركز حضروا المشخصين مسبقا والذين الصدفية مرضى من 300 على أجريت مقطعية دراسة عن لألمراض الحياة جودة دليل من العربية النسخة باستخدام الحياة جودة تقييم تم(. سنة 10.549± 35.156)هو المرضى عمر متوسط كان. بغداد إلى يشير مما ، 10 من أكثر نقاط مجموع على٪( 53.7) المرضى غالبية وحصلت ، 5.45± 11.29 هو الكلية الدرجة كان متوسط. الجلدية العالقات في تأثير أقل لوحظ بينما( 0.75± 1.66= المتوسط) والمشاعر األعراض في األكبر األثر ولوحظ. شديد في جودة حياة المرضى ضعف بينما. أفضل حياة ملموس بنوعية بشكل يرتبط الشائع الصدفية نوع إلى باإلضافة الشهري والدخل العمر ان زيادة(. 0.65± 0.51) الشخصية المرضى، حياة جودة على كبير سلبي تأثير لها الصدفية فإن كنتيجة أسوء. حياة بنوعية ملموس بشكل الخطورة ومؤشر الصدفية منطقة ارتبطت .النوع الشائع غير االخرىالصدفية وأنواع مرتفع، ونشاط مرضي الشهري المنخفض، واصحاب الدخل سنا، أصغر هم الذين أولئك بين خاصة . العراق الخطورة، ومؤشر الصدفية منطقة الجلدية، لألمراض الحياة جودة مؤشر الحياة، جودة الصدفية،: المفتاحية الكلمات introduction psoriasis is a chronic, inflammatory condition that primarily affects the skin, hair, and the joints and associated with a significant humanistic and economic consequences (1, 2). it occurs in about 1% to 3% worldwide, (3) with higher prevalence in western countries. (4) the precise cause of psoriasis stills unclears (5). psoriasis is closely related to genetics, but environmental exposure plays an essential role in the disease development. (4) obesity is another risk factor for psoriasis (6). many medicinal and chemical products, have been found to be related to the triggering and/or exacerbation of psoriasis including beta-adrenergic receptor blockers and tetracycline (7,8). 1corresponding author e-mail: sarahaider801@gmail.com received:15 /3 / 2020 accepted: 14/ 6/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp161-168 iraqi j pharm sci, vol.29(2) 2020 quality of life among patients with psoriasis 162 psoriasis is stimulated by the abnormal epidermal proliferation, leading to scaly erythematous plaques. (9) the pathophysiology of psoriasis includes an interplay between elements of the immune response, inflammatory components and an abnormal proliferation and differentiations of the keratinocytes, (10) results in scaly erythematous plaques. (9) psoriasis has many different clinical presentations. the classic one is of red plaques and silver scale, (11) associated symptoms of itch, burning and soreness. (12) psoriasis is classified as mild, moderate, or severe, relying on the percentage of the affected body surface area (bsa) and the psoriasis area and severity index (pasi). (13) the aims of treating psoriasis are to get an initial and fast control for disease symptoms, decrease the percentage of involved bsa, decrease plaque lesions, minimize adverse drug events, produce and maintain long-term remission, and improve the patient’s quality of life (qol). (11) selection of treatment in patients with psoriasis depends on disease severity, (14) the presence of comorbid conditions, such as psoriatic arthritis, and treatment history. (15) topical treatments are generally used as first-line therapy for most types of psoriasis (16) and include tar preparations, corticosteroids, calcipotriene, adarotene and anthralin. (11) moderateto-severe psoriasis is usually treated by using systemic therapies. methotrexate and biologics are the most-used systemic treatments for psoriasis. (17) biologics are often the next step in the treatment when conventional systemic therapies fail or are contraindicated. (17) biologics are very effective potent immunomodulatory drugs. (18) they are generally considered safe drugs but may be associated with rare serious adverse reactions such as malignancy and reactivation of tuberculosis. (19) the world health organization defined qol as “the individuals ‘perceptions of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns.” (20) the qol's value is important for knowing what is significant to the individuals’ qol since the principal goal for non-curative disease is to improve qol. (21) psoriasis can have a significant impact on patients’ qol and work productivity, depending on disease severity. (22) symptoms of psoriasis are not limited to physical impact, they can also have strong psychosocial effects, (23) resulting in impaired emotional state, daily activity, and body image. (24) accordingly, the aim of the current study was to evaluate qol in a sample of iraqi psoriatic patients and to determine possible association between qol and some patient’s related factors. patients the current study was a cross-sectional study that involved 300 already diagnosed psoriasis patients (mean age was 35.16 ±10.55 years) who attended to the center of dermatology and venereology, medical city/baghdad from october 2019 to january 2020. the number of male patients was 188 (62.67%), while the number of female patients was 112 (37.33%). inclusion criteria 1-patients age ≥ 18 years who willing to participate in the study. 2-the duration of the disease is ≥ 6 months. 3-prescribed pharmacological treatment for psoriasis. exclusion criteria 1patient with cognitive, speech, or a hearing deficits that would affect questions’ understanding. 2-patient with other skin disorders. 3-concomitant other significant diseases (such as diabetes mellitus, hypertension, chronic lung disease, chronic renal failure, heart disease and stroke). 4-pregnant or lactating women. 5-patients who provide incomplete information. method the questionnaires the qol was assessed by using the arabic version of dermatology life quality index (dlqi). the dlqi is a validated, dermatology-specific qol instrument that was developed in the early 1990s, (25,26) and has been used in several trials for psoriasis, (25) the dlqi questionnaire consists of 10 questions, subdivided into 6 domains related to various aspects of a person's qol: symptoms and feelings (questions one and two), daily activities (questions three and four), leisure (questions five and six), work/school (question seven), personal relationships (questions eight and nine), and treatment (question ten). higher scores indicate greater deterioration of the patient's qol and vice versa. (27) total dlqi score between 0 and 1 indicates no effect on patient's qol, 2–5 small effect, 6–10 moderate effect, 11–20 very large effect, and 21–30 indicates extremely large effect on patients' qol. (14) administration of the questionnaires when the patients arrived to the center of dermatology and venereology for checkup and to receive their medications, they were interviewed by the researcher and asked if they are willing to participate in the current study, if they agreed, then a clarification of the questionnaire was given and each patient allowed to fill the research questionnaire which takes about 5-10 minutes to be filled completely. statistical analysis the analyses were done by using the statistical package for the social science (spss, version 22, ibm, new york, usa). descriptive statistics (means, standards deviations, frequencies and percentages) of the participants and disease iraqi j pharm sci, vol.29(2) 2020 quality of life among patients with psoriasis 163 were calculated. multiple linear regression was used to measure the association between total dlqi score and multiple independent variables (sociodemographic and disease characteristics). results sociodemographic features of patients are represented in the table 1. table 1. the sociodemographic features of the patients item subcategor y frequenc y (n) % gender male 188 62.7 female 112 37.3 education level illiterate 20 6.7 primary school 84 28.0 secondary school 104 34.7 college degree 92 30.7 social status single 80 26.7 married 207 69.0 divorced 8 2.7 widow 5 1.7 having children yes 106 35.3 no 194 64.7 living place rural 46 15.3 urban 254 84.7 work status employee 92 30.7 student 26 8.7 retired 91 30.3 selfemployed 6 2.0 wife house 85 28.3 monthly income (iraqi million dinars) < 0.5 117 39.1 0.5-1.0 84 28.1 > 1.0 98 32.8 smoker yes 84 28.0 no 216 72.0 alcohol drinker yes 17 5.7 no 283 94.3 the most common type of psoriasis was vulgaris (77.7%) which followed by erythrodermic and psoriatic arthritis types (6.3%) (figure 1). v u l g a r i s e r y t h r o d e r m i c p s o r i a t i c a r t h r i t i s p u s t u l a r g u t t a t e p a l m o p l a n t a r g e n i t a l 0 2 0 4 0 6 0 8 0 1 0 0 p s o r i a s i s t y p e s % o f p a ti e n ts 7 7 . 7 % 6 . 3 % 6 . 3 % 4 . 0 % 3 . 7 % 1 . 7 % 0 . 3 % figure 1. the types of psoriasis affected the participating patients the most commonly used medications to treat psoriasis were topical steroids (63%), systemic etanercept (biological therapy) (61.7%) and vaseline (41.3%) (table 2). table 2. the prescribed medications to treat psoriasis medication frequency (n) percent (%) topical steroids 189 63.0 vaseline 124 41.3 phototherapy 7 2.3 cinnamon shampoo 6 2.0 zinc oxide ointment 6 2.0 topical calcineurin inhibitors 3 1.0 topical coal tar 2 0.7 systemic treatment etanercept 185 61.7 methotrexate 68 22.7 folic acid 68 22.7 cyclosporine 17 5.7 acitretin 5 1.7 antihistamines 6 2 isoniazid 1 0.3 aprelimast 1 0.3 adalimumab 1 0.3 although the averages pasi and bsa were not high, the psoriasis had very large effect on about half of the patient (49.3%) quality of life (average dlqi=11.29) (table 3 and figure 2). table 3 descriptive characteristics of the disease and patients. characteristic minimum maximum mean std. deviation disease duration (years) 0.5 43.0 13.12 8.60 total dlqi 0 25.0 11.29 5.45 severity index (pasi) 0.5 36.0 8.74 7.15 )2body surface area (m 1.0 90.0 17.25 17.39 total n=300. dlqi =dermatology life quality index. pasi =psoriasis area severity index.. iraqi j pharm sci, vol.29(2) 2020 quality of life among patients with psoriasis 164 0 1 0 2 0 3 0 4 0 5 0 6 0 n o e f f e c t s m a l l e f f e c t m o d e r a t e e f f e c t v e r y l a r g e e f f e c t e x t r e m e l y l a r g e e f f e c t % o f p a t i e n t s a f f e c t i n g q o l 1 . 7 % 1 3 . 3 % 3 1 . 3 % 4 9 . 3 % 4 . 3 % figure 2. effects of the disease severity on patient quality of life the total scores with their sub-scores of patient’s dlqi are shown in table 4. since the average of total dlqi scores was more than 10 (11.2±5.49), that indicates that the patient's qol is being seriously impacted by their skin disease. table 4. the means of dermatology life quality index (dlqi) item mean std. deviation “symptoms and feelings” 1.66 0.75 “daily activities” 1.5 1.52 “leisure activities” 0.71 0.77 “going to work and school” 0.63 0.66 “personal relationships” 0.51 0.65 “treatment difficulties” 1.61 0.97 “total dlqi score” 11.20 5.49 according o dlqi, more than half of the participants experienced itchy, sore, painful, stinging skin (55.7%) and/or embarrassment due to the skin disease (56.0%) in the last week. consequently, the skin disease (psoriasis) negatively impacted many patients’ daily activities (48.3%), clothes wearing (63.4%) and even social/leisure activities (36.6%). additionally, psoriasis disease prevented about one-quarter of the patients from going to work or studying (23%) and caused work/study problems for another third (34.7%) of the participants. more than half of the participants admitted that psoriasis made their home messy (57.3%) over the last week. however, the disease had low impact on patients’ sexual activities (8.7%) and their relations with close friends (20%) (table 5). table 5. patients answers to dermatology life quality index (dlqi) disease consequences in the last week not at all, n (%) a little, n (%) a lot, n (%) very much, n (%) not relevant, n (%) itchy, sore, painful or stinging skin 17 (5.7) 116 (38.7) 104 (34.7) 63 (21.0) embarrassment due to the skin disease 51 (17.0) 81 (27.0) 102 (34.0) 66 (22.0) skin interference with shopping or looking after home/garden 67 (22.3) 88 (29.3) 109 (36.3) 36 (12.0) skin influences the clothes wearing 53 (17.7) 57 (19.0) 125 (41.7) 65 (21.7) skin effects on any social or leisure activities 113 (37.7) 76 (25.3) 64 (21.3) 46 (15.3) 1 (0.3) skin influences on sport/exercise 75 (25.0) 31 (10.3) 20 (6.7) 10 (3.3) 164 (54.7) skin prevents you from working/ studying. no 144 (48.0) yes 69 (23.0) 87 (29.0) skin causes a problem at work/ studying 94 (65.3) 39 (27.1) 11 (7.6) skin creates problems with your partner or any close friends/ relatives 179 (59.9) 55 (18.4) 47 (15.7) 13 (4.3) 5 (1.7) skin causes any sexual difficulties 149 (49.8) 44 (14.7) 15 (5.0) 11 (3.7) 80 (26.8) the treatment for your skin making your home messy 47 (15.7) 79 (26.3) 115 (38.3) 57 (19.0) 2 (0.7) iraqi j pharm sci, vol.29(2) 2020 quality of life among patients with psoriasis 165 multiple linear regression analyses were used to measure the association between different sociodemographic and disease characteristics (independent variables) and total dlqi score (dependent variables) of the participants. age, monthly income and vulgaris type of psoriasis have significant negative association with total dlqi score. while pasi has significant positive association with the total dlqi score (table 6). table 6. association of total dlqi score with socio-demographic and disease characteristics variables of the participants bsa=body surface area; pasi (psoriasis area surface index). *significant association (p-value < 0.05). gender (0=female vs 1 =male). ns=nonsignificant (p-value > 0.05). we excluded some highly non-significant variables (p-value > 0.6) (without mentioning ß) in some regression analyses to fit the model better. types of psoriasis is binary variable = (0= non-vulgaris / 1= vulgaris). discussion psoriasis is a serious dermatological disease extending beyond the physical symptoms experienced by the patient, and seriously affecting the way in which the patient sees him /herself and the way he/she is seen by others. (28) in general, the commonly used measures of psoriasis activity are pasi and bsa. (29) the most widely used qol questionnaire is the dlqi which is short and easy to use. (30) in the current study, more than one-half (53.7%) of the patients had a dlqi score of more than 10, which is more than that reported by a study done by azura mohd. et. al, where about 33.1% of patients had a dlqi score greater than 10, (4) which indicates a more deterioration in qol for patients involved in the current study. the mean of total dlqi score is (11.29± 5.45) which is higher than that reported by cacilda s. et. al, where the mean of dlqi score was (6.5 ± 6.9). (31) the largest impact was found in symptoms and feelings domain (mean score = 1.66±0.75). similar findings were illustrated by cacilda s. et. al, and nilce´ia lopes. et. al, where the greatest effect was noted for symptoms and feelings domain, asserting that emotion play an essential role in the qol of patients with psoriasis, because the skin is largely responsible for an patient’s appearance. (1, 31) this mean that psoriasis has been associated with painful feelings of social anxiety and stigmatization, and fear of rejection. (32) the lowest impact was noted in personal relationships (score = 0.51± 0.65) which may be related to the fact that psoriasis is not contagious, so that the patient’s family and partner can help patients and encourage them to face the disease and come with them to doctor and take medication. in contrast to the result of the current study, a study done in brazil by nilce´ia lopes. et. al., showed that the lowest impact was in work and school. (1) in addition, the majority of the studied patients did not feel embarrassed during sexual activities regardless of the disease severity which is similar to the result reported by amany youssef. et. al. (28) this may be due that most husbands (wives) understand the non-infectious nature of the disease and encourage their husbands(wives) to cure and recover. however; contrary to current results, głowacka et. al., illustrated that psoriasis limit patients' sexual life because of their embarrassment (28). studying the association between total dlqi score with various socio-demographic and disease characteristics variables of the participants revealed a significant negative association between age and total dlqi score (table 6). similar finding had been reported from a study done in korea by sang woong youn. et. al. (29) this may be explained by that younger patients pay more attention to their shape than elderly patients, so the disease effect on their qol is greater. however; results of study done total dlqi score ßeta coefficients p-value age -.135 .048* gender ns social status .074 .273 having children ns educational level ns living place ns work status ns monthly income -.161 .006* smoker .067 .314 alcohol drinker .043 .470 pasi 3.075 .002* )2bsa(m .301 .764 face included 1.966 .050 disease duration(yrs.) .340 .734 types of psoriasis -.163 .005* biologics .003 .959 iraqi j pharm sci, vol.29(2) 2020 quality of life among patients with psoriasis 166 by amany youssef. et. al., illustrated that age played insignificant role. (28) additionally, it was noted that patients with higher income was significantly associated with lower total dlqi score which mean better qol (table 6). patients with higher monthly income can buy expensive medications which may be unavailable at public hospitals all the time and this might be reflected positively on their qol. the results of this study showed a direct association between pasi and total dlqi score (table 6), which is similar to the study done in korea by sang woong youn. et. al., where a greater impairment in qol showed in patients with more severe psoriasis. (29) the predominant psoriasis type was plaque psoriasis (77.7%), which is consistent to the other studies done by liliana g. et. al., (32) and azura mohd. et. al. (4), where the plaque psoriasis was the most common type of psoriasis. vulgaris type of psoriasis has negative association with total score of dlqi, this means that vulgaris type has lower negative effect on qol. this may be explained by the natural course of each type. for example, erythrodermic psoriasis is characterized by abrupt development of erythematous, inflammatory skin plaques with edema (33). also the pustular type is a potentially lifethreatening variant of psoriasis characterized by a sudden and widespread development of superficial pustules that lead to bad appearance and cause anxiety and restlessness. (34-36). in addition, psoriatic arthritis can cause irreversible damage to joints and supporting tissue. (37) which might be reflected negatively on quality of life of patients. conclusions in conclusion, results of the current study document that psoriasis can exerts a significant, negative impact on patients’ qol, especially among those with younger age, lower monthly income, high disease activity, and types of psoriasis other than vulgaris. accordingly, routine assessment is recommended for patients with psoriasis to detect and monitor the impact of the disease and treatment on patients’ qol with subsequent development of care plan to improve it especially in severely affected patients. limitations of the study the first limitation was incorporating psoriatic patients from only one center in baghdad city, so the data does not fully represent all iraqi patients. in addition, the cross-sectional nature of the study makes it impossible to confirm causal conclusions. references 1. lopes n, dias l, azulay-abulafia l, oyafuso l, suarez m, fabricio l,et al., humanistic and economic impact of 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patient-reported outcomes assessment tools for use in psoriasis in spain: a systematic review. actas dermosifiliográficas,english ed. 2019;110(7):56184. 31. souza cs, de castro cc, carneiro fr, pinto jm, fabricio lh, azulay‐abulafia let al. metabolic syndrome and psoriatic arthritis among patients with psoriasis vulgaris: quality of life and prevalence. j. dermatol. 2019;46(1):3-10. 32. jankovic s, raznatovic m, marinkovic j, jankovic j, kocev n, tomic-spiric v,et al. health-related quality of life in patients with psoriasis. j. cutan med. surg. 2011;15(1):2936. 33. rendo m, boster j, dalton sr, yun h. an uncommon presentation of erythrodermic psoriasis in a patient without a history of psoriasis. cureus. 2019;11(7). 34. ly k, beck km, smith mp, thibodeaux q, bhutani t. diagnosis and screening of patients with generalized pustular psoriasis. psoriasis: targets ther. 2019;9:37-42. 35. gooderham mj, van voorhees as, lebwohl mg. an update on generalized pustular psoriasis expert rev. clin. immunol. 2019 2;15(9):907-19. iraqi j pharm sci, vol.29(2) 2020 quality of life among patients with psoriasis 168 36. twelves s, mostafa a, dand n, burri e, farkas k, wilson r, et al. clinical and genetic differences between pustular psoriasis subtypes. j. allergy clin. immunol. 2019;143(3):1021-6. 37. kaine j, song x, kim g, hur p, palmer jb. higher incidence rates of comorbidities in patients with psoriatic arthritis compared with the general population using us administrative claims data. j manag care spec pharm. 2019;25(1):122-32. creative commons attribution is licensed under a bijpsbaghdad iraqi journal pharmaceutical sciences by .university of baghdad -copyrights© 2015 college of pharmacy .international license 4.0 http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 detec tion o f choles tero l in su aed a bacca ta 29 detection of cholesterol in suaeda baccata (chenopodiaceae) suhad s. al-mohammadi *1 *de pa rtme nt o f pharm cognac y,co lleg e of pharmacy, unive rsity o f bagh dad, baghda d-iraq abstract this s tudy de tects the presence of choleste rol in a n iraqi pla nt named suaeda ba cca ta forsk of the family che nopodiacae , wildly and widely grown in ira q. the abs ence of any publica tion c oncerning the sterol content of this suaeda s pec ie , and the indus tria l importa nce of choles te rol depending on its role as a precursor in the synthes is of some hormones , like progesterone , acquired this s tudy its value . the inves tiga tions revealed the prese nce of cholesterol tha t was proved by tlc together with the s ta nda rd compound cholesterol, and anisaldehyde spray reagent using three different solvent sys te ms, the n authe ntic ated by hplc, in whic h the retention time of both the s tandard c holes te rol and the pla nt extrac t cholesterol were identical. key words: cholesterol, suae da bacca t and chenopodiaceae الخالصة مى بـ ول في نبات عراقي مس ستير ول سة تختص بالكشف عن مادة الك را bacca هذه الد ta suaeda (chenopodia ceae) ق را ريا و بشكل واسع في الع وب ورية تتناول المحتوى .الذي ينم ود اي مجلة علمية او د ان عدم وج ول للصناعة الد ستير ول مية الك عض الهرمونات الستيرويدي لهاذا الصنف من النبات وأه ائية لتصنيع ب ونة المادة االبتد وائية بك ستيرون مة هذة الدراسة,مثل البروج ود مادة الكولستيرول والذي اكد بواسطة استخدام . يبين قي بينت التجارب البحثية وج ماتوغرافيا الطبقة الرقيقة واسطة ثالثة أنواع من المحاليل الناقلة tlcتقنية كرو كيدي لمادة كما تم إجراء .ب ص التأ الفح ول القياسي hplcالكولستيرول في المستخلص النباتي بواسطة ستير ول ق الك .والذي طاب in troduction the chenopodiac eae (go osefoot fa mily) is a la rge fa mily includ ing about 1 02 ge ne ra , and 1400 sp ec ies of low growing plants. (1) me mb ers o f this fa mily inc luding sua eda baccata mos tly grow naturally in soils co ntaining much s alt (halop hyte s) (2). sua eda bacca ta specie is distrib uted in iraq , in so uth of ja zira distric t, so uthern de se rt district, we stern des ert ce ntra l alluvial plain district a nd eas te rn alluvial distric t (3).the photo of this p la nt is demonstrated bellow lite ra ture survey indicated tha t d ifferent spec ies of the ge nus suae da co ntain several different comp ounds , including s te ro ls (4, 5), th us it was d eeme d desirable to find out the sterol conte nt o f this p lant. s te ro ids constitu te a natural p roduc t class of c omp ounds tha t is wid ely distributed thro ughout nature. (6) the che mical s tructure of steroids is ba se on (a) perhydrocyclopenta p henanthre ne nucleus. the steroida l nuc le us is deriv ed from is ope ntyl pyrop hosphate as fo llows : aceta te me valonate isope ntenyl p yrophosphate sq ualene cho lesterol pa thway .(7,8) 1corresp ond in g author ema ilsu hadsami04@yahoo.comsu hadsami04@yahoo.com rece ived 28-1-2006 accepted 4 -11-2006 mailto:suhadsami04@yahoo.com ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 detec tion o f choles tero l in su aed a bacca ta 30 accata] suae da) [pho togra phy o f figure (1 the firs t steroids isolated fro m nature were a se ries of c2 7 –c2 9 alco hols tha t were found in lipid fractio n of ma ny tis sues . these co mpounds were solids and therefore name d sterols fro m the gree k stereos, me aning so lid. the most widely occurring sterol is c holesterol, (greek word, c ho le , me aning b ile ). it was first isolated from huma n ga llstones . until rece ntly choles te rol was thought to be res tric ted to the anima l kingdom; however, it has now bee n identifie d in p la nts.(7, 9, 10). the re is a wide sprea d b elie f among the public a nd ev en a mong the chemists tha t plants do not c ontain choles te ro l and this is a n erro r, further mo re e ven some refe rence s re fe r to this mistake (11, 12). the fa ct is that the cho le sterol wid es pread in the plant kingdom (a ltho ugh othe r related sterols, s uch as β-sitosterol ge nerally o ccur in highe r qua ntity) (13, 14). cho le sterol o cc urs in b oth free a nd e sterified. it occ urs as a c ompo ne nt o f plant memb ra ne s and a s part of the s urfa ce lipids of lea ve s whe re it is s ome time s the ma jo r sterol. the qua ntity o f choles te ro l is ge nerally sma ll when exp re ss ed a pe rc ent o f to ta l lipid. while cho le sterol a verages p erhap s 50 mg/kg total lipid in p la nts, it can b e as high as 5 g/kg (or more) in animals. (15). the qua ntities o f choleste rol in a numbe r of vegetab le (plant) oils are giv en in the fo llowing tab le . ta ble (1) cho le sterol conte nt o f so me plant oils source cholste rol (mg/kg ) re fe re nces 1 pa lm oil 20 16 2 pa lm ke rnel 17 17 3 coconut o il 14 17 4 cotto n se ed oil 45 17 5 soybean o il 29 17 6 corn oil 55 17 7 p eanut oil 24 17 8 s un flo wer oil 14 17 9 cano la oil 53 17 1 0 av ogadro oil < 30 18 1 1 olive oil 0.5-2 19 ,2 0 1 2 .s esame o il ca.1 19 ,2 0 chole sterol and its e sters a re imp orta nt constituents o f plant me mbrane s. the follo wing ta ble re prese nts so me da ta on the sterol frac tion o f s ome plant o rgane lles . ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 detec tion o f choles tero l in su aed a bacca ta 31 table (2) s ub-ce llu la r distribution of choles te ro l in plant while c hole sterol is us ua lly a minor co nstituent of the sterol fraction in plants , it is the major constituent of s ome p la nt s urfa ce a s shown in table (3) table (3) ste ro l co nte nt of rape (canola) (23) so urce choles te rol % sitos te rol % 1. leaves a. s urfa ce 71.5 0.6 b. intra ce llular 15 30 2 . se e ds a. s urfa ce 7 .2 62 b. intra ce llular 0 .7 67 3 . se e dpods a. s urfa ce 35 21 the propo rtion of choles te ro l in the sterol frac tion o f lilia cea e, solanac eae a nd scrop hulariace ae familie s is e sp ecially la rge. (24, 25) chole sterol (c27 h46 o), 3β-chole …..s ta-5-e n3ol, mol.wt. 3 86.66 , is practica lly inso luble in water, s lightly s oluble in alco hol, more solub le in hot a lc ohol, one gram diss olves in 2.8 ml of ether, in 4 .5 ml of chlo ro fo rm, in 1.5ml of pyridine, also it is solub le in pe trole um ethe r, benze ne(26) . choles te ro l s truc ture is rep re se nted b ello w. (26) figure (2) c hole ste rol s truc ture altho ugh tota l synthes is o f so me med icinal steroids is e mployed co mmercially, there is also a great d ema nd for natural p ro ducts which will se rve as a s tarting ma te rial for the ir p artial synthe sis (2) acc ordingly, c holes terol has s erve a s a precurs or for the synthes is of p roges te ro ne (27) as repres ente d in the follo wing diagram, and this a cq uire d this study its impo rtance. so urce free cho le s tero l% cho le ste ro l ester% re fer ence 1. gre en bean leav es a . whole 1 1 2 1 b. chloroplasts 2 4 33 c. mito chondria ---- d. mic ro so mes 1 28 2 .etio la ted be an leav es a . whole 6 23 2 1 b. chloroplasts 2 7 26 c. mito chondria ---- d. mic ro so mes 6 34 3 . orga nelles o f day maiz e -21 shoo ts a . n ucle i 2 2 76 2 2 b. chloroplasts 2 52 c. mito chondria 1 32 d. mic ro so mes 1 32 ch3 ch3h3c ch3 ch3 ho a b c d ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 detec tion o f choles tero l in su aed a bacca ta 32 figure (3 ) :conve rsion o f choles te ro l to pro ge ste rone (27) material a nd meth ods the p la nt materia l (a eria l pa rt) wa s collec te d during mo nths o f july, augus t, and s epte mber 200 5. f ro m the high wa ys of baghdad c ity. the p la nt was identified by the de pa rtment of pha rmaco gno sy , c olle ge of pha rmacy/university of baghda d ; and authentica te d b y the na tional he rb arium of iraq . bo ta ny directorate a t abu-ghra ib , ira q. forty grams o f the dried a eria l pa rts were first mac erated with 50 0 ml o f n-hexane for 2 4 ho urs. the res id ual plant pa rt then wa s drie d at ro om te mperature, s oa ked in wa te r fo r 2 4 ho urs, d ried , and then refluxe d with 2 nhcl solution fo r two hours . afte r filtra tio n by buc hne r funnel, 5% ammonia so lution was add ed to the re sidual plant part, and then washed b y distilled wate r se veral times , until neutral. this plant pa rt a fte r drying ov er night will be e xtracted again with 25 0 ml of petroleum e ther (60 0c -800c ) for ten hours by the use o f soxhlet a ppa ra tus. the pe troleum ether filtrate will then b e ev apo ra te d to dryness to b e re ady for the id entifica tion o f s te ro id by tlc. (28) the following d ia gram re pres ents the extraction proc ed ure o f ste roids from sua eda bac ca ta . ho ho oh oh ho o o o ho o o o o2 na dph o2 na dph nad + keto-e nol t automerism enol-keto tautomerism p rogesteron e cholesterol preg nen olo ne ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 detec tion o f choles tero l in su aed a bacca ta 33 40 g m. of the d ried aerial p lant part ma ce ra tion with 50 0 ml n-he xa ne fo r 24 hr. res id ual plant pa rt filtrat 1 . drie d at room te mp. 2. s oak in wa te r for 2 4hr. 3. reflux with 2nhcl fo r 2hr. f iltra te residual plant part add 5% nh3 wash several times with d.w plant p art 1 dried ov er night at roo m te mp 2 reflux with p etro le um ethe r (60-80 0)fo r 10 hr. (s oxhlet). marc filtra te ev apo ra te to d ryness tlc de te ctio n for s te ro ids bacc ata suae da sche matic proce dure for the e xtrac tion o f ste roids from)figure (4 id entification of th e stero id (ch ole ster ol) id entific atio n wa s p erfo rme d firs t by tlc, us ing silic a gel g, anisalde hyde sp ra y re agent (29,30)[0.5ml anisa ld ehyde is mixe d with 10 ml glac ia l ac etic acid, fo llowe d by 8 5ml metha nol and 5 ml c oncentrate d s ulphuric ac id in that order. the tlc plates a re s pra ye d with 10 ml, he ated at 1 100 for 5-10 minute s.], s tand ard choleste rol, a nd diffe re nt s olve nt s ys te ms that were: (31,32) solve nt (1 ): toluene: ethyl ac etate (9 0:10) solve nt (2 ): benzen: ac etone (9 0:10) solve nt (3): pe troleum e ther (600-800): e thyl ac etate (7 5:25) the n this ide ntifica tion wa s authe nticated b y hplc result a nd disc ussions cho le sterol can b e fo und in p la nt eithe r in the free state or c onjugate d as s imp le glyco side(33) therefore the e xtra ctio n proce dure inc luded the us e o f wa ter and ac id , ne ces sa ry fo r the clea vage of the glyc osidic linkage and the re le as e of the a glyc one p art (cholesterol), and the pos sible suga r site atta chment is c3a to m in its structure. as the c holes terol is so luble in petroleum ethe r, the re fo re this s olve nt was use d in its e xtra ctio n. the identific ation of c holes terol was performed b y tlc using thre e diffe re nt solve nt sys tems s1 , s2 , s3 . as rep re sente d in the fo llowing diagrams : ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 detec tion o f choles tero l in su aed a bacca ta 34 a b b a b=ae rial plant e xtract b a the rf. va lue of e ach solvent system of the standard c holesterol and the plant e xtra ct cho lesterol, a re represente d in the fo llowing ta ble. ta ble (4) [rf va lues o f bo th, the ae rial plant part e xtrac t and standard cho leste rol] solve nt s ys te m rf. of standard c holes te ro l rf. o f the plant extra ct s1 (toluene: ethyl ace ta te (9 0:10) 0.73 9 0.730 s2(benze n:acetone (90 :10) 0.19 2 0.192 s3 (petro le um ether (6 00-800): ethyl ac etate (75 :25) 0.51 3 0.521 figure (5 ): tlc plate of the aeria l plant extra ct, a nd standard, using s 1 mobile pha se a=standard cho le sterol b=ae rial plant e xtract figure (6) :tlc plate o f the ae rial pla nt extra ct, and standard, using s2 mo bile pha se ) a=s tandard choles te rol b=ae rial plant e xtract figure (7 ): tlc plate of the aeria l pla nt e x tract, a nd standard, us ing s3 mo bile phase a=sta nda rd c holes tero l b=ae rial plant e xtract ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 detec tion o f choles tero l in su aed a bacca ta 35 furthe r id entifica tio n to the choles te ro l in the plant extract was perfo rmed b y hplc. in which the retention ti me of bo th the s ta nd ard choles te rol and the plant extrac t c holesterol were identical a s rep rese nted in the cha rt bellow. table (5) :ta ble of the hlpc conditions conditions of c hole ste ro l hplc (19) mob ile phase ace to nitrile:methanol 50 :50 containing 3% d.w column c18 detec to r 2 10 flow rate 1 ml/mi n inje ction volume fig ure (8) :hplcchart of pla nt e xtrac t figure (9) :hplc c hart o f standard cho le ste rol conclusion sua eda bac ca ta f orsk (che nop odia cae ), a wild iraq i plant, c onta ins cho le sterol a s one of its constituents . refe ren ces 1. rowley gd, ja co bse n h. (ed ):”a hand book of s ucc ulent plants .” vol.1, lo ndo n: bla nd ford pres s. 1 960 , p.1 . 2. tre as e and eva ns ;” pha rma cogno sy” unive rs ity of nottingha m, nottingham, uk, 15thed. 200 2, p .2 2, 2 90 . 3. ali-alrawi; “ wild plant of iraq ” minis try of agric ulture , direc to ra te general of agric ulture res earch and pro je cts. 196 4, p17 6 4. rizk, a.m.; heiba , h.i; ma’ayergi, h, a; batanouny, k.h.;”co ns tituents of plants gro wing in qa ta r.” s ci. appl. res .c ent. unive rs ity. qatar, doha, argent. 1 986 , 57 (1 ), 3-9(eng). 5. s ubrah mangam, c.; rao , k.bha skara; ra o, c.venkates ara;”chemica l examina tion of the mangro ve s pec ie s s uae da maritima and sua eda monic a.”(dep . org. chem. f ood s, drugs water, andhra univ ersity vis ha kha pa tnam, 53 000 3 ind ia ). ac ta ciene. indica chem. 199 2, 1 8(1), 7-8(eng). 6. varro e.tyler, lynn r. bra dy and james e. robb ers:”p harmac ognos y” philade lp hia, u.s .a. 9thed. 1 99 7, p .1 56. 7. ibid pp . 1 58,16 0. ir aq i j.pha rm.sc i., vol.15 (2 ) ,2006 detec tion o f choles tero l in su aed a bacca ta 36 8. erich h.”bios ynthes is of p la nt steroids ” jo urnal. na tura l prod uct (lloyd ia ) 19 68, vol.31, 1-4 p.29 3, 1 71. 9. koji n, to shio g., shoi, shinsa ku n., and shigeo n.” na tura l p ro duc ts che mis try” kod ans ha ltd. aca demic pres s, inc. new york and london, 19 74, p.422 . 10. j . b. ha rb ornc” phyto chemica l me thod ” london cha pman a nd ha ll 197 3, pp.11 0, 1 13. 11. horto n, h.r.; moran, l. a., ochs, r.s .; rawn, j .d., s crimgeo ur, k.g." princ iple s of bio che mis try" 3rd ed , p re ntic e hall; up per sad dle river, 2 002 ; p p 275 . 12. zuba y, g.l.; parso n, w.w.; vance , d.e." principles o f biochemistry" 199 5 pp3 85. 13. s hukla vks et al., "j aocs " 200 2,camelina oil, 79, 965 . 14. noda m et al., "lipids " 19 88, 23, 439 . 15. be hrama n e.j . and venkat gopa la n"j. of chemica l educa tion", ohio s ta te unive rs ity, columbus , 20 05,vo l.82 , no .1 2, . 16. gurr, m.i., "role of fats in f ood and nutrition" 2nd edn.; els ev ie r,londo n, 19 92, pp.3 6. 17. ross ell, j.b." analys is o f oils ee ds, fa t, and fatty food s", els evier, london 19 91, chapter7, tab le 7 .1 1. 18. itoh, t.;ta murat t.;mas umoto, t., dup agine p."fruits"1 97 5, 3 0, p p.687 69 8. 19. casta ng j . ana . f alsif. exp ert chim. 19 81, 74, pp 6 97-700 . 20. kochha r, s.p."prog. lipid. res ." 198 3, 22, pp 1 61-188 . 21. bra ndt r.d.; benve niste, p." biochemistry and bio physics " 19 72, 282 , 85 -92 . 22. ke mp, r.j; me rc er e.i." bio chemistry j."1 968 , 11 0,119 -1 25. 23. no da , m.; ta na ka, m. ,se to y., aib a, t."lipids ", 199 8, 2 3, 2 39-44 4. 24. akihisa t.; kokke, w.c.; tamura , t." physiology a nd bio che mistry of sterol" american oil chemist's so ciety, cha mpaign, 1 991 ; chap te r 7 . 25. ha rtma nn, m.a."lipid metabo lism and membrane biogene sis" dann g., ed n, springe r, berlin 20 04, chapte r 5. 26. me rc k and co., inc. ra hwa y, n.j .:” the merc k inde x” 8 th ed. u.s .a. 19 66, pp. 369 . 27. de wik, p .m. "med ic inal natural products, a bios ynthetic ap proac h" j ohn wile y and sons ltd, chi che ster, reprinted 19 97 . 28. ab dul-wahe d m. “ inves tiga tio n o f the steroidal s ap oge nins of tribulus sp p. wild ly gro wn in iraq” b.s c 200 2 p.38 . 29. wa gne r, w.; bla dt, s. and zga inski, e.m: “plant drug ana lysis” s pringe r-ve rlag be rlin heidelberg new yo rk-to kyo 1 98 4 p.299 -3 04. 30. s ta hl e. “ thin layer chromatograp hy” springe rverla g berlin p . 19 99 pp .3 23, 325 . 31. tagree d mh. ” plant tis sue culture of trigo ne lla foe num-gra ec um for dios genin pro ductio n” 19 97 p .4 8. 32. p ushpa k. and sa tish c.j.”dios ge nin, gitogenin and tigogenin from trigonella fo enum-gra ce um tis sue cultures .” lloyd ia 197 3,vol.36 p.36 , 33. j .b. ha rb ornc”phytochemical metho d” london chap man a nd ha ll 1973, p.110 , 1 13. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 74 conjugation of steroidal and non – steroidal anti inflammatory drugs as possible mutual prodrug muthanna d. saud* receive d 30-12-2002 ac cepted 15-5-2005 abstract prednis olone (s aid) was c onjugated with ibup ro fe n (ns aid) thro ugh an a mino ac id (glycine) as a s pac er arm to s ynthe size the fo llowing c omp ound: pre dniso lo ne – g ly cine – ibupro fe n. the me thod e mploye d co nsists o f co nve rting the carbo xylic ac id functio n of (r,s) – ib up rofen – glycine to the highly reac tiv e acid c hloride and s ub se que nt rea ction with the c21 hydroxyl group of prednis olone . this re active intermed ia te was found to re act as we ll with the c17 te rtia ry hyd ro xyl group of the s te ro id to fo rm thre e c omp ounds and e ight d ia stereo mers. thes e res ults w ere confirmed by t.l.c, and the des ired c omp ound was s epa ra te d by co lumn chro matogra phy. the id entity of the p re pared c omp ound was e stab lished using u.v sp ec tros cop y, ir sp ec tros cop y and eleme ntal mic roa na lysis.the partition co effic ie nt (pc) fo r this co mpound wa s es tima te d and fo und to b e more so luble in the organic p has e (n – octanol). prelimina ry kinetic stud y indica te d that the comp ound ne eds more than 1 5 hours fo r significa nt hydrolys is in p hos phate buffer ph 7.8. خالصة ال عقار الستيرويدي مضاد (مع عقار االيبوبروفين ) مضاد لاللتهابعقار ستيرويدي (تضمنت هذه الدراسة ربط عقار البردنزولون  –كاليسـين   –باستخدام حامض أميني هو الكاليسين كذراع مباعدة فراغية٬ لتحضير المركب النهائي وهو بردنزولون ) لاللتهاب ب  . ايبوبروفرين ي مرـك وبروفين   إن الطريقة المستخدمة للربط قد اعتمدت على تحويل مجموعة الكاربوكسيل ـف كاليسـين   –االيـب ولقد تبين أن هـذه المجموعـة   . إلى مجموعة كلورايد الكاربوكسيل الفعالة جدا كمجموعة باحثة عن االلكترونات) المحضر سابقا( 2باإلضافة إلى تفاعلها مع مجموعة الهيدروكسيل على ذرة الكاربون  في جزيئة البردنزولون لتكوين آصرة االستر٬ فأنها كذلك  1 .في جزيئة البردنزولون 17اعل مع مجموعة الهيدروكسيل على ذرة الكاربون تتف ذي    إن النتائج المستحصلة قد تم التأكد منها باستخدام تقنية كروماتوغرافيا الطبقة الرقيقة٬ وكذلك فلقد تم فصل المركب النهـائي اـل 2ة الكاربون ترتبط فيه مجموعة الكاربوكسيل الفعالة مع مجموعة الهايدروكسيل على ذر بشكل كمي ونقي٬ وباستخدام تقنية  1 .كروماتوغرافيا العمود لقد تم التأكد من صحة التركيب الكيمياوي لهذا المركب باستخدام طيف األشعة فوق البنفسجية٬ وطيف األشعة تحت الحمراء٬ وكذلك  مركب المحضر حيث تبين أن هذا المركب أكثر ذوبانا لقد تم حساب مقدار معامل التجزئة لل. بالتحليل الكمي الدقيق لعناصر المركب ول الفوسـفات          . في المذيب العضوي منه في المذيب المائي ي محـل ذا المركـب ـف ل ـه ة لسـرعة تحـل كما تبين من خالل دراسـة ابتدائـي 1إن تحلل هذا المركب يتطلب أكثر من , 7,8الدوارئي ذي األس الهيدروجيني  .ساعة 5 introduction drug ta rgeting to spe cific rec eptors or spe cific orga ns ha s bee n one of the main objec tive s o f the med ic inal and pha rma ce utic al c he mis ts from the be ginning of the p as t ce ntury. howev er, only in the pas t 30 years or s o hav e there be en a ny p ro mis ing de ve lo pments in a chiev ing this goa l(1). the s ite – s pec ific delivery of d rug is ind ee d a v ery attrac tiv e goa l be cause this prov id es one of the mo st significant potential wa ys to improve the therape utic ind ex of the drugs(2). when a drug is delive red p re fe re ntially to the s ite of the ac tion by v irtue of this de sire d differential distribution, it will sp are the re st of the bod y; thus it will b e s ignifica ntly re duc e the ov erall toxic ity while maintaining its the ra peutic be ne fits(3). one o f the a pproa ches for site – spe cific d rug de live ry is the che mical ap pro ac h or s o ca lled site – s pe cific c he mic al de live ry systems (cds s) which prov id e a wid e v arie ty of pos sibilitie s fo r s ite – e nhanc ed or s ite – spe cific d eliv ery(1,4,5). re actions by whic h the p arent drug is cov alently co upled with o ne or mo re c arrier moietie s. by d es ign, a fter d eliv ery the cds will unde rgo a v arie ty o f enzymatic convers ions, which prod uce inte rme diates a ll hav ing diffe re nt physica l prop erties and varying rates of formatio n a nd eliminatio n, thus ultima te ly allowing a p re fe re ntia l and fa vorab le d is tribution o f the prec urso r prodrug at the s ite of the a ctio n where ultima te ly the drug is re le as ed(6,7). co lo n – spe cific de live ry of bioa ctiv e c omp ounds re ce iv ed exte nsive inves tigatio ns , utilizing the s ignifica ntly variable bioe nvironments of the diffe re nt p arts of the g.i.t.(8,9). * dep artmen t of phar mace utic al chemistry, co lleg e of ph arma cy,un iver sity o f bagh dad , bagh dad ir aq . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 75 cortico steroids we re c urre ntly use d for the trea tme nt o f infla mmatory bowe l dise ase s. the y are us ed eithe r alone or in c ombina tio n with other d rugs(10,11). in a rece nt inv estiga tion in this lab oratory dexame thas one (said) was c onjugated to metro nidazole through a phosp hod ie ster linkage . this p oss ible p ro drug, which wa s fo und to be insolub le in the a queous med ium at low ph, was sugge sted to be a ble to re ac h the lo wer p art of the g.i.t. in which it will gain e nough aqueous s olubility to b e hyd ro lyze d through e nzymatic and /o r no n – enzyma tic proc ess es to libe ra te its ac tiv e moietie s(12). in this inv es tiga tion we wo uld like to re po rt the s ynthes is o f the follow ing conjuga te : prednisolone – glycine – ibupr ofen ib uprofen – glyc ine conjuga te , which wa s previously synthe sized (13), was c onverted to the a cid chloride through rea ctio n with thio nyl chlo ride . this re ac tive intermed ia te wa s allo wed to re act with the c21 hydroxyl group of prednis olone to form the final c onjugate. the rea ctiv e intermed ia te wa s found to re ac t, though to a muc h le ss er exte nt, with c1 7 hyd ro xyl group o f the cortis one a s will b e des crib ed in the follo wing se ctions . experimental section materials: the amino a cid glycine wa s purchas ed from hop kins a nd williams ltd. engla nd. pre diso lo ne and ib uprofen we re a gift from the jo rd anian p ha rma ceutic al ma nufac turing comp any ltd. the ide ntity and purity o f thes e co mpound s were c hec ke d a cco rd ing to the b.p and merc k inde x. n,n' – dic yclohexylcarbo diimide (dcc) was from acros usa. the remaining c he mic als were o f re age nt grade , a nd were us ed a s such without further purifica tion, sinc e they we re of the highe st commercially a vailable p urity. ge neral me thods: all re ac tions, througho ut this work that ne ed a co ns ta nt tempe ra ture , were carrie d out in a thermos ta ted do uble jac ke te d flas k connec ted to a co nsta nt temp era ture circ ulator a nd re frigerator o f ultra – te mp 200 0 jullab lo vc. chiller, germany. melting po ints were meas ured us ing a n electrothe rma l melting point a ppa ra tus and were uncorre cted . thin layer chro matogra phy (tlc) us ing silica gel coa te d glas s plates was p erfo rme d to follow up chemica l re actions. the purity o f the p re pared c omp ounds wa s checked by thin layer c hroma to graphy plates of (20x20) s ilica ge l (60 f254) with 0 .2 5mm layer thic kne ss o btaine d from me rc k, ge rmany. chromatogra ms were eluted by o ne of the following s olvent s ys te ms: a: me ntho l: ammonia (100 :1 .5 v:v) b: benzene: ether: ac etic a cid: me ntho l (12 0:60:18 :1 v:v) c: chloroform d: acetone: n – hexane (33 :6 7 v:v) e: benzene: ether: menthol (6 0:35:5 v:v) the chro matogra phic s pots we re rev ea le d b y e ithe r re activity with iodine v ap or or by ob se rving them under uv light. ir sp ec tra were reco rd ed on perkin – elmer s pe ctro sc opy, engla nd. uv sp ec tra we re c arried out a t the na tional cente r for p ha rma ceutic al re se arch and quality control, baghda d, us ing cec il l – 41 1, f ra nc e. column chroma tograp hy were carrie d o ut using glas s c olumn (75 cmx2 0mm) p re pa cka ge d with 5 0gm of s ilica gel (kies elgel 6 0) s us pe nde d in 10 0ml of c hloroform. ele menta l micro analysis (chn) was p erfo rme d a t the univ ersity of mouse l, colle ge of s cienc e using (chn) a nalyzer type 11 06 ca rlo erba. the ph va lues we re me as ured using pye unica m p h meter (philips), holla nd. chemical synthesis: synthe sis of ibupr ofe n – glycine acid c hlor ide , (n – [2(4 – isobutyl phenyl) propionyl] – glycyl c hloride), compound i(14 )., (scheme 1): ibuprofen – glucine (1.5gm, 5.7m mo l) w hich was prev io us ly synthe size d(13), was d is so lv ed in 1 0ml chlo ro fo rm a nd the s olution was coo le d to 5ºc. an e xc ess thio nyl c hlorid e (2.5ml) was a dde d d ro p w is e with co ntinuo us stirring, during which the te mperature of the re ac tion mixture was kep t below 10ºc. the mixture was then refluxed for more tha n 2hrs . until the e vo lution of gas eous s o2 and hcl w ere c ea se d. the so lv ent wa s e va porated to d ryne ss in va cuo and the re sidue was red is so lv ed in c hloroform a nd ev apo ra te d. this proce ss wa s repe ated s eve ra l time s in o rd er to remo ve exce ss thionyl c hlorid e. ibuprofen – glyc ine a cid c hlorid e was o btaine d as a faint yello w o ily residue and w as used as s uc h for rea ctio n with p re dnis olone . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 76 synthesis of ibupr ofen – glycine – pre dnisolone (n –[ 2 –(4 – isobutyl phe nyl) propionyl] – glycyl – (11,17α – dihydroxy pre gna – 1,4 – diene – 3,20 – dione – 21 – yl) – e ste r) (compound ii): ib up rofen – glycine ac id chlo ride (1 .4 gm, 5mmo l) was diss olved in 1 0ml of dry dichlo romethane, and the solution wa s coo le d to 0 ºc. pre dnis olone (2.52 gm, 7 mmole) wa s diss olved in 2 00ml a cetone a nd 1.5ml of triethyla mine was a dd ed. the cortis one s olution wa s add ed d ro p wis e to the co oled ac id chloride so lution o ve r p erio d of 3 – 4hrs with co ntinuo us stirring. the re ac tion mixture was the n stirred at 25ºc for 48hrs . afte r that it was filtered to remov e trie thyl ammo nium c hlorid e and the filtrate wa s ev apo ra te d to d rynes s under v ac uo to a v ery thick brown p as t. the pa sty re sidue was dis solve d in e thyl ac etate and washed with 0.1n hcl, then with distilled wa ter, the n with 5 % nahco3 so lution, twic e with dis tille d w ater. the e thyl ac etate laye r wa s drie d ov er a nhydrous ca lcium chlo ride a nd filtered. comp ound ii was the n sep arated a s dias te re ome ric mixture b y column chro matograp hy using s olvent s ystem e as the mob ile pha se (15). many a tte mpts were pe rformed to crys ta llize the pas t p ro duc t but a ll were faile d. the purity a nd identity of this c ompo und were co nfirmed us ing t.l.c., u.v. sp ectros cop y (figure 1 ) i.r s pe ctro sco py a nd c.h.n analysis . (nujol) : 3 600 – 3 300 , broa d (n – h, o – h s tre tc h. hyd rogen b ond ing). 307 0 (c – h aromatic stre tch), 2 950 (c – h) alip ha tic stretch), 1 750 (c=o, ester), 17 20 – 171 0 (c=o), ke to ne), 1 660 – 1 650 (n – c=o amide ), 1 380 , 13 50 (gem – d imethyl c – h be nd.), 16 20(c=c s tretch.), 1 220 – 1 190 (c – c – o s tre tc h.), 142 0 (c – n stretch), 700 (c – h a ro matic o ut o f plane b end .), 90 0 n – h wagging. eleme ntal a nalys is, c alculate d for c36 h 4 7 no 7 – h2o: c; 69 .3 4, h; 0 7.86, n; 2.24, fo und: c;70.12 ,h;0 7,52,n;2.55 .t.l.c; rf va lues; 0.83(a); 0.35 and 0.4 (b); 0 ,1 8(c); 0.75 (d); 0 .5 2 and 0.61(e). fig(1) uv spe ctrum of co mpou nd ii de te rmina tion o f pa rtition co e fficie nt: partition c oeffic ie nt (pc) fo r a s olute could be de te rmined us ing the follo wing relatio n: p c = where co = the co nce ntratio n of the s olute in organic p ha se, and cw = the c onc entration of the so lute in the a queous phase . partition co efficient for c ompo und ii has b een performed by a dding 2 5mg of the s olute to a sep aratory funnel c onta ining 25 ml of water pre – saturate d with octa nol and 2 5ml of octanol pre satura te d with wa te r. the sep aratory funnel was inv erte d se veral times during 30 min., a fter that it wa s le ft fo r complete sep aration of the two p ha ses . the aq ueo us p has e was a na lyzed fo r the s olute. a sta nda rd curv e had be en constructed by meas uring the a bso rb anc e of diffe re nt conce ntra tions of c omp ound ii, (figure 2). the cw co ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 77 pa rtition co efficient fo r our c ompo und wa s fo und to b e 82.3 . fig .2 abso rba nce of c ompo und ii at λ 3 70 ve rs us c once ntratio n (solve nt e tha nol) on the othe r ha nd the s ta bility of c ompo und ii in phosp ha te b uffe r (0 .1 m, ph 7.8) wa s determine d ov er a p erio d of 24 hrs b y incub ation of 10 0mg of c omp ound ii in 10ml of a queous phosp ha te b uffe r at 37ºc. an aliq uot (2 ml) of e ach s amp le wa s ta ke n at c erta in inte rv als (3 0min, 10hrs , 2 0hrs, and 25hrs ) and was me asure d fo r the remaining amount o f compound ii b y converting the abs orbance to the c orre spo nd ing conce ntra tion. a p lo t was constructed , c onc entratio n of the re maining amo unt of co mpound ii v ersus time (figure 3 ). fig.3 showing the hydrolys is of compo und ii in pho sphate buffe r (0.1 m , ph 7.89 ) at diffe re nt time inte rvals result and discussion synthesis of ibupr ofe n – glycine acid chloride: the goal for this inv es tiga tion was to co njuga te the c arbo xyl moiety of ibup ro fe n – glycine with the c21 hyd roxyl group of prednis olone thro ugh e ster linkage. to a chie ve this go al the c arboxylic ac id functionality should be a ctiv ated . the activation ma y either b e thro ugh the fo rma tion of a cid a nhydrid e us ing (dcc) as the dehydrating agent(16,17), o r though conversion to the ac id chlo ride whic h is ve ry reac tive inte rme diate. in this inve stigation ib up rofen – glycine was converted to the ac id chlo ride (scheme 1) thro ugh rea ction with thio nyl chloride. the a dva ntages of thio nyl chlo ride o ver o ther chlorinating a ge nts lie s in the fac t the byp ro duc ts o f the reac tio n (so 2 and hcl) are gas es a nd ca n be ea sily remo ved througho ut the c ourse of the re ac tion. more ov er any exc es s of the low – b oiling thionyl c hloride (7 9ºc) is eas ily remo ved by distillation. the ac id chloride thus formed is highly re active electop hilic spe cies , whic h we nt add ition at the ca rb onyl group by an a mine or hyd ro xyl groups follo wed by elimina tion of the chloride re sidue, to form an amide or e ster linkage s. synthesis of ibuprofen – glycine – prednisolone (compound ii) (sc heme 1): this w as p erfo rme d b y reac tion of ib uprofen – glycine a cid chloride with the c 21 hyd ro xyl group of p re dnis olone in the p re se nce of trie thylamine to ab stract the lib erated hcl. pre dnis olone (sc he me 1 ) ha s thre e hydroxyl group s, a prima ry hydroxyl a t c21, a s eco nda ry hyd ro xyl group at c 11 a nd a te rtia ry one a t c17. bec aus e of the high rea ctiv ity o f ac id c hlorid e, re action with c17 hydroxyl group could also be occ urre d. on the o ther hand re action with the c11hydroxyl gro up ha d be en exclud ed be ca use c11 hyd ro xyl group is sterica lly hinde re d due to the pres ence of two methyl groups at c 10 and c 13 (18). acco rd ingly, comp ound ii is not the o nly one that is fo rme d through es te rific atio n, comp ound iii is a ls o formed, though to a less er extend, by e sterification with c17 hydroxyl group . in add ition to that esterifica tion o f bo th c21, a nd c17 wa s fo und to be occ urre d to form comp ound iv (sc he me 1). rea rrangeme nt of the c17 e ster (c ompo und iii) to the mo re s ta ble c21 es te r might ta ke s place in the p re sence o f aqueo us or non – aq ue ous medium. this rea rrange ment is fa ster than hyd ro lysis to the pa re nt s teroid(19). ste re oche mistry : ibuprofen use d in this inv es tiga tion conta ins one chiral c enter a nd exist a s a ra cemic mixture compos ed of e qual amounts of two enantiome rs hav ing r and s configura tions. acc ordingly, ib uprofen – ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 78 glyc ine ac id c hlorid e (c ompo und i, scheme 1) will exist in thes e tw o configura tions. prednis olone, o n the othe r hand , is an optically ac tive molec ule w hich has mo re than one chiral cente r. bec ause the rea ctio n we are de aling with d oe s no t affe ct a ny chiral ce nter a t prednis olone molec ule, the co nfiguratio ns of the se chiral ce nters rema in co nsta nt. as a re sult, the sp ecific rotation of p re dniso lo ne will not b e changed and for s implific ation we a rb itrary co nside re d it to has an s co nfiguration. bas ed o n thes e co ns id era tio ns, it was exp ec te d that the major product (co mpound ii, sc he me i) will e xist in two d is te re ome ric fo rms , (s ,s ), and (s,r). on the othe r ha nd, whe n este rifica tio n o cc urs at c17 hydroxyl group of prednis olone (c omp ound iii), it will a ls o gene ra te two dias te re ome rs . fina lly, whe n b oth c17 and c21 hyd ro xyl groups of p re dniso lo ne ha ve be en es te rifie d (c omp ound iv), this s itua tio n w ill res ult in the fo rma tio n of fo ur d ia stereo mers (s,s ,s ), (s ,s ,r), (s,r,s), a nd (s,r,r). acc ordingly, there will be eight d ia stereomers re sulted from this re action. in a previous work, whe n dicyclohe xylc arbod iimide had be en used fo r co njugatio n of hydroc ortiso ne with ib up rofen, es terific ation was found to oc cur exclusive ly at c21 hydroxyl gro up of hydroco rtisone(13,18). se paratio n of dias te re o me rs: of a number of t.l.c. s olve nt systems experienc ed in this inve stigation, system e wa s fo und to giv e the be st se paration of dias te re ome rs . this s olv ent system ha d b ee n us ed s ucc es sfully b y othe r inve stigators for the se paration of different d ia stereo mers(15). using this s ys te m, the fina l p ro duc t, a fter purifica tio n, showed eight spo ts of significantly diffe re nt rf v alues : 0.37 , 0.44, 0.52 , 0.61 , 0.71 , 0.7 7, 0 .8 5 and 0.91. beca us e of the low po la rity of this so lv ent system, the re la tive ly non – pola r distereo mers of co mpound iv will e xpe cted to mo ve fas te r and be in the uppe r p art o f the plate. the dias te re ome rs of c omp ound iii be ing hav ing the highe st p olarity in c ompa riso n w ith the othe r two c ompo unds, will mov e the sho rter distance s and a ppe ar a t the lower p art of the plate . comp ound ii dia stereomers which hav e re la tive ly me dium polarity will e xpe cted to oc cup y the middle situation on the t.l.c. plate. the se rationa l expe ctatio ns had b een the ba sis fo r sep aratio n of the dias te re ome rs of co mpound ii thro ugh column c hromato gra phy which we re further confirmed by ir sp ectrosc op y and c.h.n analys is . comp ound ii tha t wa s synthe size d and id entified througho ut this wo rk, was found to has relative ly high partition co efficient which gav e a n indication of low s olub ility in the aqueous ga stric fluid(20). more ov er it has re la tive ly high molec ular weight a rea so n that will de crea se the pos sibility of its ab so rp tion through g.i.t. a preliminary inve stigatio n of its s tability at aqueous phosp hate buffer, p h 7.8, indicated that its significant hydrolys is too k abo ut 1 5 – 25 hours , during which it will e xpe cted to re ach the lowe r parts of g.i.t whe re it may libe ra te its ac tive s pe cies the re in. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 79 references 1. bodo r, n., ann.n.y. aca d. sc i., 1 987 , 5 07, 285 . 2. bodo r, n., and forag, h., j . med . ch em. 1 983 , 26 , 313 . 3. gregoria dis, g., na tu re, 197 7 , 2 65, 4 07 . 4. s inkula , aa, an n. n.y acad . s ci., 1 987 , 50 7 , 281 . 5. s te lla v.j . med . ch em. 1 980 , 23, 127 5 6. s te lla v. j . and oliyai r., ann. rev. phar mac ol. to xicol. 1 993 , 33 , 52 1. 7. bund ga ard , h., des ig n of prod ru gs, n.y. elsev ie r, 198 5. 8. redd y, s.m., shina, v.r., a nd red dy, d.s., drug s of tod ay 19 99 , 3 5, 5 37 . 9. mo oter, v., symyn, c., and kinge t, r., int. j . ph ar m. 19 92 , 8 7, 37 . 10. mc le od, a.d., friend d.r., and to ze r, t.n., int. j . phar m. 19 93 , 9 2, 1 05 . 11. f riend d. r., a nd chang g. w., j. med. chem., 1 984 , 27, 261 . 1 2. s uad , m. d., a nd ghani, s. m., un pub lish ed re su lts. 1 3. s uad , m. d., ph.d, th es is , baghda d, 1 997 . 1 4. s chwa rz, h., a nd bumpus, f . m., j. am. ch em. soc . 19 59 , 81, 890 . 1 5. ke mmerer, j . m., rubio, f. a., mc cla in, r. m, and koec hlin, b. a., j. phar m. sc i. 19 79 , 68 , 12 74 . 1 6. s mith, m., mo ffate, j. g., khorana, h. g., j . am. che m. s oc., 195 8, 8 0, 6 204 . 1 7. de ta r, d. f., a nd silvers te in, r., j . am. ch em. soc ., 19 66, 88, 101 2. 1 8. f ie ser, l. f., a nd fies er, m., ad van ce d or ga nic che mis try, re inhold pub. co rp ., n.y,1 961 , 29 0. 1 9. bund gaa rd , h., and ha nien, j., int. j. phar m., 198 1, 7, 1 97. 2 0. leo, a ha ns ch, and elikins , d., ch em. rev., 1 971 , 71 , 5 25. iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir doi: http://dx.doi.org/10.31351/vol27iss1pp20-29a 20 dissolution enhancement of raltegravir by hot melt extrusion technique ahmed s. abdul jabbar*, 1 *department of pharmaceutics, college of pharmacy, university of basrah, basrah, iraq. abstract the objective of the study to develop an amorphous solid dispersion for poorly soluble raltegravir by hot melt extrusion (hme) technique. a novel solubility improving agent plasdone s630 was utilized. the hme raltegravir was formulated into tablet by direct compression method. the prepared tablets were assessed for all pre and post-compression parameters. the drugexcipients interaction was examined by ftir and dsc. all formulas displayed complying with pharmacopoeial measures. the study reveals that formula prepared by utilizing drug and plasdone s630 at 1:1.5 proportion and span 20 at concentration about 30mg (trail-6) has given highest dissolution rate than contrasted with various formulas of raltegravir. keywords: hot melt extrusion, raltegravir, plasdone s630. زيادة معدل التحلل لعقار الرالتيجريفر بواسطة تقنية الصهر الحار القاذف 1،*احمد سامي عبد الجبار قسم الصيدالنيات ، كلية الصيدلة ، جامعة البصرة ، البصرة ، العراق . * الخالصة . مادة القاذف تقنية الصهر الحارالذوبان بواسطة قليللعقار الرالتيجريفر الهدف من هذه الدراسة تطوير التشتت الصلب غير المتبلور سطة بوا رالرالتيجريفر المحضرة بواسطة تقنية الصهر الحار القاذف تحض استخدمت كماده معززه للذوبانية. اقراص 036البالسدون س مواد ق من وجود اي تفاعل بين العقار والتم التحق كتم تقييم االقراص المحضرة لجميع االختبارات قبل وبعد الكبس. كذل الكبس المباشر. بالسدون س 1:1:) 0المضافة. النتائج اثبتت ان جميع االقراص المحضرة هي ضمن المعايير المتضمنة في دساتير االدوية. وجد ان الصيغة الصيغ االخرى. ( هي من افضل الصيغ المحضرة ألنها تمتلك اعلى واسرع معدل تحلل مقارنة مع06ملغم من السبان 36و 036 .063الكلمات المفتاحية : تقنية الصهر الحار القاذف, رالتيجريفر, بالسدون س introduction the oral route is the most widely recognized and favored rout for drug administration because of its suitableness and simplicity to ingestion. at the point when medication given orally in solid dosage form like tablet, capsules; at first will undergoes dissolution in the gi fluid before absorption (1). for various poorly soluble medications bioavailability is constrained by the dissolution rate. therefore, numerous techniques were developed to enhance solubility of poor water soluble medications(2). the absorption of drug from solid dosage forms generally occurs when dissolved drug was transport of the across the gastrointestinal membrane(3). in the biopharmaceutical classification system (bcs) medications are sorted on the basis of aqueous solubility and membrane permeability. numerous poor water soluble medications come to ii and iv according to bcs classes(4). the medication solubility is straightly relative to its dissolution rate and subsequently solubility is an important parameter of a medication for assurance of its absorption and dissolution and bioavailability. parameters, for example, particle size, salt form, solubility, wettability, complexation, polymorphism and so forth influence the rate of dissolution(5). aqueous solubility of a medication is a potential factor to assess the oral bioavailability of orally administered poorly water soluble medications. the adjustment in the dissolution profile of these lipophilic medication particles without changing the molecular structure could be conceivable by several methods utilized to improvement medication candidate’s aqueous solubility(6). many techniques such as particle size decrease, solid dispersion, crystal modification, lipid based system, ph adjustment can be used to enhance solubility(7).glass solution is made when at least two components are totally miscible in molten state and cooled to form amorphous one phase system. melt extrusion studies were conducted to improve dissolution rate and bioavailability of medication, controlling/modifying release of medication and masking bitter taste of medication. 1corresponding author e-mail: ahmed_1983_sami@yahoo.com received: 12/10/2017 accepted: 1/1/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp20-29a http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir 21 benefits of melt extrusion include using small tool economic technique and scale up resilience, solvent free fabricating, high mixing aptness and simply controlled route parameters. while disadvantage includes thermal route (medication/polymer stability), flow properties of polymers are necessary to processing , limited number of polymers and melt technique process could not be resorted to heat sensitive materials because of high temperature contained(8). raltegravir is an antiretroviral medication (figure 1), utilized to treat with hiv infection. raltegravir chemically known as is n-[(4fluorophenyl) methyl]-1, 6-dihydro-5-hydroxy1-methyl-2-[1-methyl-1-[[(5-methyl-1, 3, 4oxadiazol-2-yl) carbonyl] amino] ethyl]-6-oxo-4pyrimidinecarboxamide. it is targets integrase, an hiv enzyme that integrates the viral genetic material into human chromosome. it is a climacteric step in the pathogenesis of hiv. raltegravir is categorized as a bcs class ii compound, i.e. high permeability and low solubility at physiological ph. its pka is 6.3(11). figure 1. chemical structure of raltegravir materials and method materials raltegravir was supplied by pharmatrain, hyderabad. plasdone s 630, sskillet 20, aerosil, croscarmellose sodium, hydroxy propyl cellulose, mcc ph 112, lactose monohydrate, sodium steryl fumrate, magnesium stearate and banana powder were found from s.d. fine chemicals limited, hyderabad. all chemicals utilized were of analytical reagent grade. methods saturation solubility solubility of raltegravir was evaluated in 0.1n hcl, acetate buffer (ph 4.5), phosphate buffer (ph 6.8) and distilled water. an extra amount of medication was added to 50 ml conical flask and was saved under shaking for 72 hrs in the rotary shaker.saturated solution was filtered cross 0.45 μ membrane filter. absorbance of filtered solutions was examined and amount of medication solubilized was computed. standard calibration curve of raltegravir in distilled water standard stock solution: a stock solution comprising 1000 µg/ml of pure drug was prepared by dissolving 100 mg of raltegravir in enough water to get 100 ml solution in a volumetric flask. serial dilutions: ten milliliters of the stock solution was further diluted to 100 ml with water. aliquots of 0.5, 1.0, 1.5, 2.0 and 2.5 ml of diluted stock solution were pipette out into 10 ml volumetric bottles. the volume was made up to the sign with water. these dilutions give 5, 10, 15, 20, and 25 µg/ml concentration of raltegravir individually. the absorbance was evaluated in the uv-visible spectrophotometer at 315 nm utilizing purified water as blank and the concentration vs. absorbance was graphed. preparation of raltegavir immediate release tablets by using hot melt extrusion technique ten batches of immediate release raltegravir tablet were prepared depending on different drug to polymer proportion by hot melt technique as shown in table 1 .raltegravir, plasdone s630, aerosil crossed through 30 mesh sieve and granulated in rapid blender granulator with span 20 for 3 minutes. pass this intra granular blend cross hot melt extruder at 1000c, 1150c, 1300c and 1350c zones. then extrudes were powdered utilizing co-mill at 15000 rpm. except sodium stearyl fumarate, all extra granular blends sieve cross 30 mesh and blended for 10 mints. sodium stearyl fumarate is going cross 60 mesh and blended all mixture for 5 mints then compressed into tablet. iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir 22 table 1. hme raltegravir immediate release tablet composition ingredient trail1 trail2 trail3 trail4 trail5 trail6 trail7 trail8 trail9 trail10 intragranular raltregavir 400 400 400 400 400 400 400 400 400 400 plasdone s 630 0 200 400 600 600 600 600 600 600 600 span 20 30 30 30 30 50 30 30 30 15 30 aerosil 10 10 10 10 10 10 10 10 10 10 croscaramellose sodium 10 10 10 10 10 10 10 10 10 10 extragranular croscaramellose sodium 30 30 30 30 30 60 90 60 60 60 hydroxy propyl cellulose 10 10 10 10 10 10 10 20 10 10 microcrystalline cellulose 105 105 105 105 105 105 105 105 105 75 sodium stearyl fumarate 15 15 15 15 15 15 15 15 15 15 total weight 610 810 1010 1210 1230 1240 1270 1250 1225 1210 all amounts given in above table are in milligram characterizations of hot melt extrudates ft-ir spectroscopic analysis medication polymer reactions were considered by ft-ir spectroscopy. ten milligrams of raltegravir and mixture of medication and polymer were weighed and combined correctly with potassium bromide uniformly. a small amount of the powder was compressed into a thin semitransparent pellet by applying pressure. the irspectrum of the pellet from 450-4000cm-1 was read taking air as the reference and collating to study any interference. differential scanning calorimetry (dsc) differential scanning calorimetry was per made utilizing dsc-60 (shimadzu, tokyo, japan) calorimeter to ponder the thermal behavior of drug alone and mixture of drug and polymer. the testers were warmed in closed aluminum pans under nitrogen stream (80ml/min) at a scanning level of 10 degree centigrade/minute from 25 to 450 degree centigrade. empty aluminum pan was utilized as reference. micromeritic properties of pre-compressed powder flow properties measurement of the various batches pre-compressed powder was evaluated by defining their angle of repose utilizing fixed-base cone method. a glass funnel was secured with its tip situated at fixed height (h) above chart paper put on a horizontal surface. the sample was tapped cross the funnel until the apex of the conical pile touched to the funnel tip. the heap height and radius was evaluated [12]. the angle of repose (tan θ) was calculated using the equation; angle of repose[θ] =tan-1(h/r) h = funnel height, r = radius of circular base created by the granules on the ground. bulk and tapped densities measurement of the pre-compressed powder were assessed by using the bulk density apparatus. known weights of formulated granules were moved into a 50cc graduated measuring cylinder. the cylinder was stable on bulk density device and the timer knob was put for 500 tapings. then, the initial bulk volume and last volume after 500 tapings were noted(13). the respective densities of various batches of granules were computed by utilizing the following formulas; 𝐁𝐮𝐥𝐤 𝐝𝐞𝐧𝐬𝐢𝐭𝐲 [𝐠𝐦 /𝐦𝐥] = 𝑴𝒂𝒔𝒔 𝒐𝒇 𝒕𝒉𝒆 𝒔𝒂𝒎𝒑𝒍𝒆 (𝒈) 𝑩𝒖𝒍𝒌 𝒗𝒐𝒍𝒖𝒎𝒆 (𝒎𝒍) 𝐓𝐚𝐩𝐩𝐞𝐝 𝐝𝐞𝐧𝐬𝐢𝐭𝐲 [𝐠𝐦 /𝐦𝐥] = 𝑴𝒂𝒔𝒔 𝒐𝒇 𝒕𝒉𝒆 𝒔𝒂𝒎𝒑𝒍𝒆 (𝒈) 𝑻𝒂𝒑𝒑𝒆𝒅 𝒗𝒐𝒍𝒖𝒎𝒆 (𝒎𝒍) compressibility index or carr’s index value of precompressed was computed according to the following equation; compressibility index or iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir 23 carr’s index value of precompressed was computed according to the following equation; carrs index (ci%) = tapped density − bulk density tapped density × 100 hausner’s ratio of pre-compressed powder was calculated by collating the tapped density to the bulk density by utilizing the equation; hausner’s ratio = tapped density / bulk density evaluation of immediate release hme raltegravir tablets weight variation, hardness and friability the uniformity of weights of tablets was examined according to the method cited in usp [14]. weighed 20 tablets individually in an electronic balance and their average weight were computed; 𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑤𝑒𝑖𝑔ℎ𝑡(𝑔𝑚) = 𝑇𝑜𝑡𝑎𝑙 𝑜𝑓 𝑡ℎ𝑒 𝑡𝑎𝑏𝑙𝑒𝑡𝑠 20 for each formulation, the hardness of 10 arbitrarily chosen tablets was examined utilizing a pfizer hardness tester. the tablet hardness or crushing strength was evaluated in kg/cm2. the percentage of friability was evaluated by utilizing roche friabilator. ten or twenty tablets from each batch were weighed and placed in the plastic chamber. the chamber rotated for 4 minutes or 100 revolts. after 100 revolts tablets were moved from the chamber and reweighed [15]. the percentage of weight loss or friability was examined by the following formula; friability (%) = loss in weight of tablets / initial weight x 100 content uniformity five tablets of each batch were weighed and powdered. the amount of the powder equal to 10 mg of raltegravir was dissolving in 100 ml of water comprising 10 ml of methanol. the resulting solution was moved into a locked funnel bottle and the bottle was shaken for interval of 12 h by utilizing a mechanical shaker at room temperature. the following day it was stirred for 15 minutes. the solution was filtered, later suitable dilution; the medication content in the filtrate was examined at 315nm utilizing uvvisible spectrophotometer (16). in-vitro drug release studies various batches of raltegravir compressed tablet was subjected to estimate drug release in the water up to 60 minutes by utilizing dissolution test device usp xiii paddle type, 50 rpm in 900 ml dissolution medium (what media) and maintained at 37 ± 0.5 ºc. samples (5ml) were withdrawn at interval time of 5, 10, 15, 20, 25, 30, 35, 40, 45 and 60 minutes time interval. after each sampling, equal volume of the medium was substituted with similar volume of fresh medium. the tester was filtered cross 0.45µ membrane filter and diluted with suitable dilution with respective medium. then estimate the raltegravir concentration in the solution by utilizing uvvisible spectrophotometry measured at 315 nm. the absorbance of the samples was evaluated at various time intervals and the concentration, amount of medication released and the percentage of medication released were computed (17). results and discussion saturation solubility the saturation solubility of raltegravir in different media illustrated in the table 2. the results show increase in solubility of raltegravir with increasing ph. it is likely that the drug obtains a negative charge at higher ph by deprotonating of the hydroxyl group at the 5 position of the 6-oxo-1,6-dihydropyrimidine ring. this negative charge would result in increasing the solubility of the drug in aqueous buffer (18). table 2. saturation solubility of raltegravir media saturation solubility (mg/ml) 0.1 n hcl 0.031 acetate buffer (ph 4.5) 0.049 phosphate buffer (ph 6.8) 0.18 distilled water 0.31 construction of standard calibration curve of raltegravir in distilled water the absorbance of the solution was evaluated at 315nm, utilizing uv spectrometer with water as blank. figure 3 of absorbance vs. concentration was plotted which indicated in compliance to beer’s law in the concentration range 5 to 25 µg/ml. iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir 24 figure 3. calibration curve for raltegravir in distilled water micromeritic properties of pre-compressed powder blends powder blends of various formulations were evaluated for angle of repose, bulk density, tapped density, carr’s index, and hausner’s ratio. from table 3; all the formulas powder blends illustrate angle of repose (<40) demonstrating fair flow properties due to rise of bonding between microcrystals of medication and diluent(19). the bulk and tapped density values were found in the suitable range demonstrating good pack capability. compressibility index and hausner’s ratio values are found in the range of 7.27 to 10 and 1.08 to 1.11 individually. this specifies that the prepared powder blends having better compressibility and good flow properties(20). table 3. powder flow properties of (trial 1 –trial 10) formulations bulk density tapped density hausner’s ratio carr’s index angle of repose trail-1 0.80 0.89 1.11 10.00 30 trail-2 0.77 0.83 1.08 7.69 28.23 trail-3 0.80 0.89 1.11 10.00 30.02 trail-4 0.73 0.78 1.08 7.27 27.81 trail-5 0.77 0.83 1.08 7.69 27.42 trail-6 0.75 0.82 1.08 7.55 28.13 trail-7 0.78 0.85 1.09 7.84 28.87 trail-8 0.74 0.82 1.10 9.26 29.12 trail-9 0.78 0.85 1.09 7.84 28.91 trail-10 0.80 0.87 1.09 8.00 28.63 post compression studies table 4; shows that the weight difference of the tablets perceived within pharmacopeia limit submitted lower than ±5% w/w of stander deviation from the average. hardness of the formulated tablets was found within suitable range of 8.0 to 12 kg/cm2. friability values were found to be lower than1% in all prepared formulations and though to be reasonable [20]. the content of raltegravir existent in the formulated tablets was detected in the range of 98.25 to 101.27 %. trial 6 show best post compression parameters. table 4. post compression parameters of raltegravir hme tablets formulations weight variation hardness kg/cm2 disintegration time (min.) friability content uniformity trail-1 pass 8 12 0.281 99.13 trail-2 pass 8.5 17 0.146 98.61 trail-3 pass 8.5 16 0.432 100.03 trail-4 pass 10 14 0.318 98.25 trail-5 pass 9 12 0.516 99.57 trail-6 pass 10.5 10 0.125 101.25 trail-7 pass 11 10 0.314 100.57 trail-8 pass 12 14 0.264 100.63 trail-9 pass 12 13 0.468 99.15 trail-10 pass 12 16 0.312 99.87 iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir 25 ft-ir spectroscopy for assessing any conceivable chemical interactions between the medication and polymers, ftir spectra of raltegravir, physical mixture, and hme formulations were observed. ir spectrum of raltegravir introduced typical peaks at 1675.7, 1072.2, 3149.5, 1632.0, and 1270 according to c=n and c=c, c-n and c-o, n-h, (figure 4). as illustrated in figure 5 and 6, the spectra of physical mixture and hme formulations are analogous. the raltegravir skeleton stretching vibrations are not impacted by the polymer adding, assuming no reactions between the polymer and medication in the physical and hme mixtures. plasdone s-630 has two groups (=nand c=o) that could possibly create hydrogen bond with raltegravir in the hme formulations. the carbonyl group is more promising for hydrogen bonding and intermolecular interactions than the nitrogen atom due to steric hindrance (figure 7). for hme formulations, the n-h stretching bands broadened and the band intensity reduced, demonstrating specific degree of reactions between the proton donating group (-nh) of raltegravir and the proton accepting group (c=o) in the plasdone s630 polymer. this states that the drug was molecularly dispersed in the polymers or in drug loaded formulations thus thereby demonstrating the absence of any interactions. figure 4. ftir spectra of raltegravir figure 5. ftir of physical mixture iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir 26 figure 6. ftir spectra of raltegravir hme best formulation (trial 6) figure 7.ftir spectra of raltegravir and plasdone s630 differential scanning calorimetry (dsc) dsc thermogram of raltegravir, illustrated sharp endothermic melting point at 277°c (figure 8). thermograms of raltegravir hme (figure 9) illustrated a little, slightly wide endotherm that specified the reduced crystallinity of raltegravir. this might be because of interaction of drug and polymer in dsc pan during heating ramp. on heating polymer gets melted far before drug’s melting point so drug starts interacting with rubbery polymer. when temperature rises to melting point of drug, drug has already been solubilized into molten polymer and hence exhibits broad melting endotherm (21). iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir 27 figure 8. dsc spectrum of raltegravir figure 9. dsc spectrum of raltegravir hme dissolution studies high dissolution rate in the hme formulation state is accredited to the amorphous case of the medication (some of drug exist in amorphous state) that present a lesser thermodynamic barrier to dissolution and the creation of a glassy solution where the medication is molecularly dispersed in the polymer. the higher apparent solubility and increase in dissolution rate for amorphous materials is well recognized and has been widely known. the improvement in solubility is the result of the disordered structure of the amorphous solid (22). iraqi j pharm sci, vol.27(1) 2018 hot melt extrusion technique of raltegravir 28 raltegravir hot melt extrudes tablet illustrated noticeability higher dissolution more than 80 % release in 45 min (figure 10 and 11). improvement in dissolution is because of reducing in crystallization of raltegravir in hot melt extrudes. additional solubilizing agent plasdone s-630 raises the dissolution of raltegravir by improving its wettability. the drug solubility and dissolution rate were not improved by simple physical mixing with the polymer. higher apparent medication 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(eds.). 21. vavia p et al. preparation of oxcarbazepine solid dispersion by hot melt extrusion for enhanced dissolution: downstream processing to tablets. american journal of pharmtech research, 2013; 3(1): 557-569. 22. hancock bc, parks m. what is the true solubility advantage for amorphous pharmaceuticals pharm res, 2000; (4):397– 404. 23. ritesh fule et al., solubility and dissolution rate enhancement of lumefantrine using hot melt extrusion technology with physicochemical characterization. journal of pharmaceutical investigation, 2013; 43:305321. iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. doi: https://doi.org/10.31351/vol29iss2pp139-151 139 separation and identification of phenolic acid from borago officinalis (f:boraginaceae) cultivated in iraq ashwaq t. kareem*,1 and maha n. hamad* *department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad, iraq abstract the plant borago officinalis, which belongs to the boraginaceae family and celebrated as borage, is one of the useful medicinal plants cultivated in iraq. it was used in olde medicine in iraq, irane, syria and europe for management of various diseases. it is commonly used as a tonic, tranquilliser, management of cough,sore throat , pneumonia,urinary tract infection, rheumatoid arthrites antioxidant, and anticancer. this project provides the first comprehensive research done in iraq to study the phytochemicals and the methods of extraction and isolation of active constituents from borago officinalis cultivated in iraq. the plant was harvested in spring from al-rifai, nassiriyah city/ iraq in february 2019. the aerial parts were washed carefully, dried under dark for two weeks, and milled in a mechanical grinder to a fine powder.the plant was extracted by cold extraction methods using 85% methanol solvent for three days then fractionation with petrollium ethere, chloroform, ethyl acetate and n-butanol(n.b) to separate the active constituentse according to the change in polarities. the ethyle acetate fraction and n-butanol fraction were used for identification and isolation of phenolic compounds by tlc,plc,hplc and lc/mass. results of the phytochemical screening exposed the presence of, phenols, tannins, fatty acid, in the plant extract. the phenolic acid(sinapic acide, rosmarinic acides, caffeic acid) were separated and purified by plc.the isolated compounds were subjected to several chemical ,chromatographic and spectral analytical techniques for their identification such as tlc, hplc,uv andlc/mass. keywords: sinapic acid, rosmarinic acid, caffeic acid, hplc, lc/mass. في نبات لسان الثور المستزرع في العراق ةالموجودفصل وتحديد االحماض الفينولية *و مها نوري حمد 1*،اشواق طالب كريم .العراق،بغداد ، جامعة بغداد ،كلية الصيدلة ،*فرع العقاقير والنباتات الطبية الخالصة والتي استزرعت ةالنباتات الطبيه المعروف والمعروف بخبز النحل او الحمحم وهو من (boraginaceae) نبات لسان الثور من عائلة لمعالجة العديد من االمراض حديثا في العراق ويستعمل في الطب الشعبي في العراق وسوريا وايران وبالد الشام ومناطق البحر المتوسط وكندا واوربا ت المفاصل الروماتيزي وكمضاد وااللتهابات الرئوية والتهابات المسالك البولية والتهابا حيث يستعمل كمقوي ومهدئ للسعال ولعالج التهاب الحلق . تم للتاكسد والسرطانات . يعتبر هذا البحث اول بحث شامل في العراق لدراسة الموادالكيمياويه الموجودة في النبات وطرق استخالصها وفصلها سبوعان وطحنه بالمطحنه وتم غسل و تجفيف النبات لمده ا2019جمع النبات من قضاء الرفاعي في محافظة ذي قار في شهر شباط من سنة ميثانول لمدة ثالثة ايام. تمت عملية التجزئيه باستخدام عدة مذيبات %85الميكانيكيه وتمت عملية االستخالص بالطريقة الباردة طريقة نقع النبات في ادا على االختالف في القطبية بين هذه المكونات هي بالتتابع: االثير البترولي, الكلوروفورم ,خالت االثيل والبيوتانول لفصل المركبات الفعالة اعتم .استخدمت طبقتي خالت االثيل والكحول البيوتانولي في التعرف وعزل االحماض الفينولية بعدة تقنيات مثل كروماتوغرافيا الطبقه الرقيقة ائل والطيف الكتلي وكانت نتيجة الكشوفات الكيميائيه كروماتوغرافيا السائل عالية االداء وكروماتوغرافيا السووكروماتوغرافيا الطبقه التحضيرية حامض الروزمارينك وحامض السينابيك وتم كشف وعزل االحماض الفينولية ) وجود مواد فينوليه واحماض دهنيه ومواد دابغة في المستخلص. ,مطياف االشعه فوق البنفسجيه ,كروماتوغرافيا السائل كروماتوغرافيا السائل عالية االداءوكروماتوغرافيا الطبقه الرقيقة بواسطة وحامض الكافيك( .والطيف الكتلي كروماتوغرافياالسائل والطيف الكتلي.، كروماتوغرافيا السائل عالية االداء،حامض السنابيك,حامض الكافييك، حامض الروزماريك الكلمات المفتاحية: introduction borage (borago officinalis l.) is an annual plant belonging to the family. boraginaceae. it originates from western regions of mediterranean area and grows nearly in whole america, europe, canada and iran (1,2). the plant grows during november to january and reaches a height of 70 to 100 cm (3,4) its stem is shielded with hairs that secrete a strong smell nearly the aroma of fresh cucumbers while on the tops of the shoots there are the star shaped inflorescences which initially are pink, later turn blue, seldom white (5,6). aerial partse have been used in old medicine in iraq as atonic, tranquillizer, management of cough, pneumonia, sore throat, swelling and inflammatory diseases. the leaves and flower possess biological activities for cancer and heart diseases prevention (7) and have antibiotic properties (8), condense cardiovascular diseases (9) and provide benefits for improving healthe due to their various biological events (10). 1corresponding author e-mail: ashotalib@gmail.com received: 6 / 2 /2020 accepted: 20/ 5/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp139-151 iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 140 results reported by previous studies shown the presences of phenolics acid, flavonoids (quercetin, myricetin, luteolin and rutin) and isoflavonoid besides, the dominant individual fatty acids of methanolic extract as oleic acid which is an unsaturated fatty acid (omega-9), linoleic fatty acids (omega-6i) and hexadecanoic acid. thet methanolic extract was more biologically active than ethanolic extract (11). figure 1. borago officinal plant cultivated in iraq.(12) material and method plant collection borago officinalis plant was harvested from al-rifai, nassiriyah city/ iraq in february 2019. the aerial plant was driede in the shadow for two weeks and powdered. the plant was identifiede and authenticated by prof. dr israai mohammed department of biology /college of sciences/ university of baghdad. extraction and fractionation of the different active constituent two hundred and fifty grams of the powdered plant material was soaked in 1500ml, with 85% methanol and shaking, at roome of temperature. after three days, the methanol soluble materials was filtered off. the filtrate was evaporated until dryness unders vacuum using a rotatory evaporator. a dark greenish residue was obtained. then suspended in 500ml water and partitioned successively with petroleum ether (b.p. 30-60 ℃), chloroform, ethyl acetate, and n-butanole (3×500ml) for each fraction the first three fractions dried over sodium sulfate anhydrous, filtered, and evaporated to dryness(13,14) . the scheme of fractionation is shown in (figure 2). figure 2. schematic diagram for fractionations aerial part crude extracts of borago officinalis (14 ) iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 141 identification of phenolic compounds in borago officinalis plant extract 1. preliminary phytochemicals showing of the phenolic compounds using a methanolic extract from the plant using a naoh test, lead acetate, and ferric chloride test (15,16). 2. isolation and purification of phenolic compounds from the fractions ethyl acetate and n.butanoleei by preparative layer chromatography. isolation of phenols was done by using preparative tlc; 2 grame of each fraction dissolve in 10 mlof methanole and applied to the number of plc plates as a semi concentrated solution in streak using a capillary tube on each plate, then the plate placed inside glass tank which contained the s1 solvent system. the band had been scrapped off, eluted with methanol and then filtered, the filtrate evaporated to dryness, in a vacuum as shown in figure 3 and 4. 3. thin-layer chromatography. in this qualitative identification, a ready-made aluminium plates of silica gel of 245 with developing solvent systems were used for detection the plant phenols in fractions ethyl acetate and n.butanoleei with standards, as listed in table (1). table 1. developing solvent systems using in the separation of phenols in borago officinalis. no. composition references s1 chloroform: ethyl acetate : methanol :formic acid (70:14:14:10) (17) s2 dichloromethan:acetonitrile :formic acid(9.5:0.5:0.1) (17) s3 chloroform:methanol (90: 10). (17) 4. qualitative estimate of ethyl acetate and n.butanolee fractions by high-performance liquid chromatography (hplc) the expected phenols in fractions e were separated by hplc method and detected in comparison with standard compounds . the mobile phase consisted 1 % aq. acetic acid solution (a) and acetonitrile (b) solvents, the flow rate was adjusted to 0.7 ml/min, the column was thermostatically controlled at 280c, and the injection volume was kept at 20 μl. gradient elution was accomplished by varying the proportion of b to a solvents. the gradients elution was changed from 10 % to 40% b in a linear fashione for 28 min, from (40 to 60) % b in 39 min, after that from ( 60 to 90) % b solution in 50 min. the mobile phase mixture back to the initial state of solvents (b: a: 10: 90) in 55 min and permitted to run for another ten min. 5. lc-ms analytical was done using the agilent systems joined to an appliede biosystems api 2000 mass spectrometry.mobile phase solvents acetonitrile and wate a columne of 0.19mm external diameter (75μm i.d.) and 200mm length was packede with thermo scientifice hypersil gold c18 with 5μm particle size. samples were run under the following conditionse: m/z range was 250 to 10001, 200k resolution, top 5 configurationse with one ms scane and five ms/ms scans, and dynamic exclusion set to 1 with a limit of 90 seconds. a 2.5 hour lcms separatione was used for all blanke and standard samples. results and discussion borago officinalis plant active constituents in this studyi, cold extraction method was done by 85% absolute methanol to extract the active constituenti depend on the nature of these active constituents.each 250 g of plant extract yielded 32 g residue preliminary qualitative phytochemicale analysis the results of the phytochemical analysis of polyphenol in methanolic crude extract given in ( table 2). table 2. results of the phytochemical analysis of polyphenol in the methanolic crude extract active group test reaction result poly phenol lead acetate test white ppt positive polyphenol ferric chloride test dark green colour positive poly phenol naoh test yellow colour after addition naoh solution positive the isolation of phenols were done by using preparative tlc, in jar contained the s1 solvent system. as in figure 3 and 4. figure 3. preparative thin layer chromatography plate for ethyl acetate fraction on silica gel gf254 developed in the s1 system, detection by uv light at 254nm. iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 142 figure4. preparative thin-layer chromatography plate for n.b fraction on silica gel gf254 developed in the s1 system, detection by uv light at 366nm. table 3. tlc profile of isolated compound (cpd) number 1 compared with standard rosmarinic acide using the mobile phase solvents ( s1, s2, s3). solvent system rf of standard rosmarinic acid.. rf of isolated phenolic acid number 1 s1 0.247 0.246 s2 0.13 0.13 s3 0.2 0.2 table 4. tlc profile of isolatedi compound number 2 compared with sinapic acide standard using the mobile phase solvents ( s1, s2, s3) solvent system rf of standard sinapic acidee… rf of isolated phenolic acid number 2 s1 0.71 0.71 s2 0.47 0.47 s3 0.5 0.51 table 5. tlc profile of isolatedi compound number 3 compared with caffeic acid standard using the mobile phase solvents ( s1, s2, s3). solvent system rf of standard caffeic acide rf of isolated phenolic acid number 3 s1 0.517 0.517 s2 0.182 0.182 s3 0.294 0.293 a b figure 5. tlc chromatography (a) for separated compound number (1) from ethyl acetate fraction and rosmarinic acid std using silica gel gf254 as adsorbant and s1 as mobile phase and (b) separated compound after detected by spraying with five %ferric chlorides. figure 6. tlc chromatography for separated compound number (1) from ethyl acetate fraction and r.a standard using silica gel gf254 as adsorbant and mobile phase s2. iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 143 figure 7. tlc chromatography for isolated compound number (1) from ethyl acetate fraction and rosmarinic acid standard using silica gel gf254 as adsorbant and mobile phase s3. a b figurei8. tlc chromatography(a) for isolated compound number (2) from ethyl acetate fraction and sinapic acid standard using silica gel gf254 as adsorbant and mobile phase s1 (b) separated compound after detected by spraying with five %ferric chlorides. figurei9. tlc chromatography for isolatede compound number (2) from ethyl acetate fraction and standard s.a using silica gel gf254 as adsorbant and mobile phase s2. figure 10. tlc chromatography for isolated compound number (2) from ethyl acetate fraction and s.a standard using silica gel gf254 as adsorbant and s3 as mobile phase . iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 144 a b figure 11. tlc chromatography (a) for separated compound number (3) from ethyl acetate fraction and caffeic acid std using silica gel gf254 as adsorbant and s1 as mobile phase and (b) separated compound number 3 after detected by spraying with five %ferric chloride. figure 12. tlc chromatography for isolatede compound number (3) from ethyl acetate fraction and caffeic acidi standard using silica gel gf254 as adsorbant and mobile phase s2 . figure 13. tlc chromatography for isolatede compound number (3) from ethyl acetate fraction and caffeic acide standard using silica gel gf254 as adsorbant and s3 as mobile phase hplc analysis the result gained from hplc analysis method. 1. the retention time of rosmarinic acide standard match with a retention time of isolatedi compound number 1 and uv spectrum of separated compound number 1 match with uvs spectrum of rosmarinic acide standard as in figure 14and 15 and table 6 . 2. the retention time of the isolated compound number 2 match with a retention time of sinapic standard and uv spectrum of the separated compound number 2 match with standard sinapic acide as in figure 16 and 17 and table 6. 3. the retention time of the isolated compound number 3 match with a retention time of caffeic acidi standard and uv spectrum of the separated compound number 3 match with caffeic acid standard as in figure 18 and 19 and table 6. figure 14. hplc chromatogram of isolated compound number 1 and rosmarinic acide standard. iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 145 figure 15. the uv spectrum of isolated compound number1 and rosmarinic acide standard. figure 16. hplc chromatogram of the isolatedi compound number 2 and sinapic acide standard. figure 17. the uv spectrum of isolated compound number 2 and sinapic acide standard. iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 146 figure 18. hplc chromatogram of caffeic acid standard and isolatedi compound number 3. figure 19. uv spectrum of isolated compound number 3 and caffeic acid standard. table 6.retention times in minutes for standards (rosmarinic acide,sinapic acide ,caffeic acid)and isolated cpd 1,2,3. retention time of rosmarinicacid standard. retention time for isolated cpd number 1. 20 20 retention time of sinapic acid standard. retention time for isolated cpd number 2. 15 15 retentiontime of caffeic acid standard. retention time for isolated cpdnumber3. 10 10 lc/mass the result gained from lc/mass. 1. lc/mass chromatograme as shown in( figure 20) the isolated compound number 1 is rosmarinic acide with molecular weight 360.31 gram/mol as shown in figure (21). 2. lc/mass chromatograme as shown in( figure 22) the isolated compound number 2 is sinapic acid with molecular weight 224.21 gram/mol as shown in figure(23) . 3. lc/mass chromatogrami as shown in( figure 24) the isolated compound number 3 is caffeic acidi with molecular weight 180.16 gram/mol as shown in figure (25). iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 147 figure 20. lc/ms chromatogram of isolated compound number 1 molecular ion peak at m/z 360 that correspond to a molecular formula of c18h16o8(3,4dihydroxyphenyllactic acid) rosmarinic acid and molecular ion peak at m/z359[m-h] of rosmarinic acid. figure 21. chemical structure of rosmarinic acide with molecular weight 360.31 gram/mol.(18) iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 148 figure 22. lc/ms chromatogram of isolated compound number 2. molecular ion peak at m/z 223 that correspond to the molecular formula of c11h12o5 (3,5-dimethoxy-4hydroxycinnamic acid) sinapic acid. figure 23. chemical structure of sinapic acide with molecular weight 224 gram/mol.(19) iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 149 figure 24. lc/ms chromatogram of isolated compound number 3. molecular ion peak at m/z 181 that correspond to the isotope of a molecular formula of c9h8o4 (3,4dihydroxycinnamic) caffeic acid, and molecular ion peak m/z 179.3 [m-h]for caffeic acid. figure 25. chemical structure of caffeic acide with molecular weight 180 gram/mol.(20) iraqi j pharm sci, vol.29(2) 2020 borago officinalis l. 150 conclusion in the light of the results obtained, the study concluded the following: 1. phytochemical screening of new iraqi plant borago officinalis was done for aerial part of the plant nearly two hundred and fifty grams of the powdered plant material, and the results include the separation and identification of phenolic acids (rosmarinic acid, caffeic acidi and sinapic acid). 2. rosmarinic acid with mwt 360 gram /mol isolated from ethyle acetate and n.butanol fraction with high quantity nearly 13.3 mg. 3. sinapic acid with molecular weight 224 gram/mol isolated from ethyle acetate fraction with small quantity nearly 6 mg. 4. caffeic acide with molecular weight 180 gram/mol isolated from ethyle acetate fraction with high quantity nearly 9 mg. 5. all isolated phenolic acids were identified by tlc, preparative tlc, hplc, uv, lc/mass. acknowledgements the authors are grateful to acknowledge the college of pharmacy -university of baghdad for providing the necessary facilities to carry out this study. references 1. ghahreman a. flora of iran. tehran: research 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patients with diabetes mellitus, hypertension or both diseases 29 assessing quality of life among patients with diabetes mellitus, hypertension or both diseases in al-najaf province /iraq ali s. al-ibrahimy*,1 and haydar f.al-tukmagi** * department of clinical pharmacy, college of pharmacy, university of kufa, al-najaf , iraq. ** department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad iraq. abstract with growing prevalence, diabetes mellitus will be possibly the most principal cause for morbidity and mortality in next years. the predominance of diabetes mellitus has been increased over the last decades, where the occurrence of disease is anticipated to increase to 592 million at 2035. essential hypertension is chronic non-communicable disease which considered the major risk factor for many diseases. the world health organization estimates that the hypertensive patients will reach 1 billion or more at year 2025. the purpose of insertion of quality of life as indicator for health outcome is due to sensitivity of this measure for patients’ evaluations of their health status after taken treatment and its health outcome. this study is a cross-sectional survey. the total number of participants in this study was 775 individuals which divided into four groups: healthy control group, patients with diabetes mellitus only, hypertension only and patients with both diabetes mellitus and hypertension. the questionnaire used to assess quality of life is arabic version of (whoqolbrief). the mean scores of the four domains of qol instrument for diabetic, hypertensive and diabetic hypertensive patients were statistically significant lower than corresponding domains of control group. in conclusion, one chronic disease affects quality of life and combination of two chronic diseases affect quality of life to greater extent. keywords: diabetes mellitus, hypertension, quality of life, whoqolbrief. مرضى داء السكري من النوع الثاني تقييم نوعية الحياة الصحية لدى العراق -الدم في محافظة النجف األشرف و إرتفاع ضغط ا /و **جي هو حيدر فخري التكم 1*,االبراهيميعلي شالش فرع الصيدلة السريرية ، كلية الصيدلة ، جامعة الكوفه ، النجف ، العراق .* الصيدلة السريرية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . عفر ** لخالصة ا المتزايد، فإن مرض السكري ربما يكون السبب الرئيسي لالمراض والوفيات في السنوات المقبلة. وساد هذا المرض مع االنتشار أما ارتفاع 5302مليون في عام 295المصابين بهذا المرض إلى بصورة كبيرة في العقود األخيرة ، حيث من المتوقع أن يزداد عدد تقدر منظمة الصحة حيث ة غير المعدية التي تعتبر عامل الخطر الرئيسي لكثير من األمراضمن األمراض المزمن ضغط الدم األساسي هو يرجع الغرض من إدراج دراسة نمط الحياة كمؤشر .5352مرضى ارتفاع ضغط الدم سيصل إلى مليار أو أكثر في عام عدد العالمية أن حيث ان هذه دراسة شاملة مستعرضة المرضى لحالتهم الصحية بعد اخذ العالجللنتائج الصحية إلى قدرة هذا العامل على الكشف عن تقييم ومجموعة مرضى التي تشمل المجموعة الضابطة موعاتشخص مقسمين على اربعة مج 772لمرضى الضغط والسكري اجريت على ستبيان منضمة الصحة العالمية حول م النسخة العربية الموجزة الااستخد تم الضغط و مجموعة السكري ومجموعة الضغط والسكري معا. قد تأثرت بدرجة يعتد بها ظهرت هذه الدراسة ان معدل الدرجات للمجاالت االربعة )البدني والنفسي واالحتماعي والبيئي (أنمط الحياة. ان المرض المزمن ب .وبذلك يمكن االستنتاج شخاص االصحاء في المجموعة الضابطةاحصائيا مقارنة مع درجات هذه المجاالت عند األ ثران ؤثر على كافة مجاالت الحياة التي قيست في هذا االستبيان بدرحة معتد بها احصائيا اما اذا اجتمع مرضان مزمنان فانهما يؤالواحد ي بدرجة اكبر. .ة العالميةمرض السكري ، ارتفاع ضغط الدم ، نوعية الحياة ، النسخة الموجزة الستبان منظمة الصح الكلمات المفتاحية : introduction diabetes mellitus (dm) is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both (1). the longterm effects of these metabolic irregularities lead to appearance of chronic complications of diabetes mellitus (2). these complications can affect many organ systems. the diabetic complications are divided into macrovascular and microvascular complications (3). the predominance of dm has been increased over the last decades ,where the occurrence of disease is anticipated to increase to 592 million at 2035 (4). according to world health organization (who) eastern mediterranean region, the prevalence of dm in iraq was (668,000) by 2000 and is expected to increase to (2009, 000) by 2030. 1corresponding author e-mail: alipharm1989@yahoo.com received:29 /7/2017 accepted: 21/9/2017 mailto:alipharm1989@yahoo.com iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 30 there are many subtypes of dm present, but the type-1 diabetes mellitus (t1dm) and type-2 diabetes mellitus (t2dm) are the most common form of the disease and account for approximately 95 ٪ of overall cases (5). the t1dm takes 5-10٪of the cases; while, t2dm accounts for ~90% of cases (6). hypertension is chronic non-communicable disease in which there is persistent elevation of systolic and/or diastolic blood pressure of ≥ 140/90 mm hg; it may be considered the major risk factor for cardiovascular, cerebrovascular and renovascular diseases (7-9). in 2008 the prevalence of hypertension worldwide is about 40% in adults of 25 years and above (10). the who estimate that the hypertensive patients will reach 1 billion or more at year 2025 (11). the world health organization defines quality of life as “an individual's perception of their position in life in the context of the culture and value systems in which they live, and in relation to their goals, expectations, standards, and concerns (12). the purpose of insertion of quality of life (qol) as indicator for health outcome is attributed to the sensitivity of this measure for the evaluation of patients health status after taken treatment and its health outcome; where, the evaluation of quality of life is important because this evaluation can determine the aspects that are significant for quality of life of patients (13, 14) because the ultimate goal for treatment of chronic noncurable diseases like diabetes mellitus and hypertension is to improve the quality of life of such patients (15). qol's value is important for knowing what is significant to the individuals’ qol (13, 14) due to the principal goal for noncurative disease is to improve qol (15). diabetes mellitus (dm) as indicated by many studies to be independently associated with reduced levels of quality of life as evidenced by negative relationship between dm and many aspects of life like physical, mental, and social, financial aspects of individuals (16-18). the diabetic patients require controlling the symptoms of disease and adhering to complex regimens of treatment (19). thus, the impact of diabetes mellitus on quality of life can be analyzed into two interdependent aspects: the consequences attributed to the disease-related stressors and the burden imposed by the treatment demanding thus both the disease-related stressors and the burden if treatment of this disease may increase the risk making person more susceptible to the poor qol (19). several investigators have indicated that hypertension can seriously affect qol and patients well-being; where, it was shown that the known hypertensive patients have poorer qol because the diagnosis of hypertension increases the sensitivity of patients toward bodily symptoms and make an otherwise “healthy” person ill (20). method this study is a cross-sectional survey conducted to determine the impact of dm and hypertension on qol in iraqi patients in alnajaf al-ashraf province. the total number of participants in this study was 775 individuals which divided into four groups including healthy control group (190 persons), type-2 diabetic patients (194 patients), hypertensive patients (195 patients) and patients with both t2dm and hypertension (196 patients). the study was approved by scientific committee of the college of pharmacy–baghdad university. the participants in this study including known t2dm and hypertensive patients who attended at community pharmacies in urban and rural areas. after explaining the aims of the study to patients, the agreement of participation was obtained and the questionnaire provided to eligible individual. the identification of diabetic or hypertensive patients ensued after asking the patients to answer the question “do you have any of the following diseases diagnosed by doctor: dm, hypertension or dm plus hypertension“. the inclusion criteria involve all patients with t2dm, hypertension or both diseases who visited community pharmacy. the diabeticand hypertensivepatients should be treated at least six month before enrollment in the study. the exclusion criteria include pregnantor breast feeding women, patients with any other comorbidity not related to disease and patients who cannot complete the qol measures because of psychiatric or cognitive impairments that affect memory or judgment. the questionnaire was provided to all patients to be self-reported with exception of illiterate patients that were interviewed by trained community pharmacists who also report the treatment that has been taken by the patients. furthermore, such questionnaire contain organized questions such as demographic information (gender, age, education level, marital status, occupation, residence), duration of disease, -family history, and smoking status. the questionnaire for the assessment of qol of persons is the arabic version of world health organization qol– short version questionnaire (whoqol-brief) was utilized (21, 22). this questionnaire comprised of four domains each one measure specific aspect of life namely physical, psychological, iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 31 social and environmental domains. in addition, it also contains two other questions that ask about the general health and general quality of life. consequently this instrument has 26 questions. each item rated according to five points likert scale with arrangement in positive direction thus, high score refer to better qol; while, low score mean lower quality of life. the total score of domain was calculated by summation of scores of items that included in this domain after reverse the direction of three items in this questionnaire which are questions (3, 4 and 26). then calculate the mean of scores for each domain and use this mean to convert the domain score to (4-20) range by multiplying the mean by 4. then transformation to (0-100) range occur by use this formula ((score – 4) * 100/16). the sum of scores of four domains produces overall value of quality of life. the duration of study continued from november (2016) to march (2017). the statistical analysis performed by using ibm spss statistics version 23. the categorical variables represented by descriptive statistics like percentages and frequencies. the continuous variables were presented as (means ± sd). the internal reliability of questionnaire is evaluated by cronbach's alpha while student’s ttest , mann-whitney test and kruskal wallis test are used to compare between means of unpaired groups. when more than 20% of data was missed from assessment, the assessment was discarded. when the score of single item was missed the mean of other items was used to substitute the missing value. when two items were coded missing , the domain score was not computed with the exception of domain 3, where the domain should only be calculated if < 1 item is missing) ( 23). results the internal reliability of questionnaire based on cronbach's alpha value is (0.88) and for domains is ranged from (0.65) for social domain to (0.85) for physical domain indicates good internal consistency. also, pearson correlation coefficient show significant correlation between each item and the domain that comprise it. the mean age diabetic, hypertensive, dm plus hypertension patients and control group include 51.18 ± 10.220, 51.90 ± 10.928, 56.03 ± 8.878 and 50.05 ± 9.20 years respectively. the male participants for all groups are greater than female participants (table 1). table (1): age of the study groups (mean ±sd) study groups age (mean ±sd years) range ( years) diabetic patients 51.18 ± 10.220 32-78 hypertensive patients 51.90 ± 10.928 25-84 diabetic hypertensive patients 56.03 ± 8.878 37-80 control group 50.05 ± 9.20 3367 table(2): distribution of participants according to rural or urban area study groups number of participants in rural area ( alabasia) (7 community pharmacy) number of participants in urban area (30 community pharmacy) diabetic patients 29 ( 14.9) 165 (85.1) hypertensive patients 37 ( 19) 158 (81) diabetic hypertensive patients 18 (9.2) 178 (90.8) control group 22 (11.6) 168(88.4) the majority of type 2 diabetic patients (35.6 %) have secondary educational level; while, the illiterate patients take highest percent (29.7 %) among hypertensive patients but majority of (38.8 %) hypertensive diabetic patients have primary school educational level and finally the most of control group individuals (44 %) have college educational level (table 3). the majority of participants of all groups were married (table 3). the mean scores of the four domains of qol instrument for diabetic patients were statistically significant lower (p<0.001) than corresponding domains of control group (table 5) also the mean scores of the four domains of qol instrument for hypertensive patients were statistically significant lower (p<0.001) than corresponding domains of control group (table 6 ). the mean scores of the four domains of qol instrument for diabetic hypertensive patients were statistically iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 32 significant lower (p<0.001) than corresponding domains of control group (table 7). the overall qol scores (oqol) for all patients group were significantly lower (p<0.001) than that of control group (table 8). the comparison of mean of overall quality of life values between patients group showed that the effect of t2dm and hypertension were non-significantly different (p>0.05); while the presence of t2dm and hypertension together reduce the value of mean overall quality of life (oqol) significantly (p<0.05) when compared with t2dm alone and hypertension alone (table 9 ). figures (1, 2, 3, and 4) illustrated the response to first two questions about general health and qol. table (3): demographic characteristics of study participants variable diabetic hypertensive diabetic hypertensive control pvalue gender male 108(55.7) 101 (51.8) 101 (51.5) 98 (51.6) 0.813 female 86 (44.3) 94 (48.2) 95 (48.5) 92 (48.4) education level illiterate 42 (21.6) 58 (29.7) 48 (24.5) 11 (5.8) 0.001 primary school 63 (32.5) 53 (27.2) 76 (38.8) 19 (10.0) secondary school 69 (35.6) 51 (26.2) 55 (28.1) 75 (39.5) collage 20 (10.3) 33 (16.9) 17 (8.7) 85 (44.7) marital status single 4 (2.1) 6 (3.1) 4 (2.0) 10 (5.3) 0.165 married 172 (88.7) 153 (78.5) 162 (82.7) 155 (81.6) widowed 13 (13.7) 31 (15.9) 26 (13.3) 20 (10.5) divorced 5 (2.6) 5 (2.6) 4 (2.0 ) 5 (2.6) duration of disease less than 1 year 11 (5.7) 10 (5.1) 5 (2.6)* 8 (4.1)** ---- 0.001 15 year 72 (37) 91 (46.7) 45(22.9)* 75(38.3)** ---- 6 – 10 years 46 (24) 54 (27.7) 58(29.6)* 54(27.5)** ---- more than 10 years 65 (33.3) 40 (20.5) 88(44.9)* 59(30.1)** ---- residence urban 165 (85.1) 158 (81) 178 (90.8) 168(88.4) 0.030 rural 29 ( 14.9) 37 ( 19) 18 (9.2) 22 (11.6) occupation employed 43 (22.2) 48 (24.6) 37 (18.9) 72 (37.9) 0.001 non employed 151 (77.8) 147 (75.4) 159 (81.1) 118 (62.1) iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 33 continued table (3) *mean duration of diabetes mellitus , ** mean duration of hypertension table (4): number and type (s) of drug(s) taken by diabetic patients drug number percent % drug class(s) percent % one drug 58 (29.8) sulfonylurea 11 (18.9) biguanide 16 (28.3) insulin 31 ( 52.8) two drugs 123 (63.5) insulin + biguanide 62 (50.4) sulfonylurea + biguanide 58 ( 47.2) dpp4i+ biguanide 2 ( 1.6) insulin +sulfonylurea 1 (0.8) three drugs 13 (6.7) insulin+sulfonylurea+biguanide 7 (53.8) dpp4i+sulfonylurea+biguanide 6 (46.2) dpp4i:dipeptidyl peptidase -4 inhibitor variable diabetic hypertensive diabetic hypertensive control p-value family history yes 128 (66) 149 (76.4) 132 (67.3) ----- 0.051 no 66 (34) 46 (23.6) 64 (32.7) ----- smoking habit smoker 39 (20.1) 40 (20.5) 46 (23.5) 44(23.2) 0.752 non smoker 155(79.0) 155 (79.5) 150 (76.5) 146 (76.8) treatment diet only 3 (1.6) 9 ( 4.6) 1 (0.5) ----- 0.001 one drug 58 (29.8) 109 (55.9) ---------- two drugs 123(63.5) 58 (29.8) 43 (21.9) ----- three drugs 13 (6.7) 16 (8.2) 103 (52.5) ----- four drugs -----2 (1.0) 42 (21.5) ----- five drugs -----1 (0.5) 5 (2.6) ----- six drugs ----------2 (1.0) ----- iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 34 figure (1) :the proportion of responses of diabetic group for first two items of whoqol-bref about the perception of general health and quality of life. whoqol-bref = world health organization quality of lifebrief questionnaire . figure (2) :the proportion of responses of hypertensive group for first two items of whoqolbref about the perception of general health and quality of life. whoqol-bref = world health organization quality of lifebrief questionnaire iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 35 figure (3) :the proportion of responses of diabetic hypertensive group for first two items of whoqol-bref about the perception of general health and quality of life. whoqol-bref = worldhealth organization quality of lifebrief questionnaire figure (4) :the proportion of responses of control group for first two items of whoqol-bref about the perception of general health and quality of life. whoqol-bref = world health organization quality of lifebrief questionnaire. iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 36 table( 5): comparison of qol domain scores between dm patients and control group scale domain diabetic group control group pvalue 4-20 physical 11.32 ± 3.59 14.50 ± 1.86 < 0.001 psychological 12.29 ± 2.60 14.27 ± 1.58 < 0.001 social 12.78 ± 2.77 14.73 ± 2.05 < 0.001 environmental 11.61 ± 2.31 13.44 ± 1.41 < 0.001 0-100 physical 45.80 ± 22.45 65.68 ± 11.60 < 0.001 psychological 51.82 ± 16.28 64.18 ± 9.93 < 0.001 social 54.89 ± 17.31 67.10 ± 12.85 < 0.001 environmental 47.59 ± 14.47 59.01 ± 8.86 < 0.001 qol: quality of life , dm : diabetes mellitus table (6): comparison of qol domain scores between hypertensive patients and control group scale domain hypertensive group control group pvalue 4-20 physical 11.52 ± 2.69 14.50 ± 1.86 < 0.001 psychological 12.76 ± 2.38 14.27 ± 1.58 < 0.001 social 13.43 ± 3.04 14.73 ± 2.05 < 0.001 environmental 11.89 ± 2.01 13.44 ± 1.41 < 0.001 0-100 physical 47.03 ± 16.81 65.68 ± 11.60 < 0.001 psychological 54.78 ± 14.91 64.18 ± 9.93 < 0.001 social 58.97 ± 19.01 67.10 ± 12.85 < 0.001 environmental 49.34 ± 12.58 59.01 ± 8.86 < 0.001 qol: quality of life iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 37 table (7): comparison of qol domain scores between diabetic hypertensive patients and control group. scale domain dm plus hypertension control group pvalue 4-20 physical 10.20 ± 3.32 14.50 ± 1.86 < 0.001 psychological 11.74 ± 2.87 14.27 ± 1.58 < 0.001 social 12.41 ± 3.01 14.73 ± 2.05 < 0.001 environmental 11.04 ± 2.24 13.44 ± 1.41 < 0.001 0-100 physical 38.77 ± 20.77 65.68 ± 11.60 < 0.001 psychological 48.38 ± 17.94 64.18 ± 9.93 < 0.001 social 52.59 ± 18.81 67.10 ± 12.85 < 0.001 environmental 44.03 ± 14.02 59.01 ± 8.86 < 0.001 qol: quality of life , dm : diabetes mellitus table( 8): comparison of overall qol value (oqol) between patients groups and control group patients group value of (oqol) for patients group value of (oqol) for control group pvalue dm patients 50.03 ± 14.94 63.99 ± 8.19 < 0.001 hypertensive patients 52.53 ± 11.86 63.99 ± 8.19 < 0.001 dm plus hypertension 45.94 ± 15.10 63.99 ± 8.19 < 0.001 oqol : overall quality of life , dm : diabetes mellitus table( 9): comparison value of (oqol) between patients groups first group oqol (mean ±sd) other group oqol (mean ±sd) pvalue dm 50.03 ±14.94 hypertension 52.53±11.86 0.08 dm plus hypertension 45.94± 15.10 dm 50.03 ±14.94 0.02 dm plus hypertension 45.94± 15.10 hypertension 52.53±11.86 < 0.001 oqol: overall quality of life, dm: diabetes mellitus iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 38 discussion this study showed that dm has significant impact on all domains of qol as compared to control group and this supported by other studies like ashraf eljedi et al. (24) and zivcicova et al. (25) and the physical domain was affected to greater extent than other domains and this consistent with ahari et al. (26) and boonhow chew et al. (27). the social domain is affected to minor extent than other domains probably due to prevalence of social support for these persons and this also supported by boonhow chew et al. (27). hypertension affects all aspects of qol. the scores of four domains in hypertensive patients are significantly (p < 0.001) lower than the scores of control group where the physical domain is affected more while the social domain is least affected among all domains of whoqol-bref questionnaire and this result is consistent with the result obtained in brazil (28) and xianglong xu et al. that represent that the hqol is significantly affected by hypertension although it use another instrument for measurement of qol like sf-36 (29). the presence of t2dm and hypertension in same individual further lower the scores of qol than dm or hypertension alone where the scores of all domains is significantly very lower than the scores of these domains in healthy individuals due to burden of both disease and its complications and treatment on patients quality of life. when the patient suffers from single chronic illness, the only perception of being have chronic disease is enough to compromise the qol (30, 31). the t2dm alone and hypertension alone produce comparable effect on qol where the mean of overall qol (oqol) of diabetic patients is (50.03) whereas the mean of (oqol) of hypertensive patients is (52.53) and the (p > 0.05) and this is supported by tamara poljicanin et al. study (32). the mean score of (oqol) of diabetic hypertensive patients is poorer than that for patients with t2dm alone or hypertension alone and this consistent with the result obtained by hwee-lin wee et al. (33). the present study indicate that the combination of t2dm and other chronic non-communicable disease like hypertension can further lower the scores of qol domains especially physical domain which is decreased to greater extent than other domains and this is supported by otiniano me et al. (34) and oldridge nb et al. (35). also the participants of this study were drawn from general population so the findings of this study can be easily generalized to large scale population (36). also the results of this study indicate that the presence of t2dm and hypertension not only rise the healthcare costs (37) and mortality (38) but also increase the physical and psychological load of these diseases on patients. the strengths of this study are use of generic questionnaire which explain the wider effect of dm and hypertension on various aspects of life. the whoqol-bref was characterized by good reliability and acceptable validity in cross-sectional studies over 23 nations (39). other factors that strength this study is relatively large size sample and presence of control group. conclusion according to the results obtained from this study, it can be concluded that patients with t2dm alone have significant lowering of mean scores of all domains of qol and mean of overall qol compared to control group with physical domain was highly affected and social domain was least affected. also, hypertension alone has significant negative impact on qol. while the diabetic hypertensive patients have poorer qol than control group. the hypertension and t2dm have comparable impact on qol; while the presence of t2dm plus hypertension negatively affects all domains of qol to greater extent than t2dm alone or hypertension alone. the benefits of this study are: (1) determination of impact of chronic diseases like diabetes mellitus or hypertension on health related quality of life which considered as indicator for health outcome, (2): comparison of effect of presence of two chronic diseases in same patients versus one chronic disease (3) determine the aspects of life that were influenced by presence of diabetes and hypertension and determine which aspect is affected to greater extent (4) indicate that great attention is required for patients with diabetes or hypertension by activation of screening system for early detection of diabetes and hypertension and so enable for prevention, prevent the progression , and treatment of such diseases and its complications (5) this study determine that patients with diabetes or hypertension have poor quality of life and so further investigation is required to determine the causes and so improve the qol of such patients. acknowledgements i would like to thank for all community pharmacists who participate in collection of data. iraqi j pharm sci, vol.26(2) 2017 patients with diabetes mellitus, hypertension or both diseases 39 references 1. american dm association. definition of dm mellitus. diabetes care volume 37, supplement 1, january 2014. 2. alvin c. powers. harrison’s principles of internal medicine, 19th edition, mcgrawhill education , london.copyright © 2015; p : 2399-2400. 3. brian k. alldredge, robin l. corelli, michael e. ernst, b. joseph guglielmo,pamala a. jacobson, wayne a. kradjan, bradley rwilliams. applied therapeutics: the clinical use of drugs , tenth edition. lippincott williams&wilkins, philadelphia,copyright 2013; p: 1223-1225. 4. guariguata, l; whiting, d; hambleton, i; beagley, j; linnenkamp, u; eshaw, j . global estimates of dm prevalence for 2013 and projections for 2035. dm research and clinical practice. elsevier. 2014; 103:137-149. 5. world health organisation. definition and diagnosis of dm mellitus and intermediate hyperglycaemia. report of a who/idf consultation; 2006.available from: http://www.who.int/dm/publications/diagno sis_dm2006/en/. 6. public health england. adult obesity and type 2 dm; 2014. available from: https://www.gov.uk/government/publication s/adult 7. larry, e fields, vicki l. burt, jeffery a. cutler, jeffrey hughes, edward j. roccella, paulsorli. the burden of adult hypertension in the united states 1999 to 2000. hypertension. 2004; 44(4):398-404. 8. licy l yanesjane, f. reckelhoff. postmenopausal hypertension .am j hypertens. 2011; 24 (7): 740-749. 9. kwok leung ong, bernard m.y. cheung, yu bun man, chu pak lau, karen s.l.lam. prevalence, awareness, treatment, and control of hypertension among united states adults 1999–2004.hypertension. 2007; 49:69-75. 10. .saleh, farah r., razhan s. othman, and karzana.omar."prevalence of hypertension among young and adults in the kurdistan region."journal of chemical, biological and physical sciences (jcbps). 2016; 6(2): 344. 11. yongqing, z., ming, w., jian, s., pengfei, l., xiaoqun, p., meihua, d., ...& ping, l. prevalence, awareness, treatment and control of hypertension and sodium intake in jiangsu province, china: a baseline study in 2014. bmc public health.2016; 16(1): 56. 12. komal verma1, meenal dadarwal. diabetes and quality of life: a theoretical perspective. j soc health diabetes 2017;5:5-8 13. komal verma, meenaldadarwal.dm and quality of life: a theoretical perspective. j soc health dm. 2017; 5(1):5-8. 14. robinson, peter g., alison j. carr, irene j. higginson. 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(http://www.idf.org/ eatlas/home/index.cfm?node=104). 37. o'brien ja, patrick ar, caro jj. cost of managing complications resulting from type 2 dm mellitus in canada. bmc health serv res. 2003; 3:7. 38. wang sl, head j, stevens l, fuller jh. excess mortality and its relation to hypertension and proteinuria in diabetic patients. the world health organization multinational study of vascular disease in dm. dm care. 1996; 19:305-312. 39. skevington sm, lotfy m, o’connell ka. the world health organization’s whoqol-bref quality of life assessment: psychometric properties and results of the international field trial: a report from the whoqol group. qual life res. 2004; 13 (2): 299-310. effect of additives on the solubility and dissolution of piroxicam iraqi j pharm sci, vol.21(1) 2012 additives and solubility of piroxicam 117 effect of additives on the solubility and dissolution of piroxicam from prepared hard gelatin capsule eman b. h. al-khedairy* ,1 *department of pharmaceutics , college of pharmacy,university of baghdad, baghdad, raq. abstract piroxicam is a non-steroidal anti-inflammatory drug (nsaid) used in the treatment of musculo-skeletal and joint disorders. the problem with this drug is its poor solubility in water and hence poor bioavailability after oral administration. in order to improve its solubility and dissolution behavior, hydrophilic additives such as starch, lactose, superdisintegrants including crospovidone (c.p), cross carmellose sodium (ccs), and sodium starch glycolate (ssg) were physically dry mixed with the drug by simple trituration. the improvement in the solubility in 0.1 n hcl was obtained as the amount of starch or lactose increased in the physical mixture, while for superdisintegrants, they further improve the solubility when they are present in small amount and the best improvement was gained with ssg. to study the effect of these additives on the dissolution of the drug, piroxicam capsules were prepared by simple trituration of the drug with starch or lactose or combination of lactose: starch (2:1)by weight with or with out the presence of ssg. the dissolution profiles of these preparations were analyzed using similarity factor f2. best results were obtained when the drug was triturated with starch and ssg or with acombinaton of lactose: starch (2:1) by weight with ssg . the dissolution profiles of these preparations were similar to that of the marketed feldene ® pfizer 20 mg capsules with f2 74.6 and 80.37 respectively. key words: piroxicam, hydrophilic additives, superdisintegrant مه الكبسوالت الجيالتيىية الصلبةدواء البيروكسيكام وتحرر تأثير المواد المضافة على ذوباوية م الخضيري*زايمان بكر حا ,1 .انعشاق ،بغذاد د،خبيعت بغذا ،كهٍت انصٍذنت ،* فشع انصٍذالٍَبث الخالصة انًشكهت انبٍشٔكسكبو ْٕ يٍ يعبداث االنتٓبببث انغٍش ستٍشٌٔذٌت انتً تستخذو فً عالج آالو انًفبصم ٔاَالو انععهٍت . ٍٍ يع ْزا انذٔاء ًْ اَّ لهٍم انزٔببٌ ببنًبء ٔببنتبنً ٌكٌٕ انتٕفش انحٍٕي نّ ظئٍال عُذ تُبٔنّ كشكم دٔائً عٍ غشٌك انفى . نزنك ٔنتحس ، crospovidone (c.p)يثم انُشبء ، انالكتٕص ، يٕاد يفككت لٌٕتٔتحشسِ يٍ انشكم انذٔائً تى اظبفت يٕاد يحبت نهًبء يثم ّرٔببَ cross carmellose sodium (ccs) ٔsodium starch glycolate (ssg) ٔانتً تى يضخٓب يع انذٔاء بشكم خبف ٔبطشٌمت ( عُذ 1.0فً انٕسػ انحبيعً ) حبيط انٍٓذسٔكهٕسٌك رٔ عٍبسٌت . تى انحصٕل عهى تحسٍ فً انزٔببٍَت انبسٍػ انفٍضٌبٔي انسحٍ انمٌٕت فمذ ٔخذ آَب تحسٍ انزٔببٍَت عُذ تاي يُٓى فً انًضٌح، ايب ببنُسبت نهًٕاد انًفكك صٌبدةانالكتٕص يع ٔء يع انُشب اايضج انذٔ انًٕاد انًعبفت عهى سشعت تحشس . نغشض دساست تأثٍش ْزِ ssgتى انحصٕل عهٍّ ببستعًبل تحسٍٔاٌ اكثشٔخٕدْب بكًٍت لهٍهت ضج انذٔاء بطشٌمت انسحٍ انبسٍػ يع انُشب أ انالكتٕص أ يضٌح يٍ كبسٕالث عٍ غشٌك ي شانذٔاء يٍ انشكم انذٔائً ، تى تحعٍ احسٍ . f2. نمذ تى تحهٍم َتبئح تحشسانذٔاء ببستعًبل يعبيم انتشببّ ssgٔخٕد و( بٕخٕد أ عذ0:1) ٔصٍَت انالكتٕص ٔانُشب بُسبت حٍث ssg( يع 0:1) ٔصٍَت انالكتٕص ٔانُشب بُسبتأ عُذ سحُّ يع يضٌح ssgانُتبئح تى انحصٕل عهٍٓب عُذ سحٍ انذٔاء يع انُشب ٔ حٍث اٌ يعبيم يهغى نششكت فبٌضس 11نتحشسِ يٍ انًُتح انًسٕق نكبسٕالث انفهذٌٍ يشببّاٌ تحشس انذٔاء يٍ ْزِ انكبسٕالث كبٌ .ببنتعبلب 71.26 ٔ 63.7كبٌ f2انتشببّ مواد مفككة قوية . الكلمات المفتاحية : البيروكسيسام , مواد مضافة محبة للماء, introduction sufficient solubility is a prerequisite for effective oral delivery of any therapeutic agent. however, drugs with low solubility and high permeability fall into biopharmaceutics classification system (bcs) class ii (1) for which the dissolution is usually the ratelimiting step for gastrointestinal absorption. to enhance the dissolution rate and thus oral absorption of such drugs numerous formulation strategies have been developed (2) . proxicam is a nsaid which is widely used for treatment of musculo-skeletal and joint disorders (3) . its absolute bioavailability is unknown since no intravenous dosage data is available in man. it is practically insoluble in water (3,4) . hence when this drug is administered orally it may cause bioavailability problems arises from its low water solubility and law dissolution rate in acid medium where the absorption takes place (5) . 1 corresponding author email : emanalkhedairy@yahoo.com received : 27/2/2012 accepted : 16/5/2012 mailto:emanalkhedairy@yahoo.com iraqi j pharm sci, vol.21(1) 2012 additives and solubility of piroxicam 118 various techniques have been used in an attempt to improve the solubility and dissolution rate of piroxicam including liquisolid compacts (6) , salt formation with ethanolamines (7) , solid dispersion using pvp k30 (8) or peg 4000 (9) as a water soluble carriers. in addition, surface solid dispersion of piroxicam in microcrystalline cellulose and in potato starch was also prepared by coevaporation method (10) . moreover, it was found that the solubility and dissolution of poorly water soluble drugs can be markedly improved by the use of superdisintegrants using solid dispersion technique (11,12) or by preparation of ordered mixing or as physical mixture (11, 13) . the aim of this study was to determine the impact of simple physical mixing of piroxicam with different amount of starch, lactose, superdisintegrants including crospovidone, cross carmellose sodium, and sodium starch glycolate each alone on the solubility of drug in 0.1n hcl .in addition the effect of combination of piroxicam with one or more of the above additives on its dissolution from these physical mixtures enclosed in hard gelatin capsules was also explored and compared with that of pure drug and with the commercially available feldene (r) 20 mg pfizer capsules. materials and methods materials piroxicam, sodium starch glycolate (ssg) ( samara drug industry (sdi), iraq) crospovidone (c.p), cross carmellose sodium (ccs) ( dar al-dawa pharmaceutical, manufacturing co., jordan), lactose, starch (riedel-dehean, ag seelzehannover, germany), hcl ( bdh chemicals ltd.,pool, england), feldene ( r ) 20 mg pfizer capsules methods solubility study determination of piroxicam solublity the solubility of piroxicam in 0.1n hcl was measured using standardized shake flask method (14) . in this method 60 mg of the drug was added to 50 ml of 0.1n hcl (saturated solution) (15) and the mixture was shaken at 37°c for 48 hours, filtered through ordinary filter paper and the concentration of piroxicam in the filtrate, following suitable dilution, was assayed spectrophotometrically at 333 nm for piroxicam (16) . effect of additives on the solubility of piroxicam starch, lactose, c.p, ccs, ssg were used to study the effect of additives on the solubility of piroxicam. certain amounts ranging from 60480 mg of one of the above additives was added to a bottle containing 60 mg drug. manual bottle tumbling was used to prepare simple physical mixture, then 50 ml of 0.1n hcl was added to the mixture to determine the solubility of piroxicam in presence of specific amount of additives by shake flask method as mentioned previously. preparation of piroxicam capsules according to the results of solubility study, six different formulas were prepared, in which proxicam was geometrically triturated with certain amount of one or more of the additives (table 1). the resultant powder was filled in hard gelatin capsule size 0 as 20mg/capsule. table 1: composition of piroxicam capsules ingredients* formula number f1 f2 f3 f4 f5 f6 piroxicam 20 20 20 20 20 20 starch 480 -- 160 420 -- 140 lactose -- 480 320 -- 420 280 ssg -- -- -- 60 60 60 total 500 500 500 500 500 500 *all ingredients are in milligram dissolution studies the dissolution profiles of the drug alone, encapsulated drug, the prepared capsules and feldene (r) pfizer capsules as a reference capsules were studied using apparatus 1 (usp, basket). dissolution medium was 900 ml 0.1n hcl maintained at 37°c ± 0.1°c and stirred at 50 r.p.m for one hour. five ml of sample were withdrawn at specified time intervals and were replaced with an equal volume of fresh dissolution medium to maintain sink condition. the samples, following suitable dilution were assayed spectrophotometrically at 333 nm. (16) . all dissolution studies were carried in triplicate dissolution data analysis the dissolution profiles of the prepared formulas were compared using ƒ2 similarity factor. the similarity factor is a logarithmic reciprocal square-root transformation of the sum of squared error and iraqi j pharm sci, vol.21(1) 2012 additives and solubility of piroxicam 119 is a measurement of the similarity in the percentage of dissolution between two curves. { ∑ } where n is the sampling number, rt and tt are the percent dissolved of the reference and test products at each time point t. two dissolution profiles are considered similar when the ƒ2 value is greater than or equal to 50 (17) . results and discussion effect of additives on the solubility of piroxicam the effect of type and amount of additives on the solubility of piroxican in 0.1n hcl is summarized in table (2). the solubility of piroxicam was highly improved in presence of the hydrophilic superdisintegrants more than with lactose, a soluble carrier or with starch which have limited solubility and swelling properties. this improvement in solubility can be explained to be due to the surface adsorption of drug in the physical mixtures of all carriers (10) ; the wetted surface of the carrier promotes wettability and solubility of the drug (18, 19) . in addition, it was found that within the superdisintegrants, the order of improvement in the solubility is ssg > ccs > c.p which may be due to the differences in their physical properties including hydrophilicity and swelling property. on the other hand, the results in table (2) show that increasing the amount of superdisintegrants results in decreasing the solubility of the drug. this could be attributed to the formation of viscous barrier resultant from absorption of water by the superdisintegrants that decrease the wettability and thus the solubility of the drug ( 13) . table 2: effect of type and amount of additives on the solubility of piroxicam (mg/50 ml) at 37°c in 0.1 n hcl amount of additive (mg) type of additives starch lactose c.p ccs ssg 0 6.36 ± 0.46 6.36 ± 0.46 6.36 ± 0.46 6.36 ± 0.46 6.36 ± 0.46 60 6.39 ± 0.02 10.23 ± 0.04 11.47 ± 0.12 13.42 ± 0.01 14.02 ± 0.42 120 7.87 ± 0.25 10.39 ± 0.18 12.51 ± 1.09 12.65 ± 0.15 12.97 ± 0.52 180 10.89 ± 0 11.67 ± 0.17 12.01 ± 0.13 12.05 ± 0.12 12.96 ± 0.2 240 10.88 ± 1.17 12.02 ± 0.52 11.71 ± 1.09 10.88 ± 0.57 11.84 ± 0.31 300 10.97 ± 1.32 12.4 ± 0 --------- 360 11.26 ± 0 12.29 ± 0.08 --------- 420 11 ± 0.64 12.29 ± 0 --------- 480 11.04 ± 0.06 12.16 ± 0 --------- all values are expressed as mean ± standard deviation (n=3) preparation of piroxicam capsules six different formulas (table 1) of piroxicam capsules were prepared using starch, lactose or combination of lactose: starch (2:1) by weight (20) with or without the addition 60 mg ssg (the minimum amount among the superdisintegrants that cause the highest solubility) since the presence of any of the above additives have no adverse effect on the solubility of the drug. dissolution studies effect of gelatin shell the dissolution of drug from hard gelatin capsule was faster than that from pure powdered drug as shown in figure (1). pure powdered drug added to dissolution medium remains as agglomerate floats on the surface of the dissolution medium. the enhancement effect of gelatin may be due to its hydrophilic nature that increases wetting and dissolution of the drug. this result can be supported by that obtained by chono s. et al (21) who found that gelatin enhances the dissolution of poorly soluble drugs and their absorption from g.i.t. figure 1: effect of gelatin shell on the dissolution of piroxicam in 0.1 n hcl at 37°c 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time minutes % d ru g r e le a s e d drug/powder encapsulated drug iraqi j pharm sci, vol.21(1) 2012 additives and solubility of piroxicam 120 effect of type of diluents dissolution profiles of powdered drug, encapsulated drug, and formula (f1, f2 and f3) which contain starch, lactose and lactose: starch (2:1) by weight respectively are shown in figure (2). it is evident that all additives increase the dissolution of the drug. in addition the dissolution profiles of these formulas are not similar. dissolution profiles comparison (table 3) between f1 and f2 yield f2 39.75, while dissolution profiles comparison between f2 and f3 yield f2 48.9. the higher effect was obtained with f2 mainly for the first 20 minutes which is in agreement with the result obtained by ampolsuk c. et al (22) who stated that deposition of hydrophobic drug in a molecular subdivision on excess powdered lactose (due to frictionally prepared triturate) is an effective method of increasing surface of the drug and hence, its dissolution. since lactose is soluble in simulated gastric fluid, its presence in the dissolution medium would physicall separate drug particles, preventing their agglomeration and enhancing their dissolution. on the other hand, the less enhancing effect of f1 and f3 at the first 20 minutes may be due to the presence of starch which has less solubility in the dissolution medium. figure2: effect of type of diluents on the dissolution of piroxicam in 0.1 n hcl at 37°c effect of addition of ssg trying to further enhance the release of piroxicam from the prepared capsules, ssg was added in combination with the used diluents to prepare f4, f5 and f6. the dissolution profiles shown in figures 3a and 3c are non similar with f2 39.17 and 47.47 respectively which indicate that ssg enhances the release of the drug when it is present in combination with starch (f4) or with lactose: starch (2:1) (f6) .on the other hand, combination of ssg with lactose (f5) gives similar dissolution profiles as shown in figure 3b with f2 59.57, so no further improvement in dissolution . these results indicate that the presence of the ssg in the dissolution medium kept the drug particles in dispersed condition i.e it prevents the agglomeration of drug particles and promote its wetting and dissolution (23) .this action is most effective when it is combined with insoluble or slightly soluble diluents (24) , since the water soluble diluents may increase the viscosity of the penetrating fluid which tends to reduce the effectiveness of ssg while the insoluble diluents produce rapid disintegration with adequate of disintegrant (25) . figure 3.a : effect of combination of ssg with starch on the dissolution of piroxicam in 0.1 n hcl at 37°c figure 3.b: effect of combination of ssg with lactose on the dissolution of piroxicam in 0.1 n hcl at 37°c figure 3.c: effect of combination of ssg with lactose: starch (2:1) on the dissolution of piroxicam in 0.1 n hcl at 37°c 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time minutes % d ru g r e le a s e d encapsulared drug f1 f2 f3 drug/powder 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time minutes % d ru g r e le a s e d f4 f1 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time minutes % d r u g r e le a s e d f6 f3 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 time minute s % d ru g r e le a s e d f5 f2 iraqi j pharm sci, vol.21(1) 2012 additives and solubility of piroxicam 121 comparision of the prepared capsules with the marketed capsule all the prepared capsules of f1, f2, f4, and f6 met the usp specification for the release of the drug in simulated gastric fluid (not less than 75% of the drug is dissolved in 45 minutes) (16) . the prepared capsules from f4 and f6 which showed fastest dissolution were compared with the marketed felden (r) pfizer capsules (figure 4). similar release profiles were obtained between f4 and feldene (r) pfizer capsules yield f2 74.6, and that between f6 and feldene (r) pfizer capsules yield f2 80.37. in addition, to similar dissolution profiles these preparations contain simple, safe (26) , available materials that enhance the solubility and the dissolution of the drug. this gives superiority of using these diluents on sodium lauryl sulfate (a solubilizing agent) that is used in pfizer product (27) for preparation of piroxcam capsules. figure 4: comparison of the release profile of the prepared capsules with the marketed feldene ( r) pfizer capsules in 0.1 n hcl at 37°c table 3: values for the similarity factor f2 for the release profiles in 0.1 n hcl formula no. f2 f3 f4 f5 f6 felden cap. pfizer ( r ) f1 39.7504 54.85394 39.17509 49.29237 40.85396 39.12536 f2 48.90846 60.2949 59.57132 68.30451 61.21903 f3 43.99996 66.79376 47.47173 44.28989 f4 51.63288 73.76971 74.60086 f5 56.26644 51.46837 f6 80.37825 conclusion the results of this study indicate that the simple physical dry mixing of the piroxicam with starch or with lactose:starch (2:1) by weight in presence of ssg can enhance its solubility as well as its dissolution characteristics to be similar to that of felden (r) pfizer . this physical dry mixing of hydrophobic drug with suitable type and amount of additives may be considered as a 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of digoxin-lactose and hydrocortisone triturate i. j. pharm. sci. 1974; 63(1): 117-118 23. makiko f., hideko o. yusuke s., honami t. masuo k. and yoshiteru w. preparation, characterization, and tableting of a solid dispersion of indomethacin with crospovidone. int. j. pharm. 2005; 293:145-53 24. product sheet 25. chebli c. and cartlizer l. cross linked cellulose as a tablet excipient: a binding or disitegranting agent. int. j.pharm. 1998; 171: 101-110 26. pifferi g. and restani p. the safety of pharmaceutical recipients. il farmaco 58 2003; 541_/550 27. feldene (r) pfizer capsules material safety data sheet iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles doi: https://doi.org/10.31351/vol30iss2pp143-152 143 formulation and characterization of nimodipine nanoparticles for the enhancement of solubility and dissolution rate areej w. alhagiesa*,1 and mowafaq m. ghareeb** *department of pharmaceutics, college of pharmacy, university of kufa, najaf, iraq **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract nimodipine (nmd) is a dihydropyridine calcium channel blocker useful for the prevention and treatment of delayed ischemic effects. it belongs to class ⅱ drugs, which is characterized by low solubility and high permeability. this research aimed to prepare nimodipine nanoparticles (nmd nps) for the enhancement of solubility and dissolution rate. the formulation of nanoparticles was done by the solvent anti-solvent technique using either magnetic stirrer or bath sonicator for maintaining the motion of the antisolvent phase. five different stabilizers were used to prepare nmd nps( tpgs, soluplus®, hpmc e5, pvp k90, and poloxamer 407). the selected formula f2, in which soluplus® has been utilized as a stabilizer, has a particle size (77 nm) and polydispersity index (pdi) (0.016). the formulas with the smallest particle size were freeze dried with the addition of 1 % w/w mannitol as cryoprotectant. the saturation solubility of nmd in the prepared nanoparticles was increased twenty four-folds, and the complete dissolution was achieved at 90 minutes compared with pure nmd, which reaches only 6%. the formation of hydrogen bonding between nmd and the polymer or the cryoprotectant, as confirmed by the ftir study. in conclusion, the preparation of nmd as polymeric nanoparticles is a useful technique for enhancing the solubility and dissolution rate. keywords: nimodipine nanoparticles, solvent antisolvent precipitation, solubility enhancement. تصييغ وتقييم الجسيمات النانوية لعقار النيمودبين لتحسين الذوبانية **موفق محمد غريب و 1*، سىالحاج عيوهاب اريج فرع الصيدالنيات ،كلية الصيدلة ، جامعة النهرين ، بغداد ، العراق . * . بغداد،العراق بغداد، جامعة الصيدلة، كلية ، الصيدالنيات فرع** الخالصة النيمودبين هو ديهيدروبيريدين مغلق قناة الكالسيوم مفيد للوقاية والعالج من اآلثار اإلقفارية المتأخرة في الدماغ . ديبينية وية النيموهو ينتمي إلى عقاقير من النوع الثاني التي تتميز بانخفاض قابلية الذوبان ونفاذية عالية. يهدف هذا البحث إلى إعداد الجسيمات النان ان المغناطيسي لتعزيز الذوبانية ومعدل الذوبان. تم تكوين الجسيمات النانوية باستخدام تقنية الترسيب للمذيب ومضاد المذيب باستخدام إما جهاز الدور ت النانوية وتتضمن هذه أوالحمام المائي ذو الموجات الصوتية للحفاظ على حركة الطور المضاد. تم استخدام خمسة مثبتات مختلفة ألعداد الجسيما المثبتات .( tpgs, soluplus®, hpmc e5, pvp k90, and poloxamer 407) اظهرت النتائج ان سوليوبلس هو االفضل بتقليل حجم الجسيمات حيث كانت افضل صيغة وهي )الصيغة الثانية( لها حجم تم تحقيق الذوبان الكامل بعد تسعين دقيقة للجسيمات النانوية بينما النيمودبين نانوميتر( . ازدادت الذوبانية بمقدار اربع وعشرين مرة و ۷۷جسيمي ) % فقط خالل هذه المدة. كما اظهرت النتائج باالشعة تحت الحمراء تكوين االواصر الهيدروجينية بين النيمودبين والبوليمر وبذلك 6الخام قد وصل الى من ذوبانية ومعدل ذوبان النيمودبين.يمكن ان نستنتج ان الجسيمات النانوية قد حسنت الكلمات المفتاحية : الجسيمات النانوية للنيمودبين ، تقنية الترسيب بالمذيب ومضاد المذيب ، تحسين الذوبانية . introduction many new pharmaceutical entities, approximately 40 % of them, are lipophilic compounds which have low water solubility and raise a clinical issue regarding their dissolution and absorption from their administration sites(1). the solubility of drugs in aqueous media is an essential consideration to be dealt with early in the drug discovery process , and several formulation strategies have been proposed to enhance the solubility, such as complexation, ph adjustment, and using co-solvents(2). nanoparticles (nps) are solid colloidal particles that usually lie in the 100 nm size range(3). they exhibit many advantages of better stability, tremendous enhancement in solubility and dissolution rate of poorly soluble drugs, high drug loading, targeting different organs and tissues, and they can be incorporated in various dosage forms(4). nanoparticles can be prepared from liquid nanosuspension (ns) after drying by a suitable method like spray drying, vacuum drying, and the most widely used freeze-drying. 1corresponding author e-mail: areej.w.alhagiesa@uokufa.edu.iq received: 18/7/2020 accepted:11 /4 /2021 published online first: 2021-12-11 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss2pp143-152 iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles 144 the transformation of liquid ns into solid nps may provoke stressful conditions on the prepared nps. it may cause aggregation and agglomeration into larger particles and lose its unique property of nano range particle size. freezedrying is the process of removing water by sublimation and desorption under a high vacuum. it is better to mention that the solidification process is dependent mainly on the surface hydrophobicity and cohesive energy of the drug to produce a stable, dried ns. a cryoprotectant may be added to preserve the dispersibility of a ns(6). nmd is a dihydropyridine calcium channel blocker useful for the prevention and treatment of delayed ischemic effects due to cerebral vasospasm and subarachnoid hemorrhage(7). it has a low bioavailability of around 13 % because of its low water solubility (4.14 µg/ml) and extensive first-pass metabolism. it belongs to class ⅱ drugs ( low solubility and high permeability) in the biopharmaceutical classification system(8). many attempts have been applied to enhance its bioavailability like solid dispersion(9)(10), cyclodextrin complexation(11), and nanotechnology approaches like nanoemulsion(12)(13), solid lipid nanoparticles(14), nanoliposomes(15), and nanocrystals(16). nmd is chemically described as (3-(2methoxyethyl) 5-propan-2-yl 2,6-dimethyl-4-(3nitrophenyl)-1,4-dihydropyridine-3,5dicarboxylate) . its chemical structure is shown in figure (1). figure 1. the chemical structure of nimodipine nmd is a yellow crystalline powder with a melting point of 125℃, pka 5.4, and a partition coefficient (log p) 3.05. it has a molecular formula and weight of c21h26n2o7 and 418.4 gm/mol, respectively(8). this research aims to enhance the solubility and dissolution rate of the poorly water soluble drug nimodipine. materials nimodipine pure powder, tocopheryl polyethylene glycol succinate (tpgs) poloxamer 407 ( pxm 407), hydroxypropyl methylcellulose ( hpmc e5) and polyvinyl povidone (pvp k90) were purchased from hyperchem, china. soluplus® was bought from basf, germany. brij-35 was obtained from himedia, india. disodium hydrogen phosphate (na2hpo4) and (potassium dihydrogen phosphate (kh2po4)were bought from thomas baker, india. sodium chloride (nacl) was purchased from lad, india. hydrochloric acid (hcl) was obtained from chem limited, india. dialysis membrane; mwco 12000 -14000 da was puechased from (usa), ethanol was brought from chemlab, belgium. mannitol was obtained from england. method preparation of nimodipine nanoparticles nimodipine nps were prepared by a solvent – antisolvent precipitation method (nanoprecipitation method). this method involves dissolving 30 mg of nmd in 3 ml ethanol ( solvent ) and allowed to be added dropwise using syringe pump as shown in figure(2 ) at a speed of 1 ml/min into a beaker containing 27 ml distilled water (antisolvent in presence of (60 mg) of the following stabilizers ( tpgs, soluplus, hpmc e5, pvp k90, poloxamer 407) and this process was done using either magnetic stirrer at a speed of 300 rpm or bath sonicator(17). precipitation of solid nanoparticles occurred immediately. the resultant nanosuspension is left for one hour under magnetic stirrer to allow the organic solvent to evaporate. nanosuspensions with the smallest particle size were lyophilized using labconco freeze dryer (usa) after the addition of 1% w/w mannitol as a cryoprotectant to obtain the nanoparticle powder(18). figure 2. preparation of nimodipine nanoparticles using a syringe pump the composition and the variables of the prepared nmd nps are listed in the table(1) iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles 145 table 1.the composition of the prepared nmd nanoparticles. formula name polymer name nmd: polymer ration magnetic stirrer bath sonicator f1 tpgs 1:2 300 rpm f2 soluplus® 1:2 300 rpm f3 pxm 407 1:2 300 rpm f4 hpmc e5 1:2 300 rpm f5 pvp k90 1:2 300 rpm f6 tpgs 1:2 3 minutes f7 soluplus® 1:2 3 minutes f8 pxm 407 1:2 3 minutes f9 hpmc e5 1:2 3 minutes f10 pvp k90 1:2 3 minutes measurement of the particle size and polydispersity index of nimodipine nanosuspension samples of all prepared nanoparticles were analyzed using abt-9000 nanolaser particle size analyzer, the average particle size and polydispersity index (pdi) for each sample were recorded(19). characterization of the lyophilized powder determination of drug content in the lyophilized powder for the determination of nmd content in the dried nanoparticles, 18 mg (which is equivalent to 3 mg of nmd) of the lyophilized powder for the accepted smallest particle size formula was allowed to dissolve in 100 ml ethanol in a dry volumetric flask and sonicated for 10 minutes, then 2 ml of this solution were taken and diluted with ethanol twenty five times. the solution filtered, and the absorbance was measured using a uv-visible spectrophotometer at a λmax (236.8 nm). (20). the experiment was performed in triplicate, and the average value was calculated. the percentage of drug content was calculated according to the following equation: %drug content = (actual drug content)/ (theoretical drug content) x100…. eq (1). measurement of the particle size and polydispersity index after drying the particle size of the dried powder was done by dispersing an equivalent amount to 10 mg of nmd as dried nanoparticles in 9 ml distilled water then sonicated for two minutes. this procedure was done so that the concentration of the drug in the redispersed suspension is the same as the concentration of the drug in the nanosuspension before lyophilization. the particle size and pdi were measured using the abt-9000 nanolaser particle size analyzer, and the results were recorded(21) in vitro dissolution of the prepared nanoparticles in vitro dissolution was done for the prepared nmd nanoparticles with the smallest particle size and pure nmd. it was performed using dissolution apparatus 2 (paddle type) containing simulated salivary fluid (ssf) with 0.5% brij-35 (to maintain sink condition) as a dissolution media, the rotation speed was 75 rpm, and the temperature was 37°c±0.5. the dissolution was done by placing an nmd nps equivalent to 30 mg and 30 mg pure nmd separately in a dialysis membrane with a molecular weight cutoff of 12000-14000 dalton. 5 ml samples were withdrawn for analysis and substituted with an equal volume of fresh media to maintain constant volume for 120 min. the samples were filtered using 0.45 μm and analyzed using uvspectrophotometer at λmax (238 nm). the experiments were performed in triplicate, and the average value was calculated. the accumulative percentage of drug dissolved was calculated and drawn against time (22). for the statistical analysis of the dissolution study for the pure nmd and nmd nps, the similarity factor f2 was employed. the pure nmd was considered to be the reference, while the nps were supposed to be the test. the release profiles are considered to be similar when the value of f2 is between 50 and 100 f2 can be calculated from equation 2 𝑓2 = 50 × log⁡{[1 + ( 1 𝑛 ) ∑  𝑛𝑡=1 𝑤𝑡(𝑅𝑡 − 𝑇𝑡) 2] −0.5 × 100} (2) where; rt, tt is the percentage of the drug dissolved of the reference and test profile, respectively, at time t; n is the number of sampling (23). screening of pure nimodipine and nimodipine nanoparticles saturation solubility saturation solubility of pure nmd and nmd nps was measured in 0.1 n hcl ( ph 1.2) and ssf ( ph 6.8 ) in a shaking water bath at a temperature of 37±0.5 ˚c for 48 hrs. their solubility was also screened in distilled water at a temperature of a 25±0.5 ˚c for 48 hrs. then each sample was filtered, and its absorbance was measured at λmax (238 nm). fourier transform infrared (ftir) spectroscopy iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles 146 the fourier transform infrared spectroscopy (ftir) spectra were obtained using ftir shimadzu 8300 japan. samples of pure nmd, soluplus®, mannitol, and nmd nps of the selected formula were compressed with potassium bromide. the spectrum obtained was between the wavenumber of 4000-400 cm-1(24). results and discussion evaluation of the prepared nimodipine nanosuspension analysis of particle size and polydispersity index all the samples of nmd ns were analyzed by the abt-9000 nanolaser particle size analyzer, and the particle size distribution of all formulas was recorded, as shown in table (2). pdi is an essential means to evaluate the particle size distribution within the sample. it is crucial in determining the uniformity of particle size, which is valuable in the stability of a nanosuspension. monodisperse samples have lower pdi values than the polydisperse samples. pdi values in the range of (0-0.05) are considered to be (monodisperse standard), (0.050.08) is (nearly monodisperse), (0.08 -0.7) is (midrange polydispersity) and more than0.7 is (very polydisperse)(25). table 2. the particle size and pdi of the prepared nimodipine nps formula name particle size (nm) pdi f1 555± 25 0.008±0.0005 f2 77± 9 0.016±0.002 f3 1070 ± 10 0.003±0.001 f4 702 ± 4.9 0.008±0.0005 f5 872 ± 20 0.015±0.005 f6 367±15.6 0.021±0.01 f7 32.9±18.2 0.3±0.1 f8 305.3±5 0.006±0.001 f9 603±50.6 0.002±0.0005 f10 755±15 0.006±0.0005 the effect of polymer type on the particle size and pdi five different stabilizers (tpgs, soluplus®, pxm 407, hpmc e5, and pvp k90) were used to give the formulas (f1-f5) ,which were prepared by using magnetic stirrer as shown in figure (3). figure 3. the effect of polymer type on the particle size of the prepared nmd nps by magnetic stirrer the smallest particle size was obtained when using soluplus® as a stabilizer (f2 77 nm).soluplus® is a graft copolymer with amphiphilic properties. the hydrophilic part is represented by the polyethylene glycol backbone and the hydrophobic part by vinyl caprolactam/ vinyl acetate side chain. this amphipathic nature makes it an excellent surface-active and wetting agent that reduces the interfacial tension between the hydrophobic surface of nmd particles and the aqueous antisolvent. soluplus® allows the surface water interaction and maintains the small particle size of the prepared ns(26). the other four polymers show a fair particle size reduction (5551070 nm). tpgs is a watersoluble analog of vitamin e; it can stabilize the ns by hydrophobic ( vander waals) interaction between the particles(27). while pxm 407 is a hydrophilic nonionic surfactant, and it has been widely used as a coating agent for the nps. hpmc e5 and pvp k90 stabilize the newly formed nmd nps by a steric mechanism that prevents the freshly formed nps from aggregation and particle growth(28). this variation in particle size was due to the efficiency of these different stabilizers to envelop and stabilize the newly formed nmd nps. pdi of all these five formulas was in the range of (0.003-0.016), which indicates that nmd nanoparticles are monodispersed standard. the effect of using a bath sonicator on particle size and pdi formulas f6-f10 were prepared to evaluate the effect of using bath sonicator instead of magnetic stirrer on the particle size and pdi of the prepared nmd nps as shown in figure (4). iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles 147 figure 4. the effect of using bath sonicator instead of magnetic stirrer on the particle size of the prepared nmd nps the particle size of all formulas prepared by this method was significantly (p<0.05) decreased as compared with formulas prepared using a magnetic stirrer and the smallest particle size was obtained by f7 (32.9 nm) in which soluplus® was used as a stabilizer . the formation of the nanoparticles under the influence of ultrasonic waves is influenced by the higher energy and rapid miscibility of the organic solvent (ethanol) and the aqueous antisolvent, which increases the polarity of ethanol and reduce the solubility of nmd hence the rapid nucleation of nps. also, the sonication process produces high energy, high temperature, and shock waves, which inhibits the growth of newly formed nanoparticles(29). pdi of the formulas prepared by this method was in the range (0.02-0.006), which indicates that these formulas were in the limit of monodispersed standards except for the f6 in which soluplus® is the stabilizer; pdi is 0.3 which show mid-range polydispersity of the prepared nps which is not useful to maintain the stability of the prepared ns. from the results presented in this study, it appears that soluplus® is the better stabilizer and the preferred one for reducing the particle size of the prepared nps. characterization of the lyophilized nmd nanoparticles determination of drug content in the lyophilized powder the nmd content of the lyophilized powder for f2 was found to be equal to (102.25±12) % and for f7 is (92.15±10.32) % .the percentage of drug content was ranged from (92-102)%, which is an indication of the excellent and applicable way for loading the nmd into the prepared nanoparticles. particle size and pdi after lyophilization the particle size of the lyophilized powder was measured using the abt-9000 nanolaser particle size analyzer, and the results are shown in table(3), along with pdi. table 3.the particle size and pdi of nmd nps after lyophilization. formula name particle size pdi f2 45±32 0.04±0.04 f7 327±190 0.024±0.007 the particle size after lyophilization varies considerably because the drying process has a profound effect on the aggregation of the nps. this aggregation is affected substantially by the process parameters as the freezing rate, freezing temperature, and the presence of cryoprotectant(30). the particle size of f7 increased from 33 nm to 327 nm after drying, as illustrated in figure(5). this increment may be due to the relatively high pdi value ( 0.3) of the liquid nanosuspension, which indicates the presence of larger particles, and upon drying, these particles aggregate and increased the overall particle size distribution. on the other hand, the particle size of the formula f2 after drying was close to its original ps, which indicates minimal aggregation of the prepared nmd nps during lyophilization. figure 5. the particle size of the prepared nmd nps before and after lyophilization in vitro dissolution of nmd nanoparticles in vitro dissolution study was done to the formulas (f2, f7) after lyophilization and nmd pure powder using a dialysis membrane with a molecular weight cutoff 12000-14000 dalton. the study was done in a dissolution apparatus type ⅱ ( paddle type ) at a 75 rpm, and the temperature was adjusted at 37±0.5 ℃. the media was simulated salivary fluid (ssf) ( ph 6.8) containing 0.5 % brij35. the results of the dissolution profile of the dried nmd nps and pure nmd are shown in figure(6). iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles 148 figure 6. the dissolution profile of the prepared nmd nps and pure nmd in ssf containing 0.5 % brij-35. it was found the f2 (which composed of 30 mg nmd, 60mg soluplus and 90mg mannitol) reaches a complete dissolution after 90 minutes from the starting of the dissolution study, and it was the fastest formula compared with f7 and pure nmd. this formula has the smallest particle size; hence it has a faster dissolution rate according to noyes whitney equation because it has a higher surface area. the values of f2 for the nmd nps formulas were 7.1 and 11.44 for f2 and f7 respectively. screening the saturation solubility of pure nimodipine and nimodipine nanoparticles the solubility of nmd and nmd nps was done in d.w., 0.1 n hcl (ph 1.2), and ssf (ph 6.8).the solubility of nmd was increased several folds, as illustrated in the table (4) and figure (7). table 4, the saturation solubility of nmd nanoparticles in different dissolution media dissolution media ph temperature ℃ saturated solubility of pure nmd (µg/ml) saturated solubility of nmd nps (µg/ml) number of increment folds 0.1 n hcl 1.2 37 ± 0.5 10.9 25.6 2.3 ssf 6.8 37 ± 0.5 3.4 46.12 13.56 water 7-8 25 ± 0.5 4.14 100.7 24.3 figure 7. solubility of pure nmd and nmd nanoparticles in different dissolution media the enhanced solubility can be explained by ostwald–freundlich equation. the saturation solubility of nmd increases as the particle size reaches the nanoscale range. another explanation for the solubility enhancement is due to disruption of the ideal structure of the drug microparticles into the nanoparticles. this disruption causes high energy of interfacial tension, which enhances the solubility of nanoparticles (31,32) fourier transform infrared (ftir) spectroscopy ftir spectra were obtained for pure nmd, soluplus®, mannitol, and nmd nps, as shown in figures (8,9,10 and 11 respectively). this study was done to evaluate the compatibility between the drug and the other excipients in the prepared nps. iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles 149 figure 8. the ftir spectrum of pure nmd figure 9. ftir spectrum of soluplus® iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles 150 figure 10. ftir spectrum of mannitol figure 11. ftir spectrum of nmd nps the ftir spectrum of nmd shows many main peaks about 3300 cm-1 due to n-h stretching, 3226 cm-1 and 3095 cm-1 due to aromatic c-h stretching, 2981 cm-1 due to aliphatic c-h stretching, 1695 cm-1 due to c=o stretching in ester, 1647 cm-1 due to n-h bending, 1523 cm-1 and 1494 cm-1 due to c=c ring stretching and 1346 cm-1 due to c–c(=o)–o stretching of α,β-unsaturated ester. these peaks are in very close math to the reference peaks(33). the nmd nps spectrum also showed the main peaks of nimodipine ( circled in red). n-h stretching and n-h bending have been broadened, and c=o stretching frequencies have been reduced in intensity, and this is mainly due to the formation of hydrogen bonds between the iraqi j pharm sci, vol.30(2) 2021 nimodipine nanoparticles 151 hydroxyl group of soluplus® and nimodipine. it is well established that 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http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.30(1) 2021 effects of ebf on infants growth doi: https://doi.org/10.31351/vol30iss1pp76-80 76 effect of exclusive breast feeding on infants growth and comparison with other types of feeding in sulaimani city, kurdistan, iraq. sardar m. weli*,1 *nursing department, technical college of health, research center, sulaimani polytechnic university, sulaimani, iraq. abstract breastfeeding (bf) serves as a complete nutritional source for the first six months of infant’s life. breast milk contains all essential nutrients that necessary for the physiological growth and development of infants. the aim of this study was to compare the physiological growth of infants including weight, length (height) and head circumference who were exclusively breastfed for 6 months and those who were given bottle-fed or mixed fed and to find percentage of exclusive breastfeeding among mothers who contributed in this study. this study was carried out in sulaimani city/ kurdistan region of iraq and the cases were enrolled between the first of june 2018 and first of october 2019. infants’ weight, length (height) and head circumstances were measured at different age levels (at age two, six, nine and fifteen months). the results of this study found that among 198 mothers who were contributed in this study; 92 (46.5%) of mothers had ebf while 90 (45.5%) had mixed feeding and only 16 (8%) had exclusive formula feeding (eff) in the first six months of baby’s life. infant’s weight at age 2 months showed no differences between types of feeding. however, infants weight at ages 6 and 9 months were significantly higher in infants who breastfed compared to formula fed but no differences were found between exclusive breast feeding and mixed feeding. at age 15 months weights of babies were again no differences were found between all types of feeding. for length (height) parameter, infants who were exclusively breastfed for six months were significantly higher than those of formula fed at age 2, 6, 9 and 15 months. regarding head circumferences no significant differences between types of feeding at age 2 months were showed. nevertheless, at age 6 and 15 months were significantly high in infants who breastfed than formula fed. the present study concluded that infants who breast fed for first six months of life have a higher weight, height and head circumferences than infants who exclusively formula fed. the percentage of ebf among kurdish mothers were similar with other governorates of iraq but was low compared to the recommended rate of who. keywords: breastfeeding, bottle feeding, infants’ length, infant's weight, infant's head circumference. تاثير الرضاعة الطبيعية الحصرية على نمو األطفال ومقارنتها بأنواع أخرى من الرضاعة في مدينة السليمانية ، كردستان العراق 1*،سردار محمد ولي . العراق ،السليمانية ،جامعة السليمانية التقنية،مركز االبحاث ،كلية الصحة التقنية ،قسم التمريض* الخالصة الرضاعة الطبيعية هي بمثابة مصدر غذائي كامل لألشهر الستة األولى من حياة الرضيع. يحتوي لبن األم على جميع العناصر الغذائية الهدف من هذه الدراسة هو مقارنة النمو الفسيولوجي للرضع بما في ذلك الوزن والطول ومحيط الضرورية للنمو الفسيولوجي للرضع ونموهم. كان أشهر وأولئك الذين تم إطعامهم بالزجاجة أو اإلطعام المختلط وإيجاد نسبة من الرضاعة الطبيعية 6الرأس الذين تم إرضاعهم من الثدي حصريًا لمدة هذه الدراسة في مدينة السليمانية. أجريت هذه الدراسة في مدينة السليمانية / العراق وتم تسجيل الحاالت الحصرية بين األمهات اللواتي ساهمن في . تم قياس وزن الرضع وطولهم ومحيط الرأس عند مختلف المستويات العمرية )في سن الثانية ، ستة 2019وأول أكتوبر 2018بين أول حزيران ٪( من األمهات لديهن تغذية 46.5) 92أمهات ساهمت في هذه الدراسة 198هذه الدراسة أنه من بين وتسعة وخمسة عشر شهرا(. وجدت نتائج ٪( فقط لديهم تغذية زجاجة الرضاعة حصرية في األشهر الستة األولى من حياة 8) 16٪( لديهم تغذية مختلطة و 45.5) 90طبيعية حصرية بينما كانت مرتفعة بشكل كبير عند الرضع الذين 9و 6اختالفات بين أنواع الرضاعة. ومع ذلك ، في سن الطفل. لم يكن وزن الرضيع عند عمر شهرين ة. في يرضعون رضاعة طبيعية مقارنة بالصيغة التي يتم تغذيتها ولكن لم يتم العثور على اختالفات بين تغذية طبيعية حصرية والتغذية المختلط فات بين جميع أنواع التغذية. بالنسبة للطول ، كان الرضع الذين يرضعون رضاعة طبيعية حصريًا لمدة ستة شهًرا لم يتم العثور على اختال 15عمر شهًرا. فيما يتعلق بمحيط الرأس لم تظهر فروق 15و 9و 6و 2أشهر أعلى بكثير من أولئك الذين تم تغذيتهم بأنواع أخرى من الرضاعة في سن شهًرا كانت مرتفعة بشكل كبير عند الرضع الذين يرضعون من 15و 6غذية في عمر شهرين. ومع ذلك ، في سن ذات داللة إحصائية بين أنواع الت طول الرضاعة الطبيعية. خلصت الدراسة الحالية إلى أن الرضع الذين يرضعون رضاعة طبيعية خالل األشهر الستة األولى من العمر لديهم وزن ، و ين يتغذون بشكل حصري على حليب األطفال من الزجاجة. كانت نسبة تغذية طبيعية حصرية بين األمهات ، ومحيط الرأس أعلى من الرضع الذ الكرديات مماثلة مع مدن أخرى في العراق لكنها كانت منخفضة مقارنة بالمعدل الموصى به المنظمة الصحة العالمية. .للرضيع ، محيط الرأسارضيع ، وزن رضيعطول الالكلمات المفتاحية: الرضاعة الطبيعية ،زجاجة الرضاعة ، *corresponding author e-mail: sardar.weli@spu.edu.iq received: 30/7 /2020 accepted: 6/ 9/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp76-80 iraqi j pharm sci, vol.30(1) 2021 effects of ebf on infants growth 77 introduction exclusive breast feeding (ebf) means that the infant gets only breast milk and no other foods are given. breastfeeding (bf) serves as a complete nutritional source for the first six months of infant’s life. breast milk contains all essential nutrients that necessary for the physiological growth and development of infants. it is important for the growing of infant’s brain and develops cognitive function in children due to its constituents of essential fatty acids. these fatty acids are not available in other types of milk (1, 2). the breast milk has more health benefits and nutritional advantageous than other type of milks. it offers children a rapid growth by development, maintenance and maturation of tissues and organs (3). exclusive breast feeding(ebf) for at least 4 months can reduce the risk of many allergic diseases such as asthma, atopic dermatitis and suspected allergic rhinitis up to 2 years old (4). the initial milk produced by mothers called colostrum contains a large amount of immunoglobulin, this is important to protect the newborns until their immune system working suitably. colostrum act as laxative and cover gastrointestinal tract and prevent jaundice by inhibit bilirubin build up (5). ebf or predominant bf during 6 months of life protects infants from diarrhea and acute respiratory diseases (6). in addition, breastfeeding has progressive effects on infant’s cognitive development and emotional stability and prevent infants from chronic diseases and infants were not likely to be overweight later in life. on the other hands, breast feeding has beneficial effects for mothers; it reduces their risk of chronic diseases, such as hypertension, hyperlipidemia, and type 2 diabetes and protects them against onset of breast and ovarian cancers (7; 8). according to a study in america, black women have a higher rate of breast cancer than white women because they have a lower rate of breast feeding (9). this study was conducted in health units in sulaimani city to determine the physiological growth of infants including weight, length (height) and head circumference who were exclusively breastfed for 6 months and those who were given bottle-fed or mixed fed and to find a percentage of exclusive breastfeeding among those mothers who contributed in this study. methods location and participants this study was carried out in sulaimani city/ kurdistan region of iraq. one hundred ninety eight healthy and full term infants were included in this study. the criteria for entry to this study: all babies were visiting child health care units in sulaimani city on a regular basis for vaccinations at age two, six, nine and fifteen months. same infants were used to measure their weight, length (height) and head circumferences at different age level. infant’s mothers were asked about feeding their babies either exclusive breast feeding (infants get only breast milk and no other foods are given) or exclusive bottle feeding (infants receive only bottle milk and not breast milk) or mixed feeding (infants get both breast milk and bottle milk) during first 6 months of infants life. data collection and methods healthy infants were enrolled between the first of june 2018 and first of october 2019. the infants’ weight, length (height) and head circumferences were measured at different age levels (at age two, six, nine and fifteen months). infant’s weights were measured by digital baby weighting scale. infant’s length is measured from the top of their head to the bottom of one of their heels. it's the same as their height, but height is measured standing up, whereas length is measured while the baby is lying down. ruler and vernier caliper were used to measure length (height). head circumferences were measured by using a flexible tape measure. wrap the tape around the forehead to the widest part of the back of the head. data analysis data was entered into statistical package for social sciences “spss” version 26 for storage and statistical analysis. one way anova was applied to test the differences, with a p value of 0.05 or less considered as significant. results among 198 mothers who were contributed in this study; 92 (46.5%) of mothers had exclusive breast feeding (ebf) while 90 (45.5%) had mixed feeding and only 16 (8%) had exclusive formula feeding (eff) in the first six months of baby’s life, table 1. table 1. distribution of infants according to the type of feeding. type of feeding no (%) exclusive breast feeding (ebf) 92(46.5%) exclusive formula feeding (eff) 16(8 %) mixed feeding 90(45.5%) total 198 (100%) the results indicated that infant’s weight at age 2 months were significantly no differences between types of feeding. however, at ages 6 and 9 infant’s weight were significantly higher (p <0.05) in infants who breastfed compared to formula fed but significantly no differences were found between ebf and mixed feeding. at age 15 months weights of infants were again no significant differences were found between all types of feeding, table 2. iraqi j pharm sci, vol.30(1) 2021 effects of ebf on infants growth 78 table 2. distribution of the infant's weight according to age and type of feeding. infant’s age (months) infant’s weight exclusive breast feeding (ebf) no. 92(46.5%) exclusive formula feeding (eff) no. 16(8 %) mixed feeding no. 90(45.5%) two 5.642 ± 0.355 a 4.213 ± 0.253 a 5.397 ± 0.414 a six 8.432± 0.383 a 6.55±0.29 b 7.789± 0.409 a nine 9.353 ± 0.314 a 7.638± 0.239 b 8.889± 0.237 a fifteen 10.634 ± 0.372 a 8.581± 0.337 a 9.861± 0.351 a values are presented as means ± sd different letters denote significant differences between groups (p<0.05). for height parameter, infants who exclusively breastfed for six months were significantly higher (p <0.05) than those of formula fed at age 2, 6, 9 and 15 months .but no significant differences were found between breasts and mixed fed, table 3. table 3. distribution of infant's length according to age and type of feeding. infant’s age (months) infant’s length exclusive breast feeding (ebf) no. 92(46.5%) exclusive formula feeding (eff) no. 16(8 %) mixed feeding no. 90(45.5%) two 60.086 ± 3.661 a 55.938 ± 2.175 b 56.253 ± 2.916 ab six 68.129 ± 2.871 a 64.625±1.668 b 65.066 ± 2.728 ab nine 73.011±1.964 a 66.875±1.821 b 68.099±2.017 ab fifteen 78.28± 2.482 a 74.563±1.094 b 76.000±2.124 ab values are presented as means ± sd different letters denote significant differences between groups (p<0.05). regarding to head circumference the results found no significant differences between types of feeding at age 2 months. nevertheless, at age 6 and 15 months’ infant’s head circumferences were significantly higher (p <0.05) in infants who breastfed than formula fed. no significant differences were found between exclusively breasts fed and mixed fed, table 4. table 4. distribution of infant's head circumference according to age and type of feeding. infant’s age (months) infant’s head circumference exclusive breast feeding (ebf) no. 92(46.5%) exclusive formula feeding (eff) no. 16(8 %) mixed feeding no. 90(45.5%) two 40.505±0.963 a 38.375±0.719 a 40.121±0.976 a six 44.667±0.742 a 41.938±0.772 b 43.637±0.863 ab fifteen 47.452±0.745 a 44.563±1.094 b 45.967±0.781 ab values are presented as means ± sd different letters denote significant differences between groups (p<0.05). discussion the present study investigated the effects of different types of infants feeding (exclusive breast (ebf) feeding, mixed feeding and exclusive formula feeding (eff)) during first six months of life on many parameters that are related to growth of infants include weight, length (height) and head circumference at two, six, nine and fifteen months age of infants in sulaimani city. the percentage of ebf, mixed feeding and eff among participants were 46.5%, 45.5% and 8% respectively. these percentages are approximately similar to studies done in baghdad. they found that 48%, 41% and 11% were ebf, mixed feeding and eff (10). in addition, a study in misan city showed that ebf among mothers was 45.6% which was less than the recommendation of who (11). nevertheless, another study in baghdad showed that the rate of exclusive breast feeding was very high in the first few month of life (64.6%) but declined rapidly to (28.3%) at age 6 months due to milk insufficiency in mothers. other factors such as mother’s age, job and education level played role to determine ebf among iraq’s mothers (12). on the other hand, breast feeding were very high 77.2% and 76.2 among syrian and jordanian women respectively (13). iraqi j pharm sci, vol.30(1) 2021 effects of ebf on infants growth 79 the current study established that weights of infants were significantly higher in ebf than eff at age six and nine months but at age 15 no differences were found between ebf with eff. this is in parallel with a study that confirmed ebf results in sufficient weight gain in infants of low birth weight (14). another study reported that infant’s malnutrition was prevented by the role of breastfeeding and it also prevents overweight and obesity (15). this may be explained by the effects of human milk composition which contain many growth factors that contribute to infant’s health, growth and development (16). the present study found that infants who exclusively bottle fed were significantly lower weight at age six and nine months compared to ebf. this finding is consistent with a study done in ethiopia showed that infants terminated from ebf before six months of age were more likely to get diseases such as acute respiratory diseases, fever and diarrhea which are associated with increased underweight and wasting in infants (17). however, this is inconsistence with a study done previously which suggested that bottle feeding associated with infant’s weight gain because bottle feed is more concentrated and mothers encouraged infants to finish the bottle (18). another study in brazil also explained that children who were bottle-fed were at high risk to be overweight and obesity at age 12-24 months (19). the height of exclusively breast fed infants was significantly higher compared to bottle fed and mixed fed infants. this result is in agreement with a study done on malawian infants. they confirmed that infants who were breastfed until 6 months age were longer and heavier than non-exclusively breastfed infants (20). this may be due to the rates of diarrhea, fever, respiratory and gastro intestinal infections among non-exclusively breastfed infants which is higher than ebf (6). however, this finding is disagreeing with a study in iran and they found no significant differences between weight and height of infants who were breastfed and bottlefed between 4 and 6 months (21). the current study found that head circumferences of exclusively breast fed infants were higher compared to exclusively bottle fed infants at age six and fifteen months. one study done in bu-ali sina hospital in iran found significant differences between infants who breastfed with those of formula fed at 6 months of age, however they also found that no differences at age 9 and 12 months (22). in addition, other study showed that exclusive breast feeding for at least 4 months associated with increase in head circumferences and head circumferences were low in infants who were exclusively breast fed for less than 30 days (23). additionally, a study on turkish infants during 6 months of life showed that head circumferences were higher in breast fed infants compared to formula fed and mixed fed infants (24). conclusion the present study concluded that infants who exclusively breast fed for first six months of life have a higher weight, length (height) and head circumferences than infants who exclusively formula fed. but no significant differences were found between exclusive breast fed and mixed fed. the percentage of ebf among kurdish mothers were similar with other governorates of iraq but was low compared to the recommended rate of who. acknowledgments i would like to express my special thanks to staffs of shahid hama-rash and shahid shamal health units in sulaimani for their kind help and cooperation. i would also like to thanks razhan, rozhgar, baxan and zryan (technical college of health/ sulaimani polytechnic university students) for their help in collecting some of the data. references 1. jedrychowsk w, perera f, jankowski j, butscher m, mroz e, flak e, kaim i, lisowskamiszczyk i, skarupa a, sowa a. effect of exclusive breastfeeding on the development of children’s cognitive function in the krakow prospective birth cohort study. eur j pediatr 2012; 171(1): 151-158. 2. elyas l, mekasha a, admasie a, assefa a. exclusive breastfeeding practice and associated factors among mothers attending private 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effect of type of breastfeeding on overweight and obesity in children aged 12-24 months. cad. saúde pública 2016; 32(12): 1-10. 20. kuchenbecker j, jordan i, reinbott a, herrmann j, jeremias t, kennedy g, muehlhoff e, mtimuni b, krawinkel mb. exclusive breastfeeding and its effect on growth of malawian infants: results from a crosssectional study. paediatrics and international child health 2015; 35(1):14-23. 21. khadivzadeh t, parsai s. effect of exclusive breastfeeding and complementary feeding on infant growth and morbidity. eastern mediterranean health journal 2004; 10(3): 289 294. 22. gorohi f, shiemorteza m, nori mm. comparison of height, weight and head circumference index and the incidence of infectious and gastrointestinal diseases in breast-fed and formula-fed infants at 0 to 1 year old in bu-ali sina hospital. biomedical & pharmacology journal 2018; 11(3): 1717-1730. 23. ferreira hs, junior af, assunc ml¸ santos ea, horta bl. effect of breastfeeding on head circumference of children from impoverished communities. breastfeeding medicine 2013; 8(3): 294-301. 24. donma mm, donma o. the influence of feeding patterns on head circumference among turkish infants during the first 6 months of life. brain and development 1997; 19(6): 393-397. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 53 gender differences of serum leptin hormone levels in iraqi population be sma i.mustafa received 16-6-2004 accepted 21-10-2004 abstract to e va luate and comp are se rum leptin ho rmo ne leve l be twe en iraqi male & female and the re la tion betwee n this ho rmo ne & bmi in the se two groups. a to ta l of 44 normal ma le & fema le sub je cts were inc luded in this s tudy { gro up 1 : 2 2 fe male } , { gro up 2 : 2 2 male} . serum leptin hormone ,bmi &fas ting b lo od gluc os e were mea sure d fo r bo th gro ups . se rum leptin le vel in gro up 1 wa s (8 .8 2 + 2.9 μg/l) where as in group 2 it was (4.65 + 3 .2 μ g/l) . the se c ha nge s were statistically significant. fa sting blood gluco se lev els were technica lly within the no rma l va lue (1 16.43 + 3 .4 mg /dl) fo r group 1 & (11 8.52 + 2.9 mg /dl) for gro up 2 . bmi leve ls we re co mparab le in bo th gro ups d uring the stud y, with slight eleva tio n in group 1 [ 24 + 1.73 kg /m2 for group 1 & 23 + 1 .9 8 kg /m2fo r group 2 ] which within the ac ce ptable limit as far a s sa fe ty conce rn. le ptin, a n ad ip ocyte -d eriv ed ho rmo ne know n to p la y an impo rtant role in bod y-weight regula tion, the re sult of this s tudy s hows that lep tin is pres ented d ifferentially in iraqi men a nd women ; in whic h it is significantly higher in wo men than in men s erum . thes e obs erva tions are po te ntia lly impo rtant for the understanding o f diffe rence s be twe en me n and wo men in re gula tion o f food intake, we ight gain, and bod y fa t distrib utio n.the relation be tween leptin & the bmi in ma le and fe male in this s tudy may ope n anothe r ap proac h fo r this ho rmo ne to be inv olved in –fertility & in pub erta l de velop ment. الخالصة  ن مون اللبتي هر ة رن مقا غرض قياس و مع نسبة كتلة ) leptin(ل ر مذكو مون ال هر مستوى ال وعالقة ق را ي الع إلناث ف كور وا بين الذ ن bmiالجسم . في كال الجنسي سة را ملت الد 4ش موعتين 4 مج ى هم إل خص ٬ تم تقسيم ت : ش ة الثانية تضمنت 22األولى ضم موع را 22أنثى والمج اس . ذك تم قـي وعتين ـج ال الم حالة الصيام لك ي ر الدم ف سك وى ومست ن اللبتين ونسبة كتلة الجسم رمو ي . مستوى ه ون ـف هرمـ اس ال ائج قـي رت نـت أظهـ عة األولى مو 8المج .8 2  2 غرام 9. و كر ة . لتر /ماي عة الثاني جمو ون في الم رم ى اله كان مستو ن حي ي رو غرام  3.2 4.65ف / مايك ن الفر ة لتر ٬ حيث أ رة واضح معنويا بصو ة . ق كان من القيم الطبيعي كر الدم ض مستوى س ن كا 11: و 6.43  3.4 رام مل 100/ملغ عة األولى و مو 118للمج .5 2  2.9 غرام 10/مل ة 0 وعة الثاني جم ود .مل للم ع وج عتين م مو ة بين المج رب سم متقا ة الج كتل ة وكانت نسب الولى ة ا موع ع قليل في المج ل 2م/ كغم  1.73 24(ارتفا 23 1مقاـب .9 ة ) 2م/ كغـم 8 ة الثانـي موعـ مج ويات ) لل مسـت من ال ي ضـ ـه و ة عي . الطبي وزن الحسم وم بتنظيم هنية ليق مشتق من الخاليا الد ن مو هو هر ن اللبتين و ون اللبتين . ا رم مستوى ه ة ان هذه الدراس ج رت نتائ لقد اظه حيث ا عراقيين ٬ حة بين الرجال والنساء ال واض روقا هر ف جال يظ هو في الر ما وياته اعلى في النساء م ست مة . ن م مه حظات ان هذه المال ن في الجسم هو ع الد زي زن وتو وزيادة الو ي تنظيم تقبل الطعام ساء ف رجال والن مستوى . للمساعدة في فهم الفروقات بين ال ن عالقة بي ان ال ة ق هذه الدراس ي من الرجال والنساء ف جسم في كل ة كتلة ال سب ون ن مو خصوبة الهر ة مع درج ن رمو وى هذا اله ست ة عالقة م مهد لدراس د ي ال الجنسين غ في ك طور البلو . وت dep artmen t of clin ic al labo rator y sc ie nce s ,colle ge o f ph arma cy,un iver sity of baghdad , baghdad –i raq . ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 54 introduction leptin is a rece ntly disc ove re d ho rmo ne1 that is mainly s ynthes ized b y ad ip ose c ells and se creted into the bloo ds tream in a mounts re lativ e to the q ua ntity of bod y fa t. plas ma le ptin leve ls in humans a re s trongly co rrelated with b ody ma ss inde x (bmi) and to tal fat ma ss (figure 1) a nd are elev ated in obe se s ub je cts.2 fig.1. s che matic re pre s e ntatio n of ce ntral and pe riphe ral ac tions o f le ptin on bo dywe ight re g ulatio n and o n me tabolic and e ndoc rine para me te rs lepr = le ptin re ce pto r; npy = ne urope ptide y; glp -1 = gluc ago n-like pe ptide -1; msh = me lanocy te -s timulating ho rmo ne ; lh = lute iniz ing hormone ; fs h = follicle stimulating ho rmo ne ; acth = adre noco rtic otro pic hormone ; tsh = thyroid-s timula ting ho rmone . adapte d and update d from sc ott j. ne w c ha pte r fo r the fa t co ntrolle r. lep tin is tho ught to ha ve an "a dipos ta t" func tion;that is, it ac ts a s a signal informing the brain ab out the amo unt of fat stored in the bod y, through which the brain can regulate energy intake and e ne rgy exp enditure in orde r to keep bo dy weight constant (f igure 1 ).3,4 the re fo re , whe n lep tin le ve ls are low, ap petite will be s timula te d with limited use of energy, a nd w hen le ptin leve ls are high, ap petite is re duc ed a nd e ne rgy us e is stimulated . in rod ent e xp erime nts, leptin ad minis tration lead s to weight lo ss through re duc tion in food intake and increas ed e nergy exp end iture .5 in humans, howe ve r, le ptin le vels are ve ry high in o bes e peo ple , which sugge sts the exis tenc e of a lep tin – resis ta nce state , a nalogous to insulin-re sistance in type 2 diabe te s,4 the exac t me chanis ms for this re sistance are not c le ar yet. the lep tin rece ptors in brain is ma inly pre se nt in the hypo thalamus a nd choro id plexus,6 where fo od inta ke is regulated through the modula tion o f sev eral ne urotra ns mitter pathwa ys , s uch a s ne urope ptid e -y, gluc agonlike pep tide -1 , a nd melanocyte -s timula ting hormone pathways . le ptin may als o modula te pituitary se cretion of thyro id -s timula ting hormone, ad re no cortic otro pic hormone , and gonado trop ins,thus influencing indirectly the sec re tion of triio dothyro nine / thyroxine, cortis ol, and se x ho rmo nes , res pec tiv ely, a ll of which have e ffec ts on e nergy b alance7. materials and methods: fo rty four a pp arently hea lthy male & fema le (a ge be tw een 2 3 and 3 5 ye ars) we re inc luded in this s tudy and co nse de red a s two group s to determine s erum le ptin co nc entratio n, bmi and fas ting blood gluco se for e ac h individua l . exclusion criteria: any individua l that ha s hype rglyce mia or bmi > 25 wa s exclude d from this s tudy. methods veno us bloo d s amp le s (6 ml) were ta ken from ea ch subjec t to mes ure serum leptin and fa sting blood gluco se lev els. the sa mple was transferre d into a clean p la in tube, le ft at roo m te mperature for 30 minutes for clotting, centrifuged , a nd then the serum fro m all blood samp le s was sep arated a nd s to re d at –20 c for sub se que nt study .  de ter mination of fasting blood gluc ose : enzyma tic & c olorime tric me thod ( gluc ose oxida se god/ pe roxidas e pod ) , the princ iple of this me thod d epe nds on enzymatic determination of gluc os e ac co rd ing to the fo llowing rea ctio n9 : glucos e + o2 + h2o god gluconic acid + h2o2 2h2o2 +4 aminoantipyrine + p henol p od q uino neimine + 4 h2 o ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 55  dete rmination of serum leptin (elisa): enzyme immunoa ssa y (microtite r strip s) for the qua ntita tive de terminatio n o f lep tin in human s erum. 10 statistical analysis: mea sure ment of variables do ne by us ing numbers, pe rc enta ge , me ans +/s tand ard de viatio n. diffe re nc es b etwee n variables were mea sure d by us ing anova tes t when it ne ede d. result the s tudied v aria bles are lis te d in table 1. serum lep tin ho rmo ne leve ls a re p re se nted in ta ble 2 (8.82 + 2.9 μg/l) in gro up 1 & (4 .6 5 + 3.2 μg/l) in gro up 2 ,the va riation be tween the tw o gro ups wa s statistic ally s ignific ant (p < 0.05 ) . gender d ifferenc es be tw een the two groups in se rum lep tin shown c learly in figure 2,while fig 3 sho ws the difference in se rum lep tin leve ls betwee n iraq i women and the referenc e va lues for fe mal s ub je cts. table (1) the s tudie d va riable s are lis te d table (2 )serum le ptin ho rmo ne leve ls are prese nte d in table 2 fig 2 : ge nde r diffe re nces in le ptin le ve l be twee n the two groups fig 3 : comparis on be twee n ira qi (1)and othe r normal fe ma le ( 2) in le ptin le ve ls cussion:dis this stud y is one of the ne wes t stud ie s that discuss the variatio n in one of the ne wly disco vered hormones (le ptin) be twe en male & fe male subjects in iraq .the knowled ge of this re la tion is very impo rtant to und erstand vario us problems a ss ociated with ob esity, pub erty & infertility8. fro m the re sults (tab le 2 &fig 2 )of this study serum le ptin le vels are significa ntly highe r in women tha n in men (p< 0.05 ). this study which is do ne in iraq to ev alua te the value s of le ptin ho rmo ne in norma l ma le & fema le sub je cts to b e c omp ared with the s tudy of reitman & co workers the re sults s ho ws a highe r va lues in iraq i fe male (me an =8.82 ug/l) tha n other female v alue s (me an = 7 .4 va riable s group –1(n=22 )fe ma le group –2 (n=2 2)male age (ye ars) 27 .4 ± 4.18 30 .5 6 ± 3.26 bmi (k g/m2) 2 4 ± 1 .73 23 ± 1.98 f asting b lo od gluco se ( mg /d l) 3.4 +116.43 2.9 +1 18 .52 group –1(n=22)fe male group –2(n=22)male le ptin hormone (μg/l) 8.82 ± 2.9 4.65 ± 3.2 re fe rence value 3.2 +7.4 2.8+ 3.9 0 1 2 3 4 5 6 7 8 9 10 1 2 l e p ti n l e v e l (u g /l ) 0 1 2 3 4 5 6 7 8 serum leptin (ug /l) 1 2 groups ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 56 ug/l) 3; thes e difference s may be d ue to the fa ct tha t iraq i wome n hav e highe r fa t mas s than the o ther women. a co mpa ra ble res ults was found in male with different s tudies . at firs t, such diffe re nc es be tween iraqi male & fe male subjects were thought to re flec t the difference s in b ody comp os itio n be twe en me n and women. wome n in general have a higher pe rc entage o f fa t mas s for the s ame bod y weight or bmi. sinc e le ptin reflec ts ma inly the amo unt of bod y fa t, this see med to be a lo gical explana tion for the ob se rv ed gende r difference s11. ho weve r, the se gender difference s a nd their relations hip to bod y co mpos ition we re e xamined ,and lep tin lev els were found to be s ignific antly higher in wome n with c ompa ra ble bmi or fa t ma ss. a s eco nd exp la na tion was thought in the difference s in fa t distrib utio n be twe en me n and wome n. women ge nerally hav e more pe riphe ra l fat a cc umulation (es pec ia lly at the le ve l of the hips ), wherea s obe se me n hav e more abd omina l (e spe cially v is ceral) fat. it was shown in s ev eral in v itro s tudies that subcuta ne ous adipo cytes pro duce more leptin than fat c ells de rive d from the ome ntal fat de pot; this is es pec ia lly true in wo men.12,13 in vivo s tudies a ls o showed that lep tin lev els ha d a s tro nge r ass oc ia tion with pe ripheral14 or subcuta ne ous fa t than with intra ab domina l or visc eral fa t, as meas ured b y co mpute d to mogra phy s ca n.15 thus, the fac t tha t wome n ha ve more sub cuta ne ous fat, which s ec re te s more leptin, s ee ms an a dd itiona l reas on for the higher lep tin leve ls in wo men. howe ve r, eve n after co rrec ting for the amount of subcuta ne ous fa t, le ptin le vels a re s till found to be s ignifica ntly highe r in wome n.10 conclusion the res ults of this s tudy are p otentially importa nt fo r the understanding o f difference s be twe en me n and women in regulatio n of foo d intake , weight gain, a nd bo dy fat d is tribution; but sinc e these d ifferenc es in bod y co mpos ition b etween men a nd wome n may not be the o nly re as on to explain the diffe re nce s in le ptin lev els c omp le te ly, othe r fa ctors must play a role, like ste ro id hormones b ut the exact mec hanism o r inte ra ction is not ye t known. the se findings se em to indicate that this re ce ntly d is co vered hormone ma y play a role obe sity, infe rtility & o ther d is ea se s ta te s. references 1. zhang y, p ro enc a r, ma ffei m, et a l, p ositio na l cloning of the mous e ob ese gene a nd its huma n homologue . nature; . 199 4 3 72,42 5-431 . 2. ma ffei m, ha la as j, ra vussin e, et a l. lep tin le ve ls in human and rod ent: me as ureme nt o f plas ma leptin and ob rna in obe se a nd weight-reduce d subje cts. nat med; 199 5,1,115 5-116 1. 3 . reitma n m ,ga vrilova o lep tin & its role in p re gna nc y a nd fetal dev elopme nt,bio chemica l so ciety trans ac tion ,2 001 ,2 9,68-72 ,6 . 4 . ostlund re, ya ng j w, klein s, gingerich r. (relation betwe en plas ma le ptin c onc entration and bod y fa t, ge nde r, diet, age, and me tabo lic co va riates . j clin endo crinol me tab, 199 6,81,39 09 -39 13. 5 . halaas j , gajiwa la k, maffei m, et a l. ,weight-red ucing e ffe cts of the plasma pro tein enc ode d by the obe se gene . s cienc e; 199 5,269 ,5 43-54 6. 6 . tarta glia la , dembs ki m, weng x , e t al id entifica tion and e xp re ssion cloning o f a le ptin rece ptor, ob-r. cell 199 5,83:12 63127 1. 7 . wa uters m, msc , and luc va n gaal, md, phd. gender differenc es in le ptin lev els and physiology: a role fo r leptin in huma n reprod uction ;the j ourna l o f ge nde r spe cific med ic ine , 1 999 , 2[5],46-51 . 8. ro na ld c.k. new hormo nes o ffer und erstanding & p ro mis ing tre atment for diabe te s & obe sity.inte rnatio na l journal of obe sity ad vance d online public ation, 200 4,14,10 : 1-12 9. syrb io d ia gnostic re age nts for labo ra to ries und er license of eurobio la boratories –p aris 200 2. 10.considine r.v.,s inha m.k.; se rum immuno rea ctive le ptin concentra tions in normal we ight a nd obe se humans, the ne w engla nd jo urna l of medicine , 1 996 ,12 3,23. 11. wa uters m, me rtens i, cons idine r, e t al. are le ptin leve ls de pende nt on bod y fat distribution in ob es e men a nd wo men? journal of ea ting a nd weight diso rde rs ; 19 98 ,3 ,1 24130 . 12. couilla rd c, mauriege p , prud'homme d, et al. plas ma le ptin c onc entratio ns : gender differences a nd as soc ia tions with metab olic risk ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 57 fa ctors for c ardiova sc ular d is ea se. diabe to lo gia; 1 997 ,40,11 78-11 84. 13.va n harmelen v, reynisdo ttir s, eriks so n p, e t al. le ptin s ecretion from subc utane ous and visc eral adipo se tiss ue in women. diabe te s; 199 8,47,91 3-917 . 14. niska ne n lk , haffner s , karhunen lj , et al. se rum lep tin in ob es ity is related to gender and b ody fat to pogra phy b ut d oes not pre dict suc ce ss ful weight los s. eur j endo crinol; 1 997 ,1 37:61 -6 7. 15. vettor r , vic ennati v, ga mbine ri a , et al.,lep tin and the hypo thalamic– pituita ry– adrenal a xis activity in wome n with diffe re nt obe sity p he notyp es . int j obes ity re la t metab dis ord 199 7,21,70 8-711 . iraqi j pharm sci, vol.27(2) 2018 bromocriptine mesylate as liquid self-nano emulsifying doi: https://doi.org/10.31351/vol27iss2pp93-101 93 formulation and characterization of bromocriptine mesylate as liquid self-nano emulsifying drug delivery system zainab a. hussein*,1 and nawal a. rajab** * ministry of health and environment, baghdad, iraq. * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract bromocriptine mesylate is a semisynthetic ergot alkaloid derivative with a potent dopaminergic activity, used in the treatment of pituitary tumors, parkinson's disease (pd), hyperprolactinemia, neuroleptic malignant syndrome, and type 2 diabetes, the oral bioavailability is approximately 6%.therefore, the aim is the preparation and evaluation of bromocriptine mesylate as a liquid self-nano emulsifying drug delivery system to enhance its solubility, dissolution and thermodynamic stability of the formulation. solubility study was made in different vehicles to select the best one for dissolving bromocriptine mesylate. pseudo-ternary phase diagrams were constructed at 1:1, 2:1, 3:1 and 4:1ratios of surfactant and co-surfactant. four formulations were prepared, using various concentrations of castor oil, tween 80 and ethanol. all the prepared formulations were evaluated for particle size distribution, polydispersity index, drug content, thermodynamic stability, dispersibility and emulsification time, robustness to dilution and in vitro drug dissolution. it was found that, the rate and extent of release for all prepared formulations were significantly higher (p ≤ 0.05) than that in crude drug powder. from the study, it was concluded that self-nano emulsifying drug delivery system is a promising approach to improve solubility, dissolution and stability of the formulation. keywords: bromocriptine mesylate, pseudo-ternary phase diagram, dissolution rate, snedds. نالتكوي تلقائي دقيق سائل نانوي كمستحلب ميسيالت بروموكربتين دواء وتقييم إعداد ** رجبنوال عياش و 1،* زينب علي حسين وزارة الصحة والبيئة، بغداد، العراق. * .فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد، العراق** الخالصة ة يستخدم في عالج أورام الغد قوي، وت شبه اصطناعي مع نشاط دوباميناميجربتين ميسيليت هومشتق شبه قلوي ابروموكر , التوافر البيولوجي 2ا لدم،المتالزمة الخبيثة للذهان، وداءالسكري من النوع فرط نشاط هرمون الحليب في ، النخامية , مرض باركنسون وبالتالي نهدف الى إعداد وتقييم بروموكريبتين ميسيليت بشكل نظام سائل االستحالب نانوي ذاتي التكوين . ٪ 6الفموي هوما يقارب ذوبان ،الحل واالستقرار. أجريت دراسة قابلية الذوبان في مركبات مختلفة لتحديد أفضل السواغات لحل بروموكريبتين . تم بناء لتعزيز ال عتحضيرأرب . تممن نسبة الفاعل بالسطح و المادة الخافضة للتوتر السطحي 1: 4و 1: 3، 1: 2، 1: 1المخططات الطورية الزائفة في يمات الجس وحجم التشتت بمؤشر المعدة التركيبات جميع تقييم وايثانول . تم 80الخروع , توين زيت من اكيزمختلفةتر باستخدام تركيبات الجسيمات حجم لتوزيع المعدة , القابلية دواءال المختبر , محتوى في الدواء وتفكيك التخفيف في , السهولة الحراري الديناميكي , الثبات معدل اإلطالق ومدى كل التركيبات المحضرة كانت أعلى بشكل ملحوظ من مسحوق الدواء العادي. نستخلص وجد أن االستحالب . ووقت .من الدراسة ان نظام االستحالب النانو الذاتي هو نهج واعد لتحسين الذوبان , حل و استقرار بروموكربتين ميسيليت . sneddsالحل، معدل ، الثالثية ئفةالزا المرحلة مخطط ميسيليت، بروموكريبتين: الكلمات المفتاحية introduction self nano emulsifying drug delivery system (snedds) is isotropic uniform preconcentrate mixtures of oil , surfactant , co-surfactant and drug that rapidly form fine oil-in-water (o/w) nanoemulsions when introduced into the aqueous medium under mild agitation (1). 1corresponding author e-mail: zainab111hussein@gmail.com received: 2 / 8 /2018 accepted: 7 / 10 /2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp93-101 mailto:zainab111hussein@gmail.com http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 bromocriptine mesylate as liquid self-nano emulsifying 94 recently, attention has been drawn toward self-nano emulsifying drug delivery systems (snedds) and solid nanosuspensions (ns) for improving the oral bioavailability of biopharmaceutics classification system (bcs) class ii drugs through enhancing their solubility. snedds can form nanoemulsion with 20-200 nm size range upon dilution with no need to perform a dissolution step. snedds spread instantly in the gastrointestinal tract (git) fluids and its motility provide the necessary dispersion of the nanoemulsion(2,3). after administering orally, lingual and pancreatic lipases act on the oily phase of the snedds that result in the formation of emulsified mono-glycerides, diglycerides and fatty acids, resulting in the formation of intestinal mixed micelles in the presence of bile acids.when these mixed micelles pass through the enterocytes, chylomicrons are formed. these can drain the drug into the lymphatic vessels and not in the blood vessels. thus, bypassing the first pass effect, the oral bioavailability will be increased (4). snedds improve the oral bioavailability of the poorly water-soluble drug by enhancing the solubility and keeping the drug in a dissolved state, in small globules of oil, during its transit through the gastrointestinal tract (5). bromocriptine mesylate is a semisynthetic ergot alkaloid derivative with a potent dopaminergic activity. it is a dopamine agonist that used in the treatment of pituitary tumors, parkinson's disease (pd), hyperprolactinemia, neuroleptic malignant syndrome, and type 2 diabetes(6). it is practically insoluble in water, freely soluble in methanol, soluble in ethanol (96 %), sparingly soluble in methylene chloride, belonging to class ii according to biopharmaceutical classification system (bcs)(7). bromocriptine mesylate is rapidly absorbed after oral administration. the absorption from the gastrointestinal tract is only about 28% to 37%. in addition to that, the drug undergoes first-pass metabolism in the liver, such that the oral bioavailability is only approximately 6% (8). thus, this research aimed to formulate and evaluate bromocriptinemesylate as liquid self-nano emulsifying drug delivery system to enhance its solubility, wettability, dissolution and stability of dosage form for better delivery of bm through the oral cavity. materials and methods materials bromocriptine mesylate was purchased from aopharm (china). castor oil was brought from now food (usa). tween 80 was purchased from pure chemistry (germany). ethanol, methanol were purchased from sigmaaldrich (germany). hydrochloric acid was purchased from avantor performance materialsind. netherlands. allother chemicals used were of analytical grade. methods differential scanning calorimetry (dsc) the dsc technique was used to provide qualitative information about the physicochemical status of the drug in the solid snedds formula and compatibility problems. this test was done to assess the thermotropic properties and thermal behavior of the drug (bm). procedures include taking about ten mg of sample, sealing it in an aluminum pan in dsc instrument, and heated at the rate of 10°c/min, covering a temperature range of 40 to 300°c(9). solubility studies the saturation solubility test was made to detect the best vehicle for dissolving bm.the solubility of bm in (various oil phases, surfactants, co-surfactant/co-solvents mixtures, 0.1n hcl solution) was determined by dissolving an excess amount of drug in 2 ml of each of selected vehicle contained in stoppered vials (10 ml capacity) separately.the resultant liquids were mixed using a vortex and then sonicated for 10 min , then were shaken using a water bath shaker at 25±1°c for 48h to reach equilibrium. the equilibrated samples were removed from the shaker and centrifuged (3000 rpm) for 20 min. the supernatants were taken out and filtered through a membrane (0.45 μm millipore filter) (10, 11). the concentration of bm in various phases was determined by uv spectrophotometer at their respective λ max ( 309 nm in methanol ,y =0.0136x-0.0064 and 306 nm in 0.1n hcl , y = 0.013x-0.002). construction of pseudo-ternary phase diagrams based on the individual solubility studies and the hydrophilic-lipophilic balance (hlb) value, the oil, surfactant and cosurfactant were chosen for the construction of pseudo-ternary phase diagramusing aqueous titration method(12). the surfactant and cosurfactant are mixed in different ratios (1:1, 2:1,3:1 and 4:1 ) ; these s mix ratios were chosen in increasing concentration of surfactant with respect to co-surfactant. phase diagrams are constructed for each ratio; the data obtained was subjected to chemix software for construction of the ternary plot. iraqi j pharm sci, vol.27(2) 2018 bromocriptine mesylate as liquid self-nano emulsifying 95 preparation of bromocriptinemesylate liquid self-nanoemulsifying drug delivery systems bm was prepared as liquid self-nano emulsifying drug delivery systems using tween 80 as surfactant and ethanol as co-surfactant in ratios (1:1, 2:1, 3:1 and 4:1) respectively and oil: s mix ratio keeping constant as 1:9 illustrated in (table 1). bm was dissolved in castor oil in a screw-capped glass container and mixed with other components at the concentration of (2.5 mg / 0.3 ml) and heated in a water bath at (50 -60°c) to facilitate homogenization. the components were mixed by vortex mixing for 5 min to obtain a clear, uniform mixture and again cooled to room temperature followed by equilibrating the mixture on a sonication at a room temperature for 10 min, after that the formulations were kept under visual observation for at least 48h (at room temperature) and examined for any signs of turbidity or phase separation prior to droplet size distribution studies (13-15) . table 1.composition of bromocriptine mesylate liquid self-nanoemulsifying drug delivery systems (% w/w) formulas–code s mix ratio oil: smix ratio castor oil % tween80% ethanol % snedd-1 1: 1 1: 9 10 45 45 snedd-2 2: 1 1: 9 10 60 30 snedd-3 3: 1 1: 9 10 67.5 22.5 snedd-4 4: 1 1: 9 10 72 18 evaluation of the prepared bromocriptine mesylate liquid self-nanoemulsifying drug delivery systems thermodynamic stability studies all the prepared formulations were subjected to different thermodynamic stability tests (centrifugation, heating-cooling cycle and freeze-thaw cycle). all the prepared snedds formulations were centrifuged at 3500 rpm for 30 min and observed for phase separation, creaming and cracking. formulations which are stable, were subjected to heating-cooling cycle. six cycles between refrigerator temperature 4 and 45o c with storage at each temperature for not less than 48 h. formulations, that are stable at these temperatures, were subjected to freeze-thaw cycle. three freeze-thaw cycles between -21oc and +25oc with storage at each temperature for not less than 48h was carried out. formulations, which passed these thermodynamic stress tests, were taken for further studies (16). droplet size measurement and polydispersity index (pdi) mean droplet size and polydispersity index (p.i.) were estimated for all stable snedds formulations by applying 100 μl of snedds which was diluted to 250 ml 0.1n hcl in a beaker and gently mixed using a glass rod at 25°c. the estimations were performed using particle size analyzer instrument (brookhaven corp 90 plus, ny, usa) in which the light scattering was monitored at 25ºc and 90 angles (16, 17,18). robustness to dilution all the prepared snedds formulations were diluted to 50, 100, 1000 and3000 fold with distilled water, phosphate buffer ( ph 6.8) and 0.1n hcl in three separated glass vials. the diluted formulations were shaken and then visually inspected after 24 hours ( at room temperature ) for any form of phase separation, coalescence of droplets or drug precipitation (19). dispersibility tests and self-nano emulsification time the efficiency of dispersibility and selfnano emulsification time was assessed using a usp xxii dissolution apparatus ii. from each snedds formulation, 0.5ml was added to 500 ml of distilled water maintained at 37 ± 0.5oc, with paddle rotating at 50rpm for gentle agitation. the in vitro efficacy of the formulations was visually assessed until a transparent homogenous system was seen to determine the time (in a min.) required for completing nano emulsification using the grading system as shown in table 2 (20,21). iraqi j pharm sci, vol.27(2) 2018 bromocriptine mesylate as liquid self-nano emulsifying 96 table 2. grading system of in vitro performance of the snedds (dispersibility and self-nano emulsification time) grade time required for nanoemulsion formation appearance a rapidly forming (within 1 min) nanoemulsion, having a clear or bluish appearance b rapidly forming (within 1 min) slightly less clear emulsion, having a bluish white appearance c formed within 2min fine milky emulsion d slow to emulsify (longer than 2 min). dull, greyish white emulsion having slightly oily appearance e slow to emulsify (longer than 2 min). exhibiting either poor or minimal emulsification with large oil globules present on the surface. drug content snedds formulation containing bromocriptine mesylate equivalent to one dose (2.5mg) was added in 100 ml volumetric flask containing methanol (100 ml) and mixed it well. the extracted solution was suitably filtered, diluted and analyzed for drug content using uv-spectrophotometer at its λ max nm in methanol (22). in vitro dissolution study the in vitro drug release profile of all the prepared snedds formulations were determined using usp dissolution apparatus-ii (paddle method). the dissolution medium, according to the monograph of bromocriptine mesylate in usp (2009), is 0.1n hcl as the dissolution media (500 ml), at 37±0.5°c and 50 rpm, using dialysis bag technique (molecular cut off 12000 da)(23). snedds formulation containing bromocriptine mesylate equivalent to one dose (2.5mg) was placed in the dialysis bag and five ml of dissolution medium was withdrawn every 10 min over 60 min (10, 20, 30, 40, 50, 60 ) and replaced with fresh media ( 0.1n hcl ) in each withdrawal. the quantity of dissolved drug was determined using uvspectrophotometer method at its λ max in 0.1n hcl (306nm)(22). statistical analysis the results of the experiments were given as a mean of triplicate samples ± standard deviation and were analyzed according to the one way analysis of variance (anova) to determine if the changes in the applied factors are statistically significant at level of (p ≤ 0.05) and non-significant at level of (p>0.05). results and discussion differential scanning calorimetry the dsc technique has been performed to determine the thermal stability of drug. dsc thermogram of bm illustrated in (figure 1).pure bm showed a characteristic sharp endothermic peak at (204.03°c) which corresponding to its melting point ,which is near the reported one (192-196) (24). 100.00 200.00 300.00 temp [c] 0.00 10.00 20.00 30.00 40.00 50.00 mw dsc 204.03x100c 211.61x100c figure 1. dsc thermogram of bromocriptinemesylate iraqi j pharm sci, vol.27(2) 2018 bromocriptine mesylate as liquid self-nano emulsifying 97 determination of saturation solubility of bromocriptine mesylate in different oils, surfactant, and co-surfactant it was found that, the solubility of the drug at acidic media was significantly higher than that obtained at phosphate buffer (ph 6.8) as given by the (table 3), this is due to the nature of the drug (6), as it will be in an ionized form in the acidic medium. while upon increasing ph, the unionized species predominated, which explains the sharp decrease in the solubility at buffer (ph 6.8) that can be predicted by the application of the henderson-hassel balch equation (25).amongst the various oils that were screened (table 3), castor oil could solubilize the dose of bm (2.5mg) at relatively small volume and bm shows high solubility in this oil. as shown in (table 3), tween 80 showed a high ability to dissolve bm and therefore was selected for the study.from the results of saturation solubility, castor oil is used as an oil component, tween 80 as surfactant and ethanol as co-surfactant. table 3. saturation solubility of bromocriptine mesylate in different media medium solubility (mg/ml) mean ±sd* coconut oil 3.744± 0.048 sunflower oil 6.366 ± 0.075 peppermint oil 5.15 ± 0.074 castor oil 67.135 ± 0.15 oleic acid oil 59.85 ± 0.1 tween 80 125.098 ± 0.07 0.1 n hcl 0.19 ± 0.02 phosphate buffer ph 6.8 0.0254±0.0004 pseudoternary phase diagram construction pseudo-ternary phase diagrams were constructed to optimize the concentration of the oil (castor oil ), surfactant (tween 80) and cosurfactant (ethanol) to identify their effect on the nanoemulsion formation. in the pseudoternary phase plot, the shaded area represents the area of nanoemulsions while unshaded area represents the area of the emulsion. the plot with a larger shaded area indicates the presence of perfect nano emulsifying activity of formulated nanoemulsions and beneficial interaction among the s mix, oil and aqueous phase (26). pseudo-ternary phase diagram plot for different s mix ratio ( tween 80 : ethanol 1:1, 2:1, 3:1, 4:1 )are shown in figures 2 respectively. as ratio of tween increase give larger shaded area indicates the presence of perfect nano emulsifying activity of formulated nanoemulsions, usually, the addition of surfactant alone cannot lower the oil/water interfacial film sufficiently to form nanoemulsion and addition of short to medium chain length alcohol is imperative as a cosurfactant (27). figure 2 .pseudo ternary phase diagrams for different s mix ratio (tween 80: ethanol 1:1, 2:1, 3:1, and 4:1) iraqi j pharm sci, vol.27(2) 2018 bromocriptine mesylate as liquid self-nano emulsifying 98 preparation of bromocriptinemesylate liquid self-nano emulsifying drug delivery systems all the snedd formulations show yellow and clear mixtures without phase separation orvisually noticed drug precipitation, resulting in four successful formulations. evaluation of the prepared liquid self-nano emulsifying drug delivery system thermodynamic stability studies all of snedds formulations were passed the thermodynamic stability testing as there was no sign of phase separation or drug precipitation at the end of all cycles. this suggested that the formulations were persisted against the extreme storage conditions. the thermodynamic results of the prepared formulation were shown in table 4 . table 4. thermodynamic stability studies of bromocriptine mesylate liquid self-nano emulsifying drug delivery systems. formula code centrifuga -tion test heating -cooling cycles test freezethawing cycles test snedd s-1 pass pass pass snedd s-2 pass pass pass snedd s-3 pass pass pass snedd s-4 pass pass pass droplet size measurement and polydispersity index (pdi) the droplet size of the nanoemulsion determines the absorption and bioavailability of the drug, smaller droplets diameter provide a larger surface area, leading to faster drug release into the aqueous medium (28). it was found that the optimal droplet diameter was in the range of 100–500 nm (29), all the prepared snedd formulas show droplet diameter more than 100 nm and the polydispersity index below 0.3, this indicates good uniformity in the droplet size distribution after dilution with water(30,31). droplet size measurement and polydispersity index (pdi) results showed in table 5. table 5. droplet size measurement and polydispersity index (pdi) of bromocriptine mesylate liquid self-nanoemulsifying drug delivery systems formula – code mean droplet diameter (nm) polydispersity index (pdi) snedds-1 262.2 0.210 snedds-2 329.5 0.005 snedds-3 181.2 0.131 snedds-4 271.6 0.005 robustness to dilution the dilution capability of the formulations was tested to determine the capability of the formulation to withstand possibly infinite dilutions. this was because upon ingestion, the gastrointestinal fluids are responsible for the dilution, and it is impossible to accurately identify the amount of water present to form the emulsion with the formulation, robustness to dilution was performed dilution with an excess of water, standard phosphate buffer ph 6.8 and 0.1n hcl and was stored for 24h. all snedds formulas showed no precipitation or phase separation as illustrated in table 6.the ability of snedds formulation to withstand aqueous dilution was found to be fascinating. the phenomenon was attributed to the high solubilizing properties of the excipients, and also the capability to form a relatively stable emulsion with small droplet sizes. this implied, that these formulations were stable at infinite water dilution (32). there was no significant effect of ph on the snedds formulations, as non-ionic surfactants are less affected by changes in ph and ionic strength compared to ionic surfactants. it confirms that the preparations were robust to high dilution and variations in ph (33). table 6. robustness to dilution of various bromocriptine mesylate liquid self-nanoemulsifying drug delivery systems formula code phase separation drug precipitation 0.1n hcl phosphate buffer ph 6.8 distilled water 0.1n hcl phosphate buffer ph 6.8 distilled water snedds-1 pass pass pass pass pass pass snedds-2 pass pass pass pass pass pass snedds-3 pass pass pass pass pass pass snedds-4 pass pass pass pass pass pass iraqi j pharm sci, vol.27(2) 2018 bromocriptine mesylate as liquid self-nano emulsifying 99 dispersibility tests and self-nano emulsification time emulsification studies are an essential method to evaluate the self-emulsifying properties of designed formulations. when subjected to aqueous dilution under mild agitation, snedds should completely and rapidly disperse (34). the rate of emulsification is an important index for the assessment of the efficiency of emulsification. since the free energy required to form an emulsion is very low, the formation is thermodynamically spontaneous(35,36). all the prepared snedds formulations have formed the nanoemulsion in less than 1 min with grade a as illustrated in table 7. noticed that as the surfactant concentration was increased as the emulsification time was decreased, because surfactants present in the snedds reduce the interfacial tension between oil and aqueous phases and facilitate dispersion and formation of oil in water emulsion (34). table 7. dispersibility and self-nano emulsification time of bromocriptine mesylate liquid selfnanoemulsifying drug delivery systems formulacode grade emulsification time(sec) formulacode grade emulsification time(sec) snedds-1 a 21 snedds-3 a 17 snedds-2 a 19 snedds-4 a 14 drug content drug content of the all prepared bm snedds was more than 97% (from bm loaded dose 2.5mg), and there was no significant difference among the various formulations, which meet united states pharmacopeia (usp) requirements and were within an acceptable range (90%110%)(37) indicating that, there was no precipitation of drug in any of the prepared formulations. the content percents of bm snedds were illustrated in table 8. table 8. the drug content percent of a bromocriptine mesylate liquid self-nano emulsifying drug delivery system (mean ±sd) n=3. formula-code drug content % formula-code drug content % snedds-1 97.67 ± 0.125 snedds-3 98.55 ± 0.163 snedds-2 99.42 ± 0.095 snedds-4 99.33 ± 0.081 in vitro dissolution study dialysis membranes were used in this test, since they are less susceptible to blockage and the size of the pores is very small (38). they were washed with deionized water to get rid of the preservatives and then soaked in the dissolution medium ( 0.1n hcl ) overnight to achieve equilibration state (39). moreover, the size of bag chosen was about (12000 da) to ensure a large surface area exposed to the dissolution medium and avoid acting as a barrier to release of bm from formulation (23, 40). the in vitro drug release studies were made in order to ensure the fast release of the drug in the dissolution medium. the in vitro drug release profile of f1to f 4 and pure bm powder were evaluated in 0.1 n hcl and shown in figure 3. figure 3. a comparative dissolution profile of bm snedds formulas (f1, f2, f3, f4 ) and pure bm. the pure drug showed (29.45%) drug release at the end of 60 min due to its poor aqueous solubility. while prepared bm snedds formulations showed more than 97% of drug release at the end of 60 min. as the drug particles are converted into a dissolved state in the snedds, as the release faster compared to the pure drug. the faster release rate may be attributed to fine particle size and high surfactant mixture concentration, which can easily emulsify the oil rapidly for finer globule (41). all the prepared snedds formulas have iraqi j pharm sci, vol.27(2) 2018 bromocriptine mesylate as liquid self-nano emulsifying 100 no significant difference in the rate and extent of release profile ( p > 0.05), they have a higher significant difference with the rate and extent of the release profile of crude bm powder ( p ≤ 0.05). finally, the snedds formulation resulted in the spontaneous formation of a nanoemulsion with a small droplet diameter size, which permitted a faster rate of drug release into the aqueous phase, significantly much faster than pure drug powder. conclusions from this study, we can conclude that snedds provided a useful dosage form for the oral water-insoluble drug. snedds that was prepared from castor oil, tween 80 and ethanol was a promising approach to improve the solubility, wettability, dissolution rate and stability of bromocriptine mesylate. snedds of bromocriptine mesylate was successfully developed and assessed for it's in vitro performance. the nano size of these formulations is responsible for the 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of lovastatin. inter j of pharm sci and res.2015; 6(1):29-49. 39. panwar p, pandey b, lakhera pc, singh kp. preparation, characterization, and in vitro release study of albendazole encapsulated nanosize liposomes. inter j of nanomedicine.2010; 5:101-108. 40. dudhipala n, veerabrahma k. candesartan cilexetil loaded solid lipid nanoparticles for oral delivery: characterization, pharmacokinetic and pharmacodynamic evaluation. drug delivery. 2016; 23(2):395404. 41. deshmukh a and kulkarni s. novel self micro-emulsifying drug delivery systems (smedds) of efavirenz. j chempharma res. 2012 ; 4(8): 3914-3919. iraqi j pharm sci, vol.29(2) 2020 physicians' assessment of the clinical pharmacist doi: https://doi.org/10.31351/vol29iss2pp194-201 194 assessment of the clinical pharmacists' role by physicians at baghdad hospitals ahmed h. saihood*,1 and ali f. hasan *department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract in iraq, there is a limited research work exploring the clinical pharmacists' role from the point of view of other healthcare professionals. to investigate physicians' assessment of clinical pharmacy services at baghdad hospitals, and compare junior physicians with senior physicians' point of view. the study was conducted in twelve governmental hospitals in baghdad, iraq. data was collected from a sample of two hundred physicians, and through a validated, self-administered questionnaire, which comprised twenty statements in addition to a non-personal information form that precedes the questionnaire. two hundred questionnaires were completely filled. the total average score of the participants' responses was 2.945±0.99, 59% of the participants had an average score ≥3. junior physicians had more positive responses (average score=3.12±0.422), compared to senior physicians (average score=2.721±0.2662) the study findings reveal a somewhat positive point of view towards clinical pharmacists' performance in baghdad hospitals. physicians are satisfied with clinical pharmacists' recommendations regarding their prescriptions, and their availability for consultation. however, they are dissatisfied with or unaware of the unconventional services that are not directly related to the medical prescription. junior physicians are more appreciative of the clinical pharmacist's role than senior physicians. keywords: physician, clinical pharmacist, baghdad, assessment, services. تقييم األطبّاء لدور الّصيدالنّي الّسريرّي في مستشفيات بغداد **فارس حسن عليو 1*،صيهودأحمد حسين فرع الّصيدلة الّسريريّة، كليّة الّصيدلة، جامعة بغداد، بغداد، العراق.* كليّة الّصيدلة، جامعة بغداد، بغداد، العراق.فرع األدوية والّسموم، ** الخالصة تدارس محدودة. -من وجهة نظر غيرهم من مختّصي الرعاية الّصحية -تعتبر األبحاث الّتي تستكشف دور الّصيادلة الّسريريّين في العراق لمقيمين والمقيمين تقييم األطبّاء لخدمات الّصيدلة الّسريرّية في مستشفيات بغداد، والمقارنة بين وجهة نظر األطبّاء األخصائّيين ووجهة نظر األطبّاء ا ئتي طبيب، وعن طريق األقدمين. تّم إجراء الدّراسة في اثني عشر مستشفى حكومّي في بغداد، العراق، وتّم جمع البيانات من عيّنة مكّونة من م لكامل. استبيان مثبت الّصالحيّة، يشتمل على عشرين عبارة، باإلضافة إلى استمارة معلومات غير شخصيّة تسبق االستبيان. مئتا استبيان ُملئت با طبّاء المقيمين والمقيمين . إجابات األ3≤من المشاركين كان معدّل نقاطهم %59(, 0.99 ±2.945معدّل النّقاط الكلّّي إلجابات المشاركين هو ) (. نتائج الدّراسة تكشف 0.2662±2.721( مقارنة باألطباء األخّصائيّين )معدّل النّقاط=0.422±3.12األقدمين كانت أكثر إيجابيّة )معدل النّقاط= تعبّر عن رضاهم عن توصيات الّصيادلة ألداء الّصيادلة الّسريريّين في مستشفيات بغداد. إجابات األطبّاء -إلى حدٍّّ ما -عن وجهة نظر إيجابيّة الّسريريّين بخصوص الوصفات الّطبيّة، وتواجدهم المستمّر من أجل االستشارة. من جانبٍّ آخر، معظمهم غير راضين عن أو غير مدركين لوجود ون األقدمون أبدوا تقديًرا أكبر لدور الّصيدالني الّسريرّي، الخدمات غير التّقليدّية اّلتي ال تتعّلق بالوصفة الّطبيّة بشكل مباشر. األطبّاء المقيمون والمقيم مقارنة باألطبّاء األخصائيّين. كلمات مفتاحيّة: طبيب، صيدالنّي سريرّي، بغداد، تقييم، خدمات. introduction the concept of clinical pharmacy is based on the evolution of pharmacy practice from a passive role to an active role when it comes to providing patient care, which calls for a stable and effective interaction with other health care providers (1). in iraq, such interaction has always been hindered by the pharmacists' hesitance to take initiative and other health care members' unwillingness to involve the clinical pharmacist in their medicine related decisions and processes (2). ideally, the clinical pharmacist's job comprises collaboration with other health care providers, direct interaction with the patient for the assessment and monitoring of drug therapy, making any necessary therapeutic modification to warrant the safe and cost-effective use of medications, make any necessary arrangements with the community pharmacist for a seamless patient care, and being always available for consultation regarding medicine information and patient responses (3). calculating doses and monitoring medication blood levels are also among the services provided by clinical pharmacists. so the major role of the clinical pharmacists takes place in hospital wards and acute care settings, and the services that they provide tend to be more patient-oriented when compared to the community pharmacists (4). ahmed.hussein@copharm.uobaghdad.edu.iq :mail-corresponding author e1 received: 25/4 /2020 accepted: 12/ 7/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp194-201 iraqi j pharm sci, vol.29(2) 2020 physicians' assessment of the clinical pharmacist 195 to be registered as a clinical pharmacist in iraq, the newly graduated pharmacists must enroll in a one year clinical pharmacy program at a governmental teaching hospital, after which they would be lawfully committed to finish no less than five years of clinical pharmacy practice at a governmental institution (5). the program is implemented by the ministry of health, and it is a quite beneficial way of introducing the clinical pharmacy branch to hospitals in iraq, and the program was, and still is, fairly successful. however, it has some major drawbacks, it is not a specialized program, the ministry of higher education does not officially recognize it, and its capacity to keep up with the advancements in medicine is questionable. in 2000, the idea for a board certification program was presented, but due to the subsequent events that ringed the country, and the rejection by the arabic board for medical specializations, the idea was not implemented until 2011, when a 4-year training program was developed as a division of the iraqi board of medical specializations, which is recognized by the iraqi ministry of higher education and scientific researches, and was a major step forward towards a better more focused clinical pharmacy services (6). the purpose of this study is to evaluate the physicians' observation of the clinical pharmacist's role in different hospitals in baghdad governorate, and assess the difference between junior and senior physicians' point of view. subjects and methods study design a cross sectional study was conducted in twelve governmental hospitals over a period of about three months from the 21st of september 2019 to the 3rd of january 2020. the option to conduct the study around a specific institution was considered, but then the choice was made to expand the range of research spanning different locations in the city of baghdad, iraq. all divisions and wards of the twelve governmental hospitals were included. hospitals where the research was conducted are: baghdad medical city complex, al-yarmouk teaching hospital, al-karkh general hospital, pediatrics' central teaching hospital, al-forat general hospital, al-shaikh zayed hospital, ibn al-nafees hospital, al-numan general hospital, alkadhimiya teaching hospital, martyr al-sadir general hospital, ibn al-bitar hospital, and alkarama teaching hospital. sample selection two hundred physicians with different specialties have participated. the heavy schedule of the participants was complied with, avoiding presenting the questionnaire at busy hours, which can lead to rushed responses, and no time to explain the nature of the questionnaire properly. judging by the sample size, which was decided based on a previous study (7), and the inclusion of different professional classes and specialties, it can be assumed that this sample duly represents the physicians' society in baghdad hospitals. participants should meet the following criteria: ward-based physicians, employed at a governmental hospital with its own pharmacy department, situated in the city of baghdad, at least one year of experience, and were still employed at the time the study took place. questionnaire development initially, the questionnaire that was constructed comprised thirty-six statements that described various clinical pharmacy activities based on a number of literature references and journal articles (4,8), but two hospital visits were more than enough to affirm the need for a shorter more focused questionnaire, as most participants were confused and chose to omit some of the unclear and repetitive statements. so, modifications were made, and the resulted questionnaire comprised twenty statements that were variations of the original thirty-six, in addition to new statements catering to physicians, all of which were edited and revised using the broad expertise of five clinical pharmacy doctors and one pharmacology doctor. the questionnaire was written in english, and was subjected to a pilotscale experiment that was conducted at baghdad medical city, over the span of three weeks, in order to validate its reliability and internal consistency. printed questionnaires were given to the participants, to which they responded by choosing the degree of their agreement on a likert measurement scale (which was used to construct the questionnaire) from 1 to 5, 1 being "never", and 5 being "always". non-personal information form preceded the questionnaire, and included professional title, specialty, years of practice, age, gender, and the name of institution, to ensure the variety in the sample chosen. data analysis for the summarization and analysis of data, statistical package for social sciences (spss) version 23.0 and microsoft office excel 2013 were used. numeric data were expressed as mean± standard deviation, whereas categorical data were expressed as numbers and percentages. pearson correlation coefficient (r) was used to compare between test and retest to determine the reliability of the questionnaire, while internal consistency was examined for each group of questions using cronbach's alpha parameter, the results of which lie between (0-1). unpaired student t-test was performed for each group pair, and statistical significance was defined as (p-value ≤0.05). pearson correlation coefficient (r) was used to determine the type of correlation between two means, if p-values ≤0.05 the relationship was considered statistically significant. iraqi j pharm sci, vol.29(2) 2020 physicians' assessment of the clinical pharmacist 196 administrative and ethical considerations a research proposal was reviewed and accepted by the clinical pharmacy scientific committee in the college of pharmacy university of baghdad, before it was submitted and officially approved. additionally, the study was approved by the ministry of health, and participants' verbal consent was obtained. results validation of the questionnaire in order to test the reliability and internal consistencies of the questionnaire, twenty participants (randomly chosen) were recruited for the pilot study. pearson correlation between the test and retest (three weeks interval) was used to validate the reliability. as shown in table 1, the reliability of the questionnaire is excellent (r = 0.9123). for the validation of the internal consistency, cronbach's alpha constant was used, and it showed excellent internal consistency for the test (α1=0.9459), and good internal consistency for the retest (α2=0.8721). the participants in the validation process were not involved in the rest of study. table 1. validation of the questionnaire cronbach's alpha (α1) for test cronbach's alpha (α2) for re-test pearson correlation(r) between time 1 and 2 scores physicians 0.9459 0.8721 0.9123 number of participants for test and re-test was = 20 demographic characteristics of the participants in total, 200 questionnaires were completely filled (any questionnaire with missing data was omitted). as shown in table 2, the average age of the participants was (30.96±3.79 years), males represent 29% (58) of the participants, while females represent 71% (141) of them. with respect to the professional classification, 84% (169) of the participants were juniors while 16% (31) were seniors. the average duration of experience of the participants was (4.52±2.2 years). table 2. demographic characteristics of the participants physicians n=200 age (years) 30.96±3.79 gender male 58(29%) female 142(71%) professional classification junior 169(84.5%) senior 31(15.5%) duration of experience(years) 4.52±2.2 participants' responses to the questionnaire the physicians' answers in response to the 20 statements are demonstrated in table (3). responses with never ranged from 8% (in statement 13) to 40% (in statement 20); responses with rarely ranged from 15% (in statement 12) to 34 % (in statement 20); responses with sometimes ranged from 15% (in statement 7) to 25% (in statement 9); responses with often ranged from 6% (in statement 20) to 37% (in statement 13); and responses with always ranged from 5% (in statement 20) to 24% (in statement 3). the average score for each statement ranged between 2.035 (in statement 20) to 3.445 (in statement 13). these results provide an overall impression of a moderately acceptable response for the role of clinical pharmacists in hospital wards, since responses (sometimes, often and always), when combined, are almost near responses (never and rarely). however, more objective analysis and interpretation will be presented later. number of participants = 200 table 3. physicians' assessment of clinical pharmacist services physicians n e v e r r a r e ly s o m e ti m e s o ft e n a lw a y s a v e r a g e s c o r e q1/ the clinical pharmacist provides information concerning essentially similar medications and interchange possibilities to physicians n 20 49 33 51 47 3.28 % 10% 25% 17% 26% 24% q2/ the clinical pharmacist arranges the interchange of essentially similar medications, with respect to their accessibility, with physicians in the wards n 17 45 41 54 43 3.305 % 9% 23% 21% 27% 22% iraqi j pharm sci, vol.29(2) 2020 physicians' assessment of the clinical pharmacist 197 table 3. continued physicians' assessment of clinical pharmacist services. physicians n e v e r r a r e ly s o m e ti m e s o ft e n a lw a y s a v e r a g e s c o r e q3/ the clinical pharmacist tour with the physician, on a daily basis, to optimize patient treatment n 29 40 42 41 48 3.195 % 15% 20% 21% 21% 24% q4/ the clinical pharmacist enquires information on medicines and presents it to physicians n 30 55 36 51 28 2.96 % 15% 28% 18% 26% 14% q5/ the clinical pharmacist collects and assesses the literature data about medicines and presents his/her statements to the physicians n 32 50 37 51 30 2.985 % 16% 25% 19% 26% 15% q6/ the clinical pharmacist collects and assesses medication data in order to develop and submit solutions for minimizing medication costs n 43 43 37 50 27 2.875 % 22% 22% 19% 25% 14% q7/ the clinical pharmacist takes an active part in creating pharmacotherapeutic guidelines n 32 52 30 52 34 3.02 % 16% 26% 15% 26% 17% q8/ the clinical pharmacist develops and proposes systems for improving the safety of procedures of preparation and administration of medicines n 32 46 40 51 31 3.015 % 16% 23% 20% 26% 16% q9/ the clinical pharmacist collects and assesses information on the use of medical devices and suggests solutions for minimizing costs of their use n 48 40 50 39 23 2.745 % 24% 20% 25% 20% 12% q10/ the clinical pharmacist provides medication history and a list of patient’s medications at admission n 45 44 42 50 19 2.77 % 23% 22% 21% 25% 10% q11/ the clinical pharmacist detects medication contraindications, and suggests changes to the physician when necessary n 20 35 33 70 42 3.395 % 10% 18% 17% 35% 21% q12/ the clinical pharmacist detects drug interactions, and suggests changes to the physician when necessary n 20 29 41 72 38 3.395 % 10% 15% 21% 36% 19% q13/ the clinical pharmacist reviews the prescribed doses, and suggests dose adjustments in therapy to the physician when required n 15 34 38 73 40 3.445 % 8% 17% 19% 37% 20% q14/ the clinical pharmacist is always available for consultation about pharmacotherapy n 20 35 43 55 47 3.37 % 10% 18% 22% 28% 24% q15/ the clinical pharmacist gives advice to the physician about compatibility, stability of parenteral medications n 36 46 43 49 26 2.915 % 18% 23% 22% 25% 13% q16/ the clinical pharmacist gives advice about the selection of the most appropriate enteral or parenteral nutrition product concerning the patient’s condition n 60 48 33 39 20 2.555 % 30% 24% 17% 20% 10% q17/ the clinical pharmacist performs therapeutic drug monitoring and advises the physician on therapy optimization n 49 44 37 39 31 2.795 % 25% 22% 19% 20% 16% iraqi j pharm sci, vol.29(2) 2020 physicians' assessment of the clinical pharmacist 198 table 3. continued physicians' assessment of clinical pharmacist services interpretation of the role of clinical pharmacists an interpretation of the role of clinical pharmacists, as judged by the physicians, is presented in table 4. average scores <3 were interpreted as bad, while average scores ≥3 were interpreted as good. 59% of participants had an average score ≥3, while 41% had an average score <3, and the average score of physicians' agreement with the questionnaire items was 2.945±0.99. table 4. interpretation of the role of clinical pharmacists. domain good role for clinical pharmacist average score of ≥3 n (%) bad role for clinical pharmacist average score of < 3 n (%) mean score ±sd physicians 117.05 (59%) 82.92 (41%) 2.945±0.99 impact of demographic characteristics on the results concerning the professional classification of the participants, and when interpreting the results of the participants as good role if the average score is ≥3, and bad role if the average score is <3, there was no significant difference between the percentage seniors and juniors' responses (p-value=0.542). as illustrated in figure (3.1), 53% of senior physicians were appreciative of the clinical pharmacist role in hospital wards (average score ≥3), while 47% were not appreciative of the clinical pharmacist role (average score <3). as for the responses from junior physicians, 59% were appreciative of the clinical pharmacist role, and 41% were not. on the other hand, the difference between the overall average scores of junior physicians and senior physicians was significance (pvalue=0.0195). as presented in table 5, juniors had an average score of (3.12±0.422), while seniors had an average score of (2.721±0.2662). figure 1. interpretation of the percentages of physicians' response to the role of clinical pharmacists based on their professional classification. physicians n e v e r r a r e ly s o m e ti m e s o ft e n a lw a y s a v e r a g e s c o r e q18/ the clinical pharmacist suggests performing certain laboratory tests that can have an impact on the prescribed medication n 61 51 47 20 21 2.445 % 31% 26% 24% 10% 11% q19/ the clinical pharmacist takes part in conducting clinical trials for medicines and assesses the results n 57 61 42 24 16 2.405 % 29% 31% 21% 12% 8% q20/the clinical pharmacist can (has the power to) interchange essentially similar medications without consulting the physician when this is necessary for costor accessibility-related reasons concerning the prescribed medicine n 79 67 32 12 10 2.035 % 40% 34% 16% 6% 5% iraqi j pharm sci, vol.29(2) 2020 physicians' assessment of the clinical pharmacist 199 table 5. average scores of participants' responses to the role of clinical pharmacists based on their professional classification. juniors seniors p-value physicians 3.12±0.422 2.721±0.2662 0.0195 correlation between the average score of the participants and their demographic characteristics when testing the relationship between variables, a significant association was found between participants' age and their average score (p-value=0.042), with younger participants having a more positive assessment of the clinical pharmacist role. no significant association was found between the average score and the professional classification or the duration of experience (p-value=0.914 & 0.227 respectively) as demonstrated in table 6. table 6. correlation between the average score of the participants and their demographic characteristics characteristic average physicians score r p age -0.132 0.042 professional classification -0.0762 0.914 duration of experience (years) -0.086 0.227 discussion in today's medical environment in iraq, the position of clinical pharmacists in the wards can only be defined by the services they provide, which depend on their pharmacotherapeutic knowledge and their capacity to take initiative and communicate with other members of the medical team. a number of studies in the middle east, including iraq, have tackled the same topic with varying results, but their main focus was evaluating the expectations and the level of comfort of physicians concerning a recently introduced or soon-to-be introduced clinical pharmacy model, as opposed to evaluating their assessment of the current state of clinical pharmacy in their respective area of study (9–11). while physicians' responses to the questionnaire give an impression of a moderately acceptable overlook, they also show physicians' dissatisfaction with certain services. the highest average score was for the statement concerned with recommendations for dose adjustments (3.445), which is not surprising because it is among the more conventional pharmacist roles that are directly related to prescriptions. participants were also fairly satisfied with other conventional prescription-related interventions, including detection of any contraindications and drug-drug interactions. this goes along with one of the findings of a study conducted in kuwait where 57.8 % of the physicians agreed that pharmacists routinely provide recommendations about prescriptionrelated problems (10). while the lowest average score was for the statement that describes the clinical pharmacist as an authoritative figure with the power to switch similar medications without consulting the physician in necessary cases (2.035), which is also not surprising, mainly because of the blame culture that usually holds the physician accountable for any adverse events, and so they tend to be protective of their prescriptions, and it is unlikely for the pharmacist to take such responsibility. this is, to a certain degree, similar to one of the findings of a study conducted in ljubljana, slovenia, where most physicians did not expect pharmacists to have the authority to interchange similar medications without consulting them first (12). most participants were fairly positive about the clinical pharmacists' participation in the daily medical rounds (average score = 3.195), and even more so about clinical pharmacists' availability for consultation (average score = 3.37), which can imply that, in most cases, shortage of interventions is not necessarily due to lack of interaction, and this is contrary to the results of another study conducted in baghdad were 91.5% of the participants reported rarely interacting with pharmacists (7). this is a step forward for an efficient physician-pharmacist relationship as has been demonstrated in a study conducted in massachusetts, united states, which showed a 66% decline in adverse drug events caused by prescribing errors that was brought about by iraqi j pharm sci, vol.29(2) 2020 physicians' assessment of the clinical pharmacist 200 pharmacists' participation in the daily medical rounds (13). most participants' responses indicated that clinical pharmacists do not often perform services that are not confined within medication consultation in the traditional sense. this is concordant with a study conducted in irbid, jordan which concluded that physicians were skeptical about clinical pharmacists performing nontraditional roles (11). the results showed that junior physicians are more appreciative of the clinical pharmacist role compared to senior physicians. the age of the participants could be a factor in this regard in addition to the professional classification. this underlines the widespread belief that senior physicians do not need or accept pharmacists' interventions and recommendations, and this belief does hold some truth as demonstrated in one of the findings of a study conducted in england, where seniors rejected pharmacists' recommendations that they disapproved, while juniors were more willing to act on pharmacists' advice (14). conclusion the study findings reveal a somewhat positive point of view towards clinical pharmacists' performance in baghdad hospitals. physicians are satisfied with clinical pharmacists' recommendations regarding their prescriptions, and their availability for consultation. however, they are dissatisfied with or unaware of the unconventional services that are not directly related to the medical prescription. there is also a considerable difference between junior and senior physicians' point of view, as senior physicians are less appreciative of the clinical pharmacist role, so, strategies should be designed or adopted to close the gap between senior physicians and clinical pharmacists. study limitations despite the questionnaire's validity, the possibility of social and professional bias is still considerable. some participants had limited english skills, and even though all their inquiries were fully explained, it cannot be asserted that they understood all the statements. some participants did not seem to recognize pharmacists who went through the clinical pharmacy program or those who held a board certificate, additionally, not all wards had a clinical pharmacist, and so, some of the participants' responses reflect the performance of any pharmacist based in their respective wards. references 1. wiffen p, mitchell m, snelling m, stoner n, wiffen p, mitchell m, et al. oxford handbook of clinical pharmacy. oxford medical publications. 2007. 36–40 p. 2. al-jumaili aa, al-rekabi md, doucette w, hussein ah, abbas hk, hussein fh. factors influencing the degree of physician–pharmacist collaboration within iraqi public healthcare settings. int j pharm pract. 2017;25(6):411–7. 3. carlisle b, ives t, maddux s, taber p, city k. establishing and evaluating clinical pharmacy services in primary care. am coll clin pharm. 1994;14(6):743–58. 4. wiedenmayer k, summers; rs, mackie ca, gous ags, everard m, tromp d. developing pharmacy practice a focus on patient care. world health organization, international pharmaceutical federation. 2006. 11 p. 5. al-jumaili aa, hussain sa, sorofman b. pharmacy in iraq: history, current status, and future directions. am j heal pharm. 2013;70(4):368–72. 6. rasheed j, abbas h. clinical pharmacy training program in iraq implementation of a clinical pharmacy training program in iraqi teaching hospitals : review article. iraqi j pharm sci. 2012 jan 1;21(1):1–5. 7. hamadi sa, mohammed mm, dizaye ka, basheti ia. perceptions, experiences and expectations of physicians regarding the role of the pharmacist in an iraqi hospital setting. trop j pharm res. 2015;14(2):293–301. 8. mcrobbie d, webb d, davies jg. clinical pharmacy practice. in: hodson k, whittlesea c, editors. clinical pharmacy and therapeutics. 6th ed. elsevier; 2018. p. 2–12. 9. al-arifi mn, alghamdi b, al-saadi m, idris ae, wajid s, said r, et al. attitudes and perceptions of healthcare providers towards clinical pharmacy services at a tertiary care hospital in riyadh, saudi arabia. trop j pharm res. 2015;14(5):913–8. 10. matowe l, abahussain ea, al-saffar n, bihzad sm, al-foraih a, al-kandery aa. physicians’ perceptions and expectations of pharmacists’ professional duties in government hospitals in kuwait. med princ pract. 2006;15(3):185–9. 11. tahaineh lm, wazaify m, albsoul-younes a, khader y, zaidan m. perceptions, experiences, and expectations of physicians in hospital settings in jordan regarding the role of the pharmacist. res soc adm pharm. 2009;5(1):63–70. 12. čufar a, locatelli i, mrhar a. attitudes of physicians, nurses and pharmacists concerning the development of clinical pharmacy activities in a university hospital. acta pharm. 2014;64(4):447–61. iraqi j pharm sci, vol.29(2) 2020 physicians' assessment of the clinical pharmacist 201 13. leape ll, cullen dj, clapp md, burdick e, demonaco hj, erickson ji, et al. pharmacist participation on physician rounds and adverse drug events in the intensive care unit. j am med assoc. 1999;282(3):267–70. 14. axon dr, lim rhm, lewis pj, sandher s, thondee j, edwards k, et al. junior doctors’ communication with hospital pharmacists about prescribing: findings from a qualitative interview study. eur j hosp pharm sci pract [internet]. 2018/02/06. 2018 sep;25(5):257–61. available from: https: / / pubmed . ncbi .nlm .nih.gov/31157036. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. https://pubmed.ncbi.nlm.nih.gov/31157036 https://pubmed.ncbi.nlm.nih.gov/31157036 http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.26(1) 2017 antioxidant and inflammation in type ii diabetes mellitus 17 estimation of superoxide dismutase, matrix-metalloprotinase-9, and interleukin -18 in patients with type two diabetes mellitus shatha r. moustafa *,1 and shawn a. omar ** * academic faculty at college of pharmacy, hawler medical university, erbil, iraq. ** ministry of heath, erbil, iraq. abstract antioxidant status imbalance and inflammatory process are cooperative events involved in type 2 diabetes mellitus. this study aimed to investigate superoxide dismutase as a potential biomarkers of antioxidant imbalance, matrix-metaloprotinase-9, and interleukin -18 as biomarkers of inflammation in serum and to estimate the effects of other confounding factors gender, age and finally measuring the relation among the interested biomarkers. this case control study included 50 patients, and 45 of healthy subjects matched age – gender were also enrolled in this study as a control group. the focused parameters were measured using elisa technique. there was significant reduction in the serum superoxide dismutase level and significant elevation in serum matrixmetaloptotinase-9 and interleukin -18 levels which have been associated with diabetes. this finding may explain the role of the defect in antioxidants status leading to significant reduction in serum superoxide dismutase levels associated with increased inflammatory process leading to significant elevations of a matrixmetaloptotinase-9 and interleukin -18. these parameters added a diagnostic information and evaluated as potential tools for disease risk prediction. keywords: type 2 diabetes mellitus, superoxide dismutase, matrix metaloptotinase-9 and interleukin -18. لدى مزضي81 -واوتزلوكيه 9-ميتالوبزوتيىيزقياس صوبز اوكضايد دصميوتيز, ماتزكش الضكز مه الىوع الثاوي شذى رؤوف مصطفي ،*8 و شوان علي مصطفي ** * كلُت الصُذلت الطبُت ، اربُل ، العزاق . ، جاهعت هىلُز ** وسارة الصحت ، اربُل ، العزاق . الخالصة الخلل المائن فٍ هضاداث األكسذة والعولُت االلخهابُت هٍ أحذاد حشارن فٍ الٌىع الثاًٍ هي هزض السكزٌ. حهذف هذٍ بوثابت الوؤشزاث الحُىَت الوحخولت هي عذم الخىاسى الوضادة لألكسذة، ولُاس superoxide dismutaseالذراست الً لُاس metaloprotinase-9 وinterleukin -18 ؤشزاث حُىَت لاللخهاب، للخحمُك فٍ اِثار الوخزحبت علً غُزها هي العىاهل الجٌس كو والعوز وأخُزا الكشف عي هعاهل االرحباط بُي هذٍ الوعاَُز. هعافً سلُن هطابمت 50هزَضا، و 05هاث الوحذدٍ فٍ حطىر الوزض فٍ ىهذٍ الذراست هصووت لذراست االرحباط بُي الوعل .elisaفٍ الجٌس والعوز للخحك فٍ هذٍ الذراست كوجوىعت ححكن. حن لُاس الوعلواث الوحذد باسخخذام حمٌُت فٍ هسخىي الوصل احصائًوارحفاع كبُز superoxide dismutase كاى هٌان اًخفاض احصائٍ هلحىظ فٍ هسخىي هصل الخٍ حزحبظ هع حطىر الوزض. interleukin -18و metaloptotinase-9للوعاَُز superoxide هذا االكخشاف لذ َفسز دور خلل فٍ وضع هضاداث األكسذة هوا َؤدٌ إلً اًخفاض كبُز فٍ هسخىي هصل dismutase ُزة فٍ فٍ الذم وسَادة عولُت االلخهاب الخٍ حؤدٌ إلً ارحفاعاث كبmetaloptotinase-9 وinterleukin -18 ٍهذ . الوعاَُز أضافج للوعلىهاث الخشخُصُت وَوكي اى حمُُن كأدواث هحخولت للخٌبؤ بوخاطز الوزض. . 81 -واوتزلوكيه 9-ماتزكش ميتالوبزوتيىيز ،صوبز اوكضايد دصميوتيز ،مزض الضكز مه الىوع الثاوي :الكلمات المفتاحية introduction the pathogenesis of type 2 diabetes mellitus is associated with the induction of the os that is induced by hyperglycemia (1) . high blood glucose level induces os and decrease antioxidant defenses, thus leading to increase free radical (fr) formation so, there was as a significant difference between patients with t2dm and healthy control group concerning the total antioxidant capacity between t2dm patients and healthy control (2) . several studies have reported the lower concentration of antioxidant enzymes in type 2 diabetes such as superoxide dismutase and glutathione peroxidase (3,4) . superoxide dismutase (sod) cuznsod ec 1.15.1.1 is one of the most important intracellular antioxidant enzymes (5) . 1 corresponding author e-mail: shatha003@yahoo.com received: 15/10/2016 accepted: 14/3/2017 http://www.scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=blood+glucose http://scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=oxidative+stress http://www.scialert.net/asci/result.php?searchin=keywords&cat=&ascicat=all&submit=search&keyword=free+radical mailto:shatha003@yahoo.com iraqi j pharm sci, vol.26(1) 2017 antioxidant and inflammation in type ii diabetes mellitus 18 matrix metalloproteinases (mmps) are a family of zinc-binding proteolytic enzymes that were remodeling the extracellular matrix and then as the response of the inflammatory process pathologically attack the substrates (6) . increased activity of mmps has been reported in numerous disease processes. hyperglycemia and dm enhance expression and activity of mmps, mmp-9 has been investigated to be increased as a result of the stress hyperglycemia (7) . interleukin -18 (il-18) is a proinflammatory cytokine with multiple biologic functions. elevated levels of il-18 has been found in the serum of patients with t2dm (8) . from these information, it has been found that immunological and inflammatory processes have an important roles in an initiation and progression of the disease as the same time it was known that inflammatory process plays in etiologic and pathogenic mechanisms for t2dm. because of impaired glucose metabolism leads to os and there is an interrelationship between os and inflammatory process so, this study was aimed to determine the association of the change in serum levels of sod, mmp-9, and il-18 with t2dm development and to assess other confounding risk factors age, gender and finally detect the relation between these studied parameters. patients and methods this case-control study was designed to examine the associations between the selected parameters with in t2dm patients that was performed at hawler medical university, college of pharmacy on 50 patients of both genders who were randomly selected and newly diagnosed by specialist using clinical examination and confirmed by laboratory tests at diabetic center (al-shahida layla qassim) and after exclusion of other diseases by clinical history, laboratory investigations and clinical examination. in addition to 45 age – gender matched subjects have been also enrolled in this study as a control group who were also randomly selected. all participants completed the baseline questionnaire concerning several risk factors. this study has been done during the period between october 2013 to december 2014. the control group was confirmed to be healthy by clinical, biochemical and hematological examinations. all clinicopathological data of the patients have been collected from the clinical files. during the period of this study, the patients and the control groups were not taken anti-inflammatory drugs and antioxidants as dietary supplements. all procedures were in accordance by the established ethical standards. the protocol of this study has been approved by ethics committee of medical research at college of pharmacy / hawler medical university. oral consents have been taken from all the participants before starting the study. sample collection ten ml of the fasting blood samples have been collected and left for 30 minutes for coagulation purposes and then made centrifugation for 15 minutes at 25003500 rpm. the sera of the participants have been separated and divided into several parts and put them into several plastic plain tubes for the biochemical analysis. the sera of the patients and control groups have been stored at (-80 cº) at medical research center /hawler medical university till the day of the analysis. the sera have been prepared for measurement by thawing the frozen sera at room temperature. the studied parameters have been estimated using enzyme linked immunosorbent assay (elisa) technique at medical research center/ hawler medical university. statistical analysis the statistical study has been done using the statistical patch for social sciences (spss vi.18). the results of biochemical tests were expressed as mean ± standard deviation (sd). furthermore, student t-test was applied to compare between two means. a (p) value of ≤ 0.05 was considered as statistical significance. correlations between laboratory findings and continuous variables were evaluated using linear regression analysis. results subjects characteristics the clinical characteristics of the patients and control groups have been shown in (table 1). the patients group has been divided into two groups either as poor control and uncontrolled groups (35 patients with mean age 52.9 ± 7.8 and15 patients with mean age 63.3 ± 3 respectively) while the control group has been included 45 healthy adults with mean age was 55.9± 7.5. effect of controlling the disease on the serum levels of selected parameters serum levels of hba1c% and glucose were founded to be significantly higher in t2dm patients (uncontrolled group) as compared with poor control and healthy subjects (p< 0.001) (table 1), the serum levels of mmp-9 and il-18 were increased significantly in uncontrolled group t2dm as compared to poor control and healthy iraqi j pharm sci, vol.26(1) 2017 antioxidant and inflammation in type ii diabetes mellitus 19 individuals (p< 0.001), while the serum level of sod was decreased significantly in uncontrolled t2dm as compared to poor control and healthy subjects (p< 0.001). table ( 1): the demographic and clinical characteristics of the studied groups n mean ±sd p age (years) no dm 45 55.933 7.500 < 0.001 poor control 35 52.857 7.845 un-control 15 63.267 3.011 total 95 55.958 7.877 weight (kg) no dm 45 74.222 4.188 .168 poor control 35 73.943 7.372 un-control 15 70.400 11.319 total 95 73.516 6.986 hba1c % no dm 45 5.281 .183 < 0.001 poor control 35 8.386 .323 un-control 15 10.487 1.499 total 95 7.247 2.097 fbg mg/dl no dm 45 92.311 6.338 < 0.001 poor control 35 231.486 26.983 uncontrol 15 297.133 42.963 total 95 175.926 86.010 mmp-9 ng/ml no dm 45 1.506 .945 < 0.001 poor control 35 1.804 .943 un-control 15 4.563 1.669 total 95 2.098 1.524 il-18 pg/ml no dm 45 48.400 26.375 < 0.001 poor control 35 84.240 17.581 un-control 15 116.760 6.603 total 95 72.398 32.953 sod ug/ml no dm 45 501.447 162.781 < 0.001 poor control 35 357.320 43.149 un-control 15 171.867 51.409 total 95 396.308 165.428 p< 0.05 effect of the type 2 diabetes mellitus on the serum levels of selected parameters: the statistical study has been exhibited that the serum levels of hba1c% ,glucose, mmp-9 and il-18 were significantly increased in patients with t2dm as compared with the control group p˂ 0.001, while the serum level of sod was significantly decreased in patients with t2dm as compared with the control group p˂ 0.001 (table 2). iraqi j pharm sci, vol.26(1) 2017 antioxidant and inflammation in type ii diabetes mellitus 20 table ( 2): comparison between the studied groups regarding the focused parameters p< 0.05 gender effect concerning mmp-9, there was a significant difference between males and females in patients group p= 0.049 (table3). regarding sod and il-18 , there were no significant differences between men and women in patients group p > 0.05. while, in both studied groups, concerning mmp-9, there was a significant difference between males and females p=0.049 as well as there were no significant differences between men and women regarding hba1c , glucose , sod and il-18 p > 0.05 (table 4). table (3): comparison between males and females concerning the focused parametersin patient group sex n mean ±sd p mmp-9 ng/ml men 25 2.512 1.840 .049 women 25 1.861 1.070 il-18 pg/ml men 25 70.906 36.060 .665 women 25 73.858 29.912 sod ug/ml men 25 367.226 128.068 .090 women 25 424.785 192.370 p< 0.05 table (4): comparison between males and females concerning the focused parameters in both groups sex n mean sd p age years men 47 55.936 8.641 0.979 women 48 55.979 7.141 weight kg men 47 74.213 6.504 0.339 women 48 72.833 7.433 hba1c % men 47 7.359 2.348 0.609 female 48 7.137 1.837 fbg mg/dl men 47 176.532 90.680 0.946 women 48 175.333 82.139 mmp-9 ng/ml male 47 2.409 1.840 0.049 women 48 1.794 1.070 il-18 pg/ml men 47 70.906 36.060 0.665 women 48 73.858 29.912 sod ug/ml men 47 367.226 128.068 0.090 women 48 424.785 192.370 p<0.005 group n mean ±sd se p age years patients 50 55.980 8.277 1.171 .977 control 45 55.933 7.500 1.118 weight kg patients 50 72.880 8.775 1.241 .337 control 45 74.222 4.188 .624 hba1c % patients 50 9.016 1.288 .182 < 0.001 control 45 5.281 .183 .027 fbg mg/dl patients 50 251.180 44.227 6.255 < 0.001 control 45 92.311 6.338 .945 mmp-9 ng/ml patients 50 2.632 1.745 .247 < 0.001 control 45 1.506 .945 .141 il-18 pg/ml patients 50 93.996 21.297 3.012 < 0.001 control 45 48.400 26.375 3.932 sod ug patients 50 301.684 97.041 13.724 < 0.001 control 45 501.447 162.781 24.266 iraqi j pharm sci, vol.26(1) 2017 antioxidant and inflammation in type ii diabetes mellitus 21 age effect there was a significant negative weak correlation between age and sod in patients group ( r= 0.35 , p <0.05), as much as the age increase the serum level of sod decrease, while there were a significant positive weak correlations between age and mmp-9 and il18 ( r= 0.28 ,p<0.05 ) ( r=0.26 , p<0.05) respectively (table 5). table ( 5): the correlation between the age with sod, mmp-9 and il-18 in diabetic patients x variable y variable r p value n age sod -0.35 <0.05 50 age mmp-9 0.28 <0.05 50 age il-18 0.26 <0.05 50 correlation coefficient correlation coefficient between hba1c and studied parameters there was a strong significant inverse correlation between hba1c and sod (p< 0.001) as much as hba1c increase sod decrease which showed more os (r = 0.88, p< 0.001). moreover, there was a strong significant positive correlation between hba1c and mmp-9 (r= 0.76, p< 0.001), as much as hba1c increased the serum level of mmp-9 increased. in addition , there was a strong significant positive correlation between hba1c and il-18 (r= 0.76, p< 0.001), as much as hba1c increased the serum level of il-18 increased (table 6). table (6): the relation between hba1c with sod, mmp-9, il-18 in diabetic patients y variable x variable r p n hba1c sod 0.88 <0.001 50 hba1c mmp-9 0.76 <0.001 50 hba1c il-18 0.76 <0.001 50 correlation coefficient between studied parameters in patients group. in patients group, there were a negative strong correlations between sod with il-18 and mmp-9 (r= 0.89, p ˂0.001; r=-0.7, p ˂0.001) respectively. in addition, there was a positive strong correlations between mmp-9 with il-18 ( r= 0.7, p ˂0.001) (table 7). table (7): correlations between the focused parameters in the patients group x variable y variable r p value n sod il-18 -0.89 <0.001 50 sod mmp-9 -0.70 <0.001 50 il-18 mmp-9 0.70 <0.001 50 discussion t2dm and its complications are becoming one of the most important health problems in erbil population. therefore, it is necessary to confirm the involvement of novel pathogenic way to provide a good chance for early diagnosis and therapy. the current results has exhibited that antioxidant enzyme sod and inflammatory markers mmp-9 and il-18 are useful biomarkers for differentiating diabetic patients from healthy individuals. the results were in harmony with the hypothesis of the current study that, there were a statistical relationship between the significant decreased in the serum level of sod and significant elevation of the serum levels of mmp-9 and il-18 with development of t2dm. this study has been detected the existence of inflammation with reduction of antioxidant status in patients with t2dm. the results also suggested that the reduction of the serum antioxidant level may be presented consequence as increased os associated with t2dm. effect of controlling the disease on the serum levels of selected parameters in uncontrolled patients with t2dm, the serum levels of hba1c%, glucose, mmp-9 and il-18 were significantly increased as compared with poor control and healthy individuals (table 1), while there was a significantly decreased in the serum sod level in uncontrolled patients with t2dm as compared with poor control and healthy individuals (table 1). the result of sod has been exhibited that, os is severe through uncontrolled stage of the disease which was concordant with the previous findings (4) . effect of the type 2 diabetes mellitus on the serum levels of selected parameters the statistical study has been revealed that there was a significant decrease in the serum sod level in patients with t2dm as compared with the control group (table 2). it has been published (3) that hyperglycemia may interfere with the defensive mechanism antioxidant enzymes which induces the generation of free radicals (frs). the statistical decrease in the serum sod level which was significantly related with iraqi j pharm sci, vol.26(1) 2017 antioxidant and inflammation in type ii diabetes mellitus 22 the development of t2dm and might be used as a necessary biomarker for diagnosis t2dm. the current study has been shown an elevated os as a consequence of overproduction of frs in patients with t2dm concomitant with the significant decreased in antioxidant enzymes such as sod which might be associated to the etiology of t2dm and has been used as an important parameters of antioxidant status imbalance and has an significant role in the occurrence of t2dm. the result of the present study was concordant with the previous findings (3,4) . the explanation for the reduction of antioxidant levels in patients with t2dm because of the that antioxidant enzymes may have been diminished as an attempt to counteract the lipid dna, protein and lipid damage. moreover, the increased lipid, protein and dna oxidation may be as a consequence of a weakened antioxidant defensive system (9) , sod is considered the first defensive mechanism line against reactive oxygen species (ros), and it is thought that the activity of sod might be affected firstly before other antioxidant enzymes as a result of os (10) . damaged the activity of sod is due to over production of superoxide –free radicle in addition to the increased of other ros and induces lipid peroxidation processes in diabetes (4) , moreover, hyperglycemia is associated with loss of cu 2+ , which is considered as an important cofactor in sod activity as well as the activity of sod is diminished by glycosylation in erythrocytes (10) . the current result indicated that there was a statistical increase in the serum mmp-9 level in patients with t2dm as compared with the control group (table 2). the result of the present study was consistent with the previous findings (6, 8-13) . the current study also focused the important role of mmp-9 to predict the development of t2dm, in addition, supported the relation between the statistical increase of the serum mmp-9 level with development of the t2dm. the explanation for this significant elevation in the serum level of mmp-9 in patients with t2dm was that, high blood glucose level directly or indirectly as a consequence of os and increase glycosylation of products like lipid or protein might elevate the level of mmp-9 and it , s activity in large blood vessels (14) , moreover, os may has a necessary role for the pathogenesis of t2dm as well as mmp-9 is activated as a consequence of the frs generation, which emphasize that antioxidants have a beneficial effects in t2dm therapy. it is widely accepted that t2dm is related with low grade chronic inflammation (15) . so, increased inflammation is considered as non-dependent risk factor for the development t2dm (16) . this study was designed to detect the il-18 as an inflammatory diagnostic parameter for t2dm. so, this study emphasized that inflammation is associated with increased level of il-18 by high blood glucose level. there was a statistical increase in the serum il-18 level as compared with healthy individuals (table 2) which was in harmony with the previous studies (8,17,18,) . according to the current study that, il-18 might be associated with the pathogenesis of the t2dm as well as the data supported the role of the inflammatory process in the pathophysiology of t2dm. so, this result supported the findings of the previous researches that t2dm might be considered as a low grade chronic inflammatory status. so, the high serum il18 level might expect the development of t2dm and there was a relation between a significant elevation of the serum il-18 level with the development of t2dm. gender –effect concerning mmp-9, there was a significant difference between males and females in patients group (table3). regarding sod and il-18, there were no significant differences between men and women in patients group (table3). while, in both studied groups, concerning mmp-9, there was a significant difference between males and females as well as there were no significant differences between men and women regarding hba1c, glucose, sod and il-18 (table 4). it has been published (4) and illustrated the association between genders and oxidant / antioxidant state, it has been recognized that, the sod was increased in non-diabetic healthy men compared with non-diabetic healthy women, while in diabetic state, sod concentration was slightly reduced but there was no statistical significant difference between diabetic men and diabetic women, so the study (4) , has been shown that diabetic patients regardless of the gender, were exposed to an elevated levels of ros through enhanced lipid peroxidation. age factor effect of aging on glucose serum level in type 2 diabetes mellitus t2dm is a common burden in the elderly (19) . aging is associated with alterations in body composition, which has implications for the development of insulin resistance and diabetes. these information supported the result of the current study that iraqi j pharm sci, vol.26(1) 2017 antioxidant and inflammation in type ii diabetes mellitus 23 elderly individuals are at a high risk to develop t2dm. the average age of patients in the current study at diagnosis was 55.98± 8.277years (table 2). moreover, the fining of the current study was in harmony with the result of the previous research (20) which has been reported that, the patients with t2dm group with the average age (60-70 years) their mean blood glucose level was 173.4±54.2 mg/dl. accordingly, there was an association between old age and t2dm. in addition, other study (21) has been emphasized that an elevated incidence of this disease in elderly subjects might be as result of the variations in the homeostatic pathway that regulates the inflammatory parameters such as creactive protein (crp), il-6, tumor necrosis factor-α (tnf-α) which are associated with the chronic low grade inflammation occurs in t2dm. effect of aging on studied parameters in type 2 diabetes mellitus there was a significant negative weak correlation between age and sod in patients group, as much as the age increase the serum level of sod decrease, while there were a significant positive weak correlations between age and mmp-9 and il-18 (table 5). similarly, (4) reported that, sod lowers in diabetic age group ≥50 years compared with age groups 30–39 and 40–49 years. so, the results of (4) indicated that os impacts the aging process that may be caused by a number of factors including increased free radical production, decreased antioxidant defense system, or a decreased removal or repair. correlation coefficient the current study reinforced the possibility that high blood glucose level might be associated with elevated level of mmp-9 and hba1c (table 6). there was a positive strong statistical relationship between hba1c and mmp-9 when the serum activity of mmp9 was increased the amount of hba1c was also increased. this result was consistent with the finding of the previous research (22). furthermore, the current result reinforced the possibility that high blood glucose might be associated with reduction in the level of sod. table 6 shows, that there was a strong significant negative correlation between hba1c and sod, as much as hba1c increase sod decrease which showed more os, this result was shown that antioxidant status are linked with glycemic control, which was consistent with previous result (23). indeed, there was a significant interaction between elevated glucose and serum il-18 level. data from the current study reinforced the possibility that high blood glucose level might be associated with elevated concentration of il-18, the data has been shown a positive strong statistical relation between hba1c and il-18, when the serum activity of il-18 was increased the amount of hba1c was also increased. in addition, in patients group, there were a negative strong correlations between sod with il-18 and mmp-9. in addition, there was a positive strong correlations between mmp-9 with il18 (table7). the current study was the first study in erbil population that investigated the combined effects of antioxidant status by investigating sod and inflammation by measuring mmp-9 and il-18 markers in t2dm risk prediction. variations in the serum concentrations of these biomarkers associated with occurrence of t2dm and it is important to supply diagnostic information which might help in the management and therapy of t2dm. conclusion the results of the current study detected a statistical decrease in the activity of the sod in patients with t2dm, accordingly, it was concluded that, t2dm is associated with overproduction of frs as a consequence of hyperglycemia as well as decreased the activity of the antioxidant enzymes like sod, which emphasized to supply the therapeutics with antioxidant products. measuring sod might be used a parameter to regulate the blood glucose level. in 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[epub ahead of print]. 17. zilverschoon gr, tack cj, joosten la, kullberg bj, meer jw, netea mg. interleukin-18 resistance in patients with obesity and type 2 diabetes mellitus. int j obes (lond) 2008; 32:140714. 18. klimontov vv, tyan nv, fazullina on, myakina ne, orlov nb, konenkov vi1. acute-phase serum proteins and adipocytokines in women with type 2 diabetes mellitus: relationships with body composition and blood glucose fluctuations. ter arkh. 2016; 88 (10): 35-41. 19. meneilly gs, tessier d. diabetes in elderly adults. j gerontol a biol sci med sci 2001; 56: m5-13. 20. yasmin r, majeed a, rashid a, razak s. the association of age with glycaemic and cholesterol control in patients with type 2 diabetes mellitus. j pak med assoc. 2017; 67(1): 336. http://www.ncbi.nlm.nih.gov/pubmed/24043671 http://www.ncbi.nlm.nih.gov/pubmed/24043671 iraqi j pharm sci, vol.26(1) 2017 antioxidant and inflammation in type ii diabetes mellitus 25 21. undurti n. das. the risk of type 2 diabetes mellitus in those with hypertension. eur heart j 2008; 29 (7): 95253. 22. krzysztof c. lewandowski, ewa banach, małgorzata bieńkiewicz, and andrzej lewiński . matrix metalloproteinases in type 2 diabetes and non-diabetic controls: effects of short-term and chronic hyperglycaemia. arch med sci. 2011; 7(2): 294– 03. 23. bigagli e, raimondi l, mannucci e, colombi c, bardini g, rotellacm and lodovici m. lipid and protein oxidation products, antioxidant status and vascular complications in poorly controlled type 2 diabetes. british journal of diabetes & vascular disease 2012; 12: 339. http://eurheartj.oxfordjournals.org/search?author1=undurti+n.+das&sortspec=date&submit=submit http://www.ncbi.nlm.nih.gov/pubmed/?term=lewandowski%20kc%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=banach%20e%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=bie%26%23x00144%3bkiewicz%20m%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=lewi%26%23x00144%3bski%20a%5bauth%5d http://www.ncbi.nlm.nih.gov/pubmed/?term=lewi%26%23x00144%3bski%20a%5bauth%5d study the effect of orally –administered calcium carbonate to iraqi j pharm sci, vol.20(1) 2011 orally administered calcium and mild pre-eclampsia 87 the effect of orally –administered calcium carbonate to pregnant women with mild pre-eclampsia sura sagban * , nada n. al-shawi **,1 , alia mohammad * , ashwak talib * and utoor hasson * *department of obstetrics and gynecology ,karbala hospital, karbala, iraq, ** department of pharmacology and toxicology, college of pharmacy ,university of baghdad , baghdad , iraq abstract pre-eclampsia is the most common medical complication of pregnancy associated with increased maternal and infant mortality and morbidity. its exact etiology is not known, although several evidences indicate that various elements might play an important role in pre-eclampsia. this study was carried out to analyze and to compare the concentration of calcium, in mild pre-eclampsia and in normal pregnant women , and to determine the effect of oral supplementation with calcium on mild pre-eclampsia , and whether this effect is related to the change in the level of serum calcium. forty five women in the third trimester of pregnancy were selected to participate in this study and divided into: fifteen apparently healthy, normotensive pregnant women served as a control group; thirty clinically diagnosed patients with mild pre-eclampsia ( 15 mild pre-eclamptic un-treated group , 15 mild pre-eclamptic treated with calcium carbonate 500 mg twice daily) , the serum calcium were estimated with an atomic absorption spectrophotometer .the data were analyzed using the unpaired student’s-test.the serum calcium in mild pre-eclmpatic un-treated group was significantly lower than that in normal pregnant women (8.84 ± 1.14 vs. 9.66 ± 0.87 , p<0.05) , serum calcium level significantly increased in mild pre-eclamptic treated with calcium carbonate 500mg twice daily as compared to mild pre-eclamptic un-treated group (9.76 ±0.96 vs 8.84±1.14 ,p<0.05) . systolic ,diastolic, and mean arterial blood pressure were significantly reduced after one month of treatment with calcium carbonate 500 mg twice daily as compared to mild pre-eclamptic un –treated group.(134.83 ± 7.5 vs139.33 ± 5.30, 88.46 ± 3.27 vs 91 ± 3.38, 103.90 ± 3.8 vs106.66 ± 3.08 ,p<0.05) respectively.this study showed that serum calcium level in mild pre-eclampsia are lower than in normotensive pregnant women ,this finding support the hypothesis that hypocalcemia is a possible etiology in pre-eclampsia ; additionally this study showed the possible beneficial effect of calcium supplementation in controlling pre-eclampsia and reducing blood pressure by increasing serum calcium level . key words: mild pre-eclampsia, calcium carbonate tablet, pregnant women, serum calcium الخالصة انطثٛح نهحًم يصاحثح نضٚادج انعشس ٔانًٕخ فٙ ٚؼذ يشض اسذفاع ظغػ انذو أٔ يشض عًذيٛح انحًم يٍ أكثش انًعاػفاخ االو ٔانطفم. الٚضال انغثة انشئٛظ نًشض عًذيٛح انحًم يجٕٓال تانشغى يٍ اٌ ُْانك اثثاذاخ ذذل ػهٗ اٌ يغرٕٚاخ انؼُاصش استَٕاخ انكانغٕٛو فٙ حانح صًًد ْزِ انذساعح نثٛاٌ انرأثٛش انًحرًم نًادج ك انًخرهفح قذ ذهؼة دٔسا فٙ اسذفاع ظغػ انذو نالو انحايم. عًذيٛح انحًم انثغٛػ ػهٗ يغرٕٖ انكانغٕٛو تانًقاسَح يغ رنك انرشكٛض فٙ انُغاء انحٕايم راخ انعغػ انطثٛؼٙ ٔنرحذٚذ ْزا انرأثٛش ٍ انحًم ٔذى ذى اخرٛاس خًظ ٔاستؼٌٕ ايشأج فٙ االشٓش انثالثح االخٛشج ي ٔػالقرّ ترغٛٛش فٙ يغرٕٖ انكانغٕٛو فٙ يصم انذو نهُغاء. ذقغًٍٛٓ انٗ ثالثح يجًٕػاخ: انًجًٕػح االٔنٗ: خًغح ػشش ايشأج حايم رٔاخ ظغػ دو غثٛؼٙ اػرثشٌ كًجًٕػح عٛطشج. ( ايشاج حايم 51انًجًٕػح انثاَٛح: ثالثٌٕ ايشاج حايم يصاتاخ تغًذيٛح انحًم انثغٛػ. قغًد ْزِ انًجًٕػح انٗ يجًٕػرٍٛ ) يهغى يٍ 155( ايشاج حايم يصاتاخ تغًذيٛح انحًم انثغٛػ اػطٍٛ جشػح 51انحًم انثغٛػ ، )شخصد حذٚثا تاصاترٍٓ تغًذيٛح تُٛد َرائج ْزِ ذى قٛاط يغرٕٖ انكانغٕٛو فٙ يصم انُغاء انحٕايم فٙ انًجًٕػاخ اػالِ . حثٕب كاستَٕاخ انكانغٕٛو يشذاٌ ٕٚيٛا. خ تغًذيٛح انحًم اقم تصٕسج يؼُٕٚح ػُذ يقاسَرٓا تانحًم انطثٛؼٙ انذساعح اٌ ذشكٛض يغرٕٖ انكانغٕٛو فٙ يصم انُغاء انًصاتا كًا تُٛد انذساعح اٌ يغرٕٚاخ ظغػ ٔانز٘ صاد ٔتصٕسج يؼُٕٚح تؼذ اعرؼًال كاستَٕاخ انكانغٕٛو نًشظٗ عًذيٛح انحًم انثغٛػ. يهغى 155رؼًال كاستَٕاخ انكانغٕٛو انذو االَقثاظٙ ، االَثغاغٙ ٔظغػ انذو انششٚاَٙ قذ اَخفط تصٕسج يؼُٕٚح تؼذ شٓش يٍ اع تُٛد ْزِ انذساعح . يشذٍٛ تانٕٛو تانًقاسَح يغ يجًٕػح انُغاء انحٕايم انًشخصاخ حذٚثا ػهٗ آٍَ يصاتاخ تغًذيٛح انحًم انثغٛػ نحٕايم رٔاخ ظغػ اٌ يغرٕٖ يصم انكانغٕٛو فٙ انُغاء انحٕايم انًصاتاخ تغًذيٛح انحًم انثغٛػ ْٕ أقم يٍ يغرٕاِ نذٖ انُغاء ا م انذو انطثٛؼٙ. اٌ انُرائج انرٙ ذى انحصٕل ػهٛٓا يٍ ْزِ انذساعح ذذػى انُظشٚح اٌ قهح يغرٕٖ انكانغٕٛو فٙ انذو قذ ٚكٌٕ انغثة انًحرً طشج ػهٗ فٙ عًذيٛح انحًم ، تاالظافح انٗ رنك، تُٛد ْزِ انذساعح انٗ انرأثٛش انًفٛذ ٔانًحرًم نًادج كاستَٕاخ انكانغٕٛو فٙ انغٛ يشض عًذيٛح انحًم انثغٛػ ٔذخفٛط ظغػ انذو ٔذحغٍٛ انرذْٕس انحاصم فٙ يغرٕٖ يصم انكانغٕٛو. 1corresponding author email : nadaalshawi @ yahoo.com received : 4/10/2010 accepted : 17/5/2011 iraqi j pharm sci, vol.20(1) 2011 orally administered calcium and mild pre-eclampsia 88 introduction preeclampsia is one of the most common causes of maternal and fetal morbidities and mortalities (1) . its incidence is 4-8% of pregnancies (2) .the patho physiological mechanism is characterized by failure of the trophoblastic invasion of the spiral arteries, leading to mal adaptation of maternal spiral arterioles, which may be associated with an increased vascular resistance of the uterine artery and decreased perfusion of the placenta (3) . however, the exact etiology of preeclampsia is still unknown. on the physiological basis, calcium plays an important role in muscle contraction and regulation of water balance in cells. modification of plasma calcium concentration leads to the alteration of blood pressure. the lowering of serum calcium and the increase of cellular calcium can cause an elevation of blood pressure in pre-eclamptic mothers. therefore, the modification of calcium metabolism during pregnancy could be one of the potential causes of preeclampsia (4,5). however, the role and status of serum calcium, is still being discussed. the aims of the present study were to measure serum levels of calcium in mild pre-eclamptic pregnancy and compared with normal pregnancy and to investigate whether the oral supplementation of calcium decrease the incidence of pre-eclampsia, control the blood pressure, and affecting the plasma level of calcium. methods fortyfive women in the third trimester of pregnancy attending the karbala hospital; department of obstetrics and gynecology were selected to participate in this study with age ranged between (20-45) years (mean 30.99±0.47). diagnosis was carried out according to who criteria (1) , which are bases on clinical, laboratory diagnostic measures to detect hypertension and proteinuria in all patients. these women were classified into: 1. fifteen healthy normotensive pregnant women( blood pressure 120/80) the mean gestational age ( 32.73 ± 2.49 ) weeks and mean age (30.46 ± 6.79 )years, mean systolic blood pressure( 115.33 ± 5.4) mmhg , mean diastolic blood pressure(78.66 ± 5.49)mmhg , mean arterial blood pressure (90.78 ± 4.22) mmhg . these pregnant women served as control group. blood pressure measurement and blood samples were taken every two weeks until the day of delivery. 2. thirty preeclamptic pregnant women in the third trimester of pregnancy, after blood pressure measurement and protein in urine assessment in addition to clinical and diagnostic measures this group can be classified into two groups a. fifteen pre-eclamptic women, their gestational age mean (31.6 ± .46) weeks , age mean (31.31 ± 5.89 )years, their mean systolic blood pressure (139.33 ± 5.30) mmhg ; mean diastolic blood pressure(91 ± 3.38) ; and mean arterial blood pressure(106.66 ± 3.08) .they served as mild pre eclamptic untreated control group . b. fifteen pregnant women with mild preeclampsia in the third trimester of pregnancy , they received calcium carbonate 500 mg twice daily . their mean gestational age ( 32.6 ± 1.88 ) weeks, mean age(32.13 ± 6.15 )years ,mean systolic blood pressure (140.83±2.60) mmhg, mean diastolic blood pressure (91.70 ± 2.85 )mmhg , mean arterial blood pressure (108.05±2.20)mmhg. blood pressure measurement and blood samples were taken every two weeks after starting the treatment until the day of delivery. mid stream urine was collected from women in a clean plastic tube, and utilized to perform a test for protein. venous blood samples were collected and their sera were isolated by centrifugation . measurement of calcium in serum by colorimetric method , which based on combination of calcium with reactant o cresolphthalein (o-cpc) complexon, to form a stable , colored reaction product .the developed colored is measured at 570 nm ; serum calcium levels were expressed as mg / dl . none of the women had cardiac, hepatic or renal dysfunction .and none had any obstetrical abnormalities (diabetes mellitus, rhesus immunization). none had essential hypertension. statistical analysis data were presented as mean ± sd . comparison of means of parameter tested between groups was performed by un-paired student’s t test and p<0.05 was considered as statistically significant. results the present study enrolled 45 pregnant women. the clinical characteristics of the participant shown in table 1. there were no statistical difference between mild preeclamptic un-treated group and normotensive control group for age and gestational period. the results showed that systolic, diastolic ,and mean arterial blood pressures were iraqi j pharm sci, vol.20(1) 2011 orally administered calcium and mild pre-eclampsia 89 significantly higher in mild pre-eclamptic untreated group when compared with the normal pregnant women , serum calcium levels in mild pre-eclamptic un-treated women were significantly lower when compared to normotensive pregnant controls(p<0.05). table 1 : clinical characteristics of the study population. variables normotensiv e pregnant controls n=15 mild preeclamptic untreated group n=15 maternal age(years) 30.46 ± 6.79 31.31 ±5.89 ns systolic b.p. (mmhg) 115 .33 ± 5.4 139.33 ±5.30* diastolic b.p. (mmhg) 78.66 ± 5.49 91 ± 3.38 * mean arterial b.p.(mmhg) 90.78 ± 4.22 106.66 ± 3.08* gestational age (weeks) 32.73 ± 2.49 31.6 ± 2.46 ns serum calcium (mg/d) 9.66 ± 0.87 8.84 ± 1.14* data are shown as mean ±sd ; *: p < 0.05 compared to normotensive control group; ns:no significant differences. figure 1 shows that in mild pre-eclamptic women, mean arterial blood pressure levels were inversely correlated with serum calcium level ( r = 0.811 ) (p<0.05). figure 1: relation between mean arterial blood pressure and serum calcium in mild pre-eclamptic patients. mild pre-eclamptic women treated with oral tablets calcium carbonate 500 mg tablets twice daily showed a significant decrease in the levels of systolic , diastolic -, and mean arterial blood pressures compared to mild preeclamptic untreated control group ( p < 0.05) as shown in table 2. the levels of systolic , diastolic and mean arterial blood pressures in mild pre-eclamptic treated with calcium carbonate twice daily showed a significant difference with the corresponding levels in normotensive controls (p< 0.05).as shown in table 2 and figures 2,3 and 4. table 2: systolic,diastolic, and mean arterialblood pressures in mild pre-eclamptic women treated with calcium carbonate (500mg tablets) compared to mild pre-eclamptic un-treated control group and normotensive pregnant control groups. data shown as mean ± sd ; values with nonidentical subscripts ( a, b, c ) within each parameter are significantly different (p < 0.05). p<0.05 r = 0.811 100 105 110 115 120 0 5 10 15m e a n a rt e ri a l b lo o d p re ss u re (m m h g ) serum calcium mg/dl systolic blood pressure mmhg diastolic blood pressure mmhg mean arterial blood pressure mmhg mild pre-eclamptic treated with calcium carbonate n=15 134.83±75 c 88.46±27 c 103.90 ±3.8 c mild pre-eclamptic un treated control n=15 139.33±5.3 b 91 ± 3.38 b 106.6±0.08 b normoten-sive pregnant control n=15 115 ± 5.49 a 78.6±5.49 a 90.87 ± 4.2 a iraqi j pharm sci, vol.20(1) 2011 orally administered calcium and mild pre-eclampsia 90 figure2:the effect of treatment with calcium carbonate 500 mg tablet on systolic blood pressure levels in mild pre-eclamptic women . figure 3: the effect of treatment with calcium carbonate 500 mg tablet on diastolic blood pressure levels in mild pre-eclamptic women. figure 4: the effect of treatment with calcium carbonate 500 mg tablet on mean arterial blood pressure level in mild pre-eclamptic women . figure 5: the effect of treatment with calcium carbonate 500 mg tablet in mild pre-eclamptic women on serum calcium level. table 3: serum calcium in mild pre-eclamptic treated with calcium carbonate 500 mg tablet compared to mild pre-eclamptic un-treated and normotensive control groups. normotensive pregnant control n=15 mild pre-eclamptic untreated control n=15 mild pre-eclamptic treated with calcium carbonate 500 mg tablet n=15 serum calcium (mg/dl) 9.66±0.87 a 8.84±1.14 b 9.76±0.76 a data shown as mean ± sd ; values with nonidentical subscripts ( a,b ) within each parameter are significantly different (p < 0.05). a significant increase in the serum calcium level were seen in mild pre-eclamptic women treated with calcium carbonate 500 mg tablet compared to pre-eclamptic un –treated control group ( p< 0.05 ) , the level of serum calcium were reached levels of corresponding normotensive pregnant control group as shown in table 3. and figure 5. iraqi j pharm sci, vol.20(1) 2011 orally administered calcium and mild pre-eclampsia 91 discussion it has been proposed that the pathophysiological processes in pre-eclampsia began with a reduction in placental perfusion (6,7) and, ultimately, placental ischemia and infarction (8). the resultant placental damage is believed to result in the release of a variety of placental factors (9) such as soluble fmslike tyrosine kinase (sflt1), the angiotensin ii type1 receptor autoantibody (at1-aa), and cytokines such as tumor necrosis factor (tnf)-α that generate widespread dysfunction of the maternal vascular endothelium (10) . which in turn resulted in enhance formation of factors such as endothelin, reactive oxygen species (ros), thromboxane, , and augmentation the sensitivity to vascular angiotensin ii, in addition, preeclampsia is also associated with the decreased formation of vasodilators such as nitric oxide (no) and prostacyclin (11) .these alterations in vascular function not only lead to hypertension but multiorgan dysfunction, especially in women with early onset preeclampsia (12). in the present study , in mild cases of preeclampsia showed an elevation in systolic ,diastolic ,and mean arterial blood pressures compared to normotensive control pregnancies (p< 0.05), table1.deficient or excessive levels of blood electrolytes and trace elements can be an adverse factor on human pregnancy. the results from many clinical studies demonstrated the relationship between the aggravation of the hypertensive complication of pregnancy and the change in the serum concentration of electrolytes ( 13-15) .in the present study, mean serum calcium levels in mild pre-eclamptic un-treated women were significantly lower than normotensive pregnant women (p< 0.05), table1. this finding is similar to the previous studies (16,17) , and is contradictory to others (18-20) , where no significant differences in serum calcium levels in pre-eclampsia were observed compared to normal pregnancy. furthermore, our study showed an inverse relationship between serum calcium level and mean arterial blood pressure in mild pre-eclamptic patients, figures 1.the biochemical mechanism responsible for the possible decrease in extracellular calcium and concomitant increase in intracellular calcium is presently unclear. it has been suggested that parathyroid hormone plays a crucial role in influencing cation transport (21) . it was postulated that, in preeclampsia, the defective placenta is unable to produce sufficient levels of 1,25 (oh)2 d, resulting in inadequate gastrointestinal calcium absorption, low ionized calcium levels, and a secondary rise in pth, which in turn may increase cytoplasmic ca +2 or alter the production of endothelium – derived vasoactive factors (31) . low calcium levels may also contribute to hypertension via stimulation of renin release from the kidney (22) . also the decreased serum total calcium concentration in preeclampsia may be an alteration of the plasma protein concentration (primarily albumin ) results in parallel changes in total plasma calcium (23) .it is widely accepted that vascular smooth muscle contraction is triggered by increases in intracellular free ca +2 concentration due to ca +2 release from the intracellular stores and ca +2 entry from the extracellular space (24,25 ). several studies have investigated the role of angiotensin ii as an agonist for receptor-mediated intracellular calcium transients in vascular smooth muscle (26) . these studies have consistently shown an increase of intracellular free calcium concentration in platelets and lymphocytes in response to stimulation with angiotensin ii and vasopressin in patients with pre-eclampsia (27) . in addition ,ang ii may enhance ca +2 entry through plasma membrane ca +2 channels (28) , furthermore, there is evidence that several ion-transport pathways are highly sensitive to oxidative stress, and the resulting modulation of ion transport by ros will affect ca +2 homeostasis (29) .treatment of mild cases of pre-eclampsia with calcium carbonate 500 mg tablet twice daily for one month resulted in a significant decrease in the level of systolic ,diastolic , and mean arterial blood pressure ( p< 0.05) ,table 2. figures 2,3,and4.. our findings were similar to those reported by others (30,31). calcium supplementation enhances vasodilation and reduces blood pressure (3,4) by suppression of the parathyroid hormone (21) , which in turn reduces the intracellular calcium concentration in vascular smooth muscle cells ,diminishing their responsiveness to pressure stimuli and reducing angiotensin ii sensitivity in women with pre-eclampsia (32) .however, several different mechanisms have been proposed by which ca supplementation could reduce blood pressure in pre-eclampsia. some have focused on neural ,humoral, and renal effects ,whereas others have attempted to relate the antihypertensive action of ca +2 supplementation to improved vascular function (33) . it has been thought that the improved vascular function following ca supplementation in experimental animals has been attributed to decreased α-adrenoceptor responsiveness (34,35) , reduced permeability of plasma membrane to ca and other cations (36) , improved function of cell membrane na-k atpase (37) , improved vasodilator function of the vascular endothelium ,and to increased iraqi j pharm sci, vol.20(1) 2011 orally administered calcium and mild pre-eclampsia 92 sensitivity of the smooth muscle no (38) .an interesting link between the intake and metabolism of calcium and the control of arterial tone may be the extracellular receptor , the activation of which cause vasorelaxation via the release of hyperpolarizing mediators (39). the results of this study showed that mild preeclamptic patients treated with calcium carbonate showed a significant increase in serum calcium level (p< 0.05) table 3. ,figure 5., and the result of increasing serum calcium is consistent with the others (40) which demonstrated that calcium supplementation for women with a low baseline calcium intake was associated with an increase in serum calcium concentration. thus calcium supplementation could have a meaningful impact on calcium metabolism regulation by maintaining serum calcium level within the narrow physiological range and reducing serum pth (21) . moreover ,when calcium is present in optimal concentration, it stabilizes vascular membranes, blocks its own entry into cells and reduces vasoconstriction (41) , calcium in combination with other ions such as na+, k+ 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sealey je, alderman mh divalent cations in essential hypertension. relations between serum ionized calcium, magnesium, and plasma renin activity. n engl j med 1983; 309:888–891. 23. howlader mz, tamanna s, parveen s , shekhar hu, alauddin m, begum f. superoxide dismutase activity and the changes of some micronutrients in preeclampsia. jms 2009;15: pp. 107-113. 24. khalil ra and van breemen c. sustained contraction of vascular smooth muscle: calcium influx or c-kinase activation? j pharmacol exp ther1988;244(2):537-542. 25. khalil ra and van breemen c. mechanisms of calcium mobilization and homeostasis in vascular smooth muscle and their relevance to hypertension. in: hypertension: pathophysiology, diagnosis, and management, edited by laragh jh and brenner bm. new york: raven press, 1995, p. 523-540. 26. seki t, yokoshiki h, sunagawa m, nakamura m, and sperelakis n. angiotensin ii stimulation of ca +2 channel current in vascular smooth muscle cells is inhibited by lavendustin-a and ly294002. pflügers arch 1999; 437(3):317-323. 27. haller h, oeney t, hauck u, distler a, philipp t: increased intracellular free calcium and sensitivity to angiotensin ii in platelets of preeclamptic women. am j hypertens 1989;2:238 –243. 28. loutzenhiser k and loutzenhiser r. angiotensin ii-induced ca +2 influx in renal afferent and efferent arterioles: differing roles of voltage-gated and storeoperated ca +2 entry. circ res 2000; 87(7):551-557. 29. steinert jr, wyatt aw, jacob r, mann ge. redox modulation of ca +2 signaling in human endothelial and smooth muscle cells in pre-eclampsia. antioxidants and redox signaling 2009; 11(5): 1149-1163. 30. villar j, abdel-hallem h, merialdi m, mathai m, ali mm, zavaleta n, et al. world health organization randomized trial of calcium supplementation among low calcium intake pregnant women. am j obstet gynecol. 2006; 194: 639-49. 31. villar j , belizán jm. same nutrient, different hypotheses: disparities in trials of calcium supplementation during pregnancy. american journal of clinical nutrition 2000; 71: 1375s-1379s. 32. moutquin j, md,garner pr, burrows rf, rey e, helewa me, lange ir, rabkin sw. report of the canadian hypertension society consensus conference: 2. nonpharmacologic management and prevention of hypertensive disorders in pregnancy. can med assoc j 1997; 157: 907-19. 33. hatton dc, yue q, mccarron da.mechanisms of calcium's effects on blood pressure .semin nephrol 1995;15:593-602. 34. peulerjd,morganda,mark l. high calcium diet reduces blood pressure in dahl salt sensitive rats by neural mechanisms .hypertention 1987;9:iii159iii165. 35. hatton dc, mccarron da.dietary calcium and blood pressure in experimental models of hypertention . areview. hypertention 1994;23:513-530. 36. arvola p, ruskoaho h, ,porsti i.effect of high calcium diet on arterial smooth muscle function and electrolyte balance in mineralcorticoid-salt hypertensive rats. brj pharmacol 1993a 108:948-990. 37. makynen h, kahonen m, arvola p ,wu x, wuorela h, porsti i.endothelial function in deoxycorticosterone-nacl hypertention :effect of calcium supplementation .circulation 1996; 93: 1000-1008. 38. bukoski rd,ishibashi k,bian k.vascular actions of calcium regulating hormones. sem nephrol 1995;15:536-549. 39. ishioka n,bukoski rd.a role for narachidonylethanlamine (anandamide) as a mediator of sensory nervedependent ca2+-induced relaxation. j pharmacol exp ther 1999;289:245-250. 40. lópez-jaramillo p, narváez m, weigel m and yépez r (1989). calcium supplementation reduces the risk of pregnancy induced hypertension in an iraqi j pharm sci, vol.20(1) 2011 orally administered calcium and mild pre-eclampsia 94 andean population. british journal of obstetrics and gynaecology 1989; 96: 648-655. 41. nieto a, herrera ja, , villar j, matorras r , la manzanara cl, iarribas i, álvarez j, peiro e. association between calcium intake, parathormone levels and blood pressure during pregnancy. colomb med. 2009; 40: 185-93. 42. ishioka n,bukoski rd.a role for narachidonylethanlamine (anandamide) as a mediator of sensory nervedependent ca2+-induced relaxation. j pharmacol exp ther 1999;289:245-250. iraqi j pharm sci, vol.20(2) 2011 fibromyalgia in iraqi women 34 calcium, magnesium and phosphorous levels in serum of iraqi women with fibromyalgia ali a. kasim* ,1 * department of clinical laboratory science, college of pharmacy,university of baghdad, baghdad, iraq. abstract fibromyalgia (fm) is a common, debilitating, and chronic pain syndrome. the women are more likely to have more tender points on examination than are their male counterparts. iraqi study showed that fm occur in 1.5% among adolescents of iraqi population. in compare to normal healthy women, present study was revealed that iraqi women with fm have significant elevation of calcium (p = 0.003) with significant reduction of magnesium (p = 0.001), whereas the inorganic phosphorous was not differs (p = 0.31). in conclusion, magnesium and calcium would play a crucial role in etiopathogenesis of fibromyalgia. key words: calcium, magnesium, phosphorous, fibromyalgia. الخالصة ّقاط يَسيذ ٍِ عرضة ى أمثراىْساء .و ٍسٍِ اىٌ ٍشترك, ٍْهلهي ٍتالزٍة ٍتالزٍة أىٌ اىييف اىعضيي اىفيبروٍيالخيا او ٍِ اىباىغيِ ٍِ ٪ 5,1 اىفيبروٍيالخيا تحذث في عراقية اُ دراسة وأظهرت .ّظرائهِ ٍِ اىرخاه ٍِ اثْاء اىفحص اىسريري اىىهِ باىَقارّة ٍع ّساء غير ٍصابات , اظهرت اىذراسة اىحاىية اُ اىْساء اىعراقيات اىَصابات باىفيبروٍيالخيا يظهرُ ارتفاعا . اىعراقييِ ( في اىَصو. بيَْا ىٌ يظهرُ اختالفا p = 0.001( واّخفاضا ٍعْىيا بَستىي اىَغْيسيىً )p = 0.003سيىً )ٍعْىيا بَستىي اىناى يَنِ االستْتاج ٍِ ّتائح اىذراسة اّه ٍِ اىََنِ أُ ينىُ ىيناىسيىً واىَغْيسيىً .(p = 0.31ٍعْىيا بَستىي اىفسفىر غير اىعضىي ) دورا هاٍا في اٍراضية اىفيبروٍيالخيا . introduction fibromyalgia (fm) is a common, debilitating, and costly chronic pain syndrome characterized by widespread pain, fatigue, disrupted sleep, depression, and physical deconditioning. the lifetime prevalence of fm in women is approximately 7% (1) . fm symptoms negatively impact patients’ quality of life and their ability to remain competitively employed. the current treatments impart minimal benefit on symptoms and long-term functioning (2-4) . the estimated annual direct cost of fm exceeds $20 billion (5) . diagnostic criteria of the american college of rheumatology (6) include widespread pain and specific tender points. patients often have other unspecific symptoms including fatigue, sleep disturbances, morning stiffness, anxiety and autonomic complaints such as irritable bowel and bladder syndromes (7) . the women are more likely to have more tender points on examination than are their male counterparts (8) . prevalence of fm in the rheumatology clinic is 20% (9) , and an iraqi study showed that fm occur in 1.5% among adolescents of iraqi population (10) . pathophysiological hypotheses of fm include impairment in the functioning of the hypothalamic–pituitary axis and alterations in neuromodulators and neurotransmitters such as substance p (sp), nerve growth factor (ngf), n-methyl-d aspartate, norepinephrine (ne) and serotonin (5-ht) (11-13) . however, fm etiology and pathogenesis remain elusive (14,15) . metals like calcium and magnesium compete with each other in modulating muscle contraction as well as in regulating many enzymatic reactions involved in energy metabolism, signal transduction and brain activity. results concerning levels of calcium and magnesium in fm are to some extent controversial; some authors observed reduced serum calcium and calcitonin levels in a small sample of fm patients (16) , while others report a normal range of plasma calcium and magnesium levels in patients affected by fm together with their reduced concentrations inside polymorphonuclear leukocytes (17) .inorganic phosphorus is critical for numerous normal physiologic functions including skeletal development, mineral metabolism, cell membrane phospholipid content and function, cell signaling, platelet aggregation, and energy transfer through mitochondrial metabolism (18) . 1corresponding author email : ali_a_kasim@yahoo.com received : 17/4/2011 accepted : 8/10/2011 iraqi j pharm sci, vol.20(2) 2011 fibromyalgia in iraqi women 35 higher concentrations of phosphodiesterase (pde) products and inorganic phosphate (pi) have been observed in fm patients in comparison to controls (19) , together with a decrease in phosphocreatine, atp and phosphocreatine/inositol phosphate (ip) ratio in the quadriceps (20) .within this study, it is fundamentally aimed to investigate serum calcium, magnesium and phosphorus level in iraqi women with fm. materials and methods twenty five iraqi women who were attending the rheumatology clinic of baghdad teaching hospital, were diagnosed as having fm according to the acr 1990 criteria (6) , and 25 normal healthy individuals (nhi) were included in this study. both groups are age and sex matched.in the laboratory investigation, serum magnesium, calcium and phosphorus were measured by endpoint colorimetric spectrophotometry. all reagents used were of analytical grade. the serum calcium, magnesium and inorganic phosphorus level were measured with kits of biomaghreb® (biomaghreb, tunis, tunisia). the principle of the study is based on measuring the absorbance of a colored complex at a specific wave length. absorbance is proportional to the analyte level (21) . statistical analysis microsoft office excel software 2007 for windows, was used for all statistical analysis. differences between patients and nhi were evaluated by the student’s t test. the level of significance was set at p value less than 0.05. results in present study, the age of patients in fm group and nhi were 41.7 ± 7.9 (mean ±sd) years and 40.1 ± 9.5 (mean ± sd) years, respectively. there was no significant difference between ages of the both groups (p = 0.465). about the serum level of minerals, there was a significant decrease in serum magnesium level (p <0.05) in fm group in compare to nhi one. also there is a significant increase in serum calcium level (p <0.05) in the fm group in respect to nhi group. in addition, the study was revealed no significant difference in serum level of inorganic phosphorus between the involved groups (p < 0.05), (see table 1 and figure 1). table 1: magnesium, calcium and phosphorus level [mean ±sd] in the fm and the nhi groups: mineral serum level (mg/dl) fm [mean±sd] nhi [mean±sd] p-value magnesium 2.1±0.83 * 4.17±2.41 0.001117 calcium 11.1±1.19 * 9.78±1.57 0.003288 phosphorus 5.1 ±1.66 4.71±2.52 0.311017 * significant changes in compare to normal healthy individuals. p < 0.05. * significant changes in compare to normal healthy individuals. p < 0.05. figure 1: magnesium, calcium and phosphorus level in fm and nhi groups discussion although disturbances in the musculoskeletal system, the neuro-endocrine system and in the central nervous system have been implicated in the pathophysiology of fm, a primary mechanism underlying the etiopathogenesis of fm is unknown (17) . serum trace element levels have been researched to reveal etiopathogenesis of patients with fm (18–20) . however, the results of these studies have appeared to be ambiguous.in this study, serum magnesium was found significantly lower and serum calcium was significantly * * 0 2 4 6 8 10 12 14 mg ca ph m in e ra l s e ru m l e v e l (m g /d l) nhi fms iraqi j pharm sci, vol.20(2) 2011 fibromyalgia in iraqi women 36 higher in patients with fm when compared to nhi, but the same difference was not observed between the two groups when calcium and inorganic phosphorus levels are considered. in contrast to our study; in past years, prescott et al. reported that serum magnesium level of patients with fm did not change, but eisinger et al. found decreased levels of red blood cell magnesium in patients with fm (20) . similarly, abraham and flechas also found that magnesium deficiency plays a possible role in mechanism of pain in fm. they thought this was related to the role of magnesium in the production of atp (18) . some evidence exists supporting the possibility of deficiency of components required for atp synthesis in etiopathogenesis of fm (11) . on the other hand, several fibromyalgia manifestations such as fatigue, muscle weakness, irritable bowel and paresthesia are similar to symptoms of magnesium deficiency (18) .magnesium plays an important role in enzymatic reactions, especially in energy production. as the magnesium level decreases, the energy levels also decrease simultaneously. as a result of this situation, fatigue may occur (22) . magnesium supplementation may benefit in the treatment of chronic fatigue (22, 23) . magnesium is nature’s physiologic calcium blocker. it can compete with calcium for binding sites on proteins and membranes (24) . we can suppose that subjects with a diagnosis of fm have a scarce muscle fiber capacity to repair, presumably due to altered monoamine transmission and/or hormonal changes (25) , which, in turn, can provoke a reduced homeostasis of atp, magnesium and calcium inside cells. the increase in muscle intracellular calcium level may produce myofiber damage through calcium activated proteolytic enzyme activity (26) and cause persistent muscle contraction without electromyographic activity. this uses adenosine triphosphate/phosphorylcreatine, depletes energy stores (27) , and may result in muscle weakness. lund-olesen and lundolesen (28) put forward a similar hypothesis to explain the pathogenesis of chronic fatigue syndrome/fibromyalgia. they hypothesized that calcium channels of striated muscle cells in the conditions are injured and that an increased amount of calcium is permitted to enter the cells. this results in increased muscular tone. the association between these two elements and clinical parameters makes us to think that these elements may play role in etiopathogenesis of fm. further investigations are required about trace elements and fm etiopathogenesis. references 1. wolfe f, ross k, anderson j, russell ij, hebert l. the prevalence and characteristics of fibromyalgia in the general population. arthritis rheum 1995;38(1):19-28. 2. bennett rm. fibromyalgia and the disability dilemma. a new era in understanding a complex, multidimensional pain syndrome. arthritis rheum 1996;39(10):1627-34. 3. jones kd, burckhardt cs, clark sr, bennett rm, potempa km. a randomized controlled trial of muscle strengthening versus flexibility training in fibromyalgia. j rheumatol 2002;29(5):1041-8. 4. wolfe f, anderson j, harkness d, bennett rm, caro xj, goldenberg dl, et al. work and disability status of persons with fibromyalgia. j rheumatol 1997; 24(6) :1171-8. 5. thorson k. the fibromyalgia problem. j rheumatol 1998;25(5):1023; author reply, 1028-30. 6. wolfe, f., smythe, h.a., yunus, m.b., bennett, r.m., bombardier, c., goldenberg, d.l., tugwell, p., campbell, s.m., abeles, m., clark, p., et al., 1990. the american college of rheumatology 1990 criteria for the classification of fibromyalgia. report of the multicenter criteria committee. arthritis rheum 1990;33, 160–172. 7. thompson, m.e., barkhuizen, a. fibromyalgia, hepatitis c infection, and the cytokine connection. curr pain headache rep 2003,7, 342–347. 8. haynes bf, fauci as. disorder of immune system connective tissue and joint; kasper dl, fauci as, lon go dl, braunwald e, hauser sl, jameson jl, (eds), harrison's principles of internal medicine, (16th ed), mcgraw-hill companies inc, 2005;1907-2066. 9. freundlich b, leventhal l. diffuse pain syndrome; kipple jh, weyand cm, wartman rl, (eds), primer on the rheumatic disease, (11th ed), atlanta, georgia, arthritis foundation 1997;123-7. 10. al-rawi zs, kamil ah. prevalance of fibromyalgia syndrome among school children and aldoscents in iraq. arthritis rheum 2000; 435:212. 11. neeck g, crofford lj. neuroendocrine perturbations in fibromyalgia and chronic fatigue syndrome. rheum dis clin north am 2000;26:989–1002. 12. bazzichi l, giannaccini g, betti l, et al. alteration of serotonin transporter density iraqi j pharm sci, vol.20(2) 2011 fibromyalgia in iraqi women 37 and activity in fibromyalgia. arthritis res ther 2006;8:r99. 13. bazzichi l, rossi a, giuliano t, et al. association between thyroid autoimmunity and fibromyalgic disease severity. clin rheumatol 2007;26: 2115– 20. 14. kahn mf. fibromyalgie: où en est-on? rev prat 2003;53: 1865–72. 15. sarzi-puttini p, buskila d, carrabba m, et al. treatment strategy in fibromyalgia syndrome: where are we now? semin arthritis rheum 2008;37(6):353-65. 16. neeck g, rieder w. thyroid function in patients with fibromyalgia syndrome. j rheumatol 1992;19:1120–2. 17. magaldi m, moltoni i, biasi g, et al. changes in intracellular calcium and magnesium ions in the physiopathology of the fibromyalgia syndrome. minerva med 2000;91:137–40. 18. chen h.h. calcium and phosphate metabolism management in chronic renal disease,springer science&business media;2006, pp13. 19. sprott h, rzanny r, reichenbach jr, et al. 31p magnetic resonance spectroscopy in fibromyalgic muscle. j rheumatol 2000;39:1121–5. 20. park jh, phohmat p, oates ct, et al. use of p31 magnetic resonance spectroscopy to detect metabolic abnormalities in muscles of patients with fibromyalgia. arthritis rheum 1998;41:406–13. 21. arneson. w. & brickell j. : clinical chemistry :a laboratory perspective.f. a. davis company, philadelphia, usa. 2007. 22. keenoy bm, moorkens g, vertommen j, noe m, neve j, de leeuw i : magnesium status and parameters of the oxidant– antioxidant balance in patients with chronic fatigue: effects of supplementation with magnesium. j am coll nutr 2000;19(3):374–382. 23. clague je, edwards rht, jackson mj: intravenous magnesium loading in chronic fatigue syndrome. lancet 1992:340:124– 125. 24. john db and leslie mk: clinical nutrition of the essential trace elements and minerals-the guide for health professionals, humana press, new jersey,2000 ; p 75. 25. bagge e, bengtsson b, carlsson l, et al. low growth hormone secretion in patients with fibromyalgia — a preliminary report on 10 patients and 10 controls. j rheumatol 1998;25:145–8. 26. armstrong rb, warren gl, warren ja. mechanisms of exerciseinduced muscle fibre injury. sports med 1991;12:184-207. 27. soybel dj, morgan j, cohen l. calcium augmentation of enzyme leakage from mouse skeletal muscle and its possible site of action. res commun chem pathol pharmacol 1978;20:317-29. 28. lund-olesen lh, lund-olesen k. the etiology and possible treatment of chronic fatigue syndrome/fibromyalgia. med hypotheses 1994;43:55-8. http://www.amazon.com/clinical-nutrition-essential-elements-minerals/dp/0896035980/ref=sr_1_1?s=books&ie=utf8&qid=1300452428&sr=1-1 http://www.amazon.com/clinical-nutrition-essential-elements-minerals/dp/0896035980/ref=sr_1_1?s=books&ie=utf8&qid=1300452428&sr=1-1 http://www.amazon.com/clinical-nutrition-essential-elements-minerals/dp/0896035980/ref=sr_1_1?s=books&ie=utf8&qid=1300452428&sr=1-1 http://www.amazon.com/clinical-nutrition-essential-elements-minerals/dp/0896035980/ref=sr_1_1?s=books&ie=utf8&qid=1300452428&sr=1-1 iraqi j pharm sci, vol.20(1) 2011 acs and inflammatory markers 43 the role of some inflammatory markers (il-6 and crp) in the pathogenesis of acute coronary syndrome in iraqi ccu for heart diseases nadia gh. abdulkarim* ,1 , mohamed a. taher** and amal n. almarayati*** *ministry of health, kimadia, iraq . **department of clinical laboratory sciences ,college of pharmacy, baghdad university, baghdad, iraq. ***the iraqi center for heart disease ,baghdad, iraq. abstract in this work an enzyme linked immunosorbent assay (elisa) technique has been used for detection of some inflammatory markers in serum of acute coronary syndrome (acs)-patients admitted to the cardiac care unit (ccu) of iraqi centre for heart diseases and ibn alnafees teaching hospital. the present method includes quantitative measurement of interleukine-6 (il-6) and creactive protein (crp), as their increase during symptoms may be responsible for identifying the mechanism of myocardial damag, in addition to their best performance than other quantitative tests perhaps due to their association with atherosclerotic process that belongs to the endothelial dysfunction. aim of this study is to estimate the prevalence and correlation of il-6 with crp in acs patients presented with unstable angina/ non-st elevation myocardial infarction (ua/nstemi) symptoms to be as new diagnostic parameters in iraqi ccu. seventy (70) acspatients with mean age (58.55 year ± 9.98), from jun.2009 to feb. 2010 with diagnosis of ua/nstemi were included in this study. proper history, physical examination, electrocardiograph (ecg), and echocardiography (echo) were performed for all patients in addition to the routine laboratory works including fasting blood glucose, lipid profile, assay of transaminases activity (aspartate and alanine transaminase),and biomarkers analysis as cardiac troponin i and t, creatine kinase (ck and ck-mb) and myoglobin. blood sample was collected from all patients for quantitative assay of il-6 and crp. all patients underwent diagnostic coronary angiography, were 66 of them with abnormal coronary outcome and four patients have normal coronary arteries, study include 39male and 31 female. seventeen of 67 patients (25.4%) had elevated serum level of il-6 and fifty four of 62 patients (87.1%) had elevated serum level of crp. statistically found strong and significant (ss) correlation between il-6 and crp (assessed by spearman's rho correlation coefficient, p<0.01). the significant proportion of ua/nstemi patients that had elevated serum levels of il-6 and/or crp, in addition to the strong correlation with coronary angiographic findings make these inflammatory markers to be considered as risk stratification factors and good predictors for coronary artery disease independent of other traditional risk factors for cardiovascular disease (cvd). key words: acs. il-6. crp. الخالصة حى فٍ هزا انعًم اسخعًال اخخباس االيخظاص انًُاعٍ اإلَضًٍَ نهكشف عٍ بعغ انًؤششاث االنخهابُت فٍ أيظال يشػً )أو 4-انحشَك انخهىٌيخالصيت انششاٍَُ اإلكهُهُت انحاد انشاقذٍَ فٍ وحذاث انعُاَت بانقهب. واشخًهج انذساست عهً انطشق انكًُت نقُاط حُذ إٌ صَادحهى بانذو أرُاء حذود األعشاع يحخًم أٌ َكىٌ بشوحٍُ انطىس انحاد )أو بشوحٍُ سٍ انفعال(، انبٍُ ابُؼاػٍ( و انًسؤول عٍ انخعشَف بانُت انؼشس بعؼهت انقهب، هزا باإلػافت إنً أدائهى انًفؼم عٍ بقُت االخخباساث انكًُت ورنك نعالقخهى بخظهب بشوحٍُ سٍ انفعال و 4-شاٍَُ انزٌ َعىد انً االخخالل انىظُفٍ نهبطاَت. انهذف يٍ انذساست نخقذَش اَخشاس واسحباؽ انحشَك انخهىٌانش -stواحخشاء انعؼهت انقهبُت انغُش يظحىبت باسحفاعيخالصيت انششاٍَُ اإلكهُهُت انحاد يع أعشاع انزبحت انغُش يسخقشة فٍ يشػً segment ِاالخخباساث كأدواث حشخُض جذَذة فٍ وحذاث انعُاَت بانقهب فٍ انعشاق. شًهج انذساست سبعىٌ يشَؼا" نخكىٌ هز . حى اخز انخاسَخ انًشػٍ وإجشاء 9000ونغاَت شباؽ 9002ابخذاء" يٍ حضَشاٌ (year ± 9.98 58.55)بانًخالصيت يعذل أعًاسهى انحشَك ؾ طذي انقهب نهًشػً. عُُاث انذو جًعج يٍ كافت انًشػً نقُاط االخخباساث انفُضَائُت، حخطُؾ كهشبائُت انقهب وحخطُ بشوحٍُ سٍ انفعال. جًُع انًشػً خؼعىا نهقسطشة اإلكهُهُت انخشخُظُت انخٍ أظهشث َخائج غُش ؽبُعُت نهششاٍَُ و 4-انخهىٌ %( يٍ 93.2يشَغ ) 01أَزً. 12كش ور 10( يشَغ و أسبعت يشػً نذَهى ششاٍَُ إكهُهُت ؽبُعُت. شًهج انذساست 44االكهُهُت ل) 4 -بُكىغشاو/يم كُقطت قطع السحفاع يسخىي انحشَك انخهىٌ 93)اعخبش انخشكُض 4 -انحشَك انخهىٌنذَهى اسحفاع بًسخىي 41اطم طع يهغى/نخش كُقطت ق 1.11بشوحٍُ سٍ انفعال )باعخباس انخشكُض نذَهى صَادة بًسخىي 49%( يٍ اطم 11.0يشَغ ) 32بانذو( و ( p<0.01بشوحٍُ سٍ انفعال )و 4-انحشَك انخهىٌبشوحٍُ سٍ انفعال بانذو(. احظائُا" وجذ اسحباؽ يعُىٌ قىٌ بٍُ يسخىي نضَادة يشَغ روٌ اإلطاباث اإلكهُهُت. انُسبت انًعُىَت يٍ يشػً انزبحت انغُش يسخقشة واحخشاء 44نعًىو يشػً انًخالصيت وكزنك بٍُ ال بشوحٍُ سٍ انفعال و/ او 4-انحشَك انخهىٌانزٍَ نذَهى صَادة بًسخىَاث st-segmentيظحىبت باسحفاع انعؼهت انقهبُت انغُش واسحباؽهى انقىٌ بُخائج انقسطشة اإلكهُهُت َجعم هزِ انًؤششاث االنخهابُت كعىايم خطىسة وحىقع جُذة أليشاع انششاٍَُ اإلكهُهُت أيشاع انقهب انىعائُت. يسخقهٍُ عٍ عىايم انخطىسة انًخعاسف عهُها فٍ 1 corresponding author email : nadiaph.qusai@yahoo.com received : 31/8/2010 accepted : 12/3/2011 iraqi j pharm sci, vol.20(1) 2011 acs and inflammatory markers 44 introduction acs is "any group of clinical symptoms compatible with acute myocardial ischemia," which includes ua (angina is coronary insufficiency and an important distinction between stable angina and ua is that the former is exacerbated by activity or emotional stress and relieved by rest and/or nitroglycerin; in contrast, ua occurs at rest) and myocardial infarction (mi), with or without stelevation (nstemi is associated with myocardial necrosis and resultant release of cardiac biomarkers and the ecg usually shows stsegment depression or new t-wave inversion; in contrast, stemi, which was once characterized as q-wave mi, is associated with myocardial damage, with both elevated serum cardiac biomarker levels and st-segment elevation on ecg [1,2,3] ) [4] . the differences between ua and mi in prevalence, etiology, severity, pathophysiology, clinical presentation, treatment, and outcomes [5] . patients presenting with symptoms that are consistent with an acs require urgent evaluation because these conditions carry a high risk of avoidable complications, such as sudden death and mi [6] . it caused by one of two events: rupture of the fibrous cap of an atheromatous plaque or endothelial (intimal) erosion of the cap, both of which lead to the subsequent development of a thrombus and decreased myocardial perfusion [7-10] , but research has shown that inflammation plays a central role [8,11,12] . as shown in figure(1) demonstrating that causative agents of necrosis are the risk factors which play a great role in the inflammatory response. the risk markers that are indicative of an adverse prognosis include recurrent ischemia, extensive ecg changes at rest or during pain, the release of biochemical markers (creatine kinase or troponin) arrhythmias and hemodynamic complications during episodes of ischemia. risk stratification is important because it guides the use of more complex pharmacological and interventional treatment. in those patients with ua and non-q-wave mi, early diagnosis results in the admission of these patients to the ccu or to a monitored bed in a higher dependency area [13] .increasing age is associated with a significant increase in adverse outcomes in patients with ua/nstemi. diabetic patients with ua/nstemi are approximately 50% higher risk than non diabetics. patients with extra cardiac vascular disease (i.e., those with either cerebrovascular disease or peripheral arterial vascular disease) also appear to have approximately 50% higher rates of death or recurrent ischemic events [14] .cardiac cath. provided for detailed assessment of the anatomy and physiology of the heart and vasculature and to determine the nature and extent of a suspected cardiac problem in a symptomatic patient in whom surgical, electrophysiologic, or interventional therapy is anticipated. also coronary angiography is the most widely used imaging tool to evaluate luminal, obstructive disease in coronary arteries, but the modality is not optimal for identifying plaque [15] .the function that cytokines induce can both turn on or turn off particular immune responses [16] . il-6 is an 'upstream' pro-inflammatory cytokine that stimulates hepatocytes to synthesize acute phase response proteins such as c-reactive protein (crp) and fibrinogen [17,18] . kumari and his colleague propose a role for il-6 in the pathogenesis of coronary heart disease (chd) through a combination of autocrine, paracrine and endocrine mechanisms at which an autocrine and paracrine activation of monocytes by il-6 in the vessel wall contribute to the deposition of fibrinogen. il-6 decreases lipoprotein lipase activity and monomeric lpl levels in plasma, which increases macrophage uptake of lipids [19] . lindmark et al agreed with manginas that il-6 is a strong independent marker of increased mortality in ua patients, as il-6 with both proand anti-inflammatory mediators affecting both b-cell immunoglobulin production and tcell cytotoxic activity, and this indicates a possible role for il-6 in progression of coronary artery disease (cad) [20,21] .the local inflammatory response is accompanied by a systemic response known as the acutephase response. crp is a prototype positive acute phase protein whose serum level increase 1000fold during an acutephase response. a calcium-binding pentameric protein consisting of five identical, non-covalently linked, 23kda subunits [22,23] . its name for its ability to bind and precipitate the cpolysaccharide of pneumococcus. crp is expressed in atherosclerotic plaque [24] and may enhance expression of local adhesion molecules [25] , increase expression of endothelial plasminogen activator inhibitorone (pai-1), reduce endothelial nitric oxide bioactivity [26] , and alter low density lipoproteincholesterol (ldl-c) uptake by macrophages [27] . crp has been found within thin cap atheromas and immunohistochemical deposition of crp within plaques corroborates the concept that inflammation is an important component to plaque instability reflected by serum crp [28] . accordingly, elevated crp levels could be linked to increased long-term mortality [29] . http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true 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http://www.netce.com/coursecontent.php?courseid=551&works=true iraqi j pharm sci, vol.20(1) 2011 acs and inflammatory markers 45 also elevated levels of il-6, crp and matrix metalloproteinases (mmp-9) provide a link between inflammation, matrix degradation and the development and progression of acs. il-6 and mmp-9 appear to be released mainly from vulnerable plaque or necrotic myocardium during the acute phase of mi [30] . figure 1: events of acs (41) subjects, materials and methods subjects and methods seventy acsin hospital stay patients selected from multicenters in the period from june 2009 to february 2010. selection of acspatients (excluding ami with st elevation) by specialized physician depending on risk factors of ischemic heart disease, previously or newly diagnosed acs, ecg changes and echo findings of ischemic heart disease.risky patients diagnosed according to the widely used thrombolysis in myocardial infarction (timi) risk score, which include seven factors: (1) age 65 years or older, (2) at least three of the standard risk factors for coronary disease, (3) prior coronary stenosis of 50% or more, (4) st segment deviation on the presenting ecg, (5) at least two anginal episodes in the previous 24 hours, (6) use of aspirin in the previous week, and (7) elevated serum cardiac markers [31] .the mean age for 70patients was (58.55 ± 9.98 year) with age range (3785 year), were 31 females representing (44.3%) and 39 males (55.7%). most patients take medications upon admission to the hospital. in addition to the group of patients this study include eighteen apparently healthy control subjects their mean age was (33.27 ± 6.73 year) ranging (25 47 year), were 5 females (27.8 %), and 13 males (72.2 %), choosing this range of age because the inflammatory processes have been implicated in a diverse set of chronic conditions affecting older adults, ranging from depression, periodontal disease, pulmonary disease, osteoporosis, arthritis and cognitive impairment [32] .ten ml of venous blood specimen were collected from each patients and healthy subject, centrifuged at 3000 rpm for 10 minute, serum was separated within 30 minute from the time of blood collection, it http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true http://www.netce.com/coursecontent.php?courseid=551&works=true iraqi j pharm sci, vol.20(1) 2011 acs and inflammatory markers 46 was free of haemolysis, no turbidity and not lipemic. materials il-6 and crp elisa-kit (from drg international inc., usa/ germany) is an in vitro sandwich type assay for the quantitative measurement of human il-6 and crp in serum, plasma, urine and culture supernatants. the principle of the assay is based on a solid phase enzymelinked immunosorbent assay. the laboratory investigations was performed in the research centre for biotechnology and the specialized centre of endocrinology and diabetes. statistical analysis the results were expressed using software statistical package for social science (spss) version 11.5 and 12. for the estimation of result's differences between the studied groups consider the (s) at p < 0.05, 0.01.also by using special elisa software (4.01 biorad lab., inc.) to draw the standard curves of: il-6 and crp. results and discussion traditional risk factors of this study revealed the following data: smoking (7 (10%) smokers and 7(10%) previously smoking), p<0.01; hypertension (53(75.7%) hypertensive, p<0.01); diabetes mellitus (34(48.6%) diabetic, p>0.05) ; 28 patients have both dm and hypertension; family history of cardiac disease (47(67.1%), p<0.01. also found 66 patients with abnormal angiographic outcome and only four acs-patients with normal cath. findings. data of table (1) shows significant differences in the serum levels of il-6 and crp (p<0.05, 0.01 respectively) between acs-patients and apparently healthy persons. table (2) reveal the prevalence of elevated levels of crp in the serum of acs-patients having positive risk factors (positive family history of cardiac diseases, smokers, diabetic, and hypertensive patients); while the number of patients with positive risk factors that have increased level of il-6 was less in comparison to those with normal levels of il-6 this may be due to the usage of some medications by the patients (as anti-inflammatoryaspirin and clopidogrel, antihyperlipidemicstatines and fibrates, antihypertensive angiotensine converting enzyme inhibitor (acei) and hypoglycaemic agents) that causes these differences in the serum levels of il-6 in relation to the risk factors, or may be due to the release of crp immediately in the acute state while the optimal time for measuring cytokines is unknown because of the short half life of the cytokines in addition this findings indicate that the association between il-6 levels and future cardiovascular events was independent of major cardiac risk factors; also table (2) demonstrate that seventeen of 67 patients (25.4%) had elevated level of il-6 (25 pg/ml is the cutoff point for il-6 representing mean serum level of il-6 ± s.d in apparently healthy control and any level more than this value considered elevated) and fifty four of 62 patients (87.1%) had elevated level of crp (3.83 mg/l is the cutoff point for crp representing mean serum level of crp ± s.d in apparently healthy control and any level more than this value considered elevated) were 50 patients with abnormal cath. outcomes have elevated level of crp this results provides prognostic value to the crp in addition to the traditional cardiac risk factors. therefore, in a high-risk patient, an elevated crp level should even further alert both the physician and the patient to the need for aggressive risk-lowering strategies [33] so the aha/cdc recommends measuring crp levels in patients who appear to have a moderately elevated risk of cardiovascular events. in these patients, an elevated crp measurement would indicate that the risk may very well be much greater than "moderate." in the present study also found (ss) correlation between il-6 and crp (assessed by spearman's rho correlation coefficient, p<0.01) this (ss) correlation was also documented by biasucci et al stating that elevated crp in the plasma of most ua refer to the important role of an inflammation in acs and has indirect mark for increased il-6 production [34] . in addition this strong correlation between the inflammatory markers may refer to the underlying cause of this type of cardiac disease which is stated by many studies one of them is the results of hashmi and his colleague showing increased concentrations of the proinflammatory cytokines il-17, il-6, il-8 and crp, these findings point towards the role of inflammation in the form of increased activity of those interleukins in an ua and ami patients [35] . furthermore, elevated crp levels predict recurrent ischemia and death in patients with stable and ua [36-38] , this is because crp plays a direct pathogenic role in arterial disease [39] iraqi j pharm sci, vol.20(1) 2011 acs and inflammatory markers 47 table 1: demography of the inflammatory markers in the studied groups ** ftest and anova significance < 0.01 *significance < 0.05 table 2: correlation between the inflammatory markers and the risk factors in acs-patients *normal reference value of crp: 0.068-8.2mg/l (40) conclusion the significant proportion of patients with ua/nstemi that had elevated serum levels of il-6 and crp, and their strong correlation with coronary angiographic findings make these markers to be considered as risk stratification factors and good predictors for coronary artery disease independent of other traditional risk factors for cardiovascular disease. acknowledgment great thanks to dr. khalid al-lihaibi from the specialized centre for endocrinology and diabetes, ms. ghasaq from the research centre for biotechnology, the medical and lab. staff of iraqi center for heart disease, ms. salima and mrs. wafaa from college of pharmacy/ baghdad universityphysics and computer lab., and to all prof, teachers and functionaries of pharmacy college/ baghdad university. references 1. smith s, whitwam w. acute coronary syndromes. emerg med clin north am. 2006;24:53-89. 2. bhatheja r, mukherjee d. acute coronary syndromes: unstable angina/non-st elevation myocardial infarction. crit care clin. 2007;23:709-735. 3. van horn s jr, maniu cv. management of non-st-segment elevation myocardial infarction. med clin north am. 2007;91:683-700. 4. anderson j, adams cd, antman em, et al. acc/aha guidelines for the management of patients with ua/ nstemi. circulation 2007;116:803-877. 5. cannon cp, braunwald e. the spectrum of myocardial 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evaluation of oral disintegrating tablets of ketoprofen by dirct compression saddam j.nasser *,1 ,laith h. sameen * , mowafaq m.ghareeb * *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract ketoprofen is a non-steroidal anti-inflammatory (nsaid) drug with analgesic, antiinflammatory, and antipyretic effects. it is widely used in the treatment of inflammation and pain associated with rheumatic disorders such as rheumatoid arthritis, osteoarthritis, and in soft tissue injury. the purpose of this study was to prepare an oral disintegrating tablets of ketoprofen by simple method. the tablets were prepared by direct compression method and different ratios of various subliming agents or superdisintegrants were incorporated. then these tablets were evaluated for hardness, friability, weight variation, water absorption ratio, disintegrating time and dissolution time. the results showed that formula f11 batch had short disintegration time (18.0 sec) with good physical properties. this formula demonstrated a promised potentials for rapid absorption, improved bioavailability, effective therapy and patient compliance. key words: rapid disintegrating tablets, ketoprofen, direct compression, superdisintegrants. تحضير وتقييم حبت الكتوبروفين سريعت التفكك بالفم بطريقت الكبس المباشر صدام جمعت ناصر ،*1 * ، موفق محمد غريب*ليث حمزة سمين و ، بغذاد ، انؼراقبغذاد تجايؼ ، تانصٍذنتكهٍ ، انصٍذالٍَاثفرع * الخالصة افط نهحرارة . اَه شائغ االسخؼًال بؼالج دواء انكٍخىبروفٍٍ هى يضاد انخهاباث غٍر سخٍروٌذي يغ اثر يسكٍ وخ االنخهاباث واالالو انًصاحبت اليراض انًفاصم كانرثىي وانخهاب انؼظاو وجروح االَسجت انرخىة . هذف انذراست هى ححضٍر يىاد وحراكٍس حبىب سرٌؼت انخفخج بانفى بذوٌ اسخؼًال انًاء بطرٌقت انكبس انًباشر انبسٍطت وباسخؼًال ػذة ػهى شكم انكٍخىبروفٍٍ خضؼج انحبىب انًحضرة انى ػذة اخخباراث يُها انصالبت وانهشاشت واخخالف يخخهفت يٍ انًىاد انًخسايٍت ويسبباث انخفخج انًخطىرة. w/w( انًخكىَه يٍ حركٍس ) 11انىزٌ وَسبت ايخصاص انًاء ووقج انخفخج وانخحهم . أثبخج انُخائج اٌ انخركٍبت )ف 11%(crospovidone ٍس )وحركw/w 11% (microcrystaline cellulose ph(102)( ثاٍَت يغ 1..1نها اقصر وقج حفخج) .خىاص فٍسٌاوٌت جٍذة . وهً حركٍبت واػذة نسٌادة االيخصاص وححسٍ انخىافر انحٍىي وفؼانٍت انذواء وحقبم انًرٌط سريعة التفكك .حبوب سريعة التفكك ، كيتوبروفين ، الكبس المباشر ، مواد الكلمات المفتاحية : introduction the conventional oral dosage forms (tablet and capsule) have wide acceptance up to 50-60 % of the total dosage forms (1) . tablet is still most popular dosage form because of its convenience in terms of self administration, compactness and ease in manufacturing. however, geriatric and pediatric patients experience difficulty in swallowing conventional tablets, which leads to poor patient compliance (1) . orally disintegrating tablets are also called as orodispersible tablets (odts), quick disintegrating tablets, mouth dissolving tablets, fast disintegrating tablets, fast dissolving tablets, rapid dissolving tablets, porous tablets, and rap melts. however, of all the above terms, united states pharmacopoeia (usp) approved these dosage forms as odts. recently, european pharmacopoeia has used the term orodispersible tablet for tablets that disperses readily and within 3 min in mouth before swallowing (2) .to overcome this problem, scientists have developed innovative drug delivery systems known as mouth dissolving tablets. their characteristic advantages such as administration without water, anywhere, anytime which lead to their suitability to geriatric and pediatric patients. they are also suitable for the mentally ill, the bedridden and patients who do not have easy access to water. the benefits, in terms of patient compliance, rapid onset of action, increased bioavailability and good stability made these tablets popular as a dosage form in the current market (3,4) . a non-steroidal antiinflammatory (nsaid) has been indicated for various painful indications (5) and proved as effective as other nsaids with lower indications of gastro-intestinal adverse effects and thus, resulted in a greater compliance with treatment (6) . ketoprofen is propionic acid derivative anti-rheumatic drug with wellknown anti-inflammatory, antipyretic and analgesic properties to treat mild to moderate pain and dysmenorrhea the structure of ketoprofen as shown in (fig.1) (7, 8) . 1 corresponding author email :saddamjumaa @ yahoo.com received : 20/11/2011 accepted : 27/6/2012 iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 64 also it is an inhibitor of prostaglandin synthetase (9) . however, it is not freely soluble in water and causes systemic disturbances in gastrointestinal tract (10) . in the present study, an attempt has been made to develop mouth dissolving tablets of ketoprofen using different subliming agents on the disintegration of orodispersible tablets with good physical properties and acceptance. figure 1: ketoprofen chemical structure material and methods materials ketoprofen powder, crospovidone (cp), and croscarmellose sodium (ccs) were purchased from 3b pharmaceutical (wuhan) international co. ltd, china. microcrystalline cellulose (avicel ph 102)&(avicel ph 101) were purchased from hekma drug industry, jordan. magnesium stearate, mannitol, ammonium bicarbonate were purchased from riedel-de-haen ag seelze, germany. camphor was purchased from evans medical ltd, liverpool, england. all other materials were of analytical grade. methods formulation of orodispersible tablets of ketoprofen the orodispersible tablets of ketoprofen were prepared using the camphor and ammonium bicarbonate as subliming agent, menthol and crospovidone (cp), and croscarmellose sodium (ccs), and sodium starch glycolate (ssg) as superdisintegrant, microcrystalline cellulose (mcc) and mannitol as diluent, sodium saccharin as sweetening agent, and talc as flow promoter and magnesium stearate as lubricant, the composition of each batch is shown in table 1. the ketoprofen (50mg) and excipients were passed through sieve (#80) to ensure the better mixing. the powder was compressed using manesty, type f3 compression machine equipped with 8 mm round punch by direct compression technique. sublimation was performed from tablets contain subliming agents at 60ºc. a minimum of 50 tablets was prepared for each batch. table 1: composition of different batches of orodispersible tablets of ketoprofen material /mg formula no. f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 ketoprofen 50 50 50 50 50 50 50 50 50 50 50 50 camphor 4.5 (2.5%) w/w ammonium bicarbonate 4.5 (2.5%) w/w 9 (5%) w/w ccs 9 (5%) w/w 18 (10%) w/w 27 (15%) w/w ssg 9 (5%) 18 (10%) w/w 27 (15%) w/w cp 9 (5%) w/w 18 (10%) w/w 27 (15%) w/w (mcc102) 18 18 18 18 18 18 18 18 18 18 18 18 talc 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 sodium saccharin 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 3.6 mg stearate 1 1 1 1 1 1 1 1 1 1 1 1 mannitol q.s. to 180 180 180 180 180 180 180 180 180 180 180 180 iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 65 pre compression parameters angle of repose angle of repose was determined using funnel method (11) . the blend was poured through funnel that can be raised vertically until a maximum cone height (h) was obtained. radius of the heap (r) was measured and angle of repose was calculated using the formula; tan θ = h/r where, θ is the angle of repose, h is height of pile; r is radius of the base of pile. compressibility (carr’s) index an accurate weight of formula granules was poured into a volumetric cylinder to occupy a volume (v◦) and then subjected to a standard tapping procedure onto a solid surface until a constant volume was achieved (vf). the carr's index was calculated using following equation (11) . evaluation of the prepared orodispersible ketoprofen tablets weight variation randomly, twenty tablets were selected after compression and the mean weight was determined. none of the tablets deviated from the average weight by more than ±7.5%. uniformity of content the content of the prepared ketoprofen orodispersible tablets was determined. ten tablets were assayed individually. each tablet was crushed individually and dissolved in 100ml of methanol .the filtrated solution was diluted appropriately and the drug content was measured spectrophotometrically at 260nm using (carry uvvisible spectrophotometer) (11) . the requirement for this test is met if the amount of ingredient in each of the ten tablets lies within the range of (85-115) % of the drug present in each tablet. wetting time a piece of tissue paper (12cm x10.75cm) folded twice was placed in a petri dish (internal diameter=9cm) containing 10ml of buffer solution simulating saliva ph 6.8 and amaranth. a tablet was placed on the paper and the time taken for complete wetting was noted. three tablets from each formulation were randomly selected and the average wetting time was recorded (12) . hardness the crushing strength of the tablets was measured using a monsanto hardness tester. three tablets from each formulation batch were tested randomly and the average reading ± sd was recorded. friability twenty tablets were weighed and placed in a roche friabilator and the equipment was rotated at 25 rpm for 4 min. the tablets were taken out, dedusted and reweighed. the percentage friability of the tablets was calculated using following equation (13) . in vitro disintegration time the disintegration time was defined as the time in seconds taken for complete disintegration of the tablet with no palpable mass remaining in the apparatus was measure in second using artificial saliva as disintegration medium. six tablets were placed individually in each tube of disintegration test apparatus. the values reported are mean ± standard deviation (14, 15) . in vitro drug dissolution test dissolution rate was studied by using usp xxiii type-ii dissolution apparatus employing a paddle stirrer at 50 rpm the two experiments were done for dissolution using two different dissolution media 900 ml of ph( 6.8 ) phosphate buffer and 0.1 n hcl at 37±0.5ºc.one tablet was used in each test. a (5 ml) sample from dissolution medium was withdrawn at different time intervals (0, 1, 2, 4, 6, 8, 10, 15 and 20 min). the withdrawn sample was replaced by same amount of fresh dissolution medium to maintain sink conditions. the samples were filtered through 0.22 µm membrane filter and analyzed for drug content by measuring the absorbance at 260 nm using uv spectrophotometer (16) . statistical analysis the mean ± standard deviation of the experiments results were analyzed using one way analysis of variance (anova). iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 66 results and discussion ketoprofen tablets were prepared by direct compression method. twelve formulations were prepared with two different subliming agents or three different superdisinegrants. each superdisinegrants was used in three different concentrations (5% (w/w), 10% (w/w) and 15% (w/w)). table 2 shows the data obtained from the precompression evaluation of tablets. all batches of the tablets were evaluated for various pre and post compression parameters. precompression parameters like angle of repose, and compressibility index while post compression parameters such as hardness, friability, drug content, wetting time, water absorption ratio, and disintegration time were evaluated. post compression parameters are reported in table 3. all the above properties and value were near to boundary of standard limit except formula (f1) produced mechanically weak tablets with hardness and friability (2 kg/cm² and 1.21%) (13) .all the tablets maintained hardness in the range 3.17– 6.08 kg/cm 2 . the loss in total weight of the tablets due to friability was in the range of 0.26-0.88%. the drug content in different formulation was highly uniform and in the range of 99.60-99.92%. (13) .wetting time is used as an indicator of the ease of tablet disintegration and found to be 8.9-41sec. water absorption ratio ranged from 6.66106.1. the result in vitro disintegration time indicated that formula (f1) has shortest disintegration time this decrease in the disintegration time may be attributed to the increasing porosity of the tablet so that the porous hydrophilic matrix will easily pick up the disintegration medium and thus facilitating the wicking action of the superdisintegrant and bringing about rapid tablet disintegration and shorter wetting time (17) . but unfortunately with unacceptable hardness (2 kg/cm 2 ) and in general formulas based on subliming agent produce longer disintegration time than formulas used superdisintegrant thus for formulas (f4-f12) the shortest disintegration time was 18.04 for formula f11 which contain 10% crospovidone as superdisintegrant . an increasing in concentration of superdisintegrants over an optimum lead to increase in the disintegration time of orodispersible tablet. the reason behind this increase may be due to formulation of a viscous gel layer on the surface of tablet which prevents the further penetration of disintegration medium and hinder the disintegration (18) . this prevents the penetration of water to the core of the tablet. in contrast, cp has little tendency to form a gel (19) .the formula (f11) contain 10% of cp resulted in an improvement of both disintegration time and wetting time due to the property of cp and a significant difference (p<0.05) in the dt value of tablets in presence of the cp, ccs and ssg superdisintegrants (20) . from this can concluded that the superdisintegrant efficiency is in following descending order; crospovidone > sodium starch glycolate > croscaramellose, and 10% is the optimum percent for good disintegration.the formula (f11) was selected as the best formula that subjected for dissolution studies in comparison to conventional (toprec® tablet ) formula as shown in figure (2) .the results of dissolution study indicated that the selected formula and the conventional one shows release 100, 32.8% respectively at 10 minutes. table 2.: precompression parameters of ketoprofen orodispersible formulas formula code carr’s index (%) angle of repose(°) flow characters f1 10.00±0.50 28.75±0.39 good f2 30.29±0.41 29.41±1.55 good f3 30.83±0.76 29.41±1.55 good f4 31.71±0.62 29.87±0.76 good f5 29.83±0.28 31.60±1.27 passable f6 27.83±0.28 32.86±1.24 passable f7 33.20±0.14 30.73±1.51 passable f8 33.21±0.18 32.45±0.72 passable f9 33.61±0.34 29.42±0.76 good f10 35.70±0.81 32.00±1.92 passable f11 35.12±0.82 26.45±0.40 good f12 37.43±0.98 28.00±3.43 good iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 67 table 3.:post compression parameters of prepared ketoprofen orodispersible tablets formula code hardness (kg/cm²) friability (%) weight variation wetting times (sec.) water absorption ratio (%) in vitro disintegration times (sec.) f1 2.00 ± 0.10 1.21± 0.010 179 ± 1.14 12.25 ± 0.90 42.06 ± 27.86 14.51 ± 1.101 f2 4.00 ± 0.50 0.26± 0.008 179 ± 0.85 14.47 ± 2.54 42.88± 28.28 24.33 ± 1.506 f3 4.33 ± 0.76 0.61± 0.009 179 ± 1.14 41.00 ± 3.606 6.660 ± 3.360 51.50 ±1.761 f4 4.33 ± 0.58 0.88 ± 0.023 178 ± 1.48 21.90 ± 7.36 67.74 ± 11.87 23.95 ± 1.268 f5 3 .17 ± 0.29 0.78± 0.005 179 ± 0.94 21.01 ± 2.66 74.99 ± 0.638 34.09 ± 1.170 f6 3.33 ± 0.29 0.88± 0.013 179 ± 1.70 19.26 ± 1.58 98.68 ± 3.650 34.79 ± 0.944 f7 3.42 ± 0.38 0.84± 0.003 178 ± 1.41 21.00 ± 2.00 106.1 ± 0.630 25.06 ± 1.202 f8 3.50 ± 0.50 0.84± 0.003 178 ± 1.52 19.85 ± 2.55 104.9± 0.630 22.47 ± 3.224 f9 3.17 ± 0.29 0.85± 0.010 178 ± 1.41 22.00 ± 4.35 221.5 ± 8.146 29.17 ± 2.563 f10 6.08 ± 0.52 0.66± 0.010 178 ± 1.66 13.72 ± 2.29 92.55 ± 4.046 20.50 ± 0.548 f11 5.50 ± 0.50 0.75± 0.005 180 ± 1.79 8.950 ± 0.08 89.40 ± 1.189 18.04± 0.093 f12 5.17 ± 0.76 0.53± 0.028 179 ± 1.32 14.57 ± 2.34 98.48 ± 14.80 19.83 ± 3.267 figure 2: dissolution profile of ketoprofen odts from selected formula (f11) and conventional formula in phosphate buffer,( ph=6.8 )at 37°c ± 0.5°c and 50 r. p.m. references 1. habib w, khankari rk, hontz j. fast dissolve drug delivery systems. crit rev ther drug carrier syst 2000; 17:61-72. 2. sastry sv, nyshadham jr , fix ja . recent technological advances in oral drug delivery. a review.pharm sci technol today 2000; 3: 138-45. 3. chang r, guo x, burnside b, couch r. a review of fast dissolving tablets. pharm tech 2000; 12:52-8. 4. bi y, sunada h, yonezawa y, danjo k, iida k. preparation and evaluation of compressed tablets rapidly disintegrating in the oral cavity. chem pharm bull 1996; 44:2121-7. 5. british national formulary. 54 rd , 2007. 6. legrand e. aceclofenac in the management of inflammatory pain. exp opin pharmacother 2004; 5:134757. 7. ralph niiokaitettey-amlalo;invitro release of ketoprofen from proprietary and extemporaneously manufactured gels. thesis submitted to rhodes university in fulfilment of the requirements for the degree of master of science (pharmacy), 2005. 8. ahmed i,nafadi m, fatahalla f. formulation of a fast-dissolving ketoprofen tablet using freeze-drying in blisters technique. drug devind pharm 2006; 32:437–442. 9. methaqhamadsabar; preparation and in vitro evaluation of ketoprofen as a sustained release tablet using polyelectrolyte complex as a matrix former.thesis ,2011. 10. bhupendra g prajapati and nayan ratnakar; a review on recent patents on fast dissolving drug delivery system. international journal of pharmtech research, 2009; 1(3):790-798. 11. british pharmacopoeia commission. powder flow. london, uk: british pharmacopoeia commission; 2007, appendix xvii n. 12. hisakadzu s, yunxia b. preparation, evaluation and optimization of rapidly disintegrating tablets. powder technology. 2002; 122:188-198. 13. british pharmacopoeia 2009. 14. mohsin a.a; nimbalakr n.e; sanaullah s; aejaz a; formulation and evaluation of mouth dissolving tablets of amitriptyline hydrochloride by direct compression technique. international journal of pharmacy and pharmaceutical sciences, 2010; 2(1):204 -210. 15. eouani c., prinderre p., joachim j., determination of the in vitro disintegration profile of rapidly disintegrating tablets and iraqi j pharm sci, vol.21(2) 2012 ketoprofen oral disintegrating tablets 68 correlation with oral disintegration. international journal of pharmaceutics. 2004; 292:29-41. 16. nirav v patel; narendra p chotai1; mayur p patel; formulation design of oxcarbazepine fast-release tablets prepared by melt granulation technique. asian journal of pharmaceutics.2008,2225. 17. patel d., patel m., optimization of fast dissolving etoricoxib tablets prepared by sublimation technique. indian j. of pharmaceutical sciences. 2008; 70(1): 621625. 18. sunita a., chaudhary a., excipients updates for orally disintegrating dosage forms. int. j. pharm. sci. 2010; vol-1, issue-2, 103-107. 19. patel d., patel m., shah r., jogani l., balapatel i., studies in formulation of orodispersible tablets of rofecoxib. indian j. pharm. sci. 2004; 66: 621-625. 20. mallikarjuna settee; d. v. k. prasad; v. r.m. gupta and b. sa; development of fast dispersible aceclofenac tablets: effect of functionality of superdisintegrants. indian journal of pharmaceutical sciences, 2008; 180-185. iraqi j pharm sci, vol.21(2) 2012 chloramphenicol ocular in situ gel 98 preparation and evaluation of chloramphenicol as thermosensitive ocular insitu gel ali k. ali allah*, shaimaa n. abd-al hammid** ,1 * ministry of health, al-hashimmia hospital, babel, iraq **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract the purpose of this study was to develop poloxamer-based in-situ gel of chloramphenicol aiming to increase bioavailability and prolong corneal contact time, controlling drug release, and enhancing ocular bioavailability. the in-situ gel was prepared using different concentrations of poloxamer 407 combined with hydroxypropyl methyl cellulose (hpmc) or carbapol 940 to achieve gelation temperature about physiological temperature and improve rheological behavior and gelling properties of poloxamer gel. the prepared formulations were evaluated for their appearance, ph, and sol-gel transition temperature. the formulations f2, f3, and f5 have a gelation temperature within the accepted range 35-37 0 c and were evaluated for their isotonicity, rheological studies, ocular irritation test, sterility and release studies. the selected formulations (f2,f3, and f5) isotonic, pseudoplastic, non irritant, pass sterility test, and the in vitro release demonstrated a diffusion-erosion controlled release of chloramphenicol over a period of 4 hr., 6 hr., and 6 hr. respectively. key words: chloramphenicol, in-situ gel, ocular dosage form, poloxamer. تحضير وتقيين خارج الجسن للكلىرافينكىل كهالم هىضعي عيني هتحسس للحرارة هللا على علي كاظن * و شيواء نزار عبذ الحويذ **،1 عشاق .ان* ٔصاسة انصحت ، يسخشفى انٓبشًٍت ، بببم ، .كهٍت انصٍذنت ، جبيعت بغذاد ، بغذاد ، انعشاق ،صٍذالٍَبث ان فشع** الخالصت يٍ يبدة انكهٕسايفٍُكٕل يع بٕنًٍشاث انبٕنٍكسبيش حٓذف انى اطبنت صيٍ يٕضعً ْالوانٓذف يٍ ْزِ انذساست ْٕ نخطٌٕش انخًبط يع انقشٍَت ٔانسٍطشة عهى ححشس انذٔاء اضبفت انى ححسٍٍ انخٕافش انحٍٕي يٍ انعٍٍ. يع يبدة انٓبٌذسٔكسً بشٔبٍم يثٍم 704ًٍشاث انبٕنٍكسبيش يٍ اسخعًبل حشاكٍض يخخهفت يٍ بٕن انٓالو انًٕضعًنقذ حى ححضٍش نهحصٕل عهى دسجت حشاسة حكٌٍٕ انًبدة انٓاليٍت عهى يقشبت يٍ انذسجت انفضٌٕنٕجٍت نهجسى ٔنخحسٍٍ 070سهٍهٕص أ انكبسبببٕل ة حى حقٍٍى شكهٓب انخبسجً ٔدسجت طشٌقت جشٌبٌ انسٕائم ٔخٕاص حكٌٕ انًبدة انٓاليٍت نٓالو انبٕنٍكسبيش. انصٍغ انذٔائٍت انًحضش نذٌٓب دسجت حشاسة حكٌٍٕ انًبدة (f2, f3, f5)انحبيضٍت ٔدسجت حشاسة اَخقبل انًبدة يٍ سبئم انى ْاُلو. انصٍغ انذٔائٍت انًحضشة نسٕائم ٔاخخببس كًب حى دساست طشٌقت جشٌبٌ ا ( دسجت يئٌٕت ٔحى حقٍٍى انضغظ انخُبضحً نٓب.54-53انٓاليٍت ضًٍ انحذٔد انًقبٕنت ) حخذش انعٍٍ اضبفت انى اخخببس دسجت حطٓشْب يٍ انجشاثٍى )اخببس انعقبيٍت(. ٔدساست ححشس انذٔاء. انصٍغ انذٔائٍت انًحضشة انًخخبسة (f2, f3, f5) كبَج يخسبٌٔت انضغظ انخُبضحً ٔشبّ طٍّعّ ٔغٍش يخششت ٔيعقًت كًب اٌ ححشس انذٔاء خبسج انجسى اظٓش َفٕرٌت ( سبعبث ببنخخببع.6( سبعبث ٔ )6( سبعبث ٔ )7نخحشس يسٍطش عهٍّ نذٔاء انكهٕسايفٍُكٕل عهى فخشة طٕنٓب )ٔحآكم كلىراهفينكىل ، هالم هىضعي ، صيغت دوائيت عينيت ،البىليكساهر .تاحيت :فالكلواث الو introduction the conventional liquid ophthalmic delivery systems exhibit short pre corneal residence time and poor bioavailability due to rapid elimination induced by lachrymal flow, blinking, normal tear turnover, and solution drainage by gravity. as a result, frequent instillation of solution is needed in order to achieve the desired therapeutic effect (1) . one of the major disadvantages of eye drops is the pulsatile drug level, with a transient period of overdose followed by extended period of subtherapeutic levels before next dose is administered. this means that the infectious agent will be exposed by low concentration of the antibiotic leading to bacterial resistance (2) . various ophthalmic vehicles, such as inserts, ointments, suspensions, and aqueous gels, have been developed to lengthen the residence time of instilled dose and enhance ophthalmic bioavailability. these ocular drug delivery systems, however, have not been used extensively because of some drawbacks, such as blurred vision from ointment or low patient compliance from inserts (3) . several in situ gelling systems have been developed to prolong the precorneal residence 1 corresponding author e-mail: shaimaa_alsamarrai@yahoo.com received : 16/4/2012 accepted :7 /11/2012 iraqi j pharm sci, vol.21(2) 2012 chloramphenicol ocular in situ gel 99 time of a drug, improve patient compliance and consequently enhance ocular bioavailability. these systems exhibit sol-togel phase transitions due to a change in a specific physicochemical parameter (e.g.: ph, temperature, and ions) in the cul-de-sac (4) . the productive absorption of most ophthalmic drugs results from diffusional process across the corneal membrane. the efficiency of the absorption process is a function of the rate and extent at which the transport processes occur. the flux of any drug molecule across a biological membrane depends on the physicochemical properties of the permeating molecule and its interaction with the membrane. the absorption process is also a function of the physiological mechanism of pre-corneal fluid drainage or turnover (5) . chloramphenicol is an antibiotic used for serious infections in which the benefit of the drug out weight its uncommon but serious hematological toxicity such as infections caused by haemophilus influenza resistant to other antibiotics, meningitis in patients in whom penicillin can not be used, typhoid fever, and bacterial conjunctivitis. it inhibits bacterial protein synthesis by binding to 50s subunit of the bacterial ribosome (6) . its slightly soluble in h2o with melting point 149-153c and mwt 323 (6) . materials and method materials chloramphenicol powder from fluka biochemika (switzerland), hypromellose usp (metolose 90 sh4000sr)(hpmc4000) ex05097, soyabean-casein digest medium and carbopol 940 from himedia-mumbi (india), poloxamer (pluronic f127) (sybronic bf127) from actico, monosodium dihydrogen phosphate from laboratory rasayan sdfine. chemical limited mumbi india, disodium hydrogen phosphate, phosphoric acid , hydrochloric acid and sodium chloride from riedel-de haenagseelze hannover (germany), sodium bicarbonate from sdi (iraq), calcium chloride dehydrate and fluid thioglycolate medium from merck (germany). methods preparation of in -situ gel ten formulas were prepared as shown in table (1) using different ratios of poloxamer 407(pluronic f127) (10%, 15%) as gelling agent in combination with carbapol 940 (0.02%, 0.04%) or hpmc (0.5%, 1.5%) using as viscosfying agents. thermoreversible gels were prepared using cold technique. first of all the aqueous dispersions of selected concentrations of carbapol 940 (0.02%, 0.04%) for formulas (f2, f3, f7, and f8) and hpmc for formulas (f4, f5, f9, and f10), and pluronic f127 for formulas (f1 and f6) in phosphate buffer ph5.9 were prepared. the pluronic/carbapol combination and the pluronic/hpmc combination were prepared by dispersing the pluronic in the desired concentration of respective polymer solutions. then the partially dissolved solutions were refrigerated until thoroughly mixed (approximately 24 hrs). an appropriate amount of drug dissolved in phosphate buffer ph5.9, then benzalkonium chloride 0.01% added with continuous stirring until uniform drug solution was obtained. the drug solution was finally added to polymer solution with continuous stirring. the developed formulations were filled in amber glass containers ( 7 ) . table (1): composition of different formulas in-situ gelling systems of chloramphenicol. no. formulation code chloramphenicol % w/v pluronic f127 % w/v carbopol 940% w/v hpmc% w/v benzalkonium % chloride w/v formulation code phosphate buffer ph 5.9 1. f1 0.5 10 0.01 f1 q.s to 100ml 2. f2 0.5 10 0.02 0.01 f2 q.s to 100ml 3. f3 0.5 10 0.04 0.01 f3 q.s to 100ml 4. f4 0.5 10 0.5 0.01 f4 q.s to 100ml 5. f5 0.5 10 1.5 0.01 f5 q.s to 100ml 6. f6 0.5 15 0.01 f6 q.s to 100ml 7. f7 0.5 15 0.02 0.01 f7 q.s to 100ml 8. f8 0.5 15 0.04 0.01 f8 q.s to 100ml 9. f9 0.5 15 0.5 0.01 f9 q.s to 100ml 10. f10 0.5 15 1.5 0.01 f10 q.s to 100ml iraqi j pharm sci, vol.21(2) 2012 chloramphenicol ocular in situ gel 100 compatibility study a thin layer chromatography test was done using methanol: chloroform 60:40 as the mobile phase and aluminum paper silica gel as the stationary phase. spots were made for the prepared formula on silica gel to discover any incompatibility among ingredients. sterilization: the formulations were sterilized by filtration by passage through a sterile membrane filter of nominal pore size of 0.22 µm (millipore type). (8) measurement of sol-gel transition temperature the sol-to-gel phase transition temperature (gelation temperature) measured for all the prepared formulations according to the technique described by vadnere and gilbert (9) . an aliquot of 2ml refrigerated tested formulation was transferred to a test tube and sealed with a parafilm. the tube was maintained in a thermostatically controlled water bath at 4 0 c. the temperature of the water bath was increased gradually in increment of 3 0 c in the beginning of the experiment and then 1 0 c increment in the region of sol-gel transition temperature, the tested formulation was left to equilibrate for 10 min at each new setting (10) . the gelation is considered to be occurred when the meniscus of the formula would no longer move upon tilting through angle90 (11) . isotonicity evaluation isotonicity is an important characteristic of the ophthalmic preparation. isotonicity has to be maintained to prevent tissue damage or irritation of the eye. the three selected ophthalmic preparations are subjected to isotonicity testing by using osmometer apparatus (12) . rheological studies the three selected formulation were subjected for rheological studies. viscosity of the instilled formulation is an important factor in determining residence time of drug in the eye. the solutions were allowed to gel at physiological temperature and then the viscosity determination was carried out by using viscometer with spindle 2. the angular velocity increased gradually 6, 12, 30, 60 and the viscosity of the formulation is measured (13) . invitro release studies the in-vitro drug release from the selected formulation was studied through cellophane membrane using a modified usp xxiii dissolution testing apparatus(figure 1). the dissolution medium used was artificial tear fluid freshly prepared (ph 7.4) (14) . cellophane membrane, previously soaked overnight in the dissolution medium, was tied to one end of a specifically designed glass cylinder (open at both ends and of 2.5 cm diameter). a 1-ml volume of the formulation was accurately pipette into this assembly. the cylinder was attached to the metallic driveshaft and suspended in 200ml of dissolution medium maintained at 37±0.5 0 c so that the membrane just touched the receptor medium surface. the dissolution medium was stirred at 50 rpm. aliquots, each of 5ml volume, were withdrawn at half an hour intervals and replaced by an equal volume of fresh dissolution medium (15) . the samples were filtered through 0.45-mm syringe filters and subjected to spectrophotometric analysis at 278nm (16) . figure (1): modified usp xxiii in vitro dissolution testing apparatus sterility 2ml of the prepared formula was withdrawn with a sterile syringe then, aseptically transferred to thioglycolate medium (20ml) and soyabeancasein digest medium (20ml) separately. the liquid mixed with the media. the inoculated media incubated 14 days at 30-35 0 c in case of fluid thioglycolate medium and 20 25 0 c in case of soyabeancasein digest medium (17) . ocular irritancy test the draize irritancy test was designed for the ocular irritation potential of the ophthalmic product prior to marketing. according to the draize test, the amount of substance applied to the eye is normally 100 µl placed into the lower cul-de-sac with observation of the various criteria made at a designed required time interval of 1hr, 24 hrs, 48hrs, 72 hrs, and 1 week after administration. three rabbits (male) weighing 1.5 to 2kg are used for the study. the sterile formulation is instilled twice a day for a period of 7 days, and a cross-over study is carried out (a 3day washing period with saline was carried out before the cross-over study). rabbits are iraqi j pharm sci, vol.21(2) 2012 chloramphenicol ocular in situ gel 101 observed periodically for redness, swelling, and watering of the eye (18, 19) . statistical analysis the results of the experiments are given as a mean of triplicate samples and were analyzed according to the one way analysis of variance (anova) at the level of (p < 0.05). results and discussion compatibility study the thin layer chromatography test showed that only 5 compounds appeared; these are chloramphenicol, pluronic f127, carbapol, hpmc and benzalkonium chloride. this indicates that no chemical interaction takes place since there is no new compound appears at the silica gel layer. measurement of the sol-gel transition temperature thermoreversible polymers have been considered to be suitable for ocular delivery of chloramphenical if they are liquid at room temperature (20-25 0 c) and undergo gelation with the increase in temperature in cul-desac (35-37 0 c) (20) . poloxamer solutions are known to exhibit thermoreversible gelation depending on the polymer grade, concentration, and other included formulation components. at a certain concentration of the polymer and temperature, poloxamer molecules in aqueous solution will self-assemble to form spherical micelles with a dehydrated ppo (poly propylene oxide) core surrounded by hydrated swollen peo(poly ethylene oxide) chains, packing and entanglements of micelles with increase of temperature are said to be possible mechanisms of the gelation of poloxamer solutions. (21) the micelles would occupy a high fraction volume of solution, and come into contact and entangle with each other resulting in a three-dimension network structure and forming stiff gel. therefore, the product containing more effective concentration of pluronic f127 would contain more number of micelles. they would need lower energy to promote sol-gel transition and could perform sol-gel transition at lower temperature than products containing less f127 content. (22) significant decrease in gelation temperature occurs when carbopol added to poloxamer(p<0.05). this could be explained by the interactions between the polymers. one presumption is the formation of a three dimesnsional network between carboxyl groups of carbopol and ether groups of poloxamer due to hydrogen bonds which will lead to gelation at lower temperature. (11) in addition to that, carbopol molecules become associated with cavities between polymolecular poloxamer micelles and chains of carbopol and this would block the interaction between poloxamer chains which would also induce the lower gelation temperature (23) . drainage of ophthalmic formulations from the precorneal surface would be considerably reduced by addition of mucoadhesive polymers such as hpmc which allow attachment of the formulae to the corneal mucin and decrease the gelation temperature of the in situ forming gels. table (2) shows the sol-gel transition temperature of the prepared formulations and only three formulations (f2, f3, and f5) have gelation temperature within the acceptable range (35-37 0 c) therefore subjected for further studies. table (2): ph values and sol-gel transition temperatures of the prepared in-situ gel formulations & osmolarity values for the selected formulations (f2, f3, and f5). formulation ph after formulation sol-gel transition temp. 0 c osmolarity osmol f1 6.2 40 f2 5.9 36 0.298 f3 5.9 36 0.321 f4 6.2 38 f5 6.2 37 0.310 f6 6.2 32.5 f7 6 26 f8 5.9 24 f9 6.2 26 f10 6.3 24 isotonicity evaluation table (2) show the osmolarity for the selected formulations (f2, f3, and f5) as measured by osmometer. all values within the acceptable range (0.6-2%). no significant difference between them. rheological studies table (3) showed the viscosity values obtained for formulations f2, f3, and f5 at different angular velocity. the formulations exhibited pseudoplastic rheology, as evidenced by shear thinning and decrease in viscosity with increased angular velocity that can be observed in the figures (2, 3, 4). the viscosity was directly dependent on the polymeric content. the administration of ophthalmic preparations should influence as little as possible the pseudoplastic character of the pre-corneal film (24) .since the ocular shear rate is very high ranging from 0.03 s -1 during inter-blinking periods to 4250-28500 s -1 during blinking, viscoelastic fluids with a iraqi j pharm sci, vol.21(2) 2012 chloramphenicol ocular in situ gel 102 viscosity that is high under the low shear rate conditions and low under the high shear rate conditions are preferred for ophthalmic drug delivery (29) . table (3): viscosity values for the selected formulations (f2, f3, and f5). figure (2): viscosity versus rpm at 37 0 c for f2. figure (3): viscosity versus rpm at 37 0 c for f3. figure (4): viscosity versus rpm at 37 0 c for f5. in vitro release studies the in-vitro release studies were carried out for the selected formulations (f2, f3, and f5) using simulated tear fluids (stf ph 7.4) as the dissolution medium. the standard curve and release profiles are shown in figures (5, 6, 7 and8) respectively. it is obvious that f3 sustain the release of chloromphenicol more than f5 and the latter (f5) sustain the release more than f2, this may be potentiated by the rheological studies where the rate of drug release decrease when the viscosity increase (25) . in formulation f5, the retarding effect of the hpmc polymer could be attributed to its ability to increase the overall product viscosity. (26) also it has ability to distort or squeeze the extra-micelle aqueous channels of poloxamer micelles through which the drug diffuses, thereby delaying the release process (27) . f3 contain more carbopol 940 content than f2 therefore sustain the release more than f2. this is probably that stf (ph 7.4) led to ionization of carboxyl groups in carbopol molecules and thus repulsion of these ions. then, carbopol would be in an extended configuration and form strong threedimensional networks, therefore, f3 possess stronger gel structure and this will cause more retardation for drug release (28) . drug release data were fitted to different kinetic models like zero-order, first-order, higuchi and korsmeyerpeppas to ascertain the kinetic modeling of the drug release (29) . to confirm the diffusion mechanism, the data were fit into korsmeyer’s equation. (30) the exponent n gives information about the release mechanism=0.45 (indicates fickian diffusion-controlled drug release), 0.45 <n<0.89 indicates anomalous (non-fickian transport), and n=0.89 ,indicates (a case ii angular velocity (rpm) viscosity f2 f3 f5 6 52.1 160.6 54.4 12 26.6 69.6 26.7 30 11 10.4 13.1 60 9.62 7.4 10.5 iraqi j pharm sci, vol.21(2) 2012 chloramphenicol ocular in situ gel 103 relaxational release transport, non-fickian, zero-order release). (31) the kinetic values obtained for the selected formulations (f2, f3, and f5) are indicated in table (4). from this table, it is obvious that f2 and f5 follows higuchi kinetic and the drug releases by diffusion and erosion, while f3 follow peppas korsmeyer kinetic and the drug releases by diffusion and erosion. significant difference between the release of (f2, f5) and the release of f3(p<0.05). figure (5): calibration curve of chloramphenicol in artificial tears (ph=7.4) figure (7): the release profile of formula 3 in simulated tear fluids at 37 0 c. figure (8): the release profile of formula 5 in simulated tear fluids at 37 0 c sterility test no appearance of turbidity and no evidence of microbial growth when incubated for 14 days at 30-35 0 c in case of fluid thioglycolate medium and 20-25 0 c in case of soyabean casein digest medium. figure (6): the release profile of formula 2 in simulated tear fluids at 37 0 c. table (4): drug release kinetics of f2, f3, and f5. formula. no zero order first order higuchi peppas korsmeyer r 2 m r 2 m r 2 m r 2 m f2 0.586 32.03 0.37 0.688 0.9598 55.67 0.944 0.4638 f3 0.94 17.25 0.43 0.458 0.945 39.31 0.992 0.665 f5 0.774 20.89 0.54 0.48 0.9656 42.225 0.95 0.7 iraqi j pharm sci, vol.21(2) 2012 chloramphenicol ocular in situ gel 104 ocular irritancy studies the results of the ocular irritation studies indicated that formulations (f2, f3, and f5) were non-irritant with excellent ocular tolerance. no significant ocular damage or abnormal clinical signs to the cornea, iris or conjunctiva were visible (p≥0.05). no signs of redness, watering of the eye and swelling were observed throughout the study with the three formulations. conclusion the use of polymeric in-situ gels for controlled release of chloramphenicol provides a number of advantages over conventional dosage form: 1sustained and prolonged release of chloramphenicol. 2good stability and biocompatibility characteristics make the in situ gel dosage forms very reliable. 3use of biodegradable and water soluble polymers for the in situ gel formulations can make them more acceptable and excellent drug delivery systems. references 1. mansour m., mansour s., moetada nd. ocular poloxamer-based ciprofloxacin hydrochloride in situ forming gels. drug dev ind pharm, 2008; 34:744-752. 2. alonso, a., campanario, e., and martinez j., emergence of multi drug-resistant mutants is increased under selective pressure in pseudomoms aeruginosa. microbiology, 1999; 145: 2857-2862. 3. lee, v. h. l. review: new directions in the optimization of ocular drug delivery. j. ocul. pharmacol, 1999; 6: 157-164. 4. srividya, b., cardoza, r.m., and amin, p.d. sustaind ophthalmic delivery of ofloxacin from a ph triggered in situ gelling system. t. control. release. 2001; 73:205-211. 5. keister, j. c. et al. limited on optimizing ocular drug delivery. j. pharm. sci., 1991; 80(1):50. 6. rang h. p., dale m. m., ritter j.m., flower r. j., rang and dale’s pharmacology, 6 th edition, charchill livingstone, 2007; ch. 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up to date cases in 2012. breast cancer has turn a most warning to health of female in iraq, where it is the major cause of death among women after cardiovascular diseases, with a mortality rate of 23% related cancer. recently there is a crucial requirement to include community pharmacists in health elevation activities to support awareness and early diagnosis of cancer, specially breast cancer. the aim of this study is to assess knowledge, attitude and perceived barriers amongst iraqi community pharmacists towards health promotion of breast cancer. this study is cross sectional research. a questionnaire was given to pharmacists. the questionnaire comprised from four parts: community pharmacist’s demographics and description of practice; knowledge of signs, symptoms and risk causes; knowledge around breast cancer screening and perceived barriers.300 questionnaires were finished and returned by iraqi community pharmacists. mean score of knowledge and screening of breast cancer was 7.9 ± 1.86 and 1.69± 0.33 points respectively, categorizing the overall knowledge and screening of breast cancer among participants as poor level, while mean score for pharmacist attitude was 26.44± 3.86 points, categorizing the overall attitude as favorable. lack of time was perceived by a great proportion of pharmacists (68.2%) as a major barrier to providing patient education. keywords: breast cancer, knowledge, screening, attitude, perceived, community pharmacy. دراسة المعرفة والمىاقف والحىاجز لذي صيادلة المجتمع العراقييه حىل سرطان الثذي ظ محمذ البهادليحسه حاف ,*1 و حيذر فخري التكمه جي ** * . ، ثغذاد ، اىؼشاق وصاسح اىصحخ واىجُئخ ** فشع اىصُذىخ اىسشَشَخ ، ميُخ اىصُذىخ ، جبٍؼخ ثغذاد ، ثغذاد ، اىؼشاق . الخالصة حُش سجيذ ٍِ جَُغ حبالد اىسشطبُ، %52حىاىٍ حُش َشنو، اىؼبىٍَػيً اىصؼُذ اىْسبءٍغ رضاَذ اّزشبس سشطبُ اىثذٌ ثُِ اىشئُسٍ ىيىفبح ثُِ تَؼزجش اىسجاىؼشاق، حُش اىْسبء فٍمجُشا ىصحخ رهذَذااىثذٌ شنو سشطبُ . وقذ5175ٍيُىُ حبىخ حزً اُِ فٍ ػبً 7.21 اىَجزَغ حبجخ ٍيحخ إلدساج صُبدىخْبك . فٍ اِوّخ األخُشح هحبالد اىسشطبُ ٪ 52ٍِثَؼذه وفُبد ، اىقيت واالوػُخ اىذٍىَخاىْسبء ثؼذ أٍشاض ىزقٌُُ ٍؼشفخ وخجشح صُبدىخ اىهذف ٍِ هزٓ اىذساسخ هىاُ ، وخبصخ سشطبُ اىثذٌ. اىسشطبُ ٍجنشاورشخُص ىضَبدح اىىػٍ اىصحٍ وىذػٌ َزأىف االسزجُبُ ىيصُبدىخ. وحُش قذً قطؼُخ. ػجبسح ػِ ثحىس ٍهزٓ اىذساسخ سشطبُ اىثذٌ. حىه ُِ واىَؼىقبد اىزٍ رىاجههٌاىَجزَغ اىؼشاقُ ٍؼشفخ اىؼالٍبد واألػشاض وأسجبة اىخطش؛ اىََبسسبد اىَهُْخ؛يَجزَغ اىصُذىٍ ووصف أجضاء: اىىصف اىسنبٍّ ىاالسزجُبُ ٍِ أسثؼخ اىَجزَغ اىؼشاقٍ. ومبُ صُبدىخ ٍِ قجوثؼذ رىل وقذ رٌ االّزهبء ٍِ االسزجُبّبد وإػبدرهب اىَزىقؼخ. واىَؼىقبدواىَؼشفخ حىه فحص سشطبُ اىثذٌ رصُْف اىَؼشفخ اىشبٍيخ وفحص سشطبُ اىزىاىٍ، حُش رٌ ّقطخ ػيً 1.22 ± 7.17و 1..7± 1.7دسجخ ٍؼشفخ وفحص سشطبُ اىثذٌ ٍزىسط يً اّهب حُش رٌ رصُْفهب ػّقطخ، 1..2± 51.22دسجخ ٍىقف اىصُذىٍ ، فٍ حُِ مبُ ٍزىسط ظؼُفٍسزىي رو اىثذٌ ثُِ اىَشبسمُِ ػيً أّهب .اىَشَط اٍبً رؼيٌُ٪( محبجض سئُسٍ 5..1قجو ّسجخ مجُشح ٍِ اىصُبدىخ ) ظُبع جضء مجُش ٍِ اىىقذ ٍِمبُ َْظش إىً دسجخ ٍقجىىخ. سرطان الثذي،المعرفة ، الفحص ، المىقف ، االدراك ، صيادلة المجتمع .الكلمات المفتاحية: introduction breast cancer is the most frequent cancer in the midst of women worldwide, occupies about 25% of all cases of cancer, with a measured 1.57 million up to date cases in 2012 (1) . breast cancer has turn a most warning to health of female in iraq , where it is the major cause of death among women after cardiovascular system diseases, with a mortality rate of 23% related cancer (1-4) . early diagnosis through screening plays a vital role in reducing mortality and morbidity of breast cancer (5,6) . localized cancer diagnosis enhance survival, restore the breast, and declines the recurrence rate (7) . the growing burden of breast cancer in the eastern mediterranean region (emr) in general, and iraq in specially, focus the critical need to found full national control programs of cancer. 1 corresponding author e-mail: hassanhafid@ymail.com received: 16/9/ 2017 accepted:13 /10/2017 mailto:hassanhafid@ymail.com iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 57 of the registered methods to breast cancer control, as planned by the world health organization (who), early diagnosis and screening suggestion the immediate goal for a decline in mortality (8) . the risk factors that may have a relation with the occurrence of breast cancer are late age menopause, young age at menarche, late age at first live birth, use of oral contraceptives, hormone replacement therapy, high weight, extreme alcohol intake, and radiation exposure (9) . healthcare professionals are important sponsors in helping breast cancer awareness amongst their publics (10,11) . recently, the range of practice of pharmacy had developed to a patient-focused method rather than a product motivated method, allowing pharmacists to enlarge health services to patient-oriented actions in its place of traditional drug services (12,13) . previous reports shown that community pharmacists are probable providers to health promotion actions attractive into attention their accessibility and reliability (12-15) . as readily available health care specialists, pharmacists can evaluate breast cancer risk and possibly advantage large numbers of female, irrespective of age. pharmacists, in combination with physicians, can offer knowledge to permit women to create educated choices about screening and prevention (16) . pharmacists can tell women of their breast cancer threat and let them to work collected with all participants of the health care group to make balanced choices. patient teaching relating to breast cancer awareness and diagnosis through breast self-examination (bse), clinical breast examination (cbe), and mammography is significant (5,17,18,19) . nevertheless, inadequate number of texts evaluated the role of community pharmacists in educating public awareness about cancer disease, highlighting on early diagnosis and screening (20 22) . thus, there is a crucial requirement to include community pharmacists in health elevation activities to support awareness and early diagnosis of cancer, specially breast cancer. to reach this, it is necessary for community pharmacists to have widespread knowledge, progressive attitudes, willingness and essential tools to deliver specialized pharmaceutical care services (12,20,23,24) . the aim of this study is to assess knowledge, attitude and perceived barriers amongst iraqi community pharmacists towards health promotion of breast cancer. methods the study was conducted over an 8-month period from december 2016 to july 2017. of 375 questionnaires given to iraqi pharmacists, 300 questionnaires were finished and returned, resulting a response rate of 80%. a descriptive crosssectional research design was applied to this study. a structured questionnaire was established and modified from questionnaires used in previous studies (25-27) . the study was approved by scientific committee of college of pharmacy – baghdad university. suitability sampling was taken to employee pharmacists in multiple geographical areas in baghdad city (capital of iraq). participants who have bachelor’s degree in pharmacy or a higher educational degree were permitted to contribute in this research. this method is thought to reach a great answer rate of contribution. the questionnaire was established in english language and was given to pharmacists. the survey firstly evaluated by use face and content validity by different faculty memberships at college of pharmacy – baghdad university. significance and survey questions clarity were further assessed through a pilot study (n = 30). feedback and comments by the pilot group resulted in minor edits to the survey tool, which was considered in order to increase clarity and understanding of survey items. the sample data from pilot study were excluded from the final study. the study presented composed of 21 questions to be finished in 15–20 minute. the questionnaire comprised from four parts: (1) community pharmacist’s demographics and description of practice (2) knowledge around breast cancer signs, symptoms and risk causes; (3) knowledge around breast cancer screening and (4) perceived barriers to supporting breast cancer awareness and providing pharmaceutical care in community pharmacy locations. pharmacists answered to a 15-item scale around breast cancer risk factors in addition to signs and symptoms of the cancer. answers fluctuated between ‘correct’, ‘incorrect’ and ‘i don’t know’. then, each ‘correct’ answer was counted 1 point and each ‘incorrect’ and ‘i don’t know’ answers were both counted zero (0) point. the total level ranged from 0 to 15 points on overall knowledge of breast cancer. pharmacists with level range 0–8 were measured to have poor level of knowledge of breast cancer risk causes and symptoms, while those with 9–15 points were considered to have an acceptable level of knowledge. knowledge around breast cancer screening was evaluated over seven different questions established by researchers based on recently published guidelines and recommendations by american cancer society (acs) for screening and early detection of cancer (27) . the total level ranged 0–7 points. in the same way, answers to screening knowledge questions were spread between ‘correct’, ‘incorrect’ and ‘i don’t know’. knowledge of screening was divided into ‘poor’ if participants counted (0–3) points or ‘satisfactory’ if the level reached between 4 and 7 points. attitudes to breast cancer screening were calculated by a 7-item likert type attitudinal scale with five-point answers ranging from strongly agree to strongly disagree. the total attitude level ranged from 0 to 28 points, where higher values suggest more satisfactory attitude amongst participants. in this study, the internal reliability iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 58 coefficient (cronbach’s a) measured for attitude items was 0.734. analysis of data was done using ibm spss statistical package (ibm corp. version 21.0. armonk, ny, usa). by using descriptive statistics to report study variables. pearson’s chisquare (v2) test of independence was used to exam for correlations between categorical variables. bivariate correlation analysis was done to test for correlations between continuous variables. variances were considered to be statistically significant at p< 0.05. results demographics and description of practice demographics and practice features of community pharmacists are represented in table 1. the average age of answering pharmacists was 29.27 ± 3.165 years (median = 28.50 years) with a range from 24 to 41 years. majority of participants were male pharmacists (54%) and were under 30 years (63.7%). 235 (78.3%) of pharmacists have bachelor degree while 65 of them (21.7%) have postgraduate degree. nearly half of the pharmacists surveyed [148 pharmacists (49.3 %)] stated that pharmacy technicians were one at one shift of work. pharmacists’ view about oncology education in the undergraduate degree presented that [114 pharmacists (38.0%)] agreed to receive adequate education, while 68 of pharmacists (22.7%) disagreed and 118 of pharmacists (39.3%) providing a neutral response. 170 (56.7%) of pharmacists answered yes around the oral anticancer agents that dispensed in the community pharmacy while 128 (42.7%) and 2 (0.7%) answered no and i don’t know respectively. knowledge about breast cancer risk factors and early symptoms among community pharmacists: most of community pharmacists [ 208 pharmacists (69.3%)] agreed to the fact that breast cancer is the most usually diagnosed type of cancer among women worldwide. when asked if breast cancer should not be of concern for patient younger than 40 years, 152 (50.7%) of pharmacists agreed that this statement was incorrect. 171 (57.0%) of pharmacists agreed that approximately 50--70 % of patients with primary or metastatic breast cancer have hormone receptor --positive cancer. regarding the initial signs and symptoms of breast cancer, more than half of pharmacists [169 pharmacists (56.3%)] agreed that a painless lump is the initial sign. questioning about findings in advanced breast cancer 174 (58.0%) of respondents agreed that pain, nipple discharge and skin edema are common findings in this stage. additional results are demonstrated in table 2. in order to evaluate the knowledge of prompting factors to breast cancer risk, responses were assessed in form of level out of 15 points. mean score was 7.9 ± 1.86 points (median = 8, range 1–15), categorizing the overall knowledge of breast cancer among participants as poor level of knowledge of breast cancer risk factors and symptoms. overall assessment of pharmacists’ knowledge shown that half the pharmacists (50%) had poor knowledge, while the other half had satisfactory level of knowledge of breast cancer. nearly 172 pharmacists (57.3%) agreed that average-risk women should consider mammography at age of 40 and yearly thereafter. the overall mean score for pharmacists’ knowledge of screening strategies was 1.69± 0.33 out of a maximum score of 7 points (median = 2, range 0–7) categorizing the knowledge as poor. additional screening data are shown in table 3. description of attitudes towards breast cancer health promotion among community pharmacists community pharmacist’s attitude about involvement in breast cancer health activities is represented in table 4. 117 of pharmacists (39.0%) strongly agreed that pharmacists should be involved in breast cancer health promotion in community pharmacy settings. in addition, more than 200 pharmacists either agreed or strongly agreed that it is the pharmacist’s responsibility to provide breast cancer counselling to patients. mean score for pharmacist attitude was 26.44± 3.86 out of a maximum score of 28 points (median = 27, range 4–28), categorizing the overall attitude as favorable. perceived barriers towards breast cancer health promotion among community pharmacists perceived barriers to breast cancer health activities amongst community pharmacists are shown in figure 1. lack of time was perceived by a great proportion of pharmacists (68.2%) as a major barrier to providing patient education. other greatly documented barriers were lack of skills (65.2%), lack of privacy (64.9%) and lack of educational materials (60.3%). a small percentage of pharmacists (30.5%) stated lack of direct profit as a barrier to involvement in patient education. relationship of demographics and characteristics of practice with knowledge and attitudes towards breast cancer health promotion among community pharmacists bivariate correlation analysis of continuous variables (represented in table 5) showed that age had no significant association with overall knowledge of breast cancer (r = 0.021, p = 0.360), knowledge of breast cancer screening (r = 0.098, p = 0.045) or attitude (r = 0.010, p = 0.434) among pharmacists. instead, there was a significant positive association between pharmacist’s knowledge of breast cancer and knowledge of breast cancer screening (r = 0.285, p = 0.000). iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 59 table (1 ):demographic and practice characteristics (n = 300) characteristic n (%) age (years) <30 30—40 40—50 >50 191 (63.7) 104 (35.6) 2 (.7) ------ gender male female 162 (54.0) 138 (46.0) marital status of pharmacist single married divorced widowed 148 (49.3) 146 (48.7) 5 (1.7) 1 (0.3) education of pharmacist bachelor postgraduate degree 235 (78.3) 65 (21.7) place of graduation local public private 83 (27.7) 172 (57.3) 45 (15.0) number of years of practice <3 3—5 6—10 11—20 >20 143 (47.7)) 95 (31.6) 61 (20.3) 1 (.3) types of pharmacy chain independent 64 (21.3) 236 (78.7) description of practice staff pharmacist pharmacist in charge owner 79 (26.3) 90 (30.0) 131 (43.7) iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 60 continued table (1) employment status full time part time 54 (18.0) 246 (82.0) average number of adult patients seen in pharmacy in one shift <20 20—59 60—80 >80 64 (21.3) 177 (59) 52 (17.3) 7 (2.3) percentage of female patients seen in the pharmacy at one shift <25 25—50 51—75 >75 100 (33.3) 168 (56) 32 (10.7) --------- number of pharmacists working in the pharmacy at one shift 1 2 3 227 (75.7) 61 (20.3) 12 (4.0) number of pharmacy technicians in the pharmacy at any one shift none 1 2 >2 91 (30.3) 148 (49.3) 49 (16.3) 12 (4.0) dispensing of oral anticancer agents in the community pharmacy yes no i don’t know 170 (56.7) 128 (42.7) 2 (.7) family history of cancer disease yes no i don’t know 97 (32.3) 176 (58.7) 27 (9.0) received educate oncology education in the undergraduate degree agree disagree neutral 114 (38.0) 68 (22.7) 118 (39.3) values are expressed as n (%). iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 61 table (2) :knowledge of breast cancer signs and symptoms among participants (n =300) table ( 3):participants’ performance on breast cancer screening guidelines (n = 300) values are expressed as n (%). breast cancer knowledge item n ( % ) correct incorrect i don’t know breast cancer is the commonly diagnosed form of cancer among women worldwide (correct ) 208 ( 69.3) 54 (18.0 ) 38 (12.7 ) breast cancer should not be of concern for patients younger than 40 of age ( incorrect ) 121 (40.3) 152 (50.7) 27 (9.0) approximately 50--70 % of patients with primary or metastatic breast cancer have hormone receptor --positive tumor ( correct ) 171 (57.0) 77 (25.7) 52 (17.3) a painless lump is the initial sign of breast cancer in most women ( correct ) 169 (56.3) 80 (26.70 51 (17.0) pain , nipple discharge , retraction , or dimpling , skin edema , redness , or warmth are common findings in breast cancer patients advanced stages ( correct ) 174 (58.0) 82 (27.3) 44 (14.7) breast cancer screening item n ( % ) correct incorrect i don’t know women should be counseled about the importance to start breast self-examination (bse) starting at 30 years of age ( incorrect ) 129 (43.0) 135 (45.0) 36 (12.0) if a women chooses to do breast self-examination ( bse) , it is recommended to be done on monthly basis ( correct ) 166 (55.3) 94 (31.3) 40 (13.3) limitation of bse include the possibility of a false-positive result ( correct ) 163 (54.3) 86 (28.7) 51 (17.0) a symptomatic women aged 40 years or older should not receive cbe as part of their periodic health examination ( correct ) 136 (45.3) 101 (33.7) 63 (21.0) average-risk women should begin annual mammography at of 40 years ( correct ) 172 (57.3) 82 (27.3) 46 (15.3) there is no need to continue mammography after 50 years of age as a regular tool for the early detection of breast cancer ( incorrect ) 98 (32.7) 142 (47.3) 60 (20.0) mammography will not detect all breast cancers , and some breast cancers detected with mammography may still have a poor prognosis ( correct ) 139 (46.3) 104 (34.7) 57 (19.0) iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 62 table (4) :attitude of pharmacists regarding breast cancer health promotion (n = 300) values are expressed as n (%). table (5 ):correlation analysis for continuous variables (n = 300) age mean of pharmacists (years) 29.27 knowledge mean of breast cancer knowledge mean of screening attitude mean of pharmacists mean r p 7.8967 0.021 0.360 mean r p 1.6905 -0.098 0.045 mean r p 26.4400 0.010 0.434 -------- --------- mean of knowledge mean of screening r p 7.8967 1.6905 0.285 0.000 statement n ( % ) strongly agree agree neutral disagree strongly disagree pharmacists should be involved in breast cancer health promotion in the pharmacy 117 (39.0) 140 (46.7) 22 (7.3) 3 (1.0) 18 (6.0) it is my responsibility as a pharmacist to provide breast cancer counseling , and this can improve my professional status and satisfaction 102 (34.0) 127 (42.3) 36 (12.0) 19 (6.3) 16 (5.3) as a community pharmacist, i feel confident and prepared to provide breast cancer health promotion 94 (31.3) 138 (46.0) 31 (10.3) 20 (6.7) 17 (5.7) distributing breast cancer education materials is important in community pharmacy settings 77 (25.7) 137 (45.7) 33 (11.0) 34 (11.3) 19 (6.3) it is important to discuss breast cancer with my female patients to encourage breast cancer early screening and detection 97 (32.3) 117 (39.0) 27 (9.0) 25 (8.3) 34 (11.3) patients demand to get counseling on breast cancer screening and early detection from the community pharmacist 76 (25.3) 81 (27.0) 53 (17.7) 46 (15.3) 44 (14.7) patients appreciate efforts of community pharmacists when counselled about breast cancer 120 (40.0) 76 (25.3) 33 (11.0) 35 (11.7) 36 (12.0) iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 63 figure ( 1 ):barriers for breast cancer health as stated by community pharmacists. bars represent the percentage of community pharmacists agreeing to the barriers listed on the x-axis (n = 300). discussion the purposes of this study were to evaluate knowledge, attitudes and barriers of community pharmacists to breast cancer health educational services in iraq. the findings key of this study showed that the most of community pharmacists lack enough knowledge of breast cancer and new screening references. numerous reports from developing countries showed that breast cancer level of awareness about breast cancer and performance of screening was little amongst female population (28,29) . pharmacists are reachable healthcare specialists who are in direct contact with the public for long hours every day. recently, pharmacists are suitable progressively involved in a range of health care screenings and protecting services (12, 13, 30) . the consequences of this study established that community pharmacists in iraq hope to take an important role in breast cancer health promotion amongst the public. low level of knowledge of breast cancer screening references amongst community pharmacists in iraq can be described, in part, by lacking occurrence of continuous education plans and insufficient undergraduate teaching. though, these results established a vital need for continuing education plans intended for community pharmacists in order to be well-prepared to offer active breast cancer health education. it is also essential to study and expand oncology education in undergraduate pharmacy developments to attain the aims of improved health promotion roles for graduating pharmacists. in our study the overall attitude considered as favorable where community pharmacists perceived breast cancer counselling as a responsibility and a vital subject to discuss with their female patients. it is important to increase the community awareness of the health care services providing by community pharmacists through helpful hard work of community pharmacies with pharmaceutical families. previous instructions from developed countries showed that attitude and coordination of healthcare providers are significant determining factor for public participation in breast cancer screening programs (31 ,32) . in their report in 2001, giles et al. have assessed the results of a community pharmacy-based breast cancer awareness programmed, representing significant enhancements in rate of women carrying out selfexamination subsequent pharmacist-based intervention (33) . in this reading, iraqi pharmacists showed lack of time as the key barrier to breast cancer health promotion. where, community pharmacists are participating greater quantity of time in dispensing activities rather than given that patient education and health information. this universal barrier to substantial pharmacist educational performance can be overwhelmed by employing pharmacy technicians to bearing routine dispensing actions, thus provided that more time for patient-oriented services by registered pharmacists. other probable decisions to overcome time barriers would be to representative, with organization, most routine dispensing activities to pharmacist assistance, thus pharmacists might have additional time to offer pharmaceutical care (34,35) . absence of educational material was also observed as a barrier to breast tumor education by pharmacists in this study. for active patient education, community pharmacists should be providing with essential apparatuses, such as printed material, which can be simply providing to community in work locations. compared with qatar study earlier evaluation for involvement in breast cancer health education was conducted among community pharmacists in qatar (20) . similar to our study, assessment of breast cancer awareness among community pharmacists in qatar showed some knowledge limitations. the study among pharmacists in qatar indicated insufficient knowledge particularly for questions related to breast cancer risk factors and screening recommendations (20) . community pharmacists in qatar showed positive attitudes to be involved in breast cancer education. lack of personnel, time and privacy were considered major barriers among community pharmacists in qatar (20) . the results of our study showed a positive association between pharmacist’s knowledge of breast cancer and knowledge of breast cancer screening were associated with favorable attitude towards iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 64 involvement in breast cancer education among pharmacists. conclusion in this survey we found that iraqi community pharmacists have low level of knowledge about sign, symptoms and risk factor of breast cancer, in addition to knowledge of screening. however, the community pharmacists wish to increased their knowledge toward breast cancer. this improvement in knowledge of community pharmacy will be effective educators of the population about breast cancer. references 1. international agency for research on cancer: globocan 2012. lyon, 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eastern mediterranean region, 1st ed. cairo, world health organization regional office for the eastern mediterranean, 2010. 9. mutebi m, wasike r, mushtaq a, et al. the effectiveness of an abbreviated training program for health workers in breast cancer awareness: innovative strategies for resource constrained environments. springerplus 2013; 2: 528. 10. national institutes of health. what you need to know about breast cancer. washington, dc: national cancer institute, 1995. 11. daudt a, alberg aj, helzlsouer kj. epidemiology, prevention, and early detection of breast cancer. curr opin oncol 1996;8(6):455–61. 12. calis ka, hutchison lc, elliott me, et al. healthy people 2010: challenges, opportunities, and a call to action for america’s pharmacists. pharmacotherapy 2004; 24: 1241–1294. 13. anderson s. community pharmacy and public health in great britain, 1936 to 2006: how a phoenix rose from the ashes. j epidemiology community health 2007; 61: 844–848. 14. hourihan f, krass i, chen t. rural community pharmacy: a feasible site for a health promotion and screening service for cardiovascular risk factors. aust j rural health 2003; 11: 28–35. 15. ciardulli lm, goode jv. using health observances to promote wellness in community pharmacies. j am pharm assoc 2003; 43: 61–68. 16. armstrong k, eisen a, weder b. assessing the risk of breast cancer. n engl j med 2000;342(8):564–71. 17. hill d, white v, jolley d, et al. selfexamination of the breast: is it beneficial? meta-analysis of studies investigating breast self-examination and extent of disease in patients with breast cancer. br med j 1988;297(6643):271–5. 18. foster rs jr, lang sp, costanza mc, et al. breast self-examination practices and breastcancer stage. n engl j med 1978;299(6):265– 70. 19. baines cj. breast self-examination. cancer 1992;69(s7): 1942–6. 20. el hajj ms, hamid y. breast cancer health promotion in qatar: a survey of community pharmacists’ interests and needs. int j clin pharm 2011; 33: 70–79. 21. beshir sa, hanipah ma. knowledge, perception, practice and barriers of breast cancer health promotion activities among community pharmacists in two districts of selangor state, malaysia. asian pac j cancer prev 2012; 13: 4427–4430. 22. kachroo s. pharmacists should assume a larger role in overcoming the racial/ethnic barriers to breast cancer screening. j manag care pharm 2006; 12: 406–407. 23. holle lm, boehnke michaud l. oncology pharmacists in health care delivery: vital members of the cancer care team. j oncol pract 2014; 10: 142–145. 24. liekweg a,westfeld m, jaehde u . from oncology pharmacy to pharmaceutical care: new contributions to multidisciplinary cancer care. support care cancer 2004; 12: 73–79. 25. alkhasawneh im. knowledge and practice of breast cancer screening among jordanian nurses. oncol nurs forum 2007; 34: 1211– 1217. 26. alkhasawneh im, akhu-zaheya lm, suleiman sm. jordanian nurses’ knowledge and practice of breast self-examination. j adv nurs 2009; 65: 412–416. iraqi j pharm sci, vol.26(2) 2017 knowledge, attitudes and barriers towards breast cancer 65 27. madanat h, merrill rm. breast cancer riskfactor and screening awareness among women nurses and teachers in amman, jordan. cancer nurs 2002; 25: 276–282. 28. abu-helalah ma, al-shraideh ha, al-serhan ah, et al. knowledge, barriers and attitudes towards breast cancer mammography screening in jordan. asian pac j cancer prev 2015; 16: 3981–3990. 29. suleiman ak. awareness and attitudes regarding breast cancer and breast selfexamination among female jordanian students. j basic clin pharm 2014; 5: 74–78. 30. hassali m, awaisu a, shafie aa, et al. professional training and roles of community pharmacists in malaysia: views from general medical practitioners. malays fam physician 2009; 4: 71–76. 31. ibrahim na, odusanya oo. knowledge of risk factors, beliefs and practices of female healthcare professionals towards breast cancer in a tertiary institution in lagos, nigeria. bmc cancer 2009; 9: 76. 32. akhigbe ao, omuemu vo. knowledge, attitudes and practice of breast cancer screening among female health workers in a nigerian urban city. bmc cancer 2009; 9: 203. 33. giles jt, kennedy dt, dunn ec, et al. results of a community pharmacy-based breast cancer risk-assessment and education program. pharmacotherapy 2001; 21: 243–253. 34. awad a, waheedi m. community pharmacist’s role in obesity treatment in kuwait: a cross-sectional study. bmc public health 2012; 12: 863. 35. ghazal rm, hassan na, al-ahdab og, et al. barriers to the implementation of pharmaceutical care into the uae community pharmacies. iosr j pharm 2014; 4: 68–74. iraqi j pharm sci, vol.24(1) 2015 hypolipidemic effect of caffeic acid 18 hypolipidemic effect of caffeic acid isolated from arctium lappa cultivated in iraq, in hyperlipidemic rat model. mohammed a. taher * , dhuha a. abdulhussain ** , huda f. hasan ***,1 , zena m. fahmi ** , oday k. luaibi **** and mezaal g. ali ***** * department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmacognosy and medicinal plants, college of pharmacy, university of baghdad, baghdad,iraq. *** department of pharmacology and physiology, college of veterinary medicine, university of baghdad, baghdad,iraq. **** department of veterinary internal and preventive medicine, college of veterinary medicine, university of baghdad , baghdad,iraq. ***** ibn-alnafes hospital-ministry of health, baghdad, iraq. abstract the goal of the extant revision was to explore the influence of caffeic acid (ca) extracted from arctium lappa l. on lipid profile and histology of aorta in rats . analytical study demonstrated a high percentage of both chlorogenic and caffeic acid in the 80 % methanol extract of the aerial parts (leaves and stems) of arctium lappa l. from the family asteraceace. hypolipidemic activity of caffeic acid was studied against cholesterol induced hypercholesterolemia in wistar albino rats for thirty days. rats were separated into normal group (a), hypercholesterolemic positive controller group (b). while, the rest three groups (c, d and e) attended as hypercholesterolemic trial groups. caffeic acid was given verbally at doses of 20 mg / kg body mass in collection (c), 40 mg/kg in collection(d) then 20 mg/ kg in collection (e) after two weeks of administration of cholesterol induced hypercholesterolemia. there was an obvious histological improvement of changes induced by hypercholesteremia in aorta. furthermore, using (ca) at 20mg after 2 weeks of feeding on cholesterol rich diet led to a important decline (p<0.05) in serum whole cholesterol levels in compassion with the control and cholesterol rich diet group. moreover, there was a great improvement in the histology results convinced by the high cholesterol gorgeous diet, which can partly be attributed to an antioxidant activity of (ca). keywards: arctium lappa, caffeic acid , hypolipidimic, hypercholesterolemia. َسبة تخفَط عهي انعشاق فٌ انًضسوع االسقطَوٌ َبات يٍ انًعضول انكافََك حايط تأثَش .انذهَُة يفشطة انجشراٌ ًَورج فٌ انذو دهوٌ يحًذ عباط طاهش * ، ظحي عبذ انصاحب عبذ انحسٍَ ** ، هذى فالح حسٍ ***،1 ، صٍُة يُزساحًذ ** ، عذً كشٍى نعَبٌ **** و يضعم غَاض عهٌ ***** * ،جاهؼت بغذاد ، بغذاد ، الؼشاق .فشع الؼلىم الوخخبشَت السشَشَت ، كلُت الصُذلت ** فشع الؼمالُش والٌباحاث الطبُت ، كلُت الصُذلت ،جاهؼت بغذاد ، بغذاد ، الؼشاق . *** فشع االدوَت والفسلجت، كلُت الطب البُطشٌ ،جاهؼت بغذاد ، بغذاد ، الؼشاق . **** بغذاد ، الؼشاق .، كلُت الطب البُطشٌ ،جاهؼت بغذاد ، والىلائٍ فشع الطب الباطٌٍ ***** هسخشفً ابي الٌفُس ، وصاسة الصحت ، بغذاد ، الؼشاق . الخالصة خلص هي ًباث األسلطُىى ػلً حخفُض ًسبت الكىلسخشول فٍ الهذف هي هزٍ الذساست هى للبحذ ػي حارُش حاهض الكافاَُك الوسخ حاهضٍ الكافاَُك الذم ودساست الخغُُشاث الٌسُجُت فٍ الششَاى االبهش للجشراى. اظهشث الذساست الخحلُلُت اسحفاع فٍ ًسبت ائلت اسخشَسُا.حن دساست لابلُت والكلىسوجٌُك فٍ الوسخخلص الكحىلٍ لالجضاء الهىائُت هي ًباث األسلطُىى ) الجزع واالوساق( هي الؼ حاهض الكافاَُك لخخفُض ًسبت الذهىى فٍ الذم لوذة رالرىى َىهاً ػلً جشراى الىسخش البُضاء والخٍ حن اسخحذاد اصابخها باسحفاع ىًخشول وهجوىػت ك (a)الكىلسخشول فٍ الذم. حن حمسُن الجشراى فٍ هزٍ الخجشَت الً هجوىػت سُطشة غُش هصابت باسحفاع الذهىى كاًج الوجاهُغ الؼالجُت للجشراى الوصابت باسحفاع (c,d and e). بٌُوا الوجاهُغ الزالرت االخشي .(b)هصابت باسحفاع الكىلسخشول غن/ كغن هي 02و (c)غن/ كغن هي وصى الجسن لوجوىػت 02الكىلسخشول. حن اػطاء جشع حاهض الكافاَُك ػي طشَك الفن بجشػت غن/ كغن هي وصى الجسن للوجوىػت االخُشة بؼذ اصابخها باسحفاع الكىلسخشول باسبىػُي. حن 02بجشػت و (d) وصى الجسن لوجوىػت غن/ كغن هي وصى 02بجشػت هالحظت ححسي فٍ الخغُُشاث الٌسُجُت فٍ الششَاى االبهش. باالضافت اى اسخخذام حاهض الكافاَُك و (p<0.05)الجسن لوجوىػت االخُشة بؼذ اصابخها باسبىػُي اػطً ححسي واضح بالخغُُشاث الٌسُجُت وكزلك بٌسبت الذهىى بالذم .بالوماسًت هغ الوجاهُغ االخشي. هوا َبُي وجىد لابلُت هضاد لالكسذة فٍ حاهض الكافاَُك .االسقطَوٌ, حايط انكافََك , اَخفاض انذهَُة و وفشط انكونستَشول انكهًات انًفتاحَة: intoduction in the last few years great importance has been given to illnesses associated with high levels of blood cholesterol, plasma triglycerides with atherosclerosis and ischaemic heart disease. treatment of hyperlipidaemia is preferably dietary 1 corresponding author e-mail: dr.hudaalqaraghuli@yahoo.com received: 23/8/ 2014 accepted: 16/2/2015 iraqi j pharm sci, vol.24(1) 2015 hypolipidemic effect of caffeic acid 19 accompanied by other natural regimens. drug therapy was reversed for the more intractable conditions (1,2) . natural products has a beneficial phytochemical compound named caffeic acid (3,4dihydroxy cinnamic acid), it occurs regularly as the quinic acid ester, chlorogenic acid, it is an important ordinary dietary phenolic complex originate in plants which is an anti-oxidant. depressant the creation of leukotrienes which are complicated in immune-regulation, infection and sensitivity (3,4) . over the previous period, plant medication has develop a theme of worldwide prominence, making an influence on both biosphere fitness and global trade. curative plants stay to production a central heroine in the healthcare organization of large scopes of the world’s populace. this is chiefly factual in emerging kingdoms, where plant medication has a extended and continuous past of usage (5) . currently used hypolipidemic drugs are associated with so many adverse effects and withdrawal is associated with rebound phenomenon and this phenomenon referred to the appearance or recurrence of signs that were either lacking or measured while attractive a medication, but seem when that similar medication is discontinued, or concentrated in quantity. in the situation of recurrence, the strictness of the signs is often inferior than pretreatment levels, which is not seen with herbal preparations (6) . plant parts or plant extract are sometimes even more potent than known hypolipidemic drugs, this indicates that the research has stopped with just reporting the effect of plant derivate and the findings are not translated into clinical research (7) , taking these finding forward is mandatory to develop new drugs in this area, hence further research into identifying the active principle, conducting preclinical studies and if possible clinical studies is needed (8) . more than 70 medicinal plants have been documented to have significant hypolipidemic action. throughout the latter period, an upsurge in the use of remedial plants has been detected in metropolitan regions of advanced states. curative plants show a major part in hypolipidemic activity. advantages of herbal medicines reported are effectiveness, safety, affordability and acceptability (8-9) . materials and methods arctium lappa plant cultivated aerial parts in arctium lappa were collected from the department of the medicinal plants , college of agriculture, university of baghdad. method of extraction the authenticated plant parts were cleaned, dried in shade and exposed to maceration to catch rough powder. the indelicately powdered was extracted with methanolic alcohol in soxhlet device. the plant was vaporized to wetness under vacuity and dried out in void desiccators .the residue stored in a refrigerator at 2 – 8 0 c for use in the subsequent work (10) . method of separation the separation and purification of caffeic acid carried out using a combination of two or more chromatographic techniques.hplc analysis was carried out by (water/ireland) in the department of pharmacognosy, college of pharmacy, university of baghdad. hplc conditions for caffeic acid: -mobile phase: a: water 0.2% formylic acid; b: acetone-nitrile to methanolic acid (60:40 v/v). -column: c18 4.6mm x 250 mm. -detection: uv detector at 280 nm (11) . while the separation of caffeic acid was achieved by preparative thin layer chromatography (ptlc) in the traveling phase (ethyl acetic acid – ethanolic acid – water, 5 to 1 to5, v/v/v) in room heat. then detected under uv light (12) . animals and diet twenty five fit mature wistar albino rats weigh up (250-300 gm) were recycled in the extant study. rats were got from a laboratory animal household/ pharmacy college/ university of baghdad. experiments were achieved between march and april-2014. animals were sited in coops exposed to persistent environment circumstances. method of preparing the diet of high cholesterol was conducted by mixing 1gram of cholesterol per kilogram of body weight (gm/kg) with normal diet of animals. caffiec acid was given orally to animals at a doses of 20mg/kg/day and 40mg/kg/day. animals were separated to (4) equal collections (5 rats in every one) upon the following design: group (a): control (normal) that were fed normal nutrition. group (b): rats were given great cholesterol nutrition for 30 days. group (c): rats were given 20mg/kg/day of caffeic acid besides high cholesterol diet. group (d): rats were given 40mg/kg/day of caffeic acid besides great cholesterol diet. group (e): rats were given 20mg/kg/day of caffeic acid after two weeks of high cholesterol diet. * cholesterol (biochemical) bdh chemicals ltd poole england. http://en.wikipedia.org/wiki/symptom http://en.wikipedia.org/wiki/discontinuation iraqi j pharm sci, vol.24(1) 2015 hypolipidemic effect of caffeic acid 20 blood samples five at 2-8ºc for analysis. milliliters(5ml) of whole blood was drawn by heart puncture from each animal of all experimental groups after thirty days. the sample was transferred into plan tube, left at room heat for 15 min. for clotting, then put in centrifuge at 3000 r.p.m for 15 min., and finally serum was separated and preserved in a disposable tube and refrigerated biochemical parameters serum concentrations of tri-glyceride (tg), whole cholesterols (tc), height densities lipo-proteins-cholesterols (hdl-c) were sedate via usage kits from bio merieux, france. all procedures were followed according to the instructions of the manufacturer and rendering to lipid study clinics protocols (13) . low density lipoproteins of cholesterol was premeditated via usage of the method of friedewald et al. (14) tc –hdlc –tg/5. this formula is applicable when serum tg level is less than 400mg/dl. histopathological examination animals were ansthetized by cervical dislocation. aortic arches were stable in 10% formol, and surrounded in wax of paraffin; 5 micrometer-thick units (µm-t sections) were marked according to conventional methods, including hematoxylin and eosin(h and e). in addition, special stains were used to fix the slides as vangieson's and masson trichrome(a mixture of picric acid and acid fuchsin, which manufactured by american country) . determination of atherosclerotic area by intima-media thickness (imt) was conducted using calibrated ocular lens divided into millimeter (mm) for each histological section of the stained aorta with h and e (15) . arithmetical analysis arithmetical analysis was complete by consuming the statistical analysis system sas (16) . consequences were uttered as mean ± regular deviances (mean ± sd). one method anova-test was taken to compare limits in altered studied collections. p standards p < 0.05 were reflected statistically important. results chromatographic results concentration of the active phytochemical constituents in the sample was low and since the chemical tests and thin layer chromatography(tlc) were not enough to identify them. therefore, hplc was used to identify and provide a lot of information about the contents of the plant sample. in (figure 1) hplc chromatogram of the plant sample gave us a high peak of caffeic acid at retention time (rt 2.71) which is identical to the reference standard. from the area under the peak we calculated the percentage of caffeic acid in crude extract about 0.176mg/ml. figure (1): hplc chromatogram of caffeic acid std.(left one) and plant extract sample with high peak of caffeic acid at (rt 2.71) identical to the reference standard( right one). http://en.wikipedia.org/wiki/picric_acid http://en.wikipedia.org/wiki/acid_fuchsin iraqi j pharm sci, vol.24(1) 2015 hypolipidemic effect of caffeic acid 21 effects on serum lipid profiles table -1 revealed that whole cholesterols (tc), tri-glycerides (tg), low densities lipo-protein l.d.l and very low densities lipo-protein (v ldl) of group given cholesterol rich diet (group b) showed important increase (p<0.05) as compared with normal group, in adding the high density lipoprotein (hdl) in group b showed important decrease p<0.05 as related with normal group. (tc), (tg), (ldl) and (v ldl) in collections c, d and e displayed important decrease p <0.05 as comparison with collection b. while the hdl in collections c, d and e exhibited important increase p<0.05 as comparison with group b. the collection given 20 mg of ca after 2 weeks showed the best significant decline in serum total cholesterol p<0.05 comparison with the normal group and other preserved collections (c and d). all treated group indicated no important change p<0.05 in vldlcholesterol compared with control. ldl/ hdl ratio in group given cholesterol rich diet increases considerably p<0.05 as comparison with normal rats. table(1): effects of different doses of caffeic acid on lipids profiles in rats nursed on normal nutrition rich with high dose of cholesterol. * p<0.05 important differences from the normal group. a: p<0.05 important differences from group b between column numbers. b: p<0.05 important differences from group c and group d between column numbers. groups t.cholestero l m g/ d l triglyceride m g/ d l h.d.l cholesterol. m g/ d l l.d.lcholest. mg/ dl v.l.d.lcholeste rol. mg/ dl l.d.l /h.d.l a control 116.1± 4.12 101.13± 35.37 72.46± 2.62 23.4± 4.62 20.22± 7.07 0.32± 0.052 b cholest. rich diet 176.05± 35.57* 135.5± 75.49* 51.85± 7.75* 37.1± 13.67* 27.1± 15.09* 0.69± 0.17* c cholest. with20mgc .a. 95.52± 10.14 a 105.1± 21.83a 48.77± 4.34*a 25.7± 10.55a 21.02± 4.36a 0.52± 0.22*a d cholest. with 40mg c.a. 112.64± 14.08a 72.35± 16.11*a 61.6± 5.76 a 26.56± 8.61a 14.47± 3.22a 0.58± 0.099*a e cholest. with 20mg c.a. after 2 weeks. 85.82± 10.49*ab 84.62± 17.70*a 47.47± 5.87 *a 22.56± 8.79a 15.79± 4.22a 0.4± 0.21ab iraqi j pharm sci, vol.24(1) 2015 hypolipidemic effect of caffeic acid 22 the result of histopathological examination figure (1): group ahistological section in aorta of control rat showed normal aorta tissue (h and e.x400). figure (2): group bhistopathological section in aorta of rat given high cholesterol diet for 30 days showed degradation of elastic fiber and hyper enlargement due to infiltration of fat droplet between tunica media of aorta (h and e.x400). figure (3):group chistopathological section in aorta of rat given high cholesterol diet and treatment by caffeic acid 20 mg/kg/day for 30 days showed decrease of fat droplet between elastic fiber of tunica media of aorta (h and e.x400). figure (4):group dhistopathological section in aorta of rat given high cholesterol diet and treatment by caffeic acid 40 mg/kg/day for 30 days showed decrease size of tunica media of aorta due to disappear of fat droplet (h and e.x400). figure (5):group ehistopathological section in aorta of rat given high cholesterol diet for 30days , and treatment by caffeic acid 20 mg/kg/day after 2 weeks showed decrease in the size of tunica media and return to normal shape (h and e.x400). discussion and conclusion the results in table -1of groups given diet rich with cholesterol and treated with 20 and 40mg/kg of caffiec acid could be attributed to the activity of ca in decreasing serum total cholesterol levels in rats, in addition to any organic anion that competes with acetate can reduce the input of acetyl coa and block the first step in cholesterol synthesis and benzalbutyric acid (hypolipidemic drug ) impairs cholesterol and fatty acids synthesis by blocking acetylation of coenzyme a, the similarity in chemical structure of ca with βbenzylbutyric acid may suggest similar mechanism of action, this result agreement with result reported by bowman and rand (17) . moreover, many investigational and scientific studies are established the atherosclerosis and circumstances disposing to atherosclerosis like hypercholesterolemia, increase in pressure and patient with great smoke are related with augmented vascular creation of radical oxygen species (18) . there are another studies agreement with this result reported by nardini , et al ; yuchi, and kimura (19, 20) . β-benzylbutyric acid (21). caffeic acid (22) . iraqi j pharm sci, vol.24(1) 2015 hypolipidemic effect of caffeic acid 23 the influence of caffeic acid at the tg cholesterols and vldl-cholesterol might be attributed to ca decrease tri-glyceride creation by decrease the countenance of both (three hydroxyl three methy-glutary coenzyme a reductase) and glycerol three phosphate acyltransferase in the liver by arrangement of adenosine mono phosphate activated kinase (23) . while that increase of hdl-cholesterol, in all study groups treated with caffeic acid might be indicated that doses of ca may have beneficial effect on hdl-cholesterol, by its effect as anti-lipid oxidation and suggested that ca lead to diminish oxygen species, and thus reduces dna from impairment, which could be important in the regulation of liver function, this result agreement with result recorded by codrington, et al (24) . the result of ratio of ldl/hdl in cholesterol rich diet group agreement with result reported by danie, et al (25) , who found the low protein density/ high protein density percentage with apo-lipoproteins, apolipoprotein b/ apo-lipoprotein a1 fraction require a solid forecasters of coronary of artery diseases. the group treated with 20mg of ca after 2 weeks of feeding with cholesterol showed best improvement in the atherogenic index ratio (ldl/hdl) evidenced by better histopathological findings, this might be attributed to antioxidant activity of ca which inhibit the degeneration of cholesterols in low protein density, lead to decrease the creation of froth cell that give to atherosclerotic plaque, this results supported by viuda, et al (26) . further study is needed to test the arctium lappa containing ca to confirm the beneficial hypolipidimic effect of this medicinal plant arctium lappa (27) . in conclusion the arctium lappa has benficial effect in lowering lipid associated with improvement in histological changes occurred due to hyperlipidemia, the best group was that treated with 20mg of ca after 2 weeks of feeding with cholesterol rich diet, in which the tunica media return to normal shape. acknowledgements i would like to acknowledge dr. saged auda mohammad; 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104:213296. 21. thomas k. , angewanti chemie, 1979 ;18 ( 1): 1 – 90. 22. boerjan, w.; ralph, j. and baucher, m.. "ligninbiosynthesis". annual review of plant biology. 2003; 54: 519–46. 23. chung c. l. , ting t. o., hui p. h. andchau j. w.the inhibition of oleic acid induced hepatic lipogenesis and the promotion of lipolysis by caffeic acid via up-regulation of amp-activated kinase .2013;55:122-199. 24. codrington, a. m. ; hales, b. f. and robaire, b.: anti lipid oxidation of caffeic acid. 2007; 10-1095. 25. daniel j., rader h., michael d., h., richard.j., caplan b. and john s. pears lipid and apolipoprotein ratios: association with coronary artery disease and effects of rosuvastatin compared with atorvastatin, pravastatin, and simvastatin. american journal of cardiology. 2003; 91 (5a), 20c-24c. 26. viuda-m. m., fernándezl. j. and. pérez j.a pomegranate and its many functional components as related to human health, 2010; 22: 10-43. 27. fabricia s. p., ana l. r., joão e. c., mary a. f. and heidi j.k., antioxidative and in vitro antiproliferative activity of arctiu m lappa root extracts, 2011;24: 11-25. iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives doi: http://dx.doi.org/10.31351/vol27iss1pp69-78 69 synthesis, characterization and preliminary antimicrobial evaluation with dft study of new thiazole derivatives sumayah s.abbas* and ammar a. mahmood kubba*, 1 * department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract two compounds,[2-amino-4-(4-nitrophenyl)1,3-thiazole],(4) and [2-amino-4-(4-bromo phenyl)1,3-thiazole],(5), were synthesized by refluxing thiourea (1) with each of parantirophenacylbromide (2) and para-bromophanacyl bromides (3) respectively, in dry methanol. then, by reaction of compound [5] with 3,5-dinitrobenzoyl chloride in dimethylformamide (dmf) yielded compound (6) .on the other hand, reaction of compound (4) with chloroacetyl chloride in dry benzene afforded compound (7), which is upon treatment with thiourea in dry methanol, afforded compound (8) . the characterization of the titled compounds were performed utilizing ftir spectroscopy, 1hnmr, chns elemental analysis and by measurements of their physical properties. the synthesized compounds had been screened for their, in vitro preliminary antimicrobial activity against four gram positive bacteria (staphylococcus aureus, micrococcus luteus, bacillus subtilis and bacillus pumilus), and four gram negative bacteria (pseudomonas aeruginosa, escherichia coli, proteus mirabilis and klebsiella pneumoniae)and three fungi species: (saccharomyces cerevisiae, candida tropicalis and candida albicans) using a minimum inhibitory concentration (mic) of 100 µg\ml of a derivative in dimethylsulfoxide, by well diffusion method. compound (6) showed moderate antibacterial activity against some tested gram positive bacteria (bacillus pumilus and bacillus subtilis) and a moderate antifungal activity towards candida albicans. computational study was performed to calculate some of the thermodynamic parameters of synthesized derivatives by using density functional theory (dft). keywords: antimicrobial, thiazole, dft وظيفيةنظرية الكثافة ال المضاد للميكروبات مع دراسة االولي تقييموال و تشخيصتحضير ثيازولال لحلقة جديدة لمشتقات 1،*و عمار عبد الرزاق محمود كبه * سميه سعدي عباس .ق االعر،بغداد،بغداد ةجامع، ةفرع الكيمياء الصيدالني، ةكليه الصيدل* الخالصة من (5) [ ثيازول-1،3-( بروموفينيل-4) -4-أمينو-2] و (4) [ثيازول-1،3 -(فينيلنايترو-4) -4-أمينو-2] حضر المركبان: باستعمال ,على التوالي( 3)ومعوض برومو فيناسيل برومايد (2)برومايد معوض نايترو فيناسيل مع كل من( 1) ثيورياتصعيد ال ومن. (6) المركبلينتج مذيب دايمثيل فورماميدالفي كلورايد وبنزويلدينيتر-3،5 مع (5)مفاعله المركب تممن ثم . لجافالميثانول ا في مع الثايويوريابمفاعلته واالخير ,( 7)انتج المركب في البنزين الجاف كلورايد ستيلا وكلور مع (4)المركب تفاعل ىأخر ناحية (.8)المركبانتج الميثانول الجاف الدقيق التحليلو للبروتون المغناطيسي النوويالرنين و مطياف االشعة تحت الحمراء تعمال اسب المركبات المحضره تشخيص تم .للمواد المحضره الفيزيائية الخصائص قياس كذلك للعناصر و نقودية المكورات الع)انواع من البكتريا الموجبه لصبغه غرام, وهي: أربع للميكروبات ضد المختبري المضاد االولي النشاط تقييم تم لصبغه غرام, وهي: السالبة من البكتيريا وكذلك ضد أربعة انواع ،(عصوية بومليس العصوية الرقيقة و المكورات الدقيقة، الذهبية، و فطريات الخميرة) فطرياتال من وثالث انواع( الكليبسيلة الرئوية و االشريكية القولونية ,المتقلبة الرائعة الزائفة الزنجارية،) م من المادة مذابة في ا مل من الدايميثيل سلفوكسايد كأقل تركيز راغمايكرو100 عمالباست (والمبيضات البيض بيضات االستوائيةالم .مثبط وذلك باستعمال طريقة االنتشار معتدل نشاط و(عصوية بومليس العصوية الرقيقة و )الموجبه لصبغه غرام البكتيريا من بعض ضدمتوسط نشاط (6) مركبالأظهر الت اجراء دراسة حسابية لقياس بعض ,ريتم في االخ .البيض المبيضات للفطريات مضاد اايض عام م الديناميكية الحرارية ال .الكثافه الوظيفية باستعمال نظريه للمركبات المحضره ثيازول, نظريه الكثافه الوظيفية. ,كروبات يمضاد للم -الكلمات المفتاحية: 1corresponding author e-mail: kubbaammar1963@gmail.com received: 26/10/2017 accepted: 4/3/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp69-78 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives 70 introduction thiazole is a five membered aromatic heterocyclic ring containing sulfur and nitrogen in its structure. thiazole derivatives had received a great attention due to their remarkable reported different biological activities such as ,inhibitors of neuronal nitric oxide synthase(1), anti-cancer (2), antimicrobial (3-9), calcium-activated small conductance potassium channel blockers(10), antiulcerogenic(11), adenosine a3 receptor antagonists (12), antitumor (13), antifungal (14)and anti-inflammatory activities(15,16. the thiazole ring moiety is an integral part of many potent biologically active pharmaceutical products such as pramipexole (dopamine agonist indicated for treating parkinson's disease) (17), meloxicam\ (nsaid drug, cox-2 inhibitor) (18) and in many third generation cephalosporins\(19).thiazole ring is also found naturally in the essential vitamin b1 thiamin (20). dft calculations were used to study the quantitative structure-activity relationships (qsar). materials and methods all chemicals and solvents used during synthesis were of analytical grade and used without further purification. completion of reactions and the purity of compounds were ascertained by thin-layer chromatography (tlc), using silica gel gf254 (type 60) precoated aluminium sheets, merck (germany) exposed to uv-254nm light and the eluent used is ethyl acetate: n-hexane 4:6 for compounds (4), (7) and (8), and ethyl acetate: n-hexane 5:5 ,for compounds (5) and (6) to run tlc. melting points were determined using stuart smp3 melting point apparatus in open capillary tubes, and are uncorrected. fouriertransform infrared spectroscopy (ftir), (kbr disc) (υ,cm-1) were recorded using (biotech engineering management ftir-600, uk) at the university of baghdad /college of education for pure sciences ibn al-haitham /central service laboratory . furthermore, the elemental microanalysis of the synthesized compounds was done using (elemental vario micro cube instrument ,germany) in the university of mustansiriyacollege of pharmacy. 1hnmr spectra were recorded on bruker model ultra shield 300 mhz spectrophotometer with tetramethylsilane (tms) as an internal standard, chemical shift were expressed as (δ=ppm) and coupling constant in (hz), and it was run in al al-bayt university, amman, jordan. the synthetic method is depicted in scheme 1. chemical synthesis general method for synthesis of parent nucleus (4and 5) (21) a mixture of thiourea (1) (0.01 mol, 0.76g) and each of: 4-nitro phenacyl bromide (2) (0.005 mol, 1.22 g) or of 4-bromo phenacyl bromide (3), (0.005 mol,1.38 g) were dissolved in 100 ml of dry methanol in a round bottom flask and refluxed for 3-4 h. after completion of reaction, as monitored using tlc, the mixture was cooled to room temperature then poured into cold water. the solid separated was collected by filtration. the residue obtained was dried, recrystallized from absolute ethanol. 2-amino-4-(4-nitro phenyl thiazole) (4) orange powder; yield 80%; m.p. 288-291ᵒc; ir (kbr),(υ,cm-1): 3398 and 3305 cm-1 prim. (nh2) (str.); 3153 cm -1 ar-(c-h) str, 1641 cm-1 (c=n) str ,1593&1325 cm-1 asym. and sym. (n=o) str of no2,1539 cm -1 ( n-h) bend, 1502 cm-1 ar(c=c )str,1410 cm-1 (n=o) bend, 1107 cm-1 (c-n) str,1038 & 845cm-1 in plane & out of plane ar(c-h) bend, 717 cm-1 out of plane ar-(c=c) bend.,663cm-1 (c-s) str. 2-amino-4-(4-bromo phenyl thiazole) (5) off white to pink powder; yield 73%; m.p. 181-184ᵒc ; ir(kbr),(υ,cm-1): 3429 and 3282 cm-1 prim. (nh2) str, 3113 cm -1 ar(c-h) str, 1633 cm-1 (c=n) str ,1533 cm-1 (n-h) bend,1473 cm-1 ar(c=c) str,1198 cm-1 (c-n) str , 1038 & 906 cm-1 in plane& out of plane ar(c-h) bend., 727 cm-1 out of plane ar(c=c) bend., 669 (c-br) str, 636 cm-1 (c-s) str. general method for synthesis of (6) (22) 3,5-dinitrobenzoyl chloride (0.00078 mol,0.179 g) was added drop-wise to a stirring solution of compound (5) ,(0.00078 mol,0.2g) in dimethyl formamide (dmf). the mixture then refluxed for 4 h .after completion of reaction as monitored by tlc, the mixture was poured into distilled water to produce a precipitate which was collected by filtration. the residue was neutralized with 5% nahco3 to ph7, and subsequently washed with water, column chromatography was run using silica gel (60-120 mesh) and the mobile phase used; ethyl acetate: n-hexane (5:5) and recrystallized from aqueous methanol n-(4-(4-bromophenyl)thiazol-2-yl)-3,5dinitrobenzamide (6) light gray powder; yield 40%; m.p.165-168 ᵒc ; ir (kbr),(υ,cm-1):3410 cm-1 sec. amide (nh) str., 3066 cm-1 ar(c-h) str,1682 cm-1 (c=o) amide str, 1630 cm-1 (c=n) str,1572 cm-1 (n-h) amide bend., 1539&1346 cm-1 asym. &sym. (n=o)str, 1475cm-1 ar(c=c) iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives 71 str,1319 cm-1 (n=o) bend, 1288 & 908 cm-1 in plane& out of plane ar(c-h) bend., 1070 cm-1 (c-n) str, 729 cm-1 out of plane ar(c=c) bend. , 675 cm-1 for (c-s) str, 561 cm-1 for (cbr) str.; 1hnmr(300 mhz,acetone-d6,δ= ppm): 11.25(1h,s,nhco);9.15(2h,s,2arh);8.65(1h,s,ar-h);7.80(2h,d,br-2ar-h); 7.71(2h,d,br-2ar-h); 6.54(1h,s,h5thiazole(thz)); chns elemental microanalysis calc., for (c16h9brn4o5s) found: c%42.635, h%2.136, n%12.098, s%7.095; calc., c% 42.78,h% 2.02,n%12.47,s%7.14. general method for synthesis of (7) (23) a solution of compound (4) (0.005 mol, 1.10 g) in dry benzene (30 ml) was cooled to 0-5oc in an ice bath. chloroacetyl chloride (0.01 mol, 0.79 ml) dissolved in dry benzene (20 ml) was slowly added to the solution with vigorous stirring . when the addition was complete, the reaction mixture was stirred at room temperature for 30 min., then refluxed in a round bottom flask and a reflux condenser for 3 h. the reaction was monitored using tlc, and by using litmus paper which turns red indicative of hcl liberation. then benzene was removed in rotary evaporator. the residue was neutralized with 5% nahco3 to ph 7, and subsequently washed with water. the product was dried and recrystallized from methanol. 2-chloro-n-(4-(4-nitrophenyl)thiazol-2yl)acetamide (7) bright yellow powder; yield 82%; m.p. 214217 ᵒc ; ir(kbr)υ,cm-1: 3354 cm-1 sec. amide (n-h) str,3105 cm-1 ar(c-h) str, 3001&2947 cm-1 for asym. & sym. aliphatic (ch2) str,1701 cm-1 (c=o) amide str ,1597&1444 cm-1 asym. & sym. (n=o) str,1549 cm-1 (n-h) amide bend., 1504 cm-1 ar(c=c) str,1396 cm-1 (ch2) bend.,1331 cm-1 (n=o) bend.,1151 cm-1(c-n) str,1111&849 cm-1 in plane & out of plane ar(c-h) bend.,849 cm-1 (c-cl) str, 737 cm-1 out of plane ar(c=c) bend., 634 cm-1 for ( cs) str. ; 1hnmr(300 mhz,dmso-d6,δ= ppm):12.76(1h,s,nhco); 8.31(2h,d,no22ar-h);8.16 (2h,d,no2-2ar-h);8.06(1h,s,h5thz);4.43 (2h,s,coch2). ;chns elemental microanalysis calc., for (c12h11n5o3s2), found: c%44.66,h%2.853,n%14.13,s%10.428; calc.,c%44.38,h% 2.71,n%14.11,s%10.77. general method for synthesis of (8) (24) equimolar amount of compound (7) (0.00117mol,0.35g) and thiourea (0.00117 mol,0.089g) in dry methanol was refluxed for 12 h .the solvent was removed in rotary evaporator .the residue was neutralized with 5% nahco3 to ph 7,and subsequently washed with water ,filtered and dried. column chromatography was run using silica gel (60120 mesh) and the mobile phase used : ethyl acetate: n-hexane (4:6) to purify the titled compound, then recrystallized from aqueous methanol. n-(4-(4-nitrophenyl)thiazol-2-yl)-2thioureidoacetamide (8) light orange powder; yield 51%; m.p. 260 ᵒc( decomposed); ir(kbr),(υ,cm-1): 3398& 3303 cm-1 prim. (nh2) str,3141 cm -1 sec. amide (nh) str, 3116 cm-1 ar(c-h) str,2981 & 2927 cm1 asym.&sym. aliphatic (ch2) str, 1641 cm -1 (c=o) amide str, 1593cm-1 (n-h) amide bend.; 1537&1325 cm-1 asym. &sym. (n=o) str of (no2), 1502 cm -1 (c=n) str. overlapped with ar(c=c) str, 1038cm-1 (c-n) str. ; 1hnmr(300 mhz, dmso-d6,δ= ppm): 12.80(1h,br,nhco); 8.23(2h,d, 2ar-h); 8.04 (2h,d, 2ar-h); 7.40(2h,s,nh2); 7.25(1h,s,h5thz); 4.45(2h,s,ch2), ; chns elemental microanalysis calc. for (c12h11n5o3s2), found: c%42.300,h% 3.250, n%20.570, s%19.084 ;calc., c%42.72,h%3.29,n%20.76,s% 19.01. iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives 72 scheme 1. synthesis of titled thiazole derivatives. antimicrobial screening the antimicrobial activities of the synthesized derivatives were measured using well diffusion technique(25) with a comparison to cefotaxime sodium (cefot.) and sulfamethoxazole (sulf.) as standard antibacterial agents ,and miconazole as standard antifungal agent, using dimethylsulfoxide (dmso) as solvent and as a control, and it was run in the ministry of health / national center for drug control and research (ncdcr) /baghdad and in university of baghdad /college of education for pure sciences ibn al-haitham /central service laboratory. the synthesized compounds had been screened for their in vitro preliminary antimicrobial activity against four gram positive bacteria (staph.aureus, micrococcus luteus, bacillus subtilis and bacillus pumilus), four gram negative bacteria (pseud.aeruginosa, e.coli, proteus mirabilis and klebsiella pneumoniae) and three fungi (saccharomyces cerevisiae, candida tropicalis and candida albicans), using a minimum inhibitory concentration (mic) of 100 µg\ml of compound in dmso as shown in tables 1 and 2. results and discussion chemistry the synthesis of parent nucleus (4) and (5), was carried out according to hantzsch method, by refluxing thiourea (1) and 4iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives 73 substituted(4-bromo or 4-nitro) phenacyl bromides (2)and (3) in absolute methanol for 3 hours. they are characterized by ftir, due to appearance primary amine (nh2) stretching at 3398 &3305 cm-1 for compound (4) and 3429&3282 cm-1 for compound (5). compound (6) characterized by carbonyl amide (c=o) stretching at 1682 cm-1,while 1hnmr displayed (nhco) peak as singlet at δ=11.25 ppm, and the aromatic ring integrated for seven protons are displayed at their expected region ( see exp. part). compound (7), characterized by the appearance of (c=o) amide stretching at 1701 cm-1 and a characteristic peak, as a singlet due to (nhco) δ=12.76ppm in addition to the aromatic protons displayed at their expected region. compound (8) recorded in the ir spectrum two characteristic absorption bands of primary amine (side chain), (nh2) stretching, at 3398 and 3303 cm-1, while 1hnmr spectrum displayed a broad peak at δ=12.80 ppm, , attributed to nhco, also prominent peak at δ= 7.40ppm as a singlet due to nh2 of thiourea. it must be noted that the h5-thz recorded for the compound (7) and (8) as a singlet peak at δ= 8.06 and 7.25 ppm, respectively. antimicrobial activity from the data illustrated in tables 1 and 2, compound (6) showed moderate antibacterial activity against some tested gram positive bacteria (bacillus pumilus and bacillus subtilis), while compound (8) displayed no antimicrobial effect. in addition, compound (6) displayed moderate antifungal activity against candida albicans. table 1. antibacterial activity of the tested compounds. cpd. no. con c. µg/ ml staph. aureus micrococcus luteus bacillus pumilus bacillus subtilis pseud. aeruginosa e.coli proteus mirabilis klebsiella pneumoniae zone of inhibition (mm) (6) 100 _ _ 11.3 12 _ _ _ _ (8) 100 _ _ _ _ _ _ _ _ cefot. 100 50.11 59 41.5 45 32.5 53.2 27 28 sulf. 100 24 29 32.4 20 25 27.8 27 23 dmso _ _ _ _ _ _ _ _ _ table 2. antifungal activity of the tested compounds. compound no. conc. µg/ml saccharomyces cerevisiae candida tropicalis candida albicans zone of inhibition(mm) (6) 100 _ _ 10.8 (8) 100 _ _ _ miconazole 100 36.5 16 23.8 dmso _ _ _ _ (-)= no activity, slightly active (inhibition zone in between 5-10 mm), moderately active (inhibition zone in between 10-15 mm), highly active (inhibition zone more than 15 mm). (26-28) computational studies density function theory (dft) in order to explore the theoretical-experimental consistency, quantum chemical calculations were performed with complete geometry optimizations using standard spartan 10 software. geometry optimization was carried out by b3lyp / 6-31g* level of theory. the highest occupied molecular orbital (homo) and lowest unoccupied molecular orbital (lumo) play an important role in the molecule. these orbitals are sometimes referred to as the frontier molecular orbital. fig.1 shows the frontier molecule orbital density distributions of the investigated compounds .the homo orbital is an electron donor and the lumo orbital is the electron acceptor. the difference in energy between iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives 74 homo orbital and lumo orbital (energy gap) is a very effective property for characterizing the kinetic stability and chemical reactivity of the molecules. a molecule with a small lumo-homo energy gap is chemically more reactive and kinetically less stable. on the contrary, a large lumo homo energy gap corresponds to high kinetic stability and low chemical reactivity (29). structural and electronic properties dft calculations were performed for compounds (2 8). optimized molecular structures of the most stable form are shown in figure 1. homo lumo compound (2) homo lumo compound (3) homo lumo compound (4) iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives 75 homo lumo compound (5) homo lumo compound (6) homo lumo compound (7) iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives 76 compound (8) figure 1. highest occupied molecular orbital (homo) and the lowest unoccupied molecular orbital (lumo) for compounds (2–8). as shown in table 3, the result of theoretical calculations reveals, that in the case of the compounds (2-8), the energy of highest occupied molecular orbital (ehomo) are 10.804, -10.749, -8.339, -8.086, -2.264, -1.062 and -8.038 ev and the energy of the lumo orbital (elumo) are -7.084, -4.120, -2.943, 1.227, 1.93, 0.474 and -4.826. thus, the frontier orbital gap are about 3.72, 6.629, 5.396, 6.859, 4.194, 1.536 and 3.212 ev in the compounds (2-8), respectively. as a result, the compound with less energy gap has more kinetic stability and less chemical reactivity than others, like compound (7), whereas the compound with more energy gap has less kinetic stability and more chemical reactivity than others , for example, compound (3). the linkage between the molecular structures of compounds with its respective biological activities is central to the qsar paradigm. molecular descriptors play a crucial role in providing numerical description of the physicochemical properties of molecules. in order to properly account for these structural features, it is essential that suitable descriptors be chosen for qsar investigation. electronegativity (μ) essentially provides a measure of the asymmetric distribution of charges in a molecule (29) . it can be seen that compounds with the highest electronegativity were compound 2 > 3 > 8 >4> 5>7> 6 with values. it was observed that compound (2). such high value 8.944 is associated with the asymmetric distribution of electrons as afforded by the strong electron withdrawing nature of compound (2). log p provides a measure of a molecule’s lipophilicity where high log p value indicates high lipophilicity, while low value suggests low lipophilicity. the results indicated that compounds having the highest lipophilicity were 5 > 3 > 2 with corresponding values of 3.51, 2.82 and 1.73, respectively. the compound (5), set of a molecule possessed the highest lipophilicity.it can be observed that the energy gaps between homo and lumo of compounds (2-8) is 5>3>4 >6 >2>8>7. the larger the homo–lumo energy gap, the harder and more stable/ less reactive the molecule, for example, compounds (4), (5) and (8). electrostatic potential charges and related quantum chemical properties the distribution of the electronic density (electrostatic potential charges), related quantum chemical parameters . the highest occupied molecular orbital (homo) and lowest unoccupied molecular orbital (lumo) play an important role in the molecule,homo/lumo gap (table 3, row 4) and the partition coefficients of the compounds (log p; table 3, row 6)] were calculated for observed compounds. table 3. homo and lumo, and electronic properties units for compounds (2-8) using dft with b3lyp/6-b3lyp/6-31g* basis set. compound (8) compound (7) compound (6) compound (5) compound (4) compound (3) compound (2) parameters -2.9732 -3.3102 10.6845 5.9262 5.9258 14.3827 17.084 total energy kcal/mol: -8.038 -1,062 -2.264 -8.086 -8.339 -10.749 -10.804 homo ev -4.826 0.474 1.93 -1.227 -2.943 -4.120 -7.084 lumo ev 3.212 1.536 4.194 6.859 5.396 6.629 3.72 energy gap ev 6.432 0.294 0.132 4.657 5.641 7.435 8.944 electro negativity(μ)/ ev 3.51 2.82 1.73 log p iraqi j pharm sci, vol.27(1) 2018 synthesis and dft study of new thiazole derivatives 77 conclusion in the present work new derivatives were synthesized starting from,[2-amino-4-(4nitrophenyl) 1,3-thiazole], (4) and [2-amino-4(4-bromophenyl) 1,3-thiazole],(5) using conventional method, the titled derivatives (6) and (8) were evaluated for their preliminary antimicrobial activity using well diffusion method. compound (6) exhibited moderate antibacterial and antifungal activity against some gram-positive bacteria (bacillus pumilus , and bacillus subtilis) and one fungal species (candida albicans). the titled thiazole derivatives were studied theoretically by using dft calculations. the quantum chemistry calculations using the density function theory, (dft) method of biologically active molecules and can be used for a building of quantitative structure-activity relationship (qsar) model in the future. both experimental techniques and theoretical methods were used to 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jabbar*,1 and ali a. kasim ** * ministry of health and environment ,thiqar health directorate, nasiriya , iraq. **department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad, iraq. abstract retinol binding protein 4 (rbp4), an adipokine that participate in a lipid metabolism or insulin resistance through a complex regulatory network. recently, rbp4 was reported to be associated with many cardiovascular diseases (cvds) risk factors in type 2 diabetes mellitus (t2dm) patients. this study aims to study the correlation of serum rbp4 with some markers of glycemic control, dyslipidemia, hypertension, and obesity in t2dm iraqi patients. one hundred twenty (120) t2dm patients were enrolled in this cross-sectional study. serum rbp4 levels are higher in t2dm patients with dyslipidemia, hypertension, or obesity. the diference was statistically non significant between hypertensive and normotensive t2dm patients. serum rbp4 is positively correlated with body mass index, fasting blood glucose, glycosylated hemoglobin (hba1c), systolic blood pressure (p<0.001), and low density lipoprotein cholesterol (p<0.05), and negatively correlated with high density lipoprotein cholesterol (p<0.001). serum rbp4 is correlated with many risk factors of cvd including, poor glycemic indices, dyslipidemia, hypertension, and obesity, in t2dm iraqi patients. keywords: rbp4, type 2 diabetes mellitus, dyslipidemia, hypertension, obesity. في والسمنة الدم، ضغط ارتفاع الدم، شحوم الدم، جلوكوز مع 4 – للريتينول الرابط البروتينارتباط في العراق الثاني النوع السكري مرضى **علي عبد الحسين قاسم و 1*،جبار ثائر لطيف والبيئة ، دائرة صحة ذي قار، الناصرية،العراق وزارة الصحة* .، بغداد ، العراق الصيدلة، جامعة بغدادفرع العلوم المختبرية السريرية، كلية ** الخالصة حديثا،. معقدة تنظيمية شبكة خالل من األنسولين مقاومة أو الدهون استقالب في كبير بشكل (rbp4)) 4 الريتينول ربط بروتين يشارك إلى الدراسة هذه تهدف. 2 النوع من السكري مرضى لدى الوعائية القلبية الخطر عوامل من بالعديد rbp4 ارتباط عن التقارير العلمية تتحدث لدى والسمنة الدم ضغط وارتفاع الشحوم، اضطراب نسبة الدم، في السكر نسبة على السيطرة مؤشرات بعض مع rbp4 مصلال ارتباط دراسة .2 النوع من العراقيين السكري مرضى كانت الدم في rbp4 مستوياتالمستعرضة. الدراسة هذه في 2 النوع السكري مرضى كانوا من مشارًكا 120 مجموعه ما تسجيل تم االختالف لم يكن ذو داللة إحصائية السمنة. أو الدم، ضغط ارتفاع أو الدم، شحوم اضطراب يعانون الذين 2 النوع من السكري مرضى لدى عالية الجسم، لةكت بمؤشر إيجابيًا ارتباًطا rbp4 المصل أظهر. بين مرضى السكري المصابين ارتفاع ضغط الدم عن اوالئك ذوو الضغط المنتظم الكثافة المنخفضة الدهني ذو البروتين وكولسترول، (p <0.001) االنقباضي الدم وضغط والهيموغلوبين السكري المتراكم، في الصيام، الدم وجلوكوز (p <0.05) الكثافة العالية ذو الدهني البروتين كوليسترول مع سلًبيا وارتباطا p<0.001).) الدموية كضعف مؤشرات تنظيم سكر الدم او اضطراب الشحوم واألوعية القلب ألمراض الخطر عوامل من بالعديد rbp4 المصل يرتبط العراق. في 2النوع مرضى السكري من او ارتفاع ضغط الدم او السمنة في عينة من . السمنة الدم، ضغط ارتفاع الدم، شحوم اضطراب ،2 النوع من السكري داء ،rbp4 الكلمات المفتاحية: introduction diabetes mellitus (dm) is a common endocrinopathy which is usually presented as a set of metabolic defects characterized mainly by hyperglycemia as a result of impairment in insulin sensitivity and/or secretion (1). long-term 1corresponding author e-mail: thairjabbar9@gmail.com received: 6/7 /2020 accepted: 31/8 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp263-270 iraqi j pharm sci, vol.29(2) 2020 rbp4 and cvds risk factors in type 2 dm patients 264 hyperglycemia causes damage and dysfunction of multiple organs; including kidneys, heart, eyes, nerves, and blood vessels. the cardiovascular diseases (cvds) are the main cause of morbidity and mortality in dm patients (2), as indicated by the intensive regulation of glucose and lipid levels, in addition to blood pressure and body weight are essential to reduce the hazard of cardiovascular complications and diabetes advancement (3). type 2 diabetes (t2dm), hypertension, and dyslipidemia can occur comorbidly and all participate in the development of atherosclerosis (4), an important pathological hallmark in the cardiovascular diseases (cvds). obesity is common in about 90–95% of adult type 2 dm patients (5). it is manifested by low-grade chronic inflammation with increased systemic adrenergic activity, and perturbed metabolism of lipid and glucose (6). retinol binding protein 4 (rbp4) is an adipokine, synthesized primarily by the liver; a smaller amount is synthesized in the adipose tissue (7). increased systemic rbp4 concentrations can be seen in conditions of impaired glucose tolerance, t2dm, and obesity. the link of rbp4 with insulin resistance (ir) shows certain degree of controversy (8, 9). in the liver, rbp4 increases fasting blood glucose by augmenting the action of phosphoenolpyruvate carboxykinase (pepck) enzyme increasing hepatic glucose liberation. in skeletal muscle and white adipose tissue, rbp4 decreases the insulin receptors, as well as the alterations in phosphorylation of some signal proteins, such as insulin receptor substrate (irs), pdk1 and akt (10, 11). apart from its effects on glucose metabolism, rbp4 also affects lipid metabolism. graham et al. described that high rbp4 levels were associated with high serum triacylglycerol (tg) and low high-density lipoprotein cholesterol (hdl-c) levels in lean, obese, and in diabetic subjects (12). in a study conducted on chinese obese subjects, wang et al. showed that rbp4 affects lipid metabolism in gender dependent manner. he also reported that high serum rbp4 correlates with high total cholesterol (tc) and tg levels in chinese obese female subjects. also, females with high serum tg or tc levels have higher serum rbp4 levels (13). moreover, genetic variant of apolipoprotein a5 (apoa5 gene) enhances the association of serum levels of rbp4 and tg in t2dm patients (14). the aim of this work was to study the associations between circulatory rbp4 and different risk factors for cardiovascular diseases in a sample of iraqi t2dm patients. subjects and methods one hundred twenty (120) t2dm patients were enrolled in this cross sectional study, which was conducted in the specialized center of endocrinology and diabetes in al-nasiriya city, south of iraq from 4th october 2019 to 30th march 2020. t2dm patients were already diagnosed according to the american diabetes association criteria (15); of age ≥ 18years with mean age of them was (50 ±8) years, ranging from 33 to 69 years, and fifty-two of the diabetic subjects were males and 68 were females. with disease duration since diagnosis of at least 1 year. pregnant women, patients with chronic renal failure and hepatic failure were excluded in this study. this work was performed according to the helsinki ii declaration (16) and approved by the ethics committee of the college of pharmacy, university of baghdad. all participants were informed about the purpose and the expected benefits of the study before agreement of participation was documented. sociodemographic and medical histories were taken for each participant; venous blood samples were collected after fasting for 12 hours. ten milliliters blood samples were withdrawn, about (2 ml) of each sample was transferred into ethylene diamine tetracetic acid (edta) tubes to be stored at (+2 to +8 c) for analysis of hba1c within 1 week by high-performance liquid chromatography (17). the remaining (8 ml) samples were transferred into plane tubes and centrifuged for 10 minutes to obtain serum. the collected sera were stored frozen at (– 20oc) as aliquots in eppendorf tubes until analysis. fasting blood glucose (fbg) and lipid profile were analyzed using suitable colorimetric assays (18-22). the assay of serum rbp4 was performed by commercially available human enzyme-linked immunosorbent assay (elisa) kit (23). statistical analysis the statistical analysis was achieved using spss® v.25. data are presented as mean ± standard error. student t-test was used to compare means of two groups; while, analysis of variance (anova) test was used to compare means of more than two groups, which is followed by post hoc analysis using tukey’s test to describe differences among different groups. categorical variables were expressed as number and tested using chi-square (χ2) test. the correlation between different parameters and rbp4 was tested using pearson’s correlation. level of significance is set to p<0.05. results based on the dyslipidemia status, t2dm patients were grouped as dyslipidemic (n=66), and normolipidemic (n=54); according to “the american association of clinical endocrinologists’ (aace)” 2017(24). sociodemographic and clinical characterizes for each group are presented in table 1. there difference between the dyslipidemic and normolipidemic t2dm patients concerning gender, body mass index (bmi), smoking habit, diastolic blood pressure (dbp), and high density lipoproteins cholesterol (hdl-c) was non-significant (p>0.05). meanwhile, age, disease duration, systolic blood iraqi j pharm sci, vol.29(2) 2020 rbp4 and cvds risk factors in type 2 dm patients 265 pressure (sbp), serum rbp4, fasting blood glucose (fbg), glycosylated hemoglobin (hba1c), and low density lipoproteins cholesterol (ldl-c) were significantly higher in diabetic patients with dyslipidemia (p<0.05). table1. characteristics of dyslipidemic and normolipidemic t2dm patients p-value t2dm variable normolipidemic (n=54) dyslipidemic (n=66) 0.01* 48±1 51.6±0.9 age( years) 0.6 22/32 30/36 gender (m/f ) 0.2 28.9±0.6 29.8±0.4 bmi(kg/m2 ) 0.0001* 6±0.6 11±0.5 disease duration (years) 0.8 18 23 smokers number 0.04* 129±2 135.8±2 sbp (mmhg) 0.7 77.9±1 78.7±1 dbp (mmhg) 0.0001* 377.5±19 519.9±23 s.rbp4(pg/ml) 0.0001* 172.2±7 218.3±7 fbg (mg/dl) 0.0001* 8.2±0.2 9.6±0.2 hba1c% 0.0001* 224.8±1 43.4±2.6 vldl-c(mg/dl) 0.018 90.4±4 108.7±6 ldl-c (mg/dl) 0.06 46.1±1 42.6±1 hdl-c (mg/dl) * significant when p<0.05 in table-2, t2dm patients were grouped according to the prevalence of hypertension, diagnosed according to “the world health organization international society of hypertension guidelines” (25). there difference between the hypertensive and normotensive t2dm patients concerning gender, smoking habit, serum rbp4, fbg, hba1c%, and lipid profile parameters was nonsignificant (p>0.05). meanwhile, age, disease duration, and bmi were significantly higher in hypertensive t2dm patients (p<0.05). table2. characteristics of hypertensive and normotensive t2dm patients p-value t2dm variable normotensive (bp≤140/90 mmhg) (n=90) hypertensive (bp>140/90 mmhg) (n=30) 0.001* 48±0.8 54±1.3 age (years) 0.4 37/53 15/15 gender (m/f) 0.03* 28.9±0.4 30.7±0.7 bmi (kg/m2) 0.001* 8±0.4 11±1 disease duration (years) 0.2 28 13 smokers number 0.12 413.7±18 449±28 s.rbp4 (pg/ml) 0.5 195.2±6 204±11 fbg (mg/dl) 0.3 8.9±0.19 9.3±0.2 hba1c% 0.5 178±9 165±19 tg (mg/dl) 0.19 183±4 169±9 tc (mg/dl) 0.5 35±2 33±3 vldl-c (mg/dl) 0.3 102±4 93±8 ldl-c (mg/dl) 0.4 44.1±1 43.6±1.6 hdl-c (mg/dl) in table-3, t2dm patients were categorized into three groups, depending on bmi, into: normal weight, overweight and obese; non of the participant was underweight (26). serum rbp4 levels were significantly higher in obese t2dm patients than in normal weight patients (p=0.0001). similarly, diabetes duration, systolic bp, and the diastolic bp were significantly higher and serum hdl-c levels were significantly lower in obese t2dm patients compared to the normal weight patients (p<0.05). hba1c was significantly higher in obese t2dm patients compared to normal weight and overweight patients (p<0.05). there was no significant difference in age, gender, smoking habit, iraqi j pharm sci, vol.29(2) 2020 rbp4 and cvds risk factors in type 2 dm patients 266 fbg, total cholesterol (tc), triglyceride (tg), very low density lipoprotein (vldl-c) and ldl-c among obese, overweight and normal weight t2dm patients. table-3. characteristics of obese, overweight, and normoweight t2dm patients variable t2dm p-value obese bmi>30 n=54 overweight bmi(25-30) n=46 normoweight bmi(18.5-24.9) n=20 age (years) 49±1 51±1 49±2 0.5 gender (m/f) 25/29 21/25 6/14 0.4 disease duration (years) 10±0.7a 8.9±0.6ab 5±0.8b 0.001* smokers number 20 17 4 0.3 sbp (mmhg) 135±2a 134±2ab 121±2b 0.01* dbp (mmhg) 80±1a 77±1ab 74±2b 0.03* s.rbp4 (pg/ml) 499.5±21a 453.8±21ab 311.3±29b 0.0001* fbs (mg/dl) 196±5 196±8 171±15 0.5 hba1c% 9.5±0.2a 8.7±0.2b 8±0.4c 0.007* tg (mg/dl) 184±14 178±12 142±15 0.2 tc (mg/dl) 185±7 178±6 167±10 0.3 vldl-c (mg/dl) 36±2 35±2 28±3 0.24 ldl-c (mg/dl) 106±6 98±5 89±9 0.3 hdl-c (mg/dl) 42±1b 44±1ab 49±2a 0.02* superscripts (a,b,c) among different groups are considered significantly different (p<0.05) correlation studies of serum rbp4 with the studied variables of t2dm patients are presented in table4. serum rbp4 was positively correlated with bmi, fbg, hba1c, and sbp (p<0.001), and ldlc (p<0.05) and negatively correlated with hdl-c (p<0.001). while, there was no significant correlation between serum rbp4 levels and age, gender, dbp, or tg, vldl-c, and tc levels (p>0.05). strong positive correlation between serum rbp4 levels and diabetes duration in years was also recorded (p < 0.001). table4. pearson’s correlations of serum rbp4 levels with the studied variables variable r-value p-value age 0.14 0.13 gender -0.1 0.15 disease duration 0.5 0.000* bmi 0.31 0.001* sbp 0.2 0.02* dbp 012 0.17 fbg 0.6 0.000* hba1c 0.4 0.000* tc 0.17 0.06 tg 0.12 0.17 vldl-c 0.12 0.17 ldl-c 0.25 0.008* hdl-c -0.45 0.000* discussion in the present study, serum rbp4 has significant positive correlation with markers of glycemic control, fbg and hba1c (table 4). large mass of evidence suggest an important role played by rbp4 in the development of t2dm and its chronic complications, as well as, with biochemical markers of carbohydrate metabolism. elevated serum rbp4 levels associate with an augmented risk of transferring impaired glucose tolerance into overt t2dm (27). in addition, it is associated with ir in t2dm patients (28, 29). moreover, rbp4 expression is elevated in t2dm patients with retinopathy (30), nephropathy (31) and cvd (32). graham et al. reported positive correlation of serum rbp4 levels with fbg and with hba1c levels (12). however, serum rbp4 level and rbp4 synthesis speed were reported to be diminished in type 1 dm patients iraqi j pharm sci, vol.29(2) 2020 rbp4 and cvds risk factors in type 2 dm patients 267 compared to control individuals (33, 34). as stated earlier, rbp4 participates in ir by increasing the hepatic liberation of glucose, an action mediated by augmenting pepck, and by decreasing the insulin receptors expression and interfering with intracellular insulin signaling in muscle and adipose tissue (10, 11). unique pattern of dyslipidemia occurs in t2dm patients, characterized by hypertriglyceridemia, high blood levels of apolipoprotein b and of small dense ldl (sdldl), with low hdl-c levels. serum tc levels are high but not to the levels of tg (35). alterations in lipid profile in dm are attributed to increased lipolysis secondary to insulin resistance, and hence, there is an inappropriate spillover of tg and free fatty acids (ffas) (36). in the present study, serum rbp4 levels in dyslipidemic diabetic patients were higher than in normolipidemic patients. dyslipidemic t2dm patients were older with longer diabetes duration, higher systolic blood pressure and poorer glycemic control as compared with normolipidemic t2dm patients (table-1). moreover, serum rbp4 positively correlated with ldl-c level and negatively correlated with hdl-c levels, but there was no significant correlation with tg and vldlc levels (table4). this may be explained by the use of antilipidemic medicines by most of t2dm patients that may disguised the actual relationship between serum rbp4 and lipid profile. mounting data proposes that rbp4 contributes in lipid metabolism by means other than its role in ir. many clinical studies showed that serum rbp4 levels correlate positively with blood tg levels, but not with ir (37-39). however, some studies that examined the link between serum levels of rbp4 and tg have showed an association with ir (40, 41). a positive association between serum levels of rbp4 and atherogenic lipoproteins in t2dm patients has been reported previously (37, 42, 43). yamasaki et al. proposed that rbp4 may interfere with lipoprotein remnant metabolism (42). while, vergès et al. showed that rbp4 may reduce the catabolic rate of vldl (37). many studies showed a close association between serum rbp4 levels and blood pressure in prehypertensive or in untreated essential hypertension patients (44, 45). in the present study, the difference in serum rbp4 levels between hypertensive and normotensive t2dm patients was statistically nonsignificant (table-3). yet, serum rbp levels correlate positively with the sbp but not the dbp levels (table -4). high prevalence of hypertension in type 1 or 2 dm patients, is well established (46, 47). actually, about 40% of t2dm patients are already hypertensive when diagnosed as having dm (48). ephraim et al. reported that 37.4% of t2dm patients have systolic, but not diastolic, hypertension; and hyperglycemia in those patients was worse than in normotensive t2dm patients (49). hyperglycemia aids in vascular stiffness and raised vascular tonicity resulting in increased sbp (50). arterial stiffness develops mainly as a consequence of aging (51). pinto et al. has reported an increase in sbp in hypertensive patients, aged 50 years or older, arising either as primary event or developed after prolonged period of systolic-diastolic hypertension whether on antihypertensive treatment or not (46). still, progression of arterial stiffness can be hastened if it is co-occupied by additional risk factors, particularly elevated blood glucose levels, (52, 53). hyperglycemia, enhance development of arterial stiffness via several mechanisms that lead to endothelial dysfunction along with structural modifications of vascular extracellular matrix (54, 55). in the present study, obese t2dm patients have elevated serum levels of rbp4 when compared to normoweight t2dm patients. furthermore, obese t2dm patients have longer diabetes duration, and higher sbp, dbp and hba1c levels, when compared to normoweight t2dm patients. on the contrary, hdl-c levels in obese t2dm patients are lower than that of normoweight t2dm patients (table3). additionally, serum rbp4 levels show significant positive correlation with bmi (ttable 4).as rbp4 is an adipokine that is likely relating obesity, ir and t2dm (12, 56-58). graham et al. reported a positive correlation between serum rbp4 and bmi or waist-to-hip ratio; and normoweight individuals with ir also showed elevated levels of serum rbp4 indicating that ir was independent of obesity (12). in severely obese patients, expression of rbp4 is enhanced in the omental visceral fat (59). significant reduction in body weight, by dietary restrictions, physical workout, or by surgical interventions, leads to a reduction in rbp4 in blood and/or in adipose tissue (12, 60-62). the decrease in rbp4 in non-diabetic individuals, accompanying bodyweight reduction, positively associate with the reduction of fat content in the omental or mesenteric regions and not with the reduction in the whole body fat (61). several gene variants of rbp4 were associated with the level of adiposity and central obesity characterized by elevated bmi and waist-to-hip ratio (63). a regulatory single‐nucleotide polymorphism (snp) in its gene enhanced rbp4 expression with predisposition for obesity, by increasing lipogenesis (56). in addition, diabetes associated rbp4 haplotype carriers have been reported to overexpress visceral rbp4 (63). conclusion serum rbp4 correlates with different risk factors of cardiovascular diseases; including glycemic control, dyslipidemia, hypertension and obesity, in 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are not related to diet-induced changes in insulin sensitivity. journal of clinical endocrinology and metabolism. 2007;92:2330-5. 63. kovacs p, geyer m, berndt j, kloting n, graham t, bottcher y, et al. effects of genetic variation in the human retinol binding protein-4 gene (rbp4) on insulin resistance and fat depot-specific mrna expression. diabetes. 2007;56:3095-100. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole doi: http://dx.doi.org/10.31351/vol27iss1pp79-88 79 synthesis, characterization and antimicrobial evaluation with dft study of new two-amino-4-(4-chlorophenyl) thiazole derivatives nedaa a. a. rahim and ammar a mahmood kubba*,1 *department of pharmaceutical chemistry, college of pharmacy ,university of baghdad, baghdad, iraq. abstract 2-amino-4-(4-chloro phenyl)-1,3-thiazole (1) was synthesized by refluxing thiourea with parachloro phenacyl bromide in absolute methanol. the condensation of amine compound (1) with phenylisothiocyanate in the presence of pyridine will produce 1-(4-(4-chlorophenyl)thiazol-2-yl)-3phenylthiourea(2), which is upon treatment with 2,4 dinitrophenyl hydrazine by conventional method, afforded 1( 4 ( 4 – chlorophenyl ) thiazol – 2 – yl ) – 3 phenylhydrazonamide,n' ( 2 , 4 dinitrophenyl) ,(3).the characterization of the titled compounds were performed utilizing ftir spectroscopy, 1hnmr and chns elemental analysis, and by measurements of their physical properties. the synthesized compounds had been screened for their, in vitro preliminary antimicrobial activity against three gram-positive bacteria: (staph. aureus, micrococcus luteus and bacillus subtilis) and three gram-negative bacteria : (pseud.aeruginosa, e.coli and proteus mirabilis) ,and two fungal strains(candida albicans and candida glabrata ), using a minimum inhibitory concentration (mic) of 100 µg\ml of test compound, by well diffusion method. the derivatives showed moderate antibacterial activity against gram-positive staphylococcus aureus and bacillus subtilis & high antifungal activity against candida glabrata and candida albicans. computational study was performed to calculate some of thermodynamic parameters by using density functional theory (dft) keywords: antibacterial, 2-aminothiazole, ssynthesis and dft. من جديدة لمشتقين فيةالوظي الكثافة نظرية دراسة مع للميكروبات المضاد والتقييم تحضير [ثيازول-1،3( كلورو فينيل-4) -4-أمينو-2] 1*،كبه دعمار عبد الرزاق محموو نداء عبد الحميد عبد الرحيم العراق.،بغداد،جامعة بغداد ،كلية الصيدلة ،فرع الكيمياء الصيدالنية* الخالصة مع كلورو فيناسيل البروميد باستعمال ( من تصعيد الثيوريا1) [ثيازول-1،3( كلوروفينيل-4) -4-أمينو-2] حضر المركب: (, 2كب )بوجود البيريدين لينتج المر الفنيل ايسو ثايوسيانيتالميثانول المطلق ومن ثم مفاعلة مجموعة االمين من المركب السابق مع مع (3ثنائي نايتروفنيل هايدرازين بوجود االيثانول المطلق لينتج المركب النهائي ) -2،4( مع 2ثم التصعيد ثانيا للمركب ) لفيزيائية. االدقيق للعناصر و كذلك قياس الخصائص المركبات المحضره باستعمال : مطياف االشعة تحت الحمراء,والتحليل تشخيص تم انواع من البكتريا الموجبه لصبغه غرام, للميكروبات ضد ثالثة النشاط االولي المختبري المضاد تقييم تم للمركبات المحضره . , صبغه غرامل السالبة من البكتيريا وكذلك ضد ثالثه انواع ،(العصوية الرقيقة المكورات الدقيقة،و المكورات العنقودية الذهبية،)وهي: (المبيضات البيض ، والمبيضات الجرداء) ونوعين من الفطريات االشريكية القولونية والمتقلبة الرائعة( الزائفة الزنجارية،) وهي: وذلك باستعمال طريقة االنتشار. أجريت الدراسة الحسابية لحساب مايكروغرام من المادة المحضره كأقل تركيز مثبط 111باستعمال حرارية باستخدام نظريه الكثافة الوظيفية .بعض المعامالت ال . امينوثيازول, تحضير ,ونظريه الكثافه الوظيفيه-2الكلمات المفتاحيه: مضاد للبكتريا, introduction thiazoles are significantly important heterocyclic that represent interesting properties depending on the linked groups to the thiazole(1). 2-aminothiazoles are a subclass of thiazole family that are of high interest due to their broad biological activity., fungicidal(2), antioxidative (3), analgesic (4), bactericidal (5) and anti-inflammatory (5),anti-hiv (6), antitubercular (7), thiazole containing n=c=s moiety have been used as antiphychotics (8)and antimalarial (9), and as herbicidal (10) .thiazoles have broadly been engaged as effective γsecretase inhibitors to hinder alzheimer’s disease (11) and have a lot of derivations and because of its derivations type have a lot of application in various industries, medical science, and pharmacy.in 1887, hantzsch et al identified and established molecular structure of thiazoles. in 1889, the first derivation of thiazoles identified by prop et al and was named 2-amino thiazole (12). after that until now, many derivations identified and reported (13, 14). thiazole and related compounds are called 1 , 3 azoles ( nitrogen and one other heteroatom in a five – membered ring ). they are isomeric with the 1 , 2 – bazoles , the nitrogen , and sulfur compound being called isothiazole (15) 1corresponding author e-mail: kubbaammar1963@gmail.com received: 29/10/2017 accepted: 4/3/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp79-88 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole 80 compounds from readily available reagents is one of the major challenges in organic chemistry. therefore to meet the facile results of these tough challenges thiazole nucleus was being considered. among the wide variety of heterocycles that have been explored for developing pharmaceutically molecules (16).our study focuses on derivatization of new molecules starting from the parent nucleus 2amino-4-(4-chloro phenyl) 1,3-thiazole (1), by treatment with phenyl isothiocyanate, to yield (2), then refluxing it with to-2,4-dinitrophenyl hydrazine to produce (3), subsequently evaluation of the produced compounds in vitro for their preliminary antimicrobial activity ,also using dft study to identify the changes in electronic structure responsible for farmacological action (17) . material and methods chemicals used during the synthesis, supplied by hyperchem/china. completion of reactions and the purity of compounds were ascertained by thin-layer chromatography (tlc) , using silica gel gf254 (type 60) precoated aluminium sheets, merck (germany) exposed to uv-254nm light and the eluent used is n-hexane: ethyl acetate (6.0:4.0) for compound (1) , n-hexane: ethyl acetate (8.0:2.0) for compound (2) and n-hexane: ethyl acetate (4.0:6.0) for compound (3).melting points were measured by using stuart smp3 melting point apparatus in open capillary tubes, and are uncorrected. the infrared spectra were performed in kbr disc, (υ,cm-1), using ftir spectrophotometer (shimadzu, wqf-520, japan) at the university of baghdad college of education for pure sciences ibn al-haitham. the chns elemental microanalysis of the final synthesized products was done using (elemental vario micro cube instrument, germany) in the university of mustansiriyacollege of pharmacy. chemical synthesis general method for the synthesis of 2-amino4( 4 – chlorophenyl ) thiazole (1) method 1( 18): a mixture of p-chloro phenacyl bromide (0.01 mol,2.3g) in 100 ml of absolute alcohol and thiourea (0.02 mol, 1.5g) were taken in a round bottom flask and refluxed for 3 h. then the reaction mixture was cooled and precipitated with cold water. the solid separated was collected by filtration and the solvent was removed under vacuum. the residue obtained was dried, recrystallized from ethanol. 2-amino-4-(4-chlor phenyl thiazole) (1) off white powder, yield 95%,m.p 167-169ºc, ir(kbr),(υ,cm-1): (3438and3284) nh2 str, of prim.amine,3114 ar(ch) str,1535 ar(c=c)str,825 (c-cl) str,669 (c-s) str; 1hnmr(300 mhz,dmso-d6 ,δ = ppm ) : 7.98 (2h , d , cl 2ar –h ) ; 7.41(2h,d,cl-2arh);7.08 (1h,s,h5-thiazol);7.06(2h,s,nh2). method 2( 19) a mixture of p-chloro phenacyl bromide (0.0064 mol, 1.5 g), and thiourea (0.0064 mol, 0.489 g) in 10 ml of dmso was stirred at room temperature until completion of the reaction. the time of the reaction was monitored by a stopwatch. the progress of the reaction was monitored by thin-layer chromatography. on completion of the reaction, the reaction mixture was poured into crushed ice. the precipitated product was filtered and dried and recrystallized from ethanol. the product was pure enough (single spot on tlc) for all practical purposes. general method for the synthesis of 1-(4-(4chlorophenyl) thiazol -2-yl)-3-phenylthiourea (2)( 20) a mixture of (1), (0.01 mol, 2.1g) in pyridine (20 ml) was added to phenyl isothiocyanate (0.01 mol, 1.35g,) and the reaction mixture refluxed for 6 h. it was then cooled , and the solvent removed in vacuo. the resulting residue was triturated with water, and the solid obtained was recrystallized from ethanol . 1-(4-(4-chlorophenyl)thiazol-2-yl)-3phenylthiourea (2) yellow powder ,yield 92%,m.p 255-260 ºc, ir(kbr) (υ,cm-1): 3159 ar (nh)str,3113ar(ch)str, 1576ar(c=c) str,1196(c=s)str,658 (c-s)str; elemental micro analysis: calcd. for c16h12cln3s2 c,55. 56; h,3.50; n, 12.15, s, 18.54%. found: c,55.29; h,3.363; n, 12.08 ; s, 18.196 %. general method for the synthesis of 1-(4-(4chlorophenyl)thiazol-2-yl)-3phenylhydrazonamide n'-(2,4-dinitrophenyl ) (3) (21) a mixture of (2) (0.001mol,0.345g) and 2,4 di nitro phenyl hydrazine (0.001mol 1.98g) in absolute ethanol (30 ml) was refluxed for 7h. the product separated during reflux, was filtered off and recrystallized from ethanol as brown powder. 1-(4-(4-chlorophenyl)thiazol-2-yl)-3phenylhydrazonamide n '-(2,4-dinitrophenyl) (3) brown powder ,yield 78%, m.p 135-137ºc, ir(kbr),(υ,cm-1): 3313 (nh) str of sec. amine, 3026 ar(ch)str,1614(c=n)str, 1587ar (c=c)str, 1512 asym. no2 str,1331sym. no2 str,1196 (c-n)str.,833 (c-cl)str,660(c-s)str., elemental analysis: calcd. for c19h12cln3o2s2 c,51.82; h,3.16; n, 19.23; s, 6.29%. found: c,51.050; h,3.11; n, 18.93; s, 5.98 %. iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole 81 ir spectrum of compound 1 1hnmr of compound 1 iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole 82 scheme 1. synthesis of 2-amino thiazole derivatives (2and3) antimicrobial screening the antimicrobial activities of the synthesized derivatives were measured using well diffusion technique with a comparison to cefotaxime sodium(cefot.) and sulfamethoxazole (sulf.) as standard antibacterial agents ,and miconazole as standard antifungal agent, using dimethylsulfoxide (dmso) as solvent and as a control, and it was run in the ministry of health/ national center for drug control and research (ncdcr) / baghdad , and in university of baghdad/college of education for pure sciences ibn al-haitham /central service laboratory. the synthesized compounds had been screened for their in vitro preliminary antimicrobial activity against three grampositive bacteria: (staph. aureus, micrococcus luteus and bacillus subtilis), three gramnegative bacteria (pseud.aeruginosa, e.coli, and proteus mirabilis) and two fungi (candida albicans and candida glabrata), using a minimum inhibitory concentration (mic) of iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole 83 100 µg\ml of reference compounds (2) and (3) in dmso as shown in table 1. results and discussion chemistry the 2-amino-4-(4-chlorophenyl) thiazole (1) synthesis, was carried out according to hantzsch method 22, by reaction of phenacyl bromide with thiourea in the presence of absolute methanol for 3 hours. they are characterized by ftir, due to the appearance primary amine (nh2) stretching at 3438 and 3284 cm-1 and then reaction of (1) with phenylisothiocyanate in the presence of pyridine to produce (2) which was characterized by ftir, a band of( c=s) stretching displayed at 1254cm-1, and nh stretching band at 3159 cm-1,and finally condensation of (2) with, 2,4 dinitrophenyl hydrazine using absolute ethanol as a solvent. to afford (3) which showed the characteristic no2 ( asymmetrical and symmetrical)stretching at 1512 and 1331 cm-1, respectively, also peak attributed to(c=n) band recorded at 1614 cm-1. synthesis of compound (1) the method of parent nucleus synthesis was described by hantzsch, in 1887 the cyclization of α-halo carbonyl compounds by a great variety of reactants containing the n-c-s fragment of the ring is still the widely used method of synthesis of thiazoles ,as shown in the following scheme (2) (22). scheme 2. mechanism of synthesis of (1) synthesis of compound (2) the condensation of parent nucleus (1), with phenyl isothiocyanate, afforded the corresponding 1( 4-( 4chlorophenyl )thiazol2-yl )3 -phenylthiourea derivative ( 2 ), the unshared pair of electron of nh2 of (1) would attack the carbon (c = s), of thiocarbonyl group (23) as shown in the following scheme 3. scheme 3. mechanism of synthesis of (2) iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole 84 synthesis of compound (3) the reactivity of phenyl isothiocyanate is, determined by two active centers the nucleophilic nitrogen atom, and thiocarbonyl groups, which make them capable of participating in diverse type of addition and cyclization reactions. the unshared pair of electron of nh2 group of 2,4-dinitro phenyl hydrazine would attack thiocarbonyl carbon(c=s) of (2) and in the presence of acidic hydrogen of ethanol, h2s gas would be evolved, and the tatumeric structure of (3), would also formed (24), as shown in the scheme 4. scheme4. mechanism of synthesis of (3) anti microbial activity from the results illustrated in table1, showed that the new synthesized derivative (2 and 3) have moderate activity against grampositive bacteria staph. aureus and bacillus subtilis also , both compounds have no antibacterial activity against gram-negative bacteria.it is evident that the tested compounds demonstrated the most potent antifungal activity against candida albicans and candida glabrata. iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole 85 table1. the antibacterial and antifungal activity of the tested compounds. (-)= no activity, (+) = slightly active (inhibition zone in between 5-10 mm),(++) = moderately active (inhibition zone in between 10-15 mm), (+++)=highly active (inhibition zone more than 15 mm). computational studies density function theory (dft) study in order to explore the theoreticalexperimental consistency, quantum chemical calculations were performed with complete geometry optimizations using standard spartan 10 software. geometry optimization was carried out by b3lyp/6-31g* level of theory. the chemical reactivity descriptors calculated using dft are: total energy (e), log p and electronegativity (μ). on the basis of frontier molecular orbitals, chemical hardness corresponds to the gap between the homo and lumo , where elumo and ehomo are the lumo and homo energies. electronegativity (25) is defined as the negative of electronegativity of a molecule, physically,( μ) describes the escaping tendency of electrons from an equilibrium system (26). it is a measure of the stabilization in energy after a system accepts additional amount of electronic charge from the environment results and discussion structural and electronic properties dft calculations were performed for compound 13. optimized molecular structures of the most stable form are shown in figure 1. homo lumo compound (1) comp. no. conc. µg/ml s. aureus conc. µg/ml (g+ve) microco -ccus luteus conc.µ g/ml(g+ ve) bacillus subtilis conc.µg/ ml (g+ve) proteus mirabilis conc.µg/ ml (g-ve) pseudomonas aeruginosann nconc.µg/ml (g-ve) e. coli conc.µg/ml (g-ve) candida glabrata conc. µg/ml candida albicans conc. µg/ml 2 100 9.3 8.0 10.15 17 15.5 3 100 10.4 10.37 16 18.6 cefot. 100 44.5 34.4 36.05 33.5 35.5 56.7 sulf. 100 23 19.06 25.15 19.5 18.9 35.6 micon azole 100 17.0 16.9 dmso 100 iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole 86 homo lumo compound (2) homo lumo compound (3) figure 1. highest occupied molecular orbital (homo) and the lowest unoccupied molecular orbital (lumo) of compounds 1–3. it can be seen that the energy gaps between homo and lumo for compound 1 is 6.915, compound 2 is –5.85, compound 3 is 1.425. the lower value in the homo and lumo energy gap explains the eventual charge transfer interaction taking place within the molecules. the larger the homo–lumo energy gap, more stable/less reactive the molecule, for example, compound 1. the values of (μ ) for compounds 1, 2 and 3 are presented in (table 1 row 5). the trend in electronegativity for compounds 1-3 are: 2 > 3 >1 . the greater the electronic chemical potential, the less stable or more reactive is the compound. therefore, compound 2 is more reactive than compounds 3 and 1, respectively. therefore, compound 2 is the most reactive, and 3 and 1 are the least reactive. electrostatic potential charges and related quantum chemical properties the distribution of the electronic density (electrostatic potential charges), related quantum chemical parameters; homo/lumo gap (table 1, row 4), the trends in energy gap 1> 3> 2 .on the other hand log p provides a measure of a molecule’s lipophilicity, where high log p-value indicates high lipophilicity, while low value suggests low lipophilicity. the partition coefficients of the compounds (log p), (table 1, row6) were calculated for observed compounds 1-3. , the results indicated that compounds having the highest lipophilicity were 3 > 2 > 1 with corresponding values 6.66, 6.04 and 3.61,respectively. these values and properties are very useful and can be used in order to evaluate chemical properties and possibilities for interaction of compounds with biological macromolecules (receptors, enzymes) (27). table 1. global chemical reactivity indices for compounds 1–3 compound (3) compound (2) compound (1) parameters total energy kcal/mol 1.4619 -4.2637 23.7835 homo ev -8.052 -5.51 -6.283 lumo ev -1.137 0.34 -4.858 energy gap ev 1.425 -5.85 6.915 electrone gative μ/ ev -4.59 -2.59 -3.85 log p 3.61 6.04 6.66 iraqi j pharm sci, vol.27(1) 2018 dft study of new derivatives from thiazole 87 conclusion a new derivatives of 2-amino thiazole were successfully synthesized using conventional method and tested for their preliminary antimicrobial activity,using well diffusion method. the synthesized compounds displayed slight antibacterial activities against gram-positive types, while demonstrated no antibacterial activities against gram-negative bacteria, and high antifungal activity against tested fungal species. the quantum chemistry calculations using the density function theory (dft) method was performed to study stability of compounds 1-3. the results showed that the compound 1 is more stable than compound 3, as was indicated via calculations of the larger the homo– lumo energy gap parameter (28) . references 1. schnürch m, waldner b, hilber k, mihovilovic md. synthesis of 5-arylated narylthiazole-2-amines as potential skeletal muscle cell differen tiation promoters. bioorg. med. chem lett. 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compounds as inhibitors for mild steel corrosion in sulfuric acid medium. corrosion science. 2010;52(9):2793-2803. 28. m. a. migahed a. m. al-sabagh e.a. khamis m.abd-el-raouf e. g. zaki , antimicrobial activity and quantum chemical calculations of pyrazol-2,3dihydrothiazole sugar derivatives , chem. mater. res., 2014 ; 6 (12): 46-54. iraqi j pharm sci, vol.21(2) 2012 coumarin derivatives coupled to amino acid esters 1 synthesis of coumarin derivatives coupled to amino acid esters and studying their biological activity as antimicrobial agents rana a. al-dawaf *,1 and kawkab y. saour** * department of pharmaceutical chemistry,college of pharmacy, al-mustansiriya university, baghdad, iraq **department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq abstract a series of coumarin derivatives linked to amino acid ester side chains were synthesized and evaluated of their antibacterial and antifungal activity. the coumarin derivatives was alkylated by the ethyl bromoacetate and then using potassium carbonate to get alkylated hymecromone. conventional solution method for amide bond formation was used as a coupling method between the carboxyprotected amino acids with acetic acid side chain of coumarin derivatives. the dcc/ hobt coupling reagents were used for peptide bond formation. the proposed analogues were successfully synthesized and their structural formulas were consistent with the proposed structures as they were proved and characterized by thin layer chromatography (tlc), melting point, infrared spectroscopy (ir), and elemental microanalysis (chn). key words: coumarin, amino acid, coumarin antimicrobialal activity. للفعاليةللكومارين مزدوجة مع استرات االحماض االمينية مع دراسة مشتقات تحضير مضاد للثكتريا ك الثايولوجية الدواف رنا عوني ،*1 و كوكة يعقوب ساعور ** . ، انجايعت انًسخُصشٌت ، بغذاد ، انعشاق نتانصٍذ ت*فشع انكًٍٍاء انصٍذالٍَت ، كهٍ . ، جايعه بغذاد، بغذاد ، انعشاق تانصٍذن تٍت ، كهٍ*فشع انكًٍٍاء انصٍذالَ الخالصة بكخٍشٌت كًضاداث انحٍىٌت نها انفعانٍت ودساست لذ حى ححضٍشها يشحبطت باسخشاث االحًاض االيٍٍُت انكىياسٌٍ يشخماث سهسهت يٍ باسخخذاو كاسبىَاث انبىحاسٍىو نهحصىل عهى و بىاسطت أثٍم بشويى حايط انخهٍك ايشخماث انكىياسٌٍ لذ حى أنكهخه .ويضاداث فطشٌت وانخً ححخىي حايط انخهٍك كىياسٌٍانًحهىل انخمهٍذٌت يا بٍٍ يشخماث ان إٌ حكىٌٍ اصشة األيٍذ حى باحباع طشٌمتاسخش انهاًٌٍكشويىٌ. اللخشاٌ انًباشش بىجىد حىايط ايٍٍُت راث يجًىعاث حايضٍت يحًٍت إر أٌ طشٌمت حخهٍك اصشة انببخٍذ باسخخذاو ا كزساع جاَبً يع dcc/hobt وانخً حخًٍز بكىَها بسٍطت وفعانت وحؤدي انى ححمٍك عائذ جٍذ عُذ دسجت حشاسة انغشفت. إٌ انًشكباث انًمخشحت حى شافٍا حخهٍمها بُجاح إر كاَج انصٍغ انهٍكهٍت يخسمت يع انًشكباث انًمخشحت بعذ حًٍٍزها وإثباحها باسخخذاو انخمٍُاث انخانٍت: كشوياحىك ت انشلٍمت, يمٍاس دسجت االَصهاس , يطٍاف االشعت ححج انحًشاء, وانخحهٍم انذلٍك نهعُاصش انًكىَت.مانطب .الكومارين كمضادات مايكروتية فعالية, االحماض االمينية, : كومارينالمفتاحية الكلمات introduction coumarins are heterocyclic compounds containing a lactone group. they are also known as benzo-2-pyrones. these compounds and their derivatives represent a broad class of natural and pharmaceutical products (1) .this group of compounds has been isolated from a variety of plant sources. coumarin was first isolated in 1820 by vogel from the seeds of tonka beans (coumarouna adorata) , while an umbelliferone (7hydroxycoumarins) derivative present in many plants such as mannaash, sweet woodruff (2) .coumarin derivatives possess a wide range of pharmacological activities such as, anticoagulant (3) , antifungal (4) , antibacteria (5) , anti-inflammatory (6) , anticancer (7) , spasmolytic and hypotensive activities (8) . 7hydroxycoumarin is known for its antibiotic and antifungal activities. 8-substituted-4methyl-7-hydroxycoumarin (9,10) and 6substituted-4-methyl-7-hydroxycoumarin (11) have been investigated for complexing ability. hymecromone, (7-hydroxy-4-methylcoumarin), is one of coumarin derivatives. it is used in liver therapy as a choleretic and spasmolytic with the special action on the gall ducts and gall bladder (12,13) .coumarins have attracted interest in recent years because of their diverse pharmacological properties (14) .during the last twenty years, the study of the biological activities of coumarin derivatives has been the aim of many researchers (15) . chemical synthesis a. esterification of amino acids synthesis of l-leucine methyl ester hcl (leu-o-me), compound (a.1) (16) a suspension of leucine (11 .4mmol, 1.5g) dissolved in (10ml) of absolute methanol, was cooled down to -15c then thionyl chloride was added drop wise (11.4mmol, 0.86ml), (the temperature should 1 corresponding author email : rana_aldawaf@yahoo.com received : 2/11/2011 accepted 15/5/2012 mailto:rana_aldawaf@yahoo.com iraqi j pharm sci, vol.21(2) 2012 coumarin derivatives coupled to amino acid esters 2 be keep below –10c), the reaction mixture was left at 40c for 3hr, then refluxed for 3hr and left at room temperature overnight. the solvent was evaporated to dryness under vacuum, re-dissolved in methanol and evaporated, this process was repeated several times and re-crystallize the product from methanol-diethyl ether. percent yield, physical appearance, m.p, rf values were listed in table (1), and ir characteristics absorption bands were listed in table (2). synthesis of methionine methyl ester hcl (met -o-me), compound (a.2). a suspension of methionine (10mmol, 1.5g) dissolved in (15ml) of absolute methanol and (10mmol, 0.75ml) of thionyl chloride, then complete the procedure as mentioned in the synthesis of a.1. percent yield, physical appearance, m.p, rf values were listed in table (1), and ir characteristics absorption bands were listed in table (2). b. alkylationof 4-methyl-7-hydroxycoumarin compound (b) (17) a suspension of 4-methyl-7hydroxy coumarin (39.7mmol, 7g) in acetone (70ml) was refluxed with ethyl bromoacetate (59.55mmol, 6.5ml) and anhydrous k2co3 (218.3mmol, 30g) for 16 hr. after cooling, the mixture was evaporated to dryness. the residue was recrystallized from acetone to give off-white powder of 4-methyl-7-(methoxy ethyl acetate) coumarin.percent yield, physical appearance, m.p, rf values were listed in table (1), and ir characteristics absorption bands were listed in table (2). c. synthesis of 4-methyl-7-(carboxy methoxy) coumarin, compound (c) (18) compound (b) (26.7mmol, 7g) was dissolved in (100ml) of absolute ethanol and (42ml) of 5% naoh solution was added, the reaction mixture was stirred overnight at room temperature, then the solvent evaporated, and the residual was dissolved in water and acidified with 6 n of hcl. the white precipitate was filtered and crystallized from ethanol. percent yield, physical appearance, m.p, rf values were listed in table (1), and ir characteristics absorption bands were listed in table (2). d. coupling method and reagents conventional solution method was used as a coupling method between the carboxyprotected amino acid and carboxylic acid of coumarin derivatives. dcc (dicyclohexyl carbodiimide) was used as a coupling reagent, while hobt (1-hydroxy benzotriazole) was used to decrease racemization and to increase the yields. (19) 1) synthesis of 4-methyl coumarin-7-o-acetyl leucine methyl ester, compound (d.1) (20) to a stirred solution of leucine methyl ester hcl (compound a.1) (1.96mmol, 0.357g) in (6ml) of dmf, (1.96mmol, 0.2ml) of n-methyl morpholine (nmm) was added with stirring for 10 min., then (1.96mmol, 0.45 g) of (comp c) was also added, and the mixture was cooled down to (-10c) then (3.9mmol, 0.62g) of hobt and (1.96mmol, 0.4g) of dcc were added with stirring, which was continued for 2days at 0c and then at room temperature for 6 days. the crude product was evaporated to exclude dmf and redissolved in chloroform from which the n,n–dicyclohexyl urea (dcu) was filtered off , and the clear filtrate washed twice with 5% sodium carbonate solution, 0.1n hcl, once with water, and with saturated sodium chloride solution.the chloroform layer was dried with anhydrous magnesium sulfate and evaporated under vacuum; the resulted product was collected, recrystallized from (methanol: chloroform) (5:1). percent yield, physical appearance, m.p, rf values were listed in table (1), ir characteristics absorption bands were listed in table (2) and elemental microanalysis data are listed in table(3). 2) synthesis of 4-methyl coumarin-7-o-acetyl methionine methyl ester, compound (d.2) to a stirred solution of methionine methyl ester hcl (compound a.2) (2.5mmol, 0.5g) in (7.5ml) of dmf, (2.5mmol, 0.27ml) of n-methyl morpholine (nmm) was added with stirring for 10 min., then (2.5mmol, 0.58 g) of (comp c) was also added, and the mixture was cooled down to (-10c) then (5mmol, 0.8g) of hobt and (2.5mmol, 0.5g) of dcc were added with stirring, which was continued for 2days at 0c and then at room temperature for 6 days. then the procedure was completed as mentioned in the synthesis of d.1. percent yield, physical appearance, m.p, rf values were listed in table (1), ir characteristics absorption bands were listed in table (2) and elemental microanalysis data are listed in table(3). iraqi j pharm sci, vol.21(2) 2012 coumarin derivatives coupled to amino acid esters 3 table 1 : the physical appearance, percent yield, melting points and rf values of the synthesized compounds and their intermediates. table 2 : ir characteristic absorption bands. comp. no. compound name ir characteristic absorption bands (v cm-1) a.1 leu -o-me (3032-2784 nh stretch of amine salt), (2958, 2924, 2872 assym. str.ch3,ch2, ch), (1739 c=o ester),(1508 nh bend), (1451 ch3,ch2 bend), (1394,1359 ch bend) , (1228 c-o ester). a.2 meth-o-me (3136-2857 nh stretch of amine salt), (2956, 2914 assym. str. ch3 , ch2), (1747 c=o of ester) , (1489 nh bend), (1444 ch3 bend), (1232 c-o of ester). b 4-methyl-7-(methoxy ethyl acetate)coumarin (3076 ch str. of aromatic c=ch), (2980, 2928 assym. str. ch3, ch2), (2872 sym. str. ch3), (1759 c=o ester), (1606, 1508 str. aromatic c=c), (1423, 1386 ch3, ch2 bend), (1220 c-o ester), (1197 c-o lacton). c 7-(carboxy methoxy)4-methyl coumarin (3200-2500 str.oh ), (3068 ch str. of aromatic c=ch), (2987, 2916 assym. str. ch3, ch2), (1755 c=o carboxylic acid), (1708 c=o lacton), (1610, 1566, 1510 str. aromatic c=c), (1427, 1390 ch3, ch2 bend), (1253 c-o carboxylic acid), (1147 c-o lacton). d.1 4-methyl-7-o-acetylleucine methyl ester (3333 nh stretch, amide ii), (2955,2928, 2852 assy. str. ch3,ch2, ch), (1739 c=o of ester and lacton), (1676 c=o str. amide), (1620 c= c aromatic), (1527 nh bend, amide ii ), (1390,1369 ch bend), (1298 c-o ester), (1151 c-o ether). d.2 4-methyl-7-o-acetylmethionine methyl ester (3362 nh stretch, amide ii), (2926, 2852 assy., sym str. ch3,ch2), (1741 c=o of ester and lacton), (1670 c=o str. amide), (1622 c= c aromatic), (1527 nh bend, amide ii ), (1440,1390 ch3,ch2 bend), (1300 c-o ester), (1155 c-o ether). table 3 : the elemental microanalysis % of final products compound physical appearance %yield melting point observed ( o c) rf value solvent system a.1 white needle shape crystals 91 146-148 0.78 chloroform 7 methanol 3 a.2 white needle shape crystals 90 148-150 0.8 chloroform 7 methanol 3 b off white powder 74 96-97 0.7 chloroform 4 methanol 6 c off white crystals 79 207-208 0.8 chloroform 2.5 methanol 2.5 ether 5 d.1 white crystals 92 103-105 0.74 d chloroform 2 methanol 8 d.2 off white to yellow crystals 89 105-108 0.76 d chloroform 2 methanol 8 mol.wt. s o n h c value type compound 361.39 26.56 27.31 3.88 4.07 6.41 6.60 63.15 64.63 calculated observed d1 379.43 8.45 8.78 25.30 26.10 3.69 3.81 5.58 5.39 56.98 58.03 calculated observed d2 iraqi j pharm sci, vol.21(2) 2012 coumarin derivatives coupled to amino acid esters 4 biological activity a preliminary antibacterial and antifungal activity has been carried out according to well diffusion method: the prepared compounds have been studyied for their antimicrobial activity in vitro against three tested bacteria (staphylococcus aureus., streptococcus spp. as gram positive bacteria and proteus spp. as gram negative bacteria) and two fungi (aspergillus spp., and candida spp.) were clinical activated and maintained on nutrient agar medium for testing antibacterial activity and sabaroud agar medium for antifungal activity.ofloxacin was used as a standard drug for antibacterial activity and ketoconazole was used as a standard drug for antifungal activity.the plates were incubated at 30 °c for 72 hours (fungai spp.) or 37 °c for 24 hours (bacteria) (21) and the antimicrobial activity was evaluated by measuring the diameter of the inhibition zone (iz) around the disc in mm, as show in table (4) and (5) respectively . result and discussion synthetic part the overall synthesis strategy based one four major lines: 1. amino acid derivatives the amino acids were activated by thionyl chloride to get acyl chloride that attacks methanol to get methyl esters of the selected amino acids. 2. alkylation of hymecromone hymecromone was alkylated by the ethyl bromoacetate and then using potassium carbonate to get ester of hymecromone. 3. hymecromone acetic acid synthesis removal of ethoxy group from the hymecromone ester can be done by sodium hydroxide to form the conjugated base, then acidified with hydrochloric acid to get hymecromone acetic acid, as show in scheme (1). 4. amide bond formation conventional solution method for amide bond formation used as a coupling method between the carboxy-protected amino acids with acetic acid side chain of hymecromone. the dcc/ hobt coupling reagents used for peptide bond formation, as show in scheme (2). 4-methyl-7-(methoxy ethyl acetate) coumarin scheme 1 : pathway of synthesis of 4-methyl coumarin carboxylic acid. iraqi j pharm sci, vol.21(2) 2012 coumarin derivatives coupled to amino acid esters 5 d1: r= ch2 ch (ch3)2 d2: r= (ch2)2 s ch3 scheme 2 : pathway of synthesis of coumarin derivative coupled to amino acid esters. iraqi j pharm sci, vol.21(2) 2012 coumarin derivatives coupled to amino acid esters 6 nutrient agar medium for testing antibacterial activity and sabaroud agar medium for antifungal activity.ofloxacin was used as a standard drug for antibacterial activity and ketoconazole was used as a standard drug for antifungal activity.the plates were incubated at 30 °c for 72 hours (fungai spp.) or 37 °c for 24 hours (bacteria) (21) and the antimicrobial activity was evaluated by measuring the diameter of the inhibition zone (iz) around the disc in mm, as show in table (4) and (5). table 4 : the antibacterial activity of the tested compounds compound no. zone of inhibition in mm staphylococcus aureus streptococcus spp. proteus spp. d1 2µg/ml 5 3 3 20µg/ml 9 10 8 50µg/ml 14 12 11 d2 2µg/ml 1 2 no activity 20µg/ml 4 3 no activity 50µg/ml 7 7 no activity ofloxacin 2µg/ml 5 6 5 20µg/ml 10 12 10 50µg/ml 16 17 16 table 5: the antifungal activity of the tested compounds. compound no. zone of inhibition in mm aspergillus spp. candidia spp. d1 5µg/ml 6 7 20µg/ml 13 12 50µg/ml 14 18 d2 5µg/ml 3 7 20µg/ml 6 10 50µg/ml 12 14 ketoconazole 5µg/ml 12 9 20µg/ml 17 25 50µg/ml 20 30 the antimicrobial activities (antibacterial and antifungal activities) have been carried out according using well diffusion methodology.the prepared compounds have been studying for their antibacterial activity in vitro against three tested bacteria (staphylococcus aureus., streptococcus spp. as gram positive bacteria and proteus spp. as gram negative bacteria) respectively using ofloxacin as standard. also, the fungal activity has been study against two types of fungi (aspergillus spp., and candida spp.) using ketoconazole as standard.all results are fixed in tables (4) and (5), compound d1 show antibacterial activity, while both compounds d1&d2 have a good antifungal activity. conclusion the proposed compounds were successfully synthesized by the conventional solution method as previously described and their structure formula were consistent with the proposed structures since conformity of their structures was achieved by using the following techniques: thin layer chromatography (tlc), melting point, infrared spectroscopy (ir) and elemental microanalysis (chn).the best activity of the studied samples appeared in the compounds d1 (4-methyl coumarin-7-oacetyl-leucine methyl ester) as antibacterial iraqi j pharm sci, vol.21(2) 2012 coumarin derivatives coupled to amino acid esters 7 agent, and antifungal agent.the mechanism of their action is not yet fully understood and correlation of effects with chemical structures is not conclusive at the moment. references 1. amador. p. , pineda. b., lopez. a., flores . h. : standard molar enthalpies of formation in the crystalline phase of 7hydroxy-4-methylcoumarin , 7ethoxy – 4 –methylcoumarin , and 6-methoxy-4 methylcoumarin . j . chem . thermo dynamics . 2011; 43: 1414-1416. 2. sethna. s.m., shah. n.m.: the chemistry of coumarins. chem.rev.1945;36: pp. 162. 3. lowenthal. j., birnbaum. h.: vitamin k and coumarin anticoagulants: dependence of anticoagulant effect on inhibition of vitamin k transport. science. 1969; 164: 181-183. 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10401-10421. 20. yajima. h., ogawa. h., ueda. h., takagi. h.: synthesis and activity of kyotorphin and its analogue. chem pharm bull. 1980; 28: pp. 1935-1938. 21. kuete. v., tangmouo. j.g., penlap. v., mofo. f., lontsi. d.: antimicrobial activity of tridesmostemon omphalocarpoides. journal of ethnopharmacology. 2006;104: 5-11. http://www.ncbi.nlm.nih.gov/pubmed?term=%22de%20celis%20er%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22de%20celis%20er%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22shayo%20c%22%5bauthor%5d http://www.sciencedirect.com/science/journal/0014827x http://www.sciencedirect.com/science/journal/0014827x http://en.wikipedia.org/w/index.php?title=p._henklein&action=edit&redlink=1 http://en.wikipedia.org/w/index.php?title=w._rapp&action=edit&redlink=1 http://en.wikipedia.org/w/index.php?title=j._peptide_chemistry&action=edit&redlink=1 iraqi j pharm sci, vol.27(1) 2018 phytochemicals in a. precatorius l. plant doi: http://dx.doi.org/10.31351/vol27iss1pp30-38 30 phytochemical investigations of iraqi abrus precatorius linn. plant zahra’a s. nassir*,1 and enas j. khadem* *department of pharmacognosy and medicinal plants , college of pharmacy, university of baghdad, baghdad, iraq abstract the plant abrus precatorius, which belong to leguminosae (fabaceae) family and known as crab’s eyes, rosary pea with characteristic red and black seeds. it was used in folk medicine in india, china and east asian countries for treatment of various diseases. the plant was extracted by '' general method of extraction'' (harborne, 1973) using 80% aqueous ethanol as a solvent of extraction by soxhlet apparatus. preliminary qualitative phytochemical screening were performed on the crude ethanolic extract and revealed the presence of alkaloids, flavonoids ,terpenoids and phytosterols in iraqi abrus precatorius plant. three different fractions were obtained from crude extract which are fraction one (chloroform fraction), fraction two (ethyl acetate fraction), and fraction three (petroleum ether fraction) which are represent alkaloids, flavonoids and steroids respectively. the alkaloid abrine was isolated from the chloroform fraction in pure form by using preparative thin layer chromatography (ptlc) and then subjected to different physico-chemical and specteral analytical techniques to identify its chemical structure: melting point (m.p.), thin layer chromatography (tlc), high performance liquid chromatography (hplc) , fourier transforms infrared spectra (ft-ir) and elemental microanalysis (chno). keywords: abrus precatorius ,alkaloid abrine, hplc, ft-ir, chno. كيميائية لنبات عين العفريت العراقيدراسة زهراء سهيل ناصر *، 1 و ايناس جواد كاظم * . ، بغداد، العراق فرع العقاقير والنباتات الطبية ، كلية الصيدلة ، جامعة بغداد* الخالصة هو نوع من النباتات ينتمي الى العائلة البقولية وهو نبات مستزرع في شمال العراق وتحديدا في مدينة عين العفريت نبات كان يستخدم في الطب الشعبي في الهندالبازالء الوردية مع بذور ذات اللون االسود واالحمر التي يتميز بها. بالموصل ويعرف ايضاً حثالب اهذعتبريلم يخضع النبات الي نوع من الدراسة التحليلة او التشخيصية لذلك ة.والصين وبلدان شرق آسيا لعالج أمراض مختلف مضاد ك أول بحث أجري في العراق لدراسة المكونات الكيميائية النباتية التي لها اهمية طبية ونشاط الدراسة الدوائية األولية )في المختبر( لالكسدة. وفي هذه الدراسة تم استخالص وفصل وعزل وتنقية بعض المركبات الكيميائية )القلويدات والستيرويد النباتي بيتا سايتوستيرول(. من اإليثانول المائي كمذيب ٪08( باستخدام 3791تم استخالص النبات من خالل "الطريقة العامة لالستخراج"للعالم )هاربورن، فحوصات الكيميائية األولية لنواتج األيض الثانوية المختلفة من الوقد تم إجراء . (soxlet)ستخالص بواسطة جهاز سوكسليتلال خالل اختبارات كيميائية محددة على مستخلص االيثانول الخام وقد كشف عن وجود قلويدات، فالفونويدات، تربينويدات و الستيرويدات تم الحصول على ثالثة اجزاء مختلفة من المستخلص الخام والتي هي الجزء االول )جزء كذلكو. ائي العراقيفي نبات القلقل الرج الكلوروفورم(، والجزء الثاني )جزء االيثيل اسيتيت(، والجزء الثالث )جزء األثير البترولي( والتي تمثل قلويدات، الفالفونويدات من جزء الكلوروفورم )الجزء االول( في شكل نقي باستخدام كروماتوغرافيا (abrine)تم عزل قلويد ابرين والستيرويدات على التوالي. ( وقد استخدمت التقنيات الفيزيائية والكيميائية والتحليلية الطيفية لتحديد تركيبها الكيميائيpreparative tlcالطبقة الرقيقة التحضيري ) ، (hplc)( ، الكروماتوغرافيا السائل عالي االداء (tlcنصهار، كروماتوغرافيا الطبقة الرقيقة، وتشمل: نقطة االووزنه الجزيئي .(chno)والتحليل الجزئي عنصري ir-(ftمطياف االشعة تحت الحمراء ) صري حت الحمراء ، التحليل الجزئي العننبات عين العفريت ، القلويد أبرين ، كرومرتوغرافيا السائل عالي االداء، مطياف االشعة تالكلمات المفتاحية : introduction the abrus precatorius linn. of the leguminosae family is woody twinning plant widely distributed in india (1). and it is distributed in the north of iraq certainly in al mousul city and it's authenticated at first time in iraq. it's known as jequirity bean, rosary pea, and crab’s eye. abrus precatorius (figure 1) is a slender, twining vine with a woody base. it is supported generally on other plants or a fence. the leaves are pinnate and glabrous. the fruit of abrus plant , a pea-shaped pod about 1.5 inches long, splits open as it dries to reveal (35) hard-coated, brilliant scarlet, pea-sized seeds with a small enamel-black spot at the point of attachment (hilum). (2, 3). it is used traditionally to treat eye diseases, gastrointestinal aliments and as abortifacient. (4-8). 1corresponding author e-mail:zahraasuhailn@gmail.com received: 23/9/2017 accepted: 21/1/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp30-38 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 phytochemicals in a. precatorius l. plant 31 several studies have shown that the extracts of abrus precatorius at different concentrations have antimicribial activity against g + ve and g –ve bacteria and that attributed to widespread use of the plant as local remedy for a variety of ailments ranging from ulcers to bronchitis. (9) while other studies have been revealed that the petroleum ether extract of abrus precatorius linn (peeap) possessed significant antitumor activity. (10) furthermore, it have antidiabetic (11), antioxidant (12), antifertility (13) and antiinflammatory (14) effects. abrus precatorius are known as ones of the most toxic plant worldwide especially the seeds. the toxic principle contained in abrus precatorius has been found to be the toxalbumin abrin (15). the estimated human fatal dose is 0.1 to 1 microgram/kg and ingestion of one to two crushed seeds is sufficient to cause death. (16) various alkaloids of indole type have been reported in abrus precatorius (17) .which are abrine, hypaphorine and trigonelline, figure (2). abrus plant contains other phytochemicals like flavonoids and phytosterols (betasitosterol) which have various pharmacological activities. (18, 19) figure 1. iraqi abrus precatorius plant (20) iraqi j pharm sci, vol.27(1) 2018 phytochemicals in a. precatorius l. plant 32 a b figure 2. a-chemical structure of abrine, (2s)-3-(1h-indole-3-yl)-2-(methylamino) propionic acid, (c12h14o2n2) (21), b-figure (1.4): chemical structure of hypaphorine, (3s)-3-(1h-indole-3-yl)-2-(trim ethyl azaniumyl) propionate, (c14 h18 o2n2) (22). material and methods plant collection the plant was authenticated by dr. ibrahim saleh/ head of pharmacognosy department/ college of pharmacy/ almustanserya university. the seeds and aerial parts were collected during october (2016) from almosul city which is located at the north of iraq then it dried at room temperature in the shade then pulverized by mechanical mills and weighed. extraction and fractionation of different active constituents: (23) shade-dried coarsely powdered plant (seeds and aerial parts) (500gm) were defatted by maceration with hexane for 24 hr then allowed to dry at room temperature. the defatted plant materials was extracted with 80% ethanol (2 l) in soxhlet apparatus until complete exhaustion. the alcoholic extract was evaporated under reduced pressure at a temperature not exceeding 40 هc to give a dark brown residue designated as a crude fraction. crude fraction was acidified with hydrochloric acid (5%) to ph 2 and partitioned (three times) with ethyl acetate to get two layers (aqueous acidic and ethyl acetate layer). the aqueous acidic (a-1) layer was then separated and basified with equal volume of ammonium hydroxide 15% to ph 10 and extracted with chloroform in the separatory funnel (three times) to get two layers, the chloroform layer which was separated and dried by addition of anhydrous sodium sulfate powder then evaporated under reduced pressure at a temperature not exceeding 40c to give brownish residue designated as fraction 1 (f-1) and aqueous basic layer designated as (a2). the ethyl acetate layer of the original alcoholic extract (crude fraction) was evaporated to dryness under reduced pressure and basified with 300ml of sodium hydroxide 5% to ph 10 and extracted with chloroform in the separatory funnel to get two layers, the aqueous basic layer and chloroform layer. the aqueous basic layer was separated, evaporated to dryness and acidified with 5% hydrochloric acid to ph 2 then extracted with ethyl acetate to get fraction designated as fraction 2 (f-2) .chloroform layer was also separated and evaporated to dryness under reduced pressure then partitioned with methanol 80% and petroleum ether to get two layers methanol 80% and petroleum ether fraction which is designated as fraction 3 (f-3), (figure 3). each fraction was tested using specific chemical test. identification of alkaloids in abrus pretacorius plant extract 1. preliminary phytochemical screening of alkaloids in the abrus precatorius plant using the ethanolic extracts from plants using mayer's, wagner's and the dragendorff's reagents to identify the alkaloids. (23-25). 2. thin layer chromatography (tlc): in this qualitative identification using a readymade aluminum plates of silica gel gf254 and using 3 different developing solvent systems for detection the plant alkaloids in fraction one (f1) comparing with alkaloid standard(s) and detection by spraying with dragendorff's spray reagent, and they are listed in the table (1): table 1. developing solvent systems were used in identification of expected alkaloids in (f-1) of abrus precatorius plant extract no. composition references s2k chloroform: acetone: diethyl amine (50 : 40 :10) 26 s4 k dichloromethane: methanol: water: formic acid: diethyl amine (72.3 : 25 : 2.5 : 0.1 : 0.1) 27 s5 k toluene: ethyl acetate: diethyl amine(70 : 20 : 10) 28 3. qualitative estimation of fraction one (f-1) by high performance liquid chromatography (hplc) : iraqi j pharm sci, vol.27(1) 2018 phytochemicals in a. precatorius l. plant 33 the expected alkaloids in (f-1) were separated by hplc method and identified by comparison with standard compounds using ods c 18 column (250 x 4.6 mm , 5µm particle size) and acetonitrile -0.1 % h3po4 +0.1g sodium sulphonate (35:65) as a mobile phase with flow rate 1.2 ml / min, and uv detector at 254 nm at room temperature(29). isolation and purification of alkaloid from (f1) isolation of alkaloid was carried out by using preparative tlc. fraction one (f-1) was applied on a number of preparative tlc plates as a concentrated solution in streak using capillary tube on each plate , then the plates placed inside glass tank which contained the s5k (toluene: ethyl acetate: diethyl amine (70 : 20 : 10)) solvent system. the detection was done using dragendroff's reagent. the band had been scrapped off, eluted with chloroform, and then filtered. the filtrate evaporated to dryness, in vacuo to give off-white powder, for more purification the iolated compound was dissolved in a hot ethylacetate. the hot solution was filtered and the filtrate was concentrated and cooled to give solid precipitate of (expected abrine alkaloid). figure 3. general scheme for separation of different plant constituents (23) iraqi j pharm sci, vol.27(1) 2018 phytochemicals in a. precatorius l. plant 34 results and discussion extraction method the method of extraction mostly depend on the critical input parameters; understanding the nature of plant matrix; chemistry of bioactive compounds and scientific expertise. since different plant parts contain different chemical classes of active constituents, alkaloids (basic compounds), flavonoids (acidic compounds) and steroids (neutral compounds) so the fractionation based on the conversion of basic compound to its salt by aqueous mineral acids, and when the salt of an alkaloid is treated with hydroxide ion, nitrogen gives up a hydrogen ion and the free amine is liberated which is taken or extracted by specific organic solvent like (chloroform) to get free alkaloids (f-1) leaving quaternary alkaloids and water soluble compounds in the aqueous layer (a-2). table 2. percentage of different fractions obtained from plant extraction of abrus precatorius l. dried plant weight crude extract weight weight of fractions percentage % 500 gm 50 gm f1 alkaloids f2 flavonoids f3 sterols f1 alkaloids f2 flavonoids f3 sterols 3 gm 2.4 gm 4.8 gm 0.6% 0.48% 0.96% preliminary qualitative phytochemical analysis the results of phytochemical analysis of crude ethanolic extract are given in (table 3). preliminary identification of alkaloid in abrus plant by chromatographyatlc: thin layer chromatography of different fractions f1 confirms the presence of indolic alkaloid abrine in fraction-1, and that alkaloid appeared as single spot by using the mobile phase systems (s2k, s4k and s5k). the spot of alkaloid have the same retention factor ( rf) value comparing with its corresponding standard after detection by the dragendorff's sparying reagent as shown in the figures from (4 to 6). table 3: phytochemical screening of alkaloids in the crude ethanolic extract of abrus precatorius plant. alkaloids dragendorff's reagent orange ppt + mayer's reagent white ppt + wagner’s reagent reddish brown ppt + (+) represent the presence of alkaloids table 4. the rf values of abrine alkaloid in (f-1) in abrus precatorius extract, and standard in different developing solvent systems using tlc. mobile phases rf value of abrine standard rf value of abrine alkaloid in (f-1) s2k 0.4 0.38 s4k 0.58 0.6 s5k 0.24 0.23 figure 4. tlc chromatogram of f-1 for abrus pretacorius plant using silica gel gf254nm as adsorbent and s2k [chloroform: acetone: diethylamine (50: 40:10)] as a mobile phase. detecting by spraying with dragendorrf's reagent. std: abrine standard. sample: fraction 1 (f-1). iraqi j pharm sci, vol.27(1) 2018 phytochemicals in a. precatorius l. plant 35 figure 5. tlc chromatogram of f-1 for abrus pretacorius plant using silica gel gf254nm as adsorbent and s4k [dichloromethane: methanol: water: formic acid: diethylamine (72.3: 25: 2.5: 0.1: 0.1)] as a mobile phase. detecting by spraying with dragendorrf's reagent. std: abrine standard. sample: fraction 1 (f-1). figure 6. tlc chromatogram of (f-1) for abrus pretacorius plant using silica gel gf254nm as adsorbent and s5k [toluene: ethyl acetate: diethyl amine (70: 20: 10)] as a mobile phase. detecting by spraying with dragendorrf's reagent. std: abrine standard. sample: fraction one (f-1). bhplc analysis the results gained from hplc analysis method: 1-the retention time of alkaloids (a, b and c) in (f-1) (figure 7) match with the retention time of standard alkaloids (1 trigonelline, 2 abrine and 3 hypaphorine) (figure 8) respectively. 2-an abrine alkaloid have the high percent among the other alkaloids. figure 7. hplc chromatogram of fraction one (f-1). figure 8. hplc chromatogram of alkaloid standards. characterization and identification of the isolated abrine alkaloid: 1-thin layer chromatography (tlc): the isolated alkaloid appeared as single spot having the same color and rf value as the corresponding abrine standard as shown in the figure (9). rf value 0.19 and 0.18 for abrine standard and isolated abrine respectively. iraqi j pharm sci, vol.27(1) 2018 phytochemicals in a. precatorius l. plant 36 figure 9. tlc of the band (sample) isolated by preparative tlc from fraction-1 (f-1) using silica gel gf254nm as adsorbent and s5k as a mobile phase. detection by dragendorffʼs spraying reagent. std : abrine standard. mix: abrine standard and sample mixture. 2-melting point (m.p.) the isolated alkaloid was characterized from its sharp melting point of 273 – 277℃ compared with standard alkaloid melting point 275 – 280 ℃. 3hplc analysis the isolated alkaloid was identified by comparison its retention time (4.101 min) with the retention time of abrine standard (4.087 min) as shown in the figures (10 and 11). figure 10 . hplc chromatogram of isolated alkaloid . figure 11. hplc chromatogram of abrine standard alkaloid. ftir the ir spectrum of isolated alkaloid showed the characteristic bands which are reported for abrine standard as shown in (table 5) and figure (12). table 5. the characteristic bands frequencies from ft-ir spectrum of isolated (30) functional group group frequency wave number ( in cm-1) assignment o-h 3527 o-h stretching of carboxylic acid n-h 3452 n-h stretching (2ه amine) c=c-h 3039 c-h stretching of aromatic alkene c-h 2947, 2891,2810 asymmetric and symmetric stretching of ch2 and ch3 c=o 1735 c=o stretching of carboxylic acid (dimer) c=c 1585,1614 c=c stretching of aromatic alkene and n-h bending is included c-h and c=c 821,810,738 c-h and c=c bending of aromatic (out and inplane) iraqi j pharm sci, vol.27(1) 2018 phytochemicals in a. precatorius l. plant 37 figure 12.ir spectrum of isolated alkaloid. 1. elemental microanalysis chno the isolated alkaloid was subjected to elemental microanalysis to confirm its chemical structure. the result was listed in the table (6) which is demonstrated the different percentage of carbon, hydrogen, oxygen and nitrogen found in the compound. table 6. elemental microanalysis of the isolated alkaloid. element calculated % found % c 66.09 65.49 h 6.04 5.99 n 13.22 12.91 o 14.99 14.59 all the above data coincide with that reported for abrine which might indicate that the isolated compound is the alkaloid abrine. conclusions the following points were drawn based on previous findings: 1. phytochemical screening of new iraqi plant abrus precatorius was done for seeds and aerial part of plant and the results include the presence of alkaloids. 2. the abrine is the only alkaloid was detected and isolated by preparative tlc. the other alkaloidal constituents may be present in low percent so they was demonstrated their presence by hplc analytical method comparing with their corresponding standards. 3. most of the results of this study are consistent with the results of international researches which carried out on this plant. references 1. r. sunday, o. ilesanmi, e. obuotor: acute and subacute toxicity of aqueous extract of abrus precatorius seed in wister rats. the internet journal of pharmacology 2013, vol. 11 2. lewis s. nelson, richard d. shih, michael j. balick: handbook of poisonous and injurious plants. 2nd ed .springer, 2007, pp.57-58 3. ivan a. ross: medicinal plants of the world: chemical constituents, traditional and modern medicinal uses, humana 2nd edition. press inc., 2003, vol. 1, pp.16 4. jain, s. p. and d. m. verma: medicinal plants in the folk-lore of northern circle dehradun up india. nat acad sci lett (india) 1981; 4(7)269–271. 5. das, s. k.: medicinal, economic and useful plants of india. bally seed store, west bengal, 1955. 6. burkhill, i. h.: dictionary of the economic products of the malay peninsula. ministry of agriculture and cooperatives, kuala lumpur, malaysia1966, vol. 1. 7. nath, d., n. sethi, r. k. singh and a. k. jain: commonly used indian abortifacient plants with special reference to their iraqi j 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p.515. 26. stahl e.: thin layer chromatography hand book, 1999. 27. j. flieger: thin layer (planar) chromatography /iii /alkaloids, academic press, 2000. 28. wagner h., bladt s.: plant drug analysis: a thin layer chromatography atlas. 2nd ed. springer-velag, berlin, 1996, 384p. 29. pasupuleti sreenivasa roa, majeti narsimhs vara prasad: extraction, purification and characterization of indole alkaloids from strychnos wallichiana l. an endangered medicinal plant from india. medicinal and aromatic plant science and biotechnology 2008; 2(1), 63-67. 30. silversteine r.m.,webster f.x.: spectrometric identification of organic compounds (7th ed.). john wiley and sons inc., usa, 2005. iraqi j pharm sci, vol.29(2) 2020 new marker in breast cance doi: https://doi.org/10.31351/vol29iss2pp253-258 253 estimation of serum cd200 and cd200r1 levels in a sample of iraqi women with breast cancer: their role as diagnostic and prognostic markers adnan m. ismail *,1 and eman s. saleh** * department of clinical laboratory science, college of pharmacy, university of tikrit, salahulddin, iraq. ** department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. abstract breast cancer is a disease in which cells in the breast grow out of control. cd200 is a cell surface glycoprotein expressed on many cells, it belongs to the immunoglobulin family (ig) and have a great role in the regulation of inflammation in autoimmunity. cd200 is the ligand for cd200r1 receptor. to determine if serum level of cd200 and its receptor cd200r1 can be used as a diagnostic and prognostic marker in patients with breast cancer.this case control study was carried out at oncology teaching hospital – medical city in baghdad. six groups were enrolled, four groups were confirmed with breast cancer stage (i, ii, iii and iv), fifth group (benign) and sixth group was control (healthy individual). serum is divided to measure cd200 and cd200r1 by utilizing quantitative sandwich enzyme-linked immunosorbent assay (elisa) kits. serum level of cd200 was significantly different (p=0.000088) between breast cancer patients and control only, serum level of cd200 increases with disease stage and there is a significant positive correlation. serum level of cd200r1 was different with stage, although the differences were not significant but the level of cd200r1 is lower in stage 4 patients than other stages. serum cd200 level can be used as a diagnostic marker for breast cancer. serum level of cd200r1 can be used as a prognostic marker. keywords: cd200, cd200r1, breast cancer, serum cd200. ينة من النساء العراقياتالمصل لع في 200سي دي و مستقبل الـ 200قياس مستوى سي دي كمؤشرات في التشخيص و المتابعة المصابات بسرطان الثدي: بيان دورهما **و إيمان سعدي صالح 1*،عدنان مصطفى اسماعيل صالح الدين،العراق ،تكريتجامعة فرع العلوم المختبرية السريرية ،كلية الصيدلة،* بغداد،العراق جامعة بغداد، فرع العلوم المختبرية السريرية ،كلية الصيدلة،** الخالصة هو بروتين سكري يظهر على العديد cd)200كتلة التمايز)سرطان الثدي هو مرض ينتج عن نمو خاليا الثدي بصورة غير طبيعية. ينتمي الى عائلة الجلوبيولين المناعي وله دور كبير جدا في تنظيم االلتهاب في حالة االمراض المناعية. cd200من الخاليا في الجسم، ينتمي ال cd200 هو المحفز الخاص للمستقبلcd200r1 . في تشخيص و متابعة تقادم المرض في مرضى cd200r1 و المستقبل الخاص به cd200 تهدف هذه الدراسة الى امكانية استخدام مستويات الـ .سرطان الثدي مجموعات، تم توزيع المرضى المصابين 6من تتكون هذه الدراسة .مدينة الطب في بغداد –اجريت هذه الدراسة في مستشفى االورام التعليمي المجموعة الثالثة )المرحلة الثالثة(، بسرطان الثدي على اربع مجموعات ، المجموعة األولى )المرحلةاألولى( ، )المجموعة الثانية )المرحلة الثانية( ، )المتطوعين األصحاء(. تم فصل المصل الذي وتقسيمه إلى المجموعة الرابعة )المرحلة الرابعة(، المجموعة الخامسة )حميدة( و مجموعة السيطرة كانت االختالفات في مستويات مصل الدم . elisa quantitative باستخدام الفحص المناعي الكمي cd200r1 و cd200عدة اقسام لقياس تزداد cd200مستويات الـ الصحاء فقط، ( بين مرضى سرطان الثدي و المتطوعين اp=0.000088ذات داللة إحصائية جيدة ) cd200 من الـ مختلفة، على الرغم من ان الفوارق لم تكن كبيرة لكن هذه cd200r1 كانت مستويات مع زيادة مرحلة المرض كما يوجد ارتباط إيجابي بينهم. .المستويات كانت اقل ما تكون لدى المرضى في المراحل المتقدمة من سرطان الثدي لمتابعة تقادم المرض. cd200r1كمعلًم تشخيصي حاالت سرطان الثدي، كما يمكن استخدام الـ cd200 يمكن استخدام الـ .cd200، زيادة مستويات cd2001r1(، المستقبل الخاص بكتلة التمايز cd200) 200الكلمات المفتاحية: كتلة التمايز introduction cancer is the second cause of death worldwide; about 17 million new cases of cancer were diagnosed in 2018. high mortality rate is also associated with breast cancer as it comes fifth 627000 deaths annually. according to the global cancer observery in 2018, the number of new cancer cases for both sexes of all ages in iraq was 25,023 case, breast cancer was the highest number of cancers 5,141 (20.3%) followed by lung cancer 2,123 (8.4%). the number of deaths was 2,060 (14.2%) from lung cancer and 1,727 (11.9%) from breast cancer(1). 1corresponding author e-mail: adnanpharma90@outlook.com received: 16/2 /2020 accepted: 31/8 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp253-258 iraqi j pharm sci, vol.29(2) 2020 new marker in breast cance 254 breast cancer (bc) can be divided histologically into invasive and non-invasive, ductal carcinoma in situ is the common type of non-invasive, while common type of the invasive are invasive lobular and invasive ductal carcinoma (2). molecular classification ; as two types, luminal a in which there is an expression of estrogen receptor, progesterone receptor without human epidermal growth factor receptor-2, and luminal b in which there is estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 not overexpressed (3). metastasis is a complicated pathological process; it begins with increased cell motility and cell migration then stromal invasion. subsequently, tumor cells intravasate into blood vessels and/or lymphatic system; they survive in the circulation; then extravasate, colonize in distant organs, and finally, sometimes after several years of dormancy, grow to overt metastases (4). cd200 is a cell surface glycoprotein expressed on many cells, it belongs to the immunoglobulin family and have great role in the regulation of inflammation in autoimmunity, organ transplantation and viral infections (5). cd200 is the ligand for cd200 receptor (cd200r), which is a receptor with an immune inhibitory activity expressed on myeloid and lymphoid cells and is considered an immunological checkpoint (6). multiple studies have been documented the tumor-promoting effects of cd200 in leukemia (7), there are opposing results regarding its role in solid tumor. cd200 expression is associated with increased metastatic survival of squamous cell carcinoma (8). in different circumstances, expression of cd200 by melanocytes results in reduced incidence of lung metastasis, and cd200r activation by an agonistic monoclonal antibody (ox110) is associated with decreased cd200-negative melanoma tumor formation in the lungs (9). if it is not expressed on the primary tumor, cd200 can be induced on the tumor during its progression, which supports a function as enhancer of tumor cell survival. for example, metastatic squamous cell carcinomas (scc) gain cd200 expression both in mice and men by same way during progression of the tumor (8). cd200r1 is belong to the ig superfamily transmembrane glycoprotein found on the surface of myeloid cells. cd200r1 interacts with its ligand cd200, which is also belongs to the ig superfamily transmembrane glycoprotein, which down regulate myeloid cell functions. cd200 is expressed on the surface of a variety of cells including neurons, epithelial cells, endothelial cells, fibroblasts, lymphoid cells, and astrocytes (10). cd200r-signaling increases the threshold for immune activation and is known to be highly important for restraining inflammatory responses, while cd200 expression on tumor cells is a marker for disease progression, suggesting a role in suppression of anti-tumor responses (11). this study carried out to determine if serum level of cd200 and its receptor cd200r1 can be used as a diagnostic and prognostic marker in patients with breast cancer. materials and methods subjects this case control study was carried out over the 7 months period from april 2019 until november 2019 at oncology teaching hospital – medical city in baghdad. a total number of eightyfour participants enrolled (patients and apparently healthy volunteers) in this study, they diagnosed by physician based on the histopathology analysis and mammography. ethical approval was obtained from ethics committee – ministry of health. patient selection fifty-six women with confirmed breast cancer were participated in the study with mean age (47.8 ±11.98 years). the diagnosis of cancer was confirmed by histopathology analyses and mammography dr. weam abdulfatah and dr. ahmed hussein (12). those patients were newly diagnosed and those on chemotherapy. participants are grouped as six groups, group 1: 14 women with stage i bc, group 2: 14 women with stage ii bc, group 3: 14 women with stage iii bc, group 4: 14 women with stage iv bc, group 5: 14 women with benign breast tumor, and group 6: 14 healthy women. data collection all participants completed interviewadministered questionnaires regarding family history of breast cancer and other malignancies, medical history, reproductive history, and breast feeding. other information's were collected from hospital records to determine cancer stage, hormonal receptor status and treatment protocol, the following data were obtained:  histopathological report and histological classification of cancer.  molecular classification of cancer.  age at diagnosis  chemotherapy status  tumor type, size, and grade.  lymph node status. blood sample collection venous blood specimen (5 ml) was withdrawn from each woman and placed in gelcontaining tubes, left at room temperature for at least 1 hour for clotting, the specimens then centrifuged at 3000 rpm for 8 minutes. the obtained serum was separated and divided into aliquots (which kept frozen at -80°c until their assay) to measure cd200 and cd200r1. iraqi j pharm sci, vol.29(2) 2020 new marker in breast cance 255 laboratory analysis reagent preparation for cd200 and cd200r1 a. wash buffer: aliquots of 750ml diluted washing buffer were prepared by adding 30 ml of the concentrated buffer to 720 ml of distilled water. b. standard for cd200: to prepare 8000 pg/ml of standard solution: 1 ml sample / standard dilution buffer added into one standard tube, from this tube a serious of dilutions are made to get the required concentrations as follow (4000, 2000, 1000, 500, 250, 125 pg/ml) respectively. c. standard for cd200r1: to prepare 50 ng/ml of standard solution: 1 ml sample / standard dilution buffer added into one standard tube, from this tube a serious of dilutions are made to get the required concentrations as follow (25, 12.5, 6.25, 3.13, 1.57, 0.78 ng/ml) respectively. d. preparation of biotin-labelled antibody working solution according to the manufacturer, biotin-labeled antibody were freshly prepared by diluting the antibody with the dilution buffer at 1:100 ratio. e. preparation of hrp-streptavidin conjugate (sabc) working solution: this solution prepared within 30 minutes before experiment. sabc diluted with sabc dilution buffer at 1:100 and mix them thoroughly. measurement of serum cd200 level serum cd200 level was estimated using a readymade kit (elabscince ®) an enzyme-linked immune sorbent assay (elisa) for cd200 (ox-2 membrane glycoprotein) is sandwich-type (quantitative) assay using two specific antibodies, first one anti-cd200 antibody was pre-coated onto microtiter plate provided in the kit and the second one biotin-conjugated anti-cd200 antibody was used as a detection antibody. the concentration of cd200 in the samples is then determined by comparing the optical density of the samples to the standard curve. measurement of serum cd200r1 serum cd200r1 level was estimated using (elabscince®) an enzyme-linked immune sorbent assay (elisa) for cd200r1 (cd200 receptor 1) which is a quantitative sandwich-assay using provided plate pre-coated with anti-cd200r1 antibody. the concentration of cd200 in the samples is then determined by comparing the optical density of the samples to the standard curve. the results were expressed as (ng/ml). statistical analysis statistical package for social sciences version 25 (spss v. 25) was used for data input and analysis. analysis of variance (one-way): to determine the difference in means of 3 independent samples, followed by tukey's test (post-hoc test) to identify significantly different means among the groups. sensitivity and specificity test and receiver operating characteristic (roc) to determine the sensitivity and specificity of the data. pearson's correlation coefficient (r): to evaluate the correlation between the parameters. results with (p<0.05) were considered to be significant. results the mean of serum cd200 concentration were significantly higher in breast cancer patients and in benign tumor than in control (apparently healthy) group, and the differences in the mean was highly significant with p value <0.05 table (1). analysis of variance (anova) for breast cancer stages show no significant differences in the mean of cd200 concentration with p value of (0.665) table (1). table 1. serum cd200 concentrations and significance for control, benign and stages of bc women. *highly significant (p<0.01) superscripts (a,b) refer to statically significant differences the correlation between breast cancer stages and serum cd200 concentration is positive medium (r = 0.545), as breast cancer stage progresses, the level of cd200 increases and this correlation becomes significant (p=0.00002) table (2). table 2. serum cd200 concentration correlation and significance with stages correlations cd200 concentration stage correlation 0.450** sig. 0.00002 ** correlation is significant at the 0.01 level (2tailed). sensitivity and specificity at different serum concentrations is shown in the table (3), the best cut-off point is at the concentration of (533.18 pg/ml) that shows (88%) sensitivity and (100%) specificity. area under the curve for breast cancer compared to control is (0.948) which is very high study groups n serum cd200 level (pg/ml) standard error control 14 347.40b 34.86 benign 14 776.44ab 98.31 stage 1 14 939.36a 97.15 stage 2 14 1025.74a 118.43 stage 3 14 1046.04a 128.04 stage 4 14 1181.19a 189.65 p-value for all groups 0.000088* p value for cancer stages 0.665 iraqi j pharm sci, vol.29(2) 2020 new marker in breast cance 256 and is highly significant as in table (4). better illustration of roc curve showed in the figure (1). table 3. sensitivity and specificity of cd200 concentration for bc women vs. control cd200 concentration (pg/ml) sensitivity % specificity % 324.5 98 50 380.68 96.4 64.3 462.9 89.3 71.6 509.17 89 85.7 533.18 88 100 567.5 87.5 100 figure 1. roc curve for serum cd200 for bc women vs. control table 4. area under the curve for breast cancer women vs. control area std. error asymptotic sig. 0.948 0.026 0.00001* * highly significant (p<0.01) the highest serum level of cd200r1 seen in stage 2 patients, while stage four had the lowest, stage one and three show nearly equal levels, as in the table (5). although the level of cd200r1 is different among the groups, but it was statistically not significant (p=0.293) table (5), table 5. serum cd200r1 concentration for control, benign and stages of bc women group n serum cd200r1 level (ng/ml) standard error contro l 14 36.923 6.41 benign 14 60.211 28.04 stage 1 14 78.783 18.02 stage 2 14 97.372 39.75 stage 3 14 71.699 30.39 stage 4 14 22.437 2.61 pvalue 0.293 sensitivity and specificity of serum cd200r1 level for stage 4 compared to other stages which represents the best cut-off points are shown in table (6) below; table 6. sensitivity and specificity of cd200 for stage 4 bc compared to other stages stages compare d to stage 4 cd200r1 concentrati on (ng/ml) sensitivit y % specificit y % stage 1 26.83 70 79 stage 2 25.15 71 78.6 stage 3 23.86 71 57.1 discussion breast cancer is highly heterogeneous in terms of its etiology and pathological characteristics, some cases of breast cancer showing slow development with good prognosis, whereas other cases taking a highly aggressive clinical course (13). early-stage detection of this cancer could reduce breast cancer death rates significantly in the longterm. investigators have studied many breast diagnostic approaches, including mammography, ultrasound, magnetic resonance imaging, positron emission tomography (pet), computerized tomography and biopsy (14). biomarker-based methods such as immunohistochemistry, radioimmunoassay, enzyme-linked immunosorbent assay (elisa) and fluoroimmunoassay also important and provide great information to the diagnostic requirements for breast cancer (15),(16). biomarker-based techniques are sensitive and selective, however, they have some limitations such as being expensive, needing trained people, time consuming and complex labelling process are also required (17). thus, there is an urgent need to develop a high sensitivity and label-free method for rapidly diagnosing breast cancer (18). iraqi j pharm sci, vol.29(2) 2020 new marker in breast cance 257 serum level of cd200 in patients with breast cancer increased with the progression of the disease which can be used as a diagnostic marker to differentiated breast cancer from healthy individual, a study by giuseppe a. et.al, found that cd200 was an excellent marker for the differential diagnosis of chronic lymphocytic leukemia (cd200 positive), and mantle cell lymphoma (cd200 negative)(19). cd200 is expressed in different neuroendocrine neoplasia, pancreatic islet cells and other normal neuroendocrine cells provides further support for cd200 as a general marker of neuroendocrine differentiation (20). serum cd200 level in breast cancer is related with poor prognosis and metastasis as the concentration is positively corelated with the progression of disease, several studies show variation with the prognostic value of cd200 expression, for example, cd200 expression was an independent favorable prognostic factor in patients' with non-small cell lung cancer, on the other hand, it is associated with poor prognosis in hematological malignancies. lack of cd200 expression in plasma cells has been related to more aggressive multiple myeloma (21). more recently cd200 odd expression has been proposed as an adverse prognostic factor in aml (22). a research by bahrami, a. et.al, showed a significant relationship between metastasis status and positive cea levels, which suggest that high cea levels decreased sensitivity and specificity for triple-negative breast cancer (tnbc) after received neoadjuvant chemoradiation (ncrt) (23). cd200 was over-expressed and correlated with progression of metastatic melanoma as well as acting as a potential target for therapy (24). gorczynski et al. also found a similar results, they discovers that over expression of cd200 level increased breast cancer lymph node metastasis (25). a study by jason e. love, et.al, suggests that cd200 is a general sensitive marker for neuroendocrine differentiation, with expression in 87% patient of the neuroendocrine neoplasms (nens) in the dataset, including 79 of 83 (95%) gastrointestinal luminal carcinoids, 60 of 72 (83%) pulmonary small cell carcinomas, three of four (75%) pulmonary large cell neuroendocrine carcinomas, 15 of 22 (68%) pulmonary carcinoids, 125 of 146 (86%) merkel cell carcinomas, and 56 of 60 (93%) pancreatic neuroendocrine tumor (20). cd200 is highly sensitive in breast cancer patient (88%) sensitive and (100%) specific at (533.18 pg/ml). although serum level of cd200r1 is different, stage iv and control showed the lowest level compared to other stages, it cannot be used as diagnostic marker, but it can be used to follow up the patients with confirmed breast cancer as it shows about (70%) sensitivity and different specificities at different concentrations. high expression of cd200r1 in non-small cell lung cancer patients has poor prognosis (26). conclusion the results of this study suggests that serum cd200 concentration can be used as a diagnostic marker to differentiate between healthy individual and breast cancer patients, but it cannot be used as a prognostic marker regardless of increasing in the expression with stage. on the other hand, serum expression of cd200r1 cannot be used as a diagnostic marker to differentiate breast cancer patients from healthy people, but could be used a prognostic marker as the concentration is markedly lowers with progression of the disease specially in stage four patients. reference 1. cancer [internet]. who. 2018 [cited 2018 sep 12]. 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m. serial tumour markers serum carcinoembryonic antigen and cancer antigen 15-3 assays in detecting symptomatic metastasis in breast cancer patients. 2012; 24. petermann kb, rozenberg gi, zedek d, groben p, mckinnon k, buehler c, et al. cd200 is induced by erk and is a potential therapeutic target in melanoma. j clin invest. 2007;117(12):3922–9. 25. gorczynski rm, clark da, erin n, khatri i. role of cd200 expression in regulation of metastasis of emt6 tumor cells in mice. breast cancer res treat. 2011;130(1):49–60. 26. yoshimura k, suzuki y, inoue y, karayama m, iwashita y, kahyo t, et al. abstract 1762a: cd200 and cd200r1 expressions and their prognostic roles in patients with non-small cell lung cancer. cancer res [internet]. 2018 jul 1;78(13 supplement):1762a lp-1762a. available from: http://cancerres.aacrjournals.org/content/78/13 _supplement/1762a.abstract baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(2) 2012 correlation between serum total adiponectin & hemoglobin 35 the correlation between serum total adiponectin and hemoglobin in type 2 diabetic patients without microalbuminuria ammar s. khamis * , shatha h. ali ** ,1 and khalid i. al-lehibi *** * department of clinical laboratory science, college of pharmacy, al-kufa university,najaf, iraq ** department of clinical laboratory science,college of pharmacy,university of baghdad,baghdad, iraq *** the specialist center for endocrinology & diabetes, al-kindy teaching hospital,baghdad,iraq abstract low serum total adiponectin is associated with a high incidence of type 2 diabetes or coronary artery disease in the general population. paradoxically, serum total adiponectin is elevated in patients with chronic kidney disease (ckd), such as overt diabetic nephropathy. the current study aimed to investigate whether or not anemia to be dependently associated with serum level of total adiponectin in non-albuminuric male patients with type 2 diabetes . the study included 42 type 2 diabetic male patients. anemia was defined as hemoglobin (hb) below 14.0g/dl. all the patients were without microalbuminuria, to exclude diabetic nephropathy. the diabetic patients were divided into 2 groups according to the hemoglobin level in addition to 16 healthy control group. serum total adiponectin levels were measured by a sandwich enzyme-linked immunosorbent assay. in all 42 patients with type 2 diabetes, serum total adiponectin levels were correlated positively with serum creatinine and age, whereas, negative correlations were found with hb. a stepwise regression analysis demonstrated that among several significant variables, hb had the strongest independent influence on total adiponectin (β = -0.512, at p < 0.01). in conclusion, anemia could be associated with a marked elevation in serum total adiponectin levels of diabetic patients without a detectable nephropathy (-ve microalbuminuria). key words: type2 diabetes, adiponectin, hemoglobin,microalbuminuria. هن العالقة بُن الوستىي الوصلٍ لالدَبىنكتُن الكلٍ هع صباغ الذم فٍ هزضً النىع الثانٍ السكزٌ غُز الوصابُن بشالل البىل الطفُف خوُس عوار صباح * علٍ و شذي حسُن **،1 و خالذ ابزاهُن اللهُبٍ *** . ، انؼشاق َجف، انكىفة ةجايؼ ، ةانصُذن ةكهُ ، انؼهىو انًخحبشَة انسشَشَة فشع * . ، انؼشاق، بغذاد بغذاد ةجايؼ ،ةانصُذن ةكهُ انؼهىو انًخحبشَة انسشَشَة ،فشع ** نهغذد انصى ويشضً انسكشٌ ، يسحشفً انكُذٌ انحؼهًٍُ ،بغذاد ، انؼشاق . انطبٍ ***انًشكز انحخصصٍ الخالصة ًشض انسكشٌ يٍ انُىع انثاٍَ أو يشض نالصابة بيغ َسبة ػانُة األجًانٍَشجبظ انًسحىي انًصهٍ نألدَبىَكحٍُ فٍ انًشضً انزٍَ انًسحىي انًصهٍ نألدَبىَكحٍُ األجًانٍ يشجفغ بأٌ يفاسقة، و نكٍ هُاك. انًجحًؼاتانششَاٌ انحاجٍ فٍ ػًىو وانذساسة انحانُة جهذف انً جقصٍ يا إرا كاٌ فقش انذو يشجبطا .انىاضحَؼاَىٌ يٍ يشض انكهً انًزيٍ ، يثم اػحالل انكهُة انسكشٌ شخص يٍ 24قًُا بذساسة .اء انسكشٌ يٍ انُىع انثاٍَفٍ انًشضً انًصابٍُ بذ نألدَبىَكحٍُ بشكم يسحقم يغ انًسحىي انًصهٍ و قذ جى جؼشَف غى/دل. 42أقم يٍ خضاب دووقذ جى جؼشَف فقش انذو بًسحىي انًصابٍُ بًشض انسكشٌ يٍ انُىع انثاٍَ يٍ انزكىس. خضاب انذو. جى قُاس اػحالل انكهُة انىاضح بظهىس انزالل فٍ األدساس. جى جقسُى يشضً انسكشٌ انً يجًىػحٍُ وفقا نًسحىي فٍ جًُغ انًشضً انًصابٍُ بانسكشٌ يٍ انُىع انثاٍَ ، . انًسحىي انًصهٍ نالدَبىَكحٍُ االجًانٍ بحقُُة األَزَى انًُاػٍ انًشجبظ يغ وانؼًش ، فٍ حٍُ جى انؼثىس ػهً االسجباط انسهبٍ فٍ انًصماسجبطث انًسحىَات انًصهُة نألدَبىَكحٍُ بإَجابُة يغ انكشَاجٍُُُ وَشُش جحهُم االَحذاس انًحذسج انً أٌ يٍ بٍُ انؼذَذ يٍ انًحغُشات انهاية ، خضاب انذو كاٌ نه أقىي جأثُش يسحقم ػهً .خضاب انذو ، َشجبظ فقش انذو يغ اسجفاع يهحىظ فٍ و ًَكٍ االسحُحاج بأَه. (β = -0.512, at p < 0.01) اإلجًانٍ انًسحىي انًصهٍ نألدَبىَكحٍُ .انًصهُة نألدَبىَكحٍُ اإلجًانٍ فٍ يشضً انسكشٌ ، وهزا االسجفاع هى يسحقم ػٍ وظائف انكهً.انًسحىَات النىع الثانٍ هن السكزٌ ، االدَبىنكتُن وصباغ الذم )الهُوىكلىبُن( ، سالل البىل الطفبف . : الكلوات الوفتاحُة introduction adiponectin [also known as acrp30 -adipocyte complement related protein of 30 kdaor adipoq] is a 244–amino acid protein secreted mainly by the adipose tissue. it was identified almost simultaneously by 4 different groups in the mid-1990s as an adipocytesecreted hormone but remained in obscurity until the early 2000s (1) . it circulates in multimers ; as full-length or highmolecularweight (hmw), medium-molecular-weight (or hexamer), and low-molecular-weight (or trimer) adiponectin complexes (2) . additionally, full-length adiponectin may be cleaved to form a smaller, globular fragment, which has been proposed to have greater potency than fulllength adiponectin (1) . the hmw isoform was proposed to have a stronger association with insulin resistance , metabolic syndrome , and 1 corresponding author email : hshathah@yahoo.com received : 12/12/2011 accepted : 6/6/2012 iraqi j pharm sci, vol.21(2) 2012 correlation between serum total adiponectin & hemoglobin 36 cardiovascular disease as the biologically most active form of the hormone (2) . however, several studies reported that the additional predictive value provided by hmw adiponectin in humans is minimal (2, 3) . adiponectin is synthesized primarily in white adipose tissue and, at lower concentrations, in brown adipose tissue (4) . much lower concentrations of expression have been reported in skeletal muscle, liver, colon, cardiac tissue, salivary glands, and placenta. adiponectin is even detected in cerebrospinal fluid and breast milk at much lower concentrations (4,5) . normal plasma adiponectin concentrations range between 5 and 30 µg/ml (depending on the assay used), and are inversely proportional to abdominal adiposity, insulin resistance, and type 2 diabetes (5) .adiponectin has also been shown to have distinct effects on lipid metabolism as well as antiinflammatory and antiatherogenic effects (6) . in addition to its peripheral actions, adiponectin may act centrally to modulate food intake and energy expenditure (7) . women have higher adiponectin concentrations than do men (a measure that is independent of fat mass or distribution), which is possibly linked to differences in estrogen or androgen concentrations (8) . reduced serum levels of adiponectin appears to play an important causal role in the development of insulin resistance, type 2 diabetes (t2d), and metabolic disease, thereby indirectly causing atherosclerosis.a recent meta-analysis of prospective studies with a total of 14,598 subjects and 2623 cases of type 2 diabetes indicated that higher adiponectin concentrations were associated with a lower risk of type 2 diabetes (9) . moreover, reduced adiponectin levels also directly play a causal role in the development of atherosclerosis (10) . the present study aimed to investigate whether or not anemia is associated with serum level of total adiponectin in non-albuminuric type 2 diabetic males. materials and methods this study was carried out at the endocrinology and diabetes center at alkindy teaching hospital for the period from october 2010-may 2011. the study included forty two male patients with type 2 diabetes mellitus (without overt nephropathy determined as negative microalbuminuria), twenty of the patients having anemia (i.e. hb concentration less than 14gm/dl) ,the remainder of them(i.e.22) were considered as non anemic (hb≥14gm/dl). all type 2 diabetic patients were maintained on oral hypoglycemic agents, and did not receive insulin therapy; all the patients were selected under supervision of a senior physician. additionally sixteen apparently healthy male subjects matching the age and sex of the patients were included as a control group. table (1) shows the baseline characteristics of the subjects included in the study. serum adiponectin, and ferritin were measured by elisa kits provided by demeditec diagnostics, germany (11,12) .blood hemoglobin and serum urea were measured by kits provided from cypress ® diagnostics, belgium (13,14) . whereas ,serum creatinine was measured by a kit provided from spinreact ® , spain (15) . detection of microalbuminuria was performed by utilizing first morning urine specimens( turbidimetric test for quantitative determination ) using kits purchased from human ® , germany (16) . statistical analysis was performed by student t-test and analysis of variance anova to examine the degree of significancy with p values less than 0.05. correlations were tested by regression analysis using spss, version 17. table 1: baseline characteristics of subjects. variable anemic type 2 diabetic patients non anemic type 2 diabetic patients controls number 20 22 16 age (years) 55.35 ±2.231 53.23±1.869 50.33±5.20 body mass index (kg/m2) 29.394±1.634 27.375±1.137 29.27±0.95 duration of diabetes (years ) 4.795±0.734 4.671±0.9 fasting serumglucose (mmol/l) 11.022±0.878* 11.596±0.749* 6.353 ±0.165 blood hemoglobin ( hb gm/dl) 13.292±0.202* 14.938±0.139 14.795±0.551 the data are expressed as the numbers or mean ± standard error of mean (sem). * = significant difference from their control iraqi j pharm sci, vol.21(2) 2012 correlation between serum total adiponectin & hemoglobin 37 results 1. serum total adiponectin as shown in figure-1data indicated that there was no significant difference in serum total adiponectin of the all type 2 diabetic patients (anemic and non anemic) when compared with the control group (p >0.05). while, there was a significant elevation (by about 8.7%) in serum total adiponectin of the anemic type 2 diabetic patients when compared to controls (p < 0.05). but, there was no significant difference in serum total adiponectin in non anemic type 2 diabetic patients when compared to the control (p >0.05). furthermore, a significant elevation (by about 7.2%) in serum total adiponectin of the anemic type 2 diabetic patients when compared to non anemic type 2 diabetic patients (p <0.05 ). figure 1 : serum total adiponectin data are presented as: mean ± standard error 2. blood hemoglobin table -2 demonstrated that there was no significant difference in hb concentration between type 2 diabetic patients (anemic and non anemic) compared with controls (a decrease by 4.33% ,p >0.05). data indicated that, there was a significant variation in blood hb values of the anemic type 2 diabetic patients when compared to the control (lowered by 10.16%) with p < 0.05. but, there was no significant difference in blood hb values between non anemic type 2 diabetic patients and the control group (p > 0.05). while, there was a significant difference in blood hb of the anemic type 2 diabetic patients when compared to non anemic type 2 diabetic patients (by about 11%) p < 0.05. 3. serum ferritin as shown in table -2, there was a significant difference in serum ferritin levels (an increase by about 55%) of type 2 diabetic patients (anemic and non anemic) as compared with that of the control group (p <0.05). data also indicated that, there was no significant difference in serum ferritin levels of the anemic type 2 diabetic patients when compared to the controls (p >0.05). while, there was a significant difference(an increase by about 36%) in serum ferritin level between non anemic type 2 diabetic patients when compared to the control ( p < 0.05). however, there were no significant differences have been observed in serum ferritin levels between that of the anemic diabetic patients as compared to non anemic diabetics(both of them without micoalbuminuria) ,p>0.05 . table 2: blood hemoglobin & serum ferritin concentration among studied groups controls (n = 16) non anemic type 2diabetic patients (n = 22) anemic type 2 diabetic patients (n = 20) whole type 2 diabetics patients (n = 42) 14.795±0.551 14.938±0.139 13.292±0.202 c , na 14.154±0.175 blood hemoglobin (gm/dl) 60.919±9.720 132.1±20.493 c 99.139±19.355 116.792±14.25 c serum ferritin (ng/ml) data are presented as: mean ± standard error of mean (sem) c: significant difference from control na: significant difference from non anemic 4. serum urea as shown in table -3 , a significant increase ( by about 16.6%) in serum urea concentration had been detected between type 2 diabetic patients (anemic and non anemic) and that of the control group (p < 0.05). data indicated that, there was a significant difference (an increase by about 16.45%) in serum urea level between anemic type 2 diabetic patients and controls. (p < 0.05). also there was a significant increase ( by about 16.75%) in serum urea values in non anemic type 2 diabetic patients compared to the control values (p < 0.05). furthermore, there was no significant difference in serum urea levels of the anemic diabetic patients as iraqi j pharm sci, vol.21(2) 2012 correlation between serum total adiponectin & hemoglobin 38 compared to non anemic diabetic patients( p > 0.05). 5. serum creatinine there was no significant difference in serum creatinine levels of type 2 diabetic patients (anemic and non anemic) when compared with the control group , (p >0.05), as illustrated in table -3. meanwhile, there was no significant difference in serum creatinine values of the anemic type 2 diabetic patients when compared to the control (p > 0.05). and there were no significant differences in serum creatinine between non anemic type 2 diabetic patients and controls. also there was no significant difference in serum creatinine levels of the anemic type 2 diabetic patients when compared to non anemic diabetics both of them without micoalbuminuria (p >0.05). table 3: serum urea & serum creatinine among studied group. controls (n = 16) non anemic type 2 diabetic patients (n = 22) anemic type 2 diabetic patients (n = 20) whole type 2 diabetics patients (n = 42) 36.392±2.094 43.715±2.452 c 43.559±2.395 c 43.64±1.696 c serumurea (mg/dl) 0.680±0.042 0.661±0.077 0.676±0.056 0.668±0.048 serum creatnine (mg/dl) data are presented as: mean ± standard error of mean (sem) discussion the relatively high serum ferritin level in anemic type 2 diabetic patients (although it is lower than non anemic type 2 diabetic patients, but it is still higher than controls as shown in table -2) support the fact that the cause of anemia in type 2 diabetic patients is due to inflammation or so called anemia of chronic diseases rather than an iron deficiency anemia. since the patients included in this study were non albuminuric diabetics, to exclude diabetic nephropathy and its relation to anemia through reducing erythropoietin ( epo) production (17) . the question is how precisely does iron overload exert its putative effects on the diabetic state? 1. iron accumulation in hepatocytes may interfere with the liver insulin-extracting capacity. supporting evidence comes from studies in noncirrhotic hemochromatotic patients. in these patients, insulin resistance and hyperinsulinemia appeared before pancreatic iron overload with selective b cell loss occurred. 2. iron deposition may cause insulin resistance by interfering with the ability of insulin to suppress hepatic glucose production. this theory may explain the relationship found between insulin resistance and high ferritin level. 3. iron tends to be auto-oxidized to form highly reactive, lipid soluble iron-oxygen complexes. in addition, ferrous iron also catalyzes the formation of glycosylated proteins derived free radicals. as a result, these highly reactive free radicals peroxidize lipids which change membrane properties and result in tissue damage. increased oxidation of free fatty acids was also found to diminish glucose utilization in muscle tissue and to increase gluconeogenesis in the liver, leading to increased insulin resistance. indeed, serum level of lipid peroxidation substances was high in patients with diabetes and diabetic microangiopathy. the above suggestions and evidences lead to that hyperferritinemia may be associated with type 2 diabetes mellitus (18) . a significant association between serum ferritin levels (19, 20) , high iron intake (21) , and type 2 diabetes had been reported, but the association was not well understood. low iron diets are not recommended, possible influences on ferritin levels were not excluded or taken into account in the statistical analyses of our study. they include burns, the recent use of aspirin and other nonsteroidal anti-inflammatory drugs, and recent blood donation, which lowers iron reserves and increases sensitivity to insulin (22) .concerning adiponectin , because of it's strong association with type 2 diabetes risks , preliminary data suggested that adiponectin may be moderately associated with cardiovascular morbidity and mortality. high adiponectin concentrations are associated with a favorable cardiovascular risk profile (23) . however, the relationship is more complex , some discrepancy may be related to the patient population studied (men versus women, older versus younger, prevalent cardiovascular iraqi j pharm sci, vol.21(2) 2012 correlation between serum total adiponectin & hemoglobin 39 disease). in addition, adiponectin may not directly affect cardiovascular risk but may be a marker of other risks, which may require further studies to clarify the relationship between adiponectin and cardiovascular disease (24) .however ,it remains unclear why anemia is independently associated with an increased serum adiponectin levels, especially hmw adiponectin, in patients with type 2 diabetes. one possible explanation is the influence of tissue hypoxia( caused by anemia) on the expression of adiponectin in adipose tissue, through inducing the expression of hypoxia inducible factor (hif) by cells of affected tissues (25) .a recent study performed in mice has demonstrated that hif-1 upregulates the expression of adiponectin in white adipose tissue, microvascular endothelial cells, or diabetic mouse hearts, presumably by acting on the presence of two putative hif-1 response elements (hre-1 and -2) in the promoter region of the murine adiponectin gene .thus, it is possible that hypoxia may lead to increased adiponectin production either in adipose tissue or in the heart (26) . on one hand, local production of hmw adiponectin in cardiomyocytes and microvascular endothelial cells by hif-1 may protect myocardium from hypoxia. on the other hand, production of epo is also regulated by the tissue oxygen supply and the hypoxia-dependent gene expression of epo is based on activation of the hif-1 pathway. thus, anemia may cause activation of the hif pathway, resulting in the increased production of epo, which suggests that activation of hif pathway may result in an increase in serum concentrations of both hmw adiponectin and epo (27) .however, contrasting reports have indicated that hypoxia inhibits the expression of adiponectin in cultured adipocytes or mice adipose tissue (28) . thus, there is controversy about the effects of hypoxia on the expression and metabolism of adiponectin. it is well known that a high serum adiponectin level is associated with a favorable lipid profile and improved glucose metabolism in both nondiabetic and diabetic subjects (29,30) .although blood urea concentrations increase as glomerular filtration declines, urea is a poor marker for kidney disease. unlike creatinine, urea production rates are not constant, being dependent on the activity of the urea cycle enzymes and the protein load. an increased protein load may be due to diet, gastrointestinal bleeding, or catabolic states, including corticosteroid therapy (31) . in this study diabetics have higher blood urea levels as compared to non diabetic subjects. furthermore , diabetic subjects could have significantly lowered serum total protein levels as compare to non diabetic subjects (31) . these biochemical changes may be related to the effect of having diabetes, where there is a depression of glycolytic enzymes and stimulation of gluconeogenic enzymes ,thus promoting gluconeogenesis in liver, which could further contribute to hyperglycemia, due to continuous catabolism of aminoacids leading to higher urea to be formed from urea cycle(our patients were selected to be free from liver disorders) .the current study demonstrated that hb levels showed a strong negative correlation with serum total adiponectin levels in patients with type 2 diabetes (β = -0.512, at p < 0.01), as shown in figure -2-. several previous studies, have shown that gender, age, tg, hdlcholesterol, and renal function are independent determinants of the serum total or hmw adiponectin level in non diabetic and diabetic subjects (32) . in conclusion, the presence of anemia may contribute to the elevated serum levels of total adiponectin in male diabetic patients without chronic kidney diseases. figure 2: correlation between serum total adiponectin and hemoglobin in type2 diabetic patients references 1. scherer pe, williams s, fogliano m, baldini g, lodish hf. a novel serum protein similar to c1q, produced exclusively in adipocytes. j biol chem 1995;270:26746–9. 2. heidemann c, sun q, van dam rm, et al. total and high-molecular-weight adiponectin and resistin in relation to the risk for type 2 diabetes in women. ann intern med 2008;149:307–16. 3. bluher m, brennan am, kelesidis t, et al. total and high-molecular weight adiponectin in relation to metabolic iraqi j pharm sci, vol.21(2) 2012 correlation between serum total adiponectin & hemoglobin 40 variables at 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2020 transdermal apixaban ultrafine o/w nanoemulsion based gel doi: https://doi.org/10.31351/vol29iss2pp214-222 214 preparation, characterization and ex vivo permeability study of transdermal apixaban o/w nanoemulsion based gel mustafa r. abdulbaqi*,1 and nawal a.rajab** *department of pharmaceutics, college of pharmacy, university of al-bayan, baghdad, iraq **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract this study designed to prepare ultrafine apixaban (apx) o/w nanoemulsion (ne) based gel with droplet size below 50 nm as a good method for transdermal apx delivery without using permeation enhancer, alternatively, the formulation components itself act as permeation enhancer. apx, a potent oral anticoagulant drug that selectively and directly inhibit coagulation factor xa, was selected as a good candidate for transdermal delivery as it displays poor water solubility (0.028 mg/ml) and low bioavailability (50%). apx-ne gel was prepared using triacetin, triton-x-100 and carbitol as oil phase, surfactant and cosurfactant respectively, while carbopol 940 used as a gelling agent. ex vivo permeation of apx-ne gel through human stratum corneum reveal significant (p ≤ 0.05) enhancement in permeation parameters (jss, pc and er, and shorter tlag) in comparison with the prepared pure apx gel. key words: apixaban, nanoemulsion, ex vivo permeation. عن طريق الجلد مستحلب دواء االبكسابان النانويجل النفاذية ل دراسة تحضير ، تشخيص و **نوال عياش رجبو 1*،عبد الباقيرعد مصطفى العراق.بغداد ، البيان ،جامعة ،الصيدلةكلية ،فرع الصيدالنيات* العراق. ،جامعة بغداد ،كلية الصيدلة ،فرع الصيدالنيات** الخالصة االبكسابان عبر نانومتر لتسليم 50صممت هذه الدراسة لتحضير جل لمستحلب االبكسابان النانوي المتناهي الصغر بحجم قطيرة أقل من ِّن النفاذية ، بدالً من ذلك ، تعمل مكونات الصياغة تعمل على تعزيز النفاذية . تم اختيار اء قوي مضاد ، وهو دو االبكسابان الجلد دون استخدام ُمحس 0.028، كمرشح جيد للتسليم عبر الجلد و ذلك لذوبانيته القليلة في الماء ) xa للتخثر عن طريق الفم يعمل بشكل انتقائي على تثبيط عامل التخثر والكاربيتول 100اكس مستحلب االبكسابان النانوي باستخدام ثالثي األسيتات ، تريتون ٪(. تم تحضير جل50مجم / مل( وتوافر حيوي منخفض ) لـجل(ex vivo) كعامل جل. كشفت دراسة النفاذية 940كطور زيتي ، خافضة للتوتر السطحي وخافض توتر مساعد، بينما استخدم كاربوبول ، وتم er و pc و jss في (p ≤ 0.05) نفاذية خالل الطبقة القرنية البشرية بشكل ملحوظزيادة معامالت العن مستحلب االبكسابان النانوي .النقي االبكسابان أقصر مقارنةً مع جل (lagt) الحصول على وقت ضائع النفاذية خالل الجسم الحي. ،نانوي مستحلب ،الكلمات المفتاحية: ابكسابان introduction transdermal drug delivery provides a suitable alternative to circumvent limitations associated with oral drug administration. it avoids hepatic first pass metabolism, a painless method and enable self-application by patients. however, the main objective of transdermal route is the large globule size and substantial barrier of skin to drug penetration, particularly stratum corneum in epidermis layer. nanoemulsion (ne) reflect one of the most promising techniques for transdermal carriage of drugs, as it improved the dispersibility of poorly soluble drugs and hence, improve their permeation, accepted thermodynamic stability, high loading capacity of both lipophilic and hydrophilic drugs, and efficient permeating properties of its components through biologic membranes(1, 2). nanoemulsion is isotropic mixture of water and oil stabilized by smix (surfactant and cosurfactant blended in different ratios)(3). its droplets uniformly distributed with average size range of 20 200 nm allowing higher drug flux through intracellular lipophilic pathway of skin and crafts drug depot within epidermis and stratum corneum(4). for more convenience application, ultrafine nanoemulsion formulation developed with droplet size below 50 nm that prepared by selecting appropriate oil and surfactant blend to dissolve the intended dose of drug. such system demonstrates better spreading properties caused by higher area to volume ratio of ultrafine nano-sized droplets; high drug loading capacity; ultrafine nanoemulsion formulation itself behave as penetration enhancer in absence using any chemical or physical permeation facilitating technique(5). 1corresponding author e-mail: drmustafa1986@yahoo.com received: 21/3 /2020 accepted: 19/7 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp214-222 iraqi j pharm sci, vol.29(2) 2020 transdermal apixaban ultrafine o/w nanoemulsion based gel 215 apixaban (apx) is a potent oral anticoagulant drug that selectively and directly inhibit coagulation factor xa and used as a prophylactic therapy for the prevention of venous thromboembolism (vte) following total hip or knee replacement surgery(6). it was selected as a suitable candidate for incorporation into ultrafine transdermal o/w nanoemulsion, as apx display relatively low molecular mass (459.497 g/mol); poor water solubility of 0.028 mg/ml at 24 °c; and low bioavailability of about 50% after oral administration, this low bioavailability could be attributed to the incomplete absorption of apx in the gastrointestinal tract (git), and from the effect of first-pass metabolism in gut and liver(7), low dose (2.5 or 5 mg) and suitable balanced partition coefficient of 44.7 (log p 1.65). however, the low viscosity of nanoemulsion made it is inconvenient to use transdermally, due to low retention at application site, therefore the ultrafine apx-ne was included into gel system. the present study designed to explore the feasibility of ultrafine with particle < 50 nm o/w nanoemulsion based gel as a carrier of poorly water-soluble drug apixaban for systemic transdermal delivery, and to investigate the potential of the prepared formulation to act itself as penetration enhancer through human stratum corneum without using physical or chemical permeation enhancing techniques. materials and methods materials materials used include, pure apixaban obtained from zhejiang cp chemical co., ltd; methanol lab grade solvent (sigma aldrich, usa); triacetin (hangzhou hyper chemicals limited); triton-x100 (sigma aldrich, usa); carbitol (sigma aldrich, usa). methods preparation of apx-ne according to previous related study(8), triacetin oil, triton-x-100 and carbitol selected as oil phase, surfactant and cosurfactant respectively for apixaban (apx) o/w nanoemulsion (ne) preparation. apx-ne formulations prepared at smix weight ratios of 1:1, 2:1, 3:1, 4:1, 1:2, 1:3 and 1:4. in which, the assigned dose of 5 mg apx dissolved in triacetin oil, and then add smix (surfactant/cosurfactant) with continuous vortex mixing. finally, deionized water was titrated gradually until clear isotopic apx-ne formed(9). thermodynamic stability study three thermodynamic stability tests used to assess physical stability of prepared apx-ne formulations. these tests include centrifugation test, where nanoemulsions centrifuged at 3500 rpm for 30 min, followed by six heating-cooling cycles at 45 and 4 °c for 48 h in each temperature and eventually, three freeze-thaw cycles at – 20 and 25 ˚c for 24 h at each temperature. after each test, samples discarded if demonstrate phase separation, precipitation or cracking by visual check(10). characterization of apx-ne formulations droplet size and polydispersity index (pdi) measurement photon correlation spectrophotometer (brookhaven instrument, zeta plus, usa) used to measure average droplet size of prepared apx-ne by dynamic light scattering (dls) technique, which analyze fluctuations in the light of scattering at 25 °c and scattering angle of 90°. polydispersity index (pdi) is a measure of homogeneity in droplet size which ranges from 0 to 1 and measured by electrophoretic light scattering technique(11). transmittance percent and electrical conductivity measurement transmittance percent (t%) measured for optical transparency of prepared apx-ne using uv-visible spectrophotometer (spectrumlab 752pro, china) at 650 nm keeping distilled water as blank(12), while electroconductivity measured using conductivity meter (dds-11a, china). these tests performed to confirm the type of prepared apx-ne and performed in triplicate. ph and apx content determination of apx-ne digital ph meter (bp 3001, singapore) used to measure ph values of apx-ne formulations. while apx content detected after desired dilution of 2 g sample from each apx-ne with methanol. samples then filtered via 0.45 µm filter syringe and analyzed spectrophotometrically at 278 nm (𝛌max of apx in methanol)(13). the experiments performed in triplicate. selection of optimum apx-ne formula for gel preparation due to low viscosity of nanoemulsion, and thereby easily washout upon dermal application, one apx-ne formulation selected for further fabrication into apx-ne based gel for more convenient transdermal use. the selection of most optimum formula made depending on characterization techniques of apx-ne. apx-ne gel preparation one gram of carbopol 940 dispersed in small amount of distilled water and then volume completed to 100 ml and left in dark place for 24 h to ensure complete swelling of carbopol 940 and allow escape of entrapped air bubbles. then, preprepared apx-ne added slowly to the aqueous solution of carbopol 940 (1% w/v) in a ratio of 1:1 with continuous stirring at 250 rpm. afterward, 0.5 g triethanolamine added to neutralize the gel resulting in clear homogenous viscus gel of apxne with strength of 1 mg apx in each 1 g of gel(14). apx-excipient compatibility studies fourier transform infrared (ftir) the purity and compatibility of apx with other excipient in ne and ne-gel was assessed using ftir spectroscopy (shimadzu, japan) at the iraqi j pharm sci, vol.29(2) 2020 transdermal apixaban ultrafine o/w nanoemulsion based gel 216 range 4000 – 400 cm -1 to check possible chemical interaction between their functional groups(15). ftir performed for pure apx, apx-ne and apx-ne gel each separately. x-ray diffraction (xrd) lattice nature and drug – excipients possible interactions characterized using xrd diffractometer (xrd-6000, shimadzu, japan) operated at voltage 40 kv, current 30 ma, cukα radiation at λ = 1.5406 nm, scanning speed of 5°/ min and 2θ range of 0 – 60 degree for pure apx and apx-ne gel each separately(16). microscopic morphology studies transmission electron microscopy (tem) surface images and approximate particle size of apx-ne and apx-ne gel studied using tem microscope (cm 120, philips, usa) with accelerating voltage of 100 kv by placing each sample separately on carbon coated copper grid and then left to dry and form thin film at room temperature for 60 sec. the excess was wiped out using whatman filter paper(17). ex vivo apx human skin permeation study to evaluate permeation efficiency of apxne gel through human stratum corneum, abdominal skin of human female utilized, from which outer most layer of epidermis, stratum corneum, was isolated and used as diffusional barrier. preparation of human skin an abdominal skin of human female (age: 44 years old; weight: 96.75 kg) obtained after routine plastic surgery for cutting sagging skin from abdomen of adult female in baghdad educational hospital, which then transferred into laboratory using ice bag at 4 ˚c within 2-3 hours. skin specimens first cooled to 4 ˚c and washed with phosphate buffer saline ph 7.4 solution with the aid surgical scalpel to get rid of the traces of blood, adipose tissue and subcutaneous fats remained after excision. afterward, the epidermis layer was separated from the dermis layer of skin using heat separation method, in which the whole skin piece placed in water bath at 60 0c + 0.5 0c for 60 90 sec and then epidermis peeled off carefully after its separation using anatomical forceps(18). finally, the skin was wrapped in aluminum foil and stored at -20 0c until use. at time of experiment, specimens of epidermal skin thawed and washed with phosphate buffer saline ph 7.4 solution. for isolation of stratum corneum layer from epidermis, skin pieces of epidermis layer were placed in a petri dish and soaked in 1% trypsin in pbs solution of ph 7.4 for 24 h incubation period at 4 ˚c followed by 1 h at 37 ˚c. the soaking step repeated using fresh 1% trypsin solution until stratum corneum separated and floated as a transparent thin layer over the solution. ultimately, stratum corneum transferred to another petri dish using anatomical forceps and rinsed with pbs of ph 7.4 twice to wash out any trypsin traces left. the unused stratum corneum was placed flat on aluminum foil and stored at -20 0c and used within three months(19). franz cell ex vivo permeation study through human skin the use of abdominal skin of adult human female was reviewed and approved by ethical committee of baghdad university / college of pharmacy for its application in the ex vivo permeation study experiment for pure apx gel and ultrafine apx-ne based gel. human stratum corneum isolated from adult female abdominal skin was mounted between donor and acceptor parts of franz cell at 32 + 0.5 ˚c; 600 rpm stirring magnitude and diffusional area (1.77 cm2); pbs containing 1% sls as dissolution medium. then, samples of 5 g apx-ne gel and pure apx gel placed above the skin in the donor part of franz cell for permeation evaluation(20). samples of 0.1 ml withdrawn with filtered syringe of 0.45 μm at predetermined time intervals of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 h, and substituted with same volume of fresh pbs ph 7.4 / 1% sls medium. the amount of apx permeated per unit area quantified spectrophotometrically at 280 nm and plotted against sampling time(21). the experiment performed in triplicate. ex vivo permeation data analysis permeation parameters including permeation flux (jss), lag time (tlag), permeability coefficient (pc) and enhancement ratio (er) were calculated exploiting permeation profile plotted from the cumulative amount of apx permeated per unit area (q, μg/cm2) via the stratum corneum part of human skin on y-axis against time (t, h) of experimental sampling on x-axis(22). statistical analysis spss statistical program (version 16, usa) used for data analysis in this study research with the adoption of difference or p value ≤ 0.05 as statistically significant. results preparation of apx-ne twenty-one apx-ne formulations prepared (table 1) by selecting three formulations of different smix ratio. two factors dependent for the preparation; first, amount of triacetin oil selected at 5 % weight interval (5, 10 and 15 %)(23); secondly, selecting formulations with minimum smix concentrations to avoid possible skin irritation by surfactant application(24). no obvious change noticed during formulation, i.e. phase separation, turbidity or color change, as well as no drug precipitation of apx observed during deionized water addition. iraqi j pharm sci, vol.29(2) 2020 transdermal apixaban ultrafine o/w nanoemulsion based gel 217 table 1. composition (w/w %) of apixaban nanoemulsion formulations f-code smix ratio smix% triacetin oil% water% f-code smix ratio smix% triacetin oil% water% f-1 1:1 30 5 65 f-12 4:1 35 15 50 f-2 1:1 30 10 60 f-13 1:2 30 5 65 f-3 1:1 30 15 55 f-14 1:2 30 10 60 f-4 2:1 30 5 65 f-15 1:2 30 15 55 f-5 2:1 30 10 60 f-16 1:3 30 5 65 f-6 2:1 30 15 55 f-17 1:3 30 10 60 f-7 3:1 35 5 60 f-18 1:3 30 15 55 f-8 3:1 35 10 55 f-19 1:4 30 5 65 f-9 3:1 35 15 50 f-20 1:4 30 10 60 f-10 4:1 35 5 60 f-21 1:4 30 15 55 f-11 4:1 35 10 55 thermodynamic stability study results demonstrate stability of nineteen apx-ne and discarding two (f-6 and f-21).the inherited high stability could be attributed to steric stabilization of nonionic surfactant triton-x-100(25) which confers long shelf life to nanoemulsions as compared to ordinary emulsions(26). characterization of apx-ne droplet size and polydispersity index (pdi) average droplet size measured for all formulations passed thermodynamic stability tests successfully. among tested formulations, nine apx-ne demonstrate ultrafine droplet size of less than 50 nm(27) including f-2, 7, 8, 9, 10, 11, 12, 13 and 16 with lower size obtained of 12.32 nm for f12. according to the results, as presented in table 2, a decrease in droplet size demonstrated with the increase in surfactant concentration, and consequently in smix ratio, as smaller sizes obtained at smix ratios of 4:1 and 3:1. this could be attributed to stabilization effect of the triacetin oil droplets by localization of triton-x-100 surfactant molecules at the oil/water interface resulting in higher stability and smaller droplet size(28). pdi values (table 2) of ≤ 0.3 indicates homogenous monodispersed nanoemulsion formation with good stability and uniformity in droplet size distribution upon dilution(29). table 2. results of psd, pdi, ph, electrical conductivity, t%, apx content%, for the prepared apx-ne formulations, (mean ± sd, n = 3). f-code psd (nm) pdi ph electrical conductivity (μs/cm) transmittance % % apx content f-1 672.48 0.430 5.25 + 0.02 194.58 + 1.24 99.56 + 0.02 97.05 + 0.11 f-2 38.63 0.420 5.12 + 0.05 181.11 + 0.84 99.79 + 0.01 98.23 + 0.21 f-3 281.70 0.296 5.07 + 0.01 167.08 + 0.18 97.34 + 0.03 99.01 + 0.13 f-4 106.01 0.432 5.15 + 0.02 191.64 + 1.29 99.81 + 0.01 96.94 + 0.26 f-5 283.76 0.567 5.07 + 0.05 182.79 + 0.91 98.86 + 0.01 98.84 + 0.17 f-7 26.41 0.302 5.38 + 0.05 185.84 + 1.14 97.13 + 0.01 99.63 + 0.14 f-8 23.06 0.279 5.16 + 0.01 172.63 + 0.82 97.46 + 0.02 99.37 + 0.2 f-9 15.49 0.235 4.91 + 0.04 164.34 + 0.66 99.95 + 0.01 99.65 + 0.18 f-10 37.17 0.321 5.35 + 0.03 188.57 + 0.61 99.94 + 0.01 99.56 + 0.11 f-11 35.83 0.329 5.27 + 0.04 177.46 + 0.55 97.72 + 0.06 99.73 + 0.26 f-12 12.32 0.234 5.16 + 0.01 168.18 + 1.18 98.05 + 0.03 99.98 + 0.18 f-13 16.34 0.308 5.07 + 0.01 190.89 + 1.33 97.81 + 0.02 99.67 + 0.13 f-14 114.59 0.434 4.98 + 0.03 181.47 + 0.89 98.49 + 0.07 96.88 + 0.18 f-15 150.16 0.252 4.91 + 0.03 173.34 + 0.54 99.91 + 0.04 97.04 + 0.19 iraqi j pharm sci, vol.29(2) 2020 transdermal apixaban ultrafine o/w nanoemulsion based gel 218 table 2. continued results of psd, pdi, ph, electrical conductivity, t%, apx content%, for the prepared apxne formulations, (mean ± sd, n = 3). transmittance percent and electrical conductivity all tested apx-ne exhibit high light transmittance (table 2) with close approximation to 100% indicating optically clear, transparent and nanosized droplets(30). electroconductivity reveal the formation of o/w nanoemulsions with a high degree of electrical conductivity as water represents the external phase and can conduct electrical current(31). ph and apx content determination of apx-ne ph readings summarized in table 2 and reveal comparable values with skin ph, which ranges from 4.5 to 6.5, indicating its suitable application for transdermal use without skin irritation or sensitization(32). apx content presented in table 2 and reveal acceptable values that set within official range (85 % 115 %) according to the united states pharmacopeia (usp) indicating successful apx loading without any precipitation or degradation of the drug. apx-ne gel preparation apx-ne (f-12) formulation was selected for gel preparation, as f-12 demonstrate desirable properties of droplet size, pdi, ph and apx content. the prepared apx-ne gel demonstrate consistent and viscous gel that is appropriate for transdermal use. compatibility studies fourier transform infrared (ftir) the ftir spectrum of pure apx backbone structure (figure 1) display bands at 3479 and 3313 cm-1 assign for n-h stretching asymmetric and symmetric of primary nh, while 2937 and 2837 cm1 bands refer to c-h stretching asymmetric and symmetric of ch3. bands at 1512-1400 cm -1 assign for c=c stretching of benzene ring, 1184 and 1595 cm-1 refer to n-c and -c=o stretching vibration for amide. same characteristic bands of pure apx appeared after drug formulation into ne and ne-gel with small shifting, as shown in figure 2, indicating compatible apx-excipient mixing without chemical interaction(33). figure 1. chemical structure of apixaban (apx)(6). f-code psd (nm) pdi ph electrical conductivity (μs/cm) transmittance % % apx content f-16 13.12 0.300 4.86 + 0.07 196.96 + 1.36 97.48 + 0.01 99.89 + 0.11 f-17 371.53 0.414 4.73 + 0.01 187.66 + 0.81 99.99 + 0.01 97.72 + 0.34 f-18 495.17 0.411 4.67 + 0.02 180.74 + 0.65 99.63 + 0.04 98.04 + 0.21 f-19 321.68 0.415 4.95 + 0.01 195.77 + 1.08 99.57 + 0.05 97.62 + 0.11 f-20 353.85 0.386 4.82 + 0.04 185.39 + 1.36 97.84 + 0.01 98.02 + 0.19 iraqi j pharm sci, vol.29(2) 2020 transdermal apixaban ultrafine o/w nanoemulsion based gel 219 figure 2. comparative ftir spectra of pure apx in black color with the red spectra of (a) apx-ne and (b) apx-ne based gel. x-ray diffraction (xrd) the xrd diffractograms of tested samples presented in figure 3. xrd diffractogram of pure apx revealed highly crystalline structure, as it displayed sharp intense narrow diffraction peaks noted at 2θ angles 17.0362, 22.2967 and 32.7404 degrees. same peaks also displayed in apx-ne gel diffractogram indicating apx/ne-gel components compatibility without any chemical modification of apx(34). additionally, apx-ne gel displayed no sharp peaks with significant (p ≤ 0.05) lower intensities indicating amorphous structure of apx within ne-gel(35). figure 3. comparative xrd of apx (blue) and apx-ne gel (red) at intensity (a) 100000 and (b) 3000. microscopic morphology studies transmission electron microscopy (tem) photomicrographs of apx-ne and apx-ne gel shown in figure 4 and reveal discrete, dark spherical shaped droplets with almost uniform particle size distribution of less than 50 nm. these results confirm ultrafine (< 50 nm) apx-ne gel formation and fit with droplet size analysis of apx-ne f-12 (12.32 nm) using dynamic light scattering (dls) technique. furthermore, no signs of droplet coalescence observed and thus, indicating physical stability of formulation(36). iraqi j pharm sci, vol.29(2) 2020 transdermal apixaban ultrafine o/w nanoemulsion based gel 220 figure 4. tem of (a) apx-ne and (b) apx-ne gel. ex vivo apx-ne gel permeation study human abdominal skin of adult female utilized for apx-ne gel permeability assessment to get best fit with in vivo conditions using franz cell apparatus. figure 5 present cumulative amount of apx permeated through human stratum corneum from apx-ne gel and pure apx gel as comparative diagram. results reveal complete apx permeation from apx-ne gel after 7 h, whereas 6.89 % drug permeate from pure apx gel after same time, indicating significantly (p ≤ 0.05) higher cumulative amount of drug permeated from ne-gel than that of pure apx gel with 14.5-fold increment. the reasons for this superior performance include the ultrafine (< 50 nm) sized globules of apx-ne within hydrophilic carbopol 940 gel structure, and penetration enhancing properties of triton-x-100, carbitol and triacetin oil components of apx-ne, which disrupt stratum corneum lipid bilayer thereby affecting its integrity leading to pore formation and increased apx amount fluxed across skin(37). the non-ionic surfactant triton-x-100 can solubilize lipids of stratum corneum and hence enhance apx penetration and absorption(38). carbitol binds keratin filaments resulting in corneocytes disruption and enhance drug entrance. additionally, apx present in dispersible form within ne-gel structure, resulting in either increased drug uptake directly or hold within ne as a vehicle for drug transport(39). figure 5. comparative ex vivo permeability through human stratum corneum of pure apx gel (red) and apx-ne gel (blue) ex vivo permeation data analysis permeation parameters of apx-ne gel through human stratum corneum illustrated in table 3 and indicate significant (p ≤ 0.05) faster permeation than pure apx gel with higher jss, pc and er values, and shorter tlag. table 3. permeation parameters results of apxne gel. parameter through human stratum corneum pure apx gel apx-ne gel flux (μg/cm 2 .h) 2.8914 16.741 pc (cm/h) × 10 -3 1.15656 6.696 tlag 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diffusivity and follicular targeting. pharmaceutics. 2018;10(1):1-14. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ association between gallstones and diabetics type ii iraqi patients iraqi j pharm sci, vol.20(2) 2011 gallstones and dm. type 2 38 association between gallstones and diabetics type 2 iraqi patients anmar h. altaie* ,1 *department of pharmacotherapeutics, college of pharmacy,university of al-mustansyria, baghdad,iraq abstract gallstone disease is one of the most common complications among diabetic patients especially type 2 dm. till now, there is no specific and certain factor that explain the incidence of gallstones among type 2 diabetic patients and many risk factors are taken collectively to estimate its intensity and severity compared to non diabetic counter parts. this clinical study was designed to evaluate and report the incidence and severity of gallstones among type 2 diabetics and non diabetics regarding certain factors. 20 diabetic females and 20 diabetic males were collected as patients′ group and have had gallstones while 20 females and 20 males who have had gallstones without diabetes mellitus type 2 were collected as controls′ group. the age, weight, and both of the size and number of gallstones of diabetic patients and controls were correlated to demonstrate the prevalence of gallstones among the patients′ and controls′ groups. the study showed that the mean ages of female patients is slightly higher (p<0.05) than the female controls. on the other hand there was a significant difference (p<0.05) between female and male control subjects. the body mass index (bmi) of male patients was significantly higher (p<0.05) than the male controls, while there was a significant difference (p<0.05) between female and male control subjects. the results of the study presented that the stone size of female patients was significantly higher (p<0.05) than the female and male controls, while the stone diameter of male patients was significantly higher (p<0.05) than the male and female controls. the stone number of female patients was significantly higher (p<0.05) than the female and male controls, while the stone number of male patients was significantly higher (p<0.05) than the male and female controls. these findings suggest that the incidence of gallstones was higher in type 2 diabetics both in males and females than the non diabetics and more in females than males who were non diabetics. key words: diabetes mellitus, gallstones, gender, ultrasound الخالصة الًٌْع الثّاًٖ. دخٔ اٙى، ل٘س ٌُاك عاهل هعّ٘ي ابذاء السنشٕ خصْص الوشاسة أدذ أمثش الخعق٘ذاِث ب٘ي الوشضٔهشض دصٔ العْاهَل َهأُْخْرة بشنل جواعٖ ّاى الًٌْع الثّاًٖ ابذاء السنشٕ خصْص ُّهخَأَمِّذ الزٕ ُّْٗضُخ دادثتَ دصٔ الوشاسة ب٘ي الوشضٔ . ُزٍ الذساسِت السشٗشِٗت ُصّووْج ّغ٘ش الوصاب٘ي بذاء السنشٕ الًٌْع الثّاًٖ ابذاء السنشٕ خصْص الوشضٔلخَخو٘ي مثافخَِ ّشذَّحَِ ب٘ي هي االًاد 0ٓ. ّغ٘ش الوصاب٘ي بذاء السنشٕ الًٌْع الثّاًٖ اشذَِّة دصٔ الوشاسة ب٘ي الوشضىبذاء السنشٕ خصْصحق٘٘ن لخَق٘٘ن ّل بذاء السنشٕ هي الٌْع الثاًٖ اٗضا , جوعْا معٌ٘ت هشضٔ ّماًْا هشضٔ هي الزمْس 0ّٓبذاء السنشٕ هي الٌْع الثاًٖ هشٗضاث ّحن فذصِن هخخبشٗا للخأمذ هي عذم اصابخِن بالسنشٕ هي الزمْس جوعْا معٌ٘ت 0ّٓ هي االًاد 0ٓذصٔ الوشاسة بٌ٘وا هصاب٘ي ب الوصاب٘ي بذاء السنشٕ هي الٌْع وشاسة للوشضٔدجِن ّعذِد دصٔ ال ّ الوشضٔ ّصىّ دصٔ الوشاسة فقط. ُعوش ّماى عٌذُن للًاد ( p<0.05) هي هخْسط االعواس أعلٔ قل٘لً االًادِ للوشضٔ هخْسط االعواسالذساستَ بأّى الثاًٖ ّغ٘ش الوصاب٘ي بَ اظِشث . إّى هخْسط االعواس للزمْس ّاالًاد الزٗي اخزّا معٌ٘ت ( ب٘ي p<0.05هي الٌاد٘ت األخشٓ ماى ٌُاك إخخلف ُاّم ) الزٗي اخزّا معٌ٘ت ، بٌ٘وا ماى ٌُاك إخخلف (معٌ٘تأخزّا الزٗي )الزمْس ( ِهيْ p<0.05ِهْي الوشضٔ الزمِْس أعلٔ جذاً ) بٖ .ام .إ ( دلَ٘ل مخلتَ الجسن ) دصٔ الوشاسة دجنًَخائُِج الذساسِت ّضذج. معٌ٘ت للزمْس ّاالًاد الزٗي اخزّا بٖ .ام .إ ()دل٘ل مخلت الجسن ( ب٘ي p<0.05ُاّم ) الوشضٔ الزمِْس أعلٔ دصٔ الوشاسة لذٓ بٌ٘وا قطش لذٓ االًاد ّالزمْس الزٗي اخزّا معٌ٘ت ,( p<0.05أعلٔ جذاً ) االًاد للوشضٔ هي ( p< 0.05) أعلٔ جذاً ًاداالالوشضٔ دصٔ الوشاسة لذٓ إّى عذَد الزمْس ّاالًاد الزٗي ارّا معٌ٘ت . ( ِهْي p<0.05جذاً ) الزمْس ّاالًاد الزٗي ِهْي ( p< 0.05)أعلٔ جذاً الزمْسالوشضى دصٔ الوشاسة لذٓ إّى عذَد ، بٌ٘وا الزمْس ّاالًاد الزٗي اخزّا معٌ٘ت مل فٖ الزمِْس ّاإلًاد لذٓ الوصاب٘ي بذاء السنشٕ هي الٌْع الثاًٖ فٖ أعلٔ دصٔ الوشاسةحَقخشُح ُزٍ الٌخائجِ بأّى دادثتَ ارّا معٌ٘ت. ّمزلل اعلٔ فٖ االًاد هي الزمْس هي الوصاب٘ي بذصٔ الوشاسة ّ غ٘ش هصاب٘ي بذاء السنشٕ . introduction diabetes is a chronic condition caused by a relative or an absolute lack of insulin (1) . it is associated with abnormalities in carbohydrate, fat, and protein metabolism and results in chronic complications including microvascular, macrovascular, and neuropathic disorders (2) . several distinct types of dm exist and are caused by a complex interaction of genetics, environmental factors, and life-style choices. the two broad categories of dm are designated type 1 and type2 (3) . gallstones (chole = “bile”,lithia = “stone”, and -sis = “process”) are the most common digestive disease causing huge hospitalizations annually worldwide (4) . gallstones can occur anywhere within the biliary tree, including the gallbladder and the common bile duct (5) . 1corresponding author email : altaii1978@yahoo.com received : 12/5/2011 accepted :8/10/2011 iraqi j pharm sci, vol.20(2) 2011 gallstones and dm. type 2 39 gallstones are formed by concretion or accretion of normal or abnormal bile constituents. they are divided into two major types: cholesterol stones account for 80% of the total, with pigment stones comprising the remaining 20% (3) .gallstones can occur at any age, although they are rare under the age of 10 years and most common at 50–60 years in both sexes. women are more prone to develop gallstones than men (6) ..there are several factors which directly affect the formation of gallstones as; sex, age, obesity, diet with high calories, diabetes mellitus, hemolytic anemia, estrogen and clofibrate therapy etc (7,8,9) .diabetic subjects are reported to have a two to three fold increase in the prevalence of cholesterol gallstone. inadequate emptying of gallbladder and increased fasting gallbladder volume has been reported in various studies (10) .in the present study , the incidence of gallstones in type 2 diabetics is made in correlation with age, sex and weight, the size and number of stones were taken in consideration. materials and methods this study was carried out on (80) patients. of those 40 subjects (20 males, 20 females) have gallstones without diabetes mellitus type 2 served as a control group and 40 patients(20 males, 20 females) have diabetes mellitus type 2 with gallstones. certain exclusion criteria were followed to avoid interference of any other factors like drugs or pathological conditions with this research, which include: patients with history of gallstones and received previous therapy or undergoing treatment, patients with history of clinical disorders like hyperlipidemia, hepatic disorders and those taking medications other than prescribed for dm type 2. those patients were diagnosed and treated in al-yarmook teaching hospital under supervision of specialist physicians. type 2 diabetic patients were diagnosed and selected based on american diabetes association criteria of dm (2004): fasting plasma glucose concentration ≥ 126mg/dl, 2hr postprandial glucose concentration ≥ 200mg/dl and whose age at the onset of type 2 dm was equal or greater than 40 years. gallstones identification, size and number were determined by ultrasonography which is thought to be the most appropriate technique in the diagnosis of the gallstones. statistical ِ analysis the results were expressed as mean± sd, student t-test and anova test were used to examine the degree of significance which is considered significant as p value <0.05. results both of table 1 and figure 1, show the age distribution of patients depending on their gender. the mean ages of female patients having gallstones and dm type 2 was (56.2±9.24) years, is slightly higher (p<0.05) than the female controls having gallstones without diabetes (49.5±10.62) years while the mean ages of male patients having gallstones and dm type 2 was (58±10.5) years is significantly indifferent (p<0.05) from the male controls having gallstones without diabetes (58.2±10.88) years . on the other hand there was a significant difference (p<0.05) in the mean age between female and male control subjects (49.5±10.62), (59.58±10.5) years old respectively. table 1: characteristics of study parameters for both control subjects (having gallstones without type 2 dm) and patient subjects(having gallstones and type 2 dm) type and no. of patients age (year) mean± sd bmi kg/m 2 mean± sd gallstones′ size (mm) mean± sd gallstones′ number mean± sd non diabetic females(20) 49.5±10.62 27.217±2.96# 3.37±1.31 3.0±1.0 diabetic females(20) 56.2±9.24* 27.218±2.6# 7.49±4.35*# 5.0±3.0*# non diabetic males(20) 58.2±10.88# 25.05±2.78 3.36±1.63 2.0±1.0 diabetic males(20) 58.2±10.88# 28.48±2.57* 6.4±3.48*# 5.0±3.0*# *significantly different from control (p< 0.05). # significantly different between groups (p<0.05) iraqi j pharm sci, vol.20(2) 2011 gallstones and dm. type 2 40 figure 1: the mean age of both control subjects (having gallstones without type 2 dm) and patient subjects(having gallstones and type 2 dm) as presented in table 1 and figure 2, the body mass index (bmi) of female patients having gallstones and type 2 dm (27.218±2.6) kg/ m 2 was significantly indifferent (p<0.05) than the female controls (27.217±2.96) kg/ m 2 , while the bmi of male patients having gallstones and dm type 2 (28.48±2.57) kg/ m 2 was significantly higher (p<0.05) than the male controls (25.05±2.78) kg/ m 2 . on the other hand there was a significant difference (p<0.05) between female and male controls (27.218±2.6), (25.05±2.78) kg/ m 2 respectively ,and between female patients (27.217±2.96) kg/ m 2 and male control (25.05±2.78) kg/ m 2 .the stone diameter of female patients having gallstones and dm type 2 was (7.49±4.35)mm is significantly higher (p<0.05) than the female and male controls having gallstones without diabetes (3.37±1.31), (3.36±1.63) mm respectively, while the stone diameter of male patients having gallstones and dm type 2 was (6.4±3.48) mm, is significantly higher (p<0.05) than the male and female controls having gallstones without diabetes was (3.36±1.63), (3.37±1.31) mm ,respectively as shown in both of table 1 and figure 3.table 1 and figure 4, also present that the stone number of female patients having gallstones and dm type 2 was (5.0±3.0), is significantly higher (p<0.05) than the female and male controls having gallstones without diabetes (3.0±1.0) (2.0±1.0) respectively, while the stone number of male patients having gallstones and dm type 2 was (5.0±3.0), is significantly higher (p<0.05) than the male and female controls having gallstones without diabetes (2.0±1.0), (3.0±1.0) respectively. figure 2: the bmi(kg/ m 2 ) of both control subjects (having gallstones without type 2 dm) and patient subjects(having gallstones and type 2 dm) iraqi j pharm sci, vol.20(2) 2011 gallstones and dm. type 2 41 figure 3: the gallstone diameter(mm) of both control subjects (having gallstones without type 2 dm) and patient subjects(having gallstones and type 2 dm) figure 4: the gallstone number of both control subjects (having gallstones without type 2 dm) and patient subjects(having gallstones and type 2 dm) discussion gallstone disease is one of the most common digestive diseases (4) . many studies investigate and demonstrate the relationship between gallstones and type 2 dm. ali ekecik et al (11) reported a statically significant difference (p<0.01) in the frequency of gallstones between 200 non diabetic control group and 200 diabetic patients, the incidence was 10.5%(21/200) in the control group versus 27%(54/200) in type 2 diabetic patients. one of the risk factors for cholesterol gallstones includes increasing age and female gender. in the present study as showed in table 1, the age of the patients regarding the gender plays a great role in the incidence of gallstones in that the incidence was significantly higher between female and male control subjects. on the other hand there was a significant difference (p<0.05) among females having type 2 diabetes than the controls, while age plays non-significant role in the incidence of gallstones among type 2 diabetic males than the controls. the incidence was insignificantly higher between female and male patients having type 2 dm suggesting the role of factors other than the relationship between age and gender.the mean age of onset of gallstones among both the control and patient females was totally lower than the mean age of both the control and patient males. this finding may be attributed to the fact that females are reported to have prevalence of gallstones till the age of 50 probably due to hormonal influence on bile composition and gallbladder motility (12) . the results of this study is in harmony with that done by rupali saxena et al (12) , who found that 67.5% of the diabetic patients with gallbladder disorder iraqi j pharm sci, vol.20(2) 2011 gallstones and dm. type 2 42 were females, as well as by sun h. et al (13) who stated that the incidence of gallstone was higher in females than in males and with increasing age 40-64 or ≥65years, while champan et al (14) stated that subjects of both sexes with type 2 dm, had higher prevalence of gallstones than controls. another risk factor for the increased incidence of gallstones in type 2 diabetics is obesity. table 1 shows the effect of body weight regarding gender on the incidence of gallstones where the incidence was significantly higher between female and male control subjects, while insignificant between female and male diabetic patients but again significantly different between male patients having gallstones and type 2 dm and control males. the results of this study agree with a study done by a. b. olokoba et al (15) who founded that the diabetic patients had a significantly higher mean bmi than the controls (p < 0.01) in 100 type 2 diabetic patients and 100 ageand sex-matched controls. but again obesity alone is independent risk factor among obese type 2 diabetic patients and a number of factors are collectively responsible for the increasing incidence of gallstones and type 2 dm. in diabetics, lipid concentration of plasma and bile increase. the obesity observed in diabetic patients has diverse effects on increased cholesterol synthesis and bile saturation (16,17) additionally, in the obese, gallbladders may not empty normally or completely (18) . as bmi increases, the risk for developing gallstones also rises. for women, obesity is an even stronger risk factor for developing gallstones and women with a bmi greater than 32 may be as much as three times as likely to develop gallstones as those with a bmi of 24 or 25. the risk may be seven times higher in women with a bmi above 45 than in those with a bmi under 24 (19) , and this may explain why there was insignificant difference between the control and the diabetic females. the last two parameters are the gallbladder stones′ sizes and numbers and their associated incidence among controls and type 2 diabetic patients. both of the size and number of gallstones were significantly higher in female and male patients than in the both sexes of the controls. the supersaturation of bile with cholesterol, nucleation of monohydrate crystals and dysfunction of gall bladder are the three important factors that cause gallstone formation (20,21) .hypomotility of gallbladder cause gallstone formation in diabetes mellitus and other chronic disorder like obesity (22) .the gallbladder of diabetic patients are generally large and its motility is disturbed (23) . these events are probably due to the dysfunction of the gall bladder wall . the combined effects of poor emptying and possibly decreased ejection fraction rate and increased fasting volume could lead to bile stasis within the gallbladder and the formation of stones (24) . conclusions from the results and discussion of this study, we conclude that the incidence of gallstones is higher in type 2 diabetics both in males and females than the non diabetics and more in females than males who are non diabetics and multiple factors collectively are associated with this incidence that ultimately lead to marked increase in stone size and number. references 1. lisa a. kroon, mitraassemi, betsy a. carlisle. diabetes mellitus. in: applied therapeutics the clinical use of drugs.mary annekoda-kimble,lioyd yee young, brian k.alldredge, et al. 9 th ed., lippincot.usa. 2009,p50-53. 2. stephan nd, daryl kg. insulin, oral hypoglycaemic agents, and the pharmacology of the endocrine pancreas. in: goodman and gilman's the pharmacological basis of therapeutics. hardman jg, limbird le editors.10 th ed. new york: mcgraw– hill. usa. 2001, p. 1686-7. 3. alvin c. powers. diabetes 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feldman m, feldman m jr et al. incidence of cholelithiasis, cholesterosis and liver disease in diabetes mellitus, autopsy study. diabetes. 1954;23: 305-12. 17. hozbach rt. pathogenesis and medical treatment of gallstones. in:sleisenger mh, fordtan j s , eds. gastrointestinal disease, 4 th ed. wb saunders. philadelphia,usa. 1989, p 1668-90. 18. the health risks of obesity: obesity and gallstones. by jennifer r. scott, about.com guide updated january 15, 2009. 19. dieting and gallstones. national institute of diabetes and digestive and kidney diseases. 20. paumgartner t, sauerbruch t. gallstones: pathogenesis, the lancet, 1991; 8777:1117-21. 21. sherlock k, dooley j. diseases of the liver and biliary system. in: gastrointestinal disease.sleisengermh, fordtranjs, editors.blackwell scientific publications.uk. 1993;p. 562-92. 22. hahm, j.s., park, j.y., song, s.c. et al. gallbladder motility change in late pregnancy and after delivery. korean j. intern.med. 1997; 12:16-20. 23. blomm aa, stochengeld r. diabetic cholecystomegaly.jama.1985;408:357-9. 24. takahashi t, yamamura t, yokoyama e, et al. impaired contractile motility of the gallbladder after gastrectomy. am j gastroenterol. 1986; 81: 672-77 file:///c:/bio/jennifer-r-scott-6038.htm ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 80 phosphodiester conjugation of metronidazole and dexamethasone as possible mutual prodrug muthana d. saud* and suhair m. ghani received 30-12-2002 accepted 15-5-2005 abstract as p os sible mutua l prod rug had b een s ynthes ized that contain metro nidazole and dexame thazone co njuga te d through phosp hod ie ster linkage . the ratio nale for this typ e of c onjugate is to get a prodrug with p oss ible site – s pe cific de live ry of its ac tive c ons titue nts into the lower parts of the g.i.t. this c ompo und was s ynthe size d by the rea ction of de xame thzo ne – 2 1 – phos pha te with me tronid azo le to fo rm:(1 – (de xame thaz o ne – 21 – pho sphory l) – me tro nidaz ole ) this c onjugate was p erfo rme d us ing dicyc lo he xylca rb odiimid e (dcc) as a c ondensing a gent. the id entity o f the prep ared compound had be en confirmed us ing t.l.c., u.v. sp ectros cop y, ir sp ectrosc op y and e lemental a nalysis. the p artitio n coe fficient fo r it had als o b ee n d etermined through n – octano l / wate r partitioning system. الخالصة وعـة مجم ول مـع داز ي الميترونـي كسيل ف رو وعة الهيد مجم عن طريق تكثيف رونيدازول ميت مع ال ن لقد تم ربط فوسفات الدكساميثازو ون رقم ى ذرة الكارب عل وسفات 2الف د 1 ودايئماـي رب كا سـيل وهك سايكل ون باسـتخدام الداي ميثاز كسـا ف ) dcc(في جزيئة الد كـث عامـل م ك مــذي عد وباسـتخدام البريــدين ك سـا كعامــل م وكــذلك دراســة . ب و حمــراء ة فــوق البنفســجية وتحـت ال الشــع اف ا راســة أطـي ولقــد أثبتــت د ي للعناصر كم ة الى التحليل ال ة باالضاف طبقة الرقيق وغرافيا ال مات وي, كرو كيميا حة تركيبه ال كب الجديد وص وة المر مل .نقا عا تم حساب م جزئة ال) pc(الت هما ا ن مذيبي خدام ست محضر با ركب ال والماءللم كتانول . و introduction metro nidazole (2 – me thyl – 5 – nitroimidazole – 1 – e thanol). (flagyl, others ), is a synthe tic antimicrobial a gent which was found to have pa rticula rly high activity in vitro and in vivo a gainst a wide variety of a nae ro bic protozo al p aras ites a nd ana erob ic ba cteria (1,2). re ce nt studies indicate d that a comb ination of metro nida zole and a n anti – inflamma to ry s te ro id is one o f the mos t effe ctiv e re gimen for the tre atment of inflammatory b owel dise as e inc luding ulcerative c olitis and cohn's dis eas e(3). in o rd er to optimize drug ac tio n new drug fo rmula tion have b een d eve lo ped b ase d on adv anced tec hnologica l d eliv ery sys tem(4,5) or the p ro – drugs a pproa ch(6,7). pro – d rugs should be s ee n in the light of the still growing nee d fo r de live ry s ystem whic h may enable one to trans port a ctive agent se lectively to the ta rget and c onseque ntly re le ase the drug over de sired pe riod of time . s eve ra l type s o f e ster pro – drugs for me tronid azo le ha d b een synthe size d utilizing pro – moieties that impart diffe re nt p hysicoc he mic al p ro pertie s fo r this drug through the hyd ro xyl functional gro up of it. mos t of thes e pro – drugs were synthe sized in an attemp t to inc re as e water s olubility of me tronid azo le to be use d in prepa ring pa re ntal dos age fo rms (8,9,10,11). on the other ha nd a wide v arie ty of co rticos teroids p ro – drugs ha ve b een s ynthe size d and are in clinic al use . thes e pro – drugs were synthe size d utilizing the c – 21 hydroxyl group to make e sters of diffe re nt phys ic ochemica l prop erties (12). cortico steroids were us ed in the se pro – drugs either a s p ro moiety for ta rge ting the ac tiv e s pe cies as in the cas e of a ntic anc er agents (13,14), or a s the a ctiv e spe cies which we re co njuga ted to promo ie ties to c ha nge *de par tme nt o f ph arma ce utic al ch emistry colle ge o f pha rmac y, unive rs ity of baghdad ,baghdad ir aq. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 81 the p hys ic oc hemic al p ro pertie s(12), or for ta rgeting thes e cortic os te ro id s to the de sire d tis sue (15,16). mutua l pro – d rugs fo r co rticos te ro id s were also s ynthesized through co njuga tion w ith no n – steroida l anti – inflammatory drugs using a n amino a cid as s pa cer arm(17). the pre se nt rep ort de sc ribe s the synthe sis of a co mpound c onta ining de xamethas one and metronida zole linke d thro ugh a phosp ho die ster linkage. this p oss ib le mutua l pro – drug wa s de signed, thro ugh it's inhe re nt physico che mical prope rties to be cleav ed in the lowe r pa rt of the git e sp ecially the co lo n. experimental section mater ials: metro nidazole, wa s a gift fro m s amarra drug ind ustry, d exa metha so ne so dium phosp hate wa s a gift from j ord ania n pharmace utical manufa cturing co mpany ltd. the purity of the se two co mpound s wa s checked ac cording to the b.p and me rc k inde x. n,n – dicyclohexylc arbod iimide (dcc) wa s purc has ed fro m acros, usa. the remaining chemica ls were of rea gent grad e, a nd were us ed as such without further p urification, sinc e they w ere of the highest co mmercially av aila ble purity. gener al me thods all rea ctio ns , thro ughout this wo rk that ne ed a co ns ta nt temperature, were carrie d out in a the rmo stated double jacketed flas k co nne cted to a co ns ta nt temperature circulator and refrigerator o f ultratemp – 2 000 , jullab o vc. melting po ints were me asure d us ing a n elec trothe rma l me lting po int a ppa ra tus and are unconne cted. thin – la yer c hromatogra phy (t.l.c.) using silic a gel c oate d gla ss plate s was p erfo rme d to fo llow up c he mic al rea ction. purity o f the p re pared c omp ound was c hec ke d by thin – layer chroma to graphy plate s 20x20 cm o f silic a gel 6 0 f 254 with 0.25 mm la ye r thicknes s, me rc k, germany. chroma to grams we re e lute d by the follo wing so lv ent systems : a. iso propyl alco hol – wa te r – c onc entrated ammo nium hydroxid e s olutio n (7 :2 :1 ). b. chlo ro fo rm – me thanol – wa te r – a cetic ac id (25 :1 5:4:2). the chromatographic spots were re vealed by e ither rea ctivity with iodine va por or by observing them unde r uv light. uv sp ectra were re cod ed on p ye unic am uv sp ectrophotome te r, sp 8 – 010 0 germany. ir spe ctra we re rec orded on p ye unic am s p – 300 s pe ctro pho to meter, ge rma ny. c – h – n a na lysis was pe rformed at the unive rs ity o f mo sul, co llege of sc ienc e, using (c.h.n) analyzer, typ e 110 6 carlo erb a. che mis try: synthesis of 1 – (dexamethasone 21 – phosphoryl) – me tronidazole, comound i, figure (1). fig.1 chimica l structure of c ompo und i ge ne ra l proce dure (18) : de xame thas one p hos phate, s odium sa lt, 6.169 g (12mmo l) was diss olv ed in the minimum v olume of d is tille d water. to this solution was a dd ed dilute hcl so lution d rop wis e with s tirring until the comp le te precipitatio n of the free phos pho ric ac id e ster of d examethaso ne . the suspe ns io n was filte re d, a nd the white p re cipita te o f the free acid wa s co llec te d, washed with distilled water and d ried . the fre e p hos phoric ac id es ter of dexame thas one was dis solve d in 150 m of anhyd rous pyridine . to this solution wa s ad ded 4.9g (2 4 mmole) of (dcc) a nd the mixture was stirred at 2 5ºc for 2hrs , during which dicyc lo he xylurea (dcu) was p rec ipita te d and filte re d. to the filtrate the re wa s ad ded 1 .0 26g (6 mmole) of metro nidazole and the reac tion mixture wa s stirred for two d ays a t 25 ºc. the mixture wa s eva po ra te d to drynes s, a nd then the fina l tra ces o f pyridine we re remov ed by coe vap oratio n with tolue ne (50 ml). the res id ue was treated with 200 ml of 5 0% aqueous etha no l, a nd the ins oluble dcu was re move d by filtratio n. the filtrate was eva porated to d rynes s, re disolve d in 1 00ml of 50% aq ueo us ethano l, and a pplied to a silica gel column (3.5x26c m) prep acked with 5 0% aqueous ethano l. the ma teria l was then eluted ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 82 us ing is op rop yl alcohol – water – concentra te d ammo nium hyd ro xide (7 :2 :1 ) as the mobile phase . the fractions b etwee n 65 and 1 25ml were po oled, eva po ra te d to d ryne ss. a yello wis h to white crys ta lline po wer wa s co llec te d as the ammonium s alt of c omp ound 1; m.p 12 8 – 1 30ºc, the yield was 45 %. the uv s pec trum of the c omp ound (0.1mg/ml distilled wa te r) show λmax at 3 20 nm. λ max for de xame thas one p ho sphate and me tronid azole were found to be 243nm a nd 264nm re spec tively (figure (2)). fig.2 u.v spectru m d = dexa me thason sodium phosphate m = me tronida z ol i = co mpoun d i , so lv e nt : dis tille d wa te r ir s pe ctrum (figure (3)) rev ealed the fo llowing ab so rp tion freq ue ncies , cm – 1, (nujol): 3 232 (o – h) hydroge n bo nde d; 293 9 (c – h a liphatic ); 172 0 (c=o); 1 662 , 161 2 (c=o, c=c, c=n); 14 90, 124 2 (p=o); 153 5 (no2 group); 10 72 (p – o – c); 887 (c – n) stretching fo r he te ro a ro matic comp ound. fig.3 ir spe ctrum o f compound i t.l.c for compound [i], rf v alue , solve nt sys tem, a (0.88 ), solve nt b (0.53 ). tab le (1). ele menta l analys is c alcula te d fo r c28h37o10n3 pf., nh4h2o c, 50 .8 3; h,6.50 ; n, 8 .4 7 fo und c, 51 ,26; h, 6.93 ; n, 8.13. table 1: the rf va lues for the reactants and compoun d (i) us ing two diffe re nt so lve nt syste ms , whe re:solve nt a: isopropyl alcohol – wate r conc. ammonium hydroxide (7:2:1). solvent b: chloroform – methanol – wate r – acetic acid (25:15:4:2). de te rminatio n of pa rtition coe fficie nt the pa rtition c oeffic ie nt (pc) o f a s olute is defined a s the ratio of the co nce ntratio ns of solute distribute d b etwee n two immisc ib le solve nts at eq uilibrium, and it is usual to prese nt the ratio s that in fav our o f organic pha se : c pc = -(i) cw where co = the co nce ntratio n of the s olute in the orga nic pha se , and cw = the co nce ntratio n of the s olute in the aq ueo us p has e. partition co efficient for comp ound [i] had b een performed by a dding 5 0mg of the s olute to a sep aratory funnel c onta ining 50 ml of water per – saturate d with octa nol and 5 0ml of octanol per – sa turated with water. the s epa ra tory funne l was inverted s ev eral time s during 30min after tha t s epa ra to ry funne l was left for comp le te sep aration of the two pha se s. the a queous phase was a nalyzed for the s olute. a sta nda rd curv e had be en constructed by meas uring the ab so rba nc e of diffe re nt conce ntra tions o f co mpound [i], figure (4 ). the p artitio n coe fficient for o ur comp ound was calcula ted ac cording to eq ua tion (i), a nd was fo und to be 0.42. the substance s olve nt s ys te m rf value comp ound (i) a 0.88 metro nidazole a 0.96 dexa metha so ne phosp hate a 0.72 comp ound (i) b 0.53 metro nidazole b 0.7 dexa inetha so ne phosp hate b 0.35 ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 83 fig .4 u.v a bso rbance at λ 320 o f diffe re nt c oncentra tions of compound i results and discussion co mpound [i] had be en s ynthesized thro ugh the follo wing step s: in the first step de xamethas one p hos phate diso dium sa lt was c onverted to de xamethas one phosp hate ac id es te r by acidific atio n with dilute hyd ro cho ric ac id equatio n (i) (s che me i). in the se cond step , de xa mehta sone phosp horic cid e ster wa s c onv erte d to the p hos phate anhyd ride using d ic yc lo hexylca rb odiimid e (dcc) as a d ehydrative co upling rea gent, eq. (2 ). the re ac tive anhyd ride thus fo rme d wa s allo wed to rea ct with the primary hyd ro xyl group of metronida zo le which ac ts as a nucleop hile that a tta ck the p hos phate. the metho d us ed for the synthe sis of co mpound [i] is a mo difica tion o f proce dure s well es ta blis he d in the a re a o f nuc lc otid e synthe sis(19,20), as we ll a s fo r conjuga tion of nuclcos id es with c ortico sterio des thro ugh phosp hate d ie ster linkage (21). the co upling ge nt dcc, was introduced b y kho ra na d uring nucleo side polyp hos phate synthe sis to promo te s ynthetic re actions invo lving dehyd ration(22). this rea gent was then us ed b y she eha n et al., as a co nde ns ing agent for amide bond fo rma tio n during pe ptid e synthe sis(23). pyridine , d uring this c ours e of rea ctio ns, ha s tw o role s. it a cts a s a po werful s olvent a nd as a ca ta list acc ording to the following (sc heme ii)(2 4). dcc was used s uc ces sfully in this labo ra tory as a co nd ens ing agent fo r many re ac tions that involve s ynthes is of c arboxylic acid e sters, and fo r amide b ond fo rma tion a s well(25). in a p re vious work(12), a ttemp ts at condensa tion of p re dniso lo ne 2 1 – pho sphate or pre dnis one 21 – p ho sphate with 5 – hyd ro xyl gro up of nuc le os id es in the p re se nce of dcc and pyridine at room te mpe ra ture and a t re flux were not s ucc es sful. this fa ilure could be attributed in pa rt as d ue to the metho d app lie d in whic h the pred niso lo ne 21 – pho sphate was ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 84 no t conve rted to the free ac id e ster prior to re ac tion with dcc and pyridine . in add ition to that, the pro ce dure employe d did no t allo wed s ufficient time for the p hos phate anhyd ride formation prior to the re ac tion with the alcohol functio nality, as we did in our proce dure . co nfirmation of the molec ular s truc ture of co mpound [i] was provide d by eleme ntal analysis , uv, ir and tlc. lipo philic ity is a te rm c ommonly use d to d es cribe the te nde nc y fo r a chemica l a gent to pa rtition itself b etwee n aq ueo us and organic biop has es . p artitio n co efficients p ro vide a co nve nient mea sure of lipo philic ity a nd are often used in e stablishing the relative rates with which che mical substa nce s penetra te lipoida l memb ranes or pa rtic ip ate in the formation of a hyd ro pho bic bond(26). partition c oeffic ie nt a s was p re viously de fine d , is an imp orta nt parame ter for mea suring the relativ e affinitie s of the s olute fo r an aq ue ous a nd non – aq ueo us or lipid phase . the greater the value o f pc (e quatio n (1 )), the highe r the lip id so lubility o f the so lute . on the o ther hand , the lowe r the va lue of p c the highe r the a que ous solub ility for the so lute . n – oc ta no le is the s olve nt for whic h mo st pa rtition co effic ie nt v alues hav e b ee n publis hed . it is claime d that the octa nol / water system is sa tisfac to ry mode l for the b io lo gical system b ec aus e the orga nic p has e is not co mpletely no n polar and conta ins a s ignific ant amo unt of wa ter in a stab le , hydroge n bo nde d co mplex(27). in contras t, partitioning systems such a s hexane / wate r and chloroform / water co ntain so little water in the o rganic p has e that they are po or mod es fo r the lipid bila yer / water fo und in the b ody. lipid s oluble drugs us ua lly cross ce llular bound arie s by d is solving in o r interacting with the lip id me mbrane s and diffus ing ac ros s into the intra ce llular aqueous phas e. mo st drugs are wea k o rganic elec trolyte s p re se nt in eq uilibrium betwee n two fo rms , of which o nly the no n – io nize d from po ss ess lipid s olubility. since our comp ound was fo und to hav e re la tive ly lo w p artitio n c oe fficient, it has a s a re sult re la tive ly low lipid s olubility, and a s a co nse quenc e it will p oorly abs orbed from the g.i.t after o ra l ab sorption. in a dd ition to that, it has relatively la rge molec ular s ize, whic h is a n add itiona l factor fo r poo r abs orptio n. on the other ha nd our co mpound is a phosp hate d ie ster, a nd this typ e of conjuga tion is res is ta nt to che mic al cleav age by the g.i fluid. so we expec t that our comp ound will p ersist fo r a lo ng pe riod o f time d uring which it will pas s the g.i tract and reac h the colon in a signific ant qua ntity to e xert its loca l effe ct afte r co mplete hydrolysis a nd lib eratio n of the active mo ie ties . the u.v. spe ctrum of the c ompo und (0 .1 mg/ml distilled wa ter) s how λ max at 320 nm. λmax fo r dexame thas one phos pha te and metro nidazole was found to be 2 43nm and 264 nm re sp ectively. ir aq i j.pharm.sc i., vol.15 (1 ) ,2006 85 references 1. martind ale , the exte ra pha rma co poe a , 198 2,. 96 8 . 2. johns on, p.j., pa ra sitol. tod ay, 199 3, 18 3 (1 13) ,9 . 3. ibrad en g.l., american c olle ge of gas troe nterology 64 th, annual sc ie ntific meeting in pho enix, arize , octobe r 17, 199 9. 4. juliano, r.l. (ed ) dru g de live ry sy stein, oxford unive rs ity pres s, new york, 198 0. 5. langer r.s., and wis e, d.l., (eds), me dical app lica tion s of co ntrolled re le ase d, crc pre ss , bo ca ra to n, vol. 11 , 1 984 . 6. bundga ard, h, (ed), de sign of p rod rug s, els ev ie r, amsterda m, 1 985 7. redd y, s.m., s inha , v.r., and redd y, d.s ., drug s of tod ay, 199 9, 35, 537 . 8. johansen, m., a nd la rs en, c., int. j . ph arm., 198 5, 2 6, 2 27. 9. stella , v., a nd higuc hi, j ., (ed s), pr odr ug s as no vel dru g deliv er y sy stems , ame rica n che mic al s ociety, washington, dc, 107 5, i – 118 . 10. ande rs on, b.d ., conrad i, r.a., s pilma n, c.h. and f orbes , a.d., j.ph am.sci., 19 85, 74, 382 . 11. s inkula , a.a., & ya lkows ky, s .h., j. phar m. s ci., 64 , 18 1 197 5. 12. roche, e.b., "de sign o f biop ha rma ceutic s prope rties thro ugh p rod rugs and a na lo gs " roche, e.d., (ed), ame rica n pharmace utical ass ociation, n.y., 19 77,27 – 4 6. 13. ho ng, cl, nec ha ev, a., kirs ists, a.j., buchhe it, d.j., and wes t. c.r., j . me d. chem., 198 0 ,2 3, 1 34 3. 14. ibid, 198 5, 2 8, 171 . 15. f riend, d.r., and chang g.w., j. me d. chem., 198 4, 2 7, 2 61 . 16. ibid, 198 5, 2 8, 171 . 17. j ame el, a.m., ms c thes is , unive rs ity of baghda d, ba ghdad 2 002 . 18. ho ng, cii, kiriisits, a.j., ne che ae v, a, buchhe it, d.j., and we st, c.r., j. p harm. s ci., 19 84 ,73, 278 . 19. rammler, d.h., la pidot y., and khorana, li.g., j . am. che m. so c., 19 63,85 , 1 989 20. tene r, g.m., j. am. chem. s oc., 7 6, 3 51 7 1 954 . 21. ho ng, cii, nechea v, a., a nd wes t, c.r., j . am. che m. s oc.,197 9, 22, 148 2. 22. khorana, h.g., j. am. che m. s oc., 1 954 ,76, 351 7. 23. s hee ha n, j.g., and he ss , g.p., j. am. chem. so c.,1 955 ,7 7, 1 06 7. 24. ha ss ne r, a., and alexania n, r, tetra he ddron lette rs , 1 978 , 44 75. 25. s uad , m.d., synthes is o f co njuga tions of s te ro id al and no n – s te ro id al anti – inflamma to ry age nts a s p os sible pro – d rugs , ph.d thes is , univ ersity o f baghda d, baghda d 199 7. 26. camma rata , a., a nd rogers, k.s ., j. me d. chem., 197 1, 1 4, 2 69 . 27. leo, a., hansc h, c., and elkins , d., chem. rev ., 19 71,71 , 52 5. iraqi j pharm sci, vol.26(2) 2017 impact of pyridoxine graded doses on doxorubicin cardiotoxicity. 12 impacts of graded doses of pyridoxine on the biomarkers, aspartate aminotransferase, lactate dehydrogenase and total antioxidant capacity in doxorubicin-induced cardiotoxicity in female rats doaa k. abdul ridha*,1 and nada n. al-shawi** *the national center for drug and research (ncdcr), ministry of health/environment, baghdad, iraq. ** department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq. abstract doxorubicin (dox), one of the anthracycline family; most widely used antineoplastic drugs and highly effective in treating cancer patients. the intended drug exerted its activity mainly by intercalation with dna and by this means it inducing damage to the dna and inhibiting the synthesis of macromolecules that are essential to maintain cell life but their use associated with cardiotoxicity adverse effect. pyridoxine (vitamin b6) is one of the water soluble b vitamins; converted into the active form, pyridoxal 5’-phosphate (plp). pyridoxine may have a crucial role in antioxidant mechanism. the aim of the current study was to investigate the possible protective effect of graded doses (5, 10, and 15mg/kg) of pyridoxine hydrochloride intraperitoneally (ip) injected for four consecutive days against single dose of (15mg/kg) doxorubicin-induced cardiotoxicity in female rats ip injected at the fourth day only. fifty-six (56) wistar albino female rats were utilized weighing 180-200 gm allocated into eight groups, seven rats each; and by utilizing ip injection as route of administration as follows: group i: distilled water (negative control); group ii: pyridoxine (5mg/kg); group iii: pyridoxine (10mg/kg); group iv: pyridoxine (15mg/kg); group v: doxorubicin (15 mg/kg); group vi: pyridoxine (5 mg/kg) prior to doxorubicin (15 mg/kg); group vii: pyridoxine (10 mg/kg) prior to doxorubicin (15 mg/kg); group viii: pyridoxine (15 mg/kg) prior to doxorubicin (15 mg/kg). at the 5th day (after 24 hr from the last treatment), blood was withdrawn and heart tissue homogenate obtained for laboratory evaluation. dox caused significant elevations in serum biomarker enzymes of aspartate aminotransferase (ast), lactate dehydrogenase (ldh) and significant reduction in heart tissue homogenate content of total antioxidants capacity (tac). treatment with 5mg/kg pyridoxine for four consecutive days prior to a single dose 15mg/kg doxorubicin resulted in a non-significant differences in serum enzymes level of ast, ldh, and tac heart tissue homogenate contents. besides, treatment with 10 or 15mg/kg pyridoxine for four consecutive days prior to a single dose of doxorubicin produced significant increments in tac heart tissue homogenate level compared to positive control. moreover, treatment with 15mg/kg pyridoxine for four consecutive days prior to a single dose 15mg/kg doxorubicin resulted in significant reduction in serum enzymes level of ast and ldh. in conclusion, pyridoxine supplementation might be a promising adjunctive agent for improving oxidative stress and biological markers for preventing dox-induced cardiac complications. keywords: doxorubicin (dox), cardiotoxicity, pyridoxine, total antioxidant capacity. هايدروكلوريد البيريدوكسين على األنزيماتتقييم التأثير الوقائي المحتمل ل aspartate aminotransferase ast, lactate dehydrogenase ldh المستحثالقلب لتقليل سمية total antioxidant capacity toac ومضادات األكسدة بواسطة الدوكسوروبيسين في أناث الجرذان. **ندى ناجي الشاويو 1*,عبد الرضادعاء كاظم المركز الوطني للرقابة والبحوث الدوائية / دائرة األمور الفنية / وزارة الصحة والبيئة. * . السموم ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراقو االدوية فرع ** الخالصة الدوكسوروبيسين أحد األدوية الفعالة من عائلة األنثراسيكلين، يستخدم في عالج العديد من األورام السرطانية الخبيثة لدى ي المرضى المصابين بالسرطان. ان آلية العالج الكيميائي الرئيسة لعمل الدواء اعاله تتضمن إقتحامه للتركيب الحلزوني للحامض النووي الذ بدوره 1corresponding author e-mail: dr.duaakadhim84@gmail.com received:12 /6/2017 accepted: 12/8/2017 iraqi j pharm sci, vol.26(2) 2017 impact of pyridoxine graded doses on doxorubicin cardiotoxicity. 13 لب يسبب تلف الحامض النووي وتثبيط تولد جسيمات الخلية الضرورية لحفظ ديمومة الخلية الحية. تم تقييد أستخدام هذا الدواء بسبب سمية الق , من مجموعة فيتامين )ب( الذائبة )6فيتامين ب(البيريدوكسين له التي يمكن أن تؤدي الحقا الى قصورفي القلب.الناتجة من الجرع التراكمية 6يمكن ان يمتلك الفيتامين ب. للفيتامين اعالهالذي يعد الشكل الفعال البيريدوكسال فوسفاتفي الماء. يتحول هذا الفيتامين داخل الجسم إلى ملغم 5) ,جرعات متدرجة من هايدروكلوريد البايريدوكسينالدراسة هو لتقييم التأثير الوقائي المحتمل ل الهدف من لألكسدة.تأثير مهم كمضاد ايام متتالية ضد سمية القلب المستحثة بولسطة 4كل منها استخدمت لمدة ملغم / كيلو غرام ( 05ملغم / كيلو غرام، 01/ كيلو غرام، ( جرذ انثوي مختبري , 56اعطي في اليوم الرابع كجرعة منفردة في اناث الجرذان. تم أستخدام ستة وخمسون )الدوكسوروبيسين الذي قسمت الجرذان المختبرية الي ثمانية مجموعات , كل مجموعة تتكون من سبعة جرذان وباستخدام طريقة الحقن داخل الصفاق وكما يلي: ملغم / كيلو 5) حقنت المجموعة الثانيةمرة واحدة في اليوم لمدة أربعة أيام متتالية. رام ( ,. مل / غ5) حقنت ماء مقطر االولى لمجموعةا ملغم / كيلو غرام ( 01) حقنت المجموعة الثالثةهايدروكلوريد البايريدوكسين مرة واحدة في اليوم لمدة أربعة أيام متتالية. غرام ( ملغم / كيلو غرام ( 05) حقنت المجموعة الرابعةاليوم لمدة أربعة أيام متتالية. في واحدة مرة البايريدوكسين هايدروكلوريد ماء مقطر ,. مل / غرام ( 5) حقنت الخامسة المجموعة . متتالية أيام أربعة لمدة اليوم في هايدروكلوريد البايريدوكسين مرة واحدة . ملغم / كيلو غرام ( 05) مرة واحدة في اليوم لمدة أربعة أيام متتاية وفي اليوم الرابع تم حقن الجرذان بجرعة واحدة من الدوكسوروبيسين فة الى جرعة واحدة في اليوم لمدة أربعة ايام متتالة اضاملغم / كيلو غرام ( 5) المجموعة السادسة حقنت هايدروكلوريد البايريدوكسين من الدوكسوروبيسين الذي حقن في اليوم الرابع فقط. المجموعة السابعة حقنت هايدروكلوريد ملغم / كيلو غرام ( 05) جرعة واحدة من ملغم / كيلو غرام ( 05) جرعة واحدة في اليوم لمدة أربعة ايام متتالة أضافة الى جرعة واحدة ملغم / كيلو غرام ( 01) البايريدوكسين جرعة واحدة في ملغم / كيلو غرام ( 05) المجموعة الثامنة حقنت هايدروكلوريد البايريدوكسين دوكسوروبيسين في اليوم الرابع فقط.ال (من الدوكسوروبيسين في اليوم الرابع فقط. في اليوم الخامس ملغم / كيلو غرام ( 05) اليوم لمدة أربعة ايام متتالة أضافة الى جرعة واحدة ساعة على الجرعة األخيرة( تم سحب عينات من الدم ومن نسيج القلب للجرذان لغرض اجراء التحليالت المختبرية. لوحظ 44مرور بعد في مصل الدم أضافة الى أنخفاض معنوي في مستوى مضادات االكسدة (ldh), (ast)وجود ارتفاع معنوي في مستوى االنزيمات (tac) ملة بالدوكسوروبيسين لوحده بالمقارنة مع المجموعة المعاملة بالماء المقطر فقط. نتائج الدراسة بينت أن لدى مجموعة الجرذان المعا ليس له تأثير معنوي على مستوى أيام متتالية قبل أستخدام الدوكسوروبيسين 4لمدة ملغم / كيلو غرام ( 5)أستخدام البيريدوكسين بجرعة أو ملغم / كيلو غرام ( 01) أضافة الى أن استخدام البيريدوكسين بجرعة (tac)ألكسدةأومضادات ا (ldh), (ast)األنزيمات 15mg/kg قبل جرعة الدوكسوروبيسين أدى الى أرتفاع مستوى مضادات األكسدة(tac) بشكل معنوي مقارنة مع المجموعة المعاملة له تأثير معنوي في تقليل ملغم / كيلو غرام ( 05) يريدوكسين بجرعة بالدوكسوروبيسين لوحده. نتائج الدراسة بينت أيضا أن أستخدام الب أن أستخدام البيريدوكسين وبذلك نستنتج بالمقارنة مع المجموعة المعاملة بالدوكسوروبيسين لوحده. (ast),(ldh)مستوى األنزيمات المستحث بواسطة الدوكسوروبيسين من خالل تأثيره يمكن ان يكون مركب واعد عندما يستخدم بجرعة مناسبة لمنع أو تقليل سمية القلب و تحسين المعايير البايولوجية. (oxidative stress), تحسين اإلجهاد التأكسدي (antioxidant)المضاد لألكسدة مضادات األكسدة. ،البيريدوكسين،سمية القلب ،الدوكسوروبيسين: الكلمات المفتاحية introduction doxorubicin (dox) is one of the most widely used antineoplastic drugs (1). it is highly effective in treating patients with acute lymphoblastic leukemia, hodgkinis lymphoma, aggressive non-hodgkinis lymphomas, breast carcinoma, ovarian carcinoma and many solid tumors (2). dox exerted its activity mainly by intercalation with dna and by this means it inducing damage to the dna and inhibiting the synthesis of macromolecules that are essential to maintain cell life (3). the successful use of dox has been hindered by its most important and common “cardiotoxic” adverse effect which remains the major limitation of its use with strong impaction on life quality and survival (4). the cardiotoxicity of dox is attributed to complex mechanisms that include oxidative stress through ros production and possibly cellular iron accumulation, intracellular calcium dysregulation, mitochondrial damage, and apoptosis/necrosis (1). this fact allows the researchers to develop strategies to reduce the toxic effects of dox without interfering with its antitumor properties. antioxidants, which are capable of protecting the cells from oxidative injury, should be included in the potential antioxidant therapy. therefore , there is a need for identifying alternative, natural and safer sources of antioxidants (5).pyridoxine (vitamin b6) is one of the water soluble b vitamins; converted into the active form, pyridoxal 5’phosphate (plp), which is physiologically-active coenzyme of vitamin b6, is mainly involved in the metabolism of amino acids, nucleic acids, glycogen, porphyrin, and lipids. in addition, pyridoxine may have a crucial role in antioxidant mechanism (6-11). the exact antioxidant mechanism of such vitamin may confirmed; on one hand, it may react directly with the peroxy radicals and thereby scavenge radicals and inhibit lipid peroxidation (11-15). on the other hand, pyridoxine may indirectly play an antioxidant role by serving as coenzyme in the glutathione antioxidant. besides, pyridoxal 5’phosphat (plp) serves as a coenzyme in the trans-sulfuration pathway of homocysteine to cysteine, which is an important contributor for synthesizing reduced glutathione (gsh). again, pyridoxine may directly or indirectly play a role in oxidative stress and the antioxidant defense system was confirmed by others (16). it has been reported that deficiency of pyridoxine (pr, b6) or its active form pyridoxal phosphate (plp, b6) may promote oxidative lipid iraqi j pharm sci, vol.26(2) 2017 impact of pyridoxine graded doses on doxorubicin cardiotoxicity. 14 peroxidation and exacerbates the oxidative stress. moreover, investigators demonstrated that pyridoxine is strongly inhibit xanthine oxidase (xo) activity, an enzyme responsible for the formation of uric acid and hydrogen peroxide (17). the aim of the present study was to investigate the possible protective effect of three graded doses (5mg/kg, 10mg/kg, and 15mg/kg) of pyridoxine each administered prior to a single dose (15mg/kg) doxorubicininduced cardiotoxicity in female rats. materials and methods animals the experiment was performed with the utilization of 56 wistar albino female rats (the available sex) weighing 180-200 gm (age: 4 months). rats were obtained from the animal house of the college of pharmacy/university of baghdad and from the animal house of the national center of drug control and research (ncdcr). they were maintained on normal conditions of temperature (25 ±2∘c), humidity and under a 12 h light/dark cycle. they were fed standard rodent pellet diet and they have free access to water ad libitum. the animals had no manifestation of any illness upon examination. they were left for two weeks without interference for acclimatization. the study was approved by the graduate studies and the ethical committees of the college of pharmacy, university of baghdad. experimental design rats were randomly divided into eight groups of 7 rats each as follows: group i: healthy female rats intraperitoneally (ip) injected with 0.5ml of distilled water (d.w.) once daily for four consecutive days. this group served as a healthy negative control. group ii: healthy female rats ip injected with 5mg/kg pyridoxine hydrochloride once daily for four consecutive days. group iii: healthy female rats ip injected with 10mg/kg pyridoxine hydrochloride once daily for four consecutive days. group iv: healthy female rats ip injected with 15mg/kg pyridoxine hydrochloride once daily for four consecutive days. group v: healthy female rats ip injected with 0.5ml d.w. for four consecutive days. at day 4, a single dose of doxorubicin hydrochloride (15mg/kg) was ip injected. this group served as a positive control. group vi: healthy female rats ip injected with 5mg/kg pyridoxine hydrochloride (10mg/ml) once daily for four consecutive days. at day 4, a single dose of doxorubicin hydrochloride (15mg/kg) was ip injected. group vii: healthy female rats ip injected with 10mg/kg pyridoxine hydrochloride (10mg/ml) once daily for four consecutive days. at day 4, a single dose of doxorubicin hydrochloride (15mg/kg) was ip injected. group viiihealthy female rats ip injected with 15mg/kg pyridoxine hydrochloride (10mg/ml) once daily for four consecutive days. at day 4, a single dose of doxorubicin hydrochloride (15mg/kg) was ip injected. preparation of doxorubicin hydrochloride solution. fifty milligrams (50mg) of doxorubicin hydrochloride (adriblastina® powder / actavis s.p.a. pasteur/ italy) powder for injection is dissolved in 10ml d.w to obtain 5mg/ml or (3mg/0.6ml concentration per 200g rat weight). preparation of pyridoxine hydrochloride solution. two milliliters (2ml) of pyridoxine hcl (100mg) ampoule (pyridoxine hydrochloride injection usp 100mg/2ml/strides arcolab limited/india) diluted to 10ml with d.w to obtain 10mg/ml or 1mg/0.1ml concentration. sample preparation. 24 hours after the end of treatment (i.e. at day 5), animals were euthanized by anesthetic diethyl ether (may and baker, england), blood was withdrawn from carotid artery from the neck of each rat utilized in this study, and placed in labeled centrifuging tubes, then allowed to clot for 20 min at room temperature and then centrifuged at 3000 (rpm) for 15 minutes; the supernatant separated and was used for the estimation of serum aspartate aminotransferase ast and lactate dehydrogenase ldh enzymes level(18). the heart of each animal utilized in this study was quickly excised, rinsed in chilled phosphate buffer saline (pbs) solution (ph 7.4) at 4ºc to dismount thoroughly the excess blood, then heart tissues blotted with filter paper weighed, and minced to small pieces; where, 1g heart tissue was put in tube containing 10 ml of phosphate buffer saline (pbs) solution prepared at the previously-mentioned ph value, to obtain 10% tissue homogenate. the tube containing the heart tissues was put in a beaker containing ice (ice path) then homogenized with the aid of homogenizer (success technic industries, malaysia) at set 3 for 1 minute at 4 ºc. after that, the homogenate was centrifuged by the cooled centrifuge (hittich rotanta, england) for 15 minutes at 1500×g [or 5000 revolution/minute iraqi j pharm sci, vol.26(2) 2017 impact of pyridoxine graded doses on doxorubicin cardiotoxicity. 15 (rpm)] at 4 ºc. the supernatant is utilized for the estimation of tissue homogenate contents of taoc. blood and tissue homogenate samples were stored at −20ºc until analysis process(19). analysis estimation of serum aspartate aminotransferase (ast) activity aspartate aminotransferase (ast) specifically catalyzes the transfer of the amino group of aspartic acid to ketoglutarate yielding glutamate and oxaloacetate, which is then reduced to malate by the enzyme malate dehydrogenase (mdh) with the oxidization of the coenzyme nicotinamide adenine dinucleotide reduced form (nadh) to oxidized nicotinamide adenine dinucleotide (nad). aspartate aminotransferase (ast) determination involves the following reactions (20, 21): l-aspartate + ketoglutarate ast oxalacetate + l-glutamate oxalacetate + nadh mdh malate + nad+ the absorbance reduction at 340nm, as a consequence of nadh oxidation, was determined photometrically and is direct proportional to the serum ast activity in the sample. serum activity of ast is expressed as iu/l. estimation of serum lactate dehydrogenase (ldh) activity pyruvate is reduced by nadh and this reaction is catalyzed by ldh enzyme, according to the following reaction: pyruvate + nadh + h+ ldh l-lactate + ad+ nad+ is related to the serum enzymatic activity of ldh which is measured by spectrophotometer at 340 nm. serum ldh activity was expressed as iu/l. (22) (23). estimation of total antioxidant capacity (tac) level the estimation of tac in heart tissue homogenate is based on the enzyme-linked immuno-sorbent assay (elisa) with the utilization of ready-made rat kit for this purpose. the microtiter plate provided in the kit has been pre-coated with an antibody specific to tac (mixed sod/cat /gsh-px /gsh). standards or samples are added to the appropriate microtiter plate wells then followed by the addition of the second horseradish peroxidase (hrp) conjugated tac antibody to bind the analyte and incubated for 60 minutes at 37ºc. after the addition of 3,3',5,5'tetramethylbenzidine (tmb) substrate solution and incubates for 15 minutes at 37ºc, only wells that contain tac mixture will exhibit a change in color. the enzyme-substrate reaction is terminated by the addition of sulfuric acid (h2so4) solution and the color change is spectro photometrically measured at a wavelength of 450nm.the concentration of tac mixture in the samples is determined by comparing the o.d. of the samples to the standard curve. the cоncentratiоn оf tac is expressed as iu/ml (24). statistical analysis statistical analyses were carried out by using ibm spss (statistical package for social sciences) version 23.0 program. the significance of difference between the mean values was calculated utilizing unpaired student's t-test. the numeric data were expressed as mean ± standard error of means (sem). besides, the statistical significance of the differences among various groups was determined by оne-way analysis of variance (anоva) and least significant decrease (lsd). the level of significance was set at (p<0.05) for all data presented in the results of this study. results impact of three doses 5mg/kg, 10mg/kg, and 15mg/kg pyridoxine hydrochloride, single doxorubicin hydrochloride, and each prior to doxorubicin on the activities of serum ast and ldh enzymes in female rats: table (1) and figure [(1),(2)] showed that there were non-significant differences (p>0.05) in the serum activity of ast (figure 1) and ldh (figure 2) in groups of rats ip injected with either 5mg/kg (group ii) or 10mg/kg (group iii) pyridoxine compared to the corresponding activity in negative control. moreover, significant reduction (p<0.05) in the serum activity of the intended enzymes in group of animals treated with 15mg/kg (group iv) pyridoxine compared with the negative control. besides, female rats ip injected with (15mg/kg) doxorubicin (group v) showed a significant increase (p<0.05) in the serum activity of the ast and ldh compared to the negative control animals (group i). moreover, female rats ip injected with (15mg/kg) doxorubicin (group v) showed a significant increase (p<0.05) in the serum activity of the ast and ldh compared to the negative control animals (group i). furthermore, serum ast and ldh enzyme activities in female rats ip injected with either 5mg/kg pyridoxine for four consecutive days prior to a single dose of 15mg/kg doxorubicin (group vi) or 10mg/kg pyridoxine for four consecutive days prior to a single dose of iraqi j pharm sci, vol.26(2) 2017 impact of pyridoxine graded doses on doxorubicin cardiotoxicity. 16 15mg/kg doxorubicin (group vii) were nonsignificantly different (p>0.05) compared with the corresponding serum enzymes activity in positive control animals (group v). in contrast, the serum activity of the intended enzymes in group of rats treated with 15mg/kg pyridoxine for four consecutive days prior to a single dose of 15mg/kg doxorubicin (group viii) were significantly reduced (p<0.05) compared to the corresponding serum activity of positive control rats (group v). moreover, table (1) and figure [(1),(2)] showed that there were significant reduction (p<0.05) in serum ast and ldh activity among groups of animals ip injected with increasing doses of (pyridoxine hcl 5mg/kg, 10mg/kg or 15mg/kg for four consecutive days each prior to a single dose of 15mg/kg doxorubicin hcl) when compared with each other using anova and least significant differences (lsd) analysis. impact of three doses 5mg/kg, 10mg/kg, and 15mg/kg pyridoxine hydrochloride, single doxorubicin hydrochloride, and each prior to doxorubicin on the taoc levels in heart tissue homogenate of female rats table (2) and figure (3) showed that there were non-significant differences (p>0.05) in taoc level in heart tissue homogenate of rats ip injected with 5mg/kg pyridoxine (group ii) compared to the negative control (group i). beside, significant elevation (p<0.05) in heart tissue homogenate tac levels in groups of animals treated with either 10mg/kg (group iii) or 15mg/kg (group iv) pyridoxine compared with the negative control. besides, female rats intraperitoneally (ip) injected with (15mg/kg) doxorubicin (group v) produced significant reduction (p<0.05) in the heart tissue homogenate level of the (tac) compared to the corresponding levels in negative control animals (group i). furthermore, table (2) and figure (3) showed that there were non-significant differences (p>0.05) in the levels of tac in heart tissue homogenate of female rats ip injected with 5mg/kg pyridoxine for four consecutive days prior to a single dose of 15mg/kg doxorubicin (group vi) compared with the corresponding tissue levels in positive control animals (group v). besides, the heart tissue homogenate tac levels in group of rats treated with either 10mg/kg pyridoxine for four consecutive days prior to a single dose of 15mg/kg doxorubicin (group vii) or 15mg/kg pyridoxine for four consecutive days prior to a single dose of 15mg/kg doxorubicin (group viii) were significantly elevated (p<0.05) compared to the corresponding levels in positive control rats (group v). in addition, significant elevation (p<0.05) in heart tissue homogenate tac levels were observed among groups of animals ip injected with increasing doses of pyridoxine hcl (5mg/kg, 10mg/kg or 15mg/kg for four consecutive days each prior to a single dose of 15mg/kg doxorubicin hcl) when the previously-mentioned groups compared with each other’s using anova and lsd analysis, table (2) and figure (3). table (1): effects of various treatments on serum activities of aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) enzymes in female rats (n=7). groups treatment serum ast levels (iu/l) mean ± sem serum ldh levels (iu/l) mean ± sem group i negative control [distilled water (dw)] 169.6 ± 5.3745 892.175 ± 29.547 group ii pyridoxine (5 mg/kg) 167.285 ± 8.620 815 ± 37.965 group iii pyridoxine (10 mg/kg) 164.832 ± 8.359 800.808 ± 30.625 group iv pyridoxine (15 mg/kg) 148.314 ± 2.343 * *660.857 ± 42.094 group v positive control [doxorubicin (15 mg/kg)] 315.2 ± 28.691*a *a1381.266 ± 143.353 group vi pyridoxine (5 mg/kg) + doxorubicin (15 mg/kg) 295.571 ± 4.893 aa aa1309.714 ± 98.333 group vii pyridoxine (10 mg/kg) + doxorubicin (15 mg/kg) 255.428 ± 37.196ab ab1300.07 ± 65.092 group viii pyridoxine (15 mg/kg) + doxorubicin (15 mg/kg) 199.571 ± 187.8 bc bc991.385 ± 90.857 iraqi j pharm sci, vol.26(2) 2017 impact of pyridoxine graded doses on doxorubicin cardiotoxicity. 17 each value represents mean ± standard error of means (sem). * significantly different (p < 0.05) with respect to the negative control group. values with non-identical capital letters superscripts (a, and b) are significantly different (p<0.05) in comparison with the positive control group (doxorubicin-treated animals) using unpaired student t-test. values with non-identical small letters superscripts (a, b, and c) are significantly different (p<0.05) among (vi, vii and viii) groups using anova and lsd analyses. n number of animals. group i: negative control distilled water; group ii: pyridoxine (5mg/kg); group iii: pyridoxine (10mg/kg); group iv: pyridoxine (15mg/kg); group v: doxorubicin (15 mg/kg); group vi: pyridoxine (5 mg/kg) prior to doxorubicin (15 mg/kg); group vii: pyridoxine (10 mg/kg) prior to doxorubicin (15 mg/kg); group viii: pyridoxine (15 mg/kg) prior to doxorubicin (15 mg/kg). table (2): effects of various treatments on levels of the heart tissue homogenate tac in female rats (n=7). groups treatment tissue homogenate tac levels (iu/ml) mean ± sem group i negative control [distilled water (dw)] 3.903 ± 0.276 group ii pyridoxine (5 mg/kg) 4.037 ± 0.228 group iii pyridoxine (10 mg/kg) 4.784 ± 0.281* group iv pyridoxine (15 mg/kg) 4.988 ± 0.174* group v positive control [doxorubicin (15 mg/kg)] 2.168 ± 0.248*a group vi pyridoxine (5 mg/kg) + doxorubicin (15 mg/kg) 2.634 ± 0.284aa group vii pyridoxine (10 mg/kg) + doxorubicin (15 mg/kg) 3.708 ± 0.149bb group viii pyridoxine (15 mg/kg) + doxorubicin (15 mg/kg) 3.982 ± 0.365cc each value represents mean ± standard error of mean (sem). * significantly different (p < 0.05) with respect to the negative control group. values with non-identical capital letters superscripts (a, b, and c) are significantly different (p<0.05) in comparison with the positive control group (doxorubicin-treated animals) using unpaired student t-test. values with non-identical small letters superscripts (a, b, and c) are significantly different (p<0.05) among (vi, vii and viii) groups using anova and lsd analyses. n number of animals. group i: negative control distilled water; group ii: pyridoxine (5mg/kg); group iii: pyridoxine (10mg/kg); group iv: pyridoxine (15mg/kg); group v: doxorubicin (15 mg/kg); group vi: pyridoxine (5 mg/kg) prior to doxorubicin (15 mg/kg); group vii: pyridoxine (10 mg/kg) prior to doxorubicin (15 mg/kg); group viii: pyridoxine (15 mg/kg) prior to doxorubicin (15 mg/kg). iraqi j pharm sci, vol.26(2) 2017 impact of pyridoxine graded doses on doxorubicin cardiotoxicity. 18 figure (1): bar chart showing serum levels of ast in various experimental rats' groups. figure (2): bar chart showing serum levels of ldh in various experimental rats' groups. * significantly different (p < 0.05) with respect to the negative control group. non-identical capital letters (a and b) superscripts are significantly different (p<0.05) in comparison with the positive control group (doxorubicintreated animals). nonidentical small superscript letter (a, b, and c) are significantly different (p<0.05) among (vi, vii and viii) groups within (a) ast chart, and (b) ldh chart. figure (3): bar chart showing tissue homogenate enzymes level of total antioxidant capacity (taoc) in various experimental rats' groups. * significantly different (p<0.05) with respect to the negative control group. non-identical capital letters (a, b, and c) superscripts are significantly different (p<0.05) in comparison with the positive control group (doxorubicintreated animals). non-identical small letter superscripts (a, b and c) are significantly different (p<0.05) among (vi, vii and viii) groups. discussion cardiac injury is the main limiting factor for the use of dox as anticancer agent. dox-induced generation of reactive oxygen species (ros) seems to be a leading cause of cardiomyopathy (25) (26). this study investigates the effects of pyridoxine pretreatment, which were used in different doses on dox-induced acute cardiotoxicity. the data of present study were in agreement with studies performed by others; where, serum ast (27) and ldh (28) enzymes activity were significantly elevated in association with dox treatment. the increase in serum ast and ldh activity could be attributed to the well-known cardiac toxic effects of dox, which may lead to the damage of the myocardial cell membrane or it become permeable, that resulted in the leakage of ast and ldh into the blood. this probably accounts for the increase in the activity levels of these marker enzymes in the serum (27). pyridoxine at 5mg/kg or 10mg/kg dose each produced non-significant reduction in serum ast and ldh enzymes activity. in contrast, 15mg/kg pyridoxine produced significant decrement in the intended enzymes activity levels; moreover, treatment with iraqi j pharm sci, vol.26(2) 2017 impact of pyridoxine graded doses on doxorubicin cardiotoxicity. 19 pyridoxine 15mg/kg prior to a single dose 15mg/kg dox restored the activities of enzymes by reducing the marker enzymes (ast and ldh) levels in serum. this may be attributed to the protective role of pyridoxine on the myocardium, reducing the myocardial damage, thereby, restricting the leakage of these enzymes in serum. it has been reported that tac include antioxidant enzymes superoxide dismutase (sod), catalase (cat), and glutathione peroxidase (gsh-px); exist in all oxygenmetabolizing cells to prevent cells from damage exerted by free radicals and provide a repair mechanism for oxidized components (29). superoxide dismutase (sod) dismutases superoxide, the first step generated radical, to hydrogen peroxide and oxygen. hydrogen peroxide (h2o2) is neutralized to h2o by gshpx or cat. the tissue homogenate enzymatic levels of tac were significantly reduced (p<0.05) in dox-treated group (group v) this comes in tune with khan g et al (2014) (27) and al-harthi se et al (2014) (30). the current study also showed that pyridoxine 5mg/kg produced non-significant differences in tissue homogenate taoc level; while pyridoxine at 10mg/kg or 15mg/kg each dose produced a significant increment in taoc enzyme level in comparison with the negative control group. in addition, data from this study showed that tac including enzymatic and non-enzymatic cellular antioxidant defense mechanisms in groups of animals treated with either 10mg/kg pyridoxine prior to a single dose of 15mg/kg dox (group vii) or 15mg/kg pyridoxine prior to a single dose of 15mg/kg dox (groupviii) were significantly elevated, this comes in agreements with other study taş s et al (2014)(31), in which, pyridoxine enhanced serum antioxidant paraoxonase and arylesterase enzymes activities; related to pyridoxine ability to reduce oxidative stress (31) ; moreover, pyridoxine may directly or indirectly play a role in oxidative stress and the antioxidant defense system (16). it was suggested that the intended vitamin may act as a powerful chain-breaking antioxidant in biological systems related to its ability to scavenge peroxyl radicals (14) (32) (33). conclusions according to the results obtained from this study, it could be concluded that each of the pyridoxine doses (10 and 15mg/kg) administered once daily for 4 consecutive days to female rats prior to a single dose of dox (15mg/kg) has a protective effect and can ameliorate the cardiotoxic effect of dox that evidenced by a significant reduction in the measured serum biomarkers enzymes rat ast and rat ldh with significant increment in the level of rat taoc in the heart tissue; therefore, pyridoxine may have a therapeutic value against acute cardiotoxicity induced by doxorubicin in female rats via its antioxidant effects. acknowledgements this article was abstracted from m.sc. thesis submitted to the department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad/iraq. the authors are thankful to the department of pharmacology and toxicology at the college of pharmacy, baghdad university, baghdad-iraq, and the national center for drug control and research (ncdcr), ministry of health/environment, baghdad, iraq for their continuous encouragement and support. references 1. ahmed ia, mohammed aia, khaleel kj. ameliorating the anticancer drug ”adriamycin” acute cardiotoxicity by rosuvastatin and telmisartan in rats. iraqi journal of cancer and medical genetics, 2014; 7(2): 146-153. 2. clement y. can green tea do that? a literature review of the clinical evidence. preventive medicine, 2009; 49(2-3):83-7. 3. gharanei m, hussain a, janneh o, maddock hl. doxorubicin induced myocardial injury is exacerbated following ischaemic stress via opening of the mitochondrial permeability transition pore. toxicology and applied pharmacology journal, 2013; 268:149– 156. 4. ichikawa y, ghanefar m, bayeva m, et al. cardiotoxicity of doxorubicin is 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https://doi.org/10.31351/vol29iss2pp223-230 223 the role of clinical pharmacist in reducing drug related problems in hemodialysis patients aya f. talib*,1 and zinah m.anwer ** * ministry of health and environment al-karkh health directorate, , baghdad, iraq **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq abstract prescribing drugs to patients to treat ailments or reducing their morbidity may not be enough, even if the drugs were all indicated and in the right dose. clinical pharmacists play a pivotal role in conducting information and instruction to patients and conveying feedback to treating physician when appropriate, and the final goal is in the interest of the patient. the aim of the study was detection and classification of any drug related problems among hemodialysis patients and trying to reduce them by providing suitable recommendations in collaboration with the health care providers. prospective, interventional, clinical study for 180 hemodialysis patients, and was designed as two phases, an observational phase to identify drug related problems and classifying them according to the latest pharmaceutical care network europe classification, and an interventional phase to increase the awareness of patients and the nephrologists about those problems and proposing a proper solution for each one. the main drug related problems was related to the effect of drug treatment being not optimal in 58.7%, followed by no effect in 17.8%, and least for unrelated symptoms or indications in 4.8%; causes were inappropriate combination and patients taking less drug than prescribed (both 17.4%), followed by no/or incomplete drug treatment in spite of existing indication in 12.2%, drug without indication in 10.4%. erythropoietin and calcium were the most frequently drugs with problems. acceptance and full implementation were observed in 34.3% of recommendations, while about half of the drug related problems had unknown implementation (51.3%). there were significant numbers of drug related problems among iraqi patients on hemodialysis, the use of erythropoietin, calcium carbonate and sevelamer was responsible for most of inappropriate combinations. physicians and clinical pharmacist cooperation was excellent. keywords: drug related problems, hemodialysis, clinical pharmacist. pharmaceutical care, iraq. دور الصيدلي السريري في تقليل المشاكل العالجية لمرضى الغسل الكلوي **انور زينة مظفر و 1*،اية فوزي طالب . دائرة صحة بغداد، الكرخ والبيئة ، وزارة الصحة * .، بغداد ، العراق فرع الصيدلة السريرية، كلية الصيدلة، جامعة بغداد ** الخالصة ورة صحيحةبص حتى لو تم تحديد جميع األدوية، كافيا اعراض المرض قد ال يكونأو الحد من هموصف األدوية للمرضى لعالج أمراض ا محوري ا السريريونوبالجرعة المناسبة. يلعب الصيادلة إلى رجاعيةة االالتغذي ايصالالمعلومات والتعليمات للمرضى وكحلقة وصل لنقل دور .المريض مصلحة الهدف النهائي هوف، حاجة لذلكعالج عند الالطبيب الم الرعاية الصحية. االطباء الذين يقدمونومناقشتها مع باالدويةتحديد وتصنيف المشاكل المتعلقة كل المتعلقة ، وتم تصميمها على مرحلتين، مرحلة مراقبة لتحديد المشاكلوي غسيليخضعون لمريضا 180خلية وسريرية لـ اتدتقدمية، دراسة وأطباء أمراض الكلى خلية لزيادة وعي المرضىامرحلة تد و تبعتهاوتصنيفها وفق ا ألحدث تصنيف لشبكة الرعاية الصيدالنية في أوروبا ، باالدوية ٪، 58.7يكن مثاليا في بتأثير العالج الدوائي الذي لم ارتبطت باالدويةحول تلك المشاكل واقتراح حل مناسب لكل منها.المشاكل الرئيسية المتعلقة باالدوية علىلمتعلقة اسباب المشاكل ا اشتملت. ب استخدامهالعدم ترابط االدوية مع االعراض او اسبا ٪4.8 ، ثم٪17.8في فعالية الدواءيليه عدم معين عالجتخدام اس ، متبوع ا بعدممن قبل الطبيب ٪( 17.4أقل من الموصوف ) االدوية تناول جرعةو تتناسب مع بعضها، تزامن استخدام ادوية ال يوم أكثر األدوية ٪. كان اإلريثروبويتين والكالس 10.4في لذلك مؤشربدون معين دواءاستخدام ٪ ، و 12.2في لذلك مؤشرات وجود على الرغم من المتعلقة المشاكل توصيات لحل ، بينما نصفحالة ٪34.3في التوصيات كل واجراء تقبل االطباء. ولوحظ في استخدامها من مشاكل عانتالتي ٪(.51.3) من عدمه باالدوية لم يكن يعرف تطبيقها ثروبويتين والكالسيوم وكان استعمال دواء االرغسيل الكلى، تحت بين المرضى العراقيين باالدوية كل المتعلقة كان هناك عدد كبير من المشا ا. كان التعاون بين األطباء والصيادلة السريريين ممتاوالسفيالمير االكثر مسؤولية عن التسبب بالتداخالت الدوائية .ز . باالدوية، الغسل الكلوي، الصيدلي السريري،الرعاية الصيدالنية،العراقالكلمات المفتاحية: المشاكل المتعلقة 1corresponding author e-mail: ayaftalib@google.com received: 22/5 /2020 accepted: 19/7/ 2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp223-230 iraqi j pharm sci, vol.29(2) 2020 clinical pharmacist and reducing drug related problems 224 introduction worldwide chronic kidney disease (ckd) is one of the most common noncommunicable diseases, and in 2015 it was the twelfth cause of death, responsible for 1.1 million deaths (1). end-stage renal disease (esrd) is defined as irreversible decline in a person's own kidney function, which is severe enough to be fatal in the absence of dialysis or transplantation (2). the esrd can present with a number of signs and symptoms. some include volume overload not responding to diuretics, hypertension with low response to therapy, anemia, and metabolic derangements like hyperkalemia, hyponatremia, metabolic acidosis, hypo/hypercalcemia, and hyperphosphatemia. uremic toxicity can present as loss of appetite, nausea, vomiting, bleeding tendency, neuropathy or encephalopathy, seizures, loss of consciousness, with high mortality rate and it’s an urgent indications dialysis (3). generally, the symptoms of esrd appear in stages 4 and 5 when the glomerulus filtration rate (gfr) falls below 30 ml/min, but sometimes the underlying etiology presents earlier like in cases of nephrotic syndrome and cystic renal diseases (4). drug-related problems (drps) are events or circumstances involving drug therapy that actually or potentially interfere with desired health outcomes (5). pharmaceutical care network europe (pcne) had developed a classification scheme drps in 1999 and still continuously updated, the most recent version (9.0) last updated june 2019 and included: problems, causes and interventions (6). the evidence of the benefits of the pharmacists’ interventions in patients with ckd is sparse, with variable quality and unharmonious outcomes, however, on the basis of best available evidence it may have a positive impact on outcomes of patients with ckd (7). the major drps in patients on hemodialysis may include drug interactions, adverse reactions (8), under dose and treatment failure (9). aim of the study was detection and classification of any drug related problems among hemodialysis patients and trying to reduce them by providing suitable recommendations in collaboration with the health care providers. patients and methods study design this prospective, interventional, clinical study was designed as two phases, an observational phase to determine the prevalence of drps, identifying and classifying them using the latest version of pcne (6) and an interventional phase in which the researcher pharmacist increased the awareness of patients and the nephrologists about drps and proposed a proper solution for each type of drps . this study was carried out in two hemodialysis centers one in baghdad teaching hospital in the medical city complex and the second in alimamain al-kathymain medical city. data was collected from the first of december 2019, until the 29th of february 2020. the sample size was calculated by a single population proportion formula with a 95% level of confidence and after adjusting the data for the finite sample size of 400 that were registered; the final sample size was 175 and a total of 180 patients were enrolled in the study.(10, 11) inclusion criteria 1. outpatients aged ≥20 years with end stage renal disease on regular hemodialysis session. 2. agree to participate in the study 3. are registered patients for hemodialysis. exclusion criterion 1. patients infected with hepatitis b or c. 2. pregnant or breast-feeding women. 3. patients admitted for investigational purposes without hemodialysis. data collection/ observational phase a special sheet was designed by the research team to match study goals and the information was collected from patients’ casesheets regarding their demographic data, comorbidities, laboratory investigations, medication history, hemodialysis sessions number and related drugs and by participating in daily morning tours with the physicians and the clinical pharmacist. all information was double checked with the patients themselves, caregivers, or physicians. the researcher was concerned with identifying any type of drps, then classifying it according to pcne classification scheme (6). data collection/ interventional phase this interventional phase was done concomitantly with the observational phase and included offering proper clinicalpharmacological interventions, both on patients’ level, and physicians’ level, which was based on kidney disease improving global outcomes (kdigo) guidelines (12) then assessing the acceptance of the physicians to this intervention. patients level included 2 types according to pcne: 1. offering a proper patient counseling. 2. written information was provided to the patient. iraqi j pharm sci, vol.29(2) 2020 clinical pharmacist and reducing drug related problems 225 physicians’ level proposing a proper clinical intervention according to kdigo guidelines (12) after interpretation of patient’s lab data, clinical status and comorbidities. all the prescribed medications were checked for interactions and identified using the medscape and given the code c1.4 from pcne. direct interviewing, then assessing the acceptance and implementation of the physicians to this intervention. those interventions for the physicians were accompanied with information from the references in order to persuade the physicians to make the required change suggested by the researcher. ethical considerations a research proposal was approved by the scientific committee of the college of pharmacy/ university of baghdad before it was submitted and officially approved. additionally, the study was approved by the hospitals, and participants' verbal consent was obtained. statistical analysis all the categorical data were expressed in the form of frequency numbers and percentages while the continuous data were summarized as means and standard deviations. various comparison tests were conducted for different variables between the patients and a pvalue less than 0.05 was considered significant. chi-square was used for the categorical data comparisons and replaced with fisher’s exact test in case the first was inapplicable. also, independent sample t-test for binary comparisons. all the statistical work and the graphs were done using the statistical package for the social sciences (spss) software version 22. results the most frequent age group was 60-69 years, about half of the study sample was male(52%), and 39.4% of the study group were overweight, 23.3% class i obese and only 2.8% class ii obese, as shown in table (1). table 1.basic demographic data of the study sample the main drps was related to the effect of drug treatment being not optimal, in 58.7%, followed by no effect in 17.8%, and least for unrelated symptoms or indications in 4.8%, as shown in table (2). table 2. distribution of the study sample according to main drps. main drps number % no effect of drug treatment 41 17.8 effect of drug treatment not optimal 135 58.7 untreated symptoms or indication 11 4.8 adverse drug event (possibly) occurring 21 9.1 unnecessary drugtreatment 22 9.6 total 230 100.0 drps: drug related problems. there were 21 cases (9.1%) with adverse events, which did not need further explanation for causes of drp according to the pcne. the most frequent causes were inappropriate combination and patients taking less drug than prescribed (both 17.4%), then followed by no/or incomplete drug treatment in spite of existing indication in 12.2%, then no indication for the drug in 10.4%, the details are shown in table 3. variable number % age groups 20-29 14 7.8 30-39 10 5.6 40-49 23 12.8 50-59 41 22.8 60-69 63 35.0 ≥70 29 16.1 gender male 94 52.2 female 86 47.8 bmi underweight 8 4.4 normal weight 54 30.0 overweight 71 39.4 class i obesity 42 23.3 class ii obesity 5 2.8 total 180 100.0 iraqi j pharm sci, vol.29(2) 2020 clinical pharmacist and reducing drug related problems 226 table 3. distribution of the study sample according to causes of drp causes of drp number % adverse drug event (possibly) occurring 21 9.1 inappropriate drug according to guidelines/formulary 7 3.0 no indication for drug 24 10.4 inappropriate combination of drugs, or drugs and herbal medications, or drugs and dietary supplements 40 17.4 inappropriate duplication of therapeutic group or active ingredient 1 0.4 no or incomplete drug treatment in spite of existing indication 28 12.2 drug dose too low 22 9.6 drug dose too high 12 5.2 duration of treatment too short 5 2.2 duration of treatment too long 1 0.4 prescribed drug not available 1 0.4 inappropriate timing of administration or dosing intervals 1 0.4 patient uses/takes less drug than prescribed or does not take the drug at all 40 17.4 patient uses/takes more drug than prescribed 6 2.6 inappropriate timing or dosing intervals 21 9.1 total 230 100.0 erythropoietin was the most frequently encountered, with the patients taking less than prescribed dose. followed by calcium drps, which were commonly inappropriate dosing/ timing or interactions. sevelamer was usually used inappropriately combined with folic acid. ferrous sulfate drps were caused commonly dose related. cinacalcet drps mainly caused adverse reactions. amlodipine drps mainly were patient related. omeprazole, antihistamines and antipyretic all were not or incompletely used as shown in table (4). iraqi j pharm sci, vol.29(2) 2020 clinical pharmacist and reducing drug related problems 227 table 4. distribution of medications according to cause of drp expressed as number(percentage). drugs erythropoietin calcium sevelamer ferrous sulfate cinacalcet amlodipine omeprazole anti-histamine antipyretic total adverse reactions 1(4.8) 7(33.3) 1(4.8) 1(4.8) 11(52.4) 0(0) 0(0) 0(0) 0(0) 21(100) inappropriate drug 0(0) 2(28.6) 5(71.4) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 7(100) no indication for drug 0(0) 0(0) 1(100) 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 1(100) inappropriate combination of drugs 0(0) 12(38.7) 18(58.1) 0(0) 1(3.2) 0(0) 0(0) 0(0) 0(0) 31(100) no or incomplete drug treatment 0(0) 0(0) 0(0) 0(0) 0(0) 0(0) 5(41.7) 4(33.3) 3(25) 12(100) drug dose too low 7(38.9) 0(0) 3(16.7) 8(44.4) 0(0) 0(0) 0(0) 0(0) 0(0) 18(100) drug dose too high 4(33.3) 0(0) 4(33.3) 4(33.3) 0(0) 0(0) 0(0) 0(0) 0(0) 12(100) patient uses/takes less drug than prescribed or does not take the drug at all 35(87.5) 0(0) 1(2.5) 2(5) 0(0) 2(5) 0(0) 0(0) 0(0) 40(100) patient uses/takes more drug than prescribed 0(0) 0(0) 0(0) 0(0) 0(0) 5(100) 0(0) 0(0) 0(0) 5(100) inappropriate timing or dosing intervals 1(4.8) 17(81) 1(4.8) 0(0) 2(9.5) 0(0) 0(0) 0(0) 0(0) 21(100) total 48(28.6) 38(22.6) 34(20.2) 15(8.9) 14(8.3) 7(4.2) 5(3) 4(2.4) 3(1.8) 168(100) iraqi j pharm sci, vol.29(2) 2020 clinical pharmacist and reducing drug related problems 228 acceptance and full implementation were observed in 34.3%, while about half of the drps had unknown implementation (51.3%), and no agreement occurred with 7.8% of the cases, as shown in table (5). table 5. distribution of the study sample according to acceptance per planned intervention acceptance number % intervention accepted and fully implemented 79 34.3 intervention accepted, partially implemented 10 4.3 intervention accepted but not implemented 5 2.2 intervention accepted, implementation unknown 118 51.3 intervention not accepted: no agreement 18 7.8 total 230 100.0 discussion drug related problems are common medical/ pharmaceutical outcomes, especially in patients with end stage renal disease requiring replacement therapy like hemodialysis; with increasing number of drugs and complicated medication regimens, with multiple medical conditions that might require polypharmacy, however, not all the problems is due to prescriptions errors, some are unpredictable complications, or potential errors that could be prevented with modifications of some behaviors (9). in the current study, the main drps was suboptimal drug effect followed by no effect and least for unrelated symptoms or indications. in solaimani/ iraq, ossman and colleagues (2015) studied drp in 50 ckd patients on hemodialysis, but they did not use pcne classification, and reported that commonest drps were under dose in 29%, treatment failure in 27.7% and 14.7% for incorrect administration (9). in another study done in saudi arabia by alshamrani et al., (2018), that studied polypharmacy and drps in 83 patients on hemodialysis and reported that 36% of drps were related to using drug without indication, 23% subtherapeutic doses, 15% overdosing and 10% not using indicated drugs (13). these findings were in partial concordance to the results of ramadaniati et al., (2016) in indonesia, who enrolled 105 patients and reported that there were 1026 actual and potential drps during their study period, 38.9% were non allergic adverse effects, 28.7% were suboptimal effect, and 9.8% were no effect of drug (14). it can be seen that in all the previously mentioned studies, the drps were different, this could reflect the complexity of ckd and hemodialysis who usually requires multiple medications and complications of ckd and the associated co-morbidities, and with that the burdens increase on the patients and the treating physicians, here comes the role of the clinical pharmacist in elaborating, facilitating, and actively taking role in arrangement of the drugs and guiding the treating physician towards safer treatment options. in the current study, the most frequent causes of drps were inappropriate combination and patients taking less drug than prescribed, followed by no/or incomplete drug treatment in spite of existing indication, then using drugs without indication. these findings were comparable to results of a study done by nijeri (2016) in kenya, who enroled 60 hemodialysis patients in his thesis, and he observed 271 drps, from which 21.8% were drug interactions, 18.1% not using indicated drugs, 9.2% using drugs without indications, and 7% dose is too low (15). patricia and foote (2016) in unites states, who enrolled 93 hemodialysis patients with 376 medical discrepancies and 66 drps, they reported that 37.5% of drps were not using an indicated drug, 20.3% higher than recommended dose, 18.8% using drugs without indication, and 7.8% for drug-drug interactions (16). while in india, george et al., (2017) who enrolled 79 patients undergoing hemodialysis and reported that the vast majority of drps (86.4%) were drug interaction (260 out of 301 drps), from them 13.07% were serious and 75% were moderate but significant and 11.92% were mild, and the other drps were adverse drug reactions (4.98%) (8). it can be seen that drug interactions are common among patients with ckd and this can be explained as those patients usually need calcium carbonate supplementations, and it has a considerable list of interactions, including some antibiotics (tetracyclines and quinolones), bisphosphonates (which might also be needed for some patients), calcium channel blockers, iron supplements (ferrous sulfate) and low dose of aspirin. the other cause could be polypharmacy associated with those patients (8.35 ± 2.33 drugs per prescription as reported by george el al., (8)), which complicates the situation even with the aid of the clinical pharmacist. regarding certain drps in the current study, the findings were comparable to results of ossman et al., (2015) who reported that iron supplements and erythropoietin had the highest drps which were related to treatment failure and under-dosing, followed by calcium carbonate which were related to incorrect administration and nonadherence by patients, then antihypertensive drugs related to treatment failure, other drugs included anti-histamines, sevelamer and alfacalcidol (9). lumbantobing et al., (2017) in indonesia, studied 86 patients on hemodialysis and reported a total of 337 drps, and the drugs that commonly cause these problems were calcium carbonate (52.23%), ferrous sulfate (18.69%), erythropoitin (9.5%) and iraqi j pharm sci, vol.29(2) 2020 clinical pharmacist and reducing drug related problems 229 omeprazol (9.5%) (17). from this information, it can be deducted that calcium supplements causes a lot of drps, one cause could be the large number of patients with ckd who requires calcium because of increased serum phosphorus and decreased renal production of 1,25 (oh)2 vitamin d. ferrous sulfate also is needed in large number of patients due to low or absent production of erythropoietin and the anemia of chronic disease. the interventions in the current study were accepted in 92.2% of drps and were fully implemented in 34.3%, however, 51.3% the implementations were unknown. lower acceptance rates were reported by patricia and foote (2016) in unites states, as 77% of the interventions made by the clinical pharmacist were accepted, but they mentioned that some interventions were less readily accepted, which included dosage of drugs, not using an indicated drug, and using an unindicated drug (16). in another recent study done by savitha et al., (2020) in india, who enrolled 833 patients and identified 250 drps, and reported a very high acceptance and implementation of 97.6% (18). the high percent of unknown implementation was due to difficulties in following the patients after advising them about the drps and whether they actually adhered to the given recommendations. clinical interventions were introduced to help improving patients’ outcome, through actively participating in identifying drps, and offering proper clinical interventions to the treating physician or the nurse in some situations, or directly to patients, for example improving their adherence to therapy. the corporation between the medical and pharmaceutical branches definitely yield better results. limitations the most important limitations that were encountered are the unavailable laboratory investigations in the hospitals which were necessary for calculating the required doses, like parathyroid hormone levels and iron study, also most patient were tired and have low motivation for sharing information, in addition nor relatives were allowed to enter the hemodialysis centers and last the schedule of hemodialysis session starts as early as 4 am in the morning, so it was not feasible to obtain information from all patients when. conclusions 1. there were significant numbers of drps among iraqi patients on hemodialysis, and the commonest one was suboptimal effect of drug. 2. the main causes of the drps were inappropriate combinations and patients not taking the recommended doses, and the main drugs responsible were erythropoietin, calcium carbonate and sevelamer. 3. physicians and clinical pharmacist cooperation were excellent, and high percent of unknown implementation was related to patients’ factors. references: 1. wang h, naghavi m, allen c, barber rm, bhutta za, carter a, et al. global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980–2015: a systematic analysis for the global burden of disease study 2015. the lancet. 2016;388(10053):1459-544. 2. abbasi ma, chertow gm, hall yn. end-stage renal disease. bmj clin evid. 2010. 3. whitney dg, schmidt m, bell s, morgenstern h, hirth ra. incidence rate of advanced chronic kidney disease among privately insured adults with neurodevelopmental disabilities. clinical epidemiology. 2020;12:235-43. 4. benjamin o, lappin s. end-stage renal disease. in: statpearls [internet] updated 2020 mar 25. accessed on 25/4/2020. [available from: https://www.ncbi.nlm.nih.gov/books/nbk499 861/. 5. richards cdd, wiffen p, aronson crcpj, reynolds j, coleman srcphlmj, wiffen ecejhpecppscgp, et al. oxford handbook of practical drug therapy, 2nd ed. + oxford handbook of clinical pharmacy, 2nd ed: oxford university press, usa; 2016. 6. van mil jwf, horvat n, westerlund t. pcne classification for drug related problems. zuidlaren, netherlands: pharmaceutical care network europe in january; june 2019. 7. salgado tm, moles r, benrimoj si, fernandezllimos f. pharmacists' interventions in the management of patients with chronic kidney disease: a systematic review. nephrology, dialysis, transplantation : official publication of the european dialysis and transplant association european renal association. 2012;27(1):276-92. 8. george c, jacob d, thomas p, ravinandan ap, srinivasan r, thomas j. study of drug related problems in ambulatory hemodialysis patients journal of pharmacy and biological sciences. 2017;14(4):32-6. 9. ossman dh, marouf bh, ameen kh. identification of drug-related problems in patients with chronic kidney disease maintained on hemodialysis in sulaimani city. j pharm sci innov. 2015;4(3):172-5. 10. daniel w, chad l. biostatistics: a foundation for analysis in the health sciences. 10 ed. singapore: john wily & sons; 2014. 11. pourhoseingholi ma, vahedi m, rahimzadeh m. sample size calculation in medical studies. gastroenterology and hepatology from bed to bench. 2013;6(1):14-7. 12. chan ct, blankestijn pj, dember lm, gallieni m, harris dch, lok ce, et al. dialysis initiation, modality choice, access, and iraqi j pharm sci, vol.29(2) 2020 clinical pharmacist and reducing drug related problems 230 prescription: conclusions from a kidney disease: improving global outcomes (kdigo) controversies conference. kidney international. 2019;96(1):37-47. 13. alshamrani m, almalki a, qureshi m, yusuf o, ismail s. polypharmacy and medicationrelated problems in hemodialysis patients: a call for deprescribing. pharmacy. 2018;6(3):76. 14. ramadaniati hu, anggriani y, wowor vm, rianti a. drug-related problems in chronic kidneys disease patients in an indonesian hospital: do the problems really matter. int j pharm phar sci. 2016;8(12):298-302. 15. njeri lw. assessment of medication related problems among patients with chronic kidney disease in kenyatta national hospital. university of nairobi research archives http://hdlhandlenet/11295/98984. 2016. 16. patricia nj, foote ef. a pharmacy-based medication reconciliation and review program in hemodialysis patients: a prospective study. pharmacy practice (granada). 2016;14(3):0-. 17. lumbantobing r, sauriasari r, andrajati r. role of pharmacists in reducing drug-related problems in hemodialysis outpatients. asian journal of pharmaceutical and clinical research. 2017;10(special issue october):10813. 18. savitha rs, ramesh m, shetty ms, kiran kk. drug-related problems and pharmacist interventions in inpatients with chronic kidney disease. international journal of research in pharmaceutical sciences. 2020;11(1):960-6. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.26(1) 2017 effect of coumarin derivatives on the vibrio cholera isolates 32 the effect of coumarin derivatives(compounds) on the vibrio cholerae isolates from different clinical iraqi sources fatima.r.abdul * , nehad.a.taher *,1 ,ashraf s.hassan * and enaam h. batah * * department of biology, college of science , al-mustansiryha university ,baghdad ,iraq. abstract from a large number of bacterial samples collected from different hospital in iraq in central health laboratory ,only ten isolates were identified primary as vibrio. a number of morphology and biochemical test were carried out to complete this identification that showed all bacterial isolates were related to vibrio cholerae .in this study all vibrio isolates were investigated for bio typing and the result showed that all (10) isolate were related to (eltor biotypes) .also, the susceptibility test towards eight antibiotics were carried out . results shows that ciprofloxacin , norfloxacin, erythromycin, ampicillin, ceftriaxone and amikacin were the most effective antibiotics and their resistance percentage were 20%,20%,20, 20,30% and 30% respectively ,while chloramphenicol and cotrimoxazole were less effective and their resistance percentage were 90% both of them. three (3,5,6) isolates v. cholerae were selected depending on results of antibiotics sensitivity tests as showed multiple –antibiotics resistance(100%). then tested to study the effect of coumarin derivatives compounds (1, 2, 3 ) which showed inhibitory effect on v. cholera (3,5,6) isolates and the compound ( 3 ) showed the highest antibacterial activity of (12,15,14 mm) of inhibition zone diameter against v. cholera (3,5,6) isolates respectively. also, these iraqi isolates (3,5,6) used to test the effect of acridine orange (0.1%) as acuring agent , the results showed that all (3) isolates v. cholerae were sensitive to (ciprofloxacin, ceftrixone and norfloxacin), while the rest were resistance to remained five antibiotics. the results of agarose –gel electrophoresis of both normal v. cholerae (3,5,6) and cured isolates showed the presence of chromosomal and plasmid dna bands in the normal case ,while only chromosomal dna bands occur with v. cholerae (isolate 8) treated with an acridine orange at concentration of (10 -2 to 10 -4 ). keywords: vibrio cholera, coumarin derivatives, acridine orange. المعزولة مه مصادر vibrio choleraeتأثير مركبات الكىماريه على عزالت مه بكتريا سريرية عراقية مختلفة فاطمة رمضان عبذل * ، وهاد عبذ طاهر ،*1 ، اشرف سامي حسه * اوعام حميذ بطاح و * * ػهٕو انحٍاة ،كهٍت انؼهٕو ، انضايؼت انًغخُصشٌت ، بغذاد ، انؼشاق.قغى الخالصة يٍ يغخشفٍاث يخخهفت فً انؼشاق نهفخشة يٍ فً يخخبش انصحت انًشكضي يٍ يضًٕع ػذد كبٍش يٍ ًَارس بكخٍشٌت صًؼج حى اصشاء ػذد يٍ . vibrio( ػضالث شخصج يبذئٍا ػهى آَا حؼٕد انى صُظ 11),حى انحصٕل ػهى 1/7/5119 -1/11/5118 .vibrio choleraeْزا انخشخٍص يًا اظٓش اٌ ْزِ انؼضالث حؼٕد انى لانفحٕصاث انًظٓشٌت ٔانكًٍٕحٍاحٍت ال كًا ٔقذ اظٓشث انُخائش اٌ صًٍغ ْزِ انؼضالث (bio typing)شًهج ْزِ انذساعت اخضاع ْزِ انؼضالث انى انخًٍُط انحٍٕي ( يٍ انًضاداث انحٌٍٕت , ٔاظٓشث انُخائش اٌ 8)ْزِ انؼضالث حضاِ رًاٍَت (.كًا حى قٍاط حغاعٍتeltor)انؼششة كاَج يٍ َٕع رٍشا ٔبُغبت يقأيت انغبشٔفهٕكغاعٍٍ , َٕسفهٕكغاعٍٍ ,االسرشٔياٌغٍٍ, االيبغهٍٍ ٔانغٍفشحٕكغٍٍ ٔااليٍكاعٍٍ كاَج اكزشانًضاداث حا ػهى انخٕانً, بًٍُا كاَج نهكهٕسيفٍُكٕل ٔانكٕاحشايٍكغٕل اقم حارٍشا حٍذ كاَج َغبت %30%,30%,51%,51%,51%,51حغأي ( اػخًادا ػهى َخائش انحغاعٍت نهًضاداث انحٌٍٕت 9,8,6)ًْ v. cholerae % نكهًٍٓا. حى اخخباس رالد ػضالث يٍ بكخشٌا01انًقأيت ( 6,5,1)انؼضالث انزالرت( نذساعت حأرٍش يشكباث انكٕياسٌٍ انزالرت )%( . كًا حى اخضاػٓا 111)ٔانخً اظٓشث يقأيت يخؼذدة بُغبت ( كاٌ 6)ت حٍذ اظٓشث انُخائش اٌ انًشكب كهٕسٌذ انكٕبهج, كهٕسٌذ انًُغٍُض, انًشكب انزانذ ْٕ يضٌش يٍ كهًٍٓا( ػهى انؼضالث انزالر) ( ػهى انخٕانً.9,8,6)v. cholerae يهى( ضذ ػضالث 17,18,15)نّ اػهى حارٍش ضذ بكخٍشي بقطش حزبٍط كؼايم يحٍذ (%0.1)كًا شًهج ْزِ انذساعت اخخباس حغاعٍت انًضاداث يٍ خالل دساعت حأرٍش يادة االكشدٌٍ انبشحقانً بخشكٍض عبشٔفهٕكغاعٍٍ )اػالِ ٔقذ اظٓشث انُخائش اٌ صًٍغ انؼضالث انزالرت يٍ انبكخشٌا كاَج حغاعت نهًضاداث ػهى ْزِ انؼضالث ( بًٍُا بقٍت انؼضالث كاَج يقأيت انًضاداث انخًغت انباقٍت.,عٍفٕحٕكغٍى ٔانُٕسفهٕكغاعٍٍ ( انطبٍؼٍت ٔانًحٍذة ٔصٕد حضيت 9,8,6)v. cholerae ػضالث اظٓشث َخائش انضاَب انٕسارً نهخشحٍم انكٓشبائً نكم يٍ (انًؼايهت يغ االكشدٌٍ انبشحقانً 8انؼضنت )نهبالصيٍذ فً انؼضالث انطبٍؼٍت بًٍُا ظٕٓس حضيت نهكشٔيٕعٕو فقط نؼضالث ضًاث انكٕنٍشا 11 )بخشكٍض -7 11انى -5 ) . البرتقالي . االكرديهمركبات الكىماريه ، ،vibrio cholera بكتريا الكلمات المفتاحية : 1 corresponding author e-mail: dnahad17@yahoo.com received: 1 /12/2016 accepted: 20 /6/2017 iraqi j pharm sci, vol.26(1) 2017 effect of coumarin derivatives on the vibrio cholera isolates 33 introduction cholera as a disease is an acute diarrheal illness caused by v. cholerae infection of intestine,by how may infect 3-5 million person and may cause the death to 100,000 to 120,000 for each year ,if don’t have the therapy (1) . v.choleraee is a gram negative, curved rod bacterium, colonize the small intestine ,naturally habitat abrakish or salt water (2) . when ingested, v. cholerae can cause diarrhea and vomiting in the host within several hours to 2-3 days of ingestion (3,4) . the modern genetic studies that are achieved on the cholerae bacteria (eltor biotype) showed that it contains two circular chromosomes instead of one (5) . one of the two chromosomes has large size, it consists of 3 million base pair, it contains the most necessary genes to produce cytotoxin ct as well as the proteins which enter in metabolic pathways for the cell (6) , as well as it includes the important genes to dna replication, the cell division, genes transcription,protein translation and building the cell well as well as the genes that encoded about the virulence factors such as toxin corregulated pili (tcpa) and (toxr) (7) . heinemann et al referred to the existence type of plasmids is called r – plasmid which is existed in genetic content of the cholerae bacteria, this factor is responsible for bacteria resistance for many antibiotics (8) .the transferring of r-plasmid is easy where it is transferred to bacteria (e. coli – k12) to study the effect on the bacteria which is transferred to it in terms of resistance and sensitivity to the antibiotics (9) . v. choleraee bacterial was sensitive to a large number of antibiotics until the seventh decade of the twentieth century, however, the widespread and non programmable using of antibiotics leads to the emergence of resistant strains to many antibiotics (10) . until 1970 ,v. choleraee was sensitive to each of antibiotics(tetracyclin,ampicillin,chloramphe nicol), and in the eighties, it emerged resistant isolates to many antibiotics such as ampicillin, trimethoprime andtetracycline (11) . the coumarin compound afragrant organic chemical class (warfarin) benzopyrone, which is colorless crystalline substance in standard state , it it’s a natural substance found in many plants on their biosynthesis in plants occur via hydroxylation, glycolysis and cyclization of cinnamic acid. (12, 13) the coumarin compounds have important biological applications, it has been used as antifungal agents and as anticoagulants , and also to organize the growth of plants (14,15) . acridine orange (ao) is an organic compound used in nucleic acid selective fluorescent cationic dye useful for cell cycle determination at502nm and an emission maximum shifts to460nm (blue). aowill also enter acidic compartments such aslysosomes and become pronoated and sequestered in theselow ph conditions ,the dye will emit orange light .thus acridine orange can be used to identify engulfed apoptotic cells,because it will fluoresce upon engulfment .the dye is oftenused in epi florescence microscopy and flow cytometry analysis of cellular physiology and cell cycle status (15) . materials and method samples collection ten bacterial samples used in this study were obtained from central healthy laboratory collected from different hospital in iraq. bacterial diagnosis initial diagnostic depending on gram reaction and morphological characteristic of the colonies based on bacterial growth on thiosulphate citrate bile salt sucrose agar (tcbs) and blood agar, as well as the number of biochemical test (16) ,and vitek 2 compact system. the second technique biotyping of bacterial ,the biotype of v. cholerae serogroup o1(eitor) was distinguished by the following methods; polymyxinb susceptibility ,heamolysin test and voges-proskaur test (17) . antibiotic susceptibility test disk agar diffusion according to kirby baur standardized antimicrobial susceptibility single disk method was carried out toward eight antibiotics (ciprofloxacin , norfloxacin, erythromycin, ampicillin, ceftriaxone , amikacin, chloramphenicol andcotrimoxazole ), bioanaylyse(turkey). the antibiotic resistance percentage detected to each antibiotic as = number of resistance isolate to a specific antibiotic /total number of tested isolates ×100% (18) . experimental reparation of ethyl – 6 – chloro – 1 h – isochromene – 3carboxylate (s) in this study three already synthesized coumarin derivatives were used compound (1) (co(c10h5cio4)2cl2), compound (2) (mn(c10h5cio4)2cl2) and compound (3) mix of (1) and (2) compounds, and their melting point were detected (71˚c = 344f) and prepared as follow: 5-chloro–2-hydroxyl benzaldehyde(0.56 gm , 0.0036 mole ) and diethyl malonate ( 0.64 gm , 0.004 mole 10 mol % excess ) were heated with stirring in ethanol ( 95 % , 20 ml ) until dissolution occurred . addition of piperidine ( orang . the solution was refluxed iraqi j pharm sci, vol.26(1) 2017 effect of coumarin derivatives on the vibrio cholera isolates 34 for 6 hour and on cooling a white crystalline solid formed , crystals of ethyl – 6-chloro – 1 h – isochromene – 3-carboxylate were isolated by filtration and washed form ethanol , filtered (0˚c ) , the solid was recrystallized from ethanol , filtered and washed with cold ethanol again. an ethanolic solution (10ml) of the metal salt (metal (ii) salts are hydrated chloride ; mcl2 .xh2o ; where : m=, mn ( ii ) , co ( ii ) ( : x= 6, 6 ,4 and 2, respectively ) , was add to methanolic solution of the ligand (2mmole) in methanol (15ml). the reaction mixture was then refluxed around 2 hr. at ph= (6-7) , a colored precipitate was formed the product was then filtered and re-crystallized with methanol and dried at room temperature (19) . antimicrobial activity of the coumarin derivatives compounds the agar-well diffusion method was used to evaluate the antimicrobial activity of the ( 6 – chloro – 2 – oxo – 2 hchromene – 3 – carboxylic acid and complexes ) compounds . a bacterial suspension of (1.5x10 6 cells/ ml) which obtained by transfer aloopful of bacterial suspension from serial dilutions (stosk,10 -1 , …… 10 -6 ) , then streaking on (na ) nutrient agar plates and give acolony number (150 colony / 100ml medium)/dilution ) was inoculated on mullerhinton agar plates. wells (5 mm in diameter) were made in solidified agar using a sterile end of pasteur pipette. an amount of 0.1 ml(2*10 3 m) of each compounds was then added to the wells, in addition, one well was filled with dmso solution(aspecific soluble solution) , as a control (19). the plates were put in refrigerator at 4˚c for 30 min to allow for diffusion of concentrations via the medium ,then incubated for 24 hrs at 37˚c and the antimicrobial activity of each compounds was recorded by measuring the inhibition zone around the wells. effect of curing agent (acridine orange ). in this part of study the highly v. choleraee resistant isolate were used for studying the effect of the curing agent acriding orange concentrations of (0,10 -1 ,10 -2 ,10 -3 ,10 4 ,10 -5 ,10 -10 )by dissolving 0.1mg of acridine orange in 10ml d.w.then the above concentrations were prepared and added to a plate of nutrient agar seeded with alawn of nutrient broth (18-24h.) culture of isolates (3and 5). then ,the antibiotic susceptibility were measured at each concentration (20) . dna profile by gel electrophoresis bacterial plasmid dna was extracted from cultured cells using the alkaline sds method with promega dna kit described by (21) as follow: centrifuge 25 ml of culture, resuspended pellet in 0.5ml of lysozyme solution containing(0.3m naoh ,2% sds), mix and incubated at 70˚c for 15min, add 0.08ml of acid phenol/chloroform (1:1)and mix gently, separate phases by centrifugation at 10000xg for 10min and transfer the upper aquous phase to a new eppendorff tube containing 0.07m sodium acetate and 0.7ml isopropanol, centrifuge again for2min , the pellet dissolved in 0.05ml te buffer, stored at 4˚c and samples were electrophorated using 0.7% agarose with 5v/cm for 2h, after ending of electrophoresis process,the gel was exposed to u.v. light with 340nm to observe plasmid bands. results and discussion all bacterial samples expected to be a v. choleraee grown on alkaline peptone water (apw) at ph 9.2 as enrichment media, then transferred to thiosulfate citrate bile salt sucrose agar medium (tcbs) and occurred as yellow brilliant colonies on this medium figure (1).also , it occurs as comma to s-shaped – with red color under the microscope .then ,a number of biochemical test were carried out and the results showed in table(1). table (1): biochemical tests of v. choleraee isolates . all the above results in addition to identification by vitek system ,revealed that all ten vibrio isolates were related the cholerae species (100%)(15). in this study ,all (10) v. cholerae isolates were investigated biotyping and the results showed that all v. choleraee were o1(eltor) serotype group(16).our results was in agreement with the results obtained by (22) . biochemical test results oxidase + catalase + kliger iron assay a/k no gas, no h2s mannose resistance + voges proskaur + motility + urease + cholerae red + iraqi j pharm sci, vol.26(1) 2017 effect of coumarin derivatives on the vibrio cholera isolates 35 figure (1): thiosulfate citrate bile salt sucrose agar (tcbs) medium for cultivation of v. cholerae produce a distinctive yellow colonies. antibiotics susceptibility the susceptibility tests toward eight antibiotics were carried out and results are illustrated in figure (2) showed that ciprofloxacin , norfloxacin,erythromycin, ampicillin, ceftriaxone and amikacin were the most effective antibiotics and their resistance percentage were 20%,20%, 20,30% and 30% respectively ,while chloramphenicol and cotrimoxazole were less effective and their resistance percentage were 90% both of them. three isolates v. cholerae (3,5,6)were selected depending on results of antibiotics sensitivity tests as showed multiple –antibiotics resistance(100%). figure (2): the resistance percentage of v. choleraee isolates to antibiotics. results agreed with (22) who found that the v. choleraee isolates showed a high sensitivity to each of ampicillin, ciprofloxacin, erythromycine , tetracycline, gentamicin and cephalothin, and showed a low resistance (10%) and (20%) to cefotaxime and amikacin respectively, but disagreed with her results when she found that (80%) of the environmental isolates were resistance to amikacin. the antimicrobial activityof coumarin derivatives(compounds) compounds against v. choleraee. the antimicrobial activity of three types of coumarin derivatives(compounds 1,2,3) against three high resistance v. choleraee(3,5,6) were studied and results shown in the table(2) at which the compound 3 show the highest effect at which the diameter of inhibition zone was (12,15 and 14 mm) against v. choleraee isolates,3,5,6 respectively ,while the compound 1 was have a moderately effect at which the diameter of inhibition zone was (10,12 and 12 mm) . while the compound 2 was lowest effective by producing the inhibition zone of (0,11 and 10 mm) against the above isolates respectively. our results was in agreement with coixia etal, 2014 who found that many compounds containing chromene ring moiety display broad spectrum of biological activity. 2h-chromenes have gained much attention tcbs medium iraqi j pharm sci, vol.26(1) 2017 effect of coumarin derivatives on the vibrio cholera isolates 36 because of various biological activities such as antiviral, anti-tumor, anti-bacterial, fungicidal, antiflamatory, antioxidative and activator of potassium channels effects(22).coumarin (2h1-benzopyran-2-one), a naturally occurring plant constituent, has been used in the treatment of cancer and edema, and many of its derivatives have also shown biological activity. include antibacterial (23) . table (2): antimicrobial activity of the coumarim compounds against vibrio cholera isolates. the effect of acridine orange concentrations on antibiotic sensitivity of three highly resistance v. cholerae isolates. the results of the effect of acridine orange on athree higher resistance v. cholerae (3,5,6) were studied and shown as in table (3). these results showed that three v. choleraee isolates became sensitive to ciprofloxacin, norfloxacin and ceftrixone till 10 -6 concentration of acridine orange while still resistance to the remained five antibiotics under this study. also, the results of agarose electrophoresis were shown in figure3 revealed the presence of plasmid dna bands of (472, 670, 520 & 950)bp in (3,5,6,7) isolates respectively and chromosomal band in normal cases, while the presence of one chromosomal band isolate 8 which treated with acridine orange at concentration of (10 -2 to 10 -4 ). the effect of keylating agent (acridine orange) may cause a genetic mutations due to the changes in antibiotics resistance types of curing isolates after one hour of electrophoresis table (3): the effect of acridine orange concentrations on antibiotic sensitivity of three highly resistance v. choleraee isolates(3,5,6) . co-tri. chlora. amk. ceft. amp. ery. nor. cip. antibiotic acridine orange con. rrr rrr rrr rrr rrr rrr rrr rrr 0 rrr rrr rrr sss rrr rrr sss sss 10 -1 rrr rrr rrr sss rrr rrr sss sss 10 -2 rrr rrr rrr sss rrr rrr sss sss 10 -3 rrr rrr rrr sss rrr rrr sss sss 10 -4 rrr rrr rrr sss rrr rrr sss sss 10 -5 rrr rrr rrr sss rrr rrr sss sss 10 -6 rrr rrr rrr rrr rrr rrr rrr rrr 10 -7 rrr rrr rrr rrr rrr rrr rrr rrr 10 -8 rrr rrr rrr rrr rrr rrr rrr rrr 10 -9 rrr rrr rrr rrr rrr rrr rrr rrr 10 -10 these results were in agreement with hopwood etal, 1995 and pang etal 2007 who study the role of many antibiotic resistance properties of different serotypes of v.cholera which were plasmid determined and affected by many mutagenic agents (24,25) . our results were in contrast with pavlov etal 2006 who found that genetics of actinorhodine –production by streptomyces compounds bacteria isolation (co(c10h5 clo4)2 cl2) compound (1) (mn(c10h5 clo4)2cl2) compound (2) (co(c10h5 clo4)2 cl2) and (mn(c10h5 clo4)2cl2) compound (3) inhibition zone in mm isolation no 3 10 0 12 isolation no 5 12 11 15 isolation no 6 12 10 14 iraqi j pharm sci, vol.26(1) 2017 effect of coumarin derivatives on the vibrio cholera isolates 37 coelicolor was chromosomally determined and curing isolates treated with ethidiumbromide still actinorhodine producers. (26) .another study showed the presence of only one mega plasmid band of bacillus thuringensis after 1:30h of electrophoresis while, there is a complex plasmid profile with different molecular weights occur within the same species. this reason may related to the differences of plasmid properties between strains related to the same species or even sub species level (27) . bora etal 1994 found that these differences in plasmid profile may caused by the differences in the method of dna separation, the molecular weights of plasmid and its copy number found in bacterial cells (28) .our results may help to investigate the effect of coumarin derivative on another pathogenic bacteria even anti-tumor activity, also it was important to determined the specific gene sequence of cured isolate and the effect of acridine orange on other virulence factors of more epidemiological agents in iraq. figure (3): agarose gel electrophoresis of both chromosomal and plasmid dna isolated from v. choleraee iraqi isolates. lane 3,5,6,7 represent the normal isolates; lane 4 represents repeated normal isolate (3). lane 8 represents treated isolate with acridine orange at concentration (10 -2 ); lane m represents a marker. references 1. forbes, b. a; sahm; d. f. and weissfeld, a. s. baily and scott's.diagnostic microbiology. 12th ed. mosby elsevire. 2007; p: 371-8. 2. faraque, s. m., tam, v. c.; chowdhury, n., diraphat, p.; dziejman, m; heidelberg, j. f. genomic analysis of the mozambique strainsof vibrio choleraee o1 reveals the origin of eltor strains carrying classical ctx prophage. proc. nath. acad sci. 2007; 104: 5151-6. 3. farugue, s. m. ; al bert, m.j. and mekalanos, j. j. epidemiology ,genetics, and ecology of toxigenic vibrio choleraee. microbiology and molecular biology review1998; 62 (4): 13014. 4. faruqu, s. m.; chowdhury, n.; kamruzzaman , n.; dziejman, m.; rahman , m.h.; sack, d.a.; nair, g.b.; and mekalanos, j.j. genetic diversity and virulence potential of environmental vibrio choleraee population in a cholerae–endemic area. pnas. 2004; 101(7):2123-8. 5. joshi, r.m. and albert, m.j.. hybrid eltor vibrio choleraee o1emerg. infect. dis. 2009;15 (11): 1879-80. 6. tagomori, k.; iida, t. and honda, t. comparision of genome structures of vibrios, bacteria possessing two chromosomes. j. bacteriol. 2002; 184(16):4351-58. 7. xu, q.; dziejman, m. and mekalanos, j. j. determination of the transcriptome of vibrio choleraee 1000bp 900bp 500bp 300bp iraqi j pharm sci, vol.26(1) 2017 effect of coumarin derivatives on the vibrio cholera isolates 38 during international growth and midexponenttial phase in vitro. proc. natl. acad. sci. usa. 2003; 10 (13):128691. 8. heinemann, s. a. ; thomson, f. k. and hynes, w. l.. plasmid-borne antibiotic resistance in vibrio choleraee isolated from shop'sballast water. j. microbiol. 2003;(2): 2231 38. 9. coppo, a.; colombo,m.; pazzani, c.; bruni, r.; savia, a. and miamone,f. v. choleraee in the horn of africa: epidemiology, plasmid,tetracyclinie, resistance gene implication and comparison between o1and non-o1. am. j. trop. med. hyg.1995; 53 (4):351-59. 10. basu, a.; garg, p. ; datta, s.; ghakraborty, s. ; bhatt acharya, t.; khan,a.; rammurthy. t.; bhattacharya, s. k.; yamasaki, s. ; taked, y. and ,g. b.. vibrio choleraee o139 in calcutta, 1992 1998. incidence,antibiogram and genotypes. emerg. infect. dis.2000; 6 (2): 13947. 11. wilson, g.; miles, a. and parker, m. t. topley. and wilson's principles of bacteriology, virology and immunity. 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6-chloro-2-hydroxy2-trifluoromethyl-2h-chromene-3 carboxylic acid (5-allylsulfanyl (1,3,4) thiadiazol-2-yl)amide . molbank 2014; m830; doi :10.3390 / m830 . 23. bernadette, s. creaven, denise a. egan a, kevin kavanagh, malachy mccann, andy noble, bhumika thati and maureen walsh . synthesis, characterization and antimicrobial activity of a series of substituted coumarin-3-carboxylatosilver(i) complexes , inorganica chimica acta 2006; 359 :3976–84 . 24. hopwood,d.a.;chater,k.f.and bibb,m.j. genetic regulation of antibiotic production. ciba.found. symp. 1995;118:64-74. 25. pang , b; yan , m.; cui,z.;zhang ,l.and kan ,b.genetic diversity of toxigenic and not toxigenic v. cholera serogroups o1 and o139 revealed by array-based comparative genomic hybridization.j. bacteriol . 2007 ; 189 :4837 49. 26. pavlov ,a.r.,pava, n.v. ; kozyavkin , s.a. and slesarev , a.i. thermostable dna polymerase for a wide spectrum of applications : comparison of arobust hybrid topotaq to other enzymes .in kieleczawa . j.dna sequencing ii : optimizing prepration and cleanup.2006: pp. 241-57.isbn. 27. mohammed, a.j.; abdul kareem alkazaz and ali, s.m. plasmid role in crystal protein production in locally iraqi j pharm sci, vol.26(1) 2017 effect of coumarin derivatives on the vibrio cholera isolates 39 isolated bacillus thuringesis iraqi j. of science. 2013: p301-6. 28. bora, r.s.; mutry m.g. and sekar, v. introduction to lepidopteranspecific insecticidal crystal protein gene of bacillus thuringesis subspecies kurstaki by conjugal transfer in cotton phyllo sphere. appl. environ .microbiol. 1994; 60: 214-22. iraqi j pharm sci, vol.30(1) 2021 selexipag nanosuspension doi : https://doi.org/10.31351/vol30iss1pp144-153 144 nanosuspensions of selexipag: formulation, characterization, and in vitro evaluation rusul m. alwan*,1 and nawal a. rajab** * ministry of health and environment, najaf health department, najaf, iraq. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract selexipag(slp) is an orally selective long-acting prostacyclin receptor agonist indicated for pulmonary arterial hypertension treatment. it is practically insoluble in water (class ii, according to bcs). this work aims to prepare and optimize selexipag nanosuspensions (slpns) to enhance the saturation solubility and in vitro dissolution rate. the solvent antisolvent precipitation method was used for the production of ns, and the effect of formulation parameters (stabilizer type, drug: stabilizer ratio, and use of co-stabilizer) and process parameter (stirring speed) on the particle size (p.s) and polydispersity index (pdi) were studied. the result revealed that the p.s of all prepared slpns formulation was in the nanometer range, except for the formulas that stabilized by poloxamer. the optimal slpns (f15), which is stabilized by soluplus® (slp: stabilizer ratio 1:2) and prepared at a stirring speed of 1000 rpm, showed the smallest p.s and appropriate pdi, which are 47 nm and 0.073. the formula f5 exhibits 136 folds, an increase in the saturation solubility, and an enhancement in the dissolution rate in phosphate buffer ph 6.8 (100% drug release during 60 min) compared to the pure drug. this result indicates that slpns is an efficient way for improving the saturation solubility and the dissolution rate of slp. keyword: selexipag nanosuspension, solvent antisolvent precipitation method. جزيئات المعلق النانوي للسيليكسيباغ: الصياغه و التوصيف والتقييم في المختبر **نوال عياش رجب و 1، *محمد علوانرسل العراق ،النجف ،دائرة صحة النجف ،*وزارة الصحه والبيئة العراقية العراق ،بغداد ،جامعة بغداد ،كلية الصيدلة،** فرع الصيدالنيات الخالصة والذي يوصى به لعالج ارتفاع ضغط السيليكسيباغ هو ناهض مستقبالت البروستاسيكلين طويل المفعول االنتقائي الذي يستخدم فمويا ، الدم الشرياني الرئوي. هو غير قابل للذوبان في الماء)التصنيف الثاني: وفقا لنظام تصنيف الصيدالنيات البيولوجية(. تبر.معدل الذوبان في المخ الذوبانية و النانوي للسيلكسيباغ لتحقيق زيادة فيالمعلق يهدف هذا العمل إلى تحضير وتحسين جزيئات الصياغة )نوع المثبت ، الدواء: نسبة التثبيت ، عوامل نانوي ، وتم دراسة تأثيرمعلق تم استخدام طريقة الترسيب بالمذيب و المضاد للمذيب إلنتاج التشتت. ر تعددالعملية )سرعة التحريك( على حجم الجسيمات و مؤش عواملواستخدام المثبت المشترك( و وكانت .poloxamerكانت في نطاق نانومتر ، باستثناء الصيغ التي استقرت بواسطة slpnsالنتائج الى ان حجم الجسيمات لجميع عينات كشفت دورة 1000تحريك ( والمعد بسرعة 2: 1مثبت للالدواء: ةنسب) كمثبت رئيسي ®soluplusتحضيرها بوجود والذي تم (، (f15ثلاألمالعينة ، الذوبانيةيادة في زضعفًا 136 ( f5) عينة. أظهرت ال0.073 التشتت ر تعددمؤش كاننانومتر و 47حجم للجسيمات يبلغ صغر في الدقيقة ، أ النقي. تشير هذه لدواءدقيقة( مقارنة با 60لدواء خالل تحرر كامل ل) 6.8 الرقم الهيدروجينيذي للفوسفات محلول وتحسين معدل الذوبان في .الذوبانية ومعدل الذوبانطريقة فعالة لتحسين slpnsالنتيجة إلى أن .الكلمات المفتاحية : المعلق النانوي للسيلكسيباغ, طريقه الترسيب بالمذيب و المضاد للمذيب introduction it was reported that about 40% of new chemical entities are hydrophobic compounds, and one-third of drugs documented by united states pharmacopeia(usp) are poorly water-soluble. poor aqueous solubility may trigger some issues: such as low bioavailability caused by uncontrolled precipitation & extensive variation in the absorption profile(1). nanosuspensions (ns) is defined as a unique submicron colloidal dispersion of nanosized drug particles that need to be stabilized by suitable stabilizers (polymer and/or surfactant)(2). currently, ns has been considered as an attractive approach for enhancing the bioavailability of insoluble drugs owing to increase their saturation solubility and dissolution rate(3). in general, ns can be prepared either by top-down or bottom-up methods. the topdown processes involve a breakdown of large solid particles into nanosized particles, while bottom-up approaches involve precipitation of dissolved drug molecules into nanoparticles (4). 1corresponding author e-mail: rusul90alwan@yahoo.com received:22 /7/ 2020 accepted:24 /10 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp144-153 iraqi j pharm sci, vol.30(1) 2021 selexipag nanosuspension 145 although the top-down processes are favored in the pharmaceutical industry, the bottomup processes have the advantage of providing a homogenous dispersion system (narrow particle size distribution) with lower energy input (5). the liquid solvent anti-solvent precipitation method is a type of bottom-up technique that is most commonly used to prepare ns due to it is simplicity and cost-effectiveness. the basic principle of this method is based on modifying the compound solubility by mixing a water-miscible organic solvent containing the drug with the antisolvent (in which the drug is insoluble) in the presence of the stabilizer, precipitation of nanoparticles will occur instantaneously. during precipitation, the stabilizer will be absorbed on to the surface of the newly formed nanoparticles to prevent further growth of the particles (5,6). selexipag (slp) is a selective, orally active, long-acting prostacyclin (ip) receptor agonist that has been used for long term treatment of pulmonary arterial hypertension to delay disease progression and reduce the risk of hospitalization (7). in contrast, all other licensed drugs that target the prostacyclin pathway have a short half-life. most of them need to be administered by an intravenous or subcutaneous infusion or by the inhalation route(8). after oral administration, slp is rapidly hydrolyzed into the active metabolite act-333679 (approximately 37 fold more potent than slp) following absorption. the oral bioavailability of slp is approximately 50%(9). slp is a non-hygroscopic powder which appears as a pale yellow crystalline powder. it is practically insoluble in water (<1mg/ml) and exhibits ph-dependent solubility. according to the biopharmaceutical classification system, it is considered a class ii drug (low solubility, high permeability) (10,11). this study aims to prepare and characterize slpns prepared by liquid solvent antisolvent precipitation process in an attempt to improve the saturation solubility and dissolution rate of slp. materials selexipag (slp), polyvinyl alcohol(pva), hydroxypropyl methylcellulose (hpmc e15), and poloxamer-407(px-407) were purchased from hangzhou hyper chemical limited (china). soluplus® was obtained from basf (germany). tween-80 was provided by alpha chemika(india). all other solvents and chemicals used were of analytic grade and utilized without further purification. method preparation of slpns the slpns was prepared by the liquid solvent anti-solvent precipitation method. briefly, 10 mg of slp was dissolved in 3 ml methanol. after that, this solvent phase was added dropwise at a flow rate (1ml/ min) by the aid of a syringe pump into 27 ml of an aqueous solution (water containing polymer alone or in combination with co-stabilizer) under a continuous stirrer for 1 hr at a certain speed(12). different types of stabilizers, which are hpmc e15, pva, soluplus®, and px-407, were used at different concentrations, either alone or in combination with tween-80, as shown in table (1). the effect of formulation parameters (stabilizer type, slp: polymer ratio, and costabilizer) and process parameter (stirring speed) on the p.s and pdi of slp ns were investigated to select the most stable formula. in order to increase the stability of the final product and perform some evaluation tests, the freshly prepared slpns was immediately lyophilized in the presence of cryoprotectant (mannitol) to prevent the formation of the collapsing cake at a concentration of 0.5% w/v. the dispersion was pre-frozen in a freeze at -20 ℃ for 24 hr, followed by lyophilization for 72 hr at -52 ℃ and vacuum pressure of 0.2 mbar using labocconco lyophilizer (13). table 1. composition of slpns formulation formula symbol slp (mg) pva (mg) hpmc e15(mg) soluplus® (mg) px-407 (mg) tween-80 (ml) stirrer speed f1 10 10 ----500 f2 10 20 ----500 f3 10 -10 ---500 f4 10 -20 ---500 f5 10 --10 --500 f6 10 --20 --500 f7 10 ---10 -500 f8 10 ---20 -500 f9 10 10 ---0.01 500 f10 10 -10 --0.01 500 f11 10 --10 -0.01 500 f12 10 --10 0.01 500 f13 10 20 ----1000 f14 10 -20 ---1000 f15 10 --20 --1000 f16 10 ---20 -1000 iraqi j pharm sci, vol.30(1) 2021 selexipag nanosuspension 146 characterization of slpns p.s and pdi dynamic light scattering technique was used to measure the p.s and pdi of slpnss at 25 ℃ in a detection angle of 90o using the abt-9000 nano laser particle size analyzer. all samples were measured without dilution and in triplicate. the results were expressed by a mean and standard deviation (sd) (14). the drug content in slpns all formulas that have p.s under 100 nm were subjected to the drug content estimation study by diluting 1 ml of slpns with methanol into 20 ml. each sample was assayed by uv-visible spectroscopy at 297.8 nm for at least three times to measure the actual drug content based on the following equation (15): drug content %= (observed drug content /theoretical drug content) *100 …..(eq. no.1) in vitro dissolution study the dissolution test for slpns formulas that have p.s under 100 nm was performed using usp apparatus type ii (paddle type). the volume of ns equivalent to 1 mg slp was placed in a pretreated dialysis bag (mwco 12-14 kd). this dialysis bag was fixed into the paddle and immersed into the dissolution media (300 ml phosphate buffer ph 6.8 in the presence of 1% brij-35 to maintain the sink condition). the temperature was kept at 37±0.5 ℃, while the speed of the paddle was 50 rpm. an aliquot of 5 ml was withdrawn at a scheduled time interval (5, 10, 15, 20, 30, 40, 50, 60, 90, and 120 min) and replaced by the freshly prepared dissolution media. the slp amount was assayed spectrophotometrically at specified λ max for this media, which is 305 nm. all the measurements were done in triplicate (16). the similarity factor f2 was used to measure the similarity in the percent of drug dissolution between two curves according to the following equation: f2 = 50 × log {[1 + 1 𝑛 ∑ |𝑅𝑡 − 𝑇𝑡| 2 𝑛 𝑡=1 ] −0.5 × 100} where n represents the number of sampling points, while rt and tt is the average percentage of dissolved drug in the reference and test sample at time t, the fda guideline suggests that two dissolution profiles can be considered dissimilar if f2 is less than 50 (17). saturation solubility study the shaking flask method was utilized to determine the saturation solubility. an excess amount of pure slx, lyophilized ns (of the optimal formula), was added to 10 ml of d.w and subjected to shaking at 25 ℃ for 48h using a water bath shaker. the supernatant was filtered through a 0.45ϻm syringe filter and analyzed by the uvvisible spectrophotometer(18). fourier transformed infrared spectroscopy (ftir) the ftir spectra of pure slp, soluplus®, mannitol, and freeze-drying powder of the selected formula were recorded to investigate the interaction between active pharmaceutical ingredient and excipient using ftir spectrometer (ftir-8300 shimadzu, japan) by compressing the sample into a disc using kbr. the sample was analyzed through a wavelength region between 4000400 cm-1(19). statistical analysis the results in this experiment were expressed as the mean ± (sd). one way analysis of variance (anova) and t-test student was used to analyze the difference between the samples at the level of significance (p<0.05). result and discussion p.s and pdi analysis the mean p.ss and pdi values are shown in table (2). a pdi value < 0.3 indicates a narrow size distribution, while pdi >0.3 is considered a broad size distribution(20). the mean p.s of all the prepared formulas was in the range of 47 nm3835 nm. these results indicate that it is possible to formulate slpns with small p.s by controlling the critical formulation and process parameters. the pdi values of formulas ranged from 0.005-0.073, which indicates that these formulas have a narrow p.s distribution: except the formulas f4 and f9, which have a pdi value of 0.5 and 0.4, respectively, so this system is considered to have broad size distribution. iraqi j pharm sci, vol.30(1) 2021 selexipag nanosuspension 147 table 2. p.s and pdi of the different formulation. formula no. stabilizer used drug: stabilizer: co-surfactant ratio stirrer speed p.s±sd pdi f1 pva 1:1 500 541.6±6.3 0.009 f2 pva 1:2 500 439.6±26.5 0.016 f3 hpmc e15 1:1 500 467±13.8 0.02 f4 hpmc e15 1:2 500 181.6±2.3 0.5 f5 soluplus® 1:1 500 74.6±2.6 0.015 f6 solupus® 1:2 500 73.2±4.8 0.009 f7 px-407 1:1 500 2737.6±23 0.007 f8 px-407 1:2 500 3835.3±21 0.005 f9 pva: tween80 1:1:1 500 279±0 0.4 f10 hpmc e15: tween80 1:1:1 500 453.6±16.7 0.012 f11 soluplus®: tween80 1:1:1 500 83.2±6 0.019 f12 px-407: tween80 1:1:1 500 4356±0 0.007 f13 pva 1:2 1000 632±24.2 0.029 f14 hpmc e15 1:2 1000 603±12.12 0.02 f15 soluplus® 1:2 1000 47±9.2 0.073 f16 px-407 1:2 1000 3656.6±11 0.007 optimization of slpns using different types of stabilizers and at various concentrations. the selection of suitable stabilizers and concentration is one of the most critical parameters that affect the p.s and stability of ns(21). the effect of type and concentration of polymer on slpns formulations are seen in formulas (f1f8). the formulas that stabilized by soluplus® has the smallest p.s (p<0.05) followed by ns prepared with hpmc e15, pva, and px-407, as illustrated in figure (1). this can be attributed to the natural chemical composition and superior surface activity and wettability of soluplus®. soluplus® is an amphipathic graft copolymer contains a hydrophilic part (polyethylene glycol backbone) and a lipophilic part (vinyl caprolactam/ vinyl acetate side chain). the adsorption of soluplus® onto drug particles decreases the interfacial tension of the surface particles, thus providing steric hindrance to prevent the aggregation of the newly formed nanoparticles (22). further, the stabilization of slpns by hpmc e15 or pva could be explained by the formation of a hydrogen bond between slp particle and one of these polymers (hpmc e15 comprised a high degree of substitution of the methoxy and hydroxyl group while the chemical structure of pva is abundance with hydroxyl group). these polymers can effectively adsorb onto drug particle surfaces that lead to steric stabilization of the system by providing a stable thermodynamic barrier surrounded the particle surface, which retarded particle growth(23,24). px-407 is an amphiphilic, linear tri-block copolymer comprised of the hydrophobic central segment of polypropylene oxide (ppo) and two hydrophilic side segments of polyethylene oxide(peo). the hydrophobic ppo segment has driven the adsorption of polymer onto the drug particle surface. at the same time, the hydrophilic peo chains surround the particles and provide steric hindrance, thus prevent particle aggregation and growth(25). despite this advantage and unique nature of px-407, but is ineffective in producing slpns even at high concentrations. their adsorption onto drug particle surface depends on physisorption; if hydrophobichydrophilic forces are not strong enough for surface adsorption, they cannot overcome the attractive force between two-particles that become dominant due to depletion of stabilizer between the gap of particles(25,26). it was observed that with an increasing concentration of pva and hpmc e15. there was a dramatic reduction in p.s (p<0.05). the p.s of the formulas f1 and f3 (which contain drug: polymer ratio of 1:1) was 541.6 nm and 467 nm, respectively, compared to 439.6 nm and 181.6 nm for f2 and f4, which contain drug: polymer ratio of 1:2. this result could be explained based on that with increasing stabilizer concentration, the surface tension will decrease, which facilitates the adsorption of stabilizer on the surface of the newly formed nanoparticle so prevent particle growth and recrystallization by providing steric stabilization. otherwise, at low concentration, the stabilizer was ineffective in providing sufficient stability to the system(27). while the p.s of formula f6 (73.2 nm) stabilized by soluplus® and prepared at 1:2 drug: polymer ratio was not significantly reduced iraqi j pharm sci, vol.30(1) 2021 selexipag nanosuspension 148 compared with the formula f5 (74.6 nm) that prepared at a drug: polymer ratio of 1:1. this result indicates that once the stabilizer concentration is efficiently covering newly generated surfaces, a further increase in stabilizer concentration did not significantly affect the p.s and pdi(28). figure 1. effect of polymer types and concentration on the p.s of ns formulation. effect of the addition of co-stabilizer on the p.s the liquid solvent antisolvent precipitation method was used to formulate slpns in the presence of stabilizer alone (pva, hpmc e15, soluplus®, and px-407) and in combination with co-stabilizer (tween-80, which is a nonionic surfactant that sterically stabilized the system) to study the effect of polymer-surfactant interaction on the p.s as shown in figure (2). the p.s of the formula f2 (439.6 nm) that contains pva as the primary stabilizer was significantly reduced (p<0.05) with the addition of co-stabilizer (tween-80) as seen in formula f9 (p.s is 297 nm). this means there is an efficient surface affinity for this combination (pva and tween-80), which could form a stable thermodynamic barrier at the drug particle surface interface, thus delay particle growth(29). while the p.s of the formula f4 (181.6 nm), f6 (73.2 nm), and f8 (3835.3 nm) that stabilized by hpmc e15, soluplus® and px-407 respectively increased with the addition of tween-80 as shown with f10 (453.6 nm), f11 (83.7 nm), and f12 (4356 nm). this result indicates that this combination was inappropriate for slpns. costabilizer addition might retard the interaction between the drug and stabilizer, increasing the p.s of nanosuspended particles(29). figure 2.effect of the addition of co-stabilizer on the p.s of ns formulation. effect of the stirring speed on the p.s two different stirrer speeds, 500 and 1000 rpm were used to prepare the formulas (f2, f4, f6, f8, and f13f16, respectively) to study the stirring rate's impact on the p.s of slpns as shown in figure 3. all these formulas were prepared at a drug: stabilizer ratio of 1:2. the p.s of the formula f15 (47 nm) and f16 (3656 nm), which were stabilized by soluplus® and px-407 respectively, and prepared at 1000 rpm was significantly reduced (p<0.05) compared to formula f6 (73.2 nm) and f8 (3835.3 nm) that stabilized by the same polymer but at lower stirring speed (500rpm). the high, stirring rate will create high shear stress on the particles, which leads to the breakdown of these particles into smaller ones. in contrast, the p.s of formulas f13 (632nm) and f14 (603) that stabilized by pva and hpmc e15 respectively was significantly increase (p>0.05) compared to the formulas f2 (439.6 nm) and f4 (181.6 nm) that stabilized by the same polymer but at a lower stirrer speed of 500 rpm. although the high stirring speed ensures rapid nucleation and breakdown of large particles, this robust mechanical stirrer may provide the system with excessive energy that may overcome the barrier created by the stabilizer and initiates the collision between particles causing increased the p.s of the formulation (30,31). figure 3. the effect of stirring speed on the p.s of ns formulation. iraqi j pharm sci, vol.30(1) 2021 selexipag nanosuspension 149 percent of drug content in slpns the drug content in the ns formulas (which stabilized by soluplus® as a primary stabilizer) that have p.s under 100 nm was found to range from 91.194.59%, as illustrated in table (3). the high drug loading was one of ns's advantages because this drug delivery system is free from any carrier system(32). table 3. percent drug content of slpns with soluplus® formula percent of drug content f5 93.4± 0.27 f6 94.59± 0.1 f11 91.16± 0.7 f15 91.69± 0.4 in vitro dissolution study of slpns the in vitro dissolution test was performed for the pure drug and ns formulas that have p.s under 100 nm in phosphate buffer (ph 6.8) to simulate the in vivo release in the intestine, as shown in figure 4 (33). figure 4.the dissolution profile of the pure drug and slpns formulas in phosphate buffer ph 6.8 at 37± 0.5 ℃. a comparison between the dissolution profiles of slp ns formulas and pure drug (which was used as a reference) was made using the similarity factor f2. the similarity factor value falls between 0 and 100, and two dissolution profiles are considered to be similar when f2 is greater than 50(17). while the result of the comparison between the ns formulas and the pure drug was less than 50, as seen in table (4), this indicates there is a dissimilarity between the dissolution profile of slpns and pure slp powder. table 4. similarity factor f2 for slpns formula formula name f2 in phosphate buffer ph6.8 f5 26.08 f6 24.04 f11 19.19 f15 17.81 f15 shows superior in vitro performance (complete drug dissolved within 60 minutes) compared to other ns formulas. so it was selected as the optimal formula. the enhancement in the slpns dissolution rate could be explained based on the noyes whitney equation that states with reducing the p.s, especially to the nanoscale range, the surface area will be increased, which results in enhancement of dissolution velocity. the amphiphilic stabilizer (solupus®) could also improve the surface wettability of the poorly water-soluble drug, thus leading to a faster release of drugs through ns formulation when compared to pure slx powder(34). determination of saturation solubility saturation solubility of pure slp in d.w was found to be 4.2±0.07 ϻg/ml while in the case of lyophilized ns (for the optimal formula f15), it was found to be 574.4±0.03ϻg/ml. around 136 folds increased in saturation solubility was observed in slp lyophilized ns. the higher solubility of slp in lyophilized ns could be explained based on the ostwaldfreundlich equation. the surface area will be increased by reducing the p.s of the drug to the nanoscale, enhancing the slp solubility. another reason may be attributed to amphiphilic stabilizer(soluplus®), a type of polymer that can provide extra solubilization effect on the poorly water-soluble drug(2,35). fourier transforms infrared spectroscopy (ftir) ft-ir spectra for pure slp, soluplus®, mannitol, and lyophilized slp powder of the selected formula (f15) were carried out to investigate any possible interaction among different components, as seen in figures (5-8). the characteristic bands of structural moieties of slp are shown in table (5) (36). table 5.the ft-ir absorption bands of slp. band assignment wavenumber (cm-1) n-h stretching 3360-3053 (as multiple bands) aromatic c-h stretching 3024 aliphatic c-h stretching 2988 cm-12872 c=o stretching 1724 c=c ring stretch 1556, 1477, 1350 c=n stretching 1477 so2 stretching 1350 c-n stretching 1240 c-o-c stretching 1124 while the ftir spectrum of soluplus® displays the characteristic peak of a hydroxyl group at 3437 cm-1 (o-h stretching). peaks at 1741 cm-1 and 1633 cm-1 are attributed to c=o stretching of ester group and tertiary amide group, respectively. iraqi j pharm sci, vol.30(1) 2021 selexipag nanosuspension 150 in the lyophilized powder, an intermolecular hydrogen bond may be formed between the carbonyl group of slp and hydroxyl group of soluplus®, which results in a shift of the characteristic peaks. the peak assigned to c=o stretching was shifted from 1724 cm-1 to 1735 cm-1 and became less intense(32). also, the n-h peak may be overlapped with the o-h peak of mannitol, forming a band at 3280(37). figure 5. fourier transforms infrared spectroscopy (ftir) of pure slp. figure 6. fourier transforms infrared spectroscopy (ftir) of soluplus®. iraqi j pharm sci, vol.30(1) 2021 selexipag nanosuspension 151 figure 7. fourier transforms infrared spectroscopy (ftir) of mannitol. figure 9. fourier transforms 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and dissolution enhancement of ebastine by surface solid dispersion technique lina s. hussein*,1 and eman b. h. al-khedairy** *department of clinical and laboratory sciences, college of pharmacy, thi-qar university, thi-qar, iraq **department of pharmaceutics college of pharmacy, university of baghdad, baghdad, iraq. abstract ebastine (ebs) is a non-sedating antihistamine with a long duration of action. this drug has predominantly hydrophobic property causing a low solubility and low bioavailability. surface solid dispersions (ssd) is an effective technique for improving the solubility and dissolution rate of poorly soluble drugs by using hydrophilic-water insoluble carriers. the present study aims to enhance the solubility and dissolution rate of ebs by using ssd technique. avicel® ph101, avicel® ph 102, croscarmellose sodium(ccs) and sodium starch glycolate (ssg) were used as water insoluble hydrophilic carriers. the ssd formulations of ebs were prepared by the solvent evaporation method in different drug: carrier weight ratios, then evaluated for their percentage yield, drug content, water solubility, release in 0.1 n hcl, powder x-ray diffraction (pxrd) in addition to fourier transform infrared spectroscopy (ftir) for the determination the drug-carrier interaction. most of the prepared ssd formulas showed improvement of drug solubility. the best result was obtained with formula ssd16 (ebs: ccs 1:15) that showed high percentage yield (98.5%), high drug content (98.39%) and 8.2fold increase in solubility compared to solubility of pure drug with improved dissolution rate. the drug was converted to amorphous form without chemical interaction with the carrier. so, it can be concluded that the solubility and the dissolution rate of ebs were successfully enhanced by ssd technique prepared by solvent evaporation method using hydrophilic-water insoluble carriers. keywords: ebastine, surface solid dispersion, solvent evaporation technique. تقنية الصلب المنتشر السطحياإليباستين باستخدام تحسين الذوبانية وسرعة تحرر **الخضيري حازم بكر ايمان و 1 *، نلينا سالم حسي العراق.، رذي قافرع العلوم السريرية والمختبرية ، كلية الصيدلة ، جامعة ذي قار، * العراق.، بغداد ، جامعة بغداد ، كلية الصيدلة ، فرع الصيدالنيات ** الخالصة مما يسبب انخفاض النفور من الماءخاصية له غالبا هذا الدواء .مديد الفعالية مسبب للنعاساإليباستين هو دواء مضاد للهيستامين غير األدوية ضعيفة الذوبان سرعة تحرر تقنية فعالة لتحسين الذوبان والصلب المتشتت السطحي هي .له التوافر البيولوجي قلة ، وبالتالي ذوبانهقابلية .محبة للماء-باستخدام مواد غير ذائبة سيل ييفاإل ، ph101 سيلييفستخدام اإلبا .باستخدام تقنية الصلب المنتشر السطحي إليباستينسرعة تحرراتهدف الدراسة الحالية إلى تعزيز الذوبانية و ph102، الصلب تم تحضير تركيبات.ت النشا كحامالت مائية غير قابلة للذوبان في الماءيجاليكول الصوديوم للكروسكارميلٌوز وملح الصوديوم ملح نتاجية ، ومحتوى الدواء ، لإليباستين بطريقة تبخر المذيب في نسب مختلفة من وزن الدواء إلى الناقل وتم تقييمها حسب النسبة اإل السطحي المنتشر ، كما تم فحص خاصية التبلور باستخدام حيود ورمالين 0,1 في حامض الهيدروكلوريك ذو معيارية الدواءر بان في الماء وسرعة تحرقابلية الذو .تحديد التداخل الحاصل بين الدواء والناقل باستخدام مطياف األشعة تحت الحمراء األشعة السينية و باستخدام ssd16صيغة تم الحصول على أفضل وقد .باستخدام هذه التقنية الصيغ المحضرة اغلب تحسن في قابلية الذوبان للدواء في أظهرت النتائج ( %98.5نسبة إنتاجية عالية ) التي أظهرت (للكروسكارميلٌوز الصوديوم ملح: اإليباستين) 15:1ملح الصوديوم للكروسكارميلوزر بنسبة الصيغة مع تحسن في معدل تحرر الدواء . كما أضعاف في الذوبانية مقارنة بالذوبان في الدواء النقي 8,2زيادة قدرها ( و 98.39%، محتوى دواء عالي ) تم تحسينهما بنجاح إليباستينلذلك يمكن االستنتاج أن قابلية الذوبان ومعدل الذوبان لـ .إن الدواء فقد خاصية التبلور بدون تفاعل كيميائي مع الناقل .هغير قابلة للذوبان في-لماءل محبة المحضرة بطريقة تبخير المذيبات باستخدام مواد صلب المتشتت السطحيال بواسطة تقنية تقنية التبخر بالمذيبات، الصلب المنتشر السطحي، دواء اإليباستين :المفتاحية الكلمات introduction solubility is a property of substance in a particular solvent. in quantitative terms, it is a concentration of dissolved solute in a saturated solution at a certain temperature. in qualitative terms, it means spontaneous interaction of two or more substances to form one phase, clear homogeneous molecular dispersion. dissolution is the process by which the solute dissociates in a solvent forming a molecular level, physically and chemically homogenous dispersion called a solution. solubility and dissolution are dissimilar concepts but are connected.most of the new drugs have poor aqueous solubility; thereby have a difficulty in formulating in drug delivery systems. 1corresponding author e-mail: ph.lina.salm@gmail.com received: 14/8/2020 accepted:14 /10 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp122-132 iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 123 therefore, the enhancement of solubility and dissolution rate of these drugs are important preformulation steps in the pharmaceutical product development research. in general, solubility difficulties are faced in the class ii and class iv of the biopharmaceutical classification system (bcs) where dissolution becomes the rate limiting step for the drug absorption (13). many techniques have been applied for improving the solubility of poorly soluble drugs such as salt formation, converting into prodrugs, micronization and nanonization, complexation, micelles, emulsions, solid-lipid nanoparticles, and solid dispersions. among these, solid dispersion (sd) is gaining tremendous significance to enhance solubility and dissolution of poorly soluble drugs. it can be defined as molecular mixture of poorly soluble drugs in hydrophilic carriers. these carriers play an important role in drug release according to their properties (4). surface solid dispersion (ssd) is another technique for dispersing one or more active ingredients on a water insoluble-hydrophilic carrier of extremely high surface area to achieve increased dissolution rates and bioavailability of insoluble drugs. when in contact with water, this carrier disperses immediately allowing the fast release of the drug. ssd can overcome some of the disadvantages of the conventional sds which prepared by water soluble carriers like tackiness and difficulty in handling of the product. many commonly used excipients like microcrystalline cellulose, silicon dioxide, sodium starch glycolate, potato starch and croscarmellose, have been used as carriers for preparing ssd (5, 6). ebastine (ebs) a piperidine derivative, is a nonsedating antihistamine with a long duration of action. chemical structure of ebs is shown in figure 1. it is a basic compound that contains tertiary amine group with pka 8.8 and partition coefficient of 7.64. this drug belongs to bcs class ii, thus when administered orally, it is not readily bioavailable (7, 8). the objective of this research was enhancing the solubility and dissolution rate of ebs by ssd technique, using different hydrophilic-water insoluble carriers at different ratios. figure 1. chemical structure of ebastine (9) materials and methods materials ebastine and croscarmellose sodium were purchased from hangzhou, hyperchem china. avicel® ph101, avicel® ph 102 and sodium starch glycolate (ssg) were gifted from pioneer pharmaceutical company, iraq. methods preparation of surface solid dispersion (ssd) of ebs ebastine ssds were prepared by solvent evaporation method using different hydrophilic carriers. specified amount of the drug was dissolved in methanol to obtain a clear solution. then after, an accurate amount of carrier corresponding to different drug: carrier w:w ratios were added to the drug solution (table 1). the formed suspension was continuously stirred using magnetic stirrer at room temperature till all the methanol evaporated. the dried ssd mass was pulverized and passed through sieve no. 230. then they were stored in a desiccator containing anhydrous calcium chloride as a drying agent. this agent was used to reduce the humidity in a desiccator producing a dry environment (at least 24 h) for analysis (10). table 1. composition of different ebs ssd formulas . formula number ebs:carrier w:w ratio ebastine (g) avicel® ph 101 (g) avicel® ph 102 (g) ccs (g) ssg (g) ebs1 1:1 0.5 0.5 ebs2 0.5 0.5 ebs3 0.5 0.5 ebs4 0.5 0.5 ebs5 1:2 0.5 1 ebs6 0.5 1 ebs7 0.5 1 ebs8 0.5 1 ebs9 1:5 0.5 2.5 ebs10 0.5 2.5 ebs11 0.5 2.5 ebs12 0.5 2.5 ebs13 1:10 0.5 5 ebs14 0.5 5 ebs15 1:15 0.5 7.5 ebs16 0.5 7.5 iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 124 preparation of physical mixture (pm) physical mixture was prepared for the selected ssd formula by mixing the powders geometrically in a glass mortar using spatula which was then passed through sieve no.230 then stored in the desiccator for further use (11). evaluation of ssd of ebastine determination of percentage yield the percent of yield of ssds was calculated by using the following equation (12): % of yield = 𝐖𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐒𝐒𝐃𝐬 𝐖𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐝𝐫𝐮𝐠+𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐜𝐚𝐫𝐫𝐢𝐞𝐫 × 𝟏𝟎𝟎 determination of drug content of ssd monitoring drug content uniformity is required for the control of drug quality and durability of the process. a precisely weighed quantity of ssd powder equivalent to 15 mg of ebs was weighed accurately and dissolved in 30 ml of methanol. the resulted mixture was filtered through a 0.45 µm membrane filter then suitably diluted with methanol. the absorbance of the solution was measured at 253 nm (13). experiments were repeated three times. the percentage of drug content in the ssd was counted by the following equation (14): 𝐃𝐫𝐮𝐠 𝐜𝐨𝐧𝐭𝐞𝐧𝐭 = 𝐏𝐫𝐚𝐜𝐭𝐢𝐜𝐚𝐥 𝐝𝐫𝐮𝐠 𝐜𝐨𝐧𝐭𝐞𝐧𝐭 𝐓𝐡𝐞𝐨𝐫𝐞𝐭𝐢𝐜𝐚𝐥 𝐝𝐫𝐮𝐠 𝐜𝐨𝐧𝐭𝐞𝐧𝐭 × 𝟏𝟎𝟎 determination of saturation solubility of ssd an excess amount of ebs and the prepared ssds was added to 10 ml of distilled water in plain tubes. the test tubes were tightly closed and continuously stirred on isothermal water bath shaker for 48 h at 25°c to get equilibrium. then, a millipore filter syringe of 0.45 µm was used for filtering the samples. the dissolved amount was determined by uv spectrophotometer at 256 nm (15). this study was done in triplicate. determination of hydration capacity according to the solubility study, one gram of the best carriers’ formulas was placed separately in 10 ml pre-weighed centrifuge tube. sufficient distilled water was added to make up the volume to 10 ml and the suspension was shaken manually for 5 min. the suspension was allowed to stand for 10 min and then by centrifugation for 15 min at 1000 rpm. then after, the supernatant was decanted. then a tube was reweighed, and hydration capacity was calculated by using the following equation (16): 𝐇𝐲𝐝𝐫𝐚𝐭𝐢𝐨𝐧 𝐜𝐚𝐩𝐚𝐜𝐢𝐭𝐲 % = 𝐖𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐭𝐮𝐛𝐞 𝐰𝐢𝐭𝐡 𝐬𝐞𝐝𝐢𝐦𝐞𝐧𝐭 −𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐞𝐦𝐩𝐭𝐲 𝐭𝐮𝐛𝐞 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐬𝐚𝐦𝐩𝐥𝐞 𝐨𝐧 𝐝𝐫𝐲 𝐛𝐚𝐬𝐢𝐬 × 𝟏𝟎𝟎 comparative in-vitro dissolution studies of pure and ssds of ebs the accurately weighed samples equivalent to 10 mg of ebs were placed in usp type ii dissolution apparatus. in-vitro dissolution study was done for pure drug, selected ssd formula and pm. this test was performed using 1000 ml 0.1n hcl with 100 rpm paddle speed at 37± 0.5 °c. 5 ml of sample was taken at certain time intervals 10, 15, 20, 30, 40, 50 and 60 min. and substituted with an equal volume of fresh dissolution medium. after filtering, the samples were analyzed spectrophotometrically at 257 nm. the drug dissolution profile was plotted as percentage of drug release versus time (17). fourier transform infrared (ftir) analysis the ftir spectra of pure drug, the best carrier, physical mixture pm and the selected ssd formulation were recorded by using ftir (iraffinity-1) spectrophotometer (shimadzu, japan). the samples were scanned over 4000–400 cm-1 frequency range (18). powder x-ray diffraction (pxrd) analysis the pxrd was used to detect any changes in the drug crystalline nature and to determine the possibility of any polymorphic changes of drug in the ssd formulation that may influence its dissolution. the x-ray diffractograms of the drug, the best carrier, pm and optimized ssd formulation were obtained by using x-ray diffractometer (shimadzu, japan) (19). the scanning speed was 5°/min over a 2θ range of 5–80°. statistical analysis the dissolution profiles were statistically checked using a similarity factor (f2). this factor have a value range between 0 and 100. the two dissolution profiles consider the same when f2 values more than 50 (50– 100); while f2 values less than 50 indicates that the compared profiles are not the same. the similarity factor (f2) was defined by the following equation (20). f2 = 50 × log {[1 + 1 𝑛 ∑ |𝑅𝑡 − 𝑇𝑡| 2 𝑛 𝑡=1 ] −0.5 × 100} where (n) is the number of dissolution time points. (rt),(tt) is the reference and test dissolution values as % at time (t), respectively. the other results were analyzed using paired samples t test (spss) and the level of significance was set at a p-value of 0.05: a p value ˃ 0.05 was considered to be nonsignificant. a p value ˂ 0.05 was considered to be significant results and discussion percentage yield high percentage yield was obtained from all the ssds formulas that range between 96-99% as shown in table 2. this indicates that there was no more than 4% loss in ssd obtained products which shows the suitability of the solvent evaporation method in the preparation of ebs ssds. drug content of ssds all tested formulations of ssds showed acceptable drug content ranging from 9799% w/w as observed in table 2 which was in agreement with usp requirements (90-110%) (21). these results indicated a homogeneous distribution of the prepared formulations. iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 125 table 2. percentage yield and drug content of prepared ssds formula number formula composition (drug: carrier w:w ratio) percentage yield (py %) drug content (w/w) (%) (mean ±sd), n=3 ssd1 (1:1ebs:avicel®ph101) 96.2 98.67±1.95 ssd2 (1:1ebs:avicel®ph102) 96 99.68 ± 4.15 ssd3 (1:1ebs:ccs) 99.9 99.82±2.40 ssd4 (1:1ebs:ssg) 99.3 98.39±5.87 ssd5 (1:2ebs:avicel®ph101) 98.4 98.38±1.39 ssd6 (1:2ebs:avicel®ph102) 95.5 99.80±3.44 ssd7 (1:2ebs:ccs) 98.7 98.24± 3.25 ssd8 (1:2ebs:ssg) 97 98.68±1.52 ssd9 (1:5ebs:avicel®ph101) 98.4 98.67±1.64 ssd10 (1:5ebs:avicel®ph102) 97.5 98.96±3.37 ssd11 (1:5ebs:ccs) 99.2 97.23 ±1.14 ssd12 (1:5ebs:ssg) 98.8 99.82±0.44 ssd13 (1:10ebs:avicel®ph101) 98.2 99.69±4.24 ssd14 (1:10ebs:ccs) 99.4 98.24±4.24 ssd15 (1:15ebs:avicel®ph101) 96.9 99.68± 3.07 ssd16 (1:15ebs:ccs) 98.5 98.39±6.64 saturation solubility of ebs ssds most of the prepared ssds of ebs showed a significant improvement in their water saturated solubility compared to that of pure drug as shown in table 3. this could be due to the hydrophilic nature of these carriers and surface adsorption of drug particles on these carriers in an extremely fine state of subdivision or molecular form. the resulting decrease in particle size and the increase in the interfacial area of contact between the drug particles and the solvent increased the solubility of the drug compared to the drug alone. in addition, the affinity between the hydrophilic inert carriers and the solvent facilitated the penetration of the solvent into the particles that resulted in further enhancement of the solubility of drug (22-24). this enhancement was affected by the type of the carrier and drug: carrier ratio. table 3 showed that, no improvement in the solubility of ebs was obtained by using 1:1 drug: carrier w:w ratio, but significant enhancement in drug solubility (p<0.05) at 1:2 ratio in the following sequence: ccs (ssd7) < avicel® ph101 (ssd5) < ssg (ssd8) < avicel® ph102 (ssd6) also, it was found that the saturated solubility of ebs increased significantly (p > 0.05) with an increase in drug: carrier ratio from 1:2 to 1:5 for all the used carriers with exception of ssg. a higher amount of the carrier resulted a larger amount of ebs dispersed over the carrier surface and hence improved ebs wettability and solubility (25). among carriers were ccs and avicel® ph101 exhibited highest improvement of solubility at 1:5 drug: carrier ratio in comparison with the others, since they produced 3.5, 2.9 folds increase in solubility, respectively. the small particle size of ccs ( ̴ 50µm), avicel® ph101( ̴ 50µm) compared to avicel ®ph 102 (100 µm( resulted in a larger surface available for adsorption of the ebs particles which would provide better drug wetting ability associated within them (10, 26, 27). on the other hand, ssg with very small particle size (38 µm), although it increased the saturated solubility compared to the pure drug, but there was no significant (p < 0.05) improvement in the saturated solubility by increasing the proportion of this carrier from 1:2 (ssd8) to 1:5 (ssd12). this was due to enough surface area of ssg for all drug particles at ratio 1:2 w:w drug: ssg so that, further increase in ssg at 1:5 ratio was of no benefit because all drug particles adsorbed and no more was available for extra adsorption (10, 28). therefore, ccs and avicel® ph101 were chosen preferably for ssds preparation to proceed with this study. the results of the solubility study showed that an increase in the ccs ratio (1:10 and 1:15) lead to ebs solubility improvement (6.3folds and 8.2folds, respectively as compared with the solubility of pure ebs). while the results of avicel® ph101 showed the higher improvement of drug solubility occur with 1:10 ratio ssd13 (3.4 folds) in comparison to 1:15 ratios ssd15 (3 folds). these results might be due to the differences in their hydration capacity. iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 126 table 3. the saturated solubility of ebs ssds prepared with several drug:carrier w:w ratios in distilled water at 25˚c. formula number formula composition (drug: carrier w:w ratio) saturation solubility mg/ml (mean ±sd ), n=3 pure ebs 0.0017±0.0004 ssd1 (1:1ebs:avicel®ph101) 0.0017±0.0008 ssd2 (1:1ebs:avicel®ph102) 0.0018±0.0009 ssd3 (1:1ebs:ccs) 0.0018±0.0001 ssd4 (1:1ebs:ssg) 0.0020±0.0001 ssd5 (1:2ebs:avicel®ph101) 0.0031±0.0001 ssd6 (1:2ebs:avicel®ph102) 0.0022±0.0004 ssd7 (1:2ebs:ccs) 0.0036±0.0003 ssd8 (1:2ebs:ssg) 0.0023±0.0001 ssd9 (1:5ebs:avicel®ph101) 0.0050±0.0001 ssd10 (1:5ebs:avicel®ph102) 0.0026±0.0002 ssd11 (1:5ebs:ccs) 0.0060±0.0001 ssd12 (1:5ebs:ssg) 0.0021±0.0002 ssd13 (1:10ebs:avicel®ph101) 0.0058±0.0003 ssd14 (1:10ebs:ccs) 0.0107±0.0006 ssd15 (1:15ebs:avicel®ph101) 0.0051±0.0001 ssd16 (1:15ebs:ccs) 0.0140±0.0001 hydration capacity hydration capacity test is an indicator of swelling power or for extent of solidliquid interaction. the carriers under this test were wetted by water and then evaluated for their water holding capacity. the capacity of ccs was found to be 73.92% which was superior to avicel® ph101 (34.95%). this result confirmed the previous results about the superiority of ccs over avicel® ph101 in enhancing the solubility of drug as it absorbed more water so it exhibited more wetting effect (29). comparative in-vitro dissolution studies the formulas ssd14 (1:10) and ssd16 (1:15) drug: ccs ratio were used to study the effect of ccs ratio on in-vitro dissolution profile of ebs, since the highest solubility was obtained by them. figure 2 demonstrated that both formulas improved dissolution rate in 0.1 n hcl in comparison to the pure drug. the dissolution profiles for both ratios (1:10 and 1:15 drug: carrier) were almost similar (f2=77.21) so that, no further enhancement in dissolution was obtained with an increased in carrier ratio. the dissolution enhancement effect might belong to high swelling, wicking and hydration capacities of the ccs particles that would prevent aggregation of the drug particles and facilitated dissolution (30). it was observed that drug: ccs ratio of 1:10 was sufficient to adsorb the drug and improve the dissolution process as that for 1:15 ratio (31). figure 2. effect of carrier ratio of ssd on the invitro dissolution of ebs in 0.1n hcl at 37±0.5°c. although both ssd14 and ssd16 highly improved the dissolution of ebs, but ssd16 produced more enhancement in solubility than ssd14 (8.2 folds versus 6.3 folds). so that, ssd16 (ebs:ccs 1:15) was selected to be the optimized ssd on the basis of highest drug solubility and improved dissolution of drug. as well as, the efficiency of ssd technique was demonstrated by comparing the dissolution profile of ssd16 with that of pm and of pure ebs as shown in figure 3. the profile showed that the dissolution rate of ebs in pm as well as in ssd was higher as compared to that of pure ebs. the similarity factor f2 was found to be equal to 9.02 and 40.56 in comparison of ssd16 formula and its pm to pure drug sequentially. the dissolution of pure ebs was found to be 25.81% within 60 min whereas, 44.17% of ebs was released iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 127 after 60min from the pm. this might be due to the hydrophilicity of ccs in the respective mixture which rendered this mixture hydrophilic and enhanced the wettability and solubility of drug as indicated from its saturated solubility which was found to be higher than pure drug (0.0103±0.0001 mg/ml versus 0.0017±0.0004 mg/ml) , yet it was significantly (p<0.05) lower than its corresponding ssd (29, 32, 33). on the other hand, the % release of ebs from the ssd16 was increased to 93.19% within 60 min. so that, ssd was an efficient technique to improve the dissolution rate of ebs which might be attributed to a reduction of its particle size and its deposition on the surface of a hydrophilic carrier (34). figure 3. comparative in-vitro dissolution profile of the pure ebs, ssd16 and physical mixture of ssd16 (pm) in 0.1n hcl at 37±0.5 °c. fourier transforms infrared spectroscopy (ftir) the ftir spectrum of pure ebs and ccs are shown in figures (4) and (5), respectively. the ftir spectrum of ebs exhibited characteristic bands at 3051 cm-1 for c-h stretching of the ring, 2943,2916, 2819 cm-1 for ch-stretch of ( ch3 and ch2), also strong and sharp band at 1678 cm -1 for ketonic carbonyl group (c=o stretch), 1454 cm-1 for aromatic c=c stretch of phenyl ring, and c-n stretching at 1269cm-1. these results were in agreement with the previous studies (35-37). the ftir spectrum of ccs exhibited characteristic bands at 3360 and 3240 cm-1 for alcoholic o-h groups, 2904 cm-1 for aliphatic c-h stretching of (ch2), and weak band at 1732, 1720 cm -1 for c=o of carboxylic groups. these results were in consistent with the previous studies (35, 38). the ftir spectra of pm and that of the selected ssd16 formula (figure 6 and 7) showed mostly the characteristic peaks of ccs, that overwhelmed the peaks of ebs, as ccs was mixed at a ratio of 1:15 (ebs:ccs). the only characteristic peak of ketonic carbonyl group (c=o stretch) for ebs was observed in the same position in both figures with low intensity due to dilution of drug with the high amount of ccs. the absence of new peaks in ssd16 spectrum confirmed absence of chemical interaction. figure 4 .ftir spectrum of ebs iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 128 figure 5. ftir spectrum of ccs figure 6. ftir spectrum of pm iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 129 figure 7. ftir spectrum of ssd16 powder x-ray diffraction (pxrd) the pxrd patterns of ebs, ccs, pm and ssd16 are shown in figures (8-11), respectively. the diffraction pattern of the pure ebs showed a highly crystalline nature, indicated by numerous intensive peaks at a diffraction angle of 2θ (5.84°, 11.54°, 18.68°, 19.06°, 19.39°) throughout the scanning range. these values approached the previously reported data (36, 39). on the other hand, the ccs diffractogram did not show sharp peaks, but only some peaks with low intensity at 18.41°, 22.9°, 43.95°, 64.29°, 77.38° related to its amorphous nature (40). the characteristic peaks identified in the ebs pxrd were not detected neither in pm nor in ssd16 pxrd patterns whereas, peaks corresponding to the ccs were still present in both of them, indicating the predominant effect of the high amount of the carrier in this formula. the broad shape with further decrease in intensity observed in ssd16 pxrd pattern compared to that of pm may be explained by the conversion of the drug to an amorphous form, which contributed to the enhancement of its solubility (41, 42). figure 8. pxrd diffractogram of ebs iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 130 figure 9. pxrd diffractogram of ccs figure 10. pxrd diffractogram of pm figure 11. pxrd diffractogram of ssd16 iraqi j pharm sci, vol.30(1) 2021 ebastine surface solid dispersion 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dissolution and bioavailability enhancement of gliclazide by surface solid dispersion using spray drying technique. indian j. novel drug deliv. 2012;4(2): 115-124. 42. charumanee s, okonoki s, sirithunyalug j. improvement of the dissolution rate of piroxicam by surface solid dispersion. cmu j. 2004; 3(2): 77-84. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs doi: https://doi.org/10.31351/vol27iss2pp55-65 55 enhancement of the solubility and dissolution rate of rebamipide by using solid dispersion technique (part i) muna y.ismail*, 1 and mowafaq m. ghareeb** *college of pharmacy, university of uruk , baghdad, iraq **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract solid dispersion is an attractive tool of pharmaceutical technology used to improve the physical properties of drugs, among these properties is the solubility of the drugs. rebamipide (rem) is used as potent antiulcer, mucoprotective drug, by stimulating the generation of prostoglandine enhanced mucosal protection. rem is a poorly soluble drug of class iv of biopharmaceutical classification system (bcs). in the present study, attempts were made to enhance solubility and dissolution rate of rem by solid dispersion technique. thirty six rem formulas were prepared as a solid dispersion using different polymers include pluronic f-127 (poloxamer 407), polyethylene glycol 6000 (peg6000), polyvinylpyrrolidon (pvp k30), and d-α-tocopheryl polyethylene glycol 1000 succinate (tpgs) at different drug: polymer ratios (1:9, 1:12, and1:15) by using different preparation methods include solvent evaporation, fusion, and kneading method. the prepared formulas were characterized regarding drug content, production yield, solubility study, dissolution study, ftir, dsc, pxrd, and sem. the results indicate that the used polymer show improvement in drug solubility in the following descending order; tpgs>pvp k30>peg 6000 > pluronic f-127 and the best drug: polymer ratio was 1:15 while best method was solvent evaporation. the optimum formula composes of drug: tpgs at ratio of 1:15 prepared by solvent evaporation shows 36.4 folds solubility enhancement compared to pure rem. the advance characterization of the selected formula indicates amorphousization of drug. it can be concluded that the solid dispersion technique is simple physical approach that can be followed to solve the problem of rem solubility using tpgs as hydrophilic carrier and best method is solvent evaporation method. keywords: rebamipide , solid dispersion, tpgs. تحسين قابلية الذوبان ومعدل االنحالل للريباميبايد بتقنية المنتشر الصلب )الجزء االول( **موفق محمد غريب و 1*،منى يحيى اسماعيل .كلية الصيدلة ، جامعة اوروك ، بغداد ، العراق * . فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ،العراق** الخالصة الصلب المنتشر هي أداة جذابة للتكنولوجيا الصيدالنية المستخدمة لتحسين الخصائص الفيزيائية لألدوية ، من بين هذه الخصائص هو الذوبان لألدوية. كعالج قوي للقرحة،يعمل حماية للبطانة المخاطية للمعدة من خالل تحفيز توليد لبروستوكالندين (rem) الريباميبايديستخدم (. bcsالريباميبايد هو دواء ضعيف الذوبان من الفئة الرابعة من نظام التصنيف الصيدالني الحيوي ) .الذي يعزز حماية الغشاء المخاطي أجريت محاوالت لتعزيز الذوبان ومعدل الذوبان للدواء بواسطة تقنية الصلب المنتشر.في هذه الدراسة f-127ونيك بلورتم تصييغ ست وثالثين صيغة للريباميبايد كصلب منتشر باستخدام البوليمرات المختلفة التي تشمل (poloxamer 407 والبولي ايثيلين جاليكول ،)6000 (peg6000 والبولي فينيل ، )( بيرلوريدونpvp k30 و ، )d-αtocopheryl polyethylene glycol 1000 succinate (tpgs( في نسب مختلفة من الدواء: البوليمر )1، و 1:12، 9: 1 : ء ا( باستخدام طرق تحضير مختلفة تشمل تبخر المذيبات ، االنصهار ، وطريقة العجن. تم تقييم الصيغ المعدة فيما يتعلق بمحتوى الدو15 .sem، و pxrd، و dsc، و ftirونسبةاإلنتاج ، ودراسة الذوبان ، ودراسة سرعةالذوبان ، و tpgs >pvp k30 >pegتشير النتائج إلى أن البوليمرات المستخدمة تظهر تحسن في ذوبان الدواء بالترتيب التنازلي التالي ؛ 6000 >f-127 pluronic بينما كانت أفضل طريقة هي تبخير المذيبات. الصيغة 15:1البوليمر وأفضل نسبة: كانت نسبة الدواء الى remضعف تحسين بالذوبان بالمقارنة مع 36.4أعدت بطريقة تبخر المذيبات واظهرت 15:1بنسبة tpgsالمثلى تتكون من الدواء: النقي. يشير التحليل المتقدم للصيغة المختارة إلى تحول الدواء الى اكثر امورفس. مكن االستنتاج أن تقنية الصلب المنتشرهي طريقة فيزيائية بسيطة يمكن اتباعها لحل مشكلة قابلية الذوبان للريباميبايد باستخدام ي tpgs .كحامل محب للماء ، وأفضل طريقة هي طريقة تبخر المذيب . سكسونيت 1000 كاليكول اثيلين بولي توكوفيرول، المنتشر الصلب ،الكلمات المفتاحية: الريباميبايد 1corresponding author e-mail: munayehia92@gmail.com received: 4/ 8 /2018 accepted: 2 / 10 /2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp55-65 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 56 introduction rebamipide has a chemical formula of [2 ( 4 – chlorobenzoylamino ) – 3 – [ 2 ( 1h ) – quinolinon – 4 – yl ] proponic acid], (rem) as shown in figure (1). rem is an amino acid analog of 2(1h) – quinolinone. it is a new mucoprotactive drug which was developed in japan for the treatment of peptic ulcer disease (pud) (1). figure 1. the chemical structure of rem(1) mechanisms for its therapeutic effect in pud involves stimulating the generation of prostaglandins, enhanced mucosal protection, removal of oxygen free radicals and inhibiting the production of inflammatory cytokines(2). clinical investigations have shown that it has marked effects on ulcer healing and helicobacter pylori (hp) adhesion. research has shown that the rem levels presented in the gastric mucosa and gastric mucus were a result of local penetration, and the local concentration allows it to exhibit a variety of antiulcer effects after oral administration(3) . rem is classified as a class iv based on the biopharmaceutical classification system, due to its low water solubility and permeability, the bioavailability of rem is under 10% in humans. to increase the low solubility of poor water soluble drug and improve its bioavailability, several strategies can be employed, such as size reduction, use of surfactants, ph adjustment, and complexation with cyclodextrines, emulsions and solid dispersion(4). solid dispersion technologies are used for improving oral absorption and bioavailability of bsc class ii and iv drugs, in solid dispersion drug disperse in the matrix generally a hydrophobic drug is dispersed in a hydrophilic matrix, which forms a solid dispersion. when the solid dispersion interacts with gastrointestinal fluid, the carrier or polymer which enhances solubility of drug it first dissolves and the drug release as fine colloidal particles. this result in enhanced surface area produces higher dissolution rate and bioavailability of poor water soluble drug(5). many polymers have been used for sd , like pluronic f-127 , polyethelen glycol (peg), polyvinylpyrrolidon (pvp). to enhance the solubility and then the dissolution of the drugs (6). materials and methods materials rebamipide was obtained from apopharm, china, pluronic f-127 from sigma, usa, peg 6000 from chemifine chemicals. mumbai, india , pvp k30 from hyper chem, china and (tpgs) from hyper chem, china , while methanol from sigma-aldrich co., germany. all other reagents were of analytical grade. preparation of remsolid dispersionsolvent evaporation method rebamipide solid dispersion were prepeared by solvent evaporation method. using different carriers (pluronic f-127, peg 6000, pvp k30 and tpgs) in different ratios as shown in table (1), the drug and carriers; were taken separately in different ratios(1:9,1:12and1:15) wt:wt and transferred in a beaker containing appropriate amounts of methanol (solvent).by using magnetic stirrer,then the two solutions was mixed together on magnetic stirrer. the solvent was removed by leaving it for 24 hr. at room temperature (25-30 °c).the dried solidifing mass were scraped, crushed and grinded in mortar and pastle to pass through the sieve no 20 and strored in aglass umber container in a deiccator for subsequent study(7). fusion method in fusion method, accurate amount of carriers and drug in the different ratio (1:9, 1:12, 1:15). the carrier was first melted in petri-dish at the melting point of each polymer and the drug was dispersed in the molten mixture with constant stirring then cooled it. the dried mass was crushed and grinded in mortar and pastle to pass through sieve no.20 and store in desiccator (8). a modification of the above method when used pvp k30, have been prepared by closed melting point method. this method includes the addition of water to the carrier and heated, the drug dispersed in the heated mixture with constant stirring then cooled it. the dried mass was crushed and grinded in mortar and pastle to pass through sieve no.20 and store in desiccator (9). kneading method an accurate weighted quantity of drug and corresponding water soluble carrier (table 1), are mixed together in glass mortar, and triturate for 30 minutes, with drop by drop distilled until get the structure paste, the paste was spread over suitable petri-dish and dried in oven at 40°c for 24 hours. (except in tpgs polymer, the temp. was 25°c. the dried mass was crushed and grinded in mortar and pastle to pass through sieve no.20 and store in desiccator (10). iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 57 table 1. composition of rem and carriers using solid dispersion method. formulation code water soluble carriers drug: carriers ratio (wt:wt) methods of preparation sd 1 pluronic f-127 1:9 solvent evaporation sd 2 1:12 solvent evaporation sd 3 1:15 solvent evaporation sd 4 1:9 fusion sd 5 1:12 fusion sd 6 1:15 fusion sd 7 1:9 kneading sd 8 1:12 kneading sd 9 1:15 kneading sd 10 peg 6000 1:9 solvent evaporation sd 11 1:12 solvent evaporation sd 12 1:15 solvent evaporation sd 13 1:9 fusion sd 14 1:12 fusion sd 15 1:15 fusion sd 16 1:9 kneading sd 17 1:12 kneading sd 18 1:15 kneading sd 19 pvp k 30 1:9 solvent evaporation sd 20 1:12 solvent evaporation sd 21 1:15 solvent evaporation sd 22 1:9 fusion sd 23 1:12 fusion sd 24 1:15 fusion sd 25 1:9 kneading sd 26 1:12 kneading sd 27 1:15 kneading sd 28 tpgs 1:9 solvent evaporation sd 29 1:12 solvent evaporation sd 30 1:15 solvent evaporation sd 31 1:9 fusion sd 32 1:12 fusion sd 33 1:15 fusion sd 34 1:9 kneading sd 35 1:12 kneading sd 36 1:15 kneading characterization of remsolid dispersion determination of percent production yield of prepared remsolid dispersion the percent of production yield (py %) of prepared rem solid dispersion was determined by calculating the ratio of actual weight of the obtained solid dispersion on the theoretical weight of solid dispersion using the following equation(11): 𝑃𝑌% = 𝐴𝑐𝑡𝑢𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑖𝑑 𝑑𝑖𝑠𝑝𝑒𝑟𝑠𝑖𝑜𝑛 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑜𝑙𝑖𝑑 𝑑𝑖𝑠𝑝𝑒𝑟𝑠𝑖𝑜𝑛 × 100 solubility studies of rem solid dispersion an excess amount of pure rem and prepared rem-solid dispersion were placed in contact with ten milliliters of water in closed tight container that shakes continually for 72 hr. in thermocontrol water bath at 25°c. after the removal from the shaker water bath, the tubes put in centrifuge for 10 min at 4000 rpm. the solutions were filtered through a 0.45 µm filter membrane, and the concentration of rem was determined by uv – spectrophotometer at λmax.227 nm wavelength the study was done in triplicate (12). iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 58 drug content analysis the rem solid dispersion equivalents to 100mg of rebamipid were taken and dissolved in 100 mls. of methanol, then filtered by using 0.45µm filter membrane. the filtrate was suitably diluted with methanol; the drug solution was analyzed by using uvspectrophotometer at λ max. 227 nm wavelengths. the percentage of drug content in solid dispersion was calculated by using the following equation: (13) 𝐷𝑟𝑢𝑔 𝑐𝑜𝑛𝑡𝑒𝑛𝑡% = 𝐴𝑐𝑡𝑢𝑎𝑙 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑅𝐸𝑀 𝑇ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑤𝑖𝑒𝑔ℎ𝑡 𝑜𝑓 𝑅𝐸𝑀 × 100 in-vitro dissolution studies the in-vitro release of rem in the solid dispersion was determined and compared with pure rem drug by using usp xxii rotating paddle apparatus (ii). rem-solid dispersion equivalents to 100 mg of pure drug were dispersed in dissolution medium surface. the dissolution medium employed for drug release study was 900 milliliters. of 0.1n hcl, maintained at 37.5 ±0.5°c by means of thermostatic water bath and under shaking provide by the paddle at 100 rpm (14). five milliliters were withdrawn at (1,3,5,10,15,30,45,60,90,120) minutes time interval for two hours, and each withdrawn sample was replaced with an equal volume of fresh 0.1 n hcl dissolution medium as soon as to maintain sink condition. the samples were filtered through 0.45µm filter membrane and analyzed by uv-spectrophotometer at λmax 227 nm wavelengths (15). factors affecting dissolution behavior of rem from solid dispersion the effect of different polymer types (pluronic f-127, peg 6000, pvp k30 and tpgs) on dissolution behavior of rem from solid dispersion was studies on the formulas sd1, sd10, sd19 and sd28. the effect of different drug: polymer ratio on dissolution behavior of rem from solid dispersion was studies on the formulas (sd1sd3), (sd10-sd13), (sd19-21), (sd28sd30), which they are belong to the solvent evaporation method. the effect of preparation method on dissolution behavior of rem from solid dispersion was studies on the formulas (sd1, sd4, sd7), (sd10, sd13, sd16), (sd19, sd22, sd25) and (sd28, sd31, sd34). the solubility and in-vitro dissolution profile were used for selecting the best formula which will be subjected to further analysis. evaluations of selected solid dispersion formula fouier transforms infrared spectroscopy (ftir) the ftir of pure rem and selected formula were performed to investigate drugpolymer the interaction. the samples were compressed with potassium bromide as a disc, and carried out by ftir shimadzu 8000 japan; the scanning range the spectrum obtained was in between the wave number of 4000400 cmˉ¹ (16). differential scanning calorimeter (dsc) thermal characteristic of the selected formula and tpgs characterized by an automatic thermal analyzer system (shimadzu, dsc-60, japan). approximately 5 mg of samples were placed in none hermetically aluminum pan and heated at rate 10°c/ min over temperature 25°c to 400°c(17). powder x-ray diffraction (pxrd) powder x-ray diffraction is instrument used to study the molecular structure of crystalline substance such as drug and optimum formula. the degree of crysallinity was determined by (xrd-6000, shimadzu, japan 220v/50hz). xray diffractometer with cu-ka radiation at 40 kv and 30 ma. samples were scanned over a 2θ range of 30-80 ° at step size of 0.02° (18). scanning electron microscopy (sem) scanning electron microscope of the selected formula and pure drug (rem) were analyzed using (shimadzu, japan); in order to examination the external morphology and shape of the particle. the samples coated with layer of gold at room temperature under argon gas. the sem was operated at a high vacuum with accelerating voltage of 5-15 kv. secondary electron images were recorded digitally at higher magnification (19). statistical analysis the results of the experiments were given as a mean for triplicates samples ± stander deviation and were analyzed according to oneway analysis of variance (anova) test level of (p<0.05) was considered to be statistically significant, and non-significant value with (p>0.05). results and disscution determination of rem saturated solubility the solubility of rem in different media (water, 0.1 n hcl, phosphate buffer ph 6.8) at 25 °c was determined. the results revealed that the solubility of rem is very poor in 0.1 n hcl ph (1.2), which may be attributed to it is acidic nature of the drug, so increasing the ph of the solvent using phosphate buffer lead to increase iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 59 the solubility from 1.8µg/ml (ph 1.2) to 1320 µg/ml at ph6.8 (730 x fold). and the solubility of rem in water was 23.9µg/ml as shown in table (2). table 2. saturation solubility of rem in different media at 25°c under normal atmospheric pressure. solvent solubility (μg/ml) mean± sd* description forms distilled water 23.9± 0.1 practically insoluble 0.1 n hcl 1.8± 0.1 practically insoluble phosphate buffer 6.8 1320± 1.15 slightly soluble *sd stander deviation from mean, n=3 on the other hand, the solubility of rem at phosphate buffer ph6.8 (1320µg/ml) is still with a range of slightly soluble drug category. that it’s why the mission of the study; to enhance the solubility of rem . characterization of rem solid dispersion determination of percent production yield of prepared rem-solid dispersion the rem yield percentage was calculated to investigate best method of preparation of solid dispersion granules after drying, grinding and sieved through mesh size no. 20. the solid dispersion granules were valid with percentage yield 7699%, mainly for solvents and fusion methods. this behavior may be attributed to the best entrapment or best solvation of rem particles by polymer used using melted carriers (fusion), or solvent vehicle (evaporation) methods. determination of rem content in prepared solid dispersion moreover the content of rem in solid dispersion was found in a range of 96.12% 100.2% in all prepared formulations, which is in the same range of u.s. pharmacopoeia requirements (90-110%) the above results indicated a uniform distribution of rem particles within polymers used in all prepared formulas (20). determination of saturated solubility of rem in solid dispersion the solubility study of rem as a pure powder and as a drug loaded in solid dispersion formulas was carried out using distillated water maintained at 25°c temperature. figure (2), (3) and (4) demonstrated that best result obtained, when tpgs used as a solid dispersion carriers compared with pvp k30, peg 6000 and pluronic f-127 polymers the saturated solubility were 874.6 µg/ml, 180.1µg/ml, 59.6µg/ml, and 61.7 µg/ml., respectively using (1:15) drug polymer ratios, compared with 23.9 µg/ml for pure rem powder the last behavior of above polymers is significant (p<0.05), and increasing the solubility of rem may be referred to hydrophilic nature of all polymer used, besides hydrogen bonding formation between rem and carrier polymers to enhance the solubility (21). on the other hand, tpgs was found as the best polymer carriers for rem with enhanced saturated solubility by 36.4 x fold compared with a pure rem. figure 2. effect of carrier type of rem solid dispersion at ratio (1:9) on the solubility of rem in distilled water at 25°c (results are expressed as mean, n=3) figure 3. effect of carrier type of rem solid dispersion at a ratio (1:12) on the solubility of rem in distilled water at 25°c (results are expressed as mean, n=3). figure 4. effect of carrier type of rem solid dispersion at a ratio (1:15) on the solubility of rem in distilled water at 25°c (results are expressed as mean, n=3). iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 60 factors affecting dissolution of rem within solid dispersion particles effect of polymer type figure (5) demonstrated that best results obtained for rem dissolution when tpgs polymer used among other hydrophilic polymers. rem: polymer ratio (1:12) using solvent evaporation method demonstrated, that 70% of rem released in aqueous medium (p<0.05) was increased compared with 14%, 4%, and 2% for pvp k30, peg6000 and pluronic f-127 polymers, respectively after 90 minutes. this result was attributed to that tpgs has amphiphilic structure of lipophilic alkyl tail and hydrophilic polar head that lowering surface tension between rem and solvent, improvement of wetting characteristics and micellar solubilization of drugs (22). in a focus of using surfactants like tpgs, the surface activity and self-emulsifing properties was gave the third generation type solid dispersion that’s have highest degree of solubilization together with improving drug wet ability and spread ability by decreasing the interfacial tension between the drug particles and the aqueous medium, and also the amorphization of the crystalline drugs and avoiding recrystallization that can occur after contact with aqueous medium can further enhances the saturation solubility(23). meanwhile, the dissolution profile for rem–pvp k30 solid dispersion (sd19) had maximum percentage of 15.1% within 120 minutes, although pvp k30 inhibits crystal formation of drugs and resulting amorphous nature of drug in the solid dispersion and enhance the solubility of rem, but still lower than that of tpgs use (24). figure 5. effect of polymer type of remsolid dispersion prepared by solvent evaporation method with drug: polymer ratio 1:12 on the release profile of rem in 0.1 n hcl dissolution medium at 37°c (results are expressed as mean, n=3). effect of carrier ratio the effect of different drug: polymer ratio on the dissolution behavior of rem solid dispersion was studied, as shown in figure (6). the figure represents the percent release profile of rem from solid dispersion of tpgs that prepared by solvent evaporation method. it was observed that as the ratio of tpgs increased in formulas (sd28-sd30), the drug release from solid dispersion was increased significantly (p<0.05). formula sd30 appeared to have the highest the cumulative percent of rem release of 100% comparing to sd 29 of 47.8% and sd28 of 35.3% after 30 minutes. the solid dispersions prepared with higher ratios of hydrophilic polymer could be offer more available space for surrounding of hydrophobic rem particle resulted in rapid hydration of drug molecules and consequently better wet ability and enhancement in the dissolution. this may be attributed to tpgs melts and dissolves in the dissolution medium of 37°cquickly and acts as an emulsifier of hydrophobic drugs (25). moreover, the transformation of crystalline nature of pure drug into the amorphous form as affirmed by dsc and pxrd result facilitates higher drug release rate over the pure drug (26). figure 6. effect of rem: tpgs ratio on cumulative percent drug release profile from remsolid dispersion prepared by the solvent evaporation method in 0.1 n hcl dissolution medium at 37°c. (results are expressed as mean, n=3) effect of preparation methods the effect of preparation method on the dissolution behavior of rem from prepared drugsolid dispersion was studied. figure (7) shows the cumulative percent released of rem from the formulas contain tpgs in a ratio 1:15 that prepared by solvent evaporation, fusion and kneading methods, as in sd30, sd33, and sd36. the results of dissolution of rem for individual samples (pure rem, solvent evaporation, fusion, and kneading methods) were noted, the cumulative percent of rem iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 61 released after 15 minutes was 2.3%, 96.3 %, 75.3% and 53.2%, respectively. as shown in figure (7). figure 7. effect of preparation methods of rem-tpgs solid dispersion at a ratio (1:15) in 0.1 n hcl dissolution medium at 37°c. (results are expressed as mean, n=3) the percent of rem release greatly improved with solvent evaporation method than fusion, kneading methods and pure rem as a control , this can be attributed to the fact that solid dispersion prepared by solvent evaporation method result in more uniform and homogenous distribution of rem in hydrophilic tpgs polymer matrix as compared with other methods of preparation(27). moreover, when binary system comes in contact with an aqueous dissolution medium, the hydrophilic carrier dissolves and results in precipitation of embedded drug into fine particles, which increase the exposed surface area of the drug for the available dissolution surface (28). selection of the best formula the selection of the best formula was depended on the solubility study and the cumulative dissolution profile of rem from solid dispersion granules. it could be concluded that the sd30 which contains remtpgs solid dispersion in a ratio 1:15 that prepared by solvent evaporation method was the best formula concerning higher solubility and better dissolution rate percent. therefore, it was subjected into further in-vitro evaluations study. evaluation of selected formula fourier transforms infrared spectroscopy (ftir) the ftir spectrum for the pure rem, figure (8) showed the characteristic peaks of the drug at 3276.47 cm¯¹ which are assigned to the n-h stretching vibration and 2938cm¯¹ due to c-h stretching of aromatic hydrocarbons, 1725.98 cm-1 due to c=o stretching of carboxylic acid and c=o of amide stretching at 1643.05 cm-1 and 1338.3 cm-1 due to c-n stretching, and 759.82 cm-1 due to c-h bending vibrations of aromatic ring (29). figure 8. ftir spectrum of rem the ftir spectrum of selected formula (sd30) was shown in figure (9). it was examined and matched with those of ftir of rem and tpgs spectrum separately. the obtained spectra of selected formula sd30 revealed specific bands at 1644.02 cm-1 and 1736.58 cm-1 for carbonyl groups in amide group and carboxyl functional group respectively, which demonstrated that there is no interaction between rem and tpgs when they incorporated as solid dispersion. on the other hand, another bands were noticed at 2880.17 cm-1 and weak band at 3210 iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 62 cmˉ¹ for both c-h stretching for aromatic ring and n-h (amine) stretching, respectively, that declare no interaction between rem and tpgs carrier in the sd30 formula(30). figure 9. ftir spectrum of selected formula of rem-tpgs (sd30) differential scanning calorimeter (dsc) dsc thermo grams of rem and tpgs are presented in figures (10) and (11) which showed a sharp endothermic peak at 307.06°c and 40.68°c respectively, they revealed the crystal forms nature of drug and carrier. figure (12) represents the dsc thermo gram of rem-tpgs solid dispersion prepared by solvent evaporation method, the presence of single endothermic peak in thermo gram of sd30 at 39.69°c around the polymer melting point. while the absence of endothermic peak of pure drug at 307.06°c in solid dispersion thermo gram could be due to the fact that the drug might transform from its crystalline form to amorphous form in the solid dispersion (sd30) formulation which can be further supported by pxrd (31). 100.00 200.00 300.00 400.00 temp [c] -10.00 -5.00 0.00 5.00 mw dsc 307.06x100c figure 10. dsc thermogram of rem 0.00 100.00 200.00 300.00 temp [c] -25.00 -20.00 -15.00 -10.00 mw dsc 40.68x100c figure 11. dsc thermogram of tpgs 0.00 100.00 200.00 300.00 400.00 temp [c] -5.00 0.00 mw dsc 39.69x100c figure 12. dsc thermogram of selected formula of rem-tpgs (sd 30) iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 63 powder xray diffraction (pxrd) the x-ray diffraction analysis was carried out to confirm the change in the crystalline nature of the drug in solid dispersion and pure form. the x-ray diffraction analysis of rem and solid dispersion are given in figures (13) and (14) respectively. the drug characteristics peaks were observed at 21.7°, and 27.85° at 2θ value with intensity 821, and 736 respectively, the x-ray diffraction pattern of rem-tpgs solid dispersion (1:15)peaks were observed at 21.7°at 122 and no characteristic peaks at 27.85°were obtain. the peak intensities were reduced, indicating the decreased in drug crystal structure, and converted to amorphous one, upon dispersion by solvent evaporation method (32). figure 13. xray diffraction (pxrd) pattern of pure rem figure 14. xray diffraction (pxrd) pattern of selected formula of remtpgs (sd30) scanning electron microscopy (sem) sem pictures of pure rem and selected formula (sd30) are presented in figure (15) at 2000 x magnification. the pure rem showed the crystalline appearance , while in sd30 showed irregular shaped glassy appearance in addition to size reduction and embedment. the smaller particle size as compared with pure drug size lead to great the wetted area, and hence the better the solubility (33). iraqi j pharm sci, vol.27(2) 2018 solid dispersion of rebamipide with tpgs 64 conclusions based on the results obtained from the present study, it can be concluded that the poor solubility of rem (class iv drug) was successfully enhanced using solid dispersion technique and tpgs with (1:15) ratio (sd30) formula appeared to be the best carrier compared to other hydrophilic polymers. references 1. pharmacopoeia j. society of japanese pharmacopoeia. amended chapters. 2007;35(35.2):7. 2. kawano y, ishii n, shimizu y, hanawa t. development and characterization of a suspension containing nanoparticulated rebamipide for a mouth wash for stomatitis. journal of pharmaceutical science and technology, japan. 2017;77(2):104-15. 3. shi y, zou m, an y, ji z, gao p, cheng g., a potent preparation method combining neutralization with microfluidization for rebamipide nanosuspensions and its in vivo evaluation, drug development and industrial pharmacy. 2013;39(7):9961004. 4. 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sathali aa, jayalakshmi j. enhancement of solubility and dissolution rate of olmesartan medoxomil by solid dispersion technique, journal of current chemical and pharmaceutical sciences 2013;3(2):123-134. 33. ansari mt, hussain a, nadeem s, majeed h, saeed-ul-hassan s, tariq i, mahmood q, khan ak, murtaza g. preparation and characterization of solid dispersions of artemether by freeze-dried method, biomed research international. 2015;2015:1-11. evaluation of metformin + sitagliptin versus metformin + glibenclamide on glycemic control in iraqis type 2 diabetic patients iraqi j pharm sci, vol.21(2) 2012 metformin + sitagliptin versus metformin + glibenclamide 69 evaluation of metformin + sitagliptin versus metformin + glibenclamide on glycemic control in iraqis type 2 diabetic patients hassan m. al-temimi.* , kassim j.al-shamma** ,1 and salim m.alrubaie*** * baghdad teahing hospital,medical city, baghdad ,iraq ** department of clinical pharmacy ,college of pharmacy, university of baghdad, baghdad, iraq *** college of medicine, university of baghdad, baghdad , iraq abstract combination therapy with a dipeptidyl peptidase–4 inhibitor and metformin or metformin+ glibenclamide results in substantial and additive glucoselowering effects in iraqis patients with type 2 diabetes mellitus . this study evaluated the glycemic control by using two groups of combinations of drugs metformin + glibenclamide and metformin + sitagliptin in baghdad teaching hospital / medical city. 68 t2dm patients and 34 normal healthy individuals as control group were enrolled in this study and categorized in to two treatment groups. the group 1 (34 patients ) received ( metformin 500 mg three times daily + glibenclamide 5 mg twice daily ) and the group 2 (34 patients) received (metformin 500 mg three times daily + sitagliptin 100 mg once daily ). from each patients 5 ml of blood was obtained by veinpuncture and the serum was separated and used for estimating plasma glucose level (fpg,ppg) and hba1c.the mean fasting plasma glucose and postprandial plasma glucose significantly lower for group 2 patients for 3 and 6 months of treatment (129.02 ± 1.96 and 118.4 ± 1.33), (159.38 ± 4.72 and 123.88 ± 2.41 mg / dl) respectively for fasting plasma glucose and postprandial plasma glucose respectively than in group 1 patients (150.76 ± 3.97 and 127.79 ±2.52) ,(173.25 ± 7.99 respectively and 140.67 ± 4.66 mg / dl) for fasting plasma glucose (fpg) and postprandial plasma glucose (ppg) respectively. the mean hba1c % significantly lower for group 2 patients for 3 and 6 months of treatment (6.12 ± 0.091and 5.83 ± 0.083 ) respectively compared to group 1 patients (7.1 ± 0 .63 and 6.81 ± 0.12 ). in conclusion , the combination of metformin + sitagliptin improved fasting plasma glucose , postprandial plasma glucose & hba1c % in comparison with metformin + glibenclamide combination. ker words : sitagliptin, metformin , glibenclamide ,type 2 diabetic patients. ن فعالٍة الوجوعة العالجٍة )الوتفورهٍن + الكلبنكلوٍد( ضد فعالٍة الوجوعة العالجٍة ٍتقٍ )الوتفورهٍن + السٍتكلبتٍن ( على نسبة السكر بالدم لورضى عراقٍٍن هصابٍن بورض السكري هن النوع الثانً **قاسن جلٍل الشواع، *حسن هحود عباس التوٍوً ،1 *** بٍعًسالن هحسن الرو تغذاد ، انعشاق يذٌُح انطة ،،يسرشفى تغذاد انرعهًًٍ * كهٍح انظٍذنح ، ظايعح تغذاد ، تغذاد ، انعشاقفشع انظٍذنح انسشٌشٌح ، ** ، تغذاد ، انعشاق ظايعح تغذاد ،كهٍح انطة *** نخالطحا ٍٍ + انكهثُكهًاٌذ( ذؤدي إنى ذأشٍشاخ إػافٍح ترمهٍم َسثح انسكش تانذو انًعًىعح انعالظٍح ) انًرفىسيٍٍ + انسٍراكهثرٍٍ ( او)انًرفىسي يذٌُح انطة وذحد أششاف طثٍة \نًشػى عشالٍٍٍ يظاتٍٍ تذاء انسكشي يٍ انُىع انصاًَ. هزِ انذساسح أظشٌد فً يسرشفى تغذاد انرعهًًٍ نرمٍى إيكاٍَح انسٍطشج 3123و حرى آراس 3122ساسح يٍ ذًىصاخرظاص وتًىافمح انًشػى إػافح إنى يىافمح انعهاخ انًخرظح واٌ فرشج انذ يشٌغ(ٌعاَىٌ انًشع وذى يماسَرهى ب 76عهى َسثح انسكش تانذو ,َسثح انذهىٌ , انكالٌكٍرذ هًٍىكهىتٍٍ وكزنك صٌادج كرهح انعسى. ذى اخرٍاس) 65-51يشٌغ ذشاوحد أعًاسهى تٍٍ 45األونى :( شخض يٍ األطحاء نغشع انًماسَح. ذى ذىصٌع انًشػى إنى يعًىعرٍٍ:انًعًىعح 45) يهغى يشذٍٍ 6يهغى شالز يشاخ ٌىيٍا + انكهثُكهًاٌذ 611( . إٌ هزِ انًعًىعح ذسرخذو ) انًرفىسيٍٍ 1,67± 63. 6سُح ) انًرىسط = ذسرخذو انًعًىعح هزِ ( . إٌ 1,5 ± 63 . 55) انًرىسط = سُح 65-55 تٍٍ أعًاسهى يشٌغ ذشاوحد 45انصاٍَح : انًعًىعح ٌىيٍا(. كاٌ انًعذل انحساتً نُسثح انسكش تانذو فً حانح انظٍاو وتعذ يهغى ٌىيٍا(. 211يهغى شالز يشاخ ٌىيٍا +انسٍراكهثرٍٍ 611) انًرفىسيٍٍ انذساسح كًماسَح إنى انًعًىعح األونى.انصاٍَح الم تشكم يهحىظ تعذ شالشح و سرح اشهش يٍ تذآ نهًعًىعح تانُسثح انطعاو ذُاول يٍ ساعرٍٍ يم ( تانُسثح انى َسثح انسكش تانذو فً حانح انظٍاو وتعذ \يهغ 3.5± 234.6و 5.34±265.46( و)2.4± 226.5و 235.3±2.57) ( و 3.63± 234.45و 4.54±261.47ساعرٍٍ يٍ ذُاول انطعاو وتانرراتع.إيا تانُسثح إنى انًعًىعح األونى فكاَد انُرائط ) يم ( تانُسثح إنى َسثح انسكش تانذو فً حانح انظٍاو وتعذ ساعرٍٍ يٍ ذُاول انطعاو وتانرراتع.كًا تٍُد \يهغ5.77± 251.74و 244.36±4.55) ٍ تذآ ( الم تشكم يهحىظ تانُسثح انى انًعًىعح انصاٍَح تعذ شالشح و سرح اشهش ي% hba1cانذساسح اٌ انُسثح انًؤٌح نهكالٌكٍرذ هًٍىغهىتٍٍ) 74±.4.2ايا تانُسثح إنى انًعًىعح األونى )±(6.64و± 7.23انذساسح كًماسَح إنى انًعًىعح األونى.حٍس كاَد انُرائط نهًعًىعح انصاٍَح ) ًٌكٍ االسرُراض اٌ انًعًىعح انعالظٍح يع ) انًرفىسيٍٍ + انسٍراكهثرٍٍ ( لذ شهذخ ذحسُا يهحىظا يٍ حٍس انسٍطشج عهى َسثح (.23±7.62و يماسَح انًعًىعح انعالظٍح يع انًرفىسيٍٍ + انكهثُكهًاٌذ. كًا َالحع hba1c)انسكش تانذو وكزنك انُسثح انًؤٌح نهكالٌكىكهٍرذ هًٍىغهىتٍٍ ) ذأشٍش انًراتعح وانرعهًٍاخ يٍ لثم فشٌك انعًم حٍس كاٌ نها انرأشٍش ترحسٍ انُرائط نكال انًعًىعرٍٍ. . هرض السكري هن النوع الثانً ، ٌداالكلبنكلو ،الوتفورهٍن،لبتٍن كاالسٍت الكلوات الوفتاحٍة : 1 corresponding author email :drkassim_alshamaa@yahoo.com. received : 14/5/2012 accepted : 27/6/2012 iraqi j pharm sci, vol.21(2) 2012 metformin + sitagliptin versus metformin + glibenclamide 70 introduction diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia. it is associated with abnormalities in carbohydrate, fat, and protein metabolism and results in chronic complications including microvascular, macrovascular, and neuropathic disorders. although the prevalence of type 2 dm increases with age, (1) the disorder is increasingly being recognized in adolescence. much of the increase in adolescent type 2 dm is related to an increase in adiposity and sedentary lifestyle, in addition to an inheritable predisposition. (2) the increase in insulin resistance with weight gain is directly related to the amount of visceral adipose tissue. (3,4) the classical symptoms of diabetes are polyuria (frequent urination), polydipsia (increased thirst) and polyphagia (increased hunger). (5) hba1c measurements are the gold standard for following long-term glycemic control for the previous 2 to 3 months (6) . until 1995, only two options for pharmacologic treatment were available for patients with diabetes; sulfonylurea (for type 2 dm only) and insulin (for type 1 or 2). since 1995, a number of new oral agents, injectables, and insulins have been introduced in therapy. currently, six classes of oral agents are approved for the treatment of type 2 diabetes: α-glucosidase inhibitors, biguanides, meglitinides, peroxisome proliferator-activated receptor b-agonists (which are also commonly identified as thiazolidinediones [tzds] or glitazones), dpp-iv inhibitors, and sulfonylurea. it is now known that two hormones, glucagon-like peptide-1 (glp-1) and glucose-dependent insulin-releasing peptide (gip), are responsible for more than 90% of the increased insulin secretion seen in response to an oral glucose load. in patients with type 2 diabetes glp1 levels are reduced whereas gip levels are increased. sitagliptin works to competitively inhibit the enzyme dipeptidyl peptidase – 4 (dpp-4). this enzyme breaks down the incretins glp-1 and gip,gastrointestinal hormones released in response to a meal by preventing glp-1 and gip inactivation, the are able to the increase secretion of insulin and suppress the release of glucagons by pancreas (7) . the aim of this study is to evaluate effects of combination of metformin + sitagliptin versus the effects of combination of metformin + glibenclamide on fasting plasma glucose, postprandial plasma glucose and hba1c percentage in iraqis type 2 diabetic patients. subjects and methods this study was carried out at baghdad teaching hospital / medical city & the national diabetes center for treatment and research at al-mustansuriyah university and the private clinic of consultant physician during the period of july 2011-march 2012 .the study was conducted on ( 100 ) iraqi type 2 diabetics only 68 patients completed the course of study successfully . these patients were recruited into the following groups : group (1) : includes 34 patients tested at zero time and after 3 months and 6 months.the patients were already treated by metformin 500mg three times daily & glibenclamide 5 mg twice daily. group (2) : includes 34 patients tested at zero time and after 3 months and 6 months . the patients were previously treated by metformin 500mg three times daily and sitagliptin 100 mg once daily 3-6 months before start the study and they continue on this regime of treatment . the age of patients for group ( 1 ) ranged from 40 – 59 years (52.5 ± 0.86 ), of them 20 patients (58.8 %) were male and 14 patients (41.2 %)were female .the age of patients for group ( 2 ) ranged from 44 – 59 (52.44 ± 0.9),of them 20 patients ( 58.8 % ) were male and 14 patients ( 41.2 % )were female. diagnosis was made by consultant endocrinologist; for patients as having t2dm depending on patients history ,clinical examination laboratory investigations and vital signs. for the purpose of comparison ,34 control subjects were enrolled. the age of control for group (3) ranged from 44 – 59 (52.44 ± 0.9),of them 20 patients (58.8 %) were male and 14 patients ( 41.2 % )were female.patients were excluded from this study as having the following criteria : cns disease, renal dysfunctions, liver dysfunction, pregnancy with diabetes ,concomitant endocrine disease & inflammatory disease. collection of blood sample from each patients ,5 ml of blood was obtained by veinpuncture, for fasting and hba1c.another 5 ml was withdrawn for postprandial plasma glucose. the blood sample was divided into two aliquots; 2 and 3 ml .the first aliquots was dispended in tube containing ethylene diamine tetracetic acid ( edta ) (1.5 mg /ml ). this blood was processed in less than three hours and was used for hba1c estimation and other portion ( 3 ml ) was dispended in a plane tube and left for an hour http://en.wikipedia.org/wiki/polyuria http://en.wikipedia.org/wiki/polydipsia http://en.wikipedia.org/wiki/polyphagia http://en.wikipedia.org/wiki/diabetes_mellitus#cite_note-11#cite_note-11 iraqi j pharm sci, vol.21(2) 2012 metformin + sitagliptin versus metformin + glibenclamide 71 to clot at room temperature then, it was centrifuged at 3000 rpm for 10 minutes to collect serum .the serum was separated and used for estimating plasma glucose level (fpg ). by using an enzymatic colorimetric method with a commercially available kit for determination plasma glucose level and biorad variant hemoglobin a1c for determination hba1c. (9,10) results fasting plasma glucose (fpg) (9) the results showed significant reduction in fasting plasma glucose for both groups after 3 and 6 months of treatment as compared to 1 st reading. however, there is a significant decline for group 2 treated by metformin + sitagliptin compared to group 1 treated by metformin + glibenclamide (table.1 and fig 1 ). table 1: effect of treatment with metformin 500 mg 3 times daily + glibenclamide 5 mg twice daily (group 1) versus metformin 500 mg 3 times daily + sitagliptin 100 mg once daily (group 2) on fasting plasma glucose in patients with t2dm and control normal healthy individuals(group 3 )after 1,3 and 6 months of treatment .( n = 34 individuals for each group ). values expressed as mean ± standard error of mean. a significant difference (p <0.05) in comparison with control group. b significant difference (p < 0.05) between reading. c significant difference (p < 0.05) between group 1 and 2. fpg 3 fpg 2 fpg 1 group 3 control group 1 m + g group 2 m + s 0 20 40 60 80 100 120 140 160 180 mg / dl number of reading groups fasting plasma glucose ( fpg ) group 3 control group 1 m + g group 2 m + s figure 1: histogram showing effect of treatment with metformin 500 mg 3 times daily + glibenclamide 5 mg twice daily group 1 versus metformin 500 mg 3 times daily + sitagliptin 100 mg once daily group 2 on fpg in patients with t2dm and group 3 control normal healthy subjects after 1,3 and 6 months of treatment.( n = 34 individuals for each group) . postprandial plasma glucose (ppg) (9) table 2 shows comparison between the effects of two types of treatment ( metformin + glibenclamide and metformin + sitagliptin ) on postprandial plasma glucose in patients with t2dm .there were a significant reductions in postprandial plasma glucose for both groups after 3 and 6 months of treatment as compared to 1 st reading . however, there was a significant decline for group 2 patients treated by metformin + sitagliptin compared to group 1 treated by metformin + glibenclamide. group3 mg /dl group 2 mg / dl group 1 mg /dl duration months 100 .11 ± 1.76 144.94 ± 3.25 ab 173.97 ± 5.37 a 1 96.02 ± 1.29 129.02 ± 1.96 abc 150.76 ±3.97 ab 3 94.50 ± 1.24 118.4 ± 1.33 abc 127.79 ±2.52 ab 6 iraqi j pharm sci, vol.21(2) 2012 metformin + sitagliptin versus metformin + glibenclamide 72 table 2 : effect of treatment with metformin 500 mg 3 times daily + glibenclamide 5 mg twice daily ( group 1) versus metformin 500 mg 3 times daily + sitagliptin 100 mg once daily (group 2) on postprandial plasma glucose ppg in patients with t2dm and control normal healthy individuals (group 3) after 1,3 and 6 months of treatment .( n = 34 individuals for each group ). values expressed as mean ± standard error of mean. a significant difference (p <0.05) in comparison with control group. b significant difference (p< 0.05) between reading. c significant difference (p < 0.05) between group 1 and 2. figure 2 : histogram showing effect of treatment with metformin 500 mg 3 times daily + glibenclamide 5 mg twice daily (group 1) versus group 2 metformin 500 mg 3 times daily + sitagliptin 100 mg once daily (group 2) on ppg in patients with t2dm and group 3 control normal healthy individuals (group 3) after 1,3 and 6 months of treatment.( n = 34 individuals for each group ). glycosylated hemoglobin(hba1c) (10) table.3 shows comparison between the effects of two groups of treatment (metformin + glibenclamide and metformin + sitagliptin) on hba1c in patients with t2dm. there were a significant reductions in hba1c for both groups after 3 and 6 months of treatment as compared to 1 st reading. however, there were a significant decline for group 2 patients treated by metformin + sitagliptin compared to group 1 treated by metformin + glibenclamide after 3 and 6 months of treatment . group 3 mg /dl group 2 mg / dl group 1 mg /dl duration months 108.14 ± 1.34 186.05 ± 6.2ac 203.26± 12.37a 1 104.6 ± 1.19 159.38 ± 4.72abc 173.25 ± 7.99ab 3 96.29 ± 2.41 123.88 ± 2.41abc 140.67 ± 4.66ab 6 iraqi j pharm sci, vol.21(2) 2012 metformin + sitagliptin versus metformin + glibenclamide 73 table 3 : effect of treatment with metformin 500 mg 3 times daily + glibenclamide 5 mg twice daily( group 1) versus group 2 metformin 500 mg 3 times daily + sitagliptin 100 mg once daily (group 2) on hba1c in patients with t2dm and control normal healthy individuals group (3) after 1,3 and 6 months of treatment .( n = 34 individuals for each group ) values expressed as mean ± standard error of mean. a significant difference (p <0.05) in comparison with control group. b significant difference (p< 0.05) between reading. c significant different (p < 0.05) between group 1 and 2. figure 3:histogram showing effect of treatment with metformin 500 mg 3 times daily + glibenclamide 5 mg twice daily (group 1) versus metformin 500 mg 3 times daily + sitagliptin 100 mg once daily (group 2) on hba1c (%) in patients with t2dm and control normal healthy individuals (group 3 ) after 1,3 and 6 months of treatment .( n = 34 individuals for each group ) . discussion the epidemic of type 2 diabetes and the recognition that achieving specific glycemic goals can substantially reduce morbidity have made the effective treatment of hyperglycemia a top priority (11,12) . intensive glycemic management resulting in lower a1c levels has also been shown to have beneficial effect on cardiovascular disease ( cvd ) complication in type 1 diabetes (13,14) ; however, current studies failed to demonstrate a beneficial effect of intensive diabetic therapy on cvd in type 2 diabetes (15,16) . the development of new classes of blood glucose – lowering medication to supplement the older therapies, such as lifestyle – directed interventions, insulin, sulfonylurea, and metformin, has increased in number of treatment options available to practitioners and patients has heightened uncertainty regarding the most a appropriate means of treating this wide spread disease (17) . glp-1 and glucose-dependent insulinotropic polypeptide ( gpi ),the main insulinotropic peptide of intestinal origin (incretins), are rapidly degraded by dipeptidyl peptidase four ( dpp-4 ).dpp-4 is a member of a family of cell membrane proteins that are expressed in many tissues , including immune cells (18) . the 1 st oral dpp 4 inhibitor , sitagliptin was approved by the food and drug administration in october 2006 for use as monotherapy or in combination with metformin or tzds. another dpp-4 inhibitor, vildagliptin, was approved in europe in february 2008, and several other compounds are under development. in clinical trial group 3 (%)hba1c group 2 (%) hba1c group 1 (%) hba1c duration/ months 5.33 ± 0.83 6.32 ±0.09ac 7.38 ± 0.14a 1 5.31 ± 0.86 6.12 ± 0.09ac 7.10 ±0 .63a 3 5.01 ± 0.04 5.83 ± 0.08abc 6.81 ± 0.12a 6 iraqi j pharm sci, vol.21(2) 2012 metformin + sitagliptin versus metformin + glibenclamide 74 performed to date, dpp-4 inhibitors lower a1c levels by 0.6 – 0.9 percentage points and are weight neutral and relatively well tolerated (19,20) they don’t cause hypoglycemia when used as monotherapy.a fixed–dose combination pill with metformin is available.our results regarding plasma glucose and glycosylated hemoglobin ( hba1c ) indicate that there was a successful improvement in plasma glucose levels and hba1c after treatment courses of 3 and 6 months with metformin 500 mg three times daily + glibenclamide 5 mg twice daily and combination of metformin 500 mg three times daily + sitagliptin 100 mg once daily as was shown in tables 1-3; values were improved significantly after treatment with the above mentioned drugs. however, this improvement was not enough to reach that of normal healthy individual values, in other words, there were partial improvements observed by these drugs . accordingly, and based on the comparison of the treatment groups with that of control group that continue for the same treatment period, we conclude that combination of metformin + sitagliptin significantly reduced the values of fpg, ppg and hba1c after 3 and 6 months and improve plasma glucose level and hba1c percentage compared to combination of metformin + glibenclamide. this might due to the additive effect of these two drugs i .e metformin + sitagliptin .these results were in agreement with other results that also indicate effectiveness of additive effect of metformin and sitagliptin (21) . the incretin hormones play a major role in glucose homeostasis by stimulating insulin secretion, suppressing glucagons secretion, inhibiting gastric emptying and reducing appetite and food intake (22-25) . both incretin hormones are rapidly degraded and removed from circulation by the enzyme dipeptidyl peptidase–4 (dpp-4) (26,27) . therefore, there is considerable interest in enhancing incretin action for treatment of type 2 diabetes. sitagliptin , a selective dpp-4 inhibitor , reduces both fasting and postprandial plasma glucose presumably by inhibiting the inactivation of glp-1 and gip, thereby prolonging their duration of action on pancreatic islets (28,29) .also the complementary combination therapy with sitagliptin– metformin lower glucose via enhancement of insulin secretion, suppression of glucagon secretion , and insulin sensitization. use of this combination in diabetes management will provide a greater degree of glycosylated hemoglobin – lowering than that seen with use of either drug as monotherapy (22) . in clinical trials, the dpp-4 inhibitor, sitagliptin, improved fasting and postprandial glycaemic control and measures of β-cell function in patients with type 2 diabetes, with minimal effects on measures of insulin resistance/ sensitivity (30,31) . metformin has been found to increase glp-1 levels in humans (32,33) . sulfonylurea have the advantage of being quite effective in blood glucose lowering, with an almost instant onset of the effect after start of therapy. drops in hba1c of 1–2% can be expected as a mean, with the higher the baseline hba1c, the bigger the drop. additive effects are seen when sulfonylurea are combined with metformin, and the different mechanisms of action of these two agents – one stimulating insulin secretion, the other increasing insulin sensitivity –make them the obvious couple in the dual disease that is type 2 diabetes (34) . the success story of this combination can be seen in many countries where this combination is the standard treatment in type2 diabetes. suggestions that these drugs ultimately lead to faster beta-cell failure (an observation already made in the 1970s) have not altered their popularity (34) . because sulfonylurea (sus) have been used for so many years, their safety profile and side effects are well known. they increase insulin secretion by binding to a receptor on the surface of the pancreatic betacell, resulting in a glucose-independent insulin release. their mechanism of action also implicates that sulfonylurea therapy will ultimately fail because of b-cell failure. the main disadvantage of sulfonylurea is the risk of hypoglycaemia, which rises with advanced age, poor nutrition, alcohol consumption, liver or kidney disease and polypharmacy (35) and is higher than with other oral medications (36) this is a class effect, but differences between different products have been described (37–39) . another class effect of sus is that their use leads to weight gain, typically 1–4kg with stabilization after about six months. (40) here again, data are somewhat different between the products. (41,42) . sus have a neutral effect on lipid profile or blood pressure and all current sus – in contrast to the older products, where worrying reports on cardiovascular mortality abound – are neutral to the heart. most sus are renally cleared and dose adaptations will be needed in the case of renal insufficiency. therefore, it makes sense to choose sus as the next step when metformin is not enough, but care should be taken in older patients because of the risk of hypoglycaemia. to justify its high cost, sitagliptin should be used to its maximum potential, started early in the disease process to maintain and preserve beta cell function (43) and preferably used in combination with metformin in order to iraqi j pharm sci, vol.21(2) 2012 metformin + sitagliptin versus metformin + glibenclamide 75 achieve the maximum reduction in hba1c. (44) all recent clinical trials hint to the benefit of the early use of sitagliptin, alone or in combination, of any antidiabetic medication. more specifically, glp-1 or ddp4 inhibitors, have their maximum effect observed when the diabetic process is in its early manifestations. 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28:187-218. 45. chia cw, egan jm. incretin –based therapies in type 2 diabetes mellitus. j clin endocrinol metab 2008; 93:3703-3716. 46. nissen se, wolski k. effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. n engl j med 2007; 356:2457-2471 iraqi j pharm sci, vol.24(1) 2015 captopril and bone metabolism 11 study the influence of captopril on bone metabolism in elderly hypertensive women zahraa a. mousa * , nada n. al-shawi **, 1 and ahmed a. omar * * ministry of health, al-yarmouk teaching hospital, baghdad,iraq. ** department pharmacology and toxicology, college of pharmacy, university of baghdad,baghdad,iraq. abstract widespread use of antihypertensive agents in clinical practice necessitates the knowledge of their pleiotropic effects. at the present time there are no sufficient evidences of positive effect of these medications on bone coming from randomized controlled trials; knowledge of additional effects of those drugs on the bone metabolism will allow doctors to choose optimal treatment of hypertension, taking into account the state of bone tissue. at the same time it will also allow to prevent osteoporosis in patients having osteoporosis risk factors or initial signs of bone loss. ten elderly hypertensive women age > 60 years old (64.2±3.6) treated with captopril for a 5-6 years ago while they attending al yarmouk teaching hospital in baghdad; in addition, newlydiagnosed hypertensives, and normotensive of the aged-matched women were participated in this study that were conducted during the period (januarymay 2014). measurement of serum calcium, magnesium, inorganic phosphorus, total alkaline phosphatase activity, and parathyroid hormone were done, in addition to spine mineral density and t-score of such bone density by dual energy x-ray absorptiometry. the results of this study showed that there were no significant differences in the serum levels of calcium, magnesium, inorganic phosphorus, total alkaline phosphatase activity, and parathyroid hormone in postmenopausal hypertensive women treated for 5-6 years with captopril compared to newly-diagnosed and to aged-matched normotensive women. in addition, non-significant differences were observed in the level of bone mineral density and t score of bone mineral density in all groups of the study. in conclusion, the present study provides additional knowledge concerning the influence of captopril treatment on some selected parameters of bone metabolism in elderly hypertensive women. keywords: captopril, bone metabolism, elderly women, hypertension, dual energy x-ray absorptiometry. الكبيرات في السن والالتي يعانين العظن في النساء استقالبعلى عقار الكابتىبريلدراسة تأثير هن ارتفاع ضغط الدم زهراء عامر موسي * ، ندى ناجٌ الشاوً **،1 و أحمد عبد البارً عمر * * العراق.،بغداد ،وزارة الصحت ،مستشفي الَرموك التعلَمٌ ** .، العراقجامعت بغداد،كلَت الصَدلت، فرع األدوٍت والسموم الخالصة االستخدام السرٌري الواسع لالدوٌة الخافضة لضغط الدم ٌتطلب معرفة التأثٌرات االخرى الغٌر متعارف علٌها. فً الوقت الحاضر لٌس هناك دالئل واضحة حول التأثٌرات األٌجابٌة لتلك األدوٌة على العظم كما ان المعرفة االضافٌة لتأثٌراتها على استقالب ٌار افضل دواء لعالج ارتفاع ضغط الدم مع االخذ بنظر االعتبار حالة النسٌج العظمً. وفً نفس الوقت العظم تسمح لالطباء باخت ٌمنع ذلك تخلخل العظام فً المرضى الذٌن لدٌهم عوامل خطورة تسبب تخلخل العظم أو لدٌهم أعراض أولٌة من خسارة العظم لذلك تقالب العظم فً النساء اللواتً ٌعانٌن من ارتفاع ضغط الدم، وألضافة اراء على اس صممت هذه الدراسة الستقصاء تأثٌرالكابتوبرٌل سنة وٌعانٌن من ارتفاع ضغط الدم 06الى تلك التً تمت من قبل باحثٌن بهذا الخصوص. تم مشاركة عشر نساء اعمارهن أكثر من مستشفى الٌرموك التعلٌمً فً بغداد، باالضافة سنوات خالل زٌارتهن ل 0-5عولجن من قبل أطباء أختصاص بعقار الكابتوبرٌل لمدة الى عشر نساء لدٌهن ارتفاع فً ضغط الدم شخصن حدٌثا، وعشر نساء لدٌهن ضغط دم طبٌعً وبنفس العمر. تم قٌاس معدالت ل الدم وهرمون الباراثاٌروٌد فً مص alkaline phosphataseانزٌم ال الكالسٌوم، المغنٌسٌوم، الفوسفور الالعضوي، فعالٌة باستخدام الجهاز الممتص لالشعة السٌنٌة. t-scoreو لكل النساء المشاركات فً الدراسة، كما تم قٌاس كثافة معادن العمود الفقري انزٌم أظهرت نتائج الدراسة بان لٌس هناك اختالفات معنوٌة فً معدالت الكالسٌوم، المغنٌسٌوم، الفوسفور الالعضوي، فعالٌة سنوات 0-5وهرمون الباراثاٌروٌد لدى النساء فً سن الٌأس الالتً ٌستخدمن الكابتوبرٌل لمدة alkaline phosphatase لـا عند زٌارتهن الى المستشفى والنساء حدٌثا تشخٌصه تم لعالج ارتفاع ضغط الدم بالمقارنة مع اللواتً لدٌهن ارتفاع فً ضغط الدم 1 corresponding author e-mail: nadaalshawi@yahoo.com received: 29 /9/2014 accepted: 16 /12/2014 mailto:nadaalshawi@yahoo.com iraqi j pharm sci, vol.24(1) 2015 captopril and bone metabolism 12 -tضغط دم طبٌعً وبنفس العمر. باالضافة الى ذلك، لٌس هناك اختالفات معنوٌة فً معدل كثافة معادن العظم و فً الالتً لدٌهن score ًلكثافة معادن العظم فً النساء الالتً ٌعانٌن من ارتفاع ضغط الدم وٌستخدمن الدواء بالمقارنة مع معدالته فً النساء الالت لدٌهن ارتفاع فً ضغط الدم عند زٌارتهن الى المستشفى، والنساء الالتً لدٌهن ضغط دم طبٌعً. ن هذه الدراسة اضافت معرفة حول تأثٌر عقار الكابتوبرٌل على معدالت وفقا للنتائج التً تم الحصول علٌها ٌمكن االستنتاج بأ المعاٌٌر التً اختٌرت فً الدراسة والخاصة باستقالب العظم فً النساء المسنات الالتً لدٌهن ارتفاع فً ضغط الدم. السينية . الووتص لألشعةعقار الكابتىبريل ،استقالب العظن ،النساء الكبيرات في السن ، ارتفاع ضغط الدم ، الجهاز :الكلوات الوفتاحية introduction hypertension and osteoporosis are two major age-related disorders that together account for significant morbidity and mortality in the elderly. as 50% of the hypertensive population comprises postmenopausal women at high risk of osteoporosis, hypertension represents a considerable health problem in this population. several studies suggested that high blood pressure is associated with abnormalities of calcium metabolism, leading to an increase in calcium movement from bone, thereby increasing the risk of osteoporosis (1-3) while, others did not show such association (4-6) . it has been also demonstrated that bone metabolism is closely regulated by hormones and cytokines, which have effects on both bone resorption and deposition. because the vasculature plays an important role in bone remodeling, the effect of the renin angiotensin system on bone metabolism may be partially related to the regulation of blood flow. although, the receptors for angiotensin ii is expressed in osteoblasts and osteoclasts but, the effects of such peptide hormone are controversial; however, several investigators indicated that angiotensin ii is a potent stimulator of osteoclastic bone resorption; on the contrary, others showed that angiotensin ii stimulated the proliferation of osteoblast rich populations of cells (7-8) . in vivo study demonstrated that captopril may improve osteopenia in ovariectomised rats and may promote bone formation in osteoblasts (9) . conflicting data were demonstrated by several clinical studies concerning the influence of angiotensin converting enzyme inhibitors and abnormalities of bone metabolism (10-11) . thus, this study was designed to investigate the influence of captopril on bone metabolism in elderly hypertensive women; and to add suggestions to that performed by other researchers concerning this respect. women, materials and methods ten hypertensive women on captopril therapy (daily dose range 12.5-100mg; midochemie ltd, limassol-cyprus), treated by specialist physicians while attending al yarmouk teaching hospital in baghdad (group c); ten newly-diagnosed hypertensives (group b); and ten apparently healthy (group a) were participated in this study. the study was approved by the scientific committee of the college of pharmacy-baghdad university; in addition, an ethical sheet was obtained and approved by the ministry of health, baghdadiraq. verbal consent was obtained from each woman participated in this study. the inclusion and exclusion criteria of all women in this study are summarized in table 1, and their demographic data are showed in table 2. table (1) : inclusion and exclusion criteria of women enrolled in this study inclusion criteria exclusion criteria over sixty years old normotensive women. over sixty years old patients’ women with hypertension. disease rheumatoid disease diabetic mellitus cardiovascular diseases hepatic and renal dysfunctions smoking drugs, and supplements thyroxin corticosteroids estrogen and its derivative bisphosphonates nutritional supplements diuretics iraqi j pharm sci, vol.24(1) 2015 captopril and bone metabolism 13 table (2): groups of women who participated in the present study group number of women age systolic/diastolic blood pressures mm hg body mass index (bmi) group a 10 66.4±4.6 130/86±3.1 a 28.3±4.4 a group b 10 66.1±5.8 ns 140/95±2.9 b 32.0±5.1 b group c 10 64.2±3.6 ns 130/85±3.8 a 33.2±5.2 b group a: normotensive elderly control women; group b: elderly women newly diagnosed with hypertension; group c: elderly hypertensive women treated with therapeutic dose of captopril [midochemie ltd, limassol-cyprus; (dosage range 12.5-100mg)]. values of age, systolic/diastolic blood pressures, and body mass index (bmi) are presented as mean ±standard error of mean. values with non-identical superscripts (a, and b) are considered significant. ns: non-significant difference compared to normotensive controls (group a). the body mass index (bmi) was calculated using the following formula: weight in ilogram height 2 (in meter) (12) ; while systolic/diastolic blood pressures were measured utilizing sphygmomanometer and stethoscope (13). blood sampled were collected from each women participated in the study for laboratory analysis to measure serum calcium (ca ++ ) (roche/hitachi, germany) (14) magnesium (mg ++ ) (roche/hitachi, germany) (15) , inorganic phosphorus (pi) (roche/hitachi, germany) (16) , alkaline phosphatase (alp) (roche/hitachi, germany) [17], and parathyroid hormone (pth) (roche/hitachi, germany) [18] by utilizing kits for this purpose. besides, all women were examined by x-ray to measure spine mineral density utilizing dual x-ray absorptiometry (dexa) (19) , and (t) score of such bone mineral density (20) . statistical analysis analysis of data was carried out using the statistical packages for social sciences version 22 (spss-22). the significance of difference among groups of women concerning their age, weight, height and mean arterial blood pressure was tested using pearson chi-square test ( 2 test) with application of yate's correction or fisher exact test whenever applicable. besides, the significance of difference of different means was tested using student-t-test for difference between two independent means. statistical significance was considered whenever the p value was equal or less than 0.05 in all data presented in this study. results hypertensive elderly women treated with therapeutic doses of captopril, for 5-6 years showed non-significant differences (p>0.05) in the levels of serum calcium, magnesium, and inorganic phosphorus compared to normotensive elderly controls and to newlydiagnosed hypertensive women of the agedmatched as shown in table 3. table (3): influence of captopril on serum calcium, magnesium, and inorganic phosphorus compared to normotensive elderly controls, and to newly-diagnosed elderly women. group serum calcium mg/dl serum magnesium mg/dl serum inorganic phosphorus mg/dl group a 9.00±1.05 2.13±0.11 3.01±0.63 group b 8.96±0.95 2.11±0.24 3.59±0.78 group c 8.62±0.84 2.24±0.19 3.46±0.54 group a: normotensive elderly control women; group b: elderly women newly diagnosed with hypertension; group c: elderly hypertensive women treated for 5-6 years with therapeutic dose of captopril. values are presented as mean± standard error of mean. number of women in each group is 10. iraqi j pharm sci, vol.24(1) 2015 captopril and bone metabolism 14 the data presented in table 4 showed nonsignificant differences (p>0.05) in the serum activity of alp , and the level of serum pth levels in elderly hypertensive women treated for 5-6 years with captopril compared to the corresponding levels in newly-diagnosed and to normotensive control women. table (4): influences of captopril on serum alkaline phosphatase (alp) activity and serum parathyroid hormone compared to normotensive elderly controls, and to newly-diagnosed elderly women. group alkaline phosphatase(u/l) parathyroid hormone pg/ml group a 90.00±34.55 44.24±16.89 group b 90.50±23.11 54.77±43.74 group c 71.82±20.49 47.34±15.56 group a: normotensive elderly control women; group b: elderly women newly diagnosed with hypertension; group c: elderly hypertensive women treated for 5-6 years with therapeutic dose of captopril. values are presented as mean± standard error of mean. number of women in each group is 10. the data presented in table 5 showed nonsignificant (p>0.05) differences in the levels of spine mineral density, and (t) score in elderly hypertensive women treated for 5-6 years with therapeutic dose of captopril compared to those levels in normotensive elderly controls and to newly-diagnosed hypertensive of the aged-matched women. table( 5): influence of captopril on spine, and (t) score of such bone mineral density (bmd). group spine mineral density (mg. cm -2 ) spine mineral density (t) score group a 777.60±113.52 2.74±0.92 group b 803.40±153.37 2.41±0.95 group c 841.90±169.65 2.44±1.34 group a: normotensive elderly control women; group b: elderly women newly diagnosed with hypertension; group c: elderly hypertensive women treated for 5-6 years with therapeutic dose of captopril. values are presented as mean± standard error of mean. number of women in each group is 10. discussion it has been reported that ang ii may influence calcium metabolism by decreasing ionized calcium and increasing the pth level (21) . conflicting results were obtained concerning the effects of angiotensin converting enzyme inhibitors on bone metabolism; where, patients with risk of fractures who have used angiotensin converting enzyme inhibitor (acei), no significant difference in bmd were recorded (22) ; while other study reported that patients treated with an acei showed an increased bmd and more importantly reduced fracture risks (10,11) . however, aceis also regulate the bradykinin-nitric oxide (no) pathway as well as block ang ii production, different from angiotensin receptor blockers (arbs). it was well known that the no pathway regulates local blood flow in bone marrow capillaries, which might enhance bone marrow formation. thus , the contribution of the no pathway might have a role in the prevention of osteoporosis by ace inhibition (23) . an in vitro study performed by liu et al in 2011demonstrated that captopril had potential effects of improving lumbar vertebral bone strength in aged ovariectomised (ovx) rats and promoted osteoblast bone formation (9) . moreover, the same study showed that the administration of an ace inhibitor, enalapril, did not cause significant changes in bone density, mineral content or morphometric parameters of the femur in 14-week-old ovariectomised female wistar rats. captopril acts by inhibiting the conversion of angiotensin i to ang ii; thus inhibits the synthesis and secretion of aldosterone and iraqi j pharm sci, vol.24(1) 2015 captopril and bone metabolism 15 reduces blood pressure. the drug also reduces the bradykinin degradation thus, inhibiting the generation of prostaglandins that were considered as local factors that may stimulate bone resorption (24) . it has been demonstrated that in vasculature, estrogen antagonized ang ii signaling and ang ii induced atherosclerosis, which suggest that ovx might accelerate ang ii-induced signaling. of importance, several reports suggest that estrogen may enhance the ace inhibition-mediated improvement of vascular remodeling in hypertension in female {(spontaneously hypertensive rats (shr)} with ovx through the inhibition of extracellular signal-regulated kinase 1/2. it supports the impact of a cross talk between estrogen and ang ii signaling in shrs females (25,26) . concerning magnesium (mg), the result of the present study revealed that no significant differences in the level of serum mg were observed in all elderly women groups participated in this study. such electrolyte works in concert with calcium to regulate electrical impulses in the cells; also it is largely responsible for the bone health and strong teeth. without adequate amounts of the mg, calcium will not be deposited in these hard tissues, where, mg has the ability to activate thyrocalcitonin, a hormone that under normal circumstances would send calcium to bones. furthermore, many authors demonstrated that there was a functional link between mg and calciotropic hormones such as parathyroid hormone (pth) and 1, 25(oh) 2 vitamin d3 that all responsible for the regulation of calcium homeostasis (27,28) . regarding inorganic phosphorus, the intended element is present in every cell of the body, and 85% of the body's phosphorus is found in bones and teeth. thus, it is vital to the formation of bones and teeth, and healthy bones and soft tissues require calcium and phosphorus to grow and develop throughout life (29) . the tight controlled balance of calcium and phosphorus is maintained by hormonal control of transport in the intestine, bone, and kidney; where, the latter organ acts by modulating calcium and phosphate reabsorption from the glomerular filtrate according to the body needs that is mediated by ion transporters. in addition to the classical endocrine factors (such as pth and vitamin d) that are involved in maintaining calcium and phosphate balance, other factors have been identified viz, fibroblast growth factor-23 (fgf23), which regulates urinary phosphate excretion by interacting with fgf receptors; and klotho, a transmembrane protein, facilitates this interaction, with the result of reducing phosphate reabsorption by the kidney. furthermore, dental matrix protein-1, an osteocyte product, has been shown to participate in fgf23-mediated regulation of phosphorus homeostasis (30) . furthermore, the impacts of aceis have been studied in stable ischemic heart disease; in such study, the intended group of drugs showed a beneficial effect on fgf-23 regardless of renal function (31) . the effects of captopril utilized in the present study on serum inorganic phosphorus (pi) were non-significantly different among all elderly women participated in this work which could be attributed to the above regulatory processes. thus, further works are needed to emphasize the findings of the investigators. the present study provides additional knowledge concerning the influence of captopril treatment on some selected parameters of bone metabolism in postmenopausal hypertensive women. conclusion according to the results obtained from this study, one can conclude that, there were no significant differences in the serum levels of electrolytes (calcium, magnesium, and inorganic phosphorus); parathyroid hormone,; alkaline phosphatase activity, in addition to bone mineral density in hypertensive elderly women treated with therapeutic doses of captopril, for 5-6 years compared to normotensive elderly controls and to newlydiagnosed hypertensive women of the agedmatched. acknowledgments the authors thank the laboratory technicians of al-yarmouk teaching hospital for analyzing biochemical parameters, and for dxa scanning. references 1. tsuda, k; nishio, i. and masuyama, y. bone mineral density in women with essential hypertension. am j hypertens. 2001; 14: 704-707. 2. yazici, s; yazici, m; korkmaz, u et al. relationship between blood pressure levels and bone mineral density in postmenopausal turkish women. arch med sci. 2011; 7: 264-270. 3. yang, s; nguyen, nd; center, jr. et al. association between hypertension and fragility fracture: a longitudinal study. osteoporosis international 2013: 1-7. 4. pérez-castrillón, jl; justo, i; silva, j et al. bone mass and bone modelling markers in iraqi j pharm sci, vol.24(1) 2015 captopril and bone metabolism 16 hypertensive postmenopausal women. j hum hypertens. 2003; 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21(3):239-244. 30. civitelli, r and ziambaras, k. calcium and phosphate homeostasis: concerted interplay of new regulators. j endocrinol invest. 2011 jul; 34(7 suppl):3-7. 31. udell, j.a; morrow, d. a; jarolim, p et al. fibroblast growth factor-23, cardiovascular prognosis, and benefit of angiotensin-converting enzyme inhibition in stable ischemic heart disease. j am coll cardiol. 2014; 63(22):2421-2428. doi: 10. 1016/j.jacc.2014.03.026. http://www.ncbi.nlm.nih.gov/pubmed/?term=civitelli%20r%5bauthor%5d&cauthor=true&cauthor_uid=21985972 http://www.ncbi.nlm.nih.gov/pubmed/?term=ziambaras%20k%5bauthor%5d&cauthor=true&cauthor_uid=21985972 http://www.ncbi.nlm.nih.gov/pubmed/21985972 http://www.ncbi.nlm.nih.gov/pubmed/21985972 abstract iraqi j pharm sci, vol.20(2) 2011 floating tablet of captopril 1 controlled release floating matrix tablet of captopril haithem n. abed* , alaa a. abdulrasool** and mowafaq m. ghareeb** ,1 *ministry of helth ,diala, iraq. **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract the present study was done to prepare a gastroretentive floating tablet of captopril (cap) which is an angiotensin converting enzyme inhibitor (ace-inhibitor) used in the treatment of hypertension and heart failure. cap is mainly absorbed from the proximal intestine and to a lesser extent from the stomach, also cap stability decreases as the ph raised above 1.2 and this makes it a suitable candidate for floating dosage form.effervescent floating tablets of cap were prepared in order to prolong the gastric residence time and increase the bioavailability of the drug. the floating tablets of cap were prepared by direct compression and wet granulation technique, using the polymer hydroxypropylmethylcellulose (hpmc) as the primary retarding polymer together with carboxymethylcellulose(cmc) ,ethyl cellulose(ec) and pectin as a secondary release modifying polymers in different ratios of (1:1, 3:1 and 9:1). different formulation parameters were studied such as type of diluents, amount of effervescent agent, methods of preparation and their effects on buoyancy and the in vitro drug release profile as well as their physical characteristics. the wet granulation method shows a good flow and compressibility characteristics and a better dose uniformity in comparison with direct compression technique. pectin together with hpmc in the ratio of 1:1 was found to meet the requirement for a good matrix formation and floating characteristics and the drug release was sufficiently sustained for 12 h with floating time >24h and floating lag time of 2 min. kinetic modeling of the release data for the selected formula showed that the mechanism of drug release pattern follows anomalous or non –fickian diffusion. key words: captopril, floating, matrix tablet, pectin. الخالصة انغشض يٍ ْزِ انذساست ححضيش صيغت دٔائيت طافيت راث قابهيت بقاء في انًعذة نذٔاء انكايخٕبشيم ٔانًثبظ نعًم االَضيى انكابخٕبشيم يًخص بشكم سئيسي يٍ حفاع ضغظ انذو ٔعجض انقهب.انًحٕل نالَجيٕحُسيٍ ٔيسخخذو انكابخٕبشيم نعالج ايشاض اس حقم ثباحيت انذٔاء كهًا اصدادث قاعذيت انًحيظ , يًا يجعهّ يششح يثاني نهخقُياث انذٔائيت االيعاء انذقيقت ٔبقابهيت اقم يٍ انًعذة , يٍ ة قذ حضشث ٔرنك نغشض صيادة فخشة بقائٓا في انًعذة انطافيت.انصيغ انذٔائيت انطافييت نذٔاء انكابخٕبشيم انًصُعت بانطشيقت انفٕاس انًفشدة نذٔاء انكابخٕبشيم صُعج باسخخذاو بٕنيًش انٓايذسٔكسي يثيم اجم صيادة انخٕافشيت انحيٕيت نهذٔاء . انخشكيبت انذٔائيت انطافيت ( ٔانكاسبٕكسي يثيم سهيهٕص ecهيهٕص )( كًعيق ححشس أني يع أٔ بذٌٔ يعيقاث ححشس ثإَيت يثم بٕنيًش االثيم سhpmcسهيهٕص) كًا شًهج انذساستيقاسَت عذد يٍ انعٕايم انًؤثشة عهٗ .(1:1,1:1,1:1) ( ٔبُسب يخخهفتpectin( ٔانبكخيٍ ) scmcانصٕديٕو ) انخي حسخخذو ٔانًضافاث االخشٖ ٔقذ ٔجذ اٌ انصيغت أكسيذ انكاسبٌٕ انخصييغ كطشيقت انخصييغ ٔكًيت انعايم انًٕنذ نغاص ثاَي ( حالقي انًخطهباث انالصيت نخشكيم قانب جيذ ٔخصائص طفٕ نهجشعت 1:1بٕنيًش انٓايذسٔكسي يثيم سهيهٕص يع انبكخيٍ ٔبُسبت ) 12ساعت ٔبٕقج نهطفٕ دقيقخاٌ ٔيعذل طفٕ الكثش يٍ 11حيث كاٌ يعذل ححشس انذٔاء يٍ انصيغت قذ اسخًش الكثش يٍ انذٔائيت. ( يع يخخهف انًُارج انحشكيت نخحشس انذٔاء ٔقذ نٕحظ اٌ انيت ححشس 11قت بياَاث انخحشس انذٔائي نهصيغت انًُخقاة )حى يطابساعت.ٔنقذ (.non-fickianانذٔاء حخبع ) introduction it is widely accepted that gastric emptying of a conventional dosage form in humans is affected by numerous factors and the time taken shows wide interand intrasubject variation (1) .this variability, in turn, can lead to unpredictable times to achieve peak plasma drug levels and bioavailability, since many drugs are absorbed to the greatest extent in the upper part of the small intestine (1, 2) .the residence of a drug delivery system in the upper part of the gastrointestinal tract (git) can be accomplished by several drug delivery systems, such as intragastric floating drug delivery systems (fdds) (3) , swelling and expandable systems (4) , bioadhesive systems (5) , modified shape systems (6) , high density systems (7) , delayed gastric emptying systems (8) and low density super porous systems (9) .the intragastric fdds have a bulk density less than gastric fluids and so remain buoyant in the stomach without affecting the gastric emptying rate for a prolonged period of time. while the system is floating on the gastric contents, the drug is released slowly at the desired rate from the system. after release of drug, the residual system is emptied from the stomach. this results in increasing gastric residence time (grt) and a better control of fluctuations in plasma drug concentration (10) . 1 corresponding author email : mopharmacy@yahoo.com received : 9/10/2010 accepted : 16/4/2011 iraqi j pharm sci, vol.20(2) 2011 floating tablet of captopril 2 captopril (cap) is a 1-[(2s)-3-mercapto-2methyl-1-oxopropyl]-l-proline, it is an angiotensin converting enzyme inhibitor. it has been widely used for the treatment of hypertension and congestive heart failure. it has been reported that the duration of antihypertensive action after a single oral dose of cap is only 6–8 h, so clinical use requires a daily dose of 37.5–75 mg to be taken three times (11) .the drug is freely water soluble and has elimination half-life after an oral dose of 1.7h. it is most stable at ph 1.2 and as the ph increases; it becomes unstable and undergoes a degradation reaction (12) .various attempts have been made to develop floating systems to control drug release; among them is the so called hydrodynamically balanced system (13, 14) .such a system is useful for drugs acting locally in the proximal gastrointestinal (gi) tract or for drugs that degrade in the intestinal fluid.the aim of the present investigation is to develop a sustained release single unit floating matrix tablet of cap with a view of prolonging grt and controlled release properties. materials and methods materials cap and sodium carboxymethyl cellulose (na cmc) were obtained from samara´a drug industry/iraq, metolose 90sh100000sr, a brand of hpmc obtained from nutrer farma/japan, pectin was obtained from himedia /india, ethyl cellulose (ec) obtained from pdh/england; other materials and solvents used were of analytical grade. methods preparation of cap matrix tablets the composition of different formulas of cap floating tablets is shown in table (1), effervescent floating tablets containing cap were prepared by direct compression technique using varying concentrations of polymers with sodium bicarbonate. all the ingredients were accurately weighed and blended uniformly in a mortar. after sufficient mixing of drug and excipients, magnesium stearate was added as post lubricant with further mixing for 5 minutes. the tablets were compressed using double punch and die tablet machine (manesty/england) with a diameter of 10mm biconcave punch and die.formulas f11, f12 and f13 were selected and prepared by wet granulation method using 1% solution of polyvinylpyrolidon (pvp-k30) in isopropyl alcohol as a granulating agent. a sufficient quantity of the granulation solution was added until a positive ball test consistency was resulted, then the wet mass was passed through sieve (10 mesh) and dried in pre warmed oven at 60 ºc for one hour. the dried granules were reduced in size by screening them through 12 mesh size sieve. a known weight of granules was mixed with the calculated amount of magnesium stearate and talc powder for 5 minutes. evaluation of powder blends. angle of repose flow property of the granules was evaluated by determining the angle of repose and the compressibility index. static angle of repose was measured according to fixed funnel method and free standing cone method of banker and anderson (15) . the angle of repose was calculated using the following equation, tan θ = h/r ………… (1) where (h) is the height of the cone, (r) is the radius of the cone base. compressibility index& hausner ratio carr’s compressibility index (ci) for the prepared granules was determined utilizing the equation (2), ci (%) = (vo – vf/vo)x 100 ……. (2) where (vo) is the untapped apparent volume, (vf) is the tapped apparent volume of the powder. the hausner ratio (hr) is closely related to compressibility index (ci). it can be calculated using equation (3), where (vo) is untapped apparent volume and (vf) is tapped apparent volume (16) . hr = vo / vf ………. (3) evaluation of cap tablets tablets from all of the prepared formulas were evaluated for their physical properties such as hardness, friability, content uniformity, in-vitro dissolution and the floating behaviour (floating time (ft) and floating lag time (flt). tablet hardness and friability the compression force was controlled to keep the tablet hardness 5 kg, tablet crushing strength was measured using monsanto hardness tester, the friability was determined as the percentage of weight loss after 100 revolutions of 20 tablets using erweka friabilator, germany. drug content uniformity ten tablets from each prepared formula were crushed in a mortar then a weight of one tablet were assayed for drug content using 0.1n hcl as the solvent and the samples were analyzed spectrophotometrically at 205nm λmax (17) using uv-visible spectrophotometer (carry uv, varian, australia). iraqi j pharm sci, vol.20(2) 2011 floating tablet of captopril 3 floating behaviour the floating ability was determined using usp ii apparatus (50 rpm, 37±0.5˚c, 900ml, 0.1n hcl), one tablet was placed in the medium; the time needed to go upward and float on the surface (floating lag time) was recorded also the floating duration and relative matrix integrity were measured (the results were done in triplicate and the average values were taken). the latter parameter was determined on the basis of mass loss by gravimetry and visual inspection after the floating studies . in vitro dissolution the cap release from all the prepared formulas was determined using a usp xxx paddle apparatus 2. the dissolution medium was 900 ml (0.1 n hcl, no enzyme) at 37± 0.5 °c; paddle speed 50 rpm. all experiments were done in triplicate and the average values were taken. the prepared formulas were subjected to dissolution tests for 7h. sample (5 ml) was withdrawn at predetermined time intervals, filtered through a millipore filter membrane (0.45 μ) and replaced by an equal volume of dissolution medium to maintain sink condition. drug content in the dissolution sample was determined specrophotometrically at its λmax at 205nm according to uspxxx specification (17) . selection of the best formula the selection of the best formulas was done depending on the correlation of the resulted floating matrix tablet, with respect to a reference adapted from previous research that match the release profile of gastroretentive floating dosage form of cap obtained by meka et al (18) . the similarity factor (f 2) introduced by moore and flanner (19) , was used as criterion for assessment of the best formula. … (4) where n is the number of dissolution time points, rt and tt are the reference and test dissolution values at time t. the (f2) value was found to be more than 50 (between 50 and 100), means that the two dissolution profiles are considered to be similar. kinetic modelling of drug release to analyze the cap release mechanism from the selected formula f11, the in vitro release data were fitted into various release kinetic models, first order, zero order, higuchi and korsmeyer and peppas, and the model with the highest correlation coefficient was considered to be the best model:  first order kinetic log mt = log mo + k1 t / 2.303 ………… (5)  zero order kinetic mt = mo + ko t ………… (6)  higuchi model mt / m∞ = kh t 1/2 ………… (7)  korsmeyer-peppas model (power law) mt / m∞ = kkp t n ………… (8) log [mt / m∞] = log kkp + n log t …… (9) where mt is the amount of drug released in time t, mo the initial amount of drug, m∞ is the cumulative amount of drug released at infinite time, k is respective release constant and n is the release exponent, which characterizes the mechanism of drug release. for korsmeyer and peppas equation superposes two apparently independent mechanisms of drug transport, fickian diffusion and a case-ii transport, for the description of drug release from a swelling polymer. for a matrix tablet (cylinder), when n takes the value of 0.45 it indicates diffusioncontrolled drug release and for the value 0.89, it indicates swelling-controlled drug release. values of n between 0.45 and 0.89 can be regarded as an indicator for both the phenomena (anomalous transport). (20, 21) statistical analysis the results of the experiments are given as a mean of triplicate samples ± standard deviation and were analyzed according to the one way analysis of variance (anova) at the level of (p < 0.05). results and discussion the effervescent floating tablets of cap were formulated in 13 different formulas (table 1) using direct compression technique except, f11, f12 and f13 which were prepared by wet granulation technique using 1% pvp in isopropyl alcohol as a granulating agent.the pre-compression parameters like angle of repose, compressibility index and hausner’s ratio were evaluated and all parameters are within the pharmacopoeial prescribed limits as shown in table (2).the physical property of the final blend for the selected formula (f11) showed excellent flow characteristics, the angle of repose <31º and the value of the compressibility index (c.i) which is <10 further supported the results of the flow property. the prepared tablets of all the formulas were evaluated for their post compression parameters like hardness, friability, buoyancy lag time, total floating time, drug content and in-vitro drug release as shown in table (2).the % friability was less than 1% in all of the prepared formulas ensuring that the tablets were mechanically                   100 1 1log50 5.0 1 2 2 n t tt tr n f iraqi j pharm sci, vol.20(2) 2011 floating tablet of captopril 4 stable. the drug content uniformity of the selected formula (f11) showed a value of (99.5%) which reflects a good uniformity in drug content. all the tablets passed weight variation test and they were within the pharmacopoeial limits (17) . table 1 : different formulas of captopril floating matrix tablet. ingredient in (mg) f1 f2 f3 f4 f5 f6 f7 f8 f9 f10 f11* f12* f13* cap. 50 50 50 50 50 50 50 50 50 50 50 50 50 hpmc 200 180 150 100 180 150 100 180 150 100 100 100 100 ec 20 50 100 scmc 20 50 100 pectin 20 50 100 100 100 100 nahco3 52.5 52.5 52.5 52.5 52.5 52.5 52.5 52.5 52.5 52.5 52.5 70 87.5 mcc ph102 37 37 37 37 37 37 37 37 37 37 37 19.5 2 mg. stearate 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 3.5 talc 7 7 7 7 7 7 7 7 7 7 7 7 7 * formulas were prepared by wet granulation method using 1%pvp solution in isopropyl alcohol as a granulating agent. table 2 : characterization of powder blend and the prepared captopril tablets. fo r m u la s a n g le o f r e p o se c .i .% h .r . h a r d n e ss % f r ia b il it y c o n te n t u n if o r m it y f l t (m in ) t f t (h o u r s) m a tr ix in te g r it y f1 34.9 20 1.25 5 0.215 97.6 4.5 >24 + f2 35 25 1.33 5 0.226 99.45 4.25 >24 + f3 37.5 23.3 1.3 5 0.253 90.5 4.2 20±0.5 + f4 36 20 1.25 5 0.432 93.3 3.5 17±0.6 + f5 38.8 13.3 1.15 5 0.402 90 12 >24 + f6 41.3 16.6 1.2 5 0.321 91.2 10 21±0.7 + f7 46.3 20 1.25 5 0.085 105.2 5 14±0.3 + f8 38.1 25 1.33 5 0.095 104 5 >24 + f9 41.2 21.7 1.27 5 0.167 102.5 9.5 19 + f10 42.8 21.5 1.14 5 0.428 99.5 12 14±0.5 + f11 30.9 6.6 1.07 5 0.672 99.5 1 >24 + f12 25.6 15.6 1.17 5 0.571 97.7 0.15 17±0.6 + f13 30.05 16.1 1.2 5 0.742 94.8 0 14±0.3 + +means a good matrix integrity during the test duration. the selected formula (f11) showed a flt of (1 min) and a tft of more than (24h), as would be expected slower floating and longer buoyancy duration were achieved with formulas containing smaller amount of effervescent agent and this could be due to less gases evolved and less matrix expansion, and by increasing the ratio of the effervescent agent from 15% in formula f11 to20% and 25% (formulas f12 and f13 respectively) showed a high decrease in flt, but a faster release was shown for formulas f12 and f13 (fig.1) that containing higher amount of effervescent agent, this is because the elevation of the gas-forming agent would generate larger amounts of effervescence leading to an increase in the rate of pore formation, rapid hydration of the tablets matrices and consequently a faster drug release rate (22) .all the formulas were subjected to in vitro dissolution studies. iraqi j pharm sci, vol.20(2) 2011 floating tablet of captopril 5 figure 1: effect of effervescent agent on captopril release profile in 0.1n hcl at 37ºc. the results of in vitro percentage release at different time intervals are plotted against time to obtain the release profile. a preliminary study showed that the drug to polymer (hpmc) ratio of (1:4) in f1 was the best to sustain the drug release for the desired profile. from the in vitro drug release studies, it was concluded that a partial replacement of hpmc with ec (f2, f3 and f4 in fig.2) showed an increase in final drug release rate, and the burst effect was also increased compared to f1 which contain hpmc alone, and that could be due to the presence of as little as 10% fibrous insoluble excipients which might disturb the formation of the gel layer and prevent uniform swelling (23) . figure 2: effect of ec on captopril release profile in 0.1n hcl at 37ºc . in the other hand, the partial replacement of hpmc with scmc (f5, f6 and f7 in fig.3) showed a significant decrease in final drug release (p<0.05) compared to f1, but there was no significant difference between f5 (hpmc/scmc ratio 9:1) and f6 (hpmc/scmc ratio 3:1), that might be caused by the low solubility of scmc at ph 1.2 to 3 (24) . figure 3: effect of scmc on captopril release profile in 0.1n hcl at 37ºc. formulas containing pectin together with hpmc showed different behaviour, at lower ratio (9:1) of hpmc to pectin in f8 the final drug release rate showed no significant difference compared to f1, but as the ratio of hpmc to pectin decreased to (3:1) in f9 and (1:1) in f10 the final drug release rate was also decreased (p<0.05) in addition to a little improvement in burst releasing effect (fig.4), this is due to the formation of a denser gel and slower erosion at higher pectin content (25) , formulas (f11) was prepared by wet granulation technique using 1% of pvp k30 in isopropyl alcohol showed a significant increase (p<0.05) in the last phase of drug release compared to formula (f10) that prepared with direct compression technique, as shown in (fig.5). this was because pvp-k30, which is hydrophilic in nature, allowed penetration of the medium into the matrix and a more rapid release of cap in the last phase (26) . figure 4: effect of pectin on captopril profile in 0.1n hcl at 37ºc. 0 20 40 60 80 100 120 0 200 400 600 800 time(min) c o m u l a t i v e % o f d r u g r e l e a s e d 15%nahco3(f11) 20%nahco3(f12) 25%nahco3(f13) 0 20 40 60 80 100 120 0 200 400 600 800 tme(min) c o m u l a t i v e % o f d r u g r e l e a s e d hpmc200mg(f1) hpmc180mg ec20mg(f2) hpmc150mg ec50mg(f3) hpmc100mg ec100mg(f4) 0 20 40 60 80 100 120 0 200 400 600 800 time(min) c o m u l a t i v e % o f d r u g r e l e a s e d hpmc200mg(f1) hpmc180mg scmc20(f5) hpmc150mg scmc50mg(f6) hpmc100mg scmc100mg(f7) 0 20 40 60 80 100 120 0 200 400 600 800 time(min) c o m u l a t i v e % o f d r u g r e l e a s e d hpmc200mg(f1) hpmc180mg pectin20mg(f8) hpmc150mg pectin50mg(f9) hpmc100mg pectin100mg(f10) iraqi j pharm sci, vol.20(2) 2011 floating tablet of captopril 6 figure 5: effect of the method of preparation on captopril release profile in 0.1n hcl at 37ºc. figure 6: the release profile of captopril floating matrix tablet from selected formula (f11) and the reference floating mini-matrices and conventional tablet ofcapoten®50 mg tablet (squibb), at 0.1n hcl at 37ºc. a comparison in the release profile between the suggested formula f11 and the reference standard as well as the release profile of capoten® conventional tablet is shown in (fig.6), the floating system (fig.7) was able to control the drug release for 12h compared to capoten® conventional tablet which released the drug within a 30min. table 3 : drug release kinetic parameters for captopril from selected formula (f11). model r² slope intercept zero order 0.9806 11.865 ko 6.0287 first order 0.9714 -0.1102 k/2.303 2.0278 higuchi model 0.9711 33.702 kh –9.4228 korsmeyer – peppas model 0.9791 0.8772 n 2.8546 the drug release data of the selected formula (f11) was fitted to various models like zero order, first order, higuchi’s model and korsmeyer’s model. the calculated slope, the intercept and r² are shown in table (3). this formula (f11) was best fitted for zero order model with regression value ‘r²’ of 0.9806. to know the exact mechanism of drug release the data of f11 was also fitted to korsmeyer’s model. slope value of (0.45<n<0.89) suggested that the release of cap from floating tablets followed non fickian or anomalous transport mechanism. figure 7: photographs taken during in vitro buoyancy study of formula f10 in 300 ml 0.1 n hcl at different time intervals, (a) at zero time, (b) after 2 min, (c ) after 1 hr., (d) after 8 hr., (e) after 12 hr., (f) after 24 hr. 0 20 40 60 80 100 120 0 200 400 600 800 time(min) c o m u l a ti v e % o f d r u g r e le a s e d direct compression(f10) wet granulation(f11) 0 20 40 60 80 100 120 0 100 200 300 400 500 600 700 800 time(min) c o m u l a t i v e % d r u g r e l e a s e d f11 reference capoten50mg iraqi j pharm sci, vol.20(2) 2011 floating tablet of captopril 7 conclusion the present study was done to develop a controlled release floating gastro-retentive dosage form of cap to produce an effective treatment of hypertension to reduce the fluctuation in drug level caused by conventional tablets. the result of the in vitro dissolution study shows a controlled release of cap for 12h with a zero order release and prolonged residence time. thus, results of the current study clearly indicate, a promising potential of the cap floating system as an alternative to the conventional dosage form. references 1. rouge n., buri p., doelker e., drug absorption sites in the gastrointestinal tract and dosage forms for site-specific delivery, int. j. pharm, 1996, 136; 117–139. 2. agyilirah g.a., green m., ducret r., banker g.s., evaluation of the gastric retention properties of a 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313– 339. 14. chien y.w., potential developments and new approaches in oral controlled-release drug delivery systems. drug dev. ind. pharm., 1983, 9; 1291-1330. 15. banker gs, anderson nr, tablets. in:lachman l, lieberman ha, kanig jl, (eds.),the theory and practice of industrial pharmacy, 3rd ed., 1984; 283. 16. yihong q., yisheng c., geoff g. z. zhang, developing solid oral dosage forms: pharmaceutical theory and practice, 1 st ed. academic press. 2009; 169-170. 17. united states pharmacopoeia xxx. the usp convention. 2007. 18. meka l., kesavan b., chinnala k.m., vobalaboina v., yamsani r.m., preparation of a matrix type multipleunit gastro retentive floating drug delivery system for captopril based on gas formation technique: in-vitro evaluation, aaps pharmscitech, 2008, 9, 2; 612-619. 19. moore j., flanner h., mathematical comparison of curves with an emphasis on in vitro dissolution profiles. pharm. tech., 1996, 20, 6; 64-74. 20. paulo c., jose m.s., modeling and comparison of dissolution profiles. eur. j. pharm. sci., 2001, 13; 123-133. 21. siepmann j., peppas n.a., modeling of drug release from delivery system based on hydroxypropyl methylcellulose (hpmc). advanced drug delivery reviews, 2001, 48; 139–157. 22. tadros m.i., controlled-release effervescent floating matrix tablets of ciprofloxacin hydrochloride: development, optimization and in vitro–in vivo evaluation in healthy human volunteers, eur. j. pharm. biopharm., (2009), article in press. iraqi j pharm sci, vol.20(2) 2011 floating tablet of captopril 8 23. chi l., luigi g., james l., and matthew r., the use of hypromellose in oral drug delivery, jpp, (2005) 57:533-546. 24. nokhodchi a., zadeh d.h., zadeh m.f. and zadeh n.t., effect of various surfactants and their concentration on controlled release of captopril from polymeric matrices, acta pharm., (2008)58; 151-162. 25. krogel i. and bodmeier r., development of a multifunctional matrix drug delivery system surrounded by an impermeable cylinder, j. control. rel.,(1999) 61; 43-50. 26. yu dg, shen xx, branford-white c, white k, zhu lm, bligh sw, oral fastdissolving drug delivery membranes prepared from electrospun polyvinylpyrrolidone ultrafine fibers, nanotechnology., (2009) feb 4;20(5) ; 055104. iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 1 effect of some storage conditions upon the survival of some fungal spores raghad a. al-shikli *,1 , alaa a.abdulrasool ** and mustafa m. al-hiti * * department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract folic acid and multivitamin tablets containing aspergillus flavus penicillia spp. and cladosporia spores were prepared at a compression pressure of 148 mn/m 2 and stored at 35°c under different relative humidifies (75,85, and 95)% within air tight containers, to study the effect of storage condition on them, as well as ,the estimation of the microbial level of the raw materials intended to be used in the two kinds of tablets . result showed that some raw materials derived from natural origin were heavily contaminated with microorganism compared to that of synthetic origin ,the results also indicated the effect of relative humidity , types of fungal spore , and the hygroscopic nature of exicpient upon survival. multivitamin tablets showed more survival than folic acid tablets and this is due to the presence of more nutrients. no aflatoxin was obtained from both multivitamin and folic tablets at 35°c temperature; this is due to the temperature which is not an optimum temperature for aflatoxin b1 production. key words: storage conditions of tablet, fungal spores. الخالصة ٔذى خزَٓا فً دسظح حشاسج 2يٍكا ٍَٕذٍ / و 841ذى ذحضٍش حثٕب حايض انفٕنٍك ٔيحثٕب يعًٕعح يٍ انفٍرايٍُاخ ذحد ضغظ تٍُد انُرائط ٔنقذ نهًٕاد انخاو انًضرخذيح فً ذحضٍش َٕعٍٍ يٍ انحثٕب انًٍكشٔتًو ٔسطٕتح َضثٍح يخرهفح كًا ذى حضاب انرهٕز 53° انًٕاد األٔنٍح )انخاو ( راخ األصم انحٍٕاًَ أ انُثاذٍح انرً ذذخم فً انصُاعح انًضرحضشاخ انصٍذالٍَح أكصش يهٕشح يٍ انًٕاد أٌ .. ٔقذ ذثٍٍ يٍ خالل انثحس إٌ تعض األَٕاع قذ ضٍحاألٔنٍح راخ األصم انصُاعً ياعذا انرً ذثٍٍ يٍ انثحس احرٕائٓا عهى تكرشٌا يش نكثش ٔنزنك دعد انى دساصح ظشٔف تذخ يخرهفح ٔذأشٍشْا عهى ْزِ انفطشٌاخ ٔقذ أظٓشخ انُرائط اٌ انحثٕب انرً قأيد عًهٍح ا ذحٕي يعًٕع يٍ انفٍرايٍُاخ ذظٓش تُضثح اعهى يٍ انرهٕز تضثة يا ذحٌّٕ يٍ يٕاد غزائٍح ٔاٌ انفطش اصثاسظالس أظٓش عهى َضثح .8و غٍش يالئًح إلَراض افالذٕكضٍٍ ب °53ْٔزا ٌعٕد إنى إٌ دسظح ٍفالذٕكضٍَضثح نالهرهٕز يقاسَح تثقٍح انفطشٌاخ ٔنى ٌضعم اي ن introduction the microbiological quality of nonsterile pharmaceuticals (tablets) is largely determining by the microbial contamination of a raw materials. the effect of the manufacturing process and the fate of contaminating microorganisms during storage. several infection outbreaks which would be traced back to the use of heavily contaminated raw materials of natural origin have been reported (1) (2) (3) and (4) .during manufacturing the viability of microbial cells can be significantly affected by the drying process of granules (5) and by the actual compaction (6) (7) .the availability of water probably plays an important role. as long as tablets are stored under dry conditions spoilage due to growth of micro organisms is unlikely to occur (8) . however, in regions with a hot and humid conditioned, growth of contaminating microorganisms cannot be excluded. more ever ,in such countries pharmaceutical preparations are frequently stored under uncontrolled conditions and may be dispended in non protective packaging or even without any packaging at all. few studies that investigate the effect of storage on the microbiological quality of tablets (9) (10) (11) ,but little informations are available upon the fungi as contaminants in pharmaceutical industries and possible toxicogenic power (13) (15) (16) .the aim of this study was to investigate the effect of storage under different conditions on the growth of contaminating fungal spores.. 1 corresponding author email : raghad razook@yahoo.com received : 7 /2 / 2009 accepted : 24 /5 / 2009 mailto:razook@yahoo.com iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 2 materials and methods chemicals acetonitrile, acetone , ammonium hydroxide, anhydrous sodium sulfate, benzene, chloroform , glacial acetic acid , hydrous disodium hydrogen phosphate (na2hpo4 . 12h2o) . hydrochloric acid, methanol, potassium hydroxide, potassium hydroxide, potassium chloride, sulfuric acid, sodium 1-hexana sulfonate sodium perchlorate, sodium chloride, and tween 80 (supplied by bdh england monobasic potassium phosphate from fluka-swizerland hexana supplied by merch-w germany microcrystalline cellulse (avicel ph 101, avicel ph 301), folic acid, maize starch, vitamin b1 (thiamin mononitrite), vitamin b2 (riboflavin). vitamin b6 (pyridoxine hcl). methionine, talc and magnesium stearte supplied by fmc corporation and kindly supplied from (samara drug industries sdl. iraq). microorganisms aspergillus flavus, penicillia spp. and cladsporoia cladosporoids were obtained from college of agriculture, university of baghdad. cultures were stored on sabouraud agar slants following incubation at 25°c for five days fresh cultures were prepared every four weeks. culture media sabouraud dextose agar. rose bengal agar macconkey broth solution used for dilutions and preparation of spore suspension. relative humidity of the prepared solution. extraction solvent of aflatoxin. relative humidity containers relative humidity of the prepared solution. table (1) represents the percentage of the relative humidity rh% of the prepared solutions as prescribed by al taher, 1990 extraction solvent of aflatoxin according to howell and taylor(1980) the extraction solvent of aflatoxin consists of acetonitrile : kcl 4% w/v: hcl 5n a ratio of 450 ml: 10ml. relative humidity containers relative humidity containers consist of two glass containers connected one above the other and joined together by the cover of them and the cover is punched to allow the exchange of humidity between the two containers . table 1 : relative humidity of various solution assessment of microbial levels of the raw materials and active ingredients sample of the following raw materials were collected from various sources and were subjected to microbiological assay according to the bp 1998 , as shown in table (2) in order to determine their microbial contents. three types of raw materials were examined (gelatin, talc, and starch) representing animal , mineral, and botanical origin, respectively as well as those of synthetic origin such as avicel, methionine, thiamin, pyridoxine, riboflavin and folic acid . samples were taken aseptically using the pour plate and memberane filtration techniques to determine the microbial level in the raw materials . table 2 : isolation and idetification tests for specified microorganisms (bp , 1998) eeb-m-mossel enterobacteriaceae enrichment broth mossel ;vrvgla,violet red bile agar substance % of substance in solution rh% h2so4 23% 75% kcl saturated solution 85% na2hpo4.12h2o saturated solution 95% organism enrichment primary test secondary test confirmation enterobacteriaceae lactose broth 35-37 °c for 2-5 hr eeb-mossel 35-37 °c for 24-48 hr vrbgla 35-37 °c for 16-24 hr growth of gramnegatives e.coli as above macconkey broth 43-45°c for 18-24 hr macconkey agar 43-45°c for 18-24 hr indole 43.5-44.5°c biochemical salmonella as above for 5-24 hr. tbbg broth 42-43°c for 18-24 hr. then subculture on: dca,xlda or bga 35-37 °c for 24-48 hr. tsi agar 35-37 °c for 18-24 hr. biochemical serological ps.aeruginosa saline peptone 35-37 °c for 2-5 hr casein digest broth 35-37 °c for 24-48 hr. cetrimide agar 35-37 °c for 24-48 hr. oxidase test staph.aureus as for ps.aeruginosa above as for ps.aeruginosa above baird-parker 35-37 °c for 24-48 hr. coagulase. catalase,dnase test iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 3 preparation of dried spore powder a 0.1 ml aliquots of 7 days cultures of a. flavus, cladospoa and pencillia were inoculated onto the surfaces of predried sabouraud dextrose ager plates, these were incubated at 25°c for 5 days. after this spores were clearly visible on all the plates. three millimeters of stetrile water containing 0.1% tween 80 (as a dispersing agent) were added to each plates. the spores were then dislodged by using glass spreader. the spore suspensions were obtained then stirred by using a vortex mixer for one minute. the spore suspensions were then filtered through a sterile cotton wool in order to get rid of the hypha. the filtrates were then harvested by centrifugation at (10,000xy) for 10 min ) the superntanat liquids were then decanted and the residues were resuspended in 20 ml of sterile distilled water, washing were repeated three times . the number of spores of the resultant spore suspensisons was determined by aviable count technique, spore suspensions were adjusted so that the following suspensions were obtained, as 2.16x106 spore/ml for a flavus, 1.68x106 spore/ml forpenicillia spp. and 1.98x106 spore/ml for c.cladosporoids. tablet formulation multivitamin tablets and folic acid tablets were prepared using the formulas listed in tables (3) and (4), respectively. the following excipients were used. avicel ph 301 as a direct compression excipient, starch (5% w/w) as a disintegrant, magnesium stearate and stearic acid as lubricants. they were mixed with the active ingredient and compressed directly using a single punch tabletting machine with 7-mm flat-faced punches table 3 : the formula of the prepared folic acid tablet. ingredient amount/tab. folic acid 1 mg avicel ph 301 118.6 mg maize starch 6.5 mg mg. stearte 2.6 mg talc 1.3 mg total 130 mg table 4: the formula of the prepared multivitamin tablet ingredient amount / tab tianmin mononitrite 1.5 mg riboflavin u.s.p. 2.0 mg pyridoxine hcl u.s.p 2.0 mg methionine 2.0 mg avicel ph 301 105 mg maize starch 6.5 mg taic u.s.p 6 mg stearic acid ( powdered) 3.0 mg mg.stearte ( powdered ) 2.0 mg total 130 mg preparation of contaminated tabltes: for preparation of 500 contaminated tablets (0.3 ml. 30 ml) of a. flavus (0.4 ml 40ml) of penicillia spp and (0.3ml, 30ml) of c. cladosporoids were transferred to a sterile mortars and placed in the incubator until completely evaporation of water. dried spores were scraped off and were included in direct compression formulations by dry mixing to get 10 2 spore /gram and 10 4 spore/gram for each of a. flavus pencicilia spp and c. cladosporoids respectively. ingredients including dried microorganisms spores were weighed and lightly mixed in a glass morter by the method of geometric dilution technique for 20 minutes preliminary experiments had established that this method gives a uniform distribution of the microorganisms within the formuation screen in the lubricant (magnesium stearte or stearic acid) and mixed for an additional 5 minutes . quantities each of 130 mg were accurately weighed and poured into 7-mm diameter compresed between flat –faced punces using a single punch tableting machine which was disinfected with 70% alcohol and the feed shoe was heat sterilized before use. determination of viable number of spores in the prepared tablets: viable number of spores in prepareted tablets was determined immediately after their production at different compression forces and after storage up to 8 weeks eight tablets (total wt.=1gm) were disintegrated in tryptic soy broth (9ml) according tobp 1998 using a flask shaker and suitable serial dilution in tryptic broth were prepred. one –ml sample of each dilution was poured in sterile petridish and then 15 ml of molten dextrose agar was added to the plate. iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 4 the sample and molten sabouraud dextrose agar were mixed together in forward and backward movement and swirled movement. the plate were allowed to solidify on surface . the plates were incubated at 35°c for 2-5 days. survivals as colony forming units were estimated as the mean of triplicate determination and expressed as a percentage relative to an uncompressed control samples of the contaminated formulation. physical properties of tablets: thirty tablets were prepared using different compression pressure (137.9, 144.8 and 148.3mn/m 2 ) the physical properties of the tablets were determine (plumpton, 1982), these are tablet weight, thickness, friability, hardness (breaking strength), and disintegration time assay of tablets: an hplc method was used for the assay of multivitamin tablets and folic acid tablets. assay for pyridoxine hydrochloride and thiamin multivitamin tablets the assay for pyridoxine hydrochloride and thiamin in multivitamin tablets was done according to the u.s.p xxiv method. effect of storage under different relative humidities upon the survival of a. flavus, penicilline, and cladospori in folic acid and multivitamin tablets folic acid and multivitamin tablets containing aspergillus.flavus cladosporia ,and penicillia spores prepared at a compression of 148 mn/m2 and stored at 35°c under different relative humidity's (rh). the relative humidity were 75%, 85% and 95% within airtight containers. survival of the spores within the tablets was assessed as the mean viability for each group at time 0 and after 1,2, 4,6 and 8 weeks. the total viable counts of the uninoculated (control) folic acid and multivitamin tablets were measured directly after preparation and after 4,8 weeks of storage at 35°c and 75% rh, 85% rhor 95% rh. aflatoxin assay the amount of aflatoxin produced after storage the tablets (multivitamin and folic acid) at different relative humidities (75,85 and 95)% were assed at different time intervals using a modified method of howel and taylor (1981), the modification includes the use of twenty five grams of multivitamin and flic acid tablets stored at 4,6 and 8 weeks intervals. results and discussion microbiological quality of some raw materials used in the production of tablet and tablet ingredients microbiological evaluation of the raw materials used in the production of tablets is presented in table (5), the result show that synthetic materials such as folic acid, magnesium stearate ,thiamin, riboflavin, pyridoxine, methionine and microcrystalline cellulose (avicel ph 301) had no microbial contaminants thus they meet the requirement of the b.p 98 which specify that atotal viable aerobic count bacteria should be equivalent to or less than 103 c.f u/gm and a total viable count for fungi equivalent to or to less than 102 c.f.u/gm is accepted. microcrystalline cellulose ph 101 although it is a syntheyic raw material , it showed the presence of pseudoumonas aeruginosa (2x 10 2 cfu/gm) . samples taken from different parts of the microcrystalline ph 101 container showed apresence of pathogens in upper part of the container this is because microcrystalline (mcc) is a highly hygroscopic material (17) through its capillary action when it is exposed to air .table (5) also shows that raw materials of natural origin(maize starch ,gelatin and talc) had relatively higher microbial levels which are (7*10 2 ,9*10 2 and 102 c.f.u/gm)for starch, gelatin and talc, respectively than that of the synthetic origin. this finding is similar to that of lbrahim y.k.e 1991 which is due to the fact that materials of natural origin is rich in all the necessary requirement of growth needed by the microorganism. in addition, the results indicate that raw materials derived from animal and botanical origin had a higher microbial level than that of mineral origin. this is in agreement with the reported hypothesis of bonomi and negrriti,baggerman and kannegiter (22),(23),(3) .however, the microbial level obtained still below the b.p 1998 requirements. iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 5 table 5 : microbial contamination levels in some pharmaceutical raw materials for the production of folic acid and multivitamin tablets * c.f u/gm starch gelatin talc avicel ph ph 301 101 mag steara. folic acid b1,b2,b3 methionine total viable aerobic count 710 2 910 2 bacillus 10 2 ** ** **** **** total viable count for fungi **** **** **** ** ** **** **** enterobacteriaces **** **** **** ** ** **** **** escherchia coli **** **** **** ** ** **** **** staphylucocus aureus **** **** **** ** ** **** **** pseudomonas aerugenosa **** **** **** ** ** **** **** salomonella **** **** **** ** ** **** **** tablet evaluation: folic acid and multivitamin tablets were prepared as previously mentioned (tables 3 and 4 respectively). the prepared folic acid and multivitamin tablets were evaluated physically, chemically and microbiologially. the results are shown in table 6. table 6 : physical chemical and microbiological ( control ) evaluation of folic acid and multivitamin tablet effect of relative humidity upon the survival of .a flavus in multivitamin and folic acid tablets figure1,2 show the effect of storage under different relative humidities (95,85 and 75)% upon survival of aflavus. spores in multivitamin and folic acid tablets. the results indicate that at a contamination level of 10 4 spore/gm as shown in figure(1) and storage at 75% r.h a decrease in survival over the eight weeks storage period to 13% whilest storage at 95% rh, caused an initial reduction in viability followed by a substantial increase to 86% at the end of 8 weeks in multivitamin tablets. agermination, mycelia growth and sporulation occurred.the same behavior with folic tablets but with lower percentages. visible fungal growth and sporulation were tablet evaluation multivitamin folic acid weight (7 mm ) 130 mg 130 mg c. pressure 148.3mn/m 2 148.33mn/m 2 wt. uniformity 0.9% 0.9% hardness (kp) 6.6 0.4 6.5 0.6 thickness(mm) 2.85 2.85 friability % 0.2 0/2 disintegrationtime assay 2.3 min 2 min b pyridoxine % 133.78% b1 thiamin % 148.2% folic acid % 90% microbiological quality one day after preparati less than 10 * cfu/gm after storage for 8 weeks at 35 c , 75 % rh less than 10 cfu/gm after storage for 8 weeks at 35 c , 85 % rh less than 10 cfu/gm after storage for 8 weeks at 35 c , 95 % rh less than 10 cfu/gm iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 6 apparent on the tablets after 6 weeks of storage. further storage for 8 weeks caused a tablets to afragment. storage at 85% r.h also showed visible fungal growth after 6 week. viability of the organisms decreased during the first week of storage. this was accompanied by visible signs of mycelia growth. the decreased viability was probably due to the transition from dormant to mycelia state. tablets containing hygroscopic materials are much more to physically, adsorb substantial amounts of water. avicel has the ability to pick up moisture by capillary action and loosening of inter particulate hydrogen bonds on exposure to high humidities (8) .the water requirements for microorganisms varies depending on the organism (blair,1988) as mentioned before. for different mould spores the minimum r.h required for germination varies from 70 to 98% and the optimum temperature for growth of moulds vary from (23-40) °c.for aspergillus, 80% humidity aw=0.81 (23) is essential for spore germination and the optimum temperature is (30-40) °c.multivitamin tablets show higher growth than folic acid tablets within the storage time especially 95%, r.h this may be due to presence of more nutrients like vitamins (b1.b2 and b3)carbon source (starch) and amino acids (metithionine) or what is called nutrient availability. when nutrients are abundant, growth will be sustained but when only atrace nutrients are present, growth will be minimal. fungi need various nutrients in order to meet their energy needs and to form macromolecules such as proteins and dna. since fungi cannot synthesize carbohydrate, so the substrate showed contain these compounds ; however, they can growth in a substrate rich in proteins without carbohydrates , e.g. cheese , by using amino acids as carbon source. another important nutrient is nitrogen . all fungi can assimilate organic nitrogen compounds, depending on the species , certain vitamins must also be present in substrate, while the fungus itself synthesized others. the most important factors for growth are temperature , water activity and oxygen besides the presence of nutrients. figure 2 shows the effect of storage upon the percent of survival using 10 2 spore/gm of a. flavus in multivitamin and folic acid tablets.the results indicate that storge at 75% and 85%r.h showed a decrease in number of spores to zero percent in eight weeks duration whilst storage at 95% showed increase in number of spores to 420 percent for the same time. the result also indicate that there is a significant different (p<0.05) between the three humidites as well as there are significant different between multivitamin and folic acid tablets and a significant difference between the weeks of storage. the overall effects of these three factors (humidity, time, and type of tabletnutrients) is increase in number of spores/gm tablets with increase in humidity, time and type of tablet nutritents which are r.h 95%, eight weeks storage and more nutrient (multivitamin tablet). figure 1: effect of relative humidity upon survival of a. flavus (10 4 ) spore / gm compacted in multivitamin (mv) tablet and folic acid (fa ) tablet at (148.3 mn/m 2 ) and stored at 35c . l.s.d0.05 = 13.74 . figure 2 : effect of relative humidity upon survival of a. flavus (10 2 spore / gm ) compacted in multivitamin (mv) tablet and folic acid (fa ) tablet at (148.3 mn/m 2 ) and stored at 35c . l.s.d0.05 = 8.58 . the overall effects of these three factors (humidity, time, and type of tablet-nutrients) is increase in number of spores/gm tablets with increase in humidity, time and type of tablet nutritents which are r.h 95%, eight weeks storage and more nutrient (multivitamin tablet). 1000 iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 7 the effect of r.h upon survival of penicillia spp. spores in multivitamin and folic acid tablets: figures 3,4 show the effect of relative humidities upon survival of penicillia spp. spores in multivitamin and folic acid tablets. figure 3 : effect of relative humidity upon survival of penicillia spp. (10 4 spore / gm ) compacted in multivitamin (mv) tablet and folic acid (fa ) tablet at (148.3 mn/m 2 ) and stored at 35c . l.s.d0.05 = 27.87. figure 4 : effect of relative humidity upon survival of penicillia spp. (10 2 spore / gm ) compacted in multivitamin (mv) tablet and folic acid (fa ) tablet at (148.3 mn/m 2 ) and stored at 35c . l.s.d0.05 = n.s. the results indicate that for both contamination levels of 10 4 and 10 2 spore/gm, storage at 75%rh, more decrease in survival of penicillia over the eight weeks storage period than a. flavus. was obtained i.e the decrease was to 1.8% and zero for 10 4 and 10 2 spore/gram, respectively. whilst storage at 95% r.h however caused an initial reduction in viability followed by a substantial increase as germination mycelial growth and sporulation occurred for 10 4 spore/gram. visible fungal growth and sporulation were apparent on the tablets after six weeks storage but the survival level was less than a. flavus. further storage for eight weeks caused the tablets to fragment. on the other hand, storage at 85% r.h both contamination level 10 2 and 10 4 spore/gm, no visible fungal growth was apparent on the tablets after storage for eight weeks. penicillia spp.. 80% humidity is essential for spore germination aw=0.84 (23) and the optimal temperature is (25-30) °c for most penicillia spp. the maximal temperature is (28-35) °c .the result also show that multivitamin tablets have more survival than folic acid for the three relative humidites used. so, the over all data indicate that there is a significant difference (p<0.05) between the three humilities, two types of tablets nutrients.as there is increase in anumber of spores/gm tablet with the increase in relative humidity, storage time, and type of tablet nutrients .. the effect of storage under different relative humidities survival of cladosporia cladosporoids spores in multivitamin and folic acid tablets: figures 5,6 show the effect of storage at different relative humidities upon the percent survival of c. cladosporoids spores in multivitamin and folic acid tablets. the results indicate that there is a decrease in survival for both contamination level and storage at 75,85 and 95% r.h and for both types of tablets to zero percent. this is may be due to either temperature as cladosporia is psychrophilic mould (their low optimal temperature is 8-15) °c or water since 90-95% humidity is required for spore germinayion aw=0.88 (23) this is not due to pressure because when cladosporia incubated at 25°c fungal spores were shown to retain viability over an eight weeks period (slight decrease in viability).in accordance with these water requirements tablets inoculated with a. flavus and penicillia spores spoiled due to mould growth when stored under conditions (35°c , 95% r.h), while when stored under more moderate conditions (35°c 75% r.h) the tablets were not at risk to microbial spoilage both an optimum relative humidity and an optimum temperature are required before tablets are at risk of microbiological spoilage. 100 100 iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 8 figure 5 : effect of relative humidity upon survival of penicillia spp c. clado (10 4 spore / gm ) compacted in multivitamin (mv) tablet and folic acid (fa ) tablet at (148.3 mn/m 2 ) and stored at 35c . l.s.d0.05 = n.s. figure 6 : effect of relative humidity upon survival of penicillia spp c. clado (10 2 spore / gm ) compacted in multivitamin (mv) tablet and folic acid (fa ) tablet at (148.3 mn/m 2 ) and stored at 35c . l.s.d0.05 = n.s. the obtained results are in contrast to the results obtained by fassihi and parker (24) ,who found that tablets stored at a lower temperature (25°c) and at 96% r.h did not spoil due to mould growth (aspergillus niger and penicillia spp) when stored under these condition and the viability of mould spores decreased slightly . on the other hand the results are in agreement with that of the study of bos (19) in which a visible growth of a. niger during storage at 100 and 95% r.h of sta-rx tablets and lactose/starch tablets stored at 25°c and 31°c respectively was obtained. if contaminants are introduced into tablets prior to processing (i.e. from the raw materials) then they might sitll eventually be responsible for the spoilage of the finished products . the nature of contaminating organism , the relative humidity at which tablets are stored and the tablet nutrient all contribute to survival of the organisms . aflatoxin assay aflatoxin production is highly affected by type of substrate, the presence of minerals, the humidity and temperature (26 29) .results were obtained from thin layer chromatography (tlc) and in camparison with standard showed that both multivitamin tablets and folic acid tablets contain no aflatoxin b1.if the environmental conditions (temperature, and relative humidity) are not suitable for fungal growth this will lead to decrease in aflatoxin production to a level that cannot be detected. storage of tablets at 75% rh showed no aflatoxin b1 production, this result is in agreement with who (30) which determins that 83-85% rh is an optimum rh for afb1 production by a. flavus also this result is consistent with that obtained by austwish (31) that determined 85% rh and more at temperature of 25°c is an optimum rh for a. flavus growth in addition, lakshinarasimham, and (32) determined that 20°c and rh 73.5% are considered as an optimum conditions for storage without fungal contamination. furthermore storage at 85% rh showed no aflatoxin production, although rh is considerd as optimum for a. flavus growth but storage at 35°c is not optimum temperature for aflatoxin production since the optimum temperature for aflatoxin is (25-28)°c (33, 34) .also, no aflatoxin production was noticed when storage at 95% rh although rh is considered as an optimum for a. flavus growth but 35°c is not an optimum temperature for aflatoxin production . multivitamin tablet which contanins amino acid also show no aflatoxin production this because the storage temperature is not an optimum temperature for aflatoxin production so both an optimum temperature and relative humidity required for aflatoxin production . conclusions the results showed the existence of relationship between type of the raw materials used in pharmaceutical production and its microbial level . the results showed the effect of various storage conditions upon survival, which depends upon the type of fungal spore, the hygroscopic nature of the excipient and the relative humidity of storage. multivitamin tablet showed more survival than folic acid tablet and this is due to the presence of more nutrient. and finally no aflatoxin was obtained for both multivitamin and folic acid tablets at 35°c temperature, this is due temperature 100 100 iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 9 which is not an optimum temperature for aflatoxin b1 production. reference 1. kalliugetal. kallings, l.o: ringeretz o silverstorage : and emr feldt f. microbiologgical contamination of medical preparations, actapharma suec1966: 3:219-228. 2. sinugh p1 arirastaia b, kuma. a and dubey, n.k., microb e.coli, , 2008 (56) : 555-560. 3. kimiko farmati cheskii ehumnali, surivalal of microscopic fungi mnonsierile medical prrpation and auxillary subsiances , 2006: 40: 54-56 4. obuexaeco, obekwe i.f. , ogbimi ao 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survival of stupply lococcus aureson pharmaceutical solid dosage form j. pharmsei , 1973 ,621 1317-1320. 12. goda, n. k.s v. malath and r.uu suganthi effect of some chemical and herbal como under ongrowth of aspergillus parsiticesv and of latoxin production animal feed science of technology , 2004,116:281-291. 13. abdullamh1988 the isolation of aflatoxin from acacia and the incidence of aspergillus flavus in the sudan mycopathologia, ; 1988, 104: 143-144. 14. hitokoto, h 1978 h: muruzomi, s: wauke, tsakai, s: and kuratah, fungal contaminand mycotoxindetection of powdered herbal drugs appl. 15. wall heaver k,h micrological as aspects on the subject of oral solid dosage from pharm . ind , : 1977 , 39 491-497. 16. alhiti 1998 m.m.a al janabi s. the effect of some peservits on preservative on aspergilliaus flavus growth and its aflatoxin production irqi j.pharm scivol (10): 1998. 17. aulton 2000 et., me,: tebby. h,g white p.j.p journal pharm, pharmacol, 26 suppl, 59p-60p 2000 blair, t.c graham bucton; sally f,: bloomfield on the mechanism of kill of microbial contaminants during tablet compression – int –jpharm , 1991. 72: 111115 . 18. alfonoso 1985 19. bos 89, c,e van doorne ,h lerk c.f. on microbiological stability of tablets stored under tropical conditions, : 1989 , 55: 175183 . 20. ibrahim y.k.e, 1991y.k.e; orinoloupdf. comparative microboial levels in levels in wet granulation and direct compression method of tablet production, acta helv. : 1991 66: 11. 21. bonomi, e. and nergetti, f. 1977 bonomi e. and nergetti, f. studies on the microbial content of raw materials used in pharmaceutical preparation ann. 1 st . super sonita: 1977, 13:802-832. 22. panlo. j brasillianj of microbiology , 2006 ,vol. 37.no 1. 23. northolt md and bull ermine prevention of moldgrothed and bullerman prevention of moldgroth and tex production through control of environmental condition .j. too production , 1982 ,45: 519526. 24. fassihi and parker 1977 fassihi a.r and parker m.s the of water activity and oxygen tension upon the survival of aspergillus and penicllia tablet int biodeterior bull.1977 , 13-80 25. plumpton 1982 e.j studies upon the survival of various microorganism in solid dosage froms ph.d. thesis university of manchester. 1982 26. reddy s.v m.d kiram r.m. uma, k. thir umaladai and d.v.r ready aflatoxin b different grades of chillies, 2001, 18: 553558, 27. elad. d, .risk uma, assessment grades of malicious bio contamination of food tournal food protection, 2005, 68:13021305. 28. shar pirar.o. control of mycotoxin storage and technigagus for their decontaminal my cotoxin in food detection and control. iraqi j pharm sci, vol.19(2) 2010 fungal contamination and storage conditions 10 wood head publishing cambridge u.k2004, 190-223. 29. northolt m.d. the effect water activity and temperature the production of some my cotoxines diss wageninger 1979 30. who, 2003. laboratory manual 2 nd ediction interim guidelines who. 31. austwish , p.k.c; and ayerst, g.groundaut micrflora and toxicily –chem. ind 1963 ,55 -61. 32. mukher 95 :and lakshminarasimiham a.b aflatoxin contamination of storage under controlled conditions int j med microbiol virol parassitol infect dis ., 1995 , 282(3): 387-43. 33. bennet jw, klichm my cotoxinschinical microbiology review ; 2003,16:497-516. 34. paterson rrm; venancio a; liman solution to penicillium taxonomy crucy to my eotoxin rerearch and heath research in microbiology ; 2004 ,155: 507-513. development and in vitro evaluation of bioadhesive vaginal tablet using econazole nitrate as a model drug iraqi j pharm sci, vol.20(1) 2011 econazole nitrate bioadhesive vaginal tablet 57 development and in vitro evaluation of bioadhesive vaginal tablet using econazole nitrate as a model drug dina w. ameen* ,1 *department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq . abstract in this study, a bioadhesive dosage form of eoconazole nitrate for vaginal delivery was designed using a combination of bioadhesive polymers: carbopol 941 p and sodium carboxymethylcellulose or methylcellulose in different ratios. the bioadhesive strength was evaluated by measuring the force required to detach the tablet from sheep vaginal mucosal membrane. it was found that the bioadhesive force was directly proportional to carbopol 941 p content in the different formulae. the formulae were tested for their swelling behavior using agar gel plate method. the results showed that formulae containing a combination of carbopol 941 p and sodium carboxymethylcellulose had greater swelling index than those containing a combination of carbopol 941 p and methylcellulose. in vitro drug release study showed that the release of eoconazole nitrate from formulae containing sodium carboxymethylcellulose was faster than its release from those containing methylcellulose.the dissolution profiles of the formula containing carbopol 941 p alone and those conaining various combinations of carbopol 941 p and methylcellulose could be considered similar since their calculated similarity factor values were >50. formula f3 composed of cp/nacmc in a ratio 1:1 showed moderate swelling, good bioadhesion and retardation of drug release. thus, it may be considered a good candidate for vaginal bioadhesive dosage forms. key words: bioadhesive, econazole nitrate, vaginal tablet. الخالصة ع كم يٍ ـــًـنالسرخذاو انًٓثهي. تد الَسدح انحيٕيحاتصك هرينُرشاخ االيكَٕـاصٔل ذى ذصًيى شكم دٔائي ذساسحان في ْزِ الَسدح انحيٕيح(تُسة يخرهفح ح تا)تاعرثاسْا تٕنيًشاخ الصم م سهيهٕصيثٔ انًأثيم سهيهٕص انصٕديٕو انكاستٕتٕل يع كاستٕكسي ي ح عٍ انغشاء انًخاطي انًٓثهي نهُعاج, يٍ خالل لياس انمٕج انًطهٕتح نفصم انحث ٕج األنرصاقــذى ذمييى انمنرصييغ انحثٕب انًٓثهيح. غ انًصُعح ـــــنهصي ًد دساسح لاتهيح االَرفاخذ . يا يع يحرٕٖ انكاستٕتٕل في انصيغكاَد يرُاسثح طشد لٕج االنرصاق رثيٍ اٌ ف كاستٕكســــي يثيم سهيهٕص انصٕديٕو أٌ انصيغ انًحرٕيح عهٗ خهيط يٍ انكاستٕتٕل ٔ ٔلذ اظٓشخ انُرائح .تاسرخذاو صحٌٕ االكاس من الصيغ المحتوية دساسح ذحشس انعماس أظٓشخ أٌ ذحشسانعماس .هيهٕصيٍ ذههك انًحرٕيح عهٗ خهيط انكاستٕتٕل ٔانًثيم س أَرفاخاكثشأ كاَد ليى عايم ٕص.يثيم انسهيه انصيغ انًحرٕيح عهٗ ذهك ذحشسِ يٍ يٍ اسشعكاٌ سهيهٕص انصٕديٕو اني يثيم ــــعهٗ كاستٕكس ٔتانراني يًكٍ ٠٥عهٗ يٍ أانرشاتّ انًحسٕتح نهصيغح انًحرٕيح عهٗ انكاستٕتٕل فمط ٔانصيغ انًحرٕيح عهٗ خهيط يُّ ٔانًثيم سهيهٕص ذحشساخيذا نهذٔاء ٔ اَرفاخا يعرذال ٔخاصيح انرصاق خيذج نزنك يًكٍ ( f3) ظٓشخ انصيغحأ. اعرثاس ًَط رٔتاٌ ْزج انصيغ يرشاتٓا اعرثاسْا يششحا خيذا نرطٕيش شكم دٔائي يٓثهي لاتم نالنرصاق. introduction vaginal candidiasis (vc) is now recognized as a major health problem for women of childbearing age worldwide. the majority of genitourinary tract fungal infections are caused by candida albicans. (1) approximately 75% women will have a vaginal infection with a candida strain during their life and about 40 to 50% of them will suffer a second one, and a small percentage will show a chronic course. (2) antifungal imidazole drugs are a mainstay in the treatment of fungal infections. imidazole drugs have low aqueous solubility because of their hydrophobic structures. this can have a negative impact on antifungal efficacy, side effects, pharmacokinetic variability and the development of drug resistance. (3) intravaginally delivered drugs may fail to achieve high concentrations at the site of infection because of their fairly prompt removal from the vaginal compartment through physiological secretions, thus, limiting residence time and impairing therapeutic efficacy of the drug and make multiple administrations necessary for treatment. (4,5) moreover, conventional vaginal formulations are associated with disadvantages of leakage and messiness thereby causing inconvenience to the user. (6) attempts are being made to develop novel vaginal drug delivery systems that can meet the clinical as well as the requirements of the patients. (7) therefore localized mucoadhesive dosage forms may represent a suitable formulation design to improve both the bioavailability of the drug and patient compliance. (8) 1 corresponding author email : dwahas@yahoo.com received : 10/10/2010 accepted : 5/4/2011 iraqi j pharm sci, vol.20(1) 2011 econazole nitrate bioadhesive vaginal tablet 58 bioadhesion can be defined as a phenomenon of interfacial molecular attractive forces amongst the surfaces of the biological substrate and the natural or synthetic polymers, which allows the polymer to adhere to the biological surface for an extended period of time. (9) in the pharmaceutical sciences, when the adhesive attachment is to mucus or a mucous membrane, the phenomenon is referred to as mucoadhesion. (10) the most commonly used mucoadhesive polymers that are capable of forming hydrogels are synthetic polyacrylates, polycarbophil, chitosan, cellulose derivatives, hyaluronic acid derivatives, pectin, tragacanth, carrageenan and sodium alginate. (11) econazole nitrate (en) is an imidazole antifungal agent and is mostly administered topically for the treatment of skin infections and (vc) the currently commercially available applications based on en include creams or pessaries. (8) the minimum inhibitary concentration (mic) of en for complete inhibition of fungi is 0.0011000µg/ml. (12) ghelardi et al. found that a complexation of econazole with the mucoadhesive polycarbophil significantly improved the therapeutic benefit of the drug in the topical treatment of (cv) in mice. (4) albertini et al have investigated the preparation of mucoadhesive microparticles as an innovative vaginal delivery systems for econazole nitrate (en) able to enhance the drug antifungal activity prepared by spraycongealing. (8) the aim of this study is to investigate the use bioadhesive polymers: carbopol 941 p and sodim carboxymethylcellulose or methylcellulose in different ratios to develop en vaginal tablet .the developed tablets were evaluated for their physical and bioadhesive characteristics, and in vitro release of en. materials and methods materials econazole nitrate (en) supplied by samarra drug industries, carbopol 941(cp), (goodrich,usa),sodium carboxymethylcellulose (nacmc), methylcellulose (mc), sodium lauryl sulfate (sls), and magnisium stearate (bdh chemicals, ltd),liverpool, england) ,agar no.1( oxoid limited , england), all other reagents are of analytical grade. methods preparation of en bioadhesive tablets different formulae of en bioadhesive tablets were prepared, as shown in table (1), by mixing cp alone or its mixture with either nacmc or mc at different ratios with the drug in a glass mortar. magnesium stearate equivalent to 1% was added to the mixture as lubricant then the formulations were directly compressed into tablets using flat face 12 mm punch. each tablet weighed approximately 750 mg and had thickness of about 4.2 mm. table 1 : the composition of various en bioadhesive tablet formula # en (mg) cp/na cmc cp/ mc mg stearate (mg) f1 150 3:0 7.5 f2 150 2:1 7.5 f3 150 1:1 7.5 f4 150 1:2 7.5 f5 150 2:1 7.5 f6 150 1:1 7.5 f7 150 1:2 7.5 total weight of the polymer(s) per tablet is 600mg in all of the formulae. evaluation of bioadhesive tablets hardness: the hardness of the tablet was measured with the help of a monsanto hardness tester. three tablets from each batch of formulations were tested. then average hardness and standard deviation were calculated. (13) friability : the friability test was done using roche’s friabilator. twenty tablets from each formulation were weighed (w1) and tested at a speed of 25 rpm for 4 min. after removing of dust, tablets were re-weighed (w2) and friability percentage was calculated using the following equation: (13) friability = ------eq. (1) weight variation: 20 tablets from each formulation were weighed and mean and standard deviation of the weight were determined. tablet thickness: thickness of the tablets was measured on 3 tablets with a micrometer caliper. drug content uniformity: one tablet of each formula was ground in a mortar; an accurately weighed amount of the powder equivalent to 50 mg of en was transferred into 100 ml volumetric flask and dissolved with methanol. the resulting solution is filtered and assayed spectophotometrically for en at 271 nm. (14) the drug content was determined using preconstructed calibration curve of en in iraqi j pharm sci, vol.20(1) 2011 econazole nitrate bioadhesive vaginal tablet 59 methanol as shown in fig.(1). no interference from any of the tablet components with the absorbance of en was observed under the conditions of the assay procedure. the test was performed in triplicates. figure 1 : calibration curve of ecn in methanol. surface ph tablet’s surface ph was determined by adding 2 ml of distilled water to one tablet of each formula, placed in separate beakers, they were allowed to swell at room temperature for 2 hours. ph measurement was done by contacting the electrode with the tablet surface for one minute (15) . swelling study vaginal tablets were weighed individually (w1), and placed separately in 2% agar plates at 25°c ± 0.1. the tablets were removed at ½,1,2,3, and 4 hours, excess surface moisture was removed by wiping with filter paper and then the tablets were reweighed (w2). the swelling index was calculated using the following formula: (16) . swelling index % = --eq. (2) ex vivo bioadhesive strength measurement the balance method reported by parodi (17) with some modification was used for determining the ex vivo bioadhesive strength. a female sheep was sacrificed and the sheep vaginal mucosa was obtained immediately after slaughter. the mucosal membrane was separated from other tissues, thoroughly and carefully washed with normal saline and kept frozen at – 18°c until the time of experiment. (18) at the time of experiment the tissue was thawed at room temperature in normal saline and carefully cut to fit the mouth of a glass beaker filled with citrate buffer ph 4.2. the tissue was held tightly onto the beaker with a rubber band, and then it was tightly fitted into a glass beaker filled with citrate buffer. a tablet was glued to the bottom of the left pan of a balance and the beaker with the tissue on top was positioned under the left pan so that it almost touched the tablet. a plastic container was put on the right pan. the two pans were balanced, and then a 5 gm weight was added to the left pan which caused the lowering of the pan along with the glued tablet over the tissue beneath. the assembly was kept in that position for 5 minutes, and then water (equivalent to weight) was added into the container on the right pan at a rate of 100 drops/min. water addition was continued until the tablet detachment toke place. (19, 20) the experiment was performed in triplicates and mean ± sd were reported. dertermination of solubility the solubilities of ecn in the dissolution media were determined by adding excess amount of the drug into two flasks one containing 1% sls in distilled water and the other contained 1% sls in citrate buffer ph 4.2. the flasks were stoppered and kept shaken in a water bath at 37° c for 48 hrs. samples were withdrawn, filtered, properly diluted, and determined spectrophotometrically. the solubility study was done to confirm the existence of sink condition during the dissolution of the vaginal tablet. in vitro en release study the release of en from the prepared vaginal tablets was determined using usp dissolution apparatus ii (paddle method). the dissolution media were 900 ml of either 1% sls in distilled water or 1% sls in citrate buffer ph 4.2 kept at 37° c ± 0.1. a tablet was glued to the bottom of the jar, and the apparatus was rotated at 50 rpm. two different media were used to examine the difference between the release of the drug in water and in buffer. samples (5 ml) were withdrawn at specified time intervals and replaced by an equal volume of the dissolution medium. en was determined spectrophotometrically at 271 nm. (14) dissolution was performed as triplicates. dissolution data analysis the dissolution profiles of the prepared formulae in citrate buffer were compared using ƒ2 similarity factor. (8,21) the similarity factor is a logarithmic reciprocal square-root transformation of the sum of squared error and is a measurement of the similarity in the percentage of dissolution between two curves. (21) r² = 0.9997 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 a b so rb a n ce conc. of ecn (mg/ml) iraqi j pharm sci, vol.20(1) 2011 econazole nitrate bioadhesive vaginal tablet 60 ƒ2 =50 log { ∑ } ---eq.(3) where n is the sampling number, rt and tt are the percent dissolved of the reference and test products at each time point t. two dissolution profiles are considered similar when the ƒ2 value is greater than or equal to 50. (22) en release mechanism study the mechanism of en release was determined using the following equations: (23) ----------eq.(4) --------eq.(5) where, : the fraction of drug released at time t k: release characteristic constant n: release exponent indicative of release mechanism. when n is close to 0.5, the drug is released from the polymer with a fickian diffusion mechanism (higuchi model). if 0.5 < n < 1 this indicates anomalous or non-fickian release, while if n= 1 this indicates zero-order release (case ii transport). lastly, when n > 1.0, super case ii transport is apparent. (24) fickian diffusional release occurs by the usual molecular diffusion of the drug due to a chemical potential gradient. case ii relaxational release is the drug transport mechanism associated with stresses and statetransition in hydrophilic glassy polymers, which swell in water or biological fluids. this term also includes polymer disentanglement and erosion. (25) release kinetics study in order to determine kinetics of drug release from the vaginal bioadhesive tablets , drug release data were fitted to zero order, first order, and higuchi square root. (26,27) zero-order equation: q = q0+k0t -------eq.(6) first-order equation: log q = log q0 + k1t/2.303 ---------eq.(7) higuchi´s equation: q = k2t 1/2 -------------eq. (8) where q is the amount of drug dissolved in time t, q0 is the initial amount of drug in the solution (most times, q0= 0), k0, k1, k2 are the zero, 1 st order and higuchi release constants respectively. (26) statistical analysis anova based on least significant difference test (lsd) was used for comparison of differences in means of bioadhesive strength of the tablets, swelling indices, and zero order dissolution rates. student’s t-test was used to compare the difference in mean dissolution rate constants of each formula in buffer and in distilled water. in all cases, (p<0.05) was considered significant. statistics were done using microsoft excel 2007. results and discussion physical evaluation of tablets the physical properties of the tablets and drug content are summarized in table (2). the mean weight of vaginal tablets ranged from 748.4 to 763 mg. no batch varied more than 5% of the average mass. concerning the uniformity of drug content, all of the formulations were acceptable since the amount of en in each of the tested tablets was within the range of 97.5%–101% indicating uniform mixing of the tablet formulation. these results are in compliance with the requirements of usp 28. (28) average hardness of tablets belonging to various formulae indicated high strength, that was also evident in the results of the friability test which were less than 1% for all formulae. table 2: results of the physical evaluations conducted on en bioadhesive vaginal tablets prepared formula # weight variation (mg) ± sd a friability (%) a hardness (kp) b drug content (%) ±sd b tablet thickness (mm) b surface ph b f1 748.4± 11 0.1±0.005 >13 98.5±0.2 4.4±0.02 2.75±0.01 f2 757.2±3.5 0.05±0.01 >13 99.3±0.15 4.3±0.03 3.9±0.05 f3 763±10 0.23±0.015 12.2 101±0.5 4.15±0.02 4.2±0.01 f4 757±1 0.4±0.026 8 99.2±0.7 4.2±0.01 4.8±0.02 f5 750±2 0.3±0.02 >13 96.4±0.75 4.3±0.04 3.1±0.03 f6 756±1.9 0.08±0.015 >13 97.5±0.5 4.24±0.03 4±0.07 iraqi j pharm sci, vol.20(1) 2011 econazole nitrate bioadhesive vaginal tablet 61 f7 758±3 0.05±0.007 9 99.8±0.6 4.2±0.06 3±0.05 a: n = 20 , b: n = 3 , n= number of replications surface ph the surface ph values of the prepared formulae were acidic and ranged from 2.75 4.8.formulae with ph < 4.2 are not favorable because they are too acidic and may cause tissue irritation. formula f3 has a ph value similar to that of the vaginal tract. the swelling index the swelling behavior of a bioadhesive system is an important property for uniform and prolonged release of drug and bioadhesiveness. (19) the swelling of the tablets as a function of time is shown in fig.(2), at initial stage , there was an initial rapid rise in si due to the entry of water via metastable pores in the polymer matrix of the tablets .this mechanism , known as swelling hysteresis, was followed by swelling caused by diffusion processes. (29) figure 2: swelling index of vaginal tablets formulae f1 to f7. the swelling indices (at 4 hrs) of the various vaginal tablets are summarized in table (3) the results showed that formula f1 containing cp alone had significantly lower si (p<0.05)than those containing mixtures of cp and nacmc. the si was directly proportional to the ratio of nacmc in the formulae, this appears to be due to the excessive water uptake of nacmc containing tablets attributed to greater hydrophilic nature of nacmc. (30) water causes ionization of carboxylic groups of nacmc and carbopol with subsequent repulsion and relaxation of the polymer chains that result in increase in water penetration and hence increase in si with time. (31) the structural integrity of multilayered formulations may be disrupted by the hydration of the mucoadhesive component. (32) as seen in formula f4 which showed extensive swelling that resulted in disrupting the structural integrity of the tablet. on the other hand, the inclusion of mc in the formulation caused a reduction in the si. mc hydrates on contact with water forming a viscous gel layer at the surface of the tablet, which might hinder water penetration contributing to the decreased swelling observed in formulae f5, f6, and f7. table 3 : results for the si at 4 hr and bioadhesive strength (n) formula # si (% ) at 4hr bioadhesine strength (n) f1 65.1±1.54 1.159±0.12 f2 117±1.53 0.6468±0.024 f3 129±8.4 0.6223±0.02 f4 182±3.63 0.49±0.08 f5 61±3.2 0.343±0.035 f6 56±3.9 0.392±0.05 f7 47.3±4.02 0.294±0.15 bioadhesive strength measurement the results of bioadhesive strength measurement are shown in fig.(3).it was found that formula f1 showed the maximum bioadhesion. the high bioadhesive property of cp is reported to be due to: (i) carboxyl groups present on its acrylic acid backbone, which possess an ability to interact with sialic acid molecules present in the mucus layer through hydrogen bond (30,31) and (ii) to the formation of secondary mucoadhesive bonds with mucin because of rapid swelling and interpenetration of the polymer chains in the interfacial region. while other polymers undergo only superficial bioadhesion. (15) the addition of a nacmc caused a significant reduction in the bioadhesiveness (p< 0.05), the bioadhesion decreased progressively with increasing the ratio of nacmc. similar results were reported by emami et al. (19) nacmc is a salt of an anionic polymer, which generates the carboxylic group in water and then swells and dissolves; it has excellent recognized mucoadhesive properties (8) , but the considerable swelling characterizing nacmc may be one of the factors responsible for its reduced adhesion, as swelling induces overextension of hydrogen bonds and other forces. besides, water molecules may bind to polymer groups required for bioadhesion. (33) formulae f5, f6, and f7 containing mc which is a non ionic polymerexhibited the least bioadhesive 0 50 100 150 200 0 2 4 6 s w e ll in g i n d e x % time(hr) f1 f2 f3 f4 f5 iraqi j pharm sci, vol.20(1) 2011 econazole nitrate bioadhesive vaginal tablet 62 force among all of the formulae, this may be attributed to the absence of a proton-donating carboxyl group which reduce its ability for the formation of hydrogen bonds. (33) anionic polymers have been found to form stronger adhesion when compared to neutral polymers. (9) it was shown that nacmc adhered more strongly to mucus than hydroxypropylmethyl cellulose, or mc. (34) the reduction in the bioadhesiveness of cp in the presence of mc might be attributed to the formation of hydrogen bonds between cp and mc thus reducing the number of carboxyl groups of cp available for bonding with the mucus membrane. in general, the swelling state of polymer contributes to its bioadhesive behavior. (24) however, they are not quite correlated in this study. similar findings were reported by nafee et al (33) figure 3: bioadhesive strength measured for the formulae f1 to f7. dertermination of solubility the solubilities of ecn in 1% sls in distilled water and 1% sls in citrate buffer ph 4.2,were found to be 4.37mg/ml and 4.4 mg/ml respectively. the drug concentration in the dissolution medium should not exceed 15% to 20% of saturation solubility of the drug in order to provide sink conditions. (35) in vitro en release study the results of release study of en bioadhesive tablets in citrate buffer ph 4.2 and in distilled water are shown in figures (4.a,4.b,5) respectively. generally, the release was higher in distilled water than in buffer. the release profiles for the different formulae in water were significantly different (p< 0.05) from those in buffer except for formulae f3 and f4. formula f1 containing cp alone showed very slow release in both of the dissolution media. at ph 4.2, cp carboxylic groups are mostly unionized; this is associated with high coiling and proximity of carboxylic groups leading to intramolecular hydrogen bonding. the cross linking of cp affects also elasticity of the chains as water penetrates inside the polymer network and this leads to entrapment of the drug inside the cross linked network of the polymer. (9,31) on the other hand, water causes ionization of carboxylic groups of cp with subsequent increase in water penetration. the higher the uptake of water by the polymer, the greater the amount of drug diffused from the polymer matrix. (15) these reasons may explain the greater in release of en in distilled water.formulae prepared using mixtures of cp and nacmc, exhibited faster release than formula f1. the increase in drug release was directly proportional with nacmc content, as shown in figure (4.a). figure 4.a : release profile of en in citrate buffer ph 4.2 0f formulae 1,2,3,and 4. figure 4.b : release profile of en in citrate buffer ph 4.2 of formulae f1, f5, f6 and f7. 0 20 40 60 80 100 120 0 5 10 % e n re le a se d time (hr) f1 f2 f3 0 20 40 60 0 5 10 % e n r e le a se d time (hr) f1 f5 f6 iraqi j pharm sci, vol.20(1) 2011 econazole nitrate bioadhesive vaginal tablet 63 figure 5: release profiles of en bioadhesive tablets in distilled water. as nacmc becomes hydrated and forms a swollen gel, dissolution and surface erosion of this water logged gel occur simultaneously, (36) resulting in rapid release. these results are in agreement with those obtained by emami et al. (19) formulae f5, f 6 and f7 showed slow release, as shown in figure (4.b), which may be attributed to the builds up of an excessively viscous gel around the tablet which is more resistant to water penetration and erosion. (29) a combination of polymers may show additive or synergistic release retardation. synergism in gelling ability of a combination of polymers may be due to molecular interaction between the individual polymers. (37) .the similarity factor values for the release profile in citrate buffer are summarized in table (4), which shows that formulae f1, f5, f6 and f7 have a similar dissolution profile since ƒ2 values are greater than 50.this may suggest that combining mc with cp does not have a considerable effect on the release of the drug. formulae f3 and f4 don’t resemble any other formula since their ƒ2 values were less than 50. table (5) shows the exponents n, related to drug release kinetics, which were in all cases>1except formula f4, indicating that drug release follow super case ii mechanism in which the release is time dependent and controlled by the relaxational process due to the swelling of the polymeric network. (37) while formula 4 had n = 0.786 corresponding to non fickian transport. table (6) shows the correlation coefficients for fitting the release profiles to different release kinetics, the results show that most of the formulae have zero order release kinetics except formula f4 which follow higuchi model of release and formula f6 which followed 1 st order release kinetics, the reason behind these differences may be the difference in the swelling and erosion behavior of the polymers used. table 4 : values for the similarity factor ƒ2 for the release profiles in citrate buffer. formula # f2 f3 f4 f5 f6 f7 f1 39.67169 29.07959 11.89739 73.10762 55.12816 68.22848 f2 48.08251 17.92629 43.41324 50.32749 45.33722 f3 23.31974 31.26696 35.83019 32.1486 f4 12.68356 14.27366 13.12512 f5 64.85141 82.20482 f6 67.99835 table 5: values for k, n and r 2 by regression of log mt/m∞ vs. log t. formula # k n r 2 f1 0.0214 1.487 0.99 f2 0.046881 1.365 0.99 f3 0.063533 1.344 0.994 f4 0.334195 0.786 0.994 f5 0.0281 1.377 0.988 f6 0.040926 1.246 0.964 f7 0.036308 1.26 0.988 table 6 : release kinetic parameters with correlation coefficient for designed formulae of en bioadhesive vaginal tablets in citrate buffer. formula zero order r 2 1 st order r 2 higuchi r 2 highest correlation or best fit f1 0.978 0.911 0.945 zero order 0 20 40 60 80 100 120 0 5 10 % e n r e le a se d time (hr) f1 f2 f3 f4 iraqi j pharm sci, vol.20(1) 2011 econazole nitrate bioadhesive vaginal tablet 64 f2 0.978 0.902 0.906 zero order f3 0.991 0.88 0.959 zero order f4 0.98 0.931 0.996 higuchi f5 0.957 0.982 0.874 zero order f6 0.95 0.987 0.899 1 st order f7 0.967 0.929 0.912 zero order conclusion the results of the present investigation indicate that formula f3 which is composed a mixture of cp and nacmc in a ratio of (1:1) may be considered a good candidate for vaginal bioadhesive dosage forms, since it has a suitable drug release profile, good bioadhesion, moderate swelling, and a surface ph of 4.2 simulating to that 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for buccal tablet formulation, drug development and industrial pharmacy. 2004; 30(9), 985–993. 34. a. ahuja ,r. khar, j.ali, mucoadhesive drug delivery systems. drug dev. ind. pharm.1997; 23, 489–515. 35. shahla jamzad and reza fassihi role of surfactant and ph on dissolution properties of fenofibrate and glipizide— a technical note aaps pharmscitech 2006; 7 (2) article 33 36. a. el-kamel, m. sokar, v. naggar, s. al gamal, chitosan and sodium alginatebased bioadhesive vaginal tablets, aaps pharmsci 2002;44. 37. m.varma, a.kaushal, a. grag, s. grag, factors affecting mechanism and kinetics of drug release from matrix based oral controlled drug delivery systems, am. j. drug deliv 2004; 2 (1), 43-57. 38. d. krznar, j. filipovi, b. zorc, m.zovko, dissolution of celecoxib from mucoadhesive disks based on polyaspartamide derivatives acta pharm. 2006; 56, 463–471. iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 66 belief about medications among type 2 diabetic patients attending the national diabetes center in iraq. esraa a. hussein *,1 , dheyaa j. kadhim** and tawfeeq f. al-auqbi *** * ministry of health/environment, baghdad, iraq. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. ***al-mustansiriya university, national diabetes center, baghdad, iraq. abstract diabetes mellitus is a common health problem worldwide counting about 1.2 million cases in iraq in 2015. taking in account of the patient’s beliefs about the prescribed medication had been reported to be one of the most important factors that affects adherence where holding positive beliefs about medications is a prerequisite for intentional adherence. the aim of the current study was to investigate and assess beliefs about medicines among type 2 diabetic patients and to determine possible association between this belief and glycemic control as well as some patient-specific factors. this study is a cross-sectional study carried out on 380 (mean age 56.58± 10.06 years) already diagnosed t2dm patients who attended the national diabetes center, al-mustansiriya university – baghdad/ iraq during december-2016 to march-2017. belief about medicine was assessed by using an arabic version of the questionnaire. the patients had a stronger agreement with the mean necessity scale (19.29) than the mean concern scale (14.27). the majority of the patients (76.3%) had strong beliefs in the necessity of anti-diabetic treatment for maintaining good control of diabetes (scores of specific-necessity was greater than score of specific-concern). however, (18.4%) of the patients reported strong concerns about the anti-diabetic treatment (scores of specific-concern greater than score of specific-necessity). the small number of the patients (5.3%), have equal scores for specific-necessity and specific-concern scores. the patient's belief about medicine was found to be poor predictor of good glycemic control. keywords: type 2 diabetes mellitus, beliefs about medicines, beliefs about medicines questionnaire (bmq), national diabetes center, iraq . المعتقدات عن األدوية لدى مرضى النوع الثاني من السكري المراجعين للمركز الوطني للسكري في بغداد / العراق ***، توفيق فاخر العقبي **جبار كاظمضياء ، 1*,اسراء عواد حسين ، بغداد ، العراق . وزارة الصحة والبيئة* فرع الصيدلة السريرية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق . ** المركز الوطني للسكري ، الجامعة المستنصرية ، بغداد ، العراق . *** خالصةال مليون حالة من مرض السكري قد سجلت في 2.1مرض السكري مشكلة صحية مشتركة في جميع أنحاء العالم مع حوالي يمثل واحدة من أهم العوامل التي تؤثر على االلتزام تعد . مع األخذ بعين االعتبار ان معتقدات المريض حول الدواء الموصوف1122العراق في عام المعتقدات اإليجابية حول األدوية هي شرط أساسي لاللتزام المتعمد بالعالج الموصوف للمريض . الهدف من الدراسة الحالية بالعالج ، حيث ان وتحديد االرتباط المحتمل بين هذا االعتقاد ومراقبة نسبة السكر في 1هو فحص وتقييم المعتقدات حول األدوية ، بين مرضى السكري من النوع مريض تم تشخيصهم سابقا بداء السكري 081العوامل الخاصة بالمريض. هذه الدراسة هي دراسة مستعرضة أجريت على الدم، وكذلك بعض بغداد / العراق خالل -لسكري، الجامعة المستنصرية وطني لسنة( ، الذين حضروا المركز ال 21.15± 25.28العمر النوع الثاني )متوسط ستبيان. كان لدى المرضى اتفاق قوي ال. تم تقييم المعتقدات عن االدوية باستخدام النسخة العربية من ا1122س إلى مار 1125الفترة من ديسمبر (. وكان لدى غالبية المرضى 22.12)( مقارنة بالقلق من اعراضها الجانبية والمستقبلية 21.11حول ضرورة العالج لمرض السكري بمعدل ) ة العالج المضاد للسكري للحفاظ على السيطرة الجيدة على المرض )نسبة ضرورة العالج كانت أكبر من نسبة ( معتقدات قوية في ضرور25.0٪) ( من المرضى عبروا عن مخاوف قوية بشأن العالج المضاد للسكري )نسبة القلق ٪28.2(. ومع ذلك، )لألدويةالقلق من االثار الجانبية والمستقبلية (، لديهم نسية متساوية لضرورة العالج و ٪2.0أكبر من نسبة ضرورة العالج(. عدد قليل من المرضى ) لألدويةلية من االثار الجانبية والمستقب .لتحكم الجيد في نسبة السكر في الدمكان متنبأ ضعيفا لالقلق منه. ايضا وجد أن معتقد المريض ل االدوية ، المركز الوطني للسكري .، استبيان المعتقدات حومرض السكري النوع الثاني الكلمات المفتاحية : introduction diabetes mellitus (dm) is defined by the american diabetes association (ada) as a metabolic disorders characterized hyperglycemia due to defects in insulin secretion and/ or insulin action (1). globally, an estimated 422 million adults were living with diabetes in 2014, compared to 108 million in 1980 (2). type 2 diabetes mellitus (t2dm) represents about 90 to 95% of the overall diabetes types worldwide (3). 1corresponding author e-mail: esraa_pharma87@yahoo.com received: 21/10/ 2017 accepted:11 /11/2017 iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 67 research conducted in patients with a variety of long-term conditions suggests that the key beliefs influencing patients’ commonsense evaluations of prescribed medicines can be grouped under two categories: necessity beliefs and concern beliefs (4). concern beliefs are about the adverse consequences of taking a drug, whereas necessity beliefs are about the positive effects of a drug on someone's health (5). socio-demographic factors (including gender, cultural backgrounds, and age) have been shown to be related to beliefs in medicines (6-9). in addition, clinical characteristics of individuals (including duration of disease, experience of taking medicines) have been also shown to be related to beliefs in medicines (8, 10). furthermore, taking account of the patient’s beliefs about the prescribed medication had been reported to be one of the most important factors that affects adherence where holding positive beliefs about medications is a prerequisite for intentional adherence (4, 11, 12). to measure beliefs about medicines, the beliefs about medicines questionnaire (bmq) was developed by horne et al (10). the bmq questionnaire is a selfadministered questionnaire, which focuses directly on the beliefs and concerns of the patient about the use and efficacy of medicines. it can be used for a wide range of diseases where prescribed medication is required (13). the aim of the current study was to investigate and assess beliefs about medicines, among type 2 diabetic patients and to determine possible association between this belief and glycemic control as well as some patientspecific factors. patients and method patients the current cross-sectional study was carried out on 380 (mean age 56.58± 10.06 years) already diagnosed t2dm patients who attended the national diabetes center, al-mustansiriya university – baghdad/ iraq during december-2016 to march-2017. the number of male patients was 183 (48.16%) while the number of female patients was 197 (51.84%). inclusion criteria the inclusion criteria for the current study were: 1-t2dm patients, age ≥ 30 years of either sex. 2-patients have been diagnosed with t2dm at least one year before. 3patients should be able to communicate and willing to participate in the study. exclusion criteria exclusion criteria including the following: 1-patients excluded if they did not consent to participate. 2-patient who had hearing, speech or cognitive deficits (physical or mental state) that would impair understanding of the questions. 3-female patients who are pregnant or breast feeding. sample size calculation population sample size was calculated using raosoft sample size calculator (14) . assuming the margin of error of 5% and the confidence level is 95%, the total number of diabetic patients registered at the above center were 33,408 patients at the beginning of the current study then the sample size will be of minimally 380 patients. method the questionnaires belief about medicine was assessed by using an arabic version (15) of the beliefs about medicines questionnaire (bmq) developed by horne et al (10) (figure-1). the bmq consists of two sections; general and specific. the specific section assesses patients’ beliefs about medications prescribed for a particular illness and comprises two domains assessing personal beliefs about the necessity (specific necessity) of prescribed medication for controlling their illness (5 statements) and concerns (specific concern) about the potential adverse consequences of taking it (5 statements). the general section of the bmq deals with more general beliefs about medicines and comprises also two domains , the (general overuse) scale addresses views about the way in which medicines are used by doctors (4 statements) and the (general harm) scale which assesses beliefs about the degree to which they perceive medicines as essentially harmful. patients indicate their degree of agreement with each question in subparts; belief about medicines on a five-point likert scale, where :1 = strongly disagree, 2 = disagree, 3 =uncertain, 4 = agree, and 5 = strongly agree. thus, points of each scale are summed to give a scale score. higher scores indicate stronger beliefs in the concepts of the scale. specific necessity and specific concerns scales have 5 items and scores range from 5 to 25. higher specific necessity scores represent stronger perceptions of personal need for the medication to maintain health now and in the future. higher specific concerns scores represent stronger concerns about the potential negative effects of the medication. the general overuse and the general harm scales range from 4 to 20. higher scores on the general harm scale represent more negative views about medicines as a whole and a tendency to see medicines as fundamentally harmful, addictive poisons. higher scores on the general overuse indicate more negative views about the way in which medicines are prescribed and beliefs that they are overused by doctors (16). iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 68 figure(1): the beliefs about medicines questionnaire (bmq) (10). strongly disagree disagree uncertain agree strongly agree specific necessity 1-my life would be impossible without medicine 2-without medicine i'll be very ill 3-my health , at present depend on my medicine 4-my medicine protected me from becoming worse 5-my health in the future depends on my medicine specific concern 6-i sometimes worry about the long term effect of my medicine 7-having to take medicine scares me 8-i sometimes worry about becoming too dependent on my medicine 9-my medicine disrupt my life 10-my medicines are mystery to me general-harm 11-people who take medicines should stop their treatment for a while every now and again 12-most medicines are addictive 13-medicines do more harm than good 14-all medicines are poison general-overuse 15-natural remedies are safer than medicines 16-doctors use too many medicines 17-doctors place too much trust on medicines 18-if doctors had more time with their patients they would prescribe fewer medicines study design the pilot study a pilot study was conducted on 40 t2dm patients to test wording and to optimize the arabic phrasing of the questionnaires used in the current study. these data were not included in the main study. administration of questionnaires and blood sample collection the data related to the study were collected by the researcher. the patients were interviewed (each patient spent approximately 30 minutes to fill the research questionnaires completely) after briefly explaining of study purpose. in addition, when blood sample was collected for analysis (which is a routine in this center) a sample was taken by the researcher for the analysis of glycosylated hemoglobin (hba1c) from each patient. statistical analysis anderson darling test was done to asses if continuous variables follow normal distribution, if follow normal distribution, then mean and standard deviation used, if did not follow normal distribution, then median and interquartile range (25% to 75% percentile range) was used to present the data (box plot and whisker used to present them graphically). discrete variables presented using their number and percentage, chi square test used to analyze the discrete variable or fisher exact test used to analyze the distribution between 2 groups (used instead of chi square for 2x2 table, if total sample <20 and if 2 or more with expected frequency less than 5). two samples t test used to analyzed the differences in means between two groups (if both follow normal distribution with no significant outlier). mann whitney u test used to analyzed the differences in median of two groups (if they do not follow normal distribution). linear regression analysis performed to assess the relationship between different variables, if one or both of them follow normal distribution person regression used but if both did not follow normal distribution spearman correlation will used. scatter plot used to present the regression analysis, r (correlation coefficient or standardized beta is a representative iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 69 of magnitude and direction of the relationship), r<0.25 weak, 0.25 – 0.5 mild, 0.5 – 0.75 moderate, >0.75 strong correlation. negative sign indicate inverse relationship, but positive sign represent direct relationship. receiver operating characteristic (roc) curve used to see the validity of different parameters in separating cases with torsion from none torsion and area under the curve (auc) and its p value prescribe this validity (if auc ≥ 0.9 mean excellent test, 0.8 – 0.89 means good test, 0.7 – 0.79 fair test otherwise unacceptable). trapezoidal method used for calculate the curve. in a roc curve the true positive rate (sensitivity) is plotted in function of the false positive rate (100-specificity) for different cut-off points. each point on the roc curve represents a sensitivity/specificity pair corresponding to a particular decision threshold. results personal, demographic and disease characteristics of participants the personal characteristics of the participants involved in the current study and comparison with glycemic control are shown in table-1. in all of the variables, there were no significant difference between them according to the glycemic control. table (1): comparison of sociodemographic data according to glycemic control. glycemic control poor (hba1c≥7%) good (hba1c<7%) all p value number 297 83 380 age, mean ± sd 56.3 ± 9.8 57.6 ± 11.2 56.6 ± 10.1 0.302 ≥65 years 59 (19.9%) 25 (30.1%) 84 (22.1%) <65 years 238 (80.1%) 58 (69.9%) 296 (77.9%) weight, mean ± sd 79.8 ± 11.9 79.1 ± 12.5 79.6 ± 12.0 0.648 height, mean ± sd 1.6 ± 0.1 1.7 ± 0.1 1.6 ± 0.1 0.304 body mass index, mean ± sd 29.5 ± 4.3 28.8 ± 5.0 29.3 ± 4.4 0.230 <25 38 (12.8%) 18 (21.7%) 56 (14.7%) ≥25 259 (87.2%) 65 (78.3%) 324 (85.3%) gender, no (%) 0.452 female 157 (52.9%) 40 (48.2%) 197 (51.8%) male 140 (47.1%) 43 (51.8%) 183 (48.2%) social status, no (%) 0.424 divorced 5 (1.7%) 0 (0.0%) 5 (1.3%) married 267 (89.9%) 79 (95.2%) 346 (91.1%) widowed 15 (5.1%) 2 (2.4%) 17 (4.5%) single 10 (3.4%) 2 (2.4%) 12 (3.2%) level of education 0.564 illustrate 15 (5.1%) 3 (3.6%) 18 (4.7%) primary 50 (16.8%) 18 (21.7%) 68 (17.9%) secondary 142 (47.8%) 34 (41.0%) 176 (46.3%) college 90 (30.3%) 28 (33.7%) 118 (31.1%) hba1c: glycosylated hemoglobin. the use of insulin therapy, number of medications, duration of dm , presence of comorbid diseases and emergency room admission are shown in table-2 also with a comparison of glycemic control. patient with poor glycemic control had longer median duration of diabetes mellitus compared to good glycemic control (7 versus 4 years) and it was significant. the use of insulin therapy was significantly higher in patients with poor glycemic control compared to those with good glycemic control; the rest of the variables had no significant difference between them using glycemic control. hba1c: glycosylated hemoglobin. the mean hba1c value for the sample population was (7.93 ±1.83) % as illustrated in figure-1. figure(1):mean hba1c value for the sample population. iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 70 belief about medicine table-3 highlights the results from bmqsubparts score presented according to the mean (sd), median and interquartile range (iqr). the findings showed the patients had a stronger agreement with the mean necessity scale (19.29) than the mean concern scale (14.27). furthermore, the mean score for bmq general harm is (13.8) approximately the same as the mean total score for bmq general overuse, (14.78). table( 2): comparison of insulin therapy use, medications, co-morbid disease and er admission using glycemic control glycemic control poor (hba1c≥7%) good (hba1c<7%) all p value number 297 83 380 insulin use no 183 (61.6%) 76 (91.6%) 259 (68.2%) <0.001 yes 114 (38.4%) 7 (8.4%) 121 (31.8%) dm medications singl e 125 (42.1%) 34 (41.0%) 159 (41.8%) 0.255 two 163 (54.9%) 49 (59.0%) 212 (55.8%) >two 9 (3.0%) 0 (0.0%) 9 (2.4%) hypertension no 119 (40.1%) 38 (45.8%) 157 (41.3%) 0.350 yes 178 (59.9%) 45 (54.2%) 223 (58.7%) dyslipidemia no 139 (46.8%) 42 (50.6%) 181 (47.6%) 0.540 yes 158 (53.2%) 41 (49.4%) 199 (52.4%) hypertension and dyslipidemia no 198 (66.7%) 57 (68.7%) 255 (67.1%) 0.731 yes 99 (33.3%) 26 (31.3%) 125 (32.9%) emergency room admission no 286 (96.3%) 81 (97.6%) 367 (96.6%) 0.566 yes 11 (3.7%) 2 (2.4%) 13 (3.4%) duration of diabetes (median [interquartile range]) 7.0 (7.0 – 11.5) 4.0 (3.0 – 7.0) 7.0 (4.0 – 10.0) <0.001 table (3): descriptive analysis for beliefs about medicines questionnaire. n min max mean std. dev. median interquartile range sn 380 5 25 19.29 4.51 20.0 17.0 23.0 sc 380 5 25 14.27 5.58 15.0 10.0 19.0 gh 380 4 20 13.87 4.25 14.0 12.0 17.0 go 380 5 20 14.78 3.23 15.0 13.0 17.0 bmq 380 34 84 62.21 10.26 62.0 54.3 70.0 sn: specific necessity, sc: specific concern, gh: general-harm, go: general-overuse, bmq: beliefs about medicines questionnaire. the majority of the patients (76.3%) had strong beliefs in the necessity of anti-diabetic treatment for maintaining good control of diabetes (scores bmq specific-necessity greater than score bmq specific-concern). however, (18.4%) of the patients reported strong concerns about the antidiabetic treatment (scores bmq specific-concern greater than score bmq specific-necessity). the small number of the patients (5.3%), have equal scores for bmq specific-necessity and specificconcern scores (table-4). table(4): bmq necessity – concern differential necessity – concern differential n percentage (%) necessity > concern 290 76.3 concern > necessity 70 18.4 necessity = concern 20 5.3 total 380 100.0 bmq: beliefs about medicines questionnaire. regarding specific necessity, the majority of the patients were either agree or strongly agree with median and interquartile range of 20 (17 – 23), iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 71 specific concern had a median and interquartile range of 15 (10 – 19). while the general harm had a median and interquartile range of 14 (12 – 17) and the general over use had a median and interquartile range of 15 (13 – 17) as illustrated in table-5. table(5): patient's belief about medicine (bmq) [concern, necessity, harm and overuse] median interquartile range specific necessity q1 4 (3 5) q2 4 (3 5) q3 4 (3 5) q4 4 (3 5) q5 4 (3 5) overall 20 (17 23) specific concern q6 4 (2 5) q7 3 (2 4) q8 3 (2 5) q9 2 (1 4) q10 2 (1 3) overall 15 (10 19) general harm q11 3 (1 4) q12 4 (3 5) q13 4 (2 5) q14 4 (3 5) overall 14 (12 – 17) general-overuse q15 3 (1 5) q16 4 (3 5) q17 4 (3 5) q18 5 (3 5) overall 15 (13 – 17) figure-2 represents a boxplot of patient's belief about medicine score for each component separately (the right figure),and total score (the left figure) represented by median and interquartile range for both respectively. figure(2): box plot of patient's belief about medicine (bmq) score for each component (figure in the right) and total score (left figure) figure-3 shows the distribution of total scores for the bmq which is slightly greater than the middle point of the scale. the greater the number of total score from the middle point of the scale, the higher the agreement to the conceptual proposal of belief. figure (3): -the distribution of total scores for the bmq. there was good inter-correlation between most of the components, except between (general harm or general overuse) with (specific necessity or concern), and all component correlate significant and directly with compensate score as illustrated in table-6. table(6): spearman correlation between the components of patient's belief about medicine score specific necessity specific concern general harm generaloveruse specific concern 0.273** general harm -0.086 0.096 general overuse 0.059 0.046 0.247** bmq 0.572** 0.719** 0.506** 0.468** **. correlation is significant at the 0.01 level (2tailed). bmq: beliefs about medicines questionnaire. overall there was significant direct linear correlation between hba1c and bmq score, indicating as the score increase the hba1c also will increase, however for each group (poor or good glycemic control) there was no relationship between bmq score and hba1c, as illustrated in table-7 and figure4. iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 72 table(7): linear regression analysis to predict hba1c using bmq score β p value all 0.133 0.010 poor control 0.063 0.278 good control 0.160 0.149 β: correlation coefficient figure(4):scatter plot of the relationship between hba1c and bmq score in all patients table-8 demonstrates the comparison of bmq score and it's subparts with glycemic control where a poor control patients has a higher median score compared to good control patients regarding specific necessity and it was significant ,while no significance was available with other subparts and glycemic control. table(8): comparison of bmq score and its component in their glycemic control. good poor p value specific necessity 18 (12 – 22) 21 (18 – 23) <0.001 specific concern 13 (8 – 19) 15 (10 – 19) 0.137 general harm 15 (12 – 17) 14 (12 – 17) 0.165 generaloveruse 14 (12 – 16) 15 (13 – 17) 0.240 bmq 60 (51 – 67) 63 (56 – 71) 0.018 data presented using median and interquartile range bmq: beliefs about medicines questionnaire ,to assess the ability of patient's belief about medicine in predicting good glycemic control, we noted that patient's belief about medicine had poor ability to predict good glycemic control as shown in table-9. table(9): roc analysis to determine the best predictor of good glycemic control using bmq score. score auc p value optimal cut point (days) specific necessity 0.553 poor 0.003 ≤18 specific concern 0.638 poor 0.162 ≤13 general harm 0.522 poor 0.168 >12 generaloveruse 0.542 poor 0.233 ≤13 bmq 0.585 poor 0.017 ≤56 roc: receiver operating characteristic, auc: area under the curve, bmq: beliefs about medicines questionnaire. table 10 shows that there was no significant relationship between bmq and gender, duration of dm and body mass index, while a significant positive relation found with hba1c and a negative one with age. table (10): univariate analysis between bmq and various variables beta odd ratio p value age -0.105 0.040 duration of dm -0.042 0.380 body mass index 0.020 0.695 gender a -0.006 0.994 0.31 hba1c 0.133 0.010 binary logistic regression used to find the relationship between bmq and various variables bmq: beliefs about medicines questionnaire. discussion type 2 diabetes mellitus is characterized as hyperglycemia due to insulin resistance causing reduced tissue response towards insulin(16). there is some uncertainty about the prevalence of diabetes mellitus in the iraqi population. iraq has undergone rapid economic development in the last 10 years. in december 2011, the international diabetes federation reported that, of the ten countries with the highest prevalence of diabetes in adults aged 20–79 years, six were in the middle east, i.e., kuwait (21.1%), lebanon (20.2%), qatar (20.2%), saudi arabia (20.0%), bahrain (19.9%), and the united arab emirates (19.2%).iraq is considered as having a medium prevalence (9.3%) of diabetes in the middle east based on surveys from 2006 to 2007(17). reversing this ‘‘epidemic’’ will require major lifestyle changes and remaking our health care delivery system into one focused on proactive prevention and continuous access to coordinated, evidence-based management of chronic disease(18). iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 73 in this study we assessed beliefs about medicines, among type 2 diabetic patients and to determine possible association between this belief and glycemic control as well as some patient-specific factors. in our study we used belief about medicine questionnaire (bmq) to measure patients' belief about medicines. the bmq has been translated into several languages to assess beliefs about medicines across a wide range of disease like diabetes mellitus, mental health illnesses, rheumatoid arthritis and others (15,19,20). findings of our study was that positive beliefs about the necessity of medication had recorded the highest mean among the rest of the questions and the concept of over using of the medicines and the concerns about potential adverse effects of medication recorded the same mean level of the questionnaire score while beliefs that medicines are generally harmful recorded the lowest mean level. this result was consistent with sweileh et al. study in 2014 in palestine who found that most diabetic patients strongly believe that anti-diabetic medications are necessary for their current and future health (highest score) while beliefs that medicines are generally harmful recorded the lowest mean level. however; unlike the results of current study, specificconcern score was higher than general – overuse score (21). concerns about long-term effects of using anti-diabetic medications should be assessed by healthcare providers to reduce it, thus improving medication adherence. patients with dm need to know that their medicines are not addictive and have a good safety profile for long-term use (21). aflakseir a. in 2012 suggested that when people are concerned about the negative effects of prescribed medicines, they are less likely to adhere to their medication(22). this study results showed that 76.3% of patients cleared up that patient beliefs on necessity outweighs concern, while 18.4% of patients have concern belief out-weighs necessity that need further attention. these findings are closely to that of (manan et al 2014) (23) where 70.2% of patients belief of necessity outweighs concern, and 20% have concern beliefs outweighs necessity. these negative perceptions of medication are often associated with the beliefs that the dangerous aspects of medicines are due to their chemical/unnatural origins and therefore complementary or traditional treatments are perceived to be more “natural” and safer. this explanation supported by finding that the majority of the sample patients perceived that natural remedies are safer than medicines. in an effort to assess the ability of patient's belief about medicine in predicting good glycemic control, the result of the current study revealed that patient's belief about medicine had a poor ability to predict good glycemic control, this result was consistent to that of james and john in 2009 where neither antihyperglycemic medication necessity nor concern was significantly associated with hba1c (12). the results of our study indicated that age of the patients was negatively related to patients' belief while neame et al. in 2005(20) have reported that concerns about adverse consequences medications is independent of patients’ age. most of the patients in this study strongly disagreed with the statement that "if doctors had more time with patients they would prescribe fewer medicines". shorter visits associated with increased rate of medication prescribing (24). openness between the doctor and the patient during consultations will ultimately lead to clearer understanding, both in terms of the patient’s understanding of the disease and treatment options, and in terms of the doctor’s understanding of the patient’s attitudes (25). this finding had reflected that there's a lack of patients' – healthcare provider confidence which is considered as major problem in advocating patient disease management conclusion the majority of the patients had strong beliefs in the necessity of anti-diabetic treatment for controlling their illness. however, patient's belief about medicine had poor ability to predict good glycemic control. references 1. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care. 2014; 37(suppl. 1): s81–s90. 2. global report on diabetes, world health organization, 2016. available at: www.who.int/diabetes/global-report. 3. american diabetes association. classification and diagnosis of diabetes. diabetes care.2015; 38(suppl. 1):s8–s16. 4. rob h, sarah c, rhian p, et al. understanding patients’ adherence-related beliefs about medicines prescribed for long-term conditions: a meta-analytic review of the necessity-concerns framework. plos one. 2013; 8 (12): 1-24. 5. sieta t, joost c, rosalie v, et al. medication beliefs, treatment complexity, and nonadherence to different drug classes in patients with type 2 diabetes. journal of psychosomatic research. 2014; 76 : 134–138. 6. kirsten k, hilde f, maria r, et al. beliefs about medicines among norwegian outpatients with chronic cardiovascular disease. eur j hosp pharm. 2013;0:1–3. 7. isacson d, bingefors k. attitudes towards drugs—a survey in the general population. pharm world sci. 2002;24:104–10. 8. horne r, graupner l, frost s, et al. medicine in a multi-cultural society: the effect of cultural background on beliefs about medications. soc sci med. 2004;59(6):1307-13. https://www.ncbi.nlm.nih.gov/pubmed/?term=horne%20r%5bauthor%5d&cauthor=true&cauthor_uid=15210101 https://www.ncbi.nlm.nih.gov/pubmed/?term=graupner%20l%5bauthor%5d&cauthor=true&cauthor_uid=15210101 https://www.ncbi.nlm.nih.gov/pubmed/?term=frost%20s%5bauthor%5d&cauthor=true&cauthor_uid=15210101 https://www.ncbi.nlm.nih.gov/pubmed/15210101 https://www.ncbi.nlm.nih.gov/pubmed/15210101 iraqi j pharm sci, vol.26(2) 2017 belief about medications among type 2 diabetic patients 74 9. corey s, tiffany s, amber m et al. older adults’ help-seeking attitudes and treatment beliefs concerning mental health problems. am j geriatr psychiatry. 2008; 16(12): 1010–1019. 10. horne r, weinman j, hankins m. the beliefs about medicines questionnaire: the development and evaluation of a new method for assessing the cognitive representation of medication. psychol health 1999; 14: 1–24. 11. mohamed m, salmiah m, mohd d. beliefs and adherence to medicines among malaysian malay type 2 diabetes. international journal of current research. 2014; 6 (02) :5026-5033. 12. james e, john d. diabetic patients’ medication underuse, illness outcomes, and beliefs about antihyperglycemic and antihypertensive treatments. diabetes care. 2009;32(1): 19-24. 13. konstantina t, markos g , nikolaos g, et al. beliefs about medicines questionnaire (bmq) for inflammatory bowel disease patients in greece. ιs it useful?. european journal for person centered healthcare. 2016; 4 (1) :187195. 14. raosoft sample size calculator. available at: www.raosoft.com/samplesize.html 15. raniah m, waleed m, adham s , et al. beliefs about medicines and self-reported adherence among patients with chronic illness: a study in palestine. journal of family medicine and primary care. 2014; 3 (3): 224-229. 16. marhanis s. omar, kong l. san, diabetes knowledge and medication adherence among geriatric patient with type 2 diabetes mellitus: international journal of pharmacy and pharmaceutical sciences . 2014;3 (6). 17. abbas a. mansour, ahmed aal-maliky et al. ,prevalence of diagnosed and undiagnosed diabetes mellitus in adults aged 19 years and older in basrah, iraq: diabetes, metabolic syndrome and obesity: targets and therapy 2014;7 :139–144. 18. william r. rowley, clement bezold, creating public awareness: state 2025 diabetes forecasts: population health management 2012;15 (4). 19. jo a. sirey, alexandra g. et al. , medication beliefs and self-reported adherence among community-dwelling older adults: clinical therapeutics 2013 ; 35: (2). 20. r. neame and a. hammond, beliefs about medications: a questionnaire survey of people with rheumatoid arthritis: rheumatology 2005 ; 44 : 762–767. 21. waleed m sweileh, sa’ed h zyoud, influence of patients’ disease knowledge and beliefs about medicines on medication adherence: findings from a cross-sectional survey among patients with type 2 diabetes mellitus in palestine: bmc public health 2014; 14: (94). 22. aflakseir a, role of illness and medication perceptions on adherence to medication in a group of iranian patients with type 2 diabetes: journal of diabetes 2012; 4: 243–247. 23. mohamed m. manan, salmiah m. ali,beliefs and adherence among malaysian malaly type 2 diabetics: international journal of current research 2014; 02 : (6): 5026-5033. 24. david c. dugdale ,ronald epstein, steven z. pantilat, time and the patient-physician relationship, jgim, 1999;14 (1). 25. j s chatterjee, from compliance to concordance in diabetes, j med ethics, 2006 ;32: 507-510 http://www.raosoft.com/samplesize.html synthesis and preliminary pharmacological evaluation of amine derivatives of diclofenac as a potential anti-inflammatory agents iraqi j pharm sci, vol.20(1) 2011 new analogues of diclofenac 25 synthesis and preliminary pharmacological evaluation of new analogues of diclofenac as potential anti-inflammatory agents noor h. naser*, monther f. mahdi** ,1 , tagreed n-a omar** and amar a. fadhil*** *college of pharmacy, university of kufa, najaf, iraq. **department of pharmaceutical chemistry ,college of pharmacy,university of baghdad,baghdad, iraq. ***department of pharmacology, college of pharmacy, university of baghdad., baghdad, iraq. abstract a group of amine derivatives [4-aminobenzenesulfonamide derivatives, 2-aminopyridine and 2aminothiazole] incorporated to α-carbon of diclofenac a well known non-steroidal anti-inflammatory drug (nsaid) to increase bulkiness were designed and synthesized for evaluation as a potential antiinflammatory agents with expected cox-2 selectivity. in vivo acute anti-inflammatory activity of the selected final compounds (9, 12 and 13) was evaluated in rats using egg-white induced edema model of inflammation in a dose equivalent to (3 mg/kg) of diclofenac sodium. all tested compounds produced a significant reduction in paw edema with respect to the effect of propylene glycol 50% v/v (control group). moreover, the 4-aminobenzenesulfonamide derivative (compound 9) exhibited superior antiinflammatory activity compared to diclofenac sodium at times 180-300 minutes with the same onset of action. the results of this study indicate that the incorporation of the selected aromatic amino groups in to diclofenac maintain its anti-inflammatory activity. key words: amine derivatives, anti-inflammatory, diclofenac derivatives, cox-2 selectivity. الخالصة مةةا تقمةةث وةةث خو تيةة مجستمٍ وثٍةةثل -2تمٍ وخٍرٌةة ٌم -2تمٍ ةةوخ نٌم فةةامويثمثٌ -4 قمتةةصمثز مجموعةةر مةةم تقمتةةصمثز ت مٍ ٍةةر قةة مةةممس امةةس قصمٍٍم ةةث وعوتمةة مةة ثر قنٌةةثلذ تقدةة ثم جٍةة ت ومدةةثل قاقص ةةثجٌ ي تقمعةةر قاةة تٌواونٍ ثا تقةة تر اٍةةر تق ةةصٍر امرودةثز ق تقمدةثلذ قاقص ةثج تقحةثل معثقٍرشمٍٍم تق شم قاجرذت نً تقج م تقحً ".2-أينٌم فثٌووت و ج ٍنع "ققألقص ثج ما تيصمثئٍر مصو قاصةولٌوم خجرعةر موثنةةرل تقدةٍ خثفةص تم تض ذمة شحةس تقجاة خثفص تم طرٌمر تفصح( نً تقجرذ 21 22 9)تقم صث تق ثئٍر ومجموعةر % )05 مة ثرت قاوذمةر خثقممث يةر مةا تقدةر خاٍم واٌوةو ثتي مثض ظ رزوغم(. و تقمرودثز تقم صدرذ ت /ماغ 1لتٌواونٍ ثا ) قمصةرذ لتٌواونٍ ةثاصةولٌوم ممث يةر خثق تعاةى ةثجتظ ةر نعثقٍةر مدةثلذ قاقص تمٍ ةوخ نٌم فةامويثمثٌ -4تقمروح قكذعا ذ عاى .(ضثخطر م صةث مةم تقةى ت تية مثم مجةثمٍاتظ ةرز تق صةثئ لتٌواونٍ ةثا.صةولٌوم مةا يمةا تقمعثقٍةر ت خص تئٍةر قا 155-285 تأل صدث مم تق قٍم .قاقص ثج حثنع عاى نعثقٍصر تقمدثلذ مٍم ما تق تٌواونٍ ثاتقمرودثز تقحامٍ اٍر تقحامٍ قا introduction non-steroidal anti-inflammatory drugs represent one of the most widely used classes of drugs, and are used primarily for treatment of osteoarthritis, rheumatoid arthritis and other inflammatory disorders; however, the use of nsaids is significantly limited by their ability to induce the formation of erosions and ulcers in the gastrointestinal (gi) tract (1) . the mechanism of action principally responsible for most of the nsaids seems to be inhibition of prostaglandin synthesis by causing almost complete blockade of the activity of the precursor enzymes, cyclooxygenase (2) . these are the rate-limiting enzymes in the synthesis of the inflammatory prostaglandins pge2 and pgf2α, the cytoprotective prostaglandin pge1, and the vasoactive prostanoids thromboxanea2 (txa2) and prostacyclin (pgi2). three cox iso-zymes are known; cox-1, cox-2, and cox-3 (3). the cox-1 is constitutively expressed, widely distributed and has "housekeeping" function. it is of particular importance in maintaining gastric mucosal integrity, renal function and homeostasis (4) .in contrast,cox-2 is induced in setting of inflammation by cytokines and inflammatory mediators or physiological stress (5) . cox-3 could play a role in fever and pain processes (6) . the analgesic effects of nsaids are ascribed primarily to cox-2 inhibition, whereas several adverse effects are believed to be mediated by cox-1 inhibition (7) . selective cox-2 inhibitors differ from traditional nsaids in that they are less likely to result in nsaidinduced gastropathy (8) . 1corresponding author email : dmfalameri @yahoo.com received : 29/9/2010 accepted : 3/1/2011 iraqi j pharm sci, vol.20(1) 2011 new analogues of diclofenac 26 in addition, several other clinical uses of selective cox-2 inhibitors have been investigated (9) , some of these indications including the treatment of the neurodegenerative disease like alzheimer (10) and parkinson disease (11) are now under clinical trials to validate the therapeutic possibilities of cox-2 inhibitors (12) . more ever studies show that the cox-2 enzyme would be an interesting target in the treatment of some cancers (13) , viral infection (14) , and endometriosis (15) . preferential inhibition of cox-2 is thought to be due to the additional space in the cox-2 hydrophobic channel, as well as to the presence of a side pocket in the channel (16) .this side pocket can discriminate the selective nsaids , like celecoxib (i) (12) ,valdicoxib (ii) (17) which contain amino benzene sulfonamide derivatives that occupied the side pocket (18) , from nonselective agents. in the view of this back ground we synthesized and preliminary evaluated new diclofenac derivatives by increase its bulkiness by incorporated the prerequisite (benzene sulfonamide group) for cox-2 selectivity or other aromatic amine derivatives found in potent nsaids like meloxicam (iii) and piroxicam(iv) as potent anti-inflammatory agents. experimental all reagents and anhydrous solvents were of analar type and generally used as received from the commertial suppliers (merckgermany, riedel-dehaen-germany, gccu.k,bdh-england and fluka-switzerland). diclofenac was supplied from cox.tx.lx chem. china. melting points were determined by capillary method on thomas hoover apparatus (england). thin layer chromatography (tlc) was run on dc-kartan si alumina 0.2 mm to check the purity and progress of reaction. the identification of compounds was done using iodine vapor and the chromatograms were eluted by; n-hexane: ethyl acetate: acetic acid (7: 2.5: 0.5 v/v) (19) . ft-ir spectra were recorded at (college of sciencekufa university) by using shimadzujapan spectrophotometer and the determination of the spectra were performed by using kbr discs. chnos microanalysis has been done in cleveland clinical foundation learner research institute-france, by using carlo erba elemental analyzer. a. chemistry synthesis of methyl 2[2-(2,6dichlorophenylamino) phenyl]acetate(2) (20) suspension of diclofenac (1g, 3.37mmol) in absolute methanol, was cooled down to -15 o c, then thionyl chloride (0.25ml, 3.37mmol) was added drop wise. (the temperature should be kept below -10 o c). the reaction mixture was kept at 40 o c for three hours, followed by refluxing for three hours, and left at room temperature over night. the solvent was evaporated to dryness, re-dissolved in methanol and evaporated. the process was repeated several times to ensure complete removal of thionyl chloride. the residue was collected and re-crystallized from methanolether. the percent yield, physical data and rf values were given in table (1). synthesis of methyl 2-bromo-2-[2-(2,6dichlorophenylamino)phenyl]acetate(3) (17) compound 2 (1g, 3.23mmol) was dissolved in methylene chloride (15ml), then nbs (0.57g, 3.23mmol) was added gradually with continuous stirring. the reaction was iraqi j pharm sci, vol.20(1) 2011 new analogues of diclofenac 27 allowed to proceed at room temperature with stirring for three hours, then the solvent was evaporated. ether was added to the residue, then filtered, the filtrate was dried to give compound 3 . the percent yield, physical data and rf values were given in table (1). synthesis of methyl 2[2( 2, 6 dichlorophenylamino) phenyl]2(4 sulfamoylphenylamino) acetate(4) (21) compound 3 (1g, 2.57mmol), sulfanilamide (0.44g, 2.57mmol) were placed in round flask, then dissolved with ethanol 99%:dmf (50:50) mixture (30ml). the reaction mixture was refluxed gently for three hours. the solvent was evaporated, the residue was dissolved in ethyl acetate, washed with naoh (5%, 3x), filtered over anhydrous magnesium sulfate, the filtrate was evaporated to give compound 4. the percent yield, physical data and rf values were given in table(1). synthesis of methyl 2 [ 4(nacetylsulfamoyl) phenylamino] -2 [2(2, 6 dichloro phenylamino) phenyl]acetate (5) (21) compound 3 (1g, 2.57mmol) and sulfacetamide (0.55g, 2.57mmol) were placed in round flask, then dissolved in ethanol: dmf (50:50) mixture (40ml). the reaction mixture was refluxed gently for three hours, then it was worked up as prescribed for compound 4 to liberate compound 5. the percent yield, physical data and rf values were given in table(1). synyhesis of methyl 2[ 2(2, 6dichlorophenylamino) phenyl] -2[4(n methyl sulfamoyl) phenylamino] acetate (6) (21) compound 3 (1g, 2.57mmol), 4-aminon-methylbenzene sulfonamide (0.47g, 2.57mmol) were placed in round flask, then dissolved in ethanol: dmf (50:50) mixture (40ml). the reaction mixture was refluxed gently for three hours, then it was worked up as prescribed for compound 4 to liberate compound 6.the percent yield, physical data and rf values were given in table (1). synthesis of methyl 2-[2-(2,6dichlorophenylamino) phenyl]-2-(pyridine-2ylamino)acetate(7) (21) compound 3 (0.5g, 1.28mmol) and 2aminopyridine (0.12g, 1.28mmol) were placed in round flask, then dissolved with ethanol: dmf (50:50) mixture (40ml). the reaction mixture was refluxed gently for three hours, then it was worked up as prescribed for compound 4 to liberate compound 7.the percent yield, physical data and rf values were given in table (1). synthesis of methyl 2-[2-(2,6dichlorophenylamino) phenyl]-2-(thiazol-2ylamino)acetate (8) (21) compound 3 (0.5g, 1.28mmol) and 2aminothiazol (0.128g, 1.28mmol) were placed in round flask, then dissolved with ethanol 99%:dmf (50:50) mixture (20ml). the reaction mixture was refluxed gently for three hours, then it was worked up as prescribed for compound 4 to liberate compound 8. the percent yield, physical data and rf values were given in table (1). synthesis of 2-[2-(2,6-dichlorophenylamino) phenyl]-2-(4-sulfamoyl phenyl amino) acetic acid (9) (22) compound 4 (0.3g, 0.62mmol) was dissolved in minimum volume of ethanol 99%:thf (7:1) mixture. the solution was cold to 18 o c, and then sodium hydroxide (2n, 0.37ml, 0.75mmol ) was added drop wise, with continuous stirring over a period of 30 minutes. stirring was continued at 18 o c for an additional three hours. the reaction mixture was acidified with hcl (2n, 0.37ml, 0.75mmol), excess of cold water was added and the acidic compound was precipitated, then filtered and dried to give compound 9. the percent yield, physical data and rf values were given in table (1).chnos calculated: c,51.51; h,3.67; n, 9.01; o, 13.72; s, 6.88. found: c, 51.85; h, 3.7; n, 9.36; o, 14.39; s, 7.13. synthesis of 2-[4-(n-acetylsulfamoyl) phenylamino]-2-[2-(2,6-dichloro phenyl amino) phenyl]acetic acid (10) (22) compound 5 (0.25.g, 0.47mmol) was dissolved in minimum volume of ethanol 99%:thf (20:1) mixture. the solution was cold to 18 o c, and then sodium hydroxide (2n, 0.28ml, 0.56mmol) was added drop wise, with continuous stirring over a period of 30 minutes. then the reaction mixture was worked up as prescribed for compound 9 to liberate compound 10. the percent yield, physical data and rf values were given in table (1).chnos calculated: c,51.98; h,3.77; n,8.27; o, 15.74; s,6.31. found: c,52.21; h, 3.85; n,8.57; o,16.01; s,6.54. synthesis of 2-[2-(2,6-dichlorophenylamino) phenyl]-2-[4-(n-methylsulfamoyl) phenylamino] acetic acid (11) (22) compound 6 (0.5.g, 1.24mmol) was dissolved in minimum volume of ethanol 99%:thf (20:1) mixture. the solution was iraqi j pharm sci, vol.20(1) 2011 new analogues of diclofenac 28 cold to 18 o c, and then sodium hydroxide (2n, 0.74ml, 1.49mmol) was added drop wise, with continuous stirring over a period of 30 minutes. then the reaction mixture was worked up as prescribed for compound 9 to liberate compound 11. the percent yield, physical data and rf values were given in table (1). chnos calculated: c, 52.51; h,3.99; n, 8.75;o,13.32; s, 6.68. found: c, 53.01; h, 4.12; n, 9.07; o,13.56; s, 6.62. synthesis of 2[2(2,6-dichlorophenylamino) phenyl ] -2(pyridine-2-ylamino) acetic acid(12) (22) compound 7 (0.5.g, 1.24mmol) was dissolved in minimum volume of ethanol 99%:thf (20:1) mixture. the solution was cold to 18 o c, and then sodium hydroxide (2n, 0.74ml, 1.49mmol) was added drop wise, with continuous stirring over a period of 30 minutes. then the reaction mixture was worked up as prescribed for compound 9 to liberate compound 12. the percent yield, physical data and rf values were given in table (1). chno calculated: c, 58.78; h, 3.89; n, 10.82; o, 8.24. found: c,59.21;h,4.01;n,11.28;o,8.61. synthesis of 2-[2-(2,6-dichlorophenylamino) phenyl ] -2(thiazol-2-ylamino) acetic acid (13) (22) compound 8 (0.17g, 0.42mmol) was dissolved in minimum volume of ethanol 99%:thf (15:1) mixture. the solution was cold to 18 o c, and then sodium hydroxide (2n, 0.25ml,0.5mmol) was added drop wise, with continuous stirring over a period of 30 minutes. then the reaction mixture was worked up as prescribed for compound 9 to give compound 13. the percent yield, physical data and rf values were given in table (1). chnos calculated: c,51.79; h, 3.32; n, 10.66; o, 8.12; s, 8.13. found: c, 52.23;h,3.49;n,10.2;o,8.47;s,8.49. the general routes outlined in scheme 1 were used to synthesized the target compounds and their intermediates, their characterization and physical data are presented in table 1. cl nh cl cooh cl nh cl cooch3 cl nh cl cooch3 cl nh cl cooch3 ch3oh socl2/ref lux nbs/ch2cl2 stirring, rt br + h n o o rnh2/ref lux etthanol/dmf rhn cl nh cl cooh rhn 1) naoh 2) hcl hbr (1) (2) (3) (4-8) (9-13) so2nh2 so2nhcoch3 so2nhch3 n n s (4, 9) if r= (5, 10) if r= (7, 12) if r= (6, 11) if r= (8, 13) if r= scheme1: synthesis of target compounds (9-13) and their intermediates. iraqi j pharm sci, vol.20(1) 2011 new analogues of diclofenac 29 table 1 : the characterization and physical data of the final compounds and their intermediates. d: decomposition b. pharmacology albino rats of either sex weighing (220 ± 10 g) were supplied by the animal house of the college of pharmacy, university of baghdad. and were housed in the same location under standardized conditions. animals were fed commercial chaw and had free access to water ad libitum. animals were divided into five groups (each group consist of 6 rats) as follow: group a: six rats served as control; and treated with the vehicle (propylene glycol 50% v/v). group b: six rats treated with diclofenac sodium in a dose of 3mg/ kg (23,24) .suspended in propylene glycol 50%. group c-e: six rats/group treated with the tested compounds (9, 12 and 13) respectively in doses that determined below. (suspended in propylene glycol 50%). anti-inflammatory activity the anti-inflammatory activity of the tested compounds was studied using egg-white induced edema model (25) . acute inflammation was produced by a subcutaneous injection of 0.05ml of undiluted egg-white into the planter side of the hind paw of the rats.; 30 minutes after i.p. administration of the drugs or their vehicle. the paw thickness was measured by vernea at seven time intervals (0, 30, 60, 120, 180, 240,300 minutes) after vehicle or drugs administration , the results were represented in table 2. the data are expressed as a percent of inhibition of edema thickness at each time interval, which can be calculated from the mean effect in control and treated animals according to the equation: % inhibition = 100 [(vc-vt)/vc], where vc and vt represents mean paw thickness in control and tested groups (at time t-time zero) respectively (26) , the results were represented in table 3. results and discussion the most widely used primary test to screen new anti-inflammatory agents measure the ability of a compound to reduce local edema induced in the rat paw by injection of an irritant agent (27) . the thickness of the injected paw was measured at zero time of i.p. injection of drug or vehicle) and immediately after egg-white injection (at time 30 min) and after 60, 120, 180, 240 and 300 min. compounds and intermediates empirical formula molecular weight description % yield melting point o c rf value 2 c15h13cl2no2 310 white crystals 94.9 72-75 0.88 3 c15h12brcl2no2 389 faint pink crystals 75 86-88 0.93 4 c21h19cl2n3o4s 480 yellowish powder 75.9 95-97 0.83 5 c23h21cl2n3o5s 522 faint orange powder 74.5 166-168 0.92 6 c22h21cl2n3o4s 494 faint yellow powder 74.4 71-73 0.93 7 c20h17cl2n3o2 402 white powder 68.9 95-98 0.76 8 c18h15cl2n3o2s 408 black powder 50.6 51-53 0.92 9 c20h17cl2n3o4s 466 yellow powder 25.7 100-101d 0.77 10 c22h19cl2n3o5s 508 faint yellow powder 24.6 182-184 0.87 11 c21h19cl2n3o4s 480 white powder 40.2 168-170 0.9 12 c19h15cl2n3o2 388 gray powder 23 172-173d 0.65 13 c17h13cl2n3o2s 394 black powder 31.9 161-163d 0.87 iraqi j pharm sci, vol.20(1) 2011 new analogues of diclofenac 30 table 2 : effect of compounds 9, 12 and 13, diclofenac (reference) and propylene glycol (control) on egg-white induced paw edema in rats. treatment groups paw thickness (mm) time (min) control (n=6) diclofenc (n=6) compoud 9 (n=6) compoud 12 (n=6) compoud 13 (n=6) 0 4.07±0.05 3.98±0.06 4.06±0.04 4.09±0.1 4.10±0.04 30 5.83±0.12 5.78±0.11 5.78±0.12 5.85±0.06 5.87±0.12 60 5.85±0.06 5.42±0.12* 5.26±0.13* a 5.23±0.12* a 5.80±0.11 b 120 5.5±0.11 5.11±0.04* 4.94±0.1* 4.97±0.05* 5.13±0.03* 180 5.25±0.03 4.95±0.1* a 4.59±0.05* b 4.83±0.05* a 4.96±0.05* a 240 5.03±0.1 4.69±0.12* a 4.28±0.0* b 4.6±0.11* a 4.70±0.02* a 300 4.91±0.02 4.54±0.13* a 4.15±0.1* b 4.49±0.02* a 4.56±0.06* a data are expressed in mm paw thickness as mean ± sem time (0) is the time of i.p. injection of tested compounds and propylene glycol. time (30) is the time of injection of egg-white (induction of paw edema). * significantly different compared to control (p<0.05). n=number of animals non-identical superscripts (a and b) among different groups are considered significantly different (p<0.05). table 3 show the percent of inhibition of paw edema of the three tested compounds and diclofenac (reference) with respect to the control group. all the tested compounds were effectively limited the increase in paw edema. diclofenac, compounds 9 and 12 showed inhibition that started from the first hour after drug administration, while compound 13 show delay in its onset as its inhibition effect started from the second hour after drug injection. the percent of inhibition of compound 9 is significantly differ from the reference, compounds 12 and 13 at the third, fourth and fifth hour of the experiment which indicate the superior anti-inflammatory effect, as illustrates in figure 1. figure 1: percent inhibition produced by diclofenac sodium, compounds 9, 12 and 13. table 3: percent inhibition of diclofenac, compounds 9, 12, and 13 on egg-white induced paw edema in rats. treatment groups percent inhibition time (min) diclofenac sodium (n=6) compound 9 (n=6) compound 12 (n=6) compound 13 (n=6) 60 19% 31.8% 35% 3.4% 120 20.9% 38.4% 38.4% 27.9% 180 17.7% 55% 37% 27% 240 26% 77% 46.8% 37.5% 300 33% 89% 52% 45.2% 0 10 20 30 40 50 60 70 80 90 100 60 120 180 240 300 time of inhibition (min) % o f i n h i b i t i o n diclofenac co.13 co.12 co.9 iraqi j pharm sci, vol.20(1) 2011 new analogues of diclofenac 31 conclusion in vivo anti-inflammatory study showed that the incorporation of 4aminobenzenesulfonamide, 2-aminopyridine and 2-aminothiazole into a well known antiinflammatory drug (diclofenac) maintain its anti-inflammatory activity. more over compound 9 showed more potent antiinflammatory activity than diclofenac, compounds 12 and 13 at time interval 180-300 min. with the same onset of action. acknowledgment 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formulation and evaluation of acyclovir microspheres pavani s*,1, mounika k*and naresh k* *department of pharmaceutics vaagdevi college of pharmacy, warangal, india . abstract the present study is to formulate and evaluate acyclovir (acv) microspheres using natural polymers like chitosan and sodium alginate. acv is a dna polymerase inhibitor used in treating herpes simplex virus infection and zoster varicella infections. acyclovir is a suitable candidate for sustainedrelease (sr) administration as a result of its dosage regimen twice or thrice a day and relatively short plasma half-life (approximately 2 to 4 hours). microspheres of acv were prepared by an ionic dilution method using chitosan and sodium alginate as polymers. the prepared acv microspheres were then subjected to ftir, sem, particle size, % yield, entrapment efficiency, in vitro dissolution studies and release kinetics mechanism. the ftir spectra’s revealed that, there was no interaction between polymer and acv. acv microspheres were spherical in nature, which was confirmed by sem. the particle size of microspheres was in the range of 23.8µm to 39.4µm. 72.9% drug entrapment efficiency was obtained in the formulation f3 (1:3 ratio) with a high concentration of calcium chloride (4% w/v). the in vitro performance of acv microspheres showed sustained release depending on the polymer concentration and concentration of calcium chloride. the release data was best fitted with zero order kinetics and korsemeyer-peppas release mechanism and diffusion exponent ‘n’ value of was found to be non-fickian. keywords: acyclovir, microspheres, chitosan, sodium alginate. introduction acyclovir is a guanosine analog that acts as an antimetabolite. acyclovir is converted by viral thymidine kinase to acyclovir mono phosphate, which is then converted by host cell kinases to acyclovir tri phosphate (acvtp).acv-tp, in turn, competitively inhibits and inactivates hsv-specified dna polymerases preventing further viral dna synthesis without affecting the normal cellular processes. acyclovir, bcs class iii drug is widely used in the treatment of herpes simplex virus infection as well as varicella zoster infection. acv is a guanosine analogue antiviral drug. it is the one of the most commonly used antiviral drug (1). it has short biological half-life (2-4 hours) and is usually administered orally3-4 times a day. in this work, the ionic gelation technique (2) was used due to its simplicity, reproducibility, avoidance of organic solvents and heat. sodium alginate and chitosan were employed as biodegradable polymers (3, 4). materials and methods materials acyclovir as a gift sample was procured from maithri laboratories pvt. ltd. chitosan was obtained from panvo organic pvt. ltd, sodium alginate was loba chemi, mumbai, glacial acetic acid from triveniinterchem pvt. ltd. and calcium chloride from tkm pharmaindia, secunderabad. methods preformulation studies preformulation testing is the first step in the rationale development of dosage forms of a drug substance. it can be defined as an investigation of physical and chemical properties of a drug substance alone and when combined with excipient. the main objective of preformulation testing is to generate useful information to the formulator in developing stable and bioavailable dosage forms. calibration curve for acyclovir in 0.1 n hcl 100 mg of acyclovir was dissolved in small amount of 0.1 n hcl and shaken vigorously, then volume was made up to 100 ml with 0.1 n hcl to obtain the primary stock solution. the necessary dilutions were made by using 0.1 n hcl to obtain the different concentrations of acyclovir (10 to 100µg/ml).as the first step the solution is scanned on a uv scanner between 200 to 400 nm and the maxima peak obtained was considered as λmax. the diluted solutions prepared for calibration curve were checked for their absorbance using uv-vis spectrophotometer at 252 nm against buffer as blank. standard graph was plotted between the concentration on xaxis and absorbance on yaxis. 1corresponding author e-mail:pavanisrm@gmail.com received: 30/9/2017 accepted: 8/12/2017 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp1-7 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 acyclovir microspheres 2 ftir spectroscopy identification of the chemicals procured were confirmed by ftir spectroscopy, in which ftir spectrum of the sample was compared with standard ftir spectra of the pure chemicals. preparation of acyclovir microspheres ionic-gelation method sodium alginate solutions and chitosan solutions were prepared by dissolving 300 mg in distilled water (20 ml) and 2% v/v aqueous acetic acid respectively. 200 mg of drug was added to sodium alginate solution under continuous stirring for 10 min to yield smooth dispersion. on the other hand required amount of calcium chloride (2% or 4 % w/v) was added to chitosan solution. the acyclovir-alginate dispersion was taken into 23 gauge syringe and added drop wise to the chitosan-calcium chloride solution at room temperature with continuous stirring for 1-2 hours at 800 rpm to obtain microspheres. then the microspheres were washed with distilled water and air dried (5, 6). characterization of acyclovir microspheres surface morphology scanning electron microscopy (sem) has been used to determine particle size distribution, surface topography, texture and to examine the morphology of fractured or sectioned surface. sem studies were carried out by using hitachi s-3700n scanning microscope. dry acyclovir microspheres were placed on an electron microscope brass stub. picture of acyclovir microspheres was taken by random scanning of the stub (7, 8). particle size determination a minute quantity of acyclovir microspheres was spread on a clean glass slide and average size of 100 acyclovir microspheres was determined in each batch by optical microscopy in which stage micrometer was employed (9). determination of percentage drug entrapment efficiency (pde) efficiency of drug entrapment for each batch was calculated in terms of percentage drug entrapment as per the formula practical drug content /theoretical drug content * 100(10). theoretical drug content was determined by assuming that the entire acyclovir present in the polymer solution got entrapped in acyclovir microspheres, and no loss occurs at any stage of preparation of acyclovir microspheres. practical drug content was analyzed by dissolving 50 mg of acyclovir microspheres in 50 ml of 0.1n hcl. the solution was kept overnight for the complete dissolution of acyclovir in diluted hydrochloric acid. this solution was filtered and further dilutions are made. the absorbance of the solutions was measured at 252 nm using double beam uvvisible spectrophotometer against 0.1 n hcl as blank and the percentage of drug present in the sample was calculated (11). percentage yield percentage practical yield is calculated to know about efficiency of any method, thus it helps in selection of appropriate method of production. practical yield was calculated as the weight of acyclovir microspheres recovered from each batch in relation to the amount of starting material. it is calculated by using the formula practical yield /theoretical yield * 100 in vitro drug release studies in vitro drug release studies were performed using usp type ii dissolution apparatus enclosing900ml of 0.1n hcl medium at 50 rpm maintaining 37 ± 0.5ºc. 5 ml sample was withdrawn at predetermined time intervals and replenished with same volume of fresh medium. the drug content in each sample was analyzed by uvvisible spectrophotometer at λmax 252nm (12). results and discussion microspheres of acv were prepared by an ionic gelation method using polymer like chitosan and sodium alginate. various evaluation parameters were assessed, with a view to obtain oral controlled release of acv. in the present work, total seven formulations were prepared, the detailed composition is shown in table (1). table 1. composition of acyclovir microspheres s. no formulation code drug (mg) sodium alginate (mg) chitosan (mg) calcium chloride(% w/v) 1 s1 200 300 300 2 2 s2 200 600 300 2 3 s3 200 900 300 2 4 f1 200 300 300 4 5 f2 200 600 300 4 6 f3 200 900 300 4 7 f4 200 1200 300 4 iraqi j pharm sci, vol.27(1) 2018 acyclovir microspheres 3 the prepared acv microspheres were then subjected to ftir, sem, particle size, size distribution, % yield, entrapment efficiency, in vitro dissolution and release kinetics. the melting point of acv was determined using melting point apparatus and found to be 257oc, thus indicating the purity of obtained drug sample. a standard calibration curve for the acv was obtained by measuring absorbance at λ max 252 in ph 1.2 by plotting the graph of absorbance v/s concentration which complied with standards. the standard plot of acv is shown in figure(1). figure 1. standard curve of acyclovir in 0.1 n hcl at λ max 252 nm from the spectra figure (2, 3) it was observed that all the characteristic peaks of acv were present in the sample spectrum, thus indicating compatibility of acv and other ingredients. the characteristic peaks of the acv compared with the peaks obtained for formulation are given in table (2). there was no chemical interaction between acv and polymer and it can be concluded that the characteristic bands of acv were not affected after successful formulation. table 2. drug polymer interaction (ftir) study ir spectra drug microspheres o-h stretching 3167 3344 c=c aromatic stretching 1482 1418 c-o-c ether 1045 1025 aryl ketone 1629 1604 figure 2. ftir spectra of pure drug and microspheres iraqi j pharm sci, vol.27(1) 2018 acyclovir microspheres 4 figure 3. ftir spectra of pure drug and microspheres the surface morphology of the acv microspheres was studied by sem. sem photographs of the optimized formulation f3 are shown in the figure (4 and 5). the average particle size of acyclovir microspheres was determined and results were tabulated in table (3). as polymer concentration was increased, the mean particle size of acv microspheres was also increased. the significant increase may be because of the increase in the viscosity of the droplets (may be due to the increase in concentration of polymer solution). acv microspheres having a size range of 23.8 to 39.4 μm. calcium chloride was used as cross linking agent, when low concentration of calcium chloride is used the entrapment efficiency was low. entrapment efficiency increases with an increase in the calcium chloride concentration. from the results it can be inferred that there is a proper distribution of acv in the microspheres and the deviation is within the acceptable limits. the percentage entrapment efficiency was found to be 38.21% to 72.9%. the results obtained are given in table (3). a maximum of 72.9% drug entrapment efficiency was obtained in the f3 formulation. it is observed that the drug entrapment was proportional to the acv: polymer ratio and calcium chloride concentration. the percentage yield for acv microspheres were 52.89%, 57.60%, 76.38%, 56.24%, 60.03%, 80.23% and 91.2%, for formulation s1, s2, s3, f1, f2, f3, f4 respectively were given in table (3). the increase in % yield is due to increasing in polymer concentration. the in vitro performance of acv microspheres showed prolonged and controlled release of acv. the results of the in vitro dissolution studies of formulations s1 to s3 and f1 to f4 are shown in table (4) and figure (6, 7). iraqi j pharm sci, vol.27(1) 2018 acyclovir microspheres 5 figure. 4 sem images of optimized formulation figure. 5 sem images of optimized formulation table 3. average diameter, drug entrapment efficiency and % yield of acyclovir microspheres mean ± sd where n = 3 table 4. in vitro release data of acyclovir microspheres (2 and 4 %w/v calcium chloride) s. no. time (h) cumulative % drug release s1 s2 s3 f1 f2 f3 f4 1 0 0 0 0 0 0 0 0 2 1 6.02±0.36 4.33±0.22 5.32±0.46 4.33±0.16 9.03±0.31 8.36±0.72 4.28±0.63 3 2 14.52±0.85 8.02±0.62 7.29±0.64 7.03±0.24 13.61±0.48 15.95±0.24 6.53±0.56 4 3 20.03±0.43 12.38±0.84 13.54±0.28 13.28±0.65 21.43±0.24 17.82±0.42 9.76±0.42 5 4 23.86±0.52 17.02±0.56 15.31±0.42 17.6±0.46 25.92±0.55 19.23±0.25 11.54±0.12 6 5 29.34±0.89 22.63±0.28 21.16±0.38 22.88±0.77 26.58±0.82 21.08±0.52 13.53±0.29 7 6 35.15±0.23 27.76±0.78 24.73±0.86 27.72±0.59 26.84±0.54 24.69±0.63 15.84±0.36 8 7 38.67±0.54 32.09±0.29 29.56±0.54 32.53±0.16 29.12±0.43 31.54±0.36 16.22±0.48 9 8 46.25±0.77 35.18±0.46 3260±0.23 35.64±0.52 40.03±0.22 33.38±0.92 20.08±0.71 10 9 52.23±0.21 42.08±0.27 34.59±0.37 37.43±0.21 45.82±0.41 36.72±0.46 25.03±0.62 11 10 56.25±0.68 44.11±0.19 36.03±0.84 46.64±0.36 50.08±0.73 39.83±0.73 28.43±0.19 12 11 60.08±0.32 47.30±0.48 39.89±0.67 54.38±0.52 54.03±0.34 40.06±0.44 32.27±0.37 13 12 61.66±0.56 49.33±0.43 40.65±0.74 66.42±0.18 57.34±0.42 43.75±0.28 55.83±0.33 mean ±sd where n = 3 s. no formulation code average size ± sd (μm) entrapment efficiency (%) percent yield (%) 1. s1 23.8±4.58 46.00 52.89 2. s2 28.6±6.48 54.03 57.60 3. s3 34.7±5.27 64.25 76.38 4. f1 34.5±7.23 48.38 56.24 5. f2 20.3±5.36 54.76 60.03 6. f3 36.7±7.42 72.90 80.23 7. f4 39.4±4.09 38.21 91.2 iraqi j pharm sci, vol.27(1) 2018 acyclovir microspheres 6 figure 6. drug release from s1 to s3 figure 7. drug release from f1 to f4 s3 formulation showed 40.65% cumulative drug release at the end of 12 hours while s1 formulation showed 61.66% cumulative drug release at the end of 12 hours. this is because with the increase in polymer concentration the free drug concentration on the surface of microspheres will be decreased and more drugs get entrapped in the microspheres and drug gets released slowly. the cumulative % drug release from f1 formulation is 66.42% at the end of 12 hours while cumulative % drug release from f4 formulation is 55.83% at the end of 12 hours. the drug release from s formulations and f formulations were mainly depend on amount of sodium alginate and the concentration of calcium chloride. this is because with the increase in cross linking agent concentration more drug gets entrapped in the microspheres and the microspheres formed are more intact when compared to other formulations. the microspheres and the microspheres formed are more intact when compared to other formulations. the drug release data was best explained by zero order equations, r2 for zero order and first order were obtained at 0.9896, 0.3476 (f3) respectively. based on the particle size, entrapment efficiency, drug release and r square value it was confirmed that the optimized formulation (f3) follows zero order and according to korsemeyer-peppas (n= 0.9855, r2 = 0.9489)it was non fickian. to ascertain the drug release mechanism the in vitro release data were also subjected to higuchi’s diffusion plot, hixson crowell plot and peppas plot and the correlation coefficient values were found to be 0.5871, 0.0362 and 0.9489 respectively in table (5) and figure(8,9,10,11). table 5. regression co-efficient (r2) values of different kinetic models and diffusion exponent (n) of peppas model for optimized formulation (f3) formulation zero order first order higuchi matrix korsemeyer peppas r2 value r2 value r2 value r2 value n value f3 0.9896 0.3476 0.5871 0.9489 0.9855 figure 8. zero order plot of f3 figure 9. first order plot of f3 iraqi j pharm sci, vol.27(1) 2018 acyclovir microspheres 7 figure 10. hixsoncrowell plot of f3 figure 11. higuchi plot of f3 conclusion from the above experimental results, it can be concluded that preformulation studies, like solubility and uv analysis of acv and melting point were complied with standards. the ftir spectra’s revealed that, there was no interaction between polymer and acv. the polymer used is compatible with the acv. oral controlled release of acv can be achieved by ionic gelation by using polymer chitosan and sodium alginate. the surface smoothness of the acv microspheres was increased by increasing the polymer concentration, which was confirmed by sem. as the drug to polymer ratio was increased, the mean particle size of acv microspheres was also increased. entrapment efficiency increases with an increase in the polymer concentration and also with an increase in calcium chloride concentration. from the results it can be inferred that there was a proper distribution of acv in the microspheres and the deviation was within the acceptable limits. the study also indicated that the amount of drug release decreases with an increase in the polymer concentration. the in vitro performance of acv microspheres showed prolonged and controlled release of the drug. the co-efficient of determination indicated that the release data was best fitted with zero order kinetics. from the study it is evident that promising sustained release microspheres of acv may be developed from ionic gelation techniques by using polymers chitosan and sodium alginate. 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encyclopedia of controlled drug delivery, new york, ny: john wiley and sons, inc., 1999; 493-546. iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers doi: http://dx.doi.org/10.31351/vol27iss1pp53-68 53 preparation and evaluation of darifenacin hydrobromide loaded nanostructured lipid carriers for oral administration ali k. ala allah*,1 and ahmed a. hussein** *ministry of health and environment, babylon health directorate, babylon, iraq. ** department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract darifenacin hydrobromide is a selective m3 receptor antimuscarinic drug and it is used in the management of urinary frequency, urgency, and incontinence in detrusor instability. it is slightly soluble in water, undergoes extensive hepatic first-pass metabolism and has short elimination half-life (3–4 hours). therefore, it has low bioavailability (15.4 % 18.6 %). darifenacin hydrobromide loaded nanostructured lipid carriers (nlcs) were formulated by emulsification sonication using different ratios of solid lipid to liquid lipid, different types and concentration of surfactants. formula sixteen , containing darifenacin hydrobromide 8.9 mg , solid lipid glyceryl monostearate and olic acid in a ratio equal to 77.5:22.5 , tween 80 (0.5%) , and vitamin e that is added as an antioxidant , was considered as an optimized formula based on its particle size, polydispersity index (pdi) , zeta potential and entrapment efficiency. this formula was subjected to further characterization such as dsc, ftir, xrd, afm, and release study. ftir and dsc studies indicated no interaction between drug and excipients. xrd study showed a halo pattern which is a significant pattern of amorphous form of the drug. atomic force microscopy (afm) study showed discrete lipid nanoparticles with no aggregation. release study exhibited burst release in the first hour followed by sustained and controlled release up to 12 hours. keywords: darifenacin hydrobromide, nanostructured lipid carrier, bioavailability. الحامالت الدهنية ذات البنية النانوية المحملة بالداريفناسين هايدروبرومايد المعطاة عن طريق الفم **و احمد عباس حسين 1،*علي كاظم على هللا والبيئة ، دائرة صحة بابل، بابل ، العراق.وزارة الصحة * فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ،العراق. ** الخالصة ويستخدم في عالج سلس البول. هذا العقار قليل الذوبان في m3عقار داريفناسين هايدروبرومايد يعتبر مثبط انتقائي لمستلمات النطاق بالكبد ويمتلك زمن طرح من الجسم قصير جداً من ثالثة الى اربع ساعات لذلك التوافر الحيوي الماء، يتعرض الى ايض واسع في البالزما لهذا الدواء قليل من خمسة عشر بالمئة الى ثمانية عشر بالمئة. حامالت الدهون ذات البنية النانوية المحملة بدرافيناسين صهر مع استخدام الموجات فوق الصوتية مستخدمين نسب مختلفة من الدهون الصلبة الى هايدروبرومايد صنعت بطريقة المستحلب المن الدهون السائلة، انواع مختلفة وتراكيز مختلفة من العوامل التي تقلل الشد السطحي. ملغم ( و نسبة دهون صلبة الى دهون سائلة تســـــــــــاوي 8, 9التركيبة السادسة عشر تتكون من دارافيناسين هايدروبرومايد ) التركيبة السادسة عشر تعتبر أفضل تركيبة اعتماداً يضاف كعامل مضاد لالكسدة . e( , و فيتامين %8, 5) 88توين ,22, 5: 77, 5 برت لدواء بداخلها. التركيبة السادسة عشر اختعلى حجم الجزيئات والتوزيع الحجمي للجزيئات والشحنة السطحية والقابلية على احتواء ا ( ، وكذلك دراسة afmاي اف ام ) ، حيود االشعة السينية، ( ftir ) باستخدام المسح الكالوري، مطيافية االشعة تحت الحمراء واد اخل بين الدواء والمالتركيبة السادسة عشر اختبرت باستخدام االشعة تحت الحمراء والمسح الكالوري واظهرت عدم وجود تدالتحرر. اظهر وجود جزيئات afmاالخرى في التركيبة. اختبار حيود االشعة السينية اظهر شكل غير متبلور , واختبار مجهر القوة الذريـــة دمنفصلة واليوجد تجمع للجزيئات.دراسة التحرر للدواء اظهرت تحرر سريع للدواء خالل الساعة االولى بعد ذلك تحرر بطيء الى ح اثنا عشر ساعة.اظهرت الدراسة الكلية اهمية الحامالت الدهنية ذات البنية النانوية كنواقل لزيادة التوافر الحيوي لعقار داريفناسين هايدروبرومايد مقارنة بالحبوب المعطاة عن طريق الفم . ة، التوافر الحيوي.الكلمات المفتاحية: داريفناسين هايدروبرومايد، حامالت الدهون ذات البنية النانوي introduction recently, several approaches have been investigated to develop nanosized drug delivery system such as lipid nanoparticales with a solid matrix which are divided into solid lipid nanoparticles (slns) and nanostructured lipid carriers (nlcs). slns are prepared from solid lipids only. therefore, after preparation at smallest a part of the particles crystallize in a higher energy modification (α or β). during storage, these modifications can transform to the low energy, more ordered β modification. due to high degree of order of this modification, the number of imperfections in the crystal lattice is small and this leads to drug expulsion. 1corresponding author e-mail: pharss75@gmail.com received: 31/10/2017 accepted: 3/3/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp53-68 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 54 nlcs have been developed to overcome the drawbacks associated with slns. they are considered to be the second generation of lipid nanoparticles. compared to slns, nlcs show a higher loading capacity for active compounds by creating a less ordered solid lipid matrix, i.e. by blending a liquid lipid with the solid lipid, a higher particle drug loading can be achieved. therefore, the nlcs have an increased drug loading capacity in comparison to slns and the possibility of drug expulsion during storage is less . nlcs have also a lower water content of the particle suspension and a less tendency of unpredictable gelation(1). darifenacin is a selective m3 antimuscarinic with actions similar to those of atropine. it has a greater selectivity for the muscarinic receptors of the bladder. it is subjected to extensive first-pass metabolism and has a short elimination half-life after intravenous and immediate release oral dosage forms (3-4 hr)(2). the absolute bioavailability of darifenacin from 7.5 mg and 15mg prolonged release tablet was estimated to be 15.4 % and 18.6% respectively(2). it is metabolized in the liver by cytochrome p450 isoenzymes cyp 2d6 and cyp 3a4 (2). darifenacin is a p-glycoprotein(p-gp) substrate. it is about 98% bound to plasma proteins . most of the dose is excreted as metabolites in the urine and feces(2). the objective of this study is to prepare differents darifenacin hydrobromide loaded nlcs to improve the bioavailability of darifenacin hydrobromide which undergoes extensive first-pass effect when formulated in conventional dosage form , characterization of the prepared formulas , and the selection of the best darifenacin hydrobromide loaded nlc which subjected to further characterization . after that, formulation of the best formula as a dosage form well known to the patient (capsule dosage form) was achieved in order to improve patient compliance . materials and methods materials darifenacin hydrobromide and glyceryl monostearate ( gms ) ( hangzhou hyperchemical china ) , oleic acid ( central drug house company india ) , tween80 , stearic acid and palmitic acid ( bdh chemical england ) , methanol ( romil, united kingdom ) and distilled deionized water was used. all other chemicals were reagent grade. method screening of components prior to the production of nlc formulation , lipid, oil , and surfactant screening should be performed to determine the most suitable components for the active ingredient to be incorporated in the nlc . solubility in solid lipid the solubility of darifenacin hydrobromide in different solid lipids was determined by semi-quantitative method. an accurately weighed fixed quantity (8.9 mg) of the drug was taken in a series of test tubes and solid lipids were added in increments until the drug is completely solubilized. the temperature of the test tubes was controlled at 5-10 °c above the melting point of respective lipids (3). the test tubes were intermittently mixed using cyclone mixer and observed for any drug residues. the amount of lipid (mg) required to completely solubilized the drug in the molten state was determined (3). solubility in liquid lipid an excess amount of darifenacin hydrobromide was added to 5ml of oil in a test tube and mixed using cyclone mixer. the mixture was agitated on mechanical shaker for 24 hr at room temperature for equilibration. after equilibrium, each sample was centrifuge at 10,000 rpm for 30 min to separate the undissolved drug. supernatant that obtained was pulled and filtered through 0.45 μm filter. the filtrate was diluted suitably with methanol and saturation solubility of darifenacin hydrobromide (mg/ml) in oil was determined by recording absorbance using uvvis spectrophotometer at respective λ max (4). preparation of nanostructured lipid carriers ( nlcs ) an accurately weighed solid lipid gms and liquid lipid oleic acid were mixed and then heated at 5 – 10 °c above the melting point of lipid mixture. to this lipid mixture, the drug was added to obtain a clear melting solution. an aqueous phase was prepared by dissolving surfactant in deionized water and heated to the same temperature as that of the oil phase. then, this hot aqueous phase was added dropwise to the lipid phase at a constant rate (2 ml / min) under magnetic stirring. after that, this preemulsion was sonicated for 20 minutes using probe sonicator. the resulting hot nanoemulsion was cooled to room temperature to induce crystallization. twenty-two formulas were prepared by this method as shown in table (1). vitamin e was added to the selective formula as antioxidant. the selective formula was freezedried by using cryoprotectant to convert nlc to dry powder and was filled in a hard gelatin capsule of zero size (5). iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 55 table1. formulations of darifenacin hydrobromide loaded nanostructured lipid carriers ( nlcs) formulas no. amount of drug (darifenacin hydrobromide) mg ratio of solid lipid to liquid lipid glyceryl monostearate: oleic acid type of surfactant % ( w / v ) co surfactant % ( w / v ) water tween20 tween 80 poloxamer80 span80 myverol f1 8.9 92.5 : 7.5 0.5 q.s f2 8.9 92.5 : 7.5 1 q.s f3 8.9 92.5 : 7.5 1.5 q.s f4 8.9 85 : 15 0.5 q.s f5 8.9 85 : 15 1 q.s f6 8.9 85 : 15 1.5 q.s f7 8.9 77.5 : 22.5 0.5 q.s f8 8.9 77.5 : 22.5 1 q.s f9 8.9 77.5 : 22.5 1.5 q.s f10 8.9 92.5 : 7.5 0.5 q.s f11 8.9 92.5 : 7.5 1 q.s f12 8.9 92.5 : 7.5 1.5 q.s f13 8.9 85 : 15 0.5 q.s f14 8.9 85 : 15 1 q.s f15 8.9 85 : 15 1.5 q.s f16 8.9 77.5 : 22.5 0.5 q.s f17 8.9 77.5 : 22.5 1 q.s f18 8.9 77.5 : 22.5 1.5 q.s f19 8.9 85 : 15 0.5 q.s f20 8.9 85 : 15 1 q.s f21 8.9 85 : 15 0.5 0.2 q.s f22 8.9 77.5 : 22.5 0.5 q.s iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 56 characterization and evaluation of nanostructured lipid carriers ( nlcs ) particle size and polydispersity index measurement the particle size analysis of formulas was performed using abt9000 nano laser particle size analyzer. before measurements, nlcs dispersion was diluted suitably using deionized water. data was analyzed by software and values of mean particle size, polydispersity index (pdi) and particle size distribution curve were recorded (6). zeta potential measurement the zeta potential analysis of formulas was performed using zeta sizer. before measurements, nlcs dispersion was suitably diluted (7). entrapment efficiency measurement entrapment efficiency corresponds to the percentage of drug encapsulated within the lipid matrix. certain volume of nlcs dispersion was accurately taken and subjected to centrifugation at 25000 rpm for 30 min at 4° c . after centrifugation, 1 ml of supernatant was taken and suitably diluted with methanol and the free drug concentration determined using uv-vis spectrophotometer and (%ee) measured using the following equation (8): 𝐸𝐸 ( % ) = 𝑊𝑖𝑛𝑖𝑡𝑖𝑎𝑙 − 𝑊 𝑓𝑟𝑒𝑒 𝑊𝑖𝑛𝑖𝑡𝑖𝑎𝑙 × 100 ee(%) = percentage of entrapment efficiency winitial = initial drug concentration wfree = free drug concentration ( unentraped drug ) differential scanning calorimetry ( dsc ) study the possibility of any interaction between darifenacin hydrobromide and excipients was assessed by carrying out thermal analysis of the formulation using dsc. the analysis was performed on the pure darifenacin hydrobromide, gms and lyophilized darifenacin hydrobromide nlcs . each sample was weighed accurately and kept in aluminum pans and scanned between 30 ºc – 400 ºc at a heating rate of 10 °c/min and cooling rate of 40 °c/min under nitrogen gas. an empty aluminum pan was used as reference in the study (9). ftir spectroscopy study ftir helped to confirm the identity of the drug and to detect the interaction of the drug with carriers.ftir spectral measurement for pure darifenacin hydrobromide drug, lipid glyceryl monostearate, oleic acid, tween80, vitamin e and optimized nlcs formulation were obtained on ftir using kbr disk method. the scanning range was 4004000 cm -1 (10). xray diffraction (xrd) study powder x-ray diffraction (pxrd) was performed to analyze crystalline or amorphous nature of darifenacin hydrobromide loaded nlcs. pxrd studies were performed by powder x-ray diffractometer where cuk α radiation of 1.5405°a was used as x-ray source. for the measurements , samples were kept in the glass sample holders followed by scanning from 2° to 60° with scan angular speed (2 θ /min ) of 2°/ min , 40 kv working voltage and 30 ma current. samples used for study were pure darifenacin hydrobromide, glycerl monostearate, and lyophilized dariferacin hydrobromide nlc (11). atomic force microscopy (afm) study to study morphological changes and also the particle size of nlcs , afm micrographs were imaged by using atomic force microscopy (afm). the images were obtained by measurement of interaction forces between the tip and sample surface . the experiments were done in air at room temperature (25°c) operating in noncontact mode droplets of dispersion were placed on a small mica disk. the measurements were performed in different sample locations. image data were analyzed with software (12). in-vitro drug release and release kinetic studies the in-vitro release of darifenacin hydrobromide from nlcs was carried out in 500 ml phosphate buffer solution (ph 6.8) by using the dissolution testing apparatus with rotating basket at 100 rpm and temperature 37+̅ 0.5 °c (13). this method involved placing the capsule of the selected formula inside wire basket that is rotated while immersed in the dissolution medium. five milliliters aliquots were withdrawn at 1 , 2 , 4 , 6 , 8 , 10 , and 12hr from dissolution medium and replaced with 5 ml of fresh buffer to maintain sink condition . the aliquots withdrawn were filtered by using 0.45μm filter, suitably diluted if necessary, and analyzed by using uv-vis spectrophotometer. the cumulative percentage of the released drug was plotted versus time (13). the in-vitro release profile was fitted using several kinetic models such as zero-order (cumulative percentage of drug released versus time), first – order (log cumulative percentage of drug remaining versus time), higuchi (cumulative percentage of drug released versus square root of time), and korsmeyer–peppas ( log cumulative percentage of drug released versus log time ) equations (13). statistical analysis statistical analysis of the data was carried out using one-way analysis of variance iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 57 (anova) test and the level of statistically significance difference was selected as p < 0.05. results and discussions selection of components a selection of suitable lipids and other excipients was significant to develop nlcs for slightly water-soluble darifenacin hydrobromide. to keep the drug in soluble form, it was of prime importance that drug must possess higher solubility in solid lipid and oil. selection of solid lipid solid lipid was selected by checking the solubility of the drug in melted solid lipid by means of visible observation with the naked eyes under normal light. lipids used for this study were stearic acid, palmitic acid and gms. it was found that gms showed highest darifenacin hydrobromide solubilizing capacity. table (2) shows the comparative solubility of drug in different lipids. table2. amount of solid lipid required to solubilize 8.9 mg of darifenacin hydrobromide no . lipids amount of lipid 1 stearic acid more than 1000 mg 2 palmitic acid more than 1000 mg 3 glyceryl monostearate 400 mg selection of liquid lipid liquid lipid was selected based on the maximum solubility of darifenacin hydrobromide in different liquid lipids. lipids used for this study were oleic acid, castor oil, and ethyl oleate. it was found from the result that oleic acid exhibited maximum darifenacin hydrobromide solubilizing capacity than the others as shown an table (3). therefore, it was selected as liquid lipid to make a matrix with solid lipid gms for the development of nlcs. table3. solubility of darifenacin hydrobromide in different oils no . oil saturation solubility mg / ml 1 caster oil 11.5 2 oleic acid 13.7 3 ethyl oleate 12.43 preparation of darifenacin hydrobromide loaded nanostructured lipid carriers emulsification sonication is a simple and popular method for preparation of nlcs and considered the method of choice for drugs showing high solubility in molten lipids (14). solid lipid gms and liquid lipid (oleic acid) were utilized to provide a core composed of highly lipophilic environment to accommodate darifenacin hydrobromide, thus becoming a suitable and optimum nanocarrier or reservoir for the drug. the incorporation of solid and liquid lipids mixture in the lipid matrix promoted imperfect crystallization, thus lowering the probability of the entrapped drug expulsion during storage. also, the presence of liquid lipid in formulations allowed more flexibility for modulation of drug release and better drug-entrapment efficiency (15). characterization and evaluation of nanostructured lipid carriers particle size and polydispersity index determination particle size and pdi were important characteristics in the evaluation of stability of darifenacin hydrobromide loaded nlcs (16). four darifenacin hydrobromide formulas ( f19, f20 , f21 and f22 ) from the prepared formulas were in microsize range , therefore they are not subjected to further characterization . eighteen darifenacin hydrobromide formulas in nano size range, from the prepared formulas, were successfully prepared as shown in table (4). a nanoscale particle exhibited unique physical and biological properties, making it particularly ideal for drug entrapment, and provided a large surface area for the reaction with its site of action (17). also, the nanoscale size minimized the probability of drug being phagocytized by macrophage of the mononuclear phagocytic system, hence decreasing the destruction of darifenacin hydrobromide nlcs in the body (18). particle size plays a crucial role in the gastrointestinal uptake and their clearance by the reticuloendothelial system. therefore, the precise determination of the particle size is very important where particle size less than 300 nm is advisable for the intestinal transport(19). polydispersity index (pdi) is a measurement of particle size distribution that varies from 0 to 1. the polydispersity index (pdi) of darifenacin hydrobromide loaded nlcs formulas was within the acceptable range and it indicated that all the prepared nlcs were almost in monodispersity and homogeneous with narrow size distribution as shown in table (4). the closer the value of pdi to zero, the higher the homology between the particles. the pdi of less than 0.5 indicates that there was no aggregation of the nanoparticle of darifenacin hydroiraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 58 bromide-nlcs. pdi more than 0.5 is an indication of particle aggregation. the aggregates do not interact with the site of action in the way smaller individual particles do. the aggregation or agglomeration impedes the targeting efficiency of the nanoscale particle to the target organ. also, the degree of cellular uptake might be decreased due to the presence of unwanted aggregates since , aggregation increases the particle size and lower the surface area (20-22). effect of concentration of surfactants on particle size it was observed that increasing the concentration of surfactants had statistically significant effect (p>0.05) on particle size.the particle size was found to decrease with an increase in concentration of surfactant tween80 and tween20 for formulas (f1-f18) when the ratio of solid lipid gms to liquid lipid oleic acid constant .the higher surfactants (tween 80 and tween 20) concentrations reduced the surface tension and facilitated particle partition. the decrease in the particle size is accompanied by a rapid and tremendous increase in the surface area . thus, an increase in the surfactants (tween 80 and tween 20) concentration in the primary dispersion resultsed in rapid coverage of the newly formed particle surface (23). zeta potential determination zeta potential is essential for evaluating the storage stability of colloidal dispersions(24). the zeta potential of the different formulas of darifenacin hydrobromide nlcs was found within the range of (11.78 mv to-32.44 mv) as shown in table (4). zeta potential referred to the electrostatic charges on the surface of the nanoparticle in the dispersion, which was used to predict the long term stability of the nanoparticles (24). since, the zeta potentials above 30 mv or below–30 mv were required for full electrostatic stabilization (25). many experiments demonstrated that it is not only electrostatic repulsion had an effect on the stability of any nanoparticles, but also the use of steric stabilizer that favoured the formation of stable nanoparticle dispersion (26). the steric hindrance from tween80, that was used in the production of darifenacin hydrobromide-nlcs as a stabilizer , had an additional effect in increasing the particle stability (26). also, surface charge of the nlcs has an effect on tissue permeability and cellular up take where high positive or negative zeta potential lead to superior phagocytosis (27) . effect of ratio solid lipid to liquid lipid on zeta potential the negative zeta potential value in the darifenacin hydrobromide loaded nlcs formulas related to deprotonation of carboxyl group of oleic acid. the increase in ratio of liquid lipid to solid lipid had significant effect (p>0.05) on zeta potential. the value of zeta potential increased when the ratio of oleic acid to glyceryl monostearte increased (28). entrapment efficiency determination the entrapment efficiency of the different formulas of darifenacin hydrobromide loaded nlcs is shown in table (4) . it was consistently reported that the increase in entrapment efficiency in nlcs related to the presence of solid and liquid lipids that results in great imperfections in crystal lattice providing higher space for drug accommodation (29 31). also, higher drug solubility in liquid lipid will increase the entrapment efficiency (32). effect of concentration of surfactants on entrapment efficiency it was observed that increasing the concentration of surfactants had statistically significant effect (p>0.05) on the entrapment efficiency of darifenacin hydrobromide. the entrapment efficiency of darifenacin hydrobromide loaded nlcs was found to decrease with an increase in the concentrations of surfactants (tween 80 and tween 20) for formulas (f1-f18) when the ratio of solid lipid glyceryl monostearate to liquid lipid oleic acid was constant. the high surfactants (tween 80 and tween 20) concentrations reduced the particle size and this decreased the amount of darifenacin hydrobromide entrapped in the core of darifenacin hydrobromide nlcs and adsorbed on the surface of darifenacin hydrobromide nlcs (33). iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 59 table4. particle size, zeta potential, pdi and entrapment efficiency of darifenacin hydrobromide loaded nlcs. formula no. particle size zeta potential entrapment efficiency pdi f1 98.9nm -14.32 56.27% 0.22 f2 79.1 nm -13.82 56.17% 0.22 f3 78.6 nm -15.09 53.93% 0.37 f4 87.9 nm -16.72 68% 0.15 f5 86.6 nm -14.71 55.11% 0.11 f6 19.7 nm -17.29 53.39% 0.23 f7 989 nm -18.97 83.51% 0.03 f8 436 nm -25.72 58.42% 0.01 f9 99.3 nm -21.58 47.44% 0.53 f10 191 nm -17.9 65.77% 0.1 f11 106 nm -11.78 43.97% 0.22 f12 61.9 nm -13.92 38.46% 0.01 f13 151 nm -19.23 65.78% 0.04 f14 139 nm -17.36 62.79% 0.07 f15 133 nm -17.81 61.06% 0.02 f16 249 nm -32.44 74.44% 0.2 f17 139 nm -18.46 65.38% 0.49 f18 78.7 nm -27.06 23.82% 0.33 selection of the optimum formula formula sixteen regarded as the optimum formula depending on entrapment efficiency measurement which was equal to 74.44% and zeta potential which was equal to– 32.44mv in addition to the particle size which is equal to 249 nm and polydispersity index which was equal to 0.2 . formula sixteen containing ratio of solid lipid gms to liquid lipid oleic acid equal to 77.5:22.5 , tween 80 (0.5%), darifenacin hydrobromide 8.9 mg, and vitamin e that is added as antioxidant. differential scanning calorimetry ( dsc ) study differential scanning calorimetry was performed to characterize the polymorphism and the degree of crystallinity of darifenacin hydrobromide loaded nlcs. figures (1 3) and (3) showed the dsc thermograms of darifenacin hydrobromide, gms and darifenacin hydrobromide loaded nlcs respectively. the study showed that the melting point of darifenacin hydrobromide nlcs (69.76 °c) was lower than that of the bulk material gms (76.65 °c) also disappearance of melting peak of darifenacin hydrobromide (235.17 °c) indicated that darifenacin hydrobromide was dissolved in the lipid matrix and encapsulated in the nanostructured lipid carrier. during the preparation , darifenacin hydrobromide was dissolved in the melted lipid phase. following the cooling of the dispersion to room temperature, darifenacin hydrobromide melting peak was not detected anymore. the absence of this thermodynamic transition could be due to a molecular dispersed state of darifenacin hydrobromide in the mixture (34). the decrease in the melting point of darifenacin hydrobromide nlcs (69.76 °c) which was below that of gms (76.65 °c) is described as melting point depression (35). the addition of oil (oleic acid) into the matrix provoked an additional shift of the melting point to lower temperature. decrease in melting enthalpy in darifenacin hydrobromide nlcs as compared to gms and darifenacin hydrobromide was due to less–ordered arrangement of nanoscale particles. therefore lesser amount of energy was needed to overcome the lattice force in the nlcs than gmc . also, incorporation of darifenacin hydrobromide inside the lipid matrix resulted in a further increase in the number of defects in the lipid crystal lattice (35). iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 60 100.00 200.00 temp [c] -4.00 -2.00 0.00 2.00 mw dsc 235.17x100c figure 1. dsc thermogram of darifenacin hydrobromide 50.00 100.00 150.00 200.00 250.00 temp [c] -30.00 -20.00 -10.00 0.00 mw dsc 76.65x100c figure 2. dsc thermogram of glyceryl monostearate. 100.00 200.00 temp [c] -5.00 0.00 mw dsc 69.76x100c figure 3. dsc thermogram of dariferacihydrobromide loaded nlcs. ftir spectroscopy study ftir spectra of darifenacin hydrobromide , gms , oleic acid, tween 80 , vitamin e , and darifenacin hydrobromide loaded nlcs ( f16 ) are shown in figures (4 9) illustrated that , there was no interaction between drug and excipients since, the characteristic peaks of the drug and the major constituents are still observed in ir spectrum of the selected formula . figure 4 . ir spectrum of darifenacin hydrobromide. iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 61 figure 5. ir spectrum of glyceryl monostearate (gms). figure 6 . ir spectrum of oleic acid iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 62 figure 7. ir spectrum of tween80. figure8 . ir spectrum of vitamin e. iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 63 figure 9 . ir spectrum of darifenacin hydrobromide loaded nlcs ( f16 ). x–ray diffraction study x–ray diffractograms of pure darifenacin hydrobromide , gms and freeze dried darifenacin hydrobromide nlcs were presented in figures (10-12) . the x – ray diffractogram of darifenacin hydrobromide exhibited sharp peaks at diffraction angles (2θ) with a typical crystalling patten. all major characteristic crystalline peaks ( 11.47° , 18.20° and 24.55° ) disappeared in the diffractogram of darifenacin hydrobromide nlcs. this indicated that darifenacin hydrobromide converted from crystalline to amorphous form (36). figure 10. x – ray diffractograms of darifenacin hydrobromide. iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 64 figure 11. x – ray diffractograms of glyceryl monostearate (gms ). figure 12 . x – ray diffractograms of darifenacin hydrobromide loaded nlcs iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 65 atomic force microscopy ( afm ) study the morphological analysis performed by afm showed three-dimensional structure (figure 13) and discrete lipid nanoparticles with no aggregation. the particle size equal to 260nm as shown in the histogram of particle size distribution in figure (14) (37). figure 13. three –dimensional morphology of darifenacin hydrobromide loaded nlcs ( f16) figure 14. granularity cumulation distribution of darifenacin hydrobromide loaded nlcs ( f16) in-vitro drug release and release kinetic studies the in-vitro drug release of darifenacin hydrobromide loaded nlcs showed an interesting biphasic release with an initial burst effect followed by controlled and sustained release (38) as shown in figure (15). the initial burst release of darifenacin hydrobromide might be due the presence of unentrapped darifenacin hydrobromide in the nlcs (39). another reason might be due to most of the liquid lipid ( oleic acid ) being located in the outer shell of nlcs which lead to darifenacin hydrobromide enriched shell that easily released drug by diffusion or matrix erosion (40). the third supportive factor for the burst release that if nlcs prepared with high temperature and optimum concentration of surfactant, it may produce drug burst release (40 ,41) . at the end of first hour, 30 % of drug is released, after that the drug release follow steady pattern of controlled and sustained release up to 12 hs. the kinetic of release and the mechanism of the release from nlc was evaluated by fitting the release date into first order , zero order , higuchi and korsemeyer – peppas as shown in table (5 ). the darifenacin hydrobromide loaded nlcs was fitted well with higuchi model since r2 value equal to 0.9425 . the n value ( < 0 .5 ) indicated that the release behavior of darifenacin hydrobromide loaded nlcs followed fickian diffusion mechanism (41) . figure 15. the percentage of drug release from formula sixteen per time at ph 6.8 and 37°c iraqi j pharm sci, vol.27(1) 2018 darifenacin hydrobromide loaded nanostructured lipid carriers 66 table5.the kinetic and the mechanism of the release data of darifenacin hydrobromide from nlc f o r m u la drug release kinetic krosmeyer -pepas n value zero order r2 first order r2 higuchi r2 16 0.2225 0.9298 0.9425 0.419 conclusion in this work, darifenacin hydrobromide loaded nlcs with sustained release for about 12 hours with biphasic profile effect was successfully 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magnesium) status in iraqi patients with acne vulgaris :( casecontrolled study) basil om saleh* ,1 , zainab n. h. anbar** and ali y. majid*** * department of physiological chemistry, college of medicine, university of baghdad,baghdad,iraq. ** department of biochemistry, baghdad college of pharmacy,baghdad,iraq. ***poisoning consultation center, baghdad teaching hospital, ,baghdad,iraq. abstract recently on the dermatological fields, the serum levels and the roles of zn, cu and mg have been studied especially in acne vulgaris, but the results were controversial. the aim of the present study is to investigate a relationship between the severity of acne and the serum levels of zinc (zn), copper (cu) and magnesium (mg) and to demonstrate the status of serum levels of zinc, copper, and magnesium in iraqi male patients with acne vulgaris and to compare it with those of healthy controls.this case controlled study was conducted in the department of dermatology and venerology and in the poisoning consultation center of baghdad teaching hospital between may 2009 to january 2010. fortyfive male patients with acne vulgaris, their ages ranged from 18-30 (21.82±3.77) years and 45 healthy male subjects as controls, their ages ranged from 18-30 (22.18±3.85) years were included in this study. patients were subdivided into three groups according to the severity of their acne; mild acne group (n=15), moderate (n=15) and severe acne group (n=15). investigations included serum estimation of zn, cu and mg in both patient and control groups.the data obtained from this study showed that the (mean±sd) values of serum levels of zn was significantly decreased in severe acne group compared with controls, mild and moderate type of acne group (p< 0.05). serum mg concentration was significantly lower in severe acne group compared with mild and moderate acne group (p< 0.05). with regard to serum cu, there were no significant differences among groups of patients with acne vulgaris.this study revealed a significant association between serum levels of either zn or mg with the severity of acne. key words: acne vulgaris, zinc, copper, magnesium. الخالصة ٗخبطخ ٍشع زت اىديذٝخ, اىَظبثِٞ ثبألٍشاعفٜ ٍظ٘ه اىَشػٚ ٗاىَغْٞضًٞ٘ ٗاىْسبس اىخبسطِٞ ٗإَٔٞخ أُ ٍضز٘ٙ ٌ ٍضز٘ٙ اىعْبطش اىَعذّٞخ)اىخبسطِٞ, اىْسبس, اىَغْٞضًٞ٘( ٞاىشجبة رٌ دساصزٔ ٗىنِ اىْزبئح مبّذ ٍزجبْٝخ.أُ ٕذف ٕزٓ اىذساصخ ٕ٘ رقٞ ٓ اىعْبطش ٗشذح زت ٗدساصخ اىعالقخ ثِٞ ٍضز٘ٙ ٕز اىَظبثِٞ ثَشع زت اىشجبة ٗاىزم٘س األطسبء اىعشاقِٞٞ فٜ ٍظ٘ه اىزم٘س ىقذ رٌ أخشاء ٕزٓ اىذساصخ فٜ قضٌ األٍشاع اىديذٝخ ٗ اىزْبصيٞخ ٗاىَشمز االصزشبسٛ ىيضًَ٘ فٜ ٍضزشفٚ ثغذاد اىزعيَٜٞ ىيفزشح .اىشجبة اىَعذه صْخ(, 0٠ـ ٠8رمش ٍظبة ثست اىشجبة رزشاٗذ أعَبسٌٕ ثِٞ ) ٥٤ .٢٠٠٠ٗىغبٝخ مبُّ٘ اىثبّٜ ٢ ٠٠ ٩أٝبس ٍِ 0٠ـ٠8) ( ثْفش اىفئخ اىعَشٝخاىضٞطشح رمش طسٞر)ٍدَ٘عخ ٥٤ صْخ ٗ ( ٧٧,0 – ٢ 8٢,٠) سشاف اىَعٞبسٛ ألعَبسٌٕ ٕ٘+االّ صْخ( رٌ شَ٘ىٌٖ فٜ ٕزٓ اىذساصخ. أُ ٍدَ٘عخ اىَشػٚ رٌ رقضَٖٞب 8٤,0-٠8,٢٢) صْخ(, اىَعذه +االّسشاف اىَعٞبسٛ ألعَبسٌٕ ٕ٘ , فئخ ٠٤, فئخ زت اىشجبة اىْ٘ع اىَز٘صط ٗعذدٌٕ ٠٤إىٚ ثالس فئبد زضت شذح زت اىشجبة: فئخ زت اىشجبة اىْ٘ع اىجضٞط ٗعذدٌٕ غْٞضًٞ٘ ٛ فٜ ٍظ٘ه األشخبص . اىفس٘ص رشَو رسذٝذ ٍضز٘ٙ اىخبسطِٞ, اىْسبس, ٗاى٠0َ زت اىشجبة اىْ٘ع اىشذٝذ ٗعذدٌٕ أظٖشد ّزبئح اىذساصخ عذً ٗخ٘د فشق إزظبئٜ ٍعْ٘ٛ فٜ ٍضز٘ٙ اىعْبطش)اىخبسطِٞ, اىْسبس, اىَغْٞضًٞ٘( األطسبء ٗاىَشػٚ. ٗاألطسبء. ىنِ, ٍضز٘ٙ اىخبسطِٞ قذ أّخفغ ثَضز٘ٙ إزظبئٜ ٍعْ٘ٛ ىذٙ فئخ زت اىشجبة اىْ٘ع اىشذٝذ ثِٞ ٍدَ٘عزٜ اىَشػٚ (. مزىل أظٖشد اىذساصخ اّخفبع إزظبئٜ >p 0.05 ٍدَ٘عخ األطسبء ٍٗدَ٘عخ فئخ زت اىشجبة اىْ٘ع اىجضٞط) ٍقبسّخ ٍع ٗفٜ ٍب ٝخض (.>p 0.05ٍعْ٘ٛ فٜ ٍضز٘ٙ اىَغْٞضًٞ٘ ىذٙ فئخ زت اىشجبة اىْ٘ع اىشذٝذ ٍقبسّخ ٍع فئخ زت اىشجبة اىْ٘ع اىجضٞط) ت اىشجبة . َٝنِ االصزْزبج ٍِ ٕزٓ اىذساصخ ثأُ ْٕبك عالقخ ٍعْ٘ٝخ ثِٞ ٍضز٘ٙ مو ٍضز٘ٙ اىْسبس , الٝ٘خذ فشق ٍعْ٘ٛ ثِٞ فئبد ز ٍِ اىخبسطِٞ ٗاىَغْٞضًٞ٘ فٜ ٍظ٘ه اىَشػٚ اىَظبثِٞ ثست اىشجبة ٍع شذح اىَشع. introduction acne vulgaris is the most common cutaneous disorder manifested by comedones, papules, pustules and cysts. the etiology of acne appears to be multifactorial, involving follicular hyperkeratinization, hormonal function, proliferation of propionibacterium acnes, increased sebum production and inflammation (1) . despite a significant body of scientific literature, the sequence of events leading to the production of acne lesions is not well understood (1) . specific dietary agents and certain supplements are known to enhance the health and appearance of the skin by improving immune function at the skin level and providing therapeutic bioactive agents that assist in the treatment of many skin conditions, such as psoriasis, eczema and acne (2, 3) . 1corresponding author email : basil_omsal@yahoo.com received : 21/2/2011 accepted : 9/10/2011 mailto:basil_omsal@yahoo.com iraqi j pharm sci, vol.20(2) 2011 serum trace elements and acne vulgaris 45 it has become increasingly clear that nutritional factors such as vitamins and minerals are involved in the pathogenesis of acne (4) . previous studies over the last three decades have shown that zinc(zn) levels are lower in patients with acne than healthy subjects and that oral and topical combination of zinc may be of therapeutic value (5,6) . pohit et al. in 1985 suggest that people with acne have lower-than-normal levels of zn in their bodies (7) . this fact alone does not prove that taking zinc supplements will help acne, but several small double-blind studies involving a total of more than 300 people have found generally positive results (8) .the results of elsaaiee et al. in 1983 revealed differences in the copper and iron content of the sera between 30 individuals complaining of moderate acne vulgaris type ii and healthy individuals, although they were statistically not significant. the zn content showed no changes compared to the control group (9) . recently, nasiri et al. in 2009 indicated that serum zinc levels in 30 iranian acne patients were lower than that of 35 healthy controls; however, this difference was not significant (p= 0.32) (10) . many studies including an epidemiological iraqi study had showed that acne vulgaris in general was more common in males than females (74.24%) versus (61.9%) (11,12,13) .so the aims of the present study are 1) to demonstrate the status of serum levels of zinc, copper, and magnesium in iraqi male patients with acne vulgaris and to compare it with those of healthy controls and 2) to investigate the relation between the severity of acne and the serum levels of the elements of respect. subjects and methods this case controlled study was carried out in the department of dermatology and venerology and in the poisoning consultation center of baghdad teaching hospital from may 2009 to january 2010. the study involved 45 male patients with acne vulgaris, aged range between 18-30 (mean±sd; 21.82± 3.77 years). patients were divided into three groups according to the severity of their acne. a mild acne group that included 15 patients, a moderate acne group of 15 patients and a severe acne group of 15 patients. scoring the severity of acne was according to the following rule: 1. mild acne: in which the count of papules is less than 10 and the count of pustules is less than 20. 2. moderate acne: in which the count of papules ranges from 10 to 30 and the count of pustules ranges from 20 to 40. 3. severe acne: in which the count of papules is more than 30 and the count of pustules is more than 40 (14) . exclusion criteria were intake of oral zinc, magnesium, or copper supplements or multivitamins containing such elements three months before the study, and the presence of any metabolic disease that affected serum elements levels. control group involved were 45 healthy males without acne, and were matched for age 1830 years (mean±sd; 22.18± 3.85 years), and body mass index (mean±sd; 23.04±1.38 kg/m2). five milliliters of peripheral venous blood was collected from each patient and control male in plain test tubes, left to clot, then centrifuged at 2500 rpm for 10 minute. the separated serum stored at -20ºc until the time of mineral assay. serum zinc, copper, and magnesium were determined using flame atomic absorption spectrophotometer (aa-646 shimazdzu, japan). samples were diluted 1:10 with n butanol solution as diluents (15) . levels of serum zn, cu, and mg were calculated after application of absorbancies on suitable calibration curve for each element made from standard solutions. spss version 6 for window was used for all statistical analysis. statistical significance was assessed by anova and student t-tests. the linear regression test was applied for the correlation between different parameters, and the significance of the r-value was checked using t-test. p-values of less than 0.05 were considered significant. results table 1 shows the clinical and biochemical data for healthy male subjects and male patients with acne. the results revealed that there were no significant differences in mean (±sd) values of age and bmi between healthy controls and the mild, moderate and severe type of acne. table 1, also shows the mean(±sd) values of serum zn, cu, and mg in patients with mild–, moderate–, and severe– acne types and male controls group. concerning serum zn levels in patients with severe acne type where was a significant lower levels (79.67±7.19 mg.dl) than that of healthy males (102.42±18.10 mg/dl, p=0.0001), mild acne type(116.67±12.34 mg/dl, p= 0.0001), and moderate acne type(95.67±9.58 mg/dl, p= 0.003). furthermore, patients with moderate type of acne had significantly lower levels of serum zn mean (±sd) value than that of mild acne type (p= 0.0001). the mean (±sd) value of serum cu levels did not differ significantly (p=0.085) among the acne group types and controls as well as among the acne patient iraqi j pharm sci, vol.20(2) 2011 serum trace elements and acne vulgaris 46 table 1: clinical and biochemical data for healthy male controls, mild, moderate, and severe types of acne vulgaris patients. parameters controls (n=45) mild acne (n=15) moderate acne (n=15) severe acne (n=15) age(year) 22.18±3.85 21.33±3.41 ns 22.07±4.32 ns 22.06±3.75 ns bmi(kg/m 2 ) 23.04±1.38 22.91±1.24 ns 22.6±1.33 ns 22.77±1.41 ns zn(mg/dl) 102.42±18.10 a 116.67±12.34 a 95.67±4.58 b 79.67±7.19 c cu(mg/dl) 97.56±14.48 a 102.67±22.82 a 98.67±18.85 a 95.33±15.06 a mg(mg/dl) 1.14±0.17 a 1.29±0.18 b 1.20±0.18 b 1.13±0.20 a -bmi: body mass index -ns: non significant -values with non identical superscripts(a, b and c) within each parameter were considered significant. groups themselves. with the regard to serum mg serum level, the mean (±sd) value of serum mg was significantly decreased in severe type of acne patients (1.13±0.20 mg/dl) when compared with that of mild acne type (1.29±0.18 mg/dl, p= 0.011), moderate (1.20±0.18 mg/dl, p=0.011) with no significant differences in the level of serum mg compared to control group.furthermore, the results of the present study revealed a significant correlation among the serum levels of the studied elements (zn, cu, and mg) in the mild, moderate and severe type of patients with acne vulgaris (p<0.05).as shown in the following figures: figure 1: correlation between serum levels of copper and zinc in mild acne patients figure 2: correlation between serum levels of magnesium and zinc in mild acne patients figure 3: correlation between serum levels of magnesium and copper in mild acne patients r= 0.35 p=0.039 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 20 40 60 80 100 120 140 160 serum level of copper (mg/dl) s e ru m l e v e l o f c o p p e r ( m g /d l) s e ru m l e v e l o f m a g n e s iu m (m g /d l) r= 0.274 p=0.042 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 serum level of zinc(mg/dl) r = 0.742 p=0.003 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 20 40 60 80 100 120 140 160 serum level of zinc (mg/dl) s e ru m l e v e l o f m a g n e s iu m (m g /d l) ) iraqi j pharm sci, vol.20(2) 2011 serum trace elements and acne vulgaris 47 figure 4: correlation between serum levels of copper and zinc in moderate acne patients figure 5: correlation between serum levels of magnesium and zinc in moderate acne patients figure 6: correlation between serum levels of magnesium and copper in moderate acne patients figure 7: correlation between serum levels of copper and zinc in severe acne patients figure 8: correlation between serum levels of magnesium and zinc in severe acne patients figure 9: correlation between serum levels of magnesium and copper in severe acne patients r= 0.266 p= 0.047 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 10 20 30 40 50 60 70 80 90 100 serum level of zinc (mg/dl) r= 0.344 p= 0.035 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 88 90 92 94 96 98 100 102 s e ru m l e v e l o f m a g n e s iu m ( m g /d l) r= 0.459 p=0.032 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 20 40 60 80 100 120 140 160 s e ru m l e v e l o f m a g n e s iu m (m g /d l) s e ru m l e v e l o f c o p p e r (m g /d l) r= 0.411 p=0.018 0 20 40 60 80 100 120 140 0 10 20 30 40 50 60 70 80 90 100 s e ru m l e v e l o f c o p p e r (m g /d l) r= 0.691 p=0.008 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 0 20 40 60 80 100 120 140 s e ru m l e v e l o f m a g n e s iu m ( m g /d l) serum level of zinc (mg/dl) r= 0.052 p=0.049 0 20 40 60 80 100 120 140 160 88 90 92 94 96 98 100 102 serum level of zinc (mg/dl) serum level of zinc(mg/dl) serum level of copper (mg/dl) s e ru m l e v e l o f m a g n e s iu m (m g /d l) serum level of copper (mg/dl) iraqi j pharm sci, vol.20(2) 2011 serum trace elements and acne vulgaris 48 discussion the present study showed that serum zn levels in patients with the severe type of acne were significantly lower than that of healthy controls, mild, and moderate types of acne. these data are in agreement with that reported by michaelsson et al in 1977 and amer et al. in 1982 (16,17) who showed that serum zn level was significantly reduced in severe acne male patients compared with controls. these authors suggested that low levels of zinc in the serum of patients with severe acne may provide a rational for the beneficial effect of oral zinc treatment seen in clinical practice (16) . the mineral zinc is emerging as vital nutrient for skin health and appearance. zinc nutritional status is necessary for oil gland function, local skin hormone activation, wound healing, skin inflammation control and regeneration of skin cells. zinc supplementation has been used with success in the treatment of many acne cases (18) . studies indicated that most individuals consume only 8-9 mg/day of zinc from dietary sources, whereas the recommended daily acquirement (rda) for zinc is set at 15 mg/day for adults (19) .a review reported by preston r in 2002, indicated that lack of zinc is a recipe for acne (20) . nasiri et al. in 2009 concluded from their study that zinc as antiinflammatory element may play a role in the pathogenesis of acne, and there is a need for further studies (10) .the present study also found that serum magnesium level was significantly decreased in severe type of acne compared with mild and moderate type of acne patients. magnesium is a vital element for the production of proteins and enzymes in every tissue of the body. this includes the proteins and enzymes of skin cells where new cells are constantly being produced. it is also absolutely essential for the proper use of pyridoxine, and it was suggested that taking of about 500 mg of the intended element each day is essential (20) .copper is an important element for numerous metalloenzymes and metalloproteins such as superoxide dismutase that are involved in energy and antioxidant metabolism. superoxide dismutase (cu-metalloenzyme) protects human skin cell from peroxidative damage, as human keratinocytes contain high concentrations of polyunsaturated fatty acids and also possess a significant ability to generate a reactive oxygen species (ros), mainly superoxide anion and hydrogen peroxide (21) .although, this study showed that there were no significant differences in serum copper level among patients with different type of acne with control groups. further studies are needed to show the beneficial effect of cu compounds in prevention and treatment of acne vulgaris. in conclusion; this study revealed significant association between each of zn and mg levels with the severity of acne. acknowledgments we would like to thank lecturer maysa jalal (m sc clin. biochem.) for help with statistical calculations. references 1. vora s, ovhal a, jerajani h, et al. correlation of facial sebum to serum insulin like growth factor-1 in patients with acne. british j of dermatology 2008; 159:990-991. 2. aesoph, lauri m. a holistic approach to skin protection. nutrition science news 1998; 3(4): 204-208. 3. boelsma e. nutritional skin care; health effects of micronutrients and fatty acids. american j of clinical nutrition 2001; 73(5):853-864. 4. katzman m, logan ac. acne vulgaris: nutritional factors may be influencing psychological sequelae. med hypotheses 2007; 69: 1080-1084. 5. dreno b, foulc p, reynaud a, et al. effect of zinc gluconate on propionibacterium acnes resistance to erythromycin in patients with inflammatory acne: in vitro and in vivo study. eur j dermatol 2005; 15: 152155. 6. niren nm, torok hm. nicomide improvement in clinical outcomes study (nicos): results of an 8-week trial. cutis 2006, 77(1 suppl): 17-28. 7. pohit j, saha kc, pal b. zinc status of acne vulgaris patients. j appl nutr 1985; 37; 1825. 8. verma kc, saini as, dhamija sk. oral zinc sulfate therapy in acne vulgaris: a double-blind trial. acta derm venereol 1980; 60: 337-340. 9. el-saaiea l, abdel-aal h, el-mahdy h, and abdel-aal am. serum copper, iron and zinc in cases of acne vulgaris. j med 1983; 14(2): 125-136. 10. nasiri s, ghalamkarpour f, yousefi m, and sadighha a. serum zinc levels in iranian patients with acne. clinical and experimental dermatology 2009; 34:pp e446. 11. .daniel f, dreno b, poli f, auffert n, beylot c. epidemiological study of acne in secondary school pupils in france autumn 1996. ann dermatol venereol 2000; 127: 273-8. 12. smithard a, glazebrook c, williams hc. acne prevalence knowledge about acne and iraqi j pharm sci, vol.20(2) 2011 serum trace elements and acne vulgaris 49 psychological morbidity in mid adolescence: a community – based study. br j dermatol 2001; 145:274-9. 13. al-battat ra. scarring and non scarring facial acne vulgaris and the frequency of skin diseases .a thesis submitted to the scientific council of dermatology and venereology -iraqi board for medical specializations 2006. 14. toyoda m, morhashi m. pathogenesis of acne. med electron microsc 2001;34:2940. 15. meret s, henkin k.i. clin.chem. 1971; 17:369. cited by: gowenlock h a, mcmurray r j, mclauchlan md.varly practical clinical chemistry. 1988, 6 th ed.heinemann medical books. 16. michaelsson g, vahlquist a, juhlin l. serum zinc and retinol-binding protein in acne. br j dermatol 1977; 96:283-286. 17. amer m, bahgat m, tosson z, et al. serum zinc in acne vulgaris.inter j dermatol 1982;21:481-484. 18. the doctors΄ vitamin and mineral encyclopedia(s. hendler). simonand schuster, 1990:pp 195-207(zinc). 19. nutrition for living – second edition, the benjamin/cummins puplishing companies, inc.,1988:p338. 20. preston r. acne-how to prevent and overcome acne forever. published by the international institute of nutritional research. 2002. 21. wong w y, filk g, groenen pmw, swinkels dw, thomascmg, copiuspeereboomjhj, mekushmwm and steegers-theunissen rpm .the impact of calicuim, magnesium, zinc and copper in blood. toxicology.2001;15:131-136. iraqi j pharm sci, vol.30(1) 2021 immunoglobulin levels and ovarian cancer doi: https://doi.org/10.31351/vol30iss1pp204-208 204 alteration of serum immunoglobulin levels in woman with ovarian cancer parween a. ismail*,1 and lana m. ali** *department of chemistry, college of science, university of sulaymaniyah , erbil, iraq **department of chemistry, education college, university of salahaddin, erbil, iraq abstract ovarian cancer has a high mortality and delayed diagnosis. several immunological alterations take place during ovarian carcinogenesis, and can be of value in the surveillance of the diseases. this research was conducted to evaluate serum immunoglobulin levels in women with ovarian cancer and to assess their role in disease process. the present study is composed of 85 women (mean age = 62.03±12.4 yrs) with clinically and pathologically confirmed ovarian cancer and 65 healthy females as a control group (mean age = 61±12.1 yrs). elisa test was achieved for the determination of serum [igg, iga, igm]. the findings of current study illustrated significant (p=0.001) increase in serum igg, iga, and igm levels as compared to controls. analyzing serum immunoglobulins levels might assist in identifying patients with a weak prediction, the elevation of serum immunoglobulins can be considered as an indication for disease status. key words: ovarian cancer, immunoglobulin. introduction ovarian carcinoma is one of the most common malignancy in woman (1). it has been called a hushed murder, since many ovarian carcinoma are symptomless in the untimely grade and so do not appears until the disease is at a proceed grade. most women with ovarian carcinoma are, therefore, identified and diagnosed with late stage of carcinoma (2) ovarian carcinoma has the highest death rate in cancer of a woman's reproductive organs at present (3). due to the absence of typical clinical manifestations at creation grade, most patients with ovarian carcinoma have get to a proceed grade by the time the detection is established, and even some have previously spread throughout the body (4). clinically, ovarian carcinoma is mainly healed up by removing tumors from patients with peritoneal mesothelioma and adjuvant chemotherapy in order to prevent recurrence of the tumors, particularly distant recurrence. however, for patients at a proceed grade and with a metastatic growth. (5, 6). immunoglobulins play a key role in the maintenance immunity of human body’s immune system; polypeptide chains of immunoglobulins are classified into four chains: two “light” chains and two “heavy” chains. isotypes of immunoglobulin which included (immunoglobulins a(iga), immunoglobulins d(igd), immunoglobulins g(igg), immunoglobulins e(ige), and immunoglobulins m (igm), respectively are assessed by the kind of heavy chain (7). however, some previous studies have recorded that immunoglobulins were observed to be expressed in various malignant epithelium cancer and activated in the enlargement of epithelium, but it was inversely expressed in normal epithelial cells (8, 9). heavy polypeptide chains and light polypeptide chains constant zone of immunoglobulin was present in a number of epithelial carcinoma cell lines, like (human breast cancer cell line), (human colorectal cancer cell line), (human stomach cancer cell line), a (human neck cancer cell line), and (nasopharyngeal cancer cell line) (10). furthermore, some human carcinoma cell lines could release immunoglobulins g (11,12) and immunoglobulins g and immunoglobulins a were also observed to be expressed in cancer of oral epithelial by immunohistochemical analysis which based on antibodies binding specifically to antigens in biological tissues to detect the antigens (e.g.proteins) (13). the earlier classic understanding that immunoglobulins were produced only by b lymphocytes and plasma cells were challenged when many non-lymphoid lineage cells such cancer cells were observed to have the capacity to form immunoglobulins g (14). earlier, potential defect in the secretion of immunoglobulins causes disturbance in the metabolic pathway of immunoglobulin has been proposed in the mechanisms of various carcinoma. circulating immunoglobulins concentrations were found to be linked to cancer grade and tumour mass in carcinoma such as neck carcinoma, cancer of pancreas carcinoma, primary hepatic carcinoma (15) and cancer of skin cell (16). in patients with oral carcinoma, earlier examination of circulating immunoglobulin concentrations revealed an elevate in immunoglobulins m, immunoglobulins a, immunoglobulins e, and immunoglobulins g in comparison to healthy peoples (17). 1corresponding author e-mail: parween7abdulsamad@yahoo.com received: 27/7/2020 accepted: 28/ 11/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp204-208 iraqi j pharm sci, vol.30(1) 2021 immunoglobulin levels and ovarian cancer 205 previous studies(18) refuted the prior finding and found that circulating immunoglobulins concentrations g and immunoglobulins a levels are not remarkably increased in oral carcinoma and therefore not to be utilized as a best alternative indicator for detection and prognosis. the goal of current study is to assess the immunological response in patients with ovarian carcinoma as a biochemical marker in prediction and therapy response indicators. methods and materials subjects the present study included 85 woman, aged 29–70 years, who were visited the hiwa hospital in sulaymaniyah. these females were diagnosed to have ovarian carcinoma. a total of 65 apparently healthy female without any history of ovarian carcinoma were included in the present study as a control group. ethical approval and permission for the study was taken from the ethical committee of sulaymaniyah university (iraq). informed consent was taken from all the study subjects purely for research purpose. samples collection of the blood blood was sampled before any treatment was given. six milliliters of venous blood were taken without using tornique from each individual, collected in plane polyethylene tube, allowed to stand at room temperature for thirty minutes, then the sample was centrifuged at (2000xg ) for 10 minutes, the obtained serum transferred immediately to another test tube. these samples were estimated directly for enzymes activities or frozen at – 20 c for subsequent analysis. methods assay of serum immunoglobulin (igg, iga, and igm) serum immunoglobulin levels were measured using a quantitative sandwich enzymelinked immunosorbent assay according to the kit procedure. principle of assay this elisa kit uses sandwich-elisa as the method. the microelisa stripplate provided in this kit has been pre-coated with an antibody specific to ig. standards or samples are added to the appropriate microelisa stripplate wells and combined to the specific antibody. then a horseradish peroxidase (hrp)-conjugated antibody specific for ig is added to each microelisa stripplate well and incubated. free components (unbound conjugated antibodies) are washed away. the 3,3',5,5'-tetramethylbenzidine (tmb) substrate solution is added to each well. only those wells that contain il-6 and hrp-conjugated ig antibody will appear blue in color and then turn yellow after the addition of the stop solution (0.16m h2so4). the optical density (od) is measured spectrophotometrically at a wavelength of 450 nm. the od value is directly proportional to the concentration of il-6 in standards and samples. we can calculate the concentration of ig in the samples by comparing the od of the samples to the standard curve. statistical analysis statistical analysis was performed using the software statistical package for social sciences (spss) including data evaluation and tests for significance. graph plots were done via windows excel version. results and discussion figures (1,2,3) shows the results of serum immunoglobulins (igg, iga, and igm) levels in samples of control and ovarian cancer patients. the results reflect a significant increase ((p=0.001)) in the levels of (igg, iga, and igm) in ovarian cancer patient groups in comparison to that of the control, figure 1. mean values of serum immunoglobulin a (iga) levels in control and ovarian cancer patients . figure 2. mean values of serum immunoglobulin m (igm) levels in control and ovarian cancer patients iraqi j pharm sci, vol.30(1) 2021 immunoglobulin levels and ovarian cancer 206 figure 3. mean values of serum immunoglobulin g (igg) levels in control and ovarian cancer patients the capacity to intercede and escalate the effect of the immune system to create a helpful anticancer reaction remains an area of extensive study. the progression of carcinoma may suggest a failure in the immune reactions, like mechanism of tumor get away. cancer cells show a different mechanism that makes them to control detection of the immune system and demolition, enabling the responsivity of immune system unsuccessful. throughout the progression of cancer, the capacity of the human immune system to detect and demolish developing cancer cell and as a result to act as a primary protection versus tumor has been considered for many decades. various reports now provide engrossing confirmation that specific immune response types, selectivelatory and regulatory molecules, and tracks can sometimes completely act as extraneous tumor inhibitor mechanisms (19) and hence, throughout cancer prognostication, circulating concentration of immunoglobulins are changed significantly to recompense for the altering surroundings of the carcinoma cell. levels of immune complexes are perceptible in patients with cancer of the head and neck, gastric, rectum, lungs, and in patients with malignant tumors (20) the reproducible elevation of sera iga and igg of breast cancer patients observed in this study, may be a result of the natural antibody response to the presence of antigens of breast cancer, or as a defense reaction against increasing tumor load, or may be due to the secretion of immunoglobulin by the tumor itself (21, 22). the current study is in line with that of (23). assessment of serum immunoglobulin levels in human carcinoma has been recorded by a numbers of investigators such as cancer of neck, pancreatic carcinoma (24), skin carcinoma (25) and breast carcinoma (26, 27), with altered results and thinking . in patients with oral cancer, previous researchers of circulating immunoglobulin levels demonstrate an elevate in igm, iga, ig e, and igg when compared with normal peoples. previous studies go against the prior results and found that circulating igg and iga are increased remarkably in oral carcinoma and to be utilized as a best alternative indicator for detection and prognosis. elevated concentrations of immunoglobulin m in cancer patients have been observed to associate with the clinical grades. on the contradictory a study from india (28) reported no remarkable elevate in immunoglobulin m concentrations with development of cancer. various reactions of immune defects have been linked with cancer cell and immunoglobulins are synthesized not only by b cells and cells of plasma but by other nonlymphoid lineage cells like malignant tumor. this describe the action of immune disturbance in the metabolic pathway of immunoglobulin and mechanism of ovarian carcinoma. expression of several types of immunoglobulins has been observed to associate with malignancy (29, 30)and may be correlate with formation, progression and diagnosis of the carcinoma cell as explained and identified by the american joint committee on cancer. significant elevate in immunoglobulin m was reported in breast fluid in women with breast cancer before surgery to remove all cancer tissue with diminish in immunoglobulin a and in immunoglobulin g concentrations and in immunoglobulin g concentrations found to associate with infiltration in plasma cell. conclusions the findings of the present study suggest that the elevated levels of serum immunoglobulins (igg, igm and iga) can be considered as useful biomarker in diagnosis patients with ovarian carcinoma, and alteration of circulating immunoglobulins (igg, igm and iga) levels are strongly associated with ovarian carcinoma. references 1. jemal a, siegel r, ward e, et al. cancer statistics. ca cancer j clin.; 2009;59: 225 – 249. 2. 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() cell mediated and humoral immune responses in oral submucous fibrosis. cancer .1986;58, 2628-2631 29. khana, s. and karjodkar, f.r. circulating immune complexes and trace elements (copper, iron and selenium) as markers in oral precancer and cancer: a randomised controlled clinical trial. head and face medicine . 2006 ;2, 33 30. yang, b., ma, c., chen, z., yi, w., mcnutt, m., wang, y., korteweg, c. and gu, j. correlation of immunoglobulin g expression and histological subtype and stage in breast cancer. 2013; ( 8):e58706 . baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ study of iraqi spinach leaves ( phytochemical and protective effects against methotrexate-induced hepatotoxicity in rats) iraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 8 study of iraqi spinach leaves (phytochemical and protective effects against methotrexate-induced hepatotoxicity in rats) farah k. abdul-wahab * ,1 and thukaa z. abdul jalil ** * department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq ** department of pharmacognosy, college of pharmacy, university of baghdad, baghdad, iraq abstract spinach, spinacia oleracea l is a popular vegetable belonging to the family chenopodiaceae. this study was concerned with extraction of compounds in iraqi spinach leaves, preliminary phytochemical evaluation, identification of two biological important flavonols, quercetin and kaempferol in spinach leaves and evaluation of the protective effect of aqueous spinach extract on methotrexate (mtx) induced hepatotoxicity in rats. the percentage yield of extraction procedure, identification of spinach by chemical tests and identification of flavonols by thin layer chromatography (tlc) and high performance liquid chromatography (hplc) were fully described in this study. the results indicate that the percentage of quarcetin in spinach leaves is more than the percentage of kaempferol in the same plant. the rats were divided into three groups as control, mtx group following a single dose of mtx (20 mg/kg, i.p) saline was administered for 5 days and the mtx+aqueous spinach extract group were rats received 200mg/kg orally of aqueous spinach extract 7days before and 5 days after mtx treatment. mtx administration increased the mda and decreased gsh, alp while these changes were reversed in aqueous spinach extract treated group. histological changes observed in mtx treated group was improved by aqueous spinach extract treatment. the protective effect of aqueous spinach extract against mtxinduced hepatotoxicity could be attributed to the combined effects of its constituents. key words: spinach, kaempherol , quercetin , methotrexate, oxidative stress. انىاجج عه انكبذ جسمم ضذ انىاقيةوجاثيراجها كيمياء انىباجيةان) انعراقي انسباوخ أوراق دراسة عه )في انجرران انميثىجريكسيث *فرح قيس عبذانىهاب ،1 **ركاء زهير عبذ انجهيمو . ، بغذاد ، انعشاقجبيعّ بغذاد ، نتانظٛذ تكهٛ ،فشع االدّٔٚ ٔانسًٕو * . ، بغذاد ، انعشاقجبيعّ بغذاد ،نتانظٛذ تكهٛ ،فشع انعمبلٛش ** انخالصة بسخخالصب حٓخى ْزِ انذساست chenopodiaceaeعبئهت ُخًٙ إنٗح شعبٛت خضبس عببسة عٍْٕ spinacia oleracea l انسببَخ فٙ كٕٚشسٛخٍٛ، كبًٚبفٛشٔل ٔ ببٕٚنٕجٛب انٓبيخٍٛ انفالفٌٕ، ٔححذٚذ األٔنٛت انكًٛٛبئٛت انُببحٛت، ٔحمٛٛى انعشالٙ انسببَخ أٔساق انًشكببث انفُٕٛنّٛ يٍ ٔطف انُسبّ حى( فٙ انجشراٌ . mtx) انخأثٛش انٕلبئٙ نًسخخهض انسببَغ انًبئٙ ضذ حسًى انكبذ انز٘ ٚسببّ انًٛثٕحشكسٛج ٔحمٕٚى انسببَخ أٔساق انفظم انهَٕٙ ٔ نهفظم انهَٕٙ ّٔححذٚذ انفالفٌٕ بٕاسطّ انطبمّ انشلٛم كبيم بشكم هسببَغناالخخببساث انكًٛٛبئّٛ ٚجانًئّٕٚ نهًسخخهض ٔاجش .َفس انُببث فٙ كبًٚبفٛشٔل َسبّ ْٕ أكثش يٍ انسببَخ أٔساق فٙ كٕٚشسٛخٍٛ َسبّحشٛش انُخبئج انٗ اٌ فٙ ْزِ انذساسّ. انسبئم راث انكفبءِ انعبنّٛ ( انبشٚخٌٕ يهغى / كغى فٙ 02) mtxبعذ جشعّ ٔاحذِ يٍ mtx يجًٕعتانُحٕ انخبنٙ انسٛطشِ , يجًٕعبث عهٗ إنٗ ثالد انجشراٌ حى حمسٛى يهغى / كغى( يسخخهض انسببَغ انًبئٙ 022+ يسخخهض انسببَغ انًبئٙ حهمج انجشراٌ ) mtxفٙ يجًٕعّ أٚبو 5نًذة انًحهٕل انًهحٙ حى إعطبء فٙ حٍٛ gsh ٔalpٔاَخفبع فٙ mdaصٚبدِ فٙ يسخٕٖ mtxءاعطب سبب .mtxاٚبو بعذ اعطبء 5اٚبو لبم ٔ 7عٍ طشٚك انفى mtxيجًٕعت انخٙ نٕحظج فٙ انُسٛجٛت حغٛٛش حى ححسٍٛ يعبيهت .انخغٛٛشاث فٙ انًجًٕعّ انخٙ حهمج يسخخهض انسببَغ انًبئٙ اَعكسج ْزِ انخبثٛش انًشخشن انٗ mtxضذ حسًى انكبذ انخٙ ٚسببٓب انسببَخ انًبئٙ نًسخخهض ًٔٚكٍ أٌ ٚعضٖ انخأثٛش انٕلبئٙ ببعطبء يسخخهض انسببَغ انًبئٙ .ًكَٕبث أساق انسببَغ ن .انحاكسذي االجهاد ,ميثىجريكسيث , كىيرسيحيه , ، كايمبفيرول انسباوخ :انكهمات انمفحاحية introduction spinach is a leafy green vegetable that came originally from south western asia and is now grown in most parts of the world. scientifically it is known as spinacia oleracea l. belonging to the family chenopodiaceae (figure1), locally known as ispanakh. (1) spinach is packed with vitamins, minerals and a number of antioxidants components like polyphenols, flavonoids and carotinoids which are shown to possess antiinflammatory effects, anti-mutagenic potential, anti-neoplastic effects, as well as chemo-preventive activities. (2, 3) 1 corresponding author email : farah77kais@yahoo.com received : 26/2/2012 accepted :15/5/2012 iraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 9 figure 1: spinacia oleracea at least 13 different flavonoid compounds had been discovered by researchers in spinach leaves (4) among these flavonoids, quercetin and kaempferol are the most important and wid'ely spread flavonol class (containing the typical c6-c3-c6 structure) (5) these flavonols provide the body with anti-oxidant protection, so they have significant influence against kidney damage, toxic liver damage, neurovascular complications and diabetic complications (6, 7) . methotrexate (mtx), a folic acid antagonist is widely used as a cytotoxic chemotherapeutic agent; however its associated with hepatotoxicity which is considered to be a major clinical side-effect. (8) the toxicity of mtx in the liver seems to relate to the generation of reactive oxygen species (ros) (9,10) .the present study deals with the investigation of phytochemicals found in the plant by ethanolic and aqueous spinach leaves extract and the possible protective effects of aqueous spinach extract against hepatic damage caused by methotrexate (mtx ) in rats. materials and methods plant materials fresh leaves of spinach were purchased from vegetable markets of baghdad; the plant was identified by the department of pharmacognacy, college of pharmacy/ university of baghdad; and authenticated by the herbarium of baghdad university.100gram of fresh spinach leaves collected and dried under shade, powdered and divided into two parts: the first part, 50 grams of powdered leaves were packed in a thimble of soxhlet extractor. 100 ml of petroleum ether (40-60 ºc) was used in soxhlet for 3 hours to get rid of lipids and fats to give fraction 1 (f1). the defatted powder after drying over-night were extracted with 100 ml of 96% ethanol for 72 hours by soxhlet extractor to give fraction two (f2) as shown in (figure 2) (11) . in the second part, 50 grams of powdered plant material was extracted with 100 ml of distilled water by cold maceration method, this step was repeated for three times to give fraction three (f3) as shown in (figure 2) (12) .after that, the extracts (f1, f2 and f3) were filtrated and then concentrated under reduced pressure at a temperature not exceeding 40 0 c to give greenish colored residues. figure 2: schematic procedure for first and second parts of spinach leaves extraction (11, 12) iraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 10 phytochemical evaluation phytochemical evaluation of spinach leaves were done by making general tests for fixed oils, glycosides, alkaloids, phenolic compound, tannins and flavonoids. 1. test for fixed oils (13) small quantities of various extracts (f1, f2 and f3) were separately pressed between two filter papers; the appearance of permanent spot oil on the paper indicates the presence of fixed oils. 2. test for glycosides (14) a -baljet’s test: 1ml from each extracts (f1, f2 and f3) + 1ml of picric acid make it alkaline with sodium hydroxide →orange color. bkeller-kiallian’s test: 1ml from extracts (f1,f2 and f3)+ 2ml of glacial acetic acid+ 1 drop of 0.1% ferric chloride solution → then add 1ml of h2so4 (drop by drop) → the junction between two liquid layers indicates the presence of the sugar part of glycosides. cbrontrager’s test: 1ml of f1 and f2 extract+1ml of diluted ammonia solution → pink color. while f3 was treated with chloroform and then chloroform layer was separated. to this equal amount of dilute ammonia solution added → pink color, showing the presence of glycosides. 3. test for saponins (15) each extract (f1, f2 and f3) was diluted with 10 ml of distilled water and it was agitated in test tube for 15seconds and standing for 15 minutes, the formation of not less than 1cm layer of foam shows the presence of saponins. 4. test for alkaloids (16) a small portion of extracts (f1, f2 and f3) were tested with various reagents for the presence of alkaloids. mayer’s reagent →cream precipitation. dargendroff’s reagent→ orange brown precipitation. wagner’s reagent →reddish brown precipitation. 5. test for phenolic compounds and tannins (14) the tests for phenolic compounds and tannins were carried out with following reagents: a. 5% ferric chloride solution→ deep green or deep blue. b. 10% lead acetate solution →white precipitation. c. 1% potassium dichromate solution → orange precipitate. 6. test for flavonoids (11) a. with aqueous sodium hydroxide solution anthrocyanins → blue to violet color. flavones and flavonol→ yellow color. flavonones → yellow to orange color . b. with concentrated sulphuric acid anthrocyanins → yellow orange color. flavones and flavonol→ yellow to orange color. flavonones → orange to crimson color. identification of quercetin and kaempferol as example of flavonoids spinach is loaded with flavonoids which act as antioxidants; identification of these flavonoids was performed by: 1. identification of flavonoids (quercetin and kaempferol) by tlc: (6) using ready made aluminum plates of silica gel gf254, ultraviolet light detector at 254 nm wave length as detection method, standard flavonoids in comparison with three different solvent systems s1, s2 and s3. standard flavonoids are: quercetin (fluka-austia) and kaempferol (sigmaaldrich, usa). different developing solvent systems were: s1=chloroform: acetone: formic acid (75:16.5: 8.5) s2 = chloroform: methanol (90:10) s3 = toluene: chloroform: acetone (40:25:35). 2identification of flavonoids in hplc further identification to the flavonoids in plant extracts (f2 and f3) were performed by hplc. for qualitative estimation comparison of retention time obtained at identical chromatographic conditions of plant extract with authentic standards was done. table 1: hplc conditions (6) sample mobile phase column flow rate detection quercetin acetonitrile: methanol : glacial acetic acid (70:30: 0.1) c18 5 mm x 150 mm 0.5 ml / min uv. detector at 306 ג nm kaempherol methanol : water ( 7.5 : 92.5 ) c18 ods 1.5 ml / min uv. detector at 308 ג nm for quantitative estimation the following equation was used to calculate the percentage of the compounds in the plant: (17) iraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 11 percentage of compound in the plant {( )} the weight of the plant used in the extraction was 50 gm auc = area under the curve. df = dilution factor. conc. st. = concentration of the standard used in hplc. pharmacological studies preparation of aqueous spinach extract the roots of spinach were removed by sharp knife then the remaining portion was thoroughly washed several times with tap water and finally with distilled water and the water was removed by keeping on drying cabinet for 2 hours at room temperature. these were kept on a rectangular netted plastic basket for 2 hours to remove washing water. then, the leaves and soft stalks were separated and chopped into small pieces and kept under an electric fan at room temperature then these were dried fully. the dried spinach was grinded with an electric grinder and then the grinded mass was sieved with 2mm wire sieve to get fine powder. fifteen gram of spinach powder was weighed and thoroughly mixed with 1000 ml deionized distilled water and kept for 24 hours at room temperature. after that the solution was filtered and used. (18) experimental protocol eighteen white albino rats of both sexes, weighing 200-250g were used in this study; the rats were obtained from and maintained in the animal house of the college of pharmacy, university of baghdad under conditions of controlled temperature. the animals were fed commercial pellets. the rats were divided into three groups: group 1 normal control rats. group 2mtx (20 mg/kg i.p) following a single dose of mtx saline will be administered for 5 days (19) . group 3 – mtx(20 mg/kg i.p) + spinach extract 200 mg/kg body weight orally in drinking water daily 7 days prior to and 5 days after mtx administration. biochemical estimations determination of the serum concentrations of the liver enzymes ast, alt, alp and bilirubin were measured in serum samples obtained from all groups of rats. liver tissue homogenate was prepared by standard procedure (20) and the contents of malondialdehyde (mda) (21) and reduced glutathione (gsh) (22) were analyzed in liver tissue homogenate. microscopic evaluation for the light microscopic investigations, tissue specimens from the liver were fixed with 10% formaldehyde and processed routinely for embedding in paraffin. tissue sections of 5 μm were stained with hematoxylin and eosin (h and e) (23) . statistical analysis results were expressed as mean ± sd (standard deviation). using student’s t-test, the level of statistical significance was set at p<0.05. results the dried leaves of spinacia oleraceae l. was divided into two parts, the first one defatted with petroleum ether and extracted with 96% ethanol by soxhlet apparatus while the second one extracted with distilled water by cold maceration method. the percentage yield of plant extracts was as following: petroleum ether → 1.2% ethanol→ 5.5% aqueous → 6.25% preliminary phytochemical evaluation the various extract of dried leaves were subjected for phytochemical screening which shows the presence of different compounds in plant extracts as shown in (table 2). table 2 : preliminary phytochemical evaluation of dried leaves of spinacia oleraceae. active constituents tests f1 f2 f3 fixed oil spot test + glycosides baljet’s test keller kiallan’s test brontrager’s test + + + + + + saponins foam test + + alkaloids mayer’s test dragendroff’s test wagner’s test phenolic compounds and tannins fecl3 test lead acetate test potassium dichromate test + + + + + + flavonoids dil. naoh solution conc. h2so4 + + + + where + =present ; =absent identification of flavonoids by tlc tlc of the extracts (f1, f2 and f3) obtained from dried leaves of iraqi spinach iraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 12 confirms the presence of quercetin and kaempferol in f2 and f3 only but not in f1 when comparison was made with standards, as represented in (table 3) and (figures 3, 4 and 5). table 3 : rf value of plant extracts (f2 and f3), standard quercetin and kaempferol. s1 s2 s3 solvent system 0.5 0.42 0.72 rf value of quercetin in f2 0.49 0.41 0.71 rf value of quercetin in f3 0.48 0.4 0.7 rfvalue of kaempferol standard 0.7 0.6 0.82 rf value of kaempferol f2 0.72 0.61 0.84 rf value of kaempferol f3 0.71 0.59 0.83 s1=chloroform: acetone: formic acid (75:16.5:8.5) s2 = chloroform: methanol (90:10) s3 = toluene: chloroform: acetone (40:25:35). q aq alc pet k figure 3 : tlc plate of spinach leaves extracts and standard, using s1 mobile phase k pet alc aq q figure 4: tlc plate of spinach leaves extracts and standard, using s2 mobile phase aq k q pet alc figure 5: tlc plate of spinach leaves extracts and standard, using s3 mobile phase where q=quercetin standard, k= kaempferol standard, pet=petroleum ether extract, alc=alcoholic extract and aq=aqueous extract. identification of flavonoids by hplc further identification to the quercetin and kaempferol in plant extracts (f2 and f3) were performed by hplc in which the retention time of both standards (quercetine and kaempferol) and the plant extracts (f2 and f3) were identical as represented in the figures bellow: figure 6: hplc analysis of kaempferol standard iraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 13 figure 7: hplc analysis of aqueous extract of iraqi spinach leaves figure 8: hplc analysis of alcoholic extract of iraqi spinach leaves figure 9 : hplc analysis of quercetin standard figure 10 : hplc analysis of alcoholic extract of iraqi spinach leaves figure 11: hplc analysis of aqueous extract of iraqi spinach leaves for quantitative estimation, the percentage of quercetin in both f2 and f3 were higher than the percentage of kaempferol in the same extract, as shown in table (4). table 4: data showing extractive values (perecentage w/w ) of quercetin and kaempferol in iraqi spinach leaves extraction solvent quercetin kampferol ethanolic extract 0.268 % 0.12 % aqueous extract 0.149 % 0.096 % biochemical parameters as shown in table 5, the level of mda, a major degradation product of lipid peroxidation, was found to be significantly higher and level of gsh was significantly decreased in the mtxiraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 14 treated group when compared with control group (p < 0.05).methotrexate cause no significant change in ast, alt and bilirubin level (p > 0.05) and decrease in alp level compared to control group (p < 0.05).while rats treated with an oral concentrations (200 mg / kg) of spinach extract 7 days prior to and 5 days after single i.p of methotrexate (20 mg/kg) resulted in significant decrease in mda and increase gsh and alp (p < 0.05) and produce no significant change in bilirubin, alt and ast compared to methotrexate group (p > 0.05).in the livers of rats from the control-treated rats, hepatocytes showed a normal histological appearance (figure12). in the mtxtreated rats, showed rare scattered dysplastic hepatocytic changes, periportal inflammatory cell infiltration and sinusoidal dilatation were observed (figure 13 and 14) , while in the mtx+ aqueous spinach extract treated rats affected to a significantly less degree than those in the group given mtx alone showed minimal periportal lymphocytic infiltration and lesser sinusoidal dilatation(figure 15). table 5: effect of methotrexate and aqueous spinach extract on liver biochemical parameters, mda and gsh of rats control mtx mtx+ spinach ast(u/l) 105.7 ± 19.05 121.67 ±17.45 118.67 ± 40.1 alt(u/l) 64.65 ± 24.32 45.83 ± 6.31 73.5 ± 23.25 ** alp (u/l) 519 ± 93.92 185.33 ± 68.85 * 339.5 ±179.82 ** bilirubin (u.mol/l) 0.54 ± 0.11 0.61 ± 0.02 0.61 ± 0.12 mda (nmol/gtissue) 1522.22 ± 277.08 4386.96 ±508.72 * 3069.01 ± 244.7 ** gsh (µmol/g tissue) 38.87 ± 4.28 29.21 ±1.93 * 35 ± 2.23 ** data are presented as mean ± sd. n= number of animals. *p<0.05 with respect to control group. **p<0.05 with respect to methotrexate group. figure 12: normal histological appearance of liver in control group (h and e x 200) figure 13 : rare scattered dysplastic hepatocytic changes in methotrexate treated group (h&e x 400) iraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 15 figure 14: periportal inflammatory cell infiltration (→)and sinusoidal dilatation (→)in methotrexate treated group (h&e x 200) figure 15: minimal peiportal lymphocytic infiltration and sinusoidal dilatation (→)in mtx + aqueous spinach extract treated group (h&e x 200) discussion preliminary phytochemical studies showed the presence of flavonoids, glycosides, tannins, saponins and polyphenolic compounds on both ethanolic and aqueous extracts. identification of quercetin and kaempferol as examples to flavonoids by tlc and hplc indicates the presence of these flavonoids and give the results that the percentage of quercetin in iraqi spinach leaves is more than the percentage of kaempferol in the same plant.the study examined the role of oxidative stress in an attempt to suggest a possible mechanism of mtx-inducd hepatotoxicity in a rodent model.the results of the studies indicate that mtx causes oxidative tissue damage by increasing lipid peroxidation in the liver tissue and decreasing the level of gsh. these results were consistent with other studied. (24, 25) the increased mda levels as observed in our study can be attributed to the lipid peroxidation can be induced by mtx itself or as a result of a possible increase in ros induced by mtx . (.26) the ros thus formed may lead to cellular damage by peroxidation of membrane lipids, sulfhydryl enzyme inactivation, protein cross-linking and dna breakdown . (27,25 ) it has been shown that treatment with mtx leads to a reduction in the effectiveness of antioxidant defense systems and that cellular levels of glutathione are reduced by mtx . (26) although statistically no significant differences in ast was observed, alt and bilirubin level in methotrexate treated group and this result are consistent with other studies. (25, 28) this may be due to administration of methotrexate for short duration or may need high dose to produce change in the level of these enzymes but the level of alp decreased. this reduction could be explained by the noticeable damage that affected the endothelial cells lining the blood sinusoids and blood vessels in the portal tracts, which are considered to be the main sites at which the alp acts. (29) previous studies have shown that many antioxidants, including n acetylcysteine (30) and grape seed extract (31) have protective effects in the mtx injury of the liver in rat. at microscopic level, a single dose of mtx resulted in significant liver injury. the mechanism by which mtx causes hepatotoxicity due to its binding to the enzyme dihydrofolic reductase, thus preventing conversion of folic acid to its active form, folinic acid. this in turn blocks the synthesis of nucleic acids, certain amino acids and indirectly proteins which lead to damage of organelles and plasma membranes of hepatic parenchymal cells interfering with their function (32) . aqueous spinach extract supplementation reduced mtx-induced oxidative liver damage these data are in accordance with the findings of a recent study which demonstrated that spinach leaves contain natural antioxidant system which provide free radical scavenger properties and hepatic protection against ccl 4 (33) and radiation (34) induced by oxidative stress . conclusion phytochemical investigation of the aqueous and ethanolic extracts of leaves of spinacia oleraceae showed the presence of flavanoids, glycosides, saponins, tannins, phenolic compounds and treatment of rats with aqueous spinach extract before and after mtx application provided significant protection from the hepatotoxicity of mtx could be attributed to the combined effects of its constituents as the leaves are rich in flavonoids and p-coumaric acid. references iraqi j pharm sci, vol.21(2) 2012 study of iraqi spinach leaves 16 1. subhash g p, virbhadrapp sr. and vasant o k. spinacia oleracea linn: a pharmacognostic and pharmacological overview. ijrap. 2010; 1:78-84. 2. boivin d., lamy s., lord-dufour s., jackson j., beaulieu e. and cote m., moghrabi a, barrette s, ginras d and beliveau r antiproliferative and antioxidant activities of common vegetables: acomparative study. food chem. 2009; 112: 374–380. 3. hait-darshan r., grossman s., bergman m., deutsch m., and zurgil, n. synergistic activity between a spinach-derived natural antioxidant (nao) and commercial antioxidants in a variety of oxidation systems. food. res. intel. 2009; 42: 246–253. 4. bergman m, perelman a, dubinsky, z and grossman s. scavenging of reactive oxygen spieces by anovel glucaronidated flavonoid antioxidant isolated and purified from spinach. phytochemistry. 2003; 63:753-762. 5. sultana, b. and anwar, f. flavonols (kaempferol, quercetin and myricetin) contents of selected fruits, vegetables and medicinal plants. food chem .2008; 108 : 879-884. 6. abdul.jalil,th,. saour k and nasser,a. phytochemical and antimicrobial studies of some flavonoids present in the fruits of two ammi l. spieces wildly grown in iraq. iraqi j pharm sci. 2010 ;19:48-58. 7. bergman m, vershavsky l, gottlieb he and grossman s.the antioxidant activity of aqueous spinach extract: chemical identification of active fraction. phytochemistry. 2001; 58: 143-152. 8. coleshowers cl, oguntibeju o o , ukpong m and truter e.j.effects of methotrexate on antioxidant enzyme status in a rodent model.medical technology sa . 2010; 24; 19. 9. sener g., demiralp e e., cetiner m., ercan f s¸ irvancı s , gedik, n, and yegen bc.lcarnitine a meliorates methotrexate-induced oxidative organ injury and inhibits leukocyte death. cell biol toxicol. 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j. hepatotoxic effects of methotrexate. cancer. 1966; 19: 600-6. 33. bhatia a l and jain m. spinacia oleracea l. protects against gamma radiations: a study on glutathione and lipid peroxidation in mouse liver. phytomedicine. 2004; 11: 607–615. 34. gupta r s and singh d.amelioration of ccl4-induced hepatosuppression by spinacia oleracea l. leaves in wistar albino rats. pharmacologyonline. 2006; 3: 267-278. http://www.springerlink.com/content/?author=ertugrul+uzar http://www.springerlink.com/content/?author=hasan+rifat+koyuncuoglu http://www.springerlink.com/content/?author=efkan+uz http://www.springerlink.com/content/?author=h.+ramazan+yilmaz http://www.springerlink.com/content/?author=suleyman+kutluhan http://www.springerlink.com/content/?author=serkan+kilbas http://www.springerlink.com/content/?author=fatih+gultekin iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 42 synthesis of new coumarin and 2-quinolone derivatives with expected biological activities kawkab y. saour *, ridha i. al-bayati ** and mohammed k. hadi *,1 * department of pharmaceutical chemistry, college of pharmacy, university of baghdad, iraq **department of chemistry, college of science, university of al-mustansiriya, iraq abstract a series of new coumarin and n-amino-2-quinolone derivatives have been synthesized. the reaction of coumarin (1) with excess of hydrazine hydrate 98% yielded 1-amino-2-quinolone (2), compound (2) was reacted with different sulfonyl chloride to yield sulfonamides [ n-(2-oxoquinolin1(2h)-yl) methane sulfonamide (3), n-(2-oxoquinolin-1(2h)-yl) benzene sulfonamide (4) and 4methyl-n-(2-oxoquinolin-1(2h)-yl) benzene sulfonamide (5) ], while reaction of 2-(4-methyl-2-oxo2h-chromen-7-yloxy) acetic acid (8) with different amines yielded compounds [ 2-(4-methyl-2-oxo2h-chromen-7-yloxy)-n-(2-oxoquinolin-1(2h)-yl) acetamide (9) and n-(5-methyl-1,3,4-thiadiazol-2yl)-2-(4-methyl-2-oxo-2h-chromen-7-yloxy)acetamide (10) ] through amide linkage.the reactions and purity of the products were checked by tlc. the structures of the final compounds and their intermediates were confirmed by their melting points, ir spectroscopy, and elemental microanalysis.the coumarin and n-amino-2-quinolone derivatives were evaluated for their anti bacterial and antifungal activity. key words: coumarin , 1-amino-2-quinolone, sulfonamide, amide, biological activity. جذيذج راخ فعاليح تايولوجيح متوقعحكوينولون –2 تخليق مشتقاخ كوماريه و كوكة يعقوب ساعور * الثياتي ضا اتراهيمو ر ** و محمذ كامل هادي *،1 . ، جايعه بغذاد، بغذاد ، انعراق تانصيذن ت*فرع انكيًياء انصيذالَيت ، كهي ** . خُصريت، بغذاد ، انعراقانعهىو، انجايعت انًس ترع انكيًياء ، كهيف الخالصـح ( يع زيادة يٍ 1حفاعم انكىياريٍ ) ا.حى حصُيعه كىيُىنىٌ -2-اييُى -ٌنًشخماث انكىياريٍ و يجًىعت جذيذة ( يخفاعم يع كهىريذاث سهفىَيم يخخهفت نيُخج سهفىٌ ايايذ 2(، انًركب )2كىيُىنىٌ ) -2-اييُى -1يُخج 89انهيذرازيٍ انًائي % َخج انًركبيٍ ا( يع اييُاث يخخهفت 9) acetic acid (methyl-2-oxo-2h-chromen-7-yloxy-4)-2 (، بيًُا حفاعم5،4،3) . حى يرالبت جًيع انخفاعالث وانخأكذ يٍ َماوة انًركباث بىاسطت كروياحىغرافيا انطبمت انرليمت، كًا حى ( خالل رابطت االيايذ11،8) ث انُهائيت وحًييسها يٍ خالل لياش درجاث االَصهار وانخحهيم انطيفي نالشعت ححج انحًراء، يخابعت انًركباث انىسطيت وانًركبا حى حميى فعانيخها ضذ انجراثيى و انفطرياث. كىيُىنىٌ -2-اييُى -ٌيشخماث انكىياريٍ و وانخحهيم انذليك نهعُاصر. الفعاليح الثايولوجيح .ولون ، سلفون آمايذ ،آمايذ ، ينكو -2كوماريه ، الكلماخ المفتاحيح: introduction heterocyclic chemistry is one of the largest areas of research in organic chemistry and it is growing rapidly. of all published organic chemistry literature, papers on heterocyclic synthesis accounted for around 60 % in 1998, but nowadays the fraction is much larger considering that novel heterocyclic compounds are published in different fields such as biochemistry, pharmaceuticals, materials and others (1) . a similar trend is seen for coumarin, a heterocyclic system with a very large number of different derivatives (2) . coumarin (also known as 1,2-benzopyrone or less commonly, as o-hydroxycinnamic acid-8lactone), itself is a natural heterocyclic organic aromatic compound, present in a wide variety of microorganisms and higher plants (35) , it was first isolated by vogel in 1820 by extraction from tonka beans (dipteryx odorata) specie previously known as coumarona odorata, hence the term coumarin. it was subsequently identified in a large number of plants belonging to many different families. the diverse biological activities of natural and synthetic coumarin derivatives as anticoagulants and antithrombotics are well known, so that; they are effective for the prevention and treatment of venous and arterial thrombosis (6) , some of the coumarin derivatives are also reported as antifungal and antibacterial agents (7) , antiviral and antitumor agents (8) , lipid-lowering agents (9) , anti-hiv agents (10) , 1 corresponding author email : moha_khadi77@yahoo.com received : 4/3/2012 accepted: 19/6/2012 iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 43 antioxidants and lipoxygenase inhibitor (11) , they have also been found to possess antiproliferative, vasorelaxing activities (12) , anti-inflammatory activity (13) , anthelmintic, hypnotic, insecticidal activities, and diuretic properties (14) .quinolone is one of the most popular n-heteroaromatic compounds incorporated into the structures of many pharmaceuticals. many quinolone-containing compounds exhibit a wide spectrum of pharmacological activities, such as antibacterial, antimalarial, antidepressant, anticancer and antioxidant activity (15) . many sulfonamide derivatives were synthesized, characterized and tested for antibacterial (16) , anti-carbonic anhydrase (17) , mycobacterium tuberculosis (18) , anti-inflammatory (19) , antitumour (20) ,diuretic (21) , and hypoglycemic properties (22) . experimental section materials and methods all the chemicals used in the synthesis were of analytical grade. the melting points of the compounds and their intermediates were determined (uncorrected). thin layer chromatography was performed and rf values of the intermediates and final products which showed single round spots appeared after exposing the chromatograms to iodine vapor indicating the purity and the completion of the reactions. determinations of infrared spectra were performed in kbr disc using ftir spectrophotometer shimadzu in the, college of pharmacy, university of baghdad and college of science, university of al-mustanseriya. the elemental microanalysis of the synthesized final products was done in cleveland clinical foundation learner research institute-france, by using carlo erba elemental microanalyzer. thomas hoover electronic melting point apparatus was used to determine all melting points reported in this work. the antimicrobial study of the synthesized final products was done in alkindy college of medicine / university of baghdad. synthesis of 1-amino-2-quinolone (2) (23) a solution of (1.46g, 0.01 mol) coumarin and excess hydrazine hydrate (98%) (5g, 0.1 mol) in absolute ethanol (25 ml) was refluxed for 24 h, the solvent was concentrated and the separated solid product was filtered and washed with cold ethanol, and recrystallized from chloroform, to give yellow crystals. the physical appearance, percentage yield, melting point and rf values were listed in table 1, ir characteristics absorption bands were listed in table 3. synthesis of n-(2-oxoquinolin-1(2h)-yl) methane sulfonamide (3) (24) compound (2) (0.65 g, 0.004mol) in dichloromethane (20ml) was stirred overnight at room temperature with methanesulfonyl chloride (0.48g, 0.004mol) in the presence of triethylamine (1.4ml, 0.01mol). the mixture was poured into a separatory funnel and washed with 100ml distilled water. the organic layer was dried over anhydrous sodium sulfate and the solvent was removed with a rotary evaporator. the residue was purified by flash chromatography to give brown oily product, the physical appearance, percentage yield, and rf values were listed in table 1, ir characteristics absorption bands were listed in table 3. synthesis of n-(2-oxoquinolin-1(2h)-yl) benzene sulfonamide (4) and 4-methyl-n (2 oxoquinolin-1(2h)-yl) benzene sulfonamide (5) (25) to the mixure of compound (2) (0.65g, 0.004mol) in pyridine (10 ml) benzenesulfonyl chloride (0.7g, 0.004mol) for compound (4) and ptoluenesulfonyl chloride (0.76g, 0.004mol) for compound (5) were added drop wise at 0 ◦c. the resulting solution was stirred at room temperature for 5 h. at the end of this period, the reaction solution was poured into mixture of ice and concentrated hydrochloride acid and water. the precipitate was filtered, dried, and recrystallized from the ethanol: water to give pale yellow crystals, the physical appearance, percentage yield, melting point and rf values were listed in table 1, ir characteristics absorption bands were listed in table 3. synthesis of ethyl-2-[(4-methyl-2-oxo-2hchromen-7-yl)-oxy] acetate (7) (26) mixture of 7-hydroxy-4methycoumarin (6) (1,76g, 0.01mol), ethyl bromoacetate (2.5g, 0.015 mol) and potassium carbonate (2.07g, 0.015mol) in dry acetone was refluxed for about 16 h. the mixture was filtered and solvent was removed under reduced pressure. the resulting solid was washed with excess of water. the crude product was purified by crystallization from ethanol to give off-white crystals. the physical appearance, percentage yield, melting point and rf values were listed in table 1, ir characteristics absorption bands were listed in table 3. synthesis of 2-(4-methyl-2-oxo-2h-chromen7-yloxy) acetic acid (8) (26) a solution of compound (7) (1.57g, 0.006mol) in ethanol (35ml) and 5% sodium iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 44 hydroxide (6ml) was heated under reflux for 2 h. after cooling, the solution was evaporated to dryness and the residue was dissolved in water and acidified with diluted hydrochloric acid (ph 5-6). the white precipitate was filtered, dried and crystallized from ethanol to give off-white crystals. the physical appearance, percentage yield, melting point and rf values were listed in table 1, ir characteristics absorption bands were listed in table 3. synthesis of 2-(4-methyl-2-oxo-2h-chromen7-yloxy)-n-(2-oxoquinolin-1(2h)-yl) acetamide (9) (27) to a stirred solution of compound (8) (0.7g, 0.003mol) in (20ml) of n,ndimethyl formamide (dmf), (0.48g, 0.003mol) of compound (2) was added, the mixture was cooled down to (-10c) then (0.81g ,0.006mmol) of 1-hydroxy benzotriazole (hobt) and (0.62g ,0.003mol) of n,n , dicyclohexyl carbodiimide (dcc), were added with stirring, which was continued for 2days at 0c and then at room temperature for 5days.the reaction mixture evaporated to exclude dmf and re dissolved in chloroform from which the n,n–dicyclohexyl urea (dcu) was filtered off. the clear filterate washed twice with 5% sodium bicarbonate solution, 0.1n hydrochloric acid, once with water, and with saturated sodium chloride solution. the chloroform layer was dried with anhydrous magnesium sulphate and evaporated under vacuum; the resulted product was collected, recrystallized from (chloroform:ether) (1:1) , to give beige crystals, the physical appearance, percentage yield, melting point and rf values were listed in table 1, ir characteristics absorption bands were listed in table 3. synthesis of n-(5-methyl-1,3,4-thiadiazol-2yl)-2-(4-methyl-2-oxo-2h-chromen-7yloxy)acetamide (10) to a stirred solution of compound (8) (0.7g, 0.003mol) in (20ml) of n,ndimethyl formamide (dmf),(0.4g, 0.003mol) of 5-methyl-1,3,4-thiadiazole-2-thiol was added, the mixture was cooled down to (10c) then (0.81g, 0.006mmol) of1-hydroxy benzotriazole (hobt) and (0.62g, 0.003mol) of n,n , dicyclohexyl carbodiimide (dcc), were added with stirring, which was continued for 2days at 0c and then at room temperature for 5days.then complete the procedure as mentioned in the synthesis of compound 9. a yellow crystal was obtained. the physical appearance, percentage yield, melting point and rf values were listed in table 1, ir characteristics absorption bands were listed in table 3. antimicrobial activity (28) the synthesized compounds were screened for their antibacterial activity against three strains of bacteria i.e. staphylococcus aureus, beta-hemolytic-streptococcus pyogenes, proteus spp. and two species of fungi i.e. aspergillus niger and candida albicans by disc diffusion method. nutrient agar was used as culture medium for bacteria; blood agar was used for streptococcus pyogenes, while sabouraud dextrose was used for the fungal growth agar medium. compounds were dissolved in dmso at concentration 20µg/ml, 50µg/ml, 120µg/ml. ofloxacin and ketoconozole was used as reference antibiotic and dmso as control. the zones of inhibition were determined at the end of an incubation period of 24 hr at 35° c for bacteria and 5 days at 28° c for fungi. inhibition zone were measured. results and discussion synthesis of compounds (3,4 and 5) sulfonamides (compounds 3, 4 and 5) have been synthesized by reaction of compound (2) with methanesulfonyl chloride, benzenesulfonyl chloride and ptoluenesulfonyl chloride respectively, in dichloromethane and triethylamine as a base in case of compound (3), and in pyridine in case of compounds (4) and (5) . the reaction proceeds via nucleophilic attack of the amine on sulfur atom of the sulfonylchloride with liberation of hcl, as shown in the (scheme 2).the structures of these compounds have been characterized by disappearance of symmetric and asymmetric absorption bands for (nh2) of compound (2) and appearance of new absorption band in the synthesized compounds between (3242-3260) cm -1 belong to (nh) group. other ir characteristics absorption bands were listed in table (3), melting points and rf values were listed in table (1), elemental microanalysis was listed in table (2). iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 45 scheme 1:general scheme of the synthesized compounds. scheme 2 :mechanism of sulfonamide formation iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 46 synthesis of compounds (9 10) in order to form amide bond between compound (8) and an appropriate amino compounds, the carboxyl group of compound (8) must be activated. many different ways have been accomplished for this purpose. in this work the method used was the direct coupling with dcc/hobt method. this method is characterized as being simple, efficient, and leading to a good yield at r.t. (29) .the mechanism of amide bond formation by dcc promoted condensation of carboxylic acid and amine. step 1: in the first stage of the reaction, the carboxylic acid adds to one of the double bonds of dcc to give an o–acylisourea step 2: structurally, o–acylisoureas resemble carboxylic acid anhydrides and are powerful acylating agents. in this stage the amine adds to the carbonyl group of the o–acylisourea to give a tetrahedral intermediate. step 3: the tetrahedral intermediate dissociates to amide and n, n–dicyclohexyl urea (dcu). scheme 3: mechanism of amide formation. the structures of these compounds have been characterized by disappearance of absorption bands for (c=o) and (oh) of –cooh of compound (8) and appearance of new absorption band in synthesized compounds between ( 3227-3229) cm -1 belong to (nh) group of 2 o amide. other ir characteristics absorption bands were listed in table (3), melting points and rf values were listed in table (1), elemental microanalysis was listed in table (2). the ir spectra of the synthesized compounds showed a characteristic bands of absorption which were in consistence with the chemical structures of the proposed compounds. all new compounds were analyzed for c, h, n, o and s and the results are in acceptable range. uncorrected melting points of the compounds (3-5), (9 and 10) and their intermediates were determined and were found to be different from melting points of their starting materials. as shown in table (1). iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 47 table 1: physical appearance, percentage yield, melting points and rf values of intermediates and compounds. compound no. physical appearance % yield melting point o c rf value 2 yellow crystal 75 130-132 0.20 a 3 brown oily product 45 0.89 b 4 pale yellow powder 58 151-153 0.84 b 5 pale yellow powder 55 158-160 0.79 b 7 off-white crystal 81 93-95 0.8 a 8 off-white crystal 67 204-206 0.70 d 9 beige crystal 75 188-189 0.87 c 10 yellow crystal 66 192-193 0.33 a a: (chloroform 9:1 methanol) b: (water 1:1 methanol) c: ( chloroform 1:1 methanol) d: ( chloroform 3: methanol 3: ether 4 ) table 2: elemental analysis % of the final products cpd no. molecular weight chemical formula calculated ∕ found c h n o s 4 300.33 c15h12n2o3s 59.99 60.20 4.03 4.18 9.33 9.57 15.98 15.47 10.68 11.18 5 314.36 c16h14n2o3s 61.13 60.06 4.49 4.61 8.91 8.69 15.27 15.42 10.20 10.78 9 376.36 c21h16n2o5 67.02 66.37 4.28 4.39 7.44 7.25 21.26 21.62 10 331.35 c15h13n3o4s 54.37 54.81 3.95 4.03 12.68 12.72 19.31 19.05 9.68 9.30 table 3: the characteristic ir bands of synthesized compounds. compound no. characteristic ir bands cm -1 2 ( 3299, 3200 nh2 str.) , (1643 c=o str.), (1595, 1452 c=car. str.), ( 3045 c-har.str.) (1242 c-n str.). 3 (3260 n-h str.), (3024 charo str.), (2935assy, 2865sy c-haliph. str.), (1681 c=o str. quinolone), (1614 1454 c=car str.), (1352assy, 1161sy s=o str.), (792,769 charo out of plane). 4 (3242 n-h str.), (3099, 3066 charo str.), (1685 c=o str. quinolone), (1616 1450 c=car str.), (1340assy, 1166sy s=o str.), (756-688 char out of plane). 5 (3254 n-h str.), (3024 charo str.), (2935assy, 2865sy c-haliph. str.), (1699 c=o str. quinolone), (1597 1456 c=car str.), (1340assy, 1165sy s=o str.), (813,758 char out of plane). 7 (3076 charo str.), (2980assy, 2872sy c-haliph. str.), (1759 c=o str. ester), (1708 c=o str. coumarine), (1606 1508 c=car str.), (1197 c-o str. ester), (1220assy, 1062sy ar-o-c str.). 8 (3300-2500 oh str. of cooh), (3068 charo str.),(2987assy, 2916sy c-haliph. str.), (1732 c=o str. coumarin), (1717 c=o str. cooh ), (1618 1510 c=car str.), (1253 c-o str. ester), (1207assy, 1080sy ar-o-c str.). 9 (3329 nh str.), (1714 c=o coumarin), (3040 char str. ), (2929 assy. , 2852 sy chaliph. str.), (1693 c=o amide), (1627-1514 c=c str.), (1573 nh bend. amide ii), (1153 c-o str.), (754char out of plane). 10 (3329 nh str.), (1724 c=o coumarin), (3060 char str. ), (2928 assy. , 2852 sy chaliph. str.), (1696 c=o amide), (1626 c=n str.), (1573 nh bend. amide ii), (1149 c-o str.), (719 char out of plane). (str. = stretching vibration , ar = aromatic , aliph.= aliphatic, bend. = bending vibration.) iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 48 antimicrobial activity the newly synthesized compounds were screened for their antimicrobial activity. from the result in table 4, compounds 10 and 4 showed good activity against staphylococcus aureus while compounds 5, and 10 show moderate activity against streptococcus pyogenes when 2µg/ml conc. was used. at conc. 50 µg/ml compound 10 showed significant activity against staphylococcus aureus. while compounds 4 and 9 showed moderate activity. at conc. 120 µg/ml compound 10 demonstrated good activity against staphylococcus aureus. while compounds 9 and 4 showed moderate activity against staphylococcus aureus while all tested compounds show low to no activity against streptococcus and proteus spp. when compared to oflxacin. while compound 10 showed good activity against aspergillus niger and compounds 4, 5, and 9 showed moderate activity against aspergillus niger. all remaining compounds demonstrated moderate to low activity against candida albicans when compared to ketoconazole. table 4: antimicrobial screening data (zone of inhibition in mm) for final compounds compound no. zone of inhibition in mm staphy. aureus strept. pyogenes proteus spp. aspergillus niger candidia albicans 4 2µg/ml 9 7 7 / / 50µg/ml 12 11 7 / / 120µg/ml 15 13 15 18 15 5 2µg/ml 8 9 no activity / / 50µg/ml 12 11 9 / / 120µg/ml 14 14 12 17 18 9 2µg/ml 7 7 no activity / / 50µg/ml 13 12 8 / / 120µg/ml 16 14 16 18 16 10 2µg/ml 10 9 no activity / / 50µg/ml 18 12 8 / / 120µg/ml 19 17 16 20 12 ofloxacin 2µg/ml 11 12 11 50µg/ml 16 18 16 120µg/ml 22 23 22 ketoconazole 120µg/ml 26 36 conclusion the synthesis of these proposed compounds was successfully achieved by following the stated procedures as previously described. the results obtained from this investigation indicated that the strategy adapted for the synthesis of the designed derivatives was successful, since the conformity of synthesized compounds was achieved according to the data shown by the physical and chemical analysis including (tlc, melting point, ft-ir and elemental analysis (chnso). most of these compounds show good antimicrobial activity comparable with marketable compounds. references 1. ahmed, j.; evamarie, h.; bozhana, m.; gerald, d.; emil, p.: an improved synthesis of 4-chlorocoumarin-3-sulfonyl chloride and its reactions with different 2. bidentate nucleophiles to give pyrido[1',2':2,3]-and thiazino[3',2':2,3]1,2,4-thiadiazino[6,5-c]benzopyran-6one7,7-dioxides. molecules, 2007; 12: 2017-2028. 3. forda, r.a.; hawkinsb, d.r.; may, b.c.: the in vivo dermal absorption and metabolism of [4-14c] coumarin by rats and by human volunteers under simulated conditions of use in fragrances. food and chemical toxicology, 2001; 39: 153-162. 4. wan, k.w.; hyung, s.p.; inhye, h.; mihyun, o.: natural compounds, fraxin and chemicals structurally related to fraxin protect cells from oxidative stress. experimental and molecular medicine, 2005; 37: 436-446. 5. elaine, c. p.; daniel, l. l.; josé, m. b. ; eliane, m. b.: coumarin effects on amino acid levels in mice prefrontal cortex and iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 49 hippocampus. neuroscience letters, 2009; 454: 139-142. 6. opinion of the scientific panel on food additives, flavourings, processing aids and materials in contacts with food (afc) on a request from the commission related to coumarin. the efsa journal,2004; 104:1-36. 7. dentali, f.; ageno, w.; crother, m.: treatment of coumarin-associated coagulopathy: a systematic review and proposed treatment algorithms. journal of thrombosis and haemostasis, 2006; 4: 1853–1863. 8. neveen, s. g.: novel inhibition of some pathogenic fungal and bacterial species by new synthetic phytochemical coumarin derivatives. annals of microbiology, 2009; 59 (2): 359-368. 9. nofal, z.m.; el-zahar, m.i.; abd elkarim, s.s.: novel coumarin derivatives with expected biological activity. molecules, 2000; 5: 99-113. 10. koneni, v.s.; abdhesh, k.; manoj, k.; ravi, s.b.; gitika, b.; khanna, a.k.: novel coumarin derivatives as potential antidyslipidemic agents. bioorganic & medicinal chemistry letters, 2010; 20: 4248-4251. 11. kostova, i.; raleva, s.; genova, p.; argirova, r.: structure-activity relationships of synthetic coumarins as hiv-1 inhibitors. bioinorganic chemistry and applications, 2006; pp. 1-9. 12. marina, r.; christos, k.; dimitra, h.l.; stylianos, h.; anastasia, d.: a novel synthesis of 3-aryl coumarins and evaluation of their antioxidant and lipoxygenase inhibitory activity. bioorganic and medicinal chemistry letters, 2010; 20: 3889-3892. 13. yeh, l.c.; chih, m.l.; shoiw, j.l.; daih, h. k.; li, c.c.; tai, c.w.; cherng, c.t.: synthesis, antiproliferative, and vasorelaxing evaluations of coumarin -αmethylene-ᵞ-butyrolactones. bioorganic and medicinal chemistry, 2005; 13: 57105716. 14. bansala,y.; ratraa,s.; bansala, g.; singhb, i. ; aboul-eneinc, h.y.: design and synthesis of coumarin substituted oxathiadiazolone derivatives having antiinflammatory activity possibly through p38 map kinase inhibition. j. iran. chem. soc., 2009; 6: 504-509. 15. arzu, g.; seref, k.; halil, i.u.; mustafa, b.: synthesis, complexation, and biological activity studies of 4-aminomethyl-7,8dihydroxy coumarines and their crown ether derivatives. j. heterocyclic chem., 2010; 47: 1127. 16. redha, i.a.; ahmed, a. h.; yasmien, k. a.: design, synthesis and bioassay of novel coumarins. african journal of pure and applied chemistry, 2010; 4(6): 74-86. 17. ummuhan, o.; ozdemir, a.; pınar, g.; ertan, s.; fatma, h.: synthesis, characterization and antibacterial activity of new sulfonamide derivatives and their nickel(ii), cobalt(ii) complexes. inorganica chimica acta, 2009; 362: 2613–2618. 18. virginija, d.; lina, b.; daumantas, m.: benzimidazo [1,2-c][1,2,3]thiadiazole-7sulfonamides as inhibitorsof carbonic anhydrase. bioorganic & medicinal chemistry letters, 2007; 17: 3335–3338. 19. pawar, p.; bhise, s.; rindhe, s.: synthesis of substituted sulphaquinoxalinones as antimycobacterium tuberculosis agents. international journal of pharmtech research, 2009; 1(2): 252-255. 20. varandas,l. ; fraga, c.; miranda,a.: design, synthesis and pharmacological evaluation of new nonsteroidal antiinflammatory 1,3,4-thiadiazole derivatives. letters in drug design & discovery, 2005; 2: 62-67. 21. mansour, s.; mostafa, m.; mohammed, s.: synthesis and in vitro anticancer evaluation of some novel hexahydroquinoline derivatives having a benzenesulfonamide moiety. european journal of medicinal chemistry, 2011; 46: 201-207. 22. dora, a.; pontinha, r.; carlos, s.: electrochemical oxidation of metolazone at a glassy carbon. electrode electroanalysis, 2008; 20 (23): 2531 – 2536. 23. jack, d.: oral hypoglycemic/antidiabetics: learing objectives. endocrine pharmacotherapy module, spring, 2003. 24. mazin, h.; ph.d. thesis, al-mustansiriyah university, 2006. 25. saad, r,; mohammed, e.; marium, m.: structure and acaricidal activity relationship of some sulfonamide derivatives against the two-spotted spider mite, tetranychus urticae (koch). international journal of agriculture & biology, 2006; 661–665. 26. sunila ,t.: synthesis and pharmacological screening of some benzole derivatives as anti-inflammatory agents. international journal of pharma research and development – online, 2010; 165-198. 27. najim, a. ; iman, a,; ibrahim, a.: amino acid derivatives. part i. synthesis, antiviral and antitumor evaluation of new amino acid esters bearing coumarin side chain, acta pharm. , 2006; 59, 175–188. iraqi j pharm sci, vol.21(2) 2012 new coumarin and 2-quinolone derivatives 50 28. henklein p., rapp w., comparison of microwave mediated peptide synthesis in 29. comparison to conventional peptide synthesis, j. peptide chemistry, 2008, 14(8):10401-10421. 30. eucast disk diffusion method for antimicrobial susceptibility testing. the european committee on antimicrobial susceptibility testing – eucast version 1.0, 2009 :1-16 31. carey f.a.; sunberg r. j., advance organic chemistry; part b: 198, (5 th ed.), plenum press, new york, 2008. iraqi j pharm sci, vol.20(1) 2011 pentoxifylline as radical scavenger 66 concentration-dependant antioxidant activity of pentoxifylline in nitrite-induced hemoglobin oxidation model tavga a. aziz* ,1 *department of pharmacology and toxicology,college of pharmacy, university of sulaimani, kurdistan,iraq abstract free radical formation in heme proteins is recognized as a factor in mediating the toxicity of many chemicals. the present study was designed to evaluate the dose-response relationship of the free radical scavenging properties of pentoxifylline in nitrite-induced hb oxidation. different concentrations of pentoxifylline were added at different time intervals of hb oxidation in erythrocytes lysate, and formation of methemoglobin (methb) was monitored spectrophotometrically. the results showed that in this model, pentoxifylline successfully attenuates hb oxidation after challenge with sodium nitrite; this protective effect was found to be not related to the catalytic stage of hb oxidation, though such effect was reported to be more prominent when the compound was at the same time of induction of hb oxidation with nitrite. in conclusion, pentoxifylline can effectively, in concentrationdependent pattern, attenuate sodium nitrite-induced hb oxidation in vitro. key words: pentoxifylline, radical scavenging, hemoglobin oxidation الخالصة ذعرًذ عًهٍح ذكٌٍٕ انجزٔس انحشج فً تشٔذٍٍ انذو يٍ انظٕاْش انًصاحثح نظاْشج انرسًى تانعذٌذ يٍ انًشكثاخ انكًٍٍأٌح. ذى حشج فً ًَٕرج اكسذج ذصًٍى ْزِ انذساسح نرقٍٍى انعالقح تٍٍ انرشكٍز ٔانرأثٍش نفعانٍح يشكة انثُرٕكسٍفٍههٍٍ فً اقرُاص انجزٔس ان ٔخالل فرشاخ يرعذدج خالل أكسذج انًٓغهٕتٍٍ انًسرحذز تُاٌرشاٌد انصٕدٌٕو. ذًد أضافح ذشاكٍز يخرهفح يٍ يادج انثُرٕكسٍفٍههٍٍ خضاب انذو فً يرحهم انخالٌا انحًشاء، ٔذًد يراتعح ذكٌٕ يادج انًٍثًٓغهٕتٍٍ تٕاسطح ايرصاص انطٍف انضٕئً. أظٓشخ انُرائح اٌ ْزا انًُٕرج ذًكٍ انثُرٕكسٍفٍههٍٍ تُجاح يٍ ذأخٍش عًهٍح أكسذج خضاب انذو تعذ أضافح َاٌرشاٌد انصٕدٌٕو، ٔنٕحع تأٌ ْزا انرأثٍش فً كاٌ أكثش ٔضٕحا عُذيا ذًد أضافح انثُرٕكسٍفٍههٍٍ فً َفس انٕقد انزي ذى فٍّ اسرذاز األكسذج. ٔعهٍّ ًٌكٍ األسرُراج تأٌ هٍح عهى ذأخٍش أكسذج خضاب انذو تٕاسطح َاٌرشاخ انصٕدٌٕو ٔتشكم ٌرُاسة يع ذشكٍز انًادج انًضافح.نهثُرٕكسٍفٍههٍٍ أنقات introduction pentoxifylline (ptx) [1-(59-oxohexyl)-3, 7-dimethylxanthine] is widely used as a drug since long period of time (1) . it is used in the treatment of cerebrovascular and peripheral vascular diseases (2) . ptx is known to possess anti-inflammatory properties (3) which are probably related to its ability to suppress oxygen radical production or scavenge reactive oxygen species (4) . the ability of pentoxifylline to scavenge hydroxyl radicals has been demonstrated earlier (5) . ptx is known to become metabolized to its corresponding 8oxo derivatives in the mammalian system (6) ; these metabolites (methyluric acids) are known to inhibit lipid peroxidation in human erythrocyte membranes in vitro and function as free radical scavengers (7,8) suggesting their antioxidant effects in vivo. it has also been reported that ptx has inhibitory effect on the generation of superoxide anion and hydrogen peroxide in human leukocytes in vitro and in vivo (9,10) . however, the relationship between the concentration and radical scavenging activity of ptx is not completely explained in standard in vitro system. the present study was designed to evaluate the concentrationdependence radical scavenging and membrane stabilizing activities of ptx using in vitro model of nitrite-induced oxidation of hemoglobin. materials and methods preparation of blood samples and hemolysate blood samples were obtained from healthy individuals by vein puncture and kept in (edta) containing tubes for isolation of erythrocytes and preparation of hemolysate. the blood samples were centrifuged at 2500 rpm and 4 o c for 10 minutes to remove plasma and the buffy coat of white cells. the erythrocytes were washed thrice with phosphate buffer saline (pbs, ph 7.4) and lased by suspending in 20 volumes of 20 mm phosphate buffer (pb, ph 7.4) to yield the required hemolysate concentration of 1:20 (11) . 1 corresponding author email : tavgababan@yahoo.com received : 29/12/2010 accepted : 10/4/2011 mailto:tavgababan@yahoo.com iraqi j pharm sci, vol.20(1) 2011 pentoxifylline as radical scavenger 67 preparation of pentoxifylline solutions stock solution of 80mm pentoxifylline was prepared, from which different concentrations series of 40 mm, 20 mm, 10 mm and 5mm were prepared. effect of different ptx concentrations on the time course of nitrite-induced oxidation of hemoglobin to 1.5 ml of freshly prepared hemolysate, 1 ml of each concentration of pentoxifylline (5 mm, 10 mm, 20 mm, 40 mm and 80 mm) was added concomitantly with 0.1 ml sodium nitrite, and the formation of methb was monitored spectrophotometrically at 631 nm for 45 minutes using computerized uv-visible spectrophotometer. the experiments were performed 3 times for each concentration under controlled temperature (27-30 o c). effect of pentoxifylline on the time course of methb formation at various time intervals from nitrite addition to 1.5 ml freshly prepared hemolysate, 1.0 ml of the highly effective concentration of pentoxifylline was added 10 minutes before, at time zero, 10 and 20 minutes after the addition of sodium nitrite to the hemolysate solution, and the formation of methb was monitored spectrophometrically at 631 nm. results in the presence of different concentrations of ptx (5, 10, 20, 40 and 80 mm) the timecourse of oxidation of hemoglobin shows a slow increase in light absorbance related to reduced rate of hb oxidation and inhibition of methemoglobin formation. the linear relationship was reported between ptx concentrations and inhibition of methb formation (0%, 0%, 28%, 68.3 and 76.2% respectively, figure 1 and table 1), indicating a delay in the oxidation process in a concentration-dependent manner. the time required to convert 50% of the available hemoglobin in the erythrocyte lysate to methemoglobin was 22.5 min in the absence of ptx (control), whereas it was delayed to 32, 70.9 and 94.8 min in the presence of 20, 40 and 80 mm of ptx respectively (table 1). addition of the highly effective concentration of ptx (80 mm) to the hemolysate mixture at different time intervals (10 min before nitrite, zero time, 10 min after and 20 min after nitrite addition; i.e during autocatalytic phase) decreases absorbance of light attributed to methemoglobin formation, which is an index for protection of hb against oxidation due to the addition of sodium nitrite to the lysate (57%, 76.2%, 57% and 56.6% respectively, figure 2). the time required to convert 50% of the available hemoglobin to methemoglobin was 22.5 min in the absence of ptx (control), whereas it was delayed to 52.3, 94.8, 51.8 and 46.1 min when ptx (80 mm) was added 10 min before nitrite, zero time, 10 min after and 20 min after nitrite addition respectively as shown in table 2. figure 1. concentration-effect relationship for the radical scavenging activity of pentoxifylline in nitrite-induced hb oxidation in vitro. iraqi j pharm sci, vol.20(1) 2011 pentoxifylline as radical scavenger 68 table 1. effect of different concentration of ptx on the time-course of nitrite-induced oxidation of hb and methb formation. concentration of ptx % formation of methb % inhibition of methb time to form 50% methb (min) 5mm 100 0 22.5 10mm 100 0 22.5 20mm 72 28 32 40mm 31.7 68.3 70.9 80mm 23.7 76.2 94.8 all values represent the average of 3 experiments figure 2. effect of time course of ptx addition on its radical scavenging activity in nitriteinduced hb oxidation in vitro. table 2. effect of addition of ptx at different time intervals of nitrite addition on the time course of hb oxidation and methb formation in erythrocyte lysate. all values represent the average of 3 experiments discussion in the present study, the data clearly showed that free radicals liberation due to incubation of erythrocyte lysate with sodium nitrite leads to oxidative damage and production of methb. free radicals and their derivatives are known to damage red blood cells resulting in functional and structural alterations (12) . decreased membrane fluidity, caused by an increase of lipid peroxidation, is a common consequence resulting from the influence of free radicals (13) . and functional aspects of free radical mediated damage to rbc include altered cation permeability and reduced rbc deformability (14,15) . in this in vitro study, we investigated the scavenger capacity of different concentrations of ptx; the results showed that this effect was concentration dependent and only predictable in relatively high concentrations. there is considerable evidence of the role of oxygen free radicals as important contributors to cell damage in many blood disorders (16) , acceptance of the free-radical hypothesis has contributed to an increasing interest in the use of free-radical scavengers as potential cytoprotective agents (17) . horvath et al (2002) reported a mild antioxidant activity of high concentrations of pentoxifylline in an in vitro system (18) , and the reported data in the present study are compatible with these finding. however, in the present study, ptx showed an antioxidant capacity; this effect became prominant only at very high concentration, which can be rarely reached in time for addition of 80 mm ptx % formation of methb % inhibition of methb time to form 50% methb (min) control 100 0 22.5 10 min before induction 43 57 52.3 at zero time 23.7 76.2 94.8 10 min after induction 43 57 51.8 20 min after induction 43.4 56.6 46.1 iraqi j pharm sci, vol.20(1) 2011 pentoxifylline as radical scavenger 69 the clinical practice. this result is concordant with previously reported data, which indicated that ptx had significant antioxidant capacity at very high concentration (17) , in a study employing ptx in peripheral vascular disease (19) , ptx was found to inhibit the generation of leukocyte-derived active oxygens (superpxide dismutase-inhibitable reduction of ferricytochrome) in vivo. crouch and fletcher (1992) reported superoxide anion production by polymorphonuclear cells (pmn), and a strong correlation between reduced pmn response to activated complement and plasma concentration of ptx metabolites (10) , the same study also suggested that ptx reduced oxygen radical production and protected against unwanted tissue damage in vivo via the action of its metabolites. thus, in addition to ptx alone, evaluation of the radical scavenging capacity of ptx metabolites in the nitrite-induced hb oxidation system seems to be an option to reveal the exact role of ptx in this respect. in conclusion, in this in vitro study, pentoxifylline produced strong radical scavenger effect only in relatively high concentrations, which may be of value in the protection of erythrocytes against oxidative damage; and this idea merits further investigations. acknowledgment the authors gratefully thank the university of sulaimani for supporting the project. references 1. aviado dm, porter jm. pentoxifylline: a new drug for the treatment of intermittent claudication. pharmacotherapy 1984; 6:297-307. 2. frampton je, brodgen rn. pentoxifylline (oxpentifylline). a review of its therapeutic efficacy in the management of peripheral vascular and cerebrovascular disorders. drugs aging 1995; 7:480-503. 3. satapathy sk, sakhuja p, malhotra v, sharma bc, sarin sk. beneficial effects of pentoxifylline on hepatic steatosis, fibrosis and necroinflammation in patients with non-alcoholic steatohepatitis. j gastroenterol hepatol 2007; 22:634-638. 4. oh gs, pae ho, moon mk, choi bm, et al. pentoxifylline protects l929 fibroblasts from tnf-alpha toxicity via the induction of heme oxygenase-1. biochem biophys res commun 2003; 302:109-113. 5. freitas jp, filipe pm. pentoxifylline. a hydroxyl radical scavenger. biol trace elem res 1995; 47:307-311. 6. nicklasson m, bjorkman s, roth b, jonsson m, hoglund p. stereoselective metabolism of pentoxifylline in vitro and in vivo in humans.chirality2002; 14:643-652. 7. nishida y. inhibition of lipid peroxidation by methylated analogues of uric acid. j pharm pharmacol 1991; 43:885-887. 8. schlotte v, sevanian a, hochstein p, weithmann ku. effect of uric acid and chemical analogues of oxidation of human low-density lipoprotein in vitro. free radical biol med 1998; 25:839-847. 9. kumar kv, das un, kumar gs, et al. effect of pentoxifylline on free radical generation in human peripheral leukocytes. pacific j pharmac 1992; 7:89-93. 10. crouch sp, fletcher j. effect of ingested pentoxifylline on neutrophil superoxide anion production. infect immun 1992; 60:4504-4509. 11. doyle mp, pickering ra, dykatra rl, et al. nitrite-induced hemoglobin oxidation. biochem biophys res commun 1982; 105: 127-132 12. baskurt ok. activated granulocyte induced alterations in red blood cells and protection by antioxidant enzymes. clin hemorheol micro 1996; 16:49-56. 13. sukalski ka, laberge tp, johnson wt. in vivo oxidative modification of erythrocyte membrane proteins in copper deficiency. free radic biol med 1997; 22:835-842. 14. wang x, wu z, song g, et al. effects of oxidative damage of membrane protein thiol groups on erythrocyte membrane viscoelasticities. clin hemorheol micro 1999; 21:137-146. 15. uyesaka n, hasegawa s, ishioka n, et al. effects of superoxide anions on red cell deformability and membrane proteins. biorheology 1992; 29:217-229. 16. baskurt ok, temiz a, meiselman hj. effect of superoxide anions on red blood cell rheologic properties. free radic biol med 1998; 24:102-110. 17. habon t, toth k, wittmann i, et al. the protective effect of pentoxiphylline on the free radical induced red blood cell membrane damage. clin hemorheol micro 1993; 13:316. 18. horvath b, marton z, halmosi r, alexy t, szapary l, vekasi j, biro z, habon t, kesmarky g, toth k. in vitro antioxidant properties of pentoxifylline, piracetam, and vinpocetine. clin neuropharmacol 2002; 25(1):37-42. 19. ciuffetti, g, mercuri m, ott c, lombardini r, lupattelli g, santambrogio l, mannarino e. use of pentoxifylline as an inhibitor of free radical generation in peripheral vascular disease. eur j clin pharmacol 1991; 41:511-515. iraqi j pharm sci, vol.29(2) 2020 artemisia dracunculus and psoriasis doi: https://doi.org/10.31351/vol29iss2pp176-184 176 evaluation of the effect of topical artemisia dracunculus administration on serum levels of selected interleukins and spleen index in imiquimodinduced psoriasis in male mice compared to clobetasol propionate (dermovate (r)) ointment thamer a. mohammed*, shihab h. mutlag**,1 and bahaa a. shihab*** *’ministry of health and environment, the state company for drug and medical appliances. **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad. iraq ***ministry of health and environment, dermatologists in al-suwaira general hospital, wasit,iraq. abstract psoriasis is a long-lasting autoimmune disease that is characterized by swollen skin patches. normally, these skin patches are dark, swollen, itchy and scaly. the single application of the innate toll-like receptor7/8 ligand imiquimod (imq) in mice easily induces a dermatitis that closely resemble human psoriasis, critically dependent on the axis of interleukin-23/interleukin-17. the objective of the study is to test artemisia dracunculus possible protective effect on imiquimod-induced psoriasis in mice in its topical dosage form and to compare this effect with dermovate(r) ointment. artemisia dracunculus prepared as an ointment and has been used topically to mice before imiquimod application. the real study results showed that a. dracunculus ointment as well as dermovate (r) ointment can significantly reduce the psoriasis area and severity index in both (a. dracunculus ointment + imiquimod 5%) (group iv) and (dermovate (r) ointment + imiquimod 5%) (group v) as compared with both (control group) (group i): and (vehicle ointment + imiquimod 5%) (group iii). all the results showed that a. dracunculus ameliorates psoriasis induced by imiquimod in male mice keywords: psoriasis, artemisia dracunculus, imiquimod 5%, il-23, il-17. التأثير الموضعي لنبات الشيح الطرخوني على مستويات اإلنتيرلوكينات المختارة في المصل ومؤشر الطحال في الصدفية ***و بهاء احمد شهاب 1،**شهاب حطاب مطلك، 1،*ثامر عبدهللا محمد الشركة العامة لتسويق االدوية والمستلزمات الطبية.وزارة الصحة والبيئة ، * .،العراق بغداد بغداد، جامعة ، والسموم االدوية فرع ** وزارة الصحة والبيئة ، مستشفى الصويرة العام ، واسط ، العراق . *** الخالصة الصدفية هي مرض مناعي ذاتي طويل األمد يتميز ببقع جلدية منتفخة. عادة ، تكون بقع الجلد داكنة ومتورمة وتسبب حكة ومتقشرة. إن في الفئران يحث بسهولة على التهاب الجلد الذي يشبه الصدفية البشرية بشكل كبير ، toll 7/8استخدام االميكويمود الذي يعمل على مستقبالت . الهدف من هذه الدراسة هو اختبار التأثير الوقائي المحتمل لالستخدام interleukin-23 / interleukin-17يعتمد بشكل حاسم على محور والذي تحضير الموضعي لمستخلص نبات الشيح الطرخوني على الصدفية التي يسببها االميكويمود في الفئران ومقارنة هذا التأثير مع مرهم الدرموفيت. تم تخلص نبات الشيح الطرخوني كمرهم وتم استخدامه موضعياً للفئران قبل وضع االميكويمود. أظهرت نتائج هذه الدراسة أن مرهم نبات الشيخ مس شدتها في كل من المجموعات )مرهم نبات الشيخ ومؤشر الصدفية منطقة مساحة الطرخوني وكذلك مرهم الدرموفيت يقلل وبشكل كبير مقياس ( بالمقارنة مع كل من )المجموعة 5٪()مجموعة رقم 5 ( و )درموفيت مرهم + اميكويمود4٪()مجموعة رقم 5 اميكويمود الطرخوني + (.3٪()مجموعة رقم 5 ( ومجموعة )المادة الحاملة + اميكويمود1الضابطة()مجموعة رقم لناشئة من استخدام االميكويمود في الفئران الذكور.جميع النتائج اظهرت أن مرهم نبات الشيخ الطرخوني يخفف بشكل كبير من الصدفية ا il-23 ، il17 ، ٪ 5 االميكيمود ،الشيح الطرخوني، الصدفية: المفتاحية الكلمات introduction psoriasis is one of the world's most common forms of chronic dermatitis. the latest u.s. epidemiological study showed a prevalence rate of 2.6 percent of the population, which translated into more than 6 million americans(1) . psoriasis is an organ-specific autoimmune disease that is triggered by an activated cellular immune system and is similar to other immunemediated diseases such as crohn’s disease, rheumatoid arthritis (ra), multiple sclerosis (ms) and juvenile-onset diabetes(2) . 1corresponding author e-mail: shihabalhattab@gmail.com received: 16/2 /2020 accepted: 21/6 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp176-184 iraqi j pharm sci, vol.29(2) 2020 artemisia dracunculus and psoriasis 177 because of its high prevalence, psoriasis is considered to be one of the costliest dermatological diseases. estimates of the costs per patient differ significantly depending on the severity of the disease.(3) psoriasis can start at any age, although epidemiological studies suggested that it can most commonly occur for the first time between the ages of 15 and 25.(4) moreover, its prevalence estimates vary from 0.5% to 4.6%, with rates varying from country to race. in higher latitudes, psoriasis tends to be more common than in lower latitudes and it is more caucasians than in other races.(5) at present, the combination of genetic, epigenetic and environmental influences is believed to trigger psoriasis.(6) environmental risk factors trigger the body's immune response, where naive t cells in the epidermis, particularly langerhans cells, are triggered by antigen-presenting cells (apc) which can release cytokines such as interleukin il-12 and il-23, encouraging the differentiation of naive t cells into th1 and th17 cells. (7) psoriasis vulgaris is mediated by t cells and dendritic cells. il-23 and il-12 are released by inflammatory myeloid dendritic cells to activate il17 producing t cells, th1 cells, and th 22 cells to produce abundant psoriatic cytokines il-17, ifn-γ, tnf, and il-22.(8) significant associations have also been identified in gene regions involving specific inflammatory pathways, namely il-23 signaling (il23a, il12b and il23r), immune response modulation (il4, il13) and nuclear factor kappalight-chain-enhancer of activated b cells (nfkb) signaling.(9) histopathology of various clinical types of psoriasis includes; hyperkeratosis, parakeratosis, acanthosis, elongation of rete ridges, micro-munro abscess, capillary dilatation and dermal infiltration.(10) it has been reported that the imiquimod (imq) 5% cream, which is recommended for the treatment of actinic keratosis and external genital warts; can stimulate the immune system. it was stated that antigen-presenting cells such as monocytes/macrophages and interferon-producing dendritic cells (ifns) and other cytokines and chemokines can be stimulated by imiquimod.(11) furthermore, imq was reported to stimulate immune cells through the toll-like signaling pathway 7 (tlr7)-myeloid differentiation primary response 88 (myd88).(12) the single application of the innate tlr7/8 ligand imq can easily induce dermatitis that closely resembles human psoriasis, critically dependent on the axis of il-23/il-17. this quick and convenient model makes it possible to further elucidate pathogenic mechanisms and test new psoriasis therapies.(13) genus artemisia is principally a temperate northern hemisphere plant group. a few of its 250 species, however, extend to south america and southern africa.(14) artemisia dracunculus l. (tarragon) is a common seasoning plant used in both its fresh (leaves) and dried (herbs) condition. tarragon plant is also used for medicinal purposes: it contains 0.05– 0.95 percent essential oil, flavonoids, cumarin, phenolic compounds, carotenoids, bitter tastes ,tannins, and mineral compounds.(15) the chemical composition of essential oil (eo) of a. dracunculus is estragol (methyl chavicol) in eo reached 84.9%. the other components were transbeta-ocimen (4.00%), linalool (5.09%), limonene (1.63%), (z,e)-alloocimene (2.29%), beta-ocimen (0.61%) and 3-caren (0.81%).(16) abtahi froushani sm et al. has been reported that tarragon decreases the development of il-17 and ifn-γ pro-inflammatory cytokines, as well as the reduction in splenocyte, ratios of inf-γ to il-10 and il-17 to il-10. in addition, it was observed that the amount of respiratory bursts and nitric oxide production in peritoneal macrophages and the capacity for phagocytosis of macrophages in mice treated with tarragon were reduced.(17) safari h et al (2019) has been reported that a. dracunculus extract administration ameliorates the symptoms of experimental autoimmune encephalomyelitis (eae) in mice. it also caused a decrease in the levels of inflammatory cytokines, including il-17and il-23, and caused an increase in the levels of serum antioxidants in the a. dracunculus treated mice.(18) lee sy et al (2018) have also approved that the extract of some species of artemisia (artemisia capillaries) was effective in the alleviation of psoriatic symptoms in an imiquimod-induced psoriasis models.(19) the aim of the study to test artemisia dracunculus possible protective effect on imiquimod-induced psoriasis in mice in its topical dosage form and to compare this effect with dermovate (r) ointment. materials and methods interleukin 23 and interleukin 17 kits for mice were purchased from cusabio company – china. preparation of the ointment water in oil emulsion base (w/o) was prepared according to british pharmacopoeia 2019 with the help of the pharmaceutics department /college of pharmacy /university of baghdad. seventy-five grams of wool fat were melted in a beaker on a water bath to which 25 grams of 75 ºc heated water was added with continuous stirring until congealed. then 5 gm of the steroid fraction of artemisia dracunculus extract ( which is extracted with hexane and ethanol) (20) (with the aid of pharmacognosy department) was incorporated in the prepared base using a spatula.(21) iraqi j pharm sci, vol.29(2) 2020 artemisia dracunculus and psoriasis 178 animals eight to eleven-week-old balb/c albino male mice weighing about 20 grams each were supplied from the college of pharmacy's animal house, university of baghdad. the mice were housed in five cages, 8 mice per cage, with sawdust bedding material at 20 ± 1 °c under strict hygienic conventional conditions and subjected to a 12-hour light/12-hour dark cycle at the animal house. five cages containing 8 mice per cage were used. the mice were acclimatized for one week, before and during the experiment, and provided with water and ad libitum food. the experiments were carried out in compliance with the international and national standards for ethical conduct in animal care and use and approved by the faculty of pharmacy's animal ethics committee.(22) induction of psoriasis psoriasis was induced by the topical application of a commercially available imq 5 percent cream (aldara; 3 m pharmaceuticals) on the mice's shaved dorsal skin. for nine consecutive days, a daily topical dose of 62.5 mg (5 percent) of medication was applied, translating into a daily dose of 3.125 mg of the active ingredient.(23) experimental protocol the dorsal skin of forty mice was shaved with an electric hair clipper. all mice were weighed at zero time and the thickness of their right ear was measured with a digital vernier every two days for 9 consecutive days. the mice were divided into five groups (8 mice each) for topical experiment as the follows: 1. control group (group i): mice in this group received a daily application of 62.5mg/2cm dose of the vehicle of artemisia dracunculus ointment (wool fat) on their shaved dorsal skin and (5mg) on right ear for 9 consecutive days. 2. artemisia dracunculus only group (group ii): mice received daily topical dose of artemisia dracunculus ointment (62.5mg/2cm) on their shaved dorsal skin and (5mg) on right ear for 9 consecutive days. 3. vehicle -imiquimod group (group iii): mice received daily topical dose of the vehicle of artemisia dracunculus ointment (wool fat) one hour before imiquimod 5% application (62.5mg/2cm) on their shaved dorsal skin and (5mg) on right ear for 9 consecutive days. 4. artemisia dracunculus-imq-treated group (group iv): mice received daily topical dose of artemisia dracunculus ointment one hour before imiquimod 5% application (62.5mg/2cm) on their shaved dorsal skin and (5mg) on right ear for 9 consecutive days. 5. clobetasol propionate (dermovate(r))-imqtreated group (group v): mice were received daily topical dose of 0.05% of clobetasol propionate (dermovate (r)) ointment one hour before imq 5% application (62.5mg/2cm) on their shaved dorsal skin (62.5mg/2cm) and (5mg) on right ear for 9 consecutive days. psoriasis area and severity index (pasi) calculation scoring of skin redness and dorsal skin scaling was done with an objective scoring system on a scale of 0 to 4 (0, none; 1, slight; 2,moderate; 3, marked; 4, very marked). this scoring system is based on the medical psoriasis area and severity index (pasi), except that the thickness of the skin is measured without taking into account the width of the affected area of the body.(24) changes of spleen and calculation of its index mice were weighed before being sacrificed, and then, each mouse's spleen was weighed. the spleen index (spleen index =spleen weight/ body weight) was calculated to reflect the changes of body dynamic.(25) measurement of ear thickness the ear thickness (mm) of each mouse was measured with a digital vernier caliper, at (0, 2,4,6,8 days). the change in ear thickness was used to demonstrate the severity of the inflammation.(26) measurements of skin thickness at the end of the experiment (day 10) the skin thickness of the dorsal area of each mouse was measured (in mm) with an electronic vernier caliper and the measured thickness was compared with those of the other groups.(27) preparation of serum blood was collected in an eppendorf tube by heart puncture, allowed to clot for 15-20 minutes and then centrifuged at 300 rpm for 15 minute. the supernatant, which is the serum, was separated using micropipette and stored at -20 °c until the day of analysis. the serum was utilized for the estimation of serum il-23 level (pg/ml) and serum il-17 level (pg/ml).(28) statistical analysis data were expressed as the mean ± standard deviation (sd). the significance of differences between the mean values was calculated using one way and two ways anova. p-value equal to and less than 0.05 were considered significant for all the results data of this study. results effect of various treatments on the psoriasis area and severity index (pasi) score in male mice table 1 and figure 1 show that, there was a significant elevation (p≤0.05) in pasi score in the (vehicle ointment+5% imq) group (group iii) compared to pasi score of the control group (group i). furthermore, it can be noticed that in the (a.dracunculus ointment +5% imq) group (group iv), the topical application of a.dracunculus caused significant reduction (p≤0.05) in pasi score in comparison with the (vehicle ointment + imq) group (group iii). also, in the ((clobetasol propionate (dermovate(r)) ointment + 5% imq) iraqi j pharm sci, vol.29(2) 2020 artemisia dracunculus and psoriasis 179 group (group v), the pasi score was significantly reduced (p≤0.05) in comparison with the scores of the (vehicle ointment + 5% imq) group (group iii). in the a. dracunculus ointment only group (group ii) there was no change in pasi score during the study. table 1.the comparison of psoriasis area and severity index (pasi) score mean among male mice groups psoriasis area and severity index (pasi) score control (group i) a. dracunculus ointment only (group ii) vehicle ointment + 5% imq (group iii) a. dracunculus ointment + 5% imq (group iv) clobetasol propionate (dermovate ®) ointment + 5% imq (group v) zero zero 7.12±0.64* 4.12±1.12*# 2.28±0.48*# each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. figure 1. the mean of pasi score of the groups affected by imiquimod each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. effect of various treatments on ear and dorsal skin thickness (mm) in male mice tables 2 and 3, and figures 2 and 3 show that, there was non-significant (p>0.05) difference in both ear and dorsal skin thickness (mm) in the (a. dracunculus ointment only) group (group ii) compared to the corresponding values in the control group (group i). furthermore, there was a significant increment (p≤0.05) in both ear and dorsal skin thickness (mm) in the (vehicle ointment + 5% imq) group (group iii) compared to those thickness in the control group (group i); but in the (a. dracunculus ointment + 5% im q) group (group iv), the measured ear and dorsal skin thickness (mm) were significantly reduced (p≤0.05) in comparison with those of the (vehicle ointment + 5% imq) group (group iii); while in the (clobetasol propionate (dermovate® ointment + 5% imq) group (group v), there was a significant reduction (p≤0.05) in both ear and dorsal skin thickness (mm) in comparison with those of the (vehicle ointment + 5% imq) group (group iii). table 2. comparisons of ear thickness (in mm) among various groups day control (group i) a. dracunculus ointment only (group ii) vehicle ointment + imq (group iii) a. dracunculus ointment + imq (group iv) clobetasol propionate (dermovate ®) ointment + 5% imq (group v) 0 0.22 ± 0.01 0.23 ± 0.02 0.23 ± 0.01 0.20 ± 0.02 0.20 ± 0.01# 2 0.22 ± 0.01 0.23 ± 0.02# 0.26 ± 0.01* 0.21 ± 0.01# 0.21 ± 0.01# 4 0.22 ± 0.01 0.23 ± 0.02# 0.31 ± 0.01* 0.21 ± 0.02# 0.22 ± 0.01# 6 0.22 ± 0.01 0.23 ± 0.02# 0.35 ± 0.01* 0.22 ± 0.01# 0.22 ± 0.009# 8 0.22 ± 0.01 0.23 ± 0.02# 0.40 ± 0.01* 0.22 ± 0.02# 0.22 ± 0.01# each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. iraqi j pharm sci, vol.29(2) 2020 artemisia dracunculus and psoriasis 180 figure 2. bar chart showing the comparison of ear thickness (in mm) of various experimental mice groups. each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. table 3. comparisons of dorsal skin thickness (in mm) among various groups dorsal skin thickness (in mm) comparison between the groups groups control (group i) a. dracunculus ointment only (group ii) vehicle ointment + imq (group iii) a. dracunculus ointment + 5% imq (group iv) dermovate ®ointment + 5% im q (group v) 0.50 ± 0.09 0.37 ±0.09# 0.74 ±0.13* 0.50 ±0.12# 0.51 ±0.07# each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. figure 3. bar chart showing the comparison of dorsal skin thickness (in mm) of various experimental mice groups. each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. effect of topical administration of artemisia dracunculus on the spleen index in imiquimodinduced psoriasis in mice table 4 and figure 4 show that, there was a non-significant (p>0.05) difference in the measured spleen index in the (a. dracunculus ointment only) group (group ii) compared to the corresponding measured spleen index in the control group (group i). furthermore, there was a significant increment (p≤0.05) in the measured spleen index in the (vehicle ointment + 5% imq) group (group iii) compared to those measured spleen index in the control group (group i); but in the (a. dracunculus ointment + 5% imq)) group (group iv), the measured spleen index was significantly reduced (p≤0.05) in comparison with those of the (vehicle ointment + 5% imq) group (group iii); while in the (clobetasol propionate (dermovate ® ointment) + 5% imq) group (group v), there was a significant reduction (p≤0.05) in the measured spleen index in comparison with the control group (group i). iraqi j pharm sci, vol.29(2) 2020 artemisia dracunculus and psoriasis 181 table 4. comparison of spleen index among various groups (mg/gm) spleen index comparison between the groups groups control (group i) a. dracunculus ointment only (group ii) vehicle ointment + 5% imq (group iii) a. dracunculus ointment + 5% imq (group iv) clobetasol propionate (dermovate ®) ointment + 5% imq (group v) 6.17 ± 1.78 4.81 ± 1.74# 18.67 ± 7.72* 7.44 ± 1.90# 6.12 ± 3.26# each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. figure 4. bar chart showing the comparison of spleen index in (mg/gm) of various experimental mice groups. each value represents mean ± standard deviation (sd).*= significantly different with respect to the control group (group i).# p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii).imq: imiquimod. effect of various treatments on serum il-23 and il-17 level in male mice table 5 and figure 5 show that, there was a significant elevation (p≤0.05) in both serum levels of il-23 and il-17 in the (vehicle ointment + 5% imq) group (group iii) compared to the corresponding levels in the control group (group i) mice; as well as, in the (clobetasol propionate (dermovate ®) ointment + 5% imq) group (group v). there was a significant decrease in serum il-23 and il-17 levels compared to the (vehicle+ 5% imq) group (group iii). by comparing the (a. dracunculus ointment+5% imq) group (group iv) with the (vehicle+ 5% imq) group (group iii), there was a significant reduction in serum il-23 and il-17 levels. serum il-23 and il-17 levels significantly decreased in the (a. dracunculus ointment only) group (group ii) compared with the (vehicle+ 5% imq) group (group iii). table 5. comparison of various treatments on serum il-23 and il-17 levels. groups serum il-23 level (mean ± sd)pg/ml serum il-17 level (mean ± sd) pg/ml control (group i) 170.05 ± 55.47 261.56 ± 46.34 a. dracunculus ointment only (group ii) 146.15 ± 36.56# 303.32 ± 53.47# vehicle ointment + 5% imq (group iii) 638.84 ± 50.75* 692.69 ± 75.81* a. dracunculus ointment + 5% imq (group iv) 226.53 ± 75.38# 224.41 ± 35.05# clobetasol propionate (dermovate ®) ointment + 5% imq (group v) 211.039 ± 36.43# 197.53 ± 47.04# each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. iraqi j pharm sci, vol.29(2) 2020 artemisia dracunculus and psoriasis 182 figure 5. bar chart showing the comparison of serum il_23 (pg/ml) and serum il_17 (pg/ml) levels in the various experimental mice groups. each value represents mean ± standard deviation (sd). *= significantly different with respect to the control group (group i). # p<0.05 significant in comparison with the (vehicle ointment + 5% imq) group (group iii). imq: imiquimod. discussion psoriasis is a major public health concern affecting approximately 125 million people worldwide. most of the statistics on prevalence and incidence of psoriasis was obtained from european countries, the uk. and the united states , there is therefore still a need for a leading, coherent global epidemiological tool on the epidemiology of psoriasis.(29) previous data showed that psoriasis incidence varied depending on age and geographic location, being more prevalent in countries more distant from the equator.(30) the statistical calculation of pasi score indicated that the prepared ointment of a. dracunculus significantly (p<0.05) ameliorates the psoriasis-like skin inflammation in group iv mice received topical a. dracunculus ointment 1hr before 5% imq in comparison with the (vehicle ointment + 5% imq) (group iii) as shown in table 1 and figure 1. previous studies concerning a. dracunculus indicate pharmacological properties of this plant, including anti-inflammatory, antibacterial, antidiabetic and antioxidant properties. studies have also shown that tarragon inhibited the production of pro-inflammatory cytokine (interleukin 6) and nitric oxide, indicating its antiinflammatory property and this consistent with results of the current study (31). psoriatic lesions are a result of a complex interplay of dermal infiltrates of activated dendritic cells, t cells, cytokines including but not limited to ifn-γ, tumor necrosis factor α (tnf-α), interleukin (il)-12, il-17, and il-23.(32) the immunological pathway of il-23/il-17 plays a particularly important role in promoting the onset and perpetuation of a disease. data from in vitro and clinical studies show that il-17a, a critical cytokine effector in this pathway, is primarily responsible for changes in affected tissues.(33) table 5 and figure 5 showed that there was a significant reduction in serum il_17 and il_23 levels in the (a. dracunculus ointment+5% imq) group (group iv) as compared with the (vehicle ointment + 5% imq) group (group iii). these findings indicated that a. dracunculus affects serum il-17 and il-23 levels induced by imiquimod. innate immune cells such as dendritic cells (dcs) play a critical role in psoriasis pathophysiology by supplying inflammatory / costimulative cell differentiation signals for th17 cells. a recent study (2019) showed the involvement of spleen tyrosine kinase (syk) in the inflammatory signaling cascade of dc; and the spleen tyrosine kinase (syk) has also emerged as one of the targets in the cascade of inflammatory signals whose inhibition (by substances such as tarragon) results in anti-inflammatory effects (34). results of the present study (table 4 and figure 4) are in accordance with those obtained from previous study. conclusion all the results demonstrate that a. dracunculus act as a potent anti-psoriatic agent in comparison with the dermovate® ointment topical administration. it will be a promising intervention in psoriasis in the future. overall, all the results indicated that a. dracunculus suppress serum il-17, il-23 level, as indicated by the reduction of (ear and skin thickness and spleen index) which may explain its anti-psoriatic activity. acknowledgments great thanks to the animal house officer assistant lecturer ammar.a.fathel for his support throughout my work. references 1. koo jm. current consensus and update on psoriasis therapy: a perspective from the u.s. j dermatol. 1999;26(11):723–33. 2. lowes ma, bowcock am, krueger jg. pathogenesis and therapy of psoriasis. nature. 2007;445(7130):866–73. 3. mustonen a, leino m, mattila k, koulu l, tuominen r. treatment costs of psoriasis in a 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http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil doi: https://doi.org/10.31351/vol27iss2pp123-134 123 novel combination for self-nanoemulsifying drug delivery system of candesartan cilexetil safaa f. mekhilef *, 1 and ahmed a. hussein * * department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract solubility problem of many of effective pharmaceutical molecules are still one of the major obstacle in the formulation of such molecules. candesartan cilexetil (cc) is angiotensin ii receptor antagonist with very low water solubility and this result in low and variable bioavailability. selfemulsifying drug delivery system (sedds) showed promising result in overcoming solubility problem of many drug molecules. cc was prepared as sedds by using novel combination of two surfactants (tween 80 and cremophore el) and tetraglycol as cosurfactant, in addition to the use of triacetin as oil. different tests were performed in order to confirm the stability of the final product which includes thermodynamic study, determination of self-emulsification time, particle size and zeta potential measurement, and in-vitro drug release. the results showed that the particle size of the best formula was 13.3 nm and zeta potential of -37.45 mv with approximately 100% release after 45 minutes these results suggest that the preparation of cc as sedds with the use of the above combination of surfactant and cosurfactant is a promising maneuver for oral delivery of cc in order to improve its bioavailability. key words: candesartan cilexetil, tween 80, cremophore el, triacetin, self-emulsifying. جديد من مادتين لتقليل الشد السطحي وماده مساعده للشد السطحي لمستحلب نانوي ذاتي لعقار خليط الكانديسارتان سيالكسيتيل *احمد عباس حسينو 1*،فاخر مخيلف صفاء . فرع الصيدالنيات ، كلية الصيدلة ، جامعة بغداد ، بغداد ،العراق* الخالصه مستحضرات صيدالنيه. عقار تحضيره على شكل االدويه واحده من اهم المعوقات التي تحول دونالتزال مشكله الذوبانيه للعديد من ( ولديه ذوبانيه قليله جداَ في الوسط المائي at 1( ذات النوع )iiالكانديسارتان سيالكسيتيل هو عقار يعمل على غلق مستقبالت االنجيوتنسين )نمط . المستحالبات النانونيه الذاتيه أظهرت نتائج مشجعه في محاوله التغلب على مشكله الذوبانيه للعديد من والذي نتج عنه توافر حيوي قليل ومتذبذب 80العقارات. كانديسارتان سيالكسيتيل ُحضر بشكل مستحاب نانوي ذاتي بأستخدام خليط جديد يتكون من مادتيين لتقليل الشد السطحي )التوين ماده مساعده على تقليل الشد السطحي, بأالضافه الى أستخدام زيت الترايستين.( والتتراكاليكول كelوالكريموفور مستوى وتم أجراء العديد من االختبارات لغرض اثبات ثباتيه المنتج النهائي والتي تتضمن: االستقرار الحراري, توزيع حجم الجزيئات, جهد زيتا, التحرر الدوائي. 45تحرر للدواء خالل %100( ملي فولت, وتقريبا 37.45-( نانومتر, وجهد زيتا )13.3المختاره هو ) صيغةالنتائج اظهرت انه حجم الجزيئات لل عده ادقيقه. هذه النتائج تقترح انه تحضير عقار الكانديسارتان سيالكسيتيل بشكل مستحلب نانوي ذاتي بأستخدام الخليط المذكور يعتبر طريقه و ن طريق الفم من أجل زياده التوافر الحيوي.في شكل صيدالني يعطى ع هألستخدام , تراسيتين, مستحلب ذاتي.el, كريموفور 80الكلمات المفتاحيه: كانديسارتان سيالكسيتيل, توين introduction oral rout still represent the most convenient and acceptable mean for administration of drug molecules to patients since it is associated with high rate of patient compliance in one hand and economic and flexible dosage design in others, that is why more than 70% of total dosage forms utilized by humans are tablets(1,2). one of the most important prerequisite requirements of drug molecules to be available for systemic absorption is aqueous solubility since that is the nature of git fluid. then when the drug molecules become solubilized, it has to pass the biological membrane in order to reach to systemic circulation (2). food and drug administration (fda) classifies drug molecules to belong to one of four categories based on their aqueous solubility and ability to pass through biological membrane, termed as permeability. this classification system is called biopharmaceutical classification system (bcs) (3). 1corresponding author e-mail: safaafakher@yahoo.com received: 1/ 7/2018 accepted: 21/ 10 /2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol27iss2pp123-134 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil 124 drug molecules that belong to class ii have problem in bioavailability mainly due to low aqueous solubility. in this class the rate limiting step is dissolution process and so choosing of suitable drug delivery and appropriate additives is crucial to overcome this major obstacle and improve the fraction that will reach to systemic circulation (4). many approaches were developed in order to overcome this issue with variable degree of success, from these approaches solid selfemulsifying drug delivery system (ssedds) is extensively tried. sedds is type of lipid based drug delivery system and it is isotropic mixture consist of oil, surfactant and/or co-surfactant or co-solvent that has the ability to form o/w micro or nanoemulsion when mix with water upon slight agitation (5). the simplicity of production and stability of the final product makes sedds more attractive to the formulators than ordinary emulsion(6). sedds is preconcentrate that usually filled in either soft or hard gelatin capsule. the agitation that produced by action of git peristalsis along with aqueous media is sufficient for emulsification process to complete. in addition to enhance drug solubilization, drug release and absorption also improve since, drug is already dissolved and, upon emulsification, it will produce very fine particle with large surface area (7). this system has many advantages, say: improve patient compliance and palatability since the final product filled into unit dosage form as capsule, (8) protection of drug molecules from invivo hazard condition, (9) enhanced bioavailability through improving the solubilization process, (10)quicker onset of action as the time required reaching tmax is much less in many literatures that compare sedds with other conventional dosage form, (11) and predictable therapy due to reduced variability including food effects (12). sedds has some drawbacks that limit its wider applications, from these limitations stability issues, low drug loading and volatile oil migration to gelatin capsule shell are the most frequent (13). candesartan cilexetil (cc) is a selective, reversible, competitive angiotensin ii receptor-1 antagonist, and it’s used for the management of hypertension, heart failure, and myocardial infarction. it is also used in patients with impaired left ventricular systolic function, either when ace inhibitors are not tolerated, or in addition to ace inhibitors (14, 15). cc is a prodrug (16), and following oral administration, cc undergoes hydrolysis at the ester link to form the active drug, candesartan, which is achiral. candesartan contains two acidic functional groups: a carboxyl and tetrazole moieties (pka = 5.3 for either). it is a colorless to off-white crystals or powder, with melting point range of (160-175 °c), and it is sparingly soluble in alcohol and practically insoluble in water with log partition coefficient (log p 7.43). it belongs to class ii of bcs and has molecular weight of 610.66 dalton (17, 18). materials and methods materials candesartan cilexetil powder was purchased from shenzhen nexconn pharmatechs, ltd, china, triacetin®, cremophor el, cremophor rh 100, tetraglycol, labrafil®,labrafac cc,labrafac pg, maisine 35-1, miglyol 810, miglyol 812, were purchased from hyper-chem ltd co, china,tween 20 was purchased from scrc, china, tween 40 was purchased from avondale lab, england , tween 60 was purchased from cp, china, tween 80 was purchased from pure chemistry, germany, polyethylene glycol 200 (peg 200) was purchased from bdh limited poole, england, potassium dihydrogen phosphate (kh2po4), disodium hydrogen phosphate (na2hpo4), and hydrochloric acid (hcl) were purchased from thomas baker, india. screening of sedds formulation components saturation solubility studies excess of cc was added to each vehicle (oils, surfactants and cosurfactants) and left in the water bath shaker for 72 hour (hr) under constant vibration to prepare a saturated solution. after this period, the solutions were filtered through a 0.45 micrometer "µm" millipore filter. samples were suitably diluted with ethanol and analyzed by uv/vis spectrophotometer at λ max of cc. three measurements were accomplished for each sample to calculate the solubility of cc (19). construction of pseudo-ternary phase diagrams after screening various oils, surfactants and cosurfactants, sedds were formulated with triacetin as oil, tween 80 as surfactant, and tetraglycol as co-solvent. the boundaries of the phase diagram designated the three components of the system. one of the axes represents the aqueous phase, the second axis for the oil and the third axis representing the surfactant with co-surfactant mixture. pseudo-ternary phase diagrams of oil, surfactant with co-surfactant mixture and the aqueous phase were constructed using the aqueous titration method (20). preparation of cc sedds and formulation optimization self-emulsifying drug delivery system (sedds) liquid formulations of cc were prepared using triacetin, tween 80 and/or cremophore el and tetraglycol in ratios (1:1, 1:2, 1:3, 1:4) and oil: smix at 1:9 ratios or 2:8 ratios (table 1). preparation includes mixing cc with iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil 125 triacetin oil in a screw-capped glass vial at the concentration of (8 mg/0.1 ml), and then heated in a water bath at (50-60 °c) for 20 min. to facilitate homogenization (21). the components were mixed by vortex mixing for 5 min to obtain a clear, uniform mixture and again cooled to room temperature followed by equilibrating the mixture on a sonicator at room temperature for 15 min, after that the formulations were kept under visual observation for at least 48 hr. and examined for any signs of turbidity or phase separation prior to droplet size distribution studies (22). table 1.components and percentage of sedds of cc. formula no. smix ratio oil:smix ratio triacetin %(w/w) tween 80 %(w/w) cremophor el %(w/w) tetraglyco l %(w/w) f-1 1:1 2:8 20 20 20 40 f-2 1:1 1:9 10 22.5 22.5 45 f-3 2:1 2:8 20 26.7 26.7 26.67 f-4 2:1 1:9 10 30 30 30 f-5 3:1 2:8 20 30 30 20 f-6 1:1 2:8 20 30 10 40 f-7 1:1 1:9 10 33.75 11.25 45 f-8 2:1 2:8 20 40.05 13.35 26.6 f-9 2:1 1:9 10 45 15 30 f-10 3:1 2:8 20 45 15 20 f-11 1:1 2:8 20 10 30 40 f-12 1:1 1:9 10 11.25 33.75 45 f-13 2:1 2:8 20 13.35 40.05 26.6 f-14 2:1 1:9 10 15 45 30 f-15 3:1 2:8 20 15 45 20 characterization of the optimized formulation thermodynamic study it includes the following tests: 1. centrifugation test: the sedds formulations were centrifuged at 3500 revolution per minute "rpm" for 30 min. these formulations that overcome this test and maintain a monophasic state were taken for heating/cooling cycle's test (23). 2. heating/cooling cycles test: six cycles were performed in this test. formulations were kept in refrigerator temperature of 4 °c for 48 hr and then in oven temperature of 45 °c for also 48 hr in each cycle. the formulations that pass this test were subjected to freezing-thawing cycle's test (23). 3. freezing/ thawing cycles test: formulations were kept at a temperature (–21) for overnight and then kept at room temperature (+25 °c) until they were melted completely. formulations that remain clear and not separate were selected for further studies (24). determination of self-nanoemulsification time the nanoemulsification time of cc sedds was determined using usp type ii dissolution apparatus. about 0.1 ml quantity of each formulation was added to 200 ml of 0.1 n hcl at 37°c. the samples were gently stirred at 50 rpm and visually monitored (i.e., until a transparent homogenous system was seen) to determine the time (min) for complete nanoemulsification according to the visual observation criteria for sedds formation (table 2). upper limit for formation of good (transparent) sedds was set as one min, since when emulsification occurs slowly in more than one min, milky nanoemulsion with dull appearance will be formed (25). iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil 126 table 2. sedds visual observation grades (26) grade time required for nanoemulsion formation appearance a within 1 min clear or slightly bluish b within 1 min bluish white c within 2 min bluish white, similar in appearance to milk d longer than 2 min dull, ash emulsion, slightly oily appearance e longer than 2 min poor or minimal emulsification , large oil droplets present on the surface particle size distribution and polydispersity index measurement particle size distribution and polydispersity index (pdi) measurements were performed for cc sedds formulations by using particle size analyzer instrument. (27) about 0.1 ml of each cc sedds was added to 200 ml of ph 0.1n at 50 rpm and 37°c, and then sample was taken from the resultant emulsion and filtered through 0.45 µm filter syringe and immediately measured (28). zeta potential measurement zeta potential determination was measured by zeta sizer instrument which relied on measuring electrophoretic mobility in micrometer per second (µm/second) and converting it to zeta potential by in–built software using helmholtz-smoluchowski equation. positive particles with zeta potentials above (+30 mv) or negative particles with zeta potentials lower than (-30mv) are normally considered stable. samples were prepared by the same method of measuring the particle size (29, 30). robustness to dilution robustness to dilution was evaluated by diluting all sedds formulations 100 and 1000 times with different dissolution media which were: 0.1n hcl (ph 1.2), phosphate buffer (ph 6.8) and water. the diluted products were stored for 24 hr and monitored for any signs of phase separation or drug precipitation. formula which give neither drug precipitation nor phase separation and are thus, said to be "robust" to dilution (29). in vitro drug release study of cc sedds the in vitro release of liquid sedds filled in hard gelatin capsule was performed in 900 ml of 0.5% tween 20 in 0.1 n hcl (ph 1.2), and the temperature was maintained at 37˚ c with paddle operated at 50 rpm. an aliquot of 5 ml was withdrawn at predetermined intervals of 5, 10,15,30,45 and 60 min. aliquot were analyzed after filtration through whatman filter paper (no.41), spectrophotometrically at 255 nm (31). in vitro drug release kinetics study of cc sedds to study the kinetics and mechanism of cc release from various ne formulations, data obtained from in vitro drug release study was plotted in various mathematical models including: zero order, first order, higuchi’s model and korsmeyer’s model (32). results and discussion screening of sedds formulation components suitable vehicles with maximal solubilizing potential for the drug under investigation is crucial to achieve optimum drug loading, avoid precipitation of the drug on dilution in the gut lumen and finally minimize the final volume of sedds (33). according to the saturation solubility study (table 3) triacein showed the highest solubility capacity for cc (9.37 mg/ml), and that is why it was selected for preparation of cc sedds. triacetin is considered nontoxic and nonirritant when used as excipient in oral pharmaceutical dosage form(33). tween 80 and cremophore el on other hand showed the highest solubility capacity in respect to surfactants. the high hydrophilic lipophilic balance (hlb) (≥ 12) is prerequisite to achieve o/w sedds, and since the hlb for tween is 15 and that of cremophore el is 12, so both of them considered in this study.finally transcutol p and tetraglycol showed excellent solubilizing capacity for cc with (142.94 mg/ml) for transcutol p and (173.4) for tetraglycol. cosurfactants with low lipophilicity have a faster and better ability to emulsify an oilsurfactant mixture that is in contact with water, and since the log p-values of tetraglycol and transcutol p are -1.34 and -0.43, respectively (34), then tetraglycol was selected to be the cosolvent that was used in this study. iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil 127 table 3. saturation solubility study for different oils, surfactants, and cosolvents with respect to cc. oil solubility(mg/ml) sd labrafac cc 1.71 ±0.87 labrafac pg 1.22 ±0.55 labrafil cs 2.56 ±0.56 maisine 351 2.21 ±0.96 triactin 9.37 ±0.79 miglyol 810 1.21 ±0.56 miglyol 812 2.73 ±0.37 olive oil 5.71 ±1.07 sunflower oil 7.93 ±0.89 surfactant tween 20 4.23 ±1.99 tween 40 7.95 ±1.02 tween 60 10.73 ±0.79 tween 80 32.44 ±0.75 cremophore el 29.81 ±1.11 cremophore rh 25.33 ±2.27 cosolvent peg 200 27.33 ±1.23 peg 300 31.21 ±1.09 peg 400 48.58 ±2.21 peg 600 72.44 ±1.45 transcutol p 142.94 ±2.91 tetraglycol 173.4 ±2.94 pseudo-ternary phase diagram construction the results for pseudo-ternary phase diagrams were illustrated in figures 1 3. mixing of tetraglycol with tween 80 and cremophore el to produce smix was resulted in producing clear, slightly yellowish, low viscous solution. within the nano areas, only gentle magnetic stirring leads to formation of nanoemulsion, and this is related to action of the surfactant(s) used (tween 80 and/or cremophore el in our study). surfactant(s) localized on the surface of the ne droplets reducing the interfacial free energy and providing a mechanical barrier to coalescence resulting in a spontaneous dispersion (35). mixture of surfactants showed wide nano region and among the three different ratios that were used, (1:1, 3:1, and 1:3 of tween 80 and cremophore el), (1:3 and 3:1) showed the bigger nano region. these results are expected as the phase behavior is largely influenced by the size of the molecule of the oil used. triacetin oil has a smaller size molecules compared to that of the surfactants used, and so high degree of oil penetration was expected to occur in the interfacial surfactants layer forced by the large entropy of the nanosystem. a distinct central core, which greatly disrupts the packing of the surfactants molecules in this region, could be formed leading to destabilization of the nanoemulsion resulting in reduction in its existence with different oil: smix ratios (36). figure 1. pseudo-ternary phase diagrams showing the (o/w) nanoemulsion (colored area) regions of triacetin oil (oil), tween 80 and cremophore el (1:1) ratio (surfactant mixture), tetraglycol (co-surfactant) at different smix ratios (1:1, 2:1, and 3:1) at 25 °c. figure 2. pseudo-ternary phase diagrams showing the (o/w) nanoemulsion (colored area) regions of triacetin oil (oil), tween 80 and cremophore el (3:1) ratio (surfactant mixture), tetraglycol (co-surfactant) at iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil 128 different smix ratios (1:1, 2:1, and 3:1) at 25 °c. figure 3. pseudo-ternary phase diagrams showing the (o/w) nanoemulsion (colored area) regions of triacetin oil (oil), tween 80 and cremophore el (1:3) ratio (surfactant mixture), tetraglycol (co-surfactant) at different smix ratios (1:1, 2:1, and 3:1) at 25 °c. evaluation of prepared cc sedds thermodynamic study thermodynamic study was done for all sedds formulas, and all formulas pass successfully through these extreme conditions as no phase separation and/or flocculation was observed. the result of this study illustrated in table 4 and this indicates that sudden change in temperature has on effect on the entropy of the system (37). table 4. result of thermodynamic study for all cc. formulations formul a no. centrifu gation test heating -cooling cycles test freezethawing cycles test f-1 pass pass pass f-2 pass pass pass f-3 pass pass pass f-4 pass pass pass f-5 pass pass pass f-6 pass pass pass f-7 pass pass pass f-8 pass pass pass f-9 pass pass pass f-10 pass pass pass f-11 pass pass pass f-12 pass pass pass f-13 pass pass pass f-14 pass pass pass f-15 pass pass pass determination of self-nanoemulsification time the ability of efficient selfemulsification is essential for sedds since the emulsification process is considered the rate limiting step for drug absorption, and this efficiency could be estimated through measuring the rate of emulsification time which was done by visual observation, considering one minute as maximum time for emulsification process to complete (38). the rate of emulsification depends on degree of interfacial tension reduction, phase transition and on surfactant concentration. (39) the result of this test is shown in table 5 and it showed that all the formulas pass this test successfully with grade a result. lowest time of emulsification was achieved with combination of surfactants with high percentage of cremophore el and this can be attributed to ability of them in reducing the interfacial tension and thus excess diffusion of the aqueous phase into the oil occurs, causing interfacial disruption and discharge of droplets into the bulk aqueous phase (40). iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil 129 table 5. grade and emulsification time of sedds of cc. formula no. grade emulsification time (sec.) f-1 a 27 f-2 a 26 f-3 a 27 f-4 a 19 f-5 a 20 f-6 a 24 f-7 a 23 f-8 a 29 f-9 a 29 f-10 a 24 f-11 a 33 f-12 a 31 f-13 a 16 f-14 a 16 f-15 a 15 particle size distribution and polydispersity index measurement the rate and extend of drug release in addition to drug absorption mainly depends on the particle size of sedds (table 6). therefore, particle size determination is an essential factor for sedds. in most of the cases as the concentration of surfactant increased the surfactant concentration the mean droplet size decreased and this could be attributed to the stabilization of the oil droplets as a result of localization of the surfactant molecules at the oil/water interface (41). reading of these results explicated that surfactants mixture (tween 80+cremophore el) in 1:3 ratio is best combination since it produced smallest particle size among all other maneuvers. also the results showed that as the concentration of the surfactants mixture increase in the formula, the resultant particle size decreased. in addition to that results showed that as the ratio of cremophore el increased in the mixture of surfactants smaller particle size was produced, so cremophore el has better emulsifying capacity than tween 80. there is no direct correlation between hlb value of the surfactants mixture and the resultant particle size and pdi, since the smallest particle size was produced with surfactants mixture that had lowest hlb value 13 among other used surfactants mixture ratios. on the other hand the obtained results were opposite to what was stated by osterag f. et al and qian c. et al, small molecular weight nonionic surfactants are more efficient in producing smaller particle size, since the molecular weight of tween 80 (1310 g/ mol.) lower than that of cremophore el (1630 g/ mol.) but cremophore el showed better emulsification capability and lower particle size (42, 43). the obtained result could be attributed to the theory that stats that molecular characteristics of surfactant can significantly influence the formation of sedds (44, 45). the molecular geometry of a surfactant can be described by its surfactant packing parameter (ccp), and this differ from one surfactant to another and the result of differences in surfactant packing parameters influence the interfacial characteristics of the surfactant such as the curvature of the monolayer, which is generated by surfactant molecules spontaneously associate with each other in water and has a curvature allowing the most efficient packing of the surfactant molecules (46). the change in spontaneous curvature of a surfactant during the emulsification process has been recognized to be a key factor for the nanoemulsion formulation, so there is an intermediate packing parameter which could be expected to impact the formation of fine oil droplets, and it seems that the packing parameter of cremophor el may be closer to this intermediate value than tween 80 (47). results obtained for pdi values of formulations found to be less than 0.5 (figure 4) which indicates the uniformity of droplet size distribution and homogeneity of the formed dispersion (48, 49). table 6. particale size and polydispersity index for cc formulations formula number partical size(nm) polydisersity index (pdi) f-1 68.7 0.373 f-2 46.2 0.337 f-3 59.9 0.15 f-4 21.8 0.252 f-5 21.2 0.259 f-6 71.4 0.09 f-7 95.7 0.015 f-8 87.9 0.27 f-9 77.7 0.231 f-10 21.5 0.255 f-11 42.7 0.336 f-12 34.3 0.313 f-13 24.4 0.263 f-14 19.9 0.229 f-15 13.3 0.267 iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil 130 figure 4. particle size and polydispersity index for f15. measurement of zeta potential colloidal system stabilizes itself by combination of steric and electrostatic repulsion between particles. steric stabilization can be achieved by thick layers of surfactant, which can prevent the coalescence between oil and aqueous droplets, additionally; the stability of the nano-droplets within the storage time could be due to the steric repulsion of the surfactant molecules in this system rather than electrostatic repulsion (50, 51). tween 80 and tetraglycol are both nonionic and so, they will not contribute in any charge for the nano-particles (52). the negative charge of the sedds droplets (table 7) was probably due to ionization of the free fatty acids present in cremophor el , in addition, it was deduced that increasing hlb value leads to an increase in zeta potential due to increase hydrophilic property of the system. this could be attributed to an enhancement in the number of negatively charged hydroxyl groups of water with the increase in hlb value resulted in the observed increase in the zeta potential negativity (53). table 7. zeta potential values of cc sedds formulations. formula no. zeta potential (mv) f-1 -4.09 f-2 -8.91 f-3 -0.92 f-4 -3.6 f-5 -12.66 f-6 -16.07 f-7 -8.04 f-8 -0.88 f-9 -10.7 f-10 -0.36 f-11 -28.9 f-12 -16.21 f-13 -21.9 f-14 -30.89 f-15 -37.54 robustness to dilution all formulations did not showed any signs of phase separation or drug precipitation after 24 h and 48h storage. this result proved that dilution of liquid sedds did not change the rigidity of surfactants layer at the nanodroplet interface, since dilution may cause desorption of surfactants molecules from the nano-droplet surface which acts to maintain its water phase concentration equivalent to its cmc so that preserve the solubility of cc and prevent phase separation (54, 55). in vitro drug release study the result elicited that all sedds formulations were found to release nearly 100% of the drug at the end of 60 min (figures 5-7) and there is no significant difference in dissolution through fifteen formulations of cc sedds (p>0.05). one other hand there was a significant difference in dissolution of these formulations compared with marketed cc tablet and this difference in dissolution results due to limited surface area exposed of the marketed cc oral tablet to dissolution media compare with that offered by sedds (56). also the result showed that there is a direct relationship between the particle size of the formulation and the percentage of cumulative drug release after 60 min, as the particle size of the formula decreased the percent of cumulative release increased and this related to increase the exposed surface area to the dissolution media with reduction of globule size (57). figure 5. comparative dissolution profile for formulas (f-1 to f-5) and marketed tablet of cc. in 0.1n hcl (ph 1.2) with 0.5 % tween 20 at 37 °c. f-15 iraqi j pharm sci, vol.27(2) 2018 self-nanoemulsifying drug delivery system of candesartan cilexetil 131 figure 6. comparative dissolution profile for formulas (f-6 to f-10) and marketed tablet of cc. in 0.1n hcl (ph 1.2) with 0.5 % tween 20 at 37 °c. figure 7. comparative dissolution profile for formulas (f-11 to f-15) and marketed tablet of cc. in 0.1n hcl (ph 1.2) with 0.5 % tween 20 at 37 °c. in vitro drug release kinetics study the result of release kinetic of all cc sedds formulations illustrated in table 8. the best fit (highest r2 value) was found to be korsmeyer-peppas equation and n value of above 0.5 and below 0.89 which indicated that the release mechanism was anomalous and the release was ruled by both diffusion of the drug and dissolution/ erosion. table 8. release kinetic of cc sedds formulations. formula no. zeroorder firstorder higuchi korsmeyer n release mechanism f-1 0.966 0.949 0.946 0.981 0.86 anomalous f-2 0.953 0.969 0.945 0.972 0.86 anomalous f-3 0.972 0.955 0.933 0.99 0.87 anomalous f-4 0.968 0.939 0.912 0.993 0.85 anomalous f-5 0.969 0.961 0.933 0.99 0.86 anomalous f-6 0.969 0.961 0.924 0.99 0.87 anomalous f-7 0.965 0.968 0.942 0.984 0.87 anomalous f-8 0.956 0.95 0.929 0.981 0.87 anomalous f-9 0.975 0.957 0.929 0.992 0.86 anomalous f-10 0.887 0.935 0.938 0.946 0.84 anomalous f-11 0.906 0.945 0.928 0.97 0.84 anomalous f-12 0.931 0.956 0.928 0.98 0.85 anomalous f-13 0.969 0.94 0.936 0.987 0.86 anomalous f-14 0.943 0.938 0.927 0.981 0.85 anomalous f-15 0.875 0.91 0.925 0.956 0.83 anomalous conclusion the new formulations (sedds) are a promising approach for the formulation of cc. the oral delivery of water-insoluble drugs like cc may be possible by using sedds 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hussein** *department of pharmacy, al-rasheed university college, baghdad, iraq **department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq abstract the present investigation aimed to formulate a liquid self-microemulsifying drug delivery system (smedds) of tacrolimus to enhance its oral bioavailability by improving its dispersibility and dissolution rate. four liquid smedds were prepared using maisine® cc as oil phase, labrasol® alf as surfactant and transcutol® hp as co-surfactant based on the solubility studies of tacrolimus in these components. the phase behavior of the components and the area of microemulsion were evaluated using pseudoternary phase diagrams. the formulations were also assessed for thermodynamic stability, robustness to dilution, self-emulsification time, drug content, globule size and polydispersity index. the prepared smedds formulations exhibited improved drug release compared to the pure drug powder. from this study, it was concluded that using a composition of 10% maisine® cc, 67.5% labrasol® alf and 22.5% transcutol® hp produced a liquid smedds with good thermodynamic stability and enhanced in-vitro drug release profiles compared with pure tacrolimus powder. all which supports the use of self-micro emulsifying drug delivery systems as a promising approach to improve dispersibility, dissolution and stability of poorly soluble drugs like tacrolimus. keywords: smedds, microemulsion, tacrolimus. تاكروليمستحضير وتوصيف نظام توصيل دواء ذاتي االستحالب لل **احمد عباس حسينو 1*،دعاء جعفر التميمي العراق بغداد، الجامعة ، الرشيد كلية الصيدلة، قسم * ** قسم الصيدالنيات، كلية الصيدلة، جامعة بغداد ، بغداد، العراق الخالصة عن الحيوي توافره لعقار التاكروليمس لتعزيز إيصال دواء مايكروي سائل ذاتي االستحالب نظام الهدف من الدراسة الحالية هو تحضير كوسيرفكتانت كزيت وسرفكتانت و وترانسكتولالبراسول ميسين و باستخدام سائلة مركبات أربع تحضير تم. والذوبان التشتت معدل تحسين طريق االستحالب ووقت التخفيف، ومتانة الحراري، الديناميكي الثبات تقييم تم كما. المستحلب المايكروي باستخدام مخطط ثالثي الحالة تعيين بالتعاقب وتم كان أفضل المصنعة المركبات أن معدل تحرير العقار منوجد . المتعدد لجميع المركبات التشتت ومؤشر الجزيئات، وحجم الدواء، ومحتوى الذاتي، ذوبان األدوية قليلة لتحسين كتقنية واعده ذاتي االستحالب المايكروي األدوية إيصال إمكانية استخدام نظام النقي مما يدل على الدواء مقارنة بمسحوق .الذوبانية كالتاكروليمس وزيادة استقرارها .تاكروليمس ،مستحلب مايكروي ،دوائي ذاتي االستحالبنظام : المفتاحية الكلمات introduction tacrolimus (fk506) is a selective calcineurin phosphatase inhibitor macrolide. it is the first-line agent used for preventing organ rejection in solid organ transplant patients. (1) oral dosage forms such as capsules, tablets and granules for oral suspension are available of the drug and are preferred by patients due to their convenient administration. (2) however, being a biopharmaceutics classification system (bcs) class ii drug, tacrolimus is poorly soluble in gastric ph leading to low gastrointestinal (gi) absorption and high interand intraindividual variations in pharmacokinetic parameters (3). its chemical formula is c44h69no12, and its molecular weight is 822 da , log p is 3.3. the average absolute bioavailability of tacrolimus was found to be about 21%, with a wide range of 4% to 89% (4,5). all which necessitated the development of an enhanced oral delivery system to overcome the low solubility of tacrolimus (4,5). selfmicroemulsifying drug delivery systems (smedds) are lipid-based formulations containing an isotropic mixture of oil, surfactant and cosurfactant that forms small oil-in-water (o/w) microemulsion upon contact with the aqueous medium of gi secretions under the mild agitation of peristaltic activity(6) . the spontaneous transformation of smedds along with the micro size of particles offer faster drug release profiles and a larger surface area for absorption, all leading to increased bioavailability; meaning that a smaller dose might be used to achieve the therapeutic effect with a reduction of gi irritation and other common side effects leading to improved patient compliance and overall therapeutic outcome (7) . 1corresponding author e-mail: duaa.jaafaraltamimi@yahoo.com received: 31/5/2020 accepted: 20/ 9/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp91-100 iraqi j pharm sci, vol.30(1) 2021 formulation of tacrolimus-loaded liquid smedds 92 this study aimed to formulate a tacrolimus-loaded liquid smedds with an enhanced dissolution and consequently its oral bioavailability compared to the pure drug powder. materials and methods materials tacrolimus was purchased from hyperchem ltd co (china). labrasol® alf, transcutol® hp, lauroglycol fcc lauroglycol 90, peceol and maisine® cc were donated by gattefosse co. (st. priest, france). castor oil was provided by now food, (usa). tween 20 was obtained from scrc (china). tween 80 was obtained from pure chemistry (germany). cremophor el, cremophor rh were purchased from hyper-chem ltd co (china). peg 200 was received from bdh limited poole (england). peg 400 from scrc (china). methanol and propylene glycol were bought from sigma-aldrich (germany). deionized water was purchased from j.t. baker (netherlands). all other chemicals were of analytical grade and used as supplied. methods differential scanning calorimetry (dsc) analysis the process was carried out by weighing two milligram of the pure drug, sealing it in an aluminum pan and then placing in dsc instrument. the sample was heated at temperature up to 300 °c and at a rate of 10 °c/min, using nitrogen as blank gas. solubility studies tacrolimus was added in excess amounts to glass vials containing 2ml of each vehicle separately including oils, surfactants and cosurfactants, the components were thoroughly mixed for 10 minutes with a vortex mixer then shaken using a water bath shaker for 48 hours at 25±1°c followed by centrifugation at 3000 rpm for 20 minutes to separate the undissolved drug. aliquots from the supernatants were then filtered by a 0.45 µm millipore filter. (8) the resulting solution was then diluted with 100 ml methanol and analyzed using uv-visible spectrophotometer at λmax 213 nm to measure its drug concentration. the analysis was done according the calculated λmax and calibration curve.(9) construction of pseudoternary phase diagrams pseudoternary phase diagrams of oil, surfactant/cosurfactant (smix), and water were constructed using the aqueous titration method. (10) the surfactant to cosurfactant mixtures were prepared in ratios of (1:1, 2:1, 3:1, 4:1) then mixed with oil in various weight ratios from 1:9 to 9:1 in multiple glass vials. (11) the resulting homogeneous mixtures of oil/smix were titrated with water using magnetic stirrer with 50 rpm and at room temperature. after each addition of water, the mixture was checked for phase clarity. the turbidity of different solutions samples would indicate the formation of a coarse emulsion. whereas a clear isotropic solution sample would indicate the formation of a microemulsion. the formation of microemulsion regions was checked visually for turbidity–transparency–turbidity. when the system became turbid, titration was stopped and the percentage of oil, smix, and water in 100 % w/w mixture were calculated and was utilized for the determination of the microemulsion region boundaries corresponding to the chosen value of oil and smix ratio. the percentages of oil, surfactants to cosurfactants ratios and water in each system were determined and plotted on triangular coordinated using chemix ternary plot software. (12) preparation of tacrolimus-loaded smedds tacrolimus (0.5mg) was dissolved in maisine® cc oil with different ratios of labrasol® alf and transcutol® hp in separate screw-capped glasses vials. they were stirred gently by vortex mixer (50 rpm). the resulting homogenous mixtures were then stored at room temperature. the concentration was 2mg/ml.the composition of tacrolimus liquid smedds formulations are illustrated in table 1. table 1. composition of tacrolimus liquid self-microemulsifying drug delivery systems (%w/w) formula smix ratio oil: smix ratio maisine® cc % labrasol® alf % transcutol® hp % 1 1:1 1:9 10 45 45 2 2:1 1:9 10 60 30 3 3:1 1:9 10 67.5 22.5 4 4:1 1:9 10 72 18 characterization and evaluation of tacrolimusloaded smedds thermodynamic stability studies assessing the physical stability of smedds is essential to prevent drug precipitation and excipients phase separation and ensure a good bioavailability and therapeutic drug profile upon administration. (13) therefore, the prepared formulas were serially assessed by various thermodynamic stability tests with thorough visual observation for any changes in physical appearance (14) . iraqi j pharm sci, vol.30(1) 2021 formulation of tacrolimus-loaded liquid smedds 93 centrifugation test the formulations underwent centrifugation at 3500 rpm for 30 minutes under observation. stable formulations were then subjected to the freeze-thaw cycle. heating-cooling cycle (h/c cycle) six heating-cooling cycles were performed in which the formulas were placed for 48 hours in alternating temperatures of 4 and 45 °c. the formulas that exhibited no phase separations, creaming or cracking were subjected to centrifugation test. freeze-thaw cycle the formulas were stored for 48 hours at alternating temperatures of -21 and +25 °c for three consecutive cycles. robustness to dilution and effect of ph the formulations that passed all the thermodynamic studies were diluted with deionized water and 0.1 n hcl for 50, 100, 250 and 1000 times to simulate in-vivo conditions. the formulations were stored at room temperature for 24 hours then observed visually for any changes. (15) dispersibility test and self-emulsification time the efficiency of smedds formulations was assessed using the usp dissolution apparatus ii. one ml of each formulation was mixed with 500 ml of deionized water at 37 ± 0.5 oc under stirring speed of 50 rpm.(16) the resulting solutions were visually evaluated using the grading system shown in table 2. (17) table 2. grading system of in-vitro performance of self-microemulsifying drug delivery system (dispersibility and self-microemulsification time). grade time required for microemulsion formation appearance a rapidly forming emulsion (within 1 min). having a clear or bluish appearance b rapidly forming, (within 1 min). slightly less clear emulsion, having a bluishwhite appearance c formed within 2 min. fine milky emulsion d slow to emulsify (longer than 2 min). dull, greyish-white emulsion having a slightly oily appearance e slow to emulsify (longer than 2 min). formula exhibiting either poor or minimal emulsification with large oil globules present on the surface. droplet size analysis and polydispersity index (pdi) determination fine microemulsions were formed by diluting each stable smedds formula with deionized water to 100 times under stirring with a magnetic stirrer at 37 °c. dynamic light scattering method was used to analyze the particle size using particle size analyzer apparatus (brookhaven, usa) of the resultant microemulsions and the pdi was accordingly calculated.(18) drug content determination each formula was dissolved in 100 ml methanol in a volumetric flask and thoroughly mixed. after appropriate filtration and dilution, uv–visible spectrophotometer was used to measure drug absorbance (19). in-vitro drug release studies using the usp dissolution apparatus-ii, the in-vitro release profiles of all prepared formulations along with pure drug were obtained. the dissolution medium consisted of 0.1n hcl at 37±0.5 °c and 75 rpm. (20) each tacrolimus-loaded smedds formula was placed in a dialysis bag (molecular weight cutoff of 12000 da), and a regular withdrawal of 5 ml aliquots at 10, 20, 30, 40, 50 and 60 minutes was done. equal volumes of fresh dissolution media (0.1n hcl) were added to replace the withdrawn samples in order to maintain the volume constant and keep sink condition. the amount of drug dissolved was measured using uv–visible spectrophotometer according to the calibration curve (21). statistical analysis the results of the experiments were presented as a mean of triplicate samples± standard deviation (± sd). the in-vitro dissolution studies results were statistically evaluated using the similarity factor (f2) equation. the results of the f2 test range between 0 and 100. two dissolution profiles are considered similar when the f2 value is ≥ 50. this method is more acceptable to compare dissolution profile when more than three or four dissolution time points are available(22). f2 = 50 × log {[1 + 1 𝑛 ∑ |𝑅𝑡 − 𝑇𝑡| 2 𝑛 𝑡=1 ] −0.5 × 100} results and discussion differential scanning calorimetry (dsc) the dsc was performed to determine the crystalline state of the drug and to provide specific information about the physicochemical status of tacrolimus. in addition, to evaluate the thermotropic properties and thermal behavior of tacrolimus. iraqi j pharm sci, vol.30(1) 2021 formulation of tacrolimus-loaded liquid smedds 94 the dsc thermogram of pure tacrolimus shows a sharp endothermic at 134.43 °c, corresponding to its melting point, which lies within the melting point readings of that reported in the references, which are from 126 °c to 135 °c (1), and inferring the presence of the crystalline form of the drug as shown in figure 1. figure 1. dsc thermograms of pure tacrolimus solubility studies assessing the extent of the solubility of tacrolimus in the different microemulsion components is an essential step in formulating smedds, as it greatly affects the physicochemical characteristics and drug loading capabilities of the smedds formulations. (24) in this study, the highest solubility of tacrolimus was observed in maisine® cc as oil phase, labrasol® alf as surfactant and transcutol as cosurfactant. these components were consequently chosen for further assessment. the analysis was done according the calculated λmax and calibration curve as illustrated below in figures 2 and 3. the solubility studies are shown in figure 4. figure 2. tacrolimus uv-spectrum in methanol. figure 3. tacrolimus uv calibration curve in methanol iraqi j pharm sci, vol.30(1) 2021 formulation of tacrolimus-loaded liquid smedds 95 figure 4. solubility studies of tacrolimus (a) in various oils; (b) in various surfactants; (c) in various cosurfactants. construction of pseudoternary phase diagram pseudoternary phase diagrams were utilized to determine the optimal components concentration needed to create stable smedds formulas that could withstand the aqueous dilution effects of the gastrointestinal system without losing solvent and microemulsifying capacity. (25) (26) the pseudoternanary phase diagrams of the various formulas with different smix ratios are illustrated in figure 5. the comparison of these phase diagrams shows that a higher concentration of labrasol corresponded with a larger microemulsions area indicating a higher emulsifying efficacy. this could be attributed to the fact that the surfactants stabilizes the oil-water interface and its concentration increased at the interface upon decreasing the oily content in the ternary system. thus, small size of the generated emulsions.(27) in addition, the reports indicate that a high hlb value of surfactant facilitates the formation of a stable microemulsion. (28) figure 5. pseudoternary phase diagram of smix (a) (4:1); (b) (3:1); (c) (2:1); (d) (1:1) iraqi j pharm sci, vol.30(1) 2021 formulation of tacrolimus-loaded liquid smedds 96 preparation of tacrolimus-loaded smedds different concentrations of surfactant and cosurfactant were added to maisine® cc oil in a fixed oil: smix ratio of 1:9 as shown in table 1. in all formulations, while simultaneously increasing and decreasing the concentrations of oil, surfactant and co-surfactant, respectively, smix was held at fixed ratios. the prepared formulas demonstrated a clear, homogeneous appearance with no change in the phase behavior or drug precipitation upon storage before characterization. characterization and evaluation of tacrolimusloaded smedds thermodynamic stability studies all the prepared smedds formulations passed the centrifugation, heating-cooling cycles and freeze-thawing cycles tests as shown in table 3, which indicates their thermodynamic stability under extreme conditions. table 3. thermodynamic stability studies of tacrolimus liquid self-micro emulsifying drug delivery systems formula centrifugation test heating-cooling cycles test freeze-thawing cycles test f1 pass pass pass f2 pass pass pass f3 pass pass pass f4 pass pass pass robustness to dilution and effect of ph the formulations showed excellent robustness to dilution and ph effect, as no drug or phase separation was observed in any of the prepared emulsions after 24 hours of dilution in 0.1n hcl and deionized water, as illustrated in table 4. these results reveal the high solubilizing properties of the smedds components and their resilience to changes in ph and ionic strength, probably due to their non-ionic nature. (29) the previous researches concurred with these findings, vincent jannin et al. who established a binary phase diagrams database for the development of self-emulsifying lipid-based formulations found that water-soluble surfactant labrasol alf can be associated with up to 30% of peceol oils and form a miscible mixture and this is an appropriate combination of excipients which able to dissolve the drug and form stable formulations. (30) table 4. robustness to dilution of various tacrolimus liquid self-microemulsifying drug delivery systems formula phase separation drug precipitation 0.1n hcl deionized water 0.1n hcl deionized water f1 pass pass pass pass f2 pass pass pass pass f3 pass pass pass pass f4 pass pass pass pass dispersibility test and self-emulsification time all the prepared formulas spontaneously produced clear grade a microemulsions in less than one minute as shown in table 5. higher labrasol® alf concentration was associated with reduced self-emulsification time. this may be attributed to its ability to enhance dispersion and emulsion formation by reducing the interfacial tension between the oil and aqueous phase. (31) table 5. dispersibility and self-micro emulsification time of tacrolimus liquid self-microemulsifying drug delivery systems formula grade emulsification time (sec) formula grade emulsification time (sec) f1 a 23 ± 1.12 f3 a 16 ± 1.72 f2 a 20 ± 1.96 f4 a 15 ± 2.14 droplet size analysis and polydispersity index (pdi) determination assessing droplet size and pdi values is crucial in evaluating smedds formulas as they directly affect the absorption of the drug and its uniformity after dilution (14). in this study, all prepared formulations had acceptable droplet size measurements and pdi values closer to zero indicating good homogeneity and uniformity, as shown in table 6 and presented in figure 6. similar results obtained from juno yoo et al. who studied the effect of different properties https://www.researchgate.net/profile/vincent_jannin iraqi j pharm sci, vol.30(1) 2021 formulation of tacrolimus-loaded liquid smedds 97 (labrasol to transcutol concentration is one of these factors) on self-emulsifying drug delivery system and found that when transcutol concentration increased to up to 35%, the droplet size will be increased.(32) table 6. droplet size measurement and polydispersity index (pdi) of tacrolimus liquid selfmicroemulsifying drug delivery systems formula particle size ± sd (nm) polydispersity index f1 44.3 ± 1.073 0.005 f2 32.3 ± 1.073 0.005 f3 29.5 ± 1.073 0.005 f4 34.2 ± 1.141 0.018 figure 6. droplet size and polydispersity index of of tacrolimus liquid self-microemulsifying drug delivery systems (a) f1; (b) f2; (c) f3; (d) f4. drug content determination the drug content in all the prepared formulations exceeded 98% and was within the usp-recommended range of 85%-115%. as shown in table 7. these results indicate the uniform dispersion of tacrolimus within smedds.(20) table 7. the drug content percent of tacrolimus liquid self-microemulsifying drug delivery system (mean ±sd) n=3 formula drug content % formula drug content % f1 98.84 ± 0.175 f3 99.76 ± 0.081 f2 99.53 ± 0.122 f4 98.44 ± 0.093 in-vitro drug release studies while conventional dissolution tests are useful in assessing dispersibility of smedds in the dissolution media, they are inadequate in simulating in-vivo dissolution and evaluating actual drug release profiles as they do not distinguish between the proportions of drug dissolved and those associated with the emulsion. (33) in order to evaluate the actual drug release of formulations, the proportion of drug dissolved in the aqueous medium should be separated from that associated with the emulsion (34). for this purpose, the dialysis bag method is utilized to permeate the dissolved drug only and enable a more accurate estimation of drug release from the smedds formulations. in this study, a dialysis bag with a very small pore size (molecular weight cutoff of 12000 da) was used to ensure a large surface area of particles subjected to the dissolution medium. (35) it was soaked overnight in 0.1 n hcl dissolution medium to reach equilibrium. (36) the calibration curve of tacrolimus in 0.1 n hcl shown in figure 7.the in-vitro release profiles of the prepared formulas along with that of the pure drug were assessed in 0.1 n hcl over one hour as shown in figure 8. all the prepared liquid smedds formulations had dissimilar release profiles relative to the pure drug (f2 <50). formulation f3 showed the highest release rate (98.71%) followed by f2 (97.33%) while the release rate of the pure drug (19%) was the lowest among all tested formulations. the noticeable increase in the in=vitro drug release profiles could be explained by the rapid self-emulsification properties of smedds and their ability to generate iraqi j pharm sci, vol.30(1) 2021 formulation of tacrolimus-loaded liquid smedds 98 microemulsions with fine droplet size upon dilution. (37) furthermore, it could be observed from the figures that the particle size of the produced emulsion greatly affects the drug release rate, which could be explained by the droplet size-dependent release of tacrolimus from f3 formulations (38), suggesting that formulations with smaller particles possess a higher release rate and vice versa, which explains why formulations f3 has the highest release (39,40). the in-vitro release rate and extent enhancement could be attributed to the smedds fast spontaneous emulsification properties and the production of a small globule size with a high surfactant concentration (37). figure 7. tacrolimus uv calibration curve in 0.1 n hcl. figure 8. in-vitro release profiles of tacrolimusloaded smedds formulae compared with pure tacrolimus. conclusions from this study, it is concluded that the liquid smedds containing 10% maisine® cc, 67.5% labrasol® alf and 22.5% transcutol showed good thermodynamic stability and a globule size in the nanoometric 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evaluation of rifampicin nanoemulsions. international journal of pharmaceutical sciences and drug research 2015;7(6):451–5. 39. deshmukh a, kulakrni s. novel self microemulsifying drug delivery systems (smedds) of efavirenz. j chem pharm res 2012;4:3914– 9. 40. balakrishnan p, lee b-j, oh dh, kim jo, lee y-i, kim d-d, et al. enhanced oral bioavailability of coenzyme q10 by selfemulsifying drug delivery systems. international journal of pharmaceutics 2009;374(1–2):66–72. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 quality of life in chronic obstructive pulmonary disease patients doi: https://doi.org/10.31351/vol29iss2pp169-175 169 health-related quality of life in a sample of chronic obstructive pulmonary disease patients in al-diwanyia province /iraq. akram h. kareem*,1 and dheyaa j. kadhim** * ministry of health and environment , , afak general hospital. **department of clinical pharmacy, college of pharmacy, university of baghdad, baghdad, iraq. abstract the extent to which patient's usual or expected physical, emotional and social well-being are affected by a medical condition or its treatment is known as health-related quality of life. chronic obstructive pulmonary disease (copd) is a common respiratory disease. assessment of health-related quality of life is considered important in such chronic diseases. the aim of the current study was to measure health-related quality of life in a sample of chronic obstructive pulmonary disease patients in aldiwanyia city/iraq. this study was carried out on 150 already diagnosed chronic obstructive pulmonary disease patients who attended to the center of respiratory diseases/al-diwaniyah teaching hospital during september 2019 to january 2020. the arabic version of st george’s respiratory questionnaire was used to assess the health-related quality of life. the mean symptoms score was 48.65 ±7.17, the mean activity score was 62.39 ±5.81, the mean impact score was 42.83 ±7.90 and the total score 49.58 ±4.82. symptom score was predicted by disease duration (negatively) and hospital admission (positively), activity score and impact score could not be predicted by any of independent variables and total score was predicted by forced expiratory volume in the first second (negatively) and hospital admission (positively). in conclusion, health-related quality of life decline in copd patients. increased number of hospital admissions and decreased lung function were associated with a significant worsening in health-related quality of life. keywords: health-related quality of life, copd, st george’s respiratory questionnaire, al-diwanyia, iraq. الديوانية/ العراقفي مدينة االنسداد الرئوي المزمن لدى عينة من مرضى الصحية الحياة جودة *ضياء جبار كاظم و 1*، اكرم حسين كريم . ، مستشفى افاق العام وزارة الصحة والبئية * .فرع الصيدلة السريرية ،كلية الصيدلة، جامعة بغداد، بغداد، العراق ** الخالصة الجها تعرف جودة الحياة الصحية بأنها مدى تأثر الحالة البدنية والعاطفية واالجتماعية المعتادة او المتوقعة للمريض بالحالة المرضية او ع مزمنة. . يعد مرض االنسداد الرئوي المزمن أحد أكثر أمراض الجهاز التنفسي شيوًعا. إن تقييم جودة الحياة الصحية يعتبر مهم لمثل هكذا امراض لدى عينة من مرضى االنسداد الرئوي المزمن في مدينة الديوانية / العراق. أجريت الدراسة الصحيةهدفت الدراسة الحالية إلى قياس جودة الحياة ي / مريضاً من المشخصين مسبقا بمرض االنسداد الرئوي المزمن ممن حضروا إلى مركز أمراض الجهاز التنفس 150المستعرضة الحالية على . تم تقييم جودة الحياة الصحية باستخدام النسخة العربية من 2020إلى كانون الثاني 2019مستشفى الديوانية التعليمي خالل الفترة من تشرين االول سط ، وكان متو 5.81± 62.39درجة النشاط ، وكان متوسط 7.17± 48.65درجة األعراض استبيان سانت جورج التنفسي . كان متوسط . يتم توقع درجة األعراض من خالل مدة المرض )سلبًا( ودخول المستشفى 4.82± 49.58والنتيجة اإلجمالية 7.90± 42.83درجة التأثير زفير م ال)إيجابيًا( ، وال يمكن توقع درجة النشاط ودرجة التأثير من خالل أي من المتغيرات المستقلة ويمكن توقع النتيجة اإلجمالية من خالل حج االنسداد الرئوي الصحية كانت منخفضة لدى مرضى ، فان جودة الحياة كنتيجة القسري في الثانية األولى )سلبًا( ودخول المستشفى ) بشكل ايجابي(. .الصحيةالمزمن. ارتبط ازدياد عدد مرات دخول المستشفى وانخفاض وظائف الرئة بتدهور كبير في جودة الحياة ديوانية ، العراق.ال، مدينة التنفسيسانت جورج استطالعالكلمات المفتاحية: جودة الحياة الصحية ، االنسداد الرئوي المزمن، introduction chronic obstructive pulmonary disease (copd) is a disease characterized by chronic respiratory symptoms with poorly reversible restriction of airflow that is typically progressive. copd is currently considered a major incurable worldwide health problem, and is the world's fourth largest cause of death (1). copd prevalence was (5.6 %) in 2015 and is expected to increase to (7.8%) by 2030 (2). copd affects about 600 million patients worldwide and is a growing concern in the elderly with rising numbers of copd patients aged 65 or older (3). health-related quality of life (hrqol) was adapted from the more general and wideranging concept quality of life (qol). the hrqol is defined as: "the extent to which patient's usual or expected physical, emotional and social well-being are affected by a medical condition or its treatment" (4). copd progressively impairs patients’ ability to carry out activities of daily living and reduces breathing capacity (5), so the patients will undergo a progressive decline in their lung function due to the disease’s’ symptoms (shortness of breath, wheezing, cough, etc.), which will subsequently reduce their hrqol (6). 1corresponding author e-mail: akram_hassan17@yahoo.com received:24 /3 /2020 accepted: 14/ 6/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp169-175 iraqi j pharm sci, vol.29(2) 2020 quality of life in chronic obstructive pulmonary disease patients 170 some factors such as dyspnea, poor mobility, depression, and inadequate social support adversely affect hrqol, and these factors may be amenable to treatment more than treating the impairment in lung function only (5) . health status measurement is becoming an important issue for the day-to-day management of copd patients in both primary and secondary health care (7). studying hrqol for patients with copd will provide the health care professionals with specific information concerning the problems that these patients develop. accordingly, these will enable them to implement interventions directed toward improving patients' care (5). studies report that a shorter survival is related to worse health status/hrqol (8-10). both general and disease-specific instruments have been used to measure hrqol in patients with copd (11,12). however, using disease-specific questionnaires may be more representative of the disease state than general questionnaires that do not focus on any specific disease (13). the st. george's respiratory questionnaire (sgrq) is a well-known disease-specific questionnaire for measuring hrqol in asthma and copd (14). the current study aimed to measure hrqol in patients with copd in the province of al-diwanyia /iraq. patients and method administrative arrangement and ethical considerations according to the requirements of the division of graduate studies in the college of pharmacy, university of baghdad, a research proposal that explains the purpose of the study and methods for data collection was submitted to the university committee and approval was obtained. then approval was obtained from the ministry of health. whereas consent to participate in the study was obtained verbally. patients the current cross-sectional study involved 150 already diagnosed copd patients who attended to the center of respiratory diseases/aldiwaniyah teaching hospital during september 2019 to january 2020. inclusion criteria 1-already diagnosed copd patients of either sex who give consent to participate in the study. 2-age of the patients between 18 and 70 years of age. 3-in stable phase (clinically stable condition with no physical finding or symptoms suggestive of exacerbation for at least two weeks). 4-disease duration (since diagnosis) of at least six months or more. 5-do not have co-morbid diseases such as unstable coronary heart disease, heart failure, hypertension, diabetes mellitus, kidney or liver failure. exclusion criteria 1-patient with cognitive, speech, or a hearing deficit that would affect questions understanding. 2-patient who take antidepressant drugs, or being on treatment for any neurological or psychological diseases. 3-patients providing incomplete information during completion of the questionnaire also excluded from the study. 4-pregnant or lactating mothers. study tools a questionnaire was used to collect data required for the current study which include: 1-demographic characteristics: age, gender, body mass index, social status, educational level, residency, etc. 2-disease-related variables: duration of disease, number of medication, duration of medication use. 3-measurement of hrqol using arabic version of sgrq (15). the sgrq contains 50 items in 3 key components: "symptoms (8 items), activity (16 items), and impact of disease (26 items)." the total score ranges from 0 to 100, where higher scores indicating poorer hrqol (16). the symptoms part is related to the effects, severity and frequency of respiratory symptoms; the activity part is related to daily activities that are impaired by or cause breathlessness; while the impact part is related to the psychological disturbances and social functioning associated with the respiratory disease (17). in addition, pulmonary function tests were measured by spirometer to measure forced expiratory volume in the first second (fev1), forced vital capacity (fvc), fev1 to fvc ratio, and the forced expiratory flow at 50% of fvc (fef50%). while severity of copd was classified according to the global initiative for chronic obstructive lung disease (gold) staging system as follow (18) : a-gold 1: mild where fev1 ≥ 80% predicted. b-gold 2: moderate where 50% ≤ fev1< 80% predicted. c-gold 3: severe where 30% ≤ fev1< 50% predicted. d-gold 4: very severe where fev1≤ 30% predicted. administration of the questionnaires when the patients visited the center of respiratory diseases/al-diwaniyah teaching hospital for checkup and to receive their medications, they were interviewed by the researcher and asked if they were willing to participate. if they agreed, a clarification of the questionnaire was given and each patient was allowed to fill the research questionnaire which takes about 5-10 minutes to be filled completely. statistical analysis data of the current study was analyzed using the social sciences statistical package (spss version 23) and the microsoft office excel 2010 edition. categorical variables were represented as iraqi j pharm sci, vol.29(2) 2020 quality of life in chronic obstructive pulmonary disease patients 171 (number and percentage). initially, quantitative variables were checked for normality by kolmogorov-smirnov test. then quantitative variables were presented as (mean ± standard deviation) or as median (interquartile range). multivariate regression analysis to find the predictors of quality of life score in copd patients enrolled in this study. pvalue was considered to be significant when it is ≤ than 0.05 and highly significant when it is ≤ than 0.01. results from the total of 150 patients involved in the current study, the number of male patients was 115 (76.67%) whereas the number of female patients was 35 (23.23%). the mean age of patients was (58.17 ± 7.60 years). other sociodemographic characteristics of patients are shown in table 1. while the disease characteristics of patients are shown in table 2. the disease characteristics of patients are shown in table 2 table 1. sociodemographic characteristics of copd patients enrolled in the current study characteristic result characteristic result age (years) smoking mean ±sd 58.17 ±7.62 yes, n (%) 91 (60.7 %) gender no, n (%) 59 (39.3 %) male, n (%) 115 (76.7 %) alcohol female, n (%) 35 (23.3 %) yes, n (%) 0 (0.0 %) bmi (kg/m2) no, n (%) 150 (100.0 %) mean ±sd 25.71 ±4.90 have children social status yes, n (%) 142 (94.7 %) single, n (%) 6 (4.0 %) no, n (%) 8 (5.3 %) married, n (%) 144 (96.0 %) profession education level employee, n (%) 35 (23.3 %) illiterate, n (%) 35 (23.3 %) student, n (%) 6 (4.0 %) primary, n (%) 74 (49.3 %) retired, n (%) 29 (19.3 %) secondary, n (%) 29 (19.3 %) self-employed, n (%) 39 (26.0 %) university, n (%) 12 (8.0 %) no job, n (%) 18 (12.0 %) residency housewife, n (%) 23 (15.3 %) urban, n (%) 82 (54.7 %) rural, n (%) 68 (45.3 %) n: number of cases; sd: standard deviation; bmi: body mass index table 2. disease characteristics of copd patients enrolled in the current study characteristic result characteristic result disease duration (years) fev1/fvc mean ±sd 4.09 ±2.51 mean ±sd 97.80 ±9.85 range (min.-max.) 1 -13 range (min.-max.) 73.00 -129.91 emergency admission fef50 median (iqr) 1 (1.0) mean ±sd 45.61 ±16.65 range (min.-max.) 0 -6 range (min.-max.) 19.23 -153.77 hospital admission gold stage median (iqr) 1 (0.25) mild intermitted, n (%) 0 (0.0 %) range (min.-max.) 0 -3 mild persistent, n (%) 0 (0.0 %) fev1 moderate persistent, n (%) 0 (0.0 %) mean ±sd 64.80 ±7.60 gold 2, n (%) 139 (92.7 %) range (min.-max.) 41.28 -79.47 gold 3, n (%) 11 (7.3 %) fvc treatment mean ±sd 67.61 ±11.01 protocol 1 26 (17.3 %) range (min.-max.) 31.55 -97.26 protocol 2 118 (78.7 %) protocol 3 6 (4.0 %) “n: number of cases; hs: highly significant; ns: not significant; sd: standard deviation; iqr: inter-quartile range; fev1: forced expiratory volume in the first second; fvc: forced vital capacity; pef: peak expiratory flow; fef50%: forced expiratory flow; n: number of cases; sd: standard deviation; protocol 1: as required short acting β2 agonist; protocol 2: long acting bronchodilator + inhaled steroid + (as required short acting β2 agonist); protocol 3: long acting bronchodilator + inhaled steroid + oral theophylline + oral steroid +(as required short acting β2 agonist) ”. iraqi j pharm sci, vol.29(2) 2020 quality of life in chronic obstructive pulmonary disease patients 172 results of hrqol parameters of copd patients enrolled in the current study are shown in table 3. table 3. assessment of health-related quality of life of copd patients enrolled in this study. characteristic results symptom score (mean ±sd) 48.65 ±7.17 activity score (mean ±sd) 62.39 ±5.81 impact score (mean ±sd) 42.83 ±7.90 total score (mean ±sd) 49.58 ±4.82 the mean symptoms score was 48.65 ±7.17, the mean activity score was 62.39 ±5.81, the mean impact score was 42.83 ±7.90 and the total score 49.58 ±4.82. multivariate regression analysis to find the predictors of hrqol is shown in table 4. symptom score is predicted by disease duration (negatively) and hospital admission (positively), activity score and impact score cannot be predicted by any of independent variables and total score is predicted by fev1(negatively) and hospital admission (positively). table 4. multivariate regression analysis to find the predictors of quality of life score in copd patients enrolled in this study characteristic symptoms scores activity scores impact scores total scores r p r p r p r p gender 0.032 0.726 -0.025 0.801 -0.030 0.761 0.017 0.851 age -0.049 0.631 -0.132 0.234 -0.178 0.102 -0.199 0.055 disease duration -0.217 0.025* 0.038 0.714 0.170 0.096 0.136 0.159 social status 0.069 0.455 -0.14 0.161 0.070 0.477 0.036 0.694 education level 0.152 0.090 -0.118 0.220 0.168 0.076 0.069 0.439 residency 0.124 0.148 -0.015 0.872 0.118 0.197 0.109 0.208 smoking -0.014 0.882 0.049 0.637 -0.088 0.385 -0.113 0.243 alcohol intake ---------------- have child 0.133 0.146 0.069 0.484 0.057 0.562 0.131 0.158 emergency admission -0.005 0.960 -0.066 0.532 -0.048 0.645 -0.053 0.585 hospital admission 0.264 0.028* 0.062 0.522 0.042 0.663 0.120 0.035* profession 0.190 0.089 -0.084 0.391 0.119 0.220 0.163 0.076 bmi -0.029 0.731 0.074 0.410 -0.005 0.954 -0.009 0.917 fev1 -0.045 0.803 0.156 0.424 -0.150 0.433 -0.198 0.027* fvc 0.316 0.136 0.116 0.613 0.155 0.489 0.309 0.148 fev1/fvc -0.002 0.989 0.202 0.182 0.049 0.738 0.140 0.318 fef50 -0.167 0.156 -0.034 0.791 0.034 0.788 -0.033 0.781 gold stage 0.159 0.150 0.145 0.225 -0.046 0.694 0.022 0.844 treatment protocol -0.061 0.469 0.031 0.730 -0.130 0.144 -0.253 0.003 2adjusted r 0.218 0.107 0.131 0.236 50%: fef: forced vital capacity; fvc: forced expiratory volume first second; fev1: body mass index; bmi“ forced expiratory flow; *: significant at p ≤ 0.05; **: highly significant at p ≤ 0.01”. discussion the copd's burden is increasing worldwide (19) , and it is one of the most common causes of death in most countries (20). the mean age of copd patients participating in the current study was (58.17 ±7.62 years). copd is more likely to increase with age and an old age represents a risk factor for copd (21). in the present study, (76.7 %) of the patients were males. copd is considered a male dominant disease. the higher prevalence rate in male gender may be due to higher smoking rate among men (22). in addition, occupational exposures are more frequent among men (23). in the current study, (60.7 %) of patients were smokers confirming the fact that smoking is a risk factor for copd across the world (24). the hrqol of copd patients is affected by many factors. however, the extent of effect of each factor is difficult to be predicted since many different questionnaires are used (7). many general questionnaires have been used to measure hrqol in both asthma and copd, however, using diseasespecific questionnaires may be more representative of the disease state than general questionnaires that do not focus on any specific disease (13). iraqi j pharm sci, vol.29(2) 2020 quality of life in chronic obstructive pulmonary disease patients 173 the total sgrq score in current study (49.58 ±4.82) was lower (indicating better hrqol) compared to scores of 59, 63 in spanish, and tunisian copd patients, respectively (25, 26). the differences in copd patients’ characteristics may explain the observed difference as well as the severity of disease since the current study involved only patients in stable phase (stable condition for at least two weeks). the mean impact score (42.83 ±7.90) reported in the current study was lower than mean symptom score (48.65 ±7.17) and activity score (62.39 ±5.81) (table 3). since the impact domain reflects different aspects of disturbances in psychological and social functioning, a lower score indicates a better psychosocial functioning as well as better overall hrqol (13). among all aspects affecting copd, the activity score was the highest, indicating that dyspnea is the most distressing copd symptom affecting hrqol. the same results were reported in the previous studies (27-29). multivariate regression analysis showed that symptom score was predicted by disease duration (negatively) suggesting a better control of symptoms for those who have longer disease duration. this result is inconsistent with that reported in other studies that showed copd patients with the more impairment in their hrqol are those having longer duration disease duration (30, 31). this inconsistency may be related to the mean disease duration of patients involved in the current study (4.09 ±2.51 years) was relatively short, indicating that patients are still in the early stages of the disease and have not reached its advanced and final stages, which reduce the quality of life. in addition, symptom and total scores were predicted by hospital admission (positively). obviously, more patient hospitalizations usually indicate more disease severity. hence, it can be predicted that patients with repeated hospital admission had worse hrqol. the current study confirms the adverse effect of hospital admission on hrqol in copd patients. this is consistent with findings of earlier studies in different countries (3234). lung functions as measured by fev1 showed that the lower the fev1 of copd patients, the lower the patient’s hrqol . this association of fev1 and hrqol supports the findings from previous studies (35, 36) and was expected. with impaired lung function, the normal activity will be limited or curtailed. however; spencer et al., study reported a weak association between hrqol assessed by sgrq and lung function as measured by fev1 (37). the current study showed hrqol of copd patients involved in the current study could not be predicted by that age, gender, bmi, education level, and smoking. similar findings were reported in previous studies regarding age (38, 39), gender (40, 41), and bmi (38, 42). however, other studies reported that lower level of education was associated with a worse hrqol (31, 43). in addition, contrary to the findings of the current study, ferrer et al., study showed that both impact and symptoms scores were significantly more impaired in smokers than non-smokers (16). the present study has some limitations. since this study is a cross sectional study which involved a one-time measurement of exposure and outcome, it is difficult to derive a causal relationship compared to studies that involved pre-and post-exposure assessments. furthermore, the study included only one governorate in iraq, so results cannot be generalized. conclusion the hrqol is impaired in copd patients. increasing the number of hospital admission and decreased lung function was associated with a significant worsening in hrqol. references 1. chan s, selemidis s, bozinovski s, vlahos r. pathobiological mechanisms underlying metabolic syndrome (mets)in chronic obstructive pulmonary disease (copd): clinical significance and therapeutic strategies. 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health qual life outcomes. 2006;4(31):1-9. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 1 phytochemical and pharmacological study of valepotriates in valeriana officinalis l. f.valerianeceae cultivated in iraq. zeina z. nagara *,1 and kawkab y. saour ** * department of pharmacognocy, college of pharmacy, university of baghdad, baghdad, iraq. ** department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract this study concerned with phytochemical investigation and methods of extraction and separation of active constituents from valeriana officinalis plant cultivated in iraq. due to the large number of active constituents in valeriana officinalis, it was necessary to make analytical study of its constituents to determine the chemical nature of these constituents and then determine the main classes (terpenes and iridoids) using chemical reagents specific for each class. different organic solvents like ethanol (70%) used in soxhlet apparatus and hexane, ethyl acetate and methanol were used separately to extract the main active constituents by maceration. through comparison between these solvents using thin layer chromatography (tlc), it has been found that hexane was best suited to extract most of the active constituents from the plant by maceration method. analytical study showed that terpens was separated and purified using preparative tlc and preparative hplc. identification of isolated component (valtrate) from roots of plant was obtained using hplc and phplc also depending on some material-specific constants such as infrared spectroscopy (ftir). results of analysis showed that valeriana officinalis cultivated in iraq is a good source of many constituents with different important pharmacological activities. the pharmacological study showed that the total organic plant extract has sleep induction effect when injected i.p. to experimental mice. key wards: valeriana officinalis, valepotriate, insomnia. دراسة كيميائية فعالة لنبات الناردين الطبي المسحسرع في العراق زينة زهير نَكـارا *،1 كوكة يعقوب ساعور و ** * خايؼت بغذاد، بغذاد ، انؼشاق.لسى انؼمالٍش ٔانُباحاث انطبٍت ، كهٍت انظٍذنت ، ** لسى انكًٍٍاء انظٍذالٍَت ، كهٍت انظٍذنت ،خايؼت بغذاد، بغذاد ، انؼشاق. الخالصة انؼشاق فً انًسخزسع انُباث نٓزا انفؼانت انًٕاد فظم ٔ السخخالص ٔطشائك انُباحٍت انكًٍٍائٍت نهذساست شايم بحث انذساست ْزِ حمذو انكًٍٍائٍت انطبٍؼت نخحذٌذ نًكَٕاحّ ححهٍهٍت دساست اخشاء انضشٔسي يٍ كاٌ نزا, انطبً انُاسدٌٍ َباث فً انفؼانت انًٕاد نكثشة َٔظشا . انكًٍٍائٍت انكٕاشف باسخخذاو( االساسٍت ٔانزٌٕث ٔانكالٌكٕسٍذاث انخشبٍُاث ) انفؼانت انًشكباث أطُاف ححذٌذ ثى ٔيٍ انًشكباث نٓزِ soxhletنالسخخالص بطشٌمت )( اٌثإَل% 70) االثٍهً انكحٕل ٔيُٓا انؼضٌٕت انًزٌباث يخخهف اسخخذاو حى رنك ٔبؼذ.طُف بكم انخاطت بطشٌمت انًزٌباث بٍٍ انًماسَت خالل ٔيٍ انفؼانت بطشٌمت انُمغ انباسد انًشكباث ٔانٓكساٌ ٔ خالث االثٍم ٔانكحٕل انًثٍهً السخخالص ( انُمغ بطشٌمت انُباث يٍ انفؼانت انًٕاد يٍ اكبشػذد السخخالص االَسب ْٕ اسخخذاو انٓكساٌ اٌ ٔخذَا انشلٍمت انطبمت كشٔياحٕغشافٍا انخٍشبٍُاث( يدًٕػت) كالٌكٕساٌذٌت يشكباث ٔخٕد ػٍ انشلٍمت انطبمت كشٔياحٕغشالٍا بطشٌمت انًسخخهض فحض َخائح اظٓشث انباسد. حشخٍض حى ثى انسائهت. االداء ػانٍت اػذادي كشٔياحٕغشافٍا يغ انشلٍمت انطبمت اػذادي كشٔياحٕغشافٍا باسخخذاو ٔحُمٍخٓا فظهٓا حى حٍث يغ انسائهت االداء كشٔياحٕغشافٍاػانٍت ٔيُٓا انحذٌثت انشلٍمت انطبمت انكشٔياحٕغشافٍا طشائك باسخخذاو انُباث خزٔس يٍ انًفظٕنت انًشكباث َخائح ٔبخحهٍم انحًشاء. ححج االشؼت يطٍاف يثم يادة بكم انخاطت انثٕابج بؼض ٔدساست, انسائهت االداء ػانٍت اػذادي كشٔياحٕغشافٍا يٍ نهؼذٌذ خٍذ ٔيظذس غًُ ًَٕرج ٌؼذ انؼشاق فً انًسخزسع انطبً انُاسدٌٍ َباث اٌ انمٕل ًٌكُُا ػهٍٓا حظهُا انخً انُٕػً انخمٍٍى حمٍ خالل يٍ اثباحّ ٔانًساػذة ػهى انُٕو حى األسق ػالج فً ٔفؼانٍخت انُباث نخاثٍش ٔبانُسبت انًؼشٔفت. انؼالخٍت انخاثٍشاث راث انًشكباث .ػهٍٓا حاثٍشِ ٔيشالبت انًخخبشٌت نهفٍشاٌ( انظفك) انبٍشٌخٌٕ داخم انحمٍ بطشٌمت انُباحً انًسخخهض العصبيةالكلمات المفحاحية : الناردين الطبي ، فاليبوجرايث ، عالج introduction the plant valeriana officinalis l. from valerianaceae family, figure (1a) has been used as a medicinal herb since at least the time of ancient greece and rome. its phytotherapeutical properties were described by hippocrates as sedative and anti-anxiety (1) .the part of the plant used medicinally is the root or rhizome figure (1b). the rhizome is light grayish brown, about the size of a finger joint, bearing many rootlets. the fresh root has no odor, while the dried root smells distinctly unpleasant, due to isovaleric acid. the plant itself is 50 to 150 cm tall with pinnate leaves and white or pink hermaphroditic flowers with three stamens; the stem is upright and without branches (2) . 1 corresponding author e-mail: zana20042002@yahoo.com received: 13/9/ 2014 accepted: 14/12/2014 iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 2 valerianaceae a family of 13 genera and about 360 species. genera include valeriana (over 200 spp.), valerianella (80 spp.), centranthus (12 spp.) and patrina (20 spp.). (3) the roots of members of the valeriana (valerian) species contain valepotriates, which have alkylting properties. valtrate/ isovaltrate and dihydrovaltrate are mutagenic in bacterial test systems in the presence of a metabolic activator, and their degradation products baldrinal (from valtrate) and homobaldrinal (from isovaltrate) are mutagenic even without metabolic activation. these latter compounds also have direct genotoxic effects. as far as is known, decomposition of dihydrovaltrate does not yield baldrinals. a freshly prepared tincture contains 11% of the valepotriates originally found in the root material. storage at room temperature rapidly reduces this to 3.7% after 1 week and 0% after 3 weeks. in view of this rapid degradation, it is not surprising that commercially available tincture samples yield baldrinals. there is substantial variation in the chemical constituents in plants from different sources and growing conditions, processing methods and storage conditions but the differences are small (4) . the importance of photoregulation in germination processes was studied by berbec; he confirmed that, while the light increases the germination percent, it has only a slight effect on the length of period needed for the appearance of the first germ. the effectiveness of light is dependent on temperature conditions. the effectiveness of light proved to be an optimum one at 25 °c, when the germination power (measured on 7th day) was higher by 20 % and the number of seeds which had germinated by the end of experiment was greater by 10—11 percent, compared with dark control (5) . valerian root contains bicyclic monoterpenes (valepotriates (0.5% -2.0%) – notably valtrate and dihydrovaltrate), (volatile oils(0.2 – 2.8%)valeranone, valerenal, and valerenic acids), sesquiterpenes, lignans, and(alkaloids(0.05 – 0.1%) actinidine, valerianine and alpha methyl pyrryl ketone).free amino acids, such as gamma-aminobutyric acid(gaba), tyrosine, arginine, and glutamine are also present (6,7) as shown in fig(2). epoxy iridoid esters (valepotriates) discovered in 1966, were thought to be the sole active constituents, although their decomposition products, the baldrinal , homobaldrinal and other components are understood to lend therapeutic benefit (8) . valepotriates are common in valerianaceae plant family and considered to be one of the main groups responsible for the sedative activity of valeriana preparations (9) . valerian’s mechanisms of action are not completely understood. valerian interacts with neurotransmitters such as gaba (10, 11) and produces a dose – dependent release of gaba (12) . valerian also inhibits the enzymeinduced breakdown of gaba in the brain, with concomitant sedation (13) . the greek physician, dioscorides, apparently recommended valerian root to treat myriad disorders including heart palpitations, digestive problems, and urinary tract infections (14, 15) . in 1998, valerian was the 10th most popular herbal remedy sold in the united states (16) . valerian is frequently listed among the ten most widely used herbal supplements (17) . historically, valerian has been used as antispasmodic, carminative and mild analgesic (18) . figure (1) avaleriana officinalis plant figure (1) b-dried root of valeriana officinalis iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 3 h3c ch3 o o o o o h o o h3c ch3 o valtrate valerenic acid o o ch3 o h h o oh3c och3 o o h3c ch3 didrovaltrate valerenal figure (2) active constituents of valeriana officinalis. experimental plant materials the cultivated whole plant was collected from the department of the medicinal plants, college of agriculture, university of baghdad. the plant material (root) was collected during may (2013) and dried at room temperature in the shade, then pulverized by mechanical mills and weighed instruments electrical sensitive balance: sartorius/ germany, ultraviolet light: desaga heidelberg/ germany, magnetic stirrer: heidolpha / germany, rotatory evaporator: buchi rotatory evaporator, chiller: ultratemp 2000, water bath: memmert/germany, sonicater –ultrasonic cleaner: sonication of extract was carried out using (copley scientific, uk.), ftir: shimadzo ft-ir-8400s ir spectrometer, hptlc: eike reich/camag – laboratory, phplc: phplc analysis was carried out by (jasco-fc208830)/japan,hplc: shimadzo l20204806962: hplc analysis was done using column: apex (jones chromatography, hengoed, uk) (19) . experimental animals healthy adult albino male mice were obtained from the animal house of college of pharmacy, university of baghdad. the weight of mice were 25±5 gram, the animals were housed in groups of eleven per cage, with light/dark period of 12 hours. they were provided with food and water ad libitum. all experiments were conducted between 8:00 am and 2:00 pm. all animals were carefully monitored and maintained in accordance with the ethical recommendation of college of pharmacy. chemicals all chemicals used were analytical grade, supplied from warehouse chemicals of college of pharmacy/ university of baghdad and valtrate (98%) standard from jinan boss chemical industry/ china. extraction of plant method no.1 the dried and powdered roots of valeriana officinalis (25g) were extracted in a soxhlet extractor with 70% ethanol (105 ml) (70-80 c ○ ) for 4 h. the organic extract was evaporated to dryness by using rotary evaporator. the dried and powdered roots (15g) were boiled in water (250 ml) for 30 min. the decoction was evaporated to dryness in water bath. the dried samples were kept in sealed bottles under vacuum to prevent evaporation of solvents and plant fermentation. the residues were suspended iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 4 in distilled water (3 ml for aqueous extracts and 5 ml for ethanol extracts). the two valerian extracts of ethanol and aqueous residue were extracted at room temperature with dichloromethane (3 x 25 ml, 20 min each) in an ultrasonic bath and isolated by separatory funnel. the combined extracts were filtered and evaporated to dryness and kept for chromatographic analysis (20) . method no.2 twenty gram of dried powdered of root of valeriana officinalis was extracted with nhexane (30 ml) with occasional stirring overnight by using magnetic stirrer then filtered and keep in sealed bottle in refrigerator for analysis. then the drug was extracted with ethyl acetate (30 ml) with occasional stirring overnight, filtered and keep sealed in refrigerator for analysis and finally drug was extracted with methanol (30ml) with occasional stirring overnight, filtered and kept sealed in refrigerator for chromatographic analysis (21) . general phytochemical screening by chemical tests the root part of plant has been screened for alkaloids, terpens, iridoids, saponines, tannins and essential oils. avanillin –h2so4 reagent for terpens. (22) . b2, 4 dinitrophenyl hydrazine for valepotriates. chcl-acetic acid reagent & anisldehyde reagent for valerenic acid. dreagent for alkaloids (23) . eferric chloride test for tannins. ffroth test for saponin. chemical test (detection of valepotriates) about 5 ml of dichloromethane added to 0.2 g of freshly powdered root, let stand for 5 minutes, shake several times, and filtered. rinse the marc with 2 ml of methylene chloride and added the rinse to the filtrate. collected the filtrate and washings in a test tube and blow dry to remove the solvent. dissolved the residue in 0.2 ml of methanol. added 3 ml of a mixture of equal volumes of chilled acetic acid and hydrochloric acid to 0.1 ml of the methanol. shake well. if valepotriates are present, the solution will turned in a blue color within 15 minutes (24) . isolation and identification of active constituents separation of the main active constituents of valeriana officinalis l. root was carried out using preparative tlc: tlc plates, 20x20cm and 1mm thickness of silica gel gf254 developed in solvent system (hexane : ethyl acetate : acetic acid 65:35:0.5) with standard reference. the chromatogram was visualized by uv lamp (at 254 nm and 366 nm) (25) . identification of active constituents was done by: 1-matching with standard by tlc using the following mobile phases: s1 hexane: ethyl acetate: glacial acetic acid (65:35:0.5), s2= hexane: ethyl methyl ketone (80:20). 2-high performance liquid chromatography (hplc): hplc analysis was done using column: apex (jones chromatography, hengoed, uk) ods (c18, 5 mm, 4.6 mm i.d x 250 mm) pump: shimadzu (columbia, md, us). lc-10at. detector: shimadzu spd-10a, (221) nm. the injected volume was 20 ml. the eluent was methanol/water (0.5% h3po4, ph 2) 80: 20. the flow rate was 1.5 ml/min (19) . mobile phase was methanol/water (0.5% h3po4, ph 2) 80: 20. 3preparative high performance liquid chromatography (phplc): phplc analysis was carried out by (jasco-fc2088-30)/japan, injected volume was 2 ml. the eluent was methanol/water (0.5% h3po4, ph 2) 80: 20the flow rate was 10 ml/min . 4fourier transforms infrared spectroscopy (ftir) in kbr disk. 5-high performance thin layer chromatography (hptlc): the mobile phase is hexane: ethyl acetate: acetic acid (65:35:0.5), for valtrate standard, hexane & organic extract sample (24) . pharmacological study experimental test (sodium phenobarbitalinduced sleeping time test): mice were divided into three groups (11 animals each). groups were received either 200 mg/kg i.p. extract (extract-treated group), or 10 mg/kg i.p. diazepam (diazepam treated group). in addition to the control group which received vehicle (distilled water) i.p. (control group) one hour later, all groups received 50mg/kg i.p. phenobarbital, and sleep were recorded. the time elapsed between the loss of and recovery from the righting reflex was recorded for control and treated animals (26) . results the preliminary phytochemical investigation of valeriana root revealed the presence of terpens (valepotriates), iridoids, and essential oil as main constituents while alkaloids present in a very low percentage, tannins and saponine absence, show in table (1). iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 5 table (1): the results of the general screening by chemical tests. plant plant part alkaloids terpenes iridoids tannins saponines valeriana officinalis root and rhizomes + + + identification of isolated compound (valtrate) depends on 1-qualitative analysis under uv light (254 nm) revealed the largest zone at rf 0.6 due to valtrate. as shown in fig (3). 2qualitative analysis under uv light (254 nm) revealed the rf value at 0.6 due to valtrate from organic (ethanol ) extract with reference standard as shown in fig(4). 3qualitative analysis can be done by hplc (high performance liquid chromatography) analysis by comparison of retention time of analyzed sample and valtrate standard at identical chromatographic conditions. the results are shown in figure (5) and table (2). a b figure (3) thin layer chromatography for (a) hexane extract and after spraying by anisldehyde h2so4 reagent. (b) ethyl acetate extract and after spraying by anisldehyde h2so4 reagent. with valtrate standard on silica gel gf254 developing in s1 solvent system and detection by uv light at 254nm. iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 6 figure (4) thin layer chromatography for aqueous and organic extracts with valtrate standard on silica gel gf254 developing in s1 solvent system and detection by uv light at 254nm. table 2: the retention time of isolated compound compared with that of reference standard. a b c figure (5) hplc analysis of (a) organic extract (b) valtrate standard and (c) isolated valtrate compound rt of root extract rt of valtrate standard valtrate 8.461 8.579 iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 7 4ft-ir spectroscopy: many functional groups can be identified by their characteristic vibration frequencies. as shown in table (3) and fig (6). table (3 ): ir absorption bands of isolated compound (in cm -1 ). compound approximate positions of characteristic bands. valtrate 1734.06(c=o), 1573.97-1543.10 (ar.c=cstretching),1377-1365(isopropyl), , 2916 , 2850 (c-h stretching /-ch3), ,1438 , 1365 (c-h bending /-ch3) , 2955 (c-h stretching /-ch2)1469.81 (c-h bending /-ch2) a b figure (6) ir spectrum of (a) valtrate standard (b) isolated valtrate 5hptlc was carried out for further identification of main active constituents present in hexane and ethanol extracts of valeriana officinalis roots with (valtrate) in s1 solvent system. as shown in fig (7). a b figure (7) hptlc plates of plant extract with valtrtae standard developing in s1. detection under uv light at (a) 254 nm and (b) 366 nm. 6 preparative hplc typically involves working with samples at their maximal concentrations and column loading far above the linear adsorption isotherms required for analytical purposes. as shown in fig (8) and tab (4). iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 8 table (4) retention time of organic root extract and isolated compound (valtrate) compound rt of root extract rt of valtrate standard valtrate 3.933 3.892 a b figure (8) phplc for (a) organic extract and (b) valtrate standard sodium phenobarbital-induced sleeping time test: in this study one-way anova showed significant differences in the sleeping time between the treated groups. tuckey’s post hoc test revealed that there was significant differences in the sleeping time between extract treated group compared to control group. furthermore, diazepam treated group was significantly different from control group. there was no significant differences between extract and diazepam treated groups. the results of the present study showed that animals of extract treated group slept faster than animals of other groups. righting reflex test is a simple, rapid test to assess loco motor abilities in mice. it evaluates general body strength by scoring or measuring the ability of mice to return to their four paws after having been placed in a supine position or on their side (27) . these results suggest that the cultivated valeriana officinalis in iraq that been used in this study has hypnotic effect at 200 mg/kg dose of extract. as in figure (9). figure (9) the effect of extract (200mg/kg) and diazepam (10mg/kg) on phenobarbital induced sleeping time in mice. extract as well as diazepam were significantly differ from control group *p˂ 0.05.values are expressed as mean+ sem (n=11). discussion phytochemical analysis showed that ethanol 70% was used for extraction of the main active constituents (valepotriates) by soxhlet apparatus from the dried plant material in extraction method (no.1). different solvents were used with increasing polarity (hexane, ethyl acetate and methanol) in method (no.2) to iraqi j pharm sci, vol.24(1) 2015 valepotriates in valeriana officinalis l. 9 get different fractions with different active constituents. from previous analytical data hexane and ethyl acetate extracts separated and isolated more components than organic extract in s1 solvent system due to like dissolve like rule in which s1 contained hexane and ethyl acetate portions so more components separated in tlc in addition to the nature of valeriana officinalis components in which there is a balance in lipophilic and hydrophilic groups in most of its chemical structure so extraction with hexane and ethyl acetate gave more separated components because there was different solvents ( hexanenon polar and ethyl acetate –polar) while extraction with ethanol by soxhlet apparatus ,only one polar solvent(ethanol) used so less components separated in tlc .the compounds present in the root of valeriana are involved in its pharmacological action, especially valerenic acids and valepotriates, which promote the inhibition of degradation of gamma –amino butyric acid (28) . the physiological mechanisms of the action of valeriana on cns involved in the action of gaba potentiating or a direct action at the site of receptor (29) . although valepotriates were once thought to be the active ingredients, these compounds are chemically unstable: instead, their degradation products, baldrinals, are found in number of preparations, and may account for much of valerian’s sedative effect (30) . conclusion our results demonstrated that the extracts of iraqi v. officinalis roots possess significant properties due to the structural features of the active principles they contain. extracts enhance or potentiate sleep tendency to mice, but the potency differed considerably. the different constituents of organic, aqueous, hexane and ethyl acetate extracts may be related to differences in the extraction procedure and therefore in their qualitative chemical profiles but all of these extracts indicated presence of valtrate in those extracts samples. acknowledgements i would like to acknowledge dr. saged auda mohammad; head of the medicinal plants department college of agriculture, university of 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italy jpp 2009; 61: 251– 256. 21. edvaldo rodrigues de-almeida , haroudo satiro xavier , aluizio roberto da siva , thais malheiros chaves , adelmo cavalcanti , evaluation potential of valeriana roots extract and valepotriates on behavior tests in mice , international research journal 2011;164-170 . 22. j.b. harborne, phytochemical methods, chapter three1973, pp.99. 23. fabriciaspredes; analtg ruiz; joaoe carvalho; mary a foglio; ―antioxidant and invite anti proliferative activity of arctium lappa root extracts‖, complementary and alternative medicine , 2011; 10: 11861472. 24. european pharmacopoeia, valerian root, strasbourg: council of europe department for the quality of medicines. 3rd edition ,1998. 25. wagner h, bladt s. plant drug analysis. berlin: springer verlag, 1996. 26. speroni, e., minghetti, a.neuropharmacological activity of extracts from passiflora incarnate .planta med. 1988; 54:488-491. 27. christine didonato, behavioral phenotyping for neonates: righting reflex, october 10th, 2011. 28. bos , r., woerdenbag , h.j., van putten , f. m., hendriks , h., scheffer , j.j.seasonal variation of the essential oil , valerenic acid and derivatives , and valepotriates in valeriana officinalis roots and rhizomes , and the selection of plants suitable for phytomedicines . plantamed.1998; 64:143147. 29. trainer, g., khom, s., baburin, i., benedek, b., hering, s., kopp, b.modulation of gaba receptors by valeriana extracts is related to the content of valerenic acid. plantamed.2008:74:19-24. 30. thies pw. on the chromomgenic behavior of valepotriate. 5. report on the active substances of valerian. arzneimittel forschung 1969; 19: 319-22. iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges doi: https://doi.org/10.31351/vol29iss2pp88-98 88 synthesis and evaluation of β-cyclodextrin based nanosponges of 5fluorouracil using ultrasound assisted method. ihsan k. jasim,1, shaimaa n. abd alhammidand alaa a. abdulrasool   ministry of health and environment, babylon health directorate, , babylon, iraq. department of pharmaceutics, college of pharmacy, university of baghdad, baghdad, iraq. abstract 5-fluorouracil (5-fu) is mostly used in the treatment of stomach cancer. after intravenous injection of 5-fu, it is rapidly distributed and eliminated with an apparent terminal half-life of 8-20 min. it is poorly absorbed after oral administration with an extremely variable bioavailability. hence, this study has been made to synthesize 5-fu nanosponges (ns) to increase its accumulation in gastric tumors by the help of an enhanced permeability retention effect (epr) and decrease its systemic side effects. on the other hand, 5-fu is sparingly soluble in water so its dissolution can be increased by incorporation in nanosponges as nanoparticles. cd-nanosponges were prepared by crosslinking β-cd with diphenylcarbonate (dpc) using ultrasound assisted technique. 5-fu was incorporated with ns by freeze drying, and the phase solubility study, complexation efficiency (ce) entrapment efficiency were performed. also, the particle morphology was studied using sem and afm. the in-vitro release of 5-fu from the prepared nanosponges was carried out in 0.1n hcl. 5-fu nanosponges particle size was in the nano size. the optimum formula showed a particle size of (405.46±30) nm, with a polydispersity index (pdi) (0.328±0.002) and a negative zeta potential (-18.75±1.8). also the drug entrapment efficiency varied with the cd: dpc molar ratio from 15.6 % to 30%. the sem and afm showed crystalline and porous nature of the nanosponges. the in vitro drug release study of the selected formula 5-funs2 exhibited the fastest dissolution rate which is 56% in the first hr. different molar ratios of (cyclodextrin to crosslinker) (cd: dpc) has a proficient effect on complexation efficiency (ce), apparent stability constant (kst) and entrapment efficiency of 5-fu. 5-funs2 with (1:4) molar ratio showed the best result of ce, kst and entrapment efficiency. 5funs2 gave a higher release rate than the 5-fu-βcd inclusion complex and 5-fu solution. surface morphology of the prepared nanosponges by sem, afm indicate that nanosized and highly porous nanosponges was obtained. the overall results suggest that cyclodextrin nanosponges could be a promising 5-fu delivery system utilizing the suitable formula. keywords: 5-fu, nanosponges, ultrasound assisted method, βcd, dpc, sem, afm. البيتا سايكلودكستيرين باستخدام فلورويوراسيل-5 جسيمات اإلسفنجية النانوية لعقار وتقييم تحضير الصوتية فوق بمساعدة الموجات **عالء عبد الحسين عبد الرسول و ** شيماء نزار عبد الحميد ،1*،احسان خضير جاسم بابل ،العراق. ،دائرة صحة بابل ،والبيئة وزارة الصحة * بغداد،العراق. جامعة بغداد، كلية الصيدلة، فرع الصيدالنيات ،** الخالصه مع تخلص منه يتم الفي الوريد ، يتم توزيعه بسرعة و حقنهالمعدة . بعد يستخدم بشكل شائع في عالج سرطان fu-5فلورويوراسيل 5 متغير للغاية. وبالتالي ، في حيويبعد تناوله عن طريق الفم مع توفر ضعيففلورويوراسيل بشكل 5دقيقة. يتم امتصاص 20-8 عمر نصف تبلغ نفاذيةبالحتفاظ عن طريق المساعدة في تعزيز تأثير اال أورام المعدةلزيادة تراكم الدواء في fu-5 من إسفنجيةهذه الدراسة قد تم تصنيع جسيمات (epr) عن هفلورويوراسيل شحيح الذوبان في الماء. يمكن زيادة معدل ذوبان 5. أيضا ، الناتجة عن انتشار الدواء في الجسم وتقليل اآلثار الجانبية .سيكلودكستيرين البيتا-النانوإسفنجيات في هطريق تضمين عن طريق ربط باواصر تساهمية للبيتا سايكلودكسترين مع الكربونات ثنائية وية جسيمات البيتا سيكلودكستيرين اإلسفنجية النانتم تصنيع داخل الجسيمات اإلسفنجية النانوية بواسطة طريقة التجفيف بالتجميد وكذلك تم إنجاز دراسة fu-5 تم دمجالموجات فوق الصوتية. الفينول بمساعدة جهد زيتا باستخدام محلل زيتا بلس. تمت دراسة و الجزيئات حجومتم قياس . كما الدواءحجز كفاءة و (ce) كفاءة تكوين المعقدطور الذوبانية و النانوية في محلول حامض اإلسفنجيةمن الجسيمات fu-5تحرر الدواء داخل المختبر لـ قياستم و afmو semشكل الجسيمات أيًضا باستخدام .0.1 عياريبتركيزالمخفف ك يالهيدروكلور 1corresponding author e-mail: ihsan.aljanaby@yahoo.com received: 10/1 /2020 accepted: 3/ 5/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp88-98 iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges 89 ( نانومتر 30± 405.46كان في حجم النانو ، الصيغة المثلى توضح حجم الجسيمات ) fu-5النانوية لعقار اإلسفنجية حجم الجسيمات pdi(,(0.328 ± 0.002 ( (. تباينت معدل الدواء المحتجز مع النسبة المولية )1.8± 18.75-وجهد زيتا سالبةcd: dpc من )2.6± 15.6 ٪ في للصيغة المثلى . أظهرت دراسة التحرر الدوائي اإلسفنجيةالطبيعة البلورية والمثقبة للجسيمات afmو sem٪. وأظهرت 2.3± 30إلى ٪ في الساعة األولى. 56 تحررمعدل أسرع المختبر و kstو ce( لها تأثير كبير على معدل cd: dpc( )المكون للروابط التساهميةالى دكستيرين البيتا سيكلوالنسب المولية المختلفة لـ ) funs2-5حتجاز الدواء. يوفر ونسبة ا ce ،kst( يظهر أفضل نتيجة ل 4: 1)ة نسبة المولي ذو funs2-5كمية الدواء المحتجزة .الصيغة الحجم النانو توضح و sem ،afmالنانوية المحضرة بواسطة لإلسفنجةالتشكل السطحي . fu-5ومحلول(fu-βcd-5)معدل تحرير أعلى من . ونسبة الثقوب العالية .تسليم الدواء باستخدام الصيغة المناسبةلكون نظام واعد تيمكن أن لبيتا سيكلودكستيرين النتائج اإلجمالية تشير إلى أن الجسيمات النانو ل .βcd ،dpc ،sem ،afm، طريقة الموجات فوق الصوتية المساعدة ، اإلسفنجية، الجسيمات النانوية فلورويوراسيل 5 الكلمات المفتاحية : introduction although chemotherapeutic agents can reduce tumor size and cancer remission and have a high potential to destroy cancer cells, they are not organ specific and can damage proliferative cells(1). one of the major goals of cancer therapeutics is to kill cancer cells without damaging normal tissues. one way to achieve this is the use of molecularly targeted therapy combined with chemotherapy. tissue and cell distribution of cancer therapeutic drugs can be controlled by the entrapment in sub-micron level (˂1 µm) colloidal systems, in other words known as nanoparticles. some of the desirable characteristics that are needed to deliver therapeutic agents to tumor cells include the ability to overcome drug resistance at the tumor and cellular levels and ensure an appropriate distribution, biotransformation, and clearance of the drug (2). conventional chemotherapeutic agents work by destroying rapid dividing cells, which is the main property of neoplastic cells. this is why chemotherapy also damages normal healthy cells that divide rapidly such as cells in the bone marrow, macrophages, digestive tract, and hair follicles (3). nanosponges are hyper-cross-linked cyclodextrins that can be obtained with α, β and γ cyclodextrins, either alone or as mixtures containing relevant amounts of linear dextrin, cross-linked with a suitable cross-linking molar ratio, by using an active carbonyl compound, e.g., diphenyl carbonate, by ultrasound-assisted synthesis. thus, spherical nanosponges of submicron size of cyclodextrin are connected by nanochannels to form a cage-like structure. these nanosponges can be inclusion complex drug carriers(4). nanotechnology have been applied to improve drug delivery and to overcome some of the problems of drug delivery for cancer treatment (5). nanosponges are a novel class of hyper-cross linked polymer based colloidal structures consisting of solid nanoparticles with colloidal and nanosized cavities. nanosponges solubilizes poorly water soluble drugs and provides a prolong release as well as improves the drug bioavailability by modifying the pharmacokinetic parameters of active constituents (6, 7). 5-fluorouracil (5-fu) was most commonly used in the treatment of cancers of colon, breast, stomach and pancreas (8). however, like other drugs used for chemotherapy, it affects the growth of normal body cells and often causes side effects such as hair loss, fatigue, birth defects, mouth sores, liver disease, and a temporary drop in bone marrow function (9). after intravenous injection of 5-fu, it is rapidly distributed and eliminated with an apparent terminal half-life of 8-20 min with a pka of 8, and 13 , logp(-1) (10). also 5-fu is poorly absorbed after oral administration with an extremely variable bioavailability (11). the aim of the study was synthesis and evaluation of 5-fu loaded nanosponges to enhance the dissolution rate of the sparingly soluble 5-fu. also nanosponge increases its accumulation in gastric tumors by the help of an enhanced permeability retention effect (epr) and decrease its systemic side effects. as it is intended to be formulated as floating tablet for local gastric cancer therapy in the future study. materials and methods materials 5-fluorouracil, βcd and diphenyl carbonate (dpc) were obtained from hyper-chem ltd co. (hangzhou, china). all other analytical reagents were of analytical grade. methods preparation of β-cd-nanosponges using ultrasound assisted method accurate amounts of βcd and diphenyl carbonate dpc were mixed in 100ml beaker at a different molar ratio as shown in table (1). the beaker was then placed in an oil bath and heated to 90ᵒc.then the mixture was sonicated for 4 hours at 50% amplitude using ultrasound probe capable of supplying maximum power of 500 watt at 20 khz (qsonica, usa). the reaction mixture is left to cool and the product obtained is broken up roughly. numerous needle-shaped crystals of phenols can be seen on the clear surface of the beaker as shown in figure (1) and part of the phenol developed contributes to agglomerating of the product (12). iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges 90 subsequently after cooling, the product was broken up roughly by mortar and repeatedly washed with an excess amount of distilled water (dw) through filtration by the buchner funnel to remove unreacted βcd. an additional purification step consists of soxhlet extraction in ethanol, which was performed for 24 hours to remove the unreacted dpc and phenol present as by-product of the reaction. finally, the nanosponges (ns) were dried at room temperature to obtain a fine white powder (13-16). figure1. a) structural representation of reaction for preparation of ns, b) ultrasound assisted method table1. the composition and molar ratios of cd: dpc used to prepare βcd-ns*. * β-cd β-cyclodextrin, dpc dipenyl carbonate.**1 mole of β-cd =1.135 g, 1mole of dpc = 0.2142g.***ns1-ns5 plain ns preparation of 5-fu loaded nanosponges 5-fluorouracil loaded ns were prepared by freeze drying method(17). briefly, the prepared βcd nanosponges at different cd:dpc molar ratios and an excess amount of 5-fu as a powder were mixed and the resultant mixture was suspended in 30 ml distilled water. then, the mixture was sonicated for 10 min and stirred for 24 hours using a magnetic stirrer (copley, germany). the obtained aqueous suspension was centrifuged for 10 min at 2000 rpm to separate the uncomplexed drug as a deposit. the supernatant was then lyophilized by employing a lyophilizer (copley, germany) to get 5-fu loaded (18). preparation of 5-fu inclusion complexes one formula of β-cyclodextrin inclusion complex with 5-fu (5-fu–βcd) was prepared. a weighted amount of 5-fu was finely suspended in a water solution containing an equimolar amount of βcd and 5-fu (1:1). the aqueous suspension was then stirred at room temperature in the dark place for 24 h. after centrifugation (5000 rpm, 10 min),(labent, germany) then the supernatant was freeze-dried(19). production yield of the prepared nanosponges production yield: the production yield can be determined by calculating initial weight of raw materials and final weight of nanosponges obtained (20): 𝐏𝐫𝐨𝐝𝐮𝐜𝐭𝐢𝐨𝐧 𝐲𝐢𝐞𝐥𝐝 = 𝑷𝒓𝒂𝒄𝒕𝒊𝒄𝒂𝒍 𝒎𝒂𝒔𝒔 𝒐𝒇 𝒏𝒂𝒏𝒐𝒔𝒑𝒐𝒏𝒈𝒆𝒔 𝑻𝒉𝒆𝒐𝒓𝒊𝒕𝒊𝒄𝒂𝒍 𝒎𝒂𝒔𝒔 𝒐𝒇 𝒏𝒂𝒏𝒐𝒔𝒑𝒐𝒏𝒈𝒆(𝒑𝒐𝒍𝒚𝒎𝒆𝒓 +𝒄𝒓𝒐𝒔𝒔𝒍𝒊𝒏𝒌𝒆𝒓) × 𝟏𝟎𝟎 encapsulation efficiency weighed amount of 5-fu-loaded nanosponges were dispersed in dw and sonicated for 10 min, then centrifuged at 15,000 rpm for 15 min (copley, germany) double cycle after that the supernatant was withdrawn, suitably diluted with distilled water and were subjected to uv spectroscopy for measuring the absorbance of the sample at the λmax of 5-fu (266 nm). with the help of absorbance, the concentration in the supernatant was determined by plotting the absorbance value against concentration in the standard curve. furthermore, the total drug content of 5-fu was determined by dissolving a same amount of 5-fu loaded ns in methanol and sonicated for 10 min to destroy and break the complex to calculate the total amount of 5-fu present in the 5-fu loaded ns powder . the percentage encapsulation efficiency was calculated by following equation(21): % 𝐄𝐧𝐜𝐚𝐩𝐬𝐮𝐥𝐚𝐭𝐢𝐨𝐧 𝐞𝐟𝐟𝐢𝐜𝐢𝐞𝐧𝐜𝐲 = 𝑫𝒓𝒖𝒈𝒆𝒏𝒄𝒂𝒑𝒔𝒖𝒍𝒂𝒕𝒆𝒅 𝑫𝒓𝒖𝒈𝒕𝒐𝒕𝒂𝒍 ∗ 𝟏𝟎𝟎 phase solubility studies phase solubility studies were carried out according to the higuchi–connors method(22). an accurate amount of 5-fu (100 mg) was added to a series of aqueous solutions (5 ml) containing increasing concentrations of βcd-ns, from (9.5 to 12.7) mm and in βcd (1:1). the samples were stirred in the dark at room temperature for 5 days. after equilibration, the aqueous suspensions were centrifuged and the 5-fu content in the supernatant was determined by uv spectrophotometer at 266 nm. formula s codes** * βcd:dpc* * molar ratio β-cd (g) dpc (g) ns 1 1:2 2.27 0.856 ns 2 1:4 2.27 1.713 ns 3 1:6 2.27 2.57 ns 4 1:8 2.27 3.427 ns 5 1:10 2.27 4.28 iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges 91 the phase solubility diagram was constructed by plotting the total molar concentration of 5-fu against the molar concentration of βcd-ns. stability constants (kst) from the phase solubility diagram were calculated using the equation (1): 𝐊𝐬𝐭 = 𝐬𝐥𝐨𝐩𝐞 𝑺𝟎(𝟏−𝒔𝒍𝒐𝒑𝒆) (1) where, s0 represents the solubility of 5-fu in the absence of cd. the slope was determined from the initial linear part of the concentration curves of 5fu. the complexation efficiency (ce) is the concentration ratio between cyclodextrin in a complex and free cyclodextrin, and it was calculated from the phase-solubility diagrams (15). the complexation efficiency is calculated by the slope of the phase-solubility profile using equation 2, which is referred to as the complexation efficiency (ce) (23). ce = s0k1: 1 = d cd⁄ cd = slope 1 − slope since, the numerical value of ce is only dependent on the slope of the phase-solubility profile, less variation is usually observed in the ce values compared to the stability constant kst value. characterization of the prepared 5-fu loaded nanosponges particle size, polydispersity index analysis (dynamic light scattering ) and zeta potential nanosponges sizes and polydispersity index were measured by dynamic light scattering using a 90 plus particle sizer (zetaplus particle sizing, ny, software, version 5.34), the samples were suitably diluted with water prior to measurements. zeta potential measurements were also made using an additional electrode in same instruments. the mean hydrodynamic diameter (dh) and polydispersity index (pi) of the particles were calculated in intensity using the cumulant analysis after averaging the three measurements (17, 24). fourier transform-infrared spectroscopy (ft-ir) atr–ftir spectra of 5-fu, dpc, βcd, βcdns and 5-funs were recorded on a iraffinitys1 spectrum ft-ir (shimadzu , japan) in the region of 4000–650 cm-1. it was performed, using a shimadzu spectrophotometer, to confirm the formation of βcdns and understand if there are interaction between drug and ns(15). scanning electron microscopy (sem) scanning electron microscopy (tescan mira3,france) was significant for determination of surface characteristics and size of the particle. scanning electron microscope was operated at an acceleration voltage of 15 kv(25). atomic force microscopy (afm) a further in-depth morphological analysis was performed using an atomic force microscope (angstrom advanced inc. aa3000) with a scanner of 3.1 µm with three piezo electrodes for three axes x, y and z in a noncontact mode. the sample suspensions (1% w/v) were prepared in distilled water and a drop was impregnated onto aluminum sheet (2 cm×2 cm). this was allowed to dry in a hepa filter zone and the dried region was analyzed(26). in-vitro release study of 5-fu nanosponges in-vitro release study of 5-fu from 5-fuβcd inclusion complex and the selected formula of 5-funs was performed by using an accurate amount of impregnated nanosponges equivalent to 100 mg 5-fu suspended in 5 ml of 0.1n hcl solution which was placed in the dialysis membrane (cut off 12,000 da) and the samples were individually placed in dissolution vessel containing 900 ml of 0.1n hcl, maintained at 37 ± 0.5°c at 75 rpm using a paddle dissolution apparatus (usp type ii). . at various time intervals, aliquots of 5ml were withdrawn and replaced with the same volume of fresh dissolution medium to maintain the sink conditions and the withdrawn samples were analyzed by uv spectrophotometer (emclab, germany) at 266 nm(27, 28). statistical analysis the results are reported as the mean±sd and statistical significance was determined using one-way analysis of variance (anova) and student’s t-tests as appropriate. all experiments were performed in triplicate and values were expressed as the mean standard deviation sd. values of p < 0.05 were considered statistically significant(29). results and discussion production yield the practical yield of nanosponges was found to be less for lower (β-cd: dpc) molar ratio (1:2). the practical yield increased with the increase in molar ratio up to 1:8, and at higher molar ratios (1:8 1:10), the yield was found to be almost the same for both molar ratio. this may be due to saturation of the reactive functional groups at higher concentration(30).the molar ratio significantly (p˂0.05) affected the production yield of cd ns. iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges 92 table2. production yield percent of the prepared cd ns , data are expressed as mean ± sd, n =3, standard deviation (sd). formulas no.* theoretical weight (g) production yield (g)±sd production yield % ns 1 2.702 1.85±0.19 48.1 ns 2 3.13 3.18±0.3 63.9 ns 3 3.559 4±0.1 70.2 ns 4 3.987 4.72±0.2 72.7 ns 5 4.414 5.64±0.3 74.8 *ns1-ns5= plain nanosponges. phase solubility study phase solubility studies were conducted for all the prepared nanosponges and their respective nativeβ-cyclodextrin in dw. the solubility of 5-fu was found to be about (0.02 m) in the absence of βcyclodextrins nanosponges. also, the molar concentration of ns was determined by calculated the molecular weight of ns according to the chemical formula that was mentioned in figure (1). the phase solubility diagram revealed that the solubility of 5-fu increased linearly as a function of increasing cyclodextrin nanosponges concentration indicating the phase solubility profile obtained was an “a-type” diagram, according to the higuchi and connors classification(22) . cyclodextrin-based nanosponges showed superior complexing ability than natural cyclodextrins towards many molecules. the parent βcd complex shows lower complexation efficiency(ce) and apparent stability constant (kst) (0.22 ) and (10±0.5 m-1) respectively ,as shown in table (3).these values were lower than that obtained by βcd nanosponges. this gave significant (p˂0.05) differences of ce and kst values between the prepared cdns and the parent cd. this is due to the carbonate linkage which was added to the primary hydroxyl groups of the parent βcd unit. thus, the drug molecules could be included inside the nanocavities of βcd and due to the cross-linking further interactions of the guest molecules with more βcd units might be thought. moreover, the presence of the cross-linked network might also form nanochannels in the ns structure for the polymer mesh(16). figures (2a) and (2b) shows the phase solubility diagram of 5-fu with the prepared ns and βcd, respectively. the slopes of the curves of complexes were lower than one, demonstrating the formation of 1:1 inclusion complex. figure 2.phase solubility diagram of 5-fu included in ; a) ns, b)βcd table3. different parameters of phase-solubility studies of 5-fu with the prepared ns and cd in distilled water, at 25 °c. formulas codes phase solubility study* slope (m1) intercept sₒ (m) (5-fu solubility) r² kst (m-1) mean ± sd* complexation efficiency (ce) 5-funs 1 0.3856 0.02 0.936 30.2±1.6 0.63 5-funs 2 0.6511 0.02 0.9817 89.5±3.6 1.87 5-funs 3 0.5811 0.02 0.8699 66.3±2.5 1.39 5-funs 4 0.4502 0.0199 0.9725 41.1±1.4 0.81 5-funs 5 0.4358 0.02 0.9702 37.2±3.1 0.77 5-fu-cd complex 0.18 0.02 0.987 10.7±1.2 0.22 *data are expressed as mean ± sd, n =3, standard deviation (sd). iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges 93 encapsulation efficiency among different types of nanosponges, the encapsulation efficiency of β-cdns for 5-fu was observed to be higher in 5-funs2 ( β-cdns(1:4) ) as much as 30±2.3% w/w followed by 5-funs3( βcdns(1:6) ) 25±2% and ns5 ( β-cdns(1:10) ) 22±2 % as shown in table 4. the results revealed that the degree of cross-linking affected the encapsulation efficiency of nanosponges with a significant difference (p˂0.05) between 5-funs1 and 5-funs2. it was found that at 1:2 molar ratio, the degree of crosslinking may be low, resulting in insufficient nanochannels for the guest complexation; thus 5-fu might not be encapsulated in higher amounts. particle size, polydispersity index analysis and zeta potential the dynamic light scattering (dls) related measurements were carried out after lyophilization. table 5 illustrates the particle size values of the prepared 5-fu nanosponges of cd-ns in which the smallest value was (256.3±24nm) and the largest one was (846.83±51nm). the overall sizes of ns found in the submicron range (<1μm) might be due to charge accelerated aggregation and molecular nature of relative cds, resulting in a size increment. the increased size may be due to the aggregation during the drying process (31). zeta potential predicts the long term stability of the nanosized formulations(32). zeta potential as a measure of surface charge was tested for 5-fu nanoformulations that have small particle size and lower pdi (5-funs2 and 5-funs3). the results of zeta potential obtained are presented in table 4. table 4. characterization of 5-fu nanosponges, (mean±sd) n=3 formula code cd:dpc particle size ± sd (nm)* pdi zp (mv) encapsulation efficiency 5-funs 1 1:2 545.45±23 0.492±0.01 15.6±2.6 5-funs 2 1:4 405.46±30 0.328±0.002 -18.75±1.8 30±2.3 5-funs 3 1:6 435.43±18 0.464±0.02 -16.1±1.2 25±2 5-funs 4 1:8 846.83±51 0.359±0.01 19±1.2 5-funs 5 1:10 256.3±24 1.711±0.1 22±1.7 on the basis of particles size, polydispersity index, zeta potential and encapsulation efficiency formulas 5-funs2 was chosen as the optimized formula for the preparation of nanosponges. drugexcipients compatibility studies the spectrum of 5-fu shows characteristic absorption bands in the region between 1656 and 1723 cm-1 correlated to the c=c, c=n, c=o, while the region at1247–1425 cm-1 was assigned at the vibration of the substituted pyrimidine. the bands at 470, 551, 642, 749, and 813 cm-1, as well as those between 2407 and 3100 cm-1 are due to the aromatic ring(33), as shown in figure(3). figure 3. ftir of 5-fu iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges 94 the appearance of the new peak of the carbonyl (c=o) group at 1751 cm-1 in ns spectra confirmed the successful cross-linking of relative βcds by dpc in various ratios(31). the 5-fu characteristic peaks were broadened or shifted in the formulations suggesting definite interactions between 5-fu and ns(16). the peaks correlated to the aromatic ring for the drug alone are weakened in the spectra of 5-fu loaded ns and some bands in the region between 2407 and 3100 cm-1 correlated to the aromatic ring result disappeared. these changes suggest the formation of the inclusion complexes(34),as shown in fgure(7). figure 4. ftir of di-phenyl carbonate figure 5:ftir of -cyclodextrin iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges 95 figure 6. ftir of plain ns (1:4) figure 7. ftir of 5-funs morphology studies afm has been employed to figure molecular structure of β-cd ns in the distilled water and examine their mechanical assets. the spherical crystalline ns presented the spectacular crystal planes with ordinary height of less than 400 nm. the sem images of the plain βcd nanosponges were shown in figure 8. sem analysis revealed that nanosized particles with numerous pores on its surface. figure 8. the afm of βcd nanosponges. iraqi j pharm sci, vol.29(2) 2020 5fluorouracil nanosponges 96 figure 9. the fe-sem of βcd nanosponges in-vitro release study drug release was performed in 0.1 n hcl. the 5-fu cumulative percent release of 5-fns2 (fig. 4a) showed a burst effect at the end of first hour. this fast release (56%) of the active ingredient was a result of increase in solubilization of the drug. after the first hour, the drug was released in a controlled manner indicating encapsulation of 5-fu in the nanostructures. 5-fu release from 5-funs2 was found to be higher than 5-fu-βcd complex(1:1) as compared to 5-fu solution as mentioned previously, this is belong to the carbonate linkage which was added to the primary hydroxyl groups of the parent cd unit. thus, the drug molecules could be included inside the nanocavities of cd and due to the cross-linking further interactions of the guest molecules with more cd units might be thought. moreover, the presence of the cross-linked network might also form nanochannels in the ns structure of the polymer mesh. this peculiar structural organization might be responsible for the increased solubilization and protection capacities of ns in comparison with the parent cd (16). figure 10. 5-fu release from 5-funs2, 5-fuβcd complex and 5-fu solution in 0.1n hcl conclusion different molar ratios of (cyclodextrin to crosslinker) have a proficient effect on ce, kst and entrapment efficiency of 5-fu. 5funs2 with (1:4) molar ratio shows the best result of ce, kst and entrapment efficiency. 5-funs2 gave higher release rate than 5-fuβcd inclusion complex and 5-fu solution. surface morphology of the prepared nanosponges by sem, afm and indicated nanosized and highly porous nanosponges. the overall results suggest that cyclodextrin nanosponge could be a promising 5-fu delivery system utilizing the suitable formula. acknowledgment the authors are very thankful to the college of pharmacy, university of baghdad for providing the necessary facilities to carry out this work. references 1. nabavizadeh f, fanaei h, imani a, vahedian j, amoli fa, ghorbi j, et al. evaluation of nanocarrier targeted drug delivery of capecitabine-pamam dendrimer complex in a mice 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fattorusso r, isernia c, isidori m, malgieri g, et al. alpha-and betacyclodextrin inclusion complexes with 5fluorouracil: characterization and cytotoxic activity evaluation. molecules. 2016;21(12):1644. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ invitro: a comparative activity of imipenem against bacterial skin by using uv iraqi j pharm sci, vol.20(1) 2011 mic of itraconazole against c. albicans 33 in vitro mic of itraconazole against different isolates of candida albicans inssaf i. alshemmary * , 1 * department of physiology , college of medicine,university of anbar,anbar , iraq. abstract in vitro antifungal susceptibility test of itraconazole was carried out against 38 isolates from nails, skin, oral cavity, vagina and wounds, this study was done in ramadi teaching hospital in period from january to august 2010. according to the national committee for clinical laboratory standard (nccls ) m 27a by using the broth dilution method. inoculum size was 1-5x10 3 cfu/ ml, while final concentrations of itraconazole ranged from 0.025 – 6.4 μg / ml by using rpmi – 1640 broth media and the fungus was incubated at 35 o c. no resistant stain was recorded. mic ranged from 0.05 – 6.4 μg / ml and the mean ± sem was 0.89 ± 0.28. mic for nail isolates was 0.05 – 6.4 μg/ ml, for skin isolates it was 0.05 – 6.4 μg / ml, for oral cavity isolates it was 0.05 – 0.4 μg/ ml, for vagina isolates it was 0.1 – 6.4 μg / ml and for wound isolates it was 0.1 – 1.6 μg/ ml. mic50 was 0.2 μg / ml and mic90 was 1.6μg / ml. itraconazole acts as fungistatic more than fungicidal agent; in 25( 65.8% ) isolates it acts as fungistatic and as fungicidal in 13 (34.2 % ) isolates. key words: itraconazole, c. albicans. الخالصة candida عشنت ين فطز انًبٍضاث 83واسعت انطٍف انًضادة نهفطزٌاث. تى فحص حساسٍت دواء االتزاكناسول ين االدوٌت albicans (3 نهذا انذواء خارج جزوحين ان 5ًهبم ، ين ان 5 ،ين انتجىٌف انفًىي 01 ، ين انجهذ 01 ،عشالث ين االظافز ) (. نى تظهز انعشالث يقاويت نذواء nccls)( وبطزٌقت انتخفٍف فً انىسط انسائم حسب micانجسى بىاسطت انًثبط االدنى نهنًى ) ٌساوي mic90 ،ياٌكىكزاو/ يم 1٫2ٌساوي mic50 ،ياٌكزوكزاو/ يم 6٫4–1٫15تزاوح انًثبط االدنى نهنًى ين .لاالتزاكناسو عشنت . 08عشنت وقاتم نهفطز فً 25عًم انذواء كًثبط نهنًى فً و ياٌكىكزاو/يم 0٫6 introduction itraconazole is a broad spectrum azole antifungal agent. it is a synthetic triazole drug. (1) the antifungal activity of azole drugs results from reduction of ergosterol synthesis by inhibition of fungal cytochrome p450 enzymes. the selective toxicity of azole drugs results from their greater affinity for fungal than for human cytochrome p450 enzymes. (2) the minimum inhibitory concentration (mic) is the lowest concentration of antimicrobial agent that inhibits growth of a particular test organism. (3) candida albicans ( c. albicans ) is a classic opportunistic pathogen. (4) it is a common inhabitant of the human gastrointestinal, genitourinary tract and skin and become pathogenic causing lesions in the skin, nail and mucous membrane. c. albicans is dimorphic; in addition to yeast and pseudohyphae,it can produce true hyphae. on agar media, candida grows as oval budding yeast cells (3-6 μm in size), soft white to cream – colored colonies at 37 o c for yeast form for 13 days and 12 weeks for hyphae form at 2530 o c ( 5 ,6) .the aims of the present study were (1) to determine in vitro activity (mic) of itraconazole against different clinical isolates of c. albicans (nails, skin, oral cavities, vagina, wounds) by using broth dilution method.(2) to determine if there is resistance to itraconazole from different clinical isolates.(3) to determine whether the itraconazole act as fungistatic and / or fungicidal. materials and methods 38 isolates of c. albicans were taken from different infected sites like 8 isolates from the nails (5 females, 3 males) with the age ranged from 20 – 45 years. 10 isolates from the skin (7 females, 3 males) with the age ranged from 29 – 40 years. 10 isolates from the oral cavities (5 females, 5 males) with age ranged from 2 – 8 years. 5 isolates from vagina (5 females) with the age ranged from 23 – 38 years and 5 isolates from the wounds (2 females, 3 males) with the age ranged from 18 – 32 years. excluded all isolates were taken from patients that received antifungal drugs before this study. c. albicans was identified by growth rate, morphology of the organism and germ tubes test by incubation in serum for about 90 minutes at 37 o c, yeast cell of c.albicans will begin to form true hyphae or germ tubes. ( 7,8 ) . 1 corresponding author email : dr. al-shemmary333@yahoo.com received : 6/10/2010 accepted : 17/1/2011 mailto:razook@yahoo.com iraqi j pharm sci, vol.20(1) 2011 mic of itraconazole against c. albicans 34 antifungal susceptibility test was done by broth dilution method according to the (nccls) m 27a, a reference procedure for testing yeast form. the quality control (qc) in this study was c. albicans. mic of qc was repeated 7 times (0.2 μg / ml). according to the atcc90028, the mic of itraconazole for c. albicans was 0.25 – 2 μg / ml. itraconazole ( janssen pharmaceuticals beers, belgium) 3mg was dissolved in 1ml of dimethylsulfoxide (3000 μg /ml ), then 150 μg/ml was prepared by adding 1ml of stock (3000 μg/ml ) to 19 ml of rpmi1640 media. then 51.2 μg / ml (1ml of 150 μg /ml to 1.93 ml of rpmi– 1640 ), 25.6μg /ml (1ml of 150 μg /ml to 4.86 ml of media ) were prepared. from 12.8 μg /ml (1ml of 150 μg /ml to 10.7 ml of media), we can prepare (0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3. 2, 6.4 μg/ ml) by serial dilution method. inoculum size of fungus was 15 x 10 3 cell forming unit (cfu), broth media was rpmi -1640 (sigms, st. louis, mo.), which was prepared by picking 5 colonies from pure culture, at least 1mm in diameter, then 5 ml of sterile normal saline (nacl 0.9 %) was added, mixed and standardized to 0.5 mcfarland scale (15 x10 6 cfu/ ml) by adding normal saline to the suspension until the visually match between suspension and macfarland tube occur. after that, add 1ml of suspension to 9 ml of rpmi 1460 broth media to obtain (1-5x 10 5 cfu /ml ), by 1: 10 dilution obtained (15 x10 4 cfu/ml ) and finaly (15 x10 3 cfu/ml ) for the quality control and test organism. (9,10,11) .three quality controls were used, (control: 1) consisted of only rpmi – 1640 broth media, (control: 2) consisted of 1ml of rpmi – 1640 media and 50 μl of final inocula, (control:3) consisted of 50 μl of inocula into tubes containing solvent (dimethylsulfoxide) in rpmi– 1640 media by the same method of drug preparation.the same procedure was used for preparing inoculum size, drug concentrations to qc and test organisms. (12) . take 10 test tubes and add 1 ml of 12.8 μg /ml of drug to the tube no.10, and by serial dilution prepare 9test tubes with drug concentrations (0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 μg /ml ) then add 50 μl of inoculum to all mic tubes, incubate at 35 o c for 24 hours or 48 hours. at the end point, mic was read visually to detect whether the fungus was susceptible or resistant to itraconazole. take 0.01 ml from mic tube directly after reading and culturing on the sabourauds dextrose agar and incubate at 35c o for 24 hours or 48 hours to detect whether the drug acts as fungistatic (mic) or fungicidal (mfc) according to the number of colonies. (13,14,15) results 38 isolates from nails, skin, oral cavity, vagina and wounds were susceptible to itraconazole and no resistant to the drug. mic ranged from 0.05– 6.4 μg /ml, mic50 was 0.2 μg /ml and mic90 was 1.6 μg /ml with mean ± sem o.89 ± o.28 (table 1). mic of nail isolates were 0.05 –6.4 μg /ml, skin isolates were 0.05 – 6.4 μg /ml, oral cavity isolates were 0.05 – 0.4 μg /ml, vagina isolates were 0.1 – 6.4 μg /ml and wound isolates were 0.1 – 1.6 μg /ml (table 2). this drug acted as fungistatic (mic) in 25( 65.8 % )isolates and fungicidal ( mfc ) in 5 ( 13.2 % ) isolates as complete fungicidal and incomplete fungicidal in 8 ( 21% ) isolates. itraconazole acts as fungistatic agent in 7, 6, 7, 2, 3 isolates from nails, skin, oral cavity, vagina and wounds, respectively. also itraconazole acts as fungicidal agent (mfc ) in 1, 4, 3, 3, 2 isolates from nails, skin, oral cavity, vagina and wounds, respectively (table 2 ). table 1: mic of itraconazole against yeast form of 38 isolates of c. albicans no.of isolates inoculum size cfu/ml range of drug concentration µg/ml mic rang mean ± sem mic50 mic90 nails: (8) (1 – 8) skin: (10) (918) oral cavity: (10) (19-28) vagina: (5) (29-33) wounds: (5) ( 3438) 1-5x 10 3 1-5x 10 3 1-5x 10 3 1-5x 10 3 1-5x 10 3 0.025-6.4 0.056.4 0.890.28 0.2 1.6 iraqi j pharm sci, vol.20(1) 2011 mic of itraconazole against c. albicans 35 table 2: mic of itraconazole for 38 isolates of c. albicans. isolate number mic g/ml no. of colonies* fungistatic (m ic)* fungicidal ( m f c )* complete incomplete 1 0.05 7 0.05 2 0.2 9 0.2 3 6.4 2 6.4 4 0.2 6 0.2 5 0.2 7 0.2 6 0.1 10 0.1 7 0.05 13 0.05 8 0.2 8 0.2 9 0.4 7 0.4 10 6.4 0 6.4 11 0.8 6 0.8 12 3.2 6 3.2 13 6.4 2 6.4 14 0.8 3 0.8 15 0.2 6 0.2 16 1.6 0 1.6 17 0.2 6 0.2 18 0.05 9 0.05 19 0.05 10 0.05 20 0.05 9 0.05 21 0.2 6 0.2 22 0.2 6 0.2 23 0.2 2 0.2 24 0.2 2 0.2 25 0.4 0 0.4 26 0.05 10 0.05 27 0.05 12 0.05 28 0.05 8 0.05 29 0.1 2 0.1 30 6.4 0 6.4 31 0.2 3 0.2 32 0.1 8 0.1 33 0.8 7 0.8 34 0.8 3 0.8 35 0.8 9 0.8 36 1.6 0 1.6 37 0.2 7 0.2 38 0.1 8 0.1 25 65 . 8 % 5 13 . 2 % 8 21 % * m ic = minimum inhibitory concentration * m f c = minimum fungicidal concentration * no. of colonies = 0 (complete fungicidal) ,5 or less (incomplete fungicidal), more than 5 (fungistatitic) iraqi j pharm sci, vol.20(1) 2011 mic of itraconazole against c. albicans 36 discussion mic of itraconazole against c. albicans was 0.05 – 6.4 μg / ml. mic50 was 0.2 μg / ml which means that the mic value of the strains that appeared in the 50 % of isolates, mic90 was. 1.6 μg /ml which means the mic value of the strains that appeared in the 90 % of isolates. (16,17,18). itraconazole acts as fungistatic ( mic) in 25 isolates which means that the minimum concentration of drug inhibits growth of fungus while it acts as fungicidal (mfc ) in 13 isolates which means that the minimum concentration of drug can kill the fungus. (19,20) . the present study recorded different values of mic of itraconazole against different isolates of c. albicans because c.albicans is part of normal human flora, thus candidiasis represents opportunistic infection and many antigens have been characterized, including secreted proteases, an immunodominant enolase and heat shock proteins. also predisposing factors for candidiasis are preceding surgery, immunosuppression, intravenous catheters, prolonged administration of antimicrobial agents and burns. (8,21,22) . many studies reported different values for the mic of itraconazole against c. albicans ( 0.002-32 μg/ml ,0.03-16 μg/ml and 0.25-0.5 μg/ml ) (12,23,24) .moreover differences in the site for obtaining the isolate lead to differences in their response to the effect of itraconazol , i.e. different mic values (25-27) . in conclusion, the results of the present in vitro study suggest that itraconazole has good activity against c. albicans by using the procedures recommended by nccls. further assessment of the correlation of these mic endpoints and the efficacy of this drug in vivo should be accomplished. acknowledgments i would like to thank the staff of ramadi teaching hospital for their help in carrying out this research. references 1. richard d.howland, mary j. mycek lipencotts illustrated reviews: pharmacology, 3 rd ed. lipencott williams and wilkins, 2006; p: 408 – 409. 2. bertram g. katzung basic and clinical pharmacology, 11 th ed. mcgrow – hill lange, 2009; p: 839. 3. michael e. aulton. pharmaceutics the design and manufacture of medicines, 3 rd ed. churchill livingstone elsevier. 2007 ; p: 233. 4. richard pj. weller, john aa. hunter, jhon a. savin, and mark v. dahi. clinical dermatology, 4 th ed. blackwell publishing, 2008; p: 252. 5. jawetz, melinck and adelbergs. medical microbiology,4 th ed. mcgraw-hill, 2007; p: 643. 6. rowen jl. mucocutaneous candidiasis. semin perinatol, 2003; 27: 406. 7. richard cb. slack and john forrest peutherer. medical microbiology, 4 th ed. churchill livingstone. 1992 ; p:676. 8. kwonchung kj. and john e. bennett. medical mycology. lea and febiger, philadelphia. london,1992; p 282 – 284. 9. national committee for clinical laboratory standards. references method for broth dilution antifungal susceptibility testing for yeasts. proposed standard m – 27 p, national committee for clinical laboratory standards, 1992 ;villanova, pa. 10. national committee for clinical laboratory standards. reference method for broth dilution antifungal susceptible of yeast. approved standard. document m 27a. national committee for clinical laboratory standards, 1997; wayne, pa. 11. cuenca – estrella m. tm. diaz – guerra, e. mellado, and jl. rodriguez-tudela. influnce of glucose, inoculum size on growth kinetics and antifungal susceptibility testing of candida spp. j. clin. microbial. 2001; 39: 525 – 532. 12. francesco brachiesi, arnaldo l. colombo, deanna a. mcgouch, annette w. fothergill, and michael g. rinaldi. invitro activity of itraconazole against fluconazloe – susceptibileand resistant candida albicans isolatesfrom oral cavities of patients infected with human immunodeficiency virus. j antimicrobial agent and chemotherapy, 1994 ; 38(7) : 1530 – 1533. 13. gallagher jc, dodds ashley es, drew r, perfect jr. antifungal pharmacotherapy for invasive mould infections. expert opinion in pharmacotherapy, 2003; 4 (147). 14. ismukes we, pappas pg, sobel jd. clinical mycology. oxford university press, new york, 2003. 15. mbler je. evaluation of chromagar candida for rapid identification and susceptibility testing in a district general hospital laboratory. journal clin pathol, 2001; 54: 158 – 159. 16. akins ra.an update on antifungal targets and mechanisms of resistance in candida albicans. med mycol, 2005; 43.(285). 17. dds fc and vander h. antifungal activity of itraconazole compared with hydroxyl– itraconazole in vitr. journal of antimicrobial chemotherapy, 2000 ; 45: 371 – 373. iraqi j pharm sci, vol.20(1) 2011 mic of itraconazole against c. albicans 37 18. pappas pg. infectious diseases society of americans guidelines for treatment of candidiasis.clin infect dis,2004; 38. 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(8): 2477 – 2481. 21. xiaogang he, robert n. tiballi, lidija t. zarins, suzanne f. bradley, et al. azole resistance in oropharyngeal candida albicans strains isolates from patients infected with human immunodeficiency virus. antimicrobial agent and chemotherapy,1994;38.(10): 2495 – 2497. 22. law d. denning dw. in vitro activity of schering 56592, compared with fluconazole and itraconazole against candida spp. antimicrob agent chemother. 1996; 15-18: p 115. 23. maria do rosario r. silva, mareio r. costa, andre tb. miranda, orionalda de f. lfernandes, et al. evaluation of etest and macrodilution broth method for antifungal susceptibility testing of candida sp. strains isolated from oral cavities of aids patients. rev. inst. med. trop. s. paulo. 2002; 44 (3): 121 – 125. 24. theodore c. white, scott holleman, francis dy, laurence f. mirels, et al. resistance mechanisms in clinical isolates of candida albicans. journal of antimicrobial agents and chemotherapy. 2002: 46 (6): 1704 – 1713. 25. kwok yk, tay yk, goh cl, kamarudin a, et al. epidemiology and in vitro activity of antimycotics against candidal vaginal / skin / nail infections in singapore. int j. dermatol. 1998; 37 (2): 145 – 9. 26. sandra s. richter, rudolph p. galask, shawn a. messer, richard j. hollis, et al. antifungal susceptibilities of candida species causing vulvovaginitis and epidemiology of recurrent cases. j clin. microbiol. 2005; 43 (5): 2155 – 2162. 27. elizabeth. johnson, ana espinel – langroff, adrien szekely, hans hockey and peter troke. activity of voriconazole, itraconazole, fluconazole and amphotericin b in vitro against 1763 yeasts from 472 patients in the voriconazole phase iii clinical studies. journal of antimicrobial agents . 2008; 32.issue 6: 511 – 514. iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 9 synthesis of 5-fluorouracil derivatives as possible mutual prodrugs with meloxicam and ibuprofen for targeting cancer tissues zainab a.h. dakhel * and mohammed h. mohammed *,1 *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq. abstract in the present study, five derivatives have been designed to be synthesized as possible mutual prodrugs for 5-fluorouracil (5-fu) and non steroidal anti-inflammatory drugs (nsaids) to selectively deliver the drugs into the cancer cells. the synthesis of the target compounds were accomplished following multistep reaction procedures, the chemical reaction followed up and the purity of the products were checked by tlc. the structure of the final compounds and their intermediates were confirmed by their melting points, infrared spectroscopy and elemental microanalysis, the hydrolysis of compound iii was studied using hplc technique. according to the results mentioned above, compounds (i−v) can be good candidates as possible mutual prodrugs of 5-fu and nsaids that can selectively deliver the parent drugs into the cancer cells by the effect of enzymes that elevated in tumor tissues compared with normal tissues. key wards: anticancer, 5-fluorouracil, nsaids, prodrug. الخالصة فلىرويىراسيل( واالدويح الوضادج -5هشرقاخ لرخليقها كوقذهاخ دوائيح ثٌائيح هحرولح للـ ) خوسحذن في هذٍ الذراسح ذصوين الوزكثاخ تاذثاع طزيقح الرفاعل ذن ذحضيز هذٍ ذيح اليصال االدويح وذحزيزها تأًرقائيح في الخاليا السزطاًيح.يوالسريزلاللرهاتاخ غيز هرعذد الخطىاخ، وذن هزاقثح جويع الرفاعالخ والرأكذ هي ًقاوج الوزكثاخ تىاسطح كزوهاذىغزافيا الطثقح الزقيقح، كوا ذن هراتعح د الحوزاء، الوزكثاخ الىسطيح والوزكثاخ الٌهائيح والرأكذ هي ذحضيزها هي خالل قياس درجاخ االًصهار والرحليل الطيفي لالشعح ذح وفقآ للٌرائج الوثيٌح اعالٍ، ( تأسرخذام ذقٌيح االسرشزاب السائل عالي االداءiiiوالرحليل الذقيق للعٌاصز. وقذ ذن دراسح ذحلل الوزكة) ( واالدويح الوضادج لاللرهاتاخ فلىرويىراسيل -5هزشحاخ كوقذهاخ دوائيح ثٌائيح هحرولح للـ ) (i−vيرضح تأى الوزكثاخ) ذيح لها القذرج علً ايصال االدويح تأًرقائيح للخاليا السزطاًيح وتأليح ذحزيز ذرضوي ذأثيز االًزيواخ الوىجىدج توسرىي يوسريزالغيز اعلً في االًسجح السزطاًيح هقارًح هع االًسجح الطثيعيح. introduction 5-fu is an antimetabolite of the pyrimidine analogue type, with a broad spectrum of activity against solid tumors of the gastrointestinal tract, pancreas, ovary, liver, brain, breast, etc. (1) 5-fu has an unpredictable gastrointestinal absorption and rapid degradation. (2) nsaids are useful drugs for alleviating pain, fever and inflammation, (3) nsaids have attracted considerable attention as a new type of antitumor drug. (4, 5) tumor inhibition by nsaids may be mediated by distinct cellular processes, these processes involve the ability of nsaids to restore apoptosis, induce cell-cycle arrest and inhibit angiogenesis (6) . prodrugs are bioreversible derivatives of drug molecules that undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug. (7) strategies to improve the oral bioavailability and achieve tumor-specific targeting have been the most important developments in prodrug design during the last 5 years (8) . phosphoramidate strategy has been applied to nsaids to synthesize novel nsaid derivatives as potential prodrugs for anticancer therapy or chemopreventive applications with less toxic side effects. (9) the purpose of this study was to design and synthesize of new compounds (i, ii, iii, iv, and v) as possible mutual prodrugs for targeting cancer tissue characterized by elevated levels of certain enzymes. 1 corresponding author email : dr.mhassan666666@ yahoo.com received : 2/1/2011 accepted : 29/5/2011 iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 10 materials and methods 5-fu sodium salt was purchased from ebewe pharma (avusturya); ibuprofen and meloxicam were purchased from alsafa company (iraq); phosphorus oxychloride and 2-chloroethanol were purchased from fluka (switzerland); 4-nitrophenol was purchased from searle (england); 2-phenyl ethyl amine was purchased from riedel-dehaen (seelzehannover) germany; glutathione was purchased from sigma (usa). all chemicals were reagent grade and obtained from standard commercial sources. elemental microanalysis were performed using chn analyzer 1106 carlo erba (italy); melting points (uncorrected) were measured on thomas hoover electronic melting point apparatus; and are uncorrected; infra red spectra were recorded as kbr disks on f.t.ir spectrophotometer (college of science, university of al-mustanseriya); and hplc analyzer (college of pharmacy, university of baghdad); chiller julabovc (f30) gmbh (germany). synthesis of compound ia: (10) to a stirred solution of phosphorus oxychloride (3.88 gm, 25.31 mmol) in dry chloroform (20 ml) at −20 o c, a solution of 2phenylethyl amine (3.067 gm, 25.31 mmol) in dry chloroform (20 ml) was added drop wise, then a solution of dry triethylamine (5.12 gm, 50.6 mmol) in dry chloroform (20 ml) was added drop wise to the reaction mixture with continuous stirring at the same temperature. the reaction mixture was stirred for 3.5 hours letting it warm up gradually to 10 o c, quenched with saturated ammonium chloride solution and the chloroform layer was extracted with distilled water (2 × 20 ml), the chloroform layer was dried over anhydrous sodium sulphate and filtered and the chloroform was evaporated under vacuum to give compound ia as oily material, the percent yield is (98.60%). synthesis of compound ib: (11, 12) to a stirred solution of compound ia (1.33 gm, 5.589 mmol) in freshly distilled acetonitrile (20 ml) at −20 o c, a suspension of sodium 5-fluoro-6-oxo-1,6-dihydropyrimidin2-olate (0.85 gm, 5.589 mmol) in freshly distilled acetonitrile (20 ml) was added drop wise, then the reaction mixture was stirred over night at room temperature. the mixture was filtered and the solvent of the filtrate was evaporated under vacuum to give compound ib as oily material with percent yield (34.53%). synthesis of compound i: (11, 12) to a stirred solution of compound ib (0.64 gm, 1.93 mmol) in dry chloroform (20 ml) at −20 o c, a suspension of (4-hydroxy-2methyl-n(5-methyl -1, 3thiazoiyl) benzo1, 2 thiazine -3-carboxamide-1,1-dioxide) (0.678 gm, 1.93 mmol) in dry chloroform (20 ml) and dry pyridine (2 ml) was added drop wise, then the reaction mixture was stirred over night at room temperature. the reaction mixture was filtered and the yellow precipitate was collected and recrystalized from ethanolpetroleum ether to give pale yellow powder of compound i, with percent yield (38.14%), melting point (221 o c dec.). the ir data and chn analysis were listed in tables 1 and 2 respectively. synthesis of compound ii: (11, 12) to a stirred solution of compound ib (0.529 gm, 1.595 mmol) in dry chloroform (20 ml) at −20 o c, a suspension of sodium (s)-2(4-isobutylphenyl)propanoate (0.364 gm, 1.595 mmol) in dry chloroform (20 ml) was added drop wise, then the reaction mixture was stirred over night at room temperature. the reaction mixture extracted with distilled water (2 ×20 ml), the chloroform layer was dried with anhydrous sodium sulphate and filtered, the chloroform was evaporated under vacuum to yield an oily compound, and the oil residue was triturated several times with petroleum spirit. the obtained solid compound was recrystalized from ethanol-petroleum ether to give white crystals of compound ii. the percent yield is (35.25%), melting point 128130 o c. the ir data and chn analysis were listed in tables 1 and 2 respectively. synthesis of compound iiia: (11, 12) to a stirred solution of phosphorus oxychloride (1.903 gm, 12.413 mmol) in freshly distilled acetonitrile (20 ml) at −20 o c, a suspension of sodium 4-nitrophenolate (2 gm, 12.413 mmol) in freshly distilled acetonitrile (20 ml) was added drop wise, then the reaction mixture was stirred over night at room temperature, then the mixture was filtered and the solvent of the filtrate was evaporated under vacuum to give compound iiia as oily material, the percent yield is (95.34%). synthesis of compound iiib: (11, 12) to a stirred solution of compound iiia (2.242 gm, 8.759 mmol) in dry chloroform (20 ml) at −20 o c, a suspension of sodium (s)-2(4-isobutylphenyl)propanoate (2 gm, 8.759 mmol) in dry chloroform (20 ml) was added drop wise, then the reaction mixture was stirred over night at room temperature. the mixture washed with distilled water (2× 20 ml), the chloroform layer was dried with anhydrous sodium sulphate and filtered; the chloroform was evaporated under vacuum to give compound iiib as oily material with percent yield (68.43%). iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 11 synthesis of compound iii: (11, 12) to a stirred solution of compound iiib ( 2 gm, 4.697 mmol) in freshly distilled acetonitrile (20 ml) at −20 o c, a suspension of sodium 5-fluoro-6-oxo-1,6-dihydropyrimidin2-olate (0.714 gm, 4.697 mmol) in freshly distilled acetonitrile (20 ml) was added drop wise, then the reaction mixture was stirred over night at room temperature. the mixture was filtered and the solvent of the filtrate was evaporated under vacuum to yield an oily compound. the oily residue was triturated several times with petroleum spirit. the obtained solid compound was recrystalized from ethanol-petroleum ether to give yellow powder of compound iii with percent yield (80.07%), melting point 86-88 o c. the ir data and chn analysis were listed in tables 1 and 2 respectively. synthesis of compound iva: (11, 12) to a stirred solution of compound iiia (1.456 gm, 5.691 mmol) in dry chloroform (20 ml) at −20 o c, a suspension of (4-hydroxy-2methyl-n-(5-methyl1,3thiazoiyl)benzo1,2thiazine-3-carboxamide1,1-dioxide) (2 gm, 5.691 mmol) in dry chloroform (20 ml) and dry pyridine (2 ml) was added drop wise, then the same procedure of compound iiib was carried out to give compound iva as oily material, percent yield (38.17%). synthesis of compound iv: (11, 12) to a stirred suspension of compound iva (0.534 gm, 0.935 mmol) in freshly distilled acetonitrile (20 ml) at −20 o c, a suspension of sodium 5-fluoro-6-oxo-1,6dihydropyrimidin-2-olate (0.142 gm, 0.935 mmol) in freshly distilled acetonitrile (20 ml) was added drop wise, then the same procedure of synthesis of compound iii was carried out to give pale orange powder of compound iv. percent yield (67.88%), melting point 144146 o c. the ir data and chn analysis were listed in tables 1 and 2 respectively. synthesis of compound va: (13) a solution of 2-chloroethanol (0.949 gm, 11.867 mmol) in (5 ml) ethanol was added to a solution of glutathione potassium salt (5 gm, 11.867 mmol) in (15 ml) ethanol and the reaction mixture was heated under reflux for 2 hours, allowed to cool, then acidified with 10% hcl to ph 4. evaporation of the solvent to afford an oily residue which was triturated with diethyl ether to give white powder of compound va, percent yield (33.27%), melting point (140 o c dec.). synthesis of compound vb: (14) to a solution of compound va (1.9 gm, 5.413 mmol) dissolved in (50 ml) glacial acetic acid, a solution of 30 % hydrogen peroxide (20 ml, 0.176 mol) was added. the mixture was left at room temperature for 12 hours and then evaporated to yield an oily residue. the oily residue was triturated several times with acetone to give white powder of compound vb. percent yield (56.05%), melting point (90 o c dec.). synthesis of compound vc: (11, 12) to a stirred solution of phosphorus oxychloride (0.662 gm, 4.317 mmol) in dry chloroform (20 ml) at −20 o c, a suspension of (4-hydroxy-2-methyl-n-(5-methyl1,3thiazoiyl)benzo1,2thiazine-3-carboxamide1,1-dioxide) (1.517 gm, 4.317 mmol) in dry chloroform (20 ml) and dry pyridine (2 ml) was added drop wise, then the reaction mixture was stirred over night at room temperature. then the mixture washed with distilled water (2 × 20 ml), the chloroform layer was dried with anhydrous sodium sulphate and filtered, the chloroform was evaporated under vacuum to give compound vc as oily material. percent yield (32.28%). synthesis of compound vd: (11, 12) to a stirred solution of compound vc (0.451 gm, 0.963 mmol) in freshly distilled acetonitrile (20 ml) at −20 o c, a suspension of sodium 5-fluoro-6-oxo-1,6-dihydropyrimidin2-olate (0.146 gm, 0.963 mmol) in freshly distilled acetonitrile (20 ml) was added drop wise, then the same procedure of compound iii was carried out to give compound vd as oily material. percent yield (55.80%). synthesis of compound v: (11, 12) to a stirred solution of compound vd (0.28 gm, 0.498 mmol) in dry chloroform (20 ml) at −20 o c, a suspension of compound vb (0.190 gm, 0.498 mmol) in dry chloroform (20 ml) was added drop wise, then a solution of triethylamine (0.0504 gm, 0.498 mmol) in dry chloroform(20 ml) was added drop wise into the reaction mixture with continuous stirring at the same temperature, then the same procedure of compound ii was carried out to give brown crystals of compound v. percent yield (40.42%), melting point 124-126 o c. the ir data and chn analysis were listed in tables 1 and 2 respectively. preliminary hydrolysis of nitro containing compounds this study was achieved using zorbax extend c18 column (150mm× 4.6mm, 5µm), with mobile phase consisting of methanolphosphate buffer ph 6 (20:80 v∕v), flow rate of 1ml ∕ min, and column temperature of 30 o c. uv detection was performed at 230 nm (15) . the standard material used was paminophenol, a stock solution (10mg ∕ 10ml) in methanol-phosphate buffer ph 6 (20:80 v∕v) was prepared, take 0.1ml of stock solution iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 12 diluted to 10 ml of methanol-phosphate buffer ph 6 (20:80 v∕v)(0.917μmol, 0.1mg), to be used as such for the hydrolysis procedure.the sample used was compound iii (containing nitro phenyl group) and sodium dithionite was added to it as reducing agent, a stock solution of compound iii(10mg ∕ 10ml) in methanolphosphate buffer ph 6 (20:80 v∕v) was prepared, take 0.47ml of stock solution diluted to 10 ml of methanol-phosphate buffer ph 6 (20:80 v∕v) (0.917μmol, 0.47mg). a stock solution of sodium dithionite (10mg ∕ 10ml) in methanol-phosphate buffer ph 6 (20:80 v∕v) was prepared, take 0.17ml of stock solution diluted to 10 ml of methanol-phosphate buffer ph 6 (20:80 v∕v) (0.917μmol, 0.17mg). the mixture of diluted stock solutions of compound iii and sodium dithionite was heated on a water bath at 37 o c for 1 hour (16) , to be used as such for the hydrolysis procedure. results and discussion the synthetic pathways for the designed target compounds (i− v) are illustrated in (schemes 1, 2, and 3), while the ir data and chn analysis were listed in table 1 and 2 respectively. scheme (1): chemical synthesis of compounds i&ii. iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 13 scheme (2): chemical synthesis of compounds iii&iv. iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 14 iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 15 scheme (3): chemical synthesis of compound v. iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 16 table 1 : the ir characteristic bands of the final compounds. table 2 : the elemental microanalysis of the final compounds. compound elemental microanalysis% element calculated found i c 48.29 48.689 h 3.74 3.901 n 13.00 13.349 o 17.32 17.798 s 9.92 10.569 ii c 59.88 60.491 h 5.83 5.903 n 8.38 8.799 o 15.95 16.496 s − − iii c 53.18 53.678 h 4.46 4.590 n 8.09 8.419 o 24.64 25.090 s − − iv c 43.38 43.791 h 2.73 2.811 n 12.65 13.010 o 24.08 24.599 s 9.65 10.271 v c 39.65 39.900 h 3.77 3.890 n 12.33 12.790 o 28.17 28.667 s 10.58 11.010 compounds bands(cm -1 )and their interpretation i 2931, 2831 (c−h) for ch2 stretching, 1660 (c=o of 2 o amide), 1620 (c=c of benzene), 1346 &1163 (asymmetrical & symmetrical stretching vibration of sulfone), 1267 (p=o stretching), 1064 (single c−f stretching), 941 (p−o stretching vibration of p−o−c aryl). ii 2955 & 2870 (c−h) of ch3 stretching, 2925 & 2870 (c−h) of ch2 stretching, 1651 (c=o stretching of 2 o amide), 1631, 1492 (c=c of benzene), 1384&1365 (gem dimethyl bending), 1249 (p=o stretching), 1064 (single c−f stretching), 881 (p−o stretching vibration of p−o−c aryl). iii 2893 (gem dimethyl stretching vibration), 1681 (c=o stretching of 2 o amide), 1581&1492 (c=c of benzene), 1581&1340 (asymmetric and symmetric stretching of nitro group), 1400, 1380 (gem dimethyl bending), 1300 (p=o stretching), 1114 (single c−f stretching vibration). iv 1678 (c=o of 2 o amide), 1595 (c=c of benzene), 1554 (amide ii band), 1529&1330 (asymmetrical & symmetrical stretching vibration of nitro group), 1330&1170 (asymmetrical & symmetrical stretching of sulfone group), 1296 (p=o stretching), 1066 (single c−f stretching), 941 (p−o stretching vibration of p−o−c aryl). v 3431 (nh stretching vibration of 2 o amide), 3000-2500 (oh stretching vibration of cooh), 2983, 2922 & 2850 (c−h) of ch3 &ch2 stretching vibration, 1629 (c=o stretching of 2 o amide), 1600, 1498 (c=c of benzene), 1554 (amide ii band), 1440 (oh bending of acid), 1352 & 1182 (asymmetrical & symmetrical stretching of sulfone), 1246 (p=o stretching), 1240 (c−o stretching of acid),1066 (single c−f stretching), 923 (p−o stretching vibration of p−o−c aryl). iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 17 the proposed mechanism to account for substitution processes occurring at the phosphoryl phosphorus atom for synthesis of compounds i, ii, iii and iv and their intermediates via pentavalent intermediates.the reactions were preceded through nucleophilic substitution mechanism which involves the formation of a true pentacoordinate intermediate generally thought to have trigonal bipyramidal geometry, and constructed by apical approach of the attacking nucleophile with departure of the displaced group at the opposite apex. (17) the first step in the synthesis of compound v (synthesis of intermediate va) include the reaction of potassium salt of glutathione with 2chloroethanol to form thioether (sulfide) through nucleophilic substitution reaction. (18) the second step in the synthesis of compound v (synthesis of intermediate vb) includes oxidation of thioether with hydrogen peroxide to afford sulfone compound. (18) finally, the synthesis of intermediates vc, vd, and compound v follow the same mechanism that shown for synthesis of compound i, ii, iii, and iv. hplc analysis qualitative identifications were made by using hplc technique; this was done by comparison of retention times obtained at identical chromatographic conditions of compound iii containing sodium dithionite (the sample) and p-aminophenol (the standard). the informations obtained from hplc method of analysis reveal that retention time for p-aminophenol was 2.1 minute for its peak to appear in the chart of hplc analysis. the nitro group in compound iii was reduced to amino group by using sodium dithionite as reducing agent (in vitro) which lead to release of p-aminophenol from compound iii. this was proved by using hplc analysis in which the same retention time was appeared in the chart (2.1 minute) of compound iii which indicate that the p-aminophenol was released from compound iii as shown in figure 1. expected enzymatic hydrolysis of the compounds: the expected enzymatic hydrolysis of compounds i and ii in the body include addition of hydroxyl group to the para position of the phenyl moiety due to the effect of cytochrome p450 enzymes, (19) then tyrosinase enzyme (20) catalyze the addition of another hydroxyl group and the oxidation of these phenolic substrates into the corresponding oquinone, which rearrange into o-diol followed by hydrolysis, thereby releasing the active drug. enzymatic hydrolysis of compounds iii and iv include reduction of the nitro group that was performed using sodium dithionite as previously described (16) then rearrangement reaction, followed by hydrolysis, there by releasing the active drug. finally, the expected enzymatic hydrolysis of compound v include activation by tyrosine -7 from the gst enzyme that abstracts a proton from compound v, resulting in a β-elimination reaction, subsequently generating the active drug. (21) -a b figure 1: hplc analysis of reduced compound iii (a) and p-aminophenol standard (b). references 1. arias. j. l.: novel strategies to improve the anticancer action of 5-fluorouracil by using drug delivery systems. molecules. 2008; 13: 2340-2369. iraqi j pharm sci, vol.20(2) 2011 mutual prodrug 18 2. he. y. f., wei. w., zhang. x., li. y. h., li. s., wang. f. h., lin. x. b., li. z. m., zhang. d. s., huang. h. q., hu. b., jiang. w. q.: analysis of the dpyd gene implicated in 5-fluorouracil catabolism in chinese cancer patients. j. clin. pharm. ther. 2008; 33: 307-314. 3. wang .w. h., huang. j. q., zheng. g. f., lam. s. k., karlberg. j., wong. b. c. : non-steroidal anti-inflammatory drug use and the risk of gastric cancer: a systematic review and meta-analysis. j. natl cancer inst. 2003; 95: 1784–1791. 4. hoshino. t., tsutsumi. s., tomisato. w., hwang. h. j., tsuchiya. t., mizushima. t.: prostaglandin e2 protects gastric mucosal cells from apoptosis via ep2 and ep4 receptor activation. j. biol. chem. 2003; 278: pp.12752–12758. 5. tsujii. m., sawaoka. h, tsuji. s.: prostaglandin in human breast cancer: evidence suggesting that an elevated prostaglandin production is a marker of high metastatic potential for neoplastic cells. cell. 1998; 93: 705–716. 6. thun. m. j., henley. s. j., patrono. c.: nonsteroidal anti-inflammatory drugs as anticancer agents: mechanistic, pharmacologic, and clinical issues. j natl cancer inst. 2002; 94: 252–266. 7. rautio. j., kumpulainen. h., heimbach.t. , oliyai. r., oh. d., jrvinen. t., savolainen. j.: prodrugs: design and clinical applications. nature reviews drug discovery. 2008; 7: 255–270. 8. pei-en. h., chi-feng. h., jia-you. f.: current prodrug design for drug discovery: current pharmaceutical design. 2009; 15: 2236-2250. 9. wittine. k., benci. k., rajic. z., zorc. b., kralj. m., marjanovic. m., pavelic. k., de clercq. e., andrei. g., snoeck. r., balzarini. j., mintas. m.: the novel phosphoramidate derivatives of nsaid 3hydroxypropylamides: synthesis, cytostatic and antiviral activity evaluations: european journal of medicinal chemistry. 2009; 44: 143-151. 10. kramer. k.,u.s.patent. 4, 667, 088 (2007) 11. wadsworth. w.s., larsen. jr. s., horton. h. l.: nucleophilic substitution at phosphorus. j.org.chem.1973;38: 256. 12. misiura. k., szymanowicz. d., kusnierczyk. h., wietrzyk. j., opolski. a.: isophosphoramide mustard analogues as prodrugs for anti-cancer gene-directed enzyme-prodrug therapy (gdept). acta biochimica polonica.2002; 49: 170. 13. vogal. a.: textbook of practical organic chemistry including qualitative organic analysis (4 th ed.). langman, newyork, 1979; 585. 14. johnson. b.j., trask. e.g.: the 4(methylthio) phenyl and 4(methylsulfonyl) phenyl esters in the preparation of peptides and polypeptides. synthesis of the protected heptapeptide (a82 –a88) of bovine chymotrypsinogen a. journal of organic chemistry. 1968; 33:.4523. 15. crevar. m., ivkovic. b., vladimirov. s., kuntic. v., vujic.z.: statistical optimization of reverse phase high performance liquid chromatography for the analysis of caffeine paracetamol and its degradation product p-aminophenol. acta chim. slov.2008; 55: 665. 16. vogal. a.: textbook of practical organic chemistry including qualitative organic analysis (4 th ed.). langman, newyork, 1979; pp.719-720. 17. edmundson. r. s.: phosphoric acid derivatives, in: comprehensive organic chemistry, the synthesis and reaction of organic compounds, barton.d.and ollis.w.d. 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http://jnci.oxfordjournals.org/search?author1=jia+qing+huang&sortspec=date&submit=submit http://jnci.oxfordjournals.org/search?author1=ge+fan+zheng&sortspec=date&submit=submit http://jnci.oxfordjournals.org/search?author1=shiu+kum+lam&sortspec=date&submit=submit http://jnci.oxfordjournals.org/search?author1=johan+karlberg&sortspec=date&submit=submit http://jnci.oxfordjournals.org/search?author1=benjamin+chun-yu+wong&sortspec=date&submit=submit http://www.ncbi.nlm.nih.gov/pubmed?term=%22tsutsumi%20s%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22tomisato%20w%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22tomisato%20w%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22hwang%20hj%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22tsuchiya%20t%22%5bauthor%5d http://www.ncbi.nlm.nih.gov/pubmed?term=%22mizushima%20t%22%5bauthor%5d http://www.ingentaconnect.com/content/ben/cpd;jsessionid=a1r4dm2qmttu3.alice iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 77 evaluation of trace elements in iraqi patients with rheumatoid arthritis by using atomic absorption spectrophotometer (aas) # huda m. ali* and mohammed a. al-zubaidi** ,1 * department of pharmaceutical chemistry and pharmacognosy , college of pharmacy , al-mustansiriya university, baghdad, iraq ** department of clinical laboratory sciences, college of pharmacy, almustansiriya university, baghdad, iraq abstract zinc, copper, selenium, magnesium, manganese, chromium, iron, nickel, cobalt, vanadium and germanium were determined by atomic absorption spectrophotometer (aas) in blood serum of patients with rheumatoid arthritis, (30) patients (14male and 16female) with age range (37-60) years compared with normal tensive control. the analysis of results showed that the mean value of concentration (magnesium, manganese and nickel) were significantly higher in patients with rheumatoid arthritis compared to that of healthy, while the mean levels of serum (zinc, copper, selenium, chromium, iron, cobalt and germanium) were significantly lower than controls. there were no significant changes in overall mean concentration of serum vanadium in patients with rheumatoid arthritis and control group. there were no significant variations in trace elements levels in relation to sex.our results suggest that low level of trace elements of zinc, copper, selenium, chromium, iron, cobalt and germanium , and high levels of magnesium, manganese and nickel may provide good clues to the physician about the high probability of the individual for developing of rheumatoid arthritis disease unless the imbalance of these elements in blood serum to be corrected. key words: trace elements, rheumatoid arthritis,atomic absorption spectrophotometer (aas). باستخذام ثىٌالنادرة فٍ المزضً العزاقُُن المصابُن بالتهاب المفاصل الز تقذَز العناصز مطُاف االمتصاص الذرٌ الشبُذٌ** عبذ الزضاهذي محمذ علٍ* و محمذ ،1 .، ميُح اىصُذىح ، اىداٍعح اىَسرْصشَح ، تغذاد ، اىعشاقفشع اىعقاقُش واىْثاذاخ اىطثُح * .شَح ، ميُح اىصُذىح ، اىداٍعح اىَسرْصشَح ، تغذاد ، اىعشاقفشع اىعيىً اىَخرثشَح اىسشَ ** الخالصت ح اىثاىغح فٍ وقاَح وعالج أٍشاض اىَفاصو واىرٍ ذٌ اسرخذاٍها عاىَُا عيً شنو ُذٌ قُاط اىعْاصش اىْادسج راخ االهَ ,zinc, copper, selenium, magnesium, manganese, chromium, iron, nickelعقاقُش ٍع اىفُراٍُْاخ وهٍ cobalt, vanadium and germanium تىاسطح خهاص االٍرصاص اىزسٌ اىيهثٍ وغُش اىيهثٍ, ذٌ قُاط هزٓ اىعْاصش فٍ ٍصو ( سْح, وذٌ 13-03ذرشاوذ تُِ ) اّثً( وفٍ اعَاس41 و رمش 41 ) (ٍشَط 03 دً اىَشظً اىَصاتُِ تاىرهاب اىَفاصو اىشثىٌ, ) magnesium, manganese andْادسج ٍع اىَسرىي اىطثُعٍ.أوظحد ّرائح اىرحيُو أُ ٍسرىي اىعْاصش ٍقاسّح ّرائح اىعْاصش اى nickel راخ ٍعذه ٍشذفع تشنو ٍعْىٌ ىذي اىَشظً ٍقاسّح تاألصحاء تَُْا ماُ ٍعذهzinc, copper, selenium, chromium, iron, cobalt and germanium ألصحاء. وىٌ َرٌ اىرىصو اىً فشق ظاهش ٍْخفط تشنو ٍعْىٌ عْذ ٍقاسّرها تا قذ ذعطٍ هزٓ اىعْاصش . الَىخذ هْاك اخرالف فٍ ذشمُض اىعْاصش تُِ اىزمش واالّثً.vanadiumتُِ اىَشظً واألصحاء فٍ ٍعذه ىً اىىظع دىُال واظحا ىيطثُة عِ وخىد احرَاىُح مثُشج إلصاتح األفشاد تَشض اىرهاب اىَفاصو اىشثىٌ ٍا ىٌ ذعذه هزٓ اىْسثح إ اىطثُعٍ ورىل تىاسطح اخز هزٓ اىعْاصش حسة اىحاخح. العناصز النادرة ، التهاب المفاصل الزثىٌ ،جهاس االمتصاص الذرٌ.: الكلماث المفتاحُت introduction rheumatoid arthritis had a worldwide distribution and affects all ethnic groups. rheumatoid arthritis is a chronic inflammatory, systemic disease that produces its most prominent manifestations in the diarthrodial (1,2) . typical form of the disease is symmetrical, destructive and deforming polyarthritis affecting small and large synovial joints with associated systemic disturbances, a variety of extra-particular features and the presence of circulating anti globulins antibodies (rheumatoid factor) (3) .meanwhile, trace elements are widely distributed in a variable proportion in human body and they play a vital role in growth. zinc is a part of every cell in the body and forms a part of over 300 enzymes that have functions ranging from proper action of the body hormones to cell growth. zinc deficiency can cause growth retardation (4,5) . # based on oral presentation in the eighth scientific conference of the college of pharmacy /university of baghdad held in 23-24 february 2011. 1 corresponding author email : mohammedizubaidy@yahoo.com received : 21/5/2011 accepted : 30/6/2012 iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 78 zinc is important in the maintenance of proper immune response (5) . copper is an essential part of key metalloenzymes as ceruluplasmine, cytochrome, oxidase, tyrosinase and monamine oxidase (6) . copper enters in a large number of enzymes in addition ceruloplasmin, as it will be necessary for the work of an enzyme super oxide dismutase (sod) as well as an oxidation enzyme lysyl oxidase, which is one of the necessary enzymes in the synthesis of connective tissue, it is believe that lack of this enzyme leads to decrease of copper, which leads to adverse effects in bone and connective tissue (7) . excess copper as with excess iron can cause free radical production and damage, also deficiency of copper results in poor collagen integrity with resultant blood vessel rupture (7,8) .selenium is an essential element of one of the main antioxidant enzymes in the human body, glutathione peroxidase (gsh-px). this explains its benefit in a number of “oxidative” conditions including cancer, cardiovascular disease and rheumatoid arthritis (1) , selenium is incorporated at four active site in the antioxidant enzyme glutathione peroxidase (gsh-px). this enzyme protects against free radical and oxidative damage by catalyzing the destruction of hydrogen peroxides (h2o2) and lipid peroxides, this in turn protects membrane lipids and hemoglobin against oxidation by peroxides (9,10) . magnesium is involved in at least 300 enzymatic processes; magnesium participates in a number of biochemical reactions that take place in bone. alkaline phosphatase, enzyme involved in forming a new calcium crystals, is activated by magnesium. the conversion of vitamin d to its biologically active form 1,25 dihydroxy vitamin d3, also appears to require magnesium. deficiency of magnesium can produce a syndrome of vitamin d resistance. the concentration of intracellular magnesium is very high as compared with concentration of extracellular magnesium for that reason can be attributed the increase in the output cell concentration to the damage done to the cells and then increase its concentration in the blood serum (1,11) . magnesium an important in bone structure. deficiency results in tetany and can lead to calcium deficiency. magnesium is essential to maintain both acidbase balance in the body, and healthy functioning of nerves and muscles (12) . diets that provide recommended levels of magnesium are beneficial for bone health, but further investigation on the role of magnesium in bone metabolism and osteoporosis is needed (8,13) . manganese is biochemically essential as a constituent of metalloenzymes and as an enzyme activator (14) . a deficiency of manganese concentration may affect brain health and skeletal and cartilage formation, antioxidant enzyme superoxide dismutase (sod) which prevents damage by superoxide free radicals (15,16) .chromium is one of the newer essential trace elements. have a great role in maintaining good health; chromium may have a function in the control of glucose and lipid metabolism (17) .iron carries oxygen to the cells and is necessary for the production of energy (18-19) . iron is available in both a ferrous and ferric. iron in the ferrous form is better absorbed than ferric iron. many people with iron deficiency anemia die from infection because of weak end immune systems. iron's role in maintaining immunity covers every aspect of how the systems work (20) . iron is also needed to help produce antibodies and to maintain your white blood cell count (18) .a significant amount of iron is stored as ferritine and hemosiderin. iron played a potential role in oxidative stress mediated injuries and pathologies e.g. rheumatoid arthritis (21,22) . nickel has been shown to be essential to man, is present in all tissues of the body. it is firmly attached to dna and a protein that binds to it in the blood (23) . nickel is used for increasing iron absorption, preventing iron-poor blood (anemia) and treating weak bone and bone structure, it may also be involved in iron metabolism, as it influences iron absorption from foods and may also play a role in production of red blood cell. nickel may then bind with one of the body neutral proteins, this nickel-protein complex may not be recognized by the immune system, and this may tiger signals to the body's defense mechanism to respond to the complex as if were an intruding antigen. nickel may deplete glutathione and protein-bound sulf hydryl group, resulting in the production of reactive oxygen species, such as superoxide anion, hydrogen peroxide and hydroxyl radical. other changer observed in a nickel deficient state include change in skin color, hormone imbalance and abnormal bone growth. liver function is impaired and iron metabolism is affected, resulting in poor absorption of iron. several publication review the evidence for the essentiality and proposed functions of this elements (24,25) .cobalt is essential for humans only as an integral part of vitamin b12 (cobalamin), no other function for cobalt in the body is known. deficiency of the vitamin b12 causes a megaloblastic anemia and in several cases, sub acute combined degeneration of the spinal cord (18,12) . cobalt essential for the growth and development of iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 79 healthy nervous system. deficiency of vitamin b12 and hyperplesia of the bone narrow. cobalt is one of the essential trace elements, human body need essential elements to grow. it is also necessary co-factor for making the thyroid hormone and deficiency lead to slow growth and development conditions (26) . cobalt as a co-factor in antioxidant therapy for the enzyme catalase. deficiencies are reflected in abnormal bone development (26) .vanadium biochemical function as peroxide of vanadate activated insulin receptor in animal studies (27) . vanadium is another trace mineral that we do not understand the role of very well yet. it clearly has an effect on the health of our bones. in research with goats scientists have noted skeletal deformations in the legs of vanadium a compound containing vanadium has been shown to stimulate bone cell proliferation. it was also shown to stimulate collagen (the organic part of bone that provides strength and flexibility) synthesis. vanadium compounds were shown to increase bone formation without any adverse health effect (27,28) . vanadium is necessary for bone and tooth development. high doses of vanadium improve the strength of bone and teeth in experimental (27) . it is needed for cellular metabolism and for the formation of bones. vanadium may be required for glucose, cholesterol and bone metabolism (28) . high doses of vanadium may deplete vitamin c (29) . the chemical properties of germanium are similar to those of silicon (30) . according to kidd (31) , germanium normalizes many physiological functions such as lowering high blood pressure in human. seaborn and nielsen (30) investigated whether germanium could substitute for silicon in bone formation. a most interesting characteristic of germanium is it ability to relieve a great deal of pain it does this by inhibiting the natural body enzyme that in turn inhibits production of the body endorphins (28) . germanium has the ability of stimulate immune function, supplement tissue oxygen and help inhibit rheumatoid arthritis. it has been suggested that germanium deficiency could be a contributory factor in kashin-beck disease (arthritis disease) (31) . arthritis is a disease of the immune system, commonly referred to as an (autoimmune) disorder, the membrane surrounding a joint becomes inflamed, resulting in a build up of lymphoid cells, resulting in the degeneration of bone, cartilage, ligaments and tendons. some agent, perhaps the epstein-barr virus, may tigger the initial joint inflammation, resulting in the production away results in the painful swelling and of antibodies. this immune response gone inflammation characterized by arthritis. materials and methods patients and method a shimadzu model aa6200 flame atomic absorption spectrophotometer faas and graphic furnace shimadzu flameless atomic absorption gfa6200 were used for analysis of blood serum samples, which were centrifuged through the preparation of samples then dilution the serum with deionized water for analysis. samples were taken from patients who had been admitted to baghdad teaching hospital with positive diagnosis of rheumatoid arthritis disease from (february december) 2010, the analysis of trace elements was done in ministry of science and technology labs.zinc, copper, magnesium and iron were analyzed by flame atomic absorption spectrophotometer, but selenium, manganese, chromium, nickel, cobalt, vanadium and germanium were analyzed by flameless atomic absorption spectrophotometer at their specific wavelength.standard of sample zn, cu, se mg, mn, cr, fe, ni, co, v and ge are obtained from analytical reagent using solution (aldrich, 1000µg / l) for each, and subsequent dilution is then carried out to obtain a calibration curve. all other reagent used was of analytical grade, distilled deionized water was used to ensure no leaching of any trace elements to the measured standard and samples polyethylene containers are used to maintain clean samples.serum samples were obtained from 30 patients (14 males and 16 females) their ages ranged between (37-60) years, with means ± sd (45.667±6.875). the mean duration of the disease was one year.the control group consisted of 30 healthy (14 male and 16 female) individuals with no general complications and who were receiving no medication, their ages ranged between (35-55) years, with means ± sd, ( 44.667 ± 66.874) . all statistical work and reporting of obtained data were carried out by using spss program version 10.0. difference of the means considered of significance according to the ttest at level of p<0.05 and p<0.01. result and discussion this study was performed for to compare between in apparently healthy subject with patients with rheumatoid arthritis. the role of trace metallic elements in chronic inflammatory states is of great interest because many of them are co-factors in metabolic processes involving articular tissues and immune system function (18) . however, many studies showed that there was a relationship between trace elements levels and period of iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 80 rheumatoid arthritis diseases.the role of zinc and copper in chronic inflammatory disease is of interest because they are co-factor of important enzymes involved in collagen and bone metabolism, immune functions and antioxidant protection. an excess of zinc may cause anemia or reduce bone formation. zinc also plays an important role in the catabolism of rna by regulation rnaase activity. the zinc and copper metals prevent the formation of free radicals capable of inducing mutation and have antioxidant effects (7,8,20) .results of these studies have demonstrated that both copper and zinc alteration can be explained by the active inflammatory process and that serum trace elements are measures of disease activity, and immune system function (7) .one hypothesis mentioned that a decreased zinc and increase of copper in sera of acute or chronic inflammatory processes cause an accumulation of copper and zinc in many body compartments and in the inflamed areas (7) . supporting the hypothesis that the development of inflammation induces an increase body requirement of copper and zinc. the results obtained for zinc concentrations in healthy and patients group which are shown in table (1), show high significant difference in zinc levels decreased (p<0.001). concentration of zinc is so low in patients group as compared with those in control group. the result obtained in this study are similar to those published in the literature (7,8,26) . zinc deficiency is common that effect obviously apparent in figure (2) which represented a histogram for data of table (2) means of zinc colak et al. (7) reported two effect of copper deficiency on iron metabolism, the first occurring early was an adverse effect of copper deficiency on iron absorption (or mobilization), the second was inadequate erythropoiesis, even in the presence of a abundant iron stores. serum copper level for patients and controls are shown in table (1), and figure (2) a histogram indicates decrease serum copper levels in the patients groups in comparison with controls groups in both sex.the decrease of copper level was statistically significant (p<0.001). the results of this study indicate that copper serum level decrease significantly during rheumatoid arthritis these results are in agreement with the reported (6,8) .protects cells from oxidative damage (7) . selenium has been shown to have anti (proliferative, inflammatory, viral) and immune altering effects. dietary selenium is essential for an optimum immune response, although the mechanisms of this requirement are not always fully understood (18) . figure (1) represents a histogram for data of table (1) shows the result of selenium determination in human sera. the mean±sd for control group is (102±15) serum selenium, high significant differences were found (p<0.001), decreased in all patients group compared with control group, these results proved the possible relationship between low levels of serum selenium and rheumatoid arthritis (7) .table (1) comprise the results obtained for the magnesium content in the sera of all groups involved in the present study. therefore, the difference in the mean serum magnesium concentration between the two groups is highly significant increased (p<0.001). these results are in agreement with the previous studied (8) . figure (3) shows a histogram which contains the mean values of serum magnesium concentration in control and patients groups.manganese is one of several trace elements that are necessary for bone health. one study (15) found that taking a combination of calcium, zinc, copper and manganese helped lessen spinal bone loss in a group of post menopausal women. people with arthritis tend to have low level super oxide dismutase (sod) (an oxidant that helps protect the joints from damage during inflammation). manganese is a required mineral in the metabolism of protein, fat, healthy immune also required in normal bone growth, energy production. the estimated results of serum manganese are listed in table (1), these results clearly show that high significant differences, elevated (p<0.001) in patients group with rheumatoid arthritis as compared with control group, very little information was found in the literature about this point (8) . manganese is required for the utilization of vitamin b1 and vitamin e, it is used in the formation of cartilage and synovial fluid of the joints. manganese deficiency can lead to improper bone formation and production disorder. an excess of manganese can lead to poor iron metabolism (14) .chromium is widely distributed throughout the body, infants have a higher chromium concentration than adults (1) . the basis for the suggestion that chromium may effective in preventing rheumatoid arthritis is that post menopausal women taking a chromium supplement exhibited increased plasma dehydro epiandrosterone, a precursor of estrogen which inhibits bone loss, and decreased urinary calcium and hydroxy proline excretion, which are indirect rather variable indicators of bone loss (26) . these provocative findings need to be confirmed, and the prevention of bone loss needs to be validated by the use of methods that can directly detect iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 81 changes or no changes in bone composition with chromium supplementation; chromium supplementation should view as only one of a number of speculative method that may help in maintaining healthy bones (17,28) . figure (1) shows a histogram which contains the mean values of serum chromium concentration in control and patients groups, chromium deficiency in patients with rheumatoid arthritis disease. table (1) shows the results for mean values of serum chromium concentrations of these elements, so low in patients group as compared with those in control group. the results are in agreement with the data (20) . there were no significant difference in serum trace elements level between male and female patient group according table (2) and table (3). studies show people who have both rheumatoid arthritis and anemia tend to have more severe arthritis than people without anemia. they are more likely to have serious joint symptoms; anemia is the most common problem for people with rheumatoid arthritis. studies show as many as 60% of people with rheumatoid arthritis are anemic. table 1: serum trace elements concentration in patients with rheumatoid arthritis and healthy control group in serum (ng/ml) *. trace elements studied groups no. mean(ng/ml) ± sd comparison of significance t-test p-value zn control 30 975.3 ± 170.1 30.45 0.001 patients 30 710.2 ± 200.76 cu control 30 1110 ± 250.3 20.17 0.001 patients 30 872.13 ± 146.72 se control 30 102 ± 15 6.82 0.01 patients 30 92.8 ± 12.13 mg control 30 16526.67 ± 1056 20.72 0.001 patients 30 20984.92 ± 5260 mn control 30 26.5 ± 6.25 6.45 0.01 patients 30 31.26 ± 8.14 cr control 30 46.5 ± 3 85.79 0.001 patients 30 40.2 ± 2.21 fe control 30 1150 ± 300.4 50.69 0.001 patients 30 712.33 ± 152.06 ni control 30 24.5 ± 4.7 20.12 0.001 patients 30 33.46 ± 9.88 co control 30 38.5 ± 4.3 7.87 0.001 patients 30 35.73 ± 3.28 v control 30 33 ± 1.2 0.42 0.51 patients 30 32.66 ± 2.61 ge control 30 42.1 ± 1.6 159.71 0.001 patients 30 31.73 ± 4.2 *concentration are expressed as mean± sd table 2 : mean serum levels (ng/ml) between different genders of patients * zn cu se mg mn cr male 712±220.56 878.15±151.62 93.65±12.5 22113.7±5773 32.4±8.4 40.6±2.92 female 708±180.83 866.11±143.21 91.95±10.2 19856.14±4939 30..12±7.8 39.8±3.05 *concentration are expressed as mean±sd p value >5% for all elements between male and female table 3: mean serum levels (ng/ml) between different genders of patients* fe ni co v ge male 725.82±158.4 34.77±9.23 36.65±3.57 33.48±3.13 32.81±4.5 female 698.84±146.3 32.15±8.71 34.81±2.98 31.84±2.32 30.65±4.1 *concentration are expressed as mean±sd p value >5% for all elements between male and female iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 82 0 20 40 60 80 100 120 mean serum trace elements level (ng/ml) studied groups control patients 0 200 400 600 800 1000 1200 meanserum trace elements level (ng/ml) studied groups control patients table (1) and figure (2) show a histogram which contained the mean values of serum iron concentration in control and patients groups, iron deficiency in patients with rheumatoid arthritis disease (18,28) . iron plays potential role in oxidative stress, mediated injuries and pathologies e.g. rheumatoid arthritis, decades ago it was suggested that iron may have a crucial role in progression of inflammation in rheumatoid arthritis. indeed free radical generated by iron can cause damage to lipids, proteins, carbohydrates and dna. it is this destruction process that it believe to occur in rheumatoid joint (1,18) .research showed that nickel was to be found in blood and tissue at quite consistent physiological significance. nickel is required for normal growth and reproduction in animals and presumably in human being as well it appears to have role in the modulation of the immune system. nickel plays some important role in biological system such as in enzyme activity, hormonal control also in structure or function of rna, dna and protein (24,25) .metabolism of other nutrients like calcium and vitamin b12 is also altered due to nickel deficiency. bone development, resistance to infection and immune function are some of the problems associated with nickel. table (1) figure (1) represented a histogram for mean values of serum nickel concentration in blood sera of control and patients groups.table (1) gives serum cobalt concentrations in patients with rheumatoid arthritis and control subject. attempt to measure serum cobalt levels in patients with rheumatoid arthritis disease were reported only by few investigators (18,11) .table (1) contains the results of serum vanadium concentrations in controls and patients with rheumatoid arthritis disease. as shown above these results are also included in figure (1) which represented a histogram for serum vanadium levels of control and patients groups. this study involved measurement of serum vanadium in patients described above, the results nosignificant difference decrease (p>0.05) compared with control.figure (1) represents a histogram for data of table (1) which contains all results of germanium determination in blood sera of control and patients. figure1: mean serum trace elements level (ng/ml) among the studied groups figure 2: mean serum trace elements level (ng/ml) among the studied groups se cr mn ni v co ge zn cu fe iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 83 figure 3: mean of serum trace elements level (ng/ml) among the studied groups conclusion the conclusions obtained from this study can be summarized: accurate, sensitive and reliable methods had been adopted for measurement of eleven essential and trace elements using both flame and flameless atomic absorption spectrophotometer in blood sera of normal and patients with rheumatoid arthritis.the results observed that the levels of serum zinc, copper, selenium, chromium, iron, cobalt and germanium were significantly decreased in patients with rheumatoid arthritis as compared to a control group,the results show that the level of serum magnesium, manganese and nickel were highly increased in the patients. the results of this study show no significant change in the concentrations of serum vanadium in all patients compared with normal subjects. there were no significant differences in sex related to trace elements. the alteration of trace elements in sera of patients with rheumatoid arthritis can enable us to shed more height on the role of trace elements in both physiological and pathological states. since there is a significant decrease in zinc, copper, selenium, chromium, iron, cobalt and germanium, so supplementation of these trace elements could be necessary to get a beneficial from trace elements rebalance in blood serum. we suggest that the deficiency of serum zinc, copper, selenium, chromium, iron, cobalt and germanium might be used in early diagnosis and treatments of rheumatoid arthritis. references 1. michael lb, edward pf and larry gs, "clinical chemistry” lippincott william and wilkins 2010, 403-668. 2. smith sm, donald a and webb d "complete book of vitamins and minerals" publications international, ltd 1998, 210264. 3. cerhan jr, saag kg, merlino la, mikuls tr and criswell la. "antioxidant micronutrients and risk of rheumatoid arthritis in a cohort of older women" am. j. epidiol 2003; 157(4): 345-354. 4. florianczyk b. "trace elements as constituents of antioxidative proteins", journal of pre-clinical and clinical research 2008; 2( 1): 25-27, 5. rink l and haase h. "zinc homeostasis and immunity", trends in immunology 2006; 28(1):1-4 6. ala s, shokrzadeh m, pur shoja am and saravi sss "zinc and copper plasma concentrations in rheumatoid arthritis patients from a selected population in iran" pakistan journal of biological sciences 2009; 12(14): 1041-1044. 7. colak m, bingol nk, ayhan o and avci s, "serum copper, zinc and selenium levels in rheumatoid arthritis" rhomatizma 2001; 16( 2):66-71. 8. satish kt and reshu m, “assessment of mineral status (zn, cu, mg and mn) in rheumatoid arthritis patients in chandigarh, india”, rheumatology report 2009,1(5):16-20. 9. kaplan l a, and pesce aj “clinical chemistry” 5 th edition, mosby elsevier 2008, 804-821 10. joshi yk, joshi m, singh n, benjamin j and sharma t “basics of clinical nutrition” jaypee brothers medical publishers ltd, 2 nd edition 2008, 190-202. 11. thomas md, "textbook of biochemistry", 7 th ed. john and wiley 2011, 1078-1097. iraqi j pharm sci, vol.21(2) 2012 trace elements and rheumatoid arthritis 84 12. lengman m. "safe upper levels for vitamins and minerals expert group on vitamins and minerals" 2003, 164-287. 13. nielsen fh. "trace minerals dificiencies”, by crc press llc 2002,1463-1487 14. santamaria ab. “manganese exposure, essentiality and toxicity” indian j.medzres 2003; 128: 484-500. 15. de carvalho pr, gonçalves pita mc, loureiro je, tanaka hr and ribeiro js, “manganese deficiency in bovines: connection between manganese metalloenzyme dependent in gestation and congenital defects in new born calves”, pakistan journal of nutrition 2010, 9(5): 488-503. 16. tuormaa te. “ the adverse effect of manganese deficiency on reproduction and health: a literature review”, journal of orthomolecular medicine 1996; 11( 2): 69-79. 17. jonathan cl, ” trace elements in the fetus and young infant, copper, manganese, selenium and chromium" am. j. dis.child 1981; 134: 74-81. 18. brig mn, chatterje a and shinde r, "text book medical biochemistry", 6 th ed. published by jitendar prij 2005, 178-557. 19. khanna s. “immunological and biochemical markers in oral carcinogenesis; the public health perspective”, int. j. environ. res. public health 2008; 5(5): 418-422. 20. cerhan jr, saag kg and criswell la. “antioxidant micronutrients and risk of rheumatoid arthritis in cohort of older women” am. j. epidemiol 2003; 157(4): 345-354. 21. john lb “institute of medicine, dietary reference intakes for vitamins a, k, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium and zinc”. washington dc, nutritional academy press 2001, 101-122. 22. majhi t and srivastava ak. “ iron deficiency in rheumatoid arthritic patients especially with in the middle age”, international journal of systems biology 2010; 2(1): 1-5. 23. das kk, das sn and dhundasi sa. “ nickel, its adverse health effects and oxidative stress”, indian j.med res. 2008; 128: 421-425. 24. spiewak r, pietowska j and curzytek k. “ nickel: a unique allergic from molecular structure to european legislation, epert rev. immunol. 2007; 3(6): 851-859. 25. cempel m and nikel g. ” nickel: review of its sources and environmental toxicology”, polish j. of environ. stud. 2006; 15(3): 375-382. 26. carl ab and edward ra “ tietz fundamentals of clinical chemistry”, 6 th ed., saunders an imprint of elsevier 2008, 496-507. 27. ferestein gs, larry a, cruz tf, cheng tp, banqueriso ml and boyle dl "vanadium an inhibitor of stromelysin and collagenase expression, suppresses collagen induced arthritis" j. rheumatology 2007; 34(9): 1802-1809. 28. hathcock jn, "vitamins and minerals safety", 2 nd ed. 2004, 35-49. 29. poucheter p, verma s, grynpas md and neill jh, “vanadium and diabetes”, mol. cell biochem. 2000; 208 (1-2): 167-168. 30. seaborn cd and neilson fh. "effect germanium and silicon on bone mineralization", biological trace elements research 1994; 42: 151-164. 31. kidd p m., germanium 132 (ge 132): homeolatic normalizer and immunostimulant: a review of its prevention efficacy. international clinical nutrition review 1987; 7: 11. iraqi j pharm sci, vol.28(1) 2019 silymarin microcrystals doi: https://doi.org/10.31351/vol28iss1pp1-16 1 formulation and evaluation of silymarin as microcrystals by insitu micronization technique ala'a d. noor*1 and eman b.h. al-khedairy* department of pharmaceutics,college of pharmacy, university of baghdad, baghdad, iraq. abstract silymarin (sm) is a plant extract obtained from silybum marianum( milk thistle). it is hepatoprotective drug class ii type according to biopharmaceutics classification system. it is practically insoluble with low bioavailability. micro/nanonization during crystallization, surface modification and crystal structure modification may improve the solubility and dissolution rate of poorly water-soluble drugs. the aim of this study was to enhance the water solubility and dissolution rate of sm by in-situ micronization using solvent change either by stirring or ultrasonic method. polymers like gelatin, pvp-k30, hpmc15, pulullan were used to stabilize the prepared ultra-fine crystals. effect of type and concentration of hydrophilic polymer, solvent: anti-solvent volume ratio and the effect of ultrasonic irradiation were studied. the prepared microcrystals were evaluated for their percent yield, water solubility, crystals structure by xrd, dsc, and sem. particle size and dissolution rate were also determined. silymarin microcrystals prepared by ultrasonic method and stabilized by 0.1% w/v gelatin using 1:2 solvent: anti-solvent volume ratio showed the best results with percent yield 93.4±2.7% and particle size reduction from mean diameter of 1.5µm (untreated sm) to 0.43µm with uniform morphology and enhanced solubility for about four folds with improvement in dissolution. keywords: silymarin, crystallization, in-situ micronization, solvent change method. بلورات مايكروية بتقنية التصغير اآلنيكتصييغ وتقييم السليمارين *ايمان بكر حازم الخضيري و 1،*ضياء نور االء بغداد،العراق. جامعة بغداد، فرع الصيدالنيات ،كلية الصيدلة،* الخالصه مارين هو مستخلص نباتي مستحصل عليه من عشبة سليبيوم مارينيوم )شوك الحليب(. هو دواء يقي من امراض الكبد من ضمن الصنف يالسل الصيدالنية الحيوي( والذي اليذوب في الماء عمليا, ويملك توافر حيوي منخفض بسبب انخفاض ذوبانيته. الثاني )نظام تصنيف المستحضرات النانوي أثناء التبلور وتعديل سطح وبنية البلورة قد يحسن من ذوبانية ومعدل انحالل األدوية ضعيفة الذوبان في الماء. /التصغير الميكروي ذابة ومعدل انحالل السليمارين من خالل تقنية التصغير اآلني بإستخدام تغيير المذيب إما عن طريق الهدف من هذه الدراسة هو زيادة اإل -، هيدروكسي بروبل ميثل سيليلوز30ك -التحريك أو طريقة الموجات فوق الصوتية. تم إستخدام المثبتات مثل الجالتين, بولي فينيل بيروليدون بلورات المتناهية الصغر المحضرة. حيث تم دراسة تأثير نوع وتركيز البوليمر المائي، نسبة حجم ،والبوليوالن لتحقيق اإلستقرار في ال15ي انيتها في بالمذيب:المذيب المعاكس، وتأثير اإلشعاع بالموجات فوق الصوتية. وقد تم تقييم البلورات الدقيقة المحضرة من حيث نسبة اإلنتاجية %، ذو إختبار حيود األشعة السينية، والمسح التفريقي للحرارة، والمسح المجهري اإللكتروني. كما تم إختبار حجم الجسيمات الماء، وبنيتها البلورية بواسطة ومعدل انحاللها . حجم( بإستخدام \)وزن % 0،1ان بلورات السلمارين المكروية المحضرة بواسطة طريقة الموجات فوق الصوتية والمستقرة بواسطة الجيالتين بنسبة وتخفيض حجم الجسيمات من متوسط قطر %2،7±93.4من المذيب للمذيب المعاكس أظهرت أفضل النتائج من حيث نسبة االنتاجية 2:1ة نسب مايكرون مع شكل متجانس وزيادة في الذوبانية لحوالي اربعة أضعاف مع تحسن في معدل االنحالل. 0.43مايكرون لمسحوق السلمارين الى 1.5 طريقة تغير المذيب. سليمارين، التبلور، التصغير اآلني،الكلمات المفتاحية : introduction many new drugs are poorly water-soluble, with low dissolution and bioavailability (1). one way for improving their dissolution rates is by decreasing the particle size. decreasing the particle size increases the surface area, which results in an increase in the rate of dissolution of these drugs in aqueous media such as body fluids and enhance their absorption and bioavailability(2). micro / nanonization during crystallization, surface modification and crystal structure modification may improve the dissolution rate of poorly water-soluble drugs. several studies have shown that modification of the crystalline form of the drug, by the inclusion of specific additives increases the rate of dissolution which can strongly affect the physicochemical and kinetic properties of the drug from the final product. micro/nanoparticles can be produced by several techniques such as crystallization/precipitation and solvent evaporation and by spray drying methods(3). 1corresponding author: e-mail: ala.dheia@gmail.com received: 6 / 8 /2018 accepted: 15 /9/2018 iraqi journal of pharmaceutical sciences https://doi.org/10.31351/vol28iss1pp1-16 iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 2 in situ micronization process; it is a technique in which microcrystals are prepared using a precipitation technique of a drug from solution by either ph change or by solvent change process ( using anti-solvent) in the presence of protective hydrophilic polymers(4). this technique is beneficial in these aspects as it does not require any external processing conditions except mild agitation using magnetic stirrer and the processing time is less compared to the other techniques(5). the use of the anti-solvent in crystallization reduces the solubility of a solute in the solution and generates high supersaturation that subsequently induces nucleation and simultaneous growth by condensation and coagulation.to induce rapid crystallization followed by agglomeration in case of uncontrolled growth (6). silymarin a flavonolignans extracted from the fruits and seeds of milk thistle silybum marianum (l.). (asteraceae), it is an annual herb, native to the mediterranean and north african regions; it is wildly grow throughout europe, north africa, americas and australia(7,8). silymarin (sm) is is a hepatoprotective class ii type drug according to biopharmaceutics classification system, which is practically insoluble in water, and soluble in organic solvents like methanol, ethanol, and acetone (8). it is absorbed when given orally and the peak plasma concentration is achieved in 6-8 hr. the oral absorption of silymarin is only about 23-47% resulting in low bioavailability (9).the aim of this study was to enhance the solubility of silymarin by preparing microcrystals by insitu micronization technique using solvent change process in presence of protective hydrophilic polymers. materials and methods materials silymarin (sm) purchased from hyperchemical limited,china. gelatin, polyvinylpyrrolidone (pvp-k30) from riedel de haen ag seelze, honnover, germany, hpmc15 and pullulan from hyperchemical limited,china, methanol from gcc analytical reagent, uk, all other reagents used were of analytical grade. methods determination of sm saturated solubility the solubility was measured in distilled water, by addition of an excess amount of sm powder to 10ml of distilled water in test tube and incubated in water bath shaker at 25 °c for 48 hrs. after that, the samples were centrifuged at 500 rpm for 5 min using a high-speed centrifuging machine to remove the excess insoluble sm, and then filtered through a 0.45 µm filter membrane and assayed spectrophotometrically for sm at 288nm λ max (10,11). results were recorded as the mean value of three determinations. preparation of sm microcrystals microcrystals of sm were prepared by solvent change method. in this method; organic phase was preapared by dissolving 3% of sm in methanol. aqueous phase was prepared by dissolving specific amount of polymer as stabilizing agent in distilled water (as anti-solvent). the aqueous phase was poured rapidly into the drug solution under constant stirring (400rpm) by magnetic sterrier (12) and stirring was continued for 60 min. microcrystals were formed spontaneously, collected after filtration through whatman filter paper (grade 1, 110mm diameter) by using vacuum filtration, followed by washing for three times with cold diatilled water to remove non-adsorbed excipients if any, and dried in an oven at 40°c for 1hr(13). several factors were studied to optimize the best conditions for micronization . factors affecting sm in situ micronization to select the best conditions for obtaining microcrystals with maximum yield, solubility and crystallinity the following factors were studied. effect of polymer type to select the best polymer for preparation of sm microcrystals, four formulas were prepared; mc1, mc2, mc3 and mc4 using gelatin, pvp-k30, hpmc15 and pullulan respectively as stabilizing agent with 0.1% (w/w) concentration for each as shown in table.1 effect of polymer concentration to estimate the minimum concentration of stabilizing agent (gelatin or pvp k30) required to get the maximum crystals yield and solubility, three concentrations of each stabilizing agent 0.05% (mc5 and mc7),0.1% (w/v)(mc1 and mc2) and,0.2% (w/v) (mc6 and mc8) were used respectively as shown in table.1. effect of solvent: anti-solvent ratio gelatin and pvp k 30 were selected as stabilizers to study the effect of solvent: anti-solvent ratio on the properties of the prepared sm microcrystals. table 1 shows that three solvent: anti-solvent ratios 1:2(mc9and mc11), 1:4(mc1 and mc7) and 1:6(mc10 and mc12) were used for the selection of most appropriate solvents ratio for each polymers. effect of ultrasound sonocrystallization method was used to study the effect of ultrasound irradiation on the preparation of sm microcrystals (mc9s, mc1s, mc10s) using 0.1% (w/v) gelatin as stabilizing agent with different solvent: anti-solvent ratios as shown in table.1 the properties of the resultant microcrystals are to be compared with those of the corresponding formula prepared by stirring method. by this method, the aqueous phase was poured to the organic drug solution as the previous experiments but the stirring was changed to iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 3 sonication by ultrasonic bath for 20 min. ultrasonic power used was 200 w and the frequency of ultrasound was 40 khz (14). the concentration of sm in methanol was kept constant (3%)(w/v). table (1) composition of sm microcrystals formulas. method solvent: antisolvent ratio polymer. conc. %w/v polymer organic solvent sm. conc. %w/v f. no. stirring 1:4 0.1 gelatin methanol 3 mc1 stirring 1:4 0.1 pvp-k30 methanol 3 mc2 stirring 1:4 0.1 hpmc15 methanol 3 mc3 stirring 1:4 0.1 pullulan methanol 3 mc4 stirring 1:4 0.05 gelatin methanol 3 mc5 stirring 1:4 0.2 gelatin methanol 3 mc6 stirring 1:4 0.05 pvp-k30 methanol 3 mc7 stirring 1:4 0.2 pvp-k30 methanol 3 mc8 stirring 1:2 0.1 gelatin methanol 3 mc9 stirring 1:6 0.1 gelatin methanol 3 mc10 stirring 1:2 0.05 pvp-k30 methanol 3 mc11 stirring 1:6 0.05 pvp-k30 methanol 3 mc12 sono crystallization 1:2 0.1 gelatin methanol 3 mc9 s sono crystallization 1:4 0.1 gelatin methanol 3 mc1 s sono crystallization 1:6 0.1 gelatin methanol 3 mc10 s method evaluation of the sm microcrystals determination of percent crystals yield percentage crystal yield was calculated by comparing the practical yield with respect to initial weight of drugs and polymers used in formulation of micro crystals to know about efficiency of any factors and thus it helps in the selection of appropriate micro crystallization condition. the final weights of the dried microcrystals of sm were taken and percentage crystal yield was calculated by using the formula as follows (15). 𝑷𝒆𝒓𝒄𝒆𝒏𝒕𝒂𝒈𝒆 𝒚𝒊𝒆𝒍𝒅 = 𝑨𝐜𝐭𝐮𝐚𝐥 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐬𝐢𝐥𝐲𝐦𝐚𝐫𝐢𝐧 𝐦𝐢𝐜𝐫𝐨𝐜𝐫𝐲𝐬𝐭𝐚𝐥𝐬 𝐓𝐨𝐭𝐚𝐥 𝐰𝐞𝐢𝐠𝐡𝐭 𝐨𝐟 𝐝𝐫𝐮𝐠 𝐚𝐧𝐝 𝐬𝐭𝐚𝐛𝐢𝐥𝐢𝐳𝐞𝐫 × 100 drug content a weighed quantity of the samples was dispersed in 10 ml of methanol. it was sonicated for 10 min and the samples were filtered and diluted with suitable quantity of methanol and analyzed spectrophotometrically for sm at 288nm λ max, the experiment was carried out in triplicate (16). solubility an excess amount of the resulted crystals was added to 10 ml of water in test tubes. all samples were placed in the shaking water bath, which was thermostatically controlled at 25±0.5°c, for 48 hours. after that the samples were withdrawn from the supernatant by the use of a syringe ;filtered through a 0.45-µm membrane and assayed spectrophotmetrically for sm at 288nm. results were recorded as the mean value of three determinations. (11) x-ray diffraction x-rays diffraction patterns (diffractograms) can be used to study the crystalline nature of a sample. therefore, this information was used to confirm whether the drug was in crystalline or amorphous form. the x-ray diffractograms of sm, and the selected microcrystal formulas were obtained by x-ray diffractometer (xrd-6000,shimadzu,japan) at continuous scan range between 10 80º (2θ); the operating voltage was 40 kv and 30 ma current (4) . differential scanning calorimetry (dsc) dsc can be used to determine the compatibility between the drug and polymer and also used to evaluate the crystalline state of drug especially when micronized to microparticles or nanoparticles. thermal characteristics of the samples determined by an automatic thermal analyzer system (shimadzu, dsc–60,japan). an approximately 5mg sample of sm, gelatin as a selected polymer, or selected microcrystals; were heated at a scanning rate of 10 °c/min in an aluminum pan under a nitrogen atmosphere at a temperature range of 50-300 ˚c. a similar empty pan was used as the reference (17). scanning electron microscope (sem) the morphology of pure powder sm and the selected microcrystals was observed by sem. air dried microcrystals were mounted onto metal stubs using double-sided adhesive tape, vacuum-coated iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 4 with gold and directly analyzed under sem at magnifying power of (5000×,10000×,25000×)(18). particle size a sample of sm and the selected crystals suspension were obtained after precipitation by solvent change was sonicated at 37 ˚c for 30 min and put in a 3 ml cell of abt-9000 nano laser particle size analyzer. the average particle size, and the polydispersity index (pdi), for the selected samples were measured.(17, 18) in-vitro dissolution dissolution study was carried out using the usp type ii dissolution apparatus (paddle). the dissolution media was either 900 ml 0.1n hcl (ph 1.2) or phosphate buffer ( ph6.8 ) at 37° c± 0.5 and stirring at 100 rpm. dissolution studies were performed on untreated sm (100mg) and the microcrystals of the selected formula containing an equivalent amount of the drug. aliquots of 5 ml. were withdrawn periodically and were replaced with 5 ml of fresh dissolution medium at selected time intervals (2,5, 10, 20, 30, 40, 50, 60, 90, and 120 min). the aliquots were filtered and analyzed spectrophotometrically at 288nm . each in vitro release study was performed in triplicate. the percentage drug release versus time graph was obtained and enhancement in dissolution with respect to pure drug was calculated.(19) . the dissolution data were analysed using dissolution efficiency up to 120 min (de120) which was calculated according to : de (%) = ∫ 𝑦 .𝑑𝑡 𝑡2 𝑡1 𝑦100 × (𝑡2−𝑡1) ×100 where y is the drug percent dissolved at time t (20), and dt is equal to t2-t1 . fourier transforms infrared spectroscopy (ftir) the (ftir) spectra were obtained using ftir shimadzu 8300 japan. samples of untreated sm, gelatin , and microcrystals of selected formulas, compressed with potassium bromide. the spectrum was obtained within scanning range of 4000-400 cm-1 (17,18) . statistical analysis using microsoft excel 2010, the results of the experiments were given as a mean of triplicate samples ± standard deviation and were analyzed according to the analysis of variance (anova) test and t-test. the level of significance was set at a pvalue of 0.05: p value of more than 0.05 (p> 0.05) was considered to be non-significant. p value of less than 0.05 (p< 0.05) was considered to be significant results and discussion sm saturated solubility the saturated solubility of sm in water was found to be equal to 53 ±3.1 µg/ml, so the sm considered as practically insoluble in water according to usp (21). factors affecting sm in situ micronization : effect of polymer type: gelatin, pvp-k30, hpmc 15 and pulullan stabilizers were tested by measuring percent of crystals yield. table (2) shows that gelatin and pvp k30 were the best polymers among the other, which may be due to their high affinity to the newly created drug particle surface (22), therefore they were used to study the other factors affecting the preparation of sm microcrystals table (2) effect of polymer type on sm microcrystals formulas f. no. sm. conc. %w/v organic solvent polymer polymer conc. %w/v solvent :antisolven t ratio percent yield % mc1 3 methanol gelatin 0.1 1:4 93.9± 3.3 mc2 3 methanol pvpk30 0.1 1:4 83.45±1.7 mc3 3 methanol hpmc15 0.1 1:4 no yield mc4 3 methanol pullulan 0.1 1:4 54 ± 4.5 effect of polymer concentration table 3 shows that, the minimum concentration of the stabilizer gelatin required for obtaining maximum yield with significant increase in the solubility (p <0.05) of the drug was 0.1% (w/v) . increasing the solubility of sm pure powder can be explained to be due to good adsorbed layer of hydrophilic stabilizer which induces the water solubility (23). it was also observed that as the concentration of gelatin decreased to (0.05% ) (w/v), low yield was obtained with lower solubility than that obtained from 0.1% (w/v), which may be attributed to that the amount of gelatin was not enough to form a hydrophilic stabilizer layer which leads to crystal growth with high drug content (24). furthermore, increasing the concentration of gelatin solution to 0.2% (w/v), the dried crystals stick with gelatin on whatman filter paper and no yield was obtained even by drying at room temp or using nylon filter paper. this can be explained to be due to that increasing gelatin concentration might cause efficient stabilization of drug particles until a certain concentration. further increase in concentration would lead to increase the thickness iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 5 and viscosity of the gelatin layer around each particle that may led to aggregation of many particles which were stick by drying (25). on the other hand, a fluffy powder was obtained with a significant increase (p<0.05) in solubility related to drug using pvp-k30 as stabilizer in concentration of 0.05% (w/v), and no further increase in solubility was obtained by using 0.1% (w/v) .while using 0.2% (w/v), low yield was obtained with lowest solubility than other concentrations. these results were in agreement with that obtained by jain sa and kurup ns (13). from these results, 0.1% (w/v) of gelatin and 0.05% (w/v) of pvp-k30 were the selected concentrations for further studies. table (3) effect of polymer concentration on sm microcrystals formulas f. no. polymer polymer conc.% w/v percent yield drug content % solubility µg/ml mc5 gelatin 0.05% 73.36±2.4 93.2±1.8 95± 2.5 mc1 gelatin 0.1 93.9 ± 3.3 83.95± 2.1 101.33±3.5 mc6 gelatin 0.2% 0 mc7 pvpk30 0.05% 90.2±1.4 94.6± 1.3 122± 1.5 mc2 pvpk30 0.1 83.45±1.7 90.7±1.1 128.3± 4.1 mc8 pvpk30 0.2% 85.7±1.7 87.8±2 75.5± 3.2 effect of solvent: anti-solvent volume ratio no significant difference in percent yield was obtained for all solvent : anti-solvent ratios of both polymers pvp-k30 and gelatin ( p> 0.05) which can be explained that the lowest volume of aqueous phase was enough to cause efficient change in polarity and bring out complete crystallization of both drug and polymer. while the low drug content obtained with increasing the volume of aqueous phase in both polymers may be attributed to enhance the solubilization of the drug in high aqueous phase volume in presence of hydrophilic polymer (26). the differences in solubility between batches of both polymers may be due to the different amount of the hydrophilic polymer that adsorbed on the precipitated drug particles (27). table (4) effect of solvent: anti-solvent ratio on sm microcrystals formulas. f. no. polymer polyme r conc. % solvent :antisolvent ratio percent yield drug content % solubility µg/ml mc9 gelatin 0.1 1:2 92.3 ±2.3 95.6 ±2.2 148.66±4 mc1 gelatin 0.1 1:4 93.9 ±1.6 83.95 ±2.1 101.33±3.5 mc10 gelatin 0.1 1:6 96.3 ±2.1 82 ±2.5 134.66±3.5 mc11 pvpk30 0.05 1:2 91.8 ±1.7 94.6 ±1.5 83.33±4.1 mc7 pvpk30 0.05 1:4 90.2 ±1.4 94.6 ±2.1 122±1.5 mc12 pvpk30 0.05 1:6 94.8±1.7 82.2 ±1.8 79±3 effect of sonication since microcrystals prepared using 0.1 % (w/v) gelatin as stabilizer for the three solvent: antisolvent ratios have higher solubility than those prepared by using pvp . therefore, mc1s, mc9s and mc10s were prepared to be compared with the corresponding formulas prepared by stirring. table 5 shows that all batches prepared by sonication have nearly the same percent yield and drug content to that prepared by stirring method. on the other hand, the solubility of the sonicated batches was higher than those prepared by stirring method which may be explained to the action of ultrasound waves that create microscopic turbulence within interfacial film surrounding crystals, as a result, ultrasound intensified external mass transfer and adsorption rate of gelatin which diffused rapidly and covered the surface of the sm crystals. so the polymer would be quickly reached adsorption equilibrium that would increase the solubility of sm (25). iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 6 table (5) sm microcrystals formulas prepared by two method using gelatin polymer f. no. solvent :antisolvent ratio method of preparation percent yield drug content % solubility µg/ml mc9 1:2 stirring 92.3 ±2.3 95.6 ±2.2 148.66±4 mc1 1:4 stirring 93.9 ±1.6 83.95 ±2.1 101.33±3.5 mc10 1:6 stirring 96.3 ±2.1 82 ±2.5 134.66±3.5 mc9 s 1:2 sonocrystallization 93.4±2.7 94.6±4.5 220±2.7 mc1s 1:4 sonocrystallization 95.5±2.5 85.3±3.2 120.6±1.5 mc10s 1:6 sonocrystallization 97.4±3 84.5±2.1 155±4.5 x-ray diffraction the x-ray patterns of the sm pure powder (figure.1:a) displayed the presence of numerous narrow and characteristic diffraction peaks (17°, 19°, 24°, 44°, 77°) with high intensity indicating the crystalline structure of the drug. generally, the positions of the most characteristic peak 24º of sm and microcrystals preparations were identical, although the peaks for the untreated sm crystalline powder were of highest intensity than those of the sm microcrystals. since, the peak height is affected by crystal size and crystallinity. therefore, the peaks height or intensity of the microcrystals was lower than those of sm, due to smaller size of the prepared microcrystals compared to that of sm crystals as it will be confirmed by measuring their particle size (17, 28). in addition, xrd shows that the crystallinity of the products was affected by solvent: anti solvent ratio used in the preparation of the microcrystals. microcrystals prepared with 1:2 solvent: anti solvent (figure.1:c) ratio gave peaks with highest intensity than those prepared using (1:4 or 1:6) solvent: antisolvent ratio (figure.1:d,e). furthermore, peaks with higher intensity were obtained with microcrystals prepared by sonication method (figure.1:f,g,h) compared with those prepared by stirring method, this phenomenon was observed with all solvent : anti-solvent ratios. this may be explained to ultrasound irradiation can intensify the mass transfer, accelerate molecular diffusion and, thus, reduce the induction time of crystallization, increase the nucleation rate. these effects bring considerable benefits to the crystallization process (25). ( a ) iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 7 ( b ) ( c ) ( d ) iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 8 ( e ) ( f ) ( g ) iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 9 ( h ) figure (1) x-ray diffraction patterns of silymarin (a) , gelatin(b), mc9 (c), mc1 (d), mc10 (e),mc9s (f), mc1s (g), mc10s (h) differential scanning calorimetry (dsc) dsc thermogram of untreated sm shows an endothermic peaks about 200ºc corresponds decomposition of drug compounds (figure.2:a). in addition broad endothermic event between 50°c and 120 °c corresponds to melting peak of silybin, an active constituent of sm complex (29). the thermogram of gelatin (fig.2:b) showed an intense glass transition temperature located around 98ºc. the thermogram of the selected microcrystals with different solvent: anti-solvent ratio mc9, mc1, mc10 stabilized by gelatin are shown in (figure.2:c,d,e). they reveal an induction in the intensity of endothermic peaks of sm in batches prepared with 1:2 solvent: antisolvent ratio in comparison to that of untreated sm, and microcrystals prepared with 1:4 and 1:6 solvent: anti-solvent ratio although they were available relatively at the same temperature and this may be due to increase in crystallinity due to ultrasound effect on nucleation (25,30). batches prepared by using 1:6 solvent: antisolvent volume ratio (mc10) showed a reduction in the intensity of endothermic peaks that may be due to the conversion of the drug from crystalline to amorphous state. this result agreed with increased solubility of this preparation, and confirms what was obtained from xrd results. the crystals prepared by sonication (figure.2: f,g,h) showed higher peak intensity for all solvent: antisolvent ratio in comparison to those prepared by stirring method. the higher crystallinity obtained by using 1:2 solvent: anti-solvent and by sonication method may be due to rapid nucleation rate with reduction in the induction time of crystallization (25,30). on the other hand, the disappearance of gelatin peaks in both xrd and dsc patterns of micronized sm, indicating a high drug load in these preparation (24). ( a ) iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 10 ( b ) (c) (d) iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 11 (e) (f) (g) iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 12 (h) figure (2) dsc thermogram of sm (a), gelatin (b), mc9 (c), mc1 (d), mc10 (e), mc9s (f), mc1s (g), mc10s (h). scanning electron microscope (sem) micrographs of the untreated sm crystalline powder and microcrystals of the optimum formulations prepared by two methods are shown in (figure.3). the untreated sm particles are of irregular shape, whereas the microcrystal particles mc9 which were prepared by stirring method have uniform particles nearly spherical shapes with smooth and regular surface. the microcrystals prepared by sonication (mc9s) were also homogeneous with larger crystals. obviously, the microcrystals prepared by any method were more uniform shape than that of the pure silymain drug. (a) (b) (c) figure (3) sem micrographs of untreated sm (a), mc9 (b), and mc9s (c) size distribution of microcrystals the particle size and polydispersity index (pdi) for the untreated sm and the prepared microcrystals were measured by laser particle size analyzer (brookhaven instrument), the crystals size were the largest for standard sm crystalline powder with mean diameter of 1.53µm and pdi of 0.36. the mean diameter for the crystals prepared by in situ micronization technique using stirring method (mc9) was markedly decreased to 0.43µm with 0.33 pdi, while ultrasonic irradiation method produced iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 13 crystals (mc9s) with mean diameter of 0.61 µm and pdi of 0.14. the size distribution obtained by application by sonication was more uniform as it had low pdi value which confirmed the sem. the mean size was slightly increased which can be explained to be due to the large ultrasonic energy that accelerates effectively the mass transfer in the mixture and thus enhances the driving force of crystallization .with large kinetic energy and speed, the solute molecules will have an increased opportunity to collide with each other and increase the crystals size.(31) in-vitro dissolution the in-vitro dissolution, results showed an increase in dissolution rate of both mc9 and mc9s compared to the untreated drug (figure 4,5). this effect can be explained by an increased specific surface area with hydrophilic nature due to the adsorbed hydrophilic polymers (25). significant increase (p<0.05) in dissolution efficiency was obtained by mc9s as it increased from 84% to 91.09% in hcl and from 88% to 91.02% in phosphate buffer of ph6.8 in comparison to mc9. the increase in de of mc9s may be due to its higher solubility, therefore, the formula mc9s was selected as the best formula. figure (4) dissolution profile of microcrystals mc9,mc9s and untreated powder of drug in hcl ph 1.2 at 37°c figure (5) dissolution profile of microcrystals mc9, mc9s, and untreated drug in phosphate buffer ph 6.8 at 37°c. fourier transforms infrared spectroscopy (ftir): the ftir of untreated sm, gelatin and the selected formula mc9s were studied by ftir spectroscopy using kbr disc. the data were presented in the (figure.6:a,b,c) respectively. characteristic peaks of sm appeared at 3441.35 cm1 (-oh stretching vibration), 2928.3 cm-1 (o-h stretching), 1636.3 cm-1 (c=o stretching), 1510.95,1462.74 cm-1 (skeleton vibration of aromatic c=c ring stretching),1362.46 cm-1 (-oh in plane bending),1271.82 cm-1 (c-o-c stretching),994.12 cm-1 (o-h out plane bending) and 1030.91-161.9 cm (in plane= c-h bending) (31). the results revealed that the ftir spectra of untreated sm and selected microcrystals were identical and the main absorption bands of sm still appeared in the spectra of mc9s which confirmed that no chemical modification of the drug had been taken place during in-situ micronization process (33). iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 14 ( a ) ( b) ( c ) figure (6) ftir of sm (a), gelatin (b), mc9s (c) conclusion in the light of the results, it has been concluded that solubility and dissolution rate can be increased by insitu micronization by solvent change method with using 0.1% (w/v) gelatin as hydrophilic stabilizer by sonication irradiation method. the resulted crystal in reduced size with the hydrophilic surface contribute enhance solubility and dissolution rate. references 1. lipinski c. poor solubility an industry wide problem in drug discovery. am pharm rev. 2002;5(september 2002):82–5. 2. liversidge gg, cundy kc. particle size reduction for improvement of oral bioavailability of hydrophobic drugs: i. absolute oral bioavailability of nanocrystalline danazol in beagle dogs. int j pharm. 1995;125(1):91–7. 3. bhakay a, merwade m, bilgili e, dave r n. novel aspects of wet milling for the production of microsuspensions and nanosuspensions of poorly watersoluble drugs. drug development 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screening. dissolution technologies;2010,16-21 27. reddy p.s, sujani s, reddy k.r. microcrystals: for improvement of solubility and dissolution of tinidazole. asian j. pharm. tech;2011,1(3): 6469 28. park mw, yeo s do. antisolvent crystallization of carbamazepine from organic solutions. chem eng res des. 2012;90(12):2202–8. 29. s. javed, k. kohli, m. ali, solid state compatibility between silymarin and tablet excipients by thermal and non-thermal methods, its ph stability and solubility analysis. journal of pharmacy research;2012,5(3):1300-1305 30. agrawal s. investigation and optimization of a solvent / anti-solvent crystallization process for the production of inhalation particles. phd thesis. virginia commonwealth university;2010. 31. hong li, jingkang wang, ying bao, zhichao guo, muyan zhang. rapid sonocrystallization in the salting-out process. journal of crystal growth 2003;247: 192–198 iraqi j pharm sci, vol.28(1) 2018 silymarin microcrystals 16 32. sonali d., tejal s., vaishali t., tejal g. silymarin-solid dispersions:characterization and influence of preparation methods on dissolution.acta pharm., 60, 427-443, 2010. 33. varshosaz j, talari r, mostafavi sa, nokhodchi a. dissolution enhancement of gliclazide using in situ micronization by solvent change method. powder technol. 2008;187(3):222–30. iraqi j pharm sci, vol.21(1) 2012 synthesis of thiadiazole derivatives 98 synthesis and preliminary antimicrobial study of 2-amino-5mercapto-1,3,4-thiadiazole derivatives husam a. ameen* and ahlam j. qasir *,1 *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq ** ibn-zuhr hospital ,ministry of health ,baghdad, iraq. abstract nitrogen heterocycles are of a special interest because they constitute an important class of natural and non natural products, many of which exhibit useful biological activities.among these nitrogen heterocycles are 1, 3, 4-thiadiazole containing compounds. the therapeutic effects of these derivatives have been well studied for a number of pathological conditions including inflammation, pain, or hypertension. moreover, synthesis of thiadiazoles has attracted wide-spread attention due to their diverse applications as antibacterial, anticancer, antifungal anti-inflammatory and antidepressant agents.according to this information’s new derivatives of 1, 3, 4-thiadiazole were designed and synthesized and in the hope of having some activities as antibacterial and antifungal. these are:  4-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)-2-methoxyphenol.  tert-butyl (1((2((5(methylsulfonyl) -1,3,4thiadiazol-2-yl) amino) -2oxoethyl)amino) -1oxopropan 2-yl) carbamate. key words: 1, 3, 4-thiadiazole, biological activity, peptides, schiff base. -4، 1،3-ميركابتى -5-امينى -2تخهيق ودراست تمهيذيت مضاداث انمايكروباث نمشتقاث ايادايازولث أحالو جميم قصير* و** أمين عامرحساو ،1 * فسع انكًٍٍبء انصٍدالٍَخ ،كهٍخ انصٍدنخ ، جبيعخ ثغداد ،ثغداد ، انعساق . .شْس ، ٔشازح انصحخ ، ثغداد ، انعساق اثٍ** يسزشفى الخالصــــة ، انطجٍعٍخ ٔغٍس نهًُزجبد انطجٍعٍخ فئخ ْبيخ رشكم ألَٓبرًثم أْزًبيب خبصب انٍُزسٔجٍٍ يٍ انًسكجبد انغٍس يزجبَسخ ًْ انًسكجبد انزً رحزٕي عهى حهقخ انٍُزسٔجٍٍ يٍ يٍ ثٍٍ ْرِ انًسكجبد انغٍس يزجبَسخ .ًفٍدحان األَشطخ انجٍٕنٕجٍخ ٌحًمٔكثٍس يُٓب ازرفبع أٔ، ٔاألنى ، االنزٓبة ثًب فً ذنك انحبالد انًسضٍخ نعدد يٍ يدزٔسخ انًشزقبد انعالجً نٓرِ انزأثٍس ثٍبدٌبشٔل. – 4، 3، 1 يضبد نهجساثٍى،، ثسجت رطجٍقبرّ انًزُٕعخ يثم اَزشبزأاسع االْزًبو صُبعخ ثٍبدٌبشٔل، فقد اجزرثذ ٔعالٔح عهى ذنك .ضغظ اندو رحضٍس يشزقبد رى رصًٍى ٔٔاعزًبدا عهى ْرِ انًعهٕيبد ، .، يضبد نالنزٓبثبد ٔيضبدح نالكزئبةبديضبد نهفطسٌ، يضبد نهسسطبٌ : ْٔرِ ًْ.يع رٕقع احزًبل اٌ ٌكٌٕ نٓب فعبنٍخ كًضبد ثكزٍسٌب ٔيضبد فطسي ثٍبدٌبشٔل – 4، 3، 1حهقخ جدٌدح يٍ  4-(((5-mercapto-1,3,4-thiadiazol-2-yl)imino)methyl)-2-methoxyphenol.  tert-butyl (1-((2-((5-(methylsulfonyl)-1,3,4-thiadiazol-2-yl)amino)-2-oxoethyl)amino)-1oxopropan-2-yl) carbamate. ، انفعانيت انبايىنىجيت ، بينيذاث ، قاعذة شيف . يازولاياداث – 4، 3، 1انكهماث انمفتاحيت : introduction over the past decade, drug resistance has become a growing problem in the treatment of infectious diseases caused by bacteria, fungi and viruses. in particular, resistance of bacterial pathogens to current antibiotics has emerged as a measure health problem. this is especially true in case of infectious diseases such as pneumonia, meningitis and tuberculosis, which would once have been easily treated with antibiotics, but is no longer so readily treated. at present, all widely used antibiotics, including some of the agents such as streptogramins and new generation flouroquinolones are subjected to bacterial resistance. the search for new antimicrobial agents is one of the most challenging tasks to the medicinal chemist (1) . thiadiazole thiadiazole is a five membered heterocyclic compounds that show various types of biological activity. it contains two nitrogen atoms and one sulphur atom as hetero atoms. there are several isomers of thiadiazole (fig. 1), that is a1, 2, 3 thiadiazole , b1,2,4 thiadiazole, c1,3,4 thiadiazole, d1, 2, 5 thiadiazole (2) . figure 1: structures of thiadiazole isomers 1 corresponding author email : ahlamqasir@yahoo.com received : 24/12/2011 accepted : 21/4/2012 mailto:ahlamqasir@yahoo.com iraqi j pharm sci, vol.21(1) 2012 synthesis of thiadiazole derivatives 99 it acts as a “hydrogen binding domain” and “two-electron donor system”. thiadiazole acts as a bioisosteric replacement of thiazole moiety. so, it is act as a one component of third and fourth generation cephalosporin (3) .1, 3, 4-thiadiazole is the isomer of thiadiazole series. a glance at the standard reference works shows that more studies have been carried out on the 1,3,4 thiadiazole than all the other isomers combined. members of this ring system have found their way in to such diverse applications as pharmaceuticals, oxidation inhibitors and metal complexing agents (4) . during last few years there has been intense investigation of different classes of thiadiazole compounds, many of which known to possess interesting biological properties such as antimicrobial (5) ,antituberculosis (6) , antiinflammatory (7) , analgesic (8) , anticonvulsants (9) , antihypertensive (10) , antioxidant (11) , anticancer (12) and antifungal (13) ,antiviral (4) ,antidepressant (14) activity.among them 2,5disubstituted 1,3,4-thiadiazole derivatives posses interesting biological activity probably conferred to them due to strong aromaticity of the ring system which leads to great in vivo stability and generally, a lack of toxicity for higher vertebrates, including humans when diverse functional group that interact with biological receptor are attached to aromatic ring (3,15) .schiff bases are known to have biological activities such as antibacterial (16) , antifungal (17) , antitumor (18) and antioxidant activities (19) . schiff bases appear to be important intermediates in a number of enzymatic reactions involving interaction of an. enzyme with an amino or a carbonyl group of the substrate. one of the most prevalent types of catalytic mechanisms in biochemical processes involves condensation of a primary amine in an enzyme, usually that of a lysine residue, with a carbonyl group of the substrate to form an imine, or schiff base (20) . the biological properties of thiadiazole derivatives aroused our interest in to design and synthesize new derivatives of thiadiazole then subjected them to investigate their possible biological activity (as antibacterial & antifungal activities). experimental work chemicals and equipments boc-l-alanine, carbon disulfide, dicyclohexylcarbodiimide (dcc ), hydrogen peroxide, 1-hydroxybenzotriazole (hobt), dimethylsulfoxide (dmso), glycine, methyl iodide, n-methyl morpholine (nmm), thionyl chloride, vanillin.all the solvents and materials used were of analar type and used without further purification. infrared spectral determination was performed for all compounds in kbr disk, using ftir at the collage of science (baghdad university).elemental analysis has been done using carlo erba elemental analyzer. the analysis was done in al-al bayt university in al-mafraq (jordan). chemical synthesis synthesis of 2-amino -5-mercapto -1, 3, 4thiadiazole compound (i) (21) thiosemicarbazide (4g , 0.043 mole) was suspended in absolute ethanol (30ml) in round bottom flask (250ml), anhydrous sodium carbonate (2.23g, 0.021mole) and cs2 (9.5g , 0.125mole) were then added respectively with continues stirring.the reactant mixture was refluxed for 5hours; the reaction mixture was then allowed to cool to room temperature and filtered. the filtrate was evaporated under vacuum then cold distilled water (90 ml) was added, acidification with concentrated hcl drop by drop was carried out, white –yellowish precipitate was formed, the precipitate was collected by filtration, and washed with distilled water, re-crystallized using hot distilled water. the physical appearance, percentage of yield, melting point and rf values were listed in tablet 1, the elemental analysis results are presented in table 2 while the ir data are shown in table 3. . table 1: physical appearance, percentage of yield, melting points, rf values of intermediates and final compounds compound no. physical appearance yield % melting point ( o c) rf values observed reported a b i faint yellow crystal 65% 232-234 230-232 0.40 0.33 ii yellow powder 80% 177-178 178-181 0.45 0.38 iii yellow powder 75% 205-208 0.30 0.48 iv orange powder 77% 245-246 0.55 0.28 v dark yellow powder 62% 185-188 0.22 0.29 a) chloroform: ethanol (9:1), b) acetone: ethyl acetate (1:2) (25) . iraqi j pharm sci, vol.21(1) 2012 synthesis of thiadiazole derivatives 100 table 2: elemental microanalysis of the final compounds compound molecular weight chemical formula elemental microanalysis % element calculated observed iv 267.01 c10h9n3o2s2 c 44.93 45.027 h 3.39 3.386 n 15.72 15.951 o 11.97 11.473 s 23.99 24.163 v 573.17 c26h31n5o6s2 c 54.43 55.242 h 5.45 5.493 n 12.21 12.577 o 16.73 16.086 s 11.18 10.624 table 3: characteristic ir absorptions of compounds iv compound band (cm -1 ) interpretation i 3330,3317 2614 1608 1554 1172 1058,1122 n-h stretching vibration of the primary amines. s-h stretching of thiol. c=n stretching of thiadiazole ring moiety. n-h bending. c-n stretching of the thiadiazole ring moiety. c=s stretching vibration gives evidence that compound (i) can exist in two tautomeric form, thiol form and thion form. ii 3332,3282 2945,2873 1629 1521 1373 1139 n-h stretching vibration of the primary amines. c-h stretching vibration of ch3 (symmetrical & asymmetrical). c=n stretching vibration of the thiadiazole ring moiety. n-h bending. c-h bending vibration of ch3. c-n stretching of thiadiazole ring moiety. iii 3398,3423 2939,2864 1624 1498 1423 1319, 1168 n-h stretching vibration of the primary amines. c-h stretching vibration of ch3. c=n stretching vibration of the thiadiazole ring moiety. n-h bending. c-h bending vibration of ch3. s=o stretching vibration of sulfone (symmetrical & asymmetrical). iv 3200-3500 3097-3141 2925 2554 1614 1554 1477,1363 1363 1174 1058 756 o-h stretching of phenol. c-h stretching vibration of aromatic ring. c-h stretching vibration of ch3. s-h stretching of thiol. c=n stretching. c=c stretching of aromatic ring. c-h bending of saturated carbon. o-h bending of phenol. c-o stretching of ether overlap with c=s stretching.. aromatic c-h in plane bending. aromatic c-h out of plane bending. v 3328 2930 2850 1671 1625 1574 1338,1149 1226 1180 n-h stretching of amide. c-h asymmetrical stretching vibration of ch3&ch2. c-h symmetrical stretching vibration of ch3&ch2. c=o stretching of amide and carbamate. c=n stretching. n-h bending of amide overlap with c=c stretching of aromatic ring. s=o symmetrical & asymmetrical stretching of sulfone. c-o stretching of ester. c-n stretching. iraqi j pharm sci, vol.21(1) 2012 synthesis of thiadiazole derivatives 101 synthesis of 5-(methylthio)-1, 3, 4thiadiazole-2-amine compound (ii) (22) compound (i) (1.33 g, 0.01mole) was dissolved in the minimum volume of distilled water and sufficient volume of koh (85%) solution was added under stirring at room temperature, after (5-10 minute), the solution was brought to 0°c in ice bath and then methyl iodide (0.625ml, 0.01mole) was added with vigorous stirring at a rate of 1drop every 2min, continuous stirring was maintained for 3hours. the solution then was filtered, the precipitate was washed with water, dried to give compound (ii), which was used without further purification. the physical appearance, percentage of yield, melting point and rf values were listed in tablet 1, the elemental analysis results are presented in table 2 while the ir data are shown in table 3. synthesis of 5-(methylsulfonyl)-1, 3, 4thiadiazole-2-amine, compound (iii) (23) compound (ii) (0.147 g, 1mmol) was dissolved in ethanol (95%) (30ml), h2o2 (0.068 g, 2mmol) was added with continues stirring at room temperature for 2 hrs. then the excess of solvent was evaporated to give compound (iii), which was used without further purification. the physical appearance, percentage yield, melting point and rf values were listed in tablet 1, the elemental analysis results are presented in table 2 while the ir data are shown in table 3. synthesis of 4-(((5-mercapto-1, 3, 4thiadiazol-2-yl) imino) methyl)2methoxyphenol, (compound iv) (24) compound i (0.266 g, 2 mmole) was suspended in 25 ml of absolute ethanol then vanillin (0.304 g, 2 mmole)dissolved in 25 ml of absolute ethanol solution was added. the mixture was refluxed for 8 hrs, and then left overnight. the solvent was evaporated in vacuum and the residue was re-crystallized from methanol. the physical appearance, percentage of yield, melting point and rf values were listed in tablet 1, the elemental analysis results are presented in table 2 while the ir data are shown in table 3. synthesis of tert-butyl (1-((2-((5(methylsulfonyl)-1, 3, 4-thiadiazol-2-yl) amino)-2-oxoethyl) amino)-1-oxopropan-2-yl) carbamate, (compound v) (25) to a stirred solution of the dipeptide (boc-ala-gly-oh) (0.246 g, 1 mmole) in dmf (3 ml), nmm (0.11 ml, 1 mmole) was added followed by stirring for 10 minutes. solution of compound iii (0.179 g, 1 mmole) in dmf (3 ml) was added to the reaction mixture. the mixture was then cooled to (15°c), then hobt (0.3 g, 2 mmole) was added followed by dcc (0.23 g, 1 mmole) with stirring which was continued for 72 hrs., at (0°c) and for 48 hrs., at ambient temperature. ethyl acetate (10 ml) was added to the reaction mixture which was then filtered to get rid of n, n-dicyclohexylurea (dcu). the filtrate was evaporated to dryness under vacuum, and the residue was re-dissolved in ethyl acetate (10 ml), the excess dcu which was still adhesive on the peptide residue was precipitated out and filtered. the clear filtrate was washed twice with (5 ml) hcl (0.1 n) solution, once with (10 ml) d.w. , and with (10 ml) saturated nacl solution using the sepertaory funnel. the ethyl acetate layer was dried using anhydrous magnesium sulfate then the solvent was evaporated to get compound v which was recrystallized from (ethyl acetate: petroleum ether 40-60) mixture. physical appearance, percentage of yield, melting point and rf value are listed in table 1, the elemental analysis results are presented in table 2 while the ir data are shown in table 3. antimicrobial activity (26) the synthesized compounds were screened for the presence of antibacterial constituents against four strains of bacteria i.e. staphylococcus aureus, escherichia coli, klebsiella pneumonia, beta-hemolyticstreptococcus pyogenes and one species of fungi i.e. against candida albicans by disc diffusion method. nutrient agar was used as culture medium for bacterial growth; blood agar was used as culture medium for betahemolytic-streptococcus pyogenes growth, while fungi were subcultured in sabouraud dextrose agar medium. all compounds were dissolved in dmso at concentration 0.625 mg/disc * . gentamycin [cn] (30mcg/disc for bacteria), amoxicillin/clavulanic acid [amc] (30mcg/disc for bacteria), and ketoconozole (100 units/disc for fungi) was used as reference antibiotic and dmso as control. the zones of inhibition were determined at the end of an incubation period of 24 hr at 35 ◦ c for bacteria growth and 5 days at 28 ◦ c for fungi growth. the inhibition zone values are summarized in table 4. this study is done in collage of science (baghdad university). * three different concentrations (0.25 mg/disc, 0.5 mg/disc, and 0.625 mg/disc) were tried and the above concentration 0.625 mg/disc gave the best results. iraqi j pharm sci, vol.21(1) 2012 synthesis of thiadiazole derivatives 102 table 4: antimicrobial screening data (zone of inhibition in mm) for final compounds (iv-v) compound staphylococcus aureus streptococcus pyogenes escherichia coli klebsiella pneumonia candida albicans iv 27 30 22 15 32 v 28 35 18 16 25 gentamycine 22 18 augumentin 24 20 ketoconozole 30 dmso the zone of inhibition of tested compounds shows, the 2-amino-5-mercapto-1, 3, 4 thiadiazole derivatives encompass potent bioactivities against bacterial and fungal strains, due to the strong bioactivity of our synthesized cycle and to the substituent groups that we added to the cycle. results and discussion the 2-amino-5-mercapto-1,3,4thiadiazole was synthesized through steps of reactions starting from thiosemicarbazide with carbon disulphide in basic medium.alkylation of compound (i) this step includes the synthesis of thioether or alkyl sulfide, it was done by treatment of a thiol with a base, such as koh, giving the corresponding thiolate ion (rs ). this undergoes reaction with a primary or secondary alkyl halide to give a sulfide. the reaction occurs by an sn2 mechanism, analogous to the williamson synthesis of ethers. thiolate anions are among the best nucleophiles known, and product yields are usually high in these sn2 reactions. thioethers can be oxidized to sulfoxides by one equivalent of 30% h2o2 or by many other oxidizing agents , including h2o2 –flavin catalyst; sulfoxides can be further oxidized to sulfones by another equivalent of h2o2.primary amines(compound i), add to aldehydes (vanillin) to yield imines r2c=nr,conventional solution method was used as a coupling method between the dipeptide (boc-ala-gly-oh) and compound iii for synthesizing compound v. dicyclohexylcarbodiimide (dcc) was used as a coupling reagent in amide bond formation; while 1-hydroxybenzotriazole (hobt) or nhydroxysuccinamide (hosu) were used to increase the yields of the product and suppress racemization. scheme 1: general scheme of synthesis compounds iraqi j pharm sci, vol.21(1) 2012 synthesis of thiadiazole derivatives 103 conclusion the synthesis of these proposed compounds was successfully achieved by following the stated procedures as previously described. the results obtained from this investigation indicated that the strategy adapted for the synthesis of the designed derivatives was successful, since the conformity of synthesized compounds was achieved according to the data shown by the physical and chemical analysis including (tlc, melting point, ir and elemental microanalysis (chnso). compound iv & v show good antimicrobial activity comparable with marketable compounds. the antimicrobial evaluation indicated that the newly synthesized compound iv, showed highest antimicrobial activity in comparing to augumentin for gram positive bacteria, gentamycin for gram negative bacteria and ketoconazole for fungi.compound v showed a good antimicrobial activity, highest activity against streptococcus pyogenes compared to augumentin. all the synthesized compounds have excellent antifungal activity. acknowledgment the authors gratefully thank the college of pharmacy, university of baghdad for supporting this research. . references 1. jitendra kumar gupta*, rupesh dudhey, p.k.sharma. synthesis and 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1.0 ,dec 2009;1-16. iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles doi: http://dx.doi.org/10.31351/vol27iss1pp39-52 39 preparation and characterization of domperidone nanoparticles for dissolution improvement malath h. oudah*, firas a. rahi** and mohammed s. al-lami***,1 *college of pharmacy, university of kufa , alnajaf, iraq. ** faculty of pharmacy, ibn hayyan university college, karbala, iraq. *** college of pharmacy, university of basra, al basrah, iraq abstract this study was carried out to prepare and characterize domperidone nanoparticles to enhance solubility and the release rate. domperidone is practically insoluble in water and has low erratic bioavailability range from 13%-17%. the domperidone nanoparticles were prepared by solvent/antisolvent precipitation method at different polymer:drug ratios of 1:1 and 2:1 using different polymers and grades of poly vinyl pyrolidone, hydroxy propyl methyl cellulose and sodium carboxymethyl cellulose as stabilizers. the effect of polymer type, ratio of polymer:drug, solvent:antisolvent ratio, stirring rate and stirring time on the particle size, were investigated and found to have a significant (p≤ 0.05) effect on particle size. the best formula was obtained with lowest average particle size of 84.05nm, which composed from 2:1 of pvpk15:drug and solvent/antisolvent volume ratio of 1:10. this formula was freeze dried and studied for compatibility by ftir and dsc, surface morphology by field emission scanning electron microscope (fesem) and crystalline state by xrpd. then domperidone nanoparticles were formulated into a simple capsule dosage form in order to study of the in vitro release of drug from nanoparticles in comparison pure drug and mixture of polymer:drug ratios of 2:1. the release of domperidone from best formula was highly improved with a significant (p≤ 0.05) increase. it can conclude that nanoparticles showed better in vitro dissolution profiles in comparison with pure drug keywords: domperidone, solvent/antisolvent precipitation, polymers, polyvinyl pyrrolidone, nanoparticles, dissolution rate, release. التذوب تحسينالجسيمات النانوية للدومبيريدون ل وصيفتحضير وت 1،***و محمد صبار الالمي **، فراس عزيز راهي *مالذ هاتف عودة كلية الصيدلة ، جامعة الكوفة ،النجف ، العراق .* كربالء ، العراق .كلية ابن حيان الجامعة ،قسم الصيدلة ، ** ، العراق . ، البصرة بصرةكلية الصيدلة ، جامعة ال*** ةالخالص بريدونمسرعة إطالق الدواء. الدوزيادة و يتهذوبانزيادة دومبيريدون للل الجسيمات النانوية وصيفلتحضير وتأجريت هذه الدراسة .٪٣١ -٣١ن م الحيوينقص التوافر يعاني مبريدون غير قابل للذوبان في الماء وولغثيان والقيء، الدويستخدم مضاد لهو معاكس للدوبامين و ٣:١ مر: نسببوليالباستخدام نسب مختلفه من المذيب دومبيريدون بواسطة طريقة ترسيب المذيب / مضاد للتم تحضير الجسيمات النانوية كمثبتات. (hpmc-e50, hpmc-e15, cmc-30, pvp-k30, pvp-k15) باستخدام بوليميراتودواء بوليمر لمن ال ٣:٣ ياس الجسيمات النانويه من خالل قحجم على مضاد المذيب وسرعة ووقت التحريك الىو تركيزه ونسبة المذيب البوليمر نوعتم دراسة تاثير ونسبة حجم مذيب ٣:١والتي تحتوي على نسبة بوليمرلدواء تساوي f8 للجسيمات . التوزيعحجم الجسيمات والمساحة السطحيه لها ومعامل تم تجفيفها والتحقيق فيها لدراسات نانومتر و ٨:.٥٠جسيمات للتم اختيارها كأفضل الصيغ مع متوسط حجم فقد ٣::٣لمضاد مذيب تساوي -x، الحاله البلورية للجسيمات النانويه باستخدامfesem)) باستخدام ( ، شكل الجزيئات(ftirمن خالل البوليمراتالتوافق بين الدواء و rayتأثر حسب يحجم الجسيمات النانوية انالنتائج إلى اضهرتكبسولة. النانويه فيت دومبيريدون ثم تمت صياغة جزيئا .لها ، واالستقرار حرير الدواء . وجد ان تناتجووقت تحريك ال سرعةالمذيب: نسبة المضادة للمذيبات، نسبة البوليمر، الى نوع وتركيز البوليمر، ونسبة الدواء من الدواء الخام ومزيجها مع البوليمرات . عاليهاسرع وبنسبه من الجسيمات النانوية كان ، االطالق. االذابة الكلمات المفتاحية: الدومبيريدون ، الترسيب بالمذيب ومضاد المذيب ، البوليمر ، بولي فينايل بايروليدون ، الجسيمات النانوية ، معدل introduction the solubility, dissolution rate and bioavailability of drugs are important factors for achieving in vivo effectiveness. the bioavailability of orally administered medications depends on their capability to be absorbed via gastrointestinal tract. it appears that enhancement 1corresponding author e-mail: mohsabbar@gmail.com received: 5/12/ 2017 accepted: 3/2/2018 iraqi journal of pharmaceutical sciences http://dx.doi.org/10.31351/vol27iss1pp39-52 http://bijps.com/index.php/bijps/index iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 40 of the solubility of poorly water soluble apis can translate to an increase in their bioavailability. beside the coming of new technologies in drug detection, combinatorial chemistry, and computer helped drug design, there was growing in the progress of new chemical entities with perfect therapeutic potential. however due to of the complicated chemistry, nearly 40% of the drug applicants in the development pipeline and about 60% of new apis produced by chemical synthesis are introduced with poor aqueous solubility causing in low and variable bioavailability (1,2). at the present time nanotechnology offers various methods in the area of dissolution enhancement of low aqueous soluble drugs. nanoparticles formulation technology has achieved a considerable attention by the formulation scientists. pharmaceutical nanoparticles are defined as solid, submicron-sized drug carrier that may or may not be biodegradable. the advantages of nanoparticles include lower drug toxicity, reduce the dose needed, improved bioavailability, increase drug targeting ability, decrease drug resistance ,increase patient compliance and reduced cost of treatment(3,4).the aim of this research to formulate domperidone as nanoparticles in capsule dosage form using precipitation method of solvent/antisolvent to improve dissolution rate. domperidone is antiemetic drug has chemical structure is 5chloro-1-[1-[3-(2,3-dihydro-2-oxo-1hbenzimidazole-1-yl) propyl] -4piperidinyl1] -1,3 dihydro -2h –benzimidazole -2-one) (5), domperidone is practically insoluble in water and slightly soluble in ethanol and methanol(6), and have low oral bioavailability (13-17%) is thought to be due to hepatic first-pass and intestinal metabolism and poor aqueous solubility (7). materials and method materials domperidone (science lab–india), hydroxy propyl methyl cellulose of hpmce50lv and hpmc-e15lv (gromax chemicals– usa), polyvinyl pyrrolidone of pvp-k15, pvpk30 (alpha chemika-india) na-cmc-30 (calbiochem–usa), dmf (sinopharm chemical reagent–china), disodium hydrogen orthophosphate, sodium chloride, and hydrochloric acid 37% (bdh laboratoryengland), potassium dihydrogen orthophosphate (fine chem–india) and lactose (carlo erba reagent-italy) were used as received in this study. methods preparation of domperidone nanoparticles the domperidone nanoparticles were prepared using solvent/antisolvent of precipitation technique (8). a certain amount of pure domperidone was completely dissolved in dimethyl formamide (dmf). the drug solution with specific concentration was injected at 1ml/min using ordinary syringe into water solution containing specific concentration of stabilizer of each (pvp-k15, pvp-k30, hpmce15, hpmc-e50, andcmc-30) with continuous stirring. precipitation of nanoparticles in form of colloidal solution occurred gradually upon mixing. the nanoparticles were then lyophilized to obtain the nanoparticles powder. the composition and variable conditions of preparation of different formulas are listed in table (1). table 1. composition of domperidone nanoparticles formulas formula polymer polymer: drug ratio solvent: anti solvent ratio f1 hpmc-e50 1:1 1:10 f2 hpmc-e15 1:1 1:10 f3 pvp-k15 1:1 1:10 f4 pvp-k30 1:1 1:10 f5 cmc-30 1:1 1:10 f6 hpmc-e50 2:1 1:10 f7 hpmc-e15 2:1 1:10 f8 pvp-k15 2:1 1:10 f9 pvp-k30 2:1 1:10 f10 cmc-30 2:1 1:10 f11 hpmc-e50 2:1 0.5:10 f12 hpmc-e15 2:1 0.5:10 f13 pvp-k15 2:1 0.5:10 f14 pvp-k30 2:1 0.5:10 f15 cmc-30 2:1 0.5:10 f16 hpmc-e50 2:1 2:10 f17 hpmc-e15 2:1 2:10 f18 pvp-k15 2:1 2:10 f19 pvp-k30 2:1 2:10 f20 cmc-30 2:1 2:10 particle size and poly dispersity index measurement (pdi) the abt-9000 dynamic light scattering nano laser (angstrom-usa particle size analyzer was used to measure the average particle size and poly dispersity index (pdi), as measures for the iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 41 width of the size distribution, and the specific surface area (ssa) for all prepared domperidone nanoparticles formulas. study of variables affecting on size of domperidone nanoparticles effect of type and concentration of stabilizer different stabilizer at two ratios of polymer to drug concentration of 1:1 and 2:1 were used in the preparation of domperidone nanoparticles. formulas f1-f10, were prepared and used to illustrate the effect of polymer type and concentration on the size of domperidone nanoparticles. effect of time of stirring the effect of time of stirring of 5 and 30min from finished addition of drug solution on size of formed nanoprticles was studied using polymers of pvp-k15, hpmc-e50, and cmc-30 at two ratios polymer to drug concentration of 1:1and 2:1. effect of rate of stirring the effect of stirring rate was studied using 500, 700 and 1100rpm; this effect was examined in all formulas. effect of solvent /anti solvent ratio the effect of the ratio of volume of solvent:antisolvent on the size of the formed nanoparticles was studied in three ratios of 0.5:10, 1:10 and 2:10 in all polymers at polymer:drug ratio of 2:1. characterization of lyophilized domperidone nanoparticles determination of drug content and loading efficiency. assay was carried out by taking 6mg powder of lyophilized nanoparticles and dissolved in 20ml dmf in dry volumetric flask and sonicated for 20min and then volume was completed to 60ml with same solvent and filtered on 0.45µm filter. the absorbance of filtrate was then determined using uv-visible spectrophotometer and the drug content was calculated accordingly. the loading efficiency of nanoparticles was determined from the theoretical and actual drug contents (9). this experiment was done in triplicate. %𝐋𝐨𝐚𝐝𝐢𝐧𝐠 𝐄𝐟𝐟𝐢𝐜𝐢𝐞𝐧𝐜𝐲 (𝐋𝐄) = 𝐀𝐜𝐭𝐮𝐚𝐥 𝐝𝐫𝐮𝐠 𝐜𝐨𝐧𝐭𝐞𝐧𝐭 𝐓𝐡𝐞𝐨𝐫𝐞𝐭𝐢𝐜𝐚𝐥 𝐝𝐫𝐮𝐠 𝐜𝐨𝐧𝐭𝐞𝐧𝐭 × 𝟏𝟎𝟎 determination of saturated solubility solubility of pure domperidone and domperidone nanoparticles was determined in each medium of 0.1n hcl of ph 1.2, phosphate buffer solution of ph 6.8, and distilled water using the shacking-flask method (10). plus, to ensure increase in saturated solubility of domperidone nanoparticles is due to reduction particle size, physical mixture of polymer of pvp-k15 and pure domperidone in ratio of 2:1 was used and studied. an excess amount of each powder was added in test tube containing 10ml medium, the tubes were sealed well and covered with aluminum foil then incubated in a shacking water bath at 37o c for 72h. sample of solution was drawn, filtered and the concentration of domperidone was determined spectrophometrically at the measured λmax using corresponding calibration curve equation in each media. the experiment was performed in triplicate and the average value was calculated. field emission scanning electron microscope field emission scanning electron microscope (fesem) (zeiss-germany) of pure domperidone powder, pvp-k15 powder were confirmed by direct dusting of powder on carbon tape, while fesem for liquid formula (f8), sample was done by the droplet evaporation technique. a droplet of liquid was settled on carbon tape and dried at room temperature. images were taken by secondary electrons using 1kv and different magnification powers. x-ray powder diffraction (xrpd) x-ray powder diffraction was used to study crystalline structure of drug, polymer and the prepared nanoparticles. the x-ray diffraction have the operating voltage and current were 60 (kv) and 80 (ma) respectively. fourier transform infrared spectroscopy(ftir) ftir spectra were obtained using ftir spectroscope. samples that studied were pure domperidone, pvp-k15, physical mixture of pvp-k15 and pure domperidone (at ratio of 2:1), and selected nanoparticles formula. sample was milled, mixed with potassium bromide and pressed in a form of disc of 13mm in diameter. the disc was analyzed by ftir spectroscopy at 4000-400cm-1. differential scanning calorimetry (dsc) dsc (-60, shimadzu-japan) was used to determine the crystalline state of drug particularly when converted to nanoparticles and thermal characteristics of samples were determined by an automatic thermal analyzer system. exactly weighed samples of 5mg were placed in nonhermetically aluminum pan and heated at the rate of 20º c/min against an empty aluminum pan as a reference covering a temperature range of 50 to 300º c (11). iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 42 in vitro dissolution study of domperidone nanoparticles from capsule dosage form capsules prepared by simple manual filling of hard gelatin capsule with fixed quantity domperidone and lactose as filler. then dissolution study for capsule containing domperidone nanoparticles were performed in a paddle type dissolution apparatus according to bp 2009 monograph (12). three types of capsule were prepared, containing lyophilized domperidone nanoparticles equivalent to 10mg of domperidone, 10mg of pure domperidone and physical mixture of polymer:pure domperidone using pvp-k15 in ratio of 2:1 equivalent to 10mg of domperidone. each capsule was dispersed in 900 ml of 0.1nhcl of ph1.2 with aid of special sinker of butter fly to prevent floating at 37±0.5° c and rotated paddle 50rpm. a 5ml sample was draw at specific time intervals from 5-120min for analysis and replaced with same volume of fresh media to maintain sink condition at 37±0.5° c. then sample was analyzed using uvspectrophotometer at wave length 284nm. afterward the accumulative percentage of release was calculated and draw against time. the experiment was performed in triplicate and the average value were calculated. statistical analysis the results of the experiments are given as a mean samples ± standard deviation (sd) and were analyzed for differences using one-way analysis of variance (anova) at p≤0.05 and the dissolution profiles data were fitted to f1 and f2 difference and similarity factor to determine the effect of nanoparticles formulation on the dissolution patterns of domperidone from the prepared dosage form (13). results and discussion evaluations of prepared domperidone nanoparticle particle size and polydispersity index analysis the particle size of all the prepared formulas were characterized and found within a range of nanometer sizes as shown in table (2). the most essential parameters for the produced suspended nanoparticles were the mean particle size and poly dispersity index that in turn determine and control the physicochemical properties like saturated solubility and dissolution profile (14). abt-9000 nano laser particle size analyzer is a particle size analyzer working on basis of dynamic light scattering theory (dls), was used to measure the size of domperidone nanoparticles and pdi "the common range of pdi values are 0-0.05 (monodisperse standard), 0.05-0.08 (nearly monodisperse), 0.08-0.7 (midrange polydispersity), and >0.7 (very polydisperse)'' (15). all formulas were showed monodisperse pdi except, f10 which showed a nearly monodisperse pdi while f7 and f9, showed medium range of pdi. the specific surface area (ssa) of the particles is the summation of the areas of the exposed surfaces of the particles per unit mass. result of this study showed a reduction in particle size and consequently high surface area of domperidone nanoparticles when compare with pure drug (16). particle size of formulas f1-f5 of different polymer used at same polymer:drug ratio of 1:1 gave different particle size range of 334-667.5nm, this indicates that, polymers have different affinity to domperidone particle, even at same ratio. the lowest particle size was achieved with pvp-k15 polymer in f3, the measured particle size of formula f6-f10 at polymer:drug ratio of 2:1 was significantly (p≤0.05) decreased that are in range from 84.05-265.5nm, the smallest particle size was attained with pvp-k15 polymer (f8) at this ratio. the smallest size obtained in f8 as shown in figure (1) this might be due to high affinity of pvp-k15 to domperidone and low viscosity grade than other polymers of hpmc and cmc. also that, it is found that the increase in the polymer concentration lead to a decrease in the prepared particle size of domperidone nanoparticles, as result of complete wrapping or covering and stabilize of drug particle in small size. therefore, f8 was selected and subjected for further studies. the polymers used in this study were anionic and cationic, which might be play an important role in stabilizing of the system by steric effect, this is could be achieved by adsorbing of polymer onto the surface of particle through an anchor part that is strongly interacts with the dispersed particles, while the other solvated tail part extends into the bulk medium(17). the effect of stirring time on domperidone nanoparticles as shown in figure (2), it is found that the increase in the time of stirring lead to increase size of domperidone nanoparticles. this finding agrees with that obtained by chopra (18). the higher stirring rate induces rapid nucleation toward smaller drug particles (19). the effect of stirring rate on size of the prepared domperidone nanoparticles and was found that, the increase in stirring rate led to a significant (p≤0.05) decrease in the size of the prepared domperidone nanoparticles as shown in figure (3). the effect of ratio of volume of solution containing drug (solvent) to the solution containing polymer (antisolvent) on the size of the prepared domperidone nanoparticles was studied and iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 43 results are concise in figure (4), and it was found that the solvent: antisolvent volume ratio of 1:10 gave the significant (p≤0.05) lowest mean of particle size in comparison to other ratios, that is might be result of optimum molecular distribution of drug and polymer for stabilization. the same result has been observed by dong and coworkers in the preparation of spironolactone nanoparticles using 1:10 ratio (20). figure 1. effect of type and concentration of polymers in on average size (n=3) of domperidone nanoparticles figure 2. effect of time of stirring on average size (n=3) of domperidone nanoparticles figure 3. effect of stirring rate on the average size (n=3) of domperidone nanoparticles figure 4. effect of solvent:anti solvent ratio on the average size (n=3) of domperidone nanoparticles iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 44 table 2. average of particle size ranges (n=3), averages, poly dispersity index (pdi) and specific surface area (ssa) of prepared domperidone nanoparticles. formula particle size range pdi ssa m2/g f1 472.5 0.009 4.5 f2 421 0.009 5.01 f3 334 0.014 6.3 f4 375 0.012 6.01 f5 667.5 0.014 3.32 f6 188 0.05 11.34 f7 167.5 0.15 12.49 f8 84.05 0.011 28.07 f9 118 0.3 17.07 f10 236.5 0.064 9.22 f11 530.5 0.01 4.43 f12 530.5 0.009 4.38 f13 375 0.026 5.82 f14 472.5 0.018 4.8 f15 840.5 0.036 2.62 f16 426 0.009 5.06 f17 334 0.009 6.39 f18 375 0.009 6.2 f19 421 0.021 5.19 f20 749 0.045 2.89 evaluation of selected formulas of domperidone nanoparticles drug content and loading efficiency the measured drug content result from formula f8 was 1.94±0.01mg. the loading efficiency of f8 was 97.3±0.5%, so that the solvent:antisolvent method was effective in preparing domperidone nanoparticles. saturated solubility of pure domperidone and nanoparticles. general statement of solubility increases when particle size decreases, is due to the increase of the surface area was considered. so on, the saturated solubility of domperidone nanoparticles of f8 was increased significantly (p≤0.05) in all solvent media of 0.1n hcl of ph 1.2, phosphate buffer of ph 6.8 and water as shown in table (3), and to ensure increase in saturated solubility of domperidone nanoparticles is due to reduction particle size, physical mixture of pvp-k15 and pure domperidone (at ratio of 2:1) was used and studied. table 3. the average (±sem, n=3) of saturated solubility in mg/ml of domperidone nanoparticles and pure domperidone at 37±0.5° c. 0.1n hcl of ph 1.2 phosphate buffer of ph 6.8 water pure domperidone 0. 98± 0.0033 0.02± 0.001 0.016± 0.0004 f8 2.769± 0.0003 0.08± 0.001 0.08± 0.002 physical mixture of pvpk15:pure domperidone (2:1) 1.4± 0.03 0.03± 0.001 0.035± 0.0005 field emission scanning electron microscope(fesem) fesem imaging of pure domperidone powder, pvp-k15 powder and dried liquids of f8 showed a reduction in particle size of domperidone particles to nano range. pure domperidone particles were presented with irregular shape, rough surface and large particles as shown in figure (5). polyvinyl pyrrolidone pvp-k15 showed spherical shape as in figure 6 (a and b). fesem of f8 as in figure 7, explained uniform and small particles size and this result might be caused by adsorption or capping effect of the stabilizer on drug surface and reduce size of domperidone particle to nanometer size. figure 5. fesem of pure domperidone withmagnification power of (58.98kx). iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 45 figure 6. fesem of pvp-k15 particle with magnification power of a-395x and b-1.84kx. figure 7. fesem of f8 with magnificationpower of 180.35 kx. x –ray powder diffraction analysis the obtained spectrum of x-ray diffraction test of pure domperidone showed several strong characteristic peaks at 2θ = 9.28º, 14.94º, 15.58º, 19.80º, 24.80º, and 32º as shown in figure (8), which indicates the crystalline state of pure drug. xrpd of pvp-k15 showed low intense peaks due to the amorphous nature as shown in figure (9). some domperidone peaks were still appeared with the physical mixture as in figure (10). domperidone nanoparticles of f8 as shown in figure (11) showed less number and low intense of diffraction peaks in comparison to that of pure domperidone, which indicates that the crystalline structure of domperidone was reduced and part of it converted into amorphous state, similar result was also found in candisartan nanoparticles that prepared by filipcsei’s research group (21). figure 8. xrpd spectrum of pure domperidone iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 46 figure 9. xrpd spectrum of polymer pvp-k15 figure 10. xrpd spectrum of physical mixture of pvp-k15 and pure domperidone (at ratio of 2:1)). iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 47 figure 11. xrpd spectrum of f8 fourier transform infrared spectroscopy (ftir) ftir was carried out for domperidone and nanoparticles of f8. domperidone exhibited a strong characteristic absorbance band at 1714 cm1 due to c = o stretching vibrations of amide functional group conhr, and n ̶ h bending characteristic band at 1693 cm-1, n ̶ h stretching band of secondary amine appeared at 3126 cm-1 as a single band, symmetric and asymmetric c ̶ h stretching bands appeared at 2810 and 2941 cm-1 respectively. as well as aromatic symmetric and asymmetric c ̶ h stretching bands appeared at 3024 and 3074 cm-1 respectively, and the aromatic c = c stretching band appeared at 1624 cm-1(22) as shown in figure (12). the resulted ftir of pvpk15, the physical mixtures of pvp-k15:pure domperidone (2:1) and f8 as in figures (13, 14 and 15) showed the presence of main peaks of domperidone which indicates there is no interaction or complexation between drug and polymer during preparation of nanoparticles. figure 12. ftir spectrum of pure domperidone iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 48 figure 13. ftir spectrum of pvp-k15 figure 14. ftir spectrum of physical mixture of pvp-k15:pure domperidone (2:1) figure 15 . ftir spectrum of f8 differential scanning calorimetry (dsc) dsc thermogram of domperidone as in figure (16) showed a sharp endothermic peak at 249ºc, this melting point of pure domperidone as referenced (23) and revealed that the drug has crystalline nature with high purity. dsc of pvpk15 as in figure (17) showed broad peak of water evaporation at 107º c this indicate amorphous nature of this polymer. physical mixture of pvpk15:pure domperidone (2:1) showed broad and low intensity peak of domperidone which is nearly at same position within the range of melting point in figure (18). this indicates no chemical reaction or complexation between drug and polymer, dsc of f8 in figure (19) showed remarkable reduction in peak intensity in iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 49 comparison with pure domperidone, that indicates a reduction of crystalline state of domperidone and conversion of part of it to amorphous state, this result with that obtained x-ray diffraction analysis of formula (f8) agrees with younis finding (24). figure 16. dsc thermogram of pure domperidone. figure 17. dsc thermogram of pvp-k15 iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 50 figure 18. dsc thermogram of physical mixture of pvp-k15:pure domperidone (2:1) figure 19. dsc thermogram of domperidone nanoparticles (f8) in-vitro dissolution study of domperidone nanoparticles form capsule dosage form the percentage of drug release at15min and the time required for released 100% of drug were considered for the comparison of the dissolution results between pure and nanoparticles of domperidone. the release of domperidone from f8 was faster in comparison with pure domperidone, and reached to100% of accumulative % of drug release at 15min., whereas the pure domperidone reached to 58% and 90% at 15min at the end of the study, respectively, as shown in figure (20). in addition, physical mixture of pvp-k15:pure domperidone (2:1) reached 60% and 90.9% at15 and 75min, respectively. these results indicate that, the increase in percentage of release of domperidone from nanoparticles was due to the increase in the surface area of these particles (25) and to the reduction in crystalline state of domperidone. these findings support that, the use of precipitation method was efficient and effective in preparing nanoparticles. iraqi j pharm sci, vol.27(1) 2018 domperidone nanoparticles 51 figure 20. the in-vitro release of the pure domperidone, f8, physical mixture of pvpk15:pure domperidone (2:1) in 0.1n hcl of ph 1.2 at 50rpm and 37° c (±sem, n=3). the comparison between the dissolution profiles was done using difference and similarity test of f1 and f2 respectively. the data of accumulative percentages of release of drug from selected formula was fitted using a microsoft excel program to calculate f1 and f2 and the obtained results as illustrated in table (4). it was noticed that the dissolution profiles of domperidone from nanoparticles was not similar in comparison with the pure drug as a reference, f8were not similar as f1 values was higher than 15, whereas their f2 values were lower than 50(26). from these results, it can conclude that nanoparticles showed better in vitro dissolution profiles in comparison with pure drug. table 4. difference and similarity test (f1 and f2) of selected formula and physical mixture of pvp-k15:pure domperidone (2:1) compared versus pure domperidone f1 f2 f8 37.6 29.46 physical mixture of pvp-k15:pure domperidone (2:1) 9.02 50.5 conclusions depending on obtained results, one can concludes that, pvp-k15 gave best formula (f8), the variables of type, concentration of stabilizer, volume ratio of solvent: anti solvent, time and rate of stirring showed considerable effect on decreasing of the size of domperidone nanoparticles. on increase of polymer: drug ratio, the size of produced domperidone nanoparticles 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and m. h. evaluation and comparison of different brands of domperidone tablets available in evaluation and comparison of different brands of domperidone. pakistan journal of pharmaceutical sciences 2014; 27, 935-938. iraqi j pharm sci, vol.20(2) 2011 rheumatoid factor and chloroquine phosphate 50 the correlation between rheumatoid factor, chloroquine phosphate in osteoarthritis eman s. saleh * ,1 *department of clinical laboratory science,college of pharmacy,university of baghdad, baghdad, iraq abstract osteoarthritis (oa) is a series of aggressive destructive inflammatory processes. synovitis is common both at an early and a late phase. this disease may be uniquely singular in some site but phylogenetically related at some point in time to produce a common outcome of dysfunction, disability, socioeconomic destruction and sometimes socioeconomic failure. articular cartilage, subchondral bone and synovial membrane are the site of major abnormalities in this disease process. rheumatoid factor (rf) represents one of the routine laboratory tests that made for all patients have joint complaints.chloroquine phosphate (cqp) is agent belong to disease modifying osteoathritic drugs (dmoads). chloroquine and their derivatives have been used for their anti-inflammatory effect in juvenile chronic arthritis, sjogren , s syndrome, discoid and systemic lupus erythematosus.the aim of this trial depend on using this drug in osteoarthritic patients for two months then estimate the level of rf check and rf(a,g,m).the result showed a significant correlation between cqp and rf check as well as rf type m in patients suffered from oa, so the level of these two parameters are decreased significantly in period of treatment thus leads to ameliorate the patients status and the joints pain will decreased. key words: chloroquine, osteoarthritis, rheumatoid factors. لخالصها أىفصاه اىؼظَٜ ػثارٓ ػِ سيسئ ٍِ اىؼَيٞاخ اىٖادٍٔ اىؼذائٞٔ االىرٖاتٞٔ ىيَفاصو . اىرٖاب اىغشاء اىَصيٜ ٍَنِ حص٘ىٔ فٜ اىؼظَٜ قذ تنُ٘ ٍرَٞشا ٗٗحٞذا فٜ تؼط ٍِ اسثاب حص٘ه اىفصاه اىَزاحو اىَرط٘رٓ اٗ اىَرأخزٓ ٍِ ٕذا اىَزض . ْٕاك ّ٘ػِٞ االٍامِ اٍٗؼرَذا ػيٚ اىرط٘ر اىْ٘ػٜ فٜ ٍ٘اظغ اخزٙ ٍَا ٝسثة اىؼجش ٗػذً اىنفاءٓ فٜ اداء اى٘ظائف ٗاىؼ٘س االجرَاػٜ و غثٞؼٞٔ فٞٔ. ذؼِٞٞ ػاٍاالقرصادٛ ٍؤدٝا ىفشو حزمح اىَفاصو ٗاىؼظاً اىَحٞطٔ تٖا مذىل اىغشاء اىَصيٜ ىحص٘ه ذغٞٞزاخ ال ػقار ف٘سفاخ اىني٘رمِ٘ٝ اثثد ىيَزظٚ اىيذِٝ ٝؼاُّ٘ ٍِ ٍشامو فٜ اىَفاصو.اىَؼَ٘ه تٖا اىزٍٗاذٞشً ٍِ اىرحيٞالخ اىَخرثزٝٔ فٜ اٍزاض ػذٝذٓ ٍْٖا َو ساتقاؼمؼاٍو ٍغٞز ٍٗعاد ىالىرٖاب فٜ اىفصاه اىؼظَٜ.اٍا ػقار اىني٘رامِ٘ٝ اىْقٜ ٍٗشرقاذٔ اسر اسرؼَاىٔ ٕذف ٕذٓ اىرجزتٔ ٕ٘ اسرخذاً اىَشٍْٔ اى٘الدٝٔ, ٍرالسٍح ج٘جزِٝ, داء اىذئثٔ االحَزارٝٔ اىجٖاسٛ ٗ اىقزصٜ. االىرٖاتاخ اىزث٘ٝٔ (أٛ, جٜ, أً )ف٘سفاخ اىني٘رامِ٘ٝ ىفرزج شٖزِٝ ىغزض ذؼِٞٞ ػاٍو اىزٍٗاذٞشً اىَزذثػ اىنيٜ ٗاىخاص تنو تزٗذِٞ ٍْاػٜ ّ٘ع عاً فٜ ٍسرشفٚ ٍذْٝح ٍزٝ 52ٗ األصحاءٍرط٘ػاً ٍِ 52اىرجزتح ػيٚ أجزٝدٝٔ تَْٖٞا.تاُ ْٕاك ػالقٔ ٗثٞقٔ ٍٗؼْ٘.اظٖزخ اىْرائج ىرؼِٞٞ ّسثح اىؼاٍو اىزث٘ٛ اىنيٜ ثٌ اجزاء ذحيٞو آخز ػيٚ ٗاألصحاءٍاذٞشً ٗاىرإٔٞو اىطثٜ ذٌ ذحيٞو دً اىَزظٚ اىطة/قسٌ اىزٗ ْاخ /ٍو ٍِ اىذً ىينشف ػيٚ اىَنّ٘اخ اىَفص٘ىح ٍِ اىثزٗذٍٞاٝنزٗ 52ٍِ أػيٚاىؼاٍو ٕذا اىَزظٚ اىيذِٝ ٝنُ٘ ىذٌٖٝ ذزمٞش حثح ٍٝ٘ٞاً ٍِ 5ىَذج شٖزِٝ تجزػح ِ.صزف ىيَزظٚ ػقار اىني٘رام٘ٝٗ أّ٘اػٖا ِٞ ٗذؼِٞٞ ٍسر٘ٝاذٖاتاىَْاػٞح اىَحر٘ٝح ػيٚ اىغي٘ ّسثرٖا ٍَا ٝقيو ٍِ ذٖذً ٍؼْ٘ٛ فٍٜيح٘ظ ٗ ْٕاك اّخفاض أُى٘حع ذٕا ػْٞد ذزامٞش ٕذٓ اىثزٗذْٞاخ. قثو اىطثٞة االخرصاصٜ تؼ . اظافح اىٚ ذقيٞو االىٌ ٗذحسِ حاىح اىَزظٚ اىؼظَٞح ٗاىغعزٗفٞح األّسجح introduction osteoarthritis (oa) is the most common form of arthritis, the major cause of pain and disability in older people as well as is a condition of synovial joints characterized by focal loss of articular hyaline cartilage with proliferation of new bone and remodeling of joint contour (1,2,3,4,5) . according to american college of rheumatology (acr) (6) , many signs and symptoms such as (age over 50, stiffness less than 30 minutes , crepitus , bony enlargement & tenderness ,no palpable warmth ,erythrocyte sedimentation rate less than 40 mm/ hour, rf & synovial fluid examination) at least five of them associated with oa in addition to pain.rheumatoid factors (rfs) is a laboratory test used to detect the antibodies (abs) (7) . although these factors have been associated with a person who had arthritis they are also present in normal individual (8). the titers were elevated in many cases such as viral infection, acute and chronic inflammation and lymphoproliferative disease (9,10). 1corresponding author email :dreman@yahoo.com received : 15/5/2011 accepted : 24/10/2011 iraqi j pharm sci, vol.20(2) 2011 rheumatoid factor and chloroquine phosphate 51 chloraquine cq and their derivatives were classified as anti-malarial agents or disease modifying osteoarthritic drugs (dmoads). the mechanism of these agent were complicated through interfering with many pro-and anti-inflammatory mediators that directed to breakdown cartilage also playing important role in decreasing and antibodies production as their immune effects (11,12,13,14,15). in this study attempted to use chloraquine phosphate as dmoads that has an ability to prevent, retard or reverse some signs and in oa in humans by decreasing the titer of rf check especially rf type m. patients and methods twenty-five healthy peoples (27.77)% as control and sixty-five patient (72.22%), all of them were male selected randomly by rheumatologist. the study began in january – december (2007) , the patient ’ s age range from (50 to 55) year with their mean age (54.51±2.35). detailed history of patient including : age, weight, body mass index ,occupation, duration of disease, past history of disease or operation and the use of non steroidal anti-inflammatory drug was obtained. x-ray was observed by rheumatologist and classified according to kellgren and lawrence grading criteria which start from grade (0 to iv) so the first grade represent no feature of oa and the last one is joint space greatly impaired with sclerosis of subchondral bone (16). cqp 150mg tablet were dispensed to the patients for two month, twice daily after meal. rfs value were assessed quantitatively by enzyme linked immune sorbent assay elisa (aida-rf chelal and a, m, g kits) were purchased from aida gmbh (auto immune diagnostic assay).the aim of this study is to measure the real effects of cqp on rf titer and take care dispensing corticosteroids or other agents because the age of patients related with chronic diseases such as hypertension and diabetes. serum samples were estimated by elisa rfcheck kit, if the concentration of sample is more than 20 µ/ml, the rfs agm must done in order to determine each immunoglobulin separately (17,18) .forty five (69.23%) from the total samples be above 20µ/ml. rfs check and agm were calculated as mean ± standard error of mean (m±sem) paired ±test.the analysis was done in central public health laboratory in immunological department. results in this study the presented data showed the level of rfs of patients and healthy subjects.table (1) include rfs check for all individuals and rfs agm for patients who their rfs check were above 20 µg/ml, also there are comparisons before and after 2 months of cqp treatment. figure (1) show the level of rfs check only between control, all patients before treatment and after 2 months figure (2) show the level at rfs agm for control, (69.23%) of total patients before and after using the drug. table 1: the serum level of rfs check and rfs agm for control and patients. control (c) patients before using cqp (p0) p value (c+ p0) patients after 2 months of using cqp (p2) p value (c+ p2) no. of subjects percentage(%) 25 22.77% 65 77.22% 20 30.76% rfs check µ/ml 18.73±1.22 30.49±2.89 p<0.05 s 19.12±1.27 p<0.05 s rfs check > 20 µ/ml percentage(%) 45 males from 65 (69.23%) 45 males (all) (100%) iga µ/ml 2.07±0.31 2.87±0.13 p>0.01 ns 2.39±0.13 p>0.01 ns igg µ/ml 9.99±0.45 10.12±0.79 p>0.01 ns 7.54±1.01 p<0.001 s igm µ/ml 6.76±0.68 37.95±1.02 p<0.005 s 10.45±1.62 p<0.001 s s : significant ns : non-significant iraqi j pharm sci, vol.20(2) 2011 rheumatoid factor and chloroquine phosphate 52 figure 1: levels of rf check in patients groups figure 2:levels of rfs agm in control and patients before and after 2 months of cqp treatment discussion in this study the presented data showed the level of rfs of patients and healthy subjects as control. table (1) include rfs check for all individuals and rfs agm for patients who their rfs check were above 20 µg/ml, also there are comparisons before and after 2 months of cqp treatment.figure (1) show the level of rfs check only between control(c) and all patients before treatment (p0) and after 2 months (p2) while figure (2) show the level at rfs agm for control, (69.23%) of total patients before and after using the drug.the correlation between rfs values and oa at the same time of using this agent appeared to be significant depending on the mode of action of chloroquine through ameliorating the signs and symptoms of the patients. this drug classified as weak base so has direct effect on lysosomes and stabilization of lysosomal membrane by increasing ph inside this part of cell as well as interfering with enzyme activity dependant on acidic environ, impairment of endocytosis and processing of antigen by macrophages, antigen presentation, posttranscriptional modification of protein and cytokine secretion also suppress the responsiveness of tlymphocytes and decrease polymorphnuclear cell chemotaxis, inhibit of immune complex formation through decrease the secretion of interleukin one alpha by monocyte and interleukin six by monocyte and tcell (11-15) .oa defined as a condition characterized by a series of inflammatory processes 50% synovitis is common, both as an early and late phase (20, 21) . clinically oa is a disorder of diarthrodial joints lead to pain, activity limitation (22) . radiography show the presence of osteophytes and joint space narrowing, while histopathologic find the alteration in cartilage integrity (23) . the pathologic feature of oa is a progressive loss of articular cartilage as well as affect synovial joints, subchondral bone, synovium, meniscus, ligaments, and supporting neuromuscular apparatus in addition to cartilage (2,3) . rfs are auto antibodies (aab) directed against antigenic determinants on the fragment crystalizable (fc) of igg molecule (17,23) . the production of rfs is induced during the course of many acute and chronic inflammatory diseases (2,3) where as igm rfs predominant in most of these condition, igg rfs are occasionally produced (24) .cq was previoualy used as dmoads to treat rheumatic diseases such as rheumatoid arthritis, discoid and systemic lupus erythromatosis also has anti inflammatory effects in juvenile chronic arthritis and sjogren syndrome (11-14). before five years many investigations studied the effects of cqp on oa as disease modifying agent and as decrease many pro inflammatory (il1,6,8), tumor necrosis factor-alpha (tnfα), complements, c-reactive protein (2528) . conclusion the levels of serum rfs combined and separated were determined by elisa in oa before and two months after using cqp, this factor usually used for rheumatoid arthritis but in this study for osteoarthritis with follow up of using chloroquine for two months. at zero time rf-check (combined ig agm) measure high titer due to disease (significant increase). this increasing followed by a decrement in rfs-check or rf agm after 60 days of taking the drug that play a role in acting as anti inflammatory also as a disease modifying anti rheumatic agent. references 1. doherty m, ralston sh.: muscloskeletal diseases in davidson’s principle and practice. (21 1 st ed.). nicki r., brian r., stuart h (eds.) walker, 2010; p: 1053. fig-1-levels of rf check in patients groups 0 5 10 15 20 25 30 35 1 2 3 patients groups 1(control), 2(patients before cqp), 3(patients 2 months after cqp) l e v e l s o f r f c h e c k / m l fig-2levels of rfs agm in control and patients before and after 2 months of cqp treatment 0 5 10 15 20 25 30 35 40 iga igg igm groups of control and patients l e v e ls o f r f s a g m ( /m l) control before using cqp 2 months after using cop iraqi j pharm sci, vol.20(2) 2011 rheumatoid factor and chloroquine phosphate 53 2. kumar v.y., abbas a.k., fausto n., aster j.c.: pathologic basis of diseases (8 th ed.). robbins and cotran (eds.), saunders elisevirs, 2010, p: 1. 3. kumar v.y., abbas a.k., fausto n., et al: general pathology: musclo skeletal joints, (8 th ed.). robbins (ed.), 2007; p: 818-824. 4. porth c. m.: pathophysiology: concept, of altered health status (7 th ed.) philadelphia: lippincott williams and wilkens, 2005. 5. geller a.c., rosen a.: inflammatory rheumatic introduction to clinical medicine. 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(8 th ed.) lippincott williams & wilkins, 2009; p: 565. 19. brandt k.b: osteoarthritis, 2 nd ed., oxford. oxford university press: 2003. 20. felson dt: osteoarthritis of the knee, n. engl. j. med., 2006; 354; 841. 21. fauci as, braun wald e, kasper dl, hauser sl: disorder of joints in harrison’s principle of internal medicine. 22. lippincott williams&wilkins,2007;p:100. 23. wickelgren i: policing the immune system science. 2004 ; 306; 596-599. 24. parekh rb, dwek ra, sutton bj et al: association of rheumatoid arthritis and primary osteoarthritis with changes in the glycosylation pattern of total serum igg. nature , 1985; 316; 452. 25. jawad hm, salman s. : the effect of chloroquine phosphate as a disease modifying a sent in osteoarthritis. a thesis submitted to the college of medicine and the committee of the requirements for the degree of doctor of philosophy in clinical pharmacy 2004. 26. jalab a, jawad hm ; the effect of chloroquine phosphate on pro inflammatory interleukin (1 b, 6 and 8) in knee osteoarthritis. a thesis submitted to the college of medicine and the committee of postgraduate studies in partial fulfillment of the requirement for the degree of master in clinical pharmacology , 2007. 27. saleh es, turki km, munshed mh: evaluation the role of chloroquine phosphate in a sample of iraqi patients with knee osteoarthritis: a thesis submitted to the college of medicine and the committee of postgraduate studied in partial fulfillment and the requirement for the degree of doctor of philosophy in clinical biochemistry, 2008. 28. saleh es, turki km, munshed mh: the role of chloroquine phosphate on acute phase reactant proteins in patients with knee osteoarthritis 2009; 18,56. iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 85 a comparative study of blood levels of manganese, some macroelements and heavy metals in obese and non-obese polycystic ovary syndrome patients sarah h. mhaibes*,1 , mohammed a. taher* and ala h. badr** *department of clinical laboratory science, college of pharmacy, university of baghdad, baghdad, iraq. ** kamal samarrai hospital , ministry of health/environment , baghdad, iraq. abstract polycystic ovary syndrome (pcos) is a prevalent condition in women of reproductive age. it is characterized by androgen excess and chronic anovulation. some trace elements, macroelements, and heavy metals have been linked to pathophysiological mechanisms of pcos . to study the alterations in the serum levels of the trace element manganese (mn), some macroelements, magnesium(mg) and calcium (ca), and the heavy metals cadmium (cd) and lead (pb), in obese and non-obese pcos patients; and the association of these alterations with some of the hormonal changes occurring in pcos. the study was carried out at kamal al-samarrai hospital (center for infertility treatment and in vitro fertilization "ivf") baghdadiraq. eighty-two women were enrolled in the study. fifty-four of them were diagnosed by a specialist gynecologist as pcos patients; they were subdivided into two subgroups according to their body mass index (bmi); twenty-seven obese pcos patients with bmi > 30 kg/m2, and another twenty seven non obese patients pcos with bmi <30 kg/m2. whereas, twentyeight apparently healthy women with regular menstruation and of comparable age, were selected to serve as control groups; they were subdivided into, fourteen obese women with bmi > 30kg/m2, and fourteen non obese women with bmi <30 kg/m2. blood lead and cadmium levels were significantly higher in both of the obese and the nonobese pcos groups, than in their corresponding control groups. while, serum magnesium, calcium and manganese levels were significantly lower in both of the obese and the non-obese pcos groups, as compared to their corresponding control groups. the results revealed no significant difference in the levels of the measured elements, between the obese pcos group and the non-obese pcos group. the serum fsh levels was significantly lower in obese pcos patients than in the obese and non-obese control groups. there was a positive correlation between blood lead and serum tsh levels in nonobese pcos women; and between serum total testosterone and cadmium levels in obese pcos women. finally, there was negative correlation between serum magnesium and serum lh levels in non-obese pcos women. the study has demonstrated higher blood levels of lead and cadmium; and lower serum levels of magnesium, calcium and manganese in pcos groups than control subject. there were no significant differences between obese pcos women and non-obese pcos women in the levels of the studied hormones, elements and heavy metals. keywords: pcos, trace elements, macroelements, heavy metals, infertility. فيالعناصر الرئيسية والفلزات الثقيلة بعض و من المنغنيزمستويات الدم ل ةدراسة مقارن المريضات المصابات بمتالزمة المبيض متعدد االكياس البدينات وغير البدينات من **و االء حازم بدر *حمد عباس طاهر، م 1 *,سارة هاشم محيبس بغداد ، بغداد ، العراق .فرع العلوم المختبرية السريرية ، كلية الصيدلة ، جامعة * ، بغداد ، العراق . والبيئة مستشفى كمال السامرائي ، وزارة الصحة ** الخالصة عدم االباضة و بزيادة االندروجينهي حالة سائدة لدى النساء في سن اإلنجاب. وتتميز متعدد االكياسمتالزمة المبيض آلليات المرضية الفيزيولوجية لمتالزمة المبيض قد ترتبط با الثقيلة فلزاتوال، العناصر الرئيسية بعض العناصر النزرة ان. المزمن ،بعض العناصر الرئيسية من لعنصر المنغنيزالتغيرات في مستوى المصل مع دراسة ارتباط التغيرات الهرمونية .متعدد االكياس بين النساء البدينات وغير البدينات المصاباتالكادميوم والرصاص الفلزات الثقيلة ومستوى الدم الكلي منالمغنيسوم والكالسيوم -أجريت الدراسة في مستشفى كمال السامرائي )مركز عالج العقم والتخصيب في المختبر( بغداد . كياسمتعدد اال متالزمة المبيضب ةتحت إشراف أخصائي مبيض متعدد االكياسالمتالزمة ب يضاتمنهن مر ٤٥من النساء، تم اختيار ة متطوع ٢٨العراق. شملت الدراسة لواتيال المبيض متعدد االكياسمتالزمة ب ةمريض ٨٧: الىإلى مجموعات فرعية وفقا لمؤشر كتلة الجسم تم تقسيمهن و نسائية وتوليد ، جيدة صحيةبحالة امرأة على ما يبدو ٨٢ن من السمنة المفرطة في حين تم اختيار ييعان ال اخريات٨٧ن من السمنة المفرطة و ييعان . طيف االمتصاص الذري هو طريقة تستخدم مع انتظام الدورة الشهريةالسمنة ال يعانين٤٥ويعانين من السمنة ٤٥إلى هنتم تقسيم العينات. كلالثقيلة في فلزاتالنزرة وال العناصرلقياس كمية من 1corresponding author e-mail: phsarahhashim@gmail.com received: 9/9/ 2017 accepted:27 /11/2017 mailto:phsarahhashim@gmail.com iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 86 المبيض متعدد ن من متالزمة ييعان لواتيال يضاتالمر ةالرصاص والكادميوم أعلى بكثير في مجموع اظهرت الدراسة ان المبيض متعدد متالزمة ب يضاتالمغنيسيوم والكالسيوم والمنغنيز في مر انخفاض ، في حين الطبيعياتالنساء ةمقارنة بمجموع االكياس على التوالي. لم تكشف النتائج النساء الطبيعيات البدينات وغير البدينات على حد سواء بالمقارنة مع البدينات وغير البدينات االكياس في مستوى العناصر البدينات وغير البدينات متعدد االكياس المبيض عانين من متالزمةعن وجود فرق معنوي بين النساء اللواتي ي المقاسة. يضاتالمر ةمجموعهناك عالقة طردية بين مستويات الدم من الرصاص مع مستويات المصل من الهرمون المحفز للغدة الدرقية في ينات باال ضافة الى ذلك كان هناك ارتباط طردي بين مستويات الدم من المبيض متعدد االكياس غير البدن من متالزمة ييعان لواتيال المبيض متعدد االكياس ن من متالزمة ييعان لواتيال يضاتالمر ةمجموعالكادميوم ومستويات مصل الدم من التيستوستيرون في لواتيفي النساء الlh من هرمون بين المغنيسيوم في الدم ومستويات المصل عكسيوعالوة على ذلك، كان هناك ارتباط البدينات. المبيض متعدد االكياس غير الديناتمن متالزمة ينيعان في ارتفاع مستويات الدم من الرصاص والكادميوم في حين انخفاض مستويات المصل من المغنيسيوم والكالسيوم والمنغنيز . لم تكن هناك فروق ذات النساء الطبيعيات ةمقارنة بمجموع المبيض متعدد االكياسن من متالزمة ييعان لواتيال يضاتالمر ةمجموع في المعلمات المدروسة البدينات وغير البدينات المبيض متعدد االكياسداللة إحصائية بين النساء اللواتي يعانين من متالزمة العناصر والفلزات الثقيلة.للهرمونات، . ، العقميسيةئالعناصر الر، العناصر النزرة ، المعادن الثقيلة، االكياسمتعدد ة: متالزمة المبيضالكلمات الرئيسي introduction citsycylop ovary syndrome (pcos) is symptomatic nearly 6% to 10% of women of reproductive age (12–45 years old) (1). it is a principal cause of female subfertility and the most frequent endocrine problem in women of reproductive age, although up to 70% of women with pcos may be undiagnosed (2). polycystic ovary syndrome is characterized by anovulation, insulin resistance and hyperandrogenism. anovulation causes irregular menstruation, amenorrhea, ovulationrelated infertility and polycystic ovaries. hyperandrogenism results in acne and hirsutism. insulin resistance is often correlated with obesity, type 2 diabetes, and high cholesterol level (3). the etiology of pcos is not yet entirely known. it is believed that it is a complex, multifaceted disease involving abnormalities in the hypothalamic-pituitary axis, uncontrolled ovarian steroidogenesis, excessive oxidative stress, aberrant insulin signaling and genetic/environmental factors (4,5). many studies have indicated increased oxidative stress in the patients with pcos (6,7). several characteristics and associations of pcos, including androgen excess, abdominal adiposity, insulin resistance and obesity, may contribute to the development of local and systemic oxidative stress which may reciprocally worsen these metabolic abnormalities (8). the whole dollb of heavy metals and serum levels of trace elements were altered in patients with pcos (9). environmentally relevant levels of metals are also associated with modest changes in reproductive hormone levels (10). cadmium is a heavy metal and may have a role in the formation of reactive oxygen species (ros) (11). lead may also lead to the production of ros by depleting glutathione (gsh) and protein-bound sulfhydryl groups and enhancing lipid peroxidation (12). calcium ia sn essential macroelement to body organization and structure and is the material of many indispensable substances, such as 25 hydroxyvitamin d (25(oh)d) and highdensity lipoprotein (hdl) cholesterol, which contribute to the cardiovascular system (13). magnesium, one of the abundant cations in the human body categorized as macro element, participates in enzymatic reactions and insulin secretion. magnesium is closely related to diabetes mellitus type 2 and other metabolic diseases (14). magnesium deficiency is believed to indirectly enhance the oxidative damage of biomolecules by inducing a stress response. it is possible that a magnesium deficiency stimulates catecholamine release from the adrenal glands. however, catecholamine increases the production of ros. this creates a vicious positive feedback cycle where, for example, elevated blood epinephrine levels result in a further reduction of the magnesium concentration. contrastingly, magnesium deficiency leads to the activation of the reninangiotensin system that also induces oxidative stress (15). manganese is an essential micronutrient incorporated into many metalloenzymes and proteins involved in cell metabolism and regulatory pathways controlling oxidative stress (16). subjects and methods this study was carried out at kamal al-samarrai hospital (center for infertility treatment and in vitro fertilization "ivf"), from october /2016 to april/2017. eighty-two women were enrolled in the study. fifty-four of them were patients with pcos, with the age (obese 29.04± 5.64, non-obese 26.56±4.97) and twenty-eight apparently healthy women were selected as controls, their age (obese 32.07±5.86 and non-obese 24.64±4.25). every participant woman was interviewed and requested to answer a specially designed questioning format iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 87 including; demographic data, menstrual, obstetric, medical and family histories. the diagnosis of pcos in this study was based on the revised rotterdam criteria, which require, two of the following three manifestations: (1) clinical and/or biochemical hyperandrogenism (2) oligo-and/or anovulation (cycle length >35 days), and (3) polycystic ovaries on ultrasound (polycystic ovary was defined as the appearance of more than 11 follicles in each ovary, each measuring 2-9 mm in diameter, and/or increased ovarian volume > 10 ml) (17). with the exclusion of other etiologies (androgen-secreting tumors, thyroid disorder, cushing syndrome, hyperprolactinemia, congenital adrenal hyperplasia). all of the patients were selected under the supervision of a specialist gynecologist. the studied women were divided into: afifty-four pcos patients, they were subdivided into two subgroups based on their body mass index (bmi), which was calculated as the ratio of the body weight in kilograms to the square of its height in meters (18). the two subgroups were as follows; twenty-seven obese pcos patients with bmi ≥ 30 kg/m2 (34.81±0.820), and another twenty-seven non-obese pcos patients with bmi <30 kg/m2 (26.41± 0.401). btwenty-eight apparently healthy control women were subdivided into two subgroups; fourteen obese control subjects with bmi ≥ 30kg/m2 (32.81±0.720), and fourteen nonobese control subjects with bmi <30 kg/m2 (23.90±0.678). venous blood samples (10cc) was withdrawn hcle msrf woman between days two and four of menstrual cycle after overnight fasting. blood samples were divided into two parts, one part (2.5cc) were collected in an anticoagulant containing tubes for direct measurement of lead (pb), and cadmium (cd) blood levels and the other part (7.5cc) were collected in gel and clot activator tubes without anticoagulant then samples left for 30 minutes to clot. after complete clotting, the serum was separated by centrifugation (centrifuged for 10 minutes at 3500 to 4000 rpm to obtain serum). the collected serum was divided in 10 eppendorf tubes [9 tubes kept frozen (-80˚c) until their assay, and the rest of them used for immediate measurement of fasting glucose level]. assay the whole blood levels of cadmium, lead and serum levels of magnesium, calcium, manganese were measured by atomic absorption spectrophotometer in poisoning consultation center in medical city-baghdad. serum luteinizing hormone (lh), follicle stimulating hormone (fsh) and total testosterone (tes) were tested by enzymelinked final fluorescent assay (elfa) (19,20). serum prolactin (prl) and serum thyroid stimulating hormone (tsh) were measured by a two-site sandwich immunoassay using direct chemiluminometric technology (21,22). fasting blood glucose was evaluated according to the method of barham and trindoer (23). statistical analysis the results were expressed as a mean ± standard error of the mean (sem) or percent changes. levene test was used to test the homogeneity of variances for equality of variances of the several independent groups. one-way analysis of variance (anova) and least significant tests were used for comparisons between the studied groups, and to examine the degree of significance. pearson’s correlation coefficient (r) was used to study the correlation of trace element (manganese), some macroelements and heavy metals with the measured parameters of hormones. the statistical analysis was performed using spss, version 16 (24). results the anthropometric and demographic characteristics of polycystic ovary syndrome (pcos) patients and controls are summarized in table 1. as illustrated in table 2, the serum fsh level was significantly lower in obese pcos patients than in the obese and non-obese control groups (p<0.01, p<0.05 respectively); there was no significant difference between obese and non-obese pcos (p˃0.05). serum lh, lh/fsh ratio, prolactin and total testosterone levels were significantly higher in both obese and non-obese pcos patients as compared to their corresponding control groups. yet, there was no significant difference between obese and non-obese pcos groups as well as between the control groups. tsh levels were significantly higher in the obese pcos than in the obese control (p<0.05). serum glucose levels were significantly higher in the obese pcos than in the obese and non-obese control groups (p<0.05, p<0.01 respectively), but there was no significant difference between the nonobese pcos and the non-obese control (p> 0.05). table 3 showed that, the whole blood concentrations of lead (pb) and cadmium (cd) were significantly higher in the pcos groups than in the control groups; yet, there was no significant difference between the obese and non-obese pcos groups (p> 0.05), as well as between the obese and non-obese control iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 88 groups. finally, serum concentrations of magnesium(mg), calcium(ca) and manganese (mn) were significantly lower in both of the obese and non-obese pcos groups as compared to their corresponding control groups; and there was no significant difference between the obese and nonobese pcos group. table (1): the anthropometric and demographic data of pcos patients and controls p-value controls pcos groups variables non-obese (14) obese (14) non-obese (27) obese (27) 0.204 24.64 ± 1.14 32.07 ± 1.57 26.56 ± 0.96 29.04 ± 1.09 age(years) 0.000** 23.90 ± 0.67 32.81 ± 0.72 26.41 ± 0.40 34.81 ± 0.82 bmi (kg/m2) 0.000** 76.64 ± 2.04 100.00 ± 1.96 87.76 ±1.68 103.48 ± 2.81 waist (cm) 0.000** 94.00 ± 0.70 119.4 ± 1.58 103.6 ± 1.64 116.9 ± 2.73 hip (cm) 0.000** 0.782 ± 0.02 0.838 ± 0.012 0.847 ±0.012 0.885 ± 0.011 whr 0.214 0 0 5 (18.5%) 9 (33.3%) f.hx of pcos 0.001** 0 2(14.2%) 2 (7.4%) 13 (48.1%) f.hx of obesity + + u/s for pcos 0.190 0 0 4 (14.8%) 8 (29.6%) a.n. 1.000 0 0 24 (88.9%) 24 (88.9%) hirsutism 0.564 0 0 10 (37%) 8 (29.6%) acne 0.776 0 0 9 (33.3%) 10 (37%) male pattern baldness 0.379 0 0 17 (63%) 20 (74.1%) om 0.535 0 0 8 (29.6%) 6 (22.2%) am 1.000 14 (100%) ٤٥ (100%) 2 (7.4%) 1 (3.7%) regular cycle the data are expressed as the numbers (percentage) or mean ± standard error (se). **p < 0.01 are highly significantly different. f.hx: family history, u/s: ultrasound, a.n: acanthosis nigricans, om: oligomenorrhea, am: amenorrhea table (2): descriptive statistics of studied sex hormones with comparative significant studies between groups (a) and groups (b) in serum levels of sex hormones. saliairav sluorv numbers mean ±se groups( a) groups(b) p-value* fsh (miu/ml) obese pcos (27) 4.592 ± 0.194 obese pcos obese control 0.004 (hs) non-obese pcos 0.348(ns) obese control (14) 6.499 ± 0.790 non-obese control 0.030 (s) non-obese pcos (27) 5.092 ± 0.332 obese control non-obese pcos 0.031(s) non-obese control (14) 6.010 ± 0.657 non-obese control 1.508 (ns) nonobese pcos non-obese control 0.156 (ns) lh (miu/ml) obese pcos (27) 5.006 ± 0.389 obese pcos obese control 0.001 (hs) non-obese pcos 1.000 (ns) obese control (14) 2.840 ± 0.315 non-obese control 0.000 (hs) non-obese pcos (27) 5.000 ± 0.428 obese control non-obese pcos 0.001(hs) non-obese control (14) 2.541 ± 0.259 non-obese control 0.882 (ns) nonobese pcos non-obese control 0.000 (hs) iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 89 continued table (2) variables groups numbers mean ±se groups(a) groups(b) p-value* lh/fsh ratio obese pcos (27) 1.152 ± 0.114 obese pcos obese control 0.000 (hs) non-obese pcos 0.985 (ns) obese control (14) 0.448 ± 0.068 non-obese control 0.000 (hs) non-obese pcos (27) 1.095 ± 0.112 obese control non-obese pcos 0.000 (hs) non-obese control (14) 0.465 ± 0.055 non-obese control 0.993 (ns) non-obese pcos non-obese control 0.000 (hs) prolactin (ng/ml) obese pcos (27) 27.54 ± 2.44 obese pcos obese control 0.000 (hs) non-obese pcos 0.796(ns) obese control (14) 11.30 ± 2.02 non-obese control 0.000 (hs) non-obese pcos (27) 24.06 ± 2.91 obese control non-obese pcos 0.005 (hs) non-obese control (14) 9.99 ± 1.61 non-obese control 0.956(ns) non-obese pcos non-obese control 0.001 (hs) uaar aavauvaalute (ng/ml) obese pcos (27) 3.078 ± 0.363 obese pcos obese control 0.000 (hs) non-obese pcos 0.882(ns) obese control (14) 1.229 ± 0.104 non-obese control 0.000(hs) non-obese pcos (27) 2.667 ± 0.424 obese control non-obese pcos 0.031(s) non-obese control (14) 1.175 ± 0.163 non-obese control 0.992 (ns) non-obese pcos non-obese control 0.012 (s) st (miu/l) obese pcos (27) 3.064 ± 0.868 obese pcos obese control 0.018 (s) non-obese pcos 0.054(ns) obese control (14) 0.935 ± 0.109 non-obese control 0.075 (ns) non-obese pcos (27) 1.639 ± 0.156 obese control non-obese pcos 0.428 (ns) non-obese control (14) 1.472± 0.259 non-obese control 0.598 (ns) non-obese pcos non-obese control 0.850 (ns) fsg (mmol/l) obese pcos (27) 6.530 ± 0.387 obese pcos obese control 0.012 (s) non-obese pcos 0.122(ns) obese control (14) 5.229 ± 0.149 non-obese control 0.002 (hs) non-obese pcos (27) 5.874 ± 0.312 obese control non-obese pcos 0.207 (ns) non-obese control (14) 4.936± 0.193 non-obese control 0.616 (ns) non-obese pcos non-obese control 0.068 (ns) (*) ns: non significant (p> 0.05); hs: highly significant (p<0.01); s: significant (p<0.05) iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 90 table (3): descriptive statistics of trace elements and heavy metals with significant comparative studies between groups (a) and groups (b) in blood levels of studied elements saliairav sluorv mean ±se groups(a) groups(b) p-value* lead (µg/dl) obese pcos (27) 22.48 ± 0.42 obese pcos obese control 0.000 (hs) non-obese pcos 0.360 (ns) obese control (14) 14.29 ± 0.42 non-obese control 0.000 (hs) non-obese pcos (27) 23.00 ± 0.45 obese control non-obese morbid 0.000 (hs) non-obese control (14) 14.29 ± 0.44 non-obese control 1.000 (ns) non-obese pcos non-obese control 0.000 (hs) cadmium (µg/dl) obese pcos (27) 0.317 ± 0.009 obese pcos obese control 0.000 (hs) non-obese pcos 1.000 (ns) obese control (14) 0.144 ± 0.006 non-obese control 0.000 (hs) non-obese pcos (27) 0.318 ± 0.011 obese control non-obese pcos 0.000 (hs) non-obese control (14) 0.139 ± 0.008 non-obese control 0.974 (ns) non-obese pcos non-obese control 0.000 (hs) magnesium (mg/dl) obese pcos (27) 0.979 ± 0.025 obese pcos obese control 0.000 (hs) non-obese pcos 0.721 (ns) obese control (14) 1.410 ± 0.038 non-obese control 0.000 (hs) non-obese pcos (27) 0.963 ± 0.030 obese control non-obese pcos 0.000 (hs) non-obese control (14) 1.378 ± 0.060 non-obese control 0.706 (ns) non-obese pcos non-obese control 0.000 (hs) calcium (mg/dl) obese pcos (27) 7.319 ± 0.111 obese pcos obese control 0.000 (hs) non-obese pcos 0.228(ns ) obese control (14) 9.107 ± 0.174 non-obese control 0.000 (hs) non-obese pcos (27) 7.082 ± 0.153 obese control non-obese pcos 0.000 (hs) non-obese control (14) 8.979 ± 0.228 non-obese control 0.6 (ns) non-obese pcos non-obese control 0.000 (hs) iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 91 continued table (3) variables groups mean ±se groups(a) groups(b) pvalue* manganes e (µg/dl) obese pcos (27) 0.086 ± 0.002 obese pcos obese control 0.000 (hs) non-obese pcos 0.931(n s) obese control (14) 0.225 ± 0.009 non-obese control 0.000 (hs) non-obese pcos (27) 0.084 ± 0.002 obese control non-obese pcos 0.000 (hs) non-obese control (14) 0.236 ± 0.009 non-obese control 0.837 (ns) non-obese pcos non-obese control 0.000 (hs) (*) ns: non significant (p> 0.05); hs: highly significant (p<0.01); s: significant (p<0.05) correlation studies pearson´s correlation was conducted to study the association of the alterations in the levels of the measured elements, with some of the hormonal changes occurring in pcos. the results are shown in table 4. table (4): pearson´s correlation of the measured elements and heavy metals with the hormone levels in the obese pcos and non-obese pcos groups groups hormones correlation and significant elements and heavy metals pb cd mg ca mn non-obese pcos fsh r -0.145 0.271 -0.180 0.089 0.093 p 0.471 0.171 0.368 0.660 0.643 lh r 0.344 0.066 -0.43* -0.012 0.267 p 0.079 0.742 0.027 0.954 0.642 lh\fsh r 0.366 -0.202 -0.078 -0.065 0.310 p 0.061 0.313 0.698 0.748 0.116 tsh r 0.384* -0.138 0.262 -0.028 0.245 p 0.048 0.494 0.187 0.891 0.218 prolactin r -0.005 -0.202 0.053 0.066 -0.080 p 0.981 0.312 0.846 0.744 0.690 testosterone r 0.109 0.064 0.203 0.168 0.045 p 0.589 0.751 0.309 0.402 0.824 fbs r 0.061 0.280 -0.141 0.057 -0.070 p 0.761 0.157 0.482 0.780 0.728 obese pcos fsh r 0.084 -0.118 0.141 -0.233 -0.130 p 0.677 0.559 0.483 0.241 0.518 lh r 0.012 -0.063 0.107 -0.014 0.202 p 0.953 0.756 0.597 0.945 0.313 lh\fsh r -0.009 -0.046 0.008 0.152 0.171 p 0.966 0.821 0.970 0.448 0.393 tsh r 0.122 -0.130 -0.143 -0.044 0.090 p 0.544 0.519 0.478 0.826 0.654 prolactin r 0.254 0.090 0.023 0.632 -0.371 p 0.201 0.657 0.908 0.743 -0.130 testosterone r -0.070 0.465 * -0.123 0.330 0.004 p 0.727 0.015 0.540 0.093 0.986 fbs r -0.207 -0.260 0.212 -0.060 0.078 p 0.299 0.191 0.288 0.767 0.699 *correlation is significant at the 0.05 level. ** correlation is significant at the 0.01 level. iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 92 there was positive correlation between blood lead and serum tsh levels (r 0.384, p 0.048) in non-obese pcos women (figure 1). in addition, there was positive correlation between serum total testosterone and cadmium levels (r 0.465, p 0.015) in obese pcos women (figure 2). furthermore, there was negative correlation between serum magnesium and serum lh levels (r 0.43, p 0.027) in non-obese pcos women (figure 3). figure (1): correlation between blood lead (µg/dl) and serum tsh (l/ im) (r 0.384, p 0.048) in non-obese pcos women. figure (2): correlation between serum total testosterone (ng/ml) and cadmium(cd) (µg/dl) (r 0.465, p 0.015) in obese pcos women. figure (3): correlation between serum magnesium (mg/dl) and serum lh (miu /ml) (r 0.425, p 0.027) in non-obese pcos women. discussion the diagnosis of pcos in this study was based on rotterdam criteria that are confirmed by the results obtained from analysis data related to the measured hormones (25). trace elements and heavy metals might be involved in the development of pcos (9). the present study was designed to investigate serum heavy metal, manganese and some macroelement concentrations in relation to hormone levels in pcos. in table 3, lead (pb) levels were significantly higher in pcos patients than in the control groups. and there was a positive correlation between blood lead concentration and serum tsh level in nonobese pcos patients (r 0.384, p 0.048). this occurs in accordance with singh et al. who has declared that, an increase in serum tsh levels was associated with exposure to lead, without alteration in serum t3 and t4; and proposed that lead may enhance pituitary tsh release (26). chang et al. has investigated the relationship between lead exposure and the risk of infertility in taiwan women, reporting mean blood lead levels of 35.5 and 27.8 μg/l in infertile women and pregnant women, respectively. the blood lead level was significantly higher in infertile women (27). the pathogenic effect of lead is multifactorial since it directly hinders the activity of enzymes, competitively inhibits absorption of important trace minerals and deactivates antioxidant sulfhydryl pools. free radical-induced damage by lead is accomplished by two independent, although related mechanisms. the first involves the direct formation of ros including singlet oxygen, hydrogen peroxides, and hydro peroxides, and the second mechanism is achieved by depletion of the cellular antioxidant pool. interrelations between these two mechanisms exist so that the increase in ros on one side simultaneously leads to depletion of antioxidant pools on the other (28). table 3 also showed that, there was significantly higher blood concentration of cadmium in pcos patients than in controls, and there was a positive correlation between blood concentration of cadmium and serum total testosterone levels, which is one of the main characteristics of pcos, in obese pcos patients (r 0.465, p 0.015). the toxic mechanisms of cadmium are not well understood, but it is known to act intracellularly, mainly via free radical-induced damage, particularly to reproductive organs (28). as with lead, the higher cadmium levels in pcos may contributes to the creation of ros. the possible mechanism explaining the role of cadmium in free radical generation may involve displacement of copper and iron by iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 93 cadmium from binding protein, the displaced copper can catalyze breakdown of hydrogen peroxide via the fenton reaction (28). excessive generation of ros is documented in patients with pcos (6). therefore, lead (pb) and cadmium (cd) may have a significant role in the pathogenesis of pcos linked to the oxidative stress. calcium, is a basic element in the human skeletal system, maintains body growth and is involved in muscle activity and neurotransmitter release. both of the obese and non-obese pcos patients in this study, had significantly lower levels of serum calcium than in the control subjects as can be seen in the table 3. similar findings were shown by li m et al (29). another study has showed that serum calcium levels are low in pcos patients, and these low levels can cause atherosclerosis (30). mazloomi et al. has reported that calcium positively affects pcos (31). in addition, a clinical study has found that calcium and vitamin d supplements are effective medicines for pcos (32). concerning magnesium, the results indicated a significantly lower levels, in both of the obese and non-obese pcos patients, than the levels in the control groups, as can be seen in table 3. usharani et al. has revealed a lower level of serum magnesium in pcos patients (33). hypomagnesemia alters glucose entry into the cells leading to insulin resistance, thus, dysregulation of the hypothalamus-pituitarygonadal axis (34). the serum lh level was inversely correlated with magnesium levels in non-obese pcos patients. fantidis et al. agrees with our result, by showing that low magnesium level in rats was associated with increased release of lh hormone (34). table 3 showed that, serum manganese levels in both of the obese and non-obese pcos patients, were significantly lower than that in the corresponding control subjects. manganese is a cofactor for some metalloenzymes, like manganese-containing superoxide dismutase sod (mnsod). it neutralizes the highly reactive superoxide ions to less reactive hydrogen peroxide (h2o2), which is followed by its immediate conversion to h2o by catalase and other peroxidases in the mitochondrial matrix, hence, it protects against oxidative stress (35). the result of the present study regarding manganese occurs in accordance with another study, that showed lowered serum level of manganese in pcos patients as compared to that of control subjects (36). references 1. barbosa g, de sá lb, rocha dr, et al. polycystic ovary syndrome (pcos) and fertility. open journal of endocrine and metabolic diseases. 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of metal ions. free radical biology and medicine. 1995; 18(2):321-36. https://www.dovepress.com/international-journal-of-womens-health-journal https://www.dovepress.com/international-journal-of-womens-health-journal iraqi j pharm sci, vol.26(2) 2017 blood. levels of trace elements and heavy metals in pco 94 13. li m, tang y, lin c, et al. serum macroelement and microelement concentrations in patients with polycystic ovary syndrome: a cross-sectional study. biological trace element research. 2017; 176(1):73-80. 14. evangelopoulos aa, vallianou ng, panagiotakos db, et al. an inverse relationship between cumulating components of the metabolic syndrome and serum magnesium levels. nutrition research. 2008; 28(10):659-63. 15. zhang d, luo wy, liao h, et al. the effects of oxidative stress to pcos. journal of sichuan university. medical science edition. 2008; 39(3):421-3. 16. tapiero h, tew kd. trace elements in human physiology and pathology: zinc and metallothioneins. biomedicine & pharmacotherapy. 2003; 57(9):399-411. 17. the rotterdam eshre/asrm-sponsored pcos consensus workshop group. revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (pcos). human reproduction. 2004; 19:41-7 18. rahman m, berenson ab. accuracy of current body mass index obesity classification for white, black, and hispanic reproductive age women. obstetrics and gynecology 2010; 115:982–8. 19. wide l, loraine ed j a, bell e t (eds.), human pituitary gonadotropins: hormone assays and their clinical application, 4thed, london and new york, churchill livingstone, edinburgh; 1976, pp: 78-140. 20. forest mg, cathiard am, bertrand ja. total and unbound testosterone levels in the newborn and in normal and hypogonadal children: use of a sensitive radioimmunoassay for testosterone. the journal of clinical endocrinology and metabolism. 1973; 36:1132–42. 21. owens o. steroidal hormonal evaluation for common gynecological and testicular disorders. in: kaplan la, pesce aj, editors. methods in clinical chemistry. st. louis: cv mosby, 1987. p.216– 7. 22. sc c. watts nb, endocrinology in: tietz nw editor fundamentals of clinical chemistry, third ed. philadelphia: wb saunders. 1987.p.550-1 23. barham d and trindoer p: an improved color reagent for the determination of blood glucose by the oxidative system. analyst. 1972; 97: 142-5 24. rosner b, editor. fundamentals of biostatistics. pws-kent publishing company, boston, 3rd edn, 1990. 25. teede h, deeks a, moran l. polycystic ovary syndrome: a complex condition with psychological, reproductive and metabolic manifestations that impacts on health across the lifespan. bmc medicine. 2010; 8:41. 26. singh b, chandran v, bandhu hk, et al. impact of lead exposure on pituitarythyroid axis in humans. biometals. 2000; 13(2):187. 27. chang sh, cheng bh, lee sl, et al. low blood lead concentration in association with infertility in women. environmental research. 2006;101(3):380–6. 28. jomova k, valko m. advances in metalinduced oxidative stress and human disease. toxicology. 2011; 283(2-3):65-87. 29. li m, tang y, lin c, et al. serum macroelement and microelement concentrations in patients with polycystic ovary syndrome: a cross-sectional study. biological trace element research. 2017; 176(1):73-80. 30. lerchbaum e, giuliani a, gruber hj, et al. adult‐type hypolactasia and calcium intake in polycystic ovary syndrome. clinical endocrinology. 2012; 77(6):834-43. 31. mazloomi s, sharifi f, hajihosseini r, et al. association between hypoadiponectinemia and low serum concentrations of calcium and vitamin d in women with polycystic ovary syndrome. international scholarly research network endocrinology. 2012 32. dehghani firouzabadi r, aflatoonian a, modarresi s, et al. therapeutic effects of calcium & vitamin d supplementation in women with pcos. complementary therapies in clinical practice. 2012; 18(2):85-8. 33. thys-jacobs s, donovan d, papadopoulos a, et al. vitamin d and calcium dysregulation in the polycystic ovarian syndrome. steroids. 1999; 64(6):430-5. 34. fantidis p, gancedo p, méndez j, et al. short-term and intermediate-term effects of low blood magnesium on progesterone, lh and fsh levels in rats. hormone and metabolic research. 1995; 27(03):159-60. 35. ozden o, park sh, kim hs, et al. acetylation of mnsod directs enzymatic activity responding to cellular nutrient status or oxidative stress. aging (albany ny) 2011; 3: 102-7. 36. chakraborty p, ghosh s, goswami s, et al. altered trace mineral milieu might play an etiological role in the pathogenesis of polycystic ovary syndrome. biological trace element research. 2013; 152(1):915. iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 51 uric acid as a natural scavenger of peroxynitrite in a sample of iraqi patients with rheumatoid arthritis zahraa ma. naji* ,1 , shaimaa m.mohammed * and mays f. muhammad* * department of clinical laboratory science,college of pharmacy,university of baghdad,baghdad, iraq abstract rheumatoid arthritis (ra) is a chronic inflammatory disease associated with decreased antioxidant state .this study aim to investigate the status of oxidant/antioxidant in a sample of iraqi patients with ra and the role of peroxynitrite and its natural scavenger uric acid in them .this case-controlled study was conducted at baghdad teaching hospital /baghdad from december 2010-may 2011 . twenty-five patients with mean age 39 years and 25 apparently healthy subject as controls with mean age 29 years were included in the study .investigations include estimation of serum levels of nitric oxide (no) ,peroxynitrite (pn) , malondialdehyde (mda) , and uric acid (ua) .serum pn levels were significantly elevated in ra patients as compared with control subjects , ua levels were found significantly lowered in ra patients as compared with control subjects. no significant differences were found between no and mda among patients and controls group .in conclusion :this study demonstrated decreased serum uric acid levels in patients with ra accompanied by decreased its effect as a natural scavenger of pn. key word : rheumatoid arthritis ,peroxynitrite ,uric acid. لذي نمىرج من المشضً العشاقُُن للبُشوكسُنتشات ككاسح طبُعٍ دوس حامض الُىسَك المصابُن بالتهاب المفاصل الشثىٌ صهشاء محمذ علٍ ناجٍ ،*1 محمذشُماء منزس و * محمذ و مُس فاضل * * فشع اىعيً٘ اىَخخبشٝت اىسشٝشٝت ، ميٞت اىصٞذىت ، جاٍعت بغذاد ، بغذاد ، اىعشاق . لخالصة ا ٖذف ٕزٓ اىذساست ىخقٌٞٞ ٍشحبػ ٍع اّخفاض ٍعذالث ٍعاداث االمسذة. حاىخٖاب اىَفاصو اىشث٘ٛ ٕ٘ ٍشض اىخٖابٜ ٍضٍِ ٗمزىل دٗس بٖزا اىَشضاىَشظٚ اىعشاقِٞٞ اىَصابِٞ َّ٘رج ٍِ االمسذة ىذٙ اثٍعادٗ االمسذة اىخ٘اصُ بِٞ حاىخٜ حاٍط اىٞ٘سٝل ىذٙ ٕؤالء اىَشظٚ .اجشٝج ٕزٓ اىذساست فٜ ٍسخشفٚ بغذاد اىخعيَٜٞ سح اىطبٞعٜ ىٔ ٕٗ٘ٗ دٗس اىنا اىبٞشٗمسْٞخشاث ٍشٝط ٍصاب باىخٖاب اىَفاصو 02شاسك فٜ ٕزٓ اىذساست . 0200ىغاٝت اٝاس 0202اىعشاق فٜ اىفخشة ٍِ ماُّ٘ االٗه -بغذاد– ٗحٌ حقٌٞٞ ٍعذالث سٞطشةسْت حٌ اخخٞاسٌٕ مَجَ٘عت اى03.3ٌ ٍخ٘سػ اعَاسٕشخص سيٌٞ 02ٗ سْت 93اعَاسٌٕ ٍخ٘سػ اىشث٘ٛ اٗمسٞذ اىْخشاث ,اىبٞشٗمسْٞخشاث ,اىَاىّ٘ذاٝاىذٖٝاٝذ ٗحاٍط اىٞ٘سٝل فٜ َّارج اىذً اىَسح٘بت ٍِ اىَجَ٘عخِٞ .اظٖشث اىْخائج اسحفاعا , بَْٞا ماّج ٍعذالث حاٍط اىٞ٘سٝل شةاىسٞطفٜ ٍعذالث اىبٞشٗمسْٞخشاث ىذٙ ٍجَ٘عت اىَشظٚ باىَقاسّت ٍع ٍجَ٘عت ٍعْ٘ٝا فٜ ٍعذالث اٗمسٞذ اىْخٞشاث ٍعْ٘ٛ, ماُ ْٕاك فشقا غٞش ىسٞطشةىذٙ اىَشظٚ باىَقاسّت ٍع ٍجَ٘عت ا ٍعْ٘ٝاٍْخفعت اّخفاظا اّخفاض اشاسث اىذساست اىٚ اّخفاض ٍعذالث حاٍط اىٞ٘سٝل ىذٙ ٕؤالء اىَشظٚ ٍشحبػ ٍع ٗاىَاىّ٘ذاٝاىذٖٝاٝذ بِٞ اىَجَ٘عخِٞ . دٗسحاٍط اىٞ٘سٝل مناسح غبٞعٜ ىجزس اىبٞشٗمسْٞخشاٝج . . حامض الُىسَك البُشوكسُنتشاَث ،التهاب المفاصل الشثىٌ ، تاحُة :فالكلمات الم introduction rheumatoid arthritis (ra) is a chronic inflammatory and autoimmune disease (1) , characterized by a chronic hypertrophic synovitis leading to destruction of connective tissues and functional damage of cartilage and bony structure (2) . there is evidence indicating that a low antioxidant status is associated with a higher risk of developing ra (3) . the rheumatoid inflammation is associated with an increased generation of oxidants (reactive oxygen and reactive nitrogen species ) , which play an important role in the inflammatory process and contribute to tissue destruction (4) .several mechanisms exist whereby oxygen radicals might be generated within the joint in ra ,these include the release of such species from activated synovial macrophages and polymorphs (5) ,the prostaglandin pathway (6) and xanthine oxidase mediated synovial ischemic reperfusion injury (7) . nitric oxide (no) is a biological messenger mediating many important physiological functions but also pathological process .it play a vital role in host defense and immunity by modulating inflammatory processes (8) . the increased production of no has been linked to both protective and proinflammatory mechanism associated with tissue damage in inflammatory diseases (9) . during acute and chronic inflammation ,superoxide is produced at rates that overwhelm the capacity of the endogenous superoxide dismutase (sod) enzyme defense system to remove it (10) ,superoxide interacts with and destroys the biological activity of 1 corresponding author e-mail: zahraali139@yahoo.com received :25/2/2012 accepted :23/6/2012 iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 52 no (11) ,the rate of interaction between superoxide and no is faster than the rate of removal of superoxide by sod (12) .peroxynitrite is formed by the rapid combination of no and superoxide in a reaction that is limited only bydiffusion rate of the molecule (13) . under normal physiological conditions ,there is a low production of peroxynitrite resulting in a minimal amount of oxidative damage (14) ,while during inflammatory conditions ,large amount of no and superoxide anion are produced ,leading to the formation of a strong oxidant ,peroxynitrite anion (15) . the proposed cytotoxic properties of peroxynitrite include protein nitration, lipid peroxidation, inhibition of cellular metabolic pathways & signal transduction mechanisms and dna strand breakages (15) . important targets for oxidants are the unsaturated fatty acids in cell membranes. malondialdehyde (mda) is a product of lipid peroxidation and thereby functions as a marker of oxidative stress (16) , earlier studies have shown that the level of mda is related to ra disease activity (17) .uric acid is a metabolic product of purine metabolism that may function as an antioxidant (18) .some disease states such as gout have been shown to result when ua levels in the blood are too high ,while other conditions such as neurodegenerative diseases ,may be caused by reduced serum ua levels. the manipulation of serum ua levels has become a popular strategy in the treatment of a variety of disease (19). apart from being able to scavenge peroxynitrite, ua also scavenge singlet oxygen, peroxy and hydroxyl radicals, ozon and hypochlorous acid (20) . by using rat zymosan-induced arthritis; the administration of uric acid, in addition to reducing the inflammatory parameters, also prevents the loss of articular cartilage (21) .in this study we aimed to study the role of nitric oxide and peroxynitrite in ra patients and the role of uric acid as a scavenger of these free radical in those patients and if manipulation of serum ua levels is one of the methods in the treatment of ra. patients and methods this study was conducted at baghdad teaching hospital from december 2010-may 2011. twenty-five patients with ra; their ages ranged from 17-55 years who were attending the rheumatology consultation clinic of baghdad teaching hospital, and twenty-five apparently healthy subjects as control group with age range of 19-50 years were included in the study after inform consent .ra was diagnosed on the basis of the revised criteria of the american college of rheumatology (acr) (22) . exclusion criteria were pregnancy ,the presence of active infection and the presence of cancer ,since all can affect serum no level .ten milliliter blood samples were collected from all patients by vein puncture , 2 ml of each sample were transferred to anticoagulant tube [edta (ethylene diamine tetra acetic acid)] tube for erythrocyte sedimentation rate (esr) determination according to the westergreen method (23) .the rest of 8ml were transferred to 10 ml sterile plane tube, allowed to clot for 30 minutes at room temperature and centrifuged at 3000 rpm for five minute to obtain serum .serum aliquots were divided into four 1 ml eppendroffs tubes for nitric oxide, peroxynitrite ,mda and uric acid estimation .measurement of no level was performed according to the method of miranda et al (2001), absorbance was read at 540nm using elisa reader (biotek elx50 usa) (24). serum peroxynitrite level was determined according to the method described by beckman et al (25) , cited by vanuffelen et al (26) , in which the peroxynitrite mediated nitration of phenol was measured spectrophometrically (ecm lab spectrophotometer ,germany ) at 412 nm. the levels of serum ua was measured spectrophotometrically with kit from biomaghreb company (france) .malondialdehyde level was estimated as described by hunter et al (27) ,the absorbance of the supernatant was determined at 530 nm spectrophotometrically (ecm lab spectrophotometer ,germany ) . statistical analysis statistical analysis was performed using the statistical package for social sciences version 12 (spss inc., chicago, il., usa) .mean and standard error (se) were calculated. student t-test was used to evaluate the significance (p-value) between study variables .a p-value of<0.05 was considered statistically significant. results table -1 show the demographic characteristics of the subjects .there were nonsignificance difference between patients group and control subjects regarding gender, age and body mass index. female represent 40% of total patients ,while male represent 60% . among control subjects , female represent 48% of total group and male represent 42% . the mean age of patients group was (39.19 years) with age range (17-55) years. the mean age of control subjects was (29.9 years) with age range (19-50) years. the mean bmi of patients group was (25.5kg/m 2 ) ,with range of (21.3-35.2) kg/m 2 .the mean bmi of control iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 53 subjects was (24.9kg/m 2 ) ,with range of (1836.7) kg/ m 2 . serum analysis showed significant elevation in esr levels of patients group 88.33 ±4.38 ( mean ±se) mm/hr as compared with control subjects 14±1.29 ( mean ±se) mm/hr ,(p<0.05) . there was non-significant decrease in serum no levels in patients group 163.7±23.6 (mean±se) µmol/l as compared with control subjects 164.3±9.22 (mean ±se) µmol/l,( p>0.05) ,while there was significant increase in serum levels of peroxynitrite in patients group as compared with control subjects 8.71±1.16 (mean±se) µmol/l,(0.53±0.04) (mean±se) µmol/l respectively ,(p <0.05) .there was nonsignificant increase in serum levels of mda in ra patients group 4.76 ±0.31 (mean±se) nmol/ml as compared with control subjects 4.01 ±0.47 (mean±se) nmol/ml, (p >0.05) .the serum levels of ua were significantly decreased in patients group 4.61 ±0.73 (mean±se) mg/dl as compared with control subjects 8.3 ±0.64 (mean±se) mg/dl (p <0.05) table-2 shows the levels of erythrocyte sedimentation rate, nitric oxide, peroxynitrite, malondialdehyde and uric acid in patients with rheumatoid arthritis and healthy control subjects . table1:demographic data of studied groups describe in means ±standard error parameter ra patients control number 25 25 gender f/m 10/15 12/13 age (years) 39±2.5 29.9±2.1 bmi(kg/m 2 ) 25.5±0.62 24.9±0.79 bmi: body mass index . f/m: female /male . table 2: mean±standard error of nitric oxide, peroxynitrite, malondialdehyde, uric acid and erythrocyte sedimentation rates of patients with rheumatoid arthritis and healthy controls group no: nitric oxide onoo :peroxynitrite . mda:malondialdehyde esr:erythrocyle sedimentation rate discussion despite the synovial tissue being highly vascularized , the rheumatoid joint is recognized as a site with typical biochemical features of hypoxia-induced oxidative stress (28) , the accumulation of oxidized dna ,proteins and lipids within the inflamed rheumatoid joint provides evidence for the damaging effects of radicals in this pathology (29) . it was recently reported that no by itself is protective to chondrocyte under oxidative stress in vivo, while reactive oxygen species, including peroxynitrite, promote chondrocyte death (30) . in one study taysi et al (31) found that serum mda correlated positively with disease activity score ,and deaney et al (32) have reported correlation between esr and mda . the result of our study show non-significant increase in serum mda of ra patients compared to controls and correlate with results of kajanachumpol et al study that showed no significant change in mda levels in patients with ra compared to controls (7) . while in another study ; most patients had low to moderate disease activity ,no correlation between urine mda and disease activity variables were found (33) . the serum levels of no of both groups were not different despite the expected elevation in serum no levels in patients group due to the increase of superoxide production which leads to increase in formation of peroxynitrite as it found in this study,the elevation in peroxynitrite level could explained as a result from increasing nitric oxide and super oxide reaction . uric acid is a natural antioxidant, accounting for up to 60% of the free radical scavenging activity in human blood (34) , serum urate concentration in ra correlated inversely with oxidative changes in serum albumin and immunoglobulin g, it is suggested that serum urate might have an antioxidant role under certain conditions by limiting free radical induced oxidative changes to protein during inflammation (35) . although chronic gout and rheumatoid arthritis are common clinical entities, they seldom coexist (36) . in one study the plasma level of uric acid were inversely related to indices of ra disease activity (33) . peroxynitrite ,in particular , is believed to have a significant negative impact on cellular function and survival (14) .ua may assist in the removal of superoxide by preventing against the degradation of superoxide dismutase (14) , removal of superoxide helps to prevent its reaction with nitric oxide , blocking the formation of peroxynitrite (37) .ua is also very effective at preventing peroxynitrite from nitrating the tyrosine residues of proteins , parameter patients group controls group p value no(mol/l) 163.7±23.6 164.3±9.22 0.97 onoo (mol/l) 8.71±1.16 0.53±0.04 0.00 * mda(nmol/l) 4.76±0.31 4.01±0.47 0.183 esr(mm/hr) 88.3±4.38 14±1.29 0.00 * uric acid (mg/dl) 4.61±0.73 8.3±0.64 0.003 * iraqi j pharm sci, vol.21(2) 2012 uric acid and rheumatoid arthritis 54 thereby preventing the inactivation of cellular enzymes and modification of the cytoskeleton (14) .ua also has the ability to bind iron ,and inhibit iron –dependent ascorbate oxidation ,preventing an increased production of free radicals that can further contribute to oxidative damage (38) ,thus a reduced ua concentration may decrease the ability of the body to prevent peroxynitrite and other free radicals from acting on cellular components and damaging the cell (39) .the decrease in ua levels may be attributed to its oxidation by peroxynitrite and formation of allantoin , described to occur in humans (40) .patients in this study showed a significant decrease in serum levels of uric acid when compared to control subjects , in turn this can result in decrease protective effect of uric acid as a natural scavenger of peroxynitrite which will lead to further damage in those patients. so these patients are unable to prevent free radical toxicity, leading to the development of inflammation and destruction of tissues . on the other hand, the inflammation that occur in ra leads to the consumption of ua to scavenge the excess free radicals produced ,resulting in a lower ua level .we propose for further studies to estimate the relation between ua and oxidation markers other than pn such as glutathione and also study the role of other antioxidant in ra disease process. in conclusion, we can conclude that patients with ra showed lowered serum uric acid levels accompanied by decreased its activity as natural scavenger of peroxynitrite , while the levels mda and no between both groups showed non-significant difference . references 1. phillips d c,dias h k i,kitas g d ,et al .aberrant reactive oxygen and nitrogen spesies generation in rheumatoid arthritis (ra) :causes and consequences for immune function ,cell survival ,and therapeutic intervention .antioxidants & redox signaling . 2010 ;10 (6):743-785. 2. blake dr , merry p ,unsworth j ,et al .hypoxic reperfusion injury in the inflamed human joint .lancet . 1989; 1 :289-93. 3. knekt p ,heliovaara m ,aho k ,et 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nervous system inflammation .j neuroimmunol .1997 ; :77 :1-7 . 38. davies kj ,sevanian a ,muakkassah – kelly sf ,and hochstein p .uric acid-iron ion complexes .a new aspect of antioxidant functions of uric acid .biochem j .1986 ; 235:747-754. 39. meinda k. kutzing and bonnie l .firestein . altered uric acid levels and disease states . jpet . 2008; 324 :1-7 . 40. hellsten y,tullson pc ,richter ea ,bangsbo j. oxidation of urate in human skeletal muscle during exercise .free radical biol med 1997. ;22:169-174. iraqi j pharm sci, vol.29(2) 2020 synthesis of fenoprofen hydrazones with anti-inflammatory effect doi: https://doi.org/10.31351/vol29iss2pp239-244 239 synthesis, characterization and preliminary anti-inflammatory evaluation of new fenoprofen hydrazone derivatives hayder m. abdulhamza*,1 and muthanna s. farhan* *department of pharmaceutical chemistry, college of pharmacy, university of baghdad, baghdad, iraq abstract non-steroidal anti-inflammatory drugs (nsaids) have become important as an analgesic, antipyretic and anti-inflammatory medications throughout the world. in 2006, nsaids market in the us alone was valued at $3.2 billion, and it was probably to reach $4.6 billion by 2013. nsaid use can cause a range of serious adverse effects including gastrointestinal complications, cardiovascular problems, renal failure and hypersensitivity reactions. in order to reduce the side effects and improve the anti-inflammatory activity, new derivatives of fenoprofen were synthesized which contain hydrazones moiety. the compounds then evaluated for their antiinflammatory activity by means of egg white induced paw edema method. all the synthesized target compounds were characterized by ft-ir spectroscopy, 1hnmr analysis. the synthesis of the target compounds(h1-h4) was accomplished by multistep reaction procedures. the synthesized target compounds showed an activity in reducing paw edema thickness and their anti-inflammatory effect was comparable to that of the standard (fenoprofen) except for compound h3 which shows anti-inflammatory activity higher than fenoprofen. keywords: fenoprofen hydrozone derivatives, anti-inflammatory, paw edema method. للفينوبروفين هايدروزون جديدة لمشتقات اولي مضاد لاللتهاب وتقييم صوتشخي صنيعت **مثنى سعدي فرحان و 1،*حيدر محي عبد الحمزة .العراق بغداد، بغداد، جامعة الصيدلة، كلية الصيدالنية، الكيمياء فرع* لخالصةا المستخدمة على نطاق واسع والتي تستخدم لعالج االلتهاب مضادات االلتهاب غير الستيروئيدية هي واحدة من أكثر مجموعات األدوية ٪ من جميع األدوية الموصوفة كل عام. يمكن أن يسبب استخدام مضادات االلتهاب غير الستيروئيدية 10-5واأللم. وهي مسؤولة عن ما يقرب من واألوعية الدموية والفشل الكلوي وتفاعالت فرط مجموعة من اآلثار الضارة الخطيرة بما في ذلك مضاعفات الجهاز الهضمي ومشاكل القلب الهيدرازون الحساسية. من أجل تقليل اآلثار الجانبية وتحسين النشاط المضاد لاللتهابات ، تم تصنيع مشتقات جديدة من فينوبروفين تحتوي على جزء من ض البيض. جميع المركبات المحضرة شخصت بمطياف االشعة وتم تقييم فعاليتها المضادة لاللتهاب باستخدام طريقة وذمة المخلب المستحث ببيا تحت الحمراء وجهاز الرنين النووي المغناطيسي. تم تحضيرالمركبات باتباع طرق تفاعالت متعددة الخطوات, حيث وجدت المركبات المحضرة ان ينوبروفين عدا المركب لاللتهاب اعلى من الفينوبروفين.ورم المخلب وان تأثيرها كان مشابه للف h3لها فعالية في تقليل الذي اظهر تأثيرا مضادا . الكلمات المفتاحية: مشتقات الفينوبروفين هايدروزون, مضادة لاللتهاب, طريقة وذمة المخلب introduction non-steroidal anti-inflammatory drugs (nsaids) are one of the most widely used groups of medication used to treat inflammation and pain. they are accountable for nearly 5-10% of all medicines prescribes each year (1). nsaids activity is caused by inhibition of cyclooxygenases (cox-1) and (cox-2) enzymes, which are contributed in prostaglandin synthesis, causing their analgesic, anti-inflammatory, and antipyretic effects (2). this fall in prostaglandin synthesis is related to the occurrence of several undesirable effects accompanied with the use of nsaids, particularly gastrointestinal (gi) irritation and ulceration (3). nsaids can result in gi tract damages in two different ways: the acidic moiety irritates the gastric mucosa directly, furthermore, inhibition of cox-i that reduce the levels of protecting prostaglandins (4). nsaids carboxylic acid group can be substituted with other groups while these agents still exert a powerful anti-inflammatory activity (5). fenoprofen, 2-(3-phenoxy phenyl) propionic acid, or 2-methyl2-(3-phenoxy benzene) acetic acid (figure1) is a non-steroidal anti-inflammatory, analgesic and antipyretic drug belonging to groups of nsaids commonly referred to as 2-aryl propionic acids (6). like other nsaids, fenoprofen is a cyclooxygenase (cox-1 and cox-2) inhibitor that blocks the formation of prostaglandins that are important in pain and inflammatory pathways. current indications include mild-to-moderate acute pain as well as chronic joint pain due to osteoarthritis and rheumatoid arthritis. fenoprofen is generally welltolerated, but it has the side effects of nsaids especially gastrointestinal upset and ulceration (7). figure 1. chemical structure of fenoprofen 1corresponding author e-mail: hyder_muhy@yahoo.com received: 20/ 2/2020 accepted: 9/8 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp239-244 iraqi j pharm sci, vol.29(2) 2020 synthesis of fenoprofen hydrazones with anti-inflammatory effect 240 the class of organic compounds which have the structure r1r2c=nnh2 called hydrazones. hydrazones can be synthesized through the reaction of hydrazide or hydrazine with aldehydes and ketones (8). hydrazones have shown that they exert a wide variety of biological activities antimicrobial, analgesic, anti‑inflammatory, antidepressant, anticancer, antitubercular, and antifungal (9). some of the hydrazone derivatives were developed to overcome gastrointestinal disruption and toxicity (10). in an attempt to synthesize new fenoprofen analogs with improved anti-inflammatory activity and more selective toward the cox-2 enzyme which results in decreasing gastrointestinal side effects, new fenoprofen analogs containing hydrazone moiety were synthesized and assesses their anti-inflammatory activity. experimental: fenoprofen was bought from hyper chem. company (china). solvent and other reagents that used through reaction were bought from commercial sources. the purity of products and monitoring of the reactions were done by thin-layer chromatography tlc(gf254, merkgermany) under uv light (254nm) two solvent system was used a:toluene: ethyl acetate: ethanol (3:2:1) and b: ethyl acetate: petroleum ether(1:1). melting points were incorrected and detected by using stuart smp3 melting point apparatus in open capillary tubes. ir spectra were done by thin-film technique (υ, cm1) in the university of baghdad/college of pharmacy, (shimadzu ftir spectrophotometer, japan) (11,12). 1hnmr were done in the university of tehran/central laboratory using brucker ultra shield model 300 mhz using dmso as a solvent (13,14). synthesis of ethyl 2-(3-phenoxyphenyl) propanoate (compound a) fenoprofen (r, s racemic mixture) (9.9 mmol, 2.4g) was dissolved in ethanol (20ml) and cooled to 0ºc. thionyl chloride (2.15 ml, 29.7 mmol) was added slowly over 15 minutes, the mixture then stirred for 30 min. until it reaches room temperature. after that, it refluxed for 24 hr. at (80cº) with stirring. followed by stirring overnight at room temperature. at the end of the reaction (checked by tlc (b)), the solution allows to reach room temperature, after that, approximately 50 ml of distilled water was added to the solution. saturated sodium bicarbonate solution (10% w/v) was then added for the neutralization of excess of acid. the product was extracted by dichloromethane (dcm) 20 ml for 3 times, then evaporate dcm under reduced pressure to get oil (15). yield = 85%, rf = 0.92(b) ir (v cm-1): 3066: aromatic (c-h) str., 2981: (c-h) asym. str. of ch3 and ch2, 2873: (c-h) sym. str. of ch3 and ch2, 1732(c=o) str. of ester,1180.44(c-oc) str. of ester. 1h nmr: 1.12 (3h, t, -ch2ch3 of ester), 1.37(3h, d, -ch3), 3.76(1h, q, ch), 4.06(3h, b.s. -ch2ch3 of ester), 6.89-7.43 (9h,m, ar-h). synthesis of 2-(3-phenoxyphenyl) propanehydrazide (compound b): compound (a) (10 mmol,2.42 g) dissolved in 10 ml ethanol, followed by addition of the hydrazine hydrate (80%) (3 ml,60 mmol), the reaction mixture was refluxed for 8 at (80cº) hours, then left overnight with continuous stirring, then solvent evaporation under reduced pressure to give an oily product. the product then was washed with diethyl ether five times (16). yield = 86%, rf= 0.46(b) ir (v cm-1): 3317,3217 (nh) str. of hydrazide nh2, 3035 aromatic (c-h) str.,1612 (c=o) str. of carbonyl amide, 1566(n-h) bending. 1h nmr: 1.3(3h, d, -ch3) 3.5(1h, q, ch), 4.2(2h,s. nhnh2), 6.80-7.39(9h,m,ar-h), 9.18 (1h, s, nhnh2). synthesis of final target compounds (compounds h1-h4): ethanolic solution (5 ml) of one of the following aldehydes: 4-(dimethylamino) benzaldehyde (2mmol,0.298g), 4-nitro benzaldehyde (2mmol, 0.302g), 4-chlorobenzaldehyde (2mmol,0.281g) and 4-bromobenzaldehyde (2mmol, 0.37) were prepared, followed by the addition of three drops of glacial acetic acid and the solution was stirred for 30 min. compound b (2mmol, 0.512 g) was dissolved in 10 ml of absolute ethanol then added to the solution above, then refluxed for 4 hr. at (80 cº), followed by stirring overnight at room temperature. the precipitate which is form was filtered and recrystallized from ethanol (17). (h1) n'-(4-(dimethylamino) benzylidene)-2-(3phenoxyphenyl) propanehydrazide yellowish powder, yield: 82%, m.p.: (158-160°c) rf=0.82(a), ir (v cm-1): 3163 (nh) str. of hydrazone, 3078aromatic (c-h) str., 2943: (c-h) asym. str. of ch3 and ch, 2893,2850: (c-h) sym. str. of ch3 and ch, 1662: (c=o) str. of amidic.1577: (c=n) str. 1h nmr: 1.39 (3h, d, ch3),2.89(6h, s, -n-2ch3), 3.63(1h, q, -ch), 6.687.48 (13h,m, ar-h), 8.04(1h, s, n=c 11.2(1h, s, co-nh). iraqi j pharm sci, vol.29(2) 2020 synthesis of fenoprofen hydrazones with anti-inflammatory effect 241 (h2) n'-(4-nitrobenzylidene)-2-(3 phenoxy phenyl) propanehydrazide: white powder, yield: 80%, m.p.: (157-159°c), rf=0.84(a), ir (v cm-1):3178 (nh) str. of hydrazone, 3066aromatic (c-h) str., 2951: (c-h) asymm. str. of ch3 and ch, 2893,2854: (c-h) symm. str. of ch3 and ch, 1666: (c=o) str. of amidic.1581: (c=n) str1519 a sym. (no2) str, 1338: sym. str. of (no2). 1h nmr: 1.39 (3h, d, -ch3), (3.72, q, -ch), 6.837.9 (13h, m, ar-h), 8.3(1h, s, n=ch), 11.8(1h, 2s, -co-nh). (h3) n'-(4-chlorobenzylidene)-2-(3phenoxyphenyl) propanehydrazide: off-white powder, yield: 75%, m.p.: (155-156°c) rf=0.84(a), ir (v cm-1): 3178 (nh) str. of hydrazone, 3066aromatic (c-h) str., 2947: (c-h) asym. str. of ch3 and ch, 2904,2858: (c-h) sym. str. of ch3 and ch, 1662: (c=o) str. of amide.1577: (c=n) str.. 1h nmr: 1.37 (3h, d, -ch3),3.68 (1h, q, -ch), 6.81-7.7 (13h,m, ar-h), 8.18(1h, s, n=ch), 11.36,11.6(1h, 2s, -co-nh). (h4) n'-(4-bromobenzylidene)-2-(3phenoxyphenyl) propanehydrazide: white powder, yield: 70%, m.p.: (162-164°c) rf=0.78(a), ir (v cm-1): 3178 (nh) str. of hydrazone, 3066aromatic (c-h) str., 2947: (c-h) asym. str. of ch3 and ch, 2900,2873: (c-h) symm. str. of ch3 and ch2, 1662: (c=o) str. of amidic.1577: (c=n)str.. 1h nmr: 1.37 (3h d, ch3),3.68 (1h, q, -ch), 6.81-7.84 (13h, m, ar-h), 8.17(1h, s, n=ch), , 11.6(1h,2s, -co-nh). evaluation of the anti-inflammatory activity the anti-inflammatory effects of the synthesized products (h1-h4) were evaluated using an eggwhite induced paw edema model. measuring the reduction of paw thickness as the basis for the assessment of the anti-inflammatory activity of the anticipated compounds (18). albino rats of both sexes which have the weight of (190 ± 10 g) were delivered by the animal house of the national center for drug control and research, were accommodated in the animal house of the college of pharmacy, university of baghdad, under standardized environments for 10 days for adaptation. animals were fed commercial chaw and had free access to water. animals were divided into six groups (each group was containing 6 rats): group 1: which consists of six rats that worked as a control group; and gives the solvent (dimethyl sulfoxide) (19). group 2: consists of six rats treated with fenoprofen, which is the reference(standard) drug in a dose of 20 mg/kg (20), dissolved in the (dmso). group 3-6: consist of six rats/group treated with the target compounds h1-h4, and given the dose that is equivalent by weight to 20 mg/kg of fenoprofen and also dissolved in the (dmso). the procedure was done by the administration of an intra-peritoneal (i.p.) injection of each of the fenoprofen, control and final products (h1-h4), individually to the six animal groups. thirty minutes after that subcutaneous injection (s.c.) of 0.05 ml of undiluted egg-white was injected into the plantar side of the left hind paw of the rats of every group. vernia was used to measure paw thickness at sixtime periods (0, 30, 60, 120, 180, and 240 min.), where zero time was the time at which the products, standard, and control were administered intraperitoneally (21). statistical analysis data were reported using mean ± sem. student ttest (two sample assuming equal variances) then used to calculate statistical significance between means, while anova test (two factors without replication) was used to compare between different groups. p-value < 0.05 considered statistically significant. result and discussion chemistry the pathway of synthesis for target fenoprofen hydrazones derivatives(h1-h4) was illustrated below(scheme1). fenoprofen ethyl ester (compound a) was synthesized by reaction of fenoprofen with ethanol in presence of thionyl chloride. fenoprofen hydrazide (compound b) was formed by refluxing of compound (a) with hydrazine hydrate in ethanol. the synthesis of final target compounds was involved the reaction of fenoprofen hydrazide with different aldehydes in ethanol using glacial acetic acid as a catalyst iraqi j pharm sci, vol.29(2) 2020 synthesis of fenoprofen hydrazones with anti-inflammatory effect 242 . scheme 1. multi-step reactions for the synthesis of target compounds (h1-h4) comparative analysis at time 0 and after 30 minutes, there are no significant differences between all groups. however, at time 60,120,180 and 240, there is a significant reduction of paw thickness for both fenoprofen and target compounds (h1,h2,h3,h4) compared to control. compounds (h1, h2, h4) show comparable activity to the standard while compound h3 demonstrate significantly higher activity than standard (fenoprofen) at time 60,120 and 180 which indicate rapid onset and short duration. as shown in table (1) and figure (2) below: table 1. effect of dimethyl sulfoxide (control), fenoprofen(standard) and target compounds (h1-h4) on egg-white induced paw edema 0 180 120 60 30 0 compound 5.77±0.1 5.97±0.12 6.51±0.21 6.35±0.16 5.3±0.19 3.45±0.08 control 4.79±0.24*a 5.03±0.23* 5.37±0.25*a 5.47±0.18*a 5.14±0.2 3.44±0.08 standard 4.55±0.17*a 5.06±0.11* 5.30±0.12*a 5.35±0.19*a 5.06±0.21 3.43±0.06 h1 4.63±0.15*a 4.59±0.16* 5±0.17*a 5.53±0.21*a 4.93±0.27 3.33±0.09 h2 4.69±0.16*a 4.5±0.15*b 4.7±o.14*b 5.01±0.12*b 5.2±0.13 3.33±0.04 h3 4.7±0.19*a 5.07±013* 5.37±0.14* 5.79±0.07*a 4.9±0.14 3.43±0.07 h4 #non‐identical superscripts (a, b) among different tested compounds are regarded significantly different (p < 0.05);*significantly different compared to control (p < 0.05). data are expressed in mm paw thickness as mean ± sem. n= number of rats. time (0) is the time of i.p. injection of fenoprofen, tested compounds and dmso. time (30) is the time of injection of egg white to induce edema. iraqi j pharm sci, vol.29(2) 2020 synthesis of fenoprofen hydrazones with anti-inflammatory effect 243 figure 2. effect of dimethyl sulfoxide, fenoprofen and compounds (h1-h4) on egg-white induced paw edema in rats. results are expressed as mean ± sem (n = 6 for each group). conclusion new fenoprofen hydrazones were synthesized successfully. their chemical structure was characterized using ir spectroscopy and 1hnmr. the anti-inflammatory activity of the target compound (h1-h4) was evaluated using egg white induced edema method. all synthesized compounds show effect comparable to the standard (fenoprofen), except compound h3 which shows effect superior to fenoprofen in reducing paw edema in rats. acknowledgment the authors are greatly thankful to the college of pharmacy/department of pharmaceutical chemistry for their support. great thanks also to ammar a. fadhil supervisor of the animal house for his great support and help. references 1. wongrakpanich s, wongrakpanich a, melhado k, rangaswami j. a comprehensive review of non-steroidal anti-inflammatory drug use in the elderly. aging and disease. 2018 feb;9(1):143. 2. day ro, graham gg. non-steroidal antiinflammatory drugs (nsaids). bmj. 2013 jun 27;346:f3195. 3. nugrahani 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chem. 2009;2(3):699-705. 17. abbas ah, elias an, fadhil aa. synthesis, characterization and biological evaluation of new potentially active hydrazones of naproxen hydrazide. der pharma chem. 2015;7(10):93101. 18. mahdi mf, naser nh, hammud nh. synthesis and preliminary pharmacological evaluation of new naproxen analogues having 1, 2, 4-triazole-3-thiol. int j pharm pharm sci. 2017;9(7):66-71. 19. jum'ma km, hussain sa, sulaiman aa, sigar ah. evaluation of the anti-inflammatory effect ofpioglitazone in experimental models of inflammation in rats. iraqi journal of pharmaceutical sciences. 2009;18(1 (suppl.)):1-6. 20. agotegaray ma, dennehy m, boeris ma, grela ma, burrow ra, quinzani ov. therapeutic properties, sod and catecholase mimetic activities of novel ternary copper (ii) complexes of the anti-inflammatory drug fenoprofen with imidazole and caffeine. polyhedron. 2012 feb 28;34(1):74-83. 21. alsafi mh, farhan ms. synthesis, characterization and acute anti-inflammatory evaluation of new mefenamic acid derivatives having 4-thiazolidinone nucleus. iraqi journal of pharmaceutical sciences (pissn: 1683-3597, e-issn: 2521-3512). 2019 jun 26;28(1):138-46. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(2) 2012 aloe vera gel,sesame oil &camphor oil &pseudomonas aeruginosa 18 the effects of aloe vera gel, sesame oil and camphor oil on pseudomonas aeruginosa isolated from burnt patients may t. flayyih* ,1 and raghad q. majeed* *department of biology, college of science, university of baghdad ,baghdad, iraq abstract three isolates of p. aeruginosa were isolated from burnt patients. the ability of these isolates for adhesion and formation of slime layer were tested, the result showed that all isolates were able to adherence on the smooth surface. the sensitivity of p. aeruginosa isolates for antibiotics were tested , all isolates were sensitive to gentamycin, piperacillin and amikacin ciprofloxacin, and resist to tetracyclin, amoxicillin, cephalexine , ceftriaxone. ciprofloxacin and amikacin were found effective against p. aeruginosa isolates with mic values of 3.8 μg/ ml for ciprofloxacin and 0.244 μg/ ml for amikacin the antibacterial effect of different concentrations of aloe vera gel, sesame oil and camphor oil against p. aeruginosa were determined, camphor was highly effective with concentration inhibit bacteria value of 10% followed by sesame oil (20%) and aloe vera gel (>75%). the combinations of aloe vera gel, sesame oil and camphor oil and antibiotics (ciprofloxacin and amikacin ) showed that the efficacy of the two antibiotics (ciprofloxacin and amikacin ) against p.aeroginosa isolates was improved in the presence of aloe vera gel, sesame oil and camphor oil. key words : aloe vera gel, sesame oil , camphor oil , pseudomonas aeruginosa. السوسن وزيث الكافور على بكحرياث يوز (aloe vera) االلوفيرجأثير هالم (pseudomonas aeruginosa) الحروق هرضى هن الوعسولة *هي طالب فليح ،1 *يس هجيدق رغد و ، بغذاد ، انعزاق .جايعت بغذاد ،كهٍت انعهٕو ،قسى عهٕو انحٍاة * الخالصة اخخبزث قابهٍت ْذِ انعشالث عهى يٍ يزضى انحزٔق . p.aeroginosa شالث يٍ بكخزٌاحى انحصٕل عهى رالد ع اخخبزث حساسٍت انعشالث نهًضاداث انحٍاحٍت .االنخصاق فاظٓزث انُخائج اٌ جًٍع انعشالث قابهت نالنخصاق عهى انسطٕح انًهساء انخخزاساٌكهٍٍ ًضاداثيٍكاسٍٍ ٔانسبزٔفهٕكساسٍٍ ٔيقأيت نانجُخاياٌسٍ ٔانببزاسٍهٍٍ ٔاال فظٓز اٌ جًٍع انعشالث حساست نًضاداث حٍذ عشالثاناكزز فاعهٍت ضذ يضادي انسبزٔفهٕكساسٍٍ ٔااليٍكاسٍٍ , ٔٔجذ اٌ ٔااليٕكسٍسهٍٍ ٔانسٍفانٕكسٍٍ ٔانسفخزٌاكسٌٕ . حى ياٌكٕغزاو / يم ,,0,4يٍكاسٍٍ ياٌكٕغزاو / يم ٔنًضاد اال 8,3انخزكٍش انًزبط االدَى نًضاد انسبزٔفهٕكساسٍٍ بهغج قًٍت ت حٍذ بهغ ٍححذٌذ انخارٍز انًضاد نهبكخزٌا نخزاكٍش يخخهفت يٍ ْالو االنٕفٍزا ٔٔسٌج انسًسى ٔسٌج انكافٕر فكاٌ سٌج انكافٕر اكزز فاعه زاكٍش ححج انخزكٍش %( . عُذ ديج انًضاداث بخ57 %( ْٔالو االنٕفٍزا )<40ٌهٍّ سٌج انسًسى ) %00انخزكٍش انًزبط نهبكخزٌا ٔححج ححج انخزكٍش انًزبط االدَى يع ْالو االنٕفٍزا ٔٔسٌج انسًسى ٔسٌج انكافٕر اظٓزث انًضاداث ححسُا يهحٕظا انًزبط االدَى .زٌّ ٍفً حارٍزْا عهى انعشالث انبكخ ، زيث الكافور . pseudomonas aeruginosaالوفيرا ، زيث السوسن ، الكلوات الوفحاحية : introduction in spite of considerable advances in the treatment of burns, infection continues to pose the greatest danger to burn patients. approximately 73 per cent of all death within the first five days post-burn have been shown to be directly or indirectly caused by septic processes (1) . the common pathogens isolated from burn patients include pseudomonas aeruginosa, staphylococcus aureus , klebsillea spp, and various coliform bacilli. fungi (candida albicans, aspergillus fumigatus) can also cause infection (2) . pseudomonas aeruginosa is an opportunistic gram negative pathogenic bacterium. this bacterium, in hostile conditions such as colonization in burned skin surface, produces large amounts of exopolysaccharide that bind with water and form gels (3) . infections caused by p. aeruginosa are often severe and life threatening and are difficult to treat because of the limited susceptibility to antimicrobial agents and the high frequency of an emergence of antibiotic resistance during therapy (4) . accumulation of resistance after exposure to various antibiotics and cross-resistance between agents may result in multidrugresistant (mdr) p. aeruginosa (5) .the spread of drug resistant pathogens is one of the most serious threats to successful treatment of microbial diseases,therefore, essential oils and other extracts of plants have evoked interest as sources of natural products (6 ) . 1 corresponding author email : maytalib@yahoo.com received : 1/11/2011 accepted :19/5/2012 iraqi j pharm sci, vol.21(2) 2012 aloe vera gel,sesame oil &camphor oil &pseudomonas aeruginosa 19 aloe vera which is a member of liliaceae family(400 different species) with its origin in african continent , has been used to treat various skin conditions such as cuts, burns and eczema. it is alleged that sap from aloe vera eases pain and reduces inflammation. evidence on the effects of wound healing, however, is contradictory (7) .aloe vera leaf gel can inhibit the growth of the two bacteria shigella flexneri and streptococcus pyogenies (8 ) .sesame oil, derived from sesame seeds. the seed oil of sesame spp was found to contain certain natural antibacterial agents that were effective against common skin pathogens, such as staphylococcus and streptococcus bacteria, as well as common skin fungi including the athlete's foot fungus (9) . essential oils (also called volatile oils) are aromatic oily liquids obtained from plant materials (flowers, leaves, buds, seeds etc) ,compounds. essential oils have been shown to possess antibacterial, antifungal,antiviral, insecticidal and antioxidant properties, camphor essential oil is extracted from the cinnamomum camphora (also known as laurus camphora) of the lauraceae family and dried rosemary leaves (rosmarinus officinalis), in the mint family, contain up to 20% camphor. it can also be synthetically produced from oil of turpentine, camphor oil can be used in the treatment of nervous depression, acne, inflammation, arthritis, muscular aches and pains, sprains, rheumatism, bronchitis, coughs, colds, fever, flu and infectious diseases (10, 11) .this study, aim to detect of antibacterial activity of aloe vera gel, sesame oil and camphor oil against clinical isolates of p. aeruginosa obtained from burns patients well as the potentials of their effect in combination with different antibiotics. material and methods isolation and identification of isolates specimens were collected as wound swabs from burns patients. the specimens were cultured on macconkey agar and the isolated colonies were subcultured on cetrimide agar and macconkey agar. identification of p. aeruginosa based on gram stain, colony morphology and biochemical tests ( oxidase test, catalase test , triple sugar iron (tsi) fermentation, color, pyocyanin pigment production on king a medium and an ability to grow at 4°c and 42°c ) (12) . the ability of isolates for adhesion and formation of slime layer were tested according to christensen et al. (13 ) .10ml of tryptone soya broth were inoculated with a loopful of organisms from overnight blood plate culture then incubated overnight (18-24) hours at 37ºc. the culture tube were then emptied of their contents and stained by adding 10ml of safranin stain solution. each tube was then gently rotated to ensure uniform staining of any adherent material on the inner surface and the contents gently decanted. the tubes were then placed upside down to drain. a positive result was indicated by the presence of an adherent layer of stained material on the inner surface of the tube or visible film lined the walls of the tube. ring formation at the liquidair interface was not considered indicative of slime production. antibiotic sensitivity test the sensitivity of bacteria to antibiotics (tetracyclin , amikacin ,gentamycin ,ciprofloxacin ,amoxicillin ,piperacillin ,cephalexine, ceftriaxone) were tested by using disk diffusion test were performed for all the isolates by the method recommended by clinical and laboratory standard institute (clst) (14 ) . a suspension of each isolate was made so that the turbidity was equal to 0.5 mcfarland standard and then plated onto nutrient agar plate. antibiotic disk was applied to each plate. after incubation at 37°c for 24 h, zone size was measured. the minimal inhibitory concentrations (mics) and sub minimal inhibitory concentrations (half of mic) of ciprofloxacin and amikacin were determined. this test was achieved according to morello et al. (15) , as following: sterile tubes of mueller-hinton broth were prepared; each tube contented 2ml of sterile muellerhinton broth. a serial of two-fold dilutions of antibiotics were prepared by adding of 2ml of antibiotic stock solution to the first tube of mueller-hinton broth, mixed the contents, then transferred of 2ml from this tube in to a second tube, mixed the contents of the second tube and transferred of 2ml to a third tube. the dilution process was continued until reach to the last tube. after the contents of the last tube mixed well, discarded 2ml of broth so that the final volume in all tubes was 2ml. from the nutrient agar plate culture of bacterial isolate the suspension of p. aeruginosa was prepared in 5ml of normal saline that equivalent to mcfarland 0.5 standard, 0.1ml of the bacterial suspension was transferred to the each of the serial of antibiotic-broth tubes. each tube was shacked gently to mix the tube contents and placed in the incubator at 35ºc for 18-24 hours.the experiment was included the following control tubes: a tube contents sterile broth (sterility control). a tube contents broth and bacterial isolate (growth control). http://en.wikipedia.org/wiki/rosemary http://en.wikipedia.org/wiki/rosmarinus_officinalis http://en.wikipedia.org/wiki/turpentine iraqi j pharm sci, vol.21(2) 2012 aloe vera gel,sesame oil &camphor oil &pseudomonas aeruginosa 20 a tube contents antibiotic and sterile broth. after the incubation the tubes were examined for the presence or absence of turbidity, the lowest concentration that inhibits the visible growth of bacteria was determined as mic. antibacterial activity of aloe vera gel, sesame oil and camphor oil the aloe vera gel was gotten from the leaves by pushing the plant leaf by fingers and the gel collected in sterile tube. sesame oil and camphor oil were obtained from local market. different concentrations (5-75) % of aloe vera gel, sesame oil and camphor oil were prepared by using the solvent dmso (dimethyl sulfoxide). the antibacterial effect of these different concentrations against p. aeruginosa was determined according to morello et al. (15) the effect of combinations of antibiotics with aloe vera gel, sesame oil and camphor oil the effect of two different antibiotic concentrations (subinhibitory antibiotic concentrations and sub sub mics of ciprofloxacin and amikacin in combination with sub mics and sub sub mics of aloe vera gel, sesame oil and camphor oil against p. aeruginosa were determined (15) . the combination of antibiotics with aloe vera gel, sesame oil and camphor oil were performed as mention in the table (1). the tubes contents and placed in the incubator at 35ºc for 18-24 hours. after the incubation the tubes were examined for the presence or absence of turbidity. table 1: the combination of antibiotics with aloe vera gel, sesame oil and camphor oil results and discussion three isolates of p. aeruginosa were isolated from burnt patients , a series of biochemical tests were used for identification . the isolates were positive for oxidase and catalase test and growth at4°c and 42°c (table 1). the ability of these isolates for adhesion and formation of slime layer were tested, the result showed that all isolates were able to adherence on the smooth surface (fig 1, table 2). burn patients were most commonly infected with p. aeruginosa , it is opportunistic pathogen responsible for nosocomial infections, anjuman and mir (16) reported that there is a significant increase in the number of p. aeruginosa isolated from pus followed by urine. among, 4409 burn patients samples were evaluated for microbial culture, in which 2810 (63.7%) were culture positive, the most predominant isolates in all samples was p.aeruginosa (47.7%) (17) .the sensitivity of p. aeruginosa isolates for antibiotics were tested , all isolates were sensitive to gentamycin, piperacillin and amikacin ciprofloxacin, and resist to tetracyclin, amoxicillin, cephalexine , ceftriaxone ( table 3 ) . ciprofloxacin and amikacin were found more effective against p. aeruginosa isolates with mic values of 3.8 μg/ ml for ciprofloxacin and 0.244 μg/ ml for amikacin( table 4 ). gram negative pathogens such as pseudomonas aeroginosa, and klebsiella pneumonia have become multi-drug resistant (18) . according to japoni et al.( 3 ) study, p. aeroginosa was resistant to ciprofloxacin (27.1%), ceftazidime (15.7%), cefepime (2.9%), imipeneme (67.1%), piperacilin (14.3%). the antibacterial effect of different concentrations of aloe vera gel, sesame oil and camphor oil against p. aeruginosa were determined, camphor was highly effective with concentration inhibit bacteria value of 10% followed by sesame oil (20%) and aloe vera gel (>75%) ( table 5 ) .a vera gel consists of 99.3% water. the remaining 0.7% is made up of solids with glucose and mannose constituting for a large part. these sugars together with the enzymes and amino acids in the gel give the special properties as a skin care product. the gel stimulates cell growth and as such enhances the restoration of damaged skin. it moisturizes the skin because it has a water holding capacity. this moist on the skin and also has a tube no. antibiotics in combination final volume in the tube (ml) volume of p. aeruginosa (ml) final concentration in the tube (µg/ml) ciprofloxacin or amikacin aloe vera gel , sesame oil or camphor oil volume (ml) concentration (µg/ml) volume (ml) concentration (µg/ml) 1 0.5 mic 0.5 mic 1 1 sub-mic/submic 2 0.5 sub-mic 0.5 sub-mic 1 1 sub-sub mic/sub-sub mic iraqi j pharm sci, vol.21(2) 2012 aloe vera gel,sesame oil &camphor oil &pseudomonas aeruginosa 21 cooling effect, it had shown that aloe vera leaf gel can inhibit the growth of the two bacteria shigella flexneri and streptococcus progenes. specific plant compounds such as anthraquinones and dihhydroxyanthraquinones, as well as saponins have been proposed to have direct antimicrobial activity ( 8 ,19 ) . the gc-ms phytochemical screening of methanolic extract showed the presence of carboxylic acids and phenolic groups in essential oils especially some of the most potent antioxidants like sesamol, sesamolin and sesamin. both the methanolic and ethanolic extracts have broad spectrum antimicrobial effect against all the tested micro-organisms except s.pneumoniae, candida albicans and s. aureus respectively, while the aqueous extract exhibited no inhibitory effect on s. aureus and s. pneumoniae except on c. albicans (20) . the camphor oil which extracted from fallen leaves of cinnamomum camphora by the method of distillation had antibacterial effect against e. coli , p.vulgaris ,s. aureus in some extent, and the minimum inhibitory concentration(mic) was between 0.125 to 0.25 g/ml,but the oil from fallen leaves extracted by acetone had almost no antibacterial effect against these bacteria (21) .the effect of combinations of aloe vera gel, sesame oil and camphor oil and antibiotics (ciprofloxacin and amikacin ) showed additive effect on the growth of the p.aeroginosa (table 6 and table 7) . the efficacy of the two antibiotics against p.aeroginosa isolates was improved in the presence of aloe vera gel, sesame oil and camphor oil. the enhancement in the killing effect (additivity) of the antibiotics at sub mic and sub sub mic values, suggests that sesame oil and the ketonic group in camphor oil and aloe vera gel compounds can improve the efficacy of antibiotics. the antimicrobial effects of aloe vera have been attributed to the plant’s natural anthraquinones: aloe emodin, aloetic acid, aloin, anthracine, anthranol, barbaloin, chrysophanic acid, ethereal oil, ester of cinnamonic acid, isobarbaloin, and resistannol (22) .in relatively small concentrations together with the gel fraction, these anthraquinones provide analgesic, antibacterial, antifungal, and antiviral activity; in high concentrations, they can be toxic (23) . saponins, which contain glycosides, are soapy substances that have both cleansing and antiseptic properties (24) . table 2: the results of biochemical tests of p. aeruginosa isolates biochemical tests p.aeruginosa 1 p.aeruginosa 2 p.aeruginosa 3 gram stain ve* ve ve catalase test + + + oxidase test + + + growth at4°c and 42°c + + + triple sugar iron (tsi) fermentation formation of gas formation of h 2s adherence on the smooth surface + + ++ *ve : gram negative , + : positive result , : negative result figure 1: adherence of p. aeruginosa on the smooth surface table 3: the result of antibiotics sensitivity of p. aeruginosa isolates antibiotic result diameter of inhibition zone (cm ) tetracyclin r amikacin s 3 gentamycin s 2.5 ciprofloxacin s 4 amoxicillin r piperacillin s 2 cephaloxine r ceftriaxone r r : resist s : sensitive iraqi j pharm sci, vol.21(2) 2012 aloe vera gel,sesame oil &camphor oil &pseudomonas aeruginosa 22 table 4: the result of minimal inhibitory concentrations (mics) of antibiotics antibiotic mic ( μg/ ml) sub mic ( μg/ ml) amikacin 3.8 1.9 ciprofloxacin 0.244 0.122 table 5: the result of antibacterial activity of aloe vera gel, sesame oil and camphor oil concentration inhibit bacteria (%) test > 75 aloe vera gel 20 sesame oil 10 camphor oil table 6: the effect of combinations of sub mics of antibiotics with sub mics of aloe vera gel, sesame oil and camphor oil on the growth of the p.aeroginosa result combination of sub mics no growth ciprofloxacin(0.122 μg/ ml)+ aloe vera gel( 75%) no growth ciprofloxacin(0.122 μg/ ml)+ sesame oil( 15%) no growth ciprofloxacin(0.122 μg/ ml)+ camphor oil( 5%) no growth amikacin(1.9 μg/ ml)+ aloe vera gel( 75%) no growth amikacin(1.9 μg/ ml)+ sesame oil( 15%) no growth amikacin(1.9 μg/ ml)+ camphor oil(5%) table 7: the effect of combinations of sub sub mics of antibiotics with sub sub mics of aloe vera gel, sesame oil and camphor oil on the growth of the p.aeroginosa result combination of sub sub mics no growth ciprofloxacin(0.061 μg/ml)+ aloe vera gel( 37.5%) growth ciprofloxacin(0.061 μg/ml)+ sesame oil( 7.5%) no growth ciprofloxacin(0.061 μg/ml)+ camphor oil( 2.5%) no growth amikacin(0.95 μg/ ml)+ aloe vera gel( 37.5%) growth amikacin(0.95 μg/ ml)+ sesame oil( 7.5%) no growth amikacin(0.95 μg/ ml)+ camphor oil( 2.5%) references 1. ekrami ,a and kalantar, e..bacterial infections in burn patients at a burn hospital in iran . indian j med res . 2007;126: 541-544. 2. revathi g, puria j, jaid k. bacteriology of burns. burns. 1998;24: 347-9. 3. japoni ,a.; hayati ,m.; alborzi ,a.; farshad, sh. and abbasian, s.a. in vitro susceptibility of pseudomonas aeruginosa isolated from a burn center to silver sulfadiazine and silver nitrate in shiraz, south of iran. iranian j. med. sci. 2005;30(2): 63-67 . 4. carmeli, y.; troillet, n. ; eliopoulos, g. and samore. m. h. emergence of antibiotic-resistant pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents. antimicrob. agents chemother. 1999; 43: 1379-1382. 5. saiman, l.; mehar, f.; niu, w. w. ; neu, h. c. ; shaw, k. j.; miller, g. and prince. a. antibiotic susceptibility of multiply resistant pseudomonas aeruginosa isolated from 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y. camphor oil extraction from cinnamomum camphora and its antibacterial activity. j. fujian forestry science and technology. 2006 ;4 . 22. wynn, r.l. aloe vera update for dentistry. gen.dent. 2005;53 (1):6-9. 23. davis, r.h. 1997.aloe vera: a scientific approach. new york: vantage press. 24. plaskett, l.g.1996. the health and medical use of aloe vera.tacoma, wa: life sciences press. http://en.cnki.com.cn/journal_en/d-d049-fjlk-2006-04.htm http://en.cnki.com.cn/journal_en/d-d049-fjlk-2006-04.htm iraqi j pharm sci, vol.29(2) 2020 inborn error of mitochondrial function doi: https://doi.org/10.31351/vol29iss2pp259-262 259 the frequency inborn error of mitochondrial function in mosul and kurdistan region muhammad a. ahmed*,1 , mwafak k. hasan** and ashwaq n. abbas*** *college of pharmacy, university of mosul. **college of science, university of mosul. ***college of dentistry, university of sulamiania, abstract this work aimed to estimate the frequency of mitochondrial inborn errors of metabolism (miems) in patients presenting with family history and iem-picture who referred for advance iem assay in mosul province and kurdistan region. this study was observational study conducted on 364 cases referred from different general /or private pediatric clinics with unexplained sign and symptoms and suspension of mitochondrial dysfunction. the study included 364 children with an age ranging from 1 month to 1 year. started from january 2018 to january 2020. all patients referred with their full history review, notes about their clinical examination, and laboratory investigations including blood ammonia, serum lactate/ pyruvate, arterial blood gases. in addition to the standard laboratory-tests (kidney and liver functions, blood glucose, and complete blood picture) carried out in sorain private laboratory. the results of this work show that sixteen (4.4%) of cases were positive in the iem screening test. there were 4 (1%) patients with a definitive mitochondrial related error of metabolism, 2 (0.5%) of the cases due to carnitine uptake defect and 1 (0.2%) short chain acyl coa dehydrogenase deficiency and other one patient (0.2%) case caused by 3-methylcrotonyl coa carboxylase deficiency. in conclusion, the incidence of mitochondrial inborn errors of metabolism (miems) between patients presenting with iem was higher in mosul and kurdistan region than international values keywords: mitochondria, an inborn error of metabolism, carnitine, mosul. تكرار حاالت الخلل الوراثي في وظائف المايتوكوندريا في الموصل وافليم كردستان العراق *** عباس الدين و اشواق نجم **، موفق خليل حسن 1*، محمد عبد الغفور احمد القطان كلية الصيدلة ، جامعة الموصل . * كلية العلوم ، جامعة الموصل . ** كلية طب االسنان ، جامعة السليمانية . *** الخالصة اعراض عند المرضى الذين لديهم تاريخ عائلي و بيوت الطاقة هذا العمل لتقدير تكرارأالخطاء في تمثيل الغذائي الوراثية في فيهد أجريت هذه الدراسة على .المتقدم في محافظة الموصل وإقليم كردستان تكرارأالخطاء في تمثيل الغذائي خلل في بيوت الطاقة وألذين ارسلوا لفحص مريًضا تمت إحالتهم من عيادات أطفال عامة أو خاصة مختلفة مع عالمات وأعراض غير مفسرة وتعليق بالخلل في بيوت الطاقة. تضمنت 364 . تمت 2020و انتهت في كانون الثاني 2018واحد وسنة واحدة. بدءت الدراسة في كانون الثاني طفالً تتراوح أعمارهم بين شهر 364الدراسة إحالة جميع المرضى بمراجعة كاملة لتاريخهم المرضي ، ومالحظات فحصهم السريري ، والفحوصات المخبرية بما في ذلك أمونيا الدم ، حامض انية. باإلضافة إلى التحاليل المختبرية القياسية )وظائف الكلى والكبد ، جلوكوز الدم ، صورة الدم اللبنيك / البيروفات في الدم ، غازات الدم الشري أخطاء التمثيل الغذائي الخلقية ٪( من المرضى إيجابيين مصابين باحد 4.4الكاملة( فيمختبر سوران االهلي. بينت نتائج هذا العمل ان ستة عشر ) ٪( من الحاالت بسبب عيب امتصاص الكارنيتين 0.5) 2ى الذين يعانون من خطأ استقالبي متعلق ببيوت الطاقة ، و ٪( من المرض1) 4كان هناك . سلسلة0.2) 1و نقص من )٪ acyl coa dehydrogenase ( أخرى نقص 0.2وحالة عن الناجم )٪3methylcrotonyl coa carboxylase . أالخطاء تمثيل الغذائي الخلقية في بيوت الطاقة بين المرضى الذين يعانون مننستنتج من هذا العمل ان معدل حدوث أخطاء ال في الموصل وإقليم كردستان أعلى من القيم الدولية. في تمثيل الغذائي الوراثية .الكلمات المفتاحية: بيوت الطاقة ، خطأ وراثي في التمثيل الغذائي ، كارنيتين ، الموصل introduction hundreds of single‐gene anomaly are involved in inborn errors of metabolism (1). inborn errors of metabolism are often rare multisystem disorders with neurological and non-neurological manifestations, commonly with onset during in infancy, childhood, and adulthood and many of them fatal if not treated. inborn errors of metabolism disorders in lead to abnormal metabolism and this lead to improper metabolism of nutrients and energy production(2). the disorders usually caused by the absence or abnormality of the substrate, enzyme or its cofactor, leading to either accumulation or deficiency of a specific metabolite(3). the mitochondrial error cause significant effects on neonate life by affecting different aspect of mitochondrial activities e.g. fatty acid β-oxidation in mitochondria. these patients present with clinical features may include but not restricted to(4) failure to thrive, chronic vomiting, chronic diarrhea, hypoglycemia, disturbed conscious level, seizures, 1corresponding author e-mail: ashwaq73@yahoo.com received: 22/5 /2020 accepted: 31/8/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp259-262 iraqi j pharm sci, vol.29(2) 2020 inborn error of mitochondrial function 260 delayed motor response , hypotonia or hypertonia, spasticity, mental retardation, muscle weakness, walking abnormalities, microcephaly, speech abnormalities of the patients, hepatomegaly, cardiac manifestation and ophthalmic manifestation(4). mitochondrial inborn error disorders are inherited metabolic diseases caused by either complete or partial deficiency of specific enzymes or transport proteins that involved in the mitochondrial metabolism. the mitochondrial inborn disorders have an incidence of 1 to 100,000 births(5) worldwide. however, in the middle east population, the incidence may approach 20 to 100,000(6) births with no clear data present from iraq. the mitochondrial dysfunction screening process has evolved for better understanding of these conditions, availability of diagnostic tools and treatment option development. the inclusion of specific test in newborn screening list has become controversial with varying practices and national need. aim: this work focuses on explains the exact frequency of the mitochondrial dysfunction between patients with an inborn error of metabolism in mosul and kurdistan region. patient and methods the study included three hundred and sixty-four newborn and infant with an age ranging from 1 month to 1 year. patient refers to different general and private hospitals and clinics in mosul and kurdistan region to soran private hospital laboratory started from january 2018 to january 2020 with the symptom of inborn error of metabolism that includes poor feeding, vomiting, diarrhea, and/or dehydration, temperature instability, tachypnea, apnea, seizures, hypotonia, lethargy and coma. in addition to the family history of any inborn error of metabolism and follow primary care, physician (pcp) guidelines for iem diagnosis (7) (see table-1). all patients referred to pediatrician with their full history review, notes about their clinical examination, and laboratory investigations including blood ammonia, arterial blood gases. in addition to the standard laboratory tests (kidney and liver functions, blood glucose, and complete blood picture kept for their patients record and not included in this work). all patients who included in this work have at least five above symptom. brain imaging study conducted for all cases before referral with either ct scan or mri to rule out any intracranial anomaly, diseases or trauma. the cases with their results referred back to their physician for follow up. table 1. primary care physician guidelines for iem diagnosis(7) it considered when diagnose and treat bacteremia, encephalopathy due to severe hypoxia or toxins ingestion. when laboratorytests fail to give a definitive diagnosis. symptoms did not respond even after treatment. present as acute illness or as chronic recurrent or progressive condition at any age negative family history for genetic or metabolic disorder does not exclude. neonatal death from unknown causes. the samples collected over a two-year interval. blood drop from the heel of the neonate collected before 24hrs of protein feedings the sample adsorbed to the specific type of filter paper then sample packaged in standard transport package as recommended by the manufacturer for neolab– greece for tandem mass spectrometry analysis to detection and quantification of metabolic compounds. in addition to 5 ml of whole blood were collected in plan tube for serum lactate and pyruvate estimation using cayman fluorescence-based kits (no. 700510 and no. 700470 respectively)(8,9). the results return to soran hospitals then sent it to physicians. the parents of the subjects involved in this study informed about the purpose of the study and the plan of work before they agreed to participate. statistical analysis conducted using manual methods to calculate the percentage of the obtained data of the patients. results in this study, there were 364 patients referred with unexplained symptoms with the suspension of mitochondrial inborn error of metabolism. patients referred to different general and private pediatric hospitals and clinics, with their ages ranging from 1 day to 12 months. infants of age <1 month numbered 266 (73%) and newborn aged from 2-12 months numbered 98 (27%); the number of females was 247(68%) and males represent 116 (32%). patients with positive iem consanguinity numbered 236 (65%), and 73 (20%) patients had a strong positive family history and 55 (15%) patients with no mitochondrial or other iem history. only 16 (4.4%) patient of patients were confirmed diagnosed with one of iem with only 4 (1%) cases approved to have a mitochondrial related inborn error of metabolism 2 (0.5%) of the cases due to carnitine uptake defect and 1 (0.2%) short chain acyl coa dehydrogenase deficiency (scadd) and other 1 (0.2%) case caused by 3-methylcrotonyl coa carboxylase deficiency (3-mccd) as in table 2. in addition to tandem mass spectrometry analysis serum lactate/ pyruvate ratio shows significant elevation over 20 in iraqi j pharm sci, vol.29(2) 2020 inborn error of mitochondrial function 261 only 25 (6.8%) of the cases. mitochondrial related inborn error of metabolism patient shows significant elevation over the normal range of serum lactate was 1615.2± 208.6 µmol/l in comparison with the normal newborn 1250±110 µmol/l, serum pyruvate 70.62±7 µmol/l compared to 63±0.2 and lactate to pyruvate ratio 22.8±1.24 which reflect severe mitochondrial dysfunction. table 2. mitochondrial inborn error of metabolism cases c0 = free carnitine, c2-actyle c3 = propionyl, c4 = butyryl, c5oh = 3-hydroxy, isovaleril, c6 = hexanoyl, c8 = octanoyl. discussion mitochondrial inborn error of metabolism is a part of newborn screening for iem in that approved by who. anomalies of mitochondrial function are characteristics of these metabolic disorders10. the mitochondria play a vital role in the regulation of many cellular homeostatic mechanisms; through the production of atp, reactive spices (both ros and rns), autophagy, apoptosis and biogenesis11. the signaling pathways such as sirtuins (sirt1-7) and peroxisomeproliferator-activated receptor γ co-activator-1α (pgc-1α) and antioxidant mechanisms as mndependent superoxide dismutase (mnsod) should check carefully as it is not correct to describe the relationship between phenotype and genotype in these patients as straightforward relation11. inborn error of mitochondrial metabolism patients presented with seizures with or without hypoglycemia, metabolic acidosis with the fall of sucking and coma. fatty liver associated with prolonged keto-genic stresses usually after 7 days of fibril condition hepatomegaly needs longer time (weeks) to be developed12. based on the published data, the incidence of both scadd and 3-mccd worldwide not exceeding 1 in 50,000 (0.002%) 13 while our result shows that the frequency in our sample was 0.2% which is higher than international values. similarly, the incidence of carnitine deficiency is approximately 1 in 100,000 newborns worldwide14,15. the elevated lactate/pyruvate ratio suggesting inborn errors of ketogenesis as described. the significant elevation of plasma butyryl-carnitine with or without elevation ethyl-malonic acid (c4) concentrations with a reduction in plasma free carnitine (c0) considered as a biochemical signature of inborn error of mitochondria 16 which was seen in one of the cases in this work who presented with hypotonia, seizures, metabolic acidosis after flu with hypoglycemia. conclusion to sum up, the frequency of mitochondrial iem (miems) between patients presenting with iem was higher in mosul and kurdistan region than international values. references 1. arnold gl. inborn errors of metabolism in the 21st century: past to present. ann transl med. 2018;6(24):467-467. 2. ebrahimi-fakhari d, van karnebeek c, münchau a. movement disorders in treatable inborn errors of metabolism. mov disord. 2019;34(5):598-613. 3. inborn errors of metabolism: classification uptodate. https://www.uptodate.com/contents/inbornerrors-of-metabolism-classification. accessed july 31, 2020. 4. wajner m, amaral au. mitochondrial dysfunction in fatty acid oxidation disorders: insights from human and animal studies. biosci rep. 2016;36(1):281. 5. khan na, govindaraj p, meena ak, thangaraj k. mitochondrial disorders: challenges in diagnosis & treatment. indian j med res suppl. 2015;141(jan 2015):13-26. 6. gorman gs, schaefer am, ng y, et al. prevalence of nuclear and mitochondrial dna mutations related to adult mitochondrial disease. ann neurol. 2015;77(5):753-759. 7. agana m, frueh j, kamboj m, patel dr, kanungo s. common metabolic disorder (inborn errors of metabolism) concerns in primary care practice. ann transl med. 2018;6(24):469-469. no. the metabolic error of diseases patient value reference value case no. frequency 1. short chain acyl coa dehydrogenase deficiency (scadd) c4=2.06, c4/c3=2.19 c4/c2=0.15 c4/c8=22.89 c4 < 1.02 c4/c3 < 0.65 c4/c2 < 0.05 c4/c8 < 22 1 0.2% 2. carnitine uptake defect c0=5.60 c0=6.32 c0 > 10.4 2 0.5% 3. 3-methylcrotonyl coa carboxylase deficiency (3-mccd) c5oh=2.99, c5oh/c8=16.61 c5oh/c0=0.26 c5oh < 0.41 c5oh/c0 < 0.026 c5oh/c8 < 12.5 1 0.2% iraqi j pharm sci, vol.29(2) 2020 inborn error of mitochondrial function 262 8. l-lactate assay kit | cayman chemical. https://www.caymanchem.com/product/70051 0. accessed july 29, 2020. 9. pyruvate assay kit (pyruvic acid assay kit) | cayman chemical. https: //www .caymanchem .com/product/700470/pyruvate-assay-kit. accessed july 29, 2020. 10. van rijt wj, koolhaas gd, bekhof j, et al. inborn errors of metabolism that cause sudden infant death: a systematic review with implications for population neonatal screening programmes.neonatology. 2016 ; 109 (4) :297-302. 11. ahmed m. exploring the impact of hypoxia mimetic agents on multipotent stem cell biology. keele univ. 2018. 12. rice gm, steiner rd. inborn errors of metabolism (metabolic disorders). pediatr rev. 2016;37(1):3-17. 13. lisyová j, chandoga j, jungová p, et al. an unusually high frequency of scad deficiency caused by two pathogenic variants in the acads gene and its relationship to the ethnic structure in slovakia. bmc med genet. 2018;19(1). 14. stanley ca. carnitine deficiency disorders in children. in: annals of the new york academy of sciences. vol 1033. new york academy of sciences; 2004:42-51. 15. lam c, carter jm, cederbaum sd, et al. analysis of cases of 3-methylcrotonyl coa carboxylase deficiency (3-mccd) in the california newborn screening program reported in the state database. mol genet metab. 2013;110(4):477-483. 16. short-chain acyl-coa dehydrogenase deficiency genereviews® ncbi bookshelf. https: //www .ncbi. nlm.nih .gov/ books/ nbk63582/. accessed july 31, 2020. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.29(2) 2020 naringin binding with covid-19 doi : https://doi.org/10.31351/vol29iss2pp231-238 231 docking study of naringin binding with covid-19 main protease enzyme narmin h. amin hussen*,1 *department of pharmaceutical chemistry, college of pharmacy, university of sulaimani, iraq abstract recently the pandemic coronavirus disease 2019(covid-19) has spread quickly all over the world caused by sar-cov2. in the present study, it has been used molecular docking to the binding affinity between covid-19 main protease enzyme and flavonoids with evaluations based on docking scores calculated by autodock vina. results showed that naringin interacted with covid-19 main protease, and it has the highest binding affinity than other flavonoids include quercetin, hesperetin, and naringenin. an important finding in this study is that naringin with poly hydroxyl groups can serve as an inhibitor of covid-19 main protease bind to the pocket of the protein. it is shown that residues his163, glu166, asn142, his41and gln189 participate in the hydrogen bonding interactions, the same as happened with decahydroisoquinoline as a novel structure as a protease inhibitor for sars 3cl.on the other hand, some of the known protease inhibitors and anti -influenza drugs docked with covid-19main protease, it has a low binding affinity than naringin. keywords: covid-19 main protease, flavonoids, naringin, molecular docking, protease inhibitor. introduction the novel coronavirus disease (covid19) was first identified in wuhan, china, in december 2019 caused by severe acute respiratory syndrome coronavirus 2 (sars-cov2) which can be transmitted effectively between human to human and animal to human through droplets or direct contact, causing fever, cough, shortness of breath, pneumonia and kidney failure (1,2). recently, it has been reported that the number of infected human outside of china are suddenly increased, as of may 28, 2020, there have more than 5,700,000 cases, 357,533 deaths and 2,500,000 recovered in 216 countries and territories, most of the cases, and deaths have occurred in united states of america, brazil, russia, spain, united kingdom, italy and france(3). sars-cov2 is a beta coronavirus, which is an enveloped, positive-sense, single-stranded rna virus in the family of coronaviridae. in general, coronaviruses (covs) are a large group of viruses that can be divided into four genera, including alpha, beta, delta, and gamma. alphaand beta coronaviruses mainly infect bats, but they also infect other species like humans, camels, and rabbits (46).covid-19 is closely related to two high pathogenic responsible for severe acute respiratory syndrome (sars-cov) in 2002 and middle east respiratory syndrome (mers-cov) in 2012(7-9). drug development against coronavirus includes inhibition of viral replication through acting on its critical enzymes (10). covs encode proteases such as papain-like protease (plpro) and main protease (mpro), which are involved in the proteolytic processing of the polyproteins into individual non-structural proteins (nsps) to control viral gene expression and replication (11,12). the crystallized form of covid-19 main protease (mpro) was demonstrated by a chinese researcher liu et al(13), that it is a potential drug target protein for the inhibition of sars-cov-2 replication. the mpro is a key protein required for the proteolytic maturation of the virus (14). thus, targeting mpro has the potential to provide effective treatment against sars-cov-2 by inhibition of the viral polypeptide cleavage (15). further, studies have found that sarscov-2 requires angiotensin-converting enzyme 2 (ace2) and transmembrane serine protease 2 (tmprss2), to enter lung cells, the same cellular entry receptor as sars-cov to infect humans (16 120). as of now, few antiviral strategies are being used to treat patients, lack of specific antiviral drugs or vaccines against sars-cov-2 is further aggravating the situation (21). thus, there is an urgent need to identify and develop effective antivirals against sars-cov-2 to fight this deadly virus. in this study, it has been used flavonoids with sarscov-2 main protease against covid-19 (22-24). 1corresponding author e-mail: narmin.hussen@univsul.edu.iq received: 25/3 /2020 accepted: 6/ 8/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp231-238 iraqi j pharm sci, vol.29(2) 2020 naringin binding with covid-19 232 the flavonoids, a large group of naturally occurring low molecular weight compounds widely distributed in the plant kingdom; particularly, they belong to a class of plant secondary metabolites having a polyphenolic structure, widely found in fruits, vegetables, and certain beverages. these compounds share a common structural core with two benzene rings (a and b) joined by a third heterocyclic ring (c) (figure 1) (25-27). studies have suggested that flavonoids exhibit biological activities, including anti-allergenic, antiviral, antiinflammatory, and vasodilating actions (28,29). therefore, the inhibition of proteases was proposed as a new function mechanism for flavonoids by several independent laboratories. flavonoids such as naringin, quercetin, hesperetin and naringenin possess a variable degree of antiviral activity (figure 1). (30,31). in this study, we performed molecular docking to understand the interaction between 9 flavonoids and 14 fda approved antiviral drugs such as lopinavir, indinavir, ribavirin, ritonavir, favipiravir, and remdesivir (figure1) with covid19 main protease were performed to identify these drugs inhibiting covid-19 main protease enzyme. basic structure of flavonoids (i ) naringin (ii) . quercetin (iii) hesperetin (iv) naringenin(v) lopinavir (vi) indinavir (vii) figure 1. (i) basic structure of flavonoids, (ii) structure of naringin, (iii) structure of quercetin, (iv) structure of hesperetin, (v) structure of naringenin, (vi) structure of lopinavir and (vii) structure of indinavir. methods 1. ligand preparation the two and three-dimensional models of the drug was obtained from the pub chem data base (https://pubchem.ncbi.nlm.nih.gov/) in the structure-data file (sdf). then open babel was used to converting sdf to pdb format (https :// sourceforge .net/projects/openbabel/ ). ligands used in this docking study are 9 flavonoids and 14 fda approved antiviral drugs. among these drugs, naringin is most promising, since it demonstrates the highest docking score to the covid-19 protein (table 1). https://pubchem.ncbi.nlm.nih.gov/ https://sourceforge.net/projects/openbabel/ https://sourceforge.net/projects/openbabel/ iraqi j pharm sci, vol.29(2) 2020 naringin binding with covid-19 233 2.protein preparation: protein data bank (pdb) is a structural repository for biological macromolecules such as proteins and their complexes (www.rcsb.org/pdb)(32). the crystal structure of covid-19 main protease with n3 as inhibitors(6lu7.pdb)(http://www.rcsb.org/structure /6lu7)(15)(figure2),available in protein data bank was used as a receptor. the three-dimensional structure of the target protein was retrieved from pdb by giving the pdb id in the database. protein data bank (pdb) files may have a variety of problems that need to be corrected before they can be used for docking. before docking, the entire n3as inhibitors were removed from the protein molecule. lipinski’s rule of five. the rule of five is beneficial to assess in vivo absorption abilities of the designed compounds. a ligand has a molar mass less than 500, hydrogen bond donors (-oh, nh) less than five, hydrogen bond acceptors (n, o) less than ten and calculated clogp is less than five satisfy the rule of five. clogp, the number of hydrogen donors, and number of hydrogen acceptors of the drugs were obtained from the pubchem database (https: // pubchem .ncbi.nlm.nih.gov/). all of the flavonoids in the present study satisfy the rule of five except naringin, quercetin, laurifolin and elatin. 3.molecular dockin docking between the protein and ligand was performed using autodock 4.2.6 (http://vina. scripps.edu.). autodock tools were used for preparing the input files and analyzing the result. a program for molecular docking and virtual screening is auto dock vina (33). the virtual screening program has been used is autodockvina, implemented in an application called pyrx 0.8 (https://pyrx .source forge.io/), which is open-source software to perform virtual screening. to determine the scoring function in this method, specification of search space inside the coordination system of the protein is necessary, in which different positions of the ligand should be examined. the magnitude of the search space was determined with the grid center of x: -25, y: -52, z: -4.4, and the number of points in each magnitude was x:45, y:45, z:45 in angstrom. each output file has several models ranked in the ascending order in terms of binding energy. the predicted binding energy of the ligand with the target protein is represented in kcal/mole. in each case, only the best mode is usually selected and used for subsequent analysis. 4.visualization in order to sketch, visualize, and analyze ligand molecules, a suite of applications called marvin has been used. all the marvin tools were accessible from the marvin sketch 19.9 application (https: // chemaxon .com / products / mar-vin). h-bonds interactions between ligands and amino acids of targeted protein were visualized on ucsf chimera (34). figure 2. the 3d structure of covid19 main protease results and discussion table 1 shows the binding energy of several flavonoids with covid-19 main protease sorted according to the docking scores (binding affinities) calculated from the autodock vina. naringin showed lower binding energy, and higher binding affinity than the other docked flavonoids. hydrogen-bonding contributes most to the binding affinities of all flavonoids with the receptor protein. flavonoids through hydrogen-bonding interact with covid-19 main protease, with binding energies between -10.2.to-6.0kcal/mol table 1. the docking score (kcal/mol), mw, clogp, no. of h bond donor, no. of h bond acceptor, and lipinski’s rule of five for flavonoids. flavonoids docking score (kcal/mol) mw (g/mol) <500 clogp <5 no. of h bond donor <5 no. of h bond acceptor ≤ 10 lipinski's rule of five naringin -10.2 580.5 -0.5 8 14 no quercetin -8.0 302.23 1.5 5 7 no hesperetin -7.9 302.28 2.4 3 6 yes naringenin -7.7 272.25 2.4 3 5 yes ternatin -7.0 374.3 3.1 2 8 yes hydroxyflavone -6.1 238.2 3.4 1 3 yes alvocidib -6.1 401.8 3.3 3 6 yes laurifolin -6.0 356.4 11.8 2 5 no elatin -6.0 594.5 -2.1 11 15 no https://pubchem/ https://pubchem/ iraqi j pharm sci, vol.29(2) 2020 naringin binding with covid-19 234 upon study, naringin has a high binding affinity with binding energy (-10.2 kcal/mol); however, it could not pass the rule of five criteria due to its molecular mass greater than 500 g/mol and the number of hydrogen bond acceptor exceed the allowed range. as shown in figure 3, naringin could fit well to the binding pocket of covid-19 protease through five hydrogen-bonding interactions. naringin with poly hydroxyl groups may serve as inhibitors of covid-19 protease, it is shown that residues his163, glu166, asn 142, his41, and gln 189 participate in the hydrogen bonding interaction, the same as happened with decahydroisoquinoline as a novel structure protease inhibitor (35). the one hydrogen bond is occurred between one n-h group of his163 of covid-19 interact with one hydroxyl group of naringin, distance is 1.97 å (figure 3). next hydrogen bonds between carboxyl oxygen of glu166 and asn142, chains of covid-19 protein, and the hydroxyl group of ligands, with bond length 2.43 and 3.09å, respectively (figure 3). more, one hydrogen bonds between carboxyl oxygen of gln189 of backbone and hydroxyl group of the ligand has occurred with bond length 2.63å (figure 3). finally, other hydrogen bond can be seen between n-h his41 side chain and the hydroxyl group of phenol in the ligand with bond length of 3.61 å (figure 3). an important finding in this work is that the poly hydroxyl group of naringin can function as a protease inhibitor bind to the covid19 main protease. figure 3. molecular docking of naringin has interacted with covid-19 main protease enzyme. therefore, the binding energies calculated for other flavonoids lower than naringin including quercetin, hesperetin, naringenin, ternatin, 3hydroxy flavone, and elatin are -8.0, -7.9, -7.8,-7.0 6.1 ,and -6.0 kcal/mol respectively. they bind with the same covid-19 main protease pocket, but it could not fit well to binding pocket (figure 4) because it has low poly hydroxyl group for hydrogen bonding interaction (figure 1) and also the number of hydrogen bonding interaction with the amino acid of covid-19 main protease lower than naringin. it was found the figure 4a, quercetin formed one hydrogen bonding between hydroxyl group of ligand with amino acids his 163, distance is 2.26ǻ .next hydrogen bond between the hydroxyl group of quercetin with side-chain amino acids gln 189, his41and glu 166 with bond length,2.02 ǻ,3.06 ǻ and 3.76 ǻ respectively (figure 4 a). the hydroxyl group of hesperetin formed two hydrogen bonding between the n-h group of his164 and carboxyl oxygen of gln 189 chains of covid-19 protein with bond length 4.48 ǻ and 4.67å respectively. as shown in figure 4 c, naringenin could bind with the covid-19 main protease pocket through hydrogen bonding with the amino acid glu 166 and asn 142, distances are 4.52 ǻ and 4.79 ǻ respectively.ternatin with the hydroxyl group can be formed hydrogen bonding with amino acids his 163 and gln 189, bond length 4.13 ǻ and 5.43 ǻ respectively (figure 4 d). iraqi j pharm sci, vol.29(2) 2020 naringin binding with covid-19 235 figure 4. (a) molecular docking of quercetin interacted with covid-19 main protease enzyme. (b) molecular docking of hesperetin. (c) molecular docking of naringenin. (d) molecular docking of ternatin. on the other hand, numerous recent studies have been suggested some of the drugs against covid-19 disease especially protease inhibitors drugs (36,37). to understand and compare that naringin is a reasonably better binding affinity with covid-19 main protease enzyme than other drugs; also it has been used molecular docking to the binding affinity between covid-19 main protease enzyme and 14 fda approved drugs. results showed that the low binding affinities calculated for ligands such as lopinavir, indinavir, ritonavir, and ribavirin than naringin, and they do not interact effectively with the covid-19 main protease. as shown in figure 5, all of the protease inhibitors having a different binding pockets with flavonoids. for comparison, the docking energy between the covid-19 main protease and lopinavir calculated and the score was−7.9 kcal/mol. as shown in figure 6, lopinavir could bind with the covid-19 main protease pocket through hydrogen bonding with the amino acid gln 110 and asn 151, distances are 2.77 ǻ and 3.34 ǻ respectively. the docking energy between the covid-19 main protease and indinavir, ritonavir, and ribavirin were calculated to be −7.7, -7.5, -7.0 kcal/mol respectively. all of these scores appear lower than naringin (-10.2 kcal/mol). table 2 shows the binding energy of several ligands with covid-19 main protease sorted according to the docking scores (binding energies) calculated from the autodock vina. figure 5. (a) the structure of covid-19 main protease enzyme generated by using uscf chimera with two active sites for flavonoids and protease inhibitors. (b) ligand-protein interaction for lopinavir. (c) ligand-protein interaction for naringin. iraqi j pharm sci, vol.29(2) 2020 naringin binding with covid-19 236 figure 6. molecular docking of lopinavir has interacted with covid-19 main protease enzyme table 2. the docking score (kcal/mol), mw, clogp, no. of h bond donor, no. of h bond acceptor, and lipinski’s rule of five for fda approved drugs. drug name docking score (kcal/mol) mw (g/mol) <500 clogp <5 no. of h bond donor <5 no. of h bond acceptor ≤10 lipinski's rule of five lopinavir -7.9 628.8 5.9 4 5 no indinavir -7.7 613.8 2.8 4 7 no ritonavir -7.5 720.9 6 4 9 no ribavirin -7.0 212.2 -1.8 4 7 no camostat mesilate -6.4 494.5 3 9 9 yes zanamivir -6.3 332.3 -3.2 7 8 no favipiravir -5.8 157.1 -0.6 2 4 no rimantadine -5.7 179.3 2.6 1 1 yes oseltamivir -5.5 312.4 1.1 2 5 yes simeprevir -5.3 749.9 4.8 2 10 no remdesivir -5.3 602.6 1.9 4 13 no baloxavir marboxil -5.2 571.6 3.8 0 12 no amantadine -5.2 151.2 2.4 1 1 yes boceprevir -4.7 519.7 3.1 4 5 yes conclusion as noted before, covid-19 has become a global concern, due to widespread outbreaks and lack of treatment. therefore, to contribute to this fight against covid-19, molecular docking was performed to identify novel compounds having the potential to bind main protease of covid-19. relying on this topic and repurposing concept, a procedure employing docking of flavonoids, protease inhibitors, and anti-influenza drugs was used to identify new potential molecule to bind the main protease of covid-19 and the result indicates that naringin has a high binding affinity with low energy -10.2 kcal/mol to the main protease of covid-19 than other natural molecules. however, further studies should be conducted for the validation of these compounds using in vitro and in vivo models to pave a way for these compounds in drug discovery. references 1. guangdi li and erik de clercq. therapeutic options for the 2019 novel coronavirus (2019ncov). nature reviews | drug discovery. 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they are used daily and regularly by increasing numbers of the people and the quantities consumed are increasing each year (1) .most cosmetics contain a lot of ingredients are good for microbial growth and the production of cosmetics is not a sterile process and at least the storage temperature is nearly optimal for microbial growth (2) .cosmetics products may be contaminated during manufacturing by microorganisms existing in the environment or in the raw materials , which are mostly contain water and the later form an appropriate media for microbial growth (3) . the raw materials used in cosmetics products may be grouped in to categories (table1). to avoid microbial contamination of cosmetics during use and storage , the manufacturers add preservatives to their products (5) . two different problems arise when preservatives are used in cosmetics , first is that microorganisms easily contaminate the cosmetics when the amounts of antimicrobial agents are kept low for safety and economy , and second is that serious problems of skin reactions produced by antimicrobial agents are caused when their amounts are increased for preventing microbial contamination (6) , therefore a balance needs to be established with the preservatives of choice between killing microorganisms and not injuring the cell of the consumer who uses the product, there are several different preservatives available but the cosmetic market is dominated by a few preservatives: parabens, formaldehyde, formaldehyde releasers, and methylchloroisothiazolinone/ methylisothiazolinone (7) . table 1 : raw materials categories . water acids , alkalis , salts . oils , waxes , paraffins . fatty acids , alcohol , esters . surfactants , emulsifier . talc , clay . protien , starches , botanical , gums and resin. humectants . color and pigments . preservatives , antioxidants and chelating agents . fragrances , essential oils . source adapted from orth. (4) 1 corresponding author email : h.mastermaster@yahoo.com received : 23/10/2010 accepted : 12/3/2011 iraqi j pharm sci, vol.20(1) 2011 microbial contamination of cosmetic 39 the microorganisms commonly isolated from the poorly preserved water-based products include klebsiella , enterobacter , staphylococcus , bacillus species , pseudomonas aeruginosa , penicillium and candida albicans (8) . dawson and reinhard (9) survey 15 different brands of eye shadow and they recovered 67% of them were contaminated with one or more species of microorganisms representing the genera staphylococcus , micrococcus , corynebacterium acinetobacter, bacillus and moraxella . table (2) shows some potentially pathogenic bacteria isolated from cosmetics products and some of these organisms are part of the normal human flora (10) . table 2: potentially pathogenic bacteria isolated from cosmetic preparations acinetobacter calcoaceticus escherichia coli providencia rettgeri citrobacter diversus hafnia alvei providentia stuartii citrobacter freundii klebsiella oxytoca pseudomonas cepacia clostridium spp. klebsiella pneumonia pseudomonas fluorescens enterobacter aerogenes morganella morganii serratia liquefaciens enterobacter agglomerans proteus mirabilis staphylococcus aureus enterobacter cloacea proteus vulgaris staphylococcus epidermidis enterobacter gergovia the united state pharmacopeia (usp) specifies 4 bacterial indicators for cosmetics contamination include : salmonella spp. , staphylococcus aureus , pseudomonas aeruginosa and escherichia coli , the european pharmacopeia (ep) specifies these same 4 bacterial indicators including an additional requirements for ascertaining the different levels of enterobacteriaceae (11) .the addition of organic material greatly increases the chances of growth and deposits or turbidity due to algae , mold , bacteria or yeast in a range of poorly preserved pharmacopeia solutions . emulsions can become thin , separate , decolorize or change color and become visibly heterogeneous owing the hydrolysis of the oil phase or change in ph of the aqueous phase (7) . contaminants may be seen as sediments , turbidity and pigments such as the red prodigiosin of serratia marcenscens and greenish pigments of pseudomonas and these pigments may alter the products appearance (12) , wilson and ahearn (13) have demonstrated that cosmetics may serve as a possible in transmission and persistence of microorganisms in clinical eye infections . similarly , others have reported serious infection and even death resulting from direct or indirect exposure to other microbiologically – contaminated cosmetics including mouth wash , hand cream and mascara (14) .the aim of this study is to demonstrate the microbial content of unused products at the point of the sale . the cosmetic products were manufactured in iraq and were purchased from super markets . materials and methods samples cosmetic products used in this study including 3 brands of (25) mascara , (30) lip pencil and (25) eye pencil ; all brands were purchased from different factories of iraqi supermarkets, the period of sampling was 3 months ranging from april to july 2010. firstly the visible changes were observed like pigments , turbidity and presence of sediments .after that the ph number for each brand were detected by using ph meter . aerobic plate count materials and equipments were sterilized before use and aseptic techniques were used . the caps of the products were wiped with ethanol (70%). microbiological media were reconstituted and prepared from their dehydrated powder according to manufacturer instructions . by means of a micropipette , one ml of the product was disintegrated in tryptic soy broth (9 ml) according to b.p.2010 using a flask shaker and suitable serial dilutions in tryptic soy broth were prepared . one ml sample of each dilution was poured in a sterile petridish and then 15 ml of sterile tryptic soy agar was poured on the samples , the plates were gently swirled in a round movements to allow a good mixing of the agar with the sample , then the plates were allowed to solidify on aleveled surface ,triplicates plates for each sample were used and incubated at (35°c-37°c) for 24-28 hr. for bacteria . sabouraud dextrose agar was used instead of tryptic soy agar for the detection of fungi . the prepared plates were incubated at 25°c for 5-7 days. after incubation the number of colonies was counted by estimating the total iraqi j pharm sci, vol.20(1) 2011 microbial contamination of cosmetic 40 count of the growing bacteria and fungi and then the mean of three plates were calculating . a laboratory control count was performed using negative control blank (tryptic soy broth without product) and with positive control (contaminated product). detection for specific microorganisms the detection for specific microorganisms such as escherichia coli , pseudomonas aeruginosa , salmonella spp. and staphylococcus aureus was performed following procedure under isolation and identification test for specified microorganisms ( british pharmacopeia bp2010) (14) as shown in (table 3 ) , when results showed the presence of any of these microorganisms , appropriate biochemical test were performed , the detection of fungi were depending on morphology of colonies on sabouraud dextrose agar and the direct microscopic examination by lactophenol aniline blue – stained method (15) . table 3 : isolation and identification tests for specified microorganisms (bp.2010) organism enrichment primary test secondary test confirmation enterobaceriaceae lactose broth 35-37 °c for 24-48 hr. eeb-mossel 35-37 °c for 24-48 hr. vrbgla 35-37 °c. growth of gram negatives . escherichia coli as above. macconkey broth 43-45 °c for 18-24 hr. macconkey agar 4345 °c for 18-24 hr. indole at 43.544.5 °c biochemical. salmonella spp. as above for 5-24 hr. tbbg broth 42-43 °c for 18-24hr. then subculture on :dca.xlda or bga 35-37 °c for 24-48 hr. tsi agar 35-37 °c for 18-24 hr. biochemical serological pseudomonas aeruginosa saline peptone 35-37 °c for 2-5 hr. casein digest broth 35-37 °c for 24-48 hr. cetrimide agar 35-37 °c for 24-48 hr. oxidase test staphylococcus aureus as for pseudomonas aeruginosa above as for pseudomonas aeruginosa above baird-parker 35-37°c for 24-48 hr. coagulase , catalaes , dnase test eeb-mossel : enterobacteriaceae enrichment broth –mossel; vrbgla: violet red bile agar with glucose and lactose ; tbbg: tetrathionate bile brilliant green broth ; dca: deoxycholate citrate agar ; xlda: xylose lysine deoxycholate agar ; tsi: triple sugar iron agar ; dnase : deoxyribonuclease test . results and discussion microbial contamination of cosmetic products is a matter of a great importance to the industry and it can become a major cause of both product and economic losses (16) . results shown in (table4) investigated that 32.5% of (80) products were contaminated and the mascara were more contaminated than other tested products . (table 5) shown that all products contaminated with bacteria in varying degrees including gram positive bacteria such as staphylococcus aureus , staphylococcus epidermidis and gram negative bacteria such as klebsiella pneumonia , pseudomonas aeruginosa and escherichia coli , the colony count of all detected bacteria ranging from (10 3 10 4 )c.f.u./ml. (table 6) represented that all products contaminated with fungi including penicillium spp. , aspergillus fumigatus.,and candida albicans , the colony count of fungi were ranging from (10 2 -10 4 ) c.f.u/ml . the ph number of all tested products ranging from ( 6.2-8.1) while the visible changes include color change ,pigments and sediments in some of contaminated products. poltikin and ahearn , ahearn et al ,bharauria and ahearn reported that mascaras mostly contaminated by pseudomonas aeruginosa , staphylococcus epidermidis , klebsiella pneumonia and candida parapislosis (17)(18)(19) . dawson and reinhardt reported that the genera staphylococcus , micrococcus , acinetobacter , bacillus moraxella , pseudomonas are contaminated eye pencil (9) . peter etal reported that the fungal contaminants of cosmetics consisted largely of aspergillus fumigatus , pencillium and microsporium spp. (20) .the acceptable microbiological limits are recommended in guidelines for a variety of cosmetics preparations , these limits are between 10 2 to 10 3 c.f.u/ml or gram for pathogenic and non pathogenic bacteria (3) . results obtained in recent study approximating the results of the iraqi j pharm sci, vol.20(1) 2011 microbial contamination of cosmetic 41 mentioned studies . microorganisms can grow on almost every substances existing in nature and often able to attack or even decompose them , cosmetic ingredients are rich in nutrients that provide organic substrates in the form of sugar , starch , protein , amino acids , organic acids , alcohols , lipids and etc. for microbial growth (21) , addition to that ,water is a fundamental requirements for any microorganisms likely to contaminate the cosmetics products , thus untreated or non sterile water can support microbial growth leading to contamination of cosmetics products (11) , generally microorganisms of interest in raw materials or cosmetic products grow best around neutral ph 7.0 and many yeast and molds are able to tolerate acid ph conditions (7) . according to all results obtained in recent study : most cosmetic products require the addition of preservative to prevent microbial contamination and rancidity, and cosmetic should be produced in a perfectly clean hygienic environment , equipment , instruments , storage tanks and containers should accordingly be maintained in a high standard of cleanliness. table 4: the total number of tested cosmetic products and the percentage of contamination in these products cosmetics products total no. of products no. of contaminated product percentage of contaminated products mascara 25 10 40% lip pencil 30 8 26.6% eye pencil 25 8 32% total 80 26 32.5% table 5: the diagnosed bacteria and their counts (c.f.u./ml) cosmetic products diagnosed bacteria bacterial counts mascara klebseilla pneumonia, pseudomonas aeruginosa, staphylococcus aureus, staphylococcus epidermidis. 1.5×10 5 c.f.u./ml 20×10 3 c.f.u./ml 3.7×10 4 c.f.u./ml 9.1×10 3 c.f.u./ml lip pencil escherichia coli , staphylococcus aureus, staphylococcus epidermidis. 13×10 3 c.f.u./ml 15×10 4 c.f.u./ml 3.8×10 4 c.f.u./ml eye pencil pseudomonas aeruginosa, escherichia coli , staphylococcus aureus. 1.8×10 4 c.f.u./ml 2.9×10 4 c.f.u./ml 12×10 4 c.f.u./ml table 6: the diagnosed yeasts and fungi and their counts (c.f.u./ml) cosmetic products diagnosed yeasts and fungi yeasts and fungal counts mascara candida albicans, penicillium spp. , aspergillus fumigatus . 2.5×10 3 c.f.u./ml 20×10 3 c.f.u./ml 1.6×10 4 c.f.u./ml lip pencil candida albicans 4.8×10 4 c.f.u./ml eye pencil candida albicans 6.6×10 4 c.f.u./ml conclusion microbiological safety is one of the most dynamic and critical of cosmetics qualityparameters . from this study it was found that microorganisms such as escherichia coli , klebsiella pneumonia, pseudomonas aeruginosa , staphylococcus aureus ,staphylococcus epidermidis , penicillium spp. , aspergillus fumigatus and candida albicans were contaminated mascara , iraqi j pharm sci, vol.20(1) 2011 microbial contamination of cosmetic 42 lip pencil and eye pencil in a varying degrees . the cosmetic industry has many compelling reasons to establish and maintain microbiological quality of its products . as these rarely produced under a sterile conditions , appropriate control of the many factors involved in the microbiology of the products is critical . these factors include raw material quality , hygiene and training of manufacturing personal, establishment of sanitary design and materials , application of validated cleaning and sanitization process design and control , application of general chemical /physical factors including heat, time temperature ,ph, addition of specific chemical preservation and use of appropriate barrier packaging . all of these factors are effective for the control of microbiological risks in the cosmetic products . references 1. hashim , p .; shahab , n. ; masilamani , t. ; baharom , r. and ibrahim , r. a cosmetic analysis in compliance with the legislative requirements , halal and quality control . malaysian j. chem . ,2009;11(1) : 81-87. 2. siegert , w. ; schulke and gombh , m. microbiological quality management for the production of cosmetics and toiletries . cosmet. sci. tech.,2005 . 3. naki , n. ; yekta , a; ozalp , m. ; atakan , n. and polat, m . decontamination of cosmetic products and raw materials by gamma irradiation . fabad j . pharm . sci. ,2006;31 : 198-209. 4. orth , d s. handbook of cosmetic normax f e . the microbiology ,1989;marcel dekker. new york . 5. brannan , d. and dille , j. type of closure prevents microbial contamination of cosmetics during consumer use . appl . enviro. microbiol. ,1990; 56(5) : 1476-1479. 6. aono , a. ; takahashi , k.; mori , n.; shimizu , h.; kobayashi , a.; fugiwara, n . and okada , f. calorimetric study of the antimicrobial action of various polyols used for cosmetics and toiletries . j.adv. sci. and tech. ,1999; 26(1) :2-8 . 7. razooki , r ; saeed , e and hamza , h. a study on cosmetics products marketed in iraq: microbiological aspects . iraqi j.pharm sci .,2009; 18(2):20-25. 8. perry , b. cosmetic microbiology . microbiol . today ,2000;28(1) :185-187. 9. dawsen , n l .and reinhardt , d j . microbial flora of in-use , display eye shadow tester and bacterial challenges of unused eye shadows. appl. enviro. microbiol . ,1981; 42(2). 297-302. 10. norman , f e . the cosmetic indusrty . scientific regulatory foundation . cosmet. sci. and tech. series ,1984;. 2(21):301-320 . 11. luis , j . molecular diagnosis of microbial contamination in cosmetic and pharmaceutical product . j. aoac int. ,2001; 84(3) : 671-675. 12. sutton , s . a publication of the pharmaceutical microbiology forum .pharm. microbiol. ,2008;14(3): 1-19. 13. wilson , l a . and ahearn , d g . pseudomonas – induced corneal ulcers associated with contaminated eye mascaras . am. j.ophthalmol . ,1977; 54 : 112-119. 14. british pharmacopeia , london appendix xvi b2 a409-a416, microbial examination of non sterile products ,2010. 15. winn, w. ;allen, s.; janda , w.; koneman , e.; procop ,g. ; schreckenberger , p. and woods , g. color atlas and textbook of diagnostic microbiology , 6 th edition , lippincott williams and wilkins , united state of america ,2006; 1151-1220. 16. orus , p. and leranzo , s. current trends in cosmetic microbiology . int. j. microbiol. ,2005; 8(3) :139142 . 17. poltkin , j a . and ahearn d g . evaluation of recalcitrance of mascara in a cartridge container system to microbial challenge . j. int. microbiol.,1986;1: 205207 . 18. ahearn , d g .; sanghva , j. and haller , g j . mascara contamination : in use and laboratory studies . j.soc. cosmet .chem. ,1978; 29 : 127-131. 19. bhadauria , h. and ahearn , d g . loss of effectiveness of preservative systems of mascaras with age . appl. enviro. microbiol. ,1980;39(3) : 665-667. 20. hugbo , p g .; onyekweli, a o . and igwe , i . microbial contamination and preservative capacity of some brands of cosmetic creams . tropi. j.pharm. res.,2003; 2(2):229-243. 21. franca, m . bacterial ,fungal and yeast contamination in six brands of irreversible hydrocolloid impression materials . braz. oral . res. ,2007 ;21(2) : 106-111. preparation and evaluation of atenolol floating beads as a controlled delivery system iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 70 preparation and evaluation of atenolol floating beads as a controlled delivery system lena m. thomas* and yehia i. khalil* ,1 * department of pharmaceutics , college of pharmacy , university of baghdad , baghdad , iraq. abstract this study aims to encapsulate atenolol within floating alginate-ethylcellulose beads as an oral controlled-release delivery system using aqueous colloidal polymer dispersion (acpd) method.to optimize drug entrapment efficiency and dissolution behavior of the prepared beads, different parameters of drug: polymer ratio, polymer mixture ratio, and gelling agent concentration were involved.the prepared beads were investigated with respect to their buoyancy, encapsulation efficiency, and dissolution behavior in the media: 0.1 n hcl (ph 1.2), acetate buffer (ph 4.6) and phosphate buffer (ph 6.8). the release kinetics and mechanism of the drug from the prepared beads was investigated.all prepared atenolol beads remained floating on 0.1 n hcl (ph 1.2) medium over 24 hours. besides, high yield beads of 73.0784.31% was obtained. encapsulation efficiencies were in the range of 33.10 % -79.04 %, and were found to increase as a function of increasing drug: polymer mixture ratio and the gelling agent concentrations.moreover, atenolol release profile from the beads was affected by the ph of the dissolution medium. it was found to be slowest in 0.1 n hcl (ph 1.2) and fastest in phosphate buffer (ph 6.8).the obtained results suggest that atenolol could be formulated as a controlled release beads, using ethylcellulose and alginate as polymers, using acpd method. keywords: floating beads, atenolol, controlled delivery system. الخالصة سٍطز عئٍ باسخعَاه اىجٍْاث اىص٘دًٌ٘ مْظاً ححزرٍ-طافٍت ٍِ االثٍو سيٍي٘س مزٌاثىخغيٍف االحٍْ٘ى٘ه فً ٕذٓ اىدراستحٖدف -فاُ عدة ع٘اٍو ٍثو ّسبت اىعقار ٬طزٌقت االّخشار اىَائً.ىخحسٍِ مفاءة اىخحٍَو اىدٗائً ٗ سي٘ك ححزر اىعقار ٍِ اىنزٌاث اىَحضزة ّسبت ٍشٌج اىَن٘ثزاث اضافت اىى حزمٍش اىَادة اىَٖيَْت قد حضَْج فً ٕذٓ اىدراست. ىقد حٌ اىخحزي عِ اىنزٌاث ٬اىَن٘ثزاث ٍ٘الري ىحاٍض 0.1 مفاءة اىخحٍَو اىدٗائً ٗسي٘ك اىخحزراىدٗائً فً االٗساط: ٬حضزة باىْسبت اىى قابيٍخٖا عيى اىطف٘اىَ ( ٍٗحي٘ه اىف٘سفاث اىَائً )االص 4.6ٍحي٘ه االسٍخاث اىَائً )االص اىٍٖدرٗجًٍْ ٬(1.2اىٍٖدرٗمي٘رٌل )االص اىٍٖدرٗجًٍْ جٍَع اىنزٌاث ىقد ٗجد اُاىخ٘صو اىى ٍعزفت آىٍت اىخحزراىحزمً ىيدٗاء ٍِ اىنزٌاث اىَحضزة.مَا حَج ٍحاٗىت ٬(6.8اىٍٖدرٗجًٍْ ٬ساعت 24( ىفخزة سادث عِ 1.2ٍ٘الري ىحاٍض اىٍٖدرٗمي٘رٌل )االص اىٍٖدرٗجًٍْ 0.1اىَحضزة بقٍج طافٍت فً ٗسظ حاٍضً ٬ % 30.04 3110بْسبت بقابيٍت ححٍَو دٗائً ٗ % 84.31-33.03بٍِ ٖائً ٌخزاٗح اضافت اىى اُ حيل اىنزٌاث ماّج بنٍَت ّاحج ّ ىقد ٗجد اُ اىشنو اىبٍاًّ ىخحزر . اىَادة اىَٖيَْت حزمٍشٗاىعقار:اىَن٘ثزاث شٌادة ّسبت مداىت ى مَا ٗجد اُ قابيٍت اىخحٍَو اىدٗائً حشداد ٗجًٍْ ى٘سظ اىخحزر اىدٗائً ٗاُ ابطـأ ححزر ٌخٌ فً ٗسظ االثٍو سيٍي٘س حخأثز باالص اىٍٖدر-االحٍْ٘ى٘ه ٍِ مزٌاث اىجٍْاث اىص٘دًٌ٘ (.6.8( ٗاسزعٖا فً ٍحي٘ه اىف٘سفاث اىَائً )االص اىٍٖدرٗجًٍْ 1.2ٍ٘الري ىحاٍض اىٍٖدرٗمي٘رٌل )االص اىٍٖدرٗجًٍْ 0.1 سيٍي٘ساالثٍو باسخعَاه ىخحزراُ اىْخائج اىَسخحصيت ٍِ اىدراست حقخزح باُ االحٍْ٘ى٘ه ٌَنِ حصٍٍغٔ عيى شنو مزٌاث ٍح٘رة ا . باسخعَاه طزٌقت االّخشار اىَائً مَن٘ثزاث اىجٍْاث اىص٘دًٌ٘ٗ introduction atenolol is a polar cardioselective βblocker, widely used alone or in combination with other drugs for treatment of various cardiovascular conditions. (1) it is slightly soluble in water with reported half-life of 6-7 hours. (‎2) it is considered a drug with low jejunal permeability and a low extent of absorption, therefore it has an oral bioavailability of about 46-62% (3) . thus, it seems that an increase in gastric residence time may increase the extent of absorption and bioavailability of the drug. (4) sodium alginate (na-alg) is a water-soluble salt of the naturally occurring polysaccharide alginic acid, and has received much attention in pharmaceutical preparations, particularly due to its role as a vehicle for controlled drug delivery. (5, 6) ethylcellulose (ec) is an inert, hydrophobic, organosoluble polymer and has been extensively used as a pharmaceutical vehicle in many drug delivery systems including microcapsules and microspheres to control the dissolution rate of drugs from their preparations. (7, 8) spherical gel beads of calcium alginate can be formed immediately when sodium alginate solution is added dropwise into a calcium chloride solution. the formed beads are able to entrap drug(s) in eggbox gel matrix, and thus act as a vehicle for the controlled release of many orally administered drugs. (9, 10) 1corresponding author email : ybmmaz@yahoo.com received : 27/11/2010 accepted : 23/4/2011 iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 71 such beads have also been used in the design of floating systems in order to prolong gastric retention time (11,12) the floating and sustained drug release properties of calcium alginate beads were found to be enhanced by the addition of ethylcellulose. (13) this work aims to formulate a promised controlled-release atenolol beads that are capable of floating on gastric juice to prolong gastric residence time, thereby extend the time of drug release and then enhance its oral bioavailability, using aqueous colloid polymer dispersion method. materials and methods materials atenolol powder (gift from samarra drug industry), sodium alginate (low viscosity grade; 5.5 cp in 1% solution at 25 °c; himedia lab, india), ethylcellulose (bdh chemicals ltd. poole, england), anhydrous calcium chloride (gainland chemical co. uk). all other reagents were of analytical grade. methods beads were prepared according to the method described by bodmeier et al. (14) in this method, atenolol powder (weight corresponds to drug:polymer mixture ratio, table 1) was dispersed in a previously prepared aqueous solution of sodium alginate (prepared by dissolving sodium alginate in hot (40 ○ c) water) and mixed homogeneously. an aqueous dispersion of ethylcellulose was added to this mixture and stirred well. after a good mixing, the bubble-free dispersion was dropped from a 10 ml syringe barrel (without needle) into a manually shaken beaker containing an aqueous calcium chloride solution at room temperature. the falling distance was 3.5-5 cm. the droplets instantaneously formed gelled spheres by ionotropic gelation of alginate with ca +2 ions. the formed beads were separated after 35 minutes and washed twice with distilled water as they were filtered over buchner funnel under vacuum. the beads were spread on a filter paper positioned in a petri-dish and left to dry overnight in an oven (mammert, germany) at 40 ○ c. nine formulas were prepared by this method, the composition of each formula are given in table 1:  formulas 1-4 to investigate the effect of drug: polymer mixture ratio.  formulas 4-7 to investigate the effect of sodium alginate: ethylcellulose ratio, and.  formulas 4, 8 and 9 to investigate the effect of the concentration of gelling agent (cacl2) used. table 1: the effect of different variables on floating beads of atenolol using aqueous colloidal polymer dispersion method. formula code drug: polymer ratio (% w/w) atenolol (core) (gm) (polymer mixture) gelling agent (cacl2) % w/v microencapsulation yield % yield drug loading (%) encapsulation efficiency (% ee) sodium alginate: ethylcellulose ratio (gm) theoretical (gm) actual (gm) theoretical actual 1 1:0.5 (66.6:33. 3) (8) 1:15 (0.25: 3.75) 1 12 9.45 78.75 66.66 49.40 74.18 2 1:1 (50:50) (4) 1 : 15 (0.25 : 3.75) 1 8 6.132 76.65 50 34.56 69.125 3 1:2 (33.3:66. 6) (2) 1:15 (0.25 : 3.75) 1 6 4.579 76.31 33.3 22.55 67.73 4 1:3 (25:75) (2) 1:15 (0.375 : 5.625) 1 8 6.018 75.22 25 16.65 66.60 5 1:3 (25:75) (2) 1:7.5 (0.70 : 5.30) 1 8 5.846 73.07 25 8.275 33.10 6 1:3 (25:75) (2) 1:22.5 (0.255 : 5.745) 1 8 6.00 75.00 25 12.65 50.60 7 1:3 (25:75) (2) 1:30 (0.1935 : 5.8065) 1 8 6.05 75.71 25 17.98 71.95 8 1:3 (25:75) (2) 1:15 (0.375 : 5.625) 3 8 6.279 78.48 25 18.12 72.49 9 1:3 (25:75) (2) 1:15 (0.375 : 5.625) 5 8 6.745 84.31 25 19.76 79.05 iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 72 evaluation of the prepared beads encapsulation efficiency and yield percent beads recovered after being dried were weighed and the yield was calculated as a percentage of the total weights of starting material (polymer and drug) added during preparation and the actual weight of beads obtained. thus, the yield percent was calculated using the following equation (15) : yield (%) = actual weight of beads  100 theoretical weight of beads to determine the encapsulation efficiency of the beads, 50 mg beads were crushed using a porcelain mortar and a pestle, and dispersed in 50 ml methanol. the dispersion was sonicated for 15 minutes and then the dispersion was filtered. a 1 ml sample was taken, diluted to 10 ml with methanol, and drug content assayed using a uv-visible spectrophotometer (carry uv, varian, australia) at λmax of 275 nm.the drug entrapment efficiency of prepared beads was determined by using the following equation (15) : in-vitro buoyancy test the floating ability and duration of the prepared beads were determined in usp paddle type dissolution apparatus (copley scientific ltd, england). a weight of beads equivalent to 100 mg of atenolol were dispersed over 900 ml of 0.1 n hcl (ph 1.2). the paddle speed was maintained at 50 rpm, and the temperature of the medium maintained at 37±0.5 c. buoyancy was observed visually. beads were considered to be buoyant only when all of them are floated. (12) 44 dissolution studies dissolution studies of atenolol beads were performed using usp paddle type dissolution apparatus (copley scientific ltd, england) at a rotation speed of 50 rpm. for all dissolution studies, beads (equivalent to 100 mg atenolol) were dispersed separately over 900 ml of dissolution medium maintained at 37±0.5c.for all formulas, dissolution studies were carried out using 0.1 n hcl (ph 1.2) to simulate gastric ph. furthermore, to predict drug release behavior from beads in different ph media, dissolution media of acetate buffer (ph 4.6) and phosphate buffer (ph 6.8) were used to simulate the phs pertaining to proximal and middle small intestine (duodenum and jejunum), and distal small intestine (ileum), respectively. (16, 17) the effect of ph on drug release behavior from the prepared beads was studied only for formulas 1-4.ten milliliter samples were withdrawn at specific time intervals and replaced by the same volume of fresh media. the withdrawn sample was filtered with a millipore filter (pore size 0.45 μm). the amount of atenolol released in all studied media was analyzed by a uvvisible spectrophotometer (carry uv, varian, australia) at λmax of 273 nm. analysis of drug release mechanism to establish the release kinetics and mechanism of drug release from the floating beads in different media, data obtained in the first 120 minutes of the in vitro release studies in 0.1 n hcl (ph 1.2) and acetate buffer (ph 4.6) were fitted to into zero-order, first-order, higuchi, and baker-lonsdale models. (18) meanwhile, the release profile of atenolol in phosphate buffer (ph 6.8), which is estimated in the first 60 minutes was analyzed by hopfenberg model in addition to the above mentioned models.the dissolution data were also fitted to the well-known exponential equation (korsmeyer–peppas equation), which is often used to describe the drug release behavior from polymeric systems when the mechanism is not well-known or when more than one type of release phenomena is involved. the value of the release exponent (n) in korsmeyer–peppas equation was determined and is used to indicate different release mechanisms. when n takes a value of 0.43, the drug diffuses through and is released from the polymer following fickian diffusion mechanism. a value of 0.43 < n < 0.85 represents a non-fickian (anomalous) transport; a value of n equal to 0.85 indicates case-ii transport, and a value of n above 0.85 indicates super case-ii transport. (19) the best fit model was selected as the one with the highest values of regression coefficient (r 2 ). statistical analysis the statistical analysis of results was performed by one way analysis of variance test using microsoft excel program. differences were considered to be statistically significant at p < 0.05. results and discussion encapsulation efficiency and yield percent the yield percent and encapsulation efficiency of atenolol-loaded alginate ethylcellulose beads prepared with different drug: polymer mixture ratios, using different formulation variables are listed in table 1. encapsulation efficiency (%ee) = actual drug content  100 theoretical drug content iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 73 the effect of drug: polymer mixture ratio increasing drug: polymer mixture ratio resulted in a significantly (p< 0.05) higher percentage yield and encapsulation efficiency as shown in table 1 (formulas 1-4). the higher encapsulation efficiency obtained may be attributed to the event of more available drug amounts to be encapsulated. similar results were observed with mefenamic acid (20) and indomethacin beads. (21) the effect of different ratios of polymer mixture (sodium alginate: ethylcellulose ratio) table 1 shows that the percentage yield and encapsulation efficiency was significantly increased (p < 0.05) by varying sodium alginate: ethylcellulose ratio from 1:7.5 to 1:22.5 (formulas 4-6). this indicates that at higher amounts of ethylcellulose use, and due to greater hydrophobic barrier effect, higher encapsulation efficiency was obtained due to retardation of drug migration and loss to the external aqueous phases. similar results were observed with floating cimetidine microspheres prepared by using hydroxypropylmethyl cellulose and ethylcellulose as polymers. (22) however, further reduction in sodium alginate: ethylcellulose ratio to 1:30 (formula 7) produced significantly lower (p < 0.05) percent yield and encapsulation efficiency. such an effect may be explained by the fact that at lower sodium alginate ratios, the drug particles were not encapsulated completely due to less available active calcium-binding sites in the polymeric chains and, consequently, a lower degree of cross-linking with gelling agent and formation of lower strength matrix structure. (23) the effect of gelling agent (cacl2) concentration it is observed that increasing cacl2 concentration significantly (p < 0.05) increased the drug percentage yield and encapsulation efficiency (table 1, formulas 4, 8 and 9). this may be due to better cross-linking reaction of sodium alginate present in the bead structure with the more abundant presence of calcium ions, thus a better barrier entrapping the drug inside the polymeric matrix structure of the beads is provided. similar results were observed with calcium alginate nicardipine microcapsules. (24) in-vitro buoyancy test the prepared beads showed excellent floating characters. the beads remained on top of 0.1 n hcl (ph 1.2) for duration time over 24 hr; besides, buoyancy was not affected by the changes of the formulation variables during the overall study. meanwhile, no change was observed on the whole integrity of floated beads concerning color and shape during the period of buoyancy test. the floating ability of the prepared beads is because of the use of low density materials which produced an inherently low density beads that float immediately following contact with 0.1 n hcl (ph 1.2). similar results were observed with alginateethylcellulose beads of metronidazole. (13) dissolution studies the effect of drug: polymer mixture ratio the behavior of drug release from beads having different drug: polymer mixture ratios (1:0.5, 1:1, 1:2, and 1:3) was studied in different dissolution media (0.1 n hcl (ph 1.2), acetate buffer (ph 4.6) and phosphate buffer (ph 6.8)), using an exact weight of beads equivalent to 100 mg of atenolol. the obtained release profiles are represented in figures 1, 2 and 3 respectively.for the drug release profile in acidic ph media (figures 1 and 2), a significant reduction (p <0.05) in drug release rate was observed by decreasing drug: polymer mixture ratio. the time to 50 % drug released showed a significant reduction (p < 0.05) equivalent to 3 and 5 fold from 15 to 50 minutes and from 18 to 90 minutes for 1:0.5 and 1:3 drug: polymer mixture ratio at ph (1.2) and ph (4.6), respectively.the higher drug release rates observed in the 2:1 drug: polymer mixture ratio could be attributed to inability to form a homogenous dispersion at the first step, and because of the fact that at this ratio the amount of polymer might be insufficient to coat all the drug particles present, such that unbound drug particles were randomly present in or on the surface of the beads, resulting in higher drug release rates. (24) similar findings were observed with ethylcellulose coated diclofenac sodium microcapsules. (25) the most retardant drug release effect observed with 1:3 ratio as compared to lower ratios indicates that the release rate is controlled by wall thickness: an increase in polymer ratio will increase the coat thickness surrounding the drug particles, thereby increasing the distance travelled by the drug through the coat causing a greater impedance to drug release. (26) this finding is in accordance with the results found with ethylcellulose coated isoprinosine microcapsules. (27) for the drug release profile in ph 6.8 (figure 3), varying the drug: polymer mixture ratio had a non-significant (p > 0.05) effect on drug release, and drug release profile was in an order opposite to what was observed with that for release in acidic ph. iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 74 figure 1: the release of atenolol from beads with different drug: polymer mixture ratio in 0.1 n hcl (ph 1.2) at 37 °c. figure 2: the release of atenolol from beads with different drug: polymer mixture ratio in acetate buffer (ph 4.6) at 37 °c. figure 3: the release of atenolol from beads with different drug: polymer ratio in phosphate buffer (ph 6.8) at 37 °c. the effect of varying the ph of dissolution media atenolol, being a weakly basic drug with a pka value of 9.6 (28) is expected to possess higher solubility and therefore a faster drug release rates at acidic phs than at basic phs. however, as it is evident from drug release profile in figure 4, it was seen that the drug release in acidic media (0.1 n hcl (ph 1.2) and acetate buffer (ph 4.6)) is relatively slow and equivalent to each other within the initial 2 hours compared to fast and complete drug release in phosphate buffer (ph 6.8) within 3060 minutes. it is reported that alginate matrices demonstrate a ph-dependent hydration, swelling and erosion behavior, resulting in phdependent drug release mechanisms. (29) visual observation of atenolol beads during dissolution studies revealed that those hydrated in acidic ph showed no visible evidence of erosion and reserved their shape and structural integrity. such structural stability in acidic ph may be attributed to the stability of alginate at lower phs and the conversion of ca-alginate to the insoluble but swelling alginic acid. (30) thus, a greatly retarded and limited release of atenolol from alginate–ethylcellulose matrices was observed during dissolution into an acid environment. in contrast, for dissolution in phosphate buffer (ph 6.8), contrary to what was observed in acidic media, rapid erosion and disintegration of the beads have occurred, and this greatly contributed in facilitating atenolol release rate. such bead disintegration occurs because of the exchange process of the calcium ions of the alginate beads with na + salt of the phosphate buffer, resulting in formation of monovalent sodium salts of alginates, which unlike the divalent ca 2+ salts of alginates, are water soluble. (10) in conclusion, atenolol release behavior from alginate-ethylcellulose beads was affected by the ph of the dissolution medium. such behavior is consistent with that observed with nicardipine (31) and mefenamic acid beads. (20) figure 4: the release of atenolol from beads with (1:3) drug: polymer mixture ratio in different ph media at 37 °c. iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 75 the effect of different ratios of polymer mixture (sodium alginate: ethylcellulose ratio) to study the effect of different sodium alginate: ethylcellulose ratios on atenolol release, formulas 4-7 were subjected to in vitro drug release studies in 0.1 n hcl (ph 1.2). the drug release profiles of these formulations are shown in figure 5. the results indicated that the drug release rate varied depending on the ethylcellulose content and decreased significantly (p < 0.05) as the ratio of sodium alginate: ethylcellulose was decreased from 1:7.5 (formula f5) to 1:15 (formula f4). such observation indicates that ethylcellulose, when used at higher amounts, is able to retard the release of drug from the beads due to the formation of a less permeable matrix. (32) besides, the presence of lower amounts of sodium alginate produced a highly porous matrix structure having a low gel strength, this will result in rapid diffusion of the drug from the matrix. (33) similar results were observed with floating metronidazole alginate beads containing ethylcellulose. (13) figure 5: the effect different ratios of polymer mixture (sodium alginate: ethylcellulose ratio) on the release of atenolol in 0.1 n hcl (ph 1.2) at 37 °c. the effect of gelling agent (cacl2) concentration to study the effect of gelling agent (cacl2) concentration on atenolol release, beads were formulated with three different concentrations of cacl2: 1%, 3% and 5% (w/v) (formulas 4, 8 and 9). the release profiles for these formulations are shown in figure 6.from the obtained results, it appears that increasing the concentration of cacl2 produced a nonsignificant (p> 0.05) effect on dissolution behavior. this may be attributed to the possible saturation of calcium binding sites in the guluronic acid chains of sodium alginate with the use of a 1 % (w/v) cacl2 solution which prevented further calcium ion entrapment and hence crosslinking was not altered with the higher concentrations of cacl2 solutions. (34) similar results were obtained with furosemide-loaded calcium alginate beads. (35) figure 6: the effect of calcium chloride concentration on the release of atenolol in 0.1 n hcl (ph 1.2) at 37 °c. evaluation of release kinetics in order to obtain meaningful information for atenolol release kinetics and mechanism from formulated beads, drug-release data were fitted into various drug-release kinetic models mentioned above, and the goodness of fit of the release data was assessed. tables 2-4 summarize the values of regression coefficient (r 2 ), kinetic constant (k) and release exponent (n) for the different release kinetic models in 0.1 n hcl (ph 1.2), acetate buffer (ph 4.6) and phosphate buffer (ph 6.8), respectively. in this study, as illustrated in table 2, atenolol release in 0.1 n hcl (ph 1.2) from different bead formulations showed a good fit into korsmeyer–peppas model. an exception is formula 1, which showed better fitting to baker lonsdale model. the release in all the prepared formulas is expected to occur by fickian diffusion mechanism since the value of release exponent (n) was in the range of (0.10.43); this hypothesis is further supported by visual observations that beads neither changed their shape nor disintegrated during dissolution process. for release in acetate buffer (ph 4.6), as in the case of release in 0.1 n hcl (ph 1.2), the beads preserved their shape during dissolution studies. it was observed that the drug release data of formulas having different drug: polymer mixture ratios fitted intermediate to the equations of higuchi, baker-lonsdale and korsmeyer-peppas (table3). for formulas 1-3, since higher values of regression coefficient were obtained during iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 76 data fitting to korsmeyer-peppas equation and the value of release exponent (n) were in the range of 0.585-0.656, an anomalous transport mechanism of drug release where both diffusion through swellable and non swellable polymer is expected to occur, rather than drug release behavior totally based on diffusion, which generally is the case in higuchi’s and baker-lonsdale’s models. on the other hand, for formula 4, release data fitted best to higuchi model indicating that drug release occurs by fickian diffusion mechanism. this conclusion is further supported by the value of release exponent (n =0.272) obtained from fitting to korsmeyer-peppas equation.in case of drug release in phosphate buffer (ph 6.8), simulating the drug kinetics of formulas having different drug: polymer mixture ratios (formula 1-4) with different models, it is observed that drug release fitted intermediate with hopfenberg and korsmeyer-peppas models while regression coefficients were slightly higher with the latter model (table 4). the value of diffusional exponent (n) for formulas 1-3 was in the range of 0.921-1.068, meaning that a super case ii transport mechanism is involved, which is related to polymer relaxation and erosion mechanism. a value of (n= 0.525) for formula 4 indicates a nonfickian (anomalous) transport mechanism which is an indication of both diffusion/polymer relaxation controlled drug release. these mathematical finding, supported by observations obtained during dissolution experiments showing disintegration of the bead matrix structure, proposes a mixed drug release mechanism in phosphate buffer, partially involving swelling of sodium alginate in the bead structure, bead matrix disintegration and drug diffusion out of the beads. similar drug release behavior was observed for furosemide (37) and tiaramide (38) loaded alginate beads during their dissolution in phosphate buffer. table 2: the release mechanism of atenolol from prepared beads by acpd method using different drug: polymer ratios in 0.1 n hcl (ph 1.2)*. *kinetic constant (k), correlation coefficient (r 2 ), diffusional exponent (n). shaded and bold font areas: optimum values of correlation coefficient. bold underlined areas: second best fit values of correlation coefficient. iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 77 table 3: the release mechanism of atenolol from prepared beads by acpd method using different drug: polymer ratios in acetate buffer (ph 4.6)*. *kinetic constant (k), correlation coefficient (r 2 ), diffusional exponent (n). shaded and bold font areas: optimum values of correlation coefficient. bold underlined areas: second best fit values of correlation coefficient. table 4:the release mechanism of atenolol from prepared beads by acpd method using different drug: polymer ratios in phosphate buffer (ph 6.8)*. *kinetic constant (k), correlation coefficient (r 2 ), diffusional exponent (n). shaded and bold font areas: optimum values of correlation coefficient. bold underlined areas: second best fit values of correlation coefficient. iraqi j pharm sci, vol.20(1) 2011 preparation and evaluation of atenolol floating beads 78 conclusions atenolol was successfully formulated as floating alginate-ec beads by acpd method.the drug: polymer mixture ratio, sodium alginate: ethylcellulose polymers ratio and cacl2 concentration are significant formulation factors which affected drug encapsulation efficiency.the release profile of atenolol from the alginate-ec beads increased as a function of drug: polymer mixture ratio. it is possible to achieve slow atenolol release rates from the alginate-ec beads by reducing drug: polymer mixture ratio or by increasing the amount of ethylcellulose used in the polymeric mixture.on the other hand, atenolol release from the prepared beads was found to be ph-dependant. it was slowe in acidic media. in view of the prolonged buoyancy of beads in the acidic environment, accompanied by slow drug release, entrapment of atenolol in these beads may provide a satisfactory oral controlled release drug delivery system.having a prolonged release behavior and maximum percentage encapsulation efficiency, formula 9 was selected as optimized formulation, keeping in view the 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preparation and evaluation of atenolol floating beads 80 37. das mk and senapati pc. furosemideloaded alginate microspheres prepared by ionic cross-linking technique: morphology and release characteristics. indian j. pharm. sci. 2008;70(1):77-84. 38. fathy m, safwat sm, el-shanawany sm, tous ss, otagiri m. preparation and evaluation of beads made of different calcium alginate compositions for oral sustained release of tiaramide. pharm. dev. technol. 1998; 3(3):355-364. iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs doi : https://doi.org/10.31351/vol30iss1pp133-143 133 synthesis of mutual prodrugs of secnidazole and ciprofloxacin and study their physicochemical properties marwah w. khalid*,1, leaqaa a. alrubaie *, ahmed n. abood ** * department of pharmaceutical chemistry, college of pharmacy, university of basrah, ,basrah, iraq ** department of pharmaceutics, college of pharmacy, university of basrah, basrah ,iraq abstract in present work, secnidazole and ciprofloxacin were linked as mutual prodrugs to get antibiotics with broader spectrum of activity by acting on aerobic and anaerobic bacteria, improved physicochemical properties and given by single dose to enhance patient’s compliance. furthermore, they provide structural modifications to overcome bacterial adaptation. the structures of the synthesized compounds were confirmed using ft-ir spectroscopy, mass spectrometry, elemental microanalysis (chno) and some physiochemical properties. the prodrugs were compared with the parent drugs in terms of partition coefficient, solubility, palatability and antibacterial activity. the synthesis of mutual prodrugs resulted in an increase in log p values to 1.114 for mutual i and 2 for mutual ii when compared with secnidazole (log p -0.373) and ciprofloxacin (log p -0.832). in addition, mutual i displayed 144-fold higher aqueous solubility than ciprofloxacin. while mutual ii showed 4.4fold enhancement in the aqueous solubility of ciprofloxacin. taste evaluation by panel method showed palatable taste in prodrugs compared to the parent drugs. the synthesized compounds were screened for their antimicrobial activity by well diffusion method against different bacterial strains which are; staphylococcus aureus, pseudomonas aeruginosa, escherichia coli and klebsiella pneumoniae. the prodrugs have revealed to an improvement in the antibacterial activities against all examined bacterial strains. chemical hydrolysis study at ph (1.2 and 7.4) has indicated that these compounds may pass without hydrolyzing through the stomach and produce enough stability to be absorbed from the intestine as indicated by t1/2 values. keywords: ciprofloxacin, secnidazole, mutual prodrug, the physicochemical properties, antibacterial activity. سيبروفلوكساسينالو للسيكنيدازول دوائية مشتركة مقدمات تصنيع والكيميائية الفيزيائية خصائصها ودراسة **أحمد نجم عبود و *لقاء عبد الرضا رحيم ، 1*، مروة وليد خالد ، العراق البصرة، البصرةاء الصيدالنية ، كلية الصيدلة ،جامعة يفرع الكيم* ، العراق البصرة، البصرة، كلية الصيدلة ،جامعة الصيدالنياتفرع ** الخالصة لحصول على مضادات حيوية ذات ا مشتركة لغرض كمقدمات دوائية ينسسيبروفلوكساال و السيكنيدازولتم ربط , الحالي العمل في وعالوة على .يضجرعة واحدة لتحسين امتثال المر اعطاءها بشكل لغرض .كيميائية الفيزيائية والخصائص ال لتحسينمن الفعالية و واسعطيف مطياف , ،الحمراء تحت االشعة اطيافباستخدام المحضرة وتم تأكيد هياكل المركبات .التكيف البكتيري للتغلب على هيكليةذلك، فإنها توفر تعديالت معامل حيث األصلية من األدوية مع مقدمات األدوية مقارنة تمت .وبعض الخصائص الفيزيائية الكيميائية (chno) للعناصر الدقيق التحليل، الكتلة لـ 1.114إلى log pقيم في زيادة نتج عنه مشتركةالدوائية المقدمات ال تصنيع .للبكتيريا المضاد والنشاط واالستساغةقابلية الذوبان و التقسيم mutual i لـ 2وmutual ii السيكنيدازول مقارنة مععند ال(log p -0.373) لسيبروفلوكساسين ا و(log p -0.832). ،وباإلضافة إلى ذلك أضعاف من 4.4 بمقدار أعلى مائيًا ذوبانًا mutual ii أظهر بينما .من السيبروفلوكساسين ضعفًا 144 بمقدار أعلى مائيًا ذوبانًا mutual i أظهر فحص الفعالية المضادة تم. األصلية باألدوية مقارنة الدوائية للمقدمات مستساًغا طعًما اللوحة بطريقة الطعم تقييم أظهر . السيبروفلوكساسين و staphylococcus aureus وهي المختلفة البكتيرية السالالت الحفر ضد انتشار طريقة للمركبات المحضرة بواسطة للميكروبات pseudomonas aeruginosa و escherichia coli و klebsiella pneumonia .مقدمات األدوية تحسن في األنشطة المضادة أظهرت المركبات هذه أن إلى( 7.4 و 1.2) الهيدروجيني األس عند الكيميائي المائي التحلل دراسة أشارت. ضد جميع السالالت البكتيرية المفحوصة للبكتيريا النصف . عمر قيم لذلك اشارت كما األمعاء من امتصاصها ليتم كافيًا ثباتًا وتنتج المعدة عبر تحلل بدون تمر قد .للميكروبات المضادة , الفعاليةالكيميائية و الخصائص الفيزيائيةمقدمات دوائية مشتركة ,, سيكنيدازول, ينسسيبروفلوكسا المفتاحية: الكلمات 1corresponding author e-mail: marwa.wkhalid@gmail.com received:28 / 7 /2020 accepted: 18/ 10/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp133-143 iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs 134 introduction in a clinical practice the treatment for anaerobic infections aims to treat a complex ecosystem of multiple bacterial species with an antibiotics regimen that provide effective activity against both anaerobic and facultative bacteria (1). current therapeutic interventions available for anaerobic infection relies on combining two or more antibiotics for providing adequate antimicrobial coverage for both components. in combination, these antibiotics should be synergistic or at least not antagonistic in activity against microorganisms (2). ciprofloxacin, a fluoroquinolone drug, is highly effective against many clinically vital pathogens which are responsible for variety of infections comprising gastrointestinal infections, urinary tract infections (uti), sexually transmitted diseases (std), respiratory tract infections (rti) and are also clinically useful against infections of skin, prostatitis, bones and penicillin resistant sexually transmitted disease (3,4). ciprofloxacin is active against both gram-positive and gram negative bacteria by inhibiting bacterial dna replication however, exhibits reduced activity against anaerobic pathogens (5). the effectiveness and broad spectrum activity of fluoroquinolone have made this class most heavily consumed antibacterial agent worldwide. however, due to their uncontrolled use, bacteria have evolved resistance against large groups of these compounds (6). one approach to overcome the resistance problem is the synthesis of new and ingenious antimicrobial agents. the development of new agents can be achieved by the identification of novel antibiotics or by coupling two different antibiotic molecules to make it much more difficult for bacteria to develop resistance to these molecules (7). ciprofloxacin is classified as class 4 compound according to the biopharmaceutical classification system (bcs), meaning that it possesses poor solubility as well as poor gastrointestinal permeability. this is due to the strong intermolecular bonds (van der waals and hydrogen bonding interactions) that allow the molecules to pack densely in a solid-state structure (8). secnidazole, share a common spectrum of activity and effectivity with other 5-nitroimidazoles against anaerobic micro-organisms with longer terminal elimination half-life and higher cure rates. these advantages associated with single-dose therapy of secnidazole offers an attractive alternative to multiple dosage regimens of other drugs in this class (9). although 5-nitroimidazoles are the only drugs recommended for the treatment of trichomoniasis and are the most commonly prescribed drugs for the treatment of giardiasis, their use was limited by their organoleptic properties. some of the main problems of secnidazole are its bitter taste, odor and gastrointestinal side effect (10). since secnidazole is bcs class ііі drug (highly water soluble and low permeable) (11) it is preferable to increase its lipophilicity to increase membrane permeability and impact the solubility of drug in saliva to prevent binding to taste receptors on the tongue (12). prodrug design is a choice of approach in solving many of the stability, solubility, permeability, safety and targeting problems that plague drug discovery and development. a mutual pro-drug is a form of prodrug in which two different pharmacophores linked together so that each agent acts as a promoiety/carrier for the other agent. the linkage should be cleavable under physiological conditions, via either chemical or metabolic means (13).the mutual prodrug approach is of a great interest, because the combination therapy is used for the management of many diseases where therapeutic agents can be co-administered in separate dosage forms (14).however, there are potential advantages in giving co-administered agents that applied routinely and successfully in combination therapy; and also possess the requisite functional group(s) in the form of unique chemical entity given by single dose. in the view of this background, the present study was conducted to design and synthesize mutual prodrugs of ciprofloxacin with secnidazole by ester and amine coupling. then study the synthesized compound regarding to solubility, lipophilicity, taste and antimicrobial activity and compare the results with those of the parents. preliminary chemical hydrolysis study of final compounds will be done at different ph (1.2 and 7.4) using uv method to identify the chemical stability of these compounds as they pass through the gastrointestinal tract. figure 1. chemical structures of secnidazole (15) figure 2. chemical structures of ciprofloxacin (16) iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs 135 materials and methods all reagents and anhydrous solvents were used as received from the commercial suppliers (sigma – aldrich, merk, germany; himedia, cdh, india; bdh, oxoid england; atom, u.k). ciprofloxacin base and secnidazole were supplied by hyper-chem company (china). melting points were determined by the capillary method using electro thermal ia9000, essex, uk. the thin layer chromatography (tlc) was run on silica gel (60) f254, merck (germany) to check the purity of the products as well as monitoring the progress of reactions. the identification of compounds was done using u.v. detection and chromatograms eluted with chloroform: methanol (85:15). ft-ir spectra were recorded using a shimadzu model (kyoto, japan) instrument on kbr disks. the mass spectra were obtained on a 5975c vl msd with tripe-axis detector (agilent technologies, using an ionizing potential of 70 ev) chemical synthesis the two target compounds were designed as mutual prodrugs in which secnidazole hydroxyl group is linked with ciprofloxacin from two different bonding sites (c3-carboxyl and c7 piperazine) (17) by ester and amine coupling. the steps of the synthesis were presented in scheme 1 and 2 respectively. synthesis of ciprofloxacin acid chloride (ia) (18) in a 250 ml round-bottomed flask provided with a stirrer, (2.0 g, 6.0 mmol) of ciprofloxacin was dissolved in (20 ml) dry tetrahydrofuran. thionyl chloride (2 ml, 27.6 mmol) was added dropwise during the course of 30-40 minute with cooling in ice bath under stirring. the mixture was refluxed for (4 hours) with continuous stirring and monitored by evolution of hcl gas (which is detected by changing the color of litmus paper into reddish when placed on top of the flask). the excess of thionyl chloride and solvent was removed by filtration. the deep yellow precipitate was re-dissolving in dry tetrahydrofuran (20 ml) and was refiltered several times to remove excess thionyl chloride. the product obtained was directly used for the next coupling step with secnidazole. synthesis of ciprofloxacin –secnidazole ester (mutual i) (19) the solution of above-obtained acid chloride (1.8 g, 5.12 mmol) in dry methylene dichloride (40 ml) was added drop wise to 250 ml round-bottomed flask contain a mixture of secnidazole (1g, 5.8 mmol), pyridine (1 ml) and dry methylene dichloride (40 ml) under anhydrous conditions using molecular sieves 4a0. the mixture was stirred at room temperature for 24 hrs. until the reaction was completed as evidenced by tlc, the residue which precipitated after solvent evaporation was stirred with 1m sodium carbonate solution (50 ml) for 30 minutes and extracted with chloroform three times. the chloroform extracts were combined and washed with water (3 x 50 ml), then the extract was dried over anhydrous sodium sulphate and gave faint yellow powder that washed several times with 5 ml portions of acetone. (80% yield). m.p. =223°c. rf = 0.78. ft-ir (cm-1) = 3351 (n-h) stretching of secondary amine, 3136 (c-h) stretching of aromatic, 1732 (c=o) stretching of ester, 1531 and 1365 (n=o) asymmetrical and symmetrical stretching of nitroimidazole. synthesis of ciprofloxacin methyl ester (iia) (20 ) ciprofloxacin (2.0 g, 6.0 mmol), was dissolved in methanol (50 ml), the solution was cooled in ice bath, then thionyl chloride (2 ml, 27.6 mmol) was added drop-wise during the course of 3040 minute with stirring, resulting in a yellow solution. this solution was heated under reflux for (3 hours). then left overnight at room temperature. the solution was concentrated by evaporation giving a yellow oil. the oil was stirred with 1m sodium carbonate solution (50 ml) for 15 minutes and extracted with chloroform three times. the organic layers were combined and dried over anhydrous mgso4. the solvent was removed by evaporation to give the product as a white crystalline solid. (83 % yield). m.p. 192°c. rf = 0.75. ft-ir (cm-1): 3365 (n-h) stretching of secondary amine, 3136 (c-h) stretching of aromatic, 1732 (c=o) stretching of ester. synthesis of secnidazole chloroacetate (iib) (21) the dissolved chloroacetyl chloride (0.5 ml, 5.8 mmol) in dry methylene dichloride (10ml) was added drop wise to an ice-cooled solution of a mixture of secnidazole (1g, 5.8 mmol) and tea (0.8 ml, 5.8 mmol) in dry methylene dichloride (25ml) with stirring over a period of one hour. the reaction mixture then stirred overnight at room temperature. methylene dichloride was distilled off and the residue was taken in ethyl acetate (20 ml). the ethyl acetate layer was washed with water (10 ml x 3) and dried over anhydrous sodium sulfate then the solvent was evaporated to obtain the desired product as a semisolid deep brown. (yield 76.3%). rf = 0.92. ftir (cm-1) = 3043(c-h) stretching of aromatic, 1747 (c=o) stretching of ester, 1527 and 1357(n=o) asymmetrical and symmetrical stretching of nitroimidazole. synthesis of ciprofloxacin –secnidazole amine (mutual ii) (20,22) a mixture of compound iia (2.4g, 7mmol), and compound iib (1.8g, 7mmol) was dissolved in dmf: chcl3 (25:75) mixture (40 ml), then tea (1 ml, 7mmol) was added. the reaction mixture was stirred overnight at room temperature. the product was poured into finely crushing ice with stirring. the precipitated compound was filtered then washed several times with water and recrystallized from methanol to give the final product as light iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs 136 brown crystals. (65 % yield). m.p=230°c. rf = 0.71. ft-ir (cm-1): 3016 (c-h) stretching of aromatic, 1747, 1732 (c=o) stretching of ester, 1527 and 1365 (n=o) asymmetrical and symmetrical stretching of nitroimidazole, disappearance of nh stretching of secondary amine. characterization of synthesized prodrugs determination of λ max the wavelength of maximum emission (λmax) of ciprofloxacin, secnidazole, mutual i and mutual ii in phosphate buffer (ph 7.4), 0.1n hcl (ph 1.2) and in distilled water was found by scanning 0.100mm solutions for each of them in every medium over the uv range of 200400nm. solubility measurement the solubility of mutual prodrugs in different media (0.1n hcl ph 1.2, phosphate buffer ph 7.4 and d.w.) was compared with that of both parent drugs using saturation shake flask method (23). saturated solutions were prepared separately by adding an excess of each compound to a sealed, light protected flasks containing fixed volume of vehicle (5ml). after shaking the solution for 24 hours at 37° c under constant vibration (50 rpm), the equilibrium was reached and the excess solid was allowed to settle down. the liquid phase was sampled using a filter syringe, and the filtrates were diluted volumetrically with the corresponding vehicle to be analyzed by uv-spectroscopy. three determinations were carried out for each sample to calculate the solubility. partition coefficient determination the partition coefficient is the most common way of expressing the lipophilicity of a compound, it is the ratio of the concentration of a solute in a water-saturated organic phase to its concentration in an organic-saturated aqueous phase (24). the organic solvent is usually n-octanol but occasionally other solvents such as chloroform are used. the lipophilicity each mutual prodrug was compared with that of both parent drugs by measuring the equilibrium concentration in two immiscible liquid phases chloroform and water (25). chloroform (2 ml) was added to 25 ml of distilled water. this system was shaken by a magnetic stirrer for 24 h and the phases were separated by centrifuge. one milligram of ciprofloxacin, secnidazole, mutual i and mutual ii was dissolved in the saturated aqueous phase separately. 5ml were withdrawn and each compound concentration was determined spectrophotometrically (aqueous phase 1). the remaining 20 ml of saturated aqueous solution was placed in an automatic shaking incubator with equal volumes of chloroform (20:20) ml for 24 hours. the phases were separated and the concentration of compound in the aqueous phase was determined by uv-spectroscopy (aqueous phase 2) while its concentration in chloroform was determined by subtracting the final concentration in aqueous phase 2 from its initial concentration in aqueous phase 1. the partition coefficient was calculated from the following equation (26) : partition coefficient (p) = (conc. of drug in org. phase) (conc. of drug in aq. phase2) taste evaluation palatability of each mutual prodrug was checked by panel method (27). sensory analysis was used to assess final products taste. for this purpose, six human volunteers were selected. powder was placed on tongue for 15 seconds, then spat out the compound and the bitterness degree were recorded. the volunteers were instructed not to swallow the powder, which was placed on the tongue. to indicate taste values, the following scale was used: 0=palatable, 1= normal, 2=slightly bitter, 3=bitter, 4= extremely bitter (28). chemical stability at ph 1.2 and ph 7.4 chemical stability of the synthesized prodrugs was studied in near physiological conditions in hc l (simulated gastric fluid, sgf, ph 1.2) and phosphate buffer (simulated intestinal fluid, sif, ph 7.4) at 37˚c(29). the reactions were initiated by preparing 0.02 mg/ml of each prodrug in buffer solutions. the solutions were kept in a water bath at 37˚c. samples of (3ml) were withdrawn at appropriate time interval (15, 30, 60, 120 min). the reactions were monitored by using uv spectrophotometric method at the lambda max of the prodrug by recording the decrease in absorbance of prodrugs accompanying the hydrolysis. antimicrobial activity the preliminary antibacterial activity was investigated by agar well diffusion method against four tested local bacterial isolates (escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae as gram negative bacteria and staphylococcus aureus as gram positive bacteria). three concentrations from each of ciprofloxacin, secnidazole, physical mixture of ciprofloxacin in combination with secnidazole and the synthesized prodrugs were prepared by dissolving each compound in dmso at a stock concentration of 1000 µg/ml followed by two-fold serial dilutions to 500 and 250 µg/ml. wells of 5 mm diameter were punched into an agar medium previously seeded with the test organisms. the plates were incubated for 24 h at 37 °c and the perpendicular diameter of inhibition zone in the region of each well was measured (30). results and discussion the reaction sequences for the synthesis of the mutual i was accomplished and outlined in scheme 1, that include activation the carboxyl group of ciprofloxacin by thionyl chloride to get ciprofloxacin acid chloride (ia) that attacks secnidazole (alcohol component) in a basic condition to give mutual i. iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs 137 scheme 1. synthesis pathway of ester (mutual i) the reaction sequences for the synthesis of the mutual ii was accomplished and outlined in scheme 2, in which the carboxyl group of ciprofloxacin was protected in the form of methyl ester through reaction with thionyl chloride in cold methanol to form intermediate compound (iia). intermediate compound (iib) was prepared by acylation of free hydroxyl group of secnidazole with chloroacetyl chloride, the reaction was carried out in a basic condition and led to the formation of secnidazole chloroacetate. later, the intermediate compound (iia) was reacted with the intermediate compound (iib) in a basic condition resulting in creating a new amine bond to give mutual ii. the ftir spectrum of mutual і and mutual іі showed several characteristic sharp bands. in mutual і, the appearance of the band near 3300-3400 cm-1 that represents the free nh stretching vibration of secondary amine was accompanied with the disappearance of the two peaks near 3500cm-1 of the o-h stretching modes of carboxylic acid and alcohol. this was accompanied together with the shifting of c=o stretching from lower frequency to higher frequency indicating the conversion of carboxylic acid to ester group. the c=o stretching vibration of mutual і is observed at 1732 cm-1. mutual іі exhibited a strong intensity band appears as a doublet at 1747 and 1732 cm-1 for acyl and aryl carbonyl group respectively because the c=o stretching vibration is shifted to lower frequencies when it is conjugated to aromatic system (31). the disappearance of nh stretching vibration of secondary amine and o-h stretching modes of carboxylic acid and alcohol supported the basic chemical structures of mutual іі. the mass spectra confirmed the proposed structures. iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs 138 scheme 2.synthesis pathway of amine (mutual ii) elemental microanalysis also revealed good agreement with the calculated percentages (table 1). table 1. spectral characterization of synthesized mutual prodrugs scanning drugs and prodrugs solutions (0.100mm) in d.w, 0.1 n hcl and phosphate buffer solution (ph 7.4) in the uv range 200 – 400 nm gave λmax values shown in table 2. secnidazole and ciprofloxacin results are in consistent with documented references (32,33). the synthesized mutual prodrugs present characteristic uv spectra with different maximum absorbance bands from that of ciprofloxacin and secnidazole. this difference is illustrated by a bathochromic shift in the fluoroquinolone absorbance peak due to the role of two charged centers nh2+ and cooto increase the stabilization energy by electronic delocalization (34). the solubility of the synthesized mutual prodrugs, ciprofloxacin and secnidazole was determined by direct calibration method using uv spectrophotometer. the two synthesized mutual prodrugs show an intermediate solubility between ciprofloxacin and secnidazole. higher solubility was obtained for mutual prodrugs as compared with that of ciprofloxacin. the introduction of large groups of secnidazole disrupts the rigid crystal lattice of ciprofloxacin and blocks the intermolecular hydrogen bond formed between the hydroxyl group prodrugs molecular formula mass spectral data (m/z) c.h.n.o calculated observed mutual i c24h27fn6o5 498 c (57.82%) h (5.46%) n (16.86%) o (16.05%) c (58.19%) h (5.16%) n (16.59%) o (16.33%) mutual ii c27h31fn6o7 570 c (56.84%) h (5.48%) n (14.73%) o (19.63% c (56.69%) h (5.72%), n (15.03%) o (19.27% iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs 139 in the carboxylic acid and the carbonyl of the quinolone which is likely to affect the molecular packing which contributes in ciprofloxacin limited solubility (35). moreover, the solubility of zwitterionic form of ciprofloxacin is minimum in ph near its isoelectric point which is close to neutral. these chemical modifications eliminate the zwitterionic nature of ciprofloxacin resulted in basic derivatives with a single pka (36). and led to improve the solubility in physiological ph as shown in table 3. table 2. the λ max values of starting and synthesized compounds. compound λ max (nm) d.w ph 6.8 phosphate buffer ph 7.4 ciprofloxacin 278 272 272 secnidazole 277 320 320 mutual і 282 283 283 mutual іі 282 285 285 table 3. the solubility-comparison study compound solubility (mg/ml) 0.1 n hcl ph 1.2 d.w ph 6.8 phosphate buffer ph 7.4 ciprofloxacin 25 0.088 0.16 secnidazole 44 34.5 34 mutual i 31.3 12.7 3.7 mutual ii 39.2 0.39 0.33 additionally, the prodrugs showed an increase in the lipophilic nature compared with the parent drugs as indicated by increasing numerical values of the partition coefficient (log p) as shown in (table 4). the chemical modifications on ciprofloxacin that has solid-state limited structure using flexible, lipophilic side chains resulting in a compound with less ordered crystal lattice, improved solubility and lipophilicity (8). moreover, the synthesized prodrugs are much more lipophilic than secnidazole because the addition of bulky nonpolar group. the matter that favorable for them to increase the rate of passive diffusion across the biological membrane especially when molecular mass < 500 da as in mutual i (37). table 4. the partition coefficient comparison study the increase in the lipophilicity also revealed that the synthesized mutual prodrugs may address the bitter taste of secnidazole as an evidence by sensory panel test that show a palatable taste even after the powder left in the mouth for 10 seconds. chemical stability at ph values simulating the gastrointestinal fluids is an essential requisite for a prodrug for oral delivery. consequently, the kinetics of the two synthesized mutual prodrugs were studied in aqueous buffer solutions of ph 1.2 and 7.4. the hydrolysis behaviors were monitored by uv-vis spectroscopy because under experimental conditions, the chemical hydrolysis followed the first order kinetics due to the plotting of log (residual prodrug concentration) versus time resulted in straight lines as illustrated in (fig.3 and fig.4) from the slope, the observed rate constants (kobs) were calculated and accordingly the first order kinetic half-life (t1/2) was calculated from the following equation (38): kobs=2.303/t × log (a/a-x) -----------equation (1) t1/2 = 0.693/ kobs ------------equation (2) compound concentration (mg/ml) partition coefficient log p aqueous phase 1 aqueous phase 2 chloroform layer ciprofloxacin 0.039 0.034 0.005 0.147 -0.832 secnidazole 0.037 0.026 0.011 0.423 -0.373 mutual i 0.042 0.003 0.039 13.00 1.114 mutual ii 0.043 0.0004 0.042 105 2 iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs 140 figure 3. the hydrolysis rate of mutual і at ph 1.2 and ph 7.4 figure 4.the hydrolysis rate of mutual іі at ph 1.2 and ph 7.4 where ‘a’ is initial concentration of prodrug, ‘x’ is the amount of prodrug hydrolyzed and ‘t’ is time in minutes. the data of the chemical hydrolysis is given in table 5 revealed that the two synthesized mutual prodrugs have longer half-life in acidic ph value (ph 1.2) compared with buffer ph value (ph 7.4) which means they may pass unhydrolyzed through stomach and produce enough stability to be absorbed unchanged from intestine (39). that give an advantage of possible enzymatic hydrolysis and release the parent drugs after gastrointestinal absorption (40). agar well diffusion method is a widely used method for in vitro evaluating of antimicrobial activity (41). for four important aerobic pathogens (escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae as gram negative bacteria and staphylococcus aureus as gram positive bacteria), agar well diffusion method was established using three concentrations of ciprofloxacin and secnidazole each one separately and combined in the form of physical mixture and mutual prodrugs. ciprofloxacin had the most potent bactericidal activity against pseudomonas aeruginosa and escherichia coli, the lowest response to ciprofloxacin was exhibited by staphylococcus aureus (table 6). these results match previous antimicrobial studies of ciprofloxacin (42). as expected, secnidazole show a very low effect against these aerobic pathogens. this activity returns to inhibiting virulence factors production and not due to the killing of bacterial cells (43). addition of secnidazole to ciprofloxacin in a physical mixture neither decreased nor increased the bactericidal potency of ciprofloxacin. however, the synthesized prodrugs show higher zones of inhibitions against all types of bacteria tested in all three concentrations. this may be attributed to the increased lipophilicity of the mutual prodrugs that led to an increase in permeability through lipid by-layers of the cellular membrane of these bacteria (44). table 5. kinetic data for the chemical hydrolysis of the mutual prodrugs compound ph kobs (min −1) t1/2(min) mutual i 1.2 7.4 1.381 x 10-3 1.842 x 10-3 501.8 376.2 mutual ii 1.2 7.4 0.460 x 10-3 2.072 x 10-3 1506.5 334.4 iraqi j pharm sci, vol.30(1) 2021 secnidazole and ciprofloxacin mutual prodrugs 141 table 6. inhibition zone of the studied compounds compound conc. mg /ml mean inhibition zone diameter (mm) pseudomonas aeruginosa escherichia coli staphylococcus aureus klebsiella pneumoniae. ciprofloxacin 0.25 25 30 17 19 0.5 30 33 21 22 1.0 35 39 25 28 secnidazole 0.25 0 0 0 0 0.5 7 9 8 0 1.0 9 10 9 0 physical mixture 0.25 25 30 17 19 0.5 30 33 21 22 1.0 35 39 30 28 mutual і 0.25 27 30 25 29 0.5 35 35 30 30 1.0 40 42 33 35 mutual іi 0.25 35 30 19 29 0.5 37 35 26 33 1.0 45 42 30 35 conclusions the mutual prodrugs of secnidazole and ciprofloxacin were successfully synthesized and characterized by ft-ir, mass spectrometry, elemental microanalysis (chn) and measurement of some physiochemical properties. the synthesized compounds showed improvement in the antibacterial activity, moderate solubility and palatable taste. experimental log p values indicated that the prodrugs are more lipophilic than the parent drugs. the hydrolysis of the mutual prodrugs in buffer medium ph 7.4 are higher than acidic medium ph 1.2 indicating that the conjugates are stable in an acidic condition. future recommendations 1enzymatic hydrolysis study of the synthesized prodrugs in human plasma. 2further biological characterizations for the synthesized mutual prodrugs to be formulated as an efficient dosage form. especially for mutual i as it highly enhances the solubility of ciprofloxacin and improves the lipophilicity of both 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balouiri m, sadiki m, ibnsouda sk. methods for in vitro evaluating antimicrobial activity: a review. journal of pharmaceutical analysis. 2016; 6(2):71-79. 42. chalkley lj, koornhof hj. antimicrobial activity of ciprofloxacin against pseudomonas aeruginosa, escherichia coli, and staphylococcus aureus determined by the killing curve method: antibiotic comparisons and synergistic interactions. antimicrobial agents and chemotherapy. 1985; 28(2):331-342. 43. saleh mm, abbas ha, askoura mm. repositioning secnidazole as a novel virulence factors attenuating agent in pseudomonas aeruginosa. microbial pathogenesis. 2019; 127:31-38. 44. bartzatt r, lg cirillo s, d cirillo j. design of ciprofloxacin derivatives that inhibit growth of methicillin resistant staphylococcus aureus (mrsa) and methicillin susceptible staphylococcus aureus (mssa). medicinal chemistry. 2010; 6(2):51-56. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ iraqi j pharm sci, vol.21(2) 2012 the protective effect of honey against amikacin 85 the protective effect of honey against amikacininduced nephrotoxicity in rats abeer r. abd ali * and sajida h. ismail **,1 *ministry of health, iraq **department of pharmacology and toxicology, college of pharmacy, university of baghdad, baghdad, iraq abstract drug –induced nephrotoxicity is an important cause of renal failure. aminoglycoside antibiotics, such as amikacin, which causes ototoxicity and nephrtotoxicity as a main side effects, this is focused on the use of natural materials as antioxidants against the toxic oxidative action that exert a cell damaging effect. the most important one of these materials is the honey. the aim of this work is to evaluate the antioxidant effects of honey against amikacin – induced nephrotoxicity.18 albino rats divided into 3 groups (6 rats per each group), group 1 received i.p daily dose of normal saline (control), group 2 received (35 mg/kg/day) i.p dose of amikacin ,and group 3 received (35mg/kg/day) of amikacin i.p dose in combination with oral dose of honey(500mg/kg/day) for 2 weeks. all animals (at 15 th day) were anesthetized by ether and sacrificed; blood samples were collected for the subsequent measurement of the serum creatinine, urea, malneldehyde (mda) and glutathione (gsh) while an isolated kidney was kept in 10 % of formaldehyde for the histopathological examination. this study showed that amikacin causes nephrotoxicity represented by elevation of serum level of creatinine and urea, mda and a decrease in the serum glutathione level. while the administration of honey in combination with amikacin reduced the nephro-toxic effect of amikacin that represented by a reduction of the serum creatinine and urea, mda and elevation of glutathione levels with improvement of the kidney histological findings in comparison with group 2.this study concluded that, honey decreased nephrotoxicity induced by amikacin through interference with the oxidative stress process, i.e. honey acts as free radical scavenger. key words:amikacin, honey, nephrotoxicity, oxidative stress. ألميكاسينتأثير العسل كواقي للتسمم الكلوي المستحث با 1**،و ساجدة حسين اسماعيل *عبير رياض عبد علي . ّصاسح الظحخ ، العشاق* فشع االدّٗخ ّالسوْم ، كل٘خ الظ٘ذلخ ، خبهعخ ثغذاد ، ثغذاد ، العشاق . ** الخالصت ثَ للزسون الكلْٕ رعزجش هي اُن اسجبة الفشل الكلْٕ ,ّهي ُزٍ االدّٗخ ,الوضبداد الحْ٘ٗخ هثل اى االدَّٗ الوسزح أهٌْ٘كل٘كْس٘ذ، ، ّالزٕ ٗسجت رسو٘ن أرًٖ ّكلْٕ كآثبس خبًج٘خ سئ٘س٘خ، هوب رسزذعٖ اسزخذام الوْاد الطج٘ع٘خ هوي لِب الخبطَ٘ ججَ االه٘كبس٘ي . ّاحذ هي أُن ُزٍ الوْاد الوضبدح للزؤكسذ ُْ العسل. الِذف هي الوضبدح للزؤكسذ للعول ضذ االدِذ الزؤكسذٕ الزٖ ٗس هي الدشراى 81ُزا الجحث ُْ رق٘٘ن اٙثبس الوضبدح للزؤكسذ للعسل ضذ الزسون الكلْٕ الزٕ ٗسججَ األه٘كبس٘ي . اشزول الجحث علٔ رلقذ خشعخ هي الوغزٕ الوبلح فٖ الزدْٗف الجطٌٖ )كودوْعَ 8ْعخ فئشاى فٖ كل هدوْعخ(، الودو 6هدبه٘ع ) 3الج٘ضبء هقسوخ إلٔ عْلدذ 3هلغ / كغ / ْٗه٘ب( هي االه٘كبس٘ي فٖ الزدْٗف الجشٗزًْٖ ، ّالودوْعخ 33س٘طشح(، الودوْعخ الثبًَ٘ عْلدذ ة ) /كغ/ْٗه٘ب( لوذح اسجْع٘ي. ّثعذ هغ 355هلغ/كغ/ْٗه٘ب( هي األه٘كبس٘ي فٖ الزدْٗف الجشٗزًْٖ هع خشعخ فوْٗخ هي العسل )33ة) هي الوعبلدخ ( رن رخذٗش خو٘ع الحْ٘اًبد ثبالٗثش ثن قزل الحْ٘اًبد ّ خوع عٌ٘بد هي الذم لق٘بس هسزْٓ 83اًزِبء هذح العالج )فٖ ْٗم ٪ هي 85لٔ فٖ ( فٖ هظل الذم ثٌ٘وب حفع ًوْرج هي الكgsh)الوبلًْذٗبلذُبٗذ ( ّالدلْربثْ٘ى ) mda الكشٗبرٌ٘٘ي ّالْ٘سٗب ّ ( روثل ثؤسرفبع 2الفْسهبلذِٗبٗذ ّرلك لفحض الٌس٘ح الكلْٕ . أظِشد ُزٍ الذساسخ أى الزؤث٘ش الكلْٕ لاله٘كبس٘ي )فٖ الودوْعخ هسزْٓ الكشٗبرٌ٘٘ي ّ الْ٘سٗب ّ الوبلًْذٗبلذُبٗذ ّاًخفبع فٖ هسزْٓ الدلْربثْ٘ى فٖ هظل الذم هع اثبس ّاضحَ للزلف فٖ الٌس٘ح (اًخفبضب هعٌْٗب فٖ هسزْٓ الكشٗبرٌ٘٘ي ّالْ٘سٗب ّالوبلًْذٗبلذُبٗذ هع اسرفبع 3. فٖ ح٘ي سججذ الوعبلدَ ثبلعسل ) هدوْعخ الكلْٕ .2هعٌْٕ فٖ هسزْٗبد الدلْربثْ٘ى فٖ هظل الذم , هع رحسي ّاضح فٖ ًزبئح الفحض الكلْٕ الٌس٘دٖ هقبسًخ هع دساسخ هدوْعخ زخذام العسل هع األه٘كبس٘ي قلل ّثشكل ّاضح هي عول٘خ الزؤكسذ ح٘ث عول العسل علٔ اقزٌبص الدزّس خلظذ الذساسخ إلٔ أى اس الحشح الشادٗكبل٘خ. هوب ّفش حوبٗخ للكل٘ز٘ي هي الزؤث٘ش السوٖ لاله٘كبس٘ي . . عسل ، اميكاسين ، التسمم الكلويالكلماث المفتاحيت : 1 corresponding author email : ph.sajida @ yahoo.com received : 19/2/2012 accepted : 7/7/2012 iraqi j pharm sci, vol.21(2) 2012 the protective effect of honey against amikacin 86 introduction. renal cell injury may culminate in the cell death, which may occur through necrosis, apoptosis or other pathways.chemicals in general can initiate toxicity because of their intrinsic reactivity with cellular macromolecules. they may require renal or extra renal bioactivation to reactive intermediate, or may initiate injury indirectly by inducing oxidative stress. (1) oxidative stress is caused by excessive production of reactive oxygen species and it may produce a major inter-related derangements of cellular metabolism, such as alteration of protein and nucleic acid structure, damage to dna, induction of apoptosis, increase in intracellular free calcium, damage to membrane ion transport or destruction of the cells by lipid peroxidation. (2) the antioxidants play major protective roles against the deleterious effects of oxidant agents produced in the human body. they include both enzymatic-(such as catalase, glutathione peroxidase and superoxide dismutase) and non enzymaticsubstances (such as tocopherols, phenolic compounds, flavonoids, catechins, ascorbic acid and carotenoids). (3) aminoglycosides are potent bactericidal antibiotics; they act particularly against aerobic, gram-negative bacteria . (4) amikacin is one of the aminoglycoside , mostly used for treatment of severe, hospital-acquired infections with multidrug resistant gram negative bacteria such as pseudomonas aeruginosa, acinetobacter, and enterobacter. amikacin is not more and not less ototoxic or nephrotoxic than gentamicin, (5) while mingeot(1999) found that amikacin is less nephrotoxic than gentamicin. (6) aminoglycoside induced nephro and ototoxicity,which are the limiting factors for their clinical use ,in which the oxygen free radicals have been involved. (7) wojckch lesniak, et al (2005) found that aminoglycosides, exert their adverse renal effect by generation of reactive oxygen species. (8) additionally, it has been demonstrated that aminoglycoside form a complex with mitochondrial fe + ² to catalyze the formation of free radicals. (9) honey is sweet, thick syrup made by honey bees from nectar of flowers, the flowers from which bees gather nectar largely determine the color, flavor, and aroma of honey (10) . it is basically a saturated water solution of sugar, which also includes a highly complex mixture of carbohydrates, enzymes, amino acids, organic acids, minerals, aromatic substances, pigments, wax, pollen. (11) studies reported that honey also possesses natural antioxidants through many compounds like vitamin c and polyphenols like chrysin, pinobanksin, luteolin and pinocembrin that can decrease oxidative stress in humans. (7, 11,12) the aim of this study is to evaluates the possible protective effects of honey against nephrotoxicity induced by amikacin. materials and method 18 male albino strain rats with an average weight of (150-200g) were obtained from and maintained in the animal house of the college of pharmacy/ university of baghdad under conditions of controlled temperature and humidity. the animals were fed commercial pellets and tap water. the honey used in this study was the eucalyptus honey, brought and produced by college of agriculture/ university of baghdad. it was given to animals by oral gavages tube in a dose of 500mg/kg/day. (11) experimental protocol group1six rats were treated with i.p injection of normal saline for 14 days .this group served as control. group 2– six rats were treated with i.p injection of 35 mg/kg/day of amikacin for 14 days .this group served as positive control for nephrotoxicity induced by amikacin . group 3six rats treated with oral dose of 500mg/kg/ day of honey concomitantly with i.p dose of amikacin (35mg/kg/ day) for 14 days. this group utilized to investigate the possible protective effect of honey against nephrotoxicity induced by amikacin. all animals were anesthetized by ether and sacrificed after 2 weeks of treatment. preparation of blood samples and tissue after 2 weeks of treatment, the blood samples were obtained after the animals had been sacrificed. samples were left to clot, and then centrifuged at 3000 rpm. for 15 minutes to separate serum, which was stored at -20ºc until used for the determination of creatinine (13) , urea (14) , glutathione (15) and mda (16) ; while the kidney was kept in formaldehyde(10%) and utilized for histological examination using paraffin section technique. (17) the possible histopathological changes were examined in the teaching laboratories of baghdad medical city.statistical analysis was performed using unpaired student´s t-test. data were presented as mean± sd, p-values less than 0.05 were considered significant for all data obtained from this study. http://en.wikipedia.org/wiki/multidrug_resistance http://en.wikipedia.org/wiki/gram_negative http://en.wikipedia.org/wiki/gram_negative http://en.wikipedia.org/wiki/pseudomonas_aeruginosa http://en.wikipedia.org/wiki/pseudomonas_aeruginosa http://en.wikipedia.org/wiki/acinetobacter iraqi j pharm sci, vol.21(2) 2012 the protective effect of honey against amikacin 87 results nephrotoxic effects of amikacin effect of amikacin on the renal function tests the results of this study showed significant increase (p<0.05) in the serum levels of both creatinine and urea of rats treated with 35 mg/kg/ day of amikacin (group 2) compared to the corresponding levels in the control animals ( group 1); where serum levels for creatinine was (36.8±11.73 and 54.8±11.2) and that for urea was (7.22±4.5 and 8.13±0.9) in group 1 and 2, respectively. (table 1, figure 1 and 2). effect of amikacin on the serum gsh and mda levels: this study showed significant decrease (p<0.05) in the serum level of gsh in rats treated with 35 mg/kg/ day of amikacin (group 2) compared to the corresponding level in the control animals (group 1). the serum levels of gsh were (2.638±0.742 and 1.598±0.566) in group 1 and 2, respectively; while there was significant increase (p<0.05) in the serum level of mda in rats treated with 35 mg/kg/ day of amikacin (group 2) compared to the corresponding levels in the control animals (group 1). the serum levels of mda were (4.14± 1.4 and 6.18±1.007) in group 1 and 2, respectively. (table 2, figure 2 and 3). effect of the combination of honey and amikacin on the kidney function test: there were significant decrease (p<0.05) in the serum levels of both creatinine and urea of rats treated with 35 mg/kg/ day of amikacin + 500 mg/kg/day of honey (group 3) compared to the corresponding levels of rats treated with 35 mg/kg/ day of amikacin (group 2).serum levels for creatinine were (54.8±11.2 and 42.8±7.5) and that for urea were (8.13±0.9 and 6.746±1.27) in group 2 and 3, respectively. (table 3, figure 5 and figure 6). effect of the combination of honey with amikacin on the serum gsh and mda levels: there were significant increase (p<0.05) in the serum levels of gsh of rats treated with 35 mg/kg/ day of amikacin + 500 mg/kg/day of honey(group 3) compared to the corresponding levels of rats treated with 35 mg/kg/ day of amikacin ( group 2)the serum levels of gsh were (1.598±0.566 and 3.683±0.887) in group 2 and 3, respectively ;while there were significant decrease(p<0.05) in the serum levels of mda in rats treated with 35 mg/kg/ day of amikacin + 500 mg/kg/day of honey (group3) compared to the corresponding levels of the rats treated with 35 mg/kg/ day of amikacin (group 2 ).the serum levels of mda were (6.18±1.007 and 4.51±0.465) in group 2 and 3, respectively. (table 4 and figure 8) table1: effect of amikacin on the serum urea and creatinine (n=6) parameter group1 x ± sd group2 x±sd creatinine mmol/liter 40.4±4.9 54.8±11.2* urea mmol/liter 7.2±0.45 8.13±0.9* x= mean, sd= standard deviation, n=number of animals, * =significant (p<0.05) table 2: effect of amikacin on the serum mda and gsh (n=6) parameters group1 x±sd group2 x±sd gsh µmol/l 2.638±0.742 1.598±0.566* mda µmol/l 4.14±1.4 6.18 ±1.007 x=mean sd=standard deviation, n=number of animals, *=significant (p<0.05) table 3: effect the combination of the honey with amikacin on the serum urea and creatinine (n=6) parameter group 2 x±sd group 3 x±sd creatinine mmol/liter 54.8±11.2 42.8±7.5* urea mmol/liter 8.13±0.9 6.746±1.27* x= mean, sd= standard deviation, n=number of animals, * =significant (p<0.05) table 4: effect the combination of the honey with amikacin on serum mda and gsh (n=6) parameters group2 x±sd group`3 x±sd gsh µmol/l 1.598±0.566 3.683±0.887* mda µmol/l 6.18±1.007 4.51±0.465* x=mean sd=standard deviation, n=number of animals, * =significant (p<0.05) iraqi j pharm sci, vol.21(2) 2012 the protective effect of honey against amikacin 88 figure 1: effect of amikacin on the serum urea figure2: effect of amikacin on the serum creatinine figure 3: effect of amikacin on the serum mda figure 4: effect of amikacin on the serum gsh figure 5: effect of the combination of honey with amikacin on the serum urea figure 6: effect combination of the honey with amikacin on serum creatinine iraqi j pharm sci, vol.21(2) 2012 the protective effect of honey against amikacin 89 figure 7: effect combination of the honey with amikacin on the serum mda figure8: effect of combination of the honey with amikacin on the level of serum gsh effects of combination of honey with amikacin on the histology of the kidney. kidneys of group 1(control) showed that the glomeruli consist of tuft of capillaries surrounded by bowman's capsule. the renal tubules have a normal appearance figure(9), the structure consist of proximal convoluted tubule in which lined by columnar epithelial cell and small lumen while, the distal convoluted tubule lined by occupied cell with large luminal. the collecting tubule appear large in diameter and surrounded by occupiedal epithelial cells. (figure 10) kidney of group 2 (rats treated with amikacin) showed a marked shrinkage of glomeruli structure with degeneration and necrosis of renal kidneys of group 3(rats treated with amikacin and honey) showed a mild degenerative changes of the renal tubules (proximal, distal and the collecting duct).while, there were regenerative changes of some renal tubules especially proximal tubules and columnar lining epithelial with an improvement of approximately 70% compared to the group 2 animals, and the appearance was look like the control group. (figure 11) figure 9 : section of the kidney show normal structure appearance of the glomerulai and renal tubules of control group. blue arrow represents the glomerulus. red arrow represents the pcts. white arrow represents the dcts. magnification: (100 x); staining: haematoxylline and eosin. figure :10 sections of kidney show degenerative changes and necrosis of renal tubules in amikacin treated group. blue arrow represents shrinkage of glomerular red arrow represents necrosis of pct magnification: (100 x); staining: haematoxylline and eosin. iraqi j pharm sci, vol.21(2) 2012 the protective effect of honey against amikacin 90 figure 11 : sections of kidney show slightly degenerative changes with regeneration of renal tubule in which the appearance look like the normal in amikacin with honey treted group. blue arrow represents glomeruli. red arrow represents regeneration of pcts. white arrow represents regeneration of dcts. magnification: (100 x); staining: haematoxylline and eosin. discussion aminoglycoside antibiotics have long been used as antibacterial therapy. despite their beneficial effects, aminoglycosides have considerable oto and nephro-toxic side effects (18) . it has been reported that amikacin may induce free radical production which implicates a variety of pathological processes. (19,20) in this study the marked elevation of the levels of both serum creatinine and urea in group 2 compared with group 1 were observed and give an indication to the reduction in the glomerular filteration. since serum creatinine and urea are waste products of protein metabolism that need to be excreted by the kidney; therefore such increase of serum creatinine and urea as reported in this study confirm an indication of functional damage of the kidney and these results were in consistent with other studies . (18,21) the nephrotoxicity of aminoglycoside ( represented by acute tubular necrosis) usually appeared 5 to 10 days after a toxic insult and may be seen even after discontinuation of aminoglycosides therapy. the elevation of the serum creatinine and urea may be seen in association with hypomagnesemia and hypokalemia . in general, aminoglycosides induced acute kidney injury results in non oliguric renal failure .(11,19,22) morever, the results of this study showed a significant decrease in the serum levels of gsh with significant increase in the contents of the end product of lipid peroxidation (mda) of group 2 compared with group 1.previous studies showed that in the amikacin treated rats there were had a significant increase in the serum level of mda, suggesting the involvement of oxidative stress in the nephrotoxicity. (21,23) kamil et al (2008) showed that mda is one of the wellknown secondary products generated after exposure to reactive oxygen species and free radicals, and it may be used to evaluate oxidative damage by measuring serum levels of thiobarbituric acid reactive substance . (24) glutathione (gsh) is a one of the natural antioxidant that protect cells from free radical toxins, it is found exclusively in its reduced form, since the enzyme that reveres it from its oxidized (gssg) to reduced form is constitutively active and inducible upon oxidative stress (25) ,therefore gsh is an important naturally occurring antioxidant and its level in the tissue is considered a critical determinant of the threshold for tissue injury, and an explanation for decreased gsh after amikacin treatment to the increased consumption of gsh in non-enzymatic and enzymatic removal of oxygen radicals with efflux of gssg being the major factor responsible for maintenance of redoxe ratio (26), concerning the results of this study which showed amikacin nephrotoxic effects is in agreement with similar other study in which a significant depletion of gsh in kidney cells resulting in their damage due to enhancement of lipid peroxidation. (27) aminoglycoside antibiotics are known to be transported and accumulated within lysosomes of renal proximal tubular cells and to causes proximal tubular cell injury and necrosis. the pathogenesis of aminoglycoside nephrotoxicity is postulated to be related to the capacity of these organic polycations to interact electrostatically with membrane anionic phospholipids and to disrupt membrane structure and function. (28) it was demonstrated that lipid peroxidation moieties like o·, hydrogen peroxide (h2o2) and hydroxyl radicals were increased with aminoglycoside therapy. (29,30) in addition, the results of this iraqi j pharm sci, vol.21(2) 2012 the protective effect of honey against amikacin 91 study showed a marked shrinkage of glomeruli structure in kidneys of rats in group 2 with the appearance of degeneration and necrosis in the renal epithelial cells in the proximal, distal and the collecting duct unlike the structure of the kidneys from other groups of animals. aminoglycosides cause a simultaneous inhibition of variety of different membrane protein species including sodium/potassium atpase and release of lactate dehydrogenase, resulting in an apparently multifactorial cell death process. (31) also it was found that aminoglycosides cause atp depletion from either mitochondrial damage or direct inhibition of mitochondrial oxidative phosphorylation causing an oxidative injury. (32) also renal tubular cells undergo necrosis when their cellular atp stores are severely depleted to a level incompatible with maintenance of basal metabolism and activity of membrane transport pumps. (33) results of this study showed an improvement in the serum creatinine and urea levels of rats treated with combination of honey with amikacin (group 3) compared with group 2, and these levels are near the levels in group1, and these results are in agreement with results of other study which showed that combination of cimetidine (an inhibitor of cytochrome p450) with gentamicin showed decrease in serum urea and creatinine levels. (34) the elevation of gsh levels in the serum of group 3, and decrease of mda levels in comparison with group 2, attributed to the free-radical scavenging properties of the honey where it help in maintaining the levels of reduce glutathione and mda. (35) the antioxidant effects of honey was attributed to its constituents like antioxidant trace elements and flavonoids compounds; therefore honey has been suggested to be able to decrease lipid peroxidation. (36) also the antioxidant activity of honey is due to phenolic compounds and enzymes (glucose oxidase, catalase and peroxidase). (37,38) also the content of lascorbic acid has a significant impact on total antioxidant activity of honey. (39) results of this study are in agreement with results of heba m halawa, et al (2009), which found that natural honey has protective effect against the damage in liver and kidney cells from oxidative stress induced by toxic level of lead in rats. (40) and other study which found that co administration of vitamins c and e significantly prevented the aminoglycosidesinduced nephrotoxicity demonstrated by preservation of gfr and gsh levels and prevention of the elevation of urinary enzyme activities. 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https://doi.org/10.31351/vol29iss2pp122-128 122 the effect of ginkgo biloba extracts on candida albicans isolated from healthy persons zahraa s. qasim*,1 *department of clinical laboratory science, college of pharmacy, university of mosul, mosul, iraq. abstract the objective was to study the effect of prepared ginkgo biloba (as aqueous and ethanolic) extracts against candida albicans isolated from healthy persons. after isolation and identification of c.albicans, conduct 4 tests; susceptibility test, biofilm formation test, phytochemical screening test, and antioxidant activity test. one hundred oral swabs sample were obtained from healthy persons with oral lesion attending dentistry teaching hospital in dentistry college, their age ranged from 1-30 years of both sexes. the studied samples collected through 8 months (april december / 2018). from 100 healthy person involved in this study, there were 21(21%) c. albicans isolates revealed from clinical specimens. zone of inhibition for c. albicans was higher in ethanol than aqueous extracts. three (14.2%) isolates showed positive biofilm formation in tube method, phytochemical reaction in ethanol extract showed 5 phytochemical compounds, while aqueous extract showed 4 phytochemical compounds, in addition to antioxidant activity in ethanol extract was higher than aqueous. in conclusion c. albicans is the only species from genus candida isolated from oral lesion in this study, ethanol ginkgo biloba extract have a good antifungal activity, higher number of phytochemical compounds and a higher antioxidant activity than aqueous extract. keywords: candida albicans, ginkgo biloba, susceptibility test. المعزولة من اشخاص اصحاء تأثير مستخلصات الجنكو بيلوبا على المبيضات البيضاء 1*،زهراء صديق قاسم . عراق،الموصل ، جامعة الموصل ، كلية الصيدلة ، فرع العلوم المختبرية السريرية* الخالصة البحث لدراسة تأثير مستخلصات الجنكو بيلوبا المائي وااليثانولي المحضرة ضد المبيضات البيضاء المعزولة من اشخاص الغاية من اصحاء. بعد عزل وتشخيص المبيضات البيضاء اجريت اربع فحوصات فحص الحساسية, فحص تكون الغشاء الحيوي, فحص المجاميع الكيميائية ضاد لألكسدة. مئة عينة من مسحات الفم اخذت من اشخاص اصحاء, مصابين بتقرح الفم يراجعون المستشفى التعليمي النباتية, و فحص النشاط الم (. 2018/ كانون االول –سنة لكال الجنسين. تم جمع عينات الدراسة خالل ثمانية اشهر )نيسان 30-1في كلية طب االسنان للفئة العمرية الممتدة من ( من المبيضات البيضاء من العينات السريرية. مساحة منطقة القتل للمبيضات %21)21شارك في هذه الدراسة ,عزلت شخص صحي 100من %( كانت ايجابية لفحص تكون االغشية الحيوية 14.2البيضاء كانت اعلى في مستخلص االيثانول منها في المستخلص المائي. ثالث عزالت ) المجاميع الكيميائية النباتية لمستخلص االيثانول خمسة مجاميع كيميائية نباتية, بينما المستخلص المائي اظهر اربعة بطريقة االنابيب, اظهر فحص البيضاء مجاميع كيميائية نباتية, باإلضافة لفحص النشاط المضاد لألكسدة كان اعلى في مستخلص االيثانول منه في المائي. نستنتج ان المبيضات الوحيد المعزول من جنس المبيضات من تقرح الفم في هذه الدراسة, امتلك مستخلص االيثانول للجنكو بيلوبا فعالية جيدة مضادة هو النوع للفطريات, عدد اكثر من المجاميع الكيميائية النباتية ونشاط مضاد لألكسدة اعلى من المستخلص المائي. جنكو بيلوبا, فحص الحساسية.المبيضات البيضاء, الكلمات المفتاحية : introduction a traditional chinese medicine based on herbs has gained an increased acceptance from pharmaceutical companies as a rich resource for drug discovery (1). ginkgo biloba is the oldest living tree, and one of the most popular medicinal plants with a long history of use as a herbal medicine, its leaves extracts have been widely sold as a phytomedicine in europe (1). the effects of ginkgo biloba extracts are due to its content of active phytochemicals compounds that possess pharmacological effects such as antiasthmatic, memory impairment and concentration difficulties improvement, wound healing, anticoagulant properties, and nerve cells protection (2). the presence of phytochemicals in medicinal plants has been found a significant in the production of nonstandardized phytopharmaceutical products, the most important of these phytochemicals are alkaloids, tannins, flavonoids and phenolic compounds (3). ginkgo biloba leaves extracts have 24% flavonoids, 6% terpenoids and less than 5 ppm of natural antioxidants ginkgolic acids, this constitutes the most important active substances in the extract (4). researchers are interested in antibacterial and antifungal effects that are obtained from medicinal plants sources with phytochemical activity, with advantage of no or less side effects that are often associated with synthetic antimicrobials (3). 1corresponding author e-mail: zahraasedeeq1980@gmail.com received: 20/1 /2020 accepted:20 / 5/2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol29iss2pp122-126 iraqi j pharm sci, vol.29(2) 2020 the effect of ginkgo biloba on candida albicans 123 candida albicans is a member of genus candida that is frequently isolated from mouth of healthy individuals and considered as a member of oral normal flora (5). oral cavity is a reservoir of more than 700 species of different microorganisms including candida, which considered a normal colonizer in healthy and infected human mouth that resulting in different types of candidiasis (6). in oral mucosal surface of healthy subjects candida species resist the mechanical washing action of saliva, this resistance is an important factor for adherence and then biofilm formation which is one of the most virulence factors of c. albicans pathogenicity (5). biofilm is structured of multiple cell types encased in an extracellular matrix, this biofilm phenotype of c. albicans has been shown to play a role in antifungal resistance (7). the rapid development of antifungal resistance by different candida species has increased over the past decade, which associated with a rapid emergence of resistant strains of candida species (8). in addition, toxicity of the currently used antifungal agents has necessitated the need for alternative therapy, for these reasons, studies have been directed recently to focus on development of novel antifungal medicaments from plant resources (8). the aim of this research was to study the effect of prepared ginkgo biloba (as aqueous and ethanolic) extracts against candida albicans isolated from healthy persons , also to assess 4 tests for isolated c. albicans; susceptibility test, biofilm formation test, phytochemical screening test, and antioxidant activity test. materials and methods place and duration of the study this study included 100 oral swab samples obtained from oral cavity with lesion from healthy persons (of both sexes, age ranged from 130 years) with bad oral hygiene attending dentistry teaching hospital, college of dentistry, university of mosul. samples were collected over 8 months (april december / 2018). isolation and identification c. albicans the samples were carried to the microbiology laboratory, college of pharmacy, university of mosul for isolation and identification. each swab sample was cultured on sabouraud dextrose agar. chloramphenicol was added at a concentration of 0.05 g / l to prevent any bacterial growth. then the inoculated plates were incubated aerobically for 48 –72 hours at 37 °c. colonies of candida on sabouraud dextrose agar appeared as white creamy colonies. further identification was applied including direct examination with gram stain for the presence of budding candida cells, and germ tube test which processed by transferring portion of pure cultured colony and mixed with 0.5 ml of human serum, then incubated at 37°c for 2-3 hours, loopful of the inoculated serum examined for production of germ tube as short initial hyphae. in addition to chlamydospore formation test on cornmeal with tween 80 agar, this test was processed by inoculating cornmeal tween 80 agar with tested colony of candida, then incubated the plates for 4872 hours at 37°c , later on slide was prepared by lactophenol mount for production of chlamydospore (9,5). microscopically examination of all isolated candida by gram stain, was done to confirm the identification of the c. albicans because it may confuse with the species c. dubliniensis which can give same results of tests mentioned above (10,11). preparation of ginkgo biloba extracts the dried leaves of ginkgo biloba were purchased from china, and converted into powder manually. twenty five gram of dried ginkgo biloba leaves were weighted and extracted with 250 ml of ethanol in soxhlet extractor for 12 hours. for aqueous extraction 25 gram of ginkgo biloba leaves powder were prepared by maceration with 250 ml of sterile distilled water for 3 days at room temperature with frequent agitation. the aqueous and ethanol extracts were separately filtered with filter paper, then solvents were evaporate. the crude extracts were gummy and have dark yellow to green color (3). finally the extracts of ginkgo biloba leaves were kept freeze dried in refrigerator until use (1). percentage yield of both extracts were calculated. susceptibility test susceptibility test was applied to each c. albicans isolates, which processed by transferring portion of pure culture colony of identified c. albicans and mixed with 3-5 ml sabouraud broth to prepare a yeast suspension that equal to 0.5 macfarland, then cotton swab was soaked in yeast suspension and streaked over a whole dried surface of sabouraud dextrose agar plate (3). preparation the discs a stock concentration of 200 mg/ml was prepared for the aqueous and ethanol ginkgo biloba extracts. the aqueous extract was dissolved in distilled water, while ethanol extract was dissolved in dimethyl sulfoxide (10 % dmso) (12,13). paper discs (radius 6mm) prepared from sterile filter paper (whatman no.1) were saturated with different concentrations (0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5 mg/ml, 3mg/ml) of both ginkgo biloba extracts overnight (3) . with a sterile forceps the discs were applied on the inoculated sabouraud dextrose agar with c. albicans, then the plates were incubated for 24-48 hours at 37°c . the diameter of inhibition zone were measured in millimeter with a ruler in addition to the susceptibility of the isolates to control antifungal disc voriconazole 1ug. (14). a control isolate of candida albicans (api 20 c system 6542114) used in this study, was obtained from microbiology laboratory, department of clinical laboratory science, collage of pharmacy, university of mosul. iraqi j pharm sci, vol.29(2) 2020 the effect of ginkgo biloba on candida albicans 124 biofilm formation test this test was applied to each c. albicans isolates to test if they have an ability to form a biofilm or not by tube method. ten ml of trypticase soy broth with 1% glucose inoculated with a portion of tested c. albicans colony in a test tube. the tubes were incubated at 37 °c for 16-18 h. after incubation, tubes were decanted and washed with a normal saline and left to dry. tubes were stained with crystal violet excess stain was washed with distilled water. tubes were dried in inverted position. when a visible film lined the wall and bottom of the tube, biofilm formation was considered positive. the amount of biofilm formed was scored as none, weak, moderate, and high according to results of the control isolates (15). phytochemical screening test phytochemical screening test used to detect the presence of phytochemicals including alkaloids, terpenoids, tannins, carbohydrates, saponin, coumarins glycosides, flavonoids, quinine, proteins. and steroids. these tests were applied separately to each ginkgo biloba extracts. to detect the presence of phytochemicals according to standard procedures as reported by soundararajan and coworkers, 2017, and kumar and coworkers, 2018 (16,17). 1. alkaloids: one ml of mayerʼs reagent with a small amount of each extracts in a test tubes. the formation of yellow cream precipitate indicate the presence of alkaloid 2. terpenoids: two drops of chloroform, then one ml of concentrated hydrochloric acid was added to a small amount of each extracts, then heated for two minutes. a reddish brown color was formed to show positive results for the presence of terpenoids. 3. tannins: few drops of 10% neutral lead acetate solution were added to a small amount of each extracts. white to pale yellow precipitate formation indicates the presence of tannins. 4. carbohydrates : small amount of alcoholic αnaphthol solution in a test tube , then concentrated sulphuric acid was added carefully along the side of the test tube, and a small amount of each extracts were added. violet ring formation at the junction indicated the presence of carbohydrates. 5. saponin: two to three drops of distilled water was added to a small amount of each extracts and shaking well. semi perminant foam formation indicate the presence of saponin. 6. coumarins glycosides: diluted hno3 was added to a small amount of each extracts. the presence of coumarins glycosides was indicated by the presence of yellow precipitate (17) . 7. flavonoids: few drops of fecl3 solution were added to a small amount of each extracts. green to black color formation indicates the presence of flavonoids. phenolic compounds not necessarily flavonoids. 8. quinine: small amount of each extracts were added to 1ml sodium hydroxide solution a and kept for 2 minute. forming reddish brown color indicate the presence of quinine. 9. proteins: one drop of concentrated nitric acid was added to a small amount of each extracts. formation reddish color indicate the presence of proteins. 10. steroids: small amount of each extracts were added to one ml of chloroform, and 1 ml of concentrated sulphuric acid, then shaken well and allowed to stand for five minutes. red color appearance in the lower layer indicates the presence of steroids (16) . antioxidant activity test this test is based on the scavenging activity of the stable 2,2-diphenyl-1-picrylhydrazyl (dpph) free radicals, with the antioxidant activity of ginkgo biloba extracts (18) .this test was applied separately to each ginkgo biloba extracts, which prepared by taking a three volumes (50-100-150 ul) of each ginkgo biloba extracts were added to test tubes then completed the volume to (1 ml) by distilled water (d.w), then 1ml of dpph solution was added to each tube, mixed well and incubated the test tubes at room temperature for 30 min. control was prepared from ascorbic acid solution (0.03% w/v). the absorbance (a) was measured at 517 nm using (spectrophotometer). calculation the inhibition of dpph free radical in percent (1%) was from the following equation (19) 1%= { (ac as) / ac}* 100 ac: the absorbance of the control as : the absorbance of the sample statistical analysis: number, mean, part per million, percentage. results and discussion from 100 healthy persons with oral lesion, involved in this study there were 42 males and 58 females. twenty one healthy person (21%) showed positive results for presence of c. albicans in their oral swab samples, 12 (12%) were females while 9(9%) were males (table1). different tests were used to identify the isolated c.albicans as gram stain, germ tube test , and chlamydospore formation test as shown in (figure 1). the effect obtained from aqueous and ethanol extracts of ginkgo biloba on the growth of candida albicans and control isolates, was nearly similar. iraqi j pharm sci, vol.29(2) 2020 the effect of ginkgo biloba on candida albicans 125 figure 1. chlamydospore of c. albicans by lactophenol mount. candida albicans is the main species among 20 species of genus candida inhabiting the oral cavity. it is estimated that oral candidal carriage accounts for about 35%-80% of all oral yeast isolates (20). the percentage of c.albicans infection in this study was (21%), zaremba and coworkers, 2006 found that c. albicans isolates in the oral cavity of adults with and without dentures were 17/32; 53.1% and 38/71; 53.5%, respectively (21). however the percentage of c. albicans isolated in this study was higher in females than males (12% vs. 9% respectively). in addition to that the number of isolated c. albicans was found to increase with age of the studied subjects (table1). this might be due to the fact that the number of females involved at the time of this study was higher than males. in accordance to this finding, the frequent carriers of candida species were found to be higher in females than males and increased with age (6). however, blazi and coworkers, 2005 (22) did not detect any association between age and candidal growth in between diabetic patients and healthy subjects. every 25 gram of ginkgo biloba leaves extract yielded 6.2 (24.8 %) gram for aqueous and 3.8 (15.2 %) gram for ethanol extract as shown in table 2. table 1. the presence of c. albicans in male and female according to their age. age of healthy persons sex candida albicans male female male female no. no. no. % no. % 1-5 0 2 0 0 0 0 6-10 4 7 1 1 0 0 11-15 7 10 1 1 2 2 16-20 8 11 2 2 3 3 21-25 10 13 2 2 3 3 26-30 13 15 3 3 4 4 total 42 58 9 9 12 12 table 2. weight and percentage of aqueous and ethanol of extracts from 25 gram ginkgo biloba leaves. aqueous extract of ginkgo biloba ethanol extract of ginkgo biloba wt.(gram) % wt.(gram) % 6.2 24.8 3.8 15. 2 phytochemical screening test the phytochemical screening test of both aqueous and ethanol extract was performed separately for each extract. the results of this test are shown in (table 3). in ethanol extract the phytochemical reactions were positive for alkaloids, flavonoids, tannin, carbohydrate, and coumarins glycosides. however, detection of terpenoids, saponine, quinine, proteins, steroids showed negative result. flavonoids, tannins, carbohydrates, and proteins were detected in the aqueous extract while the test did not reveal any positive finding for alkaloids, terpenoids, saponine, quinine, steroids, and coumarins glycosides. the lower antimicrobial activity of the aqueous extract might be due to the less phytochemicals (flavonoids, tannins, carbohydrate, and protein) obtained by the aqueous extraction in comparison to the alcoholic extraction. in the current study, higher number of phytochemicals was present in ethanol extract than aqueous. this explain the largest zone of inhibition for ethanol extract in antifungal susceptibility test, due to fact that ethanol is the best solvent for the active compounds extracted from plants, compared with a distilled water used as a solvent in the aqueous extract. similarly, ibrahim and coworkers, 2016 extracted (glycosides, saponines, steroids, triterpenes, tannins, flavonoids, anthraquinones, and alkaloids), these constituents have been shown to be responsible for the pharmacological activities against staphylococcus aureus, klebsiella pneumoniae, escherichia coli, c. albicans, saccharomyces cerevisiae, and geotrichum iraqi j pharm sci, vol.29(2) 2020 the effect of ginkgo biloba on candida albicans 126 candidum with zone of inhibition ranging from 2227 mm. this finding is consistent with that ginkgo biloba extracts contained active compounds that showed considerable activity on bacteria and fungi (23). table 3. phytochemical screening test of both ginkgo biloba extracts. type of extracts phytochemical reactions alk. terp fla. tan. carb. sap. qui. pro. st. cou. aqueous + + + + ethanol + + + + + susceptibility test this test was applied to 21 c. albicans isolates by using the two types (aqueous and ethanol) of prepared ginkgo biloba extracts with different concentrations. five isolates were shown resistant to all concentrations of ginkgo biloba both extracts. sixteen isolates were sensitive to both aqueous and ethanol extracts. the mean results of susceptibility test zone diameter indicated that ginkgo biloba ethanol extract showed a higher zone of inhibition than aqueous as showed in (table 4 and figure 2 a, 2b). the susceptibility test of ginkgo biloba extracts was investigated using disc agar diffusion method. sixteen c. albicans isolates were found susceptible to both ginkgo biloba extracts. the anticandidal effect of the prepared extracts was concentration dependent. the results showed a good antifungal activity of both ginkgo biloba extracts with zone inhibition ranging from 8-22 mm for aqueous extract and 10-25 mm for ethanol extract (table 4 and figures 2a, 2b). form the results obtained in our study ethanol extract had a higher growth inhibitory effects on c. albicans than aqueous extract, this might be due to the presence of a variety of active phytochemical compounds in the ethanol extract as alkaloids, flavonoids, tannins, carbohydrate, and coumarine glycosides. table 4. the mean diameter of inhibition zone in millimeter against different concentrations of both ginkgo biloba extracts and voriconazole as a control. concentration in mg/ml of extract voriconazole as control type of extracts 0.5 1 1.5 2 2.5 3 1ug mean zone diameter in (mm) aqueous extract 8 10 12 14 18 22 20 ethanol extract 10 14 18 20 25 25 22 aqueous extract (a) ethanol extract (b) figure 2. the susceptibility test of ginkgo biloba extracts against c. albicans. biofilm formation test among the 21 (21%) c. albicans isolates there were 3(14.2%) of them showed the ability to form a biofilm in tube method. one (4.7%) isolate showed weak biofilm formation indicated by faint staining, while the other 2 (9.5%) isolates were iraqi j pharm sci, vol.29(2) 2020 the effect of ginkgo biloba on candida albicans 127 moderate biofilm former. eighteen (85.7%) of the isolates didn’t form a biofilm as showed in (table 5). biofilm formation represents a significant clinical challenge in the management of microbial infections (15). the major virulence attributed to c. albicans has been shown to belong to its ability to adhere to oral epithelium then form a biofilm, these biofilm is resistant to conventional antifungal therapy (8). tube method is 73 % sensitive, 92.5 % specific and 80 % accurate for biofilm detection (15). table 5. number and percentage of the isolates that formation biofilm by tube method. number of isolates (21) biofilm formation tube method no. % high 0 0 moderate 2 9.5 weak 1 4.7 none 18 85.7 antioxidant activity test the reduction capability of dpph radical is determined by the decrease in absorbance at 517 nm induced by antioxidants activity present in both ginkgo biloba extracts. data indicated that the both extracts are able to reduce the stable radical dpph to yellow colored. the scavenging effect at high concentration (6107.14) of both extracts and ascorbic acid solution was in the following order. ethanol extract > ascorbic acid > aqueous extract as showed in table 6. the antioxidant activity of ginkgo biloba extracts is attributed to their contents of antioxidant agents, these antioxidants have a redox properties which play an important role in adsorbing and neutralizing free radicals (19). data showed that the scavenging activity of both extracts were increased with increasing in the concentration of each extract, ethanol extract in all concentrations showed a higher antioxidant activity than aqueous extract, this could be due to the fact that ethanol extract have a highly content of total phenolic and flavonoids than aqueous (19). the antioxidant activity of ethanol extract at concentration 6107.14 ppm showed a higher scavenging activity than 23 ppm of ascorbic acid, on the other hand the scavenging activity of aqueous extract at concentration 6107.14 was lower than 23 ppm ascorbic acid. constituents of plant materials that have antioxidant activity is very important in the maintenance of health and protection from diseases (19). ginkgo biloba may have chemopreventive anticancer properties that related to their antioxidant activity, anti-angiogenic and gene –regulatory action (24). table 6. the antioxidant activity of ginkgo biloba aqueous, ethanol and ascorbic acid against dpph radicals. extract concentration (ppm) scavenging activity % aqueous extract 2035.71 66.60 4071.41 80.70 6107.14 82.16 ethanol extract 2035.71 62.01 4071.41 94.20 6107.14 96.02 ascorbic acid 23 88.15 conclusion the results of this study showed that ethanolic extract of ginkgo biloba leaves, have a good antifungal, phytochemicals, and antioxidant activity. gingko biloba extract can be used in pharmaceutical herbal medicinal oral product as a tooth paste, oral wash or any other oral medication. acknowledgement the author is grateful to acknowledge the college of pharmacy – university of mosul for providing the necessary facilities to carry out this study. references 1. bahri g, lamuki m, rezae-raad m. antiproliferative effects of alcoholic and aqueous extract of ginkgo biloba green leaves on mcf7 cell line. inter j pharmaceutical med res. 2014; 2(3):8-11. 2. rimkiene l, kubiliene a, zevzikovas a, kazlauskiene d, jakstas v. variation in flavonoid composition and radical – scavenging activity in ginkgo biloba l.due to the growth location and time of harvest. j food quality. 2017; 1-8. iraqi j pharm sci, vol.29(2) 2020 the effect of ginkgo biloba on candida albicans 128 3. punnagai k, darling chellathai d, karthik vp, glory josephine i. evaluation of antifungal activity of ethanolic extract of 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adv life sci tech. 2016;46:59 -69. 15. hassan a, usman j, kaleem f, omair m, khalid a, iqbal m. evaluation of different detection methods of biofilm formation in the clinical isolates. braz j infect dis. 2011;15(4): 305-311. 16. soundararajan g, ramesh babu n g, johney j, ragunathan r. extraction of bioactive compounds from rosmarinus officinalis l. and its anticancer activity against hela cell line. inter j sci res (ijsr).2017; 6(8). 165-168. 17. kumar n, upadhyay p, saxena g. phytochemical screening of pomegranate peel using crude hydro-alcoholic extract and pharmacological activities . j sci res publications. 2018; 8(1):193 -199. 18. lee j, hwang w, lim s. antioxidant and anticancer activities of organic extracts from platycodon grandiflorum a de candolle roots. j ethnopharmacol. 2004;93(2-3):409-415. 19. hassan h, hassan n. in vitro antioxidant and free radical scavenging activities of red grape seed extracts. global j biotech & biochem. 2010;5(2): 106-115. 20. scully c, ei-kabir m, samaranayake l. candida and oral candidosis: a review. crit rev oral biol med. 1994;5(2): 125-157. 21. zaremba m, daniluk t, rozkiewicz d, cylwikrokicka d, kierklo a, tokajuk g, et al. incidence rate of candida species in the oral cavity of middle aged and elderly subjects. adv med sci. 2006;51(1): 233-236. 22. belazi m, velegraki a, fleva a, gidarakou i, papanaum l, baka d, et al. candidal overgrowth in diabetic patients: potential predisposing. mycoses. 2005;48(3):192-196 23. ibrahim m, nuhu a. phytochemical screening and antibacterial/antifungal activities of ginkgo biloba extract egb 761. j pharm biol sci (josr-jpbs) .2016;11(1): 43-49. 24. defeudis f, papadopoulos v, drieu k. ginkgo biloba extracts and cancer: a research area in infancy. fundam clin pharmacol. 2003;17(4): 405-417. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ rheological studies and in vitro release evaluation for different formulations of clotrimazole emulgel iraqi j pharm sci, vol.20(2) 2011 clotrimazole emulgel 19 preparation and evaluation of physical and, rheological properties of clotrimazole emulgel yehia i. khalil* ,1 , abeer h. khasraghi* and entidhar j. mohammed* *department of pharmaceutics , college of pharmacy , university of baghdad , baghdad , iraq . abstract recently, emulgel has emerged as one of the most interesting topical preparations in the field of pharmaceutics. in this research clotrimazole was formulated as topically applied emulgel ; different formulas were prepared. the prepared emulgels were evaluated for their physical appearance , rheological behaviour , and in vitro drug release . the influence of the type of gelling agent (carbopol 934 and methyl cellulose), the concentration of both the emulsifying agent (2% and 4% w/w of mixture of span 20 and tween 20) and the oil phase (5% and 7.5% w/w of liquid paraffin) and the type of oil phase (liquid paraffin and cetyl alcohol), on the drug release from the prepared emulgels was investigated. commercially available topical canestin® cream was used for comparison. all the prepared emulgels showed acceptable physical properties concerning colour, homogeneity, consistency, and ph value. rheological studies revealed that all emulgels formulations exhibited a shear – thinning behaviour with thixotropy, indicating structural break down of intermolecular interaction between polymeric chains. clotrimazole emulgels exhibited higher drug release than canestin® cream. the results of in vitro release showed that methyl cellulose – based emulgel gave better release than carbopol 934 – based one. also it was found that the emulsifying agent concentration had the most pronounced effect on the drug release from the emulgels, followed by the oil phase concentration, which has a retardation effect, and finally the type of the gelling agent. it was suggested that the clotrimazole emulgel formulation prepared with methyl cellulose, with low concentration of oil phase (5%w/w liquid paraffin) and high concentration of emulsifying agent (4%w/w), showed an optimum formula for highest drug release (74.4% after three hours), which followed higuchi diffusion model with a diffusion-controlled mechanism. key words: emulgel , carbopol , methyl cellulose , clotrimazole الخالصة ش نية مستهتززة رمد مالعزز تلم مستى زز ً ت ززتب ثزززم مس زز ت ززيالمسجيالتينززً حد ززذ مستغ ززشمد مس ززيذ بززضا مستغزز مغ جيالتينً ستلدة مسك ىتشميتلصوم ، ي تم ت يش عذد مب مس يغ مستخ فة حتزل تزم تييزيم ثززم مستغز لد مسجيالتينيزة مستخ فزة نززىا مس ىممززج مسجيالتينيززة تززدريشهززش مسفيضيززلوي ، عزز ىي مالنغززيلبية ، وت ززشس مسززذوم ززلس مسجغززم حزززس تتزز دسمعززة مززب يزز مست ومسطزىس مسضي زً (22و تزىيب 22% مب مضيج عز ل 4% و 2) وتشحيض حج مب عىممج مالع الب ومستثيج عي ي ىص( 434)مسكلسبىبىم ع ززً عزشعة ت زشس مسزذوم مززب مس ززشمنيب مسغزل ج و ح زىم مسغزي يج() مسطزىس مسضي ز ىانز إسززً إ زلنة %مزب مس زشمنيب مسغزل ج(5 5و 5%) أظهززشدمستغزز مسجيالتينززً وميلسن هززل مززض مستغ ززش مس جززلسي )حززشيم حلنغزز يب ( حلنززة مس ززيغ مست ززشة س تغزز مسجيالتينززً ينً حتزل سزى ع م عزشعة ت زشس مسزذوم مزب حلنزة زيغ مسهيزذسوجومألط ل ص نيضيلويزة مي ىسزة م يزة بزلس ى ، مس جزلنظ ، مسيزىم مستغ مسجيالتينً محثش مب مستغ ش مس جلسي ، حزس تم مس ىم ع ً ت شس من ج س ذوم س يغ مستثيج عي ي ىص ميلسنزة مزض زيغ تشحيض مسطىس مسضي زً ، يز مي ل وجذ م تشحيض عىممج مالع الب سه تدريش م ىظ ع ً عشعة ت شس مسذوم ي هل434مسكلسبىبىم منه ي تج ع ً معلقة ت شس مسذوم ، وم يزشم نزىا مس لمزج مسجيالتينزً حززس وجزذ م نزىا مسطزىس مسضي زً مي زل يزةرش ع زً عزشعة ت زشس % مزب 5) ز مسضيمسذوم يي شح بل مس يغة مسذوم ية س تغ مسجيالتينً مست شة بلع تلم مستثيج عي ي ىص مض مقج تشحيزض مزب مسطزىس %ب زذ 4 54) س زشس مسزذوم عزشعة مع زً إلعطزل مس يغة مستخ زلسة ث %( 4)و مع ً تشحيض مب عىممج مالع الب مس شمنيب مسغل ج( من شلس مسذوم ثً مسخطىة مست ذدة س شس مسذوم م عشعة حتل منهل ت ض ن شية ثيكىج يرالث علعلد( introduction the array of formulations and compositions employed for topical application confounds attempts at categorization . by far majority of commercial dermatologic drug products are formulated in an emulsion (or cream) base. topical formulations apply a wide spectrum of preparations both cosmetic and dermatological, to their healthy or diseased skin (1) . these formulations range in consistency nature from solid through semisolid to liquid. drug substances are seldom administered alone, but rather as part of a formulation, in combination with one or more adjuvant agents that serve varied and specialized pharmaceutical functions. drugs are administered topically for their action at the site of application, or for systemic effects (2) 1 corresponding author email : ybmmaz@yahoo.com received : 22/3/ 2011 accepted : 16/7/2011 iraqi j pharm sci, vol.20(2) 2011 clotrimazole emulgel 20 drug absorption through the skin is enhanced if the drug substance is in solution, with a favorable lipid/water partition coefficient, and nonelectrolyte. for the most part, pharmaceutical preparations applied to the skin are intended to serve some local action and, as such, are formulated to provide prolonged local contact, with minimal systemic drug absorption. drugs applied to the skin for their local action include antiseptics, antifungal agents, skin emollients, and protectants (3,4) . stratum corneum (sc) is the outermost, and least permeable, layer of the skin, it is a formidable barrier for both water transport out of the body and chemical inward permeation. in fact, the majority of drugs do not appear to penetrate the skin at a rate sufficiently high for therapeutic efficacy, and only the most potent once with appropriate physicochemical characteristics is valid candidate for transdermal delivery (5) . skin delivery is an effective for targeting therapy for topical dermatological disorder as in antifungal agents ( 6 ) . several antifungal agents are available on the market in different topical preparations (e.g., creams, ointments, and powders for the purpose of local dermatological therapy). one of these antifungal agents is clotrimazole. clotrimazole is 1-[(2-chlorophenyl) diphenylmethyl]-1h-imidazole. it has antifungal effect (7,8) .it inhibits growth of pathogenic dermatophytes .it shares with econazole, miconazole, first – choice status for topical treatment of tinea pedis, tinea cruris, and tinea corporis due to any of the a forementional organisms, candidiases due to candida albicans . it is effective for the topical treatment of vulvovaginal and oropharyngeal candidiasis (8-10) . for skin care, and the topical treatment of dermatological diseases, a wide choice of vehicles ranging from solid to semisolids and liquid preparations, is available to physician and patients. within the major groups of semisolid preparations, the use of transparent emugels has expanded, both in cosmetics and pharmaceuticals. emulgel or gellified emulsion is stable one and better vehicle for hydrophobic or water insoluble drugs (11) . it is an emulsion either of the oil in water or water in oil type, which are gelled by mixing with a gelling agent (12) . oil in water emulsions are most useful as water washable drug bases and for general cosmetic purposes, while water in oil emulsions are employed more widely for the treatment of dry skin and emollient applications (13,14) . emulgels have a high patient acceptability since they possess the advantages of both emulsions and gels. therefore, they have been recently used as vehicles to deliver various drugs to the skin (15-18 ) . many emulgels are available such as commercial voltaren emulgel (novartis pharma, basle, switzerland), containing diclofenac diethylamine.the aim of this study is to formulate emulgel containing clotrimazole using two types of gelling agent : carpobol 934 and methyl cellulose and study some variables that may affect the formulation such as the type of the gelling agent, concentration of the emulsifying agent ,the concentration and the type of the oil phase on the rheological properties besides to the in vitro release of the drug from the prepared emulgels and comparing all the obtained results with a commercial available formulation (canestin® cream ) . materials , equipments and methods materials clotrimazole powder , methyl paraben , and propyl paraben were supplied by samara drug industry . methyl cellulose , cetyl alcohol , ptoassium dihydrogen phosphate , and span 20 from ( bdh chemicals ltd , poole , england ). carbopol 934 from ( j. t. baker , indian). tween 20 from (merk – schuncherdt , germany ) . liquid paraffin from ( riedel – de haen ag seezle , hannover ) . ethanol and disodium hydrogen phosphate from ( gainland chemical company , factory ro ad , sandycroft , deeside , clwyd – u.k. ) . triethanol amine and propylene glycol from (searle company hopkin and williams , chadwell health essex , england ) and canestin® cream from bayer company. all other reagents were of analytical grade . equipments sartorius balance ( werkegmbh , type 2842 , germany ) , electrical mixer ( janke and kunkel , rf 16 ) , water bath ( memmert , germany ) , ph –meter (henna instruments ph 211 microprocessor , italy ) , usp dissolution apparatus , type 11 ( copley scientific tld , england ) , rotational viscometer ( fungilab , spain ) , spectrometer ( specord 40 , analytikjena , germany ) . methods preparation of carbopol and methyl cellulose gel fifty grams of carpobol gel was prepared by dispersing one gram of carbopol powder in 49grams purified water with aid of moderate speed stirrer ( 50 rpm ) , and then the ph was adjusted to 6 – 6.5 using triethanol amine (19) . also fifty grams of methyl cellulose gel was prepared by dispersing 3.5 grams of methyl iraqi j pharm sci, vol.20(2) 2011 clotrimazole emulgel 21 cellulose powder in 46.5 grams of heated purified water (80 °c) , and the dispersion was cooled to room temperature and left overnight to ensure hydration of the gel (20,21) . preparation of emulsion the general method was employed according to ansel h.c.et al (22) for preparation of emulsion was as follows : the oil phase was prepared by dissolving certain amount of span 20 in liquid paraffin , while the aqueous phase was prepared by dissolving the required amount of tween 20 in purified water . one gram of clotrimazole powder was dissolved in 2.5 gm of ethanol , while 0.03 gm of methyl paraben and 0.01 gm of propyl paraben were dissolved in 5 gm of propylene glycol and both were mixed with aqueous phase . both the oily and aqueous phases were separately heated to 70-80° c. then, the oil phase was added to the aqueous phase with continuous stirring at 50 rpm until cooled to room temperature (22) . preparation of clotrimazole emulgel ten formulas of clotrimazole were prepared by dispersing the obtained emulsions with the gel in 1:1 ratio with gentle stirring until homogenous emulgel was obtained (21) , as shown in table -1. table 1 : compositions of different formulas of clotrimazole emulgel ( % w/w) . evaluation of clotrimazole emulgel physical examination the prepared emulgel formulations were inspected visually for their colour , homogeneity , consistency , appearance , and ph . the ph values of 1% (w/w) aqueous solutions of the prepared emulgels were measured by a ph meter (15) . rheological study the viscosity of the different emulgel formulations was measured and rheograms were obtained at 25°c using rotational viscometer . the prepared formulas were sheared with spindle r 7 over the range of speed setting from 2 to 10 rpm with 30 seconds between each 2 successive speeds , and then in a descending order (23) . in vitro release studies a glass beaker with 2.5 cm in diameter was filled with 3 gm of each formula and canestin ® cream separately . the mouth of the beaker was covered with a filter paper which was kept in place ( sealed) with a rubber band and was inverted and immersed to about 0.5 cm of surface of phosphate buffer (24) (500 ml ) of ph 5.5 in a jar of usp dissolution test apparatus with stirring rate of 50 rpm . the study was carried out at 37 ± 0.5 °c . samples of 5 ml were withdrawn after ( 15 , 30 , 45 , 60 , 90 , 120 , and 180 minutes ) through 0.45 µm millipore filter paper and replaced with an equal volume of fresh buffer (20 ) . the drug content in the withdrawn samples was determined spectrophotometrically at λmax 260 nm using a uv spectrophotometer (25) . the commercial product canestin ® cream was used as a reference for drug release from the base . data and statistical analysis all data were represented as mean ± sd ( n = 3 ) .statistical comparisons were made using student’s t test. the differences were considered to be statistically significant when ( p < 0.05 ) . iraqi j pharm sci, vol.20(2) 2011 clotrimazole emulgel 22 results and discussion physical properties it is clearly evident that, all the prepared clotrimazole emulgel formulations were white viscous creamy preparations with a smooth and homogeneous appearance. the ph values of all prepared formulations ranged from 6.4 ± 0.04 to 6.9 ± 0.04 , which lies with the normal ph range of the skin and is considered acceptable to avoid any irritation upon application to the skin (26) . rheological properties viscosities (in poise) of clotrimazole emulgel formulations at low and high rates of shear were shown in table -2. in gel systems, consistency depends on the ratio of solid fraction, which produces structure, to liquid fraction. the difference in the type of the gelling agents result changes in structure consistency (27) . the viscosity of the emulgel formulations generally reflects its consistency (26) .carbopol 934– based formulations ( f1 , f2 , f3 , f4 , and f5 ) possessed considerably higher viscosities than the methyl cellulose – based formulations (f6 , f7 , f8 , f9 , and f10 ) , respectively . this effect may be attributed to the higher hygroscopicity of methyl cellulose compared with carbopol 934 (28) .so that , the type and the concentration of the base used play an important role in the topical preparation design since it affects the viscosity of the emulgel. meanwhile incorporation of emulsifying agent and liquid paraffin in different concentration for both types of formulas made of carbopol 934 and methyl cellulose gave marked effect on the consistency of the resulted base as a viscous or softy cream emulgel (29) . table 2 : viscosities (in poises ) of clotrimazole emulgel at low and high rates of shear formulas η max* η min** f1 470.15 1445.86 f2 364.14 1105.71 f3 619.28 2888.3 f4 587.48 2558.56 f5 1064.2 4316.11 f6 360.11 1072.65 f7 345.58 998.33 f8 385.63 1137.93 f9 367.84 1194.10 f10 401.68 1226.82 * viscosity at high rate of shear (12 rpm) **viscosity at low rate of shear ( 2 rpm) it was seen that increase the concentration of emulsifying agents ( tween 20 and span 20 ) , from 2% w/w to 4% w/w , led to increase in the viscosity of carbopol 934 – based formulations ( f3 , f4 ) as compared with ( f1 , f2 ) , respectively , at both low and high rate of shear as shown in table -2.the same effect was resulted in methyl cellulose – based formulations ( f8 , f9 ) as compared with (f6 , f7) , respectively . these results are in agreement when recombinant human growth hormone (30) and miconazol (20) were formulated using these surfactants. on the other hand increasing liquid paraffin content from 5 to 7.5 % w/w for formulas ( f2 , f4 ) at which carbopol was used as a vehicle base and formulas ( f7 , f9 ) at which methyl cellulose was used as a vehicle base , revealed a reduction in the viscosity as compared with formulas ( f1 , f3 ) and ( f6 , f8 ) , respectively , at both low and high rate of share . these results may be attributed to the ability of liquid paraffin to contribute in a formulation of emulsion with water (30) , that make the utilization of span 20 and tween 20 as a surfactants is possible and then decrease the amounts of later surfactants in each previous later formula (31) .using cetyl alcohol as oil phase instead of liquid paraffin in formulas ( f5 , f10 ) resulted in an increase in the viscosity as compared with formulas ( f1 , f6 ), respectively , so the type of the oil phase also had showed an important role in the topical preparation design. all the prepared emulgel formulations exhibited a shear thinning behaviour since the viscosity (the slope of the curve) decreased with increasing the shear rate. as the shear stress is increased , the normally disarranged molecules of the gelling material are caused to align their long axes in the direction of flow. such orientation reduces the internal resistance of the material and hence decreases the viscosity (19) . figures 1 and 2 show the rheograms ( shear rate vs. shear stress ) of ( f8 ) and ( f3 ) respectively, these formulas were chosen for this study since they gave us the highest release of clotrimazole from the emulgel. these figures show that clotrimazole emulgel formulations possessed pseudoplastic flow with thixotropic behaviour, where the down curve was displaced with regard to the up curve, showing at any rate of shear on the down curve a lower shear stress than it had on the up curve; a hysteresis loop was formed between the two curves. thixotropy, or time-dependent flow , occurs because the gel requires a finite time to rebuild its original structure that breaks down during continuous shear measurements (28, 32) . it is noteworthy that thixotropy is a desirable iraqi j pharm sci, vol.20(2) 2011 clotrimazole emulgel 23 characteristic in pharmaceutical preparations in order to deliver an initially thick product as a thinner, easily spreadable material. these findings are in agreement with chloramphenicol emulgel using carbopol 940 as the gel-forming material (15) , chlorphenesin emulgel using carbopol 934 and hpmc as gelling agents (19) , and miconazole nitrate emulgel using carbomer 941 and scmc as gelling agents (20) . figure 1 : rheogram of formula (8) at 25°c ( methyl cellulose gel base ) ( mean ± sd , n=3) figure 2 : rheogram of formula (3) at 25°c ( carbopol 934 gel base ) ( mean ± sd , n=3) effect of polymer base type the effect of gelling agent type on the release of clotrimazole was shown in figure 3.it was seen that a significant increase (p<0.05) in the amount of clotrimazole released after three hours was obtained when 3.5% w/w methyl cellulose used as a base (f6) instead of 1% w/w carbopol 934 (f1) . this result may be attributed to the physical structure of the polymer network and shape of three dimension structure of the polymer, since the entrapment of clotrimazole within these structural network revealed high capability of 1% w/w carbopol 934 compared with 3.5% w/w methyl cellulose . in addition, the result may be also due to higher viscosity of the carbopol emulgel compared with methyl cellulose emulgel as shown in table -2-.the same results were obtained for f7 , f8 , f9 , and f10 as compared with f2 , f3 , f4 , and f5 , respectively .these results are in agreement with chlorophenesin (19) , miconazole nitrate (20) , and itraconazole (21) emulgel where cellulosic derivative polymer – based formulas gave higher release than carbopol – based formulas . however the choice of appropriate base type and concentration play an important role in the topical preparation design. figure 3: the effect of gel base type on the release profile of clotrimazole 1% w/w at ph 5.5 and 37 °c ( mean ± sd , n=3) effect of surfactant added (emulsifying agents) concentration tween 20 and span 20 were used as emulsifying agents to produce emulgel formulations, the effect of increasing their concentration on the release of clotrimazole was shown in figure -4. it was seen that increasing the concentration of emulsifying agent from 2% to 4% led to significant ( p< 0.05 ) increase in the amount of clotrimazole released in dissolution medium , as seen in carbopol 934 – based formulas ( f1 , f3 ) and methyl cellulose –based formulas ( f6 , f8 ).the clotrimazole release was increased from 55.88% ( f1 ) to 67. 4% ( f3 ) and from 63.76% ( f6 ) to 74.4% ( f8 ) after three hours. this effect may be referred to the ability of these emulsifying agents to lower the interfacial tension between oily and aqueous layer in the dispersion medium (33) , indicating an increasing the hydrophilicity of emulgel 0 2 4 6 8 10 12 14 16 1000 2000 3000 4000 5000 6000 s h e a r s tre ss ( dyn e /c m 2 ) s h e a r r a t e ( s e c 1 ) up c urve d own c urve 0 2 4 6 8 10 12 14 16 4000 5000 6000 7000 8000 9000 10000 s h e a r s tre ss ( dyn e /c m 2 ) s h e a r r a t e ( s e c 1 ) up c urve d own c urve 0 10 20 30 40 50 60 70 0 50 100 150 200 t im e (m in ) % d r u g r e l e a s e d f 1(1% c arbopol 934) f 6(3.5% methy l c ellulos e iraqi j pharm sci, vol.20(2) 2011 clotrimazole emulgel 24 which in turn increase penetration of dissolution medium into the emulgel structure and then increasing the amount of clotrimazole released . this result was in consistent with that result obtained when increase the concentration of emulsifying agents from 1.5% to 2.5% in both carbopol and hydroxypropyl methyl cellulose emulgel base led to increase the release of chlorphenesin from topical emulgel (19) .also the results are in aggrement with miconazole nitrate emulgel , when increase the concentration of emulsifying agents from 2% to 4% in both carbomer 941 and scmc emulgel base led to increase the release of miconazole nitrate from topical emulgel (20) . figure 4 : the effect of emulsifying agents concentration on the release of clotrimazole 1`% w/w at ph 5.5 and 37 °c ( mean ± sd , n=3) effect of oil phase concentration the effect of paraffin concentration on the release of clotrimazole from carbopol 934 emulgel and methyl cellulose emulgel was represented in figure -5and figure -6, respectively .increasing the liquid paraffin concentration from 5% w/w to 7.5% w/w in carbopol 934 – based formulations ( f2 , f4) and in methyl cellulose based formulations (f7 , f9 ) , led to significant decrease ( p < 0.05 ) in the amount of clotrimazole released from these bases as compared with formulas (f1 , f3 ) and ( f6 , f8 ) , respectively. this result may be explained according to the concept of escaping tendency of drugs (34) , it was supposed that increasing the thermodynamic activity which can be expressed in terms of relative solubility of drug lead to enhance the releasing of drugs from vehicle (35) .the same effect was proved that the increase liquid paraffin led to retardation of miconazole nitrate release from its emulgel formulation (20) . figure 5 : the effect of paraffin concentration on the release of clotrimazole 1`% w/w from carbopol 934 emulgel at ph 5.5 and 37 °c ( mean ± sd , n=3) figure 6 : the effect of paraffin concentration on the release of clotrimazole 1`% w/w from methyl cellulose emulgel at ph 5.5 and 37 °c. ( mean ± sd , n=3) effect of type of oil phase the effect of type of the oil phase on the release of clotrimazole was shown in figure 7 .it was observed that using of cetyl alcohol as oil phase in formulas ( f5 ) and ( f10 ) causing a significant ( p < 0.05 ) reduction in the release of clotrimazole as compared with formulas that had been used liquid paraffin (f1 and f6 ) , respectively, whether the gel base was carbopol 934 or methyl cellulose . this may be attributed to the increase in viscosity of emulgel as shown in table 2 .the treatment of the obtained data with higuchi principle revealed that best fit mechanism of clotrimazole 1% w/w release from emulgel with linear relation – ship when the amount of the drug released plotted with square root of time (36) (figure -8-) , as proposed by the higuchi’s theory , indicating the diffusion controlled mechanism of drug release . this 0 10 20 30 40 50 60 70 80 90 0 50 100 150 200 t im e (m in ) % d r u g r e l e a s e d f 1(2% ea in c arbopol 934 f 3(4% ea in c arbopol 934 ) f 6(2% ea in mc ) f 8(4% ea in mc ) 0 10 20 30 40 50 60 70 80 90 0 50 100 150 200 t im e (m in ) % d r u g r e l e a s e d f 6(2% ea +5% paraffin ) f 7(2% ea +7.5% paraffin) f 8(4% ea + 5% paraffin) f 9(4% ea + 7.5% paraffin ) iraqi j pharm sci, vol.20(2) 2011 clotrimazole emulgel 25 finding indicates that the rate-controlling stage in the release process was diffusion of the dissolved drug through the gel network to the external medium. the rate release constant (k) of clotrimazole from different emulgel formulas was shown in table -3 , with a correlation coefficient ranging from 0.991 to 0.999 , which means an excellent model fit . the rate release constant ( k ) of formulas ( f8 and f3 ) was 5.879 and 6.09 %(min -1/2 ) respectively, which were the highest rates as compared with that of canestin® cream which was 2.88 %(min -1/2 ) . figure 7 : the effect of oil phase on the release of clotrimazole 1`% w/w from carbopol 934 and methyl cellulose emulgel at ph 5.5 and 37 °c. ( mean ± sd , n=3) figure 8 : the effect of emulsifying agents concentration on the release of clotrimazole 1`% w/w from emulgel ( mean ± sd , n=3) table 3 : the rate release constant (k) of clotrimazole from different emulgel bases formula k %(min -1/2 ) correlation coefficient f1 5.718 0.996 f2 3.575 0.999 f3 6.09 0.998 f4 5.152 0.998 f5 3.154 0.995 f6 5.04 0.996 f7 4.518 0.991 f8 5.879 0.997 f9 4.691 0.998 f10 3.576 0.998 canestin cream® 2.88 0.997 conclusion  clotrimazole can be formulated as emulgel with a proper consistency, exhibiting shear-thinning behaviour with thixotropy , and good release which follows higuchi diffusion model .  the factors which affect on the drug release from the emulgel can be arranged as follows: the emulsifying agent concentration > oil phase concentration and type > the gelling agent type .  methyl cellulose – based emulgel showed highest drug release when 5% w/w liquid paraffin and 4% w/w emulsifying agents were used and it is the formula of choice . references 1. lawrence h. block. medicated topicals, ch. 44 in remington , the science and practice of pharmacy 21st edition, lippincott williams and wikins , a wolters kluwer company philadelphia, new york, london ,copyright 2006 , p.879-883 . 2. rashmi m. topical gel: a review august vol. 2008; available from http:// www.pharmainfo.com. 3. sharma s. topical preparations are used for the localized effects at the site of their application by virtue of drug penetration into the underlying layers of skin or mucous membranes. pharmaceutical reviews 2008; 6:110. 4. laithy hm. and el. shaboury kmf. the development of cutina lipogels and gel microemulsion for topical administration of fluconazole. ame pharm sci. pharmscitech. 2003; 3: 10 25. 0 10 20 30 40 50 60 70 0 50 100 150 200 t im e (m in ) % d r u g r e l e a s e f 1(1% c arbopol 934 +5% paraffin) f 5(1% c arbopol 934 + 5% c ety l alc ohol) f 6(3.5 % m.c . +5% paraffin) f 10 ( 3.5% m.c . + 5% c ety l alc ohol) 0 10 20 30 40 50 60 70 80 90 0 2 4 6 8 10 12 14 16 s q u a re ro o t o f tim e (m in ) c u m u l a t i v e % d r u g r e l e a s e d f 1(2% ea in c arbopol 934 ) f 3( 4% ea in c arbopol 934 ) f 6 ( 2% ea in m.c . ) f 8(4% ea in m.c . ) http://www.pharmainfo.com/ iraqi j pharm sci, vol.20(2) 2011 clotrimazole emulgel 26 5. carmelo puglia, francesco bonina, giuseppe trapani, massimo franco, maurizio ricci. intl j. pharm. 2001; 228:79-87. 6. salama m., ghazy f., bosela a., ismail a., in vitro and clinical evaluation of chlorphensin polymeric films, alex.j.pharm.sci (1997), 11 (2), 59-64. 7. british pharmacopoeia, vol.1 , 2010 , p 552. 8. steven p. gelone. anti-infectives, ch. 90 in remington, the science and practice of pharmacy 21st edition, lippincott williams and wikins , a wolters kluwer company philadelphia, new york, london , copyright 2006, p.1670-1671. 9. the merk index, fourteenth edition. an encyclophedi of chemicals, drugs , and biologicals. maryadele j .o, neil, editor mreck and co., inc. white house station, nj, usa. 2006, 2417 10. martindale, the complete drug reference , thirty-sixth edition. edited by sean c sweetman bpharm , frpharms. published by the pharmaceutical press 2009 p. 530.1 11. guido s, fred v, martien a, cohen s, george a. oil droplet release from emulsion filled gels in relation to sensory perception. food hydrocolloids 2007; 21: 977-985. 12. hideaki t and yuya k. preparation of poly (n-isopropyl acryl amide) emulsion gels and their drug release behaviours. coll. and surf. b: bioinformatics 2008; 67:92-98. 13. lachman l. and lieberman ha .the theory and practice of industrial pharmacy. special indian edition 2009; p.503. 14. chojnicka a, sala g, cornelus g. the interaction between oil droplets and gel matrix affect the lubrication properties of sheared emulsion filled gels. food hydrocolloids 2009; 23:1038-1046. 15. abd el-bary a, shalaby s, abd el-aal s. formulation and stability of chloramphenicol gel and emulgel. bull fac pharm. 2001; 39:89-99. 16. hamed m.r., metwally s.a., el-shafey a., geneidi a.s, comparative percutaneous absorption of diclofenac emulgel preparations in normal volunteers, j. drug. res (1994), 21(1-2), 133-141. 17. hamza ye, molokhia am, soliman ii, ahmed fh, soliman na. formulation and evaluation of topical preparations containing phenol and local vesicants. az j pharm sci. 2002; 29:412-432. 18. ozguney s.i., karasulu y.h., kantarci g., transdermal delivery of diclofenac sodium through rat skin from various formulations, aaps pharm.sci.tech. (2006), 7 (4), article 88. 19. magdy i., mohamed, optimization of chlorophenesin emulgel formulation, the aaps . j, 2004; 6 (3) article 26. lubna a.s., hala t.s., and yehia i.k., an investigation release and rheological properties of miconazole nitrate from emulgel. iraqi j. pharm. sci , vol. 18 (2) 2009: 26-31 20. piyusha d., ankur j., naveen v.,hemant k., sanjay j., gellified emulsion for sustain delivery of itraconazole for topical fungal diseases. international journal of pharmacy and pharmaceutical sciences, vol. 2 , issue 1 , 2010 . 21. howard c. a. , loyd v., allen j.r. , nicholns g.p., lippincot williams & wilkins ,ansel’s pharmaceutical dosage forms and drug delivery systems , eighth edition ,copyright ( 2005 ) , p 404-423 . 22. masar b.m., formulation and evaluation of meloxicam as a topical preparation, thesis, college of pharmacy, university of baghdad, 2004. 23. british pharmacopoeia ,vol. iv , 2008 ,appendix i d , a 143 . 24. clarck’s isolation and identification of drugs in pharmaceuticals, body fluids, and postmortem material. second edition, senior consulting editor a. c. moffat. london, the pharmaceutical press 1986, p 487 25. malay k., das and abdul b.a., formulation and ex vivo evaluation of rofecoxib gel for topical application , acta poloniaedrug research , vol. 63 no. 5 pp. 461-467 , 2007 . 26. martin’s physical pharmacy and pharmaceutical sciences, fifth edition, copyright 2006, lippincott williams & wilkins, pp. 565-569 . 27. danester q., evone s. g., formulation and characterization of nystatin gel. prhsj. march 2008 vol. 27 no.1. p. 61-67. 28. wan lsc, viscosity change in salicylic acid-cetrimide system by surfactants, j.pharm.sci. 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(1962), 51, 802-804. iraqi j pharm sci, vol.30(1) 2021 gastrointestinal peptides role in appetite doi: https://doi.org/10.31351/vol30iss1pp14-21 14 eptidespastrointestinal gatiety by sppetite and aregulation of sarah h. mhaibes*,1, najwan k. fakree*, sonia i. naser * *department of clinical laboratory sciences, college of pharmacy, university of baghdad, baghdad, iraq. abstract in recent decades, global obesity has increased significantly, causing a major health problem with associated complications and major socioeconomic issues. the central nervous system (cns), particularly the hypothalamus, regulates food intake through sensing the metabolic signals of peripheral organs and modulating feeding behaviors. the hypothalamus interacts with other brain regions such as the brain stem to perform these vital functions. the gut plays a crucial role in controlling food consumption and energy homeostasis. the gut releases orexigenic and anorexigenic hormones that interact directly with the cns or indirectly through vagal afferent neurons. gastrointestinal peptides (gip) including cholecystokinin, peptide yy, nesfatin-1, glucagonlike peptide 1, and oxyntomodulin send satiety signals to the brain and ghrelin transmit hunger signals to the brain. the gip is essential for the control of food consumption; thus, explain the link between the gastrointestinal tract (git) and the brain is important for managing obesity and its associated diseases. this review aimed to explain the role of gut peptides in satiety and hunger control. keywords: obesity, gastrointestinal peptides, ghrelin, oxyntomodulin. تنظيم الشهية والشبع بواسطة الببتيدات المعدية المعوية سارة هاشم محيبس *،1 ، نجوان قيصر فخري * و سونيا عماد ناصر * . فرع العلوم المختبرية السريرية ، كلية الصيدلة ، جامعة بغداد ، بغداد ، العراق* الخالصة ، مما تسبب في مشكلة صحية كبيرة مع المضاعفات المرتبطة بها والمشاكل االجتماعية كبير بشكل زادت السمنة العالمية في العقود األخيرة ، ات واالقتصادية الرئيسية. يلعب الجهاز العصبي المركزي ، والسيما تحت المهاد ، دورا حاسما في تنظيم تناول الطعام عن طريق استشعار اشار غذية. يتفاعل المهاد مع مناطق أخرى في الدماغ مثل جذع الدماغ ألداء هذه الوظائف الحيوية. التمثيل الغذائي لألعضاء الطرفية وتعديل سلوكيات الت الجهز العصبي الطاقة. تطلق القناة الهضمية هرمونات التي تتفاعل مباشرة مع توازنتلعب القناة الهضمية دوراً حاسماً في التحكم في استهالك الغذاء و ل الخاليا العصبية . المركزي او بشكل غير مباشر من خال الجلوكاجون و أوكسيتومودولين ترسل اشارات الشبع الى الدماغ و 1البيبتيدات المعدية المعوية بما في ذلك كوليسيستوكينين ، البيبتيد ، البيبتيد العالقة شرح . هرمون الببتيد المشتق من األمعاء ضروري للسيطرة على استهالك الطعام. وبالتالي ، فإن الى الدماغ جريلين يرسل اشارات الجوع دور الببتيدات المعوية في الشبع والسيطرة وضيح إلدارة السمنة واألمراض المرتبطة بها. تهدف هذه المراجعة إلى تمهم بين الجهاز الهضمي والدماغ .على الجوع حية: السمنة ، الببتيدات المعدية المعوية ، جريلين ، أوكسينتومودولينالكلمات المفتا introduction the control of food intake in healthy individuals is done by gastrointestinal peptides (gip) that stimulate hunger or satiety. disturbance of gip metabolism can lead to obesity (1). the coordination of central and peripheral signals that control energy homeostasis is vital to understand appetite control. in the body's energy balance, the central nervous system (cns) that receives signals from the digestive tract and adipose tissue plays an indispensable role. hunger and satiety are controlled by the brain-gut axis (2). gip monitor food consumption, stomach evacuation, and bowel movements, collectively control body weight over the long term (3). several peptides originated from the gut inhibit food intake, specifically cholecystokinin (cck), peptide tyrosine (pyy), glucagon-like peptide 1 (glp-1), and nesfatin-1. in contrast, ghrelin which is centrally acting and peripherally delivered peptide stimulate food intakes (4). the worldwide spread of obesity and the major complications associated with it have induced greater necessity to understand the processes of energy balance. the present review aimed to explain the role of gip involved primarily in hunger and satiety regulation. 1corresponding author e-mail: phsarahhashim@gmail.com received: 14/7/2020 accepted:2 /11 /2020 iraqi journal of pharmaceutical science https://doi.org/10.31351/vol30iss1pp14-21 iraqi j pharm sci, vol.30(1) 2021 gastrointestinal peptides role in appetite 15 brain-gut food intake regulation the hypothalamus is critical in the relaying of afferent signals from the gut and brainstem as well as processing efferent signals that modulate food intake and energy expenditure. the hypothalamus arcuate nucleus (arc) is a structure located at the base of the hypothalamus, adjacent to the median eminence (me). the latter has a more permeable blood-brain barrier (bbb), which makes the arc neurons exposed to nutrients and gastrointestinal peptides. the arc transmits circulatory signals to other hypothalamic zones, as well as to extrahypothalamic areas such as the mesolimbic reward system and to the hunger and satiety sites in the nucleus tractus solitaries (nts) (5). the arc contains two neuronal populations in the arc implicated in the regulation of feeding. orexigenic neurons (i.e. stimulating appetite) express neuropeptide y (npy) and agouti-related protein (agrp). whilst anorexigenic neurons (i.e. inhibiting appetite) in the arc express cocaine -and amphetamine-related transcript (cart) and proopiomelanocortin (pomc). neuronal projections from these two populations then communicate with other hypothalamic areas involved in appetite regulation such as the paraventricular nucleus (pvn), dorsomedial nucleus (dmn), and lateral hypothalamic area (lha) (6) . table 1 explains the role of gip in food intake control . table1. gip activities in food intake control (6) gip receptor site of action effects on food intake ghrelin ghrelin receptor vagal nerve brain stem hypothalamus orexigenic ↑ appetite ↑ gastric motility nesfatin-1 melanocortin receptor vagal nerve brain stem hypothalamus anorexigenic ↓ appetite cck cck1 vagal nerve brain stem hypothalamus anorexigenic ↓appetite ↑ gallbladder emptying ↓ gastric emptying peptide yy y2r vagal nerve brain stem hypothalamus anorexigenic ↓gastric emptying ↓ intestinal motility glp-1 glp-1 vagal nerve brain stem hypothalamus anorexigenic ↑insulin release ↓gastric acid secretion oxm glp-1 glucagon hypothalamus anorexigenic ↑ energy expenditure ↓ gastric emptying and secretion ↑, increase; ↓, decrease; gip " gastrointestinal peptides" , git " gasterointestinal tract ", cck "cholecystokinin ", glp-1"glucagon like peptide-1", pyy "peptide yy", oxm "oxyntomodulin" , y2r " neuropeptide y2 receptor ". gastrointestinal peptides coordinating satiety and appetite 1ghrelin ghrelin is biosynthesized and secreted from the stomach, small intestine, pancreas and brain (7). ghrelin is found in the bloodstream in two forms. first is deacyl-ghrelin (desacyl-ghrelin) which is more stable and higher levels than other forms. second is the acylated form (acyl-ghrelin ag) which is the product of post-translational acylation of the hydroxyl group of the ser3 residue of the nascent ghrelin, catalyzed by ghrelin-oacyltransferase (goat), this acylated form corresponds to around 20 % of the total circulating ghrelin and is responsible for the biological activity of ghrelin. goat is responsible for the acylation of the preproghrelin and converted to proghrelin that is proteolytically cleaved by the prohormone convertase. (8). the biological activities of acylghrelin are mediated by binding to the growth hormone secretagogue receptor (ghs-r1a) (8). acyl-ghrelin has several functions in many tissues. acyl-ghrelin stimulates the secretion of growth hormones from the anterior pituitary gland and activates the hypothalamic orexigenic axis by induction the secretion of neuropeptides such as iraqi j pharm sci, vol.30(1) 2021 gastrointestinal peptides role in appetite 16 npy, stimulating food intake and reducing energy expenditure. (9) thus, ghrelin exhibits orexigenic properties and has been targeted for the treatment of obesity. chronic administration of ghrelin promotes weight gain and obesity (10). the circulating concentration of orexigenic ghrelin increased in obese patients with prader – willi syndrome (11). in addition to its orexigenic effect mediated by neuropeptide y, ghrelin contributes to obesity by stimulating gh secretion from the pituitary gland (12). the dimerization property of ghs-r1a with multiple g-protein coupled receptors allowing the cross-talk between many other neuropeptide systems of serotonin and dopamine. hence, ghrelin has the potential to engage various neuropeptide systems in mood, food, and obesity (13,14). the ghrelinergic system mediates the nonhomeostatic hedonic rewarding and motivational aspects of food intake via mesolimbic dopaminergic circuitry (15,16). ghrelin receptors spread widely throughout the body, with high levels of pituitary and hypothalamic expression with lower levels of expression in peripheral tissue, especially in the pancreas, git, immune cells, and the heart. further, ghrelin enhances git motility and diminishes insulin secretion (17). patterson et al. reviewed many studies cored about antagonizing ghrelin action by competitive inhibition or by neutralizing ghrelin, as targets for the treatment of obesity (18,19). also, the antagonizing ghrelin action may be used as a treatment of nutritional disorders like cachexia and anorexia nervosa (3). 2nesfatin-1 nesfatin-1 (nf-1) was first identified in 2006 as an anorexigenic peptide. its precursor protein, non-esterified fatty acid / nucleobinding 2 (nucb2) is expressed in cns and peripheral tissue. (20). nf-1 is secreted centrally from the hypothalamus and freely crosses the bbb. in addition, it is released peripherally from gastric mucosa, adipose tissue, pancreas, and testis tissue (21). many studies have shown that central or peripheral nf-1 injection significantly reduce food intake in rodents (22,23,24). also, nf-1 may affect absorption and digestion of food which is explained by reduced nucb2 mrna expression in git and hindered fasting gastric emptying (25). injection of nf-1 in brain of leptin-receptor mutant rats can suppress food intake by activating the melatonin system, irrespective of the leptin pathway (22). direct inhibition of an orexigenic substance is a possible mechanism for understanding the inhibition of food intake by nf-1. in vitro study has shown that nf-1 induces hyperpolarization in arc nuclei that are responsible for the secretion of npy (26). studies on various physiology parameters are ongoing to clarify differences in the nf-1 pathways. npy and the α-melanocyte-stimulating hormone (α-msh) have a controversial effect on the nf-1/nucb2 neurons of the pvn that are activated by α-msh and inhibited by npy; both effects are mediated by regulating cytosolic calcium ion levels (27). finally, nf-1 has been reported to regulate gastric motility through its effect on the pvn and the lha (28). peripheral effects of nf-1 seem to be more concerned about decreasing gastric motility and increasing the glucose-stimulated release of insulin (29,30). 3cholecystokinin cholecystokinin (cck) is biosynthesized and released by endocrine i cell in the mucosal lining of the small intestine, as well as neurons of the enteric nervous system, and neurons in the brain (31). circulating cck levels are increased at 14 minutes after food intake and persist about 3 hours; cck is inactivated by tripeptidyl peptidase ii. fat and protein-rich foods are strong release stimuli. while, duodenal bile acids are powerful biological cck release suppressors (32). cck actions are mediated via binding to two g protein-coupled receptors, ccka and cckb. ccka exists in many tissues, including git, the pancreas, hepatitis, and vagal afferents, whereas cckb is the dominant type in the cns, especially the brain (33). the cck-induced satiation by linking ccka receptors on the vagus nerve (34). so, the cck can, directly and indirectly, transmit satiation signals to the brain. the cck's physiological functions are to promote enzyme release, facilitate git motility, and defer gastric emptying. cck and leptin produce short-term food inhibition and promote long-term body weight loss, which can be considered as a potential target for obesity treatment (4). the primary clinical area of focus for ccka receptor agonists was obesity treatment (35). variability in cck-1 receptors can enhance obesity propensity. cck seems to be more important in satiation than satiety (36). the manipulation of cck-satiation signals by drugs has been found to impact eating behavior by influencing meal size but not the number of meals (37). nevertheless, the study shows that cck has an essential role as an anti-obesity counterbalanced by reducing the food intake via raising the number of meals without altering body weight (38). administration of ccka receptor agonists to obese subjects has failed to reduce body weight (39). however, coadminstration of cck and leptin in rats has shown to have a synergistic effect on weight loss (40). it is worthy to mention that both ccka and leptin receptors are expressed on vagal afferent neurons that may explain the synergistic effect between cck and leptin in weight reduction (41). 4peptide tyrosine-tyrosine peptide tyrosine-tyrosine (pyy) is anorexigenic peptide that consists of thirty-six amino acid residues. structurally, it is characterized by the iraqi j pharm sci, vol.30(1) 2021 gastrointestinal peptides role in appetite 17 presence of pancreatic polypeptide (pp)fold, similar to other peptides like npy, gip, and pp. pyy is synthesized by the intestinal enteroendocrine l-cells of the ileum and colonalong with other gut like glp-1 and oxyntomodulin (oxm), and is secreted in response to food intake (42). pyy secretion is proportional to meals' quality, and its circulatory levels increase to up to 6 hours within 2 hours of food intake (43). there are two significant forms of pyy found in circulation, pyy1-36 and pyy3-36. pyy1-36 is cleaved into pyy3-36 by dipeptidyl peptidase 4 (dpp4). pyy336 may act centrally by completive inhibition of npy y2 receptor (y2r) on npy neurons suppressing food intake. decreased endogenous levels of pyy in obese subjects when compared to lean subjects (44). obese people have lowered pyy336 levels while fasting pyy3-36 levels have been elevated since gastric bypass operation and other conditions associated with reduced appetite (43). it is suggested that increased fast and postprandial levels of pyy in massively obese individuals play a significant role in their dramatic weight loss after gastric sleeves (45). 5glucagon-like peptide-1 glucagon-like peptide-1(glp-1) formed by differential processing of the proglucagon gene in ileum cells and colon l cells (46). central and peripheral glp-1 behaviors are essential to appetite control. in normal conditions, glp-1 secretion triggered by intake of food rich with glucose and fatty acid, it is serving as an "incretin" and causing increased insulin secretion after food intake, thus, influencing glucose homeostasis. other glp-1 actions involve repression of glucagon secretion, impaired gastric emptying, and git motility suppression. the half-life of glp-1 is short due to the quick degradation of an active form into inactive form, following dipeptidyl peptidase-4 (dpp-4) disconnecting 2 terminal amino acids. (47). glp-1 has anorexigenic effects via the large-scale receptors in the git, pancreas, and brain (48). also, it reduces the rate of food absorption into circulation by decrease the gastric emptying rate (49). glp-1 was the first reliable git hormone utilized for human medicine. many preparations of glp-1 receptor agonists and dpp-iv blockers for type 2 diabetes mellitus (type 2 dm) treatment are presently available (50). the effectiveness of glp-1 receptor agonists in promoting weight loss at doses used to regulate diabetes has restricted effectiveness (51). obesity preserves the anorectic action of glp-1. consequently, diminished glp-1 secretion may lead to obesity pathogenesis. the impact of glp-1 on appetite and food intake may help weight loss (52). the glp-1 normalizes glycosylated fructosamine, decrease glycated hemoglobin, and reduces body weight after 6 weeks of treatment in type 2 dm patients. (53). therefore, glp-1 is considered good medicine to control appetite and treat obesity and type 2 dm (45). 6oxyntomodulin oxyntomodulin (oxm) is a pro-glucagon 37-amino acid peptide product, it is synthesized and secreted from the intestinal l-cells with a response to caloric consumption. oxm reduces pancreatic secretions and gastric motility and secretions and stimulates glucose metabolism (54). oxm joins both the glucagon and glp-1 receptors, while its effect on appetite mediated by glp-1 receptor since the treatment with a glp-1 receptor blocker prevents the anorectic effects of oxm (54). oxm offers an exciting possibility as an antiobesity because of its binary actions on food intake reduction and increased energy expenditure. it is acts as a glucagon receptor agonist to raise energy expenditure without effect on glucose levels. the agonist action of oxm on both glp-1 and glucagon receptors confirmed by reduced body weight without disruption of glycemic control, and an improved lipid profile in diet-induced obese animals, which gives suggestions the future expansion of obesity therapies (55, 56). obesity control with gut peptide therapies obesity is a worldwide health problem, which is a major contributor to cardiovascular disease and cancer. the world health organization has forecast an overweight of about two billion adult populations and million obese by 2015 (57). obesity is defined as an imbalance between consumption and expenditure of energy. food intake is regulated via orexigenic and anorexigenic peptides. a chronic imbalance between signals of hunger and satiety increases the risk of obesity (58). nearly every medicine and compartmental treatment for obesity lead to weight reduction and weight recovery. conversely, gastric sleeve is a treatment of obesity that provides safe weight loss control for an extended period. the mechanisms following the bariatric surgery for long-term weight loss have yet to be identified; however, numerous gut hormones are involved in this; ghrelin decreases and increases in pyy and glp-1 levels are noted after bypass surgery (59). inhibiting the response of pyy and glp-1 gave rise to appetite and increases food intake. thus, high levels of pyy and glp-1 play an essential role in weight loss after stomach bypass surgery (60). gastric bypass surgery can not consider alone for the treatment of obesity due to its costly and major complications associated with each surgery. pharmacotherapy is necessary, leading to substantial, treatable, continuous loss of weight, diabetes, and cardiovascular health improvement (60). iraqi j pharm sci, vol.30(1) 2021 gastrointestinal peptides role in appetite 18 for the following reasons, gut hormones have developed as the main type of target management of obesity. also, surgical treatment of obesity is known to increase the postprandial release of gip like glp1, oxyntomodulins, pyy, and it is supposed to lead to many of the metabolic perks of this surgery (60,61). the restriction of these hormones in patients with gastric bypass surgery inverts some advantages of surgery such as decreased appetite (62). the pyy and oxm combination causes a preferable decrease of appetite relative to the hormone alone (63). also, when giving oral pyy3-36 together with glp-1736 amide in conjunction with sodium n-caprylate to 12 healthy human-caused a significant reduction of energy consumption in the dinner served 15 minutes later (64). oxyntomodulin and pyy hormones can also reproduce the levels of post-prandial intestinal hormones shown after gastric bypass when given to 10 obese healthy individuals as a subcutaneous infusion for 10 hours (65). dual and even three-fold agonists of the intestinal hormones can serve as the basis for optimal body weight loss and a new obesity treatment strategy. however, many remedies do not produce a dramatic weight loss of over 5 percent and cause serious side effect like fatigue, heart disease, and neurological effects (66,67). thus, hormones must control food consumption, digestion, and, therefore, bodyweight without systemic administration's dangerous effects. due to the administration of nutrients or medication compounds, the enter endocrine network could raise weight reduction efficiency and mimic physiological sensors of feeling full and appetite control (68). to induce endogenous gut hormone discharge, nutritional stimulation of the enter endocrine l cell taste receptors into the distal small intestine and colon must be regarded. in reality, sweet taste receptor in vitro stimulation in immortalized and primary l cell crops induces hormone release, including oxm, glp-1, and pyy (69). thus, it is promising to specifically target nutrient receptors via oral or rectal administration with further study is required. endogenous secretion of gip may have an appropriate therapeutic choice for diabetes type 2 and obesity (70). conclusion the hypothalamus combines git signals with signals of other peripheral tissues or external environments. the processing of all input signals in cns produces different compensatory responses to maintain energy homeostasis. the role of peptide hormones derived from the git is controlling body weight and energy homeostasis. decrease appetite increases satiety with enhancing energy expenditure are criteria required to control the body weight. the characteristic of gip fulfills these criteria make them more effective therapeutic approaches against obesity and obesity-related diseases. further research needed to explain the effectiveness of gut peptide as an effective therapeutic strategy for weight gain to reduce morbidity and mortality related to obesity. acknowledgments the authors are very thankful for the support from university of baghdad, college of pharmacy, baghdadiraq. references 1. mithieux g. nutrient control of hunger by extrinsic gastrointestinal neurons. trends endocrinol metab. 2013; 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104: 15069–15074. 69. steinert re, gerspach ac, gutmann h, asarian l, drewe j, beglinger c: the functional involvement of gut-expressed sweet taste receptors in glucose-stimulated secretion of glucagon-like peptide-1 (glp-1) and peptide yy (pyy). clin nutr. 2011; 30: 524–532. 70. posovszky c, wabitsch m. regulation of appetite, satiation, and body weight by enteroendocrine cells. part 2: therapeutic potential of enteroendocrine cells in the treatment of obesity. hormone research in paediatrics. 2015;83(1):11-8. baghdad iraqi journal pharmaceutical sciences by bijps is licensed under a creative commons attribution 4.0 international license. copyrights© 2015 college of pharmacy university of baghdad. http://bijps.uobaghdad.edu.iq/index.php/bijps.com http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ type 2 dm is known to be a disease characterized by an inadequate beta-cell response to the progressive insulin resistance that typically accompanies advancing age, inactivity, and weight gain [9] iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 105 the correlation between hyperglycemia and rheumatoid factor in type 2 diabetic patients in alrisafa area, baghdad raya k. mohammed salih* ,1 , ali m. al-gharawi** and khalid i. al-lehibi*** * ministry of health, kimadia /dgmi relation section, baghdad, iraq. ** ministry of higher education , college of health and medical technology, baghdad, iraq . ***ministry of health, specialized center for endocrinology and diabetes , baghdad, iraq . abstract diabetes mellitus type 2 (t2dm) formerly called non-insulin dependent diabetes mellitus (niddm) or adult-onset diabetes is a common disease. rheumatoid factor is a well-established test used in the diagnosis and follows the prognosis of rheumatoid arthritis (ra). rheumatoid factor is sometimes found in serum of patients with other diseases including diabetes mellitus (dm), due to the presence of pro-inflammatory cytokines such as tnfα which play an important role in chronic inflammatory and autoimmune diseases like rheumatoid arthritis (ra). the aim of the study is to investigate the associations between type 2 diabetes mellitus (t2dm) and rheumatoid arthritis (ra) in scope of rheumatoid factor (rf), hyperglycemia and body mass index (bmi), in patients with t2dm lived in al-risafa area -baghdad. one hundred twenty five (125) type 2 diabetes mellitus (t2dm) patients were selected from the out patients department of the specialized center for endocrinology and diabetes, baghdad; in addition to (70) apparently healthy non diabetic, non arthritic subjects as control, during the period from sep. dec./2010. the ages of both patients and control subjects were within (35-75) years. this study focus to search for the correlation between t2dm and rf "qualitative and quantitative" in relation to body mass index (bmi) and gender. out of 125 dm-patients (73 female and 52 male), 44 (35.2 %) showed positive rf when compared with healthy controls (n=3, 4.3%). [p value =0.01 is significant] with female dominance (n=28, 63.6%) in compared to males (n=16, 36.4 %), when these diabetics with rf positive were titered for rf (8, 16, 32 and 64 iu/ml), the following results were obtained. the highest percentage of titer observed with 34.1% in those with rf titer 64 iu/ ml [p value = 0.01] when compared with healthy control. 18.2 % had rf titer of 8 iu/ ml, 20.4 % had rf titer of 16 iu/ ml, 27.3 % had rf titer of 32 iu/ ml and 34.1 % had rf titer of 64 iu/ ml. the highest percentage among the overweight, dm patients (38.9 %) have a mean titer 64 iu/ml, a percentage decrease respectively as below: 38.9 % had rf titer of 64 iu/ ml, 27.8% had rf titer of 32 iu/ ml, 16.6 % had rf titer 16 iu / ml and 16.6% had rf titer 8 iu/ ml. the highest number and percentage of dm with rf positive (n=17, 38.6 %) were located among higher age (50-59), (60-69) & (70 -79) year groups (n=17, 38.6%), (n=13, 29.5%) & (n=8, 18.2%) respectively, [pvalue < 0.01] when compared to the corresponding controls. the effect of fasting plasma glucose level of type 2 dm in patients who have rf positive titer, is found that > 7.2 mmol/l glucose in plasma contribute the highest titer (n=28, 63.6 %), in comparison with group of plasma glucose levels < 7.2 mmol/l patients (n=16, 36.4%). with a highly significant difference, p-value = 0.006.smokers diabetic patients with rf positive (n=27, 61.4%) dominate over nonsmokers with rf positive (n=17, 38.6%). the results of this study indicate that there is a reasonable increased frequency of positive rheumatoid factor (rf) in type 2 diabetic patients. poor glycemic control is associated with higher rf titer in positive cases. the titer of t2dm smoker patients is associated with positive rf values that exceed the titer of the nonsmoker rf positive patients. thus, smoking might not be correlated significantly to dm, but may contribute to its complications. key words: t2dm, rf, bmi, smoking. منطقة نعامم انرثىانً فًاننىع انثانً وظهىر ا -انعالقة بٍن ارتفاع انسكر عند مرضى انسكري بغداد انرصافه ٌا كمال محمد صانح* ر ،1 ٍبً*** ابراهٍمانغروي** و خاند محمد ، عًه انهه ٚا ٔصاسج انظحح ، * ًاد ، تغذاد ، انؼشاق . كٛ ح، تغذاد ، انؼشاق . ** ٛاخ انطٛث ح انرُم ٙ ،كٛه ى انؼان ٔصاسج انرؼٛه شكض صاسج انظحح ، اًن ٘ ، تغذاد ،انؼشاق. ***ٔ ٔانسكش ًرخظض نهغذد ان الخالصة ع انثاَٙيُٚؼذ ٍ كٌا انز٘ type 2 diabetes mellitus (t2dm) شع انسكش٘ انُٕ ٕٛن ذ ػٗه االَس ٚسًٗ ساتما داء انسكش٘ انغٛش يؼًر (niddm) ٘انؼايم انشٕثٌٔأ فحض يٍ االيشاع انشائؼح انثانغٍٛ ٘ٔأ سكش rf ًفاطمهرحمك يٍ نٚسرخذو ٕاَٙ ذشخٛض ذطٕس يشع ان ) انشث ra) rheumatoid arthritis م ع يٍ انرحٛه زا انُٕ ْ ح جاٛت ٓاب rfٚالحع ٚا ٕد يسثثاخ االنر ج رنك ٕن ٔ ٓا انسكش٘ ًُ ؼ ٚ ش يٍ االيشاع نكٛث ٍ كٛ ٕٚر ّ كًش ( α tnf-)يثم cytokineانسا ّ انزاٛذ ُاػ ٔايشاع اًن ٍ ضي ٓاب اًن ٙ االنر ًا ف ٙ ذهؼة دٔسا يٓ ٕاَٙ انر ٓاب انشث شذثط raع االنر ٔكزنكٚ كٍٛ ٕٚر ٕنٍٛ cytokine ْزا انسا يح االَس يمٔا ٔ ًُّ َا تانس ػششٌٔ ).أخرش خًسحٔ ٔ 521ٌياّئ ؼإَ ىٚ ٙ -يٍ يشع انسكش٘ ( يشٚؼا أثثد أَٓ ع انثَا انُٕ type 2 diabetes mellitus (t2dm) يُطمح ان ٙ ٘ ف ٔانسكش ٙ نهغذد انظى شكض انرخظظ ٙ اًن ٍ يشاجؼ ٗ ) -شطاّفي ًاً )07تثغذاد ، إَػافح إن سٛه ٙ َٕا ٌ انؼايم انشث ًٕه ح الٚ ٔ٘ ٍ تانسكش ى غٛش يظاٛت ك أَٓ ش يغ انرذٛل كًجًٕػح سٛطشج. rheumatoid factor (rf)تانظْا 1 corresponding author email : rayakamal38@yahoo.com received : 17/1/2012 accepted : 24/4/2012 iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 106 ؤالء ٕاَٙ ، أُدخمْ ًفاطم انشث ٓاب ان ٔانر ع انثاَٙ ٍ يشع انسكش٘ انُٕ ِز انذساسح نرحش٘ انؼال ّل ٛت ْ rheumatoid arthritis (ra)األشخاص ٙف ٕاَٙ ًال انؼايم انشث ًَّاً( rheumatoid factor (rf)تاسرؼ ك ػأً َٕ(qualitative & quantitative) ؼايم كرهح انجسى ًا ًت ػاللٓر ٔ)body mass index (bmi) ل أشٓش ) أستؼح. أُجش٘ انثحث خالل ٕٚه ٌ األٔل -أ د 2252كإَ كَا ًاس(ٔ كزنك يجًٕػّ أػ شػٗٔ ٍ انسٛطشجاًن ح ٛت ذرشٔا ى ػاو. )51 -51) ػذْد ٕاَٙ يٕجثاً % (51.2)٘أيشٚغ 44ٌأ ذثٍٛ 521يٍـ يجًٕع يشػٗ انسكش٘ٔ ى لذ أظٓش انؼايم انشث ًماسَحيُٓ يغ تان ى انسٛطشجح ػيجًٕ ٛاَسثح يهحٕظح يغ .%(4.5٘أ ) 5 ٔػذْد ٕاَٙ أكثش يٍ إطاتح انشجال [p= 0.01] . .إحظائ ساء تانؼايم انشث د إطاتح اُن 22 ٔكَا ًِٛـ53.4ّ٘أ )يشٚغ 53إٗن %(35.3٘أ )يشٚؼّ ٚمح انك ٕاَٙ تانطش ح انؼايم انشث جاٛت ٔا ٚا ؤالء انزٍٚ أظٓش ْ ُذ فحض ٙ. ػ ٕاـن ّ%( ػهٗـ انر quantitative ٚٙه نذسجح حع يا ٕاَٙ ، ٕن ش54.5ِ : انؼايم انشث ؼٚا ٓى أظٓش دسجح اًن ى أظٓش iu/ ml ٔ 25.5% 64 % يُ شِ دسجح يُٓ ؼٚا 52 اًن iu/ ml ٔ4ٔ22 ِش ؼٚا ى أظٓش دسجح اًن شِ iu/ml ٔ52.2 16% يُٓ ؼٚا ى أظٓش دسجح اًن ٗ .iu/ ml 8 % يُٓ شػ ؤالء اًن سّث يٍْ َ ٌأ أػٗه ٕا ػً ٕاَٙ ٘ر دسجح انكى كَا يٍ ثى ذمم ػٗه انرؼالة. iu/ ml 64 ٍ يجًٕػح انؼايم انشث ٚادج يهحٕظح [،ٔ ٍ .] .p= 0.01ص ٍُٛ ي أظٓش أغهة انثٚذ ى ػًٍ يجًٕػح ٕاَٙ overweight bmi (52.3%)يشػٗ انسكشْ٘ ٛاً يٍ انؼايم انشث شِ ػان ٌ (iu/ml 64) يؼٚا ٔا ػٗه انرؼالة. ٔانثإل ظٓش شِ% 52.3 ٓى أظٓش دسجح يؼٚا ِ iu/ ml ٔ 25.2 64 يُ ش ٓى أظٓش دسجح يؼٚا شِ iu/ ml ٔ 5363 52 % يُ ى أظٓش دسجح يؼٚا /iu 16% يُٓ ml ٔ 53.3ِش ى أظٓش دسجح يؼٚا ٕا ػًٍ يجًٕػح . iu/ ml 8 % يُٓ ٕاَٙ كَا ظٓش٘ انؼايم انشث ٔاًن َٔسّث يٍ يشػٗ انسكش٘ ٌإ أػٗه ػذد ح ًاس انؼاٛن )13-12)األػ ٔ)32-33( ى )52-53(ٔ َٔسثٓر ) 52.3يشٚغ، 55( ػاو ٗ 52.2، ػٗيش 2% ( ٔ ) 23.1، يشٚغ%55 (ٔ % ( ػه سّث يشػٗ انسكش٘ انرؼالة. َ كٌٕ فّٛ سكش انذو ٌأ ٔانز٘ٚ ٕاٙذ )يٙه يٕل نكم نرش 5.2≤ تذٌٔ سٛطشج ى انؼًم انشث %( أكثش 3563 ، 22ٔانزٍٚ نذٓٚ شع انسك سثح اًن َ سٛطش ػهّٛ > يٍ ٕاَٙ يٙه يٕل نكم نرش 5.2ش٘ اًن ى انؼًم انشث ٘ . (5364 ، 53) ٔانزٍٚ نذٓٚ ظاتٍٛ تانسكش ٍُٛ اًن ذخ ٌإ اًن ٕاَٙ ) ظٓش٘ انؼايم انشث ٕاَٙ )35.4، يشٚغ 25ٔاًن ظٓش٘ انؼايم انشث ٔاًن ظاتٍٛ تانسكش٘ ٍُٛ اًن ذخ ٕا اكثش يٍ غٛش اًن ، يشٚغ55%( كَا ّٛ أخشٖ ٌأ52.3 َاح يٍ ٕاَٙ ) %(ٔ. ٓى انؼايم انشث س نٚذ ٔٛن ٘ ظاتٍٛ تانسكش ٍُٛ اًن ًذخ ٓى 55.2، يشٚغ 35ػذد غٛش ان س نٚذ ٔٛن ٍُٛ ًذخ %( أكثش يٍ ان ٕاَٙ ) ٚادج يهحٕظح ػانّٛ 22.2يشٚغ ، 52انؼايم انشث ح ٙف ظٕٓس (.p.v= 0.000001 %(.)ص جاٛت ّٕن ٚا ٚاِد يؼم ُانك ص ْ ِز انذساسّ ذثثد ٌأ َرائجْ ٕاَٙ ن ٙ. -ٖذ يشػٗ انسكش٘ انؼايم انشث ع انثَا سثح انسكش ٙف انذو يغ اسذفاع انُٕ َ ٛاس انرشدد انشئٚشذثط ػؼف انسٛطشج ػٗه ٕاَٙؼ ٙف انحاالخ ث ح. ح انؼايم االٚجاٛت جاٛت ٛاس ٚا شذثٌا يؼ زا فٌا انرذخٍٛ لذ الٚ ظاتٍٛ تانسكش٘ ٓن ٔاًن ٍُٛ خ ظاتٍٛ تانسكش٘ أكثش يٍ غٛش اًن ٔاًن ٍُٛ ذخ ٕاَٙ ٙف اًن ط انشث ًا. ّ ٓت ًا تانراكٛذ راخ طه َٔا ٘ كًشع ًفاطم انشٕث ٔايشاع ان ٘ ِ تانسكش يثاشش انتدخٍن ،,rf, bmi انكهمات انمفتاحٍة : مرضى انسكري اننىع انثاًن introduction type 2 diabetes mellitus (t2dm) which was formerly called non-insulin dependent diabetes mellitus (niddm) or adult-onset diabetes, is a metabolic disorder that is characterized by high blood glucose level (1) .in t2dm, insulin concentrations may be normal or even high, there is insensitivity of the tissues to the effects of insulin(an effect termed insulin resistance) (2) .type 2 dm (t2dm) occurs as a result of chronic insulin resistance and subsequent beta-cell dysfunction that appears to be reversible, particularly in the early stages of the disease (3) . type 2 diabetes, is characterized by progressive insulin resistance that typically accompanies advancing age, inactivity, and weight gain (4) . rheumatoid factor (rf) rheumatoid factor is an antiimunglobulin with a course against fragment fc of igg human molecule. rheumatoid factor is present in (70-80 %) of patients with rheumatoid arthritis (ra), where the disease is defined as a seropositive athropathy (5) .the rfs frequently occur in a variety of other diseases such as, systemic lupus erythromatus (sle) (15-35%), systemic sclerosis (20-30%), juvenile rheumatoid (7-30%), poliomyelitis (5-10%) and infection (0-5%) (6) . rheumatoid factor is a well-established test used in the diagnosis and prognosis of rheumatoid arthritis (ra) (7) . in addition, rheumatoid factor precedes the appearance of rheumatoid arthritis. rheumatoid factor is sometimes found in serum of patients with other diseases including diabetes mellitus (dm), due to the presence of pro-inflammatory cytokines such as tnfα which play an important role in chronic inflammatory and autoimmune diseases like rheumatoid arthritis (ra). the tnfα has also been closely linked to obesity and insulin resistance (8) .substantial studies have been conducted in several iraqi regions, however; this study was planned to investigate the possible association between t2dm and rf in relation to bmi, age and smoking in alrisafa baghdad area. material and methods one hundred twenty five (125) patients with t2dm were attending the specialized center for endocrinology and diabetes, at al-risafa, baghdad, during the period from september to december/ 2010.age ranging between (35–75) years. in addition to seventy (70) age matched apparently healthy persons as controls, were selected from neighbours, friends and staff members of the college and who attended al-kindy general hospital for checking. their fasting plasma glucose (fpg) was within the normal range. all plasma specimens were submitted to fasting plasma glucose by enzymatic colorimetric method and rf – latex by slide agglutination test. assay methods 1. blood glucose determination (enzymatic). enzymatic, colorimetric method, based on glucose oxidase with reference for serum or plasma in fasting state (9) reference value of : 4.2-6.4 mmol/l 75-115 mg/dl 2. qualitative determination of rheumatoid factor [latex slide agglutination] principle of method: the rflatex is a slide agglutination test for the qualitative and semiqualitative detection of rf titer in human serum. latex particles coated with human e-globulin are agglutinated when mixed with sample http://en.wikipedia.org/wiki/metabolic_disorder http://en.wikipedia.org/wiki/blood_glucose iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 107 containing rf. the test occurs by using a kit (omega diagnostics avitex rf).uk. calibration the rf-latex sensitivity is a calibration against the word health organization (who) 1/64 rheumatoid arthritis serum. sample fresh serum should be used to detect measurable titer of anti-igg (rheumatoid factor). reading and interpretation the presence or absence of visible agglutination was observed by necked eye immediately after removing the slide from the rotator. semi-quantitative determination the semi-quantitative test was performed in the same way as the qualitative test using dilution of the serum with phosphate buffered saline as follows statistical analysis the parameters were treated and computerized by using spss version 15. p value < 0.05 is considered significant, while p value > 0.05 is considered non significant. dilutions 1/2 1/4 1/8 saline 50 µl 50 µl 50 µl serum 50 µl ─ ─ dilution serum 1/2 ─ 50 µl ─ dilution serum 1/4 ─ ─ 50 µl 8 x no of dilution 8x2 8x4 8x8 iu/ml 16 32 64 results out of diabetic patients, 44 (35.2%) are positive for rf, 16(36.4%) males and 28(63.6%) females, respectively. while the number and percentage of rf positive subjects out of healthy control are only 3 (4.3%), 1(33.33%) male and 2(66.66%) females, respectively. the differences are significant (p= 0.01). table 1: distribution of studied groups according to rf agglutination test diabetes mellitus patient healthy control comparison of significant gender rf male female total percent male female total percent p= 0.01 positive 16 28 44 35.2% 1 2 3 4.3% negative 36 45 81 64.8% 29 38 67 95.7% total 52 73 125 100% 30 40 70 100% table (2) shows the comparison of t2 dm patients & healthy control with rf positive in addition to rf negative according to their bmi(kg/m 2 ).the largest number and percentage of t2dm patients are the overweight patients 47 (37.6%). and when added to obese groups the total will be 108(86.4%).while the largest number and percentage for the control lie in the normal group of bmi 50 (71.4%), with a highly significant difference, p value < 0.01 between bmi and rf in dm as well as in control table 2: comparison of diabetic patients with healthy control who have rf positive and negative according to body mass index bmi (kg/m 2 ) diabetes mellitus patient healthy control comparison of significant rf bmi positive (n=44) negative (n=81) total (n=125) percent positive (n=3) negative (n=67) total (n=70) percent p < 0.01 under weight 16.5-18.4 0 3 3 2.4 % 0 0 0 0% normal 18.5-24.9 0 14 14 11.2 % 0 50 50 71.4 % overweight 25.0-29 18 29 47 37.6 % 1 14 15 21.4 % obese class i 30.034.9 12 16 28 22.4% 2 3 5 7.1 % obese class ii 35 40 10 12 22 17.6 % 0 0 0 0 % obese class iii over 40 4 7 11 8.8 % 0 0 0 0 % total 44 81 125 100% 3 67 70 100 % iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 108 table 3: distribution of diabetic patients who have rf positive and negative within age groups (years) compared with healthy control. diabetes mellitus patient healthy control comparison of significant rf age positive (n=44) negative (n=81) total (n=125) percent positive (n=3) negative (n=67) total (n=70) percent p =0.00l8 30-39 0 6 6 4.8% 0 7 7 10.0% 40-49 6 22 28 22.4% 0 21 21 30% 50-59 17 26 43 34.4% 1 25 26 37.1% 60-69 13 18 31 24.8% 2 9 11 15.7% 70-79 8 9 17 13.6% 0 5 5 7.1% total 44 81 125 100% 3 67 70 100% table (3) shows that the highest number and percent of t2dm with rf positive values are located within the age group (50-59) years. at the same age, healthy controls show also the highest number and percent. (pvalue =0.00l8) table 4:distribution of diabetic patients who have rf positive according to their glycemic control and rf titer. rf titers glycemic control level 8 16 32 64 total percent comparison of significant fpg > 7.2 mmol/l > (130 mg/dl) 2 5 9 12 28 63.6% p = 0.006 fpg < 7.2 mmol/l < (130 mg/dl) 6 4 3 3 16 36.4% total 8 9 12 15 44 100% table (4) summarizes the effect of fasting plasma glucose level of type 2 dm in relation to rf titer. it is found that > 7.2 mmol/l glucose in plasma contribute the highest titer (n=28, 63.6 %), in comparison with group of plasma glucose levels < 7.2 mmol/l patients (n=16, 36.4%). with a highly significant difference, p-value = 0.006. table 5: distribution of diabetic patients with rf positive according to their titer, in relationship to body mass index groups bmi (kg/m 2 ) rf titers iu/ml bmi 8 16 32 64 total percent under weight 16.5-18.4 0 0 0 0 0 0% normal 18.5-24.9 0 0 0 0 0 0% overweight 25.0-29 3 3 5 7 18 40.9% obese class i 30.034.9 4 3 3 4 14 31.8% obese class ii 35 40 1 2 3 2 8 18.2% obese class iii over 40 0 1 1 2 4 9.1% total 8 9 12 15 44 100% as indicated in table (5) it could be conclude the following two points. most of type 2 dm patients show rf positive (34.1%) high titer in 64 iu/ml: titer ( iu/ml) number percent 64 15 34.1 % 32 12 27.3 % 16 9 20.4 % 8 8 18.2 % most of overweight patients show high titer 64 iu/ml: (38.9 %) titer (iu/ml) number percent 64 7 38.9 % 32 5 27.8% 16 3 16.6 % 8 3 16.6% iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 109 table 6: distribution of smokers among diabetic patients with either rf positive or rf negative results rf smoke positive negative total percent comparison of significant positive 27 (61.4%) 18 (22.2%) 45 36.0% p= 0.000001 hs negative 17 (38.6%) 63 (77.8%) 80 64.0% total 44 (100%) 81 100%) 125 100 % a shown in table (6) smokers with rf positive t2 dm patients (n=27, 61.4%), dominate over the nonsmoker patients. the non smoker's diabetic patients who have rf negative (n=63, 77.8%) are nearly four times as many as smokers (n=18, 22.2%). table 7: distribution of rf positive diabetic patients according to smoking habit and rf titer rf titer iu/ml smoke 8 16 32 64 total percent comparison of significant positive 4 5 8 10 27 61.4% p= 0.000001 hs negative 4 4 4 5 17 38.6% total 8 9 12 15 44 100% table (7) explains the effect of smoking on t2dm/rf positive patients where the smokers are doubling as many as the nonsmokers, in titers 32 & 64 iu/ml, and the smoker's numbers are more than the non-smokers (4:4, 5:4, 8:4, and 10:5; total 27:17). the number of patients increases as the titer increases. discussion this work shows that about (35.2%) of the iraqi population sample (125 subjects) diabetic patients in al-risafa region have rf positive in their blood when compared with apparently normal subjects (4.3%) as referred in table (1). these percentages coincide with the results obtained by moustschen (10) in 1992.also coincide with searches done by al-gharawi at 2009, in medical city, baghdad, who observed that 62.5% out of dm patients showed rf positive (11) ; 49% have been recorded in al-umara city by khalawi (12) and 15.5% have been reported in al-adhamiya, baghdad city by al-hammami (13) .the differences are possibly due to geographical reason and because the attendants of the medical city are collection from different districts and most severely diseased. umara & a'dhamyah are confined areas. also they have different lifestyles, types of food & behavior. the result of this study (35.2%) rf positive dm patients seem to be affected by the same reasons stated above, and lie in between the results of the other authors. table (1) shows that 28 females and 16 males had rf positive (approximately 2:1 ratio) which coincides with the work that showed the women presented with ra more often than men, with a ratio of 3:1 (14) , indicating that hormone levels are of importance (15) . epidemiological and immunological evidence share suggested that female sex hormones could play a role in the etiology and course of chronic inflammatory diseases because of the menstrual cycle, pregnancy, and menopausal status which are important influencing factors (16) . table (2) shows the prevalence of t2dm with rf positive according to their bmi. a significantly higher level concentrated at overweight and obese groups. the t2dm is associated strongly with overweight, independent of age, gender and family history of dm. this relationship has been found consistently in other populations. (17-21) diabetes mellitus and ra are associated with an adverse cardiovascular risk profile, particularly dyslipidaemia (22, 23) and obesity is a state of lowgrade chronic inflammation, as indicated by the increased concentrations of creactive protein, il-6, and other inflammatory markers identified in the plasma of obese individuals (24,25) . indeed, pro-inflammatory cytokines (tnf-α, il-18, il-6) were found to be increased in patients with t2dm. (26, 27) the tnf-α, a pivotal proinflammatory cytokine in ra, arises from adipose tissue during chronic hyperglycemia in t2dm and has harmful effects on the pathway of insulin signaling. (28) the coincidence of bmi level and severity of t2dm resembles the work which concluded that obesity is well recognized as important risk factor for t2dm and impaired glucose tolerance (29, 30) . parallel to this idea, in same table (2) which shows that the positive rf is concentrated in the overweight and obese; while all of the normal bmi(patients & control) had negative rf.table (3) shows that increased number of t2dm patients who have rf positive are aged 50 and older, which resembles that of the american diabetes association (ada), showing that approximately 18.3% (8.6 million) of the americans aged 60 and older have diabetes (31) and t2dm patients are at ages (40-70) years, which coincide with other workers (32,33) .diabetes mellitus in these tables show that the prevalence increases ../اطروحه/4%20encyclopedia/diabetes_mellitus_2.htm#cite_note-dlife-34#cite_note-dlife-34 iraqi j pharm sci, vol.21(1) 2012 hyperglycemia and rheumatoid factor in type 2 diabetic 110 with age. in a previous review, female's dominant over males explain by the role of the menopause on pro-inflammatory cytokine activity (34) .this review focused on the increase of pro-inflammatory cytokines with the menopause (the fall of estrogens and other gonadal steroids), another review on gonadal steroids and t and b cell immunity was presented 10 years ago, but since then, a lot of new information has been generated (35) . this is particularly true with respect to chronic diseases that formerly have not been allocated to “inflammatory diseases” such as bone resorption. this is important because rate of incidence over age for osteoporosis almost matches incidence rates of inflammatory markers, as rheumatoid arthritis (ra). table (4) shows that most of diabetic patients who have rf positive (n=28 , 63.6%) lie within fpg ≥ 7.2 mmol/l category in comparison with those with lower fpg (n=16 , 36.4%) which lie in fpg < 7.2 mmol/l. these results agree with other workers who showed that 68% of dm patients had fpg > 11.1 mmol/l and 32% of them had fpg < 11.1 mmol/l (13) .glucose intolerance table (5), presents in ra and diabetes, is another parallel, and indicator for direct correlation between the degree of impaired glucose handling and inflammation (36) . the diabetic patients who have rf positive titer, which mean the severity of disease go parallel with the increase of rf titers. comparatively, the rf in blood of the control, 4.3%, is too near to the international ratio, 3 %. in this study the results are in agreement with several studies have discussed the association between chronic inflammatory disease states and disorders in intermediary metabolism (37-41) , in particular, peripheral insulin resistance (ir).the overweight groups also gain the highest score (38.9%) among the highest rf positive titer, the 44 diabetic with rf positive value agrees with other studies (12, 13) .tables (6 and 7) show the prevalence of t2dm smokers over t2dm non-smokers to have rf positive. issues of smoking and diabetes are correlated effectively in the ada technical review (42) , it is concluded that smoking might not be the causative agent for t2dm, but definitely is related to it. these tables also show the prevalence of rf positive in smokers over non-smokers, accordingly this model explains that ra results from a complex gene–environment interaction, in which ra only develops after the immune systems has been triggered by several environmental factors, a process which may take years (43) . one of the environmental factors that have been clearly shown to trigger ra is smoking (44) . references 1. giuffrida fm, reis af. genetic and clinical characteristics of maturity-onset diabetes of the young. diabetes obesmetab 2005; 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